Resistance of essential fatty acid-deficient rats to endotoxin-induced increases in vascular permeability

Resistance to endotoxin in essential fatty acid-deficient (EFAD) rats is associated with reduced synthesis of certain arachidonic acid metabolites. It was hypothesized that EFAD rats would manifest decreased vascular permeability changes during endotoxemia as a consequence of reduced arachidonic acid metabolism. To test this hypothesis, changes in hematocrit (HCT) and mesenteric localization rate of technetium-labeled human serum albumin (99mTc-HSA) and red blood cells (99mTc-RBC) were assessed in EFAD and normal rats using gamma-camera imaging. Thirty minutes after Salmonella enteritidis endotoxin, EFAD rats exhibited less hemoconcentration as determined by % HCT than normal rats (47 +/- 2% vs. 54 +/- 1% respectively, P less than 0.01). Endotoxin caused a less severe change in permeability index in the splanchnic region in EFAD rats than in normal rats (1.2 +/- 0.6 x 10(-3)min-1 vs. 4.9 +/- 1.7 x 10(-3)min-1 respectively, P less than 0.05). In contrast to 99mTc-HSA, mesenteric localization of 99mTc-RBC was not changed by endotoxin in control or EFAD rats. Supplementation with ethyl-arachidonic acid did not enhance susceptibility of EFAD rats to endotoxin-induced splanchnic permeability to 99mTc-HSA. Leukotrienes have been implicated as mediators of increased vascular permeability in endotoxin shock. Since LTC3 formation has been reported to be increased in EFA deficiency, we hypothesized that LTC3 may be less potent than LTC4. Thus the effect of LTC3 on mean arterial pressure and permeability was compared to LTC4 in normal rats. LTC3-induced increases in peak mean arterial pressure were less than LTC4 at 10 micrograms/kg (39 +/- 5 mm Hg vs. 58 +/- 4 mm Hg respectively, P less than 0.05) and at 20 micrograms/kg (56 +/- 4 mm Hg vs. 75 +/- 2 mm Hg respectively, P less than 0.05). LY171883 (30 mg/kg), an LTD4/E4 receptor antagonist, attenuated the pressor effect of LTC4, LTD4, and LTC3. Infusion of LTC4 (4 micrograms/kg/min) in normal rats induced a rise in HCT from 44 +/- 1% to 51 +/- 1% (P less than 0.01), which was greater (P less than 0.05) than the rise induced by LTC3 (47 +/- 1% to 49 +/- 1%). The results showing that EFAD rats are resistant to endotoxin-induced increases in HCT and vascular permeability raise the possibility that this may, in part, be a result of preferential LTC3 production that is less potent than LTC4.     

Arachidonic acid and docosahexaenoic acid supplemented to an essential fatty acid-deficient diet alters the response to endotoxin in rats

This study examined fatty acid profiles, triene-tetraene ratios (20:3n9/20:4n6), and nutritional and inflammatory markers in rats fed an essential fatty acid-deficient (EFAD) diet provided as 2% hydrogenated coconut oil (HCO) alone for 2 weeks or with 1.3 mg of arachidonic acid (AA) and 3.3 mg of docosahexaenoic acid (DHA) (AA + DHA) added to achieve 2% fat. Healthy controls were fed an AIN 93M diet (AIN) with 2% soybean oil. The HCO and AA + DHA diets led to significant reductions of linoleic acid, α-linolenic acid, and AA (20:4n6) and increases in Mead acid (20:3n9) in plasma and liver compared with the AIN diet; but the triene-tetraene levels remained well within normal. However, levels of 20:3n9 and 20:4n6 were lower in liver phospholipids in the AA + DHA than in HCO group, suggesting reduced elongation and desaturation in ω-9 and -6 pathways. The AA + DHA group also had significantly lower levels of 18:1n9 and 16:1n7 as well as 18:1n9/18:0 and 16:1n7/16:0 than the HCO group, suggesting inhibition of stearyl-Co A desaturase-1 activity. In response to lipopolysaccharide, the levels of tumor necrosis factor and interleukin-6 were significantly lower with HCO, reflecting reduced inflammation. The AA + DHA group had higher levels of IL-6 and C-reactive protein than the HCO group but significantly lower than the AIN group. However, in response to endotoxin, interleukin-6 was higher with AA + DHA than with AIN. Feeding an EFAD diet reduces baseline inflammation and inflammatory response to endotoxin long before the development of EFAD, and added AA + DHA modifies this response.     

Autoradiographic studies with albumin-bound 1-14C-linoleic acid in normal and essential fatty acid-deficient rats. A pilot study

Young rats fed a diet providing 0.3, 3 or 10% of the energy as essential fatty acids (EFA) were given a single intravenous dose of albumin-bound 1-14C-linoleic acid. 1 animal from each group was killed at 1 and 18 h, respectively, after the injection and submitted to whole-body autoradiography. In general, the activities in the tissues were higher in the 0.3% group than in the other groups, whereas the differences between the 3% and the 10% group were small. In all groups the highest activity occurred in the brown fat. High activities were also noted in the liver, the adrenal cortex, the diaphragm, and the gastric and intestinal mucosa. The higher activities in the tissues of the rats fed 0.3% than in the tissues of those fed 3% or 10% EFA probably reflect a higher incorporation of fatty acids of the linoleic acid series into structural and other lipids in the former group due to the lack of EFA.     

Role of endothelin-1/endothelin receptor system in endotoxic shock rats.

Endothelin (ET)-1, a potent vasoconstrictor peptide derived from the endothelium, is markedly increased in endotoxic shock, although the pathophysiological role of ET-1 under septic conditions remains obscure. To delineate the role of ET-1 and its receptor subtype in endotoxic shock, we here attempted to determine the changes of circulating levels of ET-1 and its biosynthetic intermediate big ET-1 in endotoxic shock rats, to evaluate the gene expression of ET-1 as well as the ET-1 receptor subtypes (ETA and ETB) in the heart, lung and liver, and to study the effects of ET receptor antagonists on systemic arterial blood pressure, heart rate and survival rate. Administration of bacterial lipopolysaccharide (LPS) caused profound hypotension, increased heart rate and death, and these effects were blocked by a nonselective ETA/ETB receptor antagonist (TAK044), but not by an ETA selective antagonist (BQ123). Administration of exogenous ET-1 caused a profound pressor response in control rats, but not in the LPS-pretreated rats. Injection of LPS caused marked elevation of plasma levels of both ET-1 and big ET-1, which were not affected by treatment with either ET receptor antagonist. Administration of LPS caused up-regulation of ET-1 and ETB receptor mRNA in the heart, whereas ETA receptor mRNA was markedly down-regulated in the heart, lung and liver. These data suggest differential gene regulation of ET-1 and its receptor subtypes in various organs from endotoxic shock rats, and that nonselective ETA/ETB receptor antagonist, but not ETA receptor antagonist, ameliorates endotoxin-induced hypotension and death.     

Effect of cholecystokinin on cytokines during endotoxic shock in rats

AIM To study the effect of cholecystokinin-octapeptide(CCK-8)on systemic hypotension and cytokine productionin lipopolysaccharide(LPS)-induced endotoxic shock(ES)rats.METHODS The changes of blood pressure were observedusing physiological record instrument in four groups ofrats:LPS(8mg·kg~(-1),iv)induced ES;CCK-8(40μg·kg~1,iv)pretreatment 10min before LPS(8mg.kg~(-1));CCK-8(40μg·kg~(-1),iv)or normal saline(control)groups.Differences in tissue and circulating specificity of theproinflammatory cytokines(TNF-α,IL-1β and IL-6)wereassayed with ELISA kits.RESULTS CCK-8 reversed LPS-induced decrease of meanartery blood pressure(MABP)in rats.Compared withcontrol,LPS elevated the serum level of IL-6 significantly(3567±687ng·L~(-1) vs 128±22ng·L~1,P<0.01),whilecontents of TNF-α and IL-1β elevated significantly(277±86ng.L~1 vs not detectable and 43±9ng·L~1 vs notdetectable,P<0.01)but less extent than IL-6.CCK-8significantly inhibited the LPS-induced increase in serumTNF-α,IL-1β and IL-6.LPS elevated spleen and lungcontent of IL-1β significantly(5184±85ng·L~1 vs 1047±21ng·L~1 and 4050±614ng·L~1 vs not detectable.P<0.01),while levels of TNF-α and IL-6 also rosesignificantly but in less extent than IL-1β.CCK-8 inhibitedthe LPS-induced increase of the cytokines in spleen andlung.In the heart,CCK-8 significantly inhibited LPS-induced increase of TNF-α(864±123ng·L~1 in CCK-8 LPS group vs 1599±227ng·L~1 in LPS group,P<0.01),and IL-1β(282±93ng·L~1 in CCK-8 LPS group vs 621±145ng·L~1 in LPS group,P<0.01).CONCLUSION CCK-8 reverses ES,which may be relatedto its inhibitory effect on the overproduction of cytokines.     

Increased heat shock protein 70 expression in the pancreas of rats with endotoxic shock

AIM: To investigate the ultra-structural changes and heat shock protein 70 (HSP70) expression in the pancreas of rats with endotoxic shock and to detect their possible relationship. METHODS: A total of 33 Wistar rats were randomly divided into three groups: control group (given normal saline), small dose lipopolysaccharide (LPS) group (given LPS 5 mg/kg) and large dose LPS group (given LPS 10 mg/kg). Pancreas was explanted to detect the ultra-structural changes by TEM and the HSP70 expression by immunohistochemistry and Western blot. RESULTS: Rats given small doses of LPS showed swelling and loss of mitochondrial cristae of acinar cells and increased number of autophagic vacuoles in the cytoplasm of acinar cells. Rats given large doses of LPS showed swelling, vacuolization, and obvious myeloid changes of mitochondrial cristae of acinar cells, increased number of autophagic vacuoles in the cytoplasm of acinar cells. HSP70 expression was increased compared to the control group (P<0.05). CONCLUSION: Small doses of LPS may induce stronger expression of HSP70, promote autophagocytosis and ameliorate ultra-structural injuries.     

Role of leukocyte-derived pro-opiomelanocortin peptides in endotoxic shock.

The immune and neuroendocrine systems communicate and maintain homeostasis through various mechanisms, including the use of common signal and recognition molecules and the use of similar processes. This type of integrated network has profound effects on the onset and outcome of certain disease states, including endotoxic shock, in which a cascade of mediators influence the pathophysiologic responses. We have found that some of the common signal molecules shared between the immune and neuroendocrine systems are the peptide hormones adrenocorticotropin (ACTH) and endorphins (END). Our investigations have shown that these molecules are produced in vitro by cells of the immune system treated with various stimuli, including immunological stimuli such as bacterial lipopolysaccharide (LPS; endotoxin), virus infection (Newcastle virus; NDV), and the more classical neuroendocrine stimuli corticotropin-releasing hormone (CRH). We have proposed that the production of END by the peripheral immune system contributes to the pool of opioid peptides associated with the pathophysiology of endotoxic shock. Lymphocytes from LPS-sensitive C3HeB/FeJ mice but not LPS-resistant C3H/HeJ mice produce END and ACTH both in vitro and in vivo after treatment with LPS. Purification of the in vitro produced LPS-induced END from B-lymphocyte spleen cells followed by injections into both LPS-sensitive and -resistant mice elicits changes in body temperature and respiration rate. The spleen cells from the LPS-sensitive mice process ACTH and END differently depending on the stimulus for induction and the cell type in which the processing takes place. CRH or virus induce ACTH 1-39 and beta-END, whereas inductions with LPS yield major products of ACTH 1-22 to 1-26 and gamma-END, products that are for the most part unique to the immune system. We have shown that LPS induces a novel protease that functions optimally at pH 5 to cleave ACTH 1-39 into ACTH 1-22 to 1-26. This enzyme is present in LPS, but not mock or CRH-induced B cells from LPS-sensitive mice. The LPS-resistant mice did not possess this enzyme and therefore produced only the high-molecular-weight pro-opiomelanocortin (POMC)-like molecule. The inability to produce ACTH and END, presumably by their inability to process the precursor, may account, in part, for their lack of response to the LPS. The POMC peptides also may play an indirect role in orchestrating the pathophysiologic response, since both ACTH and END were shown to induce tumor necrosis factor (TNF). Our data strongly suggest that lymphocyte POMC peptides ACTH and END are important mediators in the overall response to endotoxin.     

Leukocyte response and hypoglycemia in superlethal endotoxic shock.

This laboratory has documented a progressively developing hypoglycemia associated with systemic hypotension, hepatosplanchnic pathology, and death in endotoxin-shocked dogs. Recent data documented accelerated uptake of glucose in blood following endotoxin, with certain components of the buffy coat responsible for the increased uptake. The present study utilizing the awake dog assayed a possible protective role of leukocytes against the lethal effects of endotoxin. Animals were divided into paired groups: saline controls (Group I) and endotoxin experimentals (Group II). Group II animals were injected intravenously with sublethal doses of E. coli endotoxin on 2 successive days (Days 1 and 2), LD100 on the third day, and 2 X LD100 on Day 4. The control group received equal volumes of saline on Days 1, 2, and 3, but on Day 4 received a superlethal dose of endotoxin identical to the experimental group. The awake dog became febrile and exhibited initial leukopenia with subsequent marked leukocytosis in response to endotoxin. Lethal hypoglycemia was not seen in animals demonstrating initial leukocytosis (zero time) on the day of superlethal endotoxin challenge, while animals with initial normal leukocyte counts died with low glucose concentrations (mean, 40 mg%). Results suggest that an initial leukocytosis and sustained gluconeogenic function are important factors in survivability to endotoxin shock.     

Resistance to endotoxic shock and reduced neutrophil migration in mice deficient for the Src-family kinases Hck and Fgr

Signal transduction through the leukocyte integrins is required for the processes of firm adhesion, activation, and chemotaxis of neutrophils during inflammatory reactions. Neutrophils isolated from knockout mice that are deficient in the expression of p59/61^hck (Hck) and p58^c-fgr (Fgr), members of the Src-family of protein tyrosine kinases, have been shown to be defective in adhesion mediated activation. Cells from these animals have impaired induction of respiratory burst and granule secretion following plating on surfaces that crosslink β₂ and β₃ integrins. To determine if the defective function of hck^−/−fgr^−/− neutrophils observed in vitro also results in impaired inflammatory responses in vivo, we examined responses induced by lipopolysaccharide (LPS) injection in these animals. The hck^−/−fgr^−/− mice showed marked resistance to the lethal effects of high-dose LPS injection despite the fact that high levels of serum tumor necrosis factor α and interleukin 1α were detected. Serum chemistry analysis revealed a marked reduction in liver and renal damage in mutant mice treated with LPS, whereas blood counts showed a marked neutrophilia that was not seen in wild-type animals. Direct examination of liver sections from mutant mice revealed reduced neutrophil migration into the tissue. These data demonstrate that defective integrin signaling in neutrophils, caused by loss of Hck and Fgr tyrosine kinase activity, results in impaired inflammation-dependent tissue injury in vivo.     

Suppressor alphabeta T lymphocytes control innate resistance to endotoxic shock

A considerable amount of research has focused on elucidating the mechanisms by which cytokines synthesized by cells of the innate immune system participate in the life-threatening multiple-organ failure of endotoxic shock. We show here that alphabeta T cells, which are archetypes of the adaptive cellular immune response, suppress the proinflammatory cascade triggered during the early stages of lipopolysaccharide (LPS)-induced endotoxemia. The absence of alphabeta T cells led to the fulminant death of LPS-challenged mice, coinciding with a massive release of the proinflammatory cytokines tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma and a marked reduction in the synthesis of the immunosuppressive cytokine transforming growth factor (TGF)-beta. Cytotoxic T lymphocyte antigen (CTLA)-positive alphabeta T cells emerging shortly after LPS challenge appear to control TGF-beta synthesis. The neutralization of either TGF-beta or CTLA4 resulted in similar increases in IFN-gamma and TNF-alpha serum concentrations in LPS-challenged mice. These observations suggest that suppressor alphabeta T lymphocytes protect against the proinflammatory cascade unleashed during the innate stages of endotoxemia.     

Benoxaprofen attenuation of lethal canine endotoxic shock

Our prior work demonstrated in a canine endotoxic shock model (LD100) that the cyclooxygenase inhibitor ibuprofen given 60 minutes after endotoxin administration could improve hemodynamics but not survival over control animals. The present study was designed to examine the effect of benoxaprofen, a dual lipoxygenase and cyclooxygenase inhibitor, in the same canine endotoxic model (LD100) and compare it to ibuprofen treatment. After thiopental anesthesia (25 mg/kg IV), animals were instrumented to measure various cardiovascular parameters. Endotoxic shock was induced by injecting Escherichia coli (0111:B4) endotoxin (1 mg/kg IV). Benoxaprofen (10 mg/kg IV; N = 13), ibuprofen (12.5 mg/kg; N = 6), or saline (N = 12) was injected 60 minutes after endotoxin administration. During the treatment period, both benoxaprofen and ibuprofen increased mean arterial pressure, heart rate, and vascular resistance to the same degree over the control animals. Benoxaprofen did increase dP/dtmax while ibuprofen did not. Twenty-four-hour survival was 0% for the control animals (N = 12), 0% for the ibuprofen group (N = 6), and 61.5% for the benoxaprofen group (N = 13). In an additional set of experiments, benoxaprofen (N = 8) was given 120 minutes after endotoxin administration and demonstrated similar improvements in hemodynamics and survival. These data demonstrate that benoxaprofen could improve survival in an otherwise lethal endotoxic model and suggest that the products of the lipoxygenase pathway may contribute to the lethality of an LD100 endotoxic shock model.     

Failure of glucagon to induce hepatic phosphoenolpyruvate carboxykinase in endotoxic shock

We have determined that one reason for diminished PEPCK activity during endotoxemia is the inhibition of glucocorticoid action in hepatic cells. Since glucocorticoid and glucagon hormones act cooperatively to regulate the expression of PEPCK mRNA, we examined whether endotoxin also inhibits the action of glucagon to induce this enzyme. Treated mice were injected intraperitoneally with endotoxin and glucose after a 24 hr fast and given ad libitum access to food and water. Control mice received the same amount of glucose and access to food and water. All mice were given intravenous injections of glucagon for 3 consecutive hours before euthanasia. Blood was analyzed for glucose concentrations, and the liver was assayed for PEPCK activity. Refeeding control mice after a 24 hr fast increased plasma glucose levels to 173 +/- 14 mg/dL and decreased PEPCK activity to 20.6 +/- 2.0 units/mg liver. Subsequent administration of exogenous glucagon further increased plasma glucose to 224 +/- 17 mg/dL and hepatic PEPCK to 31.4 +/- 1.4 units/mg liver. Refeeding endotoxin-treated mice after a 24 hr fast slightly increased plasma glucose levels to 75 +/- 4 mg/dL but had no effect on PEPCK activity. Subsequent glucagon administration had no effect on plasma glucose levels (75 +/- 1.0 mg/dL) or hepatic PEPCK activities (18.8 +/- 5.0 units/mg liver). Therefore, glucagon action to increase liver PEPCK activity and plasma glucose levels was inhibited in endotoxin-treated mice.     

Male gender predisposes to development of endotoxic shock in the rat

OBJECTIVE: After intravenous (i.v.) injection of lipopolysaccharide (LPS) macrophages release nitric oxide (NO) due to the expression of the inducible NO synthase (iNOS). After LPS NO is abundantly produced also in the cardiovascular system and may contribute to the development of hypotension and shock. Since the immune response, the synthesis of NO and the regulation of blood pressure (BP) differ between males and females, in the present study the effect of LPS on BP, renal function, the plasma and urinary concentration of the metabolites of NO as well as the splenic and aortic expression of the iNOS gene were compared between male and female rats. METHODS: BP and renal function were measured in anesthetized rats following the i.v. injection of LPS (E. coli, 4 mg/kg). The NO2- and NO3- (metabolites of NO=NOx) concentration was measured by the Griess reaction. The iNOS gene expression was studied by RT-PCR. RESULTS: Four hours after LPS, BP of males (n=9) was reduced by 63+/-12 mmHg versus 10+/-4 in females (n=7, P<0.005). Aminoguanidine, a selective inhibitor of iNOS, prevented the reduction of BP in males. The plasma concentration of NOx (P(NOx)), microM) was lower in hypotensive males (128+/-20) than in normotensive females (235+/-29, P<0.005). Males also exhibited lower urinary NOx excretion (U(NOx)V) after LPS (P<0.001 vs. females). Prior castration of males provided protection against hypotension (fall of BP: -4+/-4 mmHg, n=6, P<0.02 versus males) and resulted in higher P(NOx) as well as U(NOx)V (both P<0.001 versus males and not different from females). Prior ovariectomy (n=5) had no influence on the hemodynamic and NOx response to LPS. Male rats displayed enhanced aortic iNOS/beta-actin ratio relative to females after LPS (n=3 in each group, P<0.05). CONCLUSIONS: (1) Male gender may sensitize to LPS-induced shock and (2) sensitivity of males to endotoxin is associated with an attenuated, not exaggerated total rate of NO synthesis.     

Uptake of radiolabelled alpha-linolenic, arachidonic and oleic acid in tissues of normal and essential fatty acid-deficient rats--an autoradiographic study

Rats fed diets with a low (0.3% of total energy) or normal (3%) essential fatty acid (EFA) content were injected intravenously with a single dose of 14C-labelled alpha-linolenic, arachidonic or oleic acid and the distribution of radioactivity in the tissues was compared 5 min, 1 h and 18 h after the application using whole-body autoradiography. The highest levels of labelling with all fatty acids were observed in the liver, brown fat and adrenal cortex. Specific for arachidonic acid was a high and consistent concentration of radioactivity in the myocardium at all times and for oleic acid in the white fat after 18 h. The tissue uptake of arachidonic acid was similar in both dietary groups, whereas a higher accumulation of alpha-linolenic was seen in most of the tissues in the low EFA group after 18 h compared to the normal EFA group. The uptake of oleic acid was higher in some tissues in the low EFA than in the normal EFA group after 1 h, but after 18 h these differences had disappeared almost completely.     

Administration of the calcium agonist BAY K 8644 in endotoxic shock.

Septic shock is characterized by a decreased vascular tone and a depressed myocardial function. An impairment in cellular calcium availability has been incriminated in both phenomenons. The effects of BAY K 8644, a dihydropyridine-derivative agent acting as a slow calcium-channel activator, were studied and compared to those of norepinephrine (NE) on an experimental endotoxin shock model in the dog. Thirty minutes after intravenous administration of E. coli endotoxin (3 mg/kg), fluid therapy with normal saline was initiated to restore and maintain pulmonary artery capillary wedge pressure at baseline level. In the first part of the study (8 dogs), the effects of increasing doses of 1, 2, 4, and 8 mcg/kg/min of BAY K 8644 were evaluated. BAY K 8644 administration resulted in an increase in mean arterial pressure (MAP) (65 +/- 16 to 116 +/- 24 mmHg, P less than 0.001), systemic vascular resistance (SVR) (1,451 +/- 526 to 2,632 +/- 804 dynes.sec.cm-5, P less than 0.01), and left ventricular stroke work (LVSW) (0.08 +/- 0.04 to 0.16 +/- 0.07 g.m/kg, P less than 0.05), without change in mean pulmonary artery pressure, cardiac output, O2 transport, or O2 consumption (VO2). In the second part of the study (10 dogs), BAY K 8644 and NE were administered in a randomized order at increasing doses to achieve an identical increase in MAP. For a 20 mmHg increase in MAP, BAY K 8644 increased more SVR than NE (1,284 +/- 299 to 1,717 +/- 551 vs. 1,415 +/- 312 to 1,470 +/- 603 dynes.sec.cm-5, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)