Quantitative analysis of electrolytes in frozen dried sections

The intracellular electrolyte concentrations of the bullfrog corneal epithelium have been determined in thin freeze-dried cryosections using the technique of electron-microprobe analysis. Under control conditions, transepithelial potential short-circuited and either side of the cornea incubated in Conway's solution, the mean intracellular concentrations (in mmol/kg wet weight) were 8.0 for Na, 18.4 for Cl and 117.3 for K. These values are in good agreement with ion activities previously obtained by Reuss et al. (Am. J. Physiol. 244:C336-C347, 1983) under open-circuit conditions. From a comparison of the chemical concentrations and activities of Na and K a mean intracellular activity coefficient of 0.75 is calculated. For small ions no significant differences between nuclear and cytoplasmic concentration values were detectable. The Cl concentrations in the different epithelial layers were virtually identical and showed parallel changes at varying states of Cl secretion, suggesting that the epithelium represents a functional syncytium. For Na a concentration gradient between the outer and inner epithelial layer was observed, which can be accounted for by two different models of epithelial cooperation. The behavior of the intracellular Na and Cl concentrations after removal of Na, Cl or K from the outer or inner bathing medium provides support for a passive electrodiffusive Cl efflux across the apical membrane and a Na-coupled Cl uptake across the basolateral membrane. The results are inconclusive with regard to the exact mechanism of Cl uptake, indicating either a variable stoichiometry of the symporter or the presence of more than one transport system. Furthermore, a dependence of intracellular Cl on HCO3 and CO2 was observed. Extracellular measurements in corneal stroma demonstrated that ion concentrations in this space are in free equilibrium with the inner bath.

Section: Methodsmentioning

confidence: 99%

Cl transport in the frog cornea: An electron-microprobe analysis

Rick¹,

Beck²,

Dörge³

et al. 1985

Section: Methodsmentioning

confidence: 99%

Intracellular ion concentrations in the frog cornea epithelium during stimulation and inhibition of Cl secretion

Rick¹,

Beck²,

Dörge³

et al. 1987

The intracellular electrolyte concentrations in the isolated cornea of the American bullfrog were determined in thin freeze-dried cryosections using energy-dispersive X-ray microanalysis. Stimulation of Cl secretion by isoproterenol resulted in a significant increase in the intracellular Na concentration but did not change the intracellular Cl concentration. Similar results were obtained when Cl secretion was stimulated by the Ca ionophore A23187. Inhibition of Cl secretion by ouabain produced a large increase in the intracellular Na concentration and an equivalent fall in the K concentration. Again, no increase or decrease in the intracellular Cl concentration was detectable. Clamping of the transepithelial potential to +/- 50 mV resulted in parallel changes in the transepithelial current and intracellular Na concentration, but, with the exception of the outermost cell layer, in no changes of the Cl concentration. Only when Cl secretion was inhibited by bumetanide or furosemide, together with a decrease in the Na concentration, was a large fall in the Cl concentration observed. Application of loop diuretics also produced significant increases in the P concentration and dry weight, consistent with some shrinkage of the epithelial cells. The results suggest the existence of a potent regulatory mechanism which maintains a constant intracellular Cl concentration and, thereby, a constant epithelial cell volume. Through the operation of this system any variation in the apical Cl efflux is compensated for by an equal change in the rate of Cl uptake across the basolateral membrane. Cl uptake is sensitive to loop diuretics, directly coupled to an uptake of Na, and dependent on the Na and K concentration gradients across the basolateral membrane. Isoproterenol and A23187 seem to increase the Cl permeability of the apical membrane and thus stimulate Cl efflux. Ouabain inhibits Cl secretion by abolishing the driving Na concentration gradient for Cl uptake across the basolateral membrane.

“…The application of electron-microprobe analysis to freeze-dried cryosections seemed to us a particularly useful tool for further investigation of this issue because, with this approach, direct examination of the solute composition of toad bladder epithelial cells is feasible, even down to a subcellular level (Rick, D6rge & Thurau, 1982). In this study, we attempted first, to establish the validity of approximating intracellular osmolarity with electron-microprobe analysis, second, to verify whether or not the flow of water results in an intracellular concentration gradient for solutes, and third, to evaluate the cellular specificity of the response to ADH.…”

Section: Introductionmentioning

confidence: 99%

Intracellular solute gradients during osmotic water flow: An electron-microprobe analysis

Rick

DiBona

1987

In an attempt to quantify possible intracellular water activity gradients during ADH-induced osmotic water flow, we employed energy dispersive X-ray microanalysis to thin, freeze-dried cryosections obtained from fresh, shock-frozen tissue of the toad urinary bladder. The sum of all detectable small ions (Na + K + Cl) in the cellular water space was taken as an index of the intracellular osmolarity. Presuming that all ions are osmotically active, they comprise about 90% of the cellular solutes. When the cells were exposed to dilute serosal medium, the reduction in the sum of the ions agreed well with the expected reduction in osmolarity. After inducing water flow by addition of ADH and dilution of the mucosal medium, all epithelial cells showed a fall in osmolarity. The change was more pronounced in granular cells than in basal or mitochondria-rich cells, consistent with the notion that granular cells represent the main transport pathway. Most significantly, intracellular osmolarity gradients, largely caused by an uneven distribution of K and Na, were detectable in granular cells. The gradients were not observed after ADH or mucosal dilution alone, or when the direction of transepithelial water flow was reversed. We conclude from these results that there is a significant cytoplasmic resistance to water flow which may lead to intracellular gradients of water activity. Concentration gradients of diffusible cations can be explained by a flow-induced Donnan-type distribution of fixed negative charges. With regard to transepithelial Na transport, the data suggest that ADH stimulates transport by increasing the Na permeability of the apical membranes of granular cells specifically.