Miroslav Radenković

Experimental diabetes induced by alloxan and streptozotocin: The current state of the art.

Diabetes mellitus is a chronic metabolic disorder with a high prevalence worldwide. Animal models of diabetes represent an important tool in diabetes investigation that helps us to avoid unnecessary and ethically challenging studies in human subjects, as well as to obtain a comprehensive scientific viewpoint of this disease. Although there are several methods through which diabetes can be induced, chemical methods of alloxan- and streptozotocin-induced diabetes represent the most important and highly preferable experimental models for this pathological condition. Therefore, the aim of this article was to review the current knowledge related to quoted models of diabetes, including to this point available information about mechanism of action, particular time- and dose-dependent protocols, frequent problems, as well as major limitations linked to laboratory application of alloxan and sterptozotocin in inducing diabetes. Given that diabetes is known to be closely associated with serious health consequences it is of fundamental importance that current animal models for induction of diabetes should be continuously upgraded in order to improve overall prevention, diagnosis and treatment of this pathological condition.

Characterization of adenosine action in isolated rat renal artery: Possible role of adenosine A2A receptors

Adenosine (0.1-300 microM) induced concentration- and endothelium-dependent relaxation of rat renal artery (RRA). N(G)-Nitro-L-arginine (L-NOARG, 10 microM) significantly reduced adenosine-elicited dilatation, but not the application of indomethacin (10 microM), ouabain (100 microM) or tetraethylammonium (TEA, 500 microM). In the presence of high concentration of K(+) (100 mM) or glibenclamide (1 microM), adenosine-evoked relaxation was almost abolished. 8-(3-Chlorostyril)caffeine (CSC, 0.3-3 microM), a selective A(2A)-antagonist, significantly reduced adenosine-evoked dilatation in a concentration-dependent manner (pA(2)=7.29). Conversely, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10 nM), an A(1)-antagonist, did not alter adenosine-induced relaxation. These results indicate that adenosine produces endothelium-dependent relaxation of isolated RRA. Dilatation evoked by adenosine is mediated by predominant releasing of endothelium-derived hiperpolarizing factor (EDHF) and also in one part of nitric oxide (NO) from endothelial cells. The obtained results also suggest that RRA response to adenosine is most likely initiated by activation of endothelial adenosine A(2A) receptors.

Vitamin D versus placebo in improvement of endothelial dysfunction: a meta-analysis of randomized clinical trials.

AIMS The possible effect of vitamin D administration in humans on endothelial dysfunction (ED) still remains undetermined. The current meta-analysis was performed to evaluate if vitamin D could improve ED. METHODS Randomized, double-blind, and placebo-controlled clinical trials were identified by systematic search of the PubMed, the Cochrane Library, the Web of Science and the Scopus data bases, as well as different reviews and clinical trials articles. A random effects model was used to calculate the pooled overall effect on flow-mediated dilation (FMD) linked to the vitamin D administration. Meta-regression and subgroup analyses were performed to evaluate the impact of study characteristics on the effect of vitamin D administration on FMD. RESULTS A total of eight studies with nine relevant study arms were identified. The obtained results of pooled analysis showed that vitamin D administration did not improve FMD (eight studies, 529 subjects; weighted mean difference (WMD): 0.96%, 95% CI: -1.24% to 2.06%; P = 0.09). This was probably due to significant heterogeneity in between included trials (I(2) = 84%, P < 0.00001). On the other hand, subgroup analysis demonstrated that vitamin D improved FMD in trials that lasted <16 weeks; if systolic blood pressure (SBP) was higher than 140 mmHg and in trials where diastolic blood pressure (DBP) was <80 mmHg. CONCLUSION Although the current evidence clearly demonstrates that in certain conditions vitamin D can improve ED, a larger number of clinical trials are needed to confirm this assumption to confirm or reject the final statement on this topic.

Pioglitazone and Endothelial Dysfunction: Pleiotropic Effects and Possible Therapeutic Implications

Abstract The vascular endothelium has a central role in the modulation of vascular tone with associated antioxidant, anti-inflammatory, pro-fibrinolytic, anti-adhesive, and anticoagulant effects. This is primarily accomplished by the timely release of endothelial autacoids. On the other hand, endothelial dysfunction (ED) provoked by insulin resistance has been linked with reduced nitric oxide bioavailability, increased production of reactive oxygen species, and alterations of endothelial regeneration. Pioglitazone is classified as an insulin-sensitizing, anti-hyperglycemic agent. The mechanism of action associated with pioglitazone includes the activation of peroxisome proliferator-activated receptor-gamma with stable improvement in glycemic control in diabetic patients. Today, it is known that apart from the beneficial effects on glucose homeostasis, pioglitazone exerts several pleiotropic effects, including the improvement of ED. Thus, the aim of this article was to summarize the current knowledge related to signaling mechanisms of the pioglitazone-induced improvement or reversal of ED. The relevant clinical studies and possible therapeutic implications connected to pioglitazone-related action on the endothelium were analyzed too.

Impairment of acetylcholine-mediated endothelium-dependent relaxation in isolated parotid artery of the alloxan-induced diabetic rabbit

The aim of this study was to assess the effect of type 1 diabetes mellitus (induced by a single intravenous injection of 100 mg kg(-1) of alloxan) on acetylcholine (ACh)-induced relaxation in isolated rabbit parotid gland feeding artery. Isometric force measurements and quantification of inducible nitric oxide synthase (iNOS) mRNA by real-time RT-PCR were made in parotid artery rings from diabetic and control rabbits. Acetylcholine induced concentration- and endothelium-dependent vasorelaxation that was significantly decreased in parotid artery rings from diabetic rabbits. Schild analysis of the ACh vasorelaxant effect, in the presence of selective muscarinic receptor antagonists, revealed involvement of the M(3) receptor subtype in parotid artery rings from both control and diabetic rabbits, with no change in antagonist affinity constants. The inhibitory effects of indomethacin, a non-selective inhibitor of cyclooxygenase, and of high potassium, an inhibitor of hyperpolarization, on ACh vasorelaxation were increased. The effect of N(G) -nitro-l-arginine, a non-selective inhibitor of NOS, was decreased in diabetes. S-methylisothiourea, a selective inhibitor of iNOS, significantly reduced ACh vasorelaxation only in parotid artery rings from diabetic rabbits. Also, up-regulation of iNOS mRNA expression was detected in parotid artery rings from diabetic rabbits. These results suggest that in parotid artery rings from diabetic rabbits, impaired endothelium-dependent vasorelaxation to ACh appears to be caused by the loss of a nitric oxide-mediated component and increased iNOS expression, and is unlikely to be caused by a change at the M(3) receptor level.

Altered response of human umbilical artery to 5-HT in gestational diabetic pregnancy

The aim of this investigation was to evaluate serotonin (5-HT) action on isolated human umbilical arteries (HUA) from normal and gestational diabetes mellitus (GDM) pregnancies. 5-HT caused HUA contraction in a concentration-dependent manner in both investigated groups but with lower efficacy in GDM. After endothelial denudation or in the presence of indomethacin (cyclooxygenase inhibitor), the 5-HT-evoked response was comparably augmented, but only in arteries from uncomplicated pregnancies. 5-HT contractions were unchanged by L-NOARG (NO-synthase inhibitor) or glibenclamide (K(ATP) channel blocker) in both investigated groups. Whereas nifedipine (Ca(2+) channel blocker) reduced the contractile effect of 5-HT and was more potent in GDM, ouabain (Na(+)/K(+)-ATPase inhibitor) caused the contraction of HUA prior to 5-HT addition in both groups, but with a significantly reduced effect in GDM. In vascular rings from GDM, methiothepin (a 5-HT(1)/5-HT(2) receptor antagonist) significantly reduced 5-HT-induced contraction to a similar extent as compared to uncomplicated pregnancies. Ketanserin (a 5-HT(2A) receptor antagonist) produced a concentration-dependent inhibition of the 5-HT effect in GDM. In conclusion, in normal pregnancies, 5-HT produced a concentration- and endothelium-dependent contraction of HUA, most probably via endothelial prostacyclin. In contrast, the contractile effect of 5-HT in GDM was reduced with apparent endothelial dysfunction. In both normal and diabetic pregnancies, voltage-gated Ca(2+) channels and Na(+)/K(+)-ATPase contribute to the 5-HT-evoked contraction, as well to the regulation of basal vascular tone, but those actions were notably impaired in GDM. In uncomplicated and diabetic pregnancies, the transduction mechanism of 5-HT involves activation of mixed population of 5-HT(1) and 5-HT(2A) receptors in the HUA.

Angiotensin receptor blockers & endothelial dysfunction: Possible correlation & therapeutic implications

The endothelium is one of the most important constituents of vascular homeostasis, which is achieved through continual and balanced production of different relaxing and contractile factors. When there is a pathological disturbance in release of these products, endothelial dysfunction (ED) will probably occur. ED is considered to be the initial step in the development of atherosclerosis. This pathological activation and inadequate functioning of endothelial cells was shown to be to some extent a reversible process, which all together resulted in increased interest in investigation of different beneficial treatment options. To this point, the pharmacological approach, including for example, the use of angiotensin-converting enzyme inhibitors or statins, was clearly shown to be effective in the improvement of ED. One of many critical issues underlying ED represents instability in the balance between nitric oxide and angiotensin II (Ang II) production. Considering that Ang II was confirmed to be important for the development of ED, the aim of this review article was to summarize the findings of up to date clinical studies associated with therapeutic application of angiotensin receptor blockers and improvement in ED. In addition, it was of interest to review the pleiotropic actions of angiotensin receptor blockers linked to the improvement of ED. The prospective, randomized, double-blind, placebo or active-controlled clinical trials were identified and selected for the final evaluation.

Combined Contribution of Endothelial Relaxing Autacoides in the Rat Femoral Artery Response to CPCA: An Adenosine A2 Receptor Agonist

We examined the contribution of endothelial relaxing factors and potassium channels in actions of CPCA, potent adenosine A2 receptor agonist, on isolated intact male rat femoral artery (FA). CPCA produced concentration-dependent relaxation of FA, which was notably, but not completely, reduced after endothelial denudation. DPCPX, A1 receptor antagonist, had no significant effect, while SCH 58261 (A2A receptor antagonist) notably reduced CPCA-evoked effect. Pharmacological inhibition of nitric oxide synthase or cyclooxygenase comparably reduced CPCA-evoked action, still in a lesser degree than after denudation. In the presence of buffer with high K+ (100  mM), CPCA-produced relaxations were almost abolished. TEA (nonselective KCa blocker), glibenclamide (KATP blocker), Ba++ (KIR blocker), or ouabain (Na+/K+-ATPase inhibitor) did not change CPCA-induced relaxation. Concentration-response curve for CPCA was significantly shifted to the right after the incubation of apamin (SK channel blocker). CPCA produced concentration-dependent relaxation of FA that was partly dependent on endothelial cells. Endothelium-related portion of CPCA-elicited effect was mediated by combined action of endothelial NO, prostacyclin, and EDHF after activation of endothelial A2A receptors. Small conductance KCa channels were involved in this action.

Effects of diabetes and vascular occlusion on adenosine-induced relaxant response of rat common carotid artery

BACKGROUND The aim of this study was to investigate effect of adenosine on isolated rat common carotid artery (CA) submitted to occlusion in non-diabetic or diabetic animals, and to determine whether endothelium denudation or potassium conductance block affects adenosine action. METHODS Experiments were conducted on Wistar rat CA with or without endothelium. Diabetes was induced by alloxan. Occlusion of CA was performed in randomly selected non-diabetic or diabetic animals anesthetized with urethane. Thus, experiments were performed in four groups of rats: non-operated (control) animals without or with diabetes and operated animals submitted to the occlusion of CA without or with diabetes. Concentration-response curves for adenosine were obtained in a cumulative fashion on precontracted arteries. RESULTS Adenosine produced concentration-dependent and endothelium-independent relaxation of CA with comparable maximal effects in all groups. Analysis of pEC50 values showed that responsiveness of CA decreased in following order: [diabetes (-) / occlusion (-)] = [diabetes (-) / occlusion (+)] > [diabetes (+) / occlusion (-)] > [diabetes (+) / occlusion (+)]. In the presence of high K(+) maximal relaxant response of CA from non-operated rats without diabetes was reduced. The recorded inhibition was even stronger in animals subjected to CA occlusion. Conversely, in non-operated diabetic animals obtained reduction of adenosine effect was less pronounced in regard to non-diabetic rats. CONCLUSIONS Adenosine produced equi-effective endothelium-independent relaxation of CA in all groups. Pharmacological potency of adenosine was reduced in diabetic animals solely, but even more in diabetic rats submitted to CA occlusion. The enhanced potassium transmembrane flow has certain protective role on adenosine-induced action in occluded CA from non-diabetic rats. Conversely, diabetes solely inhibited adenosine-evoked cascade connected to increased potassium conductance.

ACh- and VIP-induced vasorelaxation in rabbit facial artery after carotid artery occlusion

OBJECTIVES The influence of carotid artery occlusion (10, 30 and 60 min) on regulatory mechanisms implicated in the vasorelaxant responses of isolated glandular branch of rabbit facial artery to acetylcholine (ACh) and vasoactive intestinal polypeptide (VIP) was examined. DESIGN In organ bath studies with arterial rings precontracted with phenylephrine (1 microM), before and after carotid artery occlusion, changes in isometric tension were recorded. RESULTS Endothelium-dependent vasorelaxation by ACh and endothelium-independent vasorelaxation by VIP were significantly reduced, started from 30 and 10 min of carotid occlusion, respectively. Inhibitory effect of indomethacin on ACh vasorelaxation was enhanced whilst effect of N(G)-nitro-L-arginine reduced, started from 30 min of carotid occlusion. Sodium nitroprusside-induced vasorelaxation was not changed after carotid occlusion. Inhibition of VIP vasorelaxation by L-N(omega)-nitroarginine-2,4-L-diaminobutyric-amide, was reduced, started from 30 min of carotid occlusion. Forskolin enhanced VIP-induced vasorelaxation in control rings but this effect was reduced started from 30 min of occlusion. In the presence of VIP, vasorelaxant effect of ACh was increased; the increase was reduced, started from 10 min of carotid occlusion. CONCLUSIONS The present investigation provides evidence for the decreased responsiveness to both, ACh-endothelium-dependent and VIP-endothelium-independent vasorelaxation in rabbit facial artery after carotid occlusion. In addition, the data suggest that ischaemia alters contribution of endothelial nitric oxide (eNO) and prostaglandin to ACh, and vascular smooth muscles cAMP and neuronal NO to VIP vasorelaxant effects.