Sex hormones regulate the contribution of PKCe and PKA signalling in in¯ammatory pain in the rat Olayinka A. Dina, K. O. Aley, William Isenberg, Robert O. Messing 1 and Jon D. Levine Departments of Medicine and Oral and Maxillofacial Surgery, Division of Neuroscience and Biomedical Sciences Program, NIH Pain Center (UCSF), University of California at San, Francisco, San Francisco, CA 94143±0440, USA 1 Department of Neurology, University of California at San Francisco and Ernest Gallo Clinic and Research Center, 5858 Horton Street, Suite 200, Emeryville, CA 94608, USA Keywords: cutaneous hyperalgesia, epinephrine, gender, oestrogen, primary afferent nociceptors Abstract We have evaluated the contribution of differences in second messenger signalling to sex differences in in¯ammatory pain and its control by sex hormones. In normal male but not female rats, epinephrine-induced mechanical hyperalgesia was antagonized by inhibitors of protein kinase Ce (PKCe), protein kinase A (PKA) and nitric oxide synthetase (NOS). Similarly, in PKCe knockout mice, a contribution of PKCe to epinephrine-dependent mechanical hyperalgesia occurred in males only. In contrast, hyperalgesia induced by prostaglandin E 2 , in both females and males, was dependent on PKA and NO. In both sexes, inhibitors of mitogen- activated protein kinase/extracellular-signal related kinase kinase (MEK) inhibited epinephrine hyperalgesia. In gonadectomized females, the second messenger contributions to epinephrine hyperalgesia demonstrated the pattern seen in males. Administration of oestrogen to gonadectomized females fully reconstituted the phenotype of the normal female. These data demonstrate gender differences in PKCe, PKA and NO signalling in epinephrine-induced hyperalgesia which are oestrogen dependent and appear to be exerted at the level of the b-adrenergic receptor or the G-protein to which it is coupled. Introduction Gender and sex hormone-related differences in pain (Romero & Bodnar, 1986; Fillingim & Maixner, 1995; Unruh, 1996) and nociception (Pare, 1969; Kepler et al., 1989; Aloisi et al., 1994; Coyle et al., 1995) have been described, and an important role for sex steroids (Beatty & Beatty, 1970; Marks et al., 1972; Romero et al., 1988; Baamonde et al., 1989; Candido et al., 1992; Dawson-Basoa & Gintzler, 1993) has been suggested. Whilst most of the literature in this area has addressed the modulatory role of sex steroids on CNS mechanisms of nociception, actions at the level of peripheral nociceptive mechanisms are also probable since both oestrogen and androgen receptors are present on small-diameter dorsal root ganglion (DRG) neurons (Sohrabji et al., 1994; Papka et al., 1997; Keast & Gleeson, 1998). Moreover, in DRG neurons oestrogen regulates the expression of mRNAs encoding trkA and p75 receptors (Sohrabji et al., 1994) through which NGF signals (Kaplan et al., 1991) and presumably acts to produce its pronociceptive effects (Woolf, 1996; Okuse et al., 1997). Sex hormones also affect expression of protein kinase C (PKC) (including the e isoform, PKCe), protein kinase A (PKA) (Ansonoff & Etgen, 1998; Lavie et al., 1998; Kelly et al., 1999; Han et al., 2000) and nitric oxide synthetase activity, all of which are implicated in peripheral nociceptive mechanisms (Khasar et al., 1995; Aley et al., 1998; Aley & Levine, 1999; Khasar et al., 1999a). Whilst a mitogen-activated protein kinase/extracellular- signal related kinase kinase (MEK) second messenger signalling pathway has recently been shown to also contribute to epinephrine- induced hyperalgesia, these experiments were only performed in male rats (Aley, K.O., Martin, A., McMahon, T., Levine, J.D. & Messing, R.O., unpublished results). Taken together, these observations suggest that sex hormones and oestrogen in particular may directly in¯uence the function of primary afferent nociceptors. Epinephrine induces hyperalgesia that is mediated by PKCe and PKA. Since this action of epinephrine is mediated by action at b 2 -adrenergic receptors (Khasar et al., 1999b), whose density, agonist af®nity and coupling to second messengers is controlled by sex hormones (Hatjis et al., 1989; Shima, 1992; Ungar et al., 1993; Xu et al., 1993; Alonso et al., 1995; Yie & Brown, 1995), we tested the hypothesis that sex hormones regulate the contribution of PKCe- and PKA-mediated signalling in epinephrine-induced in¯ammatory pain. Our ®ndings indicate that PKCe, PKA and nitric oxide (NO) signalling pathways contribute to epinephrine-induced hyperalgesia in males but not in females, due to suppression by oestrogen, whilst MEK contributes in both sexes. Materials and methods Animals Behavioural experiments were performed on male and female Sprague-Dawley rats (Bantin and Kingman, Fremont, CA, USA). Twenty-one day-old rats of either sex were gonadectomized and used in experiments when they were adults. In all other cases, same aged adult (250±350 g) rats were used. Male and female mice, wild-type or lacking expression of PKCe (Khasar et al., 1999a), were employed in studies of mechanical nociception. Animals were housed in a controlled environment in the Animal Care Facility of the Correspondence: Dr Jon D. Levine, NIH Pain Center (UCSF), C522/Box 0440, 521 Parnassus Ave, University of California at San Francisco, San Francisco, CA 94143±0440, USA E-mail: levine@itsa.ucsf.edu Received 16 January 2001, revised 16 April 2001, accepted 20 April 2001 European Journal of Neuroscience, Vol. 13, pp. 2227±2233, 2001 ã Federation of European Neuroscience Societies