Raja N. Khuri

Intracellular bicarbonate in single skeletal muscle fibers.

SummaryThis is the first direct potentiometric determination of intracellular bicarbonate concentration. The new method involves the use of a double-barrelled HCO3−-selective liquid ion-exchange microelectrode that permits the simultaneous determination of intracellular [HCO3−] and membrane PD of single cells. The mean in situ intracellular [HCO3−] of single striated muscle fibers was 4.4±0.3 mM/l in the frog and 12.6±0.6 mM in the rat. Both values are inconsistent with a Donnan equilibrium distribution and can be accounted for by an active HCO3− influx or an active H+ efflux. During progressive acute hypercapnia there is an accumulative build-up of intracellular bicarbonate in rat skeletal muscle. The increase in intracellular [HCO3−] with hypercapnia is strictly proportional to the associated increase in plasma [HCO3−], thus maintaining a constant ratio of extracellular: intracellular [HCO3−]. Using the directly measured [HCO3−] in cell water, we calculate a cell pH of 7.00 for frog fibers and of 7.14 for rat fibers, both values being about 1.1 pH units on the alkaline side of those predicted for a Donnan equilibrium distribution of H+ ions across the cell membrane.

Intracellular potassium in cells of the proximal tubule of necturus maculosus

SummaryUsing double-barreled K+ selective liquid ion-exchange microelectrodes intracellular K+ activity and the peritubular potential difference (PD) were measured simultaneously in single cells of Necturus proximal tubules. Proximal tubular fluid K+ activity and the transepithelial PD were also measured simultaneously. Kidney slices analyzed by flame photometry yielded a mean K+ concentration of 103.0±1.8 mM per Kg cell water. This electrometric study yielded a mean K+ activity of 58.7±2.3 mM, thus giving a low value of 0.57 for the mean ionic activity coefficient. The electrometric mean proximal tubule fluid K+ activity of 5.4±0.1 mM and plasma K+ activity of 2.8±0.3 mM yield a fluid/plasma activity ratio of 1.9±0.2. The calculated K+ equilibrium potentials (as calculated from the activity ratios) across the whole proximal tubular epithelium, its luminal cell boundary and its peritubular cell boundary are not significantly different from their respective measured membrane PDs. This signifies that K+ is in electro-chemical equilibrium distribution across the boundaries that separate the different compartments of the proximal tubular system.

Intracellular bicarbonate in single cells of Necturus kidney proximal tubule

SummaryThe intracellular bicarbonate concentration in the cytoplasmic water of kidney cells of Necturus was determined by means of double-barreled HCO3−-selective liquid ion-exchange microelectrodes. These microelectrodes permit the simultaneous determination of intracellular HCO3− and membrane PD of single cells. The fact that these double-barreled microelectrodes yielded a normal peritubular cell membrane electrical PD (70 m V) may be taken as evidence against any significant cellular damage by the electrodes. This electrometric study yielded a mean intracellular [HCO3−] in single proximal tubule cells of Necturus of 11.1±0.6 mM, a value which is more than an order of magnitude higher than that predicted for a Donnan-type electrochemical equilibrium distribution of HCO3− ions. Thus there is a net electrochemical gradient favoring the passive efflux of HCO3− ions across both individual cell membranes. The movement of HCO3− ions from cell-to-interstitium would contribute to the renal acidification function. Across the luminal cell membrane the two possible mechanisms are either active reabsorption (lumen-to-cell) of HCO3− ions as such or active H+ secretion (cell-to-lumen). Our directly measured relatively high intracellular [HCO3−] and the associated calculated relatively alkaline kidney cell pH of 7.44 are both more consistent with the H+ secretion hypothesis.

Electrochemical potentials of potassium in proximal renal tubule of rat

SummaryBy means of double-barreled K+ selective liquid ion-exchange microelectrodes, the electrical potential differences across individual cell membranes were determined simultaneously with the K+ concentration in single cellular elements of the proximal tubular epithelium of the rat. Proximal tubular fluid [K+] and plasma [K+] were also determined electrometrically. Thin cortical slices of the rat kidney analyzed by flame photometry yielded a mean [K+] of 136.3±4.2 mM per kg cell water. This electrometric study yielded a mean intracellular [K+] of 54.4±2.5 mM, a value which is about 1/3 of the total K+ content of proximal tubule cells. The electrometric mean proximal tubule fluid (second half) [K+] was 3.7±0.1 mM while plasma [K+] was 4.3±0.1 mM, yielding a fluid/plasma concentration ratio of 0.85±0.02. The calculated K+ equilibrium potentials (EK)across the two individual from their respective measured membrane electrical PDs. This signifies that K+ exhibits an electrochemical equilibrium distribution across the luminal and peritubular cell boundaries of the proximal tubular epithelium. Thus it is no longer necessary to postulate the presence of an active K+ pump in either the luminal or peritubular cell membranes.

Electrochemical potentials of chloride in proximal renal tubule of Necturus maculosus.

Abstract 1. 1. Electrometric analysis employing double-barrelled Cl−-selective liquid ion-exchange microelectrodes yielded a mean intracellular [Cl−] of 18·7 ± 1·3 mM in single renal cells of Necturus maculosus proximal tubule. Thus two-thirds of the total intracellular Cl− content is electrometrically active. 2. 2. Thus intracellular Cl− ion is at a higher electrochemical potential than Cl− in either the luminal or peritubular phase. 3. 3. If CI− reabsorption across the proximal tubular epithelium consists of ion transport across two individual cell membranes in series, then the first reabsorptive step across the luminal cell membrane must be active while the second step across the peritubular membrane is passive.

Intracellular Electrochemical Potentials: Skeletal Muscle vs. Epithelial Steady-State vs. Kinetics

The true internal environment is the cytoplasmic aqueous solution each cell contains within its membrane. The cytosol is the cytoplasmic aqueous solution. Direct and reliable determination of the in-situ ionic composition of the intracellular environment is an essential prerequisite for our understanding of such basic phenomena as transmembrane electrical potentials, enzyme activity and membrane transport.

Effect on Mn2+ on permeability properties of frog skin

SummaryMn2+ added to the inner bathing solution of frog skin caused a transient increase in potential difference (PD) and a decrease in total skin conductance and mannitol influx. Net Na flux and short-circuit current (Is. c.) were also reduced, the isotopic net flux being reduced more than Is. c. This observed discrepancy appears to be the result of Cl− retention in the outer medium since it was not observed when the skin was bathed in a sulfate-substituted chloridefree solution. The effect of Mn2+ on the inner side of the frog skin appears to be due to a reduced permeation of Na+ and Cl− through the outer barrier of the skin.Addition of Mn2+ to the outer solution bathing the frog skin caused an increase in PD and a smaller increase in Is. c. These changes were not associated with alterations in the fluxes of Na+ or mannitol and were observed only when chloride was present in the bathing solutions. The effect of Mn2+ on this side of the frog skin may therefore be due to a net retention of Cl− in the outer solution.

Chapter 6 Intracellular Ion Activity Measurements in Kidney Tubules

Publisher Summary Knowledge of intracellular electrolyte composition is essential for the understanding of cell function, as the more important changes occur inside cells. This chapter provides a general background of the intracellular electrochemistry and renal tubular epithelium and describes the intracellular ion activity measurements of potassium, sodium, and chloride ions in kidney tubules. The intracellular electrochemical technique employs double-barreled ion-selective, liquid ion-exchange microelectrodes to simultaneously measure the intracellular electrical and chemical potentials of the same cell. The primary active event is the operation of the peritubular active Na + –K + exchange pump and includes cotransport of Na + with Cl - and HCO 3 - . While intracellular K + is all free in muscle, intracellular Na + is largely not free in muscle, and Cl - ion is passively distributed across the muscle fiber membrane. Thus, by virtue of direct and accurate determinations of intracellular ionic activity, the chapter concludes that renal epithelium differs from skeletal muscle in at least three ways with respect to the chemical potential of its cytoplasmic monovalent ions.

Electrochemical Potentials of Potassium and Chloride in the Proximal Renal Tubules of Necturus Maculosus

The renal tubular epithelium is essentially a 3-compartment system: interstitial, intracellular and luminal. These three aqueous phases are separated by two lipid plasma membranes: the outer peritubular cell membrane and the inner luminal cell membrane.

Lysine transport in turtle ventricle-I. Effect of ouabain and extracellular ions on lysine accumulation

Abstract 1. 1. Strips of turtle ventricle accumulate L -lysine to concentrations that exceed those of their bathing media by 1·5-2·0 times. 2. 2. This accumulative process is dependent on the Ca and K concentrations of the bathing medium but is not influenced by substituting choline or lithium for sodium. 3. 3. Ouabain in 10−4 M concentration inhibits lysine accumulation.This inhibition is also influenced by the electrolyte composition of the medium. It increases when calcium is raised from 1·3 to 5 mM, and decreases when 52·1 mM K is used instead of 2·1. 4. 4. A time relationship exists between the intracellular electrolyte shifts and the ouabain inhibition of amino acid uptake.