Organic Nitrates in Alleviating Chest Pain - Lab Report Example

 Laboratory Report Assignment Part A; Abstract The organic nitrates, especially GTN, are effective in alleviating chest pain resulting from ischaemia in patients with coronary artery disease (CAD) and other cardiomyopathies associated with ischaemia. The purpose of this laboratory research study was to examine the effects of phenylephrine ( PEP) used as a single agent or at submaximal doses in combination with glyceryl trinitrate (GTN), carbachol (CCH), or isoprenyline (ISO) on the vascular properties of mammalian aorta in vitro. The effects of these pharmacologic agents on vasoconstriction, expressed as the % maximal constriction of the cardiac muscle was measured at varying concentrations of these pharmacologic agents. The data showed that the aortic muscle tissue was sensitive to each of the drugs tested; moreover, combined effects were observed at similar concentration ranges for each of the drugs used to effect enhanced muscular constriction following PEP treatment. These in vitro data suggest the need for further experimentation on potential combinatorial effects of these agents in the treatment of patients with ischaemic cardiomyopathy. Part B: Introduction Organic nitrates are pharmacologic agents that have been used in the treatment of patients with chronic angina for over 100 years (Parker and Parker, 1998). Since then these drugs have also been routinely prescribed for the treatment of acute myocardial infarction and heart failure. Of these, glyceryl trinitrate (GTN) is the primary drug currently prescribed for patients with these disorders. The clinical stabilising effects of this drug are the result of its effects on the endothelial vasculature (Du, 1998). The actions of organic nitrates effect the redistribution of blood to the venous system in combination with a decrease in vascular resistance and vasodilation of coronary blood vessels. The net result is an increased oxygen supply to the myocardium. The organic nitrates are generally administered sublinguinally and are effective in alleviating chest pain resulting from ischaemia in patients with coronary artery disease (CAD) and other cardiomyopathies associated with ischaemia (Parker and Parker, 1998). In addition, they may reduce the frequency of ischaemic episodes when used in combination with other pharmacologic agents. The most common side effect is headaches which may result from vasoactive effects on the cerebral vasculature. GTN is a highly effective drug for the management of cardiac ischaemia (Miller et al, 2008). Therapeutic efficacy is believed to derive primarily from its haemodynamic effects which increase the oxygenation of the cardiac tissues. The organic nitrates appear to exert their pharmacologic effects following their conversion in the body to nitric oxide by means of the action of the cellular enzyme mitochondrial aldehyde dehydrogenase. The vasoactive effects of organic nitrates such as GTN result from their actions on guanylyl cyclase to produce cyclic GMP (Miller et al, 2008). Although organic nitrates are extremely effective in the sustained therapy of patients with cardiac ischaemia, the development of resistance to the hemodynamic effects of these drugs in long-term treatment may ultimately limit their efficacy in the treatment of patients with chronic cardiac disease (Sayed et al, 2008). This type of drug resistance is called nitrate tolerance and is defined as the loss of vascular responsiveness to organic nitrates in long-term therapeutic use. The development of drug resistance may even contribute to the mortality of patients in long-term treatment with these vasoactive agents. This may occur as the consequence of the accumulation of superoxide and peroxynitrite in drug-resistant patients which results in vascular dysfunction. Clinical research has suggested that this effect may result from oxidative stress of the cardiac vasculature independent of the primary vasoactive effects of the drugs (Sayed et al, 2008). The development of GTN induced tolerance has necessitated the search for additional pharmacologic agents that may abrogate this response or potentiate the effects of GTN (Sayed et al, 2008). Each of the pharmacologic agents used in this lab experimental study may represent a potential combined treatment approach for this application. Phenylephrine (PEP) is an alpha1-adrenoreceptor agonist used in blood pressure regulation. It is used to treat patients with hypotension to increase blood pressure. It does not affect the heart rate or contractility, which makes it a potentially useful agent in the treatment of patients with cardiomyopathy (Parker and Parker, 1998). It is a hypertensive agent and is therefore contraindicated for patients with high blood pressure. Carbachol is a cholinergic agonist that binds to the acetylcholine receptor. It is primarily used in the treatment of glaucoma (Kohashi et al, 2003). Isoprenaline is a beta-adrenergic agonist that regulates the contractile properties of myocytes by increasing the release of calcium to stimulate the contractile properties of these cells. It is often used in the treatment of bradycardia (Miller et al, 2008). The purpose of this laboratory research study was to examine the effects of PEP used as a single agent or at submaximal doses in combination with GTN, CCH, or ISO on the vascular properties of mammalian aorta in vitro. Specifically, the effects of these pharmacologic agents on vasoconstriction, expressed as the % maximal constriction of the cardiac muscle was measured at varying concentrations of these pharmacologic agents. Part C: Results The Dose/response data for PEP on the constriction of aortic tracheal muscle showed that the effective dose range was between 0.3 and 200miroM. Minimal vasoconstriction was produced at 0.3 microM concentration and the results were highly variable at this concentration, ranging from 0-25.6% constriction. The optimal PEP concentration was between 30 and 100 microM. At 30 microM the range of constriction varied considerably in repeated tests between 33.3 and 100% constriction. At 100 microM , the range of data was between 72 and 100% constriction. Moreover, the greatest consistency of repeated responses was observed at this concentration of PEP. At 200 microM all recordings were at 100%, representing saturation levels of response at this high concentration of PEP. Graph 5 shows the effects of PEP on isolated cardiac muscle constriction. There was a positive correlation between increasing concentration of PEP (LogM) and % maximal constriction of the tracheal muscle tissue for each of the 8 data sets, indicating that the drug was effective in inducing the contraction of this vascular tissue. The dose/response data show that the activity of PEP showed a positive correlation between LogM of concentration versus % constriction for the aortic muscle. !00% activity was observed at the highest doses. Repeated assays showed similar dose/ response effects although the curve was shifted to the right in repeated assays over a range of 3 logs. The results of Table 6 and Graph 6A show the % constriction remaining in the aortic muscle following PEP treatment that was further affected by the subsequent treatment with additional drugs, including carbachol (CCH), glycerol trinitrate (GTN) and isoprenaline (ISO) at varying concentrations. Treatment of the aortic muscle with carbachol produced increased constriction to 100% at a concentration of 0.01 microM. Further increases in concentration, obviously, did not produce at greater effect, although very high concentrations were somewhat inhibitory. Likewise, the addition of GTN at low concentration (0.01 microM) produced maximal constriction (100%) while higher concentrations were inhibitory. Optimal ISO activity also was observed at 0.01 microM, although increased doses were not as inhibitory as the previous two drug additions. These data are further elaborated in Graph 6. Graph 6b show that increasing doses of CCH (at approximately LogM=5) produced a negative linear correlation between dose and effects on constriction. Graph 6b shows also a linear decline in the % constriction with increasing GTN concentrations. Graph 6c shows a decline in % constriction with increasing ISO dose; however, the data are spread over a very wide range of responses to different concentrations of this drug. The data showed that the aortic muscle tissue was sensitive to each of the drugs tested; moreover, combined effects were observed at similar concentration ranges for each of the drugs used to effect enhanced muscular constriction following PEP treatment. Part D: Discussion The data obtained from this experiment indicated that each of the pharmacologic agents used in this study was vasoactive and induced the constriction of mammalian cardiac muscle in vitro. Moreover, the data showed that similar levels of vasoconstriction were achievable in cardiac muscle pre-treated with suboptimal concentrations of PEP by GTN, CCH and ISO. Extremely low concentrations of each of these agents (0.01 microM) were capable of inducing maximal constriction post-PEP treatment, suggesting that the combined treatment may produce a better pre-clinical effect than PEP alone, which requires a concentration of 30-1—microM to produce maximal constriction of this tissue. The observed combined effects of PEP with GTN suggest a potential use in the clinical treatment of GTN resistance. This possibility is suggested by the observed data as well as biochemical research that indicates that these two agents have a different mechanisms of action (Shen et al, 2006). In contrast to PEP, GTN effects the production of nitric oxide to induce vasoconstriction, whereas the pharmacologic effects of PEP occur as a result of its actions on the alpha1-adrenergic receptor. Clinical research studies by Du (1992) on patients undergoing coronary artery bypass procedures who were pre-treated with GTN, showed that subsequent addition of PEP did not alleviate GTN tolerance. This clinical result highlights the fact that it is not always possible to extrapolate the data from in vitro experiments to direct clinical applications. The addition of carbachol was also found to potentiate the action of PEP. Research studies on the effects of carbachol in rat myocytes by indicated that this agent decreases the beat rate of these cells, an effect that is inhibited by nitric oxide inhibitors (Kohashi et al, 2003). Thus, carbachol might potentiate the effects of GTN as well as PEP. Isoprenyline is a beta-adrenergic agonist whereas PEP is an alpha-adrenergic agonist. The combined effects of these agents may serve, therefore, to potentiate the vasoactive effects on cardiac tissue. In conclusion, this research experiment demonstrated that experimental pharmacologic agent may act in combined fashion to elicit vasoconstrictive responses in aortic muscle. These in vitro data suggest the need for further experimentation on potential combinatorial effects of these agents in the treatment of patients with ischaemic cardiomyopathy. References Du, Z., Buxton, B., and Woodman, O. 1992. Tolerance to glyceryl trinitrate in isolated human internal mammary arteries. J Thoracic Surg 104(5), pp. 1280-1284. Kohashi, M., Kawahara, K., and Yamauchi, Y. 2003. Carbachol-induced suppression of contraction rhythm in spontaneously beating cultured cardiac myocytes from neonatal rats. Biological Rhythm Research 34(4), pp. 367-381.  Miller, M., Grant, S., and Wadsworth, R., 2008. Selective arterial dilatation by glyceryl trinitrate is not associated with nitric oxide formation in vitro. JVasc Res (45), pp. 375-385. Parker, J., and Parker, J.O., 1998. Nitrate therapy for stable angina pectoris. New England Journal of Medicine 338(8), pp.520-531. Sayed, N., Kim, D., Fioramonti,X., Iwahashi,T., Durán, W., and Beuve, A. 2008. Nitroglycerin-induced s-nitrosylation and desensitization of soluble guanylyl cyclase contribute to nitrate tolerance. Circulation Research103, pp. 606-610. Shen, J. 2006. Isoprenaline enhances local Ca2+ release in cardiac myocytes. Acta Pharmacologica Sinica 27, pp. 927-932.   Read More