Mustafa Ark

Endothelium-dependent induction of vasorelaxation by Melissa officinalis L. ssp. officinalis in rat isolated thoracic aorta

In the current study, vasorelaxant effect produced by the aqueous extract of Melissa officinalis L. ssp. officinalis (MOO) (Lamiaceae) and its possible mechanism in isolated rat aortic rings precontracted with phenylephrine were examined. In the first series of experiments, effect of MOO on the baseline and phenylephrine (10(-5)M) precontracted arteries was investigated, while in the second group of experiments, endothelium intact or endothelium denuded effect was determined. The agents used were N(omega)-nitro-L-arginine (L-NAME), an irreversible inhibitor of nitric oxide (NO) synthase, indomethacin (10 microM), a cyclooxygenase (COX) inhibitor, and glibenclamide (10 microM), an ATP-sensitive potassium channel blocker. The extract was found to exert a vasorelaxant effect and rosmarinic acid quantity, the characteristic compound of the plant, was analyzed by reversed-phase high-performance liquid chromatography (18.75%), and was further confirmed by LC-MS analysis giving a prominent [M(+1)] molecular ion peak at m/z 365. Total phenol amount in the extract was determined using Folin-Ciocalteau reagent (0.284 mg/mg extract). Vasorelaxant effect of the extract was entirely dependent on the presence of endothelium and was abolished by pretreatment with L-NAME, whereas pretreatment with indomethacin and glibenclamide reduced the relaxation to a minor extent. Rosmarinic acid was also tested in the same manner as the extract and was found to exert vasorelaxant effect. These results suggest that the aqueous extract of MOO vasodilates via nitric oxide pathway with the possible involvement of prostacycline and endothelium-derived hyperpolarizing factor (EDHF) pathways as well.

Effects of cyclooxygenase and lipoxygenase inhibitors on digoxin-induced arrhythmias and haemodynamics in guinea-pigs

Effects of cyclooxygenase and lipoxygenase inhibitors of digoxin-induced arrhythmias and haemodynamics were studied in guinea-pigs. ECG, mean arterial blood pressure heart rate, pressure rate index and arrhythmias were recorded, starting 15 min before digoxin administration and continuing for 30 min afterwards. The cyclooxygenase inhibitor aspirin (50 mg kg-1) and the dual cyclooxygenase/lipoxygenase inhibitor BW 755C (0.25-10.0 mg kg-1) were found to produce a significant protection against the arrhythmias, whereas aspirin (100 mg kg-1) and CGS 8515 were found to be ineffective. SK&F 104 353, a potent and selective peptidoleukotriene receptor antagonist significantly attenuated the arrhythmias and mortality in a dose-dependent manner. It is concluded that production of both cyclooxygenase and lipoxygenase metabolites could favour the occurrence and/or the maintenance of digoxin-induced cardiac toxicity.

Synthesis and Pharmacological Evaluation of Some Novel Thebaine Derivatives: N-(Tetrazol-1H-5-yl)-6,14-endoethenotetrahydrothebaine Incorporating the 1,3,4-Oxadiazole or the 1,3,4-Thiadiazole Moiety

In this study, we synthesized some novel N‐(tetrazol‐1H‐5‐yl)‐6,14‐endoethenotetrahydrothebaine 7α‐substituted 1,3,4‐oxadiazole and 1,3,4‐thiadiazole derivatives as potential analgesic agents. The structures of the compounds were established on the basis of their IR, 1H NMR, 13C NMR, 2D NMR, and high‐resolution mass spectral data. The analgesic activity was evaluated by a rat‐hot plate test model and a rat tail‐flick model. Compound 12 showed analgesic activity higher than that of morphine. In addition to a histopathological and biochemical evaluation, the LD50 dose for the most active compound 12 was determined.

Chemical preconditioning effect of 3-nitropropionic acid in anesthetized rat heart.

Short ischemic episodes increase tolerance against subsequent severe ischemia in the heart. Nitropropionate (3-NP), an irreversible inhibitor of succinic dehydrogenase of the mitochondrial complex II, was shown to induce protective effect against ischemic brain injury. The aim of this study was to investigate the possible protective effect of 3-NP on regional ischemia in preconditioned rat heart in vivo. Hearts were assigned into three groups: first, in order to induce ischemic preconditioning (IP) 5 min ischemia separated by 10 min reperfusion protocol was used; second, non-preconditioned group was used as control; and third, 3-NP (20 mg/kg, i.p.) was injected 3 h before the surgical procedure in order to induce chemical preconditioning. In all these groups, 30 min regional ischemia was followed by 60 min reperfusion. Infarct size, bax expression, number of ventricular ectopic beats (VEB), duration of ventricular tachycardia (VT) and ventricular fibrillation (VF) were significantly decreased in ischemic preconditioning and 3-NP pretreatment groups, whereas bcl-2 values were not markedly changed in these groups during occlusion period. These results showed that in the anesthetized rat heart 3-NP induced chemical preconditioning by decreasing infarct size, number of VEB, duration of VT and VF. Protective effect is associated with via decreased production of bax protein expression.

Gemcitabine hydrochloride-loaded liposomes and nanoparticles: comparison of encapsulation efficiency, drug release, particle size, and cytotoxicity

Abstract The aim of this study is to formulate and compare the physicochemical properties of negatively charged liposomes and poly(lactide-co-glycolide) (PLGA) nanoparticles loaded with gemcitabine hydrochloride. The influence of the formulation variables on the liposome and nanoparticle properties on particle size, zeta potential, encapsulation efficiency, and drug release was evaluated. Although the PEGylated nanoparticles and PEGylated liposomes were of the same size (∼200 nm), the encapsulation efficiency was 1.4 times higher for PEGylated liposomes than for PEGylated nanoparticles. The optimized formulation of PEGylated liposomes and PEGylated nanoparticles had 26.1 ± 0.18 and 18.8 ± 1.52% encapsulation efficiency, respectively. The release of drug from the PEGylated liposomes and PEGylated nanoparticles exhibited a biphasic pattern that was characterized by a fast initial release during the first 2 h followed by a slower continuous release. Transmission electron microscopy (TEM) images identified separate circular structures of the liposomes and nanoparticles. The in vitro cytotoxicity of the optimized formulations was assessed in MCF-7 and MDA-MB-231 cells, and the results showed that the cytotoxicity effect of the gemcitabine hydrochloride-loaded liposomes and nanoparticles was more than commercial product Gemko® and gemcitabine hydrochloride solution.

Cardiac glycoside-induced cell death and Rho/Rho kinase pathway: Implication of different regulation in cancer cell lines.

Previously, we demonstrated that the Rho/ROCK pathway is involved in ouabain-induced apoptosis in HUVEC. In the current work, we investigated whether the Rho/ROCK pathway is functional during cardiac glycosides-induced cytotoxic effects in cancer cell lines, as well as in non-tumor cells. For that purpose, we evaluated the role of ROCK activation in bleb formation and cell migration over upstream and downstream effectors in addition to ROCK cleavage after cardiac glycosides treatment. All three cardiac glycosides (ouabain, digoxin and bufalin) induced cell death in HeLa and HepG2 cells and increased the formation of blebbing in HeLa cells. In contrast to our previous study, ROCK inhibitor Y27632 did not prevent bleb formation. Observation of ROCK II cleavage after ouabain, digoxin and oxaliplatin treatments in HeLa and/or HepG2 cells suggested that cleavage is independent of cell type and cell death induction. While inhibiting cleavage of ROCK II by the caspase inhibitors z-VAD-fmk, z-VDVAD-fmk and z-DEVD-fmk, evaluation of caspase 2 siRNA ineffectiveness on this truncation indicated that caspase-dependent ROCK II cleavage is differentially regulated in cancer cell lines. In HeLa cells, ouabain induced the activation of ROCK, although it did not induce phosphorylation of ERM, an upstream effector. While Y27632 inhibited the migration of HeLa cells, 10nM ouabain had no effect on cell migration. In conclusion, these findings indicate that the Rho/ROCK pathway is regulated differently in cancer cell lines compared to normal cells during cardiac glycosides-induced cell death.

Ouabain induces Rho-dependent rock activation and membrane blebbing in cultured endothelial cells

Small G protein Rho and its most studied effectors, ROCK I and ROCK II, are involved in several cellular fuctions including smooth muscle and non-muscle cell contractions, cell migration and apoptosis. Activation of ROCK I by caspase-3 and activation of ROCK II by granzyme B are essential for membrane blebbing in the execution phase of apoptosis. In contrast, it has been demonstrated that Rho signaling is critical for blebbing developed after serum removal. As it was shown by us previously, ouabain induces membrane blebbing and proteolitic cleavage of ROCK I and ROCK II via caspases in human umbilical endothelial cells. However, caspase inhibitors do not prevent ouabain-induced blebs. Ouabain induces concentration-dependent cell death and membrane blebbing in endothelial cells. The aim of this study was to identify the possible role of Rho in ouabain-induced membrane blebbing. Pretreatment of endothelial cells with a Rho inhibitor CT04 did not inhibit the ouabain-induced cell death but prevented the development of bleb formation. These results indicate that bleb formation is dependent on Rho activity in ouabain-induced cell death in HUVECs. Taken together, these results suggest that the mechanism of membrane bleb formation might be different depending on cell type and cell death-stimuli.

Comparison of spectrophotometric, HPLC and chemilumines-cence methods for 3-nitrotyrosine and peroxynitrite interaction

We have studied the interaction of 3-nitrotyrosine with peroxynitrite using three different methods; chemiluminescence, spectrophotometry and HPLC. Peroxynitrite-induced luminol or lucigenin chemiluminescence were significantly decreased by 3-nitrotyrosine, in concentration-dependent manners. The intensity of the peroxynitrite spectrum was also markedly reduced in the presence of 3-nitrotyrosine in the spectrophometric assay. However, there was no attenuation of the 3-nitrotyrosine signal in the HPLC assay after mixing with peroxynitrite. The interaction of 3-nitrotyrosine and hypochlorous acid (HOCI) was also studiedvia the chemiluminescence assay, where the HOCI-induced responses were markedly inhibited by 3-nitrotyrosine. These results suggest that caution should be taken when studying the levels or interactions of 3-nitrotyrosine.

The Connection Between the Cardiac Glycoside-Induced Senescent Cell Morphology and Rho/Rho Kinase Pathway

Recently drug‐induced senescence has gained momentum as a new approach in cancer therapy. It is accepted that senescent cells display typical phenotypic features including flattened, enlarged, and multinucleated cell morphology. However, it is not well elucidated how these morphological alterations occur. The current study evaluates the possible role of Rho/Rho kinase pathway in cardiac glycoside‐induced senescent cell morphology in HeLa cells. Our results indicate that the administration of cardiac glycosides, ouabain, digoxin, bufalin, to HeLa cells induced cellular senescence leading to an increase in the volume, area and maximum thickness of the cells. Although preincubation of specific Rho kinase inhibitor Y‐27632 did not inhibit the occurrence of cardiac glycoside‐induced senescence in cells, it reduced the cell area and cell volume. Inhibition of Rho by CT04 produced similar results as seen for the preincubation of Y‐27632. In addition, inhibition of Rock caused a decrease in increased actin stress fibers in senescent cells induced by ouabain. Additionally, preincubation of Y‐27632 decreased the ouabain‐induced the phosphorylation of MYPT and cofilin. In conclusion, Rock inhibition‐mediated alteration of senescent cell morphology may be associated with the decreased actin stress fibers formation. Since it is known that secretory activity is accompanied by the changes of cell morphology, these morphological alterations observed by the inhibition of Rho/Rho kinase pathway may also lead to important secretory functions of senescent cells.

Atorvastatin acutely reduces the reactivity to spasmogens in rat aorta: implication of the inhibition of geranylgeranylation and MYPT‐1 phosphorylation

Statins are known to display benefits in various diseases independently from their cholesterol lowering properties. In this study, we investigated the acute effects of atorvastatin on vascular reactivity to various spasmogens in isolated rat aorta. The responses to noradrenaline (NA, 10−8–10−4 m), endothelin‐1 (ET‐1, 10−10–10−7 m), and potassium chloride (KCl, 10–100 mm) were evaluated in aortic rings pretreated with atorvastatin (10−7–10−4 m, 30 min). To verify the mechanism of action, the effects of atorvastatin were studied in the presence of cholesterol precursor, mevalonate (10−2 m, 45 min), mevalonate‐derived isoprenoids, namely geranylgeranyl pyrophosphate (GGPP, 5 × 10−6 m, 30 min) and farnesyl pyrophosphate (FPP, 5 × 10−6 m, 30 min), and in the absence of endothelium. In parallel, aortic rings were pretreated with the specific inhibitor of Rho kinase, Y‐27632 (10−7–10−6 m). Atorvastatin significantly and concentration‐dependently reduced the contractions to spasmogens in rat aorta. This acute inhibitory effect was also evident in endothelium‐denuded rings. Pretreatment with mevalonate and GGPP, but not with FPP, reversed the inhibitory effect of atorvastatin (10−4 m) on NA and ET‐1 induced contractions. Similar to atorvastatin, pretreatment with Y‐27632 inhibited the contractions to NA and KCl in a concentration‐dependent manner. Western blot analysis revealed that both atorvastatin (10−4 m) and Y‐27632 (10−6 m) pretreatment inhibited the phosphorylation of myosin phosphatase target subunit‐1 (MYPT‐1) triggered by NA, indicating an inhibitory influence on myosin phosphatase. In conclusion, atorvastatin displayed an acute inhibitory effect on vascular contractility evoked by various spasmogens and the inhibitory effect was possibly mediated by the inhibition of mevalonate and GGPP synthesis as well as the prevention of MYPT‐1 phosphorylation induced by Rho/Rho kinase.