Changes in vascular responsiveness to a thromboxane mimetic in endotoxin tolerance.

Our previous studies have demonstrated that peritoneal macrophages obtained from endotoxin-tolerant rats exhibit altered cellular activation by endotoxin, possibly involving changes in guanine nucleotide regulatory (G) protein-coupled signal transduction pathways. Endotoxin-tolerant rats also exhibit cross tolerance and altered hemodynamic responses to thromboxane (Tx)A2 mimetics, suggesting potential changes in vascular responsiveness. We tested the hypothesis that endotoxin tolerance results in vascular hyporesponsiveness to a TxA2 mimetic via alterations in the TxA2 receptor, G protein function, and/or second messenger production. Rats were rendered endotoxin tolerant by increasing sublethal consecutive doses of Salmonella enteritidis endotoxin (100 to 5000 micrograms/kg, i.p.) for 4 days. The animals were sacrificed 2 days after the final dose of endotoxin for removal of aortas. Contractile responses of aortic rings to U46619, a TxA2 agonist, were assessed in control and tolerant rats. The EC50 values for U46619 were 14.8 +/- 6.6 nM and 32.3 +/- 3.1 nM (n = 5-7), (P < 0.05) for control and tolerant rats, respectively. Crude membranes were prepared from aortas of control and tolerant rats, and binding of I-BOP TxA2/PGH2 receptor agonist, [1S-(1 alpha, 2 beta (5Z), 3 alpha (1E, 3S*), 4 alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7- oxabicyclo-[2.2.1]heptan-2-yl]-5-heptenoic acid (I-BOP), a TxA2 agonist, was assessed by Scatchard analysis. I-BOP binding to the TxA2 receptor was saturable and revealed a single class of TxA2 receptors for both groups. There was no significant difference in control (n = 7) compared with tolerant (n = 5) Kd values (2.1 +/- 0.2 vs. 2.4 +/- 0.9 nM, respectively), or Bmax (31 +/- 6 vs. 28 +/- 12 fmol/mg protein, respectively). To assess potential changes in G protein function, aortic membrane GTpase activity was determined. GTPase activity in tolerant membranes was significantly reduced (P < 0.05) compared with control membranes (309 +/- 23 (n = 5) vs. 440 +/- 32 (n = 7) pmol/mg/protein/min, respectively). However, U46619-stimulated phosphoinositide production was similar in vascular tissue from control and tolerant rats. These observations suggest that the decreased contractile response to TxA2 mimetics in endotoxin tolerance does not result from a change in receptor number, affinity of TxA2 receptors, or changes in phosphatidylinositol metabolism but is associated with decreased vascular G protein function.

Brain blood flow patterns after the development of congestive heart failure: effects of treadmill exercise.

OBJECTIVE: Congestive heart failure (CHF) is associated with left ventricular (LV) failure, neurohormonal system activation, and diminished exercise capacity. Although alterations in systemic vascular resistive properties have been recognized to occur with CHF, whether and to what degree perfusion abnormalities occur within the brain after the development of CHF remain poorly understood. Accordingly, the present study measured brain blood flow patterns in pigs after the development of pacing-induced CHF at rest and after treadmill-induced exercise. MEASUREMENTS AND MAIN RESULTS: Adult pigs (n = 6) were studied before and after the development of pacing-induced CHF (240 beats/min, 3 wks) at rest and with treadmill exercise (3 mph, 15 degrees incline, 10 mins). At rest, LV stroke volume was reduced nearly 45% with CHF compared with normal (20+/-2 vs. 36+/-3 mL; p<.05) and was associated with a more than four-fold increase in plasma catecholamines, renin activity, and endothelin concentration. At rest, global brain blood flow was reduced with CHF compared with the normal state (1.06+/-0.13 vs. 0.81+/-0.06 mL/min/g; p<.05). At rest, blood flow to the frontal lobe, cerebellum, and medullary regions was reduced by approximately 30% in the CHF group (p<.05). With treadmill exercise, LV stroke volume remained lower and neurohormonal concentrations remained higher in the pacing CHF state. Global brain blood flow increased significantly with treadmill exercise in both the normal and CHF states (4.58+/-1.36 and 2.01+/-0.29 mL/min/g; p<.05) but remained reduced in the CHF state compared with normal values (p<.05). In the CHF group, the relative increase in blood flow with exercise was significantly blunted in the parietal and occipital regions of the cerebrum and the suprapyramidal region of the medulla. CONCLUSIONS: The development of pacing-induced CHF was associated with diminished brain perfusion under resting conditions and with treadmill exercise. These perfusion abnormalities with pacing CHF were pronounced in specific regions of the brain. The defects in brain perfusion with the development of CHF may contribute to abnormalities in centrally mediated processes of cardiovascular regulation.

8-ISO-PGF2alpha production by alveolar macrophages exposed to hyperoxia.

Oxygen exposure for a sufficient duration at high partial pressure results in pulmonary edema in humans and animals. Although the specific mediators of oxygen toxicity are unknown, evidence suggests that oxygen-based radicals such as superoxide anion (O2.) are increased in the lungs in the presence of hyperoxia and contribute to this injury. A series of isomeric prostanoid compounds, the isoprostanes, are formed by the free radical-initiated lipid peroxidation of arachidonic acid (AA). One of these isomers, 8-iso-PGF2alpha, is elevated in the bronchial alveolar lavage fluid of rats exposed to 90% oxygen for 48 h and is associated with a significant increase in protein accumulation in the pulmonary extravascular space. Alveolar macrophages (AMs) are capable of producing large quantities of (O2.), suggesting a role in pulmonary oxygen toxicity. We hypothesized that isolated rat AMs exposed to hyperoxia generate increased amount of 8-iso-PGF2alpha. AMs were exposed to air or 90% oxygen for 6, 12, 24, 48, 72, 96, and 120 h in the absence and presence of AA and/or calcium ionophore (A23187) and 8-iso-PGF2alpha was measured in the culture media. Exposure of primary cultures of AMs to 90% oxygen resulted in a significant increase in 8-iso-PGF2alpha in the media (25 +/- 2 pg/mL) compared with air-exposed controls (14 +/- 1 pg/mL). The addition of 10 microM AA and 2 microM A23187 to the culture media resulted in a marked increase in 8-iso-PGF2alpha production by AMs exposed to air and 90% oxygen. However, treatment of AMs with the combination of AA and A23187, followed by exposure to 90% oxygen for 72 h, resulted in a 27-fold increase in 8-iso-PGF2alpha compared with media alone and 90% oxygen. AMs metabolized free and phospholipid-bound AA to 8-iso-PGF2alpha, an activity enhanced in the 90% oxygen environment. Finally, acetylsalicylic acid, a cyclooxygenase inhibitor and free radical scavenger, reduced but did not abolish production of 8-iso-PGF2alpha. This study provides evidence that AMs produce a free radical-mediated isomeric prostaglandin compound that may be involved in pulmonary oxygen toxicity.

Contributory mechanisms for the beneficial effects of myocyte preconditioning during cardioplegic arrest.

BACKGROUND: Preconditioning protects the myocardium from ischemia and may be a potent means of endogenous cardioprotection during cardioplegic arrest and rewarming. However, fundamental mechanisms that potentially contribute to the beneficial effects of preconditioning during cardioplegic arrest and rewarming remain unclear. Accordingly, the overall goal of the present study was to examine the potential mechanisms by which preconditioning protects myocyte contractile function during simulated cardioplegic arrest and rewarming. METHODS AND RESULTS: Left ventricular isolated porcine myocyte contractile function was examined with the use of videomicroscopy under three conditions: (1) normothermia, maintained in cell medium (37 degrees C) for 2 hours; (2) simulated cardioplegic arrest and rewarming, incubated in crystalloid cardioplegic solution (24 mEq/L K+, 4 degrees C) for 2 hours followed by normothermic reperfusion; and (3) preconditioning/cardioplegic arrest and rewarming, hypoxia (20 minutes) and reoxygenation (20 minutes) followed by simulated cardioplegic arrest and rewarming. Cardioplegic arrest and rewarming caused a decline in steady-state myocyte shortening velocity compared with normothermic controls (22.0 +/- 1.6 versus 57.2 +/- 2.6 microns/s, respectively, P < .05), which was significantly improved with preconditioning (36.1 1.7 microns/s, P < .05). In the next series of experiments, the influence of nonmyocyte cell populations with respect to preconditioning and cardioplegic arrest was examined. Endothelial or smooth muscle cell cultures were subjected to a period of hypoxia (20 minutes) and reoxygenation (20 minutes) and the eluent incubated with naive myocytes, which were then subjected to simulated cardioplegic arrest and rewarming. Pretreatment with the eluent from endothelial cultures followed by cardioplegic arrest and rewarming improved myocyte function compared with cardioplegia-alone values (31.7 +/- 2.2 versus 24.7 +/- 1.6 microns/s, respectively, P < .05), whereas smooth muscle culture eluent pretreatment resulted in no change (23.7 +/- 4.0 microns/s, P = .81). Molecular mechanisms for the protective effects of preconditioning on myocyte contractile processes with cardioplegic arrest and rewarming were examined in a final series of experiments. Adenosine-mediated pathways or ATP-sensitive potassium channels were activated by augmenting cardioplegic solutions with adenosine (200 mumol/L) or the potassium channel opener aprikalim (100 mumol/L), respectively. Both adenosine and aprikalim augmentation significantly improved myocyte function compared with cardioplegia-alone values (53.5 +/- 1.7, 57.6 +/- 2.0 versus 25.7 +/- 1.4 microns/s, respectively, P < .05). CONCLUSIONS: The unique findings from the present study demonstrated that preconditioning provides protective effects on myocyte contractile processes independent of nonmyocyte cell populations and that these effects are mediated in part through the activation of adenosine pathways or ATP-sensitive potassium channels. Thus, preconditioning adjuvant to cardioplegia may provide a novel means of protecting myocardial function after cardioplegic arrest and rewarming.

Role of nonenzymatically generated prostanoid, 8-iso-PGF2 alpha, in pulmonary oxygen toxicity.

Oxygen delivery at higher than ambient concentrations is in frequent clinical use, yet prolonged exposure can produce pulmonary edema in humans and animals. The specific mediators of oxygen toxicity are unknown, although evidence suggests that oxygen-based radicals such as superoxide anion contribute to this injury. Recently, 8-iso-prostaglandin F2 alpha (PGF2 alpha), an F2-isoprostane formed by free radical-initiated lipid peroxidation of arachidonic acid, has been implicated in pulmonary injury. Nitric oxide (NO) also contributes to tissue oxygen radical load, and although believed to be beneficial, its metabolites may play a pathophysiological role by participating in lipid peroxidation and isoprostane formation. We hypothesized that 8-iso-PGF2 alpha and NO levels increase in high oxygen concentrations and that 8-iso-PGF2 alpha is associated with lung injury and accumulation of plasma albumin in pulmonary extravascular space. Levels of 8-iso-PGF2 alpha in bronchial alveolar lavage fluid (BALF) of rats exposed to 90% O2 at 1 atmosphere for 48 h (56 +/- 3 pg/ml) or 60 h (70 +/- 5 pg/ml) were significantly increased compared with levels in ambient air-exposed control rats (36 +/- 5 pg/ml). NO levels in BALF of rats exposed to 90% O2 at 1 atmosphere for 60 h were increased 50% compared with NO levels in BALF of rats exposed to ambient air or 48 h of 90% O2 (P < 0.05). Accumulation of radiolabeled plasma albumin in lung parenchyma of rats inhaling 8-iso-PGF2 alpha was also examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular and molecular alterations in the beta adrenergic system with cardiomyopathy induced by tachycardia.

OBJECTIVE: The aim was to examine the relationship between changes in myocyte function to changes in protein and mRNA content of components of the beta adrenergic system with tachycardia induced cardiomyopathy. METHODS: Contractile function and beta adrenergic responsiveness were measured in isolated myocytes from control pigs (n = 6) and in pigs subjected to three weeks of pacing induced supraventricular tachycardia (n = 6). beta Receptor density and affinity, the relative content of the stimulatory (Gs) and inhibitory (Gi) subunits of the G protein complex, and adenylate cyclase activity were determined from sarcolemmal preparations. In order to determine whether these changes were accompanied by alterations in steady state mRNA levels for specific components of the beta adrenergic system, mRNA content for the beta 1 adrenergic receptor and the G alpha s and G alpha i2 subunits of the G protein complex was measured. RESULTS: Chronic supraventricular tachycardia caused a 36% increase in left ventricular end diastolic dimension and a 61% decrease in left ventricular fractional shortening compared to controls. The velocity of isolated myocyte shortening was 50% lower in myocytes from hearts with tachycardia cardiomyopathy than in control myocytes. In the presence of 50 nM isoprenaline or 2 microM forskolin, the velocity of myocyte shortening was 65% lower in the myopathic myocytes than in the controls. With the development of tachycardic cardiomyopathy, beta adrenergic receptor density fell by 25% with no change in affinity, Gs decreased by 35%, and Gi increased by over 50% compared to controls. Basal adenylate cyclase activity and isoprenaline and forskolin stimulated adenylate cyclase activity fell by over 50% with supraventricular tachycardia compared to controls. The relative content of G alpha i2 mRNA increased threefold with the development of tachycardic cardiomyopathy with no change in the relative abundance of mRNA for the beta 1 receptor or G alpha s when compared with controls. CONCLUSIONS: The changes in myocyte beta adrenergic responsiveness with the development of tachycardic cardiomyopathy are due to alterations in cellular mechanisms (decreased beta receptor and Gs density, increased Gi) and in molecular mechanisms (increased Gi mRNA content).

Thromboxane A2/prostaglandin H2 receptors in streptozotocin-induced diabetes: effects of insulin therapy in the rat.

Thromboxane A2 (TXA2) and prostaglandin H2 (PGH2) are potent vasoactive and proaggregatory agents whose synthesis has been shown to be elevated in diabetes mellitus. In the present study the effects of streptozotocin (STZ)-induced uncontrolled diabetes (Severe) and insulin-treated STZ diabetes (Moderate) on TXA2/PGH2 receptor density and affinity in platelets, glomerular membranes and aortic membranes were determined using [125I]-BOP, a TXA2/PGH2 receptor agonist. The affinity and density of platelet TXA2/PGH2 receptors in Control, Moderate and Severe groups and glomerular membranes were not significantly different. However, daily insulin therapy caused significant changes in both TXA2/PGH2 receptor affinity and density of aortic membranes: Kd (nM) = 0.67 +/- 0.09, (n = 5), for Control; 0.27 +/- 0.05*, (n = 6), Moderate; and 0.74 +/- 0.16, (n = 5), Severe; Bmax (fmoles/mg protein) = 38.6 +/- 3.1, Control; 20.2 +/- 4.2*, Moderate; and 37.1 +/- 4.1, Severe: (*p < 0.05 compared to Control and Severe). Contractile responses of aortic segments to the TXA2/PGH2 receptor agonist U46619 were determined. Untreated diabetes mellitus (Severe) was associated with a decreased responsiveness of aortic segments without affecting maximum contractile responses (EC50 = 24.6 +/- 5.9* nM, (n = 10); *p < 0.05) compared to Control rats (EC50 = 11.8 +/- 1.6 nM, (n = 13)). Insulin therapy reversed the decrease seen in the Severe group to a value not different from Control (EC50 = 11.4 +/- 1.2* nM, (n = 10); *p < 0.05 compared to Severe). These results suggest that insulin therapy in the diabetic state significantly influences aortic TXA2/PGH2 receptors, as well as vascular responsiveness to TXA2/PGH2 mimetics.

Endotoxin tolerance is associated with altered GTP-binding protein function.

Previous studies have suggested that guanine nucleotide regulatory (G) proteins modulate endotoxin-stimulated peritoneal macrophage arachidonic acid (AA) metabolism. Endotoxin-stimulated metabolism of AA by peritoneal macrophages is decreased in endotoxin tolerance (Rogers et al. Prostaglandins 31: 639-650, 1986). These observations led to a study of G protein function and AA metabolism by peritoneal macrophages in endotoxin tolerance. Endotoxin tolerance was induced by the administration of sublethal doses of endotoxin. AA metabolism was assessed by measurement of thromboxane B2 (TxB2), a cyclooxygenase metabolite. NaF (5 mM), an activator of G proteins, significantly stimulated TxB2 synthesis in control macrophages from 7.7 +/- 0.2 to 19.1 +/- 0.6 (SE) ng/ml (P less than 0.05) at 2 h and was partially inhibited by pertussis toxin, suggesting a G protein-dependent mechanism. Salmonella enteritidis endotoxin (50 micrograms/ml) stimulated a similar increase in TxB2 levels (23 +/- 0.4 ng/ml, P less than 0.05). In contrast to control macrophages, macrophages from endotoxin-tolerant rats stimulated with either NaF or S. enteritidis endotoxin had TxB2 levels that were only 30 and 2% of the respective stimulated control cells. Basal guanosine-triphosphatase (GTPase) activity (33 +/- 6 pmol.mg-1.min-1) in endotoxin-tolerant macrophage membranes was significantly lower (P less than 0.05) than control basal activity (158 +/- 5 pmol.mg-1.min-1). This suppression of macrophage GTPase activity was apparent 48 h after the first in vivo sublethal endotoxin injection (100 micrograms/kg ip). The reduced GTPase activity paralleled in vitro cellular hyporesponsiveness to endotoxin-stimulated TxB2 production.(ABSTRACT TRUNCATED AT 250 WORDS)

The beneficial effects of a thromboxane receptor antagonist on spinal cord perfusion following experimental cord injury.

The eicosanoids thromboxane A2 and prostacyclin have opposing actions causing vasoconstriction and vasodilation respectively. The ratio of these two eicosanoids is thus an important determinant of circulatory homeostasis. An increase in this ratio occurs in certain inflammatory conditions with dramatic consequences in organ perfusion. In spinal cord trauma, in addition to direct physical perturbation of the spinal cord, it is likely that further structural and functional loss occurs as a result of decreased tissue perfusion precipitated by an increase in the thromboxane/prostacyclin ratio. This study evaluated hemodynamics and organ perfusion, 3 h following 24 g-cm spinal cord trauma in the rat. The role of thromboxane was investigated with an inhibitor of thromboxane synthesis (Dazoxiben) and with a receptor antagonist (13-APT). Cardiac output and blood pressure were unaffected by Dazoxiben, 13-APT, or spinal cord trauma. Injury effected approximately a 40% decrease in spinal cord perfusion from 0.41 to 0.25 ml/min/g which was not improved by the thromboxane synthase inhibitor, Dazoxiben. 13-ATP completely abrogated the decline in spinal cord blood flow flowing injury. Perfusion of other selected organs demonstrated little change as a result of the spinal trauma. Brain flow remained constant at 0.78 ml/min/g brain. Coronary blood flow, however, declined from 3.2 to 2.0 ml/min/g heart tissue. The data suggest consideration of the importance of thromboxane in therapeutic attempts to reduce secondary injury arising in spinal cord trauma.

Alterations in the myocardial capillary vasculature accompany tachycardia-induced cardiomyopathy.

Changes in capillary structure and distribution within the left ventricle (LV) occur with pressure and volume overload hypertrophy. These changes may cause an impairment in myocardial blood flow (MBF) and oxygen delivery resulting in myocyte injury and LV dysfunction. However, it is unknown whether changes in the capillary vasculature accompany the development of LV dysfunction in dilated cardiomyopathies. Accordingly, this study examined the relation between LV function, MBF and capillary architecture after the development of dilated cardiomyopathy in pigs produced by chronic supraventricular tachycardia (SVT). LV function was examined by echo-catheterization, and capillary morphometrics were computed using lectin histochemistry in two groups of pigs: sham controls (n = 8); and after 3 weeks of pacing-induced SVT (n = 8). Chronic SVT resulted in significantly increased LV end-diastolic dimension and pressure with a 50% reduction in LV fractional shortening compared to CON (p less than 0.05). Although no significant change in capillary density (2180 +/- 164 vs 2402 +/- 147/mm2, p = 0.25) occurred in the SVT group compared to CON, a significant reduction in the volume fraction of capillaries (12.2 +/- 0.5 vs 9.9 +/- 0.7%, p less than 0.05) and increased capillary diffusion distance (8.4 +/- 0.5 vs 7.5 +/- 0.3 microns, p less than 0.05) was observed. Frequency distribution analysis revealed a higher percentage of smaller diameter capillaries with chronic SVT vs CON (p less than 0.05). Ultrastructural examination revealed an increased capillary-myocyte distance with chronic SVT (0.95 +/- 0.08 vs 1.95 +/- 0.12 microns, p less than 0.05). These changes were accompanied by ultrastructural evidence of significant subendocardial injury (p less than 0.05). MBF was measured using microspheres in five additional conscious pigs in each group. MBF was significantly reduced and coronary vascular resistance increased in the SVT group compared to CON (p less than 0.05). Chronic SVT caused significant remodeling of the capillary vasculature; these changes were associated with reduced MBF, myocyte injury, and LV dysfunction.

Tachycardia-induced cardiomyopathy: effects on blood flow and capillary structure.

Chronic supraventricular tachycardia (SVT) causes a dilated cardiomyopathy. A potential factor contributing to the development of SVT-induced cardiomyopathy is abnormal myocardial blood flow (MBF). The purpose of this study was to relate changes in left ventricular (LV) function, MBF, and capillary structure with the development of SVT-induced cardiomyopathy. LV function and MBF were measured in two groups of conscious pigs: sham control (CON; n = 8) and after 3 wk of atrial pacing (SVT; 240 beats/min; n = 8) using echocardiography-catheterization and microspheres. Measurements were made under three states: 1) at rest with a basal heart rate, 2) during rapid atrial pacing (240 beats/min), and 3) during adenosine infusion (1.5 microM.kg-1.min-1) without pacing. LV capillary density, diameter, wall thickness, and capillary-myocyte distance were measured in four additional pigs from each group. LV fractional shortening was lower, and left atrial pressure was significantly higher in the SVT group compared with CON at rest, during pacing, and with adenosine (P less than 0.05). In the CON group, average LV-MBF at rest was 2.0 +/- 0.2 ml.min-1.g-1, increased with pacing to 3.0 +/- 0.2 ml.min-1.g-1 (P less than 0.05), and increased further with adenosine to 4.1 +/- 0.3 ml.min-1.g-1 (P less than 0.05). In all states, SVT LV-MBF was significantly reduced vs. CON (P less than 0.05); SVT LV-MBF was 0.8 +/- 0.2 ml.min-1.g-1 at rest, increased to 1.3 +/- 0.3 ml.min-1.g-1 with rapid pacing (P less than 0.05), and remained unchanged during adenosine (1.3 +/- 0.4 ml.min-1.g-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Endotoxin tolerance differentially alters hemodynamic responses to a thromboxane A2 mimetic and phenylephrine.

Repeated sublethal doses of endotoxin render rats tolerant to lethal doses of endotoxin and reduce thromboxane (Tx) A2 synthesis. Endotoxin-tolerant rats are also more resistant to lethal doses of U46619, a stable TxA2 mimetic. These observations raised the possibility that tolerance may alter hemodynamic responses to TxA2 via one or more mechanisms. Mean arterial pressure (MAP) responses to i.v. injections of the stable TxA2 mimetic U46619 at doses ranging from 0.17 to 8.4 micrograms/kg were determined. Despite an initial lower systemic vascular resistance in tolerant rats compared to control rats (2.4 +/- 0.3 vs 3.1 +/- 0.2 mm Hg/ml/min/100 g of body weight, respectively, p less than 0.05), the maximum pressor response to U46619 was significantly greater (p less than 0.05) at the higher doses of U46619 in tolerant rats compared to control rats. Tolerant and control rats also exhibited qualitatively different changes in MAP in response to U46619. Control rats manifested an initial hypotensive response (15 s) not observed in tolerant rats. In contrast, tolerant rats exhibited no depressor response, but a higher peak pressor response to U46619 than that seen in controls. Since prostaglandins may modulate vascular responses to U46619, subsequent studies were conducted in indomethacin-pretreated or essential fatty acid (EFA) deficient rats that were depleted of arachidonic acid substrate. Either indomethacin or EFA deficiency significantly prevented the initial hypotensive response in control rats, suggesting that prostaglandins may mediate this response to U46619. In additional studies, the MAP response in tolerant and control rats to the alpha 1-adrenergic agonist phenylephrine was determined.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of eicosanoids in mediating blood flow alterations in endotoxin shock.

The vasoactive metabolites of arachidonic acid TxA2 and sulfidopeptide-leukotrienes appear to play a role in mediating hemodynamic alterations in endotoxic shock. The ultimate goal of these and other studies in shock is, however, to understand the mechanisms underlying the pathogenic changes in human shock states in order to develop effective treatment. Recent studies (see reviews by Ball, 1988) have implicated eicosanoids in pathogenesis of shock in man confirming the directions provided by the bolus endotoxin studies and suggest additional investigations of hemodynamics following interventions designed to alter eicosanoid synthesis in shock.

The rat in sepsis and endotoxic shock.

Compared to the experimental studies, interpretation of eicosanoid levels in human sepsis is complicated by a large number of uncontrolled variables. Further clinical studies are needed to establish the relationship between the septic rat model and the human septic condition. The rat model is useful for uncovering fundamental pathophysiologic processes and should be useful in developing therapeutic interventions in human circulatory shock. Studies on the rat model offered the first indication that eicosanoids were involved in the shock syndrome (Cook et al., 1980). This led to clinical trials to determine the role of these mediators in human sepsis (Reines et al. 1982). The rat affords a model system which allows for pharmacologic manipulation and testing of a large number of hypotheses in a practical and cost effective manner. The recent evidence that leukotrienes are released during circulatory shock in the rat and that attenuation of this release alters the pathophysiologic outcome (Ball et al., 1986), lays the groundwork for future clinical studies. Finding that eicosanoids and leukotrienes are elevated in rat models of adult respiratory distress syndrome (Hammarstrom, 1983) has opened further new areas of clinical inquiry. Although rat models involving bolus endotoxin administration have not been predictive of the human septic syndrome, they have proven useful in the determination of the cellular sources and mechanisms of eicosanoid and leukotriene action. From the evidence presented, the septic shock rat model would seem to be the best model in predicting the outcome of therapies in human sepsis. Limited clinical trials have assessed the therapeutic efficacy of pharmacologic agents which alter eicosanoid and leukotriene metabolism in sepsis. In view of the continued high mortality of patients with septic shock and the failure of conventional therapies (Sprung et al., 1984; DuToit et al. 1985), further clinical and experimental studies are desirable.

Beneficial effects of a 5-lipoxygenase inhibitor in endotoxic shock in the rat.

The effects of a highly selective 5-lipoxygenase inhibitor, CGS8515 [methyl 2-[(3,4-dihydro-3,4-dioxo-1-naphthalenyl) amino]benzoate], on endotoxic shock sequelae and eicosanoid synthesis by peritoneal macrophages were evaluated in the rat. Pretreatment of peritoneal macrophages in vitro with CGS8515 significantly inhibited the synthesis (P less than .01) of immunoreactive leukotriene C4/leukotriene D4 stimulated by the calcium ionophore (A23187). Inhibition of 5-lipoxygenase produced significant shunting to immunoreactive thromboxane B2 formation (P less than .05). In rats sedated with ketamine.HCl (82.5 mg/kg) and xylazine. HCl (27.5 mg/kg), i.v. injection of Salmonella enteritidis endotoxin (25 mg/kg i.v.) produced significant decreases at 30 min in mean arterial pressure (from 89 +/- 4 to 44 +/- 8 mm Hg, N = 5, P less than .001); in white blood cell count (from 10.8 +/- 0.6 to 6.5 +/- 0.8 x 10(3)/mm3, N = 5, P less than .01); in platelet count (from 687 +/- 66 to 392 +/- 65 x 10(3)/mm3, N = 5, P less than .01); and produced an increase of hematocrit (from 46 +/- 1.2 to 57.4 +/- 1.8%, N = 5, P less than .03). CGS8515 (5 mg/kg i.v. 30 min before endotoxin injection, N = 6) blunted the endotoxin-induced hypotension by 35% (P less than .001), the leukopenia by 24% (P less than .03), the thrombocytopenia by 45% (P less than .006) and the hemoconcentration by 16% (P less than .03), compared to the shocked control rats 30 min after endotoxin injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Beneficial effects of a leukotriene antagonist on endotoxin-induced acute hemodynamic alterations.

The role of lipoxygenase metabolites of arachidonic acid as inflammatory mediators of endotoxin shock remains uncertain. In this study the effect of LY171883, a selective leukotriene D4/E4 antagonist, on the hemodynamic alterations of endotoxin shock was assessed. Adult male rats were given an intraperitoneal injection of LY171883 (30 mg/kg) or vehicle 10 minutes prior to intravenous injection of endotoxin (15 mg/kg) or vehicle. Cardiac output, mean arterial pressure, and multiple organ blood flows were determined at 30 minutes after endotoxin or vehicle administration with 85Sr-radiolabeled microspheres. Cardiac output decreased from 32.1 +/- 2.7 ml/min/100 g in the control group to 16.3 +/- 2.7 ml/min/100 g in endotoxin-treated animals (P less than .05). Pretreatment with LY171883 blunted significantly (P less than .05) this fall in cardiac output (26.3 +/- 2.6 ml/min/100 g). Endotoxin reduced mean arterial pressure from 95 +/- 8 mm Hg in controls to 57 +/- 8 (P less than .05), which was not, however, different from control values in rats receiving the LTD4/E4 antagonist. There was also significant (P less than .05) blunting of the endotoxin-induced fall in blood flow to the heart, gastrointestinal tract, and kidneys in animals pretreated with LY171883. Our data demonstrate that this selective leukotriene D4/E4 antagonist has significant salutary actions in endotoxin shock and suggest that LTD4 and/or E4 mediate, in part, the acute hemodynamic sequelae of endotoxin shock.

Improvement in organ blood flow by inhibition of thromboxane synthetase during experimental endotoxic shock in the rat.

Endotoxic shock is associated with increased metabolism of arachidonic acid to thromboxane (Tx) and prostaglandins. This investigation examined the effects of two structurally dissimilar inhibitors of (Tx) synthetase on Salmonella enteritidis endotoxin (LPS) (15 mg/kg)-induced alterations in cardiac output and organ blood flow in Long-Evans rats. An imidazole derivative, 7(1-imidazolyl) heptanoic acid (7-IHA), and sodium -(E)-3-[4-(3-pyridyl-methyl)phenyl]-2-methacrylate (OKY-1581) were injected intravenously at 30 and 5 mg/kg, respectively, 30 min before injection with endotoxin. Cardiovascular function was assessed 30 min post-LPS with Sr-85 labeled microspheres under light ether anesthesia. Injection of endotoxin caused a 60% decrease in cardiac output (34.0 +/- 2.7 ml/min/100 g body weight in control rats) and a 38.9% decrease in systolic arterial pressure. Both Tx synthetase inhibitors significantly (p less than 0.05) attenuated the decrease in cardiac output, although only 7-IHA improved blood pressure. Pretreatment with 7-IHA or OKY-1581 significantly (p less than 0.05) attenuated the LPS-induced decrease in renal perfusion. Lung nutrient blood flow (1.1 +/- 0.2 ml/min/g lung) decreased nearly 70% in shock. Both Tx synthetase inhibitors prevented this reduction. LPS shock resulted in approximately a 30% decrease in brain blood flow. 7-IHA significantly (p less than 0.05) improved flow, while OKY-1581 was without apparent effect. Splanchnic blood flow was likewise improved by 7-IHA and OKY-1581. Liver blood flow, 55% less than values of the control group in shock (p less than 0.05), was returned to values of the controls by both inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of alpha adrenergic blockade on arachidonic acid metabolism and shock sequelae in endotoxemia.

Previous studies have suggested that increased alpha adrenergic activity stimulated prostaglandin synthesis and may be involved in the modulation of eicosanoid metabolism. These observations prompted investigation of the effect of the alpha adrenergic receptor antagonist phenoxybenzamine and the cyclo-oxygenase inhibitor indomethacin on endotoxin-induced shock severity, and on plasma immunoreactive iTxB2 and i6-keto-PGF1 alpha, the stable metabolites of TxA2 and PGI2, respectively. Pretreatment with indomethacin alone blunted the endotoxin- (8 mg/kg) induced hypoglycemia. Phenoxybenzamine pretreatment also blunted endotoxin-induced mortality (LD80), hypoglycemia, hemoconcentration, and decreased plasma beta-glucuronidase (BG). The combination of phenoxybenzamine and indomethacin resulted in the improvement of all indices of shock severity. Rats pretreated with phenoxybenzamine and indomethacin alone or conjointly also exhibited significantly (P less than 0.05) enhanced survival compared to that of shocked control rats. Percent survival at 48 hr was 24, 64, 80, and 92 in untreated, indomethacin, phenoxybenzamine, and indomethacin + phenoxybenzamine treated, respectively. Mean plasma iTxB2 values at 30 min postendotoxin (15 mg/kg i.v.) were 1,532 +/- 319 pg/ml (N = 10). Phenoxybenzamine pretreatment decreased iTxB2 to 719 +/- 114 pg/ml (N = 10). Phenoxybenzamine pretreatment decreased iTxB2 to 719 +/- 114 pg/ml (N = 10) (P less than 0.05). Plasma i6-keto-PGF1 alpha was increased 4 hr after endotoxin in shocked controls to 4,161 +/- 885 pg/ml (N = 5) and attenuated by phenoxybenzamine to a value of 1,184 +/- 363 pg/ml (N = 4) (P less than 0.05). The results suggest that increased alpha adrenergic activity may be an important stimulus for arachidonic acid metabolism during endotoxemia.

Experimental endotoxemia in the rat: efficacy of prostacyclin or the prostacyclin analog iloprost.

The purpose of this study was to examine the effects of PGI2 and iloprost, a prostacyclin analog, on the sequelae of experimental endotoxic shock. Endotoxemia was produced in rats by IV injection of 20 mg/kg Salmonella enteritidis (LPS). Mean arterial blood pressure fell from 117 +/- 5 mmHg (N = 22) to 92 +/- 5 mmHg and 71 +/- 10 mmHg at 30 and 180 min post-LPS, respectively. In LPS vehicle-treated rats, blood glucose levels fell from 100 +/- 10 mg/dl to 36 +/- 8 mg/dl at 180 min. Plasma glutamate-oxaloacetate transaminase and lactate dehydrogenase (liver fraction) activities were significantly elevated above concentrations in nonshocked animals, increasing to 426 +/- 86 IU/ml and 1,019 +/- 339 IU/ml, respectively. Infusion of PGI2 or iloprost (0.5 micrograms/kg/min) did not alter the blood pressure response to LPS compared to vehicle controls. PGI2 significantly prevented the LPS-induced hypoglycemia while neither treatment significantly reduced the elevations in plasma enzymes nor prevented the lethality of LPS. Thus, the hemodynamic and biochemical indices of shock severity were not significantly improved by either prostacyclin or iloprost. It is suggested that the beneficial effects of PGI2 or prostacyclin analogs observed in other species during endotoxemia does not extend to the rat.

Exchange transfusions in rats with a perfluorated blood substitute: effect on thromboxane B2 levels during endotoxemia.

The effect of exchange transfusion with the perfluorated blood substitute (Fluosol-43) on endotoxin-induced synthesis of immunoreactive (i) thromboxane (Tx)B2, the stable metabolite of TxA2, was investigated in rats. Fluosol-43 was infused via the femoral vein with matched, incremental blood withdrawal from the carotid artery. Blood was replaced with Fluosol-43 to a final hematocrit of less than 3% in anesthetized rats maintained on 95% O2 and 5% CO2. Circulating platelet counts were reduced from 875 +/- 47 X 10(3)/mm3 in sham controls (N = 21) to 75 +/- 10 X 10(3)/mm3 in Fluosol-43 exchange transfused rats (N2 = 19, P less than 0.001). Circulating leukocytes were decreased from 105 +/- 6.3 X 10(2)/mm2 in sham controls (N = 21) to 17 +/- 1.4 X 10(2)/mm3 in the exchange transfused group (N = 19, P less than 0.001). Immunoreactive (i)TxB2 was measured in plasma or Fluosol-43 obtained from rats prior to and after injection of Salmonella enteritidis endotoxin (20 mg/kg). The iTxB2 levels at 30 minutes after endotoxin increased from 438 +/- 83 pg/ml (N = 4) to 2,895 +/- 663 pg/ml (N = 7) (P less than 0.01) in sham controls. iTxB2 also increased from 242 +/- 23 pg/ml (N = 7) to 2,213 +/- 589 pg/ml (N = 7) in the Fluosol-43 group (P less than 0.002) following endotoxin. The iTxB2 levels also remained significantly elevated (P less than 0.01) in both the sham and the Fluosol-43 groups 2 hours after endotoxin treatment. Endotoxin-stimulated iTxB2 levels at both 30 minutes and 2 hours in sham and Fluosol-43 exchange transfused rats did not vary significantly from each other. Indomethacin pretreatment (2 mg/kg) inhibited the increase in iTxB2 levels by greater than 85% in both groups (P less than 0.004). Blood and Fluosol-43 were taken from sham and exchange transfused rats and incubated ex vivo with the calcium ionophore, A23187 (10 microM). These studies demonstrated that ionophore-stimulated iTxB2 synthesis in the ex vivo Fluosol-43 samples was only 2.6% that of whole blood. Collectively these observations suggest that tissues other than blood components are potential sources of iTxB2 synthesis in endotoxin shock.