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1.
Dynein-like Mg2+-ATPase in mitotic spindles isolated from sea urchin embryos (Strongylocentrotus droebachiensis) 总被引:16,自引:13,他引:3
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《The Journal of cell biology》1980,86(3):738-745
Two distinctly different ATPases have been reported to be endogenous to the mitotic apparatus: a Mg2+-ATPase resembling axonemal dynein, and a Ca2+-ATPase postulated to be bound in membranes. To examine the nature of the Mg2+-ATPase, we isolated membrane-free mitotic spindles from Stronglylocentrotus droebachiensis embryos by rapidly lysing these in a calcium-chelating, low-ionic-strength buffer (5 mM EGTA, 0.5 mM MgCl2, 10 mM PIPES, pH 6.8) that contained 1% Nonidet P-40. The fibrous isolated mitotic spindles closely resembled spindles in living cells, both in general morphology and in birefringence. In electron micrographs, the spindles were composed primarily of microtubules, free from membranes and highly extracted of intermicrotubular cytoplasmic ground substance. As analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the pelleted spindles contain 18% tubulin, variable amounts of actin (2-8%), and an unidentified protein of 55 kdaltons in a constant weight ratio to tubulin (1:2.5). The isolated spindles also contained two polypeptides, larger than 300 kdaltons, that comigrated with egg dynein polypeptides, and ATPase activity (0.02 mumol Pi/mg . min) that closely resembled both flagellar and egg dynein. The spindle Mg2+-ATPase showed a ratio of Ca2+-/Mg2+-ATPase = 0.85, had minimal activity in KCl and EDTA, and cleaved GTP at 35% of the rate of ATP. The Mg2+-ATPase was insensitive to ouabain or oligomycin. The spindle Mg2+-ATPase was inhibited by sodium vanadate but, like egg dynein, was less sensitive to vanadate than flagellar dynein. The spindle Mg2+- ATPase does not resemble the mitotic Ca2+-ATPase described by others. We propose that the spindle Mg2+-ATPase is egg dynein. Bound carbohydrate on the two high-molecular-weight polypeptides of both egg dynein and the spindle enzyme suggest that these proteins may normally associate with membranes in the living cell. 相似文献
2.
We have purified Ca2+-ATPase from synaptosomal membranes (SM)1 from ratcerebellum by calmodulin affinity chromatography. The enzyme was identifiedas plasma membrane Ca2+-ATPase by its interaction with calmodulin andmonoclonal antibodies produced against red blood cell (RBC) Ca2+-ATPase, andby thapsigargin insensitivity. The purpose of the study was to establishwhether two regulators of the RBC Ca2+-ATPase, calmodulin and protein kinaseC (PKC), affect the Ca2+-ATPase isolated from excitable cells and whethertheir effects are comparable to those on the RBC Ca2+-ATPase. We found thatcalmodulin and PKC activated both enzymes. There were significantquantitative differences in the phosphorylation and activation of the SMversus RBC Ca2+-ATPase. The steady-state Ca2+-ATPase activity of SMCa2+-ATPase was approximately 3 fold lower and significantly less stimulatedby calmodulin. The initial rate of PKC catalyzed phosphorylation (in thepresence of 12-myristate 13-acetate phorbol) was approximately two timesslower for SM enzyme. While phosphorylation of RBC Ca2+-ATPase approachedmaximum level at around 5 min, comparable level of phosphorylation of SMCa2+-ATPase was observed only after 30 min. The PKC-catalyzedphosphorylation resulted in a statistically significant increase inCa2+-ATPase activity of up to 20-40%, higher in the SM Ca2+-ATPase.The differences may be associated with diversities in Ca2+-ATPase functionin erythrocytes and neuronal cells and different isoforms composition. 相似文献
3.
Purification and characterization of the Ca2+-ATPase of plasma membranes from Ehrlich ascites mammary carcinoma cells 总被引:1,自引:0,他引:1
R Wetzker F D B?hmer R Klinger E Müller H Hegewald M Scheven R Grosse 《Biochimica et biophysica acta》1986,854(1):117-123
Ca2+-ATPase was isolated from plasma membranes of Ehrlich ascites mammary carcinoma cells by means of calmodulin affinity chromatography. The purification procedure included removal of endogenous calmodulin from a Triton X-100 solubilizate of the membranes by DEAE ion-exchange chromatography as an essential step. With respect to its molecular mass, activation by calmodulin, Ca2+-dependent phosphorylation and highly sensitive inhibition by orthovanadate, the purified enzyme resembles the Ca2+-ATPase of erythrocyte membranes. In contrast to the strong calmodulin dependence of the isolated enzyme the Ca2+-ATPase in native Ehrlich ascites carcinoma cell membranes cannot be remarkably stimulated by added calmodulin. It is suggested that the membrane-bound Ca2+-ATPase in the presence of Ca2+ is activated by interaction with endogenously bound calmodulin. 相似文献
4.
Plasma membrane vesicles of Ehrlich ascites carcinoma cells have been isolated to a high degree of purity. In the presence of Mg2+, the plasma membrane preparation exhibits a Ca2+-dependent ATPase activity of 2 mumol Pi per h per mg protein. It is suggested that this (Ca2+ + Mg2+)-ATPase activity is related to the measured Ca2+ transport which was characterized by Km values for ATP and Ca2+ of 44 +/- 9 microM and 0.25 +/- 0.10 microM, respectively. Phosphorylation of plasma membranes with [gamma-32P]ATP and analysis of the radioactive species by polyacrylamide gel electrophoresis revealed a Ca2+-dependent hydroxylamine-sensitive phosphoprotein with a molecular mass of 135 kDa. Molecular mass and other data differentiate this phosphoprotein from the catalytic subunit of (Na+ + K+)-ATPase and from the catalytic subunit of (Ca2+ + Mg2+)-ATPase of endoplasmic reticulum. It is suggested that the 135 kDa phosphoprotein represents the phosphorylated catalytic subunit of the (Ca2+ + Mg2+)-ATPase of the plasma membrane of Ehrlich ascites carcinoma cells. This finding is discussed in relation to previous attempts to identify a Ca2+-pump in plasma membranes isolated from nucleated cells. 相似文献
5.
Regulation of Ca2+ influx during mitosis: Ca2+ influx and depletion of intracellular Ca2+ stores are coupled in interphase but not mitosis. 总被引:1,自引:0,他引:1
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Activation of a wide variety of membrane receptors leads to a sustained elevation of intracellular Ca2+ ([Ca2+]i) that is pivotal to subsequent cell responses. In general, in nonexcitable cells this elevation of [Ca2+]i results from two sources: an initial release of Ca2+ from intracellular stores followed by an influx of extracellular Ca2+. These two phases, release from intracellular stores and Ca2+ influx, are generally coupled: stimulation of influx is coordinated with depletion of Ca2+ from stores, although the mechanism of coupling is unclear. We have previously shown that histamine effects a typical [Ca2+]i response in interphase HeLa cells: a rapid rise in [Ca2+]i followed by a sustained elevation, the latter dependent entirely on extracellular Ca2+. In mitotic cells only the initial elevation, derived by Ca2+ release from intracellular stores, occurs. Thus, in mitotic cells the coupling of stores to influx may be specifically broken. In this report we first provide additional evidence that histamine-stimulated Ca2+ influx is strongly inhibited in mitotic cells. We show that efflux is also strongly stimulated by histamine in interphase cells but not in mitotics. It is possible, thus, that in mitotics intracellular stores are only very briefly depleted of Ca2+, being replenished by reuptake of Ca2+ that is retained within the cell. To ensure the depletion of Ca2+ stores in mitotic cells, we employed the sesquiterpenelactone, thapsigargin, that is known to affect the selective release of Ca2+ from intracellular stores by inhibition of a specific Ca(2+)-ATPase; reuptake is inhibited. In most cells, and in accord with Putney's capacitative model (1990), thapsigargin, presumably by depleting intracellular Ca2+ stores, stimulates Ca2+ influx. This is the case for interphase HeLa cells. Thapsigargin induces an increase in [Ca2+]i that is dependent on extracellular Ca2+ and is associated with a strong stimulation of 45Ca2+ influx. In mitotic cells thapsigargin also induces a [Ca2+]i elevation that is initially comparable in magnitude and largely independent of extracellular Ca2+. However, unlike interphase cells, in mitotic cells the elevation of [Ca2+]i is not sustained and 45Ca2+ influx is not stimulated by thapsigargin. Thus, the coupling between depletion of intracellular stores and Ca2+ influx is specifically broken in mitotic cells. Uncoupling could account for the failure of histamine to stimulate Ca2+ influx during mitosis and would effectively block all stimuli whose effects are mediated by Ca2+ influx and sustained elevations of [Ca2+]i. 相似文献
6.
Cell cycle specific variations in transport capacity of an isolated Ca2+-transport system 总被引:3,自引:0,他引:3
From sea urchin eggs as well as from mammalian cells a Ca2+-transporting system is described in its properties. One of its main components is the "mitotic" Ca2+-ATPase. If its activity is studied during the cell cycle of fertilized sea urchin eggs, fluctuations of the Ca2+-uptake capacity are found with a maximum in every cell cycle at mitosis. Additionally, only in the first cycle after fertilization, another activity increase occurs at the time of spermaster formation. This system, then, seems to qualify for one of the main regulators of the mitotic process. 相似文献
7.
(Ca2+ + Mg2+)-ATPase activity of red cells and their isolated membranes was investigated in the presence of various Ca2+ concentrations and cytoplasmic activator protein. Red cell ATPase activity was high at low Ca2+ concentrations, and low at moderate and high concentrations of Ca2+. In the case of isolated membranes, both low and moderate ca2+ concentrations produced higher (Ca2+ + Mg2+)-ATPase activity than high Ca2+ concentration. Membrane-free hemolysate containing soluble activator of (Ca2+ + Mg2+)-ATPase produced a significant increase in (Ca2+ + Mg2+)-ATPase activity only at low ca2+ concentration. Regardless of Ca2+ and activator concentrations, the enzyme activity in the membrane was lower than lysed red cells. The low level of (Ca2+ + Mg2+)-ATPase activity seen at high Ca2+ concentration can be augmented by lowering the Ca2+ concentration of EGTA in the assay medium. However, once the membrane was exposed to a high Ca2+ concentration, the activator could no longer exert it maximum stimulation at the low Ca2+ concentration brought about by addition of EGTA. This loss of activation was not attributable to the Ca2+-induced denaturation of activator protein but rather related to the alteration of (Ca2+ + Mg2+)-ATPase states in the membrane. On the basis of these data, it is suggested that only a small portion of (Ca2+ + Mg2+)-ATPase activity of isolated membranes can be stimulated by the soluble activator and that (ca2+ + Mg2+)ATPase most likely exists in various states depending upon ca2+ concentration and the presence of activator. The enzyme state exhibiting the high degree of stimulation by activator may undergo irreversible damage in the presence of high Ca2+ concentrations. 相似文献
8.
Localization of an intracellular membrane-bound Ca2+-ATPase in PtK-cells using immunofluorescence techniques 总被引:3,自引:0,他引:3
C Petzelt 《European journal of cell biology》1984,33(1):55-59
Monospecific antibodies to an intracellular membrane-bound Ca2+-ATPase were used to localize the enzyme in PtK-cells in interphase and in mitosis as well. In interphase the protein is distributed as small dots and rods in the cytoplasm with an increased concentration around the nucleus. Neither the plasma membrane nor the nuclear envelope are stained. In mitotic cells the Ca2+-ATPase is localized around the spindle rather than in it. The results are in agreement with the proposed function of enzyme as an essential part of the intracellular Ca2+-regulating system controlling Ca2+ in the respective domains of the cell. 相似文献
9.
Treatment with calcitriol of isolated cartilage cells derived from epiphyseal growth plates of rachitic chicks results in reduced intracellular calcium concentrations. The reduction in calcium was found to correlate with increased activity of Ca2+-ATPase. The activities of Na+-K+-ATPase and of Mg2+-ATPase did not change in response to the treatment with calcitriol. It is suggested that calcitriol regulates intracellular calcium by modulating the activity of the Ca2+-pumping ATPase. 相似文献
10.
Fusion of proteoliposomes and cells. ATP-dependent Ca2+ uptake into erythrocytes catalyzed by Ca2+-ATPase from skeletal muscle 总被引:1,自引:0,他引:1
Purified Ca2+-ATPase from rabbit skeletal muscle has been incorporated into intact erythrocyte membranes by a two-step procedure. The isolated protein was reconstituted into proteoliposomes composed of phosphatidylethanolamine, phosphatidylcholine, and cardiolipin (50:20:30%, respectively). The resulting proteoliposomes were fused with erythrocytes in presence of La3+, Ca2+, or Mg2+. Subsequently, 45Ca uptake into the cells could be demonstrated. It was dependent on externally added ATP, inhibited by N-ethylmaleimide and p-hydroxymercuribenzoate, and enhanced by inactivation of the endogenous Ca2+-ATPase which catalyzes Ca2+ extrusion from the cells. The insertion of the protein did not induce cell lysis, but the cells did become more fragile. Functional insertion of isolated membrane proteins into cell membranes allows a new approach to research of plasma membranes. 相似文献
11.
ATP-dependent Ca2+ transport in vesicles isolated from the bile canalicular region of the hepatocyte plasma membrane 总被引:2,自引:0,他引:2
Three plasma membrane subfractions have been isolated and characterized from rat liver cells. The high affinity Ca2+-stimulated ATPase is highly enriched in the bile canalicular subfraction. Taking into account cross-contamination by the blood sinusoidal and lateral membranes it is suggested that the high-affinity Ca2+-ATPase is located exclusively in this fraction. The high-affinity Ca2+-ATPase is coupled to Ca2+ transport, is calmodulin-insensitive, sensitive to vanadate under appropriate experimental conditions and is strongly inhibited by La3+. In the presence of Ca2+ and ATP the ATPase forms a phosphorylated intermediate of molecular mass about 200 kDa. 相似文献
12.
K Hiraga H Tahara N Taguchi E Tsuchiya S Fukui T Miyakawa 《Journal of general microbiology》1991,137(1):1-4
Plasma membrane Ca2(+)-ATPase of Saccharomyces cerevisiae was solubilized and partially purified by calmodulin-affinity chromatography. The activity of Ca2(+)-ATPase isolated from MATa cells was inhibited by physiological concentrations of the mating pheromone alpha-factor in a dose-dependent manner. The enzyme prepared from a receptor-deficient sterile mutant cells (delta ste-2) was similarly inhibited by alpha-factor, but the enzyme from MAT alpha cells was resistant to the mating pheromone. We suggest that the inhibition may be involved in the alpha-factor-induced increase of Ca2+ uptake reaction of MATa cells. 相似文献
13.
The effect of an intracellular cryoprotectant glycerol on human erythrocyte Ca2+-ATPase activity and possible involvement of calmodulin in the regulation of Ca2+-pump under these conditions were investigated. The experiments were carried out using saponin-permeabilized cells and isolated erythrocyte membrane fractions (white ghosts). Addition of rather low concentrations of glycerol to the medium increased Ca2+-ATPase activity in the saponin-permeabilized cells; the maximal effect was observed at 10% glycerol. Subsequent increase in glycerol concentrations above 20% was accompanied by inhibition of Ca2+-ATPase activity. Lack of stimulating effect of glycerol on white ghost Ca2+-ATPase may be attributed to removal of endogenous compounds regulating activity of this ion transport system. Inhibitory analysis using R24571 revealed that activation of Ca2+-ATPase by 10% glycerol was observed only in the case of inhibitor administration after modification of cells with glycerol; in the case of inhibitor addition before erythrocyte contact with glycerol, this phenomenon disappeared. These data suggest the possibility of regulation of human erythrocyte Ca2+-ATPase by glycerol; this regulatory effect may be attributed to both glycerol-induced structural changes in the membrane and also involvement of calmodulin in modulation of catalytic activity of the Ca2+-pump. 相似文献
14.
Molecular cloning of cDNAs from human kidney coding for two alternatively spliced products of the cardiac Ca2+-ATPase gene 总被引:16,自引:0,他引:16
Ca2+-ATPase molecules present in the microsomal fraction from non-muscle cells were examined immunologically. Rabbit whole brain, cerebellum, liver, kidney, and COS-1 cell microsomes all displayed a polypeptide of about 110 kDa which was immunoreactive with a polyclonal antiserum against the cardiac muscle sarcoplasmic reticulum Ca2+-ATPase molecule, but was not immunoreactive with a monoclonal antibody specific for the fast-twitch muscle Ca2+-ATPase. cDNAs encoding the full length of two Ca2+-ATPase molecules were isolated from a human kidney library using a mixture of nucleotide probes derived from both rabbit fast-twitch and cardiac muscle Ca2+-ATPase cDNAs. The human kidney cDNAs, HK1 and HK2, are the products of alternative splicing. HK2 codes for a protein identical to rabbit cardiac muscle Ca2+-ATPase, with the exception of 6 scattered amino acid replacements, whereas HK1 codes for a protein identical to that encoded by HK2, but with the carboxyl-terminal 4 amino acids replaced by an extended sequence of 49 amino acids. cDNAs of the HK1 type are by far the most abundant in the library. The partial structure of a 40-kilobase genomic DNA encoding all but the 5' end of the human cardiac Ca2+-ATPase is described. The exons which give rise to the alternatively spliced products were located by Southern blotting and sequencing, and the alternative splicing patterns were determined. 相似文献
15.
Zhou L Burnett AL Huang PL Becker LC Kuppusamy P Kass DA Kevin Donahue J Proud D Sham JS Dawson TM Xu KY 《Biochemical and biophysical research communications》2002,294(5):1030-1035
Nitric oxide (NO*) is produced endogenously from NOS isoforms bound to sarcolemmal (SL) and sarcoplasmic reticulum (SR) membranes. To investigate whether locally generated NO* directly affects the activity of enzymes mediating ion active transport, we studied whether knockout of selected NOS isoforms would affect the functions of cardiac SL (Na+ + K+)-ATPase and SR Ca2+-ATPase. Cardiac SL and SR vesicles containing either SL (Na+ + K+)-ATPase or SR Ca2+-ATPase were isolated from mice lacking either nNOS or eNOS, or both, and tested for enzyme activities. Western blot analysis revealed that absence of single or double NOS isoforms did not interrupt the protein expression of SL (Na+ + K+)-ATPase and SR Ca2+-ATPase in cardiac muscle cells. However, lack of NOS isoforms in cardiac muscle significantly altered both (Na+ + K+)-ATPase activity and SR Ca2+-ATPase function. Our experimental results suggest that disrupted endogenous NO* production may change local redox conditions and lead to an unbalanced free radical homeostasis in cardiac muscle cells which, in turn, may affect key enzyme activities and membrane ion active transport systems in the heart. 相似文献
16.
Oviedo NJ Benaim G Cervino V Proverbio T Proverbio F Marín R 《Biochimica et biophysica acta》2006,1762(3):381-385
Plasma membrane Ca2+-ATPase activity diminishes by about 50% in red blood cells during preeclampsia. We investigated whether the number of Ca2+-ATPase molecules is modified in red cell membranes from preeclamptic pregnant women by measuring the specific phosphorylated intermediate of this enzyme. Also, we isolated the Ca2+-ATPase protein from both normotensive and preeclamptic pregnant women and estimated its molecular weight, and its cross-reactions with specific polyclonal and monoclonal (5F10) antibodies against it. We measured the Ca2+-ATPase activity in a purified state and the effect of known modulators of this ATPase. It was found that the phosphorylated intermediate associated with PMCA is similar for red cell ghosts from normotensive and preeclamptic women, suggesting a similar number of ATPase molecules in these membranes. The molecular weight of the Ca2+-ATPase is around 140 kDa for both normotensive and preeclamptic membranes, and its cross-reactions with specific antibodies is similar, suggesting that the protein structure remains intact in preeclampsia. Calmodulin, ethanol, or both calmodulin plus ethanol, stimulated the Ca2+-ATPase activity to the same extent for both normotensive and preeclamptic preparations. Our results showed that the reduced Ca2+-ATPase activity of the red cell membranes from preeclamptic women is not associated with a defective enzyme, but rather with a high level of lipid peroxidation. 相似文献
17.
L Dux 《FEBS letters》1985,183(1):177-181
The vanadate-induced crystallization of Ca2+-ATPase was analyzed on sarcoplasmic reticulum vesicles isolated between 10 and 28 days of development from pectoralis muscles of chicken. After exposure to Na3VO4 in a Ca2+-free medium, Ca2+-ATPase crystals begin to appear on portions of the surface of a few vesicles, isolated at 18 days of development. Thereafter, the number of vesicles containing Ca2+-ATPase crystals rapidly increases and after 1 week of postnatal development (28 days), it reaches the adult level of about 30% of the vesicle population. These observations are discussed with reference to the mechanism of Ca2+-ATPase crystallization and the regulation of sarcoplasmic reticulum biosynthesis. 相似文献
18.
Interaction of myotoxin a with the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum 总被引:1,自引:0,他引:1
Myotoxin a is a muscle-damaging toxin isolated from the venom of Crotalus viridis viridis. Its interaction with the Ca2+-ATPase of sarcoplasmic reticulum (SR) vesicles purified from rabbit skeletal muscle was investigated. Myotoxin a inhibited Ca2+ loading and stimulated Ca2+-dependent ATPase without affecting unidirectional Ca2+ efflux. Its action was dose, time, and temperature dependent. Myotoxin a partially blocked the binding of specific anti-(rabbit SR Ca2+-ATPase) antibodies. It is concluded that myotoxin a attaches to the SR Ca2+-ATPase and uncouples Ca2+ uptake from Ca2+-dependent ATP hydrolysis. Myotoxin a also prevented the formation of decavanadate-induced two-dimensional crystalline arrays of the SR Ca2+-ATPase. 相似文献
19.
Regulation of Plant Defense Response to Fungal Pathogens: Two Types of Protein Kinases in the Reversible Phosphorylation of the Host Plasma Membrane H+-ATPase 总被引:11,自引:6,他引:5
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The role of reversible phosphorylation of the host plasma membrane H+-ATPase in signal transduction during the incompatible interaction between tomato cells and the fungal pathogen Cladosporium fulvum was investigated. Tomato cells (with the Cf-5 resistance gene) or isolated plasma membranes from Cf-5 cells treated with elicitor preparations from race 2.3 or 4 of C. fulvum (containing the avr5 gene product) showed a marked dephosphorylation of plasma membrane H+-ATPase. Similar treatment with elicitor preparations from races 5 and 2.4.5.9.11 (lacking the avr5 gene product) showed no change in dephosphorylation. Elicitor (race 4) treatment of cells, but not of isolated plasma membranes, for 2 hr resulted in rephosphorylation of the ATPase via Ca2+-dependent protein kinases. The initial (first hour) rephosphorylation was enhanced by protein kinase C (PKC) activators and was prevented by PKC inhibitors. Activity of a second kinase appeared after 1 hr and was responsible for the continuing phosphorylation of the H+-ATPase. This latter Ca2+-dependent kinase was inhibited by a calmodulin (CaM) antagonist and by an inhibitor of Ca2+/CaM-dependent protein kinase II. The activation of the Ca2+/CaM-dependent protein kinase depended on the prior activation of the PKC-like kinase. 相似文献
20.
Rough endoplasmic reticulum membranes, purified from isolated rat pancreatic acini stimulated by carbachol, had a decreased Ca2+ content and increased (Ca2+ + Mg2+)-ATPase activity. Ca2+ was regained and ATPase activity reduced to control levels only after blockade by atropine. The (Ca2+ + Mg2+)-ATPase was activated by free Ca2+ (half-maximal at 0.17 microM; maximal at 0.7 microM) over the concentration range which occurs in the cell cytoplasm. Pretreatment with EGTA, at a high concentration (5 mM), inhibited ATPase activity which, our results suggest, was due to removal of a bound activator such as calmodulin. The rate of (Ca2+ + Mg2+)-ATPase actively declined during the 10-min period over which maximal active accumulation of Ca2+ by membrane vesicles occurs. In the presence of ionophore A23187, which released actively accumulated Ca2+ and stimulated the (Ca2+ + Mg2+)-ATPase, this time-dependent decline in activity was not observed. Our data provide evidence that the activity of the Ca2+-transporting ATPase of the rough endoplasmic reticulum is regulated by both extra and intravesicular Ca2+ and is consistent with a direct role of this enzyme in the release and uptake of Ca2+ during cholinergic stimulation of pancreatic acinar cells. 相似文献