RELATIVE IMPORTANCE OF CHOLINE TRANSPORT TO SPONTANEOUS AND POTASSIUM DEPOLARIZED RELEASE OF ACh

—The importance of extracellular choline transport to spontaneous and K⁺ depolarized release of ACh was studied using mouse brain cortex minces. The results suggest that extracellular choline transport is not essential to spontaneously released ACh but is essential to K⁺ depolarized ACh release. Similar cumulative amounts of choline and ACh were found in the incubation media following incubation of minces in either Krebs or 35 mm -K⁺ Krebs suggesting the same production of free choline during both conditions. Double reciprocal plots of choline accumulation by non-depolarized cortex minces yield high and low affinity components. Conversely, similar analysis of choline accumulation by depolarized minces yields a single Michaelis constant (68 μm ) similar to the low affinity (50 μm ) Michaelis constant determined for choline accumulation by non-depolarized minces. Kinetic analysis of ACh release as a function of extracellular choline concentration during K⁺ depolarization also yields a Michaelis constant of 68 μm These data suggest a link between choline transport and ACh release during K⁺ depolarization.     

Partial Purification of Intra- and Extra-cellular Cellulases from Pyricularia oryzae: with Reference to Optimum pH at Alkaline Side

In order to elucidate the relation between the difference in cellulase activity among various strains of P. oryzae and the optimum pH at alkaline side, and also to know the relation between the intra- and extra-cellulases, the elution patterns of the enzymes from the Sephadex G–100 column were compared and the occurrence of the enzyme fractions showing the optimum pH at alkaline side was investigated.

The elution patterns of the intracellular cellulases were shown to be relatively constant, but those of the extracellular enzymes did not. The peak e appeared comparatively constant, but the peak c was considered to undergo some change during the excretion into the medium.

The optimum pH at alkaline side was shown to occur in the peak e among five peaks on Sephadex G–100 of the partially purified intra- and extra-cellular cellulases. The peak seems to be significant for P. oryzae.     

Crayfish motor nerve terminal's response to serotonin examined by intracellular microelectrode

Measurements of resting potential and action potential in presynaptic branches of the excitatory motor axon to the crayfish opener muscle were made with intracellular microelectrodes during application of serotonin (10(-9)-10(-3) M). A 5-min exposure to 10(-6) M serotonin produced enhancement of excitatory junction potentials (EJPs) lasting about 1 h. The membrane potential of the presynaptic terminal was depolarized by about 5 mV; the depolarization subsided within 1/2 h. Concomitant reduction in amplitude of the presynaptic action potential, not accompanied by spike broadening, was observed. The presynaptic depolarization, and the enhancement of EJPs, were dependent on the presence of extracellular sodium but not extracellular calcium. A possible mechanism for serotonin's effect involves initial entry of sodium into the nerve terminal, with consequent increased availability of intracellular calcium. The subsequent long-lasting phase of EJP enhancement may result from an additional effect on the metabolism of the nerve terminal.     

Electrophysiological and pharmacological properties of cultured rat thyroid cells

The electrophysiological properties of a hormone-dependent, differentiated thyroid epithelial cell strain were studied using intracellular microelectrodes. The average membrane potential of solitary, isolated cells was –78.4 ± 1.3 mV. The membrane potential depolarized 55 mV per tenfold increase in extracellular potassium concentation. Weak electrical coupling was recorded between contiguous cells. Like tyroid cells in vivo, these cells did not generate action potentials. In some cells a spontaneous, slow transition in the membrane potential from –80mV to –30 mV was accompanied by an increase in input resistance. Membrane potential transitions could be induced by perfusing cells with isotonic Hanks solutions saturated with CO₂ (pH = 5.5) or by perfusing cells with hypotonic Hanks solutions (190–290 mOsm/kg). Membrane potential transitions were due to a decreased potassium permeability. Noradrenaline elicted both a fast depolarization and a slow depolarization. The fast depolarization was due to an increase in conductance of Na⁺ channels and of Cl^– channels. Intracellular injection of Ca⁺⁺ elicited the fast depolarization. Intracellular injection of EGTA or cobalt abolished the fast depolarization. Replacemnt of extracellular Ca⁺⁺ by Mg⁺⁺ did not affect the fast depolarization. Thus, the fast depolarization was due to accumulation of intracellular Ca⁺⁺. The fast depolarization was abolished by the alpha adrenergic blocker phentolamine (10^–6 M), and was not abolished by the beta adrenergic blocker propranolol (10^–5 M).     

Anomalous Effect of Permeant Ion Concentration on Peak Open Probability of Cardiac Na+ Channels

Human heart Na⁺ channels were expressed transiently in both mammalian cells and Xenopus oocytes, and Na⁺ currents measured using 150 mM intracellular Na⁺. Decreasing extracellular permeant ion concentration decreases outward Na⁺ current at positive voltages while increasing the driving force for the current. This anomalous effect of permeant ion concentration, especially obvious in a mutant (F1485Q) in which fast inactivation is partially abolished, is due to an alteration of open probability. The effect is only observed when a highly permeant cation (Na⁺, Li⁺, or hydrazinium) is substituted for a relatively impermeant cation (K⁺, Rb⁺, Cs⁺, N -methylglucamine, Tris, choline, or tetramethylammonium). With high concentrations of extracellular permeant cations, the peak open probability of Na⁺ channels increases with depolarization and then saturates at positive voltages. By contrast, with low concentrations of permeant ions, the open probability reaches a maximum at approximately 0 mV and then decreases with further depolarization. There is little effect of permeant ion concentration on activation kinetics at depolarized voltages. Furthermore, the lowered open probability caused by a brief depolarization to +60 mV recovers within 5 ms upon repolarization to −140 mV, indicative of a gating process with rapid kinetics. Tail currents at reduced temperatures reveal the rapid onset of this gating process during a large depolarization. A large depolarization may drive a permeant cation out of a site within the extracellular mouth of the pore, reducing the efficiency with which the channel opens.     

Calcium exchange and contraction strength of guinea pig atrium in normal and hypertonic media

A Krebs-Henseleit (KH) medium made hypertonic by adding nonpermeant molecules substantially increased the isometric peak tension at steady-state contractions below 3 per sec in guinea pig atrium at 27°C. Action potential durations were decreased. KH plus 100 mM raffinose or sucrose resulted in similar and nearly maximal changes which were essentially reversible upon return to normal KH. When one active contracting atrium was used to passively stretch a second atrium, the difference in Ca ion exchange (1 min exchange with the extracellular space) between active and stretched atria significantly increased at 1 per sec and at 2 per sec in going from normal to 100 mM hypertonic KH. The calculated mean Ca ion cellular exchange per beat per 100 g of cells (a) doubled in changing from normal to 100 mM hypertonic KH, and (b) decreased slightly in changing from contractions of 1 per sec to 2 per sec in normal KH. These data are consistent with the hypothesis (a) that Ca ion entry per beat from the extracellular space is proportional to membrane depolarized time with a constant medium and a steady-state condition, and the hypothesis (b) that 100 mM hypertonicity doubles the Ca ion entry rate during depolarization. These data enable rejection of the hypothesis that the peak tension is proportional to the Ca ion entry per beat from the extracellular space under steady-state conditions, and suggest that any additional Ca ion involved in the larger contractions at higher frequencies comes from an increase in Ca ion available from intracellular stores.     

Depolarization reverses age-related decrease of spontaneous transmitter release.

The effect of increasing extracellular Ca concentration on spontaneous transmitter release was studied at soleus nerve terminals of young (10 mo) and old (24 mo) C57BL/6J mice depolarized by high extracellular K concentration ([K]o). By using intracellular recording, miniature end-plate potentials (MEPPs) were first recorded in a normal [K]o Krebs solution. Subsequently, MEPPs were recorded in high [K]o Krebs solutions with four different Ca concentrations: Ca-free/ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and 0.5, 1.5, and 2.5 mM Ca. In both the normal [K]o Krebs and the Ca-free-high [K]o Krebs solutions, MEPP frequency was lower at old than at young nerve terminals. In the three high [K]o Krebs solutions with Ca, MEPP frequency was progressively higher at old than at young nerve terminals with higher Ca concentrations. Periodic oscillations were observed in MEPP frequency of depolarized nerve terminals. The period of oscillation was inversely proportional to spontaneous transmitter release. These results demonstrate that when the nerve terminal is depolarized, permeability of the terminal membrane to Ca increases because of opening of voltage-dependent Ca channels. In the present study resting MEPP frequency was lower at old than at young terminals. On depolarization, MEPP frequency became higher at old than at young terminals. The study demonstrates that voltage-dependent Ca entry increases during aging at the soleus nerve terminal.     

Evidence for proton/sulfate cotransport and its kinetics inLemna gibba G1

Sulfate uptake into duckweed (Lemna gibba G1) was studied by means of [³⁵S]sulfate influx and measurements of electrical membrane potential. Uptake was strongly regulated by the intracellular content of soluble sulfate. At the onset of sulfate uptake the membrane potential was transiently depolarized. Fusicoccin stimulated uptake up to 165% of the control even at pH 8. It is suggested that sulfate uptake is energized in the whole pH range by a 3H⁺/sulfate cotransport mechanism. Kinetics of sulfate uptake and sulfate-induced membrane depolarization in the concentration range of 5 M to 1 mM sulfate at pH 5.7 was best described by two Michaelis-Menten terms without any linear component. The second system had a lower affinity for sulfate and was fully active only at sufficiently high proton concentrations.Abbreviations c _o extracellular sulfate concentration - c _i intracellular sulfate concentration - E _m electrical membrane potential difference - E _m sulfate-induced, maximal membrane depolarization - electrochemical proton gradient - FW fresh weight     

Effect of Alkali Metal Cations on Slow Inactivation of Cardiac Na+ Channels

Human heart Na⁺ channels were expressed transiently in both mammalian cells and Xenopus oocytes, and Na⁺ currents measured using 150 mM intracellular Na⁺. The kinetics of decaying outward Na⁺ current in response to 1-s depolarizations in the F1485Q mutant depends on the predominant cation in the extracellular solution, suggesting an effect on slow inactivation. The decay rate is lower for the alkali metal cations Li⁺, Na⁺, K⁺, Rb⁺, and Cs⁺ than for the organic cations Tris, tetramethylammonium, N-methylglucamine, and choline. In whole cell recordings, raising [Na⁺]_o from 10 to 150 mM increases the rate of recovery from slow inactivation at −140 mV, decreases the rate of slow inactivation at relatively depolarized voltages, and shifts steady-state slow inactivation in a depolarized direction. Single channel recordings of F1485Q show a decrease in the number of blank (i.e., null) records when [Na⁺]_o is increased. Significant clustering of blank records when depolarizing at a frequency of 0.5 Hz suggests that periods of inactivity represent the sojourn of a channel in a slow-inactivated state. Examination of the single channel kinetics at +60 mV during 90-ms depolarizations shows that neither open time, closed time, nor first latency is significantly affected by [Na⁺]_o. However raising [Na⁺]_o decreases the duration of the last closed interval terminated by the end of the depolarization, leading to an increased number of openings at the depolarized voltage. Analysis of single channel data indicates that at a depolarized voltage a single rate constant for entry into a slow-inactivated state is reduced in high [Na⁺]_o, suggesting that the binding of an alkali metal cation, perhaps in the ion-conducting pore, inhibits the closing of the slow inactivation gate.     

Effect of acidosis on the membrane potential of intact guinea pig aortic endothelial cells

The effects of a decrease in the extracellular pH from 7.4 to 6.9 on the membrane potential (MP) of intact non-stimulated guinea pig aortic endothelial cells and their ATP-induced electrical responses were studied using a whole-cell mode of the patch-clamp technique. Superfusion of the strip with CO₂-−HCO ₃ ⁻ -buffered acidic solution evoked endothelial depolarization of 6.1±1.0 mV. In Ca²⁺-free medium, after the MP had been stabilized at a depolarized value, there was no shift in the MP due to extracellular acidification to pH 6.9. In the case of using tris-buffered solution, the same drop in the extracellular pH in Ca²⁺-containing medium induced no change in the endothelial MP. Subsequent superfusion with CO₂−HCO ₃ ⁻ -buffered solution with pH 6.9 evoked endothelial depolarization of 7.3±1.5 mV. Changing from tris-buffered to CO₂−HCO ₃ ⁻ -buffered solution at a constant buffer pH 7.4 also induced endothelial depolarization, suggesting that intracellular pH is a possible factor that modulates leak Ca entry. The duration of ATP-induced endothelial hyperpolarization at pH 6.9 significantly dropped (76±5 sec, on average) compared with that at pH 7.4 (121±14 sec). It is concluded that modulatory effect of acidosis on the MP of endothelial cells and their ATP-induced electrical responses are caused by inhibition of leak and ATP-stimulated calcium entry into these cells.     

Ca2+ transport by intact synaptosomes: the voltage-dependent Ca2+ channel and a re-evaluation of the role of sodium/calcium exchange

The verapamil-sensitive Ca2+ channel in the synaptosomal plasma membrane is investigated. Verapamil is without effect on Ca2+ uptake or steady-state content in synaptosomes with a polarized plasma membrane, but completely inhibits the additional Ca2+ uptake following plasma-membrane depolarization by high [K+], by veratridine plus ouabain or by high concentrations of the permeant cation tetraphenylphosphonium. Verapamil-insensitive Ca2+ influx and steady-state content are identical in polarized and depolarized synaptosomes, even though the Na+ electrochemical potential is greatly decreased in the latter, indicating that Na+/Ca2+ exchange is not a significant mechanism for Ca2+ efflux under these conditions. A transient Na+-dependent Ca2+ efflux can only be observed on addition of Na+ to Na+-depleted depolarized synaptosomes. While 0.2 mM verapamil decreases the ate of 86Rb+ efflux and 22Na+ entry during depolarization induced by veratridine plus ouabain, the final steady-state Na+ accumulation is not inhibited. Ca2+ efflux from synaptosomes following mitochondrial depolarization does not occur by a verapamil-sensitive pathway.     

Effect of various ionic compositions upon the membrane potentials during activation of sea urchin eggs

The membrane potentials of sea urchin (Hemicentrotus pulcherrimus) eggs before and after fertilization and their changes during the membrane elevation induced by intracellular electrical stimulation were recorded in solutions of various ionic compositions. Upon fertilization, the membrane potential (?10 mV) depolarized and reversed polarity by a few mV, then gradually returned to a new steady level ranging between ?50 and ?60 mV. The activation potential is closely associated with a transient increase in the membrane permeability. The potential of the unfertilized egg is hyperpolarized by monovalent anions (Br^?, Cl^? and NO₃^?) and depolarized slightly by K⁺. In contrast, the membrane of the fertilized egg is markedly depolarized by K⁺. Suppression of depolarization associated with an increase of the membrane permeability was recorded in Na-free medium (Tris-HCl). The selective increase in permeability to monovalent anions is thought to alternate with the selective increase in permeability to K⁺through the mediation of a transient increase of Na⁺-permeability at the time of fertilization. No causal relationship between the membrane elevation and the depolarization was established because the breakdown of the cortical granules occurs without depolarization or an increase in membrane permeability.     

Ischemic changes in myocardial ionic contents of the isolated perfused rat hearts as studied by NMR

Using³¹P-,²³Na- and³⁹K-NMR, we assessed ischemic changes in high energy phosphates and ion contents of isolated perfused rat hearts continuously and systematically. To discriminate intra- and extracellular Na⁺, a shift reagent (Dy(TTHA)^3–) was used in²³Na-NMR study. In³⁹K-NMR study, the extracellular K⁺ signal was suppressed by inversion recovery pulse sequence in order to obtain intracellular K⁺ signal without using shift reagnets. During the early period of ischemia, increases in intracellular Na⁺ and inorganic phosphate (Pi) were observed in addition to the well-documented decreases in creatine phosphate and ATP and a fall of intracellular pH, suggesting an augmented operation of Na⁺–H⁺ exchange triggered by a fall of the intracellular pH resulted from breakdown of ATP. At around 15 min of ischemia, a second larger increase in intracellular Na⁺ and a decrease in intracellular K⁺ were observed in association with a second increase in Pi. This was accompnanied by an abrupt rise of the ventricular end-diastolic pressure. As there was a depletion of ATP at this time, the increase in intracellular Na⁺ and associated decrease in intracellular K⁺ may be explained by inhibition of the Na⁺–K⁺ ATPase due to the depletion of ATP. A longer observation with³¹P-NMR revealed a second phosphate peak (at lower magnetic field to ordinary Pi peak) which increased its intensity as ischemic time lengthened. The pH of this 2nd peak changed in parallel with the changes in pH of the bathing solution, indicating the appearance of a compartment whose hydrogen concentration is in equilibrium with that of the external compartment. Thus, the peak could be used as an index of irreversible membrane damage of the myocardium.     

The combined luminol/isoluminol chemiluminescence method for differentiating between extracellular and intracellular oxidant production by neutrophils

Abstract

To address the question why isoluminol, but not luminol, failed to detect oxidants produced intracellularly, differences between these luminophores were investigated with respect to physicochemical parameters and the character of chemiluminescence signal. Our results showed the isoluminol molecule to be more polar, more hydrophilic and possessing lower ability to form intramolecular bonds than the luminol molecule. Therefore, isoluminol: (i) only slightly pervaded biological membranes; (ii) depended essentially on extracellular peroxidase; (iii) did not produce chemiluminescence in the presence of extracellular scavengers; and (iv) it could be considered a specific detector of extracellular radicals. On the other hand, the physicochemical parameters of luminol and partial resistance of its chemiluminescence to the effect of extracellular inhibitors proved the lipo/hydrophilic character of this luminophore and thus its ability to interact with radicals both outside and inside of cells. The luminol chemiluminescence measured in the presence of extracellular scavengers and the isoluminol chemiluminescence were used with the intention to differentiate the effects of two antihistamine drugs on intra- and extracellular radical formation. In activated human neutrophils, brompheniramine inhibited the extracellular and potentiated the intracellular part of chemiluminescence signal, whereas a reducing effect of loratadine was observed in both compartments.     

Depolarization of the Electrogenic Transmembrane Electropotential of Zea mays L. by Bipolaris (Helminthosporium) maydis Race T Toxin, Azide, Cyanide, Dodecyl Succinic Acid, or Cold Temperature

The transmembrane electrical potential of root cells of Zea mays L. cv. W64A in a modified 1× Higinbotham solution was partially depolarized by semipurified toxin obtained from Bipolaris (Helminthosporium) maydis race T. At a given toxin concentration depolarization of Texas cytoplasm cells was much greater than for normal cytoplasm cells. This observation correlated directly to the differential host susceptibility to the fungus. The time course and magnitude of depolarization were dependent on toxin concentration; at high concentration the electropotential difference change was rapid. Cortex cells depolarized more slowly than epidermal cells indicating that the toxin slowly permeated intercellular regions. Toxin concentrations which affected electropotential difference were of the same magnitude as those required to inhibit root growth, ion uptake, and mitochondrial processes.

Azide, cyanide, and cold temperature (5 C) gave the same partial depolarization as did the toxin. Dodecyl succinic acid caused complete depolarization. These and other data indicate that one of the primary actions of the toxin is to inhibit electrogenic ion pumps in the plasmalemma.

     

Depolarization of the membrane potential by hyaluronan

The membrane potential is mainly maintained by the K⁺ concentration gradient across the cell membrane between the cytosol and the extracellular matrix. Here, we show that extracellular addition of high‐molecular weight hyaluronan depolarized the membrane potential of human fibroblasts, human embryonic kidney cells (HEK), and central nervous system neurons in a concentration‐dependent manner, whereas digestion of cell surface hyaluronan by hyaluronidase caused hyperpolarization. This effect could not be achieved by other glycosaminoglycans or hyaluronan oligosaccharides, chondroitin sulfate, and heparin which did not affect the membrane potential. Mixtures of high‐molecular weight hyaluronan and bovine serum albumin had a larger depolarization effect than expected as the sum of both individual components. The different behavior of high‐molecular weight hyaluronan versus hyaluronan oligosaccharides and other glycosaminoglycans can be explained by a Donnan effect combined with a steric exclusion of other molecules from the water solvated chains of high‐molecular weight hyaluronan. Depolarization of the plasma membrane by hyaluronan represents an additional pathway of signal transduction to the classical CD44 signal transduction pathway, which links the extracellular matrix to intracellular metabolism. J. Cell. Biochem. 111: 858–864, 2010. © 2010 Wiley‐Liss, Inc.     

Relationship between Energy-dependent Phosphate Uptake and the Electrical Membrane Potential in Lemna gibba G1

High rates of phosphate uptake into phosphate-starved Lemna gibba L. G1 were correlated with a high membrane potential (pd = −220 millivolts). In plants maintaining a low pd (−110 millivolts), the uptake rate was only 20% of that of high-pd plants. At the onset of phosphate transport, the membrane of high-pd plants was transiently depolarized. This effect was much smaller in low-pd plants. Light stimulated phosphate uptake and the repolarization upon phosphate-induced depolarization, especially in plants grown without sucrose. The phosphate uptake rate was optimal at pH 6 and decreased with increasing pH, corresponding to the phosphate-induced pd changes. Phosphate starvation stimulated the uptake and increased the phosphate-induced depolarization, thus indicating that phosphate uptake depends on the intracellular phosphate level. It is suggested that uptake of monovalent phosphate in Lemna gibba proceeds by an H⁺ cotransport dependent on the proton electrochemical potential difference and, hence, on the activity of an H⁺ -extrusion pump.     

Effects of Inhibitors on the Light-Induced Changes in Electric Potential in Pulvinar Motor Cells of Phaseolus vulgaris L.

To analyze the mechanism of the light-induced changes in electricpotential in motor cells of the pulvinus of Phaseolus vulgarisL., inhibitors were applied to the pulvinus by the xylem perfusionmethod. The membrane potential was –60 to –80 mV,which indicated that the polarization was less than that ofcells of a pulvinus in air. A pulse (30 s) of blue light (BL)induced transient depolarization of the membrane in the motorcells. Red light (RL) caused hyperpolarization of the membrane.The magnitude of BL pulse-induced transient depolarization wasgreater under the hyperpolarized state caused by the RL. The membrane was depolarized to –30 to –40 mV onperfusion with the respiratory inhibitor NaN₃ (1 mM) and a pulseof BL or irradiation with RL did not cause any change in thepotential in the depolarized state. Hyperpolarization of themembrane by RL was inhibited by perfusion with DCMU (50 µM),an inhibitor of electron transport in photosynthesis. However,the magnitude of the depolarization induced by the pulse ofBL was not affected. Perfusion with a proton ionophore CCCP(100µM) depolarized the membrane and no change in thepotential was induced by a pulse of BL or by RL in the depolarizedstate. The extent of the BL pulse-induced depolarization of the membranewas proportional to the magnitude of the membrane potentialat the time of which the pulse of BL was applied. It is suggestedthat the active component of the membrane potential was inhibitedby the pulse of BL. The experimental results further supportthe hypothesis that BL inhibits the activity of the proton ATPaseand, thus, causes loss of the electrogenic component of themembrane potential of the pulvinar motor cells. (Received June 22, 1992; Accepted August 24, 1992)     

Depolarization of the membrane potential decreases the ATP-induced influx of extracellular Ca2+ and the refilling of intracellular Ca2+ stores in rat thyroid FRTL-5 cells.

The aim of the present study was to investigate the effect of membrane depolarization on ATP-induced changes in intracellular Ca2+ ([Ca2+]i) and the refilling of intracellular Ca2+ stores in thyroid follicular FRTL-5 cells. Depolarizing the cells with 50 mM K+, an amount sufficient to almost totally depolarize the cells as determined by bisoxonal, significantly reduced the ATP-induced uptake of 45Ca2+. This effect was not dependent on an enhanced efflux of Ca2+, as no difference in the ATP-induced efflux of 45Ca2+ was obtained between control cells and depolarized cells. The ATP-induced transient increase in [Ca2+]i in Fura-2 loaded cells was not altered by depolarization, whereas the ATP-induced plateau in [Ca2+]i was decreased compared with control cells. Furthermore, in cells stimulated with ATP in a Ca(2+)-free buffer, readdition of Ca2+ after the termination of the ATP response induced a decreased response in [Ca2+]i in depolarized cells. Refilling of intracellular Ca2+ stores was investigated by first stimulating the cells with noradrenaline (NA). The effect of NA was then terminated with prazosin, and the cells restimulated with ATP. In cells depolarized with high K+, the response to ATP was decreased compared with that seen in control cells. The results thus suggest that both the ATP-induced influx of extracellular Ca2+ and the refilling of intracellular Ca2+ stores is decreased in depolarized FRTL-5 cells.     

Calcium fluxes in T lymphocytes.

Mechanisms controlling Ca2+ fluxes through the plasma membrane of lymphocytes have been characterized in a human T-cell clone and in the Jurkat T-cell line. Due to endogenous buffers, about 1/125 of the Ca2+ ions that enter the cell are free. Ca2+ fluxes were estimated from the variations in intracellular Ca2+ concentration ([Ca2+]i) elicited by concentration jumps in extracellular Ca2+ ([Ca2+]o). Thapsigargin was used to inhibit Ca2+ uptake into intracellular stores and to stimulate Ca2+ entry. Ca2+ extrusion was strictly due to the activity of plasma membrane Ca(2+)-ATPases since there was no detectable Na+/Ca2+ exchange activity in these cells. The rate of Ca2+ extrusion was mainly influenced by [Ca2+]i and less by [Ca2+]o but was insensitive to cell depolarization. In depolarized cells, thapsigargin-induced Ca2+ influx was reduced to 10% of the value measured in normally polarized cells, suggesting that depolarization not only reduces the electrochemical gradient for Ca2+ ions, but also inhibits Ca2+ permeation. When Ca2+ ions enter the cell, they bind to a site inside the channel, with a Kd of 3.3 mM. Stimulation of clonal T-cells with low concentrations of either anti-CD3 antibodies or thapsigargin elicited Ca2+ oscillations. Both the amplitude and the frequency of CD3-induced Ca2+ oscillations were sensitive to [Ca2+]o. These oscillations were immediately interrupted when extracellular Ca2+ was removed. The properties of Ca2+ oscillations in T lymphocytes suggest that they are mainly due to variations of Ca2+ influx, modulated by variations in [Ca2+]i.