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In the present study, the effect of endothelin-1 (ET-1) on the expression of proto-oncogene c-fos in cultured neonatal rat myocardial cells was investigated. The results are as follows: ET-1 induced c-fos expression in a dose-dependent manner. Selective ET(A) receptor antagonist blocked ET-1-induced responses. Protein kinase C(PKC) agonist PMA induced c-fos expression.PKC inhibitor staurosporine blocked ET-1 induced c-fos expression. Calcium channel blocker, nifedipine did not significantly affect the expression of c-fos induced by ET-1. These results suggest that in cultured neonatal cardiomyocytes, ET-1 induced c-fos gene expression is mediated by ET(A) receptor with the participation of protein kinase C, while the voltage-dependent L-type Ca(2+) channel is not involved.

Bovine aortic endothelial cells (BAECs) were used to study the effect of 17beta -estradiol (E(2)) on nitric oxide (NO) release, nitric oxide synthase (eNOS) mRNA expression and intracellular free calcium con~cen~tration ([Ca(2+)](I)) and modulation of the effect of E(2) by estrogen receptor (ER) antagonist tamoxifen and NOS inhibitor L-NAME. E(2) (10(-12) 10(-8) mol/L) induced NO release of BAECs in a concentration-dependent manner and the abundant expression of eNOS mRNA in BAECs increased obviously after treatment with E(2) (10(-8)mol/L) for 48 h. These effects were evidently inhibited by tamoxifen (10(-7)mol/L) and L-NAME (10(-6) mol/L). Furthermore treatment with E(2) (10(-8) mol/L) for 48 h significantly increased the resting [Ca(2+)](I) and the rise of [Ca(2+)](I) induced by ATP in BAECs. These results suggest that E(2)-induced NO release and eNOS mRNA expression in BAECs may be mediated by ER and related to calcium mobilization.

The aim of this study was to determine the regulation by MKP-1 of MAPK activity and protein expression in cardiomyocyte hypertrophic response induced by Ang II. Neonatal rat cardiomyocyte hypertrophic response was assayed by cell surface area, protein synthesis rate and protein content. MAPK activity was determined by an in-gel kinase assay. Protein expression of MAPK and MKP-1 were detected by Western blotting. The results are as follows. (1) Ang II induced promotion of (3)H-leucine incorporation and increase in cell protein content and cell surface area in a dose-dependent manner. Pretreatment with a selective AT(1) receptor antagonist CV11974 or a specific MEK inhibitor PD098059, cardiomyocyte hypertrophic response induced by Ang II was inhibited by 85% and 32.5%, respectively. (2) After pretreatment with PD098059 or CV11974, AngII-induced increases in p44MAPK and p42MAPK protein expression and enzyme activity (expressed by gamma-(32)P-ATP incorporation) were all inhibited obviously. (3) With treatment of myocytes by Ang II for 5 min, MAPK activity determined by p44MAPK and p42MAPK protein expression began to increase, while MKP-1 protein expression was detected within 30 min and lasted more than 2 h following treatment with Ang II. (4) Pretreatment of cardiomyocytes with actinomycin D (3 microgram/ml) for 30 min inhibited MKP-1 protein expression, while p44MAPK and p42MAPK protein expression was still detected 120 min after Ang II treatment. The above results demonstrate that activation of MAPK plays an important role in Ang II-induced cardiomyocyte hypertrophic response in neonatal rat cardiomyocytes through MKP-1 mediated inactivation of p44MAPK and p42MAPK.cardiomyocyte hypertrophic response in neonatal rat cardiomyocytes through MKP-1 mediated inactivation of p44MAPK and p42MAPK.

In the present study, the effects of 17 beta-estradiol on vascular reactivity of ovariectomized rats and proliferation of cultured rat aortic smooth muscle cells were studied. The vascular reactivity was significantly increased in ovariectomized rats compared with the sham-operated animals. The selective ETA receptor antagonist BQ123 inhibited the increase in [3H]-TdR incorporation in response to ET-1 on vascular smooth muscle cells (VSMCs). 17 beta-estradiol also attenuated the ET-1 effects in a dose-dependent manner. The results of RT-PCR and Western blot show that expression of ETA receptor was decreased after treatment with 17 beta-estradiol. The effect of 17 beta-estradiol was partially inhibited by estrogen receptor antagonist tamoxifen. The above results demonstrate that proliferation of VSMCs stimulated by ET-1 was mainly mediated through ETA receptor. Due to the down-regulation of ETA receptor and mediation of estrogen receptor, 17 beta-estradiol inhibits the ET-1-induced proliferation of VSMCs and decreases the vascular reactivity of ovariectomized rats.

The purpose of this study was to determine the role of nitric oxide (NO) in the angiotensin II-induced hypertrophic response in cultured neonatal rat cardiac myocytes. Angiotensin II induced significant increase of protein content, decrease of NOS activity in cultured neonatal rat cardiomyocytes and decrease of NO concentration in the culture medium. Angiotensin II decreased significantly the eNOS mRNA level of cardiomyocytes. Saralasin and PTX inhibited significantly the increased protein content, the decreased NOS activity and the decreaseed NO production of cardiomyocytes induced by angiotensin II. Sodium nitroprusside increased significantly NO concentration in the culture medium and inhibited significantly the increased protein content of cardiomyocytes induced by angiotensin II. The results suggest that angiotensin II may induce hypertrophy of cultured neonatal rat cardiomyocytes. The effect of angiotensin II is mediated by receptors which are coupled with PTX-sensitive G protein and may be related to the decreased eNOS gene expression, NOS activity and NO production. Exogenous NO can prevent the hypertrophy of cardiomyocytes induced by angiotensin II.

In this present study, the effects of ET-1 on intracellular free calcium concentration ([Ca2+]i) and the underlying mechanisms were investigated in cultured neonatal rat myocardial cells loaded with fura-2/AM. The results are as follows. ET-1 induced an increase of [Ca2+]i in a dose-dependent manner, which consisted of a transient and sustained phase. BQ123, a selective ETA receptor antagonist, blocked the ET-1 induced [Ca2+]i responses, suggesting that these responses were mediated by ETA receptors. After removal of extracellular Ca2+, ET-1 induced the transient increase of [Ca2+]i without the sustained change. Protein kinase C (PKC) agonist PMA attenuated the ET-1 induced transient [Ca2+]i increase. Amiloride and nifedipine did not block the [Ca2+]i change induced by ET-1. After pretreatment of myocardial cells with pertussis toxin, ET-1 also induced the transient increase of [Ca2+]i but did not affect the sustained increase. These results suggest that the transient [Ca2+]i increase may involve pertussis toxin-insensitive G protein and the sustained one may be caused by extracellular calcium influx, in which pertussis toxin sensitive G protein is involved. Furthermore, PKC, but not Na+/H+ exchange, plays an important role in these effects.

The aim of this study was to examine the effects of L-arginine, a nitric oxide (NO) precursor, on protein expression of endothelial nitric oxide (eNOS), nitrite/nitrate content, protein expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) and the activity of mitogen-activated protein kinase (MAPK) in cardiac tissues in renovascular hypertensive rats (RHR). The Goldblatt renovascular hypertensive model was established by two-kidney one clip method. The rats were divided into four groups, respectively treated with 50, 150 and 450 mg/kg L-arginine and 150 mg/kg L-arginine plus 10 mg/kg L-NAME (an eNOS inhibitor) (i.p.). Another group did not receive specific treatment from the 5th week after renal artery constriction. Control group was sham-operated. Mean arterial blood pressure (MABP) and the ratio of left ventricular weight to body weight (LVW/BW) were measured 8 weeks after treatment. eNOS protein expression, nitrite/nitrate content, MKP-1 protein expression and MAPK activity in cardiac tissues were detected using Western blot analysis, enzyme-reduction method and substrate in-gel kinase assay, respectively. It was found that L-arginine significantly inhibited the increase of MABP and LVW/BW, attenuated the activity of MAPK, increased protein expression of eNOS and MKP-1 and potentiated production of NO in cardiac tissue with the most effective dosage of 150 mg/kg, and these effects of L-arginine could be inhibited by L-NAME. These results suggest that MKP-1 may play an important role in the NO-induced inhibition of myocardial hypertrophy. The anti-hypertrophic effects of L-arginine may involve increase of eNOS protein expression and NO production, potentiation of MKP-1 protein expression, and inhibition of MAPK activity in the cardiac tissue of RHR.

Ventricular fibroblasts were cultured using conditioned growth medium for ventricular fibroblasts (FCGM). The rate of the total collagen synthesis of ventricular fibroblasts was measured by assaying the incorporation rate of [3H]-proline, whereas the proliferation of ventricular fibroblasts was assessed by determining the incorporation rate of [3H]-TdR and the expression of c-fos genes. FCGM significantly increased the [3H]-proline incorporation rate and [3H]-TdR incorporation rate of fibroblasts in a dose-dependent manner. Furthermore, FCGM promoted the c-fos gene expression of fibroblasts, which attained its maximum in 1 h. BQ123, an ETA receptor antagonist, partially blocked the above effects of FCGM, but AT1 receptor antagonist CV11974 and alpha-adrenergic receptor antagonist regitin did not. It is suggested that the ventricular fibroblast has an autorine function in promotion of collagen synthesis and proliferation of fibroblasts by secreting endothelin and other bioactive substances.

Using cell culture, radioimmunoassay for endothelin and RT-PCR, the effect of aldosterone on the endothelin secretion of ventricular fibroblasts was studied. The results showed that aldosterone (1 x 10(-7) mol/L) promoted the expression of ppET-1 mRNA, which began to increase in 2 hours and attained the highest level in 4 hours, thereafter decreased; aldosterone increased the endothelin level in ventricular fibroblasts and fibroblast conditioned growth medium (FCGM) as well, which was blocked by spironolactone (1 x 10(-6) mol/L), an aldosterone receptor antagonist. The results suggest that aldosterone can increase endothelin secretion by ventricular fibroblasts, which can be inhibited by its receptor antagonist spironolactone.

Endothelin-1 (ET-1) has been shown to be a potent growth factor and to induce cardiac hypertrophy. In the present study, we examined the role of G protein, protein kinase C (PKC) and Na(+)-H+ exchanger in ET-1-induced cardiac hypertrophy in cultured neonatal rat cardiac myocytes. ET-1 (10(-10)-10(-7) mol/L) induced promotion of 3H-leucine incorporation, increase in cell protein content and cell surface area in a dose-dependent manner with EC50 value of 5.2 x 10(-10), 5.2 x 10(-10) and 7.3 x 10(-10) mol/L respectively. All of these ET-1-induced cardiomyocyte hypertrophic responses were completely blocked by pretreatment with staurosporine (2 nmol/L), a protein kinase C inhibitor, and stimulated by 4-phorbol, 12-myristate, 13-acetate (PMA) (10(-8)-10(-6) mol/L), a protein kinase C activator, in a dose-dependent manner. Pretreatment of amiloride (10(-4) mol/L), a Na(+)-H+ exchange inhibitor completely inhibited the ET-1-induced, but not PMA-induced cardiomyocyte hypertrophic responses. The ET-1-induced increase in cardiomyocyte protein synthesis and cell surface area was significantly inhibited by pretreatment with pertussis toxin (150 ng/ml). These results suggest that ET-1-induced cardiomyocyte hypertrophy was linked with pertussis toxin sensitive G protein, and PKC and Na(+)-H+ exchange may be an important intracellular signaling transduction pathway during ET-1-induced cardiac hypertrophy in cultured neonatal rat cardiac myocytes.