Kazunari Higa

Molecular cloning and expression of aquapolin 1 (AQP1) in dog kidney and erythroblasts

Abstract Complementary DNA of the water channel aquapolin 1 (AQP1) was cloned from dog kidney and erythroblasts. The cDNA amplified from mRNA in dog kidney was 816 bp, the same as that in bovines, but longer by 6 bp than that in humans, mice and rats. The 235-bp fragment cDNA amplified from the mRNA in dog erythroblasts, which was differentiated from peripheral blood, was completely identical to the corresponding sequence of cDNA from the dog kidney. Thus, mature red blood cells from dog may have AQP1 in their cell membranes. The amino acid sequence in dog AQP1 was 91–94% identical to that in the other species mentioned above. Dog AQP1 has six predicted transmembrane domains, two NPA motifs, one mercury-sensitive site and four consensus phosphorylation sites, the same as the other species. However, dog and bovine AQP1 have only one N-glycosylation site, while two glycosylation sites were found in human and rodent AQP1. Xenopus oocytes injected with the mRNA of the dog AQP1 exhibited high water permeability in a hyposmotic medium. Thus, dog AQP1 performs water transport the same as in the other species.

Cation transport and regulatory volume increase in red blood cells of northern fur seals (Callorhinus ursinus)

We investigated the membrane transport of Na and K ions in red blood cells (RBCs) of northern fur seal (Callorhinus ursinus) by measurement of unidirectional fluxes. Like red blood cells of other carnivores, those of northern fur seal contain high Na and low K concentrations, which result from the lack of Na–K ATPase activity on their membranes. In physiological conditions, activities of bumetanide-sensitive Na, K–Cl cotransport and amiloride-sensitive Na/H exchange were measured. K–Cl cotransport and Na–Cl cotransport were not detected. Hypertonicity activated only Na/H exchange. We further examined the ion transport systems for regulatory volume increase (RVI) in red blood cells. In the hyperosmotic condition, shrunken RBCs restored their original cell volume in Na medium but not in Na-free medium, and this restoration with Na medium was inhibited by amiloride. From these results, it is suggested that RVI in northern fur seal RBCs are performed by amiloride-sensitive Na/H exchanger but not Na, K–Cl cotransporter.

High aspartate accumulation and defect of glutamate/aspartate transport in low potassium dog red blood cells

The accumulation of Asp in low potassium (LK) dog red blood cells (RBCs) with defective Na-dependent (ND) glutamate (Glu)/aspartate (Asp) transport was examined. In RBCs with LK and defective ND-Glu/Asp transport (LK/GAT−), the amino acid influx defect appears to be specific to Glu/Asp, since influxes of the neutral amino acids alanine (Ala) and glutamine (Gln) were similar in both LK/GAT− RBCs and LK dog RBCs without the defect (LK/GAT+). Contrary to what might be expected with this transport defect, the Asp concentration in LK/GAT− RBCs was 10-fold higher than that in LK/GAT+ RBCs. Large, light RBCs separated by specific gravity were younger than small, dense RBCs in both LK/GAT− and LK/GAT+ RBCs. The youngest 10% of the cells had a 20% higher Asp concentration than the oldest 10% of the cells in both LK/GAT+ and LK/GAT− RBCs. The Glu and Gln concentrations decreased by 37%–47% as the cells aged, whereas Asp concentration decreased by 18%–19%. Therefore, the change in Asp metabolism during cell ageing might be smaller than the changes in the other amino acids. Thus, the Asp accumulation perhaps occurs at an early stage of cell development in LK/GAT− RBCs. The most likely cause of the Asp accumulation is a defect in Asp extrusion through ND-Glu/Asp transport.

Comparison of glutamate metabolism in low K (LK), high K and high glutathione (HK/HG) and high K and low glutathione (HK/LG) dog red blood cells

The reasons for the high accumulation of glutamate (Glu), aspartate (Asp) and glutamine (Gin) in high K and high glutathione (HK/HG) dog red blood cells (DRBCs) have been explained as due to enhanced Glu/Asp influxes. However, in our study, Glu/Asp influxes in high K and low glutathione (HK/LG) DRBCs were low, whereas their cellular Asp and Gin contents were high. In low K (LK) DRBCs, there were also other variant cells with high Asp accumulation, but extremely low Glu/Asp influxes. So, the high amino acid accumulation in DRBCs of these new variants might not be due to Glu/Asp influxes. To examine the high accumulation of these amino acids in these variant DRBCs, first, LK and HK/LG DRBCs were classified into two subgroups with their Na-dependent Glu/Asp influxes; one had clear Na-dependent Glu/Asp transport (GAT+), and the other failed to have any transport (GAT−). The influxes of both Glu and Asp in HK/HG DRBCs were the highest, and the order was HK/HG>LK/GAT+>HK/LG/GAT+>>LK/GAT−=HK/LG/GAT−. LK/GAT+ cells represented normal DRBCs. Glu/Asp influxes were only trace in both LK/GAT− and HK/LG/GAT− cells, but Glu and Asp concentration was high in HK/LG/GAT− cells whereas Asp concentration was high in LK/GAT− cells. In HK/HG cells, the conversion of Glu into Gin in whole cells was several fold higher than in the other cell groups due to the differing amount of the substrate of glutamine synthetase, Glu, but glutamine synthetase activity itself was not different among these cell groups. Furthermore, glutamine synthetase and glutaminase activities were not different among the cell groups. Therefore, these enzymes were not involved in the high amino acid accumulation.

Regulatory volume decrease in northern fur seal (Callorhinus ursinus) red blood cells

The cation transport and regulatory volume decrease (RVD) were investigated in the red blood cells (RBCs) of northern fur seals (Callorhinus ursinus). Extracellular Ca-dependent Na efflux was increased to threefold by hypotonicity. K–Cl cotransport activity was not detected by hypotonic medium, but measured only by nitrite or N-ethylmaleimide stimulation. RBCs were restored to their original volume after being swollen in hypoosmotic medium with Ca, though this recovery was inhibited by the addition of quinidine. Based on these results, Na/Ca exchange transporter played the major role in the regulatory volume decrease in the RBCs of northern fur seals.

Application of a two-phase liquid culture system for dog erythroid progenitor differentiation

This study assessed a technique for the isolation and differentiation of dog erythroid progenitors. Mononuclear cells from peripheral blood were cultured in a two-phase liquid culture system. The colony burst was confirmed after the first-phase culture by using a semi-solid culture containing methylcellulose. By immunofluorescent observation or reverse transcriptase-polymerase chain reaction analysis, the cells which proliferated during the second-phase culture were found to express glycophorin, globin, and glutamate/aspartate transporter, which are markers of dog erythrocytes. This system is beneficial for the analysis of dog erythrocytes as a reasonable amount of well-matured cells (0.5×106/ml) are obtainable.