External Chloride Concentration Research Articles

Glutamate-gated inhibitory currents of central pattern generator neurons in the lobster stomatogastric ganglion

Inhibitory glutamatergic neurotransmission is an elemental "building block" of the oscillatory networks within the crustacean stomatogastric ganglion (STG). This study constitutes the initial characterization of glutamatergic currents in isolated STG neurons in primary culture. Superfusion of 1 mM L-glutamate evoked a current response in 45 of 65 neurons examined. The evoked current incorporated two kinetically distinct components in variable proportion: a fast desensitizing component and a slower component. The current was mediated by an outwardly rectifying conductance increase and reversed at -48.8 +/- 5.3 mV. Reducing the external chloride concentration by 50% deflected the glutamate equilibrium potential (Eglu) by +14 mV, while increasing external potassium threefold shifted Eglu by up to +6 mV. Ibotenic acid fully activated both components of the glutamate response. Saturating concentrations of glutamate completely occluded neuronal responses to ibotenic acid, indicating that ibotenic acid was activating the same receptor(s) as glutamate. Millimolar concentrations of quisqualic acid, kainate, AMPA, and NMDA each failed to evoke any response. Picrotoxin (10(-4)M) completely blocked the glutamate response. Niflumic acid (100 microM) blocked > 80% of the desensitizing component and congruent to 50% of the sustained component. Reduction or elimination of extracellular calcium did not abolish the response. This study extends the ionic and pharmacological analysis of glutamatergic conductances in STG neurons. The currents described are consistent with glutamatergic inhibitory synaptic and agonist-evoked responses previously described in situ. We discuss their pharmacology, ionic mechanisms, and functional significance.

Osmolality and chloride concentration in the hemolymph of subadult Penaeus chinensis subjected to different salinity levels

Hemolymph osmolality, chloride concentration and tissue water of subadult Penaeus chinensis (12.65±2.55 g) transferred from 30 ppt to 10, 20, 30 (control) and 40 ppt seawater were examined after 0.25, 0.5, 1, 2, 5 and 10 days of exposure. No shrimp died during the experimental period. Hemolymph osmolality, chloride concentration and tissue water of shrimp transferred from 30 ppt to 10, 20 and 40 ppt reached constant levels within one-half day. Percentage tissue water decreased with external osmolality at a rate of 0.091% per one ppt. Isosmotic and isoionic values were 707 mOsm/kg (25.0 ppt) and 285 mM (18.4 ppt), respectively. Slopes of 0.39 and 0.34, obtained from the relationship between hemolymph osmolality and external osmolality and between chloride concentration and external chloride concentration, indicated that subadult P. chinensis osmoregulates between 10 and 40 ppt. It exhibited hyper-osmotic or hyper-ionic regulation in salinities below isosmotic or isoionic salinities, and hypo-osmotic or hypo-ionic regulation in those above.

Electrical properties of Y-79 cells, a multipotent line of human retinoblastoma

1. Whole-cell and perforated-patch tight-seal recording techniques were used to characterize the voltage-dependent membrane conductances of the Y-79 cells, a human retinoblastoma line composed of pluripotential retinal precursor cells. 2. Membrane resistance and capacitance were measured under current clamp, yielding approximate average values of 1.8 G omega and 26 pF, respectively. The cells are electrically excitable, and depolarization above -20 mV triggers slow action potentials. 3. Step depolarization of the membrane under voltage clamp elicits a high-threshold transient inward current, followed by a sustained, larger outward current. The outward current is carried by potassium ions, as determined by its susceptibility to blockage by K-channel antagonists [tetraethylammonium (TEA), Cs, and 4-aminopyridine (4-AP)] and insensitivity to reduction of external chloride concentration. 4. The isolated inward current displayed some unusual properties: its amplitude is directly related to extracellular calcium concentration, and replacement of calcium by magnesium completely abolishes it. However, none of the calcium channel antagonists tested (cadmium, nickel, nifedipine, and amiloride) exerted a substantial blockage. In addition, removal of external sodium or superfusion with tetrodotoxin significantly reduce the size of this current. 5. A single voltage-dependent conductance appears to underlie the inward current, because a variety of manipulations, such as changes in the holding potential, in the extracellular concentration of calcium or sodium, or superfusion with tetrodotoxin, failed to reveal the presence of kinetically distinct components. 6. The results suggest that a single voltage-dependent conductance mechanism underlies the depolarization-activated inward current in Y-79 cells. This channel appears to be primarily permeable to calcium, but with a significant contribution by sodium ions. Its functioning appears to be modulated by extracellular calcium.

The voltage-dependent step of the chloride transporter of Valonia utricularis encounters a Nernst-Planck and not an Eyring type of potential energy barrier

Voltage-clamp experiments were performed on cells of the giant marine alga Valonia utricularis to study the voltage dependence of the previously postulated chloride transporter (Wang, J., G. Wehner, R. Benz, and U. Zimmermann. 1991. Biophys. J. 59:235-248). Only one exponential current relaxation (apart from the capacitive spike) could be resolved up to a clamp voltage of approximately 120 mV within the time resolution of our experimental instrumentation (100 mus). This means that the rate constants of the heterogeneous complexation, k(R) (association) and k(D) (dissociation), were too fast to be resolved. Therefore, the "Läuger" model for carrier-mediated ion transport with equilibrium heterogeneous surface reaction was used to fit the experimental results. The voltage dependence of the initial membrane conductance was used for the evaluation of the voltage dependence of the translocation rate constant of the complexed carriers, k(AS). The initial conductance was found to be independent on the clamp voltage, which means that the translocation rate constant k(AS) is a linear function of the applied voltage and that the voltage dependence of the translocation of charged carriers through the plasmalemma could be explained by a square-type Nernst-Planck barrier. The movement of the complexed form of the carrier through the membrane may be explained by a diffusion process rather than by simple first-order kinetic jump across an Eyring-type potential well. The current relaxation after a voltage clamp was studied as a function of the external chloride concentration. The results allowed an estimation of the stability constant, K, of the heterogeneous complexation reaction and a calculation of the translocation rate constants of the free and the complexed carriers, k(s) and k(AS), respectively.

Effect of cytoplasmic chloride concentration on open-state preference of glycine-activated chloride channels in cultured spinal cord cells

1. Glycine was bath-applied to outside-out patches taken from cultured mouse spinal cord neurons. Glycine-activated chloride channels had at least six open states with permeabilities of 40 (A), 68 (B), 100 (C), 145 (D), 180 (E), and 250 (F) al/s (1 attoliter = 10(-18) liter). Usually no more than two such states were seen in any given patch. The usual extracellular chloride concentration was 157 mM; chloride concentration in the electrode (on the cytoplasmic face of the patch) was varied between 7 and 170 mM, replaced at lower concentrations with either of the inert anions gluconate or methane sulphonate. 2. With normal (7 mM) chloride concentration in the electrode, openings to state D were the most probable, and the average permeability of all openings was 115 al/s. When the cytoplasmic chloride concentration was 20 mM, the most probable open state was the lower-permeability state (C), and the average permeability was 92 al/s. In symmetrical 170 mM chloride, the most probable open state was B and the mean permeability 77 al/s. 3. The glycine-activated channels had a vanishingly small permeability to fluoride. However, in experiments in which 150 mM gluconate in the pipette was replaced by fluoride, the most probable open state was B, as in the high-chloride solution. Thus the two halides had similar effects on open state preference. 4. With external and internal chloride concentrations of 7 and 157 mM, respectively, single-channel current-voltage relations could be fit with constant field relations appropriate to one or more of the permeability states.(ABSTRACT TRUNCATED AT 250 WORDS)

Thiamin and derivatives as modulators of rat brain chloride channels

Several membrane fractions were prepared from rat brain by differential and sucrose density gradient centrifugation. Most fractions took up 36Cl − rapidly at a rate linear with time during the first 30–60 s, then the rate progressively slowed down. The lowest rate of uptake was found in the mitochondrial fraction. Oxythiamin partially inhibited 36C1 − uptake in all fractions. In P2 (crude synaptosomal fraction), oxythiamin decreased the initial rate of uptake by 32%, the apparent K 1, being 1.5mM. Thiamin and amprolium were less effective as inhibitors. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (0.1–1 mM) inhibited 36Cl − uptake by 40–50%. In the presence of this compound at a concentration ≥5 × 10 −4M, oxythiamin became ineffective. 36Cl − uptake was increased by GABA (0.1 mM) and this effect was antagonized by picrotoxin as expected, but not by oxythiamin. The rate of 36Cl − uptake did not appreciably depend on the external chloride concentration and was unaffected by bumetanide or by replacement of external Na + by choline. Taken together, these data suggest that the oxythiamin-sensitive 36Cl − influx is essentially diffusional and is not related to the GABA receptor or the Na:K:2Cl co-transport. Partial replacement of external Na + by K + or treatment with 0.1 mM veratridine (which should both result in membrane depolarization) increased 36Cl − uptake by 50 and 30% respectively; the inhibitory effect of oxythiamin was enhanced to the same proportion. When a brain homogenate was preincubated in the presence of 1 mM thiamin diphosphate and a synaptoneurosomal fraction was subsequently prepared from the homogenate, the vesicles took up more 36Cl − than control vesicles; the thiamin triphosphate content of the vesicles was also increased by pretreatment with thiamin diphosphate. When 36Cl − uptake was measured in vesicles depolarized by K + or veratridine, the difference between thiamin diphosphate-treated and control vesicles disappeared. The difference also disappeared in the presence of oxythiamin. A possible interpretation of these data is that membrane depolarization favors the opening of a chloride channel which is modulated by thiamin derivatives.

Guanosine 3',5'-cyclic monophosphate regulates calcium channels in neurones of rabbit vesical pelvic ganglia.

1. The effects of dibutyryl guanosine 3',5'-cyclic monophosphate (db-cyclic GMP) were studied in vitro on calcium channels of neurones in rabbit vesical parasympathetic ganglia, using intracellular and single-electrode voltage-clamp recordings. 2. Db-cyclic GMP (100 microM) caused membrane depolarization associated with a decrease in membrane input resistance and an after-hyperpolarization associated with an increase in membrane input resistance. 3. Db-cyclic GMP (0.01-1 mM) caused a concentration-dependent, transient inward current followed by a long-lasting outward current. Membrane conductance was increased and decreased during the inward and outward currents, respectively. 4. The db-cyclic GMP-induced inward current was depressed in nominally calcium-free solutions, by cobalt (1 mM) and nicardipine (10 microM). The mean reversal potentials of the inward current were +42 and -20 mV in the presence and absence of calcium in the external solution, respectively. 5. The db-cyclic GMP-induced inward current was not altered by lowering the external sodium concentration, raising external potassium concentration or by intracellular injection of caesium. 6. A calcium-insensitive component of the db-cyclic GMP-induced current was increased by lowering the external chloride concentration and blocked by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid, a chloride channel blocker. 7. Voltage-dependent, high-threshold calcium currents were depressed during the db-cyclic GMP-induced inward current and facilitated during the outward current. 8. Cyclic GMP was less potent than db-cyclic GMP in causing both inward and outward currents or modulation of calcium currents. GTP, GDP, GMP, guanosine, 8-bromoadenosine 3',5'-cyclic monophosphate and forskolin did not alter the holding current or voltage-dependent calcium currents. 9. It is concluded that intracellular cyclic GMP causes not only activation of resting calcium and chloride channels but also a transient depression followed by long-lasting facilitation of voltage-dependent calcium currents in neurones of vesical parasympathetic ganglia.

Extracellular MgATP activates the Cl‐/HCO3‐ exchanger in single rat cardiac cells.

1. The effect of extracellular MgATP on cytosolic pH (pHi) was investigated in single rat cardiac cells loaded with the pH-sensitive probe Snarf-1. 2. Basal pHi in HEPES-buffered solution (containing 4.4 mM-NaHCO3) was 7.08. MgATP induced a transient acidification followed by an alkalinization. The latter is prevented by ethylisopropylamiloride (EIPA) and has been attributed to the activation of the Na+/H+ antiport. The MgATP-induced acidification reached a maximal value of 0.42 +/- 0.03 pH units (U pH). It was concentration dependent with a K0.5 of 2.6 microM-MgATP. This acidification was also observed with the same magnitude in the presence of the more physiological Krebs-bicarbonate buffer but was greatly reduced in nominally HCO3-free HEPES. 3. The MgATP-induced acidification was prevented by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), probenecid and ethacrynic acid but not by bumetanide. It was dependent upon the external chloride concentration. The K0.5[Cl-] was 9 mM and the maximal acidification required 60 mM-Cl-. 4. MgATP accelerated the recovery from an alkalinization triggered by a pulse of NH4Cl. The nucleotide also facilitated the efflux of HCO3- when the cell was switched from a Krebs-bicarbonate buffer gassed with 5% CO2 to an HEPES buffer. 5. The acidification was only evoked by MgATP and its poorly hydrolysable analogues but not by the other nucleotides (ADP, GTP (guanosine triphosphate), CTP (cytidine triphosphate) UTP (urodine triphosphate), ITP (inositol triphosphate) nor by adenosine. It required the presence of Mg2+ ions. 6. These results provide evidence that MgATP activates the Cl-/HCO3- exchanger and that this activation accounts for the acidification. Such an activation could not be related to the P1- or the P2-purinergic receptors since it requires triphosphate adenylic compounds and Mg2+ ions. This leads us to suggest the existence of a putative P3-type of purinergic receptor.

A potential role for guanine nucleotide-binding protein in the regulation of endosomal proton transport.

The effects of guanosine 5'-triphosphate (GTP) and GTP-gamma-S, known activators of GTP binding proteins, on proton transport were investigated in endosome-enriched vesicles (endosomes). Endosomes were prepared from rabbit renal cortex following the intravenous injection of FITC-dextran. The rate of intravesicular acidification was determined by measuring changes in fluorescence of FITC-dextran. Both GTP and GTP-gamma-S stimulated significantly the initial rate of proton transport. In contrast, GDP-beta-S, which does not activate GTP binding proteins, inhibited proton transport. The rank order of stimulation was GTP-gamma-S greater than GTP greater than control greater than GDP-beta-S. GTP-gamma-S stimulation of proton transport was also observed under conditions in which chloride entry was eliminated, i.e., 0 mM external chloride concentration in the presence of potassium/valinomycin voltage clamping. GTP-gamma-S did not affect proton leak in endosomes as determined by collapse of H+ ATPase-generated pH gradients. ADP ribosylation by treatment of endosomal membranes with pertussis toxin revealed two substrates corresponding to the 39-41 kD region and comigrating with alpha i subunits. Pretreatment of the membranes with pertussis toxin had no effect on proton transport in the absence of GTP or GTP-gamma-S. However, pretreatment with pertussis toxin blocked the stimulation of proton transport by GTP. In contrast, as reported in other membranes by others previously, pertussis toxin did not prevent the stimulation of proton transport by GTP-gamma-S. These findings, taken together, indicate that GTP binding proteins are present in endosomal membranes derived from renal cortex and that activation of G protein by GTP and GTP-gamma-S stimulates proton transport in a rank order identical to that reported for other transport pathways modulated by Gi proteins. Therefore, these studies suggest that G proteins are capable of stimulating the vacuolar H ATPase of endosomes directly.

Functional assay for GABA receptor subtypes of a cockroach giant interneuron.

gamma-Aminobutyric acid (GABA) receptors were examined in the cockroach central nervous system (CNS) using the single fiber-oil gap method applied to an identified giant interneuron. Short-lasting pressure application of 10 mM GABA developed a multiphasic response composed of a fast hyperpolarization followed by a transient depolarizing component and a stable hyperpolarization. This triphasic characteristic shape of the response was modified according to the dose of GABA injected or bath-applied and to the precise localization of the injection within the dendritic area. The transient depolarizing phase showed a negative reversal potential of -70 mV. Both hyperpolarizing phases reversed at a more negative level ranging to -80 mV. A positive shift of these values was caused by a decrease in external chloride concentration. Bath-application of 0.1 mM picrotoxin (Ptx) decreased the depolarizing phase which was progressively replaced by a stable hyperpolarization. The transient depolarizing component desensitized quickly and was the most sensitive phase to Ptx action. The Ptx-resistant response reversed at a mean value of -100 mV close to the equilibrium potential for potassium ions (EK+), suggesting that it was generated by a K(+)-channel coupled receptor. Although baclofen was unable to mimic the Ptx-resistant GABA response, the compound CGA 147823, known to bind with a high specificity to vertebrate GABAB receptors, has been successfully used to reproduce the Ptx-resistant GABA response. It is suggested that, in addition to GABA receptors linked to chloride channels, the insect CNS possesses GABA receptors sharing ionic characteristics of GABAB receptors especially those located in the vertebrate CNS, although they are insensitive to baclofen.

Influence of external chloride concentration on the kinetics of mobile charges in the cell membrane of Valonia utricularis: Evidence for the existence of a chloride carrier

Charge pulse relaxation studies were performed on cells of the giant marine alga Valonia utricularis. Two exponential voltage relaxations were recorded as found previously (Benz, R., and U. Zimmermann. 1983. Biophys. J. 43:13-26.). The parameters of the two exponential voltage decays were studied as a function of the chloride concentration in the artificial sea water. Replacement of external chloride by 2(N-morpholino)ethanesulfonate (Mes(-)) had a dramatic influence on the four relaxation parameters. This chloride dependence could not be satisfactorily explained by the simplified model used earlier. Accordingly, additional reaction steps had to be included in the model. Only two relaxation processes could be resolved under all experimental conditions. This means that the heterogeneous complexation reactions, k(R) (association), and k(D) (dissociation) were too fast to be resolved. Therefore a carrier model with equilibrium heterogeneous surface reactions was used to fit the experimental results. From the charge pulse data at different chloride concentrations the translocation rate constants of the free and complexed carriers, k(S) and k(AS), through the membrane, as well as the total surface concentration of carrier systems, N(0), could be evaluated. The results described here indicate that the cell membrane of Valonia utricularis contains an electrogenic transport system for chloride.

Effect of bradykinin on Na-K-2Cl cotransport and bumetanide binding in aortic endothelial cells.

Simultaneous measurements of potassium influx and binding of [3H]bumetanide were performed in endothelial cells cultured from bovine aortas to determine how bradykinin regulates Na-K-2Cl cotransport. [3H]Bumetanide displayed saturable binding and was displaced by low concentrations of unlabeled bumetanide. All three transported ions were required for binding and high concentrations of chloride inhibited binding, consistent with binding of bumetanide to the second chloride site of the transporter. Scatchard analysis of binding under maximal conditions (100 mM sodium, 30 mM potassium, 30 mM chloride) revealed a single class of binding sites with a binding constant of 112 nM and a density of 22 fmol/cm2 or approximately 122,000 sites/cells. Na-K-2Cl cotransport, measured as bumetanide-sensitive potassium influx, was stimulated 118 +/- 30% by bradykinin (p less than 0.01) at physiologic ion concentrations. Stimulation was inhibited by increased potassium or decreased external chloride concentrations and was not seen in conditions required for maximal binding of bumetanide. Simultaneous measurement of the binding of tracer [3H]bumetanide and its inhibition of potassium influx in medium containing 10 mM potassium and 130 mM chloride revealed a turnover number for the cotransporter of 293 +/- 68 s-1 which increased to 687 +/- 105 s-1 with bradykinin (p less than 0.001). There was no change in cell volume and only a 5.6 mM increase in intracellular sodium concentration associated with this stimulation. Bradykinin also increased the affinity of the cotransporter for bumetanide as indicated by a decrease in the Ki for potassium influx from 464 +/- 46 nM to 219 +/- 19 nM (p less than 0.005). Our results show that [3H]bumetanide can be used to quantitate Na-K-2Cl cotransporter sites in aortic endothelial cells and to determine the mechanism by which cotransport is regulated. The stimulation of cotransport in aortic endothelial cells by bradykinin is due to an increase in the activity of existing transporters rather than to an increase in the number of transporters. This, together with the increased affinity for bumetanide, strongly suggests that a change in cotransporter structure is occurring in response to bradykinin.

Heterogeneity of cAMP effect on endosomal proton transport

The effects of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) on proton transport in endocytotic vesicles (i.e., endosomes) obtained from rabbit renal cortex were investigated. Endosomes were prepared from New Zealand White rabbits following the intravenous injection of horseradish peroxidase. Under high (106 mM) chloride conditions, 5 microM 8-BrcAMP stimulated proton transport by 11 and 12% in fractions 1 and 2, but inhibited proton transport by 10, 8, and 14% in fractions 4, 5, and 6. Two hundred and fifty micromolar 8-BrcAMP inhibited proton transport progressively from 20% in fraction 1 to 50% in fraction 6. Under conditions in which chloride entry was minimized, i.e., low (6 mM) external chloride concentration plus voltage clamping, 5 microM 8-BrcAMP consistently and significantly inhibited proton transport by 40-60% in all six fractions. Under the same low chloride conditions, 250 microM 8-BrcAMP inhibited proton transport by 80-90% in all six fractions. These studies indicate that 8-BrcAMP can modify endosomal transport in a heterogeneous manner, that 8-BrcAMP inhibits the H+-ATPase directly, and that this effect is significantly modified by chloride. Last, these studies suggest that the observed heterogeneity in proton transport arises from interaction of 8-BrcAMP and chloride.

Requirements for, and Cytoplasmic Concentrations of, Sulphate and Chloride, and Cytoplasmic Volume Spaces in the Halophilic Bacterium Ectothiorhodospira mobilis

Ectothiorhodospira mobilis is a halophilic phototrophic bacterium that has been isolated from soda lakes containing high concentrations of sulphate, chloride and carbonates. It utilizes reduced sulphur compounds as photosynthetic electron donors and oxidizes them to sulphate, but can also grow photoheterotrophically with sulphate as sole sulphur source. The requirements for, and the cytoplasmic concentrations of, sulphate and chloride have been determined. High concentrations of sulphate are neither required for nor inhibit growth. Although chloride is by far the dominant anion of the environment, growth of E. mobilis occurs in the absence of added chloride. Sodium chloride can be replaced by sodium sulphate and sodium carbonate. Chloride is excluded from the cytoplasm with decreasing ratios of cytoplasmic/external chloride at increasing external chloride concentrations (under iso-osmotic conditions).

The ionic basis of the resting potential of molluscan unstriated muscle

The membrane potential (Vm) of unstriated, non-spiking fibres from the buccal retractor muscle of the opisthobranch molluscPhiline aperta is primarily determined by the distribution of the potassium ion across the membrane. In salines where potassium is varied and chloride remains constant or nearly so, the membrane potential varied with log external K+ with a slope of 50.6 (±2.3) mV per decade. In chloride-free salines the slope was 48.5 mV per decade. The experiments were conducted at temperatures of 18–20° C. A ten-fold reduction in external chloride concentration depolarised the fibres by around 10 mV, indicating that chloride permeability makes some contribution to Vm. In salines where [K]0·[Cl]0 is constant the Nernst slope was 55.8 mV per decade compared with the theoretical value of 58 mV. The experimental data suggest that the internal potassium concentration of the fibres is 247±31 mM and pNa/pK is 0.01, giving a predicted value of Vm in sea water of −72 mV. The membrane potential of 90 fibres measured in sea water was −74.2±1.3 mV. The membrane contains an electrogenic sodium pump which contributes 4–5 mV to the membrane potential.

Relative contributions of passive equilibrium and active transport to the distribution of chloride in mammalian cortical neurons.

1. Active and passive factors affecting the chloride gradient of cortical neurons were assessed using intracellular recordings from neurons in slices of cingulate cortex maintained in vitro. The chloride equilibrium potential (ECl-) was estimated indirectly from the reversal potentials of responses to perisomatic gamma-aminobutyric acid (GABA) application and the Cl(-)-dependent inhibitory postsynaptic potential (IPSP). Under control conditions the mean resting potential (Vm; -69.7 mV) was not significantly different than the mean IPSP reversal potential (EIPSP; -70.1 mV). 2. Increasing the external potassium concentration ([K+]o) from 1 to 10 mM shifted the mean EIPSP from -80.4 to -61.8 mV. The mean EIPSP was approximately equal to the mean Vm at all [K+]oS. The conditions of Donnan equilibrium are not met in [K+]o less than 10 mM. 3. Polarization of Vm up to 20 mV away from EIPSP for 4 min with maintained current injection had no significant effect on EIPSP. 4. The GABA reversal potential was maintained 37-52 mV less negative than Vm after equilibration in saline in which the external chloride concentration had been reduced from 133 to 5 mM by substitution with isethionate. Vm and input resistance were not significantly different from control values in cells recorded under these conditions. 5. We conclude that Cl- is not passively distributed in cortical neurons, perhaps due to a low resting Cl- permeability. 6. Impalement with electrodes containing 2 M KCl resulted in a rapid 10 mV depolarizing shift in EIPSP that then remained relatively constant. Intracellular iontophoresis of Cl- resulted in a further depolarizing shift of EIPSP of 5-10 mV that returned to control in less than 1 min. The time course of recovery of IPSP amplitude could be fit with a single exponential having a mean time constant of 6.9 +/- 1.5 s and was independent of the amount of Cl- injected or stimulation frequency. 7. Reductions in temperature from 37 to 32 degrees C significantly increased the mean time constant of IPSP recovery from Cl- injection to 11.1 +/- 3.3 s, corresponding to Q10 = 2.6.(ABSTRACT TRUNCATED AT 400 WORDS)

Osmotic and chloride regulation in the hemolymph of the tiger prawn Penaeus monodon during molting in various salinities

The effect of molting on osmotic and chloride concentrations in the blood of the prawn Penaeus monodon Fabricius (20±3 g) at various salinities was investigated. Prawns were obtained from ponds in Iloilo, Philippines, in 1984. They were stocked in salinities of 8, 20, 32 and 44‰, and their hemolymph was sampled during molt (Time 0), and then 0.125, 0.25, 0.5, 1, 2, 4, 6, 10 and 14 d after molting. Prawns during and immediately after molt tended to conform to the environmental osmolality. Subsequent postmolt (≧0.5 d) stages displayed more divergence from external salinity. The isosmotic point was higher (940±30 mOsm kg-1) during molt than during intermolt (663±8 mOsm/kg-1), suggesting different osmotic requirements in early molt. Hyperregulation of hemolymph chloride below 20‰ S, as well as isoionic point (301±6 mM), were independent of molting stage. At 20‰ S and above, newly molted (0 to 0.25 d post-molt) individuals tended to conform to the external chloride concentration while intermolt (≧0.5 d) post-molt individuals did not. Contribution of hemolymph chloride to hemolymph osmolality was greater during intermolt than during ecdysis, suggesting an important role for other negatively charged ions during molt. When molt occurred in 20‰ S (the test salinity most similar to the isoionic salinity), there was little or no change in hemolymph osmolality or chloride concentration from 0 to 14 d postmolt. At 8, 32 and 44‰ S, the change from molt to intermolt values in hemolymph osmotic and chloride concentrations was hyperbolic. Non-linear least-squares regression showed that prawns generally achieved intermolt values within 1 d after molting. Prawns at intermolt regulated hemolymph osmolality (620 to 820 mOsm kg-1) and chloride concentration (300 to 450 mM) at a much narrower range than during molt (520 to 1 170 mOsm kg-1 and 250 to 520 mM, respectively). Hemolymph osmolality was a more sensitive indicator of physiological response than hemolymph chloride concentration. Distribution and culture of P. monodon might be limited in low salinities by its ability to maintain a hemolymph osmolality ≧500 mOsm kg-1 during molt and ≧600 mOsm kg-1 in intermolt, and in high salinities by its capacity to reduce the hemolymph osmolality from values at molt to those in intermolt. Osmotic and chloride concentrations in the blood of P. monodon clearly varied with both molt stage and salinity of the medium. Dependence on external factors, however, gradually declined in older molt stages, suggesting a reduction in integument permeability and greater development of ion absorption/secretion mechanisms as the exoskeleton hardened.

Chloride uptake in freshwater teleosts and its relationship to nitrite uptake and toxicity

1. The relationship between rate of chloride uptake and external chloride concentration was investigated in Rainbow trout,Salmo gairdneri, and Perch,Perca fluviatilis. The relationship between nitrite uptake and external nitrite and the inhibition of chloride uptake by nitrite was also investigated. Nitrite tolerance tests were performed on a variety of freshwater animals, including Carp,Cyprinus carpio, Tench,Tinca tinca, Pike,Esox lucius, Eel,Anguilla anguilla, and tadpoles,Rana temporaria. 2. The chloride uptake mechanism is saturable, with maximum uptake rates of 368 μMh−1kg−1 and 429 μMh−1kg−1 for the trout and perch, respectively. The half saturation value (Km, the affinity constant) is 159 μMl−1 for trout and 333 μMl−1 for perch. 3. Net nitrite transport was determined in trout, net movement being into the fish against a concentration gradient, with a maximum uptake rate of 281 μMh−1kg−1; theKm is 198 μMl−1. This suggests that nitrite enters the fish via an active uptake process. 4. The data suggest that nitrite is a simple competitive inhibitor of active chloride uptake in both trout and perch. Trout are less tolerant of nitrite than perch (24-h LC50 values are 0.7 mMl−1 for trout and 1.2 mM l−1 for perch) and also have a greater affinity for nitrite. 5. The spectrum of nitrite sensitivity seen in freshwater animals is discussed in relation to chloride uptake kinetics. These data support the hypothesis that nitrite uptake is an active process and furthermore uptake is linked quantitatively with chloride uptake, suggesting that chloride and nitrite enter the fish via the same route.

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.

Elaborations of the basal surface of the cells of the Malpighian tubules of an insect

Electrical measurements of membrane potential and resistance using intracellular microelectrodes showed that the fluid-secreting parts of the Malpighian tubules of Rhodnius have superficial regions different in electrical properties from the main body of the cell. The membrane potential in these superficial regions was smaller by 30–40 mV, but showed a standard depolarization on changing the potassium concentration of the bathing medium. The response to changes in the external chloride concentration also differed in the two regions, a finding that was reinforced by different responses to the drug, furosemide. Electron microscopy of the basal regions of the cells revealed many long cellular projections that run parallel to the cell surface and interdigitate with similar projections from neighbouring cells. The degree of interdigitation was examined by marking individual cells with alcian blue or by horseradish peroxidase injection. A survey of the published micrographs of insect Malpighian tubules shows that most have similar projections on their basal surfaces and not the simple basal infoldings previously supposed.