Yoshikazu Kambayashi

Multiple Growth Factors Modulate mRNA Expression of Angiotensin II Type-2 Receptor in R3T3 Cells

Previous studies showed that angiotensin II type-2 receptor (AT2) sites were increased when R3T3 cells were growth arrested and decreased when they were stimulated with fibroblast growth factor or serum. We examined the effects of several other growth factors on the expression of AT2 mRNA to clarify the relation between the AT2 receptor and growth factors. R3T3 cells were cultured in the medium containing 10% FCS until they were confluent and then serum was removed. AT2 mRNA was increased after serum was depleted, and the expression level reached a plateau after 2 days of serum depletion. The presence of serum (10%), fibroblast growth factor (10 ng/mL), or lysophosphatidic acid (1 mumol/L) reduced the AT2 mRNA expression. Phorbol ester (1 to 100 nmol/L) also suppressed the AT2 mRNA expression in a dose-dependent manner. Interleukin-1 beta (1 ng/mL) enhanced the AT2 mRNA expression 1.6-fold and the AT2 receptor number 1.4-fold. Insulin (100 nmol/L) enhanced AT2 mRNA expression 1.4-fold and the AT2 receptor number 1.6-fold. These results suggest that AT2 mRNA expression is modulated by multiple growth factors in both positive and negative directions. The presence of potential cis DNA elements that respond to interleukin-1 beta (CCAAT enhancer binding protein site), insulin [insulin response sequence of phospho(enol)pyruvate carboxykinase gene], and phorbol ester (AP-1 site) in the promoter region of the mouse AT2 gene suggests that the effects of these growth factors and phorbol ester may be mediated via these cis DNA elements.

ANGIOTENSIN RECEPTORS: MOLECULAR BIOLOGY AND SIGNALLING

1. The active peptide hormone angiotensin II (AngII) is formed from its prohormone angiotensinogen by way of inactive angiotensin I. The highly specific protease, renin, responsible for the initiation of this system was elusive and considered unstable. We isolated it in a pure and stable form from the kidney of the pig, human, rat, and land submandibular glands of the mouse. It was shown that there is only one type of renin with highly stringent substrate specificity, except certain strains of the mouse which have two gene products.

Cloning of the cDNA and the genomic DNA of the mouse angiotensin II type 2 receptor

Of the two major isoforms of the angiotensin II receptors, type 1 (AT1) and type 2 (AT2), little is known about the structure and features of AT2. We cloned a mouse AT2 cDNA from a mouse fetus cDNA library and an AT2 genomic DNA from a 129SV mouse genomic DNA library. The amino acid sequence of the mouse AT2 (363 residues) deduced from a mouse cDNA clone showed seven membrane-spanning domains. Amino acid identity of the mouse AT2 with mouse AT1 is 37%, and 98% with rat AT2. The genomic DNA (4.4 kb) contained three exons and two introns and the entire coding region was contained in the third exon.

Molecular cloning and expression of angiotensin II type 2 receptor gene.

Angiotensin II (Ang II) has been known as one of potent vasoactive peptides which also regulates fluid homeostasis, blood pressure, drinking behavior, aldosterone release, peripheral norepinephrine release, cell proliferation and hypertrophy (1–3). These diversity of specific physiological effects of Ang II has suggested possible presence of subtypes of Ang II receptor. While the recognition of reducing agent-sensitive and -insensitive Ang II receptors had suggested the possibility of multiple subtypes (4), the question remained unresolved until the development of new peptide and non-peptidic antagonists of the Ang II receptor (5–7). These compounds turned out to be isoform specific and powerful tools for the characterization and isolation of the Ang II receptors and receptor genes.

Atrial Natriuretic Factors and Fragments

Publisher Summary Atrial natriuretic factor (ANF) or atrial natriuretic peptide (ANP) consists of mixtures of peptides of various lengths. Studies have shown that the circulating form of ANF in plasma consists of a 28-amino acid residue ANF (ANP), whereas the rat and human atrium largely stores pro-ANF (γ-ANP), containing 126 amino acid residues, formed from prepro-ANF. The pro-ANF converts to the 28-amino acid active peptide by a co-secretional mechanism. Analysis by high-performance liquid chromatography (HPLC) revealed that ANF is stored in the brain tissues in active forms consisting of 24 and 25 residues. ANF in urine increases with increased salt feeding, reflecting its well-recognized role in natriuresis. However, it has been found that urinary ANF contains 32 amino acid residues. Purification studies revealed that two additional forms of ANF exist, which are products of different genes. Therefore, three different genes have been found for ANF families, and they are termed ANP (ANF), BNP, and CNP. BNP was originally isolated from porcine brain but was subsequently shown to exist largely in the atria and ventricles of the heart, whereas CNP seems to be localized in the brain.