Anthony D. C. Macknight

Relationships between serosal medium potassium concentration and sodium transport in toad urinary bladder: III. Exchangeability of epithelial cellular potassium

SummaryThe exchangeability of toad bladder epithelial cell potassium has been investigated. An insignificant amount of cellular potassium exchanged with mucosal medium42K. From the rate of uptake of42K into the cells from the serosal medium at least two cellular potassium pools were identified. The more rapidly exchanging pool contained about one-quarter to one-third of the cellular potassium and exchanged with a half-time of about 30 min. It was from this pool that potassium was lost from cells exposed to ouabain or to a potassium-free medium. In addition, when 3.5mm rubidium replaced 3.5mm potassium in sodium Ringers the epithelial cells lost in 60 min about one-quarter of their cellular potassium in exchange for rubidium. Inhibition of transepithelial sodium transport by amiloride, 10−5mm, seemed to depress the rate of potassium uptake into the more rapidly exchanging pool without affecting total cellular potassium content. However, stimulation of transepithelial sodium transport by vasopressin appeared not to affect the rate of potassium uptake. The rate of potassium uptake into this pool seemed much less than that required for a tight 1∶1 coupling between transepithelial sodium transport and potassium uptake. The remaining cellular potassium exchanged at a much slower rate and even after 19 hours of incubation only 67% of cellular potassium was labelled. If this slower exchanging potassium represents a single pool, 99% of cellular potassium would be labelled only after incubation with42K for 56 hours.

Effects of vasopressin on the water and ionic composition of toad bladder epithelial cells

SummaryIsolated sheets of epithelial cells as well as epithelial cells scraped from paired hemibladders mounted in chambers both showed significant increases in water, sodium and chloride contents after exposure to vasopressin (100 mU/ml), without any change in potassium content. In the isolated cells these changes were prevented by amiloride (10−5m), suggesting that the gain of sodium after vasopressin occurs across the mucosal membrane. This hypothesis was confirmed in experiments in which it was found that, in hemibladders mounted in chambers and bathed on their mucosal surface by sodium Ringers with24Na, the gains of chemical sodium and24Na after vasopressin were equivalent.

Measurement of the composition of epithelial cells from the toad urinary bladder.

SummaryTwo methods are described by which epithelial cells from toad urinary bladders can be obtained for analysis of their intracellular water and electrolyte contents. In the first, a method similar to that described in 1968 by J. T. Gatzy and W. O. Berndt, sheets of epithelial cells are scraped from bladders after incubation in sodium Ringers and collagenase (400 mg/liter). The scraped cells were incubated under various conditions and their composition subsequently determined. Oxygen consumption was also measured. In the second method, epithelial cells were scraped from hemibladders removed from chambers. These cells were then analyzed without further incubation. The morphology of epithelial cells obtained by each method is illustrated. Both methods yield similar results and evidence is provided that the derived intracellular values obtained truly reflect the composition of the epithelial cells.

The site of the aldosterone induced stimulation of sodium transport

Abstract The content and concentration of major ions within the scraped mucosal epithelial cells of the urinary bladder of the toad Bufo marinus were determined using [ 14 C]- and [ 3 H]-inulin to correct for adherent extracellular fluid. In aldosterone treated hemi-bladders the radioactive sodium content and concentration increased significantly as compared with paired control hemi-bladders when both tissues were exposed to [ 24 Na] in the mucosal bathing medium. Changes in total non-inulin space sodium were not detected but the stimulation of transepithelial sodium transport by aldosterone in this set of observations was small, averaging only some 30% above control. The results are compatible with our hypothesis that the major action of aldosterone is to facilitate the entry of sodium from the mucosal medium across the apical permeability barrier of the transporting mucosal cells.

Electron probe X-ray microanalysis of intact pathway for human aqueous humor outflow

Intraocular pressure (IOP) is regulated by the resistance to outflow of the eyes aqueous humor. Elevated resistance raises IOP and can cause glaucoma. Despite the importance of outflow resistance, its site and regulation are unclear. The small size, complex geometry, and relative inaccessibility of the outflow pathway have limited study to whole animal, whole eye, or anterior-segment preparations, or isolated cells. We now report measuring elemental contents of the heterogeneous cell types within the intact human trabecular outflow pathway using electron-probe X-ray microanalysis. Baseline contents of Na(+), K(+), Cl(-), and P and volume (monitored as Na+K contents) were comparable to those of epithelial cells previously studied. Elemental contents and volume were altered by ouabain to block Na(+)-K(+)-activated ATPase and by hypotonicity to trigger a regulatory volume decrease (RVD). Previous results with isolated trabecular meshwork (TM) cells had disagreed whether TM cells express an RVD. In the intact tissue, we found that all cells, including TM cells, displayed a regulatory solute release consistent with an RVD. Selective agonists of A(1) and A(2) adenosine receptors (ARs), which exert opposite effects on IOP, produced similar effects on juxtacanalicular (JCT) cells, previously inaccessible to functional study, but not on Schlemms canal cells that adjoin the JCT. The results obtained with hypotonicity and AR agonists indicate the potential of this approach to dissect physiological mechanisms in an area that is extremely difficult to study functionally and demonstrate the utility of electron microprobe analysis in studying the cellular physiology of the human trabecular outflow pathway in situ.