Changes in regulator of G protein signaling-4 gene expression in the spinal cord of adrenalectomized rats in response to intrathecal morphine

Introduction: Regulators of G-protein signaling protein negatively control G protein -coupled receptor signaling duration by accelerating Gα subunit guanosine triphosphate hydrolysis . Since regulator of G-protein signaling4 has an important role in modulating morphine effects at the cellular level and the exact mechanism(s) of adrenalectomy-induced morphine sensitization have not been fully clarified, the present study was designed to determine the changes in the levels of RGS4 mRNA and protein in intact and adrenalectomized (ADX) morphine-treated rats. Materials and Methods: All experiments were carried out on male Wistar rats. The tail-flick test was used to assess the nociceptive threshold and corticosterone levels were measured by radioimmunoassay. The dorsal half of the lumbar spinal cord was assayed for the expression of RGS4 using semi-quantitative RT-PCR and immunoblotting. Results: Results showed that the anti-nociceptive effect of intrathecal morphine (5 μg) was significantly increased in ADX rats. The levels of RGS4 mRNA and protein in ADX rats were similar to those in intact animals. However, morphine could elicit a significant increase in both mRNA and protein levels of RGS4 following adrenalectomy. In contrast, the pattern of RGS4 gene expression did not show significant changes in the lumbar spinal cord of intact animals after morphine injection. Conclusion: Our results demonstrate that in the absence of corticosterone, morphine increases RGS4 through promoting its gene expression. 39 | Physiol Pharmacol 19 (2015) 38-45 Esmaeili-Mahani et al. All three opioid receptor subtypes (μ, δ and κ) belong to the superfamily of G-protein coupled receptors (GPCRs). Opioid receptors induce responses in the nervous system via coupling to Gαi/o proteins (Williams et al., 2001), and their activation results in adenylyl cyclase inhibition and/or the regulation of various ion channels and other effectors, leading to hyperpolarization of cells, thereby exerting an inhibitory effect (Law et al., 2000, Nestler, 2004). However, the GPCRs do not act in isolation and their activities are modulated by several accessory proteins such as arrestins, kinases, and regulators of G protein signaling (RGS) proteins (Lamberts and R Traynor, 2013). RGS proteins are a large family of highly diverse, multifunctional signaling proteins, which share a conserved signature domain (RGS domain) that binds directly to the activated Gα subunits and modulate G protein signaling. These proteins are responsible for Gα binding and markedly regulate the GTPase activity of Gα subunits leading to their deactivation and termination of downstream signals (Baltoumas et al., 2013). RGS proteins, the negative regulators of G proteins, can also modulate μ-opioid receptor (MOR) signaling and play a controlling role in the effectiveness of opioid receptor ligands both in vitro and in vivo (Clark et al., 2003, Wang et al., 2009, Lamberts and R Traynor, 2013) Several members of RGSs, in particular RGS4 and RGS9-2 have been demonstrated to affect MOR signaling and morphine-induced behaviors, including analgesia and reward. In addition, morphine modulates the expression levels of RGS proteins, especially RGS4 in a tissue and time dependent manner (Traynor, 2012). It has been demonstrated that G-protein subunits are under the coordinated control of glucocorticoids in the rat brain and are the physiological targets of glucocorticoids (Saito et al., 1989). Furthermore, glucocorticoids can regulate RGS4 expression levels in nerve tissue (Ni et al., 1999). Since RGS4 proteins have an important role in the termination of G-protein coupled receptors such as opioid receptors and glucocorticoids have regulating role on RGS4 expression levels the present study was designed to determine the impact of the changes in the levels of RGS4 mRNA and protein in adrenalectomyinduced potentiation of morphine analgesia. Materials and methods