Activation of Divalent Cation Influx into S. cerevisiae Cells by Hypotonic Downshift

Subjecting Saccharomyces cerevisiae cells to a hypotonic downshift by transferring cells from YPD medium containing 0.8 m sorbitol to YPD medium without sorbitol induces a transient rapid influx of Ca²⁺ and other divalent cations into the cell. For cells grown in YPD at 37°C, this hypotonic downshift increases Ca²⁺ accumulation 6.7-fold. Hypotonic downshift-induced Ca²⁺ accumulation and steady-state Ca²⁺ accumulation in isotonic YPD medium are differentially affected by dodecylamine and Mg²⁺. The Ca²⁺-influx pathway responsible for hypotonic-induced Ca²⁺ influx may account for about 10–35% of Ca²⁺ accumulation by cells growing in YPD. Ca²⁺ influx is not required for cells to survive a hypotonic downshift. Hypotonic downshift greatly reduces the ability of S. cerevisiae cells to survive a 5-min exposure to 10 mm Cd²⁺ suggesting that mutants resistant to acute Cd²⁺ exposure may help identify genes required for hypotonic downshift-induced divalent cation influx. Received: 14 January 1997/Revised: 20 June 1997     

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     

A9 Fibroblasts Transfected with the m3 Muscarinic Receptor Clone Express a Ca2+ Channel Activated by Carbachol,GTP and GDP

Muscarinic m3 receptor-mediated changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_l) occur by activation of Ca²⁺ release channels present in the endoplasmic reticulum membrane and Ca²⁺ entry pathways across the plasma membrane. In this report we demonstrate the coupling of m3 muscarinic receptors to the activation of a voltage-insensitive, cation-selective channel of low conductance (3.2 ± 0.6 pS; 25 mm Ca²⁺ as charge carrier) in a fibroblast cell line expressing m3 muscarinic receptor clone (A9m3 cells). Carbachol (CCh)-induced activation of the cation-selective channel occurred both in whole cell and excised membrane patches (CCh on the external side), suggesting that the underlying mechanism involves receptor-channel coupling independent of intracellular messengers. In excised inside-out membrane patches from nonstimulated A9m3 cells GTP (10 μm) and GDP (10 μm) activated cation-selective channels with conductances of approximately 4.3 and 3.3 pS, (25 mm Ca²⁺ as charge carrier) respectively. In contrast, ATP (10 μm), UTP (10 μm) or CTP (10 μm) failed to activate the channel. Taken together, these results suggest that carbachol and guanine nucleotides regulate the activation of a cation channel that conducts calcium. Received: 14 November 1996/Revised: 4 April 1997     

Activation of the Cardiac Ryanodine Receptor by Sulfhydryl Oxidation is Modified by Mg2+ and ATP

The reactive disulfide 4,4′-dithiodipyridine (4,4′DTDP) was added to single cardiac ryanodine receptors (RyRs) in lipid bilayers. The activity of native RyRs, with cytoplasmic (cis) [Ca²⁺] of 10⁻⁷ m (in the absence of Mg²⁺ and ATP), increased within ∼1 min of addition of 1 mm 4,4′-DTDP, and then irreversibly ceased 5 to 6 min after the addition. Channels, inhibited by either 1 mm cis Mg²⁺ (10⁻⁷ m cis Ca²⁺) or by 10 mm cis Mg²⁺ (10⁻³ m cis Ca²⁺), or activated by 4 mm ATP (10⁻⁷ m cis Ca²⁺), also responded to 1 mm cis 4,4′-DTDP with activation and then loss of activity. P _o and mean open time (T _o ) of the maximally activated channels were lower in the presence of Mg²⁺ than in its absence, and the number of openings within the long time constant components of the open time distribution was reduced. In contrast to the reduced activation by 1 mm 4,4′-DTDP in channels inhibited by Mg²⁺, and the previously reported enhanced activation by 4,4′-DTDP in channels activated by Ca²⁺ or caffeine (Eager et al., 1997), the activation produced by 1 mm cis 4,4′-DTDP was the same in the presence and absence of ATP. These results suggest that there is a physical interaction between the ATP binding domain of the cardiac RyR and the SH groups whose oxidation leads to channel activation. Received: 8 September 1997/Revised: 20 January 1998     

Magnesium Inhibition of Ryanodine-Receptor Calcium Channels: Evidence for Two Independent Mechanisms

The gating of ryanodine receptor calcium release channels (RyRs) depends on myoplasmic Ca²⁺ and Mg²⁺ concentrations. RyRs from skeletal and cardiac muscle are activated by μm Ca²⁺ and inhibited by mm Ca²⁺ and Mg²⁺. ⁴⁵Ca²⁺ release from skeletal SR vesicles suggests two mechanisms for Mg²⁺-inhibition (Meissner, Darling & Eveleth, 1986, Biochemistry 25:236–244). The present study investigates the nature of these mechanisms using measurements of single-channel activity from cardiac- and skeletal RyRs incorporated into planar lipid bilayers. Our measurements of Mg²⁺- and Ca²⁺-dependent gating kinetics confirm that there are two mechanisms for Mg²⁺ inhibition (Type I and II inhibition) in skeletal and cardiac RyRs. The mechanisms operate concurrently, are independent and are associated with different parts of the channel protein. Mg²⁺ reduces P _o by competing with Ca²⁺ for the activation site (Type-I) or binding to more than one, and probably two low affinity inhibition sites which do not discriminate between Ca²⁺ and Mg²⁺ (Type-II). The relative contributions of the two inhibition mechanisms to the total Mg²⁺ effect depend on cytoplasmic [Ca²⁺] in such a way that Mg²⁺ inhibition has the properties of Types-I and II inhibition at low and high [Ca²⁺] respectively. Both mechanisms are equally important when [Ca²⁺] = 10 μm in cardiac RyRs or 1 μm in skeletal RyRs. We show that Type-I inhibition is not the sole mechanism responsible for Mg²⁺ inhibition, as is often assumed, and we discuss the physiological implications of this finding. Received: 1 January 1996/Revised: 14 November 1996     

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     

Chemical Modification Reveals Involvement of Different Sites for Nucleotide Analogues in the Phosphatase Activity of the Red Cell Calcium Pump

The calcium pump of plasma membranes catalyzes the hydrolysis of ATP and phosphoric esters like p-nitrophenyl phosphate (pNPP). The latter activity requires the presence of ATP and/or calmodulin, and Ca²⁺ [22, 25]. We have studied the effects of nucleotide-analogues and chemical modifications of nucleotide binding sites on Ca²⁺-pNPPase activity. Treatment with fluorescein isothiocyanate (FITC), abolished Ca²⁺-ATPase and ATP-dependent pNPPase, but affected only 45% of the calmodulin-dependent pNPPase activity. The nucleotide analogue eosin-Y had an inhibitory effect on calmodulin-dependent pNPPase (Ki _eosin-Y= 2 μm). FITC treatment increased Ki _eosin-Y 15 times. Acetylation of lysine residues with N-hydroxysuccinimidyl acetate inactivates Ca²⁺-ATPase by modifying the catalytic site, and impairs stimulation by modulators by modifying residues outside this site [9]. Acetylation suppressed the ATP-dependent pNPPase with biphasic kinetics. ATP or pNPP during acetylation cancels the fast component of inactivation. Acetylation inhibited only partially the calmodulin-dependent pNPPase, but neither ATP nor pNPP prevented this inactivation. From these results we conclude: (i) ATP-dependent pNPPase depends on binding of ATP to the catalytic site; (ii) the catalytic site plays no role in calmodulin-dependent pNPPase. The decreased affinity for eosin-Y of the FITC-modified enzyme, suggests that the sites for these two molecules are closely related but not overlapped. Acetimidation of the pump inhibited totally the calmodulin-dependent pNPPase, but only partially the ATP-pNPPase. Since calmodulin binds to E₁, the E₁ conformation or the E₂? E₁ transition would be involved during calmodulin-dependent pNPPase activity. Received: 20 January 1998     

Calcium-dependent Modulation of the Agonist Affinity of the Mammalian Olfactory Cyclic Nucleotide-gated Channel by Calmodulin and a Novel Endogenous Factor

The calcium-dependent modulation of the affinity of the cyclic nucleotide-gated (CNG) channels for adenosine 3′,5′-cyclic monophosphate (cAMP) was studied in enzymatically dissociated rat olfactory receptor neurons, by recording macroscopic cAMP-activated currents from inside-out patches excised from their dendritic knobs. Upon intracellular addition of 0.2 mm Ca²⁺ (0.2 Ca) the concentration of cAMP required for the activation of half-maximal current (EC₅₀) was reversibly increased from 3 μm to about 30 μm. This Ca²⁺-induced affinity shift was insensitive to the calmodulin antagonist, mastoparan, was abolished irreversibly by a 2-min exposure to 3 mm Mg²⁺+ 2 mm EGTA (Mg + EGTA), and was not restored by the application of calmodulin (CAM). Addition of CAM plus 0.2 mm Ca²⁺ (0.2 Ca + CAM), further reversibly shifted the cAMP affinity from 30 μm to about 200 μm. This affinity shift was not affected by Mg + EGTA exposure, but was reversed by mastoparan. Thus, the former Ca²⁺-only effect must be mediated by an unknown endogenous factor, distinct from CAM. Removal of this factor also increased the affinity of the channel for CAM. The affinity shift induced by Ca²⁺-only was maintained in the presence of the nonhydrolyzable cAMP analogue, 8-bromo-cAMP and the phosphatase inhibitor, microcystin-LR, ruling out modulation by phosphodiesterases or phosphatases. Our results indicate that the olfactory CNG channels are modulated by an as yet unidentified factor distinct from CAM. Received: 26 December 1995/Revised: 14 March 1996     

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     

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     

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     

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     

Characterization of Ryanodine-sensitive Ca2+ Release from Microsomal Vesicles of Rat Parotid Acinar Cells: Regulation by Cyclic ADP-ribose

We have measured ryanodine (caffeine)-sensitive ⁴⁵Ca²⁺ release from isolated microsomal vesicles of endoplasmic reticulum prepared from rat parotid acinar cells. After a steady state of ATP-dependent ⁴⁵Ca²⁺ uptake, the addition of caffeine (40 mm), ryanodine (10∼500 μm) or an NAD⁺ metabolite, cyclic ADP-ribose (cADPR, 4 μm) released about 10% of the ⁴⁵Ca²⁺ that had been taken up. The ⁴⁵Ca²⁺ release was not inhibited by heparin, an antagonist of IP₃ receptor. The effects of caffeine, ryanodine and cADPR on ⁴⁵Ca²⁺ release were also tested in the presence of thapsigargin (TG), an inhibitor of microsomal Ca²⁺-ATPase. When caffeine (10∼40 mm), ryanodine (10 μm) or cADPR (1∼10 μm) was added in the medium with 100 nm TG, a significant ⁴⁵Ca²⁺ release was seen, while higher concentrations of ryanodine (>100 μm) did not cause any ⁴⁵Ca²⁺ release in the presence of TG. The initial rate of caffeine (40 mm)-induced ⁴⁵Ca²⁺ release was increased by a pretreatment with 10 μm ryanodine, whereas the caffeine-induced ⁴⁵Ca²⁺ release was strongly inhibited by the presence of a higher concentration (500 μm) of ryanodine. cADPR-induced ⁴⁵Ca²⁺ release was also inhibited by 500 μm ryanodine. Caffeine (40 mm)- or cADPR (4 μm)-induced ⁴⁵Ca²⁺ release was abolished by a presence of ruthenium red (50∼100 μm). The presence of a low concentration (0.5 μm) of cADPR shifted the dose-response curve of caffeine-induced ⁴⁵Ca²⁺ release to the left. These results indicate the presence of a ryanodine sensitive Ca²⁺ release mechanism in the endoplasmic reticulum of rat parotid acinar cells that is distinct from the IP₃-sensitive Ca²⁺ channel and is activated by caffeine, cADPR and a low concentration (10 μm) of ryanodine, but is inhibited by higher concentrations (>100 μm) of ryanodine and ruthenium red. The properties of the ryanodine-sensitive mechanism are similar to that of the ryanodine receptor as described in muscle cells. Received: 11 June 1996/Revised: 14 November 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     

Roles of Ca2+ and Protein Tyrosine Kinase in Insulin Action on Cell Volume via Na+ and K+ Channels and Na+/K+/2Cl− Cotransporter in Fetal Rat Alveolar Type II Pneumocyte

The aim of the present study was to investigate the roles of Ca²⁺ and protein tyrosine kinase (PTK) in the insulin action on cell volume in fetal rat (20-day gestational age) type II pneumocytes. Insulin (100 nm) increased cell volume in the presence of extracellular Ca²⁺ (1 mm), while cell shrinkage was induced by insulin in the absence of extracellular Ca²⁺ (<1 nm). This insulin action in a Ca²⁺-containing solution was completely blocked by co-application of bumetanide (50 μm, an inhibitor of Na⁺/K⁺/2Cl⁻ cotransporter) and amiloride (10 μm, an inhibitor of epithelial Na⁺ channel), but not by the individual application of either bumetanide or amiloride. On the other hand, the insulin action on cell volume in a Ca²⁺-free solution was completely blocked by quinine (1 mm, a blocker of Ca²⁺-activated K⁺ channel), but not by bumetanide and/or amiloride. These observations suggest that insulin activates an amiloride-sensitive Na⁺ channel and a bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter in the presence of 1 mm extracellular Ca²⁺, that the stimulatory action of insulin on an amiloride-sensitive Na⁺ channel and a bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter requires Ca²⁺, and that in a Ca²⁺-free solution insulin activates a quinine-sensitive K⁺ channel but not in the presence of 1 mm Ca²⁺. The insulin action on cell volume in a Ca²⁺-free solution was almost completely blocked by treatment with BAPTA (10 μm) or thapsigargin (1 μM, an inhibitor of Ca²⁺-ATPase which depletes the intracellular Ca²⁺ pool). Further, lavendustin A (10 μm, an inhibitor of receptor type PTK) blocked the insulin action in a Ca²⁺-free solution. These observations suggest that the stimulatory action of insulin on a quinine-sensitive K⁺ channel is mediated through PTK activity in a cytosolic Ca²⁺-dependent manner. Lavendustin A, further, completely blocked the activity of the Na⁺/K⁺/2Cl⁻ cotransporter in a Ca²⁺-free solution, but only partially blocked the activity of the Na⁺/K⁺/2Cl⁻ cotransporter in the presence of 1 mm Ca²⁺. This observation suggests that the activity of the Na⁺/K⁺/2Cl⁻ cotransporter is maintained through two different pathways; one is a PTK-dependent, Ca²⁺-independent pathway and the other is a PTK-independent, Ca²⁺-dependent pathway. Further, we observed that removal of extracellular Ca²⁺ caused cell shrinkage by diminishing the activity of the amiloride-sensitive Na⁺ channel and the bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter, and that removal of extracellular Ca²⁺ abolished the activity of the quinine-sensitive K⁺ channel. We conclude that the cell shrinkage induced by removal of extracellular Ca²⁺ results from diverse effects on the cotransporter and Na⁺ and K⁺ channels. Received: 2 September 1998/Revised: 30 November 1998     

Sodium-dependent and -independent Choline Uptake by Type II Epithelial Cells from Rat Lung

The uptake of ³H-labeled choline by a suspension of isolated type II epithelial cells from rat lung has been studied in a Ringer medium. Uptake was linear for 4 min at both 0.1 μm and 5.0 μm medium choline; at 5 μm, only 10% of the label was recovered in a lipid fraction. Further experiments were conducted at the low concentration (0.1 μm), permitting characterization of the properties of high-affinity systems. Three fractions of choline uptake were detected: (i) a sodium-dependent system that was totally inhibited by hemicholinium-3 (HC-3); (ii) a sodium-independent uptake, when Na⁺ was replaced by Li⁺, K⁺ or Mg²⁺, inhibited by HC-3; (iii) a residual portion persisting in the absence of Na⁺ and unaffected by HC-3. Choline uptake was sigmoidally related to the medium Na⁺ concentration. Kinetic properties of the uptake of 0.1 μm ³H-choline in the presence and absence of medium Na⁺ were examined in two ways. (a) Inhibition by increasing concentrations of unlabeled choline (0.5–100 μm) was consistent with the presence of two Michaelis-Menten-type systems in the presence of Na⁺; a Na⁺-dependent portion (a mean of 0.52 of the total) had a K _m for choline of 1.5 μm while K _m in the absence of Na⁺ (Li⁺ substituting) was 18.6 μm. (b) Inhibition by HC-3 (0.3–300 μm) gave K_i values of 1.7 μm and 5.0 μm HC-3 for the Na⁺-dependent and -independent fractions. The apparent K _m of the Na⁺-dependent uptake is lower than that reported previously for lung-derived cells and is in the range of the K _m values reported for high-affinity, Na⁺-dependent choline uptake by neuronal cells. Received: 18 February 1997/Revised: 7 December 1997     

Calcium-Sensitive Nonselective Cation Channel Identified in the Epithelial Cells Isolated from the Endolymphatic Sac of Guinea Pigs

We identified a Ca²⁺-sensitive cation channel in acutely dissociated epithelial cells from the endolymphatic sac (ES) of guinea pigs using the patch-clamp technique. Single-channel recordings showed that the cation channel had a conductance of 24.0 ± 1.3 pS (n= 8) in our standard solution. The relative ionic permeability of the channel was in the order K⁺= Na⁺ > Ca²⁺≫ Cl⁻. This channel was weakly voltage-dependent but was strongly activated by Ca²⁺ on the cytosolic side at a concentration of around 1 mm in inside-out excised patches. With cell-attached patches, however, the channel was activated by much lower Ca²⁺ concentrations. Treatment of the cells, under cell-attached configuration, with ionomycin (10 μm), carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 20 μm), or ATP (1 mm), which increased intracellular Ca²⁺ concentration ([Ca²⁺]_i), activated the channel at an estimated [Ca²⁺]_i from 0.6 μm to 10 μm. It is suggested that some activators of the channel were deteriorated or washed out during the formation of excised patches. Based on this Ca²⁺ sensitivity, we speculated that the channel contributes to the regulation of ionic balance and volume of the ES by absorbing Na⁺ under certain pathological conditions that will increase [Ca²⁺]_i. This is the first report of single-channel recordings in endolymphatic sac epithelial cells. Received: 24 October 2000/Revised: 10 April 2001     

Effects of 2,3-Butanedione 2-Monoxime on Ca2+ Release Channels (Ryanodine Receptors) of Cardiac and Skeletal Muscle

Single channel and [³H]ryanodine binding measurements were performed to test for a direct functional interaction between 2,3-butanedione 2-monoxime (BDM) and the skeletal and cardiac muscle sarcoplasmic reticulum Ca²⁺ release channels (ryanodine receptors). Single channel measurements were carried out in symmetric 0.25 m KCl media using the planar lipid bilayer method. BDM (1–10 mm) activated suboptimally Ca²⁺-activated (0.5–1 μm free Ca²⁺) single, purified and native cardiac and skeletal release channels in a concentration-dependent manner by increasing the number of channel events without a change of single channel conductances. BDM activated the two channel isoforms when added to either side of the bilayer. At a maximally activating cytosolic Ca²⁺ concentration of 20 μm, BDM was without effect on the cardiac channel, whereas it inhibited skeletal channel activities with IC₅₀≈ 2.5 mm. In agreement with single channel measurements, high-affinity [³H]ryanodine binding to the two channel isoforms was increased in a concentration-dependent manner at ≤1 μm Ca²⁺. BDM was without a noticeable effect at low (≤0.01 μm) Ca²⁺ concentrations. At 20 μm Ca²⁺, BDM inhibited the skeletal but not cardiac channel. These results suggest that BDM regulates the Ca²⁺ release channels from the sarcoplasmic reticulum of skeletal and cardiac muscle in a concentration, Ca²⁺ and tissue-dependent manner. Received: 31 December 1998/Revised: 9 March 1999     

Are B-type Ca2+ Channels of Cardiac Myocytes Akin to the Passive Ion Channel in the Plasma Membrane Ca2+ Pump?

The present study demonstrates that B-type Ca²⁺ channels observed in rat ventricular myocytes markedly reacted to agents known to affect the ion-motive plasma membrane Ca²⁺-ATPase (PMCA) pump. Chlorpromazine (CPZ)-activated B-type Ca²⁺ channels were completely blocked by internal application of PMCA pump inhibitors, namely La³⁺ (100 μm), eosin (10 μm) and AIF₃ (100 μm). Calmodulin (50 U/ml), the main endogenous positive regulator of PMCA, was unable to activate but significantly reduced CPZ-activated B-type channel activity. In the same manner, ATP (1 and 4 mm), the main energizing substrate of PMCA, was able to reversibly and significantly reduce this activity in a dose-dependent manner. Interestingly, anti-PMCA antibody 5F10, but not anti-Na/K ATPase antibody (used as a negative control) induced a marked Ba²⁺-conducting channel activity that shared the same characteristics with that of CPZ-activated B-type channels. 5F10-Activated channels were mostly selective towards Ba²⁺, mainly had three observed conductance levels (23, 47 and 85 pS), were observed with a frequency of about 1 out of 5 membrane patches and were completely blocked by 10 μm eosin. These results suggest that B-type Ca²⁺ channels are some form of the PMCA pump. Received: 24 July 2000/Revised: 5 October 2000     

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

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