Agonist of serotonin 5HT1A-receptors 8-OH-DPAT increases the force of contraction of rat aorta and mesenteric artery in the presence of endothelin-1 or vasopressin and causes relaxation of the vessels preconstricted with noradrenaline

Agonist 5HT1A serotonin receptors 8-OH-DPAT at 70–80% in rats relax the isolated aorta and mesenteric artery, precollapsed with noradrenaline. An inhibitor of NO-synthase L-NAME two or more times suppresses vasodilatatornyh reaction in response to the effect of 8-OH-DPAT. The addition of 8-OH-DPAT to the aorta in a state of rest or precollapsed with endothelin-1 or vasopressin causes an increase in apower reduction. A blocker of α₁-adrenoceptors prazosin almost completely suppresses the aorta collapse reaction to the effect of 8-OH-DPAT in the absence of vasoconstrictives, but does not affect the contraction force in response to 8-OH-DPAT of the aorta in the presence of endothelin-1 or vasopressin and does not shift the curve of the dependence of force collapse on the concentration of 8-OH-DPAT. Our data show the existence in the rat aorta of vasodilator and vasoconstrictive 5HT1A-receptors.     

Activation of “silent” vasoconstrictive 5-HT1A receptors as a possible mechanism of synergism in angiotensin II and serotonin effect on vascular tone

It has been shown that the agonist of 5HT1A-receptors 8-OH-DPAT induces contraction of aortic rings in the presence of angiotensin II. This effect is not associated with activation of α₁-adrenoceptors by 8-OH-DPAT as it is reproduced in the presence of prazosin which completely suppresses the nonspecific vasoconstrictive effect of 8-OH-DPAT via α₁-adrenoceptors on the aorta incubated without angiotensin II. Synergism in the action of angiotensin II and 8-OH-DPAT is completely preserved after partial desensitization of the receptors of angiotensin II. It has been found that 8-OH-DPAT increases the free cytoplasmic calcium concentration in cultured smooth muscle cells from the rat aorta. The data obtained support the hypothesis about the existence of “silent” vasoconstrictive 5HT1A-receptors. It has been suggested that activation of these receptors underlies synergism in vasoconstrictive action of serotonin and angiotensin II.     

Calmodulin inhibitors suppress calcium signaling from serotonin receptors in smooth muscle cells and abolish vasoconstrictive response on intravenous introduction of serotonin

Comparative study of the effect of calmodulin inhibitors trifluoperazine, W-12, and W-13 and the TRPV1 channel blocker capsazepine on receptor-dependent calcium metabolism in smooth muscle cells of the rat aorta and on the contraction of the isolated rat aorta was performed. Trifluoperazine almost completely abolishes an increase in free cytoplasmic calcium concentration in smooth muscle cells isolated from the rat aorta and smooth muscle cells of the A7r5 line in response to serotonin and does not affect cellular reaction to vasopressin and angiotensin II. W-12 and W-13 also do not attenuate responses to vasopressin and angiotensin II and reduces by two times free cytoplasmic calcium concentration elevation in response to serotonin. The efficiency of calcium metabolism suppression by calmodulin inhibitors correlates with the degree of inhibition of the aorta contractile response to serotonin. It was demonstrated that the inhibitory action of calmodulin antagonists on calcium metabolism in smooth muscle cells and the contractility of the isolated rat aorta during the activation of serotonin vasoconstrictive receptors are realized by a TRPV1-independent mechanism. It was demonstrated in experiments in vivo that trifluoperazine does not influence hypotensive reaction in rats (normally observed in response to intravenous serotonin injection), but removes the hypertensive effect of this neurotransmitter in rats after chronic introduction of dexamethasone. The results obtained confirm the hypothesis (that we previously stated) about the direct involvement of calmodulin in signal transmission from vasoconstrictive serotonin receptors.     

The effects of inhibitors of Rho- and tyrosine c-Src-kinases on serotonin-induced constrictions of the aorta and mesenteric artery in rats

We found that the inhibitor of Rho-kinase fasudil selectively inhibited constriction of isolated rings of the aorta and mesenteric artery in rats in response to application of the agonists of 5HT2A-(DOI and TBC-2) and 5HT1A-receptors (8-OH-DPAT) and did not influence vasoconstriction induced by serotonin. We demonstrate for the first time that application of the agonists of 5HT2C-receptors (MK 212 and SCH 23390) did not influence the tone of “intact” vessels. The marked vasoconstrictory effect of the agonists of 5HT2C-receptors was observed in the vessels preconstricted due to angiotensin II or vasopressin. We found that the inhibitor of Rho-kinase did not influence negatively on MK 212 or SCH 23390-induced constriction of isolated rings of the aorta and mesenteric artery in rats. We suppose that, in the presence of fasudil, serotonin induces constriction of vessels through the interaction with 5HT2C-receptors and signal transduction from these receptors does not involve Rho-kinase activity. We found that fasudil attenuated vasoconstriction induced by norepinephrine and vasopressin by 40%. We demonstrated that tyrosine c-Src-kinase plays the most important role in signal transduction from 5HT-receptors because its effects are specific with relation to these receptors.     

Effect of calmodulin antagonists on auxin-induced elongation

Coleoptile segments of oat (Avena sativa var Cayuse) and corn (Zea mays L. var Patriot) were incubated in different concentrations of calmodulin antagonists in the presence and absence of α-naphthaleneacetic acid. The calmodulin antgonists (chlorpromazine (CP), trifluoperazine, and fluphenazine) inhibited the auxin-induced elongation at 5 to 50 micromolar concentrations. Chlorpromazine sulfoxide, an analog of chlorpromazine, did not have significant effect on the elongation of oat and corn coleoptiles. A specific inhibitor of calmodulin N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide hydrochloride (W-7, a naphthalenesulfonamide derivative) inhibited coleoptile elongation, while its inactive analog N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) was ineffective at similar concentrations. During a 4-hour incubation period, coleoptile segments accumulated significant quantities of ³H-CP. About 85 to 90% of auxin-induced growth was recovered after 4 hours of preincubation with CP or 12 hours with W-7 and transferring coleoptiles to buffer containing NAA. Leakage of amino acids from coleoptiles increased with increasing concentration of CP, showing a rapid and significant increase above 20 micromolar CP. The amount of amino acids released in the presence of W-7 and W-5 was significantly lower than the amount released in the presence of CP. Both W-5 and W-7 increased amino acid release but only W-7 inhibited auxin-induced growth. Calmodulin activity measured by phosphodiesterase activation did not differ significantly between auxin-treated and control coleoptile segments. These results suggest the possible involvement of calmodulin in auxin-induced coleoptile elongation.     

Plasma membrane depolarization and activation of receptors for endogenous vasoconstrictors as possible mechanisms of potentiation of vasoconstrictive response to serotonin in traumatic shock in rats

The goal of this work was to study possible mechanisms underlying the potentiation of vasopressor response to serotonin observed in traumatic shock. Experiments with isolated aorta and mesenteric artery of the rat showed that vasoconstriction is caused by the activation of 5HT2A receptors. Agonists of 5HT1B, 5HT1D, 5HT2B, and 5HT4 receptors induced vasodilation. Agonists of 5HT1A receptors had a dual effect determined by interaction with α₁-adrenergic receptors and 5HT1A receptors. Plasma membrane depolarization with 15 mM KCl increased the vasoconstrictive force in response to serotonin. This effect was determined by the ability of KCl to activate voltage-gated calcium channels, as a result of which the intracellular calcium stores are replenished. Inhibition of the response to serotonin by ketanserin, a 5HT2A receptor blocker, did not depend on the presence of 15 mM KCl. Constriction in response to serotonin was potentiated after its addition to vessels preconstricted with noradrenaline or endothelin-1. The constriction response partially retained in the presence of 2 × 10^?7 M ketanserin, which completely suppressed the serotonin-induced constriction of dilated vessels both at normal membrane potential and after plasma membrane depolarization. It can be assumed that noradrenalin and endothelin-1 alter the characteristics of 5HT2A receptors and possibly 5HT1A receptors as a result of their heterodimerization with the receptors for these vasoconstrictive hormones or receptor-receptor interaction at the level of signaling systems. Along with the potentiating effect of KCl, this mechanism may underlie the enhancement of vasopressor response to serotonin in shock.     

The inhibitors of the 5HT-transporter fluoxetine and clomipramine attenuate serotonin-induced constriction of the aorta and the calcium signal in smooth muscle cells of the rat

We found that the inhibitors of the serotonin (5HT) transporter fluoxetine and clomipramine significantly inhibit 5HT-induced constriction of isolated rings of the aorta. The most prominent inhibitory effect was observed for clomipramine, which at a concentration of 2 μM, prevented aorta constriction in response to low and moderate doses of 5HT and multiply attenuated it in response to high doses (10 μM). The inhibitors of the 5HT-transporter attenuated the strength of norepinephrine-induced aorta constriction by 40–60% and eliminated long-term tonic constriction. Application of clomipramine or fluoxetine on the vessels preliminarily constricted by norepinephrine resulted in 100% relaxation, which was maintained in the presence of the NO-synthase inhibitor L-NAME. The inhibitors of the 5HT-transporter decreased but did not prevent 5HT-induced [Ca²⁺]_cyt increase in smooth muscle cells (SMCs) of the aorta even at high concentrations. Clomipramine and fluoxetine did not affect the vasopressin-induced [Ca²⁺]_cyt increase in SMCs and the strength of constriction of isolated aorta rings. We found that the sensitivity of the rat aorta to the vasoconstrictor effect of 5HT and the role of the 5HT-transporter in regulation of the vascular tone increased with aging.     

Labelling of Rat Brain ß-Adrenoceptors: (3H)CGP-12177 or (125I)Iodocyanopindolol?

Abstract

Binding of (¹²⁵I) iodocyanopindolol (ICYP) and (³H) CGP-12177 to rat brain homogenates was characterized and compared. ICYP was shown to bind to both ß-adrenergic and serotonin1B (5HT_1B) receptors whereas (³H)CGP-12177 only labelled the first ones. The addition of 10 μM serotonin (5HT) prevented ICYP binding to 5HT receptors and under these experimental conditions both ligands labelled a similar total number of ß-adrenoceptors in the different rat brain regions. ICYP displayed a higher affinity for cerebellar (mainly ß₂-subtype) than for cerebral cortex ß-adrenoceptors (mainly ß-subtype) suggesting a subtype selectivity. A multiple displacement binding approach using CGP-20712A, a ß₁-subtype ligand, as competitor revealed a 2.6 fold selectivity of ICYP for the ß₂-adrenoceptor subtype. On the other hand, (³H)CGP-12177 binds only to ß-adrenoceptors and is not subtype selective in the rat brain homogenate. Considering both its high specificity and its lack of subtype selectivity (³H)CGP-12177 seems to be a more suitable ligand than ICYP to non-selectively label ß-adrenoceptors in rat brain.     

Traumatic shock causes elevation of the serotonin 5-HT2B receptor mRNA level in rat aorta

Previously we have shown that at traumatic shock in rats the force of contraction of isolated aorta in response to angiotensin II, vasopressin, endothelin 1, or norepinephrine is decreased. On the contrary, vasoconstriction caused by serotonin is increased. A possible reason of the alterations of neuroendocrine regulation of vascular tone in shock may be a change in the expression of the receptors of these agonists in blood vessels. In the present study, using real-time PCR, we demonstrated that a day after injury the contents of mRNA encoding receptors V1A for vasopressin, ETA for endothelin 1, and AT1 for angiotensin II are not changed in aorta. There was a slight increase of the serotonin 5-HT2A receptor mRNA (36 ± 16%; p = 0.41). The level of the 5-HT2B receptor mRNA in aorta, initially low (2% of the content of the mRNA of receptors 5-HT2A), after the injury increased 15.8 ± 0.3 times (p < 0.01). However, at traumatic shock there was no contraction of aorta in response to 5-HT2B receptor agonist BW723C86, while vasodilation of the isolated aorta preconstricted with norepinephrine in response to BW723C86 was similar to that of the vessel isolated from control rats. The data obtained suggest that the observed 5-HT2B receptor overexpression is not related to the increased serotonin-induced vasoconstriction and might cause other vascular pathological changes at traumatic shock.     

Possible involvement of calmodulin and the cytoskeleton in electrofusion of plant protoplasts

Calmodulin antagonists, trifluoperazine, chlorpromazine, calmidazolium, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), strongly inhibited the electrofusion of barley (Hordeum vulgare L. cv Moor) protoplasts with a marked increase of broken fusion products, after 60 minutes of incubation. W-5, a dechlorinated analog of W-7, was found less effective for the inhibition than W-7. Ethyleneglycol-bis(β- aminoethylether)-N,N′-tetraacetic acid a Ca²⁺ chelator, La³⁺, a surface Ca²⁺ antagonist, and verapamil, a Ca²⁺ channel blocker, also inhibited electrofusion. Dielectrophoresis was inhibited by La³⁺. A microtubule inhibitor, vinblastine, inhibited electrofusion strongly while colchicine, slightly. A microfilament inhibitor, cytochalasin B, promoted fused cells to become spherical while phalloidin did not affect electrofusion.     

Identification of calmodulin in the green alga Mougeotia and its possible function in chloroplast reorientational movement

A soluble protein was isolated from Mougeotia by chloropromazine-sepharose 4 B affinity chromatography. The protein matches the properties of calmodulin in terms of heat stability, Ca²⁺-dependent electrophoretic mobility in sodium-dodecyl-sulfate polyacrylamide gels, and its ability to activate cyclic nucleotide phosphodiesterase in a Ca²⁺-dependent manner. Phytochrome-mediated chloroplast reorientational movement in Mougeotia was inhibited by the calmodulin antagonist trifluoperazine, a hydrophobic compound, or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a hydrophilic compound; 50% inhibition (IC₅₀) of chloroplast movement is caused by 20–50 mol l^-1 trifluoperazine or 100 mol l^-1 W-7. The Ca²⁺-calmodulin may act as an intermediate in the chloroplast reorientational response in Mougeotia governed by phytochrome.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - SDS sodium dodecyl sulfate - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide     

Induction of ornithine decarboxylase in guinea-pig lymphocytes. Synergistic effect of diacylglycerol and calcium

Calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5) and trifluoperazine inhibited ornithine decarboxylase induction in lymphocytes activated with phytohemagglutinin or inophore A23187. W-7, a more potent calmodulin antagonist than W-5, suppressed ornithine decarboxylase induction in a higher extent than did W-5. These results suggest that calmodulin may play an important role in ornithine decarboxylase induction in the activated lymphocytes. However, the extent of ornithine decarboxylase induction was greater in cells pretreated with Clostridium phospholipase C and then incubated with ionophore A23187 than in cells incubated with ionophore A23187 without the pretreatment. Moreover, combined treatment of cells with ionophore A23187 and tumor promotor, phorbol 12-myristate 13-acetate, caused synergistic induction of ornithine decarboxylase activity. These results, taken together, suggest that both activations of Ca2+-activated phospholipid-dependent protein kinase by diacylglycerol and of calmodulin-dependent function resulted from an elevation of cytosolic Ca2+ concentration may operate in the induction of ornithine decarboxylase in the activated lymphocytes.     

Ca2+-mediated activation of ERK in hepatocytes by norepinephrine and prostaglandin F2α: role of calmodulin and src kinases

Background

Previous studies have shown that several agents that stimulate heptahelical G-protein coupled receptors activate the extracellular signal regulated kinases ERK1 (p44^mapk) and ERK2 (p42^mapk) in hepatocytes. The molecular pathways that convey their signals to ERK1/2 are only partially clarified. In the present study we have explored the role of Ca²⁺ and Ca²⁺-dependent steps leading to ERK1/2 activation induced by norepinephrine and prostaglandin (PG)F_2α.

Results

Pretreatment of the cells with the Ca²⁺ chelators BAPTA-AM or EGTA, as well as the Ca²⁺ influx inhibitor gadolinium, resulted in a partial decrease of the ERK response. Furthermore, the calmodulin antagonists W-7, trifluoperazine, and J-8 markedly decreased ERK activation. Pretreatment with KN-93, an inhibitor of the multifunctional Ca²⁺/calmodulin-dependent protein kinase, had no effect on ERK activation. The Src kinase inhibitors PP1 and PP2 partially diminished the ERK responses elicited by both norepinephrine and PGF_2α.

Conclusion

The present data indicate that Ca²⁺ is involved in ERK activation induced by hormones acting on G protein-coupled receptors in hepatocytes, and suggest that calmodulin and Src kinases might play a role in these signaling pathways.     

The involvement of calmodulin in motile activities of fish chromatophores

1. Effects of W-7 and W-5, calmodulin antagonists, on the pigment aggregation within melanophores and coloring response of iridophores were examined in the blue damselfish, Chrysiptera cyanea.2. W-7 was found to antagonize norepinephrine-induced responses of the chromatophores, whereas W-5 had only a slight effect on inhibition of the responses.3. H-7, a specific antagonist of protein kinase C, did not arrest the responses of melanophores and iridophores at all.4. The chromatophores responded normally to norepinephrine in Ca²⁺, Mg²⁺-free saline solution.5. These results indicate that it is a Ca²⁺/calmodulin-regulated enzyme and not protein kinase C that is involved in motile activities of fish chromatophores. Ca²⁺ may be supplied from an intracellular store.     

Dancylcadaverine eliminates calmodulin stimulation of phosphodiesterase

Dansylcadaverine, which structurally resembles the calmodulin antagonists W-7 and W-5, prevented the calmodulin dependent stimulation of 3′:5′-cyclic nucleotide phophodiesterase $\text{in}$$\text{vitro}$. Dansylcadaverine and trifluoperazine sensitized cells to $\text{Pseudomonas}$$\text{aeruginosa}$ exotoxin A in apparently the same way, exept that 40 times higher concentrations of dansylcadaverine than of trifluoperazine was required.     

Calmodulin binding to platelet plasma membranes

Calmodulin copurifies with platelet plasma membranes isolated by glycerol-induced lysis and density gradient centrifugation. These membranes also bind ¹²⁵I-labeled calmodulin in vitro in the presence of Ca²⁺. Binding is largely reduced by replacing Ca²⁺ by Mg²⁺ or by addition of an excess unlabeled calmodulin. The specific component of binding is saturable, with an apparent K_d of 27 nM and a maximum of 15.9 pmol binding sites per mg of membrane protein. This is equivalent to approx. 4100 binding sites per platelet. Binding was inhibited by addition of phenothiazines, a group of calmodulin antagonists. Half-maximal inhibition was attained with approx. 20 μM trifluoperazine or 50 μM chlorpromazine. In contrast, chlorpromazine-sulfoxide which is inactive towards calmodulin, did not affect the binding. Calmodulin binding polypeptides of the plasma membrane were identified by a gel-overlay technique. A major calmodulin-binding component of molecular weight 149 000 was detected. Binding to this band was Ca²⁺-dependent and inhibited by chlorpromazine. The molecular weight of this polypeptide is similar to that of glycoprotein I and also that of the red cell (Ca²⁺ + Mg²⁺)-stimulated ATPase, which is known to bind calmodulin. The possible role of calmodulin in platelet activation is analysed.     

Effects of calcium antagonists and calmodulin inhibitors on angiotensin II-induced prostaglandin productions in the isolated dog renal arteries

Angiotensin II markedly potentiated both PGE2 and PGI2 productions in the isolated dog renal arteries. This angiotensin II-induced response was significantly reduced by the treatments of EGTA and calcium antagonists such as verapamil, nifedipine and 8-(N,N'-diethylamino)-octyl-3,4,5,-trimethoxybenzoate (TMB-8). Calmodulin inhibitors, trifluoperazine and W-7 also inhibited the angiotensin II-induced PG productions while an inactive analogue of W-7, W-5 did not have any effect. The results suggest that angiotensin II may enhance the intracellular Ca2+ level through the influx of extracellular Ca2+ and then, calmodulin activated with Ca2+ will stimulate both PGE2 and PGI2 productions via its activation of phospholipase A2 in the dog renal arteries.     

The role of various calcium and potassium channels in the regulation of somatodendritic serotonin release

We prepared slices from midbrain containing the raphe nuclei and from hippocampus of rats. The brain slices were loaded with [³H]serotonin and superfused in order to measure the release of radioactivity at rest and in response to electrical stimulation. No difference was observed in the resting and stimulated fractional release of tritium in the somatodendritic and axon terminal parts of serotonergic neurons. The selective 5-HT_1A receptor agonist 8-OH-DPAT decreased the electrically induced tritium effux from raphe nuclei slices preloaded with [³H]serotonin, and this inhibition was reversed by 5-HT_1A receptor antagonist (+)WAY-100135. The 5-HT_1B receptor agonist CGS-12066B but not 8-OH-DPAT, inhibited the stimulation-evoked tritium efflux from hippocampal slices after labeling with [³H]serotonin. The electrical stimulation-evoked tritium efflux in raphe nuclei slices incubate with [³H]serotonin was completely external Ca²⁺-dependent, and omega-conotoxin GVIA and Cd²⁺, but not diltiazem, inhibited the tritium overflow. In raphe nuclei slices 4-aminopyridine enhanced the electrical stimulation-induced trititum release in a concentration-dependent manner. The inhibition of tritium efflux by 8-OH-DPAT was abolished with 4-aminopyridine. Glibenclamide or tolbutamide proved to be ineffective. These data indicate that (1) different 5-HT receptor subtypes (5-HT_1A and 5-HT_1B) regulate dendritic and axon terminal 5-HT release; (2) serotonin release from the dendrites may be regulated by the voltage-sensitive N-type Ca²⁺ channels; (3) the 5-HT_1A receptor-mediated inhibition of serotonin release may be due to opening of voltage-sensitive K⁺ channels.     

Positive interaction between leukotriene C4 and histamine and other mediators on vascular tissues

The interaction of leukotriene C₄ (LTC₄) with the contractile activity of histamine (H), serotonin (5HT) and norepinephrine (NE) has been investigated in isolated vascular preparations. Threshold concentration of LTC₄ (5 × 10⁻⁹ M) significantly potentiated the vasoconstricting effect of these compounds on guinea-pig pulmonary artery (GPPA). This phenomenon was long-lasting for H since it was still present 40 min after LTC₄ had been washed. FPL-55712 (10⁻⁵M) counteracted the increased H response on GPPA induced by LTC₄. Potentiation of H activity due to LTC₄ was also observed on guinea-pig thoracic aorta (GPTA) indicating that LTC₄-induced hyperreactivity is not a phenomenon restricted to the pulmonary vascular bed. In the experiments carried out in presence of indomethacin (3 × 10⁻⁶M), LTC₄ still potentiated H-induced vasoconstriction on GPPA, however the time course of the phenomenon was significantly shorter than that observed in absence of the cyclooxygenase inhibitor. The contractile activity of H and NE on guinea-pig portal vein (GPPV) was not potentiated by LTC₄ These results demonstrate that LTC₄ induces hyperreactivity of the arterial vascular tissue to vasoactive compounds and suggest that cysteinyl-leukotrienes may have pathological significance in the hemodynamic changes occurring during anaphylactic reactions. Preliminary experiments carried out on human intralobar pulmonary artery strongly support this hypothesis.     

Calmodulin and protein kinase C antagonists also inhibit the Ca2+-dependent protein protease, calpain I

The calmodulin and C-kinase antagonists melittin, calmidazolium, N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W7), and trifluoperazine (TFP) also inhibit the activity of the human erythrocyte Ca2+-dependent protease, calpain I. W-5, the nonchlorinated derivative of W-7, was ineffective as an inhibitor of calpain I just as it is for calmodulin and protein kinase C. Dose response studies provided the following IC50 values: melittin, 2.6 microM; calmidazolium, 6.2 microM; trifluoperazine, 130 microM; W-7, 251 microM. These IC50 values indicate that the compounds have affinities 10 to 600 fold less for calpain I than for calmodulin; however, the affinities of the inhibitory compounds are comparable for calpain I and protein kinase C. Kinetic analysis indicates that the compounds are competitive inhibitors of calpain I with respect to substrate.