PT  - JOURNAL ARTICLE
AU  - Smith, Neil C. E.
AU  - Levi, Roberto
TI  - LLC-PK<sub>1</sub> Cells Stably Expressing the Human Norepinephrine Transporter: A Functional Model of Carrier-Mediated Norepinephrine Release in Protracted Myocardial Ischemia
DP  - 1999 Nov 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - 456--463
VI  - 291
IP  - 2
4099  - http://jpet.aspetjournals.org/content/291/2/456.short
4100  - http://jpet.aspetjournals.org/content/291/2/456.full
SO  - J Pharmacol Exp Ther1999 Nov 01; 291
AB  - 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. The American Society for Pharmacology and Experimental Therapeutics