We have previously provided evidence that the pentose phosphate pathway (PPP)/glucose-6-phosphate dehydrogenase (G6PD) and NADPH redox is involved in modulating contractile function of the coronary (CA) artery. However, the machanism(s) by which G6PD and NADPH modulates contractile function of CA are obscure. Therefore, the primary focus of this proposal will be, to elucidate the signaling pathways involved in mediating the effects of G6PD and NADPH redox on smooth muscle cell L-type Ca2+ currents and CA function. To achieve these goals, we will, in Aim #1 determine if G6PD is active in the sub- cellular fractions of resting and contracting CA, by estimating the rate of glucose oxidation, and the G6PD activity levels by biochemical and radioisotope tracer assays. Furthermore, we will identify mechanism(s) involved in contractile agents-induced-G6PD activation, by investigating the role of PKC and metabolic pathways.
In Aim #2, we will determine whether G6PD mediates L-type Ca2+ channel activity, intracellular Ca2+, and vasomotor tone in resting and contracting CA, by examining L-type Ca2+ function, measure intracellular Ca2+ changes and vasomotor function after inhibiting G6PD with pharmacological agents and siRNA transfection, and in G6PD deficient mouse aorta.
In Aim #3, we will determine whether glucose-6-phosphate dehydrogenase modulates the L-type Ca2+ channel function, intracellular Ca2+, contraction and redox changes, in CA via direct physical interaction with the ion channel proteins (alpha subunit of CaV1.2), by co-immunoprecipitation, co-localization and in-vitro binding assays. Additionally, we will determine whether direct binding of NADP+ or NADPH to the L-type Ca2+ channel protein inactivates the channel and whether changes in the levels of reduced/oxidized glutathione (GSH) or hydrogen peroxide (H2O2), induced by decrease in NADPH levels (due to the inhibition of G6PD activity), modulates L-type Ca2+ channel function, in smooth muscle cells isolated from coronary and aorta of G6PD deficient mouse. The PPP/G6PD and NADPH redox is up-regulated in diabetes, pulmonary hypertension and heart failure, thereby suggesting a potential role for G6PD and NADPH redox in profoundly impairing the contractile function of blood vessels in these diseases. This study, on completion as anticipated, will prove to be useful in developing novel therapies for the treatment of vascular dysfunction in pulmonary hypertension, diabetes, and heart failure. In the current project, we have undertaken a task to determine whether metabolic changes play a role in the development of circulatory system malfunction, which is a major cause of morbidity and mortality in the USA. This study, therefore, on completion as anticipated, will prove to be useful in developing novel therapies to treat vascular dysfunction in pulmonary hypertension, diabetes, and heart failure.
|Chettimada, Sukrutha; Gupte, Rakhee; Rawat, Dhwajbahadur et al. (2015) Hypoxia-induced glucose-6-phosphate dehydrogenase overexpression and -activation in pulmonary artery smooth muscle cells: implication in pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 308:L287-300|
|Rawat, Dhawjbahadur K; Alzoubi, Abdallah; Gupte, Rakhee et al. (2014) Increased reactive oxygen species, metabolic maladaptation, and autophagy contribute to pulmonary arterial hypertension-induced ventricular hypertrophy and diastolic heart failure. Hypertension 64:1266-74|
|Chettimada, Sukrutha; Ata, Hirotaka; Rawat, Dhwajbahadur K et al. (2014) Contractile protein expression is upregulated by reactive oxygen species in aorta of Goto-Kakizaki rat. Am J Physiol Heart Circ Physiol 306:H214-24|
|Ochi, Rikuo; Chettimada, Sukrutha; Gupte, Sachin A (2014) Poly(ethylene glycol)-cholesterol inhibits L-type Ca2+ channel currents and augments voltage-dependent inactivation in A7r5 cells. PLoS One 9:e107049|
|Chettimada, Sukrutha; Rawat, Dhwajbahadur K; Dey, Nupur et al. (2012) Glc-6-PD and PKG contribute to hypoxia-induced decrease in smooth muscle cell contractile phenotype proteins in pulmonary artery. Am J Physiol Lung Cell Mol Physiol 303:L64-74|
|Schneider, Andrew M; Rawat, Dhwajbahadur; Weinstein, L Steve et al. (2012) Effects of laparoscopic Roux-en-Y gastric bypass on glucose-6 phosphate dehydrogenase activity in obese type 2 diabetics. Surg Endosc 26:823-30|
|Gupte, Sachin A; Wolin, Michael S (2012) Relationships between vascular oxygen sensing mechanisms and hypertensive disease processes. Hypertension 60:269-75|
|Rawat, Dhwajbahadur K; Hecker, Peter; Watanabe, Makino et al. (2012) Glucose-6-phosphate dehydrogenase and NADPH redox regulates cardiac myocyte L-type calcium channel activity and myocardial contractile function. PLoS One 7:e45365|
|Gupte, Rakhee S; Ata, Hirotaka; Rawat, Dhawjbahadur et al. (2011) Glucose-6-phosphate dehydrogenase is a regulator of vascular smooth muscle contraction. Antioxid Redox Signal 14:543-58|
|Ata, Hirotaka; Rawat, Dhwajbhadur K; Lincoln, Thomas et al. (2011) Mechanism of glucose-6-phosphate dehydrogenase-mediated regulation of coronary artery contractility. Am J Physiol Heart Circ Physiol 300:H2054-63|
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