Induction by low Na⁺ or Cl⁻ of cocaine sensitive carrier-mediated efflux of amines from cells transfected with the cloned human catecholamine transporters

Abstract

1. COS-7 cells transfected with the cDNA of the human dopamine transporter (DAT cells) or the human noradrenaline transporter (NAT cells) were loaded with [³H]-dopamine or [³H]-noradrenaline and superfused with buffers of different ionic composition.

2. In DAT cells lowering the Na⁺ concentration to 0, 5 or 10 mM caused an increase in ³H-efflux. Cocaine (10 μM) or mazindol (0.3 μM) blocked the efflux at low Na⁺, but not at 0 Na⁺. Lowering the Cl⁻ concentration to 0, 5 or 10 mM resulted in an increased efflux, which was blocked by cocaine or mazindol. Desipramine (0.1 μM) was without effect in all the conditions tested.

3. In NAT cells, lowering the Na⁺ concentration to 0, 5 or 10 mM caused an increase in ³H-efflux, which was blocked by cocaine or mazindol. Desipramine produced a partial block, its action being stronger at 5 or 10 mM Na⁺ than at 0 mM Na⁺. Efflux induced by 0, 5 or 10 mM Cl⁻ was completely blocked by all three uptake inhibitors.

4. In cross-loading experiments, 5 mM Na⁺- or 0 Cl⁻-induced efflux was much lower from [³H]-noradrenaline-loaded DAT, than NAT cells and was sensitive to mazindol, but not to desipramine. Efflux from [³H]-dopamine-loaded NAT cells elicited by 5 mM Na⁺ or 0 Cl⁻ was blocked by mazindol, as well as by desipramine.

5. Thus, cloned catecholamine transporters display carrier-mediated efflux of amines if challenged by lowering the extracellular Na⁺ or Cl⁻, whilst retaining their pharmacological profile. The transporters differ with regard to the ion dependence of the blockade of reverse transport by uptake inhibitors.

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