Effects of cilnidipine on nitric oxide and endothelin-1 expression and extracellular signal-regulated kinase in hypertensive rats

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Abstract

We evaluated the effects of cilnidipine, a long-acting Ca2+ channel antagonist, on endothelial nitric oxide synthase (eNOS), preproendothelin-1 and endothelin ETA receptor expression in the left ventricle, and evaluated the relations between these effects and coronary microvascular remodeling and extracellular signal-regulated kinases belonging to one subfamily of mitogen-activated protein kinases in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Cilnidipine (DOCA-cilnidipine, 1 mg/kg/day, subdepressor dose) or vehicle (DOCA-vehicle) was given after induction of DOCA-salt hypertension for 5 weeks. The eNOS mRNA and protein expression in the left ventricle was significantly lower in DOCA-vehicle than in control rats and significantly higher in DOCA-cilnidipine than in DOCA-vehicle rats. Preproendothelin-1 and endothelin ETA receptor expression levels and phospho-p42/p44 extracellular signal-regulated kinase activities were significantly increased in DOCA-vehicle compared with control rats and significantly suppressed in DOCA-cilnidipine compared with DOCA-vehicle rats. DOCA-vehicle rats showed a significant increase in the wall-to-lumen ratio, perivascular fibrosis and myocardial fibrosis, with all these parameters being significantly improved by cilnidipine. These results led us to conclude that phospho-p42/p44 extracellular signal-regulated kinase activities may contribute to the coronary microvascular remodeling of DOCA rats and that protective effects of cilnidipine on cardiovascular remodeling may be at least in part mediated by an increased eNOS expression and a decreased endothelin-1 and endothelin ETA receptor expression in the left ventricle.

Introduction

Left ventricular hypertrophy is a strong predictor, independent of blood pressure or other risk factors, of cardiac and cerebrovascular morbidity and mortality in patients with hypertension (Verdecchia et al., 1994). In hypertensive heart disease, myocytes hypertrophy and interstitial components undergo hyperplasia, hypertrophy and remodeling (Weber and Brilla, 1991). Excess collagen production by fibroblasts increases total interstitial and perivascular fibrosis. In addition, vascular smooth muscle cells undergo hyperplasia and hypertrophy, resulting in media hypertrophy, coronary artery wall remodeling and increased coronary wall to lumen ratio (Schwarzkopff et al., 1993). These structural changes decrease vasodilator capacity and may be associated with the endothelial dysfunction. Endothelial dysfunction has been demonstrated in conditions associated with premature development of atherosclerosis and hypertension. Mitogen-activated protein kinases are an ubiquitous group of protein serine/threonine kinases and are important mediators of the signal transduction pathway, which is responsible for cellular proliferation. Extracellular signal-regulated kinases (ERKs) are a subgroup of the mitogen-activated protein kinase family and are composed of p42ERK and p44ERK (Davis, 1993). Recent evidence indicates that ERKs in cultured neonatal rat cardiac myocytes are rapidly activated by various extracellular stimuli, such as growth factors and other mitogens, and play a key role in cell growth and the regulation of various gene expressions. Recent reports on cultured cardiac myocytes support the idea that ERKs participate in the mechanism of cardiac hypertrophy and remodeling Kojima et al., 1994, Bogoyevitch et al., 1996.

The potential importance of endothelin-1 in cardiovascular disease was suggested by its production in endothelial cells and its potent vasoconstricting and growth-promoting properties (Yamazaki et al., 1999). Infusion of exogenous endothelin-1 has been reported to produce significant coronary vasoconstriction in humans, supporting a role for endothelin-1 as an important potential mediator of myocardial ischemia in states of endothelin-1 activation, as in atherosclerosis (Lerman et al., 1991). The vasoconstrictor actions of endothelin-1 are mediated by at least two receptor subtypes, including the endothelin ETA and ETB receptors. Endothelin-1 has direct effects on contractile function, protein synthesis and electrophysiological events in cardiac myocytes, and these effects are mediated primarily by the endothelin ETA receptor (Ishikawa et al., 1988). Indeed, aortic tissue endothelin-1 content acts as a local mediator of vascular dysfunction and aortic hypertrophy, and endothelin ETA receptor antagonism may have a therapeutic potential for lowering vascular endothelin-1 content, improving endothelial function and preventing structural changes in the cardiovascular hypertrophy (Barton et al., 1998).

Endothelium-derived relaxing factor, nitric oxide (NO), reduces monocyte and leukocyte adhesion to endothelial cells and is an important inhibitor of platelet aggregability and platelet and vessel wall interaction. Furthermore, NO decreases endothelial permeability and thus diminishes the transport of lipoproteins into the vessel wall and suppresses vascular smooth muscle proliferation and migration both in vivo and in vitro (Cooke and Dzau, 1997). The observation that induction of hypertension in animal models results in impaired endothelial-dependent vasodilation suggested that effective antihypertensive therapy may normalize, or at least improve, endothelial vasodilator function (Lüscher et al., 1987). Indeed, long-term treatment with an angiotensin-converting enzyme inhibitor or a Ca2+ channel antagonist improved endothelial dysfunction in a rat model of NO-deficient hypertension (Takase et al., 1996). Cilnidipine, a novel dihydropyridine Ca2+ channel antagonist, has been shown to act as a slow-onset and long-lasting antihypertensive drug in clinical and experimental studies (Tominaga et al., 1997), and to possess a potent inhibitory action on the N-type as well as on the L-type voltage-dependent Ca2+ channel in rats (Uneyama et al., 1997). In addition, cilnidipine is effective as a once-daily antihypertensive agent and causes reflex tachycardia less than does nisoldipine (Minami et al., 1998). Recently, we evaluated the effects of chronic treatment with the L-type dihydropyridine Ca2+ channel antagonists, benidipine (Kobayashi et al., 1999b) and amlodipine (Kobayashi et al., 1999c) on endothelial NO synthase (eNOS) mRNA expression in two-kidney, one-clip Goldblatt rats (Kobayashi et al., 1999b) and in rats with prolonged nitric oxide blockade-induced hypertension (Kobayashi et al., 1999c). Downregulation of eNOS mRNA expression in the left ventricle of these models was significantly increased by a subdepressor dose of these agents. However, very few studies have evaluated whether the beneficial effects of cilnidipine on coronary microvascular remodeling is associated with direct local gene expression of eNOS and endothelin-1 mRNA in the left ventricle. The purpose of the present study was to evaluate the effects of long-term treatment with a subdepressor dose of cilnidipine on gene expression of eNOS, preproendothelin-1 and endothelin ETA receptor mRNA in the left ventricle, and the relation of these effects to coronary microvascular remodeling and phospho-p42/p44 ERK activity in deoxycorticosterone acetate (DOCA)-salt hypertensive rats.

Section snippets

Animal models and experimental designs

All procedures were in accordance with institutional guidelines for animal research. Twenty-four male normotensive Wistar rats (Oriental Bioservice Kanto Ibaragi, Japan) aged 6 weeks were used, and DOCA-salt hypertension was induced in 16 rats as described previously (Takanohashi et al., 1996). Eight rats received weekly subcutaneous injections of DOCA (30 mg/kg) after right nephrectomy and were given 1% saline as drinking water (DOCA-vehicle, n=8). The remaining eight DOCA-salt rats were

Systemic hemodynamics, body weight and left ventricular weight

Systolic blood pressure in DOCA-vehicle and DOCA-cilnidipine rats was similar and significantly higher than that in sham-operated rats (186±6 and 183±5 vs. 129±3 mm Hg, sham-operated rats, P<0.01, respectively). Heart rate was similar in sham-operated rats and DOCA-vehicle rats (396±15, 389±17 bpm) and was not changed by the administration of cilnidipine (387±17 bpm). Body weight was also similar among the three groups. The left ventricular mass of the DOCA-vehicle rats was significantly

Discussion

The present study demonstrated that the production of eNOS mRNA and protein expression is downregulated, and preproendothelin-1 and endothelin ETA receptor expression and phospho-p42/p44ERK activity are upregulated in the left ventricle of DOCA-salt hypertensive rats. The chronic administration of a long-acting dihydropyridine Ca2+ channel antagonist, cilnidipine, increased eNOS mRNA and protein expression, and decreased preproendothelin-1 and endothelin ETA receptor expression. In addition,

Acknowledgements

This work was supported by a grant from the Ueda Memorial Trust Fund for Research of Heart Diseases, Tokyo, Japan. We thank Kazumi Akimoto PhD for technical assistance with RT-PCR, and Mrs Noriko Suzuki for preparing and staining tissue sections for histological investigation, and Miss Yasuko Mamada for technical assistance. The authors wish to thank UCB Japan for supplying cilnidipine.

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