Epithelial Cell Volume Regulation Research Articles

Physiological Regulation of ATP Release at the Apical Surface of Human Airway Epithelia

Extracellular ATP and its metabolite adenosine regulate mucociliary clearance in airway epithelia. Little has been known, however, regarding the actual ATP and adenosine concentrations in the thin ( approximately 7 microm) liquid layer lining native airway surfaces and the link between ATP release/metabolism and autocrine/paracrine regulation of epithelial function. In this study, chimeric Staphylococcus aureus protein A-luciferase (SPA-luc) was bound to endogenous antigens on primary human bronchial epithelial (HBE) cell surface and ATP concentrations assessed in real-time in the thin airway surface liquid (ASL). ATP concentrations on resting cells were 1-10 nm. Inhibition of ecto-nucleotidases resulted in ATP accumulation at a rate of approximately 250 fmol/min/cm2, reflecting the basal ATP release rate. Following hypotonic challenge to promote cell swelling, cell-surface ATP concentration measured by SPA-luc transiently reached approximately 1 microm independent of ASL volume, reflecting a transient 3-log increase in ATP release rates. In contrast, peak ATP concentrations measured in bulk ASL by soluble luciferase inversely correlated with volume. ATP release rates were intracellular calcium-independent, suggesting that non-exocytotic ATP release from ciliated cells, which dominate our cultures, mediated hypotonicity-induced nucleotide release. However, the cystic fibrosis transmembrane conductance regulator (CFTR) did not participate in this function. Following the acute swelling phase, HBE cells exhibited regulatory volume decrease which was impaired by apyrase and facilitated by ATP or UTP. Our data provide the first evidence that ATP concentrations at the airway epithelial surface reach the range for P2Y2 receptor activation by physiological stimuli and identify a role for mucosal ATP release in airway epithelial cell volume regulation.

Purinergic signaling underlies CFTR control of human airway epithelial cell volume

Background: Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function in cystic fibrosis (CF) causes dysregulation of multiple ion channels, water channels, and acid–base transporters in epithelia. As such, we hypothesized that dysregulation of many critical ion channels and transporters may cause defects in human airway epithelial cell volume regulation. Methods: Cell volume, regulatory volume decrease, and its regulation was assessed in real-time via Coulter Counter Multisizer III-driven electronic cell sizing in non-CF, CF, and CFTR-complemented CF human airway epithelial cells. SPQ halide fluorescence assay of hypotonicity-induced chloride efflux provided indirect validation of the cell volume assays. Results: CFTR, via autocrine ATP signaling, governs human airway epithelial cell volume regulation. Non-CF cells and wild-type (WT)-CFTR-transfected CF cells had normal regulatory volume decrease (RVD) responses that were attenuated by blockade of autocrine and paracrine purinergic signaling. In contrast, parental IB3-1 CF cells or IB3-1 cells expressing CFTR mutants (ΔF508, G551D, and S1455X) failed to RVD. CF cell RVD was rescued by agonists to P2Y G protein-coupled receptors and, more robustly, by agonists to P2X purinergic receptor channels. Conclusions: Loss of CFTR and CFTR-driven autocrine ATP signaling may underlie defective cell volume regulation and dysregulated ion, water, and acid–base transport in CF airway epithelia.

Impaired Hearing in Mice Lacking Aquaporin-4 Water Channels

A role for aquaporins (AQPs) in hearing has been suggested from the specific expression of aquaporins in inner ear and the need for precise volume regulation in epithelial cells involved in acoustic signal transduction. Using mice deficient in selected aquaporins as controls, we localized AQP1 in fibrocytes in the spiral ligament and AQP4 in supporting epithelial cells (Hensen's, Claudius, and inner sulcus cells) in the organ of Corti. To determine whether aquaporins play a role in hearing, auditory brain stem response (ABR) thresholds were compared in wild-type mice and transgenic null mice lacking (individually) AQP1, AQP3, AQP4, and AQP5. In 4-5-week-old mice in a CD1 genetic background, ABR thresholds in response to a click stimulus were remarkably increased by >12 db in AQP4 null mice compared with wild-type mice (p < 0.001), whereas ABR thresholds were not affected by AQP1, AQP3, or AQP5 deletion. In a C57/bl6 background, nearly all AQP4 null mice were deaf, whereas ABRs could be elicited in wild-type controls. ABRs in AQP4 null CD1 mice measured in response to tone bursts (4-20 kHz) indicated a frequency-independent hearing deficit. Light microscopy showed no differences in cochlear morphology of wild-type versus AQP4 null mice. These results provide the first direct evidence that an aquaporin water channel plays a role in hearing. AQP4 may facilitate rapid osmotic equilibration in epithelial cells in the organ of Corti, which are subject to large K(+) fluxes during mechano-electric signal transduction.

Role of Na(+)-H(+)-antiport in restitution of isolated guinea pig gastric epithelium after superficial injury.

In addition to its pHi regulatory function Na(+)-H(+)-antiport is also involved in volume regulation of epithelial cells, particularly in neutral conditions. It is also known that the antiport is activated after ligand binding following growth factor receptor activation. The aim of the present study was to evaluate the role of the antiport in restitution of gastric mucosa and whether its activity is dependent on the type of superficial injury. Therefore the fundic epithelium of guinea pig stomach was perfused in an Ussing chamber in neutral conditions. Na(+)-H(+)- and Cl(-)-HCO3(-)-antiports were inhibited with 1.0 mM amiloride, 1.0 mM SITS, or with HCO3- removal and Na(+)-K(+)-2Cl(2-)-cotransporter with 0.3 M furosemide during 4 hr of restitution after superficial injury induced either by 1.25 M NaCl or by 1.0% Triton. Luminal exposure of the epithelium to amiloride had no effect on restitution but serosal application abolished the process completely. The inhibitory effect of amiloride was similar after both NaCl and Triton injury. The inhibition of Cl(-)-HCO3(-)-antiport with SITS interfered with the process as well, while HCO3- removal had no significant inhibitory effect, nor did the inhibition of Na(+)-K(+)-2Cl(-)-cotransporter. The morphologic findings were in accordance with the electrophysiologic measurements in each pair of tissues. It is concluded that the Na(+)-H(+)-antiport is essential for the epithelial cells during restitution even in neutral conditions, but a functional Cl(-)-HCO3(-)-antiport is also required. The activity of Na(+)-H(+)-antiport is sensitive to basolateral amiloride and is necessary regardless of the type of chemical injury.

Effects of osmotic perturbation on [Ca2+]i and pHi in rabbit proximal tubular cells in primary culture

The effects of anisosmotic media on intracellular Ca2+ and H+ concentrations ([Ca2+]i and pHi, respectively) were studied to investigate whether these changes play a role in epithelial cell volume regulation. [Ca2+]i and pHi were measured in rabbit proximal tubular cells in primary culture using the fluorescent ratio probes fura 2 and 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Changing medium osmolarity from 300 to 150 mosmol resulted in a rapid transient increase in fura 2 ratio from 0.89 +/- 0.02 to 1.15 +/- 0.03, which lasted for several minutes and returned to base line within 10 min. The source of Ca2+ was extracellular as well as intracellular. Simultaneous with this increase in [Ca2+]i, cells slowly acidified from pHi of 7.51 +/- 0.03 to 6.86 +/- 0.02. This osmotic swelling-induced acidification could not be explained by a decrease in the rate of Na+/H+ exchange or increase in the rate of Cl-/HCO3- exchange. Subsequently increasing medium osmolarity from 150 to 500 mosmol decreased the fura 2 ratio below the initial level observed in isotonic media, while pHi increased from 6.96 +/- 0.02 to 7.37 +/- 0.03. This decrease in [Ca2+]i was due to inhibition of Ca2+ influx and to an increase in Ca2+ efflux. The osmotic shrinkage-induced alkalinization was slightly inhibited by ethylisopropylamiloride, indicative of activation of Na+/H+ exchange. To test whether an increase in [Ca2+]i causes a decrease in pHi or vice versa, pHi and [Ca2+]i were manipulated at isotonic conditions. Surprisingly, a decrease in [Ca2+]i was accompanied by a decrease in pHi, and an increase in pHi resulted in an increase in [Ca2+]i, in the absence of osmotic perturbation.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of cytoskeletal rearrangements and loss of volume regulation in epithelial cells by Treponema denticola

The early responses of oral epithelial cells to the adhesion of the oral spirochaete Treponema denticola were studied as a model of microbial perturbation of the plasma membrane. KB cell (ATCC CCL 17) monolayers were incubated with T. denticola (ATCC 35405) in α-MEM (minimal essential medium) for periods of 1–4 h at 37C without serum. Control cultures were exposed to bacteria-conditioned α-MEM without serum or bacteria or to α-MEM alone. At the end of each incubation, detached and attached epithelial cells were harvested and analysed separately. Compared with controls, T. denticola induced in 25% of cells a two-fold, time-dependent increase of detachment by 4 h. Detached cells in both T. denticola-exposed and control cultures exhibited 25% reductions in modal diameter, did not exclude propidium iodide, did not readhere, and did not form colonies. In T. denticola-exposed cultures, a larger subset (75%) of cells remained attached to the substratum, demonstrated no significant reduction of colony-forming efficiency and excluded propidium iodide. However, these cells exhibited a 21% reduction in diameter ( p < 0.05), a 60% decrease of F-actin ( p < 0.001), and a 74% reduction in the proportion expressing desmoplakin II ( p < 0.01) after exposure to T. denticola. Flow cytometry showed a small (14%) but significant ( p < 0.001) reduction in mean fluorescence intensity due to keratin expression in T. denticola-treated cultures. Exposure of cells to anisosmotic media demonstrated that, in contrast to controls, cultures challenged by bacteria failed to undergo compensatory volume regulation. These data indicate that contact of T. denticola with epithelial cells induces: (1) cell detachment and death in a small subset; (2) loss of cellular volume regulation that is associated with disruption of cytoskeletal proteins in attached cells.

Necturus gallbladder epithelial cell volume regulation and inhibitors of arachidonic acid metabolism

Inhibition of the metabolism of arachidonic acid by the epoxygenase (cytochrome P-450) pathway with the inhibitor ketoconazole results in excessive cell swelling upon exposure to hyposmolality instead of the rapid and complete regulatory volume decrease (RVD) normally observed. NaCl entry from bathing solutions to cell interior was shown to cause this swelling, with Na influx occurring across the basolateral membrane and electrically silent Cl influx across the apical membrane. Ion substitution experiments show that the KCl efflux mediating RVD was unimpaired by ketoconazole, but was overwhelmed by the NaCl influx. Measurements of transepithelial fluid flux, Cl concentration, osmolality and pH showed that gallbladders treated with ketoconazole transiently secreted fluid rather than the normal absorption. We conclude that inhibition of arachidonic acid metabolism does not directly affect RVD by Necturus gallbladder, but that blockade of the epoxygenase pathway can have a profound influence on NaCl entry into gallbladder epithelial cells.

Solute transport and epithelial cell volume regulation

1. 1. The regulation of epithelial cell volume is an essential requirement for normal tissue function and the maintenance of cellular integrity. 2. 2. Renal papillary epithelial cells utilize an organic to compensate for the shrinkage associated with exposure to hypertonic solutions. 3. 3. These cells synthesize the polyol, sorbitol, to increase their intracellular solute content. 4. 4. Sorbitol is synthesized from glucose by the enzyme aldose reductase; exposure of the cells to hypertonic media causes aldose reductase synthesis and subsequent sorbitol generation over a two or three day period. 5. 5. The intracellular signal for the formation of aldose reductase is not yet identified.

Role of intracellular calcium in cellular volume regulation.

We investigated the role of intracellular calcium in epithelial cell volume regulation using cells isolated from the toad urinary bladder. A suspension of cells was prepared by treatment of the bladder with collagenase followed by ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid. The cells retained their ion-transporting capabilities: ouabain (1 mM) and amiloride (10 microM) inhibited cellular uptake of 86Rb and 22Na, respectively. Using a Coulter counter to measure cellular volume, we found that we could swell cells either by reducing the extracellular osmolality or by adding the permeant solute urea (45 mM) isosmotically. Under both conditions, cells first swelled and then returned to their base-line volume, in spite of the continued presence of the stimulus to swell. Volume regulation was inhibited when cells were swelled at low extracellular [Ca] (100 nM) and was retarded in cells preloaded with the calcium buffer quin 2. Swelling increased the intracellular free calcium concentration ([Ca]i), as measured by quin 2 fluorescence: [Ca]i increased 35 +/- 9 nM (n = 6) after hypotonic swelling and 42 +/- 3 nM (n = 3) after urea swelling. Reducing extracellular [Ca] to less than 100 nM prevented the swelling-induced increase in [Ca]i, suggesting that the source of the increase in [Ca]i was extracellular. This result was confirmed in measurements of cellular uptake of 45Ca: the rate of uptake was significantly higher in swollen cells compared with control (1.1 +/- 0.2 vs. 0.4 +/- 0.1 fmol . cell-1 X 5 min-1). Our experiments provide the first demonstration that cellular swelling increases [Ca]i. This increase is likely to play a critical role in cellular volume regulation.

Morphological and cell volume changes in the rat lens during the formation of radiation cataracts

Earlier studies in our laboratory showed that 24 hr after X-irradiation, epithelial cells of early postnatal rat lenses increased in volume. Three days after X-irradiation, the underlying lens fibers increased in volume. This finding suggested a correlation between damage to epithelial cell volume regulation and subsequent fiber cell swelling. To test this hypothesis 4-week-old rat lenses were three-dimensionally reconstructed to determine average cell volumes of specific lens regions and wet weights of whole lenses were measured during radiation cataract formation. In addition, the differentiation of epithelial cells into lens fibers was monitored by autoradiography. Four-week-old Sprague-Dawley rats were injected with [ 3H]-thymidine and, 24 hr later, their eyes were irradiated with either 400 or 1200 rad. Lenses were examined with a slit lamp and cataracts were graded on a scale of 1 + to 4 +. Animals were killed 24 hr and 3, 5, 15 and 30 weeks after exposure. Lenses were serially sectioned at 0·75 μm and epithelial, equatorial and cortical fiber cell volumes were determined. Rats exposed to 400 or 1200 rad developed 0·5−1·5 + or 2·5−3·0 + cataracts, respectively, 10–16 weeks after X-irradiation. Epithelial and equatorial cells of both groups did not significantly increase in volume during this period. Three weeks after irradiation with 1200 rad cortical fibers were disorganized and had increased volume. By 5 and 15 weeks, cortical fibers had more normal cell volumes, although their morphology remained grossly altered. Cortical fiber volume of lenses irradiated with 400 rad were not significantly different from control lenses throughout the experimental period. By 15 weeks lenses irradiated with 400 rad showed subtle changes in morphology. Wet weight determinations indicated that the localized increase in cortical fiber volume did not result in an increase in the wet weight of the entire lens. Autoradiography showed that affected fibers had been epithelial cells at the time of X-irradiation. These results provide additional evidence that disturbances in fiber differentiation are involved with cataract formation, but do not support the initial hypothesis that a disturbance in epithelial cell volume regulation leads to fiber cell swelling. Earlier results suggesting defects in lens epithelial volume regulation in radiation cataract formation may have been complicated by ocular inflammation.

Epithelial Cell Volume Regulation Illustrated by Experiments in Frog Skin

The volume control of the syncytium of principal cells (as opposed to the mitochondria-rich cells) is largely confined to the movement of ions and water through the basolateral membrane. The apical membrane is nearly tight to water and ions except sodium. The basolateral membrane is normally tight to chloride, but its chloride channels open if the cells swell osmotically or if the membrane is depolarized. If the epithelium has lost KCl during osmotic swelling, it is recovered by a basolateral cotransport of KNaCl2.

Involvement of calcium and cytoskeleton in gallbladder epithelial cell volume regulation.

The importance of calcium and cellular cytoskeletal elements in the activation or control of volume regulation by epithelial cells was explored in Necturus gallbladder. Gallbladder cells have been previously shown to rapidly readjust their volumes to control size after osmotic perturbation of the mucosal bathing solution. Removal of calcium from the perfusates caused dramatic morphological changes that prevented assessment of the role of extracellular calcium in volume regulation. The regulatory volume increase (RVI) that follows shrinkage of the cell due to perfusion of a hypertonic mannitol solution is insensitive to agents that interfere with cell calcium- or calmodulin-mediated events (quinidine, trifluoperazine) and is not blocked by agents that cause changes in the cytoskeleton (colchicine, cytochalasin B). Osmotically induced cell swelling is followed by regulatory volume decrease (RVD), which is inhibited by agents that interfere with calcium-dependent processes (quinidine, trifluoperazine) and by the microfilament inhibitor, cytochalasin B. These results indicate that RVD depends on calcium, calmodulin, and an intact microfilament network, whereas RVI is independent of these factors.

Epithelial cell volume modulation and regulation.

Epithelial cell volume is a sensitive indicator of the balance between solute entry into the cell and solute exit. Solute accumulation in the cell leads to cell swelling because the water permeability of the cell membranes is high. Similarly, solute depletion leads to cell shrinkage. The rate of volume change under a variety of experimental conditions may be utilized to study the rate and direction of solute transport by an epithelial cell. The pathways of water movement across an epithelium may also be deduced from the changes in cellular volume. A technique for the measurement of the volume of living epithelial cells is described, and a number of experiments are discussed in which cell volume determination provided significant new information about the dynamic behavior of epithelia. The mechanism of volume regulation of epithelial cells exposed to anisotonic bathing solution is discussed and shown to involve the transient stimulation of normally dormant ion exchangers in the cell membrane.