On the Role of Calcium in the Regulatory Volume Decrease (RVD) Response in Ehrlich Mouse Ascites Tumor Cells

The putative role for Ca²⁺ entry and Ca²⁺ mobilization in the activation of the regulatory volume decrease (RVD) response has been assessed in Ehrlich cells. Following hypotonic exposure (50% osmolarity) there is: (i) no increase in cellular Ins(1,4,5)P₃ content, as measured in extracts from [2-³H]myoinositol-labeled cells, a finding at variance with earlier reports from our group; (ii) no evidence of Ca²⁺-signaling recorded in a suspension of fura-2-loaded cells; (iii) Ca²⁺-signaling in only about 6% of the single, fura-2-loaded cells at 1-mm Ca²⁺ (1% only at 0.1-mm Ca²⁺ and in Ca²⁺-free medium), as monitored by fluorescence-ratio imaging; (iv) no effect of removing external Ca²⁺ upon the volume-induced K⁺ loss; (v) no significant inhibition of the RVD response in cells loaded with the Ca²⁺ chelator BAPTA when the BAPTA-loading is performed in K⁺ equilibrium medium; (vi) an inhibition of the swelling-induced K⁺ loss (about 50%) at 1-mm Ba²⁺, but almost no effect of charybdotoxin (100 nm) or of clotrimazole (10 μm), reported inhibitors of the K⁺ loss induced by Ca²⁺-mobilizing agonists. Thus, Ca²⁺signaling by Ca²⁺ release or Ca²⁺ entry appears to play no role in the activation mechanism for the RVD response in Ehrlich cells. Received: 8 December 1996/Revised: 14 January 1997     

Separate Swelling- and Ca2+-activated Anion Currents in Ehrlich Ascites Tumor Cells

A Ca²⁺-activated (I _Cl,Ca) and a swelling-activated anion current (I _Cl,vol) were investigated in Ehrlich ascites tumor cells using the whole cell patch clamp technique. Large, outwardly rectifying currents were activated by an increase in the free intracellular calcium concentration ([Ca²⁺] _i ), or by hypotonic exposure of the cells, respectively. The reversal potential of both currents was dependent on the extracellular Cl⁻ concentration. I _Cl,Ca current density increased with increasing [Ca²⁺] _i , and this current was abolished by lowering [Ca²⁺] _i to <1 nm using 1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA). In contrast, activation of I _Cl,vol did not require an increase in [Ca²⁺] _i . The kinetics of I _Cl,Ca and I _Cl,vol were different: at depolarized potentials, I _Cl,Ca as activated in a [Ca²⁺] _i - and voltage-dependent manner, while at hyperpolarized potentials, the current was deactivated. In contrast, I _Cl,vol exhibited time- and voltage-dependent deactivation at depolarized potentials and reactivation at hyperpolarized potentials. The deactivation of I _Cl,vol was dependent on the extracellular Mg²⁺ concentration. The anion permeability sequence for both currents was I ⁻ > Cl⁻ > gluconate. I _Cl,Ca was inhibited by niflumic acid (100 μm), 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 μm) and 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS, 100 μm), niflumic acid being the most potent inhibitor. In contrast, I _Cl,vol was unaffected by niflumic acid (100 μm), but abolished by tamoxifen (10 μm). Thus, in Ehrlich cells, separate chloride currents, I _Cl,Ca and I _Cl,vol, are activated by an increase in [Ca²⁺] _i and by cell swelling, respectively. Received: 12 November 1997/Revised: 5 February 1998     

Leukotriene D4-induced Ca2+ Mobilization in Ehrlich Ascites Tumor Cells

Stimulation of Ehrlich ascites tumor cells with leukotriene D₄ (LTD₄) within the concentration range 1–100 nm leads to a concentration-dependent, transient increase in the intracellular, free Ca²⁺ concentration, [Ca²⁺] _i . The Ca²⁺ peak time, i.e., the time between addition of LTD₄ and the highest measured [Ca²⁺] _i value, is in the range 0.20 to 0.21 min in ten out of fourteen independent experiments. After addition of a saturating concentration of LTD₄ (100 nm), the highest measured increase in [Ca²⁺] _i in Ehrlich cells suspended in Ca²⁺-containing medium is 260 ± 14 nm and the EC₅₀ value for LTD₄-induced Ca²⁺ mobilization is estimated at 10 nm. Neither the peptido-leukotrienes LTC₄ and LTE₄ nor LTB₄ are able to mimic or block the LTD₄-induced Ca²⁺ mobilization, hence the receptor is specific for LTD₄. Removal of Ca²⁺ from the experimental buffer significantly reduces the size of the LTD₄-induced increase in [Ca²⁺] _i . Furthermore, depletion of the intracellular Ins(1,4,5)P₃-sensitive Ca²⁺ stores by addition of the ER-Ca²⁺-ATPase inhibitor thapsigargin also reduces the size of the LTD₄-induced increase in [Ca²⁺] _i in Ehrlich cells suspended in Ca²⁺-containing medium, and completely abolishes the LTD₄-induced increase in [Ca²⁺] _i in Ehrlich cells suspended in Ca²⁺-free medium containing EGTA. Thus, the LTD₄-induced increase in [Ca²⁺] _i in Ehrlich cells involves an influx of Ca²⁺ from the extracellular compartment as well as a release of Ca²⁺ from intracellular Ins(1,4,5)P₃-sensitive stores. The Ca²⁺ peak times for the LTD₄-induced Ca²⁺ influx and for the LTD₄-induced Ca²⁺ release are recorded in the time range 0.20 to 0.21 min in four out of five experiments and in the time range 0.34 to 0.35 min in six out of eight experiments, respectively. Stimulation with LTD₄ also induces a transient increase in Ins(1,4,5)P₃ generation in the Ehrlich cells, and the Ins(1,4,5)P₃ peak time is recorded in the time range 0.27 to 0.30 min. Thus, the Ins(1,4,5)P₃ content seems to increase before the LTD₄-induced Ca²⁺ release from the intracellular stores but after the LTD₄-induced Ca²⁺ influx. Inhibition of phospholipase C by preincubation with U73122 abolishes the LTD₄-induced increase in Ins(1,4,5)P₃ as well as the LTD₄-induced increase in [Ca²⁺] _i , indicating that a U73122-sensitive phospholipase C is involved in the LTD₄-induced Ca²⁺ mobilization in Ehrlich cells. The LTD₄-induced Ca²⁺ influx is insensitive to verapamil, gadolinium and SK&F 96365, suggesting that the LTD₄-activated Ca²⁺ channel in Ehrlich cells is neither voltage gated nor stretch activated and most probably not receptor operated. In conclusion, LTD₄ acts in the Ehrlich cells via a specific receptor for LTD₄, which upon stimulation initiates an influx of Ca²⁺, through yet unidentified Ca²⁺ channels, and an activation of a U73122-sensitive phospholipase C, Ins(1,4,5)P₃ formation and finally release of Ca²⁺ from the intracellular Ins(1,4,5)P₃-sensitive stores. Received: 9 February 1996/Revised: 15 August 1996     

Regulatory Volume Decrease by SV40-transformed Rabbit Corneal Epithelial Cells Requires Ryanodine-sensitive Ca2+-induced Ca2+ Release

The relationship between relative cell volume and time-dependent changes in intracellular Ca²⁺ concentration ([Ca²⁺] _i ) during exposure to hypotonicity was characterized in SV-40 transformed rabbit corneal epithelial cells (tRCE) (i). Light scattering measurements revealed rapid initial swelling with subsequent 97% recovery of relative cell volume (characteristic time (τ _vr ) was 5.9 min); (ii). Fura2-fluorescence single-cell imaging showed that [Ca²⁺] _i initially rose by 216% in 30 sec with subsequent return to near baseline level after another 100 sec. Both relative cell volume recovery and [Ca²⁺] _i transients were inhibited by either: (a) Ca²⁺-free medium; (b) 5 mm Ni²⁺ (inhibitor of plasmalemma Ca²⁺ influx); (c) 10 μm cyclopiazonic acid, CPA (which causes depletion of intracellular Ca²⁺ content); or (d) 100 μm ryanodine (inhibitor of Ca²⁺ release from intracellular stores). To determine the temporal relationship between an increased plasmalemma Ca²⁺ influx and the emptying of intracellular Ca²⁺ stores during the [Ca²⁺] _i transients, Mn²⁺ quenching of fura2-fluorescence was quantified. In the presence of CPA, hypotonic challenge increased plasmalemma Mn²⁺ permeability 6-fold. However, Mn²⁺ permeability remained unchanged during exposure to either: 1.100 μm ryanodine; 2.10 μm CPA and 100 μm ryanodine. This report for the first time documents the time dependence of the components of the [Ca²⁺] _i transient required for a regulatory volume decrease (RVD). The results show that ryanodine sensitive Ca²⁺ release from an intracellular store leads to a subsequent increase in plasmalemma Ca²⁺ influx, and that both are required for cells to undergo RVD. Received: 7 November 1996/Revised: 6 January 1997     

Shrinkage-induced Activation of the Na+/H+ Exchanger in Ehrlich Ascites Tumor Cells: Mechanisms Involved in the Activation and a Role for the Exchanger in Cell Volume Regulation

Amiloride-sensitive, Na⁺-dependent, DIDS-insensitive cytoplasmic alkalinization is observed after hypertonic challenge in Ehrlich ascites tumor cells. This was assessed using the fluorescent pH-sensitive probe 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). A parallel increase in the amiloride-sensitive unidirectional Na⁺ influx is also observed. This indicates that hypertonic challenge activates a Na⁺/H⁺ exchanger. Activation occurs after several types of hypertonic challenge, is a graded function of the osmotic challenge, and is temperature-dependent. Observations on single cells reveal a considerable variation in the shrinkage-induced changes in cellular pH _i , but the overall picture confirms the results from cell suspensions. Shrinkage-induced alkalinization and recovery of cellular pH after an acid load, is strongly reduced in ATP-depleted cells. Furthermore, it is inhibited by chelerythrine and H-7, inhibitors of protein kinase C (PKC). In contrast, Calyculin A, an inhibitor of protein phosphatases PP1 and PP2A, stimulates shrinkage-induced alkalinization. Osmotic activation of the exchanger is unaffected by removal of calcium from the experimental medium, and by buffering of intracellular free calcium with BAPTA. At 25 mm HCO⁻ ₃, but not in nominally HCO⁻ ₃-free medium, Na⁺/H⁺ exchange contributes significantly to regulatory volume increase in Ehrlich cells. Under isotonic conditions, the Na⁺/H⁺ exchanger is activated by ionomycin, an effect which may be secondary to ionomycin-induced cell shrinkage. Received: 2 March 1995/Revised: 29 September 1995     

Cell Swelling Activates Phospholipase A2 in Ehrlich Ascites Tumor Cells

Ehrlich ascites tumor cells, loaded with ³H-labeled arachidonic acid and ¹⁴C-labeled stearic acid for two hours, were washed and transferred to either isotonic or hypotonic media containing BSA to scavenge the labeled fatty acids released from the cells. During the first two minutes of hypo-osmotic exposure the rate of ³H-labeled arachidonic acid release is 3.3 times higher than that observed at normal osmolality. Cell swelling also causes an increase in the production of ¹⁴C-stearic acid-labeled lysophosphatidylcholine. This indicates that a phospholipase A₂ is activated by cell swelling in the Ehrlich cells. Within the same time frame there is no swelling-induced increase in ¹⁴C-labeled stearic acid release nor in the synthesis of phosphatidyl ¹⁴C-butanol in the presence of ¹⁴C-butanol. Furthermore, U7312, an inhibitor of phospholipase C, does not affect the swelling induced release of ¹⁴C-labeled arachidonic acid. Taken together these results exclude involvement of phospholipase A₁, C and D in the swelling-induced liberation of arachidonic acid. The swelling-induced release of ³H-labeled arachidonic acid from Ehrlich cells as well as the volume regulatory response are inhibited after preincubation with GDP_βS or with AACOCF₃, an inhibitor of the 85 kDa, cytosolic phospholipase A₂. Based on these results we propose that cell swelling activates a phospholipase A₂—perhaps the cytosolic 85 kDa type—by a partly G-protein coupled process, and that this activation is essential for the subsequent volume regulatory response. Received: 23 July 1996/Revised: 17 June 1997     

Protein Kinase C and Regulatory Volume Decrease in Mudpuppy Red Blood Cells

This study examined whether protein kinase C (PKC) stimulates K⁺ efflux during regulatory volume decrease (RVD) in Necturus maculosus (mudpuppy) red blood cells (RBCs). The limit of osmotic fragility increased with the general protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, 10 μm), but not with the cyclic nucleotide-dependent kinase antagonists N-(2′-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004, 10 μm) and N-2-(methylamino)ethyl-5-isoquinoline-sulfonamide (H-8, 5 μm). Consistent with these results, osmotic fragility also increased with the PKC antagonists bisindolylmaleimide I (GF-109203X or bis I, 100 nm), bisindolylmaleimide II (bis II, 100 nm), and chelerythrine (10 μm). The effect of these three antagonists and H-7 was reversed with gramicidin (5 μm in a choline Ringer), indicating PKC was linked to K⁺ efflux (gramicidin is a cationophore that was used to ensure a high K⁺ permeability). We also measured cell volume recovery from hypotonic shock (0.5× Ringer) with a Coulter counter and estimated cell volume from the hematocrit. The percent RVD compared to control decreased with H-7 (10 μm), sphingosine (100 nm), chelerythrine (10 μm), bis I (100 nm), and bis II (100 nm), but not with HA-1004 (10 μm) nor H-8 (5 μm). Inhibition of RVD by H-7, chelerythrine, bis I, and bis II was reversed with gramicidin (5 μm). Furthermore, using the patch clamp technique, we found H-7 (10 μm) reduced a whole cell conductance that was activated during cell swelling. In addition, a conductance responsible for K⁺ efflux during cell swelling was inhibited by bis I (100 nm) and bis II (100 nm). These results indicate that a conductive pathway mediating K⁺ loss during RVD is regulated, at least in part, by protein kinase C. Received: 20 January 1998/Revised: 2 September 1998     

Characterization of the Increase in [Ca2+] i During Hypotonic Shock and the Involvement of Ca2+-activated K+ Channels in the Regulatory Volume Decrease in Human Osteoblast-like Cells

The calcium indicator fura-2 was used to study the effect of hypotonic solutions on the intracellular calcium concentration, [Ca²⁺] _i , in a human osteoblast-like cell line. Decreasing the tonicity of the extracellular solution to 50% leads to an increase in [Ca²⁺] _i from ∼150 nm up to 1.3 μm. This increase in [Ca²⁺] _i was mainly due to an influx of extracellular Ca²⁺ since removing of extracellular Ca²⁺ reduced this increase to ∼250 nm. After cell swelling most of the cells were able to regulate their volume to the initial level within 800 sec. The whole-cell recording mode of the patch-clamp technique was also used to study the effect of an increase in [Ca²⁺] _i on membrane currents in these cells. An increase in [Ca²⁺] _i revealed two types of Ca²⁺-activated K⁺ channels, K(Ca) channels. Current through both channel types could not be observed below voltage of +80 mV with [Ca²⁺] _i buffered to 100 nm or less. With patch-electrodes filled with solutions buffering [Ca²⁺] _i to 10 μm both channels types could be readily observed. The activation of the first type was apparently voltage-independent since current could be observed over the entire voltage range used from −160 to +100 mV. In addition, the current was also blocked by charybdotoxin (CTX). The second type of K(Ca) channels in these cells could be activated with depolarizations more positive than −40 mV from a holding potential of −80 mV. This type was blocked by CTX and paxilline. Adding paxilline to the extracellular solution inhibited regulatory volume decrease (RVD), but could not abolish RVD. We conclude that two K(Ca) channel types exist in human osteoblasts, an intermediate conductance K(Ca) channel and a MaxiK-like K(Ca) channel. MaxiK channels might get activated either directly or by an increase in [Ca²⁺] _i elicited through hypotonic solutions. In combination with the volume-regulated Cl⁻ conductance in the same cells this K⁺ channel seems to play a vital role in volume regulation in human osteoblasts. Received: 8 February 2000/Revised: 13 July 2000     

The Role of Calcium in the Volume Regulation of Rat Lacrimal Acinar Cells

Earlier studies have suggested a role for Ca²⁺ in regulatory volume decrease (RVD) in response to hypotonic stress through the activation of Ca²⁺-dependent ion channels (Kotera & Brown, 1993; Park et al., 1994). The involvement of Ca²⁺ in regulating cell volume in rat lacrimal acinar cells was therefore examined using a video-imaging technique to measure cell volume. The trivalent cation Gd³⁺ inhibited RVD, suggesting that Ca²⁺ entry is important and may be via stretch-activated cation channels. However, Fura-2 loaded cells did not show an increase in [Ca²⁺] _i during exposure to hypotonic solutions. The absence of any changes in [Ca²⁺] _i resulted from the buffering of cytosolic Ca²⁺ by Fura-2 during hypotonic shock and therefore inhibition of RVD. The intracellular Ca²⁺ chelator, BAPTA, also inhibited the RVD response to hypotonic shock. An increase in [Ca²⁺] _i induced by either acetylcholine or ionomycin, was found to decrease cell volume under isotonic conditions in lacrimal acinar cells. Cell shrinkage was inhibited by tetraethylammonium ion, an inhibitor of Ca²⁺-activated K⁺ channels. On the basis of the presented data, we suggest an involvement of intracellular Ca²⁺ in controlling cell volume in lacrimal acinar cells. Received: 20 February 1998/Revised: 1 May 1998     

ClC-2 Activation Modulates Regulatory Volume Decrease

ClC-2 belongs to a large family of chloride channels and its expression in certain cell types is associated with the appearance of swelling-activated chloride (Cl⁻) currents. In the present report, we examined the hypothesis that ClC-2 plays a role in regulatory volume decrease by expressing ClC-2 in Sf9 cells using the baculovirus system. First, we showed that ClC-2 protein expression is associated with appearance of a Cl⁻ conductance which is activated by hypo-osmotic shock and can be distinguished from swelling-activated chloride currents endogenous to Sf9 cells on the basis of its pharmacology and specific inhibition by an anti-ClC-2 antibody. Second, we show that the rate of regulatory volume decrease is significantly enhanced in Sf9 cells expressing ClC-2 protein. Hence, our data support the hypothesis that ClC-2 is capable of mediating regulatory volume decrease. Received: 12 August/Revised: 23 October 1998     

Phosphorylation is Involved in the Regulation of the Taurine Influx Via the β-system in Ehrlich Ascites Tumor Cells

The role of 3′,5′-cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), protein kinase C (PKC) and phosphatases in the regulation of the taurine influx via the β-system in Ehrlich ascites tumor cells has been investigated. The taurine uptake by the β-system in Ehrlich cells is inhibited when PKC is activated by phorbol 12-myristate 13-acetate (PMA) and when protein phosphatases are inhibited by calyculin A (CLA). On the other hand, taurine uptake by the β-system is stimulated by an increased level of cAMP or following addition of N⁶,2′-O-dibutyryl-3′,5′-cyclic adenosine monophosphate (dbcAMP). The effect of dbcAMP is partially blocked by addition of the protein kinase inhibitor H-89, and suppressed in the presence of CLA. It is proposed that the β-system in the Ehrlich cells exists in three states of activity: State I, where a PKC phosphorylation site on the transporter or on a regulator is phosphorylated and transport activity is low. State II, where the PKC phosphorylation site is dephosphorylated and transport activity is normal. State III, representing a state with high transport activity, induced by an elevated cellular cAMP level. Apparently, cAMP preferentially stimulates taurine transport when the β-system is in State II. Received: 8 September/Revised: 9 November 1995     

Mechanosensitive Calcium Entry and Mobilization in Renal A6 Cells

Using spectrofluorescence imaging of fura-2 loaded renal A6 cells, we have investigated the generation of the cytosolic Ca²⁺ signal in response to osmotic shock and localized membrane stretch. Upon hypotonic exposure, the cells began to swell prior to a transient increase in [Ca²⁺] _i and the cells remained swollen after [Ca²⁺] _i had returned towards basal levels. Exposure to 2/3rd strength Ringer produced a cell volume increase within 3 min, followed by a slow regulatory volume decrease (RVD). The hypotonic challenge also produced a transient increase in [Ca²⁺] after a delay of 22 sec. Both the RVD and [Ca²⁺] _i response to hypotonicity were inhibited in a Ca²⁺-free bathing solution and by gadolinium (10 μm), an inhibitor of stretch-activated channels. Stretching the membrane by application of subatmospheric pressure (-2 kPa) inside a cell-attached patch-pipette induced a similar global increase in [Ca²⁺] _i as occurred after hypotonic shock. A stretch-sensitive [Ca²⁺] _i increase was also observed in a Ca²⁺-free bathing solution, provided the patch-pipette contained Ca²⁺. The mechanosensitive [Ca²⁺] _i response was by gadolinium (10 μm) or Ca²⁺-free pipette solutions, even when Ca²⁺ (2 mm) was present in the bath. Long-term (>10 min) pretreatment of the cells with thapsigargin inhibited the [Ca²⁺] _i response to hypotonicity. These results provide evidence that cell swelling or mechanical stimulation can activate a powerful amplification system linked to intracellular Ca²⁺ release mechanisms. Received: 3 August 1998/Revised: 19 November 1998     

5-Lipoxygenase Metabolites of Arachidonic Acid Regulate Volume Decrease by Mudpuppy Red Blood Cells

We examined whether metabolites of arachidonic acid (AA) regulate K⁺ efflux during regulatory volume decrease (RVD) by mudpuppy red blood cells (RBCs). Volume regulation was inhibited by the phospholipase A₂ antagonists mepacrine (10 μm) and ONO-RS-082 (10 μm); the inhibitory effect of ONO-RS-082 was reversed by gramicidin (5 μm). Eicosatetraynoic acid (ETYA, 100 μm), a general antagonist of AA metabolism, also blocked RVD. In addition, volume regulation was inhibited by the lipoxygenase pathway antagonist nordihydroguaiaretic acid (NDGA, 10 μm), the 5 lipoxygenase antagonists AA-861 (5 μm) and curcumin (20 μm), and by the 5-lipoxygenase activating protein inhibitor L-655,298 (5 μm). Inhibition by all four of these agents was reversed with gramicidin. In contrast, the 12- and 15-lipoxygenase pathway inhibitor ethyl-3,4-dihydroxy-benzylidene-cyanoacetate (EDBCA, 1 μm) and the cytochrome P-450 monooxygenase pathway blocker ketoconazole (20 μm) had no effect. On the other hand, the cyclooxygenase pathway inhibitor aspirin (100 μm) slightly enhanced RVD. Consistent with these findings, a K⁺-selective whole cell conductance responsible for K⁺ efflux during cell swelling was inhibited by ONO-RS-082 (10 μm), NDGA (10 μm), AA-861 (5 μm), curcumin (20 μm), and l-655,298 (5 μm). In contrast, EDBCA (1 μm), ketoconazole (20 μm), and indomethacin (10 μm) did not block this whole cell conductance. These results indicate that a channel mediating K⁺ loss during RVD is regulated by a 5-lipoxygenase metabolite of arachidonic acid. Received: 12 December 1996/Revised: 28 February 1997     

Distinct Mechanisms of [Ca2+]i Oscillations in HSY and HSG Cells: Role of Ca2+ Influx and Internal Ca2+ Store Recycling

This study examined [Ca²⁺]_i oscillations in the human salivary gland cell lines, HSY and HSG. Relatively low concentrations of carbachol (CCh) induced oscillatory, and higher [CCh] induced sustained, steady-state increases in [Ca²⁺]_i and K _Ca currents in both cell types. Low IP₃, but not thapsigargin (Tg), induced [Ca²⁺]_i oscillations, whereas Tg blocked CCh-stimulated [Ca²⁺]_i oscillations in both cell types. Unlike in HSG cells, removal of extracellular Ca²⁺ from HSY cells (i) did not affect CCh-stimulated [Ca²⁺]_i oscillations or internal Ca²⁺ store refill, and (ii) converted high [CCh]-induced steady-state increase in [Ca²⁺]_i into oscillations. CCh- or thapsigargin-induced Ca²⁺ influx was higher in HSY, than in HSG, cells. Importantly, HSY cells displayed relatively higher levels of sarcoendoplasmic reticulum Ca²⁺ pump (SERCA) and inositoltrisphosphate receptors (IP₃Rs) than HSG cells. These data demonstrate that [Ca²⁺]_i oscillations in both HSY and HSG cells are primarily determined by the uptake of Ca²⁺ from, and release of Ca²⁺ into, the cytosol by the SERCA and IP₃R activities, respectively. In HSY cells, Ca²⁺ influx does not acutely contribute to this process, although it determines the steady-state increase in [Ca²⁺]_i. In HSG cells, [Ca²⁺]_i oscillations directly depend on Ca²⁺ influx; Ca²⁺ coming into the cell is rapidly taken up into the store and then released into the cytosol. We suggest that the differences in the mechanism of [Ca²⁺]_i oscillations HSY and HSG cells is related to their respective abilities to recycle internal Ca²⁺ stores. Received: 30 October 2000/Revised: 26 February 2001     

Calcium-dependent Enhancement of Depletion-activated Calcium Current in Jurkat T Lymphocytes

We have obtained evidence that the Ca²⁺-selective current activated by Ca²⁺ store depletion (Ca²⁺ release-activated Ca²⁺ current; I _crac) in Jurkat T lymphocytes is augmented in a time-dependent manner by Ca²⁺ itself. Whole cell patch clamp experiments employed high cytosolic Ca²⁺-buffering conditions to passively deplete Ca²⁺ stores. Rapidly switching to nominally Ca²⁺-free extracellular buffer instantaneously reduced I _crac measured at −100 mV to leak current level. Unexpectedly, readmission of 2 mm Ca²⁺ instantaneously restored only 38 ± 5% (mean ±sem; n = 9) of the full I _crac amplitude. The remainder reappeared in a monotonic time-dependent manner over 10 to 20 sec. Rapid vs. slow intracellular Ca²⁺ chelators did not alter this process, and inorganic I _crac blockers did not regenerate it, arguing against an intracellular site of action. The effect was specific to Ca²⁺: introduction of the permeant ions, Ba²⁺ or Sr²⁺, failed to invoke time-dependent I _crac reappearance. Moreover, equimolar substitution of Ba²⁺ for Ca²⁺ initially produced Ba²⁺ current of similar magnitude to the full Ca²⁺ current, but the Ba²⁺ current decayed monotonically to <50% of its initial amplitude in <20 sec. Conversely, return to Ca²⁺ produced a time-dependent increase in I _crac to its larger Ca²⁺ permeation level. Thus Ca²⁺ appears to selectively promote a reversible transition of I _crac that results in larger current flux, and at least partially explains the selectivity of this current for Ca²⁺ over other divalent ions. Received: 30 August 1995/Revised: 7 November 1995     

Regulation of Intracellular Ca2+ by CFTR in Chinese Hamster Ovary Cells

In cystic fibrosis, the mutation of the CFTR protein causes reduced transepithelial Cl⁻ secretion. As recently proposed, beside its role of Cl⁻ channel, CFTR may regulate the activity of other channels such as a Ca²⁺-activated Cl⁻ channel. Using a calcium imaging system, we show, in adenovirus-CFTR infected Chinese Hamster Ovary (CHO) cell monolayers, that CFTR can act as a regulator of intracellular [Ca²⁺] _i ([Ca²⁺] _i ), involving purino-receptors. Apical exposure to ATP or UTP produced an increase in ([Ca²⁺] _i in noninfected CHO cell monolayers (CHO-WT), in CHO monolayers infected with an adenovirus-CFTR (CHO-CFTR) or infected with an adenovirus-LacZ (CHO-LacZ). The transient [Ca²⁺] _i increase produced by ATP or UTP could be mimicked by activation of CFTR with forskolin (20 μm) in CHO-CFTR confluent monolayers. However, forskolin had no significant effect on [Ca²⁺] _i in noninfected CHO-WT or in CHO-LacZ cells. Pretreatment with purino-receptor antagonists such as suramin (100 μm) or reactive blue-2. (100 μm), and with hexokinase (0.28 U/mg) inhibited the [Ca²⁺] _i response to forskolin in CHO-CFTR infected cells. Taken together, our experiments provide evidence for purino-receptor activation by ATP released from the cell and regulation of [Ca²⁺] _i by CFTR in CHO epithelial cell membranes. Received: 5 April 1999/Revised: 28 June 1999     

Single K+ Channels in Embryonic Leech Ganglion Cells

We investigated the properties of single K⁺ channels in the soma membrane of embryonic leech ganglion cells using the patch-clamp technique. We compared these K⁺ channels with the K⁺ channels found previously in Retzius neurons of the adult leech. In ganglion cells of 9- to 15-day-old embryos we characterized eight different types of K⁺ channels with mean conductances of 21, 55, 84, 111, 122, 132, 149 and 223 pS. The 55 pS and 84 pS channels showed flickering and were active for less than 2 min after excising the patch. The 111 pS channel was an outward rectifier, and the open state probability (p _o ) decreased in the inside-out configuration when the Ca²⁺ concentration was raised from pCa 7 to pCa 3. The 122 pS channel also showed outward rectification. This type of channel was activated after changing from the cell-attached to the inside-out configuration and it did not inactivate during more than 30 min. The p _o was Ca²⁺- and voltage-insensitive. One hundred μm glibenclamide reversibly reduced p _o . The 132 pS channel was an outward rectifier and was Ca²⁺-insensitive. The 149 pS channel inactivated in the inside-out configuration. The 149- and the 223 pS channel showed inward rectification. The 111 pS channel had similar properties to the Ca²⁺-dependent K⁺ channel and the 122 pS channel resembled the ATP-inhibited K⁺ channel found previously in Retzius neurons of the adult leech. Received: 20 April 1995/Revised: 18 January 1996