Methadone but not Morphine Inhibits Lubiprostone-Stimulated Cl− Currents in T84 Intestinal Cells and Recombinant Human ClC-2, but not CFTR Cl− Currents

In clinical trials, methadone, but not morphine, appeared to prevent beneficial effects of lubiprostone, a ClC-2 Cl^? channel activator, on opioid-induced constipation. Effects of methadone and morphine on lubiprostone-stimulated Cl^? currents were measured by short circuit current (Isc) across T84 cells. Whole cell patch clamp of human ClC-2 (hClC-2) stably expressed in HEK293 cells and in a high expression cell line (HEK293EBNA) as well as human CFTR (hCFTR) stably expressed in HEK293 cells was used to study methadone and morphine effects on recombinant hClC-2 and hCFTR Cl^? currents. Methadone but not morphine inhibited lubiprostone-stimulated Isc in T84 cells with half-maximal inhibition at 100 nM. Naloxone did not affect lubiprostone stimulation or methadone inhibition of Isc. Lubiprostone-stimulated Cl^? currents in hClC-2/HEK293 cells, but not forskolin/IBMX-stimulated Cl^? currents in hCFTR/HEK293 cells, were inhibited by methadone, but not morphine. HEK293EBNA cells expressing hClC-2 showed time-dependent, voltage-activated, CdCl₂-inhibited Cl^? currents in the absence (control) and the presence of lubiprostone. Methadone, but not morphine, inhibited control and lubiprostone-stimulated hClC-2 Cl^? currents with half-maximal inhibition at 100 and 200–230 nM, respectively. Forskolin/IBMX-stimulated hClC-2 Cl^? currents were also inhibited by methadone. Myristoylated protein kinase inhibitor (a specific PKA inhibitor) inhibited forskolin/IBMX- but not lubiprostone-stimulated hClC-2 Cl^? currents. Methadone caused greater inhibition of lubiprostone-stimulated currents added before patching (66.1 %) compared with after patching (28.7 %). Methadone caused inhibition of lubiprostone-stimulated Cl^? currents in T84 cells and control; lubiprostone- and forskolin/IBMX-stimulated recombinant hClC-2 Cl^? currents may be the basis for reduced efficacy of lubiprostone in methadone-treated patients.     

Regulation of Cl− Secretion by Extracellular ATP in Cultured Mouse Endometrial Epithelium

The present study explored regulation of electrogenic ion transport across cultured mouse endometrial epithelium by extracellular ATP using the short-circuit current (I _SC ) and the patch-clamp techniques. The cultured endometrial monolayers responded to apical application of ATP with an increase in I _SC in a concentration-dependent manner (EC₅₀ at 3 μm). Replacement of Cl⁻ in the bathing solution or treatment of the cells with Cl⁻ channel blockers, DIDS and DPC, markedly reduced the I _SC , indicating that a substantial portion of the ATP-activated I _SC was Cl⁻-dependent. Amiloride at a concentration (10 μm) known to block Na⁺ channels was found to have no effect on the ATP-activated I _SC excluding the involvement of Na⁺ absorption. Adenosine was found to have little effect on the I _SC excluding the involvement of P₁ receptors. The effect of UTP, a potent P_2U receptor agonist on the I _SC was similar to that of ATP while potent P_2X agonist, α-β-Methylene adenosine 5′-triphosphate (α-β-M-ATP) and P_2Y agonist, 2-methylthio-adenosine triphosphate (2-M-ATP), were found to be ineffective. The effect of ATP on I _SC was mimicked by the Ca²⁺ ionophore, ionomycin, indicating a role of intracellular Ca²⁺ in mediating the ATP response. Confocal microscopic study also demonstrated a rise in intracellular Ca²⁺ upon stimulation by extracellular ATP. In voltage-clamped endometrial epithelial cells, ATP elicited a whole-cell Cl⁻ current which exhibited outward rectification and delayed activation and inactivation at depolarizing and hyperpolarizing voltages, respectively. The results of the present study demonstrate the presence of a regulatory mechanism involving extracellular ATP and P_2U purinoceptors for endometrial Cl⁻ secretion.     

Apical Cl− Channels in A6 Cells

Short-circuit current (I _sc ), transepithelial conductance (G _t ), electrical capacitance (C _T ) and the fluctuation in I _sc were analyzed in polarized epithelial cells from the distal nephron of Xenopus laevis (A6 cell line). Tissues were incubated with Na⁺- and Cl⁻-free solutions on the apical surface. Basolateral perfusate was NaCl-Ringer. Agents that increase cellular cAMP evoked increases in G _t , C _T , I _sc and generated a Lorentzian I _sc -noise. The responses could be related to active, electrogenic secretion of Cl⁻. Arginine-vasotocin and oxytocin caused a typical peak-plateau response pattern. Stimulation with a membrane-permeant nonhydrolyzable cAMP analogue or forskolin showed stable increases in G _t with only moderate peaking of I _sc . Phosphodiesterase inhibitors also stimulated Cl⁻ secretion with peaking responses in G _t and I _sc . All stimulants elicited a spontaneous Lorentzian noise, originating from the activated apical Cl⁻ channel, with almost identical corner frequency (40–50 Hz). Repetitive challenge with the hormones led to a refractory behavior of all parameters. Activation of the cAMP route could overcome this refractoriness. All agents caused C _T , a measure of apical membrane area, to increase in a manner roughly synchronous with G _t . These results suggest that activation of the cAMP-messenger route may, at least partly, involve exocytosis of a vesicular Cl⁻ channel pool. Apical flufenamate depressed Cl⁻ current and conductance and apparently generated blocker-noise. However, blocking kinetics extracted from noise experiments could not be reconciled with those obtained from current inhibition, suggesting the drug does not act as simple open-channel inhibitor. Received: 20 May 1998/Revised: 8 September 1998     

A Ca2+-activated Whole-Cell Cl− Conductance In Human Placental Cytotrophoblast Cells Activated Via a G Protein

Whole-cell patch clamp experiments were performed on cultured human cytotrophoblast cells incubated for 24–48 hr after their isolation from term placentas. Cl⁻-selective currents were examined using K⁺-free solutions. Under nonstimulated conditions, most cells initially expressed only small background leak currents. However, inclusion of 0.2 mm GTPγS in the electrode solution caused activation of an outwardly rectifying conductance which showed marked time-dependent activation at depolarized potentials above +20 mV. Stimulation of this conductance by GTPγS was found to be Ca²⁺-dependent since GTPγS failed to activate currents when included in a Ca²⁺-free electrode solution. In addition, similar currents could be activated by increasing the [Ca²⁺] of the pipette solution to 500 nm. The Ca²⁺-activated conductance was judged to be Cl⁻-selective, since reversal potentials were predicted by Nernst equilibrium potentials for Cl⁻. This conductance could also be reversibly inhibited by addition of the anion channel blocker DIDS to the bath solution at a dose of 100 μm. Preliminary experiments indicated the presence of a second whole-cell anion conductance in human cytotrophoblast cells, which may be activated by cell swelling. Possible roles for the Ca²⁺-activated Cl⁻ conductance in human placental trophoblast are discussed. Received: 9 November 1995/Revised: 18 January 1996     

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     

Potentiation by Isoproterenol on Carbachol-induced K+ and Cl− Currents and Fluid Secretion in Rat Parotid

Isoproterenol (IPR) and 8-(4-chlorophenylthio)-cyclic AMP (cpt-cAMP) enhanced carbachol (CCh)-induced fluid secretion from rat parotid glands, but had no effect by themselves. The enhancement by IPR was blocked by propranolol. In dispersed parotid acinar cells, IPR and cpt-cAMP potentiated CCh-induced K⁺ and Cl⁻ currents (I _K and I _Cl). IPR at the concentration of 0.1 μm significantly potentiated the CCh-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺] _i ), but 1 mm cpt-cAMP did not. The incidence of the potentiation by IPR in CCh-induced Mn²⁺ entry was 31% and that by cpt-cAMP was 21%. The potentiation by IPR in the ionic currents and the [Ca²⁺] _i was suppressed by propranolol. These results suggest that the CCh-induced fluid secretion from rat parotid glands is enhanced by IPR through the potentiation of I _K and I _Cl mainly by the increased cyclic AMP level and partially by the potentiated Ca²⁺ influx and [Ca²⁺] _i increase, and that IPR is more effective than cpt-cAMP in the enhancement of the CCh-induced [Ca²⁺] _i increase. Received: 6 October 1997/Revised: 16 April 1998     

Lymphotoxin β Receptor Signaling Induces IL-8 Production in Human Bronchial Epithelial Cells

Asthma-related mortality has been decreasing due to inhaled corticosteroid use, but severe asthma remains a major clinical problem. One characteristic of severe asthma is resistance to steroid therapy, which is related to neutrophilic inflammation. Recently, the tumor necrosis factor superfamily member (TNFSF) 14/LIGHT has been recognized as a key mediator in severe asthmatic airway inflammation. However, the profiles and intracellular mechanisms of cytokine/chemokine production induced in cells by LIGHT are poorly understood. We aimed to elucidate the molecular mechanism of LIGHT-induced cytokine/chemokine production by bronchial epithelial cells. Human bronchial epithelial cells express lymphotoxin β receptor (LTβR), but not herpesvirus entry mediator, which are receptors for LIGHT. LIGHT induced various cytokines/chemokines, such as interleukin (IL)-6, oncostatin M, monocyte chemotactic protein-1, growth-regulated protein α and IL-8. Specific siRNA for LTβR attenuated IL-6 and IL-8 production by BEAS-2B and normal human bronchial epithelial cells. LIGHT activated intracellular signaling, such as mitogen-activated protein kinase and nuclear factor-κB (NF-κB) signaling. LIGHT also induced luciferase activity of NF-κB response element, but not of activator protein-1 or serum response element. Specific inhibitors of phosphorylation of extracellular signal-regulated kinase (Erk) and that of inhibitor κB attenuated IL-8 production, suggesting that LIGHT-LTβR signaling induces IL-8 production via the Erk and NF-κB pathways. LIGHT, via LTβR signaling, may contribute to exacerbation of airway neutrophilic inflammation through cytokine and chemokine production by bronchial epithelial cells.     

Human Mesenchymal Stem Cells Suppress the Stretch–Induced Inflammatory miR-155 and Cytokines in Bronchial Epithelial Cells

Current research in pulmonary pathology has focused on inflammatory reactions initiated by immunological responses to allergens and irritants. In addition to these biochemical stimuli, physical forces also play an important role in regulating the structure, function, and metabolism of the lung. Hyperstretch of lung tissues can contribute to the inflammatory responses in asthma, but the mechanisms of mechanically induced inflammation in the lung remain unclear. Our results demonstrate that excessive stretch increased the secretion of inflammatory cytokines by human bronchial epithelial cells (hBECs), including IL-8. This increase of IL-8 secretion was due to an elevated microRNA-155 (miR-155) expression, which caused the suppression of Src homology 2 domain–containing inositol 5-phosphatase 1 (SHIP1) production and the subsequent activation of JNK signaling. In vivo studies in our asthmatic mouse model also showed such changes in miR-155, IL-8, and SHIP1 expressions that reflect inflammatory responses. Co-culture with human mesenchymal stem cells (hMSCs) reversed the stretch-induced hBEC inflammatory responses as a result of IL-10 secretion by hMSCs to down-regulate miR-155 expression in hBECs. In summary, we have demonstrated that mechanical stretch modulates the homeostasis of the hBEC secretome involving miR-155 and that hMSCs can be used as a potential therapeutic approach to reverse bronchial epithelial inflammation in asthma.     

Chloroquine Stimulates Cl? Secretion by Ca2+ Activated Cl? Channels in Rat Ileum

Chloroquine (CQ), a bitter tasting drug widely used in treatment of malaria, is associated gastrointestinal side effects including nausea or diarrhea. In the present study, we investigated the effect of CQ on electrolyte transport in rat ileum using the Ussing chamber technique. The results showed that CQ evoked an increase in short circuit current (I_SC) in rat ileum at lower concentration (≤5×10⁻⁴ M ) but induced a decrease at higher concentrations (≥10⁻³ M). These responses were not affected by tetrodotoxin (TTX). Other bitter compounds, such as denatoniumbenzoate and quinine, exhibited similar effects. CQ-evoked increase in I_SC was partly reduced by amiloride(10⁻⁴ M), a blocker of epithelial Na⁺ channels. Furosemide (10⁻⁴ M), an inhibitor of Na⁺-K⁺ -2Cl⁻ co-transporter, also inhibited the increased I_SC response to CQ, whereas another Cl⁻ channel inhibitor, CFTR(inh)-172(10⁻⁵M), had no effect. Intriguingly, CQ-evoked increases were almost completely abolished by niflumic acid (10⁻⁴M), a relatively specific Ca²⁺-activated Cl⁻ channel (CaCC) inhibitor. Furthermore, other CaCC inhibitors, such as DIDS and NPPB, also exhibited similar effects. CQ-induced increases in I_SC were also abolished by thapsigargin(10⁻⁶M), a Ca²⁺ pump inhibitor and in the absence of either Cl⁻ or Ca²⁺ from bathing solutions. Further studies demonstrated that T2R and CaCC-TMEM16A were colocalized in small intestinal epithelial cells and the T2R agonist CQ evoked an increase of intracelluar Ca²⁺ in small intestinal epithelial cells. Taken together, these results demonstrate that CQ induces Cl⁻ secretion in rat ileum through CaCC at low concentrations, suggesting a novel explanation for CQ-associated gastrointestinal side-effects during the treatment of malaria.     

17β-Estradiol Inhibits Outward Voltage-Gated K+ Currents in Human Osteoblast-Like MG63 Cells

Previous studies have shown that 17β-estradiol has a pivotal function by blocking voltage-gated K⁺ (Kv) channels in several different types of cells such as cardiac myocytes and neurons. Outward Kv currents can also be measured in osteoblasts, although little is known about the effects of 17β-estradiol on these currents. In human osteoblast-like MG63 cells, we found that 17β-estradiol inhibits peak and end Kv currents, with IC₅₀ values of 480 and 325 nM, respectively. To elucidate the mechanism of inhibition, the kinetics of Kv currents were investigated. The half-maximum activation potential (V _1/2) was 1.3 mV and was shifted left to ?4.4 mV after application of 500 nM 17β-estradiol. For steady-state inactivation, the V _1/2 was –55.0 mV and weakly shifted left to –58.2 mV. To identify the molecular basis of outward Kv currents in MG63 cells, we performed RT-PCR analyses. The expression of Kv2.1 channels appeared to dominate over that of other Kv channels in MG63 cells. In COS-7 cells with heterologously expressed Kv2.1 channels, 17β-estradiol also inhibits macroscopic currents of Kv2.1. Our data indicate that 17β-estradiol inhibits Kv currents in human osteoblast-like MG63 cells and that Kv2.1 is a potential molecular correlate of outward Kv currents in these cells.     

Macrophages Facilitate Coal Tar Pitch Extract-Induced Tumorigenic Transformation of Human Bronchial Epithelial Cells Mediated by NF-κB

Objective

Chronic respiratory inflammation has been associated with lung cancer. Tumor-associated macrophages (TAMs) play a critical role in the formation of inflammation microenvironment. We sought to characterize the role of TAMs in coal tar pitch extract (CTPE)-induced tumorigenic transformation of human bronchial epithelial cells and the underlying mechanisms.

Methods

The expression of TAMs-specific CD68 in lung cancer tissues and paired adjacent tissues from cancer patients was determined using immunostaining. Co-culture of human bronchial epithelial cells (BEAS-2B) and macrophage-like THP-1 cells were conducted to evaluate the promotive effect of macrophages on CTPE-induced tumorigenic transformation of BEAS-2B cells. BEAS-2B cells were first treated with 2.4 µg/mL CTPE for 72 hours. After removal of CTPE, the cells were continuously cultured either with or without THP-1 cells and passaged using trypsin-EDTA. Alterations of cell cycle, karyotype, colony formation in soft agar and tumor xenograft growth in nude mice of BEAS-2B cells at passages 10, 20 and 30, indicative of tumorigenecity, were determined, respectively. In addition, mRNA and protein levels of NF-κB in BEAS-2B cells were measured with RT-PCR and western blot, respectively. B(a)P was used as the positive control.

Results

The over-expression of TAMs-specific CD68 around lung tumor tissues was detected and associated with lung cancer progression. The tumorigenic alterations of BEAS-2B cells including increase in cell growth rate, number of cells with aneuploidy, clonogenicity in soft agar, and tumor size in nude mice in vivo occurred at passage 10, becoming significant at passages 20 and 30 of the co-culture following CTPE removal in compared to BEAS-2B cells alone. In addition, the expression levels of NF-κB in BEAS-2B cells were positively correlated to the malignancy of BEAS-2B cells under different conditions of treatment.

Conclusion

The presence of macrophages facilitated CTPE-induced tumorigenic transformation of BEAS-2B cells, which may be mediated by NF-κB.     

Vasorin/ATIA Promotes Cigarette Smoke–Induced Transformation of Human Bronchial Epithelial Cells by Suppressing Autophagy-Mediated Apoptosis

BACKGROUND: Escaping cell death pathways is an important event during carcinogenesis. We previously identified anti-TNFα-induced apoptosis (ATIA, also known as vasorin) as an antiapoptotic factor that suppresses reactive oxygen species (ROS) production. However, the role of vasorin in lung carcinogenesis has not been investigated. METHODS: Vasorin expression was examined in human lung cancer tissues with immunohistochemistry and database analysis. Genetic and pharmacological approaches were used to manipulate protein expression and autophagy activity in human bronchial epithelial cells (HBECs). ROS generation was measured with fluorescent indicator, apoptosis with release of lactate dehydrogenase, and cell transformation was assessed with colony formation in soft agar. RESULTS: Vasorin expression was increased in human lung cancer tissues and cell lines, which was inversely associated with lung cancer patient survival. Cigarette smoke extract (CSE) and benzo[a]pyrene diol epoxide (BPDE)–induced vasorin expression in HBECs. Vasorin knockdown in HBECs significantly suppressed CSE-induced transformation in association with enhanced ROS accumulation and autophagy. Scavenging ROS attenuated autophagy and cytotoxicity in vasorin knockdown cells, suggesting that vasorin potentiates transformation by impeding ROS-mediated CSE cytotoxicity and improving survival of the premalignant cells. Suppression of autophagy effectively inhibited CSE-induced apoptosis, suggesting that autophagy was pro-apoptotic in CSE-treated cells. Importantly, blocking autophagy strongly potentiated CSE-induced transformation. CONCLUSION: These results suggest that vasorin is a potential lung cancer–promoting factor that facilitates cigarette smoke–induced bronchial epithelial cell transformation by suppressing autophagy-mediated apoptosis, which could be exploited for lung cancer prevention.     

A Large-Conductance Chloride Channel in Pigmented Ciliary Epithelial Cells Activated by GTPγS

A large-conductance (or maxi-) chloride channel was identified in bovine pigmented ciliary epithelial (PCE) cells using inside-out excised patch clamp recording. The channel had a mean conductance of 293 pS when excised patches were bathed in symmetrical 130 mm NaCl although the conductance decreased to 209 pS when the solution bathing the cytoplasmic face of the patch contained only 33 mm NaCl. The channel was highly selective for chloride, with a P _Cl/P _Na= 24. A flickery, reversible block was produced by the diuretic stilbene 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), while 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) produced a permanent block. The channel was rarely active in cell-attached patches and usually required several minutes of polarization before activity could be detected in excised patches, a process known as metagenesis. Once activated, the channel was voltage-dependent and was mainly open within the voltage range −30 to +30 mV closing when the membrane was polarized to larger values. GTPγS (100 μm) activated the channel with a latency of 170 sec when applied to the cytoplasmic face of patches. This activation was not reversible upon return to control solution within the duration of the experiment. We assess the available evidence and suggest a role for this channel in volume regulation. Received: 24 June 1996/Revised: 18 February 1997     

Excitation-revealed changes in cytoplasmic Cl− concentration in “Cl−-starved”Chara cells

Summary The changes in the cytoplasmic Cl^– concentration, [Cl^–] _c , are monitored at the time of withdrawal (starvation) and subsequent replacement of Cl^– in the outside medium. The measurement technique exploits the involvement of Cl^– inChara excitation. The transient clamp current due to Cl^–,I _Cl, is separated from other excitation transients through Hodgkin-Huxley (HH) equations, which have been adjusted toChara. TheI _Cl amplitude depends on HH parameters, [Cl^–] _c and the maximum membrane conductance to Cl^–, . The results are discussed in terms of these quantities.I _Cl and were found to fall after 6–10 hr of Cl^– starvation, thus supporting the hypothesis that [Cl^– _c decreases in Cl^–-free medium. The best HH fit to starved data was obtained with [Cl^– _c =3.5mm. The time-course forI _Cl decline is considerably slower than the time-course of the rise of the starvation-stimulated influx. As cells starved for periods longer than 24 hr are re-exposed to Cl^–, it is revealed that while [Cl^–] _c remains low during long starvation, increases to values greater than those of the normal cells. Such differences among cells starved for various lengths of time have not been detected previously.     

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