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Hippocampal slices have been widely used to investigate electrophysiological and metabolic neuronal parameters, as well as parameters of astroglial activity including protein phosphorylation and glutamate uptake. S100B is an astroglial-derived protein, which extracellularly plays a neurotrophic activity during development and excitotoxic insult. Herein, we characterized S100B secretion in acute hippocampal slices exposed to different concentrations of K(+) and Ca(2+) in the extracellular medium. Absence of Ca(2+) and/or low K(+) (0.2 mM KCl) caused an increase in S100B secretion, possibly by mobilization of internal stores of Ca(2+). In contrast, high K(+) (30 mM KCl) or calcium channel blockers caused a decrease in S100B secretion. This study suggests that exposure of acute hippocampal slices to low- and high-K(+) could be used as an assay to evaluate astrocyte activity by S100B secretion: positively regulated by low K(+) (possibly involving mobilization of internal stores of Ca(2+)) and negatively regulated by high-K(+) (likely secondary to influx of K(+)).

Since constitutively-high intracellular Ca(2+) ([Ca(2+)](i)) may confer hypoglossal motoneurons special vulnerability to excitoxic damage, we investigated the spatiotemporal dynamics of [Ca(2+)](i) and its relation to spike firing of rat hypoglossal motoneurons recorded under whole-cell patch clamp coupled with high resolution [Ca(2+)](i) imaging. A rise in [Ca(2+)](i), appearing in conjunction with single action potentials and becoming larger during spike trains, was first detected immediately beneath the cell membrane area, peaked 10-20 ms after each spike, and propagated to the cell core with slow decay time. Depletion of ryanodine-sensitive [Ca(2+)](i) stores by caffeine increased background [Ca(2+)](i), augmented the spike medium afterhyperpolarization, slowed down the action potential firing rate and depolarized cells (after an early hyperpolarization). The decay time constant of [Ca(2+)](i) transients was more than doubled by caffeine, although peak [Ca(2+)](i) remained unchanged. It is suggested that the main role of caffeine-sensitive stores was to buffer [Ca(2+)](i) elevated by sustained firing and to control spike accommodation.

The subcellular localization of the Ca(2+)-modulated protein, S100A11, was investigated in the renal cell line LLC-PK1 by immunofluorescence and confocal laser scanning microscopy under varying experimental conditions. In control cells, S100A11 was detected on the plasma membrane, where the protein co-localized with annexin I (ANXA1) at discrete sites, and found diffusely in the cytoplasm. Elevation of the cytosolic Ca(2+) concentration by means of the Ca(2+) ionophore, ionomycin, caused a significant fraction of S100A11 to associate with vimentin intermediate filament (IF)-bound S100B, another member of the S100 protein family. Under these conditions, ANXA1 underwent a quite different kind of relocation. Translocation of S100A11 onto vimentin IF-bound S100B was also observed upon activation of protein kinase C (PKC). Under these conditions, S100A11 appeared to associate directly with vimentin IFs at cell sites displaying low or no abundance of S100B such as cell processes, and, again, S100A11 and ANXA1 underwent a different relocation. Our data suggest the possibility that the intracellular Ca(2+) level might regulate the subcellular localization of S100A11 and its interaction with definite target proteins, and that S100A11 might serve the function of modulating S100B activities. Interestingly, in spite of the known ability of S100A11 to form heterotetramers with ANXA1, the two proteins underwent a different relocation on elevation of the cytosolic Ca(2+) concentration or activation of PKC, pointing to different regulatory activities of individual proteins in renal cells., (Copyright 2003 Wiley-Liss, Inc.)

S100 is a multigenic family of non-ubiquitous Ca(2+)-modulated proteins of the EF-hand type expressed in vertebrates exclusively and implicated in intracellular and extracellular regulatory activities. Within cells, most of S100 members exist in the form of antiparallelly packed homodimers (in some cases heterodimers), capable of functionally crossbridging two homologous or heterologous target proteins in a Ca(2+)-dependent (and, in some instances, Ca(2+)-independent) manner. S100 oligomers can also form, under the non-reducing conditions found in the extracellular space and/or within cells upon changes in the cell redox status. Within cells, S100 proteins have been implicated in the regulation of protein phosphorylation, some enzyme activities, the dynamics of cytoskeleton components, transcription factors, Ca(2+) homeostasis, and cell proliferation and differentiation. Certain S100 members are released into the extracellular space by an unknown mechanism. Extracellular S100 proteins stimulate neuronal survival and/or differentiation and astrocyte proliferation, cause neuronal death via apoptosis, and stimulate (in some cases) or inhibit (in other cases) the activity of inflammatory cells. A cell surface receptor, RAGE, has been identified on inflammatory cells and neurons for S100A12 and S100B, which transduces S100A12 and S100B effects. It is not known whether RAGE is a universal S100 receptor, S100 members interact with other cell surface receptors, or S100 protein interaction with other extracellular factors specifies the biological effects of a given S100 protein on a target cell. The variety of intracellular target proteins of S100 proteins and, in some cases, of a single S100 protein, and the cell specificity of expression of certain S100 members suggest that these proteins might have a role in the fine regulation of effector proteins and/or specific steps of signaling pathways/cellular functions. Future analyses should discriminate between functionally relevant S100 interactions with target proteins and in vitro observations devoid of physiological importance.

Members of the annexin protein family interact with members of the S100 protein family thereby forming heterotetramers in which an S100 homodimer crossbridges two copies of the pertinent annexin. Previous work has shown that S100A1 and S100B bind annexin VI in a Ca(2+)-dependent manner and that annexin VI, but not annexin V, blocks the inhibitory effect of S100A1 and S100B on intermediate filament assembly. We show here that both halves of annexin VI (i.e., the N-terminal half or annexin VI-a and the C-terminal half or annexin VI-b) bind individual S100s on unique sites and that annexin VI-b, but not annexin VI-a, blocks the ability of S100A1 and S100B to inhibit intermediate filament assembly. We also show that the C-terminal extension of S100A1 (and, by analogy, S100B), that was previously demonstrated to be critical for S100A1 and S100B binding to several target proteins including intermediate filament subunits, is not part of the S100 surface implicated in the recognition of annexin VI, annexin VI-a, or annexin VI-b. Evaluation of functional properties with a liposome stability and a calcium influx assay reveals the ability of both S100 proteins to permeabilize the membrane bilayer in a similar fashion like annexins. When tested in combinations with different annexin proteins both S100 proteins mostly lead to a decrease in the calcium influx activity although not all annexin/S100 combinations behave in the same manner. Latter observation supports the hypothesis that the S100-annexin interactions differ mechanistically depending on the particular protein partners.

The Ca2+-modulated, dimeric proteins of the EF-hand (helix-loop-helix) type, S100A1 and S100B, that have been shown to inhibit microtubule (MT) protein assembly and to promote MT disassembly, interact with the type III intermediate filament (IF) subunits, desmin and glial fibrillary acidic protein (GFAP), with a stoichiometry of 2 mol of IF subunit/mol of S100A1 or S100B dimer and an affinity of 0.5-1.0 microM in the presence of a few micromolar concentrations of Ca2+. Binding of S100A1 and S100B results in inhibition of desmin and GFAP assemblies into IFs and stimulation of the disassembly of preformed desmin and GFAP IFs. S100A1 and S100B interact with a stretch of residues in the N-terminal (head) domain of desmin and GFAP, thereby blocking the head-to-tail process of IF elongation. The C-terminal extension of S100A1 (and, likely, S100B) represents a critical part of the site that recognizes desmin and GFAP. S100B is localized to IFs within cells, suggesting that it might have a role in remodeling IFs upon elevation of cytosolic Ca2+ concentration by avoiding excess IF assembly and/or promoting IF disassembly in vivo. S100A1, that is not localized to IFs, might also play a role in the regulation of IF dynamics by binding to and sequestering unassembled IF subunits. Together, these observations suggest that S100A1 and S100B may be regarded as Ca2+-dependent regulators of the state of assembly of two important elements of the cytoskeleton, IFs and MTs, and, potentially, of MT- and IF-based activities.

We investigated the expression and the subcellular localization of S100A1 and S100B, two Ca(2+)-binding proteins of the EF-hand type, in replicating myoblasts and fused myotubes. Northern blot and reverse transcriptase-polymerase chain reaction analyses revealed the presence of S100A1 mRNA and S100B mRNA respectively, in myoblasts. Immunofluorescence and immunogold electron microscopy were used to localize individual proteins in myoblasts and myotubes. In the present report we document that: (1) in replicating myoblasts S100B is localized to intracellular membranes, including Golgi membranes, vimentin intermediate filaments (IFs) and microtubule (MT) structures; (2) in the same cells S100A1 is found associated with intracellular membranes; (3) following treatment of replicating myoblasts with colchicine, a fraction of S100B remains colocalized with bundled and collapsed vimentin IFs, whereas another fraction follows the destiny of endoplasmic membranes; (4) under the same conditions S100A1, like a fraction of S100B, follows the collapse of the endoplasmic reticulum around the nucleus; and (5) in fused myotubes S100A1 is found diffusely in the cytoplasm, whereas S100B is mostly found associated with vimentin IFs. These data suggest that in the skeletal myogenic cell line used in the present study S100A1 and S100B might share binding sites on or close to intracellular membranes, but display a significant degree of target specificity with respect of IFs and MTs. The results of these analyses suggest that expression of S100B in skeletal muscle cells may be developmentally regulated and lend support to the possibility that S100B might regulate the MT and IF dynamics.

The Ca2(+)-binding proteins of the EF-hand type, S100B and S100A1, were detected in the outer segment of bovine retina photoreceptors where they are localized to disc membranes, as investigated by immunofluorescence and immunogold cytochemistry. S100B and S100A1 stimulate a membrane-bound guanylate cyclase activity associated with photoreceptor disc membranes in dark-adapted retina in a Ca2(+)-dependent manner, although with different Ca2+ requirements, as investigated by an ultracytochemical approach. Other retinal cell types express S100B and S100A1 as well. S100B is detected in the outer limiting membrane, fine cell processes in the outer nuclear layer and the outer plexiform layer, cell bodies in the inner nuclear layer and the ganglion cell layer, and the inner limiting membrane, whereas S100A1 has a more discrete distribution. S100B and S100A1 also stimulate a membrane-bound guanylate cyclase activity in photoreceptor cell bodies and Muller cells, but their effect appears independent of the light- or dark-adapted state of the retina and is observed at relatively high Ca2+ concentrations. These data represent the ultrastructural counterpart of recent biochemical observations implicating S100B and, possibly, S100A1 in the Ca2(+)-dependent stimulation of a photoreceptor membrane-bound guanylate cyclase activity [T. Duda, R. M. Goraczniak and R. K. Sharma (1996) Molecular characterization of S100A1-S1000B protein in retina and its activation mechanism of bovine photoreceptor guanylate cyclast. Biochemistry 35, 6263-6266; A. Margulis, N. Pozdnyakov and A. Sitaramayya (1996) Activation of bovine photoreceptor guanylate cyclast by S100 proteins. Biochem. Biophys. Res. Commun. 218, 243-247]. Our data suggest that at least S100B may take part in the regulation of a membrane-bound guanylate cyclase-based signalling pathway in both photoreceptors and Muller cells.

Phase-modulation fluorescence lifetime measurements were used to study the single Trp residue of the Ca(2+)-binding protein S-100a both in the absence and in the presence of Ca2+ and/or Mg2+. Trp fluorescence decay for the protein was satisfactorily described by Lorentzian lifetime distributions centered around two components (approximately 4 ns and 0.5 ns). Lifetime values were unchanged by 2 mM Ca2+, but the fractional intensity associated with longer lifetime increased up to 75%. In the presence of Mg2+, the Ca2+ induced increase of the fractional intensity associated with longer lifetime was only 57%. For the protein in buffer, about the 85% of the recovered anisotropy was associated to a rotational correlation time of 6.7 ns. After the addition of Ca2+, this value was increased to 16.08 ns. In the presence of Mg2+, Ca+2 increased the rotational correlation time to 33.75 ns. Similar studies were performed with S-100a interacting with egg phosphatidylcholine vesicles (SUV). Our data suggest that the conformation of the protein may be influenced by structural features of the lipidic membrane. Moreover, data obtained in the presence of Mg2+ indicate some interaction between lipids and S-100, likely mediated by this ion.

Annexin II2-p11(2) (calpactin I) was tested as a potential regulator of GFAP assembly into glial filaments (GF), following the observation that it interacts with GFAP and cosediments with GF in a sedimentation assay. Under conditions where GFAP assembly is reduced, e.g., at pH values > 6.8, annexin II2-p11(2) stimulates GF formation in a Ca(2+)- and dose-dependent manner. Concomitantly, an ever larger fraction of annexin II2-p11(2) can be recovered in GF pellets as the pH is raised from 6.8 to 7.35. Monomeric annexin II also stimulates GFAP assembly, although with a smaller efficacy as compared to annexin II2-p11(2), but does not cosediment with GF to a large extent, whereas p11 neither cosediments with GF nor affects GFAP assembly. On the other hand, the in vitro reconstituted annexin II2-p11(2) heterotetramer mimics native annexin II2-p11(2), and perturbation of the integrity of annexin II2-p11(2) by a mild treatment with alpha-chymotrypsin results in the nearly complete abolition of the stimulatory effect of annexin II2-p11(2) on GFAP assembly. These data suggest that annexin II2-p11(2) might be involved in the regulation of the state of assembly of GF, possibly in concert with other proteins.

Calpactin I, a heterotetrameric, cytoskeletal protein complex composed of two copies of annexin II cross-linked by two copies of p11, an S100-like protein, binds to the glial fibrillary acidic protein (GFAP) and cosediments with glial filaments (GF) in a Ca(2+)-dependent manner, apparently without affecting GFAP polymerization under the present experimental conditions. Cosedimentation of calpactin I with GF, which occurs at micromolar free Ca2+ concentrations, is proportional to the concentrations of both calpactin I and GFAP and does not occur under conditions where GFAP assembly is maximally inhibited by, e.g., S100 protein. Annexin II also cosediments with GF and binds to GFAP, although to much smaller extents. Other annexins, such as annexins I, V, and VI, or p11 do not bind to either GF or GFAP. Calpactin I and S100 protein bind to different sites on GFAP, as investigated by fluorescence spectroscopy using acrylodan-labeled GFAP. Calpactin I and S100 protein might act, in the presence of Ca2+, in a concerted manner to determine the number and topography of GF in differentiating and/or mature glial cells.

S-100 protein, a Ca(2+)-binding protein of the EF-hand type, interacts with the glial fibrillary acidic protein (GFAP) in a Ca(2+)-dependent manner. The binding of S-100 protein to GFAP was investigated by fluorescence spectroscopy using acrylodan-S-100 protein and cross-linking experiments using the bifunctional cross-linker, disuccinimidyl suberate. The binding affinity was observed to be in the nanomolar range with a stoichiometry of 2 mol of GFAP/mol of S-100 protein (dimer). S-100 protein was found to inhibit the polymerization of GFAP in a dose- and Ca(2+)-dependent manner, with a half-maximal effect at an S-100 protein/GFAP molar ratio of 0.2 and maximal effect at a molar ratio of 0.5. Identical results were obtained irrespective of whether the unfractionated bovine brain S-100 protein mixture (S-100a plus S-100b), S-100ao, S-100a, or S-100b was used. S-100 protein was observed to be maximally effective as an inhibitor of GFAP polymerization at approximately 3 microM free Ca2+. Calmodulin neither bound to GFAP nor inhibited its polymerization. Altogether, the present results suggest that S-100 protein might be involved in the regulation of the state of assembly of glial filaments by binding to and sequestering unpolymerized GFAP.

We have used phase-modulation fluorescence lifetime measurements to study the single Trp residue of the Ca(2+)-binding protein S-100a. Trp fluorescence decay was not exponential for the protein irrespective of the absence or presence of Ca2+. Fluorescence decay was best described by Lorentzian lifetime distributions centered around two components (approx. 3 and 0.7 ns) for protein in absence of Ca2+ and one component (approx. 2.9 ns) for the protein in presence of 2 mM Ca2+. Similar studies were performed with S-100a interacting with cardiolipin, phosphatidylserine or egg phosphatidylcholine, both in absence and in presence of 2 mM Ca2+. Our data suggest that the conformation of the protein and its Ca(2+)-binding properties vary depending on the characteristics of charge and structure of phospholipids.

The S-100 protein family constitutes a subgroup of Ca(2+)-binding proteins of the EF-hand type comprising three dimeric isoforms, S-100a0, S-100a and S-100b, plus a number of structurally related proteins displaying 28-55% homology with S-100 subunits. S-100 protein was discovered in 1965; yet, its biological functions have not been fully elucidated. The present report will review the putative biological roles of S-100 protein. Both intracellular and extracellular roles have been proposed for S-100 protein. Within cells, S-100 protein has been reported to regulate protein phosphorylation, ATPase, adenylate cyclase, and aldolase activities and Ca(2+)-induced Ca2+ release. Also, cytoskeletal systems, namely microtubules and microfilaments have been reported to be regulated by the protein in the presence of Ca2+. Some molecular targets of S-100 protein within cells, have been identified. This is the case with microtubule proteins, caldesmon, and a brain aldolase. S-100 protein has been reported to be secreted; extracellular S-100 protein can stimulate neuronal differentiation, glial proliferation, and prolactin secretion. However, the mechanisms by which S-100 is secreted and stimulates the above processes are largely unknown. Future research should characterize these latter aspects of S-100 biology and find out the linkage between its intracellular effects and its extracellular activities.

Two acidic Ca2(+)-binding proteins (CaBP33 and CaBP37) purified from bovine brain have been characterized in terms of immunological properties, heat-sensitivity, electrophoretic mobility, and Ca2(+)-dependent binding to negatively charged phospholipids and to brain membranes. They were induced to bind to membranes by homogenization of brain tissue in the presence of CaCl2. The membrane-bound CaBP33/CaBP37 mixture resisted extraction with detergents and was solubilized with high concentrations of EGTA/KCl. However, apparent Ca2(+)-independent binding of the two proteins to membranes seemed to occur as well. This latter fraction of membrane-bound CaBP33 and CaBP37 could be solubilized with Triton X-100, indicating that brain membranes normally contain the two proteins as intrinsic components.

The interaction of S-100b protein with cardiolipin (CL) vesicles has been studied by electron spin resonance, pyrene fluorescence, and circular dichroism. Electron spin resonance and pyrene fluorescence data indicate that S-100b binds to the polar surface of vesicles Ca2+-independently. In the presence of Ca2+, S-100b potentiates the Ca2+-induced clustering of the polar headgroups of CL molecules and causes a further reduction in the Ca2+-dependent decrease in the lateral mobility of the pyrene inserted into the lipid bilayer, which points to an effect of the protein on the hydrophobic core of the lipid bilayer through a larger perturbation of its polar surface. Circular dichroism analyses indicate that CL vesicles cause a decrease in the alpha-helical content of S-100b, analogous to that produced by Ca2+ and that the effects of CL vesicles and of Ca2+ on the secondary structure of the protein are supra-additive. By this technique, we found that the affinity of Ca2+ for S-100b increases substantially in the presence of CL vesicles, even in the presence of physiologic concentrations of KCl, suggesting that once S-100b had interacted with CL vesicles it assumes a new conformation in which its Ca2+-binding properties are greatly enhanced. These results are discussed in relation to binding of S-100b proteins to natural membranes, and to a possible involvement of S-100b in the regulation of membrane structural organization.

S-100a0 protein, the alpha alpha-isoform of the S-100 family, stimulates Ca2+-induced Ca2+ release from terminal cisternae isolated from rat skeletal muscle cells. The stimulatory effect of S-100a0 is maximal at approximately 5 microM S-100a0 and half maximal at approximately 0.1 microM S-100a0, at 1.8 microM free Ca2+ in the presence of 5 mM Mg2+ plus 0.1 M KCl. The effect of the protein on Ca2+-induced Ca2+ release is completely inhibited by the calcium release blocker, ruthenium red.

We have examined the S-100-chlorpromazine interplay at the level of brain microtubule proteins in vitro. The results indicate that in the presence of 0.12 M KCl and 10 microM free Ca2+ the inhibitory effect of S-100 on microtubule assembly is additive to that of chlorpromazine, but S-100 fails to potentiate the disassembling effect of 0.1 mM Ca2+ if added to assembled microtubule proteins after chlorpromazine and Ca2+, probably because of inhibition of S-100 by the phenothiazine. Chlorpromazine does not compete with S-100 for binding to purified tubulin.

In the presence of the usual 0.1 M Mes buffer, pH 6.7, mM free Ca2+ levels are required for half-maximal decrease in the rate and extent of brain microtubule protein (MTP) assembly in the absence of ox brain S-100, while microM free Ca2+ levels are sufficient in the presence of S-100. At the same pH 6.7, but in the presence of 0.12 M KCl, as low as 1.5 microM free Ca2+ is sufficient for S-100 to produce half-maximal reduction in the rate of assembly, while as high as 0.5 mM free Ca2+ is required in the absence of S-100. Similar results are obtained with rat brain S-100 (S-100b), indicating that single S-100 iso forms are equipotent in affecting the MTP assembly. At pH 7.5, MTPs are remarkably resistant to Ca2+ in the absence of S-100. In the presence of S-100, not only is the free Ca2+ concentration required for complete inhibition of assembly at least one order of magnitude smaller than that required in the absence of S-100, but significant S-100-dependent inhibition of assembly occurs in the absence of Ca2+. Under the two conditions where S-100 is particularly effective in inhibiting the assembly, i.e. at pH 6.7 in the presence of KCl and at pH 7.5, S-100 increases the disassembly rate even in the presence of microM Ca2+ levels. Our results suggest that the free Ca2+ concentration regulates the way S-100 disassembles microtubules (MTs): at microM Ca2+ levels, S-100 sequesters tubulin with concomitant increase in the disassembly rate; at mM Ca2+ levels, the S-100-Ca2+ complex probably interacts with MTs producing endwise disassembly.

At alkaline pH, Ca2+ is no longer required for S-100 proteins to inhibit the assembly and to promote the disassembly of brain microtubules in vitro, though the presence of Ca2+ significantly favors the S-100 effects. These effects are inversely related to the microtubule protein concentration and directly related to the S-100 concentration and the pH. Ca2+-independent, pH-regulated inhibition of assembly of phosphocellulose-purified tubulin by S-100 is also described. The microtubule disassembling effect of S-100 is additive to that of alkali (used to raise the pH), and S-100 further disassembles microtubules after alkalinization. Thus the larger inhibitory effect of S-100 on microtubule assembly at alkaline versus acid pH depends on both a decrease in the assembly rate and an increase in the disassembly rate. Together with previous data on this topic, the present findings indicate that S-100 proteins act on microtubule protein in vitro primarily by binding to tubulin, this event being Ca2+-regulated at a given pH, and pH-regulated at a given free Ca2+ concentration.

The ability of S-100 proteins to inhibit the assembly of brain microtubule proteins (MTPs) in the presence of microM levels of Ca2+ increases as a function of pH. This seems to be due to an increasingly larger inhibitory effect of S-100 on the nucleation and, probably, on the elongation of microtubules (MTs) as the pH raises. In the presence of microM Ca2+ levels, the ability of S-100 to disassemble MTs also increases linearly with the pH, suggesting that the larger inhibitory effect of S-100 on MTP assembly at alkaline than at acidic pH may depend on both a decrease in the assembly rate and an increase in the disassembly rate. Also, S-100 inhibits the assembly of phosphocellulose-purified tubulin to a larger and larger extent as the pH raises. S-100 brings about its effect on MT assembly-disassembly probably by sequestering soluble tubulin, though additional mechanisms cannot be excluded. The present data are briefly discussed in relation to the role attributed to changes in intracellular pH in the regulation of the state of assembly of cytoplasmic MTs.