Efficacy of linezolid in treatment of experimental endocarditis caused by methicillin-resistant Staphylococcus aureus.

The efficacies of orally (p.o.) dosed linezolid and intravenously (i.v.) dosed vancomycin against methicillin-resistant Staphylococcus aureus (MRSA) in rabbits with experimental aortic-valve endocarditis were investigated. After endocarditis was established with a recent clinical MRSA isolate, rabbits were dosed for 5 days with linezolid (p.o., three times a day) at either 25, 50, or 75 mg/kg of body weight or vancomycin (i.v., twice a day) at 25 mg/kg. The 25-mg/kg linezolid group had a high mortality rate and bacterial counts in the valve vegetations that were not different from those of the controls. Linezolid dosed p.o. at 50 and 75 mg/kg and i.v. vancomycin produced statistically significant reductions in bacterial counts compared to those of the untreated controls. The reduced bacterial counts and culture-negative valve rates for the animals treated with linezolid at 75 mg/kg were similar to those for the vancomycin-treated animals. Concentrations of linezolid in plasma were determined at several points in the dosing regimen. These results suggest that the efficacy of linezolid in this infection model is related to trough levels in plasma that remain above the MIC for this microorganism. At the ineffective dose of linezolid (25 mg/kg) the concentration at sacrifice was 0.045 times the MIC, whereas the concentrations of linezolid in plasma in the 50- and 75-mg/kg groups were 2 and 5 times the MIC at sacrifice, respectively. The results from this experimental model suggest that the oxazolidinone linezolid may be effective for the treatment of serious staphylococcal infections when resistance to other antimicrobials is present.

Directed evolution of a new catalytic site in 2-keto-3-deoxy-6-phosphogluconate aldolase from Escherichia coli.

BACKGROUND: Aldolases are carbon bond-forming enzymes that have long been identified as useful tools for the organic chemist. However, their utility is limited in part by their narrow substrate utilization. Site-directed mutagenesis of various enzymes to alter their specificity has been performed for many years, typically without the desired effect. More recently directed evolution has been employed to engineer new activities onto existing scaffoldings. This approach allows random mutation of the gene and then selects for fitness to purpose those proteins with the desired activity. To date such approaches have furnished novel activities through multiple mutations of residues involved in recognition; in no instance has a key catalytic residue been altered while activity is retained. RESULTS: We report a double mutant of E. coli 2-keto-3-deoxy-6-phosphogluconate aldolase with reduced but measurable enzyme activity and a synthetically useful substrate profile. The mutant was identified from directed-evolution experiments. Modification of substrate specificity is achieved by altering the position of the active site lysine from one beta strand to a neighboring strand rather than by modification of the substrate recognition site. The new enzyme is different to all other existing aldolases with respect to the location of its active site to secondary structure. The new enzyme still displays enantiofacial discrimination during aldol addition. We have determined the crystal structure of the wild-type enzyme (by multiple wavelength methods) to 2.17 A and the double mutant enzyme to 2.7 A resolution. CONCLUSIONS: These results suggest that the scope of directed evolution is substantially larger than previously envisioned in that it is possible to perturb the active site residues themselves as well as surrounding loops to alter specificity. The structure of the double mutant shows how catalytic competency is maintained despite spatial reorganization of the active site with respect to substrate.

Progress toward the development of a safe and effective agent for treating reentrant cardiac arrhythmias: synthesis and evaluation of ibutilide analogues with enhanced metabolic stability and diminished proarrhythmic potential.

A series of ibutilide analogues with fluorine substituents on the heptyl side chain was prepared and evaluated for class III antiarrhythmic activity, metabolic stability, and proarrhythmic potential. It was found that fluorine substituents stabilized the side chain to metabolic oxidation. Many of the compounds also retained the ability to increase the refractoriness of cardiac tissue at both slow and fast pacing rates. The potential for producing polymorphic ventricular tachycardia in the rabbit model was dependent on the chirality of the benzylic carbon. The S-enantiomers generally had less proarrhythmic activity than the corresponding racemates. One compound from this series (45E, trecetilide fumarate) had excellent antiarrhythmic activity and metabolic stability and was devoid of proarrhythmic activity in the rabbit model. It was chosen for further development.

Efficacy of linezolid in a staphylococcal endocarditis rabbit model.

A rabbit endocarditis model was used to test the efficacy of oral linezolid and iv vancomycin. Twenty-four hours after catheter placement across the aortic valve, rabbits were infected with 3.5 x 10(6) cfu of Staphylococcus aureus (UC-9258). Two days after infection, control rabbits were killed, and treated rabbits were given 5 days of therapy with linezolid at 8 h intervals (tds) using either 25, 50 or 75 mg/kg/dose, or vancomycin at 12 h intervals (bd) using 25 mg/kg/dose. Linezolid at 75 and 50 mg/kg, and vancomycin significantly reduced S. aureus in aortic valve vegetations compared with the control. Linezolid at 25 mg/kg was ineffective. The efficacy of 75 and 50 mg/kg linezolid was related to maintenance of plasma drug levels near or above the linezolid MIC for UC-9258 (2 mg/L).

Initiating a structural study of 2-keto-3-deoxy-6-phosphogluconate aldolase from Escherichia coli.

2-Keto-3-deoxy-6-phosphogluconate aldolase (KDPG aldolase, E.C. 4.1. 2.14) is a member of the pyruvate/phosphoenolpyruvate aldolase family. It is also a synthetically useful enzyme, capable of catalyzing the stereoselective aldol addition of pyruvate to a range of unnatural electrophilic substrates. The recombinant protein was purified by a two-step HPLC protocol involving anion-exchange and hydrophobic chromatography. Dynamic light-scattering experiments indicated the protein to be monodisperse. Crystals were obtained using the sitting-drop vapour-diffusion method, with PEG 6K as precipitant. Diffraction data were collected on a frozen crystal to a resolution of 2.26 A on station PX9.6 at the Daresbury synchrotron. The crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 53.2, b = 77.9, c = 146.8 A.

Initiating a crystallographic study of UDP-galactopyranose mutase from Escherichia coli.

UDP-galactopyranose mutase, the enzyme responsible for the conversion of UDP-galactopyranose to UDP-galactofuranose, has been crystallized in a form suitable for X-ray diffraction studies. UDP-galactofuranose is a key component of mycobacterial cell walls. Crystals of both the native protein and a selenomethionine variant have been grown by the vapour-diffusion method in hanging drops, and diffract to beyond 3.0 A using synchrotron radiation. Equilibration was against a solution of 20%(w/v) polyethylene glycol (4K), 12%(v/v) 2--propanol, 0.1 M HEPES pH 7.6 at 293.5 K. Crystals grow as thin plates of dimensions 0.4 x 0.2 x approximately 0.02 mm. They are monoclinic [corrected], space group P21, with unit-cell dimensions a = 71. 12, b = 58.42, c = 96.38 A, beta = 96.38 degrees. 92% (native) and 94% (selenomethionine) complete data sets have been recorded to 2.9 A (Rmerge = 5.0%) and 3.0 A (Rmerge = 6.9%), respectively. The Matthews coefficient is 2.35 A3 Da-1 for a dimer in the asymmetric unit, the solvent content being 47%. Diffraction data have also been recorded on a putative platinum derivative to 3.5 A.

Tissue and species variation in the vascular receptor binding of 3H-P1075, a potent KATP opener vasodilator.

A high-affinity receptor site for 3H-P1075 previously observed in rat aorta has been proposed to mediate the vasorelaxation effects of P1075 and other ATP-sensitive K+ channel (KATP) openers. We tested this hypothesis by correlating the receptor binding of 3H-P1075 with its vasorelaxation effects in several isolated vascular preparations from three species: rat, rabbit and dog. In rat aorta and mesenteric artery, 3H-P1075 (1-5 nM) showed high amounts of specific binding (5-10 fmol/mg tissue), which was 48 to 79% of total binding. In contrast, little (< or = 17%) to no specific binding of 3H-P1075 (1-5 nM) was observed in dog coronary artery, dog mesenteric artery or rabbit mesenteric artery. However, all vascular preparations studied relaxed with P1075 (1-100 nM), showing maximal relaxations at 30 to 100 nM. The P1075 relaxation EC50 values in rat aorta, rabbit mesenteric artery and dog coronary artery ranged from 7.5 to 24.1 nM depending on the level of contractile activation. Thus, the pharmacological effect of P1075 could be correlated with the presence of specific receptor binding sites only in rat vascular preparations. These data show that there are significant differences in the characteristics of the proposed specific receptor site for 3H-P1075 in different vascular preparations from different species, and they raise questions regarding the pharmacological significance of this KATP opener binding site. Until such questions are resolved, it appears that the study of functional significance of this receptor site as well as further biochemical characterization of this receptor site may necessitate the use of only the rat vascular preparations.

Cardiovascular effects of the K-ATP channel blocker U-37883A and structurally related morpholinoguanidines.

The cardiovascular effects of the K-ATP channel blocker U-37883A and 5 related morpholinoguanidines were determined in 6 experimental preparations. In anesthetized dogs, U-37883A (0.5-8.0 mg/kg i.v.) increased mean arterial pressure (MAP; +18%) and left ventricular (LV) effective refractory period (ERP; +35%), and decreased LV contractility (-41%). Higher doses of U-37883A (16-32 mg/kg) fatally reduced MAP (-84%), heart rate (HR; -57%) and LV contractility (-72%). In anesthetized rats, U-37883A (1.0-50 mg/kg i.v.) also maximally reduced MAP, HR and LV contractility by 68, 77 and 48%, respectively. U-37883A and its analogs were diuretic in conscious rats (1.5-15 mg/kg i.v.) and blocked pinacidil in rabbit mesenteric artery (EC50 = 0.5-50 microM). In rabbit papillary muscle, 50 microM U-37883A significantly reduced force of contraction (-33%) and prolonged conduction time (+244%). Milder papillary effects were seen with the N'-OH analog U-45194A, which did not depress LV contractility in intact rats. In conscious dogs, oral U-45194A (50 mg/kg) was diuretic but reduced LV stroke volume and increased peripheral vascular resistance. These studies characterize U-37883A's systemic cardiovascular and direct myocardial effects, and identify U-45194A as a less cardiac depressant analog having U-37883A-like diuretic and functional K-ATP channel blocking activities.

Antiarrhythmic and electrophysiologic effects of intravenous ibutilide and sotalol in the canine sterile pericarditis model.

INTRODUCTION: Atrial arrhythmias are a frequent clinical complication following open heart surgery. We compared the Class III agents d,l-so-talol and ibutilide fumarate in an intravenous cross-over study using the canine atrial sterile pericarditis model. METHODS AND RESULTS: We studied pacing-induced sustained atrial flutter over a 7-day post-surgical period in conscious dogs, alternating analysis of ibutilide (1.0 to 30.0 micrograms/kg) and d,l-sotalol (0.1 to 3.0 mg/kg). Ibutilide significantly increased atrial flutter cycle length (AFL CL) 11 +/- 2 msec and atrial effective refractory period (AERP) 13 +/- 2 msec, and terminated atrial flutter in all cases (n = 12) following a mean dose of 6 +/- 2 micrograms/kg. Plasma concentrations of ibutilide were 53 +/- 13 ng/mL. Ventricular effective refractory period (VERP) was not significantly affected (4 +/- 2 msec). Following termination with ibutilide, atrial flutter could be reinitiated in 1 of 12 trials, and was nonsustained (40-sec duration). Sotalol significantly increased AFL CL 23 +/- 3 msec and terminated atrial flutter in 8 of 12 trials following a mean dose of 1.5 +/- 0.4 mg/kg. AERP and VERP were significantly increased 20 +/- 6 and 12 +/- 2 msec, respectively. The incidence of reinduced atrial flutter was 9 of 12 trials (P < or = 0.05 vs ibutilide) (7 nonsustained 57 +/- 7 sec duration, and 2 sustained). Sotalol failed to terminate atrial flutter in two dogs on days 1 and 5, despite increases in AFL CL (21 +/- 8 msec) and AERP (16 +/- 9 msec), whereas on day 3, ibutilide (20 +/- 7 micrograms/kg) terminated atrial flutter in those two dogs while increasing AFL CL and AERP 18 +/- 6 and 15 +/- 0 msec, respectively. CONCLUSION: Both sotalol and ibutilide terminate atrial flutter in this model. Ibutilide converted atrial flutter in dogs in which sotalol was not successful. Following atrial flutter termination, ibutilide had a lower incidence of reinduced arrhythmias compared to sotalol. Ibutilide produced atrial antiarrhythmic effects while having no significant electrophysiologic effects on the ventricle.

Acute intravenous conversion of canine atrial flutter: comparison of antiarrhythmic agents.

We studied the acute intravenous (i.v.) effects of the antiarrhythmic agents ibutilide, sematilide, lidocaine, and encainide in a canine Y-shaped right atrial incision model of atrial flutter. After baseline determination of atrial effective refractory period (AERP), sustained atrial flutter (AFL) was initiated by atrial pacing in 25 dogs. Each dog then received placebo, followed by sequential doses of a particular agent every 5 min until AFL was terminated or the highest dose was administered. Ibutilide increased AFL cycle length (CL: 26 +/- 6 ms) before termination of AFL in 8 of 8 dogs in 1 min after the effective dose (6 +/- 1 micrograms/kg). AERP determined immediately after termination of AFL was significantly increased (44 +/- 7 ms). AFL could not be reinitiated after termination with ibutilide. Sematilide increased AFL CL 12 +/- 6 ms, significantly increased AERP 25 +/- 6 ms, and terminated AFL in 4 of 8 dogs (0.6 +/- 0.2 mg/kg). After sematilide or pacing-induced termination, AFL was reinducible in only 1 dog. Lidocaine terminated AFL in 2 of 5 dogs (2.0 +/- 0.5 mg/kg). AERP was unchanged in both animals, and AFL CL increased by 37 +/- 3 ms. AFL was reinducible in all 5 lidocaine-treated dogs after AFL was terminated. Encainide terminated AFL in 3 of 4 dogs after a dose of 3.0 +/- 0.0 mg/kg. AERP was significantly increased 77 +/- 10 ms, and AFL CL was markedly increased by 226 +/- 11 ms. AFL could not be reinitiated after termination of AFL by encainide.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of adenosine on atrial refractoriness and arrhythmias.

OBJECTIVE: Transient atrial fibrillation is sometimes observed following adenosine administration and adenosine is known to shorten atrial action potential duration and refractory period. This study was designed to characterise the dose-response relationship of adenosine on these variables relative to arrhythmia induction with single atrial premature stimuli. The effects of adenosine during sustained atrial flutter were also determined. METHODS: Intravenous bolus doses of adenosine were given to pentobarbitone anaesthetised dogs following cervical vagotomy and autonomic blockade with atropine and nadolol. Monophasic action potential catheter recordings were obtained from the right atrium and a programmable stimulator was used for pacing. RESULTS: Placebo had no effect on monophasic action potential duration (MAPD) or atrial effective refractory period (ERP) and no arrhythmias were observed. Adenosine (0.1-1.0 mg.kg-1) produced dose related decreases in MAPD and ERP and transient atrial fibrillation (5-122 s) was repeatedly and reproducibly induced in 12 dogs. In six dogs, intravenous dipyridamole (0.25 mg.kg-1) enhanced the effects of adenosine on MAPD and ERP and increased the incidence of atrial fibrillation. In another six dogs, 8-sulphophenyltheophylline (5.0 mg.kg-1, intravenously) markedly blunted the effects of adenosine, and atrial fibrillation could no longer be induced by premature stimuli. In a separate series of experiments the effects of adenosine were evaluated in seven dogs in which sustained atrial flutter could reproducibly be induced by rapid atrial pacing. Administration of placebo never caused termination of the arrhythmia, whereas intravenous boluses of adenosine (0.1-1.0 mg.kg-1) decreased and produced variation in atrial flutter cycle length, and then terminated the arrhythmia in all cases. These effects of adenosine were also enhanced by dipyridamole and antagonised by 8-sulphophenyltheophylline. CONCLUSIONS: Adenosine produces a receptor mediated shortening of monophasic action potential duration and refractoriness which increases vulnerability to transient atrial arrhythmias. During sustained atrial flutter, these effects may also contribute to destabilisation and termination of the arrhythmia.

Comparative effects of the potassium channel openers cromakalim and pinacidil and the cromakalim analog U-89232 on isolated vascular and cardiac tissue.

ATP-sensitive potassium (K+ATP) channel openers such as cromakalim and pinacidil exhibit both potent vasodilatory and anti-ischemic properties. U-89232, a cyanoguanidine analog of cromakalim, has recently been found to exhibit myocardial protection during ischemia without altering in vivo hemodynamics. We examined the effects of U-89232, cromakalim and pinacidil in isolated vascular and cardiac tissue and tested whether glyburide, a KATP channel blocker, could antagonize their effects. All three compounds produced concentration-dependent relaxation in isolated vascular segments, with cromakalim being approximately 100-fold more potent than either pinacidil or U-89232. Glyburide completely antagonized the effects of pinacidil but merely blunted the effects of cromakalim and U-89232. In an isolated rabbit cardiac tissue preparation, U-89232 had little effect on maximum tension in cardiac muscle, whereas cromakalim and pinacidil significantly decreased maximum developed tension in a concentration-dependent manner. Glyburide effectively antagonized the effects of cromakalim and pinacidil in cardiac tissue. These data suggest that U-89232, although chemically related to cromakalim, possesses activity which is not common to known potassium channel openers.

Comparative assessment of ibutilide, D-sotalol, clofilium, E-4031, and UK-68,798 in a rabbit model of proarrhythmia.

Class III agents have been associated with development of a polymorphic ventricular tachycardia (PVT) known as torsades de pointes. We compared the class III agent ibutilide, which prolongs repolarization through enhancement of an inward sodium current, with the potassium channel blockers E-4031, UK-68,798, clofilium, and D-sotalol for proarrhythmic effects in an anesthetized rabbit model. In these animals, prolongation of repolarization during alpha 1 stimulation with methoxamine produces early after depolarizations (EADs) and a pause-dependent torsades de pointes-like PVT. Agents were compared over dosage ranges that produced maximal increases in QTc interval and monophasic action potential duration (MAPD). PVT typically developed after atrioventricular (A-V) conduction block and slowing of heart rate (HR), and was preceded by development of repolarization arrhythmias characterized by EADs and triggered activity producing extrasystolic beats. Ibutilide administration resulted in significantly lower EAD amplitudes and a lower incidence of repolarization arrhythmias and PVT as compared with administration of other class III agents. The percentage of rabbits developing PVT for each agent was ibutilide 12%, D-sotalol 70%, E-4031 56%, UK-68,798 69%, and clofilium 80%. Rabbits receiving saline vehicle instead of a class III agent never developed conduction or repolarization abnormalities or PVT. Under the conditions of this study at doses that generate maximal class III effects, ibutilide produces lesser increases in QTc interval and MAPD, and EADs of lower amplitude, resulting in a lower incidence of repolarization arrhythmias and PVT as compared with other class III agents.

Antiarrhythmic and electrophysiologic effects of ibutilide in a chronic canine model of atrial flutter.

We studied the effects of orally administered ibutilide, a class III antiarrhythmic agent, in a model of reentrant atrial flutter in conscious dogs. After baseline determination of atrial effective refractory period (AERP) and demonstration of reproducible induction of atrial flutter by rapid atrial pacing, 8 dogs received either placebo or one of six doses of ibutilide ranging from 0.1 to 5 mg/kg. Refractory periods and the ability to induce atrial flutter were then assessed at periodic intervals for 8 hours. Ibutilide produced dose-related increases in AERP which were well correlated with prevention of initiation of atrial flutter after doses > or = 0.25 mg/kg. Placebo and 0.1 mg/kg ibutilide had no effect on AERP or the ability to induce atrial flutter. Doses of 0.25 to 1.0 mg/kg ibutilide significantly increased AERP and prevented induction of atrial flutter for 4-6 h. After treatment with 2.5 or 5 mg/kg ibutilide, significant increases in AERP and prevention of induction of atrial flutter persisted throughout the 8-h study period. The cycle length of inducible atrial flutter was significantly increased after administration of 5 mg/kg ibutilide. The results demonstrate oral efficacy of ibutilide with rapid onset of action (in 30-60 min), resulting in increased AERP and prevention of induced atrial flutter in this model.

Evidence that S-nitrosothiols are responsible for the smooth muscle relaxing activity of the bovine retractor penis inhibitory factor.

Inhibitory factor (IF), an extract of the bovine retractor penis muscle, when treated with acid, becomes a vasodilator with properties similar to endothelium-derived relaxing factor (EDRF). EDRF has been proposed to be nitric oxide (NO), long known to be a potent vasodilator. Recently, biologically active IF was proposed to be NO, as well, generated by acid activation of inorganic nitrite. We compared acid-activated IF with acid-activated nitrite and found that NO formation was not sufficient to explain the properties of acid-activated IF. Endothelium-denuded rings of rabbit aorta were used to test the smooth muscle-relaxing properties of IF and nitrite. Although both IF (0.5 ml) and nitrite (1 microM) relaxed phenylephrine-contracted rabbit aorta to a similar extent after acid activation (approximately 30%), several significant differences were observed. IF was most active when acid activated by a 5-min, pH 2 step followed by neutralization; nitrite was relatively inactive when acid activated in this manner, and was most active when assayed immediately after acidification to pH 2. Purging with argon for 5 min reduced the smooth muscle-relaxing activity of 1.0 microM nitrite from 27 +/- 2 to 10 +/- 2% relaxation, whereas the activity of IF was not changed by argon purging (control, 31 +/- 2% relaxation; argon purged, 34 +/- 2% relaxation). When IF samples were assayed for nitrite content, the amount of nitrite found (0.5-5 nmol/0.5 ml sample) was not sufficient to explained the observed smooth muscle relaxing activity. Furthermore, acid-activated IF significantly stimulated cyclic GMP production by platelet-soluble guanylate cyclase from 3.2 +/- 0.2 to 12.4 +/- 0.4 pmol/min/mg protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ibutilide on spontaneous and induced ventricular arrhythmias in 24-hour canine myocardial infarction: a comparative study with sotalol and encainide.

The electrophysiologic and antiarrhythmic effects of ibutilide, sotalol, and encainide were compared in dogs 24 h after myocardial infarction. Ibutilide (0.03 to 0.3 mg/kg i.v.) prevented the induction of ventricular arrhythmias in 100% of the dogs that had demonstrated inducible ventricular arrhythmias prior to treatment. This antiarrhythmic action was associated with significant increases in ventricular refractoriness and monophasic action potential duration. Sotalol (1.0 to 10.0 mg/kg i.v.) increased the ventricular refractory period and monophasic action potential duration and prevented the induction of ventricular arrhythmias in 75% of the dogs that demonstrated inducible ventricular tachyarrhythmias at baseline. Although 10 mg/kg of sotalol was required to prevent the initiation of ventricular tachycardia, this dose produced marked cardiovascular depression and hypotension in 50% of the dogs tested. Neither ibutilide nor sotalol significantly decreased the incidence of spontaneous ventricular arrhythmias. The class IC agent encainide (0.3 to 3.0 mg/kg i.v.) was successful in preventing the induction of ventricular arrhythmias in only 20% of the dogs tested. However, in contrast to ibutilide and sotalol, encainide significantly reduced spontaneous arrhythmias. Atrial and ventricular refractoriness were significantly increased only after the highest dose of encainide tested (3.0 mg/kg). Over the dose ranges studied, the relative efficacy for prevention of pacing-induced ventricular arrhythmias was ibutilide greater than sotalol much greater than encainide. For suppression of spontaneous ventricular arrhythmias, the relative efficacy was encainide much greater than ibutilide = sotalol.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of U74006F, a novel inhibitor of lipid peroxidation, in stunned reperfused canine myocardium.

U74006F, a novel 21-amino steroid is a potent inhibitor of iron-mediated lipid peroxidation and has been shown to be of therapeutic benefit in central nervous system ischemia. As oxygen radicals have been implicated in the development of postischemic myocardial dysfunction, we examined the efficacy of U74006F to enhance the recovery of function in a canine model of stunned, reperfused myocardium. Twenty-six dogs were randomized to either a vehicle (n = 11), U74006F (n = 10), or U74006F-paced group (n = 5). U74006F (6 mg/kg i.v.) was administered 15 min prior to coronary artery occlusion. Myocardial blood flows were measured by the microsphere technique, and function data were obtained by sonomicrometry. Both U74006F-treated groups demonstrated a significant increase in posterior wall thickening as compared to the vehicle treatment (U74006F-paced, 27.0 +/- 12.8%; U74006F, 22.4 +/- 11%; vehicle, -13.5 +/- 9.9%, p less than 0.001 following 3 h of reperfusion). Enhanced function recovery was accompanied by lower heart rates in the U74006F-treated group following reperfusion (treated versus vehicle, 109 +/- 6.7 versus 131 +/- 8.8 beats/min, p = 0.004). The U74006F-paced group was maintained at the same rate as the vehicle group, with no diminution in function recovery compared to the unpaced group. No effects in systemic hemodynamics or nutrient blood flow were evident as a function of drug treatment. We conclude that pretreatment with U74006F enhances the recovery of function in stunned canine myocardium via the inhibition of oxygen radicals and lipid peroxidation products. This activity suggests that this compound represents a new therapeutic adjunct in reperfusion and recanalization therapies.

In vivo characterization of synthetic thromboxane A2 in canine myocardium.

Recently, total chemical synthesis of thromboxane was achieved. The in vitro activity of synthetic thromboxane A2 is indistinguishable from biologically generated material. The present study describes the in vivo characterization of synthetic thromboxane A2 on the regional blood flow distribution of the canine heart. Local injections of synthetic thromboxane A2 into the coronary vasculature caused marked reductions in coronary blood flow, measured by both radiolabeled microsphere injection and an electromagnetic flow device. The threshold concentration required to bring about this effect varied greatly between dogs and ranged from 0.125 microgram/kg to 2.0 micrograms/kg. Similarly, the dose of thromboxane A2 required to aggregate dog platelets in vitro varied from 30 ng/ml to 1,000 ng/ml. Bolus injections of 2 micrograms/ml thromboxane A2 into the circumflex or left anterior coronary artery resulted in a simultaneous reduction in platelet count in coronary sinus blood of 83 +/- 5.2% (mean +/- SEM, n = 4, p = .0005). Both flow reduction and platelet effects were transient and localized. The time taken from onset to recovery of the response to control levels was 77 +/- 6.0 seconds (mean +/- SEM) for flow and 70-80 seconds for platelet count. Injections of thromboxane A2 caused a small but significant increase in heart rate with no change in systemic blood pressure. In conclusion, the in vivo actions of synthetic thromboxane A2 are consistent with the vasoconstrictor and platelet aggregatory effects seen in vitro, but dogs vary considerably in their sensitivity.