Amlodipine and endothelial nitric oxide synthase activity

Time for primary review 2 days. See article by Lenasi et al. [1] (pages 844–853) in this issue. In an interesting and well crafted paper in this issue Lenasi et al. [1] have provided novel and exciting evidence on the ability of the 1,4-dihydropyridine Ca 2+ channel antagonists, amlodipine and nifedipine, to generate nitric oxide (NO) and in doing so produce relaxation in the vasculature. Admittedly, organic Ca 2+ channel antagonists produce vasorelaxation predominantly by inhibiting the influx of Ca 2+ into smooth muscle cells via the so-called voltage-gated Ca 2+ channels [2] . However, there is empirical clinical data which implies that, in part, the cardiovascular protective actions of some Ca 2+ channel antagonists cannot be readily explained purely on the basis of the inhibition of voltage-gated Ca 2+ channels. For example, it has been reported that in the Prospective Randomized Amlodipine Survival Evaluation Study Group (PRAISE) trial, that amlodipine produced substantial beneficial effects in the group of patients with dilated cardiomyopathy, and that there was a 31% ( P =0.04) reduction in combined fatal and nonfatal events, as well as, a decrease in the risk of death by 46% ( P <0.001) in patients with nonischaemic cardiomyopathy [3] . In addition, data from Prospective Randomized Evaluation of the Vascular Effects of Nordics Trial (PREVENT) has revealed that while treatment with amlodipine did not produce demonstrable effects on angiograghic progression of coronary atherosclerosis, it did result in fewer hospitalizations for unstable angina and revascularization [4] . Moreover, in the same trial it was also revealed that treatment with amlodipine slowed the progression of carotid artery atherosclerosis [4] . Taken together, these findings among others have propelled the view that perhaps other mechanisms may, in part, account for some of the clinical actions of the Ca 2+ channel antagonists. Certainly, evidence in the literature has implicated a role for the generation of NO for a portion of the vasorelaxant effects of a number of Ca 2+ channel antagonists namely, S 11568 [5] , mibefradil [6] , nifedipine, nisoldipine and nimodipine [7] , nitrendipine [8] , amlodipine [9] and benidipine [10] . More recently, Loke et al. [11] clearly demonstrated that amlodipine but not diltiazem was able to reduce myocardial oxygen consumption in mice as well, the response to amlodipine was sensitive to the inhibition by the nitric oxide synthase (NOS) inhibitor, N -nitro- l -arginine methyl ester. Furthermore, in the same study, they also demonstrated that this effect of amlodipine was absent in genetically altered mice lacking the eNOS gene (eNOS −/−) [11] . Thus indicating an integral role for the eNOS/NO cascade system in the pharmacological actions of the Ca 2+ channel antagonist, amlodipine. Indeed, a great debate has been on-going for some time now as to how some 1,4-dihydropyridine Ca 2+ channel antagonists produce NO-dependent vasorelaxation via the eNOS pathway, and whether all Ca 2+ channel antagonists possess such an ability under the right circumstances with the delimiting factor being the blood vessel. At present, the two prevailing views as to how an organic Ca 2+ channel antagonist enhances the generation and release of NO in blood vessels include (a) an increase in the free intracellular concentration of Ca 2+ in endothelial cells initiating a cascade of reactions that leads to the activation of eNOS and enhanced release of NO [8] , and (b) stimulation of B 2 -kinin receptors which will result in the activation of eNOS consequently enhancing the production of NO [12] . Needless to say that a combination of the former and latter mechanisms may also account for an enhancement of NO generation in vasculature by a Ca 2+ channel antagonist. Thus the recent investigation by Lenasi et al. [1] has elegantly demonstrated that there is yet another possible and viable mechanism by which 1,4-dihydropyridine Ca 2+ channel antagonist such as amlodipine can, under acute conditions increase the activity of eNOS and precipitate an enhancement in the generation of NO. However, the data from the latter study has also produced some intriguing findings regarding the actions of amlodipine via the B 2 -kinin receptors but has also essentially ruled out the possibility that at least amlodipine or nifedipine enhances the generation of NO via an increase in intracellular concentration of free Ca 2+ in endothelial cells [1] . Therefore, in their investigation, Lenasi and associates [1] have convincingly demonstrated that amlodipine enhances the activation of eNOS by affecting phosphorylation of Ser 1177 and Thr 495 . Effectively, this is achieved by the ability of the Ca 2+ channel antagonists inhibiting the activity of protein kinase C (PKC) in endothelial cells. It is recognized that phosphorylation of eNOS is most likely required for its Ca 2+ independent activation [13] . Convincingly and with the appropriate experimental design, Lenasi et al. [1] have argued that inhibition of PKC can acutely lead to two biochemical events that can ultimately result in an enhancement in the activity of eNOS which is then translated into an increase in the generation of NO in the endothelial cells. These events being (a) an attenuation of Thr 495 phosphorylation, and (b) an enhancement of phosphorylation of Ser 1177 . The latter being achieved possibly via an attenuation in the actions of phosphatase, PP2A [1] . More recently, Ayoagi et al. [14] using crystallographic data collection of calmodulin bound to eNOS demonstrated that phosphorylation of Thr 495 will result in a weaker calmodulin to eNOS binding thus increasing the propensity of the deactivation of eNOS. Lenasi et al. [1] also described a NO-sensitive component of amlodipine mediated relaxation that was sensitive to the actions of a B 2 -kinin receptor antagonist, icatibant. Certainly, the exact nature of mechanism(s) of the latter observation remains to be elucidated fully. This component of the vasorelaxation by amlodipine was (a) independent of that involving inhibition of PKC, and (b) not associated with the elevation of cyclic GMP [1] ; the latter observation being quite puzzling. Notwithstanding the fact that generation of NO should result in an increase in the intracellular levels of cyclic GMP levels, it is possible that the relaxant actions of amlodipine via the B 2 -kinin receptor could have been due to changes in the resting membrane potential of vascular smooth muscle cells. Needless to say that since the cellular nature of this action is an enigma, and remains to be fully established, one can easily be dismissive of this interesting and intriguing observation, and thus not place it at the forefront as a viable mechanism responsible for vascular action of this Ca 2+ channel antagonist in vivo. However, this would be a mistake. Indeed, it is quite clear that a component of amlodipine response in vasculature is mediated via the activation of B 2 -kinin receptors [9] , and data presented by Loke et al. [15] in B 2 -kinin receptor knockout mice (B 2 −/−) has given further credence to such a view. Loke et al. [15] have clearly demonstrated that amlodipine had no effects on myocardial oxygen consumption in the genetically altered B 2 -kinin receptor knockout mice (B 2 −/−). Undoubtedly, the observation by Lenasi et al. [1] related to the actions of amlodipine via the B 2 -kinin receptors must be further pursued until the key to this fascinating puzzle is uncovered. It must also be recognized that pathological changes may have an impact on the nature of the effects of Ca 2+ channel antagonists via the NO/NOS pathways. In a recent investigation the impact of NOS inhibition on vasorelaxant effects of nifedipine, diltiazem and mibefradil in aortic rings from Dahl salt-resistant normotensive and salt-sensitive hypertensive rats was assessed [16] . It was revealed that while NOS inhibition only affected the vasorelaxant actions of mibefradil in tissues from hypertensive rats, it significantly attenuated relaxation by all three Ca 2+ channel antagonists in tissue from normotensive rats. This would suggest that pathophysiological states that modify endothelial cell function [17] can also alter the vascular actions of Ca 2+ channel antagonists mediated via the NO/NOS system [16] . It seems that at present, the available clinical data on the additional beneficial cardiovascular effects of Ca 2+ channel antagonists due to enhanced generation of NO is not very compelling. Therefore, further clinical trials designed and aimed at the specific question of whether 1,4-dihydropyridine Ca 2+ channel antagonists have an additional benefit and are able to reduce morbidity and mortality in patients with cardiovascular dysfunction due to generation of NO are clearly needed.