共查询到20条相似文献,搜索用时 31 毫秒
1.
Data have been presented on the effect of serum and of cell adhesion to a solid substrate on the intracellular pH (pHi) value in anchorage-dependent Chinese hamster fibroblasts. Proliferation of cells was observed in a pHi range from 7.0 to 7.5, which exceeded by 0.3-0.4 the pHi of quiescent cells. It was shown that attachment and spreading of cells in a bicarbonate-containing media without serum produced elevation of pHi from 6.7 to 7.1 but did not provide such an alkalization value for times greater than the lag period of cell growth. By the end of the first day after plating the pHi value of the spread cells in a serum-free medium reduced to 6.75, and the cells ceased to grow. The serum without cell attachment produced only a short-time (less than 1 h) pHi increase from 6.7 to 6.88, which was inadequate for cell growth. However, the serum produced a prolongation in cytoplasm alkalization characteristic of proliferation and caused by attachment of cells to a solid substrates. Evidence also was obtained for the absence of Na-independent HCO3-/Cl- exchange, the presence of a slow HCO3- transport into the cell and the key role of Na+/H+ exchange in pHi regulation. Addition to the bicarbonate-containing medium of amiloride, a blocker of Na+/H+ exchange, resulted in acidification of the cytoplasm to 6.5, and inhibited cell attachment and proliferation. 相似文献
2.
The dynamics of intracellular pH (pHi) during proliferation of cells of LS line in bicarbonate-containing media and of its derivative LSM line adapted to grow in a monolayer has been studied. The contact of LS cells with a solid substrate was not accompanied by their spreading and by an increase in pHi. The pHi values of growing and resting LS cells were practically equal (7.03 and 6.97, respectively). The adhesion and spreading of LSM cells were accompanied by an increase in pHi. The proliferation of LSM cells occurred at different pHi values: at 7.32 on solid substrate with serum, at 7.18 on substrate without serum, at 7.13 in a serum-containing suspension, at 6.97 in a suspension without serum. The highest growth rate was observed at the increased pHi value. Cell proliferation on the substrate stopped at pHi values within 7.10 and 7.13 which were equal to or exceeded the pHi of growing cells in suspension. No difference was observed between LS and LSM cells in the activities of Na+/H+ exchange and transport of Cl- into cells that are involved in pHi regulation. Transport of HCO3- into the cytoplasm of LSM cells was more active than that of LS cells. The role of pHi in the anchorage dependence of cell proliferation is discussed. 相似文献
3.
Intracellular pH and cell adhesion to solid substrate 总被引:3,自引:0,他引:3
It was shown that activation of the Na+/H+ antiporter resulting in an increase of intracellular pH (pHi) by 0.2-0.3 is a necessary stage of cell stimulation by soluble growth factors. Solid substrate can also be formally regarded as a growth factor since adhesion stimulates proliferation of various cell types. In the present study we have found that the attachment of mouse embryo fibroblasts to solid substrate is followed by an increase of pHi by approx. 0.3 units. pH shift occurs after the cell attaches to the substrate and is obligatory for cell spreading. The evidence for Na+/H+ antiporter involvement in the increase of pHi in substrate-attached cells is presented. It is suggested that signals for cell proliferation by chemical (soluble ligands) and physical (solid substrate) growth factors are transmitted similarly. 相似文献
4.
E Koh S Morimoto S Kim T Nabata Y Miyashita T Ogihara 《Biochemistry international》1990,20(2):375-380
The effect of endothelin (ET) on the intracellular pH (pHi) of vascular smooth muscle cells (VSMC), was investigated using a fluorescent pH indicator 2',7'-bis(carboxyethyl)carboxyfluorescein (BCECF). ET at concentrations of over 10(-9) M caused dose-dependent transient acidification followed by Na(+)-dependent and amiloride-sensitive alkalization of the cells due to stimulation of Na+/H+ exchange. The alkalization induced by ET was Ca2(+)-dependent and was inhibited by a calcium channel blocker, nicardipine. Pretreatment with H-7, an inhibitor of protein kinase C, also inhibited the ET-induced cell alkalization. These results indicate that ET stimulates Na+/H+ exchange, resulting in alkalization of VSMC and that this ET-induced cell-alkalization is probably linked to Ca2+ influx and activation of protein kinase C. 相似文献
5.
E Bello-Reuss 《Renal physiology and biochemistry》1991,14(1-2):1-11
Double-barrel microelectrodes were used to determine membrane voltages and the intracellular pH (pHi) in primary cultures of cortical collecting duct cells (CCD) grown in the absence of aldosterone. Electrophysiologically, two main cell types were identified. In cell type 1, the apical membrane voltage (Va) was -60 +/- 5 mV. The fractional resistance of the apical membrane (fRa) was 0.40 +/- 0.03, and pHi was 7.21 +/- 0.04. Exposure to 50 mM K+ on the apical side depolarized Va by 21 +/- 4 mV. When Cl- was replaced by cyclamate two types of responses were observed: (a) depolarization of Va by 26 +/- 3 mV while pHi remained unchanged, and (b) no change in Va. In cell type 2, Va was -36 +/- 5 mV, fRa was 0.91 +/- 0.03 and increasing apical [K+] from 5 to 50 mM did not change Va. Two subpopulations were distinguished by the response of pHi to lowering apical [Cl-]. In one of them pHi increased from 6.99 +/- 0.05 to 7.11 +/- 0.07. In the other, pHi was significantly decreased from 7.16 +/- 0.08 to 7.03 +/- 0.07. These results are compatible with the conclusion that about 50% of the impaled cells type 2 have a Cl-/HCO-3 exchanger at the apical membrane. In summary, two different cell types can be identified electrophysiologically in CCD monolayers. Cell type 1 has the electrical characteristics of principal cells. Cell type 2 resembles the intercalated cells. The cell alkalinization observed in approximately 50% of the cells type 2 in response to Cl- removal suggests the presence of an apical Cl-/HCO-3 exchanger. Thus, these cells should be the bicarbonate-secreting cells. The remaining cells should correspond to the acid-secreting cells. 相似文献
6.
G Gaidano D Ghigo M Schena L Bergui S Treves F Turrini F C Cappio A Bosia 《Journal of immunology (Baltimore, Md. : 1950)》1989,142(3):913-918
In various mammalian cell types the stimulation of the plasma membrane amiloride-sensitive Na+/H+ exchange and the resulting increase of intracellular pH (pHi) play a key role in the initiation of cell proliferation. In the present work we have investigated whether Na+/H+ exchange is involved in normal human B cell proliferation and whether it is also operating in malignant B-chronic lymphocytic leukemia (B-CLL) lymphocytes. Our results show that: 1) normal human B cells contain an operating Na+/H+ exchanger, as inferred by their ability to recover pHi after acid-loading in a HCO3- -free medium and by evidences that LPS and phorbol ester PMA elicit a pHi rise inhibitable by either 5-(N-ethyl-N-isopropyl)amiloride (EIPA) or a Na+-free medium; 2) LPS-induced proliferation of normal human B cells is strongly inhibited when the amiloride analog EIPA (5 microM) is present in the culture medium (after 72 h the proportion of B cells incorporation bromodeoxyuridine falls from 13.9 +/- 3.9% to 2.8 +/- 1.1%); 3) EIPA does not affect BdR incorporation when B cells proliferation is induced by the co-mitogenic activity of IL-4 and low m.w. B cell growth factor (BCGF); 4) B-CLL cells, which proliferate in response to IL-4/BCGF but not to LPS, fail to increase pHi above their pHi resting levels when challenged with LPS or PMA and pHi recovery after acid-loading is highly impaired. These results lead to conclude that Na+/H+ exchange operation is necessary for LPS-(but not for IL-4/BCGF)-induced proliferation of human normal B lymphocytes and that Na+/H+ exchange activation is impaired in malignant B-CLL lymphocytes. 相似文献
7.
The course of intracellular pH (pHi) was followed in superfused (36 degrees C) single glomus (type I) cells of the freshly dissociated adult rat carotid body. The cells had been loaded with the pH-sensitive fluorescent dye 2',7'-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein. The high K(+)-nigericin method was used for calibration. The pHi of the glomus cell at pHo 7.40, without CO2, was 7.23 +/- 0.02 (n = 70); in 5% CO2/25 mM HCO3-, pHi was 7.18 +/- 0.08 (n = 9). The pHi was very sensitive to changes in pHo. Without CO2, delta pHi/delta pHo was 0.85 (pHo 6.20-8.00; 32 cells), while in CO2/HCO3- this ratio was 0.82 irrespective of whether pHo (6.80-7.40; 14 cells) was changed at constant PCO2 or at constant [HCO3-]o. The great pHi sensitivity of the glomus cell to pHo is matched only by that of the human red cell. An active Na+/H+ exchanger (apparent Km = 58 +/- 6 mM) is present in glomus cells: Na+ removal or addition of the amiloride derivative 5-(N,N-hexamethylene)-amiloride induced pHi to fall by as much as 0.9. The membrane of these cells also contains a K+/H+ exchanger. Raising [K+]o from 4.7 to 25, 50, or 140 mM reversibly raised pHi by 0.2, 0.3, and 0.6, respectively. Rb+ had no effect, but in corresponding concentrations of Tl+ alkalinization was much faster than in K+. Reducing [K+]o to 1.5 mM lowered pHi by 0.1. These pHi changes were shown not to be due to changes in membrane voltage, and were even more striking in the absence of Na+. Intrinsic buffering power (amount of strong base required to produce, in the nominal absence of CO2, a small pHi rise) increased from 3 to approximately 21 mM as pHi was lowered, but remained nearly unchanged below pHi 6.60. The fitted expression assumed the presence of one "equivalent" intracellular buffer (pK 6.41, 41 mM). The exceptional pHi sensitivity to pHo suggests that the pHi of the glomus cell is a link in the chemoreceptor's response to external acidity. 相似文献
8.
Deficient protein kinase C-dependent Na+/H+ exchanger activity in T cells from bone marrow transplantation recipients 总被引:2,自引:0,他引:2
M Izquierdo J M Redondo M A Balboa A López-Rivas E J Cragoe L Vázquez J M Fernández-Ra?ada M López-Botet 《Journal of immunology (Baltimore, Md. : 1950)》1989,143(7):2185-2192
The early Na+/H+ exchanger-mediated alkalinization of intracellular pH (pHi) was analyzed in peripheral blood T cells from 23 bone marrow transplantation (BMT) recipients (17 allogeneic and 6 autologous) and a group of 13 healthy controls, in response to stimulation of protein kinase C (PKC) with a phorbol ester. In parallel we evaluated the proliferative response of peripheral blood T cells to an anti-CD3 mAb in the presence of either IL-2 or PMA. The pHi increase (delta pHi) observed in control samples ranged from 0.14 to 0.23 pH units (X +/- SD = 0.17 +/- 0.03). In 10 allogeneic and four autologous BMT recipients the delta pHi was under the lower limit of the control range (range: 0.01 to 0.09, X +/- SD = 0.05 +/- 0.02), whereas the remaining nine cases responded similarly to control samples (range: 0.14 to 0.24, X +/- SD = 0.17 +/- 0.04). The response of the Na+/H+ antiporter to a PKC-independent osmotic stimulation appeared to be normal, thus indicating that the intrinsic Na+/H+ exchanger activity was unaltered. The anti-CD3 induced proliferative response of the group of samples displaying a suboptimal delta pHi, was significantly lower (p less than 0.01) than that detected in control samples. T cell proliferation in samples from BMT recipients displaying a normal delta pHi was undistinguishable from the control group (p greater than 0.05). Our results provide the first evidence for a defective early metabolic event, closely related to PKC activity, in T cells from BMT recipients displaying a low proliferative response to T cell mitogens. 相似文献
9.
G B Mills E J Cragoe E W Gelfand S Grinstein 《The Journal of biological chemistry》1985,260(23):12500-12507
In several cell types, proliferation initiated by growth factors is associated with a rapid increase in cytoplasmic pH (pHi). This cytoplasmic alkalinization is due to the activation of an amiloride-sensitive Na+/H+ antiport. It is unclear whether growth factor-induced activation of the antiport or the resultant increase in pHi is the trigger for proliferation, an obligatory requirement for proliferation, or simply an associated phenomenon. Interleukin 2 (IL 2) acts as a growth factor for mitogen or antigen-stimulated thymus-derived (T) lymphocytes. In this study, we established that IL 2 produces an increase in pHi and determined whether this increase in pHi plays a role in the proliferative response to IL 2. Monitoring pHi with an intracellularly trapped, pH-sensitive, fluorescent dye, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, we demonstrated that IL 2 rapidly (less than 90 s) initiates an increase in pHi in IL 2-sensitive human and murine T cells. Because intracellular alkalinization requires extracellular Na+ and is amiloride-sensitive, it likely occurs through activation of the Na+/H+ antiport. Using partitioning of a weak acid, 5,5-dimethyl-2,4-oxazolidinedione, we confirmed that the IL 2-dependent increase in pHi is sustained for several hours and returns to near base-line levels by 18 h. We also investigated the consequence of preventing Na+/H+ exchange on the proliferative response induced by IL 2. IL 2-driven proliferation occurred in nominally bicarbonate-free medium in the presence of concentrations of amiloride analogs sufficient to inhibit the Na+/H+ antiport and prevent intracellular alkalinization. These data suggest that although the antiport is activated by binding of IL 2 to its receptor, intracellular alkalinization is not essential for IL 2-dependent proliferation. It seems unlikely that either cytoplasmic alkalinization or activation of the Na+/H+ antiport are triggers for T cell proliferation. 相似文献
10.
R A Star B X Zhang P A Loessberg S Muallem 《The Journal of biological chemistry》1992,267(25):17665-17669
The technique for the simultaneous recording of cell volume changes and pHi in single cells was used to study the role of HCO3- in regulatory volume decrease (RVD) by the osteosarcoma cells UMR-106-01. In the presence of HCO3-, steady state pHi is regulated by Na+/H+ exchange, Na+ (HCO3-)3 cotransport and Na(+)-independent Cl-/HCO3- exchange. Following swelling in hypotonic medium, pHi was reduced from 7.16 +/- 0.02 to 6.48 +/- 0.02 within 3.4 +/- 0.28 min. During this period of time, the cells performed RVD until cell volume was decreased by 31 +/- 5% beyond that of control cells (RVD overshoot). Subsequently, while the cells were still in hypotonic medium, pHi slowly increased from 6.48 +/- 0.02 to 6.75 +/- 0.02. This increase in pHi coincided with an increase in cell volume back to normal (recovery from RVD overshoot or hypotonic regulatory volume increase (RVI)). The same profound changes in cell volume and pHi after cell swelling were observed in the complete absence of Cl- or Na+, providing HCO3- was present. On the other hand, depolarizing the cells by increasing external K+ or by inhibition of K+ channels with quinidine, Ba2+ or tetraethylammonium prevented the changes in pHi and RVD. These findings suggest that in the presence of HCO3-, RVD in UMR-106-01 cells is largely mediated by the conductive efflux of K+ and HCO3-. Removal of external Na+ but not Cl- prevented the hypotonic RVI that occurred after the overshoot in RVD. Amiloride had no effect, whereas pretreatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) strongly inhibited hypotonic RVI. Thus, hypotonic RVI is mediated by a Na+(out)-dependent, Cl(-)-independent and DIDS-inhibitable mechanism, which is indicative of a Na+(HCO3-)3 cotransporter. This is the first evidence for the involvement of this transporter in cell volume regulation. The present results also stress the power of the new technique used in delineating complicated cell volume regulatory mechanisms in attached single cells. 相似文献
11.
D Caracciolo A Pannocchia S Treves D Ghigo E Gallo C Tarella F Bussolino F Turrini G Tamponi A Bosia 《Journal of cellular physiology》1990,143(1):133-139
The growth of the human leukemia cell line AML-193 in a serum-free medium is strictly dependent on the presence of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), which is one of the major regulators of the myelomonocytic lineage. At present, little is known about the mechanisms by which this growth factor transduces the signal intracellularly. The results of this study demonstrate that GM-CSF needs the operation of a Na+/H+ exchanger, which is located in the plasma membrane of almost every vertebrate cell. In fact, the GM-CSF-dependent proliferation of AML-193 cells is strongly reduced in the presence of the amiloride analog EIPA, a specific inhibitor of the Na+/H+ exchanger. When acidified, AML-193 cells are able to recover the original pHi in a Na(+)-dependent and EIPA-inhibitable way; this demonstrates for the first time the presence of the Na+/H+ exchanger in these cells. Finally, GM-CSF, at doses superimposable to those needed for triggering proliferation, induces in AML-193 cells a sustained alkalinization, which is dependent on a operating Na+/H+ exchange, as it is inhibited by EIPA. These results suggest that GM-CSF, like other growth factors in other cell systems, exerts its mitogenic activity in AML-193 cells by inducing a Na+/H+ exchanger-mediated rise in pHi. 相似文献
12.
Direct measurement of intracellular pH changes in Xenopus eggs at
fertilization and cleavage 总被引:9,自引:7,他引:2
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《The Journal of cell biology》1981,91(2):562-567
We have used Thomas-type recessed-tip pH-sensitive microelectrodes to measure the intracellular pH (pHi) in Xenopus eggs during both fertilization and ionophore activation. The average pHi in unfertilized eggs is 7.33 +/- 0.11 (SD; n = 21) with a resting membrane potential of -10.1 +/- 3.5 (SD; n = 38) mV. Within 2 min after the onset of the fertilization potential, there is a slight, transient pHi decrease of 0.03 +/- (SD, n = 8), followed by a distinct, permanent pHi increase of 0.31 +/- 0.11 (SD; n = 7) beginning approximately 10 min after the start of the fertilization potential and becoming complete approximately 1 h later. The pHi remains near this level of 7.67 +/- 0.13 (SD, n = 10) through at least 10 cleavage cycles, but it is possible to discern pHi oscillations with a mean amplitude of 0.03 +/- 0.02 (SD, n = 38). Eggs perfused for at least 2 h in Na+-free solution with 1 mM amiloride exhibited all of these pHi changes, so these changes do not require extracellular Na+. Similar cytoplasmic alkalinizations that accompany the activation of metabolism and the cell cycle in a wide variety of cell types are discussed. 相似文献
13.
We studied the effects of epidermal growth factor (EGF), thyroid-stimulating hormone (TSH) and amiloride on cytoplasmic pH (pHi) in cultured porcine thyroid cells. We used 2',7'-bis(2-carboxyethyl)-5- (and 6-)carboxyfluorescein (BCECF), an internalized fluorescent pH indicator, to measure pHi. EGF stimulated thyroid cell alkalinization and proliferation, which were blocked by amiloride. EGF-stimulated thyroid cell alkalinization depended on extracellular Na+ concentrations. EGF stimulation resulted in an activation of Na+/H+ exchange, which alkalinized the cells. The results indicated that Na+/H+ exchange or cell alkalinization might function as a transmembrane signal transducer in the action of EGF. In the present system, TSH did not stimulate alkalinization or proliferation. 相似文献
14.
A K Ho C L Chik J L Weller E J Cragoe D C Klein 《The Journal of biological chemistry》1989,264(22):12983-12988
The regulation of intracellular pH (pHi) in isolated rat pinealocytes was studied using the fluorescent pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Resting pHi was 7.09 when the extracellular pH (pHe) was 7.2. Treatment of pinealocytes with the physiological regulator of pineal function, norepinephrine, resulted in a concentration-dependent increase in pHi. Further analysis indicated that norepinephrine is probably acting via an alpha 1-adrenergic----[Ca2+]i----Ca2+/phospholipid- dependent protein kinase (protein kinase C) mechanism to activate the Na+/H+ antiporter, thereby causing cytoplasmic alkalization. A potential influence of cytosolic alkalization on the responsiveness of cyclic nucleotides to adrenergic agonists was also studied. Five analogs of the antiporter inhibitor amiloride reduced norepinephrine stimulation of cGMP accumulation with the same relative potency as they act on the antiporter. In contrast, although inhibitory effects of these compounds on cAMP accumulation were detectable, they occurred at 10-100-fold higher concentrations, and the relative potency of these inhibitors did not indicate they were acting via the antiporter. These findings provide evidence that 1) alpha 1-adrenergic receptor activation increases pinealocyte pHi through Ca2+----protein kinase C-dependent activation of the Na+/H+ antiporter; and 2) norepinephrine stimulation of cGMP accumulation is pHi-dependent. It would appear that alpha 1-adrenergic regulation of pHi via the Na+/H+ antiporter may be of general importance in the control of cGMP accumulation. 相似文献
15.
P Vigne J P Breittmayer C Frelin M Lazdunski 《The Journal of biological chemistry》1988,263(34):18023-18029
Two mechanisms are involved in the regulation of the intracellular pH (pHi) of aortic smooth muscle cells: the Na+/H+ antiporter and a Na+-independent HCO3-/Cl- antiporter. The Na+/H+ antiporter acts as a cell alkalinizing mechanism. It is activated by vasopressin and by phorbol esters when cells are incubated in the presence of bicarbonate but is not affected in the absence of bicarbonate. The HCO3-/Cl- antiporter acts as a cell acidifying mechanism. Agents such as forskolin, 8-Br-cAMP, and isoproterenol which raise intracellular cAMP levels inhibit the HCO3-/Cl- antiporter by shifting its pHi dependence in the alkaline direction. Thus, within the same cell type, different hormones control pHi variations by acting on different pHi regulating systems. An increase in pHi can be achieved either by a stimulation of a cell alkalinizing mechanism or by inhibition of a cell acidifying mechanism. A change of the activity of one pHi regulating mechanism modifies the responsiveness of the other to regulatory agents. Bicarbonate turns on the HCO3-/Cl- antiporter, decreases pHi and allows its regulation by protein kinase C through the Na+/H+ antiporter. Inhibition of the HCO3-/Cl- antiporter by cAMP increases the pHi and switches off the protein kinase C-mediated regulation. 相似文献
16.
H Helbig C Korbmacher F Stumpff M Coca-Prados M Wiederholt 《Journal of cellular physiology》1988,137(2):384-389
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. 相似文献
17.
31P-NMR spectroscopy was used to monitor intracellular pH (pHi) in a suspension of LLC-PK1 cells, a renal epithelial cell line. The regulation of intracellular pH (pHi) was studied during intracellular acidification with 20% CO2 or intracellular alkalinization with 30 mM NH4Cl. The steady-state pHi in bicarbonate-containing Ringer's solution (pHo 7.40) was 7.14 +/- 0.04 and in bicarbonate-free Ringer's solution (pHo 7.40) 7.24 +/- 0.04. When pHo was altered in nominally HCO3(-)-free Ringer's, the intracellular pHi changed to only a small extent between pHo 6.6 and pHo 7.6; beyond this range pHi was linearly related to pHo. Below pHo 6.6 the cell was capable of maintaining a delta pH of 0.2 pH unit (inside more alkaline), above pH 7.6 a delta pH of 0.4 unit could be generated (inside more acid). During exposure to 20% CO2 in HCO3(-)-free Ringer's solution, pHi dropped initially to 6.9 +/- 0.05, the rate of realkalinisation was found to be 0.071 pH unit X min-1. After removal of CO2 the pHi increased by 0.65 and the rate of reacidification was 0.056 pH unit X min-1. Exposure to 30 mM NH4Cl caused a raise of pHi by 0.48 pH unit and an initial rate of re-acidification of 0.063 pH unit X min-1, after removal of NH4Cl the pHi fell by 0.58 pH unit below the steady-state pHi, followed by a subsequent re-alkalinization of 0.083 pH unit X min-1. Under both experimental conditions, the pHi recovery after an intracellular acidification, introduced by exposure to 20% CO2 and by removal of NH4+, was found to be inhibited by 53% and 63%, respectively, in the absence of sodium and 60% and 72%, respectively, by 1 mM amiloride. These studies indicate that 31P-NMR can be used to monitor steady-state intracellular pH as well a pHi transients in suspensions of epithelial cells. The results support the view that LLC-PK1 cells use an Na+-H+ exchange system to readjust their internal pH after acid loading of the cell. 相似文献
18.
BCECF fluorescence has been applied to determine intracellular pH (pHi) in NIH 3T3 fibroblasts expressing the Ha-ras oncogene (+ras) and otherwise identical cells not expressing the oncogene (-ras). In +ras cells, pHi is significantly more alkaline (6.79 +/- 0.03 n = 12) than in -ras cells (6.64 +/- 0.02, n = 8). Bradykinin (100 nmol/l) leads to intracellular alkalinization in both +ras (to 6.96 +/- 0.04, n = 12) and -ras cells (to 6.85 +/- 0.02, n = 8). The effect of bradykinin is completely abolished in the presence of dimethylamiloride (100 mumol/l), which does not modify pHi in the absence of bradykinin. Similar to bradykinin, cell shrinkage by addition of 15 mmol/l NaCl to the extracellular fluid leads to intracellular alkalinization (by 0.08 +/- 0.01, n = 15). Cell volume is significantly greater in +ras cells (2.7 +/- 0.4 pl, n = 15) than in -ras cells (2.2 +/- 0.4 pl, n = 15). Bradykinin leads to cell shrinkage in both +ras cells (by 7 +/- 1%, n = 17) and -ras cells (by 5 +/- 1%, n = 15). The effect of bradykinin on cell volume can be reversed by the reduction of extracellular NaCl concentration by 15 mmol/l NaCl in +ras cells and by 7 mmol/l NaCl in -ras cells. This maneuver completely abolishes (in -ras cells) or blunts (in +ras cells) the alkalinizing effect of bradykinin. In conclusion, +ras cells are more alkaline than -ras cells. Bradykinin leads to further intracellular alkalinization by activation of the Na+/H(+)-exchanger, at least in part secondary to hormone-induced cell shrinkage. 相似文献
19.
Regulation of intracellular pH (pHi) and protection against cytosolic acidification is primarily a function of the ubiquitous plasma membrane Na+/H+exchanger-1 (NHE1), which uses a highly conserved process to transfer cytosolic hydrogen ions (H+) across plasma membranes in exchange for extracellular sodium ions (Na+). Growth factors, which are essential regulators of morphogenesis, have also been found to be key activators of NHE1 exchanger activity; however, the crosstalk between both has not been fully evaluated during organ development. Here we report that mammary branching morphogenesis induced by transforming growth factor-alpha (TGFα) requires PI3K-dependent NHE1-activation and subsequent pHi alkalization. Inhibiting NHE1 activity after TGFα stimulation with 10 μM of the NHE1-specific inhibitor N-Methyl-N-isobutyl Amiloride (MIA) dramatically disrupted branching morphogenesis, induced extensive proliferation, ectopic expression of the epithelial hyper-proliferative marker Keratin-6 and sustained activation of MAPK. Together these findings indicate a novel developmental signaling cascade involving TGFα>PI3K>NHE1>pHi alkalization, which leads to a permissible environment for MAPK negative feedback inhibition and thus regulated mammary branching morphogenesis. 相似文献
20.
The action of low density lipoprotein on vascular smooth muscle cells involves increase in intracellular pH 总被引:1,自引:0,他引:1
E Koh S Morimoto T Nabata Y Miyashita S Kitano R Morita T Ogihara 《Biochemistry international》1990,20(1):127-133
The effect of low density lipoprotein (LDL) on the intracellular pH (pHi) of vascular smooth muscle cells (VSMC) was investigated using a fluorescent pH indicator, 2',7'-bis(carboxyethyl)carboxyfluorescein (BCECF). LDL and apoprotein B (apo-B), a binding protein for the LDL receptor, caused transient acidification followed by Na(+)-dependent and amiloride-sensitive alkalization of the cells due to stimulation of Na+/H+ exchanger. NH4Cl also caused intracellular alkalization, but independently of extracellular Na+. LDL, apo-B and NH4Cl all stimulated thymidine incorporation. These results indicate that the binding of LDL to its receptor stimulates Na+/H+ exchanger, resulting in alkalization of VSMC and suggest that this may function as a massage in stimulation of DNA synthesis evoked by LDL. 相似文献