8-bromocyclic-gmp and Pheochromocytoma

The effects of vinpocetine, an inhibitor of cyclic GMP phosphodiesterase, on ionic currents were examined in rat pituitary GH3 lactotrophs with the aid of the patch-clamp technique. In GH3 cells bathed in normal Tyrode's solution, vinpocetine (10 microM) reversibly increased the amplitude of Ca2+-activated K+ current (I(K)Ca) with an EC50 value of 4 microM. When the recording pipettes were filled with 10 mM EGTA, vinpocetine also stimulated I(K)Ca. In the cell-attached configuration, application of vinpocetine to the bath increased the activity of large-conductance Ca2+-activated K+ (BK(Ca)) channels. In excised membrane patches, application of vinpocetine (10 microM) to the bath did not change the single-channel conductance of BK(Ca) channels; however, it did increase channel activity. In the inside-out configuration, neither 8-bromo cyclic GMP nor YC-1 applied intracellularly affected BK(Ca) channel activity. The vinpocetine-induced change in the kinetic behavior of BK(Ca) channels was due to an increase in mean open time and a decrease in mean closed time. Vinpocetine (10 microM) caused a leftward shift in the midpoint for the voltage-dependent opening. Under the current-clamp mode, vinpocetine (10 microM) decreased the firing rate of spontaneous action potentials induced by thyrotropin-releasing hormone (10 microM) in GH3 cells. In pheochromocytoma PC12 cells, vinpocetine (10 microM) applied intracellularly also enhanced the activity of BK(Ca) channels without altering single-channel conductance. Thus, the present study suggests that vinpocetine-mediated stimulation of I(K)Ca may result from the direct activation of BK(Ca) channels and indirectly from elevated cytosolic Ca2+.

Topics: Animals; Calcium Channel Blockers; Calcium Channels; Cyclic GMP; Drug Interactions; Electrophysiology; Enzyme Activators; Indazoles; Kinetics; Large-Conductance Calcium-Activated Potassium Channels; Membrane Potentials; PC12 Cells; Pheochromocytoma; Pituitary Gland; Potassium Channels; Potassium Channels, Calcium-Activated; Rats; Tumor Cells, Cultured; Vinca Alkaloids

In order to identify the specific molecular mechanisms involved in neurosecretion, we investigated the mechanism of action of tetanus toxin, a potent presynaptic neurotoxin, in the rat adrenal pheochromocytoma PC12 cell line. It has recently been reported that tetanus toxin is a potent inhibitor of the release of depolarization-evoked 3H-acetylcholine (ACh) from nerve growth factor-differentiated PC12 cells (Sandberg et al., 1989a). In PC12 cells, as in many neural tissue preparations, cGMP accumulation in intact cells increased 6- to 17-fold when stimulated with veratridine (200 microM), carbachol (1 mM), Ba2+ (2 mM), or K+ (30 mM). Preincubation of the cells with tetanus toxin inhibits this accumulation by greater than 95%. The toxin dose-inhibition curves for 3H-ACh release and cGMP accumulation are similar, with half-maximal doses of tetanus toxin seen at approximately 5 nM. The time courses for the development of the effects of tetanus on 3H-ACh release and on cGMP accumulation were also similar. Protocols which elevated intracellular cGMP levels reversed the action of the toxin. For example, evoked ACh release was restored in intoxicated PC12 cells by a 15 min exposure to 100 microM 8-bromo-cGMP. The half-maximal dose was observed at 50 microM nucleotide. Examination of the nucleotide specificity revealed that only cyclic guanine analogs were effective in reversing the effects of tetanus toxin. These results suggested that the inhibition of depolarization-evoked cGMP accumulation is causally related to the action of tetanus toxin on neurosecretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcholine; Adrenal Gland Neoplasms; Animals; Atropine; Barium; Cell Line; Cyclic GMP; Dexamethasone; Electric Stimulation; Guanylate Cyclase; Kinetics; Nerve Growth Factors; Pheochromocytoma; Potassium; Rats; Tetanus Toxin; Veratridine