Overexpression of cellular glutathione peroxidase rescues homocyst(e)ine-induced endothelial dysfunction.

Homocyst(e)ine (Hcy) inhibits the expression of the antioxidant enzyme cellular glutathione peroxidase (GPx-1) in vitro and in vivo, which can lead to an increase in reactive oxygen species that inactivate NO and promote endothelial dysfunction. In this study, we tested the hypothesis that overexpression of GPx-1 can restore the normal endothelial phenotype in hyperhomocyst(e)inemic states. Heterozygous cystathionine beta-synthase-deficient (CBS((-/+))) mice and their wild-type littermates (CBS((+/+))) were crossbred with mice that overexpress GPx-1 [GPx-1((tg+)) mice]. GPx-1 activity was 28% lower in CBS((-/+))/GPx-1((tg-)) compared with CBS((+/+))/GPx-1((tg-)) mice (P < 0.05), and CBS((-/+)) and CBS((+/+)) mice overexpressing GPx-1 had 1.5-fold higher GPx-1 activity compared with GPx-1 nontransgenic mice (P < 0.05). Mesenteric arterioles of CBS((-/+))/GPx-1((tg-)) mice showed vasoconstriction to superfusion with beta-methacholine and bradykinin (P < 0.001 vs. all other groups), whereas nonhyperhomocyst(e)inemic mice [CBS((+/+))/GPx-1((tg-)) and CBS((+/+))/GPx-1((tg+)) mice] demonstrated dose-dependent vasodilation in response to both agonists. Overexpression of GPx-1 in hyperhomocyst(e)inemic mice restored the normal endothelium-dependent vasodilator response. Bovine aortic endothelial cells (BAEC) were transiently transfected with GPx-1 and incubated with dl-homocysteine (HcyH) or l-cysteine. HcyH incubation decreased GPx-1 activity in sham-transfected BAEC (P < 0.005) but not in GPx-1-transfected cells. Nitric oxide release from BAEC was significantly decreased by HcyH but not cysteine, and GPx-1 overexpression attenuated this decrease. These findings demonstrate that overexpression of GPx-1 can compensate for the adverse effects of Hcy on endothelial function and suggest that the adverse vascular effects of Hcy are at least partly mediated by oxidative inactivation of NO.

Photoresponsive Fischer 344 Rats are reproductively inhibited by melatonin and differ in 2-[125I] lodomelatonin binding from nonphotoresponsive Sprague-Dawley rats.

Many temperate-zone species use photoperiod as an environmental cue to regulate reproductive timing. Strains of laboratory rats differ in their responsiveness to photoperiod, with the Fischer 344 (F344) strain being the most responsive known. F344 rats and closely related strains that differ in photoresponsiveness may be useful models to study the mechanisms and genetic basis for photoresponsiveness. We tested two hypotheses: (i) that melatonin mediates photoresponsiveness in F344 rats, as is the case in all other mammals tested, and (ii) that the location, abundance, or affinity of melatonin receptors, as estimated by the amount and location of binding of the radioligand 2-[125I]-iodomelatonin (IMEL) in the brain, might cause variation in photoresponsiveness among rat strains. Melatonin injections 1 h before lights off in a stimulatory photoperiod (L14 : D10) induced reproductive inhibition and reduced weight gain in a manner similar to short days of L8 : D16, while injections of ethanolic saline vehicle did not. Interestingly, melatonin injections administered during an inhibitory photoperiod (L10 : D14) caused greater inhibition of both reproduction and weight gain than short photoperiod alone. Pinealectomized F344 rats implanted subcutaneously with melatonin in a silastic capsule did not differ in testis size or body weight from controls with blank implants. The brains and pars tuberalis of the pituitary from photoresponsive F344 rats and nonphotoresponsive Harlan Sprague-Dawley (HSD) rats were processed for autoradiography using IMEL. We found significantly higher specific IMEL binding in the anterior and posterior regions of the paraventricular nucleus of the thalamus (PVNt) and reuniens nucleus of the thalamus of F344 rats than in the same areas in HSD rats. There were no differences between strains in specific IMEL binding in the medial PVNt, anteroventral and anterodorsal nucleus of the thalamus, suprachiasmatic nucleus, or the pars tuberalis. These results indicate that melatonin mediates photoresponsiveness in F344 rats. In addition, they provide support for the hypothesis that F344 rats may be photoresponsive due to differences from other strains in the location, density, or affinity of melatonin receptors.

Inhibition of reproductive maturation and somatic growth of Fischer 344 rats by photoperiods shorter than L14:D10 and by gradually decreasing photoperiod.

Photoperiod is the major regulator of reproduction in temperate-zone mammals. Laboratory rats are generally considered to be nonphotoresponsive, but young male Fischer 344 (F344) rats have a uniquely robust response to short photoperiods of 8 h of light. Rats transferred at weaning from a photoperiod of 16 h to photoperiods of < 14 h of light slowed in both reproductive development and somatic growth rate. Those in photoperiods < 13 h of light underwent the strongest responses. The critical photoperiod of F344 rats can be defined as 13.5 h of light, but photoperiods of

Mechanism of nitric oxide-induced vasodilatation: refilling of intracellular stores by sarcoplasmic reticulum Ca2+ ATPase and inhibition of store-operated Ca2+ influx.

The precise mechanisms by which nitric oxide (NO) decreases free [Ca2+]i, inhibits Ca2+ influx, and relaxes vascular smooth muscle are poorly understood. In rabbit and mouse aorta, agonist-induced contractions and increases in [Ca2+]i were resistant to nifedipine, suggesting Ca2+ entry through non-L-type Ca2+ channels. Relaxations to NO were inhibited by thapsigargin (TG) or cyclopiazonic acid (CPA) indicating the involvement of sarcoplasmic reticulum ATPase (SERCA). Studies of the effect of NO on [Ca2+]i and the rate of Mn2+ influx with fura-2 fluorometry in rabbit aortic smooth muscle cells in primary culture were designed to test how SERCA is involved in mediating the response to NO. When cells were stimulated with angiotensin II (AII), NO accelerated the removal of Ca2+ from the cytoplasm, decreased [Ca2+]i, and inhibited Ca2+ and Mn2+ influx. Inhibition of SERCA abolished all the effects of NO. In contrast, inhibition of the Na+/Ca2+exchanger or the plasma membrane Ca2+ ATPase had no influence on the ability of NO to decrease [Ca2+]i. NO maximally decreased [Ca2+]i within 5 s, whereas significant inhibition of AII-induced Ca2+ and Mn2+ influx required more than 15 s. The inhibition of cation influx strictly depended on [Ca2+]o and functional SERCA, suggesting that during the delay before NO inhibits Ca2+ influx, the influx of Ca2+ and the uptake into intracellular stores are required. In the absence of [Ca2+]o, NO diminished the AII-induced [Ca2+]i transient by a SERCA-dependent mechanism and increased the amount of Ca2+ in the stores subsequently released by ionomycin. The present study indicates that the initial rapid decrease in [Ca2+]i caused by NO in vascular smooth muscle is accounted for by the uptake of Ca2+ by SERCA into intracellular stores. It is proposed that the refilling of the stores inhibits store-operated Ca2+ influx through non-L-type Ca2+ conducting ion channels and that this maintains the decrease in [Ca2+]i and NO-induced relaxation.