Germ plasm is found in germ-line cells of Xenopus and thought to include the determinant of primordial germ cells (PGCs). As mitochondria is abundant in germ plasm, vital staining of mitochondria was used to analyze the movement and function of germ plasm; however, its application was limited in early cleavage embryos. We made transgenic Xenopus, harboring enhanced green fluorescent protein (EGFP) fused to the mitochondria transport signal (Dria-line). Germ plasm with EGFP-labeled mitochondria was clearly distinguishable from the other cytoplasm, and retained mostly during one generation of germ-line cells in Dria-line females. Using the Dria-line, we show that germ plasm is reorganized from near the cell membrane to the perinuclear space at St. 9, dependent on the microtubule system.

In isolated rat pancreatic beta-cells, hypotonic stimulation elicited an increase in cytosolic Ca(2+) concentration ([Ca(2+)](c)) at 2.8 mM glucose. The hypotonically induced [Ca(2+)](c) elevation was significantly suppressed by nicardipine, a voltage-dependent Ca(2+) channel blocker, and by Gd(3+), amiloride, 2-aminoethoxydiphenylborate, and ruthenium red, all cation channel blockers. In contrast, the [Ca(2+)](c) elevation was not inhibited by suramin, a P(2) purinoceptor antagonist. Whole cell patch-clamp analyses showed that hypotonic stimulation induced membrane depolarization of beta-cells and produced outwardly rectifying cation currents; Gd(3+) inhibited both responses. Hypotonic stimulation also increased insulin secretion from isolated rat islets, and Gd(3+) significantly suppressed this secretion. Together, these results suggest that osmotic cell swelling activates cation channels in rat pancreatic beta-cells, thereby causing membrane depolarization and subsequent activation of voltage-dependent Ca(2+) channels and thus elevating insulin secretion.

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The effects of green tea catechins on glucose-stimulated insulin secretion (GSIS) were investigated in the β-cell line INS-1D. Epigallocatechin gallate (EGCG) at 10 µM or gallocatechin gallate (GCG) at 30 µM caused significant inhibitory effects on GSIS, and each of these at 100 µM almost abolished it. In contrast, epicatechin (EC) or catechin (CA) had no effect on GSIS at concentrations up to 100 µM. We thus investigated the structure-activity relationship by using epigallocatechin (EGC) and gallocatechin (GC) containing a trihydroxyl group in the B-ring, and epicatechin gallate (ECG) and catechin gallate (CG) containing the gallate moiety. EGC, GC, and ECG caused an inhibition of GSIS, although significant effects were obtained only at 100 µM. At this concentration, EGC almost abolished GSIS, whereas GC and ECG partially inhibited it. In contrast, CG did not affect GSIS at concentrations up to 100 µM. EGCG also abolished the insulin secretion induced by tolbutamide, an ATP-sensitive K channel blocker, and partially inhibited that induced by 30 mM K . Moreover, EGCG, but not EC, inhibited the oscillation of intracellular Ca 2 concentration induced by 11.1 mM glucose. These results suggest that some catechins at supraphysiological concentrations have inhibitory effects on GSIS, the potency of which depends on their structure; the order of potency was EGCG>GCG>EGC>GC≈ECG. The inhibitory effects seem to be mediated by the inhibition of voltagedependent Ca 2 channels, which is caused, at least in part, by membrane hyperpolarization resulting from the activation of K channels.

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