Mdm2-P53 Interaction Inhibitor with Cisplatin Enhances Apoptosis in Colon and Prostate Cancer Cells In-Vitro.

OBJECTIVE: To study the effect of RITA (MDM2-p53 interaction inhibitor) and its action along with genotoxic drug cisplatin was evaluated on COLO-205 colon cancer and PC-3 prostate cancer cells. METHOD: Various in-vitro parameters to determine cytotoxic and apoptotic potential of RITA with genotoxic drug cisplatin were evaluated. The potentiation of cytotoxic effect was evaluated using MTT assay and colony forming assay, mechanism of cell death by Etbr/AcO assay and the mechanism of apoptosis was determined by caspase-3 release assay. RESULTS: The findings from MTT confirmed the best possible potent combination of 5+5µM and 10+10µM concentration of Cisplatin and RITA respectively. These combinations were further evaluated for its chemo sensitizing effect which confirmed the significant reduction in number of colonies in combination as compared to monotherapy. Also, the results of Etbr/AcO assay were in line with the colony forming assay. For apoptotic activity, it was noted that increasing the concentration of cisplatin and RITA (10µM), did not affect much to apoptotic activity and was found to be equally effective to that of low dose (5µM) concentration. The same results were seen in Caspase-3 release effect on both the cell lines. CONCLUSION: Our present study provides compelling evidence that pharmacological activation of the p53 by blocking the MDM2-p53 interaction is a promising cancer therapeutic strategy and using RITA in combination with Cisplatin not only decrease the toxic effect of Cisplatin by decreasing its dose but also increasing the apoptotic effect, warrants clinical evaluation on both colon and prostate cancer.

Preconditioning offers cardioprotection in hyperlipidemic rat hearts: possible role of Dopamine (D2) signaling.

BACKGROUND: Ischemic preconditioning (IPC) induced cardioprotection has been reported to be blunted in hyperlipidemic subjects. Dopamine, via its D2 receptor signaling, appears to mimic the signaling cascade involved in myocardial preconditioning and is also involved in the inhibition of hyperlipidemia induced mediators. The present study was designed to investigate the possible involvement of D2 receptors in IPC and to see whether dopamine preconditioning can offer cardioprotection in hyperlipidemic rat hearts. METHODS: Wistar albino rats were divided into 8 groups and fed on normal or high fat diet for 4 weeks. Hyperlipidemia was confirmed after 4 weeks by serum lipid estimations. Isolated perfused hearts were subjected to ischemic preconditioning or dopamine induced pharmacological preconditioning followed by 30-min ischemic insult and 60-min reperfusion. Clozapine was administered as D2 antagonist. Coronary perfusate (basal and post-ischemic) was collected for the estimations of LDH (Lactate dehydrogenase) and CKMB (Creatine kinase MB). Hearts were then removed and frozen for infarct size measurement. RESULTS: A significant increase body weight, serum lipids except HDL was noted in high fat diet fed rats, as compared to normal rats. The level of LDH, CKMB in coronary effluent and infarct size were found to be decreased in preconditioned normal hearts, as compared to hearts treated with ischemia reperfusion. This effect was found to be blunted in hyperlipidemic animals. Dopamine (10 µM) alone and in combination with ischemic preconditioning significantly reduced the levels of LDH, CKMB and infarct size in hyperlipidemic hearts, as compared to preconditioned and non-preconditioned hyperlipidemic hearts. This effect was abolished significantly by Clozapine (D2 antagonist). CONCLUSION: The present study reveals possible involvement of D2 receptors in ischemic preconditioning and suggests that dopamine preconditioning may offer significant cardioprotection in hyperlipidemic rat hearts.

Mild alcohol intake exacerbates metabolic syndrome in rodents: a putative role of GSK-3ß.

Metabolic syndrome is characterized with abdominal obesity, insulin resistance, dyslipidemia and hepatic dysfunction. Glycogen synthase kinase-3ß (GSK-3ß) expression has been observed in adipose tissues in obese and diabetic humans, and in rodents. The aim of study was to investigate role of GSK-3ß in modulation of metabolic alterations in alcoholic fed rats. Male Wistar albino rats (180-220 g) were used. High fat diet (HFD) for 8 weeks and alcohol (2%) from third to eighth week were given. Lithium chloride (LiCl), a GSK-3ß inhibitor (60 mg/kg) was used orally from third to eighth week. HFD treatment caused significant (p < 0.05) increase in the percentage of body weight gain, BMI, Lee index, different fat pads, liver weights, serum glucose, leptin, triglyceride, LDL, VLDL, cholesterol, alanine transaminase, aspartate transaminase, tissue thio-barbituric acid reactive substances, nitrate/nitrite and significant decrease in food intake (g), serum HDL and tissue GSH in HFD control rats, as compared to normal control (NC). Administration of alcohol (2%) ad libitum potentiated the effect of normal and HFD, respectively, in NC and HFD control rats, respectively. Administration of LiCl produced significant amelioration in biochemical and pathological changes caused in the form of metabolic syndrome in HFD alone and HFD and alcohol-treated rats. The histological observations also showed similar findings in liver tissue. It may be concluded that inactivation of GSK-3ß consequently leads to increased leptin and insulin sensitivity as evidenced by the reversal of alterations caused due to metabolic syndrome in rodents fed with HFD and mild alcohol.

Protective effect and mechanism of Ginkgo biloba extract-EGb 761 on STZ-induced diabetic cardiomyopathy in rats.

UNLABELLED: Diabetes mellitus (DM) is a complex metabolic disorder which leads to development of various long-term complications including cardiomyopathy. Oxidative stress due to hyperglycemia plays a key role in the development and progression of diabetic cardiomyopathy (DC). Oxidative stress causes the opening of mitochondrial permeability transition pore (mPTP) eventually leading to myocardium dysfunction. The Ginkgo biloba extract (EGb 761) has antioxidant and mitochondrial membrane potential stabilizing property. Therefore, this study was designed to evaluate the effect of EGb 761 and its possible mechanism of action in DC. MATERIALS AND METHODS: DM was induced by single injection of Streptozotocin (STZ) (50 mg/kg, i.p.) and cardiac dysfunction was developed on 8(th) weeks after STZ injection. Cardiac dysfunction was assessed by measuring left ventricle weight/body weight (LVW/BW) ratio, left ventricle (LV) collagen content, LV protein content, serum lactate dehydrogenase (LDH) level. RESULTS: EGb 761 treatment (started after 7(th) week of STZ injection and continued for 3 weeks) attenuated cardiac dysfunction in diabetic rats as evidenced by a decrease in LV collagen content, protein content, LVW/BW ratio, serum LDH level. Moreover, EGb 761 attenuated the oxido-nitrosative stress (thiobarbituric acid reactive substances, superoxide anion generation, myocardium nitrite) and concomitantly improved the antioxidant enzyme (reduced glutathione) level as compared to untreated diabetic rats. However, protective effect of EGb 761 was inhibited by atractyloside (mPTP opener) that was given for 3 weeks, 30 min before the EGb 761 treatment. These results indicate that EGb 761 corrects diabetic cardiac dysfunction probably by its direct radical scavenging activity and its ability to inhibit the opening of mPTP channel since the cardioprotective effect of EGb 761 was completely abolished by atractyloside.

Possible modulation of FAS and PTP-1B signaling in ameliorative potential of Bombax ceiba against high fat diet induced obesity.

BACKGROUND: Bombax ceiba Linn., commonly called as Semal, is used in various gastro-intestinal disturbances. It contains Lupeol which inhibits PTP-1B, adipogenesis, TG synthesis and accumulation of lipids in adipocytes and adipokines whereas the flavonoids isolated from B. ceiba has FAS inhibitory activity. The present study was aimed to investigate ameliorative potential of Bombax ceiba to experimental obesity in Wistar rats, and its possible mechanism of action. METHODS: Male Wistar albino rats weighing 180-220 g were employed in present study. Experimental obesity was induced by feeding high fat diet for 10 weeks. Methanolic extract of B. ceiba extract 100, 200 and 400 mg/kg and Gemfibrozil 50 mg/kg as standard drug were given orally from 7th to 10th week. RESULTS: Induction with HFD for 10 weeks caused significant (p < 0.05) increase in % body wt, BMI, LEE indices; serum glucose, triglyceride, LDL, VLDL, cholesterol, free fatty acid, ALT, AST; tissue TBARS, nitrate/nitrite levels; different fat pads and relative liver weight; and significant decrease in food intake (g and kcal), serum HDL and tissue glutathione levels in HFD control rats. Treatment with B. ceiba extract and Gemfibrozil significantly attenuated these HFD induced changes, as compared to HFD control. The effect of B. ceiba 200 and 400 mg/kg was more pronounced in comparison to Gemfibrozil. CONCLUSION: On the basis of results obtained, it may be concluded that the methanolic extract of stem bark of Bombax ceiba has significant ameliorative potential against HFD induced obesity in rats, possibly through modulation of FAS and PTP-1B signaling due to the presence of flavonoids and lupeol.

Mechanism of hyperhomocysteinemia-induced vascular endothelium dysfunction - possible dysregulation of phosphatidylinositol-3-kinase and its downstream phosphoinositide dependent kinase and protein kinase B.

Imbalance of l-arginine/endothelial nitric oxide synthatase (eNOS) activity is the hallmark of vascular endothelium dysfunction. Hyperhomocysteinemia (Hhy) has been identified as a potential risk factor for vascular endothelium dysfunction that leads to cardiovascular disorders. Phosphatidylinositol-3 kinase (PI3K) is a ubiquitous enzyme involved in plethora of cell signaling including the endothelial cells and it has been reported that signaling through this enzyme and its downstream pathway viz phosphoinositide-dependent kinase (PDK)/protein kinase B (Akt) and eNOS is impaired in diseased conditions. Thus present study was designed to investigate the role of PI3K and PDK/Akt in vascular endothelium dysfunction produced by Hhy. Hhy was produced by administering l-methionine (1.7%w/w, p.o). After four weeks of l-methionine administration, vascular endothelium dysfunction was assessed in terms of attenuation of acetylcholine-induced endothelium dependent relaxation (Isolated aortic ring preparation), a decrease in serum nitrite level, mRNA expression of eNOS (rtPCR) and disruption of integrity of vascular endothelium (Electron microscopy). Administration of insulin (0.6 IU/kg/day, s.c), YS-49 (1.6 mg/kg/day, i.p), DAQB1 (5mg/kg/day, i.p) and atorvastatin (30 mg/kg/day, p.o) significantly improved acetylcholine-induced endothelium-dependent relaxation, serum nitrate/nitrite level, mRNA expression of eNOS and integrity of vascular endothelium. This ameliorative effect of insulin was blocked by wortmannin (inhibitor of PI3K), UCN-01(PDK inhibitor), API-2 (Akt inhibitor) and l-NAME (eNOS inhibitor). Thus, it may be concluded that activation of PI3K and its downstream pathways viz. PDK/Akt and eNOS improve Hhy-induced vascular endothelium dysfunction and that therapeutic interventions designed for these pathways may provide potential therapeutic strategies to combat vascular complications.

Neuroprotective role of PDE4 and PDE5 inhibitors in 3-nitropropionic acid induced behavioral and biochemical toxicities in rats.

Phosphodiesterase inhibitors have been reported to be beneficial in cognitive and motor disorders. In the present study, we have investigated the effects of RO 20-1724 (PDE4 inhibitor) and sildenafil (PDE5 inhibitor) in 3-nitropropionic acid (3-NP) induced experimental Huntington's disease in rats. 3-Nitropropionic acid was administered for 14 days (10 mg/kg i.p.) 1h following 3-NP administration, the rats were treated with either vehicle, RO 20-1724 (0.25 and 0.5 mg/kg i.p.) or sildenafil (2 and 4 mg/kg i.p.) for 14 days. Cognitive functions were assessed by using Morris water maze whereas, motor functions were assessed by spontaneous locomotor activity, limb withdrawal and suspended wire test at different time points. Biochemically, markers of oxidative stress and cell damage, such as reduced glutathione, malondialdehyde, nitrite and lactate dehydrogenase levels were assessed terminally in the brain homogenate. Chronic administration of 3-NP produced significant decrease in body weight, showed marked abnormalities in cognitive and motor functions. Further, significant oxidative-nitrosative stress and cell damage was also observed. Chronic administration of RO 20-1724 and sildenafil in 3-NP treated rats significantly and dose dependently attenuated 3-NP induced behavioral and biochemical abnormalities in rats. Both these drugs were equally effective in attenuating 3-NP induced neurotoxicity. These results suggesting that the inhibition of PDE4 and PDE5 would be therapeutic in neurodegenerative disorders associated with cognitive and motor dysfunction.

Phosphodiesterases: Regulators of cyclic nucleotide signals and novel molecular target for movement disorders.

Movement disorders rank among the most common neurological disorders. During the last two decades substantial progress has been made in understanding of the pathological basis of these disorders. Although, several mechanisms have been proposed, downregulation of cyclic nucleotide mediated signaling cascade has consistently been shown to contribute to the striatal dysfunctioning as seen in movement disorders. Thus, counteracting dysregulated cyclic nucleotide signaling has been considered to be beneficial in movement disorders. Cyclic nucleotide phosphodiesterases (PDEs) are the enzymes responsible for the breakdown of cyclic nucleotides and upregulation in PDE activity has been reported in various movement disorders. Thus, PDE inhibition is considered to be a novel strategy to restore cerebral cyclic nucleotide levels and their downstream signalling cascade. Indeed, various PDE inhibitors have been tested pre-clinically and were reported to be neuroprotective in various neurodegenerative disorders associated with movement disabilities. In this review, we have discussed a putative role of PDE inhibitors in movement disorders and associated abnormalities.

Pharmacological inhibition of GSK-3ß produces late phase of cardioprotection in hyperlipidemic rat: possible involvement of HSP 72.

The acute, as well as late, phase of cardioprotection induced by ischemic preconditioning is abolished in hyperlipidemic (HL) rat heart. The pharmacological inhibition of glycogen synthase kinase-3ß (GSK-3ß), has earlier been reported to restore this attenuated acute cardioprotective effect. However, it not known whether GSK-3ß inhibitors administered 24 h before the ischemic injury would restore the late cardioprotective in HL rat and, if yes, the role of heat shock protein 72 (HSP 72) in its modulation. Hyperlipidemia was produced in rat by feeding high-fat diet for 6 weeks. Isolated perfused rat heart was subjected to 30 min of ischemia followed by 120 min of reperfusion (I/R). Myocardial infarct size was estimated by triphenyltetrazolium chloride staining, while lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) levels were analyzed from coronary effluent. GSK-3ß inhibitors, SB 216763 (SB, 0.6 mg/kg, i.p.), and indirubin-3 monoxime (IND, 0.4 mg/kg, i.p.), administered 24 h before the isolation of heart, significantly decreased the I/R-induced myocardial infarct size and the release of LDH and CK-MB. The cardioprotective effect of GSK-3ß inhibitors was significantly attenuated by quercetin (4 mg/kg, i.p.), a HSP 72 inhibitor, administered 1 h before the administration of SB or IND. That the late phase of cardioprotection induced by pretreatment with GSK-3ß inhibitors is not attenuated/lost in HL rat heart is a new finding in our study. Our results indicate that HSP 72 acts on pathway of GSK-3ß and plays a significant role in cardioprotection.

Effect of dipyrone and thalidomide alone and in combination on STZ-induced diabetic neuropathic pain.

Diabetic neuropathy is recognized as one of the most common complications of chronic diabetes, but its pathophysiological mechanism is complex and yet to be completely explored. Monotherapy with conventional analgesics fails to provide adequate pain relief in peripheral diabetic neuropathy. There are a number of evidence suggesting that tumor necrosis factor (TNF-α) plays an important role in the pathogenesis of peripheral diabetic neuropathy. TNF-α up-regulation activates nuclear factor κB, which further up-regulates cyclooxygenase (COX)-2 leading to altered prostaglandin profile. Inhibition of TNF-α and COX-2 provides beneficial effect on diabetic neuropathy by decreasing the oxidative stress level and by preventing neuronal hypersensitivity due to an increased prostaglandin level. The present study was designed to assess the effect of dipyrone and thalidomide on streptozotocin (STZ)-induced neuropathic pain behavior in rats. STZ 50 mg/kg, i.p. was administered to induce experimental diabetes in the rats. Three weeks following STZ, dipyrone (300 and 600 mg/kg, i.p.) and thalidomide (25 and 50 mg/kg, i.p.) alone and subeffective dose combination of dipyrone and thalidomide (300 and 25 mg/kg(-1), i.p.) administered daily for 2 weeks significantly attenuated thermal hyperalgesia, mechanical allodynia, and formalin-induced phase-2 flinching response. Moreover, the subeffective dose combination of dipyrone and thalidomide and preemptive treatment with thalidomide (50 mg/kg) reduces oxidative stress in diabetic rats. In conclusion, the combination of subeffective dose of dipyrone and thalidomide prevented the development and maintenance of experimental diabetic neuropathy. The combination of thalidomide (TNF-α inhibitor) and dipyrone (COX inhibitor) may be used as a potential therapeutic agent for the treatment of diabetic neuropathy.

Ameliorative effect of daidzein: a caveolin-1 inhibitor in vascular endothelium dysfunction induced by ovariectomy.

Estrogen deficiency was produced in female Sprague-Dawley rats by surgical removal of both the ovaries and these animals were used 4 weeks later. Endothelium-dependent and endothelium-independent relaxations due to acetylcholine and sodium nitroprusside were observed respectively, in isolated rat thoracic aortic ring preparation. Extent of lipid peroxidation was measured by estimating serum TBARS. Integrity of vascular endothelium was assessed using hematoxylin and eosin staining. Generation of nitric oxide was measured indirectly, by estimating serum and urinary nitrite/nitrate concentration. Ovariectomy produced significant vascular endothelial dysfunction, measured in terms of reduced acetylcholine-induced endothelium-dependent vasorelaxation, serum and urinary nitrite/nitrate concentration and impairment of integrity of vascular endothelium. Administration of daidzein (0.2 mgkg(-1)day(-1), sc 0.4 mgkg(-1)day(-1), sc and 0.8 mgkg(-1)day(-1), sc) and Atorvastatin (30 mgkg(-1)day(-1), po Positive Control) for one week markedly improved vascular endothelial dysfunction due to increase in nitric oxide bioavailability perhaps by inhibiting caveolin-1 and activation of PI3K-AKT pathway.

Possible mechanism of protective effect of thalidomide in STZ-induced-neuropathic pain behavior in rats.

INTRODUCTION: Diabetes-induced neuropathic pain is recognized as one of the most difficult type of pain to treat and conventional analgesics are well known to be partially effective or associated with potential toxicity. Recently, it has been demonstrated that thalidomide, besides its teratogenic potential, reduced chronic pain in an SNL experimental pain model. OBJECTIVE: The present study was designed to investigate the effect of thalidomide on streptozotocin (STZ)-induced neuropathic pain in rats. MATERIALS AND METHODS: Streptozotocin (20 mg/kg, i.p, daily × 4 days) was administered to induce diabetes in the rats. Nociceptive latency was measured using tail-flick and paw-withdrawal test. Thermal hyperalgesia and mechanical allodynia were measured using planter test and dynamic aesthesiometer (Ugo-Basile, Italy), respectively. Urinary and serum nitrite concentration was estimated using Greiss reagent method. Spleen homogenate supernatant was prepared from spleen of 28th day diabetic rats and administered to normal rats (400 ul, i.v) daily for 28 days. RESULTS: Pain threshold progressively decreased in STZ-treated rats, as compared with control rats. 3 weeks after induction of diabetes, the rat exhibited thermal hyperalgesia and mechanical allodynia. The analgesic effect of morphine (8 mg/kg, s.c.) was significantly decreased in both diabetic and in SHS-treated non-diabetic rats. Administration of thalidomide (25 and 50 mg/kg, i.p), a TNF-α inhibitor, significantly prevented hyperglycemia-induced thermal hyperalgesia and mechanical allodynia and also attenuated the increase in serum and urinary nitrite concentration, as compared with untreated diabetic rats. Also, thalidomide (25 and 50 mg/kg, i.p) 1 h before or concurrently with morphine significantly restored the analgesic effect of morphine in diabetic rats. CONCLUSION: It may be concluded that thalidomide has a beneficial effect in neuropathic pain by decreasing cytokines (TNF-α) and nitric oxide level and may provide a novel promising therapeutic approach for managing painful diabetic neuropathy.

Anti-inflammatory and antihyperalgesic effects of the combination of ibuprofen and hemin in adjuvant-induced arthritis in the Wistar rat.

AIM: Although, pharmacological activation of heme oxygenase (HO)-1 has shown to produce ameliorative effects in various experimental models of inflammation, but such beneficial effects have not been observed in adjuvant-induced arthritis. Further, the upregulated activity of HO-1 has been implicated in the pathogenesis of adjuvant arthritis. The present study was designed to investigate the anti-inflammatory and antihyperalgesic effects of the prophylactic use of hemin alone and/or in combination with ibuprofen using adjuvant-induced arthritis in Wistar rat. METHODS: Arthritis was induced by an intradermal injection of complete Freund's adjuvant (CFA) into left hind paw. Paw volume, thermal hyperalgesia, mechanical allodynia, joint stiffness and mobility behaviors (score) were measured. RESULTS: Administration of ibuprofen (8.75, 17.5, 35 mg/kg/day, p.o.) and hemin (1, 5, 10 mg/kg/day, i.p.) were significantly effective in suppressing CFA-induced paw oedema, thermal and mechanical hyperalgesia, joint stiffness and mobility. The combination of low doses of ibuprofen (8.75 mg/kg, p.o.) and hemin (1 mg/kg, i.p.) significantly reduced paw volume, thermal and mechanical hyperalgesia, as compared to the individual dose of the ibuprofen and hemin alone. CONCLUSIONS: Hence, it may be concluded that the prophylactic administration of either hemin produced significantly enhanced anti-inflammatory and analgesic effects. Further, concurrent low dose administration of hemin and ibuprofen produced significantly enhanced anti-inflammatory and analgesic effects, as compared to the either treatment alone, in CFA-induced arthritis in Wistar rats.

Possible attenuation of nitric oxide expression in anti-inflammatory effect of Ziziphus jujuba in rat.

The hydroalcoholic extract of fruits of Ziziphus jujuba (ZJ) was investigated for its anti-inflammatory effect using acute and chronic models of inflammation in rat. Wistar albino rats of either sex were employed in the present study (n = 6). Acute inflammation was induced by subplantar administration of carrageenan (1%) in rat hind paw. Chronic inflammation was induced by interscapular implantation of a sterile cotton pellet (50 mg). ZJ extract as test drug and indomethacin (10 mg/kg) as standard were used. Serum nitrite/nitrate was also estimated to determine the expression of nitric oxide. In the acute study, carrageenan (1%) administration caused marked paw edema. Pretreatment with ZJ extract exhibited marked dose-dependent attenuation in edema compared to control. In the chronic study, interscapular implantation of sterile cotton pellets caused significant granuloma formation after 7 days, serving as control. ZJ extract significantly decreased granuloma tissue formation compared to control. The serum nitrite/nitrate level was significantly increased after 7 days in the control group due to chronic inflammation, but was decreased by ZJ extract. Moreover, phytochemical studies indicated the presence of jujubosides, flavonoids and terpenes, which may produce the marked anti-inflammatory effect of ZJ fruit in acute and chronic inflammation, possibly by inhibiting nitric oxide expression. The study provides a scientific and ethnopharmacological rationale for the therapeutic use of ZJ fruit as an anti-inflammatory agent.

Mechanism of attenuation of diabetes mellitus and hypercholesterolemia induced vascular endothelial dysfunction by protein tyrosine phosphatase inhibition.

The study has been designed to investigate downstream mechanisms in the PTPase inhibition mediated attenuation of diabetes mellitus and hypercholesterolemia-induced vascular endothelial dysfunction. Diabetes mellitus was induced in rats using streptozotocin (55 mg/kg, i.v. once), while hypercholesterolemia was produced by feeding high cholesterol diet. After 4 weeks of streptozotocin and Cholesterol rich diet administration, vascular endothelium dysfunction was assessed, in terms of attenuation of acetylcholine-induced, endothelium-dependent relaxation (Isolated Aortic Ring Preparation), a decrease in serum nitrate/nitrite level, as well as mRNA expression of eNOS (rtPCR) and disruption of integrity of vascular endothelium (Electron microscopy). After 14 days of daily administration, sodium orthovanadate (8 mg/kg, p.o., 16 mg/kg, p.o and 24 mg/kg, p.o) and atorvastatin (30 mg/kg, p.o) (positive control) significantly improved acetylcholine-induced endothelium-dependent relaxation, serum nitrate/nitrite level, mRNA expression of eNOS and maintained integrity of vascular endothelium. However, this ameliorative effect of SOV was significantly blocked by UCN-01, (PDK inhibitor) and L-NAME (Inhibitor of eNOS). Therefore, it may be concluded that sodium orthovanadate, a specific inhibitor of PTPase, may stimulate PDK and eNOS and consequently improve vascular endothelium dysfunction. Thus, inhibition of PTPase might be a useful approach in the therapeutics of vascular endothelium dysfunction.

Beneficial effect of insulin in hyperhomocysteinemia and diabetes mellitus-induced vascular endothelium dysfunction: role of phosphoinositide dependent kinase and protein kinase B.

A primary defect in the vascular action of insulin may be the key intermediate mechanism that links endothelial dysfunction with diabetes mellitus and hyperhomocysteinemia. This study investigated the downstream targets of insulin, involved in this process. Hyperhomocysteinemia (serum homocysteine > 10 µm/l) was produced in rats by administering L-methionine (1.7% w/w, p.o.x. 4 weeks) and diabetes mellitus (serum glucose > 140 mg/dl) was induced using streptozotocin (55 mg/kg/day, i.v. once) in another group. Four weeks after L-methionine and streptozotocin administration, vascular endothelium dysfunction was assessed in terms of attenuation of acetylcholine-induced, endothelium-dependent relaxation (isolated aortic ring preparation), decrease in serum nitrate/nitrite level, as well as mRNA expression of eNOS (rtPCR), and disruption of integrity of vascular endothelium. Both hyperhomocysteinemia and diabetes mellitus significantly attenuated acetylcholine-induced endothelial-dependent relaxation, and the increase in serum nitrite/nitrate concentration and the expression of eNOS. Insulin (0.4 and 0.6 IU/kg/day, s.c.) and atorvastatin (30 mg/kg/day, p.o.x. 4 weeks) significantly improved all these parameters. However, this ameliorative effect of insulin was blocked by 7-hydroxystaurosporine (UCN-01) [Inhibitor of phosphoinositide dependent kinase (PDK)], and triciribine (API-2) (protein kinase B/Akt inhibitor). It is suggested that amelioration of vascular endothelium dysfunction by insulin may be due to stimulation of PDK and Akt pathways.

Possible involvement of α1-adrenergic receptor and K(ATP) channels in cardioprotective effect of remote aortic preconditioning in isolated rat heart.

BACKGROUND: Remote preconditioning is a phenomenon in which brief episodes of ischemia and reperfusion to remote organs protect the target organ against sustained ischemia/reperfusion (I/R)-induced injury. Protective effects of remote aortic preconditioning (RAPC) are well established in the heart, but their mechanisms still remain to be elucidated. OBJECTIVE: This study has been designed to investigate the possible involvement of α-1-adrenergic receptor (AR) and K(ATP) channels in cardio-protective effect of RAPC in isolated rat heart. MATERIALS AND METHODS: Four episodes of ischemia and reperfusion, each comprising of 5 min occlusion and 5 min reperfusion, were used to produce RAPC. Isolated perfused rat heart was subjected to global ischemia for 30 min followed by reperfusion for 120 min. Coronary effluent was analyzed for LDH and CK-MB release to assess the degree of cardiac injury. Myocardial infarct size was estimated macroscopically using TTC staining. RESULTS: Phenylephrine (20 µ/kg i.p.), as α-1-AR agonist, was noted to produce RAPC-like cardio-protection. However, administration of glibenclamide concomitantly or prior to phenylephrine abolished cardioprotection. Moreover, prazocin (1 mg/kg. i.p), as α-1-AR antagonist and glibenclamide (1 mg/kg i.p), a K(ATP) channel blocker, abolished the cardioprotective effect of RAPC. CONCLUSION: These data provide the evidence that α-1-AR activation involved in cardioprotective effect of RAPC-mediated trough opening of K(ATP) channels.

Modulation of the cardioprotective effect of ischemic preconditioning in hyperlipidaemic rat heart.

Ischemic preconditioning (IPC) produces cardioprotection by phosphorylation of glycogen synthaes kinase-3beta (GSK-3beta) that inhibits the opening of mitochondrial permeability transition pore (MPTP), and this cardioprotective action of IPC is attenuated by hyperlipidaemia. The present study investigated the role of GSK-3beta in attenuation of cardioprotective effect of IPC, by hyperlipidaemia in the rat heart. Hyperlipidaemia was produced in rat by feeding high fat diet for six weeks. Isolated perfused rat heart was subjected to 30 min of ischemia followed by 120 min of reperfusion. Myocardial infarct size was estimated by triphenyltetrazolium chloride (TTC) staining and lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) was analyzed from coronary effluent. IPC significantly decreased the myocardial infarct size and the release of LDH and CK-MB from normal rat heart. IPC induced myocardial protection was attenuated in hyperlipidaemic rat heart. However, cardioprotective effect of pharmacological preconditioning with GSK-3beta inhibitors i.e. Lithium Chloride (LiCl) (20mM), Indirubin - 3 Monooxime (1 microM) and 3-(2, 4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2, 5-dione (SB216763) (3 microM), was not attenuated. This differential attenuation by hyperlipidaemia, of IPC and pharmacological preconditioning induced cardioprotection is a new finding in our study. GSK-3beta inhibition is reported to increase the threshold of opening for MPTP during reperfusion. Administration of atractyloside (20 microM), an opener of MPTP, significantly attenuated the cardioprotective effect of IPC in normal heart, and pharmacological preconditioning in the hyperlipidaemic rat heart. Thus, the attenuation of cardioprotective effect of IPC in hyperlipidaemic heart may be due to inhibition of protective signaling pathways upstream of GSK-3beta and inhibition of opening of MPTP.