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     

Magnesium Transport Across the Basolateral Plasma Membrane of the Fish Enterocyte

In tilapia (Oreochromis mossambicus) intestine, Mg²⁺ transport across the epithelium involves a transcellular, Na⁺- and Na⁺/K⁺-ATPase dependent pathway. In our search for the Mg²⁺ extrusion mechanism of the basolateral compartment of the enterocyte, we could exclude Na⁺/Mg²⁺ antiport or ATP-driven transport. Evidence is provided, however, that Mg²⁺ movement across the membrane is coupled to anion transport. In basolateral plasma membrane vesicles, an inwardly directed Cl⁻ gradient stimulated Mg²⁺ uptake (as followed with the radionuclide ²⁷Mg) twofold. As Cl⁻-stimulated uptake was inhibited by the detergent saponin and by the ionophore A23187, Mg²⁺ may be accumulated intravesicularly above chemical equilibrium. Valinomycin did not affect uptake, suggesting that electroneutral symport activity occurred. The involvement of anion coupled transport was further indicated by the inhibition of Mg²⁺ uptake by the stilbene derivative, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid. Kinetic analyses of the Cl⁻-stimulated Mg²⁺ uptake yielded a K _m (Mg²⁺) of 6.08 ± 1.29 mmol · l⁻¹ and a K _m (Cl⁻) of 26.5 ± 6.5 mmol · l⁻¹, compatible with transport activity at intracellular Mg²⁺- and Cl⁻-levels. We propose that Mg²⁺ absorption in the tilapia intestine involves an electrically neutral anion symport mechanism. Received: 19 January 1996/Revised: 1 August 1996     

Tonoplast Anion Channel Activity Modulation by pH in Chara corallina

The patch-clamp technique was used to investigate regulation of anion channel activity in the tonoplast of Chara corallina in response to changing proton and calcium concentrations on both sides of the membrane. These channels are known to be Ca²⁺-dependent, with conductances in the range of 37 to 48 pS at pH 7.4. By using low pH at the vacuolar side (either pH_vac 5.3 or 6.0) and a cytosolic pH (pH_cyt) varying in a range of 4.3 to 9.0, anion channel activity and single-channel conductance could be reversibly modulated. In addition, Ca²⁺-sensitivity of the channels was markedly influenced by pH changes. At pH_cyt values of 7.2 and 7.4 the half-maximal concentration (EC ₅₀) for calcium activation was 100–200 μm, whereas an EC ₅₀ of about 5 μm was found at a pH_cyt of 6.0. This suggests an improved binding of Ca²⁺ ions to the channel protein at more acidic cytoplasm. At low pH_cyt, anion channel activity and mean open times were voltage-dependent. At pipette potentials (V _p) of +100 mV, channel activity was approximately 15-fold higher than activity at negative pipette potentials and the mean open time of the channel increased. In contrast, at pH_cyt 7.2, anion channel activity and the opening behavior seemed to be independent of the applied V _p. The kinetics of the channel could be further controlled by the Ca²⁺ concentration at the cytosolic membrane side: the mean open time significantly increased in the presence of a high cytosolic Ca²⁺ concentration. These results show that tonoplast anion channels are maintained in a highly active state in a narrow pH range, below the resting pH_cyt. A putative physiological role of the pH-dependent modulation of these anion channels is discussed. Received: 14 March 2001/Revised: 16 July 2001     

Sorbitol Uptake in Plasma Membrane Vesicles Isolated from Immortalized Rabbit TALH Cells: Activation by a Ca2+/Calmodulin-dependent Protein Kinase

Apical plasma membrane vesicles were isolated from cultures of immortalized thick ascending limb of Henle's loop (TALH) cells and sorbitol uptake was investigated using a rapid filtration technique. In the presence of Mg²⁺, Ca²⁺, ATP, and GTP sorbitol equilibrated within three minutes with the intravesicular space; this uptake was reduced by 75% when the incubation temperature was decreased from 37°C to 4°C. A lower level of uptake was also observed in the presence of 100 μm quinidine and when Ca²⁺ or ATP were omitted from the medium. Membranes preincubated with Mg²⁺, Ca²⁺, ATP, and GTP showed, however, a high sorbitol uptake in ATP-free medium. Staurosporine, but only at high concentrations of 200 nm, inhibited sorbitol uptake when present during the transport experiments or during the preincubation with ATP. Similar results were obtained with 1 μm trifluoperazine. Protein kinase C inhibitory peptide was ineffective whereas 20 nm KT 5926, at low concentrations a specific inhibitor of Ca²⁺/calmodulin-dependent kinase, attenuated the activation. On the basis of these data we suggest that a Ca²⁺/calmodulin-dependent kinase is a mediator of regulation of sorbitol plasma membrane permeability in renal medullary cells. Received: 31 March 1997/Revised: 11 June 1997     

Effect of Adenosine and Intracellular GTP on KATP Channels of Mammalian Skeletal Muscle

We investigated the action of adenosine and GTP on K_ATP channels, using inside-out patch clamp recordings from dissociated single fibers of rat flexor digitorum brevis (FDB) skeletal muscle. In excised patches, K_ATP channels could be activated by a combination of an extracellular adenosine agonist and intracellular Mg²⁺-ATP and GTP or GTP-γ-S. The activation required hydrolyzable ATP and could be partially reversed with Mg²⁺, suggesting that it may involve a G-protein dependent phosphorylation of K_ATP channels. We found that K_ATP channels of the rat FDB could not be activated by Mg²⁺-ATP alone or by Mg²⁺-ATP in the presence of extracellular adenosine. Patches whose channel activity had been `rundown' by Ca²⁺ could not be recovered by adenosine, GTP or Mg²⁺-ATP. K_ATP channels activated by adenosine receptor agonists had a similar ATP sensitivity to those under control conditions; but adenosine appears to be able to switch these K_ATP channels from an inactive to an active mode. Received: 29 December 1995/Revised: 22 March 1996     

Molecular Characterization of the Glycated Plasma Membrane Calcium Pump

We have previously demonstrated (Diabetes 39:707–711, 1990) that in vitro glycation of the red cell Ca²⁺ pump diminishes the Ca²⁺-ATPase activity of the enzyme up to 50%. Such effect is due to the reaction of glucose with lysine residues of the Ca²⁺ pump (Biochem. J. 293:369–375, 1993). The aim of this work was to determine whether the effect of glucose is due to a full inactivation of a fraction of the total population of Ca²⁺ pump, or to a partial inactivation of all the molecules. Glycation decreased the V _max for the ATPase activity leaving unaffected the apparent affinities for Ca²⁺, calmodulin or ATP. The apparent turnover was identical in both, the glycated and the native enzyme. Glycation decreased the V _max for the ATP-dependent but not for the calmodulin-activated phosphatase activities. Concomitantly with the inhibition, up to 6.5% of the lysine residues were randomly glycated. The probabilistic analysis of the relation between the enzyme activity and the fraction of nonmodified residues indicates that only one Lys residue is responsible for the inhibition. We suggest that glucose decreases the Ca²⁺-ATPase activity by reacting with one essential Lys residue probably located in the vicinity of the catalytic site, which results in the full inactivation of the enzyme. Thus, Ca²⁺-ATPase activity measured in erythrocyte membranes or purified enzyme preparations preincubated with glucose depends on the remaining enzyme molecules in which the essential Lys residue stays unglycated. Received: 9 March 1999/Revised: 11 May 1999     

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     

Glucagon Induces Suppression of ATP-sensitive K+ Channel Activity Through a Ca2+/Calmodulin-dependent Pathway in Mouse Pancreatic β-Cells

Glucagon is known to increase intracellular cAMP levels and enhance glucose-induced electrical activity and insulin secretion in pancreatic β-cell perfused with Krebs-Ringer bicarbonate solution. The present experiments were aimed at evaluation of the hypothesis that changes in β-cells ATP-sensitive K⁺ (K_(ATP)) channel activity are involved in the glucagon-induced enhancement of electrical activity. Channel activity was recorded using the cell-attached configuration of the patch-clamp technique. Addition of glucagon (2.9 × 10⁻⁷ m) in the presence of 11.1 mm glucose caused closure of K_(ATP) channels followed by an increase in the frequency of biphasic current transients (action currents) due to action potential generation in the cell. Three calmodulin-antagonists (W-7, chlorpromazine, and trifluoperazine) restored with similar efficacy K_(ATP) channel activity in cells being exposed to glucagon. At 2.8 mm glucose, glucagon did not affect K_(ATP) channel activity until Ca²⁺ was released from Nitr-5 by flash photolysis, at which point channel activity was transiently suppressed. Similar effects were seen when db-cAMP was used instead of glucagon.These results support the view that glucagon and other cAMP-generating agonists enhance glucose-induced β-cell electrical activity through a Ca²⁺/calmodulin dependent-closure of K_(ATP) channels. Received: 26 May 1998/Revised: 18 September 1998     

Reactivity of Cysteines in the Transmembrane Region of the Na,K-ATPase α Subunit Probed with Hg2+

To gain insight into the structure and conformational coupling in the Na,K-ATPase, this study characterized the reaction of the α1 subunit transmembrane cysteines with a small probe. Intact HeLa cells expressing heterologous Na,K-ATPase were treated with (μm) HgCl₂ after placing the enzyme predominantly in either of two conformations, phosphorylated E2P.Na/E2P or dephosphorylated ATP.E1.K/ATP.E1. Under both conditions the treatment led to enzyme inactivation following a double exponential kinetic as determined by ouabain-sensitive K⁺ uptake measurements. However, the rate constant of the slow reacting component was ten times larger when the protein was probed in a medium that would favor enzyme phosphorylation. Enzymes carrying mutations of cysteines located in the α1 subunit transmembrane region were used to identify the reacting–SH groups. Replacement Cys104Ser reduced enzyme inactivation by removing the slow reacting component under both treatment conditions. Replacement of Cys964 reduced the inactivation rate constant of the fast reacting component (79%) and removed the slow reacting component when the dephosphorylated enzyme was treated with Hg²⁺. Moreover, Cys964Ser substituted enzyme was insensitive to Hg²⁺ when treated under phosphorylation conditions. These results indicate that Cys964 is involved in the fast inactivation by Hg²⁺. Although the double mutant Cys964, 104Ser was still partially inactivated by treatment under nonphosphorylating conditions, an enzyme devoid of transmembrane cysteines was insensitive to Hg²⁺ under all treatment conditions. Thus, this enzyme provides a background where accessibility of engineered transmembrane cysteines can be tested. Received: 13 March 2000/Revised: 23 June 2000     

Muscarinic Activation of BK Channels Induces Membrane Oscillations in Glioma Cells and Leads to Inhibition of Cell Migration

Patients with cerebral tumors often present with elevated levels of acetylcholine (ACh) in their cerebrospinal fluid. This motivated us to investigate physiological effects of ACh on cultured human astrocytoma cells (U373) using a combination of videomicroscopy, calcium microspectrofluorimetry and perforated patch-clamp recording. Astrocytoma cells exhibited the typical morphological changes associated with cell migration; polarized cells displayed prominent lamellipodia and associated membrane ruffling at the anterior of the cell, and a long tail region that periodically contracted into the cell body as the cell moved forward. Bath application of the ACh receptor agonist, muscarine, reversibly inhibited cell migration. In conjunction with this inhibition, ACh induced a dose-dependent, biphasic increase in resting intracellular free calcium concentration ([Ca²⁺] _i ) associated with periodic Ca²⁺ oscillations during prolonged ACh applications. The early transient rise in [Ca²⁺] _i was abolished by ionomycin and thapsigargin but was insensitive to caffeine and ryanodine while the plateau phase was strictly dependent on external calcium. The Ca²⁺ response to ACh was mimicked by muscarine and abolished by the muscarinic antagonists, atropine or 4-DAMP, but not by pirenzepine. Using perforated patch-clamp recordings combined with fluorescent imaging, we demonstrated that ACh-induced [Ca²⁺] _i oscillations triggered membrane voltage oscillations that were due to the activation of voltage-dependent, Ca²⁺-sensitive K⁺ currents. These K⁺ currents were blocked by intracellular injection of EGTA, or by extracellular application of TEA, quinine, or charybdotoxin, but not by apamin. These studies suggest that activation of muscarinic receptors on glioma cells induce the release of Ca²⁺ from intracellular stores which in turn activate Ca²⁺-dependent (BK-type) K⁺ channels. Furthermore, this effect was associated with inhibition of cell migration, suggesting an interaction of this pathway with glioma cell migration. Received: 17 December/Revised: 17 March 2000     

H+- and K+-Dependence of Ca2+ Uptake in Lung Lamellar Bodies

Lung lamellar bodies maintain an acidic interior by an energy-dependent process. The acidic pH may affect the packaging of surfactant phospholipids, processing of surfactant proteins, or surfactant protein A-dependent lipid aggregation. The electron-probe microanalysis of lamellar body elemental composition has previously suggested that lamellar bodies contain high levels of calcium some of which may be in ionic form. In this study, we investigated the Ca²⁺ uptake characteristics in isolated lung lamellar bodies. The uptake of Ca²⁺ was measured by monitoring changes in the fluorescence of Fluo-3, a Ca²⁺ indicator dye. The uptake of Ca²⁺ in lamellar bodies was ATP-dependent and increased with increasing concentrations of Ca²⁺. At 100 nm Ca²⁺, the uptake was almost completely inhibited by bafilomycin A1, a selective inhibitor of vacuolar type H⁺-ATPase, or by NH₄Cl, which raises the lamellar body pH, suggesting that the pH gradient regulates the uptake. The uptake of Ca²⁺ increased as the Ca²⁺ concentration was increased, but the relative contribution of bafilomycin A1-sensitive uptake decreased. At 700 nm, it comprised only 20% of the total uptake. These results suggest the presence of additional mechanism(s) for uptake at higher Ca²⁺ concentrations. At 700 nm Ca²⁺, the rate and extent of uptake were lower in the absence of K⁺ than in the presence of K⁺. The inhibitors of Ca²⁺-activated K⁺-channels, tetraethylammonium, Penitrem A, and 4-aminopyridine, also inhibited the K⁺-dependent Ca²⁺ uptake at 700 nm Ca²⁺. Thus the uptake of Ca²⁺ in isolated lung lamellar bodies appears to be regulated by two mechanisms, (i) the H⁺-gradient and (ii) the K⁺ transport across the lamellar body membrane. We speculate that lamellar bodies accumulate Ca²⁺ and contribute to regulation of cytosolic Ca²⁺ in type II cells under resting and stimulated conditions. Received: 18 August 1999/Revised: 9 November 1999     

Inositol 1,4,5-Trisphosphate But Not Ryanodine-Receptor Agonists Induces Calcium Release from Rat Liver Golgi Apparatus Membrane Vesicles

We investigated the direct effect of inositol 1,4,5-trisphosphate (IP₃) and ryanodine receptor agonists on Ca²⁺ release from vesicles of a rat liver Golgi apparatus (GA) enriched fraction, which were actively loaded with ⁴⁵Ca²⁺. Results in GA were compared with those obtained in a rat liver endoplasmic reticulum (ER) enriched fraction. The addition of IP₃ at concentrations ranging from 100 nm to 100 μm, in the presence of thapsigargin, a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPases, promoted a rapid decrease in the Ca²⁺ content of GA vesicles. The amount of Ca²⁺ released from the vesicles was a function of IP₃ concentration, reaching about 60% in both GA and ER fractions at 100 μm IP₃. Calcium release was inhibited by heparin, an antagonist of IP₃ receptors. Calcium exhibited a bell-shaped effect on IP₃-dependent Ca²⁺ released from GA vesicles: it activated Ca²⁺ release at concentrations up to 1 μm, and inhibited it at higher concentrations. In contrast to that found in the endoplasmic reticulum fraction, none of the ryanodine receptor agonists tested (cyclic ADP-ribose, caffeine and ryanodine) significantly induced Ca²⁺ release from GA fraction vesicles in the presence of thapsigargin. Our results indicate the presence of an IP₃-sensitive Ca²⁺ release mechanism in the Golgi apparatus membrane analogous to that of the ER. However, a Ca²⁺ release mechanism sensitive to ryanodine receptor agonists like that of ER is not evident in the GA membrane. Received: 13 March 2000/Revised: 13 July 2000     

Mechanotransduction in Colonic Smooth Muscle Cells

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

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     

Ca2+ Uptake Through Voltage-gated L-type Ca2+ Channels by Polarized Enterocytes from Atlantic Cod Gadus morhua

The presence and localization of voltage-gated Ca²⁺ channels of L-type were investigated in intestinal cells of the Atlantic cod. Enterocytes were loaded with the fluorescent Ca²⁺ probe, fure-2/AM and changes in intracellular Ca²⁺ concentrations ([Ca²⁺] _i ) were measured, in cell suspensions, in the presence of high potassium levels (100 mm), BAY K-8644 (5 μm), nifedipine (5 μm) or ω-conotoxin (1 μm). L-type Ca²⁺ channels were visualized on intestinal sections using the fluorescent dihydropyridine (-)-STBodipy. Depolarization of the plasma membrane produced a rapid (within 5 sec) and transient (at basal levels after 21 sec) increase in [Ca²⁺] _i . BAY K-8644 increased the [Ca²⁺] _i by 7.2%. Cells in a Ca²⁺-free buffer increased [Ca²⁺] _i after addition of 10 mm Ca²⁺, and this increase was abolished by nifedipine in both depolarizing and normal medium but not by ω-conotoxin. Single cell experiments using video microscopy revealed that enterocytes remained polarized several hours after preparation and that the Ca²⁺ entry and extrusion occurred at specific and different regions of the enterocyte outer membrane. Fluorescent staining of L-type Ca²⁺ channels in the intestinal mucosa showed the most intense staining at the brushborder membrane. These results demonstrate the presence of voltage gated L-type Ca²⁺ channels in enterocytes from the Atlantic cod. The channels are mainly located at the apical side of the cells, and there is a polarized uptake of Ca²⁺ into the enterocytes. This suggests that the L-type Ca²⁺ channels are involved in the transcellular Ca²⁺ entry into the enterocytes. Received: 21 August 1997/Revised: 15 April 1998     

Electrogenic Partial Reactions of the SR-Ca-ATPase Investigated by a Fluorescence Method

A fluorescence method was adapted to investigate active ion transport in membrane preparations of the SR-Ca-ATPase. The styryl dye RH421 previously used to investigate the Na,K-ATPase was replaced by an analogue, 2BITC, to obtain optimized fluorescence changes upon substrate-induced partial reactions. Assuming changes of the local electric field to be the source of fluorescence changes that are produced by uptake/release or by movement of ions inside the protein, 2BITC allowed the determination of electrogenic partial reactions in the pump cycle. It was found that Ca²⁺ binding on the cytoplasmic and on the lumenal side of the pump is electrogenic while phosphorylation and conformational transition showed only minor electrogenicity. Ca²⁺ equilibrium titration experiments at pH 7.2 in the two major conformations of the protein indicated cooperative binding of two Ca²⁺ ions in state E₁ with an apparent half-saturation concentration, K _M of 600 nm. In state P-E₂ two K _M values, 5 μm and 2.2 mM, were determined and are in fair agreement with published data. From Ca²⁺ titrations in buffers with various pH and from pH titrations in P-E₂, it could be demonstrated that H⁺ binding is electrogenic and that Ca²⁺ and H⁺ compete for the same binding site(s). Tharpsigargin-induced inhibition of the Ca-ATPase led to a state with a specific fluorescence level comparable to that of state E₁ with unoccupied ion sites, independent of the buffer composition. Received: 21 September 1998/Revised: 18 December 1998     

Veratridine-Mediated Ca2+ Dynamics and Exocytosis in Paramecium Cells

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

Alkaline pH and Internal Calcium Increase Na+ and K+ Effluxes in LK Sheep Red Blood Cells in Cl−-free Solutions

We examined the effects of pH, internal ionized Ca (Ca²⁺ _i ), cellular ATP, external divalent cations and quinine on Cl-independent ouabain-resistant K⁺ efflux in volume-clamped sheep red blood cells (SRBCs) of normal high (HK) and low (LK) intracellular K⁺ phenotypes. In LK SRBCs the K⁺ efflux was higher at pH 9.0 (350%) than at pHs 7.4 and 6.5, and was inhibited by external divalent cations, quinine, and cellular ATP depletion. The above findings suggest that the increased K⁺ efflux at alkaline pH is due to the opening of ion channels or specific transporters in the cell membrane. In addition, K⁺ efflux was activated (100%) when Ca²⁺ _i was increased (+A23187, +Ca²⁺ _o ) into the μm range. However, in comparison to human red blood cells, the Ca²⁺ _i -induced increase in K⁺ efflux in LK SRBCs was fourfold smaller and insensitive to quinine and charybdotoxin. The Na⁺ efflux was also higher at pH 9.0 than at pH 7.4, and activated (about 40%) by increasing Ca²⁺ _i . In contrast, in HK SRBCs the K⁺ efflux at pH 9.0 was neither inhibited by quinine nor activated by Ca²⁺ _i . These studies suggest the presence in LK SRBCs, of at least two pathways for Cl⁻-independent K⁺ and Na⁺ transport, of which one is unmasked by alkalinization, and the other by a rise in Ca²⁺ _i . Received: 23 May 1996/Revised: 6 December 1996     

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

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

I. Ion Channels in Human THP-1 Monocytes

The THP-1 human monocytic leukemia cell line is a useful model of macrophage differentiation. Patch clamp methods were used to identify five types of ion channels in undifferentiated THP-1 monocytes. (i) Delayed rectifier K⁺ current, I _DR, was activated by depolarization to potentials positive to −50 mV, inactivated with a time constant of several hundred msec, and recovered from inactivation with a time constant ∼21 sec. I _DR was inhibited by 4-aminopyridine (4-AP), tetraethylammonium (TEA⁺), and potently by charybdotoxin (ChTX). (ii) Ca-activated K⁺ current (I _SK) dominated whole-cell currents in cells studied with 3–10 μm [Ca²⁺] _i . I _SK was at most weakly voltage-dependent, with reduced conductance at large positive potentials, and was inhibited by ChTX and weakly by TEA⁺, Cs⁺, and Ba²⁺, but not 4-AP or apamin. Block by Cs⁺ and Ba²⁺ was enhanced by hyperpolarization. (iii) Nonselective cation current, I _cat, appeared at voltages above +20 mV. Little time-dependence was observed, and a panel of channel blockers was without effect. (iv) Chloride current, I _Cl, was present early in experiments, but disappeared with time. (v) Voltage-activated H⁺ selective current is described in detail in a companion paper (DeCoursey & Cherny, 1996. J. Membrane Biol. 152:2). The ion channels in THP-1 cells are compared with channels described in other macrophage-related cells. Profound changes in ion channel expression that occur during differentiation of THP-1 cells are described in a companion paper (DeCoursey et al., 1996. J. Membrane Biol. 152:2). Received: 19 September 1995/Revised: 14 March 1996