Voltage-dependent calcium channels in pituitary cells in culture. I. Characterization by 45Ca2+ fluxes

Properties of Ca2+ transport activated by depolarization of the membrane potential were investigated in the GH4C1 strain of rat pituitary cells. Membrane potential was depolarized by increasing external K+ concentration and was determined by [3H]tetraphenylphosphonium+ distribution. Depolarization by 50 mM [K+]o increased the initial rate of 45Ca2+ uptake 5-fold and the steady state 45Ca2+ content 8-fold. Stimulated 45Ca2+ uptake was not inhibited by tetrodotoxin (2 X 10(-6) M), and was insensitive to external Na+ concentration. Stimulated 45Ca2+ uptake increased with increasing external Ca2+ concentration (for [Ca2+]o less than 10 mM) and could be described by a Langmuir type expression (KCa = 4.3 mM). Initial rates of 45Ca2+ uptake increased almost linearly between -51 and -30 mV, from 2 to 12 nmol/min/mg of protein, and a maximum of 14 nmol/min/mg of protein was reached at -12 mV beyond which 45Ca2+ influx decreased, and was 11 nmol/min/mg of protein at 0 mV. Ca2+ permeability, PCa, calculated from the Goldman-Hodgkin-Katz constant field expression increased almost linearly for a wide range of membrane potential (-51 to -20 mV) and began to level off at -6 mV. Activated 45Ca2+ uptake was completely inhibited by La3+, Co2+, Mn2+, Mg2+, nifedipine, and verapamil; K1/2 values for inhibition were 1.7 X 10(-7) M, 0.1 mM, 0.1 mM, 2 mM, 1.7 X 10(-8) M, and 2 X 10(-5) M, respectively, at 0.5 mM [Ca2+]o. Ba2+ could substitute for Ca2+ in the uptake mechanism. The increase in activated 45Ca2+ uptake was transient and was turned off with time. We conclude that the initial rate of K+-stimulated 45Ca2+ uptake measured under the experimental conditions described represents uptake via voltage-dependent Ca2+ channels. Knowledge of the properties of this channel in GH4C1 cells will be essential in elucidating its role in the Ca2+-dependent actions of thyrotropin-releasing hormone.