Edward G. Shesely

Endothelial Nitric Oxide Gene Knockout Mice Cardiac Phenotypes and the Effect of Angiotensin-Converting Enzyme Inhibitor on Myocardial Ischemia/Reperfusion Injury

We tested the hypothesis that nitric oxide (NO) released by endothelial NO synthase (eNOS) is not only important in blood pressure regulation but also involved in cardiac function and remodeling and in the cardioprotective effect of angiotensin-converting enzyme inhibitors (ACEi). With the use of a 2D Doppler echocardiography system equipped with a 15-MHz linear transducer, we evaluated left ventricular (LV) morphology and function in conscious eNOS knockout mice (eNOS(-/-); n=15) and their wild-type littermates (eNOS(+/+); n=16). We also studied whether in eNOS(-/-) mice (1) myocardial ischemia/reperfusion injury is more severe and (2) the cardioprotective effect of ACEi is diminished or absent. In comparison with the wild type, eNOS(-/-) mice had significantly increased systolic blood pressure (128+/-3 versus 108+/-5 mm Hg; P<0.001) and decreased heart rate (531+/-22 versus 629+/-18 bpm; P<0.001) associated with increased LV posterior wall thickness (0.80+/-0.04 versus 0.64+/-0.02 mm; P<0.001) and LV mass (18.3+/-0.9 versus 13.1+/-0.5 mg/10 g body weight; P<0.01). Despite hypertension and LV hypertrophy, LV chamber dimension, shortening fraction and ejection fraction (indicators of LV contractility), and cardiac output did not differ between the 2 strains, which indicates that LV function in eNOS(-/-) mice is well compensated. We also found that in eNOS(+/+) mice, ACEi decreased the ratio of myocardial infarct size to area at risk from 62.7+/-3.9% to 36.3+/-1.6% (P<0. 001), whereas in eNOS(-/-) mice this effect of ACEi was almost abolished: the ratio of myocardial infarct size to area at risk was 67.2+/-2.9% in the vehicle-treated group and 62.7+/-3.9% in mice treated with ACEi. Moreover, infarct size in vehicle-treated eNOS(-/-) mice was not significantly different from eNOS(+/+) mice given the same treatment. We concluded that (1) endothelium-derived NO plays an important role in the regulation of blood pressure homeostasis; (2) NO released under basal conditions has no significant impact on cardiac function; and (3) ACEi protect the heart against ischemia/reperfusion injury in mice and that this effect is mediated in part by endothelium-derived NO.

Protein expression, vascular reactivity and soluble guanylate cyclase activity in mice lacking the endothelial cell nitric oxide synthase: contributions of NOS isoforms to blood pressure and heart rate control

OBJECTIVEnBoth disruption of the endothelial nitric oxide synthase (eNOS) gene and pharmacological inhibition of the NOS produce modest hypertension. It is unclear if and to what extent NOS isoforms other than eNOS contribute to this effect and how loss of one copy of the eNOS gene might impact on vascular reactivity or eNOS protein expression.nnnMETHODSnWe examined protein expression, vascular reactivity, activity of soluble guanylate cyclase, blood pressure and heart rate in mice completely lacking the eNOS gene (eNOS-/-), wild-type mice (eNOS+/+) and mice heterozygotic for the eNOS gene (eNOS+/-).nnnRESULTSnWhile eNOS-/- mice had mild hypertension and bradycardia, eNOS+/- mice were normotensive. In control mice, oral administration of L-NAME (approximately 100 mg/kg/day x 21 days) increased blood pressure to levels observed in eNOS-/- mice. In eNOS-/- mice, chronic oral administration of L-NAME had no effect on blood pressure, suggesting that inhibition of other NOS isoforms unlikely contribute to hypertension. L-NAME treatment induced bradycardia in both control and eNOS-/- mice, suggesting that both eNOS and other isoforms of NOS might be involved in heart rate control. Studies of aortic rings from eNOS-/- mice revealed a complete lack of endothelium-dependent vascular relaxation in response to acetylcholine and the calcium ionophore A23187 and an increase in sensitivity to phenylephrine, serotonin and nitroglycerin. Aortic rings from eNOS+/- mice demonstrated only minor alterations of responses to nitroglycerin and a normal relaxation to either acetylcholine or A23187 compared to vessels from eNOS-/+. Western analysis demonstrated that eNOS expression was virtually identical between eNOS+/+ and eNOS+/- mice and was absent in eNOS-/- mice. The activity of lung-isolated soluble guanylate cyclase was identical in the three strains of mice.nnnCONCLUSIONSnWe conclude that loss of one copy of the eNOS gene, as observed in heterozygotic animals, has no effect on vascular reactivity, blood pressure or eNOS protein expression. Isoforms of NOS, other than eNOS are unlikely involved in blood pressure regulation but may participate in heart rate control.

eNOS Mediates l-Arginine–Induced Inhibition of Thick Ascending Limb Chloride Flux

We recently reported that the rat thick ascending limb (THAL) possesses an active isoform of nitric oxide synthase (NOS) that is substrate-limited in vitro. NO produced by THAL NOS inhibits chloride flux. Protein and transcript for each of the primary NOS isoforms-endothelial (eNOS), inducible (iNOS), and neuronal (nNOS)-have been demonstrated in THALs. However, the NOS isoform that mediates NO-induced inhibition of chloride flux is unknown. We hypothesized that NO produced from eNOS in the THAL inhibits NaCl transport. THALs from male eNOS, iNOS, and nNOS knockout mice and C57BL/6J wild-type controls were perfused in vitro and the response of transepithelial chloride flux (J(Cl)) to L-arginine (L-Arg), the substrate for NOS, and spermine NONOate (SPM), an NO donor was measured. We first tested whether isolated mouse THALs could synthesize NO and whether this NO inhibits transport. Addition of 0. 5 mmol/L L-Arg to the bath decreased J(Cl) from 105.8+/-17.5 to 79. 2+/-15.8 pmol/mm per minute (P<0.01) in C57BL/6J wild-type mice, whereas addition of D-Arginine had no effects on J(Cl.) In contrast, addition of 0.5 mmol/L L-Arg to the bath did not alter J(Cl) of THALs from eNOS knockout mice. When 10 micromol/L SPM was added to the bath of eNOS knockout THALs, J(Cl) decreased from 89.1+/-8.6 to 74.8+/-7.5 pmol/mm/min (P<0.05). Thus the lack of responsiveness of eNOS knockout THALs to L-Arg was not due to an inability to respond to NO. We next evaluated the role of iNOS and nNOS in the response to L-Arg. Addition of 0.5 mmol/L L-Arg to the bath decreased J(Cl) in THALs from iNOS and nNOS knockout mice by 37.7+/-6.4% (P<0.05) and 31.8+/-8.3% (P<0.01), respectively. We conclude that eNOS is the active isoform of NOS in the THAL under basal conditions. Mouse THAL eNOS responds to exogenous L-Arg by increasing NO production, which, in turn, inhibits J(Cl).

Role of nNOS in Blood Pressure Regulation in eNOS Null Mutant Mice

The role of neural nitric oxide synthase (nNOS) in regulating blood pressure (BP) remains uncertain. Recently it was reported that in mice lacking functional endothelial NOS (eNOS) genes (-/-), acute administration of a nonselective NOS inhibitor, Nw-nitro-L-arginine, decreased mean BP, suggesting that NO released by non-eNOS isoforms increases BP. Because the inducible NOS isoform is not constitutively expressed and when induced causes hypotension, we hypothesize that it is NO produced by nNOS that increases BP in the absence of eNOS activity. To test this hypothesis, we studied the acute effect of selective and nonselective nNOS inhibitors on BP and cerebellar NOS activity in eNOS (-/-), wild-type (+/+), and heterozygous (+/-) mice as well as in +/+ mice with renovascular hypertension. Because it is not known whether the decrease in BP caused by acute NOS inhibition in -/- mice can occur chronically, we also studied the effect of chronic NOS inhibition on both BP and cerebellar NOS activity. eNOS (-/-) mice had higher BP than +/+ or +/-mice, and acute administration of the selective nNOS inhibitor 7-nitroindazole (7-NI) decreased their mean BP from 137+/-13 to 124+/-12 mm Hg (P<0.01). In +/+, +/-, or renovascular hypertensive +/+ mice, 7-NI caused a small but insignificant rise from 105+/-5 to 110+/-6 mm Hg, from 115+/-9 to 119+/-13 mm Hg, and from 146+/-6 to 150+/-6 mm Hg, respectively. Fifteen minutes after administration of 7-NI, cerebellar NOS activity decreased by 70%; however, this inhibitory effect was brief, since 2 hours after 7-NI administration NOS returned toward control values. Chronic oral or intraperitoneal administration of 7-NI did not inhibit cerebellar NOS activity, whereas the nonselective NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) decreased this activity by 50%. Therefore, we studied the effect of chronic L-NAME administration (4 weeks) on BP. In -/- mice, chronic L-NAME administration decreased BP from 135+/-4 to 120+/-3 mm Hg (P<0.05), whereas in +/+ and +/-mice, as expected, it increased BP from 109+/-2 to 125+/-3 mm Hg (P<0.001) and from 107+/-6 to 119+/-5 mm Hg (P<0.02), respectively. After L-NAME administration was stopped, BP returned to baseline. These results suggest that in eNOS -/- mice, NO derived from nNOS increases BP both acutely and chronically.

Effect of ACE Inhibitor on DOCA-Salt– and Aortic Coarctation–Induced Hypertension in Mice: Do Kinin B2 Receptors Play a Role?

Kinins have been shown to play an important role in the cardioprotective effect of ACE inhibitors (ACEi) during heart failure and ischemia-reperfusion. However, it is controversial as to whether kinins oppose the hypertensinogenic effect of deoxycorticosterone acetate plus salt (DOCA-salt) or aortic coarctation and whether they mediate both chronic antihypertensive and cardiac antihypertrophic effects of ACEi in hypertension. Using normal 129/SvEvTac mice and mice lacking the bradykinin B2 receptor gene (B2-KO), we investigated whether (1) the hypertensinogenic effect of DOCA-salt or aortic coarctation is enhanced in B2-KO mice and (2) the chronic antihypertensive and antihypertrophic effects of an ACEi (ramipril, 4 mg. kg-1. d-1) are mediated by B2 receptors in aortic coarctation (6 weeks)- and DOCA-salt (4 weeks)-induced hypertension. Before surgery, there was no difference between 129/SvEvTac and B2-KO mice in terms of blood pressure and heart weight, suggesting that kinins are not essential to maintaining normal blood pressure. DOCA-salt (volume expansion) or aortic coarctation (renin-dependent) induced similar hypertension and left ventricular hypertrophy (LVH) in 129/SvEvTac and B2-KO mice, suggesting that kinins do not play an essential role in the development of DOCA-salt- or aortic coarctation-induced hypertension. We found that B2 receptors mediate only the early (1 week) but not the late phase (4 weeks) of the chronic hypotensive effect of ACEi in DOCA-salt hypertension. On the other hand, chronic ACE inhibition prevented the development of hypertension and LVH in both 129/SvEvTac and B2-KO mice given DOCA-salt or subjected to aortic coarctation, suggesting that kinins do not participate in the chronic antihypertensive and antihypertrophic effects of ACEi in these 2 models of hypertension. Thus, in mice, kinins acting via B2 receptors do not participate in (1) maintenance of normal basal blood pressure, (2) establishment and maintenance of hypertension induced by DOCA-salt or aortic coarctation, and (3) chronic antihypertensive and cardiac antihypertrophic effects of ACEi in DOCA-salt and aortic coarctation hypertension.

Effect of Chronic Blockade of the Kallikrein-Kinin System on the Development of Hypertension in Rats

The kallikrein-kininogen-kinin system is an important vasodilator and vasodepressor component of the cardiovascular system. Acting mainly through B2 receptors, kinins may counterbalance the pressor effect of angiotensin II, salt, and mineralocorticoids plus salt. Using rats lacking the bradykinin precursors low- and high-molecular-weight kininogen or a B2 kinin receptor antagonist (icatibant), we investigated whether absence or blockade of the kallikrein-kinin system alters blood pressure (BP) in rats given (1) chronic infusion of Ang II, (2) a normal or high salt diet, or (3) chronic administration of deoxycorticosterone acetate (DOCA) plus salt. We confirmed the genotype and phenotype of Brown Norway Katholiek rats (BNK) and found that they had a G-to-A point mutation on the kininogen gene compared with Brown Norway (BN) and Sprague-Dawley (SD) rats, very low levels of high-molecular-weight kininogen (17±3 ng/mL) compared with BN and SD (1814±253 and 2397±302 ng/mL, respectively;P <0.01), and plasma low-molecular-weight kininogen concentrations below detectable limits compared with 1773±74 and 1781±140 ng/mL for BN and SD, respectively. Basal BP was the same in BNK and BN. Chronic infusion of icatibant did not alter BP in BN or Wistar rats. At doses that blocked the acute effect of bradykinin, icatibant did not potentiate the pressor effect of a chronic subpressor or pressor dose of angiotensin II in male and female Wistar rats nor that of a high salt diet (2%) plus unilateral nephrectomy in male Wistar rats. Moreover, blockade of the kallikrein-kininogen-kinin system in either BN rats given a very high dose of icatibant or kinin-deficient rats (BNK) did not potentiate the pressor effect of angiotensin II (nonpressor dose) or a high salt (3% NaCl) diet given for 2 weeks. Established DOCA-salt hypertension was not exaggerated in rats treated with icatibant but was partially attenuated by ramipril (1.5 mg · kg−1 · d−1 for 7 days;P <0.002). This antihypertensive effect was abolished by icatibant (P <0.002, ramipril versus ramipril plus icatibant). These results suggest that endogenous kinins do not participate in the maintenance of normal blood pressure or antagonize the development of hypertension induced by chronic infusion of angiotensin II, a high salt diet, or DOCA-salt. However, kinins appear to play an important role in the antihypertensive effect of angiotensin-converting enzyme inhibitors in DOCA-salt hypertension.

An Enhanced Effect of Arginine Vasopressin in Bradykinin B2 Receptor Null Mutant Mice

Under water restriction, arginine vasopressin (AVP) is released and promotes water reabsorption in the distal nephron, mainly through AVP V2-receptors. It has been proposed that renal kinins counteract the hydro-osmotic effect of AVP. We hypothesized that kinins acting through B2 receptors antagonize the urinary concentrating effect of AVP. To test this, bradykinin B2 receptor knockout mice (B2-KO) and 129/SvEv mice (controls) were placed in metabolic cages and urine collected for 24 hours (water ad libitum). After that, urine was again collected from the same mice during 24 hours of water restriction. Urinary volume (UV), urinary osmolarity (UOsm), and urinary Na+ (UNaV) and K+ (UKV) excretion were determined. On water restriction, UV in controls decreased by approximately 25%, whereas in B2-KO mice there was almost a 60% drop in urinary output (P=0.001 versus controls). In the controls, water restriction increased UOsm by 347 mOsm/kg H2O, approximately 14% above baseline (NS), whereas in knockout mice the increase was 3 times that seen in the controls: >1000 mOsm/kg H2O (P=0.001 versus controls). Compared with normohydration, UNaV and UKV in the water-restricted state increased more in controls than in B2-KO mice. This difference in electrolyte excretion could be explained by greater dehydration in the controls (dehydration natriuresis). In a second protocol, we tried to mimic the effect of endogenous AVP by exogenous administration of an AVP V2-receptor agonist, desmopressin (DDAVP). To suppress endogenous AVP levels before DDAVP administration, mice were volume-overloaded with dextrose and alcohol. UOsm was 685+/-125 and 561+/-58 mOsm/kg H2O in water-loaded controls and B2-KO mice, respectively. After DDAVP was injected subcutaneously at a dose of 1 microgram/kg, UOsm increased to 1175+/-86 mOsm/kg H2O (Delta+490 mOsm) in the controls and 2347+/-518 mOsm/kg H2O (Delta+1786 mOsm) in B2-KO mice (P<0.05 versus controls). We concluded that water restriction or exogenous administration of an AVP V2-receptor agonist has a greater urinary concentrating effect in B2-KO mice than in controls, suggesting that endogenous kinins acting through B2 receptors oppose the antidiuretic effect of AVP in vivo.

Response to Inflammation, Angiotensin II, and Hypertension

The hypothesis that inflammation plays a role in the pathogenesis of hypertension is supported by epidemiological studies indicating that inflammatory markers are associated with hypertension.1,2 However, association does not necessarily indicate cause and effect. Evidence that inflammation may participate in the pathogenesis of various models of experimental hypertension is provided by the use of immunosuppressive drugs. As indicated in the letter by Rodriguez-Iturbe et al,3 these compounds prevent inflammatory cell infiltration and decrease blood pressure. There are many other anti-inflammatory drugs, such as nonsteroidal anti-inflammatory agents or glucocorticoids, that do not ameliorate hypertension. On the contrary, they tend to antagonize the …