Adaptation to hypercapnia vs. intracellular pH in cat carotid body: responses in vitro

1996 ◽  
Vol 80 (4) ◽  
pp. 1090-1099 ◽  
Author(s):  
S. Lahiri ◽  
R. Iturriaga ◽  
A. Mokashi ◽  
F. Botre ◽  
D. Chugh ◽  
...  
Keyword(s):  
Steady State ◽  
Intracellular Ph ◽  
Baseline Level ◽  
Discharge Rate ◽  
Initial Response ◽  
Extracellular Ph ◽  
Carotid Bodies ◽  
Initial Peak ◽  
Initial Change

The hypotheses that the chemosensory discharge rate parallels the intracellular pH (pHi) during hypercapnia and that the initial change in pHi (delta pHi) is always more than the stead-state delta pHi were studied by using cat carotid bodies in vitro at 36.5 degrees C in the absence and presence of methazolamide (30-100 mg/l). Incremental acidic hypercapnia was followed by an incremental initial peak response and a greater adaptation. A given acidic hypercapnia elicited a rapid initial response followed by a slower adaptation; isohydric hypercapnia produced an equally rapid initial response but of smaller magnitude that returned to near-baseline level; alkaline hypercapnia induced a similar rapid initial response but one of still smaller magnitude that decreased rapidly to below the baseline. Methazolamide eliminated the initial overshoot, which also suggested involvement of the initial rapid pHi in the overshoot. These results show that the initial delta pHi is always greater than the steady-state delta pHi and during hypercapnia. Also, the steady-state chemoreceptor activity varied linearly with the extracellular pH, indicating a linear relationship between extracellular pH and pHi.

scholarly journals Effects of ammonium on intracellular pH in rat medullary thick ascending limb: mechanisms of apical membrane NH4+ transport.

1994 ◽  
Vol 103 (5) ◽  
pp. 917-936 ◽  
Author(s):  
B A Watts ◽  
D W Good
Keyword(s):  
Steady State ◽  
Intracellular Ph ◽  
Apical Membrane ◽  
Ammonium Transport ◽  
Thick Ascending Limb ◽  
Transport Pathway ◽  
Critical Determinant ◽  
Medullary Thick Ascending Limb ◽  
Intracellular Alkalinization

The renal medullary thick ascending limb (MTAL) actively reabsorbs ammonium ions. To examine the effects of NH4+ transport on intracellular pH (pHi) and the mechanisms of apical membrane NH4+ transport, MTALs from rats were isolated and perfused in vitro with 25 mM HCO3(-)-buffered solutions (pH 7.4). pHi was monitored using the fluorescent dye BCECF. In the absence of NH4+, the mean pHi was 7.16. Luminal addition of 20 mM NH4+ caused a rapid intracellular acidification (dpHi/dt = 11.1 U/min) and reduced the steady state pHi to 6.67 (delta pHi = 0.5 U), indicating that apical NH4+ entry was more rapid than entry of NH3. Luminal furosemide (10(-4) M) reduced the initial rate of cell acidification by 70% and the fall in steady state pHi by 35%. The residual acidification observed with furosemide was inhibited by luminal barium (12 mM), indicating that apical NH4+ entry occurred via both furosemide (Na(+)-NH4(+)-2Cl- cotransport) and barium-sensitive pathways. The role of these pathways in NH4+ absorption was assessed under symmetric ammonium conditions. With 4 mM NH4+ in perfusate and bath, mean steady state pHi was 6.61 and net ammonium absorption was 12 pmol/min/mm. Addition of furosemide to the lumen abolished net ammonium absorption and caused pHi to increase abruptly (dpHi/dt = 0.8 U/min) to 7.0. Increasing luminal [K+] from 4 to 25 mM caused a similar, rapid cell alkalinization. The pronounced cell alkalinization observed with furosemide or increasing [K+] was not observed in the absence of NH4+. In symmetric 4 mM NH4+ solutions, addition of barium to the lumen caused a slow intracellular alkalinization and reduced net ammonium absorption only by 14%. Conclusions: (a) ammonium transport is a critical determinant of pHi in the MTAL, with NH4+ absorption markedly acidifying the cells and maneuvers that inhibit apical NH4+ uptake (furosemide or elevation of luminal [K+]) causing intracellular alkalinization; (b) most or all of transcellular ammonium absorption is mediated by apical membrane Na(+)-NH4(+)-2Cl- cotransport; (c) NH4+ also permeates a barium-sensitive apical membrane transport pathway (presumably apical membrane K+ channels) but this pathway does not contribute significantly to ammonium absorption under physiologic (symmetric ammonium) conditions.

Intracellular pH in the OK cell. I. Identification of H+ conductance and observations on buffering capacity

1991 ◽  
Vol 261 (6) ◽  
pp. C1143-C1153 ◽  
Author(s):  
M. Graber ◽  
J. DiPaola ◽  
F. L. Hsiang ◽  
C. Barry ◽  
E. Pastoriza
Keyword(s):  
Steady State ◽  
Intracellular Ph ◽  
Large Cell ◽  
Buffering Capacity ◽  
Disulfonic Acid ◽  
Transport Pathways ◽  
Opossum Kidney ◽  
Direct Titration ◽  
Cell Ph

The regulation of intracellular pH (pHi) in the opossum kidney (OK) cell line was studied in vitro using the pH-sensitive excitation ratio of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Recovery from an NH4Cl acid load disclosed a Na-dependent component blocked by amiloride and a smaller Na-independent component. The Na-independent recovery rate was proportional to the H+ gradient from cell to buffer and was zero in the absence of an electrochemical gradient. The Na-independent recovery was not affected by N-ethylmaleimide, dicyclohexylcarbodiimide, HCO3, phloretin, or ZnCl2 but was accelerated in depolarized cells and by membrane-fluidizing drugs and was inhibited by glutaraldehyde. The apparent cellular buffering capacity changed in proportion to this H+ conductance. Consistent with an electrogenic H+ leak, steady-state cell pH alkalinized with depolarization and acidified with hyperpolarization. Removal of buffer Na+ produced a profound acidification, as did amiloride. In 0-Na+ buffers, extremely large cell-to-buffer H+ gradients were present and proportional to buffer pH. 4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid had no effect on steady-state pHi. Measurements of intracellular buffering capacity were derived from the change of cell pH induced by withdrawing NH4Cl. This buffering capacity was increased threefold in Na-free buffers, whereas the value measured by direct titration of cell lysate was the same or less than that of control cells. The NH4Cl-derived buffering capacity varied in direct proportion to the magnitude of the H+ leak. Drugs that changed H+ permeability produced the apparent changes of the measured buffering capacity within a few minutes. We conclude that, in HCO3-free buffer, the OK cell uses two membrane acid-base transport pathways: a Na-H antiporter active at physiological pH and a substantial passive H+ conductance. The results also reveal that the NH4Cl-derived buffering capacity is subject to artifacts, possibly due to a finite leak of ionic NH4+.

Modulation of agonist-activated calcium influx by extracellular pH in rat pancreatic acini

1989 ◽  
Vol 257 (6) ◽  
pp. G917-G924 ◽  
Author(s):  
S. Muallem ◽  
S. J. Pandol ◽  
T. G. Beeker
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Intracellular Ph ◽  
Calcium Influx ◽  
Extracellular Ph ◽  
Transport Pathways ◽  
Pancreatic Acini ◽  
Hormone Stimulation ◽  
Entry Pathway ◽  
Agonist Stimulation

The biochemical and Ca2+ transport pathways involved in generating the hormone-evoked Ca2+ signal are reported to be influenced by pH. The present study was designed to determine the effect of extracellular pH (pHo) and intracellular pH (pHi) on hormone-stimulated Ca2+ transport. We used rat pancreatic acini and measured free cytosolic Ca2+ concentration ([Ca2+]i) with fura-2, pHi with 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and Ca2+ fluxes with 45Ca2+. In the presence of external Ca2+, increasing pHo increased steady-state [Ca2+]i during sustained agonist stimulation; in the absence of external Ca2+, this increase in [Ca2+]i did not occur. The addition of an antagonist or blocking plasma membrane Ca2+ influx with La3+ in stimulated cells suspended at pHo 8.2 resulted in a reduction in [Ca2+]i. Increasing pHo increased the rate and extent of 45Ca2+ uptake into stimulated cells and the rate and extent of Ca2+ reloading of intracellular stores. The increased Ca2+ content of the intracellular stores with increased pHo indicated that at physiological pHo and pHi the agonist-mobilizable internal stores are not saturated with Ca2+. Changes in pHo affected pHi. However, changes in pHi at constant pHo had no effect on hormone-evoked [Ca2+]i increase, reduction in [Ca2+]i after hormone stimulation, or reloading of intracellular stores. We conclude that the hormone-activated plasma membrane Ca2+ entry pathway responsible for Ca2+ reloading is directly modulated by external H+.

scholarly journals Phorbol 12-myristate 13-acetate inhibits epidermal growth factor signalling in human keratinocytes, leading to decreased ornithine decarboxylase activity

1996 ◽  
Vol 319 (2) ◽  
pp. 641-648 ◽  
Author(s):  
Guo Z XUE ◽  
Zai S ZHENG ◽  
Rong Z CHEN ◽  
Mary B. LLOYD ◽  
Janet H. PRYSTOWSKY
Keyword(s):  
Biological Activity ◽  
Steady State ◽  
Growth Factor ◽  
Baseline Level ◽  
Human Keratinocytes ◽  
Enzyme Synthesis ◽  
Enzyme Protein ◽  
Epidermal Growth ◽  
Pkc Activation

Several studies have suggested that murine and human keratinocytes respond differently to phorbol 12-myristate 13-acetate (PMA). Using an in vitro assay, we found that in contrast to its effect on murine skin, PMA did not induce ornithine decarboxylase (ODC) activity in human skin biopsies. To explore the signalling induced by PMA and to determine whether an in vitro culture system could be used to predict biological activity of retinoids in human keratinocytes, we studied a simian virus 40 (SV40)-transformed human keratinocyte cell line. Epidermal growth factor (EGF) stimulates ODC activity and increases the steady-state level of ODC mRNA in a dose- and time-dependent manner in these cells [Prystowsky, Clevenger and Zheng (1993) Exp. Dermatol. 2, 125–132]. In this report, 10-10 M–10-7 M PMA induced ODC mRNA and enzyme synthesis at 7 h, but did not significantly induce ODC activity and inhibited the EGF induction of ODC activity. To explore the mechanism whereby PMA interferred with EGF signalling, the effect of PMA on EGF binding to its cell-surface receptor was studied; acute treatment with PMA (within 7 h) decreased EGF binding to 41–57% of the baseline level. In contrast, chronic treatment with PMA (24 h) increased EGF binding to 156% of the baseline level and was associated with an increase in quantity of EGF receptor protein. Protein kinase C (PKC) activation correlated with the acute decrease in EGF binding following PMA treatment. In summary, PMA induced ODC mRNA and ODC enzyme synthesis, while steady-state levels of immunoprecipitable ODC enzyme protein and ODC activity were not increased, demonstrating possible increased turnover of ODC enzyme protein. Additionally, PMA inhibited the induction of ODC by EGF through decreased EGF binding, possibly mediated by PKC activation. Finally treatment of the keratinocytes with retinoids including etretinate, Ro13-7410, etarotene, Ro40-8757, 13-cis-retinoic acid, and acitretin blocked the PMA induction of ODC mRNA, suggesting this in vitro model could be a valuable screening assay for predicting biological activity in humans.

The effect of acid–base changes on skeletal muscle twitch tension

1978 ◽  
Vol 56 (4) ◽  
pp. 543-549 ◽  
Author(s):  
David W. Fretthold ◽  
Lal C. Garg
Keyword(s):  
Carbon Dioxide ◽  
Skeletal Muscle ◽  
Intracellular Ph ◽  
Muscle Tension ◽  
Carbon Dioxide Tension ◽  
Extracellular Ph ◽  
Acid Base ◽  
Muscle Twitch ◽  
Deficient Diet

The effects of acid–base alterations produced by changing bicarbonate (metabolic type), carbon dioxide tension (respiratory type), or both bicarbonate and carbon dioxide tension (compensated type) on skeletal muscle twitch tension, intracellular pH, and intracellular potassium were studied in vitro. Hemidiaphragm muscles from normal rats and rats fed a potassium-deficient diet were used. Decreasing the extracellular pH by decreasing bicarbonate or increasing CO2 in the bathing fluid produced a decrease in intracellular pH, intracellular K+, and muscle twitch tension. However, at a constant extracellular pH, an increase in CO2 (compensated by an increase in bicarbonate) produced an increase in intracellular K+ and twitch tension in spite of a decrease in intracellular pH. The effect on twitch tension of the hemidiaphragms showed a rapid onset, was reversible, persisted until the buffer composition was changed, and was independent of synaptic transmission.It is concluded that the twitch tension of the skeletal muscle decreases with a decrease in intracellular K+. The muscle tension also decreases with an increase in the ratio of intracellular and extracellular H+ concentration. However, there is no consistent relationship between muscle tension and extracellular or intracellular pH. The muscle tension of the diaphragms taken from K+-deficient rats is more sensitive to variations in CO2, pH, and bicarbonate concentration of the medium than that of the control rat diaphragms.

scholarly journals Regulation of Intracellular pH in Single Rat Cortical Neurons in vitro: A Microspectrofluorometric Study

1993 ◽  
Vol 13 (5) ◽  
pp. 827-840 ◽  
Author(s):  
Yibing Ou-Yang ◽  
Pekka Mellergård ◽  
Bo K. Siesjö
Keyword(s):  
Steady State ◽  
Intracellular Ph ◽  
Cortical Neurons ◽  
Ph Sensitive ◽  
Disulfonic Acid ◽  
Extrusion Rate ◽  
Rat Cortical Neurons ◽  
Acid Extrusion

Intracellular pH (pHi) and the mechanisms of pHi regulation in cultured rat cortical neurons were studied with microspectrofluorometry and the pH-sensitive fluorophore 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein. Steady-state pHi was 7.00 ± 0.17 (mean ± SD) and 7.09 ± 0.14 in nominally HCO3− -free and HCO3−-containing solutions, respectively, and was dependent on extracellular Na+ and Cl−. Following an acid transient, induced by an NH1 prepulse or an increase in CO2 tension, pHi decreased and then rapidly returned to baseline, with an average net acid extrusion rate of 2.6 and 2.8 mmol/L/min, in nominally HCO3− -free and HCO3− -containing solutions, respectively. The recovery was completely blocked by removal of extracellular Na+ and was partially inhibited by amiloride or 5- N-methyl- N-isobutylamiloride. In most cells pHi recovery was completely blocked in the presence of harmaline. The recovery of pHi was not influenced by addition of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) or removal of Cl−. The rapid regulation of pHi seen following a transient alkalinization was not inhibited by amiloride or by removal of extracellular Na+, but was partially inhibited by DIDS and by removal of extracellular Cl−. The results are compatible with the presence of at least two different pHi-regulating mechanisms: an acid-extruding Na+/H+ antiporter, possibly consisting of different subtypes, and a passive Cl−/HCO3− exchanger, mediating loss of HCO3− from the cell.

scholarly journals Mechanism of Excitation of Aplysia Neurons by Carbon Dioxide

1970 ◽  
Vol 56 (5) ◽  
pp. 543-558 ◽  
Author(s):  
A. M. Brown ◽  
P. R. Berman
Keyword(s):  
Intracellular Ph ◽  
External Surface ◽  
Discharge Rate ◽  
Membrane Conductance ◽  
Giant Cells ◽  
Extracellular Ph ◽  
Direct Result ◽  
Similar Response ◽  
Pacemaker Neurons ◽  
Spike Discharge

The abdominal ganglion of Aplysia californica was perfused with artificial seawater equilibrated at different PCOCO2's and pH's for 5 min or less. 5% CO2 dropped perfusate pH from 8.0 to 6.5 and produced depolarization and increased discharge rate in visceromotor neurons. Half the giant cells studied had a similar response, whereas the other half were hyperpolarized. Pacemaker neurons showed little, if any, response to such changes in pH or CO2. Membrane conductance of responsive cells was always increased. The effect of CO2 occurred even when synaptic transmission was blocked by low calcium and high magnesium, and therefore must have been a direct result of CO2 or the concomitant fall in pH. When extracellular pH was lowered to 6.5 using HCl or H2SO4 and no CO2, the same effects were observed. Also, local application of HCl or H2SO4 to the external surface of the cell soma elicited depolarization and spike discharge. When extracellular pH was held constant by continual titration, 5–50% CO2 had no effect. Intracellular pH was probably decreased at least one pH unit under these circumstances. Thus CO2 per se, decreased intracellular pH, and increased bicarbonate ion were without effect. It is concluded that CO2 acts solely through a decrease in extracellular pH.

Effect of PCO2 on intracellular pH in in vitro frog gastric mucosa

1989 ◽  
Vol 256 (1) ◽  
pp. G206-G213 ◽  
Author(s):  
K. J. Carter ◽  
I. Saario ◽  
U. Seidler ◽  
W. Silen
Keyword(s):  
Steady State ◽  
Gastric Mucosa ◽  
Intracellular Ph ◽  
Ringer Solution ◽  
Partial Recovery ◽  
Muscularis Mucosa ◽  
Fundic Mucosa ◽  
Ethanesulfonic Acid ◽  
Oxynticopeptic Cells

Steady-state intracellular pH (pHi) in 0, 5, and 10% CO2-buffered Ringer solution in sheets of in vitro frog gastric antral or fundic mucosa has been measured using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). In tissues perfused with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-100% O2 buffer [extracellular pH (pHo) = 7.14], steady-state pHi in antral surface cells was 7.08 +/- 0.06 (n = 8), in fundic oxynticopeptic cells 6.91 +/- 0.03 (n = 13), in the muscularis mucosa 7.58 +/- 0.06 (n = 4). In mucosae perfused with 17.8 mM HCO3- -95% O2-5% CO2 buffer (pHo = 7.14), steady-state pHi in antral surface cells was 6.97 +/- 0.02 (n = 22), in fundic oxynticopeptic cells 7.00 +/- 0.04 (n = 18), and in fundic muscularis mucosa 7.39 +/- 0.05 (n = 8). In fundic oxynticopeptic cells perfused with 35.6 mM HCO3- -90% O2-10% CO2 (pHo = 7.14) steady-state pHi was 6.77 +/- 0.07 (n = 4). In tissues equilibrated initially with 100% O2 and changed to 5% CO2, antral surface cells acidified by 0.21 pH units and fundic oxynticopeptic cells by 0.10 pH units, with restoration of pHi to resting levels within 30 and 10 min, respectively. Exposure of tissues initially equilibrated with 5% CO2 to 100% O2 alkalinized antral surface cells by 0.22 pH units and fundic oxynticopeptic cells by 0.23 pH units, with only partial recovery of pHi by 30 min. These data suggest that steady-state pHi is equivalent in surface and oxynticopeptic cells and is lower than in the muscularis mucosa.(ABSTRACT TRUNCATED AT 250 WORDS)

Na+/H+ exchange in mosquito Malpighian tubules

2000 ◽  
Vol 279 (6) ◽  
pp. R1996-R2003 ◽  
Author(s):  
D. H. Petzel
Keyword(s):  
Steady State ◽  
Intracellular Ph ◽  
Specific Inhibitor ◽  
Fluid Secretion ◽  
Malpighian Tubules ◽  
Pulse Technique ◽  
Acid Load ◽  
Rate Of Recovery ◽  
Stimulation Of

Fluid secretion and intracellular pH were measured in isolated mosquito Malpighian tubules to determine the presence of Na+/H+ exchange. Rates of fluid secretion by individual Malpighian tubules in vitro were inhibited by 78% of control in the presence of 100 μM 5-( N-ethyl- n-isopropyl)-amiloride (EIPA), a specific inhibitor of Na+/H+ exchange. Steady-state intracellular pH was measured microfluorometrically by using 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in individual Malpighian tubules. Bathing the Malpighian tubules in 0 mM extracellular Na+ or in the presence of 100 μM EIPA reduced the steady-state intracellular pH by 0.5 pH units. Stimulation of the Na+/H+ exchanger by using the NH4Cl pulse technique resulted in a rate of recovery from the NH4Cl-induced acute acid load of 8.7 ± 1.0 × 10−3 pH/s. The rates of recovery of intracellular pH after the acute acid load in the absence of extracellular Na+ or in the presence of 100 μM EIPA were 0.7 ± 0.6 and −0.3 ± 0.3 × 10−3 pH/s, respectively. These results indicate that mosquito Malpighian tubules possess a Na+/H+ exchanger.

Determinants of intracellular pH in gas gland cells of the swimbladder of the European eel Anguilla anguilla

2002 ◽  
Vol 205 (8) ◽  
pp. 1069-1075 ◽  
Author(s):  
E. Sötz ◽  
H. Niederstätter ◽  
B. Pelster
Keyword(s):  
Steady State ◽  
Anion Exchange ◽  
Intracellular Ph ◽  
Specific Inhibitor ◽  
Anguilla Anguilla ◽  
Extracellular Ph ◽  
European Eel ◽  
Pulse Technique ◽  
Gland Cells ◽  
Gas Gland

SUMMARY Gas gland cells of the European eel (Anguilla anguilla) were cultured on collagen-coated coverslips, and intracellular pH was measured using the pH-sensitive fluorescent probe 2′,7′-bis-(2-carboxypropyl)-5-(6)-carboxyfluorescein (BCPCF). The contributions of various proton-translocating mechanisms to homeostasis of intracellular pH (pHi) were assessed by adding specific inhibitors of the various proton-translocating mechanisms at a constant extracellular pH (pHe)of 7.4 and after artificial acidification of the cells using the ammonium pulse technique. The greatest decrease in pHi was observed after addition of 5-(N-ethyl-N-isobutyl)-amiloride (MIA), an inhibitor of Na+/H+ exchange. Na+/H+ exchange was active under steady-state conditions at an extracellular pH of 7.4, and activity increased after intracellular acidification. Incubation of gas gland cells with 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid(DIDS), an inhibitor of anion exchange, also caused a decrease in pHi, but this decrease was not as pronounced as in the presence of MIA. Furthermore, at low pHi, the effect of DIDS was further reduced, suggesting that bicarbonate-exchanging mechanisms are involved in maintaining a steady-state pHi but that their importance is reduced at low pH. Bafilomycin A1,a specific inhibitor of the V-ATPase, had no effect on steady-state pHi. However, recovery of intracellular pH after an artificial acid load was significantly impaired in the presence of bafilomycin. Our results suggest that Na+/H+ exchange and anion exchange are important for the regulation of pHi at alkaline values of pHe. When pHi is low, a situation probably often encountered by gas gland cells during gas secretion,Na+/H+ exchange continues to play an important role in acid secretion and a V-ATPase appears to contribute to proton secretion.

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