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1.
Gao J Mathias RT Cohen IS Wang Y Sun X Baldo GJ 《Pflügers Archiv : European journal of physiology》1999,437(5):643-651
We have previously shown activation of α1-adrenergic receptors increases Na+-K+ pump current (I
p) in guinea pig ventricular myocytes, and the increase is eliminated by blockers of phosphokinase C (PKC). In this study we
examined the effect of activators of PKC on I
p. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased I
P at each test potential without shifting its voltage dependence. The concentration required for a half-maximal response (K
0.5) was 6 μM at 15 nM cytosolic [Ca2+] ([Ca2+]i) and13 nM at 314 nM [Ca2+]i. The maximal increase at either [Ca2+]i was about 30%. Another activator of PKC, 1,2-dioctanoyl-sn-glycerol (diC8), increased I
p similarly. The effect of PMA on I
P was eliminated by the PKC inhibitor staurosporine, but not by the peptide PKI, an inhibitor of protein kinase A (PKA). PMA
and α1-adrenergic agonist effects both were sensitive to [Ca2+]i, blocked by PKC inhibitors, unaffected by PKA inhibition, and increased I
p uniformly at all voltages. However, they differed in that α1-activation caused a maximum increase of 15% vs 30% via PMA, and α1-effects were less sensitive to [Ca2+]i than PMA effects. These results demonstrate that activation of PKC causes an increase in I
p in guinea pig ventricular myocytes. Moreover, they suggest that the coupling of α1-adrenergic activation to I
p is entirely through PKC, however α1-activation may be coupled to a specific population of PKC whereas PMA is a more global agonist.
Received: 21 August 1998 / Received after revision: 7 December 1998 / Accepted: 11 December 1998 相似文献
2.
Using the whole-cell voltage-clamp method to measure ATP-sensitive K+(KATP) currents, changes in cell capacitance to measure secretion and microfluorimetry to monitor intracellular Ca2+ and mitochondrial function, we have investigated the direct effect of sulphonylureas on exocytosis in pancreatic β-cells.
Tolbutamide (100 μM) and 100 nM 4-β-12-phorbolmyristate-13-acetate (PMA), which activates the protein kinase C (PKC) isoforms
found in β-cells, potentiated exocytosis in a non-additive manner. These effects were blocked by down-regulation of PKC. Our
data support the idea that tolbutamide can potentiate secretion from β-cells via a PKC-dependent pathway. Because PKC and
sulphonylureas can modulate the activity of KATP channels, we explored whether the above effects are caused by inhibition of this channel. PMA increased whole-cell KATP currents but did not affect their sensitivity to tolbutamide. Down-regulation of PKC affected neither the magnitude nor the
tolbutamide sensitivity of the KATP current. Both tolbutamide and the mitochondrial uncoupler FCCP (1 μM) mobilized intracellular Ca2+ and prolonged Ca2+ transients elicited by cholinergic mobilization of intracellular Ca2+ stores. Tolbutamide (0.1–0.5 mM), like FCCP, depolarized the mitochondrial membrane potential and activated KATP currents. We suggest that sulphonylureas can directly potentiate exocytosis by impairing mitochondrial function and Ca2+ handling, which ultimately leads to activation of Ca2+-dependent enzymes such as PKC.
Received: 1 September 1998 / Received after revision: 9 November 1998 / Accepted: 10 November 1998 相似文献
3.
R. Greger Markus Bleich Richard Warth I. Thiele John N. Forrest 《Pflügers Archiv : European journal of physiology》1999,438(1):15-22
We have examined the mechanism whereby C-type natriuretic peptide (CNP), an agonist acting through the second messenger cGMP,
enhances NaCl secretion in the rectal gland of Squalus acanthias. Single rectal gland tubules (RGT) were dissected manually, perfused in vitro and equivalent short-circuit current [I
sc=transepithelial voltage/transepithelial resistance (R
te)] as well as basolateral membrane voltage (V
bl) were measured. CNP was added to luminal and basolateral perfusates at concentrations between 1 and 1000 nmol/l and its effects on the above
parameters were compared to those of a ”stimulation cocktail” (Stim, containing dibutyryl cAMP, adenosine and forskolin) that
maximally enhances cytosolic cAMP, and other agonists and hormones such as guanylin, vasoactive intestinal peptide (VIP),
and adenosine. CNP had no effect from the luminal side (n=6). Its effects from the basolateral side consisted of a substantial increase in I
sc (–31.6±7.7 to –316±82.2 μA/cm2, n=15). CNP significantly depolarized the luminal membrane from –87.4±1.0 to –82.3±2.6 mV (n=12). V
bl was not changed (n=12) but the fractional conductance for K+ was increased (n=3). These effects were qualitatively and even quantitatively comparable to those of other agonists acting via cytosolic cAMP,
but were less marked than those caused by Stim (n=64). The effects of VIP and CNP on I
sc were not additive (n=5). The cytosolic Ca2+ concentration ([Ca2+]i) was monitored using the fura-2 fluorescence ratio (FFR 340/380 nm) and it was found that CNP, like agonists acting via cAMP,
enhances FFR significantly from 1.02±0.05 to 1.32±0.05 (n=8) with a time constant in the 1–2 min in range. Our data suggest that CNP, acting via the second messenger cGMP, induces
a marked increase in I
sc in the rectal gland. The concomitant fall in R
te corresponds to increases in the luminal membrane Cl– conductance and in the basolateral membrane K+ conductance. The latter effect is probably due to an increase in [Ca2+]i.
Received: 21 December 1998 / Received after revision: 5 February 1999 / Accepted: 9 February 1999 相似文献
4.
Angiotensin II stimulation of Ca2+-channel current in vascular smooth muscle cells is inhibited by lavendustin-A and LY-294002 总被引:1,自引:1,他引:0
T. Seki Hisashi Yokoshiki Masanori Sunagawa Mariko Nakamura Nicholas Sperelakis 《Pflügers Archiv : European journal of physiology》1999,437(3):317-323
Angiotensin II (AngII) is coupled to several important intracellular signaling pathways, and increases intracellular Ca2+. In vascular smooth muscle (VSM) cells, AngII is known to activate enzymes such as tyrosine protein kinase (Tyr-PK), phospholipase
C (PLC), protein kinase C (PKC), and phophatidylinositol-3-kinase (PI-3-K). A non-receptor Tyr-PK, pp60c-src, and PKC have been reported to stimulate the Ca2+ channels in VSM cells. However, less is known about AngII action on the voltage-gated Ca2+ channels. The Ca2+-channel currents of a cultured rat aortic smooth muscle cell line, A7r5, were recorded using whole-cell voltage clamp. Application
of 50 nM AngII significantly increased the amplitude of Ba2+ currents through the voltage-gated Ca2+ channels (I
Ba) by 34.5±9.1% (n=10) within 1 min. In the presence of lavendustin-A (5 μM), a selective inhibitor of Tyr-PK, AngII failed to stimulate I
Ba (n=5). AngII stimulation of I
Ba was also prevented by (5 μM) LY-294002, an inhibitor of PI-3-K (n=5). In contrast, H-7 (30 μM), an inhibitor of PKC, did not prevent the effect of AngII on I
Ba (n=6). These results suggest that AngII may stimulate the Ca2+ channels of VSM cells through Tyr-PK and PI-3-K under conditions that probably exclude participation of PK-C.
Received: 17 July 1998 / Received after revision: 10 September 1998 / Accepted: 29 September 1998 相似文献
5.
Protein kinase C stimulates swelling-induced chloride current in canine atrial cells 总被引:10,自引:0,他引:10
The whole-cell patch-clamp technique was used to study the effect of protein kinase C (PKC) stimulation and α-adrenergic
agonists on the swelling-induced chloride current (I
Cl,swell) in canine atrial cells. I
Cl,swell was activated by positive-pressure inflation. 4β-Phorbol 12, 13-dibutyrate (PDBu) concentration-dependently stimulated I
Cl,swell. PDBu (500 nM) increased the current density of I
Cl,swell from 9.1±1.3 to 24.2±4.8 pA/pF at +20 mV (n=4). This effect developed slowly, reaching a steady-state after more than 5 min of exposure. 4α-Phorbol 12, 13-dibutyrate
(4α-PDBu, 500 nM), an inactive analogue of PDBu, did not affect I
Cl,swell. The effect of PDBu was inhibited by bisindolylmaleimide I. After down regulation of PKC by phorbol 12-myristate 13-acetate
(PMA, 1.6 μM, 24 h), I
Cl,swell no longer responded to PDBu (n=4). Neither the basal whole-cell current (prior to cell inflation) nor inflation-induced I
Cl,swell were affected by PKC down regulation. Phenylephrine did not affect I
Cl,swell. We conclude that PKC activity stimulates and does not prevent the activation of dog atrial I
Cl,swell. These results contrast with reports of PKC-dependent inhibition of rabbit atrial I
Cl,swell and currents conducted by ClC-3, a putative clone for I
Cl,swell. The data suggest species-dependent variations in the modulation of cardiac I
Cl,swell by PKC.
Received: 19 June 1998 / Received after revision: 15 September 1998 / Accepted: 25 September 1998 相似文献
6.
A. W. Trafford M. E. Díaz D. A. Eisner 《Pflügers Archiv : European journal of physiology》1999,437(3):501-503
This paper outlines a simple method of estimating both the Ca-buffering properties of the cytoplasm and the time-course of
changes of sarcoplasmic reticulum (s.r.) Ca concentration during systole. The experiments were performed on voltage-clamped
ferret single ventricular myocytes loaded with the free acid of fluo-3 through a patch pipette. The application of caffeine
(10 mM) resulted in a Na-Ca exchange current and a transient increase of the free intracellular Ca concentration ([Ca2+]i). The time-course of change of total Ca in the cell was obtained by integrating the current and this was compared with the
measurements of [Ca2+]i to obtain a buffering curve. This could be fit with a maximum capacity for the intrinsic buffers of 114±18 μmol l–1 and K
d of 0.59±0.17 μM (n=8). During the systolic rise of [Ca2+]i, the measured changes of [Ca2+]i and the buffering curve were used to calculate the magnitude and time-course of the change of total cytoplasmic Ca and thence
of both s.r. Ca content and Ca release flux. This method provides a simple and reversible mechanism to measure Ca buffering
and the time-course of both total cytoplasmic and s.r. Ca.
Received: 14 October 1998 / Accepted: 6 November 1998 相似文献
7.
S. Lipscomb R. E. Palmer Q. Li L. D. Allhouse T. Miller J. D. Potter C. C. Ashley 《Pflügers Archiv : European journal of physiology》1999,437(2):204-212
The aim of this study was to compare the effects of increased concentrations of MgADP, inorganic phosphate (Pi) and H+ ([MgADP], [Pi] and [H+], respectively) on the rate of relaxation in two different muscle types: skinned muscle fibres from the frog Rana temporaria and myofibrillar bundles from the giant Pacific acorn barnacle Balanus nubilus. Relaxation transients are produced by the photolysis of diazo-2 and are well fitted with a double exponential curve, giving
two rate constants: k1 [5.6±0.1 s–1 for barnacle, n=30; 26.3±0.7 s–1 for frog, n=14 (mean±SEM)] and k2 [0.6±0.1 s–1 in barnacle, n=30; 10.4±1.0 s–1 in frog, n=14 (mean±SEM)], at 10°C. Decreasing the pH by 0.5 pH units did not significantly affect k1 for barnacle relaxation [5.6±0.1 s–1 (mean±SEM), n=15] compared to the decrease in k1 of 40% seen in frog. Use of the Ca2+-sensitive fluorescent label acrylodan on barnacle wild-type troponin C demonstrated that decreasing the pH from 7.0 to 6.6
only alters the pCa50 value by 0.23 in the cuvette, while stopped-flow experiments with acrylodan revealed no significant change in koff from the labelled protein [322±32 s–1 at pH 7.0 and 381±24 s–1 (mean±SEM) at pH 6.6]. Increasing [MgADP] by 20 μM (50 μM added ADP) from control values of 50 μM in frog decreased k1 to 12.3±0.4 s–1 (mean±SEM, n=8), and at 400 μM MgADP, k1=9.6±0.1 s–1 (mean±SEM, n=12). In barnacle, 500 μM MgADP had a much smaller effect on k1 (4.0±0.9 s–1, mean±SEM, n=8). Increasing the free [Pi] from the contaminant level of 0.36 mM to 1.9 mM slowed k1 by ≈15% in barnacle [4.8±0.8 s–1, mean±SEM, n=7], compared to a ≈30% reduction seen in frog. We conclude that the differences between barnacle and frog seen here are most
probably due to different isomers of the contractile proteins, and that events underlying the crossbridge cycle are the same
or similar. We interpret our results according to a model of crossbridge transitions during relaxation.
Received: 18 May 1998 / Received after revision and accepted: / 1 September 1998 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.
Satoh S Kinugawa S Tsutsui H Takeshita A 《Pflügers Archiv : European journal of physiology》1999,437(5):702-709
To investigate the mechanisms of receptor-mediated regulation of heart muscle contraction, we developed a tension-recording
system using β-escin-skinned single cardiac cells of rats and studied the effects of agonists on myofibrillar Ca2+ sensitivity and Ca2+ release from the sarcoplasmic reticulum (SR). In pCa/tension relations, 1 μM isoproterenol plus 100 μM guanosine 5′-triphosphate
(GTP) decreased the myofibrillar Ca2+ sensitivity (pCa50, the [Ca2+] required for half-maximal tension, as an indicator of the sensitivity; from 6.07 to 5.92); this effect was blocked by 1
μM metoprolol or 1 mM guanosine 5′-O-(2-thiodiphosphate) (GDPβS). Phenylephrine (10 μM) plus 100 μM GTP increased the Ca2+ sensitivity (pCa50; from 6.12 to 6.28), and this effect was blocked by 1 μM phentolamine or 1 mM GDPβS. After Ca2+ loading into the SR, 10 μM phenylephrine plus 100 μM GTP in a low-ethylene- glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA, 0.1 mM) relaxing solution induced oscillatory contractions that were attenuated by either 1 μM phentolamine
or pre-treatment with 10 μM inositol 1,4,5-trisphosphate. Our results demonstrate that β1-adrenergic stimulation decreases myofibrillar Ca2+ sensitivity and that α1-adrenergic stimulation both increases the Ca2+ sensitivity and activates Ca2+ release from the agonist-sensitive SR through GTP-binding protein-related mechanisms.
Received: 17 August 1998 / Received after revision: 23 October 1998 / Accepted: 15 December 1998 相似文献
10.
Tomohiko Ai M. Horie Kazuhiko Obayashi Shigetake Sasayama 《Pflügers Archiv : European journal of physiology》1998,436(2):168-174
To examine mechanism(s) underlying the accentuated antagonism by angiotensin II (A-II) on twitch tension, we recorded L-type
Ca2+ currents (I
Ca,L) using conventional patch-clamp techniques in single, guinea-pig, ventricular myocytes. I
Ca,L was recorded by a step-pulse protocol after eliminating K+ conductances (internal Cs+ plus tetraethylammonium chloride and K+-free extracellular solution). A-II (100 nM) did not affect basal I
Ca,L, but inhibited I
Ca,L that had been enhanced (approximately 200% of control) by (ISO, isoproterenol 100 nM). The inhibitory action of A-II was
concentration dependent (concentration eliciting 50% inhibition 88±9 pM, n=41) and the ISO-enhanced component of I
Ca,L was completely blocked by A-II at concentrations above 10 nM. CV-11974 (500 nM), an A-II type-1 receptor (AT1) antagonist, prevented the inhibitory action of A-II. Pre-incubation with pertussis toxin (PTX) abolished the inhibitory
effect of A-II. A-II also inhibited the I
Ca,L enhanced by histamine (500 nM) and forskolin (1 μM), but failed to affect I
Ca,L enhanced by intracellular cyclic adenosine monophosphate (1 mM). The inhibitory action of A-II may therefore involve AT1 receptors/PTX-sensitive, guanine nucleotide-binding (G) proteins (Gi)/adenylate cyclase and partially explains the A-II-dependent
accentuated antagonism of inotropy. 相似文献
11.
Mary K. Convery Allan J. Levi Daniel Khananshvili J. C. Hancox 《Pflügers Archiv : European journal of physiology》1998,436(4):581-590
In cardiac muscle, the electrogenic Na-Ca exchanger plays important roles in determining action potential shape and in the
beat-to-beat homeostasis of intracellular calcium. In this study we tested the actions of a putative cell-permeant blocker
of the cardiac sarcolemmal Na-Ca exchange, ”Myristyl- (Myr-) FRCRCFa”. Experiments were performed using isolated rabbit right
ventricular myocytes and whole-cell patch-clamp at 35–37°C. The Na-Ca exchange current (I
Na-Ca), L-type calcium current (I
Ca,L), inward rectifier potassium current (I
K1) and delayed rectifier potassium current (I
K) were compared in untreated cells and cells incubated in a solution containing N-myristylated FRCRCFa. With other major currents blocked, I
Na-Ca was measured as the Ni-sensitive component of current during a voltage ramp applied from the holding potential of –40 mV,
between +80 and –120 mV (ramp velocity 0.1 V s–1). In untreated cells, I
Na-Ca at +60 mV was 7.1±0.6 pA/pF and at –100 mV was –2.7±0.3 pA/pF (n=9). After a 15-min pre-incubation with 20 μM Myr-FRCRCFa, I
Na-Ca was reduced to 4.2±0.3 pA/pF at +60 mV and –1.5±0.2 pA/pF at –100 mV (P<0.02; n=7). After incubation with 20 μM Myr-FRCRCFa for 1 h, I
Na-Ca at both potentials was further reduced (2.3±0.8 pA/pF at +60 mV; –0.9±0.3 pA/pF at –100 mV; P<0.008 compared with control; n=4). Under selective recording conditions for I
Ca,L, there was little difference in I
Ca,L density between untreated and cells incubated with Myr-FRCRCFa. A Boltzmann fit to the I
Ca,L/V relation showed no significant alteration of half-maximal activation potential or slope factor of activation. I
K1 was also largely unaffected by pre-incubation of cells with Myr-FRCRCFa. I
K, measured as deactivating tail current following 1-s test depolarisations to a range of test potentials, was also not significantly
altered by Myr-FRCRCFa. The suppression of I
Na-Ca in cells incubated in Myr-FRCRCFa suggests that addition of the myristyl group to FRCRCFa peptide conveys cell permeancy
to the peptide and that Myr-FRCRCFa applied externally to rabbit ventricular myocytes is moderately effective as an I
Na-Ca blocker. I
Ca,L, I
K1 and I
K were largely unaffected by Myr-FRCRCFa. N-Myristylation of such conformationally constrained hexapeptides may, therefore, provide a means of producing cell-permeant
inhibitors of the cardiac Na-Ca exchanger.
Received: 6 February 1997 / Received after revision: 8 April 1998 / Accepted: 9 April 1998 相似文献
12.
Hussain M Drago GA Bhogal M Colyer J Orchard CH 《Pflügers Archiv : European journal of physiology》1999,437(4):529-537
We investigated the effects of a protein kinase A (PKA) inhibitor, H-89 {N-[2-(p-bromocinnamylamino)ethyl]-5-iso-quinolinesulphonamide}, on Ca2+ regulation in Fura-2-loaded ferret myocytes. H-89 (10 μmol/l) decreased the amplitude of the Fura-2 transient to 28.2±4.3%
(P<0.001) of control and prolonged its duration, characterized by a decrease in the rate of decline of Ca2+ to diastolic levels: t
1/2 increased from 311±35 ms to 547±43 ms (P<0.001, n=7). Reduced Ca2+ uptake by the sarcoplasmic reticulum (SR) in the presence of H-89 was also indicated by a decrease in the SR Ca2+ content, as assessed with caffeine. The apparent slowing of the SR Ca2+-ATPase was not caused by changes in phosphorylation of phospholamban (PLB). However, Ca2+ uptake in microsomal vesicles prepared from canine hearts and fast-twitch rat skeletal muscle (which lacks PLB) was decreased
by 34.1 and 46.8% (n=3), respectively, suggesting that H-89 has a direct inhibitory effect on the SR Ca2+-ATPase. In electrophysiological experiments, 5.0 μmol/l H-89 decreased the L-type Ca2+ current (I
Ca) by 39.5% (n=6) and slowed the upstroke of the action potential and, in some cases, caused loss of excitability without changes in the
resting membrane potential. In summary, data show that [Ca2+ ]i regulation, and hence contraction, is sustained by PKA-mediated phosphorylation, even in the absence of β-agonists. However,
the use of H-89 as a tool to study the role of this signalling pathway is limited by the non-specific effects of H-89 on the
SR Ca2+-ATPase.
Received: 4 September 1998 / Received after revision: 19 October 1998 / Accepted: 20 October 1998 相似文献
13.
Akinori Kuruma Masayasu Hiraoka S. Kawano 《Pflügers Archiv : European journal of physiology》1998,436(6):976-983
We investigated how Ca2+-sensitive transient outward current, I
to(Ca), is activated in rabbit ventricular myocytes in the presence of intracellular Na+ (Na+
i) using the whole-cell patch-clamp technique at 36°C. In cells dialysed with Na+-free solutions,the application of nicardipine (5 μM) to block L-type Ca2+ current (I
Ca) completely inhibited I
to(Ca). In cells dialysed with a [Na+]i≥5 mM, however, I
to(Ca) could be observed after blockade of I
Ca, indicating the activity of an I
Ca-independent component. The amplitude of I
Ca-independent I
to(Ca) increased with voltage in a [Na+]i-dependent manner. The block of Ca2+ release from the sarcoplasmic reticulum by caffeine, ryanodine or thapsigargin blocked I
Ca-independent I
to(Ca). In Ca2+-free bath solution I
to(Ca) was completely abolished. The application of 2 mM Ni2+ or the newly synthesized compound KBR7943, a selective blocker of the reverse mode of Na+/Ca2+ exchange, or perfusion with pipette solution containing XIP (10 μM), a selective blocker of the exchanger, blocked I
Ca-independent I
to(Ca). From these results we conclude that, in the presence of Na+
i, I
to(Ca) can be activated via Ca2+-induced Ca2+ release triggered by Na+/Ca2+ exchange operating in the reverse mode after blockade of I
Ca.
Received: 20 January 1998 / Received after revision: 6 July 1998 / Accepted: 25 July 1998 相似文献
14.
Jesús-Miguel Hernández-Guijo Luis Gandía Baldomero Lara A. G. García 《Pflügers Archiv : European journal of physiology》1998,437(1):104-113
Applying 10-s pulses of 10 mM Ba2+ to resting or K+-depolarized (70 mM) bovine adrenal chromaffin cells superfused with a nominal 0Ca2+ solution produced a large catecholamine secretory peak. In contrast, pulses of 10 mM Sr2+ or Ca2+ did not induce secretion from polarized resting cells, and induced smaller and narrower secretory peaks from depolarized
cells; the areas of the secretory peaks from depolarized cells were 1.87, 3.06 and 27.4 nA s, respectively, for Ca2+, Sr2+ and Ba2+. Ca2+ channel currents in isolated cells or in cells surrounded by other unpatched cells (cell cluster) were studied with either
the continuous-flow or the flow-stop method. When applied to an isolated cell, flow-stop reduced the amplitude of I
Ca by 19%, I
Sr by 31%, and I
Ba by 53%, compared with the current amplitude measured under continuous-flow conditions. This decrease in current amplitude
was accompanied by a pronounced slowing down of current activation and could be largely relieved by applying strong depolarizing
prepulses (facilitation). Under continuous-flow conditions, 10 μM exogenous ATP reduced (about 50%) I
Ca, I
Sr and I
Ba similarly. On the other hand, the use of Na+ as a charge carrier through Ca2+ channels, or intracellular dialysis with 1 mM BAPTA prevented the modulation of current by flow-stop. In cell clusters, activating
secretion from unpatched cells, by either 10 mM Ba2+, 100 μM acetylcholine or 70 mM K+, caused a pronounced slowing down of current activation, as well as a decrease of its magnitude in the voltage-clamped cell
immersed in the cluster. Such modulation of isolated cells was not observed. These data are compatible with the idea that
the secretory activity of adrenal medullary chromaffin cells ”in situ” controls the activity of their Ca2+ channels through autocrine/paracrine mechanisms.
Received: 29 June 1998 / Received after revision: 20 August 1998 / Accepted: 1 September 1998 相似文献
15.
P. Poronnik L. M. O’Mullane A. D. Conigrave D. I. Cook 《Pflügers Archiv : European journal of physiology》1999,438(1):79-85
HSG and HT29 cells express muscarinic receptors that increase intracellular free Ca2+ ([Ca2+]i) by activating phospholipase Cβ. In the present study, we have used the measurement of [Ca2+]i with Fura-2 to show that these receptors are of the M3 sub-type and that the increase in [Ca2+]i triggered when they are activated is not sensitive to pertussis toxin. We have also used replication-deficient adenoviruses
expressing wild-type and dominant-negative mutants of the α-subunits of the heterotrimeric G proteins, Gq and Gi2, to investigate the mechanisms by which these receptors control phospholipase Cβ. We find that the Ca2+ response to 100 μmol/l carbachol is not affected by increased expression of the wild-type α-subunit of Gq, but is blocked by the dominant-negative mutant of Gq and by both the wild-type and the dominant-negative mutant α-subunits of Gi2. Expression of α-subunits of Gi2 presumably blocks the response to carbachol by scavenging free βγ-subunits. We conclude that in HSG and HT29 cells, the Ca2+ response to M3 receptor activation is mediated by the βγ- rather than the α-subunits of Gq.
Received: 23 September 1998 / Received after revision: 15 December 1998 / Accepted: 11 January 1999 相似文献
16.
I. P. Mulligan R. E. Palmer S. Lipscomb B. Hoskins C. C. Ashley 《Pflügers Archiv : European journal of physiology》1999,437(3):393-399
The effect of phosphate on the relaxation of isometrically contracting single skinned fibres from the semitendinosus muscle
of the frog Rana temporaria has been investigated using laser pulse photolysis of the photolabile caged calcium-chelator diazo-2 to rapidly reduce the
Ca2+ (<2 ms) within the fibre and produce >90% relaxation of force. Relaxation occurred in two phases – an initial linear shoulder
which lasted approximately 20 ms followed by a double-exponential phase which gave two rate constants, k
1 (43.4±1.8 s–1, mean ±SEM, n=14) and k
2 (15.6±0.3 s–1, mean ±SEM, n=14) at 12°C. Increased phosphate concentrations did not affect the linear phase, but slowed the double-exponential phase
following photolysis of diazo-2 in a dose-dependent fashion (k
50= 0.9 mM for k
1, 1.15 mM for k
2). Reducing the concentration of contaminating phosphate (from 640 μM to 100 μM) led to an increase in the rate of the double-exponential
phase (k
1=67.1±4.4 s–1, k
2=19.7±0.6 s–1, mean ±SEM, n=12). Time-resolved measurements of sarcomere length during relaxation, both in control fibres and in the presence of a raised
phosphate concentration, reveal a <2% change throughout the whole relaxation transient, and less than 0.1% at the end of the
linear phase. This finding implies that gross changes in sarcomere length do not contribute to the decay of the relaxation
transient seen upon diazo-2 photolysis. Our results suggest that cross-bridges in states prior to phosphate release are already
committed to force generation and must relax by releasing phosphate, rather than by a reversal of the force-generating step
to a weakly bound, low-force phosphate-bound state. These findings also indicate that an increase in the phosphate concentration
within muscle fibres plays an important part in the slowing of relaxation observed in skeletal muscle fatigue and that the
relaxation transients observed upon diazo-2 photolysis represent a disengagement of the cross-bridges.
Received: 14 September 1998 / Received after revision and accepted: 20 October 1998 相似文献
17.
M. Hatch Robert W. Freel N. D. Vaziri 《Pflügers Archiv : European journal of physiology》1998,436(5):717-724
The effect of angiotensin II (ANG II) on potassium transport across the short-circuited rat distal colon was investigated
using 86Rb+ as a tracer for unidirectional K+ fluxes. The addition of high concentrations of ANG II (≥10–6 M) to the serosal bathing solution had no effect on the mucosal to serosal flux of Rb+, but significantly increased the serosal to mucosal flux and abolished the basal net absorptive Rb+ flux. These ANG-II-induced alterations in Rb+ transport were blocked by the AT1 receptor antagonist losartan and its metabolite EXP3174, which is also known to have AT1 receptor antagonistic activity. In contrast, an AT2 receptor antagonist, PD123319, did not prevent the alterations in colonic Rb+ transport induced by ANG II in vitro. At lower bath concentrations (10–7 M to 10–10 M ), ANG II had no measurable effects on Rb+ transport across this tissue but ANG II did have a bimodal effect on short-circuit current (I
sc), indicating additional effects on the electrogenic transport of other ions. Dose-dependent reductions in I
sc were observed between 10–7 M (↓ΔI
sc=1.96±0.49 μEq·cm–2·h–1, n=6) and 10–10 M (↓ΔI
sc=0.16±0.19 μEq·cm–2·h–1, n=7) ANG II, whereas I
sc was increased at the higher concentrations (↑ΔI
sc= 3.36±0.52 μEq·cm–2·h–1, n=7, at 10–4 M). The ANG-II-induced increases and decreases in I
sc were both blocked by losartan but not by PD123319. These studies are the first to demonstrate an effect of ANG II on K+ transport across rat colon that is independent of aldosterone and mediated by AT1 receptors.
Received: 13 March 1998 Received after revision and accepted: 26 May 1998 相似文献
18.
Young Chul Kim Sung Joon Kim Jae Hoon Sim Jae Yeoul Jun Tong Mook Kang Suk Hyo Suh Insuk So K. W. Kim 《Pflügers Archiv : European journal of physiology》1998,436(1):1-8
The possibility of the protein kinase C (PKC) pathway being a mechanism underlying the desensitization of carbachol- (CCh-)activated
nonselective cationic current (I
CCh) was investigated in a study of guinea-pig gastric myocytes. Using the conventional whole-cell patch-clamp technique with
symmetrical CsCl-rich solution in pipette and bath, I
CCh was induced by bath application of 50 μM CCh. With 0.5 mM EGTA [ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic
acid] in the pipette solution (0.5 mM [EGTA]i), I
CCh decayed spontaneously (desensitization of I
CCh) to around 20% within 10 min. Desensitization of I
CCh was significantly attenuated with 2 mM [EGTA]i. At a concentration of 20 μM OAG (1-oleoyl-2-acetyl-sn-glycerol), a PKC activator, inhibited I
CCh at 0.5 mM [EGTA]i but far less at 2 mM [EGTA]i (18% and 81% of control, respectively). The same cationic current induced by intracellular guanosine-5′-O-(3-thiotriphosphate) (GTP[γ-S]) was not inhibited by OAG with 0.5 mM [EGTA]i. The pretreatment of gastric myocytes with PKC inhibitors, either 1 μM chelerythrine or 1 μM peptide inhibitor, attenuated
the desensitization of I
CCh. [Ca2+]i was also measured by single cell microfluorometry using fura-2. Under CCh stimulation with 2 mM [EGTA]i, [Ca2+]i did not increase above 100 nM while it increased to around 260 nM with 0.5 mM [EGTA]i. These results suggest that the desensitization of I
CCh is partly due to the Ca2+-dependent PKC pathway in guinea-pig gastric myocytes.
Received: 27 August 1997 / Received after revision: 2 January 1998 / Accepted: 21 January 1998 相似文献
19.
The effects of adrenoceptor agonists on the transepithelial Cl– conductance (G
Cl) in the skin of several amphibian species, both toads and frogs, were studied. Epinephrine (Epi) from the serosal side selectively
and reversibly inhibited the voltage-activated G
Cl in toad skin and the short-circuit G
Cl in frog skin. The main effects of activation of the adrenoceptors must reside in the skin epithelium and not in the glands,
since measurements were made both from intact skins and split epithelia with essentially the same results. Effective concentrations
of Epi were variable among individual tissues. G
Cl was reduced to 34±17% (n=46) with 1 μmol/l Epi, but in some tissues 0.1 μmol/l inhibited more than 80% of G
Cl, whereas some preparations were little influenced at >3 μmol/l Epi. The affected receptor type was identified by the use
of the α1-agonist phenylephrine, which mimicked the response of Epi at concentrations above 30 μmol/l, whereas the α2-agonists xylazine and iodoclonidine had no effect at supramaximal concentrations. Prazosin, a specific α1-antagonist, reduced or eliminated the inhibition by Epi, but the response pattern suggests a low affinity. The α2-antagonist yohimbine, at concentrations ≤0.3 μmol/l, had a minimal effect, but reduced the inhibition by Epi at concentrations
of 1–10 μmol/l. This might indicate affinity to α1-adrenoceptors in amphibian skin. Activation of β-adrenoceptors by isoproterenol (0.1–5 μmol/l) led to a transient increase
of the baseline inactivated component of G
Cl with a slight reduction of the voltage-activated G
Cl at the higher concentrations, but the inhibitory effect of Epi was not altered. Epi, on the other hand, neither prevented
nor reversed the induction of a voltage-insensitive G
Cl in toad skin caused by application of cAMP at supramaximal concentrations (>100 μmol/l CPT-cAMP). Preincubation of the serosal
medium with Ca2+-free solution (in the presence of 2 mmol/l EGTA) for extended periods of time (>30 min) eliminated the response to Epi. It
is concluded that α1-adrenoceptors participate in the physiological control of voltage-activated Cl– conductance in amphibian skin epithelium via modulation of intracellular Ca2+, presumably by efflux from intracellular stores.
Received: 11 March 1998 / Received after revision: 2 June 1998 / Accepted: 8 June 1998 相似文献
20.
ATP acting on P2Y receptors triggers calcium mobilization in primary cultures of rat neurohypophysial astrocytes (pituicytes) 总被引:3,自引:0,他引:3
Troadec JD Thirion S Petturiti D Bohn MT Poujeol P 《Pflügers Archiv : European journal of physiology》1999,437(5):745-753
The effect of adenosine triphosphate (ATP) on the intracellular Ca2+ concentration ([Ca2+]i) of cultured neurohypophysial astrocytes (pituicytes) was studied by fluorescence videomicroscopy. ATP evoked a [Ca2+]i increase, which was dose dependent in the 2.5–50 μM range (EC50=4.3 μM). The ATP-evoked [Ca2+]i rise was not modified during the first minute following the removal of external Ca2+. Application of 500 nM thapsigargin inhibited the ATP-dependent [Ca2+]i increase. Caffeine (10 mM) and ryanodine (1 μM) did not affect the ATP-induced [Ca2+]i rise. The pituicytes responded to various P2 purinoceptor agonists with the following order of potency: ATP=ATP[γ-S]=2-MeSATP≥ADP, where ATP[γ-S] is adenosine 5′-O-(3-thiotriphosphate) and 2-MeSATP is 2-methylthio-adenosine-5′-triphosphate. Adenosine, AMP, α,β-methylene adenosine-5′-triphosphate
(α,β-MeATP), β,γ methylene adenosine-5′-triphosphate (β,γ-MeATP) and uridine 5′-triphosphate (UTP) were ineffective. The P2 purinoceptor antagonists blocked the ATP-evoked [Ca2+]i increase with the following selectivity: RB-2>suramin>PPADS, where RB-2 is Reactive Blue 2 and PPADS is pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic
acid. The ATP-evoked [Ca2+]i increase was substantially blocked by pertussis toxin treatment, suggesting that it might be mediated by a pertussis-toxin-sensitive
G protein. The phospholipase C (PLC) inhibitor U-73122 (0.5 μM) abolished the ATP-evoked [Ca2+]i rise, whereas its inactive stereoisomer U-73343 (0.5 μM) remained ineffective. Our results indicate that, in rat cultured
pituicytes, ATP stimulation induces an increase in [Ca2+]i due to PLC-mediated release from intracellular stores through activation of a pertussis-toxin-sensitive, G-protein-linked
P2Y receptor.
Received: 24 September 1998 / Received after revision: 10 December 1998 / Accepted: 18 December 1998 相似文献