Fluid transport across leaky epithelia: central role of the tight junction and supporting role of aquaporins.

The mechanism of epithelial fluid transport remains unsolved, which is partly due to inherent experimental difficulties. However, a preparation with which our laboratory works, the corneal endothelium, is a simple leaky secretory epithelium in which we have made some experimental and theoretical headway. As we have reported, transendothelial fluid movements can be generated by electrical currents as long as there is tight junction integrity. The direction of the fluid movement can be reversed by current reversal or by changing junctional electrical charges by polylysine. Residual endothelial fluid transport persists even when no anions (hence no salt) are being transported by the tissue and is only eliminated when all local recirculating electrical currents are. Aquaporin (AQP) 1 is the only AQP present in these cells, and its deletion in AQP1 null mice significantly affects cell osmotic permeability (by ∼40%) but fluid transport much less (∼20%), which militates against the presence of sizable water movements across the cell. In 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 we have developed correctly predicts experimental results only when paracellular electro-osmosis is assumed rather than transcellular local osmosis. Our evidence therefore suggests that the fluid is transported across this layer via the paracellular route by a mechanism that we attribute to electro-osmotic coupling at the junctions. From our findings we have developed 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; and 4) an AQP role in regulation rather than as a significant water pathway. These elements are remarkably similar to those proposed by the laboratory of Adrian Hill for fluid transport across other leaky epithelia.

Epithelial Fluid Transport is Due to Electro-osmosis (80%), Plus Osmosis (20%).

Epithelial fluid transport, an important physiological process shrouded in a long-standing enigma, may finally be moving closer to a solution. We propose that, for the corneal endothelium, relative proportions for the driving forces for fluid transport are 80% of paracellular electro-osmosis, and 20% classical transcellular osmosis. These operate in a cyclical process with a period of 9.2 s, which is dictated by the decrease and exhaustion of cellular Na+. Paracellular electro-osmosis is sketched here, and partially discussed as much as the subject still allows; transcellular osmosis is presented at length.

Regulatory volume increase and regulatory volume decrease responses in HL-1 atrial myocytes.

BACKGROUND/AIMS: we have investigated whether cultured cardiomyocytes of the cell line HL-1 have the ability to perform regulatory volume responses both in hypotonic and hypertonic conditions. Furthermore, we characterized those regulatory responses and studied the effects of bumetanide and DIDS in volume regulation of HL-1 cells. METHODS: we used a light scattering system to measure the transient volume changes of HL-1 cells when subjected to osmotic challenge. RESULTS: We found that HL-1 cells correct for their volume excess by undergoing regulatory volume decrease (RVD), and also respond to hypertonic stress with a regulatory volume increase (RVI). Rate of RVD was 0.08 ± 0.04 intensity/min, and rate of RVI was 0.09 ± 0.01 intensity/min. Volume recovery was 83.68 ± 5.73 % for RVD and 92.3 ± 2.3 % for RVI. Bumetanide 50 µM inhibited volume recovery, from 92.3 ± 2.3 % (control) to 24.6 ± 8.8 % and reduced the rate of RVI from 0.070 ± 0.020 intensity/min (control) to 0.010 ± 0.005 intensity/min. 50 µM DIDS reduced volume recovery to 42.93 ± 7.7 % and rate of RVI, to 0.03 ± 0.01 intensity/min. CONCLUSIONS: these results suggest that bumetanide- and DIDS-sensitive mechanisms are involved in the RVI of HL-1 cells, which points to the involvement of the Na(+)/K(+)/2Cl(-) cotransporter and Cl(-)/bicarbonate exchanger in RVI, respectively.

Comparative permeabilities of the paracellular and transcellular pathways of corneal endothelial layers.

Layers of rabbit corneal endothelial cells were cultured on permeable inserts. We characterized the diffusional permeability of the cell layer to nonelectrolyte and charged molecules and compared the diffusional and filtration permeabilities of the paracellular and transcellular pathways. We determined the rates of diffusion of (3)H- and (14)C-labeled nonelectrolyte test molecules and estimated the equivalent pore radius of the tight junction. Negatively charged molecules permeate slower than neutral molecules, while positively charged molecules permeate faster. Palmitoyl-DL-carnitine, which opens tight junctions, caused an increase of permeability and equivalent pore radius. Diffusional water permeability was determined with (3)H-labeled water; the permeabilities of the tight junction and lateral intercellular space were calculated using tissue geometry and the Renkin equation. The diffusional permeability (P(d)) of the paracellular pathway to water is 0.57 µm s(-1) and that of the transcellular path is 2.52 µm s(-1). From the P(d) data we calculated the filtration permeabilities (P(f)) for the paracellular and transcellular pathways as 41.3 and 30.2 µm s(-1), respectively. In conclusion, the movement of hydrophilic molecules through tight junctions corresponds to diffusion through negatively charged pores (r = 2.1 ± 0.35 nm). The paracellular water permeability represents 58% of the filtration permeability of the layer, which points to that route as the site of sizable water transport. In addition, we calculated for NaCl a reflection coefficient of 0.16 ≤ σ(NaCl) ≤ 0.33, which militates against osmosis through the junctions and, hence, indirectly supports the electro-osmosis hypothesis.

A single amino acid residue can determine the sensitivity of SERCAs to artemisinins.

Artemisinins are the most important class of antimalarial drugs. They specifically inhibit PfATP6, a SERCA-type ATPase of Plasmodium falciparum. Here we show that a single amino acid in transmembrane segment 3 of SERCAs can determine susceptibility to artemisinin. An L263E replacement of a malarial by a mammalian residue abolishes inhibition by artemisinins. Introducing residues found in other Plasmodium spp. also modulates artemisinin sensitivity, suggesting that artemisinins interact with the thapsigargin-binding cleft of susceptible SERCAs.

Frequency spectrum of transepithelial potential difference reveals transport-related oscillations.

How epithelia transport fluid is a fundamental issue that is unresolved. Explanations offered include molecular engines, local transcellular osmosis, local paracellular osmosis, and paracellular fluid transport. On the basis of experimental and theoretical work done on corneal endothelium, a fluid transporting epithelium, we suggest electroosmotic coupling at the level of the intercellular junctions driven by the transendothelial electrical potential difference as an explanation of paracellular fluid transport. We collect frequency spectra of that potential difference in real-time. For what we believe is the first time for any epithelium, we report that, unexpectedly, the potential difference displays oscillations at many characteristic frequencies. We also show that on both stimulating cell activity and inhibiting ion transport mechanisms, there are corresponding changes in the oscillations amplitudes that mirror changes known previously in rates of fluid transport. We believe these findings provide a novel tool to study the kinetics of electrogenic elements such as channels and transporters, which from this evidence would give rise to current oscillations with characteristic periods going from 150 ms to 8 s.

Corneal endothelium transports fluid in the absence of net solute transport.

The corneal endothelium transports fluid from the corneal stroma to the aqueous humor, thus maintaining stromal transparency by keeping it relatively dehydrated. This fluid transport mechanism is thought to be driven by the transcellular transports of HCO(3)(-) and Cl(-) in the same direction, from stroma to aqueous. In parallel to these anion movements, for electroneutrality, there are paracellular Na(+) and transcellular K(+) transports in the same direction. The resulting net flow of solute might generate local osmotic gradients that drive fluid transport. However, there are reports that some 50% residual fluid transport remains in nominally HCO(3)(-) free solutions. We have examined the driving force for this residual fluid transport. We confirm that in nominally HCO(3)(-) free solutions, 48% of control fluid transport remains. When in addition Cl(-) channels are inhibited, 30% of control fluid movement still remains. Addition of a carbonic anhydrase inhibitor has no further effect. These manipulations combined inhibit the transcellular transport of all anions, without which there cannot be any net transport of solute and consequently no local osmotic gradients, yet there is residual fluid movement. Only the further addition of benzamil, an inhibitor of epithelial Na(+) channels, abolishes fluid transport completely. Our data are inconsistent with transcellular local osmosis and instead support the paradigm of paracellular fluid transport driven by electro-osmotic coupling.

Diquafosol tetrasodium. Inspire/Allergan/Santen.

Inspire, in collaboration with Allergan and Santen, is developing an eye-drop formulation of diquafosol tetrasodium (INS-365), a second-generation uridine nucleotide analog P2Y, receptor agonist for the potential treatment of dry eye disease. In June 2003, Inspire submitted an NDA for the treatment of dry eye, and in July 2003 the FDA granted the NDA Priority Review status. FDA action is expected in December 2003, and in January 2003 launch was expected in the first half of 2004.

Water channels and their roles in some ocular tissues.

Water is a major component of the eye, and water channels (aquaporins) are ubiquitous in ocular tissues, and quite abundant at their different locations. AQP1 is expressed in corneal endothelium, lens epithelium, ciliary epithelium, and retinal pigment epithelium. AQP3 is expressed in corneal epithelium, and in conjunctival epithelium. AQP4 is expressed in ciliary epithelium and retinal Muller cells. AQP5 is expressed in corneal epithelium, and conjunctival epithelium. AQP0 is expressed in lens fiber cells. It is known that five ocular tissues transport fluid, namely: (1) Corneal endothelium; (2) Conjunctival epithelium; (3) Lens epithelium; (4) Ciliary epithelium; (5) Retinal pigment epithelium. For the corneal endothelium, aquaporins are not the main route for trans-tissue water movement, which is paracellular. Instead, we propose that aquaporins allow fast osmotic equilibration of the cell, which is necessary to maintain optimal rates of fluid movement since the cyclic paracellular water transfer mechanism operates separately and tends to create periodic osmotic imbalances (τ∼5 s).

Modulation of tight junction properties relevant to fluid transport across rabbit corneal endothelium.

Paracellular junctions could play an important role in corneal endothelial fluid transport. In this study we explored the effects of different reagents on the tight junctional barrier by assessing the translayer specific electrical resistance (TER) across rabbit corneal endothelial preparations and cultured rabbit corneal endothelial cells' (CRCEC) monolayers, the paracellular permeability (Papp) for fluorescein isothiocyanate (FITC) dextrans across CRCEC, and fluid transport across de-epithelialized rabbit corneal endothelial preparations. Palmitoyl carnitine (PC), poly-L-lysine (PLL), adenosine triphosphate (ATP), and dibutyryl adenosine 3',5'-cyclic monophosphate (dB-cAMP) were used to modulate corneal endothelial fluid transport and tight junctions (TJs). After seeding, the TER across CRCEC reached maximal values (29.2+/-1.0 Omega cm2) only after the 10th day. PC (0.1 mM) caused decreases both in TER (by 40%) and fluid transport (swelling rate: 18.5+/-0.3 microm/h), and an increase in Papp. PLL resulted in increased TER rose and Papp but decreased fluid transport (swelling rate: 10+/-0.3 microm/h). dB-cAMP (0.1 mM) and ATP (0.1 mM) decreased TER by 16% and 6%, increased Papp slightly, and stimulated fluid transport; the rates of de-swelling (in microm/h) were -5.4+/-0.3 and -12.1+/-0.4, respectively. PC might cause the junctions to open up unspecifically and thus increase passive leak. PLL is a known junctional charge modifier that may be adding steric hindrance to the tight junctions. The results with dB-cAMP and ATP are consistent with fluid transport via the paracellular route.

Lack of threshold for anisotonic cell volume regulation.

Most cells possess mechanisms that are able to detect cellular volume shifts and to signal the initiation of appropriate volume regulatory responses. However, the identity and characteristics of the detecting mechanism remain obscure. In this study, we explored the influence of hypertonic and hypotonic challenges of varying magnitude on the characteristics of the ensuing regulatory volume increase (RVI) and regulatory volume decrease (RVD) of cultured bovine corneal endothelial cells (CBCECs). The main question we asked was whether a threshold of stimulation existed that would unleash a regulatory response. CBCECs (passage 1-3) were seeded on rectangular glass coverslips and grown for 1-2 days. We used a procedure based on detection of light scattering to monitor the transient volume changes of such plated cells when subjected to osmotic challenge. The osmometric responses were asymmetric: cells shrank faster than they swelled (by a factor of 3). Complete volume regulatory responses took 10-12 min. Bumetanide (50 microM) resulted in incomplete (50%) RVI. We found no threshold as the cells examined responded to hypertonic and hypotonic stimuli as low as 1%. There was some gradation as stimuli of <4% resulted in incomplete volume regulation. The degree of activation of the volume responses grew as an exponential buildup with the strength of the anisotonic challenge. We discuss how our observations are consistent with volume sensing mechanisms based on both ionic strength and the cytoskeleton.

Bench meets bedside: a 10-year-old girl and amino acid residue glycine 75 of the facilitative glucose transporter GLUT1.

In 2000, amino acid residue G75 of the facilitative glucose transporter GLUT1 was identified by mutagenesis as being essential for transport function [Olsowski, A., et al. (2000) Biochemistry 39, 2469-74]. In 2002, we identified a heterozygous missense mutation substituting glycine at residue 75 for tryptophan in a 10-year-old girl with intractable seizures and low glucose concentrations in the cerebrospinal fluid indicative of GLUT1 deficiency. Glucose uptake into erythrocytes of the patient was 36% of controls, and GLUT1-specific immunoreactivity was normal, indicating a functional GLUT1 defect. In silico three-dimensional modeling of the G75W mutant provided a smaller gyration radius for transmembrane segment 2 as the potential pathogenic mechanism in this patient. This case illustrates a GLUT1 mutation characterized in vitro and later confirmed by disease itself and highlights the potential of basic science and clinical medicine to collaborate for the benefit of patients.

Identification of a hydrophobic residue as a key determinant of fructose transport by the facilitative hexose transporter SLC2A7 (GLUT7).

Until recently, the only facilitated hexose transporter GLUT proteins (SLC2A) known to transport fructose were GLUTs 2 and 5. However, the recently cloned GLUT7 can also transport fructose as well as glucose. Comparison of sequence alignments indicated that GLUTs 2, 5, and 7 all had an isoleucine residue at position "314" (GLUT7), whereas the non-fructose-transporting isoforms, GLUTs 1, 3, and 4, had a valine at this position. Mutation of Ile-314 to a valine in GLUT7 resulted in a loss of fructose transport, whereas glucose transport remained completely unaffected. Similar results were obtained with GLUTs 2 and 5. Energy minimization modeling of GLUT7 indicated that Ile-314 projects from transmembrane domain 7 (TM7) into the lumen of the aqueous pore, where it could form a hydrophobic interaction with tryptophan 89 from TM2. A valine residue at 314 appeared to produce a narrowing of the vestibule when compared with the isoleucine. It is proposed that this hydrophobic interaction across the pore forms a selectivity filter restricting the access of some hexoses to the substrate binding site(s) within the aqueous channel. The presence of a selectivity filter in the extracellular vestibule of GLUT proteins would allow for subtle changes in substrate specificity without changing the kinetic parameters of the protein.

Regulation of Na-K-2Cl cotransport in cultured bovine corneal endothelial cells.

We have previously demonstrated the presence of a Na(+)-K(+)-2Cl cotransporter in cultured bovine corneal endothelial cells (CBCEC) and determined that this cotransporter is located in the basolateral membrane. This transporter may contribute to volume regulation and transendothelial fluid transport. We have now investigated factors regulating the activity of the cotransporter. This activity was assessed by measuring the bumetanide-sensitive (86)Rubidium ((86)Rb) uptake in (86)Rb-containing solutions. Data were normalized to protein content determined with a Lowry protein assay. We investigated the regulation by extracellular and intracellular ion concentrations, by osmotic gradients, and by second messengers. Our results indicate that extracellular Na+ and K+ each are required for activation of the cotransporter and activate with first-order kinetics at half-maximally effective concentrations (k(1/2)) of 21.1 and 1.33 mM, respectively. Extracellular Cl- is also required for cotransport activation, but shows higher order kinetics; the k(1/2) for Cl- is 28.1 mM and the Hill coefficient 2.1. HCO(3)(-) exerts a modulating effect on cotransporter activity; at 0 HCO(3)(-) the bumetanide-sensitive K(+) uptake is reduced by 30% compared to that at 26 mm HCO(3)(-). Manipulations of the intracellular [Cl-] by preincubation in Cl- -free solution or inhibition of Cl- efflux resulted in increased uptake at low [Cl-](i) and decreased uptake at high [Cl-](i). To assess the role of protein kinases in the regulation of cotransport, we have determined the effect of protein kinase inhibitors. H-89 and KT5270, inhibitors of PKA, inhibit cotransport almost completely, while calphostin C, an inhibitor of PKC, produces a small activation of cotransport. The tyrosine kinase inhibitor genistein reduced K+ uptake while its inactive analog daidzein was without effect. The calmodulin kinase inhibitor KN-93 was without effect. We also investigated the effects of phosphatase inhibitors. Calyculin A (k(1/2)=21 nM) and okadaic acid (k(1/2)=915 nM) produced approximate doubling of K+ uptake, suggesting that phosphatase 1 is dominant. We also investigated the role of the cytoskeleton and its activation. Reduction of Ca(i)(2+) by preincubation in Ca2+ -free medium as well as by exposure to W-7, an inhibitor of the binding of Ca(2+) to calmodulin, reduced K+ uptake. Consistent with this, ML-7, a relatively specific inhibitor of the Ca2+ -calmodulin activated myosin light chain kinase, inhibited cotransport by 40%. The Ca2+ -calmodulin activated myosin light chain kinase contributes to the modulation of the cytoskeleton by regulating the actin-myosin interaction. Consistent with the above, disruption of the actin polymerization by cytochalasin D led to a decrease in K+ uptake. We conclude that extracellular Na+, K+ and Cl- are requirements for the function of the CBCEC Na(+)-K(+)-2Cl(-) cotransporter, while intracellular Cl- and extracellular HCO(3)(-) modulate its activity. Several protein kinases, including PKA, PKC, tyrosine kinase, and myosin light chain kinase, modulate the K+ uptake. Another modulating pathway for cotransport involves the state of the cytoskeleton.

Redefining the facilitated transport of mannose in human cells: absence of a glucose-insensitive, high-affinity facilitated mannose transport system.

Current evidence suggests that extracellular mannose can be transported intracellularly and utilized for glycoprotein synthesis; however, the identity and the functional characteristics of the transporters of mannose are controversial. Although the glucose transporters are capable of transporting mannose, it has been postulated that the entry of mannose in mammalian cells is mediated by a transporter that is insensitive to glucose [Panneerselvam, K., and Freeze, H. (1996) J. Biol. Chem. 271, 9417-9421] or by a transporter induced by cell treatment with metformin [Shang, J., and Lehrman, M. A. (2004) J. Biol. Chem. 279, 9703-9712]. We performed a detailed analysis of the uptake of mannose in normal human erythrocytes and in leukemia cell line HL-60. Short uptake assays allowed the identification of a single functional activity involved in mannose uptake in both cell types, with a K(m) for transport of 6 mM. Transport was inhibited in a competitive manner by classical glucose transporter substrates. Similarly, the glucose transporter inhibitors cytochalasin B, genistein, and myricetin inhibited mannose transport by 100%. Using long uptake experiments, we identified a second, high-affinity component associated with the intracellular trapping of mannose in the HL-60 cells that is not directly involved in the transport of mannose via the glucose transporters. Thus, the transport of mannose via glucose transporters is a process which is kinetically and biologically separable from its intracellular trapping. A general survey of human cells revealed that mannose uptake was entirely blocked by concentrations of cytochalasin B that obliterates the activity of the glucose transporters. The transport and inhibition data demonstrate that extracellular mannose, whose physiological concentration is in the micromolar range, enters cells in the presence of physiological concentrations of glucose. Overall, our data indicate that transport through the glucose transporter is the main mechanism by which human cells acquire mannose.

On the mechanism of fluid transport across corneal endothelium and epithelia in general.

The mechanism by which fluid is transported across epithelial layers is still unclear. The prevalent idea is that fluid traverses these layers transcellularly, driven by local osmotic gradients secondary to electrolyte transport and utilizing the high osmotic permeability of aquaporins. However, recent findings that some aquaporin knockout mice epithelia transport fluid sow doubts on local osmosis. This review discusses recent evidence in corneal endothelium that points instead to electro-osmosis as the mechanism underlying fluid transport. In this concept, a local recirculating electrical current would result in electro-osmotic coupling at the level of the intercellular junctions, dragging fluid via the paracellular route. The text also mentions possible mechanisms for apical bicarbonate exit from endothelial cells, and discusses whether electro-osmosis could be a general mechanism.

An update on corneal hydration control.

An account is provided of developments in our understanding of the mechanism of corneal hydration control, particularly as regards the possibility of an active system for its regulation. Emphasis is given to issues that are contentious, such as the role of bicarbonate in the endothelial pump and the significance of water channels in both corneal limiting cell layers.

Effects of anticonvulsants on GLUT1-mediated glucose transport in GLUT1 deficiency syndrome in vitro.

Facilitative type-1 glucose transporter (GLUT1) deficiency syndrome is caused by a defect of glucose transport into brain, resulting in an epileptic encephalopathy. Seizures respond effectively to a ketogenic diet, but a subgroup of patients require add-on anticonvulsant therapy or do not tolerate the diet. With the exception of barbiturates, which have been shown to inhibit GLUT1 function, no anticonvulsants have been investigated for possible interactions with GLUT1. Kinetic analyses of (14)C-labeled 3-O-methyl glucose (3OMG) uptake into erythroctes were performed in 11 patients and 30 controls. For in vitro inhibition studies, zero-trans influx of 3OMG (5 mmol/L) into erythrocytes was determined following preincubation with diazepam, carbamazepine, phenytoin, and chloralhydrate. In addition, the effects of ethanol on cell lysis and 3OMG transport into erythrocytes were determined. In patients, mean 3OMG influx was 53% of controls. Ethanol, diazepam, and chloralhydrate significantly inhibited GLUT1 function. Erythrocyte cell lysis was evident at concentrations of 2.5% ethanol. Diazepam, chloralhydrate, and ethanol are inhibitors of GLUT1 function in vitro and might potentiate the effects of GLUT1-mediated glucose transport in patients with GLUT1 deficiency syndrome. In contrast, no inhibitory effects were observed for carbamazepine and phenytoin, indicating that these substances might be preferable for additional seizure control in this disorder.