Epinephrine Produces a beta -Adrenergic Receptor-Mediated Mechanical Hyperalgesia and In Vitro Sensitization of Rat Nociceptors

Sachia G. Khasar, Gordon McCarter, and Jon D. Levine

Departments of Medicine and Oral and Maxillofacial Surgery, Division of Neuroscience and Biomedical Sciences Program, National Institutes of Health Pain Center (UCSF), University of California, San Francisco, California 94143-0440


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Khasar, Sachia G., Gordon McCarter, and Jon D. Levine. Epinephrine produces a beta -adrenergic receptor-mediated mechanical hyperalgesia and in vitro sensitization of nociceptor-like neurons in the rat. Hyperalgesic and nociceptor sensitizing effects mediated by the beta -adrenergic receptor were evaluated in the rat. Intradermal injection of epinephrine, the major endogenous ligand for the beta -adrenergic receptor, into the dorsum of the hindpaw of the rat produced a dose-dependent mechanical hyperalgesia, quantified by the Randall-Selitto paw-withdrawal test. Epinephrine-induced hyperalgesia was attenuated significantly by intradermal pretreatment with propranolol, a beta -adrenergic receptor antagonist, but not by phentolamine, an alpha -adrenergic receptor antagonist. Epinephrine-induced hyperalgesia developed rapidly; it was statistically significant by 2 min after injection, reached a maximum effect within 5 min, and lasted 2 h. Injection of a more beta -adrenergic receptor-selective agonist, isoproterenol, also produced dose-dependent hyperalgesia, which was attenuated by propranolol but not phentolamine. Epinephrine-induced hyperalgesia was not affected by indomethacin, an inhibitor of cyclo-oxygenase, or by surgical sympathectomy. It was attenuated significantly by inhibitors of the adenosine 3',5'-cyclic monophosphate signaling pathway (the adenylyl cyclase inhibitor, SQ 22536, and the protein kinase A inhibitors, Rp-adenosine 3',5'-cyclic monophosphate and WIPTIDE), inhibitors of the protein kinase C signaling pathway (chelerythrine and bisindolylmaleimide) and a µ-opioid receptor agonist DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin). Consistent with the hypothesis that epinephrine produces hyperalgesia by a direct action on primary afferent nociceptors, it was found to sensitize small-diameter dorsal root ganglion neurons in culture, i.e., to produce an increase in number of spikes and a decrease in latency to firing during a ramped depolarizing stimulus. These effects were blocked by propranolol. Furthermore epinephrine, like several other direct-acting hyperalgesic agents, caused a potentiation of tetrodotoxin-resistant sodium current, an effect that was abolished by Rp-adenosine 3',5'-cyclic monophosphate and significantly attenuated by bisindolylmaleimide. Isoproterenol also potentiated tetrodotoxin-resistant sodium current. In conclusion, epinephrine produces cutaneous mechanical hyperalgesia and sensitizes cultured dorsal root ganglion neurons in the absence of nerve injury via an action at a beta -adrenergic receptor. These effects of epinephrine are mediated by both the protein kinase A and protein kinase C second-messenger pathways.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A contribution of catecholamines to both inflammatory and neuropathic pain states has been suggested based on both clinical evidence and animal models (Jänig et al. 1996; Raja 1998). Although most research in the area has evaluated the contribution of the sympathetic postganglionic neuron (SPGN) and its major transmitter norepinephrine, these pain states also may be associated with increased activity of the sympathoadrenal system, leading to elevation of the plasma concentrations of epinephrine [an endogenous beta -adrenergic receptor (beta -AR) agonist] (Cryer 1980; DeTurck and Vogel 1980; Taylor et al. 1989; Wortsman et al. 1984). Although activation of beta -ARs was shown, almost two decades ago, to produce behavioral hyperalgesia (Ferreira 1980) and epinephrine can cause anginal pain in the absence of apparent ischemia (Eriksson et al. 1995), further studies of the role of beta -ARs in peripheral pain and the elucidation of mechanisms involved have been lacking. We investigated mechanisms involved in epinephrine-induced hyperalgesia, hypothesizing that epinephrine produces hyperalgesia by directly sensitizing primary afferent nociceptors.

We tested this hypothesis in vivo using the behavioral model of mechanical hyperalgesia and in vitro using cultured dorsal root ganglion (DRG) neurons. We also tested whether epinephrine enhances tetrodotoxin-resistant sodium currents (TTX-R INa), which are carried by an ion channel selectively found in primary afferent nociceptors (Akopian et al. 1996; Sangameswaran et al. 1996) and the activity of which is increased by agents that act directly on primary afferent nociceptors in vivo to produce hyperalgesia (England et al. 1996; Gold et al. 1996b). Finally, we examined the second-messenger systems that may mediate the hyperalgesic and sensitizing effects of epinephrine.


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Experimental procedures

BEHAVIORAL EXPERIMENTS. Behavioral experiments were performed on lightly restrained male Sprague-Dawley rats (250-350 g) purchased from Bantin and Kingman (Fremont, CA) and housed in the animal care facility of the University of California, San Francisco, under a 12-h light/dark cycle. Care and use of rats conformed to National Institutes of Health guidelines, and experimental protocols were approved by the University of California, San Francisco, Committee on Animal Research. The nociceptive flexion reflex was quantified by an Ugo Basile Analgesimeter (Stoelting, Chicago, IL), which applies a linearly increasing mechanical force to the dorsal surface of the rat's hind paw.

Before the rats were used for behavioral experiments, they were trained in the paw-withdrawal reflex test at 5-min intervals for 1 h each day for a period of 5 days. This training procedure reduces variability and produces a more stable baseline paw-withdrawal threshold measurement, thereby enhancing the ability to detect the effect of agents that modulate nociception (Taiwo et al. 1989). On the day of the experiments, paw-withdrawal threshold was measured (i.e., rats were exposed to the test stimulus) at 5-min intervals for 1 h. The mean of the last six paw-withdrawal thresholds was determined. This mean value is defined as the baseline paw-withdrawal threshold before the injection of a test agent. Test agents were injected intradermally into the dorsal surface of both hindpaws, in a volume of 2.5 µl. Paw-withdrawal thresholds then were redetermined at 10, 15, and 20 min postinjection. The mean of the paw-withdrawal thresholds obtained at these three time points is the mechanical nociceptive threshold at the dose of the test agent used. The effect of each dose of a test agent was calculated as the percentage change from baseline, for each paw, as follows: [(threshold in the presence of test agent minus baseline threshold)/baseline threshold] × 100. This transformation was done to account for the differences in baseline thresholds of individual rats within a group. Increasing doses of test agents, each an order of magnitude greater than the previous dose, were injected cumulatively at 25-min intervals. Because we have shown, using this protocol under similar experimental conditions, that repeated injection of small volumes of PGE2 locally, in one paw, does not affect the mechanical nociceptive threshold of the contralateral paw (Khasar and Levine, unpublished observation), each paw was treated as an independent measure.

Each experiment was performed on a separate group of rats. Each group of rats was treated with only one agonist and/or antagonist. The dose-response relationship for the effect of epinephrine was determined both for epinephrine alone and for epinephrine in the presence of propranolol (1 µg id). Propranolol was injected into the paw, and after 15 min, paw-withdrawal thresholds were determined, before the injection of epinephrine, to assess any effect of propranolol alone on baseline paw-withdrawal threshold. Thereafter the same dose of propranolol was coinjected with every other dose of epinephrine in the course of dose-response relationship determination. Similar protocols were used in tests involving distilled water (pH 7.5, vehicle for propranolol) or phentolamine against epinephrine. As with propranolol, dose-response relationships to epinephrine were determined alone or after pretreatment with indomethacin (4 mg/kg ip). Indomethacin was injected intraperitoneally, and after 30 min, paw-withdrawal thresholds were determined, before the injection of epinephrine, to assess any effect of indomethacin alone on baseline paw-withdrawal threshold. Thereafter indomethacin (1 µg), was coinjected with every other dose of epinephrine in the course of dose-response relationship determination. This protocol was used to ensure that the effect of phentolamine, propranolol, or indomethacin did not wear off while the dose-response relationship was being determined. The effect of ascorbic acid (4 mg/ml) and repeated injections of distilled water alone on basal paw-withdrawal threshold also was tested. The dose-response relationship for isoproterenol-induced hyperalgesia also was determined for isoproterenol alone and for isoproterenol in the presence of propranolol or phentolamine as described in the preceding text for epinephrine. For determination of latency to onset and duration of action, epinephrine (1 µg) was injected intradermally into the paw, and paw-withdrawal threshold was measured at 1-min intervals for the first 5 min, then 5 min later, and at 10-min intervals for another 20 min. Thereafter, paw-withdrawal threshold was measured at 30-min intervals for another 2 h to determine the duration of action. Similar protocols have been used in previous studies to determine latency to onset and duration of action of other hyperalgesic agents (Khasar et al. 1994; Taiwo and Levine 1990; Taiwo et al. 1987). To determine whether epinephrine-induced hyperalgesia, like that produced by other hyperalgesic agents (e.g., PGE2), is inhibited by opioids, epinephrine was coinjected with [D-Ala2,N-Me-Phe4,Gly5-ol] enkephalin (DAMGO; a µ-opioid receptor agonist and stimulator of inhibitory G protein signaling). The effects of 9-(tetrahydro-2-furyl) adenine (SQ 22356; an adenylyl cyclase inhibitor) (Harris et al. 1979), Rp-cAMPs or WIPTIDE [protein kinase A (PKA) inhibitors] (Dragland et al. 1985; Glass et al. 1989), chelerythrine or bisindolylmaleimide (BIM) [protein kinase C (PKC) inhibitors] (Herbert et al. 1990; Toullec et al. 1991) (all 1 µg) on epinephrine (100 ng)-induced hyperalgesia also were tested to determine the second-messenger system(s) mediating epinephrine hyperalgesia. Paws first were pretreated with SQ 22356, chelerythrine, BIM, Rp-cAMPs or WIPTIDE, then 15 min later the same agent was coinjected with epinephrine. DAMGO was coinjected with epinephrine, as previously described (Khasar et al. 1994; Levine and Taiwo 1989). Coinjection was done such that antagonist entered the paw first. This was accomplished by drawing up 2.5 µl of the agonist into a 10-µl microsyringe (Hamilton, Reno, NV), then drawing up a small amount of air into the syringe (to avoid contact and mixing of drugs in the syringe) and finally, 2.5 µl of the antagonist was drawn up into the syringe. Effects of BIM or WIPTIDE (each 1 µg) also were tested on isoproterenol-induced hyperalgesia. Rp-cAMPs and WIPTIDE injections always were preceded by injection of distilled water to produce hypo-osmotic shock, thereby enhancing cell membrane permeability to these agents (Khasar et al. 1995b; Taiwo and Levine 1989a; Tsapis and Kepes 1977; West and Huang 1980; Widdicombe et al. 1996). The dose of epinephrine used was based on a dose-response relationship demonstrating that this dose produces between 70 and 80% of maximal hyperalgesia. The doses of DAMGO, SQ 22356, Rp-cAMPs, and WIPTIDE used have been shown previously to be effective in significantly attenuating the hyperalgesia induced by PGE2 and/or other E-type prostaglandins (Khasar et al. 1995a; Taiwo and Levine 1991). Doses of chelerythrine and BIM were selected based on a dose-response relationship determined in preliminary studies (data not shown).

SYMPATHECTOMY. To test if epinephrine hyperalgesia is dependent on the presence of intact sympathetic postganglionic neurons (SPGNs), rats were sympathectomized surgically under pentobarbital anesthesia (50 mg/kg body wt, with additional doses given to maintain areflexia during surgery). After a lateral abdominal incision, the sympathetic chain was reached via an extraperitoneal approach, and the lumbar sympathetic chains, from ganglia L1 to L4, were removed bilaterally (Baron et al. 1988; Miao et al. 1995). This procedure results in complete sympathetic denervation of the hind limbs (Baron et al. 1988). Sham surgeries were performed in a similar manner, except that the sympathetic chains were left intact, after exposure. The dose-response relationship for epinephrine-induced hyperalgesia was determined 7 days after surgical sympathectomy or sham surgery.

CELL CULTURE AND ELECTROPHYSIOLOGY. To examine the effect of epinephrine on the excitability and ionic conductance of isolated DRG neurons, primary cultures of adult rat lumbar DRGs (L1-L6) were prepared as previously described (Gold et al. 1996a). Culture medium consisted of minimal essential medium (MEM) supplemented with 10% fetal bovine serum and 1,000 units per ml each of penicillin and streptomycin. Neurons were plated onto glass cover slips coated with laminin and poly-DL-ornithine and were maintained in culture medium with nerve growth factor (NGF; GIBCO BRL, Gaithesburg, MD) at 37°C under 3% CO2. Neurons were used within 24 h of plating before there was appreciable outgrowth of neurites. Small-diameter neurons (20-30 µm in diameter) were studied because they selectively express properties of nociceptors (Gold et al. 1996a). Bath solution continuously perfused the recording chamber at 1-2 ml/min. Drugs were added as described in the respective figure legends. Experiments were performed at room temperature (21-24°C).

Whole cell patch-clamp recordings were performed using an Axopatch 200B amplifier with pClamp6 acquisition and stimulation programs (Axon Instruments, Foster City, CA). Data were low-pass filtered at 5 kHz and acquired at 20 kHz. For current-clamp recordings, the perforated-patch method was employed with borosilicate glass electrodes fire-polished to 1-3 MOmega . The electrode solution contained (in mM) 30 KCl, 55 K2S04, 1 CaCl2, 2 MgCl2, 11 EGTA, and 10 HEPES, pH adjusted to 7.2 with KOH, and osmolarity adjusted to 310 mOsm with sucrose. Nystatin was dissolved in DMSO at 6 mg/100 µl for each day's experiments, and 2 µl of this stock solution was added to 300 µl of electrode solution before filling each electrode. The current-clamp bath solution contained (in mM) 130 NaCl, 3 KCl, 2.5 CaCl2, 0.6 MgCl2, 10 HEPES, and 10 glucose, pH adjusted to 7.4 with NaOH, and osmolarity adjusted to 325 mOsm with sucrose. A neuron was only used if its resting potential was more negative than -45 mV and could be maintained throughout the experiments at -60 mV by constant current injection. Stimulation current was injected in a ramp-and-plateau protocol in which the current was increased linearly for 250 ms and then held at the final level for an additional 500 ms. The final amplitude of the injected current was adjusted to depolarize the neuron enough so that one to three action potentials were triggered during the ramp-and-plateau stimulus.

Voltage-clamp experiments were performed with 2- to 5-MOmega electrodes filled with (in mM) 140 CsCl, 10 NaCl, 0.1 CaCl2, 2 MgCl2, 11 EGTA, 10 HEPES, 2 Mg-ATP, and 1 Li-ATP; pH was adjusted to 7.2 with Tris-base. Bath solution contained (in mM) 35 NaCl, 30 tetraethylammonium chloride, 65 choline chloride, 0.1 CaCl2, 5 MgCl2, 10 HEPES, and 10 glucose; pH was adjusted to 7.4 with NaOH, and osmolarity adjusted to 325 mOsm with sucrose. Tetrodotoxin (TTX, 50 nM) was added to the bath solution. Capacitance and series resistance (>80%) was compensated, and leak subtraction was performed with a P/4 protocol. After obtaining a current-voltage (I-V) relationship for TTX-R INa, a test pulse was applied every 20 s to monitor the amplitude of TTX-R INa during the experiment. A voltage that produced approximately half-maximal current activation was used for the test pulse because hyperalgesic agents produce the greatest increase in TTX-R INa at this part of the I-V curve (Gold et al. 1996b) (also see Fig. 5B).

Materials

Drugs or reagents used in this study were from Sigma (St. Louis, MO) unless otherwise noted: prostaglandin E2 (PGE2), epinephrine [an endogenous beta -adrenergic receptor (beta -AR) agonist], isoproterenol (a specific beta -AR agonist), propranolol (a specific beta -AR antagonist), phentolamine (a specific alpha -adrenergic receptor (alpha -AR) antagonist, CIBA-GEIGY, Summit, NJ), DAMGO, enkephalin (a µ-opioid receptor agonist; Research Biochemicals, Natick, MA); SQ 22536 (an adenylyl cyclase inhibitor); BIM (a PKC inhibitor) (both from Calbiochem, La Jolla, CA); chelerythrine (a PKC inhibitor; L. C. Labs., Woburn, MA); Rp-cAMPS (a PKA inhibitor; Biolog, La Jolla, CA); WIPTIDE (a PKA inhibitor; Peninsula Labs, Belmont, CA); indomethacin sodium salt (a generous gift from Merck Research Labs, Rahway, NJ). PGE2 (4 mg/ml) stock solution was made by dissolving it in 10% ethanol in normal saline; further dilutions were made by adding saline. Final concentration of ethanol was <1%. Isoproterenol, phentolamine, propranolol, chelerythrine, BIM, and WIPTIDE were dissolved in distilled water. Epinephrine (4 mg/ml) was dissolved in distilled water with an equivalent amount of ascorbic acid just before it was used and was kept on ice in subdued lighting conditions. All other drugs were dissolved in normal saline or bath solution.

Data are presented as means ± SE and analyzed statistically using one-factor ANOVA or repeated measures ANOVA as appropriate. Where ANOVA showed significant differences between groups, Fisher's protected least-significant difference (PLSD) post hoc test was used to determine the specific pairs of groups between which statistically significant differences occurred. P < 0.05 was the accepted level for statistical significance.


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Behavioral studies

Epinephrine (1 ng to 1 µg) produced a dose-dependent decrease in the mechanical nociceptive threshold (i.e., produced hyperalgesia; F = 90.7, P < 0.01; one-factor ANOVA) when injected intradermally into the dorsal surface of the hindpaw of the rat (mean baseline paw-withdrawal threshold for this group of rats was 107.5 ± 1.2 g; n = 31) (Fig. 1A). The intradermal injection of ascorbic acid (4 mg/ml) did not significantly affect basal paw-withdrawal threshold (data not shown). The latency to onset of epinephrine (1 µg)-induced hyperalgesia was brief (Fig. 2); it was significant 2 min after injection, reached peak effect by 5 min (Fig. 2A) and lasted ~2 h (Fig. 2B). Epinephrine-induced hyperalgesia was attenuated by treating the paws (mean baseline paw-withdrawal threshold 102.6 ± 2.2 g; n = 8) with propranolol, significantly shifting the dose-response curve to the right of that for epinephrine alone (Fig. 1A). Injection of propranolol alone did not alter basal paw-withdrawal threshold (data not shown). Pretreatment of paws with distilled water and subsequent coinjection with epinephrine during the course of the experiment also produced dose-dependent hyperalgesia (F = 40.76, n = 14; Fig. 1B). Repeated injections of distilled water alone did not produce hyperalgesia (F = 1.48, n = 4; Fig. 1A).



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Fig. 1. Dose-response relationship for the mechanical hyperalgesia produced by epinephrine in normal (A, ) and after pretreatment, locally in the paw, with phentolamine (1 µg; diamond ) or propranolol (1 µg; ), 30 min before determining the dose-response relationship of their effects on mechanical nociceptive threshold and again with every other dose of epinephrine during the course of the experiment and the effect of repeated injection of distilled water (pH 7.5, vehicle for epinephrine; triangle ); (B) in distilled water pretreated (, n = 14 paws), indomethacin-treated (black-lozenge ; n = 8 paws), sympathectomized (down-triangle; n = 8 paws), and sham-sympathectomized (odot ; n = 6) rats. When indomethacin was used, rats were pretreated (4 mg/kg) intraperitoneally 30 min before dose-response relationship determination and indomethacin (1 µg) also was coinjected into the paw, with epinephrine. ANOVA showed significant differences between all the groups (A and B; F = 12.32; P < 0.01). Fisher's PLSD post hoc test revealed significant differences between the effects of epinephrine alone (n = 31) and distilled water alone (n = 6; P < 0.001), epinephrine plus propranolol (P < 0.01; n = 8). Also there was a significant difference between epinephrine plus propranolol and epinephrine plus phentolamine (P < 0.05; n = 17).



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Fig. 2. Latency to onset (A) and duration of action (B) of the mechanical hyperalgesia induced by epinephrine (1 µg), injected intradermally at 0 min. Paw-withdrawal thresholds were determined every minute for the 1st 5 min, then 5 min later, and thereafter every 10 min for 20 min; then readings were taken every 30 min for another 2 h (n = 12 paws).

Because epinephrine has affinity for both alpha - and beta -ARs, we evaluated the contribution of the alpha -AR to epinephrine-induced hyperalgesia. We tested epinephrine-induced hyperalgesia against phentolamine, an alpha -AR antagonist. Earlier studies (Levine et al. 1986) showed that injection of phentolamine alone had no effect on basal paw-withdrawal threshold in the normal rat. In the current study, phentolamine did not significantly affect epinephrine-induced hyperalgesia (Fig. 1A) The mean baseline paw-withdrawal threshold of this group of rats was 107.0 ± 1.8 g; n = 17. A similar dose for phentolamine was shown to significantly reverse formalin-induced decrease in paw threshold (Levine et al. 1986) and also inhibit rolipram-induced prolongation of PGE2 hyperalgesia (Ouseph et al. 1995). All data from the groups of rats in Fig. 1, A and B, were analyzed together. ANOVA showed significant differences between the groups (F = 12.32; P < 0.01).

It has been shown that bradykinin (BK) and norepinephrine act on intermediary cells to trigger prostaglandin synthesis, which then acts on the primary afferent to sensitize it (Lembeck et al. 1976; Taiwo and Levine 1988). We therefore tested whether the inhibition of prostaglandin synthesis by indomethacin would affect epinephrine-induced hyperalgesia. Indomethacin (4 mg/kg ip, pretreatment, and 1 µg id, during the course of the experiment), did not affect the ability of epinephrine to produce hyperalgesia (Fig. 1B). The mean baseline paw-withdrawal threshold of this group of rats was 109.2 ± 6 g; n = 8. Earlier studies (Levine et al. 1986; Taiwo et al. 1990) showed that injection of indomethacin alone has no effect on basal paw-withdrawal threshold in the normal rat. Because BK and norepinephrine also act indirectly via sympathetic neurons to affect nociceptors (Levine et al. 1986), we tested the effect of sympathectomy on epinephrine-induced hyperalgesia. The elimination of sympathetic innervation of the hindpaw by sympathectomy had no effect on the hyperalgesia produced by epinephrine (Fig. 1B). The mean baseline paw-withdrawal threshold of this group of rats was 104.2 ± 3.2 g; n = 8, for the sympathectomized and 113.6 ± 3.2 g; n = 6, for the sham-sympathectomized group.

To further explore the contribution of the beta -AR to hyperalgesia, we used the beta -AR-selective agonist, isoproterenol (Ahlquist 1976). Intradermal injection of isoproterenol (1 ng to 1 µg), like epinephrine, produced dose-dependent hyperalgesia (F = 104.7, P < 0.01, one-factor ANOVA; Fig. 3). The mean baseline paw-withdrawal threshold of this group of rats was 103.1 ± 1.5 g; n = 20. Propranolol significantly attenuated the hyperalgesia produced by isoproterenol, whereas phentolamine had no effect. The mean baseline paw-withdrawal threshold of the isoproterenol + propranolol group of rats was 101.3 ± 2.7 g; n = 8 and that for the isoproterenol + phentolamine group was 111.3 ± 2 g; n = 8. 



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Fig. 3. Dose-response relationship for the hyperalgesia induced by isoproterenol, a specific beta -adrenergic receptor agonist, alone (; n = 12) and after pretreatment, locally in the paw, with phentolamine (1 µg, black-lozenge ; n = 8) or propranolol (1 µg, black-triangle; n = 8). ANOVA showed significant differences between the groups (F = 22.19; P < 0.01). Fisher's PLSD post hoc test revealed significant differences between the effects of isoproterenol alone and isoproterenol plus propranolol (P < 0.01).

The intradermal coinjection of DAMGO (1 µg) with epinephrine (100 ng) or intradermal pretreatment of rat paws with SQ 22536, chelerythrine, BIM, Rp-cAMPS (all 1 µg), or WIPTIDE (100 ng) 15 min before injection of epinephrine, significantly attenuated epinephrine-induced hyperalgesia. The same dose of chelerythrine or BIM had no effect on PGE2-induced hyperalgesia (Table 1). BIM (1 µg) also significantly attenuated isoproterenol hyperalgesia and WIPTIDE (100 ng) almost completely abolished it (Table 1). The intradermal injection of none of these agents alone affects basal paw-withdrawal threshold [Aley and Levine 1997, (for DAMGO) and unpublished observations].


                              
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Table 1. Percentage decrease in nociceptive threshold in response to epinephrine, PGE2, or isoproterenol and effects of DAMGO, SQ 22536, chelerythrine, BIM, Rp-cAMPs, or WIPTIDE

In vitro electrophysiological studies with cultured DRG neurons

To test the hypothesis that epinephrine-induced hyperalgesia was mediated by a direct effect of epinephrine on the primary afferent nociceptor, we performed whole cell patch-clamp electrophysiological experiments on dissociated DRG neurons in culture. Small-diameter (i.e., 20-30 µm) neurons were used within 12-24 h of plating as a model for peripheral nociceptor neurons. This is based on data showing similarities in the pharmacological repertoires of the cell body in vitro after 12-24 h in culture and the primary afferent nociceptor terminal in vivo (Baccaglini and Hogan 1983; England et al. 1996; Gold et al. 1996a; Pitchford and Levine 1991).

Current-clamp recordings were performed using the perforated-patch whole cell technique. The number of action potentials generated during a 750-ms ramp-and-plateau depolarizing current injection (see METHODS), as well as the latency to the first spike, were used as measures of excitability. After 5-10 min of baseline recordings, epinephrine (1 µM) was added to the bath. Figure 4A shows voltage traces from a typical neuron before and during exposure to 1 µM epinephrine, whereas Fig. 4B shows the time course of changes in the number of action potentials and the latency to the first action potential for another cell. For 11 neurons treated with 1 µM epinephrine, the average number of action potentials generated in response to the current ramp-and-plateau stimulus was 1.7 ± 0.2 before the addition of epinephrine and 5.3 ± 0.9 5 min or more after the start of drug perfusion (P < 0.01, paired Student's t-test). The mean latency from the start of current injection to the peak of the first spike was 278 ± 42 ms before epinephrine and 189 ± 21 ms after the start of drug perfusion (n = 11, P < 0.05). Of the 11 neurons tested, 9 (81%) showed an increase in spike number, and of those, 5 (45% of neurons tested) also showed a decrease (of >= 50 ms) in the spike latency. The mean resting membrane potential of these neurons was not changed by epinephrine (-60 ± 2 mV before epinephrine, -60 ± 2 mV after, n = 11, P > 0.05).



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Fig. 4. Change in excitability of cultured small-diameter dorsal root ganglion neurons after exposure to 1 µM epinephrine. In perforated-patch current-clamp experiments, action potentials were triggered by a 250-ms ramp to a 500-ms plateau of depolarizing current set to initially trigger 1-3 spikes (150-4,000 pA). A: current protocol (bottom) and sample voltage traces (top) from a typical cell 2 min before (left) and 3 min after (right) the onset of bath perfusion of 1 µM epinephrine. Scale bars represent 40 mV and 100 ms. B: time course of the change in excitability of another cell. Number of spikes increased and the latency to 1st spike decreased within 3 min of the onset of epinephrine perfusion. Neuron remained sensitized after washout of epinephrine.

Propranolol blocked the sensitization of small-diameter DRG neurons by epinephrine. When 10 µM propranolol was perfused 30 s before and with 1 µM epinephrine, there was no increase in the number of spikes (1.2 ± 0.1 before drugs vs. 1.3 ± 0.2 after, n = 7, P > 0.05) nor any decrease in the latency to the first spike (235 ± 9 vs. 233 ± 10 ms, P > 0.05).

Because hyperalgesic agents (e.g., PGE2) that sensitize nociceptors in vitro have been shown to increase TTX-R INa (England et al. 1996; Gold et al. 1996b), we performed whole cell voltage-clamp experiments to determine whether epinephrine acted similarly. Epinephrine (1 µM) caused a marked potentiation of TTX-R INa (Fig. 5). The peak current amplitude in response to a depolarizing test pulse was increased by 37 ± 5% (n = 24, P < 0.01). Comparison of the current-voltage plots before and after drug exposure indicate that activation of TTX-R INa was shifted by ~10 mV in the hyperpolarized direction (Fig. 5B). This dose of epinephrine caused an increase in TTX-R INa in 16 of 24 neurons (67%); isoproterenol caused a similar increase in TTX-R INa (Fig. 5C). There was no potentiation of TTX-R INa when epinephrine was applied in the presence of 2 µM propranolol; mean normalized current was 87 ± 5% of baseline in the third minute of exposure to epinephrine and propranolol (n = 6) (Fig. 6). The increase in current was not attenuated by 5 µM phentolamine (n = 5) (Fig. 6).



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Fig. 5. Potentiation of the tetrodotoxin-resistant sodium current (TTX-R INa) by epinephrine. A: TTX-R INa was evoked every 20 s by a 20-ms step to -15 mV (determined to be the voltage of approximately half-maximal activation of the current in this cell). Epinephrine (1 µM) was perfused into the recording chamber during the time indicated by the bar. Inset: superimposed current traces from the times indicated left-arrow ; bars denote 2 nA and 5 ms. B: current-voltage relationship of the same cell before (open circle ) and during () epinephrine exposure. Activation of the current is shifted in the hyperpolarized direction by ~10 mV, and the maximal current increased by 24% while current amplitudes at voltages more positive than 0 mV were unchanged. C: similar response in the current in a different cell to which 1 µM isoproterenol was applied.



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Fig. 6. Normalized mean response of TTX-R INa to epinephrine alone and in the presence of alpha - or beta -AR blockers. Peak inward current amplitude throughout each experiment was normalized to the average of the current during the last 3 min before the application of epinephrine. Time course of changes in the mean normalized current ± SE is plotted with data binned into 1-min intervals. In this and the following 2 figures, all experiments for a given condition were included in the data analysis, including those in which the current was unaffected by epinephrine (~20-30% of controls). Propranolol (2 µM, n = 6) or phentolamine (5 µM, n = 7) was perfused into the recording chamber 1 min before coperfusion of 1 µM epinephrine and inhibitor as indicated by the bars. Current increase was unaffected by phentolamine (P > 0.05) but was blocked by propranolol (P < 0.01) when compared with epinephrine alone (n = 24).

In the behavioral experiments, hyperalgesia caused by epinephrine was dependent on both the PKA and PKC second-messenger systems. Therefore we examined the relative roles these two second-messenger systems play in the effect of epinephrine on TTX-R INa. When Rp-cAMPS (100 µM), a competitive inhibitor of PKA types I and II, was included in the recording pipette, the increase in TTX-R INa by epinephrine was prevented (Fig. 7).



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Fig. 7. Inhibition of the cAMP-dependent protein kinase prevents the increase in TTX-R INa by epinephrine. Recordings were made with either the PKA inhibitor Rp-cAMPS (100 µM, n = 5) or sucrose (100 µM, n = 5) added to the electrode solution. Whole cell configuration was maintained for >= 10 min before epinephrine was applied to allow Rp-cAMPS to diffuse into the cell from the electrode. There was no increase in TTX-R INa when the inhibitor was present (for minutes 3-10 after onset of epinephrine perfusion) (P < 0.05) compared with epinephrine alone.

We also applied epinephrine in the presence of the PKC inhibitor, BIM. The mean normalized current increase in response to 1 µM epinephrine was significantly smaller when neurons were pretreated with BIM compared with control experiments with no BIM pretreatment (Fig. 8; P < 0.01, n = 11 for both conditions). Of the control cells, 8 of 11 responded to the epinephrine (defined as a >10% increase in current amplitude within 3 min) whereas only 5 of 11 did with BIM present. For those neurons that responded to epinephrine, there was a mean increase of 49% in the peak current for the controls compared with 32% with BIM present. Therefore, inhibition of PKC caused both a decrease in the number of neurons responding to epinephrine and a decrease in the mean magnitude of the response.



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Fig. 8. Inhibition of protein kinase C (PKC) reduces the potentiation of TTX-R INa by epinephrine. Mean normalized current during experiments in which the PKC inhibitor bisindolylmaleimide (BIM) was perfused into the chamber for 3 min before and during epinephrine (n = 11). Compared with epinephrine alone (n = 11), BIM significantly reduced the increase in TTX-R INa. (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The rapid onset of epinephrine hyperalgesia and the observation that neither sympathectomy nor indomethacin pretreatment affected it, is consistent with a direct action of epinephrine on sensory nerve terminals in the skin (Taiwo and Levine 1989b, 1990, 1992). The efficacy of our sympathectomy or indomethacin has been established in previous studies by observing an immediate increase in paw temperature of 2-3°C after sympathectomy as well as the loss of BK-induced hyperalgesia in the normal rat (Khasar et al. 1998; Miao et al. 1996). We have suggested earlier that small-diameter cultured DRG neurons are a good in vitro model for the study of nociceptors because they possess several properties of nociceptors (Gold et al. 1996a). Furthermore we have shown that agents, such as PGE2, that produce hyperalgesia by a direct action on primary afferents enhance TTX-R INa in cultured DRG neurons (Gold et al. 1996b). The observed potentiation of TTX-R INa by epinephrine may be the mechanism by which it sensitizes nociceptors (England et al. 1996; Gold et al. 1996b). The sensitivity of epinephrine- or isoproterenol-induced hyperalgesia to propranolol but not phentolamine is consistent with a beta -AR-mediated effect. These beta -ARs are most likely coupled to G proteins because epinephrine-induced hyperalgesia was attenuated by DAMGO. DAMGO previously has been shown to attenuate PGE2-induced hyperalgesia by activating a pertussis toxin-sensitive inhibitory G protein (Khasar et al. 1995b) and to prevent the potentiation of TTX-R INa by PGE2 (Gold and Levine 1996); a similar mechanism may explain inhibition of epinephrine-induced hyperalgesia. Downstream from receptor activation, the production of cAMP as well as activation of both PKA and PKC were necessary because their respective antagonists inhibited epinephrine-induced hyperalgesia.

The results of the in vitro experiments on DRG neurons are consistent with the behavioral data and definitively demonstrate a direct action of epinephrine on primary afferent neurons. Both the sensitization of small-diameter neurons and the potentiation of TTX-R INa by epinephrine, like behavioral hyperalgesia, were blocked by propranolol. The potentiation of TTX-R INa was mimicked by isoproterenol. Taken together, these results provide the first evidence for beta -ARs on DRG neurons as well as sensory nerve terminals. Potentiation of TTX-R INa by epinephrine was blocked by inhibition of PKA but also significantly attenuated by inhibition of PKC. The mechanical hyperalgesia, sensitization, and potentiation of TTX-R INa all occurred with a similar time course of onset after introduction of epinephrine, the effects peaking within 5 min.

Ferreira (1980) found that the methylxanthines, caffeine, and theophylline (which, among other actions, are inhibitors of phosphodiesterase, the enzyme that breaks down cAMP), potentiated the hyperalgesic effect of epinephrine and isoproterenol and concluded that the propranolol-sensitive hyperalgesia was mediated by the cAMP second-messenger system. Our data, from experiments using inhibitors of adenylyl cyclase, PKA and PKC, suggest that the beta -AR-mediated hyperalgesia is mediated by the PKC as well as the PKA second-messenger systems because PKC as well as PKA inhibitors significantly attenuated epinephrine hyperalgesia. PKC has been shown to play a role in nociceptor sensitization, both in vivo and in vitro (Cesare and McNaughton 1996; Leng et al. 1996). Inhibition of isoproterenol-induced hyperalgesia by BIM and WIPTIDE suggests that beta -AR activation can be mediated by both the PKA and the PKC second-messenger systems. Thus beta -AR activation alone is enough to account for the hyperalgesic action of epinephrine. The in vitro results agree with the behavioral data in that TTX-R INa potentiation by epinephrine was blocked by Rp-cAMPs and significantly attenuated by BIM.

Epinephrine does not play a significant role as a neurotransmitter in the periphery or as a local inflammatory mediator under normal circumstances. However, its release from the adrenal medulla (its main source in the periphery) is increased by acute stress (Taylor et al. 1989; Wortsman et al. 1984) as is the activity in sympathetic postganglionic neurons (Mazzeo et al. 1997). Under conditions of acute stress, circulating levels of epinephrine have been shown to increase dramatically in humans as well as in rats (Cryer 1980; DeTurck and Vogel 1980; Taylor et al. 1989; Wortsman et al. 1984) and stress has been shown to induce hyperalgesia (Kawanishi et al. 1997; Okano et al. 1997; Vidal and Jacob 1986). Although it remains to be determined whether the epinephrine levels attained under these circumstances actually contribute to primary afferent nociceptor sensitization and hyperalgesia, increases in circulating levels of epinephrine produced by stress have well-documented physiological effects (Cryer 1980; Wortsman et al. 1984). Epinephrine is the endogenous beta -AR agonist, and beta -AR antagonists can attenuate inflammation in humans with rheumatoid arthritis (Kaplan et al. 1980) and in adjuvant-induced arthritis in the rat (Coderre et al. 1990; Levine et al. 1988). Furthermore, it has been suggested that epinephrine can produce cardiac pain (angina) independent of its vasoconstrictor effects in patients with syndrome X who have normal coronary arteries (Eriksson et al. 1995). Certainly, effects of epinephrine and the role of beta -ARs in the mediation of pain and hyperalgesia deserve further study, both in the setting of nerve injury as well as inflammation, in which the sympathoadrenal axis may be activated.

In conclusion, our data suggest that epinephrine produces mechanical hyperalgesia in the rat and sensitizes cultured DRG neurons. These effects of epinephrine appear to be mediated by at least two second-messenger systems, cAMP/PKA and PKC.


    ACKNOWLEDGMENTS

We are grateful to Dr. Frederick Miao for performing surgical sympathectomies.

This study was supported by National Institute of Neurological Disorders and Stroke Grants NS-21647 and NS-21445 and The Peninsula Foundation.


    FOOTNOTES

Address for reprint requests: J. D. Levine, NIH Pain Center, Box 0440, C-522, University of California, San Francisco, CA 94143-0440.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 30 April 1998; accepted in final form 20 November 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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