Norepinephrine as a possible transmitter involved in synaptic transmission in frog taste organs and Ca dependence of its release

In order to explore the role of catecholamine and Ca2+ in the synaptic transmission from taste cells to sensory nerve terminals, the effects of various agents added to an artificial solution perfusing the lingual artery on the frog taste nerve responses were examined. The injection of reserpine or guanetidine, which are catecholamine-depleting agents, led to a great reduction of the frog taste nerve responses. The addition of catecholamines to the perfusing solution did not practically enhance the spontaneous impulse discharges, but did recover the response to all the taste stimuli examined. Norepinephrine was most effective and is the most likely candidate for the transmitter. The enhancement of the responses by norepinephrine was suppressed by desipramine, cocaine, or imipramine, which suggests that the enhancement was brought about by incorporation of norepinephrine into taste cells. In a previous paper (Nagahama, S., Y. Kobatake, and K. Kurihara, 1982. J. Gen. Physiol. 80:785), we showed that the responses to the stimuli of one group depended on Ca2+, cGMP, and cAMP added to the perfusing solution and those to the stimuli of another group did not depend on these agents. After the injection or addition of reserpine to the lingual artery, which probably modified injection or addition of reserpine to the lingual artery, which probably modified the permeability of the artery, the responses to the stimuli of the latter group also came to exhibit dependences on these agents, which indicates that the responses to all the taste stimuli have dependences on Ca2+, cGMP, and cAMP.     

Taste transduction mechanism: similar effects of various modifications of gustatory receptors on neural responses to chemical and electrical stimulation in the frog

Responses in the frog glossopharyngeal nerve induced by electrical stimulation of the tongue were compared with those induced by chemical stimuli under various conditions. (a) Anodal stimulation induced much larger responses than cathodal stimulation, and anodal stimulation of the tongue adapted to 5 mM MgCl2 produced much larger responses than stimulation with the tongue adapted to 10 mM NaCl at equal current intensities, as chemical stimulation with MgCl2 produced much larger responses than stimulation with NaCl at equal concentration. (b) The enhansive and suppressive effects of 8-anilino-1-naphthalenesulfonate, NiCl2, and uranyl acetate on the responses to anodal current were similar to those on the responses to chemical stimulation. (c) Anodal stimulation of the tongue adapted to 50 mM CaCl2 resulted in a large response, whereas application of 1 M CaCl2 to the tongue adapted to 50 mM CaCl2 produced only a small response. This, together with theoretical considerations, suggested that the accumulation of salts on the tongue surface is not the cause of the generation of the response to anodal current. (d) Cathodal current suppressed the responses induced by 1 mM CaCl2, 0.3 M ethanol, and distilled water. (e) The addition of EGTA or Ca-channel blockers (CdCl2 and verapamil) to the perfusing solution of the lingual artery reversibly suppressed both the responses to chemical stimulus (NaCl) and to anodal current with 10 mM NaCl. (f) We assume from the results obtained that electrical current from the microvillus membrane of a taste cell to the synaptic area supplied by anodal stimulation or induced by chemical stimulation activates the voltage-dependent Ca channel at the synaptic area.     

Pharmacological control of ciliary activity in the young sea urchin larva: chemical studies on the role of cyclic nucleotides

Distinct peaks in cAMP and cGMP content during early development, partly opposite to each other, may be correlated with the two main phases of gastrulation and ciliary activity. Monoamines increases cAMP formation. A transient or extended decrease follows, presumably reflecting some feedback mechanism. Muscarinic agents and Ca2+ interfere. The developmental variation in cyclic nucleotides may reflect a temporal shift in the role of various signal substances as well as feedback regulation related to Ca2+ influx. The opposite changes in cAMP and cGMP during early gastrulation may reflect a mutual dependency of the two nucleotide cyclases related to changes in Ca2+ influx.     

Essential role of calcium influx in the adrenergic regulation of cAMP and cGMP in rat pinealocytes

The role of Ca2+ in the adrenergic stimulation of pinealocyte cAMP and cGMP was investigated. In this tissue alpha 1-adrenoceptor activation, which by itself is without effect, potentiates beta 1-adrenergic stimulation of cAMP and cGMP 30- to 100-fold. The present results indicate that chelation of extracellular Ca2+ with EGTA or inhibition of Ca2+ influx with inorganic Ca2+ channel blockers (La3+, Co2+, Mn2+) markedly reduces the cyclic nucleotide response to norepinephrine, a mixed alpha 1- and beta-adrenergic agonist, but not to isoproterenol, a beta-adrenergic agonist. In addition, the potentiating effects of alpha 1-adrenergic agonists were mimicked by agents which elevate cytosolic Ca2+, including K+ (EC50 = 2 X 10(-2) M), ouabain (EC50 = 2 X 10(-6) M), ionomycin (EC50 = 3 X 10(-6) M), and A23187 (EC50 = 2 X 10(-6) M); each potentiated the effects of beta-adrenergic stimulation but had no effect alone. Together these results indicate that an alpha 1-adrenoceptor-stimulated Ca2+ influx is essential for norepinephrine to increase pinealocyte cAMP and cGMP.     

Alteration of calcium conductances and outward current by cyclic adenosine monophosphate (cAMP) in neurons ofLimax maximus

Membrane responses to cyclic adenosine monophosphate (cAMP) injections have been studied by means of voltage clamp, Ca-indicator dye, and ion substitution techniques in identified neurons from the abdominal ganglion of Limax maximus. The ventral abdominal giant cell (AGC) displayed a response consisting of a decrease in outward current usually accompanied by a smaller enhancement of voltage-gated Ca2+ influx. Both responses were eliminated by external Cd2+ or Mn2+ and required membrane voltages more positive than -40 mV for expression. The enhanced influx persisted in Ba2+-substituted saline, while the decrease in outward current was blocked. A group of dorsal neurons (RD1-3, LD1) showed a mixed Na-Ca influx induced by cAMP that could be activated over a wide range of membrane potentials (less than -100 to greater than -20 mV). This flux caused a measurable increase in internal Ca2+. The influx was insensitive to Cd2+ and Mn2+ but was reduced by prolonged exposure to Co2+. The relative magnitude of the Na-Ca flux ratio showed considerable variation between specimens. In immature animals the Ca component was absent. The results demonstrated that elevation of intracellular cAMP can cause cell-specific changes of membrane conductance within closely associated neurons.     

Cyclic nucleotide regulation of store-operated Ca2+ influx in airway smooth muscle

Sarcoplasmic reticulum (SR) Ca2+ release and plasma membrane Ca2+ influx are key to intracellular Ca2+ ([Ca2+]i) regulation in airway smooth muscle (ASM). SR Ca2+ depletion triggers influx via store-operated Ca2+ channels (SOCC) for SR replenishment. Several clinically relevant bronchodilators mediate their effect via cyclic nucleotides (cAMP, cGMP). We examined the effect of cyclic nucleotides on SOCC-mediated Ca2+ influx in enzymatically dissociated porcine ASM cells. SR Ca2+ was depleted by 1 microM cyclopiazonic acid in 0 extracellular Ca2+ ([Ca2+]o), nifedipine, and KCl (preventing Ca2+ influx through L-type and SOCC channels). SOCC was then activated by reintroduction of [Ca2+]o and characterized by several techniques. We examined cAMP effects on SOCC by activating SOCC in the presence of 1 microM isoproterenol or 100 microM dibutryl cAMP (cell-permeant cAMP analog), whereas we examined cGMP effects using 1 microM (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO nitric oxide donor) or 100 microM 8-bromoguanosine 3',5'-cyclic monophosphate (cell-permeant cGMP analog). The role of protein kinases A and G was examined by preexposure to 100 nM KT-5720 and 500 nM KT-5823, respectively. SOCC-mediated Ca2+ influx was dependent on the extent of SR Ca2+ depletion, sensitive to Ni2+ and La3+, but not inhibitors of voltage-gated influx channels. cAMP as well as cGMP potently inhibited Ca2+ influx, predominantly via their respective protein kinases. Additionally, cAMP cross-activation of protein kinase G contributed to SOCC inhibition. These data demonstrate that a Ni2+/La3+-sensitive Ca2+ influx in ASM triggered by SR Ca2+ depletion is inhibited by cAMP and cGMP via a protein kinase mechanism. Such inhibition may play a role in the bronchodilatory response of ASM to clinically relevant drugs (e.g., beta-agonists vs. nitric oxide).     

Control of blastema cell proliferation by possible interplay of calcium and cyclic nucleotides during newt limb regeneration

The effects of the divalent cation ionophore A23187, papaverine, and chlorpromazine on the mitotic index and cyclic nucleotide levels in newt limb regeneration blastemata (Notophthalmus viridescens) were assessed. The results of the experiments suggest that an intracellular increase in divalent cation (Ca2+) concentration results in elevated cGMP levels, suppressed cAMP levels, and a corresponding increase in blastema cell proliferation. The results also suggest that the converse conditions, namely, calcium efflux or inhibition of calmodulin activation (i.e., inhibition of Ca2+ binding), yields elevated cAMP levels, suppressed cGMP levels, and a corresponding decrease in blastema cell divisions.     

Pretreatment with muscarinic receptor agonist activates a calcium-inhibitable adenylate cyclase in GH3 pituitary cells

We have demonstrated previously that pretreatment of GH3 pituitary cells with muscarinic agonists may induce a higher cAMP formation in response to vasoactive intestinal peptide (VIP) or forskolin. In the present study, we further examined the adenylate cyclase (AC) that may be involved. We found that carbachol-pretreatment enhanced both VIP- and forskolin-activated AC activities. The addition of calcium ions to the incubation buffer diminished this enhancing effect. Carbachol was found to induce a decrease in intracellular calcium concentration [Ca2+]i by inhibiting calcium influx through L-type Ca2+ channels. However, the incubation of cells in Ca(2+)-free buffer or in the presence of L-type Ca2+ channel blockers had no influence on forskolin-stimulated cAMP formation, although both treatments induced decreases in [Ca2+]i as carbachol did. On the other hand, incubation in the presence of LaCl3 at a low concentration not being able to enter cells, forskolin-stimulated cAMP formation as well as the enhancing effect of carbachol-pretreatment on this response, were both suppressed. Similar phenomena were observed when membrane-bound AC activities were measured in the presence of LaCl3. Taken together, these results seem to suggest that pretreatment of GH3 cells with muscarinic receptor agonist may activate a Ca(2+)-inhibitable AC for a higher stimulated response. Low intracellular calcium concentrations are essential but not sufficient for this effect.     

Carbachol and bradykinin elevate cyclic AMP and rapidly deplete ATP in cultured rat sympathetic neurons.

The agonists carbachol (CCh) and bradykinin (BK) and 54 mM KCl (high K+) were among the most potent stimulants of cyclic AMP (cAMP) production in cultured rat sympathetic neurons, measured with the use of a high-fidelity assay developed for small samples. The rise in cAMP evoked by CCh (through muscarinic receptors), BK, and high K+ was inhibited in Ca2(+)-depleted medium (1.3 mM Ca2+ and 2 mM BAPTA or EGTA), which also prevented the sustained rise in [Ca2+]i evoked by each of these stimuli, showing that elevation of cAMP requires extracellular Ca2+ and, possibly, Ca2+ influx. Preliminary results obtained with the novel calmodulin inhibitor CGS 9343B, which blocked the elevation of cAMP, and with the cyclogenase inhibitor indomethacin, which partially blocked the actions of the agonists but not those of high K+, suggest that calmodulin and arachidonate metabolites may be two components of the signaling pathway. In addition to their effects on cAMP metabolism, CCh, muscarine, and BK, but not nicotine, caused a 30-40% decrease in ATP levels. This effect was much greater than that evoked by high K+ and was largely inhibited by CGS 9343B but slightly enhanced in the Ca(+)-depleted medium, showing that agonists are still active in the absence of [Ca2+]o. Thus, agonists that activate phosphoinositide metabolism can also increase cAMP production and substantially deplete cells of ATP. These novel actions may have to be taken into account when the mechanisms by which such agonists regulate cell function are being considered.     

Is serotonin a chemical transmitter in the frog taste organ?

By artificially perfusing the frog tongue with serotonin (5HT) and its antagonists, the possibility of 5HT as a chemical transmitter from taste cells to nerve terminals in frog taste organ was examined. Although serotonin creatinine sulfate, when perfused through the lingual artery, produced impulse discharges in the glossopharyngeal nerve, creatinine sulfate elicited a similar response. Neural responses to taste stimuli were depressed by perfusion with 5HT. Among many antiserotonergic drugs perfused through the lingual artery, LSD was the only one which modified responses to taste stimuli. LSD suppressed taste responses to NaCl, CaCl₂ and water, while LSD at a high concentration (10^?5 g/ml) enhanced responses to guinine and HCl. When PCPA (DL-p-chlorophenylalanine) was injected intraperitoneally in conbination with reserpine, the agent did not significantly change taste responses. The above results possibly suggest that 5HT would not be a chemical mediator from taste cells to nerve terminals.     

Acute effects of adenosine triphosphates, cyclic 3',5'-adenosine monophosphates, and follicle-stimulating hormone on cytosolic calcium level in cultured immature rat Ssertoli cells.

The ability of ATP and FSH to induce intracellular calcium [Ca(2+)](i) changes in Sertoli cells is imperfectly understood and reports are conflicting. We have applied the single-cell microfluorometry technique with the calcium probe indo-1 to investigate [Ca(2+)](i) in individual cultured Sertoli cells. When cells were exposed to ATP, cAMP, and FSH, a fast and biphasic increase in [Ca(2+)](i) was obtained in 100%, 70%, and 56% of cells, respectively. Caffeine did not activate Ca(2+) mobilization, while thapsigargin suppressed the peak response. External calcium free-EGTA buffer suppressed the plateau phase, while blockers of voltage-operated Ca(2+) channels did not abolish the response to cAMP and ATP. We conclude that the three messengers mobilized Ca(2+) from intracellular thapsigargin-sensitive stores, which induced a subsequent Ca(2+) influx from the extracellular medium by a voltage-independent Ca(2+) entry. The well-documented mechanisms by which these messengers act on cells support the idea that they release Ca(2+) from smooth endoplasmic reticulum by two different pathways, or that FSH and cAMP first release ATP, which then acts on cells. Among the cells, 77% and 80% responded, respectively, to FSH and cAMP by a delayed long-lasting decrease in [Ca(2+)](i) that was never recorded in the presence of ATP. This suggests that FSH and cAMP also promote a slow redistribution of [Ca(2+)](i) from the exchangeable pool to the bound nonexchangeable pools. Involvement of voltage-operated and voltage-independent calcium channels in the response of Sertoli cells to ATP, FSH, and cAMP is discussed.     

Protein kinase C: subcellular redistribution by increased Ca2+ influx. Evidence that Ca2+-dependent subcellular redistribution of protein kinase C is involved in potentiation of beta-adrenergic stimulation of pineal cAMP and cGMP by K+ and A23187

Phenylephrine is known to stimulate translocation of protein kinase C in rat pinealocytes (Sugden, D., Vanecek, J., Klein, D.C., Thomas, T.P., and Anderson, W. B. (1985) Nature 314, 359-361). In the present study, the receptor mediating this effect was found to belong to the alpha 1-adrenoceptor subclass. Activation of this receptor is also known to produce a sustained increase in [Ca2+]i by increasing net influx (Sugden, A. L., Sugden, D., and Klein, D. C. (1985) J. Biol. Chem. 261, 11608-11612), which points to the possible importance of Ca2+ influx in the subcellular redistribution (activation) of protein kinase C in intact cells. This possibility was investigated by reducing extracellular Ca2+ ((Ca2+]o) with EGTA or by inhibiting Ca2+ influx with inorganic Ca2+ blockers. These treatments reduced alpha 1-adrenoceptor-mediated translocation of protein kinase C. This suggested that elevation of Ca2+ influx alone triggers activation of protein kinase C. In support of this, it was found that treatments which elevate Ca2+ influx, including increased extracellular K+ and addition of the Ca2+ ionophore A23187, cause redistribution of protein kinase C. The effect of K+ was blocked by nifedipine and that of A23187 by EGTA, indicating that effects of these agents are Ca2+-dependent. The possible role of phospholipase C activation in these effects was examined by measuring the formation of [3H]diacylglycerol by cells labeled with [3H]arachidonic acid. Although [3H]diacylglycerol formation was easily detected in the presence or absence of an effective concentration of an inhibitor of diacylglycerol kinase, none of the agents which cause rapid translocation of protein kinase C were found to cause a rapid increase in the generation of [3H]diacylglycerol. These findings establish that an increase in Ca2+ influx is sufficient to trigger translocation of protein kinase C. In addition, we found that a very close correlation exists between translocation of protein kinase C by phenylephrine, K+, and A23187 and their ability to potentiate beta-adrenergic stimulation of cAMP and cGMP accumulation. This provides strong support to the proposal that translocation of protein kinase C is required for potentiation of beta-adrenergic stimulation of pinealocyte cAMP and cGMP accumulation.     

Effect of cyclic nucleotides on calcium dependent K+ channels, in human erythrocytes

K+ efflux has been analyzed in human erythrocytes incubated in a K+ free medium containing ouabain, bumetanide, CaCl2, and the Ca2+ ionophore A23187. In these conditions, a K+ efflux, which is exponentially dependent on the concentration of A23187 present in the medium, has been observed. This flux is almost completely abolished by either quinine or EGTA, so that, the above K+ efflux has been considered Ca2+ dependent. The effects of cAMP, and cGMP, have been tested on this flux. Ca2+ dependent K+ efflux decreases in presence of millimolar concentrations of cAMP in the medium. The addition of methyl-isobutyl-xanthine to the incubation medium containing cAMP enhances the inhibitory effect of this compound. cGMP also inhibits the Ca2+ dependent K+ efflux. Our results suggest that cyclic nucleotides may modulate the activation of Ca2+ dependent K+ channels in human erythrocytes.     

Synthesis of prostanoids and cyclic nucleotides by phagocytosing rat Kupffer cells

Rat Kupffer cells in monolayer culture were allowed to phagocytose unopsonized zymosan granules. They responded with a strongly stimulated synthesis and release of prostanoids, mainly the immunologically determined prostaglandins PGE2 and PGF2 alpha. The same response could be obtained by treatment with the calcium ionophore A23187. The effects of the ionophore and the zymosan particles were of the same magnitude but not additive. The rapid uptake of Ca2+ after contact with phagocytosable material recently described by us [(1983) Eur. J. Biochem. 131, 539-543] appears to mediate the enhanced prostaglandin synthesis. That response was suppressed not only by indomethacin but also by trifluoperazine which does not inhibit Ca2+ entry in the Kupffer cells. Similar effects by R24571 and 4-bromophenacyl bromide support the participation of calcium-calmodulin and of phospholipase A2. The calcium channel blocker Verapamil did not influence the zymosan-provoked production of prostaglandin PGE2 nor were any indications obtained for a feedback inhibition by PGE1 or PGE2. Contact with zymosan resulted in a rapid but transient rise of the intracellular levels of cAMP and cGMP: 10 nM indomethacin completely blocked the increase of both cyclic nucleotides while trifluoperazine elicited different responses in the cAMP and cGMP levels. The stimulated release of prostaglandin E2 was inhibited in a dose-dependent manner by nordihydroguaiaretic acid, an inhibitor of 5-lipoxygenase and by FPL 55712, known as a receptor antagonist for some leukotrienes. This suggests a regulatory role for its metabolites on prostaglandin synthesis.     

Ca2+ influx into lily pollen grains through a hyperpolarization-activated Ca2+-permeable channel which can be regulated by extracellular CaM

Confocal laser scanning microscopy (CLSM) and whole-cell patch-clamp were used to investigate the role of Ca2+ influx in maintaining the cytosolic Ca2+ concentration ([Ca2+]c) and the features of the Ca2+ influx pathway in germinating pollen grains of Lilium davidii D. [Ca2+]c decreased when Ca2+ influx was inhibited by EGTA or Ca2+ channel blockers. A hyperpolarization-activated Ca2+-permeable channel, which can be suppressed by trivalent cations, verapamil, nifedipine or diltiazem, was identified on the plasma membrane of pollen protoplasts with whole-cell patch-clamp recording. Calmodulin (CaM) antiserum and W7-agarose, both of which are cell-impermeable CaM antagonists, lead to a [Ca2+]c decrease, while exogenous purified CaM triggers a transient increase of [Ca2+]c and also remarkably activated the hyperpolarization-activated Ca2+ conductance on plasma membrane of pollen protoplasts in a dose-dependent manner. Both the increase of [Ca2+]c and the activation of Ca2+ conductance which were induced by exogenous CaM were inhibited by EGTA or Ca2+ channel blockers. This primary evidence showed the presence of a voltage-dependent Ca2+-permeable channel, whose activity may be regulated by extracellular CaM, in pollen cells.     

A rapid calcium influx during exocytosis in Paramecium cells is followed by a rise in cyclic GMP within 1 s.

The synchrony of trichocyst exocytosis in Paramecium allows temporal correlation of associated events. Using quenched flow we observed a Ca2+ influx concurrent with exocytosis within 80 ms after stimulation with the secretagogue aminoethyldextran. Cyclic AMP did not change in depency of stimulation. Cyclic GMP transiently increased after 500 ms, culminating at 2 s, and thus considerably lags behind exocytosis induction and influx of Ca2+. Both Ca2+ influx and rise in cGMP are known to be induceable also by Ba2+ or veratridine, allegedly via the opening of ciliary Ca2+ channels. However, only veratridine stimulated exocytosis. We conclude that both aminoethyldextran and veratridine induce an exocytosis-associated Ca2+ influx, which is responsible for the rise in cGMP, through an as yet unknown pathway.     

The identification and characterization of two cyclic nucleotide phosphodiesterases from bovine adrenal medulla

Two soluble cyclic nucleotide phosphodiesterase activities, designated Peak I (Mr = 216,000) and Peak II (Mr = 230,000), have been isolated from bovine adrenal medulla by DEAE-cellulose chromatography. Peak I has Ca2+-independent, cGMP-specific phosphodiesterase activity and Peak II has cGMP-stimulated cyclic nucleotide phosphodiesterase activity. Peak I hydrolyzes cGMP with hyperbolic kinetics and demonstrates a Km of 23 microM. Peak II hydrolyzes cGMP with hyperbolic kinetics but hydrolyzes cAMP with slightly sigmoidal kinetics and demonstrates Km values of 54 +/- 0.7 microM cGMP and 38 +/- 6 microM cAMP. Cyclic AMP and cGMP are competitive inhibitors of each other's hydrolysis, suggesting that these nucleotides may be hydrolyzed at the same catalytic site. Micromolar concentrations of cGMP cause a 5-fold stimulation of the hydrolysis of subsaturating concentrations of cAMP by the Peak II phosphodiesterase. Half-maximal activation occurs at 0.5 microM cGMP and the result of activation is a decrease in the apparent Km for cAMP. Stimulation of the hydrolysis of subsaturating concentrations of cGMP by cAMP was also detected; however, cAMP is a less potent activator of the enzyme than cGMP. Cyclic AMP causes a 1.5-fold stimulation of cGMP hydrolysis and half-maximal activation occurs at 2.5 microM cAMP.     

Effects of oxytocin and methacholine on cyclic nucleotide levels of rabbit myometrium

The effects of oxytocin and methacholine on cyclic nucleotide levels in estrogen-primed rabbit myometrium were studied in the presence and absence of 1-methyl-3-isobutyl xanthine (MIX), a phosphodiesterase inhibitor. In the absence of MIX, methacholine increased guanosine 3',5'-cyclic monophosphate (cGMP) levels at a time when contraction was decreasing, but had no influence on adenosine 3',5'-cyclic monophosphate (cAMP) levels. In contrast, oxytocin did not elevate cGMP, but rapidly decreased cAMP levels. MIX (1 mM) increased both cAMP and cGMP levels. Oxytocin or methacholine further increased cGMP, indicating activation of guanylate cyclase. Oxytocin- but not methacholine-induced stimulation of guanylate cyclase was abolished in Ca2+-free solution. Oxytocin increased cAMP over the levels produced by MIX alone, whereas methacholine decreased cAMP below the MIX control values; these effects were insensitive to indomethacin. Tissue levels of cGMP and cAMP did not directly correlate with isometric tension. The results also indicate that both oxytocin and methacholine stimulate guanylate cyclase but have opposing effects on adenylate cyclase of rabbit myometrium.     

Nitroprusside differentially inhibits ADP-stimulated calcium influx and mobilization in human platelets.

1. The effect of nitroprusside on cGMP concn., cAMP concn., shape change, aggregation, intracellular free Ca2+ concn. (by quin-2 fluorescence) and Mn2+ entry (by quenching of quin-2) was investigated in human platelets incubated with 1 mM-Ca2+ or 1 mM-EGTA. 2. Nitroprusside (10 nM-10 microM) caused similar concentration-dependent increases in platelet cGMP concn. and was without effect on cAMP concn. in the presence of extracellular Ca2+ or EGTA. 3. In ADP (3-6 microM)-stimulated platelets, nitroprusside caused 50% inhibition of shape change at 0.4 microM (+Ca2+) or 1.3 microM (+EGTA), aggregation at 0.09 microM (+Ca2+) and of increased intracellular Ca2+ at 0.02 microM (+Ca2+) or 2.1 microM (+EGTA). Entry of 1 mM-Mn2+ (-Ca2+) was inhibited by 80% by 5 microM-nitroprusside. 4. In ionomycin (20-500 nM)-stimulated platelets, nitroprusside (10 nM-100 microM) did not inhibit shape change or intracellular-Ca2+-increase responses, and only partially inhibited aggregation. 5. In phorbol myristate acetate (10 nM)-stimulated platelets, neither shape change nor aggregation was inhibited by 5 microM-nitroprusside. 6. The data demonstrate that nitroprusside inhibits ADP-mediated Ca2+ influx more potently than Ca2+ mobilization. Nitroprusside appears not to influence Ca2+ efflux or sequestration and not to affect the sensitivity of the activation mechanism to intracellular Ca2+ concn. or activation of protein kinase C.     

Endogenously released ATP mediates shear stress-induced Ca2+ influx into pulmonary artery endothelial cells

The mechanisms by which flow-imposed shear stress elevates intracellular Ca2+ in cultured endothelial cells (ECs) are not fully understood. Here we report finding that endogenously released ATP contributes to shear stress-induced Ca2+ responses. Application of flow of Hanks' balanced solution to human pulmonary artery ECs (HPAECs) elicited shear stress-dependent increases in Ca2+ concentrations. Chelation of extracellular Ca2+ with EGTA completely abolished the Ca2+ responses, whereas the phospholipase C inhibitor U-73122 or the Ca2+-ATPase inhibitor thapsigargin had no effect, which thereby indicates that the response was due to the influx of extracellular Ca2+. The Ca2+ influx was significantly suppressed by apyrase, which degrades ATP, or antisense oligonucleotide targeted to P2X4 purinoceptors. A luciferase luminometric assay showed that shear stress induced dose-dependent release of ATP. When the ATP release was inhibited by the ATP synthase inhibitors angiostatin or oligomycin, the Ca2+ influx was markedly suppressed but was restored by removal of these inhibitors or addition of extracellular ATP. These results suggest that shear stress stimulates HPAECs to release ATP, which activates Ca2+ influx via P2X4 receptors.