Effect of Various Divalent Ions on the Calcium Current of Adrenal Medullary Chromaffin Cells in the Rat

Effect of Various Divalent Ions on the Calcium Current of Adrenal Medullary Chromaffin Cells in the Rat

It is well known that chromaffin cells of adrenal medulla secrete catecholamine in response to sympathetic nerve activation and the influx of Ca²⁺ through the voltage dependent Ca²⁺ channels (VDCC) in the cell membrane do a major role in this secretory process. In this study, we explored the effect of divalent cations on VDCC of rat chromaffin cells. Rat (Sprague-Dawley rat, 150-250 gm) chromaffin cells were isolated and cultured. Standard giga seal, whole cell recording techniques were employed to study Ca²⁺ current with external and internal solutions that could effectively isolate VDCC currents (NMG in external and TEA and Cs²⁺ in internal solution). The voltage dependence and the inactivation time course of VDCC in our cells were identical to those of bovine chromaffin cells. A persistent inward current was first activated by depolarizing step pulse from the holding potential (H.P.) of -80 mV to -40 mV, increased to maximum amplitude at around +10 mV, and became smaller with progressively higher depolarizing pulses to reverse at around +60 mV. The inactivation time constant (τ), fitted from the long duration test potential (2 sec) was 1295.2±126.8 msec (n=20, 1 day of culture, mean ±S.E.M.) and the kinetic parameters were not altered along the culture duration. Nicardipine (10 μM) blocked the current almost completely. Among treated divalent cations such as Cd²⁺, Co²⁺, Ni²⁺, Zn²⁺ and ,Mn²⁺, Cd²⁺ was the most potent blocker on VDCC. When the depolarizing step pulse from -80 mV to 10 mV was applied, the equilibrium dissociation constant (K_d) of Cd²⁺ was 39 μM, K_d of Co²⁺ was 100 μM and K_d of Ni²⁺];was];780μM. The principal findings of this study are as follows. First, the majority of Ca²⁺ channels in rat chromaffin cells are well classified to L-type Ca²⁺ channel in the view of kinetics and pharmacology. Second, all divalent cations tested could block the Ca²⁺ current and the most potent blocker among the tested was Cd²⁺.