Chloride-dependence of the potency of inhibitors of the neuronal noradrenaline carrier in the rat vas deferens

Vasa deferentia obtained from reserpine-pretreated rats were exposed to 0.15 μmol 1⁻¹³H-(−)noradrenaline (with monoamine oxidase and catechol-O-methyltransferase being inhibited) and initial rates of the neuronal ³H-noradrenaline uptake as well as IC₅₀ values for inhibition of uptake by desipramine, cocaine or (−)metaraminol determined at various external Cl⁻ concentrations (0–145 mmol 1⁻¹) and a fixed high Na⁺ concentration (145 mmoll⁻¹).

When the Cl⁻ concentration in the medium was decreased neuronal uptake fell. As far as Cl⁻ concentrations ranging from 10 to 145 mmol 1⁻¹ are concerned, the dependence of uptake on Cl⁻ obeyed Michaelis-Menten kinetics with an apparent K_m and V_max of 6.2 mmol 1⁻¹ and 116 pmol g⁻¹ min⁻¹, respectively. At Cl⁻ concentrations below 10 mmol l⁻¹, uptake was higher than expected from the values of K_m and V_max, and even in the nominal absence of Cl⁻ from the medium a remainder of neuronal uptake was still detectable. Evidence is presented to show that, on incubation at Cl⁻ concentrations below 10 mmol l⁻¹, intracelluar Cl⁻ leaks out, so that the actual Cl⁻ concentrations in the extracellular fluid are probably higher than in the medium.

The potencies of desipramine and cocaine for inhibition of neuronal uptake were markedly dependent on the Cl⁻ concentration in the medium, but the type of Cl⁻ dependence differed. While the IC₅₀ for desipramine decreased, that for cocaine increased with increasing Cl⁻ concentration (2–145 mmol l⁻¹). The value of IC₅₀ for cocaine and that of 1/IC₅₀ for desipramine approached saturation (with an apparent Hill coefficient of about unity) when plotted against the Cl⁻ concentration; half-maximum values were observed at Cl⁻ concentrations of 9 and 24 mmol l⁻¹, respectively.

(−)Metaraminol (an alternative substrate of the noradrenaline carrier) remained equally potent in inhibiting neuronal uptake when the Cl⁻ concentration was decreased from 145 to 2 mmol l⁻¹. However, when Cl⁻ was omitted from the medium, the IC₅₀ for (−)metaraminol increased. Hence, the C⁻-dependence of the potency of (−)metaraminol appears to be restricted to very low extracellular Cl⁻ concentrations.

It is concluded that not only the neuronal uptake process itself, but also its inhibition by desipramine and cocaine are highly Cl⁻-dependent. Since desipramine and cocaine differ with respect to the type of Cl⁻-dependence of their inhibitory potency, they are likely to act by combining with distinctly different states of the noradrenaline carrier. It is suggested that desipramine interacts with the carrier loaded with Cl⁻ while cocaine is capable of interacting with its Cl⁻-free state.