Effects of RU486 on Glu-6-pase gene expression in hemorrhage and resuscitation.

To assess the role of glucocorticoid receptor antagonists and mediators released by Kupffer cells and other resident macrophages, we have used RU486 and gadolinium chloride to prevent the induction of glucose-6-phosphatase (Glu-6-Pase) gene expression in the liver following hemorrhagic shock (HS) and lactated Ringer's (LR) solution resuscitation. HS was induced in fasted, anesthetized, and cannulated rats by rapid phlebotomy to a mean arterial pressure of 40 mmHg and maintained for 30 min by withdrawal or infusion of blood. The LR solution group underwent induction and maintenance of HS for 30 min followed by LR resuscitation. Rats were injected with gadolinium chloride (7 mg/kg) to inhibit the phagocytic function of Kupffer cells, and with glucocorticoid receptor antagonist RU486 (20 mg/kg) prior to induction of HS. Arterial blood samples were obtained and livers were freeze clamped in liquid nitrogen and stored at -70 degrees C for subsequent analysis. Northern blot analysis indicated that Glu-6-Pase mRNA abundance increased 2-fold in HS rats and a further 2-fold with resuscitation. Gadolinium chloride administration had no significant effect on Glu-6-Pase mRNA abundance in HS or in LR solution. In contrast, RU486 pre-treatment reduced Glu-6-Pase mRNA by about one half in HS rats compared with control and that in LR solution to normal. This was associated with a normalization of Glu-6-Pase activity and plasma glucose toward pre-hemorrhage levels. These results suggest that gadolinium chloride inhibition of macrophage factor release has no effect on the induction of Glu-6-Pase mRNA during HS or in LR solution resuscitation. On the other hand, the suppression of Glu-6-Pase mRNA by RU486 suggests that glucocorticoids are responsible for the induction of the mRNA in HS and during LR resuscitation. KEYWORDS-Shock, hyperglycemia, corticosterone, gadolinium chloride, diltiazem, animal model, mRNA

Regulation of liver and kidney glucose-6-phosphatase gene expression in hemorrhage and resuscitation.

UNLABELLED: The authors have recently demonstrated that increased gene expression of glucose-6-phosphatase (Glu-6-Pase) in hemorrhagic hypotension (HH) and following lactated Ringer's resuscitation (LR) is associated with a decrease in insulin and an increase in corticosterone concentrations. OBJECTIVE: To evaluate the in-vivo role of hormones the authors used insulin (IN), phentolamine and propranolol (PP) as an adrenergic blocker, and cyclic somatostatin (CS) as a glucagon blocker to prevent the induction of Glu-6-Pase gene expression in liver and kidney following HH and LR. METHODS: Hemorrhage was induced in fasted anesthetized rats, and the reduction of blood pressure to 40 mm Hg for a duration of 30 minutes was accomplished by withdrawal or infusion of shed blood. The resuscitated group underwent hemorrhage followed by fluid resuscitation with lactated Ringer's solution. RESULTS: Neither PP nor CS treatment could block the induction of Glu-6-Pase messenger ribonucleic acid (mRNA) following either HH or LR. However, the administration of IN significantly prevented the increase of Glu-6-Pase mRNA level and activity in both liver and kidney following HH and LR. This was associated with a normalization of plasma glucose, corticosterone, and glucagon levels and glucose-6-phosphate concentrations in liver and kidney toward prehemorrhage levels. CONCLUSIONS: These results indicate that in-vivo treatment with insulin during hemorrhagic hypotension and resuscitation is capable of preventing the increase in Glu-6-Pase gene expression in liver and kidney responsible for the observed hyperglycemia.

Alterations in glucose-6-phosphatase gene expression in sepsis.

BACKGROUND: The influence of sepsis on the expression and activity of hepatic glucose-6-phosphatase (Glu-6-Pase) was examined during the early hyperglycemic phase and the later hypoglycemic phase. METHODS: Sepsis was induced in anesthetized, fasted rats by cecal ligation and puncture, and liver samples were taken at 0, 0.5, 1, 1.5, and 20 hours after cecal ligation and puncture. RESULTS: The mRNA abundance of hepatic Glu-6-Pase increased fourfold at 0.5 hours over healthy control values, two-fold after 1 hour, and returned to normal after 1.5 hours. This finding was followed by a corresponding increase in Glu-6-Pase activity and was coincident with increased plasma glucose levels and decreased liver glucose-6-phosphate (Glu-6-P) at 0.5 and 1 hours. Plasma insulin and glucagon levels remained unchanged during this period, whereas corticosterone levels increased 2.5-fold over control values. At 20 hours cecal ligation and puncture, plasma glucose levels returned to normal, coincident with a 90% reduction in Glu-6-Pase mRNA abundance. Glu-6-Pase activity and Glu-6-P concentration returned to normal levels, while insulin, glucagon, and corticosterone levels increased significantly, i.e., 40-fold, 6.5-fold, and 6-fold, respectively. CONCLUSION: The initial rise and subsequent decline in blood glucose correlate very well with a corticosterone-dependent induction of hepatic Glu-6-Pase, mRNA, and protein, followed by an insulin-dependent suppression of its expression.

Alterations in tissue glucose uptake during the hyperglycemic and hypoglycemic phases of sepsis.

The purpose of the present study was to characterize the alterations in tissue glucose uptake during the hyperglycemic, euglycemic, and hypoglycemic phases of peritonitis. Rats had vascular catheters implanted, and sepsis was induced by cecal ligation and puncture. Rates of whole-body glucose appearance (Ra), disappearance (Rd), and metabolic clearance (MCR) were determined by the constant infusion of 3H-glucose, and in vivo glucose uptake (Rg) by individual tissues was assessed by using 14C-deoxyglucose. During the hyperglycemic phase of sepsis (2 h), glucose Ra and Rd were increased, but glucose MCR was unaltered. In contrast, during the euglycemic phase (6 h), the sepsis-induced increase in glucose Ra and Rd was associated with an elevation in the MCR. Finally, during the hypoglycemic phase (24 h), sepsis decreased glucose Ra and Rd and the glucose MCR. The sepsis-induced changes in Rg for skeletal muscle and adipose tissue mimic those seen for the whole body at each time point. Rg for skin and intestine was elevated at 2 h and 6 h but was not different from control values at 24 h. In contrast, the Rg for liver, lung, and spleen was increased at all 3 time points. In a second study, there was no difference in Rg for any tissue between 2-h septic rats and control animals in which blood glucose and insulin levels were artificially elevated to the same degree. In a third study, the prevailing glucose and insulin levels in control animals were decreased, by injection of the gluconeogenic inhibitor 3-mercaptopicolinic acid, to levels seen in 24-h septic rats. There was no difference in the Rg for muscle and adipose tissue between 24-h septic rats and hypoglycemic insulinopenic control animals. However, the Rg for liver, lung, and spleen remained elevated in 24-h septic rats, compared with hypoglycemic insulinopenic control values. These data indicate that the increased tissue glucose uptake observed during the early phase of sepsis is a consequence of concomitant changes in plasma glucose and insulin. In contrast, during the euglycemic and hypoglycemic stages of sepsis, glucose uptake in macrophage-rich tissues remains elevated and is independent of changes in glucose and insulin.

Endotoxin-induced alterations in hepatic glucose-6-phosphatase activity and gene expression.

The mechanisms responsible for the glycemic changes associated with endotoxic shock are not fully understood, but are known to involve the ability of the liver to produce glucose. The purpose of the present study was to determine whether endotoxin (LPS) influences the expression and activity of glucose-6-phosphatase (Glu-6-Pase) during the early hyperglycemic phase and the later hypoglycemic phase. Rats were injected with a relatively large dose of LPS (20 mg/kg) or saline (control), and sacrificed at 1 or 5 h post-injection. Both the plasma glucose concentration and glucose production were elevated 1 h post-LPS (2-fold) and both decreased at 5 h postinjection (50%). Compared to time-matched control values, hepatic glucose-6-phosphate and fructose-6-phosphate levels were significantly decreased at both 1 and 5 h. Hepatic Glu-6-Pase activity and mRNA levels were moderately increased, 1 h after injection of LPS. At 5 h, an 88% decrease in mRNA abundance for Glu-6-Pase was associated with a 30% decrease in activity of this enzyme. Plasma insulin concentrations were not different 1 h after LPS and were elevated 2-fold from control values at 5 h. Circulating levels of glucagon and corticosterone were elevated at both time points following LPS. Our data indicate that the LPS-induced hypoglycemia and reduction in hepatic glucose production were accompanied by a depression in Glu-6-Pase activity and gene expression.

Alterations in hepatic gluconeogenesis, prostanoid, and intracellular calcium during sepsis.

OBJECTIVE: The metabolic alterations observed during sepsis may be associated with changes in local concentrations of intracellular calcium (Ca2+) and prostanoid synthesis in the liver. The authors studied hepatocyte intracellular Ca2+ and the release of glucose and prostanoid in an in-vivo murine liver perfusion model. METHODS: Sepsis was induced in anesthetized, fasted rats by cecal ligation and puncture (CLP, n = 42). Hepatic glucose release was studied in control (n = 10) and CLP (n = 10) groups using a non-recirculating liver perfusion model with and without lactate as gluconeogenic substrate. Hepatocyte intracellular Ca2+ (n = 11) was measured using the selective indicator Fura-2 under basal and epinephrine (10(-5) M) stimulated conditions. 6-Keto-prostaglandin F1alpha (6-Keto) and thromboxane B2 (TxB2) were determined from liver perfusate by radioimmunassay (n = 11). Data were analyzed using t-tests and repeated-measures ANOVA. RESULTS: Plasma glucose was significantly lower in CLP groups compared with controls (74.9+/-6.6 vs 115.7+/-4.6 mg/dL, p < 0.05). Plasma lactate was significantly higher in CLP vs controls (3.7+/-0.4 vs 1.4+/-0.1 mM, p < 0.05). Glucose release in isolated perfused livers was significantly lower in CLP vs controls (8.5 vs 16+/-1.2 microM/g/hr, p < 0.001). With the addition of lactate + pyruvate to the perfusate, glucose output in CLP livers was significantly lower following 5 (9.9+/-0.7 vs 17.7+/-1.1 microM/g/hr, p < 0.05) and 10 (11.9+/-1.2 vs 20.6+/-1.3 microM/g/hr, p < 0.001) minutes of perfusion. The basal level of intracellular calcium ([Ca2+]i) in CLP rats (460.1+/-91.6 nM) was significantly higher than in control rats (196.3+/-35.5 nM) (p < 0.05). A significant increase (p < 0.05) in [Ca2+]i occurred after the addition of epinephrine in hepatocytes in control (196.3+/-35.5 vs 331.8+/-41.4 nM) but not CLP (460.1+/-91.6 vs 489.4+/-105 nM) rats. 6-Keto was significantly lower in CLP compared with controls at 30 minutes (25.7+/-3.9 vs 33.4+/-5.5 pg/mL, p < 0.05), whereas TxB2 was not significantly altered (52.1+/-34.7 vs 87.5+/-43.2 pg/mL). CONCLUSION: These results demonstrate that CLP sepsis is associated with an increase in hepatocyte intracellular free Ca2+ concentration along with attenuation of hormone-mediated Ca2+ mobilization and hepatic gluconeogenesis.

Alterations in hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and glucose-6-phosphatase gene expression after hemorrhagic hypotension and resuscitation.

The mRNA abundance of several hepatic glycolytic and gluconeogenic enzymes and blood hormone concentrations were determined in hemorrhagic hypotension-induced rats before and after resuscitation with lactated Ringer's. Northern blot analysis of total liver RNA after 30 min of hemorrhage showed control values for phospho-enolpyruvate carboxykinase and fructose-1,6-bisphosphatase mRNA, but significantly lower values for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF2K/FBPase) as well as 2.5-fold increases in glucose-6-phosphatase (Glu-6-Pase) mRNA. The latter finding is in agreement with the greatly reduced intracellular levels of fructose-6-phosphate and glucose-6-phosphate, and the results are consistent with a rapid activation of hepatic gluconeogenesis by the concomitant decrease in 6PF2K/FBPase and increase in Glu-6-Pase. Blood insulin levels were decreased during hemorrhage and with resuscitation, whereas glucocorticoids were increased 1.5-fold in both cases. Glucagon was unchanged during hemorrhage, but was reduced with resuscitation. Lactated Ringer's resuscitation seemed to affect 6PF2K/FBPase only, which was restored to, and even exceeded, control values. In contrast, Glu-6-Pase mRNA was increased to fourfold control values. The increase in Glu-6-Pase and the decrease in 6PF2K/FBPase mRNA is probably at the level of altered transcriptional rates, because insulin, which plays a dominant role in the regulation of these genes, was decreased during hemorrhage. It remains to be determined what factors are causing further induction of Glu-6-Pase gene after lactated Ringer's resuscitation when hepatic glucose metabolism seems to have reverted to the glycolytic mode.

Renal gluconeogenesis and blood flow during endotoxic shock.

OBJECTIVES: To determine whether endotoxic shock decreases the renal gluconeogenic capacity and the renal artery blood flow. METHODS: An in-vivo, murine, nonrecirculating kidney perfusion model was studied in a trauma research laboratory. Each of 12 fasted, male Holtzman rats (shock n = 6, control n = 6) was injected with 1 mL of normal saline or endotoxin (20 mg/kg). Five hours after the injection, all the rats were anesthetized and blood samples were obtained for the determination of the plasma glucose. Right renal artery blood flow was measured by an ultrasonic small-animal flow meter. The kidney was then perfused via the renal artery with 37 degrees C, oxygenated, glucose-free Krebs-Henseleit solution in the presence of 100 mumol of phloridzin to inhibit the cellular uptake of glucose. Renal glucose production was determined by measuring glucose in both renal vein effluent and urine. After 30 minutes of equilibration, 5 mmol of lactate and 0.5 mmol of pyruvate were added to the perfusate as a gluconeogenic substrate. Renal vein and ureteral effluent samples were collected after 5, 10, and 15 minutes. RESULTS: The endotoxic shock group showed hypoglycemia (p < 0.05) as well as a decrease in renal artery blood flow (p < 0.05). Gluconeogenic stimulation was demonstrable in both the control and the endotoxic shock groups after 15 minutes of perfusion with substrate (p < 0.05). However, renal gluconeogenesis was significantly attenuated in the endotoxic shock group compared with the control. CONCLUSIONS: Renal glucose production in response to a gluconeogenic substrate is significantly attenuated during the hypoglycemic phase of endotoxic shock. Endotoxic shock also causes a significant decrease of renal artery blood flow.

Effective treatment for acute alkali injury to the esophagus using weak-acid neutralization therapy: an ex-vivo study.

OBJECTIVE: 1) To evaluate whether neutralization therapy with weak acid is effective in reducing observed histopathologic esophageal tissue injury secondary to liquid alkali, 2) to quantify the temperature change of the neutralizing agent, and 3) to determine the effect of interval to therapy on injury severity. METHODS: Harvested Sprague-Dawley rat esophagi were catheterized and placed in an oxygenated saline bath (37 degrees C) for 60 minutes and then fixed in 10% formalin. Nine groups (n = 10) were perfused with 50% sodium hydroxide (NaOH). Six of the groups were treated by neutralization with cooled orange juice (OJ) or cola that was maintained between 2 degrees C and 4 degrees C. This was performed at 0, 5, or 30 minutes after injury. In addition, two positive control groups were exposed to OJ or cola at time 0 and were not exposed to strong alkali. A third control group was exposed to strong alkali but was not administered any subsequent treatment. The temperature of the neutralizing agent was recorded prior to instillation and after exiting the esophagus. Blinded pathologic scoring of 0 (no injury) to 3 (severe) was recorded performed for six histopathologic categories: epithelial cell viability, cornified epithelial cell differentiation, granular cell differentiation, epithelial cell nuclei, muscle cells, and muscle cell nuclei. Comparisons were made among treatment times using the Kruskal-Wallis test and linear trend analysis. RESULTS: For each histopathologic category and each treatment mode, the Kruskal-Wallis test showed significant differences between the groups (p < 0.002) over time. Trend analyses showed more severe injury with delayed neutralization therapy (p < 0.05) for each treatment mode and histopathologic category. CONCLUSION: Early neutralization therapy with OJ or cola reduces acute esophageal alkali injury. Additional in-vivo study is needed before neutralization therapy is adopted for clinical use.

Histopathologic evaluation of the therapeutic efficacy of water and milk dilution for esophageal acid injury.

OBJECTIVE: To determine whether acid-induced injury to the esophagus is decreased by early dilutional therapy with water or milk. METHODS: A controlled in-vitro animal model for acid injury to the esophagus was carried out using esophagi harvested from 70 Sprague-Dawley rats of both sexes and weighing 250-350 g. One control and six experimental groups each containing ten esophagi were instilled with 1 mL of 0.5 normal solution of hydrogen chloride (N HCl). Dilution with water or milk was performed at 0, 5, or 30 minutes postinjury in the experimental groups. No dilution was performed with the control group. Specimens were maintained in an oxygenated saline bath for a 60-minute experimental period and then fixed in 10% formalin for histologic evaluation. Injury severity was rated by blinded histopathologic examination using scores of 0 (no injury), 1 (minor), 2 (moderate), and 3 (severe) for the histopathologic categories: cornified epithelial cells (CEs), granular cells (GCs), granular cell nuclei (GNs), and basal cells (BCs). Red blood cells were scored as positive or negative for lysis. RESULTS: The controls showed the most severe outcomes. Significant differences in injury occurred for all time periods and histopathologic categories, except for the GN/water and BC/milk histopathologic category/treatment groups. However, a linear trend analysis was significant for all histopathologic categories except BC. These analyses support decreased injury in the earlier treated groups. Injury severity was highest in the most superficial cell layer (CE). CONCLUSIONS: Emergency therapy with water or milk reduces acute acid injury to the esophagus. Earlier treatment is associated with decreased injury severity. This research supports the use of dilutional therapy with water or milk for acute acid injury to the esophagus.

Alterations in hepatic production and peripheral clearance of IGF-I after endotoxin.

Lipopolysaccharide (LPS) produces a rapid and sustained reduction in the circulating concentration of insulin-like growth factor I (IGF-I), which may be responsible, in part, for the alterations in protein metabolism observed in these animals. The purpose of the present study was to determine whether this drop was due to a decreased hepatic production of IGF-I and/or an increased clearance of the peptide from the blood. Four hours after intravenous injection of LPS the plasma IGF-I concentration was decreased 50%. IGF-I release by in situ perfused livers from control rats was constant throughout the 60-min perfusion period and averaged 111 +/- 3 ng/min. In contrast, hepatic IGF-I output was decreased 46% by in vivo LPS. In contrast, livers from LPS-injected rats released more IGF binding proteins-1, -2 and -4 than did control livers. Hepatic cell isolation indicated that LPS decreased the IGF-I content in Kupffer and parenchymal cells, but not endothelial cells, by approximately 45%. Pharmacokinetic analysis of blood 125I-IGF-I decay curves indicated that the half-life for whole body clearance of 125I-IGF-I from the circulation was not altered by LPS. However, LPS increased 125I-IGF-I uptake by spleen, liver, lung, and kidney while decreasing uptake by the pancreas and gastrointestinal tract. These results indicate that the LPS-induced decrease in blood IGF-I concentration is primarily due to a reduction in hepatic production, not a change in whole body peptide clearance, and that a decreased production by both parenchymal and Kupffer cells contributes to this alteration.

Glucose-6-phosphatase gene expression and activity are modulated in hemorrhagic shock: evidence for a new heat-sensitive activator.

Decreased hepatic fructose 2,6-bisphosphate levels were observed in the early phase of hemorrhagic shock. The lower sugar bisphosphatae level was a result of increased phosphoenolpyruvate levels and decreased glucose-6-phosphate and fructose-6-phosphate levels. The decreased glucose-6-phosphate levels correlated with increased activity of liver glucose-6-phosphatase and a concomitant 2.5-fold increase in glucose-6-phosphatase mRNA abundance. In addition, protein-free filtrate from hemorrhagic shock rats, but not from control rats, increased glucose-6-phosphatase activity. However, when control and hemorrhagic shock protein-free filtrates were heated, they both increased the glucose-6-phosphatase activity of the respective microsomes to the same extent. It is concluded that the early hyperglycemic phase of hemorrhagic shock is due to enhanced glucose-6-phosphatase gene expression and activity and the generation of a heat sensitive activator of the enzyme.

Therapeutic effects of water and milk for acute alkali injury of the esophagus.

STUDY BACKGROUND: Alkali ingestions cause progressive and devastating injury to the esophagus by liquefaction necrosis. However, the therapeutic efficacy of water or milk dilution for alkali-induced esophageal injury has not been determined. This study used our previously reported model of alkali-induced esophageal injury to evaluate the effectiveness of water and milk dilution. HYPOTHESIS: Early dilution with water or milk is efficacious in decreasing esophageal damage from alkali exposure. METHODS: The esopgagi of 75 Sprague-Dawley rats were harvested, and each end was cannulated with a 20-gauge catheter. Specimens were maintained in an oxygenated saline solution (at 37 degrees C) during a 60-minute experimental period and then fixed immediately in 10% Formalin solution for histologic examination. Esophagi from six experimental groups (total of 60) were perfused with 50% NaOH solution at time 0. Water or milk dilution was performed immediately at 0 minutes, 5 minutes after injury, and 30 minutes after injury. Blinded pathologic examination was performed using a score of 0 (no injury), 1 (minimal), 2 (moderate), or 3 (severe) for the following six histologic categories: epithelial viability, cornified epithelial cell differentiation, granular cell differentiation, epithelial cell nuclei, muscle cells, and muscle cell nuclei. RESULTS: Positive and negative controls showed expected outcomes. Significant progressions of injury over time were seen for every histologic category for both water and milk dilution. The injury scores for the milk-treated group at 0 minutes were less than or equal to the injury score for the water-treated group for all categories. However, these differences were significant only for the cornified epithelial cells. CONCLUSION: Early dilution therapy with water or milk reduces acute alkali injury of the esophagus and supports use of these forms of emergency treatment.

Isolation of a cDNA for the catalytic subunit of rat liver glucose-6-phosphatase: regulation of gene expression in FAO hepatoma cells by insulin, dexamethasone and cAMP.

cDNA clones coding for the catalytic subunit of rat liver glucose-6-phosphatase (EC 3.1.3.9) were isolated from a rat liver cDNA library in lambda gt11 phage. The sequence of the cDNA and the amino acid sequence derived from it were greater than 90% identical to the corresponding sequences for the mouse and human forms of liver glucose-6-phosphatase. Northern blot analysis of RNA from FAO hepatoma cells revealed that dexamethasone induced the glucose-6-phosphatase mRNA while insulin suppressed its expression. When both hormones were added together insulin completely suppressed the effect of glucocorticoid. cAMP addition alone decreased the abundance of glucose-6-phosphatase mRNA. The results demonstrate multihormonal regulation of gene expression of hepatic glucose-6-phosphatase and support a dominant role for insulin.

Diltiazem preserves hepatic gluconeogenesis following hemorrhagic shock.

Prolonged hemorrhagic shock is characterized by the progression from hyperglycemia to hypoglycemia and failure to respond to standard methods of resuscitation. Previous studies have shown that the transition to irreversible shock is accompanied by attenuation of hepatic gluconeogenic capacity and a rising level of intracellular calcium. Additionally, it has been observed that diltiazem improves survival following prolonged hemorrhagic shock in rats. We examined the effect of resuscitation containing diltiazem upon hepatic gluconeogenesis during early and late phases of hemorrhagic shock in a rat model. Fasted male Sprague-Dawley rats (250-350 g) were rapidly bled to a mean arterial pressure of 40 mm Hg for a period of 30 minutes (group A) or 120 minutes (group B). At the end of the hemorrhagic shock period, rats were randomized to resuscitation utilizing lactated Ringer's (LR) solution, or LR+diltiazem (DZ, 1.2 mg/kg). Following resuscitation, rats underwent laparotomy and in situ liver perfusion with an oxygenated 37 degrees C glucose-free Krebs solution via the portal vein. After equilibration, 5 mmol/L lactate and 0.5 mmol/L pyruvate were added to the perfusate as substrate and effluent samples collected. Serum glucose concentration and portal venous flow did not differ significantly between DZ and LR groups throughout the study periods. In group A, hepatic glucose production was significantly elevated in DZ animals when compared with controls (p < 0.05). A similar significant improvement in gluconeogenesis was observed following 120 minutes of hemorrhagic shock in group B (p < 0.05). Additionally, treated rats (DZ, both groups A and B) demonstrated improved gluconeogenic response to substrate when compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in renal gluconeogenesis and blood flow during hemorrhagic shock.

The role of renal gluconeogenesis following hemorrhagic shock was studied. Hemorrhagic shock was induced in fasted, anesthesized rats by reduction of blood pressure to 40 mm Hg for either 30 or 150 min. Plasma samples were obtained for plasma glucose determination, and right renal arterial blood flow was determined with the help of an ultrasonic transit time blood flowmeter in hemorrhagic shock and control rats. The kidney was perfused via the renal artery with 37 degrees C oxygenated, glucose-free Krebs-Henseleit solution in the presence of 100 microM phloridzin to inhibit the cellular reuptake of glucose. Renal glucose production was determined by measuring glucose in both renal vein effluent and urine. After 30 min of equilibration, 5 mM lactate and 0.5 mM pyruvate were added to the perfusate as a gluconeogenic substrate, and effluent samples were collected after 5, 10, and 15 min. Moderate hyperglycemia was observed in vivo following 30 min of hemorrhagic shock, and significant hypoglycemia (P < .05) was observed following 150 min of hemorrhagic shock. Renal arterial flow was significantly decreased at 30 min (P < .05) and 150 min (P < .05) of hemorrhagic shock. Renal glucose production with and without substrate after 30 min of hemorrhagic shock was similar compared to control. Renal glucose production after 150 min of hemorrhagic shock was significantly decreased (P < .05) compared to control.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of diltiazem on altered glucose regulation during endotoxic shock.

Endotoxic shock is associated with profound metabolic alterations including hypoglycemia and hyperlactiacidemia. We studied hepatic glucose production and the role of diltiazem in affecting these metabolic alterations in a rat model of endotoxic shock. Fasted rats were intravenously injected with saline, endotoxin (20 mg/kg), endotoxin (20 mg/kg) plus diltiazem (1.2 mg/kg), or saline plus diltiazem. Five hours after the injections, the rats were anesthetized and blood samples were obtained for plasma glucose and lactate. The livers of all rats were then perfused in situ with an oxygenated 37 degrees C glucose-free Hanks' balanced salt solution at a rate of 30 ml/min via the portal vein and effluent was collected from the inferior vena cava. After 30 min equilibration, 5 mM lactate was added to the perfusate as a gluconeogenic substrate. Endotoxic groups exhibited hypoglycemia (64.5 +/- 12.7 mg/dl) and hyperlacticacidemia (4.3 +/- 0.63 mM). Diltiazem administration improved the hypoglycemia (96.9 +/- 9.1 mg/dl) and attenuated the hyperlacticacidemia (2.2 +/- 0.7 mM) in the endotoxic group. Gluconeogenic stimulation with lactate was demonstrated in both the control (18.1 +/- 2.3 vs 12.8 +/- 1.86 microns/g/hr, P < .05) and endotoxic (16.6 +/- 2.3 vs 9.8 +/- 1.1, P < .05) groups. However, stimulation in the endotoxic groups was significantly less compared with control. Gluconeogenic stimulation in the endotoxic group was unaffected after diltiazem administration. These data suggest that diltiazem treatment in endotoxic shock improves hypoglycemia and attenuates hyperlacticacidemia. These metabolic alterations are not associated with an improvement in substrate-specific stimulation of gluconeogenesis.

Effective treatment of acute alkali injury of the rat esophagus with early saline dilution therapy.

BACKGROUND: Controversy persists regarding the appropriate treatment of acute alkali injury to the esophagus. The current study establishes a controlled model of alkali esophageal injury and examines the efficacy of saline dilution therapy. STUDY HYPOTHESIS: Early saline dilution therapy effectively reduces esophageal injury resulting from acute alkali exposure. METHODS: The esophagi were harvested from 60 Sprague-Dawley rats. Each end was cannulated with a 20-gauge catheter. Specimens were maintained in an oxygen-perfused saline bath (37 C) during a 60-minute experimental period and then fixed immediately in 10% formalin solution for histologic examination. Three experimental groups (A, B, and C) were perfused with 50% NaOH solution at time zero. Treatment with saline perfusion was performed immediately in group A, five minutes after injury in group B, and 30 minutes after injury in group C. The positive control group D was perfused with saline at time zero. A negative control, group E, was perfused with 50% NaOH at time zero. This group did not receive subsequent treatment with saline. Pathologic examination was performed in a blinded fashion using a score of 0 to 3 (0, no injury; 1, minimal; 2, moderate; 3, severe) for seven histologic criteria: epithelial viability, extent of injury, cornified epithelial cell differentiation, granular cell differentiation, epithelial cell nuclei, muscle cells, and muscle cell nuclei. RESULTS: The positive control group demonstrated scores of zero. Nonparametric analysis showed a significant difference among treatment groups for each injury category. Trend analysis revealed a significant progression of injury for each category associated with time to treatment. Discriminant analysis indicated that the muscle cells category was the most useful category with which to distinguish injury among groups. CONCLUSION: In our model, saline lavage decreased objective evidence of esophageal injury after a severe alkaline exposure, and early therapy enhanced this beneficial effect.

Altered cellular calcium regulation and hepatic glucose production during hemorrhagic shock.

The relationship between intracellular Ca2+ and glucose production in the liver during early and late states of hemorrhagic shock was studied. Rats were anesthetized with intraperitoneal sodium pentobarbital and both femoral arteries and one femoral vein were cannulated. Rats were divided into two groups. One group was subjected to hemorrhagic shock by rapid withdrawal of blood to a mean arterial pressure of 40 mm Hg and maintained in shock for either 30 or 150 min. Rats in the control group were observed for the same time period. Hepatic glucose production was evaluated in both groups by a nonrecirculating liver perfusion model with and without lactate as a substrate. Intracellular free Ca2+ in hepatocytes was measured using the Ca2+ selective indicator Fura-2, under basal and epinephrine-stimulated conditions. Hyperglycemia and hyperlacticacidemia were observed in vivo at 30 min of hemorrhagic shock, whereas hypoglycemia and hyperlacticacidemia were observed at 150 min of shock. Hepatic glucose production in isolated perfused livers was significantly depressed at 30 min in animals subjected to shock (P less than 0.05). Lactate-induced glucose production was significantly attenuated at 30 and 150 min (P less than 0.05). Basal Ca2+, in isolated hepatocytes, at 30 and 150 min of hemorrhagic shock was significantly (P less than 0.05) higher than in controls. The hemorrhagic shock rat hepatocytes failed to evaluate intracellular free Ca2+ upon stimulation with 10(-5) M epinephrine. These results demonstrate that hemorrhagic shock is associated with an increase in hepatocyte intracellular Ca2+ concentration along with attenuation of hormone-mediated mobilization of calcium and substrate specific stimulation of hepatic glucose production.

Effect of alpha-adrenergic blockage on cellular Ca2+ during endotoxic shock.

The effect of alpha-adrenergic receptor antagonists, phentolamine, and prazosin on cytosolic Ca2+ concentration, [Ca2+]i, was studied in hepatocytes during endotoxic shock. Rats were given intravenous injections of endotoxin (20 mg/kg), phentolamine (3 mg/kg) plus endotoxin (20 mg/kg), or prazosin (5 mg/kg) plus endotoxin (20 mg/kg). They were sacrificed 5 hr later, at which time the endotoxin-injected rats showed signs of shock. Isolated hepatocytes were prepared and employed for the measurement of [Ca2+]i under basal and hormone-stimulated (1 and 10 microM epinephrine) conditions by means of Quin 2 fluorescence technique. The apparent basal level of [Ca2+]i in endotoxic rat hepatocytes (mean +/- SE: 482 +/- 31 nM) was significantly higher (P less than 0.05) than in phentolamine plus endotoxin (242 +/- 73) and prazosin plus endotoxin (240 +/- 43) groups. A significant increase in hepatocyte [Ca2+]i occurred with epinephrine in the phentolamine plus endotoxin and prazosin plus endotoxin groups, but not in the group receiving endotoxin alone. Endotoxic rats showed a mortality rate of 75%, whereas phentolamine plus endotoxin and prazosin plus endotoxin groups showed a mortality rate of 38% and 20% respectively. These data suggest that the protective effect of alpha-adrenergic receptor antagonists during endotoxic shock may be mediated, in part, by attenuating the entrance of Ca2+ into endotoxic liver cells.