Occurrence of Na(+)-Ca2+ exchange in the ciliate Euplotes crassus and its role in Ca2+ homeostasis.

Ciliates possess diverse Ca2+ homeostasis systems, but little is known about the occurrence of a Na(+)-Ca2+ exchanger. We studied Na(+)-Ca2+ exchange in the ciliate Euplotes crassus by digital imaging. Cells were loaded with fura-2/AM or SBF1/AM for fluorescence measurements of cytosolic Ca2+ and Na+ respectively. Ouabain pre-treatment and Na+o substitution in fura-2/AM-loaded cells elicited a bepridil-sensitive [Ca2+]i rise followed by partial recovery, indicating the occurrence of Na(+)-Ca2+ exchanger working in reverse mode. In experiments on prolonged effects, ouabain, Na+o substitution, and bepridil all caused Ca2+o-dependent [Ca2+]i increase, showing a role for Na(+)-Ca2+ exchange in Ca2+ homeostasis. In addition, by comparing the effect of orthovanadate (affecting not only Ca2+ ATPase, but also Na(+)-K+ ATPase and, hence, Na(+)-Ca2+ exchange) to that of bepridil on [Ca2+]i, it was shown that Na(+)-Ca2+ exchange contributes to Ca2+ homeostasis. In electrophysiological experiments, no membrane potential variation was observed after bepridil treatment suggesting compensatory mechanisms for ion effects on cell membrane voltage, which also agrees with membrane potential stability after ouabain treatment. In conclusion, data indicate the presence of a Na(+)-Ca2+ exchanger in the plasma membrane of E. crassus, which is essential for Ca2+ homeostasis, but could also promote Ca2+ entry under specific conditions.

Sexual behaviour in Euplotes raikovi is accompanied by pheromone-induced modifications of ionic currents.

In the marine ciliate Euplotes raikovi, pheromone released by a complementary mating type (nonself pheromone) induces typical sexual behaviour, whereas self pheromone released by the same mating type generally has no effect. Nonself pheromone evokes a reduction of the mean walking speed by 66 %, a threefold increase in the frequency and duration of long-lasting rest phases and a doubling in the number of side-stepping reactions. Consequently, translocation is strongly reduced and the cells remain in a small area. This could increase the probability of finding a sexual partner for pair formation (conjugation). The usual pattern of rhythmic, spontaneous depolarizations controlling the walking rhythm is absent in nonself-pheromone-stimulated cells. The remaining depolarizations arise from a 4 mV hyperpolarized membrane potential and do not reach the usual amplitudes of 15-20 mV but only of 6-10 mV. In addition, the amplitudes of K+ currents are increased at depolarizations of more than 20 mV by at least 30 %. Hyperpolarization- and depolarization-activated Na+ current amplitudes are increased, whereas the Ca2+ current amplitude remains nearly unaffected.

Characterisation of the voltage-activated calcium current in the marine ciliate Euplotes vannus.

We have isolated the early Ca current (ICa) from the whole cell current that activates upon depolarisations in the marine ciliate Euplotes vannus. The peak of ICa activated within 4.2 ms at depolarisations to 5 mV with an amplitude of 2.5 +/- 0.35 nA and was reduced to 1.0 +/- 0.14 nA (n = 5) when the extracellular Ca concentration was changed from 10 to 1 mM. The voltage-dependent activation curve was steeper and shifted to more negative values when external Ca2+ was replaced by Ba2+. The early inward current inactivated with a double-exponential time course including a fast and a slow component, and no inactivation was recorded with Ba2+. The time constants for the recovery from inactivation varied between 44 and 153 ms according to the depolarisation-dependent Ca influx. At the common resting potential of -25 mV, ICa was not steady-state inactivated; ICa half-inactivated at -14.5 mV, and totally inactivated at -5 mV. ICa was inhibited by 10 mM extracellular Cd2+. The peptides omega-conotoxin-GVIA (20 microM), omega-conotoxin-MVIIC (600 nM), omega-agatoxin-IVA (60 nM) and calciseptine (900 nM) did not block ICa. The benzothiazepine-derivative diltiazem (100 microM) and the dihydropiridine nifedipine (100 microM) inhibited 51% and 33% of ICa, respectively. The naphthalene sulfonamide W7 reduced ICa with an inhibition coefficient of 33 microM.

Glutamate-activated ionic currents in cultured astrocytes from trout: evidence for the occurrence of non-N-methyl-D-aspartate receptors.

Glutamate-induced currents were recorded from cultured trout astrocytes with the whole-cell variation of the patch-clamp technique. Ninety percent of the tested cells were directly depolarized by the amino acid neurotransmitter in a concentration-dependent manner. The depolarizing effect was due to an inward current that reversed near 0 mV and was accompanied by a noise increase, indicating the opening of an ion channel. Ion substitution experiments revealed that the glutamate-induced current was mainly carried by sodium ions but not chloride or calcium ions. The glutamate-induced response could be mimicked by the neuronal glutamate receptor subtype agonists kainate and quisqualate, while N-methyl-D-aspartate was without detectable effect.

Voltage-dependent sodium and potassium currents in cultured trout astrocytes.

Voltage-gated ionic currents were recorded from cultured trout astrocytes with the whole-cell variation of the patch-clamp technique. In a subpopulation of astrocytes depolarizations above -40 mV activated a fast transient inward current that was identified as a sodium current by ion substitution experiments, its current reversal potential, and its TTX-sensitivity. Regarding threshold of activation, peak current voltage, and amplitude this current closely resembled those previously described for mammalian astrocytes. Voltage-dependence of inactivation and kinetics, however, markedly differed from the "glial-like" sodium current occurring in mammalian hippocampal or optic nerve astrocytes, since the sodium current of trout astrocytes exhibited a faster time course of activation and decay and a more depolarized steady-state inactivation curve with midpoints close to -60 mV. During a period of 2 weeks in culture the biophysical properties of the sodium current did not change significantly, albeit a continuous decrease in current density was observed. At depolarizing voltage steps positive to -40 mV, additionally voltage-gated potassium outward currents were evoked, which could be separated into a steady-state current with delayed rectifier properties and an inactivating component resembling the A-type current. Moreover, in a subpopulation of astrocytes an inward potassium current was elicited at hyperpolarizing potentials, which exhibited biophysical features consistent with the potassium inward rectifier of mammalian astrocytes.

Electrical responses of the marine ciliate Euplotes vannus (hypotrichia) to mechanical stimulation at the posterior cell end.

Electrical responses upon mechanostimulation at the posterior cell end were investigated in the marine hypotrichous ciliate Euplotes vannus. A new mechanostimulator was developed to mimic stimuli that are identical with those involved in cell-cell collisions. The receptor potential hyperpolarized by 18-35 mV within 12-25 msec, reached a peak value of -62 mV with a delay of 4-9 msec after membrane deformation, and was deactivated after 50-70 msec. Cirri were stimulated to beat accelerated backward. The corresponding receptor current exerted a similar time course with a peak of 2.4 nA. The shift of the reversal potential by 57.6 mV at a tenfold increase of [K+]o identifies potassium ions as current carriers within the development of the receptor potential. An intracellular K concentration of 355 mmol/liter was calculated for cells in a medium that was composed similar to sea-water. The mechanically activated potassium current was totally inhibited by extracellular TEA and intracellular Cs+, and partially inhibited by extracellular 4-AP. The total inhibition of the current by injected EGTA points to a Ca dependence of the posterior mechanosensitivity. It was confirmed by the increase of the peak current amplitude with rising [Ca2+]o. Sodium presumably repolarizes the receptor potential because the repolarization was delayed and after-depolarizations were eliminated in media without sodium. Since deciliation did not affect mechanosensitivity, the corresponding ion channels reside within the soma membrane.

Inward rectification by hyperpolarization-activated Na current in the marine ciliate Euplotes vannus.

The ionic mechanisms underlying inward or anomalous rectification have been studied in the marine hypotrichous ciliate Euplotes vannus. Inward-current pulses of moderate amplitude elicited time-dependent rectification that started from a hyperpolarization peak and was expressed as a depolarizing sag towards rest. Voltage-clamp analysis showed that this depolarization is caused by the activation of a complex inward current that does not inactivate with time. The current is carried by a major Na and a minor K component. The Na-current component has been identified by its concentration-dependent reduction in low extracellular Na solutions and the capability of Li+ as Na substitute to carry the current, though with a slightly reduced amplitude. The K-current component has been isolated from the total current after the replacement of Na+ within the experimental solution. It was blocked in media that contained 10 mmol/liter TEA, a well-known blocker for K inwardly rectifying currents. TEA was only effective at membrane potentials close to or negative to the potassium equilibrium potential. The inward current was reduced after the injection of the Ca chelator EGTA into the cell. Also the elimination of the ciliary membrane, by deciliating cells with ethanol, reduced the amplitude of the inwardly rectifying currents. Both experiments indicate a regulatory function of Ca2+ in inward rectification.

Voltage-dependent potassium currents in cultured trout oligodendrocytes.

Ionic currents were recorded in cultured oligodendrocytes from the brain of trout using the whole-cell configuration of the patch-clamp technique. Outward currents were evoked at membrane potentials more positive than -40 mV, which could be separated into two components according to their kinetic parameters and their sensitivity to the holding potential: a fast inactivating current which was completely suppressed by 4-aminopyridine and reduced by tetraethylammonium and a slow steady-state conductance which was similarly sensitive to both potassium channel blockers. The current reversal potential was close to the potassium equilibrium potential. In contrast to mammalian oligodendrocytes but in similarity with cultured Schwann cells, trout oligodendrocytes did not exhibit any inwardly rectifying currents at hyperpolarized membrane potentials.

Calcium-dependent transient potassium outward current in the marine ciliate Euplotes vannus.

In the marine hypotrichous ciliate Euplotes vannus, the transient K+ outward current, IK fast, was studied by use of a single-microelectrode voltage-clamp equipment. Activation and inactivation kinetics, and steady-state inactivation are comparable to the properties of A-currents. Not typical for this type of current is its insensitivity to either 4-AP or 3,4-AP and its Ca2+ dependence which was derived from its inhibition by either extracellular Cd2+, La3+, D-600, or by intracellular BAPTA. Actual amplitudes of IK fast were obtained from a composite current, by subtraction of early parts of a slowly activating K+ current, IK slow, and of the early, transient Ca2+ inward current, ICa fast, that is typical for ciliates. IK fast counteracts ICa fast during the first milliseconds after onset of depolarization such that the composite current is purely outward directed.

Calcium-dependent sodium current in the marine ciliate Euplotes vannus.

Ca and Na inward currents were recorded upon depolarizations in Euplotes after the blockage of K outward currents with intracellular Cs ions. The Na current was analyzed under voltage clamp and had the following properties: it activated to a maximum within 150 msec and partly inactivated during sustained voltage steps. It had a positive equilibrium potential between 25 and 30 mV and could be carried by Na or Li ions but not by K, choline or Tris ions. The current revealed a prominent associated inward tail current which deactivated with a single-exponential time constant of 118 msec. Both the current and its tail were strongly reduced after reduction of the extracellular Na concentration. Externally applied K channel blocker tetraethylammonium chloride did not block the current. Either EGTA injection into the cell or nonlethal deciliation with ethanol eliminated the current and its tail. These results indicate the existence of a Na conductance within the membrane of Euplotes which is activated by the intracellular level of free Ca2+.

Membrane excitability and membrane currents in the marine ciliate Euplotes vannus.

Electrical properties of E. vannus were investigated by use of constant current injection, voltage-clamp, and isoosmotic ion substitution. The resting potential of approximately -40 mV was K(+) and Ca(2+)-dependent. Spontaneous depolarizations occurred frequently with peaks around -20 mV and durations from several hundred ms to several s. External Ba(2+) or internal Cs(+) induced all-or-none action potentials. Current stimuli induced Ca(2+)-dependent graded action potentials. Sr(2+) or Ba(2+), but not Mg(2+), instead of Ca(2+) increased the regenerative response. Repolarization occurred in two steps: a first fast and a second slow one. It was strongly modified by the Ca(2+) substitutes. A voltage-dependent small Ca(2+) inward current was activated at depolarizations beyond -20 mV. It triggered a fast and a slowly activating K(+) outward current and was itself short-circuited by the fast K(+) current. Therefore, it could only be measured when K(+) currents were not activated or inhibited. A slowly activating Na(+) inward current was identified that turned to outward direction after replacement of external Na(+) by choline(+). The K(+) outward currents differed in their sensitivity to external TEA(+) and in their inactivation kinetics. All currents were correlated to the voltage-dependent influx of Ca(2+).