ARACHIDONIC-ACID INHIBITS HORMONE-STIMULATED CAMP ACCUMULATION IN THE MEDULLARY 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, microdissected from the rat kidney, which are sensitive to arginine vasopressin (AVP). In the presence of 5 mu M indomethacin, the addition of 5 mu 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 an inhibition much more pronounced in the medullary portion (MTAL) than in the cortical portion. In MTAL, the response to 10 nM AVP was inhibited by 34.4+/-9.6% (SEM) and 65.8+/-5.4% with 1 mu M and 5 mu M AA, respectively, 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 added to all media; (2) a 10-min pre-incubation and a 4-min incubation of 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 saturated or unsaturated fatty acids had no inhibitory effect on the AVP-dependent cAMP level. In fura-2-loaded MTAL samples, AA induced a slow increase of the intracellular calcium concentration ([Ca2+](i)) which reached 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 was 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 blocked 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'-tetraacetic 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) prevented AA-induced inhibition: in the presence of PTX inhibition was 24.7+/-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 induced by 5 mu M forskolin. It is concluded that AA inhibits AVP-dependent cAMP accumulation in the rat MTAL by a mechanism which implicates a GTP-dependent protein sensitive to PTX.