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Bradykinin and prostaglandins are both local mediators strongly implicated in pain and inflammation. Here, we have investigated the effects of bradykinin on the release of prostaglandin E(2) from cultured neurones derived from adult rat trigeminal ganglia. Bradykinin was a potent inducer of prostaglandin E(2) release, an effect that was likely mediated by bradykinin B(2) receptors, as the bradykinin-induced prostaglandin E(2) release was attenuated by the bradykinin B(2) receptor-selective antagonist, arginyl-L-prolyl-trans-4-hydroxy-L-prolylglycyl-3-(2-thienyl)-L-alanyl-L-seryl-D-1,2,3,4-tetrahydro-3-isoquinolinecarbonyl-L-(2 alpha, 3 beta, 7a beta)-octahydro-1H-indole-2-carbonyl-L-arginine (HOE 140), but not by the bradykinin B(1) receptor-selective antagonist, des-Arg(9),[Leu(8)]-bradykinin. Furthermore, bradykinin-induced prostaglandin E(2) release was inhibited following treatment with the phospholipase A(2) inhibitor, arachidonyltrifluoromethyl ketone (AACOCF(3)), the nonselective cyclooxygenase inhibitor, piroxicam, the mitogen-activated protein kinase kinase-1 (MEK1) inhibitor, 2'-amino-3'-methoxyflavone (PD98059), and the protein kinase C inhibitor, bisindolylmaleimide XI (Ro320432). Taken together, these data suggest that bradykinin, acting via bradykinin B(2) receptors, induces prostaglandin E(2) release from trigeminal neurones through the protein kinase C and mitogen-activated protein kinase-dependent activation of phospholipase A(2) and consequent stimulation of cyclooxygenases.

The purpose of this study was to use intravital microscopy to determine the effect of a selective adenosine A1 receptor agonist, GR79236 (1, 3 and 10 microg/kg i.v.), on neurogenic dural blood vessel dilation in anaesthetized rats. Vasodilation was evoked either by electrical stimulation of perivascular trigeminal nerves or by intravenous CGRP. GR79236 (1-10 microg/kg i.v.) caused a dose-dependent inhibition of neurogenic vasodilation, but had no significant effect on dural vasodilation caused by CGRP. GR79236 (1-3 microg/kg i.v.) had no effect on basal dural vessel diameter, but caused transient dose-dependant bradycardia and hypotension. Bradycardia was more prolonged following 10 microg/kg i.v. GR79236. Pre-treatment with the adenosine A1 receptor antagonist DPCPX (1 mg/kg i.v.) prevented the inhibitory effect of GR79236 (10 microg/kg i.v.) on neurogenic vasodilation as well as GR79236-induced bradycardia and hypotension. These data suggest that the inhibition of neurogenic vasodilation by GR79236 is mediated via the activation of prejunctional adenosine A1 receptors. Provided the systemic cardiovascular effects could be limited, such a mechanism may offer a novel approach to migraine therapy.

We have identified a migraine locus on chromosome 19p13.3/2 using linkage and association analysis. We isolated 48 single-nucleotide polymorphisms within the locus, of which we genotyped 24 in a Caucasian population comprising 827 unrelated cases and 765 controls. Five single-nucleotide polymorphisms within the insulin receptor gene showed significant association with migraine. This association was independently replicated in a case-control population collected separately. We used experiments with insulin receptor RNA and protein to investigate functionality for the migraine-associated single-nucleotide polymorphisms. We suggest possible functions for the insulin receptor in migraine pathogenesis.

1. Prostaglandins and the vasodilator neuropeptide, calcitonin-gene related peptide (CGRP), have both been implicated in the pathogenesis of migraine headache. We have used primary cultures of adult rat trigeminal neurones to examine the effects of prostanoids on CGRP release in vitro. 2. CGRP release was stimulated by prostaglandin E2 (PGE2) and the IP receptor agonist, carbaprostacyclin (cPGI2). These responses were extracellular calcium-dependent, and the PGE2-induced CGRP release was unaltered by inhibition of nitric oxide synthase (NOS), ATP receptor blockade, or the addition of adenosine deaminase. 3. Increases in CGRP levels were also observed in response to prostaglandin D2 (PGD2), and the EP2 receptor selective agonist, butaprost. No increases in CGRP release were observed in response to prostaglandin F2alpha (PGF2alpha) or the TP receptor selective agonist, U46619, or the EP3 receptor selective agonist, GR63799X. 4. The selective DP receptor antagonist, BWA868C, antagonized the PGD2-, but not PGE2- or cPGI2-induced release. Furthermore, the EP1 selective antagonist, ZM325802, failed to antagonize the PGE2-induced CGRP release from these cells. 5. These data indicate that activation of DP, EP and IP receptors can each cause CGRP release from trigeminal neurones, and suggest that the predominant EP receptor subtype involved may be the EP2 receptor. Together with evidence that the cyclo-oxygenase inhibitor, aspirin, particularly when administered intravenously is effective in treating acute migraine, these findings further suggest a role for prostaglandins in migraine pathophysiology.

Calcitonin gene-related peptide (CGRP), a potent vasodilator, has been implicated in the pathogenesis of migraine. Its release from adult rat trigeminal neurons in culture was shown to be markedly increased by the activation of adenylate cyclase with forskolin. Modulation of this secretion was investigated by a number of agents with known inhibitory effects on cAMP generation mediated via receptor coupling to G(i/o) proteins. Significantly, forskolin-stimulated CGRP release could be closely correlated with the phosphorylation of the protein kinase A (PKA) substrate cyclic AMP response element-binding protein (CREB). Forskolin-stimulated CGRP release could be potently and effectively inhibited by the adenosine A(1) receptor-selective agonist GR79236X (pIC(50) = 7.7 +/- 0.1, maximal inhibition 65 +/- 2.5% at 300 nM), whereas the A(2A) (CGS21680) and the A(3) (2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide) receptor-selective agonists were without effect. GR79236X-mediated inhibition was abolished by the A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. Immunocytochemical studies and Western analysis revealed the presence of adenosine A(1) receptors on trigeminal neurons. However, despite the additional detection of 5-hydroxytryptamine (5-HT)(1B) receptors on these cells, the clinically effective antimigraine 5-HT(1B/1D) agonist sumatriptan did not inhibit forskolin-stimulated CGRP release nor did it show any effect on the concomitant CREB phosphorylation. In contrast, the mu-opioid agonist fentanyl elicited a 74 +/- 4% reduction in CGRP levels. Forskolin-stimulated CGRP release and CREB phosphorylation could be mimicked by incubation of the cells with chlorophenylthio-cAMP and blocked by pretreatment with the PKA inhibitor myrPKI(14-22). Taken together, the present data confirm the PKA-dependence of forskolin-stimulated CGRP release and suggest that A(1) adenosine agonists may warrant further investigation in models of migraine and neurogenic inflammation.

There is controversy as to whether somatostatin sst(4) receptors internalize. In this study, CHO-K1 cells expressing human sst(4) receptor (CHOsst(4) cells) cells internalized [(125)I]-[(11)Tyr]-SRIF in a time-dependent manner, reaching a steady state at 60 min (1.4+/-0.1x10(4) molecules internalized per cell). Internalization was blocked by hypertonic sucrose (0.5 M), ATP depletion or by decreasing the temperature to 4 degrees C. Internalization of [(125)I]-[(11)Tyr]-SRIF was also inhibited (pIC(50) values) by increasing concentrations of SRIF (7.74), L-362855 (6.27) and NNC-296100 (6.50) with pIC(50) values approximately 10 fold lower than those obtained for inhibition of [(125)I]-[(11)Tyr]-SRIF binding to membrane homogenates. Internalized ligand recycled rapidly to the extracellular media (t(1/2) 3.9+/-0.7 min) with only 6.8+/-0.6% of internalized radioactivity remaining in the cell after 45 min. Confocal microscopy of permeabilized, HA-epitope tagged CHOsst(4) cells labelled with a Cy-3 conjugated antibody revealed little internal immunostaining after SRIF (1 microM) treatment, consistent with the small proportion of receptors (3.5%) estimated to be internalized by radioimmunoassay. In summary, CHOsst(4) cells internalized [(125)I]-[(11)Tyr]-SRIF in a clathrin- and ATP-dependent manner with subsequent rapid recycling to the extracellular medium. Rapid receptor recycling and the consequent low proportion of receptors internalized at any one time may explain the inability to visualize internalized receptors by confocal microscopy. It seems unlikely therefore that the marked receptor desensitization observed in CHOsst(4) cells following SRIF treatment can be accounted for by a decrease in cell surface receptor expression.

Constrained analogues 5-7 of the potent and subtype selective somatostatin mimetic 1 were prepared by incorporating conformational constraints into the nine-membered heterocyclic scaffold. Each constrained peptidomimetic showed an altered activity profile relative to lead compound 1, with compound 7 exhibiting a 25-fold and 2-fold binding enhancement against somatostatin receptor subtypes sst4 and sst5, respectively.

The cyclic octapeptide, CYN-154806, inhibited specific [(125)I]-[Tyr(11)]-SRIF binding to CHO-K1 cell membranes expressing human recombinant somatostatin (SRIF) sst(2) receptors (pIC(50) 8. 58) or rat sst(2(a)) and rat sst(2(b)) receptors (pIC(50) 8.35 and 8. 10, respectively). The affinity of CYN-154806 at other human somatostatin receptor types was at least 100 times lower (pIC(50) 5. 41-6.48). In functional studies, CYN-154806 inhibited SRIF-induced increases in extracellular acidification (EAR) in CHO-K1 cells expressing h sst(2) receptors (pK(B) 7.92) but had no effect on UTP-induced increases in EAR. CYN-154806 also blocked SRIF-induced increases [(35)S]-GTPgammaS binding in CHO-K1 cell membranes expressing h sst(2) receptors as well as rat sst(2(a)) and rat sst(2(b)) receptors (pK(B) 7.81, 7.68 and 7.96, respectively). In marked contrast, no blockade was observed at h sst(5) receptors in concentrations as high 10 microM. The antagonistic activity of CYN-154806 was also studied in isolated tissue preparations that are known to express endogenous SRIF receptors. Thus CYN-154806 blocked SRIF, but not DAMGO-induced inhibition of neurogenic contractions in rat isolated vas deferens and guinea-pig ileum (pK(B) 7.79 and 7.49, respectively). CYN-154806 had no effect on SRIF-28 induced inhibition of neurogenic contractions in guinea-pig vas deferens. The results demonstrate that CYN-154806 is a highly potent specific and selective SRIF sst(2) receptor blocking drug. Furthermore, sst(2) receptors mediate SRIF-induced inhibition of neurogenic contractions in rat vas deferens and guinea-pig ileum but not guinea-pig vas deferens which is thought to be mediated by sst(5) receptors.

We have previously demonstrated in CHO-K1 cells expressing recombinant human sst(4) receptors that somatostatin-induced increases in extracellular acidification are susceptible to a marked desensitisation after pretreatment with somatostatin, but not the somatostatin analogue, L-362855. In the present study, we have examined the human sst(4) receptor-mediated stimulation of p44/p42 mitogen-activated protein (MAP) kinase to determine whether this response is susceptible to a similar agonist-specific desensitisation. Western analysis using phosphospecific antibodies revealed that both somatostatin and L-362855 induced a transient stimulation of MAP kinase which could be desensitised by pretreatment with somatostatin, but not L-362855. The selective phosphoinositide (PI) 3-kinase inhibitor, LY 249002, blocked both the somatostatin-induced increase in MAP kinase phosphorylation and extracellular acidification. However, the MEK1 inhibitor, PD 98059, blocked only the sst(4) receptor-mediated stimulation of MAP kinase and not the extracellular acidification response. In summary, the human sst(4) receptor is selectively desensitised by somatostatin and not by L-362855 and signals through two different PI 3-kinase linked pathways., (Copyright 1999 Academic Press.)

The peptide hormone somatostatin exhibits antiproliferative activity by interacting with the G protein-coupled sst2 or sst5 receptor types. We show here that somatostatin at the human recombinant sst4 receptor induced a concentration-dependent increase in proliferation (EC50 20 nM) with a maximal response 5-fold greater than that produced by its synthetic analog, L-362,855. Analysis of the phosphorylation status of extracellular signal-regulated kinase (ERK)1 and ERK2 showed temporal differences in the changes evoked by the agonists. Phosphorylation induced by somatostatin (100 nM) peaked 10 min after the application and produced a response that continued for at least 4 h. In contrast, L-362,855 (1 microM) showed transient phosphorylation that had declined to basal levels by 1 h. However, both agonists induced rapid and sustained tyrosine phosphorylation of signal transducer and activator of transcription 3 (STAT3) which was pertussis toxin-insensitive. Serine phosphorylation of STAT3 was only apparent after somatostatin treatment and was abolished by pertussis toxin or PD 98059, together with the associated increases in proliferation. Mitogen-activated protein/ERK kinase-1 inhibition also decreased the time interval over which somatostatin-induced ERK phosphorylation was observed (<2 h). We conclude that the difference in the magnitude of the proliferative response evoked by the two agonists at the sst4 receptor can be accounted for by their differential ability to phosphorylate STAT3 on serine residues and supports the concept that selective signaling can be achieved through pharmacological diversity.

1. The coupling of the human somatostatin sst5 receptor recombinantly expressed in Chinese hamster ovary (CHO-K1) cells to adenylate cyclase was investigated using receptor selective ligands. 2. Forskolin (10 microM)-stimulated adenosine 3': 5'-cyclic monophosphate (cyclic AMP) accumulation was inhibited by somatostatin-14 and a number of receptor-selective agonists with a rank order of agonist potency typical of the sst5 receptor. L-362,855 and BIM-23056 behaved as full agonists. At higher somatostatin-14 concentrations there was sub-maximal inhibition resulting in a bell-shaped concentration-effect relationship. Pertussis toxin (PTx; 100 ng ml(-1), 18 h) pre-treatment abolished agonist-mediated inhibition of cyclic AMP accumulation and markedly enhanced stimulation of cyclic AMP at higher agonist concentrations. 3. The concentration of prostaglandin E2 (PGE2) in the incubation media was raised 14 fold by 1 microM somatostatin-14 but was insufficient to stimulate adenylate cyclase activity via endogenous prostanoid receptors. 4. Pre-treatment with cholera toxin (ChTx; 20 microg ml(-1), 18 h) markedly inhibited sst5 receptor-mediated increases in cyclic AMP formation in intact cells. Somatostatin-14-stimulated cyclic AMP accumulation was also observed in sst5 receptor containing CHO-K1 membranes and was inhibited by the synthetic peptide Galphasacetyl-354-372-amide (100 microM) by 65.9+/-3.5%, implicating a Galphas protein involvement in this response. 5. Activation of Galphas proteins by somatostatin-14 could be demonstrated with [35S]-guanosine 5'-[gamma-thio]triphosphate ([35S]-GTPgammaS) binding and subsequent immunoprecipitation of 35S labelled Galphas proteins with anti-Galphas serum. 6. These data show that the sst5 receptor is very efficiently coupled in a negative manner to adenylate cyclase. However, at higher agonist concentrations the receptor can also mediate activation of adenylate cyclase by a mechanism apparently involving Galphas protein activation.

1. The operational characteristics of somatostatin (SRIF) sst4 receptors are poorly understood. In this study, we have characterized human recombinant sst4 receptors expressed in CHO cells (CHOsst4) by radioligand binding and microphysiometry. 2. Increasing concentrations SRIF or other SRIF receptor ligands inhibited specific [125I]-Tyr11-SRIF binding in CHOsst4 cell membranes with respective pIC50 values of SRIF (8.82), L-362855 (7.40), BIM-23027 (<5.5) and MK-678 (<5.5). 3. These ligands displayed agonist activity, producing concentration-dependent increases in rates of extracellular acidification (EAR) with pEC50 values of SRIF (9.6) and L-362855 (8.0), respectively. BIM-23027 and MK-678 were at least 1000 times weaker than SRIF. The SRIF maximum was about 40% of that observed with L-362855. 4. In the presence of SRIF (0.1-1 nM), concentration-effect curves to L-362855 were displaced to the right with a progressive reduction in the L-362855 maximum. 5. When cells were only exposed to a single maximally effective concentration of SRIF or L-362855, there was no difference in the magnitude of the agonist-induced increase in EAR. However, a second agonist challenge, 30 min later showed that responses to SRIF but not L-362855 were markedly desensitized. 6. When concentration-effect curves to SRIF and L-362855 were obtained by combining data from cells exposed to only a single agonist concentration, SRIF (pEC50 9.2) was approximately 20 times more potent than L-362855 (pEC50 8.0) but the maxima were the same. Responses to both SRIF and L-362855 were abolished by pertussis toxin. 7. SRIF and L-362855-induced increases in EAR were inhibited by N-ethyl isopropyl amiloride (10 microM) but were not modified by inhibitors of PKC (Go-6976), MAP kinase (PD-98059), tyrosine kinase (genistein) or tyrosine phosphatase (sodium orthovanadate). 8. The results suggest that SRIF-induced increases in EAR in CHOsst4 cells involved activation of the Na+/H+ antiporter and were mediated via Gi/Go G proteins. Responses to SRIF, but not L-362855, were subject to marked desensitization which may be a consequence of differential activation of receptor-effector coupling pathways.

1. The mouse somatostatin (SRIF) sst2 receptor exists in two splice variants, sst2(a) and sst2(b), which differ in their intracellular carboxy-termini only. The murine sst2(b) receptor was reported to be less prone to agonist-induced desensitization as compared with the sst2(a) receptor. To determine whether a sst2(b) splice variant with similar functional characteristics exists in the rat, we have isolated a cDNA fragment from rat gastric mucosa encoding a sst2(b) receptor and expressed the full-length protein in CHO-K1 cells for functional characterization. 2. This study provides the first evidence for the occurrence in the rat of the sst2(b) receptor, which has a 15 amino acid carboxy-terminus differing in composition to the 38 amino acid C-terminus of the rat sst2(a) receptor. 3. In CHO-K1 cells expressing rat recombinant sst2(a) or sst2(b) receptors, SRIF caused concentration-dependent increases in extracellular acidification rates (EAR) with pEC50 values of 9.0 and 9.9, respectively. Pre-treatment with pertussis toxin (Ptx) caused a rightward displacement of the SRIF concentration-effect curves with pEC50 values of 8.3 (sst2(a) and 8.4 (sst2(b)). 4. SRIF (3 pM-3 nM) also caused concentration-dependent inhibition of forskolin-stimulated cyclic AMP formation in CHO-sst2(a) cells (pIC50 10.5) and CHO-sst2(b) cells (pIC50 10.4). The degree of inhibition was less with higher concentrations of SRIF resulting in bell-shaped concentration-effect curves. Following pre-treatment with Ptx, the inhibitory effect of SRIF was abolished and SRIF caused only increases in cyclic AMP formation. 5. Both the SRIF-induced increases in EAR and inhibition of cyclic AMP formation were susceptible to agonist-induced desensitization, but this was less apparent following pre-treatment with Ptx. 6. This demonstrates that the operational characteristics of the recombinant rat sst2(a) and sst2(b) receptors are broadly similar. Both isoforms couple to Ptx-sensitive as well as -insensitive G proteins and are equally prone to agonist-induced desensitization.

1. A high density of receptors for somatostatin (SRIF) exists in the anterior cingulate cortex but their function is unknown. Whole-cell patch clamp recordings were made from visualized deep layer pyramidal cells of the rat anterior cingulate cortex contained in isolated brain slices to investigate the putative effects of SRIF and to identify the receptor subtype(s) involved. 2. SRIF (1-1000 nM) produced a concentration-dependent outward current which was associated with an increased membrane conductance, was sensitive to Ba2+ (300 microM - 1 mM), and was absent in the presence of a maximal concentration of the GABA(B) receptor agonist, baclofen (100 microM). These observations suggest the outward current was carried by K+ ions. 3. SRIF analogues also elicited outward currents with a rank potency order of (EC50, nM): octreotide (1.8)>BIM-23027 (3.7)>SRIF (20)=L-362,855 (20). BIM-23056 was without agonist or antagonist activity. Responses to L-362,855 were unlike those to the other agonists since they were sustained for the duration of the application. 4. The sst2 receptor antagonist, L-Tyr8Cyanamid 154806 (1 microM), had no effect alone but partially reversed responses to submaximal concentrations of SRIF (100 nM, 44+/-6% reversal) and L-362,855 (100 nM, 70+/-6% reversal) and fully reversed the response to BIM-23027 (10 nM). In contrast, L-Tyr8Cyanamid 154806 did not antagonize the response to baclofen (10 microM). 5. We conclude that SRIF activates a K+ conductance in anterior cingulate pyramidal neurones via an action predominantly at sst2 receptors.

1. Somatostatin (SRIF) exerts antiproliferative effects, and angiopeptin (an sst2/sst5 receptor-selective analogue) has recently been evaluated in clinical trials for the prophylaxis of restenosis following coronary angioplasty. Using an in vitro model of cell growth we have examined the effects of SRIF and angiopeptin on cell proliferation in CHO-K1 cells stably transfected with the human or rat recombinant sst2 or sst5 receptor and compared these with their effects on rat aortic vascular smooth muscle cells (VSMC) expressing endogenous somatostatin receptors. 2. In CHO-KI cells, expressing either human or rat recombinant sst2 or sst5 receptors, or in rat aortic VSMC, SRIF and angiopeptin (0.1-1000 nM) had no effect on basal re-growth of cells into a denuded area of a previously confluent monolayer. In contrast, basic fibroblast growth factor (bFGF, 10 ng ml(-1)) stimulated re-growth of these cells. 3. SRIF (0.1-1000 nM) caused a concentration-dependent inhibition of the bFGF-stimulated re-growth in CHO-K1 cells expressing human sst2 (h sst2) or sst5 (h sst5) receptors (pIC50=8.05+/-0.03 and 8.56+/-0.12, respectively). In contrast, angiopeptin (0.1-1000 nM) acted as a partial agonist at the h sst2 receptor (44.6+/-2.7% inhibition of the bFGF-stimulated re-growth at 100 nM; pIC50=8.69+/-0.25) but was devoid of any agonist activity at the h sst5 receptor. 4. In CHO-K1 cells stably expressing rat recombinant sst2 (r sst2) or sst5 (r sst5) receptors, SRIF (0.1-1000 nM) was able to inhibit the bFGF-stimulated re-growth (pIC50=7.98+/-24 and 8.50+/-0.12, respectively). Angiopeptin (0.1-1000 nM) caused a concentration-dependent inhibition of bFGF-stimulated re-growth at the r sst2 receptor (pIC50=8.08+/-0.24) but acted as a partial agonist at the r sst5 receptor (maximum response= 57.7+/-3.6% inhibition of bFGF-stimulated re-growth at 100 nM; pIC50=8.60+/-0.16). 5. Although angiopeptin was inactive as an agonist at the h sst5 receptor, 100 nM angiopeptin potently antagonized the SRIF-induced inhibition of proliferation in CHO h sst5 (estimated pKB= 10.4+/-0.3). 5-Hydroxytryptamine (0.1 nM-10 microM) also inhibited bFGF-stimulated re-growth (pIC50=8.36+/-0.11) and angiopeptin had no effect on this response (pKB<7). 6. SRIF (0.1-1000 nM) caused a concentration-dependent (pIC50=8.04+/-0.08) inhibition of bFGF-stimulated re-growth in VSMC, whereas angiopeptin displayed weak agonist activity, only inhibiting bFGF-stimulated re-growth at concentrations greater than 100 nM. Angiopeptin (100 nM) caused a rightward displacement of the concentration-effect curve to SRIF with an estimated pKB value of 7.70+/-0.12. 7. These findings suggest that the low intrinsic activity of angiopeptin at the h sst2 receptor, combined with its lack of agonist activity at the h sst5 receptor, may explain the poor clinical efficacy of angiopeptin in trials for coronary artery restenosis, which contrasts with encouraging data found in equivalent in vivo animal studies.

The mechanisms responsible for somatostatin (SRIF)-induced increases in intracellular Ca2+ concentration ([Ca2+]i) and subsequent desensitisation were studied in CHO-K1 cells expressing human sst5 receptors (CHOsst5 cells). To study the nature of the desensitisation, interactions with uridine triphosphate (UTP) were examined. SRIF (pEC50 7.10) and UTP (pEC50) 5.14) caused concentration-dependent increases in [Ca2+]i but the SRIF maximum was about 40% of that to UTP. SRIF-, but not UTP-, induced increases in [Ca2+]i were transient and abolished by pertussis toxin. SRIF and UTP caused sustained increases in Ins(1,4,5)P3 but the SRIF maximum was about 30% of that to UTP. Removal of [Ca2+]e attenuated the SRIF-induced peak rise in [Ca2+]i but had no effect on the peak increases in Ins(1,4,5)P3. UTP-induced increases in [Ca2+]i and Ins(1,4,5)P3 were attenuated in the absence of [Ca2+]e. Following pre-exposure to SRIF (1 microM) or UTP (100 microM) for 5 min, subsequent SRIF responses were desensitised. Similar results were obtained in the absence of [Ca2+]e. Pre-exposure to SRIF had no effect on subsequent responses to UTP but in the absence of [Ca2+]e, responses to UTP were attenuated. The results suggest that SRIF but not UTP-induced increases in [Ca2+]i in CHOsst5 cells are mediated by pertussis toxin sensitive G proteins and are caused by an entry of extracellular Ca2+ and release from an Ins(1,4,5)P3 sensitive Ca2+ store. Homologous or heterologous desensitisation of agonist-induced increases in [Ca2+]i could be demonstrated in the presence or absence of extracellular Ca2+ respectively, and the latter appeared to involve depletion of a common intracellular Ca2+ store.

1. The aim of the present study was to determine the effect of somatostatin (SRIF) on mitogen-induced regeneration of rat aortic vascular smooth muscle cells (VSMC) and for comparison Chinese hamster ovary (CHO)-K1 cells expressing human recombinant sst5 receptors (CHOsst5), following partial denudation of a confluent cell monolayer. Regeneration was assessed by measuring areas of recovery into the denuded area and by counting total cell numbers. 2. In VSMC, SRIF (0.1 nM - 1 microM) had no effect on the basal levels of regeneration but caused a concentration-dependent inhibition (pIC50 8.0-8.6) of the stimulated regeneration induced by submaximal concentrations of basic fibroblast growth factor (bFGF, 10 ng ml[-1]), platelet-derived growth factor-BB (PDGF, 5 ng ml[-1]) or endothelin-1 (ET-1, 100 nM). SRIF (pIC50 8.8) also inhibited bFGF-induced regeneration of CHOsst5 cells. 3. In VSMC, the inhibitory action of SRIF on the regeneration induced by bFGF (10 ng ml[-1]) was due to an anti-proliferative effect, rather than an effect on cell migration, as SRIF (0.1 nM - 1 microM) abolished bFGF-induced increases in total cell numbers. The bFGF-induced increase in cell numbers was also abolished by actinomycin D (0.1 microg ml[-1]). 4. The sst5 receptor-selective agonist, L-362,855 (pIC50 10.5), was about 100 times more potent than SRIF at inhibiting bFGF-induced regeneration of both VSMC and CHOsst5 cells whilst the sst2 receptor-selective agonist, BIM-23027 (pIC50 6.8), was approximately 20 times weaker than SRIF. 5. The sst5 receptor antagonist, BIM-23056 (100 nM), antagonized SRIF-induced inhibition of bFGF-induced regeneration in both VSMC and CHOsst5 cells (estimated pKB values 8.8 and 8.3, respectively). 6. SRIF-induced inhibition of bFGF-induced regeneration of VSMC and CHOsst5 cells was abolished by pretreating cells with pertussis toxin (100 ng ml[-1]) for 20 h. 7. These findings suggest that SRIF-induced inhibition of the proliferation of rat aortic VSMC is mediated via activation of receptors which are similar to human sst5 receptors. Furthermore this inhibitory effect is transduced via pertussis toxin-sensitive Gi/Go proteins.

We studied the activation of the human somatostatin5 receptor recombinantly expressed in CHO-K1 cells by using some newly available agonists and antagonists. Somatostatin-28 bound to this receptor with a higher affinity than somatostatin-14 and was more potent in increasing [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding. Somatostatin-14-induced [35S]GTPgammaS binding to membranes from this cell line was decreased in a concentration-related manner by increasing concentrations of GDP and sodium chloride. At 50 mM (low) sodium, agonist EC50 values for stimulating [35S]GTPgammaS binding were lower than those at 150 mM (high) sodium and were closer to their respective affinity estimates (dissociation equilibrium constants) for binding to the receptor in the absence of sodium. Both agonist binding to the high affinity state of the receptor and agonist-induced [35S]GTPgammaS binding were abolished by pertussis toxin pretreatment. The putative somatostatin5 receptor-selective ligand L-362,855, unlike somatostatin-14 and somatostatin-28, showed differential intrinsic activity for stimulation of [35S]GTPgammaS binding, behaving as a partial agonist in high sodium and a full agonist in low sodium. In contrast, BIM-23056 did not behave as an agonist under any conditions studied but was able to antagonize somatostatin-14-induced [35S]GTPgammaS binding. We conclude that measurement of [35S]GTPgammaS binding mediated by somatostatin receptor activation in the presence of different concentrations of sodium chloride provides a useful functional assay for assessing the relative agonist efficacies of novel ligands identified from radioligand binding studies.

The biological profile of naratriptan (N-methyl-3-(1-methyl-4-piperidinyl)-1H-indole-5-ethane-sulphonamide), a novel 5HT1B/1D receptor agonist, was investigated in a variety of experimental models of relevance to migraine. Naratriptan has high affinity for human recombinant 5HT1B and 5HT1D receptors (pKi = 8.7 +/- 0.03 and 8.3 +/- 0.1, respectively) and causes contractions of dog isolated basilar and middle cerebral artery (EC50 values of 0.11 and 0.07 microM, respectively). Naratriptan causes small contractions of human isolated coronary arteries (EC50 value of 0.17 microM; maximum contraction equivalent to 33% of 5HT maximum). In anaesthetized dogs, naratriptan causes selective vasoconstriction of the carotid arterial bed (CD50 dose = 19 +/- 3 micrograms kg-1) and, in anaesthetized rats, naratriptan selectively inhibits neurogenic plasma protein extravasation in the dura (ID50 = 4.1 micrograms kg-1). In a variety of antinociceptive tests, naratriptan has no effect even at high doses. In conscious rats and dogs, naratriptan has high oral bioavailability (71% and 95%, respectively). The data show that naratriptan is a selective agonist at 5HT1B/1D receptors, with a pharmacological profile very similar to that of sumatriptan, albeit 2-3 fold more potent. These observations, coupled with high oral bioavailability in animals, suggest that naratriptan has the profile of an orally effective anti-migraine drug.

1. We have functionally characterized the human recombinant somatostatin (SRIF) sst5 receptor in Chinese hamster ovary-K1 (CHOsst5) cells by measuring total [3H]-inositol phosphate ([3H]-InsPx) accumulation, in the presence of 10 mM LiCl, in cells labelled with [3H]-myo-inositol. 2. In CHOsst5 cells, SRIF, SRIF-28 and the cyclic hexapeptide, L-362,855, produced time- and concentration-related increases in [3H]-InsPx accumulation, with similar potency (pEC50 values of 6.5, 6.8 and 7.2, respectively). L-362,855 behaved as a partial agonist, producing approximately 30% of the SRIF maximum response. The other peptide analogues of SRIF, BIM-23027 and BIM-23056, were inactive as agonists. 3. Increasing concentrations of L-362,855 increased [3H]-InsPx accumulation and simultaneously produced rightward shifts of SRIF concentration-effect curves, with an estimated pKp value of 7.6, confirming that it was acting as a partial agonist. 4. BIM-23056, but not BIM-23027, potently antagonized SRIF-induced [3H]-InsPx accumulation, with an estimated pKB value of 7.4. BIM-23056 did not antagonize [3H]-InsPx accumulation induced by uridine 5'-triphosphate (UTP). 5. SRIF- but not UTP-induced [3H]-InsPx accumulation was inhibited by increasing concentrations of pertussis toxin (0.01-100 ng ml-1), indicating the involvement of pertussis toxin-sensitive G-proteins. 6. These findings show that the human recombinant sst5 receptor, when stably expressed in CHO-K1 cells, is able to mediate activation of phosphoinositide metabolism in a pertussis toxin-sensitive manner. In this system L-362,855 behaved as a partial agonist while BIM-23056 was a specific antagonist. These agents should provide useful tools for functionally characterizing endogenous SRIF receptors.

We have previously shown that the somatostatin (SRIF) sst2 receptor-selective peptide, BIM-23027, is a potent antisecretory agent in rat isolated distal colonic mucosa (RDCM) and in radioligand binding studies in RDCM membranes, it only maximally inhibited approximately 40% of [125I]-Tyr11-SRIF-14 binding (McKeen ES, Feniuk W, Humphrey PPA (1995) Naunyn-Schmiedeberg's Arch Pharmacol 352:402-411). The aim of this study was to characterise the BIM-23027-sensitive and -insensitive SRIF binding sites in more detail and to compare their properties with those of the recombinant sst2 receptor stably expressed in mouse fibroblast (Ltk-) cells. SRIF-14, SRIF-28, CGP-23996 and D Trp8-SRIF-14 abolished [125I]-Tyr11-SRIF-14 binding (pIC50 values, 8.7-9.7) but the competition curves had Hill slopes which were less than unity. Octreotide and L-362,855 inhibited binding over a wide concentration range (0.1 nM-1 microM) and inhibition of binding was incomplete at the highest concentration studied. BIM-23056 (pIC50 < 6.5) was a weak inhibitor of [125]-Tyr11-SRIF-14 binding. GTP gamma S decreased [125I]-Tyr11-SRIF-14 binding by 40%. Further binding experiments with [125I]-Tyr11-SRIF-14 were carried out in RDCM in the continuous presence of BIM-23027 (1 microM). Under these conditions, seglitide had no effect on [125I]-Tyr11-SRIF-14 binding at concentrations up to 10 microM, whilst SRIF-14 and SRIF-28 abolished specific [125I]-Tyr11-SRIF-14 binding in a manner which was consistent with the ligand binding to two sites. SRIF-14 and SRIF-28 displayed high affinity (pIC50 values of 7.8 and 7.3) for the remaining sites. Octreotide, L-362,855 and BIM-23056 were weak inhibitors of [125I]-Tyr11-SRIF-14 binding (pIC50 < 6.5). [125I]-BIM-23027 labelled a single population of SRIF binding sites in RDCM membranes and mouse fibroblast (Ltk-) cells stably expressing the human recombinant sst2 receptor. There was a significant correlation between the affinity estimates of a range of SRIF analogues at inhibiting [125I]-BIM-23027 binding in RDCM membranes and binding to the recombinant sst2 receptor in Ltk- cells, suggesting that the sites labelled by [125I]-BIM-23027 in RDCM are similar to the sst2 receptor. GTP gamma S (100 microM) decreased [125I]-BIM-23027 binding in RDCM by 60%. The results from these studies demonstrate that [125I]-Tyr11-SRIF-14 labels a heterogeneous population of high affinity SRIF binding sites in RDCM membranes. The majority of these sites are insensitive to GTP gamma S and display negligible affinity for the cyclic hexapeptides, BIM-23027 and seglitide. The remaining high affinity binding sites can be selectively labelled with [125I]-BIM-23027, are sensitive to GTP gamma S and show similar characteristics to the recombinant sst2 receptor which appears to mediate the antisecretory effects of SRIF in the mucosa (McKeen ES, Feniuk W, Humphrey PPA (1995) Naunyn-Schmiedeberg's Arch Pharmacol 352:402-411).

1. The aim of this study was to determine the location and functional characteristics of the somatostatin (SRIF) receptor type(s) which mediate inhibition of acid secretion in rat isolated gastric mucosa. 2. Gastrin (1 nM-1 microM), dimaprit (10 microM-300 microM) and isobutyl methylxanthine (IBMX, 1 microM-100 microM) all caused concentration-dependent increases in acid output. Responses to gastrin were almost completely inhibited by ranitidine (10 microM) at a concentration which abolished the secretory response to dimaprit. In contrast, responses to IBMX were not changed by ranitidine suggesting that IBMX acts directly on the parietal cell and not indirectly by releasing histamine from enterochromaffin-like (ECL) cells. 3. SRIF-14 (1 nM-1 microM) had no effect on basal acid output, but inhibited acid output produced by gastrin, dimaprit and IBMX in a concentration-dependent manner with respective EC50 values of 46, 54 and 167 nM. The peptidase inhibitors, amastatin (10 microM) and phosphoramidon (1 microM), had no effect on SRIF-induced inhibition of dimaprit stimulated gastric acid secretion. 4. The inhibitory effect of a range of SRIF analogues on gastrin-, dimaprit- and IBMX-induced acid secretion was also studied. Irrespective of the secretagogue used to increase acid output, the rank order of potencies was similar (BIM-23027 = seglitide = octreotide > SRIF-14 = SRIF-28 > L-362,855). The linear peptide BIM-23056 was devoid of agonist or antagonist activity in concentrations up to 1 microM. 5. The sst2 receptor selective peptides, BIM-23027, seglitide and octreotide were the most potent inhibitors of gastrin-, dimaprit- and IBMX-induced acid secretion suggesting that SRIF receptors resembling the recombinant sst2 receptors are involved. Furthermore, since dimaprit and IBMX stimulate gastric acid secretion independently of histamine release, sst2 receptor-mediated inhibition must occur at the level of the parietal cell itself.

We have investigated the effects of somatostatin (SRIF) and the linear octapeptide BIM-23056 on changes in intracellular calcium ion concentration ([Ca2+]i) and on the formation of inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) in CHO-K1 cells transfected with the human recombinant SRIF sst5 receptor. SRIF elicited concentration-dependent increases in [Ca2+]i, with a pEC50 of 7.02 +/- 0.06, while BIM-23056 (1 x 10(-7) M) behaved not as an agonist but as a potent, surmountable antagonist of these increases in [Ca2+]i. The SRIF concentration-effect curve for increases in [Ca2+]i was shifted rightward producing an estimated pKB for the antagonist of 8.0. BIM-23056 (1 x 10(-7) M) also significantly attenuated Ins(1,4,5)P3 increases due to SRIF, but had no effect on either basal or uridine 5'-triphosphate (UTP) (1 x 10(-4) M) stimulated increases in the levels of [Ca2+]i or Ins(1,4,5)P3.

1. In order to characterize somatostatin (SRIF) receptor inhibiting spontaneous firing of rat locus coeruleus neurones, and their transduction mechanism(S), extracellular recordings were obtained from a pontine slice preparation of rat brain containing the locus coeruleus (LC). LC neurones were identified by electrophysiological and pharmacological properties; spontaneous firing (characteristically 0.5-5 Hz) was reversibly and concentration-dependently inhibited by exogenously applied noradrenaline. 2. Spontaneous firing of LC neurones was reversibly and concentration-dependently inhibited by SRIF and the N-terminally extended form, somatostatin-28 (SRIF-28), with EC50 values of 15.1 and 19.4 nM, respectively. The synthetic SRIF analogues (octreotide, MK-678, BIM-23027 and L-362,855) also caused concentration-dependent inhibition of LC neurone firing with a rank order of agonist potencies compatible with actions at a receptor resembling the recombinant sst2 receptor. The putative sst3 selective agonist, BIM-23056, was without agonist or antagonist effect. 3. Addition of 100 nM desipramine significantly increased the efficacy of exogenously applied noradrenaline (EC50 values, 2.96 and 0.13 microM, absence and presence of desipramine, respectively) but did not significantly affect SRIF-induced inhibition (EC50 values, 15.6 and 8.0 nM, respectively). Furthermore, application of phenoxybenzamine (3 microM) abolished responses to NA, but did not affect responses to SRIF (EC50 = 14.1 nM). 4. Application of the cyclic AMP analogue, 8-bromoadenosine-cyclic monophosphate (8-Br-cyclic AMP; 500 microM), significantly increased the spontaneous firing rate of all neurones tested (223 +/- 24% over basal rate). Concentration-effect curves for SRIF constructed in the absence and presence of 8-Br-cyclic AMP had similar threshold concentrations, maxima and EC50 values. 5. Incubation of pontine slices in a modified artificial CSF containing 500 ng ml-1 pertussis toxin (PTX) for 18 h prior to extracellular recording affected neither the spontaneous firing of LC neurones, nor the inhibitory responses to muscimol (EC50 2.2 and 1.2 microM, absence and presence of PTX). However, inhibitory responses to SRIF were markedly attenuated. 6. We conclude that the inhibitory actions of SRIF on spontaneous firing of LC neurones are mediated directly by activation of somatodendritic SRIF receptors, and not indirectly by release of noradrenaline. The SRIF receptors involved appear to couple via a pertussis toxin sensitive G-protein, and elicit their response by a mechanism apparently independent of inhibition of cyclic AMP formation. The agonist profile of several selective and novel SRIF analogues suggests the identity of this receptor to be similar to the recombinant sst2 receptor.

1. The human recombinant somatostatin (SRIF) receptors, sst1 and sst2, have been stably expressed in mouse fibroblast (Ltk-) cells. Two stable clones, LSSR 1/20 and LSSR 11/13, expressing sst1 and sst2 receptors, respectively, have been used to characterize these receptor types using radioligand binding assays as well as measurements of changes in extracellular acidification rates using microphysiometry. 2. [125I]-[Tyr11]-SRIF bound to sst1 and sst2 receptors expressed in Ltk- cells with high affinity, Kd values being 1.52 nM, and 0.23 nM respectively. 3. In Ltk- cells expressing sst1 receptors, SRIF, SRIF-28, [D-Trp8]-SRIF and CGP 23996 all displaced [125I]-[Tyr11]-SRIF binding with high potency (IC50 values of 0.43 - 1.27 nM) whilst seglitide, BIM-23027, BIM-23056 and L-362855 were either weak inhibitors of binding or were ineffective. 4. In contrast MK-678 (seglitide) and BIM-23027 were the most potent inhibitors of [125I]-[Tyr11]-SRIF binding in Ltk- cells expressing sst2 receptors with IC50 values of 0.014 and 0.035 nM, respectively. 5. SRIF and a number of SRIF agonists, including seglitide and BIM-23027, caused concentration-dependent increases in extracellular acidification rates in Ltk- cells expressing sst2 receptors but not in Ltk- cells expressing sst1 receptors. The maximum increase in acidification rate produced by SRIF was 11.3 +/- 0.7% above baseline (0.1-0.28 pH unit min-1). The relative potencies of the SRIF agonists examined in causing increases in extracellular acidification rates in Ltk- cells expressing sst2 receptors correlated well with their relative potencies in inhibiting [125I]-[Tyr11] -SRIF binding (r = 0.94). 6. The increase in extracellular acidification produced by SRIF was markedly inhibited by pretreatment of cells with pertussis toxin (100 ng ml-1) indicating the involvement of pertussis toxin-sensitive G proteins. 7. SRIF (1 microM) had no effect on basal cyclic AMP levels in Ltk- cells expressing sst1 or sst2 receptors nor did it inhibit forskolin stimulated increases in cyclic AMP levels in either cell type. 8. The results from the present study describe the operational characteristics of human sst2 receptors expressed in Ltk- cells where receptor activation causes increases in extracellular acidification rates. This receptor is coupled to a pertussis toxin-sensitive G protein. In contrast, activation of sst1 receptors, at a similar transfection density, did not cause increases in extracellular acidification rates.

GR127935 is the most potent 5-HT1D receptor antagonist yet described, possessing nanomolar affinity at human 5-HT1D receptors. Sumatriptan-induced contractions of the dog isolated basilar artery and saphenous vein are antagonised by GR127935 in an insurmountable manner indicative of its slow dissociation from the 5-HT1D receptor. 5-HT1D receptor-mediated hypothermia and rotational behaviour in guinea-pigs are antagonised potently, and with long duration, by GR127935, administered by a variety of routes. GR127935 also blocks central 5-HT1D autoreceptors in vitro and in vivo. GR127935 has much lower affinity at other 5-HT, and non-5-HT, receptors. In functional studies, GR127935 fails to affect 5-HT2 receptor-mediated 'wet dog shakes' in guinea-pigs and 5-HT1A receptor-mediated inhibition of 5-HT release in rat dorsal raphé nucleus. The compound has a good safety profile in all species tested. It is concluded that GR127935 is a useful pharmacological tool to characterise 5-HT1D receptor function.

The kinetic properties, steady state binding characteristics and autoradiographic distribution of the somatostatin (SRIF) sst2 receptor-selective ligand, [125I]-BIM-23027, have been investigated in the rat central nervous system. Analysis of kinetic, saturation and competition binding data in rat hippocampal membranes was consistent with [125I]-BIM-23207 binding to a single population of non-interacting binding sites. Competition studies, using different SRIF ligands suggested that [125I]-BIM-23027 was binding to sites similar to that of the recombinant sst2 receptor. The rank order of affinity for displacing specific binding was BIM-23027 = SRIF > L-362855 > > BIM-23056. There was a widespread distribution of [125I]-BIM-23027 binding sites in the rat central nervous system. The highest density of binding was observed in the dentate gyrus, medial habenular, amygdala, claustrum and lateral septum as well as in the piriform, cingulate and parietal cortex. The cervical and lumbar spinal cord also displayed moderate levels of binding localized to the substantia gelatinosa. The cellular localization of [125I]-BIM-23027 binding was found to be associated with dendritic terminal fields. In contrast, the cellular signal for sst2 receptor mRNA was restricted to cell somata. The widespread distribution of [125I]-BIM-23027 binding sites within the brain suggests that receptors similar to the recombinant sst2 receptor may mediate a variety of different physiological effects within the central nervous system.

A range of somatostatin (SRIF) analogues have been used to characterize the SRIF receptor-mediating contraction of the human saphenous vein. SRIF produced concentration-dependent contractions with an EC50 value of approximately 20 nM. The peptidase inhibitors phosphoramidon and amastatin did not alter the potency of SRIF. The sst2 receptor-selective peptide BIM-23027 was approximately three times more potent than SRIF in contracting the vein, whereas the sst5 receptor-selective peptide L-362855 was approximately 50 times weaker. The sst3 receptor-selective peptide BIM-23056 did not contract the saphenous vein. Contractions to SRIF were not antagonised by the putative SRIF receptor blocker cyclo(7-aminoheptanoyl-Phe-D Trp-Lys-Thr[Bzl]) (CPP), phentolamine, or indomethacin. Decreasing the external calcium concentration reduced the maximum contraction to SRIF in a concentration-dependent manner without altering the EC50 value. Nifedipine and verapamil also markedly reduced the SRIF-induced contraction. SRIF and several SRIF analogues caused contraction of the human saphenous vein by what appeared to be a direct effect on the smooth muscle. Their relative potencies suggest that their effects were mediated by a somatostatin receptor that is like the recombinant sst2 receptor. The receptor transduction mechanism appears to involve activation of L-type calcium channels and entry of extracellular calcium.

The aim of this study was to examine the potencies of several recently identified selective somatostatin (SRIF)-receptor ligands as inhibitors of electrogenic ion transport in the rat distal colonic mucosa with the view to identifying the SRIF receptor type involved. Under basal conditions, cumulative administration of SRIF and SRIF28 decreased short circuit current (SCC), a measure of electrogenic ion transport, with EC50 values of 4 nM and 9 nM respectively. The peptidase inhibitors, phosphoramidon (1 microM) and amastatin (10 microM), has no effect on the potencies of either SRIF or SRIF28. The inhibitory action of SRIF on basal SCC was suppressed by piretanide and diphenylamine-2-carboxylate, compatible with the assumption that the Na+K+2Cl- co-transporter and Cl- channels, respectively, may be involved in this antisecretory action of SRIF. Tetrodotoxin (1 microM) had no effect on the antisecretory action of SRIF, suggesting that the process was not neuronally mediated. All of the SRIF analogues examined, with the exception of BIM-23056, maximally inhibited basal SCC to a similar extent as SRIF. Seglitide and octreotide were both more potent antisecretory agents than SRIF (respective EC50 values, 0.4 nM and 1.5 nM) suggesting that this effect was mediated by a receptor belonging to the SRIF1 receptor group. The most distinguishing feature of the rank order of agonist potencies was the high potency of the selective sst2 receptor ligand, BIM-23027 (EC50 value 0.32 nM), the weaker potency exhibited by the selective sst5 receptor ligand, L-362855 (EC50 value 21 nM), and the lack of agonist activity displayed by the selective sst3 receptor ligand, BIM-23056 (EC50 value > 1000 nM). This profile is comparable with that observed in binding studies on the recombinant sst2 receptor. Forskolin-stimulated secretion was suppressed by SRIF analogues with the rank order of agonist potencies BIM-23027 > SRIF > L-362855 >> BIM-23056 which resembled that exhibited under basal conditions. However, the absolute potencies of these agonists were lower (respective EC50 values 2 nM, 14 nM< 38 nM and > 1000 nM) whilst the magnitude of inhibition was about three fold greater. BIM-23027 and SRIF (both 30 nM) also inhibited carbachol-stimulated increases in basal SCC by 60-70%, while a similar concentration of L-362855 inhibited these responses by 11%. BIM-23056 (1 microM) had no effect on carbachol-simulated secretion. Radioligand binding studies on rat colonic mucosal membranes using [125I]-Tyr11-SRIF suggested heterogeneity of SRIF binding sites. Thus, SRIF and SRIF28 competed for binding (IC50 values, 0.32 and 0.63 nM, respectively) with Hill slopes less than unity; while seglitide and BIM-23027 both maximally displaced only 30-40% of specific binding with apparent high affinity (respective pIC50 values, 10.1 nM and 10.0). In conclusion, SRIF decreases basal as well as both cAMP and Ca(2+)-dependent Cl- secretion in rat colonic mucosa. The rank order of agonist potencies suggests that receptors resembling the recombinant sst2 receptor mediate inhibition of basal and forskolin-stimulated secretion. Radioligand binding studies suggest that BIM-23027 interacts with a sub-population of [125I]Tyr11-SRIF binding sites in rat colonic mucosal membranes which probably corresponds to the receptors mediating the antisecretory effects described here.

1. Somatostatin (SRIF) causes a concentration-dependent inhibition of neurotransmission in guinea-pig ileum and vas deferens as well as negative inotropy in guinea-pig isolated right atrium. The SRIF receptors mediating these effects have now been further characterized by use of the peptides BIM-23027, BIM-23056 and L-362855, reported as selective for the recombinant SRIF receptor types, sst2, sst3 and sst5, respectively. 2. BIM-23027 was a highly potent agonist at causing an inhibition of neurotransmission in the guinea-pig ileum (EC50 value 1.9 nM), being about 3 times more potent than SRIF (EC50 value 6.8 nM). In contrast, in both guinea-pig vas deferens and right atrial preparations, BIM-23027 was a relatively weak agonist being at least 30-100 times weaker than SRIF. In guinea-pig atria, BIM-23027 (3 microM) antagonized the negative inotropic action of SRIF28 (apparent pKB = 5.9 +/- 0.1) but had no effect on the negative inotropic action of cyclohexyladenosine. 3. The inhibitory effect of BIM-23027 in the guinea-pig ileum was readily desensitized. Prior exposure to BIM-23027 (0.3 microM) markedly attenuated the inhibitory effect of SRIF but had no effect on the inhibitory action of clonidine suggesting that BIM-23027 and SRIF act via a common receptor mechanism. 4. L-362855 caused a concentration-dependent inhibition of neurotransmission in both the guinea-pig ileum and vas deferens as well as causing negative inotropy in the guinea-pig atrium but was at least 30-100 times weaker than SRIF. In guinea-pig isolated atria, L-362855 (3 microM) did not antagonize the negative inotropic action of SRIF28. 5. BIM-23056 in concentrations up to 1 microM was inactive as an agonist in guinea-pig isolated ileum, vas deferens and atrium and did not antagonize the inhibitory actions of SRIF in any of these preparations.6. The results from this study support our previous contention that the sst2 receptor type mediates inhibition of neurotransmission by SRIF in the guinea-pig ileum. The SRIF receptor type mediating inhibition of neurotransmission in the guinea-pig vas deferens appears different, but similar, to that mediating negative inotropy in the atrium. However the characteristics of these latter receptors appear different from that of the recombinant sst2, sst3 and sst5 receptors for SRIF described for rat and man.

The involvement of 5-HT1D receptors in the regulation of 5-HT release in the guinea-pig brain was examined using the novel 5-HT1D receptor blocking drug GR127935. Levels of 5-HT were measured in frontal cortex of anaesthetized guinea-pigs using microdialysis. The infusion of GR127935 (100 nM) through the dialysis probe into frontal cortex caused a significant increase (61 +/- 8%) in cortical extracellular levels of 5-HT. The increase was transient (approximately 40 min) even in the continuous presence of GR127935. The transient increase was abolished by tetrodotoxin (1 microM). The 5-HT1 receptor agonist GR46611 (10 mg/kg s.c.) caused a significant and sustained (> 100 min) reduction in extracellular levels of 5-HT (65 +/- 5%). This response was abolished in animals pre-treated with GR127935, 0.05 mg/kg i.p. Paradoxically, systemic administration of higher doses of GR127935 (0.1-1 mg/kg i.p.) in naive anaesthetized guinea-pigs caused significant and sustained (> 120 min) decreases (> 65%) in cortical levels of 5-HT. The increase in extracellular 5-HT seen following infusion of GR127935 into frontal cortex may be due to GR127935 blocking 5-HT terminal autoreceptors causing a subsequent increase in the outflow of 5-HT from pre-synaptic terminals. This conclusion is supported by the ability of GR127935 to block the decrease in 5-HT induced by the 5-HT1 receptor agonist GR46611.(ABSTRACT TRUNCATED AT 250 WORDS)

There is considerable controversy about the classification and nomenclature of somatostatin receptors. To date, five distinct receptor genes have been cloned and named chronologically according to their respective publication dates, but two were unfortunately given the same appellation (SSTR4). Consensually, a nomenclature for the recombinant receptors has been agreed according to IUPHAR guidelines (sst1, sst2, sst3, sst4, and sst5). However, a more informative classification is to be preferred for the future, employing all classification criteria in an integrated scheme. It is already apparent that the five recombinant receptors fall into two classes or groups, on the basis of not only structure but also pharmacological characteristics. One class (already referred to by some as SRIF1) appears to comprise sst2, sst3 and sst5 receptor subtypes. The other class (SRIF2) appears to comprise the other two recombinant receptor subtypes (sst1 and sst4). This promising approach is discussed but it is acknowledged that much more data from endogenous receptors in whole tissues are needed before further recommendations on somatostatin receptor nomenclature can be made.

1. The motor effects of somatostatin-14 (SRIF), and several SRIF peptide analogues were investigated on the rat isolated distal colon. The objective of these studies was to characterize the receptor mediating the contractile action of SRIF by comparing the relative agonist potencies of a range of SRIF analogues. 2. SRIF (1 nM-1 microM) produced concentration-dependent contractions with an EC50 value of approximately 10 nM. Contractile responses induced by SRIF were insensitive to atropine (1 microM) or naloxone (1 microM) but abolished by tetrodotoxin (1 microM). Somatostatin-28 (SRIF28), also induced concentration-dependent contractions and was equipotent with SRIF. Phosphoramidon (1 microM) and amastatin (10 microM) did not increase the potency of either SRIF or SRIF28. 3. The SRIF peptide analogues, octreotide, SRIF25, seglitide, angiopeptin and CGP23996 (1 nM-1 microM) produced contractile responses in the rat distal colon, each having similar potency and maximal activity relative to SRIF. The SSTR2 receptor-selective hexapeptide, BIM23027 (0.1 nM-1 microM), and the SRIF stereoisomer, D-Trp8-SRIF (0.1 nM-1 microM), were the most potent agonists examined being approximately 12 and 7 times more potent than SRIF, respectively. In contrast, the SSTR5 receptor-selective analogue, L362,855, was approximately 120 times weaker than SRIF, whilst the SSTR3 receptor-selective analogue, BIM23056, was inactive at concentrations up to 3 microM. 4. The putative SRIF receptor antagonist, (cyclo(7-aminoheptanoyl Phe-D-Trp-Lys-Thr[Bzl]))(CPP) (1 microM), had no agonist activity and had no effect on contractions induced by SRIF. 5. The contractile actions of BIM23027 and seglitide were subject to pronounced desensitization. Desensitization of preparations by BIM23027 (0.3 JIM) abolished the contractile action of SRIF andSRIF28 but had no effect on contractions produced by acetylcholine (0.1 nM-I1M), suggesting thatBIM23027, SRIF and SRIF28 act via a common receptor mechanism.6. In conclusion, the rat isolated distal colon contracts in response to SRIF and a number of SRIF analogues. Seglitide and octreotide exhibited similar potency and maximal activity relative to SRIF,suggesting that in the rat colon the receptor mediating contraction belongs to the SRIF,-receptor group,of which the recombinant SSTR2, SSTR3 and SSTR5 receptors appear to be subtypes. The high potency of BIM23027, the weak agonist activity of L362,855 and the lack of activity exhibited by BIM23056suggests that the SRIF receptor mediating contraction in the rat distal colon is similar to there combinant SSTR2 receptor.

The potential effects of pertussis toxin pretreatment on the inhibitory effect of somatostatin (SRIF) and the selective SRIF receptor agonist, seglitide, were studied in mouse vas deferens and these were compared with its effect on the negative chronotropic action of carbachol in mouse atria. Somatostatin and seglitide caused a concentration-dependent inhibition of neurogenically mediated contractile responses in the vas deferens (EC50 values of 15 and 0.6 nM respectively). There was no difference in their potencies in preparations removed from pertussis toxin pretreated mice. In contrast, the negative chronotropic action of carbachol in mouse atria was abolished by pretreatment with pertussis toxin. We conclude that, in contrast to muscarinic receptor activation in mouse atria, the inhibitory effect of somatostatin in the vas deferens is not mediated by a pertussis toxin sensitive G-protein. The high potency of seglitide suggests that the SRIF receptor involved is of the SRIF1 type.

The 5-HT(1) receptor agonist GR46611 (3-30 mg/kg s.c.) caused a dose-related decrease in rectal temperature in the adult guinea-pig. A lower dose (20 μg) administered directly into the lateral cerebral ventricle also caused a hypothermic response, suggesting that this effect is centrally mediated. GR46611-induced (10 mg/kg s.c.) hypothermia was not attenuated by WAY100135 (3-10 mg/kg s.c.), ritanserin (0.3-1 mg/kg s.c.), spiperone (0.1-0.3 mg/kg s.c.) and ondansetron (0.1-1 mg/kg s.c.), suggesting that 5-HT(1A), 5-HT(2A), 5-HT( 2C) and 5-HT(3) receptors are unlikely to be involved in this response. In contrast, the poorly selective 5-HT receptor antagonist, metergoline (1-10 mg/kg s.c.), and the potent 5-HT(1D) receptor antagonist, GR127935 (0.1-1 mg/kg p.o.), antagonized the effects of GR46611. The present data suggest that antagonism of GR46611-induced hypothermia may be useful for assessing the potency and duration of action of centrally-acting 5-HT( 1D) receptor antagonists in the guinea-pig.

1. Somatostatin14 (SS14) inhibits neurogenically mediated contractile responses in guinea-pig ileum and vas deferens and exerts a direct negative inotropic action in guinea-pig spontaneously beating right atrium. In this study, the receptors mediating these inhibitory effects have been characterized by comparing the potencies of several cyclic somatostatin analogues. 2. In the guinea-pig ileum, SS14, somatostatin28 (SS28), somatostatin25 (SS25) and several smaller cyclic somatostatin analogues including octreotide, angiopeptin and CGP 23996, inhibited neurogenically mediated contractile responses, each being of similar potency. 3. In contrast, in the guinea-pig vas deferens and right atrium, SS28 was about 30 times more potent than SS14. However, although angiopeptin was nearly as potent as SS14 as an agonist in the vas deferens, in guinea-pig atrium angiopeptin had low intrinsic activity and antagonized the negative inotropic action of both SS14 and SS28 (pKB values of 7.4 and 7.2, respectively). CGP 23996 was 2-7 times weaker than SS14 in guinea-pig vas deferens and atria. 4. Phosphoramidon (1 microM) and amastatin (10 microM) did not influence the potency of SS14 or SS28 in either the guinea-pig ileum or right atrium. In the guinea-pig vas deferens, phosphoramidon and amastatin did not affect the potency of SS28, but enhanced the potency of SS14 about 5 fold. Despite the presence of phosphoramidon and amastatin, SS28 was still more potent than SS14 in the vas deferens. 5. The putative somatostatin receptor blocking drug, cyclo(7-aminoheptanoyl Phe-D-Trp-Lys-Thr[Brl]) (CPP; 1 microM), did not antagonize the effects of either SS14 or SS28 in ileum, vas deferens or atrial preparations. 6. Somatostatin14 did not modify the contractile action of carbachol or alpha,beta-methylene ATP in the ileum and vas deferens respectively, suggesting that the site of the inhibitory effects on neurogenically mediated contractile responses in both preparations was pre-junctional. Consistent with this conclusion was the observation that the inhibitory effect of SS14 was markedly and inversely related to the external Ca2+concentration. The inhibitory effect of SS14 in guinea-pig atrium was only partly dependent on the external Ca2+ concentration.7. The somatostatin receptors mediating the inhibitory effect of SS14 in the ileum and vas deferens can be distinguished by the differential relative potencies of SS14 and SS28. In the former, SS14 and SS28 have similar potency whilst in the latter SS28 is much more potent. In this respect, the somatostatin receptor mediating negative inotropy in the guinea-pig right atrium appears similar to that identified in the vas deferens.8. We suggest that the somatostatin receptor mediating inhibition of neurogenic contraction in the ileum is similar to the recently cloned SSTR2 receptor. In contrast, the somatostatin receptor mediating negative inotropy in the atrium and inhibition of neurotransmission in the vas deferens appears similar to the SSTR4 receptor which recognises SS28 with higher affinity than SS14.

The effects of serotonin on ion channel activity in epithelial cells from rat choroid plexus were examined. Serotonin (50 nM, 500 nM and 1 microM) stimulated channel activity in cell-attached patches. The current-voltage (I-V) relationship for the serotonin-activated channel gave a conductance of 26.6 +/- 1.5 pS and the current reversed at an applied electrode potential (-Vp) = 15.3 +/- 3.3 mV with a KCl-rich electrode solution (n = 8). Similar I-V relationships were obtained using electrode solutions in which K+ was replaced by other cations (Na+ and N-methyl-D-glucamine), suggesting that the serotonin-activated channels are selective to Cl-. The effect of 1 microM serotonin on channel activity was inhibited by ritanserin (30 and 100 nM) which has a high affinity for serotonin 5-HT1C receptors and 5-HT2 receptors. Spiperone (30 nM), which binds weakly to 5-HT1C receptors but has a high affinity for 5-HT2 receptors, did not inhibit the actions of serotonin. These data suggest that serotonin increases Cl- channel activity by acting on the 5-HT1C receptors on the epithelium.

Somatostatin (SS) exerts a negative inotropic effect in isolated atria. Here we report that in guinea-pig isolated right atria, seglitide, a potent cyclic hexapeptide somatostatin agonist, behaves as a competitive somatostatin receptor antagonist with pA2 values against SS14, SS25 and SS28, of 6.50 +/- 0.40, 6.24 +/- 0.08 and 6.09 +/- 0.06, respectively. Seglitide had little or no effect on the negative inotropic action of carbachol or N6-cyclohexyladenosine. Our findings indicate that the receptor-response coupling characteristics of guinea-pig atria are such that in this preparation seglitide has low intrinsic activity and behaves specifically as a somatostatin receptor antagonist.

The aim of this study was to investigate the effect of the anti-migraine drug and selective 5-HT1 receptor agonist, sumatriptan, on membrane potential of guinea-pig isolated trigeminal ganglion. Ganglia were divided into three longitudinally, placed in two-compartment baths and the d.c. potential between compartments was recorded extracellularly. Drugs were applied to the Krebs superfusion fluid of one compartment. KCl (3 mmol/l) and GABA (0.1 mmol/l) caused depolarization (0.30 +/- 0.05 and 0.55 +/- 0.08 mV respectively, n = 11-19). 5-HT (1-10 mumol/l) caused small depolarizations (0.06 +/- 0.02 mV, n = 8) but sumatriptan (0.1-10 mumol/l) had no effect on trigeminal ganglion membrane potential. Collagenase pretreatment, to enhance desheathing, or modification of the composition of the Krebs solution failed to reveal any effect of sumatriptan. These data provide no evidence to suggest that sumatriptan inhibits neurotransmission in trigeminal ganglion. However, 5-HT1 receptors may be present in insufficient numbers in the trigeminal ganglion to elicit a change in membrane potential. Further studies are required to investigate the effect of sumatriptan at the level of the sensory nerve terminals within the intracranial vasculature, where 5-HT1 receptors may be concentrated.

1. The effects of selective neurokinin agents on pial artery diameter, measured with an on-line image analyser, have been studied in anaesthetized guinea-pigs in order to characterize the neurokinin receptors present on pial arteries. 2. Perivascular injection of either substance P (0.01-1 microM) or the selective NK1 receptor agonists, substance P methyl ester (SPOMe, 0.01-1 microM) and GR73632 (0.1 microM), increased pial artery diameter. 3. In contrast, the selective NK2 receptor agonist, GR64349 (1 microM), produced a small vasoconstriction while the NK3 receptor-selective agonist, senktide (1 microM) was inactive. 4. Co-administration of GR82334 (1 microM), a selective NK1 receptor antagonist, inhibited the vasodilatation produced by SPOMe (0.1 microM) but not that caused by calcitonin gene-related peptide (CGRP, 0.01 microM). 5. The results are consistent with an involvement of NK1 receptors in the neurokinin-induced increase in guinea-pig pial artery diameter.

1. Despite the fact that 5-hydroxytryptamine (5-HT)-induced contractions of the rabbit isolated renal artery are mediated by a receptor belonging to the heterogeneous 5-HT1-like category, we observed that the so-called selective 5-HT3 receptor agonist, 2-methyl-5-HT, caused a concentration-dependent contraction of this vessel. This study was therefore undertaken to analyze the effects of 2-methyl-5-HT in the renal artery segments, either quiescent or precontracted with U46619 (10(-7) M). alpha-Methyl-5-HT and 5-methoxytryptamine, which have high affinities for 5-HT2 and 5-HT4 receptors, respectively, were used for comparison. 2. In the precontracted vessel segments, the maximum contractile responses obtained with 2-methyl-5-HT, alpha-methyl-5-HT, 5-methoxytryptamine and 5-HT were similar to those in the quiescent segments. However the pD2 values were higher in the precontracted segments, making them about 4-100 fold more sensitive. 3. Neither MDL 72222 (10(-6) M) nor tropisetron (3 x 10(-6) M) suppressed renal artery contractions elicited by 5-HT, 2-methyl-5-HT, alpha-methyl-5-HT or 5-methoxytryptamine, thus ruling out the involvement of 5-HT3 as well as 5-HT4 receptors. 4. On the other hand, both methiothepin (10(-8) and 10(-7) M) and ketanserin (10(-7) and 10(-6) M) caused a rightward shift of agonist concentration-effect curves.The two antagonists had similar pA2 values against the different agonists tested on either quiescent or precontracted vessels, but ketanserin (apparent pA2: 6.6 to 7.0) was between 20-100 fold less potent than methiothepin (apparent pA2: 8.4 to 8.8).5. The results of this functional study permit us to conclude that the contractile effects of 2-methyl-5-HT as well as ct-methyl-5-HT and 5-methoxytryptamine on the rabbit isolated renal artery are mediated by a 5-HT1-like receptor. Since, in addition, the reported ligand binding affinity of 2-methyl-5-HT at 5-HT3 receptors is similar to both the ligand binding affinity and the functional pD2 at 5-HTI sites, this compound cannot be regarded as a selective 5-HT3 receptor agonist. Similarly, a-methyl-5-HT and 5-methoxytryptamine have only a limited selectivity for 5-HT2 and 5-HT4 receptors, respectively.

The action of sumatriptan, a selective 5-HT1-like receptor agonist that is effective for the acute treatment of migraine, was compared on pial vessel diameter following perivascular or intravenous administration to anaesthetised cats. Sumatriptan (0.01-10 microM), when microinjected perivascularly, caused a decrease in pial artery diameter (maximum change of -19 +/- 9%; mean +/- SD) but had no effect on the diameter of pial veins. Sumatriptan (1 microM)-induced pial artery vasoconstriction was unaffected by coadministration of ketanserin (1 microM) or ondansetron (1 microM) but was significantly (p less than 0.01) attenuated by methiothepin (1 microM). Intravenous infusion of a clinically effective dose of sumatriptan (64 micrograms/kg/10 min) caused selective carotid vasoconstriction (22 +/- 6% increase in carotid vascular resistance with little or no change in blood pressure or heart rate) and no change in pial artery diameter, although sumatriptan (1 microM) administered perivascularly in these animals before and after the infusion caused pial artery vasoconstriction. These results demonstrate that perivascularly administered sumatriptan causes pial artery vasoconstriction via activation of 5-HT1-like receptors. However, intravenously administered sumatriptan does not cause pial artery vasoconstriction, which suggests that sumatriptan does not readily penetrate the cerebrovascular intima.

1. The aim of this study was to characterize the neurokinin receptor which mediates relaxation of dog isolated middle cerebral artery by the use of selective agonists and antagonists and to establish whether substance P is involved in the neurogenically mediated relaxant response in this vessel. 2. Substance P caused concentration-related, endothelium-dependent relaxations of dog isolated middle cerebral artery, contracted with prostaglandin F2 alpha. The selective NK1 receptor agonists, GR73632 and substance P methyl ester (SPOMe), also caused relaxation with similar maximum effects to those of substance P. GR73632 and SPOMe were approximately 20 times and 6 times less potent respectively than substance P. The selective NK2 and NK3 receptor agonists, GR64349 and senktide, were only weakly active in causing relaxation being at least 425 times and 245 times less potent respectively than substance P. 3. The selective NK1 receptor antagonist, GR82334, was a potent, specific, competitive antagonist of the relaxant effects of substance P. In contrast, the selective NK2 receptor antagonist, R396 (10 microM) had no effect on the response to substance P. 4. Electrical field stimulation of dog isolated middle cerebral artery, contracted with prostaglandin F2 alpha, caused neurogenically mediated, non-adrenergic non-cholinergic (NANC) relaxations. These NANC relaxations were unaffected by endothelium removal, GR82334 (10 microM) or by capsaicin (10 microM) treatment. However, the nitric oxide synthesis inhibitor, L-NG-monomethyl arginine methyl ester (L-NMMA) (100 microM) markedly attenuated the response to electrical stimulation. 5. These results suggest that substance P causes relaxation of dog isolated middle cerebral artery via activation of NK1 receptors. However, substance P does not appear to be involved in NANC neurotransmission. In contrast, the marked inhibitory effect of L-NMMA on NANC relaxations implicates nitric oxide in NANC neurotransmission in this vessel.

1. We have investigated the mechanism of smooth muscle contraction evoked by activation of 5-HT1-like receptors in dog isolated saphenous vein. 2. In the presence of the 5-HT2 receptor antagonist, ritanserin (0.1 microM), concentration-effect curves (10 nM-300 microM) for 5-hydroxytryptamine (5-HT)-induced smooth muscle contraction were biphasic. This could be attributed to a direct action on 5-HT1-like receptors at low concentrations of 5-HT (10 nM-10 microM) and an indirect (through the release of noradrenaline from sympathetic neurones) activation of postjunctional alpha-adrenoceptors at higher 5-HT concentrations. In contrast, concentration-effect curves (100 nM-100 microM) for sumatriptan-induced contractions were not biphasic, and were due solely to activation of 5-HT1-like receptors. 3. Smooth muscle contractions evoked either by low concentrations of 5-HT or by sumatriptan were abolished by removal of extracellular calcium and were markedly inhibited, but not abolished, by the calcium channel blocker, verapamil (1-30 microM). In contrast, contractions evoked by high concentrations of 5-HT were markedly less sensitive to removal of extracellular calcium or to verapamil. 4. 5-HT and sumatriptan also inhibited (to a maximum of about 50%) prostaglandin E2 (PGE2, 5 microM)-stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation. This effect was mimicked by the alpha 2-adrenoceptor agonist, azepexole (B-HT933) but not by the alpha 1-adrenoceptor agonist, methoxamine.5. In contrast to mediation of smooth muscle contraction, the 5-HT1-like receptor-mediated inhibition of PGE2-stimulated cyclic AMP formation evoked by 5-HT or sumatriptan was not attenuated by removal of extracellular calcium or by verapamil (1 microM).6. A directly-acting inhibitor of adenylyl cyclase, 2',3'-dideoxyadenosine (1 mM) inhibited PGE2-stimulated cyclic AMP formation but did not produce smooth muscle contraction.7. These results suggest that contractile responses of dog isolated saphenous vein arising through activation of 5-HT1-like receptors are associated with both an influx of extracellular calcium ions (to a large extent via voltage-dependent channels) and an inhibition of adenylyl cyclase. However, although these two responses are coupled to the same receptor, they appear to be independent.

1. Using a number of agonist and antagonist compounds, we have attempted to characterize the responses and receptors involved in the effects of 5-hydroxytryptamine (5-HT) in the rabbit isolated renal artery. 2. In vessel segments precontracted with the thromboxane-mimetic agent, U46619 (100 nM), neither 5-HT (10(-8) to 10(-4) M) nor 5-carboxamidotryptamine (5-CT; 10(-8) to 3 x 10(-4) M) caused relaxations like those observed with methacholine. Both 5-HT and 5-CT further increased the tone of the vessels, with pD2 values of 7.1 and 7.9, respectively. 3. In the absence of U46619, both 5-HT (10(-7) to 3 x 10(-3) M) and 5-CT (10(-7) to 10(-3) M) contracted the rabbit renal artery, but with reduced potencies. The contractions to 5-HT were reproducible and the rank order of potency (pD2) of the agonists was: alpha-methyl-5-HT (5.7), sumatriptan (5.3), 5-HT (5.1), 8-hydroxy-2(di-n-propylamino)tetralin (5.0), 5-CT (4.7) and 5-methoxytryptamine (4.3). 1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane, flesinoxan and RU 24969 elicited either only small contractions or none at all. 4. The contractile effect of 5-HT was unaffected by MDL 72222 (10(-6) M) and metergoline (10(-8) and 10(-7) M), was weakly antagonized by ketanserin and phentolamine (pKB: 6.6 and 6.8, respectively), but was effectively antagonized by methiothepin (pKB: 8.6). Responses to 5-CT and sumatriptan were affected by ketanserin, phentolamine and methiothepin similarly to 5-HT-induced responses. 5. Ketanserin was ineffective against noradrenaline-induced contractions, which were antagonized by phentolamine with a pKB of 7.3. The pKB values of phentolamine against 5-HT, 5-CT or sumatriptan were about half a log unit lower than against noradrenaline.6. In vascular preparations treated with cocaine (3 x 10- I M), the potency (pKB) of phentolamine as an antagonist of the responses to noradrenaline (7.6) and 5-HT (6.7) did not differ significantly from the values in untreated preparations. However, the difference between the pKB values of phentolamine against the two agonists was now about one log unit.7. Pretreatment of the vascular strips with 6-hydroxydopamine (1.5 x 10- 3M) did not significantly affect responses to 5-HT or 5-CT, but almost eliminated those to tyramine. Cocaine (3 x 10- 5M) slightly potentiated noradrenaline-induced contractions, but did not significantly affect those induced by 5-HT.8. These data suggest that: (i) 5-HT receptors mediating vasodilatation are not present in the rabbit renal artery smooth muscle or endothelium; (ii) the contractile effect of 5-HT does not involve the release of noradrenaline from sympathetic nerve stores; (iii) the 5-HT receptor in the rabbit renal artery is not of the 5-HT2, 5-HT3 or 5-HT4 type. The pharmacological properties of this receptor most closely resemble those described for the heterogeneous 5-HT1-like category.