F.P.J. Diecke

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.

A Mathematical Model of Electrolyte and Fluid Transport Across Corneal Endothelium

To predict the behavior of a transporting epithelium by intuitive means can be complex and frustrating. As the number of parameters to be considered increases beyond a few, the task can be termed impossible. The alternative is to model epithelial behavior by mathematical means. For that to be feasible, it has been presumed that a large amount of experimental information is required, so as to be able to use known values for the majority of kinetic parameters. However, in the present case, we are modeling corneal endothelial behavior beginning with experimental values for only five of eleven parameters. The remaining parameter values are calculated assuming cellular steady state and using algebraic software. With that as base, as in preceding treatments but with a distribution of channels/transporters suited to the endothelium, temporal cell and tissue behavior are computed by a program written in Basic that monitors changes in chemical and electrical driving forces across cell membranes and the paracellular pathway. We find that the program reproduces quite well the behaviors experimentally observed for the translayer electrical potential difference and rate of fluid transport, (a) in the steady state, (b) after perturbations by changes in ambient conditions HCO3−, Na+, and Cl− concentrations), and (c) after challenge by inhibitors (ouabain, DIDS, Na+- and Cl−-channel inhibitors). In addition, we have used the program to compare predictions of translayer fluid transport by two competing theories, electro-osmosis and local osmosis. Only predictions using electro-osmosis fit all the experimental data.

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.

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.

Presynaptic modulation of sympathetic neurotransmitter release by modulators of cyclic 3',5'-guanosine monophosphate in canine vascular smooth muscle.

Extracellular calcium ion is required for neurotransmitter release consequent to electrical and receptor-mediated stimulation of sympathetic and other neurons. A large number of substances are known to modify the quanta of norepinephrine (NE) released per nerve impulse. It has been proposed that cyclic 3’5’-adenosine monophosphate (CAMP) may enhance, and endogenous cyclic 3’,5’-guanosine monophosphate (cGMP) may inhibit, neurotransmitter release. However, no consistent relationship between the effects of the several modulators of neurally mediated N E release and their effects on adenylate and guanylate cyclase is as yet apparent. This research has been hampered by the relative absence of techniques that allow the measurement of cyclic nucleotide levels in the presynaptic sympathetic nerve terminal after exposure to the putative modulators of release and consequent to nerve stimulation. Although intraneuronal cGMP or CAMP still has not been measured in intact peripheral sympathetic nerve terminals, new information on the effects of modulators of CAMP and cGMP on sympathetic neuroeffector transmission has emerged through the use of pharmacologic modulators of the cyclic nucleotides system. This review evaluates the role of CAMP and cGMP in sympathetic neurotransmitter release to vascular smooth muscle (VSM) and from adrenal chromaffin cells. The concept that emerges is that cGMP, in addition to its role as a directly acting smooth muscle relaxant, may decrease smooth muscle tone by acting as a physiologic feedback inhibitory modulator of the release of the sympathetic neurotransmitters N E and epinephrine (EPI). The release of epinephrine and norepinephrine from adrenal medullary cells by depolarization and acetylcholine (ACh), and €rom sympathetic nerve terminals innervating peripheral vascular smooth muscle during sympathetic nerve stimulation (SNS),

Interneuronal Cyclic GMP and ‘EDRF-like Substance’ Modulate Norepinephrine Release from Peripheral Sympathetic Nerves

ABSTRACT Stimulators of cyclic 3′5′ guanosine monophosphate (cGMP) or release of endothelium-derived relaxing factor (EDRF) inhibit sympathetic neurotransmission (SNS) to canine mesenteric (MA) and porcine carotid arteries (CA). Inhibitors of guanylate cyclase and 1-N-monomethylarginine (LNMMA) enhance contractions to SNS and the calcium-dependent efflux of 2- 14 C-norepinephrine (NE). In endotheliumdamaged porcine CA the responses to SNS, but not NE, are enhanced by LNMMA and inhibited by difluoromethylornithine (DFMO) an inhibitor of ornithine decarboxylase which shunts ornithine into endogenous arginine via citrulline. Thus, intraneuronal EDRF-like substance and cyclic GMP may act as endogenous inhibitory feedback modulators of calcium-dependent release of sympathetic neurotransmitter in vascular smooth muscle.