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 t he absence of extracellular Ca2+, the secretory effects of Zn2+ were e nhanced. At low concentrations (3-10 mu M), Zn2+ enhanced the secretor y responses to 10-s pulses of 100 mu M 1,1-dimethyl-4-phenylpiperazini um (DMPP, a nicotinic receptor agonist) or 100 mM K+. In the presence of DMPP, secretion was increased 47% above controls and in high-K+ sol utions, secretion increased 54% above control. These low concentration s of Zn2+ did not facilitate the whole-cell Ca2+ (I-ca) or Ba2+ (I-Ba) currents in patch-clamped chromaffin cells. Higher Zn2+ concentration s inhibited the currents (IC50 values, 346 mu M for I-ca and 91 mu M f or I-Ba) and blocked DMPP- and K+-evoked secretion (IC50 values, 141 a nd 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, 73 4, 330 and 7.4 pA, respectively. Zn2+ entry was also evidenced using t he fluorescent Ca2+ probe fura-2. This was possible because Zn2+ cause s 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 ac celerated by stimulation with DMPP or high-K+ solution. The entry of Z n2+ was concentration dependent, slightly antagonized by 1 mM Ca2+ and completely blocked by 5 mM Ni2+. The entry of Ca2+ evoked by depolari zation with high-K+ solution was antagonized by Zn2+. We conclude that inhibition by Zn2+ of evoked catecholamine secretion is associated wi th blockade of Ca2+ entry through Ca2+ channels recruited by DMPP or K +. However, the facilitation of secretion observed at low Zn2+ concent rations, or in the absence of Ca2+, may be exerted at an intracellular site on the secretory machinery. This is plausible because Zn2+ perme ates the bovine chromaffin cell Ca2+ channels and in this way gains ac cess to the cytosol. In addition, we have established conditions for m easuring Zn2+ transients in fura-2-loaded cells with a very high sensi tivity, taking advantage of the high-affinity binding of Zn2+ to fura- 2 and the modification of its fluorescence spectrum.