THE ACTIVATION OF PROTEIN-KINASE-C PREVENTS PGE(2)-INDUCED INHIBITIONOF AVP-DEPENDENT CAMP ACCUMULATION IN THE RAT OUTER MEDULLARY COLLECTING TUBULE

Previous studies have demonstrated that prostaglandin E(2) (PGE(2)) in hibits arginine vasopressin(AVP)dependent adenosine 3',5'-cyclic monop hosphate (cAMP) accumulation in microdissected rat outer medullary col lecting tubules (OMCD), by a mechanism unrelated to the inhibition of cAMP synthesis. The poten tial role of the activation of protein kinas e C (PKC) to explain the negative regulation elicited by PGE(2) was in vestigated in this study. Single OMCD samples were pre-incubated (10 m in, 30 degrees C) in the presence or absence of either activators of P KC, phorbol 12-myristate 13 acetate (PMA), 1 -oleoyl-2-acetyl-glycerol (GAG), dioc tanoylglycerol (DOG) or an inhibitor of PKC, staurosporin e (SSP). These compounds were present also with the agonists tested du ring the incubation period (4 min, 35 degrees C). In contrast to PGE(2 ), activators of PKC did not decrease AVP-dependent cAMP accumulation (mean +/- SEM): 1 nM AVP = 47.1 +/- 6.8 fmol.mm(-1) 4 min(-1); AVP + 0 .3 mu M PGE(2) = 20.1 +/- 2.7, P < 0.01 versus AVP; AVP + 10 nM PMA = 42.0 +/- 4.7, NS versus AVP; AVP + 50 mu g/ml OAG = 44.1 +/- 4.8. NS v ersus AVP, N = 5 experiments. However, 10 nM PMA prevented PGE(2)-indu ced inhibition: AVP + PGE(2) 44.2 +/- 3.5% of the response to AVP and 90.3 +/- 3.2% without and with PMA respectively, N = 16. Similar resul ts were obtained with either 50 mu g/ml OAG or 25 mu g/ ml DOG (AVP PGE(2) + OAG = 92.9 +/- 6.6% of the response to AVP, N= 8; AVP + PGE, + DOG = 94.1 +/- 5.3%, N= 7). OAG, DOG, PMA or PMA + PGE(2) had no int rinsic agonist activity in the rat OMCD and the addition of an inactiv e phorbol ester did not prevent PGE(2)-induced inhibition. SSP, 50 nM or 0.1 mu M, did not affect the inhibition due to PGE, but abolished t he reversion by PMA of PGE(2)-induced inhibition. A similar regulation was observed on forskolin-(FK)dependent cAMP accumulation: 5 mu M FK + 0.3 mu M PGE(2) = 37.7 +/- 6.2% of the response to FK; FK + PGE(2) 10 nM PMA = 89.5 +/- 6.7%; FK + PGE(2) + PMA + 0.1 mu M SSP = 43.1 +/ - 7.9%, N = 4. The inhibition induced by an alpha(2)-adrenergic agonis t, clonidine 1 mu M, was not blocked by the activation of PKC. In fura -2-loaded OMCD samples, 10 nM PMA decreased by 63.3 +/- 5.0% and by 57 .2 +/- 7.1% the peak and pla teau phases, respectively, of the increas e in intracellular calcium concentration ([Ca2+](i)) obtained with PGE (2) when compared to control responses in the same tubules (n = 12) an d did not affect the increase in [Ca2+](i) induced by 0.1 mM carbachol . It is concluded that: (1) in the rat OMCD the activation of PKC by P MA or analogues of diacylglycerol did not reproduce PGE(2)-induced inh ibition of AVP- or FK-dependent cAMP accumulation, but prevented speci fically this inhibitory action; and (2) this reversion might be the co nsequence of the effect of PKC activation which impaired the rises in [Ca2+]( )(i)induced by PGE(2).