CA2- ACTION AND INTERACTION OF PROTEIN-KINASE-A AND PROTEIN-KINASE-C(TRANSPORT BY THE LUMINAL MEMBRANE OF THE DISTAL NEPHRON )

We previously reported that parathyroid hormone and calcitonin increas e Ca2+ uptake by purified distal luminal membranes. This effect is mim icked by high concentrations of cAMP. However, both hormones stimulate adenylate cyclase and phospholipase C. The purpose of the present stu dy was to investigate the role of the phospholipase C pathway in the h ormone action, and the interrelationship between the two messengers. D istal tubules from rabbis kidneys were incubated with dibutyryl cAMP ( dbcAMP) or PMA, or both, and Ca2+ uptake by purified luminal membranes was measured by the rapid filtration technique. Incubation of the dis tal tubules with 1 mM dbcAMP significantly increased Ca2+ transport by the luminal membranes. A dose-response curve showed a half-maximal st imulation with 0.82 mM dbcAMP. In contrast, treatment of the tubules w ith 10 nM, 100 nM or 1 mu M PMA did not influence Ca2+ uptake by these membranes. However, the addition of 100 nM PMA to low concentrations of dbcAMP strongly increased this uptake. The presence of cAMP or prot ein kinase C inhibitors prevented the effects of either a high concent ration of dbcAMP alone or a low concentration of dbcAMP combined with 100 nM PMA. Our laboratory has already reported that Ca2+ uptake by th e distal luminal membranes displays two-component kinetics, dbcAMP inc reased the V-max of the low-affinity component, whereas a combination of the two messengers stimulated the V-max of both the low- and high-a ffinity components. From these results, we conclude that: (1) in the d istal tubule cells, activation of both protein kinases A and C is nece ssary for the stimulation of Ca2+ transport by the luminal membrane; ( 2) the combined effect of protein kinases A and C involves both compon ents of the Ca2+-transport kinetics.