scholarly journals Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus

1997 ◽  
Vol 110 (4) ◽  
pp. 453-465 ◽  
Author(s):  
Mark O. Bevensee ◽  
Regina A. Weed ◽  
Walter F. Boron
Keyword(s):  
Intracellular Ph ◽  
Ph Regulation ◽  
Disulfonic Acid ◽  
Bath Solution ◽  
Major Cation ◽  
Cultured Astrocytes ◽  
Buffering Power ◽  
Acid Load ◽  
Acid Extrusion ◽  
Best Fit

We studied the regulation of intracellular pH (pHi) in single cultured astrocytes passaged once from the hippocampus of the rat, using the dye 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) to monitor pHi. Intrinsic buffering power (βI) was 10.5 mM (pH unit)−1 at pHi 7.0, and decreased linearly with pHi; the best-fit line to the data had a slope of −10.0 mM (pH unit)−2. In the absence of HCO3−, pHi recovery from an acid load was mediated predominantly by a Na-H exchanger because the recovery was inhibited 88% by amiloride and 79% by ethylisopropylamiloride (EIPA) at pHi 6.05. The ethylisopropylamiloride-sensitive component of acid extrusion fell linearly with pHi. Acid extrusion was inhibited 68% (pHi 6.23) by substituting Li+ for Na+ in the bath solution. Switching from a CO2/HCO3−-free to a CO2/HCO3−-containing bath solution caused mean steady state pHi to increase from 6.82 to 6.90, due to a Na+-driven HCO3− transporter. The HCO3−-induced pHi increase was unaffected by amiloride, but was inhibited 75% (pHi 6.85) by 400 μM 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), and 65% (pHi 6.55–6.75) by pretreating astrocytes for up to ∼6.3 h with 400 μM 4-acetamide-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS). The CO2/HCO3−-induced pHi increase was blocked when external Na+ was replaced with N-methyl-d-glucammonium (NMDG+). In the presence of HCO3−, the Na+-driven HCO3− transporter contributed to the pHi recovery from an acid load. For example, HCO3− shifted the plot of acid-extrusion rate vs. pHi by 0.15–0.3 pH units in the alkaline direction. Also, with Na-H exchange inhibited by amiloride, HCO3− increased acid extrusion 3.8-fold (pHi 6.20). When astrocytes were acid loaded in amiloride, with Li+ as the major cation, HCO3− failed to elicit a substantial increase in pHi. Thus, Li+ does not appear to substitute well for Na+ on the HCO3− transporter. We conclude that an amiloride-sensitive Na-H exchanger and a Na+-driven HCO3− transporter are the predominant acid extruders in astrocytes.

Intracellular pH transients in rat diaphragm muscle measured with DMO

1978 ◽  
Vol 235 (1) ◽  
pp. C49-C54 ◽  
Author(s):  
A. Roos ◽  
W. F. Boron
Keyword(s):  
Intracellular Ph ◽  
Weak Acid ◽  
Diaphragm Muscle ◽  
Disulfonic Acid ◽  
Free Solution ◽  
Rat Diaphragm ◽  
Control Value ◽  
Acid Load ◽  
Acid Extrusion ◽  
Acid Loading

Changes of the intracellular pH of rat diaphragm muscle were monitored at 30-min intervals with the weak acid DMO (5,5-dimethyl-2,4-oxazolidinedione). Transferring the muscle from a CO2-containing to a CO2-free solution caused intracellular pH (pHi) to rise by an average of 0.18 during the first 30 min and then to level off at a slightly lower value over the next 60-90 min. Transferring the muscle from a CO2-free to a CO2-containing solution caused pHi to fall by 0.18 during the first 30 min and then to recover by 0.05 over the next 90 min. Subsequent return to the CO2-free solution caused pHi to overshoot the control value by 0.10. Both the recovery and the overshoot can be accounted for by an acid-extruding pump. Intracellular acid loading with 118 mM DMO similarly caused pHi to fall initially, to recover slowly during the acid loading, and then to overshoot the control pHi on removal of the acid load. In the absence of HCO3-/CO2, acid extrusion was reduced by about a fifth. SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid) had no effect. The absence of either Na+ or Cl- from HCO3-/CO2- free solution reduced acid extrusion by about a half.

pH regulation in barnacle muscle fibers: dependence on intracellular and extracellular pH

1979 ◽  
Vol 237 (3) ◽  
pp. C185-C193 ◽  
Author(s):  
W. F. Boron ◽  
W. C. McCormick ◽  
A. Roos
Keyword(s):  
Ph Regulation ◽  
Muscle Fibers ◽  
Extracellular Ph ◽  
Disulfonic Acid ◽  
Extrusion Rate ◽  
Barnacle Muscle ◽  
Buffering Power ◽  
Acid Extrusion ◽  
Barnacle Muscle Fibers ◽  
Very High

Intracellular pH (pHi) regulation was studied in acid-loaded barnacle muscle fibers by monitoring recovery of pHi with a pH-sensitive microelectrode. By multiplying the rate of pHi recovery by total intracellular buffering power, the acid extrusion rate was obtained. The acid extrusion rate was greatest at low values of pHi, and declined toward zero as pHi approached normal levels. It increased as the extracellular pH (pHo) was raised either by increasing external [HCO3] ([HCO3]o) at constant PCO2 or by decreasing PCO2 at constant [HCO3]o, but more so in the former case than in the latter. These observations suggest that pHo per se is an important determinant of the acid extrusion rate, but that raising [HCO3]o by itself also stimulates acid extrusion. This would be expected if acid extrusion involves the inward movement of HCO3. When fibers were exposed to HCO3-containing solutions at very low or very high pHo, pHi drifted downward or upward, respectively; thbe drifts were inhibited by 4-acetamido-4' isothiocyanostilbene-2,2' disulfonic acid (SITS). Our results are discussed in terms of possible mechanisms of acid extrusion.

scholarly journals Effect of eccentric contraction-induced injury on force and intracellular pH in rat skeletal muscles

2002 ◽  
Vol 92 (1) ◽  
pp. 93-99 ◽  
Author(s):  
E. W. Yeung ◽  
J.-P. Bourreau ◽  
D. G. Allen ◽  
H. J. Ballard
Keyword(s):  
Intracellular Ph ◽  
Eccentric Contraction ◽  
Eccentric Contractions ◽  
Optimal Length ◽  
Extrusion Rate ◽  
Reduction In Force ◽  
Buffering Power ◽  
Acid Load ◽  
Rat Soleus Muscle ◽  
Acid Extrusion

The effect of eccentric contraction on force generation and intracellular pH (pHi) regulation was investigated in rat soleus muscle. Eccentric muscle damage was induced by stretching muscle bundles by 30% of the optimal length for a series of 10 tetani. After eccentric contractions, there was reduction in force at all stimulation frequencies and a greater reduction in relative force at low-stimulus frequencies. There was also a shift of optimal length to longer lengths. pHi was measured with a pH-sensitive probe, 2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein AM. pHi regulation was studied by inducing an acute acid load with the removal of 20–40 mM ammonium chloride, and the rate of pHi recovery was monitored. The acid extrusion rate was obtained by multiplying the rate of pHi recovery by the buffering power. The resting pHi after eccentric contractions was more acidic, and the rate of recovery from acid load post-eccentric contractions was slower than that from postisometric controls. This is further supported by the slower acid extrusion rate. Amiloride slowed the recovery from an acid load in control experiments. Because the Na+/H+ exchanger is the dominant mechanism for the recovery of pHi, this suggests that the impairment in the ability of the muscle to regulate pHiafter eccentric contractions is caused by decreased activity of the Na+/H+ exchanger.

scholarly journals Role of Na-H exchangers and vacuolar H+ pumps in intracellular pH regulation in neonatal rat osteoclasts.

1995 ◽  
Vol 105 (2) ◽  
pp. 177-208 ◽  
Author(s):  
J H Ravesloot ◽  
T Eisen ◽  
R Baron ◽  
W F Boron
Keyword(s):  
Intracellular Ph ◽  
Imaging System ◽  
Ph Regulation ◽  
Neonatal Rat ◽  
Hill Coefficient ◽  
Acid Base ◽  
Buffering Power ◽  
Acid Extrusion

Osteoclasts resorb bone by pumping of H+ into a compartment between the cell and the bone surface. Intracellular pH (pHi) homeostasis requires that this acid extrusion, mediated by a vacuolar-type H+ ATPase, be complemented by other acid-base transporters. We investigated acid-extrusion mechanisms of single, freshly isolated, neonatal rat osteoclasts. Cells adherent to glass coverslips were studied in the nominal absence of CO2/HCO3-, using the pH-sensitive dye BCECF and a digital imaging system. Initial pHi averaged 7.31 and was uniform throughout individual cells. Intrinsic buffering power (beta 1) decreased curvilinearly from approximately 25 mM at pHi = 6.4 to approximately 6.0 mM at pHi = 7.4. In all polygonally shaped osteoclasts, and approximately 60% of round osteoclasts (approximately 20% of total), pHi recovery from acid loads was mediated exclusively by Na-H exchange. In these pattern-1 cells, pHi recovery was 95% complete within 200 s, and was blocked by removing Na+, or by applying 1 mM amiloride, 50 microM ethylisopropylamiloride (EIPA), or 50 microM hexamethyleneamiloride (HMA). The apparent K1/2 for HMA ([Na+]o = 150 mM) was 49 nM, and the apparent K1/2 for Na+ was 45 mM. Na-H exchange, corrected for amiloride-insensitive fluxes, was half maximal at pHi 6.73, with an apparent Hill coefficient for intracellular H+ of 2.9. Maximal Na-H exchange averaged 741 microM/s. In the remaining approximately 40% of round osteoclasts (pattern-2 cells), pHi recovery from acid loads was brisk even in the absence of Na+ or presence of amiloride. This Na(+)-independent pHi recovery was blocked by 7-chloro-4-nitrobenz-2-oxa-1,3-diazol (NBD-Cl), a vacuolar-type H+ pump inhibitor. Corrected for NBD-Cl insensitive fluxes, H+ pump fluxes decreased approximately linearly from 96 at pHi 6.8 to 11 microM/s at pHi 7.45. In approximately 45% of pattern-2 cells, Na+ readdition elicited a further pHi recovery, suggesting that H+ pumps and Na-H exchangers can exist simultaneously. We conclude that, under the conditions of our study, most neonatal rat osteoclasts express Na-H exchangers that are probably of the ubiquitous basolateral subtype. Some cells express vacuolar-type H+ pumps in their plasma membrane, as do active osteoclasts in situ.

Ionic requirements for intracellular pH regulation in rainbow trout hepatocytes

1986 ◽  
Vol 250 (1) ◽  
pp. R24-R29 ◽  
Author(s):  
P. J. Walsh
Keyword(s):  
Rainbow Trout ◽  
Intracellular Ph ◽  
Ph Regulation ◽  
Disulfonic Acid ◽  
Salmo Gairdneri ◽  
Exchange System ◽  
Exchange Processes ◽  
Acid Load ◽  
Rate Of Recovery ◽  
22Na Uptake

The ionic requirements for pH regulation in isolated rainbow trout (Salmo gairdneri) hepatocytes were determined by manipulation of intracellular pH (pHi; measured by the dimethyloxazolidinedione distribution technique) by NH4Cl prepulse and changes in external [CO2] in the presence and absence of various drugs and external ions. The presence of a Na+/H+(NH+4) exchange system is supported by the following results: 1) the rate of recovery from an acid load is decreased by amiloride (0.5 mM) or reduction of external [Na+]; 2) the rate of 22Na uptake is increased during recovery from an acid load, and this increase in amiloride sensitive. The presence of a Cl-/HCO3- exchange system is supported by the observations that 1) pHi is increased, and 2) rates of recovery of pHi from acid loading are enhanced, by exposure to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (0.5 mM) and reductions in external [Cl-]. Further studies are required to determine the role of these exchange processes during physiological pHi perturbations.

scholarly journals Intracellular pH regulation in the S3 segment of the rabbit proximal tubule in HCO3- -free solutions.

1988 ◽  
Vol 92 (3) ◽  
pp. 369-393 ◽  
Author(s):  
N L Nakhoul ◽  
A G Lopes ◽  
J R Chaillet ◽  
W F Boron
Keyword(s):  
Proximal Tubule ◽  
Intracellular Ph ◽  
Ph Regulation ◽  
Transport Systems ◽  
Total Acid ◽  
Luminal Membrane ◽  
Acid Load ◽  
S3 Segment ◽  
Acid Extrusion ◽  
Dependent Mechanism

We used the absorbance spectrum of 4',5'-dimethyl-5-(and 6) carboxyfluorescein to measure intracellular pH (pHi) in the isolated, perfused S3 segment of the rabbit proximal tubule. Experiments were conducted in HCO3- -free solutions. pHi recovered from an acid load imposed by an NH4+ prepulse, indicating the presence of one or more active acid-extrusion mechanisms. Removal of Na+ from bath and lumen caused pHi to decrease by approximately 0.6, whereas Na+ readdition caused complete pHi recovery. Removal of Na+ from the bath caused only a slow pHi decrease that was enhanced about fourfold when Na+ was subsequently removed from the lumen also. Similarly, the pHi recovery produced by the readdition of Na+ to the bath and lumen was about ninefold faster than when Na+ was returned to the bath only. Amiloride (1-2 mM) inhibited the pHi recovery that was elicited by returning 15 or 29 mM Na+ to lumen by only approximately 30%. However, in the absence of external acetate (Ac-), 1 mM amiloride inhibited approximately 66% of the pHi recovery induced by the readdition of 29 mM Na+ to the lumen only. The removal of external Ac- reduced the pHi recovery rate from an NH4+-induced acid load by approximately 47%, and that elicited by Na+ readdition, by approximately 67%. Finally, when bilateral removal of Na+ was maintained for several minutes, pHi recovered from the initial acidification, slowly at first, and then more rapidly, eventually reaching a pHi approximately 0.1 higher than the initial one. This Na+-independent pHi recovery was not significantly affected by lowering [HEPES]o from 32 to 3 mM or by adding N'N'-dicyclohexylcarbodiimide (10(-4) M) to the lumen, but it was reduced approximately 57% by iodoacetate (0.5 mM) plus cyanide (1 mM). We conclude that in the nominal absence of HCO3-, three transport systems contribute to acid extrusion by S3 cells: (a) a Na+-independent mechanism, possibly an H+ pump; (b) a Na-H exchanger, confined primarily to the luminal membrane; and (c) an Ac- and luminal Na+-dependent mechanism. The contribution of these three mechanisms to total acid extrusion, assessed by the rapid readdition of Na+, was approximately 13, approximately 30, and approximately 57%, respectively.

pH regulation in ileum: Na(+)-H+ and Cl(-)-HCO3- exchange in isolated crypt and villus cells

1991 ◽  
Vol 260 (3) ◽  
pp. G440-G449 ◽  
Author(s):  
U. Sundaram ◽  
R. G. Knickelbein ◽  
J. W. Dobbins
Keyword(s):  
Intracellular Ph ◽  
Ph Regulation ◽  
Second Messengers ◽  
Cell Types ◽  
Current Evidence ◽  
Leucine Incorporation ◽  
Trypan Blue Exclusion ◽  
Morphological Criteria ◽  
Acid Load ◽  
Separation Cell

Current evidence suggests that intestinal crypt and villus cells have different functions in electrolyte transport. To study the regulation of transporters, we isolated and separated these two cell types. This was accomplished by sequential collection of enterocytes from rabbit ileal loops incubated with buffered solutions of calcium chelators. Alkaline phosphatase and thymidine kinase activity, sodium-glucose cotransport, and morphological criteria were used to determine cell separation. Cell viability was evaluated with trypan blue exclusion, leucine incorporation into protein, and morphological features. The role of Na(+)-H+ and Cl(-)-HCO3- exchange in the regulation of intracellular pH was analyzed using an intracellular pH sensitive dye, BCECF. Removal of external Na+ or the addition of amiloride resulted in acidification of both crypt and villus cells. Removal of Cl- or the addition of DIDS resulted in alkalinization of both cell types. The cells could be acidified with NH4Cl, and recovery from this acid load was dependent on Na+ and inhibited by amiloride. Similarly, the cells could be alkalinized with propionate and recovery was Cl- dependent and DIDS sensitive. These data are consistent with the presence of Na(+)-H+ and Cl(-)-HCO3- exchange in both crypt and villus cells. Both exchanges appear to be involved in the regulation of basal pH as well as in recovery from alterations in intracellular pH. Having demonstrated the presence of Na(+)-H+ and Cl(-)-HCO3- exchange activity in both crypt and villus cells, we can now use these cells to determine the regulation of these exchangers by intracellular second messengers.

pH regulation and response to AVP in A10 cells differ markedly in the presence vs. absence of CO2-HCO3-

1990 ◽  
Vol 259 (3) ◽  
pp. C471-C483 ◽  
Author(s):  
D. Kikeri ◽  
M. L. Zeidel ◽  
B. J. Ballermann ◽  
B. M. Brenner ◽  
S. C. Hebert
Keyword(s):  
Steady State ◽  
Ph Regulation ◽  
Disulfonic Acid ◽  
Potential Contribution ◽  
Vasoactive Agents ◽  
Cell Responses ◽  
Extrusion Mechanism ◽  
Acid Extrusion ◽  
Ambient Co2 ◽  
Stimulation Of

The fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) was used to determine the effect of ambient CO2-HCO3- on the regulation of intracellular pH (pHi) and the pHi response to arginine vasopressin (AVP) in A10 vascular smooth muscle (VSM) cells. Steady-state pHi averaged 7.04 +/- 0.02 in the absence and 7.25 +/- 0.01 in the presence of CO2-HCO3-. In the absence of CO2-HCO3-, virtually all (greater than 96%) of the acid extrusion from acidification occurred by amiloride-sensitive Na(+)-H+ exchange. However, in the presence of CO2-HCO3-, acid extrusion after acidification occurred by both Na(+)-H+ exchange and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive Na(+)-dependent Cl(-)-HCO3- exchange. In CO2-HCO3(-)-containing media, amiloride-sensitive Na(+)-H+ exchange mediated 85% of acid extrusion at a pHi of 6.48, but the DIDS-sensitive acid extrusion mechanism (NA(+)-dependent Cl(-)-HCO3- exchange) was the dominant acid extrusion mechanism at a pHi of 6.94. Base exited A10 cells by a DIDS-sensitive process consistent with Na(+)-independent Cl(-)-HCO3- exchange. Both amiloride- and DIDS-sensitive processes regulated steady-state pHi in CO2-HCO3-. AVP (10(-7) M) alkalinized steady-state pHi in the absence of CO2-HCO3- (delta pHi = 0.08 +/- 0.01 pH units) by stimulating Na(+)-H+ exchange; however, AVP did not alter pHi of untreated cells in CO2-HCO3- (delta pHi = -0.01 +/- 0.01 pH units) because of concomitant stimulation of Na(+)-independent Cl(-)-HCO3-exchange. We conclude that the steady-state pHi, the mechanisms of pHi regulation, and the pHi response to AVP in A10 cells are critically influenced by the presence of extracellular CO2-HCO3-. Thus the potential contribution of pHi changes to VSM cell responses to vasoactive agents should be evaluated in the presence of CO2-HCO3-.

Na-H exchange regulates intracellular pH in isolated rat hepatocyte couplets

1987 ◽  
Vol 252 (1) ◽  
pp. G109-G113
Author(s):  
R. M. Henderson ◽  
J. Graf ◽  
J. L. Boyer
Keyword(s):  
Carbon Dioxide ◽  
Intracellular Ph ◽  
Ph Regulation ◽  
Intracellular Acidification ◽  
Rat Hepatocyte ◽  
Buffering Power ◽  
Recovery From Acidification ◽  
Regulation Mechanisms ◽  
Phi Regulation ◽  
Isolated Rat

Intracellular pH (pHi) was measured directly in isolated rat hepatocyte couplets using pH sensitive microelectrodes. The hepatocytes were maintained in a minimal salt buffer without added hormones or serum. Values of pHi (6.99 +/- 0.12, mean +/- SE) were close to their Nernst equilibria. After intracellular acidification with ammonium chloride, pH regulation was inhibited with 1 mM amiloride or by omission of external sodium, consistent with a Na-H exchange mechanism. Mean intracellular buffering power, in the nominal absence of carbon dioxide, was 34.1 +/- 11.4 mM. In the presence of external bicarbonate, amiloride or omission of sodium slowed, but did not completely inhibit recovery from acidification, indicating that additional pHi regulation mechanisms may operate in this preparation. These studies provide a direct measurement of pHi in hepatocyte couplets and indicate that Na-H exchange, together with a bicarbonate dependent system are important mechanisms for pHi regulation in this preparation.

scholarly journals Intracellular pH regulation in neurons from chemosensitive and nonchemosensitive areas of the medulla

1998 ◽  
Vol 275 (4) ◽  
pp. R1152-R1163 ◽  
Author(s):  
Nick A. Ritucci ◽  
Laura Chambers-Kersh ◽  
Jay B. Dean ◽  
Robert W. Putnam
Keyword(s):  
Intracellular Ph ◽  
Inferior Olive ◽  
Ph Regulation ◽  
Ventrolateral Medulla ◽  
Solitary Tract ◽  
Intracellular Ph Regulation ◽  
Buffering Power ◽  
Recovery From Acidification ◽  
Central Chemoreception ◽  
Regulatory Properties

Intracellular pH (pHi) regulation was studied in neurons from two chemosensitive [nucleus of the solitary tract (NTS) and ventrolateral medulla (VLM)] and two nonchemosensitive [hypoglossal (Hyp) and inferior olive (IO)] areas of the medulla oblongata. Intrinsic buffering power (βint) was the same in neurons from all regions (46 mM/pH U). Na+/H+exchange mediated recovery from acidification in all neurons [Ritucci, N. A., J. B. Dean, and R. W. Putnam. Am. J. Physiol. 273 ( Regulatory Integrative Comp. Physiol.42): R433–R441, 1997]. Cl−/[Formula: see text]exchange mediated recovery from alkalinization in VLM, Hyp, and IO neurons but was absent from most NTS neurons. The Na+/H+exchanger from NTS and VLM neurons was fully inhibited when extracellular pH (pHo) <7.0, whereas the exchanger from Hyp and IO neurons was fully inhibited only when pHo <6.7. The Cl−/[Formula: see text]exchanger from VLM, but not Hyp and IO neurons, was inhibited by pHo of 7.9. These pH regulatory properties resulted in steeper pHi-pHorelationships in neurons from chemosensitive regions compared with those from nonchemosensitive regions. These differences are consistent with a role for changes of pHi as the proximate signal in central chemoreception and changes of pHo in modulating pHi changes.

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