Regulation Of GRK2, GRK5 And JNK3 Gene Expression In Rat Brain By Morphine

Prolonged exposure to opiate induces opioid tolerance, dependence and addiction, but the mechanisms responsible for these side-effects have not been well elucidated. Many studies have revealed that desensitization of opioid receptor is one of the molecular mechanisms of opioid tolerance. Abnormal regulation of gene expression, changes in plasticity of neuron and impairment of brain structure play important roles in the development of opioid addiction.The phosphorylation of G protein-coupled receptor (GPCR) by G protein-coupled receptor kinases (GRKs) under the stimulation with their specific agonists is the critical step to desensitization of GPCR. Agonists for GPCR can regulate the expression level of GRK, and the abnormal expression of GRK in vivo can markedly influence physiological functions of organs and tissues. Both GRK2 and GRKS are the main isoforms of GRK in the phosphorylation of opioid receptors but the expression regulations of GRK2 and GRK5 in brain by opiates are unclear. JNKs are the primary kinases in the phosphorylation of c-Jun which isan important transcription factor in the regulation of gene expression. Three isoforms of JNKs have been cloned. JNK3 is characterized with its specific expression in brain and exerts pivotal roles in neuron apoptosis. Many research demonstrated that morphine could induce apoptosis of immunocytes and neuronal cells in vitro, and morphological impairment of neuron in brain as well as modification of neuronal plasticity in vivo. But whether these effects of morphine on neuronal cells are associated with the regulation of JNKs is not clarified. Using in situ hybridization in this study, we investigated the regulation of gene expression of GRK2, GRK5 and JNKS in rat brain after acute and chronic treatments with morphine, spontaneous morphine withdrawal, naloxone-precipitated withdrawal and long-term cessation of morphine.The results showed that single injection of 10 mg/Kg morphine increased GRK2 mRNA level in cerebral cortex, hippocampus and thalamus, and GRKS mRNA level in cerebral cortex and hippocampus. Twice daily injection of 10 mg/Kg morphine for 9 days decreased GRK2 mRNA level in cerebral cortex, hippocampus, thalamus and locus coeruleus, but notGRK5 mRNA level in all the observed brain regions. Spontaneous withdrawal for 48 h and naloxone-precipitated withdrawal induced overshoot of bothGRK2 mRNA and GRK5 mRNA levels in most brain regions. Both acute and chronic morphine treatments enhanced JNK3 mRNA levels in frontal cortex, but not in hippocampus, thalamus and locus coeruleus. Long-term cessation of morphine for 2 weeks after chronic morphine treatment caused delayed increases in JNK3 mRNA levels in hippocampus and thalamus. A challenge injection of morphine could not modify the increased expression levels of JNK3 mRNA in these brain regions. The different distributions between GRK2 and GRK5 mRNA were also observed in periaqueductal gray (PAG) where GRK5 mRNA-positive cells are widely and uniformly distributed in the aqueduct with higher stain but GRK2 mRNA-positive cells only distribute around dorsal and ventral parts of the aqueduct with lower stain.Our results first demonstrated that opiate could regulate the gene expression of GRK2, GRK5 in transcription level. Acute and chronic morphine treatments as well as morphine withdrawal could differently regulate both GRK mRNA levels in most brain regions. The expression patterns of GRK2 and GRK5 mRNA differ in PAG.. Opiate could regulate the gene expression of JNK3. Acute and chronic morphine treatments increase the gene expression of JNK3 in frontal cortex, but not in hippocampus, thalamus and locus coeruleus. Morphine abstinence could cause delayed up-regulation of JNK.3 gene expression in hippocampus and thalamus. Our results suggest that the up-regulation of GRK2 and GRK5 mRNA by acute morphine treatment may play important roles in acute opioid tolerance. Chronic morphine treatment may initiate inhibitory mechanism to down-regulate GRK2 gene expression and negatively regulate GRK5 gene...