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1.
Geyti CS Odgaard E Overgaard MT Jensen ME Leipziger J Praetorius HA 《Pflügers Archiv : European journal of physiology》2008,455(6):1105-1117
Renal epithelia can be provoked mechanically to release nucleotides, which subsequently increases the intracellular Ca2+ concentration [Ca2+]i through activation of purinergic (P2) receptors. Cultured cells often show spontaneous [Ca2+]i oscillations, a feature suggested to involve nucleotide signalling. In this study, fluo-4 loaded Madin–Darby canine kidney
(MDCK) cells are used as a model for quantification and characterisation of spontaneous [Ca2+]i increases in renal epithelia. Spontaneous [Ca2+]i increases occurred randomly as single cell events. During an observation period of 1 min, 10.9 ± 6.7% (n = 23) of the cells showed spontaneous [Ca2+]i increases. Spontaneous adenosine triphosphate (ATP) release from MDCK cells was detected directly by luciferin/luciferase.
Scavenging of ATP by apyrase or hexokinase markedly reduced the [Ca2+]i oscillatory activity, whereas inhibition of ecto-ATPases (ARL67156) enhanced the [Ca2+]i oscillatory activity. The association between spontaneous [Ca2+]i increases and nucleotide signalling was further tested in 132–1N1 cells lacking P2 receptors. These cells hardly showed any
spontaneous [Ca2+]i increases. Transfection with either hP2Y6 or hP2Y2 receptors revealed a striking degree of oscillations. Similar spontaneous [Ca2+]i increases were observed in freshly isolated, perfused mouse medullary thick ascending limb (mTAL). The oscillatory activity
was reduced by basolateral apyrase and substantially lower in mTAL from P2Y2
knock out mice (0.050 ± 0.020 events per second, n = 8) compared to the wild type (0.147 ± 0.018 events per second, n = 9). These findings indicate that renal epithelia spontaneously release nucleotides leading to P2-receptor-dependent [Ca2+]i oscillations. Thus, tonic nucleotide release is likely to modify steady state renal function.
C. S. Geyti and E. Odgaard contributed equally to the publication. 相似文献
2.
Park JK Kim YC Sim JH Choi MY Choi W Hwang KK Cho MC Kim KW Lim SW Lee SJ 《Pflügers Archiv : European journal of physiology》2007,454(2):307-319
Employing microfluorometric system and patch clamp technique in rabbit basilar arterial myocytes, regulation mechanisms of
vascular excitability were investigated by applying intracellular pH (pHi) changers such as sodium acetate (SA) and NH4Cl. Applications of caffeine produced transient phasic contractions in a reversible manner. These caffeine-induced contractions
were significantly enhanced by SA and suppressed by NH4Cl. Intracellular Ca2+ concentration ([Ca2+]i) was monitored in a single isolated myocyte and based the ratio of fluorescence using Fura-2 AM (R
340/380). SA (20 mM) increased and NH4Cl (20 mM) decreased R
340/380 by 0.2 ± 0.03 and 0.1 ± 0.02, respectively, in a reversible manner. Caffeine (10 mM) transiently increased R
340/380 by 0.9 ± 0.07, and the ratio increment was significantly enhanced by SA and suppressed by NH4Cl, implying that SA and NH4Cl may affect [Ca2+]i (p < 0.05). Accordingly, we studied the effects of SA and NH4Cl on Ca2+-activated K+ current (IKCa) under patch clamp technique. Caffeine produced transient outward current at holding potential (V
h) of 0 mV, caffeine induced transient outward K+ current, and the spontaneous transient outward currents were significantly enhanced by SA and suppressed by NH4Cl. In addition, IKCa was significantly increased by acidotic condition when pHi was lowered by altering the NH4Cl gradient across the cell membrane. Finally, the effects of SA and NH4Cl on the membrane excitability and basal tension were studied: Under current clamp mode, resting membrane potential (RMP)
was −28 ± 2.3 mV in a single cell level and was depolarized by 13 ± 2.4 mV with 2 mM tetraethylammonium (TEA). SA hyperpolarized
and NH4Cl depolarized RMP by 10 ± 1.9 and 16 ± 4.7 mV, respectively. SA-induced hyperpolarization and relaxation of basal tension
was significantly inhibited by TEA. These results suggest that SA and NH4Cl might regulate vascular tone by altering membrane excitability through modulation of [Ca2+]i and Ca2+-activated K channels in rabbit basilar artery. 相似文献
3.
Fransen P Van Assche T Guns PJ Van Hove CE De Keulenaer GW Herman AG Bult H 《Pflügers Archiv : European journal of physiology》2008,455(5):811-818
Acetylcholine (ACh)-induced relaxation declines in apolipoprotein E-deficient (apoE−/−) mouse aortas, but only after atherosclerotic plaque formation. This study investigated intracellular calcium concentrations
[Ca2+]i and changes in phenylephrine-induced contractions as index of baseline nitric oxide (NO) bioavailability before plaque development.
Isometric contractions of thoracic aorta rings of young (4 months) apoE−/− and C57BL/6J (WT) mice were evoked by phenylephrine (3 × 10−9–3 × 10−5 M) in the presence and absence of endothelial cells (ECs) or NO synthase (NOS) inhibitors. [Ca2+]i (Fura-2 AM) and endothelium-dependent relaxation were measured at baseline and after ACh stimulation. Segments of apoE−/− mice were significantly more sensitive and developed more tension than WT segments in response to phenylephrine. The differences
disappeared after NOS inhibition or EC removal or upon increasing [Ca2+]i in apoE−/− strips with 10−6 M cyclopiazonic acid or 10−7 M Ca2+-ionophore A23187. Expression of endothelial NOS (eNOS) mRNA was similar in apoE−/− and WT aorta segments. Basal [Ca2+]i was significantly lower in apoE−/− than in WT strips. Relaxation by ACh (3 × 10−9–10−5 M) was time- and dose-dependently related to [Ca2+]i, but neither ACh-induced relaxation nor Ca2+ mobilization were diminished in apoE−/− strips. In conclusion, basal, but not ACh-induced NO bioavailability, was compromised in lesion-free aorta of apoE−/− mice. Decreased basal NO bioavailability was not related to lower eNOS expression, but most likely related to lower basal
[Ca2+]i. These findings further point to important differences between basal and stimulated eNOS activity. 相似文献
4.
Jyoti V. Chauthaiwale Takayuki Sakai Samuel E. Taylor Indu S. Ambudkar 《Pflügers Archiv : European journal of physiology》1996,432(1):105-111
The molecular mechanism(s) involved in mediating Ca2+ entry into rat parotid acinar and other non-excitable cells is not known. In this study we have examined the kinetics of
Ca2+ entry in fura-2-loaded parotid acinar cells, which were treated with thapsigargin to deplete internal Ca2+ pools (Ca2+-pool-depleted cells). The rate of Ca2+ entry was determined by measuring the initial increase in free cytosolic [Ca2+] ([Ca2+]i) in Ca2+-pool-depleted, and control (untreated), cells upon addition of various [Ca2+] to the medium. In untreated cells, a low-affinity component was detected with K
Ca = 3.4 ± 0.7 mM (where K
Ca denotes affinity for Ca2+) and V
max = 9.8 ± 0.4 nM [Ca2+]i /s. In thapsigargin-treated cells, two Ca2+ influx components were detected with K
Ca values of 152 ± 79 μM (V
max = 5.1 ± 1.9 nM [Ca2+]i/s) and 2.4 ± 0.9 mM (V
max = 37.6 ± 13.6 nM [Ca2+]i/s), respectively. We have also examined the effect of Ca2+ and depolarization on these two putative Ca2+ influx components. When cells were treated with thapsigargin in a Ca2+-free medium, Ca2+ influx was higher than into cells treated in a Ca2+-containing medium and, while there was a 46% increase in the V
max of the low-affinity component (no change in K
Ca), the high-affinity component was not clearly detected. In depolarized Ca2+-pool-depleted cells (with 50 mM KCl in the medium) the high-affinity component was considerably decreased while there was
an apparent increase in the K
Ca of the low-affinity component, without any change in the V
max. These results demonstrate that Ca2+ influx into parotid acinar cells (1) is increased (four- to five-fold) upon internal Ca2+ pool depletion, and (2) is mediated via at least two components, with low and high affinities for Ca2+.
Received: 30 October 1995/Received after revisionand accepted: 13 December 1995 相似文献
5.
Kabbara AA Nguyen LT Stephenson GM Allen DG 《Journal of muscle research and cell motility》2000,21(5):481-489
Mechanisms of fatigue were studied in single muscle fibres of the cane toad (Bufo marinus) in which force, intracellular calcium ([Ca2+]i), [Mg2+]i, glycogen and the rapidly releasable Ca2+ from the sarcoplasmic reticulum (SR) were measured. Fatigue was produced by repeated tetani continued until force had fallen to 50%. Two patterns of fatigue in the absence of glucose were studied. In the first fatigue run force fell to 50% in 8–10 min. Fatigue runs were then repeated until force fell to 50% in <3 min in the final fatigue run. Addition of extracellular glucose after the final fatigue run prolonged a subsequent fatigue run. In the first fatigue run peak tetanic [Ca2+]i initially increased and then declined and at the time when force had fallen to 50% tetanic [Ca2+]i was 54 ± 5% of initial value. In the final fatigue run force and peak tetanic [Ca2+]i declined more rapidly but to the same level as in first fatigue runs. At the end of the first fatigue run, the rapidly releasable SR Ca2+ store fell to 46 ± 6% of the pre-fatigue value. At the end of the final fatigue run the rapidly releasable SR Ca2+ store was 109 ± 16% of the pre-fatigue value. In unstimulated fibres the nonwashable glycogen content was 176 ± 30 mmol glycosyl units/l fibre. After one fatigue run the glycogen content was 117 ± 17 mmol glycosyl units/l fibre; at the end of the final fatigue run the glycogen content was reduced to 85 ± 9 mmol glycosyl units/l fibre. [Mg2+]i did not change significantly at the end of fatigue in either the first or the final fatigue run suggesting that globally-averaged ATP does not decline substantially in either pattern of fatigue. These results suggest that different mechanisms are involved in the decline of tetanic [Ca2+]i in first compared to final fatigue runs. The SR Ca2+ store is reduced in first fatigue runs; this is not the case for the final fatigue run which is associated with a decline in glycogen and possibly related to either a non-metabolic effect of glycogen or a spatially-localised metabolic decline. 相似文献
6.
Mitochondrial Ca2+ plays important roles in the regulation of energy metabolism and cellular Ca2+ homeostasis. In this study, we characterized mitochondrial Ca2+ accumulation in Syrian hamster hearts with hereditary cardiomyopathy (strain BIO 14.6). Exposure of isolated mitochondria
from 70 nM to 30 μM Ca2+ ([Ca2+]o) caused a concentration-dependent increase in intramitochondrial Ca2+ concentrations ([Ca2+]m). The [Ca2+]m was significantly lower in cardiomyopathic (CMP) hamsters than in healthy hamsters when [Ca2+]o was higher than 1 μM and a decrease of about 52% was detected at [Ca2+]o of 30 μM (916 ± 67 nM vs 1,932 ± 132 nM in control). A possible mechanism responsible for the decreased mitochondrial Ca2+ uptake in CMP hamsters is the depolarization of mitochondrial membrane potential (Δψ
m). Using a tetraphenylphosphonium (TPP+) electrode, the measured Δψ
m in failing heart mitochondria was −136 ± 1.5 mV compared with −159 ± 1.3 mV in controls. Analyses of mitochondrial respiratory
chain demonstrated a significant impairment of complex I and complex IV activities in failing heart mitochondria. In summary,
a less negative Δψ
m resulting from defects in the respiratory chain may lead to attenuated mitochondrial Ca2+ accumulation, which in turn may contribute to the depressed energy production and myocardial contractility in this model
of heart failure. In addition to other known impairments of ion transport in sarcoplasmic reticulum and plasma membrane, results
from this paper on mitochondrial dysfunctions expand our understanding of the molecular mechanisms leading to heart failure. 相似文献
7.
N. Benning J. Leipziger R. Greger R. Nitschke 《Pflügers Archiv : European journal of physiology》1996,432(1):126-133
The effect of secondary, tertiary and quaternary methyl- and ethylamines on intracellular pH (pHi) and intracellular Ca2+ activity ([Ca2+]i) of HT29 cells was investigated microspectrofluorimetrically using pH- and Ca2+- sensitive fluorescent indicators, [i.e. 2′,7′-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2 respectively].
Membrane voltage (V
m) was studied by the patch-clamp technique. Secondary and tertiary amines led to a rapid and stable concentration-dependent
alkalinization which was independent of their pK
a value. Trimethylamine (20 mmol/l) increased pHi by 0.78 ± 0.03 pH units (n = 9) and pH remained stable for the application time. Removal led to an undershoot of pHi and a slow and incomplete recovery: pHi stayed 0.26 ± 0.06 pH units more acid than the resting value. The quaternary amines, tetramethyl- and tetraethylamine were
without influence on pHi. All tested secondary and tertiary amines (dimethyl-, diethyl-, trimethyl-, and triethyl-amine) induced a [Ca2+]i transient which reached a peak value within 10–25 s and then slowly declined to a [Ca2+]i plateau. The initial Δ[Ca2+]i induced by trimethylamine (20 mmol/l) was 160 ± 15 nmol/l (n = 17). The [Ca2+]i peak was independent of the Ca2+ activity in the bath solution, but the [Ca2+]i plateau was significantly lower under Ca2+-free conditions and could be immediately interrupted by application of CO2 (10%; n = 6), a manoeuvre to acidify pHi in HT29 cells. Emptying of the carbachol- or neurotensin-sensitive intracellular Ca2+ stores completely abolished this [Ca2+]i transient. Tetramethylamine led to higher [Ca2+]i changes than the other amines tested and only this transient could be completely blocked by atropine (10−6 mol/l). Trimethylamine (20 mmol/l) hyperpolarized V
m by 22.5 ± 3.7 mV (n = 16) and increased the whole-cell conductance by 2.3 ± 0.5 nS (n = 16). We conclude that secondary and tertiary amines induce stable alkaline pHi changes, release Ca2+ from intracellular, inositol-1,4,5-trisphosphate-sensitive Ca2+ stores and increase Ca2+ influx into HT29 cells. The latter may be related to both the store depletion and the hyperpolarization.
Received: 11 September 1995/Received after revision and accepted: 18 December 1995 相似文献
8.
Katja H. Madsen Birger Brodin R. Nielsen 《Pflügers Archiv : European journal of physiology》1999,437(2):175-181
In the present study we investigated the possible existence of a Na+/Ca2+ exchange mechanism in the basolateral membrane of the frog skin epithelium and whether such a mechanism plays a role in the
regulation of transepithelial Na+ transport. Cytosolic calcium ([Ca2+]i) was measured with the probe fura-2 in a set-up in which pieces of tissue were mounted on the stage of an epifluorescence
microscope. Na+ transport was measured as the amiloride-sensitive short-circuit current (I
sc) using a conventional voltage clamp. Basal [Ca2+]i was 65±6 nM (n=15). Removal of Na+ from the mucosal solution had no effect on [Ca2+]i. When Na+ was removed from the serosal solution, [Ca2+]i increased biphasically to a peak of 220±38 nM (n=8, P=0.006). Readdition of Na+ to the serosal solution returned [Ca2+]i to control level. The serosal Na+ gradient and changes in [Ca2+]i were closely correlated; stepwise changes in serosal Na+ were followed by stepwise changes in [Ca2+]i. These observations indicate the existence of a Na+/Ca2+ exchange mechanism in the basolateral membrane of the frog skin epithelium. The transepithelial Na+ transport decreased from 13.2±1.8 to 9.2±1.5 μA cm–2 (n=8, P=0.049) when Na+ was omitted from the serosal solution. When this protocol was repeated in the absence of serosal Ca2+, Na+ transport decreased similarly from 16.7±1.7 to 11.6 ±1.8 μA cm–2 (n=6, P=0.004). We conclude that it is unlikely that the observed decrease in I
sc after removal of serosal Na+ is due to an increase in [Ca2+]i per se.
Received: 10 July 1998 / Received after revision: 23 September 1998 / Accepted: 25 September 1998 相似文献
9.
Christine Lamont David A. Eisner 《Pflügers Archiv : European journal of physiology》1996,432(6):961-969
We performed experiments using the calcium indicator Indo-1 to determine the relative roles of the sarcolemmal mechanisms
involved in the regulation of diastolic intracellular calcium concentration ([Ca2+]i) in trabeculae from the rat heart. Ryanodine was used to eliminate sarcoplasmic reticulum (SR) function. In the functional
absence of the SR, 76.8 ± 3.9% of the calcium was extruded by the Na-Ca exchange carrier in the [Ca2+]i range of diastolic concentration ± 200–400 nM. This was assessed by measuring the recovery of [Ca2+]i from small perturbations in the presence and absence of extracellular sodium. The steady-state relationship between [Ca2+]o and [Ca2+]i was linear over the range of 1–40 mM, a 20-fold increase of [Ca2+]o produced a 1.97-fold ± 0.13-fold increase in [Ca2+]i (n = 5). In the absence of extracellular sodium raising [Ca2+]o had a variable effect. In some preparations there was little change of [Ca2+]i while in others the response was almost as large as in control conditions. We conclude that the Na-Ca exchanger contributes
≈ 77% of sarcolemmal calcium extrusion following small perturbations in [Ca2+]i and that this fraction does not diminish as the [Ca2+]i declines. In addition we have shown a sodium-independent entry of calcium into quiescent cardiac muscle under resting conditions.
Received: 16 May 1996 / Received after revision and accepted: 28 June 1996 相似文献
10.
The dependence of intracellular free calcium ([Ca2+]i) and tension on membrane potential and intracellular pH (pHi) was studied in single isolated fibres of the crayfish claw-opener muscle using ion-selective microelectrodes. Tension (T) was quantified as a percentage of the maximum force, or as force per cross-sectional area (N/cm2). In resting fibres, pHi had a mean value of 7.06. Contractions evoked by an increase extracellular potassium ([K+]0) produced a fall in pHi of 0.01–0.05 units. The lowest measured levels of resting [Ca2+]i corresponded to a pCai (= –log [Ca2+]i) of 6.8. Intracellular Ca2+ transients recorded during K+-induced contractions did not reveal any distinct threshold for force development. Both the resting [Ca2+]i and resting tension were decreased by an intracellular alkalosis and increased by an acidosis. The sensitivity of resting tension to a change in pHi (quantified as –dT/ dpHi) showed a progressive increase during a fall in pHi within the range examined (pHi 6.2–7.5). The pHi/[Ca2+]i and pHi/tension relationships were monotonic throughout the multiphasic pHi change caused by NH4Cl. A fall of 0.5–0.6 units in pHi did not produce a detectable shift in the pCai/tension relationship at low levels of force development. The results indicate that resting [Ca2+]i is slightly higher than the level required for contractile activation. They also show that the dependence of tension on pHi in crayfish muscle fibres is attributable to a direct H+ and Ca2+ interaction at the level of Ca2+ sequestration and/or transport. Finally, the results suggest that in situ, the effect of pH on the Ca2+ sensitivity of the myofibrillar system is not as large as could be expected on the basis of previous work on skinned crustacean muscle fibres. 相似文献
11.
U. Langheinrich M. Mederos y Schnitzler J. Daut 《Pflügers Archiv : European journal of physiology》1998,435(3):435-438
To study the role of endothelial ATP-sensitive K+ channels in the regulation of vascular tone we examined the intracellular calcium concentration ([Ca2+]i) in coronary capillaries consisting only of endothelial cells. Coronary capillary fragments were isolated enzymatically from
the guinea-pig heart and [Ca2+]i was determined by microfluorometry of fura-2 loaded cells. Low concentrations of the K+ channel opener diazoxide, which caused pronounced glibenclamide-sensitive hyperpolarization in capillaries, induced a rapid,
transient rise in [Ca2+]i followed by a sustained elevation of [Ca2+]i (19 of 40 experiments). [Ca2+]i in the endothelial cells increased from 32 ± 7 nM at rest to 66 ± 11 nM at the peak (n = 19). One third of the [Ca2+]i-transients showed irregular oscillations of [Ca2+]i. No significant difference in the [Ca2+]i-response induced by 100 nM or 1 μM diazoxide was found. Similar results were obtained with the K+ channel opener rilmakalim. Simultaneous measurements of the membrane potential and [Ca2+]i with fluorometric methods indicated that the hyperpolarization but not the [Ca2+]i-transient could be repeatedly induced in a single capillary by the K+ channel openers. Electrophysiological recordings of the membrane potential using the ”perforated patch” method (n = 4), showed that rilmakalim (1 μM) induced hyperpolarization of capillaries towards the K+ equilibrium potential, confirming our fluorometric measurements. In conclusion, for the first time, these data indicate that
K+ channel openers induce [Ca2+]i-transients in microvascular endothelial cells. This raises the possibility that these drugs not only act as synthetic vasoactive
factors via hyperpolarizing smooth muscle cells but also via NO release of microvascular endothelial cells. Interestingly,
only 100 nM diazoxide was sufficient for a maximal response, suggesting the expression of a new type of KATP-channel in coronary capillaries characterised by high sensitivity to diazoxide.
Received: 22 August 1997 / Received after revision and accepted: 7 November 1997 相似文献
12.
This work describes the gating of the store operated calcium entry (SOCE) in adult mammalian skeletal muscle. Flexor digitorum
brevis fibers (FDB) were isolated from adult mice and exposed to conditions to deplete the sarcoplasmic reticulum (SR). A
transient SR depletion caused either by repetitive depolarizations, chlorocresol (CMC) or, cyclopiazonic acid (CPA) induced
a bell shaped calcium entry that raised the [Ca2+]i to a maximum of 27.09 ± 4.35 nM from the resting value. The activation time to reach 10–90% of the maximum amplitude was
112 ± 10 s (n = 22). On the other hand, any mechanism that caused a permanent SR depletion (like thapsigargin, continuous CPA, or continuous
CMC) triggered a calcium entry pathway that lasted 325 ± 23 s and raised the [Ca2+]i to 129.50 ± 13.05 nM from the resting level (n = 28). Then, a prolonged depletion triggered an increase in [Ca2+]i to higher values and for a longer time than when the SR is transiently depleted (p < 0.001). Our results, in skeletal muscle, showed that calcium store depletion was the signal for SOCE activation and how
the SR got depleted was not relevant. Also, we found that SOCE deactivation was not caused by [Ca2+]i but by the SR content. Our results suggest that the SR calcium content plays an important role in SOCE gating in mammalian
skeletal muscle and a calcium sensor is located inside the SR. 相似文献
13.
A. Shmigol D. A. Eisner S. Wray 《Pflügers Archiv : European journal of physiology》1998,437(1):158-160
In myometrial smooth muscle cells the rate of decline of intracellular calcium ([Ca2+]i) is determined by Ca2+ extrusion from the cell and uptake into intracellular stores. The relative quantitative contribution of these processes however,
has not been established. We therefore examined the effect of the sarcolemmal Ca2+ pump inhibitor, carboxyeosin, on the rate of the [Ca2+]i transient decline in myocytes isolated from pregnant rat uterus. Indo-1 was used in conjunction with the whole-cell patch-clamp
technique to measure [Ca2+]i simultaneously with transmembrane calcium current (I
Ca). [Ca2+]i transients were elicited by repetitive membrane depolarization to simulate the natural pattern of uterine electrical activity.
The rate of [Ca2+]i removal was calculated from the falling phase of the [Ca2+]i transient. Pre-treatment of the cells with 2 μM carboxyeosin led to a marked decrease in the rate of [Ca2+]i transient decay, suggesting that the sarcolemmal Ca2+ pump is involved in the calcium extrusion process. Removal of the extracellular Na also decreased the rate of [Ca2+]i decay, indicating an important role for the Na+/Ca2+ exchange. When both the sarcolemmal Ca2+ pump and Na+/Ca2+ exchange were inhibited the cell failed to restore [Ca2+]i after the stimulation. Comparison of the rate constants of [Ca2+]i decay in control conditions and after carboxyeosin treatment shows that approximately 30% of [Ca2+]i decay is due to the sarcolemmal calcium pump activity. The remaining 70% can be attributed to the activity of Na+/Ca2+ exchanger and the intracellular calcium stores.
Received: 17 July 1998 / Received after revision: 23 September 1998 / Accepted: 25 September 1998 相似文献
14.
Pietrangelo T Mariggiò MA Lorenzon P Fulle S Protasi F Rathbone M Werstiuk E Fanò G 《Journal of muscle research and cell motility》2002,23(2):107-118
Receptor sites, specific for guanosine 5′-triphosphate (GTP) were characterised in myoblasts and myotubes of C2C12 mouse skeletal
muscle cells, using binding experiments and measurements of intracellular Ca2+ concentration ([Ca2+]i). We identified two GTP binding sites in myoblasts membranes: a high affinity site (K
d = 15.4 ± 4.6 μM; B
max = 1.7 ± 0.5 nmol mg−1 protein); and a low affinity site (K
d = 170 ± 94.5 μM; B
max = 14.2 ± 3.9 nmol mg−1 protein). In myotube membranes only a low affinity binding site for GTP (K
d = 169 ± 39 μM; B
max = 12.3 ± 1.4 nmol mg−1 protein) was detected. In myoblasts GTP binding was not displaced by ATP or UTP, even at high concentrations (up to of 1
mM), but it was affected by treatments with suramin or Reactive Blue 2 (RB2), the non-selective purine receptor antagonists.
In contrast, in myotubes GTP binding was partially displaced by high concentrations of ATP, but treatments with the non-selective
purine receptor antagonists, suramin or RB2, and with UTP had no effect on GTP binding. The addition of GTP to myoblasts,
and to myotubes, resulted in elevations of [Ca2+]i. The patterns of Ca2+ response however, were different in the two cell phenotypes. In myoblasts the addition of GTP induced two types of Ca2+ responses: (1) a fast increase in [Ca2+]i, followed by a sustained [Ca2+]i elevation, and (2) a slow raising and steady prolonged increase in [Ca2+]i. In myotubes, however only fast Ca2+ responses were observed following the addition of 500 μM GTP. In the myoblasts and myotubes GTP-stimulated [Ca2+]i increases were abolished by treatments with suramin or RB2 at concentrations which had no effect on the ATP-induced Ca2+ responses. We conclude, that C2C12 cells express two distinct binding sites for GTP before differentiation, but only one
after, the low affinity binding site. These results suggest a possible role of the high affinity GTP binding site in early
stage of development of skeletal muscle.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
15.
K. -G. Fischer J. Leipziger P. Rubini-Illes R. Nitschke R. Greger 《Pflügers Archiv : European journal of physiology》1996,432(4):735-740
In HT29 colonic epithelial cells agonists such as carbachol (CCH) or ATP increase cytosolic Ca2+ activity ([Ca2+]i) in a biphasic manner. The first phase is caused by inositol 1,4,5-trisphophate-(Ins P
3-) mediated Ca2+ release from their respective stores and the second plateau phase is mainly due to stimulated transmembraneous Ca2+ influx. The present study was undertaken to examine the effect of increased adenosine 3′,5′-cyclic monophasphate (cAMP) (forskolin
10 μmol/l = FOR) on the Ca2+ transient in the presence of CCH (100 μmol/l). In unpaired experiments it was found that FOR induced a depolarization and
reduced cytosolic Ca2+ ([Ca2+]i, measured as the fura-2 fluorescence ratio 340/380 nm) significantly. Dideoxyforskolin had no such effect. The effect of
FOR was abolished when the cells were depolarized by a high-K+ solution. In further paired experiments utilizing video imaging in conjunction with whole-cell patch-clamp, [Ca2+]i was monitored separately for the patch-clamped cell and three to seven neighbouring cells. In the presence of CCH, FOR reduced
[Ca2+]i uniformly from a fluorescence ratio (345/380) of 2.9 ± 0.12 to 1.8 ± 0.07 in the patch-clamped cell and its neighbours (n = 48) and depolarized the membrane voltage (V
m) of the patch-clamped cells significantly and reversibly from −54 ± 7.4 to −27 ± 5.9 mV (n = 6). In additional experiments V
m was depolarized by 15–54 mV by various increments in the bath K+ concentration. This led to corresponding reductions in [Ca2+]i. Irrespective of the cause of depolarization (high K+ or FOR) there was a significant correlation between the change in V
m and change in [Ca2+]i. These data indicate that the cAMP-mediated attenuation of Ca2+ influx is caused by the depolarization produced by this second messenger.
Received: 12 March 1996/Accepted: 2 April 1996 相似文献
16.
N. J. H. Raat G. E. C. Wetzels J. G. R. De Mey 《Pflügers Archiv : European journal of physiology》1998,436(2):262-269
We evaluated the relationship between intracellular calcium concentration ([Ca2+]i) and vasoconstriction during the presence of exogenous noradrenaline (NA) and sympathetic nerve stimulation. An imaging technique
was used to determine calcium/tension relationships in isolated rat mesenteric resistance arteries that had been mounted for
recording of isometric tension development and loaded with Fura-2/AM. Experiments were performed after depletion of vasodilator
neuropeptides and in the continuous presence of 1 μM propranolol, 3 μM indomethacin, and 30 μM nitro-l-arginine. NA (10 μM) was shown first to induce a further increase in tension, but not [Ca2+]i, during the contraction induced by 125 mM K+. Subsequently, calcium/tension relationships were determined during stimulation with graded increases in extracellular [K+] (5.9–125 mM K+), cumulative administration of NA (0.2–10 μM) and electrical field stimulation of perivascular nerves (EFS, 1–16 Hz). A basal
calcium/tension relationship without the calcium-sensitizing property of NA was constructed using a cumulative concentration/response
curve of 5.9–125 mM K+ in arteries after prior exposure to the irreversible α-adrenoceptor antagonist phenoxybenzamine (POB). K+ series before and during α-blockade were also studied using the combination of the α1-antagonist prazosin and α2-antagonist yohimbine yielding comparable results as with POB. Calcium/tension curves obtained in the presence of NA, K+ and during EFS all were shifted to the left compared with the basal condition and all showed a similar slope indicating that
neurogenically released NA is equally capable of inducing calcium sensitization in smooth muscle of mesenteric resistance
arteries as exogenously applied NA. In the presence of exogenous and endogenous NA we not only observed an elevated contractile
response for a given increase in [Ca2+]i, but also an attenuated rise in [Ca2+]i for a given intensity of stimulation. This suggests that the agonist-induced calcium-sensitization is accompanied by a reduction
of the rise in [Ca2+]i.
Received: 24 October 1997 / Received after revision: 26 January 1998 / Accepted: 26 February 1998 相似文献
17.
S. P. Cairns H. Westerblad D. G. Allen 《Pflügers Archiv : European journal of physiology》1993,425(1-2):150-155
-Adrenergic agonists increase tension production in fast-twitch skeletal muscle, but the underlying mechanism is unknown. In the present study we have exposed intact, single fibres from a mouse muscle to the
2-adrenergic agonist terbutaline. Fibres were stimulated to produce 350-ms tetani at 20–100 Hz while measuring the myoplasmic Ca2+ concentration ([Ca2+]i) and tension. The fluorescent indicator Indo-1 was used to measure [Ca2+]i. Application of terbutaline resulted in marked increases of both tetanic [Ca2+]i and tension. Terbutaline had no significant effect on myofibrillar function as judged from normal Ca2+ sensitivity and tension production at saturating [Ca2+]i. The rate of [Ca2+]i and tension decline during relaxation was not affected by terbutaline, thus indicating a normal function of the sarcoplasmic reticulum (SR) Ca2+ pumps. The effect of terbutaline developed gradually over 5–10 min when fibres were stimulated each minute; the full effect of terbutaline was also obtained after a 10-min rest period in terbutaline. The [Ca2+]i at rest was not affected by terbutaline. In conclusion, -adrenergic stimulation increases tetanic tension by enhancing SR Ca2+ release. 相似文献
18.
C. Aalkjær Frank V Mortensen Peter E Jensen Henrik Nielsen 《Pflügers Archiv : European journal of physiology》1998,436(5):705-711
In vitro both acetate and hyperosmolarity cause vasodilation, which could be physiologically important during food ingestion
and during peritoneal dialysis. The purpose of this study was to investigate the role of the intracellular calcium concentration
([Ca2+]i, measured with fura-2), membrane potential (measured with glass microelectrodes) and intracellular pH [pHi, measured with bis-carboxyethylcarboxyfluorescein (BCECF)] in the vasodilation. Hyperosmolar sodium acetate (30 mM) concentration
dependently relaxed noradrenaline-precontracted arteries. This response was associated with hyperpolarization and a fall in
[Ca2+]i. In arteries precontracted with 50 mM K+ the relaxation was associated with a decrease of [Ca2+]i but no change in membrane potential. Isoosmolar sodium acetate neither relaxed or affect [Ca2+]i of K+-precontracted arteries, but induced a small relaxation with no reduction in [Ca2+]i in noradrenaline-precontracted arteries. Hyperosmolar acetate caused a transient reduction of pHi that was unrelated to relaxation. It is concluded that the mechanisms responsible for the relaxation to hyperosmolar acetate
involve a decrease of [Ca2+]i, which is only partly explained by hyperpolarization and probably a decrease in the sensitivity of the contractile proteins
to [Ca2+]i. pHi seems not to play a role in these effects.
Received: 14 January 1998 / Received after revision: 22 April 1998 / Accepted: 11 May 1998 相似文献
19.
Peter E. Jensen Michael J. Mulvany Christian Aalkjaer 《Pflügers Archiv : European journal of physiology》1992,420(5-6):536-543
The relationship between isometric tension and free cytoplasmic calcium, [Ca2+]i, was investigated in rat isolated resistance arteries using fura-2. Depolarisation with 125 mM K+ induced a tonic contraction, while [Ca2+]i increased transiently but stabilised above resting [Ca2+]i. Furthermore, the tension/[Ca2+]i ratio was lower during activation with 125 mM K+ if the effect of endogenous noradrenaline (NA) was inhibited. Concentration/ response curves with NA and K+ indicated that NA increased the sensitivity to [Ca2+]i. Calcium concentration/response curves in the presence of 10 M NA or 125 mM K+ showed that NA could induce force at or below resting [Ca2+]i, while for any given bath calcium concentration, [Ca2+]i was similar in the presence of NA or K+. Addition of NA or vasopressin (AVP) to vessels depolarised with 125 mM K+ caused force development but no increase in [Ca2+]i, suggesting that agonists increase the efficacy of [Ca2+]i. However, during activation with AVP the efficacy of [Ca2+]i decreased time-dependently. The results suggest that in resistance arteries [Ca2+]i plays a crucial role in excitation-contraction coupling, but the tension/[Ca2+]i relationship can be modified by exogenous and endogenous agonists. 相似文献
20.
Bella Felzen Gideon Berke Phyllis Gardner O. Binah 《Pflügers Archiv : European journal of physiology》1997,433(6):721-726
We have shown previously that the interaction between cytotoxic T lymphocytes (CTL) and ventricular myocytes, an in vitro
model for heart transplant rejection, results in electrophysiological and morphological alterations indicative of overload
of the intracellular [Ca2+] ([Ca2+]i). Since these deleterious effects cannot be accounted for by increased L-type Ca2+ current (I
Ca,L), we hypothesize that [Ca2+]i overload due to Ca2+ release from intracellular stores, e.g. sarcoplasmic reticulum (SR), is initiated by CTL-induced activation of the inositol
trisphosphate (IP3) cascade. Patch-clamp and fura-2-fluorescence techniques were utilized to record transmembrane potentials and [Ca2+]i from ventricular myocytes bound to peritoneal exudate CTL (PEL). In ventricular myocyte-PEL conjugates (after 60 min), resting
potential was reduced (compared with the nonconjugated state) from –80.9 ± 0.7 to –59.9 ± 2.5 mV, action potential amplitude
from 139.5 ± 1.4 to 80.6 ± 1.7 mV and action potential duration to 50% repolarization (APD50) from 797 ± 97 to 52 ± 12 ms. The ratio of fluorescence at 340 and 380 nm (R
340/380) increased from a control value (in nonconjugated myocytes) of 0.71 ± 0.02 to 2.07 ± 0.03, 30 min after conjugate formation,
and exceeded 4.0 at 60 min, before myocyte destruction. Heparin (50 μg/ml), an antagonist of IP3-induced Ca2+ release from SR channels, or U-73122 (2 μM), a phospholipase C (PLC) inhibitor (drugs were included in the pipette solution),
prevented PEL-induced morphological and electrophysiological alterations. Accordingly, heparin attenuated the PEL-induced
increase in [Ca2+]i; after 60 min of PEL-myocyte interaction, R
340/380 was 1.15 ± 0.09 (compared with approximately 4.0 in the absence of heparin). The results indicate that CTL-mediated damage
to ventricular myocytes is, at least partially, mediated by PLC activation and IP3-induced Ca2+ release from intracellular stores. Pharmacological targeting of IP3 in heart transplant rejection is thus suggested.
Received: 3 July 1996 / Received after revision: 21 October 1996 / Accepted: 3 December 1996 相似文献