Calcitonin Gene-related Peptide Binding Research Articles

Receptors for calcitonin gene-related peptide (CGRP) in the rat adrenal cortex

We previously demonstrated the presence of adrenomedullin receptors in the rat adrenal cortex. There is evidence, however, that the actions of adrenomedullin may also be mediated by the CGRP receptor. The present study was designed to determine whether specific CGRP receptors are present in the rat adrenal cortex. Adrenal glands were, sectioned and immunostained with a primary antibody raised against the first intracellular loop of the CGRP-1 receptor. Staining was visualised using alkaline phosphatase and vector red. Immunostaining for the CGRP-I receptor was found in the zona glomerulosa and the adrenal medulla, but not in the inner adrenocortical zones. ACTH treatment caused an increase in staining intensity in the glomerulosa. Ligand binding studies suggested the existence of two populations of CGRP binding sites, one with a Kd of 0.1 nM, the second of 37 nM. Only CGRP-I and adrenomedullin displaced labeled CGRP binding. These results suggest that the CGRP-I receptor is expressed in the adrenal zona glomerulosa and that a second class of binding site is also present. The CGRP-I receptor appears to be regulated by ACTH.

Tissue-specific mRNA expression of a calcitonin receptor-like receptor during fetal and postnatal development

The distribution of calcitonin receptor-like receptor (CRLR) mRNA in developing rats was investigated by in situ hybridization. Signals were found in the piriform cortex, the central and basolateral amygdala and the amygdalostriatal transition area. Among peripheral organs, the CRLR was predominantly expressed in the lung. mRNA expression in blood vessels, liver, midgut, rectum and urethra was restricted to gestational days 16 and/or 20. The CRLR was thought to be a calcitonin gene-related peptide (CGRP) type 1 receptor (Aiyar et al., J. Biol. Chem., 271 (1996) 11325–11329). This contrasts with previously reported evidence that the CRLR is an orphan receptor with no identifiable interactions with CGRP and other related ligands (Flühmann et al., Biochem. Biophys. Res. Commun., 206 (1995) 341–347). In situ hybridization signals have not been detected in the cerebellum and the spleen known to present a high density of CGRP binding sites. The different regional distribution of CGRP receptor binding sites and CRLR mRNA implies the latter encoding a different CGRP receptor subtype.

Human pulp cells respond to calcitonin gene-related peptide in vitro

Cell monolayers derived from human pulpal explants were passaged 3 to 4 times before characterization of the response of the cells to calcitonin gene-related peptide (CGRP). Northern blot analysis of mRNA revealed the presence of transcripts for bone morphogenetic protein-2 (BMP-2). Stimulation with CGRP produced a 1.8-fold increase in BMP-2 mRNA expression by the cells. Analysis of binding of CGRP to whole cells indicated that unlabeled human CGRP competed with labeled CGRP in a dose dependent fashion with a KD, estimated from the EC50, in the range of 5 x 10(-8)M. Binding of labeled CGRP was greatly reduced by the presence of 10(-6)M CGRP but was unaffected by the presence of 10(-6)M Human PTH(1-34) or 10(-6)M calcitonin. CGRP produced a 2.8-fold increase in cyclic AMP over basal levels, which was similar to the increase produced by PTH(1-34) (2.6 fold) but slightly more than the increase produced by calcitonin (1.9 fold). The pulp-derived cells displayed a high basal level of alkaline phosphatase enzyme activity, which was not altered by treatment with CGRP or either PTH(1-34) or 1,25(OH)2D3. Stimulation with 2.5 x 10(-8)M 1,25(OH)2D3 did produce a 7.6-fold increase in osteocalcin. These results indicate that pulp cells possess the cellular machinery to respond to CGRP and that stimulation of the production of BMP-2, a factor known to be associated with induction of dentin formation, is a component of the response.

Differential effects of guanine nucleotides on [125I]-hCGRP(8-37) binding to porcine lung and human neuroblastoma cell membranes.

Calcitonin gene-related peptide (CGRP) mediates its effects by binding to specific receptors which are positively coupled to adenylyl cyclase. CGRP(8-37), a CGRP fragment devoid of the N-terminal region, was shown to be a competitive CGRP receptor antagonist. Only a limited amount of data exists on the usefulness of this ligand in studying CGRP receptors. In the present study, we used [125I]-hCGRP(8-37) to characterize CGRP receptors in porcine lung and human neuroblastoma cell (SK-N-MC) membranes. [125I]-hCGRP(8-37) displayed specific and high affinity binding in both membrane preparations. Displacement studies using [125I]-hCGRP(8-37) and the agonist CGRP revealed the presence of high and low affinity CGRP binding sites in SK-N-MC cell and porcine lung membranes. Addition of guanylimidodiphosphate [Gpp(NH)p] shifted the competition curve to the right and changed the two affinity states of the receptor to a single affinity in SK-N-MC cell membranes. On the other hand, in porcine lung membranes, the whole competition curve was shifted to the right while maintaining the two affinity states. Thus, our data indicate that the new radioligand [125I]-hCGRP(8-37) is a useful tool for characterizing CGRP receptors and their coupling to guanine nucleotide binding proteins.

A rat skeletal muscle cell line (L6) expresses specific adrenomedullin binding sites but activates adenylate cyclase via calcitonin gene-related peptide receptors.

We have previously demonstrated specific binding sites for adrenomedullin, a novel member of the calcitonin family of peptides, in rat muscles. It is unclear whether these receptors are vascular or muscular. Receptors for the structurally similar calcitonin gene-related peptide (CGRP) are present on myocytes and might be involved in the regulation of myocyte glucose metabolism and control by motor neurons. We investigated whether adrenomedullin binding sites were present on L6 myocytes. Specific [125I]adrenomedullin binding sites were demonstrated where adrenomedullin competed with an IC50 of 0.22 +/- 0.04 nM (mean +/- S.E.M.) and a concentration of binding sites (Bmax) of 0.95 +/- 0.19 pmol/mg of protein (mean +/- S.E.M.). CGRP and the specific CGRP receptor antagonist CGRP(8-37) competed weakly at this site (IC50 > 10 and 601 +/- 298 nM respectively). Binding studies with [125I]CGRP revealed a binding site for CGRP (IC50 = 0.13 +/- 0.01 nM; Bmax = 0.83 +/- 0.10 pmol/mg of protein) where both CGRP(8-37) and adrenomedullin competed with [125I]CGRP with IC50 values of 1.15 +/- 0.12 and 8.68 +/- 0.98 nM respectively. Chemical cross-linking showed the CGRP and adrenomedullin binding site-ligand complexes to have approximate molecular masses of 82 and 76 kDa respectively. Both CGRP and adrenomedullin increased adenylate cyclase activity with similar potencies. In both cases adenylate cyclase activation was blocked by CGRP(8-37). Stimulation with 10 nM adrenomedullin or CGRP caused an increase in the percentage of total activated cellular cAMP-dependent protein kinase from 38% in resting cells to 100% and 98% respectively. Therefore in L6 cells adrenomedullin can bind to CGRP receptors, activating adenylate cyclase and cAMP-dependent protein kinase.

Adrenomedullin activity in chronically hypoxic rat lungs.

Adrenomedullin (AM) is a novel vasodilator with structural similarities to calcitonin gene-related peptide (CGRP). This study investigated AM activity in the rat lung during hypoxia-induced pulmonary hypertension. Both rat AM (0.2-10 nmol) and alpha-CGRP (0.2-2 nmol) produced dose-related reductions in pulmonary artery pressure in the isolated perfused lung ventilated with 2% O2. Pretreatment with alpha-CGRP, which demonstrated tachyphylaxis, or its antagonist, CGRP-(8-37), reduced the hypotensive response to AM, suggesting that part of the response to AM is mediated by CGRP receptors. 125I-labeled AM and 125I-labeled CGRP binding was significantly increased in lung membranes from 7-day hypoxic animals (AM from 1.94 +/- 0.3 to 3.36 +/- 0.4 and CGRP from 0.06 +/- 0.01 to 0.12 +/- 0.02 pmol/mg protein), with no change in dissociation constant. Moreover, the hypotensive response to both peptides was increased in the lungs of 7-day hypoxic rats. There was no significant change in lung immunoreactive AM concentrations (hypoxic 5.04 +/- 0.48 vs. control 6.28 +/- 0.76 pmol/g wet wt of tissue) or steady-state AM mRNA levels in 7-day hypoxic rats. Nonetheless, AM may be useful for the acute pharmacological manipulation of pulmonary artery pressure in hypoxia-induced pulmonary hypertension.

Adaptation of Rainbow Trout to Seawater: Changes in Calcitonin Gene-Related Peptide Levels Are Associated with an Increase in Hormone–Receptor Interaction in Gill Membranes

Transfer of trout from freshwater to seawater produced changes in plasma calcitonin gene-related peptide (CGRP), calcitonin, and calcium levels. Three days after transfer, the plasma CGRP concentration increased from 517 ± 54 (n= 6) to 1874 ± 234 (n= 7) pg/ml. Ten hours after transfer, the plasma CT concentration in seawater-adapted trout was double the control value. Plasma calcium levels also changed: 2 days after transfer, the calcium increased 1.6-fold over the basal value. The increased plasma CGRP level was correlated with an increased specific binding of CGRP to its branchial membrane receptors. Scatchard analysis of the data revealed this increase to be mainly due to increased binding affinity, as there was no difference in binding capacity between control and transferred animals. Carbonic anhydrase activity in branchial membranes increased 2.2-fold over the control value 3 days after transfer. CGRP may thus play a role in osmoregulation and ionic balance.

CGRP innervation and receptors during aging of male and female hepatic rat portal veins

Calcitonin gene-related peptide (CGRP) innervation and binding sites were studied on hepatic portal veins of male and female rats from 19 days to 22 months of age. CGRP containing nerve fibers were present both in adventitial and medial nerve plexuses, closely apposing to or penetrating into the muscular layers. The density of CGRP innervation was estimated on whole mount preparations and compared during aging. In females, aging did not affect the number of fibers per unit length, although the vessel circumference decreased after 6 months of age. In males, the vessel circumference remained constant during aging, while the density of innervation significantly decreased. Whatever the age or sex of the animals was, no CGRP binding sites were found on portal veins sections by autoradiography. CGRP had no effect on spontaneous contractions of perfused portal veins. The difference observed in the evolution of CGRP innervation density between males and females suggests that CGRP innervation in the rat portal vein may be influenced by gonadal steroids during aging.

Characterization of binding sites for calcitonin gene-related peptide in the mollusc gill

Target organs for calcitonin gene-related peptide (CGRP) were investigated in Pecten maximus using 125I-labelled human CGRP. CGRP was shown to interact specifically with mantle and gill tissue. Receptor studies using branchial membrane preparations indicated that the binding was time dependent. Scatchard analysis of binding data showed that there was a single class of binding sites. The affinity constant was found to be 0.7 · 10 8 M −1 and the number of binding sites 2600 · 10 8/mg of protein. Salmon CT inhibited the binding of 125I-labelled CGRP to branchial membranes with a lesser efficiency than that of the unlabelled hormone. A 40% inhibition of the 125I-labelled CGRP binding was observed in the presence of 2.6 and 26 nM CGRP and salmon CT, respectively. In addition, 200 nM human CGRP inhibited 25 and 10% of the basal branchial and mantle adenylate cyclase activity, respectively. These data suggest that CGRP participates in the regulation of the branchial function in molluscs probably via a vasoconstrictor role.

Calcitonin gene-related peptide in the hamster seminal vesicle and coagulating gland: an immunohistochemical, autoradiographical, and pharmacological study.

The distribution of calcitonin gene-related peptide (CGRP)-immunoreactive nerves and CGRP binding sites, as well as the effect of CGRP on the muscle tension, was studied in the hamster seminal vesicle and coagulating gland. The use of an immunofluorescence staining technique on cryostat sections revealed that in the hamster seminal vesicle and coagulating gland, CGRP-positive nerve fibers are found in the connective interstitium and in the muscular and mucosal layers. Using an in vitro receptor autoradiographic technique, CGRP binding sites were found associated with the muscular coat. CGRP (10 pM to 1 microM) relaxed the seminal vesicle and the coagulating gland precontracted by either noradrenaline (10-30 microM) or the alpha 1-agonist, phenylephrine (10 microM). In preparations contracted by carbachol (10 microM), CGRP relaxed the seminal vesicle but not the coagulating gland. In both preparations, CGRP (1 microM) did not affect the muscle resting tension. These results suggest that CGRP may act as an inhibitory modulator of the autonomic control of contractility in the male accessory sex glands of the hamster.

Decreased endothelium-dependent pulmonary vasodilator effect of calcitonin gene-related peptide in hypoxic rats contrasts with increased binding sites.

Levels of calcitonin gene-related peptide (CGRP), a vasodilator peptide present in nerves and airway endocrine cells of the rat respiratory tract, are increased in hypoxic lung and decreased in plasma, suggesting impaired CGRP release. We wanted to determine whether there was an adaptive functional response to reduced CGRP levels in hypoxia. Density of binding sites for CGRP were compared with its vascular actions following hypoxia, and with binding following administration of the sensory neurotoxin capsaicin to deplete neural CGRP. Autoradiography of lung sections incubated with 125I-labelled CGRP and other vasoactive peptides was used to quantify their binding sites, in male Wistar rats exposed to periods of hypoxia (inspiratory oxygen fraction (FI,O2) = 0.1) ranging 0-10 days (n = 5 each), in controls, and in rats treated neonatally with capsaicin. Relaxation to CGRP was compared in pulmonary artery of control and hypoxic rats. CGRP binding was seen in the vascular endothelium and was significantly elevated after 5 days of hypoxia (mean +/- SEM: control 4.6 +/- 0.4 versus hypoxic 16.6 +/- 2.4 amol.mm-2). CGRP-induced (5 x 10(-7)M) relaxation of pulmonary artery was reduced, compared with controls, following 8 and 21 days of hypoxia (mean +/- SEM) percentage of relaxation to phenylephrine: 78 +/- 3, 36 +/- 5 and 32 +/- 3, respectively) and was abolished by removal of endothelium. Capsaicin treatment also significantly elevated vascular CGRP binding. Atrial natriuretic peptide (ANP) binding levels were decreased in smooth muscle of all blood vessels after 7 days of hypoxia, but endothelin-1 (ET-1) and vasoactive intestinal peptide (VIP) binding was unchanged. We conclude that the vasodilator effects of CGRP are endothelium-dependent and, whilst they are reduced in hypoxic lung, this is not due to reduction in receptors, thereby implicating alterations in the nitric oxide guanylyl cyclase system. Furthermore, adaptive responses in some peptide binding sites occur in hypoxia, which may be due to changes in endogenous peptide levels.

Plasticity of calcitonin gene related peptide neurotransmission in the spinal cord during peripheral inflammation.

Injection of complete Freund's adjuvant (CFA; 75 microL) into the plantar surface of the hind paw of the rat results in a mild inflammation that lasts for several days and is accompanied by hyperalgesia. Multiple components of calcitonin gene related peptide (CGRP) neurotransmission in the spinal cord are altered during the course of this peripheral inflammation. The content of immunoreactive (i) CGRP in the dorsal horn of the spinal cord, where primary afferent neurons terminate, is significantly decreased within 2 days after injection of CFA but increases to a level greater than that of the control at 8 days. The early decrease in iCGRP in the spinal cord suggests that the release of CGRP from primary afferent neurons is increased during the period of maximal hyperalgesia that accompanies peripheral inflammation. Changes in the mRNA for CGRP suggest that the increase in spinal content of iCGRP is due to an increase in synthesis of the peptide as the level of mRNA for CGRP is increased from 2 to 8 days after injection of CFA. Despite the decrease in the content of iCGRP in the spinal cord, there is no apparent decrease in the amount of iCGRP that can be released from the dorsal spinal cord by capsaicin; in fact, capsaicin-evoked release is increased at 4 days. Measurements of the binding of 125I-labelled CGRP in the dorsal spinal cord indicate that high affinity binding sites for CGRP are downregulated at 4 days after injection of CFA. In total, these data support the hypothesis that the activity of CGRP-containing primary afferent neurons is increased during peripheral inflammation. CGRP released from primary afferent neurons in the spinal cord may contribute to cellular changes that accompany peripheral inflammation.

Differential antagonism of amylin's metabolic and vascular actions with amylin receptor antagonists.

High affinity amylin binding sites are present in the rat nucleus accumbens. These sites bind [125I]amylin with an affinity of 27 pM and have high affinity for salmon calcitonin (sCT) and moderately high affinity for calcitonin gene related peptide (CGRP). N-terminally truncated peptides were tested for their ability to compete for [125I]amylin binding to these sites and to antagonize the metabolic and vascular actions of amylin. CGRP(8-37), sCT(8-32), and ac-[Asn30,Tyr32]sCT(8-32) (AC187) inhibited [125I]amylin binding to rat nucleus accumbens. Order of potency at inhibiting amylin binding (AC187 > sCT(8-32) > CGRP(8-37)) differed from the order of potency at inhibiting [125I]CGRP binding to SK-N-MC neuroblastoma cells (CGRP(8-37) > AC187 > sCT(8-32)) . AC187 was the most potent antagonist of amylin's effects on isolated rat soleus muscle glycogen metabolism, and it was more effective than either sCT(8-32) or CGRP(8-37) at reducing amylin-stimulated hyperlactemia in rats. In contrast, CGRP(8-37) was the most potent peptide at antagonizing amylin-induced hypotension in rats. Amylin's hypotensive actions appear to be mediated by a weak action at CGRP receptors, while its metabolic actions are mediated by receptors with a distinct antagonist profile. AC187 is a potent antagonist of amylin binding sites in nucleus accumbens and of amylin's metabolic actions.

Binding sites for islet amyloid polypeptide in mammalian lung: species variation and effects on adenylyl cyclase.

Islet amyloid polypeptide (IAPP) and calcitonin gene related peptide (CGRP) share a 47% sequence homology. IAPP can interact with adenylyl cyclase coupled CGRP receptors. We have examined [125I]IAPP binding in mouse, pig, and guinea pig lung membranes in competition with IAPP, CGRP, and CGRP(8-37). Three types of site were shown by order of potency: (i) mouse, IAPP > CGRP(8-37) >> CGRP; (ii) pig, CGRP > IAPP > CGRP(8-37); and (iii) guinea pig, CGRP = IAPP = CGRP(8-37). Chemical cross-linking of [125I]IAPP and [125I]CGRP binding sites in lung demonstrated that both sites had similar molecular weights in any one species but differed across species, i.e., mouse M(r) = 70,000 and 98,000; pig M(r) = 68,000, 56,000, and 47,000; and guinea pig M(r) = 106,000 and 56,000. Adenylyl cyclase activity was stimulated by forskolin and AlCl3-NaF in rat, mouse, pig, and guinea pig membranes. Only in mouse and pig were CGRP and IAPP able to stimulate adenylyl cyclase activity. In mouse lung CGRP and IAPP stimulated adenylyl cyclase activity with EC50 values of 642 +/- 222 nM (n = 4) and 325 +/- 115 nM (n = 4), respectively. In pig lung membranes EC50 values were 5.7 +/- nM (n = 4) for CGRP and 1230 +/- 1130 nM (n = 4) for IAPP. Thus IAPP either did not stimulate adenylyl cyclase activity in these lung membranes or did so with a low potency.

Developmentally regulated expression of alpha- and beta-calcitonin gene-related peptide mRNA and calcitonin gene-related peptide immunoreactivity in the rat inferior olive.

Immunohistochemical methods have revealed the transient neonatal expression of calcitonin gene-related peptide (CGRP) in olivocerebellar compartments, and it has been hypothesized that this peptide plays a role in the development of olivocerebellar connectivity. Furthermore, the distribution of the CGRP binding sites in the cerebellar cortex also favors this hypothesis. In this study, the pattern of postnatal expression of alpha- and beta-CGRP mRNAs in the inferior olive (IO) complex was analyzed using in situ hybridization histochemistry with RNA probes complementary to specific sequences of alpha- and beta-CGRP mRNAs, and the results were compared with the pattern of CGRP immunoreactivity. High levels of alpha-CGRP mRNA expression were found in specific subnuclei of the IO complex, i.e., the medial part of the dorsal fold of the dorsal accessory olive, the beta nucleus, the dorsal cap, the caudal third of the medial accessory olive, and the rostral part of the dorso-medial cell column; in the same subnuclei beta-CGRP mRNA was detected. The olivary distribution of the two CGRP mRNA coincided with that of CGRP immunoreactivity. The expressions of alpha-CGRP mRNA and CGRP immunoreactivity were restricted to the first 2 postnatal weeks, the peak being reached at the end of the first week; beta-CGRP mRNA was transiently expressed in the same olivary compartments, but only from postnatal day 6 to 9. In general, the alpha-CGRP signal was also more intense than the beta-CGRP signal. The present findings indicate that the alpha- and beta-CGRP mRNA expression in the olivary complex is under developmental control and restricted to specific olivocerebellar compartments. The data provide a basis for the transient expression of a CGRP olivocerebellar compartment and further support the hypothesis of a role for CGRP in the complex postnatal cerebellar phenomena of connectivity reshaping and synapse stabilization.

Calcitonin gene-related peptide in healthy and atheromatous human epicardial coronary arteries. Function and receptor characterization.

To examine the possible role of calcitonin gene-related peptide (CGRP) in the control of human coronary vascular tone, we have investigated the action of this peptide in healthy and atheromatous human epicardial coronary arteries and localised [125I]CGRP-binding sites in these vessels. Isolated arteries were obtained from 10 cardiomyopathic patients and 6 patients with ischaemic heart disease (IHD), who were undergoing heart transplantation. CGRP elicited concentration-dependent vasodilatation in preconstricted vessels. Both healthy and diseased coronary arteries exhibited similar maximum responses and sensitivity to this peptide. Removal of the endothelium did not diminish the vasodilator action of CGRP in non-atherosclerotic coronary arteries. [125I]CGRP bound to tissue sections in a concentration-dependent manner. Binding was similar in healthy and atheromatous vessels, with a Bmax value of 10.2 +/- 5.6 and 18.9 +/- 3.0 amol/mg protein, and dissociation constant (Kd) of 0.07 +/- 0.1 and 0.18 +/- 0.1 nM, respectively. Dense [125I]CGRP binding was mainly associated with vascular smooth muscle cells of both normal and diseased vessels with some patches of binding to regions of atherosclerotic plaque in vessels from patients with IHD. These data indicate that CGRP is a potent endothelium-independent vasodilator in the human coronary vasculature. The preservation of dilator function and CGRP receptor binding in atherosclerotic coronary arteries suggests that this peptide may play a role in the control or maintenance of vascular tone in certain disease states.

Localization of calcitonin gene-related peptide binding sites in the eye of different species.

The localization of calcitonin gene-related peptide (CGRP) binding sites in the eye of monkey, pig, cat and guinea pig was studied by autoradiography. Specific binding of CGRP was found in ciliary muscle, ciliary processes and limbal conjunctiva in all tested species. Furthermore, specific binding sites of CGRP was found in the choroidea of monkey, pig and guinea pig, in the iris of pig, cat and guinea pig, in the retina of pig and in the anterior chamber angle of cat. The number of specific binding sites varied depending on the tissue and species. The present study shows that there are specific binding sites of CGRP in the eye of monkey, pig, cat and guinea pig. CGRP binding sites found in vascular system of ciliary body, choroidea and iris further demonstrates the role of CGRP as a vasoregulatory peptide. Binding sites in the ciliary muscle, in the limbal conjunctiva and in the chamber angle area may indicate a role in the regulation of ciliary muscle tone, epithelial cell regeneration and aqueous humour outflow.

Autoradiographic localization of calcitonin gene-related peptide receptors in guinea pig respiratory tract: effect of capsaicin pretreatment.

Calcitonin gene-related peptide (CGRP) is a potent vasodilator peptide present in capsaicin-sensitive neurons innervating the respiratory tract. In this study, the autoradiographic distribution of [125I]CGRP binding sites was investigated in guinea pig airways. Extremely dense specific binding occurred over parenchymal tissue, with moderate specific binding over tracheal glands, the endothelium of pulmonary veins and arteries, and small blood vessels in the bronchial wall. The localization of binding sites for [125I]CGRP over blood vessels but not bronchial smooth muscle correlates well with the physiological actions of this peptide, although the function of the parenchymal sites is unknown. No significant difference in binding was seen in vehicle- or capsaicin-pretreated animals, suggesting that sites are not reliant on factors from capsaicin-sensitive neurons.

Differential effects of CGRP on adenylyl cyclase in adult and embryonic rat brain.

The presence of functional receptors for calcitonin gene-related peptide (CGRP) in the brain of adult rats and on nerve cell cultures was investigated. Neuronal and glial cultures were obtained from mesencephalons of embryos at gestational day 16. The response to CGRP was tested by measuring the adenylyl cyclase (AC) activity on isolated membranes. CGRP binding in adult rat brains was ineffective in activating AC, whereas a dose-dependent stimulation of AC activity was induced by the peptide both in neuronal and glial cultures. This effect was more pronounced in the glial cells where high affinity binding sites for CGRP were detected. The presence of functional CGRP receptors in embryonic mesencephalic cells, suggests a role for CGRP in the development of rat mesencephalon.

Calcitonin Gene-Related Peptide Receptors in the Gubernaculum of Normal Rat and 2 Models of Cryptorchidism

We investigated calcitonin gene-related peptide binding in the gubernaculum from a normal rat, a TS rat (congenitally cryptorchid from unknown causes) and a flutamide rat (cryptorchid secondary to prenatal antiandrogen treatment). Gubernacular sections from 2-day-old male pups were incubated with 125iodine-calcitonin gene-related peptide with various concentrations of unlabeled calcitonin gene-related peptide. After exposure to x-ray film, developed film was quantified by computerized densitometry. The binding analysis showed that concentrations of calcitonin gene-related peptide receptors were 22.9 ± 2.12, 13.5 ± 1.70 and 30.3 ± 2.63 (mean plus or minus standard error of mean, fmol./mg. polymer) for each rat, respectively, and there were significant differences between the normal and TS rats (p <0.01), and the normal and flutamide rats (p <0.05). However, there was no significant difference in the dissociation constant among the 3 models. This result shows that deficiency and excess of calcitonin gene- related peptide binding in the gubernaculum are associated with cryptorchidism. Calcitonin gene-related peptide released from the genitofemoral nerve may regulate its own receptor concentration to control gubernacular motility in vivo.