The vascular endothelium is a single cell layer that lines the lumen of the entire vasculature. It is the site of synthesis of nitric oxide (NO), a vasodilatory compound synthesized by endothelial nitric oxide synthase (eNOS). NO causes intracellular calcium sequestration of the vascular smooth muscle cells, relaxing and dilating the arteries. Age profoundly affects endothelium-dependent vasodilation, leading to specific losses of NO. We sought to determine what causes the age-specific loss of endothelial NO. This was accomplished by investigating whether there are differences in markers of eNOS post-translational regulation elements in the aortic endothelium of young (2-4 months; corresponding to an adolescent human adult) and old (32-34 months; corresponding to a 65-75 year-old human). F 344 x Brown Norway hybrid rats. Results show that maximal eNOS activity significantly declines with age (n=4;p���0.05) though there was no change in eNOS protein levels in the aortic endothelium. Endothelial NOS exists in two distinct subcellular fractions. No alterations were detected in the soluble, inactive fraction while significantly less eNOS protein is detected in the active, plasma membrane fraction of the endothelium (n=4;p���0.02). Endothelial NOS activation is also controlled by its phosphorylation state. In this work we demonstrate that free ceramides and ceramide-activated phosphatase (PP2A) activity are significantly elevated with age in the endothelium and correlate with specific alterations in eNOS phosphorylation status consistent with its inactivation. These changes were concomittent with an age-associated decline in endothelial glutathione (GSH) and increased sphingomyelinase activity which liberates ceramides from membrane sphingolipids. In previously published reports we demonstrated that the dithiol compound R-��-lipoic acid (LA) increased maximal NO synthesis in cultured endothelial cells and that LA improved age-associated loss of eNOS stimulatory phosphorylation in rats. Therefore, we administered pharmacologic doses of LA (40 mg/kg, i.p. over 24 h) to old rats to determine whether it restored NO-dependent vasomotor function. Results show that LA significantly increased endothelial GSH (p���0.05 compared to saline controls), decreased sphingomyelinase activity and reversed the age-related increase in ceramide (p���0.01) in old animals. Finally, LA significantly improved endothelium-dependent vasodilation, suggesting that it might be a good therapeutic agent for age-related vascular endothelial dysfunction. / Graduation date: 2005

I conducted electrophysiological work in C57BL/6J (B6) and PWD/PhJ (PWD) mice, with the goal of providing insight into the genetic and physiological controls of calcium intake. Prior behavioral preference tests indicated that PWD mice have higher preferences for calcium compounds compared to B6 mice, though several mechanisms could underlie this observation. I therefore measured taste-evoked chorda tympani (CT) responses in B6 and PWD mice, in order to investigate the specific role of taste sensation. A second experiment was conducted to investigate the role of the calciumsensing receptor (CaSR) is in gustatory transduction of calcium ions, using the CaSR agonist glutathione. In experiment 1, responses were significantly larger in PWD than B6 mice for CaCl2, MgCl2, citric acid and quinine, but did not differ between the strains for sucrose, KCl and NaCl. These strain differences in CT responses were especially large for tonic, rather than phasic, responding. These data suggest that differences in peripheral events, such as taste transduction, contribute to differences between B6 and PWD mice in preferences for taste solutions such as CaCl2. In experiment 2, glutathione at 100 μM had negligible effects on taste-evoked CT responses, which does not support a role for CaSR in mediating taste transduction of calcium ions. / Department of Physiology and Health Science

The present study determined whether dietary supplementation with reduced glutathione (GSH), glutathione ester (GSHE) or N-acetylcysteine (NAC) increased the mitochondrial level of GSH, the major antioxidant inside cells, in rat kidney cortex and medulla. Nine month-old female Lewis rats were given daily intraperitoneal injections of isotonic saline (n=6), or saline containing GSH (250mg or 0.81mmol/Kg of body wt; n=7), GSHE (12mg or 0.03mmol/Kg; n=8), or NAC (200mg or 1.22mmol/Kg; n=8) for four weeks. At the end of the injection period, the rats were anesthetized and the kidneys removed. The kidneys were separated into cortical and medullary sections, weighed, and homogenized. The sections were separated into cytosolic and mitochondrial fractions by differential centrifugation. The GSH levels were determined by a colorimetric assay. Cortical and medullary mitochondrial GSH levels were significantly increased by all three supplements. Cytosolic GSH levels were also significantly increased in both cortical and medullary sections. Thus, dietary supplementation can significantly increase the mitochondrial pool of GSH in the rat kidney. / Department of Physiology and Health Science

This study investigated whether exogenous supplementation with reduced glutathione (GSH) increased kidney mitochondrial and cytosolic GSH levels in young and old female Lewis rats. The young rats were 3 months of age and old rats were 22 months old. The rats were divided into a young control group (n=8), an old control group (n=5), a young experimental group (n=7), and an old experimental group (n=7). Rats in the young and old control groups did not receive any treatment, while rats in both the young and old experimental groups were injected with GSH (250 mg/Kg of body weight) into the peritoneal cavity once a day for a week. At the end of the injection period, the rats were anesthetized and kidneys were harvested. The mitochondrial and cytosolic fractions were separated from rat cortex and medulla by differential centrifugation. GSH concentrations were measured using a spectrophotometric assay. Both mitochondrial and cytosolic GSH levels in kidneys from young and old female Lewis rats were significantly increased with GSH supplementation. The results indicate that kidneys from both young and old rats respond to exogenous dietary supplementation with GSH. / Access to thesis permanently restricted to Ball State community only. / Department of Physiology and Health Science