Shichiro Maruyama

Regulation of Na+,K+-ATPase in submandibular glands of hypophysectomized male mice by steroid and thyroid hormones.

The effects of thyroid hormone, androgen, glucocorticoid, and mineralocorticoid on Na+,K+-ATPase activity and on levels of its alpha-subunit protein (alpha 1 isoform) in mouse submandibular gland (SMG) were studied by enzyme assay for ouabain-sensitive ATP hydrolysis, by quantitative densitometric scanning of Western blots, and by immunohistochemistry. To define the specific regulatory effects of various pituitary-dependent hormones on expression of Na+,K+-ATPase in the SMG, we treated hypophysectomized (hypox) male mice with triiodo-L-thyronine (T3), 5 alpha-dihydrotestosterone (DHT), dexamethasone (Dex), and aldosterone (Ald), injected singly or in combination. Na+,K+-ATPase was confined to the duct system of the SMG. In intact mice there was a gender difference in SMG Na+,K+-ATPase, with levels of the enzymes activity and of its alpha 1-subunit being less in the glands of males. In males, hypophysectomy caused a rise in levels of Na+,K+-ATPase activity and in levels of the alpha 1-subunit protein of this enzyme, and in intensity of immunocytochemical staining for this subunit but there were no such changes in the SMG of hypox females. Changes caused by hormonal replacement to hypox males in Na+,K-ATPase activity, levels of its alpha 1-subunit, or the intensity of immunocytochemical staining for this subunit were complex. Ald had no effect. T3 or dexamethasone, given alone, induced Na+,K+-ATPase activity above control values (hypox males) and increased levels of its alpha 1-subunit protein and immunohistochemical staining for this subunit. By contrast, DHT did not cause a decline in any of these parameters. However, when treatment with T3 was combined with administration of Dex or DHT, enzymatic activity of Na+,K+-ATPase decreased but levels of the alpha 1-subunit protein and immunohistochemical staining for this subunit increased. Therefore, inductions of the alpha 1-subunit of this enzyme are not always correlated with increases in levels of activity of Na+,K+-ATPase, and we propose that both enzymatic and immunochemical analyses are essential for evaluation of hormonal regulation of Na+,K+-ATPase in salivary gland and in other tissues.

In vivo modulation of proliferating cell nuclear antigen in growth plate chondrocytes from normal, hypophysectomized, growth hormone-treated hypophysectomized rats: a comparative immunohistochemical study with image analysis.

Growth hormone (GH) regulates the proliferation and maturation of chondrocytes in the epiphyseal growth plate, in which a temporal dimension is superimposed on the septal organization of the tissue. In this study we investigated the in vivo effects of hypophysectomy (Hypox) and injection of GH into Hypox animals (Hypox + GH) on the proliferative activity of the growth plate chondrocytes. We assessed the immunohistochemical expression of proliferating cell nuclear antigen (PCNA) in paraffin-embedded tissues, using monoclonal antibody PC 10 against PCNA combined with immunogold-silver staining. We subjected the immunostained sections to computer-based image analysis by ACAS 570 interactive laser cytometry employing a conventional microscopic light source. Hypox was carried out on 20 rats at 8 weeks of age, half of which received a hypodermic injection of human GH at a dose of 1 IU/kg twice a day for 1 week after the operation. Another group of five rats of the same age were used as normal controls. In normal rats, a distinct PCNA immunoreaction was observed in the proliferative zone, whereas a remarkable diminution of the number of immunoreactive cells in this zone was apparent in Hypox animals. On the other hand, marked hyperplasia of PCNA-positive cells was seen in the proliferative zone of the Hypox + GH rat growth plate. The immunoreactive cells of this zone in Hypox + GH animals exhibited increased nuclear size and staining intensity of PCNA compared with those in normal and Hypox rats. These findings were further confirmed by computer-based image analysis of the specimens in terms of cell integrated value, area, perimeter, and shape factor. These different patterns of PCNA expression may imply that GH specifically promotes the proliferation phase of the chondrocytes in the proliferative zone. The data also suggest that GH influences not only cell replication activity but also cell kinetics of chondrocytes in the growth plate during their lifespan.

[Effects of inhibitors of some mediators on the paw-edema induced by acetyl glyceryl ether phosphorylcholine in rats].

The characterization of acetyl glyceryl ether phosphorylcholine (AGEPC)-induced paw edema in rats was explored. Edema formation was maximum at 45 min after the injection of AGEPC. The dose for maximal response was 1 microgram/site, while edema was suppressed at higher doses. The systemic administration (s.c.) of cyproheptadine (CH, 10 mg/kg) did not inhibit this edema differently from serotonin-induced edema, while the local treatment of CH suppressed AGEPC-induced edema in a dose-dependent manner. The combination of local treatments with pyrilamine and methysergide also suppressed this edema. The s.c. injection of indomethacin (IM, 10 mg/kg) did not inhibit AGEPC edema differently from carrageenin-induced edema, while locally given IM suppressed it partially. The local treatment with caffeic acid and esculetin, lipoxygenase inhibitors, or with FPL 55712, an antagonist of SRS-A, suppressed AGEPC edema slightly. Various combinations of local treatment with IM and lipoxygenase inhibitors gave synergistic suppression of this edema. Dexamethasone strongly suppressed AGEPC edema when given both systemically and locally 3 hr before the injection of AGEPC. However, phospholipase A2 (PLA2) inhibitors, i.e. mepacrine, p-bromophenacyl bromide, chlorpromazine and tetracaine, did not show the same effect as dexamethasone. These results suggest that the edema formation induced by AGEPC may involve not only the combined action of histamine and serotonin but also the synergistic action of cyclooxygenase and lipoxygenase products. Dexamethasone may inhibit this edema by mechanisms other than the inhibitory action of PLA2.