FcεRI signaling in rat basophilic leukemia cells depends on phosphatidylinositol 3-kinase (PI3-kinase) and the small GTPase Rac. Here, we studied the functional relationship among PI3-kinase, its effector protein kinase B (PKB), and Rac using inhibitors of PI3-kinase and toxins inhibiting Rac. Wortmannin, an inhibitor of PI3-kinase, blocked FcεRI-mediated tyrosine phosphorylation of phospholipase Cγ, inositol phosphate formation, calcium mobilization, and secretion of hexosaminidase. Similarly, Clostridium difficile toxin B, which inactivates all Rho GTPases including Rho, Rac and Cdc42, and Clostridium sordellii lethal toxin, which inhibits Rac (possibly Cdc42) but not Rho, blocked these responses. Stimulation of the FcεRI receptor induced a rapid increase in the GTP-bound form of Rac. Whereas toxin B inhibited the Rac activation, PI3-kinase inhibitors (wortmannin and LY294002) had no effect on activation of Rac. In line with this, wortmannin had no effect on tyrosine phosphorylation of the guanine nucleotide exchange factor Vav. Wortmannin, toxin B, and lethal toxin inhibited phosphorylation of PKB on Ser473. Similarly, translocation of the pleckstrin homology domain of PKB tagged with the green fluorescent protein to the membrane, which was induced by activation of the FcεRI receptor, was blocked by inhibitors of PI3-kinase and Rac inactivation. Our results indicate that in rat basophilic leukemia cells Rac and PI3-kinase regulate PKB and suggest that Rac is functionally located upstream and/or parallel of PI3-kinase/PKB in FcεRI signaling.

This is the first demonstration of L. monoyctogenes mediated CFTR transgene transfer. The successful in vitro transfer suggests that L. monocytogenes might be a potential vector for cystic fibrosis gene therapy or alternative applications and deserves further investigation in vitro as well as in vivo.

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Isolated smooth muscle cells from the small intestine of new-born rats were prepared by enzymatic digestion. These cells re-aggregate after 1 day in culture to clusters. The re-aggregates show spontaneous rhythmical contractions at 37 degrees C with a frequency (13.1 +/- 0.8 min-1, n = 49), which is similar to that of the intact smooth muscle layer. The cholinergic agonist carbachol (5 x 10(-5) mol l-1) caused an increase in the frequency of the spontaneous contractions often ending in a permanent contraction. A similar effect was achieved with the thromboxane A2 (TXA2) agonist, U-46619 (10(-5) mol l-1). In contrast, both the TXA2 receptor blocker, Bay u3405 (5 x 10(-4) mol l-1), as well as the Ca2+ channel blocker, verapamil (5 x 10(-5) mol l-1), suppressed the spontaneous contractions. The observed contractility was insensitive against the neuronal blocker tetrodotoxin (10(-6) mol l-1). These analyses of video images were supported by the measurement of relative changes in the intracellular Ca2+ concentration with the Ca(2+)-sensitive dye, fura-2. Spontaneous contractions were paralleled by spikes in the intracellular Ca2+ concentration, which were abolished by Bay u3405, but stimulated by U-46619 or carbachol. In summary, these results obtained at re-aggregates of intestinal smooth muscle cells support the hypothesis of a role of TXA2 in the generation of spontaneous intestinal smooth muscle contractions in vitro.

The metallothionein (MT) content in hairless mouse skin was determined after topical application of various glucocorticoids or zinc oxide (ZnO). Dexamethasone (1%) in a cream base or zinc paste (20% zinc oxide) were applied twice daily, at a dose of 0.5 g, to the dorsal surface of the mouse. These experiments were conducted for 7 days. The MT content of the skin increased in a time-dependent manner after dexamethasone application with maximal values (7.1 +/- 0.29 pmol MT/mg wet weight) seen after 5 days, whereas zinc paste caused only slight increases after 3 days of treatment. The effect of hydrocortisone and triamcinolone-acetonide on the MT content of the skin was also studied. The physiological significance of these results is briefly discussed.