PubMed · 9097632
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Y Arimura, K Arimura. 1997. [Electromyography].. https://pubmed.ncbi.nlm.nih.gov/9097632/
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At fertilization, sea urchin eggs undergo a series of activation events, including a Ca2+ action potential, Ca2+ release from the endoplasmic reticulum, an increase in intracellular pH, sperm pronuclear formation, MAP kinase dephosphorylation, and DNA synthesis. To examine which of these events might be initiated by activation of phospholipase Cgamma (PLCgamma), which produces the second messengers inositol trisphosphate (IP3) and diacylglycerol, we used recombinant SH2 domains of PLCgamma as specific inhibitors. Sea urchin eggs were co-injected with a GST fusion protein composed of the two tandem SH2 domains of bovine PLCgamma and (1) Ca2+ green dextran to monitor intracellular free Ca2+, (2) BCECF dextran to monitor intracellular pH, (3) Oregon Green dUTP to monitor DNA synthesis, or (4) fluorescein 70-kDa dextran to monitor nuclear envelope formation. Microinjection of the tandem SH2 domains of PLCgamma produced a concentration-dependent inhibition of Ca2+ release and also inhibited cortical granule exocytosis, cytoplasmic alkalinization, MAP kinase dephosphorylation, DNA synthesis, and cleavage after fertilization. However, the Ca2+ action potential, sperm entry, and sperm pronuclear formation were not prevented by injection of the PLCgammaSH2 domain protein. Microinjection of a control protein, the tandem SH2 domains of the phosphatase SHP2, had no effect on Ca2+ release, cortical granule exocytosis, DNA synthesis, or cleavage. Specificity of the inhibitory action of the PLCgammaSH2 domains was further indicated by the finding that microinjection of PLCgammaSH2 domains that had been point mutated at a critical arginine did not inhibit Ca release at fertilization. Additionally, Ca2+ release in response to microinjection of IP3, cholera toxin, cADP ribose, or cGMP was not inhibited by the PLCgammaSH2 fusion protein. These results indicate that PLCgamma plays a key role in several fertilization events in sea urchin eggs, including Ca2+ release and DNA synthesis, but that the action potential, sperm entry, and male pronuclear formation can occur in the absence of PLCgamma activation or Ca2+ increase.
1. The giant presynaptic terminal of chick ciliary ganglion was used to examine how protein kinase C (PKC) modulates neurotransmitter release. Cholinergic excitatory postsynaptic currents (EPSCs) were recorded under whole-cell voltage clamp. 2. Although the EPSC was potentiated by phorbol ester (phorbol 12-myristate 13-acetate, PMA; 0.1 microM) in a sustained manner, the nicotine-induced current was unaffected. PMA increased the quantal content to 2.4 +/- 0.4 (n = 9) of control without changing the quantal size. 3. The inactive isoform of PMA, 4alpha-PMA, showed no significant effect on EPSCs. The PMA-induced potentiation was antagonized by two PKC inhibitors with different modes of action, sphingosine (20 microM) and bisindolylmaleimide I (10 microM). 4. When stimulated by twin pulses of short interval, the second EPSC was on average larger than the first EPSC (paired-pulse facilitation; PPF). PMA significantly decreased the PPF ratio with a time course similar to that of the potentiation of the first EPSC. 5. PMA did not affect resting [Ca2+]i or the action potential-induced [Ca2+]i increment in the giant presynaptic terminals. 6. The effect of PMA was less at 10 mM [Ca2+]o than at 1 mM [Ca2+]o. 7. When a train of action potentials was generated with a short interval, the EPSC was eventually depressed and reached a steady-state level. The recovery process followed a simple exponential relation with a rate constant of 0.132 +/- 0.029 s-1. PMA did not affect the recovery rate constant of EPSCs from tetanic depression. In addition, PMA did not affect the steady-state EPSC which should be proportional to the refilling rate of the readily releasable pool of vesicles. 8. These results conflict with the hypothesis that PKC upregulates the size of the readily releasable pool or the number of release sites. PKC appears to upregulate the Ca2+ sensitivity of the process that controls the exocytotic fusion probability.
During repetitive action potential firing the maintenance of synaptic transmission relies on a continued supply of synaptic vesicles for fusion with the presynaptic plasma membrane. The mechanism of transport by which vesicles are delivered to the site of fusion from a reserve pool is unknown, as are the biochemical pathways linking intracellular Ca2+ elevation with vesicle mobilization. Here, using the fluorescent tracer FM1-43 in hippocampal synaptic terminals, I show that inhibitors of myosin light chain kinase can block mobilization of the reserve pool and not the immediately releasable pool.