**4.4. Mitochondrial KATP channels target for therapy**

reperfusion injury. The effective gene delivery without toxic side effects was established by intravenous injection of the gene vectors during experiments on animal models before the ischemic insult. A significant progress in the area of kidney biology, especially in heredita‐ ry kidney disease and inflammatory and fibrotic diseases was achieved by the use of adenovirus as a vector for kidney-directed gene therapy [30]. Although some advantages make adenoviral vectors suitable for gene transfer into complex organs such as the kidney. But in contrast some disadvantages downgrade these vectors. For instance, the expression of the transfected gene is limited to weeks or months in this technique, because adenovi‐ rus does not integrate into the host genome. Secondly, the adenovirus can elicit immunolog‐ ical responses, therefore vector cannot be administered repeatedly. During emergency situations in other inflammatory renal disease states, the SOD gene therapy with adenovi‐ ral vector is recommended, however, occurrence of harmful effects maximum within a week

ALS a neurodegenerative disease leads to paralysis, muscle wasting, and death, usually within 2 - 3 years of symptom onset due to death of motor neurons. The central mechanism by which motor neuron death occurs in familial ALS is oxidative stress which is due to the mutations in the antioxidant enzyme SOD1gene. Many hypotheses studied so far using ALS mouse models. Some of these studies showed that SOD1 mutants have very low benefits (3, 35). One of the most important pharmacological outcomes obtained in ALS mouse models was increasing expression of either growth factors such glial cell-derived neurotrophic factor, IGF-1, and VEGF (11–13) or RNAi molecules by the delivery of viral vectors (14–16) to silence SOD1 mutant gene expression. In gene therapy the primary cause of toxicity (i.e. mutant SOD1 proteins) is targeted, unlike drug therapy which usually acts on cell survival or deleterious

Reduction of myocardial reperfusion injury through an effective immunization with SOD and catalase has also been hypothesised. Indeed, the cardioprotective effect of intracoronary infusion of SOD may further increase with coadministration of catalase. It is proven that calcium antagonists, rennin-angiotensin system antagonists, Na+/H+ exchanger inhibitors, nitric oxide donors and adenosine induce cardioprotective effects during primary angioplasty for the management of acute myocardial infarction. When these reagents were administrated using intracoronary infusion, their efficiency has increased. Another way to attenuate myo‐ cardial ischemia-reperfusion injury is anterograde intracoronary and intravenous adminis‐ tration of anti- P-selectin and anti- ICAM-1 antibodies. The ideal injection route for these antibodies is retrograde intracoronary infusion, which has direct access to postcapillary

Application of inhibitors of cellular redox maintaining proteins which reduce intracellular ROS is complementary to the use of pro-oxidant molecules, for example, administeration of catalase or SOD in association with various peroxidases. 2-Methoxyestradiol by increasing cellular ROS formation due to its inhibition of SOD, enhances the cytotoxic effects of apoptotic agents and displays anti-leukemic activity in culture. On the other hand it has been hypothesized, continuous mitochondrial ROS formation leading to oxidative stress and mitochondrial damage has link to degenerative diseases and aging. Based on the ROS etiology of aging the

is expected (e.g., post-transplant acute renal failure) [29].

74 Toxicology Studies - Cells, Drugs and Environment

pathways [29].

venules [29].

Potassium channel openers (KCOs) are agents, discovered in the early 1980s, that act by stimulating ion flux through K+ channels. Many drugs such as, diazoxide, nicorandil, and cromakalim have been identified as KCOs. KCOs act on two types of ion channels: Ca2+ activated K+ channels (BK channels) and ATP-regulated K+ channels (KATP channels). KCOs were first identified by their antihypertensive or antianginal mode of action. Now, they are at various stages of development as and cardioprotective agents. Preclinical and clinical evidence also supports the therapeutic role of KCOs in vascular and pulmonary hypertension, and the treatment of overactive bladder. Until recently, it was believed that the effects of KCOs were entirely attributed to the modulation of K+ channels in cell plasma membranes. However, it is now proven, that new targets for KCOs exist in intracellular membranes including those of mitochondria, zymogen granules, and sarcoplasmic reticulum. It seems that Mitochondria are particularly very important targets for KCOs, because the interaction of these compounds with mitochondria appears to mediate the cardioprotection of KCOs. The protective role of mitochondrial ion channels was recently summarized and mitochondrial targets for antiischemic drugs were recently described [32].

#### *4.4.1. Potassium channel openers and mitochondrial K+ channels*

A small-conductance potassium channel, with properties similar to those of the KATP channel from the plasma membrane, in the inner membrane of rat heart and liver mitochondria and designated the mitoKATP. The mitoKATP channel was blocked not only by ATP, but also, similarly to the plasma membrane KATP channel, by antidiabetic sulfonylureas. These obser‐ vations raised the question whether the mitoKATP channel could be activated by KCOs. In fact, an increased influx of K+ and depolarization of liver mitochondria in the presence of KCOs was observed. Also, other KCOs were shown to activate potassium ion transport into mito‐ chondria. KCOs such as levcromakalim, cromakalim, and pinacidil have been shown to depolarize cardiac mitochondria. KCO-induced membrane depolarization was associated with an increase in the rate of mitochondrial respiration and decreased ATP synthesis.More‐ over, KCOs released cytochrome *c*and calcium ions from cardiac mitochondria. Despite the effect on K+ transport, diazoxide also exhibits a direct effect on mitochondrial energy metab‐ olism by inhibition of respiratory chain complex II in liver mitochondria. Recently, mitoKATP channel opener BMS-191095 with no peripheral vasodilator activity was described.Using isolated mitochondria or proteoliposomes reconstituted with partly purified mitoKATP channel and measuring potassium flux demonstrated that heart and liver liver mitochondrial KATP channels have some pharmacological similarities with the cell membrane KATP channel, i.e., both channels are activated by KCOs. Mitochondrial KATP channels are 1000 times more sensitive to diazoxide than that of cell membrane KATP channels. This document concluded that the interaction of mitochondrial KATP channels with KCOs plays a key role in cardiopro‐ tection [32].

#### *4.4.2. Mitochondrial KATP channel: A novel target for cardioprotection*

Mitochondrial KATP channel: A Novel target for Cardioprotection. KCOs mimic hypoxic/ ischemic preconditioning in the absence of ischemia in the heart myocardial cells, the reason why antagonists of KATP channel, like 5-hydroxydecanoic acid and glibenclamide, ameliorate the positive effects of short time hypoxic/ischemic conditions on the heart myocardium. The primary postulation to justify these events includes cell membrane KATP channels. Newly, it was shown that in fact KCOs including diazoxide affect the mitoKATP channel in mitochondria. In a complementary approach, it was shown that diazoxide did not activate plasma membrane KATP channels, but induced oxidation of mitochondrial flavoproteins, due to the activation of mitoKATP channel. These findings established the fact that the target for the diazoxide protec‐ tive effects in heart myocytes is the mitochondrial KATP channel rather than the cell membrane KATP channel. It is also note worthy that evidence for mitochondrial KATP channels as effectors of cardiac myocardial preconditioning has also been proven in human subjects. The initial observations on the cardioprotective action of KCOs on mitochondria were further confirmed and developed in a series of reports. It has been shown that other KCOs such as nicorandil, cromakalim, and pinacidil modulate mitochondrial Ca2+ uptake, respiration, mitochondrial membrane potential, ATP generation, and mitochondrial Ca2+ uptake. The main question remains how the opening of the mitoKATP channel could protect cells against ischemic damage. 1) Opening of the mitoKATP channel followed by mitochondrial swelling could improve mitochondrial ATP handling and/or production. 2) The protective effect of mitoKATP activation could be mediated by lowering Ca2+ overloading of mitochondria. In fact, it was found that diazoxid preserves mitochondrial function in ischaemic rat cardiomyocyte. It is now proven that hypoxia approximately decreases mitochondrial oxygen consumption rate to 40% of the normal value, and administration of diazoxide maintains the prehypoxic mitochondrial oxygen consumption rate during hypoxia/ischemia. Cardiac ATP concentration was signifi‐ cantly raised following diazoxide treatment. Secondly, by lowering Ca2+ overloading of mitochondria the protective effect of mitochondrial KATP activation could be induced. It was shown that the opening of the mitochondrial KATP channel may increase mitochondrial reactive oxygen species (ROS) formation. This increase could lead to protein kinase C activa‐ tion, which is known to be necessary for the cardioprotection. Besides, it seems that mito‐ chondrial KATP channel is enrolled in delayed preconditioning because of an alteration in expression of "protective" proteins (3). It was that pretreatment of hippocampal neurons with cromakalim and diazoxide increases the expression level of Bcl-2 and Bcl-XL which are involved in the control of apoptosisBcl-2 [32].
