Pharmacological characterization of the 5-HT receptors mediating contraction and relaxation of canine isolated proximal stomach smooth muscle.
ABSTRACT: 1. We aimed to characterize 5-HT receptors mediating contraction and relaxation to 5-HT in dog proximal stomach longitudinal muscle (LM) strips. 2. Of the tryptamine analogues tested, 5-HT was the most potent contractile agent at basal length, while 5-CT was the most potent relaxant of PGF(2alpha)-induced contraction. Neither the contractions to 5-HT, nor the relaxations to 5-CT were influenced by tetrodotoxin, illustrating that action potential propagation is not involved. 3. The 5-HT-induced contraction was antagonized by mesulergine (0.03 to 0.3 microM) and ketanserin (2 - 20 nM), but the antagonism was not of a simple competitive nature, indicating multiple receptor involvement. Ketanserin (3 to 30 nM) and mesulergine (30 nM) competitively antagonized the alpha-Me-5-HT-induced contraction (pK(B): 8.83+/-0.09 and pA(2): 8.25+/-0.06 respectively). These affinity values are in line with literature affinities of ketanserin and mesulergine at 5-HT(2A) receptors in various bioassays. 4. The 5-CT-induced inhibition of PGF(2alpha)-induced contraction was competitively antagonized by mesulergine (pK(B) estimate: 8.52+/-0.12) and by the selective 5-HT(7) receptor antagonist SB-269970 (pK(B) estimate: 9.36+/-0.14). Both pK(B) estimates are in line with literature affinities of these compounds for 5-HT(7) receptors. Mesulergine (30 nM) and SB-269970 (10 nM) shifted the relaxant curve to 5-HT parallel to the right in the presence of ketanserin (0.3 microM) (pA(2) estimates of 8.08+/-0.10 and 8.75+/-0.14 respectively), indicative of 5-HT(7) receptor involvement. 5. It is concluded that 5-HT induces dog proximal stomach (LM) contraction via smooth muscle 5-HT(2A) receptors and relaxation via smooth muscle 5-HT(7) receptors.
Project description:1. 5-Hydroxytryptamine (5-HT) receptors mediating contraction and relaxation are present in Cynomolgus monkey isolated jugular vein denuded of endothelium. 2. In the absence of spasmogen, alpha-methyl-5-HT and sumatriptan contracted the tissues with potency values (pEC50) of 6.8 (n = 2) and 6.4 +/- 0.1 (mean +/- s.e. mean, n = 3), respectively. In contrast, 5-HT caused an initial contraction (10 nM - 1 microM), followed by relaxation (1 microM - 32 microM). The contractile effect of alpha-methyl-5-HT was antagonized by ketanserin with a pKB value of 8.1 (n = 2). 5-Carboxamidotryptamine (5-CT), 5-methoxytryptamine (5-MeOT) and 8-OH-DPAT did not contract or relax the tissues in the absence of spasmogen. 3. In tissues precontracted with U46619 (10 nM) and in the presence of 5-HT1A, 5-HT1B, 5-HT2A, 5-HT3, and 5-HT4 receptor blockade, 5-CT and 5-MeOT caused endothelium-independent relaxation with potency values of 7.5 +/- 0.1 (n = 21) and 5.7 +/- 0.1 (n = 4), respectively. The potency of 5-HT was 7.2 (n = 2) while alpha-methyl-5-HT did not start to relax the tissues below a concentration of 10 microM. 4. Relaxations elicited by 5-CT were antagonized by the following compounds (with pKB values in parentheses): methiothepin (9.7), mesulergine (8.1), metergoline (8.0), clozapine (7.8), mianserin (7.7), spiperone (7.3), ritanserin (7.1), methysergide (7.0) and ketanserin (5.7). 5. It is concluded that the 5-HT receptor mediating endothelium-independent relaxation may be a functional correlate of the putative 5-ht7 receptor.
Project description:The use of human prokinetic drugs in colic horses leads to inconsistent results. This might be related to differences in gastrointestinal receptor populations. The motor effects of 5-hydroxytryptamine (5-HT; serotonin) on the equine mid-jejunum were therefore studied. Longitudinal muscle preparations were set up for isotonic measurement. 5-HT induced tonic contractions with superimposed phasic activity; these responses were not influenced by tetrodotoxin and atropine, suggesting a non-neurogenic, non-cholinergic pathway. The 5-HT receptor antagonists GR 127935 (5-HT(1B,D)), ketanserin (5-HT(2A)), SB 204741 (5-HT(2B)), RS 102221 (5-HT(2C)), granisetron (5-HT(3)), GR 113808 (5-HT(4)) and SB 269970 (5-HT(7)) had no influence on the 5-HT-induced response; the 5-HT(1A) receptor antagonists NAN 190 (pK(b)=8.13+/-0.06) and WAY 100635 (pK(b)=8.69+/-0.07), and the 5-HT(1,2,5,6,7) receptor antagonist methysergide concentration-dependently inhibited the 5-HT-induced contractile response. The 5-HT(1,7) receptor agonist 5-carboxamidotryptamine (5-CT) induced a contractile response similar to that of 5-HT; its effect was not influenced by tetrodotoxin and atropine, and SB 269970, but antagonised by WAY 100635. 8-OHDPAT, buspiron and flesinoxan, which are active at rat and human 5-HT(1A) receptors, had no contractile influence. These results suggest that the contractile effect of 5-HT in equine jejunal longitudinal muscle is due to interaction with muscular 5-HT receptors, which cannot be characterised between the actually known classes of 5-HT receptors.
Project description:This study aimed to determine, quantify and explain regional differences in the relaxant response to the selective 5-HT(1) and 5-HT(7) receptor agonist 5-carboxamidotryptamine (5-CT) throughout the canine stomach. Longitudinal muscle strips from eight gastric corpus regions and six antrum regions were mounted for isotonic measurement. The 5-CT-induced relaxation was examined on a prostaglandin F(2alpha)-induced submaximal response, expressed as percentage of this response and fitted to the operational model of agonism (OMOA). 5-HT(7) receptor messenger RNA (mRNA) expression was compared by means of quantitative PCR. 5-CT inhibited PGF(2alpha)-induced tonic contraction (corpus) and increase of phasic contraction amplitude (antrum). The consistent antagonism produced by the selective 5-HT(7) receptor antagonist SB-269970 (10 nm, pA(2) estimates 8.2-8.9) confirmed that in every region, the inhibition by 5-CT was 5-HT(7) receptor mediated. However, variation in the maximum effect (61-108%) and pEC(50) (6.4-8.6) was observed throughout the different regions. The OMOA explained these differences as differences in the efficacy parameter tau (ratio of receptor density and coupling efficiency; log tau estimates ranging from 0.1 to 2.1). The log tau gradient decreases going from the lesser to the greater curvature. A proportional difference (68%) in the relative expression of 5-HT(7) receptor mRNA between the lesser and the greater curvature indicates that differences in receptor density contribute to the observed functional differences. This study illustrates that 5-HT(7) receptors are present throughout the ventral wall of the canine stomach, but the efficacy (expressed as log tau) is clearly greater close to the lesser curvature. Differences in 5-HT(7) receptor expression at least partially explain the functional differences.
Project description:We aimed to study 5-HT(4) receptors in canine stomach contractility both in vivo and in vitro. In anaesthetized Beagle dogs, the selective 5-HT(4) receptor agonist prucalopride (i.v.) induced dose-dependent tonic stomach contractions under isobaric conditions, an effect that was antagonized by the selective 5-HT(4) receptor antagonist GR 125487 (10 microg kg(-1), i.v.). Electrical field stimulation (EFS) of corpus longitudinal muscle strips resulted in atropine- and tetrodotoxin-sensitive contractions (L-NOARG (0.1 mM) present in all organ bath solutions). Prucalopride increased these contractions (maximal response after single-dose addition (0.3 microM): 165% of initial value, or after cumulative addition: 188%). In the presence of methysergide (3 microM), 5-HT also increased EFS-contractions (after single-dose addition (0.3 microM): increase to 192%, after cumulative addition: 148%). The selective 5-HT(4) receptor antagonists GR 113808 (0.1 microM) or GR 125487 (10 nM) antagonized the prucalopride (0.3 microM)-induced contraction increments. When EFS-induced contractions were blocked by atropine or tetrodotoxin, prucalopride was ineffective. In the presence of methysergide (3 microM), the contraction increases to 5-HT (0.3 microM) were prevented by GR 113808 (0.1 microM). The prucalopride curve (pEC(50) 7.9) was shifted in parallel to the right by GR 113808 3 nM (pA(2) 9.4). In the presence of methysergide (3 microM), the curve to 5-HT (pEC(50) 8.1) was competitively antagonized by GR 113808, yielding a Schild slope of 0.8+/-0.2 (pK(B) of 9.1 with unit Schild slope). In corpus circular muscle strips, the prucalopride (0.3 microM)-induced augmentation of EFS-contractions (258%) was also prevented by GR 113808 (0.1 microM) (124%). In conclusion, the effects of 5-HT(4) receptor agonists on proximal stomach motor activity in vivo can be explained by an effect on 5-HT(4) receptors on cholinergic nerves within the gastric muscle wall.
Project description:1. Using membranes from stably or transiently transfected HEK293 cells cultured in 5-HT-free medium and expressing the recombinant human 5-HT(7) receptor splice variants (h5-HT(7(a)), h5-HT(7(b)) and h5-HT(7(d))), we compared their abilities to constitutively activate adenylyl cyclase (AC). 2. All h5-HT(7) splice variants elevated basal and forskolin-stimulated AC. The basal AC activity was reduced by the 5-HT(7) antagonist methiothepin and this effect was blocked by mesulergine (neutral 5-HT(7) antagonist) indicating that the inhibitory effect of methiothepin is inverse agonism at the 5-HT(7) receptor. 3. Receptor density correlated poorly with constitutive AC activity in stable clonal cell lines and transiently transfected cells. Mean constitutive AC activity as a percentage of forskolin-stimulated AC was significantly higher for the h5-HT(7(b)) splice variant compared to the h5-HT(7(a)) and h5-HT(7(d)) splice variants but only in stable cell lines. 4. All eight 5-HT antagonists tested inhibited constitutive AC activity of all splice variants in a concentration-dependent manner. No differences in inverse agonist potencies (pIC(50)) were observed between the splice variants. The rank order of potencies was in agreement and highly correlated with antagonist potencies (pK(b)) determined by antagonism of 5-HT-stimulated AC activity (methiothepin >metergoline> mesulergine > or = clozapine > or = spiperone > or = ritanserin > methysergide > ketanserin). 5. The efficacy of inverse agonism was not receptor level dependent and varied for several 5-HT antagonists between membrane preparations of transiently and stably transfected cells. 6. It is concluded that the h5-HT(7) splice variants display similar constitutive activity and inverse agonist properties.
Project description:1. It has been suggested that the tachycardic response to 5-hydroxytryptamine (5-HT) in the spinal-transected cat is mediated by '5-HT1-like' receptors since this effect, being mimicked by 5-carboxamidotryptamine (5-CT), is not modified by ketanserin or MDL 72222, but it is blocked by methiothepin, methysergide or mesulergine. The present study was set out to reanalyse this suggestion in terms of the IUPHAR 5-HT receptor classification schemes proposed in 1994 and 1996. 2. Intravenous (i.v.) bolus injections of the tryptamine derivatives, 5-CT (0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 30 microg kg(-1)), 5-HT (3, 10 and 30 microg kg(-1)) and 5-methoxytryptamine (3, 10 and 30 microg kg(-1)) as well as the atypical antipsychotic drug, clozapine (1000 and 3000 microg kg(-1)) resulted in dose-dependent increases in heart rate, with a rank order of agonist potency of 5-CT >> 5-HT > 5-methoxytryptamine >> clozapine. 3. The tachycardic effects of 5-HT and 5-methoxytryptamine were dose-dependently antagonized by i.v. administration of lisuride (30 and 100 microg kg(-1)), ergotamine (100 and 300 microg kg(-1)) or mesulergine (100, 300 and 1000 microg kg(-1)); the highest doses of these antagonists used also blocked the tachycardic effects of 5-CT. Clozapine (1000 and 3000 microg kg(-1)) did not affect the 5-HT-induced tachycardia, but attenuated, with its highest dose, the responses to 5-methoxytryptamine and 5-CT. However, these doses of clozapine as well as the high doses of ergotamine (300 microg kg(-1)) and mesulergine (300 and 1000 microg kg(-1)) also attenuated the tachycardic effects of isoprenaline. In contrast, 5-HT-, 5-methoxytryptamine- and 5-CT-induced tachycardia were not significantly modified after i.v. administration of physiological saline (0.1 and 0.3 ml kg(-1)), the 5-HT(1B/1D) receptor antagonist, GR127935 (500 microg kg(-1)) or the 5-HT(3/4) receptor antagonist, tropisetron (3000 microg kg(-1)). 4. Intravenous injections of the 5-HT1 receptor agonists, sumatriptan (30, 100 and 300 microg kg(-1)) and indorenate (300 and 1000 microg kg(-1)) or the 5-HT4 receptor (partial) agonist cisapride (300 and 1000 microg kg(-1)) were devoid of effects on feline heart rate per se and failed to modify significantly 5-HT-induced tachycardic responses. 5. Based upon the above rank order of agonist potency, the failure of sumatriptan, indorenate or cisapride to produce cardioacceleration and the blockade by a series of drugs showing high affinity for the cloned 5-ht7 receptor, the present results indicate that the 5-HT receptor mediating tachycardia in the cat is operationally similar to other putative 5-HT7 receptors mediating vascular and non-vascular responses (e.g. relaxation of the rabbit femoral vein, canine external carotid and coronary arteries, rat systemic vasculature and guinea-pig ileum). Since these responses represent functional correlates of the 5-ht7 gene product, the 5-HT7 receptor appellation is reinforced. Therefore, the present experimental model, which is not complicated by the presence of other 5-HT receptors, can be utilized to characterize and develop new drugs with potential agonist and antagonist properties at functional 5-HT7 receptors.
Project description:This study aimed to characterize for the first time in vitro 5-HT4 receptors in the canine gastrointestinal tract. For this purpose, we used circular muscle strips of the canine isolated rectum. In the presence of methysergide (60 microM), 5-HT induced relaxation of methacholine (1 microM)-precontracted muscle strips, yielding a monophasic sigmoidal concentration-relaxation curve (pEC50 7.2+/-0.07). Tetrodotoxin (0.3 microM) did not affect the curve to 5-HT, suggesting the inhibitory 5-HT receptor is located on the smooth muscle. Granisetron (0.3 microM) did also not affect the curve to 5-HT, which excludes the 5-HT3 receptor mediating the relaxation to 5-HT. The presence of methysergide rules out the involvement of 5-HT1, 5-HT2 or 5-HT7 receptors. 5-HT, the selective 5-HT4 receptor agonists R076186, prucalopride (R093877) and SDZ HTF-919 and the 5-HT4 receptor agonists cisapride and 5-MeOT relaxed the muscle strips with a rank order of potency R076186 = 5-HT > cisapride > prucalopride > or = SDZ HTF-919 > 5-MeOT. The selective 5-HT4 receptor antagonists GR 125487, RS 39604 and GR 113808 competitively antagonized the relaxations to 5-HT, yielding pK(B) estimates of 9.7, 7.9 and 9.1, respectively. The selective 5-HT4 receptor antagonist SB 204070 shifted the curve to 5-HT rightward and depressed the maximal response (apparent pA2 10.6). GR 113808 (10 nM) produced a parallel rightward shift of the curve to the selective 5-HT4 receptor agonists R076186 (pA2 8.8). It is concluded that 5-HT induces relaxation of the canine rectum circular muscle through stimulation of a single population of smooth muscle 5-HT4 receptors. For the first time, a nonhuman species was shown to exhibit relaxant 5-HT4 receptors in the large intestine.
Project description:1. The main aim of this investigation was to delineate the distribution of the 5-HT(7) receptor in human brain. Autoradiographic studies in guinea-pig and rat brain were also carried out in order to revisit and compare the anatomical distribution of 5-HT(7) receptors in different mammalian species. 2. Binding studies were performed in rat frontal cortex membranes using 10 nm [(3)H]mesulergine in the presence of raclopride (10 microm) and DOI (0.8 microm). Under these conditions, a binding site with pharmacological characteristics consistent with those of the 5-HT(7) receptors was identified (rank order of binding affinity values: 5-CT>5-HT>5-MeOT>mesulergine approximately methiothepin>8-OH-DPAT=spiperone approximately (+)-butaclamol>>imipramine approximately (+/-)-pindolol>>ondansetron approximately clonidine approximately prazosin). 3. The autoradiographic studies revealed that the anatomical distribution of 5-HT(7) receptors throughout the human brain was heterogenous. High densities were found over the caudate and putamen nuclei, the pyramidal layer of the CA2 field of the hippocampus, the centromedial thalamic nucleus, and the dorsal raphe nucleus. The inner layer of the frontal cortex, the dentate gyrus of the hippocampus, the subthalamic nucleus and superior colliculus, among others, presented intermediate concentrations of 5-HT(7) receptors. A similar brain anatomical distribution of 5-HT(7) receptors was observed in all three mammalian species studied. 4. By using [(3)H]mesulergine, we have mapped for the first time the anatomical distribution of 5-HT(7) receptors in the human brain, overcoming the limitations previously found in radiometric studies with other radioligands, and also revisiting the distribution in guinea-pig and rat brain.
Project description:The pharmacological characterization of a 5-HT receptor-mediated contractile response in the mouse isolated ileum is described. In the presence of methysergide (1 microM), 5-hydroxytryptamine (5-HT, 0.3 - 100 microM) produced phasic concentration-dependent contractions of segments of the mouse isolated ileum with a pEC(50) value of 5.47+/-0.09. The 5-HT(3) receptor selective agonists m-chlorophenylbiguanide (0.3 - 100 microM, pEC(50) 5.81+/-0.04), 1-phenylbiguanide (3 - 100 microM, pEC(50) 5.05+/-0.06) and 2-methyl-5-HT (3 - 100 microM, pEC(50) 5.00+/-0.07) acted as full agonists to induce contractile responses. 5-methoxytryptamine (0.1 - 100 microM), RS 67506 (0.1 - 100 microM) and alpha-methyl-5-HT (0.1 - 100 microM) failed to mimic the 5-HT responses. The contractile response to 5-HT was not antagonized by either 5-HT(2) receptor antagonists ritanserin (0.1 microM) or ketanserin (1 microM) nor the 5-HT(4) receptor antagonist SB 204070 (0.1 microM). The 5-HT(3) receptor selective antagonists granisetron (0.3 - 1 nM), tropisetron (1 - 10 nM), ondansetron (10 nM - 1 microM) and MDL 72222 (10 nM - 1 microM) caused rightward displacement of the concentration-response curves to 5-HT. The lower concentrations of the antagonists caused approximate parallel rightward shifts of the concentration-response curves to 5-HT with apparent pK(B) values for granisetron (9.70+/-0. 39), tropisetron (9.18+/-0.20), ondansetron (8.84+/-0.24) and MDL 72222 (8.65+/-0.35). But higher concentrations of antagonists resulted in a progressive reduction in the maximum responses. The contractile response to 5-HT was abolished by tetrodotoxin (0.3 microM); atropine (0.1 and 1 microM) decreased the maximum response of the 5-HT concentration-response curve by approximately 65%. It is concluded that a neuronally located 5-HT(3) receptor mediates a contractile response to 5-HT in the mouse ileum. The 5-HT(3) receptor in the mouse ileum has a different pharmacological profile to that reported for the guinea-pig ileum.
Project description:Current antidepressants still display unsatisfactory efficacy and a delayed onset of therapeutic action. Here we show that the pharmacological blockade of serotonin 7 (5-HT(7)) receptors produced a faster antidepressant-like response than the commonly prescribed antidepressant fluoxetine. In the rat, the selective 5-HT(7) receptor antagonist SB-269970 counteracted the anxiogenic-like effect of fluoxetine in the open field and exerted an antidepressant-like effect in the forced swim test. In vivo, 5-HT(7) receptors negatively regulate the firing activity of dorsal raphe 5-HT neurons and become desensitized after long-term administration of fluoxetine. In contrast with fluoxetine, a 1-week treatment with SB-269970 did not alter 5-HT firing activity but desensitized cell body 5-HT autoreceptors, enhanced the hippocampal cell proliferation, and counteracted the depressive-like behavior in olfactory bulbectomized rats. Finally, unlike fluoxetine, early-life administration of SB-269970, did not induce anxious/depressive-like behaviors in adulthood. Together, these findings indicate that the 5-HT(7) receptor antagonists may represent a new class of antidepressants with faster therapeutic action.