Characterization of cAMP-dependent inhibition of LPS-induced TNF alpha production by rolipram, a specific phosphodiesterase IV (PDE IV) inhibitor.

Bacterial endotoxins (lipopolysaccharide or LPS) provoke shock and tissue injury by eliciting the release of toxic factors from reticuloendothelial cells. One of the principal endogenous factors involved in this process is tumor necrosis factor alpha (TNF alpha). In this study, inhibitors selective for different classes of phosphodiesterases (PDE), were examined for their effects on LPS-induced TNF alpha production by human monocytes. The selective cAMP-PDE IV inhibitors, rolipram and RO-20-1724 were capable of inhibiting LPS-induced TNF alpha production by human monocytes in a concentration-dependent manner. Rolipram was used to examine further the cellular pharmacology of PDE IV inhibitors on cytokine production. The IC50 for inhibition of LPS-induced TNF alpha production by rolipram was 0.1 microM, whereas production of IL-1 beta or IL-6 was unaffected. Furthermore, rolipram was equally effective in inhibiting TNF alpha production by a number of other stimuli. Inhibition of TNF alpha production by rolipram was associated with an elevation of intracellular cAMP, consistent with a mechanism involving phosphodiesterase inhibition. Rolipram was efficacious in suppressing LPS-induced TNF alpha mRNA expression, and at the protein level was also active when added to cultures post-stimulated with LPS. This indicates that rolipram may act at both the transcriptional and translational levels. Rolipram inhibited TNF alpha production in vivo in a rat endotoxemia model. Collectively, these data suggest that the prototypic inhibitor of PDE IV isozyme, rolipram, can effectively and selectively inhibit LPS-induced TNF alpha production through elevation of intracellular cAMP.

Endotoxin-mediated changes in plasma endothelin concentrations, renal endothelin receptor and renal function.

The purpose of these studies was to examine the changes in renal endothelin (ET) receptor, renal function and plasma ET (ET-1) concentration in male Sprague-Dawley rats injected with nonlethal doses of Escherichia coli endotoxin (LPS). Prior to the injection of LPS, kidney ET receptor density was 59 +/- 5 fmol/mg protein (n = 20). At 24 h after the injection of 1 or 3 mg/kg LPS, [125I]ET-1 binding to kidney membranes was increased by 70% in both LPS groups (p < 0.001). Scatchard analysis of the saturation binding experiments confirmed that the increase in [125I]ET-1 binding was due to an increase in receptor density with no change in affinity (202 pmol/l at baseline and 168 pmol/l and 246 pmol/l at 24 h after the injection of 1 and 3 mg/kg LPS, respectively). At 7 days after the injection of LPS, kidney ET-1 receptor density was still increased by 30 +/- 5% and 58 +/- 16%, respectively (p < 0.05, compared to the baseline value). Baseline values for Na+ and K+ excretion were approximately 115 muEq/h and 214 +/- mu/Eq/h respectively, and were decreased with LPS. Maximal decreases in Na+ and K+ excretion occurred at 48 h (-85%) and 30 h (-82%), respectively, following the injection of 3 mg/kg LPS and returned to baseline levels in 7 days. Following the injection of 3 mg/kg LPS, plasma immunoreactive ET-1, as measured by radioimmunoassay, increased in a time-dependent manner: the maximal increase of 60% occurred within 1 h after the injection of LPS (p < 0.05), and thereafter returned to baseline levels. Kidney tissue levels of ET-1 increased from baseline values of 2.6 fmol/mg protein to a peak of 4.6 fmol/mg protein 1 h after the injection of LPS. Tissue ET-1 levels were still significantly elevated at 6 h but not 24 h after LPS injection. These studies suggest that ET-1, either by increases in plasma concentration and/or altered receptor density, may be involved in the LPS-induced impairment of renal function.

Fluid resuscitation improves survival of endotoxemic or septicemic rats: possible contribution of tumor necrosis factor.

The present study was designed to investigate the effects of fluid administration on survival in endotoxemic or septicemic male Sprague-Dawley rats. Endotoxemia was induced by intravenous injection of Escherichia coli lipopolysaccharide (LPS), and septicemia produced by cecal ligation and puncture (CLP). In endotoxemic animals deprived of fluid resuscitation, 7-day survival following injection of LPS at doses of 1, 3, or 10 mg/kg LPS were 70% (n = 10), 30% (n = 10), and 0% (n = 10), respectively. In rats resuscitated with 3.3 ml/kg/h of 0.9% NaCl, the dose-response curve for survival was shifted 5-fold rightward in a parallel manner (p < 0.001, between the fluid-resuscitated and nonfluid resuscitated LPS groups), indicating a reduced sensitivity to the effects of LPS following fluid resuscitation. LPS increased serum tumor necrosis factor (TNF alpha) concentrations in fluid-resuscitated endotoxemic animals from a baseline value of 20 U/ml to 2,350 U/ml at 1 h, which returned to 200 U/ml at 2 h. In endotoxemic animals not receiving fluid resuscitation, serum TNF alpha levels at 1 and 2 h were 5-fold and 27-fold higher, respectively, than in fluid-resuscitated animals. There were no differences in arterial blood pressure or heart rate between the two groups of endotoxemic animals; total peripheral resistance was significantly lower at 1 h, and cardiac index was significantly greater at 3 h in the fluid-resuscitated LPS group; otherwise there were no further differences in hemodynamic parameters between the two groups. The survival rate at 4 days following CLP without fluid resuscitation was 14%, whereas CLP with fluid resuscitation improved survival to 74% (p < 0.01). TNF alpha was undetectable (i.e., < 20 U/ml) in the serum of animals subjected to CLP. The improvement in survival with fluid infusion in the LPS and CLP models cannot be attributed to catheter implantation, or to improved hemodynamic parameters in the LPS model. The improvement in survival in the LPS model with fluid infusion was associated with attenuated increases in TNF alpha levels. Furthermore, these studies illustrate that fluid-resuscitated and nonfluid-resuscitated experimental animal models should not be considered equivalent.

Single and repeated doses of the vasodilator/beta-adrenergic antagonist, carvedilol, block cirazoline- and isoproterenol-mediated hemodynamic responses in the conscious rat.

The purpose of this study was to evaluate the effects of carvedilol, a beta 1&2-adrenergic blocker and vasodilator, on cirazoline-mediated changes in arterial blood pressure and isoproterenol-mediated changes in heart rate after acute and chronic administration. Conscious, chronically instrumented male Sprague-Dawley rats were injected with carvedilol (1 mg/kg, IV), prazosin (0.3 mg/kg, IV), or propranolol (1 mg/kg, twice daily for 8 days. After administration of the first dose of carvedilol on day 1, the vasopressor response to cirazoline (60 +/- 3 mmHg predrug) and the isoproterenol-induced tachycardia (152 +/- 13 beats/min predrug) were blocked (e.g., 7 +/- 4 mmHg postdrug and 11 +/- 3 beats/min postdrug, respectively). After the administration of carvedilol on day 8, the cirazoline vasopressor response was 2 +/- 1 mmHg and the isoproterenol-induced tachycardia was 4 +/- 3 beats/min, indicating effective alpha 1- and beta-adrenergic blockade after chronic dosing with carvedilol. Prazosin blocked the cirazoline-induced vasopressor response on both days 1 and 8 but had no effect on the isoproterenol-induced tachycardia. Propranolol blocked the isoproterenol-induced tachycardia on both days 1 and 8 but had no effect on the cirazoline vasopressor response. These data indicate that only carvedilol effectively blocked both alpha- and beta-adrenergic hemodynamic responses and that the antagonism of these responses with carvedilol was not diminished after chronic dosing of twice-a-day treatment for 8 days.

Effect of endotoxicosis on plasma and tissue levels of calcitonin gene-related peptide.

This study was designed to investigate the changes in tissue content and plasma concentrations of CGRP, a 37 amino acid vasoactive peptide, in male Sprague Dawley rats injected intravenously with a nonlethal dose of 3 mg/kg E. coli endotoxin. Plasma CGRP concentrations in nonendotoxemic animals, measured by a specific RIA, were initially 30.5 +/- 3.3 pg/ml, and were significantly increased to 63.7 +/- 4.6 pg/ml 2 hr after induction of endotoxemia (P less than 0.001; n = 13). A higher dose of LPS did not further elevate plasma CGRP levels, indicating that the maximal response occurred following a dose of 3 mg/kg LPS. CGRP levels in abdominal aorta, inferior vena cava, stomach, kidney, and left ventricular myocardium (4.11, 8.5, 2.61, 0.69, and 0.25 pmol/g wet weight tissue, respectively) were not changed significantly following the injection of endotoxin. However, in lung and mesenteric artery the levels increased significantly from 1.47 +/- 0.12 and 7.97 +/- 1.32 pmol/g wet weight tissue to 1.96 +/- 0.19 (P less than 0.05, n = 11) and 15.02 +/- 2.3 pmol/g (P less than 0.01; n = 7), respectively. In contrast, CGRP levels in the duodenum were significantly decreased from 11.3 +/- 0.93 pmol/g wet weight tissue to 6.2 +/- 0.68 pmol/g (P less than 0.001; n = 6). The changes in plasma concentration and tissue content of CGRP suggest that splanchnic organs may be the source of the elevated plasma CGRP levels in endotoxemia and that selective organ CGRP levels reflect a role in the pathogenesis of the response to endotoxemia.

Pharmacological profile of G619, a new platelet aggregation inhibitor.

G619, a 4-OH-isophthalic acid derivative, was studied for its capacity to inhibit platelet aggregation. G619 dose-dependently inhibited U46619, collagen, ADP, PAF, thrombin and epinephrine-induced platelet aggregation in vitro. The IC50 values for inhibition of U46619-induced human and rabbit platelet aggregation were 39 and 43 microM, respectively. G619, at 100 microM, inhibited high concentration collagen (10 micrograms/ml)-induced aggregation of rabbit platelets pretreated with indomethacin and increased the level of cAMP in washed rabbit platelets by 30% (p less than 0.01 vs basal). However, G619, did not inhibit fibrinogen binding to GPIIb/IIIa receptor, phosphodiesterase, U46619-induced contractile responses on canine saphenous vein or rabbit aorta, calcium-induced vasoconstriction and thrombin or PAF-induced elevation of [Ca++]i in platelets in vitro. In vivo, the U46619-induced maximal thrombocytopenia in rats was reduced from 40% (vehicle) to 22% and 18% by 10 and 30 mg/kg of G619 i.v., respectively. G619 (30 mg/kg) had no effect on the U46619-induced vasopressor response or sudden death in rats, and had no effect on TxB2 formation. Our results indicate that G619 is a broad-spectrum platelet aggregation inhibitor and may have its effect on a common mechanism for platelet aggregation besides an effect on the thromboxane A2 receptor.

Cardioprotective effects of the vasodilator/beta-adrenoceptor blocker, carvedilol, in two models of myocardial infarction in the rat.

The purpose of this study was to evaluate the cardioprotective effects of carvedilol, a beta-adrenergic blocker and vasodilator, in two models of ischemic myocardial damage in the rat. Following coronary artery occlusion for 0.5 h and reperfusion for 24 h (MI/R group), left ventricular (LV) injury was determined by planimetric analysis of triphenyltetrazolium chloride-stained tissue, and polymorphonuclear leukocyte infiltration was assessed by measuring myeloperoxidase (MPO) activity. In the vehicle-treated MI/R group, infarct size was 14.2 +/- 1.3% of the LV (n = 16), and MPO activity was increased to 2.8 +/- 0.7 from 0.14 +/- 0.03 U/g tissue in the vehicle-treated sham-occluded group (p less than 0.01). Carvedilol (1 mg/kg i.v., 15 min prior to coronary artery occlusion and at 3.5 h following reperfusion) reduced myocardial infarct size to 7.5 +/- 1.2% of the LV (n = 14; p less than 0.01) and attenuated the increase in MPO activity to 1.4 +/- 0.4 U/g tissue (p less than 0.05). A lower dose of carvedilol (i.e. 0.3 mg/kg i.v.) did not limit myocardial infarct size or the increase in MPO activity. In a model of permanent coronary artery occlusion, 24-hour survival was reduced from 85% in sham-occluded animals (n = 38) to 44% in the vehicle-treated MI group (n = 84; p less than 0.01). In comparison to the vehicle-treated MI group, carvedilol (0.3 mg/kg i.v., 15 min prior to coronary artery occlusion and 1 mg/kg 4 h after occlusion) improved survival by 55% (n = 64; p less than 0.05, compared to the vehicle-treated MI group), whereas the same dose of propranolol (n = 42) had no significant effect on survival. These results indicate that carvedilol reduces myocardial ischemia/reperfusion injury, and significantly improves survival in a permanent coronary artery occlusion model of myocardial infarction.

Reduction in myocardial ischemic/reperfusion injury and neutrophil accumulation after therapeutic administration of streptokinase.

The benefit of thrombolytic agents to reduce myocardial infarct size, improve left ventricular (LV) function, and prolong survival in human subjects is generally recognized, although the precise mechanism is poorly defined. This study was designed to evaluate the cardioprotective effects of streptokinase (SK) in rats, a species less responsive to plasminogen activators, using a model of mechanical occlusion and release of the left coronary artery. Myocardial injury and polymorphonuclear leukocyte (PMN) infiltration were determined by measuring creatine phosphokinase (CPK) specific activity and myeloperoxidase (MPO) activity, respectively, in the LV free wall (LVFW). After coronary artery occlusion for 0.5 h and reperfusion for 24 h (myocardial ischemia, MI/R), CPK specific activity decreased from 7.0 +/- 0.3 U/mg protein in the sham + vehicle group to 5.6 +/- 0.5 U/mg protein in the MI/R + vehicle group (n = 19, p less than 0.01), while MPO activity increased from 0.14 +/- 0.03 U/g tissue in the sham + vehicle group to 2.8 +/- 0.7 U/g in the MI/R + vehicle group (p less than 0.001). Administration of SK (100,000 IU/kg + 50,000 IU/kg/h for 2 h beginning 15 min before coronary artery reperfusion) reduced the loss of CPK specific activity from reperfused myocardium (6.8 +/- 0.5 U/mg protein, n = 23, p less than 0.05 as compared with the MI/R + vehicle group) and attenuated the increase in MPO activity (1.3 +/- 0.4 U/g tissue, p less than 0.05 as compared with the MI/R + vehicle group). This dose of SK did not change plasma fibrinogen concentration, slightly reduced plasminogen activity (i.e., 20% from control value), and markedly reduced alpha 2-antiplasmin activity (i.e., 60% from control values). A lower dose of SK (i.e., 10,000 IU/kg + 5,000 IU/kg/h for 2 h) did not reduce myocardial injury, did not attenuate the increase in MPO activity, and had no effect on the measured hemostatic parameters. Survival in all MI/R groups ranged from 62 to 66%, and there were no differences in survival between any of the groups (p greater than 0.05). In a model of arachidonic acid-induced rat hindpaw inflammation, SK had no effect on the increase in MPO activity, suggesting that the increase in myocardial MPO activity was not due to a direct effect on inflammatory cell accumulation. In in vitro studies, SK (1-1,000 U/ml) did not scavenge superoxide anion produced by purine (10 mM) and xanthine oxidase (10 mU/ml), nor did it reduce superoxide release, beta-glucuronidase release, or neutrophil aggregation of rabbit peritoneal neutrophils activated with fMLP.(ABSTRACT TRUNCATED AT 400 WORDS)

SK&F 86002 inhibits tumor necrosis factor formation and improves survival in endotoxemic rats.

We evaluated the effects of SK&F 86002, a dual 5-lipoxygenase/cyclooxygenase inhibitor, on the responses to 30 mg/kg Escherichia coli endotoxin (bacterial lipopolysaccharides, LPS) in conscious male Sprague-Dawley rats. Injection of LPS increased serum tumor necrosis factor (TNF alpha) activity from 20 U/ml (baseline) to 1,066 +/- 430 and 2,825 +/- 1,155 U/ml (n = 9) at 30 min and 1 h after administration of LPS, respectively (p less than 0.01 as compared with the vehicle control group). This dose of LPS reduced the survival rate to 9% at 48 h (mean survival time 9.7 +/- 2.7 h), increased heart rate (HR) to 494 +/- 18 beats/min, increased the hematocrit to 56 +/- 2 vol%, reduced the circulating platelet count at 6 h to 40% of the initial value, and produced leukopenia of 30% of the initial value at 1 h, with recovery to the initial value at 6 h. Administration of 30 mg/kg SK&F 86002 (intragastric, i.g.) 1 h before injection of LPS reduced serum TNF alpha concentrations to 92 +/- 58 and 184 +/- 117 U/ml at 30 min and 1 h (p less than 0.05), respectively, and also reduced the hemoconcentration. The survival rate was improved to 60% (mean survival time 38.1 +/- 4.3 h; p less than 0.05), although there was no effect on the hemodynamic responses to LPS. SK&F 86002 significantly reduced thrombocytopenia at 30 min and 1 h (p less than 0.05), but not at 3 and 6 h, and had no effect on changes in white blood cell (WBC) count.(ABSTRACT TRUNCATED AT 250 WORDS)

SK&F S-106203 inhibits leukotriene C4, leukotriene D4 and leukotriene E4 vasopressor responses in the conscious rat.

1. The purpose of these experiments was to investigate the effects of the selective peptidoleukotriene receptor antagonist, SK&F S-106203, on leukotriene C4 (LTC4), LTD4 and LTE4 vasopressor responses in the conscious, normotensive rat. SK&F S-106203 was administered as a bolus followed by a continuous infusion in order to provide information on the relationship between antagonism of leukotriene responses and steady-state plasma concentrations. 2. Infusion of SK&F S-106203 at doses of 0.2 mgkg-1 + 1 mgkg-1 h-1, 1 mgkg-1 + 3 mgkg- h-1 or 2 mgkg-1 + 10 mgkg-1 h-1 produced dose-dependent steady-state plasma drug concentrations of 1.0, 3.2 and 23.8 micrograms ml-1, respectively. Plasma SK&F S-106203 concentrations appeared to increase in a linear fashion at doses of 1 and 3 mgkg-1 h-1; at the highest dose the increment in plasma drug concentrations (i.e., 7-8 fold) was greater than the increment in dose (i.e., 3 fold), suggesting saturation of the primary clearance mechanism(s) at this dose. 3. SK&F S-106203 (2 mgkg-1 + 10 mgkg-1 h-1) had no effect on noradrenaline-, vasopressin-, isoprenaline-, or U 46619-induced responses. 4. SK&F S-106203 produced dose-dependent rightward shifts in the LTC4 and LTE4 dose-response curves. Administration of SK&F S-106203 at doses of 0.2mg kg1 + 1 mg kg1 h-, mg kg' + 3mgkg-'h-1, or 2mgkg-' + lOmgkg-1h'- produced dose-ratios of 1.0, 3.1 and 19.9, respectively, against LTC4 responses, and dose-ratios of 1.6, 3.8 and 9.1, respectively, against LTE4 responses. 5. Against LTD4 responses, SK&F S-106203 at doses of 0.2mgkg- + mgkg-1 h-, mg kg' + 3 mg kg- 1h - ', or 2 mg kg- + 10 mg kg- h- produced dose-ratios of 2.5, 2.8, and 11.4, respectively. Administration of D-penicillamine, a non-competitive LTD4 dipeptidase inhibitor, had no effect on LTD4 responses. 6. The similarity in the LTD4 dose-ratios at the two lower infusion rates, despite increases in the plasma drug concentrations, suggests the existence of pharmacologically heterogeneous LTD4 receptors. These results indicate that SK&F S-106203 is a potent, selective and apparently competitive antagonist of LTC4, LTD4 and LTE4 vascular responses in the intact rat.

ICI 198615 is an antagonist of leukotriene C4, leukotriene D4 and leukotriene E4 vasopressor responses in the conscious rat.

The purpose of these studies was to evaluate the effects of the peptidoleukotriene (LT) receptor antagonist, ICI 198615, on the vasopressor responses produced by LTC4, LTD4 and LTE4. Conscious, normotensive rats were prepared with arterial and venous catheters for measurement of changes in arterial blood pressure and administration of drugs, respectively. Complete dose-response curves were first generated to LTC4, LTD4 and LTE4: those agents produced dose-dependent increases in arterial blood pressure, with ED20 values (i.e. dose to increase blood pressure 20 mm Hg) of 1.7 +/- 0.2, 2.1 +/- 0.2 and 19.8 +/- 3.7 nmol/kg i.v., respectively. ICI 198615 (intravenous bolus followed by a continuous infusion) produced dose-dependent, parallel shifts to the right in the LTC4 dose-response curve. At doses of 0.2 mg/kg + 1 mg/kg/h, 1 mg/kg + 3 mg/kg/h or 2 mg/kg + 10 mg/kg/h, ICI 198615 produced dose ratios of 4.5, 17.1 and 50.0, respectively. Against LTD4 responses, ICI 198615 at a dose of 0.1 mg/kg + 0.3 mg/kg/h produced a dose ratio of 3.4, whereas at doses of 0.2 mg/kg + 1 mg/kg/h, 1 mg/kg + 3 mg/kg/h or 2 mg/kg + 10 mg/kg/h ICI 198615 produced dose ratios of 16.3, 24.9 and 16.2, respectively. The difference in the dose ratios between these three groups was not statistically significant (p greater than 0.05). However, a dose of 10 mg/kg + 30 mg/kg/h produced a dose ratio of greater than 100. Against LTE4 responses, ICI 198615 at doses of 0.2 mg/kg + 1 mg/kg/h or 1 mg/kg + 3 mg/kg/h produced dose ratios of 4.1 and 11.3, respectively. The similarity in the LTD4 dose ratios despite a 3- or 10-fold increase in the dose of ICI 198615 suggests the existence of high- and low-affinity LTD4 receptor sites, whereas the responses to LTC4 and LTE4 appeared to be mediated via a single receptor population. These results indicate that ICI 198615 is a potent and competitive antagonist of LTC4, LTD4 and LTE4 vascular responses in the rat.

Antagonism of leukotriene C4, leukotriene D4 and leukotriene E4 vasoconstrictor responses in the conscious rat with the peptidoleukotriene receptor antagonist SK&F 104353: evidence for leukotriene D4 receptor heterogeneity.

The purpose of these experiments was to investigate the effects of the selective peptidoleukotriene receptor antagonist, SK&F 104353, on leukotriene (LT)C4, LTD4 and LTE4 vasopressor responses in conscious, normotensive rats. Steady-state plasma concentrations of SK&F 104353 at infusion rates of 0.2 mg/kg + 1 mg/kg/hr, 1 mg/kg + 3 mg/kg/hr or 2 mg/kg + 10 mg/kg/hr were 0.5, 1.6 and 9.4 micrograms/ml, respectively, indicating that the plasma concentrations of SK&F 104353 were related directly to the infusion rate. LTC4, LTD4 and LTE4 (0.17-170 nmol/kg i.v.) produced dose-dependent increases in mean blood pressure. The ED20 dose (i.e., dose required to increase blood pressure 20 mm Hg) of LTC4, LTD4 or LTE4 was 2.7 +/- 0.4, 2.2 +/- 0.3 and 109 +/- 17 nmol/kg, respectively. SK&F 104353 produced dose-dependent, parallel shifts to the right in the LTC4 dose-response curve. Administration of SK&F 104353 at doses of 0.2 mg/kg + 1 mg/kg/hr, 1 mg/kg + 3 mg/kg/hr or 2 mg/kg + 10 mg/kg/hr produced dose ratios (i.e., ratio of ED20 in presence of SK&F 104353 to that of the vehicle group) of 6, 12 and 26, respectively. Against LTD4 responses, SK&F 104353 at doses of 0.1 mg/kg + 0.3 mg/kg/hr or 0.2 mg/kg + 1 mg/kg/hr produced dose ratios of 3 and 9, respectively. At a dose of 1 mg/kg + 3 mg/kg/hr, there was no further increase in the dose ratio, whereas a dose of 2 mg/kg + 10 mg/kg/hr resulted in a dose ratio of greater than 100.(ABSTRACT TRUNCATED AT 250 WORDS)

Beneficial effects of a specific leukotriene receptor antagonist in splanchnic artery occlusion shock.

The purpose of this study was to examine the effects of a new potent peptidoleukotriene receptor antagonist, SK&F 104353, in splanchnic artery occlusion shock. SK&F 104353 was administered as a 1 mg/kg initial bolus followed by an infusion of 3 mg/kg per h for the entire 2 h post-reperfusion observation period. In a group of conscious rats, this dose of SK&F 104353 shifted the LTD4 dose response curve rightward 10-fold, indicating effective antagonism of peptidoleukotriene responses in the rat. Anesthetized rats subjected to splanchnic artery occlusion shock survived an average of only 98 +/- 8 min whereas all animals receiving SK&F 104353 survived the 2 h reperfusion period (P less than 0.02 from vehicle). Therefore, the survival rate of the splanchnic artery occlusion shock group of rats receiving SK&F 104353 was improved to 100% compared with 50% survival for the vehicle-treated splanchnic artery occlusion shock group (P less than 0.025). In the splanchnic artery occlusion shock + SK&F 104353 group the increase in the plasma activities of the lysosomal hydrolase, cathepsin D, and the cardiotoxic peptide, myocardial depressant factor, were significantly attenuated in comparison to the splanchnic artery occlusion shock + vehicle group (P less than 0.025). These data indicate that the peptidoleukotriene receptor antagonist, SK&F 104353 is beneficial in splanchnic artery occlusion shock, and furthermore suggests that it may be a therapeutically useful agent in bowel ischemic shock.

Selective antagonism of peptidoleukotriene responses does not reduce myocardial damage or neutrophil accumulation following coronary artery occlusion with reperfusion.

This study was designed to assess the effect of a peptidoleukotriene receptor antagonist, SK&F 104353, for limiting myocardial damage and neutrophil accumulation in rats subjected to myocardial reperfusion injury (MI/R). In conscious rats, SK&F 10,4353 (25 mg/kg, i.v.) antagonized LTD4-induced vasopressor responses by 90% and 60% at 1 and 4 hr, respectively, indicating effective blockade of peptido-leukotriene responses. In another group of animals subjected to 30 min of coronary artery occlusion with reperfusion for 24 hr, myocardial injury and neutrophil infiltration were determined by measuring creatine phosphokinase (CPK) specific activity and myeloperoxidase (MPO) activity, respectively, in the left ventricular free wall (LVFW). Myocardial CPK levels were 8.1 +/- 0.2 U/mg protein in Sham-MI/R vehicle-treated animals, and were significantly decreased to 6.4 +/- 0.6 U/mg protein in MI/R-vehicle animals. Myocardial MPO values were 1.5 +/- 0.5 U/g LVFW in Sham-MI/R vehicle-treated animals, and significantly increased to 4.3 +/- 0.6 U/g LVFW in MI/R-vehicle animals. Administration of SK&F 10,4353 (25 mg/kg, i.v.) 1 min prior to coronary occlusion and 3.5 hr post reperfusion had no effect on the loss of myocardial CPK specific activity or the increase in MPO levels (p greater than 0.05, compared to the MI/R-vehicle group). Thus, at a dose that antagonized LTD4-induced vasopressor responses, SK&F 104353 did not attenuate either the extent of myocardial injury or inflammatory cell accumulation associated with myocardial ischemia/reperfusion. These results suggest that peptidoleukotrienes do not contribute to the progression of myocardial ischemic/reperfusion injury.

Evidence for high and low affinity leukotriene D4 receptors mediating vascular responses in the conscious rat.

Leukotriene D4 (0.17-17 nmol/kg i.v.) produced a dose-dependent increase in blood pressure in the conscious rat. Infusion of the selective peptidoleukotriene receptor antagonist, SK&F 104353, produced dose-dependent shifts in the leukotriene D4 dose-response curve. However, SK&F 104353 at doses of 0.2 mg/kg + 1 mg/kg per h or 1 mg/kg + 3 mg/kg per h produced similar dose ratios of 9.2 +/- 1.1 and 9.2 +/- 1.6, respectively. The peptidoleukotriene receptor antagonist, ICI 198615, also shifted the LTD4 dose-response curve, although doses of 0.2 mg/kg + 1 mg/kg per h, 1 mg/kg + 3 mg/kg per h or 2 mg/kg + 10 mg/kg per h produced similar dose ratios of 15.7 +/- 3.4, 19.1 +/- 6.3 and 16.2 +/- 3.6, respectively. The similarity in the dose ratios observed despite increasing doses of either SK&F 104353 or ICI 198615 suggests the existence of two vascular leukotriene D4 receptor subpopulations, differentiated by high and low agonist affinity.

Hemodynamic effects of calcitonin gene-related peptide in conscious rats.

The cardiovascular effects of calcitonin gene-related peptide (CGRP) were examined in conscious, unrestrained rats. Changes in mean arterial pressure, heart rate and cardiac output were continuously monitored before and after i.v. bolus injection of CGRP (0.1-5 micrograms/kg). Injection of the peptide caused dose-dependent reductions in mean arterial pressure (-24 +/- 4 mmHg), which were accompanied by marked tachycardia. Cardiac output was significantly increased after CGRP but little change was observed in stroke volume. CGRP also reduced total peripheral resistance (-46 +/- 6%). These data indicate that the hypotensive actions of CGRP are mediated through peripheral vasodilation rather than through reductions in cardiac output. Pretreatment with propranolol significantly reduced the tachycardia responses to CGRP from 81 +/- 11 beats/min to 36 +/- 4 beats/min, but did not abolish the increase in heart rate. These data suggest that CGRP produces a tachycardia through reflex increases in cardiac sympathetic tone and through possible direct positive chronotropic effects on the heart.

The role of perfusion washout in limb revascularization procedures.

Amputated rat hindlimbs were subjected to either normothermic (26 degrees C) or hypothermic (4 degrees C) ischemia. Experimental limbs had their microcirculation washed out (either before or after the ischemic insult) with a physiologic acellular plasma substitute previously reported to enhance flap survival following extended periods of warm ischemia. Control limbs were not washed out; i.e., stagnant blood remained in these limbs. Following the ischemic interval, amputated limbs were replanted. Monastral blue B, a colloidal pigment capable of labeling leaky blood vessels, was administered systemically to all rats just prior to vascular declamping. Limb biopsies of skin and muscle were harvested 30 minutes following revascularization in order to assess Monastral labeling and, therefore, the functional integrity of the microcirculation. Results confirm that stagnant blood under conditions of warm ischemia is detrimental to the functionality of the microcirculation in both skin (p less than 0.03) and muscle (p less than 0.007). Accordingly, perfusion washout, when performed prior to the ischemic period, enhances limb survival following 6 hours of warm ischemia (p less than 0.01). Hypothermia protects against the detrimental effects of stagnant blood; perfusion offers no benefit if hypothermic conditions prevail. Physiologic mechanisms responsible for these findings are discussed.

Delayed microcirculatory hyperpermeability following perfusion washout.

Recent experimental data have demonstrated improved flap survival following perfusion washout with a synthetic, chemically defined, mammalian plasma. In an effort to define the physiology responsible for the efficacy of perfusion, the method of "labeling" hyperpermeable blood vessels with Monastral blue B in rat epigastric vascular island flaps was utilized. Results confirmed that capillary and venular hyperpermeability is an early and progressive pathophysiologic event in ischemic flap tissue and one which is reversible prior to a critical ischemic period. Perfusion washout with a physiologic, acellular plasma substitute delays the onset of vascular hyperpermeability. This may be a mechanism responsible for improving tissue survival following extended periods of warm ischemia (12 hours). It is implied that stagnant blood and products of hemolysis in the microcirculation may be detrimental to the functional and anatomic integrity of the endothelial wall.

Preischemic flap washout and its effect on the no-reflow phenomenon.

Preischemic perfusion washout with an acellular physiologic solution delays the no-reflow phenomenon and improves tissue survival in rat epigastric free flaps following 18 and 24 hours of normothermic ischemia. This implies that stagnating blood may be a causative agent in the no-reflow phenomenon. A possible mechanism for this is capillary endothelial damage secondary to the presence of formed blood cells or their products of hemolysis. Perfusion washout may improve ischemic tolerance by preventing this blood cell-induced endothelial damage and by the prevention of sludge and thrombus. Whether any of the metabolic components of the perfusate actively enhance ischemic tolerance cannot be definitively stated.