Acetylcholine-induced, calcium-dependent norepinephrine outflow from peripheral human lymphocytes

Abstract

Catecholamines (CA) were studied in peripheral human lymphocytes, as well as in the supernatants, after incubation with l-tyrosine and l-dihydroxyphenylalanine ( l-Dopa) for 1 h. The effect that the addition of acetylcholine (ACh), Veratridine, Ionomycin or KCl had on the outflow of norepinephrine (NE) from lymphocytes was also studied. The effect of the addition of methoxyverapamil (D600, a Ca 2+ channel blocker) and cholinergic antagonists had on the ACh-induced NE outflow was assessed. CA were determined by HPLC–ECD, both in the supernatant and in the cell lysates. l-Tyrosine and l-Dopa significantly ( P<0.01) increased intracellular NE. Neither l-tyrosine, l-Dopa, nor vehicle induced a detectable outflow of NE to the supernatants. ACh [120 μM], Veratridine [100 μM], Ionomycin [10 μM] and KCl [50 mM] (with or without the simultaneous addition of l-tyrosine or l-Dopa) all induced a detectable outflow of NE to the supernatant when added 5 min before the end of incubation. NE was not detectable in the supernatant when the chemicals were added 10 to 20 min before the end of the incubation. When the chemicals were added at lower concentrations, erratic secretion or no secretion whatsoever was observed. D600 [100 μM] was able to significantly ( P<0.01) reduce the ACh-induced NE outflow. Tetraethylammonium (nicotinic antagonist), but not atropine (muscarinic antagonist), significantly ( P<0.001) decreased the ACh-induced NE outflow. The outflow of NE from peripheral human lymphocytes was seen. NE secretion seems to be ACh- and calcium-dependent since Veratridine, Ionomycin and KCl are able to induce Ca 2+ entry by means of various mechanisms. The Ca 2+ channel blocker employed in this study (D600) reduced the ACh-dependent NE outflow. We can conclude that both ACh (through nicotinic receptors) and calcium are involved in the outflow of NE from peripheral human lymphocytes.