Activation of G_i induces Mechanical Hyperalgesia Post Stress or Inflammation

Introduction

A prior localized inflammatory response (Aley et al., 2000) or a period of unpredictable sound stress (Khasar et al., 2008) produce long-term changes in the duration and second messenger signaling pathway for inflammatory mediator-induced mechanical hyperalgesia. This change involves a switch from G_s to G_i in the G-protein that mediates inflammatory mediator-induced mechanical hyperalgesia (Khasar et al., 2008). While this type of switch in second messenger signaling pathways, from G_s to G_i, has previously been reported for β₂-adrenergic receptor (β₂AR) signaling in cardiac myocytes, caused by chronic exposure to receptor agonists such as epinephrine (Lamba and Abraham, 2000), in cardiac muscle cells this involves an increased G_i-mediated inhibition of adenylyl cyclase, which would be expected to attenuate rather than enhance mechanical hyperalgesia (Levine and Taiwo, 1989; Taiwo et al., 1989). In contrast, we observed that while prior inflammation at the site of nociceptive testing (Fig. 1) (Aley et al., 2000) and repeated unpredictable sound stress (Khasar et al., 2008) also cause a switch in signaling pathways in the primary afferent nociceptor, from dependence on G_s and cAMP/protein kinase A to dependence on G_i and protein kinase Cε (PKCε), in the nociceptor this switch is associated with enhanced rather than attenuated mechanical hyperalgesia. The emergence of PKCε dependence may be important to our understanding of mechanisms of chronic pain, as this novel protein kinase C (PKC) isoform plays a critical role in the phenomenon of “hyperalgesic priming,” a model of the transition from acute to chronic pain (Aley et al., 2000).

Having established an involvement of G_i in the switch to PKCε-dependence for prostaglandin E₂ hyperalgesia, in this study we evaluated whether activation of G_i is sufficient to produce mechanical hyperalgesia, if such hyperalgesia requires or is enhanced by prior inflammation at the site of nociceptive testing or by prior exposure to unpredictable stress, and if G_i-induced hyperalgesia is also PKCε dependent.

Methods

Results

While, for sound stress, we have previously shown a switch to G_i- as well as development of PKCε –dependence following recovery from prior inflammation we have not previously evaluated the switch to G_i dependence. In animals that had experienced prior inflammation at the site of nociceptive testing, prostaglandin E₂ (PGE₂) induced mechanical hyperalgesia was still unattenuated 4 hrs later (Fig. 1), a signature of hyperalgesic priming. This hyperalgesia was completely reversed by injection of Pertussis toxin (PTx; 100 ng/μl) at the site of nociceptive testing. In a control group of rats, recovered from prior inflammation, PTx alone had no effect on nociceptive threshold.

In the first experiments to evaluate the role of G_i in mechanical hyperalgesia, we found that the intradermal injection of mastoparan, in the hind paw, produced a dose-dependent decrease in mechanical nociceptive threshold (1 ng – 1 μg; n=6, each dose; Fig. 2). In rats that had been exposed to repeated unpredictable sound stress, with the last stress exposure 14 days prior, the dose-response curve for mastoparan-induced hyperalgesia was shifted to the left (p<0.001; Fig. 3A); the peak hyperalgesia was, however, unchanged (p=NS). Mastoparan-induced hyperalgesia, in both naïve and stressed rats was reversible, being no longer detectable 24 hrs after its administration (Fig. 3B).

While intradermal injection of the selective G_i inhibitor PTx (100 ng/μl) markedly attenuated mastoparan-induced hyperalgesia 14 days post stress (p<0.001; Fig. 4A), it had no effect on mastoparan-induced hyperalgesia in a control group of animals that had not been exposed to the sound stress protocol (p=NS; Fig. 4A). Of note, the decrease in mastoparan hyperalgesia produced by PTx in stressed rats, brought hyperalgesia to a level well below that induced by mastoparan in unstressed rats. A PKCε inhibitor, PKCεV_1–2 (1 μg) also markedly attenuated mastoparan-induced hyperalgesia, again this was observed in stressed rats but not in unstressed rats (Fig. 4B); in fact, at the low mastoparan dose the PKCε inhibitory, PKCεV_1–2 appears to enhance hyperalgesia in unstressed rats.

Similar to the effect of prior stress, rats that had previously experienced a localized inflammatory response, induced by carrageenan at the site of nociceptive testing, and had recovered to pre-carrageenan baseline mechanical nociceptive threshold (Aley et al., 2000), also demonstrated mastoparan-induced hyperalgesia that was inhibited by PTx and PKCεV_1–2, though the dose response curve for mastoparan-induced hyperalgesia was not shifted to the left (Fig. 5A,B, respectively), as it was by stress (Fig. 3A, 4A). Again, the magnitude of mastoparan-induced hyperalgesia, in the presence of PTx, was markedly less in rats that had experienced prior inflammation than the hyperalgesia induced by mastoparan (without PTx) in a control group of rats (i.e., in rats that had not experienced a prior inflammatory response, Fig. 4A).

Discussion

In the present study we evaluated the role of the ‘inhibitory’ heterotrimeric G- protein, G_i, in inflammatory mediator-induced mechanical hyperalgesia in the rat. We have implicated a switch in signaling, from G_s to G_i, and onset of PKCε dependence for the second messenger pathway mediating prostaglandin E₂–induced mechanical hyperalgesia, induced by prior stress (Khasar et al., 2008) or inflammation (Fig. 1; Aley et al., 2000). In the present study we evaluated whether activation of G_i was sufficient to produce PKCε-mediated mechanical hyperalgesia by determining if a G_i activator, mastoparan (Higashijima et al., 1990; Klinker et al., 1996; Chahdi et al., 2003), can alone produce mechanical hyperalgesia following prior stress or inflammation, and whether this mastoparan-induced hyperalgesia is PKCε dependent.

To demonstrate that mastoparan-induced hyperalgesia is mediated by G_i after but not before stress or inflammation, we administered PTx, a highly selective G_i inhibitor (Kaslow et al., 1987; Aktories, 1997). As predicted, the hyperalgesia induced by mastoparan post-stress or -inflammation is markedly attenuated by PTx. Thus, in the setting of prior stress or inflammation, activation of G_i appears to be sufficient to induce mechanical hyperalgesia. We also found that after stress or prior inflammation, the hyperalgesia induced by mastoparan like that induced by prostaglandin E₂ became PKCε dependent. Thus, the signaling pathway downstream of G_i similarly includes PKCε. These findings support the suggestion that as occurs for prostaglandin E₂-induced hyperalgesia, after stress and inflammation (Khasar et al., 2008) there is a switch to a GPCR-G_i-PKCε signaling pathway.

Of note, the reduction in mastoparan-induced hyperalgesia by PTx, in rats previously exposed to stress or inflammation, reduces hyperalgesia to a level well below that induced by mastoparan in control rats (see Figs. 4A and 5A). This is similar to what we have previously observed for prostaglandin E₂-induced hyperalgesia (Khasar et al., 2008). Thus this finding supports the existence of a switch in the second messenger- signaling pathway for inflammatory mediator-induced mechanical hyperalgesia, to G_i- PKCε. The finding that PTx had no effect on mastoparan-induced hyperalgesia in naïve control rats further confirms a switch from no contribution of G_i to G_i dependence, as observed for prostaglandin E₂-induced hyperalgesia after prior stress (Khasar et al., 2008) or inflammation (Fig. 1). While Ueda and colleagues have demonstrated inhibition of nociception induced pain (for review see Ueda, 2006) this switch in G-protein coupled receptor signaling from G_s to G_i, and appearance of G_i to PKCε signaling, have not been reported previously in neurons. Of note, however, such mechanisms are well established in another excitable tissue, the cardiac myocyte. Thus, in heart muscle cells, β₂- adrenergic receptor signaling can switch from G_s to G_i, (Daaka et al., 1997; Hasseldine et al., 2003; Hill and Baker, 2003; Magocsi et al., 2007) and G_i can signal to mediators known to be upstream of PKCε (Herrlich et al., 1996; Daaka et al., 1997; Steinberg, 1999; Mackay and Mochly-Rosen, 2001; Pavoine et al., 2003; Pavoine and Defer, 2005) or directly to PKCε (Fraser et al., 2000; Otani et al., 2003; Paruchuri and Sjolander, 2003).

While mastoparan did produce mechanical hyperalgesia in the absence of prior stress or inflammation, this hyperalgesia was not inhibited by PTx, and therefore not G_i- mediated, nor was it antagonized by a PKCε inhibitor. Of note in this regard, actions of mastoparan have been divided into PTx-sensitive, G_i-mediated, and PTx-insensitive, G_i- independent signaling pathways. Examples of G_i-independent mechanisms include: activation of MAP kinase (Miles et al., 2004), phospholipase C (Nakahata et al., 1990; Schnabel et al., 1997), phospholipase D (Mizuno et al., 1998; Chahdi et al., 2003) and two small molecular weight GTP-biding proteins rho and rac (Koch et al., 1991). Whether one of the previously described G_i-independent mechanisms of mastoparan action, or another signaling pathway, mediates the PTx-resistant hyperalgesia induced by mastoparan in the control rats not exposed to prior stress or inflammation remains to be elucidated. Alternatively, mastoparan may have activated G_i in the control rats but in the absence of prior stress or inflammation G_i is not coupled to PKCε, and therefore, cannot induce hyperalgesia via this pathway.

While the present study provides evidence to support the suggestion that stress and inflammation induce a neuroplastic change in the primary afferent nociceptor, in which G_i activation can induce PKCε-dependent mechanical hyperalgesia, the mechanism by which stress and inflammation induce these changes in the function of the primary afferent nociceptor, necessary to engage G_i-PKCε signaling, remains to be elucidated. We have previously shown that the effect of unpredictable sound stress on primary afferent nociceptor function, needed to produce the observed neuroplastic changes in second messenger signaling, are mediated by the action of glucocorticoids and catecholamine on glucocorticoid and β₂-adrenergic receptors, respectively, on primary afferent nociceptors (Khasar et al., 2008). That the production of hyperalgesic priming by prior stress or inflammation is mediated by changes in the primary afferent nociceptor is suggested by the fact that TNFα and IL-6, acting at their receptors on primary afferent nociceptors, each produce hyperalgesic priming (Parada et al., 2003; Dina et al., 2008).

In conclusion, the present experiments confirm the role of a G_i-PKCε-mediated signaling pathway in primary afferent nociceptor sensitization and mechanical hyperalgesia associated with prior stress and inflammation, that may occur at or below the level of the G_i heterotrimeric G-protein in the second messenger signaling pathway. It also provides support for a change in signaling pathway, a switch that is activated by stress or prior inflammation. Thus, G_i activity is sufficient for reproducing, as well as necessary in the pathway mediating the neuroplastic changes in the primary afferent nociceptor, which may contribute in the transition from acute to chronic pain. Whether the mechanisms described here impacts sensitization for other stimulus modalities (e.g. heat, cold and various chemicals) remains to be elucidated.