PubMed · 5463944
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K Yasuda. 1970. [Flame photometry].. https://pubmed.ncbi.nlm.nih.gov/5463944/
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Two forms of evoked neurotransmitter release at excitatory synapses between cultured hippocampal neurons have been described. After an action potential, it has been shown that transmitter initially is released synchronously, and this is followed by a period of "slow" asynchronous release. The "fast" synchronous component of release at these synapses has been found routinely to demonstrate paired-pulse and tetanic depression, whereas the short-term plasticity of asynchronous release has not been investigated. In the present experiments, we have used the whole-cell patch-clamp technique to record from pairs of neurons in a low-density hippocampal culture preparation to determine both the properties and underlying mechanisms of short-term plasticity of asynchronous release. It was found that an increase in miniature EPSC (mEPSC) frequency accompanied both single and multiple stimuli, and this mEPSC increase was facilitated during paired stimuli, even when the evoked synchronous release was depressed. In addition, both the activity-dependent depression of evoked EPSCs and facilitation of asynchronous mEPSC release were dependent on Ca accumulation in the nerve terminal. However, the Ca-dependent mechanisms underlying these two processes could be distinguished by the differential effects of two membrane-permeant calcium chelators, BAPTA-AM and EGTA-AM. Frequency-dependent depression of evoked EPSCs involves a rapid rise in intraterminal Ca, which likely triggers a process that proceeds in a Ca-independent manner, whereas the asynchronous release may be linked more directly to a sustained increase in intraterminal Ca.
The mechanism by which calcium-depleted intracellular stores may trigger an external calcium influx through a calcium release-activated channel was investigated by analyzing the effects of several protein tyrosine kinase inhibitors on calcium movements in Jurkat T cells. Tyrphostin A9, an inhibitor of the kinase activity of the platelet-derived growth factor (PDGF) receptor, dramatically impaired the sustained elevation of cytosolic calcium concentration, induced by either CD3 mAbs, thapsigargin, ionomycin at low (10(-7) M) concentration, or passive depletion of intracellular stores; other tested tyrphostins, lavendustin, genistein, and compound 5 lacked significant effect. Tyrphostin A9, added during the plateau phase, was able to return cytosolic calcium to resting concentration. Likewise, it abrogated manganese entry in cells stimulated by CD3 or thapsigargin, measured by the quenching of the fluorescence of Indo-1. However, it did not measurably modify kinetics of intracellular calcium releases monitored in the absence of extracellular calcium, nor did it reverse the inhibition of phosphatidylserine that occurs as a consequence of emptying intracellular stores. Analyses of tyrosine phosphorylations demonstrated that A9 inhibited the phosphorylation of proteins, which occurred every time that internal calcium stores were depleted. These phosphorylations were not impaired by chelation of external Ca2+, nor by La3+ that inhibits calcium release-induced calcium entry. We concluded that their inhibition was not a consequence, but may be a cause, of the blockade of calcium release-activated channel by tyrphostin A9.
We recently reported that Fc mu R on NK cells is a signal transducing protein that stimulates a rapid increase in the level of cytoplasmic free calcium upon binding of IgM. This study was designed to examine signal transduction via the Fc mu R on NK cells and to characterize intracellular second messengers activated by IgM. Immunoprecipitation of IgM-bound Fc mu R by IgM-specific Ab coimmunoprecipitated the zeta- and Fc epsilon RI gamma-chains. Furthermore, engagement and clustering of Fc mu R by polyclonal IgM induced tyrosine phosphorylation of the zeta- and Fc epsilon RI gamma-chains, indicating their functional association with the Fc mu R-induced signal transduction cascade. Ligand-induced clustering of the Fc mu R also induced activity of src family kinases, Lck, Fyn, Lyn, and Src, as well as their physical interaction with the receptor. Triggering via Fc mu R also induced the activity of Syk and Zap-70, tyrosine kinases demonstrated to associate with zeta and Lck. Phospholipase C-gamma 1 and phosphatidylinositol 3-kinase were identified as substrates phosphorylated on tyrosine, as down-stream components of the signaling pathway activated in NK cells by polyclonal IgM. Although the Fc mu R on NK cells has not yet been biochemically characterized, our results suggest that the zeta- and Fc epsilon RI gamma-chains are functional subunits of this as well as other important cell surface receptors and that the Fc mu R is coupled either directly or indirectly to nonreceptor tyrosine kinases, which phosphorylate and thereby activate regulatory enzymes such as phospholipase C-gamma 1 and phosphatidylinositol 3-kinase.