Role of the plasma membrane in signal transduction in human polymorphonuclear leukocytes

To more closely examine the role of the cell surface in transmembrane signal transduction in human neutrophils, sealed right-side-membrane vesicles free of organellar membrane components were used as models of the plasma membrane. These vesicles, incubated with a fluorescent analogue of the chemotactic peptide fMLP, bound this ligand similarly in extent and kinetics to intact neutrophils. Vesicles responded to this stimulation with a slow increase in internal [Ca⁺⁺] which was inhibited by EGTA but not by verapamil; the cytosolic Ca⁺⁺ transient seen in intact cells within 10 sec of stimulation was absent in vesicles. The vesicles also maintained a transmembrane potential (ψ) and were depolarized by the K⁺ ionophore valinomycin. However, unlike intact cells which hyperpolarized and then depolarized in response to fMLP, the vesicles demonstrated only a sustained hyperpolarization. Vesicles also differed from intact cells by not producing superoxide (O₂^?) in response to fMLP. Finally, fMLP caused dramatic alterations in membrane vesicle lipid metabolism: at early time points (within 5–10 sec), there was a transient production of diacylglycerol (DAG) concomitant with inositol lipid breakdown, with no apparent hydrolysis of non-inositol phospholipids. For up to 5 min after stimulation, there was no increase in the levels of phosphatidic acid or of inositol lipids. Thus, a significant portion of the signalling pathway in neutrophils is located at the cell surface or in the plasma membrane and functions independently of intracellular components. Furthermore, the plasma membrane is intimately involved in events occurring during both the early (DAG generation) and late (slow, prolonged rise in [Ca⁺⁺]) phases of cellular response. In contrast, several of the responses to fMLP (the Ca⁺⁺ transient, depolarization, generation of O₂^?, recycling of lipid metabolites) involve signalling machinery not constitutively resident on the neutrophil surface. © 1993 Wiley-Liss, Inc.     

Microvillar Ca++ signaling: A new view of an old problem

Proceeding from the recent finding that the main components of the Ca⁺⁺ signal pathway are located in small membrane protrusions on the surface of differentiated cells, called microvilli, a novel concept of cellular Ca⁺⁺ signaling was developed. The main features of this concept can be summarized as follows: Microvilli are formed on the cell surface of differentiating or resting cells from exocytic membrane domains, growing out from the cell surface by elongation of an internal bundle of actin filaments. The microvillar tip membranes contain all functional important proteins synthesized such as ion channels and transporters for energy-providing substrates and structural components, which are, in rapidly growing undifferentiated cells, distributed over the whole cell surface by lateral diffusion. The microvillar shaft structure, a bundle of actin filaments, forms a dense cytoskeletal matrix tightly covered by the microvillar lipid membrane and represents an effective diffusion barrier separating the microvillar tip compartment (entrance compartment) from the cytoplasm. This diffusion barrier prevents the passage of low molecular components such as Ca⁺⁺ glucose and other relevant substrates from the entrance compartment into the cytoplasm. The effectiveness of the actin-based diffusion barrier is modulated by various signal pathways and effectors, most importantly, by the actin-depolymerizing/reorganizing activity of the phospholipase C (PLC)-coupled Ca⁺⁺ signaling. Moreover, the microvillar bundle of actin filaments plays a dual role in Ca⁺⁺ signaling. It combines the function of a diffusion barrier, preventing Ca⁺⁺ influx into the resting cell, with that of a high-affinity, ATP-dependent, and IP₃-sensitive Ca⁺⁺ store. Activation of Ca⁺⁺ signaling via PLC-coupled receptors simultaneously empties Ca⁺⁺ stores and activates the influx of external Ca⁺⁺. The presented concept of Ca⁺⁺ signaling is compatible with all established data on Ca⁺⁺ signaling. Properties of Ca⁺⁺ signaling, that could not be reconciled with the basic principles of the current hypothesis, are intrinsic properties of the new concept. Quantal Ca⁺⁺ release, Ca⁺⁺-induced Ca⁺⁺ release (CICR), the coupling phenomen between the filling state of the Ca⁺⁺ store and the activity of the Ca⁺⁺ influx pathway, as well as the various yet unexplained complex kinetics of Ca⁺⁺ uptake and release can be explained on a common mechanistic basis. J. Cell. Physiol. 180:19–34, 1999. © 1999 Wiley-Liss, Inc.     

Electrokinetic Properties of the Sarcoplasmic Reticulum Membrane Obtained from Reconstitution Studies

Electrophoretic mobility data of SR vesicles reconstituted with uncharged and two mixtures of charged and uncharged lipids (Brethes, D., Dulon, D., Johannin, G., Arrio, B., Gulik-Krzywicki, T., Chevallier, J. 1986. Study of the electrokinetic properties of reconstituted sarcoplasmic reticulum vesicles. Arch. Biochem. Biophys. 246:355–356) were analyzed in terms of four models of the membrane-water interface: (I) a smooth, negatively charged surface; (II) a negatively charged surface of lipid bilayer covered with an electrically neutral surface frictional layer; (III) an electrically neutral lipid bilayer covered with a neutral frictional layer containing a sheet of negative charge at some distance above the surface of the bilayer; (IV) an electrically neutral lipid bilayer covered with a homogeneously charged frictional layer. The electrophoretic mobility was predicted from the numerical integration of Poisson-Boltzmann and Navier-Stokes equations. Experimental results were consistent only with predictions based on Model-III with charged sheet about 4 nm above the bilayer and frictional layer about 10 nm thick. Assuming that the charge of the SR membrane is solely due to that on Ca⁺⁺-ATPase pumps, the dominant SR protein, the mobility data of SR and reconstituted SR vesicles are consistent with 12 electron charges/ATPase. This value compares well to the net charge of the cytoplasmic portion of ATPase estimated from the amino acid sequence (-11e). The position of the charged sheet suggests that the charge on the ATPase is concentrated in the middle of the cytoplasmic portion. The frictional layer of SR can be also assigned to the cytoplasmic portion of Ca⁺⁺-ATPase. The layer has been characterized with hydrodynamic shielding length of 1.1 nm. Its thickness is comparable to the height of the cytoplasmic portion of Ca⁺⁺-ATPase. Received: 15 June 1998/Revised: 8 October 1998     

A protein activator of the plasma membrane Ca++-ATPase of heart sarcolemma

A detergent extract of dog or beef heart sarcolemmal vesicles was prepared and found to have a stimulatory effect on the Ca⁺⁺-ATPase of plasma membranes from human erythrocyte and cardiac sarcolemma. A procedure is described which enriches the activating fraction. The protein nature of the preparation is illustrated by its sensitivity to boiling and to the proteolytic enzyme(s) trypsin and chymotrypsin. SDS polyacrylamide gels indicate that the protein(s) involved have a molecular weight of 56 and 60 kDa. The sarcolemmal activator can stimulate the Ca⁺⁺-ATPase activity of the isolated enzyme more than 100% in the presence of saturating amounts of calmodulin. The activation is calcium dependent, being greatest at approximately 10µm Ca⁺⁺, free, but does not change theK _m for Ca⁺⁺. A possible physiological role for the activator is discussed.     

Water-soluble proteins of the human red cell membrane

Summary Procedures were developed for preparation of red cell membranes almost free of hemoglobin but with minimal loss of membrane proteins. Two water-soluble protein fractions are described, each constituting about 25% of the ghost protein. The first is ionically bonded and can be solubilized in water rapidly at pH 7.0 and more slowly at higher ionic strength solutions, with a minimal rate at 20mm. This fraction contains four major components with molecular weights ranging from 30,000 to 48,000. The second fraction can only be solubilized at an appreciable rate if Ca⁺⁺ is absent and at higher pH (9.0). It is predominantly a single molecular weight component (150,000). It tends to aggregate at higher ionic strength and in the presence of Ca⁺⁺. Evidence is presented suggesting that the water-soluble proteins are present at the inner face of the membrane. The lipids remain in a water-insoluble residue that contains four major protein components ranging in molecular weight from 30,000 to 100,000. The latter is the predominant component. Only the residue contains the Na⁺–K⁺-activated ATPase, the cholinesterase, antigenic activity and most of the sialic acid and carbohydrate. The first water-soluble fraction contains a Mg⁺⁺-activated ATPase. The extraction of the water-soluble proteins is accompanied by anatomical changes resulting finally in the formation of small membranous vesicles.     

THE SARCOTUBULAR SYSTEM OF FROG SKELETAL MUSCLE : A Morphological and Biochemical Study

In the frog skeletal muscle cell a well defined and highly organized system of tubular elements is located in the sarcoplasm between the myofibrils. The sarcoplasmic component is called the sarcotubular system. By means of differential centrifugation it has been possible to isolate from the frog muscle homogenate a fraction composed of small vesicles, tubules, and particles. This fraction is without cytochrome oxidase activity, which is localized in the mitochondrial membranes. This indicates that the structural components of this fraction do not derive from the mitochondrial fragmentation, but probably from the sarcotubular system. This fraction, called sarcotubular fraction, has a Mg⁺⁺-stimulated ATPase activity which differs from that of muscle mitochondria in that it is 3 to 4 times higher on the protein basis as compared with the mitochondrial ATPase, and is inhibited by Ca⁺⁺ and by deoxycholate like the Kielley and Meyerhof ATPase. We therefore conclude that the "granules" of the Kielley and Meyerhof ATPase, which were shown to have a relaxing effect, are fragments of the sarcotubular system. The isolated sarcotubular fraction has a high RNA content and demonstrable activity in incorporating labeled amino acids, even in the absence of added supernatant.     

Ca++ fluxes in resealed synaptic plasma membrane vesicles

The effect of the monovalent cations Na⁺, Li⁺, and K⁺ on Ca⁺⁺ fluxes has been determined in resealed synaptic plasma membrane vesicle preparations from rat brain. Freshly isolated synaptic membranes, as well as synaptic membranes which were frozen (?80°C), rapidly thawed, and passively loaded with K₂/succinate and ⁴⁵CaCl₂, rapidly released approximately 60% of the intravesicular Ca⁺⁺ when exposed to NaCl or to the Ca⁺⁺ ionophore A 23187. Incubation of these vesicles with LiCl caused a lesser release of Ca⁺⁺. The EC₅₀ for Na⁺ activation of Ca⁺⁺ efflux from the vesicles was approximately 6.6mM. exposure of the Ca⁺⁺-loaded vesicles to 150 mM KCl produced a very rapid (?1 sec) loss of Ca⁺⁺ from the vesicles, but the Na⁺-induced efflux could still be detected above this K⁺ - sensitive effect. Vesicles pre-loaded with NaCl (150 mM) exhibited rapid ⁴⁵Ca uptake with an estimated EC₅₀ for Ca⁺⁺ of 7–10 μM. This Ca⁺⁺ uptake was blocked by dissipation of the Na⁺ gradient. These observations are suggestive of the preservation in these purified frozen synaptic membrane preparations of the basic properties of the Na⁺Ca⁺⁺ exchange process and of a K⁺ - sensitive Ca⁺⁺ flux across the membranes.     

Calcium overload in endotoxemia

E.coli endotoxin stimulates endogenous lipolysis in the $\text{in vitro}$ perfused rat heart. Verapamil^® inhibits endotoxin- (as well as glucagon-) stimulated lipolysis. This suggests that the endotoxin used increases the availability of Ca⁺⁺ to the lipolytic system in the cardiocytes. This conclusion is supported by the observed stimulation of contractility of the heart, especially during perfusion at a low Ca⁺⁺ concentration.The endotoxin was found to inhibit ATP-dependent Ca⁺⁺ accumulation in sarcolemma vesicles prepared from rat heart. A direct Ca⁺⁺ ionophoric action of the endotoxin on these vesicles could be excluded.It is discussed that Ca⁺⁺ overload may not be confined to the cardiovascular system during endotoxemia.     

Active calcium ion uptake by inside-out and right side-out vesicles of red blood cell membranes

The relationship between active extrusion of Ca⁺⁺ from red cell ghosts and active uptake of Ca⁺⁺ by isolated red cell membrane fragments was investigated by studying the Ca⁺⁺ uptake activities of inside-out and right side-out vesicles. Preparations A and B which had mainly inside-out and right side-out vesicles, respectively, were isolated from red cell membranes and were compared with respect to Ca⁺⁺ adenosine triphosphatase (ATPase) and ATP-dependent Ca⁺⁺ uptake activities. Preparation A had nearly eight times more inside-out vesicles and took up eight times more ⁴⁵Ca in the presence of ATP compared to preparation B. Separation of the ⁴⁵Ca-labeled membrane vesicles by density gradient centrifugation showed that the ⁴⁵Ca label was localized to the inside-out vesicle fraction. In addition, the ⁴⁵Ca taken up in the presence of ATP was lost during a subsequent incubation in the absence of ATP. The rate of ⁴⁵Ca loss was not influenced by the presence of EGTA, but was slowed in the presence of La⁺⁸ (0.1 mM) in the efflux medium. The results presented here support the thesis that the active uptake of Ca⁺⁺ by red cell membrane fragments is due to the active transport of Ca⁺⁺ into inside-out vesicles.     

Inhibition of Na+-coupled solute transport by calcium in brush border membrane vesicles

Intracellular Ca⁺⁺ is known to influence Na⁺ flux in luminal membranes. Abnormally elevated Ca⁺⁺ levels in some cells is believed to be the primary pathophysiologic defect in cystic fibrosis (CF). This in turn is thought to alter Na⁺ transport which accounts for certain clinical manifestations of this disease. Two Na⁺-dependent intestinal transport mechanisms have been reported to be suppressed or missing in CF. To examine whether alterations in cell Ca⁺⁺ may account for these findings, studies were performed to examine the influence of Ca⁺⁺ on Na⁺-solute co-transport across intestinal luminal membranes. Purified brush border membrane vesicles prepared from rat small bowel were preincubated in either Ca⁺⁺-free buffer or buffer containing 2.5 mM CaCl₂. Ca⁺⁺ loaded vesicles showed marked inhibition of Na⁺ co-transport of taurocholic acid, taurochenodeoxycholic acid, glucose and valine when compared to controls. The uptake of Na⁺ was also significantly reduced by intravesicular Ca⁺⁺. These data demonstrate that intravesicular Ca⁺⁺ inhibits Na⁺-coupled solute transport as well as Na⁺ influx across intestinal brush border membranes. These data suggest that intracellular Ca⁺⁺ may suppress Na⁺-dependent solute absorption in the intestine. Results presented here further support the theory that elevated intracellular Ca⁺⁺ may account for intestinal malabsorption and other altered transport phenomena reported in CF.     

The active transport of Mg++ and Mn++ into the yeast cell

Certain bivalent cations, particularly Mg⁺⁺ and Mn⁺⁺, can be absorbed by yeast cells, provided that glucose is available, and that phosphate is also absorbed. The cation absorption is stimulated by potassium in low concentrations, but inhibited by higher concentrations. From the time course studies, it is apparent that the absorption rather than the presence of phosphate and the potassium is the important factor. Competition studies with pairs of cations indicate that binding on the surface of the cell is not a prerequisite to absorption. The absorption mechanism if highly selective for Mg⁺⁺ and Mn⁺⁺, as compared to Ca⁺⁺, Sr⁺⁺, and UO₂⁺⁺, whereas the binding affinity is greatest for UO₂⁺⁺, with little discrimination between Mg⁺⁺, Ca⁺⁺, Mn⁺⁺, and Sr⁺⁺. In contrast to the surface-bound cations which are completely exchangeable, the absorbed cations are not exchangeable. It is concluded that Mg⁺⁺ and Mn⁺⁺ are actively transported into the cell by a mechanism involving a phosphate and a protein constituent.     

Bile acid uptake and calcium flux in brush border membrane vesicles

Our laboratory has recently reported that intestinal bile acid malabsorption in cystic fibrosis (CF) is a primary mucosal cell defect. Others have suggested that elevated intracellular Ca⁺⁺ levels in other cell types in CF may represent a common primary dysfunction in Ca⁺⁺ efflux in these cells. We examined the possibility that intestinal bile acid absorption and Ca⁺⁺ efflux in mucosal cells may be linked physiologically. Brush border membrane vesicles (BBMV) prepared from guinea pig ileum served as the experimental model to test this hypothesis. Ca⁺⁺ (2.5×10^?3M) present in the incubation medium did not alter the uptake of taurocholic acid (TCA) by BBMV. Also, TCA uptake into BBMV preloaded with Ca⁺⁺ was not significantly different from that in BBMV not previously loaded with Ca⁺⁺. Furthermore, with TCA present in the incubation medium, Ca⁺⁺ efflux from preloaded BBMV was not altered. These data suggest that ileal TCA uptake, as measured by BBMV, is not dependent upon either intra- or extravesicular Ca⁺⁺. Also, Ca⁺⁺ efflux from BBMV is unaffected by TCA uptake. Although separate lines of evidence suggest that intestinal bile acid malabsorption and reduced plasma membrane Ca⁺⁺ flux are primary defects in CF, we conclude that in the normal intestine these functions are independent physiological processes.     

Membrane transport properties differ following return of serum-deprived versus Ca++-deprived human fibroblasts to a proliferative state

Human lung fibroblasts (W138) can be brought to a quiescent state by removal of serum from the medium or by lowering of the extracellular Ca⁺⁺. Upon return of Ca⁺⁺ or serum, the cells enter the G1 phase and progress to S within 15–18 hours. Since multiple G1 phase blocks have been demonstrated, we wished to determine whether the Ca⁺⁺ and serum block were equivalent since previous data suggested that these two medium components may act at a common point in the initiation of proliferation. We have evaluated the membrane transport of ⁸⁶Rb, 3-O-methylglucose, AIB, and cycloleucine following stimulation of quiescent cells by Ca⁺⁺ or serum. Serum stimulation results in large increases in the influx of all the substances tested. These increases are prevented if Ca⁺⁺ is absent upon serum stimulation or they are rapidly diminished following Ca⁺⁺ removal. In contrast, Ca⁺⁺ stimulation of Ca⁺⁺-deprived cells causes little or no enhancement of any of the transport systems, yet the cells progress to S phase in a manner similar to serum-stimulated cells. These results indicate that the Ca⁺⁺ and serum G0 or G1 block are not equivalent and that the serum-induced change in transport of these components does not appear necessary for successful G1 phase progression. Furthermore, the data suggest that the sequence in which Ca⁺⁺ or serum are presented to the cells alters the ability of Ca⁺⁺ to modulate the transport systems. Quiescent cells which are exposed to Ca⁺⁺ prior to serum possess a Ca⁺⁺ modulation of several transport systems. Cells which are exposed to Ca⁺⁺ subsequent to serum do not appear to possess this Ca⁺⁺ regulation.     

Fusion of phospholipid vesicles reconstituted with cytochromec oxidase and mitochondrial hydrophobic protein

Summary Reconstituted cytochrome oxidase liposomes were fused with liposomes reconstituted with mitochondrial hydrophobic protein, which acts as a membrane-bound uncoupler of cytochrome oxidase. Fusion was assayed by the loss of respiratory control of cytochrome oxidase as measured by the increased rate of ascorbate oxidation induced by hydrophobic protein when both proteins shared the same vesicles. Fusion was dependent on the presence of phosphatidylserine in the liposomes and Ca⁺⁺ in the aqueous medium. Phosphatidylcholine-phosphatidylserine liposomes required higher concentrations of phosphatidylserine and Ca⁺⁺ than did phosphatidylethanolamine-phosphatidylserine liposomes. Cytochrome oxidase vesicles containing high concentrations of phosphatidylserine showed little or no respiratory control, while those with lower concentrations showed high respiratory control; respiratory control could be induced by fusing cytochrome oxidase vesicles containing high phosphatidylserine with protein-free liposomes containing low phosphatidylserine concentration. If cytochrome oxidase vesicles and hydrophobic protein vesicles were prefused separately for 15 min, they lost the ability to fuse upon being subsequently mixed together. The reconstituted vesicles had diameters of about 200 Å; fusion yielded vesicles with diameters in excess of 1000 Å.     

MIGRATORY CELL LOCOMOTION VERSUS NERVE AXON ELONGATION : Differences Based on the Effects of Lanthanum Ion

The effects of lanthanum ions (La⁺⁺⁺) on the locomotion and adhesion of g lial cells and elongating nerve axons are reported. La⁺⁺⁺ increases adhesion of both glia and of nerve growth cones to a plastic substratum. La⁺⁺⁺ also markedly reduces glia locomotion, but it does not inhibit nerve elongation. Electron-opaque deposits are seen on the cell surface and within cytoplasmic vesicles of glia and nerves cultured in a La⁺⁺⁺-containing medium. Possible modes of action for La⁺⁺⁺ are discussed, particularly the possibilities that Ca⁺⁺ fluxes or Ca⁺⁺ involvement in adhesion are altered by La⁺⁺⁺. The results are consistent with the hypothesis that cell migration and nerve axon elongation differ in mechanism, with respect to both adhesive interactions and the activity of microfilament systems.     

Effects of divalent ions on vesicle-vesicle fusion studied by a new luminescence assay for fusion

Summary A new assay has been developed for vesicle-vesicle fusion based upon the mixing of intravesicular contents of two sets of vesicles. Purified firefly luciferase and MgCl₂ were incorporated into one set of vesicles (LV) and ATP into the other (AV). Vesicles were prepared from soybean phospholipids. The luminescence that resulted from hydrolysis of ATP by luciferase was measured to determine the extent of mixing of the intravesicular contents. In the absence of divalent ions, incubation of a mixture of LV and AV did not produce luminescence. However, if Ca⁺⁺ or other divalent ions were present at millimolar concentrations, luminescence occurred. The luminescence did not result from extravesicular reaction of vesicle contents that had leaked into the medium. Instead, luminescence resulted from the mixing of intravesicular spaces of AV and LV in fused vesicles. Optical density changes and negative stain electron microscopy indicated that Ca⁺⁺ induced extensive aggregation of vesicles. However, quantitation of the maximum possible luminescence indicates that only a small percentage (less than 1%) of the vesicles actually fused in a fusion experiment.Addition of EDTA to chelate Ca⁺⁺ after luminescence had been induced resulted in a two-to threefoldincrease in light emission which then rapidly decayed. These results suggest that the sudden removal of Ca⁺⁺ caused a transient increase in fusion after which subsequent fusion was inhibited. It was also found that the vesicles were relatively stable to hypotonic solutions.     

Biochemical and morphological aspects of zinc deficiency in rhodotorula gracilis

Summary Cells of Rhodotorula gracilis cultured in a liquid medium containing Zn only as impurity stop growing at a density of about 5·10⁷ cells/ml. The addition of Zn during the prestationary or the stationary phase of growth reestablishes the growth rate, thus showing that Zn⁺⁺ is a limiting factor for growth. An analysis of the changes of the fine structure and of RNA, DNA and protein levels induced by the addition of Zn to Zn-deficient cultures indicates that the most notable features of Zn deficiency are: a) the decrease of net RNA synthesis, and consequently of protein synthesis; b) the appearance of several large vacuole-like structures containing degraded cytoplasmic components, membranous whorls and amorphous material; a decrease in the number of mitochondria and in the organization of cristae. The net synthesis of DNA appears to be much less affected, and lipid synthesis is somewhat stimulated in the Zn-deficient cultures. No important effect of Zn-deficiency was observed on either oxygen uptake or intracellular amino acid level. These results are interpreted as indicating that Zn⁺⁺ is an essential element for this organism, and that the area of RNA metabolism and protein synthesis is the one primarily affected by Zn⁺⁺ deficiency.     

A comparative study of the effect of various detergents on the structure and function of sarcoplasmic reticulum vesicles

Summary The effects produced by the detergents Triton X-100, sodium dodecylsulphate and sodium cholate on sarcoplasmic reticulum vesicles have been comparatively studied. In all cases, maximal effects are found 5 min after detergent addition. Triton X-100 and SDS are approximately ten times more effective than cholate in protein and phospholipid solubilization. Both Triton X-100 and SDS maintain Ca⁺⁺ accumulation in SR vesicles at detergent concentrations below 10^–3 M; higher concentrations cause a strong inhibition. On the other hand, cholate produces a gradual inhibition of Ca⁺⁺ accumulation in the concentration range between 10^–4 M and 2.5 × 10^–2 M. Triton X-100 and SDS produce a gradual solubilization of the specific Ca⁺⁺-ATPase activity up to a 10^–3 M detergent concentration, above which a strong inactivation occurs, while the enzyme solubilization increases with the presence of cholate in the whole concentration range under study. The different behaviour of sodium cholate, when compared to SDS or Triton X-100, is discussed in relation to the surfactant molecular structures. The possibility of membrane lysis and reassembly in the presence of some detergents is also considered.Abbreviations SR sarcoplasmic reticulum - SDS sodium dodecylsulphate - DTT dithiothreitol - EGTA ethyleneglycoltetraacetate - PEP phosphoenolpyruvate     

The effect of anti-ATPase antibodies upon the Ca++ transport of sarcoplasmic reticulum

Sheep or guinea pig antisera against the purified Ca⁺⁺ transport ATPase of sarcoplasmic reticulum inhibit Ca⁺⁺ transport due to a complement-dependent damage of the membrane, which causes massive leakage of Ca⁺⁺. The Ca⁺⁺-activated ATPase activity is only slightly affected even at ten times higher antibody concentration than that required for inhibition of Ca⁺⁺ transport. Antibodies prepared against the Ca⁺⁺ binding protein (C₁ protein) have no influence upon either ATPase activity or Ca⁺⁺ transport and ferritin-labeled anti-C₁ antibodies do not bind to microsomes.     

Ca++- and Na+, K+-ATPase activities in the fungiform papilla of the tongue of Rana Esculenta (Anura Ranidae)

In the fungiform papilla of Rana esculenta (Anura Ranidae), the Ca⁺⁺-ATPase is mainly distributed on the basolateral membrane of the sensory area cells (i.e., neuroepithelial, supporting, and mucous cells). Apical membranes of all cells facing the surface present a slight enzymatic activity. Lateral wall cells have a strong Ca⁺⁺-ATPase activity on basolateral and apical membranes. Strong Na⁺, K⁺-ATPase activity occurs on the apical surface of neuroepithelial cells. Ca⁺⁺-ATPase activity is absent on the surface of endothelial cells of the capillaries located under the sensory area. These observations lead us to conclude that the sensory area of fungiform papilla is the selective way for calcium influx. Furthermore the absence of ATPase activity on the surface of the endothelial cells indicates that there is no functional barrier to calcium influx into capillary, and that calcium can be removed by vessels from the sensory area.