Pardaxin induces exocytosis in bovine adrenal medullary chromaffin cells independent of calcium

Pardaxin, an excitatory neurotoxin, is a new tool for studying the machinery of neurotransmitter secretion. At noncytotoxic concentrations (<1 x 10^-5 M), pardaxin stimulated exocytosis, as assessed by the concomitant release of catecholamines, ATP and dopamine-β-hydroxylase from bovine adrenal medullary chromaffin cells in the presence or absence of extracellular calcium. At higher concentrations (>2 x 10^-5 M), pardaxin was increasingly cytotoxic, as inferred from trypan blue uptake, release of lactate dehydrogenase and ⁵¹Cr (ED₅₀ = 100 μM). The role of intracellular calcium ([Ca⁺⁺](i)) homeostasis in pardaxin action was investigated by using the fluorescent calcium indicator Fura-2. In the presence of extracellular calcium, addition of noncytotoxic concentrations of pardaxin yielded a steady, concentration-dependent rise in [Ca⁺⁺](i) (ED₅₀ = 1 μM). Depolarization of chromaffin cells by high K⁺ reduced pardaxin binding and abolished the pardaxin-evoked rise in [Ca⁺⁺](i). In the absence of extracellular calcium, pardaxin failed to elicit an elevation of [Ca⁺⁺](i). These data suggest that, in the presence of extracellular calcium, pardaxin might cause elevations in [Ca⁺⁺](i) and neurotransmitter release, concomitant with inducing transmembranal Ca⁺⁺ influx. However, the complex concentration dependence of [Ca⁺⁺](i) and the fact that pardaxin stimulated secretion without a rise of [Ca⁺⁺](i) suggest that the toxin, in addition to being a pore-forming molecule, might directly affect exocytosis in a Ca⁺⁺-independent way. In proposing a pharmacological working model, we hypothesize that pardaxin might present a molecular structure which mimics an essential step in the endogenous docking mechanism between secretory granules and the plasma membrane.