Molecular mechanisms involved in human platelet aggregation by synergistic interaction of platelet-activating factor and 5-hydroxytryptamine

Our recent studies have shown that co-activation of Gq and Gi proteins by 5-hydroxytryptamine (5-HT) and adrenaline show synergism in human platelet aggregation. This study was conducted to examine the mechanism(s) of synergistic interaction of 5-HT and platelet activating factor (PAF) in human platelets. We show that PAF, but not 5-HT, increased platelet aggregation in a concentration-dependent manner. However, low concentrations of 5-HT (2 microM) potentiated platelet aggregation induced by subthreshold concentration of PAF (40 nM) indicating a synergistic interaction between the two agonists and this synergism was blocked by receptor antagonists to either 5-HT or PAF. 5-HT also potentiated the effect of PAF on thromboxane A2 (TXA2) formation and phosphorylation of extracellularly regulated mitogen-activated protein kinases (ERK1/2). The synergism of 5-HT and PAF in platelet aggregation was inhibited by calcium (Ca2+) channel blockers, verapamil and diltiazem, phospholipase C (PLC) inhibitor, U73122, cyclooxygenase (COX) inhibitor, indomethacin, and MEK inhibitor, PD98059. These data suggest that synergistic effect of 5-HT and PAF on human platelet aggregation involves activation of PLC/Ca2+, COX and MAP kinase pathways.

Molecular mechanisms involved in human platelet aggregation by synergistic interaction of platelet-activating factor and 5-hydroxytryptamine

Our recent studies have shown that co-activation of Gq and Gi proteins by 5-hydroxytryptamine (5-HT) and adrenaline show synergism in human platelet aggregation. This study was conducted to examine the mechanism(s) of synergistic interaction of 5-HT and platelet activating factor (PAF) in human platelets. We show that PAF, but not 5-HT, increased platelet aggregation in a concentration-dependent manner. However, low concentrations of 5-HT (2 microM) potentiated platelet aggregation induced by subthreshold concentration of PAF (40 nM) indicating a synergistic interaction between the two agonists and this synergism was blocked by receptor antagonists to either 5-HT or PAF. 5-HT also potentiated the effect of PAF on thromboxane A2 (TXA2) formation and phosphorylation of extracellularly regulated mitogen-activated protein kinases (ERK1/2). The synergism of 5-HT and PAF in platelet aggregation was inhibited by calcium (Ca2+) channel blockers, verapamil and diltiazem, phospholipase C (PLC) inhibitor, U73122, cyclooxygenase (COX) inhibitor, indomethacin, and MEK inhibitor, PD98059. These data suggest that synergistic effect of 5-HT and PAF on human platelet aggregation involves activation of PLC/Ca2+, COX and MAP kinase pathways.

Co-activation of Gi and Gq proteins exerts synergistic effect on human platelet aggregation through activation of phospholipase C and Ca2+ signalling pathways

Our previous studies have shown that subthreshold concentrations of two platelet agonists exert synergistic effects on platelet aggregation. Here we studied the mechanism of synergistic interaction of 5-hydroxytryptamine (5-HT) and epinephrine mediated platelet aggregation. We show that 5-HT had no or little effect on aggregation but it did potentiate the aggregation response of epinephrine. The synergistic interaction of 5-HT (1-5 microM) and epinephrine (0.5-2 microM) was inhibited by alpha2-adrenoceptor blocker (yohimbine; IC50= 0.4 microM), calcium channel blockers (verapamil and diltiazem with IC50 of 10 and 48 mM, respectively), PLC inhibitor (U73122; IC50=6 microM) and nitric oxide (NO) donor, SNAP (IC50=1.6 microM)). The data suggest that synergistic effects of platelet agonists are receptor-mediated and occur through multiple signalling pathways including the activation PLC/Ca2+ signalling cascades.

Cholera toxin mediated regulation of the expression of Gq alpha and G11 alpha GTP binding proteins

Previously it has been shown that persistent activation of the stimulatory adenylyl cyclase pathway with cholera toxin (CT) downregulates the Gs alpha polypeptide (80%) in a cAMP-independent manner in C6 glioma cells (Shah, 1997). This study was conducted to examine the short and long term effects of CT on the regulation of pertussis toxin-sensitive and -insensitive G proteins and their transcripts in C6 glioma cells. Treatment of C6 cells with CT (100 ng/ml) up to 16 h had no effect on either Gi or Gq/11 alpha proteins. However, prolonged exposure (24-48 h) caused increased expression of Gi (20-30%) and Gq/11 alpha proteins (40%). Urea gradient gels, which can separate Gq alpha and G11 alpha proteins, revealed that prolonged CT treatment increased the expression of both of these G proteins. The CT-mediated enhanced expression of Gq alpha and G11 alpha proteins was accompanied by increased mRNA levels of these proteins as determined by RT/PCR. Cyclic-AMP elevating agents like forskolin (10 microM) and db-cAMP (1 mM) mimicked the effect of CT on Gi but not Gq/11 alpha proteins. These studies show long term cAMP-dependent regulation of Gi and cAMP-independent expression of Gq/11 alpha proteins in C6 glioma cells.

Pharmacokinetics and renal excretion of sulfamethoxazole in sheep

Pharmacokinetics and urinary excretion of sulfamethoxazole were investigated in healthy sheep. From the plasma disappearance curves after intravenous bolus injection [50 mg/kg] the half-life and volume of distribution were 76.4 +/- 14 min and 0.409 +/- 0.178 lit/kg respectively. Body clearance was 4.06 +/- 1.03 ml/kg/min. Vey low concentrations of drug were present in plasma after 3 hours of injection and plasma level at 6 hours was only 4.4 +/- 2.0 ug/ml. The renal clearance of sulfamethoxazole [22.04 +/- 2.17 ml/min/10 kg] exceeded the creatinine clearance [9.78 +/- 1.57 ml/min/10 kg] which may be due to involvement of active tubular secretion and pH dependent back diffusion. Half of the dose of sulfamethoxazole was excreted as unchanged free drug while acetylated amine comprised of 20% within the first 6 hours of drug administration