Calcitonin Gene-related Peptide Concentrations Research Articles

Prevention of cerebral vasospasm by calcitonin gene-related peptide slow-release tablet after subarachnoid hemorrhage in monkeys.

The goal of this work was to investigate the efficacy of a calcitonin gene-related peptide (CGRP) slow-release tablet (CGRP tablet) for the prevention of cerebral vasospasm after subarachnoid hemorrhage (SAH). Experimental SAH was produced in 10 cynomolgus monkeys by placing a clot around the internal carotid artery bifurcation (Day 0). In five animals, CGRP tablets (1200 micrograms of CGRP) were then placed in the cerebrospinal fluid space (CGRP group). In two animals, placebo tablets were similarly placed (placebo group). The remaining three animals were treated with no tablets after SAH (SAH group). A series of angiographic analyses were performed, before SAH and on Days 7 and 14, to examine changes in the diameters of the ipsilateral internal carotid artery, middle cerebral artery, and anterior cerebral artery. The CGRP concentration in the cerebrospinal fluid taken before each angiogram was also determined. In the SAH and placebo groups, cerebral vasospasm developed on Day 7 (54.8% of the pre-SAH value at the internal carotid artery, 62.3% at the middle cerebral artery, 51.3% at the anterior cerebral artery, and 56.1% as an average of the three arteries). In the CGRP group, vasospasm was significantly ameliorated at the middle cerebral artery, at the anterior cerebral artery, and on average (81.7, 81.1, and 75.7%, P < 0.05, 0.03, and 0.02, respectively). The CGRP concentration was positive only on Day 7 for the CGRP group (6.5 nmol/L). The CGRP tablet prevented cerebral vasospasm after SAH and may have significant potential for the treatment of patients with SAH.

The psychotomimetic drugs D-amphetamine and phencyclidine release calcitonin gene-related peptide in the limbic forebrain of the rat.

Calcitonin gene-related peptide (CGRP) is the major product of the calcitonin gene in brain and exerts a number of actions in the central nervous system (CNS). In particular the finding that CGRP affects dopamine (DA) release and metabolism has raised the possibility that it may play a role in several neuropsychiatric disorders. Consequently, we have here studied the effects of two psychotomimetic drugs, namely, d-amphetamine (AMPH) and phencyclidine (PCP), on CGRP concentrations in brain microdialysates from freely moving rats. The animals were stereotaxically implanted with vertical concentric probes in the medial prefrontal cortex (mPFC), the ventral striatum (vSTR), or the hippocampus; and the experiments were performed 48 hr after surgery. The dialysis probes were perfused with a modified Ringer's solution at the rate of 5 microliters/min. AMPH 1.5 mg/kg, PCP 2.5 mg/kg, or NaCl 0.9% were injected s.c.; and the perfusates were collected at 60 min intervals before and after the injections and used for CGRP-like immunoreactivity (-LI) determination by radioimmunoassay (RIA). In separate experiment, KCl (100 mM), veratridine (50 microM), or tetrodotoxin (2 microM), were added to the perfusate and infused in the vSTR. Baseline levels of CGRP-LI were detected in dialysates from all three regions. Both AMPH and PCP caused a significant and sustained increase (maximum about 300%) in CGRP-LI concentrations, in particular from the mPFC and vSTR, while saline had no effect. KCl and veratridine also increased CGRP-LI in dialysates during the first posttreatment period, while tetrodotoxin induced a significant but delayed decrease in CGRP-LI levels. Finally, cervical dislocation also elevated CGRP-LI in dialysates from the mPFC and the vSTR. Our findings demonstrate that 1) CGRP-LI can be measured in vivo in microdialysates from mPFC, vSTR, and hippocampus; 2) the release in vSTR is action potential-dependent; and 3) systemic administration of AMPH or PCP results in a long-lasting release of CGRP-LI in the mPFC and vSTR, thus demonstrating a novel action of these drugs in the brain. Since other studies have shown that major antipsychotic drugs appear to reduce CGRP release in brain, our study provides, in principle, support for a role of CGRP in psychotic disorders.

The psychotomimetic drugs D‐amphetamine and phencyclidine release calcitonin gene‐related peptide in the limbic forebrain of the rat

Calcitonin gene-related peptide (CGRP) is the major product of the calcitonin gene in brain and exerts a number of actions in the central nervous system (CNS). In particular the finding that CGRP affects dopamine (DA) release and metabolism has raised the possibility that it may play a role in several neuropsychiatric disorders. Consequently, we have here studied the effects of two psychotomimetic drugs, namely, d-amphetamine (AMPH) and phencyclidine (PCP), on CGRP concentrations in brain microdialysates from freely moving rats. The animals were stereotaxically implanted with vertical concentric probes in the medial prefrontal cortex (mPFC), the ventral striatum (vSTR), or the hippocampus; and the experiments were performed 48 hr after surgery. The dialysis probes were perfused with a modified Ringer's solution at the rate of 5 μl/min. AMPH 1.5 mg/kg, PCP 2.5 mg/kg, or NaCl 0.9% were injected s.c.; and the perfusates were collected at 60 min intervals before and after the injections and used for CGRP-like immunoreactivity (-LI) determination by radioimmunoassay (RIA). In separate experiments, KCl (100 mM), veratridine (50 μM), or tetrodotoxin (2 μM), were added to the perfusate and infused in the vSTR. Baseline levels of CGRP-LI were detected in dialysates from all three regions. Both AMPH and PCP caused a significant and sustained increase (maximum about 300%) in CGRP-LI concentrations, in particular from the mPFC and vSTR, while saline had no effect. KCl and veratridine also increased CGRP-LI in dialysates during the first posttreatment period, while tetrodotoxin induced a significant but delayed decrease in CGRP-LI levels. Finally, cervical dislocation also elevated CGRP-LI in dialysates from the mPFC and the vSTR. Our findings demonstrate that 1) CGRP-LI can be measured in vivo in microdialysates from mPFC, vSTR, and hippocampus; 2) the release in vSTR is action potential-dependent; and 3) systemic administration of AMPH or PCP results in a long-lasting release of CGRP-LI in the mPFC and vSTR, thus demonstrating a novel action of these drugs in the brain. Since other studies have shown that major antipsychotic drugs appear to reduce CGRP release in brain, our study provides, in principle, support for a role of CGRP in psychotic disorders. © 1996 Wiley-Liss, Inc.

Adrenomedullin-sensitive receptors are preferentially expressed in cultured rat mesangial cells

By using cultured rat mesangial cells, we compared the effects on cyclic nucleotide levels of adrenomedullin with those of the structurally related peptides, calcitonin gene-related peptide (CGRP) and amylin. Adrenomedullin potently increased cAMP levels 7-fold in a time- and concentration-dependent manner. Its EC 50 was 3 × 10 −9 M. CGRP was less potent (2-fold) with an EC 50 of 10 −7 M, and amylin had no effect on cAMP levels. All three peptides failed to increase cGMP levels. Treatment of cells with near maximal concentrations of adrenomedullin (10 −7 M) and CGRP (10 −6 M) had no additive effect on cAMP levels. Human adrenomedullin-(22–52)-NH 2, a putative adrenomedullin receptor antagonist, inhibited the production of cAMP elicited by adrenomedullin (IC 50: 7 × 10 −8 M) and CGRP (IC 50: 5 × 10 −8 M). Human CGRP-(8–37), a CGRP receptor antagonist, conversely, reduced the cAMP elevation caused by these peptides with a lower potency (IC 50: 10 −6 M for both peptides). This demonstrated that human adrenomedullin-(22–52)-NH 2 was a more effective antagonist for adrenomedullin- and CGRP-specific receptors than human CGRP-(8–37). Results suggest that receptors sensitive to adrenomedullin are preferentially expressed in cultured rat mesangial cells. Immunohistochemical study showed almost no immunoreactive adrenomedullin and CGRP, if any, in the cells. Adrenomedullin may regulate mesangial function as either a paracrine or circulating hormone via a cAMP- but not a cGMP-dependent mechanism.

Relationship between circulating levels of calcitonin gene-related peptide, nitric oxide metabolites and hemodynamic changes in human septic shock

This study is aimed to investigate the relationship between plasma concentrations of nitrite and nitrate as a measure of ongoing nitric oxide (NO) production, the vasodilatory neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP), endotoxemia and hemodynamic changes in human septic shock. Thirteen patients with septic shock were studied within 6 h after the development of hypotension. Hemodynamic measurements and blood samples were recorded simultaneously at 2-h intervals from study admission. Eighteen normotensive patients with sepsis were included as control group of patients. On study entry, circulating levels of endotoxin did not relate to either CGRP or nitrite and nitrate plasma values. Septic shock patients had significantly higher plasma CGRP, and nitrite and nitrate concentrations, at each of the four time points, than patients with sepsis, as well as both groups of patients compared to normal subjects. No differences were found in plasma SP levels between the two groups of patients. For pooled data from all septic shock patients and measurements ( n = 52), both plasma concentrations of CGRP and nitrite and nitrate were inversely correlated, independently from each other, to systemic vascular resistance. On study admission and at 2-h intervals, plasma CGRP concentrations correlated directly with nitrite and nitrate values. Our observations, thus, point to CGRP acting in concert with NO as important mediators responsible for hypotension in human septic shock.

Effect of oxygen inhalation on systemic, central, and splanchnic haemodynamics in cirrhosis

Background/Aims: Patients with cirrhosis exhibit a hyperdynamic circulation with increased cardiac output and low arterial blood pressure. The aim of the present study was to assess the effects of oxygen inhalation on systemic, central, and splanchnic haemodynamics and vasoactive systems in patients with cirrhosis ( n=19). Results: Spirometry was normal, but the carbon monoxide diffusing capacity (transfer factor) was significantly decreased, 18.8 ml·min −1·mmHg −1 (−32% of that predicted, p<0.0001), and correlated significantly with the cardiac output ( r=0.78, p<0.0005), plasma volume ( r=0.72, p<0.001) and the central and arterial blood volume ( r=0.67, p<0.005). After inhalation of 100% oxygen over a period of 20 min, the cardiac output decreased from 7.4 to 6.6 l/min ( p<0.0005), and the systemic vascular resistance increased from 980 to 1124 dyn·s·cm −5 ( p<0.005). The change in systemic vascular resistance was significantly greater in patients with mild liver dysfunction than in those with severe liver dysfunction ( p<0.02). In contrast, no significant changes were seen in the arterial or portal venous pressures during inhalation of oxygen. Arterial concentrations of catecholamines, renin, endothelin-1, and calcitonin gene-related peptide were all increased in patients with cirrhosis, but only the catecholamine concentrations decreased significantly (noradrenaline −13%, p<0.02 and adrenaline −16%, p<0.01) in response to oxygen. Conclusion: During oxygen inhalation cardiac output decreases and systemic vascular resistance increases in association with a decrease in arterial concentrations of catecholamine, but oxygen supply in patients with cirrhosis does not normalise the hyperdynamic circulation or the low arterial blood pressure.

Calcitonin gene-related peptide content, basal outflow and electrically-evoked release from monoarthritic rat spinal cord in vitro

In this study, Freund's adjuvant-induced monoarthritis in the rat hind paw was used to induce chronic pain and inflammation. In order to compare the basal outflow, electrically-evoked release and total content of calcitonin gene-related peptide like immunoreactivity (CGRP-LI) with previously reported changes in substance P (SP-LI), the lumbar enlargement of monoarthitic (complete Freund's adjuvant-treated, CFA rat) and control (incomplete Freund's adjuvant-treated, IFA rat) spinal cords were used. During the 4-wk period after injection, neither the basal nor the evoked release of CGRP-LI from CFA cords differed from controls. By contrast, we have previously reported that SP-LI release from CFA rat spinal cords was significantly higher than from controls, 21 days after inoculation with Freund's adjuvant. Electrically-evoked CGRP-LI release from 21-day CFA rat spinal cord slices was not modified by superfusion with a GABAB antagonist, CGP 36742 (100 μM) which could greatly increase SP-LI release. However, the release of both peptides was significantly increased to the same extent in IFA and normal tissue but to a lesser extent in CFA cords, by superfusion with the opioid antagonist naloxone (1 μM). In conclusion, CGRP-LI, unlike SP-LI, did not appear to be susceptible to any changes in the lumbar enlargement of the rat spinal cord during inflammation of the hind paw. In addition, CGRP-LI release was increased by antagonism of opiate but not GABAB receptors, suggesting that during chronic inflammation of one hind paw, the GABABergic system, unlike the opioid system, might be activated to selectively inhibit the enhanced SP-LI release but not CGRP-LI release which is not changed.

Alterations in colonic arterial and venous plasma neuropeptide concentrations in horses during low-flow ischemia and reperfusion of the large colon

Abstract Objective To measure colonic arterial (CA) and colonic venous (CV) plasma neuropeptide concentrations during low-flow ischemia and reperfusion of the large colon in horses. Animals 10 adult horses. Procedure CA and CV plasma samples collected from anesthetized horses during experimentally induced low-flow colonic ischemia and reperfusion were assayed for vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), and substance P (SP), using radioimmunoassays. In 6 anesthetized horses, colonic ischemia (20% of baseline [BL]) was maintained for 3 hours, then blood flow was restored and monitored for 3 hours. Hemodynamic variables were monitored continuously and recorded at 30-minute intervals. CA resistance was calculated from colonic blood flow (Qcolon) and mean CA pressure values at each time. Blood was obtained from CA and CV catheters at 0, 1, 2, 3, 3.25, 3.5, 4, 5, and 6 hours; plasma VIP, CGRP, and SP concentrations were quantified, using radioimmunoassays. In 4 additional horses, VIP and CGRP were measured in CA and CV blood at 0, 0.25, 0.5, 0.75, and 1 hour. Results Heart rate was significantly increased at 5.5 and 6 hours; other alterations in systemic hemodynamic variables were not significant. Decrease in Qcolon during ischemia was significant; Qcolon rebounded to a value significantly greater than BL value within 5 minutes of reperfusion and was maintained above the BL value during 3 hours of reperfusion. Mean CA pressure was significantly decreased during ischemia, but returned to a value not different from the BL value by 3.25 hours. Mean CV pressure remained unchanged from the BL value during ischemia, but increased to a value significantly greater than the BL value by 3.25 hours and remained increased through 6 hours. CA resistance began to decrease during early ischemia and was significantly less than the BL value by 3.25 hours; it remained less than the BL value through 4 hours. Increase in CV VIP concentration was significant by 0.25 hour of ischemia, but decreased to a value not different from BL value by 3.25 hours. Increase in CV CGRP was significant at 3.25 hours, but this variable returned to a value not different from BL value by 3.5 hours. Conclusions CV VIP concentration increases during low-flow colonic ischemia, and CV and CA CGRP and CA SP concentrations increase during early reperfusion. (Am J Vet Res 1996;57:1200-1205)

The contribution of nitric oxide to endotoxin-induced ocular inflammation: interaction with sensory nerve fibres.

1. The actions of nitric oxide (NO) have been investigated in an endotoxin-evoked ocular inflammatory model in the rabbit, with particular emphasis on the relationship between NO, sensory nerves (C-fibres) and the C-fibre neuropeptides, calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase activating peptide (PACAP). 2. Endotoxin, injected intravitreally, evoked inflammatory responses, i.e. conjunctival hyperaemia, miosis and protein extravasation, reflected by the aqueous flare response (AFR). In control rabbits, the maximum AFR was 66.5 +/- 9.5 (arbitrary units). Pretreatment with the NO synthase (NOS) inhibitor, NG-nitro-L-arginine (L-NAME, 200 mg kg-1) given by intravenous injection, inhibited the endotoxin-evoked responses; the AFR was 16.5 +/- 1.9 (n = 8, P < 0.001) and the conjunctival hyperaemia was abolished. 3. Endotoxin-evoked ocular inflammation is associated with the release of CGRP and PACAP from C-fibres. In the eyes challenged with endotoxin, the concentrations of PACAP-27, -38 and CGRP in the aqueous humour were 58.2 +/- 10.9, 54.4 +/- 12.4 and 5526 +/- 519 (pmoll'), respectively. L-NAME inhibited the release of PACAP-27, -38 and CGRP; the concentrations were 14.3 +/- 2.5, 13.5 +/- 2.5 and 510 +/- 67 (pmoll-1), respectively (n = 8, P < 0.01 or 0.001). 4. Intravitreal injection of 0.3 nmol CGRP induced conjunctival hyperaemia and AFR; the maximum AFR was 140.2 +/- 11.4. L-NAME suppressed the response induced by CGRP; the AFR was 23.4 +/- 5.5 (n = 8, P < 0.001). L-NAME abolished the conjunctival hyperaemia induced by PACAP-27 and -38 (0.3 nmol) and reduced the AFR. 5. The inflammatory cells that infiltrated the uvea, cornea and aqueous humour in large numbers in response to intravitreal injection of endotoxin were found to express inducible NOS. L-NAME prevented the appearance of such cells. 6. Our findings suggest that NO plays an important role in the endotoxin-evoked ocular inflammation in the rabbit: NO activates C-fibres causing release of C-fibre neuropeptides into the aqueous humour. In addition, NO mediates scme of the ocular effects of CGRP and PACAP, since L-NAME suppressed the AFR induced by these peptides.

Calcitonin gene-related peptide in experimental ischemia. Implication of an endogenous anti-ischemic effect.

Ischemia resulting from flap harvesting and vascular manipulation during microsurgery may be responsible for flap ischemic sufferance and, ultimately, necrosis. Recently, the regulatory role of the sensory nervous system in ischemia has attracted much interest. Calcitonin gene-related peptide (CGRP), a neuropeptide, is a naturally occurring vasodilator with no constrictive effects. In the present study, we developed a model of partial, chronic ischemia in the rat epigastric flap and investigated the effects of ischemia on concentrations of CGRP-like immunoreactivity (-LI) in ischemic skin and in different regions of the rat brain (striatum, hippocampus, pituitary, hypothalamus, and frontal and occipital cortex). A neurovascular island flap based on the superficial epigastric vessels was raised in 10 animals. Ischemia of the flap was obtained by ligating the feeding artery so that the blood flow was reduced to 25% of the normal circulation. An electromagnetic Doppler positioned on the artery was used to monitor the blood flow reduction. Ten nonoperated animals were used as controls. Ten days after the operation, CGRP-LI was significantly increased in five of six brain regions analyzed (striatum excepted). Significantly decreased concentrations of CGRP-LI were found in seven ischemic flaps, as opposed to the control group. In the remaining three flaps, no significant changes in CGRP concentration were observed. The highest blood flux values (detected using a laser Doppler) in the flaps correlated positively with the highest concentrations of CGRP-LI in the flap tissue. The results of the present study suggest that endogenous CGRP may be involved in the adaptive response to ischemia.

Adaptation of Rainbow Trout to Seawater: Changes in Calcitonin Gene-Related Peptide Levels Are Associated with an Increase in Hormone–Receptor Interaction in Gill Membranes

Transfer of trout from freshwater to seawater produced changes in plasma calcitonin gene-related peptide (CGRP), calcitonin, and calcium levels. Three days after transfer, the plasma CGRP concentration increased from 517 ± 54 (n= 6) to 1874 ± 234 (n= 7) pg/ml. Ten hours after transfer, the plasma CT concentration in seawater-adapted trout was double the control value. Plasma calcium levels also changed: 2 days after transfer, the calcium increased 1.6-fold over the basal value. The increased plasma CGRP level was correlated with an increased specific binding of CGRP to its branchial membrane receptors. Scatchard analysis of the data revealed this increase to be mainly due to increased binding affinity, as there was no difference in binding capacity between control and transferred animals. Carbonic anhydrase activity in branchial membranes increased 2.2-fold over the control value 3 days after transfer. CGRP may thus play a role in osmoregulation and ionic balance.

Quantitative analysis of cutaneous calcitonin gene-related peptide content in response to acute cutaneous mechanical or thermal stimuli and immobilization-induced stress in rats

The effects of various stimuli on restricted skin areas or immobilization-induced stress on the calcitonin gene-related peptide (CGRP) content in rat skin were examined by radioimmunoassay (RIA) and immunohistochemistry. Various stimuli were delivered to the shaven skin of the medial thigh by pinching, brushing, or contact with a glass tube containing hot (50°C) or ice-water for 2 min. To induce immobilization stress, animals were placed in the prone position and wrapped with flexible wire gauze at room temperature. The cutaneous CGRP content determined by RIA as well as the number of CGRP-immunoreactive nerve fibers of the skin were significantly higher at sites stimulated by pinching or ice-water compared to non-stimulated areas within the same animals. However, after brushing, hot water stimulation or any period (2 min, 30 min, 2 h, 6 h, or 3 days × 6 h) of immobilization stress, no differences in cutaneous CGRP content were observed. Plasma corticosterone levels increased after immobilization stress of 30 min or greater, but plasma CGRP level did not change after any period of immobilization stress. These data suggest that some forms of cutaneous stimulation cause a rapid rise in CGRP content in the skin, while emotional stress does not influence the cutaneous CGRP content.

Regulation of motoneuronal calcitonin gene-related peptide (CGRP) during axonal growth and neuromuscular synaptic plasticity induced by botulinum toxin in rats.

The aim of this study was to examine whether changes in rat motoneuronal calcitonin gene-related peptide (CGRP) can be correlated with axonal growth and plasticity of neuromuscular synapses. Nerve terminal outgrowth was induced by local paralysis with botulinum toxin. Normal adult soleus and tibialis anterior did not show detectable CGRP content at the motor endplates. Following botulinum toxin injection there was a progressive, transient and bimodal increase in CGRP in both motoneuron cell bodies which innervated poisoned muscles and their motor endplates. CGRP content was moderately increased 1 day after paralysis and, after an initial decline, reached a peak 20 days after injection. This was followed by a gradual decrease and a return to normal levels at the 200th day. CGRP changes in intoxicated endplates were less evident in the tibialis anterior than in the soleus muscle. The CGRP content in motoneurons was positively correlated with the degree of intramuscular nerve sprouting found by silver staining. In situ hybridization revealed an increase in CGRP mRNA in spinal cord motoneurons 20 days after toxin administration. We conclude that motoneurons regulate their CGRP in situations in which peripheral synapse remodelling and plasticity occur.

Acrylamide-induced neuropathic changes in rat enteric nerves: Similarities with effects of streptozotocin-diabetes

The effect of acrylamide intoxication (a widely used model for autonomic neuropathy) on the fluorescence intensity and density of catecholamine- and peptide-containing nerve fibres and tissue content of noradrenaline and the peptides vasoactive intestinal polypeptide, calcitonin gene-related peptide, substance P and neuropeptide Y in the enteric nerves of rat ileum was examined. Histochemical and immunohistochemical techniques were used to localize catecholamine- and peptide-containing nerve fibres. The tissue content of noradrenaline was measured using high-performance liquid chromatography, and an enzyme-linked immunosorbent assay technique was used to determine the tissue content of the peptides investigated. Acrylamide intoxication caused a significant decrease in the density of catecholamine-containing nerve fibres and tissue content of noradrenaline in the myenteric plexus of rat ileum. A decrease in tissue content and immunoreactivity of calcitonin gene-related peptide and an increase in vasoactive intestinal polypeptide was seen in the myenteric plexus of ileum from acrylamide-intoxicated rats. In the submucous plexus, the acrylamide treatment caused a decrease in calcitonin gene-related peptide immunoreactivity and an increase in vasoactive intestinal polypeptide and neuropeptide Y immunoreactivity. There was no change in either tissue content or immunoreactivity of substance P in both myenteric and submucous plexuses of the treated rat ileum. These changes have a striking similarity with those found in the enteric nerves of streptozotocin-diabetic rat ileum, suggesting the possible presence of an underlying common mechanism(s) in the development of neuropathic changes in the autonomic nerves of acrylamide-intoxicated and streptozotocin-diabetic rats.

Neural Isolation of the Jejunoileum: Effect on Tissue Morphometry, Mucosal Disaccharidase Activity, and Tissue Peptide Content

The aim of this study was to determine the effects of a model of intestinal extrinsic denervation on mucosal structure and function. Six dogs underwent in situ neural isolation of the jejunoileum (Group 2); six other dogs served as operated controls (Group 1), and five nonoperated dogs were naive controls (Group 3). Thirty-centimeter segments of proximal jejunum and distal ileum were excised before (time zero) and at 2 weeks and 8 weeks postoperatively in Groups 1 and 2, while similar regions were removed at time zero in Group 3. Tissues were analyzed for morphology with quantitative morphometry, mucosal disaccharidase activities (sucrase, maltase, and lactase), and tissue content of selected regulatory peptides in transmural, mucosa/submucosa, and muscularis regions.In situneural isolation had no significant or consistent effects on morphology/morphometry or on mucosal disaccharidase activities. Tissue content of neuropeptide Y decreased markedly (P< 0.002) in all layers of the jejunal and ileal walls, but tissue content of vasoactive inhibitory polypeptide, substance P, cholecystokinin, neurotensin, met-enkephalin, neurokinin A, somatostatin, and calcitonin gene-related peptide demonstrated only minor changes. The physiologic effects of intestinal transplantation (extrinsic denervation and disruption of intrinsic, enteric neural continuity, and lymphatic drainage) have little effect on morphology, mucosal disaccharidase activity, and tissue content of most regulatory peptides. How these minor alterations might affect enteric function, however, needs to be investigated.

Intraplantar injection of nerve growth factor into the rat hind paw: local edema and effects on thermal nociceptive threshold

Nerve growth factor (NGF) is known to produce hyperalgesia as well as to stimulate synthesis of neuropeptides in dorsal root ganglia (DRG). In the present study, we wanted to determine the effects of local NGF administration and assess to which extent mast cell-dependent factors are mediating NGF responses. Rats received 1 daily unilateral intraplantar injection for 3 days. Local edema (days 1–3), changes in thermal nociceptive threshold (days 1–4), and the content of calcitonin gene-related peptide (CGRP) and substance P (SP) in the sciatic nerve (day 4), were determined. NGF injection caused edema which was absent in rats pretreated with compound 48 80 as well as in rats treated neonatally with capsaicin (‘capsaicin denervation’). NGF-induced edema was not reduced by the neurokinin-1 receptor antagonist SR140333, but attenuated by the CGRP receptor antagonist CGRP[8–37]. On each day, NGF injection caused a decrease in thermal nociceptive threshold which lasted for less than 3 h. Capsaicin denervation, but not treatment with indomethacin, abolished NGF-induced thermal hyperalgesia. Treatment with compound 48 80 attenuated hyperalgesia produced by the first, but not by subsequent, NGF injections. On day 4, 24 h after the last of 3 NGF injections, thermal nociceptive threshold was not different from control values, but at that time, CGRP and SP were elevated in the sciatic nerve. We suggest therefore that NGF-induced local edema was caused by mast cell-derived vasoactive compounds which act together with afferent neuron-derived CGRP to increase vascular permeability. NGF-induced thermal hyperalgesia most likely was caused by an increased sensitivity of peripheral endings of capsaicin sensitive afferents. This effect of NGF was not mediated by products of the cyclooxygenase pathway, and was also observed in mast cell-depleted rats.

Effect of exercise and 2-deoxyglucose on the K + channel opener action of CGRP in the guinea pig ureter

1. 1. In the guinea pig isolated ureter, a maximally effective concentration of calcitonin gene-related peptide (CGRP, 0.1 μM) produced a prompt and transient suppression of myogenic phasic contractions (twitches) evoked by direct excitation (electrical field stimulation, EFS) of the smooth muscle. This suppressant effect is prevented by glibenclamide (1 and 10 μM), indicating the importance of K + channel activation in its genesis. In the presence of either 1 or 10 μM glibenclamide, CGRP produced a partial (about 30%) and delayed inhibition of the evoked response, but failed to produce a full suppression of twitches. 2. 2. The intensity and duration of the early, glibenclamide-sensitive suppressant effect of CGRP were inversely related to the frequency at which the ureters were driven by EFS. The glibenclamide-resistant inhibitory effect of CGRP was unaffected by changes in the EFS driving frequency, and cromakalim (3 μM) suppressed twitches independently of the EFS driving frequency. 3. 3. Replacement of 80% glucose in the Krebs solution with 2-deoxyglucose (2-DOG) reduced the amplitude of the EFS-evoked twitches. In the presence of 2-DOG the inhibitory effect of CGRP was enhanced and prolonged when tested in the absence, but not in the presence, of glibenclamide. 2-DOG counteracted the inhibitory effect produced by increasing the EFS driving frequency on the response to CGRP. 4. 4. In sucrose gap, both CGRP (0.1 μM) and cromakalim (3 μM) produced prompt hyperpolarization of the membrane. During continued superfusion for 15 min in unstimulated preparations, the hyperpolarizing effect of cromakalim and CGRP was sustained. When tested within 3 min from the end of ‘exercise’, induced by application of EFS at intervals of 15 sec for 30 min, the hyperpolarization by CGRP was reduced and shortened but that produced by cromakalim was unaffected. 5. 5. These findings demonstrate that exercise and metabolic inhibition selectively influence, in opposite directions, the K + channel opener action of CGRP in the guinea pig ureter, indicating that the ability of this neuropeptide to suppress latent pacemakers in smooth muscle is markedly dependent upon degree/ frequency of cell activation. These results suggest that the ability of endogenous CGRP to suppress ureteral motility may be inversely related to the frequency of ureteral peristalsis, the effect being reduced by, for example, increase in diuresis.

Calcitonin gene-related peptide innervation of the rat hepatobiliary system.

The digestive system is densely innervated by calcitonin gene-related peptide (CGRP)-immunoreactive neurons. The present study investigated a) the distribution and origin of CGRP-immunoreactive fibers in the rat hepatobiliary tract, and b) their relation with substance P/tachykinin (SP/TK) immunoreactivity using immunohistochemical and radioimmunoassay techniques. CGRP-containing fibers form dense networks in the fibromuscular layer of the biliary tree and surrounding the portal vein. Thin, varicose fibers are present at the base of the mucosa of the ducts. In the liver, labeled fibers are restricted to the portal areas and the stromal compartment. Neonatal treatment with capsaicin, a neurotoxin for primary afferent neurons, or celiac/superior mesenteric ganglionectomy depletes CGRP-containing fibers in the biliary tract, and reduces those associated with the portal vein. In contrast, subdiaphragmatic vagotomy does not appreciably modify the density of these fibers. Radioimmunoassay studies show a reduction of CGRP-immunoreactive contents in the biliary tract and portal vein by 84% and 65%, respectively, following capsaicin treatment, and by 80% and 66%, respectively, following ganglionectomy. By contrast, CGRP concentrations in vagotomized animals are comparable to those of controls. Most CGRP-positive fibers appear to contain SP/TK immunoreactivity, as indicated by double-label studies. These results demonstrate that the rat hepatobiliary tract is prominently innervated by CGRP- and CGRP/SP/TK-immunoreactive fibers, which are likely to originate from spinal afferent neurons. The abundance of these fibers and their association with a variety of targets are in line with the involvement of these peptidergic visceral afferents in regulating hepatobiliary activities, including hemodynamic functions of the hepatic vasculature.

Relation of calcitonin gene-related peptide to systemic vasodilatation and central hypovolaemia in cirrhosis.

The aetiology of the reduced systemic vascular resistance and abnormal 'filling' of the vascular bed in cirrhosis is still obscure. As increased concentrations of the potent vasodilator calcitonin gene-related peptide (CGRP) have recently been reported in cirrhosis, we related CGRP to central and peripheral haemodynamics in patients with cirrhosis. Thirty-one cirrhotic patients and six control subjects underwent an investigation with determination of systemic haemodynamics and circulating CGRP. Circulating CGRP was significantly increased in patients with cirrhosis (P < 0.02) and covaried directly with the severity of cirrhosis (P < 0.02). The increased CGRP covaried negatively with the reduced systemic vascular resistance (P < 0.02), the reduced central blood volume (P < 0.01), and reduced central circulation time (P < 0.002) and positively with the non-central blood volume (P < 0.05). These results suggest that increased CGRP may play a role in the systemic vasodilatation in cirrhosis and may contribute to the abnormal distribution of the blood volume, which may lead to abnormal sodium and water handling.

Calcitonin gene-related peptide and nitric oxide are involved in ultraviolet radiation-induced immunosuppression

Contact hypersensitivity responsiveness to dinitrofluorobenzene is depressed in mice that are sensitized through skin sites exposed to ultraviolet (UV) radiation. Local impairment of contact hypersensitivity by UV has been associated with a reduction in antigen-presenting cell activity within UV-irradiated skin sites marked by a decrease in the density of Ia-positive epidermal Langerhans cells. Our recent studies have demonstrated that neurogenic mediators (e.g. calcitonin gene-related peptide (CGRP) and nitric oxide (NO) contribute to cutaneous inflammation following exposure of rats to high-dose UV radiation. Since CGRP and NO inhibit antigen presentation by dendritic cells in vitro, we have investigated the possible involvement of CGRP and NO in local immunosuppression in UV-irradiated rodents. Hindpaw skin of Sprague-Dawley rats and back skin of UV-susceptible C57BL/6 mice was exposed to acute UV radiation (2.0 J/cm 2 and 0.5 J/cm 2 , respectively). Alterations in cutaneous CGRP content were analyzed by a specific radioimmunoassay (RIA). In separate experiments, the CGRP receptor antagonist CGRP-(8–37) (10 −5 M) and the nitric oxide synthase inhibitor N G - nitro - L - arginine methyl ester ( L -NAME) (2 × 10 −5 M) were topically applied to UV-exposed skin before induction of contact hypersensitivity with dinitrofluorobenzene. Finally, we examined the effects of UV irradiation and epicutaneous application of CGRP on Ia-positive Langerhans cells by immunohistochemical analysis of epidermal sheets. It was found that UV exposure lead to a decrease in skin CGRP levels starting already 2 h after irradiation and reaching a minimum (less than 40% of non-irradiated control skin) at 6–12 h. Contact hypersensitivity reactions were significantly suppressed by UV radiation in rat skin (by 51%) and murine skin (by 80%). Topical administration of both CGRP-(8–37) and L -NAME before sensitization restored the capacity to respond to haptens applied to UV-exposed skin. Both UV exposure and topical CGRP reduced the density of Ia-positive epidermal cells. Our data indicate that CGRP may be released from sensory neurons following cutaneous UV irradiation and that CGRP and NO contribute to UV-induced local immunosuppression. Moreover, topical administration of CGRP or its antagonist may be able to modulate epidermal Langerhans cell activity in vivo.