G protein-coupled receptor kinase 2 regulates mitochondrial bioenergetics and impairs myostatin-mediated autophagy in muscle cells.

G protein-coupled receptor kinase 2 (GRK2) is an important protein involved in ß-adrenergic receptor desensitization. In addition, studies have shown GRK2 can modulate different metabolic processes in the cell. For instance, GRK2 has been recently shown to promote mitochondrial biogenesis and increase ATP production. However, the role of GRK2 in skeletal muscle and the signaling mechanisms that regulate GRK2 remain poorly understood. Myostatin is a well-known myokine that has been shown to impair mitochondria function. Here, we have assessed the role of myostatin in regulating GRK2 and the subsequent downstream effect of myostatin regulation of GRK2 on mitochondrial respiration in skeletal muscle. Myostatin treatment promoted the loss of GRK2 protein in myoblasts and myotubes in a time- and dose-dependent manner, which we suggest was through enhanced ubiquitin-mediated protein loss, as treatment with proteasome inhibitors partially rescued myostatin-mediated loss of GRK2 protein. To evaluate the effects of GRK2 on mitochondrial respiration, we generated stable myoblast lines that overexpress GRK2. Stable overexpression of GRK2 resulted in increased mitochondrial content and enhanced mitochondrial/oxidative respiration. Interestingly, although overexpression of GRK2 was unable to prevent myostatin-mediated impairment of mitochondrial respiratory function, elevated levels of GRK2 blocked the increased autophagic flux observed following treatment with myostatin. Overall, our data suggest a novel role for GRK2 in regulating mitochondria mass and mitochondrial respiration in skeletal muscle.

Effects of chronic desipramine treatment on alpha2-adrenoceptors and mu-opioid receptors in the guinea pig cortex and hippocampus.

The existence of a close relation between presynaptic inhibitory alpha(2)-adrenoceptor and mu-opioid receptor pathways is well established. Such interplay may occur during chronic conditions that give rise to neuroadaptive changes involving both receptor systems. The aim of this study was to examine the effect of chronic treatment with the tricyclic antidepressant drug, desipramine, on alpha(2)-adrenoceptors and mu-opioid receptors in the guinea pig brain. Guinea pigs were treated with 10 mg/kg desipramine, injected i.p. for 21 days, every 24 h. The levels of expression of alpha(2)-adrenoceptors and mu-opioid receptors, the G protein receptor regulatory kinase, GRK2/3 and signal transduction inhibitory G proteins in synaptosomes of the guinea pig hippocampus and cortex were evaluated by immunoblotting. Quantitative analysis of alpha(2)-adrenoceptor and mu-opioid receptor mRNA levels has been carried out by competitive reverse transcriptase polymerase chain reaction. The expression levels of alpha(2)-adrenoceptors and mu-opioid receptors and the respective mRNAs were found unchanged in the cortex, after chronic desipramine treatment. In these experimental conditions alpha(2)-adrenoceptor and mu-opioid receptor levels decreased, while the relevant transcripts increased, in the hippocampus. GRK2/3 levels remained unchanged and increased, respectively, in the cortex and the hippocampus, after chronic exposure to desipramine. In the same experimental conditions, Galpha(i1), Galpha(i2), Galpha(o) and Galpha(z) levels remained unchanged, while Galpha(i3) levels decreased, in the cortex; whereas, Galpha(i1), Galpha(i2) and Galpha(i3) levels significantly increased, and Galpha(o) and Galpha(z) levels remained unchanged, in the hippocampus. On the whole, the present data suggest that alpha(2)-adrenoceptor and mu-opioid receptor expression and transcription are similarly influenced by chronic treatment with desipramine, in the guinea pig cortex and hippocampus. Furthermore, alterations in the levels of regulatory GRK2/3 and of inhibitory signal transduction G proteins, relevant to activation of both receptor pathways, have been documented. The distinct pattern of adaptations of the different protein studied in response to chronic desipramine treatment in both regions is discussed.

Alteration of the somatostatinergic system in the striatum of rats with acute experimental autoimmune encephalomyelitis.

To date, the neurochemical basis underlying the motor and cognitive deficits described in patients with multiple sclerosis (MS) is unclear. Since the neuropeptide somatostatin (SRIF) and the striatum have been implicated in movement control and implicit memory, the aim of this study was to analyze the striatal somatostatinergic system in an animal model of MS, experimental autoimmune encephalomyelitis (EAE). Female Lewis rats were immunized with an emulsion containing myelin basic protein (MBP) in complete Freund's adjuvant to induce the disease. The animals were decapitated when limp tail (grade 1) or severe hind limb paralysis (grade 3) was observed. Acute EAE in grade 3 did not modify striatal somatostatin-like immunoreactivity (SRIF-LI) content but decreased the overall SRIF receptor density, without affecting the apparent affinity, in the rat striatal membranes. A selective reduction in the protein levels of the SRIF receptor subtype sst2, analyzed by Western blotting, was detected in the EAE rats, which correlated with decreased sst2 mRNA levels. The expression of the receptor subtypes sst1, sst3 or sst4 was unaltered by the disease. The decrease in the SRIF receptor density was accompanied by an attenuated capacity of SRIF to inhibit both basal and forskolin-stimulated adenylyl cyclase activity. No significant changes, however, were found in the protein levels of Gi proteins (G(ialpha1), G(ialpha2) or G(ialpha3)) nor in those of the G-protein-coupled receptor kinase subtypes GRK2, GRK5 or GRK6. Acute EAE in grade 1 did not modify any of the parameters studied. In conclusion, these data demonstrate that acute EAE, in grade 3, disrupts the rat striatal SRIF receptor-effector system. These findings provide new insight into the molecular basis of EAE which might contribute to a better understanding of multiple sclerosis in humans.