Presence Of DIDS Research Articles

Role of HCO−3Ions in Cytosolic pH Regulation in Rat Mast Cells: Evidence for a New Na+-Independent, HCO−3-Dependent Alkalinizing Mechanism

The role of external HCO−3in pHiregulation in rat mast cells was studied with BCECF. In a HCO−3-free medium cells undergo an alkalinization after the addition of 40 mM HCO3Na. This alkalinization is unaffected by pH. In a Na+-free medium, the addition of 20 mM HCO3Na induced a higher alkalinization than 20 mM HCO3K. Amiloride (1 mM), a Na+/H+exchanger inhibitor, inhibited by 45% the alkalinization induced by HCO3Na, but it did not change that induced by HCO3K. The anion exchanger inhibitor DIDS reduced 20% the alkalinization induced by both salts. An alkalinization of 0.085 units is observed after the addition of 20 mM HCO3K, even when these exchangers are inhibited (in the absence of Na+and presence of DIDS). We conclude that the Na+/H+exchanger and the Cl−/HCO−3exchangers are alkalinizing mechanisms that regulate pHiin these cells. Also, there is some HCO−3-dependent, Na+-independent mechanism responsible for the intracellular alkalinization.

PH Regulation in Horizontal Cells of the Skate Retina

To examine the mechanisms by which horizontal cells regulate intracellular pH (pHi), measurements were recorded from isolated cells enzymatically dissociated from the skate retina utilizing the pH-sensitive dye BCECF. In a HCO3−-containing Ringer solution, steady-state pHiwas 7.32±0.13 (mean±s.d.,n=70). Recovery from acidification was examined using the NH4+prepulse technique. When NH4+was removed from the extracellular solution, pHidropped rapidly to ∼0.3 pH units below the initial baseline, and then recovered at an initial rate of ∼0.072 pH units/min. During recovery of pHiafter the acid load, the removal of Na+or the addition of amiloride from a HCO3−-free extracellular solution reduced the rate of recovery by 79%±11% and 69%±14%, respectively. In the presence of DIDS, which inhibits primarily anion transport, or during the removal of Na+, the recovery from acidification was reduced by 83%±10% and 70%±11%, respectively, as compared to the control value in HCO3−-containing solution. These results suggest that the skate horizontal cell possesses a Na/H exchanger as well as a Na+-and HCO3−-dependent mechanism for removal of excess acid. Removal of HCO3−or Cl−from the extracellular solution had little effect on pHi, but removing external Na+induced a marked decrease in pHithat fell at an initial rate of ∼0.3 pH units min−1. This rate of acidification was decreased by 58%±19% in the presence of DIDS (500 μm) and reduced by 28%±13% with the addition of amiloride (2 mm). Thus, Na- and HCO3-dependent transport was about 2-fold more active than Na/H exchange during low Na+-induced acidification. The intrinsic pH-buffer capacity, determined from the pHichange induced by incremental reductions in the [NH4+] of the extracellular solution, was 24.2 mm/pH unit at the horizontal cell's resting pHi. Moreover, pHiwas relatively insensitive to changes in membrane potential; in experiments under whole-cell voltage clamp (-70 mV), intracellular pH remained constant during depolarizing voltage swings to -30 mV or +30 mV, as well as during hyperpolarizing pulses to -90 or -110 mV.

Volume-activated DIDS-sensitive whole-cell chloride currents in trout red blood cells.

1. The nystatin-perforated whole-cell recording mode of the patch-clamp technique was used to investigate the membrane conductance of trout (Oncorhynchus mykiss) red blood cells in the steady state, 5 min after exposure to hyposmotic medium and 10 min after return to normal isosmotic medium. 2. Whole-cell I-V relations showed outward rectification when red blood cells were bathed in isosmotic (320 mosmol l-1) saline solution and the patch pipette was filled with 117 mM KCl. The membrane conductance was 2.58 +/- 0.59 nS (number of experiments, n = 18) between 0 and 100 mV and 1.32 +/- 0.19 nS (n = 18) between 0 and -100 mV. Removal of Cl- from the extracellular side or incubation with the Cl- channel blocker DIDS caused a reduction in whole-cell membrane conductance by more than 50%, indicating that the membrane current was generated by Cl- ions. The remaining conductance was voltage independent and probably due to non-selective cation conductance. 3. The membrane conductance increased approximately 2-fold after cell swelling induced by exposure to hyposmotic saline solution (215 mosmol l-1). This effect was abolished in Cl(-)-free hyposmotic medium or in the presence of DIDS. 4. The return to isosmotic solution produced a fall in membrane conductance to, or below, control values. 5. We conclude that trout red blood cells possess a significant Cl- conductance in the steady state which is reversibly activated during cell swelling and contributes to volume recovery.

Electrodiffusion, Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin

Current-voltage curves for DIDS-insensitive Cl- conductance have been determined in human red blood cells from five donors. Currents were estimated from the rate of cell shrinkage using flow cytometry and differential laser light scattering. Membrane potentials were estimated from the extracellular pH of unbuffered suspensions using the proton ionophore FCCP. The width of the Gaussian distribution of cell volumes remained invariant during cell shrinkage, indicating a homogeneous C1- conductance among the cells. After pretreatment for 30 min with DIDS, net effluxes of K+ and Cl- were induced by valinomycin and were measured in the continued presence of DIDS; inhibition was maximal at approximately 65% above 1 microM DIDS at both 25 degrees C and 37 degrees C. The nonlinear current-voltage curves for DIDS-insensitive net Cl- effluxes, induced by valinomycin or gramicidin at varied [K+] o, were compared with predictions based on (1) the theory of electrodiffusion, (2) a single barrier model, (3) single occupancy, multiple barrier models, and (4) a voltage-gated mechanism. Electrodiffusion precisely describes the relationship between the measured transmembrane voltage and [K+]o. Under our experimental conditions (pH 7.5, 23 degrees C, 1-3 microM valinomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio PK/PCl is 74 +/- 9 with 10 microM DIDS, corresponding to 73% inhibition of PCl. Fitting the constant field current-voltage equation to the measured Cl- currents yields PCl = 0.13 h-1 with DIDS, compared to 0.49 h-1 without DIDS, in good agreement with most previous studies. The inward rectifying DIDS-insensitive Cl- current, however, is inconsistent with electrodiffusion and with certain single-occupancy multiple barrier models. The data are well described either by a single barrier located near the center of the transmembrane electric field, or, alternatively, by a voltage-gated channel mechanism according to which the maximal conductance is 0.055 +/- 0.005 S/g Hb, half the channels are open at -27 +/- 2 mV, and the equivalent gating charge is -1.2 +/- 0.3.

Functional identity of a purified proximal tubule anion exchanger protein: Mediation of chloride/formate and chloride/bicarbonate exchange

Based on the transport activities and inhibitor sensitivities, different functional modes of anion exchangers have been identified in the kidney proximal tubule including chloride/formate, chloride/oxalate, chloride/hydroxyl, and chloride/bicarbonate exchange. There is little information on the molecular structure and properties of the protein(s) involved in these processes. Previously, using stilbene affinity matrix and Pac Q chromatography, we partially purified a protein with anion exchange properties in brush border membranes (BBM) isolated from rabbit kidney proximal tubules. This protein has a molecular weight of 162 kDa. When reconstituted into liposomes, the fraction containing the 162 kDa protein demonstrated Cl-/Cl- exchange activity. In the current experiments, the 162 kDa protein was purified to homogeneity using a combination of affinity, ion exchange, and size exclusion chromatography. This protein has binding affinity for known inhibitors of anion exchangers. When reconstituted in liposomes, the 162 kDa protein showed anion exchange activity as assayed by 36Cl-/Cl- exchange. Functional studies in liposomes reconstituted with the purified 162 kDa protein in revealed that this protein mediates the transport of Cl-/formate and Cl-/HCO3-. The Cl-/formate and Cl-/HCO3- exchange activities in the reconstituted liposomes were inhibited in the presence of DIDS and furosemide, two known inhibitors of renal anion exchangers. We conclude that Cl-/formate exchange and and Cl-/HCO3- exchange in kidney proximal tubules are mediated via the same protein. This protein is distinct from the known anion exchanger proteins (AE1, AE2, and AE3) and may represent another isoform from this family of transporters.

Trophoblast GLUT1 expression but not activity is refractory to changes in glycemic status

Functional identity of a purified proximal tubule anion exchanger protein: Mediation of chloride/formate and chloride/bicarbonate exchange. Based on the transport activities and inhibitor sensitivities, different functional modes of anion exchangers have been identified in the kidney proximal tubule including chloride/formate, chloride/oxalate, chloride/hydroxyl, and chloride/bicarbonate exchange. There is little information on the molecular structure and properties of the protein(s) involved in these processes. Previously, using stilbene affinity matrix and Pac Q chromatography, we partially purified a protein with anion exchange properties in brush border membranes (BBM) isolated from rabbit kidney proximal tubules. This protein has a molecular weight of 162 kDa. When reconstituted into liposomes, the fraction containing the 162 kDa protein demonstrated Cl-/Cl- exchange activity. In the current experiments, the 162 kDa protein was purified to homogeneity using a combination of affinity, ion exchange, and size exclusion chromatography. This protein has binding affinity for known inhibitors of anion exchangers. When reconstituted in liposomes, the 162 kDa protein showed anion exchange activity as assayed by 36Cl-/Cl- exchange. Functional studies in liposomes reconstituted with the purified 162 kDa protein revealed that this protein mediates the transport of Cl-/formate and Cl−/HCO3−. The Cl-/formate and Cl−/HCO3− exchange activities in the reconstituted liposomes were inhibited in the presence of DIDS and furosemide, two known inhibitors of renal anion exchangers. We conclude that Cl-/formate exchange and and Cl−/HCO3− exchange in kidney proximal tubules are mediated via the same protein. This protein is distinct from the known anion exchanger proteins (AE1, AE2, and AE3) and may represent another isoform from this family of transporters.

Intracellular acidification associated with changes in free cytosolic calcium. Evidence for Ca2+/H+ exchange via a plasma membrane Ca(2+)-ATPase in vascular smooth muscle cells.

The purpose of this study was to define the mechanism whereby agonists that increase free cytosolic calcium (Cai2+) affect intracellular pH (pHi) in smooth muscle. Rat aortic vascular smooth muscle cells grown on coverslips were loaded with BCECF/AM or fura-2/AM for continuous monitoring of pHi or Cai2+, respectively, in a HCO3-/CO2- containing medium. Recovery from rapid increases in Cai2+ produced by 1 microM angiotensin (Ang) II (delta Cai2+ -229 +/- 43 nM) or 1 microM ionomycin (delta Cai2+ -148 +/- 19 nM) was accompanied by a fall in pHi (delta pHi, -0.064 +/- 0.0085 P < 0.01, and -0.05 +/- 0.012 pH units, P < 0.01, respectively). Neither the fall in pHi nor the rise in Cai2+ elicited by Ang II was prevented by pretreatment with agents which block the action of this agonist on pHi via the stimulation of the Cl/HCo3 exchangers (DIDS, 50 microM) or the Na+/H+ antiporter (EIPA, 50 microM). In the presence of DIDS and EIPA, Ang II produced a fall in pHi (delta pHi, -0.050 +/- 0.014, P < 0.01) and a rise in Cai2+ (delta Ca2+ 252 +/- 157 nM, P < 0.01). That the change in pHi was secondary to changes in Cai2+ was inferred from the finding that, when the rise in Cai2+ elicited by Ang II was prevented by preincubation with a Ca2+ buffer, BAPTA (60 microM), the fall in pHi was abolished as well (delta pHi, 0.0014 +/- 0.0046). The pHi fall produced by Ang II and ionomycin was prevented by cadmium at a very low concentration (20 nM) which is known to inhibit plasma membrane Ca(2+)-ATPase activity (delta pHi -0.002 +/- 0.0006 and -0.0016 pH units, respectively). Cadmium also blunted Cai2+ recovery after Ang II and ionomycin. These findings suggest that the fall in pHi produced by these agents is due to H+ entry coupled to Ca2+ extrusion via the plasma membrane Ca(2+)-ATPase. Our results indicate that agonists that increase Cai2+ cause intracellular acidification as a result of Ca2+/H+ exchange across the plasma membrane. This process appears to be mediated by a plasma membrane Ca(2+)-ATPase which, in the process of extruding Ca2+ from the cell, brings in [H+] and thus acidifies the cell.

ACTIVE Na+-, Cl-- AND HCO3--DEPENDENT ACID EXTRUSION IN ATLANTIC COD RED BLOOD CELLS IN WINTER ACTIVATED BY HYPERCAPNIA

The relationship between intracellular pH (pHi) and extracellular pH (pHe) was investigated in red blood cells from the Atlantic cod (Gadus morhua) in carbon dioxide/bicarbonate-buffered salines. In summer animals (August/September), similar transmembrane distribution ratios of chloride ([Cl-]i/[Cl-]e=rCl-) and protons ([H+]e/[H+]i=rH+) suggested a passive Donnan distribution of these ions across the red blood cell membrane at pHe 6.7&shy;8.4. In winter animals (February/March), a marked discrepancy occurred between rH+ and rCl- at low pHe values. The pronounced increase in rH+ resulted in significantly higher pHi values compared with those of red blood cells from summer animals and at pHe 6.7 pHi exceeded pHe by 0.3 units. The increases in rH+ values were completely abolished by cyanide and 2,4-dinitrophenol. The high disequilibrium rH+ values were sodium-, chloride- and bicarbonate-dependent. During hypercapnic acidosis, proton equivalents were extruded from the red blood cell. The resulting high rH+ values were accompanied by a reduced chloride shift into the red blood cell and a ouabain-insensitive net sodium influx. The net sodium influx into red blood cells from winter animals was significantly reduced in the presence of DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid). The results suggest the activation of a Na+-dependent Cl-/HCO3-exchanger at low pHe in the red blood cells of the Atlantic cod in winter.

CATECHOLAMINE-ACTIVATED SODIUM/PROTON EXCHANGE IN THE RED BLOOD CELLS OF THE MARINE TELEOST GADUS MORHUA

The effects of catecholamines on the pH and the cellular ion and water content were investigated in red blood cells from the Atlantic cod (Gadus morhua). Noradrenaline induced a rapid decrease in the extracellular pH (pHe) of red blood cells suspended in a CO2/bicarbonate or in a CO2/bicarbonate-free buffer system. The noradrenaline-induced changes in pHe were a saturable function of the external sodium ion concentration and were inhibited by amiloride but not by DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid, final concentration of both 10(-4) mol l-1). The catecholamine-induced extracellular acidification was accompanied by an intracellular alkalization and protons were moved from their electrochemical equilibrium. Proton extrusion was associated with an increase in the red blood cell sodium and chloride concentrations. In the presence of DIDS, the chloride movements were blocked and the net proton efflux under these conditions matched the net sodium influx. The results strongly suggested the activation of a sodium/proton exchanger by catecholamines in the red blood cells of the Atlantic cod. The red blood cell receptor affinity for adrenaline was three times higher than that for noradrenaline. Comparison with data in the literature for in vivo catecholamine concentrations indicated that adrenaline was more effective than noradrenaline in activating the red blood cell sodium/proton exchanger in the Atlantic cod in vivo.

Regulation of intracellular pH in pyramidal neurones from the rat hippocampus by Na(+)‐dependent Cl(‐)‐HCO3‐ exchange.

1. We have measured intracellular pH (pHi) in freshly isolated pyramidal neurones from the CA1 region of the rat hippocampus using the fluorescent indicator 2',7'-bis(carboxy-ethyl)-5-(and-6)-carboxyfluorescein (BCECF). 2. The neurones selected by our isolation procedure, when studied in the nominal absence of CO2-HCO3-, had a mean steady-state pHi of 6.81 +/- 0.02 (n = 163). The recovery of pHi from acid loads was very slow. The rate of recovery from acid loads was reduced by Na+ removal, but only very slightly inhibited by 1 mM amiloride. 3. The addition of 5% CO2-25 mM HCO3- caused steady-state pHi to increase from 6.74 +/- 0.05 to 7.03 +/- 0.03 (n = 28). In the presence of 5% CO2-25 mM HCO3-, the rate of pHi recovery from acid loads was much faster than in its absence. 4. The HCO(3-)-induced alkalinization was reversible, and did not occur in the absence of extracellular Na+ or in the presence of DIDS (4,4'-diisothiocyanatostilbene- 2,2'-disulphonic acid). 5. In the absence of external Cl-, successive exposures to CO2-HCO3- elicited alkalinizations that were progressively reduced in rate and amplitude. This effect, presumably due to gradual depletion of internal Cl-, was rapidly reversed by returning Cl- to the external medium. 6. We conclude that the major acid-extrusion mechanism in pyramidal CA1 neurones is the Na(+)-dependent Cl(-)-HCO3- exchanger. The Na(+)-dependent mechanism that operates in the nominal absence of HCO3- is far less active.

In vitro analysis of volume and pH regulation in the red blood cells of a primitive air-breathing fish, the bowfin, Amia calva

In the bowfin (Amia calva), a decrease in extracellular pH in vitro was associated with an increase in the water content and chloride concentration in the red blood cells that could be inhibited by the anion-exchange blocker, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). After a step increase in CO2 tension, the extracellular total CO2 concentration was also significantly reduced by DIDS. Finally, over most of the experimental pH range, the red blood cell pH observed in the presence of DIDS was significantly elevated compared with that of controls. Taken together, these results indicate that as in most other fishes, chloride–bicarbonate exchange is clearly present and functional in bowfin red blood cells. Moreover, within the physiological pH range, ion movements across the anion exchanger have a marked influence on both the volume and the pH of bowfin red blood cells. In sharp contrast to the rainbow trout (Oncorhynchus mykiss), catecholamines had no effect on the volume, pH, or intracellular sodium concentration of red blood cells in the bowfin. Following osmotic swelling, rainbow trout red blood cells were able to regulate their volume back to control levels within 2 h. In the bowfin, however, there was no regulation of red blood cell volume after osmotic swelling. Thus, in contrast to many other fishes examined to date, it would appear that in the bowfin, the physiological mechanisms involved in the adrenergic response and in the regulatory volume decrease after osmotic swelling may be less active or possibly even absent in the red blood cells.

Modification of cardiac Ca2+ release channel gating by DIDS

The effects of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) on individual cardiac sarcoplasmic reticulum Ca2+ release channels have been examined in planar lipid bilayers. A sudden step-like increase in open probability (Po) induced by DIDS was observed. At zero holding potential it proceeded with an apparent time constant of 84 s and Po was increased 15 times in the steady state. Independent of membrane voltage, DIDS induced a long-lived open state with tau o = 15 ms at 0 mV and tau o = 5 ms at -50 mV (cis (cytoplasmic) side negative). While at 0 mV the modified channel spent most of the time in this long-lived state, at -50 mV the occupancy of the long-lived state was very low and most openings occurred to the short and medium open state, leading to deactivation of the channel to only 2-3 times of control. Voltage-induced changes in the activity of modified channels were reversible and proceeded with time constants of 14 s for deactivation and 12 s for activation, that is, faster than onset of activation by DIDS. As no changes in single channel conductance of the channel were observed in the presence of DIDS and its abrupt activating action could not be removed, a covalent modification of an amino group residing in the gating structure of the channel by DIDS can be considered.

Stilbene disulfonates block ATP-sensitive K+ channels in guinea pig ventricular myocytes

Effects of stilbene disulfonates on single KATP channel currents were investigated in inside-out and outside-out membrane patches from guinea pig ventricular myocytes. All drugs tested, 4,4'-diisothiocyanatostilbene,2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), and 4,4'-diaminostilbene-2,2'-disulfonic acid (DADS), inhibited the KATP channel when they were applied to the intracellular, but not extracellular side of the membrane patch. Inhibitory actions of DIDS and SITS were irreversible, whereas those induced by DNDS and DADS were reversible. KATP channel inhibition was concentration dependent with an order of potency of DIDS > SITS approximately DNDS > DADS; the Hill coefficient was close to unity for each drug. No change in channel conductance was observed during exposure to DIDS or DNDS; however, channel kinetics was altered. Distribution of the open time within bursts and that between bursts could be described by a single exponential relation in the absence and presence of DIDS or DNDS. The time constant of the open time within bursts was not altered, but that between bursts was decreased by DIDS (from 40.0 +/- 8.1 to 29.8 +/- 6.7 msec, P < 0.05) and by DNDS (from 43.1 +/- 9.3 to 31.9 +/- 7.1 msec, P < 0.05). Distributions of closed time within bursts were also fitted to a single exponential function both in the absence and presence of drugs, while those of the closed time between bursts were fitted to a single exponential function in the absence of drugs, but a double exponential function was required in the presence of drugs. The rates of onset and development of channel inhibition by DIDS and DNDS appeared to be concentration dependent; a longer time was required to reach a new steady-state of channel activity as drug concentration was decreased. Inhibition by DIDS or DNDS was regulated by intracellular pH; inhibition was greater during acidic conditions. For DIDS (0.1 mM), the open probability (Po) expressed as a fraction of the value before drug application was 42.9 +/- 8.3% at pH 7.4 and 8.2 +/- 6.6% at pH 6.5 (P < 0.01); corresponding values for DNDS (1 mM) were 39.6 +/- 17.6 and 8.9 +/- 5.8%, respectively (P < 0.01). From these data, we conclude that stilbene disulfonates block the KATP channel by binding to their target site with one-to-one stoichiometry. Similar to glibenclamide, the binding of stilbene disulfonates may reflect interpolation in an "intermediate lipid compartment" between the cytosolic drug and the site of drug action.

Anion transport blockers inhibit DL-2-amino-4-phosphonobutyrate responses induced by quisqualate in the rat cerebral cortex.

1. Depolarizing responses to DL-2-amino-4-phosphonobutyrate (AP4) and related amino acids have been studied in the rat cerebral cortex slice following the application of quisqualate (Quis). 2. Before exposure to Quis, 500 microM DL-AP4 had little or no effect. However, following a single application of 40 microM Quis for 2 min, DL-AP4 produced depolarizing responses. With repeated applications of DL-AP4, there was a decline in response amplitude. A second application of Quis restored the depolarizing potency of DL-AP4 to a level above that for the first DL-AP4 response after the first Quis application. With a sequence of alternate applications of Quis and DL-AP4, the amplitude of DL-AP4 responses became maximal after the second Quis application. Responses to DL-AP4 could also be induced by the application of 1 microM Quis for 60 min, but were smaller in amplitude. 3. Responses to the normally inactive amino acids L-cysteine (Cys), L-cystathionine (CTN) and L-alpha-aminoadipate (AA) were also induced once Quis was applied. These responses were also maximized after a second application of Quis, except those to L-Cys, which failed to reach a plateau after three Quis applications. 4. The co-application of DL-AP4 with the first Quis application depressed the subsequent mean DL-AP4 response by 47%. Re-application of Quis restored the amplitude of DL-AP4 responses to levels comparable to control. L-alpha-AA also suppressed the induction of DL-AP4 responses, when co-applied with the first Quis exposure, reducing mean response amplitude by 98%. Unlike DL-AP4, however, the effect with L-alpha-AA persisted so that DL-AP4 responses were significantly suppressed compared to control, even after further applications of Quis. 5. The effects of the anion transport blockers, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetoamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) on the induction process and the DL-AP4 responses themselves were examined. DIDS (100 microM) significantly inhibited the DL-AP4 responses, and to a lesser extent the induction of the responses by 40 microM Quis (2 min), while SITS (300 microM) only inhibited the DL-AP4 responses. However, the induction of responses by 1 microM Quis (60 min) was significantly affected by this concentration of SITS. 6. DIDS (100 microM) had no effect on responses to alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA), but selectively potentiated those to Quis. Examination of the full concentration-response curve for Quis revealed that, while the Rmax remained constant, the Hill slope was increased and the EC50 was decreased in the presence of DIDS. SITS (300 microM), however, antagonized responses to AMPA, and had little effect on responses to Quis except at the highest concentration of Quis tested (20 microM), where a potentiation was observed, suggesting that it is a non-NMDA receptor antagonist.7. These observations indicate that the production of depolarizing responses to a number of amino acids, including DL-AP4, in the cerebral cortex is mediated via an anion transport mechanism sensitive to DIDS and SITS, and that the exchange of DL-AP4 for a sequestered excitatory amino acid receptor agonist, probably Quis, could underlie the production of these responses. Indeed, Quis is apparently sequestered via a similar process. However, the involvement of such a process in the induction of these responses remains inconclusive.

HgCI&lt;sub&gt;2&lt;/sub&gt;-lnduced Ion Transport Pathways in Ehrlich Ascites Tumor Cells

HgCl2 (40 µM) initially activates an anion-independent unidirectional Na+ influx in Ehrlich ascites tumor cells, which amounts to 61 ± 7 µmol·g dry wt-1 min-1 (± SEM, n = 5). The unidirectional Na+ influx in control cells in 13 ± 2 µmol·dry wt-1 • min-1 (± SEM, n = 5). The HgCl2-activated Na+ influx is slightly inhibited by amiloride (inhibitor of Na+/H+ exchange), but almost completely blocked by the anion-exchange inhibitor DIDS (4,4’-diisothiocyano-2,2’-stilbenedisulfonic acid). HgCl2 also causes DIDS-sensitive cytoplasmic acidification followed by DIDS-sensitive cytoplasmic alkalinization. After addition of HgCl2 the cells initially shrink, whereupon they swell again. Half-maximal effect of HgCl2 is obtained with about 0.5 and 1 µM HgCl2 on cell swelling and cell shrinkage, respectively. The volume responses are both reduced in the presence of DIDS. The unidirectional K+ and Cl– effluxes are greatly stimulated by HgCl2. The stimulation of K+ efflux results in a Cl--dependent net loss of K+. In Cl– medium the K+ loss is 84 ± 11 µmol·g dry wt–1 • min–1 ( ± SEM, n = 6). In NO–3 medium, it is reduced to 24 ± 6 µmol·g dry wt-1 • min-1 ( ± SEM, n = 4). The HgCl2 activated K+ efflux in Cl– medium is reduced to 21 ± 6 µmol·g dry wt–1 • min–1 ( ± SEM, n = 3) by DIDS. Finally, an instantaneous hyperpolarization of the cell membrane potential was observed after addition of HgCl2. It is concluded that HgCl2 in Ehrlich cells activates (i) a DIDS-sensitive, Na+-dependent anion exchange system; (ii) a DIDS-sensitive and anion-dependent K+ transport system, and (iii) K+ channels.

Thrombospondin receptor expression in human neutrophils coincides with the release of a subpopulation of specific granules.

The extracellular matrix (ECM) protein thrombospondin (TSP) binds specifically to polymorphonuclear leucocyte (PMN) surface receptors and promotes cell adhesion and motility. TSP receptor expression increases 30-fold after activation with the synthetic chemotactic peptide, N-formylmethionyl-leucylphenylalanine (FMLP) or the Ca2+ ionophore A23187, in combination with cytochalasin B. The expression of TSP receptors was correlated with the exocytosis of both specific and azurophil granules. Newly expressed TSP receptors are not derived from easily mobilized specific granules since agents that trigger some specific granule release [phorbol myristate acetate (PMA), FMLP or ionophore A23187 alone] do not increase TSP receptor expression. In this study we used the anion-channel blocker, 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS) to investigate the source of these newly expressed receptors. When PMNs were exposed to cytochalasin B and FMLP or to cytochalasin B and ionophore A23187 in the presence of 30-100 microM-DIDS, TSP receptor expression increased coincidently with vitamin B12-binding protein release from specific granules. Under these same conditions, the release of the azurophil granule component, myeloperoxidase, was significantly inhibited. Using agonists that cause release of specific granules, or both specific granules and azurophil granules, we determined that DIDS blocked the release of PMA-mobilized specific granules and cytochalasin B plus FMLP- or cytochalasin B plus ionophore A23187-mobilized myeloperoxidase-containing azurophil granules but not specific granules mobilized by cytochalasin B plus FMLP or cytochalasin B plus ionophore A23187. These results suggested that PMNs contain at least two subpopulations of specific granules: one that is easily mobilized, lacks TSP receptors and is inhibitable by DIDS, and one that is difficult to mobilize, contains a large pool of TSP receptors and the release of which is enhanced in the presence of DIDS.

Alpha-1-adrenergic modulation of K and Cl transport in bovine retinal pigment epithelium.

Intracellular microelectrode techniques were used to characterize the electrical responses of the bovine retinal pigment epithelium (RPE)-choroid to epinephrine (EP) and several other catecholamines that are putative paracrine signals between the neural retina and the RPE. Nanomolar amounts of EP or norepinephrine (NEP), added to the apical bath, caused a series of conductance and voltage changes, first at the basolateral or choroid-facing membrane and then at the apical or retina-facing membrane. The relative potency of several adrenergic agonists and antagonists indicates that EP modulation of RPE transport begins with the activation of apical alpha-1-adrenergic receptors. The membrane-permeable calcium (Ca2+) buffer, amyl-BAPTA (1,2-bis(o-aminophenoxy)-ethane-N,N,N',N' tetraacetic acid) inhibited the EP-induced voltage and conductance changes by approximately 50-80%, implicating [Ca2+]i as a second messenger. This conclusion is supported by experiments using the Ca2+ ionophore A23187, which mimics the effects of EP. The basolateral membrane voltage response to EP was blocked by lowering cell Cl, by the presence of DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) in the basal bath, and by current clamping VB to the Cl equilibrium potential. In the latter experiments the EP-induced conductance changes were unaltered, indicating that EP increases basolateral membrane Cl conductance independent of voltage. The EP-induced change in basolateral Cl conductance was followed by a secondary decrease in apical membrane K conductance (approximately 50%) as measured by delta [K]o-induced diffusion potentials. Decreasing apical K from 5 to 2 mM in the presence of EP mimicked the effect of light on RPE apical and basolateral membrane voltage. These results indicate that EP may be an important paracrine signal that provides exquisite control of RPE physiology.

Intracellular pH regulation in isolated rat bile duct epithelial cells.

To evaluate ion transport mechanisms in bile duct epithelium (BDE), BDE cells were isolated from bile duct-ligated rats. After short-term culture pHi was measured with a single cell microfluorimetric set-up using the fluorescent pHi indicator BCECF, and calibrated with nigericin in high K+ concentration buffer. Major contaminants were identified using vital markers. In HCO3(-)-free media, baseline pHi (7.03 +/- 0.12) decreased by 0.45 +/- 0.18 pH units after Na+ removal and by 0.12 +/- .04 after amiloride administration (1 mM). After acid loading (20 mM NH4Cl) pHi recovery was inhibited by both Na+ removal and amiloride (JH+ = 0.74 +/- 1.1, and JH+ = 2.28 +/- 0.8, respectively, vs. 5.47 +/- 1.97 and 5.97 +/- 1.76 mM/min, in controls, respectively). In HCO3- containing media baseline pHi was higher (7.16 +/- 0.1, n = 36, P less than 0.05) and was decreased by Na+ substitution but not by amiloride. Na+ removal inhibited pHi recovery after an intracellular acid load (0.27 +/- 0.26, vs. 7.7 +/- 4.1 mM/min, in controls), whereas amiloride reduced JH+ only by 27%. pH recovery was inhibited by DIDS (0.5-1 mM), but not by Cl- depletion. Finally, acute Cl- removal increased pHi by 0.18 pH units in the absence but not presence of DIDS. These data indicate that BDE cells possess mechanisms for Na+/H+ exchange, Na+:HCO3- symport and Cl-/HCO3 exchange. Therefore BDE may be capable of transepithelial H+/HCO3- transport.

The effect of loop diuretics on Cl‐ transport in smooth muscle of the guinea‐pig vas deferens and taenia from the caecum.

1. The role of Na+, K+, Cl- co-transport, identified in the previous paper (Aickin & Brading, 1990), has been characterized further by investigation of the effects of loop diuretics on Cl- movements in the smooth muscle cells of guinea-pig vas deferens measured by 36Cl fluxes and Cl(-)-sensitive microelectrodes. Some flux experiments were also repeated in the taenia from the guinea-pig caecum. 2. Frusemide (2 mM) reduced the steady-state Cl- content, slowed 36Cl loss into Cl(-)-free solution and both slowed and reduced Cl- accumulation by Cl(-)-depleted cells of the vas deferens. When anion exchange was inhibited by the presence of DIDS, (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid), frusemide further slowed the loss of Cl- into Cl(-)-free solution, further reduced Cl- accumulation such that Cl- uptake amounted to a level consistent with a passive distribution and halted the rise in the intracellular Cl- activity (aiCl) at levels above about 10 mM. 3. Application of the higher-affinity loop diuretics bumetanide and piretanide in vas deferns had no significant effect on 36Cl efflux into Cl(-)-free solution or on the initial rate of rise of aiCl but reduced the final level attained. In the presence of DIDS, however, both agents further slowed efflux into Cl(-)-free solution, and halted the rise in aiCl at levels above about 10 mM. Measurement of greatly slowed intracellular pH transients on removal and readdition of external Cl- (Clo-) in the presence of frusemide suggests that the larger effects of this drug are mediated by inhibition of anion exchange as well as of co-transport. 4. The relative potency of the loop diuretics, investigated in the presence of DIDS was: bumetanide greater than piretanide greater than frusemide. This sequence was found in both vas deferens, using direct measurement of aiCl, and taenia, using 36Cl uptake. 5. Comparison of data from the vas and taenia showed that 36Cl efflux into Cl(-)-free, HCO3(-)-free solution was about twice as fast in the taenia, and that bumetanide or piretanide reduced this efflux to about the same rate as that observed in the vas with or without the loop diuretic. DIDS caused a similar absolute reduction of efflux in both preparations. 6. Stimulation of 36Cl efflux on readdition, and inhibition on removal of Nao+ in the presence of DIDS, was much greater in the taenia than in vas and in both preparations was blocked by bumetanide or piretanide.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of Na+ and K+ on Cl‐ distribution in guinea‐pig vas deferens smooth muscle: evidence for Na+, K+, Cl‐ co‐transport.

1. Smooth muscle cells of the guinea-pig vas deferens after Cl- depletion actively reaccumulate ions to a level many times higher than that predicted by a passive distribution, even when anion exchange (largely responsible for Cl- movements in this preparation) is inhibited by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid). The cells therefore must possess a second mechanism for Cl- accumulation. We have now investigated the ionic requirement of this mechanism using a combination of ion analysis, 36Cl fluxes and direct measurement of the intracellular Cl- activity (aiCl). 2. In the steady state, the Cl- content of tissues was 12-16% less in Na(+)-free solution than in normal Krebs solution. 3. Loss of 36Cl into Cl(-)-free solution was slowed by the absence of Na+ and accelerated on its readdition. Uptake of 36Cl by Cl(-)-depleted tissues was also reduced in the absence of extracellular Na+, particularly at longer time intervals as uptake reached completion. These effects occurred in the presence and absence of CO2-HCO3- and in the presence of DIDS. 4. The initial rate of rise of aiCl on readdition of Cl- to Cl(-)-depleted cells was not significantly affected by the absence of Na+ in the presence of a functional anion exchange, but aiCl stabilized at a lower value than in normal solution. Readdition of Na+ stimulated a rise in aiCl to the control level. Removal and readdition of K+ under these conditions had negligible effects. 5. When anion exchange was inhibited by the presence of DIDS, removal and readdition of Na+ caused, respectively, a marked inhibition and stimulation of the rise in aiCl during Cl- reaccumulation. Under these conditions removal and readdition of K+ had similar effects. 6. The results suggest that Na+, K+, Cl- co-transport is involved in transmembrane movements of Cl- at least when the anion exchange mechanism is blocked. 7. The possibility that the marked effects of changes in external Na+ on transmembrane Cl- movements in the presence of a functional anion exchange mechanism are caused by secondary effects due to changes in intracellular pH as well as to suppression of Na+, K+, Cl- co-transport is discussed.