Passive Cl Research Articles

Rapid co‐transport of sodium and chloride ions in giant salivary gland cells of the leech Haementeria ghilianii.

1. Double-barrelled Cl(-)-selective microelectrodes were used to measure the apparent intracellular Cl- activity (aiCl) and membrane potential (Em) of leech salivary gland cells. In standard physiological solution buffered with HEPES (10 mM), intracellular Cl- activity (corrected for interference) was 38 +/- 8 mM (n = 11) compared to a value of 12.8 mM expected for passive Cl- distribution. The mean Em was -49.4 +/- 8.2 mV (n = 21) which was about 27 mV negative to the Cl- equilibrium potential. 2. Removal of external Cl- led to a slow fall in aiCl until a steady-state level of 4-11 mM was reached in 30-60 min. Recovery of aiCl on readdition of external Cl- took only 2-3 min. The uptake followed an exponential time course having a single rate constant of 1.73 +/- 0.1 min-1 (n = 5) whereas the loss appeared to occur in two phases. Changes in external Cl- produced immediate changes in Em which were the opposite of those expected for a high Cl- permeability, i.e. Cl- removal produced an immediate hyperpolarization (3-18 mV) and readdition of Cl- produced a transient depolarization (5-22 mV). 3. The intracellular accumulation of Cl- was dependent on the external Cl- activity. Even when the external Cl- concentration was reduced to 3%, the cells accumulated Cl- against an electrochemical gradient. 4. Cl- accumulation was temperature sensitive (Q10 approximately 2). 5. On removal of external Na+, aiCl fell to a level which was close to that expected for passive distribution. The active reaccumulation of Cl-, after intracellular Cl- depletion, was abolished in the absence of external Na+; aiCl slowly increased to its passive level. Steady-state aiCl or its recovery by Cl(-)-depleted cells was not affected by the absence of K+ in the bathing solution. 6. The reaccumulation of Cl- was not affected by furosemide (1-5 mM), bumetanide (10(-4) M), amiloride (10(-3) M) or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS, 10(-4) M). 7. Removal of external Cl- caused a fall in intracellular Na+ activity (aiNa, measured with Na(+)-selective microelectrodes) from 15.9 +/- 6.8 mM (n = 9) to 2.5 +/- 1.3 mM (n = 3). When external Cl- was readded, aiNa rose to 46.5 +/- 6.6 mM (n = 3) before slowly recovering towards its original value. The maximal change in aiNa was 41.7 +/- 4.5 mM (n = 3) and the rate constant for Na+ uptake was 1.8 +/- 0.4 min-1 (n = 3).(ABSTRACT TRUNCATED AT 400 WORDS)

Active and passive chloride transport by the rabbit cortical collecting duct

Dietary K intake has been shown to influence solute flux in the cortical collecting duct (CCD). We recently observed active Cl secretion by the CCD when rabbits were fed a K-rich diet. The present studies examined two different methods of K loading (KHCO3 or KCl) on net Cl flux (JNet Cl), active Cl flux (JA Cl), and passive Cl flux (JP Cl) by the rabbit CCD. These two methods of K loading were compared with a low-K diet (control or NaHCO3 diet) that had the same anion composition as the KHCO3 diet (Na for K substitution). In the low-K group there was neither significant net Cl transport nor active Cl secretion. Increasing dietary K content (KHCO3 group) resulted in significant net Cl secretion (JNet Cl = -26.3 +/- 9.3 pmol.mm-1.min-1). Moreover, K secretion was significantly greater (23.8 +/- 4.3 pmol.mm-1.min-1) and transepithelial voltage (VT) was more lumen negative (-14.8 +/- 5.9 mV) compared with the low-K group (P less than 0.05). Consequently, there was both significant passive Cl absorption and significant active Cl secretion. In the KCl group there was significant net Cl absorption (JNet Cl = 17.2 +/- 5.1 pmol.mm-1.min-1, P less than 0.05 vs. low-K diet). However, Na absorption significantly exceeded JNet Cl (JNet Na = 42.0 +/- 7.2 pmol.mm-1.min-1, P less than 0.01), and this group also exhibited the most lumen-negative voltage (VT = -35.4 +/- 5.4 mV).(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular chloride activity in glial cells of the leech central nervous system.

1. Chloride-sensitive double-barrelled microelectrodes were used to measure the intracellular Cl- activity (aicl) and membrane potential (Em) in neuropile glial cells of the leech, Hirudo medicinalis. 2. A close relation between the equilibrium potential for Cl- (ECl = -66.1 +/- 4.9 mV; mean +/- S.D.) and the resting potential (Em = -67.8 +/- 5.2 mV; n = 19) was observed in nominally CO2-HCO3(-)-free, HEPES-buffered solutions. A saline buffered with 2% CO2, 11 mM-HCO3- elicited a membrane hyperpolarization and a concomitant decrease of aCl. 3. Changes in ECl followed these of Em with a lag of less than 30 s in response to various extracellular K+ concentrations [( K+]o) or due to bath-application of carbachol or serotonin. 4. Introduction of a Cl(-)-free solution resulted in a transient depolarization indicating a substantial Cl- conductance and a rapid decrease of aiCl to an apparent value of 0.5-0.9 mM. 5. The loop diuretics furosemide (1 mM) and bumetanide (0.2 mM) did not affect the K(+)-induced changes of aiCl. 6. The results indicate a passive Cl- distribution across the membrane of leech neuropile glial cells as a result of a high Cl- conductance.

Effect of Loop Diuretics on Bullfrog Cornea Epithelium

The effect of loop diuretics on Cl transport was studied on an in vitro preparation of the bullfrog cornea. Bumetanide (10(-4) M) or furosemide (10(-3) M) added to the stromal solution decreased Cl transport measured as the short-circuit current (Isc) to values near zero. Concomitantly, transepithelial conductance (gt) decreased, whereas the intracellular potential (Vo) hyperpolarized and the fractional resistance of the apical membrane (fRo) increased. Substitution of SO4 for Cl in the tear-side solution led to prompt changes in Isc, gt, Vo, and fRo, characteristic of appreciable passive Cl movement across the apical membrane before and after inhibition. Epinephrine (10(-4) M) was similarly effective on apical membrane conductance in inhibited tissues as under control conditions, but the effective electromotive force for transepithelial Cl transport was reduced to approximately 25%. Intracellular Cl activity, measured with ion-selective microelectrodes, decreased so much that the difference in electrochemical Cl potential divided by the Faraday constant (delta mu Cl/F) was close to zero after inhibition of Isc by bumetanide. Apical Cl permeability remained essentially unchanged. Accordingly, loop diuretics inhibit Cl transport in the Cl-secreting cornea epithelium by blocking the Na-Cl symport without secondary apical effects, as believed for other Cl-reabsorbing epithelia.

Excitatory Amino Acid‐Induced Toxicity in Chick Retina: Amino Acid Release, Histology, and Effects of Chloride Channel Blockers

Acute excitotoxicity in embryonic chick retina and the ability of Cl- channel blockers to prevent toxicity were evaluated by measurement of endogenous amino acid release and histology. Treatment of retina with kainate, quisqualate, or N-methyl-D-aspartate resulted in a large dose-dependent release of gamma-aminobutyric acid and taurine, moderate release of glutamine and alanine, and no measurable release of glutamate or aspartate. Concentrations inducing maximal gamma-aminobutyric acid release were 50 microM quisquaalate, 100 microM kainate, and 100 microM N-methyl-D-aspartate. Treatment with 1 mM glutamate resulted in significant gamma-aminobutyric acid release, as well as an elevation in medium aspartate levels. Typical excitotoxic retinal lesions were produced by the agonists and, at the lower concentrations tested, revealed a regional sensitivity. There was a positive correlation between the amount of gamma-aminobutyric acid release and the extent of tissue swelling, suggesting that release may be secondary to toxic cellular events. Omission of Cl- completely blocked cytotoxic effects due to kainate or glutamate. Likewise, addition of the Cl-/bicarbonate anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonate at 600 microM protected retina from cytotoxic damage from all excitotoxic analogs and restored amino acid levels to baseline values. Furosemide, which blocks Na+/K+/2Cl- cotransport, was only minimally effective in reducing amino acid release induced by the agonists. Consistent with the latter, histological examination showed the continued presence of the lesion but with general reduction of cellular edema. These results indicate that although influx of Cl- is a central component of the acute excitotoxic phenomenon, mechanisms other than passive Cl- flux may be involved.

Carrier-mediated Cl- transport in cultured mouse oligodendrocytes.

We studied the steady state and the regulation of intracellular Cl- activity (aCl-i) and the mechanisms of KCl uptake in cultured oligodendrocytes from mouse spinal cord using Cl(-)-selective microelectrodes. The majority of oligodendrocytes actively accumulated Cl- above passive distribution (2-3 mM), few cells showed a passive Cl- distribution. To identify the carriers mediating Cl- uptake, oligodendrocytes were maintained in a solution with low extracellular Cl- concentration ([Cl-]o) which resulted in a rapid decrease in aCl-i. The recovery of aCl-i above its passive distribution in normal [Cl-]o was blocked in the absence of Na+ or in the presence of furosemide and of bumetanide, which has been reported to inhibit Na+/K+/Cl- cotransport. We therefore conclude that Cl- uptake is primarily due to the activity of a Na+K+/Cl- transport system. Cl- uptake above passive distribution was not affected in HCO3(-)-free solution or in the presence of SITS and DIDS, indicating that Cl-/HCO3- exchange is not involved in Cl- uptake by oligodendrocytes. Elevation of [K+]o induced an increase in aCl-i and, as shown earlier, intracellular K+ activity. This K+-induced Cl- uptake was not blocked by bumetanide, furosemide, SITS, or DIDS, suggesting that under conditions of raised [K+]o the combined uptake of K+ and Cl- is not mediated by a carrier, but can be explained by the entry through channels driven by Donnan forces.

Effect of loop diuretics on bullfrog cornea epithelium.

The effect of loop diuretics on Cl transport was studied on an in vitro preparation of the bullfrog cornea. Bumetanide (10(-4) M) or furosemide (10(-3) M) added to the stromal solution decreased Cl transport measured as the short-circuit current (Isc) to values near zero. Concomitantly, transepithelial conductance (gt) decreased, whereas the intracellular potential (Vo) hyperpolarized and the fractional resistance of the apical membrane (fRo) increased. Substitution of SO4 for Cl in the tear-side solution led to prompt changes in Isc, gt, Vo, and fRo, characteristic of appreciable passive Cl movement across the apical membrane before and after inhibition. Epinephrine (10(-4) M) was similarly effective on apical membrane conductance in inhibited tissues as under control conditions, but the effective electromotive force for transepithelial Cl transport was reduced to approximately 25%. Intracellular Cl activity, measured with ion-selective microelectrodes, decreased so much that the difference in electrochemical Cl potential divided by the Faraday constant (delta mu Cl/F) was close to zero after inhibition of Isc by bumetanide. Apical Cl permeability remained essentially unchanged. Accordingly, loop diuretics inhibit Cl transport in the Cl-secreting cornea epithelium by blocking the Na-Cl symport without secondary apical effects, as believed for other Cl-reabsorbing epithelia.

Activity-dependent disinhibition. II. Effects of extracellular potassium, furosemide, and membrane potential on ECl- in hippocampal CA3 neurons.

1. Single-electrode voltage-clamp recordings were made from CA3 pyramidal cells in organotypic hippocampal slice cultures for measurement of membrane currents underlying both the gamma-aminobutyric acid (GABA)-mediated, Cl- -dependent inhibitory postsynaptic potential (IPSC), evoked in response to stimulation of the mossy fiber pathway, and responses to iontophoretically applied GABA. Their reversal potentials are presumed to equal the equilibrium potential for Cl- (37). Mechanisms underlying activity-dependent increases in the intracellular concentration of Cl- ([Cl-]i) were investigated by describing active and passive pathways for Cl- influx and efflux. 2. During 99-s applications of GABA, driving force declined by 51% due to increases in [Cl-]i; thus passive Cl- influx through GABA-activated pathways can significantly affect [Cl-]i. 3. Decreasing the extracellular K+ concentration ([K+]o) from 5.8 to 1 mM caused a rapid hyperpolarizing shift in the mean IPSC reversal potential (EIPSC) from -67.6 to -81.9 mV, even when membrane potential (Vm) was maintained constant and depolarized with respect to EIPSC. 4. Decreasing [K+]o from 5.8 to 1 mM caused a rapid hyperpolarizing shift in the mean GABA reversal potential (EGABA) from -64.7 to -81.1 mV, even when Vm was maintained constant and depolarized with respect to EGABA. Reducing the extracellular Cl- concentration from 153 to 89 mM, while maintaining [K+]o constant at 1 mM, shifted the mean EGABA from -81.1 to -66.2 mV, an amount close to that predicted by the Nernst equation for Cl-. We conclude that reducing [K+]o caused a hyperpolarizing shift in EGABA and EIPSC by decreasing [Cl-]i. 5. The shift of EIPSC and EGABA upon alteration of [K+]o did not result from contamination of the responses by additional K+-mediated components because it was unaffected by block of K+ channels with intracellular Cs+. 6. Reducing the extracellular Na+ concentration from 141 to 70 mM had no effect on EGABA. 7. Furosemide, bath-applied at 5 X 10(-4) M while holding Vm depolarized with respect to EIPSC, caused a rapid, reversible decrease in IPSC driving force averaging 69%, consistent with the presence of a furosemide-sensitive outward Cl- -transport system. 8. Reducing [K+]o from 5.8 to 1 mM in the presence of 5 X 10(-4) M furosemide produced a smaller shift of EIPSC from -61.0 to -71.2 mV, however, after washout of furosemide from [K+]o = 1 mM saline, EIPSC shifted further to -89.8 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

GABA triggers a Cl − efflux from cultured mouse oligodendrocytes

γ-Aminobutyric acid (GABA) has been shown to depolarize the membranes of astrocytes and oligodendrocytes taken from different tissues and species. The mechanism mediating this depolarization was identified, in cultured rat brain astrocytes, as an activation of GABA receptor-linked Cl − channels. A subpopulation of cultured oligodendrocytes from mouse spinal cord also responded to GABA with a membrane depolarization. In the present study we demonstrate that, in oligodendrocytes, the depolarization was accompanied by a decrease in intracellular Cl − activity ([Cl −] i) as measured with ion-selective microelectrodes. At rest, [Cl −] i was elevated above the passive distribution, and upon application of GABA, [Cl −] i decreased towards the level of passive distribution. Furosemide blocked the Cl − inward carrier which led to a passive Cl − distribution; in the presence of furosemide, GABA no longer elicited a membrane depolarization or a change in [Cl −] i. We conclude that oligodendrocytes, which were depolarized by GABA, expressed GABA-activated Cl − channels. Oligodendrocytes which were unresponsive to GABA demonstrated a non-passive Cl − distribution indicating that they did express inward-directed Cl − carriers, but no GABA-activated Cl channels.

Passive NaCl transport in the flounder urinary bladder: predominance of a cellular pathway.

The urinary bladder of the winter flounder is a high-resistance epithelium that can absorb Na and Cl in an electrically silent manner. This active absorption (mucosa-to-serosa) of NaCl is, apparently uniquely, inhibited by mucosal hydrochlorothiazide (HCTZ). These experiments evaluated the notion that virtually all of the cellular Na and Cl permeation could be inhibited by mucosal HCTZ. Mucosal Ba2+ reduced the transepithelial conductance from 0.74 +/- 0.08 to 0.60 +/- 0.06 mS/cm2. Mucosal HCTZ reduced the serosa-to-mucosa flux (backflux) of Na from 0.70 +/- 0.08 to 0.29 +/- 0.03 mueq.cm-2.h-1 and the backflux of Cl from 1.92 +/- 0.22 to 0.38 +/- 0.03 mueq.cm-2.h-1. The treatment with these two agents caused the sum of the partial ionic conductances for Na and Cl to approximate the measured transepithelial conductance. In response to the imposition of a transepithelial voltage, the HCTZ-insensitive Na and Cl backfluxes behaved largely as predicted by the laws of simple ionic diffusion, although there was still a detectable cellular backflux. As judged from dilution voltages and tracer fluxes, the diffusional (paracellular) pathway(s) is nonselective for Na and Cl. The HCTZ-sensitive cellular Na and Cl backfluxes are dependent on the presence of mucosal Na and Cl. Neither backflux is significantly inhibited by serosal application of commonly used inhibitors of Na or Cl transport. The results demonstrate that the majority of passive Na and Cl flux is via a cellular pathway. The translocation across the apical membrane probably involves the same NaCl cotransport process responsible for NaCl absorption.

Sulfate transport in toad skin: Evidence for mitochondria-rich cell pathways in common with halide ions

1. 1. In short-circuited toad skin preparations exposed bilaterally to NaCl-Ringer's containing 1 mM SO 4 2−, influx of sulfate was larger than efflux showing that the skin is capable of transporting sulfate actively in an inward direction. 2. 2. This active transport was not abolished by substituting apical Na + for K +. 3. 3. Following voltage activation of the passive Cl~ permeability of the mitochondria-rich (m.r.) cells sulfate flux-ratio increased to a value predicted from the Ussing flux-ratio equation for a monovalent anion. 4. 4. In such skins, which were shown to exhibit vanishingly small leakage conductances, the variation of the rate coefficient for sulfate influx (γ) was positively correlated with the rate coefficient for Cl − influx ( x), y = 0.035 x − 0.0077 cm/sec ( r = 0.9935, n = 15). 5. 5. Addition of the phosphodiesterase inhibitor, 3-isobutyl-l-methyl-xanthine to the serosal bath of short-circuited preparations resulted in a significant stimulation of the passive Cl − and SO 4 2− permeabilities. 6. 6. It is suggested that SO 4 2− and Cl − ions are transported along the same pathway of the m.r. cells. Depending on the transport mode of the apical Cl − transport system, electro-diffusion, active transport (sulfate:bicarbonate exchange) and self-exchange diffusion take place. Irrespective of the mechanism of transport, sulfate is probably transported as a monovalent anion species.

Evidence for active chloride accumulation in normal and denervated rat lumbrical muscle.

Intracellular Cl- activity (aiCl) was measured with Cl(-)-sensitive microelectrodes in normal and denervated rat lumbrical muscle. In normal muscle bathed in normal Krebs solution, aiCl lay close to that predicted by the Nernst equation. The addition of 9-anthracene carboxylic acid, which blocks Cl- conductance, caused aiCl to increase far above that predicted by a passive distribution. Furosemide (10 microM) reversibly blocked this accumulation. After muscle denervation, aiCl progressively increased for 1-2 wk. The rise occurred in two stages. The initial stage (1-3 d after denervation) reflected passive Cl- accumulation owing to membrane depolarization. At later times, aiCl continued to increase, with no further change in membrane potential, which suggests an active uptake mechanism. This rise approximately coincided with the natural reduction in membrane conductance to Cl- that occurs several days after denervation. Na+ replacement, K+ replacement, and furosemide each reversibly blocked the active Cl- accumulation in denervated muscle. Quantitative estimates suggested that there was little difference between Cl- flux rates in normal and denervated muscles. The results can be explained by assuming that, in normal muscle, an active accumulation mechanism operates, but that Cl- lies close to equilibrium owing to the high membrane conductance to Cl-. The rise in aiCl after denervation can be accounted for by the membrane depolarization, the reduction in membrane Cl- conductance, and the nearly unaltered action of an inwardly directed Cl- "pump."

Chara buckellii, a euryhaline charophyte from an unusual saline environment. II. Membrane potential and membrane conductance at steady state

Electrophysiological experiments were performed on internodal cells of Chara buckellii cultured in saline and freshwater media to see whether the membrane potential (Evo) and membrane conductance (Gm) are dominated by passive K+ conductance (K state), passive H+ conductance (H state), or active proton pumping (P state). Unlike other euryhaline charophytes, C. buckellii cells cultured under saline conditions were not dominated by any one state, showing little dependence on external K+ concentration and pH (pH°). Chara buckellii cells cultured in freshwater share some membrane properties with other freshwater charophytes. Freshwater cells appeared to be in the P state between pH° 5 and 7 as expected but never attained the H state usually observed at high pH° (> 10). Freshwater cells also showed a temporary, threefold increase in Gm at pH° 5, which could not be explained by an increase in passive Cl− or K+ conductance. Evidence consistent with an electrogenic Cl−/2H+ symport in freshwater-grown C. buckellii at pH° 5 and 7 is also presented.

Ion transport by mitochondria-rich cells in toad skin.

The optical sectioning video imaging technique was used for measurements of the volume of mitochondria-rich (m.r.) cells of the isolated epithelium of toad skin. Under short-circuit conditions, cell volume decreased by about 14% in response to bilateral exposure to Cl-free (gluconate substitution) solutions, apical exposure to a sodium-free solution, or to amiloride. Serosal exposure to ouabain resulted in a large increase in volume, which could be prevented either by the simultaneous application of amiloride in the apical solution or by the exposure of the epithelium to bilateral Cl-free solutions. Unilateral exposure to a Cl-free solution did not prevent ouabain-induced cell swelling. It is concluded that m.r. cells have an amiloride-blockable Na conductance in the apical membrane, a ouabain-sensitive Na pump in the basolateral membrane, and a passive Cl permeability in both membranes. From the initial rate of ouabain-induced cell volume increase the active Na current carried by a single m.r. cell was estimated to be 9.9 +/- 1.3 pA. Voltage clamping of the preparation in the physiological range of potentials (0 to -100 mV, serosa grounded) resulted in a cell volume increase with a time course similar to that of the stimulation of the voltage-dependent Cl conductance. Volume increase and conductance activation were prevented by exposure of the tissue to a Cl-free apical solution. The steady-state volume of the m.r. cells increased with the clamping voltage, and at -100 mV the volume was about 1.15 times that under short-circuit conditions. The rate of volume increase during current passage was significantly decreased by lowering the serosal K concentration (Ki) to 0.5 mM, but was independent of whether Ki was 2.4, 5, or 10 mM. This indicates that the K conductance of the serosal membrane becomes rate limiting for the uptake of KCl when Ki is significantly lower than its physiological value. It is concluded that the voltage-activated Cl currents flow through the m.r. cells and that swelling is caused by an uptake of Cl ions from the apical bath and K ions from the serosal bath. Bilateral exposure of the tissue to hypo- or hypertonic bathing solutions changed cell volume without detectable changes in the Cl conductance. The volume response to external osmotic perturbations followed that of an osmometer with an osmotically inactive volume of 21%.(ABSTRACT TRUNCATED AT 400 WORDS)

Potential dependence of unidirectional chloride fluxes across isolated frog skin

Isolated frog skins were voltage clamped at transepithelial potentials ( V t) ranging from −60 mV to 60 mV to measure transepithelial 36Cl − fluxes from the apical to the basolateral bathing solution ( J 13) and in the opposite direction ( J 31). The potential dependence of fluxes obtained in Na +-free choline Ringer's indicates the presence of conductive components that probably correspond to fluxes through paracellular and cellular pathways, respectively. Rectification of fluxes with reversal of the potential reflects a structural asymmetry, presumably in surface charge density. The data are consistent with a charge density of one negative charge per 280 Å 2 on the apical side. A new model for passive Cl − transport was developed that includes surface charge asymmetry and specifically accounts for the observed variation of conductance with potential. In normal frog Ringer's, J 13 was larger than J 31 at zero potential (active Cl − transport), J 13 rose exponentially with increasing positive potential to reach a maximum at 40 mV (approximately open-circuit), and the predicted partial Cl − conductance exceeded the measured conductance leading to the conclusion that when J 13 is largely driven by Na + transport, much of the coupling occurs via nonconductive pathways. Theophylline stimulates Cl − transport that also occurs via nonconductive pathways as V t becomes more positive.

Effect of denervation on a steady electric current generated at the end-plate region of rat skeletal muscle.

An electric current flows continuously out of the synaptic region of rat lumbrical muscle fibres. It is generated apparently as a result of a non-uniform Cl- conductance (GCl), with GCl being lowest at the end-plate. We investigated the effects of denervation on this current. The current persisted with little change after denervation. This was somewhat unexpected, since GCl falls dramatically after denervation, and in acute experiments on normal muscles, the steady current is greatly reduced by agents which block GCl. The steady current was blocked in denervated muscle, as in normal muscle, by low-Cl- solutions, Na+-free and K+-free solutions, and treatment with furosemide and 9-anthracene-carboxylic acid. The current in denervated muscle appears to be generated by the same general mechanism as in normal muscle. The results suggest that the [Cl-]i is significantly higher in denervated than in normal muscle fibres. Preliminary experiments with Cl- -selective micro-electrodes have confirmed this: [Cl-]i rises from about 12 mM to about 23 mM after denervation. This has the effect of moving the Cl- equilibrium potential (ECl) in a positive direction, so that the driving force for passive Cl- efflux is increased. The increased driving force compensates for the reduced GCl, allowing the steady current to persist in denervated fibres.

Cl transport in the frog cornea: an electron-microprobe analysis.

The intracellular electrolyte concentrations of the bullfrog corneal epithelium have been determined in thin freeze-dried cryosections using the technique of electron-microprobe analysis. Under control conditions, transepithelial potential short-circuited and either side of the cornea incubated in Conway's solution, the mean intracellular concentrations (in mmol/kg wet weight) were 8.0 for Na, 18.4 for Cl and 117.3 for K. These values are in good agreement with ion activities previously obtained by Reuss et al. (Am. J. Physiol. 244:C336-C347, 1983) under open-circuit conditions. From a comparison of the chemical concentrations and activities of Na and K a mean intracellular activity coefficient of 0.75 is calculated. For small ions no significant differences between nuclear and cytoplasmic concentration values were detectable. The Cl concentrations in the different epithelial layers were virtually identical and showed parallel changes at varying states of Cl secretion, suggesting that the epithelium represents a functional syncytium. For Na a concentration gradient between the outer and inner epithelial layer was observed, which can be accounted for by two different models of epithelial cooperation. The behavior of the intracellular Na and Cl concentrations after removal of Na, Cl or K from the outer or inner bathing medium provides support for a passive electrodiffusive Cl efflux across the apical membrane and a Na-coupled Cl uptake across the basolateral membrane. The results are inconclusive with regard to the exact mechanism of Cl uptake, indicating either a variable stoichiometry of the symporter or the presence of more than one transport system. Furthermore, a dependence of intracellular Cl on HCO3 and CO2 was observed. Extracellular measurements in corneal stroma demonstrated that ion concentrations in this space are in free equilibrium with the inner bath.

Differing effects of cGMP and cAMP on ion transport across flounder intestine.

The intestinal epithelium of the winter flounder is highly cation selective and actively absorbs NaCl via a bumetanide-sensitive (Na + K + 2Cl) cotransport system; it also actively secretes K+. Combined addition of adenosine 3',5'-cyclic monophosphate (cAMP) and theophylline was previously shown to partially inhibit NaCl absorption and to increase passive Cl- permeability. Because theophylline increases cyclic GMP (cGMP) and cAMP concentrations, we compared the effects of the 8-Bromo analogues of these two nucleotides on ion transport. cGMP inhibits Cl- absorption, K+ secretion, and Cl- and K+ influx across the brush border as effectively as do furosemide and bumetanide. Even at maximal doses, cAMP is less effective than cGMP in inhibiting ion transport; however, unlike cGMP, it abolishes the cation selectivity of the epithelium by greatly increasing Cl- permeability. The effects of the two nucleotides are not additive with each other or with those of bumetanide, although cGMP or bumetanide can further inhibit transport in cAMP-treated tissues.

Cellular mechanisms and control of KCl absorption in insect hindgut.

The hindgut of the desert locust possesses an unusual chloride transport system. The isolated locust rectum absorbs chloride from the mucosal (lumen) to the serosal (haemolymph) side at a rate which is equal to the short-circuit current (Isc). Net chloride transport (JClnet) persists in nominally Na-free or HCO3(CO2)-free saline, is insensitive to normal inhibitors of NaCl co-transport and anion exchange, and is independent of the net electrochemical gradient for sodium across the apical membrane. However, active chloride transport is strongly dependent on mucosal potassium (Ka = 5.3 mM-K). Chloride entry across the apical membrane is active, whereas the net electrochemical gradient across the apical membrane is active, whereas the net electrochemical gradient across the basal membrane favours passive Cl exit from the cell. Although mucosal potassium directly stimulates 'uphill' chloride entry, there is no evidence for coupled KCl co-transport, nor would co-entry with potassium be advantageous energetically. Net chloride absorption and Isc are stimulated by a peptide hormone from the central nervous system which acts via cyclic-AMP. Cyclic-AMP increases Isc and JClnet approximately 1000% and transepithelial conductance (Gt) approximately 100%. Approximately half of the delta Gt during stimulation results from increased Cl conductance at the basal cell border. This increase is also reflected in a shift of the basal membrane e.m.f. towards the Nernst potential for chloride. The remainder of the cAMP-induced delta Gt is due to an elevation of apical membrane K conductance, which causes a 400% increase in transepithelial potassium permeability as estimated by radiotracer diffusion. Because of this stimulation of K conductance, potassium serves as the principal counterion for active chloride transport under open-circuit conditions. Very high luminal levels of K oppose the stimulatory actions of cAMP on active Cl transport and K conductance. These and other results have been incorporated into a cellular model for KCl absorption across this insect epithelium.

Intracellular calcium store and transport of elements in acinar cells of the salivary gland determined by electron probe X-ray microanalysis.

Electron probe X-ray microanalysis using freshly frozen hydrated and dried thin sections of dog submandibular gland was performed to determine the distribution of elements and water in the acinar cells of resting and stimulating states. The results obtained are as follows: (a) The secretory granules contained high concentrations of Ca and S while high concentrations of K and P were present in the cytoplasm and/or nucleus of acinar mucous cells of the gland in the resting state. (b) With pilocarpine stimulation, the concentration of Ca increased in the cytoplasm and decreased in the secretory granules, while there was an increase in the concentration of Na and Cl in both the cytoplasm and secretory granules of the cells. (c) The local dry-mass fractions of acinar cells, estimated by comparing the continuum radiation of X-ray spectrum from the frozen hydrated sections with that from the frozen dehydrated sections, were approximately 20 and 33% in the cytoplasm and secretory granules of resting acinar cells, respectively, and each value was not significantly altered under conditions of stimulation having a tendency to decrease slightly. Therefore, the passive Na and Cl influx and the cytoplasmic Ca flowed in from extracellular spaces and released from secretory granules, an intracellular calcium store, by secretory stimulation probably triggers the passive or active Na and Cl extrusion and consequently the osmotic water flux from the basal part of acinar cells to the secretory granules and the lumen, as well as the serial exocytosis of the granules in the luminal side of the acinar cells.