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 at., 1989, Am. I. Physiol., 256: C375-383; Baron et al., J. Pharmacol. Exp. Ther. 266: 8-15), phosphatidylinositol (4)-phosphate 5-kinase pl ays a critical role in the regulation of membrane concentrations of ph osphatidylinositol (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 activit y was associated with cytoskeletal proteins and only trace activity wa s detectable in isolated sarcolemma. Using two different preparations, we studied cytoskeleton-associated phosphatidylinositol (4)-phosphate 5-kinase under conditions that attempted to mimic the ionic and therm al cytoplasmic environment of living cells. The cyloskeleton-associate d enzyme, studied using phosphatidylinositol (4)-phosphate substrate c oncentrations that produced phosphatidylinositol 4,5-bisphosphate at a bout 10% of the maximal rate, was sensitive to free [Mg2+], had an abs olute requirement for phosphatidylserine, phosphatidic acid, or phosph atidylinositol, and included type I isoforms. At 0.5 mM free [Mg2+], p hysiological spermine concentrations, 0.2-0.4 mM, increased phosphatid ylinositol (4)-phosphate 5-kinase activity two to four times compared to controls run without spermine. The EC50 for spermine-evoked increas es in activity was 0.1.7 +/- 0.02 mM. Spermine-evoked enzyme activity was a function of both free [Mg2+] and substrate concentration. Cytosk eleton-associated phosphatidylinositol (4)-phosphate 5-kinase was inhi bited 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 cytos keleton-bound phosphatidylinositol (4)-phosphate 5-kinase. J. Cell. Ph ysiol. 177:161 - 173, 1998. (C) 1998 Wiley-Liss, Inc.