Exposing bovine chromaffin cells to a single 5 ns, high-voltage (5 MV/m) electric pulse stimulates Ca(2+) entry into the cells via L-type voltage-gated Ca(2+) channels (VGCC), resulting in the release of catecholamine. In this study, fluorescence imaging was used to monitor nanosecond pulse-induced effects on intracellular Ca(2+) level ([Ca(2+)](i)) to investigate the contribution of other types of VGCCs expressed in these cells in mediating Ca(2+) entry. ω-Conotoxin GVIA and ω-agatoxin IVA, antagonists of N-type and P/Q-type VGCCs, respectively, reduced the magnitude of the rise in [Ca(2+)](i) elicited by a 5 ns pulse. ω-conotoxin MVIIC, which blocks N- and P/Q-type VGCCs, had a similar effect. Blocking L-, N-, and P\Q-type channels simultaneously with a cocktail of VGCC inhibitors abolished the pulse-induced [Ca(2+)](i) response of the cells, suggesting Ca(2+) influx occurs only via VGCCs. Lowering extracellular K(+) concentration from 5 to 2 mM or pulsing cells in Na(+)-free medium suppressed the pulse-induced rise in [Ca(2+)](i) in the majority of cells. Thus, both membrane potential and Na(+) entry appear to play a role in the mechanism by which nanoelectropulses evoke Ca(2+) influx. However, activation of voltage-gated Na(+) channels (VGSC) is not involved since tetrodotoxin (TTX) failed to block the pulse-induced rise in [Ca(2+)](i). These findings demonstrate that a single electric pulse of only 5 ns duration serves as a novel stimulus to open multiple types of VGCCs in chromaffin cells in a manner involving Na(+) transport across the plasma membrane. Whether Na(+) transport occurs via non-selective cation channels and/or through lipid nanopores remains to be determined.

1. Experiments were carried out to quantify the stimulation-evoked overflow of catecholamines and purines (ATP, ADP, AMP and adenosine) from an in vitro sympathetic nerve-smooth muscle preparation of the guinea-pig vas deferens and from isolated bovine adrenal chromaffin cells. The superfused preparations were stimulated for 60 s with electrical field stimulation (EFS; vas deferens), dimethylphenylpiperazinium (chromaffin cells) or KCI (both preparations). 2. Samples of superfusate were taken at 10 s intervals during the 60 s stimulation period for analysis of purines by HPLC-fluorescence detection and catecholamines by HPLCelectrochemical detection. 3. The evoked overflow of catecholamines and purines from chromaffin cells occurred with the same time course and in a constant ratio of approximately 4: 1 (catecholamine to purine). These findings are compatible with the release of catecholamines and purines from a homogeneous population of exocytotic vesicles in the chromaffin cells. 4. The evoked overflow of purines and noradrenaline (NA) from the vas deferens preparation differed from the pattern of overflow from chromaffin cells and there was also some temporal disparity in the overflow of the two cotransmitters. The evoked overflow of ATP exceeded that of NA. In addition, the overflow of NA was tonic while the overflow of ATP and the other purines was phasic. 5. The EFS-evoked overflow of NA and the purines from the guinea-pig vas deferens preparation was examined after treatment with the neuronal amine-uptake inhibitors desipramine and cocaine, the a,-adrenoceptor agonist methoxamine, the a1-adrenoceptor antagonist prazosin, the a2-adrenoceptor antagonists idazoxan and yohimbine, the noradrenaline-depleting drug reserpine and the adrenergic neuron-blocking agent guanethidine. The results of these studies, together with an analysis of the metabolic degradation of extracellular ATP, indicated that the temporal disparity in the overflow of NA and ATP is unlikely to be due to differences in the clearance of the cotransmitters or to the release of purines from non-neuronal sites. These results indicate that evoked overflow of the cotransmitters accurately reflects release from nerves. This pattern of release from nerves suggests that the two cotransmitters are released from two separate populations of exocytotic vesicles.6. Superfusion of the vas deferens with exogenous e-ATP, a fluorescent derivative of ATP, revealed that there was essentially no metabolism of the nucleotide over 60 s unless the tissue was subjected to EFS. Upon EFS, there was a rapid and nearly complete degradation of ATP with a corresponding increase in ADP, AMP and adenosine. This indicates the presence of a nerve stimulation-dependent metabolism of ATP.

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Vasoactive intestinal peptide (VIP) increased catecholamine biosynthesis in bovine adrenal chromaffin cells by 50-200%. Six related peptides produced no effects. In addition, VIP increased tyrosine hydroxylase (TH) activity measured in gel-filtered supernatants prepared from homogenates of treated cells. The hypothesis that cyclic AMP is the second messenger involved in these effects of VIP was also evaluated. VIP led to an elevation of cyclic AMP levels, and this increase occurred over a similar concentration range and time course as the activation of TH and the increase in catecholamine biosynthesis. Each measure reached maximal levels at 10-20 microM VIP within 1 min and remained elevated for at least 16 min. These changes produced by VIP were paralleled by enhanced phosphorylation of TH, and this phosphorylation occurred on a single tryptic peptide that was the same peptide whose phosphorylation has been previously shown to be stimulated by forskolin. In contrast to VIP and forskolin, 12-O-tetradecanoylphorbol 13-acetate, a phorbol ester known to activate protein kinase C, increased the phosphorylation on a total of three tryptic peptides of TH. Our results indicate that VIP stimulates catecholamine biosynthesis in chromaffin cells through the phosphorylation and activation of TH and support the conclusion that a cyclic AMP-dependent phosphorylation of TH is responsible for these effects.