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.

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).

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.

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.

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.

Nicotinamide is not a substrate of the facilitative hexose transporter GLUT1.

It has been proposed that GLUT1, a membrane protein that transports hexoses and the oxidized form of vitamin C, dehydroascorbic acid, is also a transporter of nicotinamide (Sofue, M., Yoshimura, Y., Nishida, M., and Kawada, J. (1992) Biochem. J. 288, 669-674). To ascertain this, we studied the transport of 2-deoxy-D-glucose, 3-O-methyl-D-glucose, and nicotinamide in human erythrocytes and right-side-out and inside-out erythrocyte membrane vesicles. The transport of nicotinamide was saturable, with a K(M) for influx and efflux of 6.1 and 6.2 mM, respectively. We found that transport of the hexoses was not competed by nicotinamide in both the erythrocytes and the erythrocyte vesicles. Likewise, the transport of nicotinamide was not affected by hexoses or by inhibitors of glucose transport such as cytochalasin B, genistein, and myricetin. On the other hand, nicotinamide blocked the binding of cytochalasin B to human erythrocyte membranes but did so in a noncompetitive manner. Using GLUT1-transfected CHO cells, we demonstrated that increased expression of GLUT1 was paralleled by a corresponding increase in hexose transport but that there were no changes in nicotinamide transport. Moreover, nicotinamide failed to affect the transport of hexoses in both control and GLUT1-transfected CHO cells. Therefore, our results indicates that GLUT1 does not transport nicotinamide, and we propose instead the existence of other systems for the translocation of nicotinamide across cell membranes.