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Background Coronary computed tomography angiography (CTA) has demonstrated high diagnostic accuracy for detection of coronary artery stenosis, and healthcare providers can detect coronary artery disease in earlier stages before it develops into more serious clinical conditions such as acute myocardial infarction (AMI). We hypothesized that the mortality ratio of AMI in regions with a higher density of coronary CTA is lower than that in regions with a lower density of coronary CTA. Methods This ecological and cross-sectional study using secondary data targeted all secondary medical service areas (SMSAs) in Japan (n = 349). We obtained the numbers of cardiologists, institutions with coronary CTA, and institutions with a cardiac catheterization laboratory (CCL) as medical resources, socioeconomic factors, lifestyle factors, exercise habit factors, and AMI mortality data from a Japanese national database. We evaluated the association between the number of these medical resources and the standardized mortality ratio (SMR) of AMI in each SMSA using a hierarchical Bayesian model accounting for spatial autocorrelation (i.e., a conditional autoregressive model). We assumed a Poisson distribution for the observed number of AMI-related deaths and set the expected number of AMI-related deaths as the offset variable. Results The number of institutions with coronary CTA was negatively and significantly associated with the SMR of AMI (relative risk [RR] 0.900; 95% credible interval [CI] 0.848–0.953), while the SMR in each SMSA was not significantly associated with the number of either cardiologists (RR 0.997; 95% CI 0.988–1.004) or institutions with a CCL (RR 1.026; 95% CI 0.963–1.096). Conclusions We observed a significant association between the number of institutions with coronary CTA and the SMR of AMI. Effective allocation of coronary CTA in each region is recommended, and it would be important to clarify the standing position of coronary CTA in regional networking for AMI treatment in the future. Electronic supplementary material The online version of this article (10.1186/s12942-018-0133-0) contains supplementary material, which is available to authorized users.

Parathyroid hormone-related protein (PTHrP) is detected in many aggressive tumors and involved in malignant conversion; however, the underlying mechanism remains obscure. Here, we identified PTHrP as a mediator of epidermal growth factor receptor (EGFR) signaling to promote the malignancies of oral cancers. PTHrP mRNA was abundantly expressed in most of the quiescent oral cancer cells, and was significantly upregulated by EGF stimulation via ERK and p38 MAPK. PTHrP silencing by RNA interference, as well as EGFR inhibitor AG1478 treatment, significantly suppressed cell proliferation, migration, and invasiveness. Furthermore, combined treatment of AG1478 and PTHrP knockdown achieved synergistic inhibition of malignant phenotypes. Recombinant PTHrP substantially promoted cell motility, and rescued the inhibition by PTHrP knockdown, suggesting the paracrine/autocrine function of PTHrP. These data indicate that PTHrP contributes to the malignancy of oral cancers downstream of EGFR signaling, and may thus provide a therapeutic target for oral cancer.

Objective— C-reactive protein (CRP), an obesity-related inflammatory marker, is a promising predictor for cardiovascular disease and may be a mediator for atherogenesis. It has been reported that diet-induced weight loss lowered CRP levels. However, the effect of exercise training, another therapy that can reduce weight, on CRP is still unclear. We examined effects of exercise training with weight loss on CRP levels and conventional cardiovascular risks. Methods and Results— A total of 227 apparently healthy women were recruited, and 199 subjects (average age 52 years) completed a 2-month weight reduction program consisting of supervised aerobic exercises. After the program, weight was reduced from 65.8 to 62.8 kg ( P P Conclusions— Exercise training with weight reduction disproportionately lowered CRP levels. Considering inflammatory status, there might be an optimal pace of exercise with weight loss.

In the hearts of spontaneously hypertensive rats, mRNA of the renin-angiotensin system (renin, angiotensinogen, and angiotensin converting enzyme) was expressed. Level of angiotensinogen and renin mRNA expressed in ventricles, and angiotensinogen mRNA expressed in fibroblasts from SHR were higher than those from WKY. ACE mRNA was also more strongly expressed in the ventricles and fibroblasts from SHR compared with those of WKY. We investigated the effects of angiotensin II on cardiac collagen synthesis in cardiac fibroblasts of 10-week-old spontaneously hypertensive rats and age-matched Wistar-Kyoto rats. Basal collagen synthesis in cardiac fibroblasts from spontaneously hypertensive rats was 1.6-fold greater than that in the cell of Wistar-Kyoto rats. Angiotensin II stimulated collagen synthesis in cardiac fibroblasts in a dose-dependent manner. The responsiveness of collagen production to angiotensin II was significantly enhanced in cardiac fibroblasts from spontaneously hypertensive rats (100 nM angiotensin II resulted in 185 ± 18% increase above basal levels, 185 ± 18 versus 128 ± 19% in Wistar-Kyoto rats, p < 0.01). This effect was receptor-specific, because the competitive inhibitor saralasin and MK 954 blocked it. These results indicate that collagen production was enhanced in cardiac fibroblasts from spontaneously hypertensive rats, that angiotensin II had a stimulatory effect on collagen synthesis in cardiac fibroblasts, and that cardiac fibroblasts from spontaneously hypertensive rats were hyperresponsive to stimulation by angiotensin II. These findings suggest that the cardiac renin-angiotensin system may play an important role in collagen accumulation in hypertensive cardiac hypertrophy.

Although the types of pathophysiological stimulation that initiate an overexpression of OPN have yet to be determined, we hypothesized that mechanical stress is one of the candidates which initiates OPN expression in vascular smooth muscle cells. Cell proliferation assay indicated that a pure atmospheric pressure of 160 mmHg activated cell proliferation by 11% in human aortic smooth muscle cells (HASMC) compared to nonpressurized controls. Immunoblot analysis probed with an anti-OPN antibody demonstrated a 50% increase in OPN. Dual-luciferase reporter assay demonstrated that OPN promoter, corresponding to the −771 through −1 region of OPN gene, was highly responsive to pure atmospheric pressure by ten times that of the control. From these observations, we concluded that pure atmospheric pressure directly promotes an expression of OPN in HASMC, with these results also suggesting that high blood pressure-mediated mechanical compression is involved in the process of atherosclerosis and remodeling via OPN expression in HASMC.

This study examined the effects of long-term treatments with the angiotensin-converting enzyme inhibitor, enalapril, and the calcium antagonist, amlodipine, on the morphologic changes, progressive left ventricular dysfunction, and gene expression of the ryanodine receptor (RyR) and phospholamban (PLN) in dilated cardiomyopathy. From the ages of 5 through 20 weeks, dilated cardiomyopathic hamsters, BIO53.58 (BIO), and control hamsters, F1b, orally received either enalapril or amlodipine. Cardiac function was assessed by echocardiography. At the age of 20 weeks, the collagen volume fractions were analyzed by the stereologic method. RyR and PLN messenger RNAs (mRNAs) were examined by Northern blot in the amlodipine group. In BIO, the reduction of left ventricular percentage of fractional shortening was attenuated in the enalapril group (p < 0.05) and amlodipine group (p < 0.001), and the increase in the collagen volume fraction and the loss of myocytes were suppressed in the amlodipine group compared with the untreated group. RyR mRNA level decreased in BIO (p < 0.01) compared with F1b, but PLN mRNA level was unchanged. RyR and PLN mRNA levels were unaffected by the treatment with amlodipine. Enalapril and amlodipine prevent progressive remodeling and reduce cardiac dysfunction in BIO. Amlodipine prevents fibrosis and cell death without modifying RyR and PLN mRNA levels in BIO.

Hypertensive cardiac hypertrophy is associated with the accumulation of collagen in the myocardial interstitium. Previous studies have demonstrated that this myocardial fibrosis accounts for impaired myocardial stiffness and ventricular dysfunction. Although cardiac fibroblasts are responsible for the synthesis of fibrillar collagen, the factors that regulate collagen synthesis in cardiac fibroblasts are not fully understood. We investigated the effects of angiotensin II on cardiac collagen synthesis in cardiac fibroblasts. Cardiac fibroblasts of 10 week old spontaneously hypertensive rats and age-matched Wistar-Kyoto rats were prepared and maintained in culture medium supplemented with 10% fetal calf serum. The expression of mRNA of the renin-angiotensin system (renin, angiotensinogen, angiotensin converting enzyme) was determined by using a ribonuclease protection assay. Basal collagen synthesis in cardiac fibroblasts from spontaneously hypertensive rats was 1.6 fold greater than that in the cell of Wistar-Kyoto rats. Angiotensin II stimulated collagen synthesis in cardiac fibroblasts in a dose-dependent manner. The responsiveness of collagen production to angiotensin II was significantly enhanced in cardiac fibroblasts from spontaneously hypertensive rats (100 nM angiotensin II resulted in 185 ± 18% increase above basal levels, 185 ± 18 versus 128 ± 19% in Wistar-Kyoto rats p < 0.01). This effect was receptor-specific, because it was blocked by the competitive inhibitor saralasin and MK 954. These results indicate that collagen production was enhanced in cardiac fibroblasts from spontaneously hypertensive rats, that angiotensin II had a stimulatory effect on collagen synthesis in cardiac fibroblasts, and that cardiac fibroblasts from spontaneously hypertensive rats were hyper-responsive to stimulation by angiotensin II. Level of angiotensin and renin mRNA expressed in ventricles, and angiotensinogen mRNA expressed in fibroblasts from SHR were higher than those from WKY. These findings suggest that the cardiac renin-angiotensin system may play an important role in collagen accumulation in hypertensive cardiac hypertrophy.

To examine the contribution of the renin-angiotensin system to hypertrophic cardiomyopathy (HCM), we studied 96 patients with HCM (mean age 50 years, 55% male), 105 of their unaffected siblings and offspring, and 160 healthy subjects without known hypertension and left ventricular hypertrophy (LVH) who were frequency matched to cases by age and sex. Patients were divided into familial or sporadic HCM (FHCM or SHCM) groups with or without affected members of their family. The region of interest in the angiotensinogen (AGT) gene, the missense mutation with methione-to-threonine amino acid substitution at codon 235 in angiotensinogen (M235T), was amplified by polymerase chain reaction with the use of allele-specific oligonucleotide primers flanking the polymorphic region of the AGT gene to amplify template deoxyribonucleic acid prepared from peripheral leukocytes. The T allele frequency was higher in the SHCM group than in unaffected siblings and offspring (88% vs 78%, X2 = 4.6, p < 0.05). The M allele frequency was higher in unaffected siblings and offspring than in patients with SHCM (23% vs 12%, X2 = 4.6, p < 0.05). The T allele frequency among unaffected siblings and offspring was similar to that observed in healthy subjects (78% vs 78%). We conclude that HCM, especially in sporadic cases, is partially determined by genetic disposition. The molecular variant of angiotensinogen T235 seems to be a predisposing factor for cardiac hypertrophy in HCM and carries an approximately twofold increased risk.

Congestive heart failure (CHF) patients share several similar features, such as reduced cardiac contractility and neurohumoral activation to compensate the impaired cardiac function. In CHF patients, the cardiac renin-angitensin (RA) system, receptors, GTP-binding proteins, and their effector molecules are inevitably exposed to chronically elevated neurohumoral stimulation. A widely recognized concept is that a chronic increase in such stimulation can desensitize target cell receptors and the post-receptor signal transducing pathway. Recently, reports of several studies have indicated that the inhibitory GTP-binding protein (Gi) can be increased in CHF patients and animal models. Although direct evidence for a change in catalytic protein of adenylyl cyclase has not been found, limited information has suggested a reduced catalytic activity in terminally failing hearts. In this paper, we have assessed the changes in beta AR, GTP-binding protein, catalytic protein and beta ARK. We also examined angiotensinogen mRNA expression in failing heart. It was detected not only in the liver, but also in both the atrial and ventricular heart tissues, suggesting that angiotensinogen is synthesized in the human heart. Immunohistochemical studies revealed a stronger reaction in the endocardial layer of the human left ventricle than in the epicardial layer, and intense immunoreactivity in the conduction system and right atrium. Our experiments revealed a widespread immunopositive reaction for angiotensinogen in the left ventricle of diseased hearts. In the non-diseased heart, ACE and AT1 receptor RNA are present in ventricular muscles. Renin and Ao mRNA could not be detected in the subendocardium of non-diseased left ventricle, but both were present in the left ventricle of diseased hearts. These data indicate that the cardiac RA system plays an important role in the deterioration of cardiac function.

The effects of transforming growth factor beta-1 on concentration of intracellular Ca2+ changes in the presence of isoproterenol were studied in cardiac fibroblasts. Transforming growth factor beta-1 effect on cyclic-AMP production and ligand assay of beta-adrenergic receptors were also examined. Production of cyclic-AMP in fibroblasts preincubated with transforming growth factor beta-1 decreased compared with non-transforming growth factor beta-1-treated fibroblasts in the presence of isoproterenol. An increase of intracellular Ca2+ concentration in the presence of isoproterenol was also inhibited in transforming growth factor beta-1-treated fibroblasts. Islet-activating protein did not restore these reactions of transforming growth factor beta-1. Concentration change of intracellular calcium under the treatment of transforming growth factor beta-1 was unaffected by forskolin addition. Binding of [3H]CGP-12177 was decreased to 47% of control preincubated for 24 h with transforming growth factor beta-1 in fibroblasts. Scatchard plots suggested a decrease in beta-adrenergic receptor number without specific change in receptor affinity. These results suggested that transforming growth factor beta-1 modulates the signal transduction through beta-adrenergic receptor and intracellular Ca2+ concentration by regulating the number of receptor in fibroblasts.

Extrahepatic synthesis and localization of angiotensinogen (ATN) have been described in animals, thus establishing the tissue renin-angiotensin (RA) system. However, there had been no reports of tissue RA systems in human organs, including the heart. In earlier, we have reported the possibility of ATN synthesis in the human heart using ribonuclease protection assay system. ATN mRNA was detected not only in the liver, but also in both the atrial and ventricular heart tissues, suggesting that ATN is synthesized in the human heart. In this report, we looked for the distribution of ATN in diseased human heart. Northern blot hybridization of cDNA with total RNA extracted from human liver, brain, kidney, atrial and ventricular tissues revealed that ATN mRNA exists in cardiac ventricule. Immunohistochemical studies using a specific antibody to ATN revealed a stronger reaction in the endocardial layer of the human left ventricle, than in the epicardial layer, and intense immunoreactivity in the conduction system and right atrium. This distribution pattern was similar to that of human atrial natriuretic peptide (hANP), which functions a smooth muscle relaxant. Double immunostaining of ATN and hANP demonstrated that all myocytes in the right atrium had immunopositive reactions to ATN, hANP or both of ATN and hANP. Double immunoelectron staining enabled us to show more detailed localization of ATN and hANP; hANP only existed in the specific granules and ATN existed in the myofibril, but not in the granule. Furthermore, our experiments provide evidence of ATN in healthy human hearts and also reveal a widespread immunopositive reaction for ATN in the left ventricle of diseased hearts.

The accumulation of inositol 1,4,5-trisphosphate (IP3) after hormonal stimulation has a physiological role, possibly by alteration of Ca2+ levels in cardiac myocyte. However, this accumulation has not been studied under pathophysiological conditions. In this report, we examine phosphatidylinositol metabolism during cellular response to norepinephrine in pressure-overloaded hypertrophic rat heart. After stimulation with norepinephrine, the accumulations of IP3 and diacylglyceride significantly increased in isolated myocytes from stroke-prone spontaneously hypertensive rat (SHRSP) heart, indicating phosphatidylinositol-specific phospholipase C activity increased in SHRSP heart cells. Protein kinase C activity was also enhanced in SHRSP, with a marked increase in particulate activity. We determined the intracellular calcium concentration and found it to be higher in SHRSP than in Wistar-Kyoto (WKY) rats at 30-40 weeks of age. Ca2+ influx was also elevated in SHRSP stimulated by norepinephrine. In SHRSP heart, cytosolic Ca2+ concentration may rise quickly in response to some stimuli, such as alpha 1-adrenergic stimulation, which is shown to be one of the pathways that increases cytosolic Ca2+ levels in hypertrophied rat heart. These data suggest that a part of the phosphatidylinositol-turnover pathway, such as the phosphatidylinositol 4,5-bisphosphate-IP3-Ca2+ pathway or the diacylglyceride-protein kinase C pathway, may play an important role in the development of hypertrophy in SHRSP heart.

It is known that inositol (1, 4, 5)-trisphosphate (IP3) stimulates Ca2+ release from sarcoplasmic reticulum (SR) in several tissues, but in cardiac myocytes this phenomenon has not been confirmed. The purpose of the present study was to confirm the effect of (1, 4, 5)-IP3 on Ca2+ release from SR in cardiac myocytes. The effect of IP3 on Ca2+ release from SR in hypertrophic cardiac cells was also determined. We examined the effects of IP3 on Ca2+ release from cardiac myocyte SR by the digital-image method in a single cell. We also determined the effect of IP3 on calcium release from isolated SR. SR was prepared from spontaneous hypertensive rat hearts and Wistar kyoto rat hearts. The SR was prelabeled with 45Ca2+, and then incubated with the indicated concentrations of IP3 for 1 min at 37 degrees C. In cardiac myocytes treated with saponin, Ca2+ release stimulated by 10 microM (1, 4, 5)-IP3 was detected by fura-2. In 45Ca2+ prelabeled SR, the maximal Ca2+ release was achieved at 10 microM IP3 incubated for 1 min. The release of Ca2+ was higher in SR of SHR than in the SR of WKY. IP3 stimulates Ca2-release from cardiac SR, and this release is greater in SHR than in WKY. However, it is uncertain whether this phenomenon plays a role in cardiac hypertrophy.

The accumulations of inositol-1,4,5-trisphosphate (IP3) and inositol-1,3,4,5-tetrakisphosphate (IP4) after hormonal stimulation may have a physiological role, possibly by alteration of Ca2+ levels in cardiac tissue. But the accumulation of inositol polyphosphate in a pathophysiological condition has not been studied. We investigated phosphatidylinositol-4,5-bisphosphate (PIP2) metabolism in hypertrophic cardiac myocytes, and clarified that the accumulations of IP3, IP4 and diacylglyceride after stimulation with norepinephrine were significantly enhanced in isolated myocytes from spontaneously hypertensive rat heart. Phospholipase C activity increased with age in SHRSP heart cells. These data suggest that PI turnover pathways, which can be mediated by both phosphatidylinositol-4,5-bisphosphate and diacylglyceride, may play an important role in development of hypertrophy in the hearts of rats with spontaneous hypertension.

BACKGROUND There have been no reports on the presence of the tissue renin-angiotensin system in the human heart, although the presence of angiotensinogen has been described in the animal heart. METHODS AND RESULTS To determine whether angiotensinogen is synthesized in the human heart, we examined angiotensinogen messenger RNA (mRNA) synthesis in autopsy hearts by using ribonuclease protection assay. As a result, angiotensinogen mRNA was detected in the atrial muscle, muscles of the conduction system, and the left ventricular wall. In the left ventricular wall, mRNA expression was more prominent in the subendocardial muscles than in the midcardial or epicardial muscles. Using a monoclonal antibody to human angiotensinogen in immunoblotting experiments, we detected two closely spaced bands at approximately 70 kd in the heart, which was quite consistent with the human angiotensinogen molecule. Immunohistochemical studies with this monoclonal antibody demonstrated intense immunoreactivity in the atrial muscles, the muscles of the conduction system, and those of the subendocardial layers. CONCLUSIONS We conclude that angiotensinogen was synthesized in the human heart. It was evident that the localization of angiotensinogen was not ubiquitous in the cardiac muscles, showing its predilection for the atrial muscles, muscles of the conduction system, and subendocardial layer of the left ventricle.

Cell death during hypoxia rose to 80% after 6 h. Calpain activity increased to 4 units during hypoxia, much higher than the 0.7 units seen in aerobic condition at 6 h. This activity was markedly inhibited by calpain-specific inhibitor I ( n -acetyl-leucyle-leucyle-norleucinal). β-Adrenergic blocking agents and calcium antagonists suppressed the calpain activity and decreased cell death during hypoxia. On the other hand, α-adrenergic blocking agents did not affect calpain activity and cell death under hypoxic conditions. These results prove that β-adrenergic blocking agents and calcium antagonists prevent protein degradation during hypoxic cell injury.

This study was undertaken to investigate the correlation between hypoxic cell injury and protease activity, as well as the effects of alpha 1-, and beta-adrenergic blocking agents and calcium antagonists during hypoxia. Cell death during hypoxia rose to 80% after 6h. Calpain activity increased to 4 units during hypoxia, much higher than the 0.7 units seen in aerobic condition at 6h. This activity was markedly inhibited by calpain-specific inhibitor I (n-acetyl-leucyle-leucyle-norleucinal). alpha 1-adrenergic blocking agents did not affect calpain activity and cell death under hypoxia. On the other hand, beta-adrenergic blocking agents and calcium antagonists suppressed the calpain activity and decreased cell death during hypoxia. These beta-adrenergic blocking agents and calcium antagonists also inhibited intracellular calcium-influx during hypoxia. These results suggest the beta-adrenergic receptor stimulation activates adenylate cyclase activity and enhances calcium-influx during hypoxia. The elevated intracellular calcium concentration then stimulates calpain activity under hypoxia. These results prove that beta-adrenergic blocking agents and calcium antagonists prevent protein degradation during hypoxic cell injury.

Hypertension is a major risk factor for atherosclerosis, and elevated pressure (i.e., mechanical pressure stresses) has been known to modulate vascular remodeling, possibly by affecting the tissue renin-angiotensin system.In the present study we applied pulsatile pressure to human aortic smooth muscle cells (HASMCs) and investigated whether mechanical pressure stress affects cell proliferation and/or the angiotensin-converting enzyme (ACE), and we tested whether the administration of an angiotensin II (AII) receptor blocker has a favorable effect.Three hours of pulsatile atmospheric pressure resulted in an approximately 8% increase in cell proliferation of human aortic smooth muscle cells. The cell surface ACE level, enzyme activity and mRNA expression were all elevated (37%, 110% and 17%, respectively) under pressurized conditions, and co-administration of AII reduced all these values. The reductions in these three parameters resulting from the administration of AII were all abolished by AII receptor blocker co-administration and values were increased (11%, 62% and 12%, respectively) under pressurized conditions. Pulsatile atmospheric pressure increased the amount of phosphorylated extracellular signal-regulated kinase (ERK) by approximately 54% in HASMCs. The administration of PD98059 (10 microM) resulted in a decrease in phosphorylated ERK and ACE activity in HASMCs compared to those of the pressurized control.From these observations, we conclude that pulsatile mechanical pressure is one of the mediators of ACE production in vascular smooth muscle cells and that AII receptor blocking may prevent negative feedback. The present findings may provide a potential therapeutical target beyond lowering blood pressure in hypertensive patients.

We studied the effects of atrial natriuretic factor (ANF) on the conversion of angiotensin I to angiotensin II. Pulmonary artery endothelial cells converted 1.22 nmol/min per dish [125I]angiotensin I to angiotensin II, but ANF suppressed the conversion by 0.475 nmol/min per dish. The maximum rate of inhibition of angiotensin converting enzyme (ACE) activity by ANF was 60% at 10(-6) mol/l ANF compared with control conditions. The amino acid fragments of ANF were studied to determine whether its specific structure was necessary for inhibiting ACE in endothelial cells. We found that atriopeptin II (fragment 5-27) inhibited ACE activity as much as ANF. Fragment 7-25 had the same rate of inhibition, but fragment 13-27 had no effect on the conversion of angiotensin I to angiotensin II. These results suggest that the amino acid sequence of Cys (7)-Cys (23) in ANF is important for the inhibition of ACE in pulmonary endothelial cells.

In the present experiment, we demonstrated that IP3 kinase activity was increased in SHRSP heart compared to WKY heart. IP3 kinase activity in the heart was highest in the cytosolic fraction in both SHRSP and WKY. Its activity progressively increased with age in 5- to 20-week SHRSP. The activity reached about three times the level of 5-week-old SHRSP in 40-week-old SHRSP. On the other hand, in WKY it was 1.3-fold at 40 weeks compared with that at 5 weeks. We determined the effect of divalent cations on IP3 kinase activity. Ca2+ stimulated its activity in a dose-dependent manner at 10(-9) to 10(-6) M. In SHRSP it was enhanced about 2.1-fold at 10(-6) M of Ca2+, but in WKY it was 1.5-fold at 1.0(-6) M of Ca2+. Mn2+ also stimulated IP3 kinase activity in both groups of animals, while, Fe2+, Zn2+, and Cu2+ inhibited IP3 kinase activity. In our experiment IP3 kinase activity was increased in SHRSP and its activity was markedly affected by divalent cations. These data suggest that the accumulations of IP3 and IP4 after hormonal stimulation play a physiologic role, possibly by alteration of Ca2+ levels in cardiac tissue.

We studied the effects of endothelin on the conversion of angiotensin I to angiotensin II in pulmonary artery endothelial cells. Endothelin had a novel effect on angiotensin I conversion. When endothelin was added to pulmonary artery endothelial cells, the conversion of angiotensin I to angiotensin II was enhanced about two-fold. The maximum stimulation was achieved at 10(-8) M of endothelin. This stimulatory effect was suppressed by angiotensin converting enzyme inhibitors such an enalapril. When the calcium antagonist, nifedipine, was incubated with 10(-8) M of endothelin, the conversion of angiotensin I to angiotensin II stimulated with endothelin was slightly suppressed by nifedipine. Enalapril (10(-6) M) completely inhibited the conversion of angiotensin I to angiotensin II in the presence of endothelin. These results suggest that endothelin may play an important role in regulating vascular tone by modulating the conversion of angiotensin I to angiotensin II.

We studied the effects of platelet activating factor (PAF) on angiotensin-converting enzyme (ACE). PAF (1 · 10 −10 to 1 · 10 −6 M) had a novel effect on angiotensin I conversion. Pulmonary artery endothelial cells converted 1 nmol/dish of 125 I-angiotensin I to angiotensin II in the absence of PAF. ACE activity was increased to 2.5 nmol/dish by the addition of 1 · 10 −6 M of PAF. To clarify the mechanism of this stimulatory effect of PAF on ACE, Ca 2+ influx and inositol 1,4,5-trisphosphate (IP 3 ) release in pulmonary artery endothelial cells were determined. PAF stimulated Ca 2+ influx in a dose-dependent manner. PAF also stimulated phospholipase C (PLC) activity and released IP 3 . To study the relationship between PLC activity and ACE activity, neomycin was added. The Ca 2+ influx and IP 3 release stimulated by 10 −6 M of PAF were suppressed by about 60–70%. ACE activity was also inhibited up to 70% in the presence of PAF (10 −10 − 10 −6 M) by 50 M of neomycin. These results suggest that ACE was stimulated by PAF, and that its activityl in endothelial cells may be mediated by the PI-turnover pathway via changes in PLC activity and IP 3 -mediated Ca 2+ release from intracellular stores.

Mechanical forces related to pressure and flow are important for cell hypertrophy and proliferation. There are still a few studies that examine responses of human vascular smooth muscle cells to pure pressure stress (transmural pressure). It is unclear as to which mechanisms are involved in cellular responses to pressure elevation. On the other hand, although the involvement of the local renin-angiotensin system (RAS) in pressure-induced responses was reported, the results were contradictory. It still remains to be determined whether RAS in human vascular smooth muscle cells is activated by pure pressure stress. We studied the upstream signal transduction events of extracellular signal kinase (ERK) in response to atmospheric pressure stress and involvement of angiotensin II in pressure-induced cell proliferation in human aortic smooth muscle cells (HASMC). A pressure-loading apparatus was set up to examine the effects of atmospheric pressure on human aortic smooth muscle cells. Pressure application of 160 mmHg for 3 h produced cell proliferation and activated ERK and c-JUN N-terminal kinase (JNK). ACE inhibitor suppressed all of them. ERK kinase (MEK) inhibitor also suppressed cell proliferation stimulated by pure pressure. The phosphorylated c-Src was increased by pure pressure stress. The treatment with c-Src kinase inhibitor suppressed pressure-induced proliferative response. In summary, our study found that ERK activation mediated pure pressure-induced proliferative response of HASMC. This activation was partly mediated by c-Src.

Contraction of skeletal muscle generates pressure stimuli to intramuscular tissues. However, the effects of pressure stimuli, other than those created by electricity or nerve impulse, on physiological and biochemical responses in skeletal muscles are unknown. The purpose of this study is to examine the effects of a pure pressure stimulus on metabolic responses in a skeletal muscle cell line. Atmospheric pressure was applied to L6 myoblasts using an original apparatus. Succinate dehydrogenase (SDH) activity was evaluated by colorimetric assay using tetrazolium monosodium salt. The amounts of 2-deoxy-[3H]glucose uptake and lactate release were measured. SDH activity was 2.6- to 2.9-fold higher in pressurized L6 cells than in nonpressurized L6 cells ( P < 0.01), and 2-deoxy-[3H]glucose uptake was 2.2-fold higher ( P < 0.001). In addition, the amount of released lactate decreased from 6.8 to 3.7 μmol/dish when pressure was applied ( P < 0.001). In contrast, the intracellular lactate contents of the pressurized cells were higher than those of nonpressurized cells ( P < 0.01). However, the total amount of released lactate and intracellular lactate was lower in the pressurized cells than in nonpressurized cells. These findings demonstrate that a pure pressure stimulus enhances aerobic metabolism in L6 skeletal muscle cells and raise the possibility that elevated intramuscular pressure during muscle activity may be an important factor in stimulating oxidative metabolic responses in skeletal muscles.

Hypertension is a major risk factor for atherosclerosis and the genesis of cardiovascular and cerebrovascular diseases. Therefore, the protection of atherosclerosis progression is one of the purpose of an anti-hypertensive treatment in vascular system. Nitric oxide (NO)-releasing drugs have been reported to have an inhibitory effect on shear stress-induced extracellular signal-regulated kinase (ERK) activation in endothelial cells. For further understanding of the effects of these drugs, the present study focused on the effects on intracellular signal transduction and cell proliferation in cultured human aortic smooth muscle cells (HASMC) under high atmospheric pressure. Three hours of 160-mmHg atmospheric pressure resulted in an approximately 380% increase in cell proliferation compared to non-pressurized controls. Nipradilol (3,4-dihydro-8-(2-hydroxy-3-isopropylaminoproxy)-3-nitroxy-2 H -1-benzopyran) (10 −6 M) demonstrated approximately 40% reduction in cell proliferation compared to that shown by pressurized HASMC as a vehicle control. Three hours of 160-mmHg atmospheric pressure resulted in a 25% increase in the amount of activated ERKs. Nipradilol (10 −6 M) demonstrated approximately a 26% reduction in the amount of activated ERKs. NO x concentration under the presence of nipradilol (10 μM) with HASMC resulted in a 7.2 μM of NO production and was 2.4-fold more than that from no dug control (3.0 μM). An administration of L-NAME (10 −4 M) supplemented with Nipradilol (10 −6 M) did not show any significant effect on cell proliferation. From these observations, we concluded that nipradilol has an anti-proliferative effect on HASMC under high atmospheric pressure. Nipradilol may act as a nitric oxide inducer from HASMC and suspected to work as a supplement to mitigate the impaired endothelial cell function caused by hypertension.

To elucidate the intracellular function and localization of the heterotrimeric G-protein beta3 subunit (Gbeta3) in the heart, we studied the effects of subtype-specific beta-adrenergic receptor (beta-AR) stimulation on Gbeta3 localization using isoform-specific antibodies. The amount of Gbeta3 in the cytosol dramatically decreased in hearts perfused with isoproterenol (ISO) alone or ISO with ICI 118551, a beta2-AR antagonist. Propranolol or CGP 20712A, a beta1-AR antagonist, blocked the ISO-induced decrease in the Gbeta3 content of the cytosol. In contrast, Gbeta3 content of the membrane fraction significantly increased in hearts perfused with ISO alone or ISO with ICI 118551. We conclude that stimulation of the beta1-AR induces isoform-specific translocation of Gbeta3 from the cytosol to the membrane fraction in rat hearts.

Chronic mechanical stress of the heart secondary to arterial hypertension is a primary cause of left ventricular hypertrophy (LVH). The renin-angiotensin system (RAS) plays an important role in the cardiovascular system, regulating the expression of cardiac hypertrophy, in part, independent of the effects of systemic hypertension. A major component of RAS is angiotensin converting enzyme (ACE), which is upregulated in pressure overload-induced cardiac hypertrophy as well as heart failure. In a recent study, we found that the T allele of the M235T polymorphism of the angiotensinogen gene in sporadic hypertrophic cardiomyopathy (HCM) patients is associated with LVH. The present study was designed to assess the contribution of the polymorphisms of the angiotensin II type 1 receptor (AGT1R A1166C) genes on development of left ventricular hypertrophy. Patients with hypertensive LVH and relatives of HCM without manifesting the disease, showed higher C allele frequency compared to patients with HCM (11.3% vs 4.2%, chi 2 = 5.3, p < 0.05 and 10.5% vs 4.2%, chi 2 = 5.3, p < 0.05, respectively), but healthy controls did not (11.3% vs 7.5%, chi 2 = 1.42, NS and 10.5% vs 7.5%, chi 2 = 1.2, NS). The strong interaction between ACE I/D and AGT1R A1166C gene polymorphisms has been found in groups of relatives of HCM patients; odds ratio associated with ACE D allele was significant in subjects carrying the AGT1R C allele (OR = 7.3, 95% CI 1.6-33.1; chi 2 = 7.9, p < 0.02) compared with healthy subjects. We conclude that the molecular variant of the AGT1R A1166C gene is not contributing to the development of cardiac hypertrophy in hypertensive LVH and HCM patients, whereas carriers of both C and D alleles had a four-fold increase in the odds ratio for family history of HCM without manifesting the disease.

Angiotensinogen mRNA expression in human tissues was detected not only in the liver, but also in both atrial and ventricular heart tissues, suggesting that angiotensinogen is synthesized in the human heart. Angiotensinogen was present in the endocardial layer of the human left ventricle, rather than in the epicardial layer, and in the conduction system and right atrium. Furthermore, we found angiotensinogen to be widespread in the left ventricle of diseased hearts. We examined the involvement of collagen in the occurrence and progression of cardiomyopathy. In the early phase of cardiomyopathy, the extracellular matrix of the myocardium is rich in type III collagen, which is similar to immature tissues. In the later phase, the matrix resembles that of hard tissues in which collagen is mainly of type I and is insoluble. We also examined the relationship between angiotensin II and collagen synthesis. Previous studies have demonstrated that myocardial fibrosis accounts for impaired myocardial stiffness and ventricular dysfunction in hypertensive cardiac hypertrophy. Basal collagen synthesis in cardiac fibroblasts from spontaneously hypertensive rats (SHR) was 1.6 fold greater than that in Wistar-Kyoto rats. The responsiveness of collagen production to angiotensin II was significantly enhanced in SHR. This effect was angiotensin receptor specific because it was blocked by the competitive inhibitor. These results indicate angiotensin II may play an important role in collagen accumulation in hypertensive cardiac hypertrophy.

Recent reports have shown that angiotensin-converting enzyme inhibitors have a role in cardiac remodeling and have beneficial effects on congestive heart failure. Several studies have described alterations in sarcoplasmic reticulum gene expression in the failing heart. These results indicate that calcium homeostasis in myocytes may be disturbed in congestive heart failure. The present study examines the effects of long-term treatments with the angiotensin-converting enzyme inhibitor Enalapril and Ang II subtype 1 receptor antagonist TCV-116 on the morphological changes in the extracellular matrix, progressive left ventricular dysfunction in cardiomyopathic hamsters. Between age 5–20 weeks, 24 BIO53.58 hamsters (model of dilated cardiomyopathy) received 10mg/kg per day orally either of TCV-116 or no treatment. Between age 5–30 weeks, 24 BIO53.58 hamsters (model of dilated cardiomyopathy) received 20mg/kg per day orally either of Enalapril or no treatment. During the study period, cardiac function was assessed by echocardiography in a noninvasive manner. At 20 or 30 weeks of age, each heart was fixed with 10% formalin, embedded in paraffin, and serial sections were stained with Gomori’s aldehyde fuchsin using the Masson-Goldner method. High framerate ultrasonoscopic echocardiograms revealed that the left ventricular percent fractional shortening (%FS) tended to improve in the Enalapril group (22 ± 4% vs 20 ± 3%) and TCV-116 group (24 ± 4% vs 21 ± 4%). The fibrous tissue volume significantly decreased in Enalapril group (25.2 ± 0.5 mm3, P < 0.05) compared with the untreated group (27.6 ± 2.3mm3). TCV-116 did not significantly decrease the fibrous tissue volume. Enalapril can prevent cardiac remodeling, but TCV-116 is not as effective as Enalapril. Enalapril may nevertheless, suppress fibrosis.

The purpose of this study was to characterize the collagen in hereditary dilated cardiomyopathic hamster hearts, and to examine the participation of the collagen in the occurrence and progression of cardiomyopathy. BIO 53.58 hamsters (5, 10, 20 weeks old) were used as the model of dilated cardiomyopathy. Flb hamsters were used as controls. The collagen content was almost constant at any age in the Flb hamsters, but increased with age in BIO 53.58 hamsters. Type III collagen increased significantly in BIO 53.58 hamsters at 10 weeks. The acetic acid solubility of collagen decreased in BIO 53.58 hamsters as the fibrosis progressed, but was unchanged in controls. Reducible crosslinks showed a tendency to decrease progressively in BIO 53.58 hamsters. There were no differences between Flb and BIO 53.58 hamsters at 5 weeks, but its expression in BIO 53.58 hamsters at 10 and 20 weeks of age increased compared to Flb controls. These findings indicate that in the early phase of cardiomyopathy the extracellular matrix of the myocardium is rich in type III collagen. In the later phase, the matrix resembles that of hard tissues, whose collagen is mainly of type I collagen and is insoluble. These data suggest that the increased collagen synthesis may impair the cardiac function in the development of cardiomyopathy.

We cloned an entire encoding sequence of beta-adrenergic receptor kinase 1 (beta ARK1) cDNA from the hearts of Syrian hamsters through reverse transcription and subsequent polymerase chain reaction. The cloned cDNA contained 2067 nucleotides coding 689 amino acids. The sequence had 95% homology to rat beta ARK1 and 90% homology to human homologue. Cardiomyopathic Syrian hamster, BIO53.58, has been used as a model animal of congestive heart failure. M-mode echocardiography confirmed that left ventricular contractility of 20-week-old BIO53.58 was markedly reduced. The expression of beta ARK1 mRNA in the hearts of BIO53.58 was significantly increased compared to control hamsters, F1b, suggesting that the enhanced beta ARK1 expression is acting as a negative feedback mechanism in order to maintain intracellular homeostasis against accelerated stimulation by catecholamines via phosphorylation of beta-adrenergic receptor.

To examine the contribution of the angiotensin-converting enzyme (ACE) gene to hypertrophic cardiomyopathy (HCM), we determined the ACE insertion/deletion (I/D) polymorphism in 80 patients with HCM and 88 of their unaffected siblings and children. Patients were divided into familial or solitary HCM (FHCM or SHCM) groups with or without affected family members. Genotypes were identified by the polymerase chain reaction (PCR) with oligonucleotide primers flanking the polymorphic region in intron 16 of the ACE gene to amplify template DNA prepared from peripheral leukocytes. D-allele frequencies were 0.38 in all subjects, 0.42 in patients with HCM, and 0.35 in relatives (p < 0.05). The probability ratios were 1.98, 1.46, and 2.97 in patients with HCM, FHCM, and SHCM, respectively. The D allele frequency was higher in SHCM than in FHCM (p < 0.05). The findings suggest that HCM, especially in solitary cases, is partially determined by genetic disposition. Findings imply that the ACE D allele is one of the genetic contributing factors associated with cardiac hypertrophy in HCM.

Extrahepatic synthesis and localization of angiotensinogen have been described in animals, thus establishing the tissue renin-angiotensin system. We examined angiotensinogen messenger RNA synthesis by northern blotting. It was detected not only in the liver, but also in both the atrial and ventricular heart tissues, suggesting that angiotensinogen is synthesized in the human heart. Immunohistochemical studies using a specific antibody to angiotensinogen revealed a stronger reaction in the endocardial layer of the human left ventricle, than in the epicardial layer, and intense immunoreactivity in the conduction system and right atrium. Furthermore, our experiments revealed a widespread immunopositive reaction for angiotensinogen in the left ventricle of diseased hearts. We examined the participation of the collagen in the occurrence and progression of cardiomyopathy. The acetic acid solubility of collagen and reducible crosslink decreased in cardiomyopathic hamsters as the fibrosis progressed, but was unchanged in controls. These findings indicate that in the early phase of cardiomyopathy the extracellular matrix of the myocardium is similar to immature tissues. In the later phase, the matrix resembles that of hard tissues, and is insoluble. Furthermore, we examined the relationship between angiotensin II and collagen synthesis. Basal collagen synthesis in cardiac fibroblasts from spontaneously hypertensive rats was 1.6-fold greater than that in Wistar-Kyoto rats. The responsiveness of collagen production to Ang II was significantly enhanced in SHR. This effect was angiotensin receptor-specific, because it was blocked by the competitive inhibitor. These results indicate angiotensin II may play an important role in collagen accumulation in hypertensive cardiac hypertrophy.

To understand the significance of the tissue renin-angiotensin system in the heart, we examined the expression of renin and angiotensin-converting enzyme (ACE) in autopsied human hearts. Samples were taken from organs obtained at autopsy from 15 patients without heart disease and 3 patients with heart disease (old myocardial infarctions, acute myocardial infarctions, and hypertrophic cardiomyopathy). We examined the expression of renin and ACE mRNA by using the reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR showed the expression of renin in the right atria in all patients. However, expression of renin mRNA in the left ventricles was not found in any of the 15 hearts without heart disease. In contrast, renin mRNA was detected in the left ventricles in hearts with heart disease. ACE mRNA was detected in both the atria and the ventricles in normal hearts, and its expression did not alter in diseased hearts. These findings suggest that renin mRNA is expressed mainly in the right atria in normal hearts, but that its expression in the left ventricle can be activated in some pathological conditions.

We attempted to determine the effects of transforming growth factor beta-1 on intracellular Ca2+ concentration changes in the presence of isoproterenol in cardiac fibroblasts. Transforming growth factor beta-1 inhibited the increase of intracellular Ca2+ concentration in the presence of isoproterenol in fibroblasts. It also inhibited the production of cyclic-AMP in fibroblasts in the presence of isoproterenol. Islet-activating protein did not block these reactions of transforming growth factor beta-1. Forskolin did not affect the intracellular calcium concentration change resulting from treatment with transforming growth factor beta-1. Binding of [3H]CGP-12177 was decreased to 47% of control preincubated for 24 hours with transforming growth factor beta-1 in fibroblasts. Scatchard plots suggested a decrease in beta-adrenergic receptor number without specific change in receptor affinity. These results suggested that transforming growth factor beta-1 modulates the signal transduction through beta-adrenergic receptor and intracellular Ca2+ concentration by regulating the number of receptors in fibroblasts.

Recently, it has been reported that the signal through the (β-adrenergic signal transduction system in failing heart is reduced. This includes the reduction of (β-adrenergic receptors and Gs-protein, decreased adenylyl activity, and increased Gl-protein (Figure 1). But it is still not clear whether the signal through α-adrenergic receptor changes in failing hearts. From this point of view, we attempted to determine the signal transduction systems in cardiomyopathy using cardiomyopathic hamster hearts. Syrian cardiomyo-pathic hamsters (BIO 14.6 and BIO 53.58) display hereditary abnormalities of the cardiac and skeletal muscles that are inherited as an autosomal recessive trait [1] BIO 14.6 cardiac involvement results in initial myocardial hypertrophy that is followed by cardiac dilatation and death due to congestive heart failure [2]. It is thought that the cardiomyopathic hamster provides a useful model for studying human cardiac diseases, such as hypertrophic cardiomyopathy [3,4].

We demonstrated that phosphatidylinositide-specific phospholipase C (PLC) activity was greater in cardiomyopathic hamster hearts (BIO 14.6 and BIO 53.58) then in hamster controls (F1b). Inositol trisphosphate (IP3) production was markedly greater in both of the cardiomyopathic hamsters, BIO 14.6 and BIO 53.58. We have also determined the sarcoplasmic reticulum (SR) function of heart. Calcium uptake into SR markedly increased in BIO 14.6. On the other hand, it significantly decreased in BIO 53.58 compared with F1b. It is well known that IP3 stimulates calcium release from SR. In BIO 14.6, calcium relrease from SR stimulated by IP3 increased, but its effect decreased in BIO 53.58 compared with F1b. These results suggest that PI response may produce high intracellular calcium levels in both BIO 14.6 and BIO 53.58 myocytes. In addition, in the BIO 53.58 hamster the sarcoplasmic reticulum deteriorate in function. It was concluded from these results that a prolonged high intracellular calcium level may lead to the death of BIO 53.58 myocytes. The expression of angiotensinogen mRNA was observed in the hamster heart. There was no differences in its expression level between F1b, BIO 14.6 and BIO 53.58. There was no effect of ages on its expression in these hamster hearts. We have also determined the distribution of angiotensinogen in these hamsters. At 4 weeks of age, the immunohistochemical study revealed that angiotensinogen was widely distributed in subendcardium in these hamsters. There was no difference in its distribution between F1b, BIO 14.6 and BIO 53.58. But at 20 weeks old of age its immunoreactivity decreased in BIO 53.58. There was no effect of age on its reactivity in F1b and BIO 14.6. We have detected angiotensinogen in heart, but its role is still not clear. A further study should be done to clarify its role in cardiac hypertrophy and cell damage.

It is well known that failing hearts show diminished responsiveness (desensitization) to catecholamines. In this study, 2 different animal models were used to investigate the alterations in the hormone sensitive cardiac adenylyl cyclase system in a congestive heart failure. In the first model, cardiomyopathic Syrian hamsters (BIO53.58), we found reduced activity of the catalytic protein of adenylyl cyclase; this reduction was more prominent in an older animals (28-week-old vs 16-week-old). At both ages, the amount of inhibitory GTP-binding protein (Gi) was markedly increased in BIO53.58 compared to control healthy hamsters. Moreover the increased Gi was shown to be fully functional in the inhibitory pathway of the adenylyl cyclase system. In the second model, chronically norepinephrine-infused rats, we found a decrease in beta-adrenergic receptor density at an early stage of injection (3 days), while the activity of catalytic protein decreased beyond 14 days of injection, and the amount of Gi increased after 7 days of injection. These results suggest that increased plasma catecholamine concentrations in the setting of congestive heart failure might be a major trigger for qualitative and quantitative alterations observed in various components of cardiac adenylyl cyclase system, and that GTP-binding proteins and catalytic protein of adenylyl cyclase are involved in the mechanism of desensitization especially after chronic in vivo stimulation of adrenergic receptors.

The accumulation of both Inositol-(1,4,5)-trisphosphate (IP3) and Inositol-(1,3,4,5)-tetrakisphosphate (IP4) after hormonal stimulation has a physiological role, possibly in altering Ca2+ levels in cardiac tissue. However, the accumulation of inositol polyphosphate under pathophysiological conditions has not been studied. In our experiments the metabolism of phatidylinositol and IP3 in cardiac myocytes as investigated. It was shown that basal levels of cytosolic phosphatidylinositol specific phospholipase C (PI-PLC), phosphatidylinositol-(4,5)-bisphosphate specific phospholipase C (PIP2-PLC) activities markedly increased in stroke-prone spontaneously hypertensive rats (SHRSP) with age compared with age matched Wistar Kyoto rats (WKY). IP3 kinase and IP3 phosphatase activities also increased in SHRSP hearts with age. Their activities increased in WKY, but to a lesser extent than in SHRSPs. These data suggest that a PI turnover pathway such as the phosphatidylinositol 4,5-bisphosphate-IP3-Ca2+ pathway or the diacylglyceride-protein kinase C pathway may have an important role in the development of hypertrophy in SHRSP heart.

The renin-angiotensin system acts to constrict vessels and enhance renal retention of sodium and water to raise the blood pressure. Angiotensinogen (ang-n) is synthesized in the liver and released into the blood. It is cut by renin, which is produced by the kidneys, and becomes angiotensin I. Angiotensin I is cleaved by angiotensin converting enzyme (ACE) into angiotensin II, which is an active pressor substance. Recently, the presence of cardiac renin-angiotensin system has been reported in some animals [1,2,3] but it has not been investigated fully in the human heart. Angiotensin II appears to be a stimulus for cardiac hypertrophy [4]. In clinical studies, ACE inhibitor has been reported to cause regression of cardiac hypertrophy [5]. Bearing these findings in mind, we studied the presence of ang-n, renin mRNAs and ang-n protein in human autopsy hearts and discussed its relation to cardiac hypertrophy.

Various diseases and clinical settings can induce congestive heart failure (CHF). As such, it is unlikely that a single mechanism can explain the cardiac dysfunction observed in CHF. However, CHF patients (or animals) share several similar features: reduced cardiac contractility, increased sympathetic nerve activity, and activation of several hormonal systems (e.g., renin-angiotensin system, vasopressin, and atrial natriuretic factor) to compensate for the impaired cardiac function. In this setting, cardiac receptor, GTP-binding proteins, and their effector molecules (adenylyl cyclase, phospholipase C, several ion channels) are inevitably exposed to chronically elevated neurohormonal stimulation. A widely recognized concept is that a chronic increase in such stimulation can desensitize target cell receptors and post-receptor signal transducing pathways (1). It is therefore likely that in CHF one or more types of desensitization contributes to a reduction in signal transducing efficiency in order to maintain intracellular homeostasis. To date, several laboratories have reported a decrease in β-adrenergic receptor (βAR) density in CHF patients and model animals (2–4, 5 for review). Recently, reports of several studies have indicated that the inhibitory GTP-binding protein (Gi) can be increased in CHF patients (6,7) and animal models (8). Although direct evidence for a change in the catalytic protein of adenylyl cyclase has not been available, limited information have suggested a reduced catalytic activity in terminally failing hearts (9). In this study, we have suggested changes of βAR, GTP-binding protein and catalytic protein using two different animal models with an emphasis on defining the kinetics for the desensitization process of adenylyl cyclase system.

In cardiac hypertrophy,collagen accumulation in the myocardial interstitium is associated with hypertrophy of cardiomyocytes [1]. Collagen remodeling causes impaired ventricular compliance,abnormal electrical conduction and impaired oxygenation of the cardio-myocytes [2].At present,factors that regulate cardiac collagen metabolism are not elucidated.Eghbali et al [3] found that cardiac fibroblasts are mainly responsible for the synthesis of fibrillar type I and III co11agen.Recent studies have demonstrated that angiotensin II (A II),in addition to its inotropic and chronotropic actions,may exert effects by acting as a growth factor in the car¬diovascular system and that there may be local autocrine or paracrine renin-angiotensin system in several tissues [4].There are no data concerning the effects of A II on cardiac nonmyocytes.In this study we examined the effects of A II on collagen synthesis by cardiac fibroblasts in normotensive and hypertensive rats.

It has been demonstrated that components of the renin-angiotensin system such as renin, angiotensinogen (ATN), angiotensin-converting enzyme (ACE) and angiotensin II receptor exist within the heart and function independently from circulating renin-angiotensin system [1–4]. The potent myotropic actions of angiotensin II, as well as its effects on positive inotropism and chronotropism, make it likely that the heart is the site of a locally active renin-angiotensin system. It is suggested that angiotensin II is one of the contributing factors to regulation of cell growth, moreover, ACE inhibitors regress left ventricular hypertrophy. The purpose of this study is to determine whether intrinsic renin-angiotensin system play a role for the development and the regression of left ventricular hypertrophy in spontaneously hypertensive rats (SHR).

We demonstrated that the activities of phosphatidylinositide-specific phospholipase C, inositol 1,4,5-trisphosphate (IP3) kinase, and IP3 phosphatase were enhanced in cardiomyopathic hamster hearts (BIO 14.6 and BIO 53.58) in comparison to control hamsters (F1b). Release of both arachidonic acid and prostacyclin was markedly enhanced by norepinephrine in the cardiomyopathic hamsters. Phospholipase C in heart has high substrate specificity to phosphatidylinositol. IP3 production was markedly enhanced in the cardiomyopathic hamsters. We also determined the intracellular calcium concentration, which was higher in BIO 53.58 hamsters than in BIO 14.6 hamsters at 5-20 weeks of age. There was no significant difference in the intracellular calcium level between F1b and BIO 14.6 hamsters at 5 weeks of age. These results suggest that phosphatidylinositol turnover stimulated by norepinephrine may produce high intracellular calcium levels in both BIO 14.6 and BIO 53.58 myocytes. In addition, in BIO 53.58 hamsters, some mechanism such as the sarcoplasmic reticulum, which controls the intracellular calcium level, may deteriorate in function. We concluded from these results that a prolonged high intracellular calcium level may lead to the death of BIO 53.58 myocytes.

The effects of increased free fatty acid (FFA) levels on ventricular arrhythmias remain controversial. Using ventricular fibrillation threshold (VFT), we examined the relationship between FFA levels and ventricular arrhythmias. Isolated rat hearts were perfused with palmitate bound to either albumin or fatty acid binding protein (FABP) by Langendorf's method. The VFT was determined by electrical stimulation. Perfusion with 0.12 mM albumin alone, 0.12 mM palmitate bound to 0.12 mM albumin, and 0.36 mM palmitate bound to 0.12 mM albumin did not lower the VFT significantly. However, 0.60 mM palmitate bound to 0.12 mM albumin lowered VFT from 2.19 +/- 0.20 mA to 1.56 +/- 0.13 mA. The perfusion of 0.36 mM palmitate bound to 0.12 mM FABP lowered the VFT from 2.05 +/- to 0.19 mA to 1.47 +/- 0.23 mA, but 0.12 mM FABP alone did not affect the VFT. Perfusion with 0.36 mM palmitate bound to 0.12 mM FABP caused the VFT to fall more than perfusion with 0.36 mM palmitate bound to 0.12 mM albumin. Then the effects of verapamil perfusion or a low concentration of perfusate Ca2+ on VFT were examined. VFT was determined by electrical stimulation. Palmitate (0.6 mM) bound to 0.12 mM albumin lowered VFT. Verapamil 10(-7) M perfusion and a low concentration of Ca2+ (Ca2+ 1.67 mM) suppressed the FFA-induced fall of VFT. These results suggested that the arrhythmogenic action of FFA was related to Ca2+ overload in myocardial cells.

To test the hypothesis that structural abnormalities exist in the kidney membrane of spontaneously hypertensive rats, we examined the effect of long-term administration of coenzyme Q10 on membrane lipid alterations in the kidney of stroke-prone spontaneously hypertensive rats (SHRSP). As compared with normotensive Wistar-Kyoto rats, renal membrane phospholipids, especially phosphatidylcholine and phosphatidylethanolamine, decreased and renal phospholipase A2 activity was enhanced with age in untreated SHRSP. Treatment with coenzyme Q10 attenuated the elevation of blood pressure, the membranous phospholipid degradation, and the enhanced phospholipase A2 activity. These results suggest that one factor contributing to the progress of hypertension is a structural membrane abnormality that alters the physical and functional properties of the cell membrane, and coenzyme Q10 might protect the renal membrane from damage due to hypertension in SHRSP.

We studied the effects of endothelin on angiotensin converting enzyme (ACE) in cultured pulmonary artery endothelial cells. ACE activity was increased 2.5-fold by the addition of 1 x 10(-8) mol/l endothelin-1. Endothelin-1 also stimulated calcium influx and phospholipase C activity in a dose-dependent manner. Calcium influx, phospholipase C and ACE activity were suppressed 60-70% in the presence of endothelin-1 (10(-10) to 10(-6) mol/l) by 50 microliters neomycin. These results suggest that ACE was stimulated by endothelin-1 and that its activity may be closely related to phosphatidylinositol turnover stimulated by endothelin-1.

We studied the effects of platelet-activating factor (PAF) on the conversion of angiotensin I to angiotensin II in pulmonary artery endothelial cells. PAF had a novel effect on angiotensin I conversion. The apparent Vmax and Km for angiotensin converting enzyme (ACE) were 2.5 nmol/min per dish and 50 mumol/l, respectively. This activity was enhanced by the addition of PAF to cells. When PAF was added to pulmonary artery endothelial cells, the conversion of angiotensin I to angiotensin II was enhanced about twofold at 10(-6) mol/l PAF. Maximal stimulation was achieved at 10(-5) mol/l PAF. This stimulatory effect was suppressed by ACE inhibitors such as enalapril and PAF antagonist CV3988. When cells were incubated with 10(-6) mol/l PAF, the conversion of angiotensin I to angiotensin II stimulated with PAF was suppressed by CV3988. Enalapril (10(-6) mol/l) completely inhibited the conversion of angiotensin I to angiotensin II in the presence of PAF. Bradykinin also suppressed ACE activity, but phosphatidylcholine and lysophosphatidylcholine did not affect its activity. These results suggest that PAF may have an important role in regulating vascular tone by modulating angiotensin conversion.