Serotonin 5-HT(7) receptors mediate relaxation of porcine pial veins.

Isolated porcine pial veins in the presence of active muscle tone have been shown to exhibit rhythmic contractions (RC) that are inhibited by serotonin (5-HT) in a concentration-dependent manner. The 5-HT inhibition of RC is mediated by an as yet unidentified 5-HT receptor subtype located on the vascular smooth muscle. 5-carboxamidotryptamine, which is a potent but nonselective agonist at 5-HT(7) receptors, has been shown to be the most potent inhibitor of RC in porcine pial veins. Therefore, the present study was designed to determine if the 5-HT-mediated inhibition of RC in pial veins is mediated by 5-HT(7) receptors and if 5-HT(7) receptor mRNA is expressed in endothelium-denuded pial veins; the study was done with the use of an in vitro tissue bath and RT-PCR techniques. Our findings indicated that 5-HT inhibition of RC in porcine pial veins was prevented by 5-HT(7)-receptor antagonists (clozapine, pimozide, and LY-215840) in a concentration-dependent manner. Furthermore, a strong PCR signal for the 5-HT(7) receptor was consistently detected in endothelium-denuded pial veins. Sequence analysis of the amplified products confirmed their high degree of homology with the porcine and/or human 5-HT(7)-receptor gene. Taken together, these data suggest that the 5-HT-induced inhibition of RC in porcine pial veins is at least in part mediated by 5-HT(7) receptors located on the venous smooth muscle.

Nitric oxide is the predominant mediator for neurogenic vasodilation in porcine pial veins.

The innervation pattern and the vasomotor response of the potential transmitters in the porcine pial veins were investigated morphologically and pharmacologically. The porcine pial veins were more densely innervated by vasoactive intestinal polypeptide (VIP)- and neuropeptide Y-immunoreactive (I) fibers than were calcitonin gene-related peptide (CGRP)-I, choline acetyltransferase-I, Substance P (SP)-I, and NADPH diaphorase fibers. Serotonin (5-HT)-I fibers, which were not detected in normal control pial veins, were observed in isolated pial veins after incubation with 5-HT (1 microM). 5-HT-I fibers, however, were not observed when incubation with 5-HT was performed in the presence of guanethidine (1 microM), suggesting that 5-HT was taken up into the sympathetic nerves. In vitro tissue bath studies demonstrated that porcine pial veins in the presence of active muscle tone relaxed on applications of exogenous 5-HT, CGRP, SP, VIP, and sodium nitroprusside, whereas exogenous norepinephrine and neuropeptide Y induced only constrictions. Transmural nerve stimulation (TNS) did not elicit any response in pial veins in the absence of active muscle tone. However, in the presence of active muscle tone, pial veins relaxed exclusively on TNS. This tetrodotoxin-sensitive relaxation was not affected by receptor antagonists for VIP, CGRP, 5-HT, or SP but was blocked by L-glutamine (1 mM) and abolished by Nomega-nitro-L-arginine (10 microM) and Nomega-nitro-L-arginine methyl ester (10 microM). The inhibition by L-glutamine, Nomega-nitro-L-arginine, and Nomega-nitro-L-arginine methyl ester was reversed by L-arginine and L-citrulline but not by their D-enantiomers. These results demonstrate that the vasomotor effect of all potential transmitters except 5-HT in the pial veins examined resembles that in cerebral arteries. Although porcine pial veins receive vasodilator and constrictor nerves, a lack of constriction on TNS suggests that the dilator nerves that release nitric oxide may play a predominant role in regulating porcine pial venous tone.

L-citrulline conversion to L-arginine in sphenopalatine ganglia and cerebral perivascular nerves in the pig.

The presence of nitric oxide synthase (NOS), argininosuccinate synthetase (ASS), and argininosuccinate lyase (ASL) and their coexistence with NADPH-diaphorase (NADPHd), a marker for NOS, in the porcine sphenopalatine ganglia (SPG), pial veins, and the anterior cerebral arteries was examined using immunohistochemical and histochemical staining techniques. NOS-immunoreactive (I), ASS-I, and ASL-I fibers were found in pial veins and the anterior cerebral arteries. NOS, ASS, and ASL immunoreactivities were also found in neuronal cell bodies in the SPG. Almost all neuronal cell bodies in the SPG and nerve fibers in pial veins and the anterior cerebral arteries that were reactive to ASS, ASL, and NOS were also stained positively with NADPHd, suggesting that ASS, ASL, and NOS were colocalized in the same neurons in the SPG and perivascular nerves. With the use of in vitro tissue bath techniques, L-citrulline but not D-citrulline reversed inhibition of neurogenic vasodilation in isolated porcine pial veins produced by NOS inhibitors such as NG-nitro-L-arginine methyl ester. In the presence of L-aspartate, L-arginine was synthesized from L-citrulline in homogenates of SPG and endothelium-denuded cerebral arteries and pial veins. These results provide evidence indicating that perivascular nerves in pial veins like cerebral arteries can convert L-citrulline to L-arginine for synthesizing nitric oxide. The conversion is most likely via an argininosuccinate pathway.

Dopamine constricts porcine pial veins.

Dopamine has been shown to induce pial arterial relaxation and constriction in several species. Its mode of action on pial veins, however, remains unclarified. The vasomotor effect of dopamine on porcine pial veins was, therefore, examined using an in vitro tissue bath technique. The results indicated that dopamine constricted exclusively isolated ring segments of pial veins in the presence or absence of active muscle tone. The constriction induced by dopamine was not affected by N(omega)-nitro-L-arginine (L-NNA, 2 x 10(-5) M) or indomethacin (10(-5) M). Only in few preparations was the constriction induced by maximum concentration of dopamine potentiated by L-NNA, suggesting that dopamine at high concentrations may release NO or a NO-related substance. In the presence of L-NNA (2 x 10(-5) M), dopamine-induced constriction was inhibited by phentolamine and yohimbine (but not prazosin) in a concentration-dependent manner with maximum inhibition at 10(-6) M. SKF38393 and 6-bromo-APB (selective dopamine D1 receptor agonists) and LY171555 (a selective dopamine D2 receptor agonist) also induced pial venous constriction exclusively in the presence of L-NNA. The constriction was not affected by phentolamine (10(-6) M). The order of potency for these agonists in the presence of phentolamine, propranolol, guanethidine and L-NNA was: 6-bromo-APB > SKF38393 > dopamine > LY171555. The dopamine-induced constriction in the presence of phentolamine was further inhibited by both SCH23390 (a selective dopamine D1 receptor antagonist) and sulpiride (a selective dopamine D2 receptor antagonist), but was not affected by dopamine D3 or D4 receptor antagonists. These results indicate that dopamine at low and high concentrations induces exclusively constriction of isolated porcine pial veins. The constriction is mediated by postsynaptic alpha2-adrenoceptors, and dopamine D1 and D2 receptors.

Norepinephrine attenuates serotonin inhibition of pial venous tone.

Serotonin (5-hydroxytryptamine, 5-HT) has been shown to inhibit the rhythmic constrictions, accompanied by an increase in cAMP synthesis, in porcine pial veins. Since porcine pial veins contain predominant postsynaptic alpha 2-adrenoceptors which are coupled to Gi-protein, the possibility that the inhibitory effect of 5-HT is antagonized by norepinephrine was examined pharmacologically, using tissue bath techniques. The results indicated that norepinephrine (0.1-1 microM) attenuated 5-HT-induced inhibition of rhythmic constriction. This effect of norepinephrine was mimicked by clonidine (an alpha 2-adrenoceptor agonist), but not by methoxamine (an alpha 1-adrenoceptor agonist). Furthermore, the effect of norepinephrine was prevented by yohimbine (an alpha 2-adrenoceptor antagonist) and pertussis toxin, but was not prevented by prazosin (an alpha 1-adrenoceptor antagonist). In parallel studies, the basal concentration of cAMP and that induced by 5-HT in the pial veins were inhibited by norepinephrine (0.3 microM). These results are consistent with the previous findings that 5-HT-induced inhibition of rhythmic constriction in the porcine pial veins is associated with an increase in vascular cAMP synthesis and suggest that norepinephrine attenuates 5-HT-induced inhibition of rhythmic constriction in part by negatively coupling to adenylate cyclase via alpha 2-adrenoceptors.

Inhibition of cerebral neurogenic vasodilation by L-glutamine and nitric oxide synthase inhibitors and its reversal by L-citrulline.

The possibility that L-citrulline is able to reverse inhibition of neurogenic vasodilation in isolated porcine cerebral arteries produced by nitric oxide synthase (NOS) inhibitors and Gln was examined by using in vitro tissue bath techniques. The results indicated that transmural nerve stimulation elicited a frequency-dependent and tetrodotoxin-sensitive vasodilation in isolated ring segments of arteries with or without endothelial cells. The dilation was abolished by L-N-omega-nitro-L-Arg and L-N-omega-L-Arg methyl ester and was completely reversed by L-citrulline, but not by D-citrulline. In parallel studies, the transmural nerve stimulation-induced relaxation was blocked in part by Gln. The blockade was completely reversed by L-citrulline and L-Arg, but not by their D-enantiomers. The time courses of relaxation after L-citrulline reversal of inhibition of vasodilation produced by Gln and NOS inhibitors were identical to that of relaxation in the control. The residual relaxation after L-citrulline reversal was abolished by L-NNA and L-N-omega-nitro-L-Arg. At 1 mM, L-glutamate, tau-aminobutyric acid or NH4Cl did not block transmural nerve stimulation-induced relaxation, nor did Gln inhibit sodium nitroprusside-induced relaxation or neuronal NOS activity. These results provide pharmacological evidence that cerebral perivascular nerves can recycle L-citrulline to L-Arg for synthesizing nitric oxide to induce neurogenic vasodilation. Although the exact mechanism of action of Gln in inhibiting nitric oxidergic neurovascular transmission remains undetermined, Gln does not seem to act by releasing ammonium ions, inhibiting NOS or modifying the nitric oxide-cyclic GMP pathway. Cerebral metabolism of Gln may play an important role in regulating nitric oxidergic neurogenic vasodilation.

A novel 5-HT1-like receptor subtype mediates cAMP synthesis in porcine pial vein.

The 5-hydroxytryptamine (5-HT) receptor subtype mediating 5-HT inhibition of spontaneous rhythmic contractions (SRC) in the porcine pial vein was characterized. Results from pharmacological studies using in vitro tissue bath techniques indicated that the inhibitory effects of 5-HT on SRC were qualitatively and quantitatively mimicked by 5-HT1-like agonists 5-methoxytryptamine (5-MT) and 5-carboxamidotryptamine (5-CT). 5-HT-, 5-MT-, and 5-CT-induced inhibitions of SRC were attenuated in a concentration-dependent manner by methysergide, which yielded similar pA2 values against these three agonists, suggesting that 5-HT, 5-MT, and 5-CT act on the same 5-HT1-like receptors. 5-MT inhibition of SRC was not affected by blocking 5-HT2 (with ketanserin and spiperone), 5-HT3 (with MDL-72222 and ICS-205-930), or 5-HT4 (with ICS-205-930) receptors. Neither was 5-MT inhibition of SRC affected by blocking 5-HT1A (with propranolol and spiperone), 5-HT1B (with propranolol), or 5-HT1C (with ketanserin) receptors. Furthermore, 5-HT and 5-MT inhibitions of SRC were enhanced by cilostazol [a selective adenosine 3',5'-cyclic monophosphate (cAMP) phosphodiesterase inhibitor] and were diminished by KT-5720 (a cAMP-dependent protein kinase inhibitor) but were not affected by M&B-22948 [a selective guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase inhibitor] or KT-5823 (a cGMP-dependent protein kinase inhibitor). Biochemical studies further demonstrated that 5-HT inhibition of SRC in porcine pial veins was accompanied by an increase in cAMP, but not cGMP, synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca(++)-independent relaxation mediated by beta adrenoceptor in Ca(++)-independent contraction of uterine smooth muscle.

Isoproterenol (ISO)-induced relaxation of oxytocin-induced Ca(++)-independent contraction of the rat uterus was examined. Oxytocin induced Ca(++)-dependent phasic contraction in a solution containing Ca++ (normal contraction) and Ca(++)-independent sustained contraction in Ca(++)-free solution (Ca(++)-free contraction). Both contractions were completely suppressed by cyclic AMP-elevating relaxants such as ISO, dibutyryl cyclic AMP and forskolin. Moreover, the ISO concentration needed to inhibit the Ca(++)-free contraction was lower than that needed for normal contraction, although the relaxing effect of dibutyryl cyclic AMP and forskolin during Ca(++)-free contraction was not significantly different from that during Ca(++)-dependent contraction. The ISO-induced relaxation of the uterus in Ca(++)-free solution may involve three mechanisms. The first is cyclic AMP-dependent relaxation shown by high concentrations (more than 1 nM) of ISO. The second is stabilization via K+ channels by intermediate concentrations (10 pM to 1 nM) of ISO. These two actions appear to be mediated through beta-1 adrenoceptors. The third is, however, via an unknown subtype of adrenoceptor stimulated by extremely low concentrations (1 pM to 10 pM) of ISO. All of these relaxing mechanisms are independent of Ca++.

Ca(2+)-dependent inhibition of Ca(2+)-independent contraction in uterine smooth muscle.

Uterine smooth muscle of the rat shows Ca(2+)-independent contraction in response to oxytocin in Ca(2+)-free medium. Micromolar Ca2+ inhibits this contraction. We now tested whether Ca2+ itself is the cause of this inhibition. The ratio of fura-2 fluorescence, the indicator of the intracellular level of Ca2+, was increased in parallel with the degree of inhibition by Ca2+. When inhibition was elicited by Ca2+, EGTA released the inhibition. Comparison of the dose-response curve for oxytocin in Ca(2+)-free solution and that in the medium with 1 microM Ca2+ showed that the inhibition by Ca2+ is non-competitive. EGTA chelation of the intracellular Ca2+ by loading of EGTA as its acetoxymethylester resulted in diminution of inhibition by Ca2+. EGTA suppressed the Ca(2+)-induced contraction but did not affect Ca(2+)-independent contraction. It is concluded that the inhibition is induced by intracellular Ca2+ itself.

Calcium ion itself inhibits Ca-free contraction of various smooth muscles in response to adrenergic and cholinergic stimulations and to TPA in the absence of Ca channel blocker and EGTA.

1. Smooth muscles such as thoracic aorta, stomach and vas deferens contract tonically in the absence of Ca ion (Ca) in response to various agonists such as norepinephrine, carbachol and TPA. 2. Addition of Ca alone to the Ca-free medium relaxes the muscle that has been contracting in response to the agonist in the Ca-free medium. 3. Ca itself has an inhibitory action on the contraction. 4. Acidification of the medium relaxes the muscle as Ca does, but weakly.