Shunichi Kobayashi

Mechanism of cAMP regulation of renin gene transcription by proximal promoter.

Renin is produced mainly by the kidney, and cAMP is a main positive regulator of its synthesis. This study was undertaken to analyze the molecular mechanism of cAMP-mediated regulation of Ren-1C gene transcription by the proximal promoter. We first showed that the promoter region from -365 to +16 of the mouse renin gene (Ren-1C) mediated the cAMP-induced chloramphenicol acetyltransferase gene expression in embryonic kidney-derived 293 cells. Deletion analysis and heterologous promoter assay disclosed that the proximal promoter region from -75 to +16 was able to activate chloramphenicol acetyltransferase expression by cAMP, and indicated that the proximal promoter element from -75 to -47 (RP-2 element) overlapping the TATA-like region was able to confer cAMP responsiveness. Electrophoretic mobility shift assay and DNase I footprinting analysis demonstrated that novel nuclear factors in 293 cells interacted with the RP-2 element, and that cAMP increased the binding activity of these nuclear factors to the RP-2 element. Furthermore, we demonstrated that cAMP enhanced the binding of nuclear factors derived from juxtaglomerular cells, the main production site of renin in the kidney, to the RP-2 element in vivo. These results suggest that the RP-2 element plays an important role in the cAMP-mediated regulation of Ren-1C gene transcription through the proximal promoter.

Molecular mechanism of adipogenic activation of the angiotensinogen gene.

Angiotensinogen gene expression is controlled in a tissue- and development-specific manner. Interestingly, the angiotensinogen gene is abundantly expressed in adipose tissues other than the liver, where it is mainly produced. We investigated the molecular mechanism of angiotensinogen gene expression in a 3T3-L1 preadipocyte-adipocyte system. Although angiotensinogen mRNA was barely detectable in preadipocytes, its levels increased significantly during differentiation. As a whole, the pattern of the change in transcriptional activity of the angiotensinogen promoter was similar to that of the angiotensinogen mRNA levels during adipogenic differentiation, indicating that the activation of the angiotensinogen promoter might be involved in the adipogenic differentiation-coupled gene expression. The proximal promoter region, from -96 to +22 of the transcriptional start site, was sufficient to confer adipogenic activation, and the proximal element from -96 to -52 of the transcriptional start site was necessary for this promoter stimulation. DNA-protein binding experiments showed that this proximal element specifically bound to a nuclear factor induced by adipogenic differentiation. These results suggest that the proximal promoter element from -96 to -52 plays a role in adipogenic activation of the angiotensinogen promoter.

Long-term effects of ezetimibe-plus-statin therapy on low-density lipoprotein cholesterol levels as compared with double-dose statin therapy in patients with coronary artery disease

OBJECTIVE To assess the mechanism of long-term LDL-C-lowering effect of ezetimibe-plus-statin. METHODS Coronary artery disease patients whose LDL-C ≥ 70 mg/dL after treatment with atorvastatin 10 mg/day or rosuvastatin 2.5 mg/day were randomly assigned to receive ezetimibe 10 mg/day + statin (n = 78) or double-dose statin (n = 72) for 52 weeks. RESULTS Greater LDL-C reduction was observed and maintained until 52 weeks in ezetimibe-plus-statin, while LDL-C levels re-increased after 12 weeks in double-dose statin. Although lathosterol/TC increased, campesterol/TC decreased more in ezetimibe-plus-statin. In contrast, lathosterol/TC unchanged and campesterol/TC increased, increasing campesterol/lathosterol ratio for 52 weeks in double-dose statin. Plasma PCSK9 levels were higher in double-dose statin than in ezetimibe-plus-statin at 12 weeks, but similar at 52 weeks. CONCLUSION Although the difference in PCSK9 between 2 groups was transient, that in both campesterol and lathosterol persisted until 52 weeks. These results demonstrated simultaneous inhibition of cholesterol absorption and synthesis provides stable and greater decrease in LDL-C levels.

Effect of Genetic Deficiency of Angiotensinogen on the Renin-Angiotensin System

This study examined expression of renin-angiotensin system (RAS) component mRNAs in angiotensinogen gene knockout (Atg-/-) mice. Wild-type (Atg+/+) and Atg-/- mice were fed a normal-salt (0.3% NaCl) or high-salt (4% NaCl) diet for 2 weeks. Angiotensinogen, renin, angiotensin-converting enzyme (ACE), angiotensin II type la receptor (AT1A), and angiotensin II type 2 receptor (AT2) mRNA levels were measured by Northern blot analysis. In Atg+/+ mice, activities of circulating RAS and renal angiotensinogen mRNA level were decreased by salt loading, whereas levels of renal and cardiac ACE; renal, brain, and cardiac AT1A; and brain and cardiac AT2 mRNA were increased by salt loading. Although activities of circulating RAS were not detected in Atg-/- mice, salt loading increased blood pressure in Atg-/- mice. In Atg-/- mice, renal renin mRNA level was decreased by salt loading; in contrast, salt loading increased renal AT1A and cardiac AT2 mRNA levels in Atg-/- mice, and these activated levels in Atg-/- mice were higher than those in Atg+/+ mice fed the high-salt diet. Thus, expression of each component of the RAS is regulated in a tissue-specific manner that is distinct from other components of systemic and local RAS and that appears to be mediated by a mechanism other than changes in the circulating or tissue levels of angiotensin peptides.

Increased Cardiac Angiotensin II Receptors in Angiotensinogen-Deficient Mice

Two subtypes of angiotensin II (Ang II) receptors, type 1 (AT1-R) and type 2 (AT2-R), have been identified in the heart. However, little is known about the regulation of cardiac AT1-R and AT2-R by Ang II in vivo. Thus, we examined cardiac AT1-R and AT2-R in angiotensinogen-deficient (Atg-/-) mice that are hypotensive and lack circulating Ang II. Cardiac Ang II receptors (Ang II-R) were assessed by radioligand binding with 125I-[Sar1,Ile8]-Ang II in plasma membrane fractions. AT1-R and AT2-R were distinguished using their specific antagonists CV-11974 and PD123319, respectively. Total densities of Ang II-R and AT1-R density were significantly greater in the Atg-/- mice than Atg+/+ mice (31.1+/-2.8 versus 18.8+/-2.1, 28.7+/-3.0 versus 16.9+/-2.3 fmol/mg protein, P<.01, respectively), and AT2-R showed a slight but not significant increase in Atg-/- mice relative to Atg+/+ control animals. Kd values were not different between the two groups. In contrast to binding experiments, levels of Ang II type 1a receptor (AT1a-R) and AT2-R mRNA did not differ between Atg-/- and Atg+/+ mice. These results suggest that lack of Ang II may upregulate AT1-R through translational and/or posttranslational mechanisms in Atg-/- mice.

ANGIOTENSIN II RECEPTORS IN CARDIAC LEFT VENTRICLES OF DAHL RATS

1. Dahl Iwai salt‐sensitive (DS) rats have been reported as becoming hypertensive with left ventricular hypertrophy (LVH) and heart failure when on a high‐salt diet. Their circulating renin–angiotensin system (RAS) has been reported to be suppressed. To evaluate the role of angiotensin II (AngII) type 1 and type 2 receptors (AT1 and AT2, respectively) in LVH, we compared cardiac AT1 and AT2 receptors in 10‐week‐old DS rats and Dahl Iwai salt‐resistant (DR) rats.

DNA fingerprint analysis of Dahl-Iwai salt-sensitive rats (S) and salt-resistant rats (R)

Using DNA fingerprint analyses, extensive molecular heterogeneity has been found between spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY), suggesting a doubtful value of simply comparing these rats in the study of the pathogenesis of genetic hypertension. Therefore, we evaluated the genetic similarity between Dahl salt-sensitive and salt-resistant rats newly inbred by Dr.Iwai (Dahl-Iwai S and R rats), by use of DNA fingerprint analysis. Fingerprint patterns were generated by probing Hinf I- or Alu I-digested DNA with an oligonucleotide corresponded to the tandem repeat sequence of the human myoglobin 33.6 minisatellite. These fingerprint patterns were same within each strain. S and R rts shared 82 percent of the bands in Hinf I-digested DNA and 93 percent of those in Alu I-digested DNA. Although Dahl-Iwai S and R rats are more closely related than SHR and WKY rats, multiple genetic differences still exist between these Dahl strains.

Effects of follicle-stimulating hormone on endothelin receptors in cultured rat ovarian granulosa cells.

Two subtypes of the endothelin (ET) receptor (ETA and ETB) were studied in cultured ovarian granulosa cells. Immature 21-day-old female Wistar-Imamichi rats were implanted with diethylstilbestrol (DES) pellets for 5 days and granulosa cells were collected by repeated puncturing. Viable cells (2.5 or 5 x 10(5)) were cultured with 50--400 ng/ml of ovine NIH follicle-stimulating hormone (FSH) in the presence or absence of [125I-Tyr13]ET-1 (50 pM) in 1 ml McCoys 5a medium for 72 h. FSH gradually increased the [125I-Tyr13]ET-1 binding to granulosa cells, whereas FSH-untreated granulosa cells had no significant changes. The dose of 200 ng/ml of FSH was most effective for [125I-Tyr13]ET-1 binding for 48-h culture, thereafter revealing a plateau. After 48 h of culture with 200 ng/ml of FSH, granulosa cells were further incubated with [125I-Tyr13]ET-1 (10 pM-1 nM) and/or [125I]IRL1620, the selective ETB receptor agonist (10 pM-1 nM) for 2 h for equilibrium study, and then the dissociation constant and the maximal binding capacity between receptors and ligands were determined by saturation curve and Scatchard plot analysis. ETA + ETB, ETB, and ETA (sites/cells) showed a 4.4-, 2.6-, and 7.5-fold increase, respectively. As for steroidogenesis, ET-1 (100 nM) or ET-3 (100 nM) suppressed FSH-induced progesterone and 17 beta-estradiol production. These results indicate that FSH upregulates both ETA and ETB receptors in DES-treated immature rat granulosa cells, with no significant differences between ET-1 and ET-3, and that ET-1 or ET-3 suppresses FSH-induced steroidogenesis. ETs may affect the granulosa cell function through the ETA and ETB receptors, and the increase in amount of ET binding does not reflect ET effects on granulosa cell function. The ET receptor plays an important role in the development of the ovary.

Dde I restriction fragment length polymorphism of the alpha 2-adrenoceptor gene does not correlate with blood pressure in the F2 generation obtained from crossing stroke-prone spontaneously hypertensive rats and Wistar-Kyoto rats.

Objective A pathogenetic role of altered α2-adrenoceptors in essential hypertension has been suggested, based on studies in humans and animals. To examine the role of the α2-adrenoceptor in genetically hypertensive rats, we compared the α2-adrenoceptor genes of stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto (WKY) rats by restriction fragment length polymorphism analysis using human α2-adrenoceptor probes (α2-C10) and Dde I restriction endonuclease, and conducted a genetic cosegregation study. Method Five female WKY rats were bred with five male SHRSP. Eight pairs of F1 rats were mated in brother-sister pairs to yield an F2 population of 84 rats. Systolic blood pressure was determined by tail-cuff sphygmomanometry. Direct arterial blood pressure was taken under ether anaesthesia just before the rats were killed. Southern blots were performed using α2C10 as a probe and the DMA from the F2 generation. Results A restriction fragment length polymorphism of the SHRSP allele of a 1.6-kb fragment and a WKY rat allele of a 0.9-kb fragment with a common band of 1.3 kb in SHRSP and WKY rats was found, as reported previously. The distribution of the genotype based on restriction fragment length polymorphism conformed to a 1:2:1 ratio in F2 rats, as expected for a Mendelian trait. There was no significant difference in the blood pressure of F2 rats with respect to α2-adrenoceptor genotype. Conclusion This study demonstrated that the α2-adrenoceptor gene restriction fragment length polymorphism distribution is a Mendelian trait in the F2 rats of crossed SHRSP and WKY rats, but failed to show genetic cosegregation of this restriction fragment length polymorphism with blood pressure in this generation.

LONG-TERM EFFECT OF EZETIMIBE-STATIN COMBINATION THERAPY ON LOW-DENSITY LIPOPROTEIN CHOLESTEROL LOWERING IN PATIENTS WITH CORONARY ARTERY DISEASE; FOCUS ON CHOLESTEROL ABSORPTION AND SYNTHESIS

Authors: Kozo Okada, Kazuki Fukui, Hideo Himeno, Tutomu Endo, Makoto Shimizu, Shunichi Kobayashi, Tomohiko Shigemasa, Yukiko Morita, Atsushi Wada, Tomoaki Shimizu, Yasuyuki Mochida, Reimin Sawada, Tomoaki Ishigami, Kazuaki Uchino, Noriaki Iwahashi, Kazuo Kimura, Satoshi Umemura, Division of Cardiology, Yokohama City Univercity Medical Center, Yokohama , AL, Japan, Department of Cardiology, Yokohama City Univercity Hospital, Yokohama, AL, Japan