Effects of arachidonic acid on the gap junctions of neonatal rat heart cells

Myocytes were isolated from neonatal rat hearts and grown in tissue-culture dishes for 1–2 days. Spontaneously formed cell pairs were used to study the conductance of gap junctions. The experiments involved a double voltage-clamp approach and whole-cell, tight-seal recording. Exposure to arachidonic acid (AA) produced a quasi dose-dependent decrease in junctional conductance, g_j (binding constant, K_d=4 M; Hill coefficient, n = 0.75). AA-dependent uncoupling was reversible. Addition of 1 mg/ml albumin to the bath solution accelerated the recovery. During control, cell pairs exhibited a gradual decrease in g_j (16.4 % in 6 min). Exposure to 20 M 4-bromophenacyl bromide, a phospholipase inhibitor, suppressed the decay in g_j (1.8% in 6 min), suggesting that endogenous AA may be involved in spontaneous uncoupling. The effect of AA on g_j was specific. Arachidic acid (100 M) and arachidonamide (10 M), structural analogues of AA, had no effect on g_j. Currents recorded shortly before complete uncoupling caused by AA, or early during recovery from uncoupling, revealed random opening and closing of single channels. The single channel conductance, _j, was not affected by the concentration of AA (1 M–100 M). The mean _j turned out to be 33.5 pS. The results suggest that AA-dependent uncoupling was caused via decrease in open channel probability, presumably mediated by a direct action on channel proteins.