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1.
Current-clamp studies of cultured leech Retzius cells revealed inward rectification in the form of slow voltage sags in response to membrane hyperpolarization. Sag responses were eliminated in Na+-free saline and blocked by Cs+, but not Ba2+. Voltage clamp experiments revealed a Cs+-sensitive inward current activated by hyperpolarization negative to −70 mV. Cs+ decreased the frequency of spontaneous impulses in Retzius cells of intact ganglia. Plateau potentials were evoked in Retzius cells following block of Ca2+ influx with Ni2+ and suppression of K+ currents with internal tetraethylammonium. Plateau potentials continued to be expressed with Li+ as the charge carrier, but were eliminated when Na+ was replaced with N-methyl-d-glucamine. A persistent Na+ current with similar pharmacology that activated positive to −40 mV and reached its peak amplitude near −5 mV was identified in voltage-clamp experiments. Inactivation of the persistent Na+ current was slow and incomplete. The current was revealed by slow voltage ramps and persisted for the duration of 5-s voltage steps. Persistent Na+ current may underlie Na+-dependent bursting recorded in neurons of intact ganglia exposed to Ca2+-channel blockers. Accepted: 22 September 1998  相似文献   

2.
The ovulation hormone-producing caudo-dorsal cells (CDC) of Lymnaea stagnalis have three states of excitability (active, inhibited, and resting), which are related to the egg-laying cycle. Active state CDC produce a firing pattern of prolonged spiking activity (1 spike/2 s), which in the animal occurs shortly before egg laying. In preparations it is evoked as an afterdischarge upon repetitive stimulation of CDC. The afterdischarge is not synaptically driven, but rests on a pacemaking mechanism. CDC are silent in the inhibited and resting states, which follow egg laying. In these states the membrane potential is mainly dependent on [K+]0. In the active state the ratio of the K+, Na+, and Ca2+ permeabilities has changed considerably, probably resulting from an increased permeability to Na+ and Ca2+. The firing rate in the afterdischarge is dependent on the membrane potential, which is confirmed experimentally by varying [K+]0.[Na+]0 and [Ca2+]0 directly influence the firing rate. Firing stops in Na+-free saline, but is enhanced by Ca2+-free or high-Mg2+ saline. TTX does not affect firing. Relatively high concentrations of Co2+ and La3+ (2 × 10?3M) strongly inhibit CDC. Regular firing can be changed into bursting by various means, such as high K+ or addition of 1 mM Ba2+. Bursting normally occurs at the beginning of the afterdischarge. Postburst hyperpolarizations are reduced in Ca2+-free saline and by low Co2+ (10?4-5 10?4M). Active CDC are driven by a pacemaking mechanism constituted by a voltage-dependent Na+/Ca2+ channel and a Ca2+-dependent K+ channel, thus resembling that of bursting pacemakers. The pacemaking mechanism is inactive in the resting and inhibited state.  相似文献   

3.
In order to identify defects in Na+-Ca2+ exchange and Ca2+-pump systems in cardiomyopathic hearts, the activities of sarcolemmal Na+-dependent Ca2+ uptake, Na+-induced Ca2+ release, ATP-dependent Ca2+ uptake and Ca2+-stimulated ATPase were examined by employing cardiomyopathic hamsters (UM-X7.1) and catecholamine-induced cardiomyopathy produced by injecting isoproterenol into rats. The rates of Na+-dependent Ca2+ uptake, ATP-dependent Ca2+ uptake and Ca2+-stimulated ATPase activities of sarcolemmal vesicles from genetically-linked cardiomyopathic as well as catecholamine-induced cardiomyopathic hearts were decreased without any changes in Na+-induced Ca2+-release. Similar results were obtained in Ca2+-paradox when isolated rat hearts were perfused for 5 min with a medium containing 1.25 mM Ca2+ following a 5 min perfusion with Ca2+-free medium. Although a 2 min reperfusion of the Ca2+-free perfused hearts depressed sarcolemmal Ca2+-pump activities without any changes in Na+-induced Ca2+-release, Na+-dependent Ca2+ uptake was increased. These results indicate that alterations in the sarcolemmal Ca2+-efflux mechanisms may play an important role in cardiomyopathies associated with the development of intracellular Ca2+ overload.  相似文献   

4.
The ionic dependency and transmission of epidermal action potentials have been examined from tailbud to hatching stages of newt embryos. Previously we have reported that the epidermal action potential is composed of a fast- and slow-action component; only the slow-action component, however, is transmitted to other cells. We address in this report the mechanism by which these responses are mediated. The slow-action potential is not produced in Na+-free saline, tricaine saline, or following the application of TTX, and thus appears to be Na+ dependent. The fast-action potential on the other hand is blocked by application of Co2+ and verapamil saline and thus appears to be Ca2+ dependent. The slow-action potentials appear to be chemically transmitted since they are transmitted even to those cells which are electrically uncoupled at low intracellular pH (NaHCO3 + HCl, pH 6.2). Furthermore 1 μM curare and atropine are inhibitory to transmission of the slow potential. Epidermal cells of the newt embryo are sensitive to acetylcholine (ACh) applied by hydrostatic ejection through a micropipet. The latter observation further suggests that propagation of the slow-action potential is, in part, a chemical event.  相似文献   

5.
The weakly electric fish Gymnotus carapo emits a triphasic electric organ discharge generated by muscle-derived electrocytes, which is modified by environmental and physiological factors. Two electrode current clamp recordings in an in vitro preparation showed that Gymnotus electrocytes fired repetitively and responded with plateau potentials when depolarized. This electrophysiological behavior has never been observed in electrocytes from related species. Two types of plateaus with different thresholds and amplitudes were evoked by depolarization when Na+-dependent currents were isolated in a K+- and Ca2+-free solution containing TEA and 4-AP. Two electrode voltage clamp recordings revealed a classical fast activating–inactivating Na+ current and two persistent Na+-dependent currents with voltage-dependencies consistent with the action potential (AP) and the two plateaus observed under current clamp, respectively. The three currents, the APs and the plateaus were reduced by TTX, and were absent in Na+-free solution. The different Na+-dependent currents in Gymnotus electrocytes may be targets for the modifications of the electric organ discharge mediated by environmental and physiological factors.  相似文献   

6.
Summary Previous current/voltage (I/V) investigations of theChara K+ state have been extended by increasing the voltage range (up to +200 mV) through blocking the action potential with La3+. A region of negative slope was found in theI/V characteristics at positive PD's, similar to that already observed at PD's more negative than the resting level. These decreases in membrane currents at PD's more negative than –150 mV and at PD's close to 0 or positive are thought to arise from the K+ channel closure. Both the negative slope regions could be reversibly abolished by 0.1mm K+, 20mm Na+, more than 10mm Ca2+ or 5mm tetraethylammonium (TEA). The K+ channels are therefore blocked by TEA, closed by low [K+] o or high [Ca2+] o and are highly selective to K+ over Na+. With the K+ channels closed, the remainingI/V profile was approximately linear over the interval of 400 mV (suggesting a leakage current), but large rectifying currents were observed at PD's more positive than +50 mV. These currents showed a substantial decrease in high [Ca2+] o , sometimes displayed a slight shift to more positive PD's with increasing [K+] o and were unaffected by TEA or changes in [Na+] o . The slope of the linear part of theI/V profile was steeper in low [K+] o than in TEA or high [Na+] o (indicating participation of K+, but not Na+, in the leak current). Diethylstilbestrol (DES) was employed to inhibit the proton pump, but it was found that the leakage current and later the K+ channels were also strongly affected.  相似文献   

7.
Summary Glucose-induced electrical activity in canine pancreatic islet B cells is distinct from that in rodent islets, though both display Ca2+-dependent insulin secretion. Rodent islet B cells undergo regular bursts of Ca2+-dependent action potentials, while canine islet B cells generate isolated Na+-dependent action potentials which often give way to a plateau depolarization. Here we present evidence to reconcile the species difference in electrical activity with the similarity of Ca2+ dependence of secretion. (i) In canine B cells increasing glucose concentrations produce membrane depolarization and increasing frequency of Nao-dependent action potentials until a background membrane potential (-40mV) is reached where Na+ currents are inactivated. (ii) Voltage-dependent Ca2+ currents are present which are activated over the voltage excursion of the action potential (–50 to +20 mV) and inactivate slowly, (over seconds) in the range of the plateau depolarization (–40 to –25 mV). Hence, they are available to contribute to both phases of depolarization. (iii) Tetrodotoxin (TTX) reduces by half an early transient phase of glucosestimulated insulin secretion but not a subsequent prolonged plateau phase. The transient phase of secretion often corresponds well in time to the period of initial high frequency action potential activity. These latter results suggest that in canine B cells voltagedependent Na+ and Ca2+ currents mediate biphasic glucose-induced insulin secretion. The early train of Na+-dependent action potentials, by transiently activating Ca2+ channels and allowing pulsatile Ca2+ entry, may promote an early transient phase of insulin secretion. The subsequent sustained plateau depolarization, by allowing sustained Ca2+ entry, may permit steady insulin release.  相似文献   

8.
The electrophysiological properties of cultured human melanocytes were investigated using the whole-cell configuration of the patch-clamp technique. Depolarizations to membrane potentials more positive than -30 mV resulted in the rapid development (<1 ms to peak) of an inward current. The maximum peak current was observed at +10 mV and reached an average amplitude of about 270 pA. During the depolarizations, the current inactivated with a time constant of about 2 ms. The current was abolished by the addition of 0.3 μM tetrodotoxin, a blocker of voltage-gated Na+-channels, and disappeared when Na+ was omitted from the extracellular medium. In addition, the melanocytes contain at least two types of outward K+-current. The first type, observed in every cell, was highly sensitive (Ki 1 mM) to the K+-channel blocker TEA, required depolarizations beyond zero to be activated and did not inactivate. The second type was less regularly observed (10% of the cells). This current activated at more negative voltages (–20 mV), was resistant to TEA (20 mM) but was blocked by 2 mM 4-aminopyridine and inactivated rapidly during depolarizations. We conclude that human melanocytes are equipped with voltage-dependent Na+-channels, a delayed rectifying K+-current and a K+-current similar to the A-current in neurones.  相似文献   

9.
The Ca2+-conducting pathway of myocytes isolated from the cricket lateral oviduct was investigated by means of the whole-cell patch clamp technique. In voltage-clamp configuration, two types of whole cell inward currents were identified. One was voltage-dependent, initially activated at –40 mV and reaching a maximum at 10 mV with the use of 140 mM Cs2+-aspartate in the patch pipette and normal saline in the bath solution. Replacement of the external Ca2+ with Ba2+ slowed the current decay. Increasing the external Ca2+ or Ba2+ concentration increased the amplitude of the inward current and the current–voltage (I–V) relationship was shifted as expected from a screening effect on negative surface charges. The inward current could be carried by Na+ in the absence of extracellular Ca2+. Current carried by Na+ (I Na) was almost completely blocked by the dihydropyridine Ca2+ channel antagonist, nifedipine, suggesting that the I Na is through voltage-dependent L-type Ca2+ channels. The other inward current is voltage-independent and its I–V relationship was linear between –100 mV to 0 mV with a slight inward rectification at more hyperpolarizing membrane potentials when 140 mM Cs+-aspartate and 140 mM Na+-gluconate were used in the patch pipette and in the bath solution, respectively. A similar current was observed even when the external Na+ was replaced with an equimolar amount of K+ or Cs+, or 50 mM Ca2+ or Ba2+. When the osmolarity of the bath solution was reduced by removing mannitol from the bath solution, the inward current became larger at negative potentials. The I–V relationship for the current evoked by the hypotonic solution also showed a linear relationship between –100 mV to 0 mV. Bath application of Gd3+ (10 M) decreased the inward current activated by membrane hyperpolarization. These results clearly indicate that the majority of current activated by a membrane hyperpolarization is through a stretch-activated Ca2+-permeable nonselective cation channel (NSCC). Here, for the first time, we have identified voltage-dependent L-type Ca2+ channel and stretch-activated Ca2+-permeable NSCCs from enzymatically isolated muscle cells of the cricket using the whole-cell patch clamp recording technique.Abbreviations I Ca Ca2+ current - I Na Na+ current - I–V current–voltage - NSCC nonselective cation channel Communicated by G. Heldmaier  相似文献   

10.
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, Ca2+-tolerant and exhibited normal action potentials accompanied by contractions. We analyzed 45Ca2+ uptake data in terms of instantaneous, fast and slow compartments. 69% of total exchangeable Ca2+ 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 45Ca2+ 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 45Ca2+ uptake is affected by [Na+]o, [Na+]i and [K+]o is compatible with the Na+-Ca2+ exchange mechanism. Our preparation should prove useful in studies of regulation of Ca2+ transport in cardiac muscles.  相似文献   

11.
Shuba  M. F.  Vladimirova  I. A.  Philyppov  I. B. 《Neurophysiology》2003,35(3-4):224-233
Nonadrenergic inhibitory and excitatory junction potentials (IJP and EJP) in the intestinal smooth muscle cells are of a complex transmitter and ion nature. The IJP consist of two components; the initial, fast, component is of a purinergic nature. Low-conductance Ca2+-dependent potassium channels (SK(Ca)) are involved in generation of the initial component of IJP because this component can be specifically and reversibly blocked by apamin. Probably, local Ca2+ release from the InsP3-sensitive store can be a link between the P2Y receptors and activation of the SK(Ca) channels because inhibition of the activity of phospholipase C (PLC) decreases IJP. The second, slow, component of IJP is nitric oxide-dependent. Such a component of IJP develops due to activation of high-conductance Ca2+-dependent potassium channels (BK(Ca)) because this component can be blocked by TEA and charybdotoxin. The release of Ca2+ from the ryanodine-sensitive store is responsible for activation of the BK(Ca) channels and generation of the second component of IJP. Thus, it appears that Ca2+ released from one of the intracellular stores can activate only a certain type of the Ca2+-dependent K+ channels involved in the generation of IJP.  相似文献   

12.
Experiments on the effects of varying concentrations of Ca2+ on the Mg2+ + Na+-dependent ATPase activity of a highly purified preparation of dog kidney (Na+ + K+)-ATPase showed that Ca2+ was a partial inhibitor of this activity. When Ca2+ was added to the reaction mixture instead of Mg2+, there was a ouabain-sensitive Ca2+ + Na+-dependent ATPase activity the maximal velocity of which was 30 to 50% of that of Mg2+ + Na+-dependent activity. The apparent affinities of the enzyme for Ca2+ and CaATP seemed to be higher than those for Mg2+ and MgATP. Addition of K+, along with Ca2+ and Na+, increased the maximal velocity and the concentration of ATP required to obtain half-maximal velocity. The maximal velocity of the ouabain-sensitive Ca2+ + Na+ + K+-dependent ATPase was about two orders of magnitude smaller than that of Mg2+ + Na+ + K+-dependent activity. In agreement with previous observations, it was shown that in the presence of Ca2+, Na+, and ATP, an acid-stable phosphoenzyme was formed that was sensitive to either ADP or K+. The enzyme also exhibited a Ca2+ + Na+-dependent ADP-ATP exchange activity. Neither the inhibitory effects of Ca2+ on Mg2+-dependent activities, nor the Ca2+-dependent activities were influenced by the addition of calmodulin. Because of the presence of small quantities of endogenous Mg2+ in all reaction mixtures, it could not be determined whether the apparent Ca2+-dependent activities involved enzyme-substrate complexes containing Ca2+ as the divalent cation or both Ca2+ and Mg2+.  相似文献   

13.
Summary Intracellular Ca2+ has been suggested to play an important role in the regulation of epithelial Na+ transport. Previous studies showed that preincubation of toad urinary bladder, a tight epithelium, in Ca2+-free medium enhanced Na+ uptake by the subsequently isolated apical membrane vesicles, suggesting the downregulation of Na+ entry across the apical membrane by intracellular Ca2+. In the present study, we have examined the effect of Ca2+-free preincubation on apical membrane Na+ transport in a leaky epithelium, i.e., brush border membrane (BBM) of rabbit renal proximal tubule. In contrast to toad urinary bladder, it was found that BBM vesicles derived from proximal tubules incubated in 1mm Ca2+ medium exhibited higher Na+ uptake than those derived from proximal tubules incubated in Ca2+-free EGTA medium. Such effect of Ca2+ in the preincubation medium was temperature dependent and could not be replaced by another divalent cation, Ba2+ (1mm). Ca2+ in the preincubation medium did not affect Na+-dependent BBM glucose uptake, and its effect on BBM Na+ uptake was pH gradient dependent and amiloride (10–3 m) sensitive, suggesting the involvement of Na+/H+ antiport system. Addition of verapamil (10–4 m) to 1mm Ca2+ preincubation medium abolished while ionomycin (10–6 m) potentiated the effect of Ca2+ to increase BBM Na+ uptake, suggesting that the effect of Ca2+ in the preincubation medium is likely to be mediated by Ca2+-dependent cellular pathways and not due to a direct effect of extracellular Ca2+ on BBM. Neither the proximal tubule content of cAMP nor the inhibitory effect of 8, bromo-cAMP (0.1mm) on BBM Na+ uptake was affected by the presence of Ca2+ in the preincubation medium, suggesting that Ca2+ in the preincubation medium did not increase BBM Na+ uptake by removing the inhibitory effect of cAMP. Addition of calmodulin inhibitor, trifluoperazine (10–4 m) to 1mm Ca2+ preincubation medium did not prevent the increase in BBM Na+ uptake. The effect of Ca2+ was, however, abolished when protein kinase C in the proximal tubule was downregulated by prolonged (24 hr) incubation with phorbol 12-myristate 13-acetate (10–6 m). In summary, these results show the Ca2+ dependency of Na+ transport by renal BBM, possibly through stimulation of Na+/H+ exchanger by protein kinase C.  相似文献   

14.
UV irradiation has multiple effects on mammalian cells, including modification of ion channel function. The present study was undertaken to investigate the response of membrane currents in guinea-pig ventricular myocytes to the type A (355, 380 nm) irradiation commonly used in Ca2+ imaging studies. Myocytes configured for whole-cell voltage clamp were generally held at −80 mV, dialyzed with K+-, Na+-free pipette solution, and bathed with K+-free Tyrode’s solution at 22°C. During experiments that lasted for ≈ 35 min, UVA irradiation caused a progressive increase in slowly-inactivating inward current elicited by 200-ms depolarizations from −80 to −40 mV, but had little effect on background current or on L-type Ca2+ current. Trials with depolarized holding potential, Ca2+ channel blockers, and tetrodotoxin (TTX) established that the current induced by irradiation was late (slowly-inactivating) Na+ current (INa). The amplitude of the late inward current sensitive to 100 μM TTX was increased by 3.5-fold after 20–30 min of irradiation. UVA modulation of late INa may (i) interfere with imaging studies, and (ii) provide a paradigm for investigation of intracellular factors likely to influence slow inactivation of cardiac INa.  相似文献   

15.
The aim of the present study was to investigate the roles of Ca2+ and protein tyrosine kinase (PTK) in the insulin action on cell volume in fetal rat (20-day gestational age) type II pneumocytes. Insulin (100 nm) increased cell volume in the presence of extracellular Ca2+ (1 mm), while cell shrinkage was induced by insulin in the absence of extracellular Ca2+ (<1 nm). This insulin action in a Ca2+-containing solution was completely blocked by co-application of bumetanide (50 μm, an inhibitor of Na+/K+/2Cl cotransporter) and amiloride (10 μm, an inhibitor of epithelial Na+ channel), but not by the individual application of either bumetanide or amiloride. On the other hand, the insulin action on cell volume in a Ca2+-free solution was completely blocked by quinine (1 mm, a blocker of Ca2+-activated K+ channel), but not by bumetanide and/or amiloride. These observations suggest that insulin activates an amiloride-sensitive Na+ channel and a bumetanide-sensitive Na+/K+/2Cl cotransporter in the presence of 1 mm extracellular Ca2+, that the stimulatory action of insulin on an amiloride-sensitive Na+ channel and a bumetanide-sensitive Na+/K+/2Cl cotransporter requires Ca2+, and that in a Ca2+-free solution insulin activates a quinine-sensitive K+ channel but not in the presence of 1 mm Ca2+. The insulin action on cell volume in a Ca2+-free solution was almost completely blocked by treatment with BAPTA (10 μm) or thapsigargin (1 μM, an inhibitor of Ca2+-ATPase which depletes the intracellular Ca2+ pool). Further, lavendustin A (10 μm, an inhibitor of receptor type PTK) blocked the insulin action in a Ca2+-free solution. These observations suggest that the stimulatory action of insulin on a quinine-sensitive K+ channel is mediated through PTK activity in a cytosolic Ca2+-dependent manner. Lavendustin A, further, completely blocked the activity of the Na+/K+/2Cl cotransporter in a Ca2+-free solution, but only partially blocked the activity of the Na+/K+/2Cl cotransporter in the presence of 1 mm Ca2+. This observation suggests that the activity of the Na+/K+/2Cl cotransporter is maintained through two different pathways; one is a PTK-dependent, Ca2+-independent pathway and the other is a PTK-independent, Ca2+-dependent pathway. Further, we observed that removal of extracellular Ca2+ caused cell shrinkage by diminishing the activity of the amiloride-sensitive Na+ channel and the bumetanide-sensitive Na+/K+/2Cl cotransporter, and that removal of extracellular Ca2+ abolished the activity of the quinine-sensitive K+ channel. We conclude that the cell shrinkage induced by removal of extracellular Ca2+ results from diverse effects on the cotransporter and Na+ and K+ channels. Received: 2 September 1998/Revised: 30 November 1998  相似文献   

16.
《Life sciences》1995,57(17):PL259-PL264
The effect of bradykinin (BK), in the presence of ouabain, an inhibitor of Na+-K+ ATPase, on catecholamine (CA) secretion was studied in cultured bovine adrenal chromaffin cells, to determine whether Na+, as well as Ca2+, is involved in BK-receptor mediated CA secretion. BK (10−8–10−5M)-induced CA secretion was markedly potentiated by addition of ouabain (10−5M), was blocked by a BK-B2 receptor antagonist, and was decreased in Ca2+-free medium. BK-induced increase in 45Ca2+ influx was also potentiated by addition of ouabain. The cultured cells were first incubated with BK for 30 min in Ca2+-free medium in the presence or absence of ouabain and then kstimulated for 15 min with Ca2+-medium without BK or ouabain. Prior stimulation of the cells, BK induced 22Na+ influx and increased Ca2+-induced CA secretion and these stimulatory effects of BK were potentiated by added ouabain. When the cells were stimulated with BK and ouabain in Na+-free sucrose medium, the Ca2+-induced CA secretion was greatly reduced. These results indicated that activation of the BK-B2 receptor and inhibition of the Na+ pump both increase the intracellular Na+ level, resulting in increase in Ca2+ influx and CA secretion.  相似文献   

17.
We cultured retinal pigment epithelial (RPE) cells dissociated from adult newt eye and analyzed their voltage-gated ion channels during culture using whole-cell patch-clamp techniques. The results were compared with those of retinal neurons under identical experimental conditions. After 6–9 days in culture (early stage), > 60% of RPE cells developed voltage-gated Na+ and Ca2+ channels that were not observed in freshly dissociated RPE cells. The number of cells expressing Na+ channels and Na+ current density were high after 12–15 days in culture (intermediate stage), while the number of Ca2+ channel-expressing cells and Ca2+ current density were high after 20–30 days in culture (late stage). The activation voltage of the Na+ current in the RPE cells was similar to that in neurons. The voltage dependence of Na+ current inactivation was somewhat different between two cell types. The steepness of the inactivation curve tended to be less in cultured RPE cells than in neurons, and the half-inactivation voltage was about −54 mV for the RPE cells and −45 mV for neurons. The Ca2+ current expressed in cultured RPE cells was too small to detect without replacement of external Ca2+ with Ba2+. The Ba2+ current, like Ca2+ current in neurons, was enhanced by Bay-K 8644 and blocked by nicardipine. These results suggest that the RPE cells, like neurons, expressed L-type Ca2+ channels in culture. The possibility that the development of both Na2+ and Ca2+ channels in cultured RPE cells is a manifestation of the transdifferentiation of RPE cells into neurons is discussed. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 377–390, 1997.  相似文献   

18.
Abstract: The inhibitory effects of Na+/Ca2+ exchange inhibitory peptide (XIP), which corresponds to residues 219–238 of the Na+/Ca2+ exchange protein from canine heart, were studied in both rat and human brain plasma membrane vesicles. XIP had very high potency with respect to the inhibition of the initial velocity of intravesicular Na+-dependent Ca2+ uptake in both rat brain [IC50 = 3.05 ± 0.69 µM (mean ± SE)] and human brain (IC50 = 3.58 ± 0.58 µM). The maximal inhibition seen in rat brain vesicles was ~80%, whereas human brain vesicles were inhibited 100%. XIP also inhibited extravesicular Na+-dependent Ca2+ release, and the inhibitory effect was enhanced by increasing the extravesicular Na+ concentration. In contrast, the inhibitory effect of bepridil was competitive with respect to extravesicular Na+. When XIP was added at steady state (5 min after the initiation of intravesicular Na+-dependent Ca2+ uptake), it was found that the intravesicular Ca2+ content declined with time. Analysis of unidirectional fluxes for Ca2+ at steady state showed that 50 µM XIP inhibited Ca2+ influx and efflux ~85 and 70%, respectively. This result suggested that XIP inhibited both Na+/Ca2+ exchange and Ca2+/Ca2+ exchange but had no effect on the passive release pathway for Ca2+. The results suggest structural homology among cardiac, rat, and human brain exchangers in the XIP binding domain and that the binding of Na+ or other monovalent cations, e.g., K+, is required for XIP to have its inhibitory effect on Ca2+ transport.  相似文献   

19.
Summary The relative contributions of the Na+/Ca2+ exchange and the plasma membrane Ca2+ pump to active Ca2+ efflux from stimulated rat pancreatic acini were studied. Na+ gradients across the plasma membrane were manipulated by loading the cells with Na+ or suspending the cells in Na+-free media. The rates of Ca2+ efflux were estimated from measurements of [Ca2+] i using the Ca2+-sensitive fluorescent dye Fura 2 and45Ca efflux. During the first 3 min of cell stimulation, the pattern of Ca2+ efflux is described by a single exponential function under control, Na+-loaded, and Na+-depleted conditions. Manipulation of Na+ gradients had no effect on the hormone-induced increase in [Ca2+] i . The results indicate that Ca2+ efflux from stimulated pancreatic acinar cells is mediated by the plasma membrane Ca2+ pump. The effects of several cations, which were used to substitute for Na+, on cellular activity were also studied. Choline+ and tetramethylammonium+ (TMA+) released Ca2+ from intracellular stores of pancreatic acinar, gastric parietal and peptic cells. These cations also stimulated enzyme and acid secretion from the cells. All effects of these cations were blocked by atropine. Measurements of cholecystokinin-octapeptide (CCK-OP)-stimulated amylase release from pancreatic acini, suspended in Na+, TMA+, choline+, or N-methyl-d-glucamine+ (NMG+) media containing atropine, were used to evaluate the effect of the cations on cellular function. NMG+, choline+, and TMA+ inhibited amylase release by 55, 40 and 14%, respectively. NMG+ also increased the Ca2+ permeability of the plasma membrane. Thus, to study Na+ dependency of cellular function, TMA+ is the preferred cation to substitute for Na+. The stimulatory effect of TMA+ can be blocked by atropine.  相似文献   

20.
The electrical membrane properties of cultured human cytotrophoblast were examined by means of a standard electrophysiological technique. The mean values of the membrane potential (Em) and the membrane resistance in a physiological medium were around ?49 mV and 12 MΩ, respectively. The membrane potential was dependent, to a large extent, on the external Ca2+ concentration ([Ca2+]0). Deprivation of external Ca2+ reduced membrane potential to about ?20 mV, and an increase in [Ca2+]0 caused a hyperpolarization in a saturable manner. The Ca2+-dependency of membrane potential was affected remarkably by [K+]0, but not by [Na+]0 or [Cl?]0. The intracellular Ca2+ injection hyperpolarized the membrane in a Ca2+-free medium. A Ca2+ channel blocker, verapamil, completely abolished the Ca2+-dependent Em. The Ca2+-dependent Em was also suppressed by cooling or by the application of metabolic inhibitors. It is suggested that the Ca2+-dependent Em in cultured human cytotrophoblast is caused by a Ca2+ influx which, in turn, increases the K+ conductance of the cell membrane, presumably due to stimulation of Ca2+-activated K+ channel.  相似文献   

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