IDENTIFICATION OF THE MECHANISM FOR THE INHIBITION OF NA+,K+-ADENOSINE TRIPHOSPHATASE BY HYPERGLYCEMIA INVOLVING ACTIVATION OF PROTEIN-KINASE-C AND CYTOSOLIC PHOSPHOLIPASE A(2)

Inhibition of Na+,K+ -ATPase activity by hyperglycemia could be an important etiological factor of chronic complications in diabetic patients, The biochemical mechanism underlying hyperglycemia's inhibitory effects has been thought to involve the alteration of the protein kinase C (PKC) pathway since agonists of PKC can normalize hyperglycemia-induced inhibition of Na+,K+-ATPase activity, Paradoxically, elevated glucose levels and diabetes have been shown to increase PKC activities in vascular cells, The present study tested the hypothesis that the inhibition of Na+,K+-ATPase activity is mediated by the sequential activation of PKC and cystolic phospholipase A(2) (cPLA(2)). In cultured rat vascular smooth muscle cells (VSMC),increasing glucose levels in the medium from 5.5 to 22 mM elevated cPLA(2) activity and increased [H-3]arachidonic acid release and PGE(2) production by 2.3-, 1.7- and 2-fold, respectively, Similar increases in cPLA(2) activity were also induced by elevated glucose levels in human VSMC and rat capillary endothelial cells, The activation of cPLA(2) was mediated by PKC since the increases in cPLA(2) phosphorylation and enzymatic activity were inhibited by the PKC inhibitor GFX. In contrast, elevation of glucose levels decreased Na+,K+-ATPase activity as measured by ouabain-sensitive Rb-86 uptake by twofold in rat VSMC, Surprisingly, both PMA, a PKC agonist, and GFX, a PKC inhibitor, were able to prevent glucose-induced decreases in Rb-86 uptake, Further, the PLA(2) inhibitor AACOCF(3) abolished both glucose-induced activation of cPLA(2), and the decrease in Rb-86 uptake, These data indicated that hyperglycemia is inhibiting Na+,K+-ATPase activity by the sequential activation of PKC and cPLA(2), resulting in the liberation of arachidonic acid and increased the production of PGE(2), which are known inhibitors of Na+, K+- ATPase.