Journal of the Renin-Angiotensin-Aldosterone System

There is increasing evidence to suggest endothelial dysfunction as a critical factor in vascular diseases. Genetically predisposed spontaneously hypertensive rats (SHR) treated with inhibitors of nitric oxide (NO) synthase, develop a severe hypertensive nephrosclerosis without the necessity for surgical reduction in renal mass, nephrectomy, renal infarction or nephrotoxic drugs. In these animals, endothelial dysfunction is considered a valid model for assessment of the efficacy of cardiovascular therapy. SHR were treated with either the angiotensin-converting enzyme inhibitor enalapril or the angiotensin II (Ang II) AT₁-receptor antagonist (AIIA) valsartan at sub-hypotensive doses and the effects on survival rates, cardiac and renal changes were monitored. Rats treated with valsartan, alone or in combination with enalapril, showed markedly higher survival rates (67—85%, respectively) than untreated animals (37%) or those treated with enalapril alone (55%). Valsartan at a dose which attenuated blood pressure increase led to even greater survival rates (95%). Despite these improved survival rates, at non-hypotensive doses the drugs had no effect on histological appearance, nor was kidney function improved. Plasma creatinine levels were reduced by valsartan, alone or in combination with enalapril, but proteinuria persisted with all treatments over the 12 weeks of the study. Aldosterone levels were significantly reduced by all treatments. The results suggest a beneficial role for endothelium in hypertension. Reduced renal perfusion pressure probably underlies the beneficial renal effects of high-dose valsartan.

The aim of the present work was to investigate the binding properties of the selective AT₁-receptor antagonist irbesartan to human AT₁-receptors by direct radioligand binding. For this purpose the specific binding of [³H]-irbesartan to intact Chinese Hamster Ovary (CHO) cells expressing human recombinant AT₁-receptors was determined. Specific binding of [³H]-irbesartan rapidly reached equilibrium and was saturable with a K_D of 1.94 ± 0.12 to a homogeneous class of binding sites. Its binding was inhibited by other AT₁ antagonists (AIIAs) with the same potency order as previous results from [³H]-angiotensin II and [³H]-candesartan binding to human AT₁-receptors. Whereas the dissociation rate of [³H]-irbesartan was essentially independent of the radioligand concentration, it was much slower at 12°C when compared with 37°C. Moreover, the dissociation rate was similar, as determined in washout experiments in the absence or presence of unlabelled AT₁ antagonists. At 37°C the dissociation rate constant corresponded to a half-life of approximately seven minutes, which is sufficient to explain the partially insurmountable inhibition by irbesartan in previous studies. In contrast, other phenomena such as the plasma half life and tissue-related factors are necessary to explain its sustained in vivo antihypertensive effect.

To investigate the molecular mechanism(s) of insulin action on the expression of the angiotensinogen (ANG) gene in kidney proximal tubular cells, we constructed a fusion gene, pOGH (hANG N-1064/+27), containing the 5'-flanking regulatory sequence of the human ANG gene fused with the human growth hormone (hGH) gene as a reporter and stably integrated the fusion gene into the opossum kidney (OK) cell genomes. The level of expression of pOGH (hANG N-1064/+27) was quantified by the amount of immunoreactive hGH secreted into the medium. The addition of a high level of D(+)-glucose (25 mM) or phorbol 12-myristate 13-acetate (PMA, 10^-7 M) stimulated the expression of the fusion gene in OK cells. The stimulatory effect of glucose (25 mM) was blocked by insulin and tolrestat (an inhibitor of aldose reductase). Tolrestat also inhibited the increase of cellular DAG and PKC activity stimulated by 25 mM glucose. While insulin did not affect the cellular DAG and PKC activity, it did block the stimulatory effect of high glucose (25 mM) and PMA on the expression of the fusion gene. Finally, PD98059 (an inhibitor of mitogen-activated protein kinase kinase (MEK)) enhanced the stimulatory effect of high levels of glucose and blocked the inhibitory effect of insulin on the expression of the fusion gene as well as on the phosphorylation of MEK and mitogen-activated protein kinase (MAPK). In contrast, Wortmannin (an inhibitor of phosphatidylinositol-3-kinase) did not block the inhibitory effect of insulin on the ANG gene expression. These studies demonstrate that the action of insulin, blocking the stimulatory effect of a high level of D(+)-glucose (25 mM) on the ANG gene expression is mediated, at least in part, via the 5'flanking region of the ANG gene and MAPK signal transduction pathway.

A recent study has shown that losartan, an AT₁-receptor antagonist, interacts with thromboxane A₂ (TxA₂)/prostaglandin H₂ (PGH₂) receptors in human platelets. The aim of the present study was to analyse the ability of different angiotensin II (Ang II) AT₁-receptor antagonists to inhibit TxA₂-dependent human platelet activation. Platelets were obtained from healthy volunteers and were stimulated with the thromboxane A₂ analogue, U46619 (10^-6 mol/L). U46619-stimulated platelet activation was significantly reduced by losartan in a dose-dependent manner. Only maximal doses of valsartan (5x10^-6 mol/L), reduced U46619-induced platelet activation. The active form of candesartan cilexetil, candesartan (CV-11974), failed to modify platelet activation. Losartan reduced the binding of [³H]-U46619 to platelets, an effect that was observed to a lesser extent with valsartan but not with CV-11974. These results suggest that, whilst some AT₁-receptor antagonists reduce TxA₂-dependent human platelet activation, it is not a feature common to all AT₁ antagonists.

We investigated a possible contribution of nitric oxide (NO) and prostaglandins to the inhibitory effect of losartan on contractions to Ang I (10^-6 M) and Ang II (10 ^-7 M) with or without L-NAME (10^-4 M) or indomethacin (10 ^-5 M) in the aorta of WKY, SHR and hamster (n=7 each). Rings of thoracic aorta (2-mm long) were placed in a myograph (5 ml). Endothelium-dependent vasodilations were evaluated with acetylcholine (10^-8 ~ 10 ^-6 M). After a 45-minute incubation with L-NAME under a resting tension of 2 g, only hamster aorta contracted (p<0.01). The SHR aorta showed impaired relaxations to acetylcholine compared with the WKY and hamster aorta (p<0.05). Despite the difference in the stimulated NO release, losartan completely abolished the responses to Ang I and Ang II both in WKY and SHR vessels irrespective of the presence of L-NAME. In contrast to the rat aorta, the inhibitory effect of losartan was attenuated in the presence of L-NAME in the hamster aorta (78% vs 99% inhibition, p<0.05). Indomethacin did not alter the effect of losartan in any vessels. Our results suggest that the presence of NO, particularly a basal secretion of NO, is necessary for the full expression of the inhibitory effect of losartan in the hamster, but not in WKY or SHR, aorta. Unlike NO, prostaglandins do not appear to play a role in the effect of losartan.

To determine whether cardioprotection after chronic angiotensin II (Ang II) type 1 (AT₁) receptor blockade involves Ang II type 2 (AT₂) receptor expression and protein kinase C-ε (PKCε) activation, we measured in vivo haemodynamics and left ventricular (LV) remodelling and dysfunction (echocardiogram/ Doppler) and ex vivo AT₁/AT₂-receptor expression, IP ₃R (1, 4, 5-inositol trisphosphate type 2 receptor) and PKCε proteins in dogs with acutely reperfused (90 minutes ischaemia, 90 minutes reperfusion) myocardial infarction (MI) following seven days of AT₁-receptor blockade with oral losartan or UP269-6. The animals were randomised to sham; sham + losartan or UP269-6; MI alone; MI + losartan; MI + UP269-6. More marked AT₁-receptor blockade with UP269-6 (greater inhibition of Ang II pressor responses) was associated with a smaller increase in preload, less systolic and diastolic dysfunction, less infarct expansion, and smaller LV diastolic and systolic volumes. However, both AT₁-receptor antagonists decreased infarct size. Importantly, MI decreased AT₁-receptor and AT₂-receptor expression while MI after AT₁-receptor antagonism increased AT₁-receptor (mRNA, not protein) and AT ₂-receptor (mRNA and protein) expression as well as IP₃R and PKCε proteins and cyclic guanosine 3', 5' monophosphate (cGMP). These results suggest that cardioprotection induced by chronic AT₁-receptor antagonism involves enhanced AT₂-receptor expression and possibly downstream signalling through IP₃R, PKCε and cGMP.

The objective of the present studies was to examine the interaction between brain-derived angiotensin II (Ang II) and prostaglandins in order to identify the mechanisms mediating the pressor response produced by these neuroregulators. Inhibiting synthesis of prostaglandins with indomethacin [indocin, 200 µg/ 5 µl artificial cerebrospinal fluid (aCSF)], administered intracerebroventricularly (icv) to conscious adult male Sprague-Dawley rats, reduced blood pressure to values below basal levels. When injected prior to Ang II (50 ng/5 µl aCSF; icv), indomethacin completely abolished the pressor response induced by the octapeptide. The increase in blood pressure produced by prostaglandin D₂ (PGD ₂, 20 µg/5 µl; icv), the most prominent prostaglandin in the rat brain, however, was not prevented by losartan (25 µg/5 µl; icv), an Ang II AT ₁-receptor antagonist. Collectively, these results indicate that prostaglandins produced tonically in the brain maintain resting arterial blood pressure and that the pressor action of Ang II is dependent on de novo synthesis of a prostaglandin.

Sympathetic nervous system (SNS) activity, measured by norepinephrine (NE) turnover rate, was greater in the posterior hypothalamic (PH) nuclei, the paraventricular nuclei (PVN), and the locus coeruleus (LC) of 5/6 nephrectomised (CRF) rats than of control rats. NE secretion from the PH was also greater in CRF than in control rats. These findings demonstrate that SNS activity plays an important role in the genesis of hypertension associated with CRF. The increase in central SNS activity was mitigated by increased local expression of nitric oxide synthase (NOS)-mRNA and nitric oxide (NOx) production. Because angiotensin II may stimulate the central SNS, we tested the hypothesis that losartan, a specific angiotensin II AT₁-receptor antagonist, may lower blood pressure (BP), at least in part, by central noradrenergic inhibition. To this end, we studied two groups of CRF rats. One group received losartan (10 mg/kg body weight) in drinking water between the 3rd and 4th week after nephrectomy, the second group received drinking water without losartan. SNS activity was measured by NE secretion from the PH using the microdialysis technique. NOS-mRNA gene expression was also measured by RT-PCR in the PH, PVN, and LC of CRF and control rats. Losartan reduced systolic BP from 184±3.7 to 152±3.1 mmHg and NE secretion from the PH from 340±9.7 to 247±4.8 pg/ml. CRF rats treated with losartan manifested a significant (p<0.01) increase in the expression of nNOS-mRNA in the PH (from 84±1.2 to 99±2.6), the PVN (from 44±1.5 to 63±2.1), and the LC (from 59±6.7 to 76±2.1). CRF rats also manifested a significant increase (p<0.01) in the expression of IL-1β in the PH (from 41.6±2.8 to 54.3±1.4), PVN (from 44±1.9 to 54±1.5), and LC (from 35.5±1.6 to 53.5±1.9).

In conclusion, these studies suggest that the antihypertensive action of losartan in CRF rats may be mediated, at least in part, by inhibition of central SNS outflow. The studies also suggest that the inhibitory action of losartan on the SNS may be mediated by activation of IL-1β, which, in turn, stimulates nNOS, an important modulator of central SNS activity.