A novel nociceptor signaling pathway revealed in protein kinase C epsilon mutant mice.

There is great interest in discovering new targets for pain therapy since current methods of analgesia are often only partially successful. Although protein kinase C (PKC) enhances nociceptor function, it is not known which PKC isozymes contribute. Here, we show that epinephrine-induced mechanical and thermal hyperalgesia and acetic acid-associated hyperalgesia are markedly attenuated in PKCepsilon mutant mice, but baseline nociceptive thresholds are normal. Moreover, epinephrine-, carrageenan-, and nerve growth factor- (NGF-) induced hyperalgesia in normal rats, and epinephrine-induced enhancement of tetrodotoxin-resistant Na+ current (TTX-R I(Na)) in cultured rat dorsal root ganglion (DRG) neurons, are inhibited by a PKCepsilon-selective inhibitor peptide. Our findings indicate that PKCepsilon regulates nociceptor function and suggest that PKCepsilon inhibitors could prove useful in the treatment of pain.

Comparison of prostaglandin E1- and prostaglandin E2-induced hyperalgesia in the rat.

We have studied prostaglandin E1-induced mechanical hyperalgesia in the rat hindpaw, by assessing paw-withdrawal thresholds, before and after injecting prostaglandin E1 alone or with other agents, in normal and streptozotocin-induced diabetic rats. In normal and diabetic rats, prostaglandin E1 (1-1000 ng) produced a dose-dependent decrease in mechanical nociceptive threshold. In diabetic rats, prostaglandin E1 was more potent than in normal rats, in producing hyperalgesia, whereas prostaglandin E2 hyperalgesia was not changed in normal and diabetic rats. Prostaglandin E1-induced hyperalgesia was not inhibited by E-type 1 prostaglandin receptor antagonists, SC19220 or SC51089, either in normal or diabetic rats. In fact, in the presence of SC19220, prostaglandin E1 produced enhanced hyperalgesia, in normal rats. Prostaglandin E1 hyperalgesia was not significantly modified by sympathectomy or indomethacin. Unlike prostaglandin E2, prostaglandin E1 hyperalgesia was not blocked by the inhibitor of the stimulatory guanine nucleotide-binding regulatory protein, guanosine 5'-O-(2-thiodiphosphate). It is suggested that prostaglandin E1 decreases primary afferent nociceptive threshold directly, by activating a prostaglandin receptor other than the E-type 1 prostaglandin receptor, and that this receptor is not coupled to a stimulatory guanine nucleotide-binding regulatory protein.

Peripheral nociceptive effects of alpha 2-adrenergic receptor agonists in the rat.

We have previously shown that norepinephrine can produce hyperalgesia via an alpha 2-adrenergic receptor mechanism. The alpha 2-adrenergic receptor agonist clonidine has, however, also been shown to produce peripheral analgesia. In view of the multiple alpha 2-subtypes currently known (i.e. alpha 2A, alpha 2B and alpha 2C), we evaluate the alpha 2-receptor subtypes mediating norepinephrine-induced peripheral hyperalgesia and clonidine analgesia. Norepinephrine and the alpha 2-adrenergic agonists clonidine and UK 14,304 (1-1000 ng), when co-injected with the calcium ionophore A23187 (1000 ng) produced dose-dependent hyperalgesia in the Randall-Selitto paw withdrawal test. Norepinephrine (100 ng) hyperalgesia was dose-dependently antagonized by alpha 2-adrenergic receptor antagonists. From the estimated ID50, the rank order of potency was: SK&F 104856 (alpha 2B) approximately imiloxan (alpha 2B) > rauwolscine (alpha 2C) >> BRL 44408 (alpha 2A). Norepinephrine hyperalgesia was not significantly affected by pertussis-toxin treatment. Prostaglandin E2 (100 ng) hyperalgesia was inhibited dose-dependently, by clonidine and UK 14,304. Rauwolscine was more potent in reversing the inhibitory effect of clonidine on prostaglandin E2 than imiloxan while BRL 44408 was ineffective. The inhibitory effect of clonidine on prostaglandin E2 hyperalgesia was reversed by pertussis toxin. These data suggest that alpha 2B-subtype receptors mediate (norepinephrine hyperalgesia while the antinociceptive effect of alpha 2-agonist is mediated by the alpha 2C-subtype receptor. Differential coupling of these receptor subtypes to second messenger systems and location on different cell types in the rat paw may explain, at least in part, their differential responses to alpha 2-agonist stimulation, leading to hyperalgesia and analgesia.

Inflammation modulates the contribution of receptor-subtypes to bradykinin-induced hyperalgesia in the rat.

While B2 receptors mediate pain and hyperalgesia induced by bradykinin, in normal rats, recent reports indicate that, in the setting of inflammation, B1 receptors also mediate pain and hyperalgesia. Since bradykinin-induced hyperalgesia in normal rats is mediated by prostaglandins released from the postganglionic sympathetic neurons, we have evaluated the contribution of the sympathetic nervous system to the hyperalgesia induced by bradykinin, a preferential B2-receptor agonist, and des-Arg9-bradykinin, a major metabolite of bradykinin and a selective B1-receptor agonist. Mechanical hyperalgesia was quantified by the Randall-Selitto paw-withdrawal method. Inflammation was induced by injecting Complete Freund's Adjuvant into the left hindpaw of the rat and testing mechanical nociceptive threshold in the right hindpaw after injecting B1 or B2 agonists and/or antagonists. Sympathectomy was achieved by surgically removing sympathetic ganglia L1-L4. Rats were used 48 h post-adjuvant injection. In the normal rat, intradermal injection of bradykinin but not des-Arg9-bradykinin, into the dorsal surface of the hindpaw, produced a dose-dependent decrease in mechanical nociceptive threshold. NPC 17731, a B2-receptor antagonist, but not des-Arg9-[Leu8]-bradykinin, a B1-receptor antagonist, almost completely inhibited the decrease in mechanical threshold, suggesting that bradykinin hyperalgesia in the normal rat hindpaw was mediated by B2 receptors. In rats whose left paws were treated, 48 h earlier, with adjuvant, intradermal injection of bradykinin or des-Arg9-bradykinin, into the right paw produced dose-dependent hyperalgesia. Bradykinin hyperalgesia was partially inhibited by NPC 17731, and the residual part by des-Arg9,[Leu8]-bradykinin. des-Arg9-bradykinin hyperalgesia was inhibited by des-Arg9,[Leu8]-bradykinin but not by NPC17731. These results suggest that in the setting of inflammation, bradykinin hyperalgesia was mediated by both B1 and B2 receptors, and that des-Arg9-bradykinin hyperalgesia was mediated by the B1 receptor. Forty-eight hours after injection of complete Freund's adjuvant, in sympathectomized rats, bradykinin or des-Arg9-bradykinin failed to produce hyperalgesia, suggesting that intact sympathetic postganglionic neurons are required for the hyperalgesia produced by these agents in this model. These results are consistent with the suggestions that B2 receptors mediate bradykinin-induced cutaneous hyperalgesia in the normal rat hindpaw. The hyperalgesia induced by bradykinin, 48 h post injection of complete Freund's adjuvant is mediated by both B1 and B2 receptors, that by des-Arg9-bradykinin is mediated by B1 receptors. The hyperalgesia induced by both agents is dependent on the presence of intact sympathetic postganglionic neurons.

Multiple second messenger systems act sequentially to mediate rolipram-induced prolongation of prostaglandin E2-induced mechanical hyperalgesia in the rat.

In this study we have evaluated the mechanisms mediating the prolonged hyperalgesia induced by administration of prostaglandin E2 plus rolipram, an inhibitor of type IV phosphodiesterase. The Randall-Selitto paw pressure device was employed to measure the effect of intradermal injection of test agents on the time course of the decrease in mechanical nociceptive threshold produced by prostaglandin E2 plus rolipram in the hairy skin of the hindpaw of the rat. The intradermal injection of prostaglandin E2 produced a dose-dependent decrease in the nociceptive threshold which lasted approximately 2 h. While rolipram alone had no significant effect on nociceptive threshold, it enhanced and prolonged (> 72 h) prostaglandin E2-induced hyperalgesia. WIPTIDE, a protein kinase A inhibitor, when administered 30 min after prostaglandin E2, or with prostaglandin E2 plus rolipram, a time when prostaglandin E2-induced hyperalgesia was at its peak, produced a significant reduction in hyperalgesia. However, at 90 or at 180 min after injection of prostaglandin E2 plus rolipram, WIPTIDE was found to be without effect. H-8, a protein kinase G inhibitor, and okadaic acid, a protein phosphatase inhibitor, when administered 30 min after prostaglandin E2, or 180 min after prostaglandin E2 plus rolipram, produced no significant effect. However, when administered 90 min after prostaglandin E2 plus rolipram, each produced a significant reduction in the hyperalgesia induced by prostaglandin E2 plus rolipram.(ABSTRACT TRUNCATED AT 250 WORDS)

Sensitization of C-fibres by prostaglandin E2 in the rat is inhibited by guanosine 5'-O-(2-thiodiphosphate), 2',5'-dideoxyadenosine and Walsh inhibitor peptide.

Behavioral studies have shown that mechanical hyperalgesia induced by intradermal injection of prostaglandin E2 is blocked by inhibitors of the cAMP second messenger system. Similarly, injection of prostaglandin E2 also induces a decrease in mechanical threshold and an increase in the number of action potentials elicited by test stimuli in most C-fibre nociceptors. This change is called sensitization. To further evaluate the degree of correlation between primary afferent sensitization and mechanical hyperalgesia, we conducted a study to evaluate the effect of agents known to block the cAMP second messenger system and behavioral manifestations of mechanical hyperalgesia following injection of prostaglandin E2. The agents tested were guanosine 5'-O-(2-thiodiphosphate), an inhibitor of stimulatory guanine nucleotide-binding regulatory proteins; 2',5'-dideoxyadenosine, an inhibitor of adenylyl cyclase; and Walsh Inhibitor Peptide, an inhibitor of cAMP-dependent protein kinase. Single fibre electrophysiologic studies of 138 C-fibres, innervating the dorsum of the hind paw, was done in male Sprague-Dawley rats. The number of spikes evoked by a 10 s application of a threshold von Frey hair were determined before and after intradermal injection of test agents administered alone and in combination with prostaglandin E2. Injection of prostaglandin E2 with the test agent vehicle (saline or distilled water) resulted in a significant decrease in von Frey hair threshold and an increase in the number of spikes generated in response to threshold von Frey hairs. In contrast, co-injection of prostaglandin E2 with guanosine-5'-O-(2-thiodiphosphate), 2',5'-dideoxyadenosine or Walsh inhibitor peptide did not result in a significant decrease in von Frey hair mechanical threshold or increase in the number of spikes generated to the threshold stimuli, compared with vehicle/prostaglandin E2. It is suggested that guanosine 5'-O-(2-thiodiphosphate), 2',5'-dideoxyadenosine and Walsh inhibitor protein inhibited prostaglandin E2 sensitization of primary afferent C-fibres by inhibiting a stimulatory guanine nucleotide-binding regulatory protein, adenylyl cyclase, and protein kinase A, respectively. These results support the hypothesis that primary afferent sensitization by prostaglandin E2 underlies prostaglandin E2-induced hyperalgesia.

Fasting is a physiological stimulus of vagus-mediated enhancement of nociception in the female rat.

The vagus nerve modulates nociception by a mechanism dependent upon gonadal hormones and the adrenal medulla. In the present study we tested the hypothesis that this modulation is dynamically controlled by physiological stimulation of structures innervated by the subdiaphragmatic vagus. Specifically, food deprivation (fasting) was employed to increase activity in the subdiaphragmatic vagus, and the experiments were performed mainly in female rats because our previous observations suggested that baseline activity in the pathway is lower in females than in males. Consistent with the hypothesis, after a 48-h fast, female rats exhibited increased nociceptive behavior in the formalin test. In contrast, fasting had no effect on formalin-evoked nociceptive behavior in male rats. The fasting-induced effect on nociception appears to be mediated by the vagus nerve since it is prevented by subdiaphragmatic vagotomy. Also similar to the previously characterized vagus-mediated modulation, the effect of fasting in the female is blocked by gonadectomy or adrenal medullectomy, and hormone replacement with 17beta-estradiol in gonadectomized female rats restored the effect of fasting. Decreased glucose metabolism apparently does not play a significant role in the effect of fasting on nociception, since the effect was unchanged when 5% glucose was provided in the drinking water throughout the fasting period. On the other hand, increasing the bulk content of the stomach (without providing nutrients) by infusion of petrolatum significantly attenuated the effect of fasting during the interphase period of the formalin response, suggesting that decreased gut distention, and possibly motility, are important in fasting-induced enhancement of nociception. These results indicate that fasting is a physiological activator of the vagus-mediated pain modulation pathway. This suggests the possibility that, especially in females, natural periodic changes in gut distention and motility may control an ongoing vagus-mediated adjustment in the organism's nociceptive sensitivity.

Is there more than one prostaglandin E receptor subtype mediating hyperalgesia in the rat hindpaw?

Five synthetic prostaglandin E analogs (11-deoxyPGE1, 17-phenyl-ol-trinor prostaglandin E2, enisoprost, MB28767 and misoprostol) have been evaluated for their ability to produce mechanical hyperalgesia in rats. The Randall-Selitto paw withdrawal model of mechanical hyperalgesia was used. Following intradermal injections (2.5 microliters) into the dorsal surface of the hindpaw, each prostaglandin E analog produced a dose-dependent (1-1000 ng) decrease in nociceptive threshold (i.e. hyperalgesia). Hyperalgesia produced by 17-phenyl-ol-trinor prostaglandin E2 and MB28767, was inhibited by the prostaglandin E1 antagonist SC19220 (7.5 ng), while the hyperalgesia produced by 11-deoxyprostaglandin E1, enisoprost and misoprostol was not inhibited by this antagonist. Hyperalgesia produced by all five analogs was significantly attenuated or completely blocked by inhibiting stimulatory guanine nucleotide-binding regulatory protein with guanosine 5'-O-(2-thiodiphosphate), adenylyl cyclase with 2'5'-dideoxyadenosine and protein kinase A with WIPTIDE. These results suggest the presence of more than one prostaglandin E-receptor subtype, which mediate hyperalgesia, predominantly via the cAMP second messenger system, in the hindpaw of the rat.

Mu-opioid agonist enhancement of prostaglandin-induced hyperalgesia in the rat: a G-protein beta gamma subunit-mediated effect?

Guanine nucleotide-binding regulatory protein stimulation of adenylyl cyclase has been shown to be an important second messenger system for many processes, including mechanical hyperalgesia. Recently, interactions between guanine nucleotide-binding regulatory protein subunits and adenylyl cyclase affecting the level of cyclic adenosine 3',5'-monophosphate accumulation have been demonstrated. In this study we evaluated such an interaction by measuring paw-withdrawal thresholds to mechanical stimuli in Sprague-Dawley rats in the presence of two direct-acting hyperalgesic agents, prostaglandin E2 and the adenosine A2-agonist, CGS21680. The effects of two agents expected to liberate inhibitory guanine nucleotide-binding regulatory protein subunits were also studied: [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (a mu-opioid receptor agonist) and N6-cyclopentyladenosine (an A1-adenosine agonist). Injection of [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin immediately before prostaglandin E2 or CGS21680 significantly attenuated the hyperalgesia subsequently induced by these agents, i.e. the sensitivity to these hyperalgesic agents was decreased. On the other hand, injection of [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin 5 min after prostaglandin E2 or CGS21680 significantly enhanced the hyperalgesia observed. Injection of the adenosine A1-agonist N6-cyclopentyladenosine immediately before and 5 min after prostaglandin E2 or CGS21680 had a similar effect to [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin. The decrease in sensitivity to prostaglandin E2- and CGS21680-induced hyperalgesia by preadministration of [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin or N6-cyclopentyladenosine and the enhancement by postadministration were all reversed by pertussis toxin, an inhibitor of inhibitory guanine nucleotide-binding regulatory protein, suggesting the involvement of an inhibitory guanine nucleotide-binding regulatory protein.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of vagal visceral afferents in the control of nociception.

We have shown that activity in subdiaphragmatic vagal afferents modulates mechanical hyperalgesic behavior in the rat. Subdiaphragmatic vagotomy decreases paw-withdrawal threshold to mechanical stimulation (baseline and after intradermal injection of bradykinin), thus enhancing mechanical hyperalgesic behavior. Most of this decrease is generated by an endocrine signal released by the adrenal medullae because denervation or removal of the adrenal medullae prevents or reverses these changes. This novel mechanism may imply that: (a) the brain is able to regulate sensitivity of nociceptors all over the body by a neuroendocrine mechanisms, (b) sensitivity of nociceptors can be influenced by changes in parts of the body which are remote from the location of the sensitized nociceptors and (c) circulating catecholamines can influence nociceptors in a way which is different from those reported so far (see Jänig and McLachlan, 1994; Jänig, 1996a; Jänig et al., 1996).

Gender and gonadal hormone effects on vagal modulation of tonic nociception.

We studied the influence of gender and gonadal hormones on modulation of tonic nociception exerted by vagal activity. In male rats, subdiaphragmatic vagotomy resulted in significantly reduced nociceptive behavior during phase 2 of the formalin test. Whereas gonadectomy alone had no effect, it completely eliminated the suppressive effect of subdiaphragmatic vagotomy; however, sex hormone replacement with either testosterone or dihydrotestosterone did not restore the ability of subdiaphragmatic vagotomy to suppress nociceptive behavior. These results suggest that, in males, a gonad-dependent but androgenic gonadal hormone-independent mechanism contributes to pronociceptive effects of vagal afferent activity. Although neither gonadectomy nor subdiaphragmatic vagotomy alone affected the response to formalin in females, gonadectomy plus vagotomy resulted in significantly reduced nociceptive behavior during phase 2. Reconstitution with 17 beta-estradiol implants in gonadectomized females not only prevented suppression of nociceptive behavior seen with gonadectomy plus vagotomy, but also led to increased nociceptive behavior in the interphase between phases 1 and 2. However, placement of 17 beta-estradiol implants in gonad-intact females had no effect on formalin-induced nociceptive behavior. The finding that estrogen produced an increase in nociceptive behavior in gonadectomized female rats after vagotomy but not in normal female rats (with intact gonads and subdiaphragmatic vagus) suggests that the interaction between estrogen and nociceptive afferent activity is suppressed by vagal function. In conclusion, a nonandrogenic action of testicular function in male rats and estrogen in females seems to influence the effect of vagal activity on formalin-induced nociceptive behavior.

Neonatal capsaicin attenuates mechanical nociception in the rat.

The contribution of chemosensitive neurons to mechanical nociception and hyperalgesia was studied by evaluating mechanical nociceptive threshold and the effect of three directly-acting hyperalgesic agents (prostaglandin E2, prostaglandin E1 and the A2-adenosine agonist, CGS21680) in rats treated neonatally with capsaicin. Mechanical nociceptive threshold was quantified by the Randall-Selitto paw-withdrawal method. The baseline mechanical paw-withdrawal threshold of the capsaicin-treated rats was 40% higher than that of the untreated rats. In the capsaicin-treated rats mechanical hyperalgesia was not induced by prostaglandin E2, prostaglandin E1 or CGS21680. These results are consistent with the suggestion that mechanical nociception and hyperalgesia induced by inflammatory substances is mediated by action on capsaicin-sensitive nociceptors.

Comparison of intradermal and subcutaneous hyperalgesic effects of inflammatory mediators in the rat.

In recent studies, the superfusion of the corium side of the skin with inflammatory mediators failed to produce sensitization of nociceptors to mechanical stimuli. We have studied the effects of intradermal (i.d.) and subcutaneous (s.c.) injections of prostaglandin E2 (PGE2) and bradykinin (BK) in a behavioral model of hyperalgesia. PGE2 or BK was injected into the rat hind-paw, and paw-withdrawal thresholds in response to noxious mechanical stimulation before and after the drug were compared. Subcutaneous injection of PGE2 (1-1000 ng), a hyperalgesic inflammatory mediator, did not significantly alter paw-withdrawal thresholds, under the same conditions in which i.d. injections dose-dependently lowered paw-withdrawal thresholds. Similarly, BK (1-1000 ng), another hyperalgesic mediator, given s.c. failed to significantly alter paw-withdrawal thresholds while i.d. injections dose-dependently lowered paw-withdrawal thresholds. The prostaglandin E-type, EP1 receptor antagonist SC19220 (750 ng), given s.c. prior to PGE2 (i.d.) did not significantly change PGE2-induced hyperalgesia. However, SC19220 significantly attenuated PGE2 hyperalgesia when both were injected i.d. Also, s.c. administration of the mu-opioid antagonist, DAMGO, before PGE2 did not inhibit PGE2-induced hyperalgesia as opposed to i.d. injection. These results suggest that the inability of s.c. injection of PGE2 or BK to reach its receptor site on the terminals of primary afferent nociceptors may be responsible for the ineffectiveness of these hyperalgesic mediators to sensitize cutaneous nociceptors to mechanical stimuli in the rat and underscore the importance of the site of application and site of action of hyperalgesic agents in the study of hyperalgesic mechanisms.

Selective attenuation of mu-opioid receptor-mediated effects in rat sensory neurons by intrathecal administration of antisense oligodeoxynucleotides.

To test the hypothesis that the expression of specific proteins on peripheral terminals of primary afferents can be attenuated by intrathecal administration of antisense oligodeoxynucleotides (ODNs), we administered ODNs antisense to the mu-opioid receptor to male Sprague-Dawley rats via chronically implanted intrathecal cannulae. Antisense but not mismatch ODN treatment significantly decreased peripheral (D-Ala2, N-Me-Phe4, Gly5-ol)-enkephalin (DAMGO) inhibition of prostaglandin E2 (PGE2) hyperalgesia. Antisense treatment affected neither the magnitude of PGE2 hyperalgesia nor the antinociception produced by a peripherally administered adenosine A1-agonist. The antinociceptive effects of DAMGO was fully recovered 8 days after cessation of ODN treatment. DAMGO-induced inhibition of voltage-gated Ca2+ currents (VGCC), in cultured dorsal root ganglion (DRG) neurons from rats treated with ODNs, was also significantly reduced by antisense but not mismatch ODNs. Taken together, these observations suggest that intrathecal administration of antisense ODNs can be used to study the function of proteins present in the peripheral terminals of primary afferent neurons.

A tetrodotoxin-resistant sodium current mediates inflammatory pain in the rat.

We report evidence for a contribution of tetrodotoxin-resistant sodium current (TTX-R INa) to prostaglandin E2 (PGE2)-induced hyperalgesia. Behavioral experiments were performed in rats chronically implanted with spinal cannulae. The study employed intrathecal administration of oligodeoxynucleotide (ODN) antisense to the recently cloned channel underlying TTX-R INa (PN3/SNS). The nociceptive flexion reflex was employed to determine changes in mechanical stimulus-induced paw-withdrawal threshold. Administration of antisense but not of sense or mismatch ODN, led to a decrease in PGE2-induced hyperalgesia. PGE2-induced hyperalgesia returned to normal 7 days after the last injection of antisense ODN. Antisense ODN selectively and significantly reduced TTX-R INa current density in cultured sensory neurons. Our observations support the hypothesis that modulation of TTX-R INa, present in peripheral terminals of primary afferent nociceptors, contributes, at least in part, to inflammatory hyperalgesia. Since TTX-R INa is found only in primary afferent nociceptors, our findings suggest TTX-R INa as a promising target for novel therapeutic interventions for the treatment of inflammatory pain.

Tachyphylaxis develops to bradykinin-induced plasma extravasation in the rat.

Bradykinin, an inflammatory mediator produced from plasma kallikreins, has potent effects on vascular functions, including increasing plasma extravasation and vasodilation. Attenuation in the response (desensitization to maintained exposure or tachyphylaxis to repeated administration) to bradykinin actions on synovial vasculature, a critical variable with respect to the role of bradykinin in sustained or chronic synovial inflammation, has not been elucidated. In the present study, we determined if tachyphylaxis and desensitization for bradykinin-induced plasma extravasation in the knee joint occur. Bradykinin-induced plasma extravasation into the knee joint cavity was determined spectrophotometrically by measuring the concentration of Evans blue dye extravasation into the joint perfusate. To examine for the development of tachyphylaxis, perfusion of bradykinin (160 ng/ml) was repeated after a 40-min wash with normal saline. Continuous intra-articular perfusion of bradykinin produced an increase in plasma extravasation that remained relatively stable with only a small, approximately 15 percent, decrease over 170 min. On the other hand, the levels of plasma extravasation produced by intermittent perfusion of bradykinin were dramatically lower than that induced by the first exposure (i.e., tachyphylaxis). We conclude that bradykinin-induced plasma extravasation develops marked tachyphylaxis but only minimal desensitization.

Activation of Gi induces mechanical hyperalgesia poststress or inflammation.

In studies of the role of primary afferent nociceptor plasticity in the transition from acute to chronic pain we recently reported that exposure to unpredictable sound stress or a prior inflammatory response induces long-term changes in the second messenger signaling pathway, in nociceptors, mediating inflammatory hyperalgesia; this change involves a switch from a G(s)-cAMP-PKA to a G(i)-PKCepsilon signaling pathway. To more directly study the role of G(i) in mechanical hyperalgesia we evaluated the nociceptive effect of the G(i) activator, mastoparan. Intradermal injection of mastoparan in the rat hind paw induces dose-dependent (0.1 ng-1 microg) mechanical hyperalgesia. The highly selective inhibitors of G(i), pertussis toxin, and of protein kinase C epsilon (PKCepsilon), PKCepsilonV(1-2), both markedly attenuate mastoparan-induced hyperalgesia in stressed rats but had no effect on mastoparan-induced hyperalgesia in unstressed rats. Similar effects were observed, at the site of nociceptive testing, after recovery from carrageenan-induced inflammation. These studies provide further confirmation for a switch to a G(i)-activated and PKCepsilon-dependent signaling pathway in primary mechanical hyperalgesia, induced by stress or inflammation.

Epinephrine produces a beta-adrenergic receptor-mediated mechanical hyperalgesia and in vitro sensitization of rat nociceptors.

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 mu-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.

Vagotomy-induced enhancement of mechanical hyperalgesia in the rat is sympathoadrenal-mediated.

We have recently shown that subdiaphragmatic vagotomy enhances bradykinin-induced hyperalgesic behavior and decreases baseline paw withdrawal threshold to mechanical stimulation of the hindpaw skin in rats by a peripheral mechanism. To elucidate the underlying mechanism, we studied whether lesions of efferent neuroendocrine pathways could prevent or reverse the potentiating effect of vagotomy. In groups of sham-vagotomized or vagotomized rats, we surgically removed or denervated the adrenal medulla. Bradykinin was injected intradermally into the skin of the dorsal surface of the rat hindpaw. Threshold of paw withdrawal to mechanical stimulation of the skin was measured. Vagotomy induced a decrease in mechanical baseline paw withdrawal threshold and enhancement of bradykinin-induced mechanical hyperalgesic behavior, both of which were maintained over the 5 week testing period. Adrenal enucleation or denervation of the adrenal gland by suprarenal ganglionectomy prevented vagotomy-induced decrease in baseline paw withdrawal threshold and enhancement of bradykinin-induced hyperalgesia. In animals that had a demonstrated decrease in baseline paw withdrawal threshold and enhancement of bradykinin-induced hyperalgesia 2 weeks after vagotomy, additional denervation of the adrenal medulla significantly reversed these effects over a 3 week period. These results imply that both the decrease in baseline paw withdrawal threshold and enhancement of bradykinin-induced hyperalgesic behavior after vagotomy are dependent on a hormonal signal released from the adrenal medulla and suggest a novel mechanism of sensitization of cutaneous nociceptors.

Modulation of bradykinin-induced mechanical hyperalgesia in the rat by activity in abdominal vagal afferents.

Bradykinin-induced plasma extravasation and mechanical hyperalgesia are sympathetic-dependent components of inflammation. Noxious stimulation has been found to inhibit bradykinin-induced plasma extravasation by activating the hypothalamo-pituitary-adrenal axis. The sensitivity of this nociceptive-neuroendocrine feedback control of inflammation is modulated by activity in subdiaphragmatic vagal afferents. In the present study, we tested the hypothesis that activity in the subdiaphragmatic vagus also modifies bradykinin-induced mechanical hyperalgesia in the rat, using the Randall-Selitto method. Following subdiaphragmatic vagotomy, the baseline paw-withdrawal threshold to mechanical stimulation decreased and bradykinin-induced mechanical hyperalgesia was enhanced. Mechanical hyperalgesia produced by prostaglandin E2, a direct-acting hyperalgesic agent, was not significantly affected by vagotomy. The effect of subdiaphragmatic vagotomy on bradykinin-induced hyperalgesia, but not on baseline paw-withdrawal threshold, was mimicked by coeliac branch vagotomy. Indomethacin blocked the hyperalgesia in normal rats, but not in vagotomized rats, suggesting that bradykinin-induced hyperalgesia in normal rats is mediated by prostaglandins, whose role was unexpectedly diminished after vagotomy. Bradykinin-induced hyperalgesia in normal rats was abolished by lumbar sympathectomy but not by sympathetic decentralization (cutting the preganglionic axons). In rats that were both vagotomized and sympathectomized, hyperalgesia induced by low-dose bradykinin was no longer present. These results demonstrate that vagotomy induces a decrease in baseline mechanical paw-withdrawal threshold and an enhancement of bradykinin-induced mechanical hyperalgesia and suggest that these phenomena are generated by actions in peripheral tissues.