Role of NAD(P)H oxidase in the regulation of cardiac L-type Ca channel function during acute hypoxia

Abstract

Objective: The role of NAD(P)H oxidase in regulating cellular production of reactive oxygen species (ROS) and the L-type Ca 2+ channel during acute hypoxia was examined in adult ventricular myocytes from guinea pig. Methods: The fluorescent indicator dihydroethidium (DHE) was used to detect superoxide and the response of the L-type Ca 2+ channel to hadrenergic receptor stimulation was used as a functional reporter since hypoxia increases the sensitivity of the L-type Ca 2+ channel (I Ca-L ) to isoproterenol (Iso). Results: Hypoxia caused a 41.2 T 5.2% decrease in the rate of the DHE signal (n = 21; p < 0.01). Of the classical NAD(P)H oxidase inhibitors, DPI but not apocynin mimicked the effect of hypoxia on the sensitivity of I Ca-L to Iso. However, the potent NAD(P)H oxidase agonist angiotensin II had no effect on cellular superoxide or the sensitivity of I Ca-L to Iso. Although DPI inhibits NAD(P)H oxidase, it also decreased superoxide in isolated mitochondria in a concentration-dependent manner. Partial inhibition of mitochondrial function with nanomolar concentrations of FCCP or myxothiazol mimicked the effect of hypoxia on cellular superoxide and the sensitivity of I Ca-L to Iso. In addition, hypoxia caused a 69.3 T 0.8% decrease in superoxide in isolated mitochondria (n = 4; p < 0.01), providing direct evidence for a role for the mitochondria. Conclusions: Our data suggest that mitochondria appear to be involved in oxygen sensing, regulation of cellular ROS, and the function of I Ca-L during acute hypoxia in cardiac myocytes and NAD(P)H oxidase does not appear to contribute substantially.

Transient Exposure to Hydrogen Peroxide Causes an Increase in Mitochondria-Derived Superoxide As a Result of Sustained Alteration in L-Type Ca 2+ Channel Function in the Absence of Apoptosis in Ventricular Myocytes

“…10,16 Generation of superoxide was assessed in 24-hour guinea pig myocytes or 36-hour contracting neonatal myocytes using the fluorescent indicator dihydroethidium (DHE) (5 mol/L, 515-to 560-nm excitation filter, 590 long pass emission; Molecular Probes), as previously described. 17 Application of the superoxide scavengers N-tert-butyl-␣-phenyl-nitrone and superoxide dismutase significantly decreased DHE signal, confirming specificity of the indicator for superoxide (nϭ4). 17 Comparisons using inhibitors were made with cells exposed to 30 mol/L H 2 O 2 for 5 minutes followed by 10 U/mL catalase for 5 minutes and were performed on the same day.…”

Endothelin-1 Regulates Cardiac L-Type Calcium Channels via NAD(P)H Oxidase-Derived Superoxide

“…ROS generation was determined using the oxidant-sensitive fluorogenic probe DHE (excitation wavelength, 488 nm; emission wavelength, 610 nm) essentially as described elsewhere (Hool et al, 2005). In brief, primary cultured cardiomyocytes were loaded with 100 nM DHE for 30 min at 37°C.…”

Oxidative stress and oxidant signaling in obstructive sleep apnea and associated cardiovascular diseases

“…Wright et al [92] showed that prolyl hydroxylation is the oxygen-sensing mechanism in cardiac myocytes, which in turn regulates HIF-1, heme oxygenase-1, and GLUT1. While the role of NADPH oxidase in O 2 sensing in cardiac myocytes has not been reported, this enzyme has been shown to be expressed and play a role in the development of cardiac hypertrophy and in the transition to heart failure [93][94][95][96][97][98][99][100]. The concept that the heart directly senses changes in oxygen tension has recently been supported by results from our laboratory that 20 min of intermittent hypoxia can elicit significant changes in gene expression and protein modifications in mouse hearts [101].…”