Nitric oxide inhibits the pannexin 1 channel through a cGMP-PKG dependent pathway.

Nitric oxide (NO), a major gaseous signaling molecule, modulates several ion channels and receptors. Here we show that NO attenuates pannexin 1 (Panx1) mediated currents in HEK-293 cells. NO exerts its effect by activating a cGMP-protein kinase G (PKG) dependent pathway. NO donors, sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO), reduced Panx1 currents by 25-41%. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase (sGC), blocked the inhibition completely, whereas sGC activator YC-1 mimicked the effect of NO, suggesting the involvement of a cGMP dependent pathway. Supporting this, NO had no effect in the presence of the PKG inhibitor, KT5823. Further, immuno-precipitated Panx1 was recognized by an anti-phosphoserine antibody in Western blot. Phosphorylation was enhanced significantly when cells were treated with SNP. The target for phosphorylation is possibly Ser 206 of Panx1, as its mutation to Ala completely abolished the NO mediated inhibition.

Nitric oxide and oral cancer: a review.

Nitric oxide (NO), a short-lived, endogenously produced gas, plays key role in various physiological as well as pathological processes. NO-inducing cell signaling events within the cell producing it and the diffusibility of it in other cells have led to the discovery of various physiological functions of NO including vasodilation, respiration, cell migration, immune response and apoptosis. On the other hand, excessive and unregulated NO synthesis has been implicated in many pathophysiological conditions including cancer. Research on NO, during the past few years is one of the growing areas in cancer biology. The high incidence of oral cancer and precancer has been linked with habits of tobacco chewing and smoking and NO has been said as the "messenger of death" in tobacco related diseases. NO seems to play a part in various stages of carcinogenesis from initiation to progression. However, there is considerable controversy and confusion in understanding its role in cancer biology. It is said to have both, tumoricidal as well as tumor promoting effects and these depend on its timing, location and concentration. Further, NO has also been shown to have antitumor, chemopreventive and therapeutic abilities. Here is an overview in which efforts are made to understand the role of this molecule in oral carcinogenesis.

P2X7 receptor-pannexin 1 hemichannel association: effect of extracellular calcium on membrane permeabilization.

Activation of P2X(7) receptor (P2X(7)R) and pannexin have been implicated in membrane permeabilization associated with ischemic cell death and many other inflammatory processes. P2X(7)R has a unique property of forming large pore upon repeated or prolonged application of agonist like ATP or 2', 3'-(4-benzoyl) benzoyl ATP. It has been proposed that pannexin 1 (panx1) hemichannel associates with P2X(7)R to form large pore, though the actual mechanism is not yet understood. Calcium concentration in extracellular milieu drops in many patho-physiological conditions, e.g. ischemia, when P2X(7)R/pannexin is also known to be activated. Therefore, we hypothesize that extracellular calcium ([Ca(2+)](o)) plays an important role in the coupling of P2X(7)R-panx1 and subsequent membrane permeabilization. In this study we show that membrane permeability of the P2X(7)R and panx1 expressing N2A cell increases in ([Ca(2+)](o))-free solution. In [Ca(2+)](o)-free solution, fluorescent dye calcein trapped cells exhibited time-dependent dye leakage resulting in about 50% decrease of fluorescence intensity in 30 min. Control cells in 2 mM [Ca(2+)](o) did not show such leakage. Like N2A cells, mixed culture of neuron and glia, derived from hippocampal progenitor cells showed similar dye leakage. Dye leakage was blocked either by pannexin-specific blocker, carbenoxolone or P2X(7)R antagonists, Brilliant Blue G, and oxidized ATP. Furthermore P2X(7)R and panx1 were co-immunoprecipitated. The amount of P2X(7)R protein pulled-down with panx1, increased by twofold when cells were incubated 30 min in [Ca(2+)](o)-free buffer. Taken together, the results of this study demonstrate the activation and association of P2X(7)R-panx1, triggered by the removal of [Ca(2+)](o).

Evaluation of the role of nitric oxide in acid sensing ion channel mediated cell death.

Acid sensing ion channels (ASICs) are widely expressed in central and peripheral nervous system. They are involved in a variety of physiological and pathophysiological processes: synaptic transmission, learning and memory, pain perception, ischemia, etc. During ischemia, metabolic acidosis causes the drop of extracellular pH (pHe) which in turn activates ASICs. Activation of calcium permeable ASIC1a has been implicated in neuronal death. ASICs are modulated by several redox reagents, divalent cations and nitric oxide (NO). Although NO potentiates ASIC mediated currents, the physiological significance of such modulation has not been studied in detail. We have evaluated the role of endogenous NO in cell death at different pH, mediated by the activation of ASICs. At pH 6.1, death rates of ASIC1 expressing Neuro2A (N2A) cells are significantly higher in comparison to the cells that do not express ASICs. Amiloride, a blocker of ASICs protects the cell from acid-injury. Sodium nitroprusside, a potent NO donor not only increases the ASIC mediated currents but also increases cell death at low pH. L-Arg, the precursor of NO also potentiates ASICs in a pH dependent manner. L-Arg-induced NO production and potentiation of ASICs were observed at pHs 7.4, 7.2, 7.0 and 6.8. Lowering the pH below 6.8 did not result in significant production of NO or potentiation of ASICs upon L-Arg stimulation. Our results suggest that potentiation of ASICs by NO and subsequent cell death in vivo depends on the severity of acidosis. During mild and moderate acidosis, NO promotes cell death by potentiating ASICs, whereas this potentiation subsides in severe acidosis due to inhibition of NO synthase.

Urinary CCN2 (CTGF) as a possible predictor of diabetic nephropathy: preliminary report.

BACKGROUND: It is currently impossible to reliably predict which diabetic patients will develop nephropathy and progress to kidney failure. Microalbuminuria, often regarded as a predictor of overt diabetic renal disease is, in fact, an indicator of established glomerular damage. We have shown that glomerular expression of the prosclerotic cytokine CCN2 (CTGF) is greatly up-regulated early in experimental and in human diabetes and mesangial cell exposure to CCN2 increases its production of extracellular matrix (ECM) molecules responsible for glomerulosclerosis. As an early marker, we therefore investigated the presence of CCN2 in urine and the relationship to diabetes and/or renal disease in an experimental model of diabetes and in a limited patient population. METHODS: Urine samples from (1) healthy rats, (2) rats made diabetic by streptozotocin (STZ), (3) healthy human volunteers, (4) diabetic patients with renal disease, and (5) diabetic patients without renal disease were examined by Western blotting and/or enzyme-linked immunosorbent assay (ELISA) for qualitative and quantitative analysis of the of CCN2. RESULTS: Low levels of urinary CCN2 were present in healthy, control rats, but were increased approximately sevenfold overall in STZ-diabetic animals. CCN2 levels were the highest at week 3 of diabetes, then decreased with time, but remained significantly elevated over controls even after 32 weeks. Consistently low levels of urinary CCN2 were also detected in healthy volunteers (mean value, 7.1 CCN2/mg creatinine). However, levels were elevated approximately sixfold in the majority of diabetic patients with nephropathy. A small number of the diabetic patients not yet exhibiting evidence of renal involvement demonstrated CCN2 urinary levels that were ninefold greater than controls. The remaining normoalbuminuric diabetic patients demonstrated CCN2 levels indistinguishable from those of healthy volunteers. Analysis by Western blotting confirmed the identity of the urinary CCN2. A molecular species equivalent to full-length CCN2 (37/39 kD doublet) was present in healthy controls. In contrast, the nephropathic group demonstrated multiple CCN2 bands. CONCLUSION: These findings support our hypothesis that CCN2 is up-regulated early in the evolution of glomerulosclerosis, including that of diabetes. We contend that urinary CCN2 may both stage nephropathy and predict those patients who are destined for progressive glomerulosclerosis and end-stage renal disease (ESRD). Cross-sectional and prospective studies of larger, well-defined diabetic patients groups will be required to prove this hypothesis, and are ongoing.