title: LLC-PK(1) cells stably expressing the human norepinephrine transporter: A functional model of carrier-mediated norepinephrine release in protracted myocardial ischemia.
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-PK(1) cell line stably transfected with human NET cDNA (LLC-NET). LLC-NET cells transported [(3)H]NE and [(3)H]N-methyl-4-phenylpyridinium ([(3)H]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 (3)H-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, [(3)H]MPP(+) proved preferable to [(3)H]NE as an NET substrate; therefore, only [(3)H]MPP(+) was used for subsequent studies. The K(+)/H(+) ionophore nigericin (10 microM) evoked a large efflux of [(3)H]MPP(+). This efflux was potentiated by the Na(+),K(+)-ATPase inhibitor ouabain (100 microM), was sensitive to desipramine, and was blocked by the NHE inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; 10 microM). In contrast, EIPA failed to inhibit the [(3)H]MPP(+) efflux elicited by the Na(+) ionophore gramicidin (10 microM). Furthermore, [(3)H]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.
authors.affiliation: Department of Pharmacology, Cornell University, Weill Medical College, New York, New York.
authors.forename: N C
authors.initials: NC
authors.lastname: Smith
authors.forename: R
authors.initials: R
authors.lastname: Levi
mesh_terms: D015655:1-Methyl-4-phenylpyridinium / Q000378:metabolism; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active; D003891:Desipramine / Q000494:pharmacology; D004347:Drug Interactions; D006096:Gramicidin / Q000494:pharmacology; D006801:Humans; D054755:Imidazoline Receptors; D018374:LLC-PK1 Cells; D008454:Mazindol / Q000494:pharmacology; D008954:Models, Biological; D017202:Myocardial Ischemia / Q000378:metabolism*; D009550:Nigericin / Q000494:pharmacology; D009638:Norepinephrine / Q000378:metabolism*; D011955:Receptors, Drug / Q000378:metabolism; D012964:Sodium / Q000502:physiology; D013552:Swine; D013997:Time Factors
chemical_list: D054755:Imidazoline Receptors; D011955:Receptors, Drug; D006096:Gramicidin; D000584:Amiloride; D012964:Sodium; D008454:Mazindol; D015655:1-Methyl-4-phenylpyridinium; D009550:Nigericin; D003891:Desipramine; C039614:ethylisopropylamiloride; D009638:Norepinephrine