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Embryonic chick intestine in organ culture. A unique system for the study of the intestinal calcium absorptive mechanism.

Duodena from 20-day-old chick embryos can be maintained in large scale organ culture on specially designed stainless-steel grids in contact with serum-free medium for 48 h with excellent preservation of mucosal structure at both the light and electron microscope levels. Although mitotic rate was subnormal, several other factors attest to the essential viability of the cultured intestine: L-leucine incorporation into protein, as well as the synthesis of a specific vitamin D3-induced calcium-binding protein (CaBP), increased over a 48-h culture period, and the electropotential gradient across the intestine was maintained throughout the culture period as was a concentration gradient for calcium. The tissue responded to vitamin D3 in the medium by synthesizing the calcium-binding protein within 6 h and by exhibiting enhanced 45Ca uptake within 12–24 h. Concentrations of vitamin D3, or its 25-hydroxylated derivative, higher than necessary for CaBP induction, also increased the activity of alkaline phosphatase. The 1,25-dihydroxylated derivative of vitamin D3, at a level extremely potent in CaBP induction, did not stimulate alkaline phosphatase. Mucosal to serosal transport of 45Ca could also be measured in everted duodenal sacs, subsequent to culture under similar conditions, and was also increased by vitamin D3 in the medium. Other embryonic organs, esophagus, stomach, liver, pancreas, lung, skin, and muscle, did not produce CaBP in response to vitamin D3 in the culture medium. However, CaBP-synthesizing capacity was present in the entire intestinal tract, exclusive of the rectum. 59Fe and 32P uptake by cultured duodenum were also stimulated by vitamin D3. The system has proven quite useful in the study of the vitamin D-mediated calcium absorptive mechanism but should be applicable to the study of the absorption of other nutrients, drugs, hormones, etc., as well as other studies of intestinal function.


INTRODUCTION
Since the discovery of the action of vitamin D on measured using methods of oral intubation (2,3), the enhancement of intestinal calcium absorption in situ perfusion (2,4,5), in situ ligated loops (1, by  (1), a variety of in vivo 2, 6, 7), and Thiry-Vella fistulas (8,9) . and in vitro techniques have been employed to In vitro, the everted intestinal sac technique study the transport mechanism(s) involved . In of Wilson and Wiseman (10) has been extensively the living animal, calcium absorption has been used in calcium absorption studies (11)(12)(13)(14) . Others have employed intestinal ringlets (15,16) or squares (17) and intestinal tissue mounted in a variety of Ussing-type (18) chambers (14,17,19,20) . These and other studies, too numerous to mention, have provided considerable insight into the phenomenon of vitamin D-mediated calcium transport (21) . However, a major difficulty existed in this research : it was never possible to be certain that perturbation of the system was a direct result of the physiological, nutritional, pathological, or pharmacological means employed . This was true of the in vitro as well as in vivo studies since the vitamin D status of the intestine, and any alterations thereof, had been established in vivo before the measurement of transport in vitro . An unrecognized technical problem of considerable importance also existed : survival of the structural integrity of the tissue in vitro is brief. Recent studies, for example, have shown that the mucosal structure of the everted rat intestine is completely lost within 60 min of being immersed in a buffer solution (22) . The fine structural integrity of the everted hamster intestine is well preserved for at least I h (23) but, at best, this still puts a severe time limitation on the types of agents or other variables to which the intestine could be subjected in vitro . An alternative approach, which would allow close control of a wider range of variables influencing calcium absorption and be more likely to allow separation of direct and indirect effects, is organ culture. However, this technique, as pioneered most notably by Strangeways and Fell (24), although extensively applied by them and many others in morphological, developmental, and biochemical studies of a number of tissues (25), had not been applied to the study of the vitamin D-mediated calcium transport mechanism of the intestine until recently (26) . In that recent report was documented "the first demonstration of an in vitro physiological effect of vitamin D a on . . . the intestine ." Initial attempts to develop an intestinal organ culture system were motivated by the observation that the vitamin D-mediated, intestinal calciumbinding protein, CaBPI (27), was not present in The duodenal loop was excised from the 20-day chick embryo and the pancreas removed . (Earlier I Abbreviations used in this paper : Ca-ATPase, calciumstudies had shown that duodena from chick embryos dependent ATPase ; CaBP, calcium-binding protein ; at least as early as day 15 can also be cultured and are 25-OHD3, 25-hydroxycholecalciferol ;1,25-(OH) 2D3i responsive to vitamin D3 .) The duodenum was then 1, 25-dihydroxycholecalciferol ; TCA, trichloroacetic either slit lengthwise with fine scissors or everted on a acid . 25-gauge platinum-iridium (30%) wire (Engelhard the chick duodenum during embryogenesis but only appeared at the time of hatching (28) . The embryonic duodenum, a normal tissue presumably microbe-free, therefore, seemed a logical choice for organ culture . Upon incubation of this tissue in medium containing vitamin D a, it was found that CaBP was induced, that the induction was de novo and that, accompanying CaBP induction, there was enhanced radiocalcium uptake by the intestine (26), all features of the in vivo calcium absorptive mechanism in the chick . The technique employed in those early studies was to simply immerse the embryonic gut in the medium in a sealed flask . Gaseous exchange and pH control were, thus, severely limited. It is the purpose of the present report to describe a much improved organ culture technique, and some results obtained using it .

Culture Medium
McCoy's 5A (modified) medium without calcium (Grand Island Biological Co ., Grand Island, N .Y.) was reconstituted from the dry powder within I mo of use and sterile filtered through a 0 . 22 ) . The gut was placed on a rectangular grid (51 X 63.5-mm surface with 6 .35mm legs formed by making 90°angle bends on the long dimension) of 60-mesh stainless steel (Newark Wire Cloth Co., Newark, N .J .) in a polystyrene petri dish containing approximately 37 ml culture medium (see above) . In the case of slit-open duodena, the gut was actually slit while resting on the grid and arranged so that the mucosal surface was uppermost . A total of four duodena (300-400 mg fresh weight) were placed on each grid in bare contact with the medium .
The petri dishes were kept in an incubator at 37 .5°C and approximately 90% relative humidity usually for 2 days . The incubator was continuously gassed with 5% CO2 in air . The pH of the medium at the start of incubation was 7.4 and never dropped below 7 .1 . At the end of the culture period, the duodena were either homogenized in a motor-driven, ground glass, tapered homogenizer (Tri-R Instruments, Inc ., Rockville Centre, N.Y .) in 2 vol of a Tris buffer (0 .0137 M Tris-HCI, 0.12 M NaCl, and 4.74 mM KCl ; pH 7.4) preparatory to various assays, or subjected to measurement of radiocalcium uptake and/or transport as described below .

Assay Procedures
TOTAL PROTEIN : Portions of duodenal homogenates were solubilized in 100 vol of 1 N NaOH (2 h at room temperature) . Total protein assay was performed by the procedure of Lowry et al . (29) using an automatic analyzer (Technicon Instruments Corp ., Tarrytown, N.Y .) and crystallized bovine albumin (Pentex Biochemical, Kankakee, Ill .) in 1 N NaOH as a standard .
ALKALINE PHOSPHATASE ACTIVITY : Portions of duodenal homogenates were solubilized in 50 vol of 1% sodium dodecyl sulfate (4 h at 37 .5°C) . Alkaline phosphatase activity was assessed by measurement of the hydrolysis of p-nitrophenylphosphate (Sigma Kit 104s) .
caaP ASSAY : Portions of duodenal homogenates were analyzed for CaBP concentration by radial immunodiffusion . The procedure consisted of filling the wells of a Model FB-20 Disposo-Tray (Linbro Chemical Co ., Inc., New Haven, Conn .) with 0 .5 ml of a buffered agar solution (1 .5% agar, 7 .5% glycine, and 0 .02% thimerosal (all wt/vol) in barbital buffer, ionic strength 0 .1 at pH 8 .6) at 48°C containing 2% (vol/vol) rabbit antiserum to purified chick intestinal CaBP . After gelling, a sample hole (2 mm) was cut into the center of each agar well and 20 µl of duodenal homogenate dispensed into it . After a reaction period of 24 h in a humidified chamber, visible precipitin rings formed around the central sample hole . The diameters of these rings were measured microscopically . 66 THE JOURNAL OF CELL BIOLOGY . VOLUME 58, 1973 A linear relationship was found between ring diameter and concentration of purified CaBP over a range of concentrations between 2 .5 and 30 µg CaBP/ml . The visibility of precipitin rings could be enhanced by either soaking the trays in 0 .02% (wt/vol) cadmium acetate solution (up to 24 h) or for 15 min in a solution consisting of 0 .18% (wt/vol) of Buffalo Black NBR (Allied Chemical Corp ., Specialty Chemicals Div., Morristown, N.J .) in methanol : H2O : acetic acid (5 :5 :1) followed by destaining in the same solvent for up to 24 h . DNA : Portions of duodenal homogenates were heated in a H2O bath at 95°C in 50 vol of 5% trichloroacetic acid (TCA) for 30 min . After cooling, 2 ml of the diphenylamine reagent prepared exactly as described by Burton (30) were added, the solution mixed, and the tubes sealed and allowed to stand at room temperature for 18-24 h . Standards, over the range of 10-300 µg DNA/ml, were prepared from calf thymus DNA, Na salt, (Mann Research Labs .) .in 5 mM NaOH and treated exactly as the homogenates .
(Preliminary trials proved that initial nucleoprotein extraction, as described by Schneider (31), was unnecessary since values ~or duodenal homogenate DNA obtained by both methods were virtually identical . ) 45 ca UPTAKE : At the end of the culture period, duodena which had been slit open were briefly rinsed with a low-sodium buffer solution (13) at room temperature and transferred to 3 ml of the same buffer containing 0 .5 µCi 45Ca/ml in a 25-m1 Erlenmeyer flask maintained at 37 .5°C in a shaking water bath . The flasks were sealed with rubber stoppers . After a 30-min incubation period, the guts were removed from the flasks, rinsed thoroughly with ice-cold buffer, blotted lightly on tissue, and weighed . They were then transferred to counting vials, I ml of NCS solubilizer added (Amersham/Searle Corp ., Arlington Heights, Ill .), the vials sealed and heated at 50°C in an oven overnight. After cooling the vials, 50 p1 of concentrated acetic acid were added followed, after mixing, by 20 ml of a cocktail composed of 5 g 2,5diphenyloxazole and 0.1 g 1,4-bis[2-(5-phenyloxazolyl)]benzene (Amersham/Searle Corp .) per liter of toluene. The samples were assayed for 45 Ca activity in' a liquid scintillation counter (Nuclear-Chicago Mark I ; Nuclear-Chicago Corp., Des Plaines, Ill .) . Channels ratio measurement using a 133 Ba external standard was always monitored but quench correction was not necessary since there was minimal variable quench in the samples . 45 Ca uptake was calculated as percent of total 45Ca in the 3 ml of incubation buffer/100 mg fresh weight of duodenum. The data were statistically analyzed in this form, but then converted to percent of zero control for presentation . (1 uCi/ml) to assess its time-dependent incorporation into protein as one measure of the functional integrity of the duodenum in culture . At time intervals after the initiation of culture, duodena were homogenized and the protein precipitated from portions with 50 vol of 10% TCA . After centrifugation, the precipitates were washed with 200 vol of 10% TCA and recentrifuged . (Preliminary trials proved that the recovery of protein from the whole homogenates in the TCA precipitates was better than 92% .) The precipitates were then dissolved in I -ml NCS solubilizer, processed and counted as above (see 45 Ca Uptake) . Additional portions of whole homogenate were analyzed for total protein by the method outlined above (see Total Protein) . [

H]THYMIDINE INCORPORATION : [Methyl-
3 H]thymidine (Amersham/Searle Corp.) of specific activity 23 Ci/mmole, was included in the culture medium (1 µCi/ml) to assess its time-dependent incorporation into DNA in an attempt to estimate the degree of cell replication of the duodenum in culture . At time intervals after the initiation of culture, duodena were homogenized and the nucleoprotein precipitated from portions with 175 vol of ice-cold 7% TCA . After centrifugation, the precipitates were washed with 200 vol of ice-cold 70/0 TCA and recentrifuged . (Preliminary trials proved that the recovery of DNA from the whole homogenates in the TCA precipitates was better than 96% .) The precipitates were then dissolved in 1-ml NCS solubilizer, processed and counted as above (see 45 Ca Uptake) . Additional portions of whole homogenate were analyzed for DNA by the method outlined above (see DNA) .
MEASUREMENT OF ELECTROPOTENTIAL DIF-FERENCE : As another indicator of the viability of the cultured duodenum, the electropotential gradient across cultured everted sacs (see Culture Technique above) was measured as a function of time in culture . Agar bridges were prepared by filling thin-wall Teflon tubing (0 .56-mm ID . ; 0 .25-mm wall) with a molten 3% (wt/vol) agar solution containing 20 g KCI . The -bridges were connected to a DC voltmeter, model 412 A"(Hewlett-Packard Co ., Palo Alto, Calif .) via calomel reference electrodes, model 40250 (Beckman Instruaround the end of one agar bridge, the whole remaining immersed in the culture medium . The end of the other agar bridge was also immersed in the culture medium . The potential gradient existing across the intestine was then measured . A correction was made for the potential existing between the agar bridge tips when both were immersed in the culture medium .

Assessments of the Organ Culture Technique SUITABILITY OF SERUM-FREE MEDIUM :
A series of preliminary experiments proved the suitability of serum-free medium for use in this culture technique . Of course the use of serumfree medium is an advantage since more clearly defined conditions are possible . Not only was serum found unnecessary, but several additives, proven useful as serum substitutes by others working with cell culture, were also found unnecessary. The following serum substitutes were tried : albumin (Pentex Biochemical) at I g/liter and/or dextran (Sigma Chemical Co., 100,000-200,000 daltons) at 10 g/liter ; fraction VI glycoprotein (Pentex Biochemical) at 10 mg/liter and/or dextran at 10 g/liter (32) . As will be seen, the embryonic chick duodenum responds to vitamin D 3 in the serum-free culture medium by synthesizing CaBP and exhibiting enhanced radiocalcium uptake and transport . The use of serum or serum substitutes did not increase this responsiveness . Histologically, at the light microscopic level, the tissue is quite well preserved even after 48 h of incubation at the surface of serum-free medium ( Fig . 1) . At the electron microscopic level, preliminary results with a few sections indicate no change in the fine structure of duodenal absorptive cells from either uncultured duodena or duodena cultured for as long as 48 h . These studies are continuing and being extended to include investigation of the was into a sac effects of various treatments on duodenal fine the observations of others (33) . Preliminary structure.
studies have been undertaken in which colchicine MITOSIS : Mitotic figures were less frequently was introduced into the culture medium to a seen in the cultured duodenum in agreement with level of 1 .5 °M 3 h before the termination of a FIGURE 1 40-day embryonic chick intestine both (A) uncultured and (B) maintained in organ culture for 48 h in contact with serum-free medium . Tissues were fixed in a 0.1 M phosphate-buffered glutaraldehyde fixative, dehydrated in methanol, embedded in glycol methacrylate ; 1-°m sections were prepared and stained with acid fuchsin-toluidin blue (48) . The only definite change in mucosal structure at the light microscopic level was a possible shortening and thickening of the viii in the cultured intestine, probably induced by gravity as the tissue rested on the grid with the villi uppermost . specific time interval of culture to force an accumulation of metaphase figures. Strictly quantitative data are not yet available, but initial histological examinations revealed the presence of many mitotic figures even after 48 h of organ culture (colchicine introduced at 45 h) clearly indicating that, while cell proliferation is reduced in organ-cultured duodenum, it does proceed . The [ 3H]thymidine incorporation studies (see below) suggest a similar conclusion .
[ 3H]LEUCINE INCORPORATION : As shown in Fig . 2, the incorporation of [ 3H]leucine into duodenal protein increased directly as a function of time of incubation, but tended to taper off towards the end of the experiment . The protein synthetic mechanism was undoubtedly functioning, although not at peak efficiency, throughout the culture period . Synthesis of the specific vitamin D-induced CaBP followed a similar course as can be seen in Fig . 3  Slit-open embryonic chick duodena were incubated mucosal side up in the presence of vitamin D 3 in the medium (26 AM) for varying periods of time . At each time interval, duodena were assayed for total protein, CaBP, and 95Ca uptake as described above (Materials and Methods) . CaBP was detected in duodena cultured in medium containing vitamin D 3 as early as 6 h after the start of incubation and increased steadily with increasing incubation time (Fig . 3 A) . A significant increase in 95Ca uptake occurred within 24 h (within 12 h in an earlier experiment) after the start of incubation and a further increase was observed at 36 h after which there was no further rise (Fig. 3 B) . There were no signifi- Methods) . CaBP could be detected in duodena cultured in the presence of as little as 6 .5 nM vitamin D 3 or 25-OHD 3 in the medium (Fig . 5 B) . With increasing sterol concentration, CaBP increased but 25-OHD3 was up to twice as potent as vitamin D 3 . There was a significant increase in 95Ca uptake in duodena incubated in medium containing 32 .5 nM 25-OHD 3 or 65 nM vitamin D 3 (Fig . 5 A) . Maximal stimulation of 95Ca uptake was observed at 650 nM of either sterol ; there was a hint of a slightly higher response to vitamin D 3 than to 25-OHD 3 at this level but in the present experiment it was not statistically significant. There was no further increase in radiocalcium uptake with increasing sterol concentration .
The extraordinary potency of 1, 25-(OH) 2D 3 in this system is shown in Fig . 6. CaBP was detected in intestine cultured in medium containing as little as 6 .5 pM 1, 25-(OH) 2D 3 (Fig . 6 B) . Increasing 1, 25-(OH) 2 D 3 concentration resulted in increasing CaBP production . At the highest level of 1, 25-(OH) 2 D 3 that could be tested (65 nM), intestinal CaBP content was slightly greater than that found in intestine cultured in 26 µM 25-OHD 3 , a 4,000-fold higher sterol concentration . In spite of this high potency for CaBP induction, stimulation of 45Ca uptake, approximately equivalent to the maximum achieved by either vitamin D 3 or 25-OHD 3 (Fig . 5 A), was achieved only at 65 nM I , 25-(OH) 2D 3 (Fig . 6 A) . There may have been a slight increase at the lowest level of 1, 25-(OH) 2D 3 tested, 6 .5 pM, but this has not yet been verified .
In several other experiments, alkaline phosphatase activity was also measured in organcultured intestine as a function of medium con centration of vitamin D 3, 25-OHD 3 45 Ca uptake increased significantly in everted duodenal sacs, but mucosal to serosal transport was increased as well (Table II) . The portion of the everted gut mucosa in contact with the medium had lost some of its structural integrity but, on the basis of the transport data obtained, this did not affect the responsiveness of the preparation to vitamin D3 .

Specificity of Tissue Response to Vitamin D 3
Small pieces of other embryonic chick organs were explanted on grids in contact with the medium. Esophagus, stomach, liver, pancreas, lung, skin, and muscle did not contain CaBP either before or after incubation in the presence of vitamin D3 in the medium (26 µM (26 )uM) . Embryonic chick kidney contains CaBP as early as day 11 3 and, therefore, its induction by vitamin D 3 in vitro was not evaluated .

Absorption of Nutrients other than Calcium
Embryonic chick duodena were cultured mucosal side up in the presence of vitamin D 3 in the medium (26 pM) for 48 h . They were then transferred to Erlenmeyer flasks containing 3 ml of low-sodium buffer and tracer amounts of either 65Zn, 63 Ni, 80Co, 59 Fe, 54Mn, 45Ca, or 32P. Duodena incubated in the presence of beta-emitting isotopes ( 63Ni, 45Ca, 32P) were processed and counted at optimal counter settings as described above (Materials and Methods ; 45Ca Uptake) . Tissues containing gamma emitters ( 85Zn, BOCo, 59Fe, and 64Mn) were dissolved in 10% NaOH and assayed at optimal counter settings in a Nuclear-Chicago Auto Gamma Counter (Nuclear-Chicago Corp .) . As expected, 45Ca uptake was enhanced by vitamin D 3 (Table III) . In addition, SsFe uptake was also stimulated by vitamin D 3 . 32P uptake was increased in tissue cultured in the presence of vitamin D 3 , and equally as much in the presence or absence of calcium in the buffer solution (Table III) . 3 A preliminary report of this finding has already appeared : A . N. Taylor and R . H.  . Vitamin D-induced calcium-binding protein (CaBP) in chick kidney during embryonic development and growth . Fed. Proc. 30 :407. (Abstr.)

DISCUSSION
The data reported here establish three important facts : first, duodena from 20-day chick embryos can be cultured on specially designed grids in fairly large quantities (at least 300-400 mg tissue) ; second, over a 48-h culture period, mucosal structure is well preserved even in serum-free medium ; and, third, the cultured tissue responds to the presence of vitamin D 3 , and two of its metabolites, in the medium by synthesizing CaBP and exhibiting enhanced uptake and transport of radiocalcium. From all available evidence, the system is one of tissue maintenance rather than tissue proliferation . Nevertheless, the system described is a valuable adjunct to other methods for the study of the intestinal calcium absorptive mechanism.
In a number of significant respects, the culture system mimics in vivo conditions. For example, not only does the tissue respond to vitamin D 3 but it does so with a characteristic lag period (Fig. 3) . The first clue to the reason for this lag in responsiveness to vitamin D was obtained when it was shown that actinomycin D inhibited the action of vitamin D (34,35) . This suggested that the initial action of vitamin D was to induce protein synthesis necessary for stimulation of calcium absorption . Subcellular distribution studies lent credence to this concept since vitamin D 3 and its metabolites were preferentially localized in intestinal cell nuclei (36)(37)(38)(39)(40) . Other work showed that early actions of vitamin D 3 in the intestine were to stimulate DNA template activity (41) and RNA synthesis (42,43), presumably, preparatory to synthesis of new protein .
The discovery of the intestinal, vitamin D-induced calcium-binding protein (27) seemed to confirm the predictions of these studies . CaBP was found to be synthesized de novo on the basis of amino acid-labeling studies (3,44) and studies which showed that CaBP synthesis in rachitic chicks injected with vitamin D 3 was prevented by prior injection of actinomycin D (45) . Substantial correlative evidence has been accumulated linking CaBP to the vitamin D 3 stimulation of calcium transport (46)(47)(48)(49)(50) . Most recently, direct evidence was obtained in preliminary "reconstitution" experiments using the organ culture technique . When intact, everted duodena from embryonic chicks were cultured in medium containing purified chick intestinal CaBP but no vitamin D3, increased absorption and mucosal- R Fig. 3 A) . After 12-24 h, a significant increase in 96Ca uptake was observed . In vivo work has shown a similar pattern of early synthesis of CaBP in response to vitamin D3 after an initial lag period, followed by enhancement of calcium absorption (3,44) .
A portion of the lag period in vivo may also be the result of conversion of vitamin D3 to more active metabolites, 25-OHD 3 formed in the liver (52) and, to some extent, in the kidney (53), and 1, 25-(OH) 2D 3 formed in the kidney (54) . It has been contended that metabolism of vitamin D3, via 25-OHD 3 to 1 , 25-(OH) 2D 3, is a prerequisite of vitamin D 3 action (55) . However, other work has shown that, at least for embryonic chick intestine maintained in organ culture, metabolism of vitamin D 3 is unnecessary (56) .
Another significant parallel exists between in vivo and in vitro responses to vitamin D 3 metabolites . It has been shown that 25-OHD3 and 1, 25-(OH) 2D 3 are faster acting and more potent than vitamin D3 in vivo (57, 58) . While a time study was not conducted in the present case, it is evident that 25-OHD 3 and most especially, 1, 25-(OH) 2D 3 are more potent than vitamin D 3 in vitro (Figs . 5 and 6) . While 45Ca uptake was stimulated by all vitamin D3 analogues tested at a minimum of 65 nM in the culture medium, it was clear that induction of CaBP synthesis was the more sensitive response . This provides support for in vivo observations showing that CaBP synthesis is a preliminary response to vitamin D3 analogues, after which there is enhancement of calcium absorption (3,44) . The data in Fig . 3 bear this out directly, in the case of vitamin D 3 itself, for the organ culture system . In vivo studies have shown that vitamin D 3 stimulates the activity of a calcium-dependent ATPase (Ca-ATPase) in brush borders isolated from chick intestinal mucosal cells (59) . The time-course of stimulation of Ca-ATPase activity correlated well with vitamin D 3 enhancement of calcium transport . A similar correlation between brush border alkaline phosphatase activity and calcium transport was also shown (60) . Later, 7 4 THE JOURNAL OF CELL BIOLOGY • VOLUME 58,1973 it was shown that intestinal Ca-ATPase and alkaline phosphatase were probably the same enzyme although, again, a good correlation between vitamin D-enhanced enzyme activity and calcium transport was observed (61,62) . In the in vitro studies reported herein intestinal alkaline phosphatase activity was increased by the presence of vitamin D 3 or 25-OHD3 in the culture medium (Table I) but at levels higher than required to demonstrate enhancement of 46Ca uptake (Fig .  5 A) . Also, at a level of 1, 25-(OH) 2D 3 in the medium which stimulated maximal enhancement of 45 Ca uptake, 65 nM ( Fig . 6 A), alkaline phosphatase was not stimulated (Table I) . Thus, the correlation between alkaline phosphatase (and/or Ca-ATPase) and calcium absorption may not, in fact, reflect an involvement of the enzyme(s) in calcium absorption . There are in vivo results which support this conclusion . When a high strontium diet was fed to previously normal chicks, there was a rapid and complete inhibition of CaBP and a parallel inhibition of 97Ca (and "Sr) absorption (48,49) . Alkaline phosphatase activity also declined at a similar rate . However, if the chicks were returned to a normal diet, after 7 days on the high strontium diet, CaBP was rapidly restored to normal (49) . By the end of a week, CaBP had, in fact, risen to well above normal . 85Sr absorption, a well-established correlate of calcium absorption, was also reinstated to a normal and, finally, a supranormal level in very close correlation with CaBP synthesis . On the other hand, alkaline phosphatase activity rose to normal within 2 days and merely stayed at this level through the week . In another study, the vitamin D3-Stimulated increase in intestinal brush border alkaline phosphatase (or Ca-ATPase) activity was inhibitable by inclusion of phenylalanine in the assay medium (62) . However, the corresponding vitamin D 3 enhancement of 95 Ca uptake and mucosal-to-serosal transport by everted ileal sacs was not inhibited by the inclusion of phenylalanine in the mucosal bathing solution during incubation . On the basis of these in vivo results, and the present in vitro data, it must be concluded that the correlations observed between alkaline phosphatase (or Ca-ATPase) and the functioning of the calcium absorptive mechanism may be only coincidental .
The data of Table II are of interest because they established that the organ-cultured intestine not only responded to vitamin D 3 by exhibiting enhanced 45Ca uptake, but also maintained a mucosal-to-serosal transport system for calcium, mimicking precisely the in vivo situation. The relative simplicity of the culture procedure, along with the ability to rigorously control the environment of the tissue, makes the use of cultured, everted intestine an almost ideal system for the study of intestinal function . It is certainly well suited to investigation of the vitamin D-mediated calcium absorptive mechanism, but it should also be adaptable to studies of the absorption of other nutrients, drugs, hormones, etc.
As one indication of the culture system's adaptability, the data of Table III are (64,65) . The data presented here also support that conclusion . Reference to Table III shows that essentially similar s2P uptakes were obtained in intestine cultured in the presence of vitamin D 3, whether or not calcium was present in the incubation solution . Thus, 82P absorption, at least in this system, would appear to be independent of calcium . 4 Of course, at 26 µM vitamin D 3 in the culture medium, alkaline phosphatase activity was stimulated, and CaBP induced, but there is no unequivocal evidence to conclude that this enzyme, or for that matter, CaBP, is involved in phosphate transport . Iron and cobalt absorption have been shown to be enhanced by vitamin D 3 in vivo (66) but no follow-up work has been done . There is, however, interest in cobalt and iron absorption per se (67)