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1.
The effects of jasmonic acid (JA) on elongation growth of coleoptile segments from etiolated maize (Zea mays L.) were investigated in the presence and absence of auxin. When supplied alone, at physiological concentrations (10−9, 10−8, and 10−5 m), JA (or methyl-JA) inhibited growth. JA at a similar range of concentrations also inhibited auxin-induced elongation growth. To determine whether this effect on growth depended on endogenous abscisic acid (ABA), we grew maize coleoptiles in the presence of norflurazon (an inhibitor of carotenoid biosynthesis) that results in reduced endogenous ABA levels. Growth of etiolated coleoptile segments from these plants was inhibited by JA (or methyl-JA) in both the absence and presence of auxin. Previously, we have observed a correlation between elongation growth and cytosolic pH (pHi), in which auxin lowers pHi, and growth inhibitors such as ABA raise pHi. We examined the effect of low concentrations of methyl-JA on pHi with dual emission dye, carboxy seminaphthorhodafluor-1, and confocal microscopy. To confirm these studies, we also used in vivo 31P NMR spectrometry to ascertain the changes in pHi after addition of jasmonate to maize coleoptiles. Coleoptiles grown in either the absence or presence of norflurazon responded to methyl-JA or JA by increases in pHi of approximately 0.2 pH unit. This response occurs over a period of 15–20 min and appears to be independent of endogenous ABA. This alkalization induced by JA is likely to form a permissive environment for JA signal transduction pathway(s). Received February 5, 1999; accepted August 25, 1999  相似文献   

2.
A. Brüdern  G. Thiel 《Protoplasma》1999,209(3-4):246-255
Summary Protoplasts are frequently isolated from maize coleoptiles with cell-wall-degrading enzymes such as pectolyase (PEC), mazerozyme, and cellulase. Incubation of coleoptiles with these enzymes caused rapid depolarizations of the membrane voltage (V M ). The depolarizing effect of 0.5% (w/v) mazerozyme or 1.5% (w/v) cellulase was unaffected by denaturation of the enzymes. In the case of pectolyase (0.1%, w/v), however, the active enzyme was significantly more potent than the denaturated enzyme in depolarizing coleoptile cells. Exposure to 0.1% active PEC but not to inactive PEC also caused an oxidative burst in coleoptiles and enhanced K+ efflux. Together this suggests that pectic breakdown products of the cell wall act as signal for wounding. Typically addition of 10 M 1-naphthylene acetic acid (NAA) to coleoptiles causes a transient depolarization followed by a slow hyperpolarization of V M . However, in the presence of PEC, V M only depolarized in NAA. After PEC-treated coleoptiles were washed free of the enzyme, NAA caused only small fluctuations of V M . A similarly small V M response to NAA appeared in coleoptiles pretreated with heatdenaturated supernatant (SUP) from a protoplast isolation buffer, the latter suspected to contain the PEC-generated wounding signal. Comparable pretreatment of coleoptiles with PEC or SUP had no significant effect on the spontaneous and NAA-evoked acidification of the incubation medium. Pretreatment with SUP also had no significant effect on the NAA-stimulated elongation of coleoptile segment. Hence, PEC treatment of coleoptile tissue affects the membrane transport properties of the cells. This effect is partly maintained after removal of the enzyme from the incubation medium, an effect not significant for NAA-generated acidification and cell elongation.Abbreviations V M membrane voltage - Vred redox voltage - PEC pectolyase - SUP supernatant from cell wall digestion - NAA 1-naphthylene acetic acid  相似文献   

3.
A role for cytosolic pH (pHi) in hormonal signalling and transport control in plants has long been mooted. Yet, while changes in pHi are a common consequence of hormonal stimuli in plant cells and contribute to hormonally evoked ion channel control, the origins of these changes remain unknown. To examine a possible role for the tonoplast and vacuolar compartment in these events, pHi was measured in the presence of auxins and during cytosolic H+ loading with weak acid in vacuolate and evacuolate protoplasts, both from mesophyll and guard cells of Vicia faba L. Evacuolate protoplasts were obtained following ultracentrifugation on Percoll gradients, and pHi of single protoplasts was recorded in both vacuolate and evacuolate preparations using fluorescence ratio microphotometry and the pH-sensitive dye BCECF. External pH measurements indicated a roughly twofold increase in the rate of net H+ secretion in evacuolate compared with vacuolate protoplasts, and showed that evacuolate protoplasts retained the characteristic stimulation of H+ secretion in the presence of auxin. BCECF fluorescence recording gave resting pHi values near 7.5, and evacuolation had no significant effect on this parameter. Reversible decreases of 0.1–0.2 units in pHi were evoked in vacuolate protoplasts by 10 μM concentrations of the auxins 1-naphthalene acetic acid and 3-indoyl-acetic acid, and not by the inactive (anti-auxin) analogue 2-naphthalene-acetic acid. However, auxin treatments failed to evoke a change in pHi in all but one of 12 experiments with evacuolate protoplasts. Evacuolation also appeared to reduce the transient, dynamic H+ buffering capacity of the protoplasts in the face of acid pHi loads imposed by adding Na+-butyrate to the bath. These results implicate the tonoplast or vacuolar compartment in short-term pHi homeostasis and generation of hormonally evoked H+ signalling in plant cells; they also conform with the view that the decrease in pHiper se is not a primary determinant in the stimulation of H+ secretion by auxin.  相似文献   

4.
Intraerythrocytic malaria parasites produce vast amounts of lactic acid through glycolysis. While the egress of lactate is very rapid, the mode of extrusion of H+ is not known. The possible involvement of a Na+/H+ antiport in the extrusion of protons across the plasma membrane of Plasmodium falciparum has been investigated by using the fluorescent pH probe 6-carboxyfluorescein. The resting cytosolic pH was 7.27 ± 0.1 in ring stage parasites and 7.31 ± 0.12 in trophozoites. Spontaneous acidification of parasite cytosol was observed in Na+-medium and realkalinization occurred upon addition of Na+ to the medium in a concentration-dependent manner, with no apparent saturation. The rate of H+-at the ring stage was higher than that at the trophozoite stage due to the larger surface/volume ratio of the young parasite stage. Na+-H+-was: 1) inhibited by the Na+/H+ inhibitors amiloride and 5-(N-ethyl-isopropyl) amiloride (EIPA), though at relatively high concentrations; 2) augmented with rising pH6 (pHi = 6.2 [Na+]o = 30 mM); and 3) decreased with increasing pHi (pHo = 7.4; [Na+]o = 30 mM). The pHi and the pHo dependencies of H+-were almost identical at all parasite stages. Only at pHi > 7.6 efflux was totally obliterated. The target of this inhibitory effect is probably other than the antiport. Results indicate that H+-is mediated by a Na+/H+ antiport which is regulated by host and parasite pH and by the host cytosol sodium concentration. The proton transport capacity of the antiport can easily cope with all the protons of lactic acid produced by parasite's glycolysis. © 1993 Wiley-Liss, Inc.  相似文献   

5.
Summary The intracellular pH (pH i ) of Ehrlich ascites tumor cells, both in the steady state and under conditions of acid loading or recovery from acid loading, was investigated by measuring the transmembrane flux of H+ equivalents and correlating this with changes in the distribution ratio of dimethyloxazolidine-2,4-dione (DMO). The pH i of cells placed in an acidic medium (pH o below 7.15) decreases and reaches a steady-state value that is more alkaline than the outside. For example when pH o is acutely reduced to 5.5, pH i falls exponentially from 7.20 ± 0.06 to 6.29 ± 0.04 with a halftime of 5.92 ± 1.37 min, suggesting a rapid influx of H+. The unidirectional influx of H+ exhibits saturation kinetics with respect to extracellular [H+]; the maximal flux is 15.8 ± 0.05 mmol/(kg dry wt · min) andK m is 0.74 ± 0.09 × 10–6 m.Steady-state cells with pH i above 6.8 continuously extrude H+ by a process that is not dependent on ATP but is inhibited by anaerobiosis. Acid-loaded cells (pH i 6.3) when returned to pH o 7.3 medium respond by transporting H+, resulting in a rapid rise in pH i . The halftime for this process is 1.09 ± 0.22 min. The H+ efflux measured under similar conditions increases as the intracellular acid load increases. An ATP-independent as well as an ATP-dependent efflux contributes to the restoration of pH i to its steady-state value.  相似文献   

6.
Summary We have studied the kinetic properties of rabbit red cell (RRBC) Na+/Na+ and Na+/H+ exchanges (EXC) in order to define whether or not both transport functions are conducted by the same molecule. The strategy has been to determine the interactions of Na+ and H+ at the internal (i) and external (o) sites for both exchanges modes. RRBC containing varying Na i and H l were prepared by nystatin and DIDS treatment of acid-loaded cells. Na+/Na+ EXC was measured as Na o -stimulated Na+ efflux and Na+/H+ EXC as Na o -stimulated H+ efflux and pH o -stimulated Na+ influx into acid-loaded cells.The activation of Na+/Na+ EXC by Na o at pH i 7.4 did not follow simple hyperbolic kinetics. Testing of different kinetic models to obtain the best fit for the experimental data indicated the presence of high (K m 2.2 mM) and low affinity (K m 108 mM) sites for a single- or two-carrier system. The activation of Na+/H+ EXC by Na o (pH i 6.6, Na i <1 mM) also showed high (K m 11 mM) and low (K m 248 mM) affinity sites. External H+ competitively inhibited Na+/Na+ EXC at the low affinity Na o site (K H 52 nM) while internally H+ were competitive inhibitors (pK 6.7) at low Na i and allosteric activators (pK 7.0) at high Na i .Na+/H+ EXC was also inhibited by acid pH o and allosterically activated by H i (pK 6.4). We also established the presence of a Na i regulatory site which activates Na+/H+ and Na+/Na+ EXC modifying the affinity for Na o of both pathways. At low Na i , Na+/Na+ EXC was inhibited by acid pH i and Na+/H+ stimulated but at high Na i , Na+/Na+ EXC was stimulated and Na+/H+ inhibited being the sum of both pathways kept constant. Both exchange modes were activated by two classes of Na o sites,cis-inhibited by external H o , allosterically modified by the binding of H+ to a H i regulatory site and regulated by Na i . These findings are consistent with Na+/Na+ EXC being a mode of operation of the Na+/H+ exchanger.Na+/H+ EXC was partially inhibited (80–100%) by dimethyl-amiloride (DMA) but basal or pH i -stimulated Na+/Na+ EXC (pH i 6.5, Na i 80 mM) was completely insensitive indicating that Na+/Na+ EXC is an amiloride-insensitive component of Na+/H+ EXC. However, Na+ and H+ efflux into Na-free media were stimulated by cell acidification and also partially (10 to 40%) inhibited by DMA: this also indicates that the Na+/H+ EXC might operate in reverse or uncoupled modes in the absence of Na+/Na+ EXC.In summary, the observed kinetic properties can be explained by a model of Na+/H+ EXC with several conformational states, H i and Na i regulatory sites and loaded/unloaded internal and external transport sites at which Na+ and H+ can compete. The occupancy of the H+ regulatory site induces a conformational change and the occupancy of the Na i regulatory site modulates the flow through both pathways so that it will conduct Na+/H+ and/or Na+/Na+ EXC depending on the ratio of internal Na+:H+.  相似文献   

7.
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   

8.
9.
The stimulation of dicotyledonous leaf growth by light depends on increased H+ efflux, to acidify and loosen the cell walls, and is enhanced by K+ uptake. The role of K+ is generally considered to be osmotic for turgor maintenance. In coleoptiles, auxin‐induced cell elongation and wall acidification depend on K+ uptake through tetraethylammonium (TEA)‐sensitive channels (Claussen et al., Planta 201, 227–234, 1997), and auxin stimulates the expression of inward‐rectifying K+ channels ( Philippar et al. 1999) . The role of K+ in growing, leaf mesophyll cells has been investigated in the present study by measuring the consequences of blocking K+ uptake on several growth‐related processes, including solute accumulation, apoplast acidification, and membrane polarization. The results show that light‐stimulated growth and wall acidification of young tobacco leaves is dependent on K+ uptake. Light‐stimulated growth is enhanced three‐fold over dark levels with increasing external K+, and this effect is blocked by the K+ channel blockers, TEA, Ba++ and Cs+. Incubation in 10 mm TEA reduced light‐stimulated growth and K+ uptake by 85%, and completely inhibited light‐stimulated wall acidification and membrane polarization. Although K+ uptake is significantly reduced in the presence of TEA, solute accumulation is increased. We suggest that the primary role of K+ in light‐stimulated leaf growth is to provide electrical counterbalance to H+ efflux, rather than to contribute to solute accumulation and turgor maintenance.  相似文献   

10.
Robert E. Cleland 《Planta》1976,128(3):201-206
Summary The fungal toxin fusicoccin (FC) induces both rapid cell elongation and H+-excretion in Avena coleoptiles. The rates for both responses are greater with FC than with optimal auxin, and in both cases the lag after addition of the hormone is less with FC. This provides additional support for the acid-growth theory. The FC responses resemble the auxin responses in that they are inhibited by a range of metabolic inhibitors, but the responses differ in three ways. First auxin, but not FC, requires continual protein synthesis for its action. The auxin-induced H+-excretion is inhibited by water stress or by low external pH, while the FC-induced H+-excretion is much less sensitive to either. It is concluded that auxin-induced and FC-induced H+-excretion may occur via different mechanisms.Abbreviations FC fusicoccin - DNP dinitrophenol - CCCP carbonylcyanide m-chlorophenylhydrazone - CHl cycloheximide - IAA indoleacetic acid  相似文献   

11.
Summary The present study describes a new perfusion technique—based on the use of a routine spectrofluorometer—which enables fluorometric evaluation of polarity, regulation and kinetics of Na+/H+ exchange at the level of an intact monolayer. Na+/ H+ exchange was evaluated in bicarbonate-free solutions in OK (opossum kidney) cells, a renal epithelial cell line. Na+/H+ exchange activity was measured by monitoring changes in intracellular pH (pH i ) after an acid load, using the pH-sensitive dye 27-bis (carboxyethyl) 5–6-carboxy-fluorescein (BCECF). Initial experiments indicated that OK cells grown on a permeable support had access to apical and basolateral perfusion media. They also demonstrate that OK cells express an apical pH i , recovery mechanism, which is Na+ dependent, ethylisopropylamiloride (EIPA) sensitive and regulated by PTH. Compared to resting conditions (pH i =7.68; pH o =7.4) where Na+/H+ exchange is not detectable, transport rate increased as pH i decreased. A positive cooperativity characterized the interaction of internal H+ with the exchanger, and suggests multiple H+ binding sites. In contrast, extracellular [Na+] increased transport with simple Michaelis-Menten kinetics. The apparent affinity of the exchanger for Na+ was 19mM at an intracellular pH of 7.1 and 60mM at an intracellular pH of 6.6. Inhibition of Na+/H+ exchange activity by EIPA was competitive with respect to extracellular [Na+] and theK i was 3.4 M. In conclusion, the technique used in the present study is well suited for determination of mechanisms involved in control of epithelial cell pH i and processes associated with their polarized expression and regulation.  相似文献   

12.
The effect of ANG II on pHi, [Ca2+]i and cell volume was investigated in T84 cells, a cell line originated from colon epithelium, using the probes BCECF-AM, Fluo 4-AM and acridine orange, respectively. The recovery rate of pHi via the Na+/H+ exchanger was examined in the first 2 min following the acidification of pHi with a NH4Cl pulse. In the control situation, the pHi recovery rate was 0.118 ± 0.001 (n = 52) pH units/min and ANG II (10−12 M or 10−9 M) increased this value (by 106% or 32%, respectively) but ANG II (10−7 M) decreased it to 47%. The control [Ca2+]i was 99 ± 4 (n = 45) nM and ANG II increased this value in a dose-dependent manner. The ANG II effects on cell volume were minor and late and should not interfere in the measurements of pHi recovery and [Ca2+]i. To document the signaling pathways in the hormonal effects we used: Staurosporine (a PKC inhibitor), W13 (a calcium-dependent calmodulin antagonist), H89 (a PKA inhibitor) or Econazole (an inhibitor of cytochrome P450 epoxygenase). Our results indicate that the biphasic effect of ANG II on Na+/H+ exchanger is a cAMP-independent mechanism and is the result of: 1) stimulation of the exchanger by PKC signaling pathway activation (at 10−12 – 10−7 M ANG II) and by increases of [Ca2+]i in the lower range (at 10−12 M ANG II) and 2) inhibition of the exchanger at high [Ca2+]i levels (at 10−9 – 10−7 M ANG II) through cytochrome P450 epoxygenase-dependent metabolites of the arachidonic acid signaling pathway.  相似文献   

13.
In vivo treatment of maize (Zea mays L.) coleoptile segments with auxin (indole-3-acetic acid; IAA) and fusicoccin (FC) followed by plasma-membrane isolation was used to characterize the effects of these treatments on the plasma-membrane H+-ATPase. Both IAA and FC increased H+ extrusion and elongation rate of the coleoptile segments, FC more strongly than IAA. Plasma membranes isolated after in-vivo treatment with FC showed a twofold stimulation of ATP hydrolysis and a several-fold stimulation of H+ pumping, whereas no effect was observed after IAA treatment, irrespective of whether the plasma membranes were prepared by two-phase partitioning or sucrose-gradient centrifugation. A more detailed investigation of the kinetic properties and pH dependence of the enzyme showed that FC treatment led to a twofold increase in V max, a decrease in K m for ATP from 1.5 mM to 0.24 mM, and a change in pH dependence resulting in increased activity at physiological pH levels. Again, IAA treatment showed no effects. Quantitation of the H+-ATPase by immunostaining using four different antibodies revealed no difference between IAA-and FC-treated material, and controls. From these data we conclude that (i) neither IAA nor FC gives rise to an increase in the amount of H+ -ATPase molecules in the plasma membrane that can be detected after membrane isolation, and (ii) if the H+-ATPase is activated by IAA, this activation is, in contrast to FC activation, not detectable after membrane isolation.Abbreviations BTP 1,3-bis(tris[hydroxymethyl]methylamino)-propane - FC fusicoccin - lyso-PC lysophosphatidylcholine - Mes 2-(N-morpholino)ethanesulfonic acid This paper is dedicated to Prof. Dieter Klämbt on the occasion of his 65th birthdayWe thank Ann-Christine Holmström and Adine Karlsson for excellent technical assistance, Professor Ramón Serrano (Instituto de Biologia Molecular y Celular de Plantas, UPV-CSIC, Universidad Politecnica, Valencia, Spain) for a generous gift of antisera to the H+-ATPase and Professor Wolfgang Michalke (Institut für Biologie III, Albert-Ludwigs-Universität, Freiburg, Germany) for kindly providing the monoclonal antibody to the H+-ATPase. This work was supported by the Swedish Natural Science Research Council, the Deutsche Agentur für Raumfahrtangelegenheiten (DARA, Bonn) via AGRAVIS (Bonn) and by the Ministerium für Wissenschaft und Forschung (MWF, Düsseldorf). Thomas Jahn received scholarships from the Deutsche Graduiertenförderung des Landes Nordrhein-Westfalen and the Deutscher Akademischer Austauschdienst (DAAD, Bonn).  相似文献   

14.
Summary Recently we proposed that cytoplasmic acidification of low K+ (LK) sheep erythrocytes may stimulate ouabain-resistant Cl-dependent K+ flux (K+Cl cotransport), also known to be activated by cell swelling, treatment with N-ethylmaleimide (NEM), or removal of cellular bivalent cations. Here we studied the dependence of K+ transport on intracellular and extracellular pH (pH i , pH o ) varied either simultaneously or independently using the Cl/HCO 3 exchange inhibitor 4,4, diisothiocyanatostilbene-3,2-disulfonic acid (DIDS). In both control and NEM-treated LK cells volumes were kept near normal by varying extracellular sucrose. Using DIDS as an effective pH clamp, both K+ efflux and influx of Rb+ used as K+ congener were strongly activated at acid pH i and alkaline pH o . A small stimulation of K+ (Rb+) flux was also seen at acid pH i in the absence of DIDS, i.e., when pH i pH o . Anti-L l serum, known to inhibit K+Cl cotransport, prevented the pH i -stimulated K+ (Rb+) fluxes. Subsequent to NEM treatment at pH 6, K+ (Rb+) fluxes were activated only by raising pH, and thus were similar to the pH activation profile of K+ (Rb+) fluxes in DIDS-treated cells with pH o varied at constant physiologic pH i . Anti-L l , which inhibited NEM-stimulated K+ (Rb+) fluxes, failed to do so in NEM-plus DIDS-treated cells. Thus, NEM treatment interferes with the internal but not with the external pH-sensitive site.  相似文献   

15.
We previously demonstrated that the progesterone‐ (P) initiated human sperm acrosome reaction (AR) was dependent on the presence of extracellular Na+ (Na+o). Moreover, Na+o depletion resulted in a decreased cytosolic pH (pHi), suggesting involvement of a Na+‐dependent pHi regulatory mechanism during the P‐initiated AR. We now report that the decreased pHi resulting from Na+o depletion is reversible and mediated by a Na+/H+ exchange (NHE) mechanism. To determine the role of an NHE in the regulation of pHi, capacitated spermatozoa were incubated in Na+‐deficient, bicarbonate/CO2‐buffered (0NaB) medium for 15–30 min, which resulted in an intracellular acidification as previously reported. These spermatozoa were then transferred to Na+‐containing, bicarbonate/CO2‐buffered (NaB) medium; Na+‐containing, Hepes‐buffered (NaH) medium; or maintained in the 0NaB medium. Included in the NaH medium was the NHE inhibitor 5‐(N‐ethyl‐N‐isopropyl) amiloride (EIPA). The steady‐state pHi was then determined by spectrofluorometric measurement of bis(carboxyethyl)‐5(6)‐carboxyfluoroscein (BCECF) fluorescence. EIPA (0.1 μM) significantly (P < 0.05) inhibited the pHi recovery produced by NaH medium. Moreover, the pHi in NaH medium was not significantly (P < 0.05) different than NaB medium. These results indicate that a Na+‐dependent, bicarbonate‐independent pHi regulatory mechanism, with a pharmacological characteristic consistent with an NHE, is present in capacitated spermatozoa. In support of the involvement of a sperm NHE, we also demonstrated specific immunoreactivity for a 100 kDa porcine sperm protein using an NHE‐1 specific monoclonal antibody. Interestingly, no significant (P = 0.79) effect was seen on the P‐initiated AR when EIPA was included in either the NaH or NaB medium. While these findings suggest that inhibition of NHE‐dependent pHi regulation in capacitated spermatozoa is not sufficient to block initiation of the AR by P, they do not preclude the possibility that an NHE mediates the regulation of capacitation or sperm motility. Mol. Reprod. Dev. 52:189–195, 1999. © 1999 Wiley‐Liss, Inc.  相似文献   

16.
Abstract: The mechanism of recovery from an acid load in primary cultures of rabbit choroid plexus epithelium (CPE) was examined, with emphasis on Na+-dependent antiports. Cells were incubated in saline solutions buffered to pH 7.38 with either HEPES or HCO3? plus 95% O2/5% CO2. Intracellular pH (pHi) was determined from the steady-state distribution of [14C]benzoate. Recovery after acidification with NH4Cl was rapid (t1/2= 5 min) and was dependent on external Na+ (EC50= 12 mM). Hexamethyleneamiloride and ethylisopropylamiloride, potent inhibitors of the Na+/H+ antiport, blocked 80% of recovery when [Na+] was 5 mM with IC50 values of 100 nM. However, neither drug blocked recovery in normal [Na+]. 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), an inhibitor of Cl?/HCO3? antiports, blocked recovery of pHi in a dose-related fashion in the presence of bicarbonate, but not in the presence of HEPES. No inhibition occurred with benzamil, an amiloride congener with high affinity for the Na+ channel, nor with dimethylbenzamil, an inhibitor of Na+/Ca2+ exchange. The carbonic anhydrase inhibitor acetazolamide also did not alter recovery from acidification. In CPE that had been pH-clamped with nigericin and KCl, the initial rate of 22Na+ uptake was very rapid (227 pmol/μg of DNA/min at pH 6.2), was dependent on external [Na+] with an EC50 value of 8 mM, and was inversely related to the pH of the medium. The maximal inhibition of 22Na+ uptake by hexamethyleneamiloride was 60% with an IC50 value of 76 nM. We conclude that both the Na+/H+ antiport and a DIDS-sensitive bicarbonate-dependent antiport are important mechanisms of regulation of the internal pH of rabbit CPE under acidifying conditions. Further, our data suggest that the rabbit choroid plexus Na+/H+ exchanger can be classified as amiloride insensitive, suggesting that this antiport may play a greater role in controlling transport mechanisms than does the pH of the CNS.  相似文献   

17.
A ouabain sensitive inward current occurs in Xenopus oocytes in Na+ and K+ -free solutions. Several laboratories have investigated the properties of this current and suggested that acidic extracellular pH (pHo) produces a conducting pathway through the Na+/K+ pump that is permeable to H+ and blocked by [Na+]o. An alternative suggestion is that the current is mediated by an electrogenic H+-ATPase. Here we investigate the effect of pHo and [Na+]o on both transient and steady-state ouabain-sensitive current. At alkaline or neutral pHo the relaxation rate of pre-steady-state current is an exponential function of voltage. Its U-shaped voltage dependence becomes apparent at acidic pHo, as predicted by a model in which protonation of the Na+/K+ pump reduces the energy barrier between the internal solution and the Na+ occluded state. The model also predicts that acidic pHo increases steady-state current leak through the pump. The apparent pK of the titratable group(s) is 6, suggesting that histidine is involved in induction of the conductance pathway. 22Na efflux experiments in squid giant axon and current measurements in oocytes at acidic pHo suggest that both Na+ and H+ are permeant. The acid-induced inward current is reduced by high [Na+]o, consistent with block by Na+. A least squares analysis predicts that H+ is four orders of magnitude more permeant than Na+, and that block occurs when 3 Na+ ions occupy a low affinity binding site (K 0.5=130±30 mM) with a dielectric coefficient of 0.23±0.03. These data support the conclusion that the ouabain-sensitive conducting pathway is a result of passive leak of both Na+ and H+ through the Na+/K+ pump.  相似文献   

18.
Effects of inorganic solutes on the binding of auxin   总被引:5,自引:5,他引:0       下载免费PDF全文
The binding of α-naphthaleneacetic acid (14C-NAA) to pelletable particulates from corn (Zea mays) coleoptiles was found to be influenced by inorganic solutes. La3+, Ca2+, and Mg2+ increased the binding whereas monovalent cations did not. The concentrations of CaCl2 which increased auxin binding were similar to those which inhibited coleoptile elongation in the presence of auxin. These results are interpreted as suggesting that the alteration of hormonal effectiveness by some inorganic solutes involves alterations in the attachment of the hormone to binding sites in the cell.  相似文献   

19.
Summary H+-coupled transport in plant and fungal cells is relatively insensitive to external pH (pH o ). H+-coupled Cl transport at the plasma membrane ofChara corallina was studied to explore the phenomena responsible for this insensitivity. Raising pH o from a control value of 7.5 to 9.0 results in a modest (2.5-fold) decline inJ max and increase inK m . Further increase in pH o results in a selective increase inJ max, in accordance with predictions from a reaction kinetic model of the transport system (Sanders, D., Hansen, U.-P., 1981.J. Membrane Biol. 58:139–153). Increase in cytosolic Cl concentration ([Cl] c ) also results in a selective decrease inJ max at pH o =7.5.Quantitative kinetic modeling of the results is not possible if it is assumed that the sole effect of pH o isvia mass action on the binding of external H+ to a transport site. If, instead, the dependence of cytosolic pH (pH c ) on pH o (Smith, F.A., 1984,J. Exp. Bot. 35:1525–1536) is taken into account along with the dependence of Cl influx on pH c (Sanders, D., 1980,J. Membrane Biol. 53:129–141), then the observed modest changes in Michaelis parameters can be accommodated by a reaction kinetic model. The quantitative parameters of the model yield respective pK a s of the internal and external H+-binding sites=7.85 and 7.2, respective dissociation constants of the internal and external Cl-binding sites=160 and 40 m, and an additional, kinetically transparent, H+-binding site with a pK a >8.0. The quantitative model independently predicts the response ofJ max andK m to acidic conditions.The results are discussed in terms of the general physiological requirement that fluxes through H+-coupled transport systems are relatively insensitive to environmental variation in pH o . It is proposed that (i) the weak (but finite) dependence of pH c on pH o , coupled with (ii) the strong dependence of H+-coupled transport on pH c are instrumental in endowing H+-coupled transport systems with a relative insensitivity to variation in pH o . This hypothesis might also explain why pH c in plants and fungi is not acutely controlled with respect to variation of pH o .  相似文献   

20.
Summary Passive proton permeability of gastrointestinal apical membrane vesicles was determined. The nature of the pathways for proton permeation was investigated using amiloride. The rate of proton permeation (k H + was determined by addition of vesicles (pH i = 6.5) to a pH 8.0 solution containing acridine orange. The rate of recovery of acridine orange fluorescence after quenching by the acidic vesicles ranged from 4 × 10–3 (gastric parietal cell stimulation-associated vesicles; SAV) and 5 × 10–3 (duodenal brush-border membrane vesicles; dBBMV) to 11 × 10+–3 sec–1 (ileal BBMV; iBBMV). Amiloride, 0.03 and 0.1 mm, significantly reduced the rate of proton permeation in dBBMV and iBBMV, but not gastric SAV. The decreases in k H + were proportionately greater in iBBMV as compared with dBBMV. The presence of Na+/H+ exchange was demonstrated in both dBBMV and iBBMV by proton-driven (pH i < pH o ) 22Na+ uptake. Evidence was also sought for the conductive nature of pathways for proton permeation. Intravesicular acidification, again determined by quenching of acridine orange fluorescence, was observed during imposition of K+-diffusion potential ([K+] i [K+ o ). In dBBMV and iBBMV, intravesicular acidification was enhanced in the presence of the K+-ionophore valinomycin, indicating that the native K+ permeability is rate limiting. In the presence of valinomycin, the K+-diffusion potential drove BBMV intravesicular acidification to levels close to the electrochemical potential. In gastric SAV, acidification was not limited by the K+ permeability. Valinomycin was without effect, but the K+/H+ ionophore nigericin enhanced acidification in gastric SAV, illustrating the low proton permeability of these membranes. Amiloride, 0.03–1 mm, resulted in concentration-dependent reductions of K+-diffusion potential-driven acidification in dBBMV and iBBMV but not in gastric SAV. These data demonstrate that proton permeation in the three membrane types is rheogenic. The sensitivity of the proton-conductive pathways in intestinal BBMV to high concentrations of amiloride correlated with the presence of the Na+/H+ antiport and indicates that this transmembrane protein may represent a pathway for proton permeation.We thank Ruth Briggs for assistance with the Na/H exchange experiments. This work was supported by a grant from the Medical Research Council (G8418056CA).  相似文献   

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