The ACh-evoked,Ca2+-activated Whole-cell K+ Current in Mouse Mandibular Secretory Cells. Whole-cell and Fluorescence Studies

In our previous studies on sheep parotid secretory cells, we showed that the K⁺ current evoked by acetylcholine (ACh) was not carried by the high-conductance voltage- and Ca²⁺-activated K⁺ (BK) channel which is so conspicuous in unstimulated cells, notwithstanding that the BK channel is activated by ACh. Since several studies from other laboratories had suggested that the BK channel did carry the ACh-evoked K⁺ current in the secretory cells of the mouse mandibular gland, and that the current could be blocked with tetraethylammonium (TEA), a known blocker of BK channels, we decided to investigate the ACh-evoked K⁺ current in mouse cells more closely. We studied whether the ACh-evoked K⁺ current in the mouse is inhibited by TEA and quinine. Using the whole-cell patch-clamp technique and microspectrofluorimetric measurement of intracellular Ca²⁺, we found that TEA and quinine do inhibit the ACh-evoked K⁺ current but that the effect is due to inhibition of the increase in intracellular Ca²⁺ evoked by ACh, not to blockade of a K⁺ conductance. Furthermore, we found that the K⁺ conductance activated when ionomycin is used to increase intracellular free Ca²⁺ was inhibited only by quinine and not by TEA. We conclude that the ACh-evoked K⁺ current in mouse mandibular cells does not have the blocker sensitivity pattern that would be expected if it were being carried by the high-conductance, voltage- and Ca²⁺-activated K⁺ (BK) channel. The properties of this current are, however, consistent with those of a 40 pS K⁺ channel that we have reported to be activated by ACh in these cells [16]. Received: 9 January 1996/Revised: 17 April 1996     

Cytological effects of urecholine stimulation on the rat pancreas

Summary Stimulation of the exocrine pancreas by the secretagogue urecholine causes degranulation of the acinar cells. Under in vivo conditions, this degranulation is not uniform throughout the tissue. Indeed some of the acini are almost completely depleted of their granules while others display the appearance of resting acini. A noticeable feature is that all the cells of the same acinus display a comparable degree of degranulation. Moreover, groups of neighbouring acini seem to respond simultaneously suggesting that the secretory stimulus is propagated from one acinus to the other. In vitro stimulation of dispersed acini also showed that some of the acini are more responsive than others indicating that this phenomenon can not be attributed to accessibility of the secretagogue to its receptor. These observations lead us to the concept that the response of the pancreatic acinar cell is controlled at the level of the acinus.     

Candidate Inhibitor of the Volume-Sensitive Kinase Regulating K-Cl Cotransport: The Myosin Light Chain Kinase Inhibitor ML-7

K-Cl cotransport, KCC, is activated by swelling in many cells types, and promotes volume regulation by a KCl efflux osmotically coupled to water efflux. KCC is probably activated by swelling-inhibition of a kinase, permitting dephosphorylation, and activation of the cotransporter by a phosphatase. The myosin light chain kinase (MLCK) inhibitor ML-7 inhibits transporters activated by shrinkage. In red blood cells from three mammalian species, ML-7 stimulated KCC in a volume-dependent manner. Relative stimulation was greatest in more shrunken cells. Stimulation was reduced by moderate cell swelling and abolished by further swelling. The half-maximal stimulation is at ∼20 μm ML-7, 50-fold greater than the IC₅₀ for inhibition of MLCK in vitro. Stimulation of KCC by ML-7 did not require cell Ca, while MLCK does. Therefore the target of ML-7 in stimulating KCC in red cells is probably not MLCK. The evidence favors stimulation of KCC by ML-7 by inhibiting the volume-sensitive kinase. Qualitatively similar effects of ML-7 on KCC in red cells from three mammalian species suggest a general mechanism. Received: 17 March 2000/Revised: 28 July 2000     

A role for AQP5 in activation of TRPV4 by hypotonicity: concerted involvement of AQP5 and TRPV4 in regulation of cell volume recovery

Regulation of cell volume in response to changes in osmolarity is critical for cell function and survival. However, the molecular basis of osmosensation and regulation of cell volume are not clearly understood. We have examined the mechanism of regulatory volume decrease (RVD) in salivary gland cells and report a novel association between osmosensing TRPV4 (transient receptor potential vanalloid 4) and AQP5 (aquaporin 5), which is required for regulating water permeability and cell volume. Exposure of salivary gland cells and acini to hypotonicity elicited an increase in cell volume and activation of RVD. Hypotonicity also activated Ca2+ entry, which was required for subsequent RVD. Ca2+ entry was associated with a distinct nonselective cation current that was activated by 4alphaPDD and inhibited by ruthenium red, suggesting involvement of TRPV4. Consistent with this, endogenous TRPV4 was detected in cells and in the apical region of acini along AQP5. Importantly, acinar cells from mice lacking either TRPV4 or AQP5 displayed greatly reduced Ca2+ entry and loss of RVD in response to hypotonicity, although the extent of cell swelling was similar. Expression of N terminus-deleted AQP5 suppressed TRPV4 activation and RVD but not cell swelling. Furthermore, hypotonicity increased the association and surface expression of AQP5 and TRPV4. Both these effects and RVD were reduced by actin depolymerization. These data demonstrate that (i) activation of TRPV4 by hypotonicity depends on AQP5, not on cell swelling per se, and (ii) TRPV4 and AQP5 concertedly control regulatory volume decrease. These data suggest a potentially important role for TRPV4 in salivary gland function.     

Rapid Swelling of a CHO-K1 Aspartate/Glutamate Transport Mutant in Hypo-osmotic Medium

Two Chinese hamster ovary cell (CHO-K1) mutants selected for defective glutamate transport via system X⁻ _AG are also highly permeable to small neutral molecules. Light microscopy demonstrated that exposure of one of these mutants, Ed-A1, to hypo-osmotic medium led to extremely rapid swelling, presumably due to increased water flux. When placed in 20% saline, Ed-A1 cells swelled to three times their original volume within 15 sec, a sixfold larger increase than parental CHO-K1. In spite of this rapid volume increase, mutant and wild-type cells remained viable for 20 min in dilute saline. A regulatory volume decrease in Ed-A1, and the continual swelling of CHO-K1, resulted in the two cells achieving equal size after 5 min in 20% saline. The time course of these volume changes permitted analysis of large numbers of cells by a hydrodynamic technique, steric field flow fractionation (FFF). Steric FFF demonstrated the expected inhibition of osmotic swelling of human erythrocytes by the mercurial, p-chloromercuribenzenesulfonic acid (PCMBS). However, PCMBS increased the apparent swelling rate of Ed-A1 and CHO-K1, suggesting that an aquaporin-like molecule is not responsible for any significant fraction of the water fluxes into either line. PCMBS also strongly inhibited aspartate transport by system X⁻ _AG. By taking advantage of their different swelling rates in hypotonic medium, steric FFF can separate mixtures of CHO-K1 and Ed-A1. Received: 2 August 1996/Revised: 25 October 1996     

Visualization of 'water secretion' by confocal microscopy in rat salivary glands: possible distinction of para- and transcellular pathway

Visualization of water transport in cells, tissues and organs is an important, yet still difficult, task in morphological science. By using confocal microscopy and the fluid-phase fluorescent tracer technique, we visualized water secretion and estimated the routes of water transport across the acinar epithelia in rat parotid and submandibular glands. Confocal microscopy of whole glands perfused arterially with Lucifer yellow revealed a bright fluorescence at the basolateral space of acini. Luminal space was devoid of fluorescence, but revealed it after isoproterenol pretreatment, ductal infusion of fluorescent dextrans into the lumen, or tissue dissociation by collagenase. Under these conditions, stimulation of fluid secretion with carbachol caused a rapid decline of the luminal fluorescence intensity, indicating that the secreted water washed out the fluorescent probes in the acinar lumen. In the stimulated dissociated acini, the luminal fluorescence disappeared by 15 sec, but reappeared at 30-45 sec to maintain a low plateau level. By assuming that the tight junction was 'paralyzed' by the collagenase digestion and that the paracellular fluid transport could not influence the dilution of Lucifer yellow, we estimated that the initial water secretion by CCh occurs via the transcellular pathway, while later than 30-45 sec the additional water permeates through the paracellular pathway.     

Bending the MDCK Cell Primary Cilium Increases Intracellular Calcium

We tested the hypothesis that the primary cilium of renal epithelia is mechanically sensitive and serves as a flow sensor in MDCK cells using differential interference contrast and fluorescence microscopy. Bending the cilium, either by suction with a micropipette or by increasing the flow rate of perfusate, causes intracellular calcium to substantially increase as indicated by the fluorescent indicator, Fluo-4. This calcium signal is initiated by Ca²⁺-influx through mechanically sensitive channels that probably reside in the cilium or its base. The influx is followed by calcium release from IP₃-sensitive stores. The calcium signal then spreads as a wave from the perturbed cell to its neighbors by diffusion of a second messenger through gap junctions. This spreading of the calcium wave points to flow sensing as a coordinated event within the tissue, rather than an isolated phenomenon in a single cell. Measurement of the membrane potential difference by microelectrode during perfusate flow reveals a profound hyperpolarization during the period of elevated intracellular calcium. We conclude that the primary cilium in MDCK cells is mechanically sensitive and responds to flow by greatly increasing intracellular calcium. Received: 4 April 2001/Revised: 28 June 2001     

A Computational Study of the Development of Epithelial Acini: I. Sufficient Conditions for the Formation of a Hollow Structure

Normal hollow epithelial acini are 3-dimensional culture structures that resemble the architecture and functions of normal breast glands and lobules. This experimental model enables in vitro investigations of genotypic and molecular abnormalities associated with epithelial cancers. However, the way in which the acinar structure is formed is not yet completely understood. Gaining more information about consecutive stages of acini development—starting from a single cell that gives rise to a cluster of randomly oriented cells, followed by cell differentiation that leads to a layer of polarised cells enclosing the hollow lumen—will provide insight into the transformations of eukaryotic cells that are necessary for their successful arrangement into an epithelium. In this paper, we introduce a two-dimensional single-cell-based model representing the cross section of a typical acinus. Using this model, we investigate mechanisms that lead to the unpolarised cell growth, cell polarisation, stabilisation of the acinar structure and maintenance of the hollow lumen and discuss the sufficient conditions for each stage of acinar formation. In the follow-up paper (Rejniak and Anderson, A computational study of the development of epithelial acini. II. Necessary conditions for structure and lumen stability), we investigate what morphological changes are observable in the growing acini when some assumptions of this model are relaxed.     

Short-Chain Fatty Acid (SCFA) Volume Regulation in Proximal and Distal Rabbit Colon is Different

SCFAs increase the volume of many different cell types rarely exposed to significant concentrations of these weak electrolytes. SCFAs swell isolated cells from colonic carcinoma cell lines, but the mechanism(s) of volume regulation in normal colonocytes, which are generally exposed to >100 mm SCFAs, has not been well characterized. Aims: To determine the effect of SCFAs on volume regulation in proximal and distal rabbit colonocytes. Methods: Isolated colonocytes were plated on coverslips and placed in a perfusion apparatus that permitted fluid changes. Cells were continuously monitored by video-microscopy; volume was estimated by measured changes in the radius of individual cells. Results: Distal colonocytes (DC) consistently had a slightly greater basal volume than proximal colonocytes (PC): [14.2 pl/fl:9.8 pl/fl] In HEPES-buffered solutions, an isotonic change to a 90 mm NaCl/50 mm Na propionate solution elicited a significant increase in cell volume within 10 min, but no noticeable regulatory volume decrease over 30 min: V/Vo in DC: 1.29 ± .09; in PC: 1.25 ± .05. In HCO₃-buffered solutions, 50 mm PROP caused significantly greater cell swelling; in DC: 1.74 ± .21; in PC: 1.52 ± .08. In DC both amiloride and EIPA blocked the SCFA-induced increase in cell volume. A hypotonic challenge confirmed that these cells were capable of swelling. In contrast, amiloride did not significantly inhibit SCFA-induced swelling in PC: control, 1.25 ± .05; amiloride, 1.36 ± .10. Cell volume increased in PC perfused with an isosmotic 50 mm propionate, Na-free solution: 1.22 ± .04. Conclusions: (i) SCFAs induce significant cell swelling, but no regulatory volume decrease, in isolated colonocytes; (ii) HCO₃ augments SCFA-induced cell swelling; (iii) volume increase in DC is dependent on Na-H exchange, but in PC appears to be Na-independent. Significance: There are fundamental differences in how proximal and distal colon respond to isosmotic volume challenge of SCFAs. Received: 1 September 1995/Revised: 9 November 1995     

Release of Calcium from Mitochondrial and Nonmitochondrial Intracellular Stores in Mouse Pancreatic Acinar Cells by Hydrogen Peroxide

In the present study we have studied how [Ca²⁺] _i is influenced by H₂O₂ in collagenase-dispersed mouse pancreatic acinar cells and the mechanism underlying this effect by using a digital microspectrofluorimetric system. In the presence of normal extracellular calcium concentration, perfusion of pancreatic acinar cells with 1 mm H₂O₂ caused a slow sustained [Ca²⁺] _i increase, reaching a stable plateau after 10–15 min of perfusion. This increase induced by H₂O₂ was also observed in a nominally calcium-free medium, reflecting the release of calcium from intracellular store(s). Application of 1 mm H₂O₂ to acinar cells, in which nonmitochondrial agonist-releasable calcium pools had been previously depleted by a maximal concentration of CCK-8 (1 nm) or thapsigargin (0.5 μm) was still able to induce calcium release. Similar results were observed when thapsigargin was substituted for the mitochondrial uncoupler FCCP (0.5 μm). By contrast, simultaneous addition of thapsigargin and FCCP clearly abolished the H₂O₂-induced calcium increase. Interestingly, co-incubation of intact pancreatic acinar cells with CCK-8 plus thapsigargin and FCCP in the presence of H₂O₂ did not significantly affect the transient calcium spike induced by the depletion of nonmitochondrial and mitochondrial agonist-releasable calcium pools, but was followed by a sustained increase of [Ca²⁺] _i . In addition, H₂O₂ was able to block calcium efflux evoked by CCK and thapsigargin. Finally, the transient increase in [Ca²⁺] _i induced by H₂O₂ was abolished by an addition of 2 mm dithiothreitol (DTT), a sulfhydryl reducing agent. Our results show that H₂O₂ releases calcium from CCK-8- and thapsigargin-sensitive intracellular stores and from mitochondria. The action of H₂O₂ is likely mediated by oxidation of sulfhydryl groups of calcium-ATPases. Received: 15 May 2000/Revised: 4 October 2000     

The Role of Calcium in the Volume Regulation of Rat Lacrimal Acinar Cells

Earlier studies have suggested a role for Ca²⁺ in regulatory volume decrease (RVD) in response to hypotonic stress through the activation of Ca²⁺-dependent ion channels (Kotera & Brown, 1993; Park et al., 1994). The involvement of Ca²⁺ in regulating cell volume in rat lacrimal acinar cells was therefore examined using a video-imaging technique to measure cell volume. The trivalent cation Gd³⁺ inhibited RVD, suggesting that Ca²⁺ entry is important and may be via stretch-activated cation channels. However, Fura-2 loaded cells did not show an increase in [Ca²⁺] _i during exposure to hypotonic solutions. The absence of any changes in [Ca²⁺] _i resulted from the buffering of cytosolic Ca²⁺ by Fura-2 during hypotonic shock and therefore inhibition of RVD. The intracellular Ca²⁺ chelator, BAPTA, also inhibited the RVD response to hypotonic shock. An increase in [Ca²⁺] _i induced by either acetylcholine or ionomycin, was found to decrease cell volume under isotonic conditions in lacrimal acinar cells. Cell shrinkage was inhibited by tetraethylammonium ion, an inhibitor of Ca²⁺-activated K⁺ channels. On the basis of the presented data, we suggest an involvement of intracellular Ca²⁺ in controlling cell volume in lacrimal acinar cells. Received: 20 February 1998/Revised: 1 May 1998     

Kinetics of Water Transport in Eel Intestinal Vesicles

Brush border membrane vesicles, BBMV, from eel intestinal cells or kidney proximal tubule cells were prepared in a low osmolarity cellobiose buffer. The osmotic water permeability coefficient P _f for eel vesicles was not affected by pCMBS and was measured at 1.6 × 10⁻³ cm sec⁻¹ at 23°C, a value lower than 3.6 × 10⁻³ cm sec⁻¹ exhibited by the kidney vesicles and similar to published values for lipid bilayers. An activation energy E _a of 14.7 Kcal mol⁻¹ for water transport was obtained for eel intestine, contrasting with 4.8 Kcal mol⁻¹ determined for rabbit kidney proximal tubule vesicles using the same method of analysis. The high value of E _a , as well as the low P _f for the eel intestine is compatible with the absence of water channels in these membrane vesicles and is consistent with the view that water permeates by dissolution and diffusion in the membrane. Further, the initial transient observed in the osmotic response of kidney vesicles, which is presumed to reflect the inhibition of water channels by membrane stress, could not be observed in the eel intestinal vesicles. The P _f dependence on the tonicity of the osmotic shock, described for kidney vesicles and related to the dissipation of pressure and stress at low tonicity shocks, was not seen with eel vesicles. These results indicate that the membranes from two volume transporter epithelia have different mechanisms of water permeation. Presumably the functional water channels observed in kidney vesicles are not present in eel intestine vesicles. The elastic modulus of the membrane was estimated by analysis of swelling kinetics of eel vesicles following hypotonic shock. The value obtained, 0.79 × 10⁻³ N cm⁻¹, compares favorably with the corresponding value, 0.87 × 10⁻³ N cm⁻¹, estimated from measurements at osmotic equilibrium. Received: 28 January 1999/Revised: 15 June 1999     

Methylglyoxal Causes Swelling and Activation of a Volume-Sensitive Anion Conductance in Rat Pancreatic β-Cells

Membrane potential and whole-cell current were studied in rat pancreatic β-cells using the `perforated patch' technique and cell volume measured by a video-imaging method. Exposure of β-cells to the α-ketoaldehyde methylglyoxal (1 mm) resulted in depolarization and electrical activity. In cells voltage-clamped at −70 mV, this effect was accompanied by the development of inward current noise. In voltage-pulse experiments, methylglyoxal activated an outwardly rectifying conductance which was virtually identical to the volume-sensitive anion conductance previously described in these cells. Two inhibitors of this conductance, 4,4′-dithiocyanatostilbene-2,2′-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), also inhibited the depolarization and inward current evoked by methylglyoxal. Methylglyoxal increased β-cell volume to a relative value of 1.33 after 10 min with a gradual return towards basal levels following withdrawal of the α-ketoaldehyde. None of the effects of methylglyoxal was observed in response to t-butylglyoxal which, unlike methylglyoxal, is a poor substrate for the glyoxalase pathway. Methylglyoxal had no apparent effect on β-cell K⁺ channel activity. It is suggested that the metabolism of methylglyoxal to d-lactate causes β-cell swelling and activation of the volume-sensitive anion channel, leading to depolarization. These findings could be relevant to the stimulatory action of d-glucose, the metabolism of which generates significant quantities of l-lactate. Received: 15 May 1998/Revised: 25 September 1998     

Regulation of Cell Volume by β2-adrenergic Stimulation in Rat Fetal Distal Lung Epithelial Cells

Cell-volume changes induced by terbutaline (a specific β₂-agonist) were studied morphometrically in rat fetal distal lung epithelium (FDLE) cells. Cell-volume changes qualitatively differed with the concentration of terbutaline. Terbutaline of 10⁻¹⁰–10⁻⁸ m induced transient cell swelling. Terbutaline of 10⁻⁷ m induced transient cell swelling followed by slow cell shrinkage. Terbutaline of 10⁻⁶–10⁻⁵ m induced rapid cell shrinkage followed by slow cell shrinkage. Terbutaline of 10⁻³ m induced transient cell shrinkage; then cell volume oscillated during stimulation. Benzamil of 10⁻⁶ m suppressed the cell swelling induced by 10⁻¹⁰–10⁻⁸ m terbutaline and quinine of 10⁻³ m inhibited the cell shrinkage induced by 10⁻⁶–10⁻⁵ m terbutaline. These results suggest that cell swelling would be induced by NaCl influx and the cell shrinkage is by KCl efflux. Dibutyryl cyclic AMP (DBcAMP) also induced similar cell-volume changes over a wide range of concentrations (10⁻⁹–10⁻³ m): a low concentration induced transient cell swelling; a high concentration, rapid and slow cell shrinkage. Forskolin (10⁻⁴ m), like terbutaline (10⁻⁵ m), induced rapid cell shrinkage followed by slow cell shrinkage, and this decrease in the cell volume was enhanced by the presence of benzamil. On the other hand, cell shrinkage was induced by ionomycin (even low concentration; 3 × 10⁻¹⁰ m ionomycin), and after that cell volume remained at a plateau level. Removal of extracellular Ca²⁺ abolished the cell swelling caused by terbutaline of 10⁻¹⁰–10⁻⁸ m. With removal of extracellular Ca²⁺, the initial, rapid cell shrinkage induced by 10⁻⁵ m terbutaline became transient, but we still detected slow cell shrinkage similar to that in the presence of extracellular Ca²⁺. Overall, at low concentrations (10⁻¹⁰–10⁻⁸ m), terbutaline induced benzamil-sensitive cell swelling in FDLE cells, which was cAMP- and Ca²⁺-dependent; high concentrations (≥⁻⁶) induced quinine-sensitive rapid cell shrinkage, which was Ca²⁺-dependent; high concentrations (≥⁻⁷) induced slow cell shrinkage, which was cAMP-dependent. These findings suggest that terbutaline regulates cell volume in FDLE cells by cytosolic cAMP and Ca²⁺ through activation of Na⁺ and K⁺ channels. Received: 13 March 1995/Revised: 17 January 1996     

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     

Millisecond analyses of Ca2+ initiation sites evoked by muscarinic receptor stimulation in exocrine acinar cells.

High speed laser confocal microscopy (8 ms/image) was applied to the dissociated parotid acini as a model to study Ca2+ signaling mechanisms in non-excitable exocrine secretory cells. Immunofluorescence microscopy showed the localization of IP3 receptor type 2 along the apical membrane region. Muscarinic stimulation with carbachol evoked a rise in [Ca2+]i that was initiated from apical region and propagated into basal region as Ca2+ waves. This was most clearly observed when extracellular Ca2+ was omitted. Carbachol also triggered the abrupt increase of [Ca2+]i simultaneously at both basal and apical regions in many acini. Within an acinus, each cell responded synchronously. The present results suggest that one Ca2+ initiation site in the rat parotid acinar cell is apical region, corresponding to the localization of IP3 receptors. Another Ca2+ initiation site is basal region, which seems to be related to Ca2+ entry from extracellular medium and/or Ca2+ release from basally located organelles such as nuclei and endoplasmic reticulum.     

Electrical coupling and uncoupling of exocrine acinar cells

The electrical communication network in the mouse pancreatic acinar tissue has been investigated using simultaneous intracellular recording with two separate microelectrodes and direct microscopical control of the localizations of the microelectrode tips. All cells within one acinus were electrically coupled, and the coupling coefficient (the electrotonic potential change in a cell neighboring to the cell into which current is injected [V2] divided by the electrotonic potential change in the cell of current injection [V1]) between two cells near each other (less than 50 micron) was always close to 1. Cells farther apart (50-100 micron) were, in some cases, coupled; in other cases, there was no coupling at all. Coupling coefficients varied between 0 and 1. There was rarely electrical coupling over distances of more than 110 micron. Using microiontophoretic acetylcholine (ACh) application, it was possible to evoke almost complete electrical uncoupling of two previously coupled pancreatic or lacrimal acinar cells from different acini or within one acinus. The effects were fully and quickly reversible. While the ACh-evoked uncoupling in the pancreas was associated with membrane depolarization, ACh caused hyperpolarization in the lacrimal acinar cells. The uncoupling was associated with a very marked reduction in electrical time constant, indicating a reduction in input capacitance (effective surface cell membrane area). The concentrations of stimulants needed to evoke reduction in pancreatic cell-to-cell coupling were 1 micron for ACh, 0.14 nM for caerulein, and 3 nM for bombesin. These concentrations are smaller than those required to evoke maximal enzyme secretion.     

Voltage-dependent Conductance Changes in a Nonvoltage-activated Sodium Current from a Mast Cell Line

Nonexcitable cells do not express voltage-activated Na⁺ channels. Instead, selective Na⁺ influx is accomplished through GTP-activated Na⁺ channels, the best characterized of which are found in renal epithelia. We have described recently a GTP-dependent Na⁺ current in rat basophilic leukemia (RBL) cells that differs from previous reported Na⁺ channels in several ways including selectivity, pharmacology and mechanism of activation. In this report, we have investigated the biophysical properties of the RBL cell Na⁺ current using the whole cell patch-clamp technique. Following activation by 250–500 μm GTPγS, hyperpolarizing steps to a fixed potential (−100 mV) from a holding potential of 0 mV evoked transient inward Na⁺ currents that declined during the pulse. If the holding potential was made more positive (range 0 to +100 mV), then the amplitude of the transient inward current evoked by the hyperpolarization increased steeply, demonstrating that the conductance of the channels was voltage-dependent. Using a paired pulse protocol (500 msec pulses to −100 mV from a holding potential of 0 mV), it was found that the peak amplitude of the current during the second pulse became larger as the interpulse potential became more positive. In addition, increasing the time at which the cells were held at positive potentials also resulted in larger currents, indicating a time-dependent conductance change. With symmetrical Na⁺ solutions, outward currents were recorded at positive potentials and these demonstrated both a time- and voltage-dependent increase in conductance. The results show that a nonvoltage activated Na⁺ channel in an electrically nonexcitable cell undergoes prominent voltage-dependent transitions. Possible mechanisms underlying this voltage dependency are discussed. Received: 12 March 1998/Revised: 5 June 1998     

The Ach-evoked Ca2+-activated K+ Current in Mouse Mandibular Secretory Cells. Single Channel Studies

Although acetylcholine (ACh) is able to activate voltage- and Ca²⁺-sensitive K⁺ (BK) channels in mouse mandibular secretory cells, our recent whole cell studies have suggested that these channels, like those in sheep parotid secretory cells, do not contribute appreciably to the conductance that carries the ACh-evoked whole cell K⁺ current. In the present study, we have used cell-attached patch clamp methods to identify and characterize the K⁺ channel type responsible for carrying the bulk of this current. When the cells were bathed in a NaCl-rich solution the predominant channel type activated by ACh (1 μmol/l or 50 nmol/l) had a conductance only of 40 pS; it was not blocked by TEA but it was sensitive to quinine and it conducted Rb⁺ to an appreciable extent. BK channels, which could be seen in some but not all patches from resting cells, also showed increased activity when ACh was added to the bath, but they were much less conspicuous during ACh stimulation than the 40-pS channels. When the cells were bathed in a KCl-rich rather than a NaCl-rich solution, a small-conductance K⁺ channel, sensitive to quinine but not to TEA, was still the most conspicuous channel to be activated by ACh although its conductance was reduced to 25 pS. Our studies confirm that the ACh-evoked whole-cell K⁺ current is not carried substantially by BK channels and show that it is carried by a small-conductance K⁺ channel with quite different properties. Received: 28 September 1995/Revised: 26 December 1995