Acetaminophen increases the risk of arsenic-mediated development of hepatic damage in rats by enhancing redox-signaling mechanism.

We evaluated whether the commonly used analgesic-antipyretic drug acetaminophen can modify the arsenic-induced hepatic oxidative stress and also whether withdrawal of acetaminophen administration during the course of long-term arsenic exposure can increase susceptibility of liver to arsenic toxicity. Acetaminophen was co-administered orally to rats for 3 days following 28 days of arsenic pre-exposure (Phase-I) and thereafter, acetaminophen was withdrawn, but arsenic exposure was continued for another 28 days (Phase-II). Arsenic increased lipid peroxidation and reactive oxygen species (ROS) generation, depleted glutathione (GSH), and decreased superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glutathione reductase (GR) activities. Acetaminophen caused exacerbation of arsenic-mediated lipid peroxidation and ROS generation and further enhancement of serum alanine aminotransferase and aspartate aminotransferase activities. In Phase-I, acetaminophen caused further GSH depletion and reduction in SOD, catalase, GPx and GR activities, but in Phase-II, only GPx and GR activities were more affected. Arsenic did not alter basal and inducible nitric oxide synthase (iNOS)-mediated NO production, but decreased constitutive NOS (cNOS)-mediated NO release. Arsenic reduced expression of endothelial NOS (eNOS) and iNOS genes. Acetaminophen up-regulated eNOS and iNOS expression and NO production in Phase-I, but reversed these effects in Phase-II. Results reveal that acetaminophen increased the risk of arsenic-mediated hepatic oxidative damage. Withdrawal of acetaminophen administration also increased susceptibility of liver to hepatotoxicity. Both ROS and NO appeared to mediate lipid peroxidation in Phase-I, whereas only ROS appeared responsible for peroxidative damage in Phase-II.

The voltage-dependent L-type Ca2+ (CaV1.2) channel C-terminus fragment is a bi-modal vasodilator.

Voltage-dependent L-type Ca(2+) channels (CaV1.2) are the primary Ca(2+) entry pathway in vascular smooth muscle cells (myocytes). CaV1.2 channels control systemic blood pressure and organ blood flow and are pathologically altered in vascular diseases, which modifies vessel contractility. The CaV1.2 distal C-terminus is susceptible to proteolytic cleavage, which yields a truncated CaV1.2 subunit and a cleaved C-terminal fragment (CCt). Previous studies in cardiac myocytes and neurons have identified CCt as both a transcription factor and CaV1.2 channel inhibitor, with different signalling mechanisms proposed to underlie some of these effects. CCt existence and physiological functions in arterial myocytes are unclear, but important to study given the functional significance of CaV1.2 channels. Here, we show that CCt exists in myocytes of both rat and human resistance-size cerebral arteries, where it locates to both the nucleus and plasma membrane. Recombinant CCt expression in arterial myocytes inhibited CaV1.2 transcription and reduced CaV1.2 protein. CCt induced a depolarizing shift in the voltage dependence of both CaV1.2 current activation and inactivation, and reduced non-inactivating current in myocytes. Recombinant truncated CCt lacking a putative nuclear localization sequence (92CCt) did not locate to the nucleus and had no effect on arterial CaV1.2 transcription or protein. However, 92CCt shifted the voltage dependence of CaV1.2 activation and inactivation similarly to CCt. CCt and 92CCt both inhibited pressure- and depolarization-induced vasoconstriction, although CCt was a far more effective vasodilator. These data demonstrate that endogenous CCt exists and reduces both CaV1.2 channel expression and voltage sensitivity in arterial myocytes. Thus, CCt is a bi-modal vasodilator.

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.

Role of nitric oxide and carbon monoxide in N(omega)-Nitro-L-arginine methyl ester-resistant acetylcholine-induced relaxation in chicken carotid artery.

The current study examined the hypothesis that acetylcholine-induced N(omega)-Nitro-L-arginine methyl ester (L-NAME)-resistant endothelium-dependent relaxations in the chicken carotid artery are mediated by nitric oxide and carbon monoxide. Acetylcholine (1 nM-3 microM) caused a concentration-dependent relaxation (pD(2) 6.81+/-0.05, R(max) 115+/-3%) of the artery segments precontracted with phenylephrine (3 microM). L-NAME (1 mM) decreased the sensitivity (pD(2) 6.44+/-0.06), but not the efficacy (R(max) 108+/-3%) of acetylcholine. It also partially decreased the acetylcholine (3 microM)-stimulated nitrite release. While treatment with N(omega)-Nitro-L-arginine (l-NNA; 1 mM) plus L-NAME (1 mM) decreased the acetylcholine-stimulated nitrite release to the basal level, it moderately inhibited (R(max) 77+/-3%) the maximal relaxation elicited with the muscarinic agonist. 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO; 100 microM) a specific scavenger of nitric oxide (NO) plus the two NOS inhibitors further decreased the acetylcholine-evoked relaxation (R(max) 34+/-2%). Although soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM) markedly inhibited the acetylcholine-stimulated increase in tissue cGMP to less than the basal levels, it only decreased the sensitivity, but not the efficacy of the agonist either in the presence or absence of L-NAME (1 mM). Zinc Protoporphyrin-IX (ZnPP; 10 microM), a hemeoxygenase (HO) inhibitor, partially inhibited (R(max) 72+/-3%) the L-NAME-resistant acetylcholine-induced relaxations. A combined treatment of the arterial rings with L-NAME, l-NNA, PTIO and ZnPP nearly abolished (R(max) 7+/-0.9%) the vasodilator responses to acetylcholine. Endothelium removal abolished the relaxation response to acetylcholine. In conclusion, it is suggested that the acetylcholine-induced L-NAME-resistant relaxation is primarily, mediated by NO with a small but significant contribution from endothelium-derived carbon monoxide in the chicken carotid artery.

Role of voltage-dependent potassium channels and myo-endothelial gap junctions in 4-aminopyridine-induced inhibition of acetylcholine relaxation in rat carotid artery.

The present study examined the role of voltage-gated potassium (K(v)) channels and myo-endothelial gap junctions in 4-aminopyridine-induced inhibition of acetylcholine-evoked endothelium-dependent relaxation and NO release in the rat carotid artery. The acetylcholine-induced relaxation was drastically inhibited by 94% and 82%, respectively in the presence of either 100 microM N(G)-nitro-l-arginine methyl ester (L-NAME) or 10 microM 1H-[1,2,4]oxadiazolo[4,3,a]quinoxalin-1-one (ODQ), while it was abolished following endothelium removal. 4-aminopyridine (1 mM), a preferential blocker of the K(v) channels significantly decreased the vasodilator potency, as well as efficacy of acetylcholine (pD(2) 5.7+/-0.09, R(max) 86.1+/-3.5% versus control 6.7+/-0.10 R(max) 106+/-3.5%, n=6), but had no effect on the relaxations elicited by either sodium nitroprusside (SNP) or 8-bromo-cyclic guanosine monophosphate (8-Br-cGMP). 4-AP (1 mM) also inhibited acetylcholine (3 microM)-stimulated nitrite release in the carotid artery segments (99.4+/-4.93 pmol/mg tissue weight wt; n=6 versus control 123.8+/-7.43 pmol/mg tissue weight wt, n=6). 18alpha-glycyrrhetinic acid (18alpha-GA, 5 microM), a gap junction blocker, completely prevented the inhibition of acetylcholine-induced relaxation, as well as nitrite release by 4-AP. In the pulmonary artery, however antagonism of acetylcholine-evoked relaxation by 4-AP was not reversed by 18alpha-GA. These results suggest that 4-AP-induced inhibition of endothelium-dependent relaxation and NO release involves electrical coupling between vascular smooth muscle and endothelial cells via myo-endothelial gap junctions in the rat carotid artery, but not in the pulmonary artery. Further, direct activation of 4-AP-sensitive vascular K(v) channels by endothelium-derived NO is not evident in the carotid blood vessel, while this appears to be an important mechanism of acetylcholine-induced relaxation in the pulmonary artery.

Involvement of inducible nitric oxide synthase and dimethyl arginine dimethylaminohydrolase in Nω-nitro-L-arginine methyl ester (L-NAME)-induced hypertension.

Chronic administration of Nω-nitro-L-arginine methyl ester (L-NAME) in rats is a chemical method to study the induction and progression of nitric oxide (NO) deficiency-induced endothelial dysfunction. Male Wistar rats received L-NAME (50 mg/kg/day in drinking water) or no drug for 6 weeks. Mean arterial pressure (MAP) was measured on Day 43 by carotid artery cannulation. Plasma interleukin 1ß (IL-1ß) level was measured by enzyme-linked immunosorbent assay. Aorta and carotid artery were isolated for determination of basal nitrite, cGMP production, soluble guanylylcyclase (sGC) activity, phosphodiesterase-5 (PDE5) activity, and dimethylarginine dimethylaminohydrolase (DDAH) activity. mRNA expression studies were done by real time-polymerase chain reaction. L-NAME induced an increase in MAP and plasma IL-1ß. The treatment had varied effect on endothelial nitric oxide synthase (eNOS), sGC, and PDE5 but showed an increase in inducible NOS (iNOS) mRNA expression and plasma asymmetric dimethyl arginine levels. Basal nitrite, cGMP levels, sGC activity, and DDAH activity were significantly decreased in the tissues. Brief incubation of tissues in vitro with 1400 W, a specific iNOS blocker, partially reversed sGC activity, and cGMP levels. The results of this study showed that L-NAME-mediated inhibition of eNOS is only partially responsible for the vascular pathology observed in this model. Secondary effects that include an increase in iNOS and a decrease in DDAH activity are likely to be the causative factors for the progression of vascular dysfunction.

Effects of acetaminophen on reactive oxygen species and nitric oxide redox signaling in kidney of arsenic-exposed rats.

We examined whether acetaminophen could alter renal oxidative stress induced by arsenic; also whether withdrawal of acetaminophen treatment can increase susceptibility of kidney to arsenic toxicity. Acetaminophen (400 and 1600 mg/kg) was co-administered orally to rats for 3 days after preexposure to arsenic (25 ppm) for 28 days (Phase-I) and thereafter, acetaminophen was withdrawn, but arsenic exposure was continued for another 28 days (Phase-II). Acetaminophen enhanced arsenic-induced lipid peroxidation, GSH depletion and ROS production and further decreased superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities. Increased peroxidation did not alter kidney weight, but increased serum urea nitrogen and creatinine. Arsenic did not alter basal, iNOS-mediated NO production or iNOS expression. Arsenic decreased cNOS-mediated NO release and eNOS expression in Phase-II. Acetaminophen increased their expressions and NO production in Phase-I. In Phase-II, arsenic-mediated effects on NO remained mostly unaffected with acetaminophen. Results reveal that acetaminophen enhanced the risk of arsenic-mediated oxidative stress in kidney. Discontinuation of acetaminophen administration also increased the susceptibility of kidney to nephrotoxic effect of arsenic. It appeared ROS were primarily responsible for oxidative stress in both the phases. NO may have a minor role in Phase-I, but does not contribute to redox signaling mechanism in Phase-II.

Pro-healing potential of hemin: an inducer of heme oxygenase-1.

Hemin induces heme oxygenase (HO), an enzyme which degrades heme in a rate-limiting manner and has an important role in cellular protection against oxidative stress and apoptosis. This HO inducer may be of potential therapeutic value in wound healing and inflammation. To identify the beneficial activity of HO vis a vis wound healing, hemin was used as inducer of HO in rats using a full-thickness cutaneous wound model. Hemin treatment increased cellular proliferation and collagen synthesis as evidenced by increase in wound contraction and hydroxyproline and glucosamine contents. mRNA expression of cytokines endorsed fast healing as was indicated by inhibition of pro-inflammatory cytokines such as ICAM-1 and TNF-alpha and up-regulation of anti-inflammatory cytokine IL-10.

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 N-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 100microM 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 microM)-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.

Influence of heat stress on the reactivity of isolated chicken carotid artery to vasoactive agents.

Cerebral ischaemia is considered to be an important cause of central nervous system dysfunction in heat stress. We hypothesized that heat stress would alter the reactivity of isolated carotid artery to vasoactive agents. Carotid arteries were isolated from broiler chickens maintained either at 23-24 degrees C with 55-65% humidity (control conditions) or exposed to 40 +/- 1 degrees C with 35% humidity for 4 h (heat stress). Contractions were elicited with vasoconstrictors such as 5-HT, phenylephrine, guanfacine and CaCl(2) (K(+)-depolarized) in endothelium-denuded arterial rings. Heat stress significantly increased the potency of 5-HT, but had no effect on the sensitivity of the vessel to phenylephrine or guanfacine. In contrast, it markedly decreased the potency and efficacy of CaCl(2). Vasodilator responses to ACh (endothelium-intact) and sodium nitroprusside (endothelium-denuded), however, were unaffected. Although cyclopiazonic acid (10 microm) significantly decreased 5-HT responses in both the conditions, the agonist was still more potent in heat stress. Extracellular Ca(2)(+) removal had no effect on contractions caused by 5-HT in control conditions, but it significantly decreased the agonist potency in heat stress. Interestingly, nifedipine (1 microm) markedly inhibited 5-HT-induced contractions both in control conditions and in heat stress, implying an inhibitory effect on both Ca(2)(+) influx and release. Thus, nifedipine had a markedly greater inhibitory effect on 5-HT-induced contractions in heat stress compared with control conditions. The results suggest that heat stress increased the vasoconstrictor responses to 5-HT by a mechanism that involved extracellular Ca(2)(+) influx through nifedipine-sensitive L-type calcium channels.