Selectivity of BMS-180448 on myocardial versus brain blood flow in dogs and ferrets.

Openers of adenosine triphosphate (ATP)-sensitive potassium channels relax vascular smooth muscle and protect ischemic myocardium. Cromakalim and BMS-180448 are examples of this class of compounds. They are equipotent in their cardioprotective activity, but cromakalim and related compounds are extremely hypotensive, an activity that limits their use. The effects of cumulative i.v. doses of BMS-180448 or cromakalim on hemodynamics and regional blood flow (radiolabeled microspheres) were evaluated in pentobarbital-anesthetized dogs and ferrets. Both compounds significantly reduced mean arterial blood pressure, cromakalim after 0.03-0.04 mg/kg in both species, and BMS-180448 only after 10 mg/kg in dogs and 30 mg/kg in ferrets. Neither drug affected cardiac output. BMS-180448, like cromakalim, increased blood flow in the heart, with augmented regional left ventricular blood flow occurring more in the subepicardium than in the subendocardium. The effect of BMS-180448 on myocardial blood flow, in both the dog and ferret, occurred at doses that were less hypotensive than those of cromakalim. The most striking difference between the actions of these agents was seen in the brain where cromakalim, but not BMS-180448, increased blood flow in all regions. The results of these studies further demonstrate the myocardium-specific vasodilator activity of BMS-180448. Moreover, the cerebral vasodilator effect of K(ATP) openers, which has been thought responsible for the occurrence of headache in clinical trials, has been found lacking in BMS-180448; this difference may represent a clear advantage in the pharmacologic profile of the agent.

Endogenous catecholamines are not necessary for ischaemic preconditioning in the isolated perfused rat heart.

OBJECTIVE: The mechanism of the protective effect of ischaemic preconditioning in the myocardium is not yet known. The aim of this study was to test the hypothesis that endogenous myocardial catecholamines may be mediators of preconditioning. METHODS: To test whether endogenous catecholamines are involved in preconditioning, experiments were performed in hearts from rats which had been catecholamine depleted with either reserpine or 6-hydroxydopamine. Experiments were also done to determine if noradrenaline can mimic preconditioning. RESULTS: Catecholamine depletion with either reserpine or 6-hydroxydopamine had no effect on preischaemic coronary flow or cardiac function. Ischaemic preconditioning (four episodes of 5 min global ischaemia and 5 min reperfusion) resulted in a significant increase in postischaemic cardiac function and a 50% decrease in lactate dehydrogenase (LDH) release following 30 min ischaemia and 30 min reperfusion compared with non-preconditioned hearts. Reserpine pretreatment did not affect the response to ischaemia or to preconditioning, although LDH release tended to be greater than in normal hearts, especially in the non-preconditioned group. Although 6-hydroxydopamine significantly increased postischaemic cardiac function in the preconditioned group, no other index of ischaemic damage (for example, LDH release, left ventricular end diastolic pressure) was affected. Further studies showed that 10 nmol.min-1 noradrenaline did not affect the severity of ischaemia, indicating that it does not mimic preconditioning. CONCLUSIONS: Endogenous catecholamines are not necessary for ischaemic preconditioning in isolated rat hearts and play little or no role in the functional responses to ischaemia.

Inhibition of nitric oxide synthesis does not affect ischemic preconditioning in isolated perfused rat hearts.

Endothelium-derived nitric oxide (NO) has recently been reported to be a mediator of ischemic preconditioning in dog hearts. The aim of the present study was to determine the role of NO in ischemic preconditioning in isolated perfused rat hearts. Rat hearts were perfused at either constant pressure (80 mmHg) or constant flow. After aerobic perfusion (37 degrees C) for 10 min, hearts were treated with N omega-nitro-L-arginine methyl ester (L-NAME; 30 microM), which is an inhibitor of NO synthase, or vehicle. Ten minutes later, the hearts were preconditioned (4 episodes of 5 min of global ischemia and 5 min of reperfusion) or perfused normally before a 30-min global ischemic period. All hearts were reperfused for 30 min. Coronary flow or perfusion pressure plus heart rate and contractile function were measured continuously. Hearts perfused at constant pressure and treated with 30 microM L-NAME, a concentration that effectively inhibits endogenous NO synthesis, exhibited decreased coronary flow after 10 min, and flow remained decreased throughout the experiment. Ischemic preconditioning before 30 min of global ischemia resulted in a doubling of contractile function and a reduction of lactate dehydrogenase release at the end of the 30-min reperfusion period compared with nonpreconditioned hearts. The protective effect of preconditioning was not different in L-NAME-treated hearts. In addition, inhibition of NO synthase had no effect on the severity of ischemia in nonpreconditioned hearts. Similar results were obtained in preconditioned hearts that were perfused at constant flow, indicating that the flow reductions caused by L-NAME did not influence the results.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of cromakalim on skeletal muscle function and blood flow in the ferret ischemic hindlimb.

Cromakalim, an agent that opens ATP-modulated potassium channels, has recently been reported to reduce skeletal muscle contractile force during anoxia in vitro. To determine whether this activity occurs in vivo, cromakalim was tested for its ability to influence muscle function and blood flow in a model of skeletal muscle ischemia. In anesthetized ferrets the muscles of the hindlimb were stimulated electrically via the sciatic nerve and isometric force of contraction was measured. Under normal perfusion conditions, contractile force peaked (324 +/- 33 g) within 1 or 2 min and gradually declined to about 85% of the peak over 20 min. Following this initial period, the abdominal aorta was partially occluded to reduce femoral blood pressure to 35-40 mm Hg, and infusion of cromakalim or vehicle was started. After 60 min of treatment, a second exercise challenge was performed. In the vehicle-treated group, peak force was reduced by 33% (p < 0.05), and over the 20-min stimulation period, the area under the force-time curve (AUC) was 22.8 +/- 2.6% of that seen during the normal flow period. Compared to vehicle, cromakalim infusions of 1.33 or 3.0 micrograms/kg/min reduced mean arterial blood pressure by 7 and 45%, but had no significant effect on either peak force or AUC. Although the higher dose of cromakalim significantly reduced vascular resistance in resting, normally perfused skeletal muscle of the forelimbs, neither dose affected blood flow in the ischemic skeletal muscles of the hindlimb during exercise. These results suggest that cromakalim does not influence skeletal muscle blood flow or function during an acute ischemic insult.

ATP-sensitive potassium channels and skeletal muscle function in vitro.

Compounds that act at ATP-modulated potassium channels (KATP) were tested in an in vitro model of skeletal muscle ischemia. The extensor digitorum longus muscles were removed from anesthetized rats and placed in tissue baths, and contractions were elicited by electrical field stimulation at 0.2 Hz. During normoxia, the force of contraction gradually decayed to about 55% of the peak over 85 min. None of the KATP openers tested, cromakalim (300 microM), P-1075 (10 microM) and pinacidil (100 microM), affected twitch force during normoxia. However, when the muscles were made anoxic, all three compounds greatly accelerated the loss of function in a concentration-related manner. For example, the cromakalim/vehicle ratios of the area under the force-time curve during anoxia were 0.98 +/- 0.03, 0.77* +/- 0.03 and 0.72* +/- 0.04 for cromakalim at 30, 100 and 300 microM, respectively (*P < .05). Upon reoxygenation, muscles treated with the KATP openers recovered twitch force to a greater extent than those treated with vehicle. Glyburide (1 or 10 microM) had no effect on its own, but it was able to prevent fully the effects of KATP openers during both anoxia and reoxygenation, indicating that the effects of the KATP openers were mediated by KATP. These results suggest that KATP openers would not be beneficial in the treatment of skeletal muscle ischemia in vivo but that they may be useful in preserving skeletal muscle function in cases of ischemia followed by reperfusion.

Effect of in vivo inhibition of neutrophil adherence on skeletal muscle function during ischemia in ferrets.

Neutrophils are reported to play an important role in the genesis of tissue damage during reperfusion after periods of ischemia in a variety of organs and may also be involved in loss of tissue function during ischemia. To test this hypothesis, the monoclonal antibody, MoAb 60.3, which prevents the adhesion of ferret neutrophils to cultured human endothelial cells at a concentration of 30 micrograms/ml, was tested in a model of peripheral vascular disease to determine whether it would preserve skeletal muscle function during ischemia. In an anesthetized ferret the muscles of the hindlimb were stimulated electrically to contract isometrically and the force of contraction was measured. Under normal perfusion conditions the contractile force peaked within 1 or 2 min of initiation of stimulation and gradually declined to approximately 80% of peak force after 20 min. When femoral arterial pressure was reduced to 45 mmHg by partial occlusion of the abdominal aorta, peak force was reduced by 25 +/- 7%, and within 5 min the force decayed to approximately 20% of the original peak, resulting in an area under the force-time curve (AUC) of 32 +/- 5% of that seen during the normal flow period. During ischemia after treatment with MoAb 60.3 (2 mg/kg iv), peak force was 94 +/- 3% and AUC was 49 +/- 5% of that observed during the normal flow period, a significant protective effect compared with the untreated control group (P = 0.0294). When the nonneutrophil-directed monoclonal antibody, L6, was tested in this model, no protective effects were evident.(ABSTRACT TRUNCATED AT 250 WORDS)

Drug effects on function in the ferret ischemic hindlimb.

In this article a new model of peripheral occlusive arterial disease is described. The lower hindlimb of an anesthetized ferret was fixed to a holder, the distal end of the Achilles tendon attached to an isometric force transducer, and a passive preload of 100 g was applied to the muscle preparation. The hindlimb was stimulated to contract isometrically via supramaximal electrical stimulation of the sciatic nerve. During the initial period, when femoral blood pressure equaled aortic blood pressure, net contractile force peaked within 1 or 2 min (372 +/- 24g, n = 20) and gradually declined to about 85% of peak over 20 min. Following 60 min of ischemia (induced by partial occlusion of the abdominal aorta), when blood pressure in the contralateral femoral artery was about 45 mm Hg, the 15-min area under the force-time curve (AUC) was 33.2 +/- 2.5% (n = 4) of the initial value. To validate the utility of this model in the search for treatment of peripheral vascular diseases, two drugs were tested. Pentoxifylline, which is clinically effective, attenuated the loss of function observed during ischemia in a dose-related manner, but nifedipine, which is without clinical benefit, had no effect at a dose that was extremely hypotensive. Because femoral perfusion pressure was controlled, systemic hemodynamic effects of test compounds are minimized as potential mechanisms of action, simplifying the evaluation of novel pharmacotherapy for treatment of ischemic diseases.

Effects of low doses of aspirin and dipyridamole on platelet aggregation in the dog coronary artery.

The circumflex coronary artery of pentobarbital-anesthetized dogs was partially obstructed with an externally applied rigid plastic band. Platelet aggregation at the site of stenosis caused a gradual decline in blood flow in the artery, which was monitored with an electromagnetic flow probe placed proximally to the obstructor. The effects of drugs on platelet aggregation were evaluated by monitoring changes in both the rate and the degree of decline in blood flow. In most dogs, aspirin inhibited intravascular platelet aggregation (ED50 = 1 mg/kg). Dipyridamole, even at doses that severely depressed blood pressure (1 mg/kg), had no effect on platelet aggregation. However, in dogs that had been pretreated with a low dose of dipyridamole (0.2 mg/kg), the antiaggregatory activity of aspirin was enhanced. This potentiation was evident only at low doses of aspirin (0.03 and 0.1 mg/kg), where the drug was 10 times more active; at high aspirin doses, which depressed vascular cyclooxygenase, no potentiation was seen. Further evidence that the mechanism of this synergism may depend on endogenous prostacyclin production at the site of the partial obstruction was seen when cyclooxygenase inhibitors applied topically on the exposed artery eliminated the antiaggregatory effect of low doses of aspirin. It is important to note that the protective effect of dipyridamole and low-dose aspirin was less than that seen at the high doses of aspirin alone, suggesting that the theoretical benefits of platelet-specific doses of aspirin may be overstated.

Effect of pulmonary and renal circulations on activity of atrial natriuretic factor.

The effect of passage through the pulmonary and renal circulation on the activity of extracted and synthetic atrial natriuretic factors was determined by monitoring the amount of the vascular relaxing activity surviving passage through the organ. When crude atrial extract was infused through isolated perfused lungs of the guinea pig, approximately 75% of its activity survived. The activity of a 23-amino acid synthetic atrial peptide was decreased 21% on passage through these lungs, but this loss of activity was not significantly different from the crude extract. Analogous experiments in vivo showed that passage through the pulmonary circulation of the dog did not alter the activity of either the crude atrial extract or of synthetic atrial peptide. In contrast to the lack of effect of the pulmonary circulation, approximately 80% of the activity of crude atrial extract and synthetic atrial peptide was removed by the isolated perfused kidney of the rabbit, and in vivo the dog kidney removed approximately 80% of the activity of both these atrial substances. By surviving transit through the pulmonary circulation, these materials exhibit a necessary property of a circulating hormone stored in the right atrium, and the fact that the renal circulation extracts most of these materials is consistent with the kidney being a target organ of this putative hormone.

Transcapillary transport of solutes in the myocardium: effects of nitroglycerin, isoproterenol and histamine on permeability-surface area products of inulin and sucrose.

Three vasoactive drugs (nitroglycerin, isoproterenol and histamine) were examined for their effects on microsolute transport across capillary walls in the myocardium. Coronary arteries of the isolated rabbit heart were perfused at constant pressure with Tris-buffered Ringer's solution (pH = 7.4, 37 degrees C) with and without drug present in the perfusion fluid. A mixture of [3H]inulin and [14C]sucrose was injected into the left ventricular wall. From measured clearance rates, capillary permeability-surface area products (PS) (in milliliters per minute per 100 g) were computed for both solutes by the method of Gosselin and Stibitz (Pflügers Arch. 318: 85-98, 1970). Mean control PS values were 60.7 and 14.1 for sucrose and inulin, respectively. This computation required experimental determination of the myocardial volume of distribution (in milliliters per gram) for each reference solute. Values of myocardial volume of distribution obtained in the presence of nitroglycerin, isoproterenol and histamine did not differ significantly from controls. In paired clearance trials, isoproterenol and nitroglycerin significantly increased coronary flow, but neither drug influenced PS-inulin, PS-sucrose or the ratio PS inulin/PS sucrose. In contrast, histamine caused an apparently irreversible decrease in flow. Furthermore, in the presence of histamine, PS inulin/PS sucrose increased from 0.28 +/- 0.03 to 0.40 +/- 0.05 (P less than 0.003). This rise is consonant with a widening of diffusion channels between neighboring endothelial cells in the capillary wall. Thus, histamine (and presumably substances capable of histamine release) appears to increase myocardial permeability to microsolutes, in addition to its well known ability to enhance protein transport across postcapillary venules.

The failure of serum albumin to affect capillary permeability in the isolated rabbit heart.

The method of local tissue clearance was used to measure capillary permeability-surface area products (PS) for [3H]inulin and [14C]sucrose in the left ventricular wall of the isolated rabbit heart. As soon as a heart was excised, its coronary arteries were perfused with Ringer solution at 37 degrees for at least 30 min before clearance trials were begun. In paired trials, Ringer perfusion fluid containing 1% bovine serum albumin (Sigma) was compared with protein-free Ringer solution in terms of sucrose PS (PSs), inulin PS (PSi), and the PS ratio (Pi/Ps). With or without protein, the mean Pi/Ps was significantly less than the ratio of the free diffusion coefficients. With the untreated albumin, flow resistance rose markedly, and the PSs of both solutes fell but not Pi/Ps. To remove the unidentified vasoactive contaminant (which apparently resisted dialysis), the albumin was "defatted" by the procedure of R. F. Chen (1967, J. Biol. Chem. 242, 173-181). Defatted albumin (1% in the perfusion fluid) did not affect the volume of distribution (lambda) of sucrose or inulin in the myocardium, the heart rate, coronary flow, flow resistance, PSs, PSi, or Pi/Ps. Apparently bovine serum albumin does not influence capillary permeability in the rabbit heart. A protein effect on permeability, however, could have been missed if it has a long latent period (more than 15 min) or a long persistence (more than 30 min).

Capillary permeability in the isolated rabbit heart as measured by local tissue clearance.

The relatively simple method of local tissue clearance was used to measure capillary permeability-surface area products (PS) in the isolated, Ringer-perfused rabbit heart. Ten microliters of a mixture of [3H]inulin and [14C]sucrose was injected at a depth of 2 mm into the left ventricular myocardium and clearance rate constants (k in min-1) were determined by analyzing the draining perfusion fluid. PS (ml/min per 100 ml) for each solute was calculated by the following equation: PS = -F ln(1 - lambda k/F), where F is perfusate flow (ml/min per 100 ml) and lambda is the equilibrium tissue/Ringer partition coefficient. At a perfusion pressure of 40 mm Hg, F = 133 +/- 9.7 (mean +/- SEM), PSsucrose = 77 +/- 8.0, and PSinulin = 13.9 +/- 0.7. These PS products are within the range of values previously reported by others using several different techniques. The mean inulin/sucrose permeability ratio was 0.189 +/- 0.018 which is significantly less than the separately measured free diffusion coefficient ratio (= 0.41 +/- 0.005), thus indicating that sucrose and inulin crossed myocardial capillary walls by restricted diffusion. The reasons why some investigators did not find similar evidence of restricted diffusion are discussed.

A single-dose protocol for azaserine initiation of pancreatic carcinogenesis in the rat.

Previously, the induction of pancreatic carcinogenesis in the rat using azaserine has involved a multiple-dose treatment protocol. The objective of the present study was to determine the effect of multiple azaserine treatments on pancreatic DNA synthesis and to develop a protocol for a single-dose initiation of pancreatic carcinogenesis by azaserine in the rat. Pancreatic DNA synthesis in young rats, which was determined by measuring the amount of [3H]-thymidine incorporation into DNA, was found to be elevated at 4.3 weeks of age and to decrease to a baseline level by 6.3 weeks. Treatment of 4-week-old rats with azaserine resulted in a dose-dependent inhibition of [3H]-thymidine incorporation into both pancreatic and liver DNA. Maximum inhibition was seen at 10 mg/kg body weight. This inhibition was followed by a gradual return of incorporation to normal values over a 48 h period. One week following pretreatment with four weekly injections of azaserine at 30 mg/kg, [3H]-thymidine incorporation into pancreatic and liver DNA was significantly elevated, suggesting that multiple injection protocols caused enhanced DNA synthesis which could have a co-carcinogenic and/or promotional effect. Single-doses of azaserine (10, 30 and 60 mg/kg) given at 7 weeks of age caused the appearance of more atypical acinar cell nodules (AACN) than when given at 5 weeks of age. The most effective dose was 30 mg/kg. Using alkaline elution, we determined that this response was due to the occurrence of more DNA damage in the 7-week-old animals. Thus, these results demonstrate a rationale for the use of single-dose initiation protocols in the pancreas. An effective single-dose protocol for induction of AACN in azaserine-treated rats fed semi-synthetic diet is presented.