Net Fluorescein Flux Across Corneal Endothelium Strongly Suggests Fluid Transport is due to Electro-osmosis.

We have presented prior evidence suggesting that fluid transport results from electro-osmosis at the intercellular junctions of the corneal endothelium. Such phenomenon ought to drag other extracellular solutes. We have investigated this using fluorescein-Na2 as an extracellular marker. We measured unidirectional fluxes across layers of cultured human corneal endothelial (HCE) cells. SV-40-transformed HCE layers were grown to confluence on permeable membrane inserts. The medium was DMEM with high glucose and no phenol red. Fluorescein-labeled medium was placed either on the basolateral or the apical side of the inserts; the other side carried unlabeled medium. The inserts were held in a CO2 incubator for 1 h (at 37 °C), after which the entire volume of the unlabeled side was collected. After that, label was placed on the opposite side, and the corresponding paired sample was collected after another hour. Fluorescein counts were determined with a (Photon Technology) DeltaScan fluorometer (excitation 380 nm; emission 550 nm; 2 nm bwth). Samples were read for 60 s. The cells utilized are known to transport fluid from the basolateral to the apical side, just as they do in vivo in several species. We used 4 inserts for influx and efflux (total: 20 1-h periods). We found a net flux of fluorescein from the basolateral to the apical side. The flux ratio was 1.104 ± 0.056. That difference was statistically significant (p = 0.00006, t test, paired samples). The endothelium has a definite restriction at the junctions. Hence, an asymmetry in unidirectional fluxes cannot arise from osmosis, and can only point instead to paracellular solvent drag. We suggest, once more, that such drag is due to electro-osmotic coupling at the paracellular junctions.

[A new approach to the electrostatic stabilization of cations in the aqueous cavity of K+ channel: the role of nonlocal-electrostatic effects].

Methods of nonlocal electrostatics, applied to ion channels in our previous papers, are used to analyze the stability of K+ in the aqueous cavity of K+ channel. Other authors used formulas of classical electrostatics to calculate the energy of K+ in the aqueous cavity of KcsA. Using a new formulation of nonlocal-electrostatic theory, we obtained a formula for the image force energy acting on K+, which is in the center of a spherical cavity of K+ channel. It is shown that nonlocal-electrostatic effects in the aqueous cavity of K+ channel leads to a decrease in the energy K+ on 4kT compared with the calculation by the formulas of classical electrostatics.

[Role of image force in a water pore of K+ channel in the channel permeability--nonlocal electrostatic effects].

A modified Parsegian formula for the calculation of the mage force in a water pore, which takes into account nonlocal electrostatic effects, has been applied to the analysis of changes in the permeability of K+ channels. It has been shown that the channel permeability increases 3 x 10(3) times as the width of the pore increases from 4 to 10 angstroms when the energy is calculated by the modified Parsegian formula and only 16 times if the classical Parsegian formula is used. We conclude that the image force in a water pore within K+ channels plays an important part in the change of channel permeability. Accounting for nonlocal electrostatic effects indicates that the permeability of the K+ channel is more strongly related to the size of the water pore than previously assumed by the original Parsegian formula.

The Role of the Tight Junction in Paracellular Fluid Transport across Corneal Endothelium. Electro-osmosis as a Driving Force.

The mechanism of epithelial fluid transport is controversial and remains unsolved. Experimental difficulties pose obstacles for work on a complex phenomenon in delicate tissues. However, the corneal endothelium is a relatively simple system to which powerful experimental tools can be applied. In recent years our laboratory has developed experimental evidence and theoretical insights that illuminate the mechanism of fluid transport across this leaky epithelium. Our evidence points to fluid being transported via the paracellular route by a mechanism requiring junctional integrity, which we attribute to electro-osmotic coupling at the junctions. Fluid movements can be produced by electrical currents. The direction of the movement can be reversed by current reversal or by changing junctional electrical charges by polylysine. Aquaporin 1 (AQP1) is the only AQP present in these cells, and its deletion in AQP1 null mice significantly affects cell osmotic permeability but not fluid transport, which militates against the presence of sizable water movements across the cell. By contrast, AQP1 null mice cells have reduced regulatory volume decrease (only 60% of control), which suggests a possible involvement of AQP1 in either the function or the expression of volume-sensitive membrane channels/transporters. A mathematical model of corneal endothelium predicts experimental results only when based on paracellular electro-osmosis, and not when transcellular local osmosis is assumed instead. Our experimental findings in corneal endothelium have allowed us to develop a novel paradigm for this preparation that includes: (1) paracellular fluid flow; (2) a crucial role for the junctions; (3) hypotonicity of the primary secretion; (4) an AQP role in regulation and not as a significant water pathway. These elements are remarkably similar to those proposed by the Hill laboratory for leaky epithelia.

Epithelial fluid transport: protruding macromolecules and space charges can bring about electro-osmotic coupling at the tight junctions.

The purpose of the present work is to investigate whether the idea of epithelial fluid transport based on electro-osmotic coupling at the level of the leaky tight junction (TJ) can be further supported by a plausible theoretical model. We develop a model for fluid transport across epithelial layers based on electro-osmotic coupling at leaky tight junctions (TJ) possessing protruding macromolecules and fixed electrical charges. The model embodies systems of electro-hydrodynamic equations for the intercellular pathway, namely the Brinkman and the Poisson-Boltzmann differential equations applied to the TJ. We obtain analytical solutions for a system of these two equations, and are able to derive expressions for the fluid velocity profile and the electrostatic potential. We illustrate the model by employing geometrical parameters and experimental data from the corneal endothelium, for which we have previously reported evidence for a central role for electro-osmosis in translayer fluid transport. Our results suggest that electro-osmotic coupling at the TJ can account for fluid transport by the corneal endothelium. We conclude that electro-osmotic coupling at the tight junctions could represent one of the basic mechanisms driving fluid transport across some leaky epithelia, a process that remains unexplained.

Characterization of regulatory volume behavior by fluorescence quenching in human corneal epithelial cells.

An in-depth understanding of the mechanisms underlying regulatory volume behavior in corneal epithelial cells has been in part hampered by the lack of adequate methodology for characterizing this phenomenon. Accordingly, we developed a novel approach to characterize time-dependent changes in relative cell volume induced by anisosmotic challenges in calcein-loaded SV40-immortalized human corneal epithelial (HCE) cells with a fluorescence microplate analyzer. During a hypertonic challenge, cells shrank rapidly, followed by a temperature-dependent regulatory volume increase (RVI), tau(c) = 19 min. In contrast, a hypotonic challenge induced a rapid (tau(c) = 2.5 min) regulatory volume decrease (RVD). Temperature decline from 37 to 24 degrees C reduced RVI by 59%, but did not affect RVD. Bumetanide (50 microM), ouabain (1 mM), DIDS (1 mM), EIPA (100 microM), or Na(+)-free solution reduced the RVI by 60, 61, 39, 32, and 69%, respectively. K+, Cl- channel and K(+)-Cl(-) cotransporter (KCC) inhibition obtained with either 4-AP (1 mM), DIDS (1 mM), DIOA (100 microM), high K+ (20 mM) or Cl(-)-free solution, suppressed RVD by 42, 47, 34, 52 and 58%, respectively. KCC activity also affects steady-state cell volume, since its inhibition or stimulation induced relative volume alterations under isotonic conditions. Taken together, K+ and Cl- channels in parallel with KCC activity are important mediators of RVD, whereas RVI is temperature-dependent and is essentially mediated by the Na(+)-K(+)-2Cl(-) cotransporter (Na(+)-K(+)-2Cl(-)) and the Na(+)-K(+) pump. Inhibition of K+ and Cl- channels and KCC but not Na(+)-K(+)-2Cl(-) affect steady-state cell volume under isotonic conditions. This is the first report that KCC activity is required for HCE cell volume regulation and maintenance of steady-state cell volume.

Functional and molecular characterization of a volume-activated chloride channel in rabbit corneal epithelial cells.

We characterized the functional and molecular properties of a volume-regulated anion channel (VRAC) in SV40-immortalized rabbit corneal epithelial cells (tRCE), since they mediate a robust regulatory volume decrease (RVD) response during exposure to a hypotonic challenge. Whole-cell patch clamp-monitored chloride currents and light-scattering measurements evaluated temporal cell-volume responsiveness to hypoosmotic challenges. Exposure to 200 mOsm medium elicited an outwardly-rectifying current (VACC), which was reversible upon reperfusion with isotonic (300 mOsm) medium. VACC and RVD were chloride-dependent because either chloride removal or application of NPPB (100 microM) suppressed these responses. VACC behavior exhibited voltage-dependent inhibition in the presence of DIDS (500 microM), whereas inhibition by both NPPB (100 microM) and niflumic acid (500 microM) was voltage-independent. VACC was insensitive to glibenclamide (250 microM), verapamil (500 microM) or removal of extracellular calcium. Phorbol dibutyrate, PDBu, (100 nM) had no effect on activated VACC. However, preincubation with PDBu prior to hypotonic challenge prevented VACC and RVD responses as well as prolonged characteristic time. An inactive phorbol ester analogue had no effect on RVD behavior. Moreover, Northern blot analysis verified expression of ClC-3 gene transcripts. The presence of ClC-3 transcripts along with the correspondence between the effects of known ClC-3 inhibitors on VACC and RVD suggest that ClC-3 activation underlies these responses to hypotonic-induced cell swelling.

Hypertonicity-induced p38MAPK activation elicits recovery of corneal epithelial cell volume and layer integrity.

In hypertonicity-stressed (i.e., 600 mOsm) SV40-immortalized rabbit and human corneal epithelial cell layers (RCEC and HCEC, respectively), we characterized the relationship between time-dependent changes in translayer resistance, relative cell volume and modulation of MAPK superfamily activities. Sulforhodamine B permeability initially increased by 1.4- and 2-fold in RCEC and HCEC, respectively. Subsequently, recovery to its isotonic level only occurred in RCEC. Light scattering revealed that in RCEC 1) regulatory volume increase (RVI) extent was 20% greater; 2) RVI half-time was 2.5-fold shorter. However, inhibition of Na-K-2Cl cotransporter and Na/K-ATPase activity suppressed the RVI response more in HCEC. MAPK activity changes were as follows: 1) p38 was wave-like and faster as well as larger in RCEC than in HCEC (90- and 18-fold, respectively); 2) increases in SAPK/JNK activity were negligible in comparison to those of p38; 3) Erk1/2 activity declined to 30-40% of their basal values. SB203580, a specific p38 inhibitor, dose dependently suppressed the RVI responses in both cell lines. However, neither U0126, which inhibits MEK, the kinase upstream of Erk, nor SP600125, inhibitor of SAPK/JNK, had any effect on this response. Taken together, sufficient activation of the p38 limb of the MAPK superfamily during a hypertonic challenge is essential for maintaining epithelial cell volume and translayer resistance. On the other hand, Erk1/2 activity restoration seems to be dependent on cell volume recovery.

Evidence for a central role for electro-osmosis in fluid transport by corneal endothelium.

The mechanism of transepithelial fluid transport remains unclear. The prevailing explanation is that transport of electrolytes across cell membranes results in local concentration gradients and transcellular osmosis. However, when transporting fluid, the corneal endothelium spontaneously generates a locally circulating current of approximately 25 microA cm(-2), and we report here that electrical currents (0 to +/-15 microA cm(-2)) imposed across this layer induce fluid movements linear with the currents. As the imposed currents must be approximately 98% paracellular, the direction of induced fluid movements and the rapidity with which they follow current imposition (rise time < or =3 sec) is consistent with electro-osmosis driven by sodium movement across the paracellular pathway. The value of the coupling coefficient between current and fluid movements found here (2.37 +/- 0.11 microm cm(2) hr(-1) microA (-1), suggests that: 1) the local endothelial current accounts for spontaneous transendothelial fluid transport; 2) the fluid transported becomes isotonically equilibrated. Ca(++)-free solutions or endothelial damage eliminate the coupling, pointing to the cells and particularly their intercellular junctions as a main site of electro-osmosis. The polycation polylysine, which is expected to affect surface charges, reverses the direction of current-induced fluid movements. Fluid transport is proportional to the electrical resistance of the ambient medium. Taken together, the results suggest that electro-osmosis through the intercellular junctions is the primary process in a sequence of events that results in fluid transport across this preparation.

Differential expression of Na:K:2Cl cotransporter, glucose transporter 1, and aquaporin 1 in freshly isolated and cultured bovine corneal tissues.

Little is known about whether culturing corneal limiting layers causes changes in the expression of their membrane transporter proteins from those present in fresh tissues. Accordingly, we compared mRNA abundance of three well-described types of transporters: water channel aquaporin 1 (AQP1), glucose transporter (GLUT1), and Na:K:2Cl cotransporter (NKCC), as well as NKCC protein levels in fresh bovine corneal epithelium and endothelium with those in their cultured counterparts. Abundance of mRNA encoding AQP1, GLUT1, and NKCC was quantified by a lysate nuclease protection assay. NKCC transcription was further characterized by Northern blotting. All data were normalized to cell DNA and protein contents. In the fresh epithelium, in all three cases mRNA levels were two to four times higher than in the endothelium. Expression of AQP1 and GLUT1 was 10 to 12 times higher than that of NKCC. After the third passage, the endothelial cell mRNA abundance in each case decreased 2- to 3-fold. Passage-dependent decreases were also observed in NKCC protein expression in the epithelial cells. In both corneal layers, there was a qualitative correlation between NKCC mRNA and protein levels. Both in fresh and cultured epithelial and endothelial cells, a shark NKCC1 DNA probe hybridized with mRNAs of two different lengths (about 5.0-5.5 and 7.0-7.5 kb). An anti-NKCC T4 monoclonal antibody recognized two major proteins with apparent molecular masses of 190 to 200 and 150 to 160 kDa. In summary, membrane transporter function in culture may not be always indicative of their role in fresh tissue since in cultured cells AQP1, GLUT1, and NKCC mRNA levels declined. Furthermore, in both epithelial and endothelial cells, there is expression of two different proteins and mRNAs that possibly encode for secretory (NKCC1) and absorptive (NKCC2) isoforms.

A three-dimensional model of the human facilitative glucose transporter Glut1.

The human facilitative transporter Glut1 is the major glucose transporter present in all human cells, has a central role in metabolism, and is an archetype of the superfamily of major protein facilitators. Here we describe a three-dimensional structure of Glut1 based on helical packing schemes proposed for lactose permease and Glut1 and predictions of secondary structure, and refined using energy minimization, molecular dynamics simulations, and quality and environmental scores. The Ramachandran scores and the stereochemical quality of the structure obtained were as good as those for the known structures of the KcsA K(+) channel and aquaporin 1. We found two channels in Glut1. One of them traverses the structure completely, and is lined by many residues known to be solvent-accessible. Since it is delimited by the QLS motif and by several well conserved residues, it may serve as the substrate transport pathway. To validate our structure, we determined the distance between these channels and all the residues for which mutations are known. From the locations of sugar transporter signatures, motifs, and residues important to the transport function, we find that this Glut1 structure is consistent with mutagenesis and biochemical studies. It also accounts for functional deficits in seven pathogenic mutants.

Fluid transport by human nonpigmented ciliary epithelial layers in culture: a homeostatic role for aquaporin-1.

We report for the first time that cultured nonpigmented human ciliary epithelial (NPE) cell layers transport fluid. Cells were grown to confluence on permeable membrane inserts, and fluid transport across the resulting cell layers was determined by volume clamp at 37 degrees C. These cell layers translocated fluid from the apical to the basal side at a steady rate of 3.6 microl x h(-1) x cm(-2) (n = 4) for 8 h. This fluid movement was independent of hydrostatic pressure and was completely inhibited by 1 mM ouabain, suggesting it arose from fluid transport. Mercuric chloride, a nonspecific but potent blocker of Hg(2+)-sensitive aquaporins, and aquaporin-1 antisense oligonucleotides both partially inhibited fluid transport across the cell layers, which suggests that water channels have a role in NPE cell homeostasis. In addition, these results suggest that of the two ciliary epithelial layers in tandem, the NPE layer by itself can transport fluid. This cultured layer, therefore, constitutes an interesting model that may be useful for physiological and pharmacological characterization of ciliary epithelial fluid secretion.

Mercurial sensitivity of aquaporin 1 endofacial loop B residues.

The water channel protein aquaporin-1 (AQP1) has two asparagine-proline-alanine (NPA) repeats on loops B and E. From recent structural information, these loops are on opposite sides of the membrane and meet to form a pore. We replaced the mercury-sensitive residue cysteine 189 in AQP1 by serine to obtain a mercury-insensitive template (C189S). Subsequently, we substituted three consecutive cysteines for residues 71-73 near the first NPA repeat (76-78) in intracellular loop B, and investigated whether they were accessible to extracellular mercurials. AQP1 and its mutants were expressed in Xenopus laevis oocytes, and the osmotic permeability (P(f)) of the oocytes was determined. C189S had wild-type P(f) but was not sensitive to HgCl(2). Expression of all three C189S cysteine mutants resulted in increased P(f), and all three mutants regained mercurial sensitivity. These results, especially the inhibitions by the large mercurial p-chloromercunbenzene-sulfonic acid (pCMBS) ( approximately 6A wide), suggest that residues 71-73 at the pore are accessible to extracellular mercurials. A 30-ps molecular dynamics simulation (at 300 K) starting with crystallographic coordinates of AQP1 showed that the width of the pore bottleneck (between Connolly surfaces) can vary (w(avg) = 3.9 A, sigma = 0.75; hydrated AQP1). Thus, although the pore width would be > or = 6 A only for 0.0026 of the time, this might suffice for pCMBS to reach residues 71-73. Alternative explanations such as passage of pCMBS across the AQP1 tetramer center or other unspecified transmembrane pathways cannot be excluded.

Immunocytochemical localization of aquaporin-1 in bovine corneal endothelial cells and keratocytes.

For immunocytochemistry, cultured bovine corneal endothelial cells (CBCEC) and bovine corneal cryosections were utilized. Preparations were fixed, permeabilized, and incubated with primary rabbit anti-rat aquaporin 1 (AQP1) antibody followed by rhodamine-conjugated secondary antibody, and were counter-stained with Sytox nuclear acid stain. Confocal microscopy of CBCEC in the x, y, and z planes showed rhodamine fluorescence, indicating the presence of AQP1 antibody localized to the apical and basolateral domains of the plasma membrane, but not to the membranes of intracellular compartments or other subcellular locations. Preabsorption with control antigenic peptide yielded no positive staining. Similar results were obtained using freshly dissected bovine corneas; in addition, these images showed AQP1 distributed to the plasma membranes of keratocytes. No AQP1 staining was seen in corneal epithelium, and no staining was observed in CBCEC layers exposed to AQP3, AQP4, and AQP5 antibodies.

Corneal endothelial NKCC: molecular identification, location, and contribution to fluid transport.

Although Na(+)-K(+)-2Cl(-) cotransport has been demonstrated in cultured bovine corneal endothelial cells, its presence and role in the native tissue have been disputed. Using RT-PCR we have now identified a partial clone of the cotransporter protein in freshly dissected as well as in cultured corneal endothelial and epithelial cells. The deduced amino acid sequence of this protein segment is 99% identical to that of the bovine isoform (bNKCC1). [(3)H]bumetanide binding shows that the cotransporter sites are located in the basolateral membrane region at a density of 1.6 pmol/mg of protein, close to that in lung epithelium. Immunocytochemistry confirms the basolateral location of the cotransporter. We calculate the turnover rate of the cotransporter to be 83 s(-1). Transendothelial fluid transport, determined from deepithelialized rabbit corneal thickness measurements, is partially inhibited (30%) by bumetanide in a dose-dependent manner. Our results demonstrate that Na(+)-K(+)-2Cl(-) cotransporters are present in the basolateral domain of freshly dissected bovine corneal endothelial cells and contribute to fluid transport across corneal endothelial preparations.

Fluid transport by cultured corneal epithelial cell layers.

BACKGROUND/AIMS: Fluid transport across the in vitro corneal epithelium is short lived, hence difficult to detect and characterise. Since stable rates of fluid transport across several cultured epithelial cell layers have been demonstrated, the behaviour of confluent SV40 transformed rabbit corneal epithelial cells (tRCEC) grown on permeable supports was examined. METHODS: Fluid transport was determined with a nanoinjector volume clamp; the specific electrical resistance of the layers was 184 (SEM 9) Omega cm(2). tRCEC layers transported fluid (from basal to apical) against a pressure head of 3 cm H(2)O for 2-3 hours. RESULTS: In the first hour, the rate of fluid transport was 5.2 (0.5) microl/h/cm(-2) (n=23), which is comparable with that found in other epithelia. Fluid transport was completely inhibited in 15-30 minutes by either 100 microM ouabain (n=6), 50 microM bumetanide (n=6), or 1 microM endothelin-1 (ET-1; n=6). Preincubation with 10 microM BQ123 (an ET(A) receptor antagonist) obviated inhibition by ET-1 (n=6). ET-1 also caused a 22% decrease in specific resistance. CONCLUSIONS: Fluid transport appears to depend on transepithelial Cl(- )transport since (1) their directions are the same (stroma-->tear), and (2) both bumetanide and ouabain inhibit it with similar time course. tRCEC appear useful to investigate aspects of the physiology and pharmacology of fluid transport across this layer, including receptor mediated control of this process.

Fluoxetine inhibits K(+) transport pathways (K(+) efflux, Na(+)-K(+)-2Cl(-) cotransport, and Na(+) pump) underlying volume regulation in corneal endothelial cells.

We have studied regulatory volume responses of cultured bovine corneal endothelial cells (CBCEC) using light scattering. We assessed the contributions of fluoxetine (Prozac) and bumetanide-sensitive membrane ion transport pathways to such responses by determining K(+) efflux and influx. Cells swollen by a 20% hypo-osmotic solution underwent a regulatory volume decrease (RVD) response, which after 6 min restored relative cell volume by 98%. Fluoxetine inhibited RVD recovery; 20 microM by 26%, and 50 microM totally. Fluoxetine had a triphasic effect on K(+) efflux; from 20 to 100 microM it inhibited efflux 2-fold, whereas at higher concentrations the efflux first increased to 1.5-fold above the control value, and then decreased again. Cells shrunk by a 20% hyperosmotic solution underwent a regulatory volume increase (RVI) which also after 6 min restored the cell volume by 99%. Fluoxetine inhibited RVI; 20 microM by 25%, and 50 microM completely. Bumetanide (1 microM) inhibited RVI by 43%. In a Cl(-)-free medium, fluoxetine (50-500 microM) progressively inhibited bumetanide-insensitive K(+) influx. The inhibitions of RVI and K(+) influx induced by fluoxetine 20 to 50 microM were similar to those induced by 1 microM bumetanide and by Cl(-)-free medium. A computer simulation suggests that fluoxetine can interact with the selectivity filter of K(+) channels. The data suggest that CBCEC can mediate RVD and RVI in part through increases in K(+) efflux and Na-K-2Cl cotransport (NKCC) activity. Interestingly, the data also suggest that fluoxetine at 20 to 50 microM inhibits NKCC, and at 100-1000 microM inhibits the Na(+) pump. One possible explanation for these findings is that fluoxetine could interact with K(+)-selective sites in K(+) channels, the NKC cotransporter and the Na(+) pump.

Transport of fluid by lens epithelium.

We report for the first time that cultured lens epithelial cell layers and rabbit lenses in vitro transport fluid. Layers of the alphaTN4 mouse cell line and bovine cell cultures were grown to confluence on permeable membrane inserts. Fluid movement across cultured layers and excised rabbit lenses was determined by volume clamp (37 degrees C). Cultured layers transported fluid from their basal to their apical sides against a pressure head of 3 cmH2O. Rates were (in microliter. h-1. cm-2) 3.3 +/- 0.3 for alphaTN4 cells (n = 27) and 4.7 +/- 1.0 for bovine layers (n = 6). Quinidine, a blocker of K+ channels, and p-chloromercuribenzenesulfonate and HgCl2, inhibitors of aquaporins, inhibited fluid transport. Rabbit lenses transported fluid from their anterior to their posterior sides against a 2.5-cmH2O pressure head at 10.3 +/- 0.62 microliter. h-1. lens-1 (n = 5) and along the same pressure head at 12.5 +/- 1.1 microliter. h-1. lens-1 (n = 6). We calculate that this flow could wash the lens extracellular space by convection about once every 2 h and therefore might contribute to lens homeostasis and transparency.