Tomasz Przygodzki

Effects of resorcylidene aminoguanidine (RAG) on selected parameters of isolated rat liver mitochondria.

In the present investigation, we attempted to study possible mechanisms of the interactions of resorcylidene aminoguanidine (RAG), the agent with a recognized anti-glycation and antioxidative activity, with rat liver mitochondria. We hypothesized that RAG affects organization of the lipid bilayer in mitochondrial membranes and thus impairs transmembrane Ca(2+) redistribution, transmembrane potential, and respiration capacity. Isolated mitochondria were exposed to RAG (50-200 microM) and several parameters of their function monitored employing spectrofluorimetric, cytometric, and respirometric techniques. Mitochondrial membrane potential and membrane fluidity were tracked using the staining with rhodamine 123 (Rh123) and 1,6-diphenyl-1,3,5-hexatriene (DPH), respectively. Mitochondrial respiration and oxidative phosphorylation was monitored with a high-resolution respirometry, and mobilization of Ca(2+) was detected using spectrofluorimetry with Calcium Green 5-N. RAG depolarized and fluidized mitochondrial membrane, as deduced from reduced fluorescence of intramitochondrial Rh123 and decreased DPH fluorescence anisotropy. The slight inhibitory effect of 100-200 microM RAG on mitochondrial respiratory capacity was observed merely when monitored in the presence of ADP. The reduced sensitivity of mitochondria to calcium-induced depolarization was significant only at higher RAG concentrations (100-200 microM). Moreover, RAG induced pronounced conformational changes in two model proteins: bovine serum albumin and cytochrome c. These findings indicate that regardless of its depolarizing and fluidizing properties, RAG does not largely affect the mitochondrial respiration, although it may significantly lower oxidative phosphorylation when used at higher concentrations.

1-methylnicotinamide effects on the selected markers of endothelial function, inflammation and haemostasis in diabetic rats.

1-methylnicotinamide (MNA) is a primary metabolite of nicotinamide. In recent years several activities of MNA have been described, such as anti-inflammatory activity in skin diseases, induction of prostacyclin synthesis via COX-2, aortal endothelium protection in diabetes and hypertriglyceridaemia and increasing survival rate of diabetic rats. The aim of the present study was to verify whether the increased survival rate of diabetic animals could be explained by anti-hyperglycaemic activity of MNA and/or by its protective effects on vascular endothelium. We used Sprague-Dawley male rats with an experimental streptozotocin diabetes. The animals received either MNA or pure drinking water. At the particular time intervals groups of rats were sacrificed and the blood was collected. We have shown that MNA increases levels of PGI2 in diabetic rats, but the effect is limited only to the early stage of diabetes. We were unable to prove anti-inflammatory effects of MNA, as it did not affect increased TNF-alpha in diabetic animals. We have confirmed our previous observations that MNA improved survival of diabetic animals, but contrary to our previous study, this effect was not accompanied by improvement in the parameters of long-term glycaemic control. Overall, we conclude that anti-diabetic activity of MNA manifested in the improved lifespan of diabetic animals is rather due to MNA pro-prostacyclin activity, and it may not be substantially related to glycaemic control in diabetes. Still other potential mechanism(s) await further elucidation.

Can metabolic impairments in experimental diabetes be cured with poly(amido)amine (PAMAM) G4 dendrimers? – In the search for minimizing of the adverse effects of PAMAM administration

Poly(amido)amine (PAMAM) G4 dendrimers, given intraperitoneally to diabetic rats, have been reported to scavenge excessive blood glucose and minimize the effects of hyperglycaemia, however, at the cost of reduced survival. This paper is the first to compare the effectiveness of three different routes of PAMAM G4 administration with regard to minimizing the adverse effects of hyperglycaemia in rats. Hence, the aim of the study is to identify the most effective and the least harmful method of dendrimer administration. Control and streptozotocin-diabetic Sprague-Dawley rats were exposed to PAMAM G4 (0.5 μmol/kg b.w.) for 60 days, administered intraperitoneally, intragastrically or subcutaneously. Intraperitoneal and subcutaneous administration of PAMAM G4 was found to be most effective in suppressing the long-term markers of hyperglycaemia, while the intragastric route appeared the least effective. Otherwise, the greatest incidence of adverse effects was associated with intraperitoneal and the lowest with subcutaneous delivery. Harmful effects of intragastrical administration were much lower compared to intraperitoneal route, but at the cost of reduced hypoglycaemizing potential. Otherwise, subcutaneous injection represents the best compromise of moderate PAMAM dendrimer toxicity and effective reduction in the markers of long-term severe hyperglycaemia in chronic experimental diabetes.

COX-2-derived prostaglandins do not contribute to coronary flow regulation in diabetic rats: distinct secretion patterns of PGI2 and PGE2.

The role of cyclooxygenase-2 (COX-2) in restoring the functions of impaired endothelium is attracting considerable attention, notably the function of COX-2-derived vasodilatory prostaglandins is disputed in the context of the regulation function in the impaired vascular beds. We have examined the hypothesis that COX-2 activity contributes more to vasodilation in hyperglycemic animals than in healthy counterparts, and that COX-2 derived vasodilatory prostaglandins (PGI(2) and PGE(2)) are responsible for this effect. Using the Langendorff heart perfusion system, the effects of COX-2 inhibition were monitored on both basal and bradykinin-induced coronary flows in Sprague-Dawley rats given 8-week streptozotocin-induced diabetes and in age-matched controls (n=15). Secretions of PGI(2) and PGE(2), both total and the COX-2 dependent pools, have also been compared. The selective COX-2 inhibitor, N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398), had no effect on coronary flow in the diabetic group of animals. Thus, the compensatory role of COX-2 in regulation of vascular tone in experimental diabetes found in other experimental models was not confirmed. However, COX-2 activity significantly contributed to PGI(2) synthesis in healthy rats, with prostacyclin secretion being two-fold decreased by NS-398. Contrary to our hypothesis, neither prostacyclin nor PGE(2) production differed between the experimental groups under the basal conditions. Bradykinin had no effect on the secretion of PGI(2) in either group, but increased PGE(2) synthesis in healthy animals, although not in the streptozotocin group. PGE(2) production in response to bradykinin was COX-2-dependent in control animals. We conclude that, in rats with 8-week streptozotocin-induced diabetes, the activity of COX-2 in coronary vasculature is not significantly enhanced.

β-Resorcylidene aminoguanidine (RAG) dilates coronary arteries in an endothelium-independent manner.

BACKGROUND β-Resorcylidene aminoguanidine (RAG), a highly reactive derivative of aminoguanidine, possesses antithrombotic activity which involves the activation of the vascular COX-2/PGI2 pathway. This endothelium-dependent effect suggests that RAG may demonstrate vasomotor activity in arterial vessels. The aim of the present study was to investigate a possible vasoactive action of RAG in coronary arteries of rat heart. METHODS Isolated rat hearts were perfused in the Langendorff model. To investigate the dose dependency of the effect of RAG on coronary flow, the hearts were perfused with RAG at increasing concentrations. Mechanisms of RAG-mediated vasodilation were subsequently tested using selective inhibitors of the endothelium-dependent and endothelium-independent mechanisms responsible for regulation of vascular tone. RESULTS RAG dilated coronary arteries at concentrations above 10(-5)mol/l. Inhibition of the endothelium-dependent mechanism of vasodilation by NG-nitro-L-arginine methyl ester, indomethacin and aminobenzotriazole did not affect RAG-mediated vasodilation. Other compounds also had no impact on the vasodilating effect of RAG: the NO-dependent guanylate cyclase inhibitor - 1H-[1,2,4]oxadiazolo[4,3]quinoxalin-1-one, the cAMP-dependent protein kinase inhibitor - PKAi, and the K(+) channel blockers - glibenclamide, tetraethylammonium, charybdotoxin, and apamin. CONCLUSIONS RAG is a strong vasodilator that exerts its effect via endothelium-independent mechanisms.

Facts and Artifacts in the Evaluation of the Anti-diabetic Activity of Spent Hop Extract in Rat Hearts in the"Experimental Model of Diabetes"

The present study was undertaken to demonstrate the anti-diabetic mode of action of Humulus lupulus L. extract and its protective influence against changes in the respiratory capacity and coronary flow of the heart in experimental diabetes.

Enhanced adhesion of blood platelets to intact endothelium of mesenteric vascular bed in mice with streptozotocin-induced diabetes is mediated by an up-regulated endothelial surface deposition of VWF – In vivo study

Abstract Numerous in vitro experiments have confirmed that a dysfunctional endothelium is characterized by, inter alia, a higher affinity for binding of platelets and leukocytes. However, there is still no direct evidence for greater interaction between platelets and intact endothelium in in vivo animal models of diabetes. Therefore, the present study examines the pro-adhesive properties of endothelium change in vivo as an effect of streptozotocin (STZ)-induced diabetes and the role of two key platelet receptors: GPIb-IX-V and GPIIb/IIIa. Mice of C57BL strain with streptozotocin-induced diabetes were used in the study. Flow cytometry was used to assess basal activation and reactivity of platelets. Adhesion of platelets to the vascular wall was visualized with the use of intravital microscopy in mesentery. The contribution of GPIIb/IIIa and GPIb-IX-V was evaluated by the injection of Fab fragments of respective antibodies. The integrity of the endothelium and vWf expression were evaluated histochemically. Basal activation and reactivity of platelets in streptozotocin-diabetic mice were elevated. Blood platelets adhered more often to the vascular wall of diabetic mice than nondiabetic animals: 11.9 (6.4; 32.8) plt/min/mm2 (median [IQR]) vs 2.7 (1.3; 6.4) plt/min/mm2. The injection of anti-GPIbα antibodies decreased the number of adhering platelets from 89.5 (34.0; 113.1) plt/min/mm2 (median [IQR]) in mice treated with isotype antibodies to 3.1 (1.7; 5.6) plt/min/mm2 in mice treated with blocking antibodies. The effect of GPIIb/IIIa blockage was not significant. Immunohistochemistry revealed a higher expression of vWF in the endothelium of STZ mice, but no substantial changes in endothelial morphology were detected. To conclude, the study shows that the platelets interact more frequently with the mesenteric vascular bed in mice with 1-month STZ-induced diabetes than in healthy mice. These interactions are mediated via platelet GPIb-IX-V and are driven by increased expression of vWF in endothelial cells.

Flow cytometry analysis reveals different activation profiles of thrombin- or TRAP-stimulated platelets in db/db mice. The regulatory role of PAR-3

INTRODUCTION Recent studies have shown that it may be the concentration of thrombin, which is discriminative in determining of the mechanism of platelet activation via protease activated receptors (PARs). Whether the observed phenomenon of differentiated responses of mouse platelets to various thrombin concentrations in non-diabetic db/+ and diabetic db/db mice depends upon the concerted action of various PARs, remains to be established. RESULTS We found elevated reactivity of platelets, as well as the enhanced PAR-3 expression in response to both the used concentrations of AYPGKF in db/db mice, as compared to db/+ heterozygotes. At low concentration of thrombin platelets from diabetic mice demonstrated hyperreactivity, reflected by higher expression of PAR-3. For higher thrombin concentration, blood platelets from db/db mice appeared hyporeactive, compared to db/+ animals, while no significant differences in PAR-3 expression were observed between diabetic and non-diabetic mice. CONCLUSIONS The novel and previously unreported finding resulting from our study is that the increased expression of PAR-3 in response to either TRAP for PAR-4 or low thrombin (when PAR-4 is not the efficient thrombin receptor) may be one of the key events contributing to higher reactivity of platelets in db/db mice.