共查询到20条相似文献,搜索用时 31 毫秒
1.
Hideaki Katsuyama Shinichi Ito Take Itoh Hirosi Kuriyama 《Pflügers Archiv : European journal of physiology》1991,419(5):460-466
To study the essential features of acetylcholine (ACh)-and caffeine-sensitive cellular Ca2+ storage sites in single vascular smooth muscle cells of the porcine coronary artery, the effects of ryanodine on both ACh- and caffeine-induced Ca2+ mobilization were investigated by measuring intracellular Ca2+ concentration ([Ca2+]i) using Fura 2 in Ca2+-containing or Ca2+-free solution. The resting [Ca2+]i of the cells was 122 nM in normal physiological solution and no spontaneous activity was observed. In a solution containing 2.6 mM Ca2+, 10 M ACh or 128 mM K+ produced a phasic, followed by a tonic, increase in [Ca2+]i but 20 mM caffeine produced only a phasic increase. In Ca2+-free solution containing 0.5 mM ethylenebis(oxonitrilo)tetraacetate (EGTA), the resting [Ca2+]i rapidly decreased to 102 nM within 5 min, and 10 M ACh or 20 mM caffeine (but not 128 mM K+) transiently increased [Ca2+]i. Ryanodine (50 M) greatly inhibited the phasic increase in [Ca2+]i induced by 10 M ACh or 5 mM caffeine and increased the time to peak and to the half decay after the peak in the presence or absence of extracellular Ca2+. By contrast, ryanodine (50 M) enhanced the tonic increase in [Ca2+]i induced by 128 mM K+ and also by 10 M ACh in Ca2+-containing solution. In Ca2+-free solution containing 0.5 mM EGTA, ACh (10 M) failed to increase [Ca2+]i following application of 20 mM caffeine. The level of [Ca2+]i induced by 20 mM caffeine was greatly reduced, but not abolished, following application of 10 M ACh in Ca2+-free solution. These results suggest that both ACh and caffeine release Ca2+ from the ryanodine-sensitive sarcoplasmic reticulum (SR) in smooth muscle cells of the porcine coronary artery. The finding that ryanodine significantly increased the resting [Ca2+]i and inhibited the rate of decline of [Ca2+]i following wasthout of high K+ or ACh in Ca2+-containing solution suggests that SR may negatively regulate the resting [Ca2+]i in smooth muscle cells of the porcine coronary artery. 相似文献
2.
Yoshihisa Kurachi Hiroyuki Ito Tsuneaki Sugimoto 《Pflügers Archiv : European journal of physiology》1990,416(1-2):216-218
Concentration-dependent effects of intracellular GTP on activation of the muscarinic K+ channel were examined in inside-out patches of cardiac atrial myocytes. The pipette solution contained 0.1 M ACh. GTP (0.01–30 M) and 0.5 mM MgCl2 were applied to the inside side of the patch membrane. K+ channels were activated with GTP concentration above 0.1 M. Channel activation reached a maximal value with 1–3 M GTP. It decreased at GTP concentrations larger than 3 M, probably due to desensitization. The dependence of the open probability of the channel on intracellular GTP showed a sigmoidal relationship with a Hill coefficient of around 3. A positive cooperative effect of intracellular GTP on the K+ channel may play an important role in amplifying the signal from the membrane receptor to the K+ channel. 相似文献
3.
Kevin Pritchard C. C. Ashley 《Pflügers Archiv : European journal of physiology》1987,410(4-5):401-407
Isolated smooth muscle cells (SMC) from guinea pig taenia coli were employed. Suspension of cells were externally loaded in saline with the fluorescent calcium indicators quin-2/AM or fura-2/AM at 20–40 M or 4 M respectively, resulting in an estimated intracellular concentration of 100–200 M for quin-2 or 10–20 M fura-2 (free acid). On addition of 100 M carbachol or high K
o
+
(80 mM) depolarization, fura-2 loaded cells contracted (104±47 m,n=121 rest: 39±13 m,n=59 contracted) identically to control (103±35 m,n=232 rest: 39±16 m,n=89 contracted) cells, whereas quin-2 loaded cells were unresponsive to these protocols and there was no significant length change. The Ca
i
2+
of fura-2 loaded cells was 100±18 nM (mean±SD,n=15) and was not significantly different from quin-2 loaded cells 107±26 nM (n=13). Treatment of fura-2 loaded cells with 100 M ouabain saline for 10–60 min progressively elevated the Ca
i
2+
to a mean of 266±83 nM (n=15). Reduction of Na
p
+
(96% Li+ replaced) significantly increased Ca
i
2+
to 317±77 nM (n=8). After pretreatment with ouabain (100 M), Na
o
+
replacement (Li+) increased Ca
i
2+
at a significantly faster rate [3.6 nM min–1 (control) cf. 19.8 nM min–1 (ouabain)]. 相似文献
4.
William Van der Kloot 《Pflügers Archiv : European journal of physiology》1988,412(3):258-263
Pretreatment with hypertonic solutions, insulin, or adrenaline increases the size of quanta at the frog neuromuscular junction, as determined by measurements of miniature end plate potentials or currents (Van der Kloot and Van der Kloot 1985, 1986). The increase in quantal size apparently is due to an increase in acetylcholine (ACh) content of individual quanta. These treatments, therefore, can be used to study the packaging of ACh. Previously, I reported that increases are blocked by an inhibitor of active ACh uptake into vesicles (Van der Kloot 1986b, 1987b). The present study shows that the increases in quantal size were antagonized by inhibiting the Na+–K+ exchange pump with 100 M ouabain, 10 M dihydroouabain, or K+-free solutions. The increases in quantal size were also antagonized by 10 M monensin, a Na+ ionophore, or by 5 M aconitine, which opens Na+ channels at normal resting potentials. Apparently a rise in intracellular [Na+] inhibits the addition of ACh to quanta. The mechanism by which a rise in intracellular Na+ inhibits ACh packing is unknown, but apparently it is not due to inhibition of choline reuptake into the terminals.Also consistent with the above hypothesis is that the increase in quantal size following depolarization for 2 h in elevated [K+]out was substantially enhanced when tetrodotoxin (TTX) was present, suggesting that in the absence of TTX there is a rise in [Na+]in that antagonizes the incorporation of additional ACh into the quanta. 相似文献
5.
Dr. H. Oberleithner A. Schwab H. -J. Westphale L. Wojnowski 《Journal of molecular medicine (Berlin, Germany)》1992,70(9):816-824
Summary Intracellular pH (pHi) plays a critical role in the entry of cells into the DNA-synthesis phase of the cell cycle. Alterations in pHi may contribute to abnormal proliferative responses such as those seen in tumorigenic cells. We observed that alkaline stress leads to genomic transformation of Madin-Darby canine kidney (MDCK) cells. Transformed cells (F cells) form foci in culture, lack contact inhibition, and are able to migrate, typical characteristics of dedifferentiated tumorigenic cells. F cells exhibit spontaneous biorhythmicity. Rhythmic transmembrane Ca2+ flux activates plasma membrane K+ channels and Na+/H+ exchange. This leads to periodic changes of membrane voltage and pHi at about one cycle per minute. We conclude that endogenous oscillatory activity could be a trigger mechanism for DNA synthesis, proliferation, and abnormal growth of renal epithelial cells in culture.Abbreviations CICR
calcium-induced calcium release
- IP3a
inositol triphosphate
- MDCK
Madin-Darby canine kidney
- pHia
intracellular pH
Dedicated to our friend and teacher Prof. Dr. Gerhard Giebisch in gratitude for his long standing support 相似文献
6.
T. Zuidema M. Kamermans J. Siegenbeek van Heukelom 《Pflügers Archiv : European journal of physiology》1986,407(3):292-298
Mucosal glucose addition evokes in goldfish intestinal epithelium a fast depolarization of the mucosal membrane potential (mc = 12 mV) followed by a slower repolarization (mc = –7 mV). The intracellular sodium activity, aiNa+, rises from 13.2±2.4 meq/l by 6.7±0.5 meq/l within 5 min, aiCl– rises about 3 meq/l above the control value of 37.7±2.2 meq/l, while aiK is constant (97.7 ±7.4 meq/l). The potassium activity measured in the submucosal interstitium near the basal side of the cells (aiK+) is 5.2±0.2 meq/l in non-absorbing tissue compared to 4.2 meq/l in the bathing solution and shows a transient increase due to glucose absorption (1.1±0.1 meq/l).In chloride-free media asK+=4.2±0.1 meq/l and mc hyperpolarizes by –13 mV. The depolarization due to glucose absorption increases (mc = 14.1 ± 1.4 mV) and the repolarization (
mc
repol
) disappears. In addition, aiNa+ rises from 16.3±2.4 meq/l by 9.9±1.5 meq/l within 5 min, aiK+ remains constant and equal to the value in chloride containing solutions (88.5±2.8 meq/l); asK+ increases transiently (1.1±0.1 meq/l).Serosal Ba2+ (5 mM) depolarizes mc (+14.2±1.0 mV) and abolishes the repolarization. Increased serosal or mucosal potassium activity depolarizes mc and abolishes the repolarization.These effects are discussed in terms of changes of ion activities, the basolateral potassium conductance, the influence of intracellular Ca2+, the functional state of the Na/K-pump, and modulation of membrane permeabilities by extracellular potassium. 相似文献
7.
S. L. Abrahamse R. J. M. Bindels C. H. van Os 《Pflügers Archiv : European journal of physiology》1992,421(6):591-597
The presence of an H+/K+-ATPase and its contribution to the regulation of intracellular pH (pHi) was investigated in Caco-2 cells. The H+/K+-ATPase was detected immunologically using the monoclonal antibody 5-B6, which was raised against hog gastric H+/K+-ATPase. Cell pH was determined using the pH-sensitive dye 2,7-bis(carboxyethyl)-carboxyfruorescein. Control pHi, measured in HCO
3
–
-free medium, was 7.62±0.03 (n=27) when cells were cultured for 14 days and decreased to 7.40±0.03 (n=18) after 35 days in culture. Recovery of pHi following a NH
4
+
/NH3 pulse could be reduced by either 100 M SCH 28080 or 1 mM amiloride, or by removing extracellular Na+. The inhibitory effects of SCH 28080 and amiloride were additive, demonstrating the involvement of a gastric-like H+/K+-ATPase and a Na+/H+ exchanger in regulating pHi. Recovery rates at pHi 6.8 were not significantly different in cells cultured for up to 21 days, but were significantly lower in cells cultured for 28 and 35 days. This decrease in recovery rate was due to a decrease in the SCH-28080-insensitive recovery, indicating a reduction of the relative importance of Na+/H+ exchange to the recovery. Recovery of pHi was also inhibited by 1 mM N-ethylmaleimide. However, it is unlikely that N-ethylmaleimide inhibited a vacuolar type of H+-ATPase, since bafilomycin A1 had no effect on pHi recovery. In conclusion, Caco-2 cells contain a SCH-28080-sensitive mechanism for regulating pHi, which is most conveniently studied after 28 days in culture, when the relative contribution of a Na+/H+ exchanger to pHi regulation is decreased. 相似文献
8.
Gervaise Loirand Pierre Pacaud 《Pflügers Archiv : European journal of physiology》1995,430(3):429-436
Effects of exogenous adenosine 5-triphosphate (ATP) were studied by measurements of intracellular Ca2+ concentration ([Ca2+]i) and membrane currents in myocytes freshly isolated from the human saphenous vein. At a holding potential of –60 mV, ATP (10 M) elicited a transient inward current and increased [Ca2+]i. These effects of ATP were inhibited by ,-methylene adenosine 5-triphosphate (AMPCPP, 10 M). The ATP-gated current corresponded to a non-selective cation conductance allowing Ca2+ entry. The ATP-induced [Ca2+]i rise was abolished in Ca2+-free solution and was reduced to 30.1±5.5% (n=14) of the control response when ATP was applied immediately after caffeine, and to 23.7±3.8% (n=11) in the presence of thapsigargin. The Ca2+-induced Ca2+ release blocker tetracaine inhibited the rise in [Ca2+]i induced by both caffeine and ATP, with apparent inhibitory constants of 70 M and 100 M, respectively. Of the ATP-induced increase in [Ca2+]i 29.3±3.9% (n=8) was tetracaine resistant. It is concluded that the effects of ATP in human saphenous vein myocytes are only mediated by activation of P2x receptor channels. The ATP-induced [Ca2+]i rise is due to both Ca2+ entry and Ca2+ release activated by Ca2+ ions that enter the cell through P2x receptor channels. 相似文献
9.
W. V. Breuer E. Mack A. Rothstein 《Pflügers Archiv : European journal of physiology》1988,411(4):450-455
In dissociated MDCK cells, activators of the cyclic AMP system cause depolarization detectable by changes in fluorescence of the membrane potential sensitive dye bisoxonol. Addition of forskolin (60 M), vasopressin (2 M), 8-bromo-cyclic AMP (0.5 mM) or l-epinephrine (10 M) depolarized the cells substantially in low Cl– (5 mM) but had little effect in high Cl– (140 mM) solution. These results are consistent with cyclic AMP activation of Cl– channels. The Ca2+-ionophore ionomycin (1 M) produced a rapid hyperpolarization in low and high Cl– solutions, consistent with K+ channel opening. Using a clonal subline, MDCK-14, the magnitude of the ionomycin hyperpolarization was roughly proportional to the concomitant rise in [Ca2+]i as measured with the intracellular Ca2+ probe indo-I. Both l-epinephrine and isoproterenol appeared to activate the Cl– channels. However only l-epinephrine produced a [Ca2+]i rise and a transient hyperpolarization (due to K+ channel opening), which preceeded the depolarization due to Cl– channel opening. The l-epinephrine-induced [Ca2+]i response of the heterogeneous MDCK cell population but not of the clonal subline MDCK-14 was inhibited by removal of extracellular Ca2+. In the latter only the slow secondary phase of the [Ca2+]i rise was affected by Ca2+ removal. It is concluded that l-epinephrine activates K+ and Cl– channels in a sequential manner in MDCK cells by Ca2+ and cAMP signals, presumably via - and -adrenergic receptors located on the same cell.Abbreviations MDCK cells
Madin Darby Canine Kidney cells
- [Ca2+]i
intracellular calcium concentration
- [Cl–]i
intracellular chloride concentration
- [Cl–]o
extracellular chloride concentration
- [Na++K+]i
intracellular concentration of Na+ and K+
- [Na++K+]o
extracellular concentration of Na+ and K+
- EM
transmembrane potential
- ECl
chloride equilibrium potential
- EK
potassium equilibrium potential
- bis-oxonol
[bis(1,3-diethylthio-barbiturate)] trimethine oxonol
- DMSO
dimethylsulfoxide
- EDTA
ethylenediaminetetraacetic acid
- EGTA
ethylene glycol bis (-aminoethyl ether) N,N-tetraacetic acid
- Hepes
4-(2-hydroxyethyl)-1 piperazineethanesulfonic acid
- NMG+
N-methylglucamine
- RPMI medium
Rosewell Park Memorial Institute medium 相似文献
10.
Christina Schwanstecher Uwe Panten 《Pflügers Archiv : European journal of physiology》1994,427(1-2):187-189
On the somata of GABAergic spiny neurons in the caudate nucleus of the rat an ATP-sensitive K+ channel (KATP-channel) was identified. The KATP-currents in cell-attached patches were activated both by energy-depleting conditions (200 M cyanide) and by diazoxide (300 M) and were reversibly blocked by tolbutamide (EC50=5 M). In inside-out patch membranes both ATP (1 mM) and its non-hydrolyzable analog AMP-PNP (adenylylimidodiphosphate; EC50=27M) reversibly inhibited channel activity. These results demonstrate that the KATP-channel in spiny neurons displays properties characteristic of the KATP-channel in hippocampal, neocortical and nigral neurons and in pancreatic ß-cells. 相似文献
11.
The interaction of angiotensin II (ANG II) and atrial natriuretic peptide (ANP) on intracellular pH (pHi) and calcium ([Ca2+]i) was investigated in T84 cells (a permanent cell line derived from human colon epithelium) using the fluorescent stains BCECF/AM and Fluo 4/AM, respectively. pHi recovery rate mediated by the Na+/H+ exchanger (NHE) was examined following an NH4Cl pulse. Under control conditions pHi recovered at 0.114±0.005 pH units/min (n=35). ANG II (10–12 or 10–9 M) increased this value, whilst ANG II (10–7 M) decreased it. These effects of ANG II were impaired by simultaneous addition of 1 M or 25 M HOE-694, indicating that the stimulatory and inhibitory effects of ANG II on pHi recovery are mediated in part via the NHE1 and NHE2 isoforms. ANG II increased [Ca2+]i concentration-dependently. ANP (10–6 M) or dimethyl-BAPTA/AM (50 M) blocked the effects of ANG II on [Ca2+]i and on the rate of pHi recovery. Thapsigargin (10–5 M) enhanced the effect of ANG II on [Ca2+]i and reversed its stimulatory effect on the rate of pHi recovery to an inhibitory one. External Ca2+-free solution did not affect the effects of ANG II on these parameters. These data suggest that the [Ca2+]i increase induced by ANG II is dependent on intracellular calcium stores. They are compatible with the demonstration of two sites on the C-terminal of the Na+/H+ exchanger, one stimulating Na+/H+ activity by increases of [Ca2+]i in the lower range (at 10–12 or 10–9 M ANG II) and the other inhibiting this activity at high [Ca2+]i levels (at 10–7 M ANG II). ANP or dimethyl-BAPTA/AM, by impairing the pathway mediating the increase in [Ca2+]i, block both the stimulatory and inhibitory effects of ANG II. 相似文献
12.
Thrombin-induced cytosolic alkalinization in human platelets occurs without an apparent involvement of HCO
3
−
/Cl− exchange 总被引:1,自引:0,他引:1
Norbert Clemens Winfried Siffert Peter Scheid 《Pflügers Archiv : European journal of physiology》1990,416(1-2):68-73
We have estimated the changes in cytosolic pH (pHi) that occur when human platelets are stimulated by thrombin. Changes in pHi were estimated (i) from the H+ efflux across the plasma membrane using an extracellular pH electrode and (ii) using an intracellular pH-sensitive fluorescent dye (BCECF). Stimulation of platelets with thrombin (0.5 unit/ml) resulted in an H+ efflux that averaged 7.7±1.6 mol/1011 platelets (means±SD) leading to an increase in pHi, from 7.05±0.04 to 7.45±0.05. Both H+ efflux and pHi changes were unaffected by 0.1 mM 4,4-diisothiocyanostilbene-2,2 disulphonate (DIDS), 0.1 mM 4-acetamido 4-isothiostilbene-2,2-disulphonic acid (SITS), or 0.5 mM bumetanide, suggesting no involvement of anion transport systems, e.g. an HCO
3
–
/Cl– exchange. Removal of HCO
3
–
or Cl– from the suspending buffer had no effect on the extent of the rise in pHi. After blockade of Na+/H+ exchange by 100 M ethylisopropylamiloride (EIPA), thrombin induced a decrease in pHi the rate of which averaged 0.39 unit/min in HCO
3
–
-containing medium, and 0.57 unit/min in HCO
3
–
-free medium. The cytosolic buffer capacity for H+ was determined by the nigericin/ NH4Cl technique in BCECF-loaded platelets and averaged 25.3 mmol/(1xpH) in buffer containing 8 mM HCO
3
–
, but only 17.2 mmol/(1xpH) in HCO
3
–
-free buffer. The total amount of H+ transferred by Na+/H+ exchange can be estimated from our measurements at 10 mmol/l platelet cytosol in the absence of HCO
3
–
and to 14 mmol/l platelet cytosol in the presence of HCO
3
–
, and is in good agreement with the estimated amount of Na+ uptake by ADP-stimulated platelets. We conclude that net extrusion of H+ from stimulated platelets is predominantly mediated by Na+/H+ exchange without an apparent contribution of HCO
3
–
/Cl– exchange. 相似文献
13.
De Santo Natale G. Capasso Giovambattista Kinne Rolf Moewes B. Carella Carlo Anastasio Pietro Giordano Carmelo 《Pflügers Archiv : European journal of physiology》1982,394(4):294-301
Hypothyroid rats reconstituted with 10 g/kg b.w. per day of tri-iodothironine (T3) for 4 days resulted in normal free T3 and TSH levels. FT3 levels were: 0.53±0.3 pg/ml in hypothyroid rats; 2.78±1.21 pg/ml in hormone reconstituted rats and 2.90±0.90 pg/ml in euthyroid rats. TSH levels were 3,508±513 g/ml in hypothyroid rats; 1,008±204 g/ml in reconstituted rats and 270±184 ng/ml in euthyroid rats.When hypothyroid rats were reconstituted with 50 g T3/kg b.w. per day, TSH levels were nearly normal after 4 days (1,157±621 ng/ml). However FT3 levels after 1–4 days were always higher than in euthyroid rats.Hypothyroid rats show a decrease in isotonic fluid reabsorption (J
v) in the proximal tubule (1.50±0.08 versus 4.96±0.23 10–2 nl·mm–1·s–1 in euthyroid animals). 1 day after T3 (10 g/kg b.w./day) injectionJ
v was increased significantly to 2.05±0.20 10–2 nl·mm–1·s–1 and continued to increase during 4 days of T3 reconstitution.When 50 g T3/kg b.w./day was used,J
v increased to 2.75±0.07 after 1 day and to 3.10±0.42 10–2 nl·mm–1·s–1 after 4 days.J
v was never reaching a value close to that of euthyroid rats because the tubular radius in hypothyroid rats (14.7±1.8 m) is less than that of euthyroid rats (19.2±0.5 m). The radius in hypothyroid rats treated with T3 was unchanged over a 4 day course with either high or low doses of T3.Na+–K+-ATPase activity was found to be 2.91±0.16 M Pi/h×mg protein in homogenates of kidney cortex from hypothyroid rats. Treatment of hypothyroid rats with 10 g or 50 g of T3 resulted in an initial decrease in ATPase activity, followed by an increase to base level in hypothyroid rats with 10 g and a significantly higher level with 50 g. This decrease in ATPase activity was contrasted to the increase inJ
v.These data indicate that there is a dissociation between the effects of physiological doses of thyroid hormones on proximal tubular reabsorption and the effects of T3 on Na+–K+-ATPase activity of kidney cortex. This leads to question the relationship between sodium transport and ATPase activity under physiological doses of thyroid hormones. An early effect of physiological doses of thyroid hormones on brush border Na+ permeability is suggested. 相似文献
14.
Horst Helbig Christoph Korbmacher Susanne Berweck Dagmar Kühner Michael Wiederholt 《Pflügers Archiv : European journal of physiology》1988,412(1-2):80-85
Uptake studies with22Na were performed in cultured bovine pigmented ciliary epithelial cells, in order to characterize mechanisms of Na+ transport. A large part of Na+ uptake was sensitive to amiloride, quinidine and harmaline. Na+ uptake was stimulated by intracellular acidification (using the NH
4
+
prepulse technique), and was inhibited with increasing extracellular proton concentration. Decreasing extracellular pH from 7.5 to 7.0 increased the apparentK
M for Na+ from 38 to 86 mM without considerable changes inV
max. In the presence of 5 mM Na+ half maximal inhibition of amiloride sensitive Na+ uptake by extracellular protons was observed at a hydrogen concentration of 50 nM. In the presence of 50 mM Na+ the proton concentration necessary for 50% inhibition was 139 nM. Thus, the mode of inhibition of extracellular H+ seemed to be competitive with aK
i of 20–40 nM. 10 M amiloride increased the apparentK
M for Na+ from 33 mM to 107 mM, whileV
max remained nearly unchanged. IC50 for amiloride was 6 M at 5 mM Na+ and 36 M in the presence of 150 mM Na+. Thus, amiloride behaves as a competitive inhibitor with aK
i of about 5 M. The affinities of Na+ to the transport site (K
M16 mM), to the inhibitory site for protons (K
M21 mM), and to the inhibitory site for amiloride (K
M26 mM) were in the same order of magnitude.In summary, we have presented evidence for the presence of a Na+/H+ exchanger in cultured bovine pigmented ciliary epithelial cells. The kinetic data suggest the presence of only one common extracellular binding site for Na+, H+ and amiloride. 相似文献
15.
Brain J. Harvey Jordi Ehrendeld 《Pflügers Archiv : European journal of physiology》1986,406(4):362-366
We have investigated the role of the electrogenic hydrogen ion pump in the regulation of intracellular sodium ion activity (a
Na
i
) and intracellular pH (pHi) in frog skin epithelial cells using double-barreled ion sensitive microelectrodes. WhenRana esculenta skin is mounted in an Ussing chamber and bathed in 1 mM Na2SO4 buffered to pH 7.34 with imidazole on the apical side and in normal Ringer on the serosal side, the apical addition of the carbonic anhydrase inhibitor, ethoxzolamide (10–4M) blocks net H+ ion excretion and Na absorption, producing a depolarization of 25–30 mV of the apical membrane, potential (mc). We demonstrate the these changes are accompanied by a fall ina
Na
i
from 6.2±0.5 mmol/l to 3.4±0.6 mmol/l and an increase in pHi from 7.20±0.03 to 7.38±0.08 (n=12 skins). Voltage clamping mc to its control value in the presence of ethoxzolamide restoreda
Na
i
but the pHi remained alkaline. Furthermore, the fall ina
Na
i
produced by ethoxzolamide could be mimicked by voltage clamping mc towards the value of the Nernst potential for Na at the apical membrane. These results indicate that the maintenance of the cellular Na+ transport pool is dependent on a favourable electrical driving force and counter-current generated by an electrogenic H+ pump at the apical membrane.Addition of amiloride (10–5 mol/l) or substitution of external Na+ by Mg2+ or K+ caused a hyperpolarization of mc and a fall ina
Na
i
. These effects were accompanied by an inhibition of H+ excretion and a fall in pHi of 0.14 ±0.08 units (n=6 skins). However, when the effect, of Na+ transport inhibition on mc was prevented by imposing a voltage clamp no effects of amiloride on H+ excretion or pHi were observed. Moreover, the effect of amiloride on pHi could be reproduced in control skins by voltage clamping mc to –100 mV. The metabolic inhibitors vanadate (1 mmol/l) and di-cyclo hexyl carbodiimide (5×10–5 mol/l) inhibited H+ excretion and decreased pHi from 7.28±0.08 to 7.02±0.06 and from 7.30±0.06 to 7.12±0.05 (n=6 skins), respectively.These results indicate that an apical membrane H+ ATPase plays a role in regulating pHi and the mechanism is sensitive to membrane potential. 相似文献
16.
Demonstration of a novel apamin-insensitive calcium-activated K+ channel in mouse pancreatic B cells
Carina Ämmälä Krister Bokvist Olof Larsson Per -Olof Berggren Patrik Rorsman 《Pflügers Archiv : European journal of physiology》1993,422(5):443-448
The whole-cell configuration of the patchclamp technique was used to characterize the biophysical and pharmacological properties of an oscillating K+-current that can be induced by intracellular application of GTP[S] in mouse pancreatic B cells (Ämmälä et al. 1991). These K+ conductance changes are evoked by periodic increases in the cytoplasmic Ca2+ concentration ([Ca2+]i) and transiently repolarize the B cell, thus inhibiting action-potential firing and giving rise to a bursting pattern. GTP[S]-evoked oscillations in K+ conductance were reversibly suppressed by a high (300 M) concentration of carbamylcholine. By contrast, 2-adrenoreceptor stimulation by 20 M clonidine did not interfere with the oscillatory behaviour but evoked a small sustained outward current. At 0 mV membrane potential, the oscillating K+-current elicited by GTP[S] was highly sensitive to extracellular tetraethylammonium (TEA; 70% block by 1 mM). The TEA-resistant component, which carried approximately 80% of the current at –40 mV, was affected neither by apamin (1 M) nor by tolbutamide (500 M). The current evoked by internal GTP[S] was highly selective for K+, as demonstrated by a 51-mV change in the reversal potential for a sevenfold change in [K+]o. Stationary fluctuation analysis indicated a unitary conductance of 0.5 pS when measured with symmetric ( 140mM) KCl solutions. The estimated singlechannel conductance with physiological ionic gradients is 0.1 pS. The results indicate the existence of a novel Ca2+-gated K+ conductance in pancreatic B cells. Activation of this K+ current may contribute to the generation of the oscillatory electrical activity characterizing the B cell at intermediate glucose concentrations. 相似文献
17.
Kovalev IV Baskakov MB Anfinogenova YD Borodin YL Kilin AA Minochenko IL Popov AG Kapilevich LV Medvedev MA Orlov SN 《Bulletin of experimental biology and medicine》2003,136(2):145-149
The effect of Na+,K+,2Cl- cotransport inhibitor bumetanide on action potentials and contractions of smooth muscle cells in the ureter of guinea pigs evoked by electrical stimulation was studied by the method of double sucrose bridge. Bumetanide (10-100 M) dose-dependently suppressed action potential and contractions of smooth muscle cells induced by 1-10 M histamine, 10 M mesatone, 5 mM tetraethylammonium, and 100 M sodium nitroprusside. Our findings suggest that test substances modulate Na+,K+,2Cl- cotransport in smooth muscle cells. 相似文献
18.
We studied the effect of isoproterenol on the Ca2+-activated K+(BK) channel in smooth muscle cells isolated from the basilar artery of the guinea pig. Cells were studied in a whole-cell configuration to allow the clamping of intracellular Ca2+ concentration, [Ca2+]i. Macroscopic BK channel currents were recorded during depolarizing test pulses from a holding potential (V
H) of 0 mV, which was used to inactivate the outward rectifier. The outward macroscopic current available from aV
H of 0 mV was highly sensitive to block by external tetraethylammonium·Cl (TEA) and charybdotoxin, and was greatly augmented by increasing [Ca2+]i from 0.01 to 1.0 M. With [Ca2+]i between 0.1 and 1.0 M, 0.4 M isoproterenol increased this current by 58.6±17.1%, whereas with [Ca2+]i at 0.01 M a sixfold smaller increase was observed. With [Ca2+]i0.1 M, 100 M dibutyryl-adenosine 3:5: cyclic monophosphate (cAMP) and 1 M forskolin increased this current by 58.5±24.1% and 59.7±10.3%, respectively. The increase with isoproterenol was blocked by 4.0 M propranolol extracellularly, and by 10 U/ml protein kinase inhibitor intracellularly. Single-channel openings during depolarizing test pulses from aV
H of 0 mV recorded in the whole-cell configuration under the same conditions (outside-outwhole-cell recording) indicated a slope conductance of 260 pS. In conventional outside-out patches, this 260-pS channel was highly sensitive to block by external TEA, and in inside-out patches, its probability of opening was greatly augmented by increasing [Ca2+]i from 0.01 to 1.0 M. Outside-out-whole-cell recordings with [Ca2+]i0.1 M indicated that 100 M dibutyryl-cAMP increased the probability of opening of the 260-pS channel by 152±115%. In inside-out patches, the catalytic subunit of protein kinase A increased the probability of opening, and this effect also depended on [Ca2+]i, with a 35-fold larger effect observed with 0.1–0.5 M Ca2+ compared to 0.01 M Ca2+. We conclude that the BK channel in cerebrovascular smooth muscle cells can be activated by-adrenoceptor stimulation, that the effect depends strongly on [Ca2+]i, and that the effect is mediated by cAMP-dependent protein kinase A with no important contribution from a direct G-protein or phosphorylation-independent mechanism. Our data indicate that the BK channel may participate in-adrenoceptor-mediated relaxation of cerebral vessels, although the importance of this pathway in obtaining vasorelaxation remains to be determined. 相似文献
19.
Pekka Mellergård Yi-bing Ouyang Bo K Siesjö 《Experimental brain research. Experimentelle Hirnforschung. Expérimentation cérébrale》1993,95(3):371-380
We studied the regulation of intracellular pH (pHi) and the mechanisms of pHi regulation in cultured rat astrocytes using microspectrofluorometry and the pH-sensitive fluorophore 2,7-bis(carboxyethyl-)-5,6-carboxyfluorescein. Control pHi was 7.00±0.02 in HCO
3
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containing solutions at an extracellular pH of 7.35. Addition of 4, 4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS) or amiloride decreased pHi, as did removal of extracellular Na+, while removal of extracellular Cl- was followed by an increase in pHi. Following exposure to an acid transient induced by increasing the CO2 content from 5 to 15%, pHi rapidly returned to base line, with an average initial rate of recovery of 0.10 pH units min-1 (corresponding to a mean acid extrusion rate of 6.3±0.36 mmolo 1-1 min-1). Regulation of pHi was impaired when either amiloride or DIDS was added or Cl- was removed. This inhibition was enhanced when both DIDS and amiloride were present, and pHi regulation was completely blocked in the absence of extracellular Na+. The rapid regulation of pHi normally seen following a transient alkalinisation was not inhibited by amiloride or removal of Na+, but was partially inhibited by DIDS and by the absence of extracellular Cl-. The results are compatible with the presence of at least three different pHi-regulating mechanisms: a Na+/H+ antiporter, a Na+-dependent HCO
3
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/Cl- exchanger (both regulating pHi during a transient acidification), and a passive Cl-/HCO
3
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exchanger (regulating pHi during transient alkalinisation). The results fail to provide firm evidence of the presence of an electrogenic Na+/HCO
3
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symporter. 相似文献
20.
We used isolated ventricular myocytes to study45Ca2+ transport in the presence of three concentrations of ouabain (10 nM, 1 M, and 100 M) in Tyrode solution containing 1 mM CaCl2. The cells were quiescent and during45Ca2+ uptake and45Ca2+ efflux experiments 10 nM ouabain decreased Ca2+ content, 1 M, didn't change it appreciably, and 100 M increased it significantly. Qualitatively, the same results were obtained at 22°C and 35°C. Ouabain did not significantly affect the electrical activity of isolated, electrically stimulated myocytes, but it increased the amplitude of shortenings of these myocytes in a dose-dependent manner. Thus, the positive inotropic effect of ouabain at therapeutic doses (10 nM) occurs in spite of decreased Ca2+ content, while at high toxic doses the positive inotropic effect is accompanied by an increment in Ca2+ content. These data support the hypothesis that the mechanisms of positive inotropy of ouabain are different at therapeutic and toxic concentrations of this drug. Finally, our study demonstrates that the effects of low doses of ouabain are independent of the release of endogenous catecholamines. 相似文献