5-HT1-like and 5-HT2A receptors mediate 5-hydroxytryptamine-induced contraction of rabbit isolated mesenteric artery

The contractions induced by 5-hydroxytryptamine (5-HT) and the 5-HT₁-like receptor agonist, sumatriptan, were investigated in the open ring preparations of rabbit mesenteric artery in order to characterize the 5-HT receptors. 5-HT induced concentration-dependent contractions. Sumatriptan did not induce any contraction of unstimulated rings, whereas it elicited concentration-dependent contractions in preparations given a moderate tone by a threshold concentration of prostaglandin F₂ (PGF₂). Pargyline, cocaine or normetanephrine were without significant effect on the contractions induced by 5-HT and sumatripan. The 5-HT concentration-effect curve was clearly biphasic. Methiothepin (0.01 M) shifted the both phases of the concentration-effect curve to the right. Ketanserin (0.1 M) shifted the second, low affinity, phase and prazosin did not alter concentration-effect curve to 5-HT. The sumatriptan concentration-effect curve was shifted by methiothepin (0.01 M) to the right (pK_B = 9.19) but not by ketanserin (1 M). Concentration-effect curves to 5-HT and sumatriptan were not affected by the 5-HT₃ receptor antagonist tropisetron (1 M). These results suggest that 5-HT₁-like type receptors are responsible for the first phase of 5-HT-induced contraction and 5-HT_2A receptor for the second phase, in rabbit mesenteric artery. Sumatriptan-induced contractions appear to be mediated by 5-HT₁-like type receptors in this artery. These results also suggest that this kind of amplification may be a common feature of vascular 5-HT₁-like type receptor as has been shown in other vascular segments such as rabbit femoral, iliac and renal arteries, and guinea-pig iliac artery.     

Molecular structural basis of ligand selectivity for 5-HT2 versus 5-HT1C cortical receptors

Summary A molecular structural criterion of ligand selectivity for the 5-HT₂ versus 5-HT_1C receptor was hypothesized on the basis of radioligand binding data. Despite the large number of compounds which have been tested at both receptors, analysis of published data led to the identification of only five agents which are greater than 10-fold selective for the 5-HT₂ versus the 5-HT_1C receptor. Comparison of the two-dimensional structures revealed that, although these five compounds represent three distinct structural classes, they share a common structural feature located in the region hypothesized to be involved in receptor binding: a carbonyl or carboxyl oxygen interposed spatially between an aromatic ring and nitrogen atom. This structural feature was used to predict the relative selectivity of compounds that had not previously been analyzed at both the 5-HT₂ and 5-HT_1C receptors.All six drugs tested which contain the identified reactive carbonyl or carboxyl group were found to be selective for the 5-HT₂ versus the 5-HT_1C receptor with selectivity ratios ranging from 26 to 380. By contrast, three agents which are structurally similar but do not contain the reactive carbonyl or carboxyl group displayed equally high affinity for both receptor binding sites. Since the physiological roles of the 5-HT₂ and 5-HT_1C receptor are markedly different, it would be of potential clinical and scientific value to utilize this molecular structural feature to further identify chemical compounds which would selectively interact with only one of the two receptors. Send offprint requests to S. J. Peroutka at the current address     

Functional 5-HT receptors in human occipital artery

5-HT receptors were studied in human occipital arteries, obtained from patients during neurosurgery. We detected mRNA for the following receptors (incidence): 5-HT_1B (14/18), 5-HT_1D (15/18), 5-HT_2A (16/18), 5-HT_2B (8/8), 5-HT_4(a) (13/18), 5-HT_4(b) (5/18), 5-HT_4(g) (7/18), 5-HT_4(i) (1/18), 5-HT_7(a/b) (10/18) and 5-HT_7(d) (12/18). 5-HT contracted and relaxed arterial rings at low (–logEC₅₀ M=7.0) and high (–logEC₅₀ M=4.2) concentrations, respectively. 5-HT-evoked contractions were antagonized partially by both 5-HT_1B-selective SB224289 (200 nM) and 5-HT_2A-selective ketanserin (1 M) but not by 5-HT_1D-selective BRL15572 (500 nM) or prazosin (1 M). Sumatriptan caused contractions (–logEC₅₀ M=6.8, intrinsic activity with respect to 5-HT=0.3). Sumatriptan-evoked contractions were antagonized by SB224289 with high potency (pK_B=9.4) but not by BRL15572. 5-HT-induced relaxations were resistant to blockade by 5-HT_1B-selective SB224289 (1 M), 5-HT_1D-selective BRL15572, 5-HT_2B-selective SB204741 (1 M), 5-HT₄-selective GR113808 (100 nM) and 5-HT₇-selective SB269970 (1 M), and a combination of SB204741 and SB269970, inconsistent with an involvement of 5-HT_1B, 5-HT_1D, 5-HT_2B, 5-HT₄ and 5-HT₇ receptors. Triton X-100 treatment of the arteries abolished acetylcholine-induced relaxations of rings precontracted by prostaglandin F₂, but a reduction of the relaxant effects of 5-HT did not reach significance. Nitro-L-arginine (1 mM) reduced 5-HT-induced relaxations, suggesting a contribution of nitric oxide released from endothelial cells. Ketanserin (1 M) prevented the relaxant effects of 5-HT. We conclude that 5-HT contracts human occipital artery through 5-HT_1B receptors at low concentrations and through 5-HT_2A receptors at high concentrations. Sumatriptan contracts mostly through 5-HT_1B receptors. These results are consistent with the 5-HT_1B and 5-HT_2A mRNA data. 5-HT-induced relaxation is mediated, in part, through ketanserin-sensitive receptors, but 5-HT_1B, 5-HT_1D, 5-HT_2B, 5-HT₄ and 5-HT₇ receptors appear not to be involved.     

Anxiolytic-like effect of a serotonergic ligand with high affinity for 5-HT1A, 5-HT2A and 5-HT3 receptors

S-(-)-2-[[4-(napht-1-yl)piperazin-1-yl]methyl]-1,4-dioxoperhydropyrrolo[1,2-alpha]-pyrazine (CSP-2503) is a serotonin (5-HT) receptor ligand with selectivity and high affinity for 5-HT1A, 5-HT2A and 5-HT3 receptors. CSP-2503 reduced rectal temperature and 5-HT neuronal hypothalamic activity in mice, decreased electrical activity of raphe nuclei cells in rats and blocked the enhancement of adenylate cyclase activity induced by forskolin in HeLa cells transfected with the human 5-HT1A receptor. This compound also blocked head-twitches induced by the 5-HT(2A/2C) receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI). Contractions of guinea pig ileum induced by the 5-HT3 receptor agonist 2-methyl-5-HT were prevented by CSP-2503. Moreover, it reduced the bradycardia reflex induced by 2-methyl-5-HT in anaesthetized rats. In the light/dark box and social interaction tests, CSP-2503 presented anxiolytic activity, an action shared by 5-HT1 agonists and 5-HT3 antagonists. Taken together, these results suggest that CSP-2503 is a new 5-HT1 receptor agonist with 5-HT2A and 5-HT3)receptor antagonist activities that might be useful in a number of conditions associated with anxiety.     

Evidence for 5-HT2 receptor mediation in quipazine anorexia

Doses ofd-amphetamine (3.2 mg/kg), fenfluramine (10 mg/kg) and quipazine (8 mg/kg) cause a significant reduction in food intake during a 30-min daily feeding session in food-deprived rats. Pirenperone and ritanserin, 5-HT₂ receptor antagonists, significantly blocked the anorectic effect of quipazine, whiled-amphetamine and fenfluramine effects were not modified. Metergoline, a non-specific blocker of 5-HT receptors, significantly blocked the anorectic effects of fenfluramine and quipazine, but not thed-amphetamine effect. Pretreatment with alpha- and beta-adrenergic receptor antagonists (prazosin, propranolol and pindolol), dopamine receptor antagonists (haloperidol and pimozide), the catecholamine synthesis inhibitor alphamethyl-para-tyrosine, and the opioid receptor antagonist naloxone failed to modify the anorectic effects of all three agents, with the exception that quipazine-induced anorexia was significantly reduced by pimozide. These results suggest that the quipazine anorexia is largely mediating through 5-HT₂ receptors, although the effect of pimozide remains to be explained. Consistent with previous studies, the fenfluramine effect appears to be mediated through 5-HT_1B receptors. Receptors involved in the anorectic effect of higher doses ofd-amphetamine are still unidentified by this analysis. Further investigation is required to define the mechanisms by which quipazine and larger doses ofd-amphetamine bring about a reduced appetite for food.     

Characterization of the contraction to 5-HT in the canine colon longitudinal muscle

1. Although conscious dogs have often been used for colonic motility studies with 5-hydroxytryptamine (5-HT), the effects of 5-HT on the isolated colon have not been thoroughly characterized yet. The current study was undertaken to characterize the response to 5-HT of the canine isolated colon longitudinal muscle.
2. Longitudinal strips of canine midcolon deprived of (sub)mucosa were prepared for isotonic measurement. 5-HT induced contractions from 3 nM onwards, which were not affected by selective inhibition of 5-HT re-uptake, monoamine oxidase or blockade of α-adrenoceptors. Tetrodotoxin (0.3 μM) did not affect the responses to 5-HT, suggesting that smooth muscle 5-HT receptors are involved. The selective 5-HT₄ receptor antagonist SB 204070 (10 nM) slightly enhanced contractions to 5-HT and therefore it was included in the organ bath solution in all further experiments. The 5-HT₁ and 5-HT₂ receptor antagonist methysergide (0.1 μM) depressed the curve to 5-HT, but the selective 5-HT₃ receptor antagonist granisetron (0.3 μM) had no effect.
3. Besides 5-HT, α-methyl-5-HT (α-Me-5-HT), 5-methoxytryptamine (5-MeOT), 2-methyl-5-HT (2-Me-5-HT) and 5-carboxamidotryptamine (5-CT) also induced contractions, with the following rank order of potency (pEC₅₀ values in parentheses): 5-HT (6.9)=α-methyl-5-HT (6.9)>2-Me-5-HT (5.8)=5-MeOT (5.7)=5-CT (5.6), indicative of 5-HT₂ receptor involvement. α-Me-5-HT produced a bell-shaped curve, which was not affected by α-adrenoceptor blockade. 5-HT, 5-MeOT, 2-Me-5-HT and 5-CT produced a monophasic concentration-response curve, consistent with an interaction with a single receptor site. 8-Hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) and tryptamine only induced contractions at a concentration exceeding 1 μM.
4. The selective 5-HT_2B receptor antagonist SB 204741 (0.3 μM) did not affect the curve to 5-HT. Ketanserin, cisapride and spiroxatrine behaved as competitive antagonists with pK_b values of, respectively, 8.4, 8.1 and 6.7. Spiroxatrine (1 μM) shifted the curve to 5-MeOT rightward yielding an apparent pA₂ of 7.1. Other antagonists at 5-HT_2A receptors also surmountably inhibited the contractions to 5-HT (apparent pA₂ value in parentheses): mesulergine (8.2), cinanserin (8.2), yohimbine (6.2) and mianserin (8.6). However, as well as a rightward shift, methiothepin (8.3), pizotifen (8.6) and spiperone (8.8) also caused a depression of the curve, indicative of ‘pseudo-irreversible'' antagonism. Taken together, the above mentioned affinity estimates most closely corresponded to literature affinity values for 5-HT_2A receptors.
5. It was concluded that 5-HT induces contractions of the canine midcolon longitudinal muscle primarily by stimulation of smooth muscle 5-HT_2A receptors. The presence of inhibitory 5-HT₄ receptors cannot be ruled out.

     

Actions of non-peptide ergot alkaloids at 5-HT1-like and 5-HT2 receptors mediating vascular smooth muscle contraction

Summary This report describes the actions of the non-peptide ergot alkaloids methysergide, methylergometrine and ergometrine at two types of 5-HT receptor mediating vascular contraction; the well established 5-HT₂ receptor in rabbit aorta and a non-5-HT₂ receptor in rabbit saphenous vein which resembles the 5-HT₁-like receptor in dog saphenous vein.In the rabbit aorta ergometrine (1 mol/l) and methylergometrine (0.3 mol/l), but not methysergide, produced small contractions (14% and 7% respectively of the maximal response to 5-HT). This contraction was not related to activation of 5-HT₂ receptors since it was resistant to blockade by ketanserin (0.3 mol/l). When examined as antagonists of 5-HT-induced contractions of rabbit aorta, each ergot displayed nanomolar affinity at the 5-HT₂ receptor but only methysergide behaved as a simple competitive antagonist (pK_B = 8.25). Methylergometrine and ergometrine produced surmountable blockade which was accompanied by a non-parallel displacement of the 5-HT concentration-effect curves. The selective 5-HT₁-like receptor agonist GR43175 ( 30 mol/l) was devoid of affinity at the 5-HT₂ receptor in rabbit aorta.In the rabbit saphenous vein each of the ergots produced concentration-dependent contractions which resulted in overtly biphasic concentration-effect curves. Only the first phase of contraction mimicked the effects of 5-HT and GR43175 since contractions were not blocked by MDL 72222 (1 mol/l), but were surmountably antagonised by methiothepin (10 nmol/1), ketanserin (0.3 mol/l) and spiperone (0.3 mol/l). These results are expected for interactions at the 5-HT₁-like receptor in this preparation (Martin and MacLennan 1990). The mechanism(s) underlying the second phase of contraction with the ergots remains to be established. Receptor inactivation studies using the alkylating agent benextramine tetrahydrochloride enabled each agonists' affinity and efficacy at the 5-HT₁-like receptor to be estimated. Affinity estimates (pK_A) decreased in the order: methylergo metrine (7.79), ergometrine (7.75), 5-HT (7.19), methysergide (6.76), GR43175 (6.20), whereas efficacies () decreased in the order: 5-HT (3.28), methylergometrine (2.24), GR43175 (2.14), ergometrine (1.94), methysergide (0.99). Of particular interest, methysergide was significantly lower in affinity and efficacy than its primary demethylated metabolite methylergometrine. Evidently, at the 5-HT₁-like receptor mediating vascular contraction the ergots ergometrine and methylergometrine are both higher in affinity than, and comparable in efficacy to, the natural receptor agonist 5-HT. This contrasts with their actions at the 5-HT₂ receptor in rabbit aorta where they demonstrated a higher affinity but much lower intrinsic efficacy than 5-HT. These results favour the view that vascular contraction induced by these ergots is more likely to be mediated by 5-HT₁-like, rather than 5-HT₂ receptors. These results are discussed in relation to the therapeutic applications of these ergots, particularly in obstetrics and in migraine, and to their utility as diagnostic agents in patients with Prinzmetal's variant form of angina.Send offprint requests to S. J. MacLennan at the above address     

Competitive antagonism by recognised 5-HT2 receptor antagonists at 5-HT1C receptors in pig choroid plexus

Summary The effects of several antagonists, known to interact with 5-HT₂ receptors (ritanserin, LY 53857, ICI 169,369, methysergide, mesulergine and ketanserin), were tested against 5-HT-stimulated production of inositol phosphate in pig choroid plexus, a 5-HT_1C receptor model. These antagonists produced dextral shifts of the concentration response curve to 5-HT in a parallel manner, without depressing significantly the maximal response. The following pA₂ values (in parentheses) were obtained: mesulergine (8.88), methysergide (8.85), LY 53857 (8.69), ritanserin (8.69), ICI 169,369 (7.86), and ketanserin (6.57). These pA₂ values were in good agreement with the pK_D values determined in radioligand binding studies performed in pig choroid plexus with [³H]mesulergine. The present data demonstrate that several drugs described as 5-HT₂ receptor selective antagonists (e.g. ritanserin, LY 53857 and ICI 169,369) are also potent, competitive and surmountable antagonists at 5-HT_1C receptors. Thus, the results provide further evidence for the pharmacological similarity of 5-HT_1C and 5-HT₂ receptors. However, in contrast to the situation described with methysergide, ritanserin and LY 53857 in several 5-HT₂ receptor models, none of these antagonists acted in a non-competitive or unsurmountable fashion at 5-HT_1C receptors. These results suggest, but do not firmly rule out, that at least in the presence of the drugs tested in the present study, 5-HT_1C receptors in the choroid plexus do not undergo an allosteric modulation; these findings are apparently in contrast to a model proposed previously for 5-HT₂ receptors (Kaumann and Frenken 1985, Naunyn-Schmiedeberg's Arch Pharmacol 328: 295–300) Send offprint requests to P. Schoeffter at the above address     

Potentiation of 5-HT3 receptor function by the activation of coexistent 5-HT2 receptors in trigeminal ganglion neurons of rats

5-HT receptor subtypes are widely expressed in primary sensory neurons, yet so far little is known about the interaction among them. This study aimed to investigate whether the activation of 5-HT2 and 5-HT1 receptors could modulate 5-HT3 receptor mediated current in rat trigeminal ganglion (TG) neurons using whole-cell patch clamp technique. The majority of TG neurons examined responded to 5-HT (10(-7)-10(-3) M) with a fast activating and rapid desensitizing inward current (77.2%, 71/92). This 5-HT activated current (I(5-HT)) was blocked by ICS 205-930 and mimicked by 2-methyl-5-HT, indicating that it was mediated by 5-HT3 receptor. With alpha-methyl-5-HT applied prior to 5-HT application, I(5-HT) was potentiated in a concentration-dependent manner, with the maximal modulatory effect at 10(-9) M of alpha-methyl-5-HT. The concentration-response curve for I(5-HT) pretreated with alpha-methyl-5-HT shifts upwards compared with that for I(5-HT) without alpha-methyl-5-HT pretreatment, the maximal I(5-HT) value having increased by (60.3 +/- 5.7)% of its control while the EC50 values of the two curves being very close, i.e. (2.0 +/- 0.3) x 10(-5) M vs (1.7 +/- 0.2) x 10(-5) M, respectively. The alpha-methyl-5-HT potentiation of I(5-HT) was removed by intracellular dialysis of either GDP-beta-S, a non-hydrolyzable GDP analog, or GF109203X, a selective PKC inhibitor, almost completely. Preapplication of R-(+)-UH-301, a selective agonist of 5-HT(1A) receptor, had no modulatory effect on I(5-HT). These results suggest that in the membrane of TG neurons, the activation of 5-HT2 receptors can exert an enhancing effect on the function of coexistent 5-HT3 receptors while that of 5-HT(1A) receptors cannot.     

Evidence for presynaptic location of inhibitory 5-HT1D\-like autoreceptors in the guinea-pig brain cortex

The effects of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists on tritium overflow evoked by high K⁺ were determined in superfused synaptosomes and slices, preincubated with [³H]5-HT, from guinea-pig brain cortex. In addition, we estimated the potencies of 5-HT receptor ligands in inhibiting specific [³H]5-HT binding (in the presence of 8-hydroxy-2(di-n-propylamino)tetralin and mesulergine to prevent binding to 5-HT_1A and 5-HT_2C sites) to guinea-pig cortical synaptosomes and membranes.5-HT receptor agonists inhibited the K⁺-evoked tritium overflow from synaptosomes and slices. In synaptosomes the rank order of potencies was 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indole-3-yl] ethylamine (L-694,247) >5-carboxamidotryptamine (5-CT) > oxymetazoline (in the presence of idazoxan) 5-HT > sumatriptan 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole (RU 24969). The potencies of the agonists in inhibiting tritium overflow from slices correlated with those in synaptosomes, suggesting that the same site of action is involved in both preparations. In synaptosomes the nonselective antagonist at cloned human 5-HT_1D, and 5-HT_1D receptors, methiothepin, shifted the concentration-response curve for 5-CT to the right (apparent pA₂: 7.87). In contrast, ketanserin at a concentration which should block the 5-HT_1D, but not the 5-HT_1D\, receptor did not alter the inhibitory effect of 5-CT on tritium overflow. In cortical synaptosomes and membranes, [³H]5-HT bound to a single site with high affinity. In competition experiments, 5-HT receptor agonists and antagonists inhibited specific [³H]5-HT binding. In synaptosomes the rank order was L-694,247 > methiothepin >5-CT >5-methoxytryptamine >5-HT sumatriptan oxymetazoline > RU 24969 > ketanserin > ritanserin. A very similar rank order was obtained in cerebral cortical membranes. The potencies of the 5-HT receptor agonists in inhibiting tritium overflow from synaptosomes and slices correlated with their potencies in inhibiting [³H]5-HT binding to synaptosomes and membranes.In conclusion, the 5-HT receptors mediating inhibition of 5-HT release in the guinea-pig cortex are located on the serotoninergic axon terminals and, hence, represent presynaptic inhibitory autoreceptors. The [³H]5-HT binding sites in cerebral cortical synaptosomes and membranes exhibit the pharmacological properties of 5-HT_1D receptors. The correlation between the functional responses and the binding data confirms the 5-HT_1D character of the presynaptic 5-HT autoreceptors. According to the results of the interaction experiment of ketanserin and methiothepin with 5-CT on 5-HT release, the presynaptic 5-HT autoreceptors can be subclassified as 5-HT_1D\-like.     

Evidence that blockade of post-synaptic 5-HT1 receptors elicits feeding in satiated rats

The effects of nine central 5-HT antagonists on food intake in free feeding male rats were examined. The 5-HT₂ antagonists ritanserin and ketanserin and the selective 5-HT₃ antagonists ICS 205-930 and MDL 72222 had no effect on food intake. In contrast, the non-selective 5-HT antagonists metergoline, methiothepin, mesulergine, mianserin and methysergide (all of which have high affinity for various 5-HT₁ receptor subtypes), dose-dependently increased food intake during a 4-h daytime test. Furthermore, metergoline dose dependently increased food intake over a 24-h period. Suprisingly, mesulergine decreased food intake over a 24-h period at the same doses that increased daytime food intake. This may indicate that the increase in daytime feeding produced by mesulergine is a non-specific response. Although the antagonists used have varying degrees of selectivity for 5-HT receptor subtypes, the pattern of results suggests that postsynaptic 5-HT₁ receptors (possibly of the 5-HT_1C type) play an important role in the control of feeding in rats.     

5-Hydroxytryptamine-induced contractions of the human isolated saphenous vein: involvement of 5-HT2 and 5-HT1D-like receptors,and a comparison with grafted veins

Summary The receptors mediating the contractile effect of 5-hydroxytryptamine (5-HT) on the human isolated saphenous vein, obtained from 42 patients undergoing coronary bypass surgery, have been further characterized using a number of 5-HT-related drugs. The rank order of agonist potency was 5-carboxamidotryptamine (5-CT) 5-HT > methysergide sumatriptan -methyl-5-HT 5-methoxy-3-(1,2,3,6-tetrahydropyridin-4-yl)-1-Hindolesuccinate (RU 24969) 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) > 2-methyl-5-HT > 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT). Flesinoxan was inactive as an agonist. Ketanserin (1 mol/l) hardly affected sumatriptan-induced contractions but it caused a rightward shift of the upper part of the concentration-response curve of 5-HT and 5-CT. The same concentration of ketanserin caused a parallel rightward shift of the concentration-response curves of -methyl-5-HT and DOI with pK_B values of 7. 1 and 7.1, respectively. The responses to sumatriptan were antagonized by methiothepin (0.1 mol/l), metergoline (0.1 and 1 mol/l), rauwolscine (1 mol/l) and cyanopindolol (1 mol/l); the calculated pK_B values were 7.3, 6.9, 7.3, 6.7 and 6.5, respectively. Contractions to 5-HT were antagonized by methysergide (1 mol/l), methiothepin (0.1 mol/l; pKB = 7.1), ICS 205-930 (1 mol/l; pK_B = 5.9) and flesinoxan (30 mol/l; pK_B = 5.3). Remarkably, the contractions elicited by 2-methyl-5-HT were not attenuated by ICS 205-930, but were antagonized by methiothepin (0.1 mol/l) and, more markedly, by ketanserin (1 mol/l).There was a high correlation between the functional pD₂ values of 5-HT₁-like receptor agonists (5-CT, 5-HT, methysergide, sumatriptan, RU 24969 and 8-OH-DPAT) and their reported binding affinities for the 5-HT_1D receptor in human or calf brain membranes. Such a correlation for the antagonism of sumatriptan-induced responses was less marked than for the agonists, but of the 5-HT₁-like receptor subtypes it was the highest for the 5-HT_1D receptor identified in human or calf brain membranes.In 3 patients, undergoing heart transplantation, saphenous vein which had previously functioned as a graft for 6–11 years, was dissected out from the heart. Though the contractions to potassium were significantly smaller in the grafted veins, the pD₂ and E_max values (calculated as percentage of potassium-induced contractions) for 5-HT and sumatriptan were similar to those found in the veins obtained directly from the lower leg.It is concluded that contractions in the human isolated saphenous vein induced by 5-HT are mediated by 5-HT₂ receptors as well as by a 5-HT₁-like receptor resembling the 5-HT_1D subtype found in brain membranes. It is also to be noted that 2-methyl-5-HT, considered selective for the 5-HT₃ receptor, contracts the saphenous vein mainly via 5-HT₂ receptors.This study was supported by the Netherlands Heart Foundation, grant 89.252 Send offprint requests to W. A. Bax at the above address     

Mitochondrial monoamine oxidase-A-mediated hydrogen peroxide generation enhances 5-hydroxytryptamine-induced contraction of rat basilar artery

BACKGROUND AND PURPOSE

We evaluated the role(s) of monoamine oxidase (MAO)-mediated H₂O₂ generation on 5-hydroxytryptamine (5-HT)-induced tension development of isolated basilar artery of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats.

EXPERIMENTAL APPROACH

Basilar artery (endothelium-denuded) was isolated for tension measurement and Western blots. Enzymically dissociated single myocytes from basilar arteries were used for patch-clamp electrophysiological and confocal microscopic studies.

KEY RESULTS

Under resting tension, 5-HT elicited a concentration-dependent tension development with a greater sensitivity (with unchanged maximum tension development) in SHR compared with WKY (EC₅₀: 28.4 ± 4.1 nM vs. 98.2 ± 9.4 nM). The exaggerated component of 5-HT-induced tension development in SHR was eradicated by polyethylene glycol-catalase, clorgyline and citalopram whereas exogenously applied H₂O₂ enhanced the 5-HT-elicited tension development in WKY. A greater protein expression of MAO-A was detected in basilar arteries from SHR than in those from WKY. In single myocytes and the entire basilar artery, 5-HT generated (clorgyline-sensitive) a greater amount of H₂O₂ in SHR compared with WKY. Whole-cell iberiotoxin-sensitive Ca²⁺-activated K⁺ (BK_Ca) amplitude measured in myocytes of SHR was approximately threefold greater than that in WKY (at +60 mV: 7.61 ± 0.89 pA·pF⁻¹ vs. 2.61 ± 0.66 pA·pF⁻¹). In SHR myocytes, 5-HT caused a greater inhibition (clorgyline-, polyethylene glycol-catalase- and reduced glutathione-sensitive) of BK_Ca amplitude than in those from WKY.

CONCLUSIONS AND IMPLICATIONS

5-HT caused an increased generation of mitochondrial H₂O₂ via MAO-A-mediated 5-HT metabolism, which caused a greater inhibition of BK_Ca gating in basilar artery myocytes, leading to exaggerated basilar artery tension development in SHR.     

芳烷酮哌嗪类化合物SCP-1和SCP-2与5-HT1,2受体的结合实验

目的研究芳烷酮哌嗪类化合物SCP-1和SCP-2与5-HT1和5-HT2受体体外是否有结合作用.方法5-HT1受体取材于大鼠大脑皮质,5-HT2受体取材于大鼠海马.采用受体-配体结合测定法测定10-3mol·L-1的SCP-1和SCP-2对5-HT1,5-HT2受体的最大结合率、半数抑制浓度(IC50)和Hill系数.结果10-3mol·L-1的SCP-1和SCP-2与5-HT1受体的最大结合率分别为75%和63%.SCP-1和SCP-2与5-HT1受体结合的IC50分别为1.584和5.495μmol·L-1,二者与5-HT1受体结合的Hill系数分别为0.98和1.02.10-3mol·L-1的SCP-1和SCP-2与5-HT2受体的最大结合率分别为92%和87%.SCP-1和SCP-2与5-HT2受体结合的IC50分别为1.0和2.512μmol·L-1,二者与5-HT2受体结合的Hill系数分别为0.86和0.88.结论SCP-1和SCP-2与5-HT1受体的结合方式是与单一受体位点结合,并且这种结合服从质量作用定律(Hill系数接近于1).SCP-1和SCP-2与5-HT2受体呈现不规则性结合,也可能有负相互作用或多种结合位点(Hill系数偏离1).     

5-HT7, but not 5-HT2B, receptors mediate hypotension in vagosympathectomized rats

This study evaluated the possible involvement of 5-HT(2B) receptors in long-lasting hypotension to 5-hydroxytryptamine (5-HT), which is predominantly mediated by 5-HT7 receptors, in anaesthetised vagosympathectomized rats. Intravenous injections of 5-HT and 5-carboxamidotryptamine (5-CT) elicited a dose-dependent hypotension that was dose-dependently antagonised by (R)-1-[(3-hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl) ethyl] pyrrolidine (SB-269970; a selective 5-HT7 receptor antagonist), but not by saline. Interestingly, alpha-methyl-5-(2-thienylmethoxy)-1H-indole-3-ethanamine (BW723C86; a 5-HT(2B) receptor agonist) produced vasopressor responses without affecting hypotension to 5-HT. These results suggest that hypotension to 5-HT and 5-CT is mainly mediated by 5-HT7 receptors, whilst the role of 5-HT(2B) receptors seems unlikely.     

Evidence for the involvement of 5-HT4 receptors in the 5-hydroxytryptamine-induced pattern of migrating myoelectric complex in sheep

1. The effects induced by 5-hydroxytryptamine (5-HT) on gastrointestinal myoelectric activity in conscious sheep were recorded through electrodes chronically implanted and analysed by computer. The 5-HT receptors and the cholinergic neuronal pathways involved in these actions were investigated.
2. The intravenous (i.v.) administration of 5-HT (2, 4 and 8 μg kg⁻¹ min⁻¹, 5 min) induced an antral inhibition concomitant with a duodenal activity front that migrated to the jejunum, followed by a period of intestinal inactivity. This myoelectric pattern closely resembled that observed in the phases III and I of the migrating myoelectric complex (MMC) in sheep. The 0.5 μg kg⁻¹ min⁻¹ dose evoked the same pattern in only two out of the six animals used. Likewise, the 1 μg kg⁻¹ min⁻¹ dose similarly affected four of the six animals. In addition, a transient stimulation was observed in the antrum and jejunum when the two highest doses were used.
3. The 5-HT₁ antagonist, methiothepin (0.1 mg kg⁻¹), the 5-HT₂ antagonists, ritanserin (0.1 mg  kg⁻¹) and ketanserin (0.3 mg  kg⁻¹), the 5-HT₃ antagonists, granisetron (0.2 mg kg⁻¹) and ondansetron (0.5 mg kg⁻¹), as well as the 5-HT₄ antagonist, {"type":"entrez-nucleotide","attrs":{"text":"GR113808","term_id":"238362519","term_text":"GR113808"}}GR113808 (0.2 mg kg⁻¹), did not modify the spontaneous gastrointestinal myoelectric activity. However, the cholinoceptor antagonists, atropine (0.2 mg kg⁻¹) and hexamethonium (2 mg kg⁻¹), inhibited gastrointestinal activity.
4. When these antagonists were injected i.v. 10 min before 5-HT (2 or 4 μg kg⁻¹ min⁻¹, 5 min), only {"type":"entrez-nucleotide","attrs":{"text":"GR113808","term_id":"238362519","term_text":"GR113808"}}GR113808, atropine and hexamethonium were able to modify the 5-HT-induced actions, all of them being completely blocked by the three antagonists.
5. Our data show that 5-HT initiates a MMC-like pattern in the gastrointestinal area in sheep through 5-HT₄ receptors. Furthermore, these actions are mediated by cholinergic neural pathways involving muscarinic and nicotinic receptors. However, our results do not indicate a role for either 5-HT₁, 5-HT₂ or 5-HT₃ receptors in the 5-HT-induced effects.

     

Acute and chronic tryptophan depletion differentially regulate central 5-HT1A and 5-HT2A receptor binding in the rat

Rationale Tryptophan depletion is used to reduce central serotonergic function and to investigate its role in psychiatric illness. Despite widespread clinical use, its effects on serotonin (5-HT) receptors have not been well characterized. Objective The aim of this study was to examine the effect of acute (ATD) and chronic tryptophan depletion (CTD) on free-plasma tryptophan (TRP), central TRP and 5-HT and brain 5-HT_1A and 5-HT_2A receptor binding in the rat. Methods TRP and 5-HT were measured by high-performance liquid chromatography and receptor levels determined by homogenate radioligand binding and in-vitro receptor autoradiography. Results Free-plasma TRP, central TRP and central 5-HT levels were significantly and similarly reduced by ATD and 1- and 3-week CTD compared to controls. ATD significantly reduced 5-HT_1A binding in the dorsal raphe (14%) but did not significantly alter postsynaptic 5-HT_1A binding (frontal cortex, remaining cortex and hippocampus) or 5-HT_2A binding (cortex and striatum). One-week CTD did not significantly alter cortical 5-HT_2A binding or postsynaptic 5-HT_1A binding. Furthermore, 3-week CTD did not significantly alter 5-HT_1A binding but significantly increased cortical 5-HT_2A binding without affecting striatal or hippocampal levels. In the CTD 1 and 3-week groups, rat body weight was significantly decreased as compared to controls. However, weight loss was not a confounding factor for decreased cortical 5-HT_2A-receptor binding. Conclusion ATD-induced reduction in somatodendritic 5-HT_1A autoreceptor binding may represent an intrinsic ‘homeostatic response’ reducing serotonergic feedback in dorsal raphe projection areas. In contrast, the increase in 5-HT_2A receptor after CTD may be a compensatory response to a long-term reduction in 5-HT.     

A PET study of brain 5-HT2 receptors and their correlation with platelet 5-HT2 receptors in healthy humans

RATIONALE: Platelets share many properties with brain serotonergic neurons such as active 5-hydroxytryptamine (5-HT) transport, 5-HT2 receptors, and mitochondrial monoamine oxidase. OBJECTIVES: We measured brain 5-HT2 receptors and platelet 5-HT2 receptors in healthy volunteers to determine if there was any correlation between the two measures. METHODS: Ten healthy volunteers with no lifetime history of psychiatric illness or family history in first-degree relatives were recruited. 5-HT2 receptor binding was determined for each subject with positron emission tomography and [18F]setoperone scan in the brain and with 3H-LSD binding in platelets. RESULTS: We found no significant correlation between 5-HT2 binding potential (BP) in platelets (Bmax/Kd) and a semiquantitative estimate of 5-HT2 BP in frontal, parietal, and temporal cortical regions. SPM voxel based analysis also showed no significant correlation between the 5-HT2 BP in platelets and in the brains of the study subjects. CONCLUSIONS: Brain 5-HT2 receptor binding was not significantly correlated to platelet 3H-LSD binding in healthy subjects. This raises questions about the validity of generalizing findings from platelet studies to 5-HT neurons in the brain.     

5-Hydroxytryptamine potentiates neurogenic contractions of rat isolated urinary bladder through both 5-HT(7) and 5-HT(2C) receptors

Serotonin (5-HT) enhances the neurogenic contractile response induced by electrical field stimulation (EFS) in the rat isolated urinary bladder. The aim of this study was to functionally characterize the receptors involved in this effect by using a range of 5-HT receptor subtype selective agonists and antagonists. 5-HT produced a concentration-dependent potentiation of contractile responses to EFS with a pEC₅₀ value of 6.86 ± 0.24. SB-269970 (0.01, 0.1 and 1 μM), a selective 5-HT₇ receptor antagonist, caused a concentration-dependent rightward shift of the 5-HT-induced response. The pA₂ value was 8.16 with a slope of 0.46 ± 0.08. Neither ketanserine nor SB-204741, 5-HT_2A and 5-HT_2B receptors antagonists, respectively, affected the concentration–response curve to 5-HT. However, 5-HT response was antagonized by the selective 5-HT_2C receptor antagonist SB-242084 (0.1 and 1 μM). In the presence of 1 μM of both antagonists SB-269970 and SB-242084, 5-HT response was almost fully inhibited. 5-CT, a 5-HT₇ receptor agonist, induced a biphasic concentration-dependent potentiation of neurogenic contractions. SB-269970 concentration-dependently antagonized the first phase of 5-CT response with a pA₂ value of 8.77 and a slope not significantly different from unity (0.91 ± 0.11) that suggests a competitive antagonism. WAY-161503, a 5-HT_2C receptor agonist (0.01–10 μM), induced a concentration-dependent potentiation of contractile response to EFS while DOI (a selective 5-HT_2A agonist) had no effect. SB-242084 (0.1 and 1 μM) antagonized the effect of WAY-161503 in a concentration-dependent manner. The current results demonstrate that 5-HT potentiates neurogenic contractions of rat isolated detrusor muscle through both 5-HT₇ and 5-HT_2c receptors.