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     

Characterization of Volume-activated Chloride Currents in Endothelial Cells from Bovine Pulmonary Artery

We have measured the kinetic and pharmacological properties of volume-activated Cl⁻ currents (I _Cl,vol) in endothelial cells, and tried to correlate them with those of the already described volume-activated current I _Cln. Both conductances show a similar permeability sequence for monovalent anions, and they are blocked by extracellular ATP. In the present report, we demonstrate by Western blot and RT-PCR that cultured endothelial cells from bovine pulmonary artery (CPAE) contain pI _Cln. The expression of this protein has been shown to be closely associated with the I _Cln current. I _Cl,vol showed however, in contrast with I _Cln, no striking inactivation at positive potentials. This property is also at variance with that of the volume-activated current related to MDR-1. Activation of I _Cl,vol at potentials more negative than −80 mV was not time dependent, which excludes a major contribution of a ClC-2 related current. The antiviral nucleoside analogue AZT (3′-azido-3′-deoxythymidine) inhibited I _Cl,vol by 21 ± 2.7% (n = 10), at a concentration of 100 μm. Another antiviral drug, acyclovir (ACV, 9-[(2-hydroxyethoxy)methyl]guanine) blocked I _Cl,vol by 27 ± 6.2% at 100 μm (n = 11). Both blocking effects are much smaller than those reported for I _Cln. The phenol derivative gossypol, which blocks I _Cln-related currents, efficiently inhibited I _Cl,vol in CPAE cells (67 ± 2.1% at 1 μm, n = 7, K _I = 0.4 μm). The presence of pI _Cln in CPAE cells and the similar qualitative pharmacological profile of I _Cl,vol and I _Cln support the hypothesis that pI _Cln is a good molecular candidate for I _Cl,vol in endothelial cells. The discrepant kinetic properties may indicate that these time-dependent currents at high positive or negative potentials are not intrinsic properties of the channels, but are caused by time-dependent depletion/accumulation phenomena due to the large amplitudes of these currents. Received: 8 May 1995/Revised: 12 October 1995     

Reversible Interruption of Gap Junctional Communication by Testosterone Propionate in Cultured Sertoli Cells and Cardiac Myocytes

A direct cell-to-cell exchange of ions and molecules occurs through specialized membrane channels built by the interaction of two half channels, termed connexons, contributed by each of the two adjacent cells. The electrical and diffusional couplings have been investigated by monitoring respectively the cell-to-cell conductance and the fluorescence recovery after photobleaching, in Sertoli and cardiac cells of young rat. In both cell types, a rapid impairment of the intercellular coupling has been observed in the presence of testosterone propionate. This interruption of the cell-to-cell communication through gap junction channels was dose-dependent, observed in the concentration range 1 to 25 μm and was progressively reversed after withdrawing the testosterone ester. Pretreatment with cyproterone acetate, an antiandrogen which blocks the nuclear testosterone receptor by binding, did not prevent the uncoupling action of the androgen ester. This observation, together with the rapid time course of the uncoupling and recoupling, and the rather high effective concentration (micromolar) of the steroid compound, suggests a nongenomic mechanism of action. The uncoupling concentrations were very similar to those of other steroid compounds known to interrupt gap junctional communication. The uncoupling could result from a direct interaction of the steroid with the proteolipidic structure of the membrane, that might alter the conformation of the gap junction channels and their functional state. Received: 10 April 1995/Revised: 27 October 1995     

Two Types of Pharmacologically Distinct Ca2+ Currents with Voltage-dependent Similarities in Zona Fasciculata Cells Isolated from Bovine Adrenal Gland

Voltage-activated Ca²⁺ currents, in zona fasciculata cells isolated from calf adrenal gland, were characterized using perforated patch-clamp recording. In control solution (Ca²⁺: 2.5 mm) a transient inward current was followed, in 40% of the cells, by a sustained one. In 20 mm Ba²⁺, 61% of the cells displayed an inward current, which consisted of transient and sustained components. The other cells produced either a sustained or a transient inward current. These different patterns were dependent upon time in culture. Current-voltage relationships show that both the transient and sustained components activated, peaked and reversed at similar potentials: −40, 0 and +60 mV, respectively. The two components, fully inactivated at −10 mV, were separated by double-pulse protocols from different holding potentials where the transient component could be inactivated or reactivated. The decaying phase of the sustained component was fitted by a double exponential (time constants: 1.9 and 20 sec at +10 mV); that of the transient component was fitted by a single exponential (time constant: 19 msec at +10 mV). Steady-state activation and inactivation curves of the two components were superimposed. Their half activation and inactivation potentials were similar, about −15 and −34 mV, respectively. The sustained component was larger in Ba²⁺ than in Sr²⁺ and Ca²⁺. Ni²⁺ (20 μm) selectively blocked the transient component while Cd²⁺ (10 μm) selectively blocked the sustained one. (±)Bay K 8644 (0.5 μm) increased the sustained component and nitrendipine (0.5–1 μm) blocked it selectively. The sustained component was inhibited by calciseptine (1 μm). Both components were unaffected by ω-conotoxin GVIA and MVIIC (0.5 μm). These results show that two distinct populations of Ca²⁺ channels coexist in this cell type. Although the voltage dependence of their activation and inactivation are comparable, these two components of the inward current are similar to T- and L-type currents described in other cells. Received: 12 July 1999/Revised: 5 October 1999     

Chloride Currents in Skeletal Muscles of Bufo arenarum

Cl⁻ currents (I _Cl) were measured in short fibers (1–2 mm) from the lumbricalis muscle of toads (Bufo arenarum) with two microelectrodes (15°C). Initially the fibers were equilibrated in a high K⁺-containing solution: (mm) K₂SO₄ 68; Na₂SO₄ 20; KCl 60; CaSO₄ 8; MgSO₄ 1; HEPES 2.5. Constant pulses were applied when all the external K⁺ was replaced by Cs⁺: Cs₂SO₄ 68; Na₂SO₄ 20; CsCl 60; CaSO₄ 8; HEPES 2.5 (pH 7.5). Under these conditions about 80–90% of the current is carried by Cl⁻. The current-voltage relation is almost linear implying constant conductance and hence voltage-independent permeability. The voltage dependence of the net Cl⁻ current could be fitted by constant field equation with a P _Cl of 3.3 × 10⁻⁶ cm/sec. In a separate group of experiments a two-pulse technique was used to estimate the availability and the inactivation of the initial I _Cl during a test pulse. After returning the potential to the holding potential for various times, test pulses of the same amplitude and duration of the prepulses were applied. The initial current during the test pulse was 70% of the initial current during the prepulse and the recovery was complete in less than 300 msec with a linear relationship between the current during the test pulse and the amplitude of the preceding prepulse. When the test pulses were preceded by a positive prepulse, the initial current for any given test pulse was larger than with a negative prepulse. If we assumed that the initial current during the test pulse is a measure of the number of channels open at the end of the prepulse, these results suggest that hyperpolarizing pulses inactivate and depolarizing prepulses activate the I _Cl. Received: 31 March 1995/Revised: 27 October 1995     

Serotonergic Agonists Inhibit Calcium-Activated Potassium and Voltage-Dependent Sodium Currents in Rat Taste Receptor Cells

Recently we reported that rat taste receptor cells respond to the neurotransmitter serotonin with an inhibition of a calcium-activated potassium current [17]. In the present study, this observation is confirmed and extended by studying the effects of an array of serotonergic agonists on membrane properties, calcium-activated potassium current, and voltage-dependent sodium current in taste receptor cells using the patch-clamp recording technique in the whole-cell configuration. Serotonergic inhibition of calcium-activated potassium current was mimicked by the agonists N-(3-trifluoromethylphenyl)piperazine and by (±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene. Both produced reversible inhibition of K _Ca as well as significantly increasing the input resistance of the cell. The agonists 1-(1-naphthyl)piperazine and buspirone (both serotonin receptor 1A agonists) were similarly effective in reducing K _Ca . Outward current was unaffected by application of phenylbiguanide, a serotonin receptor 3 agonist, though current was affected by subsequent application of (±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene. Two agonists—N-(3-trifluoromethylphenyl)piperazine and (±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene—were also tested on voltage-dependent sodium currents; both were effective and reversible in reducing its magnitude at a variety of applied potentials. These data are consistent with the notion that serotonin effects in rat taste receptor cells are mediated by serotonin 1A receptors, though other receptor subtypes may be additionally expressed. Serotonin may affect the taste cell electrical properties during active stimulation in a paracrine fashion. Received: 10 May 1999/Revised: 27 September 1999     

Cyanide Inhibition of Chloride Conductance Across Toad Skin

The effect of cyanide (CN⁻) on voltage-activated or cAMP-induced passive chloride conductance (G ^Cl ) was analyzed in isolated toad skin. Comparatively low concentrations of CN⁻ inhibited G ^Cl almost completely and fully reversibly, regardless of whether it was applied from the mucosal or serosal side. The IC₅₀ was 180 ± 12 μm for voltage-activated G ^Cl and 305 ± 30 μm for the cAMP-inducted conductance. At [CN] <100 μm, the initial inhibition frequently declined partly in the continuous presence of CN⁻. Inhibition was independent of the presence of Ca²⁺. Inhibition was stronger at more alkaline pH, which suggests that dissociated CN⁻ is the effective inhibitor. The onset of the inhibition of voltage-activated or cAMP-induced G ^Cl by CN⁻ occurred with half-times of 34 ± 10 sec, whereas reversibility upon washout was twice as fast (18 ± 7 sec). If [CN⁻] <200 μm was applied under inactivating conditions (serosa −30 mV), the reduction of G ^Cl was stronger upon subsequent voltage-activation than under steady-state activated conditions. This effect was essentially complete less than 30 sec after apical addition of CN⁻, but G _t recovered thereafter partially in the continuous presence of CN⁻. Dinitrophenol inhibited G ^Cl similarly, while omission of oxygen did not affect it. These observations, as well as the time course of inhibition and the full reversibility, suggest that interference of CN⁻ with oxidative phosphorylation and subsequent metabolic depletion is not the reason for the inhibition of G ^Cl . We propose that the inhibition is directly on G ^Cl , presumably by competition with Cl⁻ at a rate-limiting site in the pathway. Location and molecular nature of this site remain to be identified. Received: 8 February 1999/Revised: 22 September 1999     

Fluid Flow Modulates Calcium Entry and Activates Membrane Currents in Cultured Human Aortic Endothelial Cells

Human aortic endothelial cells (HAEC) respond to flow with Ca²⁺ entry, activation of a nonselective cation channel, activation of a chloride channel, and activation of a calcium-activated potassium channel. Conversely, human capillary endothelial cells were unaffected by similar flow rates. In HAEC the flow induced cytosolic free calcium increase ([Ca²⁺] _i ) and the ionic currents associated with it were sustained for up to 15 min after perfusion was stopped. In the absence of extracellular Ca²⁺, fluid flow was unable to evoke the [Ca²⁺] _i increase or the increase in membrane currents but the response could be restored by addition of extracellular Ca²⁺. Surprisingly, the flow response was inhibited in 50% of the cells by inhibitors of nitric oxide production. The results suggest that the sustained flow response in HAEC may be partially mediated by nitric oxide production and release. Received: 29 January 1999/Revised: 2 June 1999     

Extracellular GTP Causes Membrane-Potential Oscillations through the Parallel Activation of Mg2+ and Na+ Currents in Paramecium tetraurelia

Paramecium tetraurelia responds to extracellular GTP (≥ 10 nm) with repeated episodes of prolonged backward swimming. These backward swimming events cause repulsion from the stimulus and are the behavioral consequence of an oscillating membrane depolarization. Ion substitution experiments showed that either Mg²⁺ or Na⁺ could support these responses in wild-type cells, with increasing concentrations of either cation increasing the extent of backward swimming. Applying GTP to cells under voltage clamp elicited oscillating inward currents with a periodicity similar to that of the membrane-potential and behavioral responses. These currents were also Mg²⁺- and Na⁺-dependent, suggesting that GTP acts through Mg²⁺-specific (I _Mg) and Na⁺-specific (I _Na) conductances that have been described previously in Paramecium. This suggestion is strengthened by the finding that Mg²⁺ failed to support normal behavioral or electrophysiological responses to GTP in a mutant that specifically lacks I _Mg (``eccentric'), while Na<sup>+</sup> failed to support GTP responses in ``fast-2,' a mutant that specifically lacks I _Na. Both mutants responded normally to GTP if the alternative cation was provided. As I _Mg and I _Na are both Ca²⁺-dependent currents, the characteristic GTP behavior could result from oscillations in intracellular Ca²⁺ concentration. Indeed, applying GTP to cells in the absence of either Mg²⁺ or Na⁺ revealed a minor inward current with a periodicity similar to that of the depolarizations. This current persisted when known voltage-dependent Ca²⁺ currents were blocked pharmacologically or genetically, which implies that it may represent the activation of a novel purinergic-receptor–coupled Ca²⁺ conductance. Received: 28 October 1996/Revised: 24 December 1996     

Histamine Increases [Ca2+] in and Activates Ca-K and Nonselective Cation Currents in Cultured Human Capillary Endothelial Cells

We characterized the effects of histamine on intracellular Ca²⁺ and activation of ionic currents in human capillary endothelial cells. Histamine produced both a transient and sustained increase in intracellular Ca²⁺. The transient response was mediated largely through intracellular Ca²⁺ release and the sustained response was due to extracellular Ca²⁺ entry. The increase in intracellular Ca²⁺ by histamine was not affected by the H2 blocker cimetidine. But was entirely blocked by the H1 antagonist diphenhydramine showing that the histamine response in these cells is mediated through the H1 receptor. A transient ionic current is coactivated with the histamine-induced increase in intracellular Ca²⁺ and this current has several properties of a nonselective, Ca²⁺ permeable, cation channel (NSC). The magnitude of the NSC current does not strictly correlate with intracellular Ca²⁺ levels. A Ca²⁺-activated K⁺ current (BKCA) is activated by the increase in intracellular Ca²⁺ and this current is blocked by the selective BKCA blocker iberiotoxin. Received: 16 June 1999/Revised: 22 September 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     

Heterogeneity of Volume-sensitive Chloride Channels in Basolateral Membranes of A6 Epithelial Cells in Culture

A new technique allowing single-channel patch-clamp recordings from basolateral membranes of A6 renal epithelial cells in culture was developed. Using this technique we studied the chloride channels activated in these basolateral membranes during hypo-osmotic stress. Four different types of channel were identified and classified according to their current/voltage (I/V) relationships as observed in the on-cell configuration of the patch-clamp technique. Three of these channels had linear I/V relationships with unitary conductances of 12, 30 and 42 pS. The fourth type had an outwardly rectifying I/V curve with inward and outward conductances of 16 and 57 pS respectively. The kinetic properties of each class of channel were studied and kinetic models developed for two of them: the 42 pS channel and the outward rectifier. These models permitted the study of the evolution of the kinetic parameters during hypo-osmotic shock and revealed two different kinetic schemes of channel activation. The results of experiments made on the basolateral membranes were also compared with those of a set of analogous patch-clamp experiments carried out on isolated A6 cells. In these latter, the frequency of successful observations of active channels in a patch was 13%, whereas it was 31% for basolateral membranes. Also, of the four types of channel observed in basolateral membranes, two were never found in isolated cells, only the 12 pS channel and the outward rectifier were present in these isolated cells. Received: 17 April 1996/Revised: 26 June 1996     

The Chloride Conductance of Tight Junctions of Rat Ileum Can Be Increased by cAMP But Not by Carbachol

It is well known, that in mammalian small intestine, cAMP increases Cl⁻ permeability of the apical membrane of enterocytes as part of its secretory action. Paradoxically, this is usually accompanied by an increase of the transepithelial resistance. In the present study we report that in the presence of bumetanide (to block basolateral Cl⁻ uptake) cAMP always decreased the transepithelial resistance. We examined whether this decrease in resistance was due to a cAMP-dependent increase of the paracellular electrolyte permeability in addition to the increase of the Cl⁻ permeability of the apical cell membrane. We used diffusion potentials induced by serosal replacement of NaCl, and transepithelial current passage to evoke transport number effects. The results revealed that cAMP (but not carbachol) could increase the Cl⁻ permeability of the tight junctions in rat ileum. Moreover, we observed a variation in transepithelial resistance of individual tissue preparations, inversely related to the cation selectivity of the tissue, suggesting that Na⁺ permeability of the tight junctions can vary between preparations. Received: 7 September 1996/Revised: 5 November 1996     

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     

A Cyclic Nucleotide-dependent Chloride Conductance in Olfactory Receptor Neurons

Whole-cell membrane currents were recorded from olfactory receptor neurons from the neotenic salamander Necturus maculosus. Cyclic nucleotides, released intracellularly by flash photolysis of NPE-caged cAMP or NPE-caged cGMP, activated a transient chloride current. The chloride current could be elicited at constant voltage in the absence of extracellular Ca²⁺ as well as in the presence of 3 mm intracellular Ca²⁺, suggesting that the current did not require either voltage or Ca²⁺ transients for activation. The current could be elicited in the presence of the protein kinase inhibitors H-7 and H-89, and in the absence of intracellular ATP, indicating that activation was independent of protein kinase A activity. These results suggest that Necturus olfactory receptor neurons contain a novel chloride ion channel that may be directly gated by cyclic nucleotides. Received: 12 November 1996/Revised: 4 April 1997     

Induction of Ca2+-Activated K+ Current and Transient Outward Currents in Human Capillary Endothelial Cells

Human capillary endothelial cells (HCEC) in normal media contain noninactivating outwardly rectifying chloride currents, TEA-sensitive delayed rectifier K⁺ currents and an inward rectifier K⁺ current. Two additional ionic currents are induced in HCEC when the media are allowed to become conditioned: A Ca²⁺-activated K⁺ current (BKCA) that is sensitive to iberiotoxin is induced in 23.5% of the cells, a transient 4-AP-sensitive K⁺ current (A current) is induced in 24.7% of the cells, and in 22.3% of the cells both the transient and BKCA currents are coinduced. The EC₅₀ for Ca²⁺ activation of the BKCA current in HCEC from conditioned media is 213 nM. RNA message for BKCA (hSlo clone) is undetecable after PCR amplification in control cells but is seen in those from conditioned cells. The induction of BKCA current is not blocked by conditioning with inhibitors of nitric oxide synthase, cyclo-oxgenase or lypo-oxygenase pathways. Apparently the characteristics of human endothelial cells are highly malleable and can be easily modified by their local environment. Received: 21 May 1998/Revised: 23 September 1998     

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     

Activation of A3 Adenosine Receptor Induces Calcium Entry and Chloride Secretion in A6 Cells

We have previously demonstrated that in A₆ renal epithelial cells, a commonly used model of the mammalian distal section of the nephron, adenosine A₁ and A_2A receptor activation modulates sodium and chloride transport and intracellular pH (Casavola et al., 1997). Here we show that apical addition of the A₃ receptor-selective agonist, 2-chloro-N⁶-(3-iodobenzyl)-adenosine-5′-methyluronamide (Cl-IB-MECA) stimulated a chloride secretion that was mediated by calcium- and cAMP-regulated channels. Moreover, in single cell measurements using the fluorescent dye Fura 2-AM, Cl-IB-MECA caused an increase in Ca²⁺ influx. The agonist-induced rise in [Ca²⁺] _i was significantly inhibited by the selective adenosine A₃ receptor antagonists, 2,3-diethyl-4,5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5-carboxylate (MRS 1523) and 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS 1191) but not by antagonists of either A₁ or A₂ receptors supporting the hypothesis that Cl-IB-MECA increases [Ca²⁺] _i by interacting exclusively with A₃ receptors. Cl-IB-MECA-elicited Ca²⁺ entry was not significantly inhibited by pertussis toxin pretreatment while being stimulated by cholera toxin preincubation or by raising cellular cAMP levels with forskolin or rolipram. Preincubation with the protein kinase A inhibitor, H89, blunted the Cl-IB-MECA-elicited [Ca²⁺] _i response. Moreover, Cl-IB-MECA elicited an increase in cAMP production that was inhibited only by an A₃ receptor antagonist. Altogether, these data suggest that in A₆ cells a G _s /protein kinase A pathway is involved in the A₃ receptor-dependent increase in calcium entry. Received: 9 March 2000/Revised: 14 August 2000     

Effect of Adenosine on Na+ and Cl− Currents in A6 Monolayers. Receptor Localization and Messenger Involvement

The effect of adenosine regulation on sodium and chloride transport was examined in cultured A₆ renal epithelial cells. Adenosine and its analogue N⁶-cyclopentyladenosine (CPA) had different effects on short-circuit current (I _sc) depending on the side of addition. Basolateral CPA addition induced an approximately threefold increase of the I _sc that reached a maximum effect 20 min after addition and was completely inhibited by preincubation with either an A₂ selective antagonist, CSC, or the sodium channel blocker, amiloride. Apical CPA addition induced a biphasic I _sc response characterized by a rapid fourfold transient increase over its baseline followed by a decline and a plateau phase that were amiloride insensitive. The A₁ adenosine antagonist, CPX, completely prevented this response. This I _sc response to apical CPA was also strongly reduced in Cl⁻-free media and was significantly inhibited either by basolateral bumetanide or apical DPC preincubation. Only basolateral CPA addition was able to induce an increase in cAMP level. CPA, added to cells in suspension, caused a rapid rise in [Ca²⁺] _i that was antagonized by CPX, not affected by CSC and prevented by thapsigargin preincubation. These data suggest that basolateral CPA regulates active sodium transport via A₂ adenosine receptors stimulating adenylate cyclase while apical CPA regulates Cl⁻ secretion via A₁ receptor-mediated changes in [Ca²⁺] _i .