Sirtinol attenuates hepatic injury and pro-inflammatory cytokine production following trauma-hemorrhage in male Sprague-Dawley rats.

BACKGROUND: Although studies have demonstrated that sirtinol administration following adverse circulatory conditions is known to be protective, the mechanism by which sirtinol produces the salutary effects remains unknown. We hypothesized that sirtinol administration in male rats following trauma-hemorrhage decreases cytokine production and protects against hepatic injury. METHODS: Male Sprague-Dawley rats underwent trauma-hemorrhage (mean blood pressure 40 mmHg for 90 min, then resuscitation). A single dose of sirtinol (1 mg/kg of body weight) or vehicle was administered intravenously during resuscitation. Twenty-four hours thereafter, tissue myeloperoxidase (MPO) activity (a marker of neutrophil sequestration), cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-3, intercellular adhesion molecule (ICAM)-1, and interleukin (IL)-6 levels in the liver and plasma alanine aminotransferase (ALT) concentrations were measured (n=6 Sprague-Dawley rats/group). RESULTS: Trauma-hemorrhage increased hepatic MPO activity, CINC-1, CINC-3, ICAM-1, and IL-6 levels and plasma ALT concentrations. These parameters were significantly improved in the sirtinol-treated rats subjected to trauma-hemorrhage. CONCLUSION: The salutary effects of sirtinol administration on attenuation of hepatic injury following trauma-hemorrhage are, at least in part, related to reduction of pro-inflammatory mediators.

Oxidized low density lipoprotein induces apoptosis via generation of reactive oxygen species in vascular smooth muscle cells.

OBJECTIVE: Death of vascular smooth muscle cell (VSMC) induced by oxidized LDL (oxLDL) can occur by both necrosis and apoptosis which may contribute to plaque instability and rupture. Reactive oxygen species (ROS) induces apoptosis in VSMC and is involved in oxLDL action, we tested the hypothesis here that a coupling exists between ROS generation and apoptosis of oxLDL-treated VSMC. METHODS: Cultured VSMC from rat aorta were treated with oxLDL, apoptosis and necrosis were distinguished by using FITC-annexin V label and propidium iodide stain, analyzed by flow cytometry. ROS generation of VSMCs was detected by the fluorescence intensity of DCF. Apoptosis was also determined by cleavage of procaspase-3. RESULTS: OxLDL induced apoptosis (3 h) in a dose-dependent manner and reached maximum (with near-basal necrosis) at a concentration of 300 microg/ml. At this and lower (100 microg/ml) concentration, oxLDL, but not native LDL, stimulated ROS production rapidly (< or =5 min) and ROS level remained elevated for at least 45 min. Catalase and deferoxamine reduced both oxLDL-induced apoptosis and ROS generation. Superoxide dismutase and benzoic acid neither reduced the oxLDL-induced ROS generation nor inhibited apoptosis. Since oxLDL-induced ROS generation were inhibited by nordihydroguaiaretic acid and rotenone, lipoxygenase and mitochondrial pathways could be involved. In addition, catalase, deferoxamine, and N-acetylcysteine inhibited oxLDL-induced cleavage of procaspase-3 as well. CONCLUSIONS: ROS generation and apoptosis are tightly coupled in oxLDL-treated VSMCs. Antioxidants that reduced ROS level inhibited apoptosis, those that did not reduce ROS level were ineffective. Both mitochondrial and lipoxygenase activities may be involved.

17Beta-estradiol inhibits oxidized low density lipoprotein-induced generation of reactive oxygen species in endothelial cells.

Increase of intracellular reactive oxygen species (ROS) has been proposed to cause endothelial injury, and oxidized LDL (oxLDL) actions are associated with an early increase of ROS. Estrogen protects vascular cells partly via its antioxidant effects and by preventing lipid peroxidation. However, whether it can inhibit oxLDL-induced stimulation of ROS generation in endothelial cells is unknown. We utilized the fluorescent dye (DCFH-DA) to measure ROS generation and compared the stimulant effect of tert-butylhydroperoxide (TBH) and oxLDL in human umbilical vein endothelial cells (HUVECs). We found that TBH, H2O2, and oxLDL rapidly stimulated ROS generation, and in a dose-dependent manner with TBH. A concentration of estrogen effective in preventing lipid peroxidation was employed either by pretreatment of cells 18 h prior to or by direct co-incubation (30 min) with HUVEC and oxLDL. Estrogen (54 microM) pretreatment significantly suppressed both TBH- and oxLDL- induced stimulation of ROS generation. Both 1 and 54 microM concentration of estrogen could directly inhibit oxLDL-induced ROS production in HUVECs. Thus, either 18 h pretreatment or 30 min co-incubation with estrogen reduced stimulated ROS generation, suggesting that both cellular and direct actions of estrogen may be involved.

Lysophosphatidylcholine induces apoptotic and non-apoptotic death in vascular smooth muscle cells: in comparison with oxidized LDL.

Oxidized low-density lipoprotein (oxLDL) plays a key role in the development of atherogenesis, partly by causing injury to vascular cells. However, different preparations of LDL, methods of oxidation, and/or active components often produce cellular effects of various degrees. To explore the quantitative relationship between dose and level of oxidation of the oxLDL utilized, we employed combinations of different levels of oxidation and concentrations of oxLDL to induce cell death in cultured vascular smooth muscle cells (VSMC). We also examined the effect of lysophosphatidylcholine (lysoPC), a putative active component of oxLDL, on VSMCs by determining, in parallel with a cytotoxicity test (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay), DNA fragmentation ([3H]thymidine release), and flow cytometric analyses. We found that oxLDL caused cytotoxicity in an oxidative level- and dose-dependent manner, lysoPC also caused dose-dependent cytotoxicity with or without serum. Fragmentation of DNA was observed in both oxLDL- and lysoPC-treated VSMCs. Furthermore, lysoPC-induced DNA ladder was also demonstrated by gel electrophoresis at a concentration of 25 micromol/l or higher. Flow cytometric analysis yielded similar results for oxLDL- and lysoPC-treated VSMC; namely, an accumulation in the fraction of cells in G(0)/G(1) phase with a reciprocal change in S-phase fraction. Membrane phosphatidylserine exposure, detected by annexin V staining, provided additional evidence that lysoPC induced significant apoptosis in VSMC. Taken together, the degree of oxLDL-induced cytotoxicity/apoptosis of VSMC depended on combined effects of oxLDL concentration and oxidative level. Moreover, lysoPC also elicited a dose-dependent apoptosis in addition to cytotoxicity.

Aminoguanidine attenuates hemodynamic and microcirculatory derangement in rat intestinal ischemia and reperfusion.

BACKGROUND: Nitric oxide (NO) participates in the regulation of hemodynamic and microcirculatory changes in intestinal ischemia and reperfusion (I/R). However, the nature of the involvement of an inducible NO release has been controversial. This study evaluates the impact of an inducible NO synthase inhibitor, aminoguanidine, used as a treatment in a rat intestinal I/R model. METHODS: We investigated the hemodynamics by measuring the mean arterial pressure (MAP), and the microcirculatory responses of the intestine and liver to systemically administered aminoguanidine by use of laser-Doppler flowmetry (LDF), in vivo microscopy, and flow cytometry. RESULTS: During the 30-min ischemia of the selected 20-cm ileal segment, no MAP change was noted. At reperfusion, a marked decrease of MAP was noted and the lowest levels were noted 3 hours after reperfusion (67 +/- 4% vs. 99 +/- 5% in sham-operated control animals). A marked decrease in liver perfusion as measured by LDF was noted 1 hour after reperfusion and remained low at 5 hours (72 +/- 4% vs. 97 +/- 3% in sham-operated control animals). A marked decrease in intestine perfusion was noted by using LDF 1 hour after reperfusion and remained low at 5 hours (43 +/- 3% vs. 92 +/- 4% in sham-operated control animals). The flow velocity of the postcapillary venules of the intestine was markedly decreased (1.01 +/- 0.62 vs. 2.67 +/- 0.34 mm/s in sham-operated control animals) at 5 hours after reperfusion. The flow velocity of the postsinusoidal venules of the liver was also markedly decreased (1.01 +/- 0.62% vs. 2.67 +/- 0.34% in sham-operated control animals). Leukocyte-endothelial interaction (adhesion) was increased in the postcapillary venules of the intestine (54 +/- 12 vs. 6 +/- 4/microm2 in sham-operated control animals) and in the postsinusoidal venules of the liver (32 +/- 8 vs. 2 +/- 2/microm2 in sham-operated control animals). Concomitantly, the granulocyte count was increased (9.1 +/- 0.6 vs. 2.1 +/- 0.3% of total circulating leukocytes in sham-operated control animals), with an increase of CD 11b expression. Aminoguanidine administration (1 mg/kg) 0.5 hour before ischemia and 1 hour after reperfusion significantly increased MAP, increased intestine and liver perfusion, decreased adhesion, and decreased circulating granulocytes and CD 11b expression. CONCLUSION: Inhibition of an inducible NO release by aminoguanidine in intestinal I/R can attenuate hemodynamic and microcirculatory derangement.

Arginine-nitric oxide pathway in plasma membrane of rat hepatocytes during early and late sepsis.

OBJECTIVE: To study the transported L-arginine in rat hepatocytes during different stages of sepsis. DESIGN: A prospective, controlled study. SUBJECTS: Thirty-six Sprague-Dawley male rats (250 to 300 g) were anesthetized and studied. INTERVENTIONS: Early sepsis was produced 9 hrs after cecal ligation and puncture (CLP) and late sepsis developed 18 hrs after CLP. The control group underwent sham operation. Plasma membrane of rat hepatocytes was prepared by differential centrifugation. The [3H] L-arginine uptake of plasma membrane vesicles during sepsis was measured and inhibition studies employing omega-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine were performed. MEASUREMENTS AND MAIN RESULTS: L-arginine transport was saturable, increased linearly with plasma membrane protein concentration, and increased with uptake time up to 5 mins. [3H] L-arginine uptake increased by 77% to 121% (p < .05) during early sepsis, with no significant changes during late sepsis. Comparing inhibitors of nitric oxide synthase, L-NAME was effective in inhibiting L-arginine transport while aminoguanidine was not. CONCLUSIONS: L-arginine transport was enhanced in rat hepatocytes during the early stage of sepsis. The increased uptake of L-arginine could contribute to the increase production of nitric oxide by hepatocyte during sepsis.

Leukocyte-endothelial adherence correlates with pancreatic nitric oxide production in early cerulein-induced pancreatitis in rats.

The role of nitric oxide (NO) in microcirculation during the development of acute pancreatitis was not clear. An in vivo microscopic technique was used for evaluating leukocyte-endothelial adherence in the pancreatic microcirculation after induction (cerulein) of acute pancreatitis. Microdialysis was performed to detect pancreatic nitrate concentration (NO level) by high-performance liquid chromatography. Cerulein caused significantly reduced flow velocity in 1 h (31 %) and increased the number of sticking leukocytes in 2 h; both persisted for at least 3 h. Pancreatic NO level was found to be significantly elevated (2.5-fold) in 1 h and also persisted for 3 h. Both microcirculatory changes and NO elevation were significantly alleviated in cerulein-induced animals pretreated with NO synthase inhibitor (NG-nitro-L-arginine), indicating that elevation of NO could precede and account for a major portion of the observed microcirculatory changes. Furthermore, there was a strong positive correlation between numbers of adherent leukocytes and pancreatic NO level, suggesting that during the development of acute pancreatitis, NO could play an adverse role in microcirculation.

Changes of adenosine triphosphate-dependent calcium uptake in microsomal fractions of rat liver during sepsis.

BACKGROUND: Intracellular calcium concentration is an important regulator of cellular metabolism. Endoplasmic reticulum membranes play an important role in the regulation of cytoplasmic calcium in the mammalian liver. The characterization of the changes of calcium uptake in endoplasmic reticulum may contribute to the potential intracellular mechanisms for cellular dysfunction during sepsis. METHODS: The effects of sepsis on the calcium uptake in rough endoplasmic reticulum of rat liver were studied. Sepsis was induced by means of cecal ligation and puncture (CLP). The control rats underwent sham operation. Microsomal fractions were isolated from the liver with differential centrifugation. RESULTS: The calcium uptake by liver endoplasmic reticulum was decreased by 30% to 35% (p < 0.05) during early sepsis (9 hours after CLP) and by 38% to 43% (p < 0.05) during late sepsis (18 hours after CLP), respectively. The maximum velocity values for adenosine triphosphate (ATP) and for Ca2+ were also decreased by 25% to 37% (p < 0.05) during early sepsis and by 35% to 42% (p < 0.05) during late sepsis. The Michaelis-Menten constant for ATP and Ca2+ transport had no difference among three groups. The magnesium stimulation and vanadate inhibitory activity were also decreased by 17% to 38% (p < 0.05) during early sepsis and by 34% to 50% (p < 0.05) during late sepsis. CONCLUSIONS: These data demonstrate that ATP-dependent calcium uptake in rough endoplasmic reticulum of rat liver was impaired during early and late sepsis. Because the low intracellular calcium concentration plays an important role in the regulation of cellular function, an impairment in the ATP-dependent calcium uptake by endoplasmic reticulum during early and late sepsis may have a pathophysiologic significance in contributing to the development of altered hepatic metabolism during sepsis.

Inhibitory effect of estrogen on vascular contraction from male rats of different ages.

Both age and sex influence vascular contractile functions and play significant roles in cardiovascular regulation. Recent research has focused on the action of sex steroids on blood vessels and characterized both endothelium-dependent as well as-independent effects in agonists-induced contractile response. However, to study the combined influences of age and sex steroids present a complicated situation. In the current investigation, we studied the role of both these physiological factors in an in vitro approach. We found that high concentrations of estrogen (E2, 17 beta-estradiol) significantly inhibited contractile response induced by KCI in endothelium-denuded aorta derived from both young (24 wk) and aged (53 wk) male rats. The contractile responses in aorta from aged rats were significantly less than that from young adults in the absence or presence of E2 indicating that the vascular effect of E2 could be demonstrated in both young and aged male rats. Taken together, our data suggest that E2's action on endothelium-independent, KCI-induced contractile response in vitro could serve as a simple model system to examine the interaction of aging process and gender difference on vascular functions.

Inhibition of protein synthesis by reduced K+ in cultured endothelial cells.

Total protein synthesis (as precipitable [3H] leucine incorporation) was determined under various experimental conditions to examine the relationship between cellular K+ and protein synthesis in human umbilical vein endothelial cells (HUVEC). We found that under normal external K+ concentration (5 mM), total protein synthesis was inhibited by cycloheximide and ouabain, with estimated values of IC50 of 0.41 microM and 0.60 mM, respectively. Cellular K+ concentrations were determined (102 +/- 4 mM for control cells) and found to be significantly increased (P < 0.01) by high external K+ (25 mM) and significantly decreased (P < 0.001) by low external K+ (0.5 mM) as well as by ouabain (2 mM). Under high external K+, total protein synthesis and the inhibitory responses of cycloheximide and ouabain were not altered. On the contrary, cellular K+ and protein synthesis were both further reduced by about half (P < 0.001) under low external K+. While ouabain further reduced cellular K+ by half (P < 0.001), protein synthesis was only slightly reduced (P < 0.05) under low external K+ and thus the relative reduction on protein synthesis was much less than that for cellular K+. These results indicate that while elevated intracellular K+ did not alter protein synthesis, reduced intracellular K+ correlated with a reduced cellular protein synthesis in HUVEC such that K+ may play a permissible role in the regulation of protein synthesis.

Mechanism of extracellular ATP-induced proliferation of vascular smooth muscle cells.

The mitogenic effect of extracellular ATP was examined in cultured rat aortic smooth muscle cells (VSMCs). ATP, 2-methylthio-ATP, and ADP stimulated [3H]thymidine and [3H]leucine incorporation and cell growth. AMP, adenosine, UTP, and P2x agonists showed little of these effects. Reactive blue 2, a P2Y purinoceptor antagonist, was effective in suppressing the mitogenic effect of ATP and 2-methylthio-ATP, indicating that extracellular ATP-induced VSMC proliferation is mediated by P2Y purinoceptors. The P2Y purinoceptor activation was coupled to a pertussis toxin (PTX)-insensitive G protein (Gq) and triggered phosphoinositide hydrolysis with subsequent activation of protein kinase C (PKC), Raf-1, and mitogen-activated protein kinase (MAPK) in VSMCs. In response to ATP, both 42-and 44-kDa MAPKs were activated, and tyrosine was phosphorylated. Western blot analysis using PKC isozyme-specific antibodies indicated that VSMCs express PKC-alpha, PKC-delta, and PKC-zeta. A complete down-regulation of PKC-alpha and PKC-delta was seen after 24-hr treatment with 12-O-tetradecanoylphorbol-13-acetate. When cells were pretreated with 12-O-tetradecanoyl-phorbol-13-acetate for 24 hr and subsequently challenged with ATP, Raf-1 activation and 42-kDa as well as 44-kDa MAPK tyrosine phosphorylation failed to be induced. These results demonstrate that ATP-induced Raf-1 and MAPK activations involve the activation of PKC-alpha and PKC-delta. P2Y purinoceptor stimulation with ATP also caused accumulation of c-fos and c-myc mRNAs. Both Reactive blue 2 and staurosporine significantly blocked this increase by ATP. In conclusion, the mitogenic effect of ATP seemed to be triggered by activation of the Gq protein-coupled P2Y purinoceptor that led to the formation of inositol trisphosphate and activation of PKC. PKC and, in turn, Raf-1 and MAPK were then activated, leading eventually to DNA synthesis and cell proliferation.

Nitric oxide inhibits migration of cultured endothelial cells.

Endothelial cell migration is an important event in both physiological and pathophysiological processes. Although nitric oxide (NO) plays a critical role in regulating vascular functions, it is not known whether NO modulates migration of endothelial cells. We show here that chemically-derived NO inhibited the serum-induced migration of cultured human umbilical vein endothelial cells (HUVEC) in a time- and dose-dependent manner. The sensitivity of inhibition by S-nitroso-N-acetylpenicillamine (SNAP, a NO donor) was 2.36 +/- 1.032 x 10(-4) M (n = 4). This effect was attributed to NO since (1) other NO donor (e.g., sodium nitroprusside) also exhibited antimigratory effect, (2) pre-incubated SNAP (72 h) had no effect, (3) hemoglobin, a NO scavenger, eliminated the effect; while (4) superoxide dismutase, a NO protector, enhanced the antimigratory effect. Furthermore, 8-bromo-cGMP also inhibited the serum-induced migration of HUVEC. These data appear to support the notion that NO may serve as an important signaling molecule for neovascularization.

Microcirculatory vasoconstrictor response: relationship with vital capacity and smoking.

Vasoconstrictor response (VR) induced by inspiratory gasp exhibited a strong positive correlation with vital capacity (VC) which reflects the magnitude of the input stimulus for VR (Lau et al., Clin. Sci. 89:233-237, 1995). Whether a stoichiometric relationship existed between VC and VR is not known. We examined this question in two studies by determining VC and microcirculatory blood flow with laser Doppler flowmetry in healthy subjects. We first studied 40 non-smokers of different gender and age and found that the variation in VR cannot be eliminated by normalization with VC. In the second study we examined 10 young male smokers as well as matched non-smokers of identical VC, we found that smokers had reduced VR. Taking together, the present studies demonstrated that vasoconstrictor response (VR) was not determined by vital capacity alone and that smoking adversely affected VR in the absence of altered VC.

Erythrocyte Li-Na countertransport in uremic patients: effect of urea.

Abnormalities of erythrocyte cation transport systems including Li-Na countertransport have been described in uremic patients. Dialysis appears to eliminate the difference in some of the patients. We investigated the rate of Li-Na countertransport from erythrocytes of normal healthy subjects and uremic patients undergoing hemodialysis treatment. We found that erythrocytes from uremic patients exhibited a significantly elevated rate of Li-Na countertransport. Furthermore, urea (30 mM) did not affect the rate of Li-Na countertransport or the electrolyte contents for Li loaded erythrocytes. Our findings showed that while both plasma and urea concentration and erythrocyte Li-Na countertransport were elevated in uremic patients, urea was not the major cause of the enhanced activity of the transporter.

Density of muscarinic receptors in rat myocardium during early sepsis.

The muscarinic receptor changes in two subcellular fractions of rat myocardium during sepsis, the sarcolemma (SL) and light vesicles (LV), were studied. [3H]-quinuclidinyl benzilate ([3H]-QNB) was used as a radioligand. Sepsis was induced by cecal ligation and puncture (CLP). The septic rats had higher pulse rates and slightly higher blood glucose levels than control rats. The marker enzyme assays revealed that the SL fraction was enriched with 5'-nucleotidase and the Na(+)-K(+)-ATPase activity increased over 20-fold, while the LV fraction showed very little enrichment when compared with the homogenate. [3H]-QNB binding studies showed that Bmax increased by 58.8% in SL with no changes in LV during early sepsis (9 h post-CLP), but there was no significant change in the Kd value. These data indicate that muscarinic cholinergic receptors in rat heart SL increase during early sepsis. Since the muscarinic cholinergic receptors mediate parasympathetic modulation of myocardial contractility, changes in the number of muscarinic receptors in the cardiac SL may have a pathophysiologic significance in the development of hemodynamic changes during sepsis.

Changes in inositol 1,4,5-triphosphate binding in microsomal fractions from the rat liver during sepsis.

Inositol 1,4,5-triphosphate has been proposed as a second messenger for calcium mobilization. The addition of inositol 1,4,5-triphosphate at a low concentration has been shown to cause calcium release from intracellular microsomal stores in rat hepatocytes. The effects of sepsis on the inositol 1,4,5-triphosphate binding from microsomal fractions of rat liver were investigated. Sepsis was induced by cecal ligation and puncture (CLP). Control rats were sham operated. Three microsomal fractions (rough, intermediate, and smooth I) were isolated from the rat liver. The study of inositol 1,4,5-triphosphate receptor binding was performed with tritium-labeled inositol 1,4,5-triphosphate. Our results showed that the Bmax of inositol 1,4,5-triphosphate binding in early septic, late septic, and control groups was 14.9 +/- .9 fmol/mg, 9.8 +/- 1.0 fmol/mg, and 17.2 +/- 1.3 fmol/mg, respectively. The binding activity was unaffected during early sepsis but was significantly depressed by 40-50% (p < .05, vs. control) during late sepsis (18 h after CLP) in all three subfractions of endoplasmic reticulum. Because the inositol 1,4,5-triphosphate binding plays an important role in the regulation of intra-cellular calcium homeostasis in hepatocytes, an impairment of the calcium release due to depressed inositol 1,4,5-triphosphate binding in the endoplasmic reticulum may have a pathophysiological significance in contributing to altered hepatic metabolism during septic shock.