Guanine nucleotides and Ca2(+)-dependent lysosomal secretion in electropermeabilised human platelets.
1. 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 microM Ca2+. A similar effect is observed on addition either of 1,2-dioctanoin or of GTP in in the presence or absence of thrombin. 2. 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 microM. 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. 3. 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 secretion are significantly different from those characterising enhancement of the secretion of beta-N-acetylglucosaminidase and beta-galactosidase. 4. 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. 5. 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. 6. 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.
Athayde
C M
CM
Division of Biomolecular Sciences, King's College, London, England.
Scrutton
M C
MC
D000118:Acetylglucosaminidase / Q000378:metabolism*; D001665:Binding Sites; D001711:Biotransformation / Q000187:drug effects; D001792:Blood Platelets / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology*; D002463:Cell Membrane Permeability / Q000187:drug effects; D004563:Electrochemistry; D019204:GTP-Binding Proteins / Q000378:metabolism; D005696:Galactosidases / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006596:Hexosaminidases / Q000378:metabolism*; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D013917:Thrombin / Q000494:pharmacology; D001616:beta-Galactosidase / Q000378:metabolism*
D006150:Guanine Nucleotides; D006160:Guanosine Triphosphate; D005696:Galactosidases; D006596:Hexosaminidases; D001616:beta-Galactosidase; D000118:Acetylglucosaminidase; D013917:Thrombin; D019204:GTP-Binding Proteins; D002118:Calcium