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1.
U. Lönnendonker 《European biophysics journal : EBJ》1991,20(3):135-141
The use-dependent phasic blockage of sodium channels by tetrodotoxin (TTX) and saxitoxin (STX) was examined in frog nodes of Ranvier using trains of depolarizing pulses. The decline of the peak Na+ current from its initial value (I
0) before the train to a stationary value (I
) after the train was more pronounced at more negative holding potentials. The relationship betweenI
/I
0 and holding potential was fitted by a sigmoid function which yielded values for the steepness of the voltage dependencies of around –15 mV for TTX and – 8 mV for STX. Similar values were obtained at toxin concentrations of 4 and 8 nM. The higher voltage sensitivity of STX versus TTX is interpreted in terms of the higher charge and the faster binding kinetics of STX. These differences also explain the frequency dependence of the decline of Na+ currents with STX (between 0.5 and 2 Hz) and the frequency independence with TTX. Variation of the pulse amplitude in a train of conditioning pulses revealed that the magnitude of the use-dependent actions of STX parallels the steady-state Na+ inactivation curveh
. Inhibition of inactivation, by pre-treatment with chloramine-T, did not, however, abolish the use dependence. Instead, it introduced a change in the time constants of the decline of the Na+ currents and the magnitude became independent of the holding potential. 相似文献
2.
Single ventricle cells were dissociated from the hearts of two-, three-, four- or seven-day-old chick embryos, and were maintained in vitro for an additional 6 to 28 hr. Rounded 13 to 18 micron cells with input capacitance of 5 to 10 pF were selected for analysis of fast sodium current (INa). Voltage command protocols designed to investigate the magnitude, voltage dependence, and kinetics of INa were applied with patch electrodes in the whole-cell clamp configuration. INa was present in over half of the 2d, and all 3d, 4d and 7d cells selected. The current showed no systematic differences in activation kinetics, voltage dependence, or tetrodotoxin (TTX) sensitivity with age or culture conditions. Between the 2d and 7d stages, the rate of current inactivation doubled and channel density increased about eightfold. At all stages tested, INa was blocked by TTX at a half-effective concentration of 0.5 to 1.0 nM. We conclude that the lack of Na dependence of the action potential upstroke on the second day of development results from the relatively depolarized level of the diastolic potential, and failure to activate the small available excitatory Na current. The change from Ca to Na dependence of the upstroke during the third to the seventh day of incubation results partly from the negative shift of the diastolic potential during this period, and in part from the increase in available Na conductance. 相似文献
3.
R. Krattenmacher Rosita Voigt W. Clauss 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1990,160(2):161-165
Summary Transepithelial electrogenic Na transport (INa) was investigated in the colon of the frog Xenopus laevis with electrophysiological methods in vitro. The short circuit current (Isc) of the voltage-clamped tissue was 24.2±1.8 A·cm-2 (n=10). About 60% of this current was generated by electrogenic Na transport. Removal of Ca2+ from the mucosal Ringer solution stimulated INa by about 120%. INa was not blockable by amiloride (0.1 mmol·l-1), a specific Na-channel blocker in epithelia, but a fully and reversible inhibition was achieved by mucosal application of 1 mmol·l-1 lanthanum (La3-). No Na-self-inhibition was found, because INa increased linearly with the mucosal Na concentration. A stimulation of INa by antidiuretic hormones was not possible. The analysis of fluctuations in the short circuit current (noise analysis) indicated that Na ions pass the apical cell membrane via a Ca-sensitive ion channel. The results clearly demonstrate that in the colon of Xenopus laevis Na ions are absorbed through Ca-sensitive apical ion channels. They differ considerably in their properties and regulation from the amiloride-sensitive Na channel which is typically found in the colon of vertebrates.Abbreviations
G
T
transepithelial conductance
-
I
sc
short circuit current
-
I
Na
transepithelial Na-current
-
m
mucosal
-
s
serosal
-
PDS
power density spectrum
-
f
frequency
-
f
c
corner frequency of the Lorentzian component of the PDS
-
S(f)
power density of the Lorentzian component of the PDS
- So
plateau value of the Lorentzian component of the PDS 相似文献
4.
(+)-MK801, a noncompetitive NMDA receptor antagonist, was reported to exhibit anticonvulsive and neuroprotective activities during the postischemic period. Intravenous administration of (+)-MK801 produced tachycardia in rats, but bradycardia in pigs. We examined the mechanical and electrophysiological effects of (+)-MK801 on rat cardiac tissues. (+)-MK801 dose-dependently increased (3–100 µM) twitch tension in rat atria and ventricular strips. The spontaneous beating rate in rat right atria, however, was dose-dependently decreased by (+)-MK801. The inotropic effect of (+)-MK801 was affected neither by 1-antagonist (1 µM prazosin) nor by 1-adrenoceptor antagonist (3 µM atenolol), but significantly by a transient outward K+ channel blocker (3 mM 4-aminopyridine). (+)-MK801 did not cause any significant change of intracellular cAMP content. Electrophysiological study in rat ventricular cells revealed that (+)-MK801 concentration-dependently prolonged the action potential duration with a concomitant decrease in the maximum rate of the action potential upstroke (Vmax) and an increase in the recovery time constant of Vmax. Voltage clamp study showed that (+)-MK801 (3 µM) reduced inward Na+ current (INa), along with a slowing of its recovery from inactivation and a slight negative shift of its voltage-dependent steady-state inactivation curves. At a much higher concentration (30 µM), (+)-MK801 slightly reduced the amplitude of L-type calcium inward current (ICa), although the voltage dependence of its steady-state inactivation was unaffected. For the potassium currents in rat ventricular cells, 3 µM of (+)-MK801 reduced the peak transient outward current (Ito), steady-state outward current (Iss) and inward current through K1 channels. The inhibition of Ito was associated with a prominent negative shift in the voltage dependence of its steady-state inactivation curve. The outward current through K1 channels was unaffected. These results indicate that (+)-MK801 may be a strong INa and Ito blocker with some ICa blocking activity. The inhibition of Ito and other K+ efflux would prolong action potential duration, produce positive inotropic action and contribute to the negative chronotropic effect of (+)-MK801. 相似文献
5.
J. K. W. Blandino M. P. Viglione W. A. Bradley H. K. Oie Y. I. Kim 《The Journal of membrane biology》1995,143(2):153-163
Sodium channels of human small-cell lung cancer (SCLC) cells were examined with whole-cell and single-channel patch clamp methods. In the tumor cells from SCLC cell line NCI-H146, the majority of the voltage-gated Na+ channels are only weakly tetrodotoxin (TTX)-sensitive (K
d
=215 mm). With the membrane potential maintained at –60 to –80 mV, these cells produced all-or-nothing action potentials in response to depolarizing current injection (>20 pA). Similar all-ornothing spikes were also observed with anodal break excitation. Removal of external Ca2+ did not affect the action potential production, whereas 5 m TTX or substitution of Na+ with choline abolished it. Action potentials elicited in the Ca2+-free condition were reversibly blocked by 4 mm MnCl2 due to the Mn2+-induced inhibition of voltage-dependent sodium currents (I
Na). Therefore, Na+ channels, not Ca2+ channels, underlie the excitability of SCLC cells. Whole-cell I
Na was maximal with step-depolarizing stimulations to 0 mV, and reversed at +45.2 mV, in accord with the predicted Nernst equilibrium potential for a Na+-selective channel. I
Na evoked by depolarizing test potentials (–60 to +40 mV) exhibited a transient time course and activation/ inactivation kinetics typical of neuronal excitable membranes; the plot of the Hodgkin-Huxley parameters, m and h, also revealed biophysical similarity between SCLC and neuronal Na+ channels. The single channel current amplitude, as measured with the inside-out patch configuration, was 1.0 pA at –20 mV with a slope conductance of 12.1 pS. The autoantibodies implicated in the Lambert-Eaton myasthenic syndrome (LES), which are known to inhibit I
Ca and I
Na in bovine adrenal chromaffin cells, also significantly inhibited I
Na in SCLC cells. These results indicate that (i) action potentials in human SCLC cells result from the regenerative increase in voltage-gated Na+ channel conductance; (ii) fundamental characteristics of SCLC Na+ channels are the same as the classical sodium channels found in a variety of excitable cells; and (iii) in some LES patients, SCLC Na+ channels are an additional target of the pathological IgG present in the patients' sera.Department of Biomedical EngineeringThis study was supported by National Institutes of Health grant NS18607 and a research grant from the Muscular Dystrophy Association. Dr. Y.I. Kim is the recipient of a Javits Neuroscience Investigator Award from the National Institute of Neurological Disorder and Stroke. 相似文献
6.
Y. -K. Ju D. A. Saint G. D. S. Hirst P. W. Gage 《The Journal of membrane biology》1995,145(2):119-128
Cells in the pacemaker region of toad (Bufo marinus) sinus venosus had spontaneous rhythmic action potentials. The rate of firing of action potentials, the rate of diastolic depolarization and the maximum rate of rise of action potentials were reduced by TTX (10 nm to 1 m). Currents were recorded with the whole cell, tight seal technique from cells enzymatically dissociated from this region. Cells studied were identified as pacemaker cells by their characteristic morphology, spontaneous rhythmic action potential activity that could be blocked by cobalt but not by TTX and lack of inward rectification. When calcium, potassium and nonselective cation currents (If) activated by hyperpolarization were blocked, depolarization was seen to generate transient and persistent inward currents. Both were sodium currents: they were abolished by tetrodotoxin (10 to 100 nm), their reversal potential was close to the sodium equilibrium potential and their amplitude and reversal potential were influenced as expected for sodium currents when extracellular sodium ions were replaced with choline ions. The transient sodium current was activated at potentials more positive than –40 mV while the persistent sodium current was obvious at more negative potentials. It was concluded that, in toad pacemaker cells, TTX-sensitive sodium currents contributing both to the upstroke of action potentials and to diastolic depolarization may play an important role in setting heart rate.We thank the Australian National Heart Foundation for their support. D.A.S. is an NHMRC Senior Research Officer. 相似文献
7.
Background
The SCN5A encoded sodium current (INa) generates the action potential (AP) upstroke and is a major determinant of AP characteristics and AP propagation in cardiac myocytes. Unfortunately, in cardiac myocytes, investigation of kinetic properties of INa with near-physiological ion concentrations and temperature is technically challenging due to the large amplitude and rapidly activating nature of INa, which may seriously hamper the quality of voltage control over the membrane. We hypothesized that the alternating voltage clamp-current clamp (VC/CC) technique might provide an alternative to traditional voltage clamp (VC) technique for the determination of INa properties under physiological conditions.Principal Findings
We studied INa under close-to-physiological conditions by VC technique in SCN5A cDNA-transfected HEK cells or by alternating VC/CC technique in both SCN5A cDNA-transfected HEK cells and rabbit left ventricular myocytes. In these experiments, peak INa during a depolarizing VC step or maximal upstroke velocity, dV/dtmax, during VC/CC served as an indicator of available INa. In HEK cells, biophysical properties of INa, including current density, voltage dependent (in)activation, development of inactivation, and recovery from inactivation, were highly similar in VC and VC/CC experiments. As an application of the VC/CC technique we studied INa in left ventricular myocytes isolated from control or failing rabbit hearts.Conclusions
Our results demonstrate that the alternating VC/CC technique is a valuable experimental tool for INa measurements under close-to-physiological conditions in cardiac myocytes. 相似文献8.
Charge Movement Associated with the Opening and Closing of the Activation Gates of the Na Channels 总被引:26,自引:37,他引:26
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The sodium current (INa) that develops after step depolarization of a voltage clamped squid axon is preceded by a transient outward current that is closely associated with the opening of the activation gates of the Na pores. This "gating current" is best seen when permeant ions (Na and K) are replaced by relatively impermeant ones, and when the linear portion of capacitative current is eliminated by adding current from positive steps to that from exactly equal negative ones. During opening of the Na pores gating current is outward, and as the pores close there is an inward tail of current that decays with approximately the same time-course as INa recorded in Na-containing medium. Both outward and inward gating current are unaffected by tetrodotoxin (TTX). Gating current is capacitative in origin, the result of relatively slow reorientation of charged or dipolar molecules in a suddenly altered membrane field. Close association with the Na activation process is clear from the time-course of gating current, and from the fact that three procedures that reversibly block INa also block gating current: internal perfusion with Zn2+, prolonged depolarization of the membrane, and inactivation of INa with a short positive prepulse. 相似文献
9.
Summary The influence of nerve growth factor (NGF) on Na currents of rat dorsal root ganglia (DRG) was studied in neurons obtained from newborns and cultured for 2–30 hr inserum-free defined medium (SFM). Cell survival for the period studied was 78–87% both with and without NGF. Na currents were detected in all cells cultured for 6–9 hr. They were also detected after 2 hr in culture in 21.5% of the cells cultured without NGF (–NGF cells), and in 91.5% of the cells cultured with NGF (+NGF cells). Current density of the -NGF cells was 2.3 and 2 pA/m2 after growth for 2 and 6–9 hr, respectively, compared to 3.0 and 3.9 pA/m2 for the +NGF cells. The +NGF cells were separated into fast (F), Intermediate (I) and slow (S) cells, based on the Na current they expressed, while -NGF cells were all of theI type.F, I andS currents differed in their voltage-dependent inactivation (Vh
50=–79, –28 and –20 mV), kinetics of inactivation (tau
h
=0.55, 1.3 and 7.75 msec), and TTX sensitivity (K
i=60, 550 and 1100nm). All currents were depressed by [Ca]
o
with aKd
Ca of 22, 17 and 8mm forF, I andS currents, respectively. Current density ofF andS currents was 5.5 and 5 pA/m2 for theI current. The concentration-dependent curve ofI currentvs. TTX indicated thatI current has two sites: one withF-like and another withS-likeK
i for TTX. Hybridization ofF andS currents yieldI-like currents. Thus, the major effect of NGF on Na currents in SFM is the accleration of Na current acquisition and diversity, reflected in an increase of either theS orF type in a cell. 相似文献
10.
Claire Poulet Erich Wettwer Morten Grunnet Thomas Jespersen Larissa Fabritz Klaus Matschke Michael Knaut Ursula Ravens 《PloS one》2015,10(6)
Slowly inactivating Na+ channels conducting “late” Na+ current (INa,late) contribute to ventricular arrhythmogenesis under pathological conditions. INa,late was also reported to play a role in chronic atrial fibrillation (AF). The objective of this study was to investigate INa,late in human right atrial cardiomyocytes as a putative drug target for treatment of AF. To activate Na+ channels, cardiomyocytes from transgenic mice which exhibit INa,late (ΔKPQ), and right atrial cardiomyocytes from patients in sinus rhythm (SR) and AF were voltage clamped at room temperature by 250-ms long test pulses to -30 mV from a holding potential of -80 mV with a 100-ms pre-pulse to -110 mV (protocol I). INa,late at -30 mV was not discernible as deviation from the extrapolated straight line IV-curve between -110 mV and -80 mV in human atrial cells. Therefore, tetrodotoxin (TTX, 10 μM) was used to define persistent inward current after 250 ms at -30 mV as INa,late. TTX-sensitive current was 0.27±0.06 pA/pF in ventricular cardiomyocytes from ΔKPQ mice, and amounted to 0.04±0.01 pA/pF and 0.09±0.02 pA/pF in SR and AF human atrial cardiomyocytes, respectively. With protocol II (holding potential -120 mV, pre-pulse to -80 mV) TTX-sensitive INa,late was always larger than with protocol I. Ranolazine (30 μM) reduced INa,late by 0.02±0.02 pA/pF in SR and 0.09±0.02 pA/pF in AF cells. At physiological temperature (37°C), however, INa,late became insignificant. Plateau phase and upstroke velocity of action potentials (APs) recorded with sharp microelectrodes in intact human trabeculae were more sensitive to ranolazine in AF than in SR preparations. Sodium channel subunits expression measured with qPCR was high for SCN5A with no difference between SR and AF. Expression of SCN8A and SCN10A was low in general, and lower in AF than in SR. In conclusion, We confirm for the first time a TTX-sensitive current (INa,late) in right atrial cardiomyocytes from SR and AF patients at room temperature, but not at physiological temperature. While our study provides evidence for the presence of INa,late in human atria, the potential of such current as a target for the treatment of AF remains to be demonstrated. 相似文献
11.
Lawrence G. Palmer Isidore S. Edelman Bernd Lindemann 《The Journal of membrane biology》1980,57(1):59-71
Summary The basal-lateral surface of the epithelium of the urinary bladder of the toad (Bufo marinus) was depolarized by exposure of the serosal surface to 85mm KCL and 50mm sucrose. The extent of depolarization appeared to be virtually complete, as evaluated by the invariance in the transepithelial electrical potential difference and conductance on addition of nystatin (a monovalent cation ionophore) to the serosal medium. The Na-specific current (I
Na) was defined as the current sensitive to the removal of Na from the mucosal medium or inhibitable by addition of amiloride to this medium. In the presence of the high K-sucrose serosal medium, rapid, serial, stepwise clamping of the transepithelial voltage (V) yielded a curvilinear dependence ofI
Na onV; which is taken to represent theI–V curve of the apical Na channels. The constant field equation (Goldman, D.E. 1943;J. Gen. Physiol.
27:37) fits theI–V data points closely, allowing estimates to be made of the permeability to Na of the apical membrane (P
Na) and of the intracellular Na activity (Na
c
). Exposure of the apical surface to amiloride (5×10–7
m) decreasedP
Na in proportion to the decrease inI
Na (i.e., 70%) but decreased Na
c
only 25%. In contrast, an equivalent lent reduction inI
Na elicited by exposure of the basallateral surface to ouabain was accompanied by only a 20% decrease inP
Na and a sixfold increase in Na
c
. The effects of amiloride onP
Na and ouabain on Na
c
are consistent with the primary pharmacological actions of these drugs. In addition,P
Na appears to be under metabolic control, in that 2-deoxyglucose, a specific inhibitor of glycolysis, decreasedI
Na andP
Na proportionately, and lowered Na
c
marginally, effects indistinguishable from those obtained with amiloride. 相似文献
12.
Chunping Qian Jihua Ma Peihua Zhang Antao Luo Chao Wang Zhiqiang Ren Linghao Kong Shuo Zhang Xiaojing Wang Ying Wu 《PloS one》2012,7(12)
Background/Aims
Resveratrol has been demonstrated to be protective in the cardiovascular system. The aim of this study was to assess the effects of resveratrol on hydrogen peroxide (H2O2)-induced increase in late sodium current (I Na.L) which augmented the reverse Na+-Ca2+ exchanger current (I NCX), and the diastolic intracellular Ca2+ concentration in ventricular myocytes.Methods
I Na.L, I NCX, L-type Ca2+ current (I Ca.L) and intracellular Ca2+ properties were determined using whole-cell patch-clamp techniques and dual-excitation fluorescence photomultiplier system (IonOptix), respectively, in rabbit ventricular myocytes.Results
Resveratrol (10, 20, 40 and 80 µM) decreased I Na.L in myocytes both in the absence and presence of H2O2 (300 µM) in a concentration dependent manner. Ranolazine (3–9 µM) and tetrodotoxin (TTX, 4 µM), I Na.L inhibitors, decreased I Na.L in cardiomyocytes in the presence of 300 µM H2O2. H2O2 (300 µM) increased the reverse I NCX and this increase was significantly attenuated by either 20 µM resveratrol or 4 µM ranolazine or 4 µM TTX. In addition, 10 µM resveratrol and 2 µM TTX significantly depressed the increase by 150 µM H2O2 of the diastolic intracellular Ca2+ fura-2 fluorescence intensity (FFI), fura-fluorescence intensity change (△FFI), maximal velocity of intracellular Ca2+ transient rise and decay. As expected, 2 µM TTX had no effect on I Ca.L.Conclusion
Resveratrol protects the cardiomyocytes by inhibiting the H2O2-induced augmentation of I Na.L.and may contribute to the reduction of ischemia-induced lethal arrhythmias. 相似文献13.
Summary Effects of extracellular pH on the sodium current (I
Na) of single rat ventricular cells were examined under conditions of voltage clamp and internal perfusion. In this way, pH
i
was controlled while pH
o
was changed. The combined suction pipette-microelectrode method was used. The suction pipette passed current and perfused the cell's interior; the microelectrode measured membrane potential. Increasing extracellular H+ depressedI
Na and slowed inactivation. The current-voltage curves forI
Na and Slowed inactivation. The current-voltage curves forI
Na were shifted to positive and negative potentials at low and high pH
o
, respectively. Similar potential shifts were observed in both the conductance voltage curve and the steadystate inactivation voltage curve (h
). Conduction was also depressed at low pH
o
. The shifts were probably due to surface charge effects, while the impaired conduction was probably due to protonation of a site in the Na channel. 相似文献
14.
Background
Although fibroblast-to-myocyte electrical coupling is experimentally suggested, electrophysiology of cardiac fibroblasts is not as well established as contractile cardiac myocytes. The present study was therefore designed to characterize ion channels in cultured human cardiac fibroblasts.Methods and Findings
A whole-cell patch voltage clamp technique and RT-PCR were employed to determine ion channels expression and their molecular identities. We found that multiple ion channels were heterogeneously expressed in human cardiac fibroblasts. These include a big conductance Ca2+-activated K+ current (BKCa) in most (88%) human cardiac fibroblasts, a delayed rectifier K+ current (IKDR) and a transient outward K+ current (Ito) in a small population (15 and 14%, respectively) of cells, an inwardly-rectifying K+ current (IKir) in 24% of cells, and a chloride current (ICl) in 7% of cells under isotonic conditions. In addition, two types of voltage-gated Na+ currents (INa) with distinct properties were present in most (61%) human cardiac fibroblasts. One was a slowly inactivated current with a persistent component, sensitive to tetrodotoxin (TTX) inhibition (INa.TTX, IC50 = 7.8 nM), the other was a rapidly inactivated current, relatively resistant to TTX (INa.TTXR, IC50 = 1.8 µM). RT-PCR revealed the molecular identities (mRNAs) of these ion channels in human cardiac fibroblasts, including KCa.1.1 (responsible for BKCa), Kv1.5, Kv1.6 (responsible for IKDR), Kv4.2, Kv4.3 (responsible for Ito), Kir2.1, Kir2.3 (for IKir), Clnc3 (for ICl), NaV1.2, NaV1.3, NaV1.6, NaV1.7 (for INa.TTX), and NaV1.5 (for INa.TTXR).Conclusions
These results provide the first information that multiple ion channels are present in cultured human cardiac fibroblasts, and suggest the potential contribution of these ion channels to fibroblast-myocytes electrical coupling. 相似文献15.
Using a very low noise voltage clamp technique it has been possible to record from the squid giant axon a slow component of gating current (I
g
) during the inactivation phase of the macroscopic sodium current (I
Na
) which was hitherto buried in the baseline noise. In order to examine whether this slowI
g
contains gating charge that originates from transitions between the open (O) and the inactivated (I) states, which would indicate a true voltage dependence of inactivation, or whether other transitions contribute charge to slowI
g
, a new model independent analysis termed isochronic plot analysis has been developed. From a direct correlation ofI
g
and the time derivative of the sodium conductance dg
Na/d
the condition when only O-I transitions occur is detected. Then the ratio of the two signals is constant and a straight line appears in an isochronic plot ofI
g
vs. dg
Na/d
. Its slope does not depend on voltage or time and corresponds to the quantal gating charge of the O-I transition (q
h
) divided by the single channel ionic conductance (). This condition was found at voltages above – 10 mV up to + 40 mV and a figure of 1.21e
– was obtained forq
h
at temperatures of 5 and 15°C. At lower voltages additional charge from other transitions, e.g. closed to open, is displaced during macroscopic inactivation. This means that conventional Eyring rate analysis of the inactivation time constant
h
is only valid above – 10 mV and here the figure forq
h
was confirmed also from this analysis. It is further shown that most of the present controversies surrounding the voltage dependence of inactivation can be clarified. The validity of the isochronic plot analysis has been confirmed using simulated gating and ionic currents.Abbreviations
I
g
gating current
-
I
Na
sodium ionic current
-
g
Na
macroscopic sodium conductance
-
single channel conductance
- C, O, I
closed, open, inactivated state occupancy of channels
-
g
h
quantal charge displaced in a single O-I transition of Na channel
-
e
–
equivalent electron charge
-
h
index referring to inactivation process
-
S
l
limiting slope in isochronic plot see Eq.(3)
-
fractional distance, see Fig. 4 and (4, 5)
- TMA
tetramethylammonium
- TTX
tetrodotoxin
- Tris
tris(hydroxymethyl)aminomethane
- HEPES
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid 相似文献
16.
Timothy R. Traynor Scott M. O'Grady 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1992,162(1):47-53
Summary Porcine distal colon epithelium was mounted in Ussing chambers and bathed in plasma-like Ringer solution. Tissue conductances ranged from 10 to 15 mS and the short-circuit current (Isc) ranged from-15 to 220 A·cm-2. Variations in basal Isc resulted from differences in the amount of amiloride (10M mucosal addition)-sensitive Na+ absorption. Ion substitution and transepithelial flux experiments showed that 10 M amiloride produced a decrease in the mucosal-to-serosal (M-S) and net Na flux, and that this effect on Isc was independent of Cl- and HCO
3
-
replacement. When the concentration of mucosal amiloride was increased from 10 to 100 M, little change in Isc was observed. However, increasing the concentration to 1 mM produced a further inhibition, which often reversed the polarity of the Isc. The decrease in Isc due to 1 mM amiloride was dependent on both Cl- and HCO
3
-
, and was attributed to reductions in the M-S and net Na+ fluxes as well as the M-S unidirectional Cl- flux. Ion replacement experiments demonstrated that Cl- substitution reduced the M-S and net Na fluxes, while replacement of HCO
3
-
with HEPES abolished net Cl- absorption by reducing the M-S unidirectional Cl- flux. From these data it can be concluded that: (1) Na+ absorption is mediated by two distinct amiloride-sensitive transport pathways, and (2) Cl- absorption is completely HCO
3
-
-dependent (presumably mediated by Cl-/HCO
3
-
exchange) and occurs independently of Na+ absorption.Abbreviations Gt
tissue conductance
- HEPES
tris (hydroxymethyl) aminomethane
- (tris)
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
- Isc
short-circuit current
- Jr
residual flux
- M-S
mucosal-to-scrosal
- S-M
serosal-to-mucosal
- TTX
tetrodotoxin 相似文献
17.
Michael J. McLean Paul B. Bennett Ronald M. Thomas 《Molecular and cellular biochemistry》1989,80(1-2):95-107
Summary Electrophysiological and pharmacological properties distinguished subtypes of adult mammalian dorsal root ganglion neurons (DRGn) in monolayer dissociated cell culture. By analogy of action potential waveform and duration, neurons with short duration (SDn) and long duration (LDn) action potentials resembled functionally distinct subtypes of DRGn in intact ganglia. Patch clamp and conventional intracellular recording techniques were combined here to elucidate differences in the ionic basis of excitability of subtypes of DRGn in vitro. Both SDn and LDn were quiescent at the resting potential. Action potentials of SDn were brief (< 2 msec), sensitive to tetrodotoxin (TTX, 5–10 nM), exhibited damped firing during long depolarizations, and did not respond to algesic agents applied by pressure ejection. Action potentials of LDn were 2–6 msec in duration, persisted in 30 µM TTX, and fired repetitively during depolarizing current pulses or exposure to algesic agents (e.g., capsaicin, histamine and bradykinin). Whole-cell recordings from freshly dissociated neurons revealed two inward sodium currents (INa; variable with changes in sodium but not calcium concentration in the superfusate) in various proportions: a rapidly activating and inactivating, TTX-sensitive current; and, a slower, TTX (30 M)-resistant INa. Large neurons, presumable SDn, had predominantly TTX-sensitive current and little TTX-resistant current. The predominent inward current of small neurons, presumably LDn, was TTX-resistant with a smaller TTX-sensitive component. By analogy to findings from intact ganglia, these results suggest that fundamentally different ionic currents controlling excitability of subtypes of DRGn in vitro may contribute to functional differences between subtyes of neurons in situ. 相似文献
18.
Summary We have investigated the pacemaker properties of aggregates of cells dissociated from the atria and ventricles of 10 to 14-day-old chick embryonic hearts using a two-microelectrode current and voltage-clamp technique. These preparations usually beat spontaneously and rhythmically in tissue culture medium containing 1.3mm potassium with a beat rate typically in the range of 15–60 beats per minute. The beat rate results show considerable variability, which precludes any statistically significant comparison between the spontaneous activity of atrial and ventricular cell preparations at 10–14 days of development. However, the shapes of pacemaker voltage changes do exhibit differences characteristic of cell type. Spontaneous atrial preparations rapidly depolarize from maximum diastolic potential (–90 mV) to a plateau range of pacemaker potentials (–80 to –75 mV). The membrane subsequently depolarizes more gradually until threshold (–65 mV) is reached. In contrast, spontaneously beating ventricular cell preparations slowly hyperpolarize after maximum diastolic potential to the –100 to –95 mV range before gradually depolarizing toward threshold. Voltage-clamp analysis reveals a virtual lack of any time-dependent pacemaker current in atrial preparations. These preparations are characterized by an approximately linear background current (I
bg) having a slope resistance of 100 K cm2. Ventricular preparations have a potassium ion pacemaker current with slow kinetics (I
K
2), and a second time-dependent component (I
x) which is activated at potentials positive to –65 mV. The background current of these preparations displays inward rectification. Computer simulations of pacemaking reveal that the initial rapid phase of pacemaker depolarization in atrial cells is determined by the membrane time constant, which is the product of membrane capacitance and the slope resistance ofI
bg. The hyperpolarization after maximum diastolic potential of ventricular cells is caused byI
K
2. The final slow phase of depolarization in both cell types is caused in part by the steady-state amplitude of the fast inward sodium current (I
Na). This component has negative slope conductance which effectively increases the slope resistance in the vicinity of threshold compared to TTX-treated preparations. This mechanism is sufficient to produce interbeat intervals several seconds in duration, even in the absence of time-dependent pacemaker current, provided that the background current is at the appropriate level. 相似文献
19.
Summary The unidirectional influx of Na from the mucosal solution into the epithelium ofin vitro descending rabbit colon (J
me
Na
) determined under short-circuit conditions, is comprised of two components: one represents entry of Na into transporting epithelial cells and is abolished by amiloride which also abolishes Na absorption (J
net
Na
). The other represents diffusional Na entry into paracellular pathways traversing the epithelium. In all instances, exposure of the mucosal surface to amphotericin B increased tissue conductance andJ
me
Na
and elicited K secretion. Tissues showing a spontaneousI
sc of approximately 4 eq/cm2hr did not respond to amphotericin B with increasedI
sc andJ
net
Na
. However, in tissues characterized by a lowerI
sc under control conditions, amphotericin B increasedI
sc andJ
net
Na
to approximately 4eq/cm2hr. These findings suggest that amphotericin increasesJ
net
Na
and elicits K secretion by disrupting the normal permselectivity of the mucosal membrane. Under these conditions the extrusion of Na from cell-to-serosal solution becomes the rate limiting step in transepithelial Na transport. Finally, a close correlation betweenJ
me
Na
andJ
net
Na
was observed when the rate of Na absorption varied either spontaneously or experimentally with amiloride, suggesting that the backflux of Na from cell-to-mucosal solution is undetectably small. 相似文献
20.
Summary Segments of descending colon obtained from rabbits, that had been maintained on drinking water containing 25mm NaCl and an artificial diet which contains 1% Na and is nominally K-free, respond to aldosterone in vitro (after a 30 to 60-min lag period) with a marked increase in the short-circuit current (I
sc
), an equivalent increase in the rate of active Na absorption (J
net
Na
) and a decline in tissue resistance (R
t
). Aldosterone also brings about a marked increase in the unidirectional influx of Na into the cells across the mucosal membrane (zero-time rate of uptake) which does not differ significantly from the increase inI
sc
. Treatment of control tissues with amphotericin B brings about sustained increases inI
sc
andJ
net
Na
to levels observed in aldosterone-treated tissues. However, addition of amphotericin B to the mucosal solution of aldosteronetreated tissues does not result in a sustained increase inI
sc
orJ
net
Na
and these values do not differ markedly from those observed in control tissues treated with amphotericin B. These findings, together with other evidence that Na entry in the presence of amphotericin B is sufficiently rapid to saturate the active Na extrusion mechanism at the baso-lateral membrane, are consistent with the notion that the aldosterone-induced protein increases the permeability of the mucosal membrane to Na but does not increase the saturation level of the active Na pump within the time-frame of these studies (3 hr).Finally, aldosterone has no effect on the bidirectional or net transepithelial movements of K under short-circuit conditions, suggesting that the enhanced secretion of K observed in vivo is the result of increased diffusion of K from plasma to lumen via paracellular pathways in response to an increased transepithelial electrical potential difference (lumen negative). 相似文献