THE EFFECTS OF ADRENALINE,  ANGIOTENSIN II, AND ITS BLOCKER ON BOVINE CORONARY ARTERIES

Dr. Norsidah Ku Zaifah[a]

INTRODUCTION

Angiotensin II receptor blocker is one of the most recent drugs used in the treatment of hypertension. Its ability to completely block the angiotensin II effect via the angiotensin II type-1 (AT1) receptor puts it at least on par with the angiotensin converting enzyme inhibitor (ACEI) for the treatment of patients with heart failure. The renin angiotensin system (RAS) and the sympathetic system contribute to the development of coronary heart disease. In view of this, it would be of value to determine the effects of angiotensin II, the catecholamine (adrenaline), and angiotensin II receptor blocker on arterial  function. This is best conducted in an  isolated system since in an in vivo study interference from the systemic neurohormonal system may complicate data interpretation. The present experiment utilized the isolated tissue technique in an organ bath. In an isolated system, the target drug or hormone effects can be studied without interference from various systemic neuroendocrine influences that are normally present in an in vivo situation.

OBJECTIVES OF THE STUDY

The objective of the study was to evaluate the response of isolated bovine coronary artery to angiotensin II, angiotensin II receptor blocker, and adrenaline

 MATERIALS AND METHODS

Materials

This in vitro study consisted of two parts. The first part was the determination of the  potassium chloride concentration that caused 70 percent of maximal coronary artery contraction (KCl EC70). The second part was the investigation of the effects of angiotensin II, adrenaline, and Valsartan on the isolated bovine coronary arteries. The Krebs Hanselit solution used in this experiment was prepared fresh each time it was required. The bovine (Bos Taurus) coronary arteries used in this study were obtained from cattle aged approximately two and a half years of either sex. In the laboratory, the coronary artery was submerged in the Krebs Hanselit solution and was infused continuously with carbogen gas. It was isolated gently from the adherent fat and connective tissue. It was then helically cut into six or seven pieces measuring approximately 15 mm x 3-4 mm. Both ends were tied with threads. One end was fixed to a glass holder while the other was connected to the Windaq transducer system. The whole tissue was then placed in the 50 ml glass chamber. The Windaq transducer system detected isometric contraction of the artery pieces and automatically recorded the data. The strip was put under 1.0 g tension at the initial phase appropriate for its length andwidth. Each piece went through a 30-45 minutes stabilizing process before other processes took place. The Krebs Hanselit solution was changed every 15 minutes. The experiment was conducted in an organ bath. The three chemicals used in this experiment were prepared fresh each time they were required and all agents used were at five times the normal human plasma level.

Determination of KCL response curve (n=8)

Once the tissue was stabilized, 0.4 ml of KCl at 0.2 gm/ml was introduced slowly into the 50 ml container. Once a plateau was reached, no further KCl was added. Based on this finding, the concentration at which 70 percent contraction (EC70) occurs was used to precontract the other specimens.

Determination of angiotensin II, Valsartan and adrenaline effects.

For each coronary artery sample, following precontraction, the following steps  were carried out: first, the stabilization of the tissue that took 30 to 45 minutes, and then the introduction of KCl solution at EC70. A time gap of another 10 minutes was allowed for the KCl-induced contraction after which the experimental drugs were introduced into the chamber. This was then followed by another 15 minutes’ observation period. At the end of this period the tissue was washed and was allowed to ‘rest’ for 30 minutes before the next step was done. For the experiment that used Valsartan, the angiotensin II receptor blocker was introduced three minutes prior to the introduction of the other drug into the chamber to allow antagonist receptor blocker to bind to the receptor.

Statistical analysis: The Wilcoxon signed rank test was used to test for significant change in contraction.

RESULTS

The results are divided into two parts. The first part was the KCl EC70 curves determination and the second part is drug vs KCl, and drug vs dru

KCL response curve determination of EC₇₀

The minimum concentration to achieve 70 % contraction was 0.16 gm/ml and the maximum concentration was 0.45 gm/ml. The mean concentration to achieve 70 % contraction was 0.3 gm/ml (see figure 4.2).

Part 2 results

Drug vs KCl

Drug vs drug

1. Valsartan does not change the adrenaline effects on the precontract  isolated bovine coronary artery.

2. Angiotensin II does not change adrenaline effect on the precontract isolated bovine coronary artery

3. In the presence of  both Valsartan and angiotensin II, the adrenaline does not significantly change response of precontract isolated bovine coronary artery.

4. Valsartan and angiotensin II dilate precontract isolated bovine coronary artery.

5. Angiotensin II does not change the precontract isolated bovine coronary artery response in the presence of adrenaline

6. Valsartan and adrenaline do not change precontract isolated bovine coronary artery response to angiotensin

DISCUSSION

Despite the high concentration used in this setting, adrenaline caused vasodilation of the artery. There are two possible reasons for this outcome. Firstly, the alpha-receptor density in the bovine coronary artery probably is less than the beta-receptor thus the beta response was elicited. This resulted in relaxation instead of constriction. Furthermore, for cattle of  approximately 180 kg weight, the concentration of adrenaline used may still be low. Secondly, the beta receptors are known to respond at low concentrations of catecholamine compared to at higher concentration (Bowman and Rand, 1980). It has been shown that the endothelium plays an important role in regulating the alpha-adrenergic effect (Vanhoutte, 2001). An intact endothelium helps oppose alpha-adrenergic vasoconstriction. Angiotensin II, apparently did not cause significant change of the potassium-induced contraction. Broome et al. in their study used angiotensin II at more than five times the normal human plasma concentration. They found that even at this concentration, the angiotensin II used failed to cause significant change in coronary vascular tone. No significant change of the systolic and diastolic function variables of heart rate, end systolic elastance, preload recruitable stroke work and the time constant for isovolumetric relaxation was noted. They concluded that both the positive inotropic and chronotropic effects of angiotensin II in their study were mediated through extracardiac actions of angiotensin II. This reflects the lack of local angiotensin II effect on the coronary artery (Broome et al., 2002). Valsartan significantly caused vasodilation in the presence of angiotensin II. There is a possibility that angiotensin II, though on its own did not cause contraction, actually contributes to the potassium induced contraction. It is unlikely for Valsartan to act through other receptors or act as agonist as these are not its pharmacodynamic properties. Furthermore at present there is no data regarding the effect of Valsartan on bovine coronary artery or human subtypes of AT1, i.e. AT1A or AT1B. The two AT1-receptor genes subtypes however have been isolated in rodents and canine (Kiowski, 2002).

LIMITATIONS OF THE STUDY

If the storage time of specimens is shortened, more artery samples can be used in this study. Consequently, more data that are significant may be generated. The validity of the results however were maintained since individual strip of coronary artery was used as its own control.

CONCLUSION

This study looked at a tiny part of complex biological properties of the coronary artery. Not withstanding this, all the complex activities that take place in vivo, be it at the organ or the system level, start from many processes at the tissue or cellular level. Starting from ligand binding to receptor, cascades of activities, which bring about changes in tissue morphology, in organ functions and finally the whole system  in an animal or human take place. Drugs were introduced into an in vitro environment and through receptor binding, directly or indirectly, induce smooth muscle contraction that was recorded by the soft ware programme in the computer system. As shown earlier, at five time the basal human plasma concentration, adrenaline alone and Valsartan in the presence of angiotensin II caused significant relaxation of isolated bovine coronary artery. No significant interaction was noted between angiotensin II and adrenaline. However, one should not forget that at the systemic level they are closely related. Furthermore, since angiotensin II was shown to have intracellular effects/intracrine actions i.e. interaction with specific nuclear receptors to regulate gene transcription through genetic studies, the prospect of cardiac and vascular protection especially via gene therapy is there. Pathogenesis of myocardial ischaemia or its clinical presentation, angina, is not as simple as described herein. Despite this, it is obvious that the RAS, in particular angiotensin II, plays a lot of important function at various stages of disease development. Thus clinically, angiotensin II receptor blocker may have a role in the prevention of myocardial ischaemia

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