Rat Calcitonin Gene-related Peptide Research Articles

Adrenomedullin, a novel vasoactive hormone, binds to mouse astrocytes and stimulates cyclic AMP production.

We have examined the effects of adrenomedullin (AM), a novel hypotensive peptide first isolated from human pheochromocytoma, on receptor binding and cyclic AMP (cAMP) generation in primary cultures of mouse astrocytes. Competition binding studies showed that rat adrenomedullin (rAM) displaced the specific binding of [125I]rAM in a dose-dependent manner, with an estimated IC50 of 33 nM. Rat calcitonin gene-related peptide (rCGRP), which interacts with AM receptors in some vascular tissues, did not produce significant displacement of [125I]rAM at concentrations up to 3.3 microM. rAM stimulated cAMP production in mouse astrocytes in a dose-dependent manner, with an EC50 of 74 nM and a maximal stimulatory concentration of 1 microM. CGRP8-37, a CGRP receptor antagonist, failed to inhibit the cAMP response to rAM, although it attenuated CGRP-stimulated cAMP production. These data indicate that cultured mouse astrocytes possess specific AM receptors which are coupled to adenylate cyclase but do not interact with CGRP. AM may function as a neuropeptide and may play a role in the central regulation of blood pressure and body fluid balance.

Comparison of the in vitro and in vivo pharmacology of adrenomedullin, calcitonin gene-related peptide and amylin in rats

Adrenomedullin has been reported to be structurally similar to a group of peptides that includes amylin, calcitonin and calcitonin gene-related peptide (CGRP). Human and rat adrenomedullin displaced [ 125I]CGRP from membranes of SK-N-MC cells (CGRP receptors) with affinities intermediate between those of rat amylin and rat CGRPα ( K i values 0.12 ± 0.06, 0.017 ± 0.007, 3.83 ± 1.14 and 0.007 ± 0.001 nM, respectively). In contrast, K i values for displacement of [ 125I]rat amylin from nucleus accumbens membranes (amylin receptors), and [ 125I]salmon calcitonin from T47D cells (calcitonin receptors) were lower than with rat amylin or rat CGRPα in these preparations (51 ± 5, 34 ± 2, 0.024 ± 0.002, 0.31 ± 0.07 nM, respectively, at amylin receptors; 33 ± 5, 69 ± 29, 2.7 ± 1.5 and 13 ± 3 nM, respectively, at calcitonin receptors). In anesthetized rats, the hypotensive potency of adrenomedullin was between that of amylin and CGRPα. In contrast, for amylin or calcitonin agonist actions (inhibition of [ 14C]glycogen formation in soleus muscle, hyperlactemia, hypocalcemia and inhibition of gastric emptying), human adrenomedullin was without measurable effect. Thus, in its binding behaviour and in its biological actions, adrenomedullin appeared to behave as a potent CGRP agonist, but as a poor amylin or calcitonin agonist.

Peptidergic Modulation of Synaptic Transmission in the Parabrachial NucleusIn Vitro: Importance of Degradative Enzymes in Regulating Synaptic Efficacy

This study examined the effects of substance P (SP) and calcitonin gene-related peptide (CGRP) on synaptic transmission in a pontine slice containing the parabrachial nucleus (PBN). Stimulation of the ventral, external lateral portion of the PBN elicited glutamate-mediated EPSCs in cells recorded using the nystatin perforated-patch recording technique in the external lateral, external medial, and central lateral subnuclei of the PBN. Bath application of SP or CGRP dose-dependently and reversibly attenuated the evoked EPSC. The attenuation of the EPSC induced by both of these peptides was not accompanied by changes in input resistance of PBN cells over a wide voltage range, nor did these peptides alter the inward current induced by a brief bath application of AMPA. The combined application of subthreshold concentrations of these peptides revealed a synergistic interaction in reducing the evoked EPSC. The substance P neurokinin-1 receptor antagonist CGP49823 completely and reversibly blocked both the SP- and the CGRP-induced attenuation of the EPSC. However, the rat CGRP receptor antagonist human-CGRP8-37 did not block the actions of CGRP or SP on the EPSC. Using a metabolically stable analog of SP, SP (5-11), or an endopeptidase inhibitor, phosphoramidon, we were able to demonstrate that CGRP enhances the SP effect by inhibiting an SP endopeptidase. Application of phosphoramidon also revealed an endogenous SP "tone" apparently made effective by blockade of the endopeptidase. These results suggest that SP (and CGRP indirectly through an inhibition of the SP endopeptidase) acts on presynaptic NK-1 receptors to cause an inhibition of excitatory transmission in the PBN. These results indicate an important role of endopeptidases in regulating synaptic modulation by peptides.

Rapid nitric oxide- and prostaglandin-dependent release of calcitonin gene-related peptide (CGRP) triggered by endotoxin in rat mesenteric arterial bed.

1. Our objective was to determine whether endotoxin (ETX) could directly trigger the release of calcitonin gene-related peptide (CGRP) from perivascular sensory nerves in the isolated mesenteric arterial bed (MAB) of the rat and to determine whether nitric oxide (NO) and prostaglandins (PGs) are involved. 2. ETX caused time- and concentration-dependent release of CGRP, and as much as a 17 fold increase in CGRP levels in the perfusate at 10-15 min after the administration of ETX (50 micrograms ml-1). 3. CGRP-like immunoreactivity in the perfusate was shown to co-elute with synthetic rat CGRP by reverse-phase h.p.l.c. 4. Pretreatment of MAB with capsaicin or ruthenium red inhibited ETX-induced CGRP release by 90% and 71%, respectively. ETX-evoked CGRP release was decreased by 84% during Ca2(+)-free perfusion. 5. The release of CGRP evoked by ETX was enhanced by L-arginine by 43% and inhibited by N omega-nitro-L-arginine (L-NOARG) and methylene blue by 37% and 38%, respectively. L-Arginine reversed the effect of L-NOARG. 6. Indomethacin and ibuprofen also inhibited the ETX-induced CGRP release by 34% and 44%, respectively. No additive inhibition could be found when L-NOARG and indomethacin were concomitantly incubated. 7. The data suggest that ETX triggers the release of CGRP from capsaicin-sensitive sensory nerves innervating blood vessels. The ETX-induced CGRP release is dependent on extracellular Ca2+ influx and involves a ruthenium red-sensitive mechanism. Both NO and PGs appear to be involved in the ETX-induced release of CGRP in the rat mesenteric arterial bed.

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.

Receptor subtypes mediating renal actions of calcitonin gene-related peptide

We previously reported that the renal arterial infusions of non-hypotensive doses of calcitonin gene-related peptide (CGRP) caused renal vasodilatation and increases in glomerular filtration rate at a low dose, but renal vasoconstriction, natriuresis and kaliuresis at a high dose. In the present study, we examined the effects of the specific CGRP 1 receptor antagonist CGRP-(8–37) (1 and 10 nmol/kg) and the putative CGRP receptor antagonist, [Tyr 0]CGRP-(28–37) (3 and 30 nmol/kg), on the renal vascular and tubular effects of CGRP in inactin-anaesthetized Sprague-Dawley rats. Renal arterial infusion of single doses of CGRP (0.3–300 pmol/kg per min) did not significantly alter mean arterial pressure or heart rate. However, during the continuous renal arterial infusion of either CGRP-(8–37) or [Tyr 0]CGRP-(28–37), a high dose of CGRP (300 pmol/kg per min) paradoxically reduced arterial pressure and increased heart rate. CGRP-(8–37) completely but [Tyr 0]CGRP-(28–37) incompletely inhibited the vasodilatation and increments in glomerular filtration rate elicited by low doses of CGRP. Both blockers abolished the renal vasoconstriction but did not inhibit diuresis, natriuresis and kaliuresis elicited by a high but non-hypotensive dose of CGRP. On the basis that CGRP-(8–37) is a competitive CGRP 1 receptor antagonist, our results suggest: (1) the renal vascular effect of CGRP is completely mediated via the activation of CGRP 1 receptors, (2) the renal tubular effects of CGRP are not mediated via CGRP 1 receptors, and (3) [Tyr 0]CGRP-(28–37) is a CGRP 1 receptor antagonist with potency and efficacy less than those of CGRP-(8–37).

Basal tail skin temperature elevation and augmented response to calcitonin gene-related peptide in ovariectomized rats

Hyper-release of calcitonin gene-related peptide (CGRP) plays a direct and pivotal role in the induction of menopausal hot flushes (HFs), in which a drastic increase in skin temperature occurs. However, it is not possible to investigate whether CGRP induces skin temperature increase and whether skin temperature response to CGRP changes and contributes to the occurrence of HFs in postmenopausal women who are in oestrogen deficiency. By using rats' tail skin temperature (TST), a good marker to evaluate skin temperature regulation, we examined the effects of CGRP and calcitonin (3, 10 and 30 micrograms/kg, i.v.) on TST in female rats and further investigated the TST change induced by CGRP (10 micrograms/kg, i.v.) in ovariectomized (OVX) rats compared with that in sham-operated (Sham) rats. We found that CGRP, but not calcitonin, induced a TST increase in a dose-dependent manner and that the TST change induced by CGRP (0.6 +/- 0.2 degrees C for OVX rats vs 0.3 +/- 0.1 degree C for Sham rats, P < 0.05) and also the basal TST (26.0 +/- 0.2 degrees C for OVX rats vs 25.5 +/- 0.1 degree C for Sham rats) were significantly greater in OVX rats (P < 0.05). Furthermore, treatment with oestradiol (30 micrograms/kg, s.c.) for 8 days partially inhibited the augmented TST response to CGRP in OVX rats and almost completely inhibited (P < 0.05) the basal TST elevation, with the concomitant recovery of the serum oestradiol level to that in Sham rats. These results suggest that the augmented skin temperature response to CGRP and the elevation of basal skin temperature that are found in OVX rats, animals which are oestradiol deficient, may also occur in menopausal women and contribute to their HFs.

Receptors for calcitonin gene related peptide (CGRP) in gastrointestinal epithelia

A pharmacological comparison of calcitonin gene related peptide (CGRP) receptors expressed in normal rat colon mucosa and two human adenocarcinoma cell lines has been undertaken. Using voltage-clamp techniques electrogenic ion transport was continuously monitored across either mucosal preparations or confluent epithelial monolayers grown on permeable supports. The data presented at this meeting show that CGRP receptors are preferentially located on the basolateral epithelial surface and that their stimulation by a variety of CGRP analogues results in enhanced CI- secretion mediated via a cyclic AMP dependent mechanism. Responses to rat alpha CGRP in rat descending colon mucosa and in the adenocarcinoma cell line Colony-29 are insensitive to the inhibitory effects of the C-terminal fragment human CGRP(8-37); however, significant inhibition of rat alpha CGRP responses was observed in the parent epithelial cell line HCA-7. This together with the subtle differences seen in agonist orders of potency in the three preparations indicates that different CGRP receptor subtypes exist in the basolateral domains of HCA-7 compared with rat colon and Colony-29 epithelia.

In vivo central actions of rat amylin

The purpose of the present study was to examine and compare the profile of neurobehavioral effects of rat amylin (r-amylin) and rat calcitonin gene-related peptide (rCGRP), two peptides having a 50% structural homology. The effects of synthetic r-amylin and rCGRP administered in several doses (0.312–80.0 μg) into the lateral cerebro-ventricle of rats on spontaneous activity, muscular tone, body temperature, nociception, food intake as well as their potential for inducing catalepsy, were investigated. Intraventricular administration of r-amylin or rCGRP significantly reduced spontaneous motor activity and markedly increased body temperature of animals in a dose-dependent related fashion. rCGRP produced a significant increase in muscular tone and induced cataleptic effect in animals, but r-amylin had no effect on these variables. Furthermore, neither r-amylin nor rCGRP were able to induce any significant effect on nociceptive response time of animals in the tail immersion test even with doses as large as 80.0 μg. Finally, the two peptides did not affect ad libitum food intake, but significantly reduced food consumption in 22 h food-deprived animals. Together, the results of the present study suggest that amylin may be involved in a diversity of neurophysiological processes but displays a different profile of neurobehavioral effects to that of CGRP which may involve different receptors.

Pulmonary neuroendocrine cells in neonatal rats with congenital diaphragmatic hernia

Lung hypoplasia and persistent pulmonary hypertension are the principal causes of high mortality and morbidity in infants with congenital diaphragmatic hernia (CDH). Amine-and peptide-producing pulmonary neuroendocrine cells (PNEC), widely distributed throughout the airway mucosa, are thought to play an important role in both pulmonary development and regulation of pulmonary vascular tone. Furthermore, recent studies show increased levels of calcitonin gene-related peptide (CGRP), a pulmonary vasodilator produced by PNEC, during chronic hypoxia. The article reports data on morphometric analysis of CGRP immunoreactive PNEC clusters (neuroepithelial bodies, NEB) in a rat model of CDH. CDH was induced in neonatal Sprague Dawley rats by oral administration of 2,4-dichloro-phenyl-p-nitrophenylether (Nitrofen; Rohm Haas, Philadelphia, PA) to the mother at 10 days of gestation. Sections of lungs from term neonatal rats with and without CDH and controls were immunostained for CGRP (marker of NEB) with specific antibody against rat CGRP. NEB size and number of NEB/area of lung were assessed using a semiautomatic image analysis system. In lungs of neonatal rats with CDH, the number of NEB per surface area of lung parenchyma was significantly increased compared with the age-matched controls. Although the mean size of NEB was larger in CDH, the differences were not significant. This is the first study of PNEC in CDH. Whether the phenomenon observed in this study results in altered NEB function including imbalance in vasoactive mediators requires further studies, especially in the human being.

Role of endogenous CRF in the mediation of neuroendocrine and behavioral responses to calcitonin gene-related peptide in rats.

In the present study, the possible role of cortocotropin-releasing hormone (CRF) in the action of calcitonin gene-related peptide (CGRP) on the pituitary-adrenal axis and open-field activity of rats was tested. CGRP administered into the lateral brain ventricle led to a dose-dependent increase in plasma corticosterone level, which could be blocked by pretreatment with CRF antiserum. CGRP injected into the lateral brain ventricle increased the grooming and rearing activity and decreased the locomotor activity in an open field. On pretreatment with CRF antiserum, the action of CGRP on grooming was blocked, while the action on locomotion and rearing activity was unchanged. The results suggest that the CGRP-induced action on pituitary-adrenal activation is mediated by CRF. The action of CGRP on grooming in an open field is related to the pituitary-adrenal activation, and either CRF or adrenocorticotropic hormone or both may be involved in mediating the action of CGRP. The action of CGRP on rearing and locomotion is not affected by CRF antiserum, indicating that the two behavioral actions are regulated by different mechanisms.

Effects of calcitonin gene-related peptide on intracellular calcium concentration in longitudinal muscle of guinea pig ileum

The effect of rat calcitonin gene-related peptide (rCGRP) on intracellular free calcium concentration ([Ca 2+] i) and its relationship with muscle relaxation were examined in plexus-free longitudinal muscle (LM) of the guinea pig ileum using a [Ca 2+] i-tension simultaneous recording technique. Tissue was stimulated with either histamine (0.5 μM) or KCl (30 m M). rCGRP at a concentration of 263 n M (which displayed maximal relaxation of the LM) caused a small and brief but significant decrease in the [Ca 2+] i in histamine-treated tissue. On the other hand, the same concentration of rCGRP relaxed the muscle without affecting the [Ca 2+] i in KCl-treated tissue. rCGRP caused a dissociation between the changes in [Ca 2+] i and tension. The effects of forskolin (0.5 μ M) and nifedipine (10 n M) on both [Ca 2−] i and tension were examined in comparison with rCGRP. Forskolin lowered the tension to greater extent than the [Ca 2+] i whereas nifedipine (10 n M) diminished both the [Ca 2+] i and tension in a parallel manner in histamine-treated tissues. These results suggest that rCGRP may not reduce [Ca 2+] i as its primary mechanism of relaxation and it may change the calcium sensitivity of the contractile elements of the smooth muscle.

Effects of calcitonin gene-related peptide on cyclic AMP production and relaxation of longitudinal muscle of guinea pig ileum

The effects of rat calcitonin gene-related peptide (rCGRP) on the content of cyclic nucleotides in the cells of plexus-free longitudinal muscle (LM) of guinea pig ileum relative to smooth muscle relaxation were investigated. In the absence of isobutyl-methyl-xanthine (IBMX), a nonspecific inhibitor of phosphodiesterases, rCGRP significantly increased cyclic 3′5′-adenosine monophosphate (cAMP) content that correlated temporally to the relaxation of the muscle. Cyclic 3′5′-guanosine monophosphate (cGMP) content was not affected by this peptide. IBMX potentiated both the increase in cAMP content and the muscle relaxation induced by rCGRP. rCGRP increased cAMP content in a concentration-dependent manner, with an ED 50 value of 7.5 × 10 −9 M. Both the rCGRP-stimulated increase in cAMP content and the rCGRP-induced muscle relaxation were blocked by hCGRP(8–37), a selective antagonist of CGRP receptors. The pA 2 value of hCGRP(8–37) for rCGRP-induced inhibition of tension was calculated to be 6.48. These results suggest that CGRP relaxes the LM through its specific receptors and involves the generation of cAMP, but not the generation of cGMP.

Molecular characterization of calcitonin gene-related peptide (CGRP) in a rat medullary thyroid carcinoma cell line.

The rat medullary thyroid carcinoma cell line, CA-77, is known to express the calcitonin gene and the cell line has been used for characterization of procalcitonin. The present investigations concentrate on a molecular characterization of the calcitonin gene-related peptide (CGRP) expressed by a subclone of this cell line. The investigations demonstrate that this subclone produces significantly more CGRP compared to calcitonin. Gel chromatography of cell extracts demonstrates heterogeneity for both CGRP and calcitonin, but a significant amount of immunoreactivity elutes corresponding to the elution position for synthetic CGRP and calcitonin, respectively. The gel chromatogram for CGRP demonstrates four immunoreactive peaks with Kd of 0.42, 0.53, 0.68, and 0.85. The immunoreactive peak with Kd 0.42 elutes corresponding to synthetic rat CGRP. The four immunoreactive peaks were characterized by high pressure liquid chromatography followed by sequence analysis and mass spectrometry. The immunoreactive peak with Kd 0.42 was identified as rat alpha-CGRP as was the peak with Kd 0.53. The peak with Kd 0.68 was identified as 19-37 rat alpha-CGRP and the peak with Kd 0.85 as 28-37 rat alpha-CGRP. In summary, we find that the CA-77 cell line expresses large quantities of normally processed amidated alpha-CGRP and specific fragments thereof. However, the cell line does not express detectable levels of rat beta-CGRP. The findings indicate that the CA-77 cell line can be useful for studies of calcitonin/CGRP gene expression.

Calcitonin Gene-Related Peptide Relaxes Cholecystokinin-Induced Tension in Iguana iguana Gallbladder Strips

The presence of a calcitonin gene-related peptide (CGRP)-like material was demonstrated in the iguana, Iguana iguana, gallbladder using immunocytochemistry. An intense reaction was observed in nerves located in the smooth muscle layers and associated blood vessels but no immunoreactive cell bodies were found. In vitro tension studies using gallbladder strips showed that chicken CGRP was more potent in relaxing cholecystokinin-induced tension than either human or rat CGRP. The use of glibenclamide and l - N G-nitro-arginine methyl ester suggested that this relaxation is mediated by either nitric oxide release from nerves stimulated by CGRP or by CGRP acting directly on the gallbladder smooth muscle.

283 PULMONARY NEUROENDOCRINE CELLS (PNEC) IN NEONATAL RATS WITH CONGENITAL DIAPHRAGMATIC HERNIA (CDH)

Lung hypoplasia and persistent pulmonary hypertension are the principal causes of high mortality and morbidity in infants with CDH. Amine and peptide producing PNEC, widely distributed throughout airway mucosa, are thought to play an important role in both pulmonary development and in regulation of pulmonary vascular tone. Furthermore, recent studies show modulation during chronic hypoxia of calcitonin gene-related peptide (CGRP), a pulmonary vasodilator produced by PNEC (Anat Rec 1993,236:96). We report data on morphometric analysis of CGRP immunoreactive PNEC clusters (neuroepithelial bodies, NEB) in a neonatal rat model of CDH induced by oral administration of Nitrofen to pregnant rats at 10 days of gestation (J Ped Surg 1990,25:850). Sections of lungs from term neonatal rats with CDH and controls were immunostained for CGRP (marker of NEB) with specific antibody against rat CGRP. NEB size and number of NEB/area of lung were assessed using semiautomatic image analysis system.Our findings show that in lungs of neonatal rats with CDH, there is an increase in relative freguency of NEB per surface area of lung parenchyma, while the size of NEB is not significantly affected. Whether this results in altered NEB function including imbalance in vasoactive Mediators requires further studies.(Supported by NAF 91.56).

Characterization of CGRP receptors in various regions of gerbil brain

The distribution of specific calcitonin gene-related peptide (CGRP) binding sites was investigated in gerbil brain regions such as cortex, cerebellum, hypothalamus, and hippocampus. The binding of [ 125I] hCGRP to membranes prepared from these regions of the gerbil brain was rapid, saturable and specific with dissociation constants (Kd) between 10–40 pM and maximum binding (B max) between 10–30 fmol/mg protein. Human and rat CGRP and hCGRP 8–37 competed for [ 125I]hCGRP binding in a concentration-dependent manner with a Ki of 10–100 pM. Salmon calcitonin was very weak in competing for [ 125I]hCGRP binding (ki > 10μM). CGRP did not stimulate adenylate cyclase in these brain regions. Cross-linking of [ 125I] hCGRP to the brain membrane fractions with disuccinimidyl suberate revealed specific incorporation of [ 125I] hCGRP to a protein band of approximate molecular weight 52 kDa.

The role of activation of the sympathetic nervous system in the central pressor action of calcitonin gene-related peptide in conscious rats.

The effects of intracerebroventricular (i.c.v.) administration of calcitonin gene-related (CGRP) on blood pressure and heart rate (HR), and the underlying mechanisms were studied in conscious rats. CGRP (0.1-3.0 nmol i.c.v.) increased mean arterial blood pressure (MABP) and HR. CGRP (3.0 nmol i.c.v.) also significantly increased both plasma norepinephrine and epinephrine concentrations. Pretreatment with 16.5 nmol i.c.v. CGRP(8-37), a specific CGRP receptor antagonist, significantly inhibited the i.c.v. CGRP (1.0 nmol)-induced increases in MABP and HR. Phenoxybenzamine inhibited the i.c.v. CGRP-induced increase in MABP, while propranolol suppressed the tachycardiac response to i.c.v. CGRP. Chemical sympathectomy by 6-hydroxydopamine inhibited the increases in MABP and HR produced by i.c.v. CGRP. These results suggest that the central pressor and tachycardiac effects of i.c.v. CGRP are mediated by catecholamine release due to stimulation of sympathetic nervous system activity, possibly via specific CGRP receptors in the central nervous system.

Changes of spinal substance P, calcitonin gene-related peptide, somatostatin, Met-enkephalin and neurotensin in rats in response to formalin-induced pain

Changes of substance P (SP)-, calcitonin gene-related peptide (CGRP)-, somatostatin (SS)-, Met-enkephalin (Met-Enk)- and neurotensin (NT)- immunoreactive materials on two sides of the lumbar dorsal horn were inspected microscopically and quantified with a computer-assisted image processing system in rats with intact or totally transected spinal cord 2 h after injection of 0.2 ml of 0.5% formalin into the right hindpaw subcutaneously. The results showed that the SP-like immunoreactivity (SP-LI), CGRP-LI, SS-LI, Met-Enk-LI, and NT-LI were significantly higher in fibers and terminals in superficial laminae of the dorsal horn ipsilateral to the formalin injection in both of the experimental groups. It is supposed that the increased contents of these peptides reflect an increased biosynthesis, transport, and release of these peptides in primary afferents and spinal intrinsic neurons in response to the long-lasting inflow of noxious messages, and that these changes seem to be produced even in the condition when the supraspinal effects have been excluded.

Specific receptors for adrenomedullin in cultured rat vascular smooth muscle cells

The effects of synthetic rat adrenomedullin (rAM), a novel vasorelaxant peptide originally isolated from human pheochromocytoma, on receptor binding and cAMP generation were studied in cultured rat vascular smooth muscle cells (VSMC). A binding study using [ 125I]rAM revealed the presence of a single class of high-affinity ( K d1.3 × 10 −8 M) binding sites for rAM in VSMC. The apparent K i of rat calcitonin gene-related peptide (rCGRP) was 3 × 10 −7 M. Affinity labeling of VSMC membranes with [ 125I]rAM revealed two distinct labeled bands with apparent molecular weights of 120 and 70 kDa, both of which were abolished by excess unlabeled rAM or rCGRP. rAM stimulated cAMP formation with an approximate EC 50 of 10 −8 M, the effect of which was additive with isoproterenol, but not with rCGRP. The rAM-induced cAMP response was unaffected by propranalol, indomethacin, or quinaerine, but inhibited by a CGRP receptor antagonist, human CGRP[8–37]. These data suggest that VSMC possesses specific AM receptors functionally coupled to adenylate cyclase with which CGRP interacts.