Roles of exercise and pharmacokinetics in cerivastatin-induced skeletal muscle toxicity.

Three-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors are associated with adverse skeletal muscle effects, but the underlying mechanisms remain unclear. To determine whether toxicity involves the level of drug exposure in muscle tissue and to test the effect of exercise on cerivastatin (CVA)-induced skeletal muscle damage, female rats were administered vehicle or CVA at 0.1, 0.5, and 1.0 mg/kg/day by gavage for two weeks and exercised or not on treadmills for 20 min/day. Clinical chemistry and plasma and tissue pharmacokinetics were evaluated; light and transmission electron microscopy (TEM) of Type I and Type II fiber-predominant skeletal muscles were performed. Serum levels of AST, ALT, CK, and plasma lactic acid were significantly elevated dose-dependently. CVA treatment decreased psoas and quadriceps weights. At 1 mg/kg all muscles except soleus demonstrated degeneration. Exercise-exacerbated severity of CVA-induced degeneration was evident in all muscles sampled except soleus and quadriceps. Early mitochondrial involvement in toxicity is suggested by the numerous membranous whorls and degenerate mitochondria observed in muscles at 0.5 mg/kg. No significant differences in CVA concentrations between either EDL and soleus or plasma and muscle were found. We found that CVA had no effect on cleaved caspase 3. In summary, we found that treadmill exercise exacerbated the incidence and severity of CVA-induced damage in Type II fiber-predominant muscles. Tissue exposure is likely not the key factor mediating CVA-induced skeletal muscle toxicity.

Regulation of angiotensin II type 1A receptor intracellular retention, degradation, and recycling by Rab5, Rab7, and Rab11 GTPases.

Previous studies have demonstrated that the interaction of the angiotensin II type 1A receptor (AT(1A)R) carboxyl-terminal tail with Rab5a may modulate Rab5a activity, leading to the homotypic fusion of endocytic vesicles. Therefore, we have investigated whether AT(1A)R/Rab5a interactions mediate the retention of AT(1A)R.beta-arrestin complexes in early endosomes and whether the overexpression of Rab7 and Rab11 GTPases influences AT(1A)R lysosomal degradation and plasma membrane recycling. We found that internalized AT(1A)R was retained in Rab5a-positive early endosomes and was neither targeted to lysosomes nor recycled back to the cell surface, whereas a mutant defective in Rab5a binding, AT(1A)R-(1-349), was targeted to lysosomes for degradation. However, the loss of Rab5a binding to the AT(1A)R carboxyl-terminal tail did not promote AT(1A)R recycling. Rather, it was the stable binding of beta-arrestin to the AT(1A)R that prevented, at least in part, AT(1A)R recycling. The overexpression of wild-type Rab7 and Rab7-Q67L resulted in both increased AT(1A)R degradation and AT(1A)R targeting to lysosomes. The Rab7 expression-dependent transition of "putative" AT(1A)R.beta-arrestin complexes to late endosomes was blocked by the expression of dominant-negative Rab5a-S34N. Rab11 overexpression established AT(1A)R recycling and promoted the redistribution of AT(1A)R.beta-arrestin complexes from early to recycling endosomes. Taken together, our data suggest that Rab5, Rab7, and Rab11 work in concert with one another to regulate the intracellular trafficking patterns of the AT(1A)R.

Regulation of G protein-coupled receptor endocytosis and trafficking by Rab GTPases.

G protein-coupled receptors (GPCRs) are integral membrane proteins that, in response to activation by extracellular stimuli, regulate intracellular second messenger levels via their coupling to heterotrimeric G proteins. GPCR activation also initiates a series of molecular events that leads to G protein-coupled receptor kinase-mediated receptor phosphorylation and the binding of beta-arrestin proteins to the intracellular face of the receptor. beta-Arrestin binding not only contributes to the G protein-uncoupling of GPCRs, but also mediates the targeting of many GPCRs for endocytosis in clathrin-coated pits. Several GPCRs internalize as a stable complex with beta-arrestin and the stability of this complex appears to regulate, at least in part, whether the receptors are dephosphorylated in early endosomes and recycled back to the cell surface as fully functional receptors, retained in early endosomes or targeted for degradation in lysosomes. More recently, it has become appreciated that the movement of GPCRs through functionally distinct intracellular membrane compartments is regulated by a variety of Rab GTPases and that the activity of these Rab GTPases may influence GPCR function. Moreover, it appears that GPCRs are not simply passive cargo molecules, but that GPCR activation may directly influence Rab GTPase activity and as such, GPCRs may directly control their own targeting between intracellular compartments. This review provides a synopsis of the current knowledge regarding the role of beta-arrestins and Rab GTPases in regulating the intracellular trafficking and function of GPCRs.

Rab5 association with the angiotensin II type 1A receptor promotes Rab5 GTP binding and vesicular fusion.

Previous studies have demonstrated that the internalization of the angiotensin II type 1A receptor (AT(1A)R) may be mediated by both beta-arrestin-sensitive and -insensitive mechanisms. Therefore, we have used the AT(1A)R carboxyl-terminal tail to screen a rat brain yeast two-hybrid expression library for novel AT(1A)R-interacting proteins that might contribute to the regulation of AT(1A)R internalization. We have identified Rab5a as an AT(1A)R-binding protein that selectively associates with the AT(1A)R and not with the beta2-adrenergic receptor. A Rab5a-S34N mutant defective in GTP binding does not prevent the internalization of the AT(1A)R but does prevent the trafficking of the AT(1A)R into larger hollow cored vesicular structures. Agonist activation of the AT(1A)R promotes both the formation of Rab5a.AT(1A)R protein complexes and Rab5a GTP binding. Rab5a interactions with the AT(1A)R are mediated in part by the last 10 amino acid residues of the AT(1A)R carboxyl-terminal tail, and although a mutant receptor lacking these residues internalizes normally, it does not redistribute into larger hollow vesicles. Our data suggest that AT(1A)R activation modulates Rab5a activity leading to the homotypic fusion of endocytic vesicles. These observations suggest that vesicular cargo proteins, such as the AT(1A)R, may control their targeting between intracellular compartments by directly regulating the activity of components of the intracellular trafficking machinery such as Rab5a.