The Ca(2+)-modulated protein, S100B, is expressed in high abundance in and released by astrocytes. At the low levels normally found in the brain, extracellular S100B acts as a trophic factor, protecting neurons against oxidative stress and stimulating neurite outgrowth through its binding to the receptor for advanced glycation end products (RAGE). However, upon accumulation in the brain extracellular space, S100B might be detrimental to neurons. At relatively high concentrations, S100B stimulates NO release by microglia in the presence of lipid A or interferon-gamma (IFN-gamma). We analyzed further the S100B-microglia interaction to elucidate the molecular mechanism by which the protein brings about this effect. We found that S100B increased NO release by BV-2 microglia by stimulating reactive oxygen species (ROS) production and activating the stress-activated kinases, p38 and JNK. However, S100B stimulated NO production to the same extent in microglia overexpressing a transduction-incompetent mutant of RAGE and in microglia overexpressing full-length RAGE, with a significantly smaller effect in mock-transfected microglia. This suggests that the RAGE transducing activity has little or no role in S100B-stimulated NO production by microglia, whereas RAGE extracellular domain is important, probably serving to concentrate S100B on the BV-2 cell surface. On the other hand, S100B stimulated NF-kappaB transcriptional activity in BV-2 microglia in a manner that was strictly dependent on RAGE transducing activity, pointing to additional, RAGE-mediated effects of the protein on microglia that remain to be investigated.

We investigated the cellular distribution of annexin V (CaBP33) in rat tissues by immunohistochemistry. Several cell types were shown to express the protein. Glial cells in the cerebellum and in the optic nerve, the cornea! epitheium, the posterior epitheium in the iris, chondrocytes, skeletal muscle cells and cardiomyocytes, the capillary endotheial cells in many organs, the muscularis mucosae and the muscular layer in the intestinal tract, hepatocytes, Muller cells in the retina, the lens fibers, Sertoi and Leydig cells in the testis, and smooth muscle cells in the epididymis and bronchi displayed intense immunostaining. In the adrenal gland, only the cortex showed inununoreaction product. In the kidney, no apparent staining ofrenal cells was observed, whereas

The distribution of annexin V isoforms (CaBP33 and CaBP37) and of annexin VI in bovine lung, heart, and brain subfractions was investigated with special reference lo the fractions of these proteins which are membrane.bound. In addition to EGTA.extmetable pools of the above proteins, membranes from lung, heart, and brain contain EGTA-resistant annexins V and VI which can be solubilizcd whh detergents (Triton X-100 or Triton X-114). A strong base like Na:CO~, which is usually effective in extracting peripheral membrane proteins, only partially solubilizes the membranebound. EGTA-resistant annexins analyzed here. Also, only 50-60% of the Triton X-114-soluble ann¢xins partition in the aqueous phase, the remaining fraetion~ being recovered in the detergent-rich phase. Altogether, these findings suggest that, by an as ),et unknown mechanism, following Ca:'-dependent association of annexin V isoforms and annexin VI with membranes, substantial fractions of tbe~e proteins remain bound to membranes in a Ca:*-indepcndent way and behave like integral membrane proteins. These results further support the possibility that the above annexins might play a role in membrane trafficking and/or in the regulation of the structural organization of membranes.Annexin V (CaBP33 and CaBP37); Annexin Vl; Membrane; Binding; Calcium; Lun~g; Heart; Brain I, INTRODUCTIONProteins of the annexin family share the ability to bind to acidic phospholipids and to natural membranes in the presence of Ca:* (for reviews see [1][2][3]). Each annexin is made of an N-terminal tail of variable length and unique to individual annexins, which is supposed to play a role in the diversification of the biological functions of single species, and of a core. This latter is made of four, in the ease of the 32-37 kDa annexins, or eight, in the case of the 67-73 kDa annexins, internal repeats 70 residues in length, each of which contains a highly conserved consensus sequence, the endonexin fold, which is suggested to take part in the coordination of binding of both Ca 2+ and phospholipids [3][4][5]. Unlike the Ca-'+-binding pro~:eins of the EF-hand type, Ca-'*-binding to annexins does not induce the exposure of hydrophobic domains; rather~ Ca 2. would cross-bridge any annexin to the negatively charged headgroups of acidic phospholipids and/or certain annexins to target proteins [1][2][3]. Given the above model, it is expected that chelation of Ca 2÷ will result in the complete reversal of annexin binding. While this has been proven true whenever annexin binding to liposomes or proteins had been investigated in reconstitution experiments in vitro [6][7][8][9][10][11][12][13][14][15], different results were obtained with certain annexins whenever tissue or cell subfractionation was used to investigate the binding of endogenous annexins to natural membranes. So, fractions ofannexins I, 1I, IV-VII were reported to exist in several cell types in a membrane-bound form, to resist extraction with EGTA, and to require detergents for their solubilization [10,[16][17][18][19][2...

Porcine heart was observed to express annexins V (CaBP33) and VI in large amounts, and annexins III and IV in much smaller amounts. Annexin V (CaBP33) in porcine heart was examined in detail by immunochemistry.Homogenization and further processing of heart in the presence of EGTA resulted in the recovery of annexin V (CaBP33) in the cytosolic fraction and in an EGTA-resistant, Triton X-IOO-soluble fraction from 2+ cardiac membranes.Including Ca in the homogenization medium resulted in a significant decrease in the annexin V (CaBP33) content of the cytosolic fraction with concomitant increase in the content of this protein in myofibrils, mitochrondria, the sarcoplasmic reticulum and the sarcolemma. The amount of annexin V (CaBP33) in each of these subfractions depended on the free Ca*+ concentration in the homogenizing medium. At the lowest free Ca2+ concentration tested, 0.8 PM, only the sarcolemma appeared to contain bound annexin V (CaBP33). Membrane-bound annexins V (CaBP33) and VI partitioned in two fractions, one EGTA-resistant and Triton X-IOO-extractable, and one Triton X-loo-resistant and EGTA-extractable.Altogether, these data suggest that annexins V and VI are involved in the regulation of membrane-related processes.

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