Evidence against a contribution by Na+-Cl- cotransport to chloride accumulation in rat arterial smooth muscle

1. Chloride accumulation into rat saphenous arterial smooth muscle has been examined using chloride-sensitive microelectrodes, to assess the contribution of Na+-Cl- cotransport. 2. Bumetanide (10 mu M) produced a fall in intracellular chloride ([Cl-](i)), and a hyperpolarization of membrane potential (E(m)). However, [Cl-], remained above the equilibrium level predicted from the membrane potential, indicating a residual accumulation. 3. Replacement of extracellular sodium with N-methyl-D-glucamine or choline caused a fall in [Cl-](i) similar to that observed with bumetanide, but the hyperpolarization of E(m) was larger. In Na+-free media, bumetanide had no effect. [Cl-](i) remained significantly above equilibrium. 4. In the presence of bumetanide, chlorothiazide produced a further dose-dependent fall in [Cl-](i), and hyperpolarization of E(m). However, although [Cl-](i) fell more than with bumetanide alone, it remained significantly above equilibrium. Metolazone was without effect at 100 mu M. 5. In the presence of bumetanide, ethacrynic acid and N-ethyl maleimide caused a dose-dependent hyperpolarization of E(m) and a fall in [Cl-](i) to equilibrium. 6. The third inward chloride pump in rat saphenous arterial smooth muscle appears not to be a form of Na+-Cl- cotransport. The potency series of thiazide diuretic action (acetazolamide > chlorothiazide > metolazone) differed significantly from that published for Na+-Cl- cotransport, and there is no sodium dependence.