Effect of Nitric Oxide on Acid Inducing Cell Death



Ischemia is a Greek word which means (isch- is restriction, hema or haema is blood) a restriction in blood supply, it is generally due to factors in the blood vessels, with resultant damage or dysfunction of tissue.. In India more than million people are suffering from cerebral ischemia or cardiac ischemia. There are several types of ischemia such a global ischemia, focal ischemia, cerebral ischemia, cardiac ischemia and so on. Ischemia of heart muscle produces angina pectoris. Since oxygen is mainly bound to hemoglobin in red blood cells, insufficient blood supply causes tissue to become hypoxic, or anoxic, if no oxygen is supplied at all. which might cause necrosis (i.e. cell death). In very aerobic tissues such as heart and brain, necrosis due to ischemia usually takes about 3-4 hours before becoming irreversible. This time period and typically some collateral circulation to the ischemic area, accounts for the efficacy of "clot-buster" drugs such as Alteplase, given for stroke and heart-attack.

However, complete cessation of oxygenation of such organs for more than 20 minutes typically results in irreversible damage. Heart kidneys, and brain are among the organs that are the most prone to inadequate blood supply. For example, ischemia in brain tissue, due to stroke or head injury, causes a process called the ischemic cascade to be unleashed, in which proteolytic enzymes, reactive oxygen species, and other harmful chemicals, damage and may ultimately kill brain tissue. Restoration of blood flow after a period of ischemia can actually be more damaging than the ischemia. Reperfusion of oxygen causes a greater production of damaging free radicals, resulting in reperfusion injury due to which necrosis can be greatly accelerated.

Mechanisms Of Cell Injury After Ischemia

Cerebral ischemia may be either transient and followed by reperfusion, or essentially permanent where in a region of the brain may be affected, as occurs during an arterial or venous stroke, or the entire brain may become globally ischemic, as occurs during a cardiac arrest. In addition to such conditions where ischemia is the primary insult, ischemia may also contribute secondarily to brain damage in case of mass lesions, hemorrhage, or trauma. Within seconds of cerebral ischemia, local cortical activity ceases which can be detected by electroencephalography; if the ischemia is global, unconsciousness rapidly ensues (witness the Stokes-Adams attack). This massive shutdown of neural activity is induced by K+ efflux from neurons, mediated initially by the opening of voltage-dependent K+ channels and later by ATP-dependent K+ channels, leading to transient plasma membrane hyper polarization.

Despite this energy sparing response, a few minutes later, , an abrupt and dramatic redistribution of ions occurs across the plasma membrane, associated with membrane depolarization (efflux of K+ and influx of Na+, Cl–, and Ca2+). This “anoxic depolarization” results in the excessive release of neurotransmitters, in particular, glutamate, promoting further spatial spread of cellular depolarization, depletion of energy stores, and advancement of injury cascades.

Glutamate Induced Neuronal Death

The main excitatory neurotransmitter throughout the central nervous system (CNS) is the dicarboxylic amino acid, glutamate. Reflecting this ubiquitous role in cell-cell signaling, average whole brain glutamate concentrations are on the order of 10 mM, with presumably much higher concentrations within synaptic vesicles. Under ischemic conditions, transmitter glutamate is massively released (initially mediated by vesicular release from nerve terminals, and later by reverse transport from astrocytes), reaching near millimolar concentrations in the extracellular space. Unfortunately, such concentrations of glutamate are neuro-toxic, and substantial evidence now implicates the toxicity of glutamate (excitotoxicity) in the pathogenesis of neuronal death after ischemia and other acute insults. Extracellular glutamate accumulating under ischemic conditions overstimulates N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate-type glutamate receptors, promoting Na+ influx and K+ efflux through glutamate receptor-activated membrane channels.

NMDA receptor–gated ion channels are additionally highly permeable to Ca2+ and mediate Ca2+ influx into neurons. The gating of glutamate receptor–activated channels effectively achieves membrane shunting, which spreads in waves (spreading depression) from the ischemic core out toward the margins of the ischemic zone (ischemic penumbra). Spreading depression increases metabolic demand and energy failure, thus further enhancing glutamate release. As Na+ and Ca2+ entry is joined by the influx of Cl– and water, marked neuronal cell body swelling and dendrite swelling occur, which are hallmarks of necrosis death.. Elevations in neuronal intracellular free Ca2+ ([Ca2+]i), mediated both directly by NMDA receptors and indirectly via membrane depolarization–activated voltage-gated Ca2+ channels and reverse operation of the Na+-Ca2+ exchanger, bear particular responsibility for promoting spreading depression and triggering deleterious cytotoxic cascades.

Role of Nitric Oxide in Ischemic Brain Injury

There is increasing evidence that nitric oxide (NO), a free radical that can act both as a signaling molecule and a neurotoxin, is involved in the mechanisms of cerebral ischemia. Although early investigations yielded conflicting results, the introduction of more-selective pharmacological tools and the use of molecular approaches for deletion of genes encoding for NO synthase have provided a better understanding of the role of NO in the mechanisms of ischemic brain damage. However it is still not clear whether it is neuro protective or neurotoxic. There are evidences suggesting that NO is protective or destructive depending on the stage of evolution of the ischemic process and on the cellular source of NO. Defining the role of NO in cerebral ischemia provides the rationale for new neuro-protective strategies based on modulation of NO production in the post-ischemic brain.

Mechanism of Brain Damage by Nitric Oxide

NO, like other free radicals, can damage DNA by base deamination (Nguyen et al., 1992 Wink et al., 1991). Zhang et al., (1994) demonstrated that NO activates poly (adenosine 5'-diphosphoribose) synthetase (PARS) in association with damage to DNA, and PARS inhibitors prevent NMDA neuro-toxicity with relative potencies paralleling their inhibition of the enzyme. PARS activation can lead to cell death associated with depletion of ATP and a change in the NAD+/NADH redox state (Gaal et al., 1987). Inhibition of NOS during ischemia should retard this pathway and decrease consumption of both ATP and oxidized NAD+. An increase in ATP availability during ischemia certainly favors cell function, including pH regulation involving the NA+/H+ exchanger and the HCO-3/Cl- transporter, which are energy-dependent. In addition, lack of PARS activation should decrease cellular consumption of NAD+ and reduce the drive for NADH regeneration coupled to lactic acid synthesis. Free radical-mediated damage is another key common pathway of injury.

When NO reacts with super oxide anions (O-2) it forms peroxynitrite anions (ONOO-) (Beckman and Crow, 1993; Lipton et al., 1993). These ions decompose to yield highly damaging hydroxyl free radicals, which have been suggested to initiate lipid peroxidation (Watson, 1993). In addition to producing active metabolites, peroxidation may alter cell membrane fluidity , with potential detrimental effects on membrane associated H+ transporter and antiporter which are involved in brain pH regulation. Lei et al. , showed that NO may be neuro-protective against NMDA toxicity, while others have shown no neuro-protective effects . It has been reported that NO may exert both neuro-destructive and neuro-protective effects, depending on its oxidation-reduction status, with NO- being neuro-destructive and NO+ being neuro-protective. Another possible mechanism( what mechanism? Useful or harmful?) is the stimulation by NO of the S-nitrosylation of various proteins . NO can also modify GADPH, an important enzyme of glycolysis . Inhibition of GAPDH will also suppress lactate formation and could reduce brain tissue acidosis. NO has also been reported to inhibit iron-sulfur enzymes of the citric acid cycle and respiratory chain


In spite of several studies carried on , the effect of NO in ischemic cell death is is not well understood .The role of NO in cerebral ischemia is still in debate.

The present study is focused on the following objectives

(a) To study the nitric oxide dose response in ischemic condition and to determine cell death in various nitric oxide concentrations.

(b) To determine whether NO influences ASIC to induce cell death.

(c) To analyze whether the cell death in low pH is due to ASICs.


Ischemia can be described as restriction in blood supply to a of the body. It is caused due to blockage of blood vessels. Ischemia is of several types such as global ischemia, cerebral ischemia, focal ischemia, cardiac ischemia and so on. Cerebral ischemia is an ischemic condition where the parts of the brain do not receive enough blood flow to maintain normal neurological function. The normal brain requires complete oxidation of glucose to full fill its energy requirements and in ischemic conditions oxygen depletion forces the brain to switch to anaerobic glycolysis which causes the accumulation of lactic acid which in turn causes pH to fall by production of protons by ATP hydrolysis Major factors which are responsible for ischemia are oxygen depletion, low pH,opening of ASICs, high concentration of calcium ions, abnormal synthesis of Nitric oxide(NO).NO is an important signaling molecule but however it has been postulated as brain pathogenesis.

The dual role of NO is still debatable. In this present study the effect of NO is determined on the ischemic cell by LDH assay. Neuroblastoma cells were exposed to various concentration of NO using (NO donors) and it was found that NO in low concentration is neuroprotective and high concentration of NO is triggering ASIC to cause cell death. Further to determine whether the acid inducing cell death is due to ASIC cells were treated with and LDH assay was carried out for cell injury determination which gave significant results as treatment with amiloride decreases the cell death.






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