Masayo Nakagawa

Aldosterone Induces Angiotensin-Converting-Enzyme Gene Expression in Cultured Neonatal Rat Cardiocytes

Background—The cardiac renin-angiotensin-aldosterone system is activated in failing hearts in proportion to the severity of the disease. We hypothesized that a positive feedback mechanism might exist within this system and contribute to the progression of the heart failure. Methods and Results—To test this hypothesis, we examined whether angiotensin II or aldosterone induces the expression of angiotensin-converting-enzyme (ACE) mRNA in cultured neonatal rat ventricular cardiocytes. Expression of ACE mRNA was detected and quantified using real-time reverse transcription-polymerase chain reaction. Exposure to angiotensin II (10−5 mol/L) for 24 hours had no significant effect on the expression of ACE mRNA (0.7±0.5-fold versus control, P =NS), but similar treatment with aldosterone (10−5 mol/L) induced a 23.3±7.9-fold increase (P <0.01) in ACE mRNA expression. The effect of aldosterone was both time- (maximal effect, 24 hours) and dose-dependent (EC50, 4×10−7 mol/L), and it was significantly (P <0.01) inhibited by spironolactone, a specific mineralocorticoid receptor antagonist. Conclusions—Aldosterone upregulates ACE mRNA expression, which is blocked by spironolactone in neonatal rat cardiocytes. Thus, spironolactone may suppress the progression of heart failure by blocking the effects of aldosterone and angiotensin II.

Growth-Dependent Induction of Angiotensin II Type 2 Receptor in Rat Mesangial Cells

Angiotensin II acts on at least two receptor subtypes, AT1 and AT2. Although the physiological role of the AT2 receptor is still poorly defined, it may be implicated in inhibition of cell growth, vasorelaxation, and apoptosis. In the present study, to investigate the role of the AT2 receptor in the kidney and its implication in hypertensive states, we examined its expression using cultured mesangial cells (MC) from normotensive Wistar-Kyoto rats (WKY) and from stroke-prone spontaneously hypertensive rats (SHRSP). Receptor binding assays were performed using a nonselective ligand, [Sar1,Ile8]angiotensin II, or AT2-selective CGP42112A. Binding assays revealed that MC from WKY exhibited both AT1 and AT2 receptors, the ratio of which was confluence-dependent. In contrast, MC from SHRSP, whose proliferation activity was much higher than those from WKY, showed only the AT1 subtype. In receptor binding and Northern blot analyses, expression of the AT2 receptor of WKY-MC was low in the growing state but significantly induced upon confluence to become abundant in the post-confluent state, whereas that of SHRSP-MC was undetectable in either state. Gene expressions of AT1A and AT1B receptors were not significantly altered in either strain during the time in culture. These results indicate that the mesangial AT2-receptor expression is growth-dependent and suggest a role in the inhibition of MC growth in WKY. Much lower expression of the AT2 receptor in MC from SHRSP may suggest involvement in their higher proliferation activity and possibly in consequent renal disorders.

Identification of a novel R642C mutation in Na/Cl cotransporter with Gitelman's syndrome

Gitelmans syndrome, a variant of Bartters syndrome, is an inherited disorder characterized by hypokalemic metabolic alkalosis, hypomagnesemia, and hypocalciuria, and these abnormalities have recently been linked to the thiazide-sensitive Na/Cl cotransporter (TSC) gene. We evaluated three unrelated patients affected with this syndrome whose diagnosis was made based on clinical and biochemical features. The data of clearance studies in these patients were compatible with Gitelmans syndrome. We then investigated possible mutations of the TSC gene. In one patient whose parents are consanguineous, we identified a novel missense mutation in the TSC gene, which causes alteration of arginine to cysteine at codon 642 (R642C mutation) located in the cytoplasmic tail of the product. This mutation results in the loss of an MspI site in exon 15 of the TSC gene. MspI digestion analysis of genomic DNA fragments from the family was consistent with the autosomal recessive inheritance of the disorder, and presence of this mutation correlated with the clinical manifestations. Such mutation was not detected in 47 normal healthy subjects. In the second patient, we found another missense mutation in one allele of the TSC gene, which results in alteration of arginine to glutamine at codon 955. In the third patient, no mutation causing amino acid substitution was found in the TSC gene. These results indicate that the R642C mutation in TSC is critically important for impairment of this cotransporter function and also suggest the necessity of further investigations in the genetic background of Gitelmans syndrome.

PREPARATION OF A MONOCLONAL ANTIBODY AGAINST MOUSE BRAIN NATRIURETIC PEPTIDE (BNP) AND TISSUE DISTRIBUTION OF BNP IN MICE

1. In order to explore the significance of brain natriuretic peptide (BNP), a cardiac hormone secreted from the ventricle, in mice, we prepared a monoclonal antibody against mouse BNP (mBNP) and established a specific radioimmunoassay (RIA) for mBNP.

ALTERED GENE EXPRESSION OF NATRIURETIC PEPTIDE RECEPTOR SUBTYPES IN THE KIDNEY OF STROKE‐PRONE SPONTANEOUSLY HYPERTENSIVE RATS

1. To elucidate the physiological and pathophysiological role of the natriuretic peptide system in the progression of hypertensive renal disease, we examined the gene expression of natriuretic peptide receptor subtypes, guanylate cyclase‐A (GC‐A), guanylate cyclase‐B (GC‐B) and clearance receptor (C receptor), in the kidney of stroke‐prone spontaneously hypertensive rats (SHRSP) at 8 and 20 weeks of age, and compared them with their gene expression in age‐matched Wistar‐Kyoto (WKY) rats.

Monoclonal antibody against brain natriuretic peptide and characterization of brain natriuretic peptide-transgenic mice

Objective Brain natriuretic peptide (BNP) is a ventricular hormone with natriuretic, diuretic and vasodilatory actions. Acute infusion of BNP reduces cardiac pre- and after-load in healthy and diseased subjects, but its long-term therapeutic usefulness remains unclear. Design We prepared a monoclonal antibody specific to mouse BNP, and characterized transgenic mice overexpressing BNP in the liver (BNP-Tg mice) as a model of its chronic overproduction. Methods Radioimmunoassay and neutralization experiments using the monoclonal antibody, KY-mBNP-I, were performed in BNP-Tg mice in conjunction with examinations of blood pressure (BP) and other markers for body fluid homeostasis. Results We developed highly sensitive radioimmunoassay to mouse BNP. In BNP-Tg mice, the plasma BNP concentration increased more than 100-fold, while ventricular BNP concentration did not alter, suggesting that ventricular BNP production was not down-regulated in BNP-Tg mice. The BNP concentration in the kidneys was 10-fold higher than nontransgenic (nonTg) littermates, accompanied with marked reduction in the atrial natriuretic peptide (ANP) concentration, that may be due to binding of circulating BNP to the natriuretic peptide receptors. BNP-Tg mice showed significantly low arterial BP, and a bolus intraperitoneal administration of KY-mBNP-I completely abolished enhanced cGMP excretion in the urine and significantly increased the systolic BP. Conclusion These results suggested that biological actions of BNP last and reduce cardiac overload in its long-term overproduction in the transgenic mouse model.