S. K. Agulian

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.

Potassium in the rat kidney proximal tubules in situ: Determination by K+-selective liquid ion-exchange microelectrodes

SummaryIntraluminal K+ concentration in the rat kidney proximal tubule was measured in situ by means of K+-selective liquid ion-exchange microelectrodes. By virtue of its independence of sampling techniques, the in situ measurement is more accurate in dealing with fluids flowing through microtubular structures. Using K+-selective liquid ion-exchange microelectrodes it was observed that the K+ concentration falls along the length of the proximal convoluted tubule. The mean (TF/P)K concentration ratio of 0.89±0.01 for the first convolution is significantly greater (p<0.001) than the mean ratio of 0.81±0.01 for the last convolution of the proximal tubule. A description is given of the construction and calibration of the K+-selective microelectrode with a new liquid ion-exchanger.

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.

A single unit pH glass ultramicro electrode

SummaryThe single unit glass ultramicro electrode can measure the pH of a sample of less than 0.05 μl in volume. The single unit electrode is advantageous in in vivo micropuncture work. It eliminates the need for double penetration of the tissue with both the indicator and the reference electrode. The physical combination of the glass indicator half-cell with the reference half-cell permits the pair to establish contact with the solution at almost the same point, thus avoiding the incorporation of spatial potentials not due to cationic activities.

Agglutination kinetics of normal and diabetic adult rat hepatocytes

This paper describes Concanavalin A-induced agglutionation of viable rat hepatocytes obtained by collagenase perfusion from normal and streptozocin-treated diabetic rats. An irreversible cell-to-cell agglutination model is proposed to explain hepatocyte flocculation. The rate of agglutination is concentration dependent with respect to Concanavalin A, and is twice as fast in normal as compared to diabetic hepatocytes. Sticking probability constants ranging from 18.48×107 to 4.6×107 cm−1 · hepatocyte−1 and 8.32×107 to 2.31×107 cm−1 · hepatocyte−1 are calculated as a measure of agglutination for normal and diabetic cells respectively. These findings suggest that the cytoplasmic membranes of normal cells possess agglutinin receptors which are more numerous and/or differently arranged than those existing in diabetic cells.

Internal capillary glass microelectrodes with a glass seal for pH, sodium and potassium.

SummaryA description is given of the construction and utilization of glass ultramicro electrodes of the internal capillary suction-type that employ a glass-to-glass seal. These electrodes are capable of measuring pH, sodium or potassium on samples of fluid as small as 0.01 μL in volume. They are useful in measuring ionic concentrations of biological fluids both in vivo and in vitro. The characterization and the performance of the microelectrodes is described.

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.