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1.
Evidence for two modes of Ca2+ entry following muscarinic stimulation of a human salivary epithelial cell line 总被引:1,自引:0,他引:1
Xinjun He Xiaozai Wu R. James Turner Bruce J. Baum 《The Journal of membrane biology》1990,115(2):159-166
Summary We have investigated muscarinic receptor-operated Ca2+ mobilization in a salivary epithelial cell line, HSG-PA, using an experimental approach which allows independent evaluation of intracellular Ca2+ release and extracellular Ca2+ entry. The carbachol (Cch) dose response of intracellular Ca2+ release indicates the involvement of a single, relatively low-affinity, muscarinic receptor site (K
0.510 or 30 m, depending on the method for [Ca2+]
i
determination). However, similar data for Ca2+ entry indicate the involvement of two Cch sites, one consistent with that associated with Ca2+ release and a second higher affinity site withK
0.52.5 m. In addition, the Ca2+ entry response observed at lower concentrations of Cch (2.5 m) was completely inhibited by membrane depolarization induced with high K+ (>55mm) or gramicidin D (1 m), while membrane depolarization had little or no effect on Ca2+ entry induced by 100 m Cch. Another muscarinic agonist, oxotremorine-M (100 m; Oxo-M), like Cch, also induced an increase in the [Ca2+]
i
of HSG-PA cells (from 72±2 to 104±5nm). This response was profoundly blocked (75%) by the inorganic Ca2+ channel blocker La3+ (25–50 m) suggesting that Oxo-M primarily mobilizes Ca2+ in these cells by increasing Ca2+ entry. Organic Ca2+ channel blockers (verapamil or diltiazem at 10 m, nifedipine at 1 m), had no effect on this response. The Oxo-M induced Ca2+ mobilization response, like that observed at lower doses of Cch, was markedly inhibited (70–90%) by membrane depolarization (high K+ or gramicidin D). At 100 m Cch the formation of inositol trisphosphate (IP3) was increased 55% above basal levels. A low concentration of carbachol (1 m) elicited a smaller change in IP3 formation (25%), similar to that seen with 100 m Oxo-M (20%). Taken together, these results suggest that there are two modes of muscarinic receptor-induced Ca2+ entry in HSG-PA cells. One is associated with IP3 formation and intracellular Ca2+ release and is independent of membrane potential; the other is less dependent on IP3 formation and intracellular Ca2+ release and is modulated by membrane potential. This latter pathway may exhibit voltage-dependent gating. 相似文献
2.
Bent Vestergaard-Bogind Per Stampe Palle Christophersen 《The Journal of membrane biology》1987,95(2):121-130
Summary The conductance of the Ca2+-activated K+ channel (g
K(Ca)) of the human red cell membrane was studied as a function of membrane potential (V
m
) and extracellular K+ concentration ([K+]ex). ATP-depleted cells, with fixed values of cellular K+ (145mm) and pH (7.1), and preloaded with 27 m ionized Ca were transferred, with open K+ channels, to buffer-free salt solutions with given K+ concentrations. Outward-current conductances were calculated from initial net effluxes of K+, correspondingV
m
, monitored by CCCP-mediated electrochemical equilibration of protons between a buffer-free extracellular and the heavily buffered cellular phases, and Nernst equilibrium potentials of K ions (E
K) determined at the peak of hyperpolarization. Zero-current conductances were calculated from unidirectional effluxes of42K at (V
m
–E
K)0, using a single-file flux ratio exponent of 2.7. Within a [K+]ex range of 5.5 to 60mm and at (V
m
–E
K) 20 mV a basic conductance, which was independent of [K+]ex, was found. It had a small voltage dependence, varying linearly from 45 to 70 S/cm2 between 0 and –100 mV. As (V
m
–E
K) decreased from 20 towards zero mVg
K(Ca) increased hyperbolically from the basic value towards a zero-current value of 165 S/cm2. The zero-current conductance was not significantly dependent on [K+]ex (30 to 156mm) corresponding toV
m
(–50 mV to 0). A further increase ing
K(Ca) symmetrically aroundE
K is suggested as (V
m
–E
K) becomes positive. Increasing the extracellular K+ concentration from zero and up to 3mm resulted in an increase ing
K(Ca) from 50 to 70 S/cm2. Since the driving force (V
m
–E
K) was larger than 20 mV within this range of [K+]ex this was probably a specific K+ activation ofg
K(Ca). In conclusion: The Ca2+-activated K+ channel of the human red cell membrane is an inward rectifier showing the characteristic voltage dependence of this type of channel. 相似文献
3.
Bodrova ME Brailovskaya IV Efron GI Starkov AA Mokhova EN 《Biochemistry. Biokhimii?a》2003,68(4):391-398
At low Ca2+ concentrations the pore of the inner mitochondrial membrane can open in substates with lower permeability (Hunter, D. R., and Haworth, R. A. (1979) Arch. Biochem. Biophys., 195, 468-477). Recently, we showed that Ca2+ loading of mitochondria augments the cyclosporin A-dependent decrease in transmembrane potential () across the inner mitochondrial membrane caused by 10 M myristic acid but does not affect the stimulation of respiration by this fatty acid. We have proposed that in our experiments the pore opened in a substate with lower permeability rather than in the classic state (Bodrova, M. E., et al. (2000) IUBMB Life, 50, 189-194). Here we show that under conditions lowering the probability of classic pore opening in Ca2+-loaded mitochondria myristic acid induces the cyclosporin A-sensitive decrease and mitochondrial swelling more effectively than uncoupler SF6847 does, though their protonophoric activities are equal. In the absence of Pi and presence of succinate and rotenone (with or without glutamate) cyclosporin A either reversed or only stopped decrease induced by 5 M myristic acid and 5 M Ca2+. In the last case nigericin, when added after cyclosporin A, reversed the decrease, and the following addition of EGTA produced only a weak (if any) increase. In Pi-containing medium (in the presence of glutamate and malate) cyclosporin A reversed the decrease. These data show that the cyclosporin A-sensitive decrease in by low concentrations of fatty acids and Ca2+ cannot be explained by specific uncoupling effect of fatty acid. We propose that: 1) low concentrations of Ca2+ and fatty acid induce the pore opening in a substate with a selective cation permeability, and the cyclosporin A-sensitive decrease results from a conversion of to pH gradient due to the electrogenic cation transport in mitochondria; 2) the ADP/ATP-antiporter is involved in this process; 3) higher efficiency of fatty acid compared to SF6847 in the Ca2+-dependent pore opening seems to be due to its interaction with the nucleotide-binding site of the ADP/ATP-antiporter and higher affinity of fatty acids to cations. 相似文献
4.
The effects of catecholamines on intracellular Ca2+concentrations ([Ca2+]i) in single acutely dissociated bovine adrenal medulla endothelial cells (BAMECs) were measured using the intracellular fluorescent probe Fluo-3 AM. 100 m epinephrine or norepinephrine induced a biphasic [Ca2+]i rise with an initial peak followed by a delayed phase. 10 m phenylephrine (1-adrenergic agonist) caused a [Ca2+]i rise similar to that evoked by catecholamines. The increase in [Ca2+]i induced by 10 m phenylephrine was reverted by 10 m phenoxybenzamine (-adrenergic antagonist). Neither isoproterenol (-adrenergic agonist) nor clonidine (2-adrenergic agonist) induced [Ca2+]i rise. The initial peak was insensitive to zero external Ca2+ and it was abolished after Ca2+ internal storages were emptied by 10 mM caffeine. The delayed phase was reduced to near zero by external Ca2+ removal. These results indicate that BAMECs possess 1-adrenergic receptors associated to both the release of caffeine-sensitive intracellular Ca2+ stores and the entry of extracellular Ca2+ We suggest that chromaffin cell secretion may activate BAMECs in vivo through an increase in [Ca2+]i which could induce the secretion of vasoactive factors allowing a rapid entry of hormones into the circulation. (Mol Cell Biochem 000: 000-000,1999) 相似文献
5.
We have examined the effect of the Ca2+ (Mg2+)-ATPase inhibitors thapsigargin (TG) and vanadate on ATP-dependent 45Ca2+ uptake into IP3-sensitive Ca2+ pools in isolated microsomes from rat pancreatic acinar cells. The inhibitory effect of TG was biphasic. About 40–50% of total Ca2+ uptake was inhibited by TG up to 10 nm (apparent Ki4.2 nm, Ca2+ pool I). An additional increase of inhibition up to 85–90% of total Ca2+ uptake could be achieved at 15 to 20 nm of TG (apparent Ki12.1 nm, Ca2+ pool II). The rest was due to TG-insensitive contaminating plasma membranes and could be inhibited by vanadate (apparent Ki10 m). In the absence of TG, increasing concentrations of vanadate also showed two phases of inhibition of microsomal Ca2+ uptake. About 30–40% of total Ca2+ uptake was inhibited by 100 m of vanadate (apparent Ki18 m, Ca2+ pool II). The remaining 60–70% could be inhibited either by vanadate at concentrations up to 1 mm (apparent Ki300 m) or by TG up to 10 nm (Ca2+ pool I). The amount of IP3-induced Ca2+ release was constant at 25% over a wide range of Ca2+ filling. About 10–20% remained unreleasable by IP3. Reduction of IP3 releasable Ca2+ in the presence of inhibitors showed similar dose-response curves as Ca2+ uptake (apparent Ki 3.0 nm for IP3-induced Ca2+ release as compared to 4.2 nm for Ca2+ uptake at TG up to 10 nm) indicating that the highly TG-sensitive Ca2+ pump fills the IP3-sensitive Ca2+ pool I. At TG concentrations >10 nm which blocked Ca2+ pool II the apparent Ki values were 11.3 and 12.1 nm, respectively. For inhibition by vanadate up to 100 m the apparent Ki values were 18 m for Ca2+ uptake and 7 m for Ca2+ release (Ca2+ pool II). At vanadate concentrations up to 1 mm the apparent Ki values were 300 and 200 m, respectively (Ca2+ pool I). Both Ca2+ pools I and II also showed different sensitivities to IP3. Dose-response curves for IP3 in the absence of inhibitors (control) showed an apparent Km value for IP3 at 0.6 m. In the presence of TG (inhibition of Ca2+ pool I) the curve was shifted to the left with an apparent Km for IP3 at 0.08 m. In the presence of vanadate (inhibition of Ca2+ pool II), the apparent Km for IP3 was 2.1 m. These data allow the conclusion that there are at least three different Ca2+ uptake mechanisms present in pancreatic acinar cells: TG- and IP3 insensitive but highly vanadate-sensitive Ca2+ uptake occurs into membrane vesicles derived from plasma membranes. Two Ca2+ pools with different TG-, vanadate- and IP3-sensitivities are most likely located in the endoplasmic reticulum at different cell sites, which could have functional implications for hormonal stimulation of pancreatic acinar cells.This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 246. The authors wish to thank Dr. KlausDieter Preuß for valuable discussions and Mrs. Gabriele Mörschbächer for excellent secretarial help. 相似文献
6.
Dennis A. Przywara Sanjiv V. Bhave Anjali Bhave Pertha S. Chowdhury Taruna D. Wakade Arun R. Wakade 《The Journal of membrane biology》1992,125(2):155-162
Summary We studied the effects of lanthanum (La3+) on the release of 3H-norepinephrine(3H-NE), intracellular Ca2+ concentration, and voltage clamped Ca2+ and K+ currents in cultured sympathetic neurons. La3+ (0.1 to 10 m) produced concentration-dependent inhibition of depolarization induced Ca2+ influx and 3H-NE release. La3+ was more potent and more efficacious in blocking 3H-NE release than the Ca2+-channel blockers cadmium and verapamil, which never blocked more than 70% of the release. At 3 m, La3+ produced a complete block of the electrically stimulated rise in intracellular free Ca2+ ([Ca2+]
i
) in the cell body and the growth cone. The stimulation-evoked release of 3H-NE was also completely blocked by 3 m La3+. However, 3 m La3+ produced only a partial block of voltage clamped Ca2+ current (I
Ca). Following La3+ (10 m) treatment 3H-NE release could be evoked by high K+ stimulation of neurons which were refractory to electrical stimulation. La3+ (1 m) increased the hyperpolarization activated, 4-aminopyridine (4-AP) sensitive, transient K+ current (I
A
) with little effect on the late outward current elicited from depolarized holding potentials. We conclude that the effective block of electrically stimulated 3H-NE release is a result of the unique ability of La3+ to activate a stabilizing, outward K+ current at the same concentration that it blocks inward Ca2+ current. 相似文献
7.
Summary Treatment of Allium cepa L. cellsuspension cultures with a biotic elicitor derived from the fungus Botrytis cinerea, resulted in phytoalexin synthesis. Two phytoalexins, 5-octylcyclopenta-1,3-dione and 5-hexyl-cyclopenta-1,3-dione, were accumulated in cultured onion cells. Removal of extracellular Ca2+ by the calcium chelator ethylene glycol bis(b-aminoethyl ether) N,N-tetraacetic acid abolished the elicitor-mediated phytoalexin synthesis. The calcium channel blockers, verapamil and 8-N,N-(dimethylamino)octyl-3,4,5-trimethoxybenzoate caused similar effects, whereas the addition of the Ca2+ ionophore A23187 enhanced the accumulation of phytoalexins in the absence of the elicitor. Increase in the cytoplasmic Ca2+ concentration in elicitor-treated onion cells was observed as monitored by the fluorescent calcium indicator indo-1. These observations suggest that Ca2+ acts as a second messenger in the regulation of phytoalexin synthesis in cultured onion cells.Abbreviations A23187
4-bromo-calcium ionophore
- cAMP
adenosine 3,5-cyclic monophosphate
- [Ca2+]cyt
cytoplasmic Ca2+ concentration
- EGTA
ethylene glycol bis(b-aminoethyl ether) N,N-tetraacetic acid
- EtOH
ethanol
- Et2O
diethyl ether
- fr.wt
fresh weight
- HR
hypersensitive response
- PIPES
piperazine N,N-bis-(2-ethanesulfonic acid)
- TMB-8
[8-N,N-(dimethylamino)] octyl-3,4,5-trimethoxy-benzoate
- Tsl
tsibulin 相似文献
8.
In the aquatic liverwort Riccia fluitans, the uptake of 14C-labeled 3-O-methyl glucose (3-OMG) and membrane depolarization (
m
) caused by different hexoses has been studied as a function of time and concentration of hexose, K+ and H+, respectively. The rate of uptake of the non-metabolized 3-OMG shows two components: (A)A pH-dependent saturable uptake with a km value around 0.1 mM which saturates at 2.1 and 7.2 mol G
DW
-1 h-1 at pH 6.8 and 5.0, respectively; and (B) a pH-insensitive uptake component which increases linearly with the external 3-OMG concentration and does not saturate 4 mM. Hexoses rapidly depolarize the plasmalemma of the thallus cell and increase its electrical conductance. The maximal
m
was 60±2 mV, the concentrations (mM) for half-maximal
m
were 0.24 glucose, 0.32 galactose, 0.37 2-deoxy glucose, 0.38 3-OMG, 0.57 mannose, and 34 fructose. In terms of a hexose carrier model and an equivalent circuit for the hexose-induced depolarized state of the membrane, it is proposed that a hexose carrier operates either electrogenically in its protonated, pH-and voltage-sensitive state, or by transmembrane diffusion of its uncharged state.Symbols and Abbreviations
m
membrane potential (mV)
-
g
m
membrane (slope) conductance (Sm-2)
- 3-OMG
3-O-methyl glucose 相似文献
9.
Summary A systematic study was made of the action of 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid (SITS) and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) on active Ca2+ transport of human erythrocytes. Pumping activity was estimated in inside-out vesicles (IOV's) by means of Ca2+-selective electrodes or use of tracer45Ca2+. The stilbenes exhibited an approximately equal inhibitory potency and their action could be overcome by carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP) at low but not at high stilbene concentrations. In the absence of DIDS. Ca2+ transport was not affected upon addition of valinomycin, but it was appreciably reduced when vesicles were preincubated with low DIDS concentrations. Such an effect was strictly dependent on the external K+ concentration and it was abolished when valinomycin was added together with FCCP. Similar results were obtained using IOV's prepared from intact cells which had been previously exposed to the stilbene. The findings clearly demonstrate the presence in human red cells of a partially electrogenic Ca2+ pump, exchanging one Ca2+ ion for one proton. 相似文献
10.
Frank Löhr Markus Blümel Jürgen M. Schmidt Heinz Rüterjans 《Journal of biomolecular NMR》1997,10(2):107-118
A triple-resonance NMR technique suitable for the determination ofcarbonyl-related couplings in polypeptide systems is introduced. Theapplication of three novel pulse sequences to uniformly13C/15N-enriched proteins yields E.COSY-likemultiplet patterns exhibiting either one of the3J(Ci–1,Hi
), 3J(Ci–1,Ci
) and3J(Ci–1,Ci)coupling constants in the indirectly detected 13Cdimension, depending on the passive spin selected. The experiments aredemonstrated with oxidized flavodoxin from Desulfovibrio vulgaris. On thebasis of the J-values measured and the backbone -angles derived from ahigh-resolution X-ray structure of the protein, the three associated Karplusequations were reparametrized. The root-mean-square differences between theexperimental coupling constants and those predicted by the optimized Karpluscurves are 0.41, 0.33 and 0.32 Hz for3J(Ci–1,Hi
),3J(Ci–1,Ci
) and3J(Ci–1,Ci),respectively. The results are compared with the Karplus parameters previouslypublished for the same couplings. 相似文献
11.
Summary The voltage dependence for outward-going current of the Ca-activated K+ conductance (g
k (Ca)) of the human red cell membrane has been examined over a wide range of membrane potentials (V
m) at constant values of [K+]ex, [K+]c and pHc, the intact cells being preloaded to different concentrations of ionized calcium. Outward-current conductances were calculated from initial net effluxes of K+ and the corresponding (V
m-Ek) values. The basic conductance, defined as the outward-current coductance at (V
m-Ek) 20 mV and [K+]ex 3mM (B. Vestergaard-Bogind, P. Stampe and P. Christophersen,J. Membrane Biol.
95:121–130, 1987) was found to be a function of cellular ionized Ca. At all degrees of Ca activationg
K(Ca) was an apparently linear function of voltage (V
m range –40 to +70 mV), the absolute level as well as the slope decreasing with decreasing activation. In a simple two-state model the constant voltage dependence can, at the different degrees of Ca activation, be accounted for by a Boltzmann-type equilibrium function with an equivalent valence of 0.4, assuming chemical equilibrium atV
m=0 mV. Alternatively, the phenomenon might be explained by a voltage-dependent block of the outward current by an intracellular ion. Superimposed upon the basic conductance is the apparently independent inward-rectifying steep voltage function with an equivalent valence of 5 and chemical equilibrium at the givenE
K value.Abbreviations CCCP
carbonyl cyanidem-chlorophenylhydrazone
- DIDS
4,4-diisothiocyanostilbene-2,2-disul 相似文献
12.
Summary The ATP-dependent Ca2+ transport activity (T. Takuma, B.L. Kuyatt and B.J. Baum,Biochem. J.
227:239–245, 1985) exhibited by inverted basolateral membrane vesicles isolated from rat parotid gland was further characterized. The activity was dependent on Mg2+. Phosphate (5mm), but not oxalate (5mm), increased maximum Ca2+ accumulation by 50%. Half-maximal Ca2+ transport was achieved at 70nm Ca2+ in EGTA-buffered medium while maximal activity required >1 m Ca2+ (V
max=54 nmol/mg protein/min). Optimal rates of Ca2+ transport were obtained in the presence of KCl, while in a KCl-free medium (mannitol or sucrose) 40% of the total activity was achieved, which could not be stimulated by FCCP. The initial rate of Ca2+ transport could be significantly altered by preimposed membrane potentials generated by K+ gradients in the presence of valinomycin. Compared to the transport rate in the absence of membrane potential, a negative (interior) potential stimulated uptake by 30%, while a positive (interior) potential inhibited uptake. Initial rates of Ca2+ uptake could also be altered by imposing pH gradients, in the absence of KCl. When compared to the initial rate of Ca2+ transport in the absence of a pH gradient, pH
i
=7.5/pH
o
=7.5; the activity was 60% higher in the presence of an outwardly directed pH gradient, pH
i
=7.5/pH
o
=8.5; while it was 80% lower when an inwardly directed pH gradient was imposed, pH
i
=7.5/pH
o
=6.2. The data show that the ATP-dependent Ca2+ transport in BLMV can be modulated by the membrane potential, suggesting therefore that there is a transfer of charge into the vesicle during Ca2+ uptake, which could be compensated by other ion movements. 相似文献
13.
We showed that 5 M acetylcholine (ACh) and 100 M norepinephrine (NE) cause increases in the total Ca2+ content in acinar cells by 30 and 87% and in the exocytosis intensity by 15 and 20%, respectively. Application of 5 M ACh and 100 M NE increased the free cytosolic Ca2+ concentration ([Ca2+]
i
) by 87 ± 2 and 140 ± 7 nM, respectively. Application of ACh and NE in a Ca2+-free external solution caused a [Ca2+]
i
increase that was 40 and 67% lower than in physiological solution. We postulate that the exocytosis developing upon neural stimulation of the gland results from generation of Ca2+ transients that are spreading from the basal to the apical region of the exocrine cell, where secretory granules are concentrated. 相似文献
14.
Summary The presence of a coupled Na+/Ca2+ exchange system has been demonstrated in plasma membrane vesicles from rat pancreatic acinar cells. Na+/Ca2+ exchange was investigated by measuring45Ca2+ uptake and45Ca2+ efflux in the presence of sodium gradients and at different electrical potential differences across the membrane (=) in the presence of sodium. Plasma membranes were prepared by a MgCl2 precipitation method and characterized by marker enzyme distribution. When compared to the total homogenate, the typical marker for the plasma membrane, (Na++K+)-ATPase was enriched by 23-fold. Markers for the endoplasmic reticulum, such as RNA and NADPH cytochromec reductase, as well as for mitochondria, the cytochromec oxidase, were reduced by twofold, threefold and 10-fold, respectively. For the Na+/Ca2+ countertransport system, the Ca2+ uptake after 1 min of incubation was half-maximal at 0.62 mol/liter Ca2+ and at 20 mmol/liter Na+ concentration and maximal at 10 mol/liter Ca2+ and 150 mmol/liter Na+ concentration, respecitively. When Na+ was replaced by Li+, maximal Ca2+ uptake was 75% as compared to that in the presence of Na+. Amiloride (10–3 mol/liter) at 200 mmol/liter Na+ did not inhibit Na+/Ca2+ countertransport, whereas at low Na+ concentration (25 mmol/liter) amiloride exhibited dose-dependent inhibition to be 62% at 10–2 mol/liter. CFCCP (10–5 mol/liter) did not influence Na+/Ca2+ countertransport. Monensin inhibited dose dependently; at a concentration of 5×10–6 mol/liter inhibition was 80%. A SCN– or K+ diffusion potential (=), being positive at the vesicle inside, stimulated calcium uptake in the presence of sodium suggesting that Na+/Ca2+ countertransport operates electrogenically, i.e. with a stoichiometry higher than 2 Na+ for 1 Ca2+. In the absence of Na+, did not promote Ca2+ uptake. We conclude that in addition to ATP-dependent Ca2+ outward transport as characterized previously (E. Bayerdörffer, L. Eckhardt, W. Haase & 1. Schulz, 1985,J. Membrane Biol.
84:45–60) the Na+/Ca2+ countertransport system, as characterized in this study, represents a second transport system for the extrusion of calcium from the cell. Furthermore, the high affinity for calcium suggests that this system might participate in the regulation of the cytosolic free Ca2+ level. 相似文献
15.
A conventional patch clamp amplifier was used to test the feasibility of measuring whole-cell ionic currents under voltage clamp conditions from -cells in intact mouse islets of Langerhans perifused with bicarbonate Krebs buffer at 37°C. Cells impaled with a high resistance microelectrode (ca. 0.150 G) were identified as -cells by the characteristic burst pattern of electrical activity induced by 11 mm glucose. Voltage-dependent outward K+ currents were enhanced by glucose both in the presence and absence of physiological bicarbonate buffer and also by bicarbonate regardless of the presence or absence of glucose. For comparison with the usual patch clamp protocol, similar measurements were made from single rat -cells at room temperature; glucose did not enhance the outward currents in these cells. Voltage-dependent inward currents were recorded in the presence of tetraethylammonium (TEA), an effective blocker of the K+ channels known to be present in the -cell membrane. Inward currents exhibited a fast component with activation-inactivation kinetics and a delayed component with a rather slow inactivation; inward currents were dependent on Ca2+ in the extracellular solution. These results suggest the presence of either two types of voltage-gated Ca2+ channels or a single type with fast and slow inactivation. We conclude that it is feasible to use a single intracellular microelectrode to measure voltage-gated membrane currents in the -cell within the intact islet at 37°C, under conditions that support normal glucose-induced insulin secretion and that glucose enhances an as yet unidentified voltage-dependent outward K+ current.The authors are pleased to thank Dr. M.X. Li for performing some of the experiments in Fig. 2, Dr. A. Sherman for many illuminating discussions, and Drs. J. Rinzel and N. Sheppard for support. D.M. was supported in part by a National Institutes of Health training grant to The Johns Hopkins University, Department of Biomedical Engineering. Thanks are also given to B. Chidakel for electronic design and construction. 相似文献
16.
Kinetic properties of the ATP-dependent Ca2+ pump and the Na+/Ca2+ exchange system in basolateral membranes from rat kidney cortex 总被引:1,自引:0,他引:1
M. P. E. van Heeswijk J. A. M. Geertsen C. H. van Os 《The Journal of membrane biology》1984,79(1):19-31
Summary Basolateral plasma membranes from rat kidney cortex have been purified 40-fold by a combination of differential centrifugation, centrifugation in a discontinuous sucrose gradient followed by centrifugation in 8% percoll. The ratio of leaky membrane vesicles (L) versus right-side-out (RO) and inside-out (IO) resealed vesicles appeared to be LROIO=431. High-affinity Ca2+-ATPase, ATP-dependent Ca2+ transport and Na+/Ca2+ exchange have been studied with special emphasis on the relative transport capacities of the two Ca2+ transport systems. The kinetic parameters of Ca2+-ATPase activity in digitonin-treated membranes are:K
m
=0.11 m Ca2+ andV
max=81±4 nmol Pi/min·mg protein at 37°C. ATP-dependent Ca2+ transport amounts to 4.3±0.2 and 7.4±0.3 nmol Ca2+/min·mg protein at 25 and 37°C, respectively, with an affinity for Ca2+ of 0.13 and 0.07 m at 25 and 37°C. After correction for the percentage of IO-resealed vesicles involved in ATP-dependent Ca2+ transport, a stoichiometry of 0.7 mol Ca2+ transported per mol ATP is found for the Ca2+-ATPase. In the presence of 75mm Na+ in the incubation medium ATP-dependent Ca2+ uptake is inhibited 22%. When Na+ is present at 5mm an extra Ca2+ accumulation is observed which amounts to 15% of the ATP-dependent Ca2+ transport rate. This extra Ca2+ accumulation induced by low Na+ is fully inhibited by preincubation of the vesicles with 1mm ouabain, which indicates that (Na+–K+)-ATPase generates a Na+ gradient favorable for Ca2+ accumulation via the Na+/Ca2+ exchanger. In the absence of ATP, a Na+ gradient-dependent Ca2+ uptake is measured which rate amounts to 5% of the ATP-dependent Ca2+ transport capacity. The Na+ gradient-dependent Ca2+ uptake is abolished by the ionophore monensin but not influenced by the presence of valinomycin. The affinity of the Na+/Ca2+ exchange system for Ca2+ is between 0.1 and 0.2 m Ca2+, in the presence as well as in the absence of ATP. This affinity is surprisingly close to the affinity measured for the ATP-dependent Ca2+ pump. Based on these observations it is concluded that in isolated basolateral membranes from rat kidney cortex the Ca2+-ATPase system exceeds the capacity of the Na+/Ca2+ exchanger four- to fivefold and it is therefore unlikely that the latter system plays a primary role in the Ca2+ homeostasis of rat kidney cortex cells. 相似文献
17.
Single Ca2+ channel records were obtained from plasma membrane-enriched fractions of wheat roots incorporated into artificial planar lipid bilayers. The channel had a unitary conductance of 15 pS for a 10 to 95 mM CaCl2 gradient (cytoplasm: outside of the cell). The voltage dependence displayed by the channel agreed with that expected for Ca2+ channels in the plasma membrane. The channel gating was strongly modified by addition of 20 M extracellular verapamil (a Ca2+ channel antagonist). Extracellular AlCl3 (70 M, pH 4.9) almost completely blocked the channel. 相似文献
18.
1. Cultured dorsal root ganglion neurons of rat pups were depolarized by exposure to 50 mM K+ and the rise of [Ca2+]i was measured using fura-2 as an indicator.2. Lead in the extracellular solution reduced the rise of [Ca2+]i in a concentration-dependent manner, with a threshold concentration of 0.25 M. More than 80% of the calcium entry was prevented by 5 M lead. The IC50 and the Hill coefficient were 1.3 M and 1, respectively.3. This effect was considered to be due to a reduction of VACCCs, since applications of NMDA did not result in any rise of [Ca2+]i.4. Since Pb2+ itself changes the fura-2 signal in a typical and characteristic manner, fura-2 is also an indicator for Pb2+. No changes in fura-2 signals were detected when lead (5 M) was applied for several minutes in the absence of calcium, indicating that Pb2+ did not enter the cells.5. Thus it is concluded that lead prevents calcium entry by reducing VACCCs but does not cross the cell membrane itself. 相似文献
19.
Short-term responses of four carrot (Daucus carota) cultivars: Cascade, Caro Choice (CC), Oranza, and Red Core Chantenay (RCC) to CO2 concentrations (C
a) were studied in a controlled environment. Leaf net photosynthetic rate (P
N), intercellular CO2 (C
i), stomatal conductance (g
s), and transpiration rate (E) were measured at C
a from 50 to 1 050 mol mol–1. The cultivars responded similarly to C
a and did not differ in all the variables measured. The P
N increased with C
a until saturation at 650 mol mol–1 (C
i= 350–400 mol mol–1), thereafter P
N increased slightly. On average, increasing C
a from 350 to 650 and from 350 to 1 050 mol mol–1 increased P
N by 43 and 52 %, respectively. The P
N
vs.
C
i curves were fitted to a non-rectangular hyperbola model. The cultivars did not differ in the parameters estimated from the model. Carboxylation efficiencies ranged from 68 to 91 mol m–2 s–1 and maximum P
N were 15.50, 13.52, 13.31, and 14.96 mol m–2 s–1 for Cascade, CC, Oranza, and RCC, respectively. Dark respiration rate varied from 2.80 mol m–2 s–1 for Oranza to 3.96 mol m–2 s–1 for Cascade and the CO2 compensation concentration was between 42 and 46 mol mol–1. The g
s and E increased to a peak at C
a= 350 mol mol–1 and then decreased by 17 and 15 %, respectively when C
a was increased to 650 mol mol–1. An increase from 350 to 1 050 mol mol–1 reduced g
s and E by 53 and 47 %, respectively. Changes in g
s and P
N maintained the C
i:C
a ratio. The water use efficiency increased linearly with C
a due to increases in P
N in addition to the decline in E at high C
a. Hence CO2 enrichment increases P
N and decreases g
s, and can improve carrot productivity and water conservation. 相似文献
20.
Using a Ca2+-sensitive fluorescent indicator, fura-2/AM, we recorded calcium transients in secretory cells of isolated acini of the rat submandibular salivary gland; these transients were induced by hyperpotassium-induced depolarization (after an increase in [K+]
e
up to 50 mM) of the plasma membrane of the above cells. Calcium transients were significantly suppressed by 50 M nifedipine. Addition of 10 M carbonyl cyanide m-chlorophenylhydrazone to the normal extracellular solution was accompanied by a rise in [Ca2+]
i
, whereas when hyperpotassium solution is used the effect was less expressed. Blockers of CA2+-ATPase in the cellular membrane and in the endoplasmic reticulum, eosin Y (5 M) and cyclopiazonic acid (CPA, 5 M), respectively, evoked a significant increase in [Ca2+]
i
and a decrease in the K+-depolarization-induced calcium transient. Extracellular application of caffeine (2, 10, or 30 mM) was accompanied by a concentration-dependent rise in [Ca2+]
i
. Therefore, potassium depolarization of the plasma membrane of acinar cells of the rat submandibular salivary gland activates both the voltage-dependent Ca2+ influx and Ca2+-induced Ca2+ release from the endoplasmic reticulum; the initial level of [Ca2+]
i
was restored at the joint involvement of Ca2+-ATPases in the plasma membrane and the membranes of the endoplasmic reticulum and mitochondria. 相似文献