text:
Fig. 1. Processing cascade for angiotensin peptides. Renin cleaves angiotensinogen to angiotensin-(110) (ANG I), which is further processed to the
biologically active peptides ANG-(1 8) (ANG II) by angiotensin-converting
enzyme (ACE), ANG-(17) by endopeptidases such as neprilysin (Nep), and
ANG-(19) by ACE2. ANG II undergoes further processing by the carboxypeptidase ACE2 to yield ANG-(17) and at the amino terminus by aminopeptidase A (APA) to form ANG-(2 8) or ANG III. ANG-(17) is metabolized by
ACE to form ANG-(15), and ANG III is further hydrolyzed by aminopeptidase N (APN) to yield ANG-(3 8) or ANG IV. The novel peptides ANG-(1
12) and ANG-(125) may be directly derived from angiotensinogen. ANG II
and ANG III recognize the ANG II types 1 and 2 receptors (AT1R and AT2R,
respectively), whereas ANG-(19) interacts with the AT2R. ANG-(17) and
Ala1-ANG-(17) recognize the Mas and Mas-related G protein-coupled receptor member D (mRG-D) receptors. ANG IV recognizes the insulin-regulated
aminopeptidase (IRAP). DC, aspartic acid decarboxylase. Inset: pathways for
ANG-(17) formation that may reflect predominantly Nep or ACE2. Adapted
from Chappell (29).
interfering substances. Tissue or cellular content of angiotensinogen can be quantified by immunoblot, provided a source
of the protein is available to standardize this approach, or,
alternatively, by addition of renin to quantify ANG I generation from intact angiotensinogen that essentially comprises a
reverse renin assay.
Human urinary levels of angiotensinogen are lower than
plasma and average 10 200 ng/mg creatinine (0.2 to 4
pmol/mg Cr) (Table 1). However, elevated excretion of angiotensinogen is considered a biomarker for an activated renal
RAS in several models of renal injury that reflects the tubular
release of angiotensinogen (5, 69, 72, 113). We applied the
total angiotensinogen ELISA and immunoblot methods to
assess angiotensinogen expression in the salt-sensitive
mRen2.Lewis rat, a congenic model of an activated ACE-ANG
II-AT1R axis, and observed a marked sex difference in urinary
angiotensinogen levels (34) (Fig. 2). Male mRen2.Lewis exhibited 200-fold higher excretion of angiotensinogen than
females (2 vs. 0.01 pmol/mg Cr) (Fig. 2). Estrogen depletion
(ovariectomy) increased angiotensinogen excretion 20-fold,
whereas a high-salt (HS) diet enhanced excretion 100-fold
compared with intact mRen2 on a normal salt diet. Although
both maneuvers increase blood pressure to a similar extent,
excretion of angiotensinogen in the HS males remained sixfold
higher than the HS females (Fig. 2). Despite the marked
increases in excretion, renal mRNA and protein levels of
angiotensinogen were not increased following ovariectomy or
the HS diet, nor did we detect enhanced release of angiotensinogen from renal cortical slices in the HS mRen2.Lewis
rat (34) (Fig. 2). The elevated urinary angiotensinogen in the
mRen2.Lewis likely reflects a greater degree of filtration of the
circulating protein rather than a renal source and is perhaps a
sensitive biomarker of glomerular damage (34).
Renin. Typically regarded as the enzyme that initiates the
RAS cascade, renin is an aspartyl-type protease (30 40 kDa)
that cleaves angiotensinogen to form the inactive peptide ANG
I (Fig. 1). The enzyme is synthesized, glycosylated, and released in both inactive (prorenin) and active forms by the
juxtaglomerular cells of the renal cortex at a higher proreninto-renin ratio. A second source of renal renin is the collecting
duct cells that secrete active renin into the tubular fluid (52, 71,
82, 92, 113). There is evidence for alternative gene products of
intracellular renin in the kidney, brain, and adrenal that may
Cells, fmol/mg (9, 38, 77, 132, 141)
*Angiotensinogen (Aogen)
Peptide values derived from radioimmunoassay (RIA)-HPLC or HPLC-mass spectrometry corrected by molecular size. Values from direct RIA or ELISA.
Tissue levels are fmol/g wet weight. Cell content is fmol/mg protein.
AJP-Heart Circ Physiol doi:10.1152/ajpheart.00618.2015 www.ajpheart.org
imply a renin-dependent pathway for the intracellular expression of angiotensin peptides (65, 76, 106, 107, 142). Apart
from its catalytic activity, prorenin may constitute an additional circulating hormone of the RAS through binding and
activation of the prorenin receptor within various tissues (52,
71, 94). A high molecular mass complex (50 60 kDa) comprised of renin and the renin binding protein N-acetyl glucosamine epimerase is typically revealed by immunoblot analysis; the complex renders renin inactive and may influence renin
secretion (64). Regarding renin measurement, the enzyme is
released by stress, dehydration, low-salt diet, RAS blockade,
and certain anesthetics (pentobarbital sodium); therefore, physiological assessment of activity should ideally be obtained by
rapid decapitation or blood withdraw by an indwelling catheter
reflects both active renin and endogenous ANG I-angiotensinogen in the circulation. Elased et al. (39) initially used surfaceenhanced laser deabsorption ionization (SELDI)-mass spectrometry (MS) to detect ANG I and demonstrate relative renin
activity in very low volumes of plasma samples. Camenzind et al.
(20) applied matrix-assisted laser deabsorption ionization-mass
spectroscopy (MALDI-MS) to quantify ANG I generation from
angiotensinogen with comparable renin activity values to that of
RIA methods. Renin activity is typically expressed as nanograms
or picomoles ANG I generated per milliliter of plasma per hour.
Typical plasma renin activity values range from 0.510
ngml1h1 (0.510 pmolml1h1), whereas total renin (prorenin/renin) is 3- to 10-fold higher reflecting the greater abundance of prorenin. Addition of exogenous angiotensinogen yields
maximal renin activity that is conventionally but inappropriately
termed plasma renin concentration. An alternative method is a
direct human renin assay using capture and detection antibodies
with the latter antibody directed to the active or open form of
renin. In this regard, addition of the nonpeptide inhibitor aliskerin
induces a conformation change in the enzyme that facilitates
detection of prorenin by this assay (139). Using this method, van
den Huevel et al. (139) reported mean values of active and
prorenin at 14 pg/ml (0.4 pM) and 66 pg/ml (1.9 pM), respectively, in human plasma.
Renin activity is also evident in the urine at 10% of the
plasma levels of the enzyme (84, 139). In contrast to the
circulation, there is little or no urinary prorenin, and this may
reflect the predominant luminal secretion of renin from collecting duct cells (71, 82, 84, 92). Danser and colleagues (113,
139) suggest that urinary renin may be an alternative measure
of an active RAS within the kidney. Quenched fluorescent
substrates based on the ANG-(114) sequence are available to
measure renin. Although this approach does not rely on the
detection of ANG I or an exogenous source of angiotensinogen, the sensitivity and specificity of the peptide substrates are
not comparable with angiotensinogen. A protease/peptidase
cocktail of inhibitors must be added to prevent nonspecific
cleavage of the quenched substrate, as well as addition of a
renin inhibitor such as aliskerin to demonstrate specificity of
the assay.
RAS peptidases. Peptidases are generally distinguished from
proteases by their preference to hydrolyze peptides of 5 to 30
amino acids in length. In contrast to renin, the peptidases
associated with the RAS do not exhibit exclusive specificity for
angiotensins, although certain enzymes may exhibit preferential kinetics such as ACE2 for ANG II (112, 145). An extensive
peptidase class is the metalloenzymes [ACE, ACE2, neprilysin
(Nep), aminopeptidase A] that require divalent cations for
activity; thus the assessment of activity in the circulation is
performed in serum in the absence of strong chelating agents
such as EDTA or phenanthroline.
ACE. The predominant pathway of the classical RAS is the
hydrolysis of ANG I to ANG II by the metallopeptidase ACE,
a dipeptidyl carboxypeptidase that cleaves two residues from
the COOH-terminus of peptides. The peptidase is a membranebound and glycosylated protein (120 180 kDa); however,
soluble forms of the enzyme are present in the circulation,
cerebrospinal fluid, lymph, and urine (15). ACE plays a key
role in the formation of ANG II, but the peptidase metabolizes
other peptides including bradykinin, substance P, acetyl-SerAsp-Lys-Pro, and ANG-(17) (16, 145). ACE activity is typ-
ically measured by small peptide substrates such as hippurylHis-Leu or furylacrylol-Phe-Ala-Gly-Gly in a buffer containing chloride (10 200 mM) and zinc (1100 M) for optimal
peptidase activity (24, 122). Quenched fluorescent substrates
Mca-Ser-Phe-Leu-Tyr-DNP or Mca-Tyr-Val-Ala-Arg-Ala-PheLys-DNP have also been applied for ACE measurement; however, these are not specific substrates and appropriate assay conditions that include an inhibitor cocktail to prevent nonspecific
hydrolysis by other peptidases must be considered, as well as
separate samples sets for an ACE inhibitor (i.e., captopril, lisinopril) to confirm assay specificity (81). Finally, the degree of
sample quenching of the fluorescent product should also be
assessed for each source of activity.
CHYMASE. Chymases comprise a family of serine peptidases
that may form ANG II by hydrolysis of the Phe8-His9 bond of
ANG I and pseudoprecursors [ANG-(112), ANG-(125)] (chymases) or metabolize ANG II at Tyr4-Ile5 to form ANG(1 4) and ANG-(5 8) (-chymases) (3, 27, 28, 89, 138).
Humans express -chymase, whereas rodents express primarily -chymases, as well as other isoforms (mouse mast cell
protease-4 and rat mast cell protease-5) that more closely
resemble -chymase in regard to the processing of ANG I to
ANG II (27, 28, 126). The human and mouse enzymes may
also play a role in the conversion of the endothelin precursor to
the active peptide, as well as the activation of various inflammatory cytokines (28, 126). Chymases (35 kDa) are synthesized and stored in an inactive proform within mast cells and
neutrophils that are released with other proteases (cathepsin G,
tryptases, renin) upon degranulation following injury or inflammation (28). Although chymases are soluble enzymes,
they associate with the cell membrane and may locate to the
extracellular surface of tissues following release. Fluorescentquenched substrates for chymase include succinyl-Ala-AlaPro-Phe-amido-4-methylcoumarin and succinyl-Leu-LeuVal-Tyr-amido-4-methylcoumarin. Addition of the serine
protease inhibitor chymostatin is typically used to demonstrate specificity; however, chymostatin inhibits other ANG
II-generating enzymes (cathepsin G, elastase-2) and more
selective chymase inhibitors should be considered (116
118, 123, 146). The extent that chymase or other peptidases
participate in the formation of circulating or tissue ANG II
through an ACE-independent pathway remains an area of
significant debate (21).
ENDOPEPTIDASES. Nep (95 kDa) and other endopeptidases
(thimet oligopeptidase, 80 kDa; and prolyl oligopeptidase, 76
kDa) process ANG I directly to ANG-(17) without the requisite formation of ANG II (Fig. 1). These peptidases hydrolyze interior bonds of the peptide and prefer aromatic and/or
hydrophobic residues, but prolyl oligopeptidase specifically
cleaves the COOH-terminus of proline and hydrolyzes the
Pro7-Phe8 bond of both ANG I and ANG II to form ANG-(17)
(29, 112, 145). Circulating forms of soluble Nep are present in
the serum, urine, and cerebrospinal fluid (111, 129). Following
chronic ACE inhibition, circulating levels of ANG-(17) are
markedly higher that reflect processing of ANG I by vascular
Nep, as well as the reduced metabolism by ACE (Fig. 1) (29).
Various quenched substrates are available to measure Nep and
other endopeptidases (81). The quenched peptide MCA-ArgPro-Pro-Gly-Phe-Ser-Ala-Phe-Lys-DNP may be an appropriate
dual substrate for ACE and Nep (M. C. Chappell, unpublished
observations). Again, the assay conditions require inhibitors to
the target protein are appropriate controls to test antibody specificity for immunoblot and immunocytochemistry methods, although these approaches are not directly comparable with other
species or different tissues. Moreover, knockdown of the functional domain does not ensure that the remaining protein will not
be recognized by the antibody. Blockade of the immunoreactive
signal by the immunizing peptide or full-length protein is an
additional approach to address specificity of the immunoblot or
immunocytochemistry signal provided a pure source of the peptide/protein is available; however, antigenic sites exposed by SDS
denaturation, formalin fixation, or antigen retrieval may not be
accessible for proteins in solution.
The characterization of angiotensin receptors is further
complicated by the fact that these proteins do not reside or
exclusively localize to the plasma membrane. In lieu of
earlier studies on intracellular peptide receptors, ANG II
and ANG-(17) receptors were characterized on isolated
nuclei from rat and sheep kidney (54). In rat nuclei, binding
studies with the nonselective antagonist 125I-[sarcosine1,
threonine8]-ANG II (sarthran) and competition with selective antagonists revealed exclusive expression of the AT1
receptor (104, 105) (Fig. 3, AC). Fluorescence-activated
cell sorting demonstrated essentially complete overlap of
the fluorescent signature for the AT1 receptor and the
nuclear marker nucleoporin; the immunoblot confirmed a
single protein band for the renal nuclei with the same AT1R
Fig. 3. Characterization of AT1Rs in isolated nuclei from the rat renal cortex. A: saturation binding with the antagonist 125I-Sar1, Thr8-ANG II (sarthran) in
isolated renal nuclei. B: scatchard analysis of sarthran binding revealed a KD of 0.7 nM and a Bmax of 270 fmol/mg protein. C: competition studies revealed
inhibition of binding with AT1 antagonists losartan (Los), candesartan (CV), but not the AT2 antagonist PD123319 (PD) or the AT7/Mas receptor antagonist
D-Ala7-ANG-(17) (DALA). D: cell sorting of isolated renal nuclei revealed essentially complete overlap of the nuclear marker nucleoporin 62 (Nup62) and the
AT1R. PE, phycoerythrin. E: immunoblot of renal nuclei with the AT1R antibody revealed a single 52-kDa band. MS, molecular size. F: ANG II stimulates
oxidative stress in renal nuclei measured by the increased fluorescence with the dichlorofluorescein (DCF) probe. ANG II-dependent stimulation was blocked
by Los and the NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI). ROS, reactive oxygen species. *P 0.05 vs. control or ANG II-treatment. Data adapted
from Pendergrass et al. (104, 105).
AJP-Heart Circ Physiol doi:10.1152/ajpheart.00618.2015 www.ajpheart.org
Fig. 4. Characterization of the AT7/Mas receptor in the kidney and isolated nuclei from
the sheep renal cortex. AC: immunofluorescent staining for the AT7/Mas receptor
(Alomone antibody) in proximal tubules
(PT), collecting duct (CD), vasa recta (VR)
and thick ascending limb (TAL) but not
glomeruli (GM) or distal tubule (DT) in the
sheep kidney. DE: absence of ICC staining
by preabsorption with the Mas immunogenic peptide in adjacent sections. F: single
protein band in isolated nuclei from sheep
cortex revealed by Alomone Mas antibody
in full-length gel. G: competition for sarthran binding on isolated nuclei from sheep
renal cortex. The AT7/Mas receptor antagonist DALA and AT1R antagonist Los exhibit
complete additivity for competition of sarthran binding. In contrast, the AT2 antagonist PD exhibits partial additivity with
DALA. Mean values of percent competition
are given above each bar. H: ANG-(17)
(A7) stimulates nitric oxide (NO) release
(increased DAF fluorescence) that was
blocked by DALA and the nitric oxide
synthase inhibitor NG-nitro-L-arginine
methyl ester but not by Los or PD. *P
0.05 vs. ANG-(17)-treatment. Data
adapted from Gwathmey et al. (57, 58).
pared with normotensive Wistar-Kyoto rats (103). Biotinylation studies combined with an AT2R antibody was used to
reveal localization of the receptor on the plasma membrane
(103). It is not currently known whether the other angiotensin
receptor subtypes exhibit a similar type of regulation. In this
regard, a careful assessment of the subcellular fractions or
intracellular organelle for angiotensin receptor expression is
warranted. Moreover, these and other studies support the intracellular expression of G protein-coupled receptors within the
kidney and other tissues, as well as emphasize the necessity for
multiple approaches in the identification and characterization
of angiotensin receptors (17, 35, 40, 74, 132, 151).
RAS Peptides
Interest in angiotensins other than ANG II and ANG III was
stimulated by identification of endogenous ANG-(17) over 25
years ago (30), as well as subsequent evidence for an ANG(17) receptor (121) and a converting enzyme (137). [Ala1]ANG II and [Ala1]-ANG-(17) were recently identified in
human plasma, likely arising from decarboxylation of the
like enzyme to ANG II (3, 89, 143). The circulating and tissue
pathways for the generation of ANG-(112) and ANG-(125)
are not currently known but are likely renin independent.
The accurate assessment and identification of angiotensins
are critical to ascertain the status of the RAS, particularly in
pathological conditions or following therapeutic intervention;
however, their evaluation is hampered by the extremely low
level of peptide expression in the fentomole per gram or
fentomole per milliliter range, interfering substances in samples that may be present at a far higher concentration, susceptibility to metabolism in sample collection and processing, the
peptides shared sequence, and the specificity of detection. All
these factors may contribute to the marked variability in
angiotensin values evident in the literature and raise concerns
on the identity of ANG II or other angiotensins detected in
tissues and the circulation, as well as the extent that altered
peptide content truly reflects or contributes to a particular
phenotype or treatment response.
RIA/ELISA. RIA and ELISA are invaluable assays to characterize angiotensins, and they remain the most used biochemical methods to quantify endogenous peptides. Under optimal
assay and extraction conditions, both methods should allow for
the sensitive and relatively specific detection of ANG II and
other angiotensins at fentomole (pg) quantities in plasma,
tissue, urine, and cells. Since angiotensins share an identical
NH2-terminus except for the Ala1-forms of ANG II and ANG(17), antibodies for ANG II, ANG-(17), ANG-(19), ANG I,
and ANG-(112) are directed against their unique COOHterminus. The ANG II and ANG-(17) antibodies, for example,
typically distinguish a single amino acid difference (Phe8)
between ANG II and ANG-(17). Importantly, these antibodies
are not selective against the NH2-terminal portion of the
peptide (30). Thus an ANG II RIA distinguishes ANG-(17)
and ANG I but will recognize ANG III, ANG IV, and ANG(4 8) (37). Similarly, an ANG-(17) antibody recognizes the
NH2-terminal metabolites ANG-(27) and ANG-(37), but not
ANG-(4 7), ANG II, or ANG I (30). These assays can be used
across species as the COOH-terminal portion is conserved for
ANG-(17), ANG II, and ANG I; however, larger peptides
such as ANG-(112) or ANG-(125) exhibit different COOHterminal residues that are species specific and require unique
antibodies for detection (89, 90). Apart from their nonselectivity for NH2-terminal metabolites of angiotensin peptides,
RIA or ELISA may detect a significant degree of nonspecific or
background immunoreactivity that reflects the sample source
and extent of purification or extraction, as well as the assay
antibodies (37). Therefore, chromatographic separation should
be routinely coupled to RIA or ELISA to verify the identity of
immunoreactive peptides, as well as account for nonspecific
material in plasma or tissue samples. Peptide fractionation by
HPLC provides this additional level of discrimination based on
the resolution and high recovery of the sample peptides, rapid
separation time, and general compatibility with RIA methods
(22, 30, 37, 78, 99). In addition, the different characteristics for
each antibody-based assay must be appreciated, particularly
those obtained from commercial sources. The stated crossreactivity of the Peninsula human ANG-(112) ELISA for
ANG I is 0%; however, the Peninsula rat/mouse ANG-(112)
ELISA cross-reacts 60% with ANG I (Peninsula Lab Int, San
Carlos, CA). Thus HPLC fractionation would be essential to
on their unique mass to charge spectra and exhibits the capability for quantification of endogenous peptides (83). Unlike
the antibody-based RIA or ELISA, MS can potentially resolve
posttranslational events such as peptide phosphorylation, amidation, acetylation, decarboxylation, or other peptide forms.
Extracted samples are initially resolved by a chromatographic
step [HPLC, ultrahigh-pressure LC (UPLC), or nano-LC], the
eluent volatized, and the peptide or resultant fragment ions
detected by MS [single or tandem, time of flight (TOF), ion
trap, or their combination]. A key issue with MS is that a
number of components are detected following the LC separation and may render very complex spectra with similar massto-charge ratios, particularly as the prior chromatographic step
does not resolve all individual peptides, and endogenous peptides are typically 103-109 less abundant than other species in
plasma and tissues (11, 83). To address this issue, Schulz et al.
(125) developed a hybrid approach to selectively enrich ANG
II from plasma extracts by absorption to an ANG II antibody
before quantification by HPLC-MS/MS. These investigators
quantified ANG II at 20 fmol/ml (20 pM) in human plasma
that is comparable wth RIA or HPLC-RIA values, although
other NH2-terminal metabolites of ANG II were not reported
(125) (Table 1). Addition of isotopically labeled ANG II is key
in the MS approach to account for extensive sample handling
and extraction, as well as the extent of peptide ionization.
Poglitsch and colleagues (60, 108) have recently developed a
fingerprint HPLC-MS/MS approach to quantify 10 angiotensins in plasma with a comparable sensitivity to RIA; however, the addition of exogenous renin is apparently required for
peptide measurements in human plasma. HPLC-MS/MS may
potentially become the standard method to quantify angiotensins, although the complexity, expertise, and cost required
overly emphasized for the accurate measurement of endogenous angiotensins. Regardless of the detection method by RIA,
HPLC-RIA, or HPLC-MS, improper sampling handling and
extraction may yield artifactually high or low peptide values
that do not reflect endogenous content. The sensitivity required
to quantify endogenous angiotensins also increases the potential for artifacts or interfering substances whether using RIA or
MS approaches. In general, animals should be maintained in a
calm environment and anesthetic agents such as pentobarbital
sodium that markedly activate the RAS avoided. Blood samples are collected directly into an inhibitor cocktail to block
renin and other peptidases that process or metabolize angiotensin peptides; the obtained plasma should be subsequently
stored at 80C or immediately extracted. Tissue samples
should be rinsed of blood and rapidly frozen in liquid nitrogen
or an ethanol-dry ice bath or immediately extracted. Pepstatin,
an aspartyl protease inhibitor, may not be sufficient to completely block renin activity, and a more specific renin inhibitor
should be considered (73, 98). Moreover, the typical inhibitor
cocktails for protein immunoblot analysis may be inadequate to
attenuate angiotensin processing of samples in physiological
Various extraction methods that rapidly abolish peptide
metabolism, deplete the sample of contaminating proteins and
other substances that may interfere with the assay and enrich
peptide content in a manageable volume include acid (HCl)ethanol, pure methanol or acetonitrile, guanidine thiocynate,
and immediate immersion in a boiling water bath with subsequent acidification and tissue homogenization (22, 24, 30, 37,
90, 130). The precipitated proteins are removed by high-speed/
ultracentrifugation or molecular cut-off filters, and the supernatant is applied to solid-phase extraction columns (C18 or
phenyl). Both angiotensin and nonangiotensin peptides are
absorbed to these columns, whereas proteins and other substances are not retained; the peptides are subsequently eluted in
an organic solvent (methanol or acetonitrile). Peptides standards (i.e., 125I-ANG II, ANG II, or isotopically labeled ANG
II) should be routinely added to determine overall recovery in
the extraction procedure for quantification methods, and particularly to account for matrix effects on MS ionization that
may vary among samples or MS analysis runs. Recovery for
plasma samples typically ranges from 70 to 90%, whereas
tissues are generally lower in the 40 70% range. Appropriate
blanks must be included that contain all components of the
extraction method (apart from the actual sample) and assayed
in parallel with samples.
Angiotensin values. The range of angiotensin values for
plasma and tissues in Table 1 reflects studies using HPLC-RIA
or HPLC-MS quantification, as well as consideration of the
appropriate sample collection and extraction methods (2224,
37, 85, 97100, 127, 128). In lieu of these approaches, the
NH2-terminally intact forms of ANG II, ANG-(17), and ANG
I are the predominant peptides identified in plasma and various
tissues. Quantitation of the cell and urinary data reflects direct
RIA- or ELISA-based methods (9, 34, 38, 45, 50, 70, 77, 115,
131, 133, 139, 141, 148).
CIRCULATION. ANG II values in human, mouse, and rat
plasma are quite low and average 550 fmol/ml (550 pM)
(Table 1). Human plasma levels of the ANG II isoforms
[Ala1]-ANG II and [Pro1, Glu2]-ANG II average 6 and 4 pM,
respectively, as quantified by MALDI (66, 67). Circulating
the medulla than the cortex (direct RIAs) (91, 104); Pendergrass et al. (104) confirmed the predominant ANG II identify in
both cortex and medulla by HPLC-RIA. In comparison, significant levels of ANG II (300 fmol/g tissue) were quantified
in the ovary and uterus by Senanayake and colleagues (37).
Apart from these tissues, central and peripheral tissues including the heart and adipose express lower and somewhat more
variable ANG II levels of 2150 fmol/g tissue (Table 1).
In regards to MS analysis, few studies have quantified ANG
II and ANG-(17) in tissue. Ali et al. (7, 8) reported ANG II
and ANG-(17) levels of 200,000 and 350,000 fmol/g in
mouse kidney and 25,000 to 60,000 fmol/g tissue in rat kidney
cortex by UPLC-QTOF (7, 8). They also quantified ANG II
and ANG-(17) content in mouse adipose tissue at 150,000 and
350,000 fmol/g tissue, respectively (7). It is difficult to reconcile that the kidney and adipose exhibit the same synthetic
capacity to generate essentially identical levels of ANG II or
ANG-(17). Moreover, extraction of tissue with a very high
protease content such as the kidney in a Tris lysis buffer at
neutral pH is likely insufficient to abolish processing and may
contribute to the high peptide content reported for kidney (7,
8). The stated limit of quantitation for the UPLC-QTOF for
ANG II (500 fmol) and ANG-(17) (800 fmol) also does not
appear adequate to quantify the authentic peptide content in
plasma and tissues (7). In contrast to the Ali studies, Wysocki
et al. (147) quantified far lower levels of ANG II and ANG(17) (293 and 95 fmol/g tissue, respectively) in the mouse
kidney by fingerprint MS; these values are clearly more consistent with the literature, although the ANG III content (185
fmol/mg tissue) was relatively high at 60% of the total ANG II
(Fig. 5, Table 1). Finally, Burger et al. (19) found a higher
ANG II-to-ANG III ratio (700 vs. 130 fmol/g tissue) in mouse
kidney using guanidine extraction and MS. In this case, guanidine may attenuate aminopeptidase and other peptidase activities more effectively to prevent the artifactual generation of
peptides during tissue extraction.
INTERSTIAL CONTENT. The extracellular or interstial content of
ANG II in heart and kidney was assessed by direct RIA and
HPLC-RIA. These studies use an implanted dialysis probe
permeable to low molecular substances that sample the local
release or formation of angiotensin peptides in the interstial
space. DellItalia and colleagues (14, 36) reported interstial
ANG II levels in the canine heart at 0.6 to 5 pmol/ml (0.6 5
nM) by HPLC-RIA. Intravenous infusion of ANG I alone or
combined with an ACE inhibitor did not impact the interstial
ANG II levels. Schuijt et al. (124) found low to undetectable
levels of ANG II (30 fmol/ml or 30 pM) in interstial fluid of
the ovine heart also quantified by HPLC-RIA. In the rat kidney,
Kemp et al. (68) report comparable ANG II and ANG III
interstial content (80 fmol/ml or 80 pM) using the HFBAHPLC system to separate the two peptides before RIA detection. This study finds a higher ANG III-to-ANG II ratio than
that in the circulation or tissues, which may reflect the significant level of ectocellular APA activity in the renal tubules and
suggests significant angiotensin processing in the interstial
space. Finally, Nishiyama et al. (95, 96) reported higher renal
interstial levels of ANG II and ANG I (13 nM) by direct
RIAs. The interstial ANG II levels were not responsive to
peripheral ACE inhibition or volume expansion, further suggesting a local mechanism of peptide expression distinct from
to be the ideal choice to quantify angiotensin peptides; however, the required expertise and overall expense of these
systems limits their widespread availability to a limited number
of laboratories at the current time. Moreover, the application of
MS to the determination of angiotensins does not necessarily
guarantee an accurate profile or an absolute value in plasma
and tissues. Thus RIA or ELISA methods will continue to be
the predominant approach to quantify angiotensin peptides and
assess alterations in peptide expression. The limitations of
these assays, as well as the methods applied for sampling
handling, extraction, and approaches for validation, must be
considered for the accurate assessment of the peptide hormones
of the RAS, as well as to establish that an altered phenotypic or
treatment response truly reflects changes in peptide expression.
Importantly, validation of these assays for either individual
samples or a sample pool by HPLC-RIA should be a requirement for the analysis of endogenous angiotensin peptides in
various compartments, and particularly in studies that assess
the cellular expression of angiotensins.
I gratefully acknowledge K. Bridget Brosnihan and Debra I. Diz of the
Hypertension and Vascular Research Center for their critical reading and
insightful comments pertaining to this review.
This study was supported in part by National Institute of Health Grants
HL-56973, HL-51952, HD-084227, HD-047584, and HD-017644 and American Heart Association Grants AHA-151521 and AHA-355741. An unrestricted grant from the Farley-Hudson Foundation (Jacksonville, NC), Groskert
Heart Fund, and the Wake Forest Venture Fund is also acknowledged.
No conflicts of interest, financial or otherwise, are declared by the author(s).
M.C.C. drafted, edited, revised, and approved final version of manuscript.
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Posts: 36
Joined: Aug 10th, 2009, 01:04
Jun 12th, 2019, 10:39
Hi, How do I bring Soundsoap into Logic 10x to get rid of noise? Do I drag the app into Logic somewhere? Thanks!
Joe A
Posts: 50
Joined: Oct 1st, 2013, 02:28
Re: Soundsoap
Jun 12th, 2019, 12:16
Instead of the standalone application, you'd use the AU plug-in version. I'd assume that (unless you unchecked those options) the installer has already installed it -- just click on an insert in the channel you need to clean up (or the stereo output for overall cleanup) and open up the SoundSoap plug-in there.
Steve H
Posts: 7
Joined: Aug 2nd, 2016, 04:28
Re: Soundsoap
Jun 12th, 2019, 12:17
Install the AU (Audio Units) version that came with your installer package. Once that's installed on your system, Logic will recognize it as a plugin and it'll appear in the plugins section on your channel strip (in Logic).
Steve H
Posts: 7
Joined: Aug 2nd, 2016, 04:28
Re: Soundsoap
Jun 12th, 2019, 12:18
Joe: You beat me to it!
Posts: 36
Joined: Aug 10th, 2009, 01:04
Re: Soundsoap
Jun 12th, 2019, 12:52
Hi, Where would the AU (Audio Units) version be, that came with my installer package. I only seem to have the Soundsoap Icon to open it. I don't see a folder or anything.
Joe A
Posts: 50
Joined: Oct 1st, 2013, 02:28
Re: Soundsoap
Jun 12th, 2019, 01:50
No need to access the actual plug-in in Finder -- as long as the Installer installed them (standard procedure) they'll appear in the list of plug-ins that comes up when you click on an Insert in a Logic channel strip. Since it's a third-party plugin, it won't be at the top of the list with all the Logic built-in plugins, it'll be at the bottom in the Audio Unit submenu under the manufacturer's name.
Posts: 36
Joined: Aug 10th, 2009, 01:04
Re: Soundsoap
Jun 12th, 2019, 06:05
Thank you!! I got it.
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Jun 17th, 2019, 05:15, Joe A
New macOS Catalina preview
Jun 15th, 2019, 11:13, Joe A
Logic 10.4.5 update is here!
Jun 13th, 2019, 01:44, Joe A
Bouncing Files With Automation
Jun 12th, 2019, 06:56, Lynn
Neutron 3
Jun 12th, 2019, 11:39, Joe A
Jun 12th, 2019, 10:39, Lynn
FCP DVD Studio Pro
Jun 11th, 2019, 04:27, Gefafye
Logic 8 Changing tempo
May 29th, 2019, 12:31, Cijudiy
How can I get multiple audio clips to playback one at a time with a click in the right time signature?
May 24th, 2019, 05:52, Joe A
have the last final cut pro software
May 18th, 2019, 10:35, iain-anderson
Recent Posts
Re: No Monitor
You can enable input monitoring by clicking on the "I" button at the bottom of the channel strip (right next to the Rec button) -- start playback after enabling "I". This will insure that the incoming audio is passed through while playing/not recording -- it's kind of a "rehearsal mode". However, if you're not able to hear the live audio while recording, then it may be something else.. LMK Caveat : I have occasionally seen some glitchiness with audio input monitoring (w Logic 10.4.4) -- If it doesn't work as I just described, you could try toggling Auto Input Monitoring, trying the "I" button with/without the Rec button also enabled, and maybe even toggling the "Input monitoring only for focused track, and record-enabled tracks" Preference (Prefs->Audio->General)..
Jun 18th, 2019, 10:41, Joe A
New macOS Catalina preview
I'm sure by now everyone's heard of Apple's new Mac Pro (and its companion monitor), but slipped into that WWDC presentation was a preview of the upcoming Catalina OS.. https://www.apple.com/macos/catalina-preview/
Jun 15th, 2019, 11:13, Joe A
Re: Logic 10.4.5 update is here!
Looking through the (lengthy) release notes, it's clear that while the track bumps are cool, the real news in this update is the many bugfixes, performance improvements, and small features that may not be as attention-getting but can really enhance workflow. We all love to carp about bugs and issues , and I've seen many people post how "Apple should stop worrying about new features and concentrate on stability and performance for a while.." -- and it seems that's just what they did here! Among the notes are mentions of a host of situations where there should be improved responsiveness -- are people noticing that?
Jun 15th, 2019, 10:50, Joe A
Re: Neutron 3
The course is definitely up there on the Ask.Audio Academy page and working/playing. If you can see it but not get past the three demo videos, then you may not have a current subscription -- you could choose one here.. https://ask.audio/academy?nleloc=plans&sess_id=ih6dvend2bjdo313ok8p8j66t5 If you do (or did) have a subscription, then there may a problem with it -- check to see if it's expired, and if you think it should still be active and allowing you access, then you'll need to open a ticket on the page linked below, to get in touch with someone who'll be able to help you suss it out.. https://ask.audio/academy?nleloc=faq&sess_id=ih6dvend2bjdo313ok8p8j66t5&afid=dYKx8u938T
Jun 14th, 2019, 05:49, Joe A
Re: have the last final cut pro software
I'm afraid this isn't possible. A sequence of commands would do what you want, something like jump to next marker, move back five seconds, mark in, move forward ten seconds, mark out, favorite, but it's not totally straightforward. You'd need a third party app to wrap that together. Command Post might be able to this, or help you do it. It might be worth asking this question on the Command Post Facebook group? Good luck!
Jun 14th, 2019, 02:39, iain-anderson
Re: Bouncing Files With Automation
Thank you!!
Jun 13th, 2019, 09:20, Lynn
Re: Soundsoap
Thank you!! I got it.
Jun 12th, 2019, 06:05, Lynn
Re: Video looks perfect in imovie but glitchy/jumpy when exported - please help!
It's possible you're exporting to a format that your Mac can't play back very well? Maybe try going to another format and see if the problem persists?
Jun 12th, 2019, 02:01, iain-anderson
Re: How can I get multiple audio clips to playback one at a time with a click in the right time signature?
I haven't used MainStage for a while, but it seems like the way to accomplish this would be to have each song in its own Playback Plugin at the Set level (if you need to switch other Patches during the song) or the Patch level, rather than at the Concert level -- I would think that changing Sets/Patches for each song would then use the correct tempo for that song..? Maybe worth a try, if you haven't already sussed it out.. :-)
Jun 12th, 2019, 09:48, Joe A
FCP DVD Studio Pro
I'm trying to burn a dvd with FCP DVD Studio Pro. The video was edited with FCPX. When I click the "Burn" button I get the message "Formatting Failed." In the Build log it says "Seq Header duplication." How can I fix this?
Jun 11th, 2019, 04:27, Gefafye
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The Canby High School turf field has been condemned for the upcoming spring season, after the Canby School District's insurer […]Read more
Category: Sports
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In Cougar basketball this week, each game has heavy implications as the playoff picture solidifies and both boys and girls […]Read more
Mixed Results for Cougar Boys and Girls Basketball This Week
The Canby boys basketball team lost their only game last week, an away match against Tigard Friday, 50-43. They've had […]Read more
Canby Honors Senior Swimmers as Districts Loom This Weekend
The Canby swim team hosted West Linn for their last dual meet of the regular season this week. The scores […]Read more
Canby Men's Swim Team Nabs First Win of the Season
In swimming, Oregon City visited Canby at the Canby Swim Center Thursday. The Cougar men came away with their first […]Read more
Canby Girls Basketball Snaps Losing Streak with Dramatic OT Win
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Canby Cougar Swimmers Have Strong Showing in Ladies Only Meet
Canby Cougars swam against St. Mary's on Jan. 17 for a ladies only meet. The Cougars hosted, but St. Mary's […]Read more
Rough Week for Canby Cougars Basketball
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The Canby swimmers fell to Tigard Dec.13th at home, but the team showed improvementoverall. Tigard women won 106 -64, while the […]Read more


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text:
March 5, 2004
MEMBERS PRESENT: Barone, Blasi, Cotter, Hirsh, Nedick, Panayotatos (Chair), Rabinowitz (Vice Chair), Swalagin (Executive Secretary)
EXCUSED: Manhoff, Rijhsinghani
ALSO ATTENDING: Furmanski (Administrative Liaison), Leath (Faculty BOG Representative), Sclar (Newark Faculty Campus Liaison
The regular meeting of the University Senate Executive Committee was held on Friday, March 5, 2004 at 1:10 p.m. in the Cap and Skull Room, Rutgers Student Center, College Avenue Campus.
1. Chairpersons Report
Vice Chairperson Rabinowitz convened the meeting at 1:20 p.m., and presided until Chairperson Panayotatos arrived at 1:30 p.m. and assumed the chair. The Chairpersons Report was deferred until after the Secretarys Report.
2. Secretary's Report
Minutes: The Minutes of the February 6, 2004 Executive Committee meeting were approved.
Communications: The following communications were presented by Secretary Swalagin, and acted upon as noted:
Chairperson Panayotatoss Chairpersons Report included comments on: formation of a University Ad Hoc Commencement Committee, on which he and Senator Hirsh will serve, and which will be chaired by Seth Gopin; his meeting with Targum staff regarding referenda and continuation of the checked-box status on the term bill, and the availability of Targums financial information online at Guidestar.com; his decision not to run for Senate chair next year; his plan to summarize the Senates accomplishments, and the lack of any record of Senate advice being adopted as University policy on best practices for use of evaluations, appointment of deans, and FASIP awards; and ongoing revision of University Regulations, including those pertaining to the Senate, which he characterized as tidying up, and not requiring Senate input at this time. There followed some discussion of the Senate bylaws revision charge currently in the University Structure and Governance Committee. Chairperson Panayotatos continued his report by noting that the University administration is willing to change University Regulations to reflect an alternative to the term enrollment management, and soliciting Executive Committee members suggestions for alternative terms. He concluded his report by summarizing his recent advice to the University Structure and Governance Committee, and giving a copy of that advice to Secretary Swalagin. The advice included recommendations on: continued senatorial status of the immediate past chair, limiting the role of the Senate secretary in several respects, and procedures for election of at-large faculty Senators.
3. Administrative Report
Executive Vice President for Academic Affairs Furmanski delivered the administrative report, which included comments on: the state budget; initiation of major stem cell research at Rutgers; and goodwill statements and commitments by the state regarding jobs, goals, and the economy, including some issues formerly associated with, but now decoupled from, the bond issue and restructuring. At this point there was discussion of possible examination of intellectual property rights by the Senate and Rutgers. Vice President Furmanski continued his report with comments on: continuing efforts to enhance collaboration between Rutgers and other institutions, including exploration of ways to create virtual restructuring; and ongoing creation and refinement of an all-funds budgeting process. Chairperson Panayotatos gave Vice President Furmanski copies of three Senate actions which were submitted to the Lawrence administration, the implementation of which Chairperson Panayotatos had discussed with President McCormick.
4. Selection and Charge of Nominating Panel for April 2004 Election of Officers
The standard charge to the Nominating Panel, as well as last years membership, was reviewed. Chairperson Panayotatos said that Senator OConnor is again willing to chair the panel. Following discussion, the following Senators were named to the 2004-05 Nominating Panel:
Faculty Affairs and Personnel Committee Response to Charge S-0309, Evaluation of Administrators, Review: Chairperson Panayotatos, co-chair of the Faculty Affairs and Personnel Committee, summarized the committees report on charge S-0309, which had been issued as follows: Evaluate the current procedures for faculty, staff and student evaluation of deans after the completion of two cycles of evaluation. Recommend changes where appropriate, and examine the feasibility of extending the process to include vice presidents and other administrators. Following discussion, the report was docketed for action at the March 26, 2004 Senate meeting.
Instruction, Curricula and Advising Committee (ICAC) Response to Charge S-0303, Winter Session Review (Revised): The Instruction, Curricula and Advising Committee report on charge S-0303, which had been revised by the ICAC to include specific recommendations for action by the administration, as requested by the Executive Committee in February, was reviewed. The original charge had been issued as follows: Review the status of recommendations made regarding Winter Session, pursuant to the Instruction, Curricula and Advising (then Educational Policy and Planning) Committee report approved by the University Senate on October 27, 2000. The report was docketed for action at the March 26, 2004 Senate meeting.
Student Affairs Committee (SAC) Response to Charge A-0315, Knight Express Card Report: The Executive Committee reviewed the Student Affairs Committees response to charge A-0315, which had been issued as follows: Review and comment on the Knight Express Card Committee Report. Following discussion, Secretary Swalagin was instructed to ask the SAC to remove the reports the last recommendation, that the University Senate accept the report of the Knight Express Card Committee and commend the hard work of its members, noting that the report was neither a Senate report, nor was it written for the Senate. If the SAC agrees to the change, the report will be docketed for action at the March 26, 2004 Senate meeting.
Summary of Route 18 Construction Project Plans: Senator Leath, who chairs the Universitys Route 18 Widening Committee, presented a summary of the projects plans, and responded to questions. A similar presentation by Senator Leath, approximately 15 minutes long, and followed by a five-minute questions-only period was docketed for the March 26, 2004 Senate meeting. Robert Spear, Director of Parking and Transportation Services, will be invited to attend the Senate meeting, and asked to remain afterward to respond to questions and receive suggestions for minimizing the disruption of services which will result from the construction. Secretary Swalagin will inform the Senate that suggestions can also be e-mailed to the Route 18 committee at firstname.lastname@example.com.
Potential Charge to Student Affairs Committee on Timing of Placement of Textbook Orders: A potential charge proposed by SAC Co-chair Marilyn Novell was discussed. It was noted that the issue was covered under a previous Senate and SAC report and resolution on textbook buyback, the acknowledgment of which had been received from President McCormick, in which he notes that The administration is committed to improving textbook buyback and has been working with faculty and the bookstores on ways to make it as easy as possible for faculty members to work through the process. We will make a special effort to work with those faculty members who have not met the deadlines for book orders in the past. No charge was issued.
Potential Charge to Student Affairs Committee on Route 18 Construction Project Effects on Student Quality of Life: A potential charge proposed by SAC Co-chair Marilyn Novell was discussed. Senator Leath will contact Senator Novell regarding her joining the student subcommittee of the Route 18 Widening Committee.
Potential Charge to Academic Standards, Regulations and Admissions Committee (ASRAC) on the Coalition on Intercollegiate Athletics (COIA) request for review, comment and suggested revision of the COIA Draft Principles, Proposed Rules, and Guidelines Concerning the Faculty Role in Campus Athletics Governance: The COIA request included a very short deadline, so the Executive Committee agreed that Chairperson Panayotatos should provide feedback in his capacity as Senate chair rather than solicit an official response from the full Senate. The Executive Committee suggested soliciting feedback for the response from selected relevant people, including ASRAC Co-chairs Brazier and Oren, Rutgers College Dean Carl Kirschner, NCAA Faculty Representative Tom Stephens, and Faculty Council Chairs Tyler Hoffman, Diana Sclar, and Haym Hirsh (who might in turn solicit feedback from their constituencies).
6. Old Business
Senator Nedick asked the committee to revisit a previously considered charge to be issued to the University Structure and Governance Committee regarding addition of a voting student representative to the Board of Governors. The originally proposed charged was amended, resulting in the following draft charge: Investigate why there is no student voting representative on the Board of Governors in the spirit of N.J.S.A.18A:64-3.1, which states that the Board of Trustees of every state college must provide for the election of two student representatives who serve two year terms and are voting members in their second year, considering that at Rutgers the Board of Governors is currently equivalent to the Board of Trustees at most New Jersey colleges. Report back as to why this is not the case and make appropriate recommendations as to if and how this issue should be pursued. Investigate the same issues relative to faculty membership on the Board of Governors. The draft charge will be reviewed by Senators Nedick and Blasi (co-chair of the University Structure and Governance Committee), revised if necessary, and brought back to the Executive Committee for reconsideration.
Senator Nedick raised the issue of potential conflicts of interest which could arise during Targum and NJPIRG referenda if members of the Senate Student Affairs Committee, which hears procedural appeals relative to referenda, are also hired as referendum employees. Noting that the referendum guidelines are currently under review by the University Structure and Governance Committee (USGC), the Executive Committee agreed that any such conflicts would be handled internally within the Student Affairs Committee, with any SAC members employed by the referendum being recused from any appeals relating to that referendum. Secretary Swalagin will forward the current SAC roster to the Targum referendum coordinator, and the USGC will include this element in their consideration of the referendum guidelines revision.
7. Adjournment
The meeting was adjourned at 3:52 p.m. by Chairperson Panayotatos.
Kenneth Swalagin
Executive Secretary of the Senate


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