共查询到20条相似文献,搜索用时 15 毫秒
1.
The cytoplasmic concentrations of Cl−([Cl−]i) and Ca2+ ([Ca2+]i) were measured with the fluorescent indicators N-(ethoxycarbonylmethyl)-6-methoxyquinilinum bromide (MQAE) and fura-2 in pancreatic β-cells isolated from ob/ob mice. Steady-state [Cl−]i in unstimulated β-cells was 34 mM, which is higher than expected from a passive distribution. Increase of the glucose concentration from 3 to 20 mM resulted in an accelerated entry of Cl− into β-cells depleted of this ion. The exposure to 20 mM glucose did not affect steady-state [Cl−]i either in the absence or presence of furosemide inhibition of Na+, K+, 2 Cl− co-transport. Glucose-induced oscillations of [Ca2+]i were transformed into sustained elevation in the presence of 4,4′ diisothiocyanato-dihydrostilbene-2,2′-disulfonic acid (H2DIDS). A similar effect was noted when replacing 25% of extracellular Cl− with the more easily permeating anions SCN−, I−, NO3− or Br−. It is concluded that glucose stimulation of the β-cells is coupled to an increase in their Cl− permeability and that the oscillatory Ca2+ signalling is critically dependent on transmembrane Cl− fluxes. 相似文献
2.
Simon A. Lewis A. Grant Butt M. Joanne Bowler John P. Leader Anthony D. C. Macknight 《The Journal of membrane biology》1985,83(1-2):119-137
Summary The effects of complete substitution of gluconate for mucosal and/or serosal medium Cl– on transepithelial Na+ transport have been studied using toad urinary bladder. With mucosal gluconate, transepithelial potential difference (V
T) decreased rapidly, transepithelial resistance (R
T) increased, and calculated short-circuit current (I
sc) decreased. CalculatedE
Na was unaffected, indicating that the inhibition of Na+ transport was a consequence of a decreased apical membrane Na+ conductance. This conclusion was supported by the finding that a higher amiloride concentration was required to inhibit the residual transport. With serosal gluconateV
T decreased,R
T increased andI
sc fell to a new steady-state value following an initial and variable transient increase in transport. Epithelial cells were shrunken markedly as judged histologically. CalculatedE
Na fell substantially (from 130 to 68 mV on average). Ba2+ (3mm) reduced calculatedE
Na in Cl– Ringer's but not in gluconate Ringer's. With replacement of serosal Cl– by acetate, transepithelial transport was stimulated, the decrease in cellular volume was prevented andE
Na did not fall. Replacement of serosal isosmotic Cl– medium by a hypo-osmotic gluconate medium (one-half normal) also prevented cell shrinkage and did not result in inhibition of Na+ transport. Thus the inhibition of Na+ transport can be correlated with changes in cell volume rather than with the change in Cl– per se. Nystatin virtually abolished the resistance of the apical plasma membrane as judged by measurement of tissue capacitance. With K+ gluconate mucosa, Na+ gluconate serosa, calculated basolateral membrane resistance was much greater, estimated basolateral emf was much lower, and the Na+/K+ basolateral permeability ratio was much higher than with acetate media. It is concluded the decrease in cellular volume associated with substitution of serosal gluconate for Cl– results in a loss of highly specific Ba2+-sensitive K+ conductance channels from the basolateral plasma membrane. It is possible that the number of Na+ pump sites in this membrane is also decreased. 相似文献
3.
4.
Masui DC Furriel RP Mantelatto FL McNamara JC Leone FA 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2003,134(4):631-640
The kinetic properties of a microsomal gill (Na+,K+)-ATPase from the blue crab Callinectes danae were analyzed using the substrate p-nitrophenylphosphate. The (Na+,K+)-ATPase hydrolyzed PNPP obeying cooperative kinetics (n=1.5) at a rate of V=125.4±7.5 U mg−1 with K0.5=1.2±0.1 mmol l−1; stimulation by potassium (V=121.0±6.1 U mg−1; K0.5=2.1±0.1 mmol l−1) and magnesium ions (V=125.3±6.3 U mg−1; K0.5=1.0±0.1 mmol l−1) was cooperative. Ammonium ions also stimulated the enzyme through site–site interactions (nH=2.7) to a rate of V=126.1±4.8 U mg−1 with K0.5=13.7±0.5 mmol l−1. However, K+-phosphatase activity was not stimulated further by K+ plus NH4+ ions. Sodium ions (KI=36.7±1.7 mmol l−1), ouabain (KI=830.3±42.5 μmol l−1) and orthovanadate (KI=34.0±1.4 nmol l−1) completely inhibited K+-phosphatase activity. The competitive inhibition by ATP (KI=57.2±2.6 μmol l−1) of PNPPase activity suggests that both substrates are hydrolyzed at the same site on the enzyme. These data reveal that the K+-phosphatase activity corresponds strictly to a (Na+,K+)-ATPase in C. danae gill tissue. This is the first known kinetic characterization of K+-phosphatase activity in the portunid crab C. danae and should provide a useful tool for comparative studies. 相似文献
5.
Gert Flik Theo J. M. Schoenmakers Jack A. Groot Carel H. van Os Sjoerd E. Wendelaar Bonga 《The Journal of membrane biology》1990,113(1):13-22
Summary Measurements of unidirectional calcium fluxes in stripped intestinal epithelium of the tilapia,Oreochromis mossambicus, in the presence of ouabain or in the absence of sodium indicated that calcium absorption via the fish intestine is sodium dependent. Active Ca2+ transport mechanisms in the enterocyte plasma membrane were analyzed. The maximum capacity of the ATP-dependent Ca2+ pump (V
m
:0.63 nmol·min–1 mg–1,K
m
: 27nm Ca2+) is calculated to be 2.17 nmol·min–1·mg–1, correcting for 29% inside-out oriented vesicles in the membrane preparation. The maximum capacity of the Na+/Ca2+ exchanger with high affinity for Ca2+ (V
m
:7.2 nmol·min–1·mg–1,K
m
: 181nm Ca2+) is calculated to be 13.6 nmol·min–1·mg–1, correcting for 53% resealed vesicles and assuming symmetrical behavior of the Na+/Ca2+ exchanger. The high affinity for Ca2+ and the sixfold higher capacity of the exchanger compared to the ATPase suggest strongly that the Na+/Ca2+ exchanger will contribute substantially to Ca2+ extrusion in the fish enterocyte. Further evidence for an important contribution of Na+/Ca2+ exchange to Ca2+ extrusion was obtained from studies in which the simultaneous operation of ATP-and Na+-gradient-driven Ca2+ pumps in inside-out vesicles was evaluated. The fish enterocyte appears to present a model for a Ca2+ transporting cell, in which Na+/Ca2+ exchange activity with high affinity for Ca2+ extrudes Ca2+ from the cell. 相似文献
6.
- 1. (1) VO3− combines with high affinity to the Ca2+-ATPase and fully inhibits Ca2+-ATPase and Ca2+-phosphatase activities. Inhibition is associated with a parallel decrease in the steady-state level of the Ca2+-dependent phosphoenzyme.
- 2. (2) VO3− blocks hydrolysis of ATP at the catalytic site. The sites for VO3− also exhibit negative interactions in affinity with the regulatory sites for ATP of the Ca2+-ATPase.
- 3. (3) The sites for VO3− show positive interactions in affinity with sites for Mg2+ and K+. This accounts for the dependence on Mg2+ and K+ of the inhibition by VO3−. Although, with less effectiveness, Na+ substitutes for K+ whereas Li+ does not. The apparent affinities for Mg2+ and K+ for inhibition by VO3− seem to be less than those for activation of the Ca2+-ATPase.
- 4. (4) Inhibition by VO3− is independent of Ca2+ at concentrations up to 50 μM. Higher concentrations of Ca2+ lead to a progressive release of the inhibitory effect of VO3−.
Keywords: Ca2+-ATPase; Vanadate inhibition; K+; Li+; (Red cell membrane) 相似文献
7.
Daisuke Ojima Roger J. Pettersen Judith Wolkers Helge K. Johnsen Even H. Jrgensen 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》2009,153(4):378-385
In a comparative experiment the effect of cortisol and growth hormone (GH) on the hypo-osmoregulatory ability of a landlocked and an anadromous strain of Arctic charr (Salvelinus alpinus) was investigated. Cortisol and GH were implanted either alone or in combination, and the fish were exposed to a 24 h seawater challenge test (SWT) on days 14 and 28 after implantation. Hypo-osmoregulatory ability, measured as plasma osmolality and chloride concentration after the SWTs, was better in the anadromous than in the landlocked strain, irrespective of treatment. However, cortisol provided a strong stimulation of hypo-osmoregualtory ability in both strains, and this stimulation seemed to be potentiated by GH in an additive manner. Improved hypo-osmoregulatory ability in GH + cortisol treated anadromous Arctic charr was accompanied by increased gill Na+, K+-ATPase activity and Na+–K+–2Cl− cotransporter protein abundance, but no changes in gill Na+,K+-ATPase α1a and α1b mRNA levels. For landlocked charr the improved hypo-osmoregulatory ability in GH +cortisol treated fish was accompanied only with an increase in gill Na+–K+–2Cl− cotransporter protein abundance. Hormone treatment caused an improvement of hypo-osmoregulatory ability that was of approximately the same magnitude in the landlocked as in the anadromous Arctic charr. This suggests that the lack of spontaneous development of hypo-osmoregulatory ability often seen in landlocked populations of Arctic charr may depend, at least partly, on a lack of the hormonal activation seen in anadromous populations. 相似文献
8.
Summary The mechanism of salt tolerance was studied using isolated internodal cells of the charophyteNitellopsis obtusa grown in fresh water. When 100 mM NaCl was added to artificial pond water (0.1 mM each of NaCl, KC1, CaCl2), no cell survived for more than one day. Within the first 30 minutes, membrane potential (Em) depolarized and membrane resistance (Rm) decreased markedly. Simultaneously, cytoplasmic Na+ increased and K+ decreased greatly. At steady state the increase in Na+ content was roughly equal to the decrease in K+ content. The Cl– content of the cytoplasm did not change. These results suggest that Na+ enters the cytoplasm by exchange with cytoplasmic K+. Both the entry of Na+ and the exit of K+ are assumed to be passive and the latter being caused by membrane depolarization. Vacuolar K+, Na+, and Cl– remained virtually constant, suggesting that rapid influx of Na+ from the cytoplasm did not occur.In 100 mM NaCl containing 10 mM CaCl2, membrane depolarization, membrane resistance decrease and changes in cytoplasmic [Na+] and [K+] did not occur, and cells survived for many days. When cells treated with 100 mM NaCl were transferred within 1 hour to 100 mM NaCl containing 10 mM CaCl2, Em decreased, Rm increased, cytoplasmic Na+ and K+ returned to their initial levels, and cells survived. Two possible mechanisms for the role of Ca2+ in salt tolerance inNitellopsis are discussed; one a reduction in plasmalemma permeability to Na+ and the other a stimulation of active Na+-extrusion. 相似文献
9.
Intracellular calcium content of human erythrocytes: Relation to sodium transport systems 总被引:2,自引:0,他引:2
Summary To study the possible role of intracellular Ca (Ca
i
) in controlling the activities of the Na+–K+ pump, the Na+–K+ cotransport and the Na+/Li+ exchange system of human erythrocytes, a method was developed to measure the amount of Ca embodied within the red cell. For complete removal of Ca associated with the outer aspect of the membrane, it proved to be essential to wash the cells in buffers containing less than 20nm Ca. Ca was extracted by HClO4 in Teflon® vessels boiled in acid to avoid Ca contaminations and quantitated by flameless atomic absorption. Ca
i
of fresh human erythrocytes of apparently healthy donors ranged between 0.9 and 2.8 mol/liter cells. The mean value found in females was significantly higher than in males. The interindividual different Ca contents remained constant over periods of more than one year. Sixty to 90% of Ca
i
could be removed by incubation of the cells with A23187 and EGTA. The activities of the Na+–K+ pump, of Na+–K+ cotransport and Na+/Li+ exchange and the mean cellular hemoglobin content fell with rising Ca
i
; the red cell Na+ and K+ contents rose with Ca
i
. Ca depletion by A23187 plus EGTA as well as chelation of intracellular Ca2+ by quin-2 did not significantly enhance the transport rates. It is concluded that the large scatter of the values of Ca
i
of normal human erythrocytes reported in the literature mainly results from a widely differing removal of Ca associated with the outer aspect of the membrane. 相似文献
10.
Freshly dissociated myocytes from nonpregnant, pregnant, and postpartum rat uteri have been studied with the tight-seal patch-clamp method. The inward current contains both INa and ICa that are vastly different from those in tissue-cultured material. INa is abolished by Na+-free medium and by 1 μM tetrodotoxin. It first appears at ∼−40 mV, reaches maximum at 0 mV, and reverses at 84 mV. It activates with a voltage-dependent τ of 0.2 ms at 20 mV, and inactivates as a single exponential with a τ of 0.4 ms. Na+ conductance is half activated at −21.5 mV, and half inactivated at −59 mV. INa reactivates with a τ of 20 ms. ICa is abolished by Ca2+-free medium, Co2+ (5 mM), or nisoldipine (2 μM), and enhanced in 30 mM Ca2+, Ba2+, or BAY-K 8644. It first appears at ∼−30 mV and reaches maximum at +10 mV. It activates with a voltage-dependent τ of 1.5 ms at 20 mV, and inactivates in two exponential phases, with τ''s of 33 and 133 ms. Ca2+ conductance is half activated at −7.4 mV, and half inactivated at −34 mV. ICa reactivates with τ''s of 27 and 374 ms. INa and ICa are seen in myocytes from nonpregnant estrus uteri and throughout pregnancy, exhibiting complex changes. The ratio of densities of peak INa/ICa changes from 0.5 in the nonpregnant state to 1.6 at term. The enhanced role of INa, with faster kinetics, allows more frequent repetitive spike discharges to facilitate simultaneous excitation of the parturient uterus. In postpartum, both currents decrease markedly, with INa vanishing from most myocytes. Estrogen-enhanced genomic influences may account for the emergence of INa, and increased densities of INa and ICa as pregnancy progresses. Other influences may regulate varied channel expression at different stages of pregnancy. 相似文献
11.
Toru Ishikawa Yoshinori Marunaka Daniela Rotin 《The Journal of general physiology》1998,111(6):825-846
The epithelial Na+ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na+ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na+ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na+ conductance was Li+ > Na+ >> K+ = N-methyl-d-glucamine+ (NMDG+). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na+ channel current, was found to be ∼5 and 8 pS when Na+ and Li+ were used as a charge carrier, respectively. K+ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (P
o), was increased by membrane hyperpolarization. Both whole-cell Na+ current and conductance were saturated with increased extracellular Na+ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nP
o) was significantly decreased when cytosolic Na+ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na+ conductance (with Li+ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca2+ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca2+ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca2+ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca2+ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca2+ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na+ and Ca2+. 相似文献
12.
J. A. Zadunaisky S. Cardona L. Au D. M. Roberts E. Fisher B. Lowenstein E. J. Cragoe Jr. K. R. Spring 《The Journal of membrane biology》1995,143(3):207-217
Transition from low salt water to sea water of the euryhaline fish, Fundulus heteroclitus, involves a rapid signal that induces salt secretion by the gill chloride cells. An increase of 65 mOsm in plasma osmolarity was found during the transition. The isolated, chloridecell-rich opercular epithelium of sea-water-adapted Fundulus exposed to 50 mOsm mannitol on the basolateral side showed a 100% increase in chloride secretion, which was inhibited by bumetanide 10–4
m and 10–4
m DPC (N-Phenylanthranilic acid). No effect of these drugs was found on apical side exposure. A Na+/H+ exchanger, demonstrated by NH4Cl exposure, was inhibited by amiloride and its analogues and stimulated by IBMX, phorbol esters, and epithelial growth factor (EGF). Inhibition of the Na+/H+ exchanger blocks the chloride secretion increase due to basolateral hypertonicity. A Cl–/HCO
3
–
exchanger was also found in the chloride cells, inhibited by 10–4
m DIDS but not involved in the hyperosmotic response. Ca2+ concentration in the medium was critical for the stimulation of Cl– secretion to occur. Chloride cell volume shrinks in response to hypertonicity of the basolateral side in sea-water-adapted operculi; no effect was found on the apical side. Freshwater-adapted fish chloride cells show increased water permeability of the apical side. It is concluded that the rapid signal for adaptation to higher salinities is an increased tonicity of the plasma that induces chloride cell shrinkage, increased chloride secretion with activation of the Na+K+2Cl– cotransporter, the Na+/H+ exchanger and opening of Cl– channels.The work was supported by the National Institutes of Health, Research Grant EYO1340 to J.A.Z. Part of this research was performed while Dr. Zadunaisky was a Scholar In Residence at the Fogarty International Center of The National Institutes of Health in Bethesda, Maryland. Ms. Dawn Roberts was a fellow of the Grass Foundation and Pew Foundation during this work. Grants from the National Science Foundation and the National Institutes of Health to the Mount Desert Island Biological Laboratory also provided assistance for this research. 相似文献
13.
Three types of ionic current essentially determine the firing pattern of nerve cells: the persistent Na+ current, the M current and the low-voltage-activated Ca2+ current. The present article summarizes recent experiments concerned with the basic properties of these currents. Keynes and Meves (Proc R Soc Lond B (1993) 253, 61–68) studied the persistent or steady-state Na+ current on dialysed squid axons and measured the probability of channel opening both for the peak and the steady-state Na+ current (PFpeak and PFss) as a function of voltage. Whereas PFpeak starts to rise at −50 mV and reaches a maximum at +40 to +50 mV, PFss only begins to rise appreciably at around 0 mV and is still increasing at +100 mV. This differs from observations on vertebrate excitable tissues where the persistent Na+ current turns on in the threshold region and saturates at around 0 mV. Schmitt and Meves (Pflügers Arch (1993) 425, 134–139) recorded M current, a non-inactivating K+ current, from NG108-15 neuroblastoma × glioma hybrid cells, voltage-clamped in the whole-cell mode, and studied the effects of phorbol 12,13-dibutyrate (PDB), an activator of protein kinase C (PKC), and arachidonic acid (AA). PDB and AA both decreased IM, the effective concentrations being 0.1–1 μM and 5–25 μM, respectively; while the PDB effect was regularly observed, the M current depression by AA was highly variable from cell to cell. The PKC 19–31 peptide, an effective inhibitor of PKC, in a concentration of 1 μM almost totally prevented the effects of PDB and AA on M current, suggesting that both are mediated by PKC. Schmitt and Meves (Pflügers Arch (1994a) 426, Suppl R 59) measured low-voltage-activated (l-v-a) and high-voltage-activated (h-v-a) Ca2+ currents on NG108-15 cells and investigated the effect of AA and PDB on both types of current. At pulse potentials > −20 mV, AA (25–100 μM) decreased l-v-a and h-v-a ICa. The decrease was accompanied by a small negative shift and a slight flattening of the activation and inactivation curves of the l-v-a ICa. The AA effect was not prevented by 50 μM eicosa-5,8,11,14-tetraynoic acid (ETYA), an inhibitor of AA metabolism, or PKC 19–31 peptide and not mimicked by 0.1–1 μM PDB. Probably, AA acts directly on the channel protein or its lipid environment. The physiological relevance of these three sets of observations is briefly discussed. 相似文献
14.
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
15.
Mortimer M. Civan Allison Oler Kim Peterson-Yantorno Kenneth George Thomas G. O'Brien 《The Journal of membrane biology》1991,121(1):37-50
Summary Activators of protein kinase C (PKC) stimulate Na– transport (J
Na) across frog skin. We have examined the effect of Ca2+ on PKC stimulation ofJ
Na. Both the phorbol ester 12-O-tetradecanoylglycerol (DiC8) were used as PKC activators. Blocking Ca2+ entry into the cytosol (either from external or internal stores) reduced the subsequent natriferic effect of the PKC activators. This negative interaction did not simply reflect saturation of activation of the apical Na+ channels, since the stimulations produced by blocking Ca2+ entry and adding cyclic AMP were simply additive.The Ca2+ dependence of the natriferic effect could have reflected either a direct action of cytosolic Ca2+ on PKC or an indirect action on the final receptor site (the Na+ channel). To distinguish between these possibilities, the TPA- and phospholipid-dependent kinase activity of broken-cell preparations was assayed. The kinase activity was not stimulated by physiological levels of Ca2+, and in fact was inhibited at millimolar concentrations of Ca2+.We conclude that the effects of Ca2+ on the natriferic response to PKC activators are indirect. Reducing cytosolic uptake of Ca2+ may have stimulated Na+ transport by a chemical modification of the apical channels observed in other tight epithelia. The usual stimulation of Na+ transport produced by PKC activators in frog skin may reflect the operation of a nonconventional form of PKC. This enzyme is Ca2+ independent and seems related to thenPKC or PKC observed in other systems. 相似文献
16.
Theo J. M. Schoenmakers Peter H. M. Klaren Gert Flik Robert A. C. Lock Peter K. T. Pang Sjoerd E. Wendelaar Bonga 《The Journal of membrane biology》1992,127(3):161-172
Summary The inhibition of Ca2–-ATPase, (Na++K+)-ATPase and Na+/Ca2+ exchange by Cd2+ was studied in fish intestinal basolateral plasma membrane preparations. ATP driven 45Ca2+ uptake into inside-out membrane vesicles displayed a K
m
for Ca2+ of 88±17 nm, and was extremely sensitive to Cd2+ with an IC50 of 8.2±3.0 pM Cd2+, indicating an inhibition via the Ca2+ site. (Na++K+)-ATPase activity was half-maximally inhibited by micromolar amounts of Cd2+, displaying an IC50 of 2.6±0.6 m Cd2+. Cd2+ ions apparently compete for the Mg2+ site of the (Na– +K+)-ATPase. The Na+/Ca2+ exchanger was inhibited by Cd2+ with an IC50 of 73±11 nm. Cd2+ is a competitive inhibitor of the exchanger via an interaction with the Ca2+ site (K
i
= 11 nm). Bepridil, a Na+ site specific inhibitor of Na+/Ca2+ exchange, induced an additional inhibition, but did not change the K
i
of Cd2+. Also, Cd2+ is exchanged against Ca2+, albeit to a lesser extent than Ca2+. The exchanger is only partly blocked by the binding of Cd2+. In vivo cadmium that has entered the enterocyte may be shuttled across the basolateral plasma membrane by the Na+/Ca2+ exchanger. We conclude that intracellular Cd2+ ions will inhibit plasma membrane proteins predominantly via a specific interaction with divalent metal ion sites.We would like to thank Dr. D. Fackre (University of Alberta, Canada) for stimulating discussions and Mr. F.A.T. Spanings (University of Nijmegen, The Netherlands) for excellent fish husbandry. The fura-2 measurements of intracellular Ca2+ concentrations in tilapia enterocytes were carried out in the Department of Physiology, School of Medicine, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Th.J.M. Schoenmakers and G. Flik were supported by travel grants from the Foundation for Fundamental Biological Research (BION) and the Netherlands Organization for Scientific Research (NWO). 相似文献
17.
Hideaki Bujo Junichi Nakai Tai Kubo Kazuhiko Fukuda Isamu Akiba Akito Maeda Masayoshi Mishina Shosaku Numa 《FEBS letters》1988,240(1-2)
Muscarinic acetylcholine receptor (mAChR) III expressed in Xenopus oocytes, like mAChR I, mediates activation of a Ca2+-dependent Cl− current, whereas mAChR IV, like mAChR II, principally induces activation of Na+ and K+ currents in a Ca2+-independent manner. mAChR III has a sensitivity to agonist of about one order of magnitude higher than that of mAChR I in mediating the Ca2+-dependent current response in Xenopus oocytes and in stimulating phosphoinositide hydrolysis in NG108-15 neuroblastoma-glioma hybrid cells. The agonist-binding affinity of mAChR III is also about one order of magnitude higher than that of mAChR I. 相似文献
18.
K. R. Robinson 《Planta》1977,136(2):153-158
The effect of external calcium and sodium ion concentrations on the calcium fluxes on the Pelvetia fastigiata De Toni egg was measured. Decreasing external [Ca2+] greatly increased the permeability of the eggs to Ca2+; at 1 mM external Ca2+ this permeability was 60 times as great as it was at the normal [Ca2+] of 10 mM. Lowering the external [Na+] also increased Ca2+ influx; at 2 mM Na+, the Ca2+ influx was 2–3 times as great as it was at the normal [Na+] if choline was used as a Na+ substitute. Lithium was less effective as a Na+ substitute in increasing Ca2+ influx. The extra Ca2+ influx in low [Na+] seemed to be dependent on internal [Na+]. The Ca2+ efflux increased transiently and then declined in low Na+ media. 相似文献
19.
Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves 总被引:3,自引:0,他引:3
Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca2+ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.45Ca2+ transport was assayed by membrane filtration technique. Results showed that Ca2+ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca2+ transport system had a high affinity for Ca2+(K
m
(Ca2+)=0.4 m) and ATP(K
m
(ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca2+ uptake in the plasma membrane vesicles was stimulated in the presence of Cl– or NO
3
–
. Quenching of quinacrine fluorescence showed that these anions also induced H+ transport into the vesicles. The Ca2+ uptake stimulated by Cl– was dependent on the activity of H+ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO
4
3–
which is known to inhibit the H+ pump associated with the plasma membrane, canceled almost all of the Cl–-stimulated Ca2+ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca2+ uptake into the vesicles. These results suggest that the Cl–-stimulated Ca2+ uptake is caused by the efflux of H+ from the vesicles by the operation of Ca2+/H+ antiport system in the plasma membrane. In Cl–-free medium, H+ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca2+ uptake into the vesicles. These results suggest that two Ca2+ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca2+ transport system (Ca2+ pump) and the other is a Ca2+/H+ antiport system. Little difference in characteristics of Ca2+ transport was observed between the plasma membranes isolated from etiolated and green corn leaves. 相似文献
20.
Nicholas Sperelakis Yoshihito Inoue Yusuke Ohya 《Molecular and cellular biochemistry》1992,114(1-2):79-89
Summary Smooth muscle cells normally do not possess fast Na2+ channels, but inward current is carried through two types of Ca2+ channels: slow (L-type) Ca2+ channels and fast (T-type) Ca2+ channels. Using whole-cell voltage clamp of single smooth muscle cells isolated from the longitudinal layer of 18-day pregnant rat uterus, depolarizing pusles, applied from a holding potential of –90 mV, evoked two types of inward current, fast and slow [8]. The fast inward current decayed within 30 ms, depended on [Na]0, and was inhibited by TTX (K0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]0, and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na2+ channel current, and that the slow inward current is a Ca2+ channel current was not evident. Thus, the ion channels which generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na+ channels and dihudropuridine-sensitive slow Ca2+ channels. The number of fast Na+ channels increased during gestation [9]. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells which possess fast Na2+ channels, and it suggested that the fast Na+ current may be involved in spread of excitation. The Ca2+ channel current density also was higher during the latter half of gestation. These results indicate that the fast Na+ channels and Ca2+ slow channels in myometrium become more numerous as term approaches, and may facilitate parturition. Isoproterenol (beta-agonist) did not affect either ICa(s) or INa(f), whereas Mg2+ (K0.5 of 12 mM) and nifedipine (K0.5 of 3.3 nM) depressed ICa(s). Oxytocin had no effect on INa(f) and actually depressed ICa(s) to a small extect. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of ICa(s), whereas that of Mg2+ can be so explained. The stimulating action of oxytocin on uterine contractions is not due to stimulation of ICa(s). 相似文献