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J H Walker

Publications and source records attributed to J H Walker.

At least 55 records · Page 3Linked to original sources
PubMed1996

Annexins in the human neuroblastoma SH-SY5Y: demonstration of relocation of annexins II and V to membranes in response to elevation of intracellular calcium by membrane depolarisation and by the calcium ionophore A23187.

The human neuroblastoma SH-SY5Y was found to express annexins I, II, IV, V, and VI by western blot analysis. Calcium-dependent membrane-binding proteins were isolated from SH-SY5Y and analysed by 2-dimensional gel electrophoresis. Proteins with Mr and Pi values similar to those of annexins I, II, III, IV, V, and VI were observed. The identity of annexins II and V was confirmed by western blotting. The membrane association of annexins II and V was studied in cells that had been stimulated to release noradrenaline by K+ depolarisation or by treatment with the ionophore A23187. Annexins II and V were both found to associate with membranes in a manner that was resistant to elution with EGTA and required Triton X-100 for their solubilisation. Homogenisation of cells in calcium-containing buffers also resulted in the formation of EGTA-resistant membrane-associated annexins II and V. The results demonstrate calcium-dependent relocation of annexins II and V to membranes in intact cells and suggest that these annexins bind in a calcium-dependent manner to non-phospholipid components of SH-SY5Y membranes. Examination of cells by immunofluorescence microscopy demonstrated that annexin II was homogeneously associated with the plasma membrane before treatment with ionophore and relocated to discrete patches of staining after treatment. Annexin V was found by immunofluorescence to be present in the cytoplasm and in the nucleus, Stimulation of the cells produced no change in the cytoplasmic staining pattern but resulted in a partial relocation of nuclear annexin V to the periphery of the nucleus. The results argue for a general role for both annexins in calcium signalling at discrete intracellular locations. The results are not consistent with the specific involvement proposed previously for annexin II in membrane fusion at sites of vesicle exocytosis.

Annexin A2↗
PubMed1996

Annexins II, IV, V and VI relocate in response to rises in intracellular calcium in human foreskin fibroblasts.

Annexins are a family of proteins implicated in a number of cellular processes involving calcium. We studied annexins I, II, IV, V and VI and found that they are all present in human foreskin fibroblasts and, from immunocytochemical studies, have distinct locations in the cell. Only annexin IV and annexin V have unstructured cytoplasmic staining patterns consistent with predominantly cytosolic locations. Annexin VI partially colocalizes with the endoplasmic reticulum. In contrast, annexins I and II are both associated with the plasma membrane with annexin II having a very homogeneous staining compared with the punctate pattern observed for annexin I. Annexins I, IV and V are all present in the nucleus at higher concentrations than in the cytoplasm. Treatment of cells with the calcium ionophore A23187 to raise intracellular calcium, results in relocations of annexin II, IV, V and VI. Intranuclear annexins IV and V relocate to the nuclear membrane whereas the cytosolic pools of these annexins relocate to the plasma membrane. Annexin II relocates to granular structures at the plasma membrane whereas annexin VI relocates to a more homogeneous distribution on the plasma membrane. These results are consistent with an important role for annexins in mediating the calcium signal at the plasma membrane and within the nuclei of fibroblasts.

Annexin A2↗
PubMed1996

Preoperative antioxidant and allopurinol therapy for reducing reperfusion-induced injury in patients undergoing cardiothoracic surgery.

Administration of vitamin E, vitamin C, and allopurinol to reduce reperfusion-induced injury remains controversial. Clinical trials are being conducted, and the results are promising. No adverse side effects have been reported with the short-term use of these drugs. The cost of the pre-operative medication is approximately $5 per week, which favors its use. The ability of nurses to be effective advocates of patients receiving these drugs is vital. What appears to be a trivial medication regimen may save myocardial cells and promote an uncomplicated postoperative period.

Aged↗
PubMed1995

Ca2+ concentration during binding determines the manner in which annexin V binds to membranes.

Annexins are a family of calcium-binding proteins that have been implicated in a wide range of intracellular processes. We have previously reported that stimulation of platelets with thrombin can induce the association of intracellular annexin V with membranes in two distinct ways. First, in such a way that it can be eluted from the membrane with EGTA and secondly in a manner such that it is tightly bound to the membrane and requires the non-ionic detergent Triton X-100 for its solubilization. We report that exposure of platelets to the calcium ionophore A23187 mimics the relocation induced by stimulation with thrombin. In separate experiments we demonstrate that a calcium ion concentration [Ca2+] of 0.8 microM is sufficient for maximum binding of the EGTA-resistant form to membranes. In contrast a higher [Ca2+] was required to induce maximal binding of the annexin V which could be extracted with EGTA. We demonstrate that following temperature-induced phase separation in Triton X-114, the membrane-associated annexin V partitions predominantly into the aqueous phase. We also show that the isoelectric point of annexin V does not change following membrane association. These observations suggest that a covalent modification, of annexin V itself, is not responsible for its association with the membrane. Millimolar [Ca2+] is required for maximal binding of purified annexin V to phospholipid vesicles. We show that binding to phospholipids can be reversed entirely by subsequent treatment with EGTA. This suggests that the EGTA-resistant form of annexin V is binding to a membrane component other than phosphatidylserine. Annexin V has previously been shown to bind to protein kinase C. Relocation of annexin V to membranes paralleled that of protein kinase C in thrombin-stimulated cells but not in cells treated with A23187, suggesting that these proteins are not functionally linked in platelet activation. Using bifunctional cross-linking reagents we have identified an 85 kDa complex containing annexin V. This may represent an association between annexin V and an annexin V-binding protein with a molecular mass of approximately 50 kDa.

Annexin A5↗
PubMed1995

The use of the human neuroblastoma SH-SY5Y to study the effect of second messengers on noradrenaline release.

1. Recent data suggesting that the human neuroblastoma SH-SY5Y is a suitable cell line in which to study the effect of second messengers on NA release are discussed in the context of current views on exocytosis. 2. Release of NA is evoked by depolarization, as well as activation of muscarinic (M3) and bradykinin (B2) receptors in SH-SY5Y cells which have not been differentiated by the addition of growth factors. 3. Evoked release is enhanced by activation of protein kinase C. 4. Activation of protein kinase C decreases the changes in intracellular calcium evoked by carbachol, bradykinin and 100 mM K+. 5. SH-SY5Y express N-type and L-type voltage sensitive Ca2+ channels. L-Type Ca(2+)-channels are coupled to NA release under conditions of weak depolarization. However with strong depolarization (100 mM K+) both L-type and N-type channels are involved. 6. Muscarinic- and neuropeptide Y receptors are coupled to the inhibition of Ca2+ channel activity.

Cell Line↗
PubMed1994

Thrombin stimulates the intracellular relocation of annexin V in human platelets.

Annexins are a family of proteins that have been implicated in a range of intracellular processes. In this paper we confirm the existence of annexin V in human platelets (0.02 +/- 0.005% of cell protein). We also demonstrate that 13.7 +/- 6.8% of intracellular annexin V becomes tightly associated with membranes in response to platelet activation by the physiological agonist thrombin and requires non-ionic detergent for solubilization. Thrombin stimulation also induces the association of annexin V (11.0 +/- 4.6% of the total) with the membrane in a manner which requires prolonged treatment with EGTA for its release from the membrane.

Annexin A5↗
PubMed1994

Characterization and immunolocalization of rat liver annexin VI.

Annexin VI has been purified to homogeneity from rat liver and monospecific antibodies have been produced. The antibodies have been used for immunoblot analysis of rat tissues. Annexin VI is present in most tissues, with particularly high concentrations in liver, spleen, muscle, and intestine. In liver, annexin VI constitutes approximately 0.25% of total cellular protein. Immunohistochemical studies have located annexin VI on plasma membranes of hepatocytes with enhanced concentration on bile canaliculi. Annexin VI binds in a Ca(2+)-dependent manner to a sub-cellular fraction containing membranes. In the presence of physiological concentrations of ATP, the free Ca2+ concentration required for half-maximal binding of annexin VI to membranes is significantly reduced. While annexin VI binds in vitro to membranes in the presence of Ca2+, in rat liver about 31% of the annexin VI is associated with membranes in a Ca(2+)-independent manner and its solubilization requires the presence of Triton X-100. However, studies using Triton X-114 showed no increase in the hydrophobicity of this fraction of the protein compared to the purified EGTA-soluble annexin VI.

Amino Acid Sequence↗
PubMed1994

A role for protein kinase C subtypes alpha and epsilon in phorbol-ester-enhanced K(+)- and carbachol-evoked noradrenaline release from the human neuroblastoma SH-SY5Y.

Protein kinase C (PKC) consists of a family of closely related subtypes which differ in their localization and activation properties. Our previous studies have suggested a role for PKC in the regulation of noradrenaline (NA) release from the human neuroblastoma SH-SY5Y. Here we have used two approaches to characterize the PKC subtypes present in SH-SY5Y cells. Firstly, the PCR was used to show that SH-SY5Y cells contain mRNA encoding PKC subtypes alpha, beta, gamma, delta, epsilon and zeta. Secondly, immunoblotting showed that SH-SY5Y cells express PKC subtypes alpha, epsilon and zeta at the protein level. Prolonged (48 h) exposure of cells to the phorbol ester phorbol 12-myristate 13-acetate (PMA; 100 nM) resulted in a marked decrease in the amounts of PKC-alpha and PKC-epsilon, with no change in levels of PKC-zeta. Prolonged PMA treatment had no significant effect on K(+)-evoked NA release from SH-SY5Y cells, whereas carbachol-evoked release was increased 2.2-fold. However, prolonged exposure to PMA completely inhibited the ability of acute (12 min) PMA treatment to enhance both K(+)- and carbachol-evoked NA release. The specific PKC inhibitor RO 31-7459 (10 microM) was found to inhibit K(+)- and carbachol-evoked release by 27% and 68% respectively. RO 31-7549 also completely inhibited the ability of acute PMA treatment to enhance release. These data suggest that PKC-alpha and/or PKC-epsilon play an essential role in the regulation of PMA-enhanced K(+)- and carbachol-evoked NA release in SH-SY5Y cells.

Base Sequence↗
PubMed1993

Structure of chicken annexin V at 2.25-A resolution.

The crystal structure of chicken annexin V has been solved by molecular replacement and refined at 2.25 A. The final R factor is 19.7% with good geometry. The chicken annexin V structure is very similar to the human annexin V structure, with four similar domains each containing five helices. The structure includes three calcium ions in domains I, II, and IV, each bound by the characteristic K-G-X-G-T-(38 residues)-D/E motif. In view of the structural similarity between human and chicken annexin V, we suggest that they have a common vital function which developed early in evolutionary history.

Amino Acid Sequence↗
PubMed1993

The sub-cellular localization of annexin V in cultured chick-embryo fibroblasts.

The Ca(2+)- and phospholipid-binding protein, annexin V, has been shown by an immune assay to represent 0.4% of total cell protein in cultured chick-embryo fibroblasts. Immunofluorescent localization studies indicate that in primary cultures the protein is abundant in the cytoplasm of the cells and also extends into the nucleus. Nuclear staining is no longer detectable, however, in approx. 25% of the cells following sub-culture. Sub-populations of annexin V are associated with cytoskeletal structures and with the inner face of the plasma membrane in a Ca(2+)-independent manner. In addition, we report results indicating the secretion of annexin V from this cell type.

Animals↗
PubMed1993

Isolation, characterization and localization of annexin V from chicken liver.

Annexin V has been purified from chicken liver; 40 mg of annexin V was obtained per kg of tissue. In contrast with mammalian liver, very little annexin VI was obtained. Surprisingly, chicken liver annexin V resembles mammalian annexin IV in its M(r) (32,500) and its isoelectric point (5.6), but amino-acid-sequence analysis demonstrates identity with chicken annexin V (anchorin CII). It binds to phospholipids in a Ca(2+)-dependent manner with free-Ca2+ concentrations for half-maximal binding to phosphatidylserine and phosphatidic acid of 10 microM; phosphatidylethanolamine of 32 microM and phosphatidylinositol of 90 microM. No binding to phosphatidylcholine was observed at Ca2+ concentrations up to 300 microM. In isolated liver membranes a significant proportion of annexin V was not extractable with EGTA but could only be extracted with Triton X-100, suggesting the existence of a tightly membrane-associated form of annexin V. A specific antiserum to chicken annexin V was used to localize the protein in adult and embryonic chicken liver. In the adult, annexin V was highly concentrated in epithelial cells lining the bile ducts, and along the bile canaliculi. In embryonic liver, strong staining of the bile-duct epithelial cells was again evident, and in addition, endothelial cells were strongly immunoreactive.

Amino Acid Sequence↗