Involvement of calcitonin gene-related peptide (CGRP) receptors in insulin-induced vasodilatation in mesenteric resistance blood vessels of rats

1. The vascular effect of insulin in the mesenteric resistance blood vessel and the role of calcitonin gene-related peptide (CGRP)-receptor in insulin-induced vascular responsiveness were investigated in rats.
2. The mesenteric vascular beds isolated from Wistar rats were perfused with Krebs solution, and perfusion pressure was measured with a pressure transducer. In preparations contracted by perfusion with Krebs solution containing methoxamine in the presence of guanethidine, the perfusion of insulin (from 0.1 to 3000 nM) caused a concentration-dependent decrease in perfusion pressure due to vasodilatation. The pD₂ value and maximum relaxation (%) were 6.94±0.22 and 43.9±5.2, respectively.
3. This vasodilator response to insulin was unaffected by 100 nM propranolol (β-adrenoceptor antagonist) plus 100 nM atropine (muscarinic cholinoceptor antagonist), 100 μM L-N^G-nitroarginine (nitric oxide synthase inhibitor), 1 μM ouabain (Na⁺-K⁺ ATPase inhibitor), or 1 μM glibenclamide (ATP sensitive K⁺-channel inhibitor).
4. In preparations without endothelium, perfusion of insulin produced a marked vasodilatation. The pD₂ value and maximum relaxation (%) were 7.62±0.21 and 81.0±4.6, respectively, significantly greater than in preparations with intact endothelium.
5. The vasodilator responses to insulin in the preparations without endothelium were significantly inhibited by CGRP[8–37], a CGRP receptor antagonist, whereas pretreatment with capsaisin, a toxin for CGRP-containing nerves, did not affect insulin-induced vasodilatation.
6. These results suggest that insulin induces non-adrenergic, non-cholinergic and endothelium-independent vasodilatation, which is partially mediated by CGRP receptors.

     

肽能神经递质在犬脊髓损伤后神经源性膀胱中的表达及意义

目的 探讨犬不同脊髓平面损伤后神经源性膀胱时肽能神经递质血管活性肠肽(VIP)、P物质(SP)、降钙素相关基因肽( CGR P)在膀胱中表达的变化规律及意义.方法 健康成年雌性家犬14只,分为4组,应用脊髓横断的方法建立犬神经源性膀胱动物模型.A组,胸8～9平面横断脊髓;B组,腰1～2平面横断脊髓;C组,腰7骶1平面横...     

Endothelium removal augments endothelium-independent vasodilatation in rat mesenteric vascular bed

BACKGROUND AND PURPOSE: The vascular endothelium regulates vascular tone by releasing various endothelium-derived vasoactive substances to counteract excess vascular response. We investigated whether the vascular endothelium regulates vasodilatation via released endothelium-derived contracting factors (EDCFs), by examining the effect of endothelium removal on responses to periarterial nerve stimulation (PNS) and various vasodilator agents. EXPERIMENTAL APPROACH: The rat mesenteric vascular bed was perfused with Krebs solution. Vasodilator responses to PNS and 5 min perfusion of vasodilator agents in preparations with endothelium were compared with those in the same preparations without endothelium. The endothelium was removed by 30 s perfusion with sodium deoxycholate. KEY RESULTS: Endothelium removal significantly augmented vasodilator responses to PNS and calcitonin gene-related peptide (CGRP), isoprenaline (beta-adrenoceptor agonist), SNP and 8-bromo-cGMP (8-Br-cGMP; cGMP analogue) but not BAY41-2272 (soluble guanylate cyclase activator). The augmentation of SNP-induced vasodilatation after denudation was much greater than that of CGRP- or isoprenaline-induced vasodilatation. In the preparations with an intact endothelium, L-NAME (nitric oxide synthase inhibitor) significantly augmented vasodilator responses to PNS and CGRP, isoprenaline, SNP and 8-Br-cGMP, but not BAY41-2272. Indomethacin (cyclooxygenase inhibitor) and seratrodast (thromboxane A(2) receptor antagonist), but not phosphoramidon (endothelin-1-converting enzyme inhibitor) or BQ-123 (selective endothelin type A receptor antagonists), significantly augmented vasodilator responses to PNS and CGRP, isoprenaline, SNP and BAY41-2272. CONCLUSION AND IMPLICATION: These results suggest that the endothelium in rat mesenteric arteries regulates and maintains vascular tone via counteracting not only vasoconstriction through releasing endothelium-derived relaxing factors, but also vasodilatation, in part by releasing an EDCF, thromboxane A(2).     

Inhibition by nociceptin of neurogenic inflammation and the release of SP and CGRP from sensory nerve terminals

Pretreatment with the novel neuropeptide nociceptin (20 μg kg⁻¹, i.p.) caused an inhibition of plasma extravasation evoked by antidromic stimulation of the saphenous nerve or by topical application of 1% mustard oil on the skin of the acutely denervated hindleg of the rat. In contrast, it did not affect non-neurogenic inflammation evoked by s.c. injection of bradykinin after chronic denervation. Release of substance P (SP) and calcitonin gene-related peptide (CGRP) from rat isolated tracheae in response to electrical field stimulation was diminished by nociceptin (100 nM). It is concluded that nociceptin inhibits the release of sensory neuropeptides from terminals of nociceptive neurones.     

Adrenergic stimulation-released 5-HT stored in adrenergic nerves inhibits CGRPergic nerve-mediated vasodilatation in rat mesenteric resistance arteries

BACKGROUND AND PURPOSE

5-HT is taken up by and stored in adrenergic nerves and periarterial nerve stimulation (PNS) releases 5-HT to cause vasoconstriction in rat mesenteric arteries. The present study investigated whether PNS-released 5-HT stored in adrenergic nerves affects the function of perivascular calcitonin gene-related peptide-containing (CGRPergic) nerves.

EXPERIMENTAL APPROACH

Rat mesenteric vascular beds without endothelium and with active tone were perfused with Krebs solution. Changes in perfusion pressure in response to PNS and CGRP injection were measured before (control) and after perfusion of Krebs solution containing 5-HT (10 µM) for 20 min. Distributions of 5-HT- and TH-immunopositive fibres in mesenteric arteries were studied using immunohistochemical methods.

KEY RESULTS

PNS (1–4 Hz) frequency dependently caused adrenergic nerve-mediated vasoconstriction followed by CGRPergic nerve-mediated vasodilatation. 5-HT treatment inhibited PNS-induced vasodilatation without affecting exogenous CGRP-induced vasodilatation, while it augmented PNS-induced vasoconstriction. Guanethidine (adrenergic neuron blocker), methysergide (non-selective 5-HT receptor antagonist) and BRL15572 (selective 5-HT1D receptor antagonist) abolished inhibition of PNS-induced vasodilatation in 5-HT-treated preparations. Combined treatment with 5-HT and desipramine (catecholamine transporter inhibitor), but not fluoxetine (selective 5-HT reuptake inhibitor), did not inhibit PNS-induced vasodilatation. Exogenous 5-HT inhibited PNS-induced vasodilatation, which was antagonized by methysergide. In immunohistochemical experiments, 5-HT-immunopositive nerves, colocalized with adrenergic TH-immunopositive nerves, were observed only in 5-HT-treated mesenteric arteries, but not in control preparations or arteries co-treated with desipramine.

CONCLUSIONS AND IMPLICATIONS

These results suggest that 5-HT can be taken up by and released from adrenergic nerves in vitro by PNS to inhibit CGRPergic nerve transmission in rat mesenteric arteries.     

Pre-synaptic nicotinic receptors evoke endogenous glutamate and aspartate release from hippocampal synaptosomes by way of distinct coupling mechanisms

Background and purpose:

Changes in extracellular fluid osmolarity, which occur after tissue damage and disease, cause inflammation and maintain chronic inflammatory states by unknown mechanisms. Here, we investigated whether the osmosensitive channel, transient receptor potential vanilloid 4 (TRPV4), mediates inflammation to hypotonic stimuli by a neurogenic mechanism.

Experimental approach:

TRPV4 was localized in dorsal root ganglia (DRG) by immunofluorescence. The effects of TRPV4 agonists on release of pro-inflammatory neuropeptides from peripheral tissues and on inflammation were examined.

Key results:

Immunoreactive TRPV4 was detected in DRG neurones innervating the mouse hindpaw, where it was co-expressed in some neurones with CGRP and substance P, mediators of neurogenic inflammation. Hypotonic solutions and 4α-phorbol 12,13-didecanoate, which activate TRPV4, stimulated neuropeptide release in urinary bladder and airways, sites of neurogenic inflammation. Intraplantar injection of hypotonic solutions and 4α-phorbol 12,13-didecanoate caused oedema and granulocyte recruitment. These effects were inhibited by a desensitizing dose of the neurotoxin capsaicin, antagonists of CGRP and substance P receptors, and TRPV4 gene knockdown or deletion. In contrast, antagonism of neuropeptide receptors and disruption of TRPV4 did not prevent this oedema. TRPV4 gene knockdown or deletion also markedly reduced oedema and granulocyte infiltration induced by intraplantar injection of formalin.

Conclusions and implications:

Activation of TRPV4 stimulates neuropeptide release from afferent nerves and induces neurogenic inflammation. This mechanism may mediate the generation and maintenance of inflammation after injury and during diseases, in which there are changes in extracellular osmolarity. Antagonism of TRPV4 may offer a therapeutic approach for inflammatory hyperalgesia and chronic inflammation.     

Influence of AMPA/kainate receptors on extracellular 5-hydroxytryptamine in rat midbrain raphe and forebrain

1. The regulation of 5-hydroxytryptamine (5-HT) release by excitatory amino acid (EAA) receptors was examined by use of microdialysis in the CNS of freely behaving rats. Extracellular 5-HT was measured in the dorsal raphe nucleus (DRN), median raphe nucleus (MRN), nucleus accumbens, hypothalamus, frontal cortex, dorsal and ventral hippocampus.
2. Local infusion of kainate produced increases in extracellular 5-HT in the DRN and MRN. Kainate infusion into forebrain sites had a less potent effect.
3. In further studies of the DRN and nucleus accumbens, kainate-induced increases in extracellular 5-HT were blocked by the EAA receptor antagonists, kynurenate and 6,7-dinitroquinoxaline-2,3-dione (DNQX).
4. The effect of infusing kainate into the DRN or nucleus accumbens was attenuated or abolished by tetrodotoxin (TTX), suggesting that the increase in extracellular 5-HT is dependent on 5-HT neuronal activity. In contrast, ibotenate-induced lesion of intrinsic neurones did not attenuate the effect of infusing kainate into the nucleus accumbens. Thus, the effect of kainate in the nucleus accumbens does not depend on intrinsic neurones.
5. Infusion of α-amino-3-hydroxy-5-methyl-4-isoxazolaproprionate (AMPA) into the DRN and nucleus accumbens induced nonsignificant changes in extracellular 5-HT. Cyclothiazide and diazoxide, which attenuate receptor desensitization, greatly enhanced the effect of AMPA on 5-HT in the DRN, but not in the nucleus accumbens.
6. In conclusion, AMPA/kainate receptors regulate 5-HT in the raphe and in forebrain sites.

     

The ORL-1 (NOP1) receptor ligand nociceptin/orphanin FQ (N/OFQ) inhibits neurogenic dural vasodilatation in the rat

The effects of the ORL-1 (NOP(1)) receptor ligand nociceptin (N/OFQ) and the nociceptin antagonists [Nphe(1)]N/OFQ-(1-13)-NH(2) (Nphe) and nocistatin (NST) on neurogenic dural vasodilatation (NDV) in the rat dura mater evoked by electrical stimulation of a closed cranial window were studied. The middle meningeal artery was visualised using intravital microscopy, and the vessel diameter analysed using a video dimension analyser. N/OFQ (1, 10, 100 nmol kg(-1); i.v., n=10) significantly and dose-dependently suppressed NDV maximally by 65% (P<0.01). Neither Nphe (100 nmol kg(-1); n=5) nor NST (100 nmol kg(-1); n=4) alone had an effect on NDV (P>0.05). Baseline vessel diameter was not significantly affected by application of N/OFQ, NST or Nphe. Application of the selective N/OFQ antagonist Nphe (10, 100 nmol kg(-1) i.v., n=8) dose-dependently and significantly (P<0.01) reversed the inhibition of NDV induced by application of N/OFQ (10 nmol kg(-1)). NST (10, 100 nmol kg(-1); n=7) failed to reverse the effects elicited by N/OFQ. Application of N/OFQ elicited a dose-dependent transient decrease in arterial blood pressure (P<0.01). Nphe dose-dependently reversed the cardiovascular effects induced by application of N/OFQ (10 nmol kg(-1)) (P<0.01),while NST did not alter the blood pressure reaction elicited by N/OFQ. The results show that N/OFQ inhibits NDV, an effect which is antagonised by Nphe, but not by NST. ORL-1 (NOP(1)) receptors located on trigeminal sensory fibres may be involved in the regulation of dural vessel diameter and hence may play a role in migraine pathophysiology.     

Local neurogenic regulation of rat hindlimb circulation: role of calcitonin gene-related peptide in vasodilatation after skeletal muscle contraction

1. The mechanism of neurogenic regulation of skeletal muscle circulation was studied in the hindlimb of anaesthetized rats in vivo. Regional blood flow (RBF) of the hindlimb was recorded with a pulsed Doppler flow probe positioned in the iliac artery.
2. A short period (1 min) of sciatic nerve stimulation at 10 Hz caused a sustained increase in RBF (from 2.0±0.2 to 3.7±0.2 kHz at the peak), but no appreciable change in either MBP or HR, suggesting that the nerve stimulation produced local vasodilatation of the peripheral vasculature. The hyperaemic response reached a peak within 15 s and characteristically remained above the basal level for more than 5 min after the cessation of nerve stimulation. The response was regarded as a secondary response brought about by the contraction of skeletal muscles since (+)-tubocurarine (0.73 μmol kg⁻¹, i.a.) almost abolished it.
3. Lignocaine (43 μmol kg⁻¹, i.a.) and capsaicin (0.33 μmol kg⁻¹, i.a.) significantly suppressed the hyperaemic response to skeletal muscle contraction, suggesting that capsaicin-sensitive sensory nerves contribute to the hyperaemia. In contrast, an inhibitor of NO synthase, N_ω-nitro-L-arginine methyl ester (1 μmol kg⁻¹ min⁻¹, i.v.), did not affect the hyperaemic response.
4. Serum levels of calcitonin gene-related peptide (CGRP) in iliac venous effluent significantly increased from 51±4 to 77±5 fmol ml⁻¹ during the hyperaemic response to skeletal muscle contraction. A bolus injection of CGRP (300 pmol kg⁻¹, i.a.) induced a long-lasting increase in RBF of the hindlimb. Moreover, CGRP(8–37) (100 nmol kg⁻¹ min⁻¹, i.v.), a specific CGRP₁ receptor antagonist, significantly suppressed the hyperaemic response, especially the sustained phase of the response which was almost abolished by this antagonist.
5. These results suggest that CGRP, which is released from peripheral endings of capsaicin-sensitive sensory nerves, partly mediates the hyperaemia evoked by skeletal muscle contraction of the rat hindlimb.

     

软脉灵对动脉粥样硬化鹌鹑血浆内皮素和降钙素基因相关肽的影响

目的:观察软脉灵对动脉粥样硬化鹌鹑血浆内皮素和降钙素基因相关肽的影响。方法:雄性日本鹌鹑60只,随机均分成6组,即正常组、动脉粥样硬化模型组、软脉灵低、中、高剂量(折合成生药7.5,15和30g.kg-1)组和阳性对照普伐他汀钠8 mg.kg-1组,每组10只。除正常组外其余5组鹌鹑给予高脂饲料16周制备动脉粥样硬化模型,给药组在造模的同时,灌胃给药,qd,持续16周。16周后采血,测血浆内皮素和降钙素基因相关肽以及血清胆固醇和三酰甘油的含量;并取主动脉,观察斑块分级及主动脉内膜厚度,制作病理切片。结果:与正常对照组比较,模型组动物在喂饲高脂饲料16周后主动脉内膜厚度显著增加,动脉粥样硬化斑块形成;其血浆内皮素升高,降钙素基因相关肽降低,血清总胆固醇和三酰甘油明显升高。与模型组比较,软脉灵组剂量依赖性的减轻动脉粥样硬化斑块的形成和主动脉内膜的厚度,降低血浆内皮素及血清总胆固醇和三酰甘油,升高降钙素基因相关肽。结论:软脉灵抗动脉粥样硬化的机制可能与其降低血浆内皮素和升高降钙素基因相关肽有关。     

Effects of dipyridamole and adenosine on vasoactive peptides calcitonin gene-related peptide and atrial natriuretic peptide in humans: role of sympathetic activation

1. It has been observed that dipyridamole (DIP) administration produces equivalent cardiovascular effects at lower systemic adenosine (ADO) plasma concentrations than those obtained with exogenous ADO infusion. This observation led to the identification of DIP for additional 'ischaemia-inducing' mechanisms, possibly based on sympathetic activation. 2. In turn, exogenous ADO administration has proven to elicit a complex neurohumoral response, including an increase in the plasma concentration of catecholamines, associated with augmented levels of the vasoactive peptides calcitonin gene-related peptide (CGRP) and atrial natriuretic peptide (ANP). More particularly, increases in CGRP seem to be dependent on sympathetic activation, while changes in ANP do not. 3. In order to clarify some aspects of the activity of DIP on neurohumoral systems, the effects of administration of DIP and ADO on plasma levels of noradrenaline (NA), CGRP and ANP were studied in healthy volunteers. Haemodynamic parameters were also monitored. 4. Infusion of exogenous ADO produced plasma levels of ADO as high as 1893+/-386 nmol/L, together with a significant increase in plasma levels of CGRP, ANP and NA. Similarly, the infusion of DIP produced augmented plasma concentrations of the examined parameters, with a peak plasma ADO concentration of 470+/-49 nmol/L. 5. At a given ADO plasma concentration of 450+/-10 nmol/L, the increase in CGRP and NA levels with DIP infusion was significantly higher than that observed following the infusion of ADO, whereas the increase in the plasma concentration of ANP following DIP infusion was very similar to that seen following ADO infusion. 6. The physiological background of these findings is based on evidence that DIP displays a greater sympathoexcitatory activity than does exogenous ADO and only the increase in plasma CGRP seems to be mediated, although indirectly, by beta-adrenoceptor stimulation. The exact mechanism of DIP-dependent sympathetic activation remains to be elucidated.     

Neuropeptide receptors as potential drug targets in the treatment of inflammatory conditions

Cross-talk between the nervous, endocrine and immune systems exists via regulator molecules, such as neuropeptides, hormones and cytokines. A number of neuropeptides have been implicated in the genesis of inflammation, such as tachykinins and calcitonin gene-related peptide. Development of their receptor antagonists could be a promising approach to anti-inflammatory pharmacotherapy. Anti-inflammatory neuropeptides, such as vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide, α-melanocyte-stimulating hormone, urocortin, adrenomedullin, somatostatin, cortistatin, ghrelin, galanin and opioid peptides, are also released and act on their own receptors on the neurons as well as on different inflammatory and immune cells. The aim of the present review is to summarize the most prominent data of preclinical animal studies concerning the main pharmacological effects of ligands acting on the neuropeptide receptors. Promising therapeutic impacts of these compounds as potential candidates for the development of novel types of anti-inflammatory drugs are also discussed.     

CGRP、AchE在功能性排便障碍大鼠模型中的表达

目的　观察降钙素基因相关肽（CGRP）和胆碱酯酶（AchE）在功能性排便障碍（FDD）大鼠模型结直肠中的表达,探讨FDD发生的可能机制。方法　SD大鼠随机分为空白对照组、低纤维饮食组、利多卡因组、模型组,空白对照组予以普通饲料饲养,其余组采用低纤维饲料饲养;利多卡因组及模型组于饲养第63天,分别予以2 mL 2%利多卡因、0.1%亚甲蓝注射液肛周及直肠周围间隙注射1次。观察大鼠粪便性状、质量及排便功能;酶联免疫吸附试验（ELISA）检测结、直肠中AchE含量;实时荧光定量PCR（qPCR）检测结、直肠中CGRP mRNA的表达,免疫组化及Western blot法检测CGRP蛋白的表达。结果　肛周注射后,与空白对照组相比,低纤维饮食组和利多卡因组的粪便干结、呈深褐色,模型组的粪便干硬、呈黑褐色,粪便质量均下降。模型组模拟球囊排出时间较其余组长,肛管直肠静息压较其余组低;结肠及直肠中AchE含量较空白对照组和低纤维饮食组低（P＜0.05）;结肠中CGRP mRNA表达水平高于空白对照组,与低纤维饮食组和利多卡因组差异无统计学意义;直肠中CGRP mRNA表达水平较其余组高;免疫组化检测CGRP蛋白表达高于其余组（P＜0.05）;而Western blot检测各组CGRP蛋白表达差异均无统计学意义。结论　大鼠结直肠中CGRP及AchE的表达异常可能与FDD发生的神经机制有关。     

5-HT1b receptors in an animal model of depression

After exposure to a 0.8 mA course of uncontrollable shocks, Sprague-Dawley rats can be differentiated into two distinct groups defined in term of their performance in a shock escape paradigm. Learned helpless (LH) rats do not learn to escape a controllable shock while non-helpless (NLH) rats learn this response as quickly as naive controls (NC) rats do. The current experiments were designed to extend our studies of 5-HT receptors in these three groups of rats. The major finding in the present study concerned post-synaptic 5-HT receptor effects in the cortex, hippocampus, septum and hypothalamus of LH rats. These included an up regulation of 5-HT1b receptors in the cortex, hippocampus and septum in LH rats. In contrast, 5-HT1b receptors in the hypothalamus of LH rats were down-regulated. These results implicate serotonergic mechanisms in the behavioral deficit caused by uncontrollable shock with a limbic-hypothalamic circuit serving as a center for adaptation to stress.     

Activation of acetylcholine receptors and 5-HT2 receptors have additive effects in the suppression of neocortical high-voltage spindles in aged rats

We investigated if activation of the muscarinic or nicotinic acetylcholine receptors and serotonin (5-hydroxytryptamine; 5-HT) subtype 2 receptors would have additive or synergistic effects on the suppression of thalamocortically generated rhythmic neocortical high-voltage spindles (HVSs) in aged rats. The 5-HT₂ receptor antagonist, ketanserin, at a moderate dose (5 mg/kg) prevented the ability of a muscarinic acetylcholine receptor agonist, (oxotremorine 0.1 mg/kg), and a nicotinic acetylcholine receptor agonist (nicotine 0.1 mg/kg), to decrease HVSs. At a higher dose (20 mg/kg), ketanserin completely blocked the decrease in HVSs produced by moderate doses of muscarinic acetylcholine receptor agonists (pilocarpine 1 mg/kg and oxotremorine 0.1 mg/kg), and by a high dose of nicotine (0.3 mg/kg), though not that produced by high doses of pilocarpine (3 mg/kg) and oxotremorine (0.9 mg/kg). The ability of a 5-HT₂ receptor agonist, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (0.1–1.0 mg/kg), to suppress HVSs was non-significantly modulated by the nicotinic acetylcholine receptor antagonist, mecamylamine (1–15 mg/kg), and the muscarinic acetylcholine receptor antagonist, scopolamine (0.03–0.3 mg/kg). The effects of the drugs on behavioral activity could be separated from their effects on HVSs. The results suggest that activation of the muscarinic or nicotinic acetylcholine receptors plus 5-HT₂ receptors has additive effects in the suppression of thalamocortical oscillations in aged rats. Received: 2 November 1996 /Final version: 7 February 1997     

The role of 5-HT1B and 5-HT1D receptors in the selective inhibitory effect of naratriptan on trigeminovascular neurons

The importance of 5-HT(1B) and 5-HT(1D) receptors in the actions of the anti-migraine drug naratriptan was investigated using the relatively selective 5-HT(1) receptor ligands SB224289 and BRL15572. Electrical stimulation of the superior sagittal sinus (SSS) in cats activated neurones in the trigeminal nucleus caudalis. Facial receptive fields (RF) were also electrically stimulated to activate the same neurones. Responses of these neurones to SSS stimulation were suppressed by iontophoretic application of naratriptan (5-50 nA). There were two distinct populations of neurones in the nucleus--those in deeper laminae in which the responses to SSS and RF stimulation were equally suppressed by naratriptan ('non-selective') and more superficial neurones in which only the SSS responses were suppressed by naratriptan ('selective'). Concurrent micro-iontophoretic application (50 nA) of the 5-HT(1D) antagonist BRL15572 antagonised the suppression by naratriptan of the response of 'selective' cells to SSS stimulation. Iontophoretic application of SB224289 (50 nA), a 5-HT(1B) antagonist, antagonised the suppression by naratriptan of responses of 'non-selective' cells to RF stimulation and, to a lesser extent, also antagonised the suppression of responses to SSS stimulation. Intravenous administration of SB224289 antagonised the suppression only of RF responses of "non-selective" neurons by naratriptan and intravenous administration of BRL15572 antagonised the suppression only of SSS responses of "selective" neurons by naratriptan. These results suggest that the response of nucleus caudalis neurons to stimulation of the sagittal sinus can be modulated by both 5-HT(1B) and 5-HT(1D) receptor activation, with the 5-HT(1D) receptors perhaps playing a greater role. The response to RF stimulation is more influenced by 5-HT(1B) receptor modulation with 5-HT(1D) receptors being less important. Therefore, this suggests that selective 5-HT(1D) agonists may be able to target the neuronal population, which is selectively involved in the transmission of dural inputs. We conclude that the central terminals of trigeminal primary afferent fibres contain 5-HT(1B) and 5-HT(1D) receptors. Primary afferents from the dura mater may predominantly express 5-HT(1D) receptors, while facial afferents may predominantly express 5-HT(1B) receptors. Activation of 5-HT(1D) receptors in particular may be important in the anti-migraine effect of naratriptan.     

Involvement of AMPA/kainate,NMDA, and mGlu5 receptors in the nucleus accumbens core in cue-induced reinstatement of cocaine seeking in rats

Rationale Nucleus accumbens glutamate transmission has been implicated in drug-seeking behavior, but the involvement of glutamate receptor subtypes in drug seeking maintained by drug-associated cues has not been fully investigated. Objective This study examined the effects of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate, N-methyl-d-aspartate (NMDA) and mGlu5 receptor blockade in the nucleus accumbens core on cue-induced reinstatement of cocaine seeking. Method Wistar rats were trained to self-administer cocaine and associate a compound stimulus (light and tone) with the drug under an FR4(FR5:S) second-order schedule of reinforcement. After extinction, during which neither cocaine nor the compound stimulus was available, responding was reinstated by contingent presentations of the compound stimulus. The effects of the intra-accumbal AMPA/kainate receptor antagonist 6-cyano-7-nitro-quinoxaline-2, 3-dione (CNQX; 0, 0.01, and 0.03 μg/side), the NMDA antagonist d-2-amino-5-phosphonopentanoate (D-AP5; 0, 1, and 2 μg/side), and the mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP; 0, 0.5, and 1 μg/side) on reinstatement were examined in a within-subjects design. Results CNQX and D-AP5 attenuated cue-induced reinstatement of cocaine seeking dose-dependently. MPEP, however, decreased cocaine seeking only relative to baseline because also the saline vehicle included in the within-subjects series of injections decreased responding, possibly reflecting conditioned anhedonic effects of MPEP. In additional experiments, none of the antagonists attenuated locomotor activity or responding for sucrose pellets. Conclusions The results suggest that cue-induced reinstatement of cocaine seeking after a period of withdrawal from cocaine is sensitive to AMPA/kainate and NMDA receptor antagonism in the nucleus accumbens core and give further evidence for the role of the accumbal glutamate transmission in modulation of drug-seeking behavior.     

Specific interaction of 5-HT-moduline with human 5-HT1b as well as 5-HT1d receptors expressed in transfected cultured cells

5-HT_1B receptors are the predominant auto- and heteroreceptors located on serotonergic and non-serotonergic terminals where they regulate the neuronal release of neurotransmitters. 5-HT-moduline (Leu-Ser-Ala-Leu) has been shown to specifically interact with a very high apparent affinity and in a non-competitive manner with 5-HT_1B receptors (Massot et al. 1996; Rousselle et al. 1996). Using transfected cells expressing either 5-HT_1B or 5-HT_1D receptors, it was shown that 5-HT-moduline prevents the binding of [³H]5-HT to 5-HT_1B as well as to 5-HT_1D receptors with similar biochemical characteristics: the IC₅₀ of the peptide was 1.2×10^–12 M for 5-HT_1B and 9×10^–13 M for 5-HT_1D receptors. The observed effect corresponds to a marked decrease of the maximal binding for [³H]5-HT on 5-HT_1B (–51.2±1%) as well as 5-HT_1D binding (–47.2±7.7% of the control binding) whereas the affinity of 5-HT is increased by a factor close to 3. No effect is observed using the “scrambled” peptide (Ala-Leu-Leu-Ser). Parallel assays using transfected cells expressing 5-HT_1A or 5-ht₆ receptors did not show any significant change induced by the peptide under similar assay conditions. The interaction of the peptide was also studied on the functional activity related to the stimulation of the receptors as measured by the increase in [³⁵S]GTPγS binding reflecting the coupling of the receptor to the G-protein. 5-HT-moduline yields an antagonistic effect on the 5-HT induced coupling with a corresponding IC₅₀=1.2±0.7×10^–12 M for 5-HT_1B and 9.8±4.0×10^–12 M for 5-HT_1D receptors, respectively. The present results demonstrate that 5-HT-moduline interacts with 5-HT_1D as well as 5-HT_1B receptors and possesses a non-competitive antagonistic activity, likely corresponding to its role of endogenous allosteric modulator, specific for both 5-HT_1B and 5-HT_1D receptors. Received: 26 March 1998 / Accepted: 22 May 1998     

Antagonism of 5-HT(2A) receptors inhibits the expression of pronociceptive mediator and enhances endogenous opioid mechanism in carrageenan-induced inflammation in rats

We have recently reported that treatment with the 5-HT_2A receptor antagonist ketanserin in the inflamed paw raises the nociceptive threshold above normal level (hypoalgesia) and this response is naloxone-reversible. The present study aimed to investigate neurochemical changes at the site of inflammation and in dorsal root ganglia (DRG) and the spinal cord following the blockade of 5-HT_2A receptors. Intraplantar injection of ketanserin (20 μg) inhibited carrageenan-induced increase in CGRP immunoreactivity-positive neurons in DRG. On the other hand, administration of ketanserin (20 μg) and 5-HT (10 μg), but not vehicle, enhanced and inhibited recruitment of β-endorphin-expressing immune cells, respectively, in subcutaneous loci of inflamed hindpaw. Moreover, the treatment with ketanserin increased the number of endomorphine-containing cells in the inflamed paw and μ-opioid receptor-expressing neurons in DRG at L4–5 but reduced the expression of endomorphine in superficial layers of the lumbar spinal cord. The present study provided evidence at the cellular level showing that the blockade of 5-HT_2A receptors inhibited inflammation-associated increase in pronociceptive mediator, and that the pronociceptive property of 5-HT is mediated by the suppression of inflammation-activated opioid mechanism. Therefore, targeting the 5-HT_2A receptors in the site of inflammation may be a promising approach to inhibit inflammatory pain.     

Activation of midbrain presumed dopaminergic neurones by muscarinic cholinergic receptors: an in vivo electrophysiological study in the rat

1. Extracellular single-unit recording and iontophoresis were used to examine the effects of different cholinoceptor agonists and antagonists on the firing rate and firing pattern of A9 and A10 presumed dopaminergic neurones in the anaesthetized rat.
2. Administration of low currents (1–5 nA) of the selective muscarinic agonists oxotremorine M (Oxo M) and muscarine and of the non-selective muscarinic/nicotinic agonist carbamylcholine (CCh) produced a dose-dependent increase in firing rate in most of the A9 and A10 presumed dopaminergic neurones tested. Oxo M-induced activation could be completely blocked by iontophoretic application of the muscarinic antagonist butyl-scopolamine or systemic administration of the muscarinic antagonist scopolamine (300 μg kg⁻¹, i.v.).
3. Iontophoretic application of the selective nicotinic agonist methylcarbamylcholine (MCCh), but not nicotine, induced a consistent increase in firing rate. Surprisingly, the excitatory effect of MCCh was significantly reduced by the selective muscarinic antagonist scopolamine (300 μg kg⁻¹, i.v.), but not by the selective nicotinic antagonist mecamylamine (2.2 mg kg⁻¹, i.v.). Mecamylamine (3 mg kg⁻¹, i.v.) was also ineffective in reducing the CCh-induced activation of presumed dopamine neurones, suggesting that both CCh and MCCh increased the activity of dopamine neurones via an interaction with muscarinic receptors.
4. Iontophoretic application of the endogenous agonist acetylcholine (ACh) had no or little effect on the firing activity of A10 presumed dopaminergic neurones. However, concomitant application of neostigmine, a potent cholinesterase inhibitor, with acetylcholine induced a substantial activation of these neurones. This activation consisted of two components; one, which was prevalent, was scopolamine (300 μg kg⁻¹, i.v.)-sensitive, and the other was mecamylamine (2 mg kg⁻¹, i.v.)-sensitive.
5. In addition to their effect on firing activity, Oxo M, muscarine and concomitant neostigmine/ACh caused a significant increase in burst firing of A10 neurones, but not of A9 neurones.
6. These data suggest that dopamine cells, both in the A9 and A10 regions, possess functional muscarinic receptors, the activation of which can increase their firing rate and, for A10 neurones, their amount of burst activity. These cholinoceptors would be able to influence the activity of the midbrain dopamine system greatly and may play a role in, and/or be a therapeutic target for, brain disorders in which dopamine is involved (e.g., Parkinson''s disease, drug addiction and schizophrenia).