Palmitic acid induces osteoblastic differentiation in vascular smooth muscle cells through ACSL3 and NF-κB, novel targets of eicosapentaenoic acid.

Free fatty acids (FFAs), elevated in metabolic syndrome and diabetes, play a crucial role in the development of atherosclerotic cardiovascular disease, and eicosapentaenoic acid (EPA) counteracts many aspects of FFA-induced vascular pathology. Although vascular calcification is invariably associated with atherosclerosis, the mechanisms involved are not completely elucidated. In this study, we tested the hypothesis that EPA prevents the osteoblastic differentiation and mineralization of vascular smooth muscle cells (VSMC) induced by palmitic acid (PA), the most abundant long-chain saturated fatty acid in plasma. PA increased and EPA abolished the expression of the genes for bone-related proteins, including bone morphogenetic protein (BMP)-2, Msx2 and osteopontin in human aortic smooth muscle cells (HASMC). Among the long-chain acyl-CoA synthetase (ACSL) subfamily, ACSL3 expression was predominant in HASMC, and PA robustly increased and EPA efficiently inhibited ACSL3 expression. Importantly, PA-induced osteoblastic differentiation was mediated, at least in part, by ACSL3 activation because acyl-CoA synthetase (ACS) inhibitor or siRNA targeted to ACSL3 completely prevented the PA induction of both BMP-2 and Msx2. Conversely, adenovirus-mediated ACSL3 overexpression enhanced PA-induced BMP-2 and Msx2 expression. In addition, EPA, ACSL3 siRNA and ACS inhibitor attenuated calcium deposition and caspase activation induced by PA. Notably, PA induced activation of NF-κB, and NF-κB inhibitor prevented PA-induction of osteoblastic gene expression and calcium deposition. Immunohistochemistry revealed the prominent expression of ACSL3 in VSMC and macrophages in human non-calcifying and calcifying atherosclerotic plaques from the carotid arteries. These results identify ACSL3 and NF-κB as mediators of PA-induced osteoblastic differentiation and calcium deposition in VSMC and suggest that EPA prevents vascular calcification by inhibiting such a new molecular pathway elicited by PA.

Loss of muscarinic and purinergic receptors in urinary bladder of rats with hydrochloric acid-induced cystitis.

OBJECTIVES: To clarify the basic mechanism involved in the pathophysiology of cystitis by characterizing the urodynamic parameters, pharmacologically relevant (muscarinic and purinergic) receptors, and the in vivo release of adenosine triphosphate (ATP) in the bladder of hydrochloric acid (HCl)-treated rats. METHODS: The muscarinic and purinergic receptors in rat tissue were measured by radioreceptor assays using (N-methyl-³H) scopolamine methyl chloride ([³H]NMS) and αß-methylene-ATP (2,8-³H) tetrasodium salt ([³H]αß-MeATP), respectively. The urodynamic parameters and ATP levels were measured using a cystometric method and the luciferin-luciferase assay, respectively. RESULTS: In the HCl-treated rats, the micturition interval and micturition volume were significantly (48% and 55%, respectively, P <.05) decreased and the number of micturitions was significantly (3.2-fold, P <.05) increased compared with those of the control rats. The maximal number of binding sites for [³H]NMS and [³H]αß-MeATP was significantly (55% and 72%, respectively, P <.001) decreased in the bladder of HCl-treated rats, suggesting downregulation of both muscarinic and purinergic receptors. In the HCl-treated rats, the inhibition constant, K(i), values for oxybutynin, solifenacin, and darifenacin were significantly (1.3-1.4-fold, P <.05) increased, but those for tolterodine and AF-DX116 were unchanged. Similarly, the inhibition constant for A-317491, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium, and MRS2273 was significantly (5.5, 11, and 7.6-fold, respectively, P <.001) increased. Furthermore, the in vivo release of ATP was significantly (P <.05) enhanced in the HCl-treated rat bladder. CONCLUSIONS: Both muscarinic and purinergic mechanisms might be, at least in part, associated with the urinary dysfunction due to cystitis.

Alteration of muscarinic and purinergic receptors in urinary bladder of rats with cyclophosphamide-induced interstitial cystitis.

We characterized muscarnic and purinergic receptors and urodynamic parameters in the bladder of cyclophosphamide (CYP)-treated rats to clarify the mechanisms involved in the pathophysiology of interstitial cystitis (IC). In the cystometry of CYP-treated rats compared with control rats, the micturition interval and micturition volume were significantly (55% and 77%, respectively) decreased and the frequency of micturition and basal pressure were significantly (3 and 2.3 times, respectively) increased. These changes in urodynamic parameters may characterize the detrusor overactivity occurring in CYP-treated rats. The maximal number of binding sites (B(max)) for specific binding of [N-methyl-(3)H]scopolamine methyl chloride ([(3)H]NMS) and alphabeta-methylene ATP [2,8-(3)H]tetrasodium salt ([(3)H]alphabeta-MeATP) was significantly (43% and 31%, respectively) decreased in the bladder of CYP-treated rats compared with control rats. On the other hand, the apparent dissociation constant (K(d)) for neither radioligand was significantly altered by the CYP treatment. K(i) value for the inhibition of bladder [(3)H]NMS binding by antimuscarinic agents (oxybutynin, tolterodine, darifenacin, and AF-DX 116) did not differ significantly between control and CYP-treated rats. The inhibition constant (K(i)) for the inhibition of bladder [(3)H]alphabeta-MeATP binding by purinergic antagonists (A-317491, PPADS) was significantly higher in CYP-treated rats than control rats. In conclusion, CYP treatment has been shown to cause down-regulation of pharmacologically relevant (muscarinic and purinergic) receptors in the bladder of rats. Thus, the present study offers further pharmacological evidence that both muscarinic and purinergic mechanisms contribute significantly to the urinary dysfunction due to IC.

Comparative evaluation of central muscarinic receptor binding activity by oxybutynin, tolterodine and darifenacin used to treat overactive bladder.

PURPOSE: We characterized muscarinic receptor binding in the mouse cerebral cortex after oral administration of anticholinergic agents used to treat overactive bladder. MATERIALS AND METHODS: Muscarinic receptors in the mouse cerebral cortex and bladder after oral administration of anticholinergic agents were measured using [(3)H]N-methylscopolamine. RESULTS: In vitro binding affinities of tolterodine and its metabolite 5-hydroxymethyl metabolite in the mouse cerebral cortex and bladder were considerably greater than those of oxybutynin and darifenacin. Also, muscarinic receptor binding affinity of oxybutynin and its metabolite N-desethyl-oxybutynin in the cerebral cortex compared with that in the bladder was 2 to 3 times higher, whereas that of tolterodine and 5-hydroxymethyl metabolite was approximately 2 times lower. Oral administration of oxybutynin (76.1 micromol/kg), tolterodine (6.31 micromol/kg) and darifenacin (59.1 micromol/kg) showed binding activity that was approximately equal to that of bladder muscarinic receptors. Oral administration of oxybutynin (76.1 micromol/kg) showed significant binding of cerebral cortical muscarinic receptors in mice, as indicated by about a 2-fold increase in K(d) values for specific [(3)H]N-methylscopolamine binding 0.5 and 2 hours later. On the other hand, tolterodine and darifenacin given at oral doses that would exert a similar extent of bladder receptor binding activity as oxybutynin showed only a low level of binding activity of central muscarinic receptors in mice. CONCLUSIONS: Significant binding of brain muscarinic receptors in mice was observed by the oral administration of oxybutynin but not tolterodine and darifenacin.