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     

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     

Ca2+/Calmodulin Kinase II and Decreases in Intracellular pH are Required to Activate K+ Channels After Substantial Swelling in Villus Epithelial Cells

To assess the activation of the charybdotoxin-insensitive K⁺ channel responsible for Regulatory Volume Decrease (RVD) after substantial volume increases, we measured intracellular pH (pH _i ), intracellular calcium ([Ca²⁺] _i ) and inhibitors of kinases and phosphoprotein phosphatases in guinea pig jejunal villus enterocytes in response to volume changes. Fluorescence spectroscopy was used to measure pH _i and [Ca²⁺] _i of cells in suspension, loaded with 2,7,bis-carboxyethyl-5-6-carboxyfluorescein and Indo-1, respectively, and cell volume was assessed using electronic cell sizing. A modest 7% volume increase or substantial 15 to 20% volume increase caused [Ca²⁺] _i to increase proportionately but the 7% increase caused alkalinization while the larger increases resulted in acidification of ≃0.14 pH units. Following a 15% volume increase, 1-N-0-bis (5-isoquinoline-sulfonyl)-N-methyl-l-4-phenyl-piperazine (KN-62, 50 μm), an inhibitor of Ca²⁺/calmodulin kinase II, blocked RVD. Gramicidin (0.5 μm) bypassed this inhibition suggesting that the K⁺ channel had been affected by the KN-62. RVD after a modest 7% volume increase was not influenced by KN-62 unless the cell was acidified. Okadaic acid, an inhibitor of phosphoprotein phosphatases 1 and 2A, accelerated RVD after a 20% volume increase; inhibition of RVD generated by increasing the K⁺ gradient was bypassed by okadaic acid. Tyrosine kinase inhibitor, genistein (100 μm) had no effect on RVD after 20% volume increases. We conclude that activation of charybdotoxin-insensitive K⁺ channels utilized for RVD after substantial (>7%) `nonphysiological' volume increases requires phosphorylation mediated by Ca²⁺/calmodulin kinase II and that increases in cytosolic acidification rather than larger increases in [Ca²⁺] _i are a critical determinant of this activation. Received: 30 March 1999/Revised: 6 July 1999     

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     

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     

Mechanotransduction in Colonic Smooth Muscle Cells

We evaluated mechanisms which mediate alterations in intracellular biochemical events in response to transient mechanical stimulation of colonic smooth muscle cells. Cultured myocytes from the circular muscle layer of the rabbit distal colon responded to brief focal mechanical deformation of the plasma membrane with a transient increase in intracellular calcium concentration ([Ca²⁺] _i ) with peak of 422.7 ± 43.8 nm above an average resting [Ca²⁺] _i of 104.8 ± 10.9 nm (n= 57) followed by both rapid and prolonged recovery phases. The peak [Ca²⁺] _i increase was reduced by 50% in the absence of extracellular Ca²⁺, while the prolonged [Ca²⁺] _i recovery was either abolished or reduced to ≤15% of control values. In contrast, no significant effect of gadolinium chloride (100 μm) or lanthanum chloride (25 μm) on either peak transient or prolonged [Ca²⁺] _i recovery was observed. Pretreatment of cells with thapsigargin (1 μm) resulted in a 25% reduction of the mechanically induced peak [Ca²⁺] _i response, while the phospholipase C inhibitor U-73122 had no effect on the [Ca²⁺] _i transient peak. [Ca²⁺] _i transients were abolished when cells previously treated with thapsigargin were mechanically stimulated in Ca²⁺-free solution, or when Ca²⁺ stores were depleted by thapsigargin in Ca²⁺-free solution. Pretreatment with the microfilament disrupting drug cytochalasin D (10 μm) or microinjection of myocytes with an intracellular saline resulted in complete inhibition of the transient. The effect of cytochalasin D was reversible and did not prevent the [Ca²⁺] _i increases in response to thapsigargin. These results suggest a communication, which may be mediated by direct mechanical link via actin filaments, between the plasma membrane and an internal Ca²⁺ store. Received: 24 March 1997/Revised: 21 July 1997     

CFTR Regulation of Intracellular Calcium in Normal and Cystic Fibrosis Human Airway Epithelia

In cystic fibrosis airway epithelia, mutation of the CFTR protein causes a reduced response of Cl⁻ secretion to secretagogues acting via cAMP. Using a Ca²⁺ imaging system, the hypothesis that CFTR activation may permit ATP release and regulate [Ca²⁺] _i via a receptor-mediated mechanism, is tested in this study. Application of external nucleotides produced a significant increase in [Ca²⁺] _i in normal (16HBE14o⁻ cell line and primary lung culture) and in cystic fibrosis (CFTE29o⁻ cell line) human airway epithelia. The potency order of nucleotides on [Ca²⁺] _i variation was UTP ≫ ATP > UDP > ADP > AMP > adenosine in both cell types. The nucleotide [Ca²⁺] _i response could be mimicked by activation of CFTR with forskolin (20 μm) in a temperature-dependent manner. In 16HBE14o⁻ cells, the forskolin-induced [Ca²⁺] _i response increased with increasing temperature. In CFTE29o⁻ cells, forskolin had no effect on [Ca²⁺] _i at body temperature-forskolin-induced [Ca²⁺] _i response in CF cells could only be observed at low experimental temperature (14°C) or when cells were cultured at 26°C instead of 37°C. Pretreatment with CFTR channel blockers glibenclamide (100 μm) and DPC (100 μm), with hexokinase (0.5 U/mg), and with the purinoceptor antagonist suramin (100 μm), inhibited the forskolin [Ca²⁺] _i response. Together, these results demonstrate that once activated, CFTR regulates [Ca²⁺] _i by mediating nucleotide release and activating cell surface purinoceptors in normal and CF human airway epithelia. Received: 3 April 2000/Revised: 30 June 2000     

Modulation of Ca2+ Influx in Leech Retzius Neurons II. Effect of Extracellular Ca2+

We investigated the cytosolic free calcium concentration ([Ca²⁺]_i) of leech Retzius neurons in situ while varying the extracellular Ca²⁺ concentration via the bathing solution ([Ca²⁺]_B). Changing [Ca²⁺]_B had only an effect on [Ca²⁺]_i if the cells were depolarized by raising the extracellular K⁺ concentration. Surprisingly, raising [Ca²⁺]_B from 2 to 10 mm caused a decrease in [Ca²⁺]_i, and an increase was evoked by reducing [Ca²⁺]_B to 0.1 mm. These changes were not due to shifts in membrane potential. At low [Ca²⁺]_B moderate membrane depolarizations were sufficient to evoke a [Ca²⁺]_i increase, while progressively larger depolarizations were necessary at higher [Ca²⁺]_B. The changes in the relationship between [Ca²⁺]_i and membrane potential upon varying [Ca²⁺]_B could be reversed by changing extracellular pH. We conclude that [Ca²⁺]_B affects [Ca²⁺]_i by modulating Ca²⁺ influx through voltage-dependent Ca²⁺ channels via the electrochemical Ca²⁺ gradient and the surface potential at the extracellular side of the plasma membrane. These two parameters are affected in a counteracting way: Raising the extracellular Ca²⁺ concentration enhances the electrochemical Ca²⁺ gradient and hence Ca²⁺ influx, but it attenuates Ca²⁺ channel activity by shifting the extracellular surface potential to the positive direction, and vice versa. Received: 23 January 2001/Revised: 23 June 2001     

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     

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     

Increases in Intracellular pH and Ca2+ are Essential for K+ Channel Activation After Modest `Physiological' Swelling in Villus Epithelial Cells

We studied the relationship between changes in intracellular pH (pH _i ), intracellular Ca²⁺([Ca²⁺] _i ) and charybdotoxin sensitive (CTX) maxi-K⁺ channels occurring after modest `physiological' swelling in guinea pig jejunal villus enterocytes. Villus cell volume was assessed by electronic cell sizing, and pH <sub>i</sub> and [Ca<sup>2+</sup>] <sub>i</sub> by fluorescence spectroscopy with 2,7, biscarboxyethyl-5-6-carboxyfluorescein and Indo-1, respectively. In a slightly (0.93 × isotonic) hypotonic medium, villus cells swelled to the same size they would reach during d-glucose or l-alanine absorption; the subsequent Regulatory Volume Decrease (RVD) was prevented by CTX. After the large volume increase in a more hypotonic (0.80 × isotonic) medium, RVD was unaffected by CTX. After modest swelling associated with 0.93 × isotonic dilution, the pH <sub>i</sub> alkalinized but N-5-methyl-isobutyl amiloride (MIA) prevented this ΔpH <sub>i</sub> and the subsequent RVD. Even in the presence of MIA, alkalinization with added NH<sub>4</sub>Cl permitted complete RVD which could be inhibited by CTX. The rate of <sup>86</sup>Rb efflux which also increased after this 0.93 × isotonic dilution was inhibited an equivalent amount by CTX, MIA or Na<sup>+</sup>-free medium. Modest swelling transiently increased [Ca<sup>2+</sup>] <sub>i</sub> and Ca<sup>2+</sup>-free medium or blocking alkalinization by MIA or Na<sup>+</sup>-free medium diminished this transient increase an equivalent amount. RVD after modest swelling was prevented in Ca<sup>2+</sup>-free medium but alkalinization still occurred. After large volume increases, alkalinization of cells increased [Ca<sup>2+</sup>] <sub>i</sub> and volume changes became sensitive to CTX. We conclude that both alkalinization of pH <sub>i</sub> and increased [Ca<sup>2+</sup>] <sub>i</sub> observed with `physiological' volume increase are essential for the activation of CTX-sensitive maxi-K⁺ channels required for RVD. Received: 30 March 1999/Revised: 6 July 1999     

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     

Differential desensitization of Ca2+ mobilization and vasoconstriction by ET(A) receptors in the gerbil spiral modiolar artery

Endothelins are known to be among the most potent endogenous vasoconstrictors. Vasoconstriction of the spiral modiolar artery, which supplies the cochlea, may be implicated in hearing loss and tinnitus. The purpose of the present study was to determine whether the spiral modiolar artery responds to endothelin, whether a change in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) mediates the response and which endothelin receptors are present. The vascular diameter and [Ca²⁺]_i were measured simultaneously by videomicroscopy and microfluorometry in the isolated spiral modiolar artery from the gerbil. ET-1 induced a transient [Ca²⁺]_i increase and a strong and long-lasting vasoconstriction. The transient [Ca²⁺]_i increase underwent rapid desensitization, was independent of extracellular Ca²⁺ and inhibited by the IP₃-receptor blocker (75 μm) 2-aminoethoxydiphenyl borate (2-APB) and by depletion of Ca²⁺ stores with 10⁻⁶ m thapsigargin. In contrast, the vasoconstriction displayed no comparable desensitization. The initial vasoconstriction was independent of extracellular Ca²⁺ but maintenance of the constriction depended on the presence of extracellular Ca²⁺. The half-maximal concentration values (EC ₅₀) for the agonists ET-1, ET-3 and sarafotoxin S6c were 0.8 nm, >10 nm and >100 nm, respectively. Affinity constants for the antagonists BQ-123 and BQ-788 were 24 nm and 77 nm, respectively. These observations demonstrate that ET-1 mediates a vasoconstriction of the gerbil spiral modiolar artery via ET_A receptors and an IP₃ receptor-mediated release of Ca²⁺ from thapsigargin-sensitive Ca²⁺ stores. The marked difference in desensitization between Ca²⁺ mobilization and vasoconstriction suggests that Ca²⁺ mobilization is not solely responsible for the vasoconstriction and that other signaling mechanisms must be present. Received: 4 January 2001/Revised: 23 April 2001     

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     

Veratridine-Mediated Ca2+ Dynamics and Exocytosis in Paramecium Cells

We analyzed [Ca²⁺] _i transients in Paramecium cells in response to veratridine for which we had previously established an agonist effect for trichocyst exocytosis (Erxleben & Plattner, 1994. J. Cell Biol. 127:935–945; Plattner et al., 1994. J. Membrane Biol. 158:197–208). Wild-type cells (7S), nondischarge strain nd9–28°C and trichocyst-free strain ``trichless' (tl), respectively, displayed similar, though somewhat diverging time course and plateau values of [Ca<sup>2+</sup>] <sub>i</sub> transients with moderate [Ca<sup>2+</sup>] <sub>o</sub> in the culture/assay fluid (50 μm or 1 mm). In 7S cells which are representative for a normal reaction, at [Ca<sup>2+</sup>] <sub>o</sub> = 30 nm (c.f. [Ca<sup>2+</sup>] <sup>rest</sup> <sub>i</sub> =∼50 to 100 nm), veratridine produced only a small cortical [Ca<sup>2+</sup>] <sub>i</sub> transient. This increased in size and spatial distribution at [Ca<sup>2+</sup>] <sub>o</sub> = 50 μm of 1 mm. Interestingly with unusually high yet nontoxic [Ca<sup>2+</sup>] <sub>o</sub> = 10 mm, [Ca<sup>2+</sup>] <sub>i</sub> transients were much delayed and also reduced, as is trichocyst exocytosis. We interpret our results as follows. (i) With [Ca<sup>2+</sup>] <sub>o</sub> = 30 nm, the restricted residual response observed is due to Ca<sup>2+</sup> mobilization from subplasmalemmal stores. (ii) With moderate [Ca<sup>2+</sup>] <sub>o</sub> = 50 μm to 1 mm, the established membrane labilizing effect of veratridine may activate not only subplasmalemmal stores but also Ca<sup>2+</sup> <sub>o</sub> influx from the medium via so far unidentified (anteriorly enriched) channels. Visibility of these phenomena is best in tl cells, where free docking sites allow for rapid Ca<sup>2+</sup> spread, and least in 7S cells, whose perfectly assembled docking sites may ``consume' a large part of the [Ca²⁺] _i increase. (iii) With unusually high [Ca²⁺] _o , mobilization of cortical stores and/or Ca²⁺ _o influx may be impeded by the known membrane stabilizing effect of Ca²⁺ _o counteracting the labilizing/channel activating effect of veratridine. (iv) We show these effects to be reversible, and, hence, not to be toxic side-effects, as confirmed by retention of injected calcein. (v) Finally, Mn²⁺ entry during veratridine stimulation, documented by Fura-2 fluorescence quenching, may indicate activation of unspecific Me²⁺ channels by veratridine. Our data have some bearing on analysis of other cells, notably neurons, whose response to veratridine is of particular and continous interest. Received: 8 December 1998/Revised: 2 March 1999     

Involvement of Protein Tyrosine Kinase in the InsP3-Induced Activation of Ca2+-Dependent Cl− Currents in Cultured Cells of the Rat Retinal Pigment Epithelium

This combined study of patch-clamp and intracellular Ca²⁺ ([Ca²⁺] _i ) measurement was undertaken in order to identify signaling pathways that lead to activation of Ca²⁺-dependent Cl⁻ channels in cultured rat retinal pigment epithelial (RPE) cells. Intracellular application of InsP3 (10 μm) led to an increase in [Ca²⁺] _i and activation of Cl⁻ currents. In contrast, intracellular application of Ca²⁺ (10 μm) only induced transient activation of Cl⁻ currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca²⁺ and to the blocker of I _CRAC, La³⁺ (10 μm), despite the fact that both maneuvers led to a decline in [Ca²⁺] _i . The InsP3-induced rise in Cl⁻ conductance could be prevented either by thapsigargin-induced (1 μm) depletion of intracellular Ca²⁺ stores or by removal of Ca²⁺ prior to the experiment. The effect of InsP3 could be mimicked by intracellular application of the Ca²⁺-chelator BAPTA (10 mm). Block of PKC (chelerythrine, 1 μm) had no effect. Inhibition of Ca²⁺/calmodulin kinase (KN-63, KN-92; 5 μm) reduced Cl⁻-conductance in 50% of the cells investigated without affecting [Ca²⁺] _i . Inhibition of protein tyrosine kinase (50 μm tyrphostin 51, 5 μm genistein, 5 μm lavendustin) reduced an increase in [Ca²⁺] _i and Cl⁻ conductance. In summary, elevation of [Ca] _i by InsP3 leads to activation of Cl⁻ channels involving cytosolic Ca²⁺ stores and Ca²⁺ influx from extracellular space. Tyrosine kinases are essential for the Ca²⁺-independent maintenance of this conductance. Received: 15 October 1998/Revised: 3 March 1999     

Characteristics of Single, Large-Conductance Calcium-Dependent Potassium Channels (BKCa) from Smooth Muscle Cells Isolated from the Rabbit Mesenteric Artery

Smooth muscle cells isolated from the secondary and tertiary branches of the rabbit mesenteric artery contain large Ca²⁺-dependent channels. In excised patches with symmetrical (140 mm) K⁺ solutions, these channels had an average slope conductance of 235 ± 3 pS, and reversed in direction at −6.1 ± 0.4 mV. The channel showed K⁺ selectivity and its open probability (P _o ) was voltage-dependent. Iberiotoxin (50 nm) reversibly decreased P _o , whereas tetraethylammonium (TEA, at 1 mm) reduced the unitary current amplitude. Apamin (200 nm) had no effect. The channel displayed sublevels around 1/3 and 1/2 of the mainstate level. The effect of [Ca²⁺] on P _o was studied and data fitted to Boltzmann relationships. In 0.1, 0.3, 1.0 and 10 μm Ca²⁺, V _1/2 was 77.1 ± 5.3 (n= 18), 71.2 ± 4.8 (n= 16), 47.3 ± 10.1 (n= 11) and −14.9 ± 10.1 mV (n= 6), respectively. Values of k obtained in 1 and 10 μm [Ca²⁺] were significantly larger than that observed in 0.1 μm [Ca²⁺]. With 30 μm NS 1619 (a BK_Ca channel activator), V _1/2 values were shifted by 39 mV to the left (hyperpolarizing direction) and k values were not affected. TEA applied intracellularly, reduced the unitary current amplitude with a K _d of 59 mm. In summary, BK_Ca channels show a particularly weak sensitivity to intracellular TEA and they also display large variation in V _1/2 and k. These findings suggest the possibility that different types (isoforms) of BK_Ca channels may exist in this vascular tissue. Received: 22 December 1997/Revised: 27 March 1998     

``Frustrated Exocytosis' — A Novel Phenomenon: Membrane Fusion without Contents Release, Followed by Detachment and Reattachment of Dense Core Vesicles in Paramecium Cells

The lipophilic fluorescent dye, FM1-43, as now frequently used to stain cell membranes and to monitor exo-endocytosis and membrane recycling, induces a cortical [Ca²⁺] _i transient and exocytosis of dense core vesicles (``trichocysts') in Paramecium cells, when applied at usual concentrations (≤10 μm) in presence of extracellular Ca<sup>2+</sup> ([Ca<sup>2+</sup>] <sub>o</sub> = 50 μm). When [Ca<sup>2+</sup>] <sub>o</sub> is kept at 30 nm (<[Ca<sup>2+</sup>]<sup>rest</sup> <sub>i</sub> ), in about one third of the population of extrudable trichocysts docked at the cell membrane, FM1-43 induces membrane fusion, visible by FM1-43 fluorescence of the vesicle membrane. However, in this system extrusion of secretory contents cannot occur in absence of any sufficient Ca<sup>2+</sup> <sub>o</sub> . Upon readdition of Ca<sup>2+</sup> <sub>o</sub> or some other appropriate Me<sup>2+</sup> <sub>o</sub> at 90 μm, secretory contents can be released (complete exocytosis). Resulting ghosts formed in presence of Ca<sup>2+</sup>, Sr<sup>2+</sup> or Mn<sup>2+</sup> are vesicular, but when formed in presence of Mg<sup>2+</sup>, for reasons to be elucidated, they are tubular, though both types are endocytosed and lose their FM1-43 stain. In contrast, in presence of [Mg<sup>2+</sup>] <sub>o</sub> = 3 mm (which inhibits contents release), the exocytotic openings reseal and intact trichocysts with labeled membranes and with still condensed contents are detached from the cell surface (``frustrated exocytosis') within ∼15 min. They undergo cytoplasmic streaming and saltatory redocking, with a half-time of ∼35 min. During this time, the population of redocked trichocysts amenable to exocytosis upon a second stimulus increases with a half-time of ∼35 min. Therefore, acquirement of competence for exocytotic membrane fusion may occur with only a small delay after docking, and this maturation process may last only a short time. A similar number of trichocysts can be detached by merely increasing [Mg²⁺] _o to 3 mm, or by application of the anti-calmodulin drug, R21547 (calmidazolium). Essentially we show (i) requirement of calmodulin and appropriate [Me²⁺] to maintain docking sites in a functional state, (ii) requirement of Ca²⁺ _o or of some other Me²⁺ _o to drive membrane resealing during exo-endocytosis, (iii) requirement of an ``empty' signal to go to the regular endocytotic pathway (with fading fluorescence), and (iv) occurrence of a ``filled' signal for trichocysts to undergo detachment and redocking (with fluorescence) after ``frustrated exocytosis'. Received: 20 January 2000/Revised: 5 May 2000     

Activation by Angiotensin II of Ca2+-dependent K+ and Cl− Currents in Zona Fasciculata Cells of Bovine Adrenal Gland

The effects of angiotensin II (100 nm) on the electrical membrane properties of zona fasciculata cells isolated from calf adrenal gland were studied using the whole cell patch recording method. In current-clamp condition, angiotension II induced a biphasic membrane response which began by a transient hyperpolarization followed by a depolarization more positive than the control resting potential. These effects were abolished by Losartan (10⁻⁵ m), an antagonist of angiotensin receptors of type 1. The angiotensin II-induced transient hyperpolarization was characterized in voltage-clamp condition from a holding potential of −10 mV. Using either the perforated or the standard recording method, a transient outward current accompanied by an increase of the membrane conductance was observed in response to the hormonal stimulation. This outward current consisted of an initial fast peak followed by an oscillating or a slowly decaying plateau current. In Cl⁻-free solution, the outward current reversed at −78.5 mV, a value close to E _K. It was blocked by external TEA (20 mm) and by apamin (50 nm). In K⁺-free solution, the transient outward current, sensitive to Cl⁻ channel blocker DPC (400 μm), reversed at −52 mV, a more positive potential than E _Cl. Its magnitude changed in the same direction as the driving force for Cl⁻. The hormone-induced transient outward current was never observed when EGTA (5 mm) was added to the pipette solution. The plateau current was suppressed in nominally Ca²⁺-free solution (47% of cells) and was reversibly blocked by Cd²⁺ (300 μm) but not by nisoldipine (0.5–1 μm) which inhibited voltage-gated Ca²⁺ currents identified in this cell type. The present experiments show that the transient hyperpolarization induced by angiotensin II is due to Ca²⁺-dependent K⁺ and Cl⁻ currents. These two membrane currents are co-activated in response to an internal increase of [Ca²⁺] _i originating from intra- and extracellular stores. Received: 29 May 1997/Revised: 4 November 1997     

Modulation of Ca2+ Influx in Leech Retzius Neurons. I. Effect of Extracellular pH

We investigated the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) of leech Retzius neurons in situ while varying the extracellular and intracellular pH as well as the extracellular ionic strength. Changing these parameters had no significant effect on [Ca²⁺]_i when the membrane potential of the cells was close to its resting value. However, when the cells were depolarized by raising the extracellular K⁺ concentration or by applying the glutamatergic agonist kainate, extracellular pH and ionic strength markedly affected [Ca²⁺]_i, whereas intracellular pH changes appeared to have virtually no effect. An extracellular acidification decreased [Ca²⁺]_i, while alkalinization or reduction of the ionic strength increased it. Correspondingly, [Ca²⁺]_i also increased when the kainate-induced extracellular acidification was reduced by raising the pH-buffering capacity. At low extracellular pH, the membrane potential to which the cells must be depolarized to evoke a detectable [Ca²⁺]_i increase was shifted to more positive values, and it moved to more negative values at high pH. We conclude that in leech Retzius neurons extracellular pH, but not intracellular pH, affects [Ca²⁺]_i by modulating Ca²⁺ influx through voltage-dependent Ca²⁺ channels. The results suggest that this modulation is mediated primarily by shifts in the surface potential at the extracellular side of the plasma membrane. Received: 23 January 2001/Revised: 15 June 2001