Koh-ichi Enomoto

Spontaneous calcium oscillations and mechanically and chemically induced calcium responses in mammary epithelial cells

Changes of intracellular calcium activity (Cai2+) in mouse mammary epithelial cells in primary culture (normal) and in an established cell line (MMT060562, cancerous) were investigated by microfluorometry and image analysis of fura-2 fluorescence. In both types of cells, some populations exhibited occasional Cai2+oscillations with a period of 50–160 s. Slight mechanical stimulation of a cell with a fine glass pipette induced a Cai2+increase, which spread from the stimulated cell to the surrounding cells with a speed of 7–12 μm/s. ATP (>1 μmol/l) and ADP, but not AMP induced a Cai2+increase in both cell types. Bradykinin was highly effective (> 10 nmol/l) only in the cancerous mammary epithelial cells. In Ca2+-free solution, all these Cai2+responses remained unchanged at the first application, and decreased abruptly at the second trial. La3+ (>0.5 mmol/l) suppressed the response to ATP but not the response to bradykinin. Addition of extracellular Mn2+ rapidly quenched the fura-2 fluorescence in the cell even in a non-stimulated state. Influx of Mn2+ did not increase during Cai2+responses. These results indicate that the sources of Cai2+responses in mammary epithelial cells are intracellular stores, which exchange Ca2+ with the extracellular medium.

Molecular cloning and heterologous expression of an α-adrenergic-like octopamine receptor from the silkworm Bombyx mori

A cDNA encoding an octopamine (OA) receptor (BmOAR1) was isolated from the nerve tissue of silkworm (Bombyx mori) larvae. Comparison of amino acid sequences showed that BmOAR1 is highly identical to OA receptors isolated from Periplaneta americana (Pa oa1), Apis mellifera (AmOA1), and Drosophila melanogaster (OAMB or DmOA1A). BmOAR1 was stably expressed in HEK‐293 cells. OA above 1 µm led to an increase in intracellular cyclic AMP concentration ([cAMP]i). The synthetic OA‐receptor agonist demethylchlordimeform also elevated [cAMP]i to the same maximal level (≈ 5‐fold over the basal level) as that induced by OA. However, other biogenic amines, tyramine and dopamine, and chlordimeform were without effects. The [cAMP]i level raised by OA was lowered by antagonists; the rank order of antagonist activity was chlorpromazine > mianserin = yohimbine. Cyproheptadine and metoclopramide had little effect. OA above 100 nm induced a transient or sustained increase in intracellular Ca2+ concentration ([Ca2+]i), depending on the concentration of OA. Sequence homology and functional analysis data indicate that BmOAR1 is an α‐adrenergic‐like OA receptor of B. mori.

Cloning and embryonic expression patterns of the zebrafish Runt domain genes, runxa and runxb.

We isolated zebrafish homologues of the Runt-related transcription factor gene family (Runx family), runxa and runxb, and analyzed their developmental expression patterns. The deduced amino acid sequence of Runxa was highly homologous to that of AML1 (also called CBFA2, PEBP2alphaB or Runx1), a critical regulator of mammalian hematopoiesis expressed in cells of the hematopoietic lineage as well as other tissues. During zebrafish development, the runxa gene was not expressed in hematopoietic tissues but in the olfactory placodes and cells attached to the otic vesicles. We identified three kinds of runxb transcripts, which encoded two types of proteins with different N-terminal regions. The Runxb proteins were highly similar to AML2 (CBFA3, PEBP2alphaC or Runx3). The expression sites of the shared region of runxb mRNAs during development were the trigeminal ganglions, dorsal neurons of the neural tube and the lateral line primordia. These findings show that expression patterns of the zebrafish Runx genes are distinct from that of the mammalian genes.

Expression Cloning and Signal Transduction Pathway of P2U Receptor in Mammary Tumor Cells

Extracellularly applied ATP, UTP and UDP induce a transient increase in the intracellular Ca2+ concentration of mammary cells via a P2U receptor. The P2U receptor in the mammary tumor cell line MMT060562 was cloned and expressed in the human leukemia cell line K-562. The deduced amino acid sequence of the mammary tumor cell P2U receptor was 98% homologous with that of mouse NG108-15 cells. It was a member of the superfamily of GTP-binding-protein-coupled receptors. ATP and UTP induced the increase in the intracellular concentrations of Ca2+ and inositol-1,4,5-trisphosphate in both mammary tumor cells and P2U-receptor-expressed K562 cells. Dose-response curves on the production of inositol-1,4,5-trisphosphate and Ca2+ by ATP and UTP were consistently similar. Injection of GTP enhanced the ATP-induced outward current and injection of GTP gamma S induced a repetitive outward current. Both pertussis and cholera toxins did not affect ATP-induced calcium increase. It was suggested that the P2U receptor coupled with pertussis- and cholera-toxin-insensitive GTP-binding proteins and activated phosphoinositide turnover.

Effects of toki-shakuyaku-san (Tsumura TJ-23) on electrical activity in neuroblastoma cells and frog neuromuscular junctions

Toki-shakuyaku-san (Tsumura TJ-23) is a Chinese medicine which has been used for the treatment of gynecological symptoms in aged women. There are several reports on the usefulness of this drug in the treatment of cognitive disorders. We studied the effects of toki-shakuyaku-san on electrical activity in NG108-15 cells, a cell line of differentiated neuroblastoma x glioma hybrid cells, and on frog neuromuscular transmission. In the hybrid cells, an extract of toki-shakuyaku-san slightly depolarized the membrane potential, and strongly decreased the peak heights of the Na+ and Ca2+ current components of the action potential. The order of potency for NG108-15 cells of the 5 ingredients in toki-shakuyaku-san was soujyutsu >> shakuyaku, takusha, toki, senkyu. In voltage-clamped NG108-15 cells, toki-shakuyaku-san and soujyutsu decreased the Na+, K+, and Ca2+ current components. Toki-shakuyaku-san and soujyutsu also induced an increase in the intracellular calcium concentration. However, toki-shakuyaku-san did not affect neuromuscular transmission in the frog sartorius muscle. The results suggest that the effects of toki-shakuyaku-san on neurons are multiple, and tissue- and species-specific, and its effect derives mainly from soujyutsu.

Thioredoxin Gene Expression in Rat Knee Articular Cartilage After Full-Thickness Injury

To study the expression of the antioxidative protein thioredoxin (TRX) in intact and injured articular cartilage, we examined the presence of trxmRNA in rat knee joints by in situ hybridization. Our results showed that in the intact knee, most cells, including articular cartilage chondrocytes, expressed trx mRNA. We examined joints at 1, 7, 14, and 28 days after the infliction of full-thickness cartilage injuries on distal femoral condyles. At 1 day after injury, no significant changes were observed in the wound or in trx expression pattern. However, at 7 to 28 days after injury, the wound became filled with repair tissue. Also, trx expression was detected in differentiating mesenchymal cells in the deeper zones of the wound but not in fibroblast-like cells in the upper part of the repair tissue, toward the joint cavity. This lack of TRX expression in the fibroblast-like cells may underlie the susceptibility of the repair tissue fibrocartilage to oxidative stress.