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 (10-1000nM), 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-HT1B/D receptor antagonist, GR127935 (100-1000 nM), and the 5-HT1D 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 microM, 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 microM did not affect the decrease in 5-HT output induced by the selective 5-HT1B/D receptor agonist, naratriptan (used at 10 microM in raphe and 0.1 microM in hypothalamus), in the raphe or the hypothalamus. In the raphe, WAY100635, a 5-HT1A receptor antagonist, at 1 microM, did not prevent naratriptan (10 microM) 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-HT1B 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-HT1B receptors are involved in the modulation of 5-HT neurotransmission.     

Pharmacological characterization of a rat 5-hydroxytryptamine type3 receptor subunit (r5-HT3A(b)) expressed in Xenopus laevis oocytes

The present study has utilized the two electrode voltage-clamp technique to examine the pharmacological profile of a splice variant of the rat orthologue of the 5-hydroxytryptamine type 3A subunit (5-HT3A(b)) heterologously expressed in Xenopus laevis oocytes. At negative holding potentials, bath applied 5-HT (300 nM - 10 microM) evoked a transient, concentration-dependent (EC50 = 1.1+/-0.1 microM), inward current. The response reversed in sign at a holding potential of -2.1+/-1.6 mV. The response to 5-HT was mimicked by the 5-HT3 receptor selective agonists 2-methyl-5-HT (EC50= 4.1+/-0.2 microM), 1-phenylbiguanide (EC50=3.0+/-0.1 microM), 3-chlorophenylbiguanide (EC50 = 140+/-10 nM), 3,5-dichlorophenylbiguanide (EC50 = 14.5+/-0.4 nM) and 2,5-dichlorophenylbiguanide (EC50 = 10.2+/-0.6 nM). With the exception of 2-methyl-5-HT, all of the agonists tested elicited maximal current responses comparable to those produced by a saturating concentration (10 microM) of 5-HT. Responses evoked by 5-HT at EC50 were blocked by the 5-HT3 receptor selective antagonist ondansetron (IC50=231+/-22 pM) and by the less selective agents (+)-tubocurarine (IC50=31.9+/-0.01 nM) and cocaine (IC50 = 2.1+/-0.2 microM). The data are discussed in the context of results previously obtained with the human and mouse orthologues of the 5-HT3A subunit. Overall, the study reinforces the conclusion that species differences detected for native 5-HT3 receptors extend to, and appear largely explained by, differences in the properties of homo-oligomeric receptors formed from 5-HT3A subunit orthologues.     

Streptokinase entrapment in interdigitation-fusion liposomes improves thrombolysis in an experimental rabbit model

1. The 5-HT receptor involved in the effect of mucosal application of 5-HT to facilitate peristalsis was investigated in the isolated guinea pig ileum. 2. An application of 5-HT (3-100 microM) to the mucosal surface (by inclusion of 5-HT in the Krebs-Henseleit solution passing through the lumen of the ileum) caused a concentration related facilitation of peristalsis characterized by a reduction in the peristaltic threshold. 3. Peristalsis was not modified by methiothepine (0.1 microM), ritanserin (0.1 microM), ondansetron (5 microM), granisetron (1 microM) or SB 204070 (0.1 microM) administered alone to the mucosal surface. 4. The concentration-response curve to mucosally applied 5-HT was not altered by the mucosally applied 5-HT1/2 receptor antagonist methiothepine (0.1 microM), the 5-HT2 receptor antagonist ritanserin (0.1 microM) or the 5-HT4 receptor antagonist SB 204070 (0.1 microM). However, the mucosally applied 5-HT3 receptor antagonists ondansetron (5 microM) and granisetron (1 microM) shifted the response curves to mucosally applied 5-HT to the right in a parallel and surmountable manner. The pD2 values in the absence and presence of ondansetron were 5.42 +/- 0.07 and 4.12 +/- 0.10, respectively, (n = 6) and that of granisetron were 5.45 +/- 0.12 and 4.50 +/- 0.10 respectively, (n = 5). 5. Serosally applied ondansetron (5 microM) or granisetron (1 microM) had no effect on the concentration-response curve to mucosally applied 5-HT. However, the serosally applied ondansetron and granisetron antagonised the facilitatory effect of serosally applied 5-HT (10 microM) when administered in the presence of serosally applied SB 204070 (0.1 microM). 6. It is concluded that the facilitatory effect of mucosally applied 5-HT to reduce the peristaltic threshold in the guinea pig ileum is mediated via a 5-HT3 receptor located on the mucosal and not the serosal side of the ileum.     

SC-53606, a potent and selective antagonist of 5-hydroxytryptamine4 receptors in isolated rat esophageal tunica muscularis mucosae

(1-S,8-S)-N-[(hexahydro-1H-pyrrolizin-1-yl)methyl]-6-chloroimi+ ++- dazo[1,2-a]pyridine-8-carboxamide hydrochloride (SC-53606) acts as an antagonist of 5-hydroxytryptamine4 (5-HT4) receptor-mediated relaxation of carbachol-induced contractions in rat esophageal tunica muscular mucosae, but does not possess 5-HT4 agonist activity. SC-53606 demonstrated a pA2 value against 5-HT in this tissue of 7.91 +/- 0.08 (Ki = 12.3 +/- 1.17 nM). Similar pA2 values of 7.68 +/- 0.06, 7.67 +/- 0.06 and 7.63 +/- 0.05 were determined for the synthetic 5-HT4 receptor agonists SC-53116, 5-methoxytryptamine and renzapride, respectively. In addition, slopes of Schild plots for antagonism of these four agonists by SC-53606 were 1.07 +/- 0.02, 0.98 +/- 0.03, 1.04 +/- 0.02 and 0.96 +/- 0.06, respectively, and did not deviate from unity. The pA2 values for 5-HT4 antagonism against 5-HT were determined to be 6.80 +/- 0.09 for tropisetron and 7.36 +/- 0.08 for 2-methoxy-4-amino-S- chlorobenzoic acid-2-(diethylamino)ethyl ester SDZ 205-557), indicating that SC-53606 is more a potent 5-HT4 antagonist than either of the reference antagonists. Radioligand binding studies also demonstrated that SC-53606 is a selective antagonist with more affinity for 5-HT4 than for other 5-HT receptors. Displacement of radioligand binding from 5-HT1 and 5-HT2 receptors by SC-53606 was less than 50% at a 10 microM concentration. Similarly, SC-53606 displayed little binding affinity at alpha 1, alpha 2 and beta adrenergic, dopamine1, dopamine2 and muscarinic cholinergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiac manifestations of polymyositis. Apropos of 2 Senegalese cases]

The relaxations mediated by the activation of 5-HT receptors in the guinea pig proximal colon were investigated. Longitudinal strips were cut from the colon segment and placed into the bath. In the presence of atropine (0.2 microM), the relaxations were evoked by adding increasing concentrations of 5-HT (1-100 microM). Noncumulative concentration-response curves were established in the absence and presence of either 5-HT or nitric oxide synthase (NOS) antagonists. Selective 5-HT3 antagonists tropisetron (10 and 100 nM) and ondansetron (1 microM) inhibited the relaxations and shifted the concentration-response curves to the right. Similar effects were observed in the presence of the NOS inhibitor N(G)-nitro-L-arginine (3.2, 10, 32 microM) and partly reversed with L-arginine (100, 320 microM). N(G)-nitro-D-arginine, serving as a negative control, was ineffective. The relaxations were further inhibited in the presence of the soluble guanylate cyclase blocker methylene blue (10 microM) or NO scavenger hemoglobin (32 microM). These results suggest that the 5-HT3 receptor plays a role in neurogenic relaxations of guinea pig proximal colon, which are at least partly mediated via release of NO from nerve endings.     

Effects of 5-HT4-receptor agonists, cisapride, mosapride citrate, and zacopride, on cardiac action potentials in guinea pig isolated papillary muscles

The purpose of this study was to examine the effects of 5-HT4-receptor agonists cisapride, mosapride citrate (mosapride), and zacopride on action potentials (APs) in guinea pig isolated papillary muscles. Cisapride (0.1-3 microM) concentration-relatedly prolonged the duration of APs (APD) without affecting the other AP parameters. Mosapride and its main metabolite M1 (des-4-fluoro-benzyl-mosapride) did not affect APs at 10 microM. Zacopride at 10 microM shortened APD, and the APD-shortening effect was not affect by GR113808 (10 microM), a 5-HT4-receptor antagonist. The cisapride (1 microM)-induced prolongation of APD was not affected by GR113808 (10 microM), ritanserin (10 microM), a 5-HT2A/2C-receptor antagonist, or prazosin (10 microM), an alpha 1-receptor antagonist. The same concentrations of GR113808, ritanserin, and prazosin did not affect APD. Clofilium, a class III antiarrhythmic agent, prolonged APD; the effect was more pronounced at a stimulus frequency of 0.3 Hz than at 2.0 Hz. Cisapride did not exert such reverse use dependence, suggesting that its mechanism of action is different from that of clofilium. These results suggest that cisapride prolongs APD without involvement of 5-HT2, 5-HT4, or alpha 1 receptors. Mosapride is unlikely to induce the prolongation of electrocardiographic QT intervals correlated with the prolongation of APD in isolated ventricular muscles.     

Sequence and functional analysis of cloned guinea pig and rat serotonin 5-HT1D receptors: common pharmacological features within the 5-HT1D receptor subfamily

This study was undertaken to investigate the pharmacology of cloned guinea pig and rat 5-hydroxytryptamine (serotonin; 5-HT)1D receptor sites. Guinea pig, rat, and mouse 5-HT1D receptor genes were cloned, and their amino acid sequences were compared with those of the human, dog, and rabbit. The overall amino acid sequence identity between these 5-HT1D receptors is high and varies between 86 and 99%. The sequence homology is slightly more divergent (13-27%) in the N-terminal extracellular region of these 5-HT1D receptors. Guinea pig and rat 5-HT1D receptors, stably and separately expressed in rat C6 glial cells, are negatively coupled to cyclic AMP formation upon stimulation with agonists, as previously found for cloned human 5-HT1D receptor sites. The cyclic AMP data show some common pharmacological features for the 5-HT1D receptors of guinea pig, rat, and human: an almost similar rank order of potency for the investigated 5-HT1D receptor agonists, stereoselectivity for the binding affinity and agonist potency of R(+)-8-hydroxy-2-(di-n-propylamino)tetralin, and equal 5-HT1D receptor-mediated antagonist potency for methiothepin and the 5-HT2 receptor antagonists ritanserin and ketanserin. In conclusion, the pharmacology of the cloned 5-HT1D receptor subtype seems, unlike the 5-HT1B receptor subtype, conserved among various mammal species such as the human, guinea pig, and rat.     

Comparison of right ventricular outflow tract and apical lead permanent pacing on cardiac output

Interactions of gastrointestinal prokinetic benzamides with 5-hydroxytryptamine (5-HT)3 and 5-HT4 receptors and the relation to their effects on gastrointestinal propulsion were investigated. Renzapride and zacopride potently inhibited 5-HT3-receptor-mediated contractions in the guinea pig colon, whereas RS67506 (1-(4-amino-5-chloro-2-methoxyphenyl)-3-[1-(2-methyl sulphonylamino)ethyl-4-piperidinyl]-1-propanone hydrochloride), a selective 5-HT4-receptor agonist, showed no inhibition. RS67506, renzapride and zacopride all exerted 5-HT4 receptor-mediated relaxation in the carbachol-precontracted rat oesophagus. In mice, RS67506 shortened the whole gut transit time, whereas renzapride and zacopride were reported to prolong it. Gastrointestinal prokinetic benzamides, which are selective for 5-HT4-receptor agonistic over 5-HT3-receptor antagonistic action, may be useful in treating gastrointestinal disorders associated with impaired lower intestinal propulsion such as constipation.     

Novel diversity in Th1, Th2 type differentiation of hemagglutinin-specific T cell clones elicited by natural influenza virus infection in three major haplotypes (H-2b,d,k)

The serotonin 5-HT3 receptor, a ligand-gated ion channel, has previously been shown to be present on a subpopulation of brain nerve terminals, where, on activation, the 5-HT3 receptors induce Ca2+ influx. Whereas postsynaptic 5-HT3 receptors induce depolarization, being permeant to Na+ and K+, the basis of presynaptic 5-HT3 receptor-induced calcium influx is unknown. Because the small size of isolated brain nerve terminals (synaptosomes) precludes electrophysiological measurements, confocal microscopic imaging has been used to detect calcium influx into them. Application of 100 nM 1-(m-chlorophenyl)biguanide (mCPBG), a highly specific 5-HT3 receptor agonist, induced increases in internal free Ca2+ concentration ([Ca2+]i) and exocytosis in a subset of corpus striatal synaptosomes. mCPBG-induced increases in [Ca2+]i ranged from 1.3 to 1.6 times over basal values and were inhibited by 10 nM tropisetron, a potent and highly specific 5-HT3 receptor antagonist, but were insensitive to the removal of external free Na+ (substituted with N-methyl-D-glucamine), to prior depolarization induced on addition of 20 mM K+, or to voltage-gated Ca2+ channel blockade by 10 microM Co2+/Cd2+ or by 1 microM omega-conotoxin MVIIC/1 microM oemga-conotoxin GVIA/200 nM agatoxin TK. In contrast, the Ca2+ influx induced by 5-HT3 receptor activation in NG108-15 cells by 1 microM mCPBG was substantially reduced by 10 microM Co2+/Cd2+ and was completely blocked by 1 microM nitrendipine, an L-type Ca2+ channel blocker. We conclude that in contrast to the perikaryal 5-HT3 receptors, presynaptic 5-HT3 receptors appear to be uniquely calcium-permeant.     

Serotonin efflux in the rat ventral lateral geniculate nucleus assessed by fast cyclic voltammetry is modulated by 5-HT1B and 5-HT1D autoreceptors

Fast cyclic voltammetry (FCV) was used to measure electrically stimulated monoamine efflux in the rat ventral lateral geniculate nucleus (vLGN). The electrochemical characteristics of the released species resembled 5-HT but not dopamine or noradrenaline. Amine efflux was abolished by the sodium channel blocker tetrodotoxin (0.1 microM), Ro 4-1284 (1.0 microM), the fast-acting reserpine analogue, and removal of Ca2+ from the superfusate. Amine efflux was unaffected by the monoamine oxidase inhibitor clorgyline (0.1 microM). Of paroxetine (0.1 microM), desipramine (50 nM) and vanoxerine (0.5 microM), selective blockers of 5-HT, noradrenaline and dopamine uptake respectively, only paroxetine increased monoamine efflux (to 194 +/- 25%, mean +/- SEM) and prolonged the removal half-life (to 638 +/- 105%). The non-specific 5-HT1 antagonist methiothepin (0.2 microM) increased 5-HT efflux on long (20 pulses at 20 Hz) but not short trains (20 pulses at 100 Hz). When tested on pseudo-one-pulse stimulations (5 pulses, 100 Hz), the selective 5-HT1A agonist 8-OHDPAT (1.0 microM) had no effect. CP 93129 (0.3 microM), the selective 5-HT1B agonist, decreased 5-HT efflux to 37 +/- 4% of control and was antagonised by the 5-HT1B blocker isamoltane (0.5 microM) and by the 5-HT1D/B antagonist GR 127935 (50 nM). The preferential 5-HT1D agonist sumatriptan (0.5 microM) also decreased 5-HT efflux, to 55 +/- 6% and was antagonised by GR 127935 (50 nM) but not isamoltane (0.5 microM). These results suggest that 5-HT released in the vLGN can be measured by FCV. Furthermore, released 5-HT is taken up by the 5-HT transporter and may be under the influence of 5-HT1B and 5-HT1D autoreceptors.     

5-HT receptors in mammalian brain: receptor autoradiography and in situ hybridization studies of new ligands and newly identified receptors

In recent years the family of mammalian serotonin receptors has grown to 14 different subtypes, characterized by pharmacological or molecular biological techniques. In parallel, new ligand molecules have been developed for their study. However, selective ligands are not yet available to study every one of them. In addition the degree of selectivity of ligands, hitherto regarded as specific for a particular receptor subtype has been called in question by their affinities for newly discovered receptors. Consequently, a re-evaluation of past ligand receptor autoradiography work is necessary in view of the redefined receptor profiles of these ligands, and the introduction of newly developed ligands. A further difficulty for the characterization of these receptors is the absence of selective antagonist ligands which, for some of the subtypes, have become available only recently. In an attempt to overcome these difficulties we have combined in situ hybridization histochemistry and receptor ligand autoradiography to study the regional and cellular localization of several serotonin receptors in the rodent brain. In addition, for some receptors, we have expanded these studies to primates, including humans. We have found that the distribution of 5-HT1A receptors in monkey brain, labelled with the agonist 3H-8-OH-DPAT and the antagonist 3H-WAY 100635 was very similar at the levels examined, and corresponded well with that observed for the cells containing mRNA coding for this receptor, confirming the somatodendritic localization of 5-HT1A receptors in monkey brain. The labelling conditions to visualize 5-HT1F receptors in guinea pig brain, namely 3H-sumatriptan in the presence of 10(-8) M 5-CT to block 5-HT1D receptors, are suitable for visualizing this receptor, since the results agreed with those observed by in situ hybridization. By using 3H-ketanserin and 3H-mesulergine in parallel with in situ hybridization using the corresponding oligonucleotides, we were able to show that these ligands label respectively 5-HT2A and 5-HT2C binding sites in monkey brain. 5-HT4 receptors were localized in the brain of several species including humans by using 125I-SB 207710. In situ hybridization experiments performed in guinea pig confirmed that 5-HT4 receptors are localized on the terminals of the striatopallidal and striatonigral projections. 5-HT7 binding sites were labelled in rat and guinea pig brains by incubating with 3H-5-CT in the presence of 100 microM WAY 100135 and 250 microM GR 127935; the distribution obtained in both species agreed, in general, with that of the corresponding mRNA coding for them. These results are an illustration of the understanding of our current knowledge of the chemical neuroanatomy of the mammalian 5-HT system.     

Electrophysiological characterization of 5-HT receptors on rat petrosal neurons in dissociated cell culture

The petrosal ganglion supplies chemoafferent pathways via the glossopharyngeal (IXth) nerve to peripheral targets which release various neurotransmitters including serotonin (5-HT). Here, we combined rapid 5-HT application with patch clamp, whole-cell recording to investigate whether 5-HT receptors are expressed on isolated petrosal neurons (PN), cultured from 7-12 day-old rat pups. In responsive cells, the dominant effect of 5-HT was a rapid depolarization associated with a conductance increase in approximately 43% of the neurons (53/123); however, in a minority population ( approximately 6%; 8/123), 5-HT caused membrane depolarization associated with a conductance decrease. In the former group, 5-HT produced a transient inward current (I5-HT) in neurons voltage-clamped near the resting potential ( approximately -60 mV); the effect was mimicked by the 5-HT3 receptor-specific agonist, 2-methyl-5-HT, suggesting it was mediated by 5-HT3 receptors. Further, I5-HT was selectively inhibited by the 5-HT3 receptor-specific antagonist MDL72222 (1-10 microM), but was unaffected by either 5-HT1/5-HT2 receptor antagonist, spiperone, or by 5-HT2 receptor-specific antagonist, ketanserin (50-100 microM). I5-HT displayed moderate inward rectification and had a mean reversal potential (+/-S.E.M.) of -4.3+/-6.6 mV (n=6). Application of 5-HT (dose range: 0.1-100 microM) produced a dose-response curve that was fitted by the Hill equation with EC50= approximately 3.4 microM and Hill coefficient= approximately 1.6 (n=8). The activation phase of I5-HT (10 microM 5-HT at -60 mV) was well fitted by a single exponential with mean (+/-S.E.M.) time constant of 45+/-30 ms (n=6). The desensitization phase of I5-HT was best fitted by a single exponential with mean (+/-S.E.M.) time constant of 660+/-167 ms (n=6). Fluctuation analysis yielded an apparent mean single-channel conductance (+/-S.E.M) of 2.7+/-1.5 pS (n=4) at -60 mV. In the minority ( approximately 6%) population of neurons which responded to 5-HT with a conductance decrease, the depolarization was blocked by the 5-HT2 receptor antagonist, ketanserin (50 microM). Taken together, these results suggest that 5-HT3 receptors are the major subtype expressed by rat petrosal neurons, and therefore are candidates for facilitating chemoafferent excitation in response to 5-HT released from peripheral targets.     

Nutritional assessment of folate and cyanocobalamin status in a Spanish elderly group

The mammalian tachykinins, neurokinin A (NKA) and NKA(4-10), along with the tachykinin NK2 receptor-selective antagonist MEN 10,376, were compared to their C-terminal free acid derivatives, NKA-OH, NKA(4-10)-OH and MEN 10,456, respectively, on several in vitro bioassays for NK1, NK2 and NK3 tachykinin receptors. NKA-OH and NKA(4-10)-OH were much weaker agonists than NKA or NKA(4-10) in the endothelium-deprived rabbit pulmonary artery (endowed with NK2A receptors) and in the guinea pig isolated bronchus (endowed with NK2A and NK1 receptors), where they produced submaximal contractile responses, and were inactive in the hamster isolated trachea (endowed with NK2B receptors) and in the rat isolated portal vein (endowed with NK3 receptors). At NK1 receptors of the guinea pig isolated ileum, NKA-OH produced weak agonist responses, whereas NKA(4-10)-OH was ineffective. In sharp contrast, MEN 10,456, while maintaining the same antagonist potency of the parent compound MEN 10,376 in the rabbit pulmonary artery and hamster isolated trachea, developed a clear-cut agonist character in the rat isolated portal vein, guinea pig isolated ileum and guinea pig isolated bronchus. The agonist responses produced by MEN 10,456 (10 microM) were reduced by MEN 10,376 in the guinea pig isolated bronchus and by the NK1 receptor antagonist GR 82,334 in the guinea pig isolated ileum. These results, although indicating the importance of C-terminal amidation for the agonist activity of natural tachykinins, suggest that the C-terminal amide group may not be directly involved in stimulation of the tachykinin receptors, but could induce agonist activity through a conformation effect.     

Pharmacologic actions of the second-generation leukotriene B4 receptor antagonist LY293111: in vitro studies

The in vitro actions were investigated of LY293111, a potent and selective leukotriene B4 (LTB4) receptor antagonist, on human neutrophils, human blood fractions, guinea pig lung membranes, and guinea pig parenchymal and tracheal strips. The IC50 for inhibiting [3H]LTB4 binding to human neutrophils was 17.6 +/- 4.8 nM. LY293111 inhibited LTB4-induced human neutrophil aggregation (IC50 = 32 +/- 5 nM), luminol-dependent chemiluminescence (IC50 = 20 +/- 2 nM), chemotaxis (IC50 = 6.3 +/- 1.7 nM), and superoxide production by adherent cells (IC50 = 0.5 nM). Corresponding responses induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine were inhibited by 100-fold higher concentrations of LY293111. LTB4 binding to guinea pig tissues and subsequent activation were also inhibited. The Ki for inhibition of [3H]LTB4 binding to lung membranes was 7.1 +/- 0.8 nM; IC50 for preventing binding of [3H]LTB4 to spleen membranes was 65 nM. The compound inhibited LTB4-induced contraction of guinea pig lung parenchyma. At 10 nM, LY293111 caused a parallel rightward shift of the LTB4 concentration-response curve. At higher concentrations, plots were shifted in a nonparallel manner, and maximum responses were depressed. LY293111 did not prevent antigen-stimulated contraction of sensitized trachea strips. At micromolar concentrations, LY293111 inhibited production of LTB4 and thromboxane B2 by plasma-depleted human blood stimulated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine and thrombin. In addition, at these higher concentrations, formation of LTB4 by A23187-activated whole blood and conversion of arachidonic acid to LTB4 by a human neutrophil cytosolic fraction were inhibited. In summary, LY293111 is a second-generation LTB4 receptor antagonist with much improved potency in a variety of functional assay systems.     

5-HT1B receptor antagonist properties of novel arylpiperazide derivatives of 1-naphthylpiperazine

A new series of arylpiperazide derivatives of 1-naphthylpiperazine of general formula 4 has been prepared and evaluated as 5-HT1B antagonists. Binding experiments at cloned human 5-HT1A, 5-HT1B, and 5-HT1D receptors show that these derivatives are potent and selective ligands for 5-HT1B/1D subtypes with increased binding selectivity versus the 5-HT1A receptor when compared to 1-naphthylpiperazine (1-NP). Studies of inhibition of the forskolin-stimulated cAMP formation mediated by the human 5-HT1B receptor demonstrate that the nature of the arylpiperazide substituent modulates the intrinsic activity of these 1-NP derivatives. Among them, 2-[[8-(4-methylpiperazin-1-yl)naphthalen-2-yl]oxy] -1-(4-o-tolylpiperazin-1-yl)ethanone (4a) was identified as a potent neutral 5-HT1B antagonist able to antagonize the inhibition of 5-HT release induced by 5-CT (5-carbamoyltryptamine) in guinea pig hypothalamus slices. Moreover, 4a was found to potently antagonize the hypothermia induced by a selective 5-HT1B/1D agonist in vivo in the guinea pig following oral administration (ED50 = 0.13 mg/kg).     

Analysis of the mechanisms underlying the biphasic responses to bradykinin in circular muscle from guinea pig ileum

Experiments were designed to further characterize the receptor mediating the biphasic response to bradykinin in circular muscle from guinea pig ileum in vitro by the use of HOE 140, a potent and specific bradykinin antagonist. D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin (HOE 140, 0.1-1000 nM) caused a graded inhibition of bradykinin (10 nM)-induced contraction and relaxation responses in circular muscle from guinea pig ileum, with IC50s of 4 and 10 nM respectively. However, the potency of HOE 140 to antagonize the bradykinin (300 nM)-induced contraction and relaxation was decreased about 6-fold (IC50 22 nM) and 57-fold (IC50 570 nM). HOE 140 (3-100 nM) caused parallel and concentration-dependent rightward displacements of bradykinin (0.1-3000 nM)-induced biphasic concentration-response curves in circular muscle from guinea pig ileum. Schild regression plots yielded straight lines with slopes not significantly different from unity and pKb values of 9.0 and 8.7 against bradykinin-induced contraction and relaxation, respectively. Similar pKb values (8.7) were obtained for HOE 140 against bradykinin-mediated contraction in the longitudinal muscle of the guinea pig ileum. The action of HOE 140 was selective for bradykinin, since response to other agonists were not affected. It is concluded that HOE 140 does not discriminate the receptors mediating the biphasic responses to bradykinin in circular muscle from guinea pig ileum, as it showed a similar selective, competitive and reversible antagonism against both components of the bradykinin response in this preparation.     

Retinoic acid and N-(4-hydroxy-phenyl) retinamide suppress growth of esophageal squamous carcinoma cell lines

The roles of endogenous serotonin (5-HT) and 5-HT receptor subtypes in regulation of acetylcholine (ACh) release in frontal cortex of conscious rats were examined using a microdialysis technique. Systemic administration (1 and 3 mg/kg, i.p.) of the 5-HT-releasing agent p-chloroamphetamine (PCA) elevated ACh output in a dose-dependent manner. Depletion of endogenous 5-HT by p-chlorophenylalanine significantly attenuated the facilitatory effect of PCA on ACh release. The PCA (3 mg/kg)-induced increase in ACh release was significantly inhibited by local application of the 5-HT4 receptor antagonists RS23597 (50 microM) and GR113803 (1 microM), while the 5-HT1A antagonist WAY-100135 (10 mg/kg, i.p.; 100 microM), 5-HT(1A/1B)/beta-adrenoceptor antagonists (-)-pindolol (8 mg/kg, i.p.) and (-)-propranolol (150 microM), 5-HT(2A/2C) antagonist ritanserin (1 mg/kg, i.p.; 10 microM) and 5-HT3 antagonist ondansetron (1 mg/kg, i.p.; 10 microM) failed to significantly modify the effect of PCA. These results suggest that PCA-induced enhancement of 5-HT transmission facilitates ACh release from rat frontal cortex at least in part through 5-HT4 receptors.     

Are inner or outer hair cells the source of summating potentials recorded from the round window?

Previous studies indicated that antidromic stimulation of capsaicin-sensitive vagal afferent fibers activated, via peripheral release of tachykinins, nonadrenergic, noncholinergic parasympathetic ganglion neurons that mediate relaxations of guinea pig trachealis. On the basis of the effects of selective agonists and inhibition with a nonselective receptor antagonist (SR 48968), we speculated that tachykinin-mediated activation of neurokinin3 (NK3) receptors might be involved. Using the recently developed NK3-selective receptor antagonist SR 142801, we further assessed the role of NK3 receptors in these relaxant responses. Relaxations of the guinea pig trachea elicited by antidromic stimulation of capsaicin-sensitive vagal afferent nerves were markedly inhibited by 0.3 microM SR 142801 and were abolished by a combination of SR 142801 and either of the NK1-selective receptor antagonists SR 140333 and CP 99994 (0.3 microM each). The NK3 receptor antagonist had similar effects on the relaxant responses elicited by capsaicin and substance P, but it had no effect on relaxations of the trachealis elicited by electrical field stimulation of the postganglionic nerves that innervate the trachealis or by stimulation of the preganglionic parasympathetic vagal nerves that innervate the trachea. These results and the observation that the ganglion neurons that mediate these responses are densely innervated by substance P-containing nerve fibers lead us conclude that stimulation of capsaicin-sensitive visceral afferent fibers activates, upon peripheral release of tachykinins, nonadrenergic, noncholinergic inhibitory neurons innervating guinea pig trachealis via activation of both NK3 and NK1 receptors.     

Facilitation of acetylcholine release in rat frontal cortex by indeloxazine hydrochloride: involvement of endogenous serotonin and 5-HT4 receptors

Effects of indeloxazine hydrochloride, an inhibitor of serotonin (5-HT) and norepinephrine (NE) reuptake with a facilitatory effect on 5-HT release, on acetylcholine (ACh) output in frontal cortex of conscious rats were characterized using an in vivo microdialysis technique. Systemic administration of indeloxazine (3 and 10 mg/kg, i.p.) increased ACh and 5-HT output in a dose-dependent manner. Depletion of endogenous monoamines by reserpine and of 5-HT by p-chlorophenylalanine, but not that of catecholamines by alpha-methyl-p-tyrosine, significantly attenuated the facilitatory effect of indeloxazine on ACh release. When applied locally by reverse dialysis, indeloxazine (10 and 30 microM) and the selective 5-HT reuptake inhibitor citalopram (10 microM), but not the NE reuptake inhibitor maprotiline (30 microM), increased cortical ACh output. Indeloxazine (10 mg/kg)-induced increase in ACh release was significantly inhibited by local application of the 5-HT4 receptor antagonists RS23597 (50 microM) and GR113803 (1 microM), while the 5-HT1A antagonist WAY-100135 (100 microM), 5-HT1A/1B/beta-adrenoceptor antagonist (-)propranolol (150 microM), 5-HT2A/2C antagonist ritanserin (10 microM) and 5-HT3 antagonist ondansetron (10 microM) failed to significantly modify this effect. Neither depletion of monoamines nor treatment with serotonergic antagonists significantly changed the basal ACh level, indicating that endogenous monoamines do not tonically activate ACh release. These results suggest that indeloxazine-induced facilitation of ACh release in rat frontal cortex is mediated by endogenous 5-HT and involves at least in part cortical 5-HT4 receptors.     

Expression and activity of 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase in the rat Purkinje neuron during neonatal life

Diadenosine tetraphosphate (AP4A) is an endogenous compound and exerts diverse physiological effects in animal systems. However, the effects of AP4A on inotropy in ventricular cardiac preparations have not yet been studied. The effects of AP4A on force of contraction (FOC) were studied in isolated electrically driven guinea pig and human cardiac preparations. Furthermore, the effects of AP4A on L-type calcium current and [Ca]i were studied in isolated guinea pig ventricular myocytes. In guinea pig left atria, AP4A (0.1-100 microM) reduced FOC maximally by 36.5 +/- 4.3%. In guinea pig papillary muscles, AP4A (100 microM) alone was ineffective, but reduced isoproterenol-stimulated FOC maximally by 29.3 +/- 3.4%. The negative inotropic effects of AP4A in atria and papillary muscles were abolished by the A1-adenosine receptor antagonist 1, 3-dipropyl-cyclopentylxanthine. In guinea pig ventricular myocytes, AP4A (100 microM) attenuated isoproterenol-stimulated L-type calcium current and [Ca]i. In human atrial and ventricular preparations, AP4A (100 microM) alone increased FOC to 158.3 +/- 12.4% and 167.5 +/- 25.1%, respectively. These positive inotropic effects were abolished by the P2-purinoceptor antagonist suramin. On the other hand, AP4A (100 microM) reduced FOC by 27.2 +/- 7.4% in isoproterenol-stimulated human ventricular trabeculae. The latter effect was abolished by 1,3-dipropyl-cyclopentylxanthine. In summary, after beta adrenergic stimulation AP4A exerts negative inotropic effects in animal and human ventricular preparations via stimulation of A1-adenosine receptors. In contrast, AP4A alone can exert positive inotropic effects via P2-purinoceptors in human ventricular myocardium. Thus, P2-purinoceptor stimulation might be a new positive inotropic principle in the human myocardium.