Progression of HIV in haemophilia

A series of permeability thresholds to Ca2+ metabolites and macromolecules, occurring at different times when cells are attacked by complement, has been established by imaging HeLa cells transiently expressing a recombinant cytosolic fusion protein of firefly luciferase and aequorin (luciferase-aequorin) to measure changes in ATP and cytosolic free Ca2+. Nuclear fluorescence of propidium was used as a measure of permeability to small molecules, and luciferase activity imaged to assess lysis. The rise in cytosolic free Ca2+ observed after C9 attack preceded by at least 60 s both the increase in propidium fluorescence, measured in single cells, and the decrease in ATP monitored by luciferase light emission. These effects were dependent on the concentration of C9. At concentrations of C9 up to 4 micrograms/ml no loss of luciferase-aequorin protein was detected at the end of the experiment. Thus the membrane integrity of the cells remained intact, even though the cells were permeable to propidium. These results confirmed our earlier observations that propidium permeability in cells attacked by complement was not a reliable measure of cell death. They also show that it is vital to take account of cellular heterogeneity if the mechanisms by which cells respond to membrane pore former attack are to be correctly interpreted.     

Colloid determination of fibrin network permeability

The effects of polymers, dextran and polyvinylpyrolidone (PVP) and of albumin on the permeability of thrombin-induced fibrin networks developed in plasma were examined. Both PVP and dextran increased the network permeability and turbidity and increased the fibrin fibre thickness. The effect was molecular weight dependent. Derivation of the dimensionless permeability (permeability/fibre radius2) indicated that the increase in network permeability was mainly from altered arrangement of fibres and not from increased fibre thickness. The effects of albumin on network structure were similar to those of the polymers. Scanning electron microscopy of networks developed in plasma under the influence of dextran and poloxamer 188 showed fibres with increased thickness and a coarse nodular appearance. There was an increased tendency for fibres to be aggregated into clumps. It is suggested that during polymerization fibrin fibres and fibrin polymerization intermediaries behave as colloidal particles. Attractive forces between the particles are generated by soluble macromolecules such as plasma proteins or polymers. Attractive forces increase the thickness of fibrin fibres and induce a more permeable arrangement of the fibres in the network. The most likely colloidal mechanism is depletion flocculation. This would account for (1) the molecular weight dependence and concentration dependence of the effects of macromolecules, (2) the effects of macromolecules which do not bind to fibrin, (3) the effects of the surfactant poloxamer 188. Depletion flocculation may be a significant mechanism for biological regulation of fibrin network permeability by non-specific macromolecules such as soluble proteins or fibrin intermediaries.     

Phosphorylation of photolyzed rhodopsin is calcium-insensitive in retina permeabilized by alpha-toxin

Light triggers the phototransduction cascade by activating the visual pigment rhodopsin (Rho --> Rho*). Phosphorylation of Rho* by rhodopsin kinase (RK) is necessary for the fast recovery of sensitivity after intense illumination. Ca2+ ions, acting through Ca2+-binding proteins, have been implicated in the desensitization of phototransduction. One such protein, recoverin, has been proposed to regulate RK activity contributing to adaptation to background illumination in retinal photoreceptor cells. In this report, we describe an in vitro assay system using isolated retinas that is well suited for a variety of biochemical assays, including assessing Ca2+ effects on Rho* phosphorylation. Pieces of bovine retina with intact rod outer segments were treated with pore-forming staphylococcal alpha-toxin, including an alpha-toxin mutant that forms pores whose permeability is modulated by Zn2+. The pores formed through the plasma membranes of rod cells permit the diffusion of small molecules <2 kDa but prevent the loss of proteins, including recoverin (25 kDa). The selective permeability of these pores was confirmed by using the small intracellular tracer N-(2-aminoethyl) biotinamide hydrochloride. Application of [gamma-32P]ATP to alpha-toxin-treated, isolated retina allowed us to monitor and quantify phosphorylation of Rho*. Under various experimental conditions, including low and high [Ca2+]free, the same level of Rho* phosphorylation was measured. No differences were observed between low and high [Ca2+]free conditions, even when rods were loaded with ATP and the pores were closed by Zn2+. These results suggest that under physiological conditions, Rho* phosphorylation is insensitive to regulation by Ca2+ and Ca2+-binding proteins, including recoverin.     

Mouse microglial cells express a plasma membrane pore gated by extracellular ATP

We have investigated responses to extracellular ATP (ATPe) in the microglial cell lines N9 and N13 and in freshly isolated mouse microglial cells. Upon stimulation with this nucleotide, N9 and N13 cells underwent an increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i), a sustained depolarization of the plasma membrane, and an uptake of extracellular markers such as ethidium bromide and lucifer yellow; increases in plasma membrane permeability were paralleled by striking morphologic changes. ATPe, as well as other nucleotides, activated a spiking Ca2+ release from intracellular stores; however, only ATPe was also able to cause a massive transmembrane Ca2+ influx. The ATP analogue 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) triggered a sustained Ca2+ influx accompanied by little release from stores. The ATP derivative oxidized ATP (oATP) strongly inhibited Ca2+ influx, minimally affecting Ca2+ release. From ATPe-sensitive microglial cell lines, we selected several ATPe-resistant clones that showed complete lack of ATPe-mediated plasma membrane permeability changes, although they retained the Ca2+ mobilization response from intracellular stores. ATPe-dependent plasma membrane permeability changes were also greatly reduced in growth-arrested microglial cells. Finally, ATPe triggered IL-1 beta release from wild-type but not ATPe-resistant microglial cells. These results show that microglial cells express at least two purinergic receptor subtypes, metabotropic (P2Y) and ionotropic (P2Z), and that the latter is modulated during cell cycle and coupled to IL-1 beta release.     

Effects of storage and homogenization methods on the hepatic recovery of dextrans determined by size-exclusion chromatography

The effects of storage and homogenization methods on the analytical recovery of dextran macromolecules from rat livers were investigated using a high-performance size-exclusion chromatographic (HPSEC) method. Livers were collected from rats dosed with fluorescein-labeled dextrans with molecular weights of 150 or 70 kD. Subsequently, the livers were subjected to different methods to study the effects of the following parameters on the hepatic recovery of dextrans: storage method (freezing the livers before homogenization or freezing the homogenates); contents of the homogenization buffer (addition of 1% Triton X-100); and sample type (HPSEC analysis of the whole homogenate or the supernatant after centrifugation). It is shown that in the absence of Triton in the homogenization buffer, the hepatic recovery of dextrans is substantially affected by all the factors studied. However, in the presence of 1% Triton in the buffer, the hepatic recoveries were maximal and independent of the storage method or sample type. These studies suggest that for optimal recovery of dextran macromolecules from the liver, a sample preparation method capable of disrupting the subcellular membranes should be used.     

Availability of amino acids from casein proteins subjected to heating under model conditions

Localization of a radiopharmaceutical agent in a "tumor" is best conceptualized in terms of the altered regional physiology attendant to the presence of the "tumor". Such localization should be expected to occur in association with other disease states characterized by similar altered regional physiology. Neoplasms, areas of inflammation, and certain phases of infarct development are characterized by increased permeability of their capillary beds to macromolecules. This is largely due to neovascularization and the large intercapillary pores associated with new growth of capillary beds in these circumstances. Often, total perfusion to such lesions is increased in comparison to surrounding normal tissue. Thus, in all three clinical conditions, the entry of macro-molecules into the interstitial fluid space from the intravascular space is increased above that seen in normal tissue. Moreover, with neoplasms and inflammatory processes, there may be a delay in new lymphatic vessel growth adding to the residence time of the macromolecules in the interstitial fluid space. In all three conditions, the increased macrophage activity associated with tissue necrosis may result in ingestion of the labeled macromolecule by the macrophage. Pinocytosis may result in ingestion of the labeled macromolecule by other cells in the lesion, or there may be specific receptor sites on the cell membrane for the macromolecule, which may lead to fixation of the labeled macromolecule on the cell surface and possible intracellular translocation of the label itself. Radiolabeled macromolecules such as albumin, fibrinogen, or gamma globulins, and radionuclides that bind to macromolecules such as radiogallium, radioindium, and other radioelements, exhibit localizing behavior in tumors, inflammatory lesions, and during certain stages of infarcts. In the case of radiogallium and radioindium, the binding macromolecule is transferrin, and it is known that some cells have specific receptor sites for transferrin-bound iron on the cell membrane. It is possible that certain cells within these lesions have cell membrane receptor sites for radiogallium- and radioindium-labeled transferrin, and the cell erroneously accepts these radioelements from the transferrin in lieu of iron in attempting to engage in heme enzyme synthesis. Another mechanism that may be operative in the localization of agents in neoplasms, infarcts, and inflammatory lesions may be the altered cell permeability found in many cells of such lesions. It is known that many agents, such as supravital dyes, are excluded from entering normal cells by the selective permeability of normal cell membranes. When cell membrane permeability is altered, such as can be seen in traumatized, dying, or dead cells, the normally excluded agent may penetrate the abnormal cell membrane and bind, and consequently accumulate in intracellular constituents.     

Excimer laser treatment of hyperopia

In a previous study we demonstrated that human vaginal mucosa was as permeable to water as was buccal mucosa. Water, however, is a very small molecule with a molecular weight of 18 d. To further explore similarities between these two types of mucosa with respect to permeability, it was decided to investigate the passage of two large, hydrophilic molecules across these epithelia. Specimens of fresh, clinically healthy human vaginal and buccal mucosa were taken from excised tissue obtained during vaginal hysterectomies and various oral surgical procedures. Seven biopsy materials from each specimen were mounted in flow-through diffusion cells (exposed area, 0.039 cm2), and their permeability to 4.4- and 12-kd fluorescein-isothiocyanate-labeled dextrans was determined through use of a continuous flow-through perfusion system. Dextran was detected by means of a fluorospectrophotometric method at excitation and emission wave lengths of 498 and 520 nm, respectively. Specimens were examined histologically before and after permeability experiments, and similarities between vaginal and buccal tissues were verified. No statistically significant differences between the flux values of the 4.4-kd dextran across vaginal and buccal mucosa were found. However, for the 12-kd dextran the flux rate across buccal mucosa was significantly higher than the rate across vaginal mucosa. These results demonstrate that human vaginal mucosa is for practical purposes as permeable as buccal mucosa to 4.4-kd hydrophilic molecules. This further supports the hypothesis that vaginal mucosa may be a useful model for studying the passage across buccal mucosa of chemical compounds and therapeutic agents that are less than approximately 4.4 kd in molecular mass. For a 12-kd dextran the flux rate across buccal mucosa is significantly higher than the flux rate across vaginal mucosa, and the model becomes inaccurate.     

Mathematical modelling of cancer invasion and metastasis: an interdisciplinary workshop held at the University of Warwick, Coventry, UK. 11-13 September 1996

A microneedle puncture of the fibroblast or sea urchin egg surface rapidly evokes a localized exocytotic reaction that may be required for the rapid resealing that follows this breach in plasma membrane integrity (Steinhardt, R.A,. G. Bi, and J.M. Alderton. 1994. Science (Wash. DC). 263:390-393). How this exocytotic reaction facilitates the resealing process is unknown. We found that starfish oocytes and sea urchin eggs rapidly reseal much larger disruptions than those produced with a microneedle. When an approximately 40 by 10 microm surface patch was torn off, entry of fluorescein stachyose (FS; 1, 000 mol wt) or fluorescein dextran (FDx; 10,000 mol wt) from extracellular sea water (SW) was not detected by confocal microscopy. Moreover, only a brief (approximately 5-10 s) rise in cytosolic Ca2+ was detected at the wound site. Several lines of evidence indicate that intracellular membranes are the primary source of the membrane recruited for this massive resealing event. When we injected FS-containing SW deep into the cells, a vesicle formed immediately, entrapping within its confines most of the FS. DiI staining and EM confirmed that the barrier delimiting injected SW was a membrane bilayer. The threshold for vesicle formation was approximately 3 mM Ca2+ (SW is approximately 10 mM Ca2+). The capacity of intracellular membranes for sealing off SW was further demonstrated by extruding egg cytoplasm from a micropipet into SW. A boundary immediately formed around such cytoplasm, entrapping FDx or FS dissolved in it. This entrapment did not occur in Ca2+ -free SW (CFSW). When egg cytoplasm stratified by centrifugation was exposed to SW, only the yolk platelet-rich domain formed a membrane, suggesting that the yolk platelet is a critical element in this response and that the ER is not required. We propose that plasma membrane disruption evokes Ca2+ regulated vesicle-vesicle (including endocytic compartments but possibly excluding ER) fusion reactions. The function in resealing of this cytoplasmic fusion reaction is to form a replacement bilayer patch. This patch is added to the discontinuous surface bilayer by exocytotic fusion events.     

Molecular size-dependent leakage of intracellular molecules from frog skeletal muscle fibers permeabilized with beta-escin

The permeability of beta-escin-treated cell membrane was characterized in terms of the permeant molecular size, by monitoring the leak of cytoplasmic molecules in frog skeletal muscle fibers. With a low concentration of beta-escin (5 microM), most of the cellular ATP was lost within 30-40 min (as revealed by rigor force generation), whereas a fluorescence-labeled dextran injected into the cytoplasm (approximately 10 kDa) and cytoplasmic proteins (14-80 kDa) slowly leaked out of the cell. A high concentration of beta-escin (50-100 microM) accelerated the leak of large molecules. Therefore, low concentrations of beta-escin may be used as a means of permeabilizing the cell membrane to relatively small molecules, while retaining a major fraction of the cellular macromolecules.     

Permeability enhancement in Caco-2 cell monolayers by sodium salicylate and sodium taurodihydrofusidate: assessment of effect-reversibility and imaging of transepithelial transport routes by confocal laser scanning microscopy

The effects of sodium salicylate and sodium tauro-24,25-dihydrofusidate (STDHF) on the aqueous permeability of confluent monolayers of Caco-2 cells were studied. Measurements of transepithelial electrical resistance (TEER) showed a concentration-dependent effect of both compounds after apical incubation for 1 hr. Reductions in TEER resulting from EC50 concentrations (2.8 mM for STDHF; 173 mM for salicylate) were reversible within 5.75 hr. The transpithelial fluxes of two hydrophilic model compounds, sodium fluorescein F (molecular weight 376) and a fluorescein isothiocyanate-labeled dextran (mean molecular weight 4000) was significantly increased by STDHF (2.8 mM). Sodium salicylate (173 mM) only enhanced the transport of sodium fluorescein significantly. At the EC50 concentrations, confocal laser scanning microscopy (CLSM) visualized both fluorescent tracers mainly in the paracellular route. With higher enhancer concentrations (373 mM sodium salicylate and 8 mM STDHF), both transport markers appeared intracellularly as a result of cell death. STDHF rapidly extracted an exogenous lipophilic membrane probe, 5-(N-hexadecanoyl)aminofluorescein (HEDAF), from the apical part of Caco-2 plasma membranes, indicating qualitatively that STDHF interacts with the lipid portion of cell membranes. These results suggest that both sodium salicylate and STDHF can be used to reversibly increase paracellular permeability of Caco-2 cell monolayers, whereby STDHF appears to be advantageous compared to sodium salicylate. By adapting the Costar cell culture system to CLSM, we have shown that this technique is suitable to study membrane interactions qualitatively and for visualizing transport routes of hydrophilic tracers through nonfixed, filter-grown monolayers.     

Regulation of rabbit erythrocyte Ca(2+)-pump sensitivity to calmodulin in experimental hyperlipidemia

Intracellular free calcium activity is in part determined by a calmodulin-regulated plasma membrane Ca(2+)-pump. Since changes in Ca2+ permeability have been implicated in atherosclerotic plaque formation, we initiated a lipid hyperalimentation protocol during which we measured various erythrocyte calcium flux parameters and early atheroma development. Adolescent New Zealand White rabbits were fed a diet with 0.5% cholesterol and 2.5% lard over a 3-month period. Plasma cholesterol and triacylglycerols increased on average 18.7- and 13.9-fold respectively, while erythrocyte membrane cholesterol content decreased 18% and total phospholipids by 54%. After 3 months of lipid hyperalimentation, 22% of the aortic arch was covered with large, early-stage, raised atheroma. Basal and calmodulin-activated (Ca2+ + Mg2+)-ATPase activities in erythrocyte membranes increased by 31% and 123%, respectively at 2 months, with a concomitant increase in calmodulin affinity (Km) from 15.6 to 4.2 nM. These differences were transient on account of changes in the control animals which exhibited a slowly developing sensitivity to calmodulin during maturation. Basal Ca2+ transport and passive Ca2+ permeability increased about 7-fold during the hyperlipidemic phase. This suggests that overt hyperlipidemia, leading to atherosclerotic plaque development, alters plasma membrane Ca2+ regulatory mechanisms including passive Ca2+ permeability. The changes in enzymatic function, membrane composition, and Ca2+ permeability seen in this red cell model system may be a reflection of early changes in cells that are directly involved in the development of atherosclerotic plaques.     

Irreversible inhibition of plasma membrane (Ca2+ + Mg2+)-ATPase and Ca2+ transport by 4-OH-2,3-trans-nonenal

4-OH-2,3-trans-nonenal (HNE), a major aldehydic lipid peroxidation product, has been shown to cause cellular toxicities and has been linked to a number of pathophysiological processes including atherogenesis. Specifically, in vitro exposure of erythrocyte plasma membrane preparations to HNE resulted in the inhibition of membrane transport function and integrity. To characterize the nature of the inhibitory effects of HNE on plasma membrane regulatory mechanisms, we investigated its effects on substrate and calmodulin (CaM) stimulation on erythrocyte Ca2+ transport and (Ca2+ + Mg2+)-ATPase activities. Concentration-effect relationship analysis in erythrocyte membrane "ghosts" and inside-out vesicles (IOVs) yielded purely noncompetitive kinetics for Ca2+, ATP, and CaM activation of (Ca2+ + Mg2+)-ATPase and Ca2+ transport. Reductions of Vmax from direct addition of 0.1 mM HNE to the assay incubation mixtures ranged from 23 to 41%. Similarly, pretreatment with HNE of both membrane ghosts and IOVs resulted in a concentration-dependent inactivation of ATPase and transport activities without changes in affinity for Ca2+, ATP, or CaM. Conversely, pretreatment of CaM itself did not impair its ability to stimulate (Ca2+ + Mg2+)-ATPase activity threefold. Moreover, HNE-pretreated membranes exhibited unaltered acetylcholinesterase activity compared to sham-pretreated membranes. Together, these results suggest that HNE may structurally, and thus irreversibly, modify one or more functionally important sites on the transport protein itself.     

SNAP-25 is required for a late postdocking step in Ca2+-dependent exocytosis

The Ca2+-activated fusion of large dense core vesicles (LDCVs) with the plasma membrane is reconstituted in mechanically permeabilized PC12 cells by provision of millimolar MgATP and cytosolic proteins. Ca2+-activated LDCV exocytosis was inhibited completely by the type E but not the type A botulinum neurotoxin (BoNT) even though both BoNTs were equally effective in proteolytically cleaving the synaptosome-associated protein of 25 kDa (SNAP-25). The greater inhibition of exocytosis by BoNT E correlated with a greater destabilization of detergent-extracted complexes consisting of SNAP-25, synaptobrevin, and syntaxin. LDCVs in permeable PC12 cells can be poised at a late postdocking, prefusion state by MgATP-dependent priming processes catalyzed by N-ethylmaleimide sensitive factor and priming in exocytosis proteins. BoNT E completely blocked Ca2+-activated LDCV exocytosis in ATP-primed cells, whereas BoNT A was only slightly inhibitory, implying that the C-terminal region of SNAP-25 (Ile181-Gln197) between the cleavage sites for BoNT E and BoNT A is essential for late postdocking steps. A required role for SNAP-25 at this stage was also indicated by inhibition of Ca2+-activated LDCV fusion in ATP-primed cells by a C-terminal peptide antibody. We conclude that plasma membrane SNAP-25, particularly residues 181-197, is required for Ca2+-regulated membrane fusion at a step beyond LDCV docking and ATP utilization.     

Localization of a brain protein metabolically linked with behavioral plasticity in the goldfish

Isotonic suspensions of human erythrocytes were exposed to single electric pulses of intensity at a few kV/cm and duration in microseconds. Upon pulsation, the cell membranes became permeable to Na+ and K+, and the erythrocytes eventually hemolysed through the colloid osmotic effect of hemoglobin. The enhanced permeability is attributed to the formation of pores in the cell membranes. These pores are formed within a fraction of a microsecond, once the transmembrane potential induced by the applied electric field reaches a critical value of 1.0 V. Increased field intensity and pulse duration, or pulsation at low ionic strengths all expand the pore size, leading to an accelerated hemolysis reaction. In contrast to this expansion process, the initial step of pore formatin is governed solely by the magnitude of the transmembrane potential: the critical value of the potential stays essentially constant in media of different ionic strengths, nor does it change appreciably with varying pulse duration. An abrupt increase in membrane permeability at a transmembrane potential adround 1 V has been observed in many cellular systems. It is suggested that a similar mechanism of pore formation may apply to these systems as well.     

Resting cytoplasmic free Ca2+ concentration in frog skeletal muscle measured with fura-2 conjugated to high molecular weight dextran

Intact frog skeletal muscle fibers were injected with the Ca2+ indicator fura-2 conjugated to high molecular weight dextran (fura dextran, MW approximately 10,000; dissociation constant for Ca2+, 0.52 microM), and the fluorescence was measured from cytoplasm (17 degrees C). The fluorescence excitation spectrum of fura dextran measured in resting fibers was slightly red-shifted compared with the spectrum of the Ca(2+)-free indicator in buffer solutions. A simple comparison of the spectra in the cytoplasm and the in vitro solutions indicates an apparently "negative" cytoplasmic [Ca2+], which probably reflects an alteration of the indicator properties in the cytoplasm. To calibrate the indicator's fluorescence signal in terms of cytoplasmic [Ca2+], we applied beta-escin to permeabilize the cell membrane of the fibers injected with fura dextran. After treatment with 5 microM beta-escin for 30-35 min, the cell membrane was permeable to small molecules (e.g., Ca2+, ATP), whereas the 10-kD fura dextran only slowly leaked out of the fiber. It was thus possible to estimate calibration parameters in the indicator fluorescence in the fibers by changing the bathing solution [Ca2+] to various levels; the average values for the fraction of Ca(2+)-bound indicator in the resting fibers and the dissociation constant for Ca2+ (KD) were, respectively, 0.052 and 1.0 microM. For the comparison, the KD value was also estimated by a kinetic analysis of the indicator fluorescence change after an action potential stimulation in intact muscle fibers, and the average value was 2.5 microM. From these values estimated in the fibers, resting cytoplasmic [Ca2+] in frog skeletal muscle fibers was calculated to be 0.06-0.14 microM. The range lies between the high estimates from other tetracarboxylate indicators (0.1-0.3 microM; Kurebayashi, N., A. B. Harkins, and S. M. Baylor. 1993. Biophysical Journal. 64:1934-1960; Harkins, A. B., N. Kurebayashi, and S. M. Baylor. 1993. Biophysical Journal. 65:865-881) and the low estimate from the simultaneous use of aequorin and Ca(2+)-sensitive microelectrodes (< 0.04-0.06 microM; Blatter, L. A., and J. R. Blinks. 1991. Journal of General Physiology. 98:1141-1160) recently reported for resting cytoplasmic [Ca2+] in frog muscle fibers.     

Role of intracellular calcium in fast and slow desensitization of P2-receptors in PC12 cells

1. Combined whole-cell patch clamp recording and confocal laser scanning microscopy of [Ca2+]i transients were performed on single PC12 cells to study any correlation between membrane currents induced by ATP and elevation in [Ca2+]i. ATP was applied by pressure from micropipettes near the recorded PC12 cells continuously superfused at a fast rate. 2. Brief (20 ms) pulses of ATP elicited monophasic inward currents and [Ca2+]i increases. Long applications (2 s) of ATP (5 mM) evoked peak currents which rapidly faded during the pulse and were followed by a large rebound current, interpreted as due to rapid desensitization and recovery of P2-receptors. The associated [Ca2+]i increase grew monotonically to a peak reached only after the occurrence of the current rebound, indicating that it is unlikely this cation has a role in fast desensitization. 3. Both membrane currents and [Ca2+]i transients were linearly dependent on holding membrane potential, suggesting that Ca2+ influx is the predominant cause of [Ca2+]i elevation. This view was supported by experiments carried out in Ca(2+)-free solution. 4. Brief pulses of ATP applied after a desensitizing pulse (2 s) of the same elicited smaller inward currents and [Ca2+]i rises indicating a role for [Ca2+]i in controlling slow desensitization of P2-receptors. 5. This notion was confirmed in experiments with various [Ca2+]i chelators which differentially affected slow desensitization in relation to their buffering capacity, while sparing fast receptor desensitization. 6. These results suggest a role for [Ca2+]i in slow rather than fast desensitization of P2-receptors, thus proposing this divalent cation as an intracellular factor able to provide an efficient and reversible control over receptor activity induced by ATP.     

Mechanical stress induces release of ATP from Ehrlich ascites tumor cells

The supernatant from a suspension of Ehrlich cells exposed to centrifugation at 700xg for 45 s induced a transient increase in the intracellular concentration of free, cytosolic Ca2+, [Ca2+]i, as well as activation of an outwardly rectifying whole-cell current when added to a suspension of non-stimulated cells. These effects were inhibited by suramin, a non-specific P2 receptor antagonist, and mimicked by ATP. Reversed phase HPLC analysis revealed that the supernatant from Ehrlich cells exposed to centrifugation contained 2. 6+/-0.2 microM ATP, and that the mechanical stress-induced release of ATP was inhibited by glibenclamide and verapamil, non-specific inhibitors of the cystic fibrosis transmembrane conductance regulator and P-glycoprotein, respectively. After trypan blue staining, less than 0.5% of the cells were unable to extrude the dye. Addition of extracellular ATP induced a suramin-sensitive, transient, concentration-dependent increase in [Ca2+]i, activation of an outwardly rectifying whole-cell current and a hyperpolarization of the plasma membrane. The ATP-induced hyperpolarization of the plasma membrane was strongly inhibited in the presence of charybdotoxin (ChTX), an inhibitor of several Ca2+-activated K+ channels, suggesting that stimulation of P2 receptors in Ehrlich cells evokes a Ca2+-activated K+ current. The relative potencies of several nucleotides (ATP, UTP, ADP, 2-MeSATP, alpha,beta-MeATP, bzATP) in eliciting an increase in [Ca2+]i, as well as the effect of repetitive addition of nucleotides were investigated. The results lead us to conclude that mechanical stimulation of Ehrlich cells leads to release of ATP, which in turn stimulates both P2Y1 and P2Y2 receptors, resulting in Ca2+ influx as well as release and activation of an outwardly rectifying whole-cell current.     

Myristoylation of hippocalcin is linked to its calcium-dependent membrane association properties

Hippocalcin, a recently identified Ca(2+)-binding protein of the recoverin family exclusively expressed in the hippocampus, has a primary structure containing three putative Ca(2+)-binding sites (EF-hands) and a possible NH2-terminal myristoylation site. 45Ca blots demonstrated that every three EF-hand domains, expressed as fusion proteins in Escherichia coli, bind Ca2+, indicating that hippocalcin binds 3 mol of Ca2+/mol of protein. To determine whether hippocalcin is myristoylated, hippocalcin mRNA was translated in vitro in the presence of [3H]myristic acid. 3H label was resistant to hydroxylamine treatment, and replacement of NH2-terminal glycine with alanine prevented 3H label incorporation, indicating that in vitro translated hippocalcin covalently bound [3H]myristic acid at the NH2-terminal glycine. In vitro translated hippocalcin is quantitatively myristoylated, as evidenced by an electrophoretic mobility shift of [35S]methionine-labeled protein on two-dimensional gels. Native hippocalcin comigrated precisely with the in vitro translated hippocalcin on two-dimensional gels, suggesting that native hippocalcin is myristoylated. Native and in vitro translated hippocalcins, but not non-myristoylated mutagenic (Gly1-Ala1) hippocalcin, displayed Ca(2+)-dependent membrane association, indicating that myristoylation participates in its Ca(2+)-dependent membrane association properties. In vitro translated hippocalcin bound to phospholipid vesicles somewhat, however, phospholipid association was insufficient for its membrane association properties, suggesting that the NH2-terminal myristoyl moiety on hippocalcin interacts with lipid bilayers and facilitates interaction with other membrane proteins.     

Receptors for the 5-oxo class of eicosanoids in neutrophils

5-Hydroxy- and 5-oxo-eicosatetraenoate (5-HETE and 5-oxoETE) activate polymorphonuclear neutrophils (PMNs) through a common, receptor-like recognition system. To define this system, we examined the interaction of these eicosanoids with human PMNs. PMNs esterified 5-[3H]HETE to glycerolipids at 37 and 4 degreesC. At 37 but not 4 degreesC, the cells also hydroxylated the label to 5, 20-[3H]diHETE. The acyl:CoA synthetase blocker, triacsin C, inhibited esterification but also led to an increase in the hydroxylation of the label. PMNs processed 5-[3H]oxoETE through the same pathways but only or principally after reducing it to 5-[3H]HETE (37 or 4 degreesC). In the presence of these varying metabolic reactions, PMNs (37 or 4 degreesC; +/- triacsin C) could not be shown to receptor bind either radiolabel. Plasma membranes isolated from PMNs esterified but unlike whole cells did not reduce or hydroxylate 5-[3H]oxoETE. Triacsin C blocked esterification, thereby rendering the membranes unable to metabolize this radiolabel. Indeed, triacsin C-treated membranes bound (Kd = 3.8 nM) 5-[3H]oxoETE specifically and reversibly to 86 pmol of sites per 25 micrograms of membrane protein. 5-OxoETE, 5-HETE, and 5,15-diHETE displaced this binding at concentrations correlating with their potency in eliciting PMN Ca2+ transients. GTP and GTPgammaS, but not ATP or ATPgammaS, also reduced 5-[3H]oxoETE binding, whereas 15-HETE, leukotriene B4, platelet-activating factor, IL-8, C5a, and N-formyl-Met-Leu-Phe lacked this effect. We conclude that PMNs and their plasma membranes use an acyl:CoA synthetase-dependent route to esterify 5-HETE and 5-oxoETE into lipids. Blockade of the synthetase uncovers cryptic plasmalemma sites that bind 5-oxoETE with exquisite specificity. These sites apparently mediate responses to the 5-oxo class of eicosanoids and are likely members of the serpentine superfamily of G protein-linked receptors.     

Kinetic uniformity of the ATP hydrolysis reaction catalyzed by Mg2+-ATPase in smooth muscle cell membrane

The kinetic properties of Mg(2+)-ATPase (EC 3.6.1.3) from myometrium cell plasma membranes have been studied. Under conditions of enzyme saturation with ATP (0.5-1.0 mM) or Mg2+ (1.0-5.0 mM) the initial maximal rates of the Mg(2+)-dependent enzymatic ATP hydrolysis, V0 ATP and V0 Mg, are 27.4 +/- 3.3 and 25.2 +/- 4.1 mumol Pi/hour/mg of protein, respectively. The apparent Michaelis constant, Km, for ATP and of the apparent activation constant, K alpha, for Mg2+ are equal to 28.1 +/- 2.6 and 107.0 +/- 26.0 microM, respectively. The bivalent metal ions used at 1.0 mM suppress the Mg(2+)-dependent hydrolysis of ATP whose efficiency decreases in the following order: Cu2+ > Zn2+ = Ni2+ > Mn2+ > Ca2+ > Co2+. Alkalinization of the incubation medium from pH 6.0 to pH 8.0 stimulates the Mg(2+)-dependent hydrolysis of ATP. It has been found that Mg(2+)-ATPase has the properties of an H(+)-sensitive enzymatic sensor which is characterized by a linear dependence between the initial maximal rate of the reaction, V0, and the pH value. The feasible role of plasma membrane Mg(2+)-ATPase in some reactions responsible for the control of proton and Ca2+ homeostasis in myometrium cells has been investigated.