Certainly, much of the pharmacology and metabolism of GTN have been unraveled over 100 years of intense investigation. Nevertheless, fundamental questions have existed pertaining to the molecular mechanisms that link the administration of minute doses of GTN in the clinic to the robust and momentary pharmacologic effects such doses elicit in patients. Various studies have indicated that eNOS is activated by GTN in endothelial cells and that eNOS substrates/cofactors contribute to maximize the effects of GTN as a vasodilator and attenuate GTN resistance [8
]. These studies have supported a role for eNOS activation in mediating the drug-induced vasodilation. In contrast, another set of investigations has argued against a fundamental role for eNOS in mediating GTN-induced pharmacologic and toxic effects upon the vasculature. These studies have claimed that metabolic routes sustain NO production from GTN and that their inactivation is causative of GTN tolerance [2
]. Although we believe that metabolic routes contribute to GTN-induced effects, particularly at higher doses, our recent observations are consistent with the first set of studies that found endogenous NO production as the cause of nitroglycerin-mediated vasodilation.
Indeed, we recently presented directed evidence demonstrating that eNOS phosphorylation occurs momentarily after GTN administration [3
] and that NO recovery from GTN-treated cells is comparable to that elicited by classical activators of signal transduction such as VEGF (this work, ). Likewise, L-NIO, an irreversible inhibitor of constitutive nitric oxide synthases significantly reduced NO production from endothelial cells exposed to GTN and VEGF ([3
], and ). Notably, the similar inhibitory effects were attained through the use of PI3K and Akt inhibitors, which are known upstream activators of agonist elicited NO production by eNOS. The relevance of the PI3K/Akt pathway for GTN-induced vasodilation was further demonstrated in through the pharmacologic inhibition of each enzyme and validated in mesenteric arteries of genetic knockout animals. Importantly, demonstrates that in either case (inhibitor studies or genetic knockout) significant attenuation of GTN effects is achieved at pharmacologically relevant doses of GTN (<50 nM) but not at higher concentrations, at which metabolic conversion of GTN to NO is likely to prevail. The studies presented in are in close agreement with previously published results that demonstrated the efficacy of NO inhibitors or endothelial removal in preventing low-dose (<50 nM) but not high-dose nitroglycerin-induced vasodilation [3
]. Not surprisingly, pronounced effects of GTN in diminishing diastolic blood pressure in rats were markedly reduced when the animals were pretreated with wortmannin or Akt inhibitor (). Taken together, these results constitute compelling evidence implicating signal transduction pathways in the mediation of GTN's pharmacological effects by activating eNOS. Indeed, studies performed with endothelial cells and presented in demonstrated that 0.5 µM GTN instantaneously induced the phosphorylation of eNOS at the activation site Ser 1177, which was fully inhibited by either PI3K or Akt inhibitor. These studies were recapitulated in human endothelial microvascular cells (). In both BAEC and HMEC, eNOS phosphorylation was temporally paralleled by Akt activation, indicating the involvement of the PI3K/Akt pathway in GTN-induced eNOS activation. Interestingly, we also found that PTEN, the enzyme that opposes PI3K activity by degrading 3,4,5-InsP3
, was rapidly inhibited by GTN. PTEN inhibition was determined through the Western blot analysis of the inhibitory site Ser 380 phosphorylation and through the quantification of the active second messenger 3,4,5-InsP3
(). PTEN inhibition was further confirmed by the measurement of PTEN activity after immunopurification from lysates of cells previously exposed to GTN (). Therefore, we propose that GTN rapidly inactivates PTEN in endothelial cells leading to the accumulation of 3,4,5-InsP3
. Higher 3,4,5-InsP3
levels arising from the unopposed PI3K activity lead to Akt and eNOS activation (schematically represented in ). Importantly, PTEN lipid phosphatase activity is dependent on the critical active residue Cys 124. In its reduced form the low-pKa
Cys 124 thiolate catalyzes the removal of the 3-phosphate group of 3,4,5-phosphatidylinositol in remarkable similarity to the proposed and widely accepted mechanism of ALDH-2 inhibition by GTN. However, different from ALDH-2, which is confined in mitochondria, PTEN, which is itself fairly sensitive to inhibition by oxidants and by electrophiles, resides predominantly in the cytosol, specifically at the vicinity of the plasma membrane, and is thus more likely to interact with diffusible xenobiotics upon their entry into the cell. Indeed, the fundamental role of ALDH-2 in GTN bioconversion to NO was claimed largely on the basis of knockout studies that showed that ALDH-2-knockout animals are less responsive to low-dose GTN than ALDH-2-competent animals. Nevertheless, depletion of ALDH-2 has been linked to increased oxidative stress and vascular dysfunction [30
] probably because of increased levels of reactive species (aldehydes and oxidants) production. Hence, with the currently available data it is impossible to distinguish whether the GTN-tolerant phenotype exhibited by the ALDH-2-knockout animal is a consequence of its inability to convert GTN to NO or, alternatively, is attributable to dysregulation of oxidant-sensitive signal transduction pathways such as the PI3K/Akt/PTEN axis.
Representation of the proposed mechanism of GTN-induced eNOS activation via PTEN inhibition.
Aldehydes and oxidants can potentially lead to persistent inactivation of PTEN [33
] and eNOS aberrant activation, which is claimed to be a cause of vascular dysfunction in several publications [34
]. eNOS and, secondary to it, endothelial dysfunction may be a consequence of ALDH-2 deficiency, explaining the unresponsive phenotype of the ALDH-2-knockout animals independent of ALDH-2 enzymatic activity. Consistent with this possibility, recent studies have demonstrated that ALDH-2 depletion causes vascular dysfunction, seemingly because of a higher superoxide radical anion production by mitochondria, which further reduces NO availability while producing the strong oxidant peroxynitrite [33
]. Therefore, a definitive role for ALDH's intermediacy in low-dose GTN-induced vasodilation is pending the verification that in ALDH-2-knockouts increased
, oxidative stress, and aldehyde accumulation do not critically affect GTN-mediated signaling or consume NO, thus limiting its biological actions. In a recent study, we directly demonstrated that GTN is capable of inducing eNOS phosphorylation at the activation site Ser 1177 in the aorta of animals and that nitric oxide inhibition is sufficient to attenuate both the decrease in blood pressure and the response of isolated aortic rings to low-dose GTN [3
]. In addition, we showed that at low doses (<50 nM) GTN-induced vasodilation is dependent on the endothelium and correlates temporally with eNOS activation in accordance with previously published work [9
]. These results, the earlier studies showing eNOS activation by GTN in cells, and the demonstrated dependence of PI3K on the GTN-induced eNOS activation reported here leave little space for any doubt about the involvement of nitric oxide synthases and signal transduction pathways in low-dose GTN-induced effects. At high concentrations (>50 nM) metabolism-driven routes are likely to be dominant, as previously shown by us and others [2
] and confirmed here by the demonstration that at high GTN doses inhibition of PI3K/Akt does not result in attenuation of GTN-induced vasodilation (). Because metabolic processes are dependent on enzymatic reactions governed by rate laws, it is expected that such pathways would be favored by high but not low doses, in which case amplification of a signal by an array of interdependent and highly efficient transducers should prevail.
In summary, we have demonstrated that by inhibiting PTEN, GTN augments Akt and eNOS activities, which mediate the low-dose effects of GTN on the vasculature (please see schematic representation in ). The mechanisms underlying the activity of GTN as a powerful vasodilator are determined by dose and depend on multiple intricate mechanisms, which involve signal transduction and metabolic bioactivation. The demonstration that GTN, like other electrophiles, is capable of inducing PI3K/Akt/eNOS activation through PTEN inhibition may serve as a cornerstone warranting further studies focused on the cellular adaptations that trigger GTN tolerance and nitroglycerin-induced vascular dysfunction by affecting cellular signaling networks.