Thakur Uttam Singh

TRPV4 channel activation leads to endothelium-dependent relaxation mediated by nitric oxide and endothelium-derived hyperpolarizing factor in rat pulmonary artery

The purpose of the present study was to characterize TRPV4 channels in the rat pulmonary artery and examine their role in endothelium-dependent relaxation. Tension, Real-Time polymerase chain reaction (Real-Time PCR) and Western blot experiments were conducted on left and right branches of the main pulmonary artery from male Wistar rats. TRPV4 channel agonist GSK1016790A (GSK) caused concentration-related robust relaxation (Emax 88.6±5.5%; pD2 8.7±0.2) of the endothelium-intact pulmonary artery. Endothelium-denudation nearly abolished the relaxation (Emax 5.6±1.3%) to GSK. TRPV4 channel selective antagonist HC067047 significantly attenuated GSK-induced relaxation (Emax 56.2±6.6% vs. control Emax 87.9±3.3%) in endothelium-intact vessels, but had no effect on either ACh-induced endothelium-dependent or SNP-induced endothelium-independent relaxations. GSK-induced relaxations were markedly inhibited either in the presence of NO synthase inhibitor L-NAME (Emax 8.5±2.7%) or sGC inhibitor ODQ (Emax 28.1±5.9%). A significant portion (Emax 30.2±4.4%) of endothelium-dependent relaxation still persisted in the combined presence of L-NAME and cyclooxygenase inhibitor indomethacin. This EDHF-mediated relaxation was sensitive to inhibition by 60mM K(+) depolarizing solution or K(+) channel blockers apamin (SKCa; KCa2.3) and TRAM-34 (IKCa; KCa3.1). GSK (10(-10)-10(-7)M) caused either modest decrease or increase in the basal tone of endothelium-intact or denuded rings, respectively. We found a greater abundance (>1.5 fold) of TRPV4 mRNA and protein expressions in endothelium-intact vs. denuded vessels, suggesting the presence of this channel in pulmonary endothelial and smooth muscle cells as well. The present study demonstrated that NO and EDHF significantly contributed to TRPV4 channel-mediated endothelium-dependent relaxation of the rat pulmonary artery.

Eicosapentaenoic acid-induced endothelium-dependent and -independent relaxation of sheep pulmonary artery.

It is known that long chain polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), have beneficial effects on cardiovascular function including pulmonary hypertension. The purpose of the present study was to examine the mechanisms involved in EPA-induced relaxation of sheep isolated pulmonary artery by measuring isometric tension. Nitric oxide (NO) derived from constitutive nitric oxide synthase (cNOS) was measured by Greiss method in the presence of the inducible nitric oxide synthase (iNOS) selective inhibitor N-[[3-(aminomethyl) phenyl]methyl]-ethanimidamide, dihydrochloride (1400 W). EPA (10(-)(7)-10(-)(4)M) caused concentration-dependent relaxation of sheep pulmonary artery with a pD(2) of 5.56+/-0.09 and E(max) of 87.40+/-3.10% (n=9). N(G)-nitro-L-arginine methyl ester (L-NAME) 100 microM significantly attenuated (E(max) 41.95+/-6.70%; n=8) EPA-induced relaxation of endothelium intact arterial rings. Similarly, endothelium denudation markedly inhibited (E(max) 17.60+/-1.21%; n=4) EPA-induced relaxation. EPA (30 microM) significantly increased the cNOS-derived NO release (10.17+/-0.96; n=8 versus control 7.43+/-0.78 pmol/mg tissue wet wt./h; n=7) in endothelium intact vessels. However, EPA-stimulated NO release was markedly blunted by either 100 microM L-NAME (7.07+/-0.54 pmol/mg tissue wet wt./h; n=8) or endothelium removal (6.97+/-0.87 pmol/mg tissue wet wt./h; n=17). In endothelium-denuded K(+) (80 mM)-depolarized arterial rings, EPA (30 microM) significantly inhibited CaCl(2)-induced contractions (E(max) 42.77+/-5.90% versus control 94.78+/-9.82%; n=5). The fatty acid also inhibited nifedipine (1 microM)-insensitive 5-HT-induced contractions in this vessel (E(max) 70.57+/-4.88% versus control 161.50+/-17.46%; n=5). In conclusion, EPA relaxes sheep pulmonary artery primarily through endothelium-dependent NO release, and the residual endothelium-independent relaxation may result from inhibition of Ca(2+)-influx through L-type calcium channels, as well as 5-HT-stimulated intracellular Ca(2+) release.

Atorvastatin restores the impaired vascular endothelium-dependent relaxations mediated by nitric oxide and endothelium-derived hyperpolarizing factors but not hypotension in sepsis.

Sepsis has been reported to impair endothelium-dependent vasodilations mediated by nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). Although some studies demonstrate that statins can improve NO-mediated response in septic animals, little is known about its effect on the EDHF response. The present study examined the effects of atorvastatin pretreatment on sepsis-induced endothelial dysfunctions and hypotension in rats. Eighteen hours after the induction of sepsis by cecal ligation and puncture, thoracic aorta and second generation pulmonary arteries were isolated to examine acetylcholine-induced endothelium-dependent dilations mediated by NO and EDHF, respectively. The messenger RNA (mRNA) expression for endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) was done by real-time polymerase chain reaction. NO was measured as nitrate/nitrite release using Griess method. Mean arterial pressure was measured by the invasive method. Sepsis significantly decreased (26%) the relaxation response to acetylcholine in the rat aorta. It also markedly inhibited the eNOS mRNA expression and acetylcholine-stimulated NO release in this vessel. Pretreatment of the rats with atorvastatin (10 mg/kg, orally) 48, 24, and 2 hours before induction of sepsis preserved acetylcholine-induced relaxation, eNOS mRNA expression, acetylcholine-stimulated NO release, and attenuated increase in the inducible NO synthase mRNA expression and basal NO production in the aorta. The maximal EDHF response mediated by acetylcholine was 25.30% ± 3.00% in the pulmonary artery. Sepsis abolished this response but atorvastatin restored it (22.55% ± 2.50%). Atorvastatin, however, failed to prevent sepsis-induced hypotension. These results suggest that atorvastatin can restore impaired endothelium-dependent vasodilations mediated by NO and EDHF but not hypotension in sepsis.

Atorvastatin prevents vascular hyporeactivity to norepinephrine in sepsis: Role of nitric oxide and α1-adrenoceptor mRNA Expression

Hyporeactivity to vasoconstrictors is one of the clinical manifestations of sepsis in man and experimental animals. The objective of the investigation was to examine whether atorvastatin can prevent hyporeactivity to norepinephrine (NE) in mouse aorta in sepsis, and if so, what are the mechanisms involved. Sepsis in mice was induced by cecal ligation and puncture. The aorta was harvested for tension experiment, nitric oxide (NO) and cyclic guanosine monophosphate measurements, and inducible NO synthase (iNOS) and &agr;1D-adrenoceptor mRNA expression studies. In comparison with sham-operated controls, sepsis significantly decreased the contractile response to NE in the mouse aorta. Pretreatment with atorvastatin of septic animals completely restored NE-induced contractions to levels similar to those of sham-operated controls and significantly increased survival time and mean arterial pressure. Atorvastatin also attenuated iNOS-induced overproduction of NO, as well as iNOS mRNA expression. Accordingly, hyporeactivity to NE was not evident in tissues pretreated with selective iNOS inhibitor 1400W in sepsis. Although basal cyclic guanosine monophosphate accumulation in the aorta was reduced in sepsis, pretreatment of the tissues with soluble guanylyl cyclase inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ) partially restored the reactivity to NE. Interestingly, hyporeactivity to NE in sepsis was associated with a decreased &agr;1D-adrenoceptor mRNA expression in the mouse aorta. Atorvastatin pretreatment, however, prevented the decrease in &agr;1D-adrenoceptor mRNA expression in septic animals. In conclusion, atorvastatin seems to prevent hyporeactivity to vasoconstrictor NE in the aorta from septic mice through attenuation of overproduction of NO as well as improved &agr;1D-adrenoceptor mRNA expression. The findings of the present study may explain the beneficial effects of atorvastatin on improved hemodynamic functions in sepsis.

Deferoxamine modulates cytokines and growth factors to accelerate cutaneous wound healing in diabetic rats

Deferoxamine has shown cutaneous wound healing potential by increased neovascularization. We hypothesized that topically applied deferoxamine facilitates wound healing in diabetic rats by modulating important cytokines and growth factors that take part in healing processes in a time-dependent manner. Diabetes was induced in male Wistar rats by streptozotocin and wound was created under pentobarbitone anesthesia. The diabetic rats were divided into two groups, of which one (control) was treated with ointment base and other with deferoxamine ointment (0.1%). Wound closure measurement and tissue collection were done on days 3, 7, 14 and 19 post-wounding. The relative expressions of hypoxia-inducible factor 1-alpha (HIF-1α), vascular endothelial growth factor (VEGF), stromal cell-derived factor 1-alpha (SDF-1α), transforming growth factor beta 1 (TGF-β1), tumor necrosis factor-alpha (TNF-α), matrix metalloproteinase-9 (MMP-9), interleukin-1 beta (IL-1β) and interleukin-10 (IL-10) mRNA and proteins were determined in the wound tissues. CD-31 staining and collagen content were evaluated by immunohistochemistry and picrosirius red staining, respectively. Histological changes were assessed by H&E staining. The per cent wound closure was significantly higher from day 7 onwards in deferoxamine-treated rats. HIF-1α, VEGF, SDF-1α, TGF-β1, IL-10 mRNA and their protein levels were significantly higher on days 3, 7 and 14 in deferoxamine-treated rats. The mRNA expression and protein levels of TNF-α, MMP-9 and IL-1β were progressively and markedly reduced in deferoxamine-treated rats. The collagen deposition and formation of blood vessels were greater in deferoxamine-treated rats. It is suggested that topical application of deferoxamine ointment might be useful in cutaneous wound healing in diabetic patients.

Inhibitory effect of essential oils of Allium sativum and Piper longum on spontaneous muscular activity of liver fluke, Fasciola gigantica.

Effects of essential oil of Allium sativum (garlic) and Piper longum (Indian long pepper) were evaluated on muscular activity of whole Fasciola gigantica and its strip preparation. The whole flukes and longitudinal strip preparations of the flukes were isometrically mounted to record the spontaneous muscular activity (SMA) and to evaluate effects of cumulative doses (0.1, 0.3, 1.0 and 3.0mg/ml) of the plant essential oils. Whole flukes and the strip preparations exhibited continuous SMA without any significant difference in its baseline tension, frequency and amplitude for 2h. Essential oil of A. sativum produced significant reduction in the frequency and the amplitude of the SMA of whole fluke at 1 and 3mg/ml concentrations. It caused complete paralysis of the fluke after 15 min of administration of 3mg/ml concentration. Similar to whole fluke, essential oil of A. sativum (3mg/ml) also produced flaccid paralysis in the strip preparations of the flukes. Essential oil of P. longum firstly induced marked excitatory effect and then there was flaccid paralysis of the whole fluke following 15 min exposure at 3mg/ml concentration. Complete flaccid paralysis of the strip preparation was also ensued after 15 min of administration of 3mg/ml concentration of P. longum. In both the essential oils, the whole fluke and strip preparations did not recover from paralysis following 2-3 washes. In conclusion, the observations demonstrated irreversible paralytic effect of essential oils of A. sativum and P. longum on F. giganticain vitro which might possibly help to developing herbal-based anthelmintic.

Atorvastatin prevents sepsis-induced downregulation of myocardial β1-adrenoceptors and decreased cAMP response in mice.

ABSTRACT Impaired cardiac &bgr;-adrenoceptor signaling is an important cause of sepsis-induced myocardial depression in man and experimental animals. We examined the effect of atorvastatin (ATR) pretreatment on myocardial &bgr;1-adrenoceptor (&bgr;1-AR) expressions and post–receptor signaling in a mouse model of sepsis (cecal ligation and puncture [CLP]). After 20 ± 2 h of surgery, hearts were isolated for the measurement of left ventricular functions (left ventricular developed pressure, dp/dtmax and dp/dtmin) using Langendorff setup. Western blot was used to determine &bgr;1-AR and G protein–coupled receptor kinase 2 protein expressions. Real-time polymerase chain reaction was done to determine &bgr;1-AR mRNA expression. Atorvastatin prevented sepsis-induced decrease in left ventricular functions, such as left ventricular developed pressure (CLP 75.90 ± 0.53 vs. ATR 100.24 ± 1.64 mmHg), dp/dtmax (CLP 3,742 ± 71 vs. ATR 4,291 ± 88 mmHg/s), and dp/dtmin (CLP −1,010 ± 24 vs. ATR −1,346 ± 84 mmHg/s). Associated with functional impairments, sepsis decreased both myocardial &bgr;1-AR protein and mRNA expressions by 52% ± 9% and 62% ± 7%, respectively. However, ATR treatment of CLP mice (ATR) preserved &bgr;1-AR protein (96% ± 11%) and mRNA (88% ± 14%) expressions comparable to sham-operated level. Furthermore, it not only attenuated sepsis-induced decrease in basal cardiac adenosine 3′,5′-cyclic monophosphate content (CLP 1.30 ± 0.27 vs. ATR 6.30 ± 0.67 pmol/mg protein), but also prevented its refractoriness to dobutamine stimulation (CLP 1.72 ± 0.27 vs. ATR 10.83 ± 1.37 pmol/mg protein). Atorvastatin also inhibited sepsis-induced increase in cardiac G protein–coupled receptor kinase 2 protein expression (CLP 1.73 ± 0.18-fold vs. ATR 1.10 ± 0.18-fold), protein kinase A activity (CLP 1.12 ± 0.14 vs. ATR 0.66 ± 0.08 U/mg protein) and plasma catecholamines (CLP 138 ± 22 vs. ATR 59 ± 2 pg/mL). In conclusion, ATR seems to improve left ventricular functions in vitro through the preservation of &bgr;1-AR signaling in sepsis.

Paralytic effect of alcoholic extract of Allium sativum and Piper longum on liver amphistome, Gigantocotyle explanatum

Objective: To investigate the effects of alcoholic extract of Allium sativum and Piper longum on the muscular activity of a parasitic amphistome, Gigantocotyle explanatum. Materials and Methods: Amphistomes were isometrically mounted to record the spontaneous muscular activity by using Chart 4 software program (Power Lab, AD Instruments, Australia) and to examine the effects of cumulative doses (100, 300, 1000, and 3000 μg/ml) of the plant extracts on the amplitude (g), frequency (per 10 min), and baseline tension (g) of the spontaneous muscular activity of the amphistome. Results: Alcoholic extract of A. sativum produced significant reduction in the frequency and amplitude of contractile activity of the amphistome at 1000 and 3000 μg/ml bath concentrations. Complete paralysis of the amphistome was observed after 15 min of addition of 3000 μg/ml concentration. Alcoholic extract of P. longum also caused paralysis following 15-20 min exposure of the amphistome to 3000 μg/ml concentration. In both the cases the amphistomes did not recover from paralysis following 2-3 washes. Conclusion: The observations demonstrate the paralytic effect of alcoholic extract of A. sativum and P. longum on G. explanatum.

Essential role of nitric oxide in sepsis-induced impairment of endothelium-derived hyperpolarizing factor-mediated relaxation in rat pulmonary artery.

Both endothelial nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) are important vasodilators in pulmonary circulation. Sepsis is known to impair endothelium-dependent dilation in the pulmonary vasculature, but the mechanisms are incompletely understood. We have examined the relative contribution of EDHF/NO to the attenuated endothelium-dependent relaxation of pulmonary artery in sepsis, and the role of inducible nitric oxide synthase (iNOS)-derived NO in this mechanism. Sepsis was induced in male adult Wistar rats by caecal ligation and puncture. At 18h after surgery, left and right branches of pulmonary arteries were isolated for tension recording, NO/cyclic guanosine monophosphate (cGMP) measurements, mRNA and protein expressions. Despite a marked decrease in the arterial endothelial nitric oxide synthase (eNOS) mRNA and phosphorylated-eNOS (p-eNOS) protein expressions in sepsis, endothelium-dependent relaxation to acetylcholine (ACh) mediated by NO, acetylcholine-stimulated NO release and tissue cGMP levels were moderately inhibited. Sepsis however abolished the N(G)-Nitro-l-arginine methyl ester (L-NAME)/indomethacin-resistant arterial relaxation (EDHF response) to acetylcholine in this vessel. In vitro treatment of the arterial rings from septic rats with 1400W, a selective inhibitor of iNOS restored the EDHF response, but had no effect on the acetylcholine-induced relaxation mediated by endothelial NO. The functional role of iNOS-derived NO in impairing EDHF-mediated relaxation was coincident with an increased basal NO production, iNOS mRNA and protein expressions in the rat pulmonary artery. In conclusion, the loss of the EDHF response may be primarily responsible for the endothelial dysfunction in sepsis, and its restoration by a selective iNOS inhibitor may improve pulmonary vasodilation.

Role of protein kinase G in nitric oxide deficiency-induced supersensitivity to nitrovasodilator in rat pulmonary artery.

The aim of the present study was to examine the role of protein kinase G (G-kinase) in the mechanism of endogenous nitric oxide (NO) deficiency-induced supersensitivity to the nitrovasodilator sodium nitroprusside (SNP) in isolated rat pulmonary artery. Tension experiments and cGMP measurements were carried out on isolated rat pulmonary artery to assess the influence of NO deficiency, caused by either NG-nitro-L-arginine methyl ester (L-NAME) treatment or endothelium removal on the vasodilator potency of SNP. Sodium nitroprusside was more potent (pD2; 8.21 ± 0.04) in relaxing arterial rings treated with 100μM L-NAME or denuded of the endothelium (pD2; 8.44 ± 0.11) compared with the endothelium-intact controls (pD2; 7.61 ± 0.05). Similarly, the tissue sensitivity to 8-Br-cGMP, a G-kinase activator, was significantly (P < 0.05) greater after L-NAME treatment (pD2; 5.04 ± 0.09) or endothelium removal (pD2; 5.28 ± 0.11) in comparison with the controls (pD2; 4.22 ± 0.17). On the other hand, dibutyryl cAMP, an activator of protein kinase A, was equipotent in dilating control (pD2; 4.14 ± 0.04) and L-NAME-treated (pD2 4.21 ± 0.05) vessels. Further, L-NAME treatment significantly (P < 0.05) decreased the basal cGMP but enhanced SNP (1 μM)-stimulated increase in the tissue cyclic nucleotide levels (271.8 ± 39.93 pmol/mg protein versus control: 66.19 ± 7.18 pmol/mg protein), indicating sensitization of soluble guanylyl cyclase to NO. The increased sensitivity of G-kinase to cGMP observed in the present study suggests a novel mechanism of supersensitivity in vascular smooth muscle to nitrovasodilators in acute NO deficiency. Further, it explains the influence of ambient cGMP in determining the sensitivity of G-kinase in vascular smooth muscle.