Toxicity of the synthetic polymeric 3-alkylpyridinium salt (APS3) is due to specific block of nicotinic acetylcholine receptors

The in vivo and in vitro toxic effects of the synthetic polymeric 3-alkylpyridinium salt (APS3), from the Mediterranean marine sponge Reniera sarai, were evaluated on mammals, with emphasis to determine its mode of action. The median lethal doses of APS3 were 7.25 and higher that 20 mg/kg in mouse and rat, respectively. Intravenous administration of 7.25 and 20 mg/kg APS3 to rat caused a significant fall followed by an increase in mean arterial blood pressure accompanied by tachycardia. In addition, cumulative doses of APS3 (up to 60 mg/kg) inhibited rat nerve-evoked skeletal muscle contraction in vivo, with a median inhibitory dose (ID₅₀) of 37.25 mg/kg. When administrated locally by intramuscular injection to mouse, APS3 decreased the compound muscle action potential recorded in response to in vivo nerve stimulation, with an ID₅₀ of 0.5 mg/kg. In vitro experiments confirmed the inhibitory effect of APS3 on mouse hemidiaphragm nerve-evoked muscle contraction with a median inhibitory concentration (IC₅₀) of 20.3 μM, without affecting directly elicited muscle contraction. The compound inhibited also miniature endplate potentials and nerve-evoked endplate potentials with an IC₅₀ of 7.28 μM in mouse hemidiaphragm. Finally, APS3 efficiently blocked acetylcholine-activated membrane inward currents flowing through Torpedo nicotinic acetylcholine receptors (nAChRs) incorporated to Xenopus oocytes, with an IC₅₀ of 0.19 μM. In conclusion, our results strongly suggest that APS3 blocks muscle-type nAChRs, and show for the first time that in vivo toxicity of APS3 is likely to occur through an antagonist action of the compound on these receptors.     

Contribution of NO,ATP-sensitive K+ channels and prostaglandins to adenosine receptor agonists-induced relaxation of the rat tail artery

The mechanism of relaxation in the rat tail artery induced by the adenosine A1 receptor-selective agonist N⁶-cyclohexyladenosine (CHA, 10 nM–300 μM) and the adenosine A1/A_2a receptor agonist 5’-N-ethylcarboxamidoadenosine (NECA, 10 nM–300 μM) has been characterized. To do this, we used α₁-receptor agonist phenylephrine to evoke contraction (10 μM), and inhibitors of nitric oxide synthase (L-NAME, 10 μM), ATP-sensitive K⁺ channels (glibenclamide, 10 μM) and prostaglandin synthesis (indomethacin, 10 μM). CHA and NECA induced relaxation of rat-tail artery by 80% and 70% in a concentration-dependent manner, respectively. The relaxation effect of NECA was completely abolished in the presence of L-NAME, while glibenclamide and indomethacin prevented CHA-induced relaxation of the rat tail artery by approximately 25% and 40%, respectively. Our results indicate that nonspecific effects such nitric oxide and prostaglandins release or the activation of potassium channels significantly contributed to the effects of CHA and NECA.     

LPS from Porphyromonas gingivalis increases the sensitivity of contractile response mediated by endothelin-B (ETB) receptors in cultured endothelium-intact rat coronary arteries

The purpose of our study was to examine if lipopolysaccharide (LPS) from Porphyromonas gingivalis (P.g.) modifies the vasomotor responses to Endothelin-1 (ET-1) and Sarafotoxin 6c (S6c) in rat coronary arteries. The arteries were studied directly or following organ culture for 24 h in absence and presence of 2.5 EU/ml LPS. The contractile responses of coronary arteries were investigated by using the selective ETB receptor agonist S6c (1 pM–0.3 μM) and ET-1 (1 pM–0.3 μM). The functional studies demonstrated an augmented contractile response only to S6c in isolated rat coronary arteries after organ culture (with or without LPS). These contractile responses by S6c were blocked by the selective ETB receptor antagonist BQ788 in both vessel groups. The augmented contractile response to S6c was supported by immunohistochemistry, where a significant increase in fluorescence intensity for ETB receptors in smooth muscle cells was observed after organ culture. The presence of LPS in the culture medium significantly increased the sensitivity of endothelium-intact coronary artery to S6c as compared to endothelium-denuded segments. Our results showed a significant increase in both ETB receptor protein levels and S6c-induced maximal contraction in coronary arteries upon 24 h of organ culture, which was further sensitized by LPS.     

Inhibitory vs. protective effects of N-acetyl-l-cysteine (NAC) on the electromechanical properties of the spontaneously beating atria of the frog (Rana ridibunda): An ex vivo study

The results of this study have shown that N-acetyl-l-cysteine (NAC), a compound used for protection of tissues or cell cultures against the deleterious effects of various environmental pollutants, has certain unusual effects on the contraction of the spontaneously beating atria of the frog isolated in saline (ex vivo): (1) NAC, 6.0 and 10.0 mM, eliminated, in a concentration-dependent manner, the contractile properties of the atria (force and frequency) within minutes, without affecting its electrical properties; (2) the IC₅₀ of NAC for the force was 5.09 ± 1.01 mM (n = 6) [4.98–5.19 mM, 95% confidence interval (CI)], significantly lower than the IC₅₀ for the frequency, 6.15 ± 1.01 mM, (6.02–6.29 mM, 95% CI), indicating that working atria cells are more sensitive to NAC than autorhythmic cells. The no-observed-effect concentration (NOEC) was 1–2 mM; (3) the pattern of NAC-induced inhibition of electromechanical activity was similar to that of verapamil, an indication that NAC possibly affects L-type voltage-gated calcium channels; (4) NAC at 2 mM protected against cadmium-induced inhibition of atria contraction. The IC₅₀ for cadmium was 17.9 ± 1.1 μM (n = 6) (16.9–19.0 μM, 95% CI), while in the presence of 2 mM NAC, it became 123.3 ± 1.0 μΜ (n = 6) (114.8–132.4 μM, 95% CI). The same concentration of NAC failed to exert any protective effects against rotenone (5 μM)-induced inhibition of atria contraction. The protective effects of NAC are probably due to chelation of cadmium, rather than scavenging of oxidants.     

Sildenafil increases the force of right atrial contractions in vitro via the NO-guanylyl cyclase pathway involving β-adrenoceptor linked mechanisms

Sildenafil, a drug used in the treatment of erectile dysfunction, is a phosphodiesterase 5A inhibitor that increases cyclic guanosine monophosphate (cGMP) levels. In addition to its vascular actions, sildenafil is also known to alter cardiac functions. This study was undertaken to elucidate the effect of sildenafil on cardiac contractility and the underlying mechanisms. The experiments were conducted on spontaneously-beating right atria isolated from adult rats. The effect of sildenafil on the isometric contractions in vitro was examined in the absence or presence of antagonists. Sildenafil (0.001–10 μM) produced a concentration-dependent increase in the atrial force of contraction without altering the atrial rate, even up to 10 μM. A concentration as low as 0.001 μM produced a significant increase (16%) in force and the increase was about 50% at 10 μM. Pretreatment with methylene blue (a guanylyl cyclase inhibitor) or N-ω-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor) blocked the force changes induced by sildenafil. Sildenafil-induced increase in force of contraction was also blocked by propranolol (a β-adrenoceptor antagonist) and diltiazem (an L-type Ca²⁺ channel antagonist). The present results demonstrate that sildenafil increases the atrial force of contraction involving cGMP-β-adrenoceptor-Ca²⁺ channel-dependent mechanisms.     

Cytotoxicity and apoptosis induction on RTG-2 cells of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and decabrominated diphenyl ether (BDE-209)

Recently, the environmental residues of polybrominated diphenyl ethers (PBDEs) have markedly increased. In particular, the levels of certain PBDE congeners in fish have raised concern regarding potential risks associated with dietary PBDEs exposures. However, little is known regarding PBDE-mediated cell injury in relevant in vitro fish cell models. In this study, the cytotoxic effects of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and decabrominated diphenyl ether (BDE-209) on RTG-2 cells were investigated. RTG-2 cells were incubated with different concentrations of BDE-47 and BDE-209 (1–100 μM) for 72 h, and a set of bioassays were conducted to measure: cell viability (evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and neutral red (NR) uptake), lactate dehydrogenase (LDH) leakage, reactive oxygen species (ROS) formation and cell apoptosis. The results showed that BDE-47 and BDE-209 inhibited the cells viability, increased LDH leakage, and induced cell apoptosis in time and concentration-dependent manner. All significant effects were observed at concentrations of 12.5 μM and above for BDE-47 and 25 μM and above for BDE-209 (P < 0.05). At the concentration of 100 μM BDE-47 and BDE-209, the cell viability of the exposed cells dropped to about 40% and 50% of the control, and the apoptotic rates were 52.6% and 34.6%, respectively. After 12 h exposure, a concentration-dependent increases of BDE-47 and BDE-209 (12.5–100 μM) in ROS formation were observed. Collectively, the results of cell viability, LDH leakage, cell apoptosis and ROS formation demonstrated that the toxic mechanism of PBDEs on RTG-2 might be mediated by oxidative stress.     

Sub-lethal concentrations of CdCl2 disrupt cell migration and cytoskeletal proteins in cultured mouse TM4 Sertoli cells

The aims of this study were to examine the effects of CdCl₂ on the viability, migration and cytoskeleton of cultured mouse TM4 Sertoli cells. Time- and concentration-dependent changes were exhibited by the cells but 1 μM CdCl₂ was sub-cytotoxic at all time-points. Exposure to 1 and 12 μM CdCl₂ for 4 h resulted in disruption of the leading edge, as determined by chemical staining. Cell migration was inhibited by both 1 and 12 μM CdCl₂ in a scratch assay monitored by live cell imaging, although exposure to the higher concentration was associated with cell death. Western blotting and immunofluorescence staining indicated that CdCl₂ caused a concentration dependent reduction in actin and tubulin levels. Exposure to Cd^2 + also resulted in significant changes in the levels and/or phosphorylation status of the microtubule and microfilament destabilising proteins cofilin and stathmin, suggesting disruption of cytoskeletal dynamics. Given that 1–12 μM Cd^2 + is attainable in vivo, our findings are consistent with the possibility that Cd^2 + induced impairment of testicular development and reproductive health may involve a combination of reduced Sertoli cell migration and impaired Sertoli cell viability depending on the timing, level and duration of exposure.     

The force of the spontaneously contracting zebrafish heart,in the assessment of cardiovascular toxicity: Application on adriamycin

The heart of the zebrafish has been used extensively to assess the cardiotoxic effect of compounds, using the frequency of heart contractions as the main index of cardiac response to drugs. In this study, the force and the frequency generated by the spontaneously contracting zebrafish heart, isolated in saline, were found to be 0.87 ± 0.05 mN and 1.54 ± 0.03 Hz (n = 6) respectively within the first hour of recording. Both values of force and frequency remained constant for over 8 h. The advantage of prolonged vitality in the assessment of cardiovascular toxicity was shown using the well-known anticancer drug adriamycin, which has severe cardiotoxic side effects. At 10.0 μM there was a 21.05 ± 4.42% (p = 0.02, n = 4) decrease in the force of contraction, while the frequency was not affected after 3 h treatment (p > 0.05). At 50.0 and 100.0 μM there was a 33.24 ± 3.0 and 46.6 ± 4.80% irreversible decrease in force (p < 0.05, n = 4), while a 18.02 ± 4.07% and 16.16 ± 4.07% reversible increase was observed in the frequency (p = 0.02, n = 4). These contradictory positive chronotropic and negative inotropic responses indicate the strong inhibitory effect of adriamycin on ventricular cardiomyocytes and its excitatory effects on auto-rhythmical pacemaker cells. If heart frequency was the only parameter used to assess the cardiotoxic effect of adriamycin, at the above range of concentrations, this compound would have been classified as non-cardiotoxic.     

Development of novel N-linked aminopiperidine-based mycobacterial DNA gyrase B inhibitors: Scaffold hopping from known antibacterial leads

DNA gyrase of Mycobacterium tuberculosis (MTB) is a type II topoisomerase that ensures the regulation of DNA topology and has been genetically demonstrated to be a bactericidal drug target. We present the discovery and optimisation of a novel series of mycobacterial DNA gyrase inhibitors with a high degree of specificity towards the mycobacterial ATPase domain. Compound 5-fluoro-1-(2-(4-(4-(trifluoromethyl)benzylamino)piperidin-1-yl)ethyl)indoline-2,3-dione (17) emerged as the most potent lead, exhibiting inhibition of MTB DNA gyrase supercoiling assay with an IC₅₀ (50% inhibitory concentration) of 3.6 ± 0.16 μM, a Mycobacterium smegmatis GyrB IC₅₀ of 10.6 ± 0.6 μM, and MTB minimum inhibitory concentrations of 6.95 μM and 10 μM against drug-sensitive (MTB H37Rv) and extensively drug-resistant strains, respectively. Furthermore, the compounds did not show any signs of cardiotoxicity in zebrafish ether-à-go-go-related gene (zERG), and hence constitute a major breakthrough among the otherwise cardiotoxic N-linked aminopiperidine analogues.     

5-HT-induced depression of the spinal monosynaptic reflex potential utilizes different types of 5-HT receptors depending on Mg2+ availability

Receptor subtypes involved in the 5-hydroxytryptamine (5-HT)-induced depression of synaptic transmission in neonatal rat spinal cords in vitro were evaluated in the absence or presence of Mg²⁺ in the medium. Stimulation of a dorsal root evoked monosynaptic reflex potential (MSP) and polysynaptic reflex potential (PSP) in the segmental ventral root in Mg²⁺-free medium where the voltage-dependent blockade of NMDAreceptors is absent. The 5-HT (0.3–50 μM) in the Mg²⁺-free medium depressed the MSPand PSP in a concentration-dependent manner. At 30 μM of 5-HT, the depression was 57% and 95% for MSP and PSP, respectively, and no further depression was seen at 50 μM. The 5-HT-induced depression of the reflexes in the Mg²⁺-free medium was blocked by ondansetron (5-HT₃ receptor antagonist), but not by spiperone (5-HT_2A/2C antagonist). In the Mg²⁺-free medium, phenylbiguanide (5-HT₃ agonist) also depressed the MSPand PSP in a concentration-dependent manner and was blocked by ondansetron. Addition of Mg²⁺ (1.3 mM) to the medium abolished the PSP and decreased the MSPby 30%. In the presence of Mg²⁺, 5-HT (1–50 μM) also depressed the MSP in a concentration-dependent manner. At 10 μM of 5-HT, there was approximately 20% depression and at 50 μM the depression was 100%. The 5-HT-induced depression of MSP in the Mg²⁺-containing medium was antagonized by spiperone (p < 0.05, two-way ANOVA), but not by ondansetron. The results indicate that the 5-HT-induced depression of MSPinvolves 5-HT₃ receptors in the Mg²⁺-free medium and 5-HT_2A/2C in the presence of Mg²⁺ when NMDA receptors are in the closed state.     

Contractile effects of endothelins on isolated ampullar segment of human oviduct in luteal phase of menstrual cycle

Endothelin-1 induces contractions of human oviduct ampullar segment in follicular phase of menstrual cycle, acting on ET_A receptors. The aim of our study was to investigate effects of endothelin-1, endothelin-2 and endothelin-3 on isolated ampullar segment of human oviducts, taken from the patients in luteal phase of menstrual cycle. Fallopian tubes were taken from 20 female patients (one tube from each patient) during abdominal hysterectomy with adnexectomy, due to extensive uterine fibroids. The oviduct ampulla was mounted in an organ bath longitudinally, and the tension of the isolated preparation was recorded with the isometric transducer. Endothelin-1 produced concentration-dependent tonic contraction of the isolated ampullar segment (EC₅₀ = 6.80 ± 1.2 × 10^?10 M), and concentration-dependent inhibition of its rhythmic contractions (EC₅₀ = 7.86 ± 2.3 × 10^?10 M). Endothelin-2 produced concentration-dependent tonic contraction of the isolated ampullar segment (EC₅₀ = 4.56 ± 0.3 × 10^?10 M), without affecting its rhythmic contractions. Endothelin-3 did not affect either tone or rhythmic contractions of the isolated preparations. Selective antagonist for ET_A receptor subtype, BQ 123, produced inhibition of endothelin-1 effects on both tone (pA₂ = 9.50) and spontaneous rhythmic contractions (pA₂ = 10.73), while selective antagonist for ET_B receptor subtype, BQ 788, produced only inhibition of endothelin-1 effects on tone (pA₂ = 9.61), while the effect of endothelin-1 on spontaneous rhythmic contractions remained unaffected. The results of our study suggest that in the luteal phase both ET_A and ET_B receptors regulate tone, and only ET_A receptors regulate rhythmic activity of human oviduct's ampullar segment.     

Comparative toxicity and apoptosis induced by diorganotins in rat pheochromocytoma (PC12) cells

As ubiquitous environmental toxicants, organotin (IV) compounds (OTC) accumulate in the food chain and potential effects on human health are disquieting. The present study compared the cytotoxicity of three diorganotins, namely, dimethyltin (DMT), dibutyltin (DBT) and diphenyltin (DPT), in rat pheochromocytoma (PC12) cells, and the molecular mechanisms responsible for their cytotoxic effects were also explored. Twenty-four hours exposure of PC12 cells to DBT and DPT resulted in a concentration-dependent decrease in cell viability with median lethal concentration (LC₅₀) of 2.97 μM and 7.24 μM, respectively. However, DMT at concentrations up to 128 μM had no obvious effect on cell viability. The mechanistic study revealed that the extent of apoptosis was greater for DBT than that for DPT, followed by DMT, as evidenced by acridine orange/ethidium bromide (AO/EB) fluorescent staining method and annexin V-FITC/PI staining flow cytometry analysis, as well as generation of intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP) disruption, release of cytochrome c (Cyt c), and consequent activation of caspase-9, and -3. These investigations suggested that the cytotoxic potency of three diorganotins in PC12 cells was in the order of DBT > DPT ≫ DMT, and these compounds could induce PC12 cells apoptosis through ROS mediated mitochondrial pathway.     

Zn2+, derived from cell preparation,partly attenuates Ca2+-dependent cell death induced by A23187, calcium ionophore,in rat thymocytes

A23187, a calcium ionophore, is used to induce Ca²⁺-dependent cell death by increasing intracellular Ca²⁺ concentration ([Ca²⁺]_i) under in vitro condition. Since this ionophore also increases membrane permeability of metal divalent cations such as Zn²⁺ and Fe²⁺ rather than Ca²⁺, trace metal cations in cell suspension may affect Ca²⁺-dependent cell death induced by A23187. Therefore, the effects of chelators for divalent metal cations, EDTA and TPEN, on the A23187-induced cytotoxicity were cytometrically examined in rat thymocytes. The cytotoxicity of A23187 was attenuated by 1 mM EDTA while it was augmented by 50 μM EDTA and 10 μM TPEN. These changes were statistically significant. The A23187-induced increase in Fluo-3 fluorescence intensity, a parameter for [Ca²⁺]_i, was significantly reduced by 1 mM EDTA while it was not the case for 50 μM EDTA and 10 μM TPEN. The intensity of FluoZin-3 fluorescence, a parameter for [Zn²⁺]_i, increased by A23187 was respectively reduced by 50 μM EDTA and 10 μM TPEN. It is suggested that the attenuation of A23187-induced cytotoxicity by 1 mM EDTA is due to the chelation of extracellular Ca²⁺ and Zn²⁺ while the augmentation by 50 μM ETDA or 10 μM TPEN is due to the chelation of extracellular Zn²⁺. The Tyrode’s solution without thymocytes contained 32.4 nM of zinc while it was 216.9 nM in the cell suspension. In conclusion, trace Zn²⁺, derived from cell preparation, partly attenuates the Ca²⁺-dependent cell death induced by A23187.     

Zinc oxide nanoparticle disruption of store-operated calcium entry in a muscarinic receptor signaling pathway

The influences of ZnO nanoparticles on cellular responses to activation of muscarinic receptors were studied in Chinese hamster ovary cells expressing the human M3 muscarinic acetylcholine receptor. ZnO particles (20 nm) induced cytotoxicity in a time and concentration-dependent manner: following a 24 h exposure, toxicity was minimal at concentrations below 20 μg/ml but virtually complete at concentrations above 28 μg/ml. ZnO particles did not affect antagonist binding to M3 receptors or allosteric ligand effects, but increased agonist binding affinity while eliminating guanine nucleotide sensitivity. At a noncytotoxic concentration (10 μg/ml), ZnO increased resting [Ca²⁺]_i from 40 to 130 nM without compromising calcium homeostatic mechanisms. ZnO particles had minimal effects on IP3- or thapsigargin-mediated release of intracellular calcium from the endoplasmic reticulum, but strongly inhibited store-operated calcium entry (capacitive calcium entry). The latter effect was seen as (1) a decrease in the plateau phase of the response and (2) a decrease in Ca²⁺ entry upon introduction of calcium to the extracellular medium following thapsigargin-induced depletion of calcium from the endoplasmic reticulum (EC50’s ≈ 2 μg/ml). Thus, ZnO nanoparticles interfere with two specific aspects of the M3 signaling pathway, agonist binding and store-operated calcium entry.     

Micromolar Zn2+ potentiates the cytotoxic action of submicromolar econazole in rat thymocytes: Possible disturbance of intracellular Ca2+ and Zn2+ homeostasis

Econazole, one of imidazole antifungals, has been reported to exhibit an inhibitory action on Mycobacterium tuberculosis and its multidrug-resistant strains under in vitro and ex vivo conditions. There is a chemotherapeutic potential of econazole against tuberculosis. We have revealed that Zn²⁺ at micromolar concentrations potentiates the cytotoxicity of imidazole antifungals by increasing membrane Zn²⁺ permeability. It is reminiscent of a possibility that econazole exhibits harmful action on human in the presence of Zn²⁺ at a physiological range when the agent is systemically administered. Because it is necessary to characterize the cytotoxic action of econazole in the presence of Zn²⁺, we have cytometrically examined the effects of econazole, ZnCl₂, and their combination on rat thymocytes. ZnCl₂ at concentrations ranging from 1 μM to 30 μM significantly increased the lethality induced by 10 μM econazole in a concentration-dependent manner. Econazole at a sublethal concentration of 1 μM significantly augmented the intensity of side scatter in the presence of micromolar ZnCl₂, suggesting the change in an intracellular circumstance by the combination of econazole and ZnCl₂. Econazole at 0.3 μM or more in the presence of ZnCl₂ increased the intensity of Fluo-3 fluorescence, an indicator for intracellular Ca²⁺. Furthermore, the intensity of FluoZin-3 fluorescence, an indicator for intracellular Zn²⁺, was also augmented by econazole at 0.1 μM or more in the presence of ZnCl₂. Results suggest that the combination of submicromolar econazole with micromolar ZnCl₂ may increase the intracellular concentration of Ca²⁺ and Zn²⁺, leading to disturbance of intracellular Ca²⁺ and Zn²⁺ homeostasis that triggers cytotoxic action.     

Interaction of mephedrone with dopamine and serotonin targets in rats

IntroductionWe described a first approach to the pharmacological targets of mephedrone (4-methyl-methcathinone) in rats to establish the basis of the mechanism of action of this drug of abuse.Experimental proceduresWe performed in vitro experiments in isolated synaptosomes or tissue membrane preparations from rat cortex or striatum, studying the effect of mephedrone on monoamine uptake and the displacement of several specific radioligands by this drug.ResultsIn isolated synaptosomes from rat cortex or striatum, mephedrone inhibited the uptake of serotonin (5-HT) with an IC₅₀ value lower than that of dopamine (DA) uptake (IC₅₀ = 0.31 ± 0.08 and 0.97 ± 0.05 μM, respectively). Moreover, mephedrone displaced competitively both [³H]paroxetine and [³H]WIN35428 binding in a concentration-dependent manner (Ki values of 17.55 ± 0.78 μM and 1.53 ± 0.47 μM, respectively), indicating a greater affinity for DA than for 5-HT membrane transporters. The affinity profile of mephedrone for the 5-HT₂ and D₂ receptors was assessed by studying [³H]ketanserin and [³H] raclopride binding in rat membranes. Mephedrone showed a greater affinity for the 5-HT₂ than for the D₂ receptors.DiscussionThese results provide evidence that mephedrone, interacting with 5-HT and DA transporters and receptors must display a similar pattern of other psychoactive drugs such as amphetamine-like compounds.     

Endothelium-dependent vasomotor effects of telmisartan in isolated rat femoral arteries

AT₁ receptor antagonists (ARBs) are drugs widely used for preventing and/or treating major cardiovascular diseases. Some of these drugs also show AT₁ receptor-independent effects that may have patho-physiological significance, such as Peroxisome Proliferator-Activated Receptors gamma (PPARγ) stimulation. Here we investigated the effect of telmisartan (that also stimulates PPARγ) on vasomotor responses of femoral arteries isolated from rat, in comparison to losartan. Femoral artery segments were mounted in a wire myograph and challenged with cumulative concentrations of phenylephrine (PE) and acetylcholine (ACh) after 30-min incubation in the absence or presence of 30 μM telmisartan or 30 μM losartan. Vasomotor responses were not significantly changed by losartan, whereas telmisartan reduced vasoconstriction to PE and increased vasodilatation to ACh. Incubation with 0.1 mM N^G-nitro-l-arginine abolished relaxation to ACh in untreated controls as well as in losartan-treated preparations, but did not in telmisartan-treated preparations (were 20% relaxation subsisted); this residual relaxing effect was abolished by indomethacin and by endothelium removal. Incubation with 30 μM GW9662 (PPARγ antagonist), 10 μM PD123319 (AT₂ antagonist) or 30 μM A779 (angiotensin(1-7)/Mas antagonist) did not change the effect of telmisartan on vasomotor responses in preparations with intact endothelium. We conclude that telmisartan modifies constriction and dilatation of isolated arteries in an endothelium-dependent manner, involving both nitric oxide and prostanoid production. The present effect of telmisartan, however, does not seem to involve PPARγ, AT₂ or angiotensin(1-7)/Mas.     

Vasorelaxant and antihypertensive effects of methanolic fraction of the essential oil of Alpinia zerumbet

Alpinia zerumbet is used in folk medicine in Brazil to treat hypertension. However, several pathways involved in the mechanism of vasorelaxation are still unclear. This study was designed to verify the antihypertensive effect of the methanolic fraction of the essential oil of A. zerumbet (MFEOAz) and to characterize its mechanism of action. The thoracic aortic rings from the Wistar rats were perfused in the organ chambers filled with Kreb's solution, where the tension of each ring was measured. The antihypertensive effect of MFEOAz was assessed in rats submitted to chronic hypertension by inhibition of nitric oxide synthesis by indirect measurement of blood pressure with indirect tail cuff method. MFEOAz relaxed phenylephrine and KCl-induced contraction of either endothelium-intact or endothelium-denuded rat aortic rings in a concentration-dependent manner. Pre-incubation with MFEOAz (100 and 300 μg/mL) in Ca^2 +-free Krebs solution attenuated phenylephrine- or caffeine-induced contraction. Pre-incubation with L-NAME, ODQ, wortmannin, atropine, indomethacin, catalase, SOD, TEA, 4-aminopyridine, glibenclamide, apamin, charybdotoxin, or iberiotoxin did not affect MFEOAz-induced relaxation. The intragastric administration of MFEOAz induced an antihypertensive effect. MFEOAz it seems inhibited the calcium influx via voltage-operated calcium channels and receptor-operated calcium channels, as well as inhibition of calcium mobilization from intracellular stores.     

Human urotensin II in internal mammary and radial arteries of patients undergoing coronary surgery

AimsInternal mammary (IMA) and radial artery (RA) have different incidence of vasospasm and long-term patency rates in arterial grafting. We compared the vasoreactivity of human urotensin II (hU-II) and its receptor with mechanism investigations in IMA and RA.MethodsIMA and RA taken from patients undergoing coronary bypass surgery were studied in organ baths. Urotensin receptor expression was determined by RT-PCR.ResultshU-II contracted IMA with pD₂ of 8.57 ± 0.41 and 45.4 ± 9.1% E_max of contraction to 100 mM KCl, whereas caused less contractile responses in RA (pD₂:8.30 ± 0.79, E_max:20.4 ± 4.8%, p < 0.05). Nifedipine inhibited hU-II-contraction in IMA. In U₄₆₆₁₉-precontraction, hU-II elicited comparable relaxation in IMA (pD₂:8.39 ± 0.43, E_max:56.1 ± 4.0%) and RA (pD₂:9.03 ± 0.46, E_max:65.2 ± 7.1%). The relaxation was abolished by endothelium denudation and by indomethacin, oxadiazoloquinoxalinone or N^ω-nitro-l-arginine, oxyhemoglobin, and Ca²⁺-activated K⁺ channel (K_Ca) blockers. Urotensin receptor mRNA was detected in both arteries.ConclusionshU-II is an important spasmogen in arterial grafts with receptors expressed in IMA and RA. hU-II elicits stronger contraction in IMA than in RA and a moderate endothelium-dependent relaxation attributable to nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor with involvement of K_Ca activation. The relaxant response of endothelium-intact IMA and RA to hU-II demonstrates the importance of preservation of endothelium in these grafts.     

Protection of S-adenosyl methionine against the toxicity of clivorine on hepatocytes

In this study, we investigated the protective effects of S-adenosyl-l-methionine (SAM), which is a precursor of cellular reduced glutathione (GSH), against the hepatotoxicity of pyrrolizidine alkaloid clivorine. MTT assay showed that SAM (5 μM) prevented the cytotoxicity of clivorine on human normal liver L-02 cells. DNA fragmentation assay showed that SAM (5 μM) improved clivorine-induced L-02 cell apoptosis, and the results of Western blot showed that SAM (5 μM) decreased clivorine-induced caspase-3 activation. Cellular GSH analysis showed that when L-02 cells were exposed to different concentrations (0, 3, 10, 30, 50 and 100 μM) of clivorine for 48 h, cellular GSH was decreased in a concentration-dependent manner, while SAM (5 μM) enhanced 50 μM clivorine decreased cellular GSH. Further MTT assay showed that 5 mM GSH and 5 mM N-acetyl-l-cysteine (NAC) both had protective effects against clivorine-induced hepatotoxicity. Our results suggest that SAM has protective effects against the hepatotoxicity of clivorine possibly by enhancing cellular GSH level and increasing cellular defensive ability against clivorine-induced cytotoxicity.