The modulation of voltage-dependent calcium channels by hormones and neurotransmitters has important implications for the control of many Ca 2+ -dependent cellular functions including exocytosis and contractility1 -7. We made use of electrophysiological techniques, including whole-cell patch-clamp recordings from dorsal root ganglion (DRG) neurones, to demonstrate a role for GTP-binding proteins (G-proteins) as signal transducers in the noradrenaline-and γ-aminobutyric acid (GABA)-induced inhibition of voltage-dependent calcium channels8 -11. This action of the transmitters was blocked by: (1) preincubation of the cells with pertussis toxin (a bacterial exotoxin catalysing ADP-ribosylation of G-proteins12); or (2) intracellular administration of guanosine 5′-O-(2-thiodiphosphate) (GDP-β-S), a non-hydrolysable analogue of GDP that competitively inhibits the binding of GTP to G-proteins 13 . Our findings provide the first direct demonstration of the G-protein-mediated inhibition of voltage-dependent calcium channels by neurotransmitters. This mode of transmitter action may explain the ability of noradrenaline and GABA to presynaptically inhibit Ca 2+ -dependent neurosecretion from DRG sensory neurones 4,5 .Recordings were obtained from primary cultures of embryonic chick DRG cells (see Fig. 1). When bathed in solutions containing 1 mM Ba 2+ and 2 mM Ca 2+ , these cells generate action potentials with a prominent calcium-dependent plateau phase. The plateau results from a regenerative inward current carried by Ba 2+ and Ca 2+ ions and is blocked by cobalt14. Previous studies have demonstrated that noradrenaline and GABA decrease the duration of these action potentials by inhibiting the depolarization-induced calcium current8 ,9 . In the present study, a saturating concentration of noradrenaline (50 µM) reduced the actionpotential duration in 75% of the cells tested by an average of 43 ± 4.7% (Fig. 1a, Table 1). The inhibitory action of noradrenaline was also observed as a decrease in the Ca current recorded from voltage-clamped DRG cells (Fig. 1b). Similar decreases in action-potential duration and Ca current were also observed during application of GABA (Table 1).Pertussis toxin (PTX) blocks G-protein-mediated responses to hormones and neurotransmitters in many cell types 12 . Specifically, PTX catalyses ADP-ribosylation of Gproteins, thereby preventing agonist-induced dissociation of the proteins into active subunits15 , 16. When applied to DRG cells, PTX inhibits the transmitter-induced decrease in action-potential duration (Table 1). Following exposure to PTX (140 ng ml −1 ), only 9 and 19% of the cells responded to noradrenaline and GABA, respectively. Note that the mean percentage decrease in action-potential duration for those PTX-treated cells which did respond to noradrenaline and GABA was also reduced relative to control. The responses recorded from PTX-treated cells were very slow in onset: the maximal decrease in actionpotential duration was observed only after continued application of noradrena...
The diacylglycerol analogue 1,2-oleoylacetylglycerol (OAG) and the phorbol ester 12-deoxyphorbol 13-isobutyrate (DPB) were tested for their effects on the voltagedependent calcium (Ca) current in embryonic chicken dorsal root ganglion neurons in vitro. OAG (0.6-60 pM) and DPB (0.01-50 FM) produced reversible decreases in Ca current. Neither drug affected resting membrane conductance, the voltage-dependent potassium current, or the Ca current-voltage relationship. The concentrations of OAG and DPB that reduced Ca current correlate well with those concentrations that have been shown, in other systems, to activate protein kinase C-dependent phosphorylation. The time course for OAG action on Ca current is also consistent with an involvement of kinase C. Incubation of dorsal root ganglion cells in 60 ,pM OAG prevented further reductions in Ca current by either 50 ILM DPB or 10 p&M norepinephrine, a known modulator of the voltage-dependent Ca channel in these cells. This evidence suggests that protein kinase C may play a role in modulating Ca channel function.Modification of voltage-dependent calcium (Ca) channel function is likely to play an important role in controlling a number of Ca-dependent cellular processes (e.g., neurotransmitter release) (1, 2). The mechanisms underlying the modulation of Ca channel activity have, therefore, become the focus of much attention. One mechanism of channel modulation that has recently gained prominence involves the activation of protein kinases by intracellular second messengers (3-5). Protein kinase C, for example, has been implicated in the control of Ca flux and intracellular Ca mobilization in a variety of tissues (6). This kinase is especially concentrated in the membrane components of brain tissue (7-9), and it is Ca-and phospholipid-dependent. Full activation of the enzyme requires the presence of diacylglycerol, a second messenger produced by the breakdown of membrane phospholipids in response to an extracellular signal (10, 11). Kinase C can also be activated by the extracellular application of 1,2-oleoylacetylglycerol (OAG), a synthetic, membrane-permeable analogue of diacylglycerol (12). In addition, the tumor-promoting phorbol esters, acting at the diacylglycerol binding site on kinase C, are well known to activate the enzyme (13-15).We have tested OAG and phorbol ester on the voltagedependent Ca current of embryonic chicken dorsal root ganglion (DRG) neurons grown in culture. OAG caused a large, reversible decrease in the peak amplitude of the Ca current, with no change in either the threshold for current activation or the zero current potential. The phorbol ester 12-deoxyphorbol 13-isobutyrate (DPB) was also found to decrease Ca current in DRG cells at concentrations similar to those reported to activate kinase C (13, 16). The sensitivity of DRG cell Ca current to OAG and DPB suggests that modulation of the Ca channel may involve protein kinase C-dependent phosphorylation. Some of these results have been reported in preliminary form (17). METHODSDRGs were di...
Dihydropyridine (DHP) calcium channel antagonists, which inhibit the slowly inactivating or L-type cardiac calcium (Ca) current, have been shown to be ineffective in blocking 45 Ca influx and Cadependent secretion in a number of neuronal preparations. In the studies reported here, however, the antagonist DHP nifedipine inhibited both the L-type Ca current and potassium-evoked substance P (SP) release from embryonic chick dorsal root ganglion (DRG) neurons. These results suggest that, in DRG neurons, Ca entry through L-type channels is critical to the control of secretion. The inhibition of Ca current by nifedipine was both voltage and time-dependent, significant effects being observed only on currents evoked from relatively positive holding potentials maintained for several seconds. As expected from these results, nifedipine failed to inhibit L-type Ca current underlying the brief plateau phase of the action potential generated from the cell's normal resting potential; likewise, no significant effect of the drug was observed on action potential-stimulated SP release evoked by electrical field stimulation. The results of this work are discussed in terms of an assessment of the role of L-type Ca channels in neurosecretion.
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