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The Journal of Neurophysiology Vol. 78 No. 6 December 1997, pp. 3154-3164
Copyright ©1997 by the American Physiological Society
1 Medical Neurobiology Program and 2 Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202
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ABSTRACT |
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 Abstract
 Introduction
Methods
Results
Discussion
References
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Lopshire, J. C. and G. D. Nicol. Activation and recovery of the PGE2-mediated sensitization of the capsaicin response in rat sensory neurons. J. Neurophysiol. 78: 3154-3164, 1997. Pro-inflammatory prostaglandins are known to enhance the sensitivity of sensory neurons to various modalities of stimulation, including the excitatory chemical agent, capsaicin. In this report, we examined the capacity of prostaglandin E2 (PGE2) to enhance the capsaicin response recorded from sensory neurons isolated from embryonic rats and grown in culture. Previous work demonstrated that the cyclic adenosine 3
,5-monophosphate pathway mediates initiation of the PGE2-induced sensitization, however, little is known about the pathways regulating the recovery from sensitization. Therefore, we examined the neuronal transduction cascades that control the duration of sensitization. Treatment with PGE2 enhanced the capsaicin-evoked current by two- to threefold, however, this sensitization was transient even in the continued presence of prostaglandin. The duration of sensitization produced by PGE2 was related inversely to the extracellular Ca2+ concentration with the shortest recovery times observed in cells exposed to 2 mM Ca2+-Ringer. Inclusion of the Ca2+ chelator, bis-(o-aminophenoxy)-N,N,N,N-tetraacetic acid, in the recording pipette greatly lengthened the period of sensitization. Pretreatment with either the nitric oxide synthase inhibitor, nitro-L-arginine methyl ester (L-NAME), or the inhibitor of the cyclic guanosine 3,5-monophosphate (GMP)-dependent protein kinase, KT-5823, before the application of PGE2 increased the duration of sensitization even in the presence of 2 mM Ca2+. In contrast, after attaining maximal sensitization in 2 mM Ca2+-Ringer containing L-NAME, the addition of either nitric oxide donors (3-morpholinosydnonimine or s-nitroso-n-acetylpenicillamine) or 8-Br-cyclic GMP led to a rapid decrease in the level of sensitization. In the absence of sensitization, nitric oxide-cyclic GMP modulating agents had no effect on the capsaicin-evoked current. Therefore, these results suggest that capsaicin-induced elevations in intracellular Ca2+ levels lead to an enhanced production of cyclic GMP, via the nitric oxide pathway, that ultimately activates cyclic GMP-dependent protein kinase. This protein kinase inactivates or terminates the sensitization produced by PGE2 by an as yet unidentified mechanism.
The sensitivity of mammalian sensory neurons to excitatory chemical agents can be enhanced greatly by exposure to pro-inflammatory prostaglandins (Foreman 1987 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 Electrophysiology
The procedures for whole cell patch-clamp recording from rat sensory neurons have been described in detail previously (Nicol and Cui 1994
Chemicals
Prostaglandins were obtained from Cayman Chemical (Ann Arbor, MI). Nitro-L-arginine methyl ester (L-NAME), s-nitroso-n-acetylpenicillamine (SNAP), 3-morpholinosydnonimine (SIN-1), and KT-5823 were obtained from Calbiochem (La Jolla, CA). All other chemicals were obtained from Sigma Chemical (St. Louis, MO). Prostaglandins, capsaicin, and KT-5823 were dissolved in 1-methyl-2-pyrrolidinone (HPLC grade, Aldrich Chemical, Milwaukee, WI) to obtain concentrated stock solutions. Amphotericin B was dissolved in dimethyl sulfoxide (Aldrich). These stock solutions then were diluted with the appropriate solution to yield the desired concentration. The extracellular concentrations of free Ca2+ were calculated from the binding constants described by Caldwell (1970) Analysis
All values are reported as the means ± SE. The concentration-response relation was described by a ligand-binding isotherm of the following form: CAPresp = 1.1/1 + ([CAP]0.5/[CAP])n, where CAPresp is the fractional response to capsaicin, [CAP]0.5 is the half-maximal effective concentration (EC50) and n is a steepness factor that is analogous to the Hill coefficient in enzyme kinetics (e.g., Davis et al. 1977 Characterization of the capsaicin response
In embryonic rat sensory neurons, the voltage response to capsaicin was characterized by a depolarization that was accompanied by the generation of action potentials. As illustrated for the representative neuron in Fig. 1A (top), the focal application of 100 nM capsaicin elicited a barrage of action potentials and a depolarization of 34 mV. In voltage clamp (
PGE2 sensitization of the capsaicin response
To examine whether prostaglandins modulate the capsaicin response, a concentration of 100 nM capsaicin was used as this value was slightly less than the estimated EC50 value. As illustrated in Fig. 3, treatment with PGE2 increased the amplitude of the inward current evoked by the focal application of capsaicin in a time-dependent manner. Figure 3A demonstrates the current traces obtained from a representative neuron before and after exposure to 1 µM PGE2. Under control conditions, 100 nM capsaicin evoked an inward current of 190 pA. After a 10-min treatment with PGE2 (middle), the current amplitude was increased to 532 pA, a 2.8-fold enhancement. The sensitization was not sustained and began to recover even in the continuous presence of PGE2, where after a 20-min exposure to prostaglandin, capsaicin elicited a current of only 205 pA (bottom). This transient sensitization of the capsaicin response was observed for both 100 nM and 1 µM PGE2, and these results are summarized in Fig. 3B. The amplitudes of the capsaicin-evoked currents after experimental treatments were normalized to the average value of their respective control responses. After exposure to 100 nM PGE2, the capsaicin-evoked current was significantly enhanced by 1.4 ± 0.1-fold (range 1.3-1.7, n = 6) compared with the control. Similarly, treatment with 1 µM PGE2 significantly increased the current by 2.1 ± 0.3-fold (range 1.3-4.2, n = 6 to 9). The times to peak sensitization were not affected by the concentration of PGE2; the average values for 100 nM and 1 µM PGE2 were 8.6 ± 1.2 min (range 6-14 min) and 8.9 ± 0.6 min (range 6-10 min), respectively. After reaching maximal values, the sensitization produced by PGE2 began to recover back to control values. As determined from the time courses of individual neurons, the times for sensitization to return to half-maximal values (t1/2 MAX) for 100 nM (5.5 ± 0.8 min, range 2.4-7.5 min) and 1 µM PGE2 (3.7 ± 0.5 min, range 1.2-5.5 min) were not significantly different. To determine whether exposure to capsaicin alone contributed to the sensitization or desensitization of these neurons, 100 nM capsaicin was applied focally every 2 min for a period of 30 min. As shown in Fig. 3C, the repeated exposure did not sensitize these sensory neurons as was observed after treatment with PGE2 (note the smaller scale for the fold-increase). Furthermore, the brief applications of 100 nM capsaicin over time did not appear to desensitize the neurons because the fold-increase relative to the zero time point did not become less than one. Therefore, these results indicate that exposure to PGE2 transiently sensitizes sensory neurons to the excitation produced by capsaicin.
Calcium dependence of the PGE2-induced sensitization
The results described above imply that activation of fewer capsaicin receptors might lengthen the period of time that these sensory neurons remain sensitized. Previous studies have indicated that the capsaicin receptor-ion channel complex is permeable primarily to Na+ and Ca2+ (Bevan and Szolcsanyi 1990
Activation of the nitric oxide-cyclic GMP pathway mediates the inactivation of sensitization
Many investigations have established that an influx of Ca2+ leads to activation of the nitric oxide pathway with the subsequent generation of cyclic GMP (Bredt and Synder 1989
Inhibition of cyclic GMP-dependent protein kinase suppresses the inactivation of sensitization
As discussed above, elevated levels of cyclic GMP appear to inactivate the sensitization produced by PGE2. To determine whether this effect resulted from cyclic GMP directly or via activation of PKG, the compound KT-5823 was used to block activation of PKG (Ito and Karachot 1990
In embryonic sensory neurons, capsaicin evokes an inward current that has properties similar to those observed in recordings from sensory neurons obtained from adult animals. Examples of this are a reversal potential near 0 mV, desensitization of the response after exposure to high concentrations of capsaicin, and suppression of the response by pretreatment with the capsaicin antagonist, capsazepine (Lopshire, unpublished observations). The concentration dependence for the amplitude of the capsaicin-evoked current has an EC50 value (163 nM) and is similar to values observed for other capsaicin-mediated events. The lower values range from 70 nM for the capsaicin-induced increase in calcium (Cholewinski et al. 1993
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
; Higgs et al. 1984; Salmon and Higgs 1987). This sensitization is observed in behavioral studies as a facilitated perception of pain (Handwerker and Kobal 1993; Handwerker and Reeh 1991) as well as in electrophysiological recordings obtained from isolated sensory neurons. For example, in cultured sensory neurons, treatment with prostaglandin E2 (PGE2) enhances the number of action potentials generated by exposure to either elevated levels of potassium (Baccaglini and Hogan 1983) or the inflammatory mediator, bradykinin (Nicol and Cui 1994). The sensitization produced by PGE2 is mediated by the cyclic adenosine 3,5-monophosphate (cAMP) transduction cascade because PGE2 increases the intracellular levels of cAMP, exogenous cAMP mimics the effects of PGE2, and inhibition of the cAMP-dependent protein kinase (PKA) blocks sensitization by PGE2 (Cui and Nicol 1995; Hingtgen et al. 1995). In contrast, very little is known about the transduction pathways that lead to the termination of PGE2-initiated sensitization.
and C fibers are believed to be involved in nociceptive signaling (Holzer 1991). Capsaicin generates an inward current that depolarizes the sensory neuron (Bevan and Forbes 1988; Bevan and Szolcsanyi 1990; Heyman and Rang 1985; Marsh et al. 1987). This inward current results from the opening of a nonselective cationic channel that is largely permeable to Na+ and Ca2+ (Bevan and Szolcsanyi 1990; Marsh et al. 1987; Oh et al. 1996; Wood et al. 1988). The capsaicin-mediated influx of Ca2+ appears to play a critical role in activating cellular pathways that give rise to the rapid and significant desensitization of the capsaicin response (Cholewinski et al. 1993; Liu and Simon 1996; Yeats et al. 1991). Indeed, recent observations suggest that activation of the Ca2+-dependent phosphatase, calcineurin, somehow modifies the activity of the capsaicin-gated channel to produce the desensitization (Docherty et al. 1996).
,5-monophosphate (GMP) (Dymshitz and Vasko 1994; Wood et al. 1989) that is dependent on the presence of extracellular Ca2+, however, cAMP levels remain unaffected (Wood et al. 1989). This raises an interesting question as to whether cyclic GMP may play a physiological role in modulating the neuronal sensitization produced by pro-inflammatory prostaglandins. In support of this idea, McGehee et al. (1992) demonstrated that elevated levels of cyclic GMP, as well as receptor occupancy, were required for desensitization of the bradykinin response in rat sensory neurons. Therefore, in this study, we examine the capacity of the pro-inflammatory prostaglandin, PGE2, to sensitize the response to capsaicin recorded from rat sensory neurons grown in culture. Furthermore, we investigate the roles of extracellular Ca2+, the nitric oxide-cyclic GMP pathway, and the cyclic GMP-dependent protein kinase (PKG) in regulating inactivation of the PGE2-induced sensitization. Our findings suggest that activation of PKG via the nitric oxide-cyclic GMP pathway controls the duration of PGE2-induced sensitization.
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). Briefly, pregnant rats (days 15-17 of gestation) were rendered unconscious by placing them in a chamber filled with CO2; animals then were killed by cervical dislocation. The dorsal root ganglia (DRG) from the embryos were dissected free and placed in a dish containing sterile calcium-free, magnesium-free Hank's balanced saline (HBSS) at 4°C. The DRGs then were incubated in HBSS containing 0.025% trypsin for 25 min at 37°C. The digestion was terminated with the addition of 0.25% trypsin inhibitor; ganglia were washed once with HBSS, centrifuged, and then resuspended in growth medium that consisted of 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). Individual cells were obtained by mechanical agitation with a fire-polished pipette until a cloudy suspension was observed. Approximately 300,000 viable cells were plated in a collagen-coated culture dish (35 mm) containing small plastic cover slips. Cells were grown at 37°C in a 95% O2-5% CO2 atmosphere and the media changed every 2 days. All procedures were approved by the Animal Care and Use Committee at Indiana University School of Medicine.
). Briefly, a cover slip with the sensory neurons (typically after 4 days in culture) was placed in a recording chamber where the neurons were superfused with normal Ringer solution of the following composition (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), and 10 glucose, pH at 7.4 with NaOH. Using the whole cell patch-clamp technique (Hamill et al. 1981), membrane voltages or currents were recorded with a List EPC-7 patch-clamp amplifier (List Electronic, Darmstadt, Germany). Recording pipettes were pulled from borosilicate disposable pipettes and typically had resistances of 2-4 M
when filled with the following solution (in mM): 140 KCl, 5 MgCl2, 4 Na2ATP, 0.3 Na3GTP, 2.5 CaCl2, 5 ethylene glycol-bis(
-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA; calculated free Ca2+ concentration of 100 nM), and 10 HEPES, pH at 7.2 with KOH. In those experiments using 10 mM bis-(o-aminophenoxy)-N,N,N,N-tetraacetic acid (BAPTA; tetrapotassium salt), the concentration of KCl was lowered to 100 mM.
(average value 3.4 ± 0.21 M). In voltage-clamp recordings, the membrane was held at 
60 mV, which is close to the value of the normal resting potential of about 55 mV (see Cui and Nicol 1995; Nicol and Cui 1994). The currents evoked by capsaicin were recorded on videotape at 3 kHz. Individual responses were played through an 8-pole Bessel filter at 100 Hz and then into the computer using the program Axotape (Axon Instruments, Foster City, CA) where they were sampled at 300 Hz. The traces shown in the figures have been filtered digitally at 40 Hz using the Fetchan program of pClamp 6.0.3 (Axon Instruments).
. Briefly, the pipette tip was filled with a solution of the following composition (in mM): 90 K-aspartate, 50 KCl, 1 MgCl2, 10 HEPES, pH adjusted to 7.2 with KOH. The electrode then was backfilled with the same solution containing amphotericin B (240 µg/ml). These electrodes had resistances between 2 and 4 M in normal Ringer solution. After attaining the cell-attached configuration, membrane partitioning of amphotericin B was assessed by applying +10 mV voltage pulses (from 60 mV) at 2-min intervals until a steady state access resistance was obtained (typically 20 min). The mean access resistance was 11.2 ± 0.6 M (n = 12). The series resistance then was compensated (mean value was 55 ± 5%) to yield a final uncompensated series resistance of 4.9 ± 0.6 M. All other conditions were the same as described above.
). In those studies investigating sensitization of the capsaicin response by prostaglandins, two control responses separated by ~2 min were obtained to the focal application of capsaicin. After the application of the sensitizing agent, capsaicin responses were obtained at 2, 6, 10, 14, and 20 min and thereafter at 5-min intervals. Responses from only a single neuron were obtained from each cover slip to avoid any possible desensitization that might occur. All experiments were done at room temperature (~21°C).
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FIG. 2.
Amplitude of the capsaicin-elicited current is concentration dependent. Amplitude of the response to focally applied capsaicin is expressed as the fraction of the maximal current evoked by the bath application of 1 µM capsaicin. For the control, the focal pipette contained normal Ringer with 1 mM trypan blue and 0.1% vehicle. Data were fit by the ligand-binding isotherm as described in METHODS. Numbers in parentheses represent the neurons comprising each mean ± SE value.
45 mV for 
4-5 min after establishing the whole cell configuration. Second, in all those experiments examining prostaglandin sensitization, the amplitudes of the two control responses obtained to the focal application of capsaicin must be within ±10% of their mean value or the recording was terminated.
.
; McGehee et al. 1992). The time course of sensitization was characterized by two parameters, the time to peak and the time for sensitization to return to half-maximal values(t1/2 MAX); these values were determined from the time courses of sensitization obtained from individual neurons. We define the time to peak sensitization as the time elapsed from the onset of PGE2 treatment to the maximally sensitized response and t1/2 MAX as the time elapsed from the peak of sensitization to the point where sensitization recovered to one-half the maximal response minus the control value. These values then were averaged to obtain the mean ± SE. Statistical differences between the control recordings and those obtained under various treatment conditions were determined by using either a two-tailed Student's t-test or a one-way analysis of variance (ANOVA). When a significant difference was obtained using ANOVA, a Student-Newman-Keuls (an all-points comparison) or Dunnett's (control vs. treatment) post hoc test was performed. Values of P < 0.05 were judged to be statistically significant.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
60 mV holding potential), the application of capsaicin to this same neuron caused an inward current that had a maximal amplitude of 192 pA (bottom). Note that with the application of capsaicin, there was a large increase in the membrane current noise, suggesting that the inward current resulted from the opening of membrane channels. In a total of eight neurons, capsaicin depolarized sensory neurons by an average value of 20 ± 3 mV. Both the time to peak and the time for recovery are similar in the voltage- and current-clamp recordings, suggesting that the capsaicin-induced inward current gives rise to the depolarization.
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FIG. 1.
Capsaicin depolarizes and elicits an inward current in embryonic sensory neurons. A, top: current clamp recording from a representative neuron (resting potential of 54 mV) where the focal application of 100 nM capsaicin for 2 s generated a burst of action potentials and depolarized the cell. In a voltage-clamp recording from this same neuron (bottom), capsaicin evoked an inward current (shown as downward) at a holding potential of 60 mV. As shown in B, the reversal potential for the capsaicin-elicited current is between 0 and +10 mV. Capsaicin (100 nM) was applied focally for a period of 1 s. The application of capsaicin is indicated by the bar below/above each trace.
60 mV, the application of capsaicin produced an inward current. The potential was shifted to 10 mV for ~15 s where the application of capsaicin elicited an inward current of 27 pA. At 0 mV, the current was reduced further (17 pA), but remained noticeably inward. However, at +10 mV, the current clearly became outward in direction (shown as the upward deflection) and had an amplitude of 26 pA. In recordings from three neurons, the average reversal potential for the current elicited by capsaicin was +5 ± 1 mV, which may suggest that the ion channels activated by capsaicin are nonselective for cations.
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FIG. 3.
Prostaglandin E2 (PGE2) enhances the capsaicin response in a time-dependent manner. A: enhancement of the capsaicin-evoked current by 1 µM PGE2 as obtained from a representative sensory neuron. Bar above each trace indicates the focal application of 100 nM capsaicin. Extent and time courses of the PGE2-induced sensitization are summarized in B. Amplitude of the capsaicin response after PGE2 was normalized to the average of 2 control responses and represented as the fold-increase relative to the control. Filled circles, those neurons treated with 1 µM PGE2 (n = 6-9); filled squares, those neurons exposed to 100 nM PGE2 (n = 6). Hatched bar, change to Ringer solution containing PGE2. C: results (n = 3) obtained for the focal application of 100 nM capsaicin every 2 min for a period of 30 min in the absence of PGE2. * Significant difference at P < 0.05 compared with control.
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FIG. 4.
Duration of PGE2-induced sensitization is increased with concentrations of focally applied capsaicin <100 nM. Fold-increase in the response to 10 nM capsaicin after sensitization by 1 µM PGE2 is shown as a function of time. Results obtained with 100 nM capsaicin (Fig. 3B) are presented as a point of reference. Hatched bar, application of PGE2. Results for 10 nM capsaicin were obtained from 5-7 neurons. * Significant difference in capsaicin responses after PGE2 compared with their respective controls.
; Marsh et al. 1987; Oh et al. 1996; Wood et al. 1988). As the influx of extracellular Ca2+ has been implicated in the desensitization of this complex (Cholewinski et al. 1993; Docherty et al. 1996; Liu and Simon 1996; Yeats et al. 1991), we investigated the role of Ca2+ in modulating sensitization of sensory neurons by lowering the concentration of Ca2+ in the Ringer solution (see Fig. 5A). At 30 µM free Ca2+ (2 mM CaCl2 and 2 mM EGTA) in the absence of PGE2, the amplitude of the peak inward current elicited by 100 nM capsaicin was not altered significantly compared with that observed for these same neurons in 2 mM Ca2+-Ringer (268 ± 128 pA compared with 273 ± 153 pA for the 2 mM Ca2+ control, n = 3). In the lowered Ca2+, 1 µM PGE2 produced a sensitization (2.9 ± 0.2-fold increase, range 2.7-3.4) that was analogous to that observed in 2 mM Ca2+-Ringer. Although the time to peak in 30 µM Ca2+ was not significantly different (11.3 ± 1.1 min, range 10-14 min) from that observed in 2 mM Ca2+-Ringer,t1/2 MAX was increased to 10.3 ± 1.6 min (range 6.8-13.7 min). Lowering the free Ca2+ concentration to 100 nM (2 mM CaCl2 and 3.5 mM EGTA), significantly enhanced the magnitude and duration of sensitization produced by 1 µM PGE2. The extent of sensitization (4.1 ± 0.9-fold, range 2.0-7.3, n = 7), the time to peak (19.6 ± 1.4 min, range 14-24 min), and t1/2 MAX (19.6 ± 1.7 min, range 14.7-26.4 min) were all increased relative to the values obtained in 2 mM Ca2+-Ringer even though the peak amplitude of the capsaicin-evoked current in the absence of PGE2 was not altered significantly in these neurons that were bathed initially in 2 mM Ca2+ (93 ± 31 pA compared with 123 ± 20 pA for the 2 mM Ca2+-Ringer control, n = 4). In contrast, raising the Ca2+ concentration to 10 mM blocked the sensitization in all neurons examined (n = 5). In the elevated Ca2+, the amplitude of the capsaicin-elicited current in the absence of PGE2 was not altered significantly (260 ± 84 pA compared with 336 ± 172 pA for the 2 mM Ca2+ controls). Therefore, these results indicate that the duration of sensitization may depend on elevations in intracellular Ca2+ concentration via influx through the capsaicin receptor-ion channel complex.
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FIG. 5.
Duration of PGE2-induced sensitization is dependent on the concentration of extracellular Ca2+. A: results obtained from sensory neurons that were superfused with modified Ringer solutions for 5 min before acquisition of the 2 control responses to 100 nM capsaicin. Neurons then were exposed to 1 µM PGE2 ( 
) in the altered Ca2+ solution as indicated. Filled circle, results previously shown in Fig. 3B for 2 mM Ca2+-Ringer and have been included as a point of reference. B: transient sensitization produced by 1 µM PGE2 () in either 2 mM or 100 nM Ca2+-Ringer for neurons in which the recordings were obtained using the perforated-patch technique. C: effects of internal perfusion with 10 mM bis-(o-aminophenoxy)-N,N,N,N-tetraacetic acid (BAPTA) on the sensitization produced by 1 µM PGE2. In these experiments, the response was elicited by the focal application of 100 nM capsaicin in 2 mM Ca2+-Ringer. D: lack of sensitization by 1 µM PGF2
in different Ca2+ concentrations as indicated. Hatched bar, addition of PGF2 to the bath. * Significant difference at P < 0.05 compared with their respective controls.
; Rae et al. 1991) to determine whether the Ca2+ buffering capacity of the pipette contents (2.5 mM CaCl2, 5 mM EGTA, calculated 100 nM free Ca2+) used in the whole cell recordings influenced the time course of sensitization. Under control conditions, 100 nM capsaicin evoked a current that had an average amplitude of 213 ± 45 pA (range 76-336pA, n = 5). As shown in Fig. 5B, treatment with 1 µM PGE2in 2 mM Ca2+-Ringer produced a transient sensitization(1.8 ± 0.1, range 1.7-2.0, n = 5) of the capsaicin-elicited current. The time to peak was 8.4 ± 0.9 min (range 6-10 min) and t1/2 MAX was 3.3 ± 0.8 min (range 1.9-5.9 min). These values were not significantly different from that obtained in the whole cell recordings. In 100 nM Ca2+-Ringer, the recordings obtained with the perforated-patch were also similar to those obtained with the whole cell configuration. Under low Ca2+ conditions, 100 nM capsaicin evoked an average response of 153 ± 30 pA (range 59-247 pA, n = 7) and was not significantly different from the response obtained in these same neurons that were bathed initially in 2 mM Ca2+ (161 ± 53 pA, range 53-449 pA). Treatment with 1 µM PGE2 caused a 2.9 ± 0.3-fold increase (range 2.0-4.1, n = 7) in the capsaicin response. As determined from the individual time courses of sensitization, neither thetime to peak (13.8 ± 2.4 min, range 10-25 min) nor t1/2 MAX(16.3 ± 3.3 min, range 7.2-28.3 min) were significantly different from values obtained with the whole cell method in 100 nM Ca2+. This suggests that with the addition of some amount of Ca2+ buffering via the pipette solution (EGTA) there may be less of a change in intracellular Ca2+. At present, it is uncertain whether the similar rate of inactivation of sensitization observed in the whole cell recordings at 100 nM Ca2+ resulted from the ineffectiveness of EGTA to buffer changes in intracellular Ca2+ (Adler et al. 1991; Huang and Neher 1996) or whether the slow relaxation represents a Ca2+-independent component of inactivation.
, which is a prostaglandin that does not sensitize sensory neurons (Hingtgen et al. 1995; Nicol and Cui 1994), to modulate the capsaicin-evoked current at different extracellular Ca2+ concentrations. These results are illustrated in Fig. 5D. Under these varied Ca2+ concentrations, exposure to 1 µM PGF2 did not alter significantly the response to capsaicin. Thus the current evoked by the focal application of capsaicin was relatively stable during the 30-min time period regardless of the extracellular Ca2+ concentration, and the response amplitude was not modified by exposure to a nonsensitizing eicosanoid.
; Garthwaite et al. 1988; Knowles et al. 1989). Because lowering the Ca2+ concentration increased the duration of sensitization, the possibility that the persistence of sensitization might depend on the production of nitric oxide was examined by using the inhibitor of nitric oxide synthase, L-NAME. These experiments were performed in 2 mM Ca2+-Ringer as this would provide for the greatest possible Ca2+-dependent activation of nitric oxide synthase (NOS). Sensory neurons were pretreated with 100 µM L-NAME for 10 min before acquisition of the two control responses to 100 nM capsaicin, and L-NAME was present throughout the exposure to PGE2. A concentration of 100 µM L-NAME was used as this concentration blocked completely the capsaicin-induced increase in cyclic GMP observed in embryonic sensory neurons grown in culture (Dymshitz and Vasko 1994). In the presence of L-NAME, the average amplitude of the control responses (277 ± 59 pA, range 110-474 pA, n = 9) was not significantly different from that obtained in 2 mM Ca2+-Ringer (449 ± 159 pA, range 100-1,300 pA,n = 9). After exposure to 1 µM PGE2 and L-NAME, the capsaicin response was sensitized and reached a maximal increase of 3.1 ± 1.3-fold (range 1.8-5.9) compared with control values (see Fig. 6A). The time to peak (13.6 ± 2 min, range 10-30 min, n = 9) was similar to that observed in 2 mM Ca2+, however, t1/2 MAX was increased significantly to 14.3 ± 3.8 min (range 5-20 min). In the absence of PGE2, L-NAME had no effect on the relative amplitude of the capsaicin-evoked inward current. These results suggest the activation of NOS and the subsequent generation of nitric oxide are involved in the inactivation of PGE2-induced sensitization.
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FIG. 6.
Nitric oxide-cyclic guanosine 3 , 5-monophosphate (GMP) pathway modulates the duration of PGE2-induced sensitization. A: inhibition of nitric oxide synthase increases the duration of sensitization. Diamonds, neurons (n = 5-6) that were pretreated with 100 µM nitro-L-arginine methyl ester (L-NAME) in 2 mM Ca2+-Ringer for 10 min before the application of 1 µM PGE2. L-NAME was present (open bar) throughout exposure to PGE2 (). Filled circles, neurons treated with PGE2 alone (same results as presented in Fig. 3B provided for reference). Filled squares, different group of sensory neurons (n = 5-6) treated with 100 µM L-NAME alone. Error bars are smaller than the symbol. B shows that addition of nitric oxide donors (NOD; 
) inactivates the PGE2-induced sensitization. Open squares and filled circles, different groups of sensory neurons that were exposed to s-nitroso-n-acetylpenicillamine (SNAP;n = 5) and 3-morpholinosydnonimine (SIN-1; n = 6), respectively. Other conditions (i.e., PGE2 and L-NAME) are the same as in A. C: shows that the addition of 100 µM 8-bromo-cyclic GMP () inactivates the PGE2-induced sensitization. Other conditions (i.e., PGE2 and L-NAME) are the same as in A. * Significant difference at P < 0.05 compared with their respective controls.
). Neurons were pretreated with 100 µM L-NAME to block any residual NOS activity. Exposure to 1 µM PGE2 produced about a twofold enhancement of the capsaicin-evoked current. At the peak of sensitization, the addition of SIN-1 or SNAP rapidly reduced the sensitization with a t1/2 MAX of 2.1 ± 0.2 min (range 1.7-2.9 min, n = 6) or 2.1 ± 0.1 min (range 1.8-2.3, n = 5), respectively. This value is similar to that observed under normal recording conditions (see Fig. 3B). Thus these results demonstrate that an increase in nitric oxide can produce a rapid inactivation of the sensitization produced by PGE2.
previously reported that the nitric oxide-cyclic GMP pathway played a causal role in the desensitization of the bradykinin response in rat sensory neurons. Based on our finding that in the presence of L-NAME the duration of sensitization was increased and that nitric oxide donors rapidly inactivated sensitization, we examined the capacity of cyclic GMP to modulate the PGE2-induced sensitization. In the presence of L-NAME, the application of 100 µM 8-Br-cyclic GMP, a membrane permeant analogue of cyclic GMP, produced a rapid inactivation of the PGE2-induced sensitization (see Fig. 6C). The 8-Br-cyclic GMP was added after 14 min in PGE2 so that the peak of sensitization had been attained (for this particular set of neurons the time to peak was 12.7 ± 0.8 min, range 10-14 min, n = 6). After the addition of 8-Br-cyclic GMP, sensitization of the capsaicin response inactivated with an average t1/2 MAX of 4.3 ± 0.7 min (range 2.7-6.9 min, n = 6) and is similar to that observed in Figs. 3B and 6B.These results suggest that the capsaicin-mediated influx of Ca2+ activates NOS, and nitric oxide, perhaps through the activation of guanylyl cyclase, leads to inactivation of PGE2-induced sensitization.
; Wood et al. 1989) and that this increase depended on an influx of extracellular Ca2+ (Wood et al. 1989). Therefore, to investigate further the role of cyclic GMP in the inactivation of sensitization, 8-Br-cyclic GMP was applied to sensory neurons after a 10-min exposure to 1 µM PGE2 in the absence of nitric oxide modulation. These experiments were performed in 100 nM free Ca2+ so that the duration of sensitization was lengthened greatly and also the potential for residual Ca2+-induced activation of guanylyl cyclase was reduced. As shown in Fig. 7A, treatment with PGE2 sensitized the neurons by about threefold (range 2.0-3.8, n = 5); at this point the application of 100 µM 8-Br-cyclic GMP caused a rapid reduction in the level of sensitization where t1/2 MAX was 3.7 ± 0.9 min (range 2.3-7.5 min). Even though the Ca2+ concentration was reduced to 100 nM, this value for t1/2 MAX was similar to that obtained in 2 mM Ca2+-Ringer (3.7 ± 0.5 min, Fig. 3B; 4.3 ± 0.7 min, Fig. 6B). After a 10-min exposure to 8-Br-cyclic GMP, the sensitization had returned to control levels. It seems unlikely that the decreased sensitization resulted from a cyclic GMP-induced desensitization to capsaicin because the normalized amplitude of the response never fell below a value of one.
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FIG. 7.
Cyclic GMP attenuates the PGE2-induced sensitization. A: sensory neurons were exposed to 100 nM free Ca2+ Ringer solution. Hatched bar, when the perfusate was changed to 100 nM free Ca2+ Ringer containing 1 µM PGE2. After a 10-min treatment period to attain near maximal levels of sensitization, 100 µM 8-Br-cyclic GMP was applied ( 
). Neurons exposed to PGE2 alone are the same as those shown in Fig. 5 and are provided as a point of reference. Values represent the means ± SE from 5 neurons. B: effects of exposure to 100 µM 8-Br-cyclic GMP () before the addition of 1 µM PGE2 (). These experiments were performed in 2 mM Ca2+-Ringer; results were obtained from 4 neurons. Results obtained for the exposure to PGE2 alone are the same as shown in Fig. 3B where the error bars have been removed for clarity. * Significant difference at P < 0.05 compared with their respective controls.
; Kase et al. 1987). These experiments were carried out in 2 mM Ca2+-Ringer to permit the optimal production of cyclic GMP. Sensory neurons were pretreated with 100 nM KT-5823 for 10 min before acquiring the two control responses to capsaicin, and KT-5823 was present throughout exposure to 1 µM PGE2. This concentration of KT-5823 was used because concentrations >1 µM inhibit the activity of PKA (Murthy and Makhlouf 1995). In the presence of KT-5823, the capsaicin-evoked current had an average amplitude of 248 ± 61 pA (range 30-460 pA, n = 6). As shown in Fig. 8, KT-5823 did not alter the capacity of PGE2 to sensitize the sensory neurons (a 2.6 ± 0.2-fold increase, range 2.4-3.4). However, inhibition of PKG led to a sustained sensitization of the capsaicin response. In fact, we were not able to determine a t1/2 MAX for these neurons because of the prolonged sensitization levels. KT-5823 alone had no effect on the capsaicin response during the 60-min time course of these experiments. Furthermore, in the presence of KT-5823, the addition of 8-Br-cyclic GMP did not produce the rapid inactivation of PGE2-induced sensitization (Fig. 8). The peak and plateau values (obtained between 13 and 38 min) for PGE2-induced sensitization in the presence of KT-5823 or KT-5823/8-Br-cyclic GMP were not significantly different. Therefore, these results indicate that elevated cyclic GMP by itself does not reverse the sensitization, but rather, it is the activation of PKG that subsequently terminates the PGE2-induced sensitization.
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FIG. 8.
Inhibition of cyclic GMP-dependent protein kinase leads to a sustained sensitization by PGE2. Filled circle, results obtained from neurons that were pretreated with 100 nM KT-5823 for 10 min (open bar) before the addition 1 µM PGE2 ( ). Results were obtained from 4-6 neurons except the points at 33 and 38 min, which represent 3 and 2 neurons, respectively. 
, results obtained from neurons (n = 5) that were pretreated with 100 nM KT-5823 for 10 min (open bar) before exposure to 1 µM PGE2 (). As indicated (), 100 µM 8-Br-cyclic GMP in the appropriate solution was applied to the bath. Open square, a different group of neurons (n = 3) that were exposed only to 100 nM KT-5823. * Significant difference at P < 0.05 compared with their respective controls.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
) or cyclic GMP (Wood et al. 1989) to 155 nM for the capsaicin-evoked release of calcitonin gene-related peptide (Hingtgen and Vasko 1994). Higher values for the EC50 range from 500 nM for the capsaicin-evoked depolarization (Marsh et al. 1987) to 1.1 µM for capsaicin activation of single channel currents (Oh et al. 1996). In addition, activation of the capsaicin receptor/ion channel complex may occur with positive cooperativity because the steepness factor, n, was determined to be 3.2. In support of this observation, previous reports determined Hill coefficients of 1.7 for the binding of resiniferatoxin, a potent analogue of capsaicin, to membranes prepared from the dorsal root ganglion (Szallasi et al. 1993) and 1.8 for activation of the capsaicin-gated ion channel (Oh et al. 1996). Therefore, the values for n and the EC50 imply that activation of the capsaicin receptor/ion channel complex might require the binding of at least two molecules of capsaicin at relatively low concentrations of agonist.
; Nicol and Cui 1994). Sensitization of the capsaicin response is somewhat different from that observed for sensitization of the bradykinin response. Although the number of action potentials elicited by bradykinin increased threefold after a 10-min exposure to PGE2, the amplitude of the bradykinin-evoked inward current was unaltered. The bradykinin results suggest that the enhanced excitability likely results from modulation of other conductances regulating the capacity of the neuron to generate an action potential. In contrast, PGE2 produced a significant enhancement of the capsaicin-evoked current, indicating that the pathway activated by PGE2 can modulate the capsaicin receptor/ion channel complex to conduct greater currents.
; Huang and Neher 1996; Stern 1992). The observed differences for the inactivation of sensitization with BAPTA and EGTA suggest that the inactivation might result from transient changes in Ca2+ concentration rather than sustained increases.
; Liu and Simon 1996; Yeats et al. 1991). We believe that inactivation of sensitization does not result from desensitization of the capsaicin receptor/ion channel complex. This idea is based on several observations. First, we use a short exposure time to 100 nM capsaicin (1-2 s), whereas those studies examining desensitization used long exposure times (20 s to 2 min) and concentrations of capsaicin ranging from 100 nM to 1 µM. Second, under normal recording conditions, the normalized amplitude of the capsaicin response, during a period of 30 min, never falls below a value of one, which would indicate desensitization of the response (see Fig. 3C). Third, inhibition of NOS suppressed the inactivation of sensitization, whereas NOS inhibitors had no effect on desensitization of the capsaicin response (Docherty et al. 1996).
) to activate this pathway.
, 1992). Taken together, these results suggest that elevations in intracellular cyclic GMP levels leading to activation of PKG may play an important role in suppressing the heightened sensitivity to various modalities of noxious stimulation associated with the inflammatory response.
) as well as the calcium current in cardiac myocytes (Méry et al. 1991) and rat pinealocytes (Chik et al. 1995). In isolated hippocampal neurons, cyclic GMP appears to inhibit a calcium current through activation of a phosphodiesterase (Doerner and Alger 1988). However, phosphodiesterase activation is not the case in the cardiac myocyte studies because inhibition of phosphodiesterase with isobutylmethylxanthine does not alter the capacity of cyclic GMP to suppress the calcium current (Méry et al. 1991; Wahler and Dollinger 1995). Alternatively, PKG might activate protein phosphatases because inhibition of either PKG with KT-5823 or protein phosphatase 2A with okadaic acid reversed the cyclic GMP-induced enhancement of the activity of the BK potassium channel (White et al. 1993; Zhou et al. 1996). Along these same lines, in the cerebellum a major substrate for PKG (G substrate) appears to be a protein that shares many of the characteristics of protein phosphatase inhibitor-1 (Detre et al. 1985). A recent study determined that PKG is found in sensory neurons at levels that are similar to the high levels found in the cerebellum, however, there was no detectable G substrate in either adult or embryonic sensory neurons (Qian et al. 1996). Thus the cellular mechanisms whereby PKG leads to inactivation of the PGE2-induced sensitization of sensory neurons are clearly an area for future investigation.
; Hingtgen et al. 1995). Our findings are similar to those reported previously for the actions of PKG on the calcium current recorded from cardiac myocytes. In these cells, the calcium current is suppressed by agents that activate PKG (cyclic GMP or a nitric oxide donor), however, this inhibition occurs only after the calcium current is enhanced by prior exposure to agents that elevate intracellular cAMP levels (Méry et al. 1991; Wahler and Dollinger 1995). This proposed mechanism is consistent with the yin yang hypothesis developed by Goldberg et al. (1975) where cAMP and cyclic GMP have antagonistic influences on the regulation of physiological processes. In addition, our observations may account for the lack of effect that nitric oxide donors and cyclic GMP have on the capsaicin-evoked release of substance P and calcitonin gene-related peptide from nonsensitized sensory neurons (Dymshitz and Vasko 1994). Therefore, taken together, these observations suggest that sensitization of the capsaicin receptor/ion channel complex may result from PKA-dependent phosphorylation. Once modified by PKA, this complex subsequently can be altered by PKG, via an unknown mechanism or pathway, to inactivate the processes contributing to the facilitated conductance. Thus activation of the cyclic GMP pathway may serve an important regulatory role in reversing the heightened sensitivity to noxious stimulation observed in neurogenic inflammation.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Michael Vasko for discussions about signaling in sensory neurons and a suggestion to try cyclic GMP and to Dr. Angela Evans for providing some of the neuronal preparations.
J. C. Lopshire was supported by the Indiana University-Purdue University, Indianapolis Research Investment Fund and a fellowship from the American Heart Association, Indiana Affiliate. This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-30527 to G. D. Nicol.
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FOOTNOTES |
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Address for reprint requests: G. D. Nicol, Dept. of Pharmacology and Toxicology, 635 Barnhill Dr., Indiana University School of Medicine, Indianapolis, IN 46202.
Received 9 May 1997; accepted in final form 30 July 1997.
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REFERENCES |
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Abstract
Introduction
Methods
Results
Discussion
References
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