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 microM) produced a fall in intracellular chloride ([Cl‐]i), and a hyperpolarization of membrane potential (Em). However, [Cl‐]i 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 Em 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 Em. However, although [Cl‐]i fell more than with bumetanide alone, it remained significantly above equilibrium. Metolazone was without effect at 100 microM. 5. In the presence of bumetanide, ethacrynic acid and N‐ethyl maleimide caused a dose‐dependent hyperpolarization of Em 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.