Characterization of interactions of methylmercury with Ca2+ channels in synaptosomes and pheochromocytoma cells: radiotracer flux and binding studies

TJ Shafer, ML Contreras and WD Atchison

Department of Pharmacology, Michigan State University, East Lansing 48824.

The interaction of methylmercury (MeHg) with neuronal Ca2+ channels in rat forebrain synaptosomes and dihydropyridine (DHP)-sensitive Ca2+ channels in rat pheochromocytoma (PC12) cells was examined using radiotracer flux assays and radioligand binding analyses. In synaptosomes, the influx of 45Ca2+ was used to examine the voltage and state dependence of block of Ca2+ channels by MeHg, as well as the effects of MeHg on apparent inactivation of 45Ca2+ influx. In addition, the differential influx of 45Ca2+, 85Sr2+, and 133Ba2+ was used to examine the effect of MeHg on the ionic selectivity of synaptosomal Ca2+ channels. The ability of MeHg to block 45Ca2+ influx via a DHP- sensitive Ca2+ channel was examined in PC12 cells. Effects of MeHg on binding of [3H]nitrendipine in synaptosomes and 125I-omega-conotoxin GVIA (CgTx) in synaptosomes and PC12 cells were measured. In synaptosomes, MeHg blocked 45Ca2+ influx in a voltage-dependent manner, inasmuch as increasing the extracellular K+ concentration increased the magnitude of block by 100 microM MeHg. When synaptosomes were incubated for 10 sec in either a nondepolarizing or a depolarizing solution before measurement of 1 sec of depolarization-induced 45Ca2+ influx, the potency and efficacy of the block of 45Ca2+ influx by MeHg were similar. Thus, block of Ca2+ channels by MeHg does not appear to be state dependent. To determine the kinetics of apparent inactivation of 45Ca2+ influx, synaptosomes were predepolarized in Ca2(+)-free high [K+] solution, for intervals varying from 1 to 10 sec, before measurement of 1 sec of K(+)-induced 45Ca2+ influx. When compared with control, MeHg (100 microM) altered the rate constant for apparent inactivation and decreased the fraction of 45Ca2+ influx that does not inactivate. Influx of 45Ca2+, 85Sr2+, and 133Ba2+ during 1 sec of depolarization was blocked in a dose-dependent manner by MeHg, with estimated IC50 values of 125, 150, and greater than 150 microM for 45Ca2+, 85Sr2+, and 133Ba2+, respectively. In triple-label experiments, the relative flux of radiolabeled Ca2+:Sr2+:Ba2+ was altered from approximately 6:2:3 to 6:1:3 in the presence of 100 microM MeHg. In undifferentiated and nerve growth factor-differentiated PC12 cells, K(+)-induced 45Ca2+ influx was blocked by the DHP nifedipine, with an approximate IC50 value of 5 nM. MeHg reduced 45Ca2+ influx in PC12 cells with an estimated IC50 value of 50 microM, and 125 microM MeHg reduced uptake by greater than 90%. [3H]Nitrendipine bound to synaptosomes with high affinity in normal and elevated [K+] solutions.(ABSTRACT TRUNCATED AT 400 WORDS)

Volume 38, Issue 1, pp. 102-113, 07/01/1990
Copyright © 1990 by American Society for Pharmacology and Experimental Therapeutics

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