Ryanodine: its possible mechanism of action in the caffeine-sensitive calcium store of smooth muscle

The caffeine-sensitive intracellular Ca store was characterized and the mechanism of action of ryanodine in the store was studied using K-depolarized guinea-pig taenia caecum. (1) After incubation of the preparation with CaCl₂ (Ca loading), caffeine was applied in Ca-deprived medium, to produce a transient contraction and to monitor the amount of the stored Ca. As duration of Ca deprivation was prolonged, the amplitude of the caffeine-induced contraction was decreased. When ryanodine was applied during Ca deprivation, the rate of the decrease was remarkably accelerated. (2) The rate of rise of the contraction induced by external Ca ((Ca)o) was slowed by preceding depletion of the stored Ca by caffeine, compared with that observed in the Ca loaded preparation. However, in the presence of ryanodine, even if stored Ca was depleted by caffeine, the rate of rise of the (Ca)o-induced contraction remained at a higher level. (3) These results suggest that ryanodine stimulates a leak of the stored Ca, and that the contraction induced by the transmembrane influxed Ca could be modulated by the amount of Ca in, or leakiness of, the caffeine-sensitive Ca store.     

Biological properties and gene expression associated with metastatic potential of human osteosarcoma

Lung metastasis has a great influence on the prognosis of patients with osteosarcoma. We previously established two high-metastatic sublines, M112 and M132, from the HuO9 human osteosarcoma cell line by in vivo selection. In this study, we newly isolated a high-metastatic subline, H3, and three low-metastatic sublines, L6, L12 and L13, from HuO9 by the dilution plating method. Three high-metastatic sublines produced more than 200 metastatic nodules in the lung, while three low-metastatic sublines produced no or few nodules after injection of 2 × 10⁶ cells into the tail vein of nude mice. There were significant differences in the motility and invasiveness between high- and low-metastatic sublines, whereas the growth rates in vitro and the tumorigenicity in vivo showed no correlation with their metastatic abilities. Early adherence to culture plates was significantly lower in two of three low-metastatic sublines, which occupied smaller surface areas on the culture plates than other sublines did. Comparison of the expression of 637 cancer-related genes by cDNA microarray revealed that seven genes were differentially expressed between high- and low-metastatic sublines. Among them, five genes (AXL, TGFA, COLL7A1, WNT5A, and MKK6) were associated with adherence, motility, and/or invasiveness. These results suggest that the differences in motility/invasiveness and adhesive abilities are key determinants of lung metastasis in osteosarcoma. This revised version was published online in July 2006 with corrections to the Cover Date.     

Establishment of human osteosarcoma cell lines with high metastatic potential to lungs and their utilities for therapeutic studies on metastatic osteosarcoma

Relevant animal models for metastasis of osteosarcoma is needed to understand the biology and to develop the treatment modality of metastasis of human osteosarcoma. Therefore, we screened six human osteosarcoma cell lines for metastatic ability in nude mice. The HuO9 cell line was identified as being metastatic to the lung after intravenous injection. We established two sublines, HuO9-M112 and HuO9-M132, with high metastatic potential to the lung from the parental HuO9 cells by in vivo selection. There were no differences between these two sublines and the parental cells in the growth rate in vitro and the tumorigenicity after subcutaneous injection in nude mice, however, mice injected with the metastatic sublines became moribund earlier than mice injected with the parental HuO9 cells did. Thus, adriamycin (ADR) and recombinant interleukin-12 (IL-12) were administered to mice injected with the HuO9-M112 subline to suppress experimental lung metastases. Production of lung colonies was significantly suppressed and the prognoses of mice were significantly improved by both ADR and IL-12 treatments. These results indicate that both ADR and IL-12 are effective agents against pulmonary metastatic osteosarcoma, and that these sublines are useful for studies on the biological behavior and treatment of pulmonary metastatic osteosarcoma. This revised version was published online in July 2006 with corrections to the Cover Date.     

Endothelin-1 induced contraction of rat aorta: contributions made by Ca2+ influx and activation of contractile apparatus associated with no change in cytoplasmic Ca2+ level

Summary The modes by which Endothelin-1 (ET) induces Ca²⁺-influx and the relative functional importance of the different sources of Ca²⁺ for ET-induced contraction were studied using fura 2-loaded and unloaded rat aortic strips. ET caused an increase in the cytosolic free Ca²⁺ level ([Ca²⁺]_i) followed by a tonic contraction in Ca²⁺-containing solution, and produced a transient elevation of [Ca²⁺]_i followed by a small sustained contraction in Ca²⁺-free medium. ET also stimulated ⁴⁵Ca influx into La²⁺-inaccessible fraction significantly. With the same change of [Ca²⁺]_i, ET caused a larger tension than that induced by high K. ET-induced contraction and [Ca²⁺]_i elevation were not significantly inhibited by 0.1–0.3 M nicardipine which nearly abolished the contraction and [Ca⁺]_i elevation produced by high K. During treatment of the strips with high K, addition of ET induced further increases in [Ca²⁺]_i and muscle tension, and vice versa. In Ca²⁺-free medium, ET-induced contraction was influenced neither by ryanodine-treatment nor by high K-treatment, although the former attenuated and the latter potentiated the [Ca²⁺]_i transient induced by ET. Further, the ET-induced sustained contraction under Ca²⁺-free conditions began to develop after the [Ca²⁺]_i level returned to the baseline. Thus, it seems that the Ca²⁺ released from the ryanodine-sensitive and -insensitive Ca²⁺ stores by ET may provide only a minor or indirect contribution, if any, to the tension development. ET might cause a contraction mainly by stimulating Ca²⁺-influx through Ca²⁺ channel(s) other than voltage-dependent Ca²⁺ channels in character, and by increasing the sensitivity of the contractile filaments to Ca²⁺ or activating them Ca²⁺-independently.Visiting from Zun Yi Medical College, China Send offprint requests to I. Takayanagi at the above address     

Lamivudine treatment in a patient with hepatitis B virus reactivation after allogenic peripheral bone marrow transplantation.

We report the case of a 52-year-old woman with hepatitis B virus (HBV) reactivation during treatment for chronic graft vs. host disease (GVHD) after peripheral bone marrow transplantation (PBSCT) to treat chronic myelocytic leukemia. She was given cyclosporine and prednisolone orally to treat chronic GVHD after PBSCT. Liver dysfunction first developed 25 months after transplantation with the appearance of hepatitis B s antigen (HBsAg), hepatitis B e antigen (HBeAg), and elevation of HBV-DNA up to 4.5 log copies/ml. Retrospective examination of her serum before PBSCT proved negative for HBsAg and HBeAg, and positive for anti-HBsAg, anti-HBeAg, anti-hepatitis B core antigen, and HBV-DNA (2.7 log copies/ml), showing that she was in a state of occult HBV infection. Nucleotide sequences of the HBV genome obtained from her serum showed no core promoter mutations at nt 1762 and 1764 and no pre-core mutation at nt 1896. Polymerase chain reaction-restriction fragment length polymorphism showed that she was infected with HBV genotype B. The administration of lamivudine, a nucleoside analog, improved her liver function and reduced HBV-DNA replication. We conclude that antiviral agents, such as lamivudine, are effective for treating hepatitis B reactivation during immunosuppressive treatment, such as for GVHD. The administration of a nucleoside analog before transplantation should also be considered in the light of HBV genotypes and mutations, even if HBsAg was negative and the viral load was low before transplantation.