Synergism between 5-HT1B/1D and 5-HT1A receptor antagonists on turnover and release of 5-HT in guinea-pig brain in vivo

The effects on 5-HT turnover (5-HIAA/5-HT ratio) and extracellular 5-HT and 5-HIAA levels (in vivo microdialysis in freely moving animals) were analysed in guinea-pig brains following the 5-HT_1B receptor antagonist, GR 127935 {N-[4-methoxy-3-(4-methyl-1-piper_{azinyl)phenyl]-2’-methyl-4’-(5-methyl-1,2,4-oxadiazol-3-}yl) [1,1-biphenyl]-4-carboxamide}, or the 5-HT_1A receptor antagonist, WAY-100635 (N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride), administered alone or in combination. GR 127935, injected alone, increased 5-HT turnover with maximal effects approximately 50% above the control levels in the four brain regions examined (hypothalamus, hippocampus, striatum and frontal cortex). GR 127935 significantly increased extracellular concentrations of 5-HT and 5-HIAA in frontal cortex (40%), whereas 5-HIAA, but not 5-HT, was elevated in striatum (20–30%). WAY-100635 did not significantly change 5-HT turnover but caused a small significant increase in the extracellular 5-HT and 5-HIAA concentrations in both striatum and frontal cortex. The combined treatment with GR 127935 and WAY-100635 resulted in an increased 5-HT turnover reaching maximal effects of 70–90% above the control values in all brain regions tested and produced a significant elevation of striatal and frontal cortex extracellular 5-HT (40% and 60%, respectively) and 5-HIAA (60% and 70%, respectively) concentrations. The synergistic effect of the two receptor antagonists on the 5-HT turnover and the terminal release of 5-HT indicate somatodendritic 5-HT release and stimulation of inhibitory 5-HT_1A receptors at this level. Extracellular 5-HIAA seems to be a better marker than 5-HT itself for the evoked 5-HT release when the reuptake mechanism is intact. Received: 2 September 1998 / Accepted: 19 November 1998     

Further characterization of the 5-HT<Subscript>1</Subscript> receptors mediating cardiac sympatho-inhibition in pithed rats: pharmacological correlation with the 5-HT<Subscript>1B</Subscript> and 5-HT<Subscript>1D</Subscript> subtypes

Serotonin (5-hydroxytryptamine; 5-HT) is capable of inhibiting the tachycardic responses elicited by sympathetic stimulation, but not by exogenous noradrenaline, in pithed rats pre-treated with desipramine. More recently, it has been shown that this cardiac sympatho-inhibitory response to 5-HT, mediated by prejunctional 5-HT₁ receptors as well as putative 5-ht_5A/5B receptors, is mimicked dose-dependently by the agonists CP 93,129 (r5-HT_1B), sumatriptan (5-HT_1B/1D) and PNU-142633 (5-HT_1D). This study analysed further the pharmacological profile of the above 5-HT₁ receptors.Continuous i.v. infusions of CP 93,129, sumatriptan or PNU-142633 (30 µg kg^–1min^–1 each) failed to modify the tachycardic responses to exogenous noradrenaline but inhibited those elicited by preganglionic (C7–T1) stimulation of the cardiac sympathetic outflow. These sympatho-inhibitory responses were unaltered after i.v. administration of physiological saline (1 ml kg^–1) or the 5-HT_1A receptor antagonist WAY 100635 (10 µg kg^–1). In contrast, the antagonist GR 127935 (5-HT_1B/1D; 100 µg kg^–1, i.v.) abolished the responses to CP 93,129, sumatriptan and PNU-142633, whilst SB224289 (5-HT_1B; 300 µg kg^–1, i.v.) abolished the responses to CP 93,129 without affecting those to sumatriptan and PNU-142633. Interestingly, BRL15572 (5-HT_1D; 300 µg kg^–1, i.v.) abolished the responses to PNU-142633 and attenuated those to sumatriptan, but not those to CP 93,129.WAY 100635, GR 127935, SB224289 and BRL15572, given alone at the above doses, failed to modify the sympathetically induced tachycardic responses. The 5-HT₁ receptors producing cardiac sympatho-inhibition in pithed rats thus display the pharmacological profile of the 5-HT_1B and 5-HT_1D receptor subtypes.     

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

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

Involvement of 5-HT1B/1D and 5-HT2A receptors in 5-HT-induced contraction of endothelium-denuded rabbit epicardial coronary arteries

1. The receptors responsible for 5-hydroxytryptamine (5-HT)-mediated contraction of rabbit isolated epicardial coronary artery denuded of endothelium was examined by bioassay.
2. A variety of 5-HT mimetics caused concentration-dependent contractions. The rank order of agonist potency was 5-carboxamidotryptamine (5-CT)>5-HT>(±)-α-methyl-5-hydroxytryptamine ((±)-α-me-5-HT)=sumatriptan. This was not consistent with relative potencies at any single recognized 5-HT receptor, suggesting the presence of a mixed receptor population. In one subset of preparations precontracted with U46619 (10–30 nM) with the endothelium intact, none of the agonists caused a relaxation.
3. Contractions to 5-HT were antagonized by ketanserin, a 5-HT_2A-selective antagonist, but the displacement of concentration-response curves was inconsistent with an interaction between 5-HT and a single receptor population; the slope of regression between antagonist log M concentration and agonist log (concentration-ratio −1) was shallow (0.57). Responses to 5-HT were also antagonized by the 5-HT_1B/1D-receptor antagonist {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 and, again, the slope of regression was shallow (0.68). These data suggest a possible involvement of 5-HT_2A and 5-HT_1B or 5-HT_1D receptors in the response to 5-HT.
4. Contractions to (±)-α-me-5-HT, which is selective for 5-HT_2A over 5-HT_1B and 5-HT_1D receptors, were competitively antagonized by low concentrations of ketanserin. The regression between antagonist log M concentration and agonist log (concentration-ratio −1) fitted the Schild equation with a slope that was not significantly different from unity (0.95), giving a pA₂ value of 9.0. {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 (3–30 nM), had no effect on the contractile response to (±)-α-me-5-HT. These data establish, unequivocally, the presence of 5-HT_2A receptors in the tissue.
5. Sumatriptan, a relatively selective 5-HT_1B/1D-receptor agonist, induced contractions that were antagonized competitively by {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 (3–30 nM), although there was a reduction in the maximum response when concentrations of {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 exceeded 3 nM. The apparent pA₂ (estimated by imposing a unit slope on the log agonist (concentration-ratio −1) value in the presence of 3 nM {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935) was 8.92. Contractions to sumatriptan were not affected by low (5-HT_2A receptor-selective) concentrations of ketanserin, but were antagonized in a competitive manner at higher concentrations (pA₂ 6.5). These data appear to confirm the presence of 5-HT_1B and/or 5-HT_1D receptors in the tissue.
6. Antagonism of 5-HT responses by {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 was reassessed after blockade of 5-HT_2A receptors with 1 μM ketanserin. Under these conditions, {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 was able to antagonize 5-HT-induced contractions fully. The slope of regression between log M antagonist concentration and log agonist (concentration-ratio −1) fitted the Schild equation with a slope not significantly different from unity (1.1) (albeit there was still a reduction in maximum response when {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 concentration exceeded 3 nM). The apparent pA₂ value was 8.8. This reinforces the evidence that 5-HT_1B and/or 5-HT_1D receptors contribute to the effects of 5-HT in the tissue.
7. In conclusion, in endothelium denuded rabbit epicardial coronary arteries, 5-HT activates 5-HT_2A and 5-HT_1D and/or 5-HT_1B receptors to cause contraction. This appears to be similar to the situation in man.

     

Characterization of putative 5-HT7 receptors mediating tachycardia in the cat

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 μg kg⁻¹), 5-HT (3, 10 and 30 μg kg⁻¹) and 5-methoxytryptamine (3, 10 and 30 μg kg⁻¹) as well as the atypical antipsychotic drug, clozapine (1000 and 3000 μg 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 μg kg⁻¹), ergotamine (100 and 300 μg kg⁻¹) or mesulergine (100, 300 and 1000 μg kg⁻¹); the highest doses of these antagonists used also blocked the tachycardic effects of 5-CT. Clozapine (1000 and 3000 μg 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 μg kg⁻¹) and mesulergine (300 and 1000 μg 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, {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 (500 μg kg⁻¹) or the 5-HT_3/4 receptor antagonist, tropisetron (3000 μg kg⁻¹).
4. Intravenous injections of the 5-HT₁ receptor agonists, sumatriptan (30, 100 and 300 μg kg⁻¹) and indorenate (300 and 1000 μg kg⁻¹) or the 5-HT₄ receptor (partial) agonist cisapride (300 and 1000 μg 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.

     

The 5-HT1-like receptors mediating inhibition of sympathetic vasopressor outflow in the pithed rat: operational correlation with the 5-HT1A, 5-HT1B and 5-HT1D subtypes

1. It has been suggested that the inhibition of sympathetically-induced vasopressor responses produced by 5-hydroxytryptamine (5-HT) in pithed rats is mediated by 5-HT₁-like receptors. The present study has re-analysed this suggestion with regard to the classification schemes recently proposed by the NC-IUPHAR subcommittee on 5-HT receptors.
2. Intravenous (i.v.) continuous infusions of 5-HT and the 5-HT₁ receptor agonists, 8-OH-DPAT (5-HT_1A), indorenate (5-HT_1A), CP 93,129 (5-HT_1B) and sumatriptan (5-HT_1B/1D), resulted in a dose-dependent inhibition of sympathetically-induced vasopressor responses.
3. The sympatho-inhibitory responses induced by 5-HT, 8-OH-DPAT, indorenate, CP 93,129 or sumatriptan were analysed before and after i.v. treatment with blocking doses of the putative 5-HT receptor antagonists, WAY 100635 (5-HT_1A), cyanopindolol (5-HT_1A/1B) or GR 127935 (5-HT_1B/1D). Thus, after WAY 100635, the responses to 5-HT and indorenate, but not to 8-OH-DPAT, CP 93,129 and sumatriptan, were blocked. After cyanopindolol, the responses to 5-HT, indorenate and CP 93,129 were abolished, whilst those to 8-OH-DPAT and sumatriptan (except at the lowest frequency of stimulation) remained unaltered. In contrast, after GR 127935, the responses to 5-HT, CP 93,129 and sumatriptan, but not to 8-OH-DPAT and indorenate, were abolished.
4. In additional experiments, the inhibition induced by 5-HT was not modified after 5-HT₇ receptor blocking doses of mesulergine.
5. The above results suggest that the 5-HT₁-like receptors, which inhibit the sympathetic vasopressor outflow in pithed rats, display the pharmacological profile of the 5-HT_1A, 5-HT_1B and 5-HT_1D, but not that of 5-HT₇, receptors.

     

Characterization of 5-HT receptors mediating constriction of porcine carotid arteriovenous anastomoses; involvement of 5-HT1B/1D and novel receptors

1. It was previously shown that porcine cranial arteriovenous anastomoses (AVAs) constrict to 5-hydroxytryptamine (5-HT), ergotamine, dihydroergotamine, as well as sumatriptan and that sumatriptan acts exclusively via 5-HT_1B/1D receptors. The present study was devoted to establish the contribution of 5-HT_1B/1D receptors in the constriction of AVAs elicited by 5-HT (in presence of 0.5 mg kg⁻¹ ketanserin), ergotamine and dihydroergotamine in anaesthetized pigs.
2. Intracarotid infusion of 5-HT (2 μg kg⁻¹ min⁻¹) and intravenous doses of ergotamine (2.5–20 μg kg⁻¹) and dihydroergotamine (3–100 μg kg⁻¹) reduced AVA and increased nutrient blood flows and vascular conductances. The vasodilator response to 5-HT, observed mainly in the skin and ear, was much more prominent than that of the ergot alkaloids.
3. Treatment with the 5-HT_1B/1D receptor antagonist {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 (0.5 mg kg⁻¹, i.v.) significantly attenuated both ergot-induced AVA constriction and arteriolar dilatation, whereas {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 only slightly affected the carotid vascular effects of 5-HT.
4. The results suggest that 5-HT constricts carotid AVAs primarily via receptors, which seem to differ from those (5-HT_1B/1D) stimulated by sumatriptan. The ergot alkaloids produce AVA constriction for a substantial part via 5-HT_1B/1D receptors, but also stimulate unidentified receptors. Both these non-5-HT_1B/1D receptors may be targets for the development of novel antimigraine drugs.
5. The moderate vasodilator response to the ergot derivatives seems to be mediated, at least in part, by 5-HT_1B/1D receptors, whereas the arteriolar dilatation caused by 5-HT may be mediated by other, possibly 5-HT₇ receptors.

     

5-Bicycloindoles as 5-HT1D agonists

In this patent, novel 3-(2-aminoethyl)-, 3-(piperidin-4-yl)- and (R)-3- (pyrrolidin-2-ylmethyl) 5-bicycloindole derivatives are claimed. They are claimed to be agonists at the 5-HT_1D receptors useful for the treatment of 5-HT_1D-mediated disorders such as migraine. The preparation of the compounds is illustrated by six schemes and exemplified by eight examples.The compounds were tested for their inhibition of [³H]-5-HT binding at 5-HT_1D and 5-HT_1B receptors. The compounds are stated to bind selectively to the 5-HT_1D receptor, relative particularly to the 5-HT_1B receptors.     

SB-216641 and BRL-15572 – compounds to pharmacologically discriminate h5-HT1B and h5-HT1D receptors

Despite only modest homology between h5-HT_1B and h5-HT_1D receptor amino acid sequences, these receptors display a remarkably similar pharmacology. To date there are few compounds which discriminate between these receptor subtypes and those with some degree of selectivity, such as ketanserin, have greater affinity for other 5-HT receptor subtypes. We now report on two compounds, SB-216641 (N-[3-(2-dimethylamino) ethoxy-4-methoxyphenyl]-2’-methyl-4’-(5-methyl-1,2,4-oxadiazol-3-yl)-(1,1’-biphenyl)-4-carboxamide) and BRL-15572 3-[4-(3-chlorophenyl) piperazin-1-yl]-1,1-diphenyl-2-propanol), which display high affinity and selectivity for h5-HT_1B and h5-HT_1D receptors, respectively. In receptor binding studies on human receptors expressed in CHO cells, SB-216641 has high affinity (pK_i=9.0) for h5-HT_1B receptors and has 25-fold lower affinity at h5-HT_1D receptors. In contrast, BRL-15572 has 60-fold higher affinity for h5-HT_1D (pK_i=7.9) than 5-HT_1B receptors. Similar affinities for these compounds were determined on native tissue 5-HT_1B receptors in guinea-pig striatum. Functional activities of SB-216641 and BRL-15572 were measured in a [³⁵S]GTPγS binding assay and in a cAMP accumulation assay on recombinant h5-HT_1B and h5-HT_1D receptors. Both compounds were partial agonists in these high receptor expression systems, with potencies and selectivities which correlated with their receptor binding affinities. In the cAMP accumulation assay, results from pK_B measurements on the compounds again correlated with receptor binding affinities (SB-216641, pK _B=9.3 and 7.3; BRL-15572, pK_B=<6 and 7.1, for h5-HT_1B and h5-HT_1D receptors respectively). These compounds will be useful pharmacological agents to characterise 5-HT_1B and 5-HT_1D receptor mediated responses. Received: 14 March 1997 / Accepted: 18 May 1997     

Activation of recombinant h 5-HT1B and h 5-HT1D receptors stably expressed in C6 glioma cells produces increases in Ca2+-dependent K+ current

The putative coupling between stably expressed recombinant h 5-HT_1B or h 5-HT_1D receptors and K⁺ channels which regulate excitability was investigated in C6 glioma cells. Outward K⁺ currents (I _K) were examined in non-transfected C6 glioma cells and in cells expressing cloned h 5-HT_1B or h 5-HT_1D receptors using the patch-clamp technique in the whole-cell configuration. I _K was elicited by a depolarizing step from a holding potential of –60 mV. In C6 glioma cells expressing either recombinant h 5-HT_1B or h 5-HT_1D receptors, sumatriptan similarly increased I _K in a concentration-dependent manner (maximum increase 19.4±7.2%, n=8, P<0.05 and 25.1±3.9%, n=6, P<0.001, respectively) with EC₅₀ values (geometric mean with 95% confidence intervals in parentheses) of 56.3 nM (7.9–140 nM) and 68.7 nM (16–120 nM), respectively. Sumatriptan failed to elicit increases in I _K in non-transfected cells, confirming a specific involvement of the respective membrane h 5-HT_1B and h 5-HT_1D receptors in transfected C6 cells. In the presence of the mixed 5-HT_1B/D receptor antagonist GR 127935 (0.1 μM), sumatriptan (1 μM) failed to significantly increase I _K in C6 cells expressing h 5-HT_1B receptors (–7.5±3.5%, P=NS, n=6), although a higher concentration of GR 127935 (1 μM) was required to significantly inhibit sumatriptan-evoked increases in I _K in C6 cells expressing h 5-HT_1D receptors (–1.8±3.5%, P=NS, n=6), confirming that sumatriptan-evoked responses were indeed mediated by h 5-HT_1B and h 5-HT_1D receptors, respectively. In C6 cells expressing either cloned h 5-HT_1B or h 5-HT_1D receptors, sumatriptan-induced increases in I _K were prevented by the calcium chelator EGTA (5 mM) when included in the patch pipette (maximum increase 0.57±0.6%, n=3, P=NS and –2.8±1.6%, n=5, P=NS, respectively). In C6 cells expressing cloned h 5-HT_1B receptors, sumatriptan (1 μM) similarly failed to significantly increase I _K in the presence of dibutyryl cAMP (10 μM) or when a nominally Ca²⁺-free medium was included in the patch pipette (–19.4±5.1%, n=5 and –5.2±4.3%, n=5, respectively, P=NS in each case). In addition, the Ca²⁺-dependent K⁺ channel blockers iberiotoxin (0.1 μM) and tetraethylammonium (TEA, 1 mM) abolished sumatriptan-induced increases in I _K (–0.5±1.0%, n=4 and –3.9±3.1%, n=4, respectively, P=NS in each case) in C6 cells expressing h 5-HT_1B receptors, confirming the involvement of Ca²⁺-dependent K⁺ channels. In C6 cells expressing cloned h 5-HT_1B receptors, sumatriptan (1 μM) similarly failed to significantly increase I _k after 30-min incubation with thapsigargin (1 μM) or when heparin (2 mg/ml) was included in the patch pipette (1.1±0.4%, n=5 and 1.2±2.4%, n=5, respectively, P=NS). In conclusion, evidence is provided that both recombinant h 5-HT_1B and h 5-HT_1D receptors stably transfected in C6 glioma cells are positively coupled to Ca²⁺-dependent K⁺ channels, and the outward hyperpolarizing current mediated by these channels is dependent upon IP₃ receptor-mediated intracellular Ca²⁺ release. Received: 15 April 1998 / Accepted: 9 September 1998     

Operational characteristics of the 5-HT1-like receptors mediating external carotid vasoconstriction in vagosympathectomized dogs

It has recently been shown that the external carotid vasoconstrictor response to 5-HT in the dog is primarily mediated by sumatriptan-sensitive 5-HT₁-like receptors; however, the fact that these receptors are not blocked by metergoline, a 5-HT_1D ligand, raises questions about their possible correlation with the 5-HT_1D receptor subtype. Since a number of drugs display high affinity for the 5-HT_1D (GR127935) and 5-HT_1F (e.g. methysergide and oxymetazoline) receptor subtypes, in this study we have used these drugs to determine whether the above vasoconstrictor 5-HT₁-like receptors correlate with the 5-HT_1D and/or 5-HT_1F receptor subtypes.One-minute intracarotid infusions of 5-HT (0.3–30 g/min), sumatriptan (1–30 g/min), oxymetazoline (0.03–3 g/min) and noradrenaline (0.3–3 g/min) resulted in dose-dependent decreases in external carotid blood flow without changes in arterial blood pressure or heart rate. These vasoconstrictor responses remained unaltered after i.v. administration of physiological saline (0.015, 0.05 and 0.15 ml/kg; n = 4) or ritanserin (1 mg/kg; n = 5). In contrast, GR127935 (1, 3 and 10 g/kg, n = 6) potently blocked the responses to 5-HT (unmasking a dose-dependent vasodilator component) and sumatriptan without affecting those to oxymetazoline or noradrenaline. Interestingly, methysergide (10, 30 and 100 g/kg, n = 5) also blocked the vasoconstrictor responses to 5-HT and sumatriptan, but unlike GR127935, did not revert the vasoconstrictor response to 5-HT; the responses to oxymetazoline remained unaffected, but those to noradrenaline were apparently attenuated by the highest dose.Taken together, the above findings suggest that the sumatriptan-sensitive 5-HT₁-like receptors mediating canine external carotid vasoconstriction resemble 5-HT_1D receptors, probably of the 5-HT_1D subtype on the basis of the resistance to blockade by ritanserin. The pharmacological profile of these receptors could be similar (bovine and human cerebral arteries, porcine carotid arteriovenous anastomoses and human coronary arteries) to other putative 5-HT_1D receptors mediating vascular responses.     

The role of 5-HT receptor subtypes in the anxiolytic effects of selective serotonin reuptake inhibitors in the rat ultrasonic vocalization test

 We evaluated whether the anxiolytic effects of selective serotonin reuptake inhibitors (SSRIs) in the rat ultrasonic vocalization (USV) test are preferentially mediated by (indirect) activation of 5-HT_1A, 5-HT_1B/1D, 5-HT_2A, 5-HT₃ or 5-HT₄ receptors. The SSRIs, paroxetine (ED₅₀ in mg/kg, IP: 6.9), citalopram (6.5), fluvoxamine (11.7) and fluoxetine (>30), dose dependently reduced shock-induced USV. The effects of paroxetine (3.0 mg/kg, IP) were not blocked by the selective 5-HT_1A receptor antagonist, WAY-100635 (3.0 mg/kg, IP), the 5-HT_1B/1D receptor antagonist, GR 127935 (30 mg/kg, IP), the nonselective 5-HT_2A receptor antagonists, ritanserin (3.0 mg/kg, IP) and ketanserin (1.0 mg/kg, IP), the 5-HT₃ receptor antagonist, ondansetron (0.1 mg/kg, IP), or the 5-HT₄ receptor antagonist, GR 125487D (3.0 mg/kg, SC). In contrast, the selective 5-HT_2A receptor antagonist, MDL 100,907 (0.1 mg/kg, IP), completely prevented the paroxetine-induced reduction of USV. Under similar conditions, WAY-100635 blocked the anxiolytic-like effects of the selective 5-HT_1A receptor agonist, 8-OH-DPAT [(±)-8-hydroxy-2-(di-n-propylamino)tetralin, 1.0 mg/kg, IP], and ritanserin, ketanserin, and MDL 100,907 blocked the anxiolytic-like effects of the mixed 5-HT_2A/2C receptor agonist, DOI [1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, 3.0 mg/kg, IP]. WAY-100635 (1.0 mg/kg, IP) in combination with ritanserin (3.0 mg/kg, IP), but not ondansetron (0.1 mg/kg, IP), GR 125487D (3.0 mg/ kg, SC), or GR 127935 (30 mg/kg, IP), attenuated the USV reducing effects of paroxetine. Although the results suggest that selective stimulation of 5-HT_1A and 5-HT_2A receptors produces a decrease of USV, we postulate that only 5-HT_2A receptors play a pivotal role in the effects of SSRIs in this model of anxiety. Received: 19 May 1997 / Final version: 21 July 1997     

Dilatation induced by 5-HT in the middle meningeal artery of the anaesthetised cat

In chloralose-anaesthetised cats, we studied the effects of intravenous and intra-carotid injections of 5-HT on the middle meningeal artery and the way these were modified by 5-HT antagonists. Cats were prepared for blood pressure recording and intravenous injections and a catheter inserted into one carotid artery via a lingual artery. The middle meningeal arteries were exposed and blood flow recorded with laser Doppler probes. Intravenous injections of 5-HT, 2–50 µg kg^–1 (5.2–129 nmole kg^–1), produced a dose-dependent fall in blood pressure, a rise in meningeal blood flow, and an associated fall in middle meningeal resistance. Resistance changes were the result of a local dilatation and not due to changes downstream of the recording probe. Intracarotid injections of 5-HT produced similar systemic and craniovascular responses, which were larger in the ipsilateral middle meningeal artery. Dose-response curves of vascular resistance changes to intravenous injection of 5-HT were not significantly affected by WAY100635 (5-HT_1A antagonist), GR127935 (5-HT_1B/1D antagonist), methiothepin (5-HT_2C and 5-HT₇ antagonist), ketanserin (5-HT_2A antagonist), SB203186 (5-HT₄ antagonist) or cervical sympathectomy, but were blocked by the 5-HT_3/4 antagonist tropisetron, the 5-HT₃ antagonist ondansetron, the ganglion-blocking drug hexamethonium and by vagotomy. These drugs and procedures did not significantly antagonise the response to intra-arterially injected 5-HT. We conclude that intravenously-administered 5-HT is a vasodilator in vivo in the cat dural circulation, and that the dilation is not mediated by 5-HT₁, 5-HT₂, 5-HT₄ or 5-HT₇ receptors, but is primarily mediated by a vagal reflex, initiated via 5-HT₃ receptor activation and brought about by an increase in parasympathetic tone to the middle meningeal artery as part of the von Bezold-Jarisch reflex. There also appears to be a direct vasodilator effect mediated by unknown receptor types, particularly after intra-arterial administration. Neither of these effects is, however, likely to be of importance in the pathophysiology of migraine or other vascular headaches.     

5-HT1B receptors modulate release of [3H]dopamine from rat striatal synaptosomes

The effect of the selective r5-HT_1B agonist 3-(1,2,5,6-tetrahydro)-4-pyridil-5-pyrrolo [3,2-b] pyril-5-one (CP93,129) on the K⁺-evoked overflow of [³H]dopamine was studied in rat striatal synaptosomes loaded with [³H]dopamine. The aim of the study was to investigate the participation of 5-HT_1B receptors in the serotonergic modulation of striatal dopaminergic transmission. The Ca²⁺-dependent, tetrodotoxin-resistant K⁺-evoked overflow of [³H]dopamine was inhibited by CP93,129 (0.01–100 μM) in a concentration-dependent manner (IC₅₀=1.8 μM; maximal inhibition by 35.5% of control). [±]8-OH-DPAT, a 5-HT_1A receptor agonist, [+/–]DOI, a 5-HT₂ receptor agonist, and 2-methyl-5-hydroxytryptamine, a 5-HT₃ receptor agonist, at concentrations ranging from 0.01 μM to 100 μM did not show any significant effect. Neither ketanserin (1 μM and 5 μM), a selective 5-HT₂/5-HT_1D receptor antagonist, nor ondansetron (1 μM), a 5-HT₃ receptor antagonist, changed the inhibitory effect of CP93,129. SB224289, GR55562, GR127935, isamoltane and metergoline, selective and non-selective 5-HT_1B receptor antagonists, in contrast, used at a concentration of 1 μM, antagonized the inhibitory effect of CP93,129 (3 μM and 10 μM). SB224289, a selective 5-HT_1B receptor antagonist, inhibited the effect of CP93,129 in a concentration-dependent manner; the calculated K _i value was 1.8 nM. Our results indicate that in rat striatal axon terminals the K⁺-evoked release of dopamine is regulated by the presynaptic 5-HT_1B heteroreceptors. Received: 7 September 1998 / Accepted: 2 November 1998     

GR 127935 reduces basal locomotor activity and prevents RU 24969-, but not D-amphetamine-induced hyperlocomotion, in the Wistar-Kyoto Hyperactive (WKHA) rat

The hyperlocomotor effect of the serotonin (5-HT)_1A,B receptor agonist 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969) has been repeatedly reported. However, 5-HT_1A receptors, 5-HT_1B receptors (or both) have been claimed to mediate this effect of RU 24969. These contradictory data possibly arise from protocol differences, especially those related to animal species, drugs, and activity assessment. Herein, the influence of a pretreatment with the selective 5-HT_1B,D receptor antagonist N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2′-methyl-4′-(5-methyl-1,2,4-oxadiozol-3-yl)-biphenyl-4-carboxamide (GR 127935; 1, 3.3 and 10 mg/kg IP) on the hyperlocomotor effect of a 5 mg/kg (IP) dose of RU 24969 was studied in Wistar-Kyoto Hyperactive (WKHA) rats. In a first series of experiments, it was confirmed that RU 24969 (2.5 and 5 mg/kg), administered 10 min after the onset of activity recordings, increases locomotion dose-dependently (cage crossings). In a second series of experiments, administration of GR 127935 10 min after the onset of activity recordings promoted a dose-dependent decrease in basal activity (and rearings) and prevented (3.3 and 10 mg/kg) RU 24969-elicited locomotor activity. On the other hand, GR 127935 was ineffective against RU 24969-induced inhibition of rearings. Lastly, it was observed that 3.3 mg/kg GR 127935 did not affect the number of cage crossings and rearings displayed by rats administered 1.5 mg/kg D-amphetamine. This study shows that 5-HT_1B receptors play a major role in the hyperlocomotor effect of RU 24969, at least under our experimental setting. Whether these receptors also play a tonic role in the high locomotor activity displayed by WKHA rats remains to be determined. Received: 11 May 1998/Final version: 3 July 1998     

5-HT autoreceptors in the regulation of 5-HT release from guinea pig raphe nucleus and hypothalamus

5-HT autoreceptors involved in the regulation of 5-HT release in the guinea pig dorsal raphe nucleus have been studied in comparison with those in the hypothalamus. In vitro release was measured in slices of raphe and hypothalamus prelabelled with [³H]5-HT, superfused with Krebs solution and depolarized electrically. The non-selective 5-HT receptor agonist, 5-carboxamidotryptamine (5-CT) (0.1–10 nM for raphe; 1–100 nM for hypothalamus) and antagonist, methiothepin (10–1000 nM), decreased and increased, respectively, the release of [³H]5-HT evoked by electrical stimulation in either of these regions when given alone. The selective 5-HT_1B/D receptor antagonist, GR127935 (100–1000 nM), and the 5-HT_1D receptor antagonist, ketanserin (300–1000 nM), had no significant effect on this release in either of these regions. Methiothepin and GR127935 (100–1000 nM) shifted to the right the concentration-effect curve of 5-CT in both the raphe and the hypothalamus. At 300 nM, ketanserin shifted to the right the concentration-effect curve of 5-CT in the raphe but did not modify the 5-CT curve in the hypothalamus. In microdialysis experiments ketanserin, applied locally at 10 μM, increased the extracellular levels of 5-HT in the dorsal raphe nucleus of the freely moving guinea pig, whereas 5-HT levels were unchanged in the hypothalamus. Ketanserin at 1 μM did not affect the decrease in 5-HT output induced by the selective 5-HT_1B/D receptor agonist, naratriptan (used at 10 μM in raphe and 0.1 μM in hypothalamus), in the raphe or the hypothalamus. In the raphe, WAY100635, a 5-HT_1A receptor antagonist, at 1 μM, did not prevent naratriptan (10 μM) from reducing the extracellular levels of 5-HT. These results suggest that, in the conditions used in this study, the release of 5-HT in the dorsal raphe nucleus is possibly modulated in part by 5-HT_1B receptors but essentially the control is through 5-HT receptors whose subtype is still to be determined. In the hypothalamus, however, it is clear that only 5-HT_1B receptors are involved in the modulation of 5-HT neurotransmission.     

Interaction of 5-HT1B/D ligands with recombinant h 5-HT1A receptors:Intrinsic activity and modulation by G-protein activation state

Many 5-HT_1B/D receptor ligands have affinity for 5-HT_1A receptors. In the present study, the intrinsic activity of a series of 5-HT_1B/D ligands was investigated at human 5-HT_1A (h 5-HT_1A) receptors by measuring G-protein activation in recombinant C6-glial and HeLa membranes, using agonist-stimulated [³⁵S]GTPγS binding. In these two membrane preparations, the density of h 5-HT_1A receptors (i.e., 246 to 320 fmol mg^–1 protein) and of their G-proteins, and the receptor: G-protein density ratio (0.08 to 0.18) appeared to be similar. It was found that: (i) the maximal [³⁵S]GTPγS binding responses induced by the 5-HT_1B/D receptor ligands in the HeLa preparation at 30 μM GDP were comparable to that of the native agonist 5-HT; (ii) as compared to 5-HT (1.00), similar potencies but lower maximal responses were observed in the C6-glial preparation at 0.3 μM GDP for zolmitriptan (0.89), dihydroergotamine (0.81), rizatriptan (0.71), CP122638 (0.69), naratriptan (0.60) and sumatriptan (0.53); and that (iii) maximal [³⁵S]GTPγS binding responses induced by 5-HT_1B/D ligands in the C6-glial preparation were either unaffected or significantly enhanced by increasing the GDP concentration from 0.3 to 30 μM and higher concentrations. These features differ from those observed with 5-HT_1A receptor agonists; the latter display the same rank order of potency and efficacy in both membrane preparations, and increasing the amount of GDP with C6-glial membranes results in an attenuation of both the agonist’s maximal effect and the apparent potency of partial agonists. The differential regulation of 5-HT_1A and 5-HT_1B/D agonist responses by GDP suggests that different G-protein subtypes are involved upon 5-HT_1A receptor activation by 5-HT_1A and 5-HT_1B/D agonists. Received: 28 October 1997 / Accepted: 14 February 1998     

Effects of selective h5-HT1B (SB-216641) and h5-HT1D (BRL-15572) receptor ligands on guinea-pig and human 5-HT auto- and heteroreceptors

Human cerebral cortical slices and synaptosomes, guinea-pig cerebral cortical slices and human right atrial appendages were used to study the effects of SB-216641, a preferential h5-HT_1B receptor ligand, and of BRL-15572, a preferential h5-HT_1D receptor ligand, on the presynaptic h5-HT_1B and h5-HT_1B-like autoreceptors in the human and guinea-pig brain preparations, respectively, and on the presynaptic h5-HT_1D heteroreceptors in the human atrium. The brain preparations, preincubated with [³H]serotonin ([³H]5-HT), and the segments of atrial appendages, preincubated with [³H]noradrenaline, were superfused with modified Krebs’ solution and tritium overflow was evoked electrically (human and guinea-pig cerebral cortex slices and human atrial appendages) or by high K⁺ (human cerebral cortex synaptosomes). The electrically evoked tritium overflow from guinea-pig cerebral cortex slices was reduced by the 5-HT receptor agonist 5-carboxamidotryptamine (5-CT). This effect was not modified by BRL-15572 (2μM; concentration 154 times higher than its K_i at h5-HT_1D receptors) but was antagonized by SB-216641 (0.1μM; concentration 100 times higher than its K_i at h5-HT_1B receptors; apparent pA₂ 8.45). SB-216641 (0.1μM) by itself facilitated, whereas BRL-15572 (2μM) did not affect, the evoked overflow. In human cerebral cortex slices SB-216641 (0.1μM) also facilitated, and BRL-15572 (2μM) again failed to affect, the electrically evoked tritium overflow. In human cerebral cortical synaptosomes, 5-CT reduced the K⁺-evoked tritium overflow. This response was unaffected by BRL-15572 (300nM) but antagonized by SB-216641 (15nM; drug concentrations 23 and 15 times higher than their K_i at h5-HT_1D and h5-HT_1B receptors, respectively). Both drugs, given alone, did not modify the K⁺-evoked tritium overflow. In human atrial appendages, the electrically evoked tritium overflow was inhibited by 5-HT in a manner susceptible to antagonism by BRL-15572 (300nM; 23 times K_i at h5-HT_1D receptors) but not by SB-216641 (30nM; 30 times K_i at h5-HT_1B receptors). Both drugs by themselves did not change the electrically evoked tritium overflow. In conclusion, SB-216641 behaves as a preferential antagonist at native human 5-HT_1B receptors and BRL-15572 as a preferential antagonist at native human 5-HT_1D receptors. These compounds are clearly useful tools for the differentiation between human 5-HT_1B and 5-HT_1D receptors in functional studies. Received: 14 March 1997 / Accepted: 18 May 1997     

RU 24969, a 5-HT1A/1B agonist, elevates brain stimulation reward thresholds: an effect reversed by GR 127935, a 5-HT1B/1D antagonist

 Recent studies suggest that serotonergic neurotransmission through the serotonin-1B (5-HT_1B) receptor is involved in reward processes. The purpose of the present studies was to investigate the effects of 5-HT_1B receptor activation and antagonism on intracranial self-stimulation (ICSS) reward using a current-threshold ICSS task. Male Wistar rats were prepared with bipolar electrodes in the lateral hypothalamus. When stable baseline thresholds were established, the effects of the mixed 5-HT_1A/1B receptor agonist, RU 24969 (0–1 mg/kg, SC), on ICSS behavior were assessed. Administration of this compound elevated ICSS thresholds without affecting response latencies, a measure of general motoric activity. The 5-HT_1B/1D receptor antagonist, GR 127935 (0–10 mg/kg, SC), had no significant effect on ICSS behavior. However, pretreatment with an intermediate dose of GR 127935 (3 mg/kg), which was previously without effect on ICSS behavior, reversed the threshold-elevating effects of RU 24969 (1 mg/kg), suggesting the involvement of the 5-HT_1B receptor in this effect of RU 24969 administration. Furthermore, pretreatment with RU 24969 (0.3 and 0.6 mg/kg), prior to 10 mg/kg cocaine hydrochloride, dose-dependently attenuated the threshold-reducing effects of cocaine. This result is interpreted as two opposing drug effects canceling each other out rather than a specific pharmacological antagonism. In conclusion, the results suggest that activation of 5-HT_1B receptors reduces brain stimulation reward. Received: 31 October 1997 / Final version: 23 July 1998     

Correlation between 5-HT7 receptor affinity and protection against sound-induced seizures in DBA/2J mice

Audiogenic seizures can be induced in DBA/2J mice following intense auditory stimulation. A number of neurotransmitters, including 5-hydroxytryptamine (5-HT), are believed to be involved in mediating this effect since it has been shown previously that depletion of 5-HT or blockade of 5-HT receptors protects DBA/2J mice from these audiogenic seizures. The present study was undertaken to determine whether antagonism of the newly identified 5-HT₇ receptor may protect DBA/2J mice from audiogenic seizures by attempting to correlate in vivo potency of compounds with their affinity at the 5-HT₇ receptor. All compounds used in the correlation were shown to be antagonists at the 5-HT₇ receptor and a statistically significant correlation was observed between 5-HT₇ affinity and doses for half-maximal response (ED₅₀) for protection of DBA/2J mice from sound-induced seizures (r = 0.80; P < 0.05). No significant correlation was observed between in vivo activity and affinity at either 5-HT_1A, 5-HT_2A or 5-HT_2C receptors. It is also unlikely that interactions between the 5-ht₅ receptor will protect DBA/2J mice from audiogenic seizures since metergoline and mesulergine which are both active in this in vivo model have no affinity for the 5-ht₅ receptor. There are similarities between the pharmacology of the 5-HT₇ receptor and that of the 5-HT_1A receptor, however the correlation between the in vivo potency in DBA/2J mice and 5-HT_1A affinity was not significant. Furthermore, the 5-HT_1A receptor antagonist WAY 100135 did not protect DBA/2J mice from audiogenic seizures at doses that antagonise 5-HT_1A receptor-mediated effects in mice. These data suggest that antagonism of 5-HT₇ receptors may protect against audiogenic seizures in DBA/2J mice although a definitive conclusion must await studies with selective 5-HT₇ antagonists. Received: 20 March 1997 / Accepted: 10 August 1997