Acid efflux from retinal glial cells generated by sodium bicarbonate cotransport
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Sodium bicarbonate cotransport was studied in freshly dissociated Müller cells of the salamander retina. Variations in intracellular and extracellular pH evoked extracellular potassium concentration ([K+]o were recorded. Intracellular pH was measured by standard ratio imaging of the pH-sensitive dye BCECF, whereas extracellular pH was monitored by imaging BCECF fixed to coverslips under dissociated cells. Increasing [K+]o from 2.5 to 50 mM resulted in an intracellular alkalinization. The rate of alkalinization, 0.047 pH units/min, was reduced to 42% of control when HEPES was substituted for HCO3- and was reduced to 36% of control by the addition of 0.5 mM DIDS, a Na+/HCO3- cotransport blocker. The K(+)-evoked alkalinization was Cl(-)-independent and was not substantially reduced by amiloride or bumetanide. Increasing [K+]o to 50 mM also produced a rapid extracellular acidification, 0.01 to 0.05 pH units in amplitude. HEPES substitution and addition of 0.5 mM DIDS reduced the acidification to 7-8% of control, respectively. These results confirm the presence of a Na+/HCO3- cotransport system in salamander Müller cells and provide definitive evidence that glial cells can generate an extracellular acidification when [K+]o is increased. The K(+)-evoked extracellular acidification measured beneath cell endfeet was 304% of the amplitude of the acidification beneath cell somata, confirming that cotransporter sites are preferentially localized to the endfoot. The carbonic anhydrase inhibitor benzolamide (2 x 10(-5) M), which is poorly membrane permeant, increased the K(+)-evoked extracellular acidification to 269% of control, demonstrating that salamander Müller cells possess extracellular carbonic anhydrase.Keywords:
Intracellular pH
DIDS
Bumetanide
Amiloride
Alkalosis
Bicarbonate
HEPES
Carbonic anhydrase inhibitor
Nigericin
Abstract: The mechanism of recovery from an acid load in primary cultures of rabbit choroid plexus epithelium (CPE) was examined, with emphasis on Na + ‐dependent antiports. Cells were incubated in saline solutions buffered to pH 7.38 with either HEPES or HCO 3 − plus 95% O 2 /5% CO 2 . Intracellular pH (pH i ) was determined from the steady‐state distribution of [ 14 C]benzoate. Recovery after acidification with NH 4 Cl was rapid ( t 1/2 = 5 min) and was dependent on external Na + (EC 50 = 12 m M ). Hexamethyleneamiloride and ethylisopropylamiloride, potent inhibitors of the Na + /H + antiport, blocked 80% of recovery when [Na + ] was 5 m M with IC 50 values of 100 n M . However, neither drug blocked recovery in normal [Na + ]. 4,4′‐Diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS), an inhibitor of Cl − /HCO 3 − antiports, blocked recovery of pH i in a dose‐related fashion in the presence of bicarbonate, but not in the presence of HEPES. No inhibition occurred with benzamil, an amiloride congener with high affinity for the Na + channel, nor with dimethylbenzamil, an inhibitor of Na + /Ca 2+ exchange. The carbonic anhydrase inhibitor acetazolamide also did not alter recovery from acidification. In CPE that had been pH‐clamped with nigericin and KCl, the initial rate of 22 Na + uptake was very rapid (227 pmol/μg of DNA/min at pH 6.2), was dependent on external [Na + ] with an EC 50 value of 8 m M , and was inversely related to the pH of the medium. The maximal inhibition of 22 Na + uptake by hexamethyleneamiloride was 60% with an IC 50 value of 76 n M . We conclude that both the Na + /H + antiport and a DIDS‐sensitive bicarbonate‐dependent antiport are important mechanisms of regulation of the internal pH of rabbit CPE under acidifying conditions. Further, our data suggest that the rabbit choroid plexus Na + /H + exchanger can be classified as amiloride insensitive, suggesting that this antiport may play a greater role in controlling transport mechanisms than does the pH of the CNS.
Nigericin
DIDS
Amiloride
Acetazolamide
Intracellular pH
Bicarbonate
HEPES
Carbonic anhydrase inhibitor
Bafilomycin
Sodium–hydrogen antiporter
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The effects of amiloride (an inhibitor of Na+/H+ antiport), DIDS (an inhibitor of Na(+)-coupled and Na(+)-independent HCO3-/Cl- exchange) and nigericin (K+/H+ ionophore) alone and in various combinations on the intracellular pH (pHi) and thermosensitivity of SCK tumor cells were studied. Hyperthermia alone at 43 degrees C for 2 h decreased pHi of SCK cells by 0.15-0.20 pH units, as measured fluorometrically using the pH-sensitive dye BCECF. When the cells were treated with 0.5 mM amiloride at 37 degrees C, the pHi declined by 0.10-0.15 pH units at an extracellular pH (pHe) of both 7.2 and 6.6. Amiloride at 0.5 mM enhanced the thermal damage to SCK cells at pHe 6.6 but not at pHe 7.2. DIDS alone at 0.1 mM exerted no effect on pHi or cellular thermosensitivity. DIDS, however, enhanced the effects of amiloride in decreasing pHi and in increasing the thermoresponse of SCK cells, particularly at pHe 6.6. Treatment of the cells with nigericin at 0.1-1.0 micrograms/ml lowered the pHi and enhanced the thermosensitivity of the cells in a dose-dependent manner. Reductions in pHi and increases in thermosensitivity by nigericin at the lower concentration at pHe 6.6 were far greater than at pHe 7.2. When a mixture of 1.0 micrograms/ml nigericin, 0.5 mM amiloride, and 0.1 mM DIDS was present in the medium, the pHi rapidly decreased by about 0.3 and 0.4 pH units at pHe 7.2 and 6.6, respectively. This drug combination was also extremely effective in sensitizing SCK cells to heat, particularly at pHe 6.6. The fact that the thermosensitization by these drugs at pHe 6.6 is more pronounced than at pHe 7.2 and that intratumor environments are known to be acidic strongly suggested that it may then be possible to enhance the thermal damage with such drugs preferentially in tumors relative to normal tissues.
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DIDS
Intracellular pH
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DIDS
HEPES
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Cells resuspended in hypotonic medium initially swell as nearly perfect osmometers, but later recover their volume with an associated KCl loss. This regulatory volume decrease (RVD) is unaffected when nitrate is substituted for Cl- or if bumetanide or 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) is added. It is inhibited by quinine, Ba2+, low pH, anticalmodulin drugs, and depletion of intracellular Ca2+. It is accelerated by the Ca2+ ionophore A23187, or by a sudden increase in external Ca2+ and at high pH. A net KCl loss is also seen after addition of ionophore A23187 in isotonic medium. Similarities are demonstrated between the KCl loss seen after addition of A23187 and the KCl loss seen during RVD. It is proposed that separate conductive K+ and Cl- channels are activated during RVD by release of Ca2+ from internal stores, and that the effect is mediated by calmodulin. After restoration of tonicity the cells shrink initially, but recover their volume with an associated KCl uptake. This regulatory volume increase (RVI) is inhibited when NO3- is substituted for Cl-, and is also inhibited by furosemide or bumetanide, but it is unaffected by DIDS. The unidirectional Cl-flux ratio is compatible with either a coupled uptake of Na+ and Cl-, or an uptake via a K+/Na+/2Cl- cotransport system. No K+ uptake was found, however, in ouabain-poisoned cells where a bumetanide-sensitive uptake of Na+ and Cl- in nearly equimolar amounts was demonstrated. Therefore, it is proposed that the primary process during RVI is an activation of an otherwise quiescent Na+/Cl- cotransport system with subsequent replacement of Na+ by K+ via the Na+/K+ pump. There is a marked increase in the rate of pump activity in the absence of a detectable increase in intracellular Na+ concentration.
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DIDS
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Osmotic concentration
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DIDS
Bumetanide
Tetrahymena pyriformis
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Studies were performed in strips of opossum lower esophageal sphincter (LES) muscle in vitro. External Cl(-)-free Krebs solution (0[Cl-]o) inhibited resting tone. Treatment with the Cl- channel blocker diphenylamine-2-carboxylate (DPC, 0.3-100 microM) caused a concentration-dependent relaxation of LES muscle, as did treatment with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 1 microM-3 mM), a Cl(-)-HCO3- exchange blocker, and bumetanide (0.3-100 microM), a blocker of the Na(+)-K(+)-2Cl- cotransport. DIDS and bumetanide are also known to cause Cl- channel block. The calculated pD2 and Emax values for DPC, DIDS, and bumetanide were 5.24 +/- 0.28 (n = 5), 3.38 +/- 0.16 (n = 5), and 4.49 +/- 0.23 M (n = 5), and 78.80 +/- 5.38, 74.80 +/- 6.54, and 83.70 +/- 10.20%, respectively. The neuronal Cl- channel activators gamma-aminobutyric acid and glycine had no effect on the resting tone. DPC, DIDS, and bumetanide appear to have acted directly on smooth muscle rather than indirectly through the release of inhibitory neurotransmitters because LES relaxation by these agents was not influenced by tetrodotoxin (10 microM), which blocks action potentials in nerves, or by omega-conotoxin (1 microM), which inhibits the release of neurotransmitters from nerve terminals. LES muscle relaxed by exposure to 0[Cl-]o, DPC, DIDS, and bumetanide contracted with the addition of carbachol (30 microM); muscle so treated was resistant to the inhibitory neurotransmitter-mediated relaxation ordinarily induced by electrical field stimulation (EFS, 0.12-32 Hz). This effect was not nonselective, as the EFS-resistant muscle relaxed fully with isoproterenol (0.1-100 microM). HCO(3-)-free Krebs in the nominal absence of CO2 did not affect the resting tone and its relaxation. The Ca2+ channel blocker nifedipine decreased resting tone but did not antagonize the relaxation of carbachol-contracted muscle induced by either EFS or isoproterenol. These studies suggest that Cl- may play an important role in LES tone and relaxation due to inhibitory neurotransmitter released from intramural nerves.
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Tetrodotoxin
Channel blocker
Muscle relaxation
Potassium channel blocker
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To investigate the electrophysiology and mechanisms of chloride (Cl-) transport across the ciliary body-epithelium (CBE) of the porcine eye. The pig is widely believed to be a good model for studying human physiology. Current results strengthen our understanding of the physiology of aqueous humor formation (AHF).Freshly isolated porcine CBE were maintained in modified Ussing-Zerahn-type chambers. The effects of the bathing anion substitution (Cl- and HCO3-) and transport inhibitors including bumetanide, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt (DIDS), heptanol, and two chloride channel inhibitors, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), and niflumic acid, on the electrical properties and transepithelial Cl- transport were investigated.Viable porcine CBE preparations were maintained in vitro. A spontaneous transepithelial potential difference (PD) of approximately 1 mV was found across the CBE (aqueous side negative). The magnitudes of the PD and short-circuit current (I(sc)) were found to be dependent on both the bathing Cl- and HCO3- concentrations. In short-circuited conditions, a significant net Cl- transport (1.01 microEq x h(-1) x cm(-2); n = 109; P < 0.001) in the stromal-to-aqueous direction (J(net)Cl) was detected. The magnitude of the Cl- current carried by the J(net)Cl was approximately 2.2 times the measured I(sc), suggesting there was cation (e.g., Na+) transport along with Cl- and/or anion transport (e.g., HCO3-) in the opposite direction. Bilateral bumetanide (0.1 mM) reduced the J(net)Cl by approximately 56% while stromal DIDS (0.1 mM) produced no inhibition. Instead, aqueous DIDS (0.1 mM) triggered a sustained stimulation of both I(sc) and J(net)Cl. Even if bilateral DIDS was used at a higher concentration (1 mM), together with bilateral dimethylamiloride (DMA, 0.1 mM), no inhibition of the I(sc) was observed. Bilateral heptanol (3.5 mM) drastically reduced the I(sc) and J(net)Cl. NPPB (0.1 mM), a common chloride channel inhibitor, did not inhibit the J(net)Cl, whereas NFA (1.0 mM) virtually abolished it.In the porcine eye, active secretion of Cl- into aqueous was identified that may act as a driving force for AHF. The bumetanide-sensitive Na+/K+/2Cl- cotransporter (NKCC) clearly contributes to the Cl- uptake into the pigmented epithelium (PE), whereas the DIDS-sensitive Cl-/HCO3- anion exchanger (AE) may exert a minor role. The intercellular gap junctions couple the porcine ciliary bilayers and thus the transepithelial Cl- transport, as in other species. The Cl- channel/efflux pathway located in the nonpigmented epithelium (NPE) is niflumic acid-sensitive but NPPB-insensitive. We also hypothesize that the AE located on the NPE may regulate the activity of a putative Cl- channel on the basolateral membrane facing aqueous via modulation of the intracellular pH (pHi). This work reinforces the general consensus that active secretion of Cl- is the major driving force of AHF in mammalian eye and further substantiates the existence of species differences in the mechanism that accomplishes transepithelial Cl- transport.
DIDS
Bumetanide
Chloride channel
Transepithelial potential difference
Niflumic acid
Ussing chamber
Amiloride
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DIDS
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1. DIDS (4,4'‐diisothiocyano‐2,2'‐stilbene disulphonic acid) reduced the CI Isc across the toad's (Bufo marinus) cornea. It acted on either the aqueous or tear side and these effects were additive. 2. The reduction in the Isc was equivalent to the decline in the undirectional flux of Cl from the aqueous to the tear side. The Cl flux from tear to aqueous was not changed. 3. DIDS did not change transmural Na transport. 4. The action of the diuretic bumetanide, which also inhibits Cl transport was additive to that of DIDS when both compounds were present on the aqueous though not when they were on the tear side of the cornea. 5. The results are consistent with the role of a Cl‐/anion exchange mechanism in active Cl transport across the cornea. 6. A hypothesis regarding the interactions and site of action of bumetanide in relation to that of DIDS, and the Cl transport process, is proposed.
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