Wortmannin interferes with thrombin-evoked secondary calcium redistribution in human platelets.

Wortmannin has previously been reported to inhibit calcium entry in thrombin-stimulated human platelets. We extend these findings by demonstrating that the redistribution of calcium from intracellular stores features two separate, consecutive phases the second of which is selectively abolished by wortmannin. The primary release of calcium from Ins 1,4,5 P3-sensitive stores remains unaffected. Hence, wortmannin is interfering with regulation of any secondary, sustained calcium accumulation in the cytosolic compartment of activated platelets, originating either from intracellular stores or from calcium entry. We assume that wortmannin blocks a common step in receptor-dependent regulation of calcium entry. We assume that wortmannin blocks a common step in receptor-dependent regulation of calcium entry and intracellular calcium circulation.

Ebselen-binding equilibria between plasma and target proteins.

The antiinflammatory drug ebselen (2-phenyl-1,2-benzisoselenazo-3(2H)-one) is known to bind covalently to thiols to form seleno disulfides that, directly or indirectly, are responsible for its pharmacological effects. Due to its reactive thiol group and high plasma concentration, albumin is a preferred target of ebselen, which it binds covalently. Ebselen should not, then, be available for intracellular actions at other target proteins. We have addressed this question, and show by difference spectroscopy that the interaction of ebselen with albumin occurs stoichiometrically under ring opening, but is readily reversible in the presence of glutathione. With intact human polymorphonuclear leukocytes (PMN), a similar stoichiometric reaction with distinct spectral features was observed with ebselen that was completely abolished by pretreatment of PMN with N-ethylmaleimide, but not by selective depletion of cellular glutathione. Human platelets, again, exhibited different spectral changes upon addition of ebselen. In agreement with results reported in the literature, we show that 14C-ebselen is in dynamic equilibrium with all accessible thiol groups and, hence, despite mostly being bound covalently to albumin, it will exchange rapidly with other target proteins in PMN or platelets.

Receptor occupancy regulates Ca2+ entry and intracellular Ca2+ redistribution in activated human platelets.

Fura-2-loaded human platelets were used to study Ca2+ release from intracellular compartments, as well as Ca2+ influx from the extracellular space. We investigated the response towards the endoperoxide/thromboxane-receptor agonist. U46619, and the inhibitor of the endoplasmic-reticulum Ca(2+)-ATPase, thapsigargin. U46619 dose-dependently depleted intracellular Ca2+ stores, followed by active sequestration of released Ca2+. Ca2+ influx induced by U46619 largely relies on receptor occupancy. Removing the thromboxane analogue from its receptor by using the endoperoxide/thromboxane-receptor antagonist BM 13177 largely blunted U46619-mediated Ca2+ influx. The Ca(2+)-ATPase inhibitor thapsigargin evoked a gradual rise in intracellular Ca2+, which was potentiated by a preceding activation of platelets with the receptor agonist U46619. This agonist-sensitizing effect also depends on receptor occupancy. Removing U46619 from its receptor by addition of the endoperoxide/thromboxane-receptor antagonist BM13177 suppressed the sensitizing effect completely. Furthermore, interrupting downstream receptor signalling events by raising intracellular levels of cyclic nucleotides (cyclic AMP, cyclic GMP) again suppressed the U46619-sensitizing effect on thapsigargin-induced Ca2+ release. This study indicates that the process of Ca2+ release followed by resequestration in response to a platelet agonist by its own is not sufficient to produce the sensitizing effect. Rather, a continuously occupied receptor triggering sustained downstream signalling events seems to be required for sensitization. The presence of a receptor agonist may induce an increased cycling of Ca2+ between the agonist-responsive and the thapsigargin-dischargeable compartment, leading to faster and more intense accumulation of Ca2+ in the cytosolic compartment after inhibition of the Ca(2+) ATPase. Suggestively, receptor occupancy increases the Ca(2+)-releasing potency of thapsigargin by coupling the thapsigargin-sensitive Ca(2+)-storing compartments with an agonist-responsive compartment that exhibits a high leakage rate in stimulated platelets.

Efflux of cyclic GMP from activated human platelets.

Activation of human platelets is associated with an increased level of cGMP, when total cGMP in individual samples is measured. However, by discriminating between intracellular and extracellular cGMP we were able to demonstrate that cGMP accumulates in the extracellular space only, whereas the level of intraplatelet cGMP actually decreases. Therefore, during the first minutes of platelet aggregation cGMP is released from the cell, and it thereby escapes hydrolysis by intracellular phosphodiesterases. In contrast, during direct activation of soluble guanylyl cyclase by nitrovasodilators, such as sodium nitroprusside, the newly synthesized cGMP remains mainly inside the cells. Elevation of intracellular calcium and activation of protein kinase C are likely to be involved in promoting cGMP efflux. Our results are discussed in contrast to the general hypothesis that the cGMP increase associated with platelet aggregation may represent a feedback mechanism designed to terminate early events of activating signal transduction. According to our data the apparent cGMP increase results from cGMP release from thrombocytes, rather than soluble guanylyl cyclase activation. This cGMP efflux provides a mechanism of decreasing the intracellular cGMP level upon stimulation with platelet agonists and thus favors platelet activation.

Reversible activation of soluble guanylate cyclase by oxidizing agents.

Soluble guanylate cyclase of human platelets was stimulated by thiol oxidizing compounds like diamide and the reactive disulfide 4, 4'-dithiodipyridine. Activation followed a bell-shaped curve, revealing somewhat different optimum concentrations for each compound, although in both cases, higher concentrations were inhibitory. Diamide at a concentration of 100 microM transiently activated the enzyme. In the presence of moderate concentrations of diamide and 4,4'-dithiodipyridine, causing a two- to fourfold activation by themselves, the stimulatory activity of NO-releasing compounds like sodium nitroprusside was potentiated. In contrast, higher concentrations of thiol oxidizing compounds inhibited the NO-stimulated activation of soluble guanylate cyclase. Activation of guanylate cyclase was accompanied by a reduction in reduced glutathione and a concomitant formation of protein-bound glutathione (protein-SSG). Both compounds showed an activating potency as long as reduced glutathione remained, leading to inhibition of the enzyme just when all reduced glutathione was oxidized. Activation was reversible while reduced glutathione recovered and protein-SSG disappeared. We propose that diamide or reactive disulfides and other thiol oxidizing compounds inducing thiol-disulfide exchange activate soluble guanylate cyclase. In this respect partial oxidation is associated with enzyme activation, whereas massive oxidation results in loss of enzymatic activity. Physiologically, partial disulfide formation may amplify the signal toward NO as the endogenous activator of soluble guanylate cyclase.

Post-translational arginylation and intracellular proteolysis.

Cellular proteins may be designated to fast degradation by their N-terminal amino acids, and especially a N-terminal arginine residue should have an extremely destabilizing effect on cytosol proteins. We investigated the post-translational arginylation of cytosol proteins and especially of ornithine decarboxylase (ODC) by the cytosolic enzyme arginyl transferase by incubation with radioactive L-arginyl-tRNA and isolation of ODC with our monoclonal antibody. Arginylated ODC had a specific radioactivity 8600 times that of the bulk of cytosolic proteins and Edman-degradation of this ODC showed that the post-translational arginylation occurred only at the L-amino-end of the enzyme. The inhibitor of arginyltransferase, L-Glutamyl-L-Valyl-L-Phenylalanine, increased the half-life of ODC in cultured hepatocytes from 39 min to more than 90 min. This post-translational arginylation of ODC and also of other cytosol proteins is reversible. At least 25 different cytosol proteins in addition to ODC can be arginylated in hepatocytes, and at least 15 different proteins can be arginylated in Dictyostelium discoideum. The arginylated proteins are much more rapidly degraded by cellular proteinases, especially by calpains, than those cytosolic proteins which are not arginylated.