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PubMed1989

In vivo chemotaxis of bovine neutrophils induced by 5-lipoxygenase metabolites of arachidonic and eicosapentaenoic acid.

The 5-lipoxygenase metabolites of arachidonic (AA) and eicosapentaenoic acid (EPA), 5S,12R-dihydroxy-6,8,10,14-eicosatetraenoic acid (LTB4), 5-hydroxyeicosatetraenoic acid (5-HETE), 5S,12R-dihydroxy-6,8,10,14,17-eicosapentaenoic acid (LTB5), and 5-hydroxyeicosapentaenoic acid (5-HEPE), were injected intradermally into the ear skin of steers to assess their in vivo potency as chemotactic factors for bovine neutrophils. A dose of 30 picomoles of LTB4 was required to elicit a significant extravascular dermal accumulation of neutrophils (P less than 0.05). In contrast, 1.0 nanomole of LTB5 was required to achieve a cellular influx equivalent to that elicited by 30 picomoles of LTB4. Nearly five times as many neutrophils were present in the bovine dermis injected with 1.0 nanomole of LTB4 compared with sites given the equivalent dose of LTB5 (245 cells/sq mm vs. 52 cells/sq mm). Six nanomoles of either 5-HETE or 5-HEPE were required before a significant neutrophil accumulation occurred. These results show clearly that 5-lipoxygenase metabolites can initiate the extravascular accumulation of bovine neutrophils in vivo. However, the chemotactic potency of the EPA metabolites is much reduced when compared with that of the homologous AA lipids. The results obtained support the premise that modification of the inflammatory response, including control of cellular influxes, by dietary supplementation with EPA is feasible.

Animals↗
PubMed1989

[Regulation of intracellular pH by Na+/H+ exchange in human lymphocytes].

Spectrofluorimetry and the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)carboxyfluorescein (BCECF) was used to measure the intracellular pH (pHi) of suspended human lymphocytes. A linear relationship exists between pHi and the fluorescent spinal-ratio (I490nm/I435nm, emission 526 nm) between pH 6.5 and pH 7.8. At the end of each experiment the ratio was calibrated using the high [K+] nigericin technique. All solutions were HEPES buffered. The pHi in resting cells was 7.27 +/- 0.02 (n = 37) at 25 degrees C. Na+ free solution caused a pHi decrease to 6.81 +/- 0.08. The ammonium prepulse technique (25 mM NH4Cl) dropped the pHi to pH 6.80. A rapid recovery of the pHi after this acidification was observed in NaCl Ringer solution. Na+ free solution completely blocked the recovery. 1 mM amiloride led to a partial block of recovery. pHi was restored to the basal value after readdition of Na+. We conclude that in HEPES buffered solutions human lymphocytes recover pHi via a mechanism dependent on extracellular Na+ and largely accomplished by an amiloride inhibitory Na+/H+ exchanger.

Acid-Base Equilibrium↗
PubMedDate not supplied

Homogeneous growth of tumor cell colonies in agar containing glass capillaries.

The capillary cloning system has been shown to have advantages over conventional cloning of human tumor cells in Petri dishes. In the present study a further optimization towards homogeneous colony distribution and high cloning efficiency is described. For reasons of reproducibility the study focused on cell lines, i.e. three human linew (MDA-231, HT-29, L363) and one rodent line (CHO-AB). Major variables investigated were the gel length, the capillary tube diameter, the tube sealing and buffer system, and the cell number. Criteria for optimal tumor colony growth included homogeneous colony distribution along the gel, mean colony size and cloning efficiency. It was found that colony distribution as well as overall colony growth depended largely on the gel length, i.e. on the volume of tumor cell containing agar applied per capillary tube. The results showed that optimal tumor cell colony growth was achieved in 100 ul capillary tubes of 1.2 mm internal diameter filled with 30ul, yielding a gel length of 27 mm. Colony formation did not significantly differ between sealed and unsealed tubes, provided that HEPES buffer was added. It was concluded that, for practical reasons, sealing of tube ends and therefore utilization of HEPES buffer is not necessary. In a head to head comparison, cloning efficiency was equal or higher in capillary tubes than in Petri dishes. The capillary cloning system is an alternative for drug development as well as for predictive drug testing. Its major advantage is the utilization of fewer tumor cells.

Agar↗
PubMed1988

Cultivation of the life cycle stages of Trypanosoma brucei sspp.

A culture system was devised for the production of the various stages in the developmental cycle of Trypanosoma brucei brucei and T. b. rhodesiense. The bloodstream forms were grown at 37 degrees C on a feeder layer of fibroblasts from embryos of Microtus montanus or CD-1 mice in HEPES-buffered Minimum Essential Medium with Earle's salts, supplemented with 15% heat-inactivated rabbit serum. When they were transferred to HEPES-buffered Cunningham's medium and incubated at 27 degrees C, they transformed into procyclic trypomastigotes, some of which developed into epimastigote and metacyclic forms in the presence of explants of Glossina morsitans or Phormia regina. Stages infective to mice were produced in cultures of procyclic forms grown with Anopheles gambiae cells in medium consisting of a mixture of 3 volumes of Cunningham's medium and 1 volume of Anopheles cell medium. In vitro-produced metacyclic trypanosomes were used to initiate cultures of bloodstream forms.

Animals↗
PubMed1988

Factors influencing the oxidation of the radioprotector WR-1065.

N-(2-Mercaptoethyl)-1,3-diaminopropane (WR-1065) is the free thiol form of the radio- and chemoprotector S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721). Interest currently exists in the clinical use of WR-2721 and WR-1065 as radio- and chemoprotectors of normal tissues. However, measurement of plasma levels of WR-1065 has proven difficult, due to rapid drug oxidation. Therefore, we studied factors influencing the oxidation of WR-1065, in Hepes-buffered saline as well as in tissue culture media containing 10% fetal bovine serum. The rate of oxygen consumption by WR-1065, as determined using the Clark oxygen electrode system, was faster in medium plus serum than in Hepes-buffered saline. That this effect is largely due to the presence of trace metal ions in tissue culture media and serum was indicated by the observation that addition of Cu2+ or Fe3+ to buffer stimulated oxygen consumption. Addition of KCN inhibited the reaction of WR-1065 with oxygen, and this effect was dependent on KCN concentration. That KCN blocked WR-1065 oxidation to the disulfide was verified using Ellman's reagent to quantitate the free thiol form. The rate of oxygen consumption was shown to be affected by temperature as well as concentration of WR-1065. Catalase reduced the rate of oxygen consumption of WR-1065, indicating that peroxide is formed in this system. Superoxide dismutase had a stimulatory effect. WR-1065 was found to stimulate the hexose monophosphate shunt in A549 cells. Since this stimulation was prevented by the presence of catalase, it appeared to be due to the response of the cells to peroxide, formed as a result of WR-1065 autooxidation.

Mercaptoethylamines↗
PubMed1988

The role of low intracellular or extracellular pH in sensitization to hyperthermia.

Cells are more sensitive to heat when they are heated in an acidic environment, and this study confirms (K. G. Hofer and N. F. Mivechi, J. Natl. Cancer Inst., 65, 621, 1980) that intracellular pH (pHi) and not extracellular pH (pHe) is responsible for the sensitization. The relationship between pHe, pHi, and heat survival of cells heated in vitro in various buffers at pHe 6.3-8.0 was investigated. Cells' adaptation to low environmental pH in terms of increases in pHi and heat survival also was investigated. Finally, we studied the relationships among pHe, pHi, and survival from heat for cells heated in sodium-free reconstructed medium. Intracellular pH was measured by the distribution of the weak acid, [2-14C]5,5-dimethyl-2,4-oxazolidinedione. Our results are summarized as follows: (1) CHO cells maintained the same relationship between pHe and pHi in four different media or buffers (McCoy's 5a medium buffered with CO2 and NaHCO3 or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) and 2-(N-morpholino)ethanesulfonic acid (Mes), Krebs-Ringer bicarbonate solution, and Krebs-Ringer phosphate solution) with pHi being 0.05 to 0.20 pH units higher than pHe as it varied from 7.0 to 6.4; furthermore, heat sensitization by acid was the same in medium buffered with NaHCO3 or Hepes and Mes. (2) The low pHe adapted cells multiplied with an increased doubling time of 20.7 +/- 0.7 h and appeared morphologically similar to the unadapted cells. However, the pHi of these cells was 0.15-0.30 pH units higher than that of the unadapted cells when pHe was varied between 7.0 and 6.3. (3) After being heated at 43.5 degrees C for 55 min or at 42.5 degrees C for 150 min at pHe 6.3-7.2, the pHi of the adapted cells increased by 0.2-0.1 pH units. However, heat caused no significant change in the unadapted cells. (4) Heat survival plotted versus pHe was 1000-fold higher for the adapted cells than for the unadapted cells at pHe of 6.3. However, heat survival plotted versus pHi was identical for the two cell types. (5) In sodium-free reconstructed McCoy's 5a medium, pHi was 0.25-0.1 pH units lower than that in the sodium-containing counterpart at pHe 6.3-7.2, and heat sensitization increased accordingly; however, heat survival plotted versus pHi was identical for the two types of media.

Animals↗
PubMed1977

Cation effects on cell shape.

We have found that human erythrocyte ghosts in 10 mM HEPES (pH 7.0) at 0 degrees C would crenate when 20-50 mM of Na+ or K+, 0.2-0.5 mM OF Ca++, Ba++, Sr++, or Mg++, or 10 muM of La+++ was added. The shape change after cation addition was faster than fixation by 1% glutaraldehyde at 4 degrees C and was readily reversible upon dilution of the cation. After incubation of ghosts in 10 mM HEPES (pH 7.0) at 37 degrees for 10-20 min there was a significant inhibition of subsequent crenation by cations. In a process that is believed to occur by a similar mechanism, whole red blood cells were observed to cup (invaginate) when 20 mM of a divalent or 0.1 mM of a trivalent cation was added. After neuraminidase treatment to remove the sialic acid charge groups, these same shape changes were observed in ghosts and whole cells. Another type of cation-induced crenation was found to follow upon the addition to whole cells of A23187 and Ca++ or Ba++ but not Mg++. This process is much slower than crenation in the ghost and is believed to be caused by a different mechanism.

Barium↗
PubMed1986

The nicotinamide adenine dinucleotides as allosteric effectors of human hemoglobin.

The oxygen binding properties of human hemoglobin are appreciably altered by the nicotinamide dinucleotides NADH, NADP+, and NADPH. These cofactors are important in the control of many metabolic pathways and in providing reductive potential for a number of enzymatic reactions, including in vivo reduction of methemoglobin. Specific binding of these cofactors to hemoglobin and their potential for acting as allosteric modifiers of hemoglobin function have not been previously recognized. Detailed oxygen binding studies utilizing a thin-layer method suggest that the nicotinamide dinucleotides bind with high affinity to the deoxyhemoglobin tetramer at the beta chain anion-binding site and stabilize the low affinity "T-state" conformation. Stripped Hb A in 0.05 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, pH 6.5, at 20 degrees C is half-saturated at a pO2 of 1.6 mm Hg. In the presence of 0.5 mM NADH, NADP+, or NADPH, the P50 is raised to 3.8, 7.1, and 12.5 mm Hg, respectively. The Bohr factor for stripped Hb A in 0.05 M HEPES buffer is sensitive to these effectors and is raised from 0.25 to about 0.65 by the addition of NADPH. The data suggest that routine use of these effectors in studies of human hemoglobin variants or the allosteric mechanism of Hb A be considered carefully. The relatively low intraerythrocytic levels of the nicotinamide dinucleotides in relation to hemoglobin dictate that these cofactors cannot significantly affect in vivo oxygen delivery. However, the converse is theoretically possible. The binding of the cofactors to hemoglobin and the preferential binding of their reduced forms may affect cofactor-dependent metabolic processes in red blood cells.

Hemoglobin A↗
PubMed1985

Preservation of human skin: a study of two media using the athymic (nude) mouse model.

This study is part of an ongoing attempt to define the optimal conditions for short-term storage of viable human skin for auto- or homotransplantation. Roswell Park Memorial Institute 1640 (RPMI) media was more convenient to use than Eagle's Minimum Essential Medium with HEPES buffer (MEM-H) but functional skin viability was not statistically different in the two media. MEM without HEPES buffer did not preserve human skin at 20 days storage. The athymic (nude) mouse provides an excellent in vivo test of stored human skin functional viability.

Animals↗
PubMed1984

Effects of divalent cations on the regulation of insulin-sensitive glucose transport and cAMP phosphodiesterase in adipocytes. Insulin-like effects of divalent cations.

Effects of divalent cations on the regulation of glucose transport and cAMP phosphodiesterase in isolated rat epididymal adipocytes were studied. EDTA (5 mM) moderately inhibited the binding of insulin to adipocytes in Krebs-Henseleit Hepes buffer. In the same buffer, A-23187 (an ionophore specific for divalent cations; 50 microM) plus EDTA (5 mM) almost completely blocked the insulin- or hydrogen peroxide-dependent stimulation of phosphodiesterase. This inhibition was not secondary to the loss of ATP. When cells that had been treated with A-23187 plus EDTA were washed and then exposed to 1-10 mM of divalent cations, the cellular phosphodiesterase activity was elevated. Mn2+ was most stimulatory, Mg2+ was next, and Ca2+ was least effective. The stimulatory effects were enhanced by insulin. In the presence of insulin, Mn2+ at 10 mM was less stimulatory than that at 1 mM. In regular Krebs-Henseleit Hepes buffer, Mn2+ greatly stimulated phosphodiesterase if cells were first exposed to A-23187. The Mn2+-dependent stimulation was blocked by treatment of cells with 2,4-dinitrophenol. Results essentially parallel to those described above were also obtained when the rate of glucose transport was determined. The above results indicate that divalent cations mildly support the extracellular binding of insulin to its receptor, facilitate the physiological actions of insulin, and mimic the hormone actions, presumably by stimulating an intracellular enzyme.

2,4-Dinitrophenol↗
PubMed1983

Stimulation of eicosapentaenoic acid metabolism in washed human platelets by 12-hydroperoxyeicosatetraenoic acid.

Washed human platelets were not able to convert eicosapentaenoic acid (EPA) to thromboxane B3 (TXB3) and 12-hydroxyeicosapentaenoic acid (AA) to washed human platelets induced conversion of EPA to TXB3 and 12-HEPE. Esculetin, a specific inhibitor of 12-lipoxygenase, prevented the effect of AA, but cyclooxygenase inhibitor did not. The conversion of AA to TXB2 was not affected by the same dose of esculetin. These data suggest that products of AA formed by 12-lipoxygenase in human platelets have stimulatory effects on EPA metabolism. When AA was preincubated with washed human platelets, its effect on EPA conversion was reduced, suggesting that a labile product of AA formed by 12-lipoxygenase is involved in the facilitation of EPA metabolism. Addition of 12-hydroperoxyeicosatetraenoic acid directly to washed human platelets caused dose-dependent synthesis of TXB3 and 12-HEPE, while addition of 12-hydroxyeicosatetraenoic acid had no effect. Thus, 12-hydroperoxyeicosatetraenoic acid formed from AA promotes the metabolism of EPA in washed human platelets.

Arachidonic Acid↗
PubMed1983

Use of non-physiological buffer systems in the analysis of methotrexate transport in L1210 cells.

Methotrexate transport parameters have been compared in L1210 cells suspended in a series of HEPES buffer systems of varying ionic compositions. While no effect was observed on the Vmax for methotrexate influx, the Kt for half-maximal influx, the steady-state level of methotrexate, and the efflux rate each varied substantially and to an extent which could be correlated directly to the anionic composition of the external medium. Buffer composition also affected the membrane potential, the ATP level of the cells, and, in one instance, the cell volume, but these changes did not exert a significant effect on the transport process. These results suggest that the integrity of L1210 cells is not adversely affected by either the presence of HEPES in the suspending medium or by the absence of certain physiological ions, and, moreover, that methotrexate transport parameters measured under these conditions, although not necessarily indicative of the quantitative events that might occur in vivo, can nevertheless provide meaningful information on the properties and mechanism of this transport system.

Adenosine Triphosphate↗
PubMed1981

Non-frozen preservation of committed hematopoietic stem cells from normal human bone marrow.

We examined the optimal conditions for maintaining normal human hematopoietic stem cells without cryopreservation. The effects of serum, HEPES, CPD and storage temperature were studied. Stem cell integrity (CFU-c, CFU-e, and BFU-e) was optimally maintained when the bone marrow cells were suspended in a medium containing 40% fetal calf serum, 25 mM HEPES, and 12.28% CPD and maintained at 4 degrees C. For example, under these storage conditions the recovery of functionally intact early erythroid stem cells, BFU-e, was 84% after 2 days, 44% after 4 days and 12% after 7 days. These results indicate that non-frozen short-term storage of human bone marrow may be of clinical utility in bone marrow transplantation.

Cell Survival↗
PubMed1995

Intracellular pH regulation in Hep G2 cells: effects of epidermal growth factor, transforming growth factor-alpha, and insulinlike growth factor-II on Na+/H+ exchange activity.

Intracellular pH (pHi) plays an important role in the metabolic activation of quiescent cells after a proliferative stimulus, and Na+/H+ exchange activity is required for growth in some extrahepatic tumors. To investigate intracellular acid/base homeostasis in hepatoma cells and the effects of putative liver growth factors on Na+/h+ exchange activity, we have studied intracellular pH (pHi) regulation in Hep G2 cells, a well-differentiated hepatoma cell line, both in resting conditions and after administration of epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), and insulinlike growth factor-II (IGF-II). The effects of fetal calf serum, TGF alpha, and amiloride on 3H-Thymidine incorporation were also studied. Amiloride (1 mmol/L) and external Na+ removal decreased baseline pHi in both HEPES and KRB. In HEPES, cells recovered from an acid load (20 mmol/L NH4Cl) by an amiloride inhibitable Na+/H+ exchange. In KRB, an additional, DIDS-inhibitable, Na(+)- and HCO3- dependent, but Cl(-)-independent acid extruder (Na:HCO3 cotransport) was activated. No evidence was found for a Cl/HCO3 exchange acting as acid loader. Administration of EGF and TGF alpha, but not of IGF-II, induced a dose-dependent, amiloride-inhibitable increase in baseline pHi, together with an increase in Na+/H+ exchange activity, shifting to the right the JH/pHi curve. Finally, 3H-thymidine incorporation in Hep G2 cells, in the presence of FCS or TGF alpha, was strongly inhibited by amiloride. In conclusion, in Hep G2 cells, pHi is mainly regulated by Na+/H+ exchange, which activity can be stimulated by EGF and TGF alpha, but not by IGF-II. Administration of TGF alpha stimulates DNA synthesis, an effect that is blocked by amiloride, an inhibitor of Na+/H+ exchanger. These data suggest that Na+/H+ exchange activation may play a critical role in the growth of some hepatic tumors.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1995

An electrogenic sodium-bicarbonate cotransport in the regulation of myocardial intracellular pH.

Experiments were performed in cat papillary muscles in order to explore the possible existence of an electrogenic Na+/HCO3- cotransport. Developed tension (DT), intracellular pH (pHi) with the pH-sensitive dye 2'-7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) and resting membrane potential (Vm) with 3M KCl filled glass microelectrodes were measured. A change from HEPES to HCO3(-)-buffered superfusate induced an immediate decrease in pHi and DT followed by a recovery in which pHi and DT stabilized at values slightly higher than in HEPES buffer. Introduction of HCO3- hyperpolarized Vm by 8 +/- 2.3 mV (P < 0.05). SITS (0.1 mM) completely abolished the hyperpolarization and attenuated the recovery of both pHi and DT. Under steady-state conditions in HCO3- buffered media, SITS induced a depolarization compatible with the suppression of the entry of negative charges. Depolarization by high Ko+ (45 mM) elicited a rise in pHi of 0.07 +/- 0.02 (P < 0.05), that was reversed by returning Ko+ to normal. The depolarization-induced rise in pHi proved to be Na(+)-dependent, SITS sensitive and still occurred after EIPA (microM) blockade. All the evidence strongly supports the existence of an electrogenic Na+/HCO3- cotransport mechanism that participates in the regulation of myocardial pHi. At pHi of 6.94 this mechanism seems to contribute almost equally to the Na+/H+ exchanger to pHi regulation. However, acid equivalent extrusion is potentiated when both the Na+/H+ exchanger and the HCO3-dependent mechanism are simultaneously regulating pHi.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1993

Antitachyphylactic effects of progesterone and oxytocin on term human myometrial contractile activity in vitro.

OBJECTIVE: To determine whether progesterone causes any changes in the action of oxytocin on the contractile activity of term human myometrium in vitro. METHODS: Myometrial biopsies from 13 term pregnant women undergoing cesarean delivery were immediately immersed in Hepes buffer or buffer containing 5 micrograms/mL progesterone. In the laboratory under a stereomicroscope, the specimens were dissected into 1 x 7.5-mm muscle strips. These were mounted into six tissue baths and superfused with Hepes buffer and buffer containing 5 micrograms/mL progesterone, 5 micrograms/mL progesterone plus 10 microU/mL oxytocin, 5 micrograms/mL progesterone plus 100 microU/mL oxytocin, 10 microU/mL oxytocin, and 100 microU/mL oxytocin. Isometric tension was recorded constantly for at least 3 hours. RESULTS: The frequency of myometrial contractions and tonus increased by superfusion with progesterone, progesterone plus oxytocin, and oxytocin alone compared to buffer. In addition, a gradual decrease in frequency was observed after 60 minutes of contractions with oxytocin alone, whereas progesterone plus oxytocin had no such decrease. The activity area of contractions was greater with 10 microU/mL oxytocin and decreased with progesterone, progesterone plus both concentrations of oxytocin, and 100 microU/mL oxytocin alone, compared to buffer. CONCLUSIONS: Progesterone stimulated the frequency of contractions and tonus of strips from the lower uterine segment of term human myometrium. A lasting increase in the frequency of contractions was observed with superfusion of progesterone plus oxytocin, suggesting that progesterone counteracted the reaction of tachyphylaxis to oxytocin.

Dose-Response Relationship, Drug↗
PubMed1993

Unexpected stimulatory effect of progesterone on human myometrial contractile activity in vitro.

OBJECTIVE: To investigate the effects of continuous exposure of progesterone on contractile activity of term human myometrium in vitro. METHODS: Myometrial biopsy specimens were obtained from 14 term pregnant women undergoing elective cesarean. The specimens were immediately immersed in Hepes buffer and buffer containing 0.33, 4.1, and 70 micrograms/mL progesterone. Muscle biopsies were dissected along the bundles of smooth muscle under stereomicroscope. These myometrial strips were mounted in tissue baths and superfused with Hepes buffer and buffer containing different concentrations of progesterone. RESULTS: The progesterone concentration of 0.33 microgram/mL had no significant effect on myometrial contractile activity. The concentrations of 4.1 and 70 micrograms/mL increased the frequency of contractions (P < .01 for each) and tonus (P < .01 for each), decreased the activity area of contractions (P < .01 for each), and delayed the onset of contractions (P < .01 and P < .05, respectively). CONCLUSION: When the myometrium is not deprived of progesterone, this hormone may have excitatory effects on the frequency of contractions and tonus of the lower uterine segment of term human myometrium.

Adult↗
PubMed1996

Acid efflux from retinal glial cells generated by sodium bicarbonate cotransport.

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.

Acids↗