Depolarization of mitochondria in neurons promotes activation of nitric oxide synthase and generation of nitric oxide

Abstract

-The diverse signaling events following mitochondrial depolarization in neurons are not clear. We examined for the first time the effects of mitochondrial depolarization on mitochondrial function, intracellular calcium, neuronal nitric oxide synthase (nNOS) activation, and nitric oxide (NO) production in cultured neurons and perivascular nerves. Cultured rat primary cortical neurons were studied on 7-10 days in vitro, and endothelium-denuded cerebral arteries of adult Sprague-Dawley rats were studied ex vivo. Diazoxide and BMS-191095 (BMS), activators of mitochondrial K ATP channels, depolarized mitochondria in cultured neurons and increased cytosolic calcium levels. However, the mitochondrial oxygen consumption rate was unaffected by mitochondrial depolarization. In addition, diazoxide and BMS not only increased the nNOS phosphorylation at positive regulatory serine 1417 but also decreased nNOS phosphorylation at negative regulatory serine 847. Furthermore, diazoxide and BMS increased NO production in cultured neurons measured with both fluorescence microscopy and electron spin resonance spectroscopy, which was sensitive to inhibition by the selective nNOS inhibitor 7-nitroindazole (7-NI). Diazoxide also protected cultured neurons against oxygen-glucose deprivation, which was blocked by NOS inhibition and rescued by NO donors. Finally, BMS induced vasodilation of endothelium denuded, freshly isolated cerebral arteries that was diminished by 7-NI and tetrodotoxin. Thus pharmacological depolarization of mitochondria promotes activation of nNOS leading to generation of NO in cultured neurons and endothelium-denuded arteries. Mitochondrial-induced NO production leads to increased cellular resistance to lethal stress by cultured neurons and to vasodilation of denuded cerebral arteries. MITOCHONDRIAL MEMBRANE POTENTIAL is a critical regulator of cellular activity and survival and is controlled by a variety of factors including the ATP-sensitive potassium (mitoK ATP ) channels located on the inner mitochondrial membrane (2). Pharmacological activators of mitoK ATP channels, such as BMS-191095 (BMS) and diazoxide, decrease the mitochondrial membrane potential by facilitating the influx of potassium from cytosol into the matrix (2). Signaling events following mitochondrial depolarization appear to be diverse in the various cell types comprising the neurovascular unit (cerebral vascular endothelium and smooth muscle, perivascular neurons, parenchymal neurons, and glia). Both BMS and diazoxide applications result in dilation of isolated cerebral arteries; however, individual effects on vascular smooth muscle and endothelium, which contribute to the overall vascular effect, are completely different (20,22,35,42). Thus BMS and diazoxide relax cerebral vascular smooth muscle cells via generation of localized calcium sparks by sarcoplasmic reticulum linked to mitochondrial depolarization, resulting in decreased global intracellular Ca 2ϩ ([Ca 2ϩ ] i ) (20, 42). Conversely, mitochondrial depolarization in cerebral endothe...

Role of Mitochondria in Cerebral Vascular Function: Energy Production, Cellular Protection, and Regulation of Vascular Tone

“…10). In a recent study, we found that preconditioning with diazoxide protected cultured neurons against oxygen-glucose deprivation (OGD) via steps beginning with mitochondrial activation and involving production and generation of NO (50,89). We had previously shown that NO is also a major component in the induction of preconditioning in the intact brain following cortical spreading depression (75) and similar results have been reported by other laboratories (142).…”

“…Although it has been suggested that mitochondria contain a variant of nitric oxide synthase (NOS) in normal cells, and thus could produce NO via this enzymatic pathway, there is significant evidence against this view (98-100). Nonetheless, NO arising from nonmitochondrial sources, such as endothelial cells, VSM, astroglia, pericytes, and neurons (88,89) can affect the functioning of the electron transport chain by reducing mitochondrial oxygen consumption and ATP production (132) and could combine with superoxide anion to form peroxynitrite (54,58,145). We have shown that peroxynitrite is a potent activator of mitochondria (101).…”

“…We have shown that peroxynitrite is a potent activator of mitochondria (101). On the other hand, we have shown that NO production from cytosolic NOS occurs due to NOS phosphorylation and/or increased cytosolic calcium in endothelial cells and neurons following pharmacological activation of mitochondria (88,89). NO appears to have a restraining effect on mitochondrial oxygen consumption in intact cerebral arteries (132).…”

“…Our findings, however, demonstrated for the first time that mitochondrial depolarization and enhanced mitochondrial ROS production are not mandatorily linked events in cells. In isolated brain mitochondria, cultured neurons, astroglia and cerebral vascular endothelium, and intact cerebral vascular endothelium and VSM, the agent BMS-191095 causes mitochondrial depolarization without ROS production and promotes diverse cellular responses such as pre-/post-conditioning and VSM relaxation; thus, mitochondrial depolarization alone is sufficient to activate distinct cellular signaling pathways (12,15,64,86,88,89). Our findings with BMS-191095 are supported by results from other laboratories (126,143,144) and by other experimental approaches in our laboratory (41).…”

“…Although it is known that extracellular ATP, from either glycolysis or oxidative phosphorylation, as well as the metabolites adenosine, AMP, and ADP (3,115) can alter cerebrovascular tone via plasmalemmal purinergic receptors (115,123), only a few studies have examined direct nonpurinergic mitochondrial influences on the diameter of cerebral resistance vessels (26,85,86,88,89,131,132,148,149). Thus, the study of mitochondrialinitiated events on the cerebral vasculature is a relatively new field.…”