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  • 1
    ISSN: 0730-2312
    Keywords: antisensense oligonucleotides ; pertussis toxin ; splenocytes ; Nb2 cells ; Giα ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In a previous report we shwed that glucocorticoed inhibition of cytosolic PLC activity correlated with a reduction in cytosolic Giα levels, suggesting that there may be a functional relationship between cytosolic PLC and cytosolic Giα. In order to establish the nature of the coupliing between cytosolic Giα and cytosolic PLC we examined the effects of Protein activators, and inhibitors on cytosolic PLC activity from rat spenocytes and the rat lymphoma cell line Nb 2, with [3H] PI and [3H]PIP2 as substrates. (1) Neither GTP nor its nonhydrolyzable analogue, GTPγS, at 100 μm had any effect on the calcium stimulated as well as the basal PLC activity. (2) Howevr, affinity purified antibodies to Giα1 and Giα2 inhibited soluble PLC activity, by 85% and 55%, respectively, with PI as substrate; with PIP2 as substrate, soluble PLC activity was inhibited 50-70% by antibodies to Gi1, whereas antibodies to Gi2 had little effect. (3)Administration of Giα1 antisense oligonucleotides to splenocytes for 48 h produced 25-40% decrease in cytosolic Giα1 levels compared to control. The soluble PLC activity with both PI and PIP2 as substrates was also reduced by 25-50% compared to control conditions. This suggest that cytosolic Giα is associated with the activation of splenocyte soluble PLC. (4) Pertussis toxin administered in vivo sugnificantly reduced cytosolic Giα immunoreactivity and soluble PLC activiry when PI was used as substrate, providing additional evidence that cytosolic Giα is associated with the activation of splencyte soluble PLC. (5) Another agent that has beeen used extensively to define G-protein coupled processes is NaF/AlCl3. NaF(4mM; with or without AlCl3 inhibited soluble PLC activity with PIP2 as substrate, in contrast ot the stimulatory effect that has been reported in the activation of membrane PLC. 6) because NaF can act as a protein phosphatase inhibitor, we also tested the effects of trifluoperzine (50 μm, TFP), an inhibitor of protein phosphatase 2B; TFP (50 μm) signigicantly inhibited soluble PLC activity PI was used as substrate. These results suggest a direct involvement of cytosolic Giα in the activation of soluble PLC form splenocytes. Other questions pertaining to the functional significance, the nature, and possible substrate preference of the splenocyte Giα coupled PLC is addressed in the second paper.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 0730-2312
    Keywords: G proteins ; sodium metavanadate ; phorbol esters ; dexamethasone ; NaF ; trifluoperizine ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have described in the first paper the coupling betweencytosolic Giα and cytosolic PLC activity in a cell free preparation. In order to establish the functional significance of the cytosolic Giα coupled soluble PLC, we examined the effects of Dex, NaF, and trifluopeerizine (TEP) on concanavalin A(Con A)-induced PI-turnover in intact slenocytes and, in parallel, on soluble PLC activity in cytosol preparations. Vytosolic PLC activity was measured with [3H]PIP and [3H]PIP2 as substrates. (1) The con A-induced increase (2-4 fold) in Pl-turnover in intact splenocytes was paralleled by an 1.2-5-fold increase in soluble PLC activity in vitro. Con A administration also increased cytosolic Giα immunoreactivity 3-6-fold as expected if cytosolic Giα was coupled to soluble PLC activation. (2) DEX (10-7 M), administered 6 h prior to Con A administration inbited the Con A-induced increase in Pl-turnover in intact splenocytes. This was paralleled by DEX inhibition of the Con A-induced increase in soluble PLC activity measured in vitro and cytosolic Giα imunoreactivity. (3) We have demonstrated in the first paper that NaF and TEP inhibited soluble PLC activity. Here we show that NaF and TFP inhibited the Con A-induced increase in PI-turnover extending the similarities between soluble PLC activity and Con A- Stimulated PLC Activity in intact splenocytes. (4) In order to examine Whether or not the Con A-induced PLC activity and Con A-stimulated PLC activity in intact splenocytes. (4) In order to examine Whether or not the Con A-induced PLC was similar to PLCγ, we measured PI-turnover induced by Con A or BaVO3 in combination with DEX and PMA. Whereas the Con A-induced PI-turnover was significantly inhibited (40-60%) by DEX, the NaVO3 -induced PI-turnover was not affected by DEX. The Con A-induced PI-turnover was not affected by PMA (50nM), But the NaVO3-induced Pi-turnover was increased over 2-fold PMA (50nM), suggesting that the Con A-induced PLC in intact splenocytes is different from NaVO3-induced PLC. Based on these results a model for the sequential activation of substrate-specific PLCs in splenocyte by mitogen is presented.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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