Internal Chloride Concentration Research Articles

GABA and glycine channels in isolated ganglion cells from the goldfish retina.

1. Adult goldfish retinas were enzymatically dissociated and ganglion cells were maintained in culture for periods of 1-5 days. Ganglion cells could be identified by their morphology, and this identification was confirmed by retrograde transport of the fluorescent dye Fast Blue injected into the optic nerve stub. 2. All the ganglion cells tested responded to 30 microM-GABA or 100 microM-glycine between 2 and 30 h after enzymatic dissociation of the retina. 3. Whole-cell responses to 30 microM-GABA or glycine declined over a period of seconds during sustained applications of the agonists, probably as a result of desensitization. There was an irreversible decline in the peak whole-cell response to repeated applications of 30 microM-GABA unless the pipette-filling solution contained 2 mM-ATP, 4 mM-Mg2+, 10 mM-EGTA and no added Ca2+. Both GABA and glycine responses also showed an irreversible decline in outside-out patches but, in this case, Mg2+, ATP, and very low Ca2+ failed to stabilize the response. 4. Whole-cell currents activated by both GABA and glycine were demonstrated to be chloride-selective by investigating the dependence of reversal potential (Vr) on internal chloride concentration ([Cl-]i). For GABA responses, the dependence of Vr on [Cl-]i could not be distinguished from that predicted by the Nernst relation. For glycine, deviations from Nernstian dependence were observed, but the permeability to Cl- was at least 20 times greater than to isethionate, SO4(2-), or monovalent cations (Na+ and Cs+). 5. Bicuculline methochloride (10 microM) selectively blocked responses to 3-30 microM-GABA without affecting responses to 30 microM-glycine. Bicuculline itself was not as selective. At agonist concentrations of 30 microM, 3 microM-bicuculline partially blocked the response to GABA but not that to glycine, but bicuculline at 10 microM blocked responses to both GABA and glycine. Strychnine (0.3-1 microM) blocked responses to 30 microM-glycine but also competitively antagonized GABA responses. Picrotoxinin (10 microM) blocked responses to 3 microM-GABA in some cells but also partially antagonized responses to 30 microM-glycine. 6. GABA channels had at least two conductance states at 10-12 degrees C in nearly symmetrical (141 mM in, 142 mM out) chloride. The slope conductance of the most frequently observed (main) state was 16 +/- 2 pS. The reversal potential for the main state was not significantly different from the chloride equilibrium potential (0 mV).(ABSTRACT TRUNCATED AT 400 WORDS)

Chloride conductance activated by external agonists and internal messengers in rat peritoneal mast cells.

1. Stimulation of mast cells by externally applied secretagogues activated a slowly developing membrane current. With high external and low internal chloride (Cl-) concentrations, the current reversed at about -40 mV, but when external Cl- was made equal to internal Cl-, the reversal potential shifted to about 0 mV, demonstrating that the current carrier was Cl-. 2. In addition to external agonists, internally applied cyclic AMP and high concentrations of intracellular calcium [Ca2+]i could also activate the Cl- current. However, elevated [Ca2+]i produced only slow and incomplete activation. This suggests that the Cl- current is not directly Ca2+ activated. Also, activation of Cl- current by external agonists and by cyclic AMP was unimpaired when [Ca2+]i was clamped to low levels with internal ethylene glycol bis-N,N,N',N'-tetraacetic acid (EGTA), indicating that elevated [Ca2+]i is not necessary for activation of the Cl- current. Although activation by cyclic AMP was faster than that produced by elevated [Ca2+]i, it still required tens of seconds; thus the effect of cyclic AMP was also likely to be indirect. 3. Internal guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) could also activate the Cl- current, suggesting the involvement of a G protein in the control of the current. 4. The variance associated with the Cl- current was small, and noise analysis gave a lower limit of about 1-2 pS for the single-channel conductance. The Cl- current was reduced by 4,4'-diisothiocyano-2,2'-stilbenedisulphonate (DIDS), and during DIDS blockade, the variance of the current increased. This suggests that DIDS enters and blocks the open channel. 5. Activation of the Cl- current would make the membrane potential negative following stimulation of a mast cell, thus providing a driving force for entry of external calcium via the stimulation-induced influx pathways described in the preceding paper (Matthews, Neher & Penner, 1989).

P 2-purinoceptor-mediated membrane currents in DDT 1 MF-2 smooth muscle cells

The electrophysiological response evoked by ATP was investigated in the DDT 1 MF-2 smooth muscle cell line using the microelectrode technique and the whole-cell patch clamp technique. Application of ATP (10 −3 M) to the bathing solution caused a small initial depolarization of the cell membrane, followed by hyperpolarization and slow depolarization. During voltage clamping (−50 mV) a triphasic response was recorded on stimulation with ATP (10 −4-10 −3 M). A short-lasting inward current was followed by a transient outward current and a slowly decreasing inward current. This response was not affected by the receptor antagonists, propranolol (3 × 10 −6 M), phentolamine (3 × 10 −6 M), atropine (3 × 10 −6 M) or theophylline (10 −3 M). The ATP-induced currents were not modified by the voltage-dependent channel blocking agents, tetraethyl ammonium (3 × 10 −3 M), 3,4-diaminopyridine (10 −3 M), tetrodotoxin (3 × 10 −7 M) or diltiazem (10 −5 M). The fast inward current was not detectable at a low ATP concentration (10 −5 M). The outward current showed a reversal potential near −76 mV, which equals the potassium equilibrium potential. This current was abolished after neutralization of the potassium electrochemical gradient. The outward current was suppressed under calcium-free conditions and also in the presence of tolbutamide (10 −4 M) or glipizide (5 × 10 −6 M). Guanosine triphosphate (5 × 10 −6 M) promoted the outward current, while this current was inhibited in the presence of guanosine diphosphate (5 × 10 −6 M). Both inward currents persisted after reduction of the external sodium concentration or the internal chloride concentration and also in the absence of external magnesium or calcium. The fast inward current, however, declined after repeated stimulation of the cell with ATP under calcium-free conditions. The results indicate that, in DDT 1 MF-2 smooth muscle cells, ATP activates purinoceptor-operated ion channels, represented by a fast calcium current followed by a transient potassium current, this effect being calcium and GTP-dependent, and a non-specific inward current.

Effects of pH, potential, chloride and furosemide on passive Na+ and K+ effluxes from human red blood cells.

Ouabain-resistant effluxes from pretreated cells containing K+/Na+ = 1.5 into K+ and Na+ free media were measured. Furosemide-sensitive cation effluxes from cells with nearly normal membrane potential and pH were lower in NO3- media than in Cl- media; they were reduced when pH was lowered in Cl- media. When the membrane potential was positive inside furosemide increased the effluxes of Na+ and K+ (7 experiments). With inside-positive membrane potential the furosemide-insensitive effluxes were markedly increased, they decreased with decreasing pH at constant internal Cl- and also when internal Cl- was reduced at constant pH. The correlation between cation flux and the membrane potential was different for cells with high or low internal chloride concentrations. The data with chloride greater than or equal to 47 mM showed a better fit with the single-barrier model than with the infinite number-of-barriers model. With low chloride no significant correlation between flux and membrane potential was found. The data are not compatible with pure independent diffusion of Na+ and K+ in the presence of ouabain and furosemide.

GABA-mediated synaptic inhibition of projection neurons in the antennal lobes of the sphinx moth, Manduca sexta.

Responses of neurons in the antennal lobe (AL) of the moth Manduca sexta to stimulation of the ipsilateral antenna by odors consist of excitatory and inhibitory synaptic potentials. Stimulation of primary afferent fibers by electrical shock of the antennal nerve causes a characteristic IPSP-EPSP synaptic response in AL projection neurons. The IPSP in projection neurons reverses below the resting potential, is sensitive to changes in external and internal chloride concentration, and thus is apparently mediated by an increase in chloride conductance. The IPSP is reversibly blocked by 100 microM picrotoxin or bicuculline. Many AL neurons respond to application of GABA with a strong hyperpolarization and an inhibition of spontaneous spiking activity. GABA responses are associated with an increase in neuronal input conductance and a reversal potential below the resting potential. Application of GABA blocks inhibitory synaptic inputs and reduces or blocks excitatory inputs. EPSPs can be protected from depression by application of GABA. Muscimol, a GABA analog that mimics GABA responses at GABAA receptors but not at GABAB receptors in the vertebrate CNS, inhibits many AL neurons in the moth.

Intracellular salt concentrations of the anaerobic halophilic eubacteria Haloanaerobium praevalens and Halobacteroides halobius

The obligately anaerobic, moderately halophilic eubacteria Haloanaerobium praevalens (isolated from Great Salt Lake, Utah) and Halobacteroides halobius (isolated from the Dead Sea) were found to contain high intracellular potassium concentrations (0.76–2.05 M, not well correlated with the external NaCl concentration), and high intracellular sodium concentrations (0.28–2.6 M, increasing with increasing extracellular NaCl concentration). The sum of intracellular potassium and sodium concentrations approximated the total cation concentration of the medium. Internal chloride concentrations in Haloanaerobium praevalens equalled the external chloride concentration. Organic solutes, such as betaine, glycerol, and different amino acids that have been shown to serve as compatible solutes in other halophilic eubacteria and eukaryotic organisms, were not found in significant concentrations. Analysis of the amino acids in the proteins of the anaerobic halophilic eubacteria showed an excess of acidic amino acids, similar to that reported in halophilic archaebacteria of the genus Halobacterium.

Slow potential changes in mammalian muscle fibers during prolonged hyperpolarization: transport number effects and chloride depletion.

Mammalian skeletal muscle fibers exhibit large slow changes in membrane potential when hyperpolarized in standard chloride solutions. These large slow potential changes are radically reduced in low chloride solutions, where the faster and smaller potential change ("creep"), usually observed in amphibian fibers, becomes apparent. The slow potential change during a hyperpolarizing current pulse leads to an increase in apparent resistance of up to nine times the instantaneous value and takes minutes to reach a steady value. It then takes a similar time to decay very slowly back to the resting membrane potential after the current pulse. The halftime for the slow potential change was found to be inversely proportional to the current magnitude. From measurements of immediate post-pulse membrane potentials, assuming constant ionic permeabilities, the internal chloride concentration was calculated to decrease exponentially towards a steady value (e.g., for one fiber from 12.3 to 6.6 mM after a 330-sec pulse). The time course and magnitude of the concentration change were predicted from chloride transport number differences, and the known and measured properties of the fibers, and were found to agree very well with the values obtained from experimental measurements. In addition, the shapes of the V2-V1 responses, measured in the three-electrode current clamp set-up with either potassium chloride or potassium citrate current electrodes, were as predicted by transport number chloride depletion effects and were at variance with the predictions of a permeability change mechanism.

The ionic mechanism of the dopamine response in retzius cells of two leech species ( Hirudo medicinalis and Haemopis sanguisuga)

1. Dopamine (DA) hyperpolarises the membrane potential of Retzius cells from the leeches Hirudo medicinalis and Haemopis sanguisuga by increasing the peability of the membrane to chloride ions. 2. The DA response could not be reversed by hyperpolarising the cell membrane. 3. The values for internal chloride concentration ( Hirudo, 8.6 ± 1.0 mM; Haemopis, 9.0 ± 1.0 mM) and chloride reversal potential ( Hirudo, 69.7 ± 3.4 mV); Haemopis, 68.6 ± 2.6 mV) calculated from the DA response did not differ signigicantly from those calculated from the 5-hydroxytryptamine (5-HT) induced chloride event. 4. It is concluded that DA and 5-HT activate the same type of chloride ionophore in the membrane of the leech Retzius cell.

Effects of monocarboxylates and external pH on some parameters of insect muscle fibres

The resting membrane potential and the conductance of flight muscle fibres of the butterfly Pieris brassicae were measured by means of two intracellular electrodes. The intracellular potassium, chloride, and hydrogen ion concentrations were estimated by measuring the concentrations in diluted muscle homogenates. Chloride in the bath was replaced in part by monocarboxylates and the pH was subsequently lowered. Replacement of chloride by propionate caused decrease in the internal chloride concentration, a marked hyperpolarization and a small increase in conductance. Lowering the pH in the propionate saline caused an additional decrease in the internal chloride concentration, an increase in the internal hydrogen ion concentration, a marked depolarization and a concurrent decrease in conductance. This was followed by an irreversible increase in conductance. With glycolate the effects on the membrane parameters were small and with pyruvate no effect was observed.

ATP-Dependent chloride influx into internally dialyzed squid giant axons.

Measurements were made of 36C1 influx into squid giant axons whose internal solutes were controlled by means of internal dialysis. When the intracellular chloride concentration was 50mM and the internal concentration of adenosine 5'-triphosphate (ATP) was 4mM, the average chloride influx was 11.6 pmoles/cm2 x sec. When the axons were dialyzed with an ATP-free solution, the average influx fell to 5.1 pmoles/cm2 x sec. The effect was fully reversible upon the return of ATP to the dialysis fluid. Chloride-36 influx in the presence and absence of ATP was found to be inversely related to the internal chloride concentration.

Membrane permeability changes during stimulation of isolated salivary glands of Calliphora by 5-hydroxytryptamine.

1. The membrane resistance of isolated salivary glands was found to decrease in response to 5-HT. The change in resistance was calcium-dependent. 2. The resistance change of the apical membrane was found to be much greater than the change in resistance of the basal membrane. 3. Potential responses under current-clamped conditions showed that one part of the biphasic response to 5-HT (attributed to an increase in chloride permeability) could be reversed and the other part (attributed to an increase in a potassium pump) could not. 4. These observations have been incorporated into a model which, on evaluation, predicts all of the observed potential changes during the action of 5-HT. It suggests that the potential responses reflect changes in the internal chloride concentration produced by the calcium-dependent increases in chloride permeability.

Factors affecting the net fluxes of ions in the isolated perfused gills of freshwaterAnguilla dieffenbachii

The net fluxes of sodium, chloride and potassium across the gills of freshwaterAnguilla dieffenbachii were measured using an isolated, perfused preparation. Reductions in the external sodium and chloride concentrations below 1.0 mMol/l resulted in an increasing net outflux of these ions from the gill (Fig. 1) whereas reductions in the internal sodium and chloride concentrations had no significant effect on the net fluxes of these ions (Fig. 2). Increases in the internal sodium and chloride concentrations above those normally found in the serum resulted in increasing net outfluxes of these ions (Fig. 2). Reductions in the internal potassium concentration below 1.0 mMol/l resulted in an increasing net outflux of sodium essentially similar to that obtained on reducing the external sodium concentration (Fig. 3). These results are discussed with reference to the possible active transport of ions across the branchial epithelium of teleosts in fresh water. In particular, the involvement of Na+/K+ exchanges in such transport processes are considered.

The ionic mechanism for 5-hydroxytryptamine inhibition on retzius cells of the leech Hirudo medicinalis

1. 1. Intracellular recordings were made from Retzius cells in the segmental ganglia of the leech, Hirudo medicinalis, and the leech, Haemopsis sp. 5-Hydroxytryptamine (5-HT) hyperpolarized the membrane potential of these cells. 2. 2. The inhibitory effect of 5-HT was due to an increase in chloride permeability of the cell membrane. No evidence was found for a potassium component in this response. The internal chloride concentration was calculated to be 8·4 mM which gave a chloride equilibrium potential of — 70 mV. 3. 3. Part of the membrane potential was due to an electrogenic pump but 5-HT did not act by stimulating this system. 4. 4. By the use of a specific 5-HT antagonist, mianserin, and by desensitization experiments it was concluded that 5-HT and strychnine (which also had an inhibitory action on these cells) acted via different receptors. 5. 5. It is concluded that 5-HT acted directly on the Retzius cell membrane to increase chloride conductance.

Activities of the sodium pump in cat pyramidal tract cell studied with intracellular injection of sodium ions.

Sodium ions were injected into cat pyramidal tract (PT) cells electrophoretically through an intracellular NaCl or Na glutamate-filled microelectrode. Following an injection there were decreases in the maximum rates of rise and fall of the spike potential and there was displacement of the inhibitory postsynaptic potential in a depolarizing direction. These changes recovered with an exponential time course, indicating concomitant changes in the internal sodium, potassium and chloride concentrations under the operation of the sodium pump in extruding excess sodium. From the exponential recovery curve, the rate constant of active sodium extrusion was estimated as about 60 hr−1 in fast PT cells and about 90 hr−1 in slow PT cells. It was suggested that the sodium pump was at least partly electrogenic, since the resting membrane was hyperpolarized by the sodium injection to the degree which depended on the amount of sodium-injecting current. Further support for this possibility was obtained by the experiment of high-frequency activation of PT cells, in which the sodium entry through the active membrane developed a slow post-tetanic hyperpolarization.

Inhibitory control of the abdominal stretch receptors of the crayfish. 3. The accessory reflex as a recurrent inhibitory feed-back.

AbstractJansen, J. K. S., A. Njå, K. Ormstad and L. Walløe, Inhibitory control of the abdominal stretch receptors of the crayfish. III. The accessory reflex as a recurrent inhibitory feed‐back. Acta physiol. scand. 1971. 81. 472–483.The autogenetic activation of the thick accessory fibre of the stretch recepton has been examined by intracellular recording and excitation of reflexly intact recepton. The steady‐state effect of this recurrent inhibitory feed‐back is to decrease the sensitivity of the receptor over its entire working range. The closed loop effect can be predicted from the mechanism of the inhibitory action and. the central coupling of the reflex. The effect of the feed‐back can be reversed by increased internal chloride concentration in the receptor. Some time‐dependent properties of the responses of the feed‐back loop are described.

The Use of Short-Term and Quasi-Steady Influx in Estimating Plasmalemma and Tonoplast Influx in Barley Root Cells at Various External and Internal Chloride Concentrations

Chloride influx measured with 36Cl over a 10 min loading period followed by a 5 min wash in inactive solution is used as an estimate of influx across the plasmalemma in barley roots. Chloride influx measured over a 60 min loading period followed by a 30 min wash is used as an estimate of the quasi-steady influx from the external solution to the vacuole

A Critical Study of the Evidence for Peripheral Inhibitory Axons in Insects

ABSTRACT The metathoracic anterior coxal adductor (a.c.a.) muscle of the locust and the grasshopper is innervated by a peripheral inhibitory axon similar to the inhibitory axon which innervates the metathoracic extensor tibiae muscles of these insects. No evidence was found to justify calling this axon an inhibitory-conditioning axon. Hyperpolarizing inhibitory postsynaptic potentials (IPSPs) are normally recorded from a.c.a. muscle fibres during stimulation of this axon, and if the bathing medium contains a high concentration of potassium ions the tonic fibres of the a.c.a. muscle relax slightly during inhibitory stimulation. The IPSPs are chloride potentials and can be converted to depolarizing responses by changing either the external or internal chloride concentration of the a.c.a. muscle fibres. Depolarizing IPSPs are frequently accompanied by small contractions of a.c.a. muscle fibres innervated by the inhibitory axon. The a.c.a. muscle fibres are permeable to potassium and chloride ions but influx of potassium chloride is much faster than efflux. Therefore when a.c.a. muscle fibres are loaded with chloride by exposing them to high-K saline (20−100 m-equiv. potassium/I.) and are then returned to normal (10 m-equiv. potassium/1.) saline the internal chloride concentration remains elevated for some time and during this period the equilibrium potential for the inhibitory response is less negative than the resting potential and the IPSPs are depolarizing. Depolarizing IPSPs are usually recorded from a.c.a. muscle fibres of locusts and grasshoppers when these fibres are transferred from their normal bathing medium, haemolymph, to 10 K saline. Probably the main reason for this reversal of the IPSPs is the entry of KC1 into the muscle fibres during dissection of the nerve-muscle preparations. Large quantities of KC1 would be released into the environment surrounding these preparations from muscle fibres cut and removed during dissection. Depolarizing IPSPs were more frequently recorded from muscle fibres of grassfed locusts than from fibres of starved locusts. The potassium concentration of haemolymph of grass fed locusts is higher than that of locust saline (10 m-equiv./l.). The potassium concentration of locust haemolymph presumably fluctuates in vivo but these fluctuations are too slow to affect the sign of the IPSP. The IPSPs are therefore always hyperpolarizing in vivo. The effect of changes in the potassium concentration of the bathing medium on the magnitude and polarity of the IPSP could account for the diverse responses recorded previously from a.c.a. muscle fibres of locusts and grasshoppers.

Some osmotic responses of the sipunculid worm Themiste dyscritum

1. 1. Themiste dyscritum, apparently stenohaline in nature, tolerates a wide range of salinities in the laboratory (168–639 mM Cl −, or 30–114% sea water). Long-term survival probably does not occur below about 50% sea water. 2. 2. T. dyscritum is a passive osmoconformer over this entire salinity range, but has an apparent ion deficit in the coelomic fluid, as indicated by low internal chloride concentrations relative to external levels. This apparent ion deficit is most marked in worms in low salinities, and may be made up by small molecular weight organic molecules. 3. 3. Water content is strongly dependent upon salinity, and the worms absorb large amounts of water osmotically after a transfer to a lower salinity. Volume regulation is restricted to worms adapted to high salinities and to transfers which are not excessive. 4. 4. Nephridial contents are little different in concentration from the coelomic fluid. 5. 5. Limited data on Phascolosoma agassizii in 84% sea water indicate the coelomic fluid is slightly hyperosmotic to the medium, but iso-ionic with respect to chloride.

Chloride Electrochemical Potentials and Membrane Resistances inNitella translucens

The chloride electrochemical potential difference between the inside of cells of Nitella translucens and the bathing medium has been measured by a direct electrical method employing Ag/AgCl electrodes. The membrane potential has been measured by means of conventional salt bridge microelectrodes. These data have been used to calculate the internal chloride concentration of the cells; the mean value obtained was 39 mM. This chloride electrochemical potential difference has been short-circuited thus causing an outward (depolarizing) electric current to flow through the cell membrane. The resulting membrane depolarization has been measured at two points along the length of the cell enabling the membrane resistance and space constant to be deduced; the respective values obtained were 24.8 KΩcm2 and 3.0 cm. It is suggested that these experiments lend additional support to the hypothesis that during the action potential in the Characeae there occurs a transient increase in the chloride conductance of the plasmalemma.

The internal chloride concentration of H and D cells in the snail brain

1. 1. A method is described of blocking microelectrodes with silver so that they become sensitive to chloride ion concentration. 2. 2. These electrodes have been used to measure the internal chloride concentration of the H cells and D cells in the brain of the snail. 3. 3. The H cells (which are hyperpolarized by acetylcholilne) have a lower chlotide concentration, 11·2 ± 0·6 mM, than the D cells (which are depolarized by acetylcholine), 24·7 ± 0·8 mM. 4. 4. It is suggested that the different actions of acetylcholine on the D and H cell is not simply to bring them to the chloride equilibrium potential, but that instead there are differences in the membranes of these types of cells and that in the H cellas, there is an increased permeability to chloride whilst in the D cell, there is an increased permeability to sodium ions.