Occupational adaptation: toward a holistic approach for contemporary practice, Part 1. A theoretical perspective designed for clinical application and based on fundamental occupational therapy principles is offered. This perspective, the occupational adaptation frame of reference, is presented as an articulation of (a) a normal developmental process leading to competence in occupational functioning; (b) the process through which the benefits of occupational therapy occur; and (c) a perspective that promotes holistic practice. The person is viewed as operating occupationally through an idiosyncratic configuration of sensorimotor, cognitive, and psychosocial systems, all of which are inevitably involved in each occupational response. This occupational functioning is described as occurring through interaction of the person with a work, play and leisure, or selfcare context that has distinctive physical, social, and cultural properties (i.e., the occupational environment). Occupational adaptation is a perspective that can influence practice, education, and research.


The use of electromyographic biofeedback in treating a client with tension headaches. EMG biofeedback was used during a 4-week treatment program to decrease upper trapezius activity related to tension headaches. The headaches were believed to be caused by general tension and anxiety and affected the patient's ability to adequately attend to activities of daily living, including child care, homemaking, and vocational activities. The program combined deep-breathing exercises, progressive muscular relaxation exercises, resisted shoulder elevation exercises, and EMG monitoring during upper extremity tasks involving shoulder flexion. An important adjunct to clinic sessions was a home exercise program to be completed 2 to 3 times per day. In this case, the EMG biofeedback program was successful in assisting the patient to eliminate tension headaches for at least an 8-week period. At the same time, the patient reported an increased ability to attend to activities of daily living, stating that she was able to "do more around the house" and had not missed a day at work in 2 months.


AMP deaminase binding in contracting rat skeletal muscle. AMP deaminase, which hydrolyses AMP to inosine 5'-monophosphate (IMP) and NH3 at high rates during excessive energy demands in skeletal muscle, is activated when bound to myosin in vitro. We evaluated AMP deaminase binding in vivo during muscle contractions to assess whether binding 1) is inherent to deamination and found only with high rates of IMP production or simply coincident with the contractile process and 2) requires cellular acidosis. AMP deaminase activity (mumol.min-1.g-1) was measured in the supernatant (free) and 10(4)-g pellet (bound) homogenate fractions of muscle of anesthetized rats after in situ contractions to determine the percent bound. In resting muscle, nearly all (approximately 90%) AMP deaminase is free (cytosolic). During contractions when energy balance was well maintained, binding did not significantly differ from resting values. However, during intense contraction conditions that lead to increased IMP concentration, binding increased to approximately 60% (P less than 0.001) in fast-twitch and approximately 50% in slow-twitch muscle. Binding increased in an apparent first-order manner and preceded initiation of IMP formation. Further, binding rapidly declined within 1 min after cessation of intense stimulation, even though the cell remained extremely acidotic. Extensive binding during contractions was also evident without cellular acidosis (iodoacetic acid-treated muscle). Thus the in vivo AMP deaminase-myosin complex association/dissociation is not coupled to changes in cellular acidosis. Interestingly, binding remained elevated after contractions, if energy recovery was limited by ischemia. Our results are consistent with myosin binding having a role in AMP deaminase activation and subsequent IMP formation in contracting muscle.


Altered kinetics of AMP deaminase by myosin binding. AMP deaminase catalyzes the deamination of AMP to inosine 5'-monophosphate (IMP) and ammonia. Factors controlling the enzyme in muscle can rapidly promote high rates of IMP formation when ATP utilization exceeds supply. We evaluated whether binding of AMP deaminase to myosin, which occurs during intense contraction conditions, alters the kinetic behavior of the enzyme. Reaction kinetics of myosin-bound and free AMP deaminase were evaluated. Reaction kinetics of the free enzyme yielded a near-linear double-reciprocal plot with an expected Km of approximately 1 mM AMP concentration (AMP). In contrast, reaction kinetics of AMP deaminase became bimodal when bound to myosin. At [AMP] less than 0.15 mM, a high-affinity Km (0.05-0.10 mM) with maximal velocity approximately 20% that of free enzyme was evident. At [AMP] greater than 0.15 mM, the Km and maximal velocity values were similar to that of the free enzyme. The 10- to 20-fold higher affinity Km would allow for a higher rate of AMP deamination at the low [AMP] found physiologically. AMP deaminase binding to myosin also induced a marked resistance to orthophosphate inhibition (10 mM) in the presence of 50 microM ADP. Results were similar for purified preparations of AMP deaminase bound to myosin subfragment 2 and crude extracts obtained from contracting muscle. Our results add further support to the hypothesis that AMP deaminase binding to myosin serves an important role in control of enzyme activity in contracting muscle.


Changes of cytosolic Ca2+ interfere with measurements of cytosolic Mg2+ using mag-fura-2. In rat pancreatic and submandibular gland acini during exposure to carbachol, changes in the fluorescence emission intensity ratio (R) of acini loaded with mag-fura-2 resemble changes in cytosolic Ca2+ concentration (Ca2+i) in acini loaded with fura-2. Furthermore, changes of R depend on the presence of extracellular Ca2+ (Ca2+o) but are much less influenced by changes in extracellular Mg2+ (Mg2+o). To evaluate interference with measurement of cytosolic Mg2+ (Mg2+i) by changes in Ca2+i, we determined the dissociation constant (Kd) and Hill coefficient (NH) of the Ca(2+)-mag-fura-2 and Mg(2+)-mag-fura-2 complexes in standard solutions, in intact acini after loading with the acetoxymethyl ester of mag-fura-2 (mag-fura-2/AM), and in lysates derived from acini loaded with mag-fura-2/AM. The Kd of the Ca(2+)-mag-fura-2 complex in acini (determined with ionomycin) was 20.49 +/- 5.20 microM, and NH was 1.44 +/- 0.16 (n = 24). Kd of the Mg(2+)-mag-fura-2 complex in acini was 2.25 +/- 0.98 mM, and NH was 1.20 +/- 0.20 (n = 25). Mean Kd values were slightly lower in acinar lysates and in solutions of standard mag-fura-2. In acini from either gland, 1,2-bis(2-aminophenoxy)ethan-N,N,N',N'-tetraacetic acid (BAPTA) suppressed carbachol-induced Ca2+i transients. The R value in stimulated acini loaded with BAPTA and mag-fura-2 increased slightly when Mg2+o was increased from less than 10 nM to 1.2 mM, suggesting that Mg2+ influx contributes to the maintenance of Mg2+i during exposure to carbachol. Under these conditions, pancreatic acinar Mg2+i is 0.53 +/- 0.14 mM (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)


Differential modulation of a sodium conductance in skeletal muscle by intracellular and extracellular fatty acids. Voltage-activated sodium channels of cultured skeletal muscle show diametrically divergent responses to intracellular vs. extracellular exposure to free fatty acids. Intracellular exposure to 1-20 microM arachidonic acid increased the magnitude of voltage-activated sodium currents, but not potassium currents, in whole cell recordings of human primary muscle cells and in the C2C12 mouse cell line. Oleic and stearic acids also stimulated increased sodium currents. In contrast, extracellular exposure to 5-10 microM arachidonic acid reversibly inhibited inward currents. Externally applied oleic acid was a less effective inhibitor, and stearic acid (up to 20 microM) produced no inhibition. The difference in sodium current responses to intracellular vs. extracellular exposure indicates that fatty acids can modulate skeletal muscle sodium channel function by at least two different pathways.


Interaction of calcium with plasma membrane of epithelial (MDCK) cells during junction formation. We have previously shown that upon transferring confluent monolayers of Madin-Darby canine kidney (MDCK) cells from low- to normal-Ca2+ medium, cytosolic Ca2+ increases and tight junctions (TJs) assemble and seal, but the increase in cytosolic Ca2+ does not seem to be necessary for junction formation. In the present work we establish that these are in fact two independent phenomena. We first measured unidirectional Ca2+ fluxes across the plasma membrane of MDCK cells to find suitable inhibitors and tested their effects on the ability of Ca2+ to seal the TJ. Likewise, we studied a variety of multivalent cations. We observed that 1) Ca2+ triggering of junction formation does not depend on its entering the cell, 2) cations like La3+ may impair the influx of Ca2+ without affecting the sealing of TJs, and 3) only Cd2+ is able to block both Ca2+ penetration and junction formation; however, 4) Cd2+ itself cannot trigger junction formation. We interpret that Ca2+ triggers junction formation by acting mainly on an extracellular membrane site and that this site has a higher Ca2+ selectivity than the mechanisms for Ca2+ translocation across the membrane.


Acceleration of growth of cultured cardiomyocytes and translocation of protein kinase C. Phorbol 12-myristate 13-acetate (PMA), norepinephrine (NE), and contraction stimulate cardiomyocyte growth (increased protein content). Differences exist in the time course and extent of protein and RNA accumulation. Cells plated at 4 x 10(6) cells/60-mm dish and arrested with 50 mM KCl demonstrated no significant growth. Treatment with PMA stimulated growth to a maximum of 17% at 48 h. In contrast, maximal stimulation of growth was 36% at 48 h and 31% at 72 h for contracting and NE-treated cells, respectively. Maximal stimulation of the capacity for protein synthesis (RNA content) was 32% for PMA-treated cells at 24 h compared with 59% and 77% for NE-treated and contracting cells, respectively, at 72 h. In support of a primary role for altered capacity in the regulation of protein synthesis, there was a significant correlation (r = 0.84) between RNA and protein contents that was independent of the stimulus used. Angiotensin II increased RNA content by 28% at 48 h but had no effect on growth up to 72 h. Growth stimulation and increased nuclear protein kinase C (PKC) activity were induced by contraction, NE, and PMA treatment and were inhibited by staurosporine (a PKC inhibitor), suggestive of a central role for PKC.


Hypoxia induces glucose transporter expression in endothelial cells. Endothelial cells in various tissues of the body are often exposed to hypoxic conditions. To examine the effects of sustained hypoxia on energy metabolism in endothelial cells, we have maintained bovine aortic and human umbilical vein endothelial cells in an atmosphere containing low oxygen concentrations (14 mmHg) for up to 96 h. We report here that endothelial cells maintained under these conditions upregulate their glucose transport activity, consume more glucose, and produce greater amounts of lactic acid than normoxic cells. Upregulation of glucose transport activity by hypoxic endothelial cells required several hours to occur, was associated with increased expression of mRNA and protein for the erythroid/brain form of the facilitative glucose transporter, and was not due to depletion of glucose from the medium. Prolonged treatment of endothelial cells with inhibitors or uncouplers of oxidative phosphorylation (antimycin, azide, dinitrophenol) under normoxic conditions also upregulated glucose transporter expression. These results suggest that reduced rates of oxidative metabolism may represent an important signal for cells to adapt metabolically to hypoxia. Furthermore, in our examination of endothelial cell energy metabolism, we discovered that endothelial cells contain phosphocreatine and express both the brain and muscle isozymes of creatine kinase.


Depletion in nuclear spermine during human spermatogenesis, a natural process of cell differentiation. Polyamines (PA), polycations present in all mammalian cells, are essential for cell proliferation and differentiation. In vitro, PA are known to bind to DNA with a high affinity. In vivo, the intimate association of endogenous PA with highly condensed chromatin has been reported. During spermatogenesis, when processes of cell proliferation and differentiation take place, the potential role of polyamines has not been studied in depth. We report here the PA levels measured in human spermatogenic cell nuclei at different stages of differentiation. Cell populations (spermatocytes and round, elongating, or elongated spermatids) were obtained after submitting human testes to a trypsin-deoxyribonuclease digestion, then to a centrifugal elutriation and Percoll gradient centrifugation. A significant and progressive nuclear spermine level decrease was observed from primary spermatocytes to elongated spermatids. This release of spermine from nuclei was concomitant with three major events in mammalian spermiogenesis: the reduction of DNA transcription activity, the replacement of histone proteins by protamines, and the compaction of chromatin. This is the first report arguing a release of nuclear spermine during an in vivo physiological cell differentiation process.


Chloride secretory response to extracellular ATP in human normal and cystic fibrosis nasal epithelia. Chloride secretion across cystic fibrosis (CF) airway epithelia is effectively regulated by pathways associated with intracellular Ca2+ metabolism, but not by mechanisms dependent on protein kinase A or C. In a search for therapeutically useful agonists, we investigated the effects of extracellular ATP on the Cl- secretory process in human normal and CF nasal epithelial cultures with double-barreled Cl- selective microelectrodes. When applied to the basolateral membrane of normal, but not CF, nasal epithelium, extracellular ATP (10(-4) M) stimulated a small increase in Cl- secretion that was primarily associated with a hyperpolarizing conductance in the basolateral membrane. In contrast, ATP applied to the apical (luminal) membrane of either normal or CF nasal epithelium stimulated a greater increase in Cl- secretion that was associated with activation of an apical membrane Cl- conductance. The increases in Cl- current and apical conductance were greater in CF tissues and attained maximal values similar to normal nasal epithelium. We conclude 1) that basolateral application of ATP indirectly induces Cl- secretion by activating a basolateral (K+) conductance and is an effective secretagogue only in normal nasal epithelium and 2) that luminally applied ATP is an effective Cl- secretagogue that activates the apical membrane Cl- conductance of normal and CF nasal epithelia to an equivalent level.


Two-dimensional 31P-chemical shift imaging of intramuscular heterogeneity in exercising human forearm muscle. Two-dimensional phosphorus chemical shift imaging (2D-31P-CSI) was used to investigate macroscopic heterogeneity within the flexor digitorum profundus (FDP) muscle of the human forearm during exercise. Subjects performed low-frequency steady-state finger flexion exercise at submaximal work levels using a bulb ergometer. The number of fingers actively involved in finger flexion exercise was varied in a total of 12 experiments. Active muscles could be determined by the increase of Pi and decrease of phosphocreatine observed in the localized spectra. Within the FDP, regions of active and inactive fibers were significantly different in levels of Pi and phosphocreatine (P less than 0.01) during flexion of individual fingers. Individual flexion of the index finger was found to involve only fibers in the deep region of the FDP. Fibers in the central region were found to be involved in flexion of the middle finger, and fibers in the superficial region were involved in flexion of the ring and little fingers. The results of this study demonstrate the potential of 2D-31P-CSI for in vivo investigation of intramuscular heterogeneity in human skeletal muscle.


Early effects of aldosterone on apical and basolateral membrane conductances of TBM cells. The early effects (1-4 h) of aldosterone on membrane conductance were studied in the TBM cells, an epithelial cell line derived from the toad urinary bladder. Aldosterone stimulated Na+ transport (measured as short-circuit current) and induced parallel increases of the amiloride-sensitive apical membrane Na+ conductance and the basolateral membrane conductance. In another set of experiments, we prevented the aldosterone-induced increase of the Na+ transport by adding amiloride to the apical solution in concentrations sufficient to reduce short-circuit current to values similar to that of matched control. The effect of aldosterone on the basolateral conductance was not inhibited. This effect was essentially due to a change of the barium-sensitive K(+)-specific conductance. We also observed that aldosterone prevented a time-dependent decline of the paracellular resistance observed in control preparations. Aldosterone induces an early increase of the basolateral membrane conductance, an effect that is not secondary to the increase of the Na+ load to the cell.


Physiological fluid shear stress causes downregulation of endothelin-1 mRNA in bovine aortic endothelium. We report here that the level of endothelin-1 (ET-1) mRNA from bovine aortic endothelial cells grown in vitro is rapidly (within 1 h of exposure) and significantly (fivefold) decreased in response to fluid shear stress of physiological magnitude. The downregulation of ET-1 mRNA occurs in a dose-dependent manner that exhibits saturation above 15 dyn/cm2. The decrease is complete prior to detectable changes in endothelial cell shape and is maintained throughout and following alignment in the direction of blood flow. Peptide levels of ET-1 secreted into the media are also reduced in response to fluid shear stress. Cyclical stretch experiments demonstrated no changes in ET-1 mRNA, while increasing media viscosity with dextran showed that the downregulation is a specific response to shear stress and not to fluid velocity. Although both pulsatile and turbulent shear stress of equal time-average magnitude elicited the same decrease in ET-1 mRNA as steady laminar shear (15 dyn/cm2), low-frequency reversing shear stress did not result in any change. These results show that the magnitude as well as the dynamic character of fluid shear stress can modulate expression of ET-1 in vascular endothelium.


Protective effect of the dimer of 16,16-diMePGB1 against KCN-induced mitochondrial failure in hepatocytes. The dimer and trimer of 16,16-dimethyl-15-dehydroprostaglandin B1 (16,16-diMePGB1) previously have been shown to have protective effects on mitochondrial function. To examine the potential mechanisms involved in protection against mitochondrial failure, we have studied the effects of the dimer of 16,16-diMe-PGB1 (dicalciphor) on mitochondrial function in hepatocytes exposed to KCN. Addition of micromolar concentrations of dicalciphor provided substantial protection against KCN-induced toxicity in a concentration- and time-dependent manner. Dicalciphor, however, had no effect on total or mitochondrial ATP losses in KCN-treated cells. The dimer prevented the marked loss of mitochondrial membrane potential (delta psi) and delta pH that occurs as a result of KCN treatment and prevented KCN-induced loading of phosphate in mitochondria. Furthermore, the dimer of 16,16-diMePGB1 also prevented KCN-induced mitochondrial and cellular swelling. These results demonstrate that dicalciphor protects against KCN-induced damage and that this protection is associated with regulation of specific mitochondrial ion transport functions.


Volume regulation during recovery from chronic hypertonicity in brain glial cells. Rat C6 glial cells undergo rapid regulatory volume increase (5-10 min) via electrolyte uptake when exposed to a hypertonic medium. With chronic exposure to hypertonicity (greater than 8 h), accumulated electrolyte is replaced partly by inositol. Inositol accumulation is brought about by upregulation of Na(+)-dependent inositol transport. When C6 cells acclimated chronically to hypertonic NaCl medium were returned to isotonic conditions, inositol levels dropped slowly from 478 nmol/mg protein towards control values (117 nmol/mg protein) in 18-24 h. Inositol loss occurred in part by efflux to the external medium via a pathway distinct from the uptake mechanism. Laser light-scattering measurements demonstrated that regulatory volume decrease (RVD) is slow under these experimental conditions. In contrast, cells exposed acutely to hypertonicity swell and then undergo a rapid and nearly complete RVD when returned to isotonic medium. These results suggest that slow inositol loss is rate limiting for RVD during recovery from chronic hypertonic stress. The slow inositol loss and RVD may be due to slow turnover of the efflux mechanism and/or slow downregulation of the hypertonically stimulated inositol uptake pathway.


TGF-beta 1 potentiates growth factor-stimulated proliferation of vascular smooth muscle cells in genetic hypertension. We have examined the interactions between transforming growth factor-beta 1 (TGF-beta 1) and epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), or platelet-derived growth factor (PDGF) isoforms PDGF-AB and PDGF-BB on the proliferation of vascular smooth muscle cells isolated from the spontaneously hypertensive rat. TGF-beta 1 alone stimulated [3H]thymidine incorporation approximately twofold without a corresponding increase in cell number. In combination, TGF-beta 1 action was synergistic in further stimulating both DNA synthesis and cell proliferation 100-300% above the responses elicited by each growth factor. To gain further insight into the mechanism responsible for this potentiation, we examined the interaction between TGF-beta 1 and EGF. The synergistic interaction between TGF-beta 1 and EGF on DNA synthesis was independent of initial cell density. This effect of TGF-beta 1 was initiated early in the G1 phase of the cell cycle and did not appear to be mediated through the mobilization of Ca2+ or alterations in c-jun mRNA expression. However, in the presence of both TGF-beta 1 and EGF, there was a sustained elevation of c-myc mRNA levels over a 24-h period. These results suggest that TGF-beta 1 may interact with other growth factors in vivo to enhance their proliferative action on vascular smooth muscle of spontaneously hypertensive rats via mechanisms dependent on c-myc mRNA expression.


Abundance, localization, and insulin-induced translocation of glucose transporters in red and white muscle. D-Glucose protectable cytochalasin B (CB) binding to subcellular membrane fractions was used to determine glucose transporter number in red (quadriceps-gastrocnemius-soleus) and white (quadriceps-gastrocnemius) rat muscle. CB binding was twofold higher in isolated plasma membranes of red than of white muscle. In contrast, the number of transporters in an isolated insulin-sensitive intracellular membrane organelle was similar in the two muscle groups. Immunoblotting and immunofluorescence microscopy with anti-GLUT4 and anti-GLUT1 antibodies indicated that both GLUT1 and GLUT4 transporter isoforms account for the higher abundance of CB binding sites in plasma membranes of red than of white muscle. Immunofluorescence localized GLUT4 to both the surface and the interior of the muscle cell and demonstrated that type I (slow twitch oxidative) and type IIa (fast twitch oxidative-glycolytic) fibers are enriched in GLUT4 protein compared with type IIb (fast twitch glycolytic) fibers. In contrast, GLUT1 reactivity was restricted to the surface of the muscle cell and was also highly enriched in the perineurial sheaths of peripheral nerves and the capsules of muscle spindles present in both red and white muscles. Insulin caused a twofold increase in CB binding in isolated plasma membranes of red or white muscles with a corresponding 40-50% decrease in CB binding in isolated intracellular membranes. These changes in CB binding were paralleled by similar changes in the membrane distribution of the GLUT4 glucose transporter isoform and in glucose transport activity measured after insulin perfusion of hindquarter muscles. In contrast, insulin did not change the distribution of either GLUT1 glucose transporters or Na(+)-K(+)-ATPase alpha 1-subunits. The molar ratio of GLUT4 to GLUT1 in red and white muscle plasma membranes was found to be 4:1 in the basal state and 7:1 in the insulin-stimulated state. These results indicate that red muscle contains a higher amount of GLUT1 and GLUT4 transporters at the plasma membrane than white muscle in the basal and insulin-stimulated states but that GLUT4 translocation does not differ between muscle types. In addition, GLUT4 expression correlates with the metabolic nature (oxidative vs. glycolytic) of skeletal muscle fibers, rather than with their contractile properties (slow twitch vs. fast twitch).


Maturational changes in respiratory control through creatine kinase in heart in vivo. The role of creatine kinase in regulation of myocardial respiration was studied in vivo as a function of maturation. Unidirectional creatine kinase flux (JCK), phosphocreatine to gamma-ATP, was measured in newborn lambs (age 3-9 days, n = 8) and mature sheep (age 30-60 days, n = 6) using 31P saturation transfer techniques, and total creatine kinase activity was measured using standard methods. Myocardial oxygen consumption (MVO2) was measured simultaneously via an extracorporeal shunt from the coronary sinus as cardiac work was increased via epinephrine (1-3 micrograms.kg-1.min-1). Findings were as follows: 1) baseline newborn JCK was markedly lower than in mature sheep despite higher levels of MVO2, and this could be related to a decrease in total creatine kinase activity; 2) JCK was substantially higher than the rate of ATP synthesis in both groups at baseline rates of oxygen consumption; and 3) JCK decreased significantly in newborns during increases in MVO2, whereas there was no change in flux rate in the mature sheep during even larger relative changes in work and oxygen consumption. These data imply that creatine kinase does not limit oxidative phosphorylation. However, this enzyme system probably maintains at least an indirect role in respiratory control that is a function of the myocardial developmental state.


Transmucosal impedance of small intestine: correlation with transport of sugars and amino acids. Transmucosal impedances of isolated perfused segments of jejunum from mice and hamsters were measured at frequencies from 10-100,000 Hz in the presence and absence of sugars and amino acids. Na-coupled transport of organic substrates caused large decreases of transmucosal impedance, reflecting contraction of cytoskeletal proteins controlling permeability of tight junctions, functional surface of basolateral membranes, and width of extracellular pathways. The observed changes of impedance were closely correlated with molar rates of Na-coupled active transport rather than with molecular species. Thus amino acids and sugars having the same molar rates of active transport also have the same effects on transmucosal impedance. It is proposed that a nonspecific increase of intracellular osmotic pressure during active transport is the first step initiating cytoskeletal contraction. Cell volume regulatory responses, including increased basolateral K+ conductance and Ca2+ influx, may be subsequent steps leading to contraction of perijunctional actomyosin, formation of junctional dilatations, and exposure of lateral membranes. Enhancement of oxygen capacity of perfusion fluids (e.g., with fluorocarbon emulsion) is required to maintain viability of isolated intestinal epithelium; in plain oxygenated Ringer-HCO3 solution, the transmucosal impedance is abnormally low and cytoskeletal contractile responses to Na-coupled transport are attenuated. An electrical circuit analog is presented that simulates almost exactly the observed transmucosal impedances and provides quantitative evaluation of the effects of Na-coupled transport of sugars and amino acids on resistances of tight junctions, capacitance of basolateral membranes, and postjunctional resistances of lateral intercellular spaces and villus cores.


Arachidonic acid and lipoxygenase metabolites uncouple neonatal rat cardiac myocyte pairs. The effects of arachidonic acid (AA) and its metabolites on the conductance (gj) of the gap junctions between neonatal rat myocardial cells was investigated. AA reduced gj in a dose- (2, 5, and 20 microM) and time-dependent fashion. Pretreatment of the cells with an inhibitor of the 5-lipoxygenase pathway, U-70344A, shifted the dose-response curve to the right; pretreatment with indomethacin, an inhibitor of the cyclooxygenase pathway, had no effect. The mean time to uncoupling was 3.7 +/- 0.3, 3.8 +/- 0.9, and 4.6 +/- 0.6 min (means +/- SE, P less than 0.05) for 5 microM AA, 5 microM AA + indomethacin, and 5 microM AA + U-70344A, respectively. Incorporation of AA into membrane phospholipids was not affected by the inhibitor. These studies suggest that complete uncoupling of the cells occurred at membrane concentrations of 3-4 mol%. The data indicate that AA and a 5-lipoxygenase metabolite uncouple neonatal rat heart cells. The data are discussed with respect to the possible underlying mechanism of uncoupling and the potential role of gap junctions in arrhythmia formation in ischemic heart disease.


Na+ alters the affinity for glucose and phosphate in rat renal brush-border membranes: a study of NMR relaxation rates. Transport of D-glucose (Glc) and Pi across renal microvilli brush-border membrane vesicles (BBMV) is coupled to Na+.Na+ may alter substrate binding and dissociation or the mobility of the translocator and its complexes. To evaluate substrate binding in NaCl or KCl solutions containing BBMV, we measured nuclear magnetic resonance transverse relaxation rates (1/T2) of [1-13C]Glc and 31P(i), as well as their temperature, magnetic field, and pH dependencies. The data indicate the following: 1) the alpha-anomer, but not the beta-anomer, of Glc binds to BBMV, more in the presence of Na+ than of K+; 2) interactions of P(i) with BBMV that increase its 1/T2 are significant in the presence of K+ but not of Na+; 3) temperature and magnetic field dependencies of the 1/T2 of P(i) bound to BBMV are consistent with more rapid exchange with free P(i) in the presence of Na+ than of K+; and 4) in the presence of Na+, [H+] promoted interactions of P(i) with BBMV that increase its 1/T2, probably by interfering with formation of rapidly dissociating Na(+)-P(i)-carrier complexes. In conclusion, Na+ promotes association of alpha-Glc to BBMV but promotes rapid exchange and dissociation of P(i) from BBMV. In both cases, Na+ effects coupling by altering the affinity of the cotransporters for the substrate.


Stored calcium modulates inositol phosphate synthesis in cultured smooth muscle cells. Cytosolic Ca2+ concentrations ([Ca2+]cyt) and [3H]inositol phosphates ([3H]InsP) were correlated while varying the Ca2+ content of the sarcoplasmic reticulum (SR) in cultured A7r5 cells at rest and during activation with [Arg8]-vasopressin (AVP). Thapsigargin (TG) raised and superfusion with 0 Ca2+ lowered [Ca2+]cyt, but both treatments decreased SR Ca2+ and AVP-evoked Ca2+ transients. Neither TG nor 0 Ca2+ affected basal [3H]InsP, but both treatments increased AVP-evoked synthesis of [3H]InsP. Exposure for several minutes to 40 mM K+ solution, BAY K 8644, or low-Na+ solution all elevated [Ca2+]cyt and, thereby, increased SR Ca2+, as manifested by augmented AVP-evoked Ca2+ transients. In all three cases, AVP-evoked, but not basal, [3H]InsP were reduced. The inhibitory effect of 40 mM K+ on AVP-evoked [3H]InsP synthesis was blocked when SR Ca2+ uptake was prevented by TG. Brief (30-s) exposures to 40 mM K+, which elevated [Ca2+]cyt but not SR Ca2+ loading, did not modify AVP-evoked [3H]InsP synthesis or Ca2+ transients. These results demonstrate an inverse relationship between SR Ca2+ content and evoked [3H]-InsP synthesis. Moreover, they suggest that SR Ca2+ may serve as a signal that modulates sarcolemmal [3H]InsP formation.


Transient myosin phosphorylation at constant Ca2+ during agonist activation of permeabilized arteries. Norepinephrine (NE) plus guanosine triphosphate (GTP) increases myofilament Ca2+ sensitivity in alpha-toxin-permeabilized smooth muscle. We used alpha-toxin-permeabilized rabbit mesenteric arteries to determine the temporal relationships among force, myosin light chain (MLC) phosphorylation, stiffness, and shortening velocity during contractions in response to Ca2+ alone and to the same [Ca2+] in the presence of NE plus GTP. The addition of NE plus GTP caused a marked increase in the tonic contraction but only transiently elevated the level of MLC phosphorylation over that observed in the presence of Ca2+ alone. NE plus GTP induced similar increases in force and stiffness, but shortening velocity depended solely on the [Ca2+]. A regulated MLC phosphatase could explain the initial increase in force and MLC phosphorylation, but not the maintenance of enhanced force while MLC phosphorylation levels fell to values similar to those in response to Ca2+ alone. Therefore, additional elements must be involved in the maintenance of the receptor and G protein-dependent increase in myofilament Ca2+ sensitivity.


Immunofluorescence localization of the Na-Ca exchanger in heart cells. We investigated the localization of the Na-Ca exchanger in fixed, isolated heart cells from rat and guinea pig using immunocytochemical methods with epifluorescence and confocal microscopy. We found that the Na-Ca exchanger is distributed throughout all membranes in contact with the extracellular space, including the sarcolemma, the transverse tubules (T-tubules), and the intercalated disks. Microscopic nonuniformities in the fluorescent labeling appear to reflect varying views of the membranes containing Na-Ca exchanger protein. Confocal thin-section imaging reveals a regular grid of discrete foci of fluorescence, which represent Na-Ca exchanger in T-tubules viewed en face. These foci are 1.80 +/- 0.01 microns apart from sarcomere to sarcomere and are aligned with the Z-line. Along each Z-line, these foci are spaced at 1.22 +/- 0.11-microns intervals. Longitudinal sections of the sarcolemma-T-tubule junction show a comblike appearance, with T-tubules extending inward from the heavily labeled sarcolemma. Our finding that the Na-Ca exchanger is widely distributed over the cell surface may provide further insight into the role of Na-Ca exchange in the heart.


Regulation of acute parathyroid hormone release in normal humans: combined calcium and citrate clamp study. The objective of the present study was to elucidate the dynamics of parathyroid hormone regulation, with particular reference to the mechanism controlling the acute parathyroid hormone release. Through utilization of the citrate clamp technique and the calcium clamp technique we were able, in a standardized way, to stimulate and suppress the parathyroid hormone secretion. Precise bedside measurements of blood ionized calcium and measurements of intact parathyroid hormone were performed. Twelve healthy young volunteers participated in two trials 6-12 wk apart, a citrate clamp (delta-blood ionized calcium -0.19 mmol/l) and a calcium plus citrate clamp (delta-blood ionized calcium +0.22 mmol/l and -0.19 mmol/l). During the citrate clamp, preceded by normal calcemia, serum intact parathyroid hormone peaked to a maximum after 5-10 min, four to six times above baseline concentration and then declined to a steady state two to three times above baseline concentration. During the citrate clamp, preceded by hypercalcemia induced by a calcium clamp, serum intact parathyroid hormone also peaked immediately to about five to nine times above its suppressed level, approximately two times above the baseline concentration. Subsequently, serum intact parathyroid hormone declined to a steady state just below the baseline concentration. In conclusion, within the range studied, the mechanism eliciting the acute serum intact parathyroid hormone release from its depot is a fall in blood ionized calcium, not the absolute concentration of ionized calcium.


Effects of epinephrine on insulin-mediated glucose uptake in whole body and leg muscle in humans: role of blood flow. In vivo insulin-mediated glucose uptake (IMGU) occurs chiefly in skeletal muscle, where it is determined by the product of arteriovenous glucose difference (delta AVG) and blood flow (BF) rate into muscle. Epinephrine (Epi) reduces the rate of IMGU in whole body. To examine whether this is due to a reduction in delta AVG across or BF into skeletal muscle we constructed insulin dose-response curves for whole body IMGU and leg muscle IMGU- using euglycemic clamp ((+)[3-3H]glucose infusion) and leg balance techniques during insulin infusions ranging from 10 to 1,200 mU.m-2.min-1. We studied six subjects [wt 70 +/- 2 (SE) kg] during an Epi infusion at a single rate of 0.002 mg.kg-1.min-1 and six subjects (70 +/- 3 kg) during a saline infusion alone. Maximum whole body glucose uptake (WBGU) was similar during Epi and saline infusions [71.4 vs. 73.6 mmol.kg-1.min-1, P = not significant (NS)]. Compared with saline, maximum delta AVG was decreased during Epi infusion (1.04 vs. 1.31 mM, P less than 0.01). Compared with saline alone maximum leg BF was increased (5.3 vs. 4.3 dl/min, P less than 0.01) during Epi infusion. Thus maximum leg glucose uptake (LGU) was similar (696 vs. 821 pmol.leg-1.min-1, P = NS) during infusion of Epi and saline, respectively. Half-maximal effective dose for insulin's effect to stimulate WBGU, delta AVG, BF, and LGU was increased two- to threefold during Epi vs. saline infusions (P less than 0.01 for all values).(ABSTRACT TRUNCATED AT 250 WORDS)


PDGF induces tyrosine phosphorylation in osteoblast-like cells: relevance to mitogenesis. We have studied the association between protein tyrosine phosphorylation and the mitogenic effect induced by platelet-derived growth factor (PDGF) in the osteoblast-like cell line MC3T3-E1. PDGF caused a dose-dependent increase in [3H]thymidine incorporation in MC3T3-E1 cells, reaching a plateau at 10 ng/ml. Vanadate, a potent phosphatase inhibitor, induced a twofold increase in thymidine incorporation. The combination of vanadate and PDGF resulted in a dose-dependent synergistic effect on thymidine incorporation. Genistein, a tyrosine kinase inhibitor, inhibited in a dose-related manner (2-20 microM) the mitogenic effect induced by either PDGF or the combination of vanadate and PDGF. These observations suggest that tyrosine kinases are involved in mediating the mitogenic effect of PDGF in these cells. PDGF treatment of MC3T3-E1 cells and subsequent immunoblotting with antiphosphotyrosine antibodies resulted in a marked phosphorylation of the PDGF receptor. Vanadate had a lesser effect on PDGF receptor phosphorylation, but given together with PDGF it induced a significant increase in the intensity of receptor phosphorylation. Preincubation with genistein abrogated these effects. Taken together, these findings indicate a direct correlation between thymidine incorporation and tyrosine phosphorylation in MC3T3-E1 cells and suggest that tyrosine phosphorylation plays a role in PDGF-induced mitogenic activity in osteoblast-like cells.


Leucine kinetics in fed low-birth-weight infants: importance of splanchnic tissues. Whole body Leu kinetics were determined in fed low-birth-weight (LBW) infants. To assess the importance of first-pass splanchnic extraction of ingested amino acids, two tracers were simultaneously infused by intravenous (L-[1-13C]Leu) and intragastric (L-[5,5,5-2H3]Leu) routes in 13 LBW infants [1,742 +/- 169 (SE) g] fed with protein-enriched human milk (protein intake 3.23 +/- 0.97 g.kg-1.day-1). Splanchnic extraction estimated from plasma [2H3]Leu appearance was 48.2 +/- 15.6% of Leu intake. Total Leu flux, endogenous Leu flux (index of protein catabolism), and nonoxidative Leu disposal (NOLD, index of protein synthesis) were 3.45 +/- 0.57, 1.90 +/- 0.74, and 2.54 +/- 0.62 mumol.kg-1.min-1, respectively. Higher estimates were obtained when using alpha-ketoisocaproate as a precursor pool. There was a wide individual variation of protein intake due to the use of human milk, and, over this range, Leu intake was correlated negatively with endogenous Leu flux (r = 0.88, P less than 0.01) whereas NOLD remained fairly constant. Thus, in LBW infants, 1) splanchnic extraction is two times as high as in adults and might reflect an elevated splanchnic protein turnover and 2) increasing protein intake probably promotes protein gain mainly by inhibiting protein catabolism.


Spatial and temporal thyrocyte response to TSH: a computer-assisted image analysis. Pseudopods at the apical pole of porcine thyroid monolayers in culture were considered as reflecting individual thyrocyte responses to thyrotropin (TSH) stimulation. Scanning electron microscopy and computer-assisted image analysis showed that whatever TSH stimulation was used, the pseudopods were characterized by two populations: P1 with small diameters (2 microns) and P2 with greater diameters (5 microns). The density of P1 rapidly increased to reach a plateau, while P2 continuously increased during stimulation. Two-dimensional pseudopod patterns were compared with random point distributions by means of two topographical parameters: the interpseudopod distances and angles. A factorial analysis of experimental distribution of pseudopods obtained after increasing stimulation times displayed a shift from a nonrandom (10-20 min) to a random (60-90 min) distribution. Clusters of three pseudopods characterized by short distances (6-9 microns) and equilateral organization (angles 40-60 degrees) were observed after a 10-min stimulation. These results suggested that early thyrocyte response to TSH stimulation is characterized by interrelations between three adjoining cells, with the thyrocyte response later appearing as random.


Platelet catecholamine contents as related to trait anxiety and aerobic fitness. Platelet catecholamines (CA) are derived from plasma and are considered as a cumulative index of sympathoadrenal activity. We investigated whether trait anxiety (TA) and aerobic fitness, two interrelated long-term determinants of sympathoadrenal activity, affect platelet CA concentration as measured in rest and after acute increments of plasma epinephrine (Epi). For that purpose, platelet CA were measured 15 min before and after Epi infusion (5-160 ng.kg-1.min-1, increased in 6 steps) in 45 healthy male students with high (n = 21) and low (n = 24) TA, of whom maximal aerobic power (VO2max) was assessed. Plasma CA and electrolytes were measured during each infusion rate. Epi infusion significantly raised platelet Epi by 67% and decreased platelet and plasma norepinephrine (NE) by 23 and 17%. Plasma potassium significantly decreased by 32% and plasma sodium slightly increased. Platelet Epi increase was inversely related to basal platelet Epi (r = -0.58, P less than 0.001). Platelet NE decrease was positively associated with basal platelet NE and plasma potassium levels (r = 0.50, P less than 0.01). TA and VO2max were not related to basal levels and responses of electrolytes and platelet NE. VO2max was correlated with platelet Epi increase in low TA (r = 0.62, P = 0.002), whereas no such relationship existed in high TA (r = 0.008). We conclude that fitness positively influences platelet Epi accumulation, but that high TA interferes with this relationship.


Epinephrine's ketogenic effect in humans is mediated principally by lipolysis. To quantify epinephrine's effects on acetoacetate and beta-hydroxybutyrate kinetics, we infused subjects with 0.3 and 2.5 micrograms/min epinephrine, either alone or with a concomitant somatostatin infusion with insulin, glucagon, and growth hormone replaced at postabsorptive levels (islet clamp). Additional subjects received no epinephrine but sequential infusions of heparin plus 10% Intralipid at rates of 0.5 and 3.0 ml/min. Both epinephrine and Intralipid increased ketone body appearance (unaffected by islet clamp), augmented the interconversion rates between ketone bodies and, during the 2.5 micrograms/min infusion, caused a marked increase in beta-hydroxybutyrate appearance. The fraction of plasma free fatty acid (FFA) flux appearing as plasma ketones increased from 6 to 7% in the basal state to 11% at the high-epinephrine infusion. This fraction was also unaffected by the islet clamp and was not different from values obtained at similar Intralipid plus heparin-induced elevations in plasma FFA levels. We conclude that epinephrine's ketogenic effect in humans is primarily the result of its lipolytic effect, is accompanied by a significantly increased rate of ketone body interconversion, is manifest largely as an increase in plasma beta-hydroxybutyrate appearance at high plasma epinephrine values, and is not limited by portal insulin at post-absorptive levels.


Forearm ketone body metabolism in normal and in insulin-dependent diabetic patients. In insulin deficiency, there is excessive arterial delivery of free fatty acid (FFA) to muscles where they are converted to acetoacetyl-CoA and acetyl-CoA. These intermediates may be metabolized further to acetoacetate and beta-hydroxybutyrate, which can be released into the venous circulation. When ketone body (KB) tracers are infused in vivo, they are diluted across muscle tissue. This dilution may be due to 1) KB newly formed within muscle (ketogenesis); 2) exchange of tracer between labeled and unlabeled acetyl-CoA and acetoacetyl-CoA, intermediates common to the metabolism of both FFA and KB (pseudoketogenesis). Thus this study assessed whether such label exchange could be detected across the human forearm and whether an increased delivery of FFA in insulin-sufficient controls provoked dilution of labeled KB tracer comparable to that observed in insulin-deficient diabetics. Five normal and five insulin-dependent diabetic (IDDM) subjects were infused with labeled [3,4-13C2]-acetoacetate. [13C]KB enrichments were lower in forearm vein than in the artery, and dilution of labeled KB was always higher than that which could be explained by arterial-venous differences of unlabeled KB. When arterial FFA concentrations in normals were raised (Intralipid+heparin) to values comparable to those of the diabetics, no additional increase in forearm arteriovenous dilution of labeled KB was observed. Neither in the basal state nor under conditions of increased plasma FFA were we able to detect venous appearance of KB labeled in the first and in the second carbon atoms, a necessary consequence of pseudoketogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)


Origin and disposal of 1,5-anhydroglucitol, a major polyol in the human body. The origin and disposal of 1,5-anhydro-D-glucitol (AG), one of the main polyols found in the human body, was studied in normal subjects and diabetic patients. AG was detected in various kinds of foods. The mean AG supplement through foods was estimated to be approximately 4.38 mg/day, which was compatible with that calculated in a food analysis (average 0.22 mg AG/100 kcal in Japanese foods) on eight healthy subjects. The mean AG excretion in urine was approximately 4.76 mg/day in these subjects. Excretion into stools was negligible. From observations on the patients without oral supplement of AG, 0.4 mg of daily de novo synthesis of AG was strongly suggested. It was also implied that urinary AG excretion occurred soon after food ingestion and that its amount was closely correlated with daily supplement through foods. Thus the fundamental kinetics of AG were recognized as follows: 1) AG in the body originates mainly from foods and is well absorbed in the intestine, 2) AG is little degraded and metabolized in the body, and 3) an equilibrium exists between oral supplement plus a small but steady amount of de novo synthesis and excretion in urine.


Amylin and insulin in rat soleus muscle: dose responses for cosecreted noncompetitive antagonists. Increasing concentrations of amylin progressively depressed the maximal insulin-stimulated radioglucose incorporation into soleus muscle glycogen, but did not substantively change the EC50 (range 0.78 to 1.52 nM); these findings show noncompetitive, insurmountable antagonism of insulin action by amylin. The results from 36 combinations of different insulin and amylin concentrations were used to construct a response surface that can be used to predict the response for any combination of insulin and amylin concentration. The predicted response to a constant ratio of insulin and amylin concentration is a bell-shaped curve. The experimentally determined response to increasing amounts of an amylin-insulin mixture (molar ratio of 0.14:1, within the range measured for pancreatic secretion and plasma levels) gave a bell-shaped response rather than the sigmoidal response seen with insulin alone. The amylin dose-response relation in the soleus system provides a useful bioassay for amylin agonists. The dose response for highly purified, synthetic human amylin obtained by measuring amylin concentrations by radioimmunoassay in the incubation medium gave an EC50 of 456 pM (+/- 0.18 log units). Human amylin had a potency greater than or equal to that of human insulin in this highly insulin-sensitive preparation.


Trophic action of local intraileal infusion of insulin-like growth factor I: polyamine dependence. Experiments were performed to determine potential growth-promoting effects of human recombinant insulin-like growth factor I (hrIGF-I) in the gastrointestinal tract. IGF-I and IGF-II, but not insulin, were potent (half-maximal effective concentration 0.3 nM) and efficacious inducers of the growth-related enzyme ornithine decarboxylase (ODC) in the gut-derived cell line IEC-6. Maximal ODC induction was observed after treatment of cells with 10 nM IGF-I. In whole animal experiments, bolus intraileal injection of 10 nM hrIGF-I in anesthetized rats induced a 300% increase in ileal mucosal ODC activity, which was sensitive to inhibition with difluoromethylornithine (DFMO). Rats were implanted intraperitoneally with osmotic minipumps filled with 0.9% NaCl or 10 nM IGF-I that was delivered to the ileal lumen by a short Silastic catheter. Sixty-six hours of 1 microliter/h intraluminal IGF-I infusion produced an approximate doubling of mucosal wet weight (NaCl 50 mg vs. IGF-I 102 micrograms/2 cm mucosa) and total mucosal RNA, DNA, and protein content over that in rats that were infused with NaCl. Intraperitoneal treatment with 200 mg/kg DFMO three times per day had little effect on ileal mucosal mass, but completely inhibited the trophic response to IGF-I infusion. IGF-I infusion had no effect on body weight.


Is there a role for the adrenals in the development of hypercholesterolemia in Zucker fatty rats? We tested the hypothesis that hypercorticosteronemia causes the hypercholesterolemia in young developing "fatty" rats. Obesity induced increases in corticosterone. Insulin, glucose, body weight, average daily food intake, plasma triglyceride, plasma phospholipids, liver weight, liver triglyceride, various adipose tissue parameters, and liver hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase activity were all ameliorated by adrenalectomy. Adrenalectomy exacerbated the hypercholesterolemia in obese animals and induced it in lean rats. Changes or lack of change in hepatic microsomal cholesterol, HMG-CoA reductase, and 7 alpha-hydroxylase, combined with the adrenalectomy-induced curtailment of tissue storage of cholesterol in adipose tissue, all contribute to the hypercholesterolemia caused by adrenalectomy. We suggest a mechanism whereby this may be related to elevated hepatic very low-density lipoprotein secretion rates. The elevated HMG-CoA reductase activity in obese rats results from the lower liver microsomal free cholesterol content. We conclude that the absence of glucocorticoids does not directly reduce plasma cholesterol in obese Zucker rats. The surprising elevation of cholesterol by adrenalectomy is due to other prevailing mechanisms in liver and adipose tissue, which curtail their capacity to store cholesterol.


Spontaneous physical activity and obesity: cross-sectional and longitudinal studies in Pima Indians. Healthy, nondiabetic Pima Indians [103 males, 77 females; 27 +/- 6 (SD) yr, 97 +/- 25 kg, 33 +/- 9% body fat] were studied in a respiratory chamber in which spontaneous physical activity (SPA) was measured by two microwave sensors. SPA, defined as the percentage of time the subjects were active, varied widely from 4.4 to 17.5%. It was higher in males (9.3 +/- 2.0%) than in females (8.6 +/- 2.3%; P less than 0.05) and was not related to body fatness in either sex. However, SPA accounted for a significant portion of the daily energy expenditure (24-h EE) in males (1,389 +/- 423 kJ/day) and females (1,163 +/- 351 kJ/day) and correlated positively with 24-h EE adjusted for differences in fat-free mass, fat mass, age, and sex (r = 0.42, P less than 0.0001). In 88 siblings, family membership accounted for 57% of the variance in SPA (r(i) = 0.57, P less than 0.02). Body composition was reassessed in a subgroup of 123 subjects (65 males, 58 females) 33 +/- 14 mo later. In males only, SPA correlated inversely to the rate of subsequent body weight change (r = -0.25, P less than 0.05) and the rate of fat-mass change (r = -0.35, P less than 0.005). We conclude that spontaneous physical activity is a familial trait that may play a role in the pathogenesis of obesity.


Differential effects of interleukin-1 and tumor necrosis factor on ketogenesis. To determine the role of cytokines in mediating the decrease in ketones associated with infection, we studied the effect of endotoxin (LPS), interleukin-1 (IL-1), and tumor necrosis factor (TNF) on serum and hepatic ketone body levels (KB), serum free fatty acids (FFA), and hepatic malonyl-CoA levels. LPS decreased serum and hepatic KB in C57Bl/6 (LPS sensitive) mice, whereas it had little effect in C3H/HeJ (LPS resistant) mice, whose macrophages lack the ability to produce IL-1 and TNF in response to LPS, suggesting that IL-1 and TNF may mediate this effect. IL-1 and TNF decreased serum KB in both strains of mice. As seen with LPS, IL-1 decreased hepatic KB, whereas TNF had no such effect. LPS, IL-1, and TNF increased hepatic malonyl-CoA levels. TNF acutely raised serum FFA, whereas LPS and IL-1 did not. Postulating that the TNF-induced increase in FFA overrides the inhibitory effect of malonyl-CoA on fatty acid oxidation and ketogenesis, we used R-2-phenylisopropyladenosine to block TNF-induced lipolysis and demonstrated that in the absence of increased fatty acid flux, TNF inhibits KB formation. As seen with LPS, IL-1, but not TNF, decreased KB in the fasting state. These data suggest that IL-1 and TNF may mediate the antiketogenic effect of infection and that IL-1 has properties closest to that of LPS.


Selective reduction of creatine kinase subunit mRNAs in striated muscle of diabetic rats. Creatine kinase (CK) is important for energy transfer and is composed of mitochondrial (mitCK), muscle (MCK), and brain (BCK) subunits, each being the product of separate nuclear genes. The concentrations of MCK and BCK mRNAs have been shown to decrease in streptozotocin-hypoinsulinemic rat hearts, and in this report, we examined in detail the diabetic effect on CK gene expression in cardiac muscle and in two types of skeletal muscle. The level of sarcomeric mitCK mRNA was not altered in the diabetic myocardium, but was reduced by 86 and 67% in diabetic slow-twitch soleus muscle and fast-twitch extensor digitorum longus (EDL) muscle, respectively. MCK mRNA was also lowered in diabetic soleus muscle by 56%, while it remained at control levels in diabetic EDL. In both skeletal muscles, at either state, BCK mRNA was not detectable. There was a 33% decrease in total CK activity in diabetic cardiac and soleus muscle, but not in EDL. Diabetes thus exerts a widespread, muscle type-dependent adverse effect on CK expression that we found to be insulin therapy revertible. This study adds to our understanding of defective energy transduction in diabetic muscle.


Suppression of muscle protein turnover and amino acid degradation by dietary protein deficiency. To define the adaptations that conserve amino acids and muscle protein when dietary protein intake is inadequate, rats (60-70 g final wt) were fed a normal or protein-deficient (PD) diet (18 or 1% lactalbumin), and their muscles were studied in vitro. After 7 days on the PD diet, both protein degradation and synthesis fell 30-40% in skeletal muscles and atria. This fall in proteolysis did not result from reduced amino acid supply to the muscle and preceded any clear decrease in plasma amino acids. Oxidation of branched-chain amino acids, glutamine and alanine synthesis, and uptake of alpha-aminoisobutyrate also fell by 30-50% in muscles and adipose tissue of PD rats. After 1 day on the PD diet, muscle protein synthesis and amino acid uptake decreased by 25-40%, and after 3 days proteolysis and leucine oxidation fell 30-45%. Upon refeeding with the normal diet, protein synthesis also rose more rapidly (+30% by 1 day) than proteolysis, which increased significantly after 3 days (+60%). These different time courses suggest distinct endocrine signals for these responses. The high rate of protein synthesis and low rate of proteolysis during the first 3 days of refeeding a normal diet to PD rats contributes to the rapid weight gain ("catch-up growth") of such animals.


Dietary protein deficiency reduces lysosomal and nonlysosomal ATP-dependent proteolysis in muscle. When rats are fed a protein deficient (PD) diet for 7 days, rates of proteolysis in skeletal muscle decrease by 40-50% (N. E. Tawa, Jr., and A. L. Goldberg. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E317-325, 1992). To identify the underlying biochemical adaptations, we measured different proteolytic processes in incubated muscles. The capacity for intralysosomal proteolysis, as shown by sensitivity to methylamine or lysosomal protease inhibitors, fell 55-75% in muscles from PD rats. Furthermore, extracts of muscles of PD rats showed 30-70% lower activity of many lysosomal proteases, including cathepsins B, H, and C, and carboxypeptidases A and C, as well as other lysosomal hydrolases. The fall in cathepsin B and proteolysis was evident by 3 days on the PD diet, and both returned to control levels 3 days after refeeding of the normal diet. In muscles maintained under optimal conditions, 80-90% of protein breakdown occurs by nonlysosomal pathways. In muscles of PD rats, this ATP-dependent process was also 40-60% slower. Even though overall proteolysis decreased in muscles of PD rats, their capacity for Ca(2+)-dependent proteolysis increased (by 66%), as did the activity of the calpains (+150-250%). Thus the lysosomal and the ATP-dependent processes decrease coordinately and contribute to the fall in muscle proteolysis in PD animals.


Soluble and particulate phenylethanolamine N-methyltransferase in hypothalamus of diabetic rats. Experimental diabetes increases total phenylethanolamine N-methyltransferase (PNMT) activity in the medulla-pons but not in the hypothalamus. In this study diabetes was induced with streptozotocin (65 mg/kg) in male Sprague-Dawley rats. Twenty-eight days after treatment there were no differences in soluble PNMT activity in the hypothalamus of diabetics and controls, but PNMT activity in a membrane-associated (particulate) fraction of hypothalamus was evaluated approximately twofold in tissues of diabetic animals compared with controls. A specific PNMT inhibitor, incubated with tissue extracts of control rats, abolished greater than 90% of particulate PNMT activity in the hypothalamus but reduced soluble PNMT activity in the hypothalamus by only 47%. These findings indicate that membrane-associated PNMT activity in rat hypothalamus differs from soluble hypothalamic PNMT in the in vitro response to an inhibitor and the in vivo response to diabetes and suggest the importance of separating subcellular hypothalamic fractions prior to assay of PNMT.


Effects of prior exercise on the action of insulin-like growth factor I in skeletal muscle. Prior exercise increases insulin sensitivity for glucose and system A neutral amino acid transport activities in skeletal muscle. Insulin-like growth factor I (IGF-I) also activates these transport processes in resting muscle. It is not known, however, whether prior exercise increases IGF-I action in muscle. Therefore we determined the effect of a single exhausting bout of swim exercise on IGF-I-stimulated glucose transport activity [assessed by 2-deoxy-D-glucose (2-DG) uptake] and system A activity [assessed by alpha-(methylamino)isobutyric acid (MeAIB) uptake] in the isolated rat epitrochlearis muscle. When measured 3.5 h after exercise, the responses to a submaximal concentration (0.2 nM), but not a maximal concentration (13.3 nM), of insulin for activation of 2-DG uptake and MeAIB uptake were enhanced. In contrast, prior exercise increased markedly both the submaximal (5 nM) and maximal (20 nM) responses to IGF-I for activation of 2-DG uptake, whereas only the submaximal response to IGF-I (3 nM) for MeAIB uptake was enhanced after exercise. We conclude that 1) prior exercise significantly enhances the response to a submaximal concentration of IGF-I for activation of the glucose transport and system A neutral amino acid transport systems in skeletal muscle and 2) the enhanced maximal response for IGF-I action after exercise is restricted to the signaling pathway for activation of the glucose transport system.


Metabolic regulation in peripheral tissues and transition to increased gluconeogenic mode during prolonged exercise. To assess the means by which peripheral metabolism facilitates the transition to a gluconeogenic state, dogs were studied during 150 min of moderate treadmill exercise. Metabolism in the working hindlimb was assessed with arteriovenous difference and isotopic techniques (n = 9). In a separate group (n = 6), hepatic metabolism was assessed using arteriovenous differences. Limb glucose uptake (LGU) and oxidation (GOX) rose from 33 +/- 10 and 5 +/- 2 to 101 +/- 20 and 54 +/- 15 mumol/min at 10 min of exercise. LGU continued to rise (151 +/- 21 mumol/min at 150 min), while GOX declined. Nonoxidative glucose metabolism (GNOX) was 28 +/- 10 mumol/min at rest and 47 +/- 24 and 108 +/- 16 mumol/min at 10 and 150 min of exercise. Limb nonglycemic (predominantly glycogen) pyruvate formation rose from 52 +/- 22 to 198 +/- 54 and 242 +/- 74 mumol/min at 10 and 150 min of exercise. The gradual increase in GNOX and the high glycogenolytic rate were paralleled by accelerated lactate, pyruvate, and glutamine releases. Limb glycerol release rose promptly and remained elevated during exercise. Plasma nonesterified fatty acids (NEFAs) rose gradually and paralleled the gradual rise in GNOX (r = 0.93). The resulting rise in hepatic NEFA delivery was highly correlated to hepatic O2 uptake (r = 0.87), hepatic vein lactate-to-pyruvate ratio (r = 0.90), and intrahepatic gluconeogenic efficiency (r = 0.96). In summary, during exercise, 1) the primary fate of the added glucose consumed by the working limb is initially oxidation, but becomes GNOX as exercise duration progresses; 2) glycogenolysis rises promptly, but attains its highest rate at the end of exercise; 3) the late increases in GNOX and glycogenolysis relate to an increased gluconeogenic precursor release from the working limb; 4) although lipolysis increases promptly and is sustained, circulating NEFAs rise only gradually; and 5) the gradual rise in plasma NEFAs is highly correlated to the shift from GOX to GNOX and the adjustments in hepatic metabolism that are necessary for the full gluconeogenic response.


Stimulation of angiotensinogen mRNA levels in rat pituitary by estradiol. Angiotensin II (ANG II) is a putative paracrine hormone in the anterior pituitary. Angiotensinogen mRNA, however, is not detectable by Northern blot hybridization, suggesting that ANG II may not be synthesized within the pituitary. An alternative explanation may be that angiotensinogen gene activity is low under normal conditions, with angiotensinogen mRNA being below the level of detection. Utilizing a sensitive solution hybridization method, we sought to determine whether angiotensinogen mRNA could be detected in pituitaries from normal male rats or ovariectomized (OVX) rats treated with estradiol (E2) for 4 days. Very low levels of angiotensinogen mRNA were detected from male or OVX rat pituitaries, but E2 treatment resulted in a marked dose-dependent increase in pituitary angiotensinogen mRNA levels. Levels of angiotensinogen within the pituitary were not significantly different after the E2 treatment. Angiotensinogen mRNA levels in liver and brain were much higher than in the pituitary but were not altered significantly by the chronic E2 treatment. These results are consistent with the local synthesis of angiotensinogen in the pituitary and further suggest that pituitary angiotensinogen gene transcription is regulated by estrogen.


Effect of aging and exercise on GLUT-4 glucose transporters in muscle. This study was conducted to investigate whether changes in muscle glucose transporter GLUT-4 protein might be associated with a previously reported deterioration in glucose tolerance with aging, and, furthermore, to determine whether exercise training could increase GLUT-4 protein levels in older animals. GLUT-4 protein concentration was measured in soleus, gastrocnemius, and extensor digitorum longus muscles of trained (10 or 15 wk treadmill running) and untrained young (6-8 mo), middle-aged (15-17 mo), and old (27-29 mo) Fischer 344 rats. All GLUT-4 protein values were expressed as a percent of the mean for the young untrained group. Two-way analysis of variance demonstrated significant main effects of both training and aging in the gastrocnemius and soleus muscles. Exercise training produced significant increases in GLUT-4 protein in the soleus muscle of young (273 +/- 32.9 vs. 100 +/- 38.5%) and middle-aged rats (215 +/- 19.9 vs. 108 +/- 33.2%) compared with sedentary controls. Similar significant increases were also found in the gastrocnemius muscle of young (169 +/- 20.1 vs. 100 +/- 5.8%) and middle-aged rats (167 +/- 46.7 vs. 60 +/- 7.9%) with training. In the oldest rats, GLUT-4 was not significantly increased with training, but the trend toward an increase was apparent in all three muscle types. The main effect of aging was primarily due to a statistically significant difference between the old trained and young trained rats. A trend toward decreased GLUT-4 with aging was apparent in the untrained animals, but this was not statistically significant.


Effect of total parenteral nutrition, systemic sepsis, and glutamine on gut mucosa in rats. The effect of the combination of total parenteral nutrition (TPN) and systemic sepsis on mucosal morphology and protein synthesis was investigated. Rats were given a standard TPN mixture consisting of glucose (216 kcal.kg-1.day-1), lipid (24 kcal.kg-1.day-1), and amino acids (1.5 g N.kg-1.day-1) for 5 days. On the 5th day the rats (n = 37) were randomized into four groups according to diet as follows: 1) control nonseptic on standard TPN, 2) control nonseptic on TPN with glutamine, 3) septic on standard TPN, and 4) septic with the TPN supplemented with glutamine. Twenty hours after the injection of Escherichia coli, the rats were given a 4-h constant infusion of [U-14C]leucine to determine the mucosal fractional protein synthesis rates. The following results were obtained. 1) Histological examination showed that systemic sepsis caused tissue damage to the ileum and jejunum. 2) Glutamine supplementation attenuated these changes. 3) There were no visible changes to the colon either from glutamine supplementation or sepsis. 4) Sepsis was associated with an increase in mucosal protein synthesis and decreased muscle synthesis. 5) Addition of glutamine to the TPN mix further increased protein synthesis in the intestinal mucosa of septic rats.


Apolipoprotein expression and cellular differentiation in Caco-2 intestinal cells. Caco-2 cells, cultured for 18 days on porous filter supports and conventional plastic culture dishes, were used to study the effects of cellular differentiation on the expression of apolipoprotein (apo) genes. Media of filter-grown cells accumulated more apo B as apo B-48 and contained three times the amount of edited apo B mRNA compared with plastic-grown cells. The accumulation of apo A-I by media of plastic-grown cells was higher than accumulation by filter-grown cells, despite similar concentrations of apo A-I mRNA. The apo A-IV was detectable in the culture media earlier with filter-grown cells compared with plastic-grown cells, despite similar apo A-IV mRNA concentrations. Plastic-grown cells contained more apo E mRNA, and their media accumulated more apo E than filter-grown cells. With the exception of apo A-I, apo gene expression changed with Caco-2 cell differentiation to resemble more closely the patterns seen in adult enterocytes. There were no effects or minimal effects of added retinoic acid, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], or thyroid hormone on apo accumulation in media of filter-grown cultures of Caco-2 cells. However, 1,25(OH)2D3 and thyroid hormone increased apo B, apo A-IV, and apo A-I mRNA concentrations, retinoic acid increased apo B mRNA concentrations alone, and all three reduced apo E mRNA concentrations. Ratios of edited to unedited apo B mRNA were unaffected. In conclusion, culture substratum importantly influences Caco-2 cell differentiation. Soluble factors that influence cellular differentiation may affect apo gene expression over and above effects mediated by the culture substratum.(ABSTRACT TRUNCATED AT 250 WORDS)


Insulin-regulated sorting of glucose transporters in 3T3-L1 adipocytes. Two glucose transporters (GLUT-4 and GLUT-1) move from within the cell to the plasma membrane (PM) when 3T3-L1 adipocytes are stimulated with insulin. To study the sorting of these two molecules, vesicles containing GLUT-4 and GLUT-1 were immunoadsorbed from basal and insulin-treated cells. Two different vesicle populations were isolated as follows: 1) a compartment that contained the majority of intracellular GLUT-4 and GLUT-1 and 2) a subpopulation of vesicles containing 43% of the intracellular GLUT-4 that was highly insulin regulatable and that contained relatively low levels of GLUT-1. After incubation at 19 degrees C, basal glucose transport was slightly increased, whereas insulin-dependent transport was blocked. Consistent with these observations, cell surface GLUT-1 levels were increased in the basal state, whereas insulin-dependent translocation of GLUT-4 to the PM was blocked at 19 degrees C. However, insulin-dependent sorting of GLUT-4 within the intracellular compartment was still evident at 19 degrees C. These data indicate that GLUT-1 and GLUT-4 are heterogeneously distributed throughout the same intracellular compartment in 3T3-L1 adipocytes. Furthermore, we have uncoupled two distinct steps in the insulin-dependent movement of GLUT-4 to the cell surface. These include movement of GLUT-4 out of its storage compartment and accumulation of GLUT-4 at the cell surface. Only the former step occurs in cells preincubated at 19 degrees C.


Estimation of the rate of appearance in the non-steady state with a two-compartment model. A simple tracer-based method for calculating the rate of appearance of endogenous substances in the non-steady state, free from the inconsistencies of Steele's equation, is still lacking. This paper presents a method based on a two-compartment model by which the rate of appearance can be calculated with only a modest increase in complexity over Steele's approach. An equation is developed where the rate of appearance is expressed as a sum of three terms: a steady-state term, a term for the first compartment, and a term for the second compartment. The formula employs three parameters and makes the relationship between rate of appearance and specific activity changes explicit. An equation is also provided for estimating the error of the method in each individual run. The algorithm can be implemented with a spreadsheet on a personal computer. Simulated and experimental data obtained by the hyperinsulinemic euglycemic glucose clamp technique were used as a test. The accuracy with which the time course of glucose production could be reconstructed was clearly better than that using Steele's equation. Marked negative values for endogenous glucose output were calculated with Steele's equation but not with the new method. The characteristics of generality, simplicity, and accuracy and the availability of an error estimate make this new method suitable for routine application to non-steady-state tracer analysis.


Role of EDRF in splanchnic blood flow of normal and chronic portal hypertensive rats. The role of endogenous endothelium-derived relaxing factor (EDRF) in splanchnic blood flow was assessed in normal and portal vein-stenosed rats (PSRs). Specific and maximal inhibition of EDRF was achieved by intravenous administration of NG-nitro-L-arginine (L-NOARG) as a 1.75 mumol/kg bolus, followed by constant infusion of 1.75 mumol/kg for 20 min. Pretreatment with L-arginine (175 mumol/kg iv) completely blocked both hypertension and the reduction in blood flow induced by L-NOARG. Pretreatment with D-arginine (175 mumol/kg iv) and prazosin (500 micrograms/kg iv) did not attenuate the pressor effect of L-NOARG. These results indicate that L-NOARG selectively blocks EDRF. The blood flow to the stomach, duodenum, jejunum, ileum, cecum, and colon in control rats was 81.1 +/- 8.7, 199.1 +/- 21.9, 153.3 +/- 20.0, 68.6 +/- 10.6, 79.4 +/- 11.8, and 59.3 +/- 7.8 ml.min-1.100 g-1, respectively, and in PSRs was 141.4 +/- 10.8, 244.0 +/- 10.4, 208.3 +/- 9.8, 126.8 +/- 13.0, 166.9 +/- 16.5, and 94.8 +/- 4.7 ml.min-1.100 g-1, respectively. Blood flow was measured using the radioactive microsphere method. L-NOARG significantly reduced blood flow to the stomach, duodenum, jejunum, ileum, cecum, and colon in control rats by 47, 44, 48, 55, 40, and 41%, respectively, and in PSRs by 30, 27, 36, 33, 28, and 23%, respectively. The magnitude of blood flow reduction in PSRs was lower than in normal rats. These results indicate that EDRF plays an important role in control of the splanchnic circulation, but its effect on the hyperdynamic circulation observed in PSRs is insignificant.


Effect of aspirin on ulcer site blood flow in cat stomachs. Radioactive microspheres were used to measure blood flow in the cat stomach during exposure to saline, 0.075 M HCl, and then 15 and 30 min after exposure to 20 or 40 mM aspirin in HCl. At the end of the experiment, the stomach wall was divided into ulcerated regions and adjacent nonulcerated areas. When exposed to saline, both regions had similar blood flow: 27 +/- 5 and 25 +/- 5 ml.min-1.100 g-1 (means +/- SE). Addition of acid caused a significant increase in blood flow to 41 +/- 7 ml.min-1.100 g-1 only at those sites that eventually ulcerated in the presence of aspirin. In the adjacent nonulcerated regions, blood flow was 31 +/- 5 ml.min-1.100 g-1 and was not significantly greater than the flow recorded during saline exposure. Aspirin caused ulcer site blood flow to increase dramatically to 89 +/- 12 and 122 +/- 18 ml.min-1.100 g-1 after 15 and 30 min, whereas the adjacent nonulcerated tissue rose to 40 +/- 6 and 44 +/- 5 ml.min-1.100 g-1, respectively. The ulcer site hyperemia with acid alone suggests higher mucosal permeability in these regions allowing back-diffusion of acid and injurious agents. The present data obtained in the cat do not support the notion that ischemia plays a role in initiating nonsteroidal anti-inflammatory drug (NSAID)-induced ulcers, but rather that acute NSAID ulcers are associated initially with a hyperemia.


Innervation of pylorus in control of motility and gastric emptying. The motility index (MI) and contractile frequency were determined for the gastric antrum, pylorus, and duodenum during digestive and interdigestive states in eight dogs before and after resection of the ramus pyloricus nervi vagi (NR) and after selective proximal vagotomy (SPV) subsequent to NR. Neither NR nor subsequent SPV altered the migrating motor complex in the interdigestive state. In the digestive state, NR decreased the MI in the antrum, pylorus, and duodenum. The MI did not further change after SPV subsequent to NR. The cholecystokinin antagonist L364,718 decreased MI in the antrum, pylorus, and duodenum. After NR, L364,718 caused a further reduction in the MI during administration. Gastric emptying was accelerated after NR. SPV subsequent to NR increased gastric emptying further. L364,718, in the absence of NR, accelerated gastric emptying but only during the initial period of emptying. After NR, L364,718 also decreased the time required for emptying of 50 and 100% of the meal. After SPV subsequent to NR, no additional acceleration of emptying occurred.


Polyamine transport systems in isolated rat hepatocytes derived from resting and regenerating livers. Polyamines and their principal metabolite, gamma-aminobutyric acid (GABA), modulate eukaryotic cell growth. To determine whether the liver possesses specific polyamine transport sites and whether changes occur to these or GABA transport sites during hepatic regeneration, suspensions of rat hepatocytes derived from in situ collagenase perfusions of livers at times 0, 24, 48, and 72 h post-partial hepatectomy were incubated at 4, 20, and 37 degrees C with various concentrations of the following ligands: [3H]putrescine, [3H]spermidine, [14C]spermine, and [3H]GABA together with or without excess unlabeled ligand, KCN, ouabain, or digitoxigenin. Of the ligands studied, only [14C]spermine and [3H]GABA were associated with specific binding to hepatocytes derived from nonregenerating livers. Spermine binding correlated with the concentration of hepatocytes in the incubation mixture and reached equilibrium within 60 min. The approximate affinity constant (KD) was 5.5 x 10(-5) mol/10(6) hepatocytes, and maximum number of binding sites (Bmax) was 1.8 +/- 1.2 x 10(-7) mol.10(6) hepatocytes-1.min-1. Binding was neither temperature nor sodium dependent and was not inhibited by KCN, ouabain, digitoxigenin, other polyamines, or GABA. Aside from a 43% decrease in spermine binding at 24 h post-partial hepatectomy [5.1 +/- 1.1 vs. 8.9 +/- 3.1 x 10(3) disintegrations per minute (dpm)/10(6) hepatocytes at time 0, P less than 0.05] and a 39% decrease in GABA binding (3.4 +/- 1.3 vs. 5.5 +/- 1.9 x 10(3) dpm/10(6) hepatocytes, P less than 0.05), there were no significant changes in ligand binding during hepatic regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)


Immunocytochemical studies suggest two pathways for enteroendocrine cell differentiation in the colon. Enteroendocrine cells represent the most heterogeneous population of terminally differentiated cells in the mouse intestinal epithelium. Each of the approximately 15 different enteroendocrine cell subpopulations shows characteristic distributions along both the cephalocaudal and crypt-to-villus (in the small intestine) or crypt-to-surface epithelial cuff (in the colon) axes of the gut. These cells provide a sensitive model for studying how the continuously renewing gut epithelium is able to establish and maintain its spatial differentiation. Enteroendocrine cells are derived from the same multipotent stem cell that gives rise to enterocytes and goblet and Paneth cells. Regional differences in enteroendocrine cell number and type reflect positional differences in the differentiation programs of this lineage. To better understand the nature of these programs, we used multilabel immunocytochemical methods to examine the accumulation of endogenous neuroendocrine products as well as the product of a liver fatty acid binding protein/human growth hormone transgene in enteroendocrine cells located in proximal colonic glands. The results suggest that serotonin, substance P-, glucagon-like peptide-1 (GLP-1)-, peptide tyrosine tyrosine (PYY)-, neurotensin-, and cholecystokinin (CCK)-producing cells can all arise from a single stem cell located within a given gland. Based on pairwise comparison of the coexpression of each of these six products in individual cells as well as their ability to support transgene expression, it appears that the enteroendocrine lineage has two branches; one branch produces substance P and serotonin cells while the other yields GLP-1, PYY, neurotensin, and CCK cells.


Cardiac performance in the portal vein-stenosed rat. Patient studies and experimental models suggest that impaired cardiac function might contribute to the altered pressor responses and compromised reflex responses to hypotension observed in liver disease. To elucidate the role that portosystemic shunting plays in cardiac impairment, right atrial chronotropic and right ventricular inotropic responses to a beta-adrenoceptor agonist were compared using isolated cardiac tissues from sham-operated and chronic portal vein-stenosed rats. Maximal chronotropic responses of sinoatrial tissue from portal vein-stenosed rats to isoproterenol were reduced 18% (P less than 0.05) with no change in sensitivity [mean effective dose (ED50)]. Basal indexes of contraction were diminished 48% (P less than 0.0025) and 60% (P less than 0.0005) for developed tension and change in force over change in time (dF/dt), respectively, in ventricular tissue from portal vein-stenosed animals. In addition, the maximal response for isoproterenol-induced ventricular contraction was smaller in portal hypertensive animals (-44%, P less than 0.0005, and -52%, P less than 0.01 for developed tension and dF/dt, respectively), but no changes in isoproterenol ED50 values were found. These results indicate that portal venous hypertension with extensive portosystemic shunting leads to impairment of basal myocardial contraction and decreased chronotropic and inotropic responsiveness to beta-adrenoceptor agonists.


Regulation of gene expression in gastric epithelial cell populations of fetal, neonatal, and adult transgenic mice. Little is known about lineage relationships and differentiation programs of various epithelial cells present in mouse gastric units. We have previously used rat liver fatty acid binding protein/human growth hormone (L-FABP/hGH) transgenes to define epithelial cell lineages relationships in the small intestine of fetal and adult mice and to examine regulation of their terminal differentiation programs along the crypt-to-villus and duodenal-to-ileal axes. We have now used these transgenes to explore similar issues in the stomach. Immunocytochemical studies of fetal and adult transgenic L-FABP/hGH animals and their normal littermates revealed that the intact endogenous mouse L-FABP gene (Fabpl) is not expressed in gastric epithelium. Nucleotides-596 to +21 of the rat L-FABP gene direct "inappropriate" expression of hGH in the gastric epithelium as early as fetal day 15. From 1 to 13 mo, L-FABP-596 to +21/hGH expression occurs only in surface mucous cells of zymogenic and mucous gastric units; the reporter is not detectable in the enteroendocrine, parietal and chief cell populations of zymogenic glands. Electron microscopic immunocytochemistry revealed that hGH is directed to apical secretory granules in surface and pit mucous cells expressing the transgene. hGH levels vary widely among surface mucous cells both within single pits and between gastric units in a given animal. The heterogeneity noted in reporter expression suggests that there are marked differences in the regulatory environments of individual cells of a single type within a given gastric unit. This raises the possibility that cell differentiation programs in the stomach may not be as tightly coupled to cellular translocation as in the small intestine. Finally, the lack of expression of L-FABP-596 to +21/hGH in gastrin- and serotonin-immunoreactive cells of the stomach contrasts with its efficient expression in comparable cell types located in the duodenum; providing a model system for examining differential regulation of gene expression in terminally differentiated cell types represented in both gastric and intestinal epithelium.


Changes in antroduodenal resistance induced by Cisapride in conscious dogs. The effects of Cisapride on gastric outflow/pressure relationships have been examined in conscious dogs. In the digestive state, after inhibition of emptying in the first 10 min, Cisapride accelerated gastric emptying for 110 min by increasing the volume (7.3 +/- 1.2 vs. 2.3 +/- 0.3 ml) rather than by decreasing the interval between flow pulses (4.9 +/- 0.2 vs. 4.5 +/- 0.1s). After non-nutrient gastric loading, Cisapride also first inhibited and then accelerated gastric emptying as the consequence of both a larger stroke volume (8.0 +/- 1.7 vs. 3.2 +/- 0.5 ml) and more frequent pulses (3.9 +/- 0.1 vs. 4.9 +/- 0.1 s). In both situations, Cisapride increased the amplitude of the antral/pyloric pressure waves. Antropyloroduodenal resistance increased substantially in the first 10 min after Cisapride then recovered to its control value. We conclude that stimulation of antropyloroduodenal motility by Cisapride can both impede and increase gastric emptying by changes in antroduodenal resistance.


Capsaicin-induced hyperemia in the stomach: possible contribution of mast cells. Topical application of capsaicin to the gastric mucosa results in marked hyperemia as a consequence of the release of vasoactive neuropeptides from sensory afferent neurons. Because many of these neuropeptides have the capacity to induce mast cell degranulation, we investigated the possible contribution of mast cells to capsaicin-induced hyperemia. Application of capsaicin to the gastric mucosa of normal rats resulted in a concentration-dependent increase in blood flow. In rats in which mastocytosis was induced by prior infection with Nippostrongylus brasiliensis, the hyperemic responses to capsaicin were significantly greater than in control rats. This augmented hyperemic response could be significantly attenuated by pretreatment with a histamine H1-receptor antagonist (pyrilamine) or with a mast cell stabilizer (doxantrazole). Depletion of mucosal mast cells through treatment with dexamethasone also significantly reduced the hyperemic response to capsaicin. Hyperemic response to capsaicin in normal rats and in rats with mucosal mastocytosis could be completely abolished by pretreatment with ruthenium red or prior ablation of the sensory afferent neurons with capsaicin. These results suggest that in rats with gastric mastocytosis, sensory neuron-dependent activation of mast cells contributes to the hyperemic response to topical capsaicin. These findings are therefore consistent with the hypothesis that there is communication between nerves and mast cells in the gastric mucosa, at least in rats previously infected with N. brasiliensis.


Differential regulation of cytochrome P-450 genes along rat intestinal crypt-villus axis. Mammalian small intestine contains cytochrome P-450-dependent monooxygenase enzymes that are capable of metabolizing a wide variety of xenobiotics and activating procarcinogens to mutagenic compounds. The epithelial cells lining the small intestine are separated into a proliferating undifferentiated compartment located in crypts and a nonproliferating differentiated compartment located on villi. The constitutive expression and induction by xenobiotics of genes that encode components of the cytochrome P-450-dependent mono-oxygenase system along the rat intestinal crypt-villus axis were investigated using isolated epithelial cells and in situ hybridization. For each gene examined, hybridization analysis of RNA obtained from isolated epithelial cells correlated with findings on in situ RNA hybridization. Cytochrome P-450IA1 mRNA (CYP1A1), the major aromatic hydrocarbon-inducible P-450, and cytochrome P-450IIB1 mRNA (CYP2B1), the major phenobarbital-inducible P-450, were constitutively expressed in villus cells with no detectable mRNA present in crypts. Treatment with several chemical inducers resulted in a marked increase in CYP1A1 mRNA in both crypt and villus cells. In contrast, although CYP2B1 mRNA was inducible in villus cells, CYP2B1 mRNA was not detected in crypts after treatment with chemical inducers. NADPH cytochrome P-450 reductase, a necessary component for the activity of all P-450 enzymes, was expressed constitutively at low levels only in villus cells. Treatment with dexamethasone induced reductase mRNA in both crypt and villus cells. Taken together, these results demonstrate that there is a complex gene-specific pattern of expression of the microsomal monooxygenase system along the crypt-villus axis of rat small intestine.(ABSTRACT TRUNCATED AT 250 WORDS)


Polyamines attenuate jejunal mucosal injury induced by oleic acid. Effects of putrescine, spermidine, and spermine on lipid-induced injury to jejunal mucosa were assessed in anesthetized rats. Mucosal epithelial integrity was continuously monitored by measuring blood-to-lumen clearance of 51Cr-labeled EDTA. Perfusion of jejunal lumen with emulsified lipid (20 mM sodium taurocholate and 40 mM oleic acid) increased 51Cr-EDTA clearance. Addition of spermidine (0.5 mM), but not putrescine (2.0 mM) or spermine (0.25 mM), to the lipid perfusate reduced the increment in 51Cr-EDTA clearance. Histological evaluation of jejunal mucosa indicated that the epithelial lining of the villous tips was damaged by emulsified oleic acid and that this injury was ameliorated by spermidine. Pretreatment of jejunal mucosa with spermidine did not prevent disruption of mucosal integrity induced by a subsequent perfusion with emulsified lipids. Intravenous infusion of spermidine to achieve an extracellular concentration of 0.5 mM did not prevent the lipid-induced increase in 51Cr-EDTA. Spermidine also ameliorated lipid-induced disruption of Caco-2 cell monolayers in culture; this protective effect was dose dependent and was observed only when spermidine was applied to the apical aspect of the monolayers. These findings indicate that spermidine must be present on the apical portion of the epithelial cell during lipid insult. Substitution of lysine or arginine for spermidine did not reduce the extent of lipid-induced injury to jejunal mucosa, indicating that spermidine's protective effects cannot simply be attributed to its cationic nature. Spermidine did not alter the turbidity of a micellar oleic acid solution, indicating that spermidine was not removing oleic acid from the soluble phase.(ABSTRACT TRUNCATED AT 250 WORDS)


Measurement of axial forces during emptying from the human stomach. Our aim was to measure axial forces in the stomach and to evaluate their relation to circumferential contractions of the gastric walls and the emptying of gastric content. We used a combination of simultaneous radioscintigraphy, gastroduodenal manometry, and an axial force transducer with an inflatable 2-ml balloon fluoroscopically placed in the antrum. In vitro studies demonstrated that the axial force transducer records only antegrade forces along the longitudinal axis of this probe in an intensity-dependent manner. In vivo studies were performed in five healthy subjects for at least 3 h after ingestion of radiolabeled meals. When administered separately, the emptying of liquids or solids from the stomach is associated with generation of antral axial forces and coincident phasic pressure activity; however, almost 20% (average) of gastric axial forces during emptying of liquids or solids are unassociated with proximal or distal antral pressure activity ("isolated" forces). High amplitude antral axial forces and pressures occur during both lag and postlag emptying phases. During emptying of liquids, there is a trend for axial forces to be coincident more often with proximal than with distal antral pressure activity and vice versa for the emptying of solids (P = 0.015). These data suggest that when placed in the antrum, the transducer can semiquantitatively record axial forces during gastric emptying. By combining these observations with the data from in vitro studies, it appears that axial forces predominantly result from traction on the balloon by the longitudinal vector resulting from circumferential gastric contractions. The combination of radioscintigraphy and measurement of antral axial forces is a promising method to evaluate mechanical forces involved in the emptying of the human stomach.


Preservation and propagation of cyclic myoelectric activity after feeding in rat small intestine. The cyclic nature and distal propagation of the fasting migrating motor complex (MMC) of the small intestine have been well described. The fate of the MMC after feeding, however, has not been elucidated. We used time series analysis with fast Fourier transforms (FFT) to study myoelectric cycling before and after feeding. Ten rats were chronically prepared with bipolar electrodes secured to the duodenum and proximal jejunum. Spikeburst frequency was recorded before and after feeding simple nutrients. During fasting, cyclic activity occurred at the MMC frequency. Although after feeding this periodicity appeared disrupted, FFT analysis showed persistent cycling at the fasted (MMC) rate. Digital filtering of data at the MMC frequency isolated cycling from background noise during both fasted and fed states and showed a depression in the amplitude of the waveform after feeding. Root-mean-square analysis of the waveform confirmed statistically significant depression of amplitude by 47-57%. The waveform propagated from the duodenum to the jejunum during both the fasted and fed state at an unchanged rate. We conclude that factors controlling myoelectric cycling during the fasted state persist after feeding, allowing continued net abroad propulsion of food.


Influence of luminal nutrient composition on hemodynamics and oxygenation in developing intestine. Age-related differences in the intestinal hemodynamic and oxygenation responses to carbohydrate, protein, and lipid were studied in 1-day-, 3-day-, 2-wk-, and 1-mo-old piglets. A branch of the mesenteric vein draining an isolated loop of jejunoileum was used to measure intestinal blood flow, arteriovenous oxygen content difference, and venous and capillary pressure and to calculate oxygen uptake and vascular resistance. Fractionated intestinal flow was measured with radiolabeled microspheres. Measurements were made before and after luminal placement of either 5% glucose, 2.3% casec, or 5% corn oil. In 1-day-old animals, unlike all older age groups, total intestinal blood flow and vascular resistance were unchanged by any nutrient. Fractionated flow to the mucosa/submucosa levels did, however, increase in the intestine of 1-day-old piglets to a similar extent as that in older age groups. Placement of lipid or protein into the lumen led to increased oxygen uptake in all age groups, whereas carbohydrate absorption resulted in no increase in intestinal oxygen consumption in 1- and 3-day-old animals. In 1-day-olds, the increased oxygen consumption was achieved by enhanced oxygen extraction with no change in total blood flow, whereas all other groups demonstrated increases in blood flow and/or oxygen extraction. Compared with a mixed meal, oxygen consumption was not significantly greater for an individual nutrient component.


Two-dimensional coupling by gap junctions in cultured gastric smooth muscle monolayers. We studied intercellular transfer in cultured rabbit gastric smooth muscle cell monolayers after microinjection of electrotonic current or the fluorescent probe Lucifer yellow CH. Because cultured gastric muscle cells proliferate in vitro and form regular arrays of parallel spindle-shaped cells, we sought to assess the role of cell shape and orientation in determining two-dimensional coupling properties. With the use of electron microscopy, gap junctions were identified between adjacent cells. Northern blot analyses using specific cDNA probes demonstrated expression of mRNA for the gap junction protein connexin43. Dye injection of Lucifer yellow resulted in 97% transfer to at least one adjacent cell, and 88% of adjacent cells received dye. Electrophysiological studies were performed using two intracellular microelectrodes to measure electrotonic current flow between cells at varying interelectrode distances. Current flow in the monolayers was modeled using a modified two-dimensional analysis. Initial assessment showed that the ratio of calculated space constants (longitudinal axis/perpendicular axis) was 4.4, indicating anisotropic conditions. However, when a geometric transform was used to normalize the spindle-shaped cells to regular hexagons, the space constants became statistically equivalent (200 microns longitudinal, 256 microns perpendicular). These results suggest that anisotropy of current flow in the monolayer of gastric smooth muscle cells was due primarily to the shape of the cells and not to intrinsic membrane properties or the distribution of gap junctions.