Mitochondrial migration and Ca-ATPase modulation in secretory ameloblasts of fasted and calcium-loaded rats

Migration of mitochondria and modulation of Ca-ATPase activity in secretory ameloblasts were investigated ultrastructurally and ultracytochemically by using lower incisors taken from normally fed, 30-hr-fasted, and calcium (Ca)-loaded rats. In normally fed rats, almost all mitochondria were localized in a narrow infranuclear compartment between the nucleus and proximal cell webs of secretory ameloblasts. In 30-hr-fasted rats, a prominent migration of many mitochondria into the supranuclear region of the cells was noted. Mitochondria returned to the infranuclear compartment and seldom appeared in the supranuclear region when fasted rats were Ca-loaded by transcardiac perfusion with physiological Ca solution. Normally, the mitochondria of secretory ameloblast exhibited moderate Ca-ATPase activity along their inner membranes. This mitochondrial Ca-ATPase was decreased by a 30-hr fast and became prominent again after Ca loading. Plasma-membrane Ca-ATPase was demonstrated in the entire cell surface of secretory ameloblasts. An especially abundant reaction was found along the invaginated cell surface of the Tomes process. This Ca-ATPase also became very weak and was almost abolished from the Tomes process after fasting, but Ca loading caused reappearance of an intense Ca-ATPase activity on the entire cell surface, including along Tomes's processes. These results suggest that 1) mitochondrial localization in secretory ameloblasts is influenced by the Ca concentration of the extracellular milieu, and 2) the level of mitochondrial and cell-membrane ATPase activity is responsive to the concentration of extracellular calcium.     

Pulmonary surfactant protein A augments the phagocytosis of Streptococcus pneumoniae by alveolar macrophages through a casein kinase 2-dependent increase of cell surface localization of scavenger receptor A

Pulmonary surfactant proteins A (SP-A) and D (SP-D), members of the collectin family, play important roles in the innate immune system of the lung. Here, we show that SP-A but not SP-D augmented phagocytosis of Streptococcus pneumoniae by alveolar macrophages, independent of its binding to the bacteria. Analysis of the SP-A/SP-D chimeras, in which progressively longer carboxyl-terminal regions of SP-A were replaced with the corresponding SP-D regions, has revealed that the SP-D region Gly(346)-Phe(355) can be substituted for the SP-A region Leu(219)-Phe(228) without altering the SP-A activity of enhancing the phagocytosis and that the SP-A region Cys(204)-Cys(218) is required for the SP-A-mediated phagocytosis. Acetylated low density lipoprotein significantly reduced the SP-A-stimulated uptake of the bacteria. SP-A failed to enhance the phagocytosis of S. pneumoniae by alveolar macrophages derived from scavenger receptor A (SR-A)-deficient mice, demonstrating that SP-A augments SRA-mediated phagocytosis. Preincubation of macrophages with SP-A at 37 degrees C but not at 4 degrees C stimulated the phagocytosis. The SP-A-mediated enhanced phagocytosis was not inhibited by the presence of cycloheximide. SP-A increased cell surface localization of SR-A that was inhibitable by apigenin, a casein kinase 2 (CK2) inhibitor. SP-A-treated macrophages exhibited significantly greater binding of acetylated low density lipoprotein than nontreated cells. The SP-A-stimulated phagocytosis was also abolished by apigenin. In addition, SP-A stimulated CK2 activity. These results demonstrate that SP-A enhances the phagocytosis of S. pneumoniae by alveolar macrophages through a CK2-dependent increase of cell surface SR-A localization. This study reveals a novel mechanism of bacterial clearance by alveolar macrophages.     

Histochemical studies of Ca-ATPase, succinate and NAD+-dependent isocitrate dehydrogenases in the shell gland of laying Japanese quails: with special reference to calcium-transporting cells

In order to elucidate the problem of which cells are involved in calcium transport and to estimate the role of mitochondria in calcium transport in the avian shell gland, the fine structure and the Ca-ATPase, succinate dehydrogenase (SDH) and nicotinamide adenine dinucleotide (NAD+)-dependent isocitrate dehydrogenase (NAD+-ICDH) activity of the shell gland of egg-laying Japanese quails were examined. The surface epithelial cells, consisting of ciliated cells with cilia and microvilli and non-ciliated cells with microvilli, had many large and electron-dense granules. The tubular-gland cells occupied the proprial layer and lacked secretory granules. When an egg was in the shell gland, the well-developed mitochondria of tubular-gland cells characteristically tended to accumulate in the apical cytoplasm, while they were scattered throughout the cytoplasm when an egg was not in the shell gland. Intense Ca-ATPase activity was found on the microvilli of tubular-gland cells, and moderate activity was found on the lateral-cell surface. In the surface epithelial cells, the basolateral cell surface showed moderate enzymatic activity. Both SDH and NAD+-ICDH activity were found in tubular-gland cells when an egg was in the shell gland. These results strongly suggest that calcium for eggshell calcification is actively transported by the tubular-gland (depending on Ca-ATPase activity) and that the mitochondria of gland cells may play an important role in this process as an energy source.     

Pretaporter,a Drosophila protein serving as a ligand for Draper in the phagocytosis of apoptotic cells

Phagocytic removal of cells undergoing apoptosis is necessary for animal development and tissue homeostasis. Draper, a homologue of the Caenorhabditis elegans phagocytosis receptor CED‐1, is responsible for the phagocytosis of apoptotic cells in Drosophila, but its ligand presumably present on apoptotic cells remains unknown. An endoplasmic reticulum protein that binds to the extracellular region of Draper was isolated. Loss of this protein, which we name Pretaporter, led to a reduced level of apoptotic cell clearance in embryos, and the overexpression of pretaporter in the mutant flies rescued this defect. Results from genetic analyses suggested that Pretaporter functionally interacts with Draper and the corresponding signal mediators. Pretaporter was exposed at the cell surface after the induction of apoptosis, and cells artificially expressing Pretaporter at their surface became susceptible to Draper‐mediated phagocytosis. Finally, the incubation with Pretaporter augmented the tyrosine‐phosphorylation of Draper in phagocytic cells. These results collectively suggest that Pretaporter relocates from the endoplasmic reticulum to the cell surface during apoptosis to serve as a ligand for Draper in the phagocytosis of apoptotic cells.     

Histochemical studies of Ca-ATPase, succinate and NAD+-dependent isocitrate dehydrogenases in the shell gland of laying Japanese quails: with special reference to calcium-transporting cells

Summary In order to elucidate the problem of which cells are involved in calcium transport and to estimate the role of mitochondria in calcium transport in the avian shell gland, the fine structure and the Ca-ATPase, succinate dehydrogenase (SDH) and nicotinamide adenine dinucleotide (NAD⁺)-dependent isocitrate dehydrogenase (NAD⁺-ICDH) activity of the shell gland of egg-laying Japanese quails were examined. The surface epithelial cells, consisting of ciliated cells with cilia and microvilli and non-ciliated cells with microvilli, had many large and electron-dense granules. The tubular-gland cells occupied the proprial layer and lacked secretory granules. When an egg was in the shell gland, the well-developed mitochondria of tubular-gland cells characteristically tended to accumulate in the apical cytoplasm, while they were scattered throughout the cytoplasm when an egg was not in the shell gland. Intense Ca-ATPase activity was found on the microvilli of tubular-gland cells, and moderate activity was found on the lateral-cell surface. In the surface epithelial cells, the basolateral cell surface showed moderate enzymatic activity. Both SDH and NAD⁺-ICDH activity were found in tubular-gland cells when an egg was in the shell gland. These results strongly suggest that calcium for eggshell calcification is actively transported by the tubular-gland (depending on Ca-ATPase activity) and that the mitochondria of gland cells may play an important role in this process as an energy source.     

Phospholamban inhibits Ca-ATPase conformational changes involving the E2 intermediate

We have used steady-state fluorescence spectroscopy in combination with enzyme kinetic assays to test the hypothesis that phospholamban (PLB) stabilizes the Ca-ATPase in the E2 intermediate state. The cardiac muscle Ca-ATPase (SERCA2a) isoform was expressed either alone or coexpressed with PLB in High-Five insect cells and was isolated as insect cell microsomes. Fluorescence studies of the Ca-ATPase covalently labeled with the probe 5-(2-((iodoacetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid showed that PLB decreased the amplitude of the Ca-ATPase E2 --> E1 conformational transition by 45 +/- 3% and shifted the [Ca2+] dependence of the transition to higher Ca2+ levels (DeltaKCa = 230 nM), similar to the effect of PLB on Ca-ATPase activity. Similarly, PLB decreased the amplitude of Ca-ATPase phosphorylation by inorganic phosphate (Pi) by 55 +/- 2% and decreased slightly the affinity for Pi (DeltaK0.5 = 70 microM). However, PLB did not affect the Ca2+-dependent inhibition of Ca-ATPase phosphorylation by Pi. Finally, PLB decreased Ca-ATPase sensitivity to vanadate, increasing the IC50 value by 300 nM. The results suggest that PLB binding to Ca-ATPase stabilizes the enzyme in a conformation distinct from E2, decreasing the number of enzymes in the E2 state capable of undergoing ligand-dependent conformational changes involving the Ca-free E2 intermediate. The inability of conformation-specific ligands to fully convert this E2-like state into E1 or E2 implies that these states are not in a simple equilibrium relationship.     

Inactivation of calcium uptake by EGTA is due to an irreversible thermotropic conformational change in the calcium binding domain of the Ca(2+)-ATPase.

Calcium uptake by rabbit skeletal sarcoplasmic reticulum (SR) is inhibited with an effective inactivation temperature (TI) of 37 degrees C in EGTA with no effect on ATPase activity. Since the Ca-ATPase denatures at a much higher temperature (49 degrees C) in EGTA, this suggests that a small or localized conformational change of the Ca-ATPase at 37 degrees C results in inability to accumulate calcium by the SR. Using a fluorescent analogue of dicyclohexylcarbodiimide, N-cyclohexyl-N'-[4-(dimethylamino)-alpha-naphthyl]-carbodiimide (NCD-4), the region of the calcium binding sites of the SR Ca-ATPase was labeled. Steady-state and frequency-resolved fluorescence measurements were subsequently performed on the NCD-4-labeled Ca-ATPase. Site-specific information pertaining to the hydrophobicity and segmental flexibility of the region of the calcium binding sites was derived from the steady-state fluorescence intensity, lifetime, and rotational rate of the covalently bound NCD-4 label as a function of temperature (0-50 degrees C). A reversible transition at approximately 15 degrees C and an irreversible transition at approximately 35 degrees C were deduced from the measured fluorescence parameters. The low-temperature transition agrees with the previously observed break in the Arrhenius plot of ATPase activity of the native Ca-ATPase at 15-20 degrees C. The high-temperature transition conforms well with the conformational transition, resulting in uncoupling of Ca translocation from ATP hydrolysis as predicted from the irreversible inactivation of Ca uptake at 31-37 degrees C in 1 mM EGTA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell surface topography controls phagocytosis and cell spreading: The membrane reservoir in neutrophils

Neutrophils exhibit rapid cell spreading and phagocytosis, both requiring a large apparent increase in the cell surface area. The wrinkled surface topography of these cells may provide the membrane reservoir for this. Here, the effects of manipulation of the neutrophil cell surface topography on phagocytosis and cell spreading were established. Chemical expansion of the plasma membrane or osmotic swelling had no effects. However, osmotic shrinking of neutrophils inhibited both cell spreading and phagocytosis. Triggering a Ca²⁺ signal in osmotically shrunk cells (by IP₃ uncaging) evoked tubular blebs instead of full cell spreading. Phagocytosis was halted at the phagocytic cup stage by osmotic shrinking induced after the phagocytic Ca²⁺ signalling. Restoration of isotonicity was able to restore complete phagocytosis. These data thus provide evidence that the wrinkled neutrophil surface topography provides the membrane reservoir to increase the available cell surface area for phagocytosis and spreading by neutrophils.     

Ca-ATPase and ALPase activities at the initial calcification sites of dentin and enamel in the rat incisor

Summary Enzymatic activities of calcium-magnesium dependent adenosine triphosphatase (Ca-ATPase) and nonspecific alkaline phosphatase (ALPase) were localized at the initial calcification sites of dentin and enamel of rat incisor teeth using electron-microscopic cytochemistry.Ca-ATPase was localized in the Golgi cisternae, cytoplasmic vesicles and along the outer surface of the presecretory and secretory ameloblasts, whereas it was totally absent from the odontoblasts in the pulp. Inversely, ALPase reaction was localized along the outer surface of the odontoblasts, but almost completely absent from the ameloblasts.Diffuse extracellular reactions of both enzymes were distributed throughout the unmineralized fibrous matrix of mantle dentin in which a large number of matrix vesicles were scattered. Both Ca-ATPase and ALPase reactions, which appeared in the matrix vesicles in the process of formation of mantle dentin, became most conspicuous at the site of initial dentin calcification. At this stage, an intense Ca-ATPase reaction also appeared along some of the collagen fibrils adjacent to the reactive matrix vesicles. No ALPase reaction was localized along these Ca-ATPase reactive collagen fibrils.Our observations suggest strongly that Ca-ATPase in the matrix vesicles originates from the inner enamel epithelium and/or preameloblasts whereas ALPase originates from the odontoblasts in the pulp. The importance of the coexistence of both enzymes for the control of initial calcification of dental hard tissues is suggested.     

Modulation of FcR function by complement: subcomponent C1q enhances the phagocytosis of IgG-opsonized targets by human monocytes and culture-derived macrophages

We have investigated the interaction of C1q, a subunit of the first component of complement, with human monocytes and culture-derived macrophages. Adherence of these mononuclear phagocytes to surfaces coated with C1q induced a marked enhancement of the phagocytosis of sheep erythrocytes opsonized with IgG anti-Forssman antibody (EA-IgG). This C1q-mediated enhancement of phagocytosis was dose dependent, and was specifically blocked by pretreatment of the C1q-coated surfaces with F(ab')2 anti-C1q. The augmentation of FcR-mediated phagocytosis by C1q was determined to be a result of the interaction between the C1q and the phagocytic effector cell, and was not due to interaction between the surface-bound C1q and the EA-IgG. Neither resting nor N-formyl-methionyl-leucyl-phenylalanine-stimulated polymorphonuclear leukocytes were induced by C1q to increase FcR-mediated phagocytosis. Experiments conducted with purified fragments of C1q suggest that the C1q phagocytosis enhancement signal resides in the collagen-like tail domain of the molecule. This region is the same portion of the molecule previously shown to interact with the cell surface C1q receptor. Native type I collagen was unable to enhance FcR-mediated phagocytosis by mononuclear phagocytes. It has been demonstrated that C1q can be localized to areas of inflammation, and additionally C1q can be secreted by macrophages in culture. In view of these findings and the results of our present study, we hypothesize that C1q could provide local, direct, and non-opsonic enhancement of phagocytosis by mononuclear phagocytes in areas of infection and inflammation.     

Effect of phagocytosis on receptor distribution and endocytic activity in macrophages

Phagocytosis requires the internalization of a significant fraction of the plasma membrane and results in the intracellular deposition of large particles. We evaluated the effect of phagocytosis on the cellular distribution of recycling receptors and uptake of ligand to determine whether phagocytosis affects receptor behavior. Phagocytosis of zymosan, latex particles, or IgG-coated red blood cells by rabbit alveolar macrophages did not decrease the number of cell surface receptors for transferrin, alpha 2-macroglobulin X protease complexes, maleylated proteins, or mannosylated proteins. The number of surface receptors for transferrin was also unaltered in J774 cells, a macrophage-like cell line. In both cell types extensive phagocytosis did not affect the rate of receptor-mediated endocytosis or the distribution of receptors between the endosome and the cell surface. However, fluid phase pinocytosis was reduced by phagocytosis. The major reduction appeared to be not in the rate of internalization but rather in the delivery of fluid to the lysosome. These results demonstrate that internalization of a significant amount of the plasma membrane during phagocytosis does not diminish the number of receptors on the cell surface and has no effect on receptor-mediated ligand uptake.     

Assessment of the role of endogenous regulators in the activation of Ca-ATPase in erythrocyte membranes

The contribution of calmodulin and protein kinases A or C to the activation of membrane Ca-ATPase was studied on saponin-permeabilized rat erythrocytes. In the presence of all endogenous regulators, the dependence of the Ca-ATPase activity of Ca2+ concentration was described by a bell-shaped curve with a maximum at 2-5 microM Ca2+; K0.5 = 0.43 microM Ca2+. Washing of erythrocyte membranes with 5-10 microM Ca2+ maintained up to 75% of the ATPase activity, while washing with EGTA (2 mM) decreased the activity, on the average, 5-fold, and increased K0.5 up to 0.54-0.6 microM Ca2+. An addition of an EGTA extract to washed membranes restored up to 75% of the original ATPase activity, while calmodulin restored about 40% of the original Ca-ATPase activity and decreased K0.5 to 0.23-0.3 microM Ca2+. The calmodulin inhibitor R24571 failed to alter the Ca-ATPase activity in permeabilized erythrocytes but slightly diminished it in reconstituted membranes. The protein kinase C inhibitors H7 and polymyxin increased the Ca-ATPase activity in permeabilized red cells and suppressed it in reconstituted membranes. The data obtained suggest that in native red cell membranes Ca-ATPase is activated by regulator(s) dependent on Ca2+ and protein kinase which are other than calmodulin.     

Effects of melittin on molecular dynamics and Ca-ATPase activity in sarcoplasmic reticulum membranes: electron paramagnetic resonance.

We have performed electron paramagnetic resonance (EPR) experiments on nitroxide spin labels incorporated into rabbit skeletal sarcoplasmic reticulum (SR), in order to investigate the physical and functional interactions between melittin, a small basic membrane-binding peptide, and the Ca-ATPase of SR. Melittin binding to SR substantially inhibits Ca(2+)-dependent ATPase activity at 25 degrees C, with half-maximal inhibition at 9 mol of melittin bound per mole of Ca-ATPase. Saturation transfer EPR (ST-EPR) of maleimide spin-labeled Ca-ATPase showed that melittin decreases the submillisecond rotational mobility of the enzyme, with a 4-fold increase in the effective rotational correlation time (tau r) at a melittin/Ca-ATPase mole ratio of 10:1. This decreased rotational motion is consistent with melittin-induced aggregation of the Ca-ATPase. Conventional EPR was used to measure the submicrosecond rotational dynamics of spin-labeled stearic acid probes incorporated into SR. Melittin binding to SR at a melittin/Ca-ATPase mole ratio of 10:1 decreases lipid hydrocarbon chain mobility (fluidity) 25% near the surface of the membrane, but only 5% near the center of the bilayer. This gradient effect of melittin on SR fluidity suggests that melittin interacts primarily with the membrane surface. For all of these melittin effects (on enzymatic activity, protein mobility, and fluidity), increasing the ionic strength lessened the effect of melittin but did not alleviate it entirely. This is consistent with a melittin-SR interaction characterized by both hydrophobic and electrostatic forces. Since the effect of melittin on lipid fluidity alone is too small to account for the large inhibition of Ca-ATPase rotational mobility and enzymatic activity, we propose that melittin inhibits the ATPase primarily through its capacity to aggregate the enzyme, consistent with previous observations of decreased Ca-ATPase activity under conditions that decrease protein rotational mobility.     

Conformational changes within the cytosolic portion of phospholamban upon release of Ca-ATPase inhibition

Phospholamban (PLB) is a major target of the beta-adrenergic cascade in the heart, functioning to modulate contractile force by altering the rate of calcium re-sequestration by the Ca-ATPase. Functionally, inhibition by PLB binding is manifested by shifts in the calcium dependence of Ca-ATPase activation toward higher calcium levels; phosphorylation of PLB by PKA reverses the inhibitory action of PLB. To investigate structural changes in the cytoplasmic portion of PLB that result from either the phosphorylation of PLB by cAMP-dependent protein kinase (PKA) or calcium binding to the Ca-ATPase, we have used frequency-domain fluorescence spectroscopy to measure the spatial separation and conformational heterogeneity between N-(1-pyrenyl)maleimide, covalently bound to a single cysteine (Cys(24)) engineered near the membrane surface of the transmembrane domain of PLB, and Tyr(6) in the cytosolic domain. Irrespective of calcium activation of the Ca-ATPase or phosphorylation of Ser(16) in PLB by PKA, we find that PLB remains tightly associated with the Ca-ATPase in a well-defined conformation. However, calcium activation of the Ca-ATPase induces an increase in the overall dimensions of the cytoplasmic portion of bound PLB, whereas PLB phosphorylation results in a more compact structure, consistent with increased helical content induced by a salt link between phospho-Ser(16) and Arg(13). Thus, enzyme activation of the Ca-ATPase may occur through different mechanisms: calcium binding to high-affinity sites within the Ca-ATPase functions to overcome conformational constraints imposed by PLB on the N-domain of the Ca-ATPase; alternatively, phosphorylation stabilizes the backbone fold of PLB to release inhibitory interactions with the Ca-ATPase.     

Identification of hydrophobic regions of the calcium-transport ATPase from sarcoplasmic reticulum after photochemical labeling with adamantane diazirine

The Ca-ATPase from skeletal muscle sarcoplasmic reticulum was labeled with [3H]adamantane diazirine. Adamantane diazirine is a hydrophobic photoactivated probe that partitions into the cell membrane and can be used to identify regions of proteins that are embedded within the membrane. Digestion of the labeled protein with trypsin and separation of the labeled tryptic fragments by SDS-polyacrylamide-gel electrophoresis indicated that all of the major tryptic fragments were labeled by the probe. The presence of glutathione in the sample buffer during photolysis did not alter the pattern of labeling, indicating that adamantane diazirine labeled the Ca-ATPase from within the lipid bilayer. These results indicate that the Ca-ATPase polypeptide must cross the membrane at least 3 times.     

Essential role for Pro21 in phospholamban for optimal inhibition of the Ca-ATPase

We have investigated the functional role of the flexible hinge region centered near the sequence TIEMP(21), which connects the N-terminal cytosolic and C-terminal membrane-spanning helical domains of phospholamban (PLB). Specifically, we ask if the conformation of this region is important to attain optimal inhibitory interactions with the Ca-ATPase. A genetically engineered PLB mutant was constructed in which Pro(21) was mutated to an alanine (P21A-PLB(C)); in this construct, all three transmembrane cysteines were substituted with alanines to stabilize the monomeric form of PLB, and a unique cysteine was introduced at position 24 near the hinge element (A24C), permitting the site-specific attachment of fluorescein-5-maleimide (FMal) to monitor structure changes. In agreement with prior measurements in cardiac SR microsomes, the calcium concentration associated with half-maximal activation (Ca(1/2)) of the Ca-ATPase, 290 +/- 10 nM, is shifted to 580 +/- 20 nM when co-reconstituted with PLB(C) (Pro21) as a result of a reduction in the cooperativity associated with the calcium-dependent structural transition. Kinetic simulations indicate that PLB(C) association with the Ca-ATPase results in a 75% reduction in the equilibrium constant associated with the formation of the second high-affinity calcium binding site. In comparison, there is a 43% reduction in KCa(1/2) upon reconstitution of the Ca-ATPase with P21A-PLB(C), which can be simulated by decreasing the equilibrium constant associated with the calcium-dependent structural activation by 50%. The diminished inhibitory action of P21A-PLB(C) is associated with alterations in the structure of the hinge element, as evidenced by the diminished solvent accessibility of FMal relative to the native structure. Likewise, increases in the alpha-helical content and decreases in the mobility of the carboxyl-terminal domain of P21A-PLB(C) are observed using circular dichroism and fluorescence spectroscopy. Collectively, these results indicate that the overall dimensions of the carboxyl-terminal domain of PLB are increased through a stabilization of secondary structural elements upon mutation in P21A-PLB(C) that result in a reduction in the ability of the amino-terminal cytosolic portion of PLB to productively inhibit the Ca-ATPase. Further, these results suggest that the unstructured characteristics of the flexible hinge region in PLB are critical for optimal inhibitory interactions with the Ca-ATPase and suggest its role as a conformational switch.     

Exposure of phosphatidylserine is a general feature in the phagocytosis of apoptotic lymphocytes by macrophages

Although different macrophages exploit different cell surface receptors to recognize apoptotic lymphocytes, indirect evidence suggested that the phosphatidylserine (PS) that appears on the surface of lymphocytes undergoing apoptosis participates in specific recognition by all types of macrophages. To test this possibility directly, annexin V, a protein that specifically binds to PS, was used to mask this phospholipid on the apoptotic cell surface. Preincubation of apoptotic lymphocytes with annexin V blocked phagocytosis by elicited mouse peritoneal macrophages, macrophages of the mouse J774 cell line and mouse bone marrow macrophages. Similarly, annexin V was able to inhibit phagocytosis of lipid-symmetric erythrocytes, another target cell upon which PS is exposed. Together these results demonstrate directly that macrophages of all types depend on the PS exposed on the surface of apoptotic lymphocytes for recognition and phagocytosis.     

Binding and phagocytosis of fibronectin-coated beads by BHK cells: receptor specificity and dynamics

Studies on the receptor specificity and dynamics involved in fibroblast phagocytosis of latex beads revealed the following: 1) Ligands other than fibronectin such as concanavalin A (ConA) and serum spreading factor, when coated on latex beads, were found to promote phagocytosis of the beads. This indicates that fibroblast phagocytosis, like spreading, is a ligand-receptor mediated phenomenon not specifically requiring fibronectin (pFN); 2) Anti-pFN antibodies were found to inhibit the ability of cells to ingest pFN-coated beads that previously were bound on the cell surfaces. Consequently, binding of beads to the cell surfaces per se is not a sufficient signal to promote ingestion of the beads; 3) Finally, divalent cations protected receptor function necessary for phagocytosis of pFN-coated beads from proteolysis by trypsin, as previously was found for receptors involved in cell attachment and spreading on pFN-coated culture dishes. Recovery experiments carried out with cells whose surface receptors had been destroyed indicated that there was an internal (or cryptic cell surface) pool of receptors that amounted to at least 50% of the receptors normally found on the cell surface. After complete destruction of the cell surface and cryptic pools of receptors, reappearance of receptors required for bead binding and phagocytosis required several hours and did not occur in the absence of new protein synthesis.     

Disappearance of macrophage surface folds after antibody-dependent phagocytosis

We have employed the method of Burwen and Satir (J. Cell Biol., 1977, 74:690) to measure the disappearance of surface folds from resident guinea pig peritoneal macrophages after antibody-dependent phagocytosis. Unilamellar phospholipid vesicles containing dimyristoylphosphatidylcholine and 1 mol % dinitrophenyl-epsilon- aminocaproyl-phosphatidylethanolamine, a lipid that possesses a hapten headgroup, were prepared by an ether injection technique. These vesicles were taken up by macrophages in a time- and temperature- dependent fashion. Vesicles that contained ferritin trapped in the internal aqueous volume were identified within macrophages by transmission electron microscopy. Scanning electron microscopy has shown that macrophage surface folds decrease dramatically after phagocytosis. The surface fold length (micrometer) per unit smooth sphere surface area (micrometer2) decreases from 1.3 +/- 0.3 micrometer- 1 to 0.53 +/- 0.25 micrometer-1 when cells are incubated in the presence of specific anti-DNP antibody and vesicles at 37 degrees C. No significant effect was observed in the presence of antibody only or vesicles only. Our studies shown that phagocytosis is associated with a loss of cell surface folds and a loss of cell surface area, which is consonant with current views of the endocytic process. On the basis of our uptake data, we estimate that approximately 400 micrometer2 of vesicle surface membrane is internalized. The guinea pig macrophage plasma membrane has a total area of approximately 400 micrometer2 in control studies, whereas the cells have roughly 300 micrometer2 after phagocytosis. These estimates of surface areas include membrane ruffles and changes directly related to changes in cell volume. We suggest that during antibody-dependent phagocytosis a membrane reservoir is made available to the cell surface.     

Different anesthetic sensitivities of skeletal and cardiac isoforms of the Ca-ATPase.

We have previously shown that low levels of the volatile anesthetic halothane activate the Ca-ATPase in skeletal sarcoplasmic reticulum (SR), but inhibit the Ca-ATPase in cardiac SR. In this study, we ask whether the differential inhibition is due to (a) the presence of the regulatory protein phospholamban in cardiac SR, (b) different lipid environments in skeletal and cardiac SR, or (c) the different Ca-ATPase isoforms present in the two tissues. By expressing skeletal (SERCA 1) and cardiac (SERCA 2a) isoforms of the Ca-ATPase in Sf21 insect cell organelles, we found that differential anesthetic effects in skeletal and cardiac SR are due to differential sensitivities of the SERCA 1 and SERCA 2a isoforms to anesthetics. Low levels of halothane inhibit the SERCA 2a isoform of the Ca-ATPase, and have little effect on the SERCA 1 isoform. The biochemical mechanism of halothane inhibition involves stabilization of E2 conformations of the Ca-ATPase, suggesting direct anesthetic interaction with the ATPase. This study establishes a biochemical model for the mechanism of action of an anesthetic on a membrane protein, and should lead to the identification of anesthetic binding sites on the SERCA 1 and SERCA 2a isoforms of the Ca-ATPase.