Introduction

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- and µ-opioid agonist inhibition of calcium currents (Macdonald and Werz, 1986; Moises et al., 1994a; Tsunoo et al., 1986; Werz and Macdonald, 1984) is thought to mediate opioid reduction of calcium-dependent neurotransmitter release from presynaptic terminals (Werz and Macdonald, 1984). - and µ-agonists selectively inhibited the same high threshold calcium currents in rat dorsal root ganglion (DRG) neurons (Moises et al., 1994b; Wiley et al., 1997). Although N-type calcium current accounted for ~75% of the total opioid-sensitive calcium current, both - and µ-agonists also inhibited P- and Q-type calcium currents (Moises et al., 1994a; Wiley et al., 1997). Opioid inhibition of calcium current was mediated specifically by coupling to the G protein -subunit G_o (Gross et al., 1990a; Moises et al., 1994b; Wiley et al., 1997). Activation of a number of other inhibitory G protein-linked neurotransmitter receptors, including muscarinic, adrenergic, somatostatin, and -aminobutyric acid_B receptors, also inhibited calcium channels in a pertussis toxin-sensitive manner (Swartz, 1993). Although receptors demonstrate specificity for G protein -subunits, the actual inhibition of calcium current may be mediated via the G protein -subunits (Herlitze et al., 1996; Ikeda, 1996), possibly by inhibiting calcium current through a direct interaction with the 1 subunit of the calcium channel (De Waard et al., 1997; Zamponi et al., 1997).

Protein kinase C (PKC) has been implicated in several in vivo studies of opioid action. Intrathecal administration of the phorbol ester 4-phorbol 12-myristate 13-acetate (PMA) blocked opioid-induced antinociception in rats (Zhang et al., 1990; Narita et al., 1997). Intrathecal administration of PKC inhibitors did not block opioid antinociception but did block acute opioid tolerance (Narita et al., 1995). Furthermore, chronic i.p. morphine increased PKC activity in the pons/medulla in rats (Narita et al., 1994).

Although in vivo tolerance is often receptor specific (i.e., homologous), heterologous (i.e., cross-receptor) tolerance has also been observed in opioid coupling to calcium channels (Kaneko et al., 1997). -Opioid receptors recently also have been shown to be heterologously desensitized by N-methyl-D-asparate in a PKC-dependent manner (Fan et al., 1998). Although PKC does not appear to be involved in agonist-induced receptor phosphorylation (Pei et al., 1995) and thus may not be directly involved in homologous desensitization, it may be important in mediating cross-tolerance between different opioid receptors and/or other neurotransmitters such as N-methyl-D-aspartate.

Activation of PKC with phorbol esters reduced calcium current inhibition by inhibitory G protein-linked neurotransmitter receptor agonists in several rat neuronal types (Swartz, 1993; Wilding et al., 1995; Zhu and Ikeda, 1994), but PKC did not appear to be involved in the actual signaling pathway leading to inhibition of calcium current induced by these agonists (Swartz, 1993). Because multiple neurotransmitter systems can activate PKC, PKC attenuation of - and/or µ-opioid receptor coupling to calcium channels could be a potential mechanism for the observed PKC effects on systemic opioid action. However, although PKC has been shown to attenuate inhibition of calcium currents by other Gi-linked neurotransmitter, it is not known whether activation of PKC can block opioid-induced inhibition of calcium currents. To investigate this possibility, we examined the effects of PKC on µ-opioid (morphine) and -opioid (dynorphin A and U50,488) inhibition of calcium channel currents in rat DRG neurons.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Preparation of Acutely Dissociated Neurons. DRG neurons were prepared from Sprague-Dawley rats, 14 to 50 days of age, of both sexes using a technique similar to that described previously (Gross et al., 1990b). Briefly, after removal from the spinal column, thoracic DRG neurons were minced, incubated with collagenase and trypsin, and then triturated to separate the cells. Cells were then plated onto uncoated 35-mm culture plates or plates coated with rat-tail collagen in minimal essential media supplemented with 16.5 mM NaHCO₃, 28.2 mM glucose, and 10% fetal calf serum. Cultures were incubated at 37°C in 95% air, 5% CO₂. Recordings were made at room temperature, typically within 10 h of plating.

Drugs and Enzymes. Constitutively active PKC catalytic subunit (PKM) purified from bovine brain (Woodgett and Hunter, 1987) was a kind gift of Dr. Michael Browning (University of Colorado, Denver, CO). Protein kinase C inhibitory peptide (PKC-I), PKC_19-31, and dynorphin A were obtained from RBI (Natick, MA). Fetal calf serum was from Life Technologies, Inc. (Grand Island, NY). Morphine, U50,488, PMA, nifedipine, -conotoxin from Conus geographus (GVIA), collagenase, trypsin, and all other chemicals were from Sigma Chemical Co. (St. Louis, MO).

Solutions and Drug Application. Stock solutions of PKM (1 µM) and PKC-I (4 mM) were stored at 80°C. Just before use, solutions of PKM, PKC-I, and/or vehicle were prepared. PKM and PKC-I were diluted into internal pipette solution (see below) at 40 nM or 4 µM, respectively, and kept on ice until use. To prepare a solution of both PKM and PKC-I, the stock solution of PKC-I was diluted to 4 µM into the working solution of PKM, and the mixture was incubated at 37°C for 15 min to inactivate PKM. When using solutions containing PKM or PKC-I, the recording pipette tip was filled with internal recording solution, and then back-filled with the peptide-containing solution. Stock solutions of all other drugs were diluted into external solution on the day of use. Nifedipine was dissolved in dimethyl sulfoxide (DMSO) (10 mM) on the day used (final DMSO concentration was less than the 1:1000). Stock solutions of dynorphin A and -conotoxin GVIA (1 mM) were dissolved in filtered distilled water, lyophilized in aliquots, and stored at 20°C until the day used. Stock solutions of morphine were prepared in distilled water (10 mM) and of PMA in DMSO (100 µM) and were stored at 20°C until used. Opioids were applied to cells using a modified U-tube application system (Greenfield and Macdonald, 1996) using a solenoid-controlled 10-s application of drug, followed by vacuum reuptake. PMA and -conotoxin GVIA were applied by pressure ejection from a blunt-tipped (20-40-µm opening) pipette positioned ~25-50 µm from the neuron.

Whole-Cell Patch Clamp Recording Techniques. Voltage-clamp recordings were made using the whole-cell variant of the patch-clamp recording technique (Hamill et al., 1981) using an Axopatch 1-B amplifier (Axon Instruments, Foster City, CA), and glass recording pipettes, micropipette tip resistances of 1-2 megaohms, and seal resistances of greater than 1 gigaohm. Patch clamp micropipettes were pulled from Labcraft micro-hematocrit capillary tubes (Curtin Matheson Scientific, Inc., Houston, TX) using a P-87 Flaming-Brown micropipette puller (Sutter Instrument Co., San Rafael, CA). Signals were low pass filtered at 2 kHz using an 8-pole Bessel filter then digitized, recorded, and analyzed using pCLAMP6 software (Axon Instruments).

Data Analysis. Leak current was estimated as the inverse of the current generated by hyperpolarizing commands of equal value to those used to depolarize the neurons. These were digitally subtracted from total currents to give leak-subtracted barium or calcium currents. Statistical comparisons of the effects of drugs, peptides, and PKM on peak current and on current rundown were performed using a two-tailed Student's t test. Comparisons between dynorphin A inhibition of calcium currents before and after treatment with drugs in the same cell were analyzed using a paired-sample t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

PMA Increased Calcium Channel Current. Effects of phorbol esters and PKM on calcium channels and -opioid signaling were studied using a tight-seal, whole-cell voltage clamp protocol on the somata of acutely dissociated rat DRG neurons. Barium was used as a charge carrier through calcium channels, and internal calcium was buffered with EGTA. Currents were elicited by voltage steps to 0 mV from a holding potential of 80 mV. Under these conditions, primarily transient high-threshold, voltage-dependent calcium channel currents were activated (Moises et al., 1994a). External application of 250 nM PMA to the neurons increased total barium current, with maximal increase within 3 min (Fig. 1, A and B). In contrast, after treatment of neurons with vehicle (1:1000 DMSO) or 250 nM 4-phorbol 12,13-didecanoate (4-PdBu), which does not activate PKC, barium current decreased or "ran down" slightly (Fig. 1, A and B). The average maximal increase in current was to 116 ± 4% of current before application of PMA (Fig. 1C). The actual increase in barium current induced by PMA appeared to be somewhat larger than this, because currents in both vehicle- and 4-PdBu-treated neurons ran down about 10% within 3 min (Fig. 1C). The largest PMA-induced increase recorded was to 136% of control current, and increases in current were seen in 8 of 11 neurons tested. Thus, the PMA-induced increase in calcium current reported here was consistent with effects reported previously of PMA in DRG neurons (Swartz, 1993), although the magnitude of the increase was smaller than that reported in other rat neuronal types such as cerebral cortical and superior cervical ganglion neurons (Swartz, 1993). The observed effect of PMA was also smaller than that produced by internal application of PKM, which we have previously shown maximally increased peak calcium current to >200% of control in DRG neurons (Hall et al., 1995).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   PMA increased current through calcium channels in rat DRG neurons. Calcium channel currents were evoked with a 200-ms pulse to 0 mV from a holding potential of 80 mV with barium as the charge carrier. Currents from three separate neurons treated with either PMA (), 4-PdBu (), or vehicle (1:1000 DMSO, ) are shown in A as peak currents plotted as a function of time after patch rupture and in B as superimposed traces at 0-4 min after treatment. Peak barium current amplitude was measured at 15 ms after pulse. In C, relative change in peak amplitude was plotted as a function of time after treatment with PMA (), 4-PdBu (), or vehicle (1:1000 DMSO, ). Points represent means ± S.E.M. Change in peak amplitude was calculated by dividing the (-Ibarium) at each point by the current before treatment (-Ibariumt = 0).

PMA Decreased and PKC-I Increased Rate of Calcium Channel Current Rundown. We reported previously that calcium currents elicited from DRG neurons routinely ran down over time; currents reached a peak at 2-5 min and then progressively decreased for the remainder of the recording (Hall et al., 1995). This phenomenon appeared to be due to dialysis out of the neuron of cellular components required for maintaining functional calcium channels. As rundown was slowed by inclusion of ATP in the recording pipette (Hall and Macdonald, unpublished data), it appeared likely that a loss of protein kinase activity was involved. All data reported here were obtained from recordings with 5 mM ATP in the pipette. In control neurons, current declined to 50% of peak value within ~20 min of patch rupture and was usually <10% of peak within 40 min of patch rupture (Fig. 2, ). We have shown previously that intracellular PKM increased peak current without affecting rundown. In contrast, while external PMA only modestly increased "peak" calcium current, it substantially decreased the rate of current rundown. Current from neurons treated with external PMA ran down at a slower rate than from control neurons (Fig. 2, ), typically with currents not reduced to 50% of peak until 30-40 min after patch rupture. In some neurons treated with PMA, currents as large as 50% of peak were recorded as long as 70 min after patch rupture. This suggested potential differences in effects of activation of endogenous PKC by PMA and introduction of a constitutively activated kinase into the neuron.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   PMA decreased and PKC-I increased the rate of calcium channel current rundown. Barium currents were evoked by a 200-ms pulse to 0 mV from a Vh of 80 mV with barium as a charge carrier in the presence of 3 µM nifedipine in control (, n = 6), PMA-treated (, n = 5), PKC-I-treated (, n = 5), and PKC-I and PMA-treated (, n = 5) DRG neurons. Rates of current rundown were compared by dividing inward currents at each time point (-Ibarium) by the maximum inward current (-Ibariummax) attained during the experiment. The rundown period was defined as the period following maximum current. PMA treatment caused a significant decrease in the rate of rundown in control and PKC-I treated neurons, whereas PKC-I increased the rate of rundown in control neurons. (Data were analyzed using a two-tailed Student's t test. Values were shown as mean ± S.E.M.

PMA Attenuated Dynorphin A Inhibition of Calcium Channel Currents. Morphine and Dynorphin A, through interaction with µ- and -opioid receptors, respectively, inhibit calcium channel currents through a rapid, reversible, voltage-dependent, pertussis toxin-sensitive mechanism (Taussig et al., 1992; Moises et al., 1994b). Acutely dissociated DRG neurons are a heterogeneous preparation, with populations of dynorphin- and/or morphine-sensitive neurons, and furthermore, dynorphin A can have effects through µ- as well as -opioid receptors. However, populations of dynorphin-sensitive and morphine-insensitive DRG neurons have been observed (Moises et al., 1994a); and studies using µ-specific H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH₂ (CTAP), -funaltrexamine) and -specific (norbinaltorphimine) antagonists also indicated that the effects of dynorphin A and morphine on calcium currents at the concentrations used in this study were due primarily to interactions with -opioid and µ-opioid receptors, respectively (Moises et al., 1994a; Wiley et al., 1997; King and Macdonald, unpublished observation). -Opioid receptor inhibition of calcium channel currents was probably mediated through pathways similar or identical with those of µ-opioid receptors, which have been shown to be rapid and membrane delimited (Wilding et al., 1995).

View larger version (30K): [in this window] [in a new window]   Fig. 3.   PMA attenuated µ- and -opioid inhibition of calcium channel current. In A, barium currents evoked by a 200-ms pulse to 0 mV from a Vh of 80 mV were shown as peak current plotted as a function of time after patch rupture and as superimposed traces before and immediately after 10-s applications of 3 µM dynorphin A (arrows marked by Dyn) and 250 nM PMA (bar). B, inhibition (shown as percentage of change in amplitude from currents immediately before application of agonists) of barium current by 3 µM dynorphin A (Dyn, n = 11), 3 µM U50,488 (U50, n = 4), 3 µM morphine (Morph, n = 8), and 100 µM pentobarbital (PentoB, n = 4) before (light column) and 3 min after (dark column) treatment of neurons with 250 nM PMA.

View larger version (31K): [in this window] [in a new window]   Fig. 4.   PKC-I blocks PMA attenuation of dynorphin A inhibition of calcium channel current. In A, barium currents evoked by a 200-ms pulse to 0 mV from a Vh of 80 mV from neurons dialyzed with 4 µM PKC-I in the recording pipette were shown as peak current plotted as a function of time after patch rupture and as superimposed traces before and immediately after 10-s applications of 3 µM dynorphin A (arrows marked by Dyn) and 250 nM PMA (bar). B, inhibition of barium current (shown as percentage of change in amplitude from currents immediately before application of dynorphin) in neurons dialyzed with 4 µM PKC-I by 3 µM dynorphin A before (light column) and 3 min after (dark column) treatment of neurons with 250 nM PMA. (n = 4, NS.)

Effect of Blockade of N-Type Current on Reduction by PMA of Calcium Channel Current Inhibition by Dynorphin A. DRG neurons contain multiple high-threshold calcium current subtypes, including N-, L-, Q-, P- and R-types (Moises et al., 1994a; Wiley et al., 1997). N-type current accounted for about 75% of the total dynorphin A-sensitive current in rat DRG neurons, although dynorphin A also inhibited a portion of the Q-, P-, and R-current (Wiley et al., 1997). To gain information as to the specific calcium channels being affected by PMA, we examined the effect of the N-channel blocker -conotoxin GVIA on the ability of PMA to attenuate dynorphin A inhibition of calcium currents (Fig. 5). Dynorphin A (3 µM) was applied to DRG neurons to determine the total dynorphin A-sensitive current (Fig. 4A, traces 1 and 2). Dynorphin A reduced the current from 2.7 to 2.2 nA (21%). After recovery, the neuron was treated with 250 nM PMA for 2 min, and dynorphin A was reapplied, which reversibly inhibited current from 2.5 to 2.4 nA (a 7% decrease) (Fig. 5A, traces 3 and 4). After recovery for 1 min, N-channels were blocked with -conotoxin GVIA, which reduced the basal current by 52% from 2.5 to 1.2 nA, and then dynorphin A was reapplied. Dynorphin A only reduced the current from 1.2 to 1.1 nA (Fig. 5A, traces 5 and 6), a decrease which represented 5% of the original current and a 9% inhibition of the residual current. A similar protocol was followed to determine the levels of dynorphin A- and -conotoxin GVIA-sensitive currents in neurons not treated with PMA. In the absence of conotoxin, dynorphin A inhibited an average of 22 ± 2% of the current before and 5 ± 2% of the current after PMA treatment (n = 11); after -conotoxin GVIA treatment, dynorphin A inhibited an average of 6 ± 1% of the current in control (n = 4) and 6 ± 2% of the current in PMA treated (n = 4) neurons (Fig. 5B). Although the effects of dynorphin A on the residual calcium currents after PMA were small, there appeared to be no difference in dynorphin sensitivity in control and PMA-treated neurons after -conotoxin GVIA treatment (Fig. 5B). -Conotoxin GVIA also did not appear to further decrease the dynorphin A-sensitive current when applied after PMA (Fig. 5B, compare striped columns). Furthermore, when PMA was applied after -conotoxin GVIA, the small residual dynorphin-sensitive current was not further reduced (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Effect of PMA and -conotoxin GVIA on dynorphin A inhibition of calcium channel currents. In A, barium currents evoked by a 200-ms pulse to 0 mV from a Vh of 80 mV in the presence of 3 µM nifedipine were shown as peak current plotted as a function of time after patch rupture and as superimposed traces at times indicated by arrows. Application of dynorphin A (3 µM) for 10 s reversibly reduced total high-threshold barium current. Application of PMA (250 nM) for 5 min had little effect on total barium current but substantially reduced the fraction of current inhibited by dynorphin A. Application of -conotoxin GVIA (3 µM) for 30 s irreversibly reduced total whole-cell barium current but had little effect on the fraction of current inhibited by dynorphin A. B, dynorphin A inhibition (shown as percentage of change) of barium current in control (n = 4) and PMA-treated (n = 4) neurons before and after treatment with -conotoxin GVIA (3 µM). PMA treatment significantly (p < .01) decreased dynorphin A inhibition of barium current. There was no difference in dynorphin A inhibition in control and PMA neurons after treatment with -GVIA, and -GVIA did not further reduce the fraction of current inhibited by dynorphin A in PMA-treated neurons.

PKM Attenuated Dynorphin A Inhibition of Calcium Channel Current. In addition to activating endogenous PKC, phorbol esters could be accompanied by other "nonspecific" effects on calcium channels, receptors, or other cellular proteins. For example, phorbol esters are known to alter the cycling and down-regulation rate of multiple receptors and ion channels and have been shown to recruit covert or previously inactive calcium channels in Aplysia neurons (Strong et al., 1987). Phorbol esters activate multiple isoforms of PKC, which may be differentially regulated in normal cells, and there is evidence that a phorbol ester-insensitive PKC isoform was involved in norepinephrine regulation of calcium channels in chick DRG neurons (Boehm et al., 1996). Furthermore, there have been conflicting reports of the effects of phorbol esters and other activators of PKC to either increase (Swartz, 1993; Zhu and Ikeda, 1994), decrease (Diverse-Pierluissi and Dunlap, 1993; Werz and Macdonald, 1987), or have no effect (Boehm et al., 1996) on calcium channel currents. Because of these concerns, we examined the effect of intracellular application of purified, constitutively active PKC, PKM, on dynorphin A inhibition of calcium channels. As demonstrated previously with DRG neurons (Hall et al., 1995), intracellular application of 20 nM PKM increased peak calcium currents compared with those from control neurons (3.4 ± 0.4 nA PKM treated, n = 6 versus 2.5 ± 0.4 nA control, n = 7, p < .05); whereas intracellular application of excess PKC-I (4 µM) with 20 nM PKM, decreased peak currents (1.5 ± 0.3 nA, n = 6, p < .05), likely as a result of inhibition of endogenous PKC.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Dynorphin A inhibition of calcium current was decreased in PKM-treated neurons. A, superimposed current traces before and immediately after 10-s treatment with dynorphin A (3 µM) in control neurons (Control), neurons dialyzed with PKM (40 nM) (PKM), and neurons dialyzed with PKM (40 nM) and PKC-I (4 µM) (PKM+PKC-I). Currents were evoked by a 200-ms pulse to 0 mV from a Vh of 80 mV, with calcium as the charge carrier, in the presence of 3 µM nifedipine. B, inhibition (shown as percentage of change) of barium current by 3 µM dynorphin A in control (n = 7, light column), PKM-treated (n = 7, dark column), and PKM + PKC-I-treated (n = 6, striped column) neurons. Data were normalized to the amplitudes of currents immediately before application of dynorphin A.

Effect of Blockade of N-Type Current on PKM Attenuation of Dynorphin A Inhibition of Calcium Channel Current Inhibition. We next investigated effects of intracellular PKM on specific calcium current subtypes. Dynorphin A inhibition of calcium currents in control and PKM-treated neurons was compared before and after blocking N-type currents with -conotoxin GVIA. Before treatment with -conotoxin GVIA, dynorphin inhibition of calcium current was significantly smaller (p < .05) in neurons dialyzed with 20 nM PKM (9 ± 4%, n = 6) than in control (23 ± 3%, n = 7) neurons (Fig. 7A, traces 1 and 2; Fig. 6B). After -conotoxin GVIA treatment, however, dynorphin A inhibition of calcium current was not significantly different in control (5 ± 4%) and PKM-dialyzed (6 ± 2%) neurons (Fig. 7A, traces 3 and 4; Fig. 6B). Furthermore, in neurons dialyzed with PKM, treatment with -conotoxin GVIA did not appear to further decrease dynorphin A effects (Fig. 7B, compare the striped columns). These findings suggested that, like PMA treatment, PKM appeared to primarily affect -opioid receptor coupling with N-type calcium currents. Also similar to PMA, intracellular PKM did not decrease the proportion of N-type current. -Conotoxin GVIA inhibited a slightly larger proportion of calcium current in PKM-treated neurons than in control neurons (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Effect of PKM and -conotoxin GVIA on dynorphin A inhibition of calcium currents. A, superimposed current traces of control and PKM-dialyzed neurons before (trace 1) and immediately after (trace 2) 10-s treatment with 3 µM dynorphin A; after treatment with 3 µM -conotoxin GVIA (trace 3); and subsequently with 10-s 3 µM dynorphin A (trace 4). Currents were evoked by a 200-ms pulse to 0 mV from a Vh of 80 mV with calcium as the charge carrier in the presence of 3 µM nifedipine. B, comparison of dynorphin A inhibition (shown as percentage of change) of calcium current (-Icalcium) in control neurons (n = 6) and neurons dialyzed with PKM (n = 7) before and after treatment with -conotoxin GVIA (3 µM). Dynorphin A inhibition of Icalcium was reduced in PKM-treated neurons (p < .05). There was no difference in dynorphin A inhibition in control and PKM-treated neurons after treatment with -GVIA, and -GVIA did not further reduce the fraction of current inhibited by dynorphin A in PKM-treated neurons.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

External PMA and Dialysis with PKM Both Attenuated - and µ-Opioid Receptor Inhibition of Calcium Channel Currents in DRG Neurons. We demonstrated that in acutely dissociated rat DRG neurons, both external treatment with the phorbol ester PMA and intracellular dialysis with activated PKC, PKM, attenuated - and µ-opioid receptor-mediated inhibition of calcium currents. The PMA attenuation of dynorphin A inhibition of calcium current was similar to effects of PMA on several types of rat neurons on inhibition of calcium currents by agonists of ₂-adrenergic, -aminobutyric acid_B, and muscarinic cholinergic receptors (Swartz, 1993; Zhu and Ikeda, 1994). The effects of PMA on calcium current inhibition appeared to be due to activation of endogenous PKC rather than nonspecific actions on membranes or cellular proteins, because the effects of PMA were blocked by dialysis of the neuron with the PKC-I peptide. Endogenous PKC activity did not appear to be necessary for dynorphin A inhibition of calcium currents because intracellular PKC-I did not block dynorphin A inhibition, consistent with effects of PKC-I on morphine- and other agonist-induced inhibition of calcium currents in rat neurons (Swartz, 1993; Wilding et al., 1995).

Differential Effects of External PMA and PKM Dialysis on Peak Calcium Current and Current Rundown. Although both PKM dialysis and external PMA application increased calcium current, they did so with different efficacy. Treatment of DRG neurons with PMA produced only a modest increase in peak calcium current (15%), which was in contrast to larger effects of PMA (50-100%) in rat cerebral cortical, hippocampal, and superior cervical ganglion neurons (Swartz, 1993; Zhu and Ikeda, 1994). Dialysis of DRG neurons with PKM resulted in a larger increase in basal calcium current (~100%) than did PMA treatment, i.e., the PKM-induced increase in calcium current in DRG neurons was comparable to that induced by activation of endogenous PKC by PMA in central neuronal types. This could reflect differences in levels of PKC activity or efficacy between DRG and central neurons. However, it is also possible that differences between effects of PMA and PKC may have been due to the relatively high level of kinase activity in neurons dialyzed with PKM, leading to the phosphorylation of nonphysiological substrates or other artifactual causes.

PKC Primarily Attenuated -Opioid Receptor Coupling to N-Type Calcium Currents. Rat DRG sensory neurons contain low-threshold transient (T-type) calcium current and multiple high-threshold calcium current subtypes. Using toxins that are specific blockers of current subtypes, L-, N-, P-, Q- and "R-" (toxin-resistant) subtypes of high-threshold voltage-dependent calcium currents have been reported in rat DRG neurons (Moises et al., 1994a; Rusin and Moises, 1995; Wiley et al., 1997). Dynorphin A did not inhibit L-type currents but did inhibit N-, P-, and Q-type currents, although N-type current accounted for ~75% of the total dynorphin A-sensitive current (Wiley et al., 1997). Neither PMA nor PKM decreased the proportion of N-type current, indicating that their effect to decrease dynorphin A inhibition of calcium currents was not simply due to a selective removal of a large part of the dynorphin A-sensitive calcium currents by PKC. Because PMA and PKM did not block dynorphin A inhibition of total calcium current completely, it is possible that PKC selectively had an effect on one or more subtype of calcium channel. Thus, the residual dynorphin A inhibition seen in the presence of activated PKC could have been due to effects of dynorphin A on PKC-insensitive channels. Blocking N-type current with -conotoxin did not further decrease dynorphin A inhibition in PMA- and PKM-treated neurons, suggesting that PKC primarily affected -opioid receptor coupling to N-type channels.

Potential Mechanisms for PKC Attenuation of -Opioid Receptor Coupling to Calcium Channels. Recently, inhibition of calcium currents by G protein-linked receptors has been concluded to be mediated by G protein -subunits, because coexpression of G protein - and -subunits, but not expression of -subunits or - or -subunits individually, were capable of inhibiting calcium currents in a manner similar to agonists (Herlitze et al., 1996; Ikeda, 1996). G protein -subunits bind directly to the ₁-subunit of the calcium channel at sites on the cytoplasmic "linker" region between transmembrane domains I and II (De Waard et al., 1997; Zamponi et al., 1997), in a region overlapping or adjacent to the putative interaction site for the calcium channel -subunit (De Waard et al., 1996). Furthermore, there was evidence that PKC directly phosphorylated the calcium channel ₁-subunit on serines and threonines within the same region and thus blocked the effect of G protein -subunits binding (De Waard et al., 1997). Thus, these findings point to PKC phosphorylation of the calcium channel as a mechanism for attenuation of G protein inhibition.

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