IOVS
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

(Investigative Ophthalmology and Visual Science. 2008;49:5517-5525.)
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.07-0435
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Herok, G. H.
Right arrow Articles by Martin, D. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Herok, G. H.
Right arrow Articles by Martin, D. K.

Role of Chloride Channels in Regulating the Volume of Acinar Cells of the Rabbit Superior Lacrimal Gland

George H. Herok,1,2 Thomas J. Millar,1,2 Philip J. Anderton,1 and Donald K. Martin3

1From the Co-operative Research Centre for Eye Research and Technology, University of New South Wales, Kensington, NSW, Australia; the 2Department of Natural Sciences, University of Western Sydney/Nepean, Sydney, NSW, Australia; and the 3Department of Medical and Molecular Biology, Faculty of Science, University of Technology, Sydney, NSW, Australia.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. To characterize the outward chloride currents (ClOR) in single acinar cells isolated from the rabbit superior lacrimal gland (RSLG) to investigate the hypothesis that ClOR may have a role in regulating the volume of RSLG acini.

METHODS. ClOR was characterized by using patch-clamp electrophysiology. Confocal microscopy was used to measure intracellular calcium concentration ([Ca2+]i) and cell volume. Cell volume was altered by superfusing the cells with a hyposmotic solution.

RESULTS. The ClOR current contributed 33% of total membrane conductance. With normal osmotic conditions, the ClOR current was activated by [Ca2+]i with an EC50 of 10–8 M. A decrease in intracellular pH from 7.4 to 6.8 totally inhibited ClOR current activity. Continuous superfusion of hyposmotic solution caused (1) an increase in cell volume that peaked within 4 minutes and gradually returned to baseline levels after 12 minutes, (2) an increase in [Ca2+]i that peaked between 6 and 8 minutes and gradually returned to baseline levels after 15 minutes, and (3) an increase the ClOR current that peaked within 6 minutes after commencement of perfusion and quickly returned to baseline levels.

CONCLUSIONS. The ClOR current appears to be triggered by an increase in cell volume and then deactivates within the period of raised [Ca2+]i during hyposmotic stress, suggesting that ClOR may be an initiating event for volume homeostasis. This effect would be important during RSLG tear secretion, which usually involves cell volume changes and is accompanied by intracellular pH changes in the presence of the raised [Ca2+]i to support secretion.

Tears provide a protective and lubricating film that covers the anterior surface of the eye. The bulk of tears is a salty serous fluid containing a variety of proteins associated with bacterial defense, which in rabbits is produced by the superior and inferior lacrimal glands. Secretion of the aqueous component of tears is dependent on the distribution and nature of ion channels in lacrimal acinar cells. Studies of ion channel activity in acinar cells from the exorbital gland of the rat1 2 3 4 5 and mouse6 7 have identified K+ and Cl– channels that are both Ca2+-dependent. However, histochemical studies have shown that the rat lacrimal gland is an acid mucin gland,8 similar to the rabbit zygomatic salivary gland,9 but quite different from the predominantly serous secreting lacrimal gland (superior and inferior) of the rabbit.10

Because this histochemical difference may indicate a difference in the secretory mechanisms, and as the rabbit is a widely used model for studying the phenomenon of dry eye associated with the disease keratoconjunctivitis sicca,11 we investigated the nature of the ion channels in the superior lacrimal gland of the rabbit. In an earlier study12 of rabbit superior lacrimal gland (RSLG) cells, we showed that TEA-sensitive K+ currents comprises a significant portion of the outward currents. In the present study, the other TEA-insensitive component of outward currents was carried by an outwardly rectifying chloride channel (ClOR). Our identification of ClOR was based on functional studies, since only a limited number of chloride channel genes have been identified.13 From the functional point of view, Cl– channels have been classified according to their gating mechanisms,14 which include (1) transmembrane voltage (the CLC family), (2) protein kinase or nucleotide mediated mechanism (CFTR), (3) an increase in intracellular Ca2+ (Ca2+-activated Cl– channels, CaCC), (4) cell swelling (volume-regulated anion channels, VRAC), or (5) binding of a ligand (GABA-activated channels). Transepithelial movement of chloride ions in many secretory epithelia may occur through any of four general classes of Cl– channels based on their modes of activation, including intracellular cAMP, cell swelling (volume changes), hyperpolarization, and intracellular Ca2+ ([Ca2+]i) levels.14 There most likely is some overlap of function, since apical Cl– channels that are involved in fluid and electrolyte secretion are primarily activated by cAMP in some epithelia15 and by Ca2+ in other epithelia.16 Also, volume sensitive Cl– channels have been identified in rat parotid cells16 and in rat lacrimal gland acinar cells.17 The voltage-gated Cl– channel known as CLC-2 is activated by hyperpolarization and is found in a variety of secretory epithelia including mouse mandibular cells18 and rat parotid acinar cells.16 The rat submandibular gland cells17 and the rat lacrimal gland cells19 were found to express CLC-3 protein and contain mRNA for CLC-3 which has a controversial role as mediating a swelling-induced Cl– current.13 We investigated the hypothesis that the ClOR channels that we identified in the RSLG acini may have a role in regulating the volume of lacrimal acinar cells, since it has been shown that salivary acinar cells undergo changes in volume during secretion.20

Most cells placed in an hyposmotic environment swell and then undergo regulatory volume decrease (RVD), which is usually accomplished by the extrusion of K+ and Cl– ions via Ca2+-activated K+ and Cl– channels.21 However, K+/Cl– cotransporters mediate the RVD regulation in fish,22 dog,23 and human24 red blood cells (RBCs). Cells exposed to a hyperosmotic environment will shrink and then undergo regulatory volume increase (RVI). Previously, ionic channels have not been implicated in RVI, which is thought to be mediated either by Na+/Cl– cotransporters,25 Na+/H+ exchangers linked to Cl–/HCO3– exchangers,26 or Na+/K+/2Cl– cotransporters.27

In the present study, we characterized the ClOR in the RSLG and showed that activation of this ClOR was triggered by an increase in cell volume. The ClOR may be involved in RVI as the initiating event for restoring volume homeostasis after hyposmotic stress or secretion.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Preparation of Lacrimal Gland Cells
New Zealand White rabbits of both sexes (1.5–2.5 kg) were killed by an overdose of pentobarbital (45 mg/kg) administered IV in accordance with Australian National Health and Medical Research Council guidelines for animal ethics and the tenets of the ARVO Statement of the Use of Animals in Ophthalmic Vision Research. The superior lacrimal glands were excised and placed in a Petri dish containing 5 mL of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with L-glutamine (0.584 g/L) and D-glucose (4.5 g/L). The glands were cut into small pieces and gently centrifuged for 1 minute at 25 g. The supernatant was discarded, and the glandular fragments were then incubated in trypsin (1 mg/mL, type IX; Sigma-Aldrich, Castle Hill, Australia) for 8 minutes at 37°C in an oscillating water bath (130 osc/min). After centrifugation of the trypsin solution, the supernatant was discarded and the cellular pellet was rinsed with soybean trypsin inhibitor (STI, 1 mg/mL, Type 1-S; Sigma-Aldrich). The tissues were then transferred to a 25-mL Erlenmeyer flask and incubated in the standard NaCl bathing solution (see Solutions and Chemicals) containing collagenase (200 U/mL, type II; Worthington Biochemicals, Templestowe, VC, Australia) and hyaluronidase (600 U/mL, type V; Sigma-Aldrich) for 48 minutes at 37°C in the oscillating water bath. The enzyme solution was then filtered through 100-µm nylon mesh and gently centrifuged for 2 minutes. The cellular pellet was then washed twice with the standard NaCl bath solution containing bovine serum albumin (BSA, 2 mg/mL; Sigma-Aldrich). After each wash, the cells were gently centrifuged for 2 minutes and the supernatant discarded. After the second wash, the cells were resuspended in 6 mL of DMEM containing fetal bovine serum (FBS, 10%; PA Biologicals Co Pty Ltd., Sydney, Australia). They were cultured in uncoated 35-mm plastic dishes (polystyrene, cat. no. 150318; Nunc, Thermo Fisher Scientific, Rochester, NY) by placing 1 mL of the resuspended cell solution into the dish and then adding 1 mL of the DMEM containing 10% FBS solution. Media were changed every 2 days. The cells settled within 24 hours. All results were recorded from these freshly isolated and plated acinar cells. The recordings were made from cells between days 3 and 8 in culture, because we found that within that period, the ion currents retained the outwardly rectifying characteristics but had become stable. For example, the conductance of the outward current in the range from 0 to 80 mV from sample cells in culture was 13.3 (day 3), 13.6 (day 6), and 13.3 (day 8) nS. The current at +80 mV for those sample cells was 1299 (day 3), 1312 (day 6), and 1289 (day 8) pA.

Patch-Clamp Recording and Analysis
Whole-cell currents were recorded by standard patch-clamp techniques28 at room temperature (20–25°C). The channel currents were amplified and filtered at 1 kHz (–3 dB; Axopatch 200A and CV201A headstage; Axon Instruments, Inc., Melbourne, Australia) and sampled online by a computer (IBM 486-compatible) using commercial software and associated A/D hardware (pClamp 5.5.1/Labmaster TL-1-125, Axon Instruments, Inc. and Scientific Solutions, Inc. Woodstock, GA). Patch pipettes (4–10 M{Omega}) were fabricated from thin-walled borosilicate glass (Vitrex Microhematocrit tubes; Modulohm I/S, Herlev, Denmark). The reference electrode was a Ag/AgCl electrode immersed in a 140 mM KCl solution which was connected to the bath via an agar bridge. In experiments where the agar bridge was not used, the pipette potential was corrected for the liquid junction potentials between the pipette solution and bath solution.29 Outward current was shown by an upward deflection in all traces by adopting the convention of positive ions leaving the pipette. The membrane conductance (Gm) was calculated by dividing the current (Im) by the electrical driving force (Vm – Vrev), with Gm = Im/(Vm – Vrev). Relaxations in the current recordings were fitted with a single exponential using the least-squares fitting routine available in the pClamp software (Axon Instruments, Inc.). The fit was commenced approximately 20 ms after each voltage step to allow enough time for the slow capacitive currents to decay.3 Results are reported as the mean ± SEM, with n the number of observations, in parentheses. Statistical comparisons were performed using Student’s t-test, with the appropriate corrections (Bonferroni) when used for multiple comparisons.

Membrane Capacitance Measurements
Voltage clamp experiments provide for measurement of the transmembrane current (I) required to maintain an applied membrane voltage. A step to a new clamp voltage (V) results in a brief transient current spike, due to the membrane capacitance, that decays exponentially. The membrane capacitance (C) was calculated from the area-under-the-curve (AUC) of that transient capacitative current spike after a voltage step ({Delta}V) in the following way:

Formula 1(1)
with

Formula 2(2)
to give

Formula 3(3)

Confocal Microscopy
Changes in [Ca2+]i concentration in single cells were measured by loading the cells with the acetoxy-methyl (AM) ester form of the Ca2+ indicator dye Fluo-3 (F-1242; Invitrogen-Molecular Probes, Eugene, OR). The cells were loaded with Fluo-3 AM (1.5 µM) by incubation in the dark for 25 minutes at room temperature. They were then washed with four changes of the extracellular bath solution to remove uncleaved Fluo-3 AM molecules. The cells and the Fluo-3-labeled [Ca2+]i were visualized with a 32x objective with a confocal laser scanning microscope (Model TCS-4D; Leica Microsystems Pty Ltd., North Ryde, NSW, Australia), using an excitation wavelength of 488 nm and detecting an emission wavelength of 525 to 530 nm.

The footprint area of cells was measured from the optical section of the cell closest to its adhesion with the surface of the culture dish (Quantimet 570 Image Analyzer; Cambridge Instruments, now owned by Leica). The footprint area was used as an indicator of cell volume,20 by raising the footprint area of the single optical section to the power of 3/2. It was not possible to measure cell volume by using z-sectioning and the oil-immersion objective lens available to us, because live cells were imaged in their (aqueous) extracellular bath electrolyte solution.

Solutions and Chemicals
The standard pipette solution (pH 7.4) contained (in mM): KCl (140) MgCl2 (1.13), HEPES (10), and EGTA (5). The standard bath solution (pH 7.4) contained (in mM): NaCl (145), MgCl2 (1.13), KCl (5), CaCl2 (1), glucose (10), and HEPES (10). In the ion substitution experiments, the following pipette and bath solutions were used. Pipette solution (pH 7.4) contained (in mM): NaCl (140), MgCl2 (1.13), HEPES (10), glucose (10), CaCl2 (0.97), and EGTA (1.92). The bath solution (pH 7.4) contained (in mM): NaCl (145), KCl (5), CsCl (5), MgCl2 (1.13), CaCl2 (1), HEPES (10), and glucose (10). To determine the ion selectivity of this channel for various anions (SCN–, I–, Br–, and NO3–) we replaced all but 16 mM of the Cl– in the bathing solution with the monovalent anions.

The free Ca2+ in the pipette solutions was adjusted by using different amounts of EGTA30 to achieve concentrations of 1.0 x 10–6 M (1.54 mM Ca2+ and 1.73 mM EGTA), 1.5 x 10–7 M (0.97 mM Ca2+ and 1.92 mM EGTA) or 1.0 x 10–9 M (no added Ca2+, 5 mM EGTA).

Hyposmotic bath solutions (217 mOsM) were prepared by adding deionized water to the standard bath solution (310 mOsM). Hyperosmotic bath solutions (398 mOsM) were prepared by adding 90 mM mannitol (Sigma-Aldrich) to the standard bath solution (310 mOsM).

Furosemide, DIDS, tetraethylammonium chloride (TEA) and Na-gluconate were obtained from Sigma-Aldrich and tetramethylammonium chloride (TMA) and diphenylamine-2-carboxylic acid (DPC) were obtained from Fluka-Sigma-Aldrich (Buchs, Switzerland). All chemicals used in the experiments were of AR grade or higher.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Identification of the Outward Chloride Currents, ClOR
The series resistance of 8.9 ± 0.3 M{Omega} (n = 17) was not compensated for electronically during the experiments, and the average cell capacitance was 9.0 ± 0.2 pF (n = 17). The resting cell membrane potential with the standard solutions in the pipette (KCl-rich) and in the bath (NaCl-rich) was –40 ± 2 mV (n = 17), similar to the resting membrane potential of –37 ± 8 mV (n = 50) obtained with intracellular electrodes.31 The inset in Figure 1A shows a typical example of whole-cell currents evoked by voltage steps between –60 mV and +80 mV. The steady state whole cell current–voltage relation (I–V) from a sample of 12 cells showed a dominant outwardly rectifying current, which activated at potentials more positive than –40 mV (Fig. 1A) . The conductance of these currents was 24 ± 1 nS (n = 12) in the range from +10 to +80 mV. The addition of 10 mM TEA extracellularly to block K+ channels shifted the I–V reversal potential from –39 ± 2 to 1 ± 2 mV (n = 8) (Fig. 1A) and reduced the chord conductances for inward currents from 3.9 ± 0.4 to 1.8 ± 0.2 nS (by 54%, n = 8) and for outward currents from 24 ± 1 to 16.2 ± 1.5 nS (by 33%, n = 8).


Figure 1
View larger version (21K):
[in this window]
[in a new window]

  		FIGURE 1. Whole-cell current responses to voltage change, TEA, and both symmetrical and asymmetrical Cl– solutions. (A) Steady state whole-cell I–V relations of single RSLG acinar cells before (•, n = 12) and after ({circ}, n = 8) the addition of 10 mM TEA to the extracellular bath solution. In each experiment, the pipette contained a KCl-rich solution (140 mM) and the bath contained an NaCl-rich solution (145 mM). Inset: whole-cell current responses of single RSLG acinar cells to voltage steps between –60 and +80 mV from a holding potential of –50 mV. (B) Steady state whole-cell I–V relations of single RSLG acinar cells, with either an extracellular bath solution of 145 mM Cl– (•, n = 25) or an extracellular bath solution containing 20 mM Cl– and 125 mM gluconate ({blacksquare}, n = 5). There was no change in the reversal potential when the Na+ ions in the pipette solution were replaced with TMA ({circ}, n = 5). In these experiments the pipette solution contained Na+ ions rather than K+ ions, and 5 mM Cs+ was present in the bath. In all the experiments, the vertical bars represent the SEM when these were larger than the symbols.

 
We studied the nature of the TEA-insensitive component of the current by substituting Na+ for K+ in the pipette solution and adding 5 mM CsCl to the bath solution. The Cl– concentrations in the bath and pipette solutions were 159 and 142 mM, respectively. Under these conditions the whole-cell I–V had a reversal potential of –1.0 ± 0.4 mV (n = 25), suggesting that it was carried by either Na+ or Cl– ions. In five subsequent experiments, replacement of pipette Na+ with the larger cation tetramethylammonium (TMA) had no effect on the reversal potential (Fig. 1B) . We concluded that the current was carried by Cl– ions, which we confirmed by substituting 125 mM of the extracellular NaCl with Na-gluconate, which shifted the reversal potential to 39 ± 2 mV (n = 5; Fig. 1B ). The Nernst potential for Cl– was 48 mV under these conditions, indicating that ClOR channels carried the TEA-insensitive component of the macroscopic whole-cell current.

Pharmacologic confirmation that this TEA-insensitive current was carried by Cl– ions was observed by the use of several known chloride channel blockers which markedly inhibited ClOR current activity (Fig. 2) . The blockers used were furosemide (1 mM) which is also known to inhibit Na+-K+-2Cl– cotransporters, DPC (1 mM), and DIDS (2 mM).


Figure 2
View larger version (21K):
[in this window]
[in a new window]

  		FIGURE 2. Inhibition of the whole-cell ClOR currents by Cl– channel blockers added to the extracellular bath solution. The cells were held at –50 mV between voltage steps ranging from 0 to +80 mV. In each experiment the pipette solution contained Na+ ions rather than K+ ions, 1.5 x 10–7 free Ca2+ ions and 5 mM Cs+ was present in the bath solution. The histograms show the mean ± SEM results from n = 4 cells for each blocker. (A) DPC (1 mM), (B) furosemide (1 mM), and (C) DIDS (2 mM).

 
Ca2+ and Voltage Activation of the ClOR Currents
Figure 3A shows the whole-cell I–V evoked by voltage steps between –60 and +80 mV when the free Ca2+ concentration in the pipette solution was either 1.0 x 10–9 (n = 6), 1.5 x 10–7 (n = 8), or 1.0 x 10–6 M (n = 6). The pipette solution for these experiments contained 5 mM CsCl with KCl replaced by NaCl. Figure 3B shows that the total membrane conductance of the outward currents increased by 117% when the [Ca2+]i was changed from 1.0 x 10–9 to 1.5 x 10–7 M, but only increased a further 16% when the [Ca2+]i was changed from 1.5 x 10–7 to 1.0 x 10–6 M. Within that range of [Ca2+]i it appeared that a half-maximum increase in conductance is on the order of 10–8 M.


Figure 3
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 3. (A) Effect of various [Ca2+]i concentrations on whole-cell Cl– currents. Steady state whole-cell I–V relations of freshly isolated single RSLG) acinar cells with free Ca2+ ion concentrations in the pipette solution of 1.0 x 10–9 (•, n = 6), 1.5 x 10–7 ({circ}, n = 8), and 1.0 x 10–6 M ({blacksquare}, n = 6). The cells were held at –50 mV between voltage steps of 10 mV increments over the range from –60 to +80 mV. (B) Relation between the membrane potential and the total membrane conductance (Gm) for the ClOR channels, for free Ca2+ concentrations in the pipette solution of 1.0 x 10–9 (•, n = 6), 1.5 x 10–7 ({circ}, n = 8), and 1.0 x 10–6 M ({blacksquare}, n = 6). The ClOR conductance increased as the membrane potential became more positive and as the Ca2+ concentration was increased. The potential at which the conductance started to increase shifted toward more negative values as the Ca2+ concentration increased, with +10 mV at 1.0 x 10–9, –20 mV at 1.5 x 10–7, and –40 mV at 1.0 x 10–6 M. In each experiment, the pipette solution contained Na+ ions rather than K+ ions and 5 mM Cs+ was present in the bath solution. In all the experiments the bars represent the SEM when these were larger than the symbols.

 
Figure 3B also demonstrates that the ClOR conductance was voltage-activated. At the [Ca2+]i of 1.0 x 10–9 M, the ClOR conductance increased at membrane potentials more positive than +10 mV. The modulating effect of Ca2+ on this voltage-activation was to shift the potential at which the ClOR conductance increased toward more negative potentials. For example, this potential was around –20 mV at 1.5 x 10–7 M [Ca2+]i and around –40 mV at 1.0 x 10–6 M [Ca2+]i.

Analysis of ClOR Current Relaxations
Figure 4 shows the relation between the "on" relaxation time constant ({tau}ON) and membrane potential with different levels of [Ca2+]i. The {tau}ON provides a measure of how quickly the ClOR channels are recruited to conduct Cl– ions in response to a step-change in the membrane potential. When the [Ca2+]i was 1.5 x 10–7 M, the time constant decreased progressively from 58 ± 7 ms (n = 21) to 14 ± 1 ms (n = 21), as the pipette potential was increased from –20 to +80 mV. A similar trend was observed when the [Ca2+]i was 1.0 x 10–6 M, whereby {tau}ON decreased from 61 ± 9 ms (n = 6) to 23 ± 3 ms (n = 6) over the same voltage range. However, there was no statistically significant difference between the effect of [Ca2+]i on {tau}ON within the voltage range –20 to +50 mV. The activation kinetics were significantly slower ({tau}ON larger) at 1.0 x 10–6 M [Ca2+]i compared with 1.5 x 10–7 M [Ca2+]i when the cell potential was clamped at +60 (P = 0.046), +70 (P = 0.04), or +80 mV (P = 0.001). The relaxation of the current could not be reliably fitted with exponential curves when the Ca2+ concentration was 1.0 x 10–9 M.


Figure 4
View larger version (13K):
[in this window]
[in a new window]

  		FIGURE 4. Relation between the relaxation time constant ({tau}ON) and membrane potential. In each experiment, the pipette and bath solutions contained Na+ ions rather than K+ ions. The solutions contained 5 mM Cs+ and either 1.5 x 10–7 M free Ca2+ ions (•, n = 8) or 1.0 x 10–6 M ({circ}, n = 6). Vertical bars: the SEM when these values were larger than the symbol. There was no significant difference between the data recorded at either Ca2+ concentration in the range of potentials between –20 and +50 mV. The activation kinetics were significantly slower ({tau}ON larger) at 1.0 x 10–6 M [Ca2+]i compared with 1.5 x 10–7 M [Ca2+]i when the cell potential was clamped at +60 (P = 0.046), +70 (P = 0.04), or +80 mV (P = 0.001).

 
Ion Selectivity of the ClOR Currents
The selectivity of the ClOR channel to other anions was derived from reversal potential measurements when all but 16 mM of the Cl– in the bathing solution was replaced with either I–, Br–, SCN– or NO3–. The magnitude of the shift in reversal potentials recorded with different anions was used to calculate the permeability ratio of the anion (X) relative to that of Cl– (PX/PCl), using the Goldman-Hodgkin-Katz equation with the assumption that the currents were carried solely through Cl– channels (Table 1) . The permeability sequence was SCN– (2.32) > Br– (1.80) > I– (1.58) > NO3– (1.32) > Cl– (1.0). The conductance sequence was determined by measuring the slope conductance from the I–V relations for the different anions relative to Cl–. The conductance sequence was: NO3– (1.30) > Br– (1.24) > SCN– (1.14) > Cl– (1.0) > I– (0.70). Three of the anions (SCN–, Br–, and NO3–) have both higher permeabilities and conductivities relative to Cl–, but I– has a higher relative permeability but a lower relative conductivity compared to Cl–.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Relative Anion Permeability and Conductivity Sequences for the ClOR Channel

 
Effect of pH on ClOR Current Activity
Figure 5A shows current profiles recorded from three cells by using pipette solutions of different pH and with the free [Ca2+]i set at 1.5 x 10–7. At pH 7.8, the time-dependent ClOR currents at depolarizing potentials were substantially larger than at pH 7.4, whereas at pH 6.8 the ClOR current was inhibited. Figure 5B is a plot of the current density as a function of membrane potential at various pH pipette solutions. The current density was determined by dividing the measured ClOR currents by the capacitance of the membrane for each cell. It was important to use current density since that allowed us to separate the pH-induced changes in the magnitude of the ClOR current from those changes due simply to variations in cell size. The capacitance, and hence size, of the cells exposed to each pH was not significantly different (P < 0.05, n = 14). The mean (±SEM) cell capacitances were 13.1 ± 0.3 pF at pH 6.8, 13.0 ± 0.2 pF at pH 7.4, and 13.2 ± 0.2 pF at pH 7.8. At pH 7.4 and pH 7.8 the I–V relationship between –60 and +80 mV displayed outward rectification with increasing depolarizing potentials. Lowering intracellular pH (pHi) from 7.4 to 6.8 markedly decreased outward current and the I–V relationship was almost linear. This inhibitory effect of lowering pH on ClOR current activity was independent of the Ca2+ concentration in the pipette solution. It was observed that the ClOR current was inhibited at pH 6.8 even at free [Ca2+]i concentrations of 1.0 x 10–5, 1.0 x 10–4, and 1.0 x 10–3 M. The results shown in Figure 5B indicate that changes in ClOR current activity are due to changes in RSLG and not due to cell size. This indicates that variation in current density between cells is not an artifact causing the changes in ClOR current activity, but in fact these variations in current activity are directly due to pH changes.


Figure 5
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 5. Effect of pH on whole-cell currents in RSLG acinar cells. (A) Typical whole-cell currents recorded from three different acinar cells using pipette solutions containing 1.5 x 10–7 M [Ca2+]i, with pH buffered at 6.8, 7.4, and 7.8. Voltage steps were applied over the range of –60 to 80 mV at 10-mV increments from a holding potential of –50 mV. In each experiment, the pipette solution contained Na+ ions rather than K+ ions, and 1.5 x 10–7 free Ca2+ ions and 5 mM Cs+ were present in the bath solution. (B) Whole-cell steady state I–V relation between current density and membrane potential for the ClOR channel in freshly isolated RSLG acinar cells activated by an increase in pHi. Currents were recorded between –60 and +80 mV in 10-mV voltage step increments at pH 6.8 ({circ}, n = 14), 7.4 (•, n = 14), and 7.8 ({blacksquare}, n = 14) with each solution containing 1.5 x 10–7 M free Ca2+ ions, and 5 mM Cs+ was present in the bath solution.

 
Effect of Osmotic Stress on ClOR Current Activity, Cell Volume, and [Ca2+]i
A sample of cells (n = 8) exposed to a hyposmotic solution (217 mOsM) showed a gradual increase in ClOR current, measured at a holding potential of +80 mV, which reached a peak at 6 minutes and then quickly reduced to the baseline level (Fig. 6A) . Analysis of the activation kinetics of the maximal CLOR current at 6 minutes showed that the time constant had decreased to 15 ± 2 ms (n = 11) from the baseline value of 68 ± 6 ms (n = 11).


Figure 6
View larger version (13K):
[in this window]
[in a new window]

  		FIGURE 6. Effect of osmotic stress on cell volume, ClOR current activity, and [Ca2+]i in RSLG acinar cells. (A) The change in ClOR current, measured at a holding potential of +80mV, in RLSG acinar cells exposed to a hyposmotic solution (217 mOsM; n = 8). In each experiment, the pipette solution contained Na+ ions rather than K+ ions, and 1.5 x 10–7 M free Ca2+ ions and 5 mM Cs+ were present in the bath solution. ClOR current (I) is shown as a fraction of the ClOR current (Io) before exposure to the hyposmotic solution. (B) The change in cell volume, as measured using confocal microscopy over time in RLSG acinar cells exposed to hyposmotic solution (217 mOsM; n = 22). Cell volume (V) is shown as a fraction of the cell volume (Vo) before exposure to the hyposmotic solution. (C) [Ca2+]iconcentration in a sample of RLSG acinar cells (n = 18) exposed to a hyposmotic solution (217 mOsM).

 
The volume, measured using confocal microscopy, increased when a sample of cells (n = 22) were exposed to the hyposmotic solution (Fig. 6B) . The cell volume reached a peak at 4 minutes and then the volume oscillated to follow a gradual return to the baseline volume at around 18 minutes after exposure to the hyposmotic solution.

[Ca2+]i in a sample of cells (n = 18) exposed to the hyposmotic solution increased to reach a peak at 6 minutes (Fig. 6C) . The [Ca2+]i oscillated to follow a gradual return to the baseline level around 15 minutes after exposure to the hyposmotic solution.


   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
In the present study, we showed that the RSLG acinar cells contain an a ClOR channel, which we found to be both Ca2+- and voltage-activated and which responds to hyposmotic stress. Those characteristics are relevant to the cell homeostasis after the secretory functions of the RSLG. By analogy, there is a decrease in cell volume during secretion in other glands.20 32

The Cl– channel that we characterized carries approximately one-third of the macroscopic outward current in the rabbit lacrimal gland acinar cells, which we identified as the ClOR current. We identified the ClOR channels by ion substitution experiments, in which the I–V reversal potential shifted from –1 to +39 mV, and then with the use of the chloride channel blockers furosemide, DIDS and DPC. The ClOR channels were activated by cell depolarization and by [Ca2+]i in the range 1.0 x 10–9 to 1.0 x 10–6 M (with an EC50 approximately 1.0 x 10–8 M). The conductance, permeability properties and activation kinetics of the ClOR channel in the RSLG distinguished it from Cl– channels in other epithelial cell types. Furthermore, the permeability sequence and sensitivity to DIDS for ClOR indicate that it is unlikely to be a member of the CLC or CFTR families of Cl– channels but is most likely a member of a class of Cl– channels that shares the characteristics of both the swelling-activated (VRAC) and Ca2+-activated (CaCC) Cl– channel families.13 The CFTR Cl– channel is sensitive neither to [Ca2+]i levels,33 nor to membrane potential changes, there are only slight time-dependent voltage effects on the Cl– conductive properties of CFTR,34 and the anion permeability sequence of CFTR is different.35 Furthermore, the CFTR Cl– conductance was not very sensitive to either DIDS or DPC.36 In respiratory airway cells,37 the Cl– channels are activated by membrane depolarization but are insensitive to [Ca2+]i. The Cl– conductance in rat pancreatic acinar cells was only activated at [Ca2+]i levels of 1.0 x 10–6 M and greater.38 The small conductance Cl– channels in rat and human pancreatic duct cells39 differ from the Cl– channels in the RSLG by their insensitivity to DIDS and differences in their anion selectivity sequence. A comparison of anion selectivity for Cl– channels in various glands is summarized in Table 2 .


View this table:
[in this window]
[in a new window]

  		TABLE 2. Relative Permeabilities for Different Anions in CI– Channels from a Variety of Epithelia

 
The Cl– channel in the rat mandibular salivary gland40 is activated by membrane depolarization and [Ca2+]i, and its permeability sequence is virtually identical with that of the ClOR channel in the RSLG. Similarly, the Cl– conductance in sheep parotid cells is activated by changes in membrane potential and [Ca2+]i and is inhibited by the Cl– channel blockers DIDS and furosemide.41 However, its anion permeability sequence (Table 2) is quite different from the ClOR channel found in the superior lacrimal gland of the rabbit. The Cl– channel in the rat lacrimal gland1 3 5 is also activated by depolarization and [Ca2+]i. However, it is less sensitive to Ca2+ than the ClOR channel, since the Cl– conductance in the rat lacrimal gland3 cells became active only when the intracellular free Ca2+ concentration exceeded 0.5 x 10–6 M. The rat lacrimal gland Cl– channel was inhibited by furosemide,4 but its anion selectivity sequence differed from the ClOR channel in the RSLG.

The activation kinetics of the ClOR channels are faster than the Cl– channels in either the sheep parotid41 or rat lacrimal gland3 acini. In sheep parotid and rat lacrimal glands, the time constant of the single exponential fitted to the "on" current relaxation ({tau}ON) was usually greater at more positive potentials than for the ClOR channels in the RSLG. However, in rat lacrimal gland cells with a large [Ca2+]i concentration (> 500 mM) the trend was for {tau}ON to decrease slightly for more positive potentials, and under these conditions {tau}ON was similar to the ClOR channels in the rabbit. We did not find significant differences between {tau}ON when the [Ca2+]i concentration was changed from 1.5 x 10–7 to 1.0 x 10–6 M. The interspecies variations in activation kinetics and the modulation of gating by Ca2+, suggest that there are differences in the structure of Ca2+- and voltage-activated Cl– channels present in different exocrine glands. These differences more importantly highlight the interspecies variation between rabbit and rat lacrimal glands.

Similar to the results presented here for the ClOR channel, the inhibition of Ca2+-activated Cl– channels by a decrease in pHi has been reported in rat lacrimal glands42 and rat parotid acinar cells.16 However, studies on T84 cells43 and on a mutant form of the cystic fibrosis transmembrane conductance channel44 showed that a decrease in pHi actually causes an increase in Cl– channel activity. Other studies43 suggest that this may be due to a decrease in the interactions between charged sites within the channel pore and the permeating ions leading to an increase in channel conductance. Conversely, the decrease in ClOR current activity in rabbit superior lacrimal acinar cells may be due to an increase in the interaction between charged sites within the pore and the permeating H+ ions, resulting in conformational changes within the protein pore, affecting its structure and inhibiting the permeation of the Cl– ions through the channel. There is limited previous evidence to suggest that pHi plays a role in regulating channel activity in lacrimal acinar cells. A recent study45 reported that overexpression of CLC-3 in HEK293T cells produced a novel Cl– current that was pH-dependent in the pH range from 6.35 to 8.2. However, small fluctuations in pHi have been observed in both lacrimal46 and salivary47 48 49 cells after stimulation with ACh, which causes a small transient intracellular acidosis followed by a sustained alkalinization. It has been suggested49 in rabbit salivary glands that the initial ACh induced transient acidosis may possibly be due to HCO3– efflux through Cl– channels.

We investigated a role for the ClOR channels in volume regulation because it has been shown in rat salivary glands20 32 that the acinar cells undergo volume changes during secretion. The response of this ClOR current to osmotic stress is interesting, in that intuitively one might expect a decrease in ClOR current under hyposmotic conditions. However, when cells are exposed to a hyposmotic solution they initially swell and subsequently undergo RVD.21 50 51 52 In many cells, this is observed by an increase in K+ and Cl– channel activity leading to an extrusion of these ions with the passive movement of water afterward. After RVD, the decrease in cell volume then triggers an increase in the inward movement of Cl– which corresponds to the large transient increase in ClOR current activity which causes an increase in cell volume. The cell volume returned to basal levels in an oscillatory manner after exposure to hyposmotic stress. This ClOR channel in the RSLG which has some properties similar to those of the rat lacrimal1 3 53 and sheep parotid41 glands may be involved in the compensatory response to RVD initially induced by hyposmotic stress resulting in the influx of Cl– ions followed by the passive influx of water due to osmotic pressure. This type of RVI cellular response after RVD has been termed post-RVD RVI.21 Our experimental results support this post-RVD RVI phenomenon induced by hyposmotic stress, whereby we show that the increase in ClOR channel activity is closely related to cell volume changes.

Confocal microscopy demonstrated that hyposmotic stress evoked an increase in [Ca2+]i concentrations which were maintained for up to 10 minutes. This increase in [Ca2+]i concentration may be the result of both release of Ca2+ from intracellular stores and an influx of Ca2+ from extracellular sources via stretch-activated cation channels located on the cell membrane. This response is not surprising since [Ca2+]i is involved in cell volume regulation54 and both Ca2+-activated Cl– and K+ channels are responsible for the oscillatory volume changes observed in returning cell volume back to basal conditions after hyposmotic shock. These changes in cell volume may be accompanied by fluctuations in pHi resulting in activation and deactivation of the ClORchannel. Studies by Saito et al.46 showed that after stimulation of mouse lacrimal acinar cells by ACh, transient acidosis occurs followed by sustained alkalinization. Of interest, our experiments showed that Ca2+-activated ClORchannels are inhibited by a decrease in pHi and activated by an increase in pHi. This continual competition between H+and Ca2+ ions may lead to fluctuations in ClOR current activity concomitant with changes seen in cell volume.

The ClOR currents in the RSLG have two distinctive characteristics that are quite different from other volume sensitive Cl– currents in other epithelial cells. First, ClOR currents were Ca2+-dependent as shown by our patch-clamp studies, and calcium imaging using confocal microscopy showed that osmotic stress evoked an increase in [Ca2+]i concentration. In contrast, patch-clamp studies in other epithelia including, human intestinal cells,55 56 human sweat glands,57 Madin Darby Canine Kidney cells,58 and T84 cells59 have identified Cl– channels that are activated by cell swelling due to hyposmotic solutions but are independent of [Ca2+]i concentrations. However, in some epithelia, cell swelling was shown to cause an increase in [Ca2+]i levels which activated K+ channels, causing RVD.55 56 58 60

Second, the ClOR current activity in the rabbit lacrimal gland is voltage dependent, with ClOR currents being activated by depolarization. A similar osmotically activated ClOR current in the rat lacrimal gland3 5 has also been found to be activated by depolarization. However, the investigators in those studies make no reference to its possible role in cell volume regulation. In contrast, volume sensitive Cl– currents in other epithelia are inactivated at depolarizing potentials.57 59 60 61 Our functional characterization of the ClOR current in the RSLG shows that it shares the characteristics of both the swelling-activated (VRAC) and Ca2+-activated (CaCC) Cl– channel families. The recent reports that overexpression of CLC-3 produced a novel pH-sensitive current in HEK293T cells45 and that CLC-3 is expressed in rat lacrimal gland acini19 raise the intriguing possibility that ClOR may also share characteristics of CLC-3. Notwithstanding, the osmotically active ClOR current that we describe in RSLG acinar cells is rather unique and different from volume-sensitive Cl– currents in other epithelial cells. Further studies are needed to accurately define the membrane domain in which the ClOR channels are localized.


   Footnotes
 
Supported by grants from the Australian Research Council and the National Health and Medical Research Council of Australia (TJM, PJA, DKM).

Submitted for publication April 12, 2007; revised September 25, 2007 and March 24, 2008; accepted October 14, 2008.

Disclosure: G.H. Herok, None; T.J. Millar, None; P.J. Anderton, None; D.K. Martin, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Donald K. Martin, Department of Medical and Molecular Biology, Faculty of Science, University of Technology, Sydney, P.O. Box 123, Broadway, NSW, 2007, Australia; donm{at}uts.edu.au.


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

  1. Marty A, Tan YP, Trautmann A. Three types of calcium-dependent channel in rat lacrimal glands. J Physiol. 1984;357:293–325.[Abstract/Free Full Text]
  2. Trautmann A, Marty A. Activation of Ca-dependent K channels by carbamylcholine in rat lacrimal glands. Proc Natl Acad Sci USA. 1984;81:611–615.[Abstract/Free Full Text]
  3. Evans MG, Marty A. Calcium-dependent chloride currents in isolated cells from rat lacrimal glands. J Physiol. 1986;378:437–460.[Abstract/Free Full Text]
  4. Evans MG, Marty A, Tan YP, Trautmann A. Blockage of Ca-activated Cl conductance by furosemide in rat lacrimal glands. Pflügers Arch. 1986;406:65–68.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  5. Kotera T, Brown PD. Calcium-dependent chloride current activated by hyposmotic stress in rat lacrimal gland. J Membr Biol. 1993;134:67–74.[Web of Science][Medline][Order article via Infotrieve]
  6. Findlay I. A patch-clamp study of potassium channels and whole-cell currents in acinar cells of the mouse lacrimal gland. J Physiol. 1984;350:179–195.[Abstract/Free Full Text]
  7. Findlay I, Petersen OH. Acetylcholine stimulates a Ca2+-dependent Cl– conductance in mouse lacrimal acinar cells. Pflügers Arch. 1985;403:328–330.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  8. Spicer SS, Duvenci J. Histochemical characteristics of mucopolysaccharides in salivary and exorbital lacrimal glands. Anat Rec. 1964;149:333–358.[Medline][Order article via Infotrieve]
  9. Oikawa M, Okano T. The relations between the lacrimal gland, infraorbital gland and zygomatic gland of the rabbit. Aichi-Gakuin J Dent Sci. 1978;16:132–138.
  10. Millar TJ, Herok GH, Koutavas H, Martin DK, Anderton PJ. Immunohistochemical and histochemical characterisation of epithelial cells of rabbit lacrimal glands in tissue sections and cell cultures. Tissue Cell. 1996;28:301–312.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  11. Gilbard JP, Rossi SR, Gray KL. A new rabbit model for keratoconjuctivitis sicca. Invest Ophthalmol Vis Sci. 1987;28:225–228.[Abstract/Free Full Text]
  12. Herok GH, Millar TJ, Anderton PJ, Martin DK. Characterization of an inwardly rectifying potassium channel in the rabbit superior lacrimal gland. Invest Ophthalmol Vis Sci. 1998;39:308–314.[Abstract/Free Full Text]
  13. Jentsch TJ, Stein V, Weinreich F, Zdebik AA. Molecular structure and physiological function of chloride channels. Physiol Rev. 2002;82:503–568.[Abstract/Free Full Text]
  14. Nilius B, Droogmans G. Amazing chloride channels: an overview. Acta Physiol Scand. 2003;177:119–147.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  15. Chan HC, Kaetzel MA, Nelson DJ, Hazarika P, Dedman JR. Antibody against a cystic fibrosis transmembrane conductance regulator-derived synthetic peptide inhibits anion currents in human colonic cell line T84. J Biol Chem. 1992;267:8411–8416.[Abstract/Free Full Text]
  16. Arreola J, Melvin JE, Begenisich T. Inhibition of Ca2+-dependent Cl– channels from secretory epithelial cells by low internal pH. J Membr Biol. 1995;147:95–104.28.[Web of Science][Medline][Order article via Infotrieve]
  17. Majid A, Brown PD, Best L, Park K. Expression of volume-sensitive Cl– channels and CIC-3 in acinar cells isolated from the rat lacrimal gland and submandibular salivary gland. J Physiol. 2001;534:409–421.[Abstract/Free Full Text]
  18. Komwatana P, Dinudom A, Young JA, Cook DI. Characterisation of the Cl– conductance in the granular duct cells of the mouse mandibular glands. Pflügers Arch. 1994;426:320–327.[CrossRef][Medline][Order article via Infotrieve]
  19. Ubels JL, Hoffman HM, Srikanth S, Resau JH, Webb CP. Gene expression in rat lacrimal gland duct cells collected using laser capture microdissection: evidence for K+ secretion by duct cells. Invest Ophthalmol Vis Sci. 2006;47:1876–1885.[Abstract/Free Full Text]
  20. Foskett JK, Melvin JE. Activation of salivary secretion: coupling of cell volume and [Ca2+] in single cells. Science. 1989;244:1582–1585.[Abstract/Free Full Text]
  21. McCarthy NA, O'Neil RG. Calcium signaling in cell volume regulation. Physiol Rev. 1992;72:1037–1061.[Abstract/Free Full Text]
  22. Cala PM. Volume regulation by flounder red blood cells in anisotonic media. J Gen Biol. 1977;69:537–552.
  23. Parker JC. Volume-activated transport systems in dog red blood cells. Comp Biochem Physiol A Comp Physiol. 1988;90:539–542.
  24. O'Neil WC. Cl-dependent K transport in a pure population of volume-regulating human erythrocytes. Am J Physiol. 1989;256:C858–C864.
  25. Hoffmann EK. Anion exchange and anion-cation co-transport systems in mammalian cells. Phil Trans R Soc Lond Biol Sci. 1982;299:519–535.[Abstract/Free Full Text]
  26. Cala PM. Volume regulation by Amphiuma red blood cells: the membrane potential and its implications regarding the nature of the ion-flux pathways. J Gen Physiol. 1980;76:683–708.[Abstract/Free Full Text]
  27. Haas M, Schmidt WF, McManus TJ. Catecholamine-stimulated ion transport in duck red cells: gradient effects in electrically neutral [Na+K+2Cl] co-transport. J Gen Physiol. 1982;80:125–147.[Abstract/Free Full Text]
  28. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflügers Arch. 1981;391:85–100.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  29. Barry PH. JPCalc, a software package for calculating liquid junction potential corrections in patch-clamp, intracellular, epithelial and bilayer measurements and for correcting junction potential measurements. J Neurosci Methods. 1994;51:107–116.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  30. Findlay I, Dunne MJ, Petersen OH. ATP-sensitive inward rectifier and voltage and calcium-activated K+ channels in cultured pancreatic islet cells. J Membr Biol. 1985;83:169–175.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  31. Kikkawa T. Secretory potentials in the lacrimal gland of the rabbit. Jpn J Ophthalmol. 1970;14:25–40.
  32. Foskett JK. [Ca2+]i modulation of Cl– content controls cell volume in single salivary acinar cells during fluid secretion. Am J Physiol. 1990;259:C998–C1004.[Medline][Order article via Infotrieve]
  33. Anderson MP, Welsh MJ. Calcium and cAMP activate different chloride channels in the apical membrane of normal and cystic fibrosis epithelia. Proc Natl Acad Sci USA. 1991;88:6003–6007.[Abstract/Free Full Text]
  34. Welsh MJ, Anderson MP, Rich DP, et al. Cystic fibrosis transmembrane regulator: a chloride channel with novel regulation. Neuron. 1992;8:821–829.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  35. Anderson MP, Gregory RJ, Thompson S, et al. Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. Science. 1991;253:202–205.[Abstract/Free Full Text]
  36. Cunningham SA, Worrell RT, Benos DJ, Frizzell RA. cAMP-stimulated ion currents in Xenopus oocytes expressing CFTR cRNA. Am J Physiol. 1992;262:C783–C788.[Medline][Order article via Infotrieve]
  37. Kunzelmann K, Pavenstadt H, Greger R. Properties and regulation of chloride channels in cystic fibrosis and normal airway cells. Pflügers Arch. 1989;415:172–182.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  38. Randriamanpta C, Chanson M, Trautmann A. Calcium and secretagogue-induced conductances in rat exocrine pancreas. Pflügers Arch. 1988;411:53–57.[CrossRef][Medline][Order article via Infotrieve]
  39. Gray MA, Pollard CE, Harris A, Coleman L, Greenwell JR, Argent BE. Anion selectivity and block of the small-conductance chloride channel on pancreatic duct cells. Am J Physiol. 1990;259:C752–C761.[Medline][Order article via Infotrieve]
  40. Martin DK. Small conductance chloride channels in acinar cells from the rat mandibular salivary gland are directly controlled by a G-protein. Biochem Biophys Res Comm. 1993;192:1266–1273.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  41. Ishikawa T, Cook DI. A Ca2+-activated Cl– current in sheep parotid secretory cells. J Membr Biol. 1993;135:261–271.[Web of Science][Medline][Order article via Infotrieve]
  42. Park K, Brown PD. Intracellular pH modulates the activity of chloride channels in isolated lacrimal gland acinar cells. Am J Physiol. 1995;268:C647–C650.[Medline][Order article via Infotrieve]
  43. Halm DR, Frizzell RA. Anion permeation in an apical membrane chloride channel of a secretory epithelial cell. J Gen Physiol. 1992;99:339–366.[Abstract/Free Full Text]
  44. Sheppard DN, Rich DP, Ostedgaard LS, Gregory RJ, Smith AE, Welsh MJ. Mutations associated with mild-disease-form Cl– channels with altered pore properties. Nature. 1993;362:160–164.[CrossRef][Medline][Order article via Infotrieve]
  45. Matsuda JJ, Filali MS, Volk KA, Collins MM, Moreland JG, Lamb FS. Overexpression of CLC-3 in HEK293T cells yields novel currents that are pH dependent. Am J Physiol Cell Physiol. 2008;294:C251–C262.[Abstract/Free Full Text]
  46. Saito Y, Ozawa T, Suzuki S, Nishiyama A. Intracellular pH regulation in the mouse lacrimal gland acinar cells. J Membr Biol. 1988;101:73–81.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  47. Pirani D, Evans LAR, Cook DI, Young JA. Intracellular pH in the rat mandibular salivary gland: the role of Na-H and Cl-HCO3 antiports in secretion. Pflügers Arch. 1987;408:178–184.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  48. Melvin JE, Moran A, Turner RJ. The role of HCO3– and Na+/H+ exchange in response of rat parotid acinar cells to muscarinic stimulation. J Biol Chem. 1988;263:19564–19569.[Abstract/Free Full Text]
  49. Steward MC, Seo Y, Case RM. Intracellular pH during secretion in the perfused rabbit mandibular salivary gland measured by 31P NMR spectroscopy. Pflügers Arch. 1989;414:200–207.[CrossRef][Medline][Order article via Infotrieve]
  50. Hoffmann EK, Simonsen LO. Membrane mechanisms in volume and pH regulation in vertebrate cells. Physiol Rev. 1989;69:315–382.[Free Full Text]
  51. Pierce SK, Politis AD. Ca2+- activated cell volume recovery mechanisms. Annu Rev Physiol. 1990;52:27–42.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  52. Sarkadi B, Parker JC. Activation of ion transport pathways by changes in cell volume. Biochimica et Biophysica Acta. 1991;1071:407–427.[Medline][Order article via Infotrieve]
  53. Evans MG, Marty A. Potentiation of muscarinic and {alpha}-adrenergic responses by an analogue of guanosine 5'-triphosphate. Proc Natl Acad Sci USA. 1986;83:4099–4103.[Abstract/Free Full Text]
  54. Speake T, Douglas IJ, Brown PD. The role of calcium in the volume regulation of rat lacrimal acinar cells. J Membr Biol. 1998;164:283–291.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  55. Hazama A, Okada Y. Ca2+ sensitivity of volume-regulatory K+ and Cl– channels in cultured human epithelial cells. J Physiol. 1988;402:687–702.[Abstract/Free Full Text]
  56. Hazama A, Okada Y. Biphasic rises in cytosolic free Ca2+ in association with activation of K+ and Cl– conductance during the regulatory volume decrease in cultured human epithelial cells. Pflügers Arch. 1990;416:710–714.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  57. Solc CK, Wine JJ. Swelling-induced and depolarization-induced Cl– channels in normal and cystic fibrosis epithelial cells. Am J Physiol. 1991;261:C658–C674.[Medline][Order article via Infotrieve]
  58. Weiss H, Lang F. Ion channels activated by swelling of Madin Darby Canine Kidney (MDCK) cells. J Membrane Biol. 1992;126:109–114.[Web of Science][Medline][Order article via Infotrieve]
  59. Worrell RT, Butt AG, Cliff WH, Frizzell RA. A volume-sensitive chloride conductance in human colonic cell line T84. Am J Physiol. 1989;256:C1111–C1119.[Web of Science][Medline][Order article via Infotrieve]
  60. Montrose-Rafizadeh C, Guggino WB. Role of intracellular calcium in volume regulation by rabbit medullary thick ascending limb cells. Am J Physiol. 1991;260:F402–F409.[Medline][Order article via Infotrieve]
  61. Valverde MA, Diaz M, Sepulveda FV, Gill DR, Hyde SC, Higgins CF. Volume-regulated chloride channels associated with the human multidrug-resistance P-glycoprotein. Nature. 1992;355:830–833.[CrossRef][Medline][Order article via Infotrieve]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Herok, G. H.
Right arrow Articles by Martin, D. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Herok, G. H.
Right arrow Articles by Martin, D. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS