Vol. 291, Issue 2, 456-463, November 1999

LLC-PK₁ Cells Stably Expressing the Human Norepinephrine Transporter: A Functional Model of Carrier-Mediated Norepinephrine Release in Protracted Myocardial Ischemia¹

Neil C. E. Smith and Roberto Levi

Department of Pharmacology, Cornell University, Weill Medical College, New York, New York

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 of ³H-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.