Vol. 274, Issue 1, F51-F62, January 1998
Characterization of binding sites for amylin, calcitonin, and
CGRP in primate kidney
Siew Yeen
Chai1,
George
Christopoulos2,
Mark E.
Cooper3, and
Patrick M.
Sexton2
2 Neurobiology Unit, St.
Vincent's Institute of Medical Research, Fitzroy 3065;
1 Howard Florey Institute of
Experimental Physiology and Medicine, Parkville 3052; and
3 Department of Medicine, University of
Melbourne, Austin and Repatriation Medical Center, Heidelberg 3084, Victoria, Australia.
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ABSTRACT |
Analysis of receptor distributions for
125I-labeled amylin,
125I-labeled calcitonin, and
125I-labeled
calcitonin gene-related peptide (CGRP) in
Macaca
fascicularis kidney by in vitro
autoradiography revealed distinct patterns of binding for each peptide.
125I-rat amylin bound primarily to
the cortex, being associated with the distal tubule, including apparent
binding to the juxtaglomerular apparatus.
125I-salmon calcitonin displayed
high-density binding in the cortex with low-density binding to the
medulla. Emulsion autoradiography indicated that binding was associated
with both distal tubule and thick ascending limb of the loop of Henle.
Intense binding was also found often over juxtaglomerular apparatus.
125I-rat CGRP-
exhibited low-
to moderate-density binding to the inner medulla/papilla with
high-density binding over small-, medium-, and large-caliber arteries.
Weak binding to the glomerulus was also seen, but no binding was
associated with cortical tubules. Competition binding studies,
performed with each of the radioligands, revealed peptide specificity
profiles for CGRP and calcitonin receptors that were similar to those
described in rat. However, the monkey amylin receptors differed from
those in rat, exhibiting relatively higher affinity for calcitonin
peptides but reduced affinity for CGRP peptides. These studies suggest
potential roles for amylin, calcitonin, and CGRP in primate renal
function.
calcitonin gene-related peptide; autoradiography; G protein-coupled
receptor
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INTRODUCTION |
AMYLIN, ALSO KNOWN AS islet amyloid polypeptide or
diabetes-associated peptide, is a 37-amino acid peptide hormone that is cosecreted from the pancreas with insulin in response to the intake of
nutrients (10). Peripherally administered amylin modulates in vivo
glucose metabolism and has been proposed to act as a metabolic partner
to insulin in metabolic regulation (37). High-affinity amylin binding
sites were first described in rat central nervous system (2) and
correspond to the C3 binding sites, originally described as
calcitonin-sensitive calcitonin gene-related peptide (CGRP) binding
sites (40). The specificity of ligand interaction at the
central C3-type amylin receptors is similar to that
described for amylin-mediated physiological responses in skeletal
muscle (3), suggesting that an equivalent receptor mediates the
metabolic actions of amylin.
Calcitonin (CT) receptors have been cloned from a number of species,
including rat and human (30, 39), and classically have highest affinity
for CTs with relatively weak interaction with the related peptides CGRP
and amylin. We have termed these receptors C1 (42), and this forms the
basis of the C1a and C1b nomenclature used for the rat CT receptor
isoforms (39). CGRP receptors have high affinity for the CGRPs with
only very weak interaction with any of the CTs (40). We have termed
these receptors C2 to distinguish them from the C3-type amylin
receptors described below. On the basis of pharmacological but not
competition binding studies, division of CGRP receptors into two
subtypes has been proposed: type 1, which is potently antagonized by
CGRP-(8
37) and which has only weak interaction with linear CGRP
analogs, or type 2, which has moderate interaction with linear analogs with poor antagonism by CGRP-(837) (14, 15). Nonetheless, neither
type 1 nor type 2 receptors have significant affinity for salmon CT
(sCT) and thus would fall into our C2 class of receptors. The C3-type
amylin receptor, in the rat, has high affinity for both amylin and sCT,
moderate to high affinity for the CGRPs, but only low affinity for rat
or human CT (2, 41, 46).
Amylin shares ~50% and 18-33% sequence homology with the CGRPs
and the CTs, respectively (Fig. 1). The
three peptides form part of a related gene family (4) and exhibit a
number of common primary and secondary structural features. These
include a disulfide bridged loop of six or seven amino acids at the
amino terminus, an amidated aromatic residue present at
the carboxy terminus, and a region of predicted amphipathic -helical
secondary structure from residues 8-18 (residues 8-22 for
sCT).
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Fig. 1.
Amino acid alignment of calcitonin (CT), calcitonin gene-related
peptide (CGRP), amylin, and analog amino acid sequences.
Top: agonist peptides with amino acids
in common with rat amylin boxed.
Bottom: antagonist peptides with amino
acids in common with salmon CT-(832) [sCT-(832)] boxed.
Shaded region represents residues absent from full-length CT sequence.
In both top and
bottom, CT sequence has been gapped
between amino acids 21 and 22 to allow alignment with amylin and CGRP
sequences.
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In concordance with the structural conservation among the peptides,
amylin can induce CT-like actions in osteoclasts and CGRP-like actions
in the vasculature (reviewed in Ref. 42). However, in both these
actions, amylin is much weaker than either CT or CGRP and is thought to
act via classic CT or CGRP receptors.
Recently, we identified high-affinity binding sites for amylin in the
renal cortex of the rat and demonstrated cognate specificity of ligand
interaction to the binding sites in brain (41, 46). Furthermore, low
doses of amylin induce rises in plasma renin in both rats (46) and
humans (11), with a corresponding hypertension in rats (25), consistent
with the hypothesis that amylin may be a causative link between insulin
resistance and hypertension in patients with syndrome X (11, 45).
Nonetheless, very little is known about the distribution and
specificity of amylin in primates (including humans). In this study, we
have localized and characterized receptors for amylin and the related
peptides CGRP and sCT in the kidney of the monkey Macaca
fascicularis and demonstrated that
distinct receptors for all three peptides exist but that divergence
occurs between the specificity of ligand interaction in monkey and
rodents.
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METHODS |
Materials.
Rat amylin, sCT, porcine CT (pCT), human CT (hCT), sCT-(832), rat
CGRP-, human CGRP- (hCGRP-), hCGRP-
, and hCGRP--(837) were from Bachem (Torrance, CA). AC-413 and AC-512
{[Lys11-Bolton-Hunter-Arg18,Asn30,Tyr32]sCT-(932)}
were a gift from Amylin Pharmaceuticals (San Diego, CA). The sequences
of peptides used in this study are illustrated in Fig. 1.
125I-labeled rat amylin
(Bolton-Hunter labeled at the amino-terminal lysine; 2,000 Ci/mmol) and
Na125I were from Amersham
(Buckinghamshire, UK).
125I-labeled sCT (~700 Ci/mmol)
and 125I-labeled rat CGRP-
(2,000 Ci/mmol) were iodinated and purified as previously described
(38, 40). Bovine serum albumin (BSA) was from Commonwealth Serum
Laboratories (Parkville, Victoria, Australia). Bacitracin was from
Sigma Chemical (St. Louis, MO). All other chemicals were
reagent grade or better.
Male monkeys were killed by an overdose of Nembutal (5 ml iv, 50 mg/ml), and the kidneys were quickly removed and frozen by immersion
into isopentane cooled with dry ice.
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Fig. 2.
Dry-film autoradiographic localization of receptor binding sites
in horizontal sections of monkey kidney.
A:
125I-labeled sCT, total binding;
B:
125I-sCT, binding in presence of 1 µM unlabeled sCT; C:
125I-labeled amylin, total
binding; D:
125I-amylin, binding in presence
of 1 µM unlabeled rat amylin; E:
125I-labeled CGRP, total binding;
F:
125I-CGRP, binding in presence of
1 µM unlabeled human CGRP- (hCGRP-). Cx, cortex; IM, inner
medulla; A, artery.
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Distribution studies.
Ten- or twenty-micrometer sections were cut in a cryostat
at 
15°C, thaw mounted onto gelatin-coated slides,
dehydrated, and then stored at 70°C until use (maximum of 4 wk).
Binding studies were performed as previously described for
125I-rat amylin (~70 pM) (41,
46) or 125I-sCT (100 pM) (38) and
125I-rat CGRP- (~70 pM) (40).
Briefly, sections were thawed and preincubated at ambient temperature
(20-22°C) for a total of 15 min by transferring through three
chambers of incubation buffer (amylin buffer: 20 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4, containing 100 mM NaCl, 1 mg/ml BSA, and 0.5 mg/ml bacitracin; sCT and CGRP buffer: 100 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 7.4, containing 2 mg/ml BSA and 0.5 mg/ml
bacitracin). Sections were then incubated for 60 min in
incubation buffer containing either
125I-rat amylin,
125I-sCT, or
125I-rat CGRP-. Nonspecific
binding was measured in the presence of
10
6 M homologous unlabeled
peptide. After incubation, sections were washed four times for 1 min in
ice-cold incubation buffer without BSA or bacitracin before being
dipped in ice-cold deionized water and dried under a stream of air.
Sections were exposed to Agfascopic CR3B X-ray film for 14-28 days
before development. After exposure, sections were stained with
hematoxylin and eosin to aid in anatomic localization of binding
sites.
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Fig. 3.
Emulsion autoradiographic localization of
125I-amylin binding to horizontal
sections of monkey kidney. A: inner
medulla (magnification, ×130), total binding;
B: inner medulla (×130), binding
in presence of 1 µM unlabeled amylin;
C: cortex (×39), total binding;
D: cortex (×130), total binding;
E: cortex (×158), total binding;
F: cortex (×98), binding in
presence of 1 µM unlabeled amylin;
G: large renal artery (×98),
total binding; H: large renal artery
(×130), binding in presence of 1 µM unlabeled amylin. Arrows
(A and
B) depict isolated cells with
high-density, nonspecific radioligand binding. bv, Blood vessel; CT,
collecting tubule; DT, distal tubule; e, endothelium; G, glomerulus;
JGA, juxtaglomerular apparatus; PT, proximal tubule; RA, renal artery;
sm, smooth muscle; TAL, thick ascending limb of loop of
Henle.
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For emulsion autoradiographic studies, sections were incubated as
described above, except that, after washing, the sections were placed
into 2.5% glutaraldehyde in phosphate-buffered saline [PBS; (in
mM) 140 NaCl, 2 KCl, 1 KH2PO4,
8 Na2HPO4],
pH 7.4, at 4°C for 30-60 min. The sections were then washed
for 1 min in PBS at 22°C, dipped into distilled deionized water,
and allowed to air dry. Before emulsion dipping, the sections were
defatted in graded alcohol and xylene. After defatting, slides were
prewarmed to ~42°C and then dipped for 1-2 s into LM-1
liquid emulsion at 42°C (Amersham) diluted 1:1 with distilled
deionized water. Excess emulsion was allowed to drain from the slides,
which were subsequently dried for 4-5 h at 25°C and >80%
humidity. The dried emulsion-covered sections were exposed in a
light-tight box with dehydrated silica gel for 1-3 mo, at 4°C,
before subsequent development, according to the manufacturer's
specifications. To aid in anatomic localization of binding sites,
sections were subsequently stained with hematoxylin and eosin.
Competition binding studies.
Serial 20-µm transverse sections were cut and incubated, as described
above, with 125I-rat amylin,
125I-sCT, or
125I-rat CGRP- containing
increasing concentrations of unlabeled peptide. Serial sections to
those used for competitive binding at each concentration examined were
taken for measurement of total binding to account for changes in
binding densities with different levels of section. As such, binding is
expressed as a percentage of total binding at each level of section.
Three sections were analyzed for each concentration of peptide. To
enable quantitation of the autoradiographs, a series of
125I-labeled radioactivity
standards were included in each cassette (40). After development, the
autoradiographs were analyzed by computerized densitometry, using the
personal computer-based microcomputer imaging device system (Imaging
Research, St. Catharines, ON, Canada).
Dissociation constant
(Kd) and
maximum binding site concentration
(Bmax) values were determined by
analysis of homologous competition binding studies, using the program
LIGAND, as adapted for the personal computer (32). The concentrations
of nonhomologous ligands yielding 50% inhibition of radioligand
binding were determined with the use of SigmaPlot (Jandel Scientific,
San Rafael, CA).
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RESULTS |
Receptor distribution.
Analysis of receptor distributions, using dry-film autoradiography,
demonstrated high-density binding of both
125I-sCT and
125I-amylin to the renal cortex
(Fig. 2, A and
C).
125I-sCT but not
125I-amylin also demonstrated
low-density binding to the inner medulla (Fig.
2A). The binding of
125I-CGRP, in contrast to the
other radioligands, was predominantly to vascular tissue, with
low-level, diffuse binding seen throughout the inner medulla/papilla
(Fig. 2E).
Microscopic analysis of the binding distributions, using emulsion
autoradiography, indicated no apparent specific binding of
125I-amylin to the inner medulla.
However, as with other parts of the kidney, a number of isolated cells
exhibited nondisplaceable 125I-amylin binding (Fig.
3, A and
B).
125I-amylin binding was also
absent from most of the transitional zone between medulla and cortex.
In the cortex, amylin binding was to tubules (Fig. 3,
C-E).
The tighter nuclear spacing of tubular cells that bound amylin
suggested that the binding was primarily to distal tubule. However, the
morphology of the tissue did not allow us to discern between distal
tubule and cortical collecting tubule. No binding to glomeruli or blood
vessels in the cortex was observed (Fig. 3,
C-E),
although low-density binding to large renal arteries in the medulla was
seen (Fig. 3G). Likewise, much of
the proximal tubule clearly had no discernible binding of
125I-amylin (Fig. 3,
C-E),
although again it was impossible to exclude binding to a population of
proximal tubule as a component of the binding. The lack of binding to
either the inner or outer medulla suggested that neither the thick limb
nor the thin limb of the loop of Henle contained amylin binding sites.
Moderate- to high-density binding of
125I-amylin was often found
adjacent to the vascular pole of the glomerulus, consistent with
binding to the juxtaglomerular apparatus (Fig.
3E).
Binding of 125I-sCT in the medulla
was confined to the thick ascending limb of the loop of Henle (Fig.
4A).
No binding was found over small-caliber vasculature, thin limb of the
loop of Henle, or collecting tubules. In the cortex, as with the amylin
binding, although it was possible to exclude binding to much of the
proximal tubule (Fig. 4, C,
E, G,
and H), it was impossible to totally exclude binding to some part of the proximal tubule as a component of
the observed binding. The extent of tubular binding of
125I-sCT in the cortex was greater
than that found for amylin and probably reflects binding of
125I-sCT to thick limb of the loop
of Henle, which is absent for 125I-amylin. As with amylin,
moderate- to high-density binding occurred to distal tubule (Fig. 4,
C, E,
G, and
H), although we could not delineate
early cortical collecting tubule from distal tubule. In many instances,
intense binding was found in close association with the glomerulus,
suggesting binding to juxtaglomerular apparatus (Fig. 4,
G and
H).
125I-sCT did not bind to either
glomeruli (Fig. 4, C,
E, G,
and H) or to renal blood vessels
(Fig. 4E).
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Fig. 4.
Emulsion autoradiography of
125I-sCT binding to horizontal
sections of monkey kidney. A: inner
medulla (magnification, ×129), total binding;
B: inner medulla (×129), binding
in presence of 1 µM unlabeled sCT;
C: cortex (×63), total binding;
D: cortex (×63), binding in
presence of 1 µM unlabeled sCT; E:
cortex (×129), total binding; F:
cortex (×129), binding in presence of 1 µM unlabeled sCT;
G: cortex (×157), total binding;
H: cortex (×314), total
binding.
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In contrast to amylin and CT binding, the highest densities of
125I-CGRP binding occurred over
arteries or arterioles (Fig.
5,
C-E) and included intense binding to the smooth muscle layer in the larger
vessels (Fig. 5D). The large renal
arteries likewise had high-density binding (Fig.
5A). As seen macroscopically (Fig. 2E), low-level, diffuse binding also
occurred in the inner medulla/papilla. This binding appeared to be
predominantly extratubular, consistent with binding to the extensive
capillary network in this region of the kidney (Fig.
5G). Whether specific binding
occurred to tubules in this region was unclear. In the cortex, binding
was absent from tubules, being confined mainly to blood vessels;
however, weak binding was seen over glomeruli (Fig. 5,
C and
E).
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Fig. 5.
Emulsion autoradiography of
125I-CGRP binding to horizontal
sections of monkey kidney. A: large
artery (magnification, ×157), total binding;
B: large artery (×129), binding
in presence of 1 µM unlabeled hCGRP-;
C: cortex (×129), total binding;
D: cortex (medium sized artery;
×314), total binding; E: cortex
(×157), total binding; F: cortex
(×129), binding in presence of 1 µM unlabeled hCGRP-;
G: inner medulla (×157), total
binding; H: inner medulla
(×129), binding in presence of 1 µM unlabeled hCGRP-.
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Competition binding studies.
The specificity of binding to CT, amylin, and CGRP receptors was
assessed by radioligand binding competition to horizontal sections of
monkey kidney.
125I-sCT.
125I-sCT bound to monkey renal
cortex with high affinity, exhibiting a
Kd in competition
binding studies of 5.88 ± 1.00 × 1010 M and a
Bmax of 113 ± 14 fmol/mg
protein. The rank order of potency for agonist peptides in competing
for binding was sCT > pCT > hCT >> rat amylin, whereas the
CGRPs essentially did not compete for binding in concentrations up to
106 M (Fig.
6A, Table
1). For antagonist peptides, AC-512 was the most potent, being almost one order of magnitude more potent than either sCT-(832) or AC-413. Human CGRP--(837) was without effect at concentrations up to 106
M (Fig. 6B, Table 1).
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Fig. 6.
Competition of 125I-sCT binding to
monkey kidney sections by unlabeled peptides.
A: agonist peptides sCT ( ), porcine
CT (pCT;  ), human CT (hCT;  ), rat amylin ( ), rat CGRP-
( ), hCGRP- ( ), and hCGRP- ( ).
B: antagonist peptides sCT (),
AC-512 ( ), AC-413 ( ), sCT-(832) ( ), and hCGRP--(837)
(solid hexagon). sCT is included in both graphs as a reference. B,
binding; Bo, total
binding.
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Table 1.
IC50 values for nonhomologous peptides in competition
for 125I-labeled sCT, 125I-labeled rat amylin,
or 125I-labeled rat CGRP- binding to
horizontal sections of monkey kidney
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125I-rat amylin.
Analysis of competition of
125I-rat amylin binding to renal
cortex by rat amylin indicated both high-affinity
(Kd 7.15 ± 0.77 × 1011 M) and
low-affinity (Kd
7.77 ± 0.12 × 109
M) binding sites, with Bmax values
of 1.40 ± 0.19 and 525 ± 59 fmol/mg protein for each site,
respectively. The relative potency of nonamylin agonist peptides in
competition for 125I-amylin
binding was sCT > pCT > hCT > hCGRP- and rat CGRP- > hCGRP- (Fig. 7A,
Table 1). For antagonist peptides, the order of potency was AC-512 > sCT-(832) > AC-413, whereas hCGRP--(837) was ineffective in
competition for binding at concentrations up to
106 M (Fig.
7B, Table 1).
125I-rat CGRP-.
Analysis of competition of
125I-CGRP binding by rat CGRP-
also indicated both a high-affinity
(Kd 1.07 ± 0.78 × 1010 M;
Bmax 6.93 ± 4.62 fmol/mg
protein) and a low-affinity
(Kd 1.49 ± 0.79 × 108
M; Bmax 258 ± 74 fmol/mg
protein) binding site. The relative potency of agonist peptides in
competition for binding was rat CGRP-, hCGRP- > hCGRP-
>> rat amylin, whereas sCT, pCT, and hCT did not compete for
binding in concentrations up to
106 M (Fig.
8A, Table 1). For
antagonist peptides, hCGRP--(837) was the most potent ligand,
being two- to fourfold more potent than AC-413 and AC-512,
respectively. sCT-(832), however, did not compete for binding, even
at 106 M (Fig.
8B, Table 1).
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DISCUSSION |
In this study, we demonstrated specific, high-affinity binding sites
for amylin, CT, and CGRP in M.
fascicularis kidney. The distribution
of binding sites in monkey was predominantly similar to those reported
in the rat (23, 38, 44, 46). However, a number of anomalies were
apparent.
The distribution of 125I-sCT
binding sites paralleled that of the rat, in which binding was
localized to cortical and medullary thick ascending limb of the loop of
Henle and distal tubule (38). This also parallels the distribution of
CT-responsive adenylate cyclase in both rat and human nephrons (28, 33,
34). Physiologically, in rats, CT decreases urinary excretion of
magnesium and calcium (5, 19, 20) primarily because of increased
reabsorption in the thick ascending limb of the loop of Henle (19). In
the loop of Henle, CT also increases reabsorption of potassium and to a
lesser extent sodium and chloride (19). In the distal tubule, in
addition to effects on calcium and magnesium, CT decreases fractional
excretion of sodium, chloride, and total solutes and decreases
secretion of potassium (18). Because the distributions of receptors in
monkey and rat are essentially equivalent, it is likely that similar
functions are performed by CT receptors in distal tubule and thick
ascending limb of the loop of Henle in primates. An additional
observation in the current study was the intense binding of
125I-sCT to structures consistent
with juxtaglomerular apparatus. The juxtaglomerular apparatus is the
principal source of circulating renin, and binding of CT to this
structure may provide an explanation for the rise in plasma renin seen
with peripherally administered sCT (9, 31, 47). Interestingly, sCT may
elevate blood pressure acutely, in association with the rise in plasma
renin activity (25), and, consequently, potential mediation of this
effect by receptors on the juxtaglomerular apparatus needs to be
considered.
The distribution of 125I-CGRP
binding sites was also similar between rat and monkey, with moderate-
to high-density binding to the renal medulla. However, the rat also
exhibited relatively high-level, punctate binding in the cortex,
indicative of glomerular binding (46). Although glomerular binding was
also seen with 125I-CGRP in the
current study, the level of binding was relatively low. In rats, on the
basis of the relative efficacy of adrenomedullin and CGRP in
stimulating cAMP production in glomeruli and the lack of inhibition of
the response by the CGRP antagonist CGRP-(837), it has been suggested
that the glomerular receptors are principally adrenomedullin receptors
(17). As such, the low level of CGRP binding may represent a decreased
affinity of these receptors for CGRP in monkey, although alternative
explanations, including low levels of either adrenomedullin or CGRP
receptors, are equally valid. In monkey, the highest levels of CGRP
binding occurred over renal arteries and arterioles and are consistent
with the potent action of CGRP as a renal vasodilator (1, 16, 21, 24),
with consequent increases in renal blood flow (1, 43). Although the
resolution of the autoradiographic procedure did not allow us to
determine whether binding occurred to endothelium as well as to smooth
muscle, physiological studies in the rat indicate that renal
vasodilatation is dependent on release of endothelium-derived nitric
oxide (21), and thus it is likely that receptors are present on
endothelial cells.
CGRP-like immunoreactivity has been localized to both the cortex and
medullopapillary portion of the human kidney (22), whereas renovascular
innervation of CGRP-containing neurons is well described in other
species (13, 29). Thus it is likely that CGRP-mediated actions in the
kidney occur via local release of CGRP from peptidergic nerves.
The distribution of 125I-amylin
binding sites in monkey presented as a subset of sites labeled with
125I-sCT and was localized to
cortical tubule structures, which included at least the distal tubule.
In this, it was consistent with binding to most other C3-type amylin
receptors (2, 41) and probably reflects the high affinity of sCT for
both C3-type amylin and C1-type CT receptors. This binding
distribution, however, contrasts with that reported for amylin
receptors in the rat, which indicated that binding was exclusively to
proximal tubules, although not all proximal tubules bound
125I-amylin (23).
A potential explanation for the discrepancy in amylin binding
distributions in rat and monkey is that, in monkey, the amylin binding
represents lower-affinity binding to C1-type CT receptors, thus
accounting for the parallelism in cortical distribution of CT and
amylin binding, with no amylin binding observed in the medulla because
of the relatively low density of CT receptors in this part of the
kidney. However, we believe this is unlikely for the following reasons.
In cells transfected with either rat or hCT receptor cDNAs,
essentially no binding is seen using
125I-amylin at concentrations used
in the current study (Ref. 26 and Sexton, unpublished data).
Furthermore, in monkey brain, the pattern of amylin receptor
distribution parallels that of the rat and, like the rat, forms a
subset of sites labeled with
125I-sCT. In this tissue, many
regions of high-density 125I-sCT
binding remain devoid of amylin binding, indicating that amylin binding
is not a consequence of low-affinity binding to C1-type CT receptors
(Ref. 8 and G. Paxinos, S. Y. Chai, G. Christopoulos, X.-F.
Huang, A. W. Toga, P. M. Sexton, unpublished observations). In addition, the specificity of amylin
receptors in the brain is similar to that described in kidney in the
current study (Paxinos et al., unpublished observations), indicating
that the renal and neuronal amylin receptors are equivalent and
represent the monkey equivalent of rat C3-type amylin receptors.
Although amylin receptors, distinct from C3-type amylin receptors, have
been described that lack interaction with sCT (12, 27), it seems
unlikely that these underlie the anomaly in rat renal amylin receptors,
because equivalent receptor distributions were demonstrated by in vitro
and in vivo techniques (23), and in vitro analysis of receptor
specificity, in competition binding studies, revealed a similar profile
to other C3-type amylin receptors, with amylin, CGRP, and sCT all
exhibiting similar potency in inhibiting 125I-amylin binding
(46). Nonetheless, the nature of the paradoxical lack of
125I-sCT binding to proximal
tubule amylin receptors in the rat remains unclear. The apparent
divergence in receptor distributions in monkey and rat suggests that
renal amylin receptors may subserve different functions in the two
species. Alternatively, the differences in distribution may be partly
due to ontological differences in receptor expression. It has been
suggested that rat cortical amylin receptors are initially expressed
during a stage of extensive differentiation and organogenesis in the
kidney and that amylin might have a mitogenic effect on proximal tubule
cells during this phase of development (23). It is possible that the
proximal tubule amylin receptors seen in the adult rat are carried over from this developmental role but are lost during maturation of monkey
kidney to the adult state. No data are available on amylin receptor
expression in developing monkey kidney. The apparent localization of
amylin receptors to juxtaglomerular apparatus in the adult monkey
kidney, however, is consistent with a direct action of amylin in
inducing renin release in humans (11). Of note, amylin is more potent
in stimulating plasma renin activity in humans (~5-fold) (11) than in
rats (~2-fold) (46), which may reflect the more developed
juxtaglomerular apparatus in primates. The physiological role of amylin
receptors in the distal tubule is unclear. The weak binding of amylin
to smooth muscle of large renal arteries, however, is likely due to a
low-affinity interaction of amylin with CGRP receptors. Studies on the
renovascular action of amylin indicate that it is much weaker than CGRP
and can be blocked by the CGRP antagonist CGRP-(837) (7), which is
consistent with this hypothesis.
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Fig. 7.
Competition of 125I-rat amylin
binding to monkey kidney sections by unlabeled peptides.
A: agonist peptides sCT (), pCT
(), hCT (), rat amylin (), rat CGRP- (), hCGRP-
(), and hCGRP- (). B:
antagonist peptides rat amylin (), AC-512 (), AC-413 (),
sCT-(832) (), and hCGRP--(837) (solid hexagon). Rat amylin is
included in both graphs as a reference.
|
|
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|
Fig. 8.
Competition of 125I-rat CGRP-
binding to monkey kidney sections by unlabeled peptides.
A: agonist peptides sCT (), pCT
(), hCT (), rat amylin (), rat CGRP- (), hCGRP-
(), and hCGRP- (). B:
antagonist peptides rat CGRP- (), AC-512 (), AC-413 (),
sCT-(832) (), and hCGRP--(837) (solid hexagon). Rat CGRP-
is included in both graphs as a reference.
|
|
Analysis of receptor specificity in competition binding studies
revealed specificity profiles for CT and CGRP receptors similar to the
profile of rat receptors studied under similar conditions (38, 40).
Although, on the basis of competition binding studies, division of CGRP
receptors into type 1 or type 2 receptors has not been
possible, most data indicate that at least the renal vascular receptor exhibits a type 1 phenotype (6, 7). Of note, however,
was the potency of AC-512 in competing for
125I-CGRP binding. This peptide
was only slightly weaker (~4-fold) than CGRP-(837) in competing for
binding, whereas sCT-(832) was completely impotent. AC-512 differs
from sCT-(832) in only three positions (Fig. 1), and the importance
of the carboxy-terminal aromatic residue in binding (35) may indicate a
significant role of the benzene ring structure in primate CGRP receptor
recognition, at least for the inactive state receptor.
Little is known about the specificity of amylin receptors outside the
rat. Our data indicate that there is considerable divergence in
receptor specificity for monkey versus rat receptors. In particular, there is an apparent increase in the efficacy of CT peptides to compete
for binding in the monkey along with a small decrease in the relative
efficacy of CGRP peptides. Analysis of homologous competition studies
indicated that the 125I-amylin
binding could be divided into two components, a high-affinity state of
~7 × 1011 M and a
low-affinity state of ~8 × 109 M. Although it is
possible that the low-affinity state reflects binding to CT receptors,
the lack of 125I-amylin binding to
high-density CT receptor sites in parts of the brain suggests that this
is unlikely. A more probable explanation is that the binding reflects
affinities for active (G protein coupled) and inactive (G protein
uncoupled) states, with binding being primarily to inactive-state (low
affinity) receptor. Indeed, in mouse -thyroid-stimulating hormone
thyrotroph cells, we demonstrated a direct effect of G protein coupling
on amylin receptor affinity (36). As such, the differences in relative
specificities between rat and monkey may, in part, reflect differences
in the availability of G protein or other binding cofactors. As with
the rat, the antagonist peptides sCT-(832) and peptide chimeras
between sCT-(832) and amylin were potent competitors of
amylin binding. However, CGRP-(837) failed to compete for binding in
concentrations of up to 1 µM. Thus, unlike rat, in which CGRP-(837)
is ~100-fold weaker than either sCT-(832) or AC-413 (46) and acts
as a weak antagonist of amylin receptors in skeletal muscle (3),
CGRP-(837) is unlikely to be an effective antagonist of amylin
receptors in monkey and consequently may act as a more specific CGRP
type 1 receptor antagonist.
In conclusion, these studies demonstrate the existence of high-affinity
receptors for amylin, CT, and CGRP within the primate kidney. The
distribution of receptor sites suggests that the peptides will exert
distinct biological actions on renal vasculature, primarily mediated
via CGRP receptors, and renal tubules, primarily mediated via amylin or
CT receptors. Furthermore, the apparent divergence in receptor
specificity between amylin receptors in monkey and rat indicates that
caution should be taken in extrapolating on functional specificity of
peptides between species.
| |
ACKNOWLEDGEMENTS |
This work was supported by grants from the National Health and
Medical Research Council of Australia, the Australian Kidney Foundation, Amylin Pharmaceuticals, and Glaxo Research. P. M. Sexton is
a research fellow of the Australian Research Council.
| |
FOOTNOTES |
Address for reprint requests: P. M. Sexton, Neurobiology Unit, St.
Vincent's Institute of Medical Research, 41 Victoria Parade, Fitzroy
3065, Victoria, Australia.
Received 29 April 1997; accepted in final form 28 August 1997.
| |
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