A partial sequence of ionic changes associated with the acrosome reaction of Strongylocentrotus purpuratus |
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Authors: | Robert W Schackmann Bennett M Shapiro |
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Institution: | Department of Biochemistry, University of Washington, Seattle, Washington 98195 USA |
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Abstract: | Relationships among several of the ion movements associated with the acrosome reaction of S. purpuratus were investigated. Egg jelly initiates 45Ca2+ and 22Na+ uptake, and K+ and H+ efflux. H+ efflux and 22Na+ uptake occur with approximately equivalent stoichiometries as rapidly as the appearance of acrosomal rods, perhaps reflecting a linked process. Most K+ loss, as measured either by 42K+ efflux or K+-ion-selective electrodes, occurs after the acrosome reaction is complete. Since an elevation of seawater K+ (from 10 to 15 mM) or the addition of 0.5 mM tetraethylammonium (TEA), an inhibitor of K+ channels, inhibits the acrosome reaction half-maximally, K+ movements or alterations of K+-dependent membrane potentials may regulate the triggering by jelly. Most, but not all, of the 45Ca2+ influx is inhibited with a mixture of 10 μM FCCP, 1 mM CN?, and 2 μg/ml oligomycin, suggesting that the mitochondria store most of the Ca2+. The extracellular Na+ concentration affects Ca2+ fluxes: sperm placed into 5 mM Na+ seawater have enhanced 45Ca2+ uptake, but do not undergo the acrosome reaction, unless 30 mM Na+ is also added. Low Na+ concentrations lead to spontaneous triggering, by allowing for both Ca2+ influx and Na+-dependent H+ efflux. At least one early Ca2+ requirement precedes the Na+ and H+ movements, as inferred from attempts at reversing the inhibitors of jelly induction of the acrosome reaction. When sperm are incubated with jelly in the absence of Ca2+, then washed and incubated with jelly in the presence of Ca2+, the acrosome reaction is triggered only upon the second incubation. However, when sperm are mixed with jelly in the presence of the other inhibitors (verapamil, TEA, 5 mM Na+ seawater, low pH, or elevated K+), they are altered so that even upon subsequent washing, jelly-mediated triggering is no longer possible. This suggests the existence of an intermediate state in the reaction pathway, that follows an event for which Ca2+ is required, but that precedes the Na+ and H+ movements, which are inhibited by all inhibitors of the acrosome reaction. These data are used to develop a partial sequence of ionic changes associated with the triggering mechanism. |
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Keywords: | TEA tetraethylammonium chloride ASW artificial seawater MES DNP 2 4-dinitrophenol FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone EGTA CaFSW |
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