Identification of exchange mechanisms for the regulation of intracellular pH in rat thyroid FRTL-5 cells

1992 ◽  
Vol 9 (3) ◽  
pp. 301-308 ◽  
Author(s):  
A. M. Wood ◽  
G. Warhurst ◽  
S. P. Bidey ◽  
J. Soden ◽  
R. Taylor ◽  
...  
Keyword(s):  
Intracellular Ph ◽  
Similar Rate ◽  
Thyroid Cell ◽  
Cell Monolayers ◽  
Ionic Substitution ◽  
Acid Load ◽  
Rate Of Recovery ◽  
Disulphonic Acid ◽  
Rat Thyroid ◽  
Free Media

ABSTRACT pH is maintained in cells by plasma membrane exchange mechanisms. In the absence of HCO3− ions, FRTL-5 cells regulate intracellular pH (pHi) by an Na+/H+ antiport but HCO3−-dependent exchangers cannot operate. We have investigated pHi regulation (by microfluorimetry and the pH sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6′)-carboxy-fluorescein) in small groups (five to six cells) of FRTL-5 thyroid cell monolayers held in kREBS—Ringer buffer (pH 7·4) with or without HCO3− ions. The exchangers were investigated with inhibitors (amiloride or its derivative dimethylamiloride for the Na+/H+ antiporter and the stilbene derivative disodium 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) for HCO3 −-dependent mechanisms), ionic substitution and by NH4+/NH3 (10mm) acid loading. Basal pHi was lower in the presence (7·3±0·058, mean±s.d., n= 14) than in the absence (7·59±0·078, n=10) of HCO3 ions. In HCO3 −-free media, cells recovered from acid load by 0·34±0·04 pH units in the first 2 min and finally reached a pHi of 7·35±0·06. This recovery was Na+-dependent and blocked by dimethylamiloride during the 15 min following intracellular acidification. In HCO3−-containing media, cells recovered from an acid load at a similar rate, but reached 99 ± 10% (n = 9) of the baseline pH; this recovery was also dependent on Na+ ions. Moreover, although dimethylamiloride and DIDS reduced the rate of recovery to 0·06±0·02 and 0·18±0·04 pH units respectively during the 2-min period, the cells returned to the basal pHi within 15 min. Removal of Na+ from HCO3−-containing media acidified the cells (ΔpH=–0·82±0·05, n=10) within 40 min; this acidification was partially blocked by either amiloride or DIDS. Removal of Cl− alkalinized the cells (ΔpH=+0·51 ± 0·06, n=10) after 40 min, and this alkalinization was totally prevented by DIDS. Furthermore, in the absence of Na+ and presence of amiloride, alkalinization was still seen on the removal of Cl−, albeit at a diminished rate (i.e. ΔpH = +0·25±0·05, n=8) after 40 min. In conclusion, FRTL-5 cells maintain pHi by two Na+-dependent exchangers, one sensitive to amiloride, the other to DIDS, and a Na+-independent, Cl−/HCO3− mechanism.

Regulation of intracellular pH in J774 murine macrophage cells: H+ extrusion processes

1995 ◽  
Vol 268 (1) ◽  
pp. C210-C217 ◽  
Author(s):  
L. C. McKinney ◽  
A. Moran
Keyword(s):  
Intracellular Ph ◽  
Initial Rate ◽  
Adherent Cells ◽  
Murine Macrophage ◽  
Bafilomycin A1 ◽  
Macrophage Cell ◽  
Recovery From Acidification ◽  
J774 Cells ◽  
Acid Load ◽  
Rate Of Recovery

Mechanisms of intracellular pH (pHi) regulation were characterized in the murine macrophage cell line J774.1, using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein to measure pHi. Under nominally HCO3(-)-free conditions, resting pHi of nonadherent J774.1 cells was 7.53 +/- 0.02 (n = 86), and of adherent cells was 7.59 +/- 0.02 (n = 97). In the presence of HCO3-/CO2, pHi values were reduced to 7.41 +/- 0.02 (n = 12) and 7.40 +/- 0.01 (n = 28), respectively. Amiloride, an inhibitor of Na+/H+ exchange, did not affect resting pHi. Inhibitors of a vacuolar type H(+)-ATPase [bafilomycin A1, N-ethylmaleimide (NEM), 7-chloro-4-nitrobenz-2-oxa-1,3-diazide (NBD), and p-chloromercuriphenylsulfonic acid (pCMBS)] reduced pHi by at least 0.2 pH units. Inhibitors of other classes of H(+)-ATPases (oligomycin, azide, vanadate, and ouabain) were without effect. Inhibition of H+ efflux, measured by the change in extracellular pH of a weakly buffered cell suspension, followed the same pharmacological profile, indicating that the reduction of pHi was due to inhibition of H+ extrusion. Mechanisms of recovery from an imposed intracellular acid load were also investigated. In NaCl-Hanks' solution, pHi recovered exponentially to normal within 2 min. The initial rate of recovery was inhibited > 90% by amiloride or by replacement of extracellular Na+ concentration by N-methyl-glucamine. Inhibitors of the vacuolar H(+)-ATPase also inhibited recovery. NEM and NBD nonspecifically inhibited all recovery. Bafilomycin A1 and pCMBS did not inhibit the initial amiloride-sensitive portion of recovery, but they did inhibit a late component of recovery when pHi was above 7.0. We conclude that the Na+/H+ exchanger is primarily responsible for recovery from an acid load but does not regulate resting pHi. Conversely, a vacuolar H(+)-ATPase regulates the resting pHi of J774 cells but contributes little to recovery from acidification.

Influence of surface pH on intracellular pH regulation in cardiac and skeletal muscle

1986 ◽  
Vol 250 (5) ◽  
pp. C748-C760 ◽  
Author(s):  
B. Vanheel ◽  
A. de Hemptinne ◽  
I. Leusen
Keyword(s):  
Intracellular Ph ◽  
Soleus Muscle ◽  
Ph Regulation ◽  
Purkinje Fiber ◽  
Intracellular Acidification ◽  
Surface Ph ◽  
Acid Load ◽  
Rat Soleus Muscle ◽  
Rate Of Recovery ◽  
Intracellular Acid

The influence of the surface pH (pHs) on the intracellular pH (pHi) and the recovery of pHi after an imposed intracellular acid load was investigated in isolated sheep cardiac Purkinje fiber, rabbit papillary muscle, and mouse and rat soleus muscle. pHs and pHi, respectively, were continuously measured by use of single- and double-barreled pH-sensitive glass microelectrodes. Surface acidosis, usually obtained by superfusion with solutions of acid pH, was also produced with low buffered (5 mM N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid) solutions at control pH. The pHs decrease (delta pHs) induced by low buffering was smallest (-0.08 pH unit) in Purkinje fiber and largest (-0.31 pH unit) in rat soleus muscle, which already had a more acid surface in control conditions. delta pHs was somewhat dependent on the superfusion rate. Higher superfusion rates decreased but did not abolish delta pHs. Surface acidosis was associated with a small intracellular acidification. Intracellular acid loads were produced by adding and subsequently withdrawing 20 meq/l NH4+ from the superfusate. In all preparations, the rate of recovery of pHi after NH4+ withdrawal was notably decreased at acidified pHs. This effect was amiloride sensitive. It is concluded that, in superfused multi-cellular preparations, pHs and therefore the buffer concentration of a superfusate can considerably influence steady-state pHi and pHi recovery from an imposed intracellular acid load.

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.

Intracellular pH regulation in isolated myocytes from adult rat heart in HCO-3-containing and HCO-3-free media

1993 ◽  
Vol 84 (2) ◽  
pp. 133-139 ◽  
Author(s):  
L. L. Ng ◽  
J. E. Davies ◽  
P. Quinn
Keyword(s):  
Intracellular Ph ◽  
Ventricular Myocytes ◽  
Ph Regulation ◽  
Specific Inhibitor ◽  
Buffering Capacity ◽  
Intracellular Acidosis ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
Acid Load ◽  
Free Media

1. Using microfluorimetry, intracellular pH, buffering capacity and intracellular pH recovery from intracellular acidosis were determined in isolated adult rat ventricular myocytes, in buffers with and without HCO-3. 2. In nominally HCO-3-free media, the intracellular pH was higher than in HCO-3-containing media. Buffering capacity at resting intracellular pH and at a pH of about 6.3 was also lower in HCO-3-free media. 3. In HCO-3-free media, recovery from an acid load after an NH4C1 prepulse was almost completely inhibited by the Na+/H+ antiport activity specific inhibitor ethylisopropyl amiloride. However, in the presence of HCO-3, H+ efflux rate was enhanced, and ethylisopropyl amiloride led to only partial inhibition of H+ efflux. Complete inhibition was achieved only with further addition of the anion-transport inhibitor 4,4′-di-isothiocyanatostilbene-2,2′-disulphonate. 4. Thus, in adult rat ventricular myocytes, recovery from intracellular acidosis in the absence of HCO-3 was almost wholly due to Na+/H+ antiport activity. In the more physiological situation with HCO-3 present, a third of the recovery from an intracellular acid load was attributed to an additional external Na+-dependent di-isothiocyanatostilbene-disulphonate-sensitive H+ efflux.

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 Trace Amine-Associated Receptor 1 Trafficking to Cilia of Thyroid Epithelial Cells

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1518
Author(s):  
Maria Qatato ◽  
Vaishnavi Venugopalan ◽  
Alaa Al-Hashimi ◽  
Maren Rehders ◽  
Aaron D. Valentine ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Human Thyroid ◽  
Apical Plasma Membrane ◽  
Thyroid Cell ◽  
Thyroid Cells ◽  
Trace Amine ◽  
Rat Thyroid

Trace amine-associated receptor 1 (rodent Taar1/human TAAR1) is a G protein-coupled receptor that is mainly recognized for its functions in neuromodulation. Previous in vitro studies suggested that Taar1 may signal from intracellular compartments. However, we have shown Taar1 to localize apically and on ciliary extensions in rodent thyrocytes, suggesting that at least in the thyroid, Taar1 may signal from the cilia at the apical plasma membrane domain of thyrocytes in situ, where it is exposed to the content of the follicle lumen containing putative Taar1 ligands. This study was designed to explore mouse Taar1 (mTaar1) trafficking, heterologously expressed in human and rat thyroid cell lines in order to establish an in vitro system in which Taar1 signaling from the cell surface can be studied in future. The results showed that chimeric mTaar1-EGFP traffics to the apical cell surface and localizes particularly to spherical structures of polarized thyroid cells, procilia, and primary cilia upon serum-starvation. Moreover, mTaar1-EGFP appears to form high molecular mass forms, possibly homodimers and tetramers, in stably expressing human thyroid cell lines. However, only monomeric mTaar1-EGFP was cell surface biotinylated in polarized human thyrocytes. In polarized rat thyrocytes, mTaar1-EGFP is retained in the endoplasmic reticulum, while cilia were reached by mTaar1-EGFP transiently co-expressed in combination with an HA-tagged construct of the related mTaar5. We conclude that Taar1 trafficking to cilia depends on their integrity. The results further suggest that an in vitro cell model was established that recapitulates Taar1 trafficking in thyrocytes in situ, in principle, and will enable studying Taar1 signaling in future, thus extending our general understanding of its potential significance for thyroid autoregulation.

scholarly journals Cytoplasmic [Ca2+] and intracellular pH in lymphocytes. Role of membrane potential and volume-activated Na+/H+ exchange.

1987 ◽  
Vol 89 (2) ◽  
pp. 185-213 ◽  
Author(s):  
S Grinstein ◽  
S Cohen
Keyword(s):  
Protein Kinase C ◽  
Membrane Potential ◽  
Protein Kinase ◽  
Intracellular Ph ◽  
Enzyme Treatment ◽  
Membrane Hyperpolarization ◽  
Kinase C ◽  
Free Media ◽  
Stimulation Of

The effect of elevating cytoplasmic Ca2+ [( Ca2+]i) on the intracellular pH (pHi) of thymic lymphocytes was investigated. In Na+-containing media, treatment of the cells with ionomycin, a divalent cation ionophore, induced a moderate cytoplasmic alkalinization. In the presence of amiloride or in Na+-free media, an acidification was observed. This acidification is at least partly due to H+ (equivalent) uptake in response to membrane hyperpolarization since: it was enhanced by pretreatment with conductive protonophores, it could be mimicked by valinomycin, and it was decreased by depolarization with K+ or gramicidin. In addition, activation of metabolic H+ production also contributes to the acidification. The alkalinization is due to Na+/H+ exchange inasmuch as it is Na+ dependent, amiloride sensitive, and accompanied by H+ efflux and net Na+ gain. A shift in the pHi dependence underlies the activation of the antiport. The effect of [Ca2+]i on Na+/H+ exchange was not associated with redistribution of protein kinase C and was also observed in cells previously depleted of this enzyme. Treatment with ionomycin induced significant cell shrinking. Prevention of shrinking largely eliminated the activation of the antiport. Moreover, a comparable shrinking produced by hypertonic media also activated the antiport. It is concluded that stimulation of Na+/H+ exchange by elevation of [Ca2+]i is due, at least in part, to cell shrinking and does not require stimulation of protein kinase C.

The polarized epithelial phenotype is dominant in hybrids between polarized and unpolarized rat thyroid cell lines

1991 ◽  
Vol 98 (1) ◽  
pp. 65-73
Author(s):  
C. Zurzolo ◽  
R. Gentile ◽  
A. Mascia ◽  
C. Garbi ◽  
C. Polistina ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Chromosome Complement ◽  
Thyroid Cell ◽  
Chromosome Loss ◽  
Epithelial Phenotype ◽  
Membrane Antigens ◽  
Dependent Growth ◽  
Rat Thyroid ◽  
Hybrid Clones ◽  
Specific Membrane

We have studied the expression of cell polarity in hybrids between two rat thyroid epithelial cells: FRT and FRTL-5. FRT cells are polarized but do not express tissue-specific properties, FRTL-5 are unpolarized and express many thyroid-specific genes. A and express many thyroid-specific genes. A pool of 170 hybrid clones and five independent clones were characterized. The chromosome complement was that expected from 1:1 fusion of the parental cells. No chromosome loss was observed for several generations. All hybrids were polarized as judged from: (1) morphology, (2) transepithelial resistance, (3) preferential secretion of several proteins either through the apical (e.g. thyroglobulin) or through the basolateral pole, and (4) basolateral trapping of iodide. On the other hand, the expression of thyroid-specific markers: thyroglobulin synthesis and secretion, trapping of iodide, thyrotropin-dependent growth and expression of specific membrane antigens, were greatly reduced or inhibited in the pool and in the isolated clones. We also found that reduction of thyroglobulin synthesis was correlated with the loss of activity of the trans-acting factor TgTF1. We conclude that cell polarity, a property of FRT cells, is dominant in the hybrids whereas thyroid differentiation is recessive.

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