Myeloperoxidase instigates proinflammatory responses in a cecal ligation and puncture rat model of sepsis.

Myeloperoxidase (MPO)-derived hypochlorous (HOCl) reacts with membrane plasmalogens to yield α-chlorofatty aldehydes such as 2-chlorofatty aldehyde (2-ClFALD) and its metabolite 2-chlorofatty acid (2-ClFA). Recent studies showed that 2-ClFALD and 2-ClFA serve as mediators of the inflammatory responses to sepsis by as yet unknown mechanisms. Since no scavenger for chlorinated lipids is available and on the basis of the well-established role of the MPO/HOCl/chlorinated lipid axis in inflammatory responses, we hypothesized that treatment with MPO inhibitors (N-acetyl lysyltyrosylcysteine amide or 4-aminobenzoic acid hydrazide) would inhibit inflammation and proinflammatory mediator expression induced by cecal ligation and puncture (CLP). We used intravital microscopy to quantify in vivo inflammatory responses in Sham and CLP rats with or without MPO inhibition. Small intestines, mesenteries, and lungs were collected to assess changes in MPO-positive staining and lung injury, respectively, as well as free 2-ClFA and proinflammatory mediators levels. CLP caused neutrophil infiltration, 2-ClFA generation, acute lung injury, leukocyte-/platelet-endothelium interactions, mast cell activation (MCA), plasminogen activator inhibitor-1 (PAI-1) production, and the expression of several cytokines, chemokines, and vascular endothelial growth factor, changes that were reduced by MPO inhibition. Pretreatment with a PAI-1 inhibitor or MC stabilizer prevented CLP-induced leukocyte-endothelium interactions and MCA, and abrogated exogenous 2-ClFALD-induced inflammatory responses. Thus, we provide evidence that MPO instigates these inflammatory changes in CLP and that chlorinated lipids may serve as a mechanistic link between the enzymatic activity of MPO and PAI-1- and mast cell-dependent adhesive interactions, providing a rationale for new therapeutic interventions in sepsis.NEW & NOTEWORTHY Using two distinct myeloperoxidase (MPO) inhibitors, we show for the first time that MPO plays an important role in producing increases in free 2-chlorofatty aldehyde (2-ClFALD)-a powerful proinflammatory chlorinated lipid in plasma and intestine-a number of cytokines and other inflammatory mediators, leukocyte and platelet rolling and adhesion in postcapillary venules, and lung injury in a cecal ligation and puncture model of sepsis. In addition, the use of a plasminogen activator inhibitor-1 (PAI-1) inhibitor or a mast cell stabilizer prevented inflammatory responses in CLP-induced sepsis. PAI-1 inhibition also prevented the proinflammatory responses to exogenous 2-ClFALD superfusion. Thus, our study provides some of the first evidence that MPO-derived free 2-ClFA plays an important role in CLP-induced sepsis by a PAI-1- and mast cell-dependent mechanism.

Tissue Concentrations of Vitamin K and Expression of Key Enzymes of Vitamin K Metabolism Are Influenced by Sex and Diet but Not Housing in C57Bl6 Mice.

BACKGROUND: There has been limited characterization of biological variables that impact vitamin K metabolism. This gap in knowledge can limit the translation of data obtained from preclinical animal studies to future human studies. OBJECTIVE: The purpose of this study was to determine the effects of diet, sex, and housing on serum, tissue, and fecal vitamin K concentrations and gene expression in C57BL6 mice during dietary vitamin K manipulation. METHODS: C57BL6 4-mo-old male and female mice were randomly assigned to conventional or suspended-wire cages and fed control [1400 ± 80 µg phylloquinone (PK)/kg] or deficient (31 ± 0.45 µg PK/kg) diets for 28 d in a factorial design. PK and menaquinone (MK) 4 plasma and tissue concentrations were measured by HPLC. Long-chain MKs were measured in all matrices by LC-atmospheric pressure chemical ionization-mass spectrometry. Gene expression was quantified by reverse transcriptase-polymerase chain reaction in the liver, brain, kidney, pancreas, and adipose tissue. RESULTS: Male and female mice responded differently to dietary manipulation in a tissue-dependent manner. In mice fed the control diet, females had ∼3-fold more MK4 in the brain and mesenteric adipose tissue than did males and 100% greater PK concentrations in the liver, kidney, and mesenteric adipose tissue than did males. In mice fed the deficient diet, kidney MK4 concentrations were ∼4-fold greater in females than in males, and there were no differences in other tissues. Males and females differed in the expression of vitamin K expoxide reductase complex 1 (Vkorc1) in mesenteric adipose tissue and the pancreas and ubiA domain-containing protein 1 (Ubiad1) in the kidney and brain. There was no effect of housing on serum, tissue, or fecal concentrations of any vitamin K form. CONCLUSIONS: Vitamin K concentrations and expression of key metabolic enzymes differ between male and female mice and in response to the dietary PK concentration. Identifying factors that may impact study design and outcomes of interest is critical to optimize study parameters examining vitamin K metabolism in animal models.

Glytan decreases portal pressure via mesentery vasoconstriction in portal hypertensive rats.

AIM: To investigate the effects of Glytan on splanchnic hemodynamics and its reduction of portal pressure in portal hypertensive rats. METHODS: Glytan (Ganluotong in Chinese), is composed of salvianolic acid B and diammonium glycyrrhizinate. Portal hypertension (PHT) was induced in the rats by common bile duct ligation (BDL). Hemodynamic studies were performed using the colored microsphere method. Radioimmunoassay (RIA) was used to determine endothelin (ET)-1 levels in the mesenteric circulation. Western blotting methods were used to investigate the effect of Glytan on ET A receptor (ETAR), ET B receptor (ETBR), endothelial NO synthase (eNOS), G-protein-coupled receptor kinase (GRK)2, and ß-arrestin 2 expression in the mesentery. The mRNA of ETAR and ETBR was determined using real-time polymerase chain reaction. RESULTS: Treatment with Glytan reduced portal pressure (PP) and portal territory blood flow (PTBF) and increased both mean arterial pressure (MAP) and splanchnic vascular resistance (SVR). Especially at 4 wk, PP decreased by about 40%, while MAP increased by 13%, SVR increased by 12%, and PTBF decreased by about 21%. The effect of blood flow reduction was greatest in the mesentery (about 33%) at 4 wk. The mesenteric circulation ET-1 levels of BDL rats were lower and negatively correlated with PP at 4 wk. Glytan can increase mesenteric ET-1 content and inhibit ETBR, eNOS, GRK2, and ß-arrestin 2 expression in the mesentery. Moreover, Glytan showed no effect on the expression of ETAR protein and mRNA. CONCLUSION: The decreased PP and PTBF observed after Glytan treatment were related to increased mesenteric vasoconstriction and increased receptor sensitivity to vasoconstrictor.

Oregonin from the Bark of Alnus japonica restrained ischemia-reperfusion-induced mesentery oxidative stress by inhibiting NADPH oxidase activation.

OBJECTIVE: NADPH oxidase activation results in ROS overproduction that is the pathological basis of I/R injury. This study aimed to investigate potential effects of ORG on I/R-induced ROS production in rat mesenteric microvasculature and underlying mechanisms. METHODS: Mesenteric I/R in Male Wistar rats (200~250 g) was induced by ligation of the mesenteric artery and vein for 10 minutes followed by reperfusion for 60 minutes by releasing of the occlusion. The rats were infused intravenously with or without ORG (5 mg/kg per hour) 10 minutes before ischemia (pretreatment) or 20 minutes after reperfusion (posttreatment). The DHR fluorescence intensity on, the leukocytes adherent to, and mast cell degranulation out of mesenteric venules were determined using an intravital microscope. NADPH oxidase subunit p47(phox) membrane translocation in intestine tissues was detected by Western blotting. RESULTS: Pre- or posttreatment with ORG inhibited I/R-induced DHR fluorescence intensity on the venular walls and leukocytes adhesion, ORG pretreatment inhibited mast cell degranulation as well. Furthermore, the translocation of p47(phox) from cytosol to membrane was suppressed markedly by ORG after I/R. CONCLUSIONS: The results suggested that ORG restrained I/R-induced ROS production, which might be correlated with its inhibitive effect on NADPH activation.

Mesenteric lymph diversion abrogates 5-lipoxygenase activation in the kidney following trauma and hemorrhagic shock.

BACKGROUND: Early acute kidney injury (AKI) following trauma is associated with multiorgan failure and mortality. Leukotrienes have been implicated both in AKI and in acute lung injury. Activated 5-lipoxygenase (5-LO) colocalizes with 5-LO-activating protein (FLAP) in the first step of leukotriene production following trauma and hemorrhagic shock (T/HS). Diversion of postshock mesenteric lymph, which is rich in the 5-LO substrate of arachidonate, attenuates lung injury and decreases 5-LO/FLAP associations in the lung after T/HS. We hypothesized that mesenteric lymph diversion (MLD) will also attenuate postshock 5-LO-mediated AKI. METHODS: Rats underwent T/HS (laparotomy, hemorrhagic shock to a mean arterial pressure of 30 mm Hg for 45 minutes, and resuscitation), and MLD was accomplished via cannulation of the mesenteric duct. Extent of kidney injury was determined via histology score and verified by urinary neutrophil gelatinase-associated lipocalin assay. Kidney sections were immunostained for 5-LO and FLAP, and colocalization was determined by fluorescence resonance energy transfer signal intensity. The end leukotriene products of 5-LO were determined in urine. RESULTS: AKI was evident in the T/HS group by derangement in kidney tubule architecture and confirmed by neutrophil gelatinase-associated lipocalin assay, whereas MLD during T/HS preserved renal tubule morphology at a sham level. MLD during T/HS decreased the associations between 5-LO and FLAP demonstrated by fluorescence resonance energy transfer microscopy and decreased leukotriene production in urine. CONCLUSION: 5-LO and FLAP colocalize in the interstitium of the renal medulla following T/HS. MLD attenuates this phenomenon, which coincides with pathologic changes seen in tubules during kidney injury and biochemical evidence of AKI. These data suggest that gut-derived leukotriene substrate predisposes the kidney and the lung to subsequent injury.

Evidence of increased oxidative stress in aged mesenteric lymphatic vessels.

BACKGROUND: We have previously shown that aging is associated with weakened rat mesenteric lymphatic vessel (MLV) contractility. However, the specific mechanisms contributing to this aging-associated contractile degeneration remain unknown. Aging is often associated with elevations in oxidative stress, and reactive oxygen species (ROS) have been shown to reduce the contractility of MLV. Thus in the present study, we sought to assess whether aging is associated with increased levels of oxidative stress and oxidative damage in MLV. METHODS AND RESULTS: MLV were isolated from 9-mo- and 24-mo-old Fischer-344 rats and subjected to the following experimental techniques: measurement of total superoxide dismutase (SOD) activity; estimation of lipid peroxidation levels via measurement of thiobarbituric acid reactive substances (TBARS); detection of superoxide and mitochondrial ROS in live MLV; Western blot analysis, and immunohistochemical labeling of the SOD isoforms and nitro-tyrosine proteins. We found that aging is associated with increased levels of cellular superoxide and mitochondrial ROS concomitant with a reduction in Cu/Zn-SOD protein expression and total SOD enzymatic activity in MLV. This increase in oxidative stress and decrease in antioxidant activity was associated with evidence of increased lipid (as indicated by TBARS) and protein (as indicated by nitro-tyrosine labeling) oxidative damage. CONCLUSIONS: Thus for the first time, we demonstrate that aging-associated increases in oxidative stress and oxidative damage is indeed present in the walls of MLV and may contribute to the aging-associated lymphatic pump dysfunction we previously reported.

Expression of retinaldehyde dehydrogenase enzymes in mucosal dendritic cells and gut-draining lymph node stromal cells is controlled by dietary vitamin A.

The vitamin A metabolite retinoic acid (RA) plays a crucial role in mucosal immune responses. We demonstrate in this study that RA-producing retinaldehyde dehydrogenase (RALDH) enzymes are postnatally induced in mesenteric lymph node (MLN) dendritic cells (DCs) and MLN stromal cells. RALDH enzyme activity in lamina propria-derived CD103(+) MLN-DCs did not depend on TLR signaling. Remarkably, RA itself could directly induce RALDH2 in both DCs and stromal cells in vitro. Furthermore, upon provision of a vitamin A-deficient diet, it was found that RA-mediated signaling was strongly reduced within the small intestines, while RALDH2 mRNA and RALDH enzyme activity in lamina propria DCs and MLN-DCs, as well as RALDH2 mRNA expression in MLN stromal cells, were strongly diminished. Moreover, supply of vitamin A to vitamin A-deficient mice restored RA-mediated signaling in the intestine and RALDH activity in lamina propria-derived CD103(+) MLN-DCs. Our results show that RA-dependent signaling within the intestine is indispensable for RALDH activity in the draining MLN.

Microvascular display of xanthine oxidase and NADPH oxidase in the spontaneously hypertensive rat.

OBJECTIVE: Oxygen free radical production in hypertension may be associated with elevated arteriolar tone and organ injury. Previous results suggest an enhanced level of oxygen free radical formation in microvascular endothelium and in circulating neutrophils associated with xanthine oxidase activity in the spontaneously hypertensive rats (SHR) compared with their normotensive controls, the Wistar Kyoto rats (WKY). The aim of this study was to gain more detailed understanding of where oxidative enzymes are located in the microcirculation. METHODS: An approach was developed to delineate the cellular distribution of two selected oxidative enzymes, xanthine oxidase and nicotinamide adenine dinucleotide phosphate (NADPH) dependent oxidase (protein 67-kDa fraction). Immunolabeling with peroxidase substrate was utilized, which permits full delineation of the primary antibody in all microvascular structures of the mesentery. RESULTS: Xanthine oxidase is present in the endothelium of all segments of the microcirculation, in mast cells, and in parenchymal cells of the mesentery. NADPH oxidase can be detected in the endothelium, leukocytes, and mast cells and with lower levels in parenchymal cells. The mesentery of WKY and SHR has similar enzyme distributions with enhancements on the arteriolar and venular side of the microcirculation that coincide with the sites of enhanced free radical production recently reported. Immune label measurements under standardized conditions indicate that both enzymes are significantly enhanced in the SHR. Adrenalectomy, which serves to reduce the blood pressure and free radical production of the SHR to normotensive levels, leads to a reduction of NADPH and xanthine oxidase to normotensive levels, while supplementation of adrenalectomized SHR with dexamethasone significantly increases the oxidase expression in several parts of the microcirculation to levels above the WKY rats. CONCLUSION: The results indicate that enhanced expression of NADPH and xanthine oxidase in the SHR depends on an adrenal pathway that is detectable in the arteriolar and venular network at high and low pressure regions of the circulation.

Pregnancy effects on rat adipose tissue lipolytic capacity are dependent on anatomical location.

Pregnancy is characterized by changes in maternal adiposity. The aim of this study was to carry out a detailed analysis of the different steps of the adrenergic pathway, lipoprotein lipase (LPL) levels and adipocyte size, in order to evaluate the response of white adipose tissue (WAT) to the metabolic changes during pregnancy depending on the anatomical location. In general, the levels of the proteins of the lipolytic pathway decreased with pregnancy. In retroperitoneal WAT adenylate cyclase (AC) levels decreased from 100% in controls to 44% by day 13 and 11% by day 20. In mesenteric WAT the alpha (2A)/beta (3)-adrenergic receptor balance seemed to be one of the main regulatory points of the lipolytic pathway and the reduction in the postreceptor element levels was clearly lower than for the other two depots (PKA levels reduced from 100% in controls to 72% by day 20, while in the other two depots it decreased to 30%, and AC and HSL levels did not show statistically significant changes in this depot). In contrast, the LPL-to-HSL ratio may be a major regulatory point in gonadal WAT. In summary, we describe regional differences in the regulation of WAT metabolism throughout pregnancy, which may be of great importance to determine the role of the different fat depots during late pregnancy. Thus, gonadal and mesenteric WAT changed to a lipolytic state to sustain the rapid foetal growth, although with differences between them in the main regulatory points, while retroperitoneal WAT could have a role later on, during lactation.

Hydroxymethylglutaryl co-enzyme A reductase inhibition attenuates endotoxin-mediated inflammatory responses.

BACKGROUND: The aim of this study was to investigate whether inhibition of hydroxymethylglutaryl co-enzyme A reductase attenuates leucocyte-endothelial cell interactions and alters expression of endothelial constitutive nitric oxide synthase (ecNOS) and inducible nitric oxide synthase (iNOS) following exposure to endotoxin. METHODS: Male Sprague-Dawley rats were randomized into control, lipopolysaccharide (LPS) and pravastatin + LPS groups (seven per group). Pravastatin sodium was gavaged at 0.4 mg per kg per day for 5 days, after which LPS 15 mg/kg was administered via the jugular vein. Intravital microscopy was used to determine leucocyte-endothelial cell interactions. RESULTS: Following the administration of LPS there was a significant reduction in leucocyte rolling velocity at 10 min (mean(s.e.m.) 69(3) versus 102(6) per cent of baseline value; P = 0.041), an increase in the number of adherent leucocytes at 10 min (4.5(0.5) versus 2.8(0.3) per 100 microm; P = 0.044) and an increase in the number of leucocytes undergoing transendothelial migration at 30 min (4.2(0.4) versus 1.7(0.4) per field; P = 0.008) compared with controls. Pretreatment with pravastatin significantly attenuated LPS-induced leucocyte-endothelial cell interactions (rolling velocity 89(6) per cent at 10 min, P = 0.038; adherent leucocytes 3.0(0.5) per 100 microm at 10 min, P = 0.038; migrating leucocytes 1.9(0.5) per field at 30 min, P = 0.001). This endothelial protection was associated with maintenance of ecNOS and reduced iNOS expression within mesenteric tissues. CONCLUSION: These data show that pravastatin produces anti-inflammatory effects in response to injurious stimuli by attenuation of leucocyte-endothelial cell interactions.

Estrogen replacement suppresses stress-induced cardiovascular responses in ovariectomized rats.

We assessed the hypothesis that chronic estrogen replacement in ovariectomized rats has the beneficial effect of suppressing stress-induced cardiovascular responses through endothelial nitric oxide synthase (eNOS). We employed a radiotelemetry system to measure blood pressure and heart rate (HR). Female Wistar rats aged 11 wk were ovariectomized and implanted with radiotelemetry devices. After 4 wk, the rats were assigned either to a placebo-treated group (Placebo; n=6) or a group treated with 17beta-estradiol (Estrogen; n=8) subcutaneously implanted with either placebo- or 17beta-estradiol (1.5 mg/60-day release) pellets under anesthesia. These rats underwent either of the two types of stress after 4 wk of estrogen or placebo treatment. Cage-switch stress and restraint stress rapidly and continuously elevated the mean arterial pressure (MAP) and HR both in the Placebo and Estrogen groups. However, the MAP and HR responses to cage-switch stress and the MAP but not HR response to restraint stress were attenuated significantly in the Estrogen group compared with the Placebo group. A NOS inhibitor, NG-nitro-L-arginine methyl ester, given in drinking water, reduced the difference in the pressor response to cage-switch between the Estrogen and Placebo groups. In addition, Western blot analysis showed that eNOS expression in the mesentery was increased in the Estrogen group compared with the Placebo group. Thus for the first time we showed that mesenteric eNOS overexpression could explain at least partly why chronic estrogen treatment suppressed the enhanced cardiovascular responses to psychological stress in the ovariectomized rat.

Conversion of renin substrate tetradecapeptide to angiotensin II by rat MAB elastase-2.

A new approach for the purification of rat mesenteric arterial bed (MAB) elastase-2 has been developed using the chromogenic substrates N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide to monitor the enzymatic activity during various stages of purification. The purified enzyme was evaluated in the presence of various inhibitors and confirmed to have angiotensin (Ang) II-forming ability. The active site-directed inhibitor acetyl-Ala-Ala-Pro-Leu-chloromethylketone (100 micromol x L(-1)), described for human pancreatic elastase-2, abolished the enzymatic activity, confirming that the enzyme is an elastase-2. Chymostatin (100 micromol x L(-1)), an inhibitor regarded as selective for chymases, also showed a remarkable inhibitory effect (94%), whereas captopril (100 micromol x L(-1)) had no effect at all on the Ang II-forming activity. The Ang II precursor renin substrate tetradecapeptide (RS-14P) was converted into Ang II by the rat MAB elastase-2 with the following kinetic constants: Km = 124 +/- 21 micromol x L(-1); Kcat = 629 min(-1); catalytic efficiency (Kcat /Km) = 5.1 min(-1) micro(mol/L)-1. In conclusion, the strategy for the purification of rat MAB elastase-2 with the chromogenic substrates proved to be simple, rapid, accurate, and highly reproducible; therefore, it can be reliably and conveniently used to routinely purify this enzyme. The kinetic parameters for the formation of Ang II from RS-14P by rat MAB elastase-2 emphasize differences in substrate specificity between this and other Ang II-forming enzymes.

A novel role for calpains in the endothelial dysfunction of hyperglycemia.

Recent studies have reported that the activity of the calcium-dependent protease calpain is increased in acute inflammatory processes of the cardiovascular system. Because diabetes is associated with vascular inflammation, we hypothesized that increased calpain activity in response to hyperglycemia may play a role in diabetic cardiovascular disease. The effects of calpain inhibition on leukocyte-endothelium interactions induced by hyperglycemia were examined by intravital microscopy. Intraperitoneal administration of the selective calpain inhibitor benzyloxycarbonyl-leucyl-leucinal (5 micromol/L) prevented the up-regulation of leukocyte-endothelium interactions in response to 25 mmol/L D-glucose via a nitric oxide-dependent mechanism. Furthermore, treatment of rats with D-glucose significantly decreased basal endothelial NO release in mesenteric post-capillary venules, a phenomenon prevented by inhibition of calpain activity. Immunoprecipitation studies revealed that glucose induces loss of NO via a calpain-dependent decrease in the association of hsp90 with endothelial nitric oxide synthase. In addition, inhibition of calpain activity decreased endothelial cell surface expression of the pro-inflammatory adhesion molecules ICAM-1 and VCAM-1 during hyperglycemia. These data demonstrate that calpains contribute to important inflammatory events during hyperglycemia and that pharmacological inhibition of calpain activity attenuates leukocyte-endothelium interactions and preserves eNOS function.

Increased heme oxygenase-1 gene expression in liver cells and splanchnic organs from portal hypertensive rats.

Heme oxygenase (HO) catalyzes the conversion of heme into biliverdin, iron, and carbon monoxide (CO). Two isoforms of HO have been identified: the inducible HO-1 and the constitutive HO-2. CO, like nitric oxide, is an endogenous vasodilator that could contribute to modulation of systemic and local vascular tone. The aim of the present study was to determine the expression of HO isoforms in liver cells and splanchnic organs from portal hypertensive (PH) and sham-operated (SO) rats. Liver cells (hepatocytes, Kupffer and stellate cells), and splanchnic organs (liver, mesentery, intestine, colon, and spleen) were isolated from PH and SO rats. Expression of HO mRNA and protein was assessed by reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. In SO rats, HO-1 mRNA expression was only detected in spleen. In contrast, in PH rats, HO-1 mRNA was expressed in hepatocytes, Kupffer cells, and in all the splanchnic organs studied. Moreover, levels of HO-1 protein in splanchnic organs were significantly higher in PH rats than in SO animals. In addition, HO-2 expression was observed in all liver cell types and splanchnic organs studied from both PH and SO rats. These results indicate that HO-2 is expressed in parenchymal and nonparenchymal liver cells, as well as splanchnic organs, of both PH and SO rats. In addition, HO-1 is up-regulated in hepatocytes and splanchnic organs of PH rats, compared with SO animals, suggesting a possible pathophysiological role of HO-1 in chronic portal hypertension.

Cytochemical evidence for potassium-dependent p-nitrophenylphosphatase activity in pavement cells of Rana esculenta mesentery.

BACKGROUND: We previously reported that during hibernation in Rana esculenta, various organs (i.e., skin, urinary bladder, kidney) change their osmoregulatory activity. Here, we considered the possible role of the frog mesentery in the ion transport, evaluating morphological and cytochemical (K+-p-nitrophenylphosphatase activity) aspects. METHODS: Pieces of mesentery from Rana esculenta collected in their natural environment during April, June, October, and January were processed to reveal ultrastructural morphology and K+-p-NPPase activity, using cerium as capture agent. RESULTS: The mesenteric mesothelium contained three types of cells: pavement, mitochondria-rich, and ciliated. Only the pavement cells expressed intense reactivity on the basolateral membranes and in the adjacent pinocytotic vesicles; some reaction product also was found on the apical membranes. Moreover, morphological and cytochemical characteristics of the pavement cells appeared to be very seasonal. CONCLUSIONS: The presence of mitochondria-rich cells and ciliated cells, generally found in structures involved in the transport of liquids, as well as K+-p-NPPase activity and pinocytosis in pavement cells, is consistent with the hypothesis that frog mesentery may be involved in seasonally variable osmoregulation.

[The intramural neural apparatus of the mesenteric lymphangion of the small intestine]. / Intramural'nyi nervnyi apparat limfangiona bryzheiki tonkoi kishki.

Concentration of nerve structures in the lymphangion wall directly correlates with myocyte number in its different regions. Maximal adrenergic and cholinergic fibres concentration was defined in valvular torus muscular cuff of the lymphangion and minimal--in the valvular sinus wall. Besides, as structural organization of adrenergic plexus does not differ from that of antichollinaestherase-positive one and electron microscopic photos display diverse cholinergic and monoaminergic mediator vesicles in same fibres a suggestion is made on the mixed mediator nature of intramural plexuses of the lymphangion.

Choline acetyltransferase-immunoreactive neurones in a prevertebral sympathetic ganglion, the inferior mesenteric ganglion.

Using immunohistochemical techniques a small population of choline acetyltransferase (ChAT) immunoreactive (IR) neurones has been identified in the inferior mesenteric ganglion (IMG) of guinea pig (4.6% of all neurones), ferret (6.4%) and rat (0.4%). A detailed study in the guinea-pig IMG revealed that the vast majority of cholinergic neurones did not express tyrosine hydroxylase (TH)-IR, indicating that they were non-catecholaminergic. The cholinergic neurones were significantly larger than the TH-positive neurones. The majority of the ChAT-IR cells (64%) was observed in small clusters which were consistently located in the caudal lobe of the IMG close to the entry of the hypogastric nerves. 83% of the ChAT-IR cells also contained neuropeptide Y (NPY). Since the vast majority of TH-negative cells were ChAT-positive (94%), the TH negativity was taken as an indirect indication for ChAT-IR. NPY-IR, somatostatin (SOM)-IR and vasoactive intestinal peptide (VIP)-IR were found in both the TH-IR cells (22, 84 and 1%, respectively) and the putative cholinergic population (95, 84 and 70, respectively). Thus the majority of cholinergic neurones in the IMG were likely to contain NPY, SOM and VIP. TH-IR cells exhibited an extensive innervation of fibers immunoreactive for ChAT, VIP, ENK and NOS. In contrast, only a sparse plexus of ChAT-, ENK-, NOS-, NPY- and SOM-positive fibres was found around the TH-negative cells. VIP-IR fibres did not appear to innervate ChAT neurones.

Increased splanchnic prostacyclin synthase and cyclooxygenase content and activity during ischemia is due to new protein synthesis.

BACKGROUND: This study examines the hypothesis that the exaggerated splanchnic release of prostacyclin is due to new synthesis of both cyclooxygenase and prostacyclin synthase (PS) in the ileum muscularis/serosa. METHODS: Sprague-Dawley rats were anesthetized and subjected to acute hemorrhage to 30 mm Hg for 30 minutes (shock) or sham shock. The superior mesenteric artery (SMA) was cannulated and removed with its end-organ intestine and perfused in vitro with Krebs-Henseleit buffer with and without cycloheximide (50 micrograms/ml) or indomethacin (20 micrograms/ml). Venous effluent was analyzed for eicosanoids by radioimmunoassay. The SMA, aorta and ileal mucosa, and muscularis/serosa were analyzed for PS and cyclooxygenase content by immunoblot analysis. RESULTS: The sham splanchnic bed released threefold more 6-keto-PGF1 alpha than prostaglandin E2 and thromboxane. Acute ischemia increased splanchnic release of 6-keto-PGF1 alpha threefold compared with sham, which was abolished by cycloheximide or indomethacin treatment. Acute ischemia increased content of PS and cyclooxygenase in the ileal muscularis/serosa twofold and PS in the aorta and SMA by 50%. CONCLUSIONS: Acute ischemia increased release of 6-keto-PGF1 alpha, which was dependent on new protein synthesis. The immunoblot data suggest that the location of the increased enzymes responsible for increased 6-keto-PGF1 alpha release is the ileal muscularis/serosa and in the aorta and SMA.

Induction by endotoxin of nitric oxide synthase in the rat mesentery: lack of effect on action of vasoconstrictors.

1. Male Sprague-Dawley or Wistar rats were injected with bacterial lipopolysaccharide (LPS; 5 mg kg-1, i.p.) and killed after 1, 3, 6, 15, and 24 h. The brains, mesenteries, spleens, lungs, livers, kidneys, hearts, aortae and diaphragms were removed and frozen immediately. Control rats were injected with sterile saline and killed after 6 h. 2. The organs were homogenized in a semi-frozen state and NO synthase (NOS) activity measured in tissues from both LPS-treated and saline-treated groups by the ability of homogenates to convert [3H]-L-arginine to [3H]-L-citrulline in a NADPH-dependent manner. 3. The NOS activity in all organs taken from control animals was found to be calcium-dependent, with the highest activity being in the brain. After LPS-treatment an induced calcium-independent NOS was detected in all tissues tested, with the exception of the brain. The spleen, lung, mesentery and liver had the highest amounts of LPS-induced NOS activity. No induction of calcium-dependent NOS was detected. 4. Induction of NOS was maximum 6 h after administration of LPS and had returned to control levels in 24 h. 5. The constitutive NOS in brain and mesentery and the LPS-induced activities in the spleen, lung, liver and mesentery were inhibited by NG-monomethyl-L-arginine (L-NMMA) or NG-nitro-L-arginine methyl ester (L-NAME) according to concentration. The IC50 for L-NAME was 2.5 microM against the constitutive NOS from brain, and 20-25 microM against the inducible NOS. For L-NMMA the IC50 was 20-25 microM against either NOS isoform. 7. The vascular responses to endothelin-I (ET-1), the thromboxane A2-mimetic 11 alpha,9 alpha-epoxymethanoprostaglandin F2alpha (U46619), phenylephrine (PE) or 5-hydroxytryptamine (5-HT) were measured in the simultaneously perfused arterial and venous mesenteric vascular beds from both control and LPS-treated(6 h) rats. Vasoconstrictor responses to all agonists tested were unaffected by LPS treatment. In the presence of L-NAME (100 microM) vasoconstrictor responses were potentiated in both the arterial and venous portion of the mesenteric beds from both control and LPS-treated rats. The potentiation of responses to U46619 was significantly greater in beds from LPS-treated rats.8. Injection of LPS i.p. is associated with induction of NOS in all organs tested, except for the brain. In the mesentery this is not accompanied by a hyporesponsiveness to constrictor agents suggesting an increased sensitivity, particularly to U46619. This may explain the poor perfusion and tissue damage in the splanchnic circulation associated with sepsis.

Differentiation of the anti-shock effect of ulinastatin from steroid hormone, by the continuous observation of microcirculation dynamics.

The effects of ulinastatin (UST) and methyl prednisolone (MPS) on endotoxin induced shock were compared by the criterion of microcirculation dynamics in rat mesenterium and plasma phospholipase A2 (PLA2) activity in modified Shwartzman reaction model. The mean arterial pressure and red cell velocity were well maintained when MPS was administered during endotoxin induced shock. In the case of UST, superior anti-shock effects, indicated by reduced vasoconstriction, were obtained when it was administered prior to endotoxin induced shock. The anti-shock effect of UST, similar to MPS, was supported by the change of serum PLA2 activity. Therefore, concerning the administration timing of anti-shock drugs, MPS should be administered after shock occurs, and UST is most effective as a prophylactic treatment. UST has an anti-shock effect like steroid hormone.