L-NAME hypertension: trying to fit the pieces together

Inhibition of nitric oxide (NO) synthase (NOS) by NG-nitro-L-arginine methyl ester (L-NAME) is a well-established model of experimental hypertension, and cardiovascular and renal disease [1–4]. The cardiovascular effects of L-NAME include endothelial dysfunction, vascular remodelling and inflammation, and myocardial hypertrophy [3–7]. The mechanism of L-NAME-induced hypertension and cardiovascular disease involves more than removal of the tonic vasodilator action of NO by NOS inhibition, and there is evidence for contributions from the renin–angiotensin system (RAS), sympathetic nervous system (SNS), prostaglandins, and superoxide anions to this process. Chronic L-NAME administration increases plasma renin levels [3,8,9] and cardiac and vascular angiotensin-converting enzyme (ACE) activity [3], and causes a transient increase in both type 1 (AT1) and type 2 (AT2) angiotensin II receptors in the heart [4]. Acute administration of ACE inhibitor (ACEI) or AT1 receptor blocker (ARB) causes only modest reduction in blood pressure of L-NAME hypertensive rats [9,10], but chronic ACEI or ARB administration normalizes blood pressure [8,11,12] and prevents the vascular remodelling and inflammation, and myocardial hypertrophy due to L-NAME administration [4,6,12]. The different responses to acute and chronic RAS inhibition suggest both functional and structural mechanisms contribute to L-NAME hypertension. L-NAME hypertensive rats have increased plasma noradrenaline and adrenaline levels [9] and show a marked fall in blood pressure with pentolinium administration [9,10,13], suggesting an important contribution by the SNS to L-NAME hypertension. Sympathectomy, and the α1 blocker, prazosin, also reverse L-NAME hypertension [14–16]. Reversal of L-NAME hypertension by both RAS and SNS inhibition suggests the hypertension may be due in part to an altered balance between NO and angiotensin II in the central nervous system, resulting in increased sympathetic tone that might be normalized by RAS inhibition [17]. L-NAME hypertension is associated with increased vascular and renal expression of cyclooxygenase-2 (COX-2), and COX-2 inhibition considerably attenuates the hypertension and proteinuria that accompany L-NAME administration [18]. L-NAME hypertension is associated with increased vascular production of superoxide anions [7,19–21]. Simultaneous administration of the anti-oxidant N-acetylcysteine prevented development of hypertension and vascular inflammation, and augmented NOS activity, in L-NAME-treated rats [7,21]. However, N-acetylcysteine had only a modest effect on blood pressure of rats with established L-NAME hypertension [7,21]. Chronic ACEI and ARB therapy prevented the increased vascular superoxide production [7,19,20], suggesting that angiotensin II-stimulated vascular NAD(P)H oxidases are responsible for the increased superoxide production that accompanies L-NAME administration. Angiotensin II-stimulated superoxide production may cause down-regulation of endothelial dihydrofolate reductase, resulting in uncoupling of endothelial NOS (eNOS) and further reducing NO bioavailability [7,20,22]. Several different strategies have been used to dissect the mechanisms of L-NAME-induced hypertension and cardiovascular disease. Hydralazine was administered to L-NAME hypertensive rats to dissociate the effects of hypertension from other actions of L-NAME. Hydralazine prevented hypertension and restored acetylcholine-mediated vasodilatation and endothelial soluble guanylyl cyclase expression, but did not affect the increased superoxide production due to L-NAME administration [5]. By contrast, prevention of superoxide production by inhibition of superoxide dismutase in vitro did not correct endothelial dysfunction [5]. In addition, hydralazine failed to prevent the cardiovascular structural changes, increased cardiac angiotensin II receptors, and vascular inflammatory changes [3,4], whereas L-arginine prevented these effects of L-NAME administration [4]. ACEI and ARB administration prevented the vascular inflammation due to L-NAME administration [6,7,20], and these anti-inflammatory effects were seen for both hypotensive and non-hypotensive doses of the ARB, losartan, indicating they were independent of change in blood pressure [7]. Vascular inflammation consequent to L-NAME administration is accompanied by induction of inducible NOS (iNOS) expression, and iNOS contributes to NO production during L-NAME hypertension [6,10,21]. Induction of iNOS by L-NAME is prevented by ARB [6]. These studies indicate that endothelial dysfunction that accompanies L-NAME hypertension is due to increased blood pressure. Moreover, some of the effects of RAS inhibition in L-NAME hypertension are due to reduction of blood pressure, and some are independent of blood pressure lowering. The studies reported by De Gennaro Colonna et al. in this issue of the journal [23] and elsewhere [24] provide a new level of complexity to our understanding of L-NAME hypertension and its response to RAS inhibition by showing that some of the effects of the ACEI, enalapril, and the ARB, losartan, in L-NAME hypertension were prevented by coadministration of the bradykinin type 2 (B2) receptor antagonist icatibant. L-NAME administration to rats produced hypertension, together with decreased eNOS mRNA in the aorta, decreased plasma nitrite/nitrate concentrations, decreased aortic release of 6-keto-PGF1α, and attenuation of the vasorelaxant effects of acetylcholine [23,24]. Enalapril or losartan administration normalized blood pressure and all other parameters, and these actions of enalapril and losartan were prevented by icatibant. These studies show that some of the effects of ACEI and ARB in L-NAME hypertension may be dependent upon, or mediated by, B2 receptor activation. However, these findings do not exclude contributions from angiotensin II and other mechanisms to L-NAME-induced hypertension and cardiovascular disease. Restoration of plasma nitrite/nitrate concentrations by enalapril and losartan, despite continued L-NAME administration [23,24], suggests the inhibition of vascular NO production by L-NAME, at the dose administered, was mainly due to reduced eNOS gene expression, with little contribution from direct inhibition of NOS activity. The correction of endothelial dysfunction by hydralazine, cited above [5], suggests L-NAME-induced reduction of eNOS mRNA levels was a consequence of hypertension. This would explain why these findings of De Gennaro Colonna et al. [23,24] differ from those of Grumbach et al. [25], who reported L-NAME to increase eNOS mRNA levels and gene transcription by endothelial cells in vitro. However, correction of hypertension does not explain why losartan increased eNOS mRNA levels above control levels [23]. Recovery of endothelial function due to correction of hypertension may also account for the restoration by enalapril and losartan of aortic release of 6-keto-PGF1α. Many issues need to be considered when interpreting the effect of icatibant on the actions of enalapril and losartan in L-NAME hypertension. As reviewed by De Gennaro Colonna et al. [23,24], there is ample evidence that bradykinin and B2 receptors participate in the actions of ACEI and ARB. ACEI increase bradykinin levels [26,27], and icatibant attenuates some of the actions of ACEI [28–32]. Losartan was reported not to increase bradykinin levels in rats [33], but recent studies showed losartan increased bradykinin levels in hypertensive humans [34]. Both animal and human studies implicate kinin peptides in the actions of ARB, possibly mediated by the stimulation of AT2 receptors by the increased angiotensin II levels that accompany ARB therapy [35–45]. There are additional mechanisms by which the actions of ACEI and ARB may be susceptible to interference by icatibant. ACEI potentiate the effects of bradykinin not only by inhibition of bradykinin metabolism, but also by a process dependent on cross talk between ACE and the B2 receptor [46]. Furthermore, additional to a role for bradykinin in mediating the effects of AT2 receptor stimulation [35–45], AT1 and B2 receptors form heterodimers that enhance AT1 receptor signalling and may contribute to increased responsiveness to angiotensin II [47]. In addition, losartan has many actions independent of the AT1 receptor [48]. Watanabe et al. [49] recently reported that in short-term studies performed in vitro, the losartan metabolite EXP3179 activated Akt and eNOS phosphorylation via the vascular endothelial growth factor receptor-2, and reduced endothelial cell apoptosis, by a mechanism independent of either the AT1 or the AT2 receptor. Several other issues require consideration when interpreting the effect of icatibant on the actions of enalapril and losartan in L-NAME hypertension. First, just as losartan may have actions independent of the AT1 receptor, icatibant and its metabolites may have actions independent of the B2 receptor, although evidence for this suggestion is currently lacking. Second, although an intact B2 receptor mechanism and tonic B2 receptor activity may be a necessary requirement for the actions of enalapril and losartan, change in B2 receptor activity may not be required for these compounds to exert their actions. Third, it is of note that icatibant had no effect when administered alone to L-NAME-treated rats [23]. This may have been because a dysfunctional endothelium was unable to respond to B2 receptor stimulation, and therefore B2 receptor blockade was ineffective. It is therefore paradoxical that some restoration of endothelial function by enalapril or losartan may have been necessary for icatibant to be effective. Fourth, the studies by De Gennaro Colonna et al. [23,24] were chronic studies. As noted above, acute ACEI and ARB administration had only modest effect on blood pressure in established L-NAME hypertension [9,10]. The changes observed by De Gennaro Colonna et al. [23,24] with chronic ACEI and ARB administration may have been due to a sequence of events; an intact B2 receptor mechanism may have been necessary for one part of the sequence. Fifth, as described above, ACEI and ARB also reduce superoxide anion production and prevent the vascular remodelling and inflammation, and myocardial hypertrophy due to L-NAME administration [4,6,7,12,19,20]. Further studies will be required to assess whether the B2 receptor has a role in mediating these additional actions of ACEI and ARB in L-NAME hypertension. The studies of De Gennaro Colonna et al. [23,24] provide a new piece of the puzzle that is L-NAME hypertension and its response to RAS inhibition, but further studies are required before we can fit the pieces together.