Depolarization-induced calcium release from sarcoplasmic reticulum fragments. II. Release of calcium incorporated with ATP.

Ca2& incorporated in vesicles of sarcoplasmic reticulum fragments (SRF) by diffusion could be released rapidly by changing the ionic environment, by dilution from methanesulfonate (MS) to chloride. This ion exchange is considered to make the membrane potential of SRF inside-negative. Much faster release of Ca2t was also observed upon osmotic change from high to low. These responses were very similar to the Ca2& release from SRF after take up using ATP, but the release rate was slow in the case of anion exchnage. The behavior of K&, Na&, sucrose, and inulin incorporated in SRF was followed upon similar treatment. These ions and nolecules were not released upon ion exchange, but were immediately released by osomtic treatment. Therefore, the Ca& release upon anion exchange was not due to the bursting of SRF, but to a direct effect such as a membrane potential change of the SRF. The behavior of anion such as C1- and propionate could not followed by the same method because of the large permability of these anions. It was also shown that Ca& release upon ion exchange was not a direct effect of pH change. Liver microsomes did not show Ca& release upon the same treatment as SRF.     

Characterization of the Release of Cholecystokinin-8 from Isolated Nerve Terminals and Comparison with Exocytosis of Classical Transmitters

In the present study, the release of the neuropeptide cholecystokinin-8 (CCK) from purified nerve terminals (synaptosomes) of the rat hippocampus was characterized with respect to the subcellular distribution, the release upon addition of various agents, the release kinetics, the Ca2+ and ATP dependence of release, and the relationship between CCK release and elevations of intraterminal free Ca2+ concentration ([Ca]i). These characteristics were compared with those for the release of classical transmitters in similar preparations. CCK-like immunoreactivity (CCK-LI) is enriched in the purified synaptosomal fraction of hippocampus homogenates and released in a strictly Ca2(+)-dependent manner upon chemical depolarization, addition of 4-aminopyridine, or stimulation with the Ca2+ ionophore ionomycin. The presence of Ca2+ in the medium significantly stimulates the basal efflux of CCK-LI from synaptosomes. The release upon stimulation develops gradually in time with no significant release in the first 10 s and levels off after 3 min of depolarization. At this time, a large amount of CCK-LI is still present inside the synaptosomes. A correlation exists between the release of CCK-LI and the elevations of [Ca]i. The release of CCK-LI is decreased, but not blocked, upon ATP depletion. These characteristics markedly differ from those for classical transmitters, which show a fast component of Ca2(+)-dependent (exocytotic) release, an absolute dependence on cellular ATP, and no marked stimulation of basal efflux in the presence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton release due to manganese binding and oxidation in modified bacterial reaction centers

The pH dependence of binding and oxidation of Mn2+ in highly oxidizing reaction centers with designed metal-binding sites was characterized by light-minus-dark optical difference spectroscopy and direct measurements of proton uptake/release. These mutants bind a Mn2+ ion that can efficiently transfer an electron to the oxidized bacteriochlorophyll dimer, as described earlier [Thielges et al. (2005) Biochemistry 44, 7389-7394]. The dissociation constant, KD, significantly increased with decreasing pH. The pH dependence of KD between pH 7 and pH 8 was consistent with the binding of Mn2+ being stabilized by the electrostatic release of two protons. The strong pH dependence of proton release upon Mn2+ binding, with a maximal release of 1.4 H+ per reaction center, was interpreted as being a result of a shift in the pKa values of the coordinating residues and possibly other nearby residues. A small amount of proton release associated with Mn2+ oxidation was observed upon illumination. These results show that functional metal-binding sites can be incorporated into proteins upon consideration of both the metal coordination and protonation states of the ligands.     

Immunomagnetic Particle Induced Lysis of Antibody-Conjugated Liposomes

Abstract

We have prepared liposomes of dioleoylphosphatidylethanolamine (DOPE) which have been stabilized by addition of 9-12 mol% N-biotinyl- phosphatidylethanolamine. Liposomes composed of DOPE/N-biotinyl-PE are quite stable and non-leaky although they exhibit strong temperature-dependent leakage following incorporation of palmitylated murine monoclonal antibodies as a targeting ligand. Addition of magnetic chromium dioxide particles coated with anti-mouse antibody to these immunoliposomes lead to their aggregation and the release of entrapped calcein. The lytic event was biphasic with an initial rapid release of 20% dye within 5 min. followed by a slower rate which reached nearly 40% release after 80 min. The rapid release phase was dependent upon the concentration of the liposomes and that of the multivalent particles. Lysis was immunospecific since no release was observed upon addition of nonspecific immunomagnetic particles to the immunoliposomes or if no antibody was incorporated into the liposome. Lysis could also be blocked by the addition of free murine antibody to the solution. The ability of these liposomes to release their contents in response to binding a multivalent antigen validates their potential for therapeutic or diagnostic applications.     

Suppression of the release of prostaglandin-like substances by hydrocortisone in vivo

Muscular exercise of the dog's hind leg evokes the release of prostaglandin-like substances / PG-like substances/ into femoral venous blood. The release of PG-like substances detected by the bioassay method was significantly greater in adrenalectomized as compared to normal dogs. To test the possibility that this difference may be related to the deficiency of adrenocortical secretion in adrenalectomized dogs, the effect of hydrocortisone / HC / and aldosterone / AS / upon the release of PG-like substances induced by muscular work of the dog's hind leg was investigated. The doses of HC and AS infused intravenously or intraarterially were close to the range of physiological secretion rate of these hormones. HC suppressed the release of PG-like material by 30 to 60%, whereas AS had no effect upon the rate and duration of the release. The rate of removal of exogenous PGE₂ in the hind limb circulation was not influenced by HC, suggesting that the diminution of PG release by HC results from the suppression of PG generation rather than from the enhancement of degradation. It is suggested that inhibitory effect of HC upon the rate of the release of PG-like substances may be related to the membrane-stabilizing properties of this hormone. The difference in the intensity of the release of PG-like substances between normal and adrenalectomized dogs suggests that, at least in some conditions, the release of endogenous PGs from tissues may be influenced by the state of adrenocortical activity.     

Defence by plant toxins in milkweed bugs (Heteroptera: Lygaeinae) through the evolution of a sophisticated storage compartment

Species of the heteropteran subfamily Lygaeinae possess special subcuticular compartments to store cardiac glycosides, plant‐derived defensive compounds, which they release upon predator attack. In adults of the large milkweed bug, Oncopeltus fasciatus, these storage compartments have previously been described as a modified integument, forming a fluid‐filled dorsolateral space. Here we use three‐dimensional imaging of serial histological sections and synchrotron radiation‐based micro‐computed tomography data to reveal the morphology of these storage compartments and the mechanisms used for the release of a cardiac glycoside‐rich fluid upon attack. Our comparative analysis revealed that the morphology and release mechanism vary among the species investigated. By reconstructing these traits on a recent molecular phylogeny of the Lygaeinae, we demonstrate that the adaptations for the storage and release of cardiac glycosides have evolved in a stepwise manner.     

Adenosine 3'',5''-cyclic monophosphate-dependent release of prolactin from GH3 pituitary tumour cells. A quantitative analysis.

The involvement of cyclic AMP in mediating regulatory peptide-controlled prolactin release from GH3 pituitary tumour cells was investigated. Cholera toxin and forskolin elicited concentration-dependent increases in both GH3 cell cyclic AMP content and prolactin release. The maximum rise in prolactin release with these agents was 2-fold over basal. 8-Bromo-cyclic AMP produced a similar stimulation of prolactin release. The phosphodiesterase inhibitor isobutylmethylxanthine also produced an increase in prolactin release and GH3 cell cyclic AMP content. However, the magnitude of the stimulated prolactin release exceeded that obtained with any other agent. Thyrotropin-releasing hormone (thyroliberin) and vasoactive intestinal polypeptide produced a concentration-dependent rise in both cell cyclic AMP content and prolactin release. However, only vasoactive intestinal polypeptide elicited an increase in cell cyclic AMP content at concentrations relevant to the stimulation of prolactin release. Vasoactive intestinal polypeptide and thyrotropin-releasing hormone, when used in combination, were additive with respect to prolactin release. Vasoactive intestinal polypeptide and forskolin, at concentrations that were maximal upon prolactin release, were, when used in combination, synergistic upon GH3 cell cyclic AMP content but were not additive upon prolactin release. In conclusion the evidence supports a role for cyclic AMP in the mediation of vasoactive intestinal polypeptide- but not thyrotropin-releasing hormone-stimulated prolactin release from GH3 cells. A quantitative analysis indicates that a 50-100% rise in cyclic AMP suffices to stimulate cyclic AMP-dependent prolactin release fully.     

The effect of diphenylhydantoin upon degradation of sulphated macromolecules in cat palatal mucosa in vitro

The effect of diphenylhydantioin (DPH) upon the degradation of in vivo [35S]-sulphate-labelled proteoglycans was studied in cat palatal mucosa during organ culture. 8-week-old cats were injected intraperitoneally with [35S]-sulphate and 24 hours later the palatal mucosa was taken to organ culture. The release of radioactivity into the culture medium was taken as a measure of degradation of sulphated macromolecules, presumably proteoglycans, and the release of hydroxyproline as an indicator for collagen degradation. A parallel decrease in the release of radioactivity and in the release of hydroxyproline was observed when the culture was done in the presence of DPH (20 mg/l). Chromatography of the culture medium upon Sephadex G-25 revealed that the reduced release of radioactivity was due to a reduction of macromolecular degradation products leaving the amounts of free sulphate in the medium unchanged. The results were interpreted using a two compartment theory for proteoglycan degradation, extracellular breakdown of the protein core resulting in the production of macromolecular degradation products and intracellular lysosomal degradation resulting in free sulphate as the identifiable product. The results indicate that DPH inhibited the extracellular enzymatic degradation of proteoglycans without influencing their intracellular degradation.     

Superoxide generation of phagocytes and nonphagocytic cells

Phagocytes release high amounts of reactive oxygen intermediates upon contact with appropriate stimuli into the environment as an important function in the immune defence against infectious agents. On the other hand nonphagocytic cells release low amounts upon stimulation, which have important functions in the inter- and intracellular signal transduction. Both systems represent a class of isoenzymes making a specific regulation possible. Fibroblasts, hepatocytes, and HeLa cells possess a superoxide system which shows higher activity with NADH than NADPH. The enzyme was purified to homogeneity in an active form, showed a molecular mass of 84 kDa and possessed a flavin and low-potential cytochrome b558.     

Galig, a novel cell death gene that encodes a mitochondrial protein promoting cytochrome c release

Galectin-3 internal gene (Galig) was recently identified as an internal gene transcribed from the second intron of the human galectin-3 gene that is implicated in cell growth, cell differentiation, and cancer development. In this study, we show that galig expression causes morphological alterations in human cells, such as cell shrinkage, cytoplasm vacuolization, nuclei condensation, and ultimately cell death. These alterations were associated with extramitochondrial release of cytochrome c, a known cell death effector. Furthermore, Bcl-xL co-transfection significantly reduced the release of cytochrome c induced by galig expression, suggesting a common pathway between the cytotoxic activity of galig and the anti-apoptotic activity of Bcl-xL. This antagonism was not observed upon co-transfection of Bcl-2 and galig. Galig encodes a mitochondrial-targeted protein named mitogaligin. Structure-activity relationship studies showed that the mitochondrial addressing of mitogaligin relies on an internal sequence that is required and sufficient for the release of cytochrome c and cell death upon cell transfection. Moreover, incubation of isolated mitochondria with peptides derived from mitogaligin induces cytochrome c release. Altogether, these results show that galig is a novel cell death gene encoding mitogaligin, a protein promoting cytochrome c release upon direct interaction with the mitochondria.     

Release and intercellular transfer of cell surface CD81 via microparticles

The human tetraspan molecule CD81 is a coreceptor in B and T cell activation and a candidate receptor for hepatitis C virus infection. We examined the surface expression of CD81 on B and T lymphocytes by quantitative flow cytometry. Upon cellular activation, CD81 surface levels were rapidly reduced. This reduction occurred as early as 1 h after activation and was linked to the release of CD81-positive microparticles into the cell culture medium. CD81 mRNA levels were not affected early after activation, but the release of CD81-positive microparticles was rapidly enhanced. In addition, intercellular transfer of CD81 was observed upon coculture of CD81-positive donor cells (Jurkat T cell line) with CD81-negative acceptor cells (U937 promonocytic cell line). This transfer was rapidly increased upon T cell activation, coinciding with enhanced CD81 release from activated Jurkat cells. We propose that the release and intercellular trafficking of CD81-positive microparticles regulate the expression of CD81 surface receptors in lymphocytes and play a role in the immune response during infections.     

l-Glutamic acid,a neuromodulator of dopaminergic transmission in the rat corpus striatum

A superfusion system was used to study the effects of neuroexcitatory amino acids upon spontaneous and depolarization-evoked release of exogenously taken up and newly synthesized [³H]dopamine by rat striatal slices. Neither l-glutamate nor other aminoacids such as l-aspartate and d-glutamate (5 × 10^?5 M) modified the spontaneous release of exogenous [³H]dopamine from rat striatal slices. In contrast, these neuroexcitatory aminoacids did potentiate spontaneous release of striatal [³H]dopamine newly synthesized from [³H]tyrosine. A different pattern of effects emerged when depolarization-evoked release of dopamine was studied. Only l-glutamate (5 × 10^?6-1 × 10^?4 M) potentiated dopamine release under these experimental conditions in a rather specific and stereoselective manner. In addition, similar results were obtained regardless of whether depolarization-induced release of exogenous or newly synthesized [³H]dopamine was studied. The effect of l-glutamate on depolarization-induced release depended both upon the degree of neuronal depolarization and upon the presence of external Ca²⁺ in the superfusion medium and it was blocked by l-glutamate diethylester. Furthermore, this effect of l-glutamate seemed quite specific with regard to regional localization within the brain as it was only demonstrated in slices from striatum and not in slices from olfactory tubercle or hippocampus. It is suggested that during depolarization a Ca²⁺-dependent event occurs at the striatal membrane level which changes the sensitivity of the dopamine release process to neuroexcitatory aminoacids in such a way as to render it relatively more specific and stereoselective towards l-glutamate stimulation. The findings reported have led us to propose that l-glutamic acid could play a role as a neuromodulator of dopaminergic transmission in the rat corpus striatum.     

Differential effect of glycolytic intermediaries upon cyclic ADP-ribose-, inositol 1',4',5'-trisphosphate-, and nicotinate adenine dinucleotide phosphate-induced Ca(2+) release systems.

We investigated the effect of glycolytic pathway intermediaries upon Ca(2+) release induced by cyclic ADP-ribose (cADPR), inositol 1',4', 5-trisphosphate (IP(3)), and nicotinate adenine dinucleotide phosphate (NAADP) in sea urchin egg homogenate. Fructose 1,6, -diphosphate (FDP), at concentrations up to 8 mM, did not induce Ca(2+) release by itself in sea urchin egg homogenate. However, FDP potentiates Ca(2+) release mediated by agonists of the ryanodine channel, such as ryanodine, caffeine, and palmitoyl-CoA. Furthermore, glucose 6-phosphate had similar effects. FDP also potentiates activation of the ryanodine channel mediated by the endogenous nucleotide cADPR. The half-maximal concentration for cADPR-induced Ca(2+) release was decreased approximately 3.5 times by addition of 4 mM FDP. The reverse was also true: addition of subthreshold concentrations of cADPR sensitized the homogenates to FDP. The Ca(2+) release mediated by FDP in the presence of subthreshold concentrations of cADPR was inhibited by antagonists of the ryanodine channel, such as ruthenium red, and by the cADPR inhibitor 8-Br-cADPR. However, inhibition of Ca(2+) release induced by IP(3) or NAADP had no effect upon Ca(2+) release induced by FDP in the presence of low concentrations of cADPR. Furthermore, FDP had inhibitory effects upon Ca(2+) release induced by both IP(3) and NAADP. We propose that the state of cellular intermediary metabolism may regulate cellular Ca(2+) homeostases by switching preferential effects from one intracellular Ca(2+) release channel to another.     

Modulation of human basophil histamine release by hemopoietic growth factors

To determine what kinds of CSF modulate human basophil function, recombinant or purified hemopoietic growth factors were tested for the effect on histamine release from basophils. Both granulocyte (G)-macrophage (M) CSF and IL-3 markedly enhanced histamine release upon stimulation with anti-IgE in a dose-dependent manner (maximal enhancement 25.5% by GM-CSF and 30.8% by IL-3 as expressed as percent increase against total cellular histamine content), whereas G-CSF, M-CSF, and IL-4 had no effect. Enhancing action of these factors was still observed in the highly enriched basophil population, suggesting that this action was not via contaminating cells. Enhancement of histamine release by both factors was not necessarily IgE mediated, because they also amplified histamine release upon stimulation with FMLP and ionophore A23187. The enhancement by both factors was temperature dependent, and took place rapidly and reached plateau levels in 15 min. GM-CSF and IL-3 achieved the similar plateau level of augmentation and no additive effects were observed between them. This finding suggests that they enhance histamine release by sharing the same pathway in the release reaction.     

Controling the rate of protein release from polyelectrolyte complexes

Extended protein release from readily prepared, water-insoluble complexes with oppositely charged polyions is explored. Using hen egg-white lysozyme as a model, its sustained release from such complexes with a number of polyanions under physiological conditions has been demonstrated and rationalized. The rate of release varies orders of magnitude and is controlled by the nature of the polyanion (decreasing upon increase in its linear charge density, length, and hydrophobicity) and the complex particle size (the larger the particles, the slower the release).     

Nitrendipine-induced stimulation of renin release by the isolated perfused rat kidney

The direct effects of the organic calcium antagonist nitrendipine upon renin release were assessed using the isolated rat kidney perfused at constant pressure. This model circumvents the indirect actions of vasodilating agents by artificially maintaining perfusion pressure constant, thereby avoiding the hypotensive effects associated with the systemic administration of such agents. Renin release as assessed by radioimmunoassay was stimulated 2.6-fold upon the administration of 10(-6) M nitrendipine. Since this stimulation of renin release occurred in the absence of any alteration in perfusion pressure, we conclude that it represents a direct action of nitrendipine. This finding is in support of the current hypothesis concerning the inverse relationship between cytosolic Ca2+ and renin secretory rate, and suggests that Ca entry into the juxtaglomerular cells of the juxtaglomerular apparatus is sensitive to blockade by organic calcium antagonists such as nitrendipine.     

On the significance of the influx of calcium ions into stimulated human blood platelets

Blood platelets, upon stimulation with various substances, take up calcium ions from the suspending medium. This influx occurs simultaneously with the release reaction, i.e. the specific secretion of a variety of substances from storage organelles and the second wave of aggregation. Various inhibitors of the release reaction inhibit this Ca²⁺ influx. Platelets previously loaded with ⁴⁵Ca show an increased efflux of the cation upon stimulation by thrombin. These results suggest that the plasma membrane acquires an increased permeability to Ca²⁺ only in a later phase of platelet activation, in most cases after the earlier release of Ca²⁺ into the cytoplasm from Ca-storing organelles. Rapid shape change and release proceed independently of external calcium, whereas clot retraction depends upon a prolonged increased permeability of the plasma membrane to this cation.     

Serotonin metabolism in thrombocytes and microvascular hemostasis in spontaneous arterial hypertension

Serotonin content and accumulation in platelets and its release from them, as well as changes in thrombus formation in mesenteric arterioles and venules of the small intestine have been investigated in control rats and rats with spontaneous hypertension (SHR). Serotonin accumulation in platelets was determined upon its incubation with platelets. Disodium ADP salt was used as an inductor of release. Laser-induced thrombosis was caused by microvessels exposure to impulse laser irradiation. The control animals revealed a significant difference between the initial serotonin platelet level and serotonin level upon incubation and release; in values, the values of basic thrombus-forming parameters were higher than in arterioles. In SHR there is a decrease in biogenic amine content in platelets, a depression in its accumulation and release, an increase in the time of thrombus growth, its size up to the separation of the first embolus and its length along the vascular wall. It is concluded that spontaneous hypertension is characterized by decreased functional activity of platelets and depressed resistance of arterioles and venules to thrombus formation.     

Salt dependence of DNA binding by Thermus aquaticus and Escherichia coli DNA polymerases

DNA binding properties of the Type 1 DNA polymerases from Thermus aquaticus (Taq, Klentaq) and Escherichia coli (Klenow) have been examined as a function of [KCl] and [MgCl(2)]. Full-length Taq and its Klentaq "large fragment" behave similarly in all assays. The two different species of polymerases bind DNA with sub-micromolar affinities in very different salt concentration ranges. Consequently, at similar [KCl] the binding of Klenow is approximately 3 kcal/mol (150x) tighter than that of Taq/Klentaq to the same DNA. Linkage analysis reveals a net release of 2-3 ions upon DNA binding of Taq/Klentaq and 4-5 ions upon binding of Klenow. DNA binding of Taq at a higher temperature (60 degrees C) slightly decreases the ion release. Linkage analysis of binding versus [MgCl(2)] reports the ultimate release of approximately 1 Mg(2+) ion upon complex formation. However, the MgCl(2) dependence for Klenow, but not Klentaq, shows two distinct phases. In 10 mm EDTA, both polymerase species still bind DNA, but their binding affinity is significantly diminished, Klenow more than Klentaq. In summary, the two polymerase species, when binding to identical DNA, differ substantially in their sensitivity to the salt concentration range, bind with very different affinities when compared under similar conditions, release different numbers of ions upon binding, and differ in their interactions with divalent cations.     

Structural dissection of ATP turnover in the prototypical GHL ATPase TopoVI

GHL proteins are functionally diverse enzymes defined by the presence of a conserved ATPase domain that self-associates to trap substrate upon nucleotide binding. The structural states adopted by these enzymes during nucleotide hydrolysis and product release, and their consequences for enzyme catalysis, have remained unclear. Here, we have determined a complete structural map of the ATP turnover cycle for topoVI-B, the ATPase subunit of the archaeal GHL enzyme topoisomerase VI. With this ensemble of structures, we show that significant conformational changes in the subunit occur first upon ATP binding, and subsequently upon release of hydrolyzed P(i). Together, these data provide a structural framework for understanding the role of ATP hydrolysis in the type II topoisomerase reaction. Our results also suggest that the GHL ATPase module is a molecular switch in which ATP hydrolysis serves as a prerequisite but not a driving force for substrate-dependent structural transitions in the enzyme.