Phorbol Ester-Induced Inhibition of Potassium Currents in Rat Sensory Neurons Requires Voltage-Dependent Entry of Calcium

Yi-Hong Zhang,1 J. L. Kenyon,2 and G. D. Nicol1

 1Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5120; and  2Department of Physiology and Cell Biology/MS352, University of Nevada School of Medicine, Reno, Nevada 89557


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Zhang, Yi-Hong, J. L. Kenyon, and G. D. Nicol. Phorbol Ester-Induced Inhibition of Potassium Currents in Rat Sensory Neurons Requires Voltage-Dependent Entry of Calcium. J. Neurophysiol. 85: 362-373, 2001. The whole cell patch-clamp technique was used to examine the effects of protein kinase C (PKC) activation (via the phorbol ester, phorbol 12,13 dibutyrate, PDBu) on the modulation of potassium currents (IK) in cultured capsaicin-sensitive neurons isolated from dorsal root ganglia from embryonic rat pups and grown in culture. PDBu, in a concentration- and time-dependent manner, reduced IK measured at +60 mV by ~30% if the holding potential (Vh) was -20 or -47 mV but had no effect if Vh was -80 mV. The PDBu-induced inhibition of IK was blocked by pretreatment with the PKC inhibitor bisindolylmaleimide I and IK was unaffected by 4-alpha phorbol, indicating that the suppression of IK was mediated by PKC. The inhibition of IK by 100 nM PDBu at a Vh of -50 mV was reversed over several minutes if Vh was changed to -80 mV. In addition, intracellular perfusion with 5 mM bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) or pretreatment with omega -conotoxin GVIA or Cd2+-Ringer, but not nifedipine, prevented the PDBu-induced suppression of IK at -50 mV, suggesting that a voltage-dependent influx of calcium through N-type calcium channels was necessary for the activation of PKC. The potassium channel blockers tetraethylammonium (TEA, 10 mM) and 4-aminopyridine (4-AP, 3 mM and 30 µM) reduced IK, but only TEA attenuated the ability of PDBu to further inhibit the current, suggesting that the IK modified by PDBu was sensitive to TEA. Interestingly, in the presence of 3 mM or 30 µM 4-AP, 100 nM PDBu inhibited IK when Vh was -80 mV. Thus 4-AP promotes the capacity of PDBu to reduce IK at -80 mV. We find that activation of PKC inhibits IK in rat sensory neurons and that voltage-dependent calcium entry is necessary for the development and maintenance of this inhibition.


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To understand the regulation of the sensitivity of sensory neurons, we have investigated the signaling pathways activated by sensitizing agents. Although recent work has focused on mechanisms activated by cAMP and protein kinase A (Cui and Nicol 1995; Evans et al. 1999), other pathways can mediate the physiological responses to sensitizing agents by mechanisms that are only poorly understood. For example, bradykinin, a potent inflammatory mediator, produces an inward current in C-type sensory neurons (nonmyelinated slowly conducting axons) through the activation of protein kinase C (PKC) (Burgess et al. 1989). In addition, Schepelmann et al. (1993) demonstrated that phorbol ester (an activator of PKC) (Mellor and Parker 1998; Nishizuka 1984, 1986) directly stimulated as well as sensitized the excitation of primary afferents in the knee joint to mechanical manipulation. This enhancement of neuronal activity would suggest that the release of neurotransmitter from sensory neurons might be facilitated. Indeed the release of substance P and calcitonin gene-related peptide from isolated sensory neurons grown in culture was augmented by pretreatment with the phorbol ester, phorbol 12,13 dibutyrate (PDBu), and this enhancement was blocked by inhibition of PKC by staurosporine (Barber and Vasko 1996). These results suggest that activation of PKC can modulate the sensitivity of sensory neurons to stimulation. Such observations raise the question as to the nature of the cellular mechanisms whereby PDBu-induced activation of PKC gives rise to the enhanced excitability of sensory neurons.

Previous work has implicated several mechanisms by which the activation of PKC can facilitate membrane excitability. In hippocampal slices, exposure to phorbol 12,13-diacetate increased the number of action potentials evoked by a depolarizing current and also suppressed the slow afterhyperpolarization, suggesting that this phorbol ester inhibited potassium currents in these CA1 neurons (Baraban et al. 1985). Also treatment with phorbol esters led to a broadening of the action potential in mouse sensory neurons (Werz and Macdonald 1987). However, the actions of PKC and its possible modulation of the isolated IK in mammalian sensory neurons have not been examined. We have shown previously that the excitability of embryonic rat sensory neurons was enhanced by prostaglandin E2, through activation of the cyclic AMP signaling pathway, and that this involved the suppression of a delayed rectifier-like IK (Evans et al. 1999; Nicol et al. 1997). Because phorbol esters facilitate both the activation of articular primary afferents (Schepelmann et al. 1993) and the release of neuropeptides (Barber and Vasko 1996), we examined the possibility that the activation of PKC, similar to the activation of protein kinase A (PKA), inhibits IK. Also we investigated the mechanism of action for PKC wherein we find a novel requirement for voltage-gated Ca2+ channel activity. Preliminary results have appeared in abstract form (Zhang and Nicol 1998).


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Isolation and culture of embryonic rat sensory neurons

The procedures for isolation and culture of rat sensory neurons have been described previously (Vasko et al. 1994). Briefly, timed-pregnant rats were rendered unconscious with CO2 and killed by cervical dislocation. The dorsal root ganglia were dissected from each embryo (E15-E17), and sensory neurons were dissociated from the ganglia with 0.025% trypsin (37°C, 25 min) and mechanical agitation. The cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Grand Island, NY) supplemented with 2 mM glutamine, 50 µg/ml penicillin and streptomycin, 10% (vol/vol) heat-inactivated fetal bovine serum, 50 µM 5-fluoro-2'-deoxyuridine, 150 µM uridine, and 250 ng/ml 7S-nerve growth factor (Harlan Bioproducts for Science, Indianapolis, IN). Approximately 150,000 cells/ml were plated in a collagen-coated culture dish containing small plastic cover slips. Cultures were maintained at 37°C in a 5% CO2 atmosphere, and the medium was changed every second day. All procedures have been approved by the Animal Use and Care Committee of the Indiana University School of Medicine.

Recording procedures

Recordings were made using the whole cell patch-clamp technique as previously described (Evans et al. 1999; Hamill et al. 1981; Nicol et al. 1997). Briefly, a cover slip with the sensory neurons (4-6 days in culture) was placed in a recording chamber where the neurons were bathed in normal Ringer solution of the following composition (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, pH at 7.4 with NaOH. Recording pipettes typically had resistances of 2-5 MOmega when filled with the following solution (in mM): 140 KCl, 5 MgCl2, 4 ATP, 0.3 GTP, 2.5 CaCl2, 5 EGTA (calculated free Ca2+ concentration of ~100 nM), and 10 HEPES, at pH 7.2 with KOH. We have not corrected for the junction potential of 3.7 mV (Barry 1994) and expect that the actual membrane potentials are 3-4 mV more negative than those listed. In experiments using bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA, tetrapotassium salt), the concentration of KCl was lowered accordingly.

To isolate IK, the cells were superfused with 140 mM N-methyl-glucamine chloride Ringer (NMG, an equimolar substitution for NaCl), pH 7.4 with KOH. We found that superfusion with NMG significantly depolarized the resting membrane potential in sensory neurons to -47 ± 0.3 (SE) mV from a control level of -56 ± 0.5 mV (n = 59) in normal Ringer solution. The membrane voltage was held at three different Vhs to ascertain the contribution of various potassium channel populations to the total IK. Initially the voltage was held at the zero-current potential for that particular neuron. A voltage-step protocol was used to examine the activation of IK, wherein voltage steps of 300 ms were applied at 5-s intervals in +10 mV increments to a maximum of +60 mV. Vh was then changed to -80 mV, and the voltage-step protocol was repeated; this sequence was then repeated for a Vh of -20 mV. In some cases, recordings at each holding potential were performed in different neurons.

Whole cell currents were recorded from sensory neurons with an Axopatch 200 (Axon Instruments, Foster City, CA) patch-clamp amplifier; the data were acquired and analyzed using pCLAMP6 (Axon Instruments). Both capacitance and series resistance compensation were used; however, no compensation was made for leak currents. The average uncompensated series resistance was 3.5 ± 0.1 MOmega (range 2.3-5.3 MOmega , n = 44). The maximum voltage error resulting from the uncompensated series resistance was calculated for each cell and averaged 7.4 ± 0.4 mV (n = 44). Cells were superfused continuously during exposure to the various test compounds. In experiments using 10 mM TEA, the extracellular NMG concentration in the Ringer solution was reduced to 130 mM. In contrast, 4-aminopyridine (4-AP) was added directly to the Ringer solution. These agents were bath applied for 3-4 min prior to the acquisition of additional current recordings. The results presented in this report were obtained from neurons that were sensitive to capsaicin (see criteria described in Evans et al. 1999; Holzer 1991). All experiments were performed at room temperature (~23°C).

Data analysis

All values represent the means ± SE . The voltage dependence for activation of the outward IK was fitted with the Boltzmann relation (see Evans et al. 1999). Statistical differences between the control currents and those obtained under various treatment conditions were determined by using a repeated-measures ANOVA (RM ANOVA) with a Tukey post hoc test; for the comparison of control to a single drug treatment, a Student's paired t-test was used. Values of P < 0.05 were judged to be statistically significant.

Chemicals

All chemicals were obtained from Sigma Chemical (St. Louis, MO). PDBu, 4alpha phorbol, and capsaicin were dissolved in 1-methyl-2-pyrrolidinone (HPLC grade) to obtain concentrated stock solutions, which were then diluted with Ringer solution to the appropriate concentration. We have demonstrated previously that the vehicle, 1-methyl-2-pyrrolidinone, had no effect on either the activation or steady-state inactivation curves obtained for potassium currents (Nicol et al. 1997).


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Ability of PDBu to inhibit IK depends on the holding potential

To investigate the role of PKC in the modulation of IK, sensory neurons were exposed to the phorbol ester, PDBu. Previous studies demonstrated that different populations of potassium currents can be isolated by using different Vhs to inactivate specific types of currents (e.g., Belluzzi et al. 1985a,b). Using this approach, sensory neurons were held at different Vhs and then treated with PDBu to determine the possible actions of PKC on the activation of specific types of IK. We chose three different Vhs: -20 mV was used to isolate the delayed rectifier-like IK, the resting potential of the neuron was used to observe the effects of PDBu under physiological conditions, and -80 mV was used to examine both the delayed rectifier-like IK and any rapidly-inactivating type IK (e.g., IA-like currents). As shown in Fig. 1, depolarizing voltage steps from a Vh of -20 mV (A) elicited a series of IK that exhibited little time-dependent inactivation, whereas when the neuron was held at more hyperpolarized levels of Vh, IK showed greater extents of inactivation during test depolarizations (B and C). These results suggest that when Vh was held at -20 mV, the elicited IK consisted primarily of a delayed-rectifier like current. The application of 100 nM PDBu to sensory neurons caused a time-dependent suppression of IK, when Vh was either -47 (the resting or 0-current potential, Fig. 1B) or -20 mV (Fig. 1A). When held at -47 mV, PDBu reduced IK in this particular neuron (measured at +60 mV) from a peak value of 2.52 to 1.77 nA (a 30% reduction). A similar result was obtained in a different neuron held at -20 mV (0.81 to 0.54 nA). In contrast, when a different neuron was held at a Vh of -80 mV, the elicited IK was unaffected by PDBu (Fig. 1C).



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Fig. 1. Phorbol 12,13 dibutyrate (PDBu) suppresses IK when the neuron was held at holding potentials of -20 and -47 mV but not at -80 mV. Representative whole cell recordings of IK obtained under control conditions (left) at the indicated holding potentials (Vh) recorded in sensory neurons. Right: the current recordings obtained after a 20-min exposure to 100 nM PDBu from the same cell. Recordings from different sensory neurons are shown for each holding potential. The voltage steps are in 20-mV increments. Left of the figures, --- represents the 0-current level for each panel.

The actions of 100 nM PDBu on IK are summarized in Fig. 2. As shown in Fig. 2A, after a 20-min exposure to PDBu, outward currents obtained at +60 mV (Vh,-20 mV) were reduced significantly (Student's paired t-test) from a control value of 0.69 ± 0.1 to 0.48 ± 0.06 nA (n = 7; also see Fig. 4, 100 nM PDBu). This inhibition developed slowly; IK was lowered to 0.61 ± 0.08 and 0.55 ± 0.07 nA after 2- and 10-min applications, respectively (data not shown). In cells held at -20 mV, PDBu significantly reduced IK recorded at potentials positive to -50 mV (Student's paired t-test), and we attribute this effect to the inhibition of membrane potassium conductance described in the following text. In addition, measurements of membrane resistance (between the -60- and -40-mV steps) indicate that after a 20-min exposure to 100 nM PDBu the input resistance was increased significantly to 416 ± 51 MOmega (n = 7, paired t-test) from a control value of 318 ± 39 MOmega . Similarly, when Vh was held at the resting potential (which averaged -47 ± 0.6 mV, n = 11), PDBu significantly suppressed IK in a time-dependent manner (see Fig. 2B) for voltage steps positive to -40 mV. This suppression was observed in all 11 neurons examined and ranged from 16 to 45% inhibition. Also the resistance measured between the -80- and -40-mV steps under control conditions was 350 ± 36 MOmega (n = 11), whereas after a 20-min exposure to PDBu, the resistance increased significantly to 421 ± 47 MOmega (paired t-test). To determine if PDBu altered the voltage dependence of activation of IK, the conductance-voltage relationship was examined and is shown in Fig. 2C. These average results were fit with the Boltzmann relation. When neurons were held at a Vh of -47 mV, only the value of G/Gmax was reduced significantly after PDBu treatment for voltage steps positive to -30 mV (at +60 mV the value was decreased to 0.71 ± 0.03), whereas V0.5 (control -3 mV versus PDBu -6 mV) and k (control and PDBu both 14 mV) were unaffected. However, in recordings from four different neurons, IK was unaffected by PDBu when the neurons were held at -80 mV (see Figs. 2D and 4). The conductance-voltage relationship is shown in Fig. 2E wherein the Boltzmann fitting parameters were not altered significantly by PDBu (G/Gmax at +60 mV was 0.95 ± 0.03); V0.5 (control -4 mV versus PDBu -6 mV) and k (control and PDBu both 14 mV) were nearly identical to that obtained at -47 mV.



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Fig. 2. Summary of the effects of PDBu on IK at the 3 different holding potentials. A: inhibition of IK obtained after a 20-min exposure to 100 nM PDBu when held at a Vh of -20 mV (mean ± SE, n = 7). Treatments with PDBu for 2 and 10 min produced inhibitions of 10 ± 3 and 9 ± 1%, respectively. B: the inhibition for neurons held at the resting potential of -47 mV. The data represent responses obtained from 11 neurons each held at its resting potential (range from -45 to -51 mV with an average value of -47 ± 2 mV, mean ± SD). Consequently, the data were grouped and labeled -47 mV. Treatments with PDBu for 2 and 10 min produced inhibitions of 9 ± 2 and 11 ± 1%, respectively. C: the effects of a 20-min exposure to 100 nM PDBu on the conductance; the points were fit by the Boltzmann relation where G/Gmax = 1/[1 + exp(V0.5 - Vm)/k]. G is the conductance, Gmax is the conductance at +60 mV obtained under control conditions, V0.5 is the voltage for half-maximal activation, Vm is the membrane voltage, and k is a factor that describes the steepness of the conductance-voltage relationship. D: the lack of inhibition of IK by PDBu obtained when the neurons were held at a Vh of -80 mV (n = 4). Treatments for 2 and 10 min produced inhibitions of 1 ± 1 and 2 ± 1%, respectively. E: the effects of PDBu on the conductance; the data points were fitted by the Boltzmann relation. In some cases, the error bars are smaller than the symbol.

The component of total IK that was inhibited by PDBu was determined by subtracting the currents remaining after 20 min in 100 nM PDBu from their respective control traces; these results are shown in Fig. 3. Figure 3A, left, illustrates the PDBu-sensitive IK obtained from a representative neuron held at -47 mV; this current exhibited little time-dependent inactivation. Figure 3A, middle, shows the average current-voltage relation for the PDBu-sensitive IK obtained from 11 neurons. Activation of IK begins at around -20 mV, and the peak current obtained at +60 mV had a mean value of 0.71 ± 0.12 nA. Figure 3A, right, demonstrates the conductance-voltage relation with the Boltzmann fit wherein V0.5 was 4 mV and k was 12 mV. The panels in Fig. 3B represent the PDBu-sensitive IK obtained at a Vh of -20 mV. The current traces from a representative neuron exhibit no inactivation; the average peak current obtained at +60 mV was 0.21 ± 0.05 nA (n = 7). These results suggest that the PDBu-sensitive current exhibited the properties characteristic of a delayed rectifier-like IK.



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Fig. 3. The PDBu-sensitive IK exhibits the properties of a delayed rectifier-like current. A: the representative PDBu-sensitive current obtained when held at a Vh of -47 mV. The PDBu-sensitive current was determined by subtracting the currents remaining after a 20-min exposure to 100 nM PDBu from their respective control traces. Middle: the current-voltage relation for the PDBu-sensitive IK. Right: the activation profile for the conductance values and are fit by the Boltzmann relation. B: the PDBu-sensitive current obtained from a different neuron when held at a Vh of -20 mV. Middle: the current-voltage relation determined for those 7 sensory neurons. Left of the figures: --- labeled with 0 represents the 0-current level for each panel.

The concentration dependence for the inhibition of IK also was examined; these results are summarized in Fig. 4. At a concentration of 1 nM, PDBu had no effect on IK, whereas 10 nM PDBu inhibited IK by 21 ± 2 and 15 ± 3% when neurons were held at -20 and -48 mV, respectively. As in the preceding text, 10 nM PDBu was without effect at -80 mV. These results indicate that the inhibition of IK by PDBu depends on concentration, time, and the holding voltage. In addition, this suppression does not result from a shift in the voltage-dependence of activation.



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Fig. 4. Concentration dependence for the inhibition of IK by PDBu at 3 different holding potentials. The bars represent the percentage of inhibition (mean ± SE) produced by the indicated concentrations of PDBu. The numbers in parentheses represent the number of neurons comprising each bar. The asterisks represent statistically significant differences from the control values (paired t-test) for P < 0.05.

The possibility that the inhibitory actions of PDBu were mediated by the activation of PKC rather than some nonspecific effect of the phorbol was examined by using an inhibitor of PKC as well as an inactive analog of the phorbol. Sensory neurons were pretreated with an inhibitor of PKC, bisindolylmaleimide I (BIM) (Toullec et al. 1991), to ascertain whether the PDBu-induced inhibition of IK was prevented by inactivation of PKC. As shown in Fig. 5, the inhibition of IK produced by 100 nM PDBu at both -20 and -45 mV was blocked by pretreatment with 1 µM BIM. However, IK obtained for voltage steps to +50 and +60 mV for the Vh of -45 mV (see Fig. 5B) was reduced by a small although significant amount after a 20-min exposure to PDBu in the presence of BIM. For example, the step to +60 mV elicited an average IK of 1.84 ± 0.18 nA after a 10-min treatment with 1 µM BIM, whereas after a 20-min exposure to 100 nM PDBu in the presence of BIM, IK was reduced significantly to 1.62 ± 0.20 nA (n = 3). After treatment with PDBu in BIM, the fraction of current remaining at +60 mV was (0.88 ± 0.02 vs. 0.71 ± 0.03, n = 3). After treatment with PDBu in BIM, the fraction of the current remaining at +60 mV (0.88 ± 0.02, n = 3) was significantly larger than that remaining in the absence of BIM (0.71 ± 0.03, n = 11). As described in the preceding text, neither BIM nor PDBu had any effect at a Vh of -80 mV (Fig. 5C). Exposure to the inactive analogue of PDBu, 4alpha -phorbol, failed to alter IK over a 20-min time course at any of the three Vhs (data not shown). For example, at Vhs of -50 and -20 mV, IK was 2.36 ± 0.39 and 0.49 ± 0.12 nA (+60-mV step), respectively, after a 20-min treatment with 100 nM 4alpha -phorbol compared with a values of 2.44 ± 0.47 and 0.59 ± 0.16 nA (n = 4) for the control condition. Therefore these findings suggest that the inhibition of IK produced by PDBu results from the direct activation of PKC rather than by a nonspecific mechanism.



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Fig. 5. The PKC inhibitor, bisindolylmaleimide I (BIM), blocks the PDBu-mediated inhibition of IK. Treatment with 1 µM BIM for 10 min before exposure to 100 nM PDBu prevents the inhibition of IK by PDBu when held at -20 mV and largely attenuates the inhibition when Vh was -45 mV (n = 3). As in the preceding text, PDBu had no effect at -80 mV.

Voltage and calcium dependence of the PDBu-induced inhibition

The inability of PDBu to suppress IK elicited from -80 mV was unexpected and we examined the voltage dependence of PDBu action using the protocol summarized in Fig. 6A. IK was activated by depolarizations to +60 mV. The holding potential was set initially to -50 mV, shifted to -80 mV, and returned to -50 mV. Hyperpolarization increased the amplitude of IK (in this cell from 2.89 to 3.61 nA) due to the removal of inactivation. With Vh set to -50 mV, addition of 100 nM PDBu caused a slowly developing inhibition of IK. After a 20-min exposure to PDBu, shifting Vh to -80 mV caused IK to increase in amplitude until it equaled that elicited by depolarization in the control condition. Thus hyperpolarization reversed the inhibition of IK caused by PDBu at a Vh of -50 mV. This recovery from inhibition was fully reversed when Vh was again set to -50 mV as IK declined to 0.71 ± 0.01 of its control value (Fig. 6A). In three cells studied using this protocol, a 20-min exposure to 100 nM PDBu significantly reduced IK to 0.80 ± 0.02 of control value (paired t-test) when Vh was -50 mV (Fig. 6C). Shifting Vh to -80 mV resulted in the recovery of IK to control values over ~6-8 min, i.e., currents elicited from -80 mV in PDBu were not significantly different from those in control (0.96 ± 0.06 of the control, paired t-test, see Fig. 6D). Thus at a Vh of -50 mV, PDBu caused an inhibition of IK that developed over tens of minutes but could be reversed and reinstated relatively rapidly (10 min). A plausible explanation for this is that the slow development of inhibition reflects the PDBu-induced translocation of PKC to the plasma membrane and assembly of the complexes necessary for activation of the enzyme (Bazzi and Nelsestuen 1987; Kishimoto et al. 1980; Mosior and Epand 1994; Mosior and Newton 1995). Once in place, PKC can be activated and deactivated quickly by changes in calcium activity resulting from the opening and closing of nearby voltage-dependent calcium channels (see following text). In this scheme, the rapid reversal and return of inhibition reflects the time course of phosphorylation/dephosphorylation of the PKC substrate (possibly the IK channels themselves) mediated by PKC and local phosphatases.



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Fig. 6. The suppression of IK by PDBu when the neuron is held at -50 mV can be reversed by hyperpolarizing the holding potential to - 80 mV. A: a representative recording from a sensory neuron wherein the peak value of IK was determined every 20 s for a voltage step from the indicated holding potential (top trace) to +60 mV. The bar on the bottom indicates the nature of the solution superfusing the neuron. The open bar represents the control condition, whereas the hatched bar represents changing the superfusion to N-methyl-glucamine chloride Ringer (NMG-Ringer) containing 100 nM PDBu. The time of exposure to PDBu at -50 mV was 20 min. B: another representative recording from a sensory neuron undergoing an identical protocol except that after obtaining the control recordings (open bar on the bottom), the neuron was exposed to NMG-Ringer containing 300 nM omega -conotoxin GVIA for ~3 min (represented by the solid bar). Peak values of IK obtained at the 2 holding voltages were again measured in the presence of conotoxin. The bathing medium was then changed to NMG-Ringer containing both conotoxin and 100 nM PDBu (represented by the hatched bar). The time of exposure to conotoxin and PDBu at -50 mV was 20 min. C (Vh -50 mV) and D (Vh -80 mV): the fractions of IK (IPDBu/ICont) remaining after a 20-min exposure to PDBu compared with their respective control values for the different treatment conditions. The standard solution indicates 100 nM PDBu in normal NMG-Ringer. For the different drug treatments, ICont was obtained in the presence of the drug. Asterisks indicate a significant decrease compared with the control value.

The mechanism underlying the voltage dependence of the inhibition of IK is unlikely to be a direct interaction between membrane potential and PKC. A plausible mechanism is a depolarization-induced calcium influx at -50 mV that raises intracellular calcium ([Ca2+]i) thereby enhancing the activity of PKC. Although the inclusion of 5 mM EGTA in our standard pipette solution will control bulk [Ca2+]i, the relatively slow ON rate for calcium binding by this buffer will allow significant changes in [Ca2+]i near active calcium channels (Stern 1992). Accordingly, we investigated the idea that calcium influx via voltage-gated calcium channels is necessary for the inhibition of IK by PDBu. We first dialyzed neurons with 5 mM BAPTA (with no added calcium) instead of 5 mM EGTA. Although the affinity for calcium is nearly the same for these two buffers, the association rate constant for BAPTA is 400-1,000 times than that for EGTA (Adler et al. 1991; Stern 1992). The faster ON rate for calcium binding by BAPTA would control [Ca2+]i much closer to the calcium channels (Stern 1992; also see DISCUSSION), suggesting that BAPTA might inhibit activation of PKC that was dependent on calcium influx. BAPTA was allowed to perfuse intracellularly for 5 min before any recordings were attempted. The magnitude of IK recorded in cells dialyzed with BAPTA was not significantly different from that in cells dialyzed with EGTA (data not shown). However, in neurons dialyzed with BAPTA, IK was unaffected by a 20-min exposure to PDBu whether the cells were held at -50 mV (0.99 ± 0.03 of control, n = 4, Fig. 6C) or -80 mV (1.01 ± 0.03 of control, Fig. 6D). Thus inhibition of IK by PDBu was abolished by the addition of a rapid intracellular calcium buffer.

The results obtained with BAPTA imply that an increase in [Ca2+]i is critical in the PKC-induced inhibition of IK. We next tested the hypothesis that calcium influx via voltage-gated calcium channels was necessary for the inhibition of IK by the activation of PKC by using agents that inhibit identified components of calcium influx. We first examined the responses of five neurons pretreated with 200 µM CdCl2, a nonselective inhibitor of voltage-gated Ca2+ channels (Kostyuk and Krishtal 1977; Lansman et al. 1986; Swandulla and Armstrong 1989). Cd2+ did not have a significant effect on the amplitude of IK (data not shown). Furthermore in the presence of Cd2+, IK was unaffected by exposure to 100 nM PDBu for 20 min whether the cells were held at -50 mV (0.96 ± 0.03 of control, Fig. 6C) or -80 mV (1.04 ± 0.05 of control, Fig. 6D). Thus calcium influx via voltage-gated Ca2+ channels was necessary for the inhibition of IK by the activation of PKC.

Evans et al. (1996) showed that 20-30% of the total calcium current in rat sensory neurons was mediated by L-type Ca2+ channels that were inhibited by 1 µM nifedipine, whereas ~70% was mediated by N-type Ca2+ channels that were inhibited by 100 nM omega -conotoxin GVIA (CTx). Accordingly, we examined the effect of PDBu on IK in the presence of nifedipine or CTx using the voltage protocol of Fig. 6A to identify the calcium channels that lead to the activation of PKC. Nifedipine (1 µM) did not effect the peak IK elicited from either -50 or -80 mV (data not shown). In the presence of nifedipine at a Vh of -50 mV, a 20-min exposure 100 nM PDBu significantly reduced IK (0.87 ± 0.02 of control, n = 3, Fig. 6C). The inhibition by PDBu in the presence of nifedipine was not significantly different from the inhibition observed in our standard conditions. Last, at a Vh of -80 mV, PDBu failed to reduce IK in the presence of nifedipine and was similar to results obtained in our standard conditions (Fig. 6D). Thus calcium influx via L-type Ca2+ channels was not necessary for the inhibition of IK by the activation of PKC.

In contrast, treatment with 300 nM CTx prevented the inhibition of IK by PDBu. Results from a representative sensory neuron are shown in Fig. 6B. With Vh set to -50 mV, the peak IK was unaffected by a ~3-min exposure to CTx. In the presence of CTx, a 20-min exposure to 100 nM PDBu had no effect on the amplitude of IK. Similarly, CTx or PDBu did not affect IK when Vh was -80 mV. In five neurons studied in the presence of CTx, IK was unaffected by exposure to 100 nM PDBu for 20 min whether the cells were held at -50 mV (0.97 ± 0.01 of control, Fig. 6C) or -80 mV (1.05 ± 0.03 of control, Fig. 6D). These data, in combination with the results obtained with BAPTA, indicate that calcium influx via N-type Ca2+ channels was necessary for the inhibition of IK by the activation of PKC.

Pharmacological characterization of the PDBu-suppressed IK

The inhibitors, TEA and 4-AP, were used to examine the nature of the potassium currents that were modulated by PDBu (Thompson 1977). The different holding voltages were used in combination with the pharmacological agents to isolate delayed rectifier-like currents from rapidly inactivating currents.

Component of IK inhibited by activation of PKC is blocked by TEA

The inhibition of IK by PDBu was limited to ~30% of the total current, suggesting that specific components of IK may be subject to regulation by PKC. To investigate the nature of these components, we examined the effects of PDBu in the presence of specific K+ channel blockers. Figure 7 shows IK elicited from a representative neuron at Vhs of -20, -47, and -80 mV under control conditions (left) and in the presence of 10 mM TEA, an inhibitor of delayed rectifier K+ channels (middle). At each of the holding potentials, TEA significantly reduced IK at test potentials positive to -20 mV (RM ANOVA). The extent of inhibition at +60 mV was 62 ± 4% when Vh was -20 mV, 70 ± 4% at a Vh of -47 mV, and 50 ± 6% at a Vh of -80 mV (n = 6). Figure 7, right, shows the time course of this TEA-sensitive current obtained by subtracting the TEA traces from their respective control traces. The TEA-sensitive currents activated rapidly and showed little inactivation during the depolarizing steps, properties typical of delayed rectifier-like IK.



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Fig. 7. Representative effects of TEA on IK obtained at the different Vhs in the same sensory neuron. Left: IK obtained under control conditions at the 3 different Vhs. Middle: the inhibitory effects of 10 mM TEA after an approximate 4-min treatment at each Vh. Right: the TEA-sensitive IK. The currents remaining after exposure to TEA were subtracted from their respective control traces to yield the TEA-sensitive currents. Left of the figures: --- labeled with 0 represents the 0-current level for each panel.

We next examined the sensitivity of the components of IK remaining in the presence of 10 mM TEA to activation of PKC by a 20-min exposure to 100 nM PDBu. Results obtained from six neurons are summarized in Fig. 8 and Table 1. At a Vh of -20 mV, addition of 10 mM TEA inhibited IK at test potentials positive to -20 mV (see Fig. 8A, left). In the presence of TEA, addition of PDBu did not cause a significant further reduction in IK. However, to reduce the variability in the current measurements with these different pharmacological treatments, the values of IK were normalized to the peak current obtained at +60 mV under control conditions (see Fig. 8A, right). In the presence of TEA, PDBu caused a small but significant reduction in the normalized values of IK only for voltages positive to +40 mV (RM ANOVA). When held at either -47 (see Fig. 8B) or -80 mV (see Fig. 8C) in the presence of TEA, PDBu produced no further inhibition of IK (see Table 1). Thus at each of the Vhs tested, addition of PDBu did not significantly reduce IK in the presence of 10 mM TEA (n = 6, RM ANOVA). This implies that TEA and activation of PKC inhibited current flow through the same population of K+ channels, a conclusion that is supported by the similar time- and voltage dependencies of the PDBu-sensitive currents (Fig. 3) and the TEA-sensitive current (Fig. 8).



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Fig. 8. The PDBu-induced inhibition of IK by is attenuated by prior exposure to TEA. Left: the inhibition of IK by 10 mM TEA and the subsequent effects of 100 nM PDBu (20-min exposure) in the presence of TEA at 3 different Vhs. When held at a Vh of -20 mV (A), IK was decreased significantly by TEA for voltages positive to -50 mV [repeated measure (RM) ANOVA]. When held at -47 mV (B), IK was reduced significantly by TEA for those voltages positive to -40 mV. When held at -80 mV (C), IK was suppressed significantly by TEA for voltages positive to -30 mV. Right: the effects of TEA and a 20-min treatment with 100 nM PDBu in the presence of TEA after the currents were normalized to the peak IK obtained at +60 mV under control conditions. In some cases, the error bars are smaller than the symbol.


                              
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Table 1. Summary of IK measured at +60 mV under control conditions (IK) for different holding potentials (Vh) and subsequent to the cumulative addition of K+ channel blockers and/or 100 nM PDBu

Component of IK inhibited by activation of PKC is not blocked by 3 mM 4-AP

A number of voltage-gated K+ channels are inhibited by 4-AP with individual channels showing characteristic sensitivities to this compound (see reviews by Chandy and Gutman 1995; Mathie et al. 1998). Accordingly, we investigated the relationship between the PDBu-sensitive component of IK and currents inhibited by relatively high (3 mM) and low (30 µM) concentrations of 4-AP. The effects of 3 mM 4-AP for the three different Vhs, are illustrated for a representative neuron in Fig. 9. IK was elicited from holding potentials of -20, -47, and -80 mV under control conditions (left) and in the presence of 3 mM 4-AP (middle). 4-AP had little effect on IK elicited from a Vh of -20 mV but significantly inhibited IK elicited from more negative holding potentials (see following text). The 4-AP-sensitive difference currents obtained at each holding potential are shown in Fig. 9, right. Interestingly, the time course of these currents did not show a marked inactivation associated with IA-like K+ currents (Connor and Stevens 1971; Neher 1971), indicating that 3 mM 4-AP inhibits more slowly inactivating components of IK in these sensory neurons.



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Fig. 9. Representative effects of 4-aminopyridine (4-AP) on IK for the different Vhs. Left: IK recorded under control conditions at each indicated Vh. Middle: the currents remaining after an approximate 4-min treatment with 3 mM 4-AP. Right: the 4-AP-sensitive IK. All recordings were obtained from the same neuron. Left of the figures: --- labeled with 0 represents the 0-current level for each panel.

We next examined the sensitivity of the components of IK remaining in the presence of 3 mM 4-AP to activation of PKC by a 20-min exposure to 100 nM PDBu. Results obtained from 10 neurons are summarized in Fig. 10 and Table 1. When held at -20 mV, 4-AP produced a small although significant reduction in IK elicited for only those voltage steps between -40 and 0 mV (Fig. 10A, left, RM ANOVA). For more depolarized steps, 4-AP had no significant effect; for example, at +60 mV the current remaining was 95 ± 6% (n = 10) of the control. In contrast, 4-AP significantly decreased IK by 44 ± 4% when neurons were held at -46 mV (n = 10, Fig. 10B, significant for voltages positive to -40 mV, RM ANOVA) and by 48 ± 2% at -80 mV (n = 9, Fig. 10C, significant for voltages positive to -40 mV, RM ANOVA).



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Fig. 10. Summary of the suppression of IK by 4-AP and the additional inhibition produced by PDBu. A, left: the effects of 3 mM 4-AP and 100 nM PDBu in the presence of 4-AP on IK when held at a Vh of -20 mV. Right: the effects of both 4-AP and a 20-min treatment with 100 nM PDBu in the presence of 4-AP on IK after normalization to the control current obtained at +60 mV. B: the effects of 4-AP and PDBu in the presence of 4-AP when held at a Vh of -46 mV (resting potential). Right: the effects of these agents on the normalized current. C: the effects of 4-AP and PDBu in the presence of 4-AP when held at -80 mV. Although PDBu did not produce a significant effect for the absolute values, PDBu did significantly reduce the normalized currents (right). In some cases, the error bars are smaller than the symbol.

The addition of 100 nM PDBu in the presence of 4-AP, at a Vh of -20 mV, reduced IK by an amount comparable to that observed in the absence of 4-AP (see Table 1). For both the absolute (Fig. 10, left) and normalized currents (right), PDBu significantly inhibited IK in the presence of 4-AP for voltages positive to -30 mV (RM ANOVA). At Vhs of -46 and -80 mV, PDBu, in the presence of 4-AP, caused a modest but significant reduction in the normalized values of IK (see Fig. 10, B and C, right, Table 1). For these Vhs, PDBu significantly inhibited IK for voltages positive to +20 mV (RM ANOVA). Thus in contrast to TEA, we find little overlap between the PDBu-sensitive component of IK and the component inhibited by 3 mM 4-AP.

4-AP (30 µM) permits inhibition of IK by PDBu at -80 mV

We examined if the capacity of PDBu to inhibit IK was modified by a lower concentration of 4-AP. The results obtained with 100 nM PDBu in the absence and presence of 30 µM 4-AP are summarized in Fig. 11 and Table 1. At a Vh of -20 mV, 30 µM 4-AP significantly reduced IK for voltages positive to -20 mV (RM ANOVA). At +60 mV this amounted to a suppression of only 9 ± 2% (n = 10, see Fig. 11A). At -47 mV, 4-AP significantly decreased IK for all voltages positive to -40 mV and this corresponded to a reduction of 14 ± 1% at +60 mV (n = 10, see Fig. 11B). At a Vh of -80 mV, 30 µM 4-AP significantly suppressed IK (between -40 and +60 mV, Fig. 11C); at +60 mV, IK was inhibited by 16 ± 3% (n = 10).



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Fig. 11. Summary of the suppression of IK by 30 µM 4-AP and the additional effect of PDBu. A, left: the inhibition of IK by 4-AP when held at a Vh of -20 mV; the PDBu-induced inhibition was significant for voltages positive to -30 mV (RM ANOVA). Right: the effects of 4-AP and a 20-min treatment with 100 nM PDBu in the presence of 4-AP after the currents were normalized to the peak IK obtained at +60 mV under control conditions. B: the effects of 4-AP and PDBu in the presence of 4-AP when held at -47 mV; PDBu significantly reduced IK for voltages positive to -50 mV (RM ANOVA). Right: the normalized currents. C: the effects of 4-AP and PDBu in the presence of 4-AP when held at a Vh of -80 mV; PDBu produced a significant inhibition of IK between -40 and +60 mV (RM ANOVA). Right: normalized currents. In some cases, the error bars are smaller than the symbol.

Exposure to 100 nM PDBu in the presence of 30 µM 4-AP further suppressed IK by a similar amount at all Vhs. At a Vh of -20 mV, the PDBu-induced inhibition of IK was 17 ± 3% (see Fig. 11A and Table 1) and was similar to that observed with 3 mM 4-AP. The normalized currents are shown in Fig. 11A (right) wherein PDBu significantly suppressed IK for values positive to -40 mV (RM ANOVA). The results obtained with PDBu at -47 mV were quite similar to those obtained at -20 mV (see Fig. 11B and Table 1), although at this Vh, the percent inhibition by PDBu was greater that that observed with 3 mM 4-AP. The normalized currents are shown in Fig. 11B (right) where current values positive to -20 mV were significantly different from those in 4-AP (RM ANOVA). In contrast to the control and TEA recordings described in the preceding text, PDBu produced further inhibition of IK at a Vh of -80 mV in the presence of 30 µM 4-AP (Fig. 11C and Table 1). For the normalized current, only values positive to +20 mV were significantly different from in 4-AP alone (RM ANOVA). Thus these results demonstrate that the IK, which is suppressed by PDBu, is not sensitive to either concentration of 4-AP.

Taken together, these findings indicate that PDBu modulates an IK that is sensitive to TEA but not 4-AP. Also our observations suggest that high and low concentrations of 4-AP somehow "unmask" or enable PDBu to inhibit IK at a Vh of -80 mV, whereas PDBu does not exhibit this capacity under control conditions or in the presence of TEA.


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We have demonstrated that the phorbol ester, PDBu, suppressed IK in nociceptive sensory neurons in a manner that depended on the holding voltage. These actions of PDBu were mediated by PKC because the suppression of IK was prevented by inhibition of PKC, whereas 4-alpha phorbol had no effect. PDBu did not reduce IK in the presence of TEA, suggesting that these treatments inhibit the same component of the current. We also found that 4-AP inhibited a current component that was distinct from that inhibited by TEA and largely unaffected by PDBu.

Our observations that the activation of PKC resulted in the suppression of IK suggest that modulation of this current by intracellular signaling pathways is an important mechanism in the regulation of sensory neuron sensitivity to different modalities of peripheral stimulation. Our findings are consistent with previous observations wherein exposure to PDBu depolarized unmyelinated rat vagus nerve (Rang and Ritchie 1988) and caused both a direct excitation as well as sensitization of different classes of articular afferent fibers in the knee joint of the cat (Schepelmann et al. 1993). Similarly, PDBu (50 nM) caused a direct release of substance P and calcitonin gene-related peptide from rat sensory neurons grown in culture, whereas lower concentrations (10 nM) sensitized the potassium- or capsaicin-evoked release of these peptides (Barber and Vasko 1996). These results indicate that the PKC signaling cascade and its regulation is important in mediating the enhancement of the sensitivity of sensory neurons to stimulation.

The suppression of IK by PDBu appeared to be mediated by the activation of a calcium-dependent isoform of PKC rather than novel or atypical isoforms (Mellor and Parker 1998) because the inhibition of IK by PDBu was prevented by intracellular BAPTA or pretreatment with omega -conotoxin, a specific blocker of N-type voltage-dependent calcium channels. These results suggest that, in the presence of PDBu, there may be sufficient calcium influx at the more depolarized holding potentials (-47 and -20 mV) to activate PKC, whereas at -80 mV, the lower influx of calcium does not permit activation of PKC. Indeed this contention is supported by electrophysiological measurements of calcium currents in sensory neurons of mouse and rat dorsal root ganglia. Current-voltage curves indicate that in these neurons the calcium current begins to activate at voltages positive to -60/-50 mV (Evans et al. 1996; Gross and Macdonald 1989; Regan et al. 1991; Scroggs and Fox 1992). The calcium dependency for the activation of PKC is consistent with the recent results of Kondratyuk and Rossie (1997) that the phosphorylation of synaptosomal sodium channels by PKC was increased by depolarization and that this phosphorylation depended on the presence of extracellular calcium. Furthermore Gross and Macdonald (1989) found that PDBu inhibited the calcium current (N and L types) in mouse sensory neurons at a holding voltage near the resting membrane potential, whereas phorbol ester was not effective at -80 mV.

Our observations provide information about the spatial organization of the calcium channels and PKC. We found that PDBu caused a PKC-dependent inhibition of IK in cells dialyzed with 5 mM EGTA but not with 5 mM BAPTA. These observations can be understood in terms of the different capacities of EGTA and BAPTA to control the free calcium concentration near calcium channels. Using the length constant formulation, diffusion coefficients, and association rate constants provided by Stern (1992), one calculates that in 5 mM EGTA, the concentration of free calcium falls with distance from a channel with a length constant of ~370 nm. In 5 mM BAPTA, the length constant is only 11 nm. Thus in 5 mM BAPTA, a molecule of PKC >44 nm (4 length constants) from an open plasma membrane channel will not be subject to an increase in calcium concentration above bulk free calcium. In 5 mM EGTA, the corresponding distance is 1.5 µm. Accordingly our observations imply that, on average, the PKC molecules regulating IK are farther than ~40 nm and nearer than ~1 µm from the source of activating calcium.

Exposure to phorbol esters can lead to an increased excitability as reflected by the enhanced number of action potentials elicited by a depolarizing current pulse in pyramidal neurons of the hippocampal slice (Baraban et al. 1985). Consistent with this idea, we demonstrated that activation of PKC suppressed the peak IK in mammalian sensory neurons; this enhanced excitability may contribute to the sensitization of neuropeptide release. Our findings are supported by the results of others wherein PDBu and other phorbol esters inhibit IK in a variety of neuronal systems. In cultured hippocampal pyramidal neurons, the phorbol ester, phorbol 12,13 diacetate, suppressed a persistent IK by ~30% (Doerner et al. 1988). Similarly, phorbol esters inhibited the IsK heterogously expressed in Xenopus oocytes (Busch et al. 1992) and the slow IK activated by acetylcholine in chick cochlear ganglia (Yamaguchi and Ohmori 1993). In observations similar to ours, Shipston and Armstrong (1996) reported that PDBu suppressed the BK-type IK in GH4C1 cells and that pretreatment with TEA prevented the actions of PDBu. Phorbol esters also inhibited the currents conducted by the cloned and expressed potassium channels Kv1.2 and Kv1.5 (Vogalis et al. 1995) as well as Kv3.1 (Kanemasa et al. 1995).

The identity of the potassium channel(s) modulated by PKC remains to be determined. Our findings indicate that the inhibitory effect of PDBu is prevented by pretreatment with the potassium channel blocker TEA. Under control conditions, IK also was suppressed by 30 µM and 3 mM 4-AP; however, the 4-AP-sensitive current differed from that inhibited by TEA in two respects. First, the inhibition by 4-AP was greater at more negative holding potentials, suggesting that the 4-AP-sensitive current exhibited a voltage-dependent inactivation that was not observed for the TEA-sensitive current. Second, in the presence of 4-AP, PDBu further reduced IK, whereas in the presence of TEA, PDBu was without effect. Based on this sensitivity to TEA, one could speculate on the nature of the gene product modulated by phorbol ester; Kv1.1, 1.6, 2.1, and 3 fit the TEA sensitivity. However, it is unlikely to be Kv3 based on the rapid inactivation exhibited by this gene product. Additional studies, which are currently ongoing, are necessary to further elucidate the specific types of potassium channel(s) that are modulated by phorbol esters as well as inflammatory prostaglandins. Interestingly, in the presence of either concentration of 4-AP, PDBu partially inhibited the remaining IK elicited from a Vh of -80 mV. The mechanism behind this action is unclear. Although it is well established that the block produced by 4-AP can be relieved with depolarized voltage steps (Castle et al. 1994; Hermann and Gorman 1981; Stephens et al. 1994; Yeh et al. 1976), this cannot account for the ability of PDBu to now inhibit IK at this hyperpolarized Vh. It is possible that an IK, which is PDBu insensitive but 4-AP sensitive, dominates the total current that is active at a Vh of -80 mV and that this current must be removed before the effects of PDBu can be observed at this particular Vh. Thus inhibition of this current by 4-AP "unmasks" the PDBu-sensitive IK. Also, this PDBu-insensitive, 4-AP-sensitive current might be inactivated at the more depolarized holding potentials because the extent of inhibition produced by PDBu at -20 and -47 mV in the presence of the low concentration of 4-AP is similar to that observed in the absence of 4-AP. However, 4-AP, at each concentration, inhibited the total IK by similar amounts at the -47 and -80 mV holding potentials. Alternatively in the absence of 4-AP, PDBu had no effect on IK when Vh was -80 mV because there may have been insufficient calcium influx to support activation of PKC. Thus a possibility is that 4-AP increased the calcium influx and led to the activation of PKC. The cellular mechanisms giving rise to the facilitation and the interrelationship between 4-AP and PDBu clearly are complex and will be areas for future investigation.

In summary, our findings demonstrate that the phorbol ester-induced activation of PKC suppresses a potassium current in rat sensory neurons. The reduction in this current may contribute to the enhanced excitability and the augmented release of neuropeptides observed after activation of PKC. Therefore the PKC signaling pathway plays an important role in modulating the excitability of sensory neurons and may contribute to the enhanced neuronal sensitivity during inflammation.


    ACKNOWLEDGMENTS

This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-30527 to G. D. Nicol.


    FOOTNOTES

Address for reprint requests: G. D. Nicol, Dept. of Pharmacology and Toxicology, 635 Barnhill Dr., Indiana University School of Medicine, Indianapolis, IN 46202-5120 (E-mail: gnicol{at}iupui.edu).

Received 19 May 2000; accepted in final form 15 September 2000.


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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society