Guanine nucleotides and Ca2+‐dependent lysosomal secretion in electropermeabilised human platelets

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Abstract
Metabolically stable analogues of GTP, e.g. guanosine 5''-[.gamma.-thio]triphosphate (GTP[S]) and guanosine 5''-[.beta.,.gamma.-imido]triphosphate (pp[NH]pG), enhance the extent of Ca2+-dependent secretion of .beta.-N-acetylglucosaminidase and .beta.-galactosidase from electropermeabilised human platelets in the presence of less than 5 .mu.M Ca2+. A similar effect is observed on addition either of 1,2-dioctanoin or of GTP in the presence or absence of thrombin. In the presence of higher Ca2+ concentrations the extent of enhancement of lysosomal secretion declines and little, or no, enhancement is observed at a [Ca2+] of 30-40 .mu.M. Addition of leupeptin or antipain prevents this decrease in lysosomal secretion and enhances the extent of Ca2+-dependent lysosomal secretion obtained in the presence or absence of guanine nucleotides thrombin or 1,2-dioctanoin. The concentration of GTP[S] or pp[NH]pG required to obtain half-maximal enhancement of lysosomal secretion is dependent on [Ca2+] for secretion of 5-hydroxytryptamine, .beta.-N-acetylglucosaminidase and .beta.-galactosidase. At two fixed [Ca2+] the median effective concentration (EC50) values for GTP[S] and pp[NH]pG which characterise enhancement of 5-hydroxytryptamine secretoin are significantly different from those characterising enhancement of secretion of .beta.-N-acetylglucosaminidase and .beta.-galactosidase. In the presence of a saturating concentration of GTP[S] marked 5-hydroxytryptamine and .beta.-N-acetylglucosaminidase secretion is observed at nanomolar [Ca2+] and these responses show little dependence on [Ca2+] over the attainable range. Secretion of .beta.-N-acetylglucosaminidase is also induced at nanomolar Ca2+ concentrations by addition of activators of protein kinase C. Guanosine 5''-[.beta.-thio]diphosphate inhibits enhancement of .beta.-N-acetylglucosaminidase secretion induced by GTP[S] but has no effect on secretion of this enzyme induced by Ca2+ when added alone. Our data provide some support for a model in which addition of metabolically stable guanine nucleotides enhances Ca2+-dependent platelet lysosomal secretion by activating a guanine-nucleotide-binding protein (GE) located close to the exocytotic site. However, not all the data are consistent with this postulate.