Nitric Oxide Synthase Blocker Research Articles

Anti-hyperalgesic effect of a benzilidine-cyclohexanone analogue on a mouse model of chronic constriction injury-induced neuropathic pain: Participation of the κ-opioid receptor and KATP

The present study investigated the analgesic effect of a novel synthetic cyclohexanone derivative, 2,6-bis-4-(hydroxyl-3-methoxybenzilidine)-cyclohexanone or BHMC in a mouse model of chronic constriction injury-induced neuropathic pain. It was demonstrated that intraperitoneal administration of BHMC (0.03, 0.1, 0.3 and 1.0mg/kg) exhibited dose-dependent inhibition of chronic constriction injury-induced neuropathic pain in mice, when evaluated using Randall–Selitto mechanical analgesiometer. It was also demonstrated that pretreatment of naloxone (non-selective opioid receptor blocker), nor-binaltorphimine (nor-BNI, selective κ-opioid receptor blocker), but not β-funaltrexamine (β-FN, selective μ-opioid receptor blocker) and naltrindole hydrochloride (NTI, selective δ-opioid receptor blocker), reversed the anti-nociceptive effect of BHMC. In addition, the analgesic effect of BHMC was also reverted by pretreatment of 1H-[1,2,4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ, soluble guanosyl cyclase blocker) and glibenclamide (ATP-sensitive potassium channel blocker) but not Nω-nitro-l-arginine (l-NAME, a nitric oxide synthase blocker). Taken together, the present study demonstrated that the systemic administration of BHMC attenuated chronic constriction, injury-induced neuropathic pain. We also suggested that the possible mechanisms include κ-opioid receptor activation and nitric oxide-independent cyclic guanosine monophosphate activation of ATP-sensitive potassium channel opening.

Tempol-nebivolol therapy potentiates hypotensive effect increasing NO bioavailability and signaling pathway

Nebivolol is a third generation beta blocker with endothelial nitric oxide synthase (eNOS) agonist properties. Considering the role of reactive oxygen species (ROS) in the uncoupling of eNOS, we hypothesized that the preadministration of an antioxidant as tempol, could improve the hypotensive response of nebivolol in normotensive animals increasing the nitric oxide (NO) bioavailability by a reduction of superoxide (O2•−) basal level production in the vascular tissue.Male Sprague Dawley rats were given tap water to drink (control group) or tempol (an antioxidant scavenger of superoxide) for 1 week. After 1 week, Nebivolol, at a dose of 3 mg/kg, was injected intravenously to the control group or to the tempol-treated group. Mean arterial pressure, heart rate, and blood pressure variability were evaluated in the control, tempol, nebivolol, and tempol nebivolol groups, as well as, the effect of different inhibitor as Nβ-nitro-l-arginine methyl ester (L-NAME, a Nitric oxide synthase blocker) or glybenclamide, a KATP channel inhibitor. Also, the expression of α,β soluble guanylate cyclase (sGC), phospho-eNOS, and phospho-vasodilator-stimulated phosphoprotein (P-VASP) were evaluated by Western Blot and cyclic guanosine monophosphate (cGMP) levels by an enzyme-linked immunosorbent assay (ELISA) commercial kit assay.We showed that pretreatment with tempol in normotensive rats produces a hypotensive response after nebivolol administration through an increase in the NO bioavailability and sGC, improving the NO/cGMP/protein kinase G (PKG) pathway compared to that of the nebivolol group.We demonstrated that tempol preadministration beneficiates the response of a third-generation beta blocker with eNOS stimulation properties, decreasing the basal uncoupling of eNOS, and improving NO bioavailability. Our results clearly open a possible new strategy therapeutic for treating hypertension.

13. Anti-inflammatory role of L-NG-Nitroarginine Methyl Ester in a chronic model of depression

The role of nitric oxide (NO) in the neurodegenerative disorders is well illustrated. The close relationship of NO level and the inflammation points out the clinical relevance of NO in depressive disorder. Present study evaluated the neuroprotective role of L-NAME, a NO synthase blocker in chronic restraint hypoxia (CRH) induced depressive behaviour. Briefly four groups of rat were exposed to CRH for 21 days. Group 1 and 2 served as normoxic control group (NOR + VEH, NOR + DRUG), group 3 and 4 served as stress exposed groups (CRH + VEH, CRH + DRUG) and the 5th group was LPS (Lipopolysaccharide) + DRUG. On the last day behavioural assessment was done and immediately brain sample were isolated for histology and biochemical parameters. IHC, ELISA and western blot were used to study the expression of stress markers following CRH exposure. Our results show that L-NAME administration to rats improved the exploratory and rearing behaviour and also reduced despair behaviour. DCX, a marker for neuronal migration and neurogenesis was increased significantly along with TRAF-2 and significant decreased in the expression of IL-1 receptor, NF-kB, iNOS, Cox-2, nNOS, H3K9 (epigenetic marker), nitric oxide and proinflammatory cytokines suggesting the role of L-NAME in buffering the stress induced behavioural impairment. From the above study it can be concluded that L-NAME treatment improves functional outcomes of rats during CRH exposure. Inhibiting NO with L-NAME increases neurogenesis and reduces inflammation.

Role of catecholamines and nitric oxide on pigment displacement of the chromatophores of freshwater snakehead teleost fish, Channa punctatus

We are reporting for the first time that the catecholamines (adrenaline and noradrenaline) inhibit the effect of nitric oxide (NO) on melanosome dispersion in freshly isolated scales of the freshwater snakehead fish, Channa punctatus. We studied the effect of NO and catecholamines on the pigment displacement by observing the changes in the melanophore index. The scales when treated with solution containing NO donor sodium nitroprusside (SNP) showed dispersion of melanosomes, whereas NO synthase blocker N-omega-Nitro-L-arginine suppresses this action of SNP. Treatment with adrenaline and noradrenaline on the isolated scales caused aggregation of melanosomes. Scales treated with solution containing catecholamines and SNP resulted in aggregation of melanosomes suggesting that catecholamines mask the effect of SNP. These results suggest that the catecholamines are inhibiting the effect of NO and causing the aggregation of the melanosomes may be via surface receptors.

OP014 Hydrogen sulfide induces relaxation independent of changes in smooth muscle cell calcium in rat small arteries

Background and purpose Hydrogen sulfide (H2S) is an endogenously produced gas with many physiological functions including involvement in regulation of the blood pressure. The present study was designed to investigate the effect of H2S in small arteries and the mechanisms underlying H2S-induced vasodilation. Experimental approach Mesenteric and pulmonary small arteries from rats were isolated and mounted in microvascular myographs for isometric tension recordings. The endogenous production of H2S was blocked with enzyme inhibitors dl-propargylglycine (PPG) and amino-oxyacetate (AOA) with and without the presence of blockers of nitric oxide (NO) synthase (nitro- l -arginine) and cyclooxygenase (indomethacin). NaHS (a H2S donor) was applied in arteries with and without endothelium as well as in the presence of different blockers. Simultaneous measurements of intracellular calcium concentration and force were conducted by fura-2 AM fluorescence. Key results In mesenteric arteries acetylcholine (ACh)-induced vasodilation was reduced in the presence of PPG and AOA both in the absence and the presence of indomethacin and nitro- l -arginine. In pulmonary arteries, the combination of indomethacin and nitro- l -arginine inhibited ACh-induced vasodilation, and there was no further effect by adding PPG and AOA. Application of NaHS relaxed mesenteric arteries with an EC50 of 385 μM. Removing endothelium, inhibition of KATP-channels or NO synthase did not affect H2S-induced vasodilation in mesenteric or pulmonary arteries. Simultaneous measurements of calcium and relaxation revealed that NaHS induces relaxation without changes in smooth muscle calcium. Conclusions and implications H2S induces vasodilation in both rat mesenteric and pulmonary arteries, but is only involved in ACh-induced vasodilatation in rat mesenteric arteries. H2S-induced vasodilation was endothelium-independent and may involve desensitization of the contractile apparatus in the vascular smooth muscle.

Antispasmodic effects and composition of the essential oils from two South American chemotypes of Lippia alba

Ethopharmacology relevanceLippia alba (Mill.) N. E. Brown (Verbenaceae) is an aromatic species used in Central and South America as eupeptic for indigestion. In Argentina, it is used by the “criollos” from the Chaco province. There are several chemotypes which differ in the chemical composition of the essential oils. Nowadays, it is experimentally cultivated in some countries of the region, including Argentina. Aim of the studyTo compare the chemical composition and pharmacology of the essential oils from two chemotypes: “citral” (CEO) and “linalool” (LEO), in isolated rat duodenum and ileum. Methods: Contractile concentration–response curves (CRC) of acetylcholine (ACh) and calcium in 40mM K+-medium (Ca2+-CRC) were done in isolated intestine portions, in the absence and presence of CEO or LEO at different concentrations. ResultsLikewise verapamil, CEO and LEO induced a non-competitive inhibition of the ACh-CRC, with IC50 of 7.0±0.3mg CEO/mL and 37.2±4.2mg LEO/mL. l-NAME, a NO-synthase blocker, increased the IC50 of CEO to 26.1±8.7mg CEO/mL. Likewise verapamil, CEO and LEO non-competitively inhibited the Ca2+-CRC, with IC50 of 6.3±1.7mg CEO/mL, 7.0±2.5mg LEO/mL and 0.24±0.04mg verapamil/mL (pIC50: 6.28). CEO was proved to possess limonene, neral, geranial and (−)-carvone as the major components, while LEO was rich in linalool. ConclusionsResults suggest that CEO has five times more potency than LEO to inhibit muscarinic contractions. The essential oils of both chemotypes interfered with the Ca2+-influx, but with an IC50 about 28 times higher than that of verapamil. Moreover, CEO partially stimulated the NO production. These results show the medicinal usefulness of both Lippia alba chemotypes, thus validating its traditional use, potency and mechanism of action.

Anti-inflammatory effect of a sulphated polysaccharide fraction extracted from the red algae Hypnea musciformis via the suppression of neutrophil migration by the nitric oxide signalling pathway

The aim of this study was to evaluate the anti-inflammatory effect of a sulphated polysaccharide fraction (PLS) extracted from the alga Hypnea musciformis and investigate the possible involvement of the nitric oxide (NO) pathway in this effect. The anti-inflammatory activity of PLS was evaluated using inflammatory agents (carrageenan and dextran) to induce paw oedema and peritonitis in Swiss mice. Samples of paw tissue and peritoneal fluid were removed to determine myeloperoxidase (MPO) activity, NO3 /NO2 levels, and interleukin-1β (IL-1β) level. The involvement of NO in the modulation of neutrophil migration in carrageenan-induced paw oedema or peritonitis was also investigated. Compared with vehicle-treated mice, mice pretreated with PLS (10 mg/kg) inhibited carrageenan-induced and dextran-induced oedema; it also inhibited total and differential peritoneal leucocyte counts in a model of peritonitis. These PLS effects were reversed by l-arginine treatment and recovered with the administration of a NO synthase blocker (aminoguanidine). Furthermore, PLS reduced the MPO activity, decreased IL-1β levels, and increased NO3 /NO2 levels in the peritoneal cavity. PLS reduced the inflammatory response by modulating neutrophil migration, which appeared to be dependent on the NO pathway.

Contribution of α-Adrenoceptor Stimulation by Phenylephrine to Basal Nitric Oxide Production in the Isolated Mouse Aorta

In the mouse aorta, contractions evoked by the α(1)-adrenoceptor agonist phenylephrine are strongly suppressed by the continuous production of nitric oxide (NO). We investigated whether phenylephrine itself stimulated NO production by activating endothelial α(2)-adrenoceptors. On a prostaglandin F(2α) contraction, the α(2)-adrenoceptor agonist 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14304) induced 29.3 ± 7.4% relaxation, which was inhibited by 0.1 μM 2-[(4,5-Dihydro-1H-imidazol-2-yl)methyl]-2,3-dihydro-1-methyl-1H-isoindole (BRL44408) with a pKB' corresponding to α(2)-antagonism. In the presence of NO synthase blockers, UK14304 elicited small contractions above 1 μM that were inhibited by 0.1 μM prazosin, but not influenced by 0.1 μM rauwolscine. At 3 μM or higher concentrations, phenylephrine caused only modest relaxation (up to 7.4 ± 2.3%) of segments constricted with prostaglandin F(2α) in the presence of prazosin, which was abolished with 0.1 μM BRL44408. Furthermore, BRL44408 did not increase contractions induced with 1 μM phenylephrine. These results confirm that α(1)- but not α(2)-adrenoceptors are expressed on aortic smooth muscle cells, whereas endothelial cells only express α(2)-adrenoceptors. Moreover, phenylephrine exerted a very modest relaxing effect through nonspecific stimulation of α(2)-adrenoceptors, but only at concentrations higher than 1 μM. It is concluded that the high basal output of NO in the isolated mouse aorta is not due to stimulation of α-adrenoceptors.

Angiotensin‐(1–7) stimulates angiogenesis in murine corpus cavernosum

Angiotensin (Ang)‐(1–7), a metabolite of Angiotensin‐converting enzyme‐2 (ACE2), is cardiovascular protective. Recent studies have implicated an important role of angiogenic progenitor cells (APCs) in the functions of Ang‐(1–7). This study evaluated the effect of Ang‐(1–7) in the tissue angiogenesis independent of APCs. Strips of corpus cavernosum (CC) or aortic rings were isolated from mice and cultured in matrigel. Formation of sprouts and tubes by endothelial cells (EC) were evaluated. In some experiments, assays were performed in CC‐strips from mice following treatment with Ang‐(1–7) (100μg/Kg, s.c., 4 weeks). ECs from sinusoidal CC have migrated into the matrix in 24 to 48 hours, and endothelial sprouts and tubes were observed in 3–5 days. Ang‐(1–7) treatment potentiated this response that was lower than the effect of VEGF (100nM). Effect of Ang‐(1–7) was inhibited by Mas receptor antagonist A‐779, and was not affected by nitric oxide synthase blocker L‐NAME. CC strips from Ang‐(1–7)‐treated mice have shown increased sprout and tube formations compared to the control. In mouse aortic rings, angiogenesis was observed in 3 to 5 days and the migration of smooth muscle cells was seen by day 4. Treatment with Ang‐(1–7) has no effect. These studies support that Ang‐(1–7) stimulates angiogenesis without the involvement of APCs by activating Mas receptor in the sinusoids of CC and not in a conduit blood vessel.

The role of nitric oxide in the regulation of neurotransmitter release and processes of exo- and endocytosis of synaptic vesicles in mouse motor nerve endings

In experiments with mouse diaphragm muscle, the effects of a nitric oxide (NO) donor, S-nitroso- N-acetyl-DL-penicillamine (SNAP), and an NO-synthase blocker, NG-nitro-L-arginine methyl ester (LNAME), on transmitter release and processes of exo- and endocytosis of synaptic vesicles in the motornerve ending were studied using electrophysiological and fluorescence techniques. During single stimulation of the motor nerve, SNAP reduced and LNAME did not change the amplitude of the endplate currents and both of the drugs did not affect spontaneous transmitter release. During high-frequency stimulation (20 Hz, 3 min) SNAP increased and LNAME slowed the depression of the amplitudes of endplate potentials (EPPs) compared to the dynamics of EPPs in the control. In experiments using the fluorescent dye FM 1–43, it was shown that the NO donor induced a decrease and LNAME induced an increase in the fluorescence intensity of motor-nerve endings loaded with dye during stimulation at a frequency of 20 Hz for 30 s compared to the control. At the same time, the rate of dye unloading from the terminals that were preloaded with FM 1–43 was higher after preliminary application of SNAP and lower after preliminary application of LNAME. It was suggested that exogenous and endogenous NO in the mouse neuromuscular synapse caused the depression of neurotransmitter release as a result of the suppression of synaptic-vesicle recycling due to a decrease in endocytosis or/and mobilization of synaptic vesicles from a recycling pool to the exocytosis sites.

The potent relaxant effect of resveratrol in rat corpus cavernosum and its underlying mechanisms

The aim of this study was to evaluate the relaxant effect of resveratrol (RVT), one of the most commonly employed dietary polyphenols, in rat corpus cavernosum, and to further investigate the contribution of possible underlying mechanisms. Strips of corpus cavernosum were used in organ baths for isometric tension studies. RVT (10(-6)-10(-4) M) produced concentration-dependent relaxation responses in rat corpus cavernosum precontracted by phenylephrine. The relaxant responses to RVT partially, but significantly inhibited by removal of endothelium. Nitric oxide (NO) synthase (NOS) blocker N-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) or soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10(-5) M) caused a significant inhibition on relaxation response to RVT, whereas cyclooxygenase inhibitor indomethacin (10(-5) M) did not significantly alter relaxant responses of corpus cavernosum strips to RVT. Corpus cavernosum contractions induced by stepwise addition to Ca2+ to high KCl solution with no Ca2+ were significantly inhibited by RVT incubation. The treatment of corpus cavernosum tissues with non-specific potassium channel inhibitor tetraethylammonium (TEA, 10(-2) M) did also significantly affect the relaxant activity of RVT. Otherwise, the relaxation response of corpus cavernosum induced by the phosphodiesterase-5 inhibitor sildenafil increased significantly in the group pretreated with 10(-5) M RVT. These results demonstrated that RVT has a potent relaxant effect on rat corpus cavernosum via endothelium-dependent and -independent mechanisms. Endothelium-dependent relaxation of corpus cavernosum to RVT is thought to be mediated primarily through NO/cGMP signaling pathway, and possibly an additional mechanism, endothelium-dependent hyperpolarization factor (EDHF). The residual endothelium-independent corpus cavernosum relaxation induced by RVT is uncertain but seems to depend on the interactions of RVT with Ca2+ entry mechanism from the extracellular space and also other undefined direct effects in this tissue.

Effects of vitamin D3 derivative – calcitriol on pharmacological reactivity of aortic rings in a rodent PCOS model

BackgroundThe aim of this study was to examine the effects of the hyperandrogenic state in dihydrotestosterone (DHT)-induced polycystic ovary syndrome (PCOS), the vascular responses to different vasoactive agents, and the modulatory role of vitamin D3. MethodsAPCOS model was induced by DHT application in 20 femaleWistar rats. Ten of the DHT treated rats simultaneously received calcitriol treatment. After 10 weeks, myographs were used to test the reactivity of isolated thoracic aortic rings to norepinephrine and acetylcholine. Thereafter, the vascular rings were incubated with the NO-synthase blocker (nitro-L-arginine methyl ester) or the cyclooxygenase inhibitor (indomethacin) for 20min, and the effects of norepinephrine and acetylcholine were re-evaluated. ResultsNorepinephrine-induced vasoconstriction was enhanced after DHT treatment, but this effect was attenuated by calcitriol administration. Vasorelaxation of DHT-treated thoracic aortic rings was impaired, but this could be partly reversed by calcitriol application. Impaired NO-dependent vasorelaxation in DHT-treated animals was mostly reversed by concomitant calcitriol administration, but this effect was diminished by prostanoid-dependent vasoconstriction. ConclusionsThese studies show that the enhanced sensitivity to vasoconstrictors and impaired NO-dependent vasorelaxation in hyperandrogenic PCOS rats could be partially reversed by calcitriol treatment.

NOS Inhibition Enhances Myogenic Tone by Increasing Rho-Kinase Mediated Ca2+ Sensitivity in the Male but Not the Female Gerbil Spiral Modiolar Artery

Cochlear blood flow regulation is important to prevent hearing loss caused by ischemia and oxidative stress. Cochlear blood supply is provided by the spiral modiolar artery (SMA). The myogenic tone of the SMA is enhanced by the nitric oxide synthase (NOS) blocker L-NG-Nitro-Arginine (LNNA) in males, but not in females. Here, we investigated whether this gender difference is based on differences in the cytosolic Ca2+ concentration and/or the Ca2+ sensitivity of the myofilaments. Vascular diameter, myogenic tone, cytosolic Ca2+, and Ca2+ sensitivity were evaluated in pressurized SMA segments isolated from male and female gerbils using laser-scanning microscopy and microfluorometry. The gender difference of the LNNA-induced tone was compared, in the same vessel segments, to tone induced by 150 mM K+ and endothelin-1, neither of which showed an apparent gender-difference. Interestingly, LNNA-induced tone in male SMAs was observed in protocols that included changes in intramural pressure, but not when the intramural pressure was held constant. LNNA in male SMAs did not increase the global Ca2+ concentration in smooth muscle cells but increased the Ca2+ sensitivity. This increase in the Ca2+ sensitivity was abolished in the presence of the guanylyl cyclase inhibitor ODQ or by extrinsic application of either the nitric oxide (NO)-donor DEA-NONOate or the cGMP analog 8-pCPT-cGMP. The rho-kinase blocker Y27632 decreased the basal Ca2+ sensitivity and abolished the LNNA-induced increase in Ca2+ sensitivity in male SMAs. Neither LNNA nor Y27632 changed the Ca2+ sensitivity in female SMAs. The data suggest that the gender difference in LNNA-induced tone is based on a gender difference in the regulation of rho-kinase mediated Ca2+ sensitivity. Rho-kinase and NO thus emerge as critical factors in the regulation of cochlear blood flow. The larger role of NO-dependent mechanisms in male SMAs predicts greater restrictions on cochlear blood flow under conditions of impaired endothelial cell function.

Sivelestat Attenuates Myocardial Reperfusion Injury during Brief Low Flow Postischemic Infusion

The neutrophil elastase inhibitor sivelestat (ONO-5046) possesses unknown mechanisms of cardioprotection when infused following global ischemia, even in the absence of neutrophils. Since myocardial ischemia-reperfusion injury is strongly associated with endothelial dysfunction and reactive oxygen species (ROS) generation during reperfusion, we have tested the hypothesis that infusion of sivelestat during postischemic low flow would preserve endothelial and contractile function and reduce infarct size through an ROS-mediated mechanism. Isolated male rat hearts, subjected to global ischemia of 25 minutes, were reperfused with low flow with or without sivelestat followed by a full flow reperfusion. Hearts treated with sivelestat showed a significant improvement of LV contractile function and a reduction in infarct size. Infusion of L-NAME (nonspecific blocker of endothelial nitric oxide synthase (eNOS)) along with sivelestat during reperfusion reversed the preservation of contractile function and infarct size. In vitro EPR spin trapping experiments showed that sivelestat treatment decreased superoxide adduct formation in bovine aortic endothelial cells (BAECs) subjected to hypoxia-reoxygenation. Similarly, dihydroethidine (DHE) staining showed decreased superoxide production in LV sections from sivelestat-treated hearts. Taken together, these results indicate that sivelestat infusion during postischemic low flow reduces infarct size and preserves vasoreactivity in association with decreased ROS formation and the preservation of nitric oxide.

Fos Immunoreactivity in the Motor Cortex of Rats Realizing Operant Movements: Changes after Systemic Introduction of a NOS Blocker

The immunoreactivity with respect to the c-Fos protein in the primary motor cortex (area M1) of rats was subjected to comparative examination. As was found, the respective immunoreactivity patterns in layers II–VI of this zone in intact animals and rats realizing operant food-procuring reflexes, when observed under control conditions and after systemic injections of a blocker of neuronal NO synthase, 7-nitroindazole (7-NI), demonstrated noticeable specificities. Mean values of the density of Fos-immunopositive (Fos-ip) neurons in layers II–III of the M1 area (at the levels +2.7 and +2.2 mm from the bregma) in both rats after preliminary 7-NI injections and in animals performing operant reflexes, but with no such injections, were significantly lower than in the control intact group. At the same time, significantly greater numbers of Fos-ip neurons were found after 7-NI injections in rats realizing operant food-procuring reflexes (at the contralateral, with respect to the working forelimb, side of the motor cortex). It is supposed that NO-generating NADPH-diaphorase-reactive neurons and afferent projections (ascending from the subcortical limbic centers), which are localized in the M1 area, are involved in the formation of specific patterns of neuronal activity in the examined cortical layers. Suppression of functioning of neuronal NOS after systemic introduction of 7-NI modulates neuronal activity in superficial and deep layers of the M1 in different directions, and this occurs in both control rats and animals realizing food-procuring reflexes.

Tacrine-induced tachyphylaxis in gastric smooth muscles

Abstract Tacrine is a medication applied in cases of mild to moderate dementia in Alzheimer’s disease. By blocking acetylcholinesterase activity the drug increases the concentration of acetylcholine, whose effects influence the functions of different organs and systems of the body. The effect of tacrine on smooth muscle preparations isolated from rat stomach was studied by isometric registration of muscle contractility. Our investigations found a specific significant systematic decrease in the strength of consecutive tacrine-induced contractions of smooth muscle preparations, a phenomenon known as tachyphylaxis. The tacrineinduced tachyphylaxis was significantly inhibited by SQ22536 (inhibitor of adenylate cyclase activity), by blockers of nitric oxide synthase and KT5823 (inhibitor of protein kinase G). The process was not influenced by cyclopiazonic acid (specific blocker of sarco/endoplasmic reticulum Ca2+-ATPase,) and atropine (blocker of M-cholinergic receptors). We hypothesize that the overlapping and different time-development of the two opposing processes: smooth muscle contraction caused by acetylcholinesterase inhibition and tacrine-induced relaxation influenced by synthesis of nitric oxide, results in tachyphylaxis.

Levosimendan Protection against Kidney Ischemia/Reperfusion Injuries in Anesthetized Pigs

Ischemia/reperfusion (I/R) injury is an important cause of acute renal failure because of oxidative, inflammatory, and apoptotic mechanisms. The aim of the present study was to examine any possible protective effects of levosimendan in an in vivo pig model of renal I/R injury. In 40 anesthetized pigs (eight groups of five pigs each), I/R was induced by clamping-reopening the left renal artery. During ischemia, in three groups of pigs, levosimendan and the multiorgan preservation solution Custodiol, alone or in combination with levosimendan, were infused in the renal artery. In two other groups of animals, levosimendan in combination with Custodiol was administered after the intrarenal nitric-oxide (NO) synthase blocker N(ω)-nitro-L-arginine methyl ester (L-NAME) or the mitochondrial ATP-sensitive K(+) channel (K(ATP) channel) inhibitor 5-hydroxydecanoate (5-HD). In the other animals, saline, L-NAME, or 5-HD were administered alone. Throughout the experiments, urinary N-acetyl-β-glucosaminidase (NAG) release was measured, and renal function was assessed. Moreover, renal biopsy samples were taken for the detection of apoptosis and tissue peroxidation. In pigs treated with levosimendan or the combination of levosimendan and Custodiol, NAG, peroxidation, and apoptotic markers were lower than in animals treated with Custodiol alone. In addition, renal function was better preserved, and cell survival and antioxidant systems were more activated. All beneficial effects were prevented by L-NAME and 5-HD. In conclusion, levosimendan alone or in combination with Custodiol exerted better protection against renal I/R injuries than Custodiol alone through antioxidant, antiapoptotic, and prosurvival actions depending on mitochondrial K(ATP) channels and NO-related mechanisms.

Endothelin‐Mediated Calcium Responses in Supraoptic Nucleus Astrocytes Influence Magnocellular Neurosecretory Firing Activity

In addition to their peripheral vasoactive effects, accumulating evidence supports an important role for endothelins (ETs) in the regulation of the hypothalamic magnocellular neurosecretory system, which produces and releases the neurohormones vasopressin (VP) and oxytocin (OT). Still, the precise cellular substrates, loci and mechanisms underlying the actions of ETs on the magnocellular system are poorly understood. In the present study, we combined patch-clamp electrophysiology, confocal Ca(2+) imaging and immunohistochemistry to study the actions of ETs on supraoptic nucleus (SON) magnocellular neurosecretory neurones and astrocytes. Our studies show that ET-1 evoked rises in [Ca(2+) ](i) levels in SON astrocytes (but not neurones), an effect largely mediated by the activation of ET(B) receptors and mobilisation of thapsigargin-sensitive Ca(2+) stores. The presence of ET(B) receptors in SON astrocytes was also verified immunohistochemically. ET(B) receptor activation either increased (75%) or decreased (25%) SON firing activity, both in VP and putative OT neurones, and these effects were prevented when slices were preincubated in glutamate receptor blockers or nitric oxide synthase blockers, respectively. Moreover, ET(B) -mediated effects in SON neurones were also prevented by a gliotoxin compound, and when changes in [Ca(2+) ](i) were prevented with bath-applied BAPTA-AM or thapsigargin. Conversely, intracellular Ca(2+) chelation in the recorded SON neurones failed to block ET(B) -mediated effects. In summary, our results indicate that ET(B) receptor activation in SON astrocytes induces the mobilisation of [Ca(2+) ](i) , likely resulting in the activation of glutamate and nitric oxide signalling pathways, evoking in turn excitatory and inhibitory SON neuronal responses, respectively. Taken together, our study supports an important role for astrocytes in mediating the actions of ETs on the magnocellular neurosecretory system.

Vasorelaxant and hypotensive effects of a hydroalcoholic extract from the fruits of Nitraria sibirica Pall. (Nitrariaceae)

Ethnopharmacological relevanceFruits of Nitraria sibirica Pall. are traditionally used in Uighur medicine to treat hypertension. This study aimed to support that folk use by defining their vasoactive and hypotensive properties. Materials and methodsThe vasorelaxant activity and the underlying mechanisms of a hydroalcoholic extract from the fruits of Nitraria sibirica Pall. (NSHE) were evaluated on thoracic aortic rings isolated from Wistar rats. In addition, the acute hypotensive effect of NSHE was assessed in anesthetized spontaneously hypertensive rats (SHR) and in their normotensive control Wistar Kyoto (WKY) rats. ResultsNSHE (0.1–10g/l) was clearly more effective to induce vasodilation of phenylephrine- (PE, 1μM) than high KCl- (60mM) pre-contracted aortic rings with respective Emax values of 82.9±2.2% and 34.8±3.6%. The removal of endothelium almost abolished the relaxant effect of the extract. In addition, pre-treatment with Nw-nitro-l-arginine-methyl ester (l-NAME, 100μM), atropine (1μM) or charybdotoxin (30nM) plus apamin (30nM), respective blockers of nitric oxide (NO) synthase, muscarinic receptors and endothelium-derived hyperpolarizing factor (EDHF), significantly reduced the observed effect of NSHE. By contrast, the cyclooxygenase (COX) inhibitor indomethacin (10μM) or the K+ channels blockers glibenclamide (10μM), iberiotoxin (30nM) and 4-amino-pyridine (4-AP, 1mM) failed to modify the vasodilation. Finally, the acute intravenous injection of NSHE (1, 5, 10, 20mg/kg) induced an immediate and transient hypotensive effect in anesthetized SHR and in WKY rats. ConclusionsThis experimental animal study suggests that hydroalcoholic extract from the fruits of Nitraria sibirica Pall. induces vasorelaxation through an endothelium-dependent pathway involving NO synthase (NOS) activation, EDHF production and muscarinic receptor stimulation. Additionally, our results determine that this vasorelaxant effect is translated by a significant hypotensive effect.

TGFβ receptor activation enhances cardiac apoptosis via SMAD activation and concomitant NO release

Transforming growth factor β (TGFβ) expression is induced in the myocardium during transition from compensated hypertrophy to heart failure. In cardiomyocytes, stimulation with TGFβ results in restricted contractile function and enhanced apoptosis. Nitric oxide (NO) also induces apoptosis and influences cardiac function. Therefore, we wanted to know whether NO is causally involved in TGFβ-induced apoptosis. In isolated ventricular cardiomyocytes of adult rat incubation with TGFβ(1) increased NO release which was inhibited by NOS inhibitor ETU but not with iNOS inhibitor (1400 W) or nNOS inhibitor (TFA). In addition, TGFβ-induced apoptosis was blocked with ETU and ODQ, but not with 1400 W or TFA. The consequent assumption that endothelial NOS is involved in TGFβ-induced NO formation and apoptosis was supported by increased phosphorylation of eNOS at serine 1177 and by the fact that TGFβ did not increase NO release in eNOS KO mice. Furthermore, TGFβ-induced apoptosis, NO formation, SMAD binding activity and SMAD2 phosphorylation were blocked by a TGFβ receptor antagonist, but only apoptosis and NO formation could be blocked with ETU. Expression of SMAD7 was increased after TGFβ stimulation and blocked with TGFβ receptor antagonist but not after blocking NO synthase with ETU. In cardiomyocytes TGFβ-induced apoptosis is mediated via TGFβ receptor activation that concomitantly activates SMAD transcription factors and the eNOS/NO/sGC pathway. Both of these pathways are needed for apoptosis induction by TGFβ. This reveals a new pathway of cardiac NO release and identifies NO as a possible contributor to heart failure progression mediated by TGFβ.