carbocyanines and 1-anilino-8-naphthalenesulfonate

Sperm from trout, like other sperm, are immotile in the seminal tract and initiate motility upon dilution into an appropriate fertilizing environment. Trout sperm motility is inhibited by high extracellular [K+] and can be activated by dilution of extracellular [K+]. Activation of trout sperm by the dilution of extracellular [K+] suggests regulation by membrane potential. Using the membrane potential-sensitive fluorescent dye 3,3'-dipropylthiocarbocyanine iodide (diS-C3-(5)) we directly measured the K+ contribution to the membrane potential. Manipulating the membrane potential with Cs+ and the ionophore valinomycin can override K+ regulation. We show that trout sperm can also be activated in the presence of inhibitory [K+] by the addition of divalent cations. Activation by divalent cations is explained by the cations' ability to mask membrane surface potential and thus alter the potential sensed by membrane voltage sensors. Using the surface potential-sensitive dye, 1-anilino-8-naphthosulfonate (ANS), we directly measure the divalent cations' ability to mask surface potential. We propose a model where membrane hyperpolarization is the trigger that initiates the cascade of events leading to trout sperm activation. An increase in intracellular pH has been suggested to be a conserved step in the activation of sperm motility. We show that increasing intracellular pH by procedures that activate sea urchin and mammalian sperm does not activate trout sperm. In contrast, there is a decrease in intracellular pH upon activation of trout sperm motility. Artificially decreasing intracellular pH is not sufficient for activation of motility in trout sperm in an inhibitory [K+]. Thus, unlike some other sperm, changes in intracellular pH do not regulate trout sperm motility.

Topics: Anilino Naphthalenesulfonates; Animals; Benzothiazoles; Carbocyanines; Cesium; Fluoresceins; Hydrogen-Ion Concentration; Magnesium; Male; Membrane Potentials; Models, Biological; Potassium; Sperm Motility; Spermatozoa; Trout; Valinomycin

We have employed a series of permeant, nontoxic, fluorescent probes to detect changes in ionic conditions within the mitotic apparatus of living endosperm cells of Haemanthus during the transition from metaphase to anaphase. Fluorescence emission intensity measurements from the spindle for chlorotetracycline (CTC) decline before the onset of anaphase, indicating a reduction in the amount of membrane-associated Ca2+ and suggesting an efflux of Ca2+ from membrane compartments into the spindle. Subsequent to the onset of anaphase, we observe increases in fluorescence with both 8-anilino-1-naphthalene sulfonate (ANS) and 3,3'-dipentyl 2,2'-dioxacarbocyanine (diO-C5(3)), sensitive to cationic and anionic charges at membrane surfaces, respectively. The increases with ANS and diO-C5(3) suggest that redistributions of ions within the spindle accompany anaphase motion. During the metaphase/anaphase transition, spindle membrane content remains constant, as evidenced by unchanging fluorescence with the hydrophobic probe, N-phenyl-1-naphthylamine (NPN). Shifts in emission intensity from the nonspindle cytoplasm or from the spindle poles do not accompany the changes in fluorescence we observe in the spindle, suggesting that any ionic fluxes responsible for the changes in fluorescence are restricted to the spindle domain.

Topics: 1-Naphthylamine; Anaphase; Anilino Naphthalenesulfonates; Calcium; Carbocyanines; Chlortetracycline; Fluorescent Dyes; Intracellular Membranes; Ions; Metaphase; Mitosis; Seeds

It is shown that during deamination of AMP with membrane-bound AMP-aminohydrolase of sarcoplasmic reticulum, which is accompanied by the pH increase, there occurs a fluorescence quenching of 1-anilino-8-naphthalinsulphonate and 3-3(1)-dipropylthio-dicarbocyanine bound to membranes. Such a direction in the fluorescence changes is observed when pH is artificially increased from 6.5 to 8.5. No changes are marked in fluorescence of the applied probes in experiments with the use of the sarcoplasmic reticulum fragment vesicles with AMP-aminohydrolase only or in deamination of AMP in a strongly buffered medium as well as under the effect of IMP or NH4Cl in concentrations up to 10 mM. The studies conducted show that the charge of sarcoplasmic reticulum membranes may change in the process of amp deamination.

Topics: Adenosine Monophosphate; AMP Deaminase; Anilino Naphthalenesulfonates; Animals; Benzothiazoles; Carbocyanines; Fluorescent Dyes; Hydrogen-Ion Concentration; Intracellular Membranes; Kinetics; Nucleotide Deaminases; Quinolines; Rabbits; Sarcoplasmic Reticulum; Spectrometry, Fluorescence