Exogenous Calcitonin Gene-related Peptide Research Articles

Evidence for CGRP re‐uptake in rat dura mater encephali

Calcitonin gene-related peptide (CGRP) is widely distributed in the trigeminovascular system and released from sensory fibres of the cranial dura mater upon noxious stimulation. Such release may be a mechanism underlying migraine headache. Based on data from guinea pig basilar artery preparations, we have here studied CGRP release and uptake in an organ preparation of the hemisected rat skull. CGRP release from the cranial dura was quantified by a commercial enzyme-linked immunoassay. CGRP was depleted using repetitive challenges of capsaicin. After incubating the tissue with CGRP for 20 min and extensive washing, another capsaicin challenge was performed. Immunohistochemistry was used to visualize CGRP immunofluorescence in dural nerve fibres. Capsaicin-induced CGRP release was attenuated by the transient receptor potential vanilloid receptor type I antagonist capsazepine or by Ca(2+)-free solutions. After the CGRP-depleted preparation had been exposed to exogenous CGRP, capsaicin-induced CGRP release was increased compared to the challenge just prior to incubation. CGRP uptake was not influenced by Ca(2+)-free solutions. Olcegepant and CGRP(8-37) (CGRP receptor antagonists) did not affect uptake of CGRP. However, a monoclonal CGRP-binding antibody decreased CGRP uptake significantly. Release of CGRP after incubation was attenuated by Ca(2+)-free solutions and by capsazepine. Immunohistochemical assays indicated a weak trend towards CGRP uptake in rat dura mater. We have presented evidence for CGRP uptake in nerves and its re-release in rat dura mater. This may have implications for the pathophysiology and treatment of migraine.

Dilation by CGRP of middle meningeal artery and reversal by sumatriptan in normal volunteers

Calcitonin gene-related peptide (CGRP) plays a fundamental role in the pathophysiology of neurovascular headaches. CGRP infusion causes headache and dilation of cranial vessels. However, it is unknown to what extent CGRP-induced vasodilation contributes to immediate head pain and whether the migraine-specific abortive drug sumatriptan, a 5-hydroxytryptamine 1B/1D agonist, inhibits CGRP-induced immediate vasodilation and headache. We performed a double-blind, randomized, placebo-controlled, crossover study in 18 healthy volunteers. We recorded circumference changes of the middle meningeal artery (MMA) and middle cerebral artery (MCA) using magnetic resonance angiography before and after infusion (20 minutes) of 1.5 μg/min human αCGRP or placebo (isotonic saline) as well as after a 6-mg sumatriptan subcutaneous injection. Compared with placebo, CGRP caused significant dilation of MMA (p = 0.006) and no dilation of MCA (p = 0.69). Sumatriptan caused a marked contraction of MMA (15%-25.2%) and marginal contraction of MCA (3.9% to 5.3%). Explorative analysis revealed that sumatriptan had a more selective action on MMA compared with MCA on the CGRP day (p < 0.0001) and on the placebo day (p = 0.007). These data suggest that exogenous CGRP dilates extracranial vessels and not intracranial, and that sumatriptan exerts part of its antinociceptive action by constricting MMA and not MCA. This study provides Class I evidence that IV GCRP causes dilation of the MMA but not the MCA in healthy volunteers, and that sumatriptan reverses the dilation of the MMA caused by CGRP.

Calcitonin gene-related peptide promotes angiogenesis via AMP-activated protein kinase

Ischemia induces angiogenesis as a compensatory response. Although ischemia is known to causes synthesis and release of calcitonin gene-related peptide (CGRP), it is not clear whether CGRP regulates angiogenesis under ischemia and how does it function. Thus we investigated the role of CGRP in angiogenesis and the involved mechanisms. We found that CGRP level was increased in the rat hindlimb ischemic tissue. The expression of exogenous CGRP by adenovirus vectors enhanced blood flow recovery and increased capillary density in ischemic hindlimbs. In vitro, CGRP promoted human umbilical vein endothelial cell (HUVEC) tube formation and migration. Further more, CGRP activated AMP-activated protein kinase (AMPK) both in vivo and in vitro, and pharmacological inhibition of CGRP and cAMP attenuated the CGRP-activated AMPK in vitro. CGRP also induced endothelial nitric oxide synthase (eNOS) phosphorylation in HUVECs at Ser1177 and Ser633 in a time-dependent manner, and such effects were abolished by AMPK inhibitor Compound C. As well, Compound C blocked CGRP-enhanced HUVEC tube formation and migration. These findings indicate that CGRP promotes angiogenesis by activating the AMPK-eNOS pathway in endothelial cells.

Crystal Structure of the Ectodomain Complex of the CGRP Receptor, a Class-B GPCR, Reveals the Site of Drug Antagonism

Dysregulation of the calcitonin gene-related peptide (CGRP), a potent vasodilator, is directly implicated in the pathogenesis of migraine. CGRP binds to and signals through the CGRP receptor (CGRP-R), a heterodimer containing the calcitonin receptor-like receptor (CLR), a class B GPCR, and RAMP1, a receptor activity-modifying protein. We have solved the crystal structure of the CLR/RAMP1 N-terminal ectodomain heterodimer, revealing how RAMPs bind to and potentially modulate the activities of the CLR GPCR subfamily. We also report the structures of CLR/RAMP1 in complex with the clinical receptor antagonists olcegepant (BIBN4096BS) and telcagepant (MK0974). Both drugs act by blocking access to the peptide-binding cleft at the interface of CLR and RAMP1. These structures illustrate, for the first time, how small molecules bind to and modulate the activity of a class B GPCR, and highlight the challenges of designing potent receptor antagonists for the treatment of migraine and other class B GPCR-related diseases.

Effects of ionotropic glutamate receptor antagonists on rat dural artery diameter in an intravital microscopy model

During migraine, trigeminal nerves may release calcitonin gene-related peptide (CGRP), inducing cranial vasodilatation and central nociception; hence, trigeminal inhibition or blockade of craniovascular CGRP receptors may prevent this vasodilatation and abort migraine headache. Several preclinical studies have shown that glutamate receptor antagonists affect the pathophysiology of migraine. This study investigated whether antagonists of NMDA (ketamine and MK801), AMPA (GYKI52466) and kainate (LY466195) glutamate receptors affected dural vasodilatation induced by alpha-CGRP, capsaicin and periarterial electrical stimulation in rats, using intravital microscopy. Male Sprague-Dawley rats were anaesthetized and the overlying bone was thinned to visualize the dural artery. Then, vasodilator responses to exogenous (i.v. alpha-CGRP) and endogenous (released by i.v. capsaicin and periarterial electrical stimulation) CGRP were elicited in the absence or presence of the above antagonists. alpha-CGRP, capsaicin and periarterial electrical stimulation increased dural artery diameter. Ketamine and MK801 inhibited the vasodilator responses to capsaicin and electrical stimulation, while only ketamine attenuated those to alpha-CGRP. In contrast, GYKI52466 only attenuated the vasodilatation to exogenous alpha-CGRP, while LY466195 did not affect the vasodilator responses to endogenous or exogenous CGRP. Although GYKI52466 has not been tested clinically, our data suggest that it would not inhibit migraine via vascular mechanisms. Similarly, the antimigraine efficacy of LY466195 seems unrelated to vascular CGRP-mediated pathways and/or receptors. In contrast, the cranial vascular effects of ketamine and MK801 may represent a therapeutic mechanism, although the same mechanism might contribute, peripherally, to cardiovascular side effects.

Lipopolysaccharide induces calcitonin gene‐related peptide in the RAW264.7 macrophage cell line

Calcitonin gene-related peptide (CGRP) is widely distributed and plays important roles in a wide array of biological functions. It is enriched in primary sensory neurons and hence involved in nociception and neurogenic inflammation. Recent studies have shown that CGRP can be produced by immune cells such as monocytes/macrophages following inflammatory stimulation, suggesting a role in innate immunity. However, it is unclear how CGRP is up-regulated in macrophages and if it plays a role in macrophage functions such as the production of cytokines and chemokines. Using enzyme-linked immunosorbent assay (ELISA) and multiplex ELISA, lipopolysaccharide (LPS) was found to induce CGRP in the RAW 264.7 macrophage cell line. LPS-induced inflammatory mediators such as nerve growth factor (NGF), interleukin-1beta (IL-1beta), IL-6, prostaglandin E(2) (PGE(2)) and nuclear factor-kappaB (NF-kappaB) signalling are involved in inducing CGRP, whereas the NGF receptor trkA and CGRP receptor signalling pathways are unexpectedly involved in suppressing LPS-induced CGRP, which leads to the fine-tune regulation of CGRP release. Exogenous CGRP and CGRP receptor antagonists, in a concentration-dependent manner, stimulated, inhibited or had no effect on basal or LPS-induced release of monocyte chemoattractant protein-1, IL-1beta, IL-6, tumour necrosis factor-alpha and IL-10 in RAW macrophages. The ligand-concentration-dependent regulation of the production of inflammatory mediators by CGRP receptor signalling is a novel mechanism underlying the stimulating and suppressing role of CGRP in immune and inflammatory responses. Together, our data suggest that monocytes/macrophages are an important source of CGRP. Inflammation-induced CGRP has a positive or negative reciprocal effect on the production of other pro- and anti-inflammatory mediators. Thereby CGRP plays both facilitating and suppressing roles in immune and inflammatory responses.

Stimuli of Sensory-Motor Nerves Terminate Arterial Contractile Effects of Endothelin-1 by CGRP and Dissociation of ET-1/ETA-Receptor Complexes

BackgroundEndothelin-1 (ET-1), a long-acting paracrine mediator, is implicated in cardiovascular diseases but clinical trials with ET-receptor antagonists were not successful in some areas. We tested whether the quasi-irreversible receptor-binding of ET-1 (i) limits reversing effects of the antagonists and (ii) can be selectively dissociated by an endogenous counterbalancing mechanism.Methodology/Principal findingsIn isolated rat mesenteric resistance arteries, ETA-antagonists, endothelium-derived relaxing factors and synthetic vasodilators transiently reduced contractile effects of ET-1 but did not prevent persistent effects of the peptide. Stimuli of peri-vascular vasodilator sensory-motor nerves such as capsaicin not only reduced but also terminated long-lasting effects of ET-1. This was prevented by CGRP-receptor antagonists and was mimicked by exogenous calcitonin gene-related peptide (CGRP). Using 2-photon laser scanning microscopy in vital intact arteries, capsaicin and CGRP, but not ETA-antagonism, were observed to promote dissociation of pre-existing ET-1/ETA-receptor complexes.ConclusionsIrreversible binding and activation of ETA-receptors by ET-1 (i) occur at an antagonist-insensitive site of the receptor and (ii) are selectively terminated by endogenously released CGRP. Hence, natural stimuli of sensory-motor nerves that stimulate release of endogenous CGRP can be considered for therapy of diseases involving ET-1.

Activation of PAR‐2 Elicits NO‐Dependent and CGRP‐Independent Dilation of the Dural Artery

The goal of this study was to determine the vascular effects of protease-activated receptor-2 (PAR-2) activation in the rat cranial vasculature. The role of PAR-2 in pain and inflammatory conditions has been established but the information available on its effects and receptor distribution in the trigeminal vascular axis is limited. We studied the dilatory function and expression of PAR-2 in the neuro-vascular circuit, critical in migraine pathogenesis. We also investigated the interaction of PAR-2 with calcitonin gene-related peptide (CGRP) and dural mast cells. We used an improved model of intravital microscopy on the closed cranial window in rats to study the vascular effects of PAR-2 activating peptides (PAR-2 APs; SLIGRL-NH(2), 2-Furoyl-LIGRLO-NH(2)) in the dural vasculature. Measurement of immunoreactive CGRP in skull halves and in trigeminal nucleus caudalis was done by using an enzyme-linked immunosorbent assay. We also analyzed the presence of PAR-2 in different migraine relevant tissues by quantitative real-time PCR and Western blot analysis. PAR-2 APs and trypsin induced a dose-dependent increase in dural artery diameter. The topical application of a nonspecific nitric oxide synthase (NOS) inhibitor, L-N(G)-Nitroarginine methyl ester, attenuated SLIGRL-NH(2) responses. Olcegepant, a CGRP receptor antagonist, did not a have significant effect on the SLIGRL-NH(2) responses, though exogenous CGRP responses were completely blocked. There was no significant release of CGRP from skull halves incubated with SLIGRL-NH(2) as compared with those incubated with the corresponding negative peptide. Chronic mast cell degranulation did not change the vascular effects of PAR-2 APs. mRNA and protein expression of PAR-2 were found throughout trigeminovasuclar axis. PAR-2 activation leads to vasodilation of dural arteries and these responses are partially mediated by nitric oxide. As PAR-2 is present throughout trigeminovasuclar axis, it may have a role in migraine pathogenesis, independent of CGRP and mast cell mediated mechanism.

Protective Effects and Anti-Inflammatory Pathways of Exogenous Calcitonin Gene-Related Peptide in Severe Necrotizing Pancreatitis

Background: Microcirculatory disturbances are known to play a pivotal role in the pathogenesis of severe necrotizing pancreatitis (SNP). Calcitonin gene-related peptide (CGRP) is a vasodilatatory neuropeptide with potential anti-inflammatory effects. This study characterizes the protective effects and the anti-inflammatory pathway of exogenous CGRP in SNP. Methods: SNP was induced in rats using the glycodeoxycholic acid model. CGRP was injected prophylactically before or therapeutically after initiation of the disease. Pancreatic damage was assessed using intravital microscopy, histology, NF-κB p50/p65 electrophoretic mobility shift assay, serum cytokine assay and ICAM-1 immunohistochemistry at 6 or 12 h after the onset of disease. Results: Pancreatic microcirculatory disturbances, nuclear NF-κB levels and pancreatic ICAM-1 expression were increased in SNP compared to controls. After CGRP application, microcirculatory disturbances, NF-κB levels and pancreatic ICAM-1 expression were attenuated compared to pancreatitis alone. Moreover, pancreatic morphologic damage was significantly reduced by both prophylactic and therapeutic application of CGRP. Conclusions: CGRP is a neuropeptide that ameliorates the development of SNP in rats and may provide new treatment options. Its anti-inflammatory effects appearto be mediated by the modulation of pancreatic microcirculation and the inflammatory cascade.

Calcitonin gene-related peptide enhances CREB phosphorylation and attenuates tau protein phosphorylation in rat brain during focal cerebral ischemia/reperfusion

Calcitonin gene-related peptide (CGRP) is a potent vasodilator and immune cell modulator. Exogenous CGRP could increase the cerebral blood flow significantly and protect the ischemic neurons, but its mechanism is not entirely clear. The effect of CGRP on the expressions of CREB and tau in the ipsilateral parietal cortex were detected in focal cerebral ischemia/reperfusion model. The expression of CREB mRNA decreased in ischemia/reperfusion group (I/R group) compared with that of the sham operation group, and it got highest in CGRP group. CREB expression was lesser in I/R group than sham group, but it became more in CGRP group than I/R group. Phospho-CREB became more in I/R group, and it got most in CGRP group in the cortex. No significant difference was observed on Tau mRNA expression in all the groups. The level of tau hyperphosphorylation at Ser199/202 site and total tau in rat parietal cortex were significantly higher in I/R group than sham group. CGRP significantly inhibited tau hyperphosphorylation and the level of total tau also significantly reduced in CGRP group than that in I/R group. CGRP can upregulate the expression of CREB and phospho-CREB and attenuate the level of tau hyperphosphorylation in the ischemic neurons of the parietal cortex during focal cerebral ischemia/reperfusion. Phosphorylating CREB and inhibiting tau phosphorylation are probably involved in the mechanism of protective effect of CGRP to ischemic neurons.

Colitis induces calcitonin gene-related peptide expression and Akt activation in rat primary afferent pathways

Previous study has shown that colitis-induced increases in calcitonin gene-related peptide (CGRP) immunoreactivity in bladder afferent neurons result in sensory cross-sensitization. To further determine the effects of colitis on CGRP expression in neurons other than bladder afferents, we examined and compared the levels of CGRP mRNA and immunoreactivity in the lumbosacral dorsal root ganglia (DRG) and spinal cord before and during colitis in rats. We also examined the changes in CGRP immunoreactivity in colonic afferent neurons during colitis. Results showed increases in CGRP mRNA levels in L1 (2.5-fold, p < 0.05) and S1 DRG (1.9–2.4-fold, p < 0.05). However, there were no changes in CGRP mRNA levels in L1 and S1 spinal cord during colitis. CGRP protein was significantly increased in L1 (2.5-fold increase, p < 0.05) but decreased in S1 (50% decrease, p < 0.05) colonic afferent neurons, which may reflect CGRP release from these neurons during colitis. In L1 spinal cord, colitis caused increases in the number of CGRP nerve fibers in the deep lamina region extending to the gray commissure where the number of phospho-Akt neurons was also increased. In S1 spinal cord, colitis caused the increases in the intensity of CGRP fibers in the regions of dorso-lateral tract, and caused the increases in the level of phospho-Akt in the superficial dorsal horn of the spinal cord. In spinal cord slice culture, exogenous CGRP increased the phosphorylation level of Akt but not the phosphorylation level of extracellular-signal regulated kinase ERK1/2 even though our previous studies showed that colitis increased the phosphorylation level of ERK1/2 in L1 and S1 spinal cord. These results suggest that CGRP is synthesized in the DRG and may transport to the spinal cord where it initiates signal transduction during colitis.

Locally Synthesized Calcitonin Gene-Related Peptide Has a Critical Role in Peripheral Nerve Regeneration

Regeneration of peripheral nerves involves complex and intimate interactions between axons and Schwann cells. Here, we show that local axon synthesis and action of the neuropeptide calcitonin gene-related peptide (CGRP) is critical for this collaboration. After peripheral sural sensory axon injury in rats, we observed an unexpectedly large proportion of axons that newly expressed CGRP during regeneration. Intense peptide expression accompanied local rises in alphaCGRP mRNA in the nerve trunk, and there was evidence of transport of alphaCGRP mRNA into regenerating axons, indicating intra-axonal peptide synthesis. Calcitonin gene-related peptide receptor and its receptor activity modifying protein were expressed onadjacent Schwann cells, where they were available for signaling. Moreover, exogenous CGRP induced proliferation in isolated adult Schwann cells. New axon outgrowth and CGRP expression depended on local peptide synthesis and were inhibited by exposure tolocal translation inhibitors. Local delivery of siRNAs to either alphaCGRP or receptor activity modifying protein 1 to sites of nerve transection was associated with severe disruption of axon outgrowth.These findings indicate that robust localized intra-axonal translation of the CGRP neuropeptide during regeneration signals Schwann cell proliferation, behavior that is critical for partnering during adult peripheral nerve regrowth.

Growth of the rat gubernaculum in vitro and localisation of its growth centre

Purpose Recent studies suggest that testicular descent is accomplished by outgrowth of the gubernaculum from the abdominal wall. The tip of the gubernaculum has been proposed as the primary site of growth, similar to an embryonic limb bud. We aimed to determine the maximum site of growth in organ culture. Methods Gubernacula from 1-day-old Sprague-Dawley rats (n = 40) were collected and divided into 4 groups as follows: whole gubernaculum (control), truncated gubernaculum (tip excised), gubernacular tip alone, and grafted gubernaculum with an extra tip on its side. Tissues were cultured with or without calcitonin gene-related peptide (CGRP) (714nmol/L) in medium for 24 hours. The area of each gubernaculum was determined by “Image J” analysis of digital photos collected via a Leica Wild M28 microscope (Leica Microsystems, Wetzler GmbH Germany) taken before and after culture. Results In organ culture, the neonatal rat gubernaculum normally shrank 10% to 15%, but this was prevented by the presence of exogenous CGRP (0.8% vs 11.8%; P < .003). By contrast, gubernacula with their tips excised were not affected by CGRP (3.4% vs 4.7%; not significant). Gubernacular tips alone did respond to CGRP (2.7% vs 13.5%; P < .03). Transplantation of the tip to another gubernaculum caused it to develop 2 tips. Conclusions These results suggest that the rat gubernaculum contains a growth centre in its distal tip that can respond to CGRP. This is consistent with a limb bud model of gubernacular growth during the inguinoscrotal descent of the testis.

Calcitonin gene–related peptide stimulates mitosis in the tip of the rat gubernaculum in vitro and provides the chemotactic signals to control gubernacular migration during testicular descent

Background/aims We investigated whether calcitonin gene–related peptide (CGRP) released from sensory genitofemoral nerve branches could stimulate rodent gubernacular growth and provide chemotactic signals for directing inguinoscrotal gubernaculum migration in vitro. Materials and Methods Neonatal rat gubernacula containing a developing cremaster sac (n = 60) were removed at days 0, 2, 4, 6, 8, and 10 (n = 10 per age; n = 5 per experimental group) and placed in organ culture for 24 hours with or without added CGRP (720 nmol/L). The gubernacula were stained for bromodeoxyuridine (BrdU) immunohistochemistry. Cells were counted (3 × 100 cells) in the mesenchymal tip of the gubernaculum to find the percentage of BrdU uptake. A further group of neonatal rat gubernacula (n = 21 per group) were placed in organ culture on an agar platform with 5 agarose beads soaked in either PBS or 10 −6 mol/L CGRP placed approximately 0.8 to 1 mm on each side of the tip of the cremaster sac. After 72 hours, the position of the gubernaculum was compared with its starting position and any deviation measured. Results Exogenous CGRP caused a significant increase in BrdU uptake in the tip of the gubernaculum in 0-day-old rats compared with control cultures. Two-way analysis of variance in the cellular proliferation pattern between gubernacula cultured ± CGRP between 0 and 10 days showed a significant difference ( P < .001). The cultures containing CGRP-impregnated beads caused significant ( P < .01) deviation of the tip of the gubernaculum toward the beads, whereas the controls demonstrated no net movement of the tip. Conclusions These studies demonstrate that mitosis in the tip of the rat gubernaculum is significantly increased in response to CGRP in vitro. Also, CGRP may provide chemotactic signals to control inguinoscrotal gubernacular migration in the rat.

Effects of calcitonin gene-related peptide on rat soleus muscle excitability: mechanisms and physiological significance

Intense exercise causes a large loss of K(+) from contracting muscles. The ensuing elevation of extracellular K(+) ([K(+)](o)) has been suggested to cause fatigue by depressing muscle fiber excitability. In isolated muscles, however, repeated contractions confer some protection against this effect of elevated K(+). We hypothesize that this excitation-induced force-recovery is related to the release of the neuropeptide calcitonin gene-related peptide (CGRP), which stimulates the muscular Na(+)-K(+) pumps. Using the specific CGRP antagonist CGRP-(8-37), we evaluated the role of CGRP in the excitation-induced force recovery and examined possible mechanisms. Intact rat soleus muscles were stimulated to evoke short tetani at regular intervals. Increasing extracellular K(+) ([K(+)](o)) from 4 to 11 mM decreased force to approximately 20% of initial force (P < 0.001). Addition of exogenous CGRP (10(-9) M), release of endogenous CGRP with capsaicin, or repeated electrical stimulation recovered force to 50-70% of initial force (P < 0.001). In all cases, force recovery could be almost completely suppressed by CGRP-(8-37). At 11 mM [K(+)](o), CGRP (10(-8) M) did not alter resting membrane potential or conductance but significantly improved action potentials (P < 0.001) and increased the proportion of excitable fibers from 32 to 70% (P < 0.001). CGRP was shown to induce substantial force recovery with only modest Na(+)-K(+) pump stimulation. We conclude that the excitation-induced force recovery is caused by a recovery of excitability, induced by local release of CGRP. The data suggest that the recovery of excitability partly was induced by Na(+)-K(+) pump stimulation and partly by altering Na(+) channel function.

Calcitonin gene-related peptide-mediated antihypertensive and anti-platelet effects by rutaecarpine in spontaneously hypertensive rats

We have previously reported that Chinese traditional medicine rutaecarpine (Rut) produced a sustained hypotensive effect in phenol-induced and two-kidney, one-clip hypertensive rats. The aims of this study are to determine whether Rut could exert antihypertensive and anti-platelet effects in spontaneously hypertensive rats (SHR) and the underlying mechanisms. In vivo, SHR were given Rut and the blood pressure was monitored. Blood was collected for the measurements of calcitonin gene-related peptide (CGRP), tissue factor (TF) concentration and activity, and platelet aggregation, and the dorsal root ganglia were saved for examining CGRP expression. In vitro, the effects of Rut and CGRP on platelet aggregation were measured, and the effect of CGRP on platelet-derived TF release was also determined. Rut exerted a sustained hypotensive effect in SHR concomitantly with the increased synthesis and release of CGRP. The treatment of Rut also showed an inhibitory effect on platelet aggregation concomitantly with the decreased TF activity and TF antigen level in plasma. Study in vitro showed an inhibitory effect of Rut on platelet aggregation in the presence of thoracic aorta, which was abolished by capsazepine or CGRP(8–37), an antagonist of vanilloid receptor or CGRP receptor. Exogenous CGRP was able to inhibit both platelet aggregation and the release of platelet-derived TF, which were abolished by CGRP(8–37). The results suggest that Rut exerts both antihypertensive and anti-platelet effects through stimulating the synthesis and release of CGRP in SHR, and CGRP-mediated anti-platelet effect is related to inhibiting the release of platelet-derived TF.

Therapeutic Potential of 1-Methylnicotinamide against Acute Gastric Lesions Induced by Stress: Role of Endogenous Prostacyclin and Sensory Nerves

1-Methylnicotinamide (MNA) is one of the major derivatives of nicotinamide, which was recently shown to exhibit antithrombotic and antiinflammatory actions. However, it is not yet known whether MNA affects gastric mucosal defense. The effects of exogenous MNA were studied on gastric secretion and gastric lesions induced in rats by 3.5 h of water immersion and water restraint stress (WRS) or in rats administered 75% ethanol. MNA [6.25-100 mg/kg intragastrically (i.g.)] led to a dose-dependent rise in the plasma MNA level, inhibited gastric acid secretion, and attenuated these gastric lesions induced by WRS or ethanol. The gastroprotective effect of MNA was accompanied by an increase in the gastric mucosal blood flow and plasma calcitonin gene-related peptide (CGRP) levels, the preservation of prostacyclin (PGI(2)) generation (measured as 6-keto-PGF1alpha), and an overexpression of mRNAs for cyclooxygenase (COX)-2 and CGRP in the gastric mucosa. R-3-(4-Fluoro-phenyl)-2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-propionic acid (RO 324479), which is the selective antagonist of IP/PGI(2) receptors, reversed the effects of MNA on gastric lesions and GBF. MNA-induced gastroprotection was attenuated by suppression of COX-1 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole; SC-560] and COX-2 [4-(4-methylsulfonylphenyl)-3-phenyl-5H-furan-2-one; rofecoxib] activity, capsaicin denervation, and by the pretreatment with CGRP(8-37) or capsazepine. Addition of exogenous PGI(2) or CGRP restored the MNA-induced gastroprotection in rats treated with COX-1 and COX-2 inhibitors or in those with capsaicin denervation. WRS enhanced MDA content while decreasing superoxide dismutase (SOD) activity in the gastric mucosa, but pretreatment with MNA reversed these changes. MNA exerts potent gastroprotection against WRS damage via mechanisms involving cooperative action of PGI(2) and CGRP in preservation of microvascular flow, antioxidizing enzyme SOD activity, and reduction in lipid peroxidation.

Calcitonin gene-related peptide-mediated cardioprotection of postconditioning in isolated rat hearts

Previous studies have demonstrated that endogenous calcitonin gene-related peptide (CGRP) plays an important role in mediation of ischemic preconditioning. In the present study, we tested whether CGRP is also involved in mediation of the protective effects of postconditioning in isolated rat hearts. Sixty minutes of left coronary artery occlusion and followed by 60 min of reperfusion caused a significant decrease in cardiac function and a significant increase in creatine kinase (CK) release and infarct size. Postconditioning with three cycles of 1-min ischemia and 1-min reperfusion produced a marked improvement of cardiac function and decreased CK release and infarct size, concomitantly with an increase in the release of CGRP release in coronary effluent. However, the cardioprotection afforded by postconditioning was abolished by CGRP 8-37 (10−7 M), a selective CGRP receptor antagonist, or pretreatment with capsaicin (50 mg/kg, s.c.), which depletes transmitters in sensory nerves. Exogenous CGRP (5×10−9 M) administration of CGRP reappeared postconditioning-like cardioprotection in the rats pretreated with capsaicin. These results suggest that the protective effects of ischemic postconditioning are related to stimulation of endogenous CGRP release in rat hearts.

Functional Antagonism between Endogenous Neuropeptide Y and Calcitonin Gene-Related Peptide in Mesenteric Resistance Arteries

To test the hypothesis that endogenous neuropeptide Y (NPY) counteracts the vasodilator effects of calcitonin gene-related peptide (CGRP), we used isolated mesenteric resistance arteries of rats and mice. With immunohistochemistry, we observed CGRP-containing fibers along and in the vicinity of a subset of NPY- or tyrosine hydroxylase-immunoreactive fibers. The CGRP1 receptor component calcitonin-related-like receptor was expressed by periarterial nerves and smooth muscle cells, whereas receptor activity-modifying protein 1 was observed primarily on the smooth muscle. In organ chambers, exogenous CGRP caused relaxations that were reversed by exogenous NPY. The effects were inhibited by 1-piperidinecarboxamide, N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]-carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)-methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl) (BIBN4096BS, a CGRP1 receptor antagonist; pK(B) = 8.54 +/- 0.52) and (R)-NZ-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]argininamide (BIBP3226, a Y1 antagonist; pK(B) = 7.00 +/- 0.49), respectively. Pretreatment with capsaicin (1 muM; 20 min) and the presence of BIBN4096BS (20 nM) increased contractile responses to K(+) (20-40 mM) and electrical field stimulation (EFS; 1-32 Hz). NPY increased contractile responses to K(+) and BIBP3226 (400 nM) reduced contractile responses to EFS. These effects were inhibited by capsaicin and BIBN4096BS, respectively. Furthermore, the relaxing effect of exogenous CGRP (10 nM) during phenylephrine-induced contraction (30 muM) was reversed by EFS, and this effect was reduced in the presence of BIBP3226. We confirmed that bioactive concentrations of endogenous CGRP and NPY can be released from periarterial sensory-motor and sympathetic nerves, respectively, and we demonstrate for the first time functional antagonism between endogenous NPY and CGRP at the level of the smooth muscle.

Exogenous calcitonin gene-related peptide perturbs the direction and length of gubernaculum in capsaicin-treated rats

Calcitonin gene-related peptide (CGRP) released from the genitofemoral nerve (GFN) is proposed to control gubernacular migration to the scrotum during inguinoscrotal descent. As previous studies suggested that both CGRP and androgens are necessary to stimulate gubernacular elongation and proliferation, this study aimed to test whether CGRP perturbed growth of the gubernaculum by exogenous injection in a rat model of GFN blockade with an intact androgen pathway. Sprague-Dawley rats (day 0, n = 34) were injected with capsaicin (sensory neurotixin) or vehicle. CGRP (25 microl of 2 x 10 5 molar concentration) Or castor oil was administered at the umbilicus 24 h later, then every other day thereafter for 10 days. Groups of rats were sacrified on days 8 and 28, and the tip of the gubernaculunx was identified in relation to the scrotum and umbilicus. The length of the processus vaginalis (PV) was measured. In group 1 (capsaicin and CGRP), at 8 days of age (n = 8) the testes were found in the intraabdominal or suprainguinal position in 87.5% (14 testes) and the tip of the gubernaculum was oriented more laterally than normal. However the mean PV length was 5.1 mm, which was shorter than control groups (9-10.8 mm) (P < 0.001, t-test). On the other hand, at day 28, the testes were located proximal to the internal inguinal ring in 66.7% (8 testes) with their gubernacular tip oriented in part towards the umbilicus. The mean of the PV length was 12.2 mm, which was significantly decreased compared to other groups (P < 0.001, t-test). In group 2 (capsaicin and castor oil) and group 3 (vehicle and CGRP) the testes of one rat in each group at 28 days of age were located in the abdomen at the internal inguinal ring with the distal gubernacular attachment lying free and directed caudally towards the scrotum, while the rest of the testes were found in the normal location. These findings suggest that chemical interruption of sensory nerves followed by CGRP injections in rats can perturb gubernacular growth and can change the direction of migration.