Mechanism and metabolic disposition of verapamil-evoked overflow of radioactivity from isolated atria preloaded with [³H]norepinephrine

In this study the mechanism and metabolic profile of verapamil-evoked release of radioactivity was investigated in the rat isolated atria preloaded with [³H]norepinephrine ([³H]NE). Verapamil (10^-7 to 10^-3 M) caused a dose-related increase in the outflow (or the fractional release) of ³H. The fractional ³H-release produced by verapamil was reduced markedly in tissues which had been preloaded with [³H]NE in the presence of cocaine (10 μM) or after pretreatment of animals with reserpine (5 mg/kg i.p., 24 hr before sacrifice). Verapamil-evoked fractional ³H-release was unchanged in the presence of of tetrodotoxin (5 x 10^-6 M) or in Ca⁺⁺-free Krebs' medium containing 2 mM ethylene glycol bis(β-aminoethyl ether)N,N'-tetraacetic acid. Whereas >90% of tissue ³H-content consisted of unchanged [³H]NE, 60 to 75% of the spontaneous outflow and the verapamil-evoked overflow consisted of [³H]-3,4-dihydroxyphenylglycol and 2 to 10% was unchanged [³H]NE. When both cocaine (10 μM) and hydrocortisone (28 μM) (uptake-1 and uptake-2 blockers, respectively) were present, although the spontaneous outflow, as well as verapamil-evoked overflow, of radioactivity was increased, the metabolic profiles remained essentially unchanged. The addition of pargyline (10 μM), a monoamine oxidase inhibitor, in addition to the uptake-1 and uptake-2 blockers to the Krebs' solution significantly depressed both the spontaneous outflow and verapamil-evoked overflow of ³H; the verapamil-evoked overflow under this condition, however, consisted of unchanged [³H]NE (>90%). The time course of evoked release of radioactivity by verapamil and its metabolic profile were compared to those of reserpine (10^-6 M) and tyramine (10^-4 M). Unlike verapamil and tyramine, reserpine produced a sustained release of ³H. However, the metabolic profile of verapamil-evoked overflow of radioactivity was found to be similar to that evoked by reserpine. With both drugs, the evoked overflow of ³H consisted mostly of [³H]-3,4-dihydroxyphenylglycol (60-75%), a deaminated metabolite. These findings indicate that 1) verapamil-evoked NE overflow originates primarily from neuronal sites, more specifically the NE storage granules, and 2) verapamil-evoked NE release does not involve initiation of action potentials (i.e., fast NA channels), is independent of the extracellular calcium concentration and does not appear to involve exocytosis. That the evoked release consisted mainly of [³H]-3,4-dihydroxyphenylglycol indicates that verapamil causes release of [³H]NE intraneuronally and thus produces a reserpine-like effect on sympathetic nerves, in addition to being a calcium channel blocker.