Effects of Na+, Ca2+, and Acetylcholine on Phosphoinositide‐ and ATP‐Phosphate Turnover in 32P‐Labeled Rabbit Iris Smooth Muscle

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Abstract
Parallel studies were carried out in the rabbit iris on (a) the effects of Na+ and/or Ca2+ on the acetylcholine-stimulated 32P labeling of phosphatidic acid (PA) and phosphatidylinositol (PI) and the breakdown of polyphosphoinositides (poly PI), and (b) the effects of these cations on the specific radioactivity of [.gamma.-32P]ATP. Incorporation of 32Pi into ATP and phosphoinositides is time-dependent, and it is remarkably dependent upon Na+ concentration in the incubation medium. The Na+ effect is reversible. Ca2+, in the absence of Na+, had no effect on the specific radioactivity of ATP in 32P-labeled iris muscle; however, it moderately stimulated the 32P labeling of PA and PI and the breakdown of poly PI. In contrast, the addition of Na+, in the presence or absence of Ca2+, significantly reduced the specific radioactivity of ATP and 32P labeling of phospholipids in the 32P-labeled iris muscle. Acetylcholine had no measurable effect on the specific radioactivity of ATP. It also stimulated the 32P labeling of PA and PI and the breakdown of poly PI in the 32P-labeled muscle only in the presence of both Na+ and Ca2+. Conceptually, in the rabbit iris, receptor-activated Ca2+ fluxes mediate or precede the effects of .alpha.-adrenergic and cholinergic muscarinic agents on phosphoinositid breakdown into 1,2-diacylglycerol and inositol phosphates and restoration of the polar head groups to the 1,2-diacylglycerol (i.e., the recovery stage) is probably associated with Na+ outflux, via the Na+-pump mechanism.