Inhibitory effect of calcitonin gene-related peptide on myometrial contractility is diminished at parturition

The uterus is innervated by calcitonin gene-related peptide (CGRP) immunoreactive neurons, and CGRP inhibits spontaneous and evoked contractions in the uterus and fallopian tubes. In the present study using isometric force measurements on myometrial strips, we determined that CGRP inhibition of acetylcholine-induced contractions was drastically reduced at parturition compared with earlier stages of pregnancy in mice. The levels of inhibition exerted by CGRP paralleled the expression of a novel protein recently implicated in CGRP receptor activation, the CGRP-receptor component protein (CGRP-RCP). The mouse CGRP-RCP complementary DNA was isolated from uterus, and expression of the CGRP-RCP was monitored during gestation by Northern and Western blot analysis. Although CGRP-RCP messenger RNA levels did not vary significantly during gestation and postpartum, CGRP-RCP protein was greatly diminished at parturition. This diminution correlated with the loss of CGRP inhibition of acetylcholine-induced contractions observed in the force experiments. A role for CGRP and CGRP-RCP in modulation of myometrial smooth muscle contractility during pregnancy and in labor is suggested.     

Suppression of nitric oxide generated by inflammatory macrophages by calcitonin gene-related peptide in aqueous humor

PURPOSE: Ocular immune privilege is mediated in part by the activity of constitutively produced immunosuppressive cytokines and neuropeptides. Aqueous humor was examined for content of calcitonin gene-related peptide (CGRP), and the potential of CGRP to mediate immunosuppressive activity within aqueous humor was determined. METHODS: The concentration of CGRP in fresh, normal rabbit aqueous humor was assayed by competitive enzyme-linked immunosorbent assay. The ability of CGRP to suppress interferon (IFN)-gamma production by antigen-stimulated, primed lymph node cells was examined by assaying supernatants of stimulated CGRP-treated, primed T-cell cultures for IFN-gamma. The anti-inflammatory activity of aqueous humor and CGRP was assayed by treating IFN-gamma-lipopolysaccharide (LPS)-activated RAW 264.7 cells (macrophages) with aqueous humor, aqueous humor plus anti-CGRP antibody, or CGRP alone. Culture supernatants of the treated macrophages were examined for nitrite by Griess reagent. The production of inducible nitric oxide synthase (NOS2) protein was examined by immunoblotting cell lysates of treated activated macrophages. RESULTS: The constitutive level of CGRP in fresh, normal rabbit aqueous humor was 5+/-1 x 10(-5) M. At its ocular concentration, CGRP did not inhibit IFN-gamma production by stimulated effector T cells, but it suppressed nitric oxide generation by activated macrophages. Neutralization of CGRP in normal rabbit aqueous humor prevented the aqueous humor from suppressing nitric oxide generation by macrophages. Neither CGRP nor aqueous humor suppressed NOS2 protein synthesis in activated inflammatory macrophages. CONCLUSIONS: Calcitonin gene-related peptide is a constitutive neuropeptide in aqueous humor. Through CGRP, aqueous humor suppresses nitric oxide production by activated macrophages. This suppression appears to result from inhibiting NOS2 enzymatic activity, rather than from suppressing NOS2 synthesis. The results imply that the ocular microenvironment has diverse immunoregulatory mechanisms that suppress induction, activation, and mediation of immunogenic inflammation.     

Calcitonin gene-related peptide promotes differentiation, but not survival, of rat mesencephalic dopaminergic neurons in vitro

The aim of this study was to investigate putative effects of calcitonin gene-related peptide on developing dopaminergic neurons in the ventral mesencephalon. To determine a time-point for a physiological role of calcitonin gene-related peptide in the development of this system, we first investigated calcitonin gene-related peptide messenger RNA expression in the ventral mesencephalon of Wistar rats at embryonic days (E) 11-19. Calcitonin gene-related peptide messenger RNA was not detectable at E11, i.e. prior to the appearance of dopaminergic neurons in this area. From E14 to E19, calcitonin gene-related peptide messenger RNA was expressed in increasing amounts. We therefore investigated the effects of calcitonin gene-related peptide on serum-free cell cultures established from the E14 midbrain floor. Addition of calcitonin gene-related peptide (200 ng/ml) every other day significantly increased neuronal differentiation, including longer tyrosine hydroxylase-positive neurites, enhanced immunoreactivity for growth-associated protein-43 and increased dopaminergic uptake per neuron. These effects were maximal after seven to eight days. Calcitonin gene-related peptide acted synergistically with fibroblast growth factor-2 on these parameters. In contrast to fibroblast growth factor-2, however, calcitonin gene-related peptide did not promote survival of tyrosine hydroxylase-immunoreactive neurons. Lack of calcitonin gene-related peptide expression in the mesencephalon at E11 was paralleled by a lack of effect of calcitonin gene-related peptide on early presumptive dopaminergic neurons in terms of eliciting this phenotype. Our data suggest that calcitonin gene-related peptide may act physiologically as a differentiation-promoting factor for phenotypically defined dopaminergic neurons during a time period when dopaminergic neurons assemble in the ventral mesencephalon and grow axons towards their targets.     

Immunocytochemical co-localization of substance P and calcitonin gene-related peptide in afferent renal nerve soma of the rat

Substance P, calcitonin gene-related peptide and somatostatin immunoreactivities have been demonstrated in putative afferent renal nerve fibers in the rat. Utilizing retrograde-tracing and immunohistochemistry, we labeled afferent renal nerve soma throughout dorsal root ganglia T9 to L1. Most (85%) of afferent renal nerve perikarya were immunoreactive for calcitonin gene-related peptide, 21% had substance P immunoreactivity and none had somatostatin immunoreactivity. All renal afferents immunoreactive for substance P also contained calcitonin gene-related peptide. These results provide evidence that calcitonin gene-related peptide and substance P are present and co-localized in afferent renal nerves, and therefore, mediate transmission of afferent renal input to the spinal cord in the rat.     

Calcitonin gene-related peptide reduces brain injury in a rat model of focal cerebral ischemia

BACKGROUND AND PURPOSE: Calcitonin gene-related peptide is an endogenous vasodilating neuropeptide with a dense concentration in the trigeminocerebrovascular system. It is hypothesized that depletion of this peptide contributes to delayed cerebral ischemia after subarachnoid hemorrhage and that an exogenous supply of calcitonin gene-related peptide will augment ischemic cerebral blood flow and reduce neuronal injury. METHODS: In this study we have investigated the effect of an intravenous infusion of calcitonin gene-related peptide (100 ng/kg per minute), started 1 hour before and continued throughout 4 hours of focal cerebral ischemia, on cerebral blood flow and the volume of brain injury in a rat model of middle cerebral artery occlusion. RESULTS: Calcitonin gene-related peptide produces a significant improvement in ischemic cerebral blood flow (32 +/- 2 compared with 13 +/- 2 mL/100 g per minute in the controls; t = 6.92, P < .0001) with a concomitant reduction in the volume of ischemic brain injury (102 +/- 22 compared with 234 +/- 19 mm3; t = 4.47, P < .001). CONCLUSIONS: These findings lend support for the potential use of this peptide in the prophylactic treatment of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage.     

Cardiac expression of genes encoding putative adrenomedullin/calcitonin gene-related peptide receptors

Recent studies have suggested that adrenomedullin (AM) may play a role in the pathophysiology of heart disease, though the specific cardiac receptors involved have not been defined. RT-PCR cloned fragments of three putative AM/calcitonin gene-related peptide (CGRP) receptors were used to established a quantitative RNase protection assay to identify and quantitate expression of receptor mRNAs in heart and in cardiac myocytes. Intact rat heart expressed mRNA encoding the putative AM/CGRP receptors RDC1 and CRLR at 37- and 15-fold higher levels, respectively, than the AM-selective receptor L1, with a qualitatively similar profile in cultured neonatal cardiac myocytes. The high level of expression of RDC1 and CRLR suggests that both AM and CGRP may have direct actions on the cardiac myocyte via common receptors that can interact with either ligand.     

Mutated connexin43 proteins inhibit rat glioma cell growth suppression mediated by wild-type connexin43 in a dominant-negative manner

To explore the role of calcitonin gene-related peptide (CGRP) in rat pregnancy, we determined the density of myometrial CGRP-encoded nerve fibre terminals and examined, in an organ bath, the relaxant effect of the peptide on uterine strips near parturition. Comparisons were made with the uterus and aorta of nonpregnant rats. In the myometrium, CGRP immunoreactive nerve fibers were abundant in nonpregnant rats and scarce at the parturient stage. In the aorta there was no variation in the density of CGRP fibres with gestation. In nonpregnant rats only, CGRP relaxed spontaneous and tetrodotoxin (TTX)-sensitive electrically-evoked uterine contractions (EC50 40 nM, Emax 80%). The effect was antagonized by CGRP[8-37] (pKB 6.47) but was not affected by either blockers of nitricoxid synthase or ATP-sensitive potassium channels. CGRP was also able to relax contractions evoked by direct depolarization of the cells (TTX-insensitive contractions) (EC50, 2 nM, Emax 70%). In aorta contracted with arginine vasopressin, CGRP-induced relaxation was the same in nonpregnant and parturient animals. It was antagonized by CGRP [8-371 (pKB 6.90) and was abolished in presence of the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME). Amylin neither relaxed the uterus nor the aorta. In pregnant rats, the relaxant effect of CGRP on the uterus was limited on day 21 and was totally absent on day 22 of gestation. We conclude that the primary relaxant effect of CGRP on the uterus occurs at the level of myometrial smooth muscle cells. In the myometrium, gestation decreases CGRP innervation and impairs the relaxant responses to CGRP. Such changes are not observed in vascular tissues like aorta.     

Attenuation of calcitonin gene expression in pregnant rat uterus leads to a block in embryonic implantation

The peptide hormone calcitonin plays a key role in calcium homeostasis in many tissues, such as bone and kidney. Our previous studies revealed that the expression of calcitonin is dramatically induced in the glandular epithelium of rat uterus between days 3-5 of pregnancy before the onset of blastocyst implantation on day 5. Calcitonin expression is switched off once implantation has progressed to day 6. The coincidence in timing suggested that calcitonin may function as a regulatory signal in the uterus during the early events leading to implantation. Here we report that the implantation stage-specific expression of calcitonin can be specifically attenuated by administering antisense oligodeoxynucleotides (ODNs) directed against exon IV of calcitonin messenger RNA into the uterine horns on day 2 of gestation. The loss of calcitonin messenger RNA and protein expression upon antisense ODN treatment is accompanied by a severe impairment in implantation of embryos. Based on the observations that 1) treatment with two different antisense ODNs possessing different base compositions produced similar phenotypes; and 2) treatment with the complementary sense ODNs did not affect either calcitonin expression or implantation, we conclude that the effects of antisense ODNs on calcitonin expression and implantation are specific and functionally linked. Our study strengthens the hypothesis that a transient expression of calcitonin in the preimplantation phase uterus is critical for blastocyst implantation.     

Vasorelaxation in isolated bone arteries. Vasoactive intestinal peptide, substance P, calcitonin gene-related peptide, and bradykinin studied in pigs

We assessed the effects of vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), substance P (SP), and bradykinin in arteries (diameter approximately 230 microns) isolated from cancellous bone from pigs. Arterial segments (2 mm long) were mounted on a myograph for measurement of isometric force development. After submaximal precontraction with norepinephrine, VIP (10(-10)-10(-7) M), CGRP (10(-11)-10(-7) M), SP (10(-6) M), and bradykinin (10(-11)-10(-6) M) were added. 44 arterial segments (23 pigs) were investigated. VIP-, CGRP-, and bradykinin induced a concentration-dependent vasorelaxation, while SP mediated a transient relaxation. After mechanical removal of the endothelium, the effects of SP and bradykinin were completely abolished, while the relaxation to CGRP was still pronounced. This indicates that the effects of SP and bradykinin are mediated by the endothelium, while CGRP mainly mediates relaxation by a direct effect on vascular smooth muscle cells. The relaxations to CGRP and bradykinin were still significant after inhibition of nitric oxide synthase with 10(-4) M N omega-nitro-L-arginine (L-NNA) and inhibition of prostaglandin synthesis with 10(-5) M indomethacin, indicating the existence of an alternative vasorelaxing pathway. Our findings support the theory of a vasoregulatory role of neuropeptides in bone.     

Effects of adrenomedullin and calcitonin gene-related peptide on contractions of the rat aorta and porcine coronary artery

1. Effects of adrenomedullin and alpha-calcitonin gene-related peptide (CGRP) on the contractions and cytosolic Ca2+ concentrations ([Ca2+]i) of the rat aorta and porcine coronary artery were investigated. Characteristics of the receptors mediating the effects of adrenomedullin and alpha-CGRP were also investigated. 2. Adrenomedullin and alpha-CGRP caused a concentration-dependent relaxation in the rat aorta contracted with noradrenaline. The IC50 values for adrenomedullin and alpha-CGRP were 2.4 nM and 4.0 nM, respectively. The relaxant effects of these peptides were abolished by removal of the endothelium and significantly attenuated by an inhibitor of nitric oxide synthase, NG-monomethyl-L-arginine (L-NMMA, 100 microM), but not by a cyclo-oxygenase inhibitor, indomethacin (10 microM). 3. Adrenomedullin and alpha-CGRP increased the endothelial [Ca2+]i in the rat aorta with endothelium, whereas they did not change [Ca2+]i in the smooth muscle. 4. An antagonist of the CGRP1 receptor, CGRP (8-37), antagonized the relaxant effects of alpha-CGRP and the beta-isoform of CGRP (beta-CGRP) but not those of adrenomedullin in the rat aorta. 5. In the porcine coronary artery contracted with U46619, adrenomedullin and alpha-CGRP caused a concentration-dependent relaxation with an IC50 of 27.6 and 4.1 nM, respectively. Removal of the endothelium altered neither the IC50 values nor the maximal relaxations induced by adrenomedullin or alpha-CGRP. When the artery was contracted with high K+ solution (72.7 mM), these peptides caused a small relaxation. 6. Adrenomedullin and alpha-CGRP increased cyclic AMP content and decreased the smooth muscle [Ca2+]i in the porcine coronary artery. 7. CGRP (8-37) significantly antagonized the relaxant effects of adrenomedullin and alpha-CGRP in the porcine coronary artery. However, it had little effect on the relaxations induced by the beta-isoform of CGRP (beta-CGRP). 8. These results suggest that in the rat aorta, adrenomedullin and alpha-CGRP increase the endothelial [Ca2+]i, activate nitric oxide synthase and release nitric oxide, without a direct inhibitory action on smooth muscle. In the porcine coronary artery, in contrast, adrenomedullin and alpha-CGRP directly act on smooth muscle, increase cyclic AMP content, decrease the smooth muscle [Ca2+]i and inhibit contraction. The rat aortic endothelium seems to express the CGRP receptor which is sensitive to alpha-CGRP, beta-CGRP and CGRP (8-37) and the adrenomedullin specific receptor. The porcine coronary smooth muscle, in contrast, seems to express two types of CGRP receptor; one of which is sensitive to alpha-CGRP, CGRP (8-37) and adrenomedullin and the other is sensitive only to beta-CGRP.     

Dimeric form of calcitonin gene-related peptide in the porcine thyroid gland

In this study we investigated peptides that increase rat platelet cAMP in porcine thyroid gland. Gel filtration of extracts from porcine thyroid gland showed high and low molecular weight activity. Low molecular weight activity contained peptides, including calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI). We isolated a high molecular weight peptide (M. W. 11,000) showing potent activity able to increase rat platelet cAMP in porcine thyroid gland. The peptide's N-terminal sequence was determined to be Ser-X-Asn-Thr-Ala-Thr- by gas phase sequencer analysis, a sequence identical to that of porcine CGRP. The peptide had CGRP immunoreactivity as well as platelet cAMP elevating activity. By gel filtration HPLC, synthetic human CGRP (M. W. 3790) was eluted in a position corresponding to M. W. 5,500. These results suggest that judging from its high molecular weight the above peptide is a dimeric form of CGRP.     

Nitric oxide-dependent and -independent hyperaemia due to calcitonin gene-related peptide in the rat stomach

1. Calcitonin gene-related peptide (CGRP) potently enhances mucosal blood flow in the rat stomach. The aim of this study was to examine whether CGRP also dilates extramural arteries supplying the stomach and whether the vasodilator action of CGRP involves nitric oxide (NO). 2. Rat CGRP-alpha (0.03-1 nmol kg-1, i.v.) produced a dose-dependent increase in blood flow through the left gastric artery (LGA) as determined by an ultrasonic transit time technique in urethane-anaesthetized rats. Blockade of NO synthesis by NG-nitro-L-arginine methyl ester (L-NAME, 20 and 60 mumol kg-1, i.v.) significantly reduced basal blood flow (BF) in the LGA and attenuated the hyperaemic activity of CGRP by a factor of 2.8-4. D-NAME tended to enhance basal BF in the LGA but had no influence on the dilator activity of CGRP. The ability of vasoactive intestinal polypeptide to increase left gastric arterial blood flow remained unaltered by L-NAME. 3. L-NAME (20 and 60 mumol kg-1, i.v.) evoked a prompt and sustained rise of mean arterial blood pressure (MAP) and caused a slight decrease in the hypotensive activity of CGRP. In contrast, D-NAME induced a delayed and moderate increase in MAP and did not influence the hypotensive activity of CGRP. 4. Rat CGRP-alpha dilated the isolated perfused bed of the rat LGA precontracted with methoxamine and was 3 times more potent in this respect than rat CGRP-beta. The dilator action of rat CGRP-alpha in this preparation was not affected by L-NAME or D-NAME (40 microM). 5. L-NAME (60 micromol kg-1, i.v.) reduced gastric mucosal blood flow as assessed by laser Doppler flowmetry and diminished the hyperaemic activity of rat CGRP-alpha in the gastric mucosa by a factor of 4.5, whereas D-NAME was without effect.6. These data show that CGRP is a potent dilator of mucosal and extramural resistance vessels in the rat stomach. Its dilator action involves both NO-dependent and NO-independent mechanisms.     

Adrenomedullin stimulates steroid secretion by the isolated perfused rat adrenal gland in situ: comparison with calcitonin gene-related peptide effects

Adrenomedullin (ADM), a vasodilatatory peptide contained in adrenal medulla, was found to induce a dose-dependent increase in aldosterone (ALDO) and corticosterone (B) release by the in situ perfused rat adrenal gland, along with a rise in the flow rate of the perfusion medium. The minimal effective dose for ALDO response was three and two orders of magnitude less than those able to evoke B and medium flow rate responses. Calcitonin gene-related peptide (CGRP), another vasodilatatory peptide contained in adrenal medulla and showing a slight homology in its amino acid sequence with ADM, elicited similar effects. CGRP (8-37), a specific antagonist of CGRP1 receptors, annulled all the effects of both ADM and CGRP, whereas l-alprenolol, a beta-adrenoceptor antagonist, partially reversed only ALDO response to the peptides. In light of these findings the following conclusions are drawn: i) ADM and CGRP stimulate rat adrenals in vivo to release B by raising blood flow rate; ii) ADM and CGRP enhance ALDO secretion via an indirect mechanism probably requiring the release of catecholamines by medullary chromaffin cells; and iii) the effects of ADM and CGRP on the rat adrenal gland are mediated by a common receptor of the CGRP1 subtype.     

Glyceryl nonivamide: a capsaicin derivative with cardiac calcitonin gene-related peptide releasing, K+ channel opening and vasorelaxant properties

In this study, the aorta vasorelaxant, coronary calcitonin gene-related peptide (CGRP) releasing, and atrial contractility effects of glyceryl nonivamide (GLNVA) were investigated in guinea pigs. In the isolated thoracic aorta, although GLNVA (0.01-50 microM) concentration dependently induced endothelium-independent relaxations and relaxed phenylephrine-(1.0 microM) induced contractions, it failed to relax 80 mM KCI-induced contractions. The GLNVA (1.0 microM) relaxation response in the aorta was significantly inhibited by tetraethylammonium (2.5-10 mM) or ouabain (5.0 microM) and was attenuated by increased extracellular potassium gradient (10-30 mM). Glibenclamide (0.01-10 microM) dose dependently antagonized the GLNVA relaxant effect. In the isolated perfused guinea pig heart, GLNVA (0.1-10 microM) increased CGRP-like immunoreactivity outflow from coronary circulation in a concentration-dependent manner. In the isolated right and left guinea pig atria, GLNVA (0.01-10 microM) produced concentration-dependent positive inotropic and chronotropic effects, but these effects were inhibited by pretreatments with ruthenium red (1.0 microM), capsazepine (10 microM), human calcitonin-gene-related peptide (CGRP(8-37)) (1.0 microM) and sensory neuron denervation, respectively. Based on these findings, we suggest that CGRP may be released by GLNVA from cardiovascular sensory neuron, and it then activates CGRP receptors on the coronary artery and atrium. The GLNVA-induced vasorelaxant effect in the vascular smooth muscle of the aorta is due to CGRP release associated K+ channel opening, and this effect eliminates capsaicin-derived excitability-associated K+ channel blocking activities.     

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.     

Isolation and structural characterization of calcitonin gene-related peptide from the brain and intestine of the frog, Rana ridibunda

Calcitonin gene-related peptide (CGRP) was isolated in a single molecular form from an extract of the whole brain and from the small intestine of the European green frog, Rana ridibunda. The primary structure of the peptide was established as: Ala-Cys-Asn-Thr-Ala-Thr-Cys-Val-Thr-His10-Arg-Leu-Ala-Asp-Phe-Leu- Ser-Arg-Ser-Gly20-Gly-Met-Ala-Lys-Asn-Asn-Phe-Val-Pro-Thr30- Asn-Val-Gly-Ser-Ala-Phe-NH2. Frog CGRP shows only two amino acid substitutions (Val22-->Met and Gly23-->Ala) compared with chicken CGRP, as deduced from the nucleotide sequence of a cloned cDNA. The Asp14 residue, which leads to enhanced biological potency in chicken CGRP, is also present in the frog peptide. The data show that the structure of CGRP has been strongly conserved during evolution of the vertebrates.