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Role of cyclic nucleotides in store-mediated external Ca2+ entry in human platelets.

ABSTRACT: This study explores the role of cyclic nucleotides (i.e. cyclic AMP and cyclic GMP) in store-regulated external Ca2+ entry in human platelets. To stimulate store-regulated external Ca2+ entry, thapsigargin was used to deplete Ca2+ from the dense tubules, and sodium nitroprusside and iloprost respectively were used to stimulate endogenous cyclic GMP and cyclic AMP formation. Pretreatment with sodium nitroprusside and iloprost (a) attenuated the thapsigargin-evoked external Ca2+ entry and (b) reduced the rate of Ca2+ release from the dense tubules. The effects on external Ca2+ entry and Ca2+ release from the dense tubules were exerted independently and were apparently mediated through activation of the respective cyclic nucleotide-dependent protein kinases. Both sodium nitroprusside and iloprost reduced tyrosine kinase phosphorylation of a number of proteins, particularly a 72 kDa protein band. Both agents also attenuated the thapsigargin-evoked tyrosine kinase phosphorylation of the 72 kDa band. Intracellular Ca2+ depletion resulted in a reduction in tyrosine kinase-mediated phosphorylation of a number of protein bands, including the 72 kDa band and the further attenuation of thapsigargin-mediated tyrosine phosphorylation of this band. The effects of the cyclic nucleotides on cellular Ca2+ homoeostasis in thapsigargin-treated platelets were not exerted via acceleration of Ca2+ extrusion or Ca2+ sequestration into the mitochondria. We conclude that cyclic nucleotides participate in store-regulated control of external Ca2+ entry by slowing down the rate of external Ca2+ entry and Ca2+ release from intracellular Ca2+ stores. These effects are apparently mediated via cyclic nucleotide-dependent protein kinases and the attenuation of protein phosphorylation by tyrosine kinases.

SUBMITTER: Nakamura K

PROVIDER: S-EPMC1135882 | biostudies-other | 1995 Aug

REPOSITORIES: biostudies-other

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Project description: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.

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Role of cyclic nucleotides in store-mediated external Ca2+ entry in human platelets.

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