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1.
We have studied the correlation between [ 3H]ouabain binding sites, (Na ++K +)ATPase (EC 3.6.1.3) activity and acetylcholine (ACh) release in different subcellular fractions of Torpedo marmorata electric organ (homogenate, synaptosomes, presynaptic plasma membranes). Presynaptic plasma membranes contained the greater number of [ 3H]ouabain binding sites in good agreement with the high (Na ++K +)ATPase activity found in this fraction. Blockade of this enzymatic activity by ouabain dose-dependently induced ACh release from pure cholinergic synaptosomes, either in the presence or absence of extracellular calcium ions. We suggest that one of the mechanisms involved in the ouabain-induced ACh release in the absence of Ca 2+
o may be an increase in Na +
i that could (a) evoke Ca 2+ release from internal stores and (b) inhibit ATP-dependent Ca 2+ uptake by synaptic vesicles. 相似文献
2.
The giant axon of the squid has been extensively used as a model for studying Ca regulation in excitable cells. Different techniques (extrusion, injection and dialysis) have been employed to characterize Ca fluxes across the axon membrane. Since both Ca efflux and influx are markedly dependent on [Ca 2+] i, considerable effort has been dedicated to determine the resting value of the [Ca 2+] i. Results from different laboratories indicate that the [Ca 2+] i, in a normal fibre, range from 20–100 nM. Under dialysis conditions (internal control), with an imposed [Ca 2+] i of 80 nM, Ca influx is balanced by an outward Ca movement of about 40 f/CS. Ca extrusion occurs through two parallel transport systems: one having a high affinity for [Ca 2+] i, dependent on ATP, not affected by Na i, Na o, Ca o, Mg o and inhibited by internal vanadate (uncoupled component), the other, more prominent at relatively high [Ca 2+] i, does not require ATP, is inhibited by Na i activated by Na o and not inhibited by vanadate. (Na o-dependent component). The existence of these two systems provide the axon with an effective way to maintain in the long term a constant low [Ca 2+] i in spite of short term fluctuations due to increased Ca influx during nervous activity. 相似文献
3.
This study investigated the effects of extracellular Mg 2+ ([Mg 2+] o) on basal and acetylcholine (ACh)-evoked amylase secretion and intracellular free Ca 2+ ([Ca 2+] i) in rat parotid acinar cells. In a medium containing 1.1 mM [Mg 2+] o, ACh evoked significant increases in amylase secretion and [Ca 2+] i. Either low (0 mM) or elevated (5 and 10 mM) [Mg 2+] o attenuated ACh-evoked responses. In a nominally Ca 2+ free medium, elevated [Mg 2+] o attenuated basal and ACh-evoked amylase secretion and [Ca 2+] i. In parotid acinar cells incubated with either 0, 1.1, 5 or 10 mM [Mg 2+] o, ACh evoked a gradual decrease in [Mg 2+] i. These results indicate that the ACh-evoked Mg 2+ efflux is an active process since Mg 2+ has to move against its gradient. Either lidocaine, amiloride, N-methyl- d-glucamine, quinidine, dinitrophenol or bumetanide can elevate [Mg 2+] i above basal level. In the presence of these membrane transport inhibitors, ACh still evoked a decrease in [Mg 2+] i but the response was less pronounced with either [Na +] o removal or in the presence of either amiloride or quinidine. These results indicate marked interactions between Ca 2+ and Mg 2+ signalling in parotid acinar cells and that ACh-evoked Mg 2+ transport was not dependent upon [Na +] o. 相似文献
4.
Abstract: We studied the effect of α-latrotoxin (αLTX) on [ 14C]acetylcholine ([ 14C]ACh) release, intracellular Ca 2+ concentration ([Ca 2+] i), plasma membrane potential, and high-affinity choline uptake of synaptosomes isolated from guinea pig cortex. αLTX (10 ?10-10 ?8M) caused an elevation of the [Ca 2+] i as detected by Fura 2 fluorescence and evoked [ 14C]ACh efflux. Two components in the action of the toxin were distinguished: one that required the presence of Na + in the external medium and another that did not. Displacement of Na + by sucrose or N-methylglucamine in the medium considerably decreased the elevation of [Ca 2+] i and [ 14C]ACh release by αLTX. The Na +-dependent component of the αLTX action was obvious in the inhibition of the high-affinity choline uptake of synaptosomes. Some of the toxin action on both [Ca 2+] i and [ 14C]ACh release remained in the absence of Na +. Both the Na +-dependent and the Na +-independent components of the αLTX-evoked [ 14C]ACh release partly required the presence of either Mg 2+ or Ca 2+. The nonneurotransmitter [ 14C]choline was released along with [ 14C]ACh, but this release did not depend on the presence of either Na + or Ca 2+, indicating nonspecific leakage through the plasma membrane. We conclude that there are two factors in the release of ACh from synaptosomes caused by the toxin: (1) cation-dependent ACh release, which is related to (a) Na +-dependent divalent cation entry and (b) Na +-independent divalent cation entry, and (2) nonspecific Na +- and divalent cation-independent leakage. 相似文献
5.
Increased intracellular calcium concentration ([Ca 2+] i) is required for smooth muscle contraction. In tracheal and other tonic smooth muscles, contraction and elevated [Ca 2+] i are maintained as long as an agonist is present. To evaluate the physiological role of steady-state increases in Ca 2+ on tension maintenance, [Ca 2+] i was elevated using ionomycin, a Ca 2+ ionophore or charybdotoxin, a large-conductance calcium-activated potassium channel (K Ca) blocker prior to or during exposure of tracheal smooth muscle strips to Ach (10 –9 to 10 –4
M). Ionomycin (5 µ M) in resting muscles induced increases in [Ca 2+] i to 500±230 n M and small increases in force of 2.6±2.3 N/cm 2. This tension is only 10% of the maximal tension induced by ACh. Charybdotoxin had no effect on [Ca 2+] i or tension in resting muscle. After pretreatment of muscle with ionomycin, the concentration-response relationship for ACh-induced changes in tension shifted to the left (EC 50=0.07±0.05 µ M ionomycin; 0.17±0.07 µ M, control, p<0.05). When applied to the muscles during steady-state responses to submaximal concentrations of ACh, both ionomycin and charybdotoxin induced further increases in tension. The same magnitude increase in tension occurs after ionomycin and charybdotoxin treatment, even though the increase in [Ca 2+] i induced by charybdotoxin is much smaller than that induced by ionomycin. We conclude that the resting muscle is much less sensitive to elevation of [Ca 2+] i when compared to muscles stimulated with ACh. Steady-state [Ca 2+] i limits tension development induced by submaximal concentrations of ACh. The activity of K Ca moderates the response of the muscle to ACh at concentrations less than 1 µ M. 相似文献
6.
A mathematical model of calcium dynamics in vascular smooth muscle cell (SMC) was developed based on data mostly from rat mesenteric arterioles. The model focuses on (a) the plasma membrane electrophysiology; (b) Ca 2+ uptake and release from the sarcoplasmic reticulum (SR); (c) cytosolic balance of Ca 2+, Na +, K +, and Cl − ions; and (d) IP 3 and cGMP formation in response to norepinephrine (NE) and nitric oxide (NO) stimulation. Stimulation with NE induced membrane depolarization and an intracellular Ca 2+ ([Ca 2+] i) transient followed by a plateau. The plateau concentrations were mostly determined by the activation of voltage-operated Ca 2+ channels. NE causes a greater increase in [Ca 2+] i than stimulation with KCl to equivalent depolarization. Model simulations suggest that the effect of [Na +] i accumulation on the Na +/Ca 2+ exchanger (NCX) can potentially account for this difference. Elevation of [Ca 2+] i within a concentration window (150-300 nM) by NE or KCl initiated [Ca 2+] i oscillations with a concentration-dependent period. The oscillations were generated by the nonlinear dynamics of Ca 2+ release and refilling in the SR. NO repolarized the NE-stimulated SMC and restored low [Ca 2+] i mainly through its effect on Ca 2+-activated K + channels. Under certain conditions, Na +-K +-ATPase inhibition can result in the elevation of [Na +] i and the reversal of NCX, increasing resting cytosolic and SR Ca 2+ content, as well as reactivity to NE. Blockade of the NCX's reverse mode could eliminate these effects. We conclude that the integration of the selected cellular components yields a mathematical model that reproduces, satisfactorily, some of the established features of SMC physiology. Simulations suggest a potential role of intracellular Na + in modulating Ca 2+ dynamics and provide insights into the mechanisms of SMC constriction, relaxation, and the phenomenon of vasomotion. The model will provide the basis for the development of multi-cellular mathematical models that will investigate microcirculatory function in health and disease. 相似文献
7.
The roles of Ca 2+ mobilization in development of tension induced by acetylcholine (ACh, 0.1–100 µ M) in swine tracheal smooth muscle strips were studied. Under control conditions, ACh induced a transient increase in free cytosolic calcium concentration ([Ca 2+] i) that declined to a steady-state level. The peak increase in [Ca 2+] i correlated with the magnitude of tension at each [ACh] after a single exposure to ACh, while the steady-state [Ca 2+] i did not. Removal of extracellular Ca 2+ had little effect on peak [Ca 2+] i but greatly reduced steady-state increases in [Ca 2+] i and tension. Verapamil inhibited steady-state [Ca 2+] i only at [ACh]<1 µ M. After depletion of internal Ca 2+ stores by 10 min exposure to ACh in Ca 2+-free solution and then washout of ACh for 5 min in Ca 2+-free solution, simultaneous re-exposure to ACh in the presence of 2.5 m M Ca 2+ increased [Ca 2+] i to the control steady-state level without overshoot. The tension attained was the same as control for each [ACh] used. Continuous exposure to successively increasing [ACh] (0.1–100 µ M) also reduced the overshoot of [Ca 2+] i at 10 and 100 µ M ACh, yet tension reached control levels at each [ACh] used. We conclude that the steady-state increase in [Ca 2+] i is necessary for tension maintenance and is dependent on Ca 2+ influx through voltage-gated calcium channels at 0.1 µ M ACh and through a verapamil-insensitive pathway at 10 and 100 µ M. The initial transient increase in calcium arises from intracellular stores and is correlated with the magnitude of tension only in muscles that have completely recovered from previous exposure to agonists. 相似文献
8.
It has been known for more than three decades that outward Kir currents (I K1) increase with increasing extracellular K + concentration ([K +] o). Although this increase in I K1 can have significant impacts under pathophysiological cardiac conditions, where [K +] o can be as high as 18 m m and thus predispose the heart to re-entrant ventricular arrhythmias, the underlying mechanism has remained unclear. Here, we show that the steep [K +] o dependence of Kir2.1-mediated outward I K1 was due to [K +] o-dependent inhibition of outward I K1 by extracellular Na + and Ca 2+. This could be accounted for by Na +/Ca 2+ inhibition of I K1 through screening of local negative surface charges. Consistent with this, extracellular Na + and Ca 2+ reduced the outward single-channel current and did not increase open-state noise or decrease the mean open time. In addition, neutralizing negative surface charges with a carboxylate esterifying agent inhibited outward I K1 in a similar [K +] o-dependent manner as Na +/Ca 2+. Site-directed mutagenesis studies identified Asp 114 and Glu 153 as the source of surface charges. Reducing K + activation and surface electrostatic effects in an R148Y mutant mimicked the action of extracellular Na + and Ca 2+, suggesting that in addition to exerting a surface electrostatic effect, Na + and Ca 2+ might inhibit outward I K1 by inhibiting K + activation. This study identified interactions of K + with Na + and Ca 2+ that are important for the [K +] o dependence of Kir2.1-mediated outward I K1. 相似文献
9.
The release of [3H]dopamine ([3H]DA) and [3H]noradrenaline ([3H]NA) in acutely perfused rat striatal and cortical slice preparations was measured at 37 °C and 17 °C under ischemic conditions. The ischemia was simulated by the removal of oxygen and glucose from the Krebs solution. At 37 °C, resting release rates in response to ischemia were increased; in contrast, at 17 °C, resting release rates were significantly reduced, or resting release was completely prevented. The removal of extracellular Ca2+ further increased the release rates of [3H]DA and [3H]NA induced by ischemic conditions. This finding indicated that the Na+/Ca2+ exchanger (NCX), working in reverse in the absence of extracellular Ca2+, fails to trigger the influx of Ca2+ in exchange for Na+ and fails to counteract ischemia by further increasing the intracellular Na+ concentration ([Na+]i). KB-R7943, an inhibitor of NCX, significantly reduced the cytoplasmic resting release rate of catecholamines under ischemic conditions and under conditions where Ca2+ was removed. Hypothermia inhibited the excessive release of [3H]DA in response to ischemia, even in the absence of Ca2+. These findings further indicate that the NCX plays an important role in maintaining a high [Na+]i, a condition that may lead to the reversal of monoamine transporter functions; this effect consequently leads to the excessive cytoplasmic tonic release of monoamines and the reversal of the NCX. Using HPLC combined with scintillation spectrometry, hypothermia, which enhances the stimulation-evoked release of DA, was found to inhibit the efflux of toxic DA metabolites, such as 3,4-dihydroxyphenylacetaldehyde (DOPAL). In slices prepared from human cortical brain tissue removed during elective neurosurgery, the uptake and release values for [3H]NA did not differ from those measured at 37 °C in slices that were previously maintained under hypoxic conditions at 8 °C for 20 h. This result indicates that hypothermia preserves the functions of the transport and release mechanisms, even under hypoxic conditions. Oxidative stress (H2O2), a mediator of ischemic brain injury enhanced the striatal resting release of [3H]DA and its toxic metabolites (DOPAL, quinone). The study supports our earlier findings that during ischemia transmitters are released from the cytoplasm. In addition, the major findings of this study that hypothermia of brain slice preparations prevents the extracellular calcium concentration ([Ca2+]o)-independent non-vesicular transmitter release induced by ischemic insults, inhibiting Na+/Cl?-dependent membrane transport of monoamines and their toxic metabolites into the extracellular space, where they can exert toxic effects. 相似文献
10.
The times required for a steady rate of miniature end-plate potential discharge to be reached in response to changes in extracellular [K +], [Na +], and [Ca ++] have been measured. In the presence of 15 m M KCl, Ca ++ raises and Na + lowers the steady-state mepp frequency; but the depressive effect on Na + is not specific: Li + can replace Na + to a large extent. Mepp frequency has been found to depend on the ratio of [Ca o
++]/[Na o
+]. It is assumed that in the steady state, intracellular sodium will change when extracellular sodium is changed. Because both intracellular and extracellular sodium at motor nerve endings affect acetylcholine release, it is proposed that mepp frequency depends on the ratio [Ca o] [Na i] 2·/[Na o] 2 Two models are proposed. Firstly, to account for the action of sodium and calcium a carrier is postulated for which Ca ++ and Na + compete. The carrier determines a maximum level of intracellular Ca ++ far lower than predicted by the Nernst equation for Ca. Secondly, to account for activation of acetylcholine release by a small influx of Ca ++, the ions are presumed to enter the nerve ending in a two stage process through a small intermediate compartment and to act on the acetylcholine release site in this region rather than after entering directly into the cell. 相似文献
11.
The aim of the present study was to investigate possible changes of inositol 1,4,5-trisphosphate (IP 3) mass in Torpedo cholinergic synaptosomes in conditions promoting stimulated acetylcholine (ACh) release. For this purpose, we used a radioreceptor IP 3 mass assay and a chemiluminescent method for ACh detection. Torpedo cholinergic synaptosomes have consistent IP 3 mass levels under resting conditions. The IP 3 mass was neither modified by changes in external Ca 2+ nor by a Ca 2+-free medium containing EGTA. IP 3 mass and ACh release, measured in the same conditions and in parallel, were increased by depolarization with high K + and by the ionophores A-23187 and gramicidin-D in a manner dependent on external Ca 2+ emphasizing that Ca 2+ entry, independently of the influx mechanism involved, leads to an IP 3 increase. The phospholipase C β inhibitors U-73122 and U-73343 reduced K +-stimulated IP 3 levels while K +-evoked ACh release was almost completely blocked suggesting an additional effect of these drugs on depolarization-neurotransmitter secretion coupling. The effect reported showing an increase of IP 3 by agents that stimulate ACh release may suggest a possible link between IP 3 metabolism and the neurotransmitter release mechanism. However, such a link is probably not a direct one as implied by the results obtained with the inhibitors of phospholipase C. Copyright © 1996 Elsevier Science Ltd 相似文献
12.
Ca 2+-selective electrodes have been used to measure free intracellular Ca 2+ concentrations in squid giant axons. Electrodes made of glass cannulas of about 20 μm in diameter, plugged with a poly(vinyl chloride) gelled sensor were used to impale the axons axially. They showed a Nernstian response to Ca 2+ down to about 3 μM in solutions containing 0.3 M K + and 0.025 M Na +. Sub-Nernstian but useful responses were obtained up to p Ca 8. The electrodes showed adequate selectivity to Ca 2+ over Mg 2+, H +, K + and Na +. To calibrate them properly, a set of standard solutions were prepared using different Ca 2+ buffers (EGTA, HEEDTA, nitrilotriacetic acid) after carefully characterizing their apparent Ca 2+ association constants under conditions resembling the axoplasmic environment. In fresh axons incubated in artificial seawater containing 4 mM Ca 2+, the mean resting intracellular ionized calcium concentration was 0.106 μM ( ). The Ca 2+-electrodes were used to investigate effects of different experimental procedures on the [Ca 2+] i. The main conclusions are: (i) intact axons can extrude calcium ions at low [Ca 2+] i levels by a process independent of external Na +; (ii) poisoned axons can extrude calcium ions at high levels of [Ca 2+] i by an external Na +-dependent process. The level of free intracellular Ca attained at these latter conditions is about an order to magnitude greater than the resting physiological value. 相似文献
13.
Experiments were performed to characterize the properties of the intrinsic Ca 2+ buffers in the sarcoplasmic reticulum (SR) of cut fibers from frog twitch muscle. The concentrations of total and free calcium ions within the SR ([Ca T] SR and [Ca 2+] SR) were measured, respectively, with the EGTA/phenol red method and tetramethylmurexide (a low affinity Ca 2+ indicator). Results indicate SR Ca 2+ buffering was consistent with a single cooperative-binding component or a combination of a cooperative-binding component and a linear binding component accounting for 20% or less of the bound Ca 2+. Under the assumption of a single cooperative-binding component, the most likely resting values of [Ca 2+] SR and [Ca T] SR are 0.67 and 17.1 mM, respectively, and the dissociation constant, Hill coefficient, and concentration of the Ca-binding sites are 0.78 mM, 3.0, and 44 mM, respectively. This information can be used to calculate a variable proportional to the Ca 2+ permeability of the SR, namely d[Ca T] SR/dt ÷ [Ca 2+] SR (denoted release permeability), in experiments in which only [Ca T] SR or [Ca 2+] SR is measured. In response to a voltage-clamp step to −20 mV at 15°C, the release permeability reaches an early peak followed by a rapid decline to a quasi-steady level that lasts ∼50 ms, followed by a slower decline during which the release permeability decreases by at least threefold. During the quasi-steady level of release, the release amplitude is 3.3-fold greater than expected from voltage activation alone, a result consistent with the recruitment by Ca-induced Ca 2+ release of 2.3 SR Ca 2+ release channels neighboring each channel activated by its associated voltage sensor. Release permeability at −60 mV increases as [Ca T] SR decreases from its resting physiological level to ∼0.1 of this level. This result argues against a release termination mechanism proposed in mammalian muscle fibers in which a luminal sensor of [Ca 2+] SR inhibits release when [Ca T] SR declines to a low level. 相似文献
14.
Patients with cerebral tumors often present with elevated levels of acetylcholine (ACh) in their cerebrospinal fluid. This
motivated us to investigate physiological effects of ACh on cultured human astrocytoma cells (U373) using a combination of
videomicroscopy, calcium microspectrofluorimetry and perforated patch-clamp recording. Astrocytoma cells exhibited the typical
morphological changes associated with cell migration; polarized cells displayed prominent lamellipodia and associated membrane
ruffling at the anterior of the cell, and a long tail region that periodically contracted into the cell body as the cell moved
forward. Bath application of the ACh receptor agonist, muscarine, reversibly inhibited cell migration. In conjunction with
this inhibition, ACh induced a dose-dependent, biphasic increase in resting intracellular free calcium concentration ([Ca 2+]
i
) associated with periodic Ca 2+ oscillations during prolonged ACh applications. The early transient rise in [Ca 2+]
i
was abolished by ionomycin and thapsigargin but was insensitive to caffeine and ryanodine while the plateau phase was strictly
dependent on external calcium. The Ca 2+ response to ACh was mimicked by muscarine and abolished by the muscarinic antagonists, atropine or 4-DAMP, but not by pirenzepine.
Using perforated patch-clamp recordings combined with fluorescent imaging, we demonstrated that ACh-induced [Ca 2+]
i
oscillations triggered membrane voltage oscillations that were due to the activation of voltage-dependent, Ca 2+-sensitive K + currents. These K + currents were blocked by intracellular injection of EGTA, or by extracellular application of TEA, quinine, or charybdotoxin,
but not by apamin. These studies suggest that activation of muscarinic receptors on glioma cells induce the release of Ca 2+ from intracellular stores which in turn activate Ca 2+-dependent (BK-type) K + channels. Furthermore, this effect was associated with inhibition of cell migration, suggesting an interaction of this pathway
with glioma cell migration.
Received: 17 December/Revised: 17 March 2000 相似文献
15.
We have investigated the kinetics of mitochondrial Ca 2+ influx and efflux and their dependence on cytosolic [Ca 2+] and [Na +] using low-Ca 2+-affinity aequorin. The rate of Ca 2+ release from mitochondria increased linearly with mitochondrial [Ca 2+] ([Ca 2+] M). Na +-dependent Ca 2+ release was predominant al low [Ca 2+] M but saturated at [Ca 2+] M around 400 μM, while Na +-independent Ca 2+ release was very slow at [Ca 2+] M below 200 μM, and then increased at higher [Ca 2+] M, perhaps through the opening of a new pathway. Half-maximal activation of Na +-dependent Ca 2+ release occurred at 5-10 mM [Na +], within the physiological range of cytosolic [Na +]. Ca 2+ entry rates were comparable in size to Ca 2+ exit rates at cytosolic [Ca 2+] ([Ca 2+] c) below 7 μM, but the rate of uptake was dramatically accelerated at higher [Ca 2+] c. As a consequence, the presence of [Na +] considerably reduced the rate of [Ca 2+] M increase at [Ca 2+] c below 7 μM, but its effect was hardly appreciable at 10 μM [Ca 2+] c. Exit rates were more dependent on the temperature than uptake rates, thus making the [Ca 2+] M transients to be much more prolonged at lower temperature. Our kinetic data suggest that mitochondria have little high affinity Ca 2+ buffering, and comparison of our results with data on total mitochondrial Ca 2+ fluxes indicate that the mitochondrial Ca 2+ bound/Ca 2+ free ratio is around 10- to 100-fold for most of the observed [Ca 2+] M range and suggest that massive phosphate precipitation can only occur when [Ca 2+] M reaches the millimolar range. 相似文献
16.
Summary Discrepancies about the role of L-type voltage-gated calcium channels (VGCC) in acetylcholine (ACh)-induced [Ca 2+] i oscillations in tracheal smooth muscle cells (TSMCs) have been seen in recent reports. We demonstrate here that ACh-induced
[Ca 2+] i oscillations in TMCS were reversibly inhibited by three VGCC blockers, nicardipine, nifedipine and verapamil. Prolonged (several
minutes) application of VGCC blockers, led to tachyphylaxis; that is, [Ca 2+] i oscillations resumed, but at a lower frequency. Brief (15–30 s) removal of VGCC blockers re-sensitized [Ca 2+] i oscillations to inhibition by the agents. Calcium oscillations tolerant to VGCC blockers were abolished by KB-R7943, an inhibitor
of the reverse mode of Na +/Ca 2+ exchanger (NCX). KB-R7943 alone also abolished ACh-induced [Ca 2+] i oscillations. Enhancement of the reverse mode of NCX via removing extracellular Na + reversed inhibition of ACh-induced [Ca 2+] i oscillations by VGCC blockers. Inhibition of non-selective cation channels using Gd 3+ slightly reduced the frequency of ACh-induced [Ca 2+] i oscillations, but did not prevent the occurrence of tachyphylaxis. Altogether, these results suggest that VGCC and the reverse
mode of NCX are two primary Ca 2+ entry pathways for maintaining ACh-induced [Ca 2+] i oscillations in TSMCs. The two pathways complement each other, and may account for tachyphylaxis of ACh-induced [Ca 2+] i oscillations to VGCC blockers. 相似文献
17.
In order to account for the time courses of both evoked release and facilitation, in the framework of the Ca 2+ hypothesis, Fogelson and Zucker (1985, Biophys. J.
48, 1003–1017) suggested treating diffusion of Ca 2+, once it enters through the Ca 2+ channels, as a three-dimensional process (three-dimensional diffusion model). This model is examined here as a refined version
of the “Ca 2+-theory” for neurotransmitter release. The three-dimensional model was suggested to account for both the time course of release
and that of facilitation. As such, it has been examined here as to its ability to predict the dependence of the amplitude
and time course of facilitation under various experimental conditions. It is demonstrated that the three-dimensional diffusion
model predicts the time course of facilitation to be insensitive to temperature. It also predicts the amplitude and time course
of facilitation to be independent of extracellular Ca 2+ concentration. Moreover, it predicts that inhibition of the [Na +] o↔[Ca 2+] i exchange does not alter facilitation. These predictions are not upheld by the experimental results. Facilitation is prolonged
upon reduction in temperature. The amplitude of facilitation declines and its duration is prolonged upon increase in extracellular
Ca 2+ concentration. Finally, inhibition of the [Na +] o↔[Ca 2+] i exchange prolongs facilitation but does not alter the time course of evoked release after an impulse. 相似文献
18.
This study investigates the effect of magnesium (Mg 2+) on the secretory responses and the mobilization of calcium (Ca 2+) and Mg 2+ evoked by cholecystokinin-octapeptide (CCK-8) in the exocrine rat pancreas. In the isolated intact perfused pancreas CCK-8 (10 –10 M) produced marked increases in juice flow and total protein output in zero and normal (1.1 mM) extracellular Mg 2+ [Mg 2+] o compared to a much reduced secretory response in elevated (5 mM and 10 mM) [Mg 2+] o Similar effects of perturbation of [Mg 2+] o on amylase secretion and 45Ca 2+ uptake (influx) were obtained in isolated pancreatic segments. In pancreatic acinar cells loaded with the fluorescent bioprobe fura-2 acetomethylester (AM), CCK-8 evoked marked increases in cytosolic free Ca 2+ concentration [Ca 2+] i in zero and normal [Mg 2+] o compared to a much reduced response in elevated [Mg 2+] o Pretreatment of acinar cells with either dibutyryl cyclic AMP (DB 2 cAMP) or forskolin had no effect on the CCK-8 induced changes in [Ca 2+] i. In magfura-2-loaded acinar cells CCK-8 (10 –8 M) stimulated an initial transient rise in intracellular free Mg 2+ concentration [Mg 2+] i followed by a more prolonged and sustained decrease. This response was abolished when sodium Na + was replaced with N-methyl-D-glucamine (NMDG). Incubation of acinar cells with 10 mM Mg 2+ resulted in an elevation in [Mg 2+] i. Upon stimulation with CCK-8, [Mg 2+] i. decreased only slightly compared with the response obtained in normal [Mg 2+] o. CCK-8 caused a net efflux of Mg 2+ in pancreatic segments; this effect was abolished when extracellular sodium [Na +] o was replaced with either NMDG or choline. The results indicate that Mg 2+ can regulate CCK-8-evoked secretory responses in the exocrine pancreas possibly via Ca 2+ mobilization. Moreover, the movement of Mg 2+ in pancreatic acinar cells is dependent upon extracellular Na +. 相似文献
19.
We developed a technique that yields isolated adult rat myocytes, 70% of which are elongated and morphologically similar to intact tissue. Electrophysiological studies showed most of these cells were quiescent, Ca 2+-tolerant and exhibited normal action potentials accompanied by contractions. We analyzed 45Ca 2+ uptake data in terms of instantaneous, fast and slow compartments. 69% of total exchangeable Ca 2+ was found in the slow compartment; the rest was almost equally divided between the instantaneous and fast compartments. Replacement of extracellular Na + by Li + or Tris increased 45Ca 2+ uptake by the fast compartment; high [K +] o increased this uptake further. These increases appeared to be related also to internal concentrations of Na +. This conclusion was supported by experiments with digitonin-treated cells. Our results indicate that the way Na +-dependent 45Ca 2+ uptake is affected by [Na +] o, [Na +] i and [K +] o is compatible with the Na +-Ca 2+ exchange mechanism. Our preparation should prove useful in studies of regulation of Ca 2+ transport in cardiac muscles. 相似文献
20.
Rises in cytosolic Ca 2+ concentration ([Ca 2+] cyt) are central in platelet activation, yet many aspects of the underlying mechanisms are poorly understood. Most studies examine how experimental manipulations affect agonist-evoked rises in [Ca 2+] cyt, but these only monitor the net effect of manipulations on the processes controlling [Ca 2+] cyt (Ca 2+ buffering, sequestration, release, entry and removal), and cannot resolve the source of the Ca 2+ or the transporters or channels affected. To investigate the effects of protein kinase C (PKC) on platelet Ca 2+ signalling, we here monitor Ca 2+ flux around the platelet by measuring net Ca 2+ fluxes to or from the extracellular space and the intracellular Ca 2+ stores, which act as the major sources and sinks for Ca 2+ influx into and efflux from the cytosol, as well as monitoring the cytosolic Na + concentration ([Na +] cyt), which influences platelet Ca 2+ fluxes via Na +/Ca 2+ exchange. The intracellular store Ca 2+ concentration ([Ca 2+] st) was monitored using Fluo-5N, the extracellular Ca 2+ concentration ([Ca 2+] ext) was monitored using Fluo-4 whilst [Ca 2+] cyt and [Na +] cyt were monitored using Fura-2 and SFBI, respectively. PKC inhibition using Ro-31-8220 or bisindolylmaleimide I potentiated ADP- and thrombin-evoked rises in [Ca 2+] cyt in the absence of extracellular Ca 2+. PKC inhibition potentiated ADP-evoked but reduced thrombin-evoked intracellular Ca 2+ release and Ca 2+ removal into the extracellular medium. SERCA inhibition using thapsigargin and 2,5-di(tert-butyl) l,4-benzohydroquinone abolished the effect of PKC inhibitors on ADP-evoked changes in [Ca 2+] cyt but only reduced the effect on thrombin-evoked responses. Thrombin evokes substantial rises in [Na +] cyt which would be expected to reduce Ca 2+ removal via the Na +/Ca 2+ exchanger (NCX). Thrombin-evoked rises in [Na +] cyt were potentiated by PKC inhibition, an effect which was not due to altered changes in non-selective cation permeability of the plasma membrane as assessed by Mn 2+ quench of Fura-2 fluorescence. PKC inhibition was without effect on thrombin-evoked rises in [Ca 2+] cyt following SERCA inhibition and either removal of extracellular Na + or inhibition of Na +/K +-ATPase activity by removal of extracellular K + or treatment with digoxin. These data suggest that PKC limits ADP-evoked rises in [Ca 2+] cyt by acceleration of SERCA activity, whilst rises in [Ca 2+] cyt evoked by the stronger platelet activator thrombin are limited by PKC through acceleration of both SERCA and Na +/K +-ATPase activity, with the latter limiting the effect of thrombin on rises in [Na +] cyt and so forward mode NCX activity. The use of selective PKC inhibitors indicated that conventional and not novel PKC isoforms are responsible for the inhibition of agonist-evoked Ca 2+ signalling. 相似文献
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