Separation of the intermediates of leucine catabolism by high-performance liquid chromatography

A method has been developed for the separation of leucine, 2-ketoisocaproic acid, isovaleryl CoA, 3-methylcrotonyl CoA, 3-hydroxy-3-methylglutaryl CoA, 3-methylglutaconyl CoA, acetyl CoA, and acetoacetic acid by ion-exchange high-performance liquid chromatography. The analysis requires 180 min. Use of this method to assess the catabolism of radiolabeled leucine in normal cultured human skin fibroblasts shows that these cells do not accumulate CoA esters, but convert leucine mainly to 2-ketoisocaproic acid, glutamate, and hydroxyisovalerate. In the fibroblasts of a patient with maple syrup urine disease, only 2-ketoiscaproic acid is produced from leucine.     

Selective endothelial dysfunction in conscious dogs after cardiopulmonary bypass

Zanaboni, Paul, Paul A. Murray, Brett A. Simon, Kenton Zehr,Kirk Fleischer, Elaine Tseng, and Daniel P. Nyhan. Selective endothelial dysfunction in conscious dogs after cardiopulmonary bypass.J. Appl. Physiol. 82(6):1776-1784, 1997.It has previously been demonstrated thatcardiopulmonary bypass (CPB) causes prolonged pulmonary vascularhyperreactivity (D. P. Nyhan, J. M. Redmond, A. M. Gillinov, K. Nishiwaki, and P. A. Murray. J. Appl.Physiol. 77: 1584-1590, 1994). Thisstudy investigated the effects of CPB on endothelium-dependent(acetylcholine and bradykinin) and endothelium-independent (sodiumnitroprusside) pulmonary vasodilation in conscious dogs. Continuousleft pulmonary vascular pressure-flow (LP-) plots were generated in conscious dogs before CPB and again in the same animals 3-4 days post-CPB. The dose of U-46619 used to acutely preconstrict the pulmonary circulation to similar levels pre- andpost-CPB was decreased (0.13 ± 0.01 vs. 0.10 ± 0.01 mg · kg¹ · min¹,P < 0.01) after CPB. Acetylcholine,bradykinin, and sodium nitroprusside all caused dose-dependentpulmonary vasodilation pre-CPB. The pulmonary vasodilator response toacetylcholine was completely abolished post-CPB. For example, at leftpulmonary blood flow of 80 ml · kg¹ · min¹acetylcholine (10 µg · kg¹ · min¹)resulted in 72 ± 15% reversal (P < 0.01) of U-46619 preconstriction pre-CPB but caused no changepost-CPB. However, the responses to bradykinin and sodium nitroprussidewere unchanged post-CPB. The impaired pulmonary vasodilator response toacetylcholine, but not to bradykinin, suggests a selective endothelialdefect post-CPB. The normal response to sodium nitroprusside indicates that cGMP-mediated vasodilation is unchanged post-CPB.

     

Anesthesia alters pulmonary vasoregulation by angiotensin II and captopril.

We investigated the effects of an intravenous (pentobarbital sodium) and inhalational (halothane) general anesthetic on the pulmonary vascular responses to angiotensin II and angiotensin-converting enzyme inhibition (CEI). Multipoint pulmonary vascular pressure-flow (P/Q) plots were generated in conscious pentobarbital- (30 mg/kg iv) and halothane-anesthetized (approximately 1.2% end-tidal) dogs in the intact (no drug) condition, during angiotensin II administration (60 ng.kg-1.min-1 iv), and during CEI (captopril 1 mg/kg plus 1 mg.kg-1.h-1 iv). In conscious dogs, angiotensin II increased (P less than 0.001) the pulmonary vascular pressure gradient [pulmonary arterial pressure--pulmonary arterial wedge pressure (PAP-PAWP)] over the empirically measured range of Q; i.e., angiotensin II caused pulmonary vasoconstriction. Pulmonary vasoconstriction (P less than 0.01) in response to angiotensin II was also observed during pentobarbital sodium anesthesia. In contrast, angiotensin II had no effect on the P/Q relationship during halothane anesthesia. In conscious dogs, CEI decreased (P less than 0.001) PAP-PAWP over the empirically measured range of Q; i.e., CEI caused pulmonary vasodilation. However, CEI caused pulmonary vasoconstriction (P less than 0.02) during pentobarbital sodium and had no effect on the P/Q relationship during halothane. Thus, compared with the conscious state, the pulmonary vasoconstrictor response to angiotensin II is unchanged or abolished, and the pulmonary vasodilator response to CEI is reversed to vasoconstriction or abolished during pentobarbital sodium and halothane anesthesia, respectively.     

Nitric oxide regulates tissue transglutaminase localization and function in the vasculature

The multifunctional enzyme tissue transglutaminase (TG2) contributes to the development and progression of several cardiovascular diseases. Extracellular rather than intracellular TG2 is enzymatically active, however, the mechanism by which it is exported out of the cell remains unknown. Nitric oxide (NO) is shown to constrain TG2 externalization in endothelial and fibroblast cells. Here, we examined the role of both exogenous and endogenous (endothelial cell-derived) NO in regulating TG2 localization in vascular cells and tissue. NO synthase inhibition in endothelial cells (ECs) using N-nitro l-arginine methyl ester (l-NAME) led to a time-dependent decrease in S-nitrosation and increase in externalization of TG2. Laminar shear stress led to decreased extracellular TG2 in ECs. S-nitrosoglutathione treatment led to decreased activity and externalization of TG2 in human aortic smooth muscle and fibroblast (IMR90) cells. Co-culture of these cells with ECs resulted in increased S-nitrosation and decreased externalization and activity of TG2, which was reversed by l-NAME. Aged Fischer 344 rats had higher tissue scaffold-associated TG2 compared to young. NO regulates intracellular versus extracellular TG2 localization in vascular cells and tissue, likely via S-nitrosation. This in part, explains increased TG2 externalization and activity in aging aorta.