Abstract

In myocardial ischemia, adrenergic terminals undergo ATP depletion, hypoxia, and intracellular pH reduction, causing the accumulation of axoplasmic norepinephrine (NE) and intracellular Na⁺ [via the Na⁺-H⁺ exchanger (NHE)]. This forces the reversal of the Na⁺- and Cl⁻-dependent NE transporter (NET), triggering massive carrier-mediated NE release and, thus, arrhythmias. We have now developed a cellular model of carrier-mediated NE release using an LLC-PK₁ cell line stably transfected with human NET cDNA (LLC-NET). LLC-NET cells transported [³H]NE and [³H]N-methyl-4-phenylpyridinium ([³H]MPP⁺) in an inward direction. This uptake was abolished by the NET inhibitors desipramine (100 nM) and mazindol (300 nM) and by extracellular Na⁺ removal. Na⁺-gradient reversal induced an efflux of³H-substrate from preloaded LLC-NET cells. Desipramine and mazindol blocked this efflux. Because of its greater intracellular stability and higher sensitivity to Na⁺-gradient reversal, [³H]MPP⁺ proved preferable to [³H]NE as an NET substrate; therefore, only [³H]MPP⁺ was used for subsequent studies. The K⁺/H⁺ ionophore nigericin (10 μM) evoked a large efflux of [³H]MPP⁺. This efflux was potentiated by the Na⁺,K⁺-ATPase inhibitor ouabain (100 μM), was sensitive to desipramine, and was blocked by the NHE inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; 10 μM). In contrast, EIPA failed to inhibit the [³H]MPP⁺ efflux elicited by the Na⁺ ionophore gramicidin (10 μM). Furthermore, [³H]MPP⁺ efflux induced by the NHE-stimulant proprionate (25 mM) was negatively modulated by imidazoline receptor activation. Our findings suggest that LLC-NET cells are a sensitive model for studying transductional processes of carrier-mediated NE release associated with myocardial ischemia.