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Much of our current understanding about neurodegenerative diseases can be attributed to the study of inherited forms of these disorders. For example, mutations in the presenilin 1 and 2 genes have been linked to early onset familial forms of Alzheimer's disease (FAD). Using the Drosophila central nervous system as a model we have investigated the role of presenilin in one of the earliest cellular defects associated with Alzheimer's disease, intracellular calcium deregulation. We show that expression of either wild type or FAD-mutant presenilin in Drosophila CNS neurons has no impact on resting calcium levels but does give rise to deficits in intracellular calcium stores. Furthermore, we show that a loss-of-function mutation in calmodulin, a key regulator of intracellular calcium, can suppress presenilin-induced deficits in calcium stores. Our data support a model whereby presenilin plays a role in regulating intracellular calcium stores and demonstrate that Drosophila can be used to study the link between presenilin and calcium deregulation.

S100A4, also known as mts1, is a member of the S100 family of Ca2+-binding proteins that is directly involved in tumor invasion and metastasis via interactions with specific protein targets, including nonmuscle myosin-IIA (MIIA). Human S100A4 binds two Ca2+ ions with the typical EF-hand exhibiting an affinity that is nearly 1 order of magnitude tighter than that of the pseudo-EF-hand. To examine how Ca2+ modifies the overall organization and structure of the protein, we determined the 1.7 A crystal structure of the human Ca2+-S100A4. Ca2+ binding induces a large reorientation of helix 3 in the typical EF-hand. This reorganization exposes a hydrophobic cleft that is comprised of residues from the hinge region,helix 3, and helix 4, which afford specific target recognition and binding. The Ca2+-dependent conformational change is required for S100A4 to bind peptide sequences derived from the C-terminal portion of the MIIA rod with submicromolar affinity. In addition, the level of binding of Ca2+ to both EF-hands increases by 1 order of magnitude in the presence of MIIA. NMR spectroscopy studies demonstrate that following titration with a MIIA peptide, the largest chemical shift perturbations and exchange broadening effects occur for residues in the hydrophobic pocket of Ca2+-S100A4. Most of these residues are not exposed in apo-S100A4 and explain the Ca2+ dependence of formation of theS100A4-MIIA complex. These studies provide the foundation for understanding S100A4 target recognition and may support the development of reagents that interfere with S100A4 function.

Annexin 2 is a calcium-dependent phospholipid-binding protein that has been implicated in a number of membrane-related events, including regulated exocytosis. In chromaffin cells, we previously reported that catecholamine secretion requires the translocation and formation of the annexin 2 tetramer near the exocytotic sites. Here, to obtain direct evidence for a role of annexin 2 in exocytosis, we modified its expression level in chromaffin cells by using the Semliki Forest virus expression system. Using a real-time assay for individual cells, we found that the reduction of cytosolic annexin 2, and the consequent decrease of annexin 2 tetramer at the cell periphery, strongly inhibited exocytosis, most likely at an early stage before membrane fusion. Secretion also was severely impaired in cells expressing a chimera that sequestered annexin 2 into cytosolic aggregates. Moreover, we demonstrate that secretagogue-evoked stimulation triggers the formation of lipid rafts in the plasma membrane, essential for exocytosis, and which can be attributed to the annexin 2 tetramer. We propose that annexin 2 acts as a calcium-dependent promoter of lipid microdomains required for structural and spatial organization of the exocytotic machinery.

Acute expression of recombinant proteins throughout a population of postmitotic bovine chromaffin cells was achieved using the Semliki Forest virus expression system (P. Liljestrom and H. Garoff (1991) Biotechnology 9:1356-1361). The virus was modified to express a green fluorescent protein, which faithfully reported the expression of the recombinant proteins. Two types of reporting virus were constructed: the first included a second subgenomic element, and the second an internal ribosome entry site. Both were used to express the recombinant proteins beta-galactosidase, 5HT3 receptor, or tetanus toxin light chain. Beta-galactosidase was used to quantify the rate of expression of recombinant protein in chromaffin cells, the 5HT3 receptor to trigger secretion, and the toxin to block secretion. The experiments clearly show that infection and expression of recombinant proteins throughout a population of chromaffin cells do not, per se, affect the rate and extent of triggered exocytosis, endocytosis, or membrane recycling pathways. The catecholamine content of the cell is unaltered, and the secretory mechanism can be accessed within a few hours after infection. This noncytopathic method of acutely expressing specific proteins at physiological levels in chromaffin cells offers a powerful new tool for dissecting the roles of many proteins implicated in exo- and endocytosis.

The intracellular requirements for membrane recapture in permeabilized chromaffin cells were compared to the requirements for exocytosis from the same cells. In permeabilized bovine chromaffin cells, calcium-driven exocytosis also triggers, with a short delay, uptake of extracellular horseradish peroxidase (HRP). This internalized HRP remains compartmentalized within the cell and migrates to a low density band on a Percoll gradient which is distinct from the heavier chromaffin granules. The amount of horseradish peroxidase internalized is similar in intact and leaky cells and is approximately equivalent to the volumes secreted. Endocytosis in both preparations is blocked by botulinum toxin, operates in a collapsed membrane potential, and is inhibited by low temperature. In permeabilized cells, exocytosis and coupled endocytosis are activated by the same concentrations of Ca2+ and MgATP. Although secretion requires Ca2+ and MgATP, once exocytosis has occurred the subsequent endocytosis can proceed in the virtual absence of Ca2+ or MgATP, and is largely unaffected by a variety of nucleotide triphosphates (including nonhydrolyzable analogues), and cyclic nucleotides. These data suggest that endocytosis can proceed, once exocytosis has been triggered, under conditions that are quite different from those necessary to support exocytosis, and that the specific requirements for Ca2+ and MgATP in secretion are for the exocytotic limb of the secretory cycle rather than for the associated endocytotic pathway.

The systems responsible for phosphorylating tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosynthesis, were investigated in situ in adrenal medullary cells made permeable to solutes of up to 1,000 dalton by exposure to brief intense electric fields. Two different phosphorylation systems were found. One is dependent on Ca2+, the other on cyclic AMP. The Ca2+-dependent system is half-maximally activated by 1-2 microM Ca2+ and 0.5 mM ATP, and follows a time course similar to that of secretion of catecholamines. Trifluoperazine (0.1 mM) does not inhibit significantly Ca2+-dependent phosphorylation of tyrosine hydroxylase in situ. The cyclic AMP-dependent system is half-maximally activated by addition of 0.5 microM cyclic AMP and about 0.3 mM ATP. Ca2+-dependent and cyclic AMP-dependent phosphorylations of tyrosine hydroxylase have roughly the same time course and are additive under conditions where one system is already saturated. Peptide maps of immunoprecipitated tyrosine hydroxylase, after in situ phosphorylation of the enzyme either in the presence of 10(-8)M Ca2+ plus 2 X 10(-5)M cyclic AMP or of 10(-5)M Ca2+, show a marked difference indicating that the enzyme contains several phosphorylation sites. At least one of these sites is phosphorylated only by the Ca2+-dependent system, whereas the other site(s) are phosphorylated by both the Ca2+- and cyclic AMP-dependent systems. The effect of in situ phosphorylation of tyrosine hydroxylase on its enzymatic activity was also investigated.

When isolated bovine adrenal medullary cells are incubated with the lipid-soluble Quin 2 acetoxymethyl ester, the ester permeates the plasma membrane and enters the cytosol, where it is hydrolysed by endogenous enzymes to yield an impermeant fluorescent indicator (Quin 2) which is sensitive to Ca2+ in the 0.1 microM range. This technique permits the average intracellular free Ca2+ level ([Ca2+]i) to be determined in a suspension of adrenal medullary cells. Unstimulated cells have a [Ca2+]i of 97 +/- 4 nM (n = 69). This level seems independent of extracellular calcium in the range 0.5-2 mM. When the extracellular calcium concentration is lowered to ca. 10(-7) M, however, [Ca2+]i decreases. A transient increase in [Ca2+]i occurs when cells are challenged with either acetylcholine or a high potassium medium. The time course of the [Ca2+]i transient rises to a maximum within seconds, and decreases to basal levels over minutes. The maximum level of [Ca2+]i associated with secretion is very variable. Hexamethonium, methyoxyverapamil, and the absence of extracellular calcium block not only the secretory response but also the [Ca2+]i transient. The action of acetylcholine leading to the Ca2+]i transient is blocked when cells are suspended in a depolarizing medium. Extracellular magnesium inhibits both the [Ca2+]i transient and the secretory response evoked by acetylcholine. Secretion is, however, more sensitive to magnesium inhibition than is calcium entry. The magnitudes of the [Ca2+]i transient and the secretory response decrease as the concentration of intracellular Quin 2 increases. Measurements of the amount of indicator titrated with calcium, as a result of an acetylcholine or potassium challenge, suggest that the increase in the apparent calcium content of the cytosol might arise from two contributing sources of calcium entry.

Enzyme digestion of rat pancreatic tissue yielded a preparation of isolated acinar cells, over 90% of which excluded trypan blue. These isolated cells responded to a variety of secretagogues, the responses being sensitive to the removal of extracellular calcium, increasing extracellular magnesium, and by trifluoperazine, an antagonist of Ca-dependent processes. When exposed to intense electric fields, isolated acinar cells became permeable to CaEGTA and MgATP, these markers gaining access to over 60% of the intracellular milieu within minutes. The accessibility to these markers seemed independent of the ionised Ca2+ level. Less than 0.5% of the cellular amylase was released when cells were rendered leaky in a medium containing about 10(-9) M Ca2+, but typically 4% was released when the Ca2+ level was subsequently raised to 10(-5)M levels, the EC50 for Ca2+ being 2 microM. This amount of amylase released was comparable to the amounts secreted from intact cells in response to a variety of agonists. The cytosolic marker lactate dehydrogenase was also released from leaky cells, but the extent was independent of Ca2+ concentration. No amylase was released at 10(-7)M Ca2+ when permeable cells were exposed to cyclic 3',5'-AMP or cyclic 3',5'-GMP. The calcium activation curve for amylase release seemed to be independent of cyclic nucleotides, but was markedly increased in both the extent of release and apparent affinity for Ca2+ in the presence of the phorbol ester 12-0-tetradecanoyl phorbol 13 acetate. These results suggest that when "functionally normal" isolated acinar cells are rendered permeable, Ca2+-but not cyclic nucleotides-acts as a second messenger for amylase secretion, and furthermore that protein kinase C may be involved in the secretory process.

Exposure of 'leaky' bovine adrenal medullary cells to the phorbol ester TPA causes a shift in the calcium-activation curve to lower calcium concentrations without altering the levels of secretion at the extremes of the activation curve. These results are consistent with a role for protein kinase C in exocytosis.

Addition of thrombin enhances secretion of both [14C]serotonin and beta-N-acetylglucosaminidase induced by Ca2+ in human platelets rendered permeable by exposure to intense electric fields. Enhancement of beta-N-acetylglucosaminidase secretion by thrombin results from an increase in the maximal extent of the response with no significant change in the median effective concentration EC50 for Ca2+. In contrast, thrombin shifts the dose/response curve for Ca2+-induced [14C]serotonin secretion to the left and has little effect on the maximal extent of this response even when this extent is reduced by use of a non-saturating concentration of MgATP2-. The relationship between extent of response and [MgATP2-] is similar for secretion of [14C]serotonin and of beta-N-acetylglucosaminidase in the presence or absence of thrombin. Similar nucleotide specificities are also observed. Activators of protein kinase C have previously been shown to mimic quantitatively the effect of thrombin on [14C]serotonin secretion induced by Ca2+ [D. E. Knight & M. C. Scrutton (1984) Nature (Lond.) 309, 66-68]. Such activators have the same qualitative effect as thrombin on the properties of beta-N-acetylglucosaminidase secretion induced by Ca2+ but are less effective. The EC50 for thrombin observed for enhancement of [14C]serotonin and beta-N-acetylglucosaminidase secretion is in the same range as that obtained for intact platelets under comparable conditions [D. E. Knight, T. J. Hallam & M. C. Scrutton (1982) Nature (Lond.) 296, 256-257]. The EC50, and the specificity of response, observed for activators of protein kinase C in these systems are consistent with those reported previously for the purified enzyme. Addition of 1-10 microM Ca2+ to permeabilised platelets in the presence of [gamma-32P] ATP causes marked enhancement of 32P incorporation into polypeptides of molecular mass 20 kDA, 45 kDA and 66 kDA. No additional polypeptides become phosphorylated in this system when thrombin is added together with 10 microM Ca2+, but some increase is observed in the extent of phosphorylation of the 45-kDa polypeptide. Addition of 1-oleyl-2-acetylglycerol + 1 - 2 microM Ca2+ causes enhanced phosphorylation of the 45-kDa polypeptide and to a lesser extent of the 20-kDa polypeptide. The dose/response curves for Ca2+-dependent phosphorylation of the 45-kDa polypeptide in the presence and absence of 1-oleyl-2-acetylglycerol are similar to those observed for Ca2+-dependent [14C]serotonin secretion under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Addition of GTP markedly enhances the ability of thrombin to cause a leftward shift in the Ca2+ dose/response curve for 5-hydroxytryptamine secretion from permeabilised human platelets. Little effect is observed on addition of GTP in the absence of thrombin. Neither ADP nor adrenaline, in the presence or absence of GTP, causes such a shift, whereas 5-hydroxytryptamine does so to a small extent but only in the presence of GTP. The leftward shift in the Ca2+ dose/response curve induced by 12-O-tetradecanoyl-phorbol-13-acetate or 1-oleyl-2-acetylglycerol is not enhanced by addition of GTP. The thrombin concentration required for half-maximal enhancement of the response to Ca2+ is markedly reduced by addition of GTP. The results support the postulate that the effects of excitatory agonists in this system correlate with their ability to activate phospholipase C and provide further evidence for a role for GTP in signal transduction between the receptor and phospholipase C.

Experimental analysis of the mechanisms of exocytosis and endocytosis has hitherto been hampered by the inaccessibility of the intracellular sites at which they are controlled. We have recently developed a technique that overcomes this problem. Cells are subjected to intense electric fields of brief duration; this renders the plasma membrane permeable without impairing its ability to participate in exocytosis and endocytosis. Working with 'leaky' bovine adrenal medullary cells, catecholamine release has a rather specific requirement for Mg-ATP, is activated by micromolar concentrations of ionized Ca and can be inhibited by Mg, detergents, trifluoperazine, high osmotic pressure and various anions. The mechanism of activation by Ca is discussed in some detail.

Cellular responses to extracellular signals are mediated by changes in the intracellular concentrations of one or more second messengers. In platelets, inhibitory agonists increase intracellular cyclic-3',5'-AMP [( cyclic AMP]i (refs 2, 3] whereas excitatory agonists increase [Ca2+]i and/or [1,2-diacylglycerol]i (refs 4-9), and in some cases decrease [cyclic AMP]i (refs 10, 11). Both activation and inhibition of platelet responses have been attributed to an increase in [cyclic-3',5'-GMP]i (refs 8, 12). The activity of protein kinase C, which is associated with the platelet secretory response, is increased by both 1,2-diacylglycerol and Ca2+ (refs 4, 7, 8). The role of cyclic AMP may involve either inhibition of Ca2+ mobilization to the cytosol or stimulation of intracellular Ca2+ uptake, and in addition inhibition of 1,2-diacylglycerol formation. The relationship between cyclic-3',5'-GMP (cyclic GMP) and other second messengers in platelet activation has not been defined. Using platelets made permeable by exposure to an intense electric field, we demonstrate here modulation of the Ca2+ sensitivity of platelet secretion by thrombin, and by 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-oleyl-2- acetylglycerol ( OAG ), both potent activators of protein kinase C. The effect of thrombin is selectively modified by cyclic GMP and cyclic AMP. The response to OAG and TPA is also modulated by cyclic AMP but to a much lesser extent.

Exposure of 'leaky' adrenal medullary cells to GTP-y-S inhibits Ca-dependent exocytosis in bovine cells, but stimulates exocytosis in chicken cells. The inhibitory action on bovine cells persists in the presence of TPA suggesting that in this tissue an inhibitory GTP-binding protein may modulate the action of protein kinase C on exocytosis.

A large body of evidence supports the concept that calcium (Ca2+) plays a pivotal role in the control of exocytosis. However, recent experiments suggest that a rise in intracellular Ca2+ does not necessarily trigger secretion, and also that secretion can occur independently of cytosolic free calcium levels. This article briefly summarizes the early evidence that has formulated the role of Ca2+ in secretion, and then examines some of the recent evidence suggesting a Ca2+-independent mechanism of exocytosis.

Measurements of intracellular Ca2+ in adrenal medullary cells suggest that a transient rise in Ca2+ leads to a transient secretory response, the rise in Ca2+ being brought about by an influx through voltage-sensitive Ca channels which subsequently inactivate. The level of Ca2+ observed is much smaller than the Ca2+ needed to trigger secretion when introduced directly into the cell. The discrepancy is removed by the presence of diacylglycerol, which increases the sensitivity of the secretory process to Ca2+. The site of action of Ca2+ and diacylglycerol is probably protein kinase C, and the different secretory responses to increases of Ca2+ and diacylglycerol can be modelled in terms of a preferential order of binding of these two substrates to the enzyme. ATP is needed for secretion: one role is possibly to confer stability to the secretory apparatus; another may involve phosphorylation of some key protein. The kinetics of secretion suggest that if Ca2+ regulates phosphorylation or dephosphorylation, then it is the rate of change of phosphorylation that controls secretion rather than the extent of phosphorylation or dephosphorylation. Guanine nucleotide-binding proteins may play a role not only at the level of signal transduction coupling, but also at or near the site of exocytosis, and the mechanism by which some Botulinum toxins inhibit secretion may be associated with these proteins.

Quantitative analysis of the effects of calcium on cell function requires methods for altering intracellular free Ca in a precise and reproducible manner. Microinjection of Ca is very unreliable largely because of the powerful Ca-binding properties of cytoplasm. Much more satisfactory are microinjection of Ca-buffers - provided enough buffer is introduced - and various forms of intracellular dialysis and perfusion which permit full equilibration of the cell interior with a defined artificial intracellular environment.

Amine secretion from electropermeabilized bovine chromaffin cells and human platelets requires Ca2+ and MgATP. There appears to be little correlation between the pH or potential of the interior of the amine storage granules of the chromaffin cells and the Ca2+ sensitivity or extent of secretion. The Ca2+ sensitivities of secretion for both preparations are increased by activators of protein kinase C. In the platelet, thrombin also increases the Ca2+ sensitivity. The thrombin-induced response is further enhanced by micromolar levels of GTP. The non-hydrolysable analogue GTP gamma S also potentiates the Ca2+-dependent secretory response, but this effect is additive to that seen by thrombin rather than synergistic, as is the case with GTP. GTP gamma S inhibits catecholamine secretion from bovine chromaffin cells. In both preparations the effects of GTP gamma S are inhibited by 10 microM GTP, even though GTP concentrations up to 1 mM are without effect when added alone. These results are consistent with there being two sites of action for the guanine nucleotides, one at the level of the agonist receptor and activated by GTP or one of its breakdown products, and the other one activated by GTP gamma S--possibly at the level of protein kinase C itself.

Calcium-dependent exocytosis in 'leaky' bovine adrenal medullary cells has a requirement for Mg-ATP. One possibility is that exocytosis depends in some way on the operation of the ATP-dependent proton pump that serves to maintain the core of the secretory vesicles both acid and at a positive potential with respect to the cytosol. This possibility has been tested in 'leaky' cells by monitoring exocytosis under conditions where the secretory vesicle pH and potential gradients are measured in situ. The results show rather clearly that exocytosis can persist, with unchanged Ca-activation kinetics, in the virtual absence both of a difference in pH between the cytosol and secretory vesicle core and also of a difference in potential across the vesicle membrane. The results do not, however, exclude a small modulating effect of vesicle pH or potential on exocytosis and shed no light on whether or not the plasma membrane potential, which is maintained close to zero in these experiments, influences exocytosis.

The intracellular requirements for membrane recapture in permeabilized chromaffin cells were compared to the requirements for exocytosis from the same cells. In permeabilized bovine chromaffin cells, calcium-driven exocytosis also triggers, with a short delay, uptake of extracellular horseradish peroxidase (HRP). This internalized HRP remains compartmentalized within the cell and migrates to a low density band on a Percoll gradient which is distinct from the heavier chromaffin granules. The amount of horseradish peroxidase internalized is similar in intact and leaky cells and is approximately equivalent to the volumes secreted. Endocytosis in both preparations is blocked by botulinum toxin, operates in a collapsed membrane potential, and is inhibited by low temperature. In permeabilized cells, exocytosis and coupled endocytosis are activated by the same concentrations of Ca and MgATP. Although secretion requires Ca and MgATP, once exocytosis has occurred the subsequent endocytosis can proceed in the virtual absence of Ca or MgATP, and is largely unaffected by a variety of nucleotide triphosphates (including nonhydrolyzable analogues), and cyclic nucleotides. These data suggest that endocytosis can proceed, once exocytosis has been triggered, under conditions that are quite different from those necessary to support exocytosis, and that the specific requirements for Ca and MgATP in secretion are for the exocytotic limb of the secretory cycle rather than for the associated endocytotic pathway. [ABSTRACT FROM AUTHOR]