Loss of anti-contractile effect of perivascular adipose tissue in offspring of obese rats

RATIONALE: Maternal obesity pre-programmes offspring to develop obesity and associated cardiovascular disease. Perivascular adipose tissue (PVAT) exerts an anti-contractile effect on the vasculature, which is reduced in hypertension and obesity. OBJECTIVE: The objective of this study was to determine whether maternal obesity pre-programmes offspring to develop PVAT dysfunction in later life. METHODS: Female Sprague–Dawley rats were fed a diet containing 10% (control) or 45% fat (high fat diet, HFD) for 12 weeks prior to mating and during pregnancy and lactation. Male offspring were killed at 12 or 24 weeks of age and tension in PVAT-intact or -denuded mesenteric artery segments was measured isometrically. Concentration–response curves were constructed to U46619 and norepinephrine. RESULTS: Only 24-week-old HFD offspring were hypertensive (P<0.0001), although the anti-contractile effect of PVAT was lost in vessels from HFD offspring of each age. Inhibition of nitric oxide (NO) synthase with 100 μM l-NMMA attenuated the anti-contractile effect of PVAT and increased contractility of PVAT-denuded arteries (P<0.05, P<0.0001). The increase in contraction was smaller in PVAT-intact than PVAT-denuded vessels from 12-week-old HFD offspring, suggesting decreased PVAT-derived NO and release of a contractile factor (P<0.07). An additional, NO-independent effect of PVAT was evident only in norepinephrine-contracted vessels. Activation of AMP-activated kinase (with 10 μM A769662) was anti-contractile in PVAT-denuded (P<0.0001) and -intact (P<0.01) vessels and was due solely to NO in controls; the AMPK effect was similar in HFD offspring vessels (P<0.001 and P<0.01, respectively) but was partially NO-independent. CONCLUSIONS: The diminished anti-contractile effects of PVAT in offspring of HFD dams are primarily due to release of a PVAT-derived contractile factor and reduced NO bioavailability

Kinetics and mechanism of jack bean urease inhibition by Hg²⁺

<p>Abstract</p> <p>Background</p> <p>Jack bean urease (EC 3.5.1.5) is a metalloenzyme, which catalyzes the hydrolysis of urea to produce ammonia and carbon dioxide. The heavy metal ions are common inhibitors to control the rate of the enzymatic urea hydrolysis, which take the Hg²⁺ as the representative. Hg²⁺ affects the enzyme activity causing loss of the biological function of the enzyme, which threatens the survival of many microorganism and plants. However, inhibitory kinetics of urease by the low concentration Hg²⁺ has not been explored fully. In this study, the inhibitory effect of the low concentration Hg²⁺ on jack bean urease was investigated in order to elucidate the mechanism of Hg²⁺ inhibition.</p> <p>Results</p> <p>According to the kinetic parameters for the enzyme obtained from Lineweaver–Burk plot, it is shown that the <it>K</it>_m is equal to 4.6±0.3 mM and <it>V</it>_m is equal to 29.8±1.7 μmol NH₃/min mg. The results show that the inhibition of jack bean urease by Hg²⁺ at low concentration is a reversible reaction. Equilibrium constants have been determined for Hg²⁺ binding with the enzyme or the enzyme-substrate complexes (<it>K</it>_i =0.012 μM). The results show that the Hg²⁺ is a noncompetitive inhibitor. In addition, the kinetics of enzyme inhibition by the low concentration Hg²⁺ has been studied using the kinetic method of the substrate reaction. The results suggest that the enzyme first reversibly and quickly binds Hg²⁺ and then undergoes a slow reversible course to inactivation. Furthermore, the rate constant of the forward reactions (<it>k</it>₊₀) is much larger than the rate constant of the reverse reactions (<it>k</it>_-0). By combining with the fact that the enzyme activity is almost completely lost at high concentration, the enzyme is completely inactivated when the Hg²⁺ concentration is high enough.</p> <p>Conclusions</p> <p>These results suggest that Hg²⁺ has great impacts on the urease activity and the established inhibition kinetics model is suitable.</p