首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 46 毫秒
1.
Summary Intracellular pH (pH i ) of the acinar cells of the isolated, superfused mouse lacrimal gland has been measured using pH-sensitive microelectrodes. Under nonstimulated condition pH i was 7.25, which was about 0.5 unit higher than the equilibrium pH. Alterations of the external pH by ±0.4 unit shifted pH i only by ±0.08 unit. The intracellular buffering value determined by applications of 25mm NH 4 + and bicarbonate buffer solution gassed with 5% CO2/95% O2 was 26 and 46mm/pH, respectively Stimulation with 1 m acetylcholine (ACh) caused a transient, small decrease and then a sustained increase in pH i . In the presence of amiloride (0.1mm) or the absence of Na+, application of ACh caused a significant decrease in pH i and removal of amiloride or replacement with Na+-containing saline, respectively, rapidly increased the pH i . Pretreatment with DIDS (0.2mm) did not change the pH i of the nonstimulated conditions; however, it significantly enhanced the increase in pH i induced by ACh. The present results showed that (i) there is an active acid extrusion mechanism that is stimulated by ACh; (ii) stimulation with ACh enhances the rate of acid production in the acinar cells; and (iii) the acid extrusion mechanism is inhibited by amiloride addition to and Na+ removal from the bath solution. We suggest that both Na+/H+ and HCO 3 /Cl exchange transport mechanisms are taking roles in the intracellular pH regulation in the lacrimal gland acinar cells.  相似文献   

2.
Summary Anion exchange transport in the mouse lacrimal gland acinar cell membrane was studied by measuring the intracellular H+ (pHi) and Cl (aCli) activities with double-barreled ion-selective microelectrodes. In a HCO 3 -free solution of pH 7.4 (HEPES/Tris buffered), pHi was 7.25 andaCli was 33mm. By an exposure to a HCO 3 (25mm HCO 3 /5% CO2, pH 7.4) solution for 15 min,aCli was decreased to 25mm and pHi was transiently decreased to about 7.05 within 1 min, then slowly relaxed to 7.18 in 15 min. Intracellular HCO 3 concentration [HCO 3 ]i, calculated by the Henderson-Hasselbalch's equation, was 11mm at 1 min after the exposure and then slowly increased to 15mm. Readmission of the HCO 3 -free solution reversed the changes inaCli and pHi. The intracellular buffering power was about 40mm/pH. An addition of DIDS (0.2mm) significantly inhibited the rates of change inaCli, pHi, and [HCO 3 ]i caused by admission/withdrawal of the HCO 3 , solution and decreased the buffer value. Replacement of all Cl with gluconate in the HCO 3 solution increased pHi, and readmission of Cl decreased pHi. The rates of these changes in pHi were reduced by DIDS by 32–45% but not by amiloride (0.3mm). In the HCO 3 solution, a stimulation of intracellular HCO 3 production by exposing the tissue to 25mm NH 4 + increasedaCli significantly. While in the HCO 3 -free solution or in the HCO 3 , solution containing DIDS, exposure to NH 4 + had little effect onaCli. All of these findings were consistent with the presence of a reversible, disulfonic stilbene-sensitive Cl/HCO 3 exchanger in the basolateral membrane of the acinar cells. The possibility of anion antiport different from one-for-one Cl/HCO 3 exchange is discussed.  相似文献   

3.
pH i recovery in acid-loaded Ehrlich ascites tumor cells and pH i maintenance at steady-state were studied using the fluorescent probe BCECF.Both in nominally HCO 3 -free media and at 25 mm HCO 3 , the measured pH i (7.26 and 7.82, respectively) was significantly more alkaline than the pH i . value calculated assuming the transmembrane HCO 3 gradient to be equal to the Cl gradient. Thus, pH i in these cells is not determined by the Cl gradient and by Cl/HCO 3 exchange.pH i recovery following acid loading by propionate exposure, NH 4 + withdrawal, or CO2 exposure is mediated by amiloride-sensitive Na+/H+ exchange in HCO3 free media, and in the presence of HCO 3 (25 mm) by DIDS-sensitive, Na+-dependent Cl/HCO 3 exchange. A significant residual pH i recovery in the presence of both amiloride and DIDS suggests an additional role for a primary active H+ pump in pH i regulation. pH i maintenance at steady-state involves both Na+/H+ exchange and Na+-dependent Cl/HCO 3 exchange.Acute removal of external Cl induces a DIDS-sensitive, Na+-dependent alkalinization, taken to represent HCO 3 influx in exchange for cellular Cl. Measurements of 36Cl efflux into Cl-free gluconate media with and without Na+ and/or HCO 3 (10 mm) directly demonstrate a DIDS-sensitive, Na+ dependent Cl/HCO 3 exchange operating at slightly acidic pH i (pHo 6.8), and a DIDS-sensitive, Na+-independent Cl/HCO 3 exchange operating at alkaline pH i (pH o 8.2).The excellent technical assistance of Marianne Schiødt and Birgit B. Jørgensen is gratefully acknowledged. The work was supported by the Carlsberg Foundation (B.K.) and by a grant from the Danish Natural Science Foundation (E.K.H. and L.O.S.).  相似文献   

4.
The pH-sensitivity of transepithelial K+ transport was studied in vitro in isolated vestibular dark cell epithelium from the gerbil ampulla. The cytosolic pH (pH iwas measured microfluorometrically with the pH-sensitive dye 2,7-bicarboxyethyl-5(6)-carboxyfluorescein (BCECF) and the equivalent short-circuit current (I sc), which is a measure for transepithelial K+ secretion, was calculated from measurements of the transepithelial voltage (V t)and the transepithelial resistance (R t) in a micro-Ussing chamber. All experiments were conducted in virtually HCO 3 -free solutions. Under control conditions, pH iwas 7.01±0.04 (n=18), V twas 9.1±0.5 mV, R t16.7±0.09 cm2, and I sc was 587±30 A/cm2 (n=49). Addition of 20 mm propionate caused a biphasic effect involving an initial acidification of pH i, increase in V tand I sc and decrease in R tand a subsequent alkalinization of pH i, decrease of V tand increase of R t. Removal of propionate caused a transient effect involving an alkalinization of pH i, a decrease of V tand I sc and an increase in R t. pH iin the presence of propionate exceeded pH iunder control conditions. Effects of propionate on V t, R tand I sc were significantly larger when propionate was applied to the basolateral side rather than to the apical side of the epithelium. The pH i-sensitivityof I sc between pH 6.8 and 7.5 was –1089 A/(cm2 · pH-unit) suggesting that K+ secretion ceases at about pH i7.6. Acidification of the extracellular pH (pH o)caused an increase of V tand I sc and a decrease of R tmost likely due to acidification of pH i. Effects were significantly larger when the extracellular acidification was applied to the basolateral side rather than to the apical side of the epithelium. The pH osensitivity of I sc between pH 7.4 and 6.4 was –155 A/(cm2 · pH unit). These results demonstrate that transepithelial K+ transport is sensitive to pH iand pH oand that vestibular dark cells contain propionate uptake mechanism. Further, the data suggest that cytosolic acidification activates and that cytosolic alkalinization inactivates the slowly activating K+ channel (I sK)in the apical membrane. Whether the effect of pH ion the I sK channel is a direct or indirect effect remains to be determined.The authors wish to thank Drs. Daniel C. Marcus, Zhjiun Shen and Hiroshi Sunose for helpful discussions. This work was supported by grants NIH-R29-DC01098 and NIH-R01-DC00212.  相似文献   

5.
Summary The present study was designed to investigate the apical and basolateral transport processes responsible for intracellular pH regulation in the thin descending limb of Henle. Rabbit thin descending limbs of long-loop nephrons were perfused in vitro and intracellular pH (pH i ) was measured using BCECF. Steady-state pH i in HEPES buffered solutions (pH 7.4) was 7.18±0.03. Following the removal of luminal Na+, pH i decreased at a rate of 1.96±0.37 pH/min. In the presence of luminal amiloride (1mm), the rate of decrease of pH i was significantly less, 0.73±0.18 pH/min. Steady-state pH i decreased 0.18 pH units following the addition of amiloride (1mm) to the lumen (Na+ 140mm lumen and bath). When Na+ was removed from the basolateral side of the tubule, pH i decreased at a rate of 0.49±0.05 pH/min. The rate of decrease of pH i was significantly less in the presence of 1mm basolateral amiloride, 0.29±0.04 pH/min. Addition of 1mm amiloride to the basolateral side (Na+ 140mm lumen and bath) caused steady-state pH i to decrease significantly by 0.06 pH units. When pH i was acutely decreased to 5.87±0.02 following NH4Cl removal (lumen, bath), pH i failed to recover in the absence of Na+ (lumen, bath). Addition of 140mm Na+ to the lumen caused pH i to recover at a rate of 2.17±0.59 pH/min. The rate of pH i recovery was inhibited 93% by 1mm luminal amiloride. When 140mm Na+ was added to the basolateral side, pH i recovered only partially at 0.38±0.07 pH/min. Addition of 1mm basolateral amiloride inhibited the recovery of pH i , by 97%. The results demonstrate that the rabbit thin descending limb of long-loop nephrons possesses apical and basolateral Na+/N+ antiporters. In the steady state, the rate of Na+-dependent H+ flux across the apical antiporter exceeds the rate of Na+-dependent H+ flux via the basolateral antiporter. Recovery of pH i following acute intracellular acidification is Na+ dependent and mediated primarily by the luminal antiporter.  相似文献   

6.
Internal pH (pHi) was determined inEmiliania huxleyi (Lohmann) using the probe 2,7-bis-(2-carboxyethyl)-5(and-6)carboxyfluoresceinacetoxymethylester (BCEF-AM) and digital imaging microscopy. The probe BCECF-AM was taken up and hydrolysed to the free acid by the cells. A linear relationship was established between pHi and the 490/450 fluorescence ratio of BCECF-AM over the pH range 6.0 to 8.0 using the ionophore nigericin. Two distinct pH domains were identified within the cell, the cytoplasmic domain (approx. pH 7.0) and the chloroplast domain (approx. pH 8.0). The average pHi was 7.29 (±0.11) for cells in the presence of 2 mM HCO 3 . In the absence of HCO 3 the pHi was decreased by 0.8 pH unit. The importance of these changes in pHi is considered in relation to inorganic-carbon uptake.Abbreviations AM acetoxymethylester - BCECF 2,7-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - pHi intracellular pH  相似文献   

7.
Summary We have chosen the MDCK cell line to investigate aldosterone action on H+ transport and its role in regulating cell membrane K+ conductance (G m K ). Cells grown in a monolayer respond to aldosterone indicated by the dose-dependent formation of domes and by the alkalinization of the dome fluid. The pH sensitivity of the plasma membrane K+ channels was tested in giant cells fused from individual MDCK cells. Cytoplasmic pH (pH i ) andG m K were measured simultaneously while the cell interior was acidified gradually by an extracellular acid load. We found a steep signoidal relationship between pH i andG m K (Hill coefficient 4.4±0.4), indicating multiple H+ binding sites at a single K+ channel. Application of aldosterone increased pH i within 120 min from 7.22±0.04 to 7.45±0.02 and from 7.15±0.03 to 7.28±0.02 in the absence and presence of the CO2/HCO 3 buffer system, respectively. We conclude that the hormone-induced cytoplasmic alkalinization in the presence of CO2/ HCO 3 is limited by the increased activity of a pH i -regulating HCO 3 extrusion system. SinceG m K is stimulated half-maximally at the pH i of 7.18±0.04, internal H+ ions could serve as an effective intracellular signal for the regulation of transepithelial K+ flux.  相似文献   

8.
Summary The role of transmembrane pH gradients on the ouabain, bumetanide and phloretin-resistant Na+ transport was studied in human red cells. Proton equilibration through the Jacobs-Stewart cycle was inhibited by the use of DIDS (125 m) and methazolamide (400 m). Red cells with different internal pH (pH i =6.4, 7.0 and 7.8) were prepared and Na+ influx was measured at different external pH (pH o =6.0, 7.0, 8.0). Na+ influx into acid-loaded cells (pH i =6.4) markedly increased when pH o was raised from 6.0 to 8.0. Amiloride, a well-known inhibitor of Na+/H+ exchange systems blocked about 60% of the H+-induced Na+ entry, while showing small inhibitory effects in the absence of pH gradients. When pH0 was kept at 8.0, the amiloride-sensitive Na+ entry was abolished as pH i was increased from 6.4 to 7.8. Moreover, measurements of H+ efflux into lightly buffered media indicated that the imposition of an inward Na+ gradient stimulated a net H+ efflux which was sensitive to the amiloride analog 5-N-methyl-N-butyl-amiloride. Furthermore, in the absence of a chemical gradient for Na+ (Na i + =Na 0 + =15mm,Em=+6.7 mV), an outward H+ gradient (pH i =6.4, pH0=8.0) promoted a net amiloride-sensitive Na+ uptake which was abolished at an external pH of 6.0. These findings are consistent with the presence of an amiloride-sensitive Na+/H+ exchange system in human red cells.  相似文献   

9.
Internal pH (pH i ) is in Vero cells regulated mainly by three antiports. Na+/H+ antiport and Na+-dependent Cl+/HCO 3 + antiport increase pH i in acidified cells, and Na+-independent Cl+/HCO 3 + antiport lowers pH i in cells after alkalinization. The activities of the antiporters were altered in cells after exposure to 41–45°C. Under such conditions the Na+/H+ antiport and the Na+-dependent Cl+/HCO 3 + antiport were both stimulated, whereas the Na+-independent Cl+/HCO 3 + antiport was inhibited in such a way that a higher pH value was required to activate it. This alteration was also induced by some other forms of cellular stress, but did most likely not involve stress proteins as protein synthesis was not required. The possibility of regulation by alteration in protein phosphorylation is discussed.We are grateful to Mrs. Jorunn Jacobsen for her skillful handling of the cell cultures. This work was supported by the Norwegian Research Council for Science and Humanities, the Norwegian Cancer Society and the Lærdal Foundation.  相似文献   

10.
Summary The activity of the main base-extruding mechanism in Vero cells, the Na+-independent Cl/HCO 3 antiport, increases 5- to 10-fold when the cytosolic pH (pH i ) is increased over a narrow range close to neutrality. We have studied the effect on this regulation of stimulation and inhibition of protein kinase C by short-term and long-term treatment with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). After short-term treatment with TPA to stimulate the kinase, the threshold value for activation of the antiport is shifted to a more acidic pH. After prolonged treatment with TPA to downregulate protein kinase C the sensitivity of the antiport to variation in proton concentration was lowered, possibly by reducing the number of essential protonbinding sites. Concomitantly, the steady state pH i of the cells was increased. The data indicate that protein kinase C is involved in the regulation of the Na+-independent Cl/HCO 3 antiport.  相似文献   

11.
Summary We have investigated the kinetic properties of the human red blood cell Na+/H+ exchanger to provide a tool to study the role of genetic, hormonal and environmental factors in its expression as well as its functional properties in several clinical conditions. The present study reports its stoichiometry and the kinetic effects of internal H+ (H i ) and external Na+ (Na o ) in red blood cells of normal subjects.Red blood cells with different cell Na+ (Na i ) and pH (pH i ) were prepared by nystatin and DIDS treatment of acid-loaded cells. Unidirectional and net Na+ influx were measured by varying pH i (from 5.7 to 7.4), external pH (pH o ), Na i and Na o and by incubating the cells in media containing ouabain, bumetanide and methazolamide. Net Na+ influx (Na i <2.0 mmol/liter cell, Na o = 150mm) increased sigmoidally (Hill coefficient 2.5) when pH i fell below 7.0 and the external pH o was 8.0, but increased linearly at pH o 6.0. The net Na+ influx driven by an outward H+ gradient was estimated from the difference of Na+ influx at the two pH o levels (pH o 8 and pH o 6). The H+-driven Na+ influx reached saturation between pH i 5.9 and 6.1. TheV max had a wide interindividual variation (6 to 63 mmol/liter cell · hr, 31.0±3, mean±sem,n=20). TheK m for H i to activate H+-driven Na+ influx was 347±30nm (n=7). Amiloride (1mm) or DMA (20 m) partially (59±10%) inhibited red cell Na+/H+ exchange. The stoichiometric ratio between H+-driven Na+ influx and Na+-driven H+ efflux was 11. The dependence of Na+ influx from Na o was studied at pH i 6.0, and Na i lower than 2 mmol/liter cell at pH o 6.0 and 8.0. The meanK m for Na o of the H+-gradient-driven Na+ influx was 55±7mm.An increase in Na i from 2 to 20 mmol/liter cell did not change significantly H+-driven net Na+ influx as estimated from the difference between unidirectional22Na influx and efflux. Na+/Na+ exchange was negligible in acid-loaded, DIDS-treated cells. Na+ and H+ efflux from acid-loaded cells were inhibited by amiloride analogs in the absence of external Na+ indicating that they may represent nonspecific effects of these compounds and/or uncoupled transport modes of the Na+/H+ exchanger.It is concluded that human red cell Na+/H+ exchange performs 11 exchange of external Na+ for internal protons, which is partially amiloride sensitive. Its kinetic dependence from internal H+ and external Na+ is similar to other cells, but it displays a larger variability in theV max between individuals.  相似文献   

12.
The influence of cytosolic pH (pHi) in controlling K+-channel activity and its interaction with cytosolic-free Ca2+ concentration ([Ca2+]i) was examined in stomatal guard cells ofVicia faba L. Intact guard cells were impaled with multibarrelled microelectrodes and K+-channel currents were recorded under voltage clamp while pHi or [Ca2+]i was monitored concurrently by fluorescence ratio photometry using the fluorescent dyes 2,7-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Fura-2. In 10 mM external K+ concentration, current through inward-rectifying K+ channels (IK,in) was evoked on stepping the membrane from a holding potential of –100 mV to voltages from –120 to –250 mV. Challenge with 0.3-30 mM Na+-butyrate and Na+-acetate outside imposed acid loads, lowering pHi from a mean resting value of 7.64 ± 0.03 (n = 25) to values from 7.5 to 6.7. The effect on pHi was independent of the weak acid used, and indicated a H+-buffering capacity which rose from 90 mM H+/pH unit near 7.5 to 160 mM H+/pH unit near pHi 7.0. With acid-going pHi, (IK,in) was promoted in scalar fashion, the current increasing in magnitude with the acid load, but without significant effect on the current relaxation kinetics at voltages negative of –150 mV or the voltage-dependence for channel gating. Washout of the weak acid was followed by transient rise in pHi lasting 3–5 min and was accompanied by a reduction in (IK,in) before recovery of the initial resting pHi and current amplitude. The pHi-sensitivity of the current was consistent with a single, titratable site for H+ binding with a pKa near 6.3. Acid pHi loads also affected current through the outward-rectifying K+ channels (IK,out) in a manner antiparallel to (IK,in) The effect on IK, out was also scalar, but showed an apparent pKa of 7.4 and was best accommodated by a cooperative binding of two H+. Parallel measurements showed that Na+-butyrate loads were generally without significant effect on [Ca2+]i, except when pHi was reduced to 7.0 and below. Extreme acid loads evoked reversible increases in [Ca2+]i in roughly half the cells measured, although the effect was generally delayed with respect to the time course of pHi changes and K+-channel responses. The action on [Ca2+]i coincided with a greater variability in (IK,in) stimulation evident at pHi values around 7.0 and below, and with negative displacements in the voltage-dependence of (IK,in) gating. These results distinguish the actions of pHi and [Ca2+]i in modulating (IK,in) they delimit the effect of pHi to changes in current amplitude without influence on the voltage-dependence of channel gating; and they support a role for pHi as a second messenger capable of acting in parallel with, but independent of [Ca2+]i in controlling the K+ channels.Abbreviations BCECF 2,7-bis (2-carboxyethyl)-5(6)-carboxy fluorescein - [Ca2+]i cytosolic free Ca2+ concentration - gK ensemble (steady-state) K+-channel conductance - IK,out, IK,in outward-, inward-rectifying K+ channel (current) - IN current-voltage (relation) - Mes 2-(N-morpholinolethanesulfonic acid - pHi cytosolic pH - V membrane potential  相似文献   

13.
Summary Changes in extracellular pH (pH o ) in human red cell suspensions were monitored in a stopped-flow rapid reaction apparatus. A 20% suspension of washed human RBC in saline at pH 7 containing NaHCO3 and extracellular carbonic anhydrase was mixed with an equal volume of buffered saline solution at pH 6.7. Sodium salicylate, when present, was added to both the erythrocyte suspension and the buffer solution. The effects of salicylate in the therapeutic to toxic concentration range on HCO 3 /Cl exchange were studied at 37°C. HCO 3 /Cl exchange flux was estimated using the extracellular buffer capacity and the difference betweendpH o /dt using a control RBC suspension and that using a suspension of RBC whose anion exchange pathway was markedly inhibited. The results show that salicylate competitively decreases the rate of HCO 3 /Cl exchange, with inhibition increasing as salicylate concentration increases.K I is 2.4mm. At a salicylate concentration of 10mm, HCO 3 /Cl exchange under the conditions of our experiments was inhibited by more than 70%. These findings are consistent with the possibility that CO2 transfer in capillary bedsin vivo may be diminished in the presence of salicylate due to slowing of red cell HCO 3 /Cl exchange.  相似文献   

14.
Summary Elodea canadensis grows over a wide range of inorganic carbon, nutrient, and light conditions in lakes and streams. Affinity for HCO 3 - use during photosynthesis ranged from strong to weak in Elodea collected from seven localities with different HCO 3 - and CO2 concentrations. The response to HCO 3 - was also very plastic in plants grown in the laboratory at high HCO 3 - concentrations and CO2 concentrations varying from 14.8 to 2,200 M. Bicarbonate affinity was markedly reduced with increasing CO2 concentrations in the growth medium so that ultimately HCO 3 - use was not detectable. High CO2 concentrations also decreased CO2 affinity and induced high CO2 compensation points (360M CO2) and tenfold higher half-saturation values (800 M CO2).The variable HCO 3 - affinity is probably environmentally based. Elodea is a recently introduced species in Denmark, where it reproduces only vegetatively, leaving little opportunity for genetic variation. More important, local populations in the same water system had different HCO 3 - affinities, and a similar variation was created by exposing one plant collection to different laboratory conditions.Bicarbonate use enabled Elodea to photosynthesize rapidly in waters of high alkalinity and enhanced the carbon-extracting capacity by maintaining photosynthesis above pH 10. On the other hand, use of HCO 3 - represents an investment in transport apparatus and energy which is probably not profitable when CO2 is high and HCO 3 - is low. This explanation is supported by the findings that HCO 3 - affinity was low in field populations where HCO 3 - was low (0.5 and 0.9 m M) or CO2 was locally high, and that HCO 3 - affinity was suppressed in the laboratory by high CO2 concentrations.Abbreviations DIC dissolved inorganic carbon (CO2+ HCO 3 - +CO 3 - ) - CO2 compensation point - K 1/2 apparent halfsaturation constant - PHCO 3 interpolated photosynthesis in pure HCO 3 - and zero CO2 - Pmax photosynthetic rate under carbon and light saturation  相似文献   

15.
The functional significance of the apical vacuolar-type proton pump (V-ATPase) in Drosophila Malpighian tubules was studied by measuring the intracellular pH (pHi) and luminal pH (pHlu) with double-barrelled pH-microelectrodes in proximal segments of the larval anterior tubule immersed in nominally bicarbonate-free solutions (pHo 6.9). In proximal segments both pHi (7.43±0.20) and pHlu (7.10±0.24) were significantly lower than in distal segments (pHi 7.70±0.29, pHlu 8.09±0.15). Steady-state pHi of proximal segments was much less sensitive to changes in pHo than pH of the luminal fluid (pHlu/pHo was 0.49 while pHi/pHo was 0.18; pHo 6.50–7.20). Re-alkaliniziation from an NH4Cl-induced intracellular acid load (initial pHi recovery rate 0.55±0.34 pH·min-1) was nearly totally inhibited by 1 mmol·l-1 KCN (96% inhibition) and to a large degree (79%) by 1 mol·l-1 bafilomycin A1. In contrast, both vanadate (1 mmol·l-1) and amiloride (1 mmol·l-1) inhibited pHi recovery by 38% and 33%, respectively. Unlike amiloride, removal of Na+ from the bathing saline had no effect on pHi recovery, indicating that a Na+/H+ exchange is not significantly involved in pHi regulation. Instead pHi regulation apparently depended largely on the availability of ATP and on the activity of the bafilomycin-sensitive proton pump.Abbreviations DMSO dimethylsulphoxide - DNP 2,4-dinitrophenol - NMDG N-methyl-D-glucamine - pHi intracellular pH - pHlu pH of the luminal fluid - pHo pH of the superfusion medium - I intrinsic intracellular buffer capacity  相似文献   

16.
The effects of D-glucose, D-glyceraldehyde, glibenclamide, D-600, NH 4 + and high concentrations of K+ on cytoplasmic pH (pH i ) were investigated in dispersed and cultured pancreatic -cells fromob/ob mice. The cytoplasmic pH was measured with the fluorescent H+-indicator quene 1. The average pH i value in resting -cells was 6.71. Addition of 20 mM of the physiological stimulus D-glucose increased pH i with 0.05 units. Both glibenclamide and high concentrations of K+ decreased pH i . The latter effects were completely reversed by D-600, supporting the notion that free cytoplasmic Ca2+ can be involved in the regulation of pH i . In contrast to D-glucose, 10mM of D-glyceraldehyde decreased pH i by 0.09 units, an effect persisting even in the presence of D-600. From the present study it is evident that D-glyceraldehyde and D-glucose have opposite effects on pH i in pancreatic -cells.  相似文献   

17.
Vascular smooth muscle intracellular pH is maintained by the Na+/H+ and Cl/HCO 3 antiporters. The Na+/H+ exchanger is a major route of H+ extrusion in most eukaryotic cells and is present in vascular smooth muscle cells in a similar capacity. It extrudes H into the extracellular space in exchange for Na+. The Cl/HCO 3 exchanger plays an analogous role to lower the pH of vascular smooth muscle cells when increases in intracellular pH occur. Its activity has also been demonstrated in A7r5 and A10 vascular smooth muscle cells. The Na+/H+ exchanger is regulated by a number of agents which act through inositol trisphosphate/diacylglycerol, to stimulate the antiporter. Calcium-calmodulin dependent protein kinase may also activate the antiporter in vivo. Phosphorylation of the Cl/HCO 3 exchanger has also been observed but its physiological role is not known. Both these antiporters exist in the plasma membrane as integral proteins with free acidic cytoplasmic termini. These regions may be important in sensing changes in intracellular pH, to which these antiporters respond.Abbreviations CaM Calmodulin - DCCD Dicylohexyl-Carbodiimide - DG Diacylglycerol - DIDS-4 4-Diisthiocyanostilbene-2,2-Disulfonic Acid - IP3 Inositol Trisphosphate - PKC protein Kinase C - SITS-4 4-Acetamido-4-Isothiocyanstilbene-2,2-Disulfonate - VSMC Vascular Smooth Muscle Cell  相似文献   

18.
Summary LLC-PK1 cells (a continuous epithelioid cell line with renal characteristics) are examined by microspectrofluorometry as single cells, in order to determine the mechanism of intracellular pH (pH i ) recovery from an acid load imposed by ammonium preincubation and removal (NH4 prepulse). Initial experiments evaluate the intracellular K+ levels through a null point analysis of total cellular K+ with flame photometry. The response of BCECF (a pH-sensitive fluorescent dye) is then calibrated, using saturating concentrations of nigericin to cause defined changes in pH i . For experiments with the microspectrofluorometer, LLC-PK1 cells were grown on either glass coverslips or filters (the latter attached to plastic coverslips with a hole under the filter). The cells on glass coverslips demonstrate a Na+-dependent recovery from an (NH4 prepulse) acid load which is sensitive to 1 M ethylisopropylamiloride. They also demonstrate a set point of activation of Na+/H+ exchange. When examined for changes in pH i due to changes in membrane potential, plasma membrane proton conductance could not be detected at resting pH i . Cells grown on filters also demonstrate a pH i recovery from an acid load which is Na+ dependent and ethylisopropylamiloride sensitive, but in this configuration, the majority of cells (22/23 preparations) require Na+ at the basolateral membrane for rapid pH i recovery. The morphology and polarity of the cells grown on permeable supports appears normal at the electron-microscopic level. The results are not affected by changes in cell seeding density or collagen treatment of the filters.  相似文献   

19.
Others have shown that exposing oocytes to high levels of (10–20 mM) causes a paradoxical fall in intracellular pH (pHi), whereas low levels (e.g., 0.5 mM) cause little pHi change. Here we monitored pHi and extracellular surface pH (pHS) while exposing oocytes to 5 or 0.5 mM NH3/NH4 +. We confirm that 5 mM causes a paradoxical pHi fall (−ΔpHi ≅ 0.2), but also observe an abrupt pHS fall (−ΔpHS ≅ 0.2)—indicative of NH3 influx—followed by a slow decay. Reducing [NH3/NH4 +] to 0.5 mM minimizes pHi changes but maintains pHS changes at a reduced magnitude. Expressing AmtB (bacterial Rh homologue) exaggerates −ΔpHS at both levels. During removal of 0.5 or 5 mM NH3/NH4 +, failure of pHS to markedly overshoot bulk extracellular pH implies little NH3 efflux and, thus, little free cytosolic NH3/NH4 +. A new analysis of the effects of NH3 vs. NH4 + fluxes on pHS and pHi indicates that (a) NH3 rather than NH4 + fluxes dominate pHi and pHS changes and (b) oocytes dispose of most incoming NH3. NMR studies of oocytes exposed to 15N-labeled show no significant formation of glutamine but substantial accumulation in what is likely an acid intracellular compartment. In conclusion, parallel measurements of pHi and pHS demonstrate that NH3 flows across the plasma membrane and provide new insights into how a protein molecule in the plasma membrane—AmtB—enhances the flux of a gas across a biological membrane.
Walter F. Boron (Corresponding author)Email:
  相似文献   

20.
Summary Cellular potential and pH measurements (pH i ) were carried out in the perfused kidney ofNecturus on proximal tubules with standard and recessed-tip glass microelectrodes under control conditions and after stimulation of tubular bicarbonate reabsorption. Luminal pH and net bicarbonate reabsorption were measured in parallel experiments with recessed-tip glass or antimony electrodes, both during stationary microperfusions as well as under conditions of isosmotic fluid transport. A mean cell pH of 7.15 was obtained in control conditions. When the luminal bicarbonate concentration was raised to 25 and 50mm, pH i rose to 7.44 and 7.56, respectively. These changes in pH i were fully reversible. Under all conditions intracellular H+ was below electrochemical equilibrium. Thus the maintenance of intracellular pH requires active H+ extrusion across one or both of the cell membranes. The observed rise in pH i and the peritubular depolarization after stimulation of bicarbonate reabsorption are consistent with enhanced luminal hydrogen ion secretion and augmentation of peritubular bicarbonate exit via an anion-conductive transport pathway.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号