Dome formation and tubule morphogenesis by Xenopus kidney A6 cell cultures exposed to microgravity simulated with a 3D-clinostat and to hypergravity.

Confluent high-density cell cultures of A6 cells derived from adult male Xenopus kidney exhibit spontaneous dome-formation at 1 g. To determine whether this morphogenetic property is altered by gravity, we used a three-dimensional (3D) clinostat to subject the cells to simulated microgravity, and a centrifuge to subject them to hypergravity. We used the generation orbit control method as the new rotation control system of the 3D-clinostat, not the random method. The growth of A6 cells was significantly enhanced by hypergravity, but significantly reduced by simulated microgravity. Dome formation by A6 cells at high confluence was inhibited under simulated microgravity conditions, whereas hypergravity promoted dome formation and induced tubule morphogenesis, compared to the control at 1 g. These results indicated that changes in gravity influence the morphogenetic properties of A6 cells, such as dome formation and tubule morphogenesis. When dome formation by A6 cells at high confluence was induced spontaneously in the control 1 g culture, the gene expression of the HGF family of pleiotropic factors, such as HGF-like protein (HLP) and growth factor-Livertine (GF-l.ivertine), an epithelial serine protease of channel activating protease 1 (CAP1), and Na+, K+-adenosine triphosphatase (ATPase), increased. Simulated microgravity increased the gene expression of activin A and reduced the gene expression of HLP, GF-Livertine, CAP1, and Na+, K+-ATPase. Hypergravity, on the other hand, decreased the gene expression of activin A and increased the gene expression of HLP, GF-Livertine, CAP1, and Na+, K+-ATPase. These results suggest that the effects of gravitational changes on expression of the HGF family member gene, CAP1, and Na+, K+-ATPase gene may be important for the cell growth, tubule morphogenesis, and dome formation of A6 cells in altered

Histamine release from mast cells of EAE rats by Gi protein-dependent and IgE-dependent pathways.

We investigated both Gi protein-dependent and IgE-dependent pathways that control release of histamine by PMCs derived from EAE or Complete Freund's Adjuvant (CFA) immunized rats. The number and histamine content of MCs per rat were the same between normal and EAE rats. Activation of Gi pathway by substance P (SP), DSA, 48/80, and mastoparan resulted in a dose-dependent increase in release of histamine by PMCs in normal, EAE-, and CFA-immunized rats. In EAE and CFA rats, however, the induction was decreased by 10-20% compared to normal rats. The histamine release induced by MP was decreased at a concentration of 3 microM, but not at 10 microM in severe active EAE rats. Activation of the IgE pathway by MAM and concanavalin A (Con A) in the presence of phosphatidylserine led to dose-dependent histamine release in normal rats, and a 10-25% lower level of induction was observed in EAE rats. In CFA rats, the induction of histamine release was equivalent to normal rats. There was an increase in intracellular calcium stores following activation of both pathways in normal rats, whereas depletion of calcium stores by ryanodine reduced the level of induction by 48/80 and MP by 9-11% in normal rats. In EAE rats, 48/80, Con A, and MAM induced a smaller increase, but SP and MP induced larger or similar increases in calcium stores compared to normal rats. It was unlikely that the calcium stores of the PMCs from EAE rats were depleted, because MP stimulated calcium movement subsequent to the release of histamine. These results suggested that the Gi pathway may not be correlated to clinical manifestation of EAE, but cold be involved in the inflammatory process, and that the IgE pathway is better associated with clinical symptoms of EAE and may be more directly related to disease outcome.