Isoprenaline-stimulated differential adrenergic response of K+ channels in skeletal muscle under hypokalaemic conditions.

The mechanism underlying the hyperpolarization induced by isoprenaline in mouse lumbrical muscle fibres was studied using cell-attached patch and intracellular membrane potential ( V(m)) recordings. Sarcolemmal inwardly rectifying K(+) channels (K(IR): 45 pS) and Ca(2+)-activated K(+) channels (BK: 181 pS) were identified. Exposure to isoprenaline closed K(IR) channels and increased BK channel activity. This increase was observed as a shift from 50 to -40 mV in the voltage dependence of channel activation. Isoprenaline prevented hysteresis of V(m) when the extracellular [K(+)] fell below 3.8 mM. This hysteresis was due to the properties of the K(IR). The effects of chloride transport and isoprenaline on V(m) did not interact purely competitively, but isoprenaline could prevent the depolarization induced by hyperosmotic media equally as well as bumetanide, which inhibits the Na(+)/K(+)/2Cl(-) cotransporter. In lumbrical muscle this leads to hyperpolarization, but this might vary among muscles. The switch from K(IR) to BK as the component of total K(+) conductance was due to isoprenaline.

Osmolality influences bistability of membrane potential under hypokalemic conditions in mouse skeletal muscle: an experimental and theoretical study.

The membrane potential in mouse skeletal muscle depends on both extracellular osmolality and potassium concentration. These dependencies have been related to two membrane transporters, Na+/K+/2Cl- co-transporter and the inward potassium rectifier channel. To investigate the relation of the Na+/K+/2Cl- co-transporter and the inward potassium rectifier channel in a qualitative way, a combined electrophysiological and modelling approach was used. The experimental results show that the bistability of the membrane potential, which is related to the conductive state of the inward potassium rectifier channel, is shifted to higher extracellular potassium values when medium osmolality is increased. These results are confirmed by the computer simulation calculations for increased co-transporter flux. The combined results indicate that the co-transporter is capable of modulating the conductive state of the inward potassium rectifier channel.

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

1. 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. 2. 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. 3. In a medium of 266 mOsm, the hyperpolarization by bumetanide was absent. 4. At 344 mOsm the half-maximal effective concentration (IC50) was 0.5 microM for bumetanide and 21 microM for furosemide. 5. In solutions containing 1.25 mM sodium the depolarization by hypertonicity was reduced to 2.3 mV. 6. 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. 7. In a solution of 344 mOsm containing 100 microM ouabain, the bumetanide-induced hyperpolarization of Vm was absent. 8. 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.

Modulation of the isoprenaline-induced membrane hyperpolarization of mouse skeletal muscle cells.

1. The hyperpolarization of the resting membrane potential, Vm, induced by isoprenaline in the lumbrical muscle fibres of the mouse, was investigated by use of intracellular microelectrodes. 2. In normal Krebs-Henseleit solution (potassium concentration: K+o = 5.7 mM, 'control'), Vm was -7.40 +/- 0.2 mV; lowering K+o to 0.76 mM ('low K+o') resulted in either a hyperpolarization (Vm = -95.7 +/- 2.9 mV), or a depolarization (Vm = -52.0 +/- 0.3 mV). 3. Isoprenaline (> or = 200 nM) induced a hyperpolarization of Vm by delta Vm = -5.6 +/- 0.4 mV in control solution. 4. When Vm hyperpolarized after switching to low K+o, the addition of isoprenaline resulted in increased hyperpolarization Vm: delta Vm = -16.3 +/- 3.2 mV to a final Vm = -110.1 +/- 3.4 mV. Adding iso-prenaline when Vm depolarized in low K+o, leads to a hyperpolarization of either by -11.6 +/- 0.5 mV to -63.6 +/- 0.8 mV or by -51.7 +/- 2.7 mV to -106.9 +/- 3.9 mV. 5. Ouabain (0.1 to 1 mM) did not suppress the hyperpolarization by isoprenaline in 5.7 mM K+o (delta Vm = -6.7 +/- 0.4 mV) or the hyperpolarization of the depolarized cells in low K+- (delta Vm = -9.7 +/- 1.5 mV). 6. The hyperpolarization is a logarithmically decreasing function of K+o in the range between 2 and 20 mM (12 mV/decade). 7.IBMX and 8Br-cyclic AMP mimicked the response to isoprenaline whereas forskolin (FSK) induced in low K+o a hyperpolarization of -7.0 +/- 0.7 mV that could be augmented by addition of isoprenaline (delta Vm = -8.2 +/- 1.8 mV). 8. In control and low K+o, Ba2+ (0.6 mM) inhibited the hyperpolarization induced by isoprenaline, IBMX or 8Br-cyclic AMP. Other blockers of the potassium conductance such as TEA (5 mM) and apamin (0.4 microM) had no effect. 9. We conclude that in the lumbrical muscle of the mouse the isoprenaline-induced hyperpolarization is primarily due to an increase in potassium permeability.

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.

Physiological aspects of absorption and secretion in intestine.

Two differently oriented approaches in intestinal physiology can be distinguished. One, mainly based on in vitro experiments, seeks explanations at the level of the epithelium itself. The other, mainly based on in vivo experiments, looks for explanations at the level of regulatory nervous and endocrine mechanisms and their interaction. These two approaches complement each other.

Influence of glucose absorption on ion activities in cells and submucosal space in goldfish intestine.

Mucosal glucose addition evokes in goldfish intestinal epithelium a fast depolarization of the mucosal membrane potential (delta psi mc = 12 mV) followed by a slower repolarization (delta psi mc = -7 mV). The intracellular sodium activity, aiNa+, rises from 13.2 +/- 2.4 meq/l by 6.7 +/- 0.5 meq/l within 5 min, aiCl- rises about 3 meq/l above the control value of 37.7 +/- 2.2 meq/l, while aiK is constant (97.7 +/- 7.4 meq/l). The potassium activity measured in the submucosal interstitium near the basal side of the cells (asK+) is 5.2 +/- 0.2 meq/l in non-absorbing tissue compared to 4.2 meq/l in the bathing solution and shows a transient increase due to glucose absorption (1.1 +/- 0.1 meq/l). In chloride-free media asK+ = 4.2 +/- 0.1 meq/l and psi mc hyperpolarizes by -13 mV. The depolarization due to glucose absorption increases (delta psi mc = 14.1 +/- 1.4) and the repolarization (delta psi repolmc) disappears. In addition, aiNa+ rises from 16.3 +/- 2.4 meq/l by 9.9 +/- 1.5 meq/l within 5 min, aiK+ remains constant and equal to the value in chloride containing solutions (88.5 +/- 2.8 meq/l); asK+ increases transiently (1.1 +/- 0.1 meq/l). Serosal Ba2+ (5 mM) depolarizes psi mc (+14.2 +/- 1.0 mV) and abolishes the repolarization. Increased serosal or mucosal potassium activity depolarizes psi mc and abolishes the repolarization. These effects are discussed in terms of changes of ion activities, the basolateral potassium conductance, the influence of intracellular Ca2+, the functional state of the Na/K-pump, and modulation of membrane permeabilities by extracellular potassium.

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

The 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 or L-amino acids resulted in a rapid depolarization of the mucosal membrane potential psi mc 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 for L-amino acids showed simple saturation. Simultaneous application of L-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 (delta Em) and serosal (delta Es) membrane. The change in Em 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 of Es 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.

The modulation by glucose transport of the electrical responses to hypertonic solutions of the goldfish intestinal epithelium.

Goldfish intestinal epithelium responds to mucosal hypertonicity with a negative biphasic transepithelial potential change and a relatively slow rise in transepithelial resistance, similar to that described for rabbit gallbladder (Wright et al. 1972; Smulders et al. 1972). In addition, the increase in resistance in goldfish intestine can be modulated by the presence or absence of glucose. E.g. during mucosal hypertonicity of 87 mosmoles/l the addition of 27.8 mmoles/l glucose to the serosal side further increased the resistance by 2.8 +/- 0.2 omega cm2, while mucosal addition reduced it by 11.2 +/- 2.6 omega cm2. Ouabain poisoning inverted this last response into a slowly and continuously rising resistance. The resistance response to mucosal glucose can be fully abolished by mucosal addition of phlorizin. The resistance change due to bilateral glucose addition is the sum of the separate mucosal and serosal responses. The effect of fructose at the serosal side resembles that of glucose added serosally; the mucosal effect of glucose could not be mimicked by fructose, but the decrease induced was of the same magnitude as the serosal effect of glucose, but of opposite sign. The effects of serosal addition of glucose and fructose and mucosal addition of fructose can be explained by different reflection coefficients of the cell membranes for glucose, fructose and mannitol. The mucosal effect of glucose is explained by a glucose-dependent influx of sodium at the mucosal side, stimulating a ouabain-sensitive pump at the baso-lateral aspects of the cell.

Microscopical determination of the filtration permeability of the mucosal surface of the goldfish intestinal epithelium.

The rate of shrinkage of the mucosal folds of goldfish intestine in response to mucosal hypertonicity was measured by microscopic means. Because of the geometry of the intestinal folds the rate of shrinkage could be directly related to the loss of volume from the fold through the brush border membranes and tight junctions. Experimentally a wide range of velocities was observed, reflecting the difficulty of rapidly establishing a uniform osmotic gradient at the preparation's mucosal surface. The initial velocity of volume loss provided a measure of the filtration permeability (Pf) of the mucosal surface. From the highest velocities observed the filtration permeability was estimated to be approximately 14 X 10(-3) cm/sec related to the folded mucosal surface and 65 X 10(-3) cm/sec related to the straight serosal surface. Consideration of the experimental errors and unstirred layer effects make it probable that the latter value is still an underestimate of the true Pf. The series barriers of the epithelium cause the total tissue Pf to be less than the Pf of the mucosal surface alone. In addition the Pf measured in the presence of an osmotic gradient may differ substantially from the tissue filtration permeability which exists in the absence of a change in osmolarity.

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

1. In goldfish intestine (perfused unstripped segments and mucosal strips) the serosal addition of ouabain (10(-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(-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.

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 serosally added sugars on the transepithelial electrical properties of the perfused goldfish intestine.

1. A study has been made of the effect of serosally added sugars on the transmural potential difference and electrical resistance of the perfused goldfish intestine. 2. Addition of glucose at the serosal side resulted in a decrease of the transmural potential difference independent of the presence or absence of glucose at the mucosal side. The transepithelial resistance did not change. 3. The serosal glucose effect persisted in the presence of phlorizin at the mucosal side. 4. With the activity transported non-metabolized glucose analogue 3-oxy-methylglucose the same effects were observed as with glucose. 5. Replacement of NaCl by cholineCl, RbCl or LiCl at both sides of the intestine had a diminishing effect on the glucose evoked potentials and on the transepithelial conductance. 6. Phlorizin in concentrations lower than 10(-4) M, at the serosal side did not influence neither the mucosal nor the serosal glucose effects. 7. Ouabain at the serosal side inhibited the serosal glucose effect and decreased the transepithelial conductance. 8. The results support the concept that sugar transport at the serosal side of the epithelial cell has features in common with the sodium-dependent sugar transport mechanism at the mucosal side.

Model approaches for evaluation of cell coupling in monolayers.

The electrical coupling of epithelial cells of the intestine of chick embryo cultured in monolayers was studied. This coupling can be evaluated by regarding the monolayer as a honeycomb structure of cells and the cells as equipotential spaces, as long as the cells are small (diameter <25µm). With help of this discrete model it was found that for the non-junctional membrane the membrane resistance isϱ m =250-2,000 Ω cm(2), and for the junctional membraneϱ m =5-50 Ω cm(2). In addition to this discrete model, a continuous model was also considered and good agreement between the two descriptions was found. With the aid of the continuous model, a value for the non-junctional membrane capacitance (C m ) was obtained: 5-50 µF/cm(2). The electrical values are not corrected for membrane folding, microvilli and the like. Tentative corrections based on electron microscopy suggest: 1,000 <ϱ m < 10,000 Ω cm(2), 10 <ϱ m < 100 Ω cm(2), 1