Norifumi Nakamura

A review of mutagenesis studies of angiotensin II type 1 receptor, the three-dimensional receptor model in search of the agonist and antagonist binding site and the hypothesis of a receptor activation mechanism.

Objective To seek the mechanism whereby agonists, competitive antagonists and insurmountable antagonists affect the receptor function differently, by reviewing recent mutagenesis studies of angiotensin II type 1 receptor (AT1) in which the binding of the agonist and antagonists and receptor signaling were affected. AT1 receptor structure and ligand binding sites We built a model of seven transmembrane spanning domains of the AT1 receptors using bacteriorhodopsin as a template. The carboxy terminal of angiotensin II binds to Lys199 in transmembrane domain 5, whereas the guanidinium group of Arg2 binds to Asp281 in transmembrane domain 7. Results of studies using mutagenesis supporting proposed ligand-docking models are discussed. Hypothesis for the ligand-induced receptor signaling mechanism We submit a set of hypotheses for a mechanism whereby the ligand binding induces changes in the receptor conformation by the rotation of transmembrane helices as the initial event for the subsequent activation of a G protein. In this mechanism antagonists are not capable of rotating the helices but agonists are able to do so, which results in the formation of a hydrogen bond between Asp74 in transmembrane domain 2 and Tyr292 in transmembrane domain 7. This mechanism also provides plausible explanation for the activation of monoamine receptors. Competitive and insurmountable antagonists Competitive antagonists share the same binding sites with agonists, but insurmountable antagonists do not, and binding of the latter does not preclude agonist binding, for example, to Asp281. Conclusion This hypothesis of the intrareceptor signaling mechanism and the receptor model indicate that some amino acid residues essential for the signaling play their roles in the intrareceptor activation mechanism, whereas others participate directly in ligand binding.

Angiotensin II can regulate gene expression by the AP-1 binding sequence via a protein kinase C-dependent pathway

An expression vector containing three copies of the AP-1 binding element (TRE) upstream of a thymidine kinase promotor which controlled the expression of the chloramphenicol acetyl transferase (CAT) gene was transiently transfected into vascular smooth muscle (VSM) cells and a human hepatocarcinoma cell line, Hep G2. Twelve hours of angiotensin (Ang) II exposure stimulated significantly CAT expression by 3.4 fold and 2.7 fold in Hep G2 and VSM cells, respectively. AngII had no effect on CAT expression of a control vector. This AngII-induced stimulation was attenuated significantly by an AngII receptor antagonist, Sar1 Ile8 AngII, and abolished completely by a PKC inhibitor, staurosporine. Our data suggest that the TRE plays a crucial role in AngII-induced gene expression that is mediated by PKC. We concluded that TRE is one of the AngII-responsive elements.

Distinct nuclear proteins competing for an overlapping sequence of cyclic adenosine monophosphate and negative regulatory elements regulate tissue-specific mouse renin gene expression.

The mouse renin locus (Ren-1d) exhibits specific patterns of tissue expression. It is expressed in kidney but not submandibular gland (SMG). This locus contains a negative regulatory element (NRE) and a cAMP responsive element (CRE) that share an overlapping sequence. In the kidney, CRE binding proteins (CREB) and NRE binding proteins (NREB) compete for binding to this sequence, with the CREB having a greater affinity. In the SMG, CREB is inactivated by an inhibitory protein, permitting NREB to bind to the sequence, thus inhibiting Ren-1d expression. We hypothesize that the competition between NREB and CREB for this sequence governs tissue-specific expression of mouse renin. We speculate that this may be a general paradigm that determines tissue-specific gene expression.

Prostaglandin E1 transported into cells blocks the apoptotic signals induced by nerve growth factor deprivation.

Abstract: Neuronal apoptosis in rat pheochromocytoma PC12 cells, which was confirmed by TUNEL (terminal transferase‐mediated dUTP‐biotin nick end‐labeling) staining and detection of chromatin condensation, appeared within 8 h after nerve growth factor (NGF) deprivation. Prostaglandin (PG) E1 (10−7‐10−6M) reduced the incidence of apoptotic cell death in PC12 cells. The genes encoding PG transporter specific to prostaglandins such as PGE2 or PGF2α were expressed in the cell lines as shown by RT‐PCR. Bromcresol green, an inhibitor of PG transporter, reversed the antiapoptotic effect of PGE1. Moreover, treatment of PC12 cells with an antisense oligonucleotide corresponding to PG transporter cDNA also blocked the inhibitory effects of PGE1 on apoptotic cell death. In addition, PGE1 counteracted the increased activities of stress‐activated protein kinase/c‐Jun N‐terminal kinase within 1–2 h after NGF deprivation in PC12 cells. These results indicated that the antiapoptotic effect of PGE1 in NGF‐deprived PC12 cells was achieved by inhibitory signals following uptake into neurons through the PG transporter.

Angiotensin converting enzyme (ACE) in the kidney: contribution to blood pressure regulation and possible role of brush-border ACE.

With regard to the concept of the local action of the renin angiotensin system (RAS) involved in blood pressure regulation, the presence of angiotensin converting enzyme (ACE) in a variety of organs suggests that locally produced angiotensin II (ANG II) shares, at least to some extent, the actions of this peptide on respective target organs of ANG II. However, renal ACE is less well understood for its relationship between blood pressure and enzyme activity. In our present studies, with a single oral administration of enalapril to spontaneously hypertensive rats, the inhibition of renal cortical and aortic ACE, but not plasma ACE, coincided with a reduction in blood pressure. Development of high blood pressure in stroke-prone, spontaneously hypertensive rats (SHRSP) from 7 to 22 weeks of age was accompanied by an increase in ACE activity in the renal cortex. Aortic and pulmonary ACE also tended to increase with age, but was less prominent. Isolated brush-border membranes contained abundant ACE, both in Wistar-Kyoto rats and SHRSP, and the levels of ACE in renal cortical homogenates closely correlated to the levels of brush-border ACE. Thus, changes in renal cortical ACE activity in response to the ACE inhibition and in cases of SHRSP in relation to aging are apparently associated with changes in blood pressure. It is likely that renal cortical ACE activity reflects the enzyme activity in the brush borders. Thus, brush-border ACE should probably be taken into account when discussing possible roles of renal ACE.

Pharmacologic profiles of GA0113, a novel quinoline derivative angiotensin II AT1-receptor antagonist.

GA0113 is a newly developed angiotensin II (Ang II) AT1-receptor antagonist having a quinoline moiety. This study was undertaken to clarify the pharmacologic profile of GA0113. In vitro profiles of GA0113 for Ang II receptors were examined in a receptor-binding assay and an Ang II-induced vasoconstriction study. Antihypertensive effects after single or repeated oral administrations were examined in conscious renal hypertensive (RH) or spontaneous hypertensive (SH) rats. Blood pressure (BP) and heart rate were measured by the tail-cuff method. GA0113 interacted with AT1 receptors in a competitive manner, but showed an insurmountable antagonistic action in Ang II-induced vasoconstriction. In RH rats, GA0113 (0.01-1 mg/kg) reduced BP with ED25 values of 0.015 mg/kg, and required 0.1 mg/kg for 24-h BP control. Repeated administration of GA0113 in SH rats (0.03-0.1 mg/kg) showed moderate onset and gradually potentiated reduction of BP, which reached a plateau after day 4 of treatment without alteration in heart rate. There was no tolerance of the hypotensive action or rebound phenomenon after cessation of the treatment. In pharmacokinetic studies, GA0113 shows excellent oral bioavailability (94%) and a long circulating half-life (12 h) in rats. These findings indicate that GA013 may serve as a highly potent and effective antihypertensive agent in humans. GA0113, with its unique chemical structure and pharmacologic and pharmacokinetic profiles may provide new possibilities in hypertension therapy.

Role of molecular biology in hypertension research. State of the Art lecture.

In this article we will examine the potential impact of molecular biology on hypertension research. We will review the available molecular techniques, which include gene cloning, transient and stable expressions, as well as the use of transgenic animals. To facilitate our discussion, we will focus primarily on research of the renin gene. Renin provides a useful model that illustrates the power of biotechnology in providing detailed structural and biochemical information on a complex protein that exists in low quantities in vivo. Studies of its messenger RNA and gene expression have resulted in an improved understanding of the biology of the renin system and in generating new hypotheses. These approaches can be generalized to studies of other vasoactive hormones, contractile protein, and other gene products related to cardiovascular regulation. To elucidate the role of a specific gene in genetic hypertension, we will discuss the use of genetic markers in cosegregation or linkage analysis. Finally, we will examine the potential of transgenic animals in the study of regulation of gene expression in the whole animal and the contribution of selective genes to hypertension. We believe that molecular biology complements the biochemical and physiological approaches and provides new opportunities for furthering our concept of hypertension mechanisms.

Glycosylation influences intracellular transit time and secretion rate of human prorenin in transfected cells.

The mouse pituitary tumour (AtT-20) cell transfected with the human renin gene has been shown to be a useful model system to study human renin biosynthesis. To investigate the influence of glycosylation on secretion of human renin from these cells, we performed pulse labelling experiments on transfected cells in the presence or absence of tunicamycin, a potent inhibitor of n-linked glycosylation. Intracellular and secreted renins were characterized by immunoprecipitation, sodium dodecyl sulphate (SDS) gel electrophoresis and fluorography. Our data showed that blockade of n-linked glycosylation reduced the intracellular transit time and increased the rate of prorenin secretion from transfected cells. We conclude that the carbohydrate moiety influences the kinetics of human renin secretion. This result provides one possible explanation for the observation that the secretion of glycosylated human renin is considerably slower than that of the unglycosylated mouse renin-2.

Renal metabolism in mice of exogenously administered 125I labelled renin.

125I labelled mouse submaxillary renin was administered intravenously to anaesthetized male Institute of Cancer Research, USA (ICR) mice in an attempt to determine the organ-related degradation of circulating renin. A specific antirenin antiserum was used for identification. The disappearance rate of labelled renin from plasma was estimated on the basis of a two-compartmental model. The mean half-time of clearance of labelled renin from plasma in intact control and 70% hepatectomized mice was 13.3 +/- 0.8 and 12.0 +/- 0.9 min respectively; these values were not significantly different. The mean half-time of clearance of labelled renin from plasma in nephrectomized mice was 40.0 +/- 2.4 min; this was significantly longer than intact controls and 70% hepatectomized mice. Fifty per cent of the administered renin accumulated in the kidney and 9% in the liver. A small amount of 125I labelled renin was identified in urine, and a large amount of 125I liberated from labelled renin was detected. Since the high performance liquid chromatography profiles showed that plasma renin rapidly moves into the kidney, clearance of circulating renin may take place mainly in this organ.

Effects of Long-term Treatment with the Calcium Antagonist Ae0047 on Cerebrovascular Autoregulation and Hypertrophy in Spontaneously Hypertensive Rats

Chronic hypertension is associated with structural and functional changes in the cerebrovascular bed, which influence cerebral circulation and its autoregulation. We examined whether or not long-term treatment of spontaneously hypertensive rats (SHRs) with the new calcium antagonist AE0047 would reverse structural changes in the cerebral vasculature and normalize the elevated lower blood pressure (BP) limit of the cerebral blood flow (CBF) autoregulation. Treating 6-month-old SHRs with a diet containing either 0.013 or 0.04% AE0047 for 8 weeks reduced BP and, at the higher dose, maintained BP at a level similar to that of age-matched Wistar-Kyoto (WKY) rats. At the end of the treatment period, CBF was measured by using the hydrogen-clearance method, and the lower limit of the autoregulation curve was estimated by repeated CBF measurement with stepwise reduction of BP through bleeding. This limit was significantly higher in untreated SHRs than in WKY rats (111 +/- 8 vs. 60 +/- 8 mm Hg). AE0047 caused a significant and dose-dependent shift in the elevated lower BP limit, which decreased to 83 +/- 9 and 75 +/- 6 mm Hg at the low and high dose, respectively. In perfusion-fixed proximal, intermediate, and distal portions of the middle cerebral artery, media thickness/external diameter (M/ED) ratios were significantly greater in untreated SHRs than in WKY rats. In AE0047-treated animals, M/ED ratios in all portions tended to be reduced halfway between those for untreated SHRs and those for WKY rats, but with no statistical significance. These results suggest that long-term treatment of patients with hypertension with AE0047 will normalize the autoregulatory threshold while preserving CBF and thereby improve tolerance to BP reduction, but the potential to ameliorate structural alterations may be small.