Adenosine stimulation of fluid transport across rabbit corneal endothelium

Summary The rate of fluid transport across rabbit corneal endothelium has been measured with an automatic volumetric method. The present resolution of the procedure is 1–3 nanoliters, and intervals of measurement can be made as small as seconds. In the presence of glucose, oxidized glutathione (GSSG), and adenosine, the maximal rates were 6.2±1.0 l/hr cm², and 8.2±0.8 l/hr cm² if a large portion of the stroma was dissected away. In the presence of glucose and GSSG only, the rates were lower, namely 3.7±0.5 l/hr cm². The rates consistently increased or decreased when adenosine was added or deleted, respectively, during given experiments. The stimulation of fluid transport by adenosine was in the order of 40–50%. The results raise the possibility that this transport mechanism might be subject to metabolic control.     

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.     

A central role for cell osmolarity in isotonic fluid transport across epithelia

Previous theoretical models for solute-solvent coupling in epithelia that dealt only with the intercellular channel did not predict isotonic transport except when very high cell membrane permeabilities were assumed. To study this issue, we have developed the formalisms for osmotic equilibration at an alternative location, the apical cell membrane (including its adjacent unstirred layer), which are somewhat simpler than those for the channel. Much as in other models, we confirm that only rather unrealistically high values of the cell membrane permeability lead to isotonic transport. We have also found, however, that isotonic transport can occur at much lower values of the cell membrane permeability if the concentration within the cell differs slightly from that in the ambient medium. This emphasizes the importance of incorporating the intracellular concentration as an integral part to any transport model, such as in the present apical membrane version of local osmosis.     

The role of corneal endothelium in macular corneal dystrophy development and recurrence

Macular corneal dystrophy(MCD) is a progressive,bilateral stromal dystrophic disease that arises from mutations in carbohydrate sulfotransferase 6(CHST6).Corneal transplantation is the ultimate therapeutic solution for MCD patients.Unfortunately,postoperative recurrence remains a significant challenge.We conducted a retrospective review of a clinical cohort comprising 102 MCD patients with 124 eyes that underwent either penetrating keratoplasty(PKP) or deep anterior lamellar keratoplasty(DALK).O...     

Priming of the fluid pump by osmotic gradients across rabbit corneal endothelium

The present study shows that the inclusion of 5% Dextran (average mol. wt. 40 000) in solutions to preserve in vitro rabbit corneal endothelium induces a sizable osmotic flow across the preparation which is superimposed on the existing fluid transport. Furthermore, even after fluid transport ceases due to in vitro deterioration, the Dextran-induced flow remains for some addition time. The osmotic permeability was 162 +/- 17 micrometer/s in the presence of glucose and 451 +/- 84 micrometer/s in its absence. The latter, comparatively high value suggests that such osmotic flow traverses the intracellular junctions. In addition, temporary (10--15 min) imposition of an osmotic gradient has a separate stimulatory 'priming' effect on the rate of fluid transport. Thus, the rate of fluid pumping increased by about 40% after challenge with Dextran. It was further noted that, after addition of Dextran, preparations in the absence of glucose escape gross deterioration for a time longer than those in the presence of glucose. On the other hand, mere addition of Dextran to a glucose-containing solution does not appear to prolong the estimated 'survival time' of the pumping mechanism. The sizable osmotic flows and the priming effect described here may provide a physiological context with which previously described Dextran effects on cornea preservation can now be compared.     

A model of epithelial water transport. The corneal endothelium.

To try to understand how an epithelial tissue can transport water between bathing solutions of equal tonicity and how intracellular solute and protein concentration are related to the structural specialization of the cell membrane at its apical, basal, and lateral margins, we have formulated and solved, using approximate analytical techniques, a new model which combines the detailed transport of local osmotic flow in extracellular channel with the multicompartment approach of thermodynamic models requiring the overall conservation of water and solute for the entire cell layer. Thus, unlike most previous models, which dealt exclusively with either the average properties of the cell layer or the local transport in the extracellular channel, we are able to solve simultaneously for the interaction of the cell with its environments across its apical, basal, and lateral cell membranes as well as the detailed transport in the extracellular channel. The model is then applied to corneal endothelium to obtain new insight into the water flow movement in this tissue under in vitro and in vivo conditions. Then in vitro solution shows that the cell at 297 mosmol/liter is slightly hypotonic to the 300-mosmol/liter external bathing solutions which drive water equally out both the aqueous (apical) and stromal (basal) cell faces. This water is replaced from the extracellular channel. There is a net flow of water because more water enters the channel through its open stromal end than through the higher resistance tight junction. In vivo, the solution predicts that the stromal swelling pressure forces water through the tight junctions towards the stroma so that there is no net flow. The interesting new features of our solution are the water recirculation pattern and the role of the osmotically active proteins in making the cell hypertonic relative to the channel.     

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.     

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 HCO₃⁻ 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 HCO₃⁻ free solutions. We have examined the driving force for this residual fluid transport. We confirm that in nominally HCO₃⁻ 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.     

Fluid transport, ATP level and ATPase activities in isolated rabbit corneal endothelium

The effect of certain biochemical parameters on transendothelial fluid transport has been studied. Cellular ATP level and (Na⁺ + K⁺)-activated as well as Mg²⁺-activated ATPase activities were measured by ultramicrotechniques using individual rabbit corneal endothelium after they had been subjected to in vitro perfusion with solutions fully supplemented or deficient singly or severally in glucose, adenosine and glutathione (GSH). With the complete medium, the transport system operates in vitro for approx. 6 h. Deletion of glucose alone, glucose and adenosine or glucose, adenosine and GSH brings about a cessation of fluid transport after 3.5 h, 2 to 2.5 h and 0.5 to 1 h, respectively. A marked decrease (62%) of the endothelial ATP level, however, occurs only when all metabolites are omitted. The favorable effect of GSH on transport activity is attributable to its capacity to sustain cellular ATP rather than to protect the functionality of (Na⁺ + K⁺)-activated ATPase. Adenosine, in the presence of GSH, maintains normal ATP levels and, additionally, exerts a protective effect on Mg²⁺-activated ATPase and possibly also on (Na⁺ + K⁺)-activated ATPase.     

Evidence for an accessory role of LFA-1 in lymphocyte-high endothelium interaction during homing

In a variety of lymphocyte interactions, lymphocyte function-associated antigen-1 (LFA-1) plays an important role as an accessory mechanism mediating cell adhesion. We tested the possibility that LFA-1 could also be involved in the specific binding of lymphocytes to high endothelial venules (HEV) during homing. Antibodies against LFA-1 but not against various other cell surface molecules (except the putative gp90 homing receptor defined by the MEL-14 antibody) were found to inhibit in vitro adherence of lymphocytes to HEV in frozen sections of lymph nodes. Binding of T cell lines to HEV was also inhibited by anti-LFA-1 antibody. Using sublines selected for differential expression of the MEL-14 antigen, MEL-14 high cells (which bind well to HEV) were less susceptible to inhibition by anti-LFA-1 than poor binders with low levels of the homing receptor, supporting the model of LFA-1 being an accessory mechanism strengthening weak interactions between cells. Parallel results were found in vivo where anti-LFA-1 antibodies reduced the migration of normal lymphocytes into lymph nodes and Peyer's patches by 40 to 60%. Localization in the lung, especially of activated lymphocytes, was also impaired, although to a lesser extent. These findings suggest that LFA-1 plays an accessory role in cellular interactions relevant for lymphocyte migration.     

Potassium channel in rabbit corneal endothelium activated by external anions

Summary The apical membrane of the rabbit corneal endothelium contains a potassium-selective ionic channel. In patch-clamp recordings, the probability of finding the channel in the open state (P _o) depends on the presence of either HCO ₃ ^– or Cl^– in the bathing medium. In a methane sulfonate-containing bath,P _o is <0.05 at all physiologically relevant transmembrane voltages. With 0mm [HCO ₃ ^– ] _o at +60 mV,P _o was 0.085 and increased to 0.40 when [HCO ₃ ^– ] _o was 15mm. With 4mm [Cl^–] _o at +60 mV,P _o was 0.083 and with 150mm Cl^–,P _o increased to 0.36. LowP _o's are also found when propionate, sulphate, bromide, and nitrate are the primary bath anions. The mechanism of action of the anion-stimulated K⁺ channel gating is not yet known, but a direct action of pH seems unlikely. The alkalinization of cytoplasm associated with the addition of 10mm (NH₄)₂SO₄ to the bath and the acidification accompanying its removal do not result in channel activation nor does the use of Nigericin to equilibrate intracellular pH with that of the bath over the pH range of 6.8 to 7.8. Channel gating also is not affected by bathing the internal surface of the patch with cAMP, cGMP, GTP--s, Mg²⁺ or ATP. Blockers of Na/H⁺ exchange, Na⁺–HCO ₃ ^– cotransport, Na⁺–K⁺ ATPase and carbonic anhydrase do not block the HCO ₃ ^– stimulation ofP _o. Several of the properties of the channel could explain some of the previously reported voltage changes that occur in corneal endothelial cells stimulated by extracellular anions.     

Evidence for a direct role of tRNA in an amino acid transport system.

The transport of phenylalanine by the general aromatic transport system in spheroplasts of Escherichia coli 9723 has been found to be stimulated by exogenous tRNA. Neither periodate-treated tRNA nor phenylalanine-charged tRNA stimulated, and the latter inhibited, phenylalanine uptake. Among preparations of specific tRNAs, tRNAPhe and tRNATyr were effective in stimulating the uptake of phenylalanine and tyrosine, respectively, and tRNAGlu and tRNAVal gave no detectable stimulation of phenylalanine or tyrosine transport. The preparation of tRNATyr was 10 times as active as unfractionated tRNA and gave as much as 167% stimulation of tyrosine transport. Correspondingly, the preparation of tRNAPhe was at least 3.5 times as active as the unfractionated tRNA and 2.5 times as active as the preparation of tRNATyr in stimulation of phenylalanine transport. Preliminary results in fractionation of the active component of tRNA for stimulating phenylalanine uptake show that the major activity resides in minor isoacceptor(s) tRNAPhe rather than the major component tRNAPhe, and the slight activity of preparations of tRNATyr is probably due to a contamination of the active tRNAPhe. Other preliminary results indicate that this type of stimulation occurs with uptake of other amino acids and their tRNA.     

Evidence for a role of a vicinal dithiol in the transport of gamma-butyrobetaine in Agrobacterium sp

An Agrobacterium sp. isolated from soil is able to use gamma-butyrobetaine as its sole source of carbon and nitrogen. The involvement of thiol groups for active transport of gamma-butyrobetaine was investigated by use of the thiol alkylating reagent N-ethylmaleimide (NEM) and the dithiol specific reagent phenylarsine oxide (PAO). Both reagents strongly inhibited gamma-butyrobetaine uptake, but also induced the release of the accumulated substrate, suggesting that the transport system either contains a dithiol-dependent protein or that a small thiol-containing molecule is implicated in the uptake phenomenon.     

Evidence for coupled transport of bicarbonate and sodium in cultured bovine corneal endothelial cells

Summary Usin gintracellular microelectrode technique, the response of the voltageV across the plasma membrane of cultured bovine corneal endothelial cells to changes in sodium and bicarbonate concentrations was investigated. (1) The electrical response to changes in [HCO ₃ ^– ] _o (depolarization upon lowering and hyperpolarization upon raising [HCO ₃ ^– ] _o ) was dependent on sodium. Lithium could fairly well be substituted for sodium, whereas potassium or choline were much less effective. (2) Removal of external sodium caused a depolarization, while a readdition led to a hyperpolarization, which increased with time of preincubation in the sodium-depleted medium. (3) The response to changes in [Na⁺] _o was dependent on bicarbonate. In a nominally bicarbonate-free medium, its amplitude was decreased or even reversed in sign. (4) Application of SITS or DIDS (10^–3 m) had a similar effect on the response to sodium as bicarbonate-depleted medium. (5) At [Na⁺] _o =151mm and [HCO ₃ ^– ] _o =46mm, the transients ofV depended, with 39.0±9.0 (sd) mV/decade, on bicarbonate and, with 15.3±5.8 (sd) mV/decade, on sodium. (6) After the preincubation of cells with lithium, replacement of Li by choline led to similar effects as the replacement of sodium by choline, though the response ofV was smaller with Li. This response could be reduced or reversed by the removal of bicarbonate or by the application of SITS. (7) Amiloride (10^–3 m) caused a reversible hyperpolarization of the steady-state potential by 8.5±2.6 mV (sd). It did not affect the immediate response to changes in [Na⁺] _o or [HCO ₃ ^– ] _o , but reduced the speed of regaining the steady-state potential after a change in [HCO ₃ ^– ] _o . (8) Ouabain (10^–4 m) caused a fast depolarization of –6.8±1.1 (sd) mV, which was followed by a continuing slower depolarization. The effect was almost identical at 10^–5 m. (9) It is suggested, that corneal endothelial cells possess a cotransport for sodium and bicarbonate, which transports net negative charage with these ions. It is inhibitable by stilbenes, but not directly affected by amiloride or ouabain. Lithium is a good substitute for sodium with respect to bicarbonate transport and is transported itself. In addition, the effect of amiloride provides indirect evidence for the existence of a Na⁺/H⁺-antiport. A model for the transepithelial transport of bicarbonate across the corneal endothelium is proposed.     

Evidence for a role of NisT in transport of the lantibiotic nisin produced by Lactococcus lactis N8

Abstract The biosynthesis, immunity and regulation of nisin, a lanthionine-containing antimicrobial peptide produced by Lactococcus lactis , is encoded by two gene clusters, nisAIZBTCIPRK and nisFEG . The mutant strain LAC46 with a deletion in the translocator gene nisT could not secrete nisin but nisin activity was detected from cell lysates. The nisT mutation was complemented by a NisT-expression plasmid resulting in restored capacity to secrete nisin. These results demonstrate that NisT is the transport protein dedicated to translocate nisin and that dehydration and lanthionine formation in nisin maturation can occur independently of transport.