Abstract-The aim of this study was to investigate the activity of the parasympathetic limb of the baroreflex arch in a canine model of obesity-related hypertension. Twelve male beagle dogs were randomized into 2 groups. Six dogs were fed with normal canine food and 6 were submitted to a 10-week high-fat diet (HFD). We have evaluated the consequences of HFD on heart rate (HR) and blood pressure (BP) circadian cycles and methylscopolamine dose-response curves. Binding of [ 3 H]-AF-DX 384 and adenylyl cyclase activity were investigated to determine the density and functionality of M 2 -cholinoceptors on right atrial membranes from control and HFD dogs. HFD induced a significant increase in body weight (15Ϯ1 vs 12Ϯ1 kg), systolic BP (161Ϯ5 vs 145Ϯ4 mm Hg), diastolic BP (92Ϯ3 vs 79Ϯ2 mm Hg), and HR (96Ϯ4 vs 81Ϯ3 bpm). Circadian rhythms of HR and BP observed in the baseline period were abolished after 9 weeks of HFD. After propranolol (1 mg/kg) pretreatment, the dose of methylscopolamine able to induce 50% maximum tachycardia was significantly increased after 9 weeks of HFD (7.4Ϯ0.3 vs 4.7Ϯ0.1 g/kg). In the control group, the experimental period failed to modify these parameters. The numbers of M 2 -cholinoceptors measured in right atrial membranes were significantly lower in HFD than in control groups (54Ϯ6 vs 27Ϯ6 fmol/mg protein). The ability of carbachol to inhibit isoproterenol-stimulated adenylyl cyclase activity was significantly lower in HFD than in control groups (IC 50 ϭ47Ϯ12 vs 6.4Ϯ1.4 mol/L). However, the basal activity of adenylyl cyclase was unchanged by HFD. HFD decreases M 2 -cholinoceptor number and function in cardiomyocytes. This could explain the abolition of circadian rhythm of HR and the changes in chronotropic effect brought about by methylscopolamine.

Nitric oxide (NO) is a potent vasodilator, but it can also modulate contractile responses of the airway smooth muscle. Whether or not endothelial (e) NO synthase (NOS) contributes to the regulation of bronchial tone is unknown at present. Experiments were designed to investigate the isoforms of NOS that are expressed in murine airways and to determine whether or not the endogenous release of NO modulates bronchial tone in wild-type mice and in mice with targeted deletion of eNOS [eNOS(-/-)]. The presence of neuronal NOS (nNOS), inducible NOS (iNOS), and eNOS in murine trachea and lung parenchyma was assessed by RT-PCR, immunoblotting, and immunohistochemistry. Airway resistance was measured in conscious unrestrained mice by means of a whole body plethysmography chamber. The three isoforms of NOS were constitutively present in lungs of wild-type mice, whereas only iNOS and nNOS were present in eNOS(-/-) mice. Labeling of nNOS was localized in submucosal airway nerves but was not consistently detected, and iNOS immunoreactivity was observed in tracheal and bronchiolar epithelial cells, whereas eNOS was expressed in endothelial cells. In wild-type mice, treatment with N-nitro-L-arginine methyl ester, but not with aminoguanidine, potentiated the increase in airway resistance produced by inhalation of methacholine. eNOS(-/-) mice were hyperresponsive to inhaled methacholine and markedly less sensitive to N-nitro-L-arginine methyl ester. These results demonstrate that the three NOS isoforms are expressed constitutively in murine lung and that NO derived from eNOS plays a physiological role in controlling bronchial airway reactivity.

Uncoupling protein-2 (UCP-2) is a mitochondrial protein expressed in adipocytes and has recently been involved in the control of energy dissipation. Because obesity is characterized by an imbalance between energy intake and expenditure and by an enhanced adipocyte-derived secretion of tumor necrosis factor-alpha (TNF-alpha), we asked whether TNF-alpha could directly influence UCP-2 expression in adipocytes. Experiments performed in differentiated 3T3F442A preadipocytes showed that TNF-alpha (10 ng/ml) induced a reduction of UCP-2 trancripts, assessed by Northern blot analysis. A significant decrease in UCP-2 expression (40%) was observed after 12 and 24 h of TNF-alpha stimulation of the cells. The characterization of the mechanisms responsible for the TNF-alpha effect on UCP-2 expression demonstrates an involvement of the TNF-alpha-induced inducible (i) nitric oxide synthase (NOS) expression. Cell treatment with the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME; 1 mmol/l) significantly diminished the TNF-alpha-mediated sustained downregulation of UCP-2 expression, whereas cell treatment with a nitric oxide (NO) donor (10(-3) mol/l S-nitroso-L-glutathione) mimicked the TNF-alpha effect on UCP-2 expression. Moreover, Western blot analysis clearly showed that TNF-alpha alone induces the expression of iNOS after 12-24 h treatment of differentiated 3T3F442A cells. These experiments demonstrate that TNF-alpha directly downregulates UCP-2 expression via NO-dependent pathways that involve the induction of iNOS expression.