Negative-feedback regulation of ATP release: ATP release from cardiomyocytes is strictly regulated during ischemia

Extracellular ATP acts as a potent agonist on cardiomyocytes, inducing a broad range of physiological responses via P2 purinoceptors. Its concentration in the interstitial space within the heart is elevated during ischemia or hypoxia due to its release from a number of cell types, including cardiomyocytes. However, the exact mechanism responsible for the release of ATP from cardiomyocytes during ischemia is not known. In this study, we investigated whether and how the release of ATP was strictly regulated during ischemia in cultured neonatal rat cardiomyocytes. Ischemia was mimicked by oxygen-glucose deprivation (OGD). Exposure of cardiomyocytes to OGD resulted in an increase in the concentration of extracellular ATP shortly after the onset of OGD (15 min), and the increase was reversed by treatment with blockers of maxi-anion channels. Unexpectedly, at 1 and 2h after the onset of OGD, the blocking of maxi-anion channels increased the concentration of extracellular ATP, and the increase was significantly suppressed by co-treatment with blockers of hemichannels, suggesting that ATP release via maxi-anion channels was involved in the suppression of ATP release via hemichannels during persistent OGD. Here we show the possibility that the release of ATP from cardiomyocytes was strictly regulated during ischemia by negative-feedback mechanisms; that is, maxi-anion channel-derived ATP-induced suppression of ATP release via hemichannels in cardiomyocytes.     

ATP is required for the binding of precursor proteins to chloroplasts

One of the first steps in the transport of nuclear-encoded, cytoplasmically synthesized precursor proteins into chloroplasts is a specific binding interaction between precursor proteins and the surface of the organelle. Although protein translocation into chloroplasts requires ATP hydrolysis, binding is generally thought to be energy independent. A more detailed investigation of precursor binding to the surface of chloroplasts showed that ATP was required for efficient binding. Protein translocation is known to require relatively high levels (1 mM or more) of ATP. As little as 50-100 microM ATP caused significant stimulation of precursor binding over controls with no ATP. Several different precursors were tested and all showed increased binding upon addition of low levels of ATP. Nonhydrolyzable analogs of ATP did not substitute for ATP, indicating that ATP hydrolysis was required for binding. A protonmotive force was not involved in the energy requirement for binding. Other (hydrolyzable) nucleotides could substitute for ATP but were less effective at stimulating binding. Binding was stimulated by ATP generated inside chloroplasts even when an ATP trap was present to destroy external ATP. We conclude that internal ATP is required for stimulation of precursor binding to chloroplasts.     

Observing proteins in their natural habitat: the living cell

Fluorescence microscopy has played a tremendous role in uncovering the morphological features of cells and the expression pattern of proteins by immunofluorescence. Since the discovery of green-fluorescent proteins (GFPs), this technique has undergone a revival in the life sciences as the spatial distribution of ectopically expressed fusion proteins inside living cells can now be followed more easily. By further exploiting the photophysical properties of the emitted fluorescence with microspectroscopic methods, spatial information on the biochemical parameters of intracellular processes and reactions can be obtained. This possibility will not only play an important role in the understanding of biochemical reactions in signal processing and fidelity but also help to uncover the molecular mechanisms of organelle and cell morphogenesis.     

Intravital microscopy: a novel tool to study cell biology in living animals

Intravital microscopy encompasses various optical microscopy techniques aimed at visualizing biological processes in live animals. In the last decade, the development of non-linear optical microscopy resulted in an enormous increase of in vivo studies, which have addressed key biological questions in fields such as neurobiology, immunology and tumor biology. Recently, few studies have shown that subcellular processes can be imaged dynamically in the live animal at a resolution comparable to that achieved in cell cultures, providing new opportunities to study cell biology under physiological conditions. The overall aim of this review is to give the reader a general idea of the potential applications of intravital microscopy with a particular emphasis on subcellular imaging. An overview of some of the most exciting studies in this field will be presented using resolution as a main organizing criterion. Indeed, first we will focus on those studies in which organs were imaged at the tissue level, then on those focusing on single cells imaging, and finally on those imaging subcellular organelles and structures.     

Watching proteins in the wild: fluorescence methods to study protein dynamics in living cells

The advent of GFP imaging has led to a revolution in the study of live cell protein dynamics. Ease of access to fluorescently tagged proteins has led to their widespread application and demonstrated the power of studying protein dynamics in living cells. This has spurred development of next generation approaches enabling not only the visualization of protein movements, but correlation of a protein's dynamics with its changing structural state or ligand binding. Such methods make use of fluorescence resonance energy transfer and dyes that report changes in their environment, and take advantage of new chemistries for site-specific protein labeling.     

Neuroglial ATP release through innexin channels controls microglial cell movement to a nerve injury

Microglia, the immune cells of the central nervous system, are attracted to sites of injury. The injury releases adenosine triphosphate (ATP) into the extracellular space, activating the microglia, but the full mechanism of release is not known. In glial cells, a family of physiologically regulated unpaired gap junction channels called innexons (invertebrates) or pannexons (vertebrates) located in the cell membrane is permeable to ATP. Innexons, but not pannexons, also pair to make gap junctions. Glial calcium waves, triggered by injury or mechanical stimulation, open pannexon/innexon channels and cause the release of ATP. It has been hypothesized that a glial calcium wave that triggers the release of ATP causes rapid microglial migration to distant lesions. In the present study in the leech, in which a single giant glial cell ensheathes each connective, hydrolysis of ATP with 10 U/ml apyrase or block of innexons with 10 µM carbenoxolone (CBX), which decreased injury-induced ATP release, reduced both movement of microglia and their accumulation at lesions. Directed movement and accumulation were restored in CBX by adding ATP, consistent with separate actions of ATP and nitric oxide, which is required for directed movement but does not activate glia. Injection of glia with innexin2 (Hminx2) RNAi inhibited release of carboxyfluorescein dye and microglial migration, whereas injection of innexin1 (Hminx1) RNAi did not when measured 2 days after injection, indicating that glial cells’ ATP release through innexons was required for microglial migration after nerve injury. Focal stimulation either mechanically or with ATP generated a calcium wave in the glial cell; injury caused a large, persistent intracellular calcium response. Neither the calcium wave nor the persistent response required ATP or its release. Thus, in the leech, innexin membrane channels releasing ATP from glia are required for migration and accumulation of microglia after nerve injury.     

The amphipathic membrane proteins associated with gangliosides: The Paul-Bunnell antigen is one of the gangliophilic proteins

Two amphipathic protein fractions soluble in organic solvents as well as in water have been isolated from the ganglioside fraction of bovine erythrocyte membranes by successive chromatography in chloroform-methanol mixture on DEAE-Sephadex, silicic acid, and α-hydroxypropylated Sephadex G50 (LH60) columns. These two fractions contained a similar low molecular weight protein but with distinctively different amino acid composition. One of these proteins has been characterized by having a strong Paul-Bunnell antigen activity and had a binding affinity to ganglioside. A similar protein without Paul-Bunnell antigen activity was isolated as the major ganglioside-associated protein.     

Involvement of cell surface ATP synthase in flow-induced ATP release by vascular endothelial cells

Endothelial cells (ECs) release ATP in response to shear stress, a mechanical force generated by blood flow, and the ATP released modulates EC functions through activation of purinoceptors. The molecular mechanism of the shear stress-induced ATP release, however, has not been fully elucidated. In this study, we have demonstrated that cell surface ATP synthase is involved in shear stress-induced ATP release. Immunofluorescence staining of human pulmonary arterial ECs (HPAECs) showed that cell surface ATP synthase is distributed in lipid rafts and co-localized with caveolin-1, a marker protein of caveolae. Immunoprecipitation indicated that cell surface ATP synthase and caveolin-1 are physically associated. Measurement of the extracellular metabolism of [(3)H]ADP confirmed that cell surface ATP synthase is active in ATP generation. When exposed to shear stress, HPAECs released ATP in a dose-dependent manner, and the ATP release was markedly suppressed by the membrane-impermeable ATP synthase inhibitors angiostatin and piceatannol and by an anti-ATP synthase antibody. Depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin (MbetaCD) disrupted lipid rafts and abolished co-localization of ATP synthase with caveolin-1, which resulted in a marked reduction in shear stress-induced ATP release. Pretreatment of the cells with cholesterol prevented these effects of MbetaCD. Downregulation of caveolin-1 expression by transfection of caveolin-1 siRNA also markedly suppressed ATP-releasing responses to shear stress. Neither MbetaCD, MbetaCD plus cholesterol, nor caveolin-1 siRNA had any effect on the amount of cell surface ATP synthase. These results suggest that the localization and targeting of ATP synthase to caveolae/lipid rafts is critical for shear stress-induced ATP release by HPAECs.     

The Fragilis interferon-inducible gene family of transmembrane proteins is associated with germ cell specification in mice

Background  

Specification of primordial germ cells in mice depends on instructive signalling events, which act first to confer germ cell competence on epiblast cells, and second, to impose a germ cell fate upon competent precursors. fragilis, an interferon-inducible gene coding for a transmembrane protein, is the first gene to be implicated in the acquisition of germ cell competence.     

Binding of ATP to TK1-like enzymes is associated with a conformational change in the quaternary structure

Human thymidine kinase 1 (hTK1) and structurally related TKs from other organisms catalyze the initial phosphorylation step in the thymidine salvage pathway. Though ATP is known to be the preferred phosphoryl donor for TK1-like enzymes, its exact binding mode and effect on the oligomeric state has not been analyzed. Here we report the structures of hTK1 and of the Thermotoga maritima thymidine kinase (TmTK) in complex with the bisubstrate inhibitor TP4A. The TmTK-TP4A structure reveals that the adenosine moiety of ATP binds at the subunit interface of the homotetrameric enzyme and that the majority of the ATP-enzyme interactions occur between the phosphate groups and the P-loop. In the hTK1 structure the adenosine group of TP4A exhibited no electron density. This difference between hTK1 and TmTK is rationalized by a difference in the conformation of their quaternary structure. A more open conformation, as seen in the TmTK-TP4A complex structure, is required to provide space for the adenosine moiety. Our analysis supports the formation of an analogous open conformation in hTK1 upon ATP binding.     

Cystic fibrosis transmembrane conductance regulator facilitates ATP release by stimulating a separate ATP release channel for autocrine control of cell volume regulation

These studies provide evidence that cystic fibrosis transmembrane conductance regulator (CFTR) potentiates and accelerates regulatory volume decrease (RVD) following hypotonic challenge by an autocrine mechanism involving ATP release and signaling. In wild-type CFTR-expressing cells, CFTR augments constitutive ATP release and enhances ATP release stimulated by hypotonic challenge. CFTR itself does not appear to conduct ATP. Instead, ATP is released by a separate channel, whose activity is potentiated by CFTR. Blockade of ATP release by ion channel blocking drugs, gadolinium chloride (Gd(3+)) and 4,4'-diisothiocyanatostilbene-2,2'disulfonic acid (DIDS), attenuated the effects of CFTR on acceleration and potentiation of RVD. These results support a key role for extracellular ATP and autocrine and paracrine purinergic signaling in the regulation of membrane ion permeability and suggest that CFTR potentiates ATP release by stimulating a separate ATP channel to strengthen autocrine control of cell volume regulation.     

Studies on the release of barley aleurone cell proteins: Autoradiography

Summary Both uptake and incorporation of radioactivity from [³H]l-leucine into gibberellic-acid (GA₃)-treated aleurone layers of barley (Hordeum vulgare L.) was enhanced by pretreatment with 5 mM potassium bromate. The effect of 5 mM KBrO₃ on amino-acid incorporation was quantitative rather than qualitative and could be partly reversed by the addition of neutralized casein hydrolysate at 10 mg/ml. Autoradiographs of GA₃-treated aleurone cells pulsed with [³H]leucine showed distribution of silver grains predominantly over the endoplasmic reticulum (ER) and aleurone grains. After chasing with carrier l-leucine for 60 min, fewer silver grains were associated with the ER and aleurone grains while nearly half of the silver was associated with the ground cytoplasm of the cell. Autoradiographs were prepared from aleurone cells previously stratified by ultracentrifugation. After a 10-min pulse of label, the silver grains were found over the central ER zone of centrifuged cells; however, with an increase in duration of the chase, label was found distributed throughout the aleurone grain and spherosome region of the cell. The silver grains which were located over the central zone of centrifuged cells at the end of the pulse were almost exclusively associated with the ER. There is no evidence for association of label with dictyosomes or with vesicles derived from dictyosomes. The experimental evidence indicates that labelled amino acids are incorporated into aleurone cells on the ER and are released from these cells without the participation of a membrane-bound vesicle.     

Transport of proteins into mitochondria: a potassium diffusion potential is able to drive the import of ADP/ATP carrier.

The transfer of cytoplasmically synthesized precursor proteins into or across the inner mitochondrial membrane is dependent on energization of the membrane. To investigate the role of this energy requirement, a buffer system was developed in which efficient import of ADP/ATP carrier into mitochondria from the receptor-bound state occurred. This import was rapid and was dependent on divalent cations, whereas the binding of precursor proteins to the mitochondrial surface was slow and was independent of added divalent cations. Using this buffer system, the import of ADP/ATP carrier could be driven by a valinomycin-induced potassium diffusion potential. The protonophore carbonylcyanide m-chlorophenyl-hydrazone was not able to abolish this import. Imposition of a delta pH did not stimulate the import. We conclude that the membrane potential delta psi itself and not the total protonmotive force delta p is the required energy source.     

Covisualization by computation optical-sectioning microscopy of integrin and associated proteins at the cell membrane of living onion protoplasts

Summary Using higher-resolution wide-field computational optical-sectioning fluorescence microscopy, the distribution of antigens recognized by antibodies against animal ₁ integrin, fibronectin, and vitronectin has been visualized at the outer surface of enzymatically protoplasted onion epidermis cells and in depectinated cell wall fragments. On the protplast all three antigens are colocalized in an array of small spots, as seen in raw images, in Gaussian filtered images, and in images restored by two different algorithms. Fibronectin and vitronectin but not ₁ integrin antigenicities colocalize as puncta in comparably prepared and processed images of the wall fragments. Several control visualizations suggest considerable specificity of antibody recognition. Affinity purification of onion cell extract with the same anti-integrin used for visualization has yielded protein that separates in SDS-PAGE into two bands of about 105–110 and 115–125 kDa. These bands are again recognized by the visualizationi antibody, which was raised against the extracellular domain of chicken ₁ integrin, and are also reconized by an antibody against the intracellular domain of chicken ₁ integrin. Because ₁ integrin is a key protein in numerous animal adhesion sites, it appears that the punctate distribution of this protein in the cell membranes of onion epidermis represents the adhesion sites long known to occur in cells of this tissue. Because vitronectin and fibronectin are matrix proteins that bind to integrin in animals, the punctate occurrence of antigenically similar proteins both in the wall (matrix) and on enzymatically prepared protoplasts reinforces the concept that onion cells have adhesion sites with some similarity to certain kinds of adhesioni sites in animals.     

Binding of ATP to nucleoside-diphosphate kinase: a kinetic study

The binding of nucleotides to pig heart nucleoside-diphosphate kinase was studied using Rose Bengal as an optical probe. ATP, in the absence of Mg2+, binds slowly to the enzyme, with a second order rate constant of about 3000 M-1 . s-1, whereas in its presence the binding is much faster. This finding suggests the regulation of the nucleoside-diphosphate kinase activity by uncomplexed ATP, and that ATP binds normally to the enzyme via a metal ion bridge.     

Using a GFP-gene fusion technique to study the cell cycle-dependent distribution of calmodulin in living cells

In this study, a green fluorescent protein (GFP)-calmodulin (CaM) fusion gene method was used to examine the distribution of calmodulin during various stages of cell cycle. First, it was found that the distribution of CaM in living cells changes with the cell cycle. CaM was found mainly in the cytoplasm during G1 phase. It began to move into the nucleus when the cell entered S phase. At G2 phase, CaM became more concentrated in the nucleus than in cytoplasm. Second, the accumulation of CaM in the nucleus during G2 phase appeared to be related to the onset of mitosis, since inhibiting the activation of CaM at this stage resulted in blocking the nuclear membrane breakdown and chromatin condensation. Finally, after the cell entered mitosis, a high concentration of CaM was found at the polar regions of the mitotic spindle. At this time, inhibiting the activity of CaM would cause a dismption of the spindle structure. The relationship between the stage-specific distribution of CaM and its function in regulating the progression of cell cycle was discussed.