5-Hydroxytryptamine (5-HT)4 receptors in post mortem human brain tissue: distribution, pharmacology and effects of neurodegenerative diseases.

1. The distribution, pharmacology and effects of neurodegenerative diseases on 5-HT4 receptors in human brain have been characterized in vitro. 2. The 5-HT4 receptor in post mortem human brain tissue was specifically labelled with [3H]-GR 113808. In human putamen, this ligand labelled a homogeneous population of sites, with an apparent affinity (-log Kd) of 10.1 and a density (Bmax) of 5.73 fmol mg-1 tissue. The pharmacology of this site was characterized by use of a series of displacing ligands, and the following rank order of apparent affinities (with mean +/- s.d. -log Ki values in parentheses) was generated: GR113808 (10.05 +/- 0.04) > SDZ 205,557 (8.65 +/- 0.08) > DAU 6285 (7.95 +/- 0.04) > BIMU-1 (7.81 +/- 0.06) > DAU 6215 (7.42 +/- 0.23) > tropisetron (7.39 +/- 0.23) > 5-HT (7.32 +/- 1.00) > BIMU-8 (7.25 +/- 0.04) > (R)-zacopride (5.82 +/- 0.04). The Hill coefficients were not significantly different from unity, consistent with an interaction at a single site. A comparison of the affinities of these compounds with those obtained from guinea-pig striatum indicated no evidence of species differences. 3. The regional distribution of 5-HT4 receptors was assessed by determining the density of binding sites for [3H]-GR 113808.(ABSTRACT TRUNCATED AT 250 WORDS)     

Actions of general anaesthetics on 5-HT3 receptors in N1E-115 neuroblastoma cells.

1. NIE-115 mouse neuroblastoma cells were studied under voltage clamp in the whole-cell patch-clamp configuration. Peak currents induced by bath application of 5-hydroxytryptamine (5-HT) were inwardly rectifying, reversed at 0.4 +/- 0.2 mV (mean +/- s.e.mean), and were approximately half-inhibited (at 1 microM 5-HT) by 2 nM of the 5-HT3 selective antagonist MDL-72222 (3-tropanyl-3,5-dichlorobenzoate). 2. Peak inward currents activated by a low concentration of 5-HT at a holding potential of -50 mV were potentiated by volatile general anaesthetics. At their human minimum alveolar concentrations (MACs), the degree of potentiation increased in the order isoflurane < halothane < enflurane < methoxyflurane. Potentiation by methoxyflurane was independent of membrane potential in the range -70 mV to +40 mV. The reversal potential was the same in the presence and absence of methoxyflurane. 3. Methoxyflurane shifted the 5-HT dose-response curve to lower 5-HT concentrations, without significantly changing the Hill coefficient or maximum response. The EC50 concentration for 5-HT decreased from 1.86 +/- 0.02 microM to 1.07 +/- 0.11 microM (means +/- s.e.mean) due to the presence of 1 MAC (270 microM) methoxyflurane. 4. In contrast to the volatile anaesthetics, the barbiturate anaesthetic, thiopentone, inhibited the 5-HT3 receptor. Hill analysis of thiopentone dose-response data gave an average IC50 = 117 +/- 8 microM thiopentone and Hill coefficient = 1.6 +/- 0.2 (means +/- s.e.mean). These parameters were not significantly different for data obtained at 5-HT concentrations above and below the control EC50 concentration for 5-HT, consistent with non-competitive inhibition. 5. The n-alcohols occupied an intermediate position between the volatile and barbiturate anaesthetics. The lower alcohols (butanol and hexanol) potentiated 5-HT responses at low alcohol concentrations but inhibited them at high concentrations. In contrast, the higher alcohols (octanol, decanol, dodecanol, tridecanol, tetradecanol and pentadecanol) produced no potentiation, but only inhibition, at all alcohol concentrations. 6. Inhibition of the 5-HT3 receptor by the n-alcohols exhibited a cutoff in potency similar to those previously found for tadpoles, luciferase enzymes and a neuronal nicotinic acetylcholine receptor channel.     

The guinea-pig distal colon--a sensitive preparation for the investigation of 5-HT4 receptor-mediated contractions.

1. Experiments were designed to characterize pharmacologically the contractile responses to 5-hydroxytryptamine (5-HT) in the guinea-pig isolated distal colon longitudinal muscle-myenteric plexus preparation (LMMP). 2. In the presence of methiothepin (100 nM) and granisetron (1 microM), 5-HT (10 pM-10 nM) produced concentration-dependent contractile responses of the guinea-pig distal colon LMMP, with a pEC50 of 9.2 +/- 0.08. 3. Responses to 5-HT were mimicked by a series of tryptamine analogues, with the following rank order of potency; 5-HT > 5-MeOT >> 5-CT > tryptamine > 2-Me-5-HT. All were found to be full agonists. 4. Responses to 5-HT were also mimicked by a series of substituted benzamide analogues. Their rank order of potency was 5-HT > renzapride > cisapride > (S)-zacopride > (R)-zacopride > metoclopramide. All were full agonists relative to 5-HT. 5. The benzimidazolone derivatives, BIMU 1 and BIMU 8 were approximately equipotent partial agonists (intrinsic activities of 0.8 +/- 0.07 and 0.5 +/- 0.08 respectively) in the guinea-pig distal colon. 6. Tropisetron produced a rightward displacement of the 5-HT concentration-effect curve, yielding an apparent pA2 of 6.4 +/- 0.1. The slope of the Schild plot (1.3 +/- 0.1) was significantly greater than unity. 7. SDZ 205,557 produced a concentration-dependent shift to the right of the 5-HT concentration-response curve, yielding an estimated pA2 of 7.8 +/- 0.1 and a slope which did not significantly deviate from unity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mediation by 5-HT1D receptors of 5-hydroxytryptamine-induced contractions of rabbit middle and posterior cerebral arteries.

1. 5-Hydroxytryptamine (5-HT) receptor-mediated contraction of endothelium denuded rabbit middle (MCA) and posterior (PCA) cerebral arteries was characterized by use of selective agonists and antagonists for different 5-HT receptor subtypes. 2. 5-HT and various 5-HT receptor agonists contracted the arteries with the following rank order of potency in MCA: 5-carboxamidotryptamine (5-CT) > 5-HT > 5-methoxytryptamine (5-MeOT) > sumatriptan > alpha-methyl-5-HT (alpha-Me-5-HT) >> 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) and in PCA: 5-CT > 5-HT > sumatriptan > 5-MeOT > alpha-Me-5-HT >> 8-OH-DPAT. With few exceptions, the maximal contractile responses of these agonists were similar to that induced by 5-HT. 3. The selective antagonists of 5-HT2A/2C (ketanserin), 5-HT4 (SDZ 205-557) and 5-HT1A/1B (S-(-)-propranolol) sites were devoid of inhibitory effect on 5-HT-mediated contraction in both MCA and PCA, thus excluding activation of the corresponding receptors. 4. In both arteries, the contraction-response curve to 5-HT was unaffected by the 5-HT3 receptor antagonist, ICS 205-930 (0.01 and 0.1 microM) whilst a small (3 and 6 fold displacement) was seen with MDL 72222 (0.1 and 1 microM). 5. The mixed 5-HT1-like/5-HT2A receptor antagonist, methiothepin (0.001-0.1 microM), was a potent antagonist of 5-HT-induced contractions in both arteries, giving pA2 values of 9.4 +/- 0.7 and 9.6 +/- 0.8 in MCA and PCA, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of 5-hydroxytryptamine-induced coronary vasodilation assessed by direct detection of nitric oxide production in guinea-pig isolated heart.

1. We assessed whether a submaximal concentration (1 microM) of 5-hydroxytryptamine (5-HT) releases nitric oxide (NO) from the coronary endothelium in guinea-pig perfused heart (n = 5 or 6/group) by direct detection of NO in coronary effluent, and determined whether this accounts for the associated coronary dilation. We also tested whether saponin is a selective and specific tool for examining the role of this mechanism in mediating agonist-induced coronary dilatation. 2. Continuous 5 min perfusion with 5-HT, or acetylcholine (ACh; 1 microM), substance P (1 nM) or sodium nitroprusside (SNP; 1 microM) increased coronary flow from baseline by 3.6 +/- 0.2, 3.4 +/- 0.2, 1.8 +/- 0.1 and 4.1 +/- 0.2 ml min-1 g-1, respectively (all P < 0.05). Coronary effluent NO content, detected by chemiluminescence, was correspondingly increased from baseline by 715 +/- 85, 920 +/- 136, 1019 +/- 58 and 2333 +/- 114 pmol min-1 g-1, respectively (all P < 0.05). 3. Continuous perfusion for 30 min with NG-nitro-L-arginine methyl ester (L-NAME) 100 microM reduced basal coronary effluent NO content by 370 +/- 32 pmol min-1 g-1 and coronary flow by 7.5 +/- 0.5 ml min-1 g-1 (both P < 0.05). Saponin (three cycles of 2 min of 30 micrograms ml-1 saponin perfusion interrupted by 2 min control perfusion) reduced basal coronary NO content by a similar amount (307 +/- 22 pmol min-1 g-1) but reduced basal coronary flow by only 0.6 +/- 0.2 ml min-1 g-1 (P < 0.05 versus the effect of L-NAME). 4. The increases in coronary flow in response to (5-HT), ACh and substance P were reduced (all P < 0.05) by 100 microM L-NAME to 1.2 +/- 0.3, 1.2 +/- 0.4 and 0.3 +/- 0.3 ml min-1 g-1, respectively. However, the flow increase in response to SNP was not reduced; it was in fact increased slightly to 4.8 +/- 0.4 ml min-1 g-1 (P < 0.05). 5. Similarly, after treatment with saponin, the increases in coronary flow in response to 5-HT, ACh and substance P were reduced to 2.1 +/- 0.3, 1.3 +/- 0.3 and 0.4 +/- 0.2 ml min-1 g-1, respectively (all P < 0.05). Again, the response to SNP was increased slightly to 4.6 +/- 0.5 ml min-1 g-1 (P < 0.05). 6. L-NAME and saponin also inhibited 5-HT, ACh and substance P-induced NO release (P < 0.05), without affecting equivalent responses to SNP. 7. For substance P, the change in coronary flow (delta CF) correlated with log10 delta NO in the presence and absence of saponin and L-NAME; delta CF = 1.2(log delta NO) 1.9; r = 0.92; P < 0.05. For 5-HT the relationship was delta CF = 2.2(log delta NO-2.7; r = 0.79; P < 0.05, indicating that 5-HT causes a disproportionately greater increase in coronary flow per release of NO. This was taken to indicate that 5-HT relaxes coronary vasculature in part by releasing NO, but in part by additional mechanisms. ACh resembled 5-HT in this respect. 8. Saponin had no effect on cardiac systolic or diastolic contractile function assessed by the construction of Starling curves with an isochoric intraventricular balloon. 9. In conclusion, despite its minimal effect on basal coronary flow, saponin is an effective tool for revealing endothelium-dependent actions of coronary vasodilator substances and has selectivity in that it does not impair endothelium-independent vasodilatation or cardiac contractile function. 5-HT dilates guinea-pig coronary arteries largely by the release of NO from the coronary endothelium.     

The pharmacology and distribution of human 5-hydroxytryptamine2B (5-HT2B) receptor gene products: comparison with 5-HT2A and 5-HT2C receptors.

1. Full length clones of the human 5-HT2B receptor were isolated from human liver, kidney and pancreas. The cloned human 5-HT2B receptors had a high degree of homology (approximately 80%) with the rat and mouse 5-HT2B receptors. 2. PCR amplification was used to determine the tissue distribution of human 5-HT2B receptor mRNA. mRNA encoding the 5-HT2B receptor was expressed with greatest abundance in human liver and kidney. Lower levels of expression were detected in cerebral cortex, whole brain, pancreas and spleen. Expression was not detected in heart. 3. Northern blot analysis confirmed the presence of 5-HT2B receptor mRNA (a 2.4 kB sized band) in pancreas, liver and kidney. An additional 3.2 kB sized band of hybridization was detected in liver and kidney. This raises the possibility of a splice variant of the receptor or the presence of an additional homologous receptor. 4. The human 5-HT2B receptor was expressed in Cos-7 cells and its ligand binding characteristics were compared to similarly expressed human 5-HT2A and 5-HT2C receptors. The ligand specificity of the human 5-HT2B receptor (5-HT > ritanserin > SB 204741 > spiperone) was distinct from that of the human 5-HT2A (ritanserin > spiperone > 5-HT > SB 204741) and 5-HT2C (ritanserin > 5-HT > spiperone = SB 204741) receptors. On the basis of a higher affinity for ketanserin and a lower affinity for yohimbine the human 5-HT2B receptor also appeared to differ from the rat 5-HT2B receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a 5-HT1B receptor on CHO cells: functional responses in the absence of radioligand binding.

1. Chinese hamster ovary (CHO) cells have been reported to be devoid of 5-HT receptors and have frequently been used as hosts for the expression of cloned 5-HT receptors. Unexpectedly, 5-HT was found to induce profound inhibition of forskolin-stimulated cyclic AMP production in these cells and the aim of this study was to classify the 5-HT receptor involved. 2. In CHO(dhfr-) cells 5-HT was a potent agonist and caused 80-100% inhibition of forskolin stimulated cyclic AMP production. A study using several 5-HT1 receptor agonists revealed the following potencies (p[A50]): RU24969 (9.09 +/- 0.17) > 5-carboxamidotryptamine (8.86 +/- 0.20) > 5-HT (8.07 +/- 0.05) > CP-93,129 (7.74 +/- 0.10) > sumatriptan (5.93 +/- 0.04). All five agonists achieved a similar maximum effect. Irreversible receptor alkylation studies yielded a pKA estimate of 7.04 +/- 0.34 for 5-HT. 3. The 5-HT1A/1B antagonist, (+/-)-cyanopindolol (4-100 nM), caused parallel rightward shifts of the 5-HT concentration-effect curve with no change in asymptote. Schild analysis yielded a pKB estimate of 8.69 +/- 0.09 (Schild slope 1.13 +/- 0.10). (+/-)-Cyanopindolol actually behaved as a partial agonist with an intrinsic activity of 0.2-0.5 and a p[A50] of 8.55. 4. 5-HT (0.01-10 microM) also elicited a concentration-dependent increase in intracellular [Ca2+] in CHO(dhfr-) cells thus demonstrating that dual coupling is not a phenomenon restricted to systems in which there is overexpression of transfected receptors. 5. This agonist and antagonist profile is consistent with the presence of a 5-HT1B receptor. 8-OH-DPAT (1 microM) and renzapride (3 microM) were without effect on forskolin-stimulated cyclic AMP production and ketanserin (0.3 microM) did not antagonize the inhibition produced by 5-HT, thus excluding the involvement of 5-HT1A, 5-HT4, and 5-HT2 receptors. 6. The possibility that expression of a 5-HT1B receptor was associated with the dhfr- mutation was excluded since RU24969, 5-HT and CP-93,129 were also potent agonists in unmutated, CHO-K1 cells: p[A50] 9.03 +/- 0.03, 8.34 +/- 0.05, 7.69 +/- 0.07 respectively, and (+/-)-cyanopindolol (0.1 microM) shifted the 5-HT curve to the right and yielded a pA2 estimate of 8.70 +/- 0.06. 7. Little or no specific binding of [3H]-5-HT (0.1-200 nM) or of the high affinity ligand [125I]-iodocyanopindolol (0.01-3 nM) to CHO(dhfr-) cell membranes could be detected. 5-HT also failed to elicit any increase in the binding of [35S]-GTP gamma S to CHO membranes. 8. In conclusion, cultured CHO cells express 5-HT1B receptors which are negatively coupled to adenylyl cyclase and positively coupled to increases in intracellular calcium. The absence of radioligand binding was unexpected in view of the high potency of 5-HT and the partial agonist activity of the normally ''silent'' competitive antagonist, (+/-)-cyanopindolol. This implies very efficient receptor-effector coupling of a low density of 5-HT1B receptors. Clearly, the absence of detectable radioligand binding cannot be assumed to mean the absence of receptors capable of eliciting a significant functional response.     

Recombinant saphenous vein 5-HT1B receptors of the rabbit: comparative pharmacology with human 5-HT1B receptors

1. The rabbit recombinant saphenous vein 5-hydroxytryptamine_1B (rb 5-HT_1B) receptor stably transfected in rat C6-glial cells was characterized by measuring adenosine 3′:5′-cyclic monophosphate (cyclic AMP) formation upon exposure to various 5-HT receptor ligands. The effects of agonists and antagonists were compared with their effects determined previously at the human cloned 5-HT_1B (h 5-HT_1B) receptor under similar experimental conditions.
2. Intact C6-glial cells expressing rb 5-HT_1B receptors exhibited [³H]-5-carboxamidotryptamine (5-CT) binding sites with a K_d of 0.80±0.13 nM and a B_max between 225 to 570 fmol mg⁻¹ protein. The binding affinities of a series of 5-HT receptor ligands determined in a membrane preparation with [³H]-5-CT or [³H]-N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-(4-pyridyl)benzamide (GR 125,743) were similar. With the exception of ketanserin, ligand affinities were comparable to those determined at the cloned h 5-HT_1B receptor site.
3. rb 5-HT_1B receptors were negatively coupled to cyclic AMP formation upon stimulation with 5-HT agonists. Of the several 5-HT agonists tested, 5-CT was the most potent, the potency rank order being: 5-CT>5-HT>zolmitriptan>naratriptan>rizatriptan>sumatriptan>R(+)-8-(hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The maximal responses of these agonists were similar to those induced by 5-HT. The potency of these agonists showed a positive correlation (r²=0.87; P<0.002) with their potency at the cloned h 5-HT_1B receptor subtype.
4. 2′-Methyl-4-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-amide (GR 127,935), methiothepin and ketanserin each behaved as silent, competitive antagonists at rb 5-HT_1B receptors; pK_B values were 8.41, 8.32 and 7.05, respectively when naratriptan was used as an agonist. These estimates accorded with their binding affinities and the potencies found on 5-HT and/or sumatriptan-mediated contraction of isolated rabbit saphenous vein segments.
5. In conclusion, the recombinant saphenous vein 5-HT_1B receptor of the rabbit shares important pharmacological similarities with the cloned h 5-HT_1B receptor. However, ketanserin is a more potent antagonist of rb 5-HT_1B receptors.

     

Characterization of the 5-hydroxytryptamine receptor type involved in inhibition of spontaneous activity of human isolated colonic circular muscle.

1. Experiments were carried out to characterize pharmacologically the 5-hydroxytryptamine (5-HT) receptor types which mediate inhibition of spontaneous contractions of the intertaenial circular muscle in human isolated colon. 2. 5-HT caused a reproducible concentration-dependent inhibition of spontaneous contractions of the circular muscle of human colon in vitro with a mean EC50 value of 0.2 microM and 95% confidence limits of 0.1-0.5 microM. No evidence for a contractile action of 5-HT was found. Tetrodotoxin (TTX, 1.5 microM) caused a rightward shift of the concentration-response curve of 5-HT with a concentration-ratio of 2.9. 3. The inhibitory response to 5-HT was mimicked by several indoles with the rank order of potency 5-HT > 5-methoxytryptamine = alpha-methyl-5-HT > 5-carboxamidotryptamine >> 2-methyl-5-HT. 5-Hydroxyindalpine was inactive. 4. The substituted benzamides were agonists with the following rank order of potency, 5-HT > renzapride > zacopride > metoclopramide > cisapride. 5. The inhibitory responses to 5-HT were not inhibited by methysergide (10 microM) or methiothepin (1 microM), which are antagonists selective for 5-HT1-like and 5-HT2 receptors, nor by ondansetron (10 microM) which is an antagonist at 5-HT3 receptors. 6. The inhibitory responses induced by 5-HT and 5-methoxytryptamine were competitively antagonized by a weak 5-HT4 receptor antagonist, tropisetron, with pKB values of approximately 6. Tropisetron had no significant effect on the inhibitory response curve produced by isoprenaline (0.01-100 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparative study of functional 5-HT4 receptors in human colon, rat oesophagus and rat ileum.

1. The pharmacological properties of 5-hydroxytryptamine (5-HT), the 5-HT4 receptor agonists, DAU 6236 and SC 53116 and the 5-HT4 receptor antagonist, GR 1130808, were studied in the rat oesophagus, rat ileum and human colon. 2. 5-HT relaxed the longitudinal muscle of the rat oesophagus and rat ileum and the circular muscle of the human colon. Absolute values of relaxation were measured and showed the order of the maximum responses, rat oesophagus >> human colon > rat ileum with EC50 values of 189 +/- 15 nM, 157 +/- 4 nM, 306 +/- 72 nM, respectively. 5-HT also inhibited the spontaneous contractions of the human colon with an EC50 value of 119 +/- 1 nM. The effect of 5-HT on the human colon was not affected by methysergide (10 microM) or ondansetron (1 microM). 3. The use of the uptake and metabolism inhibitors, cocaine (30 microM) and pargyline (100 microM), did not increase the potency of 5-HT in the rat oesophagus or human colon. In the rat oesophagus, cocaine (30 microM) produced a reduction in carbachol-induced tone of 22.2 +/- 0.6% and reduced the 5-HT maximum effect by 52.0 +/- 0.4%. 4. The compounds, DAU 6236 and SC 53116, showed a different pattern of potencies and efficacies in the rat oesophagus, rat ileum and human colon compared to 5-HT. DAU 6236 relaxed the human colonic circular muscle with an EC50 value of 129 +/- 16 nM but its efficacy was less than that of 5-HT. DAU 6236 (1 microM) also antagonized the 5-HT-induced relaxation of the human colon with a dose-ratio of 9.9. In the rat oesophagus and rat ileum, DAU 6236 was inactive in the majority of tissues. In the minority of oesophagus tissues that did respond the EC50 value was 1.2 +/- 0.7 microM. DAU 6236 also antagonized the effect of 5-HT in the rat oesophagus in a non-surmountable fashion. SC 53116 relaxed the rat oesophagus with an EC50 value of 91 +/- 4 nM, with an efficacy less than that observed to 5-HT; however, at 200 nM it did not antagonize the 5-HT-induced relaxation of the rat oesophagus. SC 53116 showed no agonist activity in the rat ileum and human colon, but at 1 microM it did antagonize the effect of 5-HT in the human colon with a dose-ratio of 11.3 +/- 0.3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Blood and urine 5-hydroxytryptophan and 5-hydroxytryptamine levels after administration of two 5-hydroxytryptamine precursors in normal man.

Six healthy male subjects received equimolar amounts of two 5-hydroxytryptamine (5-HT) precursors, 5-hydroxy-L-tryptophan (5-HTP) and gamma-L-glutamyl-5-hydroxy-L-tryptophan (glu-5-HTP), on two occasions in a randomised cross-over study. There were marked increases in urinary 5-HTP and 5-HT excretion after infusion of both compounds. Mean urinary excretion rate of 5-HT, which was < 0.7 nmol min-1 before dosing, rose to a peak value of 412 +/- 92 nmol min-1 at the end of 5-HTP infusion and 303 +/- 29 nmol min-1 after administration of glu-5-HTP. This occurred without significant changes in blood 5-HT levels measured in platelet-rich plasma. These findings provide further evidence that the increase in urine 5-HT after administration of both 5-HT precursors is largely due to 5-HT synthesised within the kidney.     

Cloning,expression and pharmacology of a truncated splice variant of the human 5-HT7 receptor (h5-HT7(b))

1. The rat 5-hydroxytryptamine (5-HT)₇ receptor displays two splice variations, a long form, and a truncated splice isoform, arising from the introduction of a stop codon near the carboxy-terminus. The human 5-HT₇ receptor gene contains at least two introns and encodes a 445 amino acid 5-HT receptor.
2. A truncated splice variation in the human 5-HT₇ receptor was isolated from a human placental cDNA library. In accordance with current NC-IUPHAR nomenclature guidelines, it is suggested that this receptor be denoted as the h5-HT_7(b) receptor and the long form of the receptor as h5-HT_7(a).
3. The h5-HT_7(b) receptor was stably expressed in HEK 293 cells and ligand affinities were determined by displacement of [³H]-5-carboxyamidotryptamine (5-CT; K_d=0.28±0.06 nM, B_max=7.3±1.7 pmol mg⁻¹ protein). The rank order of affinities (pK_i) for a series of ligands was: 5-carboxamidotryptamine (5-CT, 9.65)>5-hydroxytryptamine (5-HT, 9.41)>methiothepin (8.87)>mesulergine (7.87)>8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT, 6.85)>ketanserin (6.44).
4. The h5-HT_7(b) receptor coupled positively to adenylyl cyclase in HEK 293 cells. This response was elicited by a number of agonists with the following order of potency (pEC₅₀): 5-CT (8.7±0.11)>5-MeOT (5-methoxytryptamine; 8.1±0.20)>5-HT (7.5±0.13)>tryptamine (5.6±0.36)>8-OH-DPAT (5.3±0.28)>5-methoxytryptamine (5.0±0.06). This rank order was comparable to that observed in the radioligand binding studies.
5. In a similar fashion to that described for the 5-HT_7(a) receptor, PCR studies suggested that the 5-HT_7(b) receptor mRNA is found in great abundance throughout the brain, in the small intestine and aorta.
6. It is concluded that the h5-HT₇ receptor, like the rat receptor, exists as splice variants exhibiting similar pharmacology, signal transduction and distribution. It is thus likely that there exists a complex physiological role for alternate splicing products of the 5-HT₇ receptor gene.

     

Evaluation of the receptor selectivity of the H3 receptor antagonists, iodophenpropit and thioperamide: an interaction with the 5-HT3 receptor revealed.

1. In the present study we evaluated the receptor selectivity of the potent histamine H3 receptor antagonist, iodophenpropit (IPP) in comparison with the prototype antagonist, thioperamide. 2. IPP proved to be a potent competitive H3 receptor antagonist as measured against (R)-alpha-methylhistamine-induced inhibition of electrically-evoked contractions of the guinea-pig jejunum (pA2 = 9.12 +/- 0.06, Schild slope: 1.0 +/- 0.1, n = 8). In the same assay, thioperamide was slightly less potent (pA2 = 8.9 +/- 0.2). 3. In radioligand binding studies, IPP showed a high affinity for the H3 receptor. Displacement of [125I]-IPP binding to rat cortex membranes by unlabelled IPP resulted in a Ki value of 0.97 +/- 0.06 nM (n = 3). In contrast, IPP showed only a weak affinity for the histamine H1- and H2 receptor. Displacement of [3H]-mepyramine and [125I]-iodoaminopotentidine binding to respectively guinea-pig H1- and human H2 receptors by IPP resulted in Ki values of 1.71 +/- 0.32 microM (n = 3) and 2.28 +/- 0.81 microM (n = 3). For thioperamide the affinities for the H1-, H2- and H3 receptor were respectively > 10 microM, > 10 microM and 4.3 +/- 1.6 nM (n = 7). 4. Testing IPP and thioperamide in 39 different receptor binding assays revealed that IPP showed relatively high affinity for the 5-hydroxytryptamine 5-HT3 receptor (Ki = 11 +/- 1 nM, n = 3), the alpha 2-adrenoceptor (Ki = 120 +/- 5 nM, n = 3) and the sigma receptor (Ki = 170 +/- 70 nM, n = 3). Thioperamide showed relatively high affinity for the 5-HT3 receptor (Ki = 120 +/- 30 nM, n = 3) and the sigma receptor (Ki = 180 +/- 90 nM, n = 3). 5. Due to the low density of histamine H3 receptors in the brain, the interaction of IPP with the 5-HT3-, the alpha 2- and the sigma receptor might interfere with [125I]-IPP binding to rat cortex membranes. Yet, in this preparation [125I]-IPP binding was not influenced by ondansetron, yohimbine or haloperidol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the 5-HT4 receptor mediating tachycardia in piglet isolated right atrium.

1. In order to explore whether 5-HT4 receptor subtypes exist, we have characterized further the 5-HT4 receptor that mediates tachycardia in the piglet isolated right atrium. All experiments were carried out in the presence of propranolol (400 nM) and cocaine (6 microM). We used tryptamine derivatives, substituted benzamides and benzimidazolone derivatives as pharmacological tools. 2. Tachycardia responses to 5-hydroxytryptamine (5-HT) were mimicked by other tryptamine derivatives with the following order of potency: 5-HT > 5-methoxytryptamine alpha-methyl-5-HT = bufotenine bufotenine > 5-carboxamidotryptamine = tryptamine (after treatment with pargyline) > 5-methoxy-N,N-dimethyltryptamine > 2-methyl-5-HT. 3. The substituted benzamides were all partial agonists relative to 5-HT except (-)-zacopride which was a full agonist. The stimulant potency order was renzapride > cisapride = (-)-zacopride > metoclopramide > (+)-zacopride. 4. The benzimidazolone derivatives had contrasting effects. BIMU 8 (endo-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2,3-dihydro-(1-methyl(eth yl- 2-oxo-1H-benzimidazole-1-carboxamide hydrochloride) was a full agonist relative to 5-HT whilst BIMU 1 (endo-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2,3-dihydro-3-ethyl-2-oxo - 1H-benzimidazole-1-carboxamide hydrochloride) was a partial agonist with low intrinsic activity compared to 5-HT but had similar potency. We estimated a pKB of 7.9 for BIMU 1 antagonism of 5-HT-induced tachycardia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mediation by 5-HT3 receptors of an excitatory effect of 5-HT on dorsal vagal preganglionic neurones in anaesthetized rats: an ionophoretic study.

1. Extracellular recording were made from 141 vagal preganglionic neurones in the dorsal vagal nucleus (DVN). The effects of ionophoretic administration of 5-hydroxytryptamine (5-HT), the 5-HT3 receptor agonist, phenylbiguanide (PBG) and the antagonists, granisetron and tropisetron (ICS 205-930) on these vagal preganglionic neurones were studied in pentobarbitone sodium anaesthetized rats. 2. Ionophoretic application of 5-HT at low currents (< 10 nA) increased the activity in 46 (73%) of 63 neurones tested. Application of granisetron (5-20 nA) or tropisetron (5-20 nA) abolished or attenuated the 5-HT excitatory responses in 8 out of 11 and 5 out of 5 neurones respectively. At the currents used, neither antagonist had any effect on baseline firing rate. 3. Ionophoresis of the selective 5-HT3 receptor agonist, phenylbiguanide (0-40 nA) excited 54 (82%) of the 66 vagal neurones tested, whilst the remaining 12 neurones were unaffected. 4. Granisetron applied either ionophoretically (8/11) or intravenously (3/3),abolished or attenuated the excitations evoked by PBG. Similarly, tropisetron administered either ionophoretically (2/3) or intravenously (2/2), attenuated the PBG excitation. In contrast, the PBG excitations were unaffected by the 5-HT2 receptor antagonist, cinanserin (2/2), and the selective 5-HT1A receptor antagonist, WAY- 100802 (6/6). 5. In conclusion, excitation of vagal preganglionic neurones evoked by ionophoretic application of 5- HT is mediated in part by 5-HT3 receptors and activation of 5-HT3 receptors on and/or in the vicinity of vagal motoneurones causes excitation of these neurones.     

5-HT3 receptor antagonism by anpirtoline, a mixed 5-HT1 receptor agonist/5-HT3 receptor antagonist.

1. The aim of this study was to provide evidence that anpirtoline, which is an agonist at 5-HT1B and 5-HT1D receptors and also displays submicromolar affinity for 5-HT1A recognition sites, in addition, acts as an antagonist at 5-HT3 receptors. 2. In radioligand binding studies on rat brain cortical membranes, anpirtoline inhibited specific binding of [3H]-(S)-zacopride to 5-HT3 receptor recognition sites (pKi: 7.53). 3. In N1E-115 neuroblastoma cells in which [14C]-guanidinium was used as a tool to measure cation influx through the 5-HT3 receptor channel, the 5-HT-induced influx was concentration-dependently inhibited by anpirtoline. In this respect, anpirtoline mimicked other 5-HT3 receptor antagonists; the rank order of potency was ondansetron > anpirtoline > metoclopramide. 4. The concentration-response curve for 5-HT as a stimulator of [14C]-guanidinium influx was shifted to the right by anpirtoline (apparent pA2: 7.78). 5. In urethane-anaesthetized rats, anpirtoline inhibited (at lower potency than zacopride and tropisetron) the 5-HT- or phenylbiguanide-induced bradycardia (Bezold-Jarisch reflex), but did not induce this reflex by itself. 6. Intravenous infusion of cisplatin in the domestic pig caused a consistent emetic response which was antagonized by anpirtoline. 7. It is concluded that anpirtoline, which was previously characterized as a 5-HT1 receptor agonist also proved to be a 5-HT3 receptor antagonist in several experimental models and, hence, exhibits a unique pattern of properties at different 5-HT receptors.     

The interaction of trichloroethanol with murine recombinant 5-HT3 receptors.

1. The effects of ethanol, chloral hydrate and trichloroethanol upon the 5-HT3 receptor have been investigated by use of electrophysiological techniques applied to recombinant 5-HT3 receptor subunits (5-HT3R-A or 5-HT3R-As) expressed in Xenopus laevis oocytes. Additionally, the influence of trichloroethanol upon the specific binding of [3H]-granisetron to membrane preparations of HEK 293 cells stably transfected with the murine 5-HT3R-As subunit and 5-HT3 receptors endogenous to NG 108-15 cell membranes was assessed. 2. Ethanol (30-300 mM), chloral hydrate (1-30 mM) and trichloroethanol (0.3-10 mM), produced a reversible, concentration-dependent, enhancement of 5-HT-mediated currents recorded from oocytes expressing either the 5-HT3R-A, or the 5-HT3R-As subunit. 3. Trichloroethanol (5 mM) produced a parallel leftward shift of the 5-HT concentration-response curve, reducing the EC50 for 5-HT from 1 +/- 0.04 microM (n = 4) to 0.5 +/- 0.01 microM (n = 4) for oocytes expressing the 5-HT3R-A. A similar shift, from 2.1 +/- 0.05 microM (n = 11) to 1.3 +/- 0.1 microM (n = 4), was observed in oocytes expressing the 5-HT3R-As subunit. Trichloroethanol (5 mM) had little or no effect upon the maximum current produced by 5-HT for either recombinant receptor. 4. Trichloroethanol (5 mM) similarly reduced the EC50 for 2-methyl-5-HT from 13 +/- 0.4 microM (n = 4) to 4.6 +/- 0.2 microM (n = 4) and from 15 +/- 2 microM (n = 4) to 5 +/- 0.4 microM (n = 4) for oocytes expressing the 5-HT3R-A and 5-HT3R-As subunit respectively. Additionally, trichloroethanol (5 mM) produced a clear enhancement of the maximal current to 2-methyl-5-HT (expressed as a percentage of the maximal current to 5-HT) from 63 +/- 0.7% (n = 4) to 101 +/- 1.6% (n = 4) and from 9 +/- 0.2% (n = 4) to 74 +/- 2% (n = 4) for oocytes expressing the 5-HT3R-A and 5-HT3R-As subunit respectively. 5. Trichloroethanol (2.5 mM) had no effect upon the Kd, or Bmax, of specific [3H]-granisetron binding to membrane homogenates of NG 108-15 cells or HEK 293 cells. Similarly, competition for [3H]-granisetron binding by the 5-HT3 receptor antagonists ondansetron and tropisetron was unaffected. However, competition for [3H]-granisetron binding by the 5-HT3 receptor agonists, 5-HT, 2-methyl-5-HT and phenylbiguanide was enhanced by trichloroethanol (2.5 mM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Voltage-dependent block of NMDA responses by 5-HT agonists in ventral spinal cord neurones.

1. Modulation by 5-hydroxytryptamine receptor agonists of the NMDA responses of ventral spinal cord neurones was studied by use of the whole-cell patch-clamp technique. 2. In a Mg-free solution containing tetrodotoxin and glycine, 5-hydroxytryptamine (5-HT, 10-100 microM) reduced the NMDA response, the block increasing with hyperpolarization. Kainate responses were little affected. 3. Some classical agonists of 5-HT receptors induced similar blocking effects. At 10 microM, both a selective agonist of 5-HT2 receptors, (+/-)-2,5-dimethoxy-4 iodo amphetamine (DOI), and a selective agonist of some 5-HT1 receptors, (+/-)-8-hydroxy-2(n-dipropyl amino) tetralin (8-OH-DPAT), induced pronounced blocking effects, of 48% and 33% respectively at -100 mV, whereas another 5-HT1 agonist, 5-carboxamidotryptamine (5-CT) was ineffective. At 100 microM, 5-methoxytryptamine (5-MeOT) induced a complete block of the NMDA responses recorded at -100 mV. The order of potency was: 5-MeOT congruent to DOI > 8-OH-DPAT > 5-HT > 5-CT. 4. Neither spiperone nor ketanserin (1 microM) prevented the blocking effect of 5-HT or DOI. 5. Prolonged preincubations with 5-HT did not block the response if NMDA was applied without 5-HT. When 5-HT agonists were applied both by preincubation and with NMDA, the degree of block increased during the NMDA application. 6. Lowering the NMDA concentration (from 100 to 20 microM) slightly decreased the blocking effect of 5-MeOT. 7. External Mg2+ ions (1 mM) also reduced the blocking effects of 5-HT and 5-MeOT. 8. The blocking effects described appear to be independent of classical 5-HT receptors. Their voltage-dependence suggests a mechanism of open channel block consistent with all the results obtained.     

Agonist interactions with 5-HT3 receptor recognition sites in the rat entorhinal cortex labelled by structurally diverse radioligands.

1. The pharmacological properties of 5-HT3 receptor recognition sites labelled with [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 in membranes prepared from the rat entorhinal cortex were investigated to assess the presence of cooperativity within the 5-HT3 receptor complex. 2. In rat entorhinal cortex homogenates, [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 labelled homogeneous densities of recognition sites (defined by granisetron, 10 microM) with high affinity (Bmax = 75 +/- 5, 53 +/- 5, 92 +/- 6 and 79 +/- 6 fmol mg-1 protein, respectively; pKd = 9.41 +/- 0.04, 8.69 +/- 0.14, 8.81 +/- 0.06 and 10.14 +/- 0.04 for [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330, respectively, n = 3-8). 3. Quipazine and granisetron competed for the binding of each of the radioligands in the rat entorhinal cortex preparation at low nanomolar concentrations (pIC50; quipazine 9.38-8.51, granisetron 8.62-8.03), whilst the agonists, 5-hydroxytryptamine (5-HT), phenylbiguanide (PBG) and 2-methyl-5-HT competed at sub-micromolar concentrations (pIC50; 5-HT 7.16-6.42, PBG 7.52-6.40, 2-methyl-5-HT 7.38-6.09). 4. Competition curves generated with increasing concentrations of quipazine, PBG, 5-HT and 2-methyl-5-HT displayed Hill coefficients greater than unity when the 5-HT3 receptor recognition sites in the entorhinal cortex preparation were labelled with [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330. These competing compounds displayed Hill coefficients of around unity when the sites were labelled with [3H]-(S)-zacopride. Competition for the binding of [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 by granisetron generated Hill coefficients around unity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the ligand-binding subunit of the human 5-hydroxytryptamine1A receptor with N-(p-azido-m-[125I] iodophenethyl)spiperone, a high affinity radioiodinated photoaffinity probe

The ligand-binding subunit of the human 5-hydroxytryptamine1A (5-HT1A) receptor transiently expressed in COS-7 cells and of the native human 5-HT1A receptor derived from hippocampus and frontal cortex were identified by photoaffinity labeling with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS), previously characterized as a high affinity radioiodinated D2-dopamine receptor probe. The identity of the ligand-binding subunit was confirmed by immunoprecipitation with an antipeptide rabbit antiserum, JWR21, raised against a synthetic peptide derived from the predicted amino acid sequence of the putative third intracellular loop of the human 5-HT1A receptor. In transiently transfected COS-7 cells expressing 14 +/- 3 pmol/mg of protein human 5-HT1A receptors, a single broad 75-kDa band was photoaffinity labeled by [125I]N3-NAPS. This band displayed the expected pharmacology of the 5-HT1A receptor, as evidenced by the ability of a series of competing ligands to block [125I]N3-NAPS photoincorporation. Moreover, antiserum JWR21 specifically and quantitatively immunoprecipitated the 75-kDa photoaffinity-labeled band from a soluble extract of the transfected COS-7 cell membranes, further confirming its identity. Finally, utilizing a combination of photoaffinity labeling and immunoprecipitation, the native ligand-binding subunit of 62-64 kDa was identified in human hippocampus and frontal cortex. The availability of the high specific activity, high affinity, photoaffinity ligand [125I]N3-NAPS and of a potent immunoprecipitating antiserum (JWR21) should greatly facilitate the biochemical characterization of the human 5-HT1A receptor.