17beta-Estradiol inhibits keratinocyte-derived chemokine production following trauma-hemorrhage

Neutrophil infiltration is a key step in the development of organ dysfunction following trauma-hemorrhage (T-H). Although we have previously shown that 17beta-estradiol (E2) prevents neutrophil infiltration and organ damage following T-H, the mechanism by which E2 inhibits neutrophil transmigration remains unknown. We hypothesized that E2 prevents neutrophil infiltration via modulation of keratinocyte-derived chemokine (KC), a major attractant for neutrophils. To examine this, male C3H/HeN mice were subjected to T-H or sham operation and thereafter resuscitated with Ringer lactate and E2 (1 mg/kg body wt) or vehicle. Animals were killed 2 h after resuscitation, and Kupffer cells were isolated. Plasma levels and Kupffer cell production capacities of KC, TNF-alpha, and IL-6 were determined by BD Cytometric Bead Arrays; lung mRNA expression of KC was measured with real-time PCR; myeloperoxidase activity assays were performed to determine neutrophil infiltration, and organ damage was assessed by edema formation. Treatment with E2 decreased systemic levels and restored Kupffer cell production of KC, TNF-alpha, and IL-6, as well as KC gene expression and protein in the lung. This was accompanied with a decrease in neutrophil infiltration and edema formation in the lung. These results suggest that E2 prevents lung neutrophil infiltration and organ damage in part by decreasing KC during posttraumatic immune response.     

17 beta-Estradiol normalizes immune responses in ovariectomized females after trauma-hemorrhage

Recent studies indicate that immuneresponses in proestrus females are maintained after trauma-hemorrhagebut markedly depressed in ovariectomized females under such conditions.The current study tested the hypothesis that the decreased estrogenlevels after ovariectomy are responsible for this immune depression. Tostudy this hypothesis, ovariectomized female CBA/J mice were subjected to laparotomy (i.e., soft tissue trauma) and hemorrhagic shock (35 ± 5 mmHg for 90 min, then resuscitated) or sham operation. The micereceived either 17-estradiol (E2; 100 µg/25 g body wt) or vehiclesubcutaneously during resuscitation. Immune cells were isolated 24 h thereafter. Splenocyte proliferation and interferon-, interleukin(IL)-2, and IL-3 release were significantly depressed aftertrauma-hemorrhage in vehicle-treated mice, whereas these functions weremaintained in E2-treated mice. Peritoneal macrophage IL-1 and IL-6release and splenic macrophage IL-6 and IL-12 release were alsosignificantly depressed in vehicle-treated mice after trauma-hemorrhage, and release of these cytokines was restored by E2treatment. In summary our findings indicate that the depressed splenicand peritoneal immune responses after trauma-hemorrhage can benormalized by a single dose of E2. Thus estrogen appears to be thecausative factor in the maintenance of immunocompetence in femalesafter trauma-hemorrhage, and its administration to ovariectomized orpostmenopausal females should be helpful in preventing immunedepression under such conditions.

     

Evaluation of endothelin-1-induced pulmonary vasoconstriction following myocardial infarction

Endothelin (ET) levels are elevated in congestive heart failure secondary to myocardial infarction (MI) and correlate well with the severity of pulmonary hypertension (PH), suggesting that the ET peptide could contribute to the pathophysiology of venous PH. Alterations of pulmonary vasoreactivity to ET after MI and the respective roles of the ET(A) and ET(B) receptors (ET(A)-R and ET(B)-R) have never been evaluated, to our knowledge. MI was induced in rats. Three weeks later, small pulmonary resistance arteries were mounted on a microvascular myograph. Cumulative concentration-response curves to ET-1 and sarafotoxin 6c (S6c) were performed. Response to ET was also assessed in the presence of ET-R antagonists. Heterodimerization of receptors was evaluated by immunoprecipitation of the ET(B)-R, followed by western blotting for the expression of the ET(A)-R. Maximal vasoconstriction and sensitivity to ET-1 were similar in sham and MI with values of 88 +/- 3.9% and 80 +/- 3.8%, respectively. The response to S6c was similarly less in both sham (67 +/- 5.7%) and MI groups (60 +/- 6.6%). When administered alone, the ET(A)-R antagonist (10 nM A-147627.1) and the ET(B)-R antagonist (1 microM A-192621.1) had no significant effect. However, their combination markedly reduced vaso-constriction (52 +/- 5.3%; P < 0.001). The endothelial and medial distribution of ET-Rs was similar in sham and MI groups. In vitro studies demonstrated co-immunoprecipitation of the ET(A)-R and ET(B)-R. Vasoconstriction of isolated resistance pulmonary arteries to ET agonists is not altered after MI. Dual antagonism results in optimal blockade of vasoconstriction, possibly because the ET(A)-R and ET(B)-R can form functional heterodimers.     

Upregulation of hepatic prolactin receptor gene expression by 17beta-estradiol following trauma-hemorrhage.

Although studies show protective effects of 17beta-estradiol (E2) or prolactin (PRL) treatment in male rats after trauma-hemorrhage (TH), the mechanism of the salutary effects of these agents remains unknown. Because E2 modulates PRL receptor (PRL-R) expression in the liver, we examined whether E2 treatment after T-H has any effects on hepatic PLR-R gene expression. Male Sprague-Dawley rats were subjected to trauma (i.e., 5-cm midline laparotomy) and hemorrhage (35-40 mmHg for 90 min) followed by fluid resuscitation (Ringer lactate) or sham operation and then treated with E2 (50 microg/kg body wt sc) or vehicle immediately before resuscitation. Liver samples were collected at 3 h thereafter, and PRL-R mRNA expression was determined by PCR. Liver expression of PRL-R short-form gene was unaffected by T-H, whereas that of the long-form gene was suppressed. Treatment of T-H rats with E2 significantly increased PRL-R short-form gene expression and normalized PRL-R long-form gene expression to sham levels. In the isolated hepatocytes, PRL-R short-form gene expression was predominant compared with the long-form gene. In contrast, only the short form was detected in Kupffer cells. In vitro treatment by E2 demonstrated an increase in the PRL-R long-form gene in hepatocytes, but E2 had no effect on PRL-R short-form gene expression in either the Kupffer cells or hepatocytes. Thus E2 treatment after T-H in males appears to directly upregulate PRL-R long-form gene expression in hepatocytes. However, the upregulation of the PRL-R short form might involve the interaction of multiple cell types in the liver.     

Resveratrol improves cardiac contractility following trauma-hemorrhage by modulating Sirt1

Mitochondria play a critical role in metabolic homeostasis of a cell. Our recent studies, based on the reported interrelationship between c-Myc and Sirt1 (mammalian orthologue of yeast sir2 [silent information regulator 2]) expression and their role in mitochondrial biogenesis and function, demonstrated a significant downregulation of Sirt1 protein expression and an upregulation of c-Myc following trauma-hemorrhage (T-H). Activators of Sirt1 are known to improve mitochondrial function and the naturally occurring polyphenol resveratrol (RSV) has been shown to significantly increase Sirt1 activity by increasing its affinity to both NAD+ and the acetylated substrate. In this study we tested the salutary effect of RSV following T-H and its influence on Sirt1 expression. Rats were subjected to T-H or sham operation. RSV (8 mg/kg body weight, intravenously) or vehicle was administered 10 min after the onset of resuscitation, and the rats were killed 2 h following resuscitation. Sirtinol, a Sirt1 inhibitor, was administered 5 min prior to RSV administration. Cardiac contractility (±dP/dt) was measured and heart tissue was tested for Sirt1, Pgc-1α, c-Myc, cytosolic cytochrome C expression and ATP level. Left ventricular function, after T-H, was improved (P < 0.05) following RSV treatment, with significantly elevated expression of Sirt1 (P < 0.05) and Pgc-1α (P < 0.05), and decreased c-Myc (P < 0.05). We also observed significantly higher cardiac ATP content, declined cytosolic cytochrome C and decreased plasma tumor necrosis factor-α in the T-H-RSV group. The salutary effect due to RSV was abolished by sirtinol, indicating a Sirt1-mediated effect. We conclude that RSV may be a useful adjunct to resuscitation fluid following T-H.     

p38 MAPK-dependent eNOS upregulation is critical for 17beta-estradiol-mediated cardioprotection following trauma-hemorrhage

Studies have shown that p38 MAPK and nitric oxide (NO), generated by endothelial NO synthase (eNOS), play key roles under physiological and pathophysiological conditions. Although administration of 17beta-estradiol (E2) protects cardiovascular injury from trauma-hemorrhage, the mechanism by which E2 produces those effects remains unknown. Our objective was to determine whether the E2-mediated activation of myocardial p38 MAPK and subsequent eNOS expression/phosphorylation would protect the heart following trauma-hemorrhage. To study this, male Sprague-Dawley rats underwent soft-tissue trauma (midline laparatomy) and hemorrhagic shock (mean blood pressure 35-40 mmHg for 90 min), followed by fluid resuscitation. Animals were pretreated with specific p38 MAPK inhibitor SB-203580 (SB; 2 mg/kg), and nonselective NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME; 30 mg/kg) 30 min before vehicle (cyclodextrin) or E2 (100 microg/kg) treatment, followed by resuscitation, and were killed 2 h thereafter. Cardiovascular performance and other parameters were measured. E2 administration following trauma-hemorrhage increased cardiac p38 MAPK activity, eNOS expression and phosphorylation at Ser(1177), and nitrate/nitrite levels in plasma and heart tissues; these were associated with normalized cardiac performance, which was reversed by SB administration. In addition, E2 also prevented trauma-hemorrhage-induced increase in cytokines (IL-6 and TNF-alpha), chemokines (macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant-1), and ICAM-1, which was reversed by l-NAME administration. Administration of E2 following trauma-hemorrhage attenuated cardiac tissue injury markers, myeloperoxidase activity, and nitrotyrosine level, which were reversed by treatment with SB and l-NAME. The salutary effects of E2 on cardiac functions and tissue protection following trauma-hemorrhage are mediated, in part, through activation of p38 MAPK and subsequent eNOS expression and phosphorylation.     

17beta-Estradiol and the phytoestrogen genistein attenuate neuronal apoptosis induced by the endoplasmic reticulum calcium-ATPase inhibitor thapsigargin

Estrogenic compounds have been shown to protect neurons from a variety of toxic stimuli in vitro and in vivo and depletion of estrogen at menopause has been associated with increased risk of neurodegenerative diseases. Genistein is an isoflavone soy derivative that binds to estrogen receptors with selective estrogen receptor modulator (SERM) properties. Recent FDA recommendations of soy intake for cholesterol reduction have prompted investigation into the potentially estrogenic role of dietary soy phytochemicals in the brain. In this study, we have shown that 50nM genistein significantly reduces neuronal apoptosis in an estrogen receptor-dependent manner. The importance of apoptosis in the brain has been recognized with regard to organization of the developing brain as well as degeneration in response to disease or stroke; however, the effects of estrogenic compounds on neuronal apoptosis have not been thoroughly examined. We developed a model of apoptotic toxicity in primary cortical neurons by using the endoplasmic reticulum (ER) calcium-ATPase inhibitor, thapsigargin, to test potential anti-apoptotic effects of 17beta-estradiol and genistein. Estrogen receptor beta, but not estrogen receptor alpha, was detected in our primary neuron cultures. Thapsigargin-induced apoptosis was confirmed by loss of mitochondrial function, DNA laddering, nuclear condensation and fragmentation, and caspase activation. Both 17beta-estradiol and genistein reduced the number of apoptotic neurons and reduced the number of neurons containing active caspase-3. This effect was blocked by co-addition of ICI 182780. Our results demonstrate that genistein and 17beta-estradiol have comparable anti-apoptotic properties in primary cortical neurons and that these properties are mediated through estrogen receptors.     

Correlation between apolipoprotein B and endothelin-1-induced vasoconstriction in humans

Low-density lipoprotein (LDL) concentrations have been linked to altered responses to endogenous vasodilators and vasoconstrictors. We evaluated retrospectively the relationship between LDL and vasoconstrictor (endothelin-1, phenylephrine) responsiveness of the forearm vasculature in 15 elderly healthy volunteers with apolipoprotein B and LDL levels in the normal range. In contrast to phenylephrine, changes in forearm vascular resistance induced by endothelin-1 were correlated with apolipoprotein B, LDL, and total cholesterol concentrations in women but not in men. These findings might suggest that lipids may increase vascular tone through both impaired endothelial vasodilation and increased vasoconstriction to endothelin-1 at least in women.     

Adiponectin opposes endothelin-1-mediated vasoconstriction in the perfused rat hindlimb

Recent studies have shown that adiponectin is able to increase nitric oxide (NO) production by the endothelium and relax preconstricted isolated aortic rings, suggesting that adiponectin may act as a vasodilator. Endothelin-1 (ET-1) is a potent vasoconstrictor, elevated levels of which are associated with obesity, type 2 diabetes, hypertension, and cardiovascular disease. We hypothesized that adiponectin has NO-dependent vascular actions opposing the vasoconstrictor actions of ET-1. We studied the vascular and metabolic effects of a physiological concentration of adiponectin (6.5 μg/ml) on hooded Wistar rats in the constant-flow pump-perfused rat hindlimb. Adiponectin alone had no observable vascular activity; however, adiponectin pretreatment and coinfusion inhibited the increase in perfusion pressure and associated metabolic stimulation caused by low-dose (1 nM) ET-1. Adiponectin was not able to oppose vasoconstriction when infusion was commenced after ET-1. This is in contrast to the NO donor sodium nitroprusside, which significantly reduced the pressure due to established ET-1 vasoconstriction, suggesting dissociation of the actions of adiponectin and NO. In addition, adiponectin had no effect on vasoconstriction caused by either high-dose (20 nM) ET-1 or low-dose (50 nM) norepinephrine. Our findings suggest that adiponectin has specific, apparently NO-independent, vascular activity to oppose the vasoconstrictor effects of ET-1. The hemodynamic actions of adiponectin may be an important aspect of its insulin-sensitizing ability by regulating access of insulin and glucose to myocytes. Imbalance in the relationship between adiponectin and ET-1 in obesity may contribute to the development of insulin resistance and cardiovascular disease.     

Cholesterol modulates vascular reactivity to endothelin-1 by stimulating a pro-inflammatory pathway

Hypercholesterolemia (HC) is associated with coronary endothelial dysfunction and increased circulating levels of endothelin-1. We show that pre-treatment of intact rat aortic rings with cholesterol synergistically enhances the vasoconstriction induced by endothelin-1 suggesting that elevated levels of cholesterol may predispose to hypertension by modulating the vascular reactivity to endogenous vasoconstrictors. Moreover, we report that SB202190, a selective inhibitor of p38 MAPK, and PD98059 an inhibitor of MEK1/2 are able to abolish the vasoactive properties of cholesterol. MK-886, an inhibitor of 5-lipoxygenase is inefficient at blocking the vasoactive properties of cholesterol whereas NS-398, a selective inhibitor of cyclooxygenase-2 (COX-2) completely abolishes cholesterol-induced vasoconstriction. In intact rat aortae, cholesterol stimulates prostaglandin E(2) and prostaglandin F(2 alpha) production, an effect that can be completely prevented by inhibiting p38 MAPK, or COX-2. In vitro, cholesterol appears to stimulate a similar pro-inflammatory pathway in human cerebrovascular smooth muscle cells. Disruption of the MAPK/COX-2 pathway may represent a valuable therapy to block the hypertension associated with HC, as well as the development of atherosclerosis.     

Estrogen receptor-alpha predominantly mediates the salutary effects of 17beta-estradiol on splenic macrophages following trauma-hemorrhage

Although 17-estradiol administration following trauma-hemorrhage prevents the suppression in splenic macrophage cytokine production, it remains unknown whether the salutary effects are mediated via estrogen receptor (ER)- or ER- and which signaling pathways are involved in such 17-estradiol effects. Utilizing ER-- or ER--specific agonists, this study examined the role of ER- and ER- in 17-estradiol-mediated restoration of macrophage cytokine production following trauma-hemorrhage. In addition, since MAPK and NF-B are known to regulate macrophage cytokine production, we also examined the activation of those signaling molecules. Male rats underwent trauma-hemorrhage (mean arterial pressure of 40 mmHg for 90 min) and fluid resuscitation. The ER- agonist propyl pyrazole triol (PPT; 5 µg/kg), the ER- agonist diarylpropionitrile (DPN; 5 µg/kg), 17-estradiol (50 µg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. Twenty-four hours thereafter, splenic macrophages were isolated, and their IL-6 and TNF- production and activation of MAPK and NF-B were measured. Macrophage IL-6 and TNF- production and MAPK activation were decreased, whereas NF-B activity was increased, following trauma-hemorrhage. PPT or 17-estradiol administration after trauma-hemorrhage normalized those parameters. DPN administration, on the other hand, did not normalize the above parameters. Since PPT but not DPN administration following trauma-hemorrhage was as effective as 17-estradiol in preventing the suppression in macrophage cytokine production, it appears that ER- plays the predominant role in mediating the salutary effects of 17-estradiol on macrophage cytokine production following trauma-hemorrhage and that such effects are likely mediated via normalization of MAPK but not NF-B signaling pathways. shock; mitogen-activated protein kinase; nuclear factor-B; propyl pyrazole triol; diarylpropionitrile     

Caveolin-1蛋白在171β-雌二醇抑制内皮素-1诱导血管平滑肌细胞增殖中的作用

本工作旨在研究Caveolin-1在17β-雌二醇（17β-estradiol,E2)抑制内皮素-1（endothelin-1,ET-1)诱导血管平滑肌细胞（vascular smooth muscle cells,VSMCs前后ET-1对DNA合成和caveolin-1蛋白表达的影响。结果显示,ET-1可以刺激VSMCs增殖。E2作用24h后,可明显抑制ET-1的上述作用。免疫荧光证实,在VSMCs上有cavellin-1分布,ET-1刺激VSMCs增殖的过程中,VSMCs上caveolin-1蛋白荧光强度下,而事称给予E2可逆转这种下降。Western bolt证实,ET-1可抑制caveolin-1蛋白的表达而E2则可增加caveolin-1蛋白的表达。以上结果表明E2抑制ET-1诱导的VSMCs增殖可能与其增加caveolin-1蛋白表达有关。     

Angiotensin II enhances endothelin-1-induced vasoconstriction through upregulating endothelin type A receptor

Endothelin-1 (ET-1) is the most potent vasoconstrictor by binding to endothelin receptors (ET_AR) in vascular smooth muscle cells (VSMCs). The complex of angiotensin II (Ang II) and Ang II type one receptor (AT₁R) acts as a transient constrictor of VSMCs. The synergistic effect of ET-1 and Ang II on blood pressure has been observed in rats; however, the underlying mechanism remains unclear. We hypothesize that Ang II leads to enhancing ET-1-mediated vasoconstriction through the activation of endothelin receptor in VSMCs. The ET-1-induced vasoconstriction, ET-1 binding, and endothelin receptor expression were explored in the isolated endothelium-denuded aortae and A-10 VSMCs. Ang II pretreatment enhanced ET-1-induced vasoconstriction and ET-1 binding to the aorta. Ang II enhanced ET_AR expression, but not ET_BR, in aorta and increased ET-1 binding, mainly to ET_AR in A-10 VSMCs. Moreover, Ang II-enhanced ET_AR expression was blunted and ET-1 binding was reduced by AT₁R antagonism or by inhibitors of PKC or ERK individually. In conclusion, Ang II enhances ET-1-induced vasoconstriction by upregulating ET_AR expression and ET-1/ET_AR binding, which may be because of the AngII/Ang II receptor pathways and the activation of PKC or ERK. These findings suggest the synergistic effect of Ang II and ET-1 on the pathogenic development of hypertension.     

Eucapnic intermittent hypoxia augments endothelin-1 vasoconstriction in rats: role of PKCdelta

We reported previously that simulating sleep apnea by exposing rats to eucapnic intermittent hypoxia (E-IH) causes endothelin-dependent hypertension and increases constrictor sensitivity to endothelin-1 (ET-1). In addition, augmented ET-1-induced constriction in small mesenteric arteries (sMA) is mediated by increased Ca(2+) sensitization independent of Rho-associated kinase. We hypothesized that exposing rats to E-IH augments ET-1-mediated vasoconstriction by increasing protein kinase C (PKC)-dependent Ca(2+) sensitization. In sMA, the nonselective PKC inhibitor GF-109203x (3 microM) significantly inhibited ET-1-stimulated constriction in E-IH arteries but did not affect ET-1-stimulated constriction in sham arteries. Phospholipase C inhibitor U-73122 (1 microM) also inhibited constriction by ET-1 in E-IH but not sham sMA. In contrast, the classical PKC (cPKC) inhibitor G?-6976 (1 microM) had no effect on ET-1-mediated vasoconstriction in either group, but a PKCdelta-selective inhibitor (rottlerin, 3 microM) significantly decreased ET-1-mediated constriction in E-IH but not in sham sMA. ET-1 increased PKCdelta phosphorylation in E-IH but not sham sMA. In contrast, ET-1 constriction in thoracic aorta from both sham and E-IH rats was inhibited by G?-6976 but not by rottlerin. These observations support our hypothesis that E-IH exposure significantly increases ET-1-mediated constriction of sMA through PKCdelta activation and modestly augments ET-1 contraction in thoracic aorta through activation of one or more cPKC isoforms. Therefore, upregulation of a PKC pathway may contribute to elevated ET-1-dependent vascular resistance in this model of hypertension.     

Survivin expression induced by endothelin-1 promotes myofibroblast resistance to apoptosis

Fibrosis of the lungs and other organs is characterized by the accumulation of myofibroblasts, effectors of wound-repair that are responsible for the deposition and organization of new extracellular matrix (ECM) in response to tissue injury. During the resolution phase of normal wound repair, myofibroblast apoptosis limits the continued deposition of ECM. Mounting evidence suggests that myofibroblasts from fibrotic wounds acquire resistance to apoptosis, but the mechanisms regulating this resistance have not been fully elucidated. Endothelin-1 (ET-1), a soluble peptide strongly associated with fibrogenesis, decreases myofibroblast susceptibility to apoptosis through activation of phosphatidylinositol 3′-OH kinase (PI3K)/AKT. Focal adhesion kinase (FAK) also promotes myofibroblast resistance to apoptosis through PI3K/AKT-dependent and -independent mechanisms, although the role of FAK in ET-1 mediated resistance to apoptosis has not been explored. The goal of this study was to investigate whether FAK contributes to ET-1 mediated myofibroblast resistance to apoptosis and to examine potential mechanisms downstream of FAK and PI3K/AKT by which ET-1 regulates myofibroblast survival. Here, we show that ET-1 regulates myofibroblast survival by Rho/ROCK-dependent activation of FAK. The anti-apoptotic actions of FAK are, in turn, dependent on activation of PI3K/AKT and the subsequent increased expression of Survivin, a member of the inhibitor of apoptosis protein (IAP) family. Collectively, these studies define a novel mechanism by which ET-1 promotes myofibroblast resistance to apoptosis through upregulation of Survivin.     

17beta-Estradiol supplementation reduces tubulointerstitial fibrosis by increasing MMP activity in the diabetic kidney

We previously reported that supplementation with 17beta-estradiol (E2) attenuates albuminuria, glomerulosclerosis, and tubulointerstitial fibrosis in diabetic nephropathy. The present study examined the mechanisms by which E2 regulates extracellular matrix (ECM) metabolism, a process that contributes to the development of glomerulosclerosis and tubulointerstitial fibrosis. The study was performed in female nondiabetic (ND), streptozotocin-induced diabetic (D), and diabetic with E2 supplementation (D+E2) Sprague-Dawley rats for 12 wk. Diabetes was associated with an increase in the renal expression of collagen alpha type IV [ND, 0.22 +/- 0.02; D, 0.99 +/- 0.09 relative optical density (ROD); P < 0.05] and fibronectin protein (ND, 0.36 +/- 0.08; D, 1.47 +/- 0.08 ROD; P < 0.05), as measured by Western blot analysis. E2 supplementation partially attenuated this increase in collagen alpha type IV (D+E2, 0.47 +/- 0.10 ROD) and fibronectin (D+E2, 0.71 +/- 0.16 ROD) protein expression associated with D. Diabetes was also associated with a decrease in the expression of matrix metalloproteinase (MMP) isoform MMP-2 (ND, 0.79 +/- 0.01; D, 0.62 +/- 0.06 ROD; P < 0.05) and MMP-9 protein (ND, 0.49 +/- 0.02; D, 0.33 +/- 0.03 ROD; P < 0.05). E2 supplementation restored MMP-2 and MMP-9 protein to levels similar or even greater than in the ND kidneys (MMP-2, 0.75 +/- 0.06; MMP-9, 0.73 +/- 0.01 ROD). The activities of MMP-2 (ND, 7.88 +/- 0.44; D, 5.60 +/- 0.54 ROD; P < 0.05) and MMP-9 (ND, 29.9 +/- 1.8; D, 12.9 +/- 2.3 ROD; P < 0.05), as measured by zymography, were also decreased with D. E2 supplementation restored MMP-2 and MMP-9 activity to levels similar to that in ND kidneys (MMP-2, 7.66 +/- 0.35; MMP-9, 21.4 +/- 2.9 ROD). We conclude that E2 supplementation is renoprotective by attenuating glomerulosclerosis and tubulointerstitial fibrosis by reducing ECM synthesis and increasing ECM degradation.     

Endothelium-independent and -dependent contractions induced by endothelin-1 in canine basilar arteries

Endothelium-dependence of contractile responses to endothelin-1 was examined in isolated canine basilar arteries. Within 2 hrs after mounting tissue preparations, endothelin-1 (10(-9) M) caused a monophasic tonic contraction that developed very slowly and was sustained in intact and endothelium-removed arteries. More than 5 hrs after tissue mounting, endothelin-1 (10(-9) M) caused a biphasic contraction consisting of phasic and tonic components in intact arteries, and caused a monophasic tonic contraction in endothelium-removed arteries. This phasic component was significantly decreased by aspirin (5 x 10(-5) M,), OKY-046 (10(-5) M) (a TXA2 synthetase inhibitor) and ONO-3708 (10(-8) M) (a TXA2 antagonist). The present experiments demonstrate that endothelin-1 causes an endothelium-independent tonic contraction and an endothelium-dependent phasic contraction in canine basilar arteries, and suggest that TXA2 plays a role as an endothelium-derived contracting factor.     

Rat gastric relaxation induced by stimulation of endothelin-1 selective receptors.

We have investigated the smooth muscle activity of ET-1 and ET-3 on rat fundus strips in vitro as well as the effects of the peptides on gastric motility in vivo. In the isolated tissue with no precontraction ET-1 and ET-3 were potent spasmogens which produced half maximal contractions at concentrations 4.5 and 8.0 nM, respectively. In contrast, under conditions where the isolated tissue was precontracted to approx. 50% of maximum by prostaglandin E2, ET-1 dose-dependently (5 x 10(-10) - 10(-8) M) and temporarily relaxed the fundus strip, whereas ET-3 further increased the contraction. The relaxing capacity of ET-1 was absent when the tissue was precontracted by potassium yet was resistant to pretreatments with tetrodotoxin, capsaicin, propranolol, indomethacin, NG-methyl-L-arginine or glibenclamide. In addition in vivo ET-1 and ET-3 (less than 1 nmol/kg) showed opposite effects on gastric motility as the former reduced basal tonus and spontaneous activity, whereas the latter increased the motor activity of the gastric ventricle. The results support the notion that ET-1 may induce gastric relaxation by stimulation of selective receptors whereas stimulation of nonselective receptors may promote gastric smooth muscle contraction.     

Long-lasting vasoconstriction and efficient regional extraction of endothelin-1 in human splanchnic and renal tissues

Six healthy subjects were given endothelin-1, intravenously in a dose of 4 pmol.kg-1.min-1 for 20 min. Blood samples were drawn from arterial, hepatic and renal vein catheters for determinations of splanchnic and renal blood flows and the extraction of endothelin-1 in these vascular beds. Intravenous infusion of endothelin-1 increased the mean arterial blood pressure by 6.8 +/- 2.0 mm Hg (p less than 0.05) and reduced splanchnic and renal blood flows by 34% (p less than 0.005) and 26% (p less than 0.001) respectively. Return to basal flow values occurred after about 1 hr for the splanchnic and 3 hrs for the renal blood flow. The fractional extractions of endothelin-1-like immunoreactivity corresponded to 75 +/- 2% and 60 +/- 2% in the splanchnic and renal vascular beds, respectively. The disappearance curve in plasma and two half-lives of 1.4 +/- 0.1 min and 35 +/- 2.8 min respectively.     

Mechanism of the salutary effects of 17beta-estradiol following trauma-hemorrhage: direct downregulation of Kupffer cell proinflammatory cytokine production

Kupffer cells have been reported as a major source of proinflammatory cytokines (i.e. IL-6, TNF-alpha), which have been implicated in the pathogenesis of trauma-hemorrhage. Previous studies have shown a protective effect of 17beta-estradiol on immune function and physiological responses following trauma-hemorrhage. In this study, we investigated whether 17beta-estradiol has a direct effect on Kupffer cell cytokine production following trauma-hemorrhage. Male Sprague-Dawley rats were subjected to trauma (midline laparotomy) and hemorrhage (35-40 mmHg for 90 min followed by fluid resuscitation) or sham operation. Two hours later, Kupffer cells were isolated and cultured with 17beta-estradiol in the presence and absence of lipopolysaccharide stimulation. Kupffer cell IL-6 and TNF-alpha production increased following trauma-hemorrhage. Incubation with 17beta-estradiol attenuated the production of IL-6 by cells from both sham and trauma-hemorrhage animals in a dose-dependent manner. The suppression of IL-6 production by 17beta-estradiol was paralleled by a decrease in mRNA levels. In contrast to IL-6, the effects of 17beta-estradiol on TNF-alpha production were minimal. In conclusion, these results indicate the direct downregulation of Kupffer cell IL-6 production by 17beta-estradiol at a molecular level, which might explain in part the previously observed salutary effects of estradiol treatment following trauma-hemorrhage.