Bacterial toxins activation of abbreviated urea cycle in porcine cerebral vascular smooth muscle cells.

Nitric oxide (NO) overproduction via induction of inducible nitric oxide synthase (iNOS) is implicated in vasodilatory shock in sepsis, leading to septic encephalopathy and accelerating cerebral ischemic injury. An abbreviated urea-cycle (l-citrulline-l-arginine-NO cycle) has been demonstrated in cerebral perivascular nitrergic nerves and endothelial cells but not in normal cerebral vascular smooth muscle cell (CVSMC). This cycle indicates that argininosuccinate synthase (ASS) catalyzes l-citrulline (l-cit) conversion to form argininosuccinate (AS), and subsequent AS cleavage by argininosuccinate lyase (ASL) forms l-arginine (l-arg), the substrate for NO synthesis. The possibility that ASS enzyme in this cycle was induced in the CVSMC in sepsis was examined. Blood-vessel myography technique was used for measuring porcine isolated basilar arterial tone. NO in cultured CVSMC and in condition mediums were estimated by diaminofluorescein (DAF)-induced fluorescence and Griess reaction, respectively. Immunohistochemical and immunoblotting analyses were used to examine iNOS and ASS induction. l-cit and l-arg, which did not relax endothelium-denuded normal basilar arteries precontracted by U-46619, induced significant vasorelaxation with increased NO production in these arteries and the CVSMCs following 6-hour exposure to 20µg/ml lipopolysaccharide (LPS) or lipoteichoic acid (LTA). Pre-treatment with pyrrolidine dithiocarbamate (PDTC) and salicylate (SAL) (NFκB inhibitors), aminoguanidine (AG, an iNOS inhibitor), and nitro-l-arg (NLA, a non-specific NOS inhibitor) blocked NO synthesis in the CVSMC and attenuated l-cit- and l-arg-induced relaxation of LPS- and LTA-treated arteries. Furthermore, immunohistochemical and immunoblotting studies demonstrated that expression of basal iNOS and ASS in the smooth muscle cell of arterial segments denuded of endothelium and the cultured CVSMCs was significantly increased following 6-hour incubation with LPS or LTA. This increased iNOS- and ASS-proteins expression in both preparations was inhibited by SAL, but was further increased by AG. These results indicate that LPS and LTA induce the l-cit-l-arg-NO cycle via induction of iNOS and ASS in the CVSMCs, accounting for massively increased NO-production and cerebral vasodilation in septic shock. Simultaneous inhibition of both pathways and NFκB-activation may be necessary to efficiently decrease or normalize NO production in the CVSMCs in this disease condition, and/or prevention and treatment of cerebral vessel-related brain dysfunctions. Our results further suggest to avoid using iNOS inhibitors alone which may cause upregulation of iNOS and ASS resulted from feedback-inhibition of iNOS activity. Accordingly, combined treatments with specific iNOS-activity inhibitor and inhibitor for iNOS genomic expression may provide a strategy in optimally managing brain sepsis and related encephalopathy associated with enhanced iNOS expression and NO overproduction.

The effects of nicotine replacement on cognitive brain activity during smoking withdrawal studied with simultaneous fMRI/EEG.

Impaired attention ('difficulty concentrating') is a cognitive symptom of nicotine withdrawal that may be an important contributor to smoking relapse. However, the neurobiological basis of this effect and the potentially beneficial effects of nicotine replacement therapy both remain unclear. We used functional MRI with simultaneous electroencephalogram (EEG) recording to define brain activity correlates of cognitive impairment with short-term smoking cessation in habitual smokers and the effects of nicotine replacement. We found that irrespective of treatment (ie nicotine or placebo) EEG α power was negatively correlated with increased activation during performance of a rapid visual information processing (RVIP) task in dorsolateral prefrontal, dorsal anterior cingulate, parietal, and insular cortices, as well as, caudate, and thalamus. Relative to placebo, nicotine replacement further increased the α-correlated activation across these regions. We also found that EEG α power was negatively correlated with RVIP-induced deactivation in regions comprising the 'default mode' network (ie angular gyrus, cuneus, precuneus, posterior cingulate, and ventromedial prefrontal cortex). These α-correlated deactivations were further reduced by nicotine. These findings confirm that effects of nicotine on cognition during short-term smoking cessation occur with modulation of neuronal sources common to the generation of both the blood oxygen-level-dependent and α EEG signals. Our observations thus demonstrate that nicotine replacement in smokers has direct pharmacological effects on brain neuronal activity modulating cognitive networks.

Metabolite ligands of estrogen receptor-beta reduce primate coronary hyperreactivity.

Previous reports showed that 17beta-estradiol implants attenuate in vivo coronary hyperreactivity (CH), characterized by long-duration vasoconstrictions (in coronary angiographic experiments), in menopausal rhesus monkeys. Prolonged Ca2+ contraction signals that correspond with CH in coronary vascular muscle cells (VMC) to the same dual-constrictor stimulus, serotonin + the thromboxane analog U-46619, in estrogen-deprived VMC were suppressed by >72 h in 17beta-estradiol. The purpose of this study was to test whether an endogenous estrogen metabolite with estrogen receptor-beta (ER-beta) binding activity, estriol (E3), suppresses in vivo and in vitro CH. E3 treatment in vivo for 4 wk significantly attenuated the angiographically evaluated vasoconstrictor response to intracoronary serotonin + U-46619 challenge. In vitro treatment of rhesus coronary VMC for >72 h with nanomolar E3 attenuated late Ca2+ signals. This reduction of late Ca2+ signals also appeared after >72 h of treatment with subnanomolar 5alpha-androstane-3beta,17beta-diol (3beta-Adiol), an endogenous dihydrotestosterone metabolite with ER-beta binding activity. R,R-tetrahydrochrysene, a selective ER-beta antagonist, significantly blocked the E3- and 3beta-Adiol-mediated attenuation of late Ca2+ signal increases. ER-beta and thromboxane-prostanoid receptor (TPR) were coexpressed in coronary arteries and aorta. In vivo E3 treatment attenuated aortic TPR expression. Furthermore, in vitro treatment with E3 or 3beta-Adiol downregulated TPR expression in VMC, which was blocked for both agonists by pretreatment with R,R-tetrahydrochrysene. E3- and 3beta-Adiol-mediated reduction in persistent Ca2+ signals is associated with ER-beta-mediated attenuation of TPR expression and may partly explain estrogen benefits in coronary vascular muscle.

Medroxyprogesterone acetate and dihydrotestosterone induce coronary hyperreactivity in intact male rhesus monkeys.

Coronary hyperreactivity (CH), characterized by persistent severe vasoconstrictions in response to vasoconstrictor challenge, is oppositely influenced by progesterone (P) and medroxyprogesterone acetate (MPA) treatment in surgically menopausal primates. In this study we tested whether multiweek MPA or dihydrotestosterone (DHT) exposure induced CH in intact male rhesus monkeys. Coronary angiographic experiments with intracoronary serotonin and the thromboxane A(2) analog U46619 stimulated brief vasoconstriction (for 1-3 min) in large epicardial coronaries in untreated male monkeys. In contrast, MPA- and DHT-treated monkeys displayed long-duration constrictions (>5 min), with significantly greater reductions in the minimal diameters of epicardial coronaries. Immunocytochemistry demonstrated androgen receptors (AR) and P receptors in aorta and coronary arteries, and immunocytochemistry and Western blotting showed AR and P receptors in rhesus coronary vascular muscle cells. In vivo, MPA or DHT increased thromboxane prostanoid (TP) receptor expression in the aorta. In vitro, MPA or DHT increased, whereas P did not change, TP receptor expression in primary coronary vascular muscle cell. This MPA- or DHT-mediated increase in TP receptor expression was attenuated by the AR antagonist flutamide. MPA or DHT induction of CH in intact adult male primates, hypothesized to occur via androgenic up-regulation of vascular muscle TP receptor expression, could predispose to CH-mediated myocardial ischemia.

Prevention of coronary hyperreactivity in preatherogenic menopausal rhesus monkeys by transdermal progesterone.

OBJECTIVE: To test if transdermal progesterone (P) confers coronary vascular protection in surgically menopausal preatherosclerotic rhesus monkeys. METHODS AND RESULTS: Ovariectomized rhesus monkeys fed an atherogenic diet (AD) for 19 months were treated with an investigational transdermal P cream (n=7) or identical placebo cream (n=5) for 4 weeks. Aorta and carotids showed fatty streaks and Oil Red O staining demonstrated lipid deposition. Serum P levels in P-treated rhesus monkeys (0.6 ng/mL) were significantly greater than placebo (0.2 ng/mL). Significant elevation of cholesterol, LDL cholesterol, and HDL cholesterol, was noted in all animals. Lp(a) was significantly attenuated in the AD-fed P-treated monkeys. Coronary angiographic experiments stimulating vasoconstriction by intracoronary injections of serotonin plus U46619 showed exaggerated prolonged actions amplified by AD, but significant protection against severe prolonged vasoconstriction in P-treated monkeys. Immunocytochemistry confirmed co-expression of P and thromboxane prostanoid (TP) receptors in coronaries and aorta. Western blotting demonstrated TP receptor attenuation in vascular muscle after P treatment. CONCLUSIONS: Coronary hyperreactivity, a putative component of coronary artery disease mediated via increased vascular muscle thromboxane prostanoid receptors, can be prevented by subphysiological levels of P, not only in nonatherosclerotic (previously shown) but also in preatherosclerotic primates.