Inhibition of alloxan action in isolated pancreatic islets by superoxide dismutase, catalase, and a metal chelator.

The possible participation of superoxide anions, hydrogen peroxide, and hydroxyl radicals in the action of alloxan was investigated using isolated rat pancreatic islets. Exposure of islets for 5 min to alloxan (0.15 or 0.2 mg/ml) inhibited subsequent glucose-stimulated insulin release. The presence of superoxide dismutase (1000 U/ml), catalase (50 microgram/ml), or a metal chelator diethylenetriaminepentacetic acid (1 mM) markedly attenuated this effect of alloxan. Use of these agents afforded complete protection from the lower concentration of alloxan and partial protection from the higher concentration of the toxic compound. Inactivation of the enzymes or addition of excess iron to the chelating agent before its use with alloxan eliminated the protective action of these agents. The results are consistent with the proposal that hydroxyl radicals, generated via reactions that involve superoxide anions, hydrogen peroxide, and iron, mediate the deleterious effect of alloxan in pancreatic islets.

Impaired insulin release after exposure of pancreatic islets to autooxidizing dihydroxyfumarate.

Autooxidizing dihydroxyfumarate (DHF) is a known generator of superoxide anions and by hydroxyl free radicals. Isolated rat pancreatic islets were exposed for 5 min to 0.4 mg/ml DHF in an oxygenated buffer solution. As a result of this exposure, the islets exhibited a 60% reduction in glucose-stimulated insulin release. The presence of superoxide dismutase, catalase, or diethylenetriaminepentaacetic acid, a metal chelator, during the exposure period protected the islets from the effects of autooxidizing DHF. This suggests that superoxide anions, hydrogen peroxide, and, ultimately, hydroxyl free radicals play a role in the insulin inhibitory effects caused by DHF exposure. Glucose (5 mg/ml), but not galactose, was also capable of protecting islets from DHF. The effects of DHF exposure on isolated islets appear to be similar to those previously reported for alloxan and lend support to the concept that hydroxyl free radicals generated during the oxidation of certain compounds can alter endocrine cell function.

Blockade of human and porcine myocardial 5-HT4 receptors by SB 203186.

We investigated the blockade of the positive inotropic effects of 5-hydroxytryptamine (5-HT) by SB 203 186 (piperidinoethyl-indole-3-carboxylate hydrochloride) and its affinity for 5-HT4 receptors of human right atrium and piglet left atrium. We also compared the blocking effects of SB 203 186 against 5-HT-evoked tachycardia in anaesthetised adult Yucatan minipigs as well as new-born Camborough piglets. SB 203 186 caused competitive antagonism of the positive inotropic effects of 5-HT in electrically paced atrial preparations of man (pKB = 8.9) and piglet (pKB = 8.5) at concentrations (up to 0.3 micromol/l) which were devoid of depressant or stimulant effects. The affinity of SB 203 186 for atrial 5-HT4 receptors was 30-160 times higher than that of tropisetron. 5-HT caused tachycardia with similar potency and efficacy in Yucatan minipigs and new-born Camborough piglets. SB 203 186 (0.1-3 mg/kg, i.v.) surmountably antagonised 5-HT-evoked tachycardia in anaesthetised Yucatan minipigs or new-born Camborough piglets with similar potency. The blocking potency of SB 203 186 in Yucatan minipigs was 17 times higher than that of tropisetron. Intraduodenally administered SB 203 186 (0.3-3 mg/kg) to new-born Camborough piglets produced blockade of 5-HT-evoked tachycardia which was maximal after 20 min and lasted for more than 3 h with 0.3 mg/kg. The antagonism produced by the SB 203 186 administration in new-born Camborough piglets was dose-related and threefold greater through the intravenous route than through the intraduodenal route. We conclude that SB 203 186 is an antagonist with nanomolar affinity for both human and porcine atrial 5-HT4 receptor. The in vivo results demonstrate that the sinoatrial 5-HT4 receptors function is similar in new-born Camborough piglets and adult Yucatan minipigs. Both porcine breeds are valid models for human atrial 5-HT4 receptors as demonstrated with the antagonist SB 203 186.

Spin trapping of ibuprofen radicals: evidence that ibuprofen is a hydroxyl radical scavenger.

The purpose of this study was to use electron paramagnetic resonance (EPR) spectroscopy to determine if ibuprofen, [2-(4-isobutylphenyl) propanoic acid], a potent nonsterodial anti-inflammatory agent, could modify hydroxyl radicals generation in vitro. Ibuprofen (IBU; 0.1-50 mM) in water or water alone was added to EPR tubes containing ferrous sulfate (0.5-2.0 mM), and either 5,5-dimethyl-1-pyrroline-N-oxide (DMPO; 40 mM) or alpha-phenyl N-tert-butyl nitrone (PBN; 48 mM). Hydrogen peroxide (1 mM) was added to inititate the Fenton reaction, and the systems were then analyzed by EPR spectroscopy to determine the type and relative quantity of free radical(s) produced. IBU caused a dose-dependent decrease of signal intensity of the hydroxyl radical adduct of DMPO (DMPO-OH) which is an indication that IBU either scavenges the hydroxyl radical and/or chelates iron. In addition, other radicals (presumably IBU radicals) produced in these systems were trapped by both DMPO (aN = 16.1 G, aH beta = 24.0 G) and PBN (aN = 15.7 G, aH beta = 4.4 G and aN = 17.0 G, aH beta = 2.1 G). The signal height of these IBU radicals increased in systems containing ferrous sulfate (1 mM), hydrogen peroxide (1 mM), PBN (48 mM), and increasing IBU concentrations. Therefore, we conclude that IBU scavenges the hydroxyl radical. If IBU chelated iron, then less hyroxyl radicals would be generated, less IBU radical formed and the signal height of IBU radicals trapped by PBN would have decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Endotoxin-induced mortality in rats is reduced by nitrones.

The goal of these investigations was to determine if nitrone spin-trapping agents can alter mortality associated with endotoxemia in the rat. Reactive free radicals attack nitrone spin-trapping agents forming relatively reactive, persistent free radical spin adducts. We administered 85 mM (10 ml/kg) of alpha-phenyl N-tert-butyl nitrone (PBN), alpha-4-pyridyl-N-oxide N-tert-butyl nitrone (4-POBN), 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), or vehicle (saline i.p.) 30 min before endotoxin (25 mg/kg i.p.) or vehicle to Sprague-Dawley (SD) or Holtzman virus-free (HVF) rats (n = 10-17/group). All vehicle-treated rats receiving endotoxin were dead by 1 day. At 7 days, 83% of PBN-treated SD, 42% of PBN- or POBN-treated HVF, and 25% of DMPO-treated HVF rats were alive. The difference in survival of PBN-treated animals between strains may reflect the higher susceptibility of HVF rats to endotoxin. The observed reduction in mortality may be related to the well-established capacity of spin-trapping agents to capture reactive free radicals that may be generated in target tissues in response to endotoxin, and that would otherwise react with cell components and produce tissue injury.

GMP-140 mediates adhesion of stimulated platelets to neutrophils.

In vivo, platelets associate with neutrophils at sites of hemorrhage or inflammation. In vitro, stimulated platelets bind to neutrophils in a Ca2(+)-dependent manner. GMP-140, an integral membrane glycoprotein found in secretory granules of platelets and endothelium, is rapidly translocated to the cell surface after cellular activation. It shares sequence similarity with two leukocyte adhesion molecules, ELAM-1 and a lymphocyte homing receptor. We have recently shown that neutrophils bind to purified GMP-140 in a Ca2(+)-dependent fashion, and that GMP-140 participates in adhesion of neutrophils to activated endothelium. In this study we demonstrate that GMP-140 also mediates adhesion of neutrophils to stimulated platelets. Fixed thrombin-activated human platelets, but not unstimulated platelets, formed rosettes around neutrophils in the presence of Ca2+. The binding of platelets to neutrophils was inhibited by a monoclonal antibody to GMP-140 and by purified GMP-140. By promoting close cell-cell contact, GMP-140 may recruit both platelets and neutrophils to sites of tissue injury as well as modulate the function of each cell type by the other.

A new model of myocardial infarction in Yucatan minipigs.

Myocardial infarction studies in pigs have been complicated by the use of antiarrhythmic drugs or the high incidence of ventricular fibrillation. We report on a new model of experimental myocardial infarction in thiamylal-anesthetized Yucatan minipigs. These studies were performed in the absence of intravenous antiarrhythmic drugs. No animals required resuscitation during either surgery or reperfusion and only 19% were resuscitated during occlusion. Extensive systemic hemodynamic, regional contractility and coronary blood flow measurements were continuously obtained during left anterior descending coronary artery (LAD) occlusion (45 min) and reperfusion (240 min). Mean arterial blood pressure and left ventricular + dP/dt decreased during occlusion, and both declined further upon reperfusion. Persistent dysfunction (segmental shortening from 24.8 +/- 1.3 to 3.9 +/- 0.9% (p less than 0.001); pre-occlusion and 5 min post-occlusion, respectively) occurred immediately after occlusion in the myocardium perfused by the LAD, while late declines in segmental shortening (19.6 +/- 0.9 to 17.2 +/- 1.2%; pre-occlusion and 240 min post-reperfusion, respectively) were observed in myocardium perfused by the left circumflex coronary artery. While heart rate did not change during occlusion, tachycardia occurred at the onset of reperfusion. Although initial reactive hyperemia following reperfusion was manually inhibited, high LAD blood flow following reperfusion occurred early (0 to 60 min) but returned below pre-occlusion values late (180 to 240 min). The area at risk represented 23.1 +/- 0.9% (n = 34) of the left ventricle and 39.0 +/- 3.2% of this area was infarcted. Therefore, 9.2 +/- 0.9% of the left ventricle was infarcted. These data suggest that myocardical infarction in anesthetized minipigs can be achieved without the aid of intravenous antiarrhythmic drugs and reduced cardioversion. Therefore, this new model can be utilized in the evaluation of therapeutic compounds focused on altering the detrimental consequences of myocardical infarction.

Hemodynamic effects of naloxone on hemorrhagic shock in the beagle.

Naloxone reverses the hypotension in various types of hemorrhagic shock models. What has yet to be firmly established is the mechanism by which naloxone reverses the hypotension. In a canine hemorrhagic shock model, impedance cardiography and invasive methods were used to measure various cardiovascular parameters. All dogs (beagles, 10-15 kg) were bled to and maintained at a mean arterial blood pressure (MAP) of 60 mmHg for 90 min and were then given either naloxone (2 mg/kg; n = 6) or an equivalent volume of saline (n = 6) intravenously (IV). After another 90 min observation period, the shed blood was reinfused. No significant differences in the preshock and shock cardiodynamics were noted between the naloxone and the control animals. During the treatment period, MAP was significantly increased in the naloxone group. There was no increase in cardiac output (CO), stroke volume (SV), end diastolic volume (EDV), dP/dt max, dP/dt/P, or HI (the impedance contractility index) over control animals. The most significant parameter improvement was total peripheral resistance (TPR). The data suggest that naloxone in this hemorrhagic shock model improves hemodynamics primarily by increasing vascular resistance.

Effects of osmotic gradients on water and solute transport: in vivo studies in rat duodenum and ileum.

We examined effects of luminal osmolality on net water and solute movements in rat duodenum and ileum. Solutions of sodium chloride (permeating solute) or mannitol (nonpermeating solute) at hypo-, iso-, or hyperosmotic concentrations were recirculated through in situ segments. Luminal osmolality increased towards that of plasma with hyposmotic solutions of both solutes. With isosmotic solutions, luminal osmolality did not change with sodium chloride, but increased with mannitol. With hyperosmotic solutions, luminal osmolality always decreased toward that of plasma with sodium chloride; with mannitol, however, decreases were significant only when initial concentrations were above 400 mosmol/kg. The decrease in osmolality of hyperosmotic sodium chloride resulted from sodium absorption and water secretion. Thus, both hypo- and hyperosmotic solutions of sodium chloride adjusted toward isomolality with plasma by the usual mechanisms of water and solute movement. With mannitol, however, osmotic adjustment of hypertonic luminal contents was restricted or even absent due to movement of sodium down its concentration gradient and reduced hydraulic conductivity of the gut.

The effects of vasoactive mediator antagonists on endotoxic shock in dogs. I.

Many vasoactive mediators have been implicated in causing or maintaining the hypotension of endotoxic shock. What has yet to be firmly established is the relative importance of each of these mediators in a given shock model. In a canine endotoxic shock model (LD100), we studied the effects of opiate and prostaglandin inhibition 60 min after endotoxin administration. After thiopental anesthesia, the animals were instrumented to measure various cardiovascular parameters. Endotoxic shock was induced by injecting E. coli endotoxin (0111:B4) (1 mg/kg i.v.). Drug intervention occurred 60 min after endotoxin administration. Naloxone (2 mg/kg i.v.) improved mean arterial pressure (MAP) transiently. A more significant increase of MAP (85% of preshock levels) was attained after ibuprofen (12.5 mg/kg i.v.) administration secondary to an increase in total peripheral resistance (TPR). All groups had 0% 24-hour survival. These data suggest that the endogenous opioids, presumably inhibited by naloxone, seem to contribute little to this lethal canine endotoxic shock model. By contrast, the prostanoids which are inhibited by ibuprofen appear to be more hemodynamically significant in this model.

Myocardial protection with carvedilol.

Carvedilol is a multiple-action cardiovascular agent that is both a beta-adrenoceptor antagonist and a vasodilator and has recently been made available for the treatment of mild-to-moderate hypertension. Clinical trials are ongoing to establish the efficacy of carvedilol in angina and congestive heart failure. beta-Adrenoceptor antagonists are known to reduce myocardial work secondary to reductions in heart rate and contractility; accordingly, they have been shown to be cardioprotective in animals and in humans. Because carvedilol has beta-adrenoceptor antagonist activity, it also should provide significant cardioprotection. The additional vasodilating activity of carvedilol, which will further reduce myocardial work by decreasing afterload and myocardial wall tension, should provide more salvage of ischemic myocardium than that afforded by a pure beta-adrenoceptor antagonist, such as propranolol. We investigated the ability of carvedilol and propranolol to reduce infarct size in experimental models of acute myocardial infarction in the rat, pig, and dog. The left anterior descending coronary artery was occluded for 30 (rat) or 45 min (pig) and then reperfused for 24 h (rat) or 4 h (pig). In the dog, the left circumflex coronary artery was occluded for 60 min and reperfused for 24 h. Vehicle, carvedilol, or propranolol was administered intravenously 15 min before ischemia (and, in the rat only, repeated 4 h after ischemia). An additional group of dogs was subjected to permanent left anterior descending coronary artery occlusion for 6 h, and carvedilol or propranolol was given 15 min after occlusion. Infarct size was examined on stained tissue sections using quantitative image analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Carvedilol (Kredex) reduces infarct size in a canine model of acute myocardial infarction.

Carvedilol (Kredex) is a multiple action, antihypertensive agent that may also prove to be useful in the treatment of angina and congestive heart failure. Carvedilol combines in one molecule both beta-adrenoceptor blocking and vasodilating activities. Inasmuch as beta-adrenoceptor blocking agents are known to be cardioprotective and thereby reduce infarct size, it is logical to assume that carvedilol, likewise, would possess this desirable activity. Furthermore, the additional vasodilating activity of carvedilol could contribute to further reductions in infarct size by reducing myocardial work (and therefore myocardial oxygen demand) through reductions in both afterload and myocardial wall tension. As such, we have investigated the ability of carvedilol to reduce infarct size in a canine model of acute myocardial infarction. Carvedilol (1 mg/kg i.v.) or its vehicle, dimethylformamide, were administered 15 min before left circumflex coronary artery (LCX) occlusion. Following 1 h of LCX occlusion, dogs were reperfused through a critical stenosis and then allowed to recover for 24 h. Carvedilol-treated animals exhibited a 78% reduction in infarct size compared to vehicle controls, such that the percentage of the left ventricle infarcted was reduced significantly from 16.2 +/- 4.1% in control animals to 3.6 +/- 1.3% in animals treated with carvedilol (p = 0.017, n = 6). Stained tissue sections of the left ventricle were photographed, digitized and color-enhanced using an Image Analysis Computer System, and three-dimensional reconstruction of the left ventricle, including the infarcted areas, was performed.(ABSTRACT TRUNCATED AT 250 WORDS)

Benoxaprofen attenuation of lethal canine endotoxic shock.

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

An assessment of plasma histamine concentrations during documented endotoxic shock.

Recent reviews of the literature involving histamine release during sepsis and endotoxemia have reported that the majority of the studies are inconclusive due to inadequate assays or experimental protocols. In a controlled experimental setting we have employed a specific and sensitive radioenzymatic assay to determine plasma histamine concentrations temporally during documented endotoxin-induced shock in the conscious rat. Cardiovascular and metabolic measurements for the control group (n = 7) were normal during the study period. Endotoxin (n = 8, LD/90-24 hrs.) induced an early transient hypotensive episode and increase in systemic vascular resistance and a sustained decrease in cardiac index and central venous pressure and increase in heart and respiratory rates. Hypoglycemia and hyperlacticemia were present at the end of the four-hour study period. The small intestine was severely hemorrhaged in all animals given endotoxin. Histamine concentrations for the control group were unchanged throughout the study period. Contrary to previous reports, this model of endotoxemia revealed unchanging histamine concentrations during the first 30 minutes which were temporally coincident with the characteristic early hypotensive episode evoked by endotoxin. The histamine concentrations at 60 and 240 minutes following endotoxin were increased two and three-fold, respectively, compared to the control group. Three of the 8 rats given endotoxin died before four hours; histamine concentrations in plasma taken when death appeared certain were 42, 91, and 174, compared to the control value of approximately 8 ng/ml. There was no clear association of the increases in plasma histamine with any of the parameters measured in this study: however, established histamine effects may have been masked by the pre-existing effects of other mediators known to be active during endotoxemia. In separate groups of animals endotoxin (n = 5) elicited early increases in plasma concentrations of norepinephrine (5-fold) and epinephrine (8-fold) that remained elevated for the 4-hour study period while the control group (n = 4) remained stable. This study establishes that a) plasma histamine concentrations are increased during endotoxemia, b) plasma histamine is not elevated during the initial hypotension episode following endotoxin, c) plasma histamine increases during the progression of endotoxic shock, and d) plasma histamine concentrations are extremely high prior to death.