Effects of Corticotropin-(1?24)-Tetracosapeptide on Polyphosphoinositide Metabolism and Protein Phosphorylation in Rabbit Iris Subcellular Fractions

Abstract

Effects of the neuropeptide corticotropin-(1-24)-tetracosapeptide (ACTH) on the endogenous and exogenous phosphorylation of lipids and endogenous phosphorylation of proteins were investigated in microsomes and a 110,000 .times. g supernatant fraction [30-50% (NH4)2SO4 precipitate; ASP30-50] obtained from rabbit iris smooth muscle. Subcellular distribution studies revealed that both of these fractions are enriched in diphosphoinositide (DPI) kinase. The 32P labeling of lipids and proteins was measured by incubation of the subcellular fractions with [.gamma.-32P]ATP. The labeled lipids, which consisted of triphosphoinositide (TPI), DPI, and phosphatidic acid (PA) were isolated by TLC. The microsomal and ASP30-50 fractions were resolved into 6 and 9 labeled phosphoprotein bands, respectively, by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The basal labeling of both lipids and proteins was rapid (30-60 s), and it was dependent on the presence of Mg2+ in the incubation medium; in general it was inhibited by high concentrations (> 0.2 mM) of Ca2+. ACTH stimulated the labeling of TPI and inhibited that of PA in a dose-dependent manner, with maximal effect observed at 50-100 .mu.M of the peptide. ACTH appears to increase TPI labeling by stimulating the DPI kinase. Under the same experimental conditions ACTH (100 .mu.M) inhibited significantly the endogenous phosphorylation of 6 microsomal phosphoproteins (100K [kDalton], 84K, 65K, 53K, 48K, and 17K). In the ASP30-50 fraction, ACTH inhibited the phosphorylation of 3 phosphoproteins (53K, 48K, and 17K) and stimualted the labeling of 6 phosphoprotein bands (117K, 100K, 84K, 65K, 42K, and 35K). The effects of ACTH on lipid and protein phosphorylation are probably Ca2+-independent; thus the neuropeptide effects were not influenced by either 1 mM EGTA [ethyleneglycol bis[.beta.-aminoethyl ether) N,N,N'',N''-tetraacetic acid] or low concentrations of Ca2+ (50 .mu.M). A relationship may exist between polyphosphoinositide metabolism and protein phosphorylation in the rabbit iris smooth muscle.