J. Graf

Cell membrane and transepithelial voltages and resistances in isolated rat hepatocyte couplets.

SummaryThe basic electrical properties of an isolated rat hepatocyte couplet (IRHC) system have been analyzed using classical techniques of epithelial electrophysiology, including measurement of electric potentials, resistances and intracellular ion activities. Applications of these techniques are discussed with respect to their limitations in small isolated cells. Mean intracellular and intracanalicular membrane potentials ranged from −23.7 to −46.7 and −4.3 to −5.9 mV, respectively. Membrane resistances were determined using an equivalent circuit analysis modified according to the geometry of the IRHC system. Resistances of the sinusoidal (basolateral) and canalicular (luminal) cell membranes and tight junctions averaged 0.15 and 0.78 GΩ and 25mΩ, respectively. The cells are electrically coupled via low resistance intercellular communications (∼58 MΩ). Intracellular ion activities for Na+, K+ and Cl− averaged 12.2, 88.1 and 17.7 mmol/liter, respectively. The basolateral membrane potential reveals a permeability sequence ofPK>PCl>PNa. The luminal potential showed minimal dependence on changes in transjunctional ion gradients, indicating a poor ion selectivity of the paracellular pathway. The electrogenic (Na+−K)-ATPase contributes little to the luminal and cellular negative electric potential. Therefore, the luminal potential probably results from the secretion of impermeant ions and a Donnan distribution of permeant ions, a mechanism which provides the osmotic driving force for bile formation. By providing the unique opportunity to measure luminal potentials, this isolated hepatocyte system permits study of secretory mechanisms for the first time in a mammalian gland using electrophysiologic techniques.

Intracellular sodium activity and sodium transport inNecturus gallbladder epithelium

SummaryIon-sensitive glass microelectrodes, conventional microelectrodes and isotope flux measurements were employed inNecturus gallbladder epithelium to study intracellular sodium activity, [Na]i, electrical parameters of epithelial cells, and properties of active sodium transport. Mean control values were: [Na]i: 9.2 to 12.1mm; transepithelial potential difference,Ψms: −1.5 mV (lumen negative); basolateral cell membrane potential,Ψes: −62 mV (cell interior negative); sodium conductance of the luminal cell membrane,gNa: 12 μmho cm−2; active transcellular sodium flux, 88 to 101 pmol cm−2 sec−1 (estimated as instantaneous short-circuit current). Replacement of luminal Na by K led to a decrease of the intracellular sodium activity at a rate commensurate to the rate of active sodium extrusion across the basolateral cell membrane. Mucosal application of amphotericin B resulted in an increase of the luminal membrane conductance, a rise of intracellular sodium activity, and an increase of short-circuit current and unidirectional mucosa to serosa sodium flux. Conclusions: (i) sodium transport across the basolateral membrane can proceed against a steeper chemical potential difference at a higher rate than encountered under control conditions; (ii) the luminal Na-conductance is too low to accommodate sodium influx at the rate of active basolateral sodium extrusion, suggesting involvement of an electrically silent luminal transport mechanism; (iii) sodium entry across the luminal membrane is the rate-limiting step of transcellular sodium transport and active sodium extrusion across the basolateral cell membrane is not saturated under control conditions.

Na-H exchange regulates intracellular pH in isolated rat hepatocyte couplets

Intracellular pH (pHi) was measured directly in isolated rat hepatocyte couplets using pH sensitive microelectrodes. The hepatocytes were maintained in a minimal salt buffer without added hormones or serum. Values of pHi (6.99 +/- 0.12, mean +/- SE) were close to their Nernst equilibria. After intracellular acidification with ammonium chloride, pH regulation was inhibited with 1 mM amiloride or by omission of external sodium, consistent with a Na-H exchange mechanism. Mean intracellular buffering power, in the nominal absence of carbon dioxide, was 34.1 +/- 11.4 mM. In the presence of external bicarbonate, amiloride or omission of sodium slowed, but did not completely inhibit recovery from acidification, indicating that additional pHi regulation mechanisms may operate in this preparation. These studies provide a direct measurement of pHi in hepatocyte couplets and indicate that Na-H exchange, together with a bicarbonate dependent system are important mechanisms for pHi regulation in this preparation.

Preparation and specific applications of isolated hepatocyte couplets.

Publisher Summary This chapter discusses the preparation and specific applications of isolated hepatocyte couplets. A number of different methods are available for isolating hepatocytes in suspension either for short-term experiments or as preliminary steps in the preparation of hepatocyte monolayer culture systems. Hepatocyte couplets, which are plated at lower cell density then in monolayer preparations, represent a primary secretory unit that enables direct observations of the process of canalicular bile formation at its site of origin. This cell system thus permits the application of a variety of techniques and methodologies available for studying cell function in tissue culture, including phase and Nomarski microscopy and cinephotomicrogaphy as well as fluorescent and electrophysiologic applications. Specific applications include (1) determination of transepithelial potential profile, (2) determination of the resistive elements of ionic current flow (basolateral and lumenal membrane, paracellular pathway), and (3) determination of partial ionic conductances of the basolateral cell membrane.

Effect of intracellular pH on potassium conductance in liver

Introduction Disturbances of acid-base balance have long been known to be associated with changes of the internal equilibrium of potassium [2,11]. These changes of plasma K + concentration are largely ascribed to shifts of K + between skeletal muscle and blood but there is also evidence that liver cells may lose K + during acidosis []2] or gain K + during alkalosis [7]. The mechanisms of these changes of hepatocellular K + content have not been studied. It was noted though that acidosis leads to cell membrane depolarisation [5,12] whereas alkalosis causes hyperpolarisation [I]. Changes of intracellular potassium concentration ([K+]i) may contribute to these effects on membrane potential (V m) to some extent, but it has also been suggested that intracellular pH (pH i ) regulates membrane K + conductance (gK) [12]. If correct, these effects on gK would counteract the observed changes of K + equilibrium, alkalosis leading to an increased efflux of K + and acidosis promoting K + retention. Thus, other mechanisms would have to account for K + release during acidosis. This study examines the effect of pH i on gK in isolated hepatocytes of rat and mouse.

Properties of an Anion Channel in Rat Liver Canalicular Membranes

After incorporation of cLPM into the bilayer an anion channel was frequently observed. FIGURE 1 shows the ionic current through the channels observed at different holding potentials. A single channel conductance of 40 pS at 0 mV was determined in the presence of the 2 : 1 KCl gradient. A reversal potential greater than +15 mV corresponded closely to the C1gradient. The current voltage relationship showed Goldman rectification with a permeability ratio of p c , / p ~ greater than 13 : 1 (FIG. 2). The anion selectivity of the channel was investigated by the addition of different potassium salts to the trans-compartment. Only a small selectivity between several halides could be detected: zp! > P B ~ > PF. Channel kinetics were voltage dependent, with higher open probability at negative holding potentials. In summary, an anion selective channel has been identified in cLPM with properties similar to those of intermediate, nonrectifying C1channels described in other epithelia.5 The right-side out orientation of the vesicles suggests that the trans-compartment corresponds to the intracellular space.6 With such an assump-