Characterization and modulation of antigen-induced effects in isolated rat heart.

The response to antigen (trinitro-phenyl-haptenized ovalbumin) and the modulatory role of several antiallergic drugs was studied in isolated hearts from actively sensitized rats. Antigen induced a triphasic effect on coronary flow (CF) and left ventricular pressure (LVP) characterized by short-term increase (0-1.5 min = phase 1) and a severe decrease (1.5-7.5 min = phase 2) followed by a less pronounced long-lasting decrease (7.5- greater than 20 min = phase 3). The first phase was accompanied with a substantial release of 5-hydroxytryptamine (5-HT), histamine, and leukotrienes measured in cardiac effluents. The histamine2 (H2)-receptor antagonist cimetidine (60 microM) reversed the antigen-induced increase in CF to a decrease. In contrast, H1-receptor blockade by mepyramine (6 microM) had no effect. Methysergide (10 microM) and ketotifen (0.1 microM) evoked a mild suppression during all three phases. Indomethacin (10 microM) was almost inactive while tolfenamic acid (1 microM) was slightly active in this respect during phase 2. Addition of the 5-lipoxygenase inhibitor AA 861 (1 microM) resulted in complete suppression of the antigen-induced decrease in CF. The leukotriene antagonist FPL 55712 (5 and 50 nM) evoked a dose-dependent suppression with respect to the anaphylactic phases 2 and 3. A similar reduction was obtained with sodium cromoglycate (1 mM). AA 861, FPL 55712, and sodium cromoglycate also suppressed the antigen-induced decrease in LVP. The antigen-induced histamine release was not affected by the aforementioned drugs. Our results provide evidence that H2-receptor blockade during cardiac anaphylaxis enhances coronary constriction and may be detrimental in this condition. On the other hand, leukotriene antagonists and 5-lipoxygenase inhibitors may exert beneficial effects during cardiac anaphylaxis. Further experiments in this area are needed to clarify the precise role of mast cell-generated mediators in cardiac anaphylaxis possibly leading to new therapeutic approaches in this life-threatening disorder.

Cardiovascular responses to the stereoisomers of dobutamine in isolated rat hearts 48 hours after acute myocardial infarction.

Effects of acute myocardial infarction (48 h) on cardiovascular responses to (+/-)-, (-)-, and (+)-dobutamine were studied in isolated perfused rat hearts. The effects of the racemate and the isomers on ventricular pressure were measured simultaneously in infarcted left ventricle and noninfarcted right ventricle. Administration of (+/-)-, (-)-, and (+)-dobutamine resulted in dose-dependent increases in inotropic parameters and coronary flow (CF) in both control and in infarcted hearts. As compared with the (+)-isomer, the racemate and the (-)-isomer in both control and infarcted hearts were approximately 1.5 and 15 times weaker with respect to inotropic parameters. For (+/-)-, (-)-, and (+)-dobutamine, there was no significant difference in pD2 values in any of the inotropic measurements between the infarcted group and the corresponding control group. The maximal obtainable responses were significantly lower in the infarcted groups as compared with their corresponding control group. In control hearts, effects of isoproterenol and methoxamine as compared with (+/-)-dobutamine were approximately 2 log units more and 2 log units less in potency, respectively. The inotropic effects of (-)-dobutamine but not those of methoxamine were completely antagonized by propranolol (0.3 microM). Although the results provide evidence for the existence of myocardial alpha 1-adrenoceptors, all forms of dobutamine exerted positive inotropic effects through activation of beta-adrenoceptors both in control and in infarcted in rat hearts.

Effects of milrinone, sulmazole and theophylline on adenosine enhancement of antigen-induced bronchoconstriction and mediator release in rat isolated lungs.

The effects of adenosine and some of its analogues on bronchoconstriction and mediator release were studied in isolated lungs of actively sensitized rats. The influence of two novel cardiotonic drugs, milrinone and sulmazole on these adenosine-induced effects was compared with that of theophylline, a well known adenosine antagonist. Adenosine (ADO) and its analogues N-ethyl-carboxamide-adenosine (NECA) and R-phenyl-isopropyladenosine (R-PIA), dose-dependently enhanced antigen-induced bronchoconstriction. The enhancement of anaphylactic bronchoconstriction by adenosine and its analogues was accompanied by a rise in histamine release. The rank order of potency for adenosine and analogues with respect to enhancement of anaphylactic bronchoconstriction, was NECA greater than or equal to R-PIA greater than ADO. An unequivocal classification of the adenosine receptor involved, was therefore not possible. Dipyridamole and S-(p-nitrobenzyl-6-thioinosine) (NBTI), both inhibitors of adenosine uptake, had no inhibitory influence on the adenosine-induced enhancement of anaphylactic bronchoconstriction, indicating that this enhancement is mediated by an extra-cellular receptor. Theophylline, milrinone and sulmazole inhibited the enhancement of anaphylactic bronchoconstriction, without affecting preformed mediator release. Theophylline and sulmazole were both more effective as inhibitors of adenosine-enhanced bronchoconstriction than as inhibitors of antigen-induced bronchoconstriction, suggesting adenosine antagonism. Milrinone was equi-effective as inhibitor of both types of bronchoconstriction. Since adenosine antagonism has been associated with the side effects of theophylline it will be interesting to further investigate the therapeutic merits of novel cyclic nucleotide phosphodiesterase inhibitors in the treatment of asthma.

Cardiovascular responses to milrinone in pertussis toxin-pretreated pithed rats.

The modulating effects of pertussis toxin on angiotensin II and B-HT 920-evoked hemodynamic changes were compared with those of milrinone to evaluate the possible role of guanine nucleotide regulatory proteins (G proteins) in the mechanism of action of milrinone. Both milrinone and pertussis toxin shifted the blood pressure dose-response curves of B-HT 920 to the right, but the responses to angiotensin II were decreased after milrinone pretreatment only. The increase in cardiac frequency evoked by milrinone and isobutylmethylxanthine (IBMX) was not sensitive to pertussis toxin. In contrast, the decrease in systolic blood pressure elicited by milrinone could be prevented by pertussis toxin pretreatment, suggesting the involvement of a regulatory protein. Milrinone and IBMX did not influence the effects of arecoline on blood pressure or heart rate in either normal or pertussis toxin-pretreated rats. It is concluded that milrinone may affect a G protein, but not the adenylate cyclase-associated inhibitory protein, Gi.

Modulation of adrenoceptor-mediated cardiovascular effects by short-term in vivo infusion of isoproterenol in rats.

After a 16-h in vivo infusion period of isoproterenol (400 micrograms/kg/h) from a minipump implanted subcutaneously (s.c.) in rats, we observed an increase in heart weight due to tissue edema. In isolated perfused heart preparations, the EC50s of isoproterenol to induce positive inotropy and increase in coronary flow were increased approximately 7 and 4 times, respectively. Isoproterenol dose-response curves performed in trachea preparations, were shifted to the right by about fivefold as compared with the control group. In the presence of phentolamine, the isoproterenol induced maximal relaxation in the isolated aorta from isoproterenol-pretreated animals was reduced from 46.5 to 9.2% as compared with saline-pretreated rats. In the absence of phentolamine, the epinephrine (EPI) and norepinephrine (NE) cumulative dose-response curves in this preparation were not affected by isoproterenol pretreatment with respect to EC50 and maximal effect. However, in the presence of phentolamine, the contractile response to a supramaximal dose of NE (10 microM) amounted to 30 and 70% in the saline- and isoproterenol-pretreated rats, respectively. In the presence of both phentolamine and propranolol, a similar response of 70% was observed by this supramaximal dose of agonist in the saline-pretreated rats as well. In anesthetized rats, 120 min after removal of the minipumps, sodium nitroprusside induced increase in heart rate (HR) was reduced after isoproterenol pretreatment, whereas for salbutamol the decrease in diastolic blood pressure (DBP) was also reduced. As compared with salbutamol, a marked increase was observed in the ratio of mean arterial blood pressure (MAP) to HR for SNP after isoproterenol pretreatment. The phenylephrine-induced increase in MAP was increased after isoproterenol pretreatment. We conclude that in the pathogenesis of heart failure the beta-adrenoceptor-mediated effects of catecholamines are attenuated and alpha-adrenoceptor-mediated effects become progressively important.

Adenosine enhances antigen-induced bronchoconstriction and histamine release in rat isolated lungs.

The effects of adenosine and some of its analogues on bronchoconstriction and mediator release were studied in isolated lungs of actively sensitized rats. Adenosine (ADO) and its analogues R-phenyl-iso-propyl-adenosine (R-PIA) and N-ethyl-carboxamide-adenosine (NECA), enhanced antigen-induced bronchoconstriction in a dose-dependent manner. The enhancement of anaphylactic bronchoconstriction by adenosine and its analogues was accompanied by a rise in histamine release. The observed rank order of potency for adenosine and analogues (NECA greater than or equal to R-PIA greater than ADO) did not permit an unequivocal classification of the adenosine receptor involved. Dipyridamole and S-(p-nitrobenzyl-6-thioinosine) (NBTI), both inhibitors of adenosine uptake, had no inhibitory influence on the adenosine-induced enhancement of anaphylactic bronchoconstriction. Therefore, this enhancement was likely to be mediated through an extra-cellular receptor. Theophylline inhibited the enhancement of anaphylactic bronchoconstriction by adenosine in a concentration-dependent manner, without affecting preformed mediator release.

Modulation of histamine release by rat peritoneal mast cells in a superfusion set-up.

A new method is presented for studying mediator release in isolated peritoneal cells of the rat. Using a superfusion technique, cells can be maintained in a continuously cleared medium. Cells were stimulated with antigen, compound 48/80 or A23187 to characterize this preparation. In addition, inhibitory effects of theophylline, isoprenaline and cromoglycate on compound 48/80-induced histamine release were studied. We conclude that superfusion is a valid alternative to study mediator release from isolated peritoneal cells.

Plasma elimination of milrinone in rats in relation to its hemodynamic effects.

Milrinone (1,6-dihydro-2-methyl-6-oxo[3,4'-bipyridine]-5-carbonitrile) is a cardiotonic drug, currently under clinical investigation for the treatment of congestive heart failure. Its positive inotropic properties and its strong vasodilative action contribute to a favorable therapeutic effect. Plasma elimination of milrinone is generally by renal excretion of the unchanged drug. Since hemodynamic effects might influence the pharmacokinetics of renally eliminated drugs, we have investigated the plasma elimination of milrinone after different iv bolus doses of the compound in healthy rats. Half-times were 20.6 +/- 5.5 min (0.3 mg/kg milrinone), 17.0 +/- 1.8 min (1.0 mg/kg), 31.4 +/- 4.5 min (3.0 mg/kg), and 95.5 +/- 18.4 min (10.0 mg/kg). Clearances of milrinone showed a positive correlation with doses. Hemodynamic effects were dose dependent and (plasma) concentration dependent, and the maximum decrease in diastolic blood pressure was 54.5 +/- 0.5 mmHg. The half-effective plasma concentration of milrinone was 1.0 +/- 0.2 microM. The maximum increase in heart rate was 15%. We conclude that milrinone plasma elimination in rats is dose dependent, probably resulting from its strong vasodilator properties.

Density of beta adrenoceptors in rat heart and lymphocytes 48 hours and 7 days after acute myocardial infarction.

Down regulation of the beta adrenoceptor is thought to play an important role in the diminished response to catecholamines in heart failure. beta Adrenoceptor densities were measured on membrane homogenates of rat right ventricle and lymphocytes 48 h or 7 d after experimental myocardial infarction, and in rats exposed to a continuous infusion of isoprenaline (400 micrograms.kg-1.h1). The performance of the rat hearts was also evaluated 48 h post infarction in an isolated retrograde perfused heart preparation. In contrast to a 60% down regulation in right ventricle and a 20% down regulation in lymphocyte membranes after isoprenaline infusion, there was no change in right ventricle and lymphocyte beta adrenoceptor densities after myocardial infarction. Left ventricular contractile performance was significantly depressed 48 h after myocardial infarction. Mean basal left ventricular pressure decreased from 108(SEM 3) to 63(4) mm Hg while the maximal response to dobutamine was decreased from 204(4) to 105(12) mm Hg (n = 8). No correlation was found between the receptor densities of right ventricular and lymphocyte membranes. We conclude that diminished response to beta sympathomimetics after myocardial infarction cannot be attributed to a loss of surface beta adrenoceptors, and that the lymphocyte beta adrenoceptor does not provide an adequate system to monitor small receptor changes on the myocardium.

In vitro method for measurement of cardiac performance and responses to inotropic drugs after experimentally induced myocardial infarction in the rat.

In this paper, a method is described for the measurement of the performance of rat hearts with an experimentally induced myocardial infarction of the left ventricle. After ether anesthesia of the animals and left thoracotomy, the left coronary artery was ligated, and the thorax was rapidly closed. The whole procedure took no more than 2 min. Forty-eight hours after the operation, the hearts were prepared for retrograde constant pressure perfusion, according to the Langendorff technique. The effects of the betasympathomimetic drug dobutamine and of the novel phosphodiesterase inhibitor milrinone on the contractile force of the right ventricle and the infarcted left ventricle, as well as on the total coronary flow, were quantified. Sham operated animals were used as control. The maximal obtainable stimulation of the contractility of infarcted hearts by dobutamine was significantly reduced from control. Milrinone increased the contractility in control animals, although to a much lesser extent. This increase was significantly smaller in infarcted hearts. The stimulation of the coronary flow by dobutamine and milrinone was significantly reduced in hearts with an infarction. Milrinone potentiated the effect of isoprenaline significantly. The results of the present study indicate the usefulness and reproducibility of this model for the evaluation of the efficacy of positive inotropic agents on the heart in the presence of a myocardial infarction.

Anti-allergic effects of milrinone and sulmazole in isolated rat lungs in comparison with theophylline.

Milrinone and sulmazole, two recently developed drugs, inhibit specific fractions of the phosphodiesterase (PDE) isozyme system. Since theophylline aspecifically inhibits the PDE complex, we compared the effects of milrinone and sulmazole with those of theophylline on antigen-induced bronchoconstriction, vasoconstriction, mediator release and leukotriene production. In the isolated perfused and ventilated lung of actively sensitized rats, we elicited antigen-induced bronchoconstriction, vasoconstriction and release of mediators like histamine, 5-hydroxytryptamine (5-HT) and slow-reacting substance of anaphylaxis (SRS-A). Milrinone, sulmazole and theophylline inhibited antigen-induced bronchoconstriction and vasoconstriction in a dose-dependent manner with minor differences in potency. Antigen-induced release of preformed mediators like histamine and 5-HT was inhibited only at high concentrations of milrinone, whereas sulmazole failed to inhibit mediator release. Theophylline also failed to inhibit 5-HT release. However, SRS-A synthesis was markedly reduced by these drugs in relatively low concentrations. It is concluded that milrinone and sulmazole have anti-allergic effects similar to those of theophylline and that all three PDE inhibitors reduce SRS-A synthesis.

In vivo anaphylaxis in the rat: effects of phosphodiesterase inhibitors.

On the basis of their inhibitory capacities on the phosphodiesterase enzyme system, we studied the anti-anaphylactic effect of milrinone and sulmazole in comparison with theophylline. For this purpose anaphylactic shock was induced in actively sensitised, spontaneously breathing rats. Milrinone, sulmazole and theophylline reduced anaphylactic bronchoconstriction without affecting the antigen induced fall in blood pressure. Surprisingly, sulmazole reduced mortality significantly.

Comparison of the anti-anaphylactic effects of milrinone, sulmazole and theophylline in the rat.

Milrinone and sulmazole, two recently developed cardiotonic drugs, inhibit specific fractions of the phosphodiesterase (PDE) isozyme system, while theophylline inhibits the PDE complex aspecifically. Since these drugs were shown to have anti-allergic effects in isolated lungs, we wanted to study milrinone and sulmazole in comparison with theophylline in an in vivo model of anaphylaxis. For this purpose, actively sensitized rats were intravenously challenged with antigen, while tracheal pressure, oesophageal pressure, blood pressure and airflow were continuously monitored. Antigen challenge induced a transient bronchoconstriction and a persistent fall in blood pressure until death occurred. Anaphylactic shock was consistently followed by metabolic acidosis. Milrinone, 3 mg/kg, and theophylline, 40 mg/kg (but not 10 mg/kg), reduced anaphylactic bronchoconstriction. Sulmazole, 40 mg/kg, almost abolished bronchoconstriction. Although milrinone, sulmazole and theophylline, 40 mg/kg, caused a fall in blood pressure prior to antigen challenge, total fall in blood pressure (drug-induced and antigen-induced) was not affected by these drugs. Surprisingly, sulmazole reduced mortality significantly. This phenomenon is probably related to the less severe metabolic acidosis in the presence of sulmazole.

Positive inotropic effects of milrinone, sulmazole and AR-L100 on isolated normal and infarcted hearts of the rat.

The recent development of new positive inotropic agents, such as milrinone, sulmazole and AR-L100 might provide new insights for the treatment of congestive heart failure. Although it has been reported that milrinone and sulmazole have cardiac phosphodiesterase III inhibiting properties, the pharmacological action of these drugs cannot be explained by these mechanisms alone. Despite the presence of cardiac phosphodiesterase III in the rat, the effect of milrinone on the rat heart has been reported to be small or even absent. In this study we have compared the effects of milrinone and sulmazole with these of AR-L100 on isolated hearts of normal rats and of rats with an experimentally induced myocardial infarction. A perfused heart preparation was used to assess the direct positive inotropic effect of milrinone, sulmazole and AR-L100 as well as their effect on isoprenaline-evoked increase of contractile force. The increase in left ventricular systolic pressure (LVP) in hearts of control animals amounted to 42.0 +/- 3.9 mm Hg, 18.2 +/- 2.4 mm Hg and 8.0 +/- 1.7 mm Hg for AR-L100, milrinone and sulmazole, respectively. The average initial LVP was 70.4 +/- 5.3 mm Hg. In infarcted hearts, the increase in LVP was 25.5 +/- 5.9 mm Hg, 12.6 +/- 2.3 mm Hg and 6.7 +/- 1.2 mm Hg for AR-L100, milrinone and sulmazole, respectively. In infarcted hearts, the average initial LVP was 38.6 +/- 1.6 mm Hg. These data show that these drugs have positive inotropic effects on rat hearts, although the effects of milrinone and AR-L100 on hearts from myocardial infarcted rats are smaller. However, interaction studies with isoprenaline showed a stronger potentiating effect of milrinone and sulmazole in infarcted hearts than in control hearts. Only in this respect sulmazole was more active than milrinone, while AR-L100 had no potentiating effect at all. These results suggest that milrinone and sulmazole are effective agents in conditions where higher levels of circulating catecholamines exist. Therefore, they might be particularly effective in the treatment of congestive heart failure.

Rat alpha 2 macroglobulin is a selective inhibitor of antigen-induced leucotrienes in rat isolated lungs.

Exogenously administered, purified rat alpha 2 macroglobulin (alpha 2M, recognized as an acute phase reactant with anti-inflammatory properties) greatly inhibits the increase of the pulmonary resistance during the antigen-induced bronchoconstriction in rats in vivo, whereas a BaSO4 pretreatment (a method to induce a broad spectrum of serum acute phase reactants, including alpha 2M) covers a broader bronchoprotection: suppression of the decrease of the dynamic lung compliance as well. To explain these differences we studied the influence of both alpha 2M and BaSO4 on the antigen-induced bronchoconstriction in rat isolated lungs in relation to the mediator release in lung-effluents. We report here that in this model alpha 2M only inhibits the antigen-induced SRS-A release, whereas the concomitant release of histamine and 5-HT was unaffected. As distinct from alpha 2M the BaSO4 pretreatment suppressed both the antigen-induced bronchoconstriction and the histamine, 5-HT and SRS-A release to a high extent. These data suggest that alpha 2M can be considered as a selective inhibitor of leucotrienes, which offers an explanation for several anti-inflammatory properties of alpha 2M, including protection against the antigen-induced increase of the pulmonary resistance in vivo.

Pharmacokinetics of lisinopril in hypertensive patients with normal and impaired renal function.

The pharmacokinetics of lisinopril was studied after administration of single and multiple doses of 5 mg to hypertensive patients with normal and impaired renal function. In patients with severe renal failure the peak concentrations were higher, the decline in serum concentration was slower and the time to peak concentration was extended. Accumulation of lisinopril was highly correlated with the creatinine clearance. The effective half-life was doubled and tripled in patients with mild and severe renal impairment, respectively, as compared to patients with a normal renal function. Lisinopril lowered blood pressure in all three groups over 24 h. It is suggested that smaller doses of lisinopril should be administered to patients with severe renal failure.

The inhibitory effect of fentanyl, nicomorphine and 6-nicotinoyl morphine on phrenic nerve activity in relation to their cardiovascular effects in the anaesthetized cat.

The inhibitory effects of the morphine-like drugs fentanyl, nicomorphine (3,6-dinicotinoyl morphine, Vilan) and its active metabolite 6-nicotinoyl morphine (6-NM) on phrenic nerve activity (PNA) were quantified. Therefore, the drugs were simultaneously infused into the left and right vertebral artery of anaesthetized cats. Previously we demonstrated that drugs, administered via these arteries, accumulate within the pontomedullary region, whereas only insignificant amounts reach higher brain areas and peripheral structures. The results were compared with the effects of i.v. administration. It is shown that fentanyl already inhibits PNA after 60 ng via the vertebral arteries. Nicomorphine and its metabolite have much less influence on respiration (factor 564 and 47, respectively). The difference in potency between nicomorphine and 6-NM was less after i.v. injection, indicating that nicomorphine needs metabolization in order to unfold full biological activity. Haemodynamic parameters are not affected after central administration even when PNA is almost completely depressed. After i.v. injection of relatively high doses, blood pressure falls, but probably not by an interaction with opiate receptors in the lower brain stem, since it could not be reversed by intravertebral naloxone.