Is nitric oxide involved in 5-HT2B receptor-mediated contraction in the rat stomach fundus?

Although contraction of the rat stomach fundus by 5-HT is known to be mediated by the 5-HT2B receptor, the second messenger pathways involved in this response remain unclear. Since nitric oxide (NO) has been associated with contraction of certain gastrointestinal smooth muscle, the purpose of this study was to determine if NO is involved in 5-HT-induced contraction in the rat stomach fundus. The arginine analogs L-NAME and L-NMMA, at a concentration (100 μM) established to inhibit NO synthase in the rat stomach fundus by inhibiting depolarizationinduced relaxation in this tissue, had no effect on 5-HT contraction. Furthermore, the NO donors sodium nitroprusside and SNAP did not contract rat stomach fundus under basal tone, whereas 5-HT was a potent contractile agonist. These data do not support a role for NO in 5-HT_2B receptormediated contraction in the rat stomach fundus.     

The effect of chirality on serotonin receptor affinity.

α-Methylation of phenethylamines or tryptamine appears to have very little effect on serotonin (5-HT) receptor affinity when racemates are examined. Examination of resolved materials, using an isolated rat stomach fundus preparation, reveals that the R-isomers of DOM and MDA and the S-isomer of α-methlytryptamine possess a higher 5-HT receptor affinity than do their enantiomers. While S(+)-DOB possesses a lower affinity than its racemate, R(-)-DOB produces an appreciable agonistic response which interferes with the measurement of p_A2 values.     

Further evidence that the serotonin receptor in the rat stomach fundus is not 5HT1A or 5HT1B

The nature of the receptor mediating serotonin contraction in the rat stomach fundus has not been clearly associated with either 5HT1 or 5HT2 receptors. We have explored the possibility that such receptors in the rat fundus may better correlate with 5HT1A or 5HT1B receptor subtypes as defined by radiolabeled ligand binding studies with brain cortical membranes. Meta chlorophenylpiperazine (CPP) and meta trifluoromethylphenylpiperazine (TFMPP), selective ligands for the 5HT1B receptor and LY165163, a selective ligand for the 5HT1A receptor, have been evaluated for their agonist and antagonist activity at serotonin receptors in the rat stomach fundus. CPP and TFMPP were partial agonists in the rat stomach fundus whereas LY165163 showed no agonist activity in this smooth muscle in concentrations up to 10(-4)M. All three phenylpiperazines antagonized serotonin-induced contractions in the rat stomach fundus. The affinity for serotonin receptors in the rat fundus was similar for all three phenylpiperazines in spite of the reported selectivity of MCPP and TFMPP for 5HT1B and of LY165163 for 5HT1A receptors. Furthermore, the affinity of these agents for serotonin receptors in the rat stomach fundus did not agree with their reported affinity for either 5HT1A or 5HT1B binding sites in rat cortical membranes. Thus, the similarity in affinities of these phenylpiperazine derivatives for serotonin receptors mediating contraction in the rat fundus along with their different affinities for 5HT1A and 5HT1B binding sites argues against the possibility that the serotonin receptor in the rat fundus is of the 5HT1A or 5HT1B subtype of serotonin receptor.     

Comparison of serotonin agonistic and antagonistic activities of a new antidepressant agent Trelibet (EGYT-475) and its metabolite EGYT-2760 on isolated rat fundus.

The effects of Trelibet (EGYT-475, N-benzyl-piperazine-picolinyl-fumarate) and its active metabolite (EGYT-2760, N-benzyl-piperazine) on the serotoninergic responses of rat stomach fundus were investigated and compared with those of MCPP (m-chlorophenyl-piperazine) which is the common metabolite of the arylpiperazine antidepressants Trazodone and Etoperidone. The contraction inhibitory potencies of the agents were determined on the equipotent contractions (EC50) to serotonin (5-HT) and prostaglandin F2 alpha (PGF2 alpha). Isotonic contractile responses to 5-HT were not affected by EGYT-475, however, both EGYT-2760 and MCPP produced concentration related and reversible inhibition of the serotoninergic responses. The IC50 values for EGYT-2760 and MCPP were 40.5 +/- 7.5 mumol/l and 125 +/- 35 nmol/l, respectively. The inhibition was selective for the serotoninergic responses, as the equipotent responses to PGF2 alpha were not affected. EGYT-2760 and MCPP displayed not only 5-HT antagonistic, but also partial agonistic activities on the rat fundus preparation. Maximum contractile response of the fundus preparation to MCPP was approximately 25%, to EGYT-2760 was 10% of the maximum response to 5-HT.     

Serotoninergic control of somatostatin and gastrin release from the isolated rat stomach

The influence of exogenous serotonin on the secretion of gastric somatostatin and gastrin was investigated under in vitro conditions using an isolated, vascularly perfused rat stomach preparation. Serotonin stimulated gastrin release, maximal effects were observed at 10(-6) M which increased gastrin levels by 78%; on the contrary, somatostatin secretion was inhibited (maximal inhibition of 56% at 10(-6) M). Changes in hormone secretion in response to serotonin were reversed by combined blockade of 5-HT1 and 5-HT2 receptors by methysergide and blockade of 5-HT2 receptors by ketanserin (10(-5) and 10(-6) M, respectively), and of cholinoreceptors by atropine (10(-5) M). It is concluded that in rats in vitro serotonin inhibits release of gastric somatostatin and stimulates gastrin secretion via specific serotonin receptors but muscarinic cholinergic receptors are also involved.     

Identification and expression analyses of a novel serotonin receptor gene, 5-HT2β, in the field cricket, Gryllus bimaculatus

Biogenic amine serotonin (5-HT) modulates various aspects of behaviors such as aggressive behavior and circadian behavior in the cricket. In our previous report, in order to elucidate the molecular basis of the cricket 5-HT system, we identified three genes involved in 5-HT biosynthesis, as well as four 5-HT receptor genes (5-HT1A, 5-HT1B, 5-HT2α, and 5-HT7) expressed in the brain of the field cricket Gryllus bimaculatus DeGeer [7]. In the present study, we identified Gryllus 5-HT2β gene, an additional 5-HT receptor gene expressed in the cricket brain, and examined its tissue-specific distribution and embryonic stage-dependent expression. Gryllus 5-HT2β gene was ubiquitously expressed in the all examined adult tissues, and was expressed during early embryonic development, as well as during later stages. This study suggests functional differences between two 5-HT2 receptors in the cricket.     

Participation of cholinergic pathways in α-hederin-induced contraction of rat isolated stomach strips

The dry extract of Hedra helix leaves and its main active compounds, predominantly α-hederin and hederacoside C, has been traditionally believed to act spasmolytic. However, it has been recently proved that both, the extract of ivy and triterpenoid saponins, exhibit strong contractile effect on rat isolated stomach smooth muscle strips. It turned out that the most potent contractile agent isolated from the extract of ivy leaves is α-hederin. Thus, it seems reasonable to estimate the mechanism of the contractile effect of this saponin. The presented study was aimed at verifying the participation of cholinergic pathways (muscarinic and nicotine receptors) in α-hederin-induced contraction. The experiments were carried out on rat isolated stomach corpus and fundus strips under isotonic conditions. The preparations were preincubated with either atropine or hexamethonium and then exposed to α-hederin. All results are expressed as the percentage of the response to acetylcholine - a reference contractile agent. The obtained results revealed that the pretreatment of isolated stomach strips (corpus and fundus) with atropine neither prevented nor remarkably reduced the reaction of the preparations to α-hederin. Similarly, if the application of saponin was preceded by the administration of hexamethonium, the strength of the contraction of stomach fundus strips induced by α-hederin was not modified. Concluding, it can be assumed that the cholinergic pathways do not participate in α-hederin-evoked contraction of rat isolated stomach preparations.     

The toadfish serotonin 2A (5-HT(2A)) receptor: molecular characterization and its potential role in urea excretion

Based on early pharmacological work, the serotonin 2A (5-HT(2A)) receptor subtype is believed to be involved in the regulation of toadfish pulsatile urea excretion. The goal of the following study was to characterize the toadfish 5-HT(2A) receptor at a molecular level, to determine the tissues in which this receptor is predominantly expressed and to further investigate the pharmacological specificity of toadfish pulsatile urea excretion by examining the effect of ketanserin, a 5-HT(2A) receptor antagonist, on resting rates of pulsatile urea excretion. The full-length toadfish 5-HT(2A) receptor encodes a 496 amino acid sequence and shares 57-80% sequence identity to 5-HT(2A) receptors of other organisms, with 100% conservation among important ligand-binding residues. Toadfish 5-HT(2A) receptor mRNA expression was highest in the swim bladder and gonad, followed by the whole brain. All other tissues tested (esophagus, stomach, anterior intestine, posterior intestine, rectum, liver, kidney, heart, muscle and gill) had mRNA expression levels that were significantly less than whole brain. Toadfish 5-HT(2A) receptor mRNA expression within the brain was highest in the hindbrain, telencephalon and midbrain/diencephalon regions. Treatment with the 5-HT(2A) receptor antagonist, ketanserin, resulted in a significant decrease in the pulsatile component of spontaneous urea excretion due to a reduction in urea pulse size with no significant change in pulse frequency. These results lend further support for the 5-HT(2A) receptor in the regulation of pulsatile urea excretion in toadfish.     

Serotonin receptor interactions of harmaline and several related β-carbolines

The isolated rat fundus preparation was used to determine the serotonin (5-HT) receptor affinities (pA₂ values) of harmaline, harmine and several other related β-carbolines. Replacement of a 7-methoxy group by a hydroxy group decreases affinity; the 6-methoxy β-carbolines possess two to three times the affinity of their 7-methoxy isomers. A new model is proposed for the interaction of these β-carbolines with 5-HT receptors.     

The 5-HT2 serotonin receptor gene Htr-2 is tightly linked to Es-10 on mouse chromosome 14.

Clones coding for the 5-HT2 serotonin receptor were isolated from rat brain cDNA libraries. Using one of the cDNA clones as the probe, mouse genomic DNAs from intersubspecific backcrosses were analyzed by Southern blot hybridization for a restriction fragment length polymorphism. The 5-HT2 serotonin receptor gene, Htr-2, was mapped to mouse Chromosome 14 and is closely linked with the marker Es-10.     

Isolation and sequence of a rat chymotrypsin B gene

A cDNA clone encoding part of chymotrypsin B was isolated from a cDNA library prepared from rat pancreatic mRNA and used as a probe to isolate the chymotrypsin B gene. The nucleotide sequence of this gene is presented. The 4709-base pair transcribed portion of the isolated gene was inferred from the cDNA and gene sequence, and the 5' border was determined by primer extension on pancreatic polyadenylated RNA. The coding portion of the gene is interrupted by six introns. The active site residues His 57, Asp 102, and Ser 195 are encoded by separate exons. Moreover, two regions of the enzyme which form the substrate-binding pocket are also encoded by separate exons. Thus, the substrate specificity and catalytic activity of the enzyme are produced by joining several exons encoding protein segments that are intrinsically catalytically inactive. The number and location of the intron/exon junctions of the chymotrypsin gene as compared to those of other serine protease genes, as well as the location of the genes on separate chromosomes, suggest that the duplication that resulted in the formation of the chymotrypsin gene was an ancient evolutionary event.