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.
Footnotes
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Send reprint requests to: Roberto Levi, M.D., Department of Pharmacology, Cornell University, Weill Medical College, 1300 York Ave., New York, NY 10021. E-mail: rlevi{at}med.cornell.edu
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↵1 This work was supported by National Institutes of Health Grants HL34215 and HL46403. A preliminary version of these findings was presented at the 71st Scientific Sessions of the American Heart Association, November 1998, and was published in abstract form inCirculation (1998) 98:I-681.
- Abbreviations:
- NE
- norepinephrine
- NHE
- Na+-H+ exchanger
- COMT
- catechol-O-methyl transferase
- DMI
- desipramine
- hNET
- human NET
- EIPA
- 5-(N-ethyl-N-isopropyl)-amiloride
- LLC-NET
- LLC-PK1 cells transfected with human norepinephrine transporter cDNA
- MPP+
- N-methyl-4-phenylpyridinium
- Nao
- extracellular Na+
- TTX
- tetrodotoxin
- NET
- norepinephrine transporter
- pHi
- intracellular pH
- Received March 18, 1999.
- Accepted July 6, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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