Addition Of Calcitonin Gene-related Peptide Research Articles

Survival and neurotransmitter plasticity in cultured rat colonic myenteric neurons

The enteric nervous system is of great importance for maintenance and proper function of the gastrointestinal tract. The aim of this study was to quantify myenteric neuronal subpopulations expressing calcitonin gene-related peptide (CGRP), galanin, neuropeptide Y (NPY), somatostatin, vasoactive intestinal peptide (VIP) and nitric oxide synthase (NOS) in rat colon in vivo and after culturing. Further we investigated if culturing in the presence of CGRP, galanin, VIP, S-nitroso- N-acetyl- d, l-penicillamine (SNAP, a NO donor) or N-nitro- l-arginine methyl ester ( l-NAME, a NOS inhibitor) affect neuronal survival. After 4 days of culturing the proportions of neurons expressing CGRP, NPY, somatostatin or VIP increased as compared to in vivo, while the proportions of neurons expressing galanin or NOS did not change. Neuronal survival was unaffected after culturing in media enriched with CGRP, galanin, VIP, SNAP or l-NAME. Neither did addition of CGRP, galanin nor VIP to the cultures affect the relative numbers of neurons expressing CGRP, galanin or VIP respectively. Addition of SNAP or l-NAME did not change the percentage of neurons expressing NOS. In conclusion, cultured rat colonic myenteric neurons increase their expression of CGRP, NPY, somatostatin and VIP, suggesting that these neuropeptides are of importance for neuronal survival.

Orexin 1 Receptor Activation Attenuates Neurogenic Dural Vasodilation in an Animal Model of Trigeminovascular Nociception

The pathophysiology underlying the pulsating quality of the pain of a migraine attack is not fully understood, although trigeminal vascular afferents containing the sensory neuropeptide calcitonin gene-related peptide (CGRP) must have a role. Antimigraine drugs, such as triptans, serotonin 5-hydroxytryptamine(1B/1D) receptor agonists, reproducibly block neurogenic vasodilation associated with CGRP release. We examined the effects of the hypothalamic neuropeptides orexin A and orexin B on neurogenic dural vasodilation, dissecting out the receptor pharmacology with the novel orexin 1 (OX1) receptor antagonist N-(2-methyl-6-benzoxazolyl)-N''-1,5-naphthyridin-4-yl urea (SB-334867). Electrical stimulation of dural afferents (50-300 microA) resulted in reproducible dural vasodilation of 136 +/- 9%. Orexin A 30 microg kg(-1), but not 3 and 10 microg kg(-1), inhibited the dilation brought about by electrical stimulation over 60 min and maximally after 15 min by 60% (t7= 7.138; P < 0.001; n = 8). This response was reversed by pretreatment with the OX1 receptor antagonist SB-334867. Addition of CGRP(8-37) at the point of maximal effect of orexin A produced a further significant decrease in neurogenic dural vasodilation compared with orexin A only. CGRP administration (1 microg kg(-1)) produced a reproducible dural blood vessel dilation of 145 +/- 7% that was not inhibited by intravenous administration of orexin A (30 microg kg(-1)). Orexin B had no significant effect even at the highest dose. The current study demonstrates that orexin A is able to inhibit neurogenic dural vasodilation via activation of the OX1 receptor, resulting in inhibition of prejunctional release of CGRP from trigeminal neurons.

Calcitonin gene-related peptide decreases expression of HLA-DR and CD86 by human dendritic cells and dampens dendritic cell-driven T cell-proliferative responses via the type I calcitonin gene-related peptide receptor.

These studies were performed to establish whether functional receptors for calcitonin gene-related peptide (CGRP) are present on human dendritic cells (DCs) and to investigate potential immunomodulatory effects of CGRP on DCs other than Langerhans cells. Reverse transcriptase-PCR revealed expression of mRNA for a type 1 CGRP receptor by mature and immature blood-derived DCs. Sequence analysis confirmed the identity of the type 1 CGRP receptor (CGRP-R1). Addition of CGRP (10-7 M) to mature and immature DCs resulted in mobilization of intracellular calcium. Treatment of immature DCs with CGRP (10-7 M), before and after maturation in monocyte-conditioned medium, resulted in decreased cell surface expression of HLA-DR MHC class II and the costimulatory molecule, CD86. Treatment of immature DCs with CGRP (10-7 M) also resulted in decreased expression of CD86, but expression of HLA-DR was unchanged. When CGRP-treated mature DCs were used to stimulate allogeneic T cells, proliferative responses were dampened (approximately 50%), especially at low DC:T cell ratios (1:360). This effect was not observed with CGRP-treated, immature DCs. In contrast, CGRP-treated mature or immature DCs were no less efficient than untreated DCs in driving syngeneic T cell-proliferative responses to staphylococcal enterotoxin B. We conclude that mature and immature DCs express type 1 CGRP receptors and that signaling through these receptors may dampen mature DC-driven T cell proliferation most likely via down-regulation of CD86 and HLA-DR.

Calcitonin gene-related peptide as a GH secretagogue in human and rat pituitary somatotrophs

To elucidate the role of calcitonin gene-related peptide (CGRP) in regulating pituitary function, we investigated the effects of CGRP and the related peptide adrenomedullin (AdM) on the secretion of growth hormone (GH) in vitro from human pituitary adenoma cells, rat pituitary tumor (GH 3) cells, and normal rat pituitary cells. In 3 of 5 human somatotroph adenomas, GH secretion was stimulated by CGRP (1–100 nM). In one case of somatotroph adenoma, GH release was observed following the addition of 10 nM GHRH and 10 nM CGRP. The addition of CGRP or AdM (1 pM–10 nM) evoked GH secretion from GH 3 cells with a bell-shaped distribution curve. CGRP (100 pM) caused the maximum increase of GH secretion (172±14 (mean±S.D.)% of control). The addition of CGRP 8–37, an antagonist of CGRP type 1 receptors, inhibited the stimulatory effect of AdM but did not inhibit the effect of CGRP. The addition of CGRP and AdM evoked moderate GH secretion from normal rat pituitary cells. These results suggested that CGRP is a new GH secretagogue in human and rat pituitary tumor cells.

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.

Actions of calcitonin gene-related peptide in guinea pig gallbladder ganglia.

The actions of calcitonin gene-related peptide (CGRP) have been determined from intracellular recordings obtained from gallbladder neurons in intact whole mount preparations. In most cells, pressure microejection of CGRP elicited a slow, monophasic depolarization, 4 mV in amplitude, that was associated with a decrease in input resistance and increased excitability. The CGRP-induced depolarization was attenuated in a low-Na+ solution and had a reversal potential of -8 mV. In 10% of the cells, microejection of CGRP elicited a biphasic response that was composed of a rapid transient depolarization followed by a slow depolarization that was similar to the monophasic response. Addition of CGRP (1-10 nM) to the bathing solution elicited a monophasic depolarization and desensitized the cells to applications of CGRP by microejection. Forskolin, applied either by microejection or bath application, also depolarized gallbladder neurons and produced cross-desensitization to CGRP. Responses to substance P were not enhanced by CGRP, and CGRP did not affect fast synaptic responses. It is concluded that CGRP may contribute to a local axon reflex response in gallbladder ganglia.

Homologous desensitization of calcitonin gene-related peptide response in rat glomerular mesangial cells in culture.

Addition of calcitonin gene-related peptide (CGRP) to rat glomerular mesangial cells in culture resulted in an increase in cyclic adenosine monophosphate (cAMP) accumulation in a concentration-dependent fashion with an EC50 of approximately 3 nM. Inclusion of CGRP8-37, a CGRP1 selective antagonist shifted CGRP concentration-response curve to the right, suggesting the presence of CGRP1 subtype receptors in these cells. Pretreatment of these cells with CGRP followed by washing and rechallenge with CGRP or isoproterenol or forskolin resulted in selective attenuation of CGRP-mediated cAMP accumulation by 55-60% without affecting isoproterenol or forskolin-mediated accumulation, suggesting that CGRP pretreatment of mesangial cells induced homologous desensitization. CGRP-mediated desensitization of cAMP accumulation was found to be both concentration- and time-dependent. Desensitization did not affect the EC50 of CGRP for stimulation of cAMP accumulation, but resulted in a 55-60% reduction in maximal response. CGRP-mediated desensitization was fast, with half-maximal desensitization occurring as early as 5 min after pretreatment with CGRP. In addition, CGRP-mediated desensitization was blocked by the CGRP receptor antagonist, CGRP8-37, when included in the pretreatment protocol. These data suggest that rat mesangial cells display CGRP1 subtype receptors and that prolonged pretreatment of these cells with CGRP resulted in homologous desensitization.

CGRP and ANF cause relaxation of opossum internal anal sphincter via different mechanisms

This investigation examined and compared the role of cyclic nucleotides in the mediation of internal anal sphincter (IAS) relaxation caused by the addition of neuropeptide calcitonin gene-related peptide (CGRP) and atrial natriuretic factor (ANF). The studies were performed in vitro on smooth muscle strips of opossum IAS. The relaxation produced by CGRP and ANF was examined before and after the addition of tetrodotoxin (TTX) (1 x 10(-6)M). At this concentration, TTX did not have any significant effect on the relaxation produced by either CGRP or ANF, suggesting that these peptides act directly on the smooth muscle. Addition of CGRP (3 x 10(-6) M) produced the maximal relaxation and significantly increased cAMP content without changing cGMP. On the other hand, addition of ANF (3 x 10(-6) M) caused a similar fall in IAS tension that was accompanied by a significant elevation in cGMP without any change in cAMP content. The rises in the levels of cyclic nucleotides preceded the onset of fall in the resting tension of IAS. Our results demonstrate that CGRP and ANF relax isolated strips of opossum IAS by their action directly at the smooth muscle and that this relaxation is associated with an increase in cAMP and cGMP, respectively. The studies suggest the presence of both cAMP and cGMP pathways in the IAS and that the relaxation of IAS smooth muscle in response to different peptides may occur via a specific intracellular biochemical pathway.

Functional responses of different muscle types of the female rat urethra in vitro

The influence of muscle type on functional responses of the female rat urethra was investigated using morphological and functional in-vitro techniques. The urethral submucosa was found to contain longitudinally or obliquely oriented smooth muscle cells. The muscularis layer is composed of circularly oriented muscle cells. Near the bladder orifice smooth muscle fibres dominate, but in the mid-urethra the vast majority is circularly oriented striated muscle cells. Circular preparations responded to electrical field stimulation in vitro with a rapid contraction. Stimulation with single impulses resulted in a twitch response; frequencies exceeding 5-10 Hz induced a summation and tetanus. The response was unaffected by phenoxybenzamine, propranolol and scopolamine and had a low sensitivity to calcium-free solution but was sensitive to suxamethonium and tetrodotoxin. Using longer impulse trains stimulation evoked also a slow contraction, sensitive to calcium-free solution. In longitudinal preparations stimulation induced a relaxation followed by a contraction, responses much smaller than those seen in the circular preparations. Both preparations relaxed on addition of calcitonin gene-related peptide or capsaicin. The relaxation to calcitonin gene-related peptide was larger than that to capsaicin in longitudinal preparations but equally large in the circular ones. Substance P and 5-hydroxytryptamine contracted both preparations. The longitudinal urethra showed a larger contraction to 5-hydroxytryptamine than to substance P, whereas both substances induced similar responses in the circular preparations. The study shows a similar muscle arrangement in the female rat urethra as described in humans and further points to a functional differentiation between the different types of muscle.

Positive inotropic effect of calcitonin gene-related peptide mediated by cyclic AMP in guinea pig heart.

The mechanism of cardiac actions of rat calcitonin gene-related peptide (CGRP) was analyzed on isolated guinea pig hearts. CGRP exerted a positive inotropic effect in a dose-dependent manner on the electrically driven left atria but not on the ventricles. Immunohistochemical studies demonstrated that CGRP-like immunoreactive nerves were distributed densely in the myocardia of the atria but only sparsely in those of the ventricles. The CGRP-induced augmentation of the contraction was accompanied by the shortening of the time to peak force and the increase in the relaxation velocity. The positive inotropic response to CGRP was significantly enhanced by isobutylmethylxanthine and was attenuated by adenosine. CGRP increased the action potential amplitude and prolonged action potential duration at the level of 50% repolarization in the left atria. In the preparations, which were partially depolarized with an increase in extracellular potassium, CGRP induced slow response action potentials. These electrophysiological results indicate that CGRP causes an increase in the slow inward Ca2+ current. The cyclic AMP content in the left atria significantly increased following the addition of CGRP, the time course of which was nearly consistent with that of the augmentation of the contractile force. In the membrane preparation of the atria, the activity of adenylate cyclase was enhanced by CGRP in a dose-dependent manner. These effects of CGRP are qualitatively similar to those of beta-adrenoceptor stimulation. It is concluded that the CGRP-induced response in the guinea pig atria is attributed to the activation of adenylate cyclase via stimulation of its specific receptor and the subsequent increase in the intracellular cyclic AMP level.

Stimulation and Homologous Desensitization of Calcitonin Gene-Related Peptide Receptors in Cultured Beating Rat Heart Cells*

Addition of calcitonin gene-related peptide (CGRP), 1 X 10(-7) M, to cultured neonatal rat heart cells resulted in rapid increases in beating rate and cellular concentrations of cAMP. Calcitonin (1 X 10(-7) M), in contrast, had no significant effect on heart cell beating rate or cAMP content. CGRP-stimulated increases in heart cell cAMP content were rapid, transient, dose dependent, and potentiated by isobutyl-methylxanthine (1 X 10(-4) M). Half-maximal increases in heart cell cAMP content occurred at 1 X 10(-8) M CGRP. Heart cell adenylate cyclase responses to CGRP were desensitized in a rapid (i.e. within 5 min) and dose-dependent manner by prior exposure to CGRP. Complete and half-maximal desensitization of heart cells to CGRP occurred at 1 X 10(-8) and 3 X 10(-10) M CGRP, respectively. Desensitization of heart cells to CGRP did not modify the cAMP response of heart cells to beta-adrenergic agonist stimulation, and beta-adrenergic agonist desensitization of heart cells did not modify responses to CGRP. Heart cell cAMP responses to CGRP were additive to those of the beta-adrenergic agonist isoproterenol and occurred in the presence of beta-adrenergic blockade. These observations demonstrate that CGRP exerts specific and potent agonist actions in cardiac myocytes and that regulation of myocardial responses to CGRP may occur by mechanisms involving increases in cAMP and receptor desensitization.