Elsevier

Neuropharmacology

Volume 43, Issue 5, October 2002, Pages 809-816
Neuropharmacology

Acute and chronic morphine treatments and morphine withdrawal differentially regulate GRK2 and GRK5 gene expression in rat brain

https://doi.org/10.1016/S0028-3908(02)00147-8Get rights and content

Abstract

Opioid agonist stimulates activation of G protein-coupled receptor kinase (GRK) and causes desensitization of opioid signaling, which plays an important role in opioid tolerance. The current study investigated the potential regulatory effects of acute and chronic morphine administration and withdrawal on GRK2 and GRK5 gene expression in rat brain. Our results showed that the initial morphine treatment (10 mg/kg) significantly increased GRK mRNA levels in cerebral cortex, hippocampus, and lateral thalamic nuclei. A significant decrease in GRK5 mRNA levels was observed in periaqueductal gray. In strong contrast, repeated administration of morphine for 9 days failed to cause any significant increase in GRK5 mRNA in any of these brain regions. Chronic morphine treatment resulted in 30–70% down-regulation of GRK2 expression in cerebral cortex, hippocampus, thalamus, and locus coeruleus, opposite to what observed with the single morphine administration. Moreover, spontaneous and naloxone-precipitated morphine withdrawal resulted in aberrant increases in GRK2 and GRK5 mRNA levels in these brain regions. Taken together, our study suggests that opioid not only induces rapid negative feedback regulation on opioid signals through activation of GRK but also exerts its impact, via controlling levels of GRK gene expression, on the regulatory machinery itself over a longer period of time in brain.

Introduction

G protein-coupled receptor (GPCR) kinase (GRK) is a family of serine/threonine kinases. GRKs are activated upon agonist stimulation and they subsequently catalyze phosphorylation of numerous GPCRs with various vital functions (Ferguson et al., 1996, Penn et al., 2000). The agonist-stimulated, GRK-induced GPCR phosphorylation is an initial step in the process of homologous desensitization of GPCRs and GRKs play an important role in regulation of GPCR-transduced signals. At least seven subtypes of GRKs (GRK1-7) have been cloned (Benovic and Gomez, 1993, Weiss et al., 1998, Penn et al., 2000). Among these GRKs, GRK2, GRK3, GRK5 and GRK6 are expressed in a wide range of tissues such as heart, brain, lung and placenta, while GRK1, GRK4 and GRK7 are expressed exclusively in retina and testis, respectively (Penn et al., 2000). Disruption of GRK2 gene is lethal to mice (Jaber et al., 1996). Transgenic animals overexpressing GRK2 or GRK5 in cardiac tissue showed attenuated β-adrenergic receptor-mediated signaling and cardiac contractility. Transgenic mice overexpressing GRK inhibitors exhibit the opposite effects. (Koch et al., 1995, Rockman et al., 1996, Chen et al., 2001). The wide expression of GRK2, GRK3, GRK5 and GRK6 in the most rat brain regions (Arriza et al., 1992, Erdtmann-Vourliotis et al., 2001) suggests that these GRKs may play important roles in regulation of neuronal signal transduction. However, studies on physiological functions and expression regulation of diverse GRK isoforms in central nervous system in response to GPCR agonist are lacking.

Opioid receptors, a family of G protein-coupled neuropeptide receptors, play essential roles in pain modulation and other neural functions in the central nervous system. Interaction of endogenous or synthetic opioids with opioid receptors produces strong analgesia and thus opiates are used as analgesics clinically. However, long-term application of opioid drugs results in desensitization of opioid receptors and opioid tolerance and dependence. The critical role of opioid induced desensitization of opioid receptors in opioid tolerance has been demonstrated (Bohn et al., 1999, Bohn et al., 2000).

Opioid-stimulated receptor phosphorylation is a critical step leading to desensitization and internalization of opioid receptor (Pei et al., 1995) and GRKs are important protein kinases in this process (Zhao et al., 1997, Zhang et al., 1998, Guo et al., 2000, Xiang et al., 2001). These results implicate that GRKs play an important role in the regulation of opioid-induced analgesia. Among the GRK isoforms expressed in brain, GRK2, GRK3 and GRK5 have been shown to be able to regulate the opioid signaling (Kovoor et al., 1998, Zhang et al., 1998). GRK2 and GRK3 share high structural homology and belong to the same GRK subfamily (Arriza et al., 1992). GRK2 is more abundant than GRK3 in brain and functions of GRK2 in regulating signaling of opioid receptor as well as other GPCRs are well characterized (Penn et al., 2000; Jaber et al., 1996; Arriza et al., 1992; Erdtmann-Vourliotis et al., 2001; Walker et al., 1999). Our earlier research has demonstrated that GRK2 and GRK5 phosphorylate opioid receptors in neural cells (Zhao et al., 1997). In the present research, we investigated the effects of acute and chronic morphine administration and morphine withdrawal on GRK2 and GRK5 gene expression in brain.

Section snippets

Animals

Male Sprague–Dawlay rats (160–200 g, Shanghai Experimental Animal Center, Chinese Academy Sciences) were housed at 20–25°C on a 12-h light/dark cycle with food and water freely accessible. All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals.

Morphine treatments

For acute treatment, rats were injected s.c. with saline or 10 mg/kg morphine hydrochloride (Shengyang Pharmaceutical Company) and the animals were sacrificed at

Expression of GRK2 and GRK5 genes in various regions of the brain

Our results from in situ hybridization with GRK subtype-specific probes revealed that GRK2 and GRK5 expressed in most regions of rat brain. GRK2- and GRK5-positive cells with high staining intensity were present in pyramidal cell layers of cingulate cortex, motor cortex, and somatosensory cortex, and CA1-CA3/4 fields and dentate gyrus of hippocampus formation (Fig. 1). GRK2- and GRK5-positive cells with moderate to high staining intensities were observed in the thalamic nuclei, mesencephalic

Discussion

In this study, we have shown that initial morphine treatment induced significant up-regulation of GRK2 and GRK5 gene expression within hours in many brain regions including cerebral cortex, hippocampus and thalamus. But chronic administration of morphine at the same dosage failed to elevate GRK5 mRNA levels in these regions and in strong contrast to what observed with single administration of morphine, it down-regulated the transcript levels of GRK2 (60–70% lower than saline-treated control

Acknowledgements

We thank Dr Lin Lu for helpful discussion and Yan Liu, Shiduo Lu and Yingchun Cai for technical help. This work was supported in part by grants from the National Natural Science Foundation of China (39825110 and 30000050), the Ministry of Science and Technology (G1999054003), the Ministry of Education, and Shanghai Municipal Commission for Science and Technology.

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