Abstract
The egg peptide speract increases intracellular pH (pHi) and cyclic nucleotides in sperm of the sea urchin Strongylocentrotus purpuratus by a mechanism dependent on seawater Na+ but not Ca2+ (Hansbrough, J. R., and Garbers, D. L. (1981) J. Biol. Chem. 256, 2235-2241; Repaske, D. R., and Garbers, D. L. (1983) J. Biol. Chem. 258, 6025-6029). Using the Ca2+ indicators quin2 and indo-1, we show that speract stimulates a transient rise in intracellular [Ca2+] ([a2+]i) when millimolar Ca2+ is present in seawater. The rise is increased and extended by the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX), which also enhances 22Na+ uptake with or without Ca2+. Without MIX, speract initiates a rise in [Ca2+]i that peaks within approximately 5 s and decreases with a t1/2 of approximately 9 s. Activation of Na+:H+ exchange without speract by either Na+ addition to sperm in Na+-free seawater (NaFASW) or by monensin also increases [Ca2+]i, but neither change is transient. Inhibition of Na+:H+ exchange by increased seawater [K+] prevents the rise in [Ca2+]i initiated by either speract or Na+ addition to sperm in NaFASW. Increasing pHi by adding 10 mM NH4+ or by addition of Li+ to sperm in NaFASW does not increase [Ca2+]i. The data suggest that speract binding leads to rapid activation of Na+:H+ exchange; and, as a consequence, [Ca2+] entry increases transiently through either Na+:Ca2+ exchange or else through a verapamil-insensitive Ca2+ channel. MIX prevents the inactivation of this entry mechanism.
Highlights
From the Section on Metabolic Regulation, Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda,Maryland 20892
We have previously demonstrated by following H+ efflux induced by this ionophore [7] thatit first collapses the cell K+ gradient (K+/H+ exchange) and causes H+ uptake followed by a slow Na+dependent H+ efflux and pHi increase
Speract Initiates a Transient Rise in [CU~+]~-F~2~sh. ows that speract initiates a transient increase in the fluorescence of the Ca2+indicator indo-1 when millimolar [Ca"] is in the seawater
Summary
Speract Initiates a Transient Rise in [CU~+]~-F~2~sh. ows that speract initiates a transient increase in the fluorescence of the Ca2+indicator indo-1 when millimolar [Ca"] is in the seawater. The speract-induced transient reaches a half-maximal value at -2 mM [Ca2+],(Fig. 2 B ). Speract added to sperm in seawater containing Ca2+gives rise to the transient increase in [Ca2+],(Fig. 4a). The speractinduced [Ca2+Iincrease, which is approximately %fold larger than without MIX (Fig. 70), requires -40 s to reach the final steady state. This time course nearly follows first-order rate
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