To determine the type of receptors involved in the actions of ADM in
the cat iris sphincter, we examined the effects of receptor antagonists
and performed binding studies. At present, there is controversy about a
receptor(s) responsible for ADM-induced
vasorelaxation.
18 23 An issue we tried to address in the
present work is whether the observed effects of ADM and CGRP are
mediated by the same or by different receptors. Two types of ADM
receptors have been characterized.
27 One type of ADM
receptor, which binds ADM but not CGRP or CGRP (8–37) with high
affinity, is present in rat tissue endothelial cells and rat vascular
smooth muscle cells.
7 28 This is consistent with the
cloned ADM receptor, which bound [
125I]ADM with
high affinity and elevated cAMP threefold. In contrast, 1 μM CGRP had
no effect on cAMP, and the increase in cAMP caused by 10 nM ADM was not
antagonized by CGRP (8–37).
29 A second type of ADM
receptor was identified in bovine aortic endothelial cells,
neuroblastoma cells, and L6 cells, which binds ADM, CGRP, and CGRP
(8–37) with high affinity.
8 26 In this case, ADM elevates
cAMP, but the increase in cAMP is antagonized by CGRP (8–37). This may
represent the CGRP type 1 receptor, which was cloned and has 56%
homology with the calcitonin receptor.
30 The data
presented here suggest that cat iris sphincter contains the second type
of ADM receptor, namely the CGRP
1 receptor. This
conclusion is supported by the following findings in the present work:
(1) The cAMP responses to ADM and CGRP are inhibited markedly by
treatment with the competitive CGRP antagonist, CGRP (8–37)
(Fig. 3) .
CGRP (8–37) inhibited ADM- and CGRP-induced cAMP formation in a
concentration-dependent manner with IC
50 values
of 32 nM
(Fig. 3A) and 22 nM
(Fig. 3B) , respectively. CGRP (8–37) was
considerably more potent in its inhibitory effects than ADM (26–52)
(Fig. 3) . CGRP (8–37) inhibited the vasodilator response to ADM in the
isolated perfused mesenteric bed
18 and in the isolated rat
heart,
31 indicating that the effect of ADM in these
tissues is via CGRP
1 receptors. (2) Addition of
ADM and CGRP together did not result in an additive effect on cAMP
accumulation, suggesting that these peptides exert their effects
through a common receptor
(Fig. 4) . (3) The present study has for the
first time shown the presence of ADM binding sites in ocular tissues
(Fig. 5) . The Scatchard plot was linear indicating that[
125I]ADM bound with high affinity
(
K d = 0.51 ± 0.17 nM) to a single
class of sites (
B max = 93 ± 20
fmol/mg protein)
(Fig. 5A) . Both ADM and CGRP displaced the binding of[
125I]ADM to iris sphincter membranes
effectively, with IC
50 values of 0.81 and 1.15
nM, respectively
(Fig. 5B) . In the present work we have observed an
apparent difference between the IC
50 values of
ADM (26–52) and CGRP (8–37) on ADM-binding and on ADM-induced cAMP
formation
(Figs. 3 4) . This could be explained by the fact that the
binding experiments were carried out on isolated membranes
(Fig. 5) ,
whereas the cAMP studies were performed on whole muscle
(Fig. 3) . The
finding that the specific [
125I]ADM binding was
strongly inhibited by CGRP suggest that most of the[
125I]ADM binding detected here is primarily
due to interaction with CGRP receptors. In rat spinal cord microsomes[
125I]ADM binding showed high affinity
(
K d = 0.45 ± 0.06 nM) and sites were
abundant (B
max = 723 ± 71 fmol/mg
protein).
32 Scatchard plots of[
125I]ADM binding in human brain (cerebral
cortex) gave a
K d of 0.17 ± 0.03 nM
and maximal binding of 99.3 ± 1.9 fmol/mg protein.
33 In smooth muscle, pharmacologically distinct binding sites for ADM were
reported in rat uterus (
K d = 0.08 ±
0.006 nM;
B max = 21 ± 2 fmol/mg
protein),
34 and in cultured vascular smooth muscle
cells.
35 ADM interacts with the CGRP receptor in several
tissues, including cultured human neuroblastoma SK-N-MC
cells,
26 cultured human breast cancer
cells,
22 rat cardiac myocytes and
nonmyocytes,
25 rat vascular smooth muscle
cells,
35 and rat mesenteric beds.
7 In
contrast, cultured endothelial cells of human umbilical vein possess
specific ADM receptors coupled with the adenylate cyclase that may have
little affinity with CGRP,
23 the renal action of ADM is
not mediated via the activation of CGRP
1 receptors, specific ADM binding sites that differ from CGRP
receptors
36 exist in brain,
33 and cultured
mouse astrocytes possess specific ADM receptors that are coupled to
adenylate cyclase but do not interact with CGRP.
37 These
observations indicate that the distribution, characteristics, and
biological effects of ADM and CGRP overlap in many tissues. (4) ADM and
CGRP inhibited, presumably via stimulation of adenylate cyclase,
carbachol-induced contraction in the cat sphincter in a
concentration-dependent manner with IC
50 values
of 10 and 90 nM, respectively
(Fig. 6) . These results suggest that in
the iris sphincter signal transduction mechanisms responsible for
relaxation caused by ADM are similar to those caused by CGRP.