|Budget Amount *help
¥6,800,000 (Direct Cost: ¥6,800,000)
Fiscal Year 1990: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1989: ¥5,600,000 (Direct Cost: ¥5,600,000)
To study intracellular signaling mechanism of cell proliferation, we analyzed an increase in intracellular calcium ions (Ca response) as an immediate response upon application of proliferation stimulating factors. Two systems were analyzed : Mammalian eggs stimulated by sperm at fertilization and undifferentiated blood cells stimulated by cytokines. Intracellular Ca was measured by image analysis using fura 2.
Our previous studys has suggested that signal transduction of sperm-egg interaction in hamster eggs involves activation of G protein, polyphosphoinositide turnover, production of inositol 1, 4, 5-trisphosphate (IP_3), IP_3-induced Ca release from intracellular stores. In the Present work. We confirmed the existence of IP_3-induced Ca release (IICR) and Ca-induced Ca release in hamster eggs. Iontophoretic microinjection of either IP_3 or Ca ions into unfertilized mature eggs caused a localized Ca rise when injection current pulses were less than 0.7 nA (1-2 sec). Only a slight incr
ease in injection pulses resulted in an all-or-none type, propagating Ca release throughout the egg. The peak Ca concentration reached 500-600 nM. Once Ca release in the entire egg was induced, there was a refractory period of 90-120 sec, probably corresponding to the time required for recharging Ca stores with Ca. IICR was inhibited by heparin. CICR which is well Known in the sarcoplasmic reticulum is sensitized by caffeine and is inhibited by ryanodine. In hamster eggs these drugs had any significant effect, suggesting a novel CICR. We investigated the development of Ca releas mechanism during oocyte maturaion. We found that injection of IP_3 into immature oocytes causes only a local Ca release and IICR gradually develops during oocyre maturation. The all-ornone type, propagating Ca release mechanism develops at later maturation stage, as one of the factor of the establishment of fertilizability.
We examined whether cytokines induce Ca response in leukemic cells. A cell line derived from megakariyoblastic leukema cells were used, and interleukin 3 and GM-CSF (granulocyte macrophage colony stimulating factor) were applied. In preliminary experiments these cytokines did not cause any significant Ca response. Experiments are now continuing, because much time was required for improving the Ca imaging system. Less