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The mesenteric vascular bed from the streptozotocin (STZ) diabetic model was used in this thesis to elucidate the mechanisms underlying diabetic vascular dysfunction. Treatment strategies targeting oxygen free radicals, angiotensin II, and lipids were investigated. In phenylephrine-preconstricted preparations, maximum vasodilation to acetylcholine, progressively deteriorated over 8 weeks of diabetes both before and after NO synthase inhibition which isolated the EDHF component. Chronic preventive treatment with silymarin, a free radical scavenger, or allopurinol, a xanthine oxidase inhibitor, partially protected against the development of 4-week diabetic deficits of the NO and EDHF systems. On the other hand, treatment with the semicarbizide-sensitive amine oxidase (SSAO) inhibitor, MDL74972A, only significantly improved the NO component. Preventive treatment with the transition metal chelator, trientine, produced significant protection of the NO and EDHF responses. Furthermore, intervention treatment not only protected against the development of an 8-week but also reversed some of the 4-week diabetic deficit. Both preventative and intervention treatments targeting angiotensin II production through either angiotensin-converting enzyme (ACE) inhibition with lisinopril or AT1 receptor blockade with candesartan provided some protection against the diabetic-induced decline in acetylcholine relaxations. Most notably, candesartan preventive treatment completely protected against a deficit in the EDHF response. Preventive treatment with rosuvastatin, a lipid-lowering drug, partially protected against the development of NO and EDHF deficits. The results show that experimental diabetes had deleterious effects on NO and EDHF-mediated vasodilation and suggest a role for free radicals, angiotensin II and lipids in this dysfunction.

During catalysis the flavoprotein domain of neuronal nitric-oxide synthase (nNOS) shuttles NADPH-derived reducing equivalents from FAD to FMN and then to the P450-heme enabling heme-based oxygen activation and subsequent NO-synthesis. The binding of Ca�����-activated calmodulin (Ca�����-CaM) to nNOS alleviates inhibition of flavin mediated electron transfer within the diflavin domain, which is demonstrated by the increase in the rate of 2,6-dichioroindoiphenol (DCIP) reduction by 2 to 3 fold and that of cytochrome c����� by 10 to 20 fold. To investigate the effect of the Ca�����-CaM on the nNOS reductase activity, the steady-state kinetics of basal and CaM-stimulated reduction of these two substrates was studied. Parallel initial velocity patterns indicated that both substrates are reduced in a ping-pong mechanism. Product and dead-end inhibition data with DCIP as the electron acceptor were consistent with a di iso ping-pong bi-bi mechanism. In contrast, product and dead-end inhibition studies with cytochrome c����� as the second substrate were consistent with an iso (two-site) ping-pong mechanism. Ca�����-CaM did not alter the proposed kinetic mechanisms; however, it did effect to varying degrees the (k[subscript cat]/K[subscript]m) for the various substrates. The pH-dependence of basal and CaM-stimulated reduction of DCIP revealed that ionizable groups involved in the binding of substrates and catalysis are not altered by Ca�����-CaM. However, the activated cofactor does influence catalytic rate constants and/or ionizable groups involved in cytochrome c����� reduction. nNOS was found to abstract the pro-R (A-side) hydrogen from NADPH. Primary deuterium isotope effects (NADP(D)) and solvent isotope effects (SKIE) suggests that of the two half reactions, the reductive half reaction involving NADPH oxidation limits the overall reaction rate, but that hydride transfer to FAD is not the slow step. A small value of [supercript D](V/K)[subscript NADPH] (1.2-1.6) suggests hydride transfer is not the rate-limiting step within the reductive half-reaction. Large solvent kinetic isotope effects (SKIE) were observed on (V/K)[subscript cytc] for basal and CaM stimulated reduction of cytochrome c����� suggesting that proton uptake from the solvent limits the rate of the oxidative half-reaction. A small SKIE on V and (V/K)[subscript NADPH] indicates that proton uptake does not limit the overall reaction rate. Proton inventory analysis revealed multiple transition-state protons contributed to the observed SKIE. / Graduation date: 2001

Nitric oxide (NO) is a short lived free radical regulating bone turnover and bone cell function (1, 2). Osteoclasts are multinucleated bone resorbing cells which form by fusion of pre-osteoclasts. In addition, NO is a signaling molecule in mechanical loading of the bone (3), and in orthodontic tooth movement (OTM) (4). In OTM, force is applied to the tooth and transferred to the bone resulting in bone remodeling leading to tooth movement. This project has two parts: 1) NO in osteoclastogenesis: a) An intense NO signal was observed in pre-osteoclasts preceding cell fusion. b) Osteoclastogenesis increased when cells were exposed to the NOS inhibitor, L-NMMA, during their differentiation phase. c) In contrast, pre-osteoclast fusion decreased in presence of to L-NMMA during the fusion phase. d) NOS inhibitors, decreased osteoclast formation. e) The inhibitory effect of L-NMMA on osteoclast formation was abolished with increasing concentrations of sRANKL. f) NO donors increased osteoclast formation. g) An increase in NO production coincided with pre-osteoclasts fusion. h) Inhibiting fusion decreased osteoclast formation and NO production. i) L-NMMA decreased, while NO donors increased actin free barbed ends. Conclusion: While NO initially negatively regulates pre-osteoclast differentiation, it later facilitates the fusion of mononuclear pre-osteoclasts, possibly by up regulating actin remodeling. 2) Involvement of NO in OTM: Differential expression of NOS isoforms was investigated in periodontal ligament (PDL) and bone in tension and pressure sides using immunohistochemistry with NOS isoforms in a rat model of OTM. a) Expression of all isoforms was increased in the tension side. b) iNOS and nNOS expressions in the pressure side with the cell free zone were decreased while in the pressure side without the cell free zone were increased. c) The intensity of eNOS staining was increased in the tension side. d) Duration of force only changed the pattern of nNOS expression. e) Osteocyte NOS expression did not change. Conclusion: All NOS isoforms are involved in OTM with different expression patterns between the tension and pressure with nNOS being more involved in early OTM. PDL cells, rather than osteocytes are the mechanosensors in early OTM with regards to NO signaling.

Nitric oxide (NO) is a short lived free radical regulating bone turnover and bone cell function (1, 2). Osteoclasts are multinucleated bone resorbing cells which form by fusion of pre-osteoclasts. In addition, NO is a signaling molecule in mechanical loading of the bone (3), and in orthodontic tooth movement (OTM) (4). In OTM, force is applied to the tooth and transferred to the bone resulting in bone remodeling leading to tooth movement. This project has two parts: 1) NO in osteoclastogenesis: a) An intense NO signal was observed in pre-osteoclasts preceding cell fusion. b) Osteoclastogenesis increased when cells were exposed to the NOS inhibitor, L-NMMA, during their differentiation phase. c) In contrast, pre-osteoclast fusion decreased in presence of to L-NMMA during the fusion phase. d) NOS inhibitors, decreased osteoclast formation. e) The inhibitory effect of L-NMMA on osteoclast formation was abolished with increasing concentrations of sRANKL. f) NO donors increased osteoclast formation. g) An increase in NO production coincided with pre-osteoclasts fusion. h) Inhibiting fusion decreased osteoclast formation and NO production. i) L-NMMA decreased, while NO donors increased actin free barbed ends. Conclusion: While NO initially negatively regulates pre-osteoclast differentiation, it later facilitates the fusion of mononuclear pre-osteoclasts, possibly by up regulating actin remodeling. 2) Involvement of NO in OTM: Differential expression of NOS isoforms was investigated in periodontal ligament (PDL) and bone in tension and pressure sides using immunohistochemistry with NOS isoforms in a rat model of OTM. a) Expression of all isoforms was increased in the tension side. b) iNOS and nNOS expressions in the pressure side with the cell free zone were decreased while in the pressure side without the cell free zone were increased. c) The intensity of eNOS staining was increased in the tension side. d) Duration of force only changed the pattern of nNOS expression. e) Osteocyte NOS expression did not change. Conclusion: All NOS isoforms are involved in OTM with different expression patterns between the tension and pressure with nNOS being more involved in early OTM. PDL cells, rather than osteocytes are the mechanosensors in early OTM with regards to NO signaling.