Extracellular Chloride Concentration Research Articles

The membrane potential of human platelets

The membrane potential of the human platelet was investigated using the membrane potential probes 3,3′-dipropyl-2,2′-thiadicarbocyanine iodide and tritiated triphenylmethylphosphonium bromide. The membrane potential in physiologic buffer was estimated to be 52–60 mV inside negative. The membrane was depolarized when extracellular potassium or hydrogen ion concentrations were increased. Changes in extracellular sodium, chloride, or calcium ion concentration had no measurable effect on membrane potential. Elevated extracellular potassium has been shown to increase platelet sensitivity to the aggregating agent, adenosine diphosphate. Our results show that changes in extracellular ion concentrations that depolarize platelets increase platelet sensitivity to aggregating agents. These results suggest that membrane potential changes may play a role in modulating the response of platelets to aggregating agents.

Voltage-activated and calcium-activated currents studied in solitary rod inner segments from the salamander retina.

1. Solitary rod inner segments were obtained by enzymatic dissociation of the tiger salamander (Ambystoma tigrinum) retina. Their membrane currents were studied with the single-pipette voltage-clamp technique. Individual currents were isolated with the aid of pharmacological agents.2. Extracellular caesium blocked a current activated by hyperpolarization from -30 mV. Changing external sodium and potassium concentrations altered the value of the reversal potential in a manner consistent with the current being carried equally by both ions.3. Extracellular tetraethylammonium (TEA) blocked a current activated by depolarization from -70 mV. In normal medium this current had a reversal potential of -72 mV. Changing the external potassium concentration altered the value of the reversal potential in a manner consistent with the current being carried predominantly by potassium.4. Extracellular cobalt blocked a current activated by depolarization that had an initial inward and a later outward component.5. After EGTA was injected into an inner segment the outward component was suppressed. Cobalt then blocked an inward current. This current is believed to be carried predominantly by calcium. The conductance increased with depolarization from -45 mV and reached a maximum at approximately 0 mV. Following a step of depolarization the current activated rapidly (< 20 msec) and then remained constant for at least several seconds without evidence of inactivation.6. Injecting caesium into an inner segment eliminated a calcium-activated outward current believed to be carried by potassium ions.7. After the injection of caesium there remained another calcium-activated current with a reversal potential of -17 mV. Changing extracellular chloride concentration altered the value of the reversal potential in a manner consistent with chloride carrying at least 70% of the current. Another anion may carry the balance.8. When the five currents mentioned in items 2, 3, 5, 6 and 7 were blocked, the membrane resistance between -90 and -25 mV was linear, time-independent, and had a high value (2.1 GOmega).9. The five identified currents can all be activated in the physiological range of voltage in which salamander rods normally operate.

The external anion binding site of the human erythrocyte anion transporter: DNDS binding and competition with chloride

The interaction between chloride and the anion transport inhibitor DNDS (4,4'-dinitro stilbene-2,2'-disulfonate) at the external anion binding site of the human erythrocyte anion transporter was examined by two techniques: a) chloride tracer flux experiments in the presence of varying concentrations of DNDS, and b) DNDS equilibrium binding experiments in the presence of varying concentrations of intracellular and extracellular chloride, Cli and Clo. DNDS inhibited competitively the Clo-stimulated chloride efflux from intact red cells at 0 degree C and pH 7.8 with an inhibitor constant of 90 nM. Under the same conditions DNDS bound reversibly to one class of binding sites on intact cells with a capacity of 8.5 X 10(5) molecules/cell. Clo competitively inhibited DNDS binding with an inhibitor constant of 6 mM. In the absence of Clo the DNDS binding constant was 84 mM. The competition between chloride and DNDS was also tested in nystatintreated cells in which Clo always equaled Cli. Under these conditions the values of the DNDS binding constant and the chloride inhibitor constant were significantly larger. All these data were in quantitative agreement with a single-site, alternating access kinetic scheme with ping-pong-type kinetics that we have previously developed for modeling chloride exchange transport. The data also served to rule out special cases of an alternative two-sited sequential-type kinetic scheme. DNDS binding experiments were also performed at 10 and 20 degree C. We found that neither the DNDS binding constant nor the Clo inhibitor constant were significantly changed compared to 0 degree C.

Biphasic response of hippocampal pyramidal neurons to GABA

GABA released either iontophoretically or synaptically near pyramidal neurons in the CA1 region of the rat hippocampal slice could produce a biphasic response: a hyperpolarization followed by a depolarization. The depolarizing component elicited by either method was accompanied by an increased membrane conductance, and a reduction in neuronal discharge. The depolarization was reversed at a potential which was less negative than the resting membrane potential; it was blocked by antagonists of GABA action such as picrotoxin; it was sensitive to manipulation of extracellular chloride concentration; and it persisted in the presence of concentrations of cobalt or manganese which were sufficient to block evoked synaptic activity. Iontophoresis of GABA near the apical dendrites elicited an initial depolarization rather than an initial hyperpolarization, suggesting a dendritic origin for the depolarizing component. Together, these results suggest that GABA can produce, in the same neuron, both hyperpolarizing and depolarizing responses which depend at least in part upon changes in chloride conductances.

The action of apamin on guinea-pig taenia caeci

Apamin (10 −7 M), a substance extracted from bee venom ( apis mellifica) causes stimulation of the taenia caeci as seen from an increase in spike activity. The inhibitory effect of ATP or adrenaline (Adr) was reflected by hyperpolarization of the muscle cell, cessation of spike activity and relaxation of the muscle. The 42K efflux and the membrane conductance were enhanced in the presence of these substances. Apamine converted the hyperpolarization caused by ATP or Adr into a transient depolarization which produced contraction of the muscle cells. The changes in membrane conductance and 42K efflux were diminished by the bee toxin. Furthermore, the potassium-dependent phase of the action potential was lengthened by apamine. Reduction of the extracellular chloride or sodium concentration, blockade of the nervous system by TTX (3 × 10 −7 M) or inhibition of spike activity by D600 (3 × 10 −6 M) did not affect the excitatory and blocking action of apamin. A high concentration of the calcium antagonist D600 (10 −4 M) or omission of extracellular calcium was needed to reduce the transient depolarization evoked by ATP or Adr in the presence of apamin. It is concluded that apamin prevents the opening of the ATP- and Adr-sensitive and voltage-dependent potassium channels in guinea-pig taenia caeci.

Influence of chloride, potassium, and tetraethylammonium on the early outward current of sheep cardiac Purkinje fibers.

In voltage clamp studies of cardiac Purkinje fibers, a large early outward current is consistently observed during depolarizations to voltages more positive than -20 mV. After the outward peak of the current, the total membrane current declines slowly. Dudel et al. (1967. Pfluegers Arch. Eur. J. Physiol. 294:197--212) reduced the extracellular chloride concentration and found that the outward peak and the decline of the current were abolished. They concluded that the total membrane current at these voltages was largely determined by a time- and voltage-dependent change in the membrane chloride conductance. We reinvestigated the chloride sensitivity of this current, taking care to minimize possible sources of error. When the extracellular chloride concentration was reduced to 8.6% of control, the principal effect was a 20% decrease in the peak amplitude of the outward current. This implies that the membrane chloride conductance is not the major determinant of the total current at these voltages. The reversal potential of current tails obtained after a short conditioning depolarization was not changed by alterations in the extracellular chloride or potassium concentrations. We suspect that the tail currents contain both inward and outward components, and that the apparent reversal potential of the net tail current largely reflects the kinetics of the outward component, so that this experiment does not rule out potassium as a possible charge carrier. The possibility that potassium carries much of the early outward current was further investigated using tetraethylammonium, which blocks potassium currents in nerve and skeletal muscle. This drug substantially reduced the early outward current, which suggests that much of the early outward current is carried by potassium ions.

Voltage-dependent calcium current induced by serotonin.

CALCIUM ions have a central role in the regulation of neural activity1,2. Changes in intracellular calcium concentration affect membrane permeability to other ions and thereby alter membrane excitability3. Furthermore, calcium is required for transmitter release from nerve terminals4. A neurotransmitter that specifically induces an inward calcium current might have profound effects. We report here that in some neurones of Aplysia, serotonin can induce such a calcium current. A previous investigation5 revealed that iontophoretically applied serotonin elicited an unusual excitatory response in some neurones of Aplysia. At hyperpolarised potentials the neurotransmitter produced a slow excitation (component 1) that behaved simply as an increase in sodium conductance, similar to the A′ response of Gerschenfeld and Paupardin–Tritsch6. At depolarised potentials (>−30 mV), the response was voltage dependent and behaved as a regenerative inward current (component 2). At all potentials, the slow excitatory response to serotonin was insensitive to changes in extracellular chloride or potassium concentration. However, in sucrose-substituted sodium-free solutions, the response was greatly attenuated. Although a calcium current was not excluded, it was tentatively suggested that sodium was responsible for the regenerative inward current as well as for component 1. Further study has now shown that the regenerative inward current component is carried by calcium ions.

The dependence of membrane potential on extracellular chloride concentration in mammalian skeletal muscle fibres.

1. The steady-state intracellular membrane potential of fibres in thin bundles dissected from mouse extensor digitorum longus or soleus muscles or rat sternomastoid muscles was measured with 3 M-KCl glass micro-electrodes. The steady-state membrane potential was found to depend on the extracellular concentrations of Na, K and Cl ions. 2. The resting membrane potential (3.5 mM-[K]o, 160 mM-[Cl]o) was -74 +/- 1 mV (mean +/- S.E.) and a reduction in [Cl]o to 3.5 mM caused a reversible steady-state hyperpolarization to -94 +/- 1 mV (mean +/- S.E.). 3. The steady-state membrane potentials recorded in fibres exposed to different [K]o and zero [Cl]o were consistent with potentials predicted by the Goldman, Hodgkin & Katz (GHK) equation for Na and K. The results of similar experiments done with Cl as the major external anion could not be fitted by the same equation. 4. The GHK equation for Na, K and Cl did fit data obtained from fibres in solutions containing different [K]o with Cl as the major external anion if the intracellular Cl concentration was allowed to be out of equilibrium with the steady-state membrane potential. 5. It is suggested that an active influx of Cl ions controls the intracellular Cl concentrations in these fibres and hence maintains the Cl equilibrium potential at a depolarized value with respect to the resting membrane potential. 6. The steady-state membrane potential of rat diaphragm fibres was independent of [Cl]o and it seems likely that the intracellular Cl concentration of these fibres is not controlled by active Cl transport.

Chloride conductance of the amphiuma red cell membrane.

Like most other red cells, the giant erythrocytes of Amphiuma means possess a system for rapid exchange of chloride across the membrane. Also, there are indications that the net transport of chloride in these cells is slow. The size of Amphiuma erythrocytes allows direct measurements of membrane potential with microelectrodes. The present work exploits the possibility that such measurements can be used to give a quantitative estimate of the chloride conductance (GCl) of the Amphiuma red cell membrane. The membrane potential was measured as a function of extracellular chloride concentration (5-120mM), using an impermeant anion (Para-amino-hippurate) as a substitute. Furthermore, the effect of different pH values (6.0-7.2) was studied. For each extracellular chloride concentration the membrane potential was determined at a pH at which hydroxyl, hydrogen, and bicarbonate ions were in electrochemical equilibrium. From these membrane potentials and the corresponding chloride concentrations in the medium (at constant intracellular ion concentrations), the GCl of the membrane was calculated to be 3.9 x 10-7 omega-1 cm-2. This value is some six orders of magnitude smaller than that calculated from the rate of tracer exchange under equilibrium conditions. The experimental strategy used gives the values for a "partial transference number" which takes into account only ions which are not in electrochemical equilibrium. Whereas this approach gives a value for GCl, it does not permit calculation of the overall membrane conductance. From the calculated value of GCl it is possible to estimate that the maximal value of the combined conductances of hydroxyl (or proton) and bicarbonate ions is 0.6 x 10-7 omega-1 cm-2. The large discrepancy between the rate of exchange of chloride and its conductance is in agreement with measurements on human and sheep red cells employing the ionophore valinomycin to increase the potassium conductance of the membrane. The results in the present study were, however, obtained without valinomycin and an accompaning assumption of a constant field in the membrane. Therefore, the present measurements give independent support to the above mentioned conclusions.

Dependency of the resting membrane potential on the extracellular chloride concentration in identified neurons of Helix pomatia

In identified neurons of Helix pomatia, a withdrawal of extracellular Cl − led (i) to a transient hyperpolarization and to a following permanent depolarization of the normal resting membrane potential (RMP), (ii) to a decrease of the membrane resistance and (iii) to a decline of the intracellular Cl − and K + activity. After readmission of control solution a further depolarization occurred transiently. It is concluded that membrane potential changes under Cl − free solution are caused by an increase in K + conductance and a decrease of the K + equilibrium potential.

On the mechanism for radiosensitization of microorganisms by sodium chloride with special reference to their DNA repair capacity.

Some biological aspects of radiosensitization of microorganisms by NaCl at comparatively higher concentrations were studied. Among different strains of bacteria and yeasts, the radioresistant vegetative form was particularly sensitive to NaCl present during irradiations. With E. coli, such radiosensitizing effect of NaCl was abolished by mutations at the pol and rec loci or by the addition of certain solutes at the time of irradiation. The effective extracellular chloride concentrations for E. coli B/r were not less than 0.1 m. When actinomycin D and chloramphenicol were added during reincubation of E. coli B/r following irradiation in the presence of NaCl, the radiation lethal effect was enhanced, the extent of which was almost independent of doses of irradiation. From these results together with the previous data, the basic mechanism for NaCl radiosensitization was discussed with special references to a possible role of Cl2− radical anions as attacking species and to the inhibition of pol- and rec-depen...

Interaction of the complex between cloacin and its immunity protein and of cloacin with the outer and cytoplasmic membranes of sensitive cells.

1 The interaction of a complex of [14C]cloacin and its immunity protein (cloacin complex) and of [14C]cloacin itself with the outer and cytoplasmic membranes of sensitive cells has been studied to elucidate the difference in specific killing activity of both bacteriocin preparations [Eur. J. Biochem. 73, 107–114 (1977)]. 2 No significant difference exists between cloacin complex and cloacin with respect to the specificity of adsorption, the adsorption characteristics and the kinetics of binding to the outer membrane receptors of sensitive cells grown in brain/heart infusion (about 425 receptors per cell). 3 The adsorption of the bacteriocin onto the receptors shows a temperature optimum between 35°C and 40°C, but is not affected by variation in pH between 5.0 and 8.0 or extracellular potassium chloride up to a concentration of 0.5 M. An apparent association constant of 0.6 × 108 M−1 is calculated at 37°C and pH 7.0. 4 At 37°C and pH 7.0 virtually all the cloacin molecules adsorbed by sensitive cells can be digested by trypsin during at least 3 h after adsorption, in contrast to the adsorbed molecules of cloacin complex which become gradually less accessible to trypsin. 5 No significant difference exists between the affinity of cloacin complex and cloacin for cytoplasmic membrane vesicles isolated from sensitive cells. At 37°C and pH 7.0 about 105 bacteriocin molecules can bind per vesicle with an apparent association constant of about 8 × 108 M−1. 6 These results indicate that most probably the removal of immunity protein from the cloacin complex in vitro results in a strong reduction of the transfer of biologically active cloacin molecules from the cell surface receptors to the cytoplasmic membrane in vivo. 7 This conclusion is confirmed by the observation that, in vivo, cloacin itself is less effective in the induction of a leakage of potassium ions across the cytoplasmic membrane compared to the cloacin complex. Concomitant with the reduction of the leakage of potassium ions a decrease in killing activity is observed. Increasing concentrations of extracellular potassium chloride gradually reduce the killing activity of cloacin complex to 3.1% and of cloacin to 45.5% in the presence of 0.2 M potassium chloride.

Influence of chloride and sodium pump activity on carbachol- and acetylcholine-induced depolarizations in denervated rat diaphragm

The effect was measured of low chloride medium and ouabain on carbachol- and acetylcholine-induced depolarizations in denervated rat diaphragm. Membrane potentials were measured in rat diaphragm muscle fibres 10–14 days after denervation. Depolarizations induced by carbachol and acetylcholine were increased when the extracellular chloride concentration was diminished from 110 to 40 or 10 mM. Sodium pump inhibition by ouabain (10 −4 M) dramatically enhanced the carbachol depolarizations. In 40 mM Cl − solutions, dose—response relations were determined with ACh in the absence or presence of dTC. For ACh a pD 2 value of 2 was found; the pA 2 value for dTC appeared to be about 6.4. Therefore, the binding properties of the post-denervational receptors do not appear to be influenced by the chloride gradient. It was concluded that drug-induced depolarizations of the denervated rat diaphragm are short-circuited by chloride ions and counteracted by sodium pumping.

The transport of chloride in Ehrlich ascites tumor cells.

The steady state transport and distribution of chloride between the intracellular and extracellular phases was investigated when the extracellular chloride concentration was varied by isosmotic replacement with nitrate, bromide and acetate. The results of these experiments show that chloride transport, measured by uptake of 36Cl, is sensitive to the replacement anion. In the presence of nitrate, chloride transport is a linear function of the extracellular chloride concentration. The relationship between chloride transport and extracellular chloride in the presence of bromide is concave upward which suggests that this anion inhibits chloride movement. However, when acetate replaces chloride, the relationship between chloride transport and extracellular chloride is concave downward. The chloride distribution ratio of cells incubated in 145-155mM chloride medium is 0.386 and is not effected by the replacement of chloride with nitrate, bromide or acetate. These findings are consistent with the assertion that chloride transport is composed of two parallel pathways, a diffusional plus a saturating, mediated component. Of the total chloride flux (9.1 mmoles Cl-/kg dry weight per minute) measured in chloride medium (145-155 mM Cl-), the mediated component represents 40% and the diffusional component 60%.

The transport of sulfate ions across the membrane of the ehrlich ascites tumor cell

The applicability of the membrane fixed charge hypothesis to anion transport in Ehrlich ascites tumor cells was studied by investigating the dependence of steady state sulfate transport on the extracellular pH, chloride and sulfate concentration. When the extracellular sulfate was maintained at 10 mM both cellular sulfate and sulfate transport increased with decreasing pH and chloride concentration. The dependence of sulfate transport on the cellular sulfate concentration suggests a saturation phenomenon. The relationship between sulfate transport and cellular sulfate was also studied as a function of extracellular sulfate, both in the presence and absence of chloride. In both cases, sulfate transport is a saturable function of the cellular sulfate. However, in the presence of chloride the maximal flux is twice that in its absence. The discrepancy between the maximal fluxes suggests that the transport system mediates chloride-sulfate exchange in addition to sulfate self exchange. Unidirectional sulfate effluxes into chloride and sulfate-free medium; into 50 mM sulfate medium or 50 mM chloride medium were: 0.38, 1.95 and 3.91 nmoles/10-7 cells min-minus1, respectively. These results indicate that in the absence of either sulfate or chloride the net efflux of sulfate is low. However, chloride or sulfate on the trans side of the membrane is effective in accelerating unidirectional sulfate efflux. Taken together, the results of this investigation cannot be explained in terms of the membrane fixed charge hypothesis. Rather, they support the contention that sulfate transport across the tumor cell membrane is a carrier-mediated process.

Transmembrane potential and contraction of human heart in relation to potassium and ouabain.

The effects of extracellular potassium chloride concentrations (0, 2, 4, 4.6, 5, and 8 mM.) on simultaneously recorded transmembrane action potentials and contractions of human papillary muscle were investigated. In a potassium-free solution the early changes observed were a shortening of the action potential duration (APD) at the 25% level of repolariza-tion, and no change or a slight increase in the APD at the 90% level of repolarization. A temporal relation between the shortening of the APD at the 25% and 50% levels of repolarization and an increase in the force of contraction was observed. A direct relation between the increase in extracellular potassium concentration and an increase in the APD as-sociated with a corresponding decrease in the force of contraction was observed. Ouabain (10-9 and 10-8 M.) produced a shortening of the APD and an increase in the contraction similar to KCl-free solution. Ouabain was ineffective in producing its effects on the muscles prein-cubated in KCl-free solution. Exposure of ouabain treated muscles to a KCl-free solution containing ouabain resulted in a small increase in the contraction and a small shortening of the APD of the muscles. The combined effects of ouabain and KCl-free solution were not greater than the effects of a KCl-free solution alone. It is suggested that the changes in APD and force of contraction might be associated with potassium or oua-bain-induced changes in the adenosinetriphosphatase activity of the heart muscle.

Relationships between potassium, chloride, intracellular and extracellularH in dogs.

1. Total body, intracellular, and extracellular electrolyte and acid-base changes were studied following sodium nitrate-induced potassium and chloride depletion and subsequent selective chloride repletion. 2. It was found that potassium and chloride depletion was associated with markedly decreased extracellular Na, K, Cl and water content. Intracellular Na increased, intracellular K decreased and there was a large transcellular redistribution of acid, i.e. extracellular alkalosis and intracellular acidosis ensued. 3. Selective chloride repletion was associated with return of extracellular pH towards normal. Extracellular K and intracellular parameters showed very little change. Further chloride administration was followed by extracellular acidosis, but extracellular K remained low. 4. There was close relationship between extracellular chloride and hydrogen ion concentrations ([Cl−]e and [H+]e) and extracellular potassium and intracellular hydrogen ion ([K+]e and [H+]i) but not between any of the other parameters. The close relationship between [H+]e and [Cl−]e may be explained by the chemical relationship between [H+]e, Pco2, and [HCO3−]e and empirical relationships between [Cl−]e and [HCO3−]e, and Pco2 and [HCO3−]e.

Similarities between the vascular smooth muscle responses to sudden changes in external potassium, sodium and chloride ion concentrations.

AbstractThe effects of graded reductions in extracellular potassium, sodium and chloride concentrations on the spontaneous electrical and mechanical activity of the isolated rat portal vein have been studied. The immediate responses obtained on marked reduction in these ion concentrations and on readministration of normal solution were common to the three ion concentrations and on readministration of normal solution were common to the three ion species and differed from the long‐term effect of the ionic environment. Thus abrupt large reductions in the concentration of any of the three ions resulted in a transient excitation whereas return to normal Krebs solution led to a temporary complete inhibition of muscle activity. These initial, transient responses could in many cases not be predicted on the basis of the “constant field equation” by merely inserting the actual ion concentration values. However, if the concentrations of chloride and potassium ions (or chloride and sodium ions) were changed in a reciprocal manner no such transient responses occurred. Some possible explanations for this difference between the immediate and long‐term effects of a changed ionic environment are discussed

Similarities between the Vascular Smooth Muscle Responses to Sudden Changes in External Potassium, Sodium and Chloride Ion Concentrations

AbstractThe effects of graded reductions in extracellular potassium, sodium and chloride concentrations on the spontaneous electrical and mechanical activity of the isolated rat portal vein have been studied. The immediate responses obtained on marked reduction in these ion concentrations and on readministration of normal solution were common to the three ion species and differed from the long‐term effect of the ionic environment. Thus abrupt large reductions in the concentration of any of the three ions resulted in a transient excitation whereas return to normal Krebs solution led to a temporary complete inhibition of muscle activity. These initial, transient responses could in many cases not be predicted on the basis of the “constant field equation” by merely inserting the actual ion concentration values. However, if the concentrations of chloride and potassium ions (or chloride and sodium ions) were changed in a reciprocal manner no such transient responses occurred. Some possible explanations for this difference between the immediate and long‐term effects of a changed ionic environment are discussed.

Effects of chloride and bicarbonate on methemoglobin reduction in mouse erythrocytes

A methemoglobinemia generated by phenylhydroxylamine persists longer in mouse red cells suspended in Krebs-Ringer-phosphate-glucose (K-R-PO 4-G) than in vivo or in shed blood. Heparin or bovine serum albumin added to K-R-PO 4-G did not shorten the duration of the methemoglobinemic response to phenylhydroxylamine, and phosphate added to shed blood did not prolong it. When a Krebs-Ringer-bicarbonate-glucose (K-R-HCO 3-G) medium was used instead of K-R-PO 4-G, the pattern of the methemoglobinemic response to phenylhydroxylamine was altered to resemble that of shed blood. When methemoglobinemic cells originally exposed to nitrite are washed, methemoglobin reductase activity, as assessed by net decreases in methemoglobin concentration, is inversely related to the extracellular chloride concentration. This effect of chloride in prolonging a methemoglobinemia is shared by nitrate. Rates of methemoglobin reduction were not influenced by changes in red cell volume as modified by alterations in extracellular osmolality. The addition of bicarbonate to a chloride-free medium did not further stimulate reductase activity, but in the presence of a critical extracellular chloride concentration, bicarbonate stimulates methemoglobin reduction. Since these effects are still seen when lactate is substituted for glucose, they are apparently mediated through the spontaneous NADH-linked reductive system (diaphorase). These findings suggest that the chloride shift may regulate the rate of methemoglobin reduction in mouse red cells.