Effects of Ca2+, Mg2+, and depolarizing agents, on the 32Pi-labeling and degradation of phosphatidylinositols in rat brain synaptosomes.
In isolated synaptosomes from rat brain, 100 microM antimycin A and 10 microM oxamic acid inhibit the 32Pi-labeling of phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidylinositol-4-phosphate (PIP) by 90% and 95-99% respectively. 10 mM sodium fluoride inhibits the labeling by 50-60% and 10 mM A23187 inhibits the labeling by 63-70%. Phospholipase A2 inhibits the labeling of PIP2 and PIP by 93-94% and stimulates their degradation by 84-92%. Depolarization of synaptosomes with 75 mM K+ or 100 microM veratrine decreases the labeling of PIP2 and PIP by 66-74%. The decreased labeling results in large part from the Ca(2+)-dependent degradation of 32P-labeled PIP2 and PIP as shown by pulse-chase experiments in which PIP2 and PIP were prelabeled with 32Pi. Depolarization of synaptosomes results in the stimulation of 45Ca2+ uptake with the concomitant hydrolysis of PIP and PIP2. Addition of 1 mM Ca2+ accounts for 25% of the enhanced degradation whereas depolarization with 75 mM K+ accounts for 75% of the enhanced degradation of PIP2 and PIP. Depolarization with 100 mM veratrine results in a 223% increase in inositol trisphosphate as evidenced by stimulation of 45Ca2+ uptake. EGTA (10 mM) and Mg2+ (5-10 mM) inhibit the degradation of PIP and PIP2 and counteract the action of 1 mM Ca2+. Our data demonstrate that 45Ca2+, Mg2+, and membrane depolarization play an important role in the turnover of membrane phosphatidylinositols.
Department of Biochemistry, University of Rochester Medical Center, New York 14642.
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Marinetti
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Morris
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Leaky
D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D004533:Egtazic Acid / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology*; D008297:Male; D010072:Oxamic Acid / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols / Q000378:metabolism*; D010741:Phospholipases A / Q000494:pharmacology; D054467:Phospholipases A2; D010761:Phosphorus Radioisotopes; D051381:Rats; D017207:Rats, Sprague-Dawley; D012969:Sodium Fluoride / Q000494:pharmacology; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism*
D010710:Phosphates; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D010761:Phosphorus Radioisotopes; C037178:phosphatidylinositol 4-phosphate; D000001:Calcimycin; D004533:Egtazic Acid; D000968:Antimycin A; D012969:Sodium Fluoride; D010741:Phospholipases A; D054467:Phospholipases A2; D008274:Magnesium; D010072:Oxamic Acid; D002118:Calcium