Endothelium-derived relaxing factor inhibits thrombin-induced platelet aggregation by inhibiting platelet phospholipase C.

Endothelium-derived relaxing factor (EDRF) inhibits platelet function, but the mechanism underlying this inhibitory effect is not known. To examine this, cultured acetylsalicylic acid (ASA)-treated endothelial cells (EC) from bovine aorta (BAEC) or from human umbilical vein (HUVEC) were incubated with washed, ASA-treated human platelets. Incubation of platelets with either BAEC or HUVEC resulted in inhibition of thrombin-induced platelet aggregation that was dependent on the number of EC added. This effect was potentiated by superoxide dismutase and reversed by treating EC with NG-nitro-L-arginine or by treating platelets with methylene blue, indicating that the inhibition of platelet aggregation was due to the release of EDRF by EC. EC significantly blocked the thrombin stimulated breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) and the production of phosphatidic acid in [32P]orthophosphate-labeled platelets and of inositol trisphosphate in [3H]myoinositol-labeled platelets. In addition, the thrombin-mediated activation of protein kinase C (PKC) and phosphorylation of myosin light chain were inhibited in the presence of EC. Finally, thrombin stimulated an increase in cytosolic ionized calcium concentration ([Ca2+]i) in fura2-loaded platelets that was abolished by concentrations of EC which also blocked thrombin-induced aggregation. These data indicate that EDRF blocks thrombin-induced platelet aggregation by inhibiting the activation of PIP2-specific phospholipase C and thereby suppressing the consequent activation of PKC and the mobilization of [Ca2+]i.     

Alpha 1-adrenergic potentiation of vasoactive intestinal peptide stimulation of rat pinealocyte adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate: evidence for a role of calcium and protein kinase-C

alpha 1-Adrenergic agonists have recently been found to potentiate vasoactive intestinal peptide (VIP) stimulation of rat pinealocyte cAMP and cGMP. alpha 1-Adrenergic agonists also elevate pineal intracellular Ca2+ [( Ca2+]i) and activate protein kinase-C. In the present study, the possible involvement of Ca2+ and protein kinase-C in the alpha 1-adrenergic potentiation of VIP-stimulated cAMP and cGMP accumulation was examined with agents that alter [Ca2+]i or activate protein kinase-C. It was found that treatment with a Ca2+ chelator or with inorganic Ca2+ channel blockers inhibited alpha 1-adrenergic potentiation of VIP-stimulated cAMP and cGMP responses. Increasing [Ca2+]i by treatment with A23187, ouabain, or K+ potentiated VIP stimulation of cAMP and cGMP response. These observations indicate that Ca2+ mediates the alpha 1-adrenergic potentiation of VIP-stimulated cAMP and cGMP accumulation, as is true for the alpha 1-adrenergic potentiation of beta-adrenergic stimulated cAMP and cGMP accumulation. Activators of protein kinase-C mimicked the large effect alpha 1-adrenergic agonists have on cAMP accumulation in VIP-treated pinealocytes and had a small effect on cGMP accumulation in VIP-treated cells. These effects were not blocked by the Ca2+ chelator EGTA. However, the effects of a protein kinase-C activator on the cGMP response in VIP-stimulated cells were amplified by K+ (15 mM) or ouabain (1 microM), presumably through an action causing an increase in [Ca2+]i. These results suggest protein kinase-C is involved in the alpha 1-adrenergic potentiation of VIP-stimulated cAMP accumulation, as is the case for the alpha 1-adrenergic potentiation of beta-adrenergic stimulated cAMP. Protein kinase-C is also involved in cGMP accumulation, provided that there is a modest increase in [Ca2+]i.     

Glycoprotein IIb-IIIa complex and Ca2+ influx into stimulated platelets

Changes in intracellular Ca2+ concentrations [( Ca2+]i) in platelets stimulated with aggregating agents were measured with the fluorescent indicator dye quin 2. Ca2+ influx, but not intracellular mobilization, in response to adenosine diphosphate (ADP), platelet aggregating factor (PAF-acether), and sodium arachidonate was significantly inhibited by monoclonal antibodies against the glycoprotein (GP) IIb-IIIa complex; inhibition of thrombin-stimulated influx was inhibited to a lesser extent and reached statistical significance only at thrombin concentrations of 0.1 U/mL and below. Anti-GP Ib and HLA-ABC monoclonal antibodies had no effect on Ca2+ influx in response to any agonist. Thrombasthenic platelets gave normal [Ca2+]i responses to ADP and thrombin, which were not inhibited by an anti-GP IIb-IIIa antibody. It is suggested that Ca2+ influx in response to weak agonists occurs predominantly via a channel closely adjacent to the GP IIb-IIIa complex, but that higher concentrations of thrombin and A23187 also stimulate influx via another pathway.     

Platelet cAMP and cGMP in essential hypertension.

Vasodilator substances released in the blood vessel wall, such as the endothelium-derived relaxing factor (EDRF) and prostacyclin (PGI2), may participate in the regulation of arterial blood pressure. However, their role in the pathogenesis of human essential hypertension to date remains unclear. For some of these factors affecting vascular smooth muscle cells, blood platelets represent a second target tissue. Thus, EDRF and PGI2 inactivate platelets by stimulation of cyclic guanosine-5'-monophosphate (cGMP) and cyclic adenosine-5'-monophosphate (cAMP) synthesis, respectively. In the present study, platelet cAMP (n = 68) and cGMP (n = 60) were determined in a control group of healthy subjects (C) and in 12 patients with untreated essential hypertension (EH). In the control group, platelet cAMP and cGMP content averaged 13.52 +/- 0.38 and 1.48 +/- 0.06 pmol/10(9) platelets and no dependence of either variable on sex or age could be established. Furthermore, cGMP levels were similar in EH as compared to the control group (1.38 +/- 0.11 pmol/10(9) platelets). However, intracellular concentrations of cAMP were significantly lower in EH as compared to C (11.22 +/- 1.37 pmol/10(9) platelets; P < .01). In addition, we investigated the stimulatory effect on cAMP of the stable PGI2 analog iloprost (10(-9), 5 x 10(-9), 10(-8), 5 x 10(-8) mol/L) in the platelets of 12 control subjects (C12) and EH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of endothelin on human platelet aggregation and prostacyclin generation from human vascular endothelial cells in culture.

The effects of endothelin (ET) on the function of cultured human umbilical vein endothelial cells (HUVEC) and that of human platelets were investigated with reference to endothelium-derived relaxing factor (EDRF) and PGI2. Considering the platelets, ET had no effect on platelet-rich plasma (PRP) aggregation, the generation of thromboxane A2 ([TXA2]) from platelets, and cytosolic free calcium ion concentration ([Ca++]i), cAMP content ([cAMP]i) or cGMP content ([cGMP]i) in platelets. In contrast, the addition of the solution in which HUVEC had been incubated with ET to PRP produced a decrease in PRP aggregation, [TXA2], and [Ca++]i, and an increase not only in [cAMP]i but also in [cGMP]i in platelets. In the HUVEC pretreated with acetylsalicylic acid (aspirin), this increase of [cGMP]i was not affected, but the HUVEC-mediated decrease in PRP aggregation, [TXA2], and [Ca++]i induced by ET were not completely abolished. However, the pretreatment of HUVEC with a combination of aspirin and L-NG-monomethyl arginine (LNMMA) as an inhibitor of EDRF completely abolished the HUVEC-mediated decrease in PRP aggregation, [TXA2] and [Ca++]i induced by ET, and also abolished the enhancement of [cGMP]i and [cAMP]i in platelets. The PGI2 of HUVEC was enhanced by ET with no changes in [Ca++]i, [cAMP]i and [cGMP]i. The ET-induced enhancement was remarkably attenuated by pretreating the HUVEC with aspirin, but not with LNMMA. We conclude that ET attenuates the aggregation of platelets through a decrease in [TXA2] by an increase in [cAMP]i via the increase in PGI2 of HUVEC, and by an increase in [cGMP]i via EDRF.     

Characterization of prostaglandin and thromboxane receptors expressed on a megakaryoblastic leukemia cell line, MEG-01s.

MEG-01s, an established human megakaryoblastic leukemia cell line, exhibited specific high-affinity binding sites for [3H]iloprost, a stable prostaglandin (PG) I2 analogue, for [3H]SQ-29548, a stable thromboxane (TX) A2 antagonist and, for [3H]PGE2/PGE1, but not for [3H]PGD2. In the MEG-01s cells, iloprost/PGI2, or PGE1 stimulated cAMP production with ED50 values practically identical to the IC50 values for the [3H] iloprost binding. STA2 and U46619, TXA2/PGH2 agonists, PGE2/PGE1, iloprost/PGI2, and thrombin elevated the intracellular concentrations of Ca2+ ([Ca2+]i), as determined by Fura-2 fluorescence signals. Elevation of [Ca2+]i by PGE2/PGE1 and iloprost, but not that by TX-agonists or thrombin, was totally dependent on the presence of extracellular Ca2+. This effect by PGE2/PGE1 was partially inhibited by prior treatment of the cells with islet-activating protein (IAP), while that by TX-agonists or by PGI2/iloprost was not affected. We tentatively conclude from these results that: (1) MEG-01s cells express (a) PGI2/PGE1 receptor(s) coupled to adenylate cyclase and Ca2+ influx, a TXA2/PGH2 receptor coupled to the phosphatidylinositol-turnover-Ca2+ system, and the PGE2/PGE1 receptor coupled to Ca2+ influx; (2) the receptors for TXA2/PGH2 and iloprost and those for PGE2/PGE1 and thrombin are coupled to IAP-insensitive and IAP-sensitive GTP-binding proteins, respectively, and function in a different manner to elevate [Ca2+]i. Thus, the MEG-01s cell line is a pertinent model for studying eicosanoid receptor-mediated signal transduction in platelet/megakaryocyte systems.     

Inhibition of PAR4 signaling mediates ethanol-induced attenuation of platelet function in vitro

BACKGROUND: Reduction in coronary heart disease morbidity in response to moderate consumption of alcoholic beverages may be partly mediated by ethanol-induced inhibition of platelet function. However, the precise mechanisms by which ethanol modulates platelet activation induced by thrombin, which plays a central role in hemostasis, remain unclear. The goal of this study was to investigate ethanol-induced changes in platelet function and clarify the underlying mechanisms including PAR1 and PAR4 activity and [Ca2+]i dynamics in vitro. METHODS: Platelet aggregation, increase in intracellular calcium ([Ca2+]i), and release of platelet factor 4 and beta-thromboglobulin induced by alpha-thrombin, PAR1-agonist peptide (AP), or PAR4-AP were assessed in the presence or absence of ethanol. RESULTS: Ethanol exposure inhibited low-dose thrombin (0.5 nM)-induced aggregation but not an increase in [Ca2+]i. In contrast, ethanol had no effect on high-dose thrombin (10 nM)-induced aggregation or the [Ca2+]i increase. Ethanol did not significantly inhibit thrombin-induced release of platelet factor 4 and beta-thromboglobulin. Ethanol reduced PAR1-AP-induced aggregation, but did not affect the spike form of [Ca2+]i increase. In contrast, ethanol inhibited the increase in [Ca2+]i as well as the aggregation in response to PAR4-AP and resulted in delayed [Ca2+]i peak time. Furthermore, ethanol inhibited both PAR1-AP- and PAR4-AP-induced platelet factor 4 and beta-thromboglobulin release. CONCLUSIONS: These data suggest that ethanol inhibits platelet aggregation via inhibition of PAR4 signaling and subsequent inhibition of Ca2+ influx and granule release. This phenomenon may contribute to the reduction in coronary heart disease morbidity in response to consumption of alcoholic beverages.     

Inhibitory action of ethanol on L-type Ca2+ channels and Ca(2+)-dependent guanosine 3',5'-monophosphate accumulation in rat pinealocytes.

It has previously been shown that the K+ potentiation of vasoactive intestinal peptide-stimulated cAMP and cGMP responses was inhibited by ethanol in rat pinealocytes, suggesting an inhibitory action of ethanol on the voltage-dependent Ca2+ channels (VDCC). In this study, using the whole cell version of the patch clamp technique, we found that ethanol reduced the amplitude, but did not change the voltage dependence or the time course of activation or inactivation of the L-type VDCC. The inhibitory effect of ethanol on this current was concentration dependent, and ethanol (100 mM) resulted in a 40% inhibition of this current. However, in fura-2-loaded cells, total increases in intracellular Ca2+ ([Ca2+]i) caused by ethanol and BayK 8644 did not differ from the [Ca2+]i signal caused by BayK 8644 alone, suggesting that the inhibitory action of ethanol on VDCC may not be related to a reduction in [Ca2+]i. Although there was no change in the total [Ca2+]i signal, ethanol (25-200 mM) dose-dependently inhibited the potentiation effects of depolarizing concentrations of K+ and BayK 8644 on the isoproterenol-stimulated cGMP, but not the cAMP, response. Therefore, the cGMP response appears to be more sensitive to the inhibitory action of ethanol, and a site distal to elevation of [Ca2+]i of importance to the potentiation mechanism may be inhibited by ethanol. This was confirmed by the finding that ethanol was effective in inhibiting the A23187 potentiation of isoproterenol-stimulated cGMP response. These results suggest that 1) the L-type VDCC was inhibited by ethanol; 2) the Ca(2+)-mediated potentiation of the isoproterenol-stimulated cGMP response was sensitive to the inhibitory action of ethanol; and 3) although ethanol inhibits the VDCC, it alone cannot explain the inhibitory effect of ethanol on BayK 8644- and K(+)-mediated potentiation of the isoproterenol-stimulated cGMP response.     

Differential requirements for platelet aggregation and inhibition of adenylate cyclase by epinephrine. Studies of a familial platelet alpha 2-adrenergic receptor defect

We describe a family whose members have impaired platelet aggregation and secretion responses to epinephrine with normal responses to adenosine diphosphate and collagen. Platelet alpha 2-adrenergic receptors (measured using 3H methyl-yohimbine) were diminished in the propositus (78 sites per platelet), his two sisters (70 and 27 sites per platelet), and parents (37 and 63 sites per platelet), but not in two maternal aunts (12 normal subjects, 214 +/- 18 sites per platelet; mean +/- SE). However, the inhibition of cyclic adenosine monophosphate (cAMP) levels by epinephrine in platelets exposed to 400 nmol/L PGI2 was similar in the patients and five normal subjects (epinephrine concentration for 50% inhibition, 0.04 +/- 0.01 mumol/L v 0.03 +/- 0.01 mumol/L; P greater than .05). In normal platelets, the concentration of yohimbine (0.18 mumol/L) required for half maximal inhibition of aggregation induced by 2 mumol/L epinephrine was lower than that for inhibition of its effect on adenylate cyclase (1.6 mumol/L). In quin2 loaded platelets, thrombin (0.1 U/mL) stimulated rise in cytoplasmic Ca2+ concentration, [Ca2+]i, was normal in the two patients studied. The PGI2 analog ZK 36,374 completely inhibited thrombin-induced rise in [Ca2+]i; the reversal of this inhibition by epinephrine was normal in the two patients. Thus, despite the impaired aggregation response to epinephrine, platelets from these patients have normal ability to inhibit PGI2-stimulated cAMP levels. These patients with an inherited receptor defect provide evidence that fewer platelet alpha 2-adrenergic receptors are required for epinephrine-induced inhibition of adenylate cyclase than for aggregation.     

Involvement of cyclic GMP in nitric-oxide-induced gastric relaxation Comparison of the actions of cyclic GMP and cyclic AMP

BACKGROUND: Smooth muscle relaxation induced by various agents that increase the cellular levels of cyclic nucleotides (cAMP and cGMP) is accompanied by a decrease in intracellular Ca2+ concentration. However, little is known about the differences between the inhibitory effects of cAMP and cGMP on the contraction of smooth muscle. OBJECTIVE: To compare the effects and underlying mechanisms of cAMP and cGMP on the inhibition of gastric smooth muscle contraction, cyclic nucleotide promoting agents, as well as cell membrane permeable cyclic nucleotides were used. METHODS: Isometric contraction was measured from circular muscle strips prepared from the fundus of cat stomach in a cylinder-shaped chamber filled with Krebs-Ringer solution (pH 7.4, temperature 36 degrees C) bubbled with 5% CO2 in O2. The level of inositol phosphates (IPs) was measured. RESULTS: Forskolin and sodium nitroprusside significantly inhibited acetylcholine (ACh)-induced gastric smooth muscle contraction and increased the cellular levels of cAMP and cGMP, respectively. Direct application of 8-Br-cAMP and 8-Br-cGMP also significantly inhibited ACh-induced contraction. Both verapamil and TMB-8 inhibited ACh-induced contraction. The combined inhibitory effect of verapamil and TMB-8 was significantly greater than the effect of either one, separately. Forskolin or sodium nitroprusside similarly augmented the effect of verapamil. However, the inhibitory effect of TMB-8 was augmented only by 8-Br-cGMP or sodium nitroprusside but not by 8-BrcAMP or forskolin. Forskolin and 8-Br-cAMP significantly inhibited the formation of inositol phosphates stimulated by ACh. CONCLUSIONS: cAMP inhibits the contraction mechanism associated with intracellular Ca2+ mobilization as well as extracellular Ca2+ influx, while cGMP inhibits contraction by inhibiting the mechanism associated with extracellular Ca2+ influx.     

Free intracellular Ca2+ concentration ([Ca2+]i) and growth hormone release from purified rat somatotrophs. I. GH-releasing factor-induced Ca2+ influx raises [Ca2+]i.

This study was carried out to investigate the role of free intracellular Ca2+ ([Ca2+]i) in the action of GH-releasing factor (GRF) by determining whether GRF causes and increase in [Ca2+]i and whether this increase results from changes in Ca2+ influx/efflux and/or mobilization of intracellular Ca2+ stores. We used a purified preparation of normal rat somatotrophs and examined the changes in 45Ca uptake, [Ca2+]i measured with indo-1, intracellular cAMP, and GH release induced by GRF. GRF stimulated a concentration-related biphasic increase in [Ca2+]i. Both the GRF-dependent increase in [Ca2+]i and GH release were blocked by incubation in low Ca2+ medium and by the organic Ca2+ antagonists nifedipine and diltiazem. The measurement of 45Ca uptake, in both steady state and nonsteady state conditions, demonstrated directly that GRF stimulates Ca2+ influx into somatotrophs. These data demonstrate that the GRF-stimulated increase in [Ca2+]i is dependent on Ca2+ influx. Redistribution of intracellularly stored Ca2+ could not be detected, even though intracellular Ca2+ stores were present. Therefore, the increase is due to Ca2+ influx, and the biphasic nature of the increase in [Ca2+]i induced by GRF is due to a difference in the rate of activation of Ca2+ influx and Ca2+ removal from the cytosol.     

Endothelium-derived Relaxing Factor Inhibits Shear Stress-induced Platelet Aggregation

The effect of endothelium-derived relaxing factor (EDRF) and related compounds on platelet aggregation in response to physiological and pathological levels of arterial wall shear stress (30-120 dyne/cm(2)) was investigated. Platelets in plasma, or washed platelets, aggregated markedly in response to shear stresses generated by a cone-plate viscometer. Pre-treatment of platelets with the S-nitrosothiol compounds S-nitroso-N-acetylcysteine or S-nitrosocysteine, or with nitric oxide (NO) or SIN-1 (which is non-enzymatically metabolized to NO), resulted in decreased platelet aggregation in response to shear stress. Non-hydrolyzable analogues of cyclic guanosine 3',5'-monophosphate (cGMP) also inhibited shear stress-induced platelet aggregation, and specific pharmacological manipulations of NO and cGMP (with methylene blue or the cGMP phosphodiesterase inhibitor M&B 22 984) resulted in alterations of intraplatelet levels of cGMP that correlated with the degree of inhibition of shear stress-induced platelet aggregation. These results demonstrate that EDRF and related compounds inhibit platelet aggregation that is initiated by shear stress, and suggest that this physiologically relevant mechanism of platelet aggregation may be regulated by intraplatelet cGMP.     

Growth inhibition and apoptosis induced by P2Y2 receptors in human colorectal carcinoma cells: involvement of intracellular calcium and cyclic adenosine monophosphate

Extracellular nucleotides induce apoptosis and inhibit growth of colorectal cancer cells. To understand the underlying signaling pathways, we investigated the role of nucleotide-sensitive P2 receptors and focused on the receptor-mediated signaling of intracellular Ca2+ and cyclic adenosine monophosphate (cAMP) in two colorectal carcinoma cell lines (HT29, Colo320 DM). Expression and functionality of P2 receptor subtypes evaluated by RT-PCR and [Ca2+]i imaging revealed that solely metabotropic P2 receptors of the subtype P2Y2 were expressed on a functional level in both cell lines. Short-term stimulation of P2Y2 receptors caused Ca2+ mobilization from intracellular stores and a subsequent transmembrane Ca2+ influx. The receptor-induced [Ca2+]i elevation was shown to increase basal-stimulated [cAMP]i moderately and to potentiate forskolin-stimulated [cAMP]i vigorously, since the effects were dose-dependently inhibited by preloading the cells with the [Ca2+]i chelator BAPTA. In contrast, activation of protein kinase C (PKC) did not contribute to a receptor-mediated rise in [cAMP]i, since the PKC inhibitor staurosporine completely failed to reduce P2Y2 receptor-induced increases in [cAMP]i. Prolonged application of P2Y2 receptor agonists induced a time-dependent increase in apoptosis (up to 50% above control values) in both cell lines and caused dose-dependent inhibition of cell proliferation of up to 85% (Colo320 DM) or 64% (HT29). Chelating [Ca2+]i with BAPTA almost completely abolished P2Y2 receptor-induced cell death. Rises in [cAMP]i elicited by either forskolin or cAMP derivatives inhibited growth in both cell lines, too. In line with the potentiating effect of P2Y2 receptors on forskolin-stimulated [cAMP]i increases, costimulation with forskolin and P2Y2 receptor agonists led to synergistic antiproliferative effects. Moreover, a synergistic growth inhibition was observed when coincubating the cells with the P2Y2 receptor agonist ATP and the cytostatic drug 5-fluorouracil, which forms the basis for most currently applied chemotherapeutic regimes in colorectal cancer treatment. Our results demonstrate the growth inhibitory potency of P2Y2 receptors in colorectal carcinoma cells. Receptor-induced [Ca2+]i signaling appears to play a major role in the observed antiproliferative and apoptosis-inducing effects.     

Intracellular Ca2+ mobilization by thyrotropin, carbachol, and adenosine triphosphate in dog thyroid cells

The effect of TSH, carbachol (CC), and ATP on intracellular calcium concentration ([Ca2+]i) in primary cultures of dog thyroid cells was examined using the fluorescent Ca2+ indicator fura-2. TSH caused an increase in [Ca2+]i at 37 C, but not 22 C, while it increased cAMP formation in these cells at both 22 and 37 C. CC and ATP increased [Ca2+]i at both 22 and 37 C. The CC-induced increase in [Ca2+]i was under muscarinic receptor control, and it was biphasic, with an initial spike followed by a sustained increase at a lower level. TSH and ATP were weaker agonists compared to CC, since maximal doses of TSH (100-500 mU/ml) and ATP (100-500 microM) increased [Ca2+]i by 40-70% over basal levels, compared to a 2- to 4-fold increase in [Ca2+] induced by maximal doses of CC (10-50 microM). The TSH-induced increase in [Ca2+]i was transient, returning to basal levels within 1-2 min after application of the agonist. All three agents were able to transiently increase [Ca2+]i to be internal stores. In the presence of the inorganic Ca2+ channel blockers La3+, Ni2+, and Co2+, the peak [Ca2+]i change was little affected, while the persistent response to CC and ATP was blocked, indicating dependence of this phase on influx of Ca2+. Paradoxically, these channel blockers abolished the effect of TSH on [Ca2+]i. TSH stimulation of cAMP formation was also inhibited 80-90% by these blockers, but not in Ca2+-free/EGTA buffer. These results suggest that the Ca2+ channel blockers may have actions in addition to inhibition of Ca2+ entry in these cells. TMB-8 [8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate HCl] specifically blocked both the initial and sustained increase induced by CC, while having no effect on ATP or TSH-induced [Ca2+]i, suggesting that TMB-8 may not be a general antagonist of Ca2+ mobilization. Activators of protein kinase-C, such as phorbol esters or an analog of diacylglycerol, inhibited the [Ca2+]i rise induced by all the three agonists used, indicating a regulatory role of protein kinase-C activation on [Ca2+]i in these cells. In FRTL-5 cells, [Ca2+]i was also increased by TSH and ATP, but not by CC. ATP, however, was a more effective agonist than in dog thyroid cells, while TSH increased [Ca2+]i by a similar magnitude in both cell types. The results of the present study demonstrate that TSH, albeit of lesser potency than CC, increases [Ca2+]i by causing intracellular Ca2+ mobilization in cultured dog thyroid cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

S-nitrosocysteine inhibition of human platelet secretion is correlated with increases in platelet cGMP levels.

Platelet inhibition by exogenous and endogenous nitrovasodilators has been shown to be associated with increases in cGMP, but proof of a role for cGMP in this process is lacking. We therefore studied the effects of cGMP and guanylate cyclase stimulation on human platelet secretion by pharmacologically modulating intraplatelet cGMP levels. The endothelium-derived relaxing factor (EDRF)-like activator of guanylate cyclase, S-nitrosocysteine (SNOC), led to a dose-dependent inhibition of secretion in intact human platelets (IC50 = 10(-6) M). The cGMP phosphodiesterase inhibitor M&B 22,948 augmented SNOC-induced inhibition of secretion through elevations in cGMP without affecting cAMP levels (from 50% to 81% inhibition versus control, p = 0.02). Methylene blue reversed the inhibitory effects of SNOC on platelet secretion (p = 0.03). Dibutyryl-cGMP and 8-bromo-cGMP also significantly inhibited secretion in this system. Incubation of platelets with exogenous cGMP to achieve intraplatelet cGMP levels comparable to those after SNOC treatment resulted in similar degrees of inhibition of secretion (32% inhibition versus control, p = 0.01) and was also potentiated by M&B 22,948 (from 32% to 68% inhibition, p = 0.003). In addition, a highly significant correlation between intraplatelet cGMP levels and the degree of inhibition of secretion was demonstrable in these studies (r = 0.94, p = 0.016). These data demonstrate that elevation of intraplatelet cGMP levels by the EDRF-like compound SNOC is correlated with inhibition of human platelet secretion.     

Hypertonic saline solutions attenuate agonist-induced changes of intracellular calcium

The proper fluid for resuscitation of hemorrhagic shock is still controversial. Hypertonic saline solutions would cause an impairment of platelet function, aggravating blood loss in case of uncontrolled hemorrhage. This work examines the in vitro effect of hypertonic NaCl solutions on the changes in [Ca2+]i induced by 100 microM ADP, 0.1 IU/ml thrombin or 0.5 microM ionomycin in human platelets. Furthermore, it was investigated if the addition of NaCl reduces the mobilization from intracellular stores or the calcium entry from extracellular media. In a solution containing 1 mM CaCl2, the increase of [Ca2+]i produced by thrombin was 93, 75 or 55% of the 300 mosM control when osmolarity of the solution was 350, 400, or 500 mosM, respectively. The calcium signal induced by ADP decreased more rapidly in hypertonic media than in isotonic solution. In a calcium-free solution, the mobilization of stored calcium produced by thrombin was reduced when osmolarity was increased from 300 mosM to 350, 400 or 500 mosM by 86, 75 or 45% of the control, respectively. The increase of [Ca2+]i produced by subsequent introduction of 1 mM extracellular calcium was also reduced. Similar effects were found when platelets were stimulated by ADP. Instead, the capacitative calcium entry induced by ionomycin was increased in 500 mosM media by a 138% of the isotonic control. The decrease in the Ca2+ signal produced by receptor agonists in hypertonic media may play a role in the reduction of platelet responses such as aggregation or shape change when hypertonic resuscitation fluids are utilized.     

Recombinant interleukin-8 induces changes in cytosolic Ca2+ in human neutrophils.

Activation of polymorphonuclear leukocytes (PMNL) by most soluble stimulants is associated with a marked increase in cytosolic free Ca2+ ([Ca2+]i). Interleukin-8 (IL-8), a monocyte-derived neutrophil chemotactic factor and potent neutrophil-activating cytokine, effectively enhanced the resting free [Ca2+]i within human PMNL in a dose-dependent manner (maximal effect with 100 ng/mL). The increase in [Ca2+]i was substantially (55%) inhibited in the absence of extracellular Ca2+. Thus, the increase was due to extra- and intracellular cooperative mobilization of Ca2+, as supported by the reduced effect of IL-8 on [Ca2+]i after quenching with Mn2+. Granulocyte-macrophage colony-stimulating factor and interferon-gamma failed to induce a change in [Ca2+]i, suggesting that they may operate through different signal pathways. Pretreatment with Bordetella pertussis toxin largely inhibited the IL-8-induced change in [Ca2+]i. Thus, IL-8-induced cooperative mobilization of intra- and extracellular Ca2+ leads to a net Ca2+ influx into the cytoplasm through a process mediated by a guanosine triphosphate-binding protein.     

External calcium facilitates signalling, contractile and secretory mechanisms induced after activation of platelets by collagen

Platelet activation leads to the initiation of intracellular signalling processes, many of which are triggered by Ca2+. We have studied the involvement of exogenous Ca2+ in platelet response to collagen activation. Platelet suspensions were prepared with and without adding external calcium in the suspension buffers. Activation with collagen (Col-I) was carried out, before and after incubation with cytochalasin B (Cyt-B) to block the actin assembly and the cytoskeletal reorganization. We evaluated changes in (i) tyrosine phosphorylation of proteins, in platelet lysates and associated with the cytoskeletal fraction, (ii) the association of contractile proteins to the cytoskeleton, (iii) expression of intraplatelet substances at the surface, and (iv) cytosolic Ca2+ levels ([Ca2+]i). Ultrastructural evaluation of platelets by electron microscopy was also performed. Platelet activation by Col-I in the absence of added Ca2+ was followed by mild association of actin and other contractile proteins, low phosphorylation of proteins at tyrosine residues, lack of expression of intraplatelet substances at the membrane, and absence of aggregation. In the presence of millimolar Ca2+, Col-I induced intense actin filament formation with association of contractile proteins with the cytoskeleton, resulting in profound morphological changes. Under these conditions, Col-I induced signalling through tyrosine phosphorylation, with increases in the [Ca2+]i, release of intragranule content and aggregation. Inhibiting actin polymerization with Cyt-B prevented all these events. Our data indicates that platelet activation by collagen requires external Ca2+. Studies with Cyt-B indicate that assembly of new actin and cytoskeleton-mediated contraction, both dependent on exogenous Ca2+, are key events for platelet activation by collagen. In addition, our results confirm that entrance of exogenous Ca2+ depends on a functional cytoskeleton.     

Inhibitory mechanism of human platelet aggregation by nafamostat mesilate

We found that nafamostat mesilate (NM) inhibits platelet aggregation induced by all agonists tested, including ADP, collagen, arachidonic acid, thromboxane A analog, A23187, phorbol 12-myristate 13-acetate (PMA), NaF and thrombin. The IC50 values were in the range of 9.3-17.8 microM. NM inhibited agonists-induced aspirin-treated platelet aggregation at >10 microM, suggesting that the action site lies beyond thromboxane (TXA)2 formation. However, NM inhibited thrombin (0.5 IU/ml)-induced TXB2 formation (IC50 = 1.9 +/- 0.6 microM, mean +/- SD). Intracellular Ca2+ mobilization was also inhibited only when platelets were challenged by thrombin, but the effect was found at NM concentrations >50 microM. This finding suggests that NM reduces the responses to thrombin by inhibiting its proteolytic activity on the platelet thrombin receptor (PAR1). NM did not affect the intracellular cAMP concentration or A-kinase activity. Agonists-induced surface expression of activated glycoprotein (GP)IIb-IIIa was inhibited by 10 microM NM and was completely inhibited by 50 microM NM. Since this inhibitory effect was parallel to the inhibition of platelet aggregation, the main inhibitory mechanism of NM against platelet aggregation seemed to be the suppression of activated GPIIb-IIIa expression, which makes it able to bind fibrinogen.     

High molecular weight kininogen inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets.

In this study we show that high molecular weight kininogen (HK) inhibited alpha-thrombin-induced aggregation of human platelets in a dose-dependent manner with complete inhibition occurring at plasma concentration (0.67 mumol/L) of HK. HK (0.67 mumol/L) also completely inhibited thrombin-induced cleavage of aggregin (Mr = 100 Kd), a surface membrane protein that mediates adenosine diphosphate (ADP)-induced shape change, aggregation, and fibrinogen binding. The inhibition of HK was specific for alpha- and gamma-thrombin-induced platelet aggregation, because HK did not inhibit platelet aggregation induced by ADP, collagen, calcium ionophore (A23187), phorbol myristate acetate (PMA), PMA + A23187, or 9,11-methano derivative of prostaglandin H2 (U46619). These effects were explained by the ability of HK, at physiologic concentration, to completely inhibit binding of 125I-alpha-thrombin to washed platelets. As a result of this action of HK, this plasma protein also completely inhibited thrombin-induced secretion of adenosine triphosphate, blocked intracellular rise in Ca2+ in platelets exposed to alpha- and gamma-thrombin, inhibited thrombin-induced platelet shape change, and blocked the ability of thrombin to antagonize the increase in intracellular cyclic adenosine monophosphate (cAMP) levels induced by iloprost. Because elevation of cAMP is known to inhibit binding of thrombin to platelets, we established that HK did not increase the intracellular concentration of platelet cAMP. Finally, HK did not inhibit enzymatic activity of thrombin. To study the role of HK in the plasma environment, we used gamma-thrombin to avoid fibrin formation by alpha-thrombin. Platelet aggregation induced by gamma-thrombin was also inhibited by HK in a dose-dependent manner. The EC50 (concentration to produce 50% of the maximum rate of aggregation) of gamma-thrombin for washed platelets was 7 nmol/L and increased to 102 nmol/L when platelets were suspended in normal human plasma. The EC50 for platelet aggregation induced by alpha-thrombin in plasma deficient in total kininogen was 40 nmol/L. When supplemented with HK at plasma concentration (0.67 mumol/L), the EC50 increased to 90 nmol/L, a value similar to that for normal human plasma. These results indicate that (1) HK inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets; (2) HK is a specific inhibitor of platelet aggregation induced by alpha- and gamma-thrombin; and (3) HK plays a role in modulating platelet aggregation stimulated by alpha-thrombin in plasma.