Thesis (doctoral)--Lund University, 1996. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1996. / Added t.p. with thesis statement inserted.

Thesis (Ph.D.)--Ohio University, November, 2006. / Includes bibliographical references (leaves 155-168).

Nitric oxide (NO) is a key physiological messenger, but an excessive production of this molecule can be detrimental, leading to the onset or worsening of many pathological conditions. Dimethylarginine dimethylaminohydrolase (DDAH) is a key enzyme in the NO pathway, involved in the metabolism of asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA), which are both endogenous inhibitors of NO synthesis. Two isoforms of DDAH have been identified in humans, namely DDAH-1 and DDAH-2. DDAH inhibition represents a promising strategy in the treatment of NO overproduction under pathological conditions without affecting the homeostatic role of this messenger. In this work I described the design and synthesis of 12 novel potential DDAH inhibitors together with the development of a new UPLC-MS based assay to measure the activity of HEK293T cell lysates overexpressing recombinant human DDAH-1 in metabolizing ADMA into dimethylamine and L-citrulline. The same assay was used to assess the potential of the novel compounds, as well as of the well-known DDAH inhibitor L-257, to inhibit DDAH-1 catalyzed L-citrulline formation from ADMA. Three of the novel molecules (compounds 10a, 14a and 14b) showed very interesting inhibitory activity: in particular, the methylacylsulfonamide analogue of L-257 (10a) resulted in 13-fold higher inhibition potency than L-257 itself (98% of inhibition at 1mM, IC50 = 3±3 μM and Ki = 1±0 μM). This molecule was chosen for molecular dynamics simulations to study the putative mechanism for 10a inhibition of DDAH-1 activity. Furthermore, DDAH-1 and DDAH-2 were engineered introducing a FLAG-tag at the C-terminal of the proteins to allow their purification from the lysate components by immunoprecipitation. Although the purification protocol requires some further improvement, the fusion proteins did not show to be functionally affected by the modification.

These studies explored interactions between flavanols and nitric oxide in order to investigate implications for vascular health. Study 1 investigated acute effects of flavanol consumption on cutaneous microvascular endothelial function in young and older individuals along with chronic exposure in older individuals. This was accomplished by assessing skin blood flow response to local heating (thermal reactivity, TR); skin was clamped at 34°C and 40°C and values were normalized to those attained at 43°C. Older individuals demonstrated an attenuated TR at baseline during the entire local heating phase (58.4 ± 2.5% versus 49.3 ± 2.6%, p<0.05). Acutely following flavanol ingestion there was a significant increase in TR (52.4 ± 2.1% versus 56.1 ± 2.0%, p=0.05) that was not different with age. There was no effect of chronic flavanol exposure on TR in older individuals; however, there was a significant decrease in mean arterial pressure (95 ± 3 mmHg versus 91 ± 3 mmHg, p<0.001). These results contribute to research regarding flavanols increasing NO bioavailability; acutely via an improvement in cutaneous microvascular endothelial function and chronically via a reduction in blood pressure. Study 2 investigated the acute effects of flavanol consumption on cerebrovascular endothelial function in young and older individuals along with chronic flavanol exposure in older individuals. This was accomplished by assessing basal cerebral blood flow indices (cerebral vascular conductance index, CVCi) and CBF response to hypercapnia (cerebral vasomotor reactivity; CVMR). At baseline older individuals demonstrated a reduced CVCi (0.85 ± 0.04 cm/s*mmHg versus 0.55 ± 0.04 cm/s*mmHg p=0.001) and CVMR (8.6 ± 0.6 versus 6.9 ± 0.4, p=0.05). An unexpected finding was that flavanol ingestion led to an acute decrease in CVCi (0.71 ± 0.04 cm/s*mmHg versus 0.62 ± 0.04 cm/s*mmHg p<0.05) and CVMR (8.6 ± 0.6 versus 6.1 ± 0.5, p=0.001) that was not different with age. In older individuals, chronic exposure led to a significant increase in CVCi (0.60 ± 0.05 cm/s*mmHg versus 0.72 ± 0.06 cm/s*mmHg, p<0.05) but had no effect on CVMR. These data provide evidence for an improvement in cerebral hemodynamics following chronic exposure in older individuals. / text

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