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PubMedDate not supplied

Intracellular pH regulation in ventral horn neurones cultured from embryonic rat spinal cord.

Intracellular pH was measured with the pH-sensitive fluorescent probe BCECF in spinal cord neurones cultured from rat embryos. At an external pH of 7.3, the average steady-state pHi was 7.18 +/- 0.03 (SEM, n = 97) and 7.02 +/- 0.01 (n = 221) in HEPES-buffered and in bicarbonate-buffered medium, respectively. In both external media, pHi was strongly dependent on external pH (pHe). In HEPES-buffered medium, pHi recovery following an acid load induced by transient application of ammonium required external Na+ and was inhibited by amiloride, indicating the presence of a Na+/H+ exchange. Na(+)- and HCO3(-)-dependent, DIDS-sensitive alkalinizing mechanisms also contributed to pHi regulation in CO2/bicarbonate-buffered medium. The presence of an electrogenic Na(+)-HCO3- cotransporter was confirmed by the alkalinizing effect of KCl application. The fact that pHi is lower in CO2/bicarbonate- than in HEPES-buffered medium and the alkalinization observed upon suppression of external Cl- suggest that the acidifying Cl-/HCO3- transporter plays an important role in defining pHi.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2003

Irradiation of cells with ultraviolet-A (320-400 nm) in the presence of cell culture medium elicits biological effects due to extracellular generation of hydrogen peroxide.

Biological effects of ultraviolet A (UVA) irradiation have been ascribed to the photochemical generation of singlet oxygen. Not all effects described in the literature, however, are explicable solely by the generation of singlet oxygen, but rather resemble effects elicited by hydrogen peroxide (H2O2). Here, we show that when cells are kept in cell culture media during exposure to UVA, stress kinases, including ERK 1 and ERK 2 as well as Akt (protein kinase B), are activated, whereas there is no or only minor activation when cells are kept in phosphate-buffered saline during irradiation. Indeed, the exposure of cell culture media to UVA (30 J/cm2) results in the generation of significant amounts of H2O2, with concentrations of about 100 microM. H2O2 concentrations are at least three-fold higher in HEPES-buffered culture media after UVA irradiation. From experiments with solutions of riboflavin, tryptophan or HEPES, as well as combinations thereof, it is concluded that riboflavin mediates the photooxidation of either tryptophan or HEPES, resulting in the generation of H2O2. Thus, if signaling effects of UVA radiation are to be investigated in cell culture systems, riboflavin and HEPES/tryptophan should be avoided during irradiation because of artificial H2O2 generation. It should be taken into account, however, that in vivo tryptophan and riboflavin might play an important role in the generation of reactive oxygen species by UVA as both substances are abundant in living tissues.

Animals↗
PubMed1979

The effects of acidosis and bicarbonate on action potential repolarization in canine cardiac Purkinje fibers.

Studies were performed on canine cardiac Purkinje fibers to evaluate the effects of acidosis and bicarbonate (HCO3) on action potential repolarization. Extracellular pH (pHe) was reduced from 7.4 to 6.8 by increasing carbon dioxide (CO2) concentration from 4 to 15% in a HCO3-buffered solution or by NaOH titration in a Hepes-buffered solution. Both types of acidosis produced a slowing of the rate of terminal repolarization (i.e., period of repolarization starting at about -60 mV and ending at the maximum diastolic potential) with an attendant increase in action potential duration of 10--20 ms. This was accompanied by a reduction in the maximum diastolic potential of 2--8 mV. In contrast, if the same pH change was made by keeping CO2 concentration constant and lowering extracellular HCO3 from 23.7 to 6.0 mM, in addition to the slowing of terminal repolarization, the plateau was markedly prolonged resulting in an additional 50- to 80-ms increase in action potential duration. If pHe was held constant at 7.4 and HCO3 reduced from 23.7 mM to 0 (Hepes-buffered solution), the changes in repolarization were nearly identical to those seen in 6.0 mM HCO3 except that terminal repolarization was unchanged. This response was unaltered by doubling the concentration of Hepes. Reducing HCO3 to 12.0 mM produced changes in repolarization of about one-half the magnitude of those in 6.0 mM HCO3. These findings suggest that in Purkinje fibers, HCO3 either acts as a current that slows repolarization or modulates the ionic currents responsible for repolarization.

Action Potentials↗
PubMedDate not supplied

Long-term intermittent compressive stimulation improves the composition and mechanical properties of tissue-engineered cartilage.

Tissue engineering of articular cartilage is a promising alternative for cartilage repair. However, it has been difficult to develop tissue in vitro that mimicks native cartilage. Cartilaginous tissue formed in vitro does not accumulate enough extracellular matrix, is deficient in collagen, and possesses only a fraction of the mechanical properties of native cartilage. In this study, we investigated whether long-term intermittent compressive stimulation would improve the quality of the generated tissue. Chondrocyte cultures were established on the surface of porous calcium polyphosphate substrates and allowed to form cartilaginous tissue. In vitro-formed tissues were subjected to different stimulation protocols for 1 week. The optimal mechanical stimulation parameters identified in this short-term study were then applied to the cultures for up to 4 weeks. Mechanical stimulation applied at a 5% compressive amplitude at a frequency of 1 Hz for 400 cycles every second day resulted in the greatest increase in collagen synthesis (37 +/- 9% over control) while not significantly affecting proteoglycan synthesis (2 +/- 8% over control). This condition, applied to the chondrocyte cultures for 4 weeks, resulted in a significant increase in the amount of tissue that formed (stimulated, 2.4 +/- 0.2 mg dry wt; unstimulated, 1.61 +/- 0.08 mg dry wt). Stimulated tissues contained approximately 40% more collagen (stimulated, 590 +/- 58 microg; unstimulated, 420 +/- 42 microg), and 30% more proteoglycans (stimulated, 393 +/- 34 microg; unstimulated, 302 +/- 32 microg) as well as displaying a 2- to 3-fold increase in compressive mechanical properties (maximal equilibrium stress: stimulated, 10 +/- 1 kPa; unstimulated, 5 +/- 1 kPa; maximal equilibrium modulus: stimulated, 80 +/- 23 kPa; unstimulated, 24 +/- 6 kPa). The results of this study demonstrate that intermittent mechanical stimulation can increase collagen synthesis and, when applied over a 4-week period, can accelerate extracellular matrix accumulation as well as improve the material properties of the developed tissue. Interestingly, only short periods of mechanical stimulation (6 min every second day) were needed to affect the quality of cartilaginous tissue formed in vitro.

Adaptation, Physiological↗
PubMed1984

Short-term exposure of two-cell hamster embryos to collection media is detrimental to viability.

Hamster 2-cell embryos were transferred to recipients to provide information on the block to development in vitro. Previous attempts to elucidate this block have focused on possible deficiencies in the culture medium. In this study the possibility was examined that the media used for embryo collection are actually embryotoxic. Two-cell embryos obtained from superovulated mated hamsters were exposed to collection media [either a Hepes-buffered modified Tyrode's solution (TALP-Hepes) or PB1] during either a "fast transfer" procedure (time elapsed from collection to transfer 3.9 +/- 0.7 min or 3.7 +/- 0.9 min, respectively) or a "slow transfer" procedure (time elapsed 19.1 +/- 4.4 min or 20.8 +/- 5.7 min, respectively). In each transfer experiment, equal numbers of embryos from the same donor pool were transferred to one oviduct (fast transfer) and to the contralateral oviduct (slow transfer) of the same recipient. Embryos were recovered 33-36 h after transfer; embryos developing at least to the 8-cell stage were considered viable. Significant differences were found (P less than 0.005) between the viability of embryos subjected to fast and slow transfer procedures using either TALP-Hepes (embryo survival 60.8% vs. 22.2%, respectively, difference 38.6%) or PB1 (survival 50.6% vs. 10.5%, respectively, difference 40.1%). These data show that length of exposure to collection media is an important variable in determining subsequent developmental ability of 2-cell hamster embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

The effect of oxygen and carbon dioxide on tumor cell endothelin-1 production.

Endothelin-1 (ET-1) is produced by some tumor cells, but the dependence of this production on pO2 and pCO2, conditions relevant within the tumor microenvironment, has not been described. HT29 colon adenocarcinoma cells and DU145 prostate carcinoma cells produce similar amounts of ET-1 in vitro under normal cell culture conditions of 21% O2/5% CO2 (normoxia). Exposure of HT29 cells to either 2% O2 or 0.2% O2 significantly reduced ET-1 production compared to cells in normoxia. In contrast, production of ET-1 by DU145 cells was usually unaffected by hypoxia and was even slightly increased in cells exposed to 2% O2 in HEPES-buffered EMEM (HEPES-EMEM). Exposure of cells to either 2.2% CO2 or 7.1% CO2 had no effect on the production of ET-1 by cells in bicarbonate-buffered EMEM (EMEM). However, in HEPES-EMEM, ET-1 production by both cell lines was reduced in 7.1% CO2. A slight reduction in ET-1 produced by DU145 cells was also observed in 2.2% CO2. These results illustrate that changes in ET-1 production by tumor cells in response to hypoxia and hypercapnia are tumor-dependent. It is clear that the production of ET-1 by tumor cells under normal culture conditions may not accurately reflect production within the tumor microenvironment. A greater insight into the in vivo situation, however, may be possible by modifying the cell culture conditions.

Adenocarcinoma↗
PubMed1995

Idebenone protects hepatic microsomes against oxygen radical-mediated damage in organ preservation solutions.

The ability of the benzoquinone coenzyme Q-10 or its derivative QSA-10 (idebenone) to protect against lipid peroxidation and protein damage mediated by the pro-oxidative system NADPH/ADP/Fe3+ was tested in a rat liver microsomal model incubated in University of Wisconsin (UW) or histidine-tryptophan-ketoglutarate (HTK) solutions. Lipid peroxidation, as followed by direct determination of lipid hydroperoxides and by monitoring of malondialdehyde equivalents, was 1.8-fold enhanced in HTK and 3-fold attenuated in UW compared with HEPES buffer. Function and integrity of microsomal enzymes were investigated using glutathione S-transferase and cytochrome P-450 IIIA activity as assessed by lidocaine N-deethylation to monoethylglycinexylidide as well as by Western blot analysis of the cytochrome P-450 IIIA protein. Glutathione S-transferase activity was reduced by about 70% in HEPES compared with 50% in HTK and 36% in UW. Cytochrome P-450 IIIA was inactivated by about 75% in HEPES and HTK, compared with 55% in UW. The enzyme inactivation was paralleled by a loss of immunoreactive cytochrome P-450 IIIA protein. Supplementation of HTK with 0.1 mumol/L QSA-10 offered complete protection against lipid peroxidation, compared with 100 mumol/L with Q-10. QSA-10 (20 mumol/L) prevented protein damage in both preservation solutions, whereas Q-10 (20 mumol/L) offered only partial protection in UW and had no effect in HTK. The use of QSA-10 during liver transplantation may therefore have the potential of increasing the efficacy of organ preservation, maintaining donor organ quality, and preventing reperfusion injury. It is suitable for human use and has energy-conserving properties in addition to its antioxidant nature.

Adenosine↗
PubMed1992

Identification and egg hatching activity of monohydroxy fatty acid eicosanoids in the barnacle Balanus balanoides.

Monohydroxy fatty acids (MHFAs) were isolated from homogenates of the barnacle Balanus balanoides and identified by gas chromatography-mass spectrometry (GC-MS) as 14- and 17-hydroxy docosahexaenoic acids, 8-, 11-, 12-, 15- and 18-hydroxy eicosapentaenoic acids, 13- and 16-hydroxyoctadecatrienoic acids and 9-, 13- and 15-hydroxyoctadecadienoic acids. Each monohydroxy fatty acid was tested for egg hatching activity in a bioassay using Elminius modestus egg masses, but 8-hydroxy-5, 9, 11, 14, 17-eicosapentaenoic acid (8-HEPE) was the only MHFA with barnacle egg hatching activity. Studies on the egg hatching activity of MHFAs prepared from the oxidation of polyunsaturated fatty acids showed that activity was confined to the 8-hydroxy isomer of eicosapentaenoic acid and arachidonic acid, and that unsaturation at C5 and C14, but not C17, was essential for activity. In addition, the 8(R) conformation is necessary for activity, as 8(R)-HEPE caused egg hatching at 10(-7) M whereas the enantiomer 8(S)-HEPE was inactive.

Animals↗
PubMed1991

Incorporation of [3H]arachidonic and [14C]eicosapentaenoic acids into glycerophospholipids and their metabolism via lipoxygenases in isolated brain cells from rainbow trout Oncorhynchus mykiss.

The incorporation of [3H]arachidonate [( 3H]AA) and [14C]eicosapentaenoate [( 14C]EPA) into glycerophospholipids was studied in isolated brain cells from rainbow trout, a teleost fish whose lipids are rich in (n-3) polyunsaturated fatty acids (PUFAs). EPA was incorporated into total lipid to a greater extent than AA, but the incorporation of both PUFAs into total glycerophospholipids was almost identical. The incorporation of both AA and EPA was greatest into phosphatidylethanolamine (PE). However, when expressed per milligram of individual phosphoglycerides, both AA and EPA were preferentially incorporated into phosphatidylinositol (PI), the preference being significantly greater with AA. On the same basis, significantly more EPA than AA was incorporated into phosphatidylcholine (PC). When double-labelled cells were challenged with calcium ionophore A23187, the 3H and 14C released from the cells closely paralleled each other, peaking at 10 min after addition of ionophore. The 12-monohydroxylated derivative was the pre-dominant lipoxygenase product from both AA and EPA with a rank order of 12-hydroxyeicosatetraenoic acid (12-HETE) greater than leukotriene B4 (LTB4) greater than 5-HETE greater than 15-HETE for the AA products and 12-hydroxyeicosapentaenoic acid (12-HEPE) greater than 5-HEPE greater than LTB5 greater than 15 HEPE for EPA products. The 3H/14C (dpm/dpm) ratios in the glycerophospholipids, total released radioactivity, and the lipoxygenase products suggested that PC rather than PI was the likely source of eicosanoid precursors in trout brain cells.

Animals↗
PubMed1991

Brain microvessel 12-hydroxyeicosatetraenoic acid is the (S) enantiomer and is lipoxygenase derived.

12-Hydroxyeicosatetraenoic acid (12-HETE) production from arachidonic acid by cerebral microvessels isolated from perfused adult murine brain was reduced by the lipoxygenase inhibitors baicalein, esculetin, gossypol, nordihydroguaiaretic acid, and quercetin. Except for quercetin and gossypol, the IC50 did not exceed 10 microM. Each inhibitor, except baicalein, also decreased microvessel prostaglandin production when present in concentrations above their IC50 value for 12-HETE. In contrast, inhibitors of the cytochrome P450 monooxygenase system, clotrimazole, metyrapone, and proadifen (SKF-525A), had little effect on microvessel 12-HETE production. Chiral phase HPLC analysis revealed that only the (S) enantiomer of 12-HETE was formed. The major microvessel metabolite of eicosapentaenoic acid co-eluted with 12-hydroxyeicosapentaenoic acid (12-HEPE) on reverse-phase HPLC and the (S) enantiomer of 12-HEPE on chiral phase HPLC. Furthermore, like 12-HETE, 12-HEPE production was blocked by lipoxygenase inhibitors. These studies demonstrate that brain microvessels produce only the (S) enantiomeric 12-hydroxy derivatives of both arachidonic acid and eicosapentaenoic acid by the action of a lipoxygenase that can be selectively inhibited by baicalein. Since arachidonic acid and eicosapentaenoic acid are available to cerebral blood vessels in certain pathological settings, these 12-hydroxy acid lipoxygenase products may mediate some of the cerebrovascular dysfunction that occurs following stroke, brain trauma, or seizures.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1979

Non-passive chloride distribution in mammalian heart muscle: micro-electrode measurement of the intracellular chloride activity.

1. Liquid ion-exchanger Cl- -sensitive micro-electrodes were used to make continuous measurements of the intracellular Cl activity, aCli, of quiscent sheep cardiac Purkinje fibres in vitro. 2. aCli was higher than that expected from a passive distribution, (which would have been about 5 mM). It was 3--4 times hiable; EC1 was about 35 mV positive to Em. It was over twice as high in the nominal absence of bicarbonate/CO2 (when the buffer-system was HEPES/O2) but was not always so stable, and ECl was about 20 mV positive to Em. 3. Experiments designed to assess the maximum possible error likely to occur in the measurement of aCli showed that this could not be large and that the estimates of ECl were accurate to within 8 mV. 4. The ability of Cl to move down both concentration and potential gradients was established by demonstrating a loss of aCli in Cl-free solutions and a gain when Em was depolarized positive to ECl in high-K solutions. In both cases, the changes were complete within about 100--160 min. 5. The decline of aCli in Cl-free solutions (glucuronate-substituted) was not significantly affected by changes of [Ca]o from 0 to 12 mM or by the depolarizations of Em of up to 60 mV that sometimes occurred in low or zero [Ca]o. 6. Only 2--3 mM-aClo was sufficient to impede substantially the ready loss of aCli in HEPES-buffered solutions. 7. In high-K solutions (45 mM), Cl appeared to be passively distributed since, at equilibrium, Em and ECl differed by less than 2 mV. 8. In HEPES-buffered Tyrode, ECl of quiescent papillary muscle of the guinea-pig was, on average, 39 mV positive to Em. 9. It is concluded that liquid ion-exchanger Cl- -sensitive micro-electrodes are suitable for studying the Cl regulation of sheep Prukinje fibres, and probably of other cardiac tissues. The measurements of resting aCli are quite accurate when using either HEPES or bicarbonate-buffered Tyrode. The results are discussed in relation to estimates of the apparent membrane Cl permeability under various conditions and the possible existence of an inwardly directed 'Cl pump'.

Animals↗
PubMed1987

The regulation of intracellular pH by identified glial cells and neurones in the central nervous system of the leech.

1. Double-barrelled, neutral-carrier pH-sensitive micro-electrodes were used to measure the intracellular pH (pHi) and the pHi regulation of neuropile glial (n.g.) cells and of identified neurones of the leech Hirudo medicinalis. 2. The distribution of H+ in the n.g. cells and in the neurones was found not to be in electrical equilibrium. The mean pHi of the n.g. cells was 6.87 +/- 0.13 (mean +/- S.D. of mean here and for all following data n = 27) in HEPES-buffered leech saline (pHo = 7.4) and 7.18 +/- 0.19 (n = 13) in 2% CO2-11 mM-HCO3(-)-saline. The mean pHi was 7.28 +/- 0.1 (n = 20) in Retzius neurones and 7.32 +/- 0.15 (n = 12) in noxious neurones in HEPES-buffered leech saline, and 7.20 +/- 0.15 (n = 10) and 7.27 +/- 0.16 (n = 6) in 2% CO2-11 mM-HCO3(-)-buffered saline in these two types of neurones, respectively. 3. The cytoplasmic buffering power, as calculated by the change in pHi following the change from 2% CO2-11 mM-HCO3- to 5% CO2-22 mM-HCO3- in the leech saline, was 20-30 mM/pH unit in the n.g. cells and between 12 and 33 mM/pH unit in the neurones. 4. The recovery of pHi in n.g. cells from an experimentally induced acidification (addition and removal of 20 mM-NH4Cl) was dependent on the presence of external Na+. Independent of the buffer system used, pHi recovery was inhibited when external Na+ was exchanged by N-methyl-D-glucamine. Amiloride (2-3 mM) reduced the rate of pHi recovery by about 50% in these n.g. cells. 5. In CO2-HCO3(-)-free saline, or in the presence of the anion exchange blocker 4-acetamido-4'-isothiocyanostilbene-2, 2'-disulphonic acid (SITS, 0.5 mM), pHi recovery from an acid load was often slowed by up to 50% in n.g. cells. This suggests that there is a significant contribution of a HCO3(-)-dependent membrane transport to pHi regulation in n.g. cells. 6. When a HEPES-buffered saline was exchanged by a 2% CO2-11 mM-HCO3(-)-buffered saline, the pHi of n.g. cells increased by 0.31 pH units. This alkaline shift was reversible upon removal of the CO2-HCO3- and was absent in the Na+-free saline. It was not inhibited by 1 mM-furosemide or by 0.5 mM-SITS.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1988

Changes in the surface pH of voltage-clamped snail neurones apparently caused by H+ fluxes through a channel.

1. The surface and intracellular pH of snail neurones was recorded with microelectrodes while the membrane potential was reduced in 10 mV steps for a few seconds each or to positive values for periods of several minutes. 2. Depolarizations to positive membrane potentials caused rapid falls in surface pH (pHs) which varied from cell to cell and from one point to another on the surface of the same cell. 3. When pHi was normal or alkaline, the first few 10 mV steps of depolarization often caused a small pHs increase which changed to a decrease as the depolarization increased. The threshold potential at which the pHs increase changed to a decrease varied with pHi in a linear manner, so that at acid pHi values the threshold potential approached the normal resting potential. There was good agreement between the threshold and H+ equilibrium potentials calculated from pHi and pHs. 4. The size of the pHs decrease observed at a given pHi and depolarization depended on extracellular buffering power in a non-linear manner. Solutions buffered with 20 mM-NaHCO3 had similar surface buffering power to CO2-free solutions buffered with only 1-2 mM-HEPES, pH 7.5. 5. In 1 mM-HEPES pHs changes were larger, and pHi increases slower, than those seen in cells depolarized to the same potential in 20 mM-HEPES. The slowing of the rate of pHi increase suggests that the pHs changes occur all over the cell surface, and not only at the recording site. 6. With long-lasting depolarizations the size of the pHs decrease was proportional to the rate of pHi increase and thus, assuming a constant intracellular buffering power, to the rate of efflux of H+. 7. The results provide further evidence that snail neurones possess a channel permeable to H+ which is opened on depolarization. H+ efflux through this channel could cause rapid acidification of a confined extracellular space.

Animals↗
PubMed1988

Intracellular pH regulation in resting and contracting segments of rat mesenteric resistance vessels.

1. The pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF) was used to measure intracellular pH (pHi) in segments of rat resistance vessels (internal diameter about 200 microns) with the vessels mounted in a myograph for simultaneous measurements of isometric contraction. 2. BCECF loaded slowly into the vessels over 1 h and did not affect the maximal contractility of the vessels. There was a loss of dye with time which, however, was very slow when the segments were only excited for 2 s/min, suggesting that the loss was mainly due to dye bleaching with only a very slow leak. 3. The ratio of the emissions (at 540 nm) with excitation at 495 and 450 nm was calibrated in terms of pH using the K+-H+ ionophore nigericin. This calibration gave a pHi value of 7.15 +/- 0.02 (n = 20), suggesting that hydrogen ions are not in electrochemical equilibrium in these vascular smooth muscles which have a membrane potential of about -60 mV. 4. Addition of 10 mM-NH4Cl caused a transient alkalinization and wash-out of 10 mM-NH4Cl a transient acidification. Increasing CO2 with maintained bicarbonate caused a rapid acidification followed by an incomplete recovery. Removal of CO2 and bicarbonate (HEPES-buffered solution) with constant extracellular pH caused a transient alkalinization but steady-state pHi was not significantly altered. 5. In bicarbonate-free buffer the Na+-H+ exchange blocker 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and sodium-free conditions caused a slow acidification. In bicarbonate buffer (PSS) EIPA had no detectable effect after 10 min but the anion exchange blocker diisothio-cyanatostilbenedisulphonic acid (DIDS) caused a small acidification over that time course. 6. The rate of recovery after an acid load was about 50% lower in HEPES buffer compared to PSS and it was inhibited by EIPA. In PSS amiloride and EIPA each had a small inhibitory effect on the pH recovery after an acid load. DIDS also inhibited the recovery from an acid load in PSS and this effect was additive to that of EIPA. DIDS and EIPA also had additive inhibitory effects on the 22Na+ influx stimulated by the acid loading, while in HEPES buffer DIDS had no effect on either pH recovery or 22Na+ influx. These results suggest that a Na+-H+ exchange and an influx of bicarbonate coupled to sodium influx are of importance for pHi control in these vessels.(ABSTRACT TRUNCATED AT 400 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

Intracellular pH and its regulation in isolated type I carotid body cells of the neonatal rat.

1. The dual-emission pH-sensitive fluoroprobe carboxy-SNARF-1 (carboxy-seminaptharhodofluor) was used to measure pHi in type I cells enzymically dispersed from the neonatal rat carotid body. 2. Steady-state pHi in cells bathed in a HEPES-buffered Tyrode solution (pH 7.4) was found to be remarkably alkaline (pHi = 7.77) whereas cells bathed in a CO2-HCO3(-)-buffered Tyrode solution (pH 7.4) had a more 'normal' pHi (pHi = 7.28). These observations were further substantiated by using an independent nullpoint test method to determine pHi. 3. Intracellular intrinsic buffering (beta, determined by acidifying the cell using an NH4Cl pre-pulse) was in the range 7-20 mM per pH unit and appeared to be dependent upon pHi with beta increasing as pHi decreased. 4. In cells bathed in a HEPES-buffered Tyrode solution, pHi recovery from an induced intracellular acid load (10 mM-NH4Cl pre-pulse) was inhibited by the Na(+)-H+ exchange inhibitor ethyl isopropyl amiloride (EIPA; 150 microM) or substitution of Nao+ with N-methyl-D-glucamine (NMG). Both EIPA and Nao+ removal also caused a rapid intracellular acidification, which in the case of Nao+ removal, was readily reversible. The rate of this acidification was similar for both Nao+ removal and EIPA addition. 5. Transferring cells from a HEPES-buffered Tyrode solution to one buffered with 5% CO2-HCO3- resulted in an intracellular acidification which was partially, or wholly, sustained. The rate of acidification upon transfer to CO2-HCO3- was considerably slowed by the membrane permeant carbonic anhydrase inhibitor, acetazolamide, thus indicating the presence of the enzyme in these cells. 6. In CO2-HCO3(-)-buffered Tyrode solution, pHi recovery from an intracellular acidosis (NH4+ pre-pulse) was only partially inhibited by EIPA or amiloride whereas Nao+ removal completely inhibited the recovery. The stilbene DIDS (4,4-diisothiocyanatostilbenedisulphonic acid, 200 microM) also partially inhibited pHi recovery following an induced intracellular acidosis. Furthermore, the pre-treatment with 200 microM-DIDS of a pre-acidified cell in Na(+)-free Tyrode solution completely inhibited pHi recovery when Nao+ was reintroduced together with concomitant addition of 150 microM-EIPA. We conclude, that in the presence of CO2-HCO3-, a Na(+)- and HCO3-dependent (DIDS inhibitable) mechanism aids acid extrusion.(ABSTRACT TRUNCATED AT 400 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

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

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

Adenosine Triphosphate↗
PubMed1992

Role of bicarbonate in pH recovery from intracellular acidosis in the guinea-pig ventricular myocyte.

1. Intracellular pH (pHi) was recorded ratiometrically in isolated guinea-pig ventricular myocytes using the pH-sensitive fluoroprobe, carboxy-SNARF-1 (carboxy-seminaphthorhodafluor). 2. Following an intracellular acid load (10 mM NH4 Cl removal), pHi recovery in HEPES-buffered Tyrode solution was inhibited by 1.5 mM amiloride (Na(+)-H+ antiport blocker). In the presence of amiloride, switching from HEPES buffer to HCO3-/CO2 (pHo of both solutions = 7.4) stimulated a pHi recovery towards more alkaline levels. 3. Amiloride-resistant, HCO(3-)-dependent pHi recovery was inhibited by removal of external Na+ (replaced by N-methyl-D-glucamine), whereas removal of external Cl- (replaced by glucuronate, leading to depletion of internal Cl-), removal of external K+, or decreasing external Ca2+ by approximately tenfold had no inhibitory effect. These results suggest that the amiloride-resistant recovery is due to a Na(+)-HCO3- cotransport into the cell. 4. The stilbene derivative DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid, 500 microM) slowed Na(+)-HCO(3-)-dependent pHi recovery. 5. Intracellular pH increased in Cl(-)-free solution and this increase still occurred in Na(+)-free solution indicating that it is not caused via Na(+)-HCO3- symport and is more likely to be due to Cl- efflux in exchange for HCO3- influx on a sarcolemmal Cl(-)-HCO3- exchanger. The lack of any significant pHi recovery from intracellular acidosis in Na(+)-free solution suggests that this exchanger does not contribute to acid-equivalent extrusion. 6. Possible voltage sensitivity and electrogenicity of the co-transport were examined by using the whole-cell patch clamp technique in combination with SNARF-1 recordings of pHi. Stepping the holding potential from -110 to -40 mV did not affect amiloride-resistant pHi recovery from acidosis. Moreover, following an intracellular acid load, the activation of Na(+)-HCO3- co-influx (by switching from HEPES to HCO3-/CO2 buffer) produced no detectable outward current (outward current would be expected if the coupling of HCO3- with Na+ were > 1.0). 7. Intracellular intrinsic buffering power (beta i) was assessed as a function of pHi (beta i computed from the decrease of pHi following reduction of extracellular NH4 Cl in amiloride-containing solution). beta i in the ventricular myocyte increases roughly linearly with a decrease in pHi according the following equation: beta i = -28(pHi) +222.6. 8. Comparison of acid-equivalent efflux via Na(+)-HCO3- symport and Na(+)-H+ antiport showed that, following an intracellular acidosis, the symport accounts for about 40% of total acid efflux, the other 60% being carried by the antiport.(ABSTRACT TRUNCATED AT 400 WORDS)

Amiloride↗
PubMed1993

The control of intracellular pH in cultured avian chondrocytes.

1. Mechanical loading of cartilaginous tissue generates an increase in the concentration of cations in the extracellular matrix. This includes a decrease of the extracellular pH (pHo), which is known to affect the intracellular pH (pHi), thereby modifying the intracellular metabolism. Thus, the regulation of pHi is essential for the physiological function of cartilage. The fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF AM) was employed in order to assess the mechanisms responsible for control of the pHi in an embryonic avian chondrocyte cell suspension. 2. Steady-state pHi in the absence of physiological HCO3- was 7.15 +/- 0.01 pH units as compared to a pHi of 6.94 +/- 0.02 pH units in its presence (P < 0.01). The intrinsic buffering power of chondrocytes (beta i) was 38.9 mM/pH unit and the total buffering capacity (beta T) was 65.8 mM/pH unit. 3. Cells maintained in a Hepes-buffered solution were exposed to an intracellular acid load by the NH4+ prepulse technique (20 mM NH4Cl). The initial rate of pHi recovery was 0.106 pH units/min (n = 18). Amiloride (0.33 mM), an inhibitor of the Na(+)-H+ exchanger, or replacement of external sodium [Na+]o with choline induced a 60% inhibition of the recovery rate, indicating a predominant involvement of this antiporter in the response to intracellular acidification. 4. H(+)-ATPase inhibitors (oligomycin 20 micrograms/ml; N,N;-dicyclohexylcarbodiimide (DCC), 0.5 mM; N-ethylmaleimide (NEM), 0.25 mM) and iodomycin (2 mM), a metabolic cell suppressor, reduced acid extrusion by 25% as measured by the NH4Cl prepulse in Hepes-bathed cells. 5. Chondrocytes transferred from a Hepes-buffered solution to a 5% CO2-25 mM HCO3- medium (HCO3- solution) underwent a pHi decrease of approximately 0.20 pH units, followed by a regulatory alkalinizing response of 0.118 pH units/min. The Na(+)-H+ exchanger was responsible for only 15% of this alkalinization (amiloride, 0.33 mM), in contrast to its primary role in HCO(3-)-free solution. 6. The activity of a Na(+)-dependent Cl(-)-HCO3- exchanger in physiological HCO3- solution was estimated by addition of the inhibitors 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS; 0.5 mM) or diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS; 100 microM) and by the suspensions of chondrocytes in a Na(+)-free solution. Acidification performed under these conditions resulted in a 45% inhibition of the recovery rate as compared to control rates.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗