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.     

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     

Downregulation of Epithelial Sodium Channel (ENaC) by CFTR Co-expressed in Xenopus Oocytes is Independent of Cl− Conductance

Defective regulatory interactions between the cystic fibrosis conductance regulator (CFTR) and the epithelial sodium channel (ENaC) have been implicated in the elevated Na⁺ transport rates across cystic fibrosis airway epithelium. It has recently been proposed that ENaC downregulation by CFTR depends on the ability of CFTR to conduct Cl⁻ into the cell and is negligible when Cl⁻ flows out of the cell. To study the mechanisms of this downregulation we have measured amiloride-inhibitable Na⁺ current (I _amil ) in oocytes co-expressing rat ENaC and human wild-type CFTR. In oocytes voltage-clamped to −60 mV, stimulating CFTR with 1 mm IBMX reduced I _amil by up to 80%, demonstrating that ENaC is inhibited when Cl⁻ is conducted out of the cell. Decreasing the level of CFTR stimulation in a single oocyte, decreased both the degree of I _amil downregulation and the CFTR-mediated plasma membrane Cl⁻ conductance, suggesting a direct correlation. However, I _amil downregulation was not affected when Cl⁻ flux across oocyte membrane was minimized by holding the oocyte membrane potential near the Cl⁻ reversal potential (67% ± 10% inhibition at −20 mV compared to 79% ± 4% at −60 mV) demonstrating that I _amil downregulation was independent of the amount of current flow through CFTR. Studies with the Ca²⁺-sensitive photoprotein aequorin showed that Ca²⁺ is not involved in I _amil downregulation by CFTR, although Ca²⁺ injection into the cytoplasm did inhibit I _amil . These results demonstrate that downregulation of ENaC by CFTR depends on the degree of CFTR stimulation, but does not involve Ca²⁺ and is independent of the direction and magnitude of Cl⁻ transport across the plasma membrane. Received: 15 December 1998/Revised: 5 March 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     

A Bicarbonate- and Weak Acid-permeable Chloride Conductance Controlled by Cytosolic Ca2+ and ATP in Rat Submandibular Acinar Cells

A Ca²⁺-activated Cl⁻ conductance in rat submandibular acinar cells was identified and characterized using whole-cell patch-clamp technique. When the cells were dialyzed with Cs-glutamate-rich pipette solutions containing 2 mm ATP and 1 μm free Ca²⁺ and bathed in N-methyl-d-glucamine chloride (NMDG-Cl) or Choline-Cl-rich solutions, they mainly exhibited slowly activating currents. Dialysis of the cells with pipette solutions containing 300 nm or less than 1 nm free Ca²⁺ strongly reduced the Cl⁻ currents, indicating the currents were Ca²⁺-dependent. Relaxation analysis of the ``on' currents of slowly activating currents suggested that the channels were voltage-dependent. The anion permeability sequence of the Cl⁻ channels was: NO⁻ ₃ (2.00) > I⁻ (1.85) ≥ Br⁻ (1.69) > Cl⁻ (1.00) > bicarbonate (0.77) ≥ acetate (0.70) > propionate (0.41) ≫ glutamate (0.09). When the ATP concentration in the pipette solutions was increased from 0 to 10 mm, the Ca²⁺-dependency of the Cl⁻ current amplitude shifted to lower free Ca²⁺ concentrations by about two orders of magnitude. Cells dialyzed with a pipette solution (pCa = 6) containing ATP-γS (2 mm) exhibited currents of similar magnitude to those observed with the solution containing ATP (2 mm). The addition of the calmodulin inhibitors trifluoperazine (100 μm) or calmidazolium (25 μm) to the bath solution and the inclusion of KN-62 (1 μm), a specific inhibitor of calmodulin kinase, or staurosporin (10 nm), an inhibitor of protein kinase C to the pipette solution had little, if any, effect on the Ca²⁺-activated Cl⁻ currents. This suggests that Ca²⁺/Calmodulin or calmodulin kinase II and protein kinase C are not involved in Ca²⁺-activated Cl⁻ currents. The outward Cl⁻ currents at +69 mV were inhibited by NPPB (100 μm), IAA-94 (100 μm), DIDS (0.03–1 mm), 9-AC (300 μm and 1 mm) and DPC (1 mm), whereas the inward currents at −101 mV were not. These results demonstrate the presence of a bicarbonate- and weak acid-permeable Cl⁻ conductance controlled by cytosolic Ca²⁺ and ATP levels in rat submandibular acinar cells. Received: 9 January 1996/Revised: 20 May 1996     

ADH Action on Whole-Cell Currents by Cytosolic Ca2+-dependent Pathways in Aldosterone-treated A6 Cells

We studied the characteristics of the basal and antidiuretic hormone (arginine vasotocin, AVT)-activated whole cell currents of an aldosterone-treated distal nephron cell line (A6) at two different cytosolic Ca²⁺ concentrations ([Ca²⁺] _c , 2 and 30 nm). A6 cells were cultured on a permeable support filter for 10 ∼ 14 days in media with supplemental aldosterone (1 μm). At 30 nm [Ca²⁺] _c , basal conductances mainly consisted of Cl⁻ conductances, which were sensitive to 5-nitro-2-(3-phenylpropylamino)-benzoate. Reduction of [Ca²⁺] _c to 2 nm abolished the basal Cl⁻ conductance. AVT evoked Cl⁻ conductances at 2 as well as 30 nm [Ca²⁺] _c . In addition to Cl⁻ conductances, AVT induced benzamil-insensitive nonselective cation (NSC) conductances. This action on NSC conductances was observed at 30 nm [Ca²⁺] _c but not at 2 nm [Ca²⁺] _c . Thus, cytosolic Ca²⁺ regulates NSC and Cl⁻ conductances in a distal nephron cell line (A6) in response to AVT. Keeping [Ca²⁺] _c at an adequate level seems likely to be an important requirement for AVT regulation of ion conductances in aldosterone-treated A6 cells. Received: 6 May 1996/Revised: 28 June 1996     

The Swelling-Activated Anion Conductance in the Mouse Renal Inner Medullary Collecting Duct Cell Line mIMCD-K2

Swelling-activated Cl⁻ currents (I _Cl,swell ) have been characterized in a mouse renal inner medullary collecting duct cell line (mIMCD-K2). Currents activated by exposing the cells to hypotonicity exhibited characteristic outward rectification and time- and voltage-dependent inactivation at positive potentials and showed an anion selectivity of I⁻ > Br⁻ > Cl⁻ > Asp⁻. NPPB (100 μm) inhibited the current in a voltage independent manner, as did exposure to 10 μm tamoxifen and 500 μm niflumic acid (NFA). In contrast, DIDS (100 μm) blocked the current with a characteristic voltage dependency. These characteristics of I _Cl,swell in mIMCD-K2 cells are essentially identical to those of heterologously expressed cardiac CLC-3. A defining feature of CLC-3 is that activation of PKC by PDBu inhibits the conductance. In mIMCD-K2 cells preincubation with PDBu (100 nm) prevented the activation of I _Cl,swell by hypotonicity. However, PDBu inhibition of I _Cl,swell was reversed after PDBu withdrawal, but this was refractory to subsequent PDBu inhibition. Activation of either the cystic fibrosis transmembrane conductance regulator (CFTR) or Ca²⁺ activated Cl⁻ conductance (CaCC), which are coexpressed in mIMCD-K2 cells prior to PDBu treatment, abolished the PDBu inhibition of I _Cl,swell . Control of I _Cl,swell by PKC therefore depends on the physiological status of the cell. In intact mIMCD-K2 layers in Ussing chambers, forskolin stimulation of an inward short-circuit current (due to transepithelial Cl⁻ secretion via apical CFTR) was inhibited by cell swelling upon hypotonic exposure at the basolateral surface. Activation of I _Cl,swell is therefore capable of regulating transepithelial Cl⁻ secretion and suggests that I _Cl,swell is located at the basolateral membrane. PDBu exposure prior to or during hypotonic challenge was ineffective in reversing the swelling-activated inhibition of Cl⁻ secretion, but tamoxifen (100 μm) abolished the hypotonic inhibition of forskolin-stimulated short-circuit current (I _sc ). RT-PCR analysis confirmed expression of mRNA for members of the CLC family, including both CLC-2 and 3, in the mIMCD-K2 cell line. Received: 24 February 2000/Revised: 26 May 2000     

Cl− Currents Activated by Extracellular Nucleotides in Human Bronchial Cells

The perforated-patch technique was used to study the response of human bronchial cells to extracellular nucleotides. ATP or UTP (100 μm) elicited a complex response consisting of a large transient membrane current increase followed by a relatively small sustained level. These two phases were characterized by different current kinetics. Throughout the transient phase (2–3 min) the membrane current (I _p ) displayed slow activation and deactivation kinetics at depolarizing and hyperpolarizing potentials respectively. At steady-state (I _s ) the relaxation at hyperpolarizing potential disappeared whereas at positive membrane potentials the current became slightly deactivating. The I _s amplitude was dependent on the extracellular Ca²⁺ concentration, being completely inhibited in Ca²⁺-free medium. Cell pre-incubation with the membrane-permeable chelating agent BAPTA/AM prevented completely the response to nucleotides, thus suggesting that both I _p and I _s were dependent on intracellular Ca²⁺. The presence of a hypertonic medium during nucleotide stimulation abolished I _s leaving I _p unchanged. On the contrary, niflumic acid, a blocker of Ca²⁺-activated Cl⁻ channels, prevented completely I _p without reducing significantly I _s . 1,9-dideoxyforskolin fully inhibited I _s but also reduced I _p . Replacement of extracellular Cl⁻ with aspartate demonstrated that the currents activated by nucleotides were Cl⁻ selective. I _p resulted five times more Cl⁻ selective than I _s with respect to aspartate. Taken together, our results indicate that ATP and UTP activate two types of Cl⁻ currents through a Ca²⁺-dependent mechanism. Received: 15 August 1996/Revised: 6 December 1996     

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     

l-Arginine Effects on Na+ Transport in M-1 Mouse Cortical Collecting Duct Cells—A Cationic Amino Acid Absorbing Epithelium

The effect of l-arginine on transepithelial ion transport was examined in cultured M-1 mouse renal cortical collecting duct (CCD) cells using continuous short circuit current (I _SC ) measurements in HCO₃ ⁻/CO₂ buffered solution. Steady state I _SC averaged 73.8 ± 3.2 μA/cm² (n= 126) and was reduced by 94 ± 0.6% (n= 16) by the apical addition of 100 μm amiloride. This confirms that the predominant electrogenic ion transport in M-1 cells is Na⁺ absorption via the epithelial sodium channel (ENaC). Experiments using the cationic amino acid l-lysine (radiolabeled) as a stable arginine analogue show that the combined activity of an apical system y⁺ and a basal amino acid transport system y⁺L are responsible for most cationic amino acid transport across M-1 cells. Together they generate net absorptive cationic amino acid flux. Application of l-arginine (10 mm) either apically or basolaterally induced a transient peak increase in I _SC averaging 36.6 ± 5.4 μA/cm² (n= 19) and 32.0 ± 7.2 μA/cm² (n= 8), respectively. The response was preserved in the absence of bath Cl⁻ (n= 4), but was abolished either in the absence of apical Na⁺ (n= 4) or by apical addition of 100 μm amiloride (n= 6). l-lysine, which cannot serve as a precursor of NO, caused a response similar to that of l-arginine (n= 4); neither L-NMMA (100 μm; n= 3) nor L-NAME (1 mm; n= 4) (both NO-synthase inhibitors) affected the I _SC response to l-arginine. The effects of arginine or lysine were replicated by alkalinization that mimicked the transient alkalinization of the bath solution upon addition of these amino acids. We conclude that in M-1 cells l-arginine stimulates Na⁺ absorption via a pH-dependent, but NO-independent mechanism. The observed net cationic amino acid absorption will counteract passive cationic amino acid leak into the CCD in the presence of electrogenic Na⁺ transport, consistent with reports of stimulated expression of Na⁺ and cationic amino acid transporters by aldosterone. Received: 11 September 2000/Revised: 6 December 2000     

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     

The Essential Role of Cytosolic Cl− in Ca2+ Regulation of an Amiloride-Sensitive Channel in Fetal Rat Pneumocyte

An amiloride-sensitive, Ca²⁺-activated nonselective cation (NSC) channel in the apical membrane of fetal rat alveolar epithelium plays an important role in stimulation of Na⁺ transport by a beta adrenergic agonist (beta agonist). We studied whether Ca²⁺ has an essential role in the stimulation of the NSC channel by beta agonists. In cell-attached patches formed on the epithelium, terbutaline, a beta agonist, increased the open probability (P _o ) of the NSC channel to 0.62 ± 0.07 from 0.03 ± 0.01 (mean ±se; n= 8) 30 min after application of terbutaline in a solution containing 1 mm Ca²⁺. The P _o of the terbutaline-stimulated NSC channel was diminished in the absence of extracellular Ca²⁺ to 0.26 ± 0.05 (n= 8). The cytosolic Ca²⁺ concentration ([Ca²⁺] _c ) in the presence and absence of extracellular Ca²⁺ was, respectively, 100 ± 6 and 20 ± 2 nm (n= 7) 30 min after application of terbutaline. The cytosolic Cl⁻ concentration ([Cl⁻] _c ) in the presence and absence of extracellular Ca²⁺ was, respectively, 20 ± 1 and 40 ± 2 mm (n= 7) 30 min after application of terbutaline. The diminution of [Ca²⁺] _c from 100 to 20 nm itself had no significant effects on the P _o if the [Cl⁻] _c was reduced to 20 mm; the P _o was 0.58 ± 0.10 at 100 nm [Ca²⁺] _c and 0.55 ± 0.09 at 20 nm [Ca²⁺] _c (n= 8) with 20 mm [Cl⁻] _c in inside-out patches. On the other hand, the P _o (0.28 ± 0.10) at 20 nm [Ca²⁺] _c with 40 mm [Cl⁻] _c was significantly lower than that (0.58 ± 0.10; P < 0.01; n= 8) at 100 nm [Ca²⁺] _c with 20 mm [Cl⁻] _c , suggesting that reduction of [Cl⁻] _c is an important factor stimulating the NSC channel. These observations indicate that the extracellular Ca²⁺ plays an important role in the stimulatory action of beta agonist on the NSC channel via reduction of [Cl⁻] _c . Received: 11 August 2000/Revised: 4 December 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     

Chloride Conductance and P i Transport are Separate Functions Induced by the Expression of NaPi-1 in Xenopus Oocytes

Expression of the protein NaPi-1 in Xenopus oocytes has previously been shown to induce an outwardly rectifying Cl⁻ conductance (G_Cl), organic anion transport and Na⁺-dependent P _i -uptake. In the present study we investigated the relation between the NaPi-1 induced G_Cl and P _i -induced currents and transport. NaPi-1 expression induced P _i -transport, which was not different at 1–20 ng/oocyte NaPi-1 cRNA injection and was already maximal at 1–2 days after cRNA injection. In contrast, G_Cl was augmented at increased amounts of cRNA injection (1–20 ng/oocyte) and over a five day expression period. Subsequently all experiments were performed on oocytes injected with 20 ng/oocytes cRNA. P _i -induced currents (Ip) could be observed in NaPi-1 expressing oocytes at high concentrations of P _i (≥ 1 mm P _i ). The amplitudes of Ip correlated well with G_Cl. Ip was blocked by the Cl⁻ channel blocker NPPB, partially Na⁺-dependent and completely abolished in Cl⁻ free solution. In contrast, P _i -transport in NaPi-1 expressing oocytes was not NPPB sensitive, stronger depending on extracellular Na⁺ and weakly affected by Cl⁻ substitution. Endogenous P _i -uptake in water-injected oocytes amounted in all experiments to 30–50% of the Na⁺-dependent P _i -transport observed in NaPi-1 expressing oocytes. The properties of the endogenous P _i -uptake system (K _m for P _i > 1 mm; partial Na⁺- and Cl⁻-dependence; lack of NPPB block) were similar to the NaPi-1 induced P _i -uptake, but no Ip could be recorded at P _i -concentrations ≤3 mm. In summary, the present data suggest that Ip does not reflect charge transfer related to P _i -uptake, but a P _i -mediated modulation of G_Cl. Received: 22 October 1997/Revised: 24 March 1998     

An Effect of Ca2+ on the Intrinsic Cl−-conductance of Rat Kidney Cortex Brush Border Membrane Vesicles

Brush-border membrane vesicles (BBMV) were prepared from superficial rat renal cortex by a divalent²⁺-precipitation technique using either CaCl₂ or MgCl₂. The dependence of the initial [¹⁴C]-d-glucose (or [³H]-l-proline) uptake rate and the extent of the overshoot of d-glucose or l-proline uphill accumulation from solutions containing 100 mm Na⁺ salt, was found to be dependent upon the precipitating divalent cation. With Mg²⁺ precipitation the initial uptake and overshoot accumulation of either d-glucose or l-proline were enhanced compared to BBMV prepared by Ca²⁺ precipitation. When the anion composition of the media was varied (uptake in Cl⁻ media in comparison to gluconate⁻-containing media) it was found that the Cl⁻-dependent component of the initial uptake was markedly depressed with Ca²⁺-prepared BBMV (104.99 ± 33.31 vs. 13.83 ± 1.44 pmoles/sec/mg protein for Mg²⁺ and Ca²⁺ prepared vesicles respectively). When Ca²⁺ was loaded into Mg²⁺ prepared BBMV using a freeze-thaw technique, it was found that the magnitude and Cl⁻ enhancement of d-glucose transport was reduced in a dose-dependent manner. Neomycin, an inhibitor of phospholipase C, had no effect on the reduction of d-glucose uptake by Ca²⁺ in Mg²⁺ prepared vesicles. In contrast, phosphatase inhibitors such as vanadate and fluoride were able to partially reverse the Ca²⁺ inhibition of d-glucose uptake and restore the enhancement due to Cl⁻ media. In addition, inhibitors of protein phosphatase 2B, deltamethrin (50 nm) and trifluoperazine (10 μm), caused partial reversal of Ca²⁺-dependent inhibition of d-glucose uptake. Direct measurement of changes in the bi-ionic (Cl⁻ vs. gluconate⁻) transmembrane electrical potential differences using the cyanine dye, 3,3′-dipropylthiodicarbocyanine iodide DiSC₃-(5) confirmed that Cl⁻ conductance was reduced in Ca²⁺-prepared vesicles. We conclude that a Cl⁻ conductance coexists with Na⁺ cotransport in rat renal BBMV and this may be subject to negative regulation by Ca²⁺ via stimulation of protein phosphatase (PP2B). Received: 14 December 1994/Revised: 27 November 1995     

Vacuolar Chloride Transport in Mesembryanthemum crystallinum L. Measured Using the Fluorescent Dye Lucigenin

To study vacuolar chloride (Cl⁻) transport in the halophilic plant Mesembryanthemum crystallinum L., Cl⁻ uptake into isolated tonoplast vesicles was measured using the Cl⁻-sensitive fluorescent dye lucigenin (N,N′-dimethyl-9,9′-bisacridinium dinitrate). Lucigenin was used at excitation and emission wavelengths of 433 nm and 506 nm, respectively, and showed a high sensitivity towards Cl⁻, with a Stern-Volmer constant of 173 m ⁻¹ in standard assay buffer. While lucigenin fluorescence was strongly quenched by all halides, it was only weakly quenched, if at all, by other anions. However, the fluorescence intensity and Cl⁻-sensitivity of lucigenin was shown to be strongly affected by alkaline pH and was dependent on the conjugate base used as the buffering ion. Chloride transport into tonoplast vesicles of M. crystallinum loaded with 10 mm lucigenin showed saturation-type kinetics with an apparent K _m of 17.2 mm and a V _max of 4.8 mm min⁻¹. Vacuolar Cl⁻ transport was not affected by sulfate, malate, or nitrate. In the presence of 250 μm p-chloromercuribenzene sulfonate, a known anion-transport inhibitor, vacuolar Cl⁻ transport was actually significantly increased by 24%. To determine absolute fluxes of Cl⁻ using this method, the average surface to volume ratio of the tonoplast vesicles was measured by electron microscopy to be 1.13 × 10⁷ m⁻¹. After correcting for a 4.4-fold lower apparent Stern-Volmer constant for intravesicular lucigenin, a maximum rate of Cl⁻ transport of 31 nmol m⁻² sec⁻¹ was calculated, in good agreement with values obtained for the plant vacuolar membrane using other techniques. Received: 18 February 2000/Revised: 30 June 2000     

Chloride Currents Activated by Calcitonin and cAMP in Primary Cultures of Rabbit Distal Convoluted Tubule

Chloride (Cl⁻) conductances were studied in primary cultures of the bright part of rabbit distal convoluted tubule (DCTb) by the whole cell patch clamp technique. The bath solution (33°C) contained (in mm): 140 NaCl, 1 CaCl₂, 10 N-2-hydroxy-ethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.4 and the pipette solution 140 N-methyl-d-glucamine (NMDG)-Cl, 5 MgATP, 1 ethylene-glycol-bis(b-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 10 HEPES, pH 7.4. We identified a Cl⁻ current activated by 10⁻⁵ m forskolin, 10⁻³ m 8-bromo adenosine 3′,5′-cyclic monophophosphate (8 Br-cAMP), 10⁻⁶ m phorbol 12-myristate 13-acetate (PMA), 10⁻³ m intracellular adenosine 3′,5′-cyclic monophophosphate (cAMP) and 10⁻⁷ m calcitonin. The current-voltage relationship was linear and the relative ion selectivity was Br⁻ > Cl⁻≫ I⁻ > glutamate. This current was inhibited by 10⁻³ m diphenylamine-2-carboxylate (DPC) and 10⁻⁴ m 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and was insensitive to 10⁻³ m 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). These characteristics are similar to those described for the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ conductance. In a few cases, forskolin and calcitonin induced an outwardly rectifying Cl⁻ current blocked by DIDS. To determine the exact location of the Cl⁻ conductance 6-methoxy-1-(3-sulfonatopropyl) quinolinium (SPQ) fluorescence experiments were carried out. Cultures seeded on collagen-coated permeable filters were loaded overnight with 5 mm SPQ and the emitted fluorescence analyzed by laser-scan cytometry. Cl⁻ removal from the apical solution induced a Cl⁻ efflux which was stimulated by 10⁻⁵ m forskolin, 10⁻⁷ calcitonin and inhibited by 10⁻⁵ m NPPB. In 140 mm NaBr, forskolin stimulated an apical Br⁻ influx through the Cl⁻ pathway. Forskolin and calcitonin had no effect on the basolateral Cl⁻ permeability. Thus in DCTb cultured cells, exposure to calcitonin activates a Cl⁻ conductance in the apical membrane through a cAMP-dependent mechanism. Received: 5 July 1995/Revised: 21 December 1995     

Inhibition by cAMP of Calcium-Activated Chloride Currents in Cultured Sertoli Cells from Immature Testis

We have characterized a Ca²⁺-dependent Cl⁻ current (Cl_Ca) in cultured Sertoli cells from immature rat testis by using the whole cell recording patch-clamp technique. Cells dialyzed with pipette solutions containing 3 mm adenoside-triphosphate (ATP) and 1 μm free Ca²⁺, exhibited outward currents which were inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and anthracene-9-carboxylic acid (9-AC) but insensitive to tetraethylammonium (TEA). Dialysis of cells with pipette solutions containing less than 1 nm free Ca²⁺ strongly reduced the currents indicating that they were Ca²⁺ dependent. With cells dialyzed with Cs⁺ glutamate-rich pipette solutions containing 0.2 mm EGTA, 10 μm ionomycin induced outward currents having properties of Ca²⁺-activated Cl⁻ currents. With ATP-free pipette solution, the magnitude of currents was not modified suggesting the direct control by Ca²⁺. By contrast, addition of 0.1 mm cAMP in the pipette solution or the superfusion of cells by a permeant analogue of cAMP strongly reduced the currents. These results may suggest that Cl_Ca is inhibited by cAMP-dependent protein kinase. Finally, our results do not agree with the model of primary fluid secretion by exocrine cells, but are in agreement with a hyperpolarizing effect of cAMP in primary culture of Sertoli cells and the release of a low Cl⁻ and bicarbonate-rich primary fluid by these cells. Received: 30 November 1998/Revised: 2 March 1999     

Pharmacological Evidence that Calcium is Not Required for P2-Receptor-Stimulated Cl− Secretion in HT29-Cl.16E

Extracellular ATP at micro- to millimolar concentrations activates Cl⁻ conductance and increases cytosolic calcium ([Ca] _i ) in many epithelial cells, including the colonic epithelial cell line HT29-Cl.16E. Therefore, [Ca] _i has been postulated to be the intracellular messenger for Cl⁻ channel activation. HT29-Cl.16E is a highly differentiated cell line that forms confluent monolayers and secretes mucins and Cl⁻. The involvement of [Ca] _i in the purinergically-stimulated Cl⁻ secretion was investigated pharmacologically in this cell line by whole-cell patch-clamp and Ussing chamber techniques, as well as [Ca] _i measurements in fura-2 loaded cells. The calmodulin inhibitors W13 (5 μm) and chlorpromazine (50 μm) abolished increases in ATP-stimulated [Ca] _i -increases by 90% and 80%, respectively. However, these inhibitors had no effect on the ATP-stimulated Cl⁻ conductance measured in either individual cells or confluent monolayers. As controls, the effects of W13 and chlorpromazine on Ca²⁺-ionophore stimulated Cl⁻ conductance was measured. In this case, the two compounds inhibited whole cell Cl⁻ conductance and monolayer Isc by 90% and 100%, respectively. These data demonstrate: (1) The purinergically-stimulated increase in Cl⁻ current does not require an increase in [Ca] _i , suggesting the involvement of either another signaling pathway or direct activation of Cl⁻ channels by purinergic receptors. (2) A calmodulin or a calmodulinlike binding site that is sensitive to W13 and chlorpromazine participates in the regulation of the [Ca] _i increase by purinergic receptors in HT29-Cl.16E. Received: 4 December 1995/Revised: 16 August 1996     

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