Chemotaxis von Seeigel-Spermien - kinetische Messungen intrazellulärer Botenstoffe

The egg-petide resact induces a chemotactical response of sperm from sea urchin Arbacia punctulata. By bindig to a receptor-guanylylcyclase on the flagellar membrane resact activates a signaling-cascade. This leads to an increase in cGMP-/cAMP-concentration, Ca$^{2+}$- concentration an pH. The sequence and thereby the causal relations of the single physiological reactions were unkown before. In order to reveal the sequence of the signaling events I have established two methods which allowed time-resolved measurements (e.g. in millisecond time-scale) of the physiological reactions. The quenched-flow-method was used to detect the time-course of the cGMP-/cAMP-concentration. Therefore it was combined with cGMP-/cAMP-sensitive Radioimmunoassays. The stopped-flow-method was used to record the timecourse of the Ca$^{2+}$- or pH-sensitive fluorescence-indicators. Both methods enabled me to measure for the first time the sequence of the physiological reactions. The measurements showed, that resact induces a fast and high increase of the cGMP-concentration. The cGMP-concentration starts rising within the first 25 ms and reaches its half-maximal concentration within 200 ms. A cGMP-increase is already induced at picomolar resact-concentrations whereas a cAMP-increase is only induced by nanomolar resactconcentrations. Furthermore the cAMP-increase is slighter and slower compared to the cGMP-increase. Arround 250 ms after the stimulation with resact the Ca$^{2+}$-concentration increases. This Ca$^{2+}$- signal can be devided in an „early“ and a „late“ Ca$^{2+}$-signal. While the early Ca$^{2+}$-signal can be already triggered by single resact-molecules the late Ca$^{2+}$-signal is less sensitive to the eggpetide. The early Ca$^{2+}$-signal is either directly or through other signaling events triggered by cGMP. The cAMP-increase is – at least in the presence of the PDE-inhibitor IBMX – slower than the late Ca$^{2+}$-signal. Therby cAMP could only trigger the late Ca$^{2+}$-signal. In contrast to the late Ca$^{2+}$-signal the early Ca$^{2+}$-signal is induced at the same time as the resact-induced change of the swimming-behavior. Thus the early Ca$^{2+}$-signal represents the crucial reaction in the resact-induced signaling-cascade. Like the early Ca$^{2+}$-signal, the pH responds to resact-concentrations over more than 6 orders of magnitude. The delay of the pH-change is only at high resact-concentrations faster than the delay of the Ca$^{2+}$-signal. At low resact-concentrations the pH increases after the Ca$^{2+}$-concentration. Contrary to other publications, this results shows that the pH-increase does not trigger the Ca$^{2+}$-increase

Revisiting the role of H+ in chemotactic signaling of sperm

© 2004 Solzin et al. This article is distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License. The definitive version was published in Journal of General Physiology 124 (2004): 115-124, doi:10.1085/jgp.200409030.Chemotaxis of sperm is an important step toward fertilization. During chemotaxis, sperm change their swimming behavior in a gradient of the chemoattractant that is released by the eggs, and finally sperm accumulate near the eggs. A well established model to study chemotaxis is the sea urchin Arbacia punctulata. Resact, the chemoattractant of Arbacia, is a peptide that binds to a receptor guanylyl cyclase. The signaling pathway underlying chemotaxis is still poorly understood. Stimulation of sperm with resact induces a variety of cellular events, including a rise in intracellular pH (pHi) and an influx of Ca2+; the Ca2+ entry is essential for the chemotactic behavior. Previous studies proposed that the influx of Ca2+ is initiated by the rise in pHi. According to this proposal, a cGMP-induced hyperpolarization activates a voltage-dependent Na+/H+ exchanger that expels H+ from the cell. Because some aspects of the proposed signaling pathway are inconsistent with recent results (Kaupp, U.B., J. Solzin, J.E. Brown, A. Helbig, V. Hagen, M. Beyermann, E. Hildebrand, and I. Weyand. 2003. Nat. Cell Biol. 5:109–117), we reexamined the role of protons in chemotaxis of sperm using kinetic measurements of the changes in pHi and intracellular Ca2+ concentration. We show that for physiological concentrations of resact (<25 pM), the influx of Ca2+ precedes the rise in pHi. Moreover, buffering of pHi completely abolishes the resact-induced pHi signal, but leaves the Ca2+ signal and the chemotactic motor response unaffected. We conclude that an elevation of pHi is required neither to open Ca2+-permeable channels nor to control the chemotactic behavior. Intracellular release of cGMP from a caged compound does not cause an increase in pHi, indicating that the rise in pHi is induced by cellular events unrelated to cGMP itself, but probably triggered by the consumption and subsequent replenishment of GTP. These results show that the resact-induced rise in pHi is not an obligatory step in sperm chemotactic signaling. A rise in pHi is also not required for peptide-induced Ca2+ entry into sperm of the sea urchin Strongylocentrotus purpuratus. Speract, a peptide of S. purpuratus may act as a chemoattractant as well or may serve functions other than chemotaxis.This work was supported by a grant from the Deutsche Forschungsgemeinschaft

A sperm-activating peptide controls a cGMP-signaling pathway in starfish sperm☆

AbstractPeptides released from eggs of marine invertebrates play a central role in fertilization. About 80 different peptides from various phyla have been isolated, however, with one exception, their respective receptors on the sperm surface have not been unequivocally identified and the pertinent signaling pathways remain ill defined. Using rapid mixing techniques and novel membrane-permeable caged compounds of cyclic nucleotides, we show that the sperm-activating peptide asterosap evokes a fast and transient increase of the cGMP concentration in sperm of the starfish Asterias amurensis, followed by a transient cGMP-stimulated increase in the Ca2+ concentration. In contrast, cAMP levels did not change significantly and the Ca2+ response evoked by photolysis of caged cAMP was significantly smaller than that using caged cGMP. By cloning of cDNA and chemical crosslinking, we identified a receptor-type guanylyl cyclase in the sperm flagellum as the asterosap-binding protein. Sperm respond exquisitely sensitive to picomolar concentrations of asterosap, suggesting that the peptide serves a chemosensory function like resact, a peptide involved in chemotaxis of sperm of the sea urchin Arbacia punctulata. A unifying principle emerges that chemosensory transduction in sperm of marine invertebrates uses cGMP as the primary messenger, although there may be variations in the detail

Kinetic Mechanism of the Ca2+-Dependent Switch-On and Switch-Off of Cardiac Troponin in Myofibrils

The kinetics of Ca2+-dependent conformational changes of human cardiac troponin (cTn) were studied on isolated cTn and within the sarcomeric environment of myofibrils. Human cTnC was selectively labeled on cysteine 84 with N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole and reconstituted with cTnI and cTnT to the cTn complex, which was incorporated into guinea pig cardiac myofibrils. These exchanged myofibrils, or the isolated cTn, were rapidly mixed in a stopped-flow apparatus with different [Ca2+] or the Ca2+-buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid to determine the kinetics of the switch-on or switch-off, respectively, of cTn. Activation of myofibrils with high [Ca2+] (pCa 4.6) induced a biphasic fluorescence increase with rate constants of >2000 s−1 and ∼330 s−1, respectively. At low [Ca2+] (pCa 6.6), the slower rate was reduced to ∼25 s−1, but was still ∼50-fold higher than the rate constant of Ca2+-induced myofibrillar force development measured in a mechanical setup. Decreasing [Ca2+] from pCa 5.0–7.9 induced a fluorescence decay with a rate constant of 39 s−1, which was approximately fivefold faster than force relaxation. Modeling the data indicates two sequentially coupled conformational changes of cTnC in myofibrils: 1), rapid Ca2+-binding (kB ≈ 120 μM−1 s−1) and dissociation (kD ≈ 550 s−1); and 2), slower switch-on (kon = 390s−1) and switch-off (koff = 36s−1) kinetics. At high [Ca2+], ∼90% of cTnC is switched on. Both switch-on and switch-off kinetics of incorporated cTn were around fourfold faster than those of isolated cTn. In conclusion, the switch kinetics of cTn are sensitively changed by its structural integration in the sarcomere and directly rate-limit neither cardiac myofibrillar contraction nor relaxation

Kinetic Mechanism of Ca2+-controlled Changes of Skeletal Troponin I in Psoas Myofibrils

Conformational changes in the skeletal troponin complex (sTn) induced by rapidly increasing or decreasing the [Ca2+] were probed by 5-iodoacetamidofluorescein covalently bound to Cys-133 of skeletal troponin I (sTnI). Kinetics of conformational changes was determined for the isolated complex and after incorporating the complex into rabbit psoas myofibrils. Isolated and incorporated sTn exhibited biphasic Ca2+-activation kinetics. Whereas the fast phase (k(obs)similar to 1000 s(-1)) is only observed in this study, where kinetics were induced by Ca2+, the slower phase resembles the monophasic kinetics of sTnI switching observed in another study (Brenner and Chalovich. 1999. Biophys. J. 77:2692-2708) that investigated the sTnI switching induced by releasing the feedback of force-generating cross-bridges on thin filament activation. Therefore, the slower conformational change likely reflects the sTnI switch that regulates force development. Modeling reveals that the fast conformational change can occur after the first Ca2+ ion binds to skeletal troponin C (sTnC), whereas the slower change requires Ca2+ binding to both regulatory sites of sTnC. Incorporating sTn into myofibrils increased the off-rate and lowered the Ca2+ sensitivity of sTnI switching. Comparison of switch-off kinetics with myofibril force relaxation kinetics measured in a mechanical setup indicates that sTnI switching might limit the rate of fast skeletal muscle relaxation