Diastolic coronary resistance in the isolated rabbit heart during and after ischaemia: contribution of extravascular forces.

Coronary blood flow is influenced by contraction of the myocardium. The contribution of extravascular forces to coronary resistance during and after a period of low pressure perfusion or hypoxia and after a period of global ischaemia was studied in experiments on rabbit hearts perfused by the Langendorff method. End diastolic flow, perfusion pressure, developed tension, and resting tension were measured. Under control conditions the relation between end diastolic flow and perfusion pressure was almost linear (r greater than 0.92). There was an initial fall in coronary resistance on initiation of hypoxia or low pressure perfusion, but subsequently coronary resistance increased concomitantly with the rise in resting tension. After 60 min total global ischaemia and 30 min reperfusion coronary resistance increased by 410(99)% and resting tension by 382(92)%. Increasing the potassium concentration of the perfusate to 19.2 mmol X litre-1 under control conditions caused the coronary resistance to increase by 336(74)%, whereas resting tension was unchanged. The increase in resistance with potassium could be reduced but not abolished by adenosine (1.87 X 10(-5) mol X litre-1), indicating that the effect was largely attributable to vasoconstriction. Increased stimulation frequency (30 Hz) raised coronary resistance by 37(10)% and resting tension by 303(53)%. Perfusion with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (1 X 10(-6) mol X litre-1) increased coronary resistance by 169(27)% and resting tension by 782(11)6% and relaxed isolated epicardial coronary arteries. The effects of carbonyl cyanide p-trifluoromethoxyphenylhydrazone and increased stimulation frequency on coronary resistance were due to myocardial contracture.(ABSTRACT TRUNCATED AT 250 WORDS)

The resistance of low density lipoprotein to oxidation promoted by copper and its use as an index of antioxidant therapy.

The measurement ex vivo of the resistance of low density lipoprotein (LDL) to oxidation promoted by copper is now being used in surveys of human populations at risk of developing atherosclerosis. However, it is not known whether a relationship between LDL oxidisability measured in this way and the development of atherosclerotic lesions exists. Using Watanabe rabbits as a model of the disease, we have found that dietary supplementation with the antioxidants, probucol and alpha-tocopherol, increased the resistance of LDL isolated from small volumes of plasma to oxidation. The antioxidant effects of probucol incorporated into LDL through dietary supplementation were greater than when incorporated ex vivo. When dietary supplementation was extended to a period of three months, the well established anti-atherosclerotic effects of probucol were confirmed and a highly significant relationship between the probucol content of the LDL particle and the extent of the atherosclerotic lesion in the aorta emerged. These results suggest that the assessment of the resistance of LDL isolated from plasma to oxidation promoted by copper may reflect the response of the arterial atherosclerotic process to antioxidant therapy.

The adventitia and atherogenesis: removal initiates intimal proliferation in the rabbit which regresses on generation of a 'neoadventitia'.

Removal of the carotid artery adventitia from rabbits induced the formation of an intimal hyperplastic lesion. In rabbits fed a normal diet, the lesion (measured as the intimal:medial ratio) was maximal by day 14 (0.456 +/- 0.079, n = 5, P < 0.01) and thereafter, regressed towards control dimensions (0.037 +/- 0.003, n = 14) by day 28 (0.080 +/- 0.025, n = 7, P = 0.14). In rabbits fed a high cholesterol diet, the lesion was again maximal by day 14 (0.376 +/- 0.056, n = 8, P < 0.01). Although some regression was seen, the lesion persisted to day 42 (0.272 +/- 0.052, n = 8, P < 0.01). Electron microscopy and immunocytochemistry showed two types of lesion, (a) smooth muscle cell predominant on normal diet and, (b) macrophage predominant on high cholesterol diet. Smooth muscle cell predominant lesions underwent almost complete regression, whereas macrophage predominant lesions persisted. We propose that lesion formation may be initiated following the development of arterial wall hypoxia, secondary to excision of the adventitial vasa vasorum. Furthermore, we have devised a novel method to restore a highly vascular 'neoadventitia' to an artery whose adventitia has previously been removed, using loosely placed PVC tubing. We suggest this 'neoadventitia' was able to inhibit the formation of an intimal hyperplastic lesion and to promote regression of an already established lesion by restoring arterial wall oxygenation.

Purinoceptors in the rat heart.

The effects of an intracoronary bolus of adenosine triphosphate (ATP), alpha, beta-methylene ATP (APCPP), beta, gamma-methylene ATP (APPCP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and adenosine on coronary tone and ventricular myocardial contraction were investigated in the perfused rat heart. Adenine nucleotides, given by bolus injection were negatively inotropic in amounts greater than 3 X 10(-7) mol. The potency order was ATP greater than ADP greater than AMP. Adenosine (less than 1 X 10(-5)mol) had no effect on ventricular myocardial contraction. Adenine nucleotides and adenosine (1 X 10(-10)-1 X 10(-7) mol) reduced coronary tone. The potency order was ATP greater than ADP greater than AMP = adenosine. The ATP analogue APPCP was less active than ATP at reducing coronary tone, and APCPP had no vasodilator effect. This suggests the presence of a P2-purinoceptor, subclass P2Y, which mediates vasodilation. ATP and ADP increased the concentration of prostacyclin (measured as 6-keto prostaglandin F1 alpha) in the perfusate, but only after injection of greater than 3 X 10(-7) mol, suggesting that the vasodilator responses to ATP and ADP were not mediated by prostacyclin. AMP and adenosine had no effect, even at 1 X 10(-5) mol. At a dose of 3 X 10(-9) mol, approximately 40% of ATP and 70% of ADP was converted to AMP and adenosine whilst passing through the heart. The amounts of AMP and adenosine formed, however, were insufficient to account for the vasodilator effects of ATP and ADP. 6 Vasodilatation mediated by AMP and adenosine was inhibited by an infusion of 8-phenyltheophylline (8-PT; 2 x 10-5 M) indicating interaction with a P1-purinoceptor. Vasodilatation induced by ATP (at doses at which AMP and adenosine had no action) was also depressed by 8-PT indicating either an action of ATP on PI-purinoceptors, or an effect of 8-PT on P2y receptors. 7 Vasodilatation induced by AMP was unaltered during an infusion of alpha,beta-methylene ADP (2 x 10-6 M, which inhibited breakdown of AMP to adenosine by 54.2 +/- 1.5%, n = 4). This suggests that AMP acted directly, and it did not require conversion to adenosine to induce vasodilatation. 8 The ATP analogues APCPP (1 x 10-9_1 x 10-8 mol) and APPCP (1 x 10-8_l x 10-7mol) increased coronary tone, as did high doses (I x 10-5 mol) ofATP and ADP, indicating the presence of an additional P2-purinoceptor, subclass P2X, mediating vasoconstriction.

The effects of radiographic contrast media on myocardial contractility and coronary resistance: osmolality, ionic concentration, and viscosity.

With the ongoing development of new contrast agents, questions develop concerning the cardiac effects of these drugs. We used the perfused rat heart model to investigate the effects on cardiac and coronary function of hypertonic ionic (sodium chloride) and nonionic (glucose) solutions and conventional and low osmolality radiographic contrast media (RCM). We also evaluated the concurrent effects of RCM on prostacyclin and adenine nucleotide/nucleoside release. Hypertonic solutions of glucose had little effect on myocardial contraction (increase up to 7.7 +/- 0.9%), while NaCl solutions of similar osmolality were negatively inotropic (contractile force decreased up to 76.1 +/- 9.2%). Conventional RCM were negatively inotropic (decrease of 59.6 +/- 5.6% with Conray (Mallinckrodt Pharmaceuticals, St. Louis, MO), 32.2 +/- 3.2% with Angiovist 282 (Berlex Laboratories, Cedar Knolls, NJ]; two nonionic RCM, Iopamidol and Iotrol had little effect on myocardial contraction (reduction of 6.9 +/- 1.4% and increase of 12.0 +/- 2.9%, respectively). Hypertonic solutions of glucose and NaCl reduced coronary resistance in direct relationship to hyperosmolality. Conventional RCM also reduced coronary resistance, while the nonionic media caused minor alteration. None of the solutions tested altered prostacyclin or adenine nucleotide/nucleoside efflux from the heart. A solution of Ficoll 70 with a viscosity similar to that of RCM increased myocardial contraction by 9.6 +/- 3.6% and had no effect on coronary resistance, indicating that viscosity per se did not contribute to the negative inotropic effects or the reduction in coronary resistance. Hypertonic solutions, including conventional RCM, reduce coronary resistance as a result of their hyperosmolality Negative inotropic effects, however, are more related to high ionic concentration than to osmolality.

Purines and prostaglandins as regulators in the coronary circulation.

In the isolated rat heart perfused at constant flow a bolus injection of an adenine nucleotide reduced coronary perfusion pressure and was negatively inotropic (ATP greater than ADP greater than AMP). Adenosine had no effect on perfusion pressure or contractile force. ATP and ADP stimulated production of PGI2 from the coronary bed. Perfusion with indomethacin completely inhibited PGI2 production but had no effect on the reduction in perfusion pressure and cardiac function produced by ATP. Purinoceptors in the perfused rat heart are of the P2 type and responses produced by purinoceptor activation are not mediated by PGI2.

Diastolic coronary resistance after ischaemia in the isolated rabbit heart: effect of nifedipine.

The effect of nifedipine on tension, coronary flow and perfusion pressure was studied in the Langendorff rabbit heart after 15 and 60 min global ischaemia. Nifedipine (1.44 X 10(-8) M) added to the perfusate before 15 min ischaemia prevented the increase of diastolic coronary resistance which occurred on reperfusion in the absence of the drug. The recovery of force of contraction was unaltered. There was no change in the increase in resistance on reperfusion if nifedipine (1.44 X 10(-8) M) was given at the time of reperfusion after 15 min ischaemia. Ventricular fibrillation on reperfusion was prevented if nifedipine was added before 15 min of ischaemia. After a more prolonged period of ischaemia (60 min) the rise of resting tension on reperfusion was not prevented by giving nifedipine (1.44 X 10(-8) M or 1.44 X 10(-7) M) before ischaemia, although the rise during ischaemia was delayed. Both concentrations of nifedipine reduced the increase in diastolic coronary resistance which occurred on reperfusion. These results suggest that nifedipine, in a concentration close to the therapeutic range, increases coronary reperfusion after global ischaemia. This represents one mechanism by which nifedipine can have a beneficial effect on the ischaemic myocardium.

Involvement of the renin-angiotensin system in ischemic damage and reperfusion arrhythmias in the isolated perfused rat heart.

We have investigated the contribution of the renin-angiotensin system to the damage caused by 40-min global ischemia in the isolated rat heart. A converting enzyme inhibitor, enalaprilat (70 nM), an angiotensin II receptor antagonist, compound 89 (2 microM), and an inhibitor of rat renin, CGP 44099A (20 nM), given before ischemia reduced the median duration of ventricular fibrillation on reperfusion to a similar extent (5.53, 5.72, and 5.14 min, respectively, compared to 13.98 min in the control group) but had no effect on creatine phosphokinase release (22.2 +/- 2.6, 22.1 +/- 6.8, and 24.1 +/- 3.6, IU/30 min, respectively, compared to 19.9 +/- 1.9 IU/30 min) or recovery or left ventricular developed pressure (67 +/- 6, 73 +/- 7 and 71 +/- 6%, respectively, compared to 66 +/- 3% after 30 min reperfusion). The increase in coronary resistance and left ventricular diastolic pressure on reperfusion was not affected by any of the agents. All three agents also tended to reduce the duration of ventricular fibrillation when given only on reperfusion. We conclude that angiotensinogen is present in the rat heart and it is converted to angiotensin I by a renin or a renin-like aspartic proteinase. The angiotensin I is converted to angiotensin II by converting enzyme. The angiotensin II formed is an important mediator of postreperfusion ventricular fibrillation in the isolated rat heart but does not contribute to the reduction in mechanical function produced by global ischemia in this preparation.

Mechanical response of rabbit myocardium and coronary arteries to leukotriene D4. Failure to demonstrate a role in the pathophysiology of hypoxia.

We have studied the effect of leukotriene D4 (LTD4) on rabbit and rat myocardial contractility and on rabbit coronary arteries. A concentration of 2 X 10(-7) M caused only a small reduction of myocardial contractility, but caused a contraction of smooth muscle in coronary arteries similar to that obtained with potassium (30 mmoles/liter). LTD4 (2 X 10(-7) M) added to the perfusate 10 min before or at the time of reoxygenation after a period of 30 min of hypoxia did not alter contractility or resting tension. LTD4 is unlikely to be a contributing factor in the initiation of cell necrosis on reoxygenation of hypoxic myocardium.

Absence of a relationship between extracellular potassium accumulation and contractile failure in the ischemic or hypoxic rabbit heart.

Ischemia and hypoxia both cause a rapid loss of potassium from myocardial cells. We have investigated the relationship between the accumulation of potassium in the extracellular fluid and the early loss of contractility. Experiments were performed on the isolated rabbit heart perfused with physiological saline at 36 degrees C, paced at 3 Hz. Tension was recorded from the apex. Extracellular potassium concentration [( K+]o) was recorded with small ion-selective electrodes. After the onset of global ischemia, [K+]o rose within 15 sec and reached 9.5 +/- 1.1 mmoles/liter after 5 min. Developed tension (T) fell to 9 +/- 2% of control over the same period. During substrate-free hypoxia, T declined at a similar rate, and [K+]o rose slowly to 5.5 +/- 0.1 mmoles/liter after 5 min. The relationship between [K]o and T during normal perfusion and oxygenation was investigated by incrementally increasing the perfusate [K+]. T dropped to 78.6 +/- 4.5% of control at a [K+]o of 9 mmoles/liter. Comparison of the relationship between [K+]o and T during high-potassium perfusion, ischemia, and hypoxia shows that extracellular potassium accumulation per se makes almost no contribution to the decline of contractile function in ischemia or hypoxia. (Values are means +/- S.E., N = 5.)

Changes in the caldesmon isoform content and intimal thickening in the rabbit carotid artery induced by a silicone elastomer collar.

The presence of a silicone elastomer collar around one carotid artery of a rabbit induces thickening of the tunica intima. We used immunoblotting to study quantitatively changes in the isoforms of caldesmon, a protein implicated in the regulation of contractility in smooth muscle, while also monitoring the histological changes during 28 days after collaring. Control rabbit carotid arteries (n = 28) contained 245 +/- 6.4 nmol/g protein of the larger isoform of caldesmon (CDh) and 68.3 +/- 3.6 nmol/g protein of the smaller isoform (CD1). Four days after collaring, intimal thickening was slight, but 44% of arterial CDh had been lost; this loss of CDh was therefore from the tunica media. At 10 days, CDh fell to 37% of the control level. Immunofluorescence using CDh-specific antibodies showed that the CDh level was diminished but remained uniform across the wall of collared arteries. At 14 days, when intimal thickening was maximal, there was 30% more CD1 than in controls. At 28 days, the neointima had thinned, and CD1 had fallen to below control levels. Thus, CD1 levels reflected the development and regression of neointima. Changes in caldesmon isoforms showed that smooth muscle cell phenotypic changes occurred throughout the arterial wall.

Kinetics of adenine nucleotide catabolism in coronary circulation of rats.

We have used the rat isolated, perfused heart to study the metabolism of adenine nucleotides on a single passage through the coronary circulation. Low doses (3-30 nmol) of ATP, ADP, or AMP injected as a bolus were extensively catabolized by ectoenzymes. Increasing doses of each nucleotide demonstrated saturability of catabolism that occurred at significantly lower doses of AMP than of ADP or ATP. The patterns of catabolites formed in each case were consistent with the major pathway of metabolism being sequential dephosphorylation of ATP----ADP----AMP----adenosine, although from experiments in which [3H]ATP was co-injected with unlabeled ADP, it appears that some direct conversion of ATP----AMP can occur. Furthermore, particularly in the presence of excess unlabeled ATP, [3H]ADP was phosphorylated to [3H]ATP, indicating that ectoenzymes capable of interconverting nucleotides are present. By evaluating recovery and metabolism in serial samples collected rapidly after bolus injection, we were able to use the integrated form of the Michaelis-Menten equation as developed by Bronikowski et al. (Math. Biosci. 61: 237-266, 1982) to derive Michaelis constant (Km) and maximum velocity times capillary plasma volume (Amax) values for adenosinetriphosphatase, adenosine diphosphatase, and 5'-nucleotidase (450, 300, and 93 microM; and 5.3, 5.9, and 1.7 mumol/min, respectively). This analysis also indicated that there is a high degree of heterogeneity of path lengths within the coronary circulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular inhibition of human neutrophil elastase by orally active pyrrolidine-trans-lactams.

Described are the acylation binding of trans-lactam 1 to porcine pancreatic elastase, the selection of the SO2Me activating group for the lactam N which also confers metabolic stability in hamster liver microsomes, the introduction of aqueous solubility through the piperidine salt 9, the in vivo oral activity of 9 and its bioavailability, and the introduction of 9 as an intracellular neutrophil elastase inhibitor.

The discovery of a potent, intracellular, orally bioavailable, long duration inhibitor of human neutrophil elastase--GW311616A a development candidate.

The discovery of a potent intracellular inhibitor of human neutrophil elastase which is orally active and has a long duration of action is described. The pharmacodynamic and pharmacokinetic properties of a trans-lactam development candidate, GW311616A, are described.