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J Thorac Cardiovasc Surg 1997;113:108-113
© 1997 Mosby, Inc.

SURGERY FOR ACQUIRED HEART DISEASE

DIRECT VASODILATOR EFFECT OF MILRINONE, AN INOTROPIC DRUG, ON ARTERIAL CORONARY BYPASS GRAFTS

Supported by grants from National Health and Medical Research Council of Australia, Canberra, and Austin Hospital Medical Research Foundation, Heidelberg.

Received for publication August 24, 1995 Revisions requested Nov. 29, 1995 Revisions received April 15, 1996 Accepted for publication July 1, 1996 Address for reprints: James J. Liu, MD, PhD, Vascular Biology Unit, Department of Cardiac Surgery, University of Melbourne, Austin Hospital, Heidelberg, VIC 3084, Australia.

Abstract

Milrinone is an inotropic drug with vasodilator activity that has been shown to be useful in increasing cardiac output and decreasing wedge pressure. Despite these advantages, it is unknown whether this drug can be used for the treatment of perioperative spasm of coronary bypass grafts. This study was undertaken to investigate the in vitro vascular effect of milrinone on internal thoracic arteries obtained from patients undergoing coronary artery bypass grafting. The results showed that milrinone produced a potent, concentration-dependent, preventive effect on the norepinephrine-induced contraction of internal thoracic arteries, as well as reversing contraction of internal thoracic arteries by receptor-dependent agents, including the thromboxane A₂ mimetic U46619, the vasoconstrictor peptide endothelin-1, and the ₁-adrenal receptor agonist phenylephrine. The relaxing effect of milrinone was weaker, however, on internal thoracic arteries contracted with 25 mmol/L potassium chloride. Comparison of milrinone with other vasodilators, including papaverine, nitroprusside, and glyceryl trinitrate, showed milrinone to be more potent than papaverine but less potent than nitroprusside and glyceryl trinitrate. The inhibitory effect of milrinone on internal thoracic artery contraction appeared as a reduction in contractile force, not as an increase in the values of concentrations of the agonists causing 50% maximal contraction, which indicates that milrinone exerts its vasodilator effect directly on the smooth muscles, not on the membrane receptors. The results also showed no significant difference in relaxing effect between internal thoracic artery rings with and without endothelium. In conclusion, this study provides experimental evidence that milrinone is a potent, endothelium-independent, direct vasodilator of the human internal thoracic artery and provides the scientific rationale for a future clinical trial with this drug for the perioperative treatment of internal thoracic artery spasm in cardiac surgical patients. (J Thorac Cardiovasc 1997;113:108-13)

Milrinone, a phosphodiesterase inhibitor, is a noncatecholamine, nonglycosidic inotropic agent with vasodilator activity. ¹ This combination of positive inotropic and vasodilator activity has been shown to be beneficial in increasing cardiac output and decreasing left ventricular end-diastolic pressure, pulmonary wedge pressure, right atrial pressure, and systemic vascular resistance in patients with severe congestive heart failure. ^2,3 It is well tolerated. ² Despite these advantages, it is unknown whether this drug can be used for the treatment of perioperative spasm of coronary artery bypass grafts.

In addition, fewer data are available concerning direct vasodilator activity of milrinone than on its cardiotonic effect. It is important to understand the vasoactivity of this drug on the bypass grafts before it is used clinically.

Internal thoracic artery (ITA) grafts have greater late patency and longevity than do saphenous vein grafts. ^4,5 ITA is also more resistant to the development of atherosclerosis than are the native coronary arteries. ⁶ ITA spasm may contribute to early myocardial ischemia, however, increasing perioperative morbidity and mortality. ^7,8 This spasm may be caused by injury of the endothelium of the ITA during surgical procedure and by drugs administered systemically to support the circulation. Prevention and treatment of ITA spasm by vasodilator drugs are important issues in coronary artery bypass grafting. Milrinone has both cardiotonic and vasodilator activities. The use of milrinone to treat perioperative patients may be beneficial in the prevention of ITA spasm and in the recovery of cardiac function. This study was undertaken to investigate the in vitro effect of milrinone on the human ITA to obtain scientific evidence before the clinical application.

Methods

Preparation of vessels
Preparation of vessels was undertaken as described elsewhere. ^9-11 Briefly, human ITA graft segments were taken from patients undergoing coronary artery bypass grafting. The distal segments (but far before the branch points) were collected, cleaned of connective tissues, and cut into 3 mm long rings. In some rings the endothelium was removed gently by forceps. The rings were suspended on wire hooks in organ chambers containing Krebs' solution with the following composition: sodium ion, 144 mmol/L; potassium ion, 5.9 mmol/L; calcium ion, 2.5 mmol/L; magnesium ion, 1.2 mmol/L; chloride ion, 128.7 mmol/L; bicarbonate ion, 25 mmol/L; sulfate ion, 1.2; phosphate ion, 1.2 mmol/L; and glucose, 11 mmol/L. The solution was aerated with a gas mixture of 95% oxygen and 5% carbon dioxide at 37° ± 0.1° C. After the rings were equilibrated without tension on the wire hooks for 1 hour, a normalizing procedure was performed. The resting tension applied to each ring was equivalent to that required to stretch the ring to 90% of its internal circumference when distended with a transmural pressure of 100 mm Hg. In this study, the average force of the resting tension was 4.06 ± 0.27 g. A computer system was used to monitor the organ chamber experiments. The maximal contractile response was obtained with 3 x 10^-6 mol/L norepinephrine. The presence or absence of functional endothelium was tested by the response to acetylcholine (3 x 10^-6 mol/L) in rings precontracted with norepinephrine (3 x 10^-7). Mean curves were obtained from at least six different ITA rings from six different patients. Each ring was used to obtain the concentration-response curve for only one tested agent, but relevant agents and different concentrations were tested at the same time with separate artery rings from the same patient.

Data analysis
The effective concentration of a contractile or relaxing agent that caused 50% of maximal contractile force or relaxation (EC₅₀) and the maximal response induced by a constrictor (Max) were determined from each curve by a logistic, curve-fitting program called FLEXIFIT. ¹² Statistical significance was determined by an unpaired Student's t test for two-group comparisons and a two-way analysis of variance for multiple-group comparisons. Values are mean values (± standard error of the mean [SEM]). Significance was taken at p values lower than 0.05.

Drugs and chemicals
Milrinone (1,6-dihydro-2-methyl-6-oxo-[3,4'-bipyridine]-5-carbonitrile) was provided by Sterling-Winthrop (Rensselaer, N.Y.). U46619 (9,11-dideoxy-11,9-epoxymethano-prostaglandin F₂) was a gift from The Upjohn Company (Kalamazoo, Mich.). Endothelin-1 (ET-1) was purchased from American Peptide Company (Sunnyvale, Calif.). Papaverine, nitroprusside, and glyceryl trinitrate were purchased from David Bull Laboratories (Melbourne, Australia). All other drugs and chemicals were purchased from Sigma Chemical Co. (St. Louis, Mo.).

Results

Prevention of ITA contraction
A 15-minute pretreatment of the ITA rings with increasing concentrations (10^-7 to 10^-5 mol/L) of milrinone significantly prevented norepinephrine-induced contraction in a concentration-dependent manner (Fig. 1). Values of EC₅₀ were as follows: 6.82 ± 0.11 -log mol/L for the control group and 6.92 ± 0.13 -log mol/L, 6.73 ± 0.12 -log mol/L, and 6.73 ± 0.10 -log mol/L for groups treated with 10^-7 mol/L, 10^-6 mol/L, and 10^-5 mol/L milrinone, respectively. There were no significant differences in the values of EC₅₀ between the control group and each treatment group, nor among different treatment groups. Values of Max were as follows: 100.1% ± 1.5% for the control group and 80.9% ± 4.7%, 53.3% ± 3.2%, and 19.9% ± 2.1% for the groups treated with 10^-7 mol/L, 10^-6 mol/L, and 10^-5 mol/L milrinone, respectively. Milrinone significantly reduced the values of Max (p < 0.05 for 10^-7 mol/L vs control, p < 0.01 for 10^-6 mol/L vs control, and p < 0.01 for 10^-5 mol/L vs control). The significance levels of comparisons for each dose are shown in Fig. 1.

View larger version (22K): [in this window] [in a new window]   Fig. 1. Graphs showing preventive effect of milrinone on contraction of human ITAs induced by norepinephrine (noradrenaline). Mean concentration-contraction curves for norepinephrine were obtained when 10-7 mol/L, 10-6 mol/L, or 10-5 mol/L milrinone was added 15 minutes before starting norepinephrine curves. Percentage of maximal contraction is defined as percentage of maximal response to 3 x 10-6 mol/L norepinephrine. Values of EC50 and Max are shown in the text. Values for each dose in the figure are mean ± SEM (n = 6). Asterisks denote significant differences versus control (*p < 0.05, **p < 0.01). Milrinone reduced norepinephrine-induced contraction in a dose-dependent manner.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Graphs showing reversal by milrinone of precontraction of human ITAs by various agents. ITA rings were precontracted with the thromboxane A2 mimetic U46619 (10-8 mol/L), the vasoconstrictor peptide ET-1 (3 x 10-9 mol/L), the 1-adrenal receptor agonist phenylephrine (3 x 10-7 mol/L), or 25 mmol/L potassium chloride, followed by the addition of increasing concentrations of milrinone (1 x 10-9 mol/L to 1 x 10-4 mol/L). Values of EC50 are shown in text. Values for each dose in figure are mean ± SEM (n = 6). Asterisks denote significant differences versus control *p < 0.05, **p < 0.01). Difference between milrinone's effect on potassium-induced contraction and its effect on contraction induced by receptor-dependent vasoconstrictors was significant. There were no significant differences among receptor-dependent vasoconstrictors.

View larger version (25K): [in this window] [in a new window]   Fig. 3. Graphs showing lack of involvement of endothelium in milrinone-induced relaxation of human ITAs. ITA rings with (+EC) or without (-EC) endothelium were precontracted with the thromboxane A2 mimetic U46619 (10-8 mol/L), followed by the addition of increasing concentrations of milrinone (1 x 10-9 mol/L to 1 x 10-4 mol/L). Original traces are representative of six separate experiments. There was no significant difference in relaxing effects of milrinone on rings with and without endothelium (p > 0.05). EC, Endothelium.

View larger version (23K): [in this window] [in a new window]   Fig. 4. Graphs showing comparison of milrinone with other vasodilators. ITA rings were precontracted with the thromboxane A2 mimetic U46619 (10-8 mol/L), followed by addition of increasing concentrations (1 x 10-9 mol/L to 1 x 10-4 mol/L) of milrinone, papaverine, nitroprusside, and glyceryl trinitrate. Values of EC50 are given in text. Values for all doses in figure are mean ± SEM (n = 6). Asterisks and plus signs denote significant differences versus control (*p < 0.05, +p < 0.05, **p < 0.01, ++p < 0.01). Relaxing effect of milrinone was significantly stronger than that of papaverine (*) but significantly weaker than those of nitroprusside and glyceryl trinitrate (+). There were no significant differences between nitroprusside and glyceryl trinitrate.

This study demonstrates that the cardiotonic drug milrinone produces a potent, concentration-dependent, inhibitory effect on ITA contraction. It also exerts a relaxing effect on ITAs contracted with receptor-dependent agents. The mechanisms are not dependent on endothelium. The potency of milrinone is higher than that of papaverine and lower than those of nitroprusside and glyceryl trinitrate.

Selection of potassium chloride, U46619, ET-1, and phenylephrine as contracting agents in this study was made on the basis of the belief that intracellular free calcium (for contraction) will be raised by (1) a calcium ion influx through voltage-operated channels in response to membrane depolarization or (2) a release of calcium ion into the cytoplasm from bound intracellular calcium stores or from extracellular stores, triggered by receptors activated on the membrane (receptor-operated channels). Similar to the previous studies, ⁹ potassium ion served in this study as a membrane depolarizing agent to open voltage-operated channels. The other agonists were employed as receptor-dependent agents to open receptor-operated channels. In this study, it was clearly shown that milrinone is highly effective in vitro in the inhibition of the contraction associated with receptor-operated channels but is weaker in inhibition of the contraction associated with voltage-operated channels. It is known that most vasoconstrictors in human body are receptor dependent, and the high (25 mmol/L) potassium ion concentration used in the study as a pharmacologic evaluation approach is above normal values of potassium ion in human plasma. The weak effect of milrinone on contraction induced by 25 mmol/L potassium is therefore of limited clinical relevance.

The inhibitory effect of milrinone on ITA contraction appeared only as a reduction in the values of Max, not as an increase in EC₅₀. These results indicate that the vasodilator effect of milrinone is exerted directly on the smooth muscles or by noncompetitive inhibition of the membrane receptors. If the effect of milrinone were achieved by competitive blocking of norepinephrine receptors, EC₅₀ would have increased and Max would have not changed.

Milrinone is an inhibitor of the cyclic guanosine monophospate–inhibited phosphodiesterase III. ¹³ It results in an increase in cyclic adenosine monophosphate levels. ^1,13 Studies have shown that the time course for this increase does not seem to correspond to the increase in muscle-developed tension, ¹ however, suggesting that it is unlikely to be the only mechanism responsible for the effect of the drug. Endothelium-dependent mechanisms were therefore tested in this study. Endothelium-dependent relaxation is achieved by a combination of endothelium-derived prostacyclin, nitric oxide, and endothelium-dependent hyperpolarizing factor. The results of this study showed no significant difference in the relaxing effect of milrinone with and without endothelium. This indicates that the endothelium-dependent mechanisms are not involved in the vasodilator effect of milrinone. Because the vasodilator effect is independent of endothelium, milrinone may be useful in the treatment of spasm arising from endothelial dysfunction caused by surgical procedure, atherosclerosis, or drugs.

In this study, milrinone was more potent than papaverine, a vasodilator commonly used for preparation of ITAs during coronary artery bypass grafting. Papaverine is usually used at high concentration, and its solution is acidic. Whether milrinone can be used to replace papaverine for the preparation of ITAs during coronary artery bypass grafting awaits further clinical investigation. Although milrinone was less potent than nitroprusside and glyceryl trinitrate, it is well known that the latter two agents have some disadvantages. It may be difficult to control their rapid, strong, and short effects. Glyceryl trinitrate is associated with tachyphylaxis to its vasodilator effect. Both nitroprusside and glyceryl trinitrate are pure vasodilators and often require combination with other drugs, leading to use of multiple drugs. Milrinone has several theoretic advantages as an inotropic agent. In particular, it is a direct vasodilator, does not increase myocardial oxygen consumption, and is well tolerated. In addition, there are no reports of tachyphylaxis during the use of milrinone. Milrinone thus has the potential to be a superior drug for the perioperative treatment of cardiac patients.

Application of the results from in vitro studies to the clinical situation requires caution. This study does not provide direct clinical data for application of milrinone. The experimental data do, however, provide a scientific basis for a future clinical trial with this drug to prevent and to treat perioperative spasm of coronary artery bypass grafts. The concentrations used in the laboratory study cannot be directly translated to clinical dosages because conditions in vitro are significantly different from those in vivo. Bailey and colleagues, ¹⁴ in their report of a pharmacokinetic study on milrinone in cardiac patients, demonstrated that a milrinone dose of 50 µg/kg at an infusion rate of 0.5 µg x kg^-1 x min^-1 consistently maintained therapeutic plasma concentrations. The value of EC₅₀ in their study was a plasma milrinone concentration of 167 ng/ml.

In conclusion, milrinone is a potent, endothelium-independent dilator of the human ITA. With its combination of positive inotropic and vasodilator activities, it may be beneficial in the perioperative treatment of patients undergoing coronary artery bypass grafting. This study provides valuable experimental evidence that milrinone is a prime candidate for further clinical testing for this purpose.

References

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