Characterization of Na+/H+ exchange in FRTL-5 thyroid cells. Evidence for dependence on activation of protein kinase C.

Na+/H+ exchange activity was investigated in cultured rat thyroid follicular FRTL-5 cells using the pH sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Basal intracellular pH (pHi) was 7.13 +/- 0.10 in cells incubated in Hepes-buffered saline solution. The intracellular buffering capacity beta i was determined using the NH4Cl-pulse method, yielding a beta i value of 85 +/- 12 mM/pH unit. The relationship between extracellular Na+ and the initial rate of alkalinization of acid-loaded cells showed simple saturation kinetics, with an apparent Km value of 44 +/- 26 mM, and an Vmax value of 0.3 +/- 0.01 pH unit/min. The agonist-induced activation of Na+/H+ exchange was investigated in cells acidified with nigericin. Addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) or ATP induced rapid cytosolic alkalinization in acid-loaded cells. The action of both TPA and ATP was abolished by preincubating the cells with 100 microM amiloride, by substituting extracellular Na+ with equimolar concentrations of choline+, and by pretreating the cells with TPA for 24 h. Chelating extracellular Ca2+, or depleating intracellular Ca2+ pools did not affect the ATP-induced alkalinization. The results indicate, that FRTL-5 cells have a functional Na+/H+ exchange mechanism. Furthermore, stimulation of protein kinase C activity is of importance in activating the antiport.     

Cytoplasmic pH is differently regulated in the monoblastic U-937 and erythroleukemic K-562 cell lines

Regulation of cytoplasmic pH (pHi) of the human monoblastic U-937 and erythroleukemic K-562 cell lines was investigated. The apparent resting pHi, as assessed by the fluorescent pH probe quenel, were 6.61 and 6.75 for the U-937 and K-562 cells, respectively. When extracellular Na+ was substituted by equimolar choline+, pHi decreased by about 0.2 units. The protein kinase C activating beta-form of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 10(-10) and 10(-7) M) induced a dose-dependent alkalinization in both cell types of 0.03-0.12 units, whereas the alpha-form was inactive. The response was detectable after about 2 min and reached steady-state 10-15 min later. In the K-562 cells the alkalinization was mediated by Na+/H+ exchange as it was accompanied by stimulation of H+ extrusion and abolished by Na+ removal. The TPA response in the U-937 cells, however, was unaffected by Na+ removal, not accompanied by H+-efflux, and thus unrelated to Na+/H+ exchange. Since electron microscopy indicated development of multivesicular bodies with an acidic interior, the alkalinization can probably be accounted for by an intracellular mechanism. Ionomycin (10(-5) M) induced a rapid increase in the cytoplasmic Ca2+ concentration of both cell types and this response was accompanied by acidification followed by a Na+-dependent recovery. In the U-937, but not in the K-562, cells this recovery was followed by a net alkalinization. It is concluded that both cell types possess a Na+/H+ exchange of importance for pHi but that this mechanism is regulated differently in the U-937 and K-562 cells.     

Intracellular pH changes of cultured bovine aortic endothelial cells in response to ATP addition

The changes of the intracellular pH (pHi) of cultured bovine aortic endothelial cells were fluorometrically monitored using 2',7'-bis(carboxyethyl)carboxyfluorescein (BCECF). A biphasic pHi change was observed by addition of ATP: an initial acidification followed by an alkalinization of about 0.2 pH unit above the resting level of pHi 7.23. The alkalinization was dependent on [Na+]o and [H+]o, and was inhibited by 5-(N,N-hexamethylene)amiloride, indicating that the alkalinization is mediated by the Na+/H+ exchanger. The 50% effective concentration of ATP was about 1.4 microM. ADP similarly induced pHi changes, whereas AMP and adenosine were inactive. The pHi changes induced by ATP were dependent on the extracellular Ca2+, and the addition of calcium ionophore A23187 induced similar pHi changes. The results indicate that ATP activates the Na+/H+ exchanger in cultured bovine aortic endothelial cells and the activation is mediated by the P2-purinergic receptor and is dependent on the extracellular Ca2+.     

Angiotensin II-stimulated Na+/H+ exchange in cultured vascular smooth muscle cells. Evidence for protein kinase C-dependent and -independent pathways

Angiotensin II, a potent vasoconstrictor, is known to stimulate Ca2+ mobilization and Na+ influx in vascular smooth muscle cells (VSMC). The fact that the Na+/H+ exchange inhibitor, amiloride, blocks angiotensin II-stimulated Na+ influx and is itself a vasodilator suggests that Na+/H+ exchange may play a role in the angiotensin II-mediated effects on VSMC. We have used a pH-sensitive fluorescent dye to study Na+/H+ exchange in cultured rat aortic VSMC. Basal intracellular pH was 7.08 in physiological saline buffer. Angiotensin II stimulation caused an initial transient acidification, followed by a Na+-dependent alkalinization. Angiotensin II increased the rate of alkalinization with apparent threshold, half-maximal, and maximal effect of 0.01, 3, and 100 nM, respectively. Angiotensin II stimulation appeared to be mediated by a shift in the Km of the Na+/H+ exchanger for extracellular Na+. Since angiotensin II activates phospholipase C in VSMC, we tested the possibility that angiotensin II increased Na+/H+ exchange by activation of protein kinase C via stimulation of diacylglycerol formation. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated Na+/H+ exchange in VSMC cultured for 24 h in serum-free medium, and the subsequent angiotensin II response was inhibited. However, VSMC grown in serum and treated for 24 h with TPA to decrease protein kinase C activity showed no inhibition of angiotensin II-stimulated Na+/H+ exchange. TPA caused no intracellular alkalinization of VSMC grown in serum, while the angiotensin II response was actually enhanced compared to VSMC deprived of serum for 24 h. We conclude that angiotensin II stimulates an amiloride-sensitive Na+/H+ exchange system in cultured VSMC which is mediated by protein kinase C-dependent and -independent mechanisms. Angiotensin II-mediated Na+ influx and intracellular alkalinization may play a role in excitation-response coupling in vascular smooth muscle.     

Involvement of calcium and protein kinase C in the activation of the Na+/H+ exchanger in cultured bovine aortic endothelial cells stimulated by extracellular ATP

We have studied the activation of the Na+/H+ exchanger which leads to the intracellular alkalinization in cultured bovine aortic endothelial cells stimulated by extracellular ATP. The alkalinization induced by ATP was largely dependent on extracellular Ca2+ and the rate of alkalinization was decreased by about 60% in the absence of extracellular Ca2+. ATP caused a rapid and transient increase and a subsequent sustained increase of the intracellular Ca2+ concentration ([Ca2+]i) in the Ca2+ buffer, while only the rapid and transient increase of [Ca2+]i was observed in the absence of extracellular Ca2+. The Ca2+-depleted cells prepared by incubation in Ca2+-free buffer containing 0.1 mM EGTA showed only a slight increase of [Ca2+]i with no alkalinization on stimulation by ATP. The alkalinization was inhibited by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), an inhibitor of protein kinase C, but not by another isoquinoline analogue (HA 1004), which has a less inhibitory effect on the kinase. Phorbol 12-myristate 13-acetate also induced the alkalinization by the activation of the Na+/H+ exchanger. Neither dibutyryl cyclic AMP nor dibutyryl cyclic GMP affected the alkalinization induced by ATP. Treatment of the cells by pertussis and cholera toxins had no effect on the alkalinization. The results suggest that the increase in [Ca2+]i is essential for the ATP-induced activation of the Na+/H+ exchanger in cultured bovine aortic endothelial cells and a protein kinase C-dependent pathway is involved in the activation.     

Na+/H+ exchange regulates intracellular pH in a cell clone derived from bovine pigmented ciliary epithelium

The regulation of intracellular pH (pHi) was monitored in a virus-transformed cell clone derived from bovine ciliary body exhibiting characteristics of pigmented ciliary epithelium. Data were obtained from confluent monolayers grown on plastic coverslips in nominally bicarbonate-free media using the pH-sensitive absorbance of 5- (and 6-) carboxy-4',5'-dimethylfluorescein. Under resting conditions, pHi averaged 6.98 +/- 0.01 (SEM; n = 57). When cells were acid loaded by briefly exposing them to Ringer containing NH4+ and then withdrawing the NH4+, pHi spontaneously regained its initial value. In the presence of 1 mM amiloride or in the absence of Na+, this process was blocked, indicating the involvement of an Na+/H+ exchanger in the regulation of pHi after an acid load. Removing Na+ during resting conditions decreased cytoplasmatic pH. This acidification could be slowed by amiloride, which is evidence for reversal of the Na+/H+ countertransport exchanging intracellular Na+ for extracellular protons. Application of 1 mM amiloride during steady state led to a slow acidification. Thus the Na+/H+ exchanger is operative during resting conditions extruding protons, derived from cellular metabolism, or from downhill leakage into the cell. Addition of Na+ to Na+ -depleted cells led to an alkalinization, which was sensitive to amiloride, with an IC50 of about 20 microM. This alkalinization was attributed to the Na+/H+ exchanger and exhibited saturation kinetics with increasing Na+ concentrations, with an apparent KM of 29.6 mM Na+. It is concluded that Na+/H+ exchange regulates pHi during steady state and after an acid load.     

Evidence that Na+/H+ exchange regulates angiotensin II-stimulated diacylglycerol accumulation in vascular smooth muscle cells

Angiotensin II stimulation of vascular smooth muscle cells results in initial, rapid diacylglycerol (DG) formation from the polyphosphoinositides accompanied by intracellular acidification, as well as a more sustained DG accumulation which is accompanied by a prolonged intracellular alkalinization. To determine whether intracellular pH (pHi) modulates DG accumulation, NH4Cl and potassium acetate were used to alter pHi and DG formation was measured. NH4Cl (10 mM) increased pHi from 7.15 +/- 0.05 to 7.34 +/- 0.02 pH units and markedly enhanced the sustained (5 min), but not the initial (15 s), phase of DG formation in response to 100 nM angiotensin II (65 +/- 13% increase). Conversely, intracellular acidification with Na+-free buffer and potassium acetate (20 mM) decreased pHi to 6.93 +/- 0.08 and reduced subsequent angiotensin II-induced sustained DG formation by 82 +/- 9%. In intact cells, inhibition of angiotensin II-stimulated alkalinization by incubation in Na+-free buffer or by addition of the Na+/H+ exchange inhibitor dimethylamiloride (10 microM) decreased the ability of the cell to sustain DG formation, suggesting that active Na+/H+ exchange is necessary for continued DG formation. Thus, it seems that sustained, angiotensin II-induced diacylglycerol accumulation is regulated by intracellular alkalinization secondary to Na+/H+ exchange in cultured vascular smooth muscle cells.     

Cytoplasmic pH regulation in normal and abnormal neutrophils. Role of superoxide generation and Na+/H+ exchange

The cytoplasmic pH of human neutrophils was determined fluorometrically using carboxylated fluorescein derivatives. When normal neutrophils were activated by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) in Na+-containing medium, the cytoplasmic pH initially decreased but then returned to near normal values. In Na+-free media or in Na+ medium containing amiloride, TPA induced a marked monophasic intracellular acidification. The cytoplasmic acidification is associated with net H+ equivalent efflux, suggesting metabolic acid generation. The metabolic pathways responsible for the acidification were investigated by comparing normal to chronic granulomatous disease neutrophils. These cells are unable to oxidize NADPH and generate superoxide. When treated with TPA in Na+-free or amiloride-containing media, chronic granulomatous disease cells did not display a cytoplasmic acidification. This suggests that in normal cells NADPH oxidation and/or the accompanying activation of the hexose monophosphate shunt are linked to the acidification. Unlike normal neutrophils, chronic granulomatous disease cells treated with TPA in Na+-containing medium displayed a significant cytoplasmic alkalinization. The alkalinization was Na+-dependent and amiloride-sensitive, indicating activation of Na+/H+ exchange. Thus, the Na+/H+ antiport, which can be indirectly stimulated by the metabolic cytoplasmic acidification, is also directly activated by the phorbol ester.     

Thimerosal induces calcium mobilization, fructose 2,6-bisphosphate synthesis and cytoplasmic alkalinization in rat thymus lymphocytes

The effect of thimerosal on intracellular calcium ([Ca2+]i), pH (pHi) and fructose 2,6-bisphosphate (Fru 2,6-P2) in thymus lymphocytes was investigated. The effect of thimerosal on cell growth was also examined. Thimerosal produced a dose-dependent increase in [Ca2+]i, pHi and in the level of fructose 2,6-bisphosphate. Thimerosal was, however, unable to produce cell proliferation and inhibited [3H]thymidine incorporation when cells were challenged with PHA and costimulator. In the absence of external calcium, thimerosal produced only a slight increase in [Ca2+]i. In Na(+)-containing buffer, thimerosal induced an initial acidification (0.05 +/- 0.01 pH units), followed by an alkalinization of 0.08 pH units/min, whereas in Na(+)-free media, pHi decreased 0.2 +/- 0.02 units and this acidification was maintained for more than 40 min. When external calcium was removed the initial acidification was unchanged and no further increase in pHi was observed. Polymyxin B, an inhibitor of protein kinase C, did not modify the initial thimerosal-induced acidification although pH returned to basal levels after 10 min. It was concluded that alkalinization induced by thimerosal is probably due to activation of the Na+/H+ exchanger and that changes in internal Ca2+, pH and metabolic rate are not sufficient to induce cellular proliferation. The mechanism by which thimerosal inhibits thymocyte proliferation remains to be clarified.     

Na+-H+ exchange at the apical membrane of Necturus gallbladder. Extracellular and intracellular pH studies

The mechanism of luminal solution acidification was studied in Necturus gallbladder by measurement of mucosal solution and intracellular pH with glass electrodes. When the gallbladder was bathed by a Na-Ringer's solution it acidified the luminal side by a Na+-dependent, amiloride- inhibitable process. In the presence of ouabain, acidification was reduced but could be stimulated to a rate greater than that under control conditions by the imposition of an inwardly directed Na+ gradient. These results suggest that luminal acidification results from Na+-H+ exchange at the apical membrane and not by diffusion of metabolic CO2. Li+ can substitute for Na+ but K+, Rb+, Cs+, and tetramethylammonium (TMA+) cannot. The maximal rate of exchange was about five times greater for Na+ than for Li+. Intracellular pH (pHi) was measured with recessed-tip glass microelectrodes; with the tissue bathed in Na-Ringer's solution (pH 7.75), pHi was 7.51 +/- 0.04. After inhibition of Na+-H+ exchange by mucosal perfusion with amiloride (1 mM) or by complete Na+ replacement with TMA+, phi fell reversibly by 0.15 and 0.22 pH units, respectively. These results support the conclusion that Na+-H+ exchange at the apical membrane is the mechanism of luminal acidification and is involved in the maintenance of steady state pHi.     

The relationship between mitogen-induced changes in the cytoplasmic pH and free Ca2+ concentration in rat thymocytes

The relationship between pHi and [Ca]i signals generated in rat thymocytes by the mitogen Con A has been investigated. It is shown that the mitogen-induced [Ca]i rise is dependent on Na+/H+ exchange or some other Na(+)-sensitive process. This conclusion is based on the following findings: (i) [Ca]i response to Con A weakens upon decreasing the concentration of extracellular Na+, or inhibiting Na+/H+ exchange; (ii) agents that alkalinize the cytoplasm (the phorbol ester TPA, the Na+/H+ ionophore monensin and NH4Cl) cause an increase in [Ca]i (Klip, A., Rothstein, A. and Mack, E. (1984) Biochem. Biophys. Res. Commun. 124, 14-22; Grinstein, S. and Goetz, J.D. (1985) Biochim. Biophys. Acta 819, 267-270); (iii) The effects of Con A, TPA and monensin on [Ca]i are not additive. The last observation suggests that all these agents activate the same Na+/H+ (Na+ and/or H+)-dependent system of Ca2+ transport. It is found that the pH i and [Ca]i responses in rat thymocytes are sensitive to changes in the intracellular levels of cyclic nucleotides, ATP and in temperature. These regulatory effects on the ionic signals are different for Con A, TPA and monensin. In particular, both the stimulation of Na+/H+ antiport and the [Ca]i rise brought about by Con A or TPA are inhibited upon elevating the cellular cAMP. In contrast, the monensin-induced [Ca]i signal is almost independent of cAMP but is highly sensitive to changes in cGMP and temperature. Reducing the ATP level eliminates both the pHi and [Ca]i responses to Con A but not to monensin. These different characteristics of [Ca]i signals elicited by the mitogen and the Na+/H+ ionophore indicate that these agents use different mechanisms to activate the Na+/H(+)-dependent Ca2+ transporting system. A [Ca]i response to monensin has been obtained in some other cell types, namely, in lymphoblastoid Raji cells, Ehrlich ascites tumor cells and also in platelets.     

Modification of the internal pH sensitivity of the Na+/H+ antiporter by parathyroid hormone in a cultured renal cell line

Sodium-proton antiporter activity can be modulated through changes Vmax and/or intracellular proton sensitivity of the antiporter. To characterize a parathyroid hormone (PTH)-induced decrease in antiporter activity in a continuous renal cell line (opossum kidney cells), the extracellular sodium and intracellular proton dependence of amiloride-inhibitable 22Na uptake was studied. The Km for extracellular sodium at intracellular pH 6.32 was 28 mM and was unaltered by PTH, whereas the Vmax was decreased by 26%. When intracellular pH was set over the range 5.87-7.57 by the potassium-nigericin method, antiporter activity increased as intracellular pH decreased. Hill analysis revealed Hill coefficients of 1.25 and 1.01 and half-maximal antiporter activity at intracellular pH values of 6.90 and 6.35 for control and PTH-treated cells, respectively. PTH decreased the apparent Vmax at low pH by 15% and the intracellular pH at which Na+/H+ exchange is half-maximal by 0.55 pH units.     

Intracellular Ca2+ potentiates Na+/H+ exchange and cell differentiation induced by phorbol ester in U937 cells

The human cell line U937 differentiates to monocyte macrophage-like cells in response to tumour-promoting phorbol esters. This effect is attributed to activation of protein kinase C. We show here that U937 cell differentiation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) is associated with cytoplasmic alkalinization. Ethyl-isopropyl-amiloride (EIPA), a potent inhibitor of Na+/H+ exchange, blocked both cytoplasmic alkalinization and cell differentiation. Cell acidification by addition of 2-4 mM sodium propionate also blocked TPA-induced U937 cell differentiation. These results suggest that a sustained cell alkalinization mediated by activation of Na+/H+ exchange is essential for TPA-induced differentiation in U937 cells. The increase of cytoplasmic free calcium concentration ([Ca2+]i) by addition of the calcium ionophore ionomycin enhanced TPA-induced alkalinization by increasing the apparent affinity of the Na+/H+ antiporter for intracellular H+. Treatment with ionomycin also potentiated differentiation of U937 cells induced by TPA. This synergism suggests that [Ca2+]i either potentiates the activation of protein kinase C or triggers additional transducing mechanisms. The key events of this interaction occur during the first 30 min of treatment, even though cell differentiation manifests much later.     

Na+ effects on intracellular pH of isolated, perfused rabbit gastric mucosal surface cells.

The effects of extracellular Na+ on intracellular pH were studied by perfusing BCECF loaded gastric mucosal surface cells adherent to glass coverslips held in a spectrophotofluorometer. Removal of Na+ from a NaCl Ringer perfusate (pH 7.4) resulted in progressive intracellular acidification, which was partially blocked by amiloride. An H+ conductance did not appear to be present. Acidification induced either by Na+ removal or by a NH4 prepulse was reversed by extracellular Na+, but this effect was not completely prevented by amiloride. Amiloride significantly, but not completely, inhibited Na22 uptake by gastric mucosal surface cells. The data suggest that extracellular Na+ maintains intracellular pH of gastric mucosal surface cells through amiloride-sensitive and -insensitive pathways. In the absence of extracellular Na+, cellular acidification seemed to be partially due to Na+/H+ exchange.     

ATP-dependent proton uptake by proteoliposomes reconstituted with purified Na+,K+-ATPase

Proton transport catalyzed by the sodium pump was demonstrated using proteoliposomes reconstituted with purified pig kidney Na+,K+-ATPase. Intravesicular pH was monitored with fluorescence from fluorescein isothiocyanate dextran introduced into the vesicles. An ATP-induced ouabain-sensitive acidification of the intravesicular medium was observed, when the vesicles were incubated with ATP and without Na+. The ATP-induced acidification was blocked by either extravesicular Na+ or pretreatment of the enzyme with ouabain before reconstitution. Protonophores, X-537A or carbonyl cyanide m-chlorophenylhydrazone, abolished the intravesicular acidification. The acidification was not inhibited by 3 mM tetra-n-butylammonium. The initial rate of the H+ uptake was increased with a decrease in pH of the extravesicular medium, and the maximum rate was obtained at pH 5.5-5.6. It is concluded that H+ can be transported in place of Na+ by the sodium pump.     

Priming effect of hyperosmotic stress on TRH-induced activation of Na+/H+ exchange in GH4C1 rat pituitary cells

The effect of mild hyperosmotic stress on cytosolic pH (pHi) alone, and in combination with thyrotropin-releasing hormone (TRH) or the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was investigated in GH4C1 cells at resting pHi. Hyperosmotic stress induced by addition of 50 mM choline was without an effect on pHi. In cells stimulated with either TRH or TPA after choline, pHi increased 0.15 +/- 0.05 and 0.14 +/- 0.03 pH units, respectively (mean +/- SD). A similar response was obtained if TRH or TPA was added prior to choline. The effect was abolished by replacing extracellular Na+ with choline+, and by pretreatment of the cells with amiloride, indicating that the change in pHi probably was dependent on activation of Na+/H+ exchange. The results thus indicate that, in GH4C1 cells, hyperosmotic stress in combination with TRH or TPA can activate Na+/H+ exchange at resting pHi levels.     

Characterization of Na+/H+ exchange in a rabbit corneal epithelial cell line (SIRC)

Continuous intracellular pH (pHi) measurements were performed in SIRC rabbit corneal epithelial cells using the pH-sensitive absorbance of intracellularly trapped 5(and 6)-carboxy-4',5'-dimethylfluorescein. Steady-state pHi in nominally bicarbonate free Ringer's solution averaged 6.87 +/- 0.02 (mean +/- S.E., n = 53). After intracellular acidification induced by the NH4Cl-prepulse technique, there was a sodium-dependent pHi recovery towards the normal steady-state pHi. The initial pHi recovery rate was a saturable function of extracellular sodium concentration with an apparent Km for external sodium of about 25 mM and a Vmax of about 0.28 pH units/min. Virtually no pHi recovery was observed in the absence of extracellular sodium. Sodium removal during steady state acidified the cells by 0.36 +/- 0.05 pH units (mean +/- S.E., n = 13) within 5 min. There was a dose-dependent inhibition of pHi recovery after NH4Cl prepulse by amiloride with an IC50 of about 15 microM. Amiloride in a concentration of 1 mM almost completely abolished pHi recovery. Amiloride (1 mM) applied during steady state induced an intracellular acidification of 0.2 +/- 0.03 pH units (mean +/- S.E., n = 7) within 5 min. These findings suggest that a Na+/H+ exchange is present in SIRC rabbit corneal epithelial cells. Na+/H+ exchange seems to be the major process involved in pHi recovery in SIRC cells after an intracellular acid load. Na+/H+ exchange also plays a role in the maintenance of steady-state pHi.     

Characterization of the renal epithelial cell line, PKE 5, using 31P- and 13C-NMR spectroscopy

In order to characterize intracellular pH regulation and cellular metabolism in PKE 5 cells, a mutant of the renal epithelial cell line LLC-PK1 supposed to lack Na+-H+ exchanger activity, 31P and 13C-NMR studies were conducted. The 31P studies on intact cell suspensions revealed that these cells have an ATP content and an ATP/ADP ratio similar to the parent cell line. Their intracellular pH, in the presence of 5 mM HCO3-, was 7.17 +/- 0.04 (n = 5) - identical to that of LLC-PK1 cells. After acid loading the cells with 15% CO2, the initial rate of realkalinization was 0.027 pH units/min (n = 6), 50% lower than in the parent cells. The recovery rate was not affected by the removal of extracellular sodium or by the addition of 1 mM amiloride. These results indicate that PKE 5 cells are devoid of Na+-H+ exchange activity, but are able to regulate their intracellular pH by amiloride-insensitive, sodium-independent mechanisms. Extracts prepared from PKE 5 cells incubated with [13C]lactate showed 13C spectra identical to those of the parent cell line. In particular, no synthesis of 13C-labeled D-glucose was observed.     

Depolarization of the membrane potential decreases the ATP-induced influx of extracellular Ca2+ and the refilling of intracellular Ca2+ stores in rat thyroid FRTL-5 cells.

The aim of the present study was to investigate the effect of membrane depolarization on ATP-induced changes in intracellular Ca2+ ([Ca2+]i) and the refilling of intracellular Ca2+ stores in thyroid follicular FRTL-5 cells. Depolarizing the cells with 50 mM K+, an amount sufficient to almost totally depolarize the cells as determined by bisoxonal, significantly reduced the ATP-induced uptake of 45Ca2+. This effect was not dependent on an enhanced efflux of Ca2+, as no difference in the ATP-induced efflux of 45Ca2+ was obtained between control cells and depolarized cells. The ATP-induced transient increase in [Ca2+]i in Fura-2 loaded cells was not altered by depolarization, whereas the ATP-induced plateau in [Ca2+]i was decreased compared with control cells. Furthermore, in cells stimulated with ATP in a Ca(2+)-free buffer, readdition of Ca2+ after the termination of the ATP response induced a decreased response in [Ca2+]i in depolarized cells. Refilling of intracellular Ca2+ stores was investigated by first stimulating the cells with noradrenaline (NA). The effect of NA was then terminated with prazosin, and the cells restimulated with ATP. In cells depolarized with high K+, the response to ATP was decreased compared with that seen in control cells. The results thus suggest that both the ATP-induced influx of extracellular Ca2+ and the refilling of intracellular Ca2+ stores is decreased in depolarized FRTL-5 cells.     

Regulation of intracellular pH in human neutrophils

The intracellular pH (pHi) of isolated human peripheral blood neutrophils was measured from the fluorescence of 6-carboxyfluorescein (6-CF) and from the equilibrium distribution of [14C]5,5-dimethyloxazolidine -2,4-dione (DMO). At an extracellular pH (pHo) of 7.40 in nominally CO2-free medium, the steady state pHi using either indicator was approximately 7.25. When pHo was suddenly raised from 7.40 to 8.40 in the nominal absence of CO2, pHi slowly rose by approximately 0.35 during the subsequent hour. A change of similar magnitude in the opposite direction occurred when pHo was reduced to 6.40. Both changes were reversible. Intrinsic intracellular buffering power, determined by using graded pulses of CO2 or NH4Cl, was approximately 50 mM/pH over the pHi range of 6.8-7.9. The course of pHi obtained from the distribution of DMO was followed during and after imposition of intracellular acid and alkaline loads. Intracellular acidification was brought about either by exposing cells to 18% CO2 or by prepulsing with 30 mM NH4Cl, while pHo was maintained at 7.40. In both instances, pHi (6.80 and 6.45, respectively) recovered toward the control value at rates of 0.029 and 0.134 pH/min. These rates were reduced by approximately 90% either by 1 mM amiloride or by replacement of extracellular Na with N-methyl-D-glucamine. Recovery was not affected by 1 mM SITS or by 40 mM alpha-cyano-4-hydroxycinnamate (CHC), which inhibits anion exchange in neutrophils. Therefore, recovery from acid loading is probably due to an exchange of internal H for external Na. Intracellular alkalinization was achieved by exposing the cells to 30 mM NH4Cl or by prepulsing with 18% CO2, both at a constant pHo 7.40. In both instances, pHi, which was 7.65 and 7.76, respectively, recovered to the control value. The recovery rates (0.033 and 0.077 pH/min, respectively) were reduced by 80-90% either by 40 mM CHC or by replacement of extracellular Cl with p-aminohippurate (PAH). SITS, amiloride, and ouabain (0.1 mM) were ineffective.(ABSTRACT TRUNCATED AT 400 WORDS)