P133. Oocyte dysfunction in diabetes mellitus: Role of nitric oxide

Posters / Nitric Oxide 14 (2006) A57–A66 Methods: Cardiac function was examined with echocardiography and invasive pressure-volume analysis after challenge with saline (C) or endotoxin (LPS: Escherichia coli 0111:B4). Cell shortening (CS) and [Ca2+]i were measured by video-edge detection and Fura-2 fluorescence, respectively. Myofilament sensitivity to Ca2+ was determined in skinned myocytes by measuring the change in sarcomere length (DSL) in response to varying calcium concentrations. Myocardial oxidative stress was estimated by multiple methods including lucigenin-enhanced chemiluminescence, dihydroethidium staining of frozen sections, dichlorodihydrofluorescein staining of isolated cardiomyocytes, and nitrotyrosine immunoblots. Impact of NOS3 was further examined in mice subjected to colon ascendens stent peritonitis (CASP), a polymicrobial model of sepsis. Results: Endotoxin decreased LV fractional shortening and a load-independent measure of LV function dP/dtmax divided by instantaneous pressure (dP/dtmax/IP) in WTLPS and KOLPS but not in TGLPS. While [Ca2+]i transients were depressed similarly in myocytes isolated from WTLPS and TGLPS, %CS was depressed only in myocytes isolated from WTLPS. DSL was greater in skinned myocytes from TGLPS than in those from WTLPS suggesting higher myofilament sensitivity to Ca2+ in TGLPS. Co-treatment with a thiol reducing agent, dithiothreitol, augmented the calcium sensitivity in skinned WTLPS myocytes, but not in TGLPS myocytes, suggesting that NOS3 protects against myofilament thiol-modification. Production of reactive oxygen species (ROS) was increased in WTLPS but not TGLPS myocardium in part due to increased xanthine oxidase (XO) activity. Administration of a XO inhibitor, allopurinol, or a NO-donor, molsidomine, prevented myocardial dysfunction in WTLPS. Myocardial function was impaired in WT but not in TG 24 h after CASP. Myocyte-specific overexpression of NOS3 markedly improved survival of mice after endotoxin challenge or CASP. Conclusions: These results suggest that increased myocardial NO levels attenuate endotoxin-induced ROS production and increase myofilament sensitivity to calcium thereby reducing myocardial dysfunction and mortality in murine models of septic shock. Improved survival of TG in sepsis suggests that preserved myocardial function is a key determinant of survival in severe septic shock. doi:10.1016/j.niox.2006.04.198

P132. Serum, urinary, and salivary nitric oxide in rheumatoid arthritis: complexities of interpreting nitric oxide measures J. Brice Weinberg a, Thomas Lang b, William E. Wilkinson c, David S. Pisetsky d, E. William St. Clair c a Medicine, Duke and VA Medical Centers b University of Maryland School of Medicine c Duke University Medical Center d Duke and VA Medical Centers Nitric oxide (NO) may play important roles in rheumatoid arthritis (RA). RA is an inflammatory disease involving joints and other systems including salivary glands. To assess NO production in RA patients, we compared levels of serum, urine, and salivary nitrite and nitrate (NOx) in patients with RA and normal subjects, and we examined the relationships of these measures to disease activity. Serum, urine, and salivary NOx levels as well as renal creatinine and NOx clearance rates were compared in 25 RA patients and 20 age- and gender-matched healthy controls. Subjects were hospitalized for 3 days and placed on a NOx-restricted diet. NOx was assayed using nitrate reductase and the Griess reagent. RA activity was assessed using standard clinical and laboratory measures. While consuming a restricted diet for 3 days to eliminate the effects of oral intake on NOx, 24 h urinary NOx excretion decreased in both RA patients and healthy controls. Urine NOx levels at all time points were not significantly different between RA patients and normal subjects. Serum NOx levels also decreased during the 3 days of NOx restriction, but RA patients had higher serum NOx levels at all time points compared with the control group. Likewise, serum NOx/creatinine ratios were higher in RA patients than in controls. Although basal salivary flow rate and tear flow were lower

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in RA patients, salivary NOx levels did not differ between normal and RA subjects. Renal creatinine clearance was not different between the two groups, but we found that RA patients had lower renal NOx clearance. Thus, the higher serum NOx levels could be due to decreased renal NOx clearance in RA. After correction of p values for multiple comparisons, there were no significant relationships for the RA group between measures of disease activity and the urinary NOx, serum NOx, or urinary NOx clearance. Despite interest in the use of NO as a marker of disease activity, alterations in renal nitrate clearance in RA make it difficult to assess in vivo NO production even with strict dietary restriction of NOx intake. doi:10.1016/j.niox.2006.04.199

P133. Oocyte dysfunction in diabetes mellitus: Role of nitric oxide Anuradha P. Goud a, Pravin T. Goud b, Michael P. Diamond b, Husam Abu-Soud b a CS Mott Center for Human Growth and Development, Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI, USA b Wayne State University, Detroit, MI, USA Metabolic alterations in diabetes mellitus (DM) cause a severe impact during early conception leading to abnormal embryo development and could be related to oocyte dysfunction. We recently demonstrated a protective role of NO in delaying oocyte aging. Current study was undertaken to find if diminished NO bioavailability is operative in oocytes from diabetic mice. Methods: In experiment set 1, oocytes retrieved from superovulated ALS/LtJ diabetic mice and B6D2F1 non-diabetic mice at 13.5, 16 and 18 h post-hCG (set 1, groups A, B and C, n = 83). In set 2, oocytes at 16 h post-hCG were treated with NO donor (S-nitroso-N-acetyl-penicillamine, SNAP 0.23 lM/min, 3 h, n = 24) or the NO synthase inhibitor, N-omega-nitro-L-arginine methyl ester (L-NAME, 1 mM, 3 h, n = 21). Zona pellucida dissolution time (ZPDT), ooplasmic microtubule dynamics (OMD) and premature cortical granule (CG) loss were studied as parameters for oocyte aging using confocal microscopy. Results: Similar ZPDT were noted among oocytes from group A (18.5 ± 3.7 versus 17.3 ± 3.9 s), a significant increase in ZPDT was noted in relatively old oocytes from diabetic versus non-diabetic mice (group B: 35.2 ± 4.6 versus 46.2 ± 6.1 s, group C: 51.5 ± 6.1 vs 80.3 ± 6.4 sec, P < 0.001 for both). Significantly more oocytes from diabetic mice exhibited markedly increased OMD in all groups, while CG loss was predominantly noted in groups B and C (P < 0.05). In set 2, a remarkable diminution with SNAP (P = 0.01), and a significant increase with L-NAME was observed for ZPDT (P < 0.01), OMD as well as CG loss in both, diabetic as well as non-diabetic mice (P < 0.05). Conclusions: Oocytes from DM mice exhibit enhanced post-ovulatory aging, indicating a narrowing of the temporal window for optimal fertilization. Oocytes from diabetic mice are exquisitely sensitive to NOS inhibitor-induced aging, while NO has an anti-aging effect. Thus, oocyte ‘NO-insufficiency’ may be a possible causative mechanism for embryo failure in DM. doi:10.1016/j.niox.2006.04.200

P134. Cardiomyocyte-specific overexpression of nitric oxide synthase 3 improves left ventricular remodeling in mice with chronic pressure overload Emmanuel S. Buys a, Davinder S. Jassal b, Tom G. Neilan b, Michael J. Raher b, Fumito Ichinose b, Stefan Janssens c, Michael H. Picard b, Kenneth D. Bloch b, Marielle Scherrer-Crosbie b a Cardiovascular Research Center, Massachusetts General Hospital b Massachusetts General Hospital c Catholic University of Leuven-Flanders Interuniversity Institute for Biotechnology