ARACHIDONIC-ACID INHIBITS HORMONE-STIMULATED CAMP ACCUMULATION IN THEMEDULLARY THICK ASCENDING LIMB OF THE RAT-KIDNEY BY A MECHANISM SENSITIVE TO PERTUSSIS TOXIN

The possible regulation of adenosine 3',5'-cyclic monophosphate (cAMP) accumulation by arachidonic acid (AA) was studied in segments, microd issected from the rat kidney, which are sensitive to arginine vasopres sin (AVP). In the presence of 5 mu M indomethacin, the addition of 5 m u M AA did not impair AVP-dependent cAMP accumulation (measured during 4 min at 35 degrees C) in the cortical or outer medullary collecting tubule, but decreased this response in the thick ascending limb with a n inhibition much more pronounced in the medullary portion (MTAL) than in the cortical portion. In MTAL, the response to 10 nM AVP was inhib ited by 34.4+/-9.6% (SEM) and 65.8+/-5.4% with 1 mu M and 5 mu M AA, r espectively, N=5 experiments. AVP-, glucagon- and calcitonin-sensitive cAMP levels in MTAL were inhibited by 5 mu M AA to a similar extent. AA-induced inhibition was unaffected by the presence of inhibitors of AA metabolism: (1) either 10 mu M indomethacin or 50 mu M ibuprofen ad ded to all media; (2) a 10-min pre-incubation and a 4-min incubation o f MTAL samples with 10 mu M eicosa-5,8,11,14-tetrayonic acid, (3) a 1- h preincubation with either 30 mu M SKF-525A, 20 mu M ketoconazole, or 20 mu M nordihydroguariaretic acid. In contrast to AA, 11 other satur ated or unsaturated fatty acids had no inhibitory effect on the AVP-de pendent cAMP level. In fura-2-loaded MTAL samples, AA induced a slow i ncrease of the intracellular calcium concentration ([Ca2+](i)) which r eached 21.0+/-3.8 nM and 92.9+/-21.4 nM over basal values (n=11) at 2 min and 4 min, respectively, after the beginning of the superfusion of 5 mu M AA. AA-induced inhibition of AVP-dependent cAMP accumulation w as due neither to the increase in [Ca2+](i) elicited by AA, nor to an activation of protein kinase C because this inhibition: (1) was not bl ocked when MTAL samples were incubated either in zero Ca2+ medium, or in the presence of 1,2-bis(2-aminophenoxy)ethane-N, N, N', N'-tetraace tic acid (BAPTA) to chelate [Ca2+](i) and (2) it was not reproduced by a pre-treatment of MTAL segments with a phorbol ester. Pre-incubation of MTAL (6 h at 35 degrees C) with 500 ng/ml pertussis toxin (PTX) pr evented AA-induced inhibition: in the presence of PTX inhibition was 2 4.7+/-6.6% vs 10 nM AVP, as compared to 81.6+/-4.0% in control groups, i.e in the absence of PTX, N=6. AA had no effect on the cAMP level in duced by 5 mu M forskolin. It is concluded that AA inhibits AVP-depend ent cAMP accumulation in the rat MTAL by a mechanism which implicates a GTP-dependent protein sensitive to PTX.