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PubMed1999

Localization and ultrastructure of pulmonary dendritic cells during delayed-type hypersensitivity reactions in mice.

Dendritic cells (DCs) are widely distributed in the airways and can serve as potent antigen-presenting cells. To clarify their involvement in the cell-mediated immune responses of the lung, we immunohistochemically investigated their distribution and kinetics during pulmonary delayed-type hypersensitivity (DTH) reactions induced in sensitized mice by intratracheal instillation of hapten. Cellular infiltrate appeared around the bronchiole and its accompanying blood vessel at 12 h after elicitation and progressively expanded by 48 h. As quantitated by computer-assisted morphometry, I-A(+) DCs and CD4(+) Th cells significantly increased in number around the bronchiole to a maximum at 24 h, whereas F4/80(+) macrophages were predominantly accumulated around the accompanying vessel with a peak at 48 h. Serial-section analysis revealed that DCs were colocalized with Th cells in the inflamed peribronchiolar tissue. Immunoelectron microscopy demonstrated that DCs found inside and around the capillaries and venules of peribronchiolar interstitium displayed round forms, indicating their emigration from here, while those situated far from the microvessels were elongated, often in close apposition to the lymphocytes. Mitosis of DCs was rarely seen. The present results suggest that peribronchiolar accumulation of DCs resulting from accelerated influx of blood-borne immature DCs and the interaction with T cells at the application site may play inducing roles in the development of pulmonary DTH reactions by enhancing the recruitment of macrophages.

Animals↗
PubMed2006

Imaging of multiple mRNA targets using quantum dot based in situ hybridization and spectral deconvolution in clinical biopsies.

Gene expression mapping using microarray analysis has identified useful gene signatures for predicting outcome. However, little of this has been translated into clinically effective diagnostic tools as microarrays require high quality fresh-frozen tissue samples. We describe a methodology of multiplexed in situ hybridization (ISH) using a novel combination of quantum dot (QD)-labeled oligonucleotide probes and spectral imaging analysis in routinely processed, formalin-fixed paraffin embedded human biopsies. The conditions for QD-ISH were optimized using a poly d(T) oligonucleotide in decalcified bone marrow samples. Single and multiplex QD-ISH was performed in samples with acute leukemia and follicular lymphoma using oligonucleotide probes for myeloperoxidase, bcl-2, survivin, and XIAP. Spectral imaging was used for post hybridization tissue analysis, enabling separation of spatially colocalized signals. The method allows quantitative characterization of multiple gene expression using non-bleaching fluorochromes. This is expected to facilitate multiplex in situ transcript detection in routinely processed human clinical tissue.

Biopsy↗
PubMed2004

Distribution of alpha1, alpha4, gamma2, and delta subunits of GABAA receptors in hippocampal granule cells.

GABAA receptors are pentamers composed of subunits derived from the alpha, beta, gamma, delta, theta, epsilon, and pi gene families. alpha1, alpha4, gamma2, and delta subunits are expressed in the dentate gyrus of the hippocampus, but their subcellular distribution has not been described. Hippocampal sections were double-labeled for the alpha1, alpha4, gamma2, and delta subunits and GAD65 or gephyrin, and their subcellular distribution on dentate granule cells was studied by means of confocal laser scanning microscopy (CLSM). The synaptic versus extrasynaptic localization of these subunits was inferred by quantitative analysis of the frequency of colocalization of various subunits with synaptic markers in high-resolution images. GAD65 immunoreactive clusters colocalized with 26.24+/-0.86% of the alpha1 subunit immunoreactive clusters and 32.35+/-1.49% of the gamma2 subunit clusters. In contrast, only 1.58+/-0.13% of the alpha4 subunit immunoreactive clusters and 1.92+/-0.15% of the delta subunit clusters colocalized with the presynaptic marker GAD65. These findings were confirmed by studying colocalization with immunoreactivity of a postsynaptic marker, gephyrin, which colocalized with 27.61+/-0.16% of the alpha1 subunit immunoreactive clusters and 23.45+/-0.32% of the gamma2 subunit immunoreactive clusters. In contrast, only 1.90+/-0.13% of the alpha4 subunit immunoreactive clusters and 1.76+/-0.10% of the delta subunit clusters colocalized with gephyrin. These studies demonstrate that a subset of alpha1 and gamma2 subunit clusters colocalize with synaptic markers in hippocampal dentate granule cells. Furthermore, all four subunits, alpha1, alpha4, gamma2, and delta, are present in the extrasynaptic locations.

Animals↗
PubMed2026

Biochemical analysis of the TPS-b subfamily reveals a cineole-centered monoterpene biosynthetic module in Medicago truncatula.

Terpenoids constitute one of the largest and most structurally diverse classes of plant specialized metabolites, with diversity generated by terpene synthases (TPSs) and downstream tailoring enzymes. In Medicago truncatula, the TPS-b subfamily comprises five putative synthases, two of which are embedded within a previously uncharacterized genomic locus containing a cytochrome P450 (CYP) and a BAHD-type acyltransferase. Here, we present a comprehensive biochemical analysis of the M. truncatula TPS-b subfamily and define a cineole-centered monoterpene biosynthetic module. Heterologous expression and in vitro assays with multiple prenyl diphosphate substrates revealed three catalytically active TPS-b enzymes with distinct substrate preferences and product profiles. MtTPS4 functions as a dedicated (E)-β-ocimene synthase, whereas MtTPS15 exhibits substrate-dependent bifunctionality, producing (E)-β-ocimene from geranyl diphosphate and α-farnesene from farnesyl diphosphate. MtTPS36 generates 1,8-cineole as the predominant product alongside α-terpineol from geranyl and neryl diphosphate. Genome analysis revealed that MtTPS36 is colocalized with a cytochrome P450 belonging to the CYP736 family. Biochemical characterization of this CYP identified a previously undescribed plant cineole hydroxylase that catalyzes oxidation of 1,8-cineole to yield 2α-hydroxy-1,8-cineole (also known as 2-exo-hydroxy-1,8-cineole), establishing a TPS-CYP biosynthetic module. These results define the gene-to-metabolite relationships within the TPS-b subfamily in M. truncatula and expand the known enzymatic biosynthetic capacity underlying oxygenated monoterpene biosynthesis in plants.

1,8-Cineole↗
PubMed2000

Membrane-anchorage of Cripto protein by glycosylphosphatidylinositol and its distribution during early mouse development.

cripto is the original member of the family of EGF-CFC genes, recently recognized as novel extracellular factors essential for vertebrate development. During the early stages of mouse gastrulation, cripto mRNA is detected in mesodermal cells; later, cripto mRNA is detected only in the truncus arteriosus of the developing heart. Here we describe the in vivo distribution of Cripto protein throughout mouse embryo development and show that cripto mRNA and protein colocalize. By means of immunofluorescence analysis and biochemical characterization, we show that Cripto is a membrane-bound protein anchored to the lipid bilayer by a glycosylphosphatidylinositol (GPI) moiety. We suggest that presentation of Cripto on the cell surface via a GPI-linkage is important in determining the spatial specificity of cell-cell interactions that play a critical role in the early patterning of the embryo.

Animals↗
PubMed2005

Multiplexed hybridization detection with multicolor colocalization of quantum dot nanoprobes.

We demonstrate a hybridization detection method using multicolor oligonucleotide-functionalized quantum dots as nanoprobes. In the presence of various target sequences, combinatorial self-assembly of the nanoprobes via independent hybridization reactions leads to the generation of discernible sequence-specific spectral codings. Detection of single-molecule hybridization is achieved by measuring colocalization of individual nanoprobes. Genetic analysis for anthrax pathogenicity through simultaneous detection of multiple relevant sequences is demonstrated using this novel biosensing method as proof-of-concept.

Bacillus anthracis↗
PubMed2001

Role of stem cell factor and mast cells in the progression of chronic glomerulonephritides.

BACKGROUND: Mast cells (MCs) have been implicated in the pathogenesis of atherosclerosis and tissue fibrosis. However, the role of MC in the development of renal fibrosis has not been fully elucidated. Stem cell factor (SCF; the ligand for MC c-kit receptor) is thought to attract and activate MCs. METHODS: The intensity of MC infiltration and SCF expression in renal biopsies from 56 patients with different forms of primary and secondary glomerulonephritis and five controls were investigated by immunohistochemistry, using a monoclonal anti-human MC tryptase antibody and a polyclonal antihuman SCF antibody. RESULTS: A large number of MCs were detected in the renal interstitium of the diseased kidneys. Immunostainable SCF was detected in tubular as well as interstitial cells. MC infiltration was significantly higher in glomerulonephritis (16.9 +/- 10.2 cells/field) compared with controls (2.8 +/- 2.1 cells/field, P = 0.03). Similarly, immunostainable SCF was 0.6 +/- 0.3% for controls and 3.3 +/- 2.1% in the glomerulonephritis group (P = 0.02). MC infiltration was highly correlated with SCF expression in diseased kidneys (r = 0.93, P = 0.0001). Double immunostain showed them to colocalize in some interstitial cells. Analysis of MC proliferation [proliferating cell nuclear antigen (PCNA) positivity] and apoptosis (in situ end labeling of DNA) showed these cells to be terminally differentiated. Both MCs and SCF were correlated with interstitial fibrosis (R = 0.71 for MC and R = 0.62 for SCF, P = 0.0001) and interstitial alpha-smooth muscle actin (R = 0.69 for MC and R = 0.60 for SCF P = 0.0001). Using regression analysis, the number of MC infiltration was found to be a very powerful determinant of interstitial fibrosis in the glomerulonephritis group (R2 = 91.4%). CONCLUSION: MCs as an infiltrating hematopoietic cell and its growth factor (SCF) seem to be up-regulated in glomerulonephritis, and may play a role in the development of renal fibrosis.

Adolescent↗
PubMed2001

Identification of an autoimmune serum containing antibodies against the Barr body.

Transcriptional inactivation of one X chromosome in mammalian female somatic cells leads to condensation of the inactive X chromosome into the heterochromatic sex chromatin, or Barr body. Little is known about the molecular composition and structure of the Barr body or the mechanisms leading to its formation in female nuclei. Because human sera from patients with autoimmune diseases often contain antibodies against a variety of cellular components, we reasoned that some autoimmune sera may contain antibodies against proteins associated with the Barr body. Therefore, we screened autoimmune sera by immunofluorescence of human fibroblasts and identified one serum that immunostained a distinct nuclear structure with a size and nuclear localization consistent with the Barr body. The number of these structures was consistent with the number of Barr bodies expected in diploid female fibroblasts containing two to five X chromosomes. Immunostaining with the serum followed by fluorescence in situ hybridization with a probe against XIST RNA demonstrated that the major fluorescent signal from the autoantibody colocalized with XIST RNA. Further analysis of the serum showed that it stains human metaphase chromosomes and a nuclear structure consistent with the inactive X in female mouse fibroblasts. However, it does not exhibit localization to a Barr body-like structure in female mouse embryonic stem cells or in cells from female mouse E7.5 embryos. The lack of staining of the inactive X in cells from female E7.5 embryos suggests the antigen(s) may be involved in X inactivation at a stage subsequent to initiation of X inactivation. This demonstration of an autoantibody recognizing an antigen(s) associated with the Barr body presents a strategy for identifying molecular components of the Barr body and examining the molecular basis of X inactivation.

Animals↗
PubMed1999

Trafficking of endothelial nitric-oxide synthase in living cells. Quantitative evidence supporting the role of palmitoylation as a kinetic trapping mechanism limiting membrane diffusion.

To examine endothelial nitric-oxide synthase (eNOS) trafficking in living endothelial cells, the eNOS-deficient endothelial cell line ECV304 was stably transfected with an eNOS-green fluorescent protein (GFP) fusion construct and characterized by functional, biochemical, and microscopic analysis. eNOS-GFP was colocalized with Golgi and plasma membrane markers and produced NO in response to agonist challenge. Localization in the plasma membrane was dependent on the palmitoylation state, since the palmitoylation mutant of eNOS (C15S/C26S eNOS-GFP) was excluded from the plasma membrane and was concentrated in a diffuse perinuclear pattern. Fluorescence recovery after photobleaching (FRAP) revealed eNOS-GFP in the perinuclear region moving 3 times faster than the plasmalemmal pool, suggesting that protein-lipid or protein-protein interactions are different in these two cellular domains. FRAP of the palmitoylation mutant was two times faster than that of wild-type eNOS-GFP, indicating that palmitoylation was influencing the rate of trafficking. Interestingly, FRAP of C15S/C26S eNOS-GFP but not wild-type eNOS-GFP fit a model of protein diffusion in a lipid bilayer. These data suggest that the regulation of eNOS trafficking within the plasma membrane and Golgi are probably different mechanisms and not due to simple diffusion of the protein in a lipid bilayer.

Acylation↗
PubMed2000

The adapter type protein CMS/CD2AP binds to the proto-oncogenic protein c-Cbl through a tyrosine phosphorylation-regulated Src homology 3 domain interaction.

CMS/CD2AP is a cytoplasmic protein critical for the integrity of the kidney glomerular filtration and the T cell function. CMS contains domains and motifs characteristic for protein-protein interactions, and it is involved in the regulation of the actin cytoskeleton. We report here that the individual SH3 domains of CMS bind to phosphotyrosine proteins of approximately 80, 90, and 180 kDa in cell lysates stimulated with epidermal growth factor. The second SH3 domain of CMS bound specifically to a tyrosine-phosphorylated protein of 120 kDa, which we identified as the proto-oncoprotein c-Cbl. The c-Cbl-binding site for CMS mapped to the carboxyl terminus of c-Cbl and is different from the proline-rich region known to bind SH3-containing proteins. CMS binding to c-Cbl was markedly attenuated in a tyrosine phosphorylation-defective c-Cbl mutant indicating that this interaction is dependent on the tyrosine phosphorylation of CMS. It also implies that CMS interacts with c-Cbl in an inducible fashion upon stimulation of a variety of cell-surface receptors. Immunofluorescence analysis revealed that both proteins colocalize at lamellipodia and leading edges of cells, and we propose that the interaction of CMS with c-Cbl offers a mechanism by which c-Cbl associates and regulates the actin cytoskeleton.

Actins↗
PubMed2003

Dissecting the cellular functions of annexin XI using recombinant human annexin XI-specific autoantibodies cloned by phage display.

Functional studies of cellular proteins are often complicated by the lack of well-defined monoclonal antibodies, the production of which is hampered by the highly conserved nature of these cellular proteins across species. Annexin XI, a member of the Ca2+-dependent, phospholipid-binding protein family, is an example of such a protein and was used in studies to devise a strategy using human autoimmune phage display libraries to generate reagents for biological studies of conserved cellular proteins. An IgG phage display library was generated from bone marrow of an autoimmune patient with high serum antibody titer against annexin XI, which was identified recently as an autoantigen targeted by autoantibodies in several systemic autoimmune diseases. From this phage library, a panel of human monoclonal annexin XI-specific Fabs were isolated and applied to studies of the cellular functions of annexin XI. Confocal microscopy showed a cell cycle-specific redistribution of annexin XI from the cytoplasm to the mitotic spindle. In metaphase, annexin XI was up-regulated and costained with alpha-tubulin. The subcellular distribution of annexin XI in COS-7 cells was shown to be Ca2+-dependent, and exhibited a predominantly nuclear pattern at low concentrations and a cytoplasmic pattern at high Ca2+ concentrations. Calcyclin, found previously to bind annexin XI in vitro, in vivo coated the nuclear membrane of cultured cell lines and did not colocalize with annexin XI. Ultrastructural analysis by immunoelectron microscopy revealed that annexin XI associated with specific granules in both neutrophils and eosinophils, suggesting a role in the exocytotic pathway. Our results illuminate the multifunctional nature of human annexin XI, provide the first evidence that annexin XI associates with the mitotic spindles and might play a role in cell division, and clearly illustrate the potential of phage display-derived human autoantibodies in broader analyses of the function of highly conserved cellular proteins.

Amino Acid Sequence↗
PubMed2006

Nuclear Rho kinase, ROCK2, targets p300 acetyltransferase.

Rho-associated coiled-coil protein kinase (ROCK) is an effector for the small GTPase Rho and plays a pivotal role in diverse cellular activities, including cell adhesion, cytokinesis, and gene expression, primarily through an alteration of actin cytoskeleton dynamics. Here, we show that ROCK2 is localized in the nucleus and associates with p300 acetyltransferase both in vitro and in cells. Nuclear ROCK2 is present in a large protein complex and partially cofractionates with p300 by gel filtration analysis. By immunofluorescence, ROCK2 partially colocalizes with p300 in distinct insoluble nuclear structures. ROCK2 phosphorylates p300 in vitro, and nuclear-restricted expression of constitutively active ROCK2 induces p300 phosphorylation in cells. p300 acetyltransferase activity is dependent on its phosphorylation status in cells, and p300 phosphorylation by ROCK2 results in an increase in its acetyltransferase activity in vitro. These observations suggest that nucleus-localized ROCK2 targets p300 for phosphorylation to regulate its acetyltransferase activity.

Animals↗
PubMed1991

Modulation of hexokinase association with mitochondria analyzed with quantitative three-dimensional confocal microscopy.

Hexokinase isozyme I is proposed to be associated with mitochondria in vivo. Moreover, it has been suggested that this association is modulated in coordination with changes in cell metabolic state. To test these hypotheses, we analyzed the subcellular distribution of hexokinase relative to mitochondria in paraformaldehyde-fixed astrocytes using immunocytochemistry and quantitative three-dimensional confocal microscopy. Analysis of the extent of colocalization between hexokinase and mitochondria revealed that approximately 70% of cellular hexokinase is associated with mitochondria under basal metabolic conditions. In contrast to the immunocytochemical studies, between 15 to 40% of cellular hexokinase was found to be associated with mitochondria after fractionation of astrocyte cultures depending on the exact fractionation conditions. The discrepancy between fractionation studies and those based on imaging of distributions in fixed cells indicates the usefulness of using techniques that can evaluate the distributions of "cytosolic" enzymes in cells whose subcellular ultrastructure is not severely disrupted. To determine if hexokinase distribution is modulated in concert with changes in cell metabolism, the localization of hexokinase with mitochondria was evaluated after inhibition of glucose metabolism with 2-deoxyglucose. After incubation with 2-deoxyglucose there was an approximate 35% decrease in the amount of hexokinase associated with mitochondria. These findings support the hypothesis that hexokinase is bound to mitochondria in rat brain astrocytes in vivo, and that this association is sensitive to cell metabolic state.

Animals↗
PubMed1997

PSTPIP: a tyrosine phosphorylated cleavage furrow-associated protein that is a substrate for a PEST tyrosine phosphatase.

We have investigated proteins which interact with the PEST-type protein tyrosine phosphatase, PTP hematopoietic stem cell fraction (HSCF), using the yeast two-hybrid system. This resulted in the identification of proline, serine, threonine phosphatase interacting protein (PSTPIP), a novel member of the actin- associated protein family that is homologous to Schizosaccharomyces pombe CDC15p, a phosphorylated protein involved with the assembly of the actin ring in the cytokinetic cleavage furrow. The binding of PTP HSCF to PSTPIP was induced by a novel interaction between the putative coiled-coil region of PSTPIP and the COOH-terminal, proline-rich region of the phosphatase. PSTPIP is tyrosine phosphorylated both endogenously and in v-Src transfected COS cells, and cotransfection of dominant-negative PTP HSCF results in hyperphosphorylation of PSTPIP. This dominant-negative effect is dependent upon the inclusion of the COOH-terminal, proline-rich PSTPIP-binding region of the phosphatase. Confocal microscopy analysis of endogenous PSTPIP revealed colocalization with the cortical actin cytoskeleton, lamellipodia, and actin-rich cytokinetic cleavage furrow. Overexpression of PSTPIP in 3T3 cells resulted in the formation of extended filopodia, consistent with a role for this protein in actin reorganization. Finally, overexpression of mammalian PSTPIP in exponentially growing S. pombe results in a dominant-negative inhibition of cytokinesis. PSTPIP is therefore a novel actin-associated protein, potentially involved with cytokinesis, whose tyrosine phosphorylation is regulated by PTP HSCF.

3T3 Cells↗
PubMed1995

Murine CD14 gene expression in vivo: extramyeloid synthesis and regulation by lipopolysaccharide.

A murine model system was used to study the distribution and regulation of CD14 gene expression in vivo. Western blot analysis failed to detect CD14 in plasma from untreated CB6 (BALB/c x C57Bl6) mice, but showed markedly increased levels of CD14 in plasma from mice treated with lipopolysaccharide (LPS). Plasma levels of CD14 increased in a time- and dose-dependent manner, reaching a maximum between 8 and 16 h. Northern blot analysis of total RNA extracted from mouse tissues revealed low, but significant, levels of CD14 mRNA in many tissues of untreated animals with the highest levels in uterus, adipose tissue, and lung. After intraperitoneal injection of LPS, induction of CD14 gene expression was detected in all organs examined with the extent of induction varying between organs. Induction of CD14 mRNA was both time and dose dependent. Maximum induction in the heart and lung was observed 2-4 h after injection of LPS, while liver and kidney showed maximal induction between 8 and 16 h. In situ hybridization showed that CD14 mRNA was expressed in myeloid cells in many tissues, and that expression in these cells was upregulated by LPS. Unexpectedly, CD14 mRNA was also detected in other cells within tissues, including epithelial cells, and expression in these cell types also was upregulated by LPS. Immunochemical analysis revealed that CD14 antigen colocalized to the cytoplasm of cells expressing CD14 mRNA. These studies demonstrate that CD14 gene expression is not restricted to myeloid cells, and that the level of expression of CD14 is influenced by exposure to LPS.

Animals↗
PubMed1998

Intracellular localization of migration inhibitory factor-related protein (MRP) and detection of cell surface MRP binding sites on human leukemia cell lines.

The migration inhibitory factor-related proteins (MRPs) MRP-8 and MRP-14 were detected in differentiated human leukemia cell lines (THP-1 and HL-60) by immunocytochemical analysis. They were induced and colocalized in the cytoplasm and in lesser amounts in the nucleus when THP-1 and HL-60 cells were induced to differentiate by 1alpha,25-dihydroxyvitamin D3 or retinoic acid. In a search for a protein capable of binding MRPs, both MRPs were individually produced in insect cells (Sf21) infected with recombinant baculovirus. The purified recombinant MRPs were electrophoretically and antigenically indistinguishable from the native proteins, and their ability to form the MRP8/14 complex was retained. The presence of MRP binding sites was investigated by a binding assay using recombinant MRPs and specific monoclonal antibodies. MRP binding sites were detected on the cell membrane of the human leukemia cell lines THP-1, Raji, and MOLT-4. HL-60 cells treated with 1alpha, 25-dihydroxyvitamin D3 did not express MRP binding sites on the cell membrane, but a high level of MRPs accumulated in the cells. The occurrence of MRP binding sites on the cell surface of leukemia cell lines of monocyte and lymphocyte origin suggests that MRPs, released from neutrophils under certain conditions, may contribute to the activation and recruitment of effector cells to inflammatory lesions.

Animals↗
PubMed2003

Regulation of ribosomal RNA synthesis during the final phases of porcine oocyte growth.

In porcine oocytes, acquisition of meiotic competence coincides with a decrease of general transcriptional activity at the end of the oocyte growth phase and, specifically, of ribosomal RNA (rRNA) synthesis in the nucleolus. The present study investigated the regulation of rRNA synthesis during porcine oocyte growth. Localization and expression of components involved in regulation of the rRNA synthesis (the RNA polymerase I-associated factor PAF53, upstream binding factor [UBF], and the pocket proteins p130 and pRb) were assessed by immunocytochemistry and semiquantitative reverse transcription-polymerase chain reaction and correlated with ultrastructural analysis and autoradiography following [3H]uridine incubation in growing and fully grown porcine oocytes. In addition, meiotic resumption, ultrastructure, and expression of p130, UBF, and PAF53 were analyzed in growing and fully grown porcine oocytes cultured with 100 microM butyrolactone I (BL-I), a potent inhibitor of cyclin-dependent kinases, to gain insight concerning the regulation of rRNA transcription during meiotic arrest. Immunocytochemical analysis demonstrated that p130 became colocalized with UBF and PAF53 and that the intensity of the PAF53 labeling decreased toward the end of the oocyte growth phase. These data suggest that the decrease in rRNA synthesis is regulated through inhibition of UBF by p130 as well as by decreased availability of PAF53. Moreover, expression of mRNA encoding PAF53 was decreased at the end of the oocyte growth phase. At the morphological level, these events coincided with inactivation of the nucleolus, as visualized by the transformation of the fibrillogranular nucleolus to an electron-dense fibrillar sphere with remnants of the fibrillar centers at the surface. Meiotic inhibition with 100 microM BL-I had a detrimental effect on the ability of porcine oocytes to resume meiosis and on nucleolus morphology, resulting in a lack of RNA synthetic capability as the fibrillar components, where rRNA transcription and initial processing occur, condensed or even disintegrated.

4-Butyrolactone↗
PubMed2005

The T-tubule membrane ATP-operated P2X4 receptor influences contractility of skeletal muscle.

Evidence indicates that extracellular ATP may have relevant functions in skeletal muscle, even though the physiological role and distribution of specific signaling pathway elements are not well known. The present work shows that P2X4 receptor, an extracellular ATP-regulated cell membrane channel permeable to Ca2+, is expressed in several tissues of the rat, including skeletal muscle. A specific antibody detected a protein band of approximately 60 kDa. Immunofluorescence demonstrated that P2X4 has an intracellular localization, and confocal analysis revealed that the receptor colocalizes with the T-tubule membrane DHP receptor. Considering that the natural agonist of P2X4 is ATP, we explored if changes of extracellular ATP levels could occur in contracting skeletal muscle to regulate the channel. In vitro experiments showed that substantial ATP is released and rapidly hydrolyzed after electrical stimulation of rat muscle fibers. Results show that the presence of ATP-degrading enzymes (hexokinase/apyrase), inhibitors of P2X receptors or Ca2+-free conditions, all abolished the progressive twitch tension potentiation produced in soleus muscle by low-frequency (0.05 Hz) stimulation. These data reveal that ATP-mediated Ca2+ entry, most likely through P2X4 receptor, may play an important role in modulating the contractility of skeletal muscle.

Adenosine Triphosphate↗