We used amperometry to analyze the role of several second messengers and drugs in the exocytotic kinetics of bovine chromaffin cells. Activation of PKG produces a slowing down of exocytosis, which is not generally accompanied by changes in the net granule content of catecholamines. These effects are also observed after mild PKA activation. However, strong PKA stimulation also causes an increase in the apparent granule content of catecholamines, suggesting the presence of composed fusion. Conversely, PKC activation promotes acceleration of the exocytotic process. We also analyzed the contribution of different Ca(2+) channel subtypes to the exocytotic kinetics at the single event level. Although N-subtype channels do not contribute to total catecholamine release, their blockade produces a slowing down of exocytosis without changes in granule content. However, L or P/Q blockade causes, in addition, a reduction in the apparent granule content. The L-type agonist BAY-K-8644 produces giant secretory amperometric spikes, indicating that Ca(2+) favors composed fusion prior to exocytosis. Our data suggest that second messengers continuously regulate exocytotic kinetics and granule content. In addition, several well-known antihypertensive agents, such as sodium nitroprusside, organic nitrates, hydralazine, or Ca(2+) antagonists, could be acting through these novel mechanisms on sympathetic synapses by changing the synaptic performance, thereby producing additional vasodilatory effects.

Abstract-The effects of the antihypertensive agent hydralazine (1 to 100 nmol/L) on the exocytotic process of single adrenal chromaffin cells have been studied using amperometry. Hydralazine does not reduce the frequency of exocytotic spikes but rapidly slows the rate of catecholamine release from individual exocytotic events by reducing the quantal size of catecholamine exocytosis. Confocal and standard epifluorescence microscopy studies show that hydralazine rapidly accumulates within secretory vesicles. The blockade of the vesicular H ϩ pump with bafilomycin A 1 inhibits hydralazine uptake. Experiments with permeabilized cells show that hydralazine displaces catecholamines from secretory vesicles. The drug also displaces vesicular Ca 2ϩ , as shown by fura-2 microfluorimetry. These data suggest that hydralazine acts, at least partially, by interfering with the storage of catecholamines. These effects of hydralazine occurred within seconds, and at the tissue concentrations presumably reached in antihypertensive therapy; these concentrations are a thousand times lower than those described for relaxing vascular tissues in vitro. We proposed that these novel effects could explain many of the therapeutic and side effects of this drug that are likely exerted in sympathetic nerve terminals.