Biochemical and Biophysical Research Communications
ATP depletion in human platelets caused by permeabilization with saponin does not prevent serotonin secretion induced by collagen
Abstract
Saponin (5 to 25 μg/ml) produced a concentration-dependent decrease in the cellular content of total ATP and [32P]ATP in 32P-labeled human platelets. In platelets whose ATP had been profoundly decreased by saponin, Ca2+ produce phosphomonoesteratic cleavage of the polyphosphoinositides with a concomitant accumulation of phosphatidylinositol. Collagen still induced secretion of serotonin in platelets that had been treated with saponin in the presence or absence of Ca2+. This effect of collagen occurred in the absence of the formation of cyclooxygenase metabolites. In platelet permeabilized with saponin, agonist-induced secretion and aggregation seems to be unrelated to protein phosphorylation, breakdown of the inositol phospholipids by phospholipase C and formation of cyclooxygenase metabolites.
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Cited by (17)
Techniques and concepts in exocytosis: focus on mast cells
1991, BBA - Reviews on BiomembranesInositol 1,4,5-trisphosphate (InsP3) is a second messenger responsible for Ca2+ release from an internal store whose nature and location remains undefined. To get more information on this intracellular Ca2+ store, a post-nuclear particulate fraction was prepared from bovine adrenal cortex and its Ca2+ uptake and release activities were monitored with the fluorescent indicator Fura-2. In the presence of Mg2+ (2 mM), the particulate preparation showed high ATP-dependent Ca2+ sequestering activity and decreased the ambient Ca2+ concentration to about 150 nM. In the absence of Mg2+, Ca2+ was still sequestered but less efficiently, reaching a level around 170 nM. In the presence of Mg2+, the Ca2+ released by a maximal dose of InsP3 (2 μM) was completely resequestered whereas in the absence of Mg2+, no resequestration occurred even after complete degradation of InsP3. The use of selective agents such as oligomycin, saponin, ionomycin and biliary salts indicated that Ca2+ was stored in three different pools which are distinct from the mitochondria and from inside-out membrane vesicles. Our data also indicate that InsP3 releases Ca2+ from a pool which is filled up by a Mg2+-dependent Ca2+ ATPase.
Ca<sup>2+</sup>-induced secretion by electropermeabilized human neutrophils. The roles of Ca<sup>2+</sup>, nucleotides and protein kinase C
1990, BBA - Molecular Cell ResearchStudies of stimulus-response coupling have benefitted from the availability of permeabilization techniques, whereby putative second messengers and intracellular modulators can be introduced into the cell interior. Electropermeabilization, which uses high-intensity electric fields to breach the plasma membrane, creates small pores, permitting access of solutes with molecular masses below 700 KDa. Neutrophils permeabilized by this technique, but not intact cells, discharged lysosomal constituents when exposed to micromolar levels of Ca2+. Secretion by electroporated neutrophils was significantly enhanced by the presence of Mg-ATP (0.3–1.0 mM). Contrary to expectations, it was determined that ATP was not the only nucleotide which enhanced Ca2+-induced secretion in the presence of Mg2+. Not only could GTP, XTP, ITP, UTP or ADP partially or completely replace ATP, but even non-hydrolyzable nucleotides such as ADPβS ATPγS, and App[NH]p were effective. GTPγS and GDPβS were inhibitory, while Gpp[NH]p was inactive. None of these nucleotides induced secretion on its own. In contrast, neutrophils which were permeabilized and then washed, were only slightly activated by Mg-ATP and other nucleotides; even the response to Ca2+ alone was less. This hyporesponsiveness of washed cells proved to be due to a time-dependent deactivation of the permeabilized neutrophils taking place at 4° C. In an effort to assess the role for protein kinase C (PKC) in secretion in this system, we examined the effects of phorbol myristate acetate (PMA), a PKC agonist. PMA enhanced degranulation induced by Ca2+ by lowering the requirement for this divalent cation; enhancement by PMA was not dependent upon exogenous ATP. Three inhibitors of PKC with varying specificity, namely H-7, K-252a, and staurosporine, all abrogated PMA-enhanced secretion. These agents also inhibited secretion stimulated by Ca2+ plus ATP in parallel with that induced by Ca2+ plus PMA, strongly suggesting a role for PKC in modulation of degranulation by ATP. Our results show that electroper-meabilized neutrophils provide a convenient, useful model for stimulus-secretion coupling. These data also suggest that the ‘requirement’ for Mg-ATP, which has been observed in other permeabilized cell systems, is not simply for metabolic energy or as a substrate for kinases. It is possible that these nucleotides all interact with a recently described neutrophil receptor for adenine nucleotides or with a recently postulated exocytosis-linked G-protein.
Regulation of Membrane Fusion during Exocytosis
1990, International Review of CytologyThis chapter describes the regulation of membrane fusion during exocytosis. Exocytosis involves the fusion of a secretory organelle with the cell membrane. It is important to discriminate between regulated (triggered) exocytosis and constitutive (untriggered) exocytosis. Specific signals and signal-transfer mechanisms occur only with regulated exocytosis. The mechanisms of membrane-fusion regulation are different from these two types of exocytosis. Exocytosis is of the PF–PF fusion type; plasmatic membrane faces fuse. It always follows the focal fusion scheme: fusion is restricted to a small area of ≤ 10 nm, before the exocytotic opening expands to release the secretory contents. Adenosine triphosphate (ATP) is required for intracellular transport of secretory organelles; it might also be required for a rearrangement of microfilamentous elements on the cell periphery during secretion. The protein phosphorylation, another phenomenon generally occurring during secretory activity, clearly requires ATP. The hydrolysis of membrane-bound ATP has been proposed to be a fusogenic process. Guanosine triphosphate (GTP) analogs stimulate membrane fusion in some systems; they might act by various mechanisms.
Protein kinase C is not involved in secretion by permeabilized human neutrophils
1989, Cellular SignallingThe generally accepted sequence of intracellular signal transduction involves: (1) cell surface receptor-ligand interaction; (2) activation of G-proteins; (3) activation of phospholipase C, leading to inositol phosphate (IP3) and diacylglycerol production; (4) parallel mobilization of intracellular Ca2+ by IP3, and; (5) activation of protein kinase C (PKC) by diacylglycerol and Ca2+, leading to; (6) cellular responses. Human neutrophils appear to utilize this cascade, at least in general, and some, but not all, elements of the intracellular signal cascade known to be operating in intact cells also function in permeabilized cell systems. We have previously shown that permeabilized neutrophils can be induced to secrete lysosomal enzymes in response to elevated levels of Ca2+ alone and this secretion can be synergistically enhanced by the presence of guanine nucleotides. We now show that Ca2+, in the presence and absence of guanine nucleotides, can stimulate the production of solubel inositol phosphates. Furthermore, neomyxin, a putative inhibitor of phospholipase C, can block Ca2+-induced secretion. These data thus suggest a role for phospholipase C activity or its products in the transduction process. The next enzymatic activity ‘downstream’ is PKC. Consequently, we looked at the role Mg-ATP, one of the substrates of PKC, plays in degranulation by permeabilized neutrophils. We found no obligatory role for this nucleotide in the secretory process. We then looked at the activity of oleoyl-acetyl-glycerol (OAG), synthetic diacylglycerol and PKC agonist, on degranulation. We found that OAG was largely additive with Ca2+. Another PKC agonist, phorbol myristate acetate (PMA), also did not display notable synergy. Finally, inhibitors of PKC activity were not capable of blocking secretion, either in the presence or absence of guanine nucleotides. Thus, while circumstantial evidence seems to point towards a requirement for phospholipase C activation and diacylglycerol production in secretion, we were unable to demonstrate the next putative step in signal transduction, namely activation of PKC.
Effect of exogenously added acylphosphatases on inositol lipid metabolism in human platelets
1988, FEBS LettersIn this paper we demonstrate that human platelets contain an acylphosphatase isoenzyme. We then investigated the of exogenously added human muscle and erythrocyte acylphosphatases on inositol lipid content in human platelets permeabilized with saponin. Alterations in the level of the polyphosphoinositides were observed: in particular, the levels of phosphatidylinositol 4,5-bisphosphate, and of phosphatidylinositol 4-monophosphate were decreased, whereas the level of phosphatidylinositol was increased. These results suggest that acylphosphatases promote polyphosphoinositide dephosphorylation, possibly through intracellular Ca2+ mobilization.