Tsuneyoshi Funai

Increased level of atrial natriuretic peptide messenger RNA in the hypothalamus and brainstem of spontaneously hypertensive rats.

Objective: The aim of this study was to investigate atrial natriuretic peptide (ANP) gene expression in the central nervous system (CNS) during hypertension. Methods: We measured and compared immunoreactive atrial natriuretic peptide (irANP) and ANP messenger RNA (mRNA) in the hypothalamus and brainstem of 17-week-old spontaneously hypertensive rats (SHR) with those of age-matched Wistar-Kyoto (WKY) rats using ribonuclease (RNase) protection assay for ANP mRNA and a specific radioimmunoassay for irANP. Results: RNase protection assay revealed that the concentrations of ANP mRNA in the hypothalamus and brainstem of SHR were higher than those of WKY rats. IrANP concentrations in the hypothalamus and brainstem of SHR were determined by a specific radioimmunoassay and found to be higher than those of WKY rats. Elevated mRNA levels in the hypothalamus and brainstem of SHR indicated that increased level of irANP in the CNS resulted from increased synthesis of ANP. Conclusion: We propose that increased synthesis of brain ANP in SHR may reflect a compensatory mechanism induced by hypertension.

Regulation by glucagon of serine: pyruvate/alanine: glyoxylate aminotransferase gene expression in cultured rat hepatocytes.

The effects of glucagon on serine: pyruvate/alanine: glyoxylate aminotransferase (SPT/AGT) gene expression were studied in primary cultured rat hepatocytes. When hepatocytes had been precultured for 16-18 h under serum- and hormone-free conditions, the addition of glucagon caused (after a lag period of about 2 h) a remarkable increase in the cellular level of SPT/AGT mRNA by 4 h in a time- and dose-dependent manner. The induced mRNA was that for mitochondrial SPT/AGT, as judged by ribonuclease protection analysis. A nuclear run-on assay revealed that activation of transcription is responsible for the increase in mitochondrial SPT/AGT mRNA and that the maximal rate of transcription occurs 1.5 h after glucagon addition. The effect of glucagon was mimicked by 8-bromo-cAMP and suppressed by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide, an inhibitor of cAMP-dependent protein kinase (protein kinase A), while both 12-O-tetradecanoylphorbol-13-acetate and A23187 were without effect in elevating the SPT/AGT mRNA level, suggesting that the cAMP/protein kinase A system is involved in the regulation of SPT/AGT gene expression. In hepatocytes precultured for 16-18 h under serum- and hormone-free conditions, the glucagon-induced transcription was severely inhibited by cycloheximide. When the preculture was for 2 h, on the other hand, the activation of transcription by glucagon was more rapid, and the inhibition by cycloheximide was less than that observed with cells precultured for 16-18 h, suggesting that a short-lived protein factor is involved in the hormonal regulation. The glucagon-induced expression of the SPT/AGT gene was also turned off by dexamethasone.

Peroxisomal and mitochondrial targeting of serine:pyruvate/alanine:glyoxylate aminotransferase in rat liver.

Serine: pyruvate/alanine:glyoxylate aminotransferase (SPT or SPT/AGT) of rat liver is a unique enzyme of dual subcellular localization, and exists in both mitochondria and peroxisomes. To characterize a peroxisomal targeting signal of rat liver SPT, a number of C-terminal mutants were constructed and their subcellular localization in transfected COS-1 cells was examined. Deletion of C-terminal NKL, and point mutation of K2 (the second Lys from the C-terminus), K4 and E15 caused accumulation of translated products in the cytoplasm. This suggests that the PTS of SPT is not identical to PTS1 (the C-terminal SKL motif) in that it is not restricted to the C-terminal tripeptide.In vitro synthesized precursor for mitochondrial SPT was highly sensitive to the proteinase K digestion, whereas peroxisomal SPT (SPTp) was fairly resistant to the protease. In in vitro import experiment with purified peroxisomes, however, STPp recovered in the peroxisomal fraction was very sensitive to the protease. These results suggest that the mitochondrial precursor is synthesized as an unfolded form and is translocated into the mitochondrial matrix, whereas SPTp is synthesized as a folded form and its conformation changes to an unfolded form just before translocation into peroxisomes.

INDUCTION BY PEROXISOME PROLIFERATORS AND TRIIODOTHYRONINE OF SERINE:PYRUVATE/ALANINE:GLYOXYLATE AMINOTRANSFERASE OF RAT LIVER

In rat liver, a single serine:pyruvate/alanine:glyoxylate aminotransferase (SPT or SPT/AGT) gene is transcribed from two transcription initiation sites. Transcription from the upstream site generates the mRNA encoding the precursor for mitochondrial SPT (pSPTm) and is markedly enhanced by the administration of glucagon or cAMP. In this report we show the increase in the downstream transcript, the peroxisomal SPT (SPTp) mRNA, caused by peroxisome proliferators and triiodothyronine (T3). In the case of T3, the pSPTm mRNA was also increased 72 h after a single administration of the hormone in addition to an earlier increase in SPTp mRNA.

A liver enzyme, serine:pyruvate/alanine:glyoxylate aminotransferase and its mutant in a primary hyperoxaluria type 1 case

In the liver of herbivorous animals and man, serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT) locates in peroxisomes, and plays an important role of removing glyoxylate by transamination to Gly. Primary hyperoxaluria type 1 (PH1), a congenital metabolic disease characterized by increased oxalate production and precipitation of calcium oxalate crystals in many tissues, is caused by a defect in SPT/AGT. In a PH1 case we are studying, there is a point mutation of T to C in exon 6 of the SPT/AGT gene, encoding a Ser to Pro substitution at residue 205. A remarkable feature of the SPT/AGT deficiency in the PH1 case was that the mutant SPT/AGT appeared to be actively synthesized in the patient’s liver, but the enzyme was very low with respect to not only the activity but also the protein detectable on Western blot analysis. This discrepancy was explained by the finding that the mutant enzyme is decomposed much faster than normal in transfected cells and in vitro,and the degradation in vitro with a reticulocyte lysate system is ATP-dependent. The mutant SPT/AGT was purified as a fusion with maltose-binding protein (MBP), followed by cleavage with factor Xa protease. The mutant enzyme thus purified did not show the enzyme activity under the assay conditions used. However, it was not possible to conclude that the mutant enzyme is inactive, because the mutant enzyme or its fusion with MBP aggregated considerably rapidly, and after the aggregation, even normal SPT/AGT did not exibit the enzyme activity. We then attempted to determine whether or not the mutation site (Ser-205) is in a neighborhood of the pyridoxal phosphate (PLP)-binding Lys. Comparison of the predicted secondary structures of SPT/AGT with those of three other aminotransferases suggested that Lys-209 is the PLP-binding residue. Upon BrCN cleavage of the NaBH4-reduced and pyridylethylated recombinant rat SPT/AGT, a 22–23 kd fragment was recovered as main product as expected, but most products with a molecular weight over 10 kd, including the 22–23 kd product, showed pairs of bands on SDS-PAGE. Amino acid sequences of the 22–23 kd pair were determined to be the same, except that Lys was released from only one of the pair on the 14th cycle corresponding to Lys-209, suggesting that the paired bands are derived from apo-and holoSPT/AGT. These results in turn suggested that Lys-209 is indeed the PLP-binding residue. Based on these data, we are inclined to think that mutant SPT/AGT in the PH1 case is not only susceptible to the ATP-dependent intracellular degradation but also enzymically inactive.

Natriuretic Peptide mRNA in SHR and WKY

Atrial natriuretic peptide (ANP) is a recently discovered hormone from the cardiac atria [1]. The potent diuretic, natriuretic and vasorelaxant activities of ANP suggested the involvement of this cardiac hormone in the regulation of blood pressure, body fluid and electrolytes. Originally, studies using radioimmunoassay (RIA) and blot hybridization technique revealed the presence of large quantities of immunoreactive ANP (IR-ANP) and ANP messenger RNA (ANP mRNA) in the atrium but not in the ventricle [2,3]. However, following studies indicated the synthesis of ANP in various extraatrial tissues including the ventricle [4]. Its biological actions directed much attention to the implication of ANP in hypertension. Changes of IR-ANP concentrations in plasma and tissue of hypertensive rats were investigated [5,6]. Elevated ANP mRNA levels in atria and ventricles of spontaneously hypertensive rats (SHR) were also reported [7]. Recently, Sudoh et al. isolated and determined brain natriuretic peptide (BNP) from porcine brain, which derived from a distinct gene from ANP and possesses diuretic, natriuretic and vasorelaxant activities [8]. This natriuretic peptide family, ANP and BNP, seems to construct a complex regulatory system in mammalian circulation. In this study, to elucidate the pathophysiological role of natriuretic peptide family in hypertension, we tried to determine IR-ANP, IR-BNP and ANP mRNA levels in SHR and Wistar Kyoto rats (WKY).