J. Siegenbeek van Heukelom

Effects of chloride transport on bistable behaviour of the membrane potential in mouse skeletal muscle

The lumbrical skeletal muscle fibres of mice exhibited electrically bistable behaviour due to the nonlinear properties of the inwardly rectifying potassium conductance. When the membrane potential (Vm) was measured continuously using intracellular microelectrodes, either a depolarization or a hyperpolarization was observed following reduction of the extracellular potassium concentration (K+o) from 5.7 mm to values in the range 0.76–3.8 mm, and Vm showed hysteresis when K+o was slowly decreased and then increased within this range. Hypertonicity caused membrane depolarization by enhancing chloride import through the Na+–K+–2Cl− cotransporter and altered the bistable behaviour of the muscle fibres. Addition of bumetanide, a potent inhibitor of the Na+‐K+‐2Cl− cotransporter, and of anthracene‐9‐carboxylic acid, a blocker of chloride channels, caused membrane hyperpolarization particularly under hypertonic conditions, and also altered the bistable behaviour of the cells. Hysteresis loops shifted with hypertonicity to higher K+o values and with bumetanide to lower values. The addition of 80 μM BaCl2 or temperature reduction from 35 to 27 °C induced a depolarization of cells that were originally hyperpolarized. In the K+o range of 5.7–22.8 mm, cells in isotonic media (289 mmol kg−1) responded nearly Nernstianly to K+o reduction, i.e. 50 mV per decade; in hypertonic media this dependence was reduced to 36 mV per decade (319 mmol kg−1) or to 31 mV per decade (340 mmol kg−1). Our data can explain apparent discrepancies in ΔVm found in the literature. We conclude that chloride import through the Na+–K+–2Cl− cotransporter and export through Cl− channels influenced the Vm and the bistable behaviour of mammalian skeletal muscle cells. The possible implication of this bistable behaviour in hypokalaemic periodic paralysis is discussed.

The influence of bumetanide on the membrane potential of mouse skeletal muscle cells in isotonic and hypertonic media

Increasing the medium osmolality, with a non‐ionic osmoticant, from control (289 mOsm) to 319 mOsm or 344 mOsm in the lumbrical muscle cell of the mouse, resulted in a depolarization of the membrane potential (Vm) of 5.9 mV and 10.9 mV, respectively. In control medium, the blockers of chloride related cotransport bumetanide and furosemide, induced a hyperpolarization of −3.6 and −3.0 mV and prevented the depolarization due to hypertonicity. When bumetanide was added in hypertonic media Vm fully repolarized to control values. In a medium of 266 mOsm, the hyperpolarization by bumetanide was absent. At 344 mOsm the half‐maximal effective concentration (IC50) was 0.5 μm for bumetanide and 21 μm for furosemide. In solutions containing 1.25 mm sodium the depolarization by hypertonicity was reduced to 2.3 mV. Reducing chloride permeability, by anthracene 9 carboxylic acid (9‐AC) in 289 mOsm, induced a small but significant hyperpolarization of −2.6 mV. Increasing medium osmolality to 344 mOsm enlarged this hyperpolarization significantly to −7.6 mV. In a solution of 344 mOsm containing 100 μm ouabain, the bumetanide‐induced hyperpolarization of Vm was absent. The results indicate that a Na‐K‐2Cl cotransporter is present in mouse lumbrical muscle fibre and that its contribution to Vm is dependent on medium osmolality.

The electrophysiological characteristics of glucose absorption of the goldfish intestine as compared to mammalian intestines.

Abstract o 1. A new experimental set-up and procedure are described for the measurement of some electrophysiological properties of the isolated goldfish intestine ( Carassius auratus ). 2. With this apparatus it is possible to perform experiments over a 3 hr period without deterioration of the tissues structure. 3. The glucose-evoked potential (GEP) is probably the result of a rheogenic process as no change in the epithelial resistance occurred with or without glucose, mannitol or phlorizin in the mucosal solution. 4. The apparent K m for the GEP is 4 mM. 50% inhibition of the GEP was determined at a phlorizin concentration of 10 −5 M; while 10 −5 M ouabain caused an inhibition of 85%. These results are comparable with those given in the literature for mammalian intestine.

ROLE OF THE ANOMALOUS RECTIFIER IN DETERMINING MEMBRANE POTENTIALS OF MOUSE MUSCLE FIBRES AT LOW EXTRACELLULAR K

1. The membrane potential (Vm) of fibres of the extensor digitorum longus (EDL) of the mouse, measured at 35 degrees C and with extracellular potassium concentration (K+o) 5.7 mM, was Vm = ‐76 mV. 2. Lowering K+o below 1 mM could lead to either a hyperpolarizing or a depolarizing response. When Vm was lower than ‐75.5 mV in the control medium, a reduction of K+o to 0.76 mM led to a hyperpolarization of Vm (‐95.0 +/‐ 0.7 mV, n = 40); otherwise a depolarization occurred (Vm = ‐47.2 +/‐ 1.1 mV, n = 21). 3. The difference in Vm responses did not correlate consistently with functional differences in cell types, as cells that originally hyperpolarized, could later depolarize. 4. The observed phenomena could be explained if the properties of the anomalous rectifier, AR (or inward‐going rectifier), are considered to be similar to those observed in cardiac cells. 5. Apparently caesium acted as a competitive inhibitor; when the inhibition was strong enough the non‐linear properties of the AR regeneratively amplified the depolarization to the full‐blown depolarized state (Vm = ‐46.7 +/‐ 1.3 mV, n = 15). 6. Ouabain (10(‐4) M) reduced Vm (to ‐45 +/‐ 3 mV, n = 5) and reduced dramatically the selectivity of the cell membrane for potassium over sodium. These effects could be reversed readily by washing out the ouabain. 7. Adrenaline (2 microM) added to the medium hyperpolarized Vm (delta Vm = ‐4.6 +/‐ 1.4 mV, n = 9) and increased the changes induced by lowered K+o (from ‐14.3 +/‐ 0.5 mV, n = 5 to ‐18.0 +/‐ 0.8 mV, n = 9); the cells that originally depolarized when K+o was lowered could hyperpolarize after adrenaline addition.

A mechanistic explanation of the effect of potassium on goldfish intestinal transport

Partial replacement of sodium by potassium or rubidium in the solution used to perfuse isolated intestinal segments of goldfish causes an increase in transmural electrical resistance. Serosal replacements have a stronger effect than mucosal replacements.A 70% inhibition of the glucose-evoked transmural electrical current is brought about by serosal replacement of 40 mM sodium by potassium. Transmural mucosal to serosal flux of 3-O-methyl-D-glucose is also strongly inhibited by serosal potassium. These inhibitory effects of potassium do not occur when the intestinal mucosa is stripped free from the intestinal muscular layers. It is concluded that potassium-induced muscular contractures cause a decrease in transport area by pressing the mucosal folds closer against each other.Certain effects of high potassium concentrations that have been reported in mammalian intestinal preparations may involve a similar mechanism.

Effects of glucose and ouabain on transepithelial electrical resistance and cell volume in stripped and unstripped goldfish intestine

Abstract1.In goldfish intestine (perfused unstripped segments and mucosal strips) the serosal addition of ouabain (10\t-4 M) resulted in a vanishment of the transepithelial potential difference and in a continuous increase in transepithelial resistance.2.Incubation of mucosal strips with ouabain resulted in an increase in sodium content which was greater than the decrease in potassium content. The resulting increase in cation content was accompanied by an increase in chloride content and an increase in water content.3.Histological examination showed that exposure to ouabain resulted in a swelling of the epithelial layer as compared to the control situation.4.The ouabain induced resistance increase is greater in the presence of glucose, 3-OMG or fructose than in the presence of mannitol. Phlorizin (10\t-4 M) inhibits the extra resistance increase induced by mucosal glucose but is without effect on the fructose induced extra resistance increase.The initial velocity and the magnitude of the glucose induced extra resistance increase depends on the glucose concentration.5.The results suggest that in goldfish intestine ouabain induces cellular swelling with a concomitant collapse of the lateral intercellular spaces, which is the cause of the increased transepithelial resistance. The additional changes in resistance induced by sugars suggest that the cell membrane is more permeable to glucose, 3-OMG and fructose than to mannitol. The resulting changes in osmotically active material within the epithelial cell influence the cross-sectional area and consequently the conductivity of the paracellular shunt pathway. The hypothesis that these sugars do not induce a resistance change in the absence of ouabain is discussed.

The role of the potassium inward rectifier in defining cell membrane potentials in low potassium media, analyzed by computer simulation.

Abstract A model is presented that describes the contributions of the potassium conductance and the Na + /K + pump to the steady state magnitude of the plasma membrane potential, V rest , at different concentrations of potassium in the extracellular fluid: K o . The particular properties of the potassium inward rectifier, IKR, are shown to explain that V rest frequently depolarises on lowering K o below a critical value, because the IKR is only conductive when V m is near to the potassium equilibrium potential or more negative. The model aims at a generic description of the process based on compartmental analysis. It is worked out to describe V rest in mouse muscle fibres. Sensitivity analysis demonstrates that the process of switching off the IKR depends critically on the margin between V rest and E K , allowed for the IKR to remain open and the power of the Na/K pump to keep this margin small during the reduction of K o . However, cells exposed to K o just higher than the critical value may eventually also depolarize due to excessive loss of potassium. The model also demonstrates that loss of potassium membrane selectivity after blocking the Na/K pump by ouabain requires a mechanism additional to the above mentioned properties of the inward rectifier.

Circuit analysis of membrane potentials changes due to electrogenic sodium-dependent sugar transport in goldfish intestinal epithelium

The effect of glucose on the electrical potential profile in the stripped goldfish intestine was measured and analyzed in terms of cell membrane electromotive forces.In substrate free conditions, the mucosal membrane potential difference ψmc averaged −54 mV, cell interior negative. The addition of 27.8 mMd-glucose to the mucosal side of the epithelium evoked a rapid depolarization (Δψmc = +11.5 mV) followed by a slower repolarization (Δψmc = −6.0 mV) to a new steady state value of −48 mV. The membrane resistance ratioRm/Rs averaged 0.99. During the depolarization phase it was reduced by 18%, returning to the pre-glucose value within 2.5 min. The transepithelial resistanceRms was 19.7 Ωcm2 and did not change.Addition of ouabain induced a depolarization of ψmc without significant change inRms orRm/Rs during the first 15 min. Application of glucose to the mucosal solution a few minutes after ouabain exposure resulted in a rapid depolarization of ψmc without repolarization. The glucose induced transepithelial potential change reached only about 60% of the original value. From the ouabain experiments the relative shunt conductance was calculated to be 95%. In mannitol conditionsRm=Rs=197 Ωcm2, while the shunt resistanceR1=20.7 Ωcm2.Assuming no large changes in the potential through the shunt pathway, the analysis of the changes in electromotive forces (EMF) at both cell borders yields the following values ΔEm=+23.7 mV and ΔEs=−17.4 mV.Analysis of the changes in the electromotive force across the mucosal membrane as a function of the glucose concentration revealed the presence of a Na+-dependent glucosetransport system composed of two saturating processes.

Cellular and transepithelial responses of goldfish intestinal epithelium to chloride substitutions

SummaryIn goldfish intestine chloride was substituted by large inorganic anions (gluconate or glucuronate) either mucosally, serosally or bilaterally. Changes in intracellular activities of chloride (aiCl−), sodium (aiNa+) and potassium (aiK+), pHi, relative volume, membrane and transepithelial potentials, transepithelial resistance and voltage divider ratio were measured. Control values were:aiCl−=35 meq/liter, aiNa+=11 meq/liter and aiK+=95 meq/liter. During bilateral substitution the latter two did not change while aiCl− dropped to virtually zero.Mucosal membrane potentials (ψms) were: control,-53 mV; serosal substitution,-51 mV; bilateral substitution,-66 mV; while during mucosal substitution a transient depolarization occurred and the final steady state ψms was-66 mV.During control and bilateral substitution the transepithelial potentials (ψms) did not differ from zero. During unilateral substitutions ψms was small, in the order of magnitude of the errors in the liquid junction potentials near the measuring salt bridges.During bilateral substitution pHi increased 0.4 pH units. Cellular volume decreased during mucosal substitution to 88% in 40 min; after serosal substitution it transiently increased, but the new steady-state value was not significantly above its control value.Three minutes after mucosal substitution anaiCl− of approx. 10 meq/liter was measured.Chemical concentrations of Na, K and Cl were determined under control conditions and bilateral substitution. Cl concentrations were also measured as a function of time after unilateral substitutions.The data indicate an electrically silent chloride influx mechanism in the brush border membrane and an electrodiffusional chloride efflux in the basolateral membrane. A substantial bicarbonate permeability is present in the basolateral membrane. The results are in agreement with the observed changes in membrane resistances, volume changes and pH changes.

Sodium-dependent sugar and amino acid transport in isolated goldfish intestinal epithelium: electrophysiological evidence against direct interactions at the carrier level.

AbstractThe effects of mucosal application of monosaccharides and amino acids on transepithelial and membrane potentials in isolated goldfish intestinal epithelium were investigated.Isosmotic replacement of mucosal mannitol by sugars orl-amino acids resulted in a rapid depolarization of the mucosal membrane potential ψmc followed by a slow repolarization. Phlorizin inhibited the responses to sugar but not those to amino acids. d-Amino acids did not induce any electrical response in the epithelium. Dose-response curves forl-amino acids showed simple saturation.Simultaneous application ofl-amino acid and glucose induced transepithelial responses of about 80% of the sum of the separate responses to the application of amino acid or glucose alone. Simultaneous application of different amino acids in saturating concentrations did not increase the magnitude of the electrical responses.From the measured changes in potentials we calculated the change in electromotive force across the mucosal (ΔEm) and serosal (ΔEs) membrane. The change inEm induced by combined application of alanine and glucose was 90% of the sum of the calculated values induced by glucose and alanine alone. The response ofEs to both substrates was accelerated with respect to that of separate substrates alone.We conclude that by application of glucose in addition to alanine the influx of sodium is increased, thereby stimulating the basolaterally located electrogenic Na+/K+-pump. There are no indications for direct interaction of sugars and amino acids at the mucosal membrane of the intestinal epithelial cell.