The Journal of Neurophysiology Vol. 78 No. 2 August 1997, pp. 780-789
Copyright ©1997 by the American Physiological Society

Modulation of Ca²⁺ Currents by Various G Protein-Coupled Receptors in Sympathetic Neurons of Male Rat Pelvic Ganglia

Yu Zhu and Jerrel L. Yakel

Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Zhu, Yu and Jerrel L. Yakel. Modulation of Ca²⁺ currents by various G protein-coupled receptors in sympathetic neurons of male rat pelvic ganglia. J. Neurophysiol. 78: 780-789, 1997. The modulation of voltage-gated calcium (Ca²⁺) channels by various G protein-coupled receptor pathways was investigated in sympathetic neurons of the male rat major pelvic ganglion (MPG). Standard whole cell patch-clamp recording techniques were used to record Ca²⁺ currents from acutely dissociated neurons. The activation of muscarinic receptors, which uses a G protein pathway that was not blocked by either pertussis toxin (PTX) or cholera toxin (CTX), inhibited both N-type and L-type Ca²⁺ channels. The activation of ₂ noradrenergic receptors with the selective agonist UK14304, which used primarily a PTX-sensitive G protein pathway, inhibited only N-type Ca²⁺ channels. The activation ofvasoactive intestinal polypeptide (VIP) receptors, which used a CTX-sensitive G protein pathway, also inhibited only N-type Ca²⁺ channels. UK14304 and VIP induced a bell-shaped inhibition of the Ca²⁺ current with a peak inhibition at around +10 mV and decreasing inhibition at more positive potentials. In contrast, the muscarine-induced Ca²⁺ current inhibition was not bell shaped and was more prominent at more positive potentials. Furthermore, a large depolarization, which relieved the current inhibition by UK14304 and VIP, did not relieve the inhibition by muscarine. Besides inhibiting the Ca²⁺ current, UK14304 and VIP also slowed the activation kinetics, an effect not seen with muscarine. Replacing external Ca²⁺ with Ba²⁺ and replacing internal ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) with high bis-(o-aminophenoxy)-N,N,N,N-tetraacetic acid (BAPTA) completely blocked the inhibitory effect of muscarine. However, the inhibitory effects of both UK14304 and VIP were unaffected under these conditions. Surprisingly, the facilitation of the Ca²⁺ current was eliminated under these strong calcium-buffering conditions. The activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) increases the amplitude of the Ca²⁺ current, diminishes facilitation, and reduces the inhibition of this current by UK14304 and VIP. However, PKC activation did not reduce the muscarine-induced Ca²⁺ current inhibition. In summary, our data suggest that muscarine uses a mechanism different from UK14304 and VIP to modulate the N-type Ca²⁺ channels in sympathetic neurons of the MPG. Although VIP and UK14304 use different G protein pathways, these two different pathways most likely converge downstream to compete for the same target site on the N-type Ca²⁺ channels.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Male rat major pelvic ganglia (MPGs) receive preganglionic input from the hypogastric and pelvic nerves and innervate various urogenital organs such as the bladder, the prostate, and the penis (Dail 1993; Langworthy 1965). MPGs contain both sympathetic and parasympathetic neurons (Dail 1993); sympathetic neurons can be identified because they selectively express a transient T-type calcium (Ca²⁺) current (Zhu et al. 1995). Ca²⁺ channels of sympathetic neurons respond to neurotransmitter modulation differently from those of parasympathetic neurons. For example, UK14304, a specific ₂ noradrenergic receptor agonist, inhibited a large portion of Ca²⁺ current of sympathetic neurons but had marginal effects on parasympathetic neurons (Zhu et al. 1995).

Although the modulation of voltage-gated Ca²⁺ channels has been characterized in sympathetic neurons of the superior cervical ganglion (SCG), the modulation of Ca²⁺ channels by neurotransmitters in MPG neurons has not been characterized even though the MPG is known to contain a rich repertoire of neurotransmitters such as noradrenaline, acetylcholine, substance P, neuropeptide Y, and vasoactive intestinal polypeptide (VIP) (Dail 1993). The sympathetic neurons of male rat MPG appear to be different compared with sympathetic neurons of other rat autonomic ganglia. First, they are anatomically short noradrenergic neurons because they lie in close proximity to the target organs. Second, these short sympathetic neurons are different from conventional long sympathetic neurons in their physiological and pharmacological properties. For example, the short noradrenergic neurons are more resistant to the action of reserpine, a norepinephrine-depleting agent (Owman et al. 1974). Third, the sympathetic neurons of male rat MPG express low-threshold T-type Ca²⁺ channels, which have not been observed in other sympathetic neurons (Marrion et al. 1987; Plummer et al. 1989; Schofield and Ikeda 1988). Moreover, it has been reported that the modulation of Ca²⁺ channels by muscarinic receptor agonists in rat SCG sympathetic neurons was mediated through two different pathways; one is mediated by M₄ muscarinic receptors with the use of a fast, membrane-delimited and pertussis toxin (PTX)-sensitive pathway and the other is transduced via M₁ muscarinic receptors, appears to be slow and PTX insensitive, and requires a diffusable intracellular messenger (Hille 1994). Our studies have shown that the modulation of Ca²⁺ channels by muscarinic receptor agonists in sympathetic MPG neurons was primarily mediated through the slow and PTX-insensitive pathway, with little involvement of the fast membrane-delimited pathway (Zhu and Yakel 1997).

Here we present a comprehensive investigation of the modulation of voltage-gated Ca²⁺ channels by various neurotransmitter receptors known to be present in MPG to compare this information with that from other sympatheticganglia such as SCG. In SCG neurons, the activation of muscarinic receptors induces a PTX-insensitive as well as a PTX-sensitive Ca²⁺ current inhibition (Beech et al. 1991, 1992; Bernheim et al. 1991), the activation of ₂ noradrenergic receptors induces primarily a PTX-sensitive Ca²⁺ current inhibition (Beech et al. 1992; Schofield 1990, 1991), and the activation of VIP receptors induces a cholera toxin (CTX)-sensitive Ca²⁺ current inhibition (Zhu and Ikeda 1994b). Here, in sympathetic MPG neurons, the effects of muscarine, UK14304 (a specific ₂ noradrenergic agonist), and VIP on the voltage-gated Ca²⁺ channel currents were examined and the different signal transduction pathways used were characterized.

	METHODS

Abstract Introduction Methods Results Discussion References

Single neurons of MPG were isolated from 12- to 16-wk-old male Wistar rats with the use of an enzymatic method as previously described (Zhu et al. 1995). The neurons were then plated on glass coverslips coated with poly-D-lysine and short-term (<24 h) cultured in a humidified atmosphere containing 5% CO₂ in air at 37°C.

The whole cell variant of the patch-clamp technique (Hamill et al. 1981) was used to record Ca²⁺ currents from single MPG neurons as previously described (Zhu et al. 1995). Patch pipettes were made from Corning 7052 glass capillary (Garner Glass) and had resistances of 1.5-2.8 M when filled with the solutions described below. The cell membrane capacitance and series resistance (usually <6 M) were compensated by >80% with the use of the Axopatch-1D amplifier (Axon Instruments). Voltage protocols were generated with the use of the pCLAMP6 software (Axon Instruments). Traces were digitized and filtered at 5 kHz with the use of a four-pole Bessel filter in the clamp amplifier. Data from the neurons expressing T-type current were collected. The amplitudes of Ca²⁺ currents were measured isochronally 10 ms after depolarization. Data are expressed as means ± SE where appropriate. Student's t-test and analysis of variance were applied to determine the significance in differences. P < 0.05 was considered significant.

The pipette solution contained (in mM) 120 N-methyl-glucamine (NMG)-CH₃SO₃, 20 tetraethylammonium (TEA) chloride, 11 ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA), 1 CaCl₂, 10 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 4 Mg-ATP, 0.3 guanosine 5-triphosphate (GTP), and 14 creatine phosphate, pH 7.2, estimated free Ca²⁺ 80 nM. In experiments designed to examine the effect of different Ca²⁺ buffers, 11 mM EGTA was replaced with either 0.1 or 20 mM bis-(o-aminophenoxy)-N,N,N,N-tetraacetic acid (BAPTA) without the addition of CaCl₂. We observed that the Ca²⁺ current rundown was very rapid in the pipette solutions containing 0.1 mM BAPTA (decreased to <50% within 5 min). In contrast, the Ca²⁺ currents were relatively stable in the internal solution containing 11 mM EGTA (typically lasting 20-30 min with <10% rundown). Unless otherwise indicated, all the experiments reported here were performed with the use of the internal solutions containing 11 mM EGTA. The external solution contained (in mM) 145 NMG-CH₃SO₃, 10 CaCl₂, 10 HEPES, 15 glucose, and 0.0001 tetrodotoxin, pH 7.4. The external bath solution was continuously flowing (~1 ml/min) during the experiment. In a few experiments, 10 mM CaCl₂ was replaced with 5 mM BaCl₂. Various agents were added to either the pipette or external solution as needed. VIP and protein kinase C (PKC) pseudosubstrate inhibitor (PKC_19-36) were from Bachem (King of Prussia, PA); UK14304, phorbol 12-myristate 13-acetate (PMA), muscarine-Cl, somatostatin-28, PTX, CTX, and Bay K 8644 were from RBI (Natick, MA); and -conotoxin (-CgTx) GVIA and MVIIC were from Peptide Institute (Osaka, Japan). UK14304 was prepared daily from a frozen 50 mM stock solution in 0.1 M HCl. All experiments were performed at room temperature (21-24°C). The solutions containing test agents were applied to neurons through large-bore quartz tubing (310 µm ID) placed 300 µm away from the neuron under study. The application and termination of drug-containing solutions was controlled by computer-driven valves (General Valve, Fairfield, NJ).

	RESULTS

Abstract Introduction Methods Results Discussion References

Voltage dependence in Ca²⁺ current inhibition

Calcium (Ca²⁺) currents were recorded from single sympathetic neurons (i.e., those expressing transient T-type Ca²⁺ current) (Zhu et al. 1995) of the rat MPG in solutions containing 10 mM Ca²⁺ externally and 11 mM EGTA (with 1 mM added Ca²⁺) internally. Superimposed Ca²⁺ current traces after depolarization from a holding potential of 80 mV to different test potentials (30 to +50 mV) in the absence and presence of 10 µM muscarine are shown in Fig. 1A. The transient currents elicited by depolarization to 30 mV were primarily T-type currents, because they were diminished by changing the holding membrane potential from 80 to 50 mV and were partially inhibited by a nonspecific T-type Ca²⁺ channel blocker, amiloride (data not shown) (see Zhu et al. 1995). Muscarine had little effect on the Ca²⁺ current amplitude at 30 mV (indicating no effect on T-type currents), but increasingly reduced the amplitude of the Ca²⁺ current at more positive potentials (~33% reduction at +10 mV, compared with ~50% at +50 mV). The inhibition of the Ca²⁺ current by muscarine was completely blocked by atropine (10 µM, n = 4, data not shown).

View larger version (19K): [in this window] [in a new window]   View larger version (16K): [in this window] [in a new window]   FIG. 1. Inhibition of Ca2+ currents by muscarine, UK14304, and vasoactive intestinal polypeptide (VIP). A: superimposed Ca2+ current traces recorded from neuron expressing T-type Ca2+ current in absence and presence of 10 µM muscarine. Neuron was held at 80 mV and depolarized to various potentials as indicated at top. B: superimposed Ca2+ current traces recorded from neuron in absence and presence of 5 µM UK14304. C: superimposed Ca2+ current traces recorded from neuron in absence and presence of 10 µM VIP. D: voltage-dependent Ca2+ current inhibition by muscarine (n = 6), UK14304 (n = 6), and VIP (n = 5). Current amplitude was determined isochronally 10 ms after each pulse. Current inhibition was calculated as (1  Idrug/Icontrol) × 100 and plotted as function of test potential.

The inhibition of the Ca²⁺ current by the selective activation of ₂ noradrenergic receptors with the agonist UK14304 (10 µM) (Turner et al. 1985) is shown in Fig. 1B. Similar to muscarine, UK14304 had no effect on the T-type current, but inhibited the Ca²⁺ current at more positive potentials. However, in contrast to muscarine, the amount of inhibition by UK14304 was greater at +10 mV than at +50 mV. Besides reducing the amplitude of the Ca²⁺ current, UK14304 also slowed the activation kinetics, an effect not seen with muscarine. VIP (10 µM) inhibited the Ca²⁺ current in a fashion nearly identical to UK14304 (Fig. 1C).

The ability of these three agents to inhibit the Ca²⁺ current and the dependence on voltage is shown in Fig. 1D. The inhibition by muscarine was not significantly voltage dependent between +10 and +40 mV and was greater at +60 mV than at +10 mV (P < 0.05, paired t-test). This is in contrast to the "bell-shaped" inhibition by UK14304 and VIP; the largest inhibition was between 0 and +10 mV, and inhibition was significantly less at more positive potentials.

Earlier studies have shown that the inhibition of Ca²⁺ current by the activation of some G protein-coupled receptors can be partially relieved by a large preceding depolarization (Elmslie et al. 1990; Ikeda 1992). In Fig. 2, a double-pulse voltage protocol was used to determine whether the inhibition by any of the three agents was partially relieved by depolarization. The neurons were first depolarized to +10 mV for 25 ms (i.e., pre current, , see voltage protocol in Fig. 2A), then to +80 mV for 50 ms, then returned to 80 mV for 10 ms, and then redepolarized to +10 mV (i.e., post current, ). Under control conditions in the absence of agonist, the magnitude of the post current was greater than that of the pre current; this process is referred to as tonic facilitation. It is thought that the large conditioning depolarization to +80 mV relieves the tonic inhibition of Ca²⁺ currents by endogenous G proteins, resulting in the facilitation of the Ca²⁺ current (Ikeda 1991; Kasai 1991).

View larger version (30K): [in this window] [in a new window]   FIG. 2. Relief of current inhibition by large depolarization. A left: superimposed Ca2+ current traces recorded with double-pulse voltage protocol (top) in absence and presence of 10 µM muscarine. A, middle: time course of muscarine-induced current inhibition. Current amplitude was determined isochronally 10 ms after each pulse for both pre and post currents and plotted as function of time. A, right: summary of pre and post current inhibition induced by muscarine. Numbers in parentheses: number of neurons examined. Double asterisk: P < 0.01. Triple asterisk: P < 0.001. B: effect of 5 µM UK14304 on Ca2+ current. C: effect of 10 µM VIP on Ca2+ current.

The inhibition of the Ca²⁺ current by muscarine (10 µM) was reversible and relatively slow (reached maximal inhibition within 1 min), and was not relieved by a large depolarization (Fig. 2A). Muscarine inhibited both the pre and post current; however, the inhibition of the post current was significantly more (38 ± 4%, mean ± SE, n = 19) than that of the pre current (30 ± 4%, Fig. 2A). Because the post current inhibition by muscarine was greater and more consistent than the pre current inhibition, the current inhibition by muscarine for the rest of the paper specifically refers to the post current inhibition unless otherwise indicated.

In contrast to muscarine, the inhibition of the Ca²⁺ current by UK14304 (5 µM), which was rapid (reached maximal inhibition within 10 s) and reversible, was relieved by a large depolarization (Fig. 2B). UK14304 inhibited both the pre and post currents; however, the inhibition of the pre current (53 ± 6%, n = 19) was significantly greater than that of the post current (18 ± 4%, P < 0.001, Fig. 2B).

Similar to UK14304, the inhibition of the Ca²⁺ current by VIP was partially relieved by a large depolarization, and the post current inhibition (7 ± 1%) was significantly less than the pre current inhibition (30 ± 3%, P < 0.001, n = 5, Fig. 2C). However, the action of and recovery from VIP was relatively slower than for UK14304 (Fig. 2C).

Ca²⁺ dependence of current inhibition and tonic facilitation

The kinetics, time course, and voltage dependence of the muscarine-induced Ca²⁺ current inhibition in MPG neurons are similar to those of the M₁ receptor-mediated inhibition of the Ca²⁺ current in SCG neurons, which requires an intracellular diffusable messenger and low Ca²⁺-buffering solutions (Bernheim et al. 1991, 1992). Therefore the effects of different Ca²⁺-buffering solutions on the muscarine-induced Ca²⁺ current inhibition in MPG were examined. With the use of a double-pulse protocol, and with replacement of external Ca²⁺ with Ba²⁺ and internal EGTA (and added Ca²⁺) with high BAPTA (20 mM), muscarine no longer inhibited the Ba²⁺ current flowing through the Ca²⁺ channels (Fig. 3A). The effects of different Ca²⁺ buffers on the muscarine-induced Ca²⁺ current inhibition are summarized in Fig. 3C. There was no significant difference in the muscarine-induced current inhibition in solutions containing either 11 mM EGTA (38 ± 4%, n = 35) or 0.1 mM BAPTA(43 ± 7%, n = 8) internally. However, the effect of muscarine was reduced to 16 ± 3% (n = 25) when the internal BAPTA concentration was raised to 20 mM (P < 0.001 vs. 11 EGTA, unpaired), and nearly totally eliminated by the further replacement of external Ca²⁺ with Ba²⁺ (3 ± 1%,n = 6).

View larger version (33K): [in this window] [in a new window]   FIG. 3. Ca2+-dependent current inhibition and facilitation. A: superimposed Ca2+ channel (Ba2+ current) traces recorded with double-pulse voltage protocol (see Fig. 2) in absence and presence of 10 µM muscarine (top), 5 µM UK14304 (middle), and 10 µM VIP (bottom). External solutions contained 5 mM Ba2+ and internal solutions contained 20 mM bis-(o-aminophenoxy)-N,N,N,N-tetraacetic acid (BAPTA) with no Ca2+ added. B: summary of tonic facilitation observed under different conditions. Charge carrier in external solution was either 10 mM Ca2+ or 5 mM Ba2+, and Ca2+ buffer in internal solution was 0.1 mM ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA), 11 mM EGTA, or 20 mM BAPTA. Facilitation is expressed as (post current/pre current  1) × 100. C: summary of current inhibition induced by muscarine observed under different conditions. Current inhibition was calculated as (1  Idrug/Icontrol) × 100. D: summary of current inhibition induced by UK14304 under different conditions. E: summary of current inhibition induced by VIP under different conditions.

Surprisingly, tonic facilitation (the increase in the post vs. pre current amplitude) was eliminated under strong Ca²⁺-buffering conditions. The extent of facilitation averaged 32 ± 8% (n = 35) in solutions containing 11 mM EGTA internally and 10 mM Ca²⁺ externally (Fig. 3B). When EGTA was replaced with 0.1 mM BAPTA in the internal solutions, there was no significant change in facilitation. However, when the internal BAPTA concentration was increased from 0.1 to 20 mM, facilitation was significantly reduced to 10 ± 4% (n = 25, P < 0.01, unpaired). The further removal of Ca²⁺ from the external solution by replacement with Ba²⁺ completely abolished facilitation (4 ± 2%, n = 6).

In contrast to muscarine, the inhibitory effects of either UK14304 or VIP were unaffected under strong Ca²⁺-buffering conditions, i.e., replacing external Ca²⁺ with Ba²⁺ and replacing internal EGTA with high BAPTA (Fig. 3). However, the current inhibition by UK14304 was reduced in a low Ca²⁺-buffering solution (i.e., in the internal solution containing 0.1 mM BAPTA, Fig. 3D); VIP was not tested under these conditions.

Inhibition of N- and L-type Ca²⁺ channels

Previous studies have shown that in MPG neurons, the N-type Ca²⁺ channel currents constitute the majority(60-70%) of the whole cell Ca²⁺ current, whereas 10% of the whole cell Ca²⁺ current arises from the L-type Ca²⁺ channels; no P-type Ca²⁺ channels were found (Zhu et al. 1995). We wanted to identify the channel types inhibited by these three agents. The time course of Ca²⁺ current inhibition by muscarine (10 µM) before and after the application of 10 µM -CgTx GVIA, a selective N-type Ca²⁺ channel blocker (McCleskey et al. 1987), is shown in Fig. 4A. The percent inhibition of current was calculated by dividing the absolute decrease in current amplitude (in nA) due to muscarine by the control amplitude value obtained immediately before the first application of muscarine and before the exposure to -CgTx GVIA. Muscarine inhibited the current by 53% before and 15% after exposure to -CgTx GVIA. After the application of -CgTx GVIA, whose inhibition of the Ca²⁺ current persisted minutes after its washout, -CgTx MVIIC (10 µM) caused little further inhibition of Ca²⁺ current, indicating that there are no Q-type Ca²⁺ channels in the sympathetic neurons of the male rat MPG. The application of -CgTx MVIIC alone inhibited the Ca²⁺ currents (55 ± 3%, n = 3, data not shown) to the same extent as-CgTx GVIA (56 ± 5%, n = 5).

View larger version (28K): [in this window] [in a new window]   FIG. 4. Inhibition of N- and/or L-type Ca2+ channels. A: Ca2+ currents were recorded with double-pulse voltage protocol. Current amplitudes of both pre and post currents were determined and plotted against time. Neuron was externally superfused with 10 µM muscarine (solid bars), 10 µM-conotoxin GVIA (-GVIA, hatched bar), 10 µM -conotoxin MVIIC (-MVIIC, open bar). B: summary of current inhibition by muscarine, UK14304, and VIP before and after 10 µM -conotoxin. Note that, as indicated in text, % inhibition after -GVIA treatment was compared with initial control. C: superimposed current traces recorded before and during application of 10 µM Bay K 8644 (top left) and in presence of Bay K 8644 with or without 5 µM UK14304 (top right), 10 µM muscarine (bottom left), or 10 µM VIP (bottom right). Neurons were held at 50 mV to inactivate T-type currents and depolarized to +10 mV. D: summary of inhibition of tail currents induced by 10 µM Bay K 8644 in presence of muscarine, UK14304, or VIP. Tail current amplitudes were determined isochronally 5 ms after return to holding potential.

-CgTx GVIA exposure similarly reduced the inhibitory effect of both UK14304 and VIP on the Ca²⁺ current (Fig. 4B). On average, -CgTx GVIA reduced the percent inhibition of the Ca²⁺ currents by muscarine from 37 ± 7% to6 ± 3% (n = 5), by UK14304 from 50 ± 3% to 9 ± 1%(n = 5), and by VIP from 30 ± 3% to 4 ± 1% (n = 3). These results suggest that the N-type Ca²⁺ channels are the major target of neurotransmitter-induced Ca²⁺ current inhibition by muscarine, UK14304, and VIP.

The Ca²⁺ current inhibition by these three agents observed after -CgTx GVIA exposure may be due to an inhibition of the L-type Ca²⁺ channels, because ~10% of the whole cell Ca²⁺ current arises from these channels. Bay K 8644, an L-type Ca²⁺ channel agonist, was used to amplify theL-type channel current before the inhibitory action of these agents was examined. Neurons were held at 50 mV to inactivate the T-type Ca²⁺ channels, and a single pulse depolarization to +10 mV was applied (see voltage protocol in Fig. 4C). Bay K 8644 (10 µM) increased the step current by 24% and induced a large and slowly deactivating tail current (Fig. 4C, top left); this tail current is thought to be generated primarily from the L-type Ca²⁺ channels. The tail current amplitude was determined 5 ms after return to the holding potential. Muscarine inhibited the step current by 34% and the tail current by 49% in this neuron (Fig. 4C, bottom left). Both UK14304 and VIP inhibited the step currents but had little or no effect on the tail currents generated by Bay K 8644. The average inhibition of the tail current by muscarine was 37 ± 4% (n = 8); by UK14304, 4 ± 1% (n = 3); and by VIP, 1 ± 1% (n = 3, Fig. 4D).

G-protein-mediated current inhibition

To examine the possible G protein involvement in Ca²⁺ current inhibition induced by these three agents, guanosine 5-diphosphate (GDP)S (2 mM), a nonhydrolyzable analogue of GDP and competitive inhibitor of GTP binding to G-subunits (Eckstein et al. 1979), was substituted for 0.3 mM GTP in the pipette solution. After dialysis of the neuron with GDPS for >5 min, the large depolarization to +80 mV no longer facilitated the Ca²⁺ current (Fig. 5A, inset), and the current inhibition induced by muscarine and UK14304 was abolished (Fig. 5A). A similar effect was observed with VIP; all data are tabulated in Fig. 5B. The average current inhibition induced by muscarine was reduced from 38 ± 3% (n = 19) to 4 ± 1% (n = 5) with GDPS dialysis, from 47 ± 4% (n = 19) to 5 ± 2% (n = 5) by UK14304, and from 30 ± 3% (n = 5) to 1 ± 1% (n = 3) by VIP.

View larger version (28K): [in this window] [in a new window]   FIG. 5. G-protein-coupled current inhibition. A: Ca2+ currents were recorded with double-pulse voltage protocol. Current amplitudes of both pre and post currents were determined and plotted against time. Neuron was dialyzed internally with 2 mM guanosine5-diphosphate (GDP)S and externally superfused with 5 µM UK14304 (open bar) or 10 µM muscarine (solid bar). Recording was made 5 min after establishment of whole cell configuration. Inset: superimposed current traces recorded in absence (1 and 2) and presence of muscarine (3 and 4). B: summary of current inhibition by muscarine, UK14304, and VIP in neurons with or without internal GDPS. C: Ca2+ currents were recorded and amplitudes were determined as in A from neuron dialyzed internally with 0.3 mM guanosine 5-O-(3-thiotriphosphate) (GTPS). Neuron was externally superfused with UK14304 (5 µM) or muscarine (10 µM). Recording was made 7 min after establishment of whole cell configuration. Inset: superimposed current traces recorded in absence (1 and 2) and presence of muscarine (3 and 4). D: summary of current inhibition by muscarine, UK14304, or VIP in neurons with or without internal GTPS.

Further evidence of G protein involvement was obtained by dialyzing neurons with GTPS (0.3 mM), a nonhydrolyzable analogue of GTP that is thought to activate various G protein pathways and inhibit the Ca²⁺ current in the absence of receptor activation (Elmslie 1992). GTPS dialysis for >5 min inhibited the amplitude of the Ca²⁺ current and decreased the activation kinetics in a manner similar to UK14304 or VIP (Fig. 5C, inset). In addition, the inhibition by GTPS was voltage dependent in that a large depolarization to +80 mV relieved most of this inhibition (Ikeda 1991); the average facilitation was greatly increased under these conditions, to 125 ± 20% (n = 5) from a control value of 32% (see above). With GTPS dialysis, the application of UK14304 and VIP produced no further inhibition of the Ca²⁺ current (Fig. 5, C and D). Interestingly, muscarine still inhibited the Ca²⁺ current under these conditions, but to a much greater extent than under control conditions; 68 ± 3% (n = 5) for GTPS-dialyzed neurons, compared with 38 ± 3% (n = 19, P < 0.01) for controls (Fig. 5C). Furthermore, the inhibition by muscarine was irreversible.

PTX or CTX sensitivity of current inhibition

PTX is known to catalyze the ADP ribosylation of the -subunit of G_o/i proteins and disrupts the coupling of the receptor and G protein (Hepler and Gilman 1992). In neurons incubated with PTX (0.5 µg/ml) for 14-16 h, the current inhibition by UK14304 was reduced from 47 ± 8% (n = 11) to only 5 ± 1% (n = 7, Fig. 6A, top), suggesting a PTX-sensitive pathway for UK14304-induced current inhibition. Neither the muscarine- nor the VIP-induced current inhibitions were sensitive to PTX (Fig. 6, A and B).

View larger version (29K): [in this window] [in a new window]   FIG. 6. Pertussis toxin (PTX) and cholera toxin (CTX) sensitivity. A: superimposed Ca2+ current traces recorded with double-pulse voltage protocol in absence and presence of 5 µM UK14304 (top) or 10 µM VIP (bottom) from neurons preincubated with 500 ng/ml PTX for 14-16 h. B: summary of current inhibition by muscarine, UK14304, or VIP in control neurons and in neurons treated with PTX for 14-16 h. C: superimposed Ca2+ current traces recorded as in A in absence and presence of 10 µM VIP (top) or 5 µM UK14304 (bottom) from neuron preincubated with 500 ng/ml CTX for 16 h. D: summary of current inhibition by muscarine, UK14304, or VIP in control neurons and in neurons treated with CTX for 14-16 h.

CTX catalyzes the ADP ribosylation of the -subunit of G_s, G_olf, and transducin, which inhibits the GTPase activity of the -subunit of G_s, resulting in a persistent activation of these G proteins (Hepler and Gilman 1992). Previous studies have shown that VIP uses a CTX-sensitive pathway (i.e., via a G_s) to inhibit the Ca²⁺ currents of SCG neurons (Zhu and Ikeda 1994b). In the present study we examined the effect of CTX pretreatment on current inhibition by these three agents. In a neuron incubated with 0.5 µg/ml CTX for 14-16 h, the Ca²⁺ current amplitude appeared inhibited and the activation kinetics slowed, effects that were relieved by depolarization to +80 mV (Fig. 6C); these effects were similar to that seen with UK14304 and VIP, indicating that the Ca²⁺ channel inhibitory pathway was most likely preactivated by CTX. Furthermore, the application of VIP produced no further inhibition of the Ca²⁺ current. The effect of muscarine was not significantly reduced by CTX; however, current inhibition by UK14304 was significantly reduced (Fig. 6, C and D). The reduction in the UK14304-induced Ca²⁺ current inhibition by CTX is mostly likely due to the fact that the inhibitory pathway(s) leading to Ca²⁺ channel inhibition induced by VIP and UK14304 exhibit convergence (Zhu and Ikeda 1994b).

Convergence or nonadditivity between the three inhibitory pathways was further investigated by the coapplication of agonists and by comparing the inhibition of the Ca²⁺ current with that produced by the particular agonists alone. In five cells, VIP inhibited the Ca²⁺ current by 24 ± 4%, UK14304 inhibited current by 43 ± 9%, and the coapplication of both VIP and UK14304 inhibited current by 43 ± 10% (Fig. 7A). Thus the mean inhibition of the Ca²⁺ current induced by UK14304 was unaltered by its coapplication with VIP, suggesting that the actions of VIP and UK14304 are not additive, and further suggesting convergence in these inhibitory pathways. The additivity of Ca²⁺ current inhibition induced by muscarine and UK14304 was examined next. Muscarine and UK14304 inhibited the pre current by 22 ± 8% (n = 4) and 50 ± 4%, respectively (Fig. 7B, top), whereas the combined inhibition was 64 ± 4%. Apparently, some overlap in pre current inhibition existed between muscarine and UK14304. In contrast, muscarine and UK14304 inhibited the post current by 31 ± 7% (n = 4) and 20 ± 6%, respectively, and the combined inhibition was 51 ± 8% (Fig. 7B, bottom). This suggests that the post current inhibition by these two agents is fully additive, suggesting little or no convergence in these Ca²⁺ channel inhibitory pathways.

View larger version (21K): [in this window] [in a new window]   FIG. 7. Additive or nonadditive inhibitory action. A: summary of current (pre) inhibition induced by VIP (10 µM) alone, UK14304 (5 µM) alone, or VIP plus UK14304. B: summary of both pre and post current inhibition induced by muscarine (10 µM) alone, UK14304 (5 µM) alone, or muscarine plus UK14304.

Effects of PKC activation

Previous studies on SCG and some central neurons have shown that the activation of PKC increases the current amplitude, diminishes the tonic facilitation, and reduces Ca²⁺ current inhibition induced by norepinephrine, muscarine, VIP, and -aminobutyric acid (Swartz 1993; Swartz et al. 1993; Zhu and Ikeda 1994a). We investigated the effects of PKC activation on Ca²⁺ channel modulation in MPG neurons. In Fig. 8, the neuron was first exposed to muscarine and then UK14304. After complete recovery from this current inhibition, the neuron was then exposed to PMA (0.5 µM) for 10 min. As a result of PMA application, the pre current amplitude increased by 65% while the post current amplitude increased by only 18%; facilitation was totally abolished. PMA application also reduced the UK14304-induced inhibition of Ca²⁺ current from 62% to 15% in this neuron. In contrast, the muscarine-induced inhibition was not affected by PMA treatment (52% before and 53% after PMA, Fig. 8). To further substantiate the effect of PMA on PKC activation, neurons were dialyzed with the PKC pseudosubstrate inhibitor PKC_19-36 (0.2 mM). After dialysis with PKC_19-36, PMA had little effect on either the Ca²⁺ current or its modulation by muscarine and UK14304 (Fig. 9).

View larger version (25K): [in this window] [in a new window]   FIG. 8. Effect of protein kinase C (PKC) activation by phorbol 12-myristate 13-acetate (PMA). Ca2+ currents were recorded as in Fig. 2. Neuron was externally superfused with 10 µM muscarine, 5 µM UK14304, and then 500 nM PMA. After PMA application, neuron was superfused with UK14304 and muscarine again. Inset: superimposed current traces recorded before (1 and 2) and after (3 and 4) PMA application.

View larger version (23K): [in this window] [in a new window]   FIG. 9. Summary of actions of PMA. A: summary of current amplitude changes. Ca2+ currents were recorded as in Fig. 8. Neurons were exposed to various external solutions for 10 min: 1) control, 2) 0.5 µM PMA, 3) 0.5 µM PMA plus internal PKC19-36, and 4) 5 µM 4-phorbol; amplitudes of pre current were determined before and after application of various external solutions. Current amplitude changes are defined as (Iafter  Ibefore)/Ibefore × 100. B: summary of tonic facilitation. Facilitation is defined as in Fig. 3B and was determined 10 min after application of various external solutions: 1) control, 2) 0.5 µM PMA, 3) 0.5 µM PMA plus internal PKC19-36, and 4) 5 µM 4-phorbol. C: summary of muscarine-induced current inhibition determined 10 min after application of various external solutions: 1) control, 2) 0.5µM PMA, 3) 0.5 µM PMA plus internal PKC19-36,and 4) 5 µM 4-phorbol. D: summary of UK14304-induced current inhibition determined 10 min after application of various external solutions: 1) control, 2) 0.5 µM PMA, 3) 0.5 µM PMA plus internal PKC19-36, and 4) 5 µM 4-phorbol. E: summary of VIP-induced current inhibition determined 10 min after application of various external solutions: 1) control and 2) 0.5 µM PMA.

The data showing the effects of altering PKC activity are summarized in Fig. 9. Under control conditions, the pre current amplitude decreased by 5 ± 4% (n = 5) over a period of 10 min, representing a typical time-dependent current rundown. PMA significantly increased the pre current amplitude by 42 ± 6% (n = 7), an effect completely blocked by dialysis with PKC_19-36 (1 ± 4%, n = 5, Fig. 9A). The application of 4-phorbol, a phorbol ester that is unable to activate PKC, had no effect on the Ca²⁺ current. PMA completely abolished tonic facilitation, from 25 ± 6%(n = 5) for controls to 2 ± 3% (n = 9) after PMA application (Fig. 9B). Again dialysis with PKC_19-36 completely blocked this effect of PMA. Facilitation was not significantly altered by 4-phorbol. As mentioned above, the muscarine-induced inhibition of Ca²⁺ current was totally unaffected by PMA, PKC_19-36, or 4-phorbol (Fig. 9C), whereas both the UK14304- and VIP-induced inhibitions were reduced significantly by PMA (Fig. 9, D and E).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Our results show that Ca²⁺ channels in sympathetic neurons of the male rat MPG are subject to various neurotransmitter-induced inhibitory processes via coupling to different G proteins. The activation of ₂ noradrenergic receptors with UK14304, which uses a PTX-sensitive G protein, andVIP, which uses a CTX-sensitive G protein, inhibited only N-type and not L-type Ca²⁺ channels. However the activation of muscarinic receptors, which uses a G protein pathway that was not blocked by either PTX or CTX, inhibited both N-type and L-type Ca²⁺ channels. The inhibition of theL-type Ca²⁺ channels in rat sympathetic MPG neurons is consistent with the inhibition of these channels by activation of muscarinic receptors in rat sympathetic SCG neurons (Mathie et al. 1992) and mouse pancreatic B cells (Gilon et al. 1997). In addition, because norepinephrine and muscarine both activate at least two distinct G protein pathways to inhibit Ca²⁺ channels in rat SCG neurons (Hille 1994), but both appear to activate a single pathway in sympathetic MPG neurons, these sympathetic MPG neurons may prove to be a useful and simpler model for studying how these various G-protein-mediated signal transduction pathways modulate voltage-gated Ca²⁺ channels.

Voltage dependence

UK14304 and VIP induced a bell-shaped voltage-dependent inhibition of the Ca²⁺ current, consistent with the findings from SCG neurons (Beech et al. 1992; Schofield 1991; Zhu and Ikeda 1994b). However, the muscarine-induced Ca²⁺ current inhibition had a different voltage dependence in that the inhibition by muscarine was more prominent at more positive potentials. A qualitatively similar voltage dependence was observed in SCG neurons for the M₁ muscarinic receptor-mediated Ca²⁺ current inhibition (Beech et al. 1992). Furthermore a large depolarization, which relieved the current inhibition by UK14304 and VIP, did not relieve the muscarine inhibition. Instead, the large depolarization enhanced the muscarine effect, suggesting a different mechanism of action on the coupling between the various G protein pathways and Ca²⁺ channel regulation.

Ca²⁺ dependence

It is interesting to note that the tonic facilitation of the Ca²⁺ channel current (the increase in the post vs. pre current amplitude due to a large depolarization in the absence of neurotransmitter) was dependent on Ca²⁺, a result not previously reported. When Ca²⁺ was removed from the external solution (and replaced with Ba²⁺) and the Ca²⁺-buffering capacity of the internal solution was greatly increased with the use of high (20 mM) BAPTA, tonic facilitation was abolished; the mechanism for this effect is currently unknown. Consistent with the findings from SCG neurons, the facilitation in sympathetic MPG neurons was dependent on the activation of G proteins (e.g., GDPS diminished and GTPS enhanced facilitation) and was reduced by the activation of PKC (Ikeda 1991; Swartz 1993; Zhu and Ikeda 1994a). It has been observed in SCG neurons (Swartz 1993; Zhu and Ikeda 1994a) and here in MPG neurons that agents such as GDPS and PMA, which diminish the facilitation, also attenuate the current inhibition by UK14304 and VIP. The relationship between the extent of tonic facilitation and current inhibition by neurotransmitters is not clear. The present study shows that the removal of Ca²⁺ abolished the facilitation but had little effect on the UK14304- and VIP-induced Ca²⁺ current inhibition. Therefore the extent of facilitation and the ability of neurotransmitters to inhibit the Ca²⁺ current may not be intrinsically related.

Our results show that the muscarine-induced Ca²⁺ current inhibition in MPG neurons was not significantly different when the pipette solution contained either 0.1 mM BAPTA or 11 mM EGTA. This is different from the results reported for the SCG neurons, where the M₁ muscarinic receptor-mediated current inhibition was diminished with an internal solution containing 10 mM EGTA (Beech et al. 1991). Nevertheless, the inhibition of Ca²⁺ currents by muscarine in the MPG neurons was reduced when 20 mM BAPTA was used in the internal solution, consistent with the results from SCG neurons (Beech et al. 1991). Despite some modest differences in the Ca²⁺ buffer sensitivity of the muscarine-induced Ca²⁺ current inhibition between sympathetic MPG and SCG neurons, it appears that these two inhibitory responses are similar (Bernheim et al. 1991, 1992). The second-messenger pathway responsible for this inhibition is still unknown, but it appears to be a Ca²⁺-dependent process.

G protein-mediated current inhibition

Our data suggest that the Ca²⁺ current inhibition induced by muscarine, UK14304, and VIP is mediated by G protein-coupled receptors because GDPS abolishes these responses. Interestingly, GTPS dialysis mimicked the inhibition induced by UK14304 and VIP, but not by muscarine. This suggests that the G proteins mediating the UK14304 and VIP responses may have a high endogenous cycling rate and are maximally activated in the absence of receptor activation by 0.3 mM GTPS, whereas the G protein mediating the muscarine response has a slower endogenous cycling rate and is not readily activated by GTPS in the absence of receptor activation.

The present study shows that the muscarine response in MPG neurons is not sensitive to PTX or CTX, which contrasts with the SCG neurons, where muscarinic receptor-mediated current inhibition uses both PTX-sensitive and PTX-insensitive pathways (Beech et al. 1992). The lack of a PTX-sensitive muscarinic pathway in sympathetic MPG neurons may indicate a scarcity of M₄ muscarinic receptors or very poor coupling between M₄ receptor and PTX-sensitive G proteins in sympathetic MPG neurons. Our data do not suggest a paucity of PTX-sensitive G proteins, because UK14304 produces a large and PTX-sensitive current inhibition in sympathetic MPG neurons. Consistent with the findings from SCG neurons (Zhu and Ikeda 1994b), VIP induces a CTX-sensitive current inhibition in MPG neurons. Interestingly, CTX pretreatment mimics the VIP-induced current inhibition and abolishes any further inhibition by VIP in MPG neurons; in SCG, CTX pretreatment abolishes VIP-induced current inhibition but does not mimic the VIP response (Zhu and Ikeda 1994b). The reason for such a difference between MPG and SCG neurons is not clear. CTX exerts its effect by inhibiting the GTPase activity of the -subunit of G_s, resulting in persistent activation. We speculate that CTX does not mimic the effect of VIP in SCG neurons because of a fast CTX-induced desensitization of the G_s pathway (Zhu and Ikeda 1994b).

Effects of phosphorylation

The present study shows that the activation of PKC increases the amplitude of the Ca²⁺ current, diminishes tonic facilitation, and reduces the inhibition of this current by UK14304 and VIP; these results agree with those reported in SCG and central neurons (Swartz 1993; Swartz et al. 1993; Zhu and Ikeda 1994a). Recently Shapiro et al. (1996) also reported that the activation of PKC attenuated the membrane-delimited inhibition of N-type Ca²⁺ currents in rat sympathetic SCG neurons induced by norepinephrine and somatostatin. However, unlike in the present and previous (Swartz 1993; Zhu and Ikeda 1994a) studies, the effects of PKC reported by Shapiro et al. (1996) were not blocked by dialysis with the PKC pseudosubstrate inhibitor PKC_19-36. The reason for this difference is presently unknown.

Because muscarine most likely uses a different G protein-coupled mechanism to modulate Ca²⁺ channels, it is not surprising that PKC activation does not affect the muscarine-induced Ca²⁺ current inhibition. It was recently reported that the inhibition of Ca²⁺ currents in rat SCG neurons by muscarine is also not affected by PKC (Shapiro et al. 1996). Although the exact site regulated by PKC has not been determined, it is possible that the PKC-dependent phosphorylation site(s) is located on the N-type Ca²⁺ channel itself. The ₁ subunit of the N-type channel possesses multiple consensus sites for PKC-dependent phosphorylation (Williams et al. 1992), and the purified N-type Ca²⁺ channels can be phosphorylated in vitro by PKC (Ahlijanian et al. 1991). Recently Zamponi et al. (1997) have shown that PKC phosphorylates the ₁ subunit of the N-type Ca²⁺ channels (_1B) in the I-II cytoplasmic linker domain near one of the G subunit binding sites, and the PKC-dependent phosphorylation of this site antagonizes the G-induced inhibition of expressed N-type Ca²⁺ channels. In the present study the inhibition of PKC with the pseudosubstrate inhibitor (PKC_19-36) did not affect the inhibition of the Ca²⁺ current induced by either UK14304 or muscarine, indicating that PKC is not mediating these inhibitory responses.

In summary, our data suggest that muscarine uses a mechanism different from UK14304 and VIP to modulate the N-type Ca²⁺ channels in sympathetic neurons of the MPG. The evidence supporting this is as follows: the muscarine-induced inhibition of the Ca²⁺ current 1) has a different voltage dependence and is not relieved by a large depolarization to +80 mV, 2) is Ca²⁺ dependent, 3) is not mimicked by GTPS, 4) is neither PTX nor CTX sensitive, 5) is not affected by PKC activation, and 6) is additive with the action of UK14304. On the other hand, although VIP and UK14304 use different G protein pathways, these two different pathways most likely converge downstream to compete for the same target site on the N-type Ca²⁺ channels. Ehrlich and Elmslie (1995), who were investigating the ₂ noradrenergic/G_o and the VIP/G_s pathways in rat sympathetic SCG neurons, also came to the same conclusion. The evidence supporting this notion in the present study are as follows: both inhibitory responses 1) have a similar voltage dependence and are partially relieved by a large depolarization, 2) are mimicked by internal GTPS, 3) are reduced by PKC activation, and 4) are not additive. The specific identity of the G protein subunit(s) mediating the inhibition of theN-type and L-type Ca²⁺ channels is presently unknown. Recent studies in the rat SCG (for native N-type Ca²⁺ channels) have shown that the inhibition of these Ca²⁺ channels is mediated by the subunits of G proteins (Herlitze et al. 1996; Ikeda 1996). Perhaps the different G protein pathways activated by VIP (i.e., G_s) and UK14304 (i.e., G_o) use the same subunits, or else the different subunits may compete for the same target site to modulate the N-type Ca²⁺ channels in sympathetic neurons.

	ACKNOWLEDGEMENTS

  We thank S. Jones and D. Armstrong for critical reading of this manuscript.

	FOOTNOTES

  Address for reprint requests: J. L. Yakel, National Institute of Environmental Health Science F2-08, PO Box 12233, 104 T. W. Alexander Drive, Research Triangle Park, NC 27709.

  Received 30 December 1996; accepted in final form 18 April 1997.

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