Previous studies have demonstrated that prostaglandin E
2 (PGE
2) inhibits arginine vasopressin-(AVP)dependent adenosine 3,5-cyclic monophosphate (cAMP) accumulation in microdissected rat outer medullary collecting tubules (OMCD), by a mechanism unrelated to the inhibition of cAMP synthesis. The potential role of the activation of protein kinase C (PKC) to explain the negative regulation elicited by PGE
2 was investigated in this study. Single OMCD samples were pre-incubated (10 min, 30°C) in the presence or absence of either activators of PKC, phorbol 12-myristate 13-acetate (PMA), 1-oleoyl-2-acetyl-glycerol (OAG), dioctanoylglycerol (DOG) or an inhibitor of PKC, staurosporine (SSP). These compounds were present also with the agonists tested during the incubation period (4 min, 35°C). In contrast to PGE
2, activators of PKC did not decrease AVP-dependent cAMP accumulation (mean ±SEM): 1nM AVP=47.1±6.8 fmol · mm
–1· 4 min
–1; AVP + 0.3 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 g/ml OAG=44.1±4.8. NS versus AVP,
N= 5 experiments. However, 10 nM PMA prevented PGE
2-induced 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 results were obtained with either 50 g/ml OAG or 25 g/ ml DOG (AVP + PGE
2 + OAG=92.9±6.6% of the response to AVP,
N= 8; AVP + PGE
2 + DOG=94.1 ±5.3%,
N= 7). OAG, DOG, PMA or PMA + PGE
2 had no intrinsic agonist activity in the rat OMCD and the addition of an inactive phorbol ester did not prevent PGE
2-induced inhibition. SSP, 50 nM or 0.1 M, did not affect the inhibition due to PGE
2 but abolished the reversion by PMA of PGE
2-induced inhibition. A similar regulation was observed on forskolin-(FK)dependent cAMP accumulation: 5 M FK + 0.3 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 M SSP=43.1±7.9%,
N= 4. The inhibition induced by an
2-adrenergic agonist, clonidine 1 M, was not blocked by the activation of PKC. In fura-2-loaded OMCD samples, 10nM PMA decreased by 63.3±5.0% and by 57.2±7.1% the peak and plateau phases, respectively, of the increase in intracellular calcium concentration ([Ca
2+]
i) obtained with PGE
2 when compared to control responses in the same tubules (
n=12) and did not affect the increase in [Ca
2+]
i induced by 0.1 mM carbachol. It is concluded that: (1) in the rat OMCD the activation of PKC by PMA or analogues of diacylglycerol did not reproduce PGE
2-induced inhibition of AVP- or FK-dependent cAMP accumulation, but prevented specifically this inhibitory action; and (2) this reversion might be the consequence of the effect of PKC activation which impaired the rises in [Ca
2+]
i induced by PGE
2.
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