Effects of polyamines and calcium and sodium ions on smooth muscle cytoskeleton-associated phosphatidylinositol (4)-phosphate 5-kinase.
In many different cell types, including smooth muscle cells (Baron et al., 1989, Am. J. Physiol., 256: C375-383; Baron et al., J. Pharmacol. Exp. Ther. 266: 8-15), phosphatidylinositol (4)-phosphate 5-kinase plays a critical role in the regulation of membrane concentrations of phosphatidylinositol (4,5)-bisphosphate and formation of inositol (1,4,5)-trisphosphate. In unstimulated porcine trachealis smooth muscle, 70% of total cellular phosphatidylinositol (4)-phosphate 5-kinase activity was associated with cytoskeletal proteins and only trace activity was detectable in isolated sarcolemma. Using two different preparations, we studied cytoskeleton-associated phosphatidyl inositol (4)-phosphate 5-kinase under conditions that attempted to mimic the ionic and thermal cytoplasmic environment of living cells. The cytoskeleton-associated enzyme, studied using phosphatidylinositol (4)-phosphate substrate concentrations that produced phosphatidylinositol 4,5-bisphosphate at about 10% of the maximal rate, was sensitive to free [Mg2+], had an absolute requirement for phosphatidylserine, phosphatidic acid, or phosphatidylinositol, and included type I isoforms. At 0.5 mM free [Mg2+], physiological spermine concentrations, 0.2-0.4 mM, increased phosphatidylinositol (4)-phosphate 5-kinase activity two to four times compared to controls run without spermine. The EC50 for spermine-evoked increases in activity was 0.17 +/- 0.02 mM. Spermine-evoked enzyme activity was a function of both free [Mg2+] and substrate concentration. Cytoskeleton-associated phosphatidylinositol (4)-phosphate 5-kinase was inhibited by free [Ca2+] over a physiological range for cytoplasm--10(-8) to 10(-5) M, an effect independent of the presence of calmodulin. Na+ over the range 20 to 50 mM also inhibited this enzyme activated by 5 mM Mg2+ but had no effect on spermine-activated enzyme. Na+, Ca2+, and spermine appear to be physiological modulators of smooth muscle cytoskeleton-bound phosphatidylinositol (4)-phosphate 5-kinase.
Chen
H
H
Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, USA.
Baron
C B
CB
Griffiths
T
T
Greeley
P
P
Coburn
R F
RF
D000180:Acrylic Resins; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002458:Cell Fractionation; D003599:Cytoskeleton / Q000201:enzymology; D003902:Detergents; D004983:Ethanolamines; D006493:Heparin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology; D009130:Muscle, Smooth / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D010712:Phosphatidic Acids / Q000494:pharmacology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000494:pharmacology; D010718:Phosphatidylserines / Q000494:pharmacology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011073:Polyamines / Q000494:pharmacology*; D011188:Potassium / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D011700:Putrescine / Q000494:pharmacology; D012508:Sarcolemma / Q000201:enzymology; D012964:Sodium / Q000494:pharmacology*; D013095:Spermidine / Q000494:pharmacology; D013096:Spermine / Q000494:pharmacology; D013379:Substrate Specificity; D013552:Swine
D000180:Acrylic Resins; D000925:Anticoagulants; D003902:Detergents; D004983:Ethanolamines; D010712:Phosphatidic Acids; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D010718:Phosphatidylserines; D011073:Polyamines; C037239:Sephacryl Superfine; D013096:Spermine; D000255:Adenosine Triphosphate; D006493:Heparin; D012964:Sodium; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D008274:Magnesium; D011188:Potassium; C007369:2-diethylaminoethanol; D002118:Calcium; D013095:Spermidine; D011700:Putrescine