0 SK&F 97426-A is a novel bile acid sequestrant that is threefold more potent than cholestyramine at increasing bile acid excretion in the hamster. SK&F 97426-A is a quaternary alkylammonium polymethacrylate that was selected for comparison with cholestyramine in vivo because of its superior in vitro bile acid binding properties. Association, dissociation, affinity, and capacity experiments were performed under physiologically relevant conditions with the most abundant bile acids found in human bile. The bile acids came to equilibrium with SK&F 97426-A and cholestyramine within ∼30 min and 6 min, respectively. SK&F 97426-A and cholestyramine had similar capacities for all the bile acids (between 2.5 and 4 mmol/g) and both had similar, very high affinities and slow dissociation rates for the dihydroxy bile acids. However, SK&F 97426-A had much higher affinities for the trihydroxy bile acids glycocholic acid and taurocholic acid than did cholestyramine. Dissociation of glycocholic acid and taurocholic acid from SK&F 97426-A was also much slower (27 and 25%, respectively, dissociated after 60 min) than from cholestyramine (89 and 84%, respectively, dissociated after 60 min). The higher affinities and slower dissociation rates of the trihydroxy bile acids for and from SK&F 97426-A probably account for the increased potency of SK&F 97426-A over cholestyramine in vivo.

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 p1 values similar to those of annexins I, II, Ill, 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 Kdepolarisation 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 nonphospholipid 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.

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The annexins are a family of structurally related calcium dependent membrane-binding proteins [ I ] which can be shown to bind to phospholipid membranes in the presence of calcium [2].The annexins have been identified in a wide range of tissues from many species yet their physiological roles have not been clearly elucidated. They have been implicated in a variety of important cellular processes [3]. In particular annexin I1 seems to be a regulatory factor in the control of exocytosis [4,5].Annexin 11 is one of the best characterised components of the annexin family, occurring as a 36kD monomer and as a 90kDheterotetramer complex, containing two molecules of the 36kD heavy chain and two molecules of an 1 I k D light chain [6]. Evidence for the involvement of annexin 11 in the regulation of exocytosis is provided by the observations that the 36kD molecule can restore secretion in digitonin permeablised chromaffin cells [4] and that the annexin heterotetramer can restore secretion in streptolysin-0 permeabilised chromaffin cells, but must be phosphorylated by protein kinase C (PKC) to be effective [ 5 ] . The annexin 11 heavy chain is a major substrate for P K C and is phosphorylated at Ser-25 in the NH2 terminal region [7]. Annexin 11 has also been shown to promote the aggregation of chromaffin cell secretory granule membranes in a calcium dependent manner [8], and electron microscopic studies have shown that annexin 11 is concentrated between chromaffin granules and the plasma membrane in chromaffin cells [9].Previous studies by our group have shown that the human neuroblastoma SH-SY5Y expresses depolarisation and muscarinic M, receptor subtype evoked release of noradrenaline [lo]. Acute pretreatment with the PKC activating phorbol ester 12-0tetradecanoylphorbol-13-acetate (TPA) enhanced noradrenaline release [ I I], whereas addition of the selective PKC inhibitors R o 31 -7549 and Ro 31 -8220 inhibited noradrenaline release, indicating that PKC plays an important role in the release of noradrenaline from SH-SYSY.T h e first step in the investigation of the role of annexins in release of noradrenaline from SH-SY5Y was to determine which annexins were present in the cell. Annexins were isolated from SH-SYSY utilising the reversible calcium dependent binding properties of annexins to membrane phospholipids [ 121. These isolated annexins were separated on a two dimensional SDS PAGE gel and analysis of protein molecular weight and isoelectric points enabled the tentative identification of annexins l,ll,lV,V and VI by comparison with bovine lung annexins [12]. These results were confirmed by Western blot analysis of SH-SY5Y total homogenate. SH-SY5Y cells were harvested in Laemmli buffer and proteins separated by 12.5% SDS PAGE [I31 before transfer to polyvinylidene difluoride membranes. After blocking with 5% non fat milk solution, membranes were incubated with monoclonal antibodies to annexins I,II.IV and VI or with an affinity purified polyclonal antibody to annexin V. Immunolabelled proteins were identified by a p...