PERMEATION BY ZINC OF BOVINE CHROMAFFIN CELL CALCIUM CHANNELS - RELEVANCE TO SECRETION

Zn2+ increased the rate of spontaneous release of catecholamines from bovine adrenal glands. This effect was Ca2+ independent; in fact, in the absence of extracellular Ca2+, the secretory effects of Zn2+ were enhanced. At low concentrations (3-10 mu M), Zn2+ enhanced the secretory responses to 10-s pulses of 100 mu M 1,1-dimethyl-4-phenylpiperazinium (DMPP, a nicotinic receptor agonist) or 100 mM K+. In the presence of DMPP, secretion was increased 47% above controls and in high-K+ solutions, secretion increased 54% above control. These low concentrations of Zn2+ did not facilitate the whole-cell Ca2+ (I-ca) or Ba2+ (I-Ba) currents in patch-clamped chromaffin cells. Higher Zn2+ concentrations inhibited the currents (IC50 values, 346 mu M for I-ca and 91 mu M for I-Ba) and blocked DMPP- and K+-evoked secretion (IC50 values, 141 and 250 mu M, respectively). Zn2+ permeated the Ca2+ channels of bovine chromaffin cells, although at a much slower rate than other divalent cations. Peak currents at 10 mM Ba2+, Ca2+, Sr2+ and Zn2+ were 991, 734, 330 and 7.4 pA, respectively. Zn2+ entry was also evidenced using the fluorescent Ca2+ probe fura-2. This was possible because Zn2+ causes an increase in fura-2 fluorescence at the isosbestic wavelength for Ca2+, i.e. 360 nm. There was a slow resting entry of Zn2+ which was accelerated by stimulation with DMPP or high-K+ solution. The entry of Zn2+ was concentration dependent, slightly antagonized by 1 mM Ca2+ and completely blocked by 5 mM Ni2+. The entry of Ca2+ evoked by depolarization with high-K+ solution was antagonized by Zn2+. We conclude that inhibition by Zn2+ of evoked catecholamine secretion is associated with blockade of Ca2+ entry through Ca2+ channels recruited by DMPP or K+. However, the facilitation of secretion observed at low Zn2+ concentrations, or in the absence of Ca2+, may be exerted at an intracellular site on the secretory machinery. This is plausible because Zn2+ permeates the bovine chromaffin cell Ca2+ channels and in this way gains access to the cytosol. In addition, we have established conditions for measuring Zn2+ transients in fura-2-loaded cells with a very high sensitivity, taking advantage of the high-affinity binding of Zn2+ to fura-2 and the modification of its fluorescence spectrum.