Human Alpha-calcitonin Gene-related Peptide Research Articles

The vascular role of CGRP: a systematic review of human studies.

Intravenous infusion of human alpha calcitonin gene-related peptide (h-α-CGRP) has been applied to explore migraine pathogenesis and cerebral hemodynamics during the past three decades. Cumulative data implicate h-α-CGRP in regulating the vascular tone. In this systematic review, we searched PubMed and EMBASE for clinical studies investigating the vascular changes upon intravenous infusion of h-α-CGRP in humans. A total of 386 studies were screened by title and abstract. Of these, 11 studies with 61 healthy participants and 177 participants diagnosed with migraine were included. Several studies reported hemodynamic effects including flushing, palpitation, warm sensation, heart rate (HR), mean arterial blood pressure (MABP), mean blood flow velocity of middle cerebral artery (mean VMCA), and diameter of superficial temporal artery (STA). Upon the start of h-α-CGRP infusion, 163 of 165 (99%) participants had flushing, 98 of 155 (63%) participants reported palpitation, and 160 of 165 (97%) participants reported warm sensation. HR increased with 14%-58% and MABP decreased with 7%-12%. The mean VMCA was decreased with 9.5%-21%, and the diameter of the STA was dilated with 41%-43%. The vascular changes lasted from 20 to >120 min. Intravenous infusion of h-α-CGRP caused a universal vasodilation without any serious adverse events. The involvement of CGRP in the systemic hemodynamic raises concerns regarding long-term blockade of CGRP in migraine patients with and without cardiovascular complications.

Pharmacological Characterization of Novel α-Calcitonin Gene-Related Peptide (CGRP) Receptor Peptide Antagonists That Are Selective for Human CGRP Receptors

Human alpha-calcitonin gene-related peptide (CGRP) is a 37-residue neuropeptide that produces a variety of cardiovascular and other effects via activation of specific CGRP receptors that produce cAMP. Functional CGRP receptors are a heterodimeric complex composed of the heptahelical calcitonin receptor-like receptor and the single transmembrane receptor activity-modifying protein 1. Based on the known structures of the antagonist CGRP((8-37)) and the human CGRP receptor, we designed novel CGRP receptor peptide antagonists with modifications to promote high affinity and selectivity for human CGRP receptors. Antagonist affinity (K(B)) at CGRP receptors was determined using the mouse thoracic aorta and human SK-N-MC cells. In aorta, CGRP((8-37)), [N-alpha-benzoyl]human alpha-CGRP((8-37)) [bzl-CGRP((8-37))], and [N-alpha-benzoyl-His(10)-benzyl]human alpha-CGRP((8-37)) [bzl-bn-CGRP((8-37))] caused rightward shifts in the concentration-response relaxation curve for CGRP with K(B) values of 1000, 88, and 50 nM, respectively. In human SK-N-MC cells, CGRP((8-37)), bzl-CGRP((8-37)), and bzl-bn-CGRP((8-37)) caused rightward shifts in the concentration-response curve for CGRP-stimulated cAMP production with K(B) values of 797, 15, and 0.63 nM, respectively. Thus, CGRP((8-37)) had the same affinity for human and mouse CGRP receptors, whereas bzl-CGRP((8-37)) and bzl-bn-CGRP((8-37)) displayed 6- and 80-fold higher affinities, respectively, for human CGRP receptors. In addition, the selectivity of the antagonists for human CGRP receptors was highly correlated with the antagonist hydrophobicity index. These relatively high-affinity, species-selective peptide antagonists provide novel tools to differentiate structural and functional features that are unique to the human CGRP receptor. Thus, these analogs may be useful compounds for development of drugs to treat migraine headache and other cardiovascular diseases.

Development and potential of non-peptide antagonists for calcitonin-gene-related peptide (CGRP) receptors: evidence for CGRP receptor heterogeneity.

Calcitonin-gene-related peptide (CGRP) is a 37-amino-acid vasodilatory peptide, of which two isoforms, alpha CGRP and beta CGRP, have been described. The use of C-terminal fragments of CGRP peptide, such as human alpha CGRP-(8-37), has led to the pharmacological subdivision of CGRP receptors into CGRP-1 [high potency for binding of human alpha CGRP-(8-37)] and CGRP-2 (lower potency) receptors. We have recently developed BIBN4096BS, the first non-peptide CGRP antagonist, which has sub-nanomolar affinity for primate CGRP receptors. The use of BIBN4096BS has led to the discovery of further functional CGRP receptor heterogeneity in rat tissues. To further exploit BIBN4096BS as a pharmacological tool, we used BIBN4096BS in pig left anterior descending coronary vessels and cerebral basilar arteries, and compared functional with molecular data, characterizing CGRP receptor components. Our data show that, apart from a subdivision into CGRP-1 and -2 receptors, BIBN4096BS reveals additional functional differences between the actions of alpha CGRP and beta CGRP. However, evidence for CGRP receptor heterogeneity on a molecular level is scarce.

Structural determinants for binding to CGRP receptors expressed by human SK-N-MC and Col 29 cells: studies with chimeric and other peptides.

Structure-activity relationships for the binding of human alpha-calcitonin gene-related peptide 8-37 (halphaCGRP8-37) have been investigated at the CGRP receptors expressed by human SK-N-MC (neuroblastoma) and Col 29 (colonic epithelia) cells by radioligand binding assays and functional assays (halphaCGRP stimulation of adenylate cyclase). On SK-N-MC cells the potency order was halphaCGRP8-37 > halphaCGRP19-37 = AC187 > rat amylin8-37 > halpha[Tyr0]-CGRP28-37 (apparent pKBs of 7.49+/-0.25, 5.89+/-0.20, 6.18+/-0.19, 5.85+/-0.19 and 5.25+/-0.07). The SK-N-MC receptor appeared CGRP1-like. On Col 29 cells, only halphaCGRP8-37 of the above compounds was able to antagonize the actions of halphaCGRP (apparent pKB=6.48+/-0.28). Its receptor appeared CGRP2-like. halpha[Ala11,18]-CGRP8-37, where the amphipathic nature of the N-terminal alpha-helix has been reduced, bound to SK-N-MC cells a 100 fold less strongly than halphaCGRP8-37. On SK-N-MC cells, halphaCGRP8-18,28-37 (M433) and mastoparan-halphaCGRP28-37 (M432) had apparent pKBs of 6.64+/-0.16 and 6.42+/-0.26, suggesting that residues 19-27 play a minor role in binding. The physico-chemical properties of residues 8-18 may be more important than any specific side-chain interactions. M433 was almost as potent as halphaCGRP8-37 on Col 29 cells (apparent pKB=6.17+/-0.20). Other antagonists were inactive.

Development of calcitonin gene-related peptide slow-release tablet implanted in CSF space for prevention of cerebral vasospasm after experimental subarachnoid haemorrhage.

The calcitonin gene-related peptide (CGRP), a known potent intrinsic cerebral vasodilator, is contained in the sensory nerves from trigeminal ganglia that inervate the cerebral arteries. We previously reported that human alpha CGRP (hCGRP) dilates spastic cerebral arteries after experimental subarachnoid haemorrhage (SAH) in rabbits. In the present study, we investigated the prophylactic potential of a sustained higher cerebrospinal fluid level of hCGRP against experimental cerebral vasospasm. An hCGRP slow-release tablet (hCGRP s-r tablet) was developed for cisternal implantation. Experimental SAH was induced by percutaneous cisternal injection of autologous arterial blood. Angiography was initiated on day 1 (before SAH) and performed everyday. The hCGRP s-r tablet was implanted into the cisterna magna on day 2 in the treated groups. The spastic response of the basilar artery was maximized on day 4 in the non-treated (80.7% of day 1) and the placebo-treated (79.3%) groups. In contrast, the arterial diameters on day 4 were 96.1% and 90.5% of day 1 in the groups implanted with hCGRP 24 micrograms and 153 micrograms s-r tablets, respectively. We also measured the concentration of hCGRP in the cerebrospinal fluid (CSF) following implantation of the hCGRP 24 micrograms s-r tablet in the cisterna magna. The hCGRP concentration before implantation was below the dectable level. Following implantation, the hCGRP level in the CSF was 23.12 nmol/L on the second day and remained at elevated levels until the fifth day. These experiments suggest that the intrathecal single implantation of the hCGRP s-r tablet could produce an elevated concentration of hCGRP in the CSF over five days and have prevented the cerebral vasospasm after SAH in the rabbit. The hCGRP s-r tablet may be clinically applicable in the treatment of patients with SAH against cerebral vasospasm.

The inhibitory modulation of guinea-pig intestinal peristalsis caused by capsaicin involves calcitonin gene-related peptide and nitric oxide

The effect of capsaicin-induced stimulation of afferent neurons on peristalsis and the possible neural mediators involved in this action were examined in the guinea-pig isolated ileum. The intraluminal pressure threshold for eliciting peristaltic waves was used to quantify facilitation (decrease in threshold) or inhibition (increase in threshold) of peristalsis. Capsaicin (0.1-1 microM) caused an initial short-lasting stimulation of peristalsis followed by a prolonged inhibition of peristaltic activity. Capsaicin (1 microM) was ineffective when the gut segments had been pretreated with 3.3 microM capsaicin, which is indicative of an afferent neuron-dependent action of the drug. In contrast, the abolition of peristalsis caused by a high concentration of capsaicin (33 microM) was fully reversible on removal and reproducible on readministration of capsaicin, a feature characteristic of a nonspecific depression of smooth muscle excitability. Baseline peristalsis and the excitatory/inhibitory effect of capsaicin (1 microM) on peristalsis remained unaltered by a combination of the tachykinin NK1 receptor antagonist (+)-(2S, 3S)-3-(2-methoxybenzylamino)-2-phenyl piperidine (CP-99,994; 0.3 microM) and the tachykinin NK2 receptor antagonist (L(-)-N-methyl-N[4-acetylamino-4-phenyl-piperidine-2-(3,4- -dichlorophenyl)butyl]-benzamide (SR-48,968; 0.1 microM). Further experiments, performed in the presence of a low concentration of atropine (10 nM) showed that the calcitonin gene-related peptide (CGRP) antagonist human alpha-calcitonin gene-related peptide (8-37) [hCGRP(8-37); 10 microM] attenuated the delayed inhibitory effect of capsaicin on peristalsis, but did not influence baseline peristaltic activity and the capsaicin-induced facilitation of peristalsis. Blockade of nitric oxide (NO) synthesis by NG-nitro-L-arginine methylester (L-NAME, 300 microM) facilitated baseline peristaltic activity and reduced the delayed inhibition of peristalsis caused by capsaicin (1 microM) without affecting the initial peristalsis-stimulating action of capsaicin. The effects of L-NAME were prevented by L-arginine (1 mM). The data of the current study indicate that capsaicin-sensitive afferent neurons do not participate in the neural pathways subserving peristalsis in the guinea-pig small intestine, but modulate peristaltic activity upon stimulation with capsaicin. The initial stimulant action of capsaicin on peristalsis is independent of tachykinins acting via NK1 or NK2 receptors, while the delayed capsaicin-induced depression of peristalsis involves CGRP and NO.

Role of cyclic AMP and protein kinase A in K+ channel activation by calcitonin gene-related peptide (CGRP) in the guinea-pig ureter.

1. The aim of this study was to assess whether agents that interfere with the intracellular actions of cAMP and activation of protein kinase A (PKA) prevent the inhibitory action of human alpha-calcitonin gene-related peptide (CGRP) in the guinea-pig ureter smooth muscle. The action of CGRP was compared to that of the K+ channel opener, cromakalim, and the adenylyl cyclase activator, forskolin, toward electrical field stimulation- (EFS) induced myogenic twitch contractions of the ureter. To further verify the role of cAMP in the action of CGRP, we also studied the effect of stable cAMP analogues and of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). 2. Maximally effective concentrations of CGRP (0.1 microM) or forskolin (10 microM) produced a transient suppression of twitches. Cromakalim (3 microM) likewise produced a prompt suppression of twitches that in most cases exceeded 15 min. The early suppressant effect of CGRP or forskolin was inhibited by 1 or 10 microM glibenclamide; about 30% of the effect of CGRP was glibenclamide-resistant. The effect of cromakalim was totally suppressed by glibenclamide. 3. The inhibitory effect of CGRP was concentration-dependently reduced by low concentrations of barium chloride (IC50 63 microM), which blocked with similar potency the inhibitory action of cromakalim (IC50 60 microM). Glibenclamide (10 nM-10 microM) concentration-dependently inhibited the effect of CGRP and cromakalim with IC50S of 0.13 and 0.72 microM, respectively. 4. The cAMP analogues dibutyrye-cAMP (1-3 mM), 8-(4-chlorophenylthio)cAMP (0.3-1 mM) and Sp-cAMP monophosphothioate (0.1-0.3 mM) were either ineffective or poorly effective in inhibiting twitches. The cGMP analog, 8Br-cGMP (100-300 microM) produced a slowly developing, glibenclamide (1 microM)-resistant partial inhibition (25-30%) of twitches. 5. IBMX (1-300 microM) produced a concentration-dependent inhibition of twitches (EC50 16 microM). IBMX (100 microM) produced a large (peak 91%) and transient inhibition: glibenclamide (1 microM) blocked the early peak of the inhibitory action of IBMX, similar to the effect observed toward CGRP and forskolin.

Interaction of human adrenomedullin 13–52 with calcitonin gene‐related peptide receptors in the microvasculature of the rat and hamster

1. Adrenomedullin (ADM), a recently discovered circulating hypotensive peptide, shares limited sequence homology with the sensory nerve-derived vasodilator, calcitonin gene-related peptide (CGRP). This study compared the vasodilator effect of sequence 13-52 of human adrenomedullin (ADM13-52) with that of human alpha CGRP (CGRP), in the microvasculature of the hamster cheek pouch and rat skin in vivo. 2. Single arterioles (20-40 microns diameter) in the hamster cheek pouch were visualised by intravital microscopy and video recording, and measured by image analysis. Both ADM13-52 (1 pmol-0.4 nmol) and CGRP (0.1 pmol-1 nmol) evoked dose-related increases in the diameter of preconstricted arterioles (n = 6). ADM13-52 (ED50 14 pmol) was 20 fold less active than CGRP (ED50 0.71 pmol). The kinetics of onset and decline of vasodilator responses to both peptides were similar, with vasodilator responses to both peptides reaching a maximum at ca. 2 min, and reversing after 10-15 min (n = 5-7). The submaximal increase in blood flow evoked by ADM13-52 was significantly inhibited (P < 0.05; n = 6) by the CGRP1 receptor antagonist, CGRP8-37, at a dose (300 nmol kg-1, i.v.) that we have previously shown to inhibit significantly equivalent vasodilator responses to CGRP in this preparation. 3. In experiments measuring changes in local blood flow in rat skin by a 133xenon clearance technique, intradermal injection of both ADM13-52 (3-300 pmol) and CGRP (0.1-30 pmol) evoked dose-related increases in local blood flow. ADM13-52 (ED50 27 pmol) was 17 fold less potent than CGRP (ED501.6 pmol) (n = 6). The submaximal increase in blood flow evoked by both peptides was significantly inhibited (P<0.02; n = 5) by CGRP837 (100 nmol kg-1, i.v.).4. We conclude that ADM13-52 is a potent vasodilator in the microvasculature of the hamster and rat invivo. It mediates its vasodilator effect by arteriolar dilatation and this effect is due, at least in part, to the stimulation of CGRPI receptors.

Multiple mechanisms in the smooth muscle relaxant action of calcitonin gene-related peptide (CGRP) in the guinea-pig ureter

We have investigated the ability of human alpha CGRP (CGRP) to inhibit the electrically-evoked myogenic contractions of the guinea-pig ureter, in comparison with the K channel opener, cromakalim, and the adenylate cyclase activator, forskolin. CGRP (0.1 nM-0.1 microM) produced a concentration-dependent inhibition of the evoked contractions; its action was prevented by the CGRP receptor antagonist, CGRP(8-37) (1 microM), while it was unaffected by the nitric oxide (NO) synthase inhibitor, L-nitroarginine (30 microM). The effect of CGRP was antagonized in a noncompetitive manner (depression of Emax, no change in EC50) by glibenclamide (1-10 microM), a blocker of ATP-sensitive potassium channels (KATP). A substantial fraction of the inhibitory effect of CGRP was glibenclamide-resistant, however. Glibenclamide also blocked the inhibitory action of cromakalim (0.1-10 microM) without affecting the inhibition produced by forskolin (0.1-30 microM). When tested in a low-K medium (extracellular K reduced from 5.9 to 1.2 mM), the inhibitory effects of CGRP, cromakalim and forskolin were enhanced. The inhibitory effect of forskolin was partly antagonized by glibenclamide when tested in a low-K medium. CGRP (0.1 microM), cromakalim (3 microM) and forskolin (10 microM) inhibited the contractile response to KCl (80 mM), which is characterized by a distinct phasic and tonic component: cromakalim selectively inhibited the phasic response to KCl with CGRP and forskolin inhibited both components. The inhibitory effect of CGRP on the phasic contraction to KCl was partly glibenclamide-(1 microM) sensitive, while that on the tonic contraction was glibenclamide-resistant. The inhibitory action of forskolin on both components of the response to KCl was unchanged by glibenclamide.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenomedullin mediates vasodilation via CGRP1 receptors.

Adrenomedullin is a recently discovered 52 amino acid polypeptide with potent hypotensive activity. The peptide possesses 21% homology with the amino acid sequence of human calcitonin gene-related peptide-alpha (hCGRP-alpha). In 125I-hCGRP-alpha receptor binding experiments using membranes from human neuroblastoma cells (SK-N-MC) adrenomedullin is a potent competitor with a Ki of 0.37 nM. In SK-N-MC cells hCGRP-alpha and adrenomedullin concentration-dependently increase cAMP levels with -logEC50 values of 9.65 and 7.75, respectively. Both responses were attenuated in the presence of 30 nM CGRP[8-37], a CGRP1 receptor antagonist. In isolated rat hearts, perfused at constant flow, bolus infusion of adrenomedullin (1 to 100 nM) resulted in a concentration-dependent, pronounced and long-lasting vasodilation with an approximate EC50 of about 3 nM. This effect was markedly attenuated in the presence of 100 nM CGRP[8-37]. In this model, bolus infusion of hCGRP-alpha (0.01 to 100 nM) evoked a comparable vasodilation with an approximate EC50 of 0.5 nM. This effect was also potently inhibited in the presence of CGRP[8-37]. These results suggest that adrenomedullin-mediated vasodilation is linked to the activation of CGRP1 receptors in the coronary vascular system.

Inhibition by SR140333 of NK1 tachykinin receptor‐evoked, nitric oxide‐dependent vasodilatation in the hamster cheek pouch microvasculature in vivo

1. This study investigated tachykinin-evoked vasodilatation in the microvasculature of the hamster cheek pouch in vivo. Arterioles and venules were observed by intravital microscopy with video recording, and vasodilatation and constriction, defined as changes in blood vessel diameter, measured by image analysis. All agents were applied topically by superfusion. None of the agents tested had a significant effect on venule diameter. 2. When arterioles were preconstricted (by ca. 50%) with endothelin-1 present in the superfusing medium, substance P (0.3-30 nM) was a potent vasodilator, being 10 fold more active than both neurokinin A and the NK1 receptor-selective agonist, substance P methyl ester. The NK2 receptor-selective agonist, [beta-Ala8]-NKA(4-10)(0.1-10 microM) was active only at high concentrations, and the NK3 receptor-selective agonist senktide (0.1-10 microM) was virtually inactive (n = 8 hamsters). Dilatation evoked by tachykinins and analogues was rapid in onset (< 0.5 min) and readily reversible. 3. At low concentrations (1-10 nM), the non-peptide tachykinin NK1 receptor antagonist SR140333 ((S)1-(2-[3(3,4-dichlorophenyl)-1-(3-iso-propoxyphenylacetyl)pi peridin-3- yl]ethyl)-4-phenyl-1-azoniabicyclo[2.2.2]octone, chloride) had no effect on the diameter of preconstricted arterioles per se, but potently inhibited dilator responses to substance P methyl ester (apparent pKB 9.9 +/- 0.2; n = 5 hamsters, n = 10 estimates). SR140333 (10 nM) did not inhibit submaximal dilator responses evoked by human alpha calcitonin gene-related peptide (alpha CGRPh; 1.0 nM; P > 0.05; n = 5). 4 The nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 10 microM) caused a51.3 +/- 5.4% arteriolar constriction. In the presence of L-NAME, submaximal vasodilator responses to substance P (10-I00 nM) and carbachol (0.1-1.0 microM) were significantly attenuated (n = 5 hamsters;P<0.05) as compared to responses obtained in preparations that were preconstricted to a similar extent by endothelin-l (48.0 +/- 5.6%). L-NAME (10 M) was without effect on submaximal vasodilator responses to alpha CGRPh (0.1 nM) or sodium nitroprusside (1O nM) (n = 5 hamsters; P> 0.05).5 We conclude that tachykinin-evoked arteriolar vasodilatation in the hamster cheek pouch is mediated via NK, receptor activation and depends, at least in part, on the release of nitric oxide. The NKI receptors mediating vasodilatation can be blocked by topical application of SR140333; which may therefore be useful in the investigation of the role of NK1 receptors in neurogenic inflammation in the microvasculature.

Inhibitory transmitter action of calcitonin gene‐related peptide in guinea‐pig ureter via activation of glibenclamide‐sensitive K channels

1. In single sucrose gap, electrical field stimulation (EFS, 1-5 Hz) produced graded hyperpolarization of the membrane of the guinea-pig ureter smooth muscle, which was blocked by tetrodotoxin (0.3 microM) or in vitro capsaicin desensitization (3 microM for 15 min). Capsaicin itself produced a transient hyperpolarization of the membrane on its first application. 2. Superfusion with human alpha calcitonin gene-related peptide (CGRP, 30-300 mM) likewise produced a transient hyperpolarization of the membrane, mimicking the neurogenic inhibitory junction potential (i.j.p.). The hyperpolarization by CGRP was unaffected by tetrodotoxin, indicating a postjunctional site of action. 3. Both the EFS-evoked i.j.p. and the CGRP-induced hyperpolarization were inhibited by the CGRP receptor antagonist, CGRP(8-37) (0.3-3 microM) which did not affect the hyperpolarization produced by the KATP channel opener, cromakalim (0.3 microM). 4. The KATP channel blocker, glibenclamide (1 microM) blocked both the EFS-evoked i.j.p. and the CGRP-induced hyperpolarization. 5. When evoked in a low K medium (1.2 mM, KCl being replaced by an equimolar amount of NaCl), the EFS-evoked i.j.p. and the CGRP-induced hyperpolarization were both markedly enhanced, consistent with the idea that opening of K channels underlies both responses. 6. The present findings provide direct electrophysiological evidence for a neurotransmitter role of CGRP, released from the peripheral endings of capsaicin-sensitive primary afferent neurones, in the guinea-pig ureter. The action of both exogenous and endogenous CGRP involves the activation of glibenclamide-sensitive (KATP) potassium channels.

Studies on the mechanism of the contractile action of rat calcitonin gene-related peptide and of capsaicin on the guinea-pig lleum: Effect of hCGRP (8–37) and CGRP tachyphylaxis

Both the neuropeptide calcitonin gene-related peptide (CGRP) and the sensory stimulant drug capsaicin exert excitatory as well as inhibitory actions in the guinea-pig ileum. It has been known that their excitatory effects are due to activation of myenteric neurons. In the present study it has been established that a fairly high concentration (3 μM) of the CGRP antagonist human alpha CGRP (8–37) [hCGRP (8–37)], which has been known to reverse the inhibitory effects of rCGRP, failed to inhibit longitudinal contractions of ileal longitudinal muscle-myenteric plexus strips due to rat (r) CGRP. Instead, a slight enhancement of the contractions was observed, probably due to the inhibition of the relaxant effect. Tachyphylaxis to rCGRP was able to markedly reduce the contractile action of rCGRP but did not influence contractions due to capsaicin (1 μM). It is concluded that myenteric neuronal excitation due to rCGRP involves receptors not sensitive to hCGRP (8–37). Moreover, neuronal excitation by capsaicin does not seem to involve a CGRP-like mediator.

The contribution of calcitonin gene-related peptide (CGRP) to neurogenic vasodilator responses.

The neuropeptide calcitonin gene-related peptide (CGRP) is a potent vasodilator in the microcirculation of many tissues including the skin and joint. In order to elucidate the mechanism of endogenous CGRP release, we have used a multiple site 133Xe clearance technique to measure local blood flow changes in response to agents injected intradermally in the rabbit. Capsaicin (100 nmol/site) and human alpha CGRP (3 pmol/site) stimulated similar increases in blood flow and, in both cases, the effect was totally abolished by the CGRP antagonist, CGRP8-37 (1 nmol/site). By contrast, the nitric oxide synthase inhibitor L-nitro arginine methyl ester (L-NAME, 30 nmol/site) had little effect on human alpha CGRP-induced vasodilation, but caused significant inhibition of the response to capsaicin (p < 0.05). These results show that increased blood flow in rabbit skin caused by exogenous CGRP is independent of nitric oxide. In addition, however, they suggest that nitric oxide is required for either the release of endogenous CGRP from capsaicin-sensitive nerves or its subsequent activity.

Conformational characterization by circular-dichroism spectroscopy of various fragments and analogs of calcitonin-gene-related peptide.

A conformational study by circular-dichroism spectroscopy of calcitonin-gene-related peptide (CGRP) and related fragments and analogs was carried out in structure-promoting solvent mixtures. The structural characterization of rat CGRP alpha and the two isoforms of human CGRP, alpha and beta, revealed that these peptides possess very similar conformational features. The far-ultraviolet circular-dichroism spectra, in pure water, of human CGRP alpha, (hCGRP alpha), [Acm-Cys2,7]hCGRP alpha, various fragments and analogs indicated that these peptides exhibited predominantly a random-coil conformation. The addition of increasing concentrations of 1,1,1,3,3,3-hexafluoro-2-propanol to the peptide solutions resulted in a transition from a random-coil conformation to a stabilized alpha-helical structure. The substantial loss of helical content measured with [Acm-Cys2,7]hCGRP alpha, [Acm-Cys2,7]hCGRP-(1-24)-CONH2 and hCGRP-(8-37), compared to hCGRP alpha, suggested that the N-terminal disulfide bridge of hCGRP alpha is essential for adopting a highly stabilized alpha-helical conformation. Moreover, the lower helical content of hCGRP-(8-37), as compared to [Acm-Cys2,7]hCGRP alpha, as well as spectroscopic results measured with various fragments and analogs of hCGRP-(8-37) revealed that N-terminal residues found in the peptide segment 1-12 are important for the full conservation of the amphiphilic alpha-helix. In addition, the similar alpha-helical content of hCGRP-(8-37) and hCGRP-(8-18) indicated that the C-terminal segment 19-37 is not essential for the stabilization of the alpha-helix structure.

Lack of effect of sumatriptan and UK-14,304 on capsaicin-induced relaxation of guinea-pig isolated basilar artery.

1. The objectives of this study were to assess the effects of sensory neuropeptide antagonists and presynaptically acting receptor agonists on capsaicin-induced relaxations of guinea-pig isolated basilar artery (GPBA). 2. Capsaicin, human alpha-calcitonin gene-related peptide (CGRP) and substance P (SP) caused concentration-related relaxations of GPBA which had been pre-contracted with prostaglandin F2 alpha (PGF2 alpha). Responses to capsaicin were not modified by the peptidase inhibitors, phosphoramidon (1 microM) and bestatin (100 microM). 3. The relaxant responses to capsaicin were blocked in a selective manner by ruthenium red (3 microM) and by the CGRP antagonist, CGRP8-37 (1 microM). CGRP8-37 also selectively inhibited the relaxant effects of CGRP. 4. The selective NK1 receptor antagonist, GR82334 (10 microM), inhibited SP-induced relaxations but had little effect on capsaicin-induced relaxations. 5. The 5-HT1 receptor agonist, sumatriptan, produced small contractions of GPBA under conditions of resting tone. In the presence of PGF2 alpha, sumatriptan had no further contractile effect. Sumatriptan (0.3 and 3 microM) did not modify capsaicin-induced relaxations of GPBA. 6. The alpha 2-adrenoceptor agonist, UK-14,304 (0.1 microM), had no effect on basal or PGF2 alpha-induced tone. UK-14,304 did not modify capsaicin-induced relaxations. 7. These results suggest that capsaicin causes relaxation of GPBA via a release of CGRP. This process is amenable to blockade by CGRP8-37 and ruthenium red, but not to modulation by either sumatriptan or UK-14,304.

Human alpha-calcitonin gene-related peptide stimulates adenylate cyclase and guanylate cyclase and relaxes rat thoracic aorta by releasing nitric oxide.

1. The signal transduction pathway for vasorelaxation induced by human alpha-calcitonin gene-related peptide (human alpha-CGRP) was studied in rat thoracic aortic rings preconstricted with noradrenaline (10(-7) M). 2. Vasorelaxation by human alpha-CGRP was inhibited by haemoglobin (10(-6) M) and methylene blue (10(-5) M) but was unaffected by ibuprofen (10(-5) M). 3. Acetylcholine caused a 16 fold increase in levels of guanosine 3':5'-cyclic monophosphate (cyclic GMP) with levels of adenosine 3':5'-cyclic monophosphate (cyclic AMP) being unaltered. Human alpha-CGRP caused a 12 fold increase in levels of cyclic GMP but, in contrast to acetylcholine, evoked a 2.5 fold rise in levels of cyclic AMP. The rises in cyclic nucleotides evoked by human alpha-CGRP and acetylcholine were dependent on the presence of an intact endothelium. 4. NG-nitro-L-arginine (L-NOARG: 10(-5) M), which inhibits nitric oxide synthetase, inhibited the relaxant response to human alpha-CGRP and cyclic GMP accumulation without affecting the cyclic AMP accumulation. 5. The data presented in this paper suggests that human alpha-CGRP relaxes the rat thoracic aorta by releasing nitric oxide and stimulating guanylate cyclase. The stimulation of adenylate cyclase by human alpha-CGRP probably precedes the activation of nitric oxide synthase but could be unrelated to the relaxant response.

Involvement of multiple receptors in the biological effects of calcitonin gene-related peptide and amylin in rat and guinea-pig preparations.

1. The activity of rat alpha and beta calcitonin gene-related peptide (CGRP) as compared to the structurally related peptide, rat amylin, has been investigated in the guinea-pig isolated left atrium (electrically driven), in mucosa-free strips from the base of the guinea-pig urinary bladder and in the rat isolated vas deferens (pars prostatica). The antagonist activity of the C-terminal fragment of human alpha CGRP, alpha CGRP(8-37), was also investigated. 2. In the guinea-pig isolated left atrium the three peptides produced a concentration-related positive inotropic effect, amylin being about 16 and 31 times less potent than alpha or beta CGRP, respectively. Human alpha CGRP(8-37) produced a rightward displacement of the log concentration-response curve to the three agonists tested, without depression of maximal response attainable. Apparent pKB values calculated on the basis of the displacement produced by 1 microM human alpha CGRP(8-37) indicated an agonist-independent affinity of the antagonist (6.66 +/- 0.11 for alpha CGRP, 6.42 +/- 0.17 for beta CGRP and 6.95 +/- 0.11 for amylin). 3. In the guinea-pig isolated urinary bladder, alpha or beta CGRP or amylin produce a concentration-related inhibition of twitch contractions evoked by train electrical field stimulation (10 Hz frequency, 0.25 ms duration at 100 V for 0.5 s every 60 s). Amylin was about 100 times less potent than alpha or beta CGRP. Human alpha CGRP(8-37) (3 microM) did not significantly affect the inhibitory action of the three agonists tested.4. In the rat isolated vas deferens, a or PCGRP or amylin produced a concentration-related inhibition of twitch contractions evoked by electrical field stimulation (0.2 Hz frequency, 0.5 ms duration at 60 volts). Amylin was about 100 times less potent than a or PCGRP. Human aCGRP(8-37) at 3 microM did not significantly affect the inhibitory action of amylin and at 3 microM antagonized the responses to rat a and PCGRP with apparent pKB values of 5.86 +/- 0.15 and 6.11 +/- 0.13, respectively.5. These findings indicate that multiple receptors mediate the actions of peptides of the CGRP/amylin family in the preparations investigated. In the guinea-pig atrium both a and P forms of rat CGRP as well as amylin act by stimulating a single class of receptors which are sensitive to the inhibitory action of human aCGRP(8-37). In rat isolated vas deferens, at least two receptors could be present, one activated by a and PCGRP and partially sensitive to human aCGRP(8-37) and another which is sensitive to amylin but not recognised by human aCGRP(8-37). This latter type of receptor could be entirely responsible for the action of the agonists in the guinea-pig urinary bladder.

Pituitary adenylate cyclase activating polypeptide is a potent vasodilator and oedema potentiator in rabbit skin in vivo.

1. The effects of pituitary adenylate cyclase activating polypeptide (PACAP) on microvascular blood flow and plasma protein leakage were investigated in rabbit skin in vivo. 2. Intradermal injection of PACAP38, the 38 amino acid form of the peptide, caused a dose-dependent increase in blood flow measured by a 133Xe clearance technique. An equivalent increase in blood flow was induced by 10(-12) mol per site of PACAP38, 10(-12) mol per site of human alpha-calcitonin gene-related peptide (CGRP) and 10(-10) mol per site of vasoactive intestinal polypeptide (VIP). 3. The vasodilator activity of PACAP38 was not significantly different from that of the 27 amino acid form of the peptide, PACAP27, when measured with a laser Doppler flow meter, causing a 104 +/- 14% compared with 110 +/- 18% increase above basal blood flow at 10(-12) mol per site respectively. 4. At 10(-12) mol per site the effect of PACAP38 was longer lasting than that of CGRP. Blood flow remained significantly increased above control at 2 h with PACAP38 (P less than 0.05) whereas blood flow after intradermal CGRP had returned to control values by this time. 5. PACAP38 injected alone had no significant effect on microvascular leakage of 125I-labelled albumin. However, PACAP38 significantly potentiated bradykinin-induced oedema where it was approximately 100 fold more potent than VIP. 6. Oedema potentiation induced by PACAP38 was not inhibited by indomethacin at a dose which did inhibit potentiation of bradykinin-induced oedema by arachidonic acid.7. PACAP38 is at least as potent as other peptides which have been postulated to be involved in the inflammatory response when tested in rabbit skin in vivo. PACAP may contribute to both the hyperaemia and oedema components of inflammation.

In Vivo Effect of Calcitonin Gene Related Peptide on Middle Cerebral Artery Blood Flow Velocity In Humans

A double blind, placebo controlled, escalating dose study was undertaken in five healthy, conscious volunteers to investigate the effects of human synthetic alpha calcitonin gene-related peptide (CGRP) on middle cerebral artery (MCA) blood flow velocity using transcranial Doppler sonography. During placebo infusion, there was no significant change in any of the parameters studied. During CGRP infusion, all subjects showed flushing of the face and neck. Infusion of CGRP caused a significant increase in arterial pulse pressure and heart rate and a fall in diastolic and mean arterial blood pressure when compared to baseline. Peak and mean MCA velocity did not change significantly. There was a significant increase in pulsatility index though interpretation of this was confounded by the central systemic effects of CGRP. The observed haemodynamic changes may be explained by a prompt sympathetic nervous system response in order to maintain mean arterial blood pressure. Our data suggest that if cerebral vasodilatation had occurred, it was not associated with increased blood flow as an increase in MCA velocity might have been expected. An alternative explanation is that, at the doses employed, CGRP did not cause dilatation of normal cerebral vessels in healthy subjects. This does not however exclude the fact that CGRP may cause dilatation of a spastic artery as seen in cerebral vasospasm following subarachnoid haemorrhage.