Mitko Mladenov

Role of KCNQ Channels in Skeletal Muscle Arteries and Periadventitial Vascular Dysfunction

KCNQ channels have been identified in arterial smooth muscle. However, their role in vasoregulation and chronic vascular diseases remains elusive. We tested the hypothesis that KCNQ channels contribute to periadventitial vasoregulation in peripheral skeletal muscle arteries by perivascular adipose tissue and that they represent novel targets to rescue periadventitial vascular dysfunction. Two models, spontaneously hypertensive rats and New Zealand obese mice, were studied using quantitative polymerase chain reaction, the patch-clamp technique, membrane potential measurements, myography of isolated vessels, and blood pressure telemetry. In rat Gracilis muscle arteries, anticontractile effects of perivascular fat were inhibited by the KCNQ channel blockers XE991 and linopirdine but not by other selective K+ channel inhibitors. Accordingly, XE991 and linopirdine blocked noninactivating K+ currents in freshly isolated Gracilis artery smooth muscle cells. mRNAs of several KCNQ channel subtypes were detected in those arteries, with KCNQ4 channels being dominant. In spontaneously hypertensive rats, the anticontractile effect of perivascular fat in Gracilis muscle arteries was largely reduced compared with Wistar rats. However, the vasodilator effects of KCNQ channel openers and mRNA expression of KCNQ channels were normal. Furthermore, KCNQ channel openers restored the diminished anticontractile effects of perivascular fat in spontaneously hypertensive rats. Moreover, KCNQ channel openers reduced arterial blood pressure in both models of hypertension independent of ganglionic blockade. Thus, our data suggest that KCNQ channels play a pivotal role in periadventitial vasoregulation of peripheral skeletal muscle arteries, and KCNQ channel opening may be an effective mechanism to improve impaired periadventitial vasoregulation and associated hypertension.

Simple 2,4-Diacylphloroglucinols as Classic Transient Receptor Potential-6 Activators—Identification of a Novel Pharmacophore

The naturally occurring acylated phloroglucinol derivative hyperforin was recently identified as the first specific canonical transient receptor potential-6 (TRPC6) activator. Hyperforin is the major antidepressant component of St. Johns wort, which mediates its antidepressant-like properties via TRPC6 channel activation. However, its pharmacophore moiety for activating TRPC6 channels is unknown. We hypothesized that the phloroglucinol moiety could be the essential pharmacophore of hyperforin and that its activity profile could be due to structural similarities with diacylglycerol (DAG), an endogenous nonselective activator of TRPC3, TRPC6, and TRPC7. Accordingly, a few 2-acyl and 2,4-diacylphloroglucinols were tested for their hyperforin-like activity profiles. We used a battery of experimental models to investigate all functional aspects of TRPC6 activation, including ion channel recordings, Ca2+ imaging, neurite outgrowth, and inhibition of synaptosomal uptake. Phloroglucinol itself was inactive in all of our assays, which was also the case for 2-acylphloroglucinols. For TRPC6 activation, the presence of two symmetrically acyl-substitutions with appropriate alkyl chains in the phloroglucinol moiety seems to be an essential prerequisite. Potencies of these compounds in all assays were comparable with that of hyperforin for activating the TRPC6 channel. Finally, using structure-based modeling techniques, we suggest a binding mode for hyperforin to TRPC6. Based on this modeling approach, we propose that DAG is able to activate TRPC3, TRPC6, and TRPC7 because of higher flexibility within the chemical structure of DAG compared with the rather rigid structures of hyperforin and the 2,4-diacylphloroglucinol derivatives.

Effects of interleukin-6 on the bio-electric activity of rat atrial tissue under normal conditions and during gradual stretching

Using the micro-electrode technique we studied the effects of interleukin-6 on bio-electric activity of rat atrial tissue under normal conditions and after gradual stretching. It was shown that IL-6 caused increasing of the duration of the action potential at the levels of 25, 50, and 90% re-polarization. The hump-like depolarization at APD90 appeared 7-10 min after initial stretching and transformed into single extra-potentials after tension removing. Perfusion with IL-6 for more than 20 min led to the appearance of atrial fibrillation even with the application of slight tension. Close observation of the IL-6 induced mechanisms and stretch induced APD alteration, confirmed the existence of a tight link between examined cytokine and stretch induced mechanisms.

Interfacial Properties of Three Different Bioactive Dentine Substitutes

Three different bioactive materials suitable as dentine substitutes in tooth repair have been studied: glass-ionomer cement, particulate bioglass, and calcium-silicate cement. On 15 permanent human molars, Class V cavities were prepared and the bottom of each cavity was de-mineralized by an artificial caries gel. After the de-mineralization, the teeth were restored with: (1) Bioglass®45S5 and ChemFil® Superior; (2) Biodentine™ and ChemFil® Superior; and (3) ChemFil® Superior for a complete repair. The teeth were stored for 6 weeks in artificial saliva, then cut in half along the longitudinal axis: the first half was imaged in a scanning electron microscope (SEM) and the other half was embedded in resin and analyzed by SEM using energy-dispersive X-ray analysis. The glass-ionomer and the bioglass underwent ion exchange with the surrounding tooth tissue, confirming their bioactivity. However, the particle size of the bioglass meant that cavity adaptation was poor. It is concluded that smaller particle size bioglasses may give more acceptable results. In contrast, both the glass-ionomer and the calcium-silicate cements performed well as dentine substitutes. The glass-ionomer showed ion exchange properties, whereas the calcium silicate gave an excellent seal resulting from its micromechanical attachment.

Cytokine Effects on Mechano-Induced Electrical Activity in Atrial Myocardium

ABSTRACT The role of cytokines as regulators of stretch-related mechanisms is of special importance since mechano-sensitivity plays an important role in a wide variety of biological processes. Here, we elucidate the influence of cytokine application on mechano-sensitivity and mechano-transduction. The atrial myocardial stretch induces production of interleukin (IL)-2, IL-6, IL-13, IL-17A, and IL-18 with exception of tumor necrosis factor α (TNF-α), IL-1β, and vascular endothelial growth factor B (VEGF-B). Positive ionotropic effect was specific for VEGF-B, negative ionotropic effects were specific for TNF-α, IL-1β, IL-2, IL-6, IL-13, IL-17A and IL-18, while IL-1α doesn’t show direct ionotropic effect. The IL-2, IL-6, IL-17A, IL-18, and VEGF-B cause elongation of the APD, in comparison with the reduced APD caused by the IL-13. The TNF-α, IL-1β, and IL-18 influences L-type Ca2+ channels, IL-2 has an inhibitory effect on the fast Na+ channels while IL-17A and VEGF-B were specific for Kir channels. With exception of the IL-1α, IL-2, and VEGF-B, all analyzed cytokines include nitric oxide dependent signaling with resultant combined effects on mechano-gated and Ca2+ channels. The relationships between these pathways and the time-dependence of their activation are of important considerations in the evaluation of cytokine-induced electrical abnormality, specific for cardiac dysfunctions. In general, the discussion presented in this review covers research devoted to counterbalance between different cytokines in the regulation of stretch-induced effects in rat atrial myocardium. Abbreviations: APs: action potentials; APD25: action potential durations to 25% of re-polarization; APD50: action potential durations to 50% of repolarization; APD90: action potential durations to 90% of repolarization; MGCs: mechanically gated channels

IL-1 provokes electrical abnormalities in rat atrial myocardium

Using a micro-electrode technique we studied the effects of interleukin 1α and interleukin 1β on bio-electric activity of rat atrial myocardium under normal conditions and after gradual stretching. Perfusion with interleukin 1α increased the duration of the action potential at the level of 90% re-polarization. Stretch induced tachy-arrhythmia in the presence of interleukin 1α is mainly regulated via stretch increased nitric oxide production, while the ionotropic effect of the interleukin-1α during stretching is not pronounced. The perfusion with interleukin 1β did not change the values of the duration of the action potentials at the levels of 25, 50 and 90% repolarization. The interleukin lβ caused an appearance of extra-systolic patterns which turned into normal rhythm, alternating with periods of normal activity. The total intracellular nitric oxide level induced by both interleukin 1β and stretching is balanced by interleukin-1β induced cation influx.

L-citrulline supplementation reverses the impaired airway relaxation in neonatal rats exposed to hyperoxia

BackgroundHyperoxia is shown to impair airway relaxation via limiting L-arginine bioavailability to nitric oxide synthase (NOS) and reducing NO production as a consequence. L-arginine can also be synthesized by L-citrulline recycling. The role of L-citrulline supplementation was investigated in the reversing of hyperoxia-induced impaired relaxation of rat tracheal smooth muscle (TSM).MethodsElectrical field stimulation (EFS, 2–20 V)-induced relaxation was measured under in vitro conditions in preconstricted tracheal preparations obtained from 12 day old rat pups exposed to room air or hyperoxia (>95% oxygen) for 7 days supplemented with L-citrulline or saline (in vitro or in vivo). The role of the L-citrulline/L-arginine cycle under basal conditions was studied by incubation of preparations in the presence of argininosuccinate synthase (ASS) inhibitor [α-methyl-D, L-aspartate, 1 mM] or argininosuccinate lyase inhibitor (ASL) succinate (1 mM) and/or NOS inhibitor [Nω-nitro-L-arginine methyl ester; 100 μM] with respect to the presence or absence of L-citrulline (2 mM).ResultsHyperoxia impaired the EFS-induced relaxation of TSM as compared to room air control (p < 0.001; 0.5 ± 0.1% at 2 V to 50.6 ± 5.7% at 20 V in hyperoxic group: 0.7 ± 0.2 at 2 V to 80.0 ± 5.6% at 20 V in room air group). Inhibition of ASS or ASL, and L-citrulline supplementation did not affect relaxation responses under basal conditions. However, inhibition of NOS significantly reduced relaxation responses (p < 0.001), which were restored to control level by L-citrulline. L-citrulline supplementation in vivo and in vitro also reversed the hyperoxia-impaired relaxation. The differences were significant (p <0.001; 0.8 ± 0.3% at 2 V to 47.1 ± 4.1% at 20 V without L-citrulline; 0.9 ± 0.3% at 2 V to 68.2 ± 4.8% at 20 V with L-citrulline). Inhibition of ASS or ASL prevented this effect of L-citrulline.ConclusionThe results indicate the presence of an L-citrulline/L-arginine cycle in the airways of rat pups. L-citrulline recycling does not play a major role under basal conditions in airways, but it has an important role under conditions of substrate limitations to NOS as a source of L-arginine, and L-citrulline supplementation reverses the impaired relaxation of airways under hyperoxic conditions.

Effects of interleukin-17A on the bioelectric activity of rat atrial myocardium under normal conditions and during gradual stretching.

Using microelectrode technique we studied the effects of interleukin-17A on the activity of rat atrial myocardium under normal conditions and after gradual stretching of the tissue. Perfusion with interleukin-17A for 35 min without stretch, led to an increase in APD25, APD50 and APD90. The effect on the frequency and force of the contraction was absent. Stretching during interleukin-17A perfusion led to an increase only at the level of APD90. In the same condition, the repetition frequency of the action potentials did not change as well. Close observation of the cytokine induced mechanisms, confirmed that IL-17A act on different levels and induce different signaling pathways involved in the regulation of cardiac function.

Evaluation of Bone Healing After Osteotomies Prepared With Er:YAG Laser in Contact and Noncontact Modes and Piezosurgery--An Animal Study.

PURPOSE To analyze the healing of bone tissue treated with Er:YAG laser contact and noncontact modes of and piezosurgery in a rat model using triangular laser profilometry. MATERIALS AND METHODS Twenty-four 10-week-old adult male Wistar rats were used in the study. Three osteotomies on the medial part of tibia were performed in each animal, 1 in the right tibia and 2 in the left tibia. The osteotomies were performed with a piezoelectric device set at maximal power and the Er:YAG laser in contact mode (power, 7.5 W; pulse energy, 375 mJ; repetition rate, 20 Hz; MSP mode) and noncontact mode (power, 7.5 W; pulse energy, 750 mJ; repetition rate, 10 Hz; QSP mode) with a novel type of circular, digitally controlled handpiece (x-Runner). After surgery, 6 animals were immediately euthanized (group 1), and the others were euthanized after 1 week (group 2, n = 6), 2 weeks (group 3, n = 6), and 3 weeks (group 4, n = 6). Bone healing after osteotomy was analyzed using a 3-dimensional laser scanning technique (ie, laser triangulation profilometry). RESULTS The volume reduction rates are similar for all 3 techniques (0.2 to 0.25 mm(3) per week). Greater volume reduction of 0.25 mm3 per week was observed for the Er:YAG laser in noncontact mode (x-Runner). After 3 weeks, almost complete healing of the prepared osteotomy was observed. CONCLUSION Within the limitations of this study, the osteotomies performed by the Er:YAG laser in digitally controlled noncontact mode healed the fastest.

Cystathionine gamma-lyase of perivascular adipose tissue with reversed regulatory effect in diabetic rat artery

The aim of this study is to reveal the regulatory role of cystathionine gamma-lyase (CSE), the main source of hydrogen sulphide (H2S) in perivascular adipose tissue (PVAT), of diabetic rats. Diabetes was induced in male rats by a single intraperitoneal injection of streptozotocin. Animals with glucose levels above 20 mmol/L were determined as diabetic. The rat gracilis arteries (a. gracilis) were dissected with or without PVAT. In all in vitro experiments endothelium-denuded preparations were used for isometric contraction measurements. Increasing concentrations of 5-hydroxytryptamine (5-HT) from 10−10 to 10−5 mol/L were applied to induce gradual increase in force of contractions of circular artery segments. The relaxing effect of CSE was inhibited by DL-propargyl glycine (PGG). The presence of PVAT decreases the contractile response to 5-HT of a. gracilis from control rats. This response is reversed in contraction studies in the same rat artery from diabetic rats. DL-PPG (1 mmol/L) induced significant increase of the force of contraction in artery preparations with PVAT from control rats in the whole range of 5-HT. In contrast, PGG had a relaxing effect in high concentrations of 5-HT (10−6 and 10−5 mol/L) in diabetic rat arteries with PVAT. It is concluded that in skeletal muscle artery from diabetic rats, a mediator related to H2S is released from PVAT. This paracrine mediator increases the maximal force of contraction of endothelium-denuded preparations at higher concentrations of 5-HT.