Mechanisms underlying neuronal death induced by chromogranin A-activated microglia.

The neurotoxic effects of activated microglia in neurodegenerative diseases are well established. We recently provided evidence that chromogranin A (CGA), a multifunctional protein localized in dystrophic neurites and in senile plaques, induces an activated phenotype and secretion of neurotoxins by rat microglia in culture. In the present study, we focused on the mechanisms underlying neuronal degeneration triggered by CGA-activated microglia. We found that neuronal death exhibits apoptotic features, characterized by the externalization of phosphatidylserine and the fragmentation of DNA. Microglial neurotoxins markedly stimulate the phosphorylation and activity of neuronal p38 mitogen-activated protein kinase and provoke the release of mitochondrial cytochrome c, which precedes apoptosis. Inhibition of p38 kinase with SB 203580 partially protects neurons from death induced by CGA-activated microglia. Furthermore, neurons are also protected by Fas-Fc, which antagonizes the interactions between the death receptor Fas and its ligand FasL and by cell-permeable peptides that inhibit caspases 8 and 3. Thus, CGA triggers the release of microglial neurotoxins that mobilize several death-signaling pathways in neurons. Our results further support the idea that CGA, which is up-regulated in many neuropathologies, represents a potent endogeneous inflammatory factor possibly responsible for neuronal degeneration.

Chromogranin A induces a neurotoxic phenotype in brain microglial cells.

Chromogranin A (CGA) belongs to a multifunctional protein family widely distributed in secretory vesicles in neurons and neuroendocrine cells. Within the brain, CGA is localized in neurodegenerative areas associated with reactive microglia. By using cultured rodent microglia, we recently described that CGA induces an activated phenotype and the generation of nitric oxide. These findings led us to examine whether CGA might affect neuronal survival, expression of neurofilaments, and high affinity gamma-aminobutyric acid uptake in neurons cultured in the presence or absence of microglial cells. We found that CGA was unable to exert a direct toxic effect on neurons but provoked neuronal injury and degeneration in the presence of microglial cells. These effects were observed with natural and recombinant CGA and with a recombinant N-terminal fragment corresponding to residues 1-78. CGA stimulated microglial cells to secrete heat-stable diffusible neurotoxic agents. CGA also induced a marked accumulation of nitric oxide and tumor necrosis factor-alpha by microglia, but we could not establish a direct correlation between the levels of nitric oxide and tumor necrosis factor-alpha and the neuronal damage. The possibility that CGA represents an endogenous factor that triggers the microglial responses responsible for the pathogenesis of neuronal degeneration is discussed.

Chromogranin A triggers a phenotypic transformation and the generation of nitric oxide in brain microglial cells.

Chromogranin A is an ubiquitous 48,000 mol. wt secretory protein stored and released from many neuroendocrine cells and neurons. In human brain, chromogranin A is a common feature of regions that are known to be affected by various neurodegenerative pathologies such as Alzheimer's, Parkinson's and Pick's diseases. Brain degenerative areas are generally infiltrated by activated microglial cells, the resident macrophage cell population within the central nervous system. Here, we report that both recombinant human chromogranin A and chromogranin A purified from bovine chromaffin granules trigger drastic morphological changes in rat microglial cells maintained in culture. Microglial cells exposed to chromogranin A adopted a flattened amoeboid shape and, this change was associated with an accumulation of actin in the subplasmalemmal region, as observed by immunocytochemistry and confocal laser microscopy. In single microglial cells loaded with indo-1, chromogranin A elicited a rapid and transient increase in [Ca2+]i which preceded the reorganization of actin cytoskeleton. The activity of nitric oxide synthase was estimated by measuring the accumulation of nitrite in the culture medium. Both recombinant human chromogranin A and bovine chromogranin A triggered an important accumulation of nitrite comparable to that induced by lipopolysaccharide, a well-known activator of microglia. The effect of chromogranin A was dose dependent, inhibited by N omega-nitro-L-arginine methyl ester, a competitive inhibitor of nitric oxide synthase, and by cycloheximide, an inhibitor of protein synthesis. These findings suggest that chromogranin A induces an activated phenotype of microglia, and thus may have a role in the pathogenesis of neuronal degeneration in the brain.

Immunocytochemical localization of protein kinases Yes and Src in amoeboid microglia in culture: association of Yes kinase with vimentin intermediate filaments.

Amoeboid microglia isolated from primary cultures of neonatal rat brain correspond to a transient form of activated microglia, a resident population of macrophage-like cells. In order to understand the molecular aspects of microglial activation, we have studied amoeboid microglia in primary culture for the presence of Yes and Src protein tyrosine kinases, two kinases which have been implicated in signal transduction process during monocyte/macrophage activation. Immunofluorescence with an antibody raised against the peptide from unique N-terminal domains of Yes and Src demonstrated that Yes and Src kinases are enriched in perinuclear areas of amoeboid microglia. In addition, the antibody to c-yes peptide had a cytoplasmic distribution which coincided with the distribution of vimentin-containing intermediate filaments. Preadsorption of anti-c-yes antibody with an excess of antigenic peptide inhibited anti-c-yes immunofluorescence, while vimentin immunofluorescence remained unchanged. Double immunofluorescence images analyzed with the two-dimensional intensity distribution program (2-D scattered histograms) on Zeiss confocal scanning laser microscope demonstrate the colocalization of c-yes with vimentin. The extent of colocalization was more prominent after exposure of intact cultured microglia to dibutyryl cyclic AMP (dBcAMP), or to phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) or to okadaic acid, an inhibitor of protein phosphatases. The findings suggest that vimentin might serve as molecular support for Yes kinase and, since previous studies have shown that vimentin in amoeboid microglia is one of the major protein substrates of serine/threonine protein kinases, this function could be regulated by phosphorylation.

Bacterial endotoxin induces [Ca2+]i transients and changes the organization of actin in microglia.

We have employed amoeboid microglia purified from primary cultures of neonatal rat brain to examine the effect of bacterial lipopolysaccharide (LPS), a potent activator of immune cells, on intracellular calcium concentration ([Ca2+]i) in brain macrophages. In single brain macrophages loaded with indo 1, pulse administration of LPS elicited a rapid and transient increase in [Ca2+]i. From a total of 70 cells examined, all responded to LPS with a similar [Ca2+]i transient, indicating a good homogeneity of the cell population with regard to the LPS response. It was concluded that the rise of cytosolic [Ca2+]i originated from intracellular stores because the response to LPS occurred similarly in the presence or in the absence of extracellular Ca2+. A second administration of LPS to the same cells resulted in a second but reduced [Ca2+]i transient. In contrast to the first response to LPS, this second response was totally dependent on the presence of Ca2+ in the extracellular medium. The first response to LPS was strongly inhibited by ruthenium red and could be suppressed in a reversible manner by preincubating the cells with caffeine in the absence of Ca2+ in the extracellular medium. These results indicate that caffeine-sensitive intracellular Ca2+ stores may be the major source of Ca2+ in the response of brain macrophages to LPS. The possible release of Ca2+ from phosphatidylinositol(3,4,5)-trisphosphate (IP3)-sensitive stores in brain macrophages was also evaluated by stimulating cells with the IP3-mobilizing agonist histamine. Brain macrophages were heterogeneous with regard to the histamine response since histamine induced a [Ca2+]i rise in only 30% of cells examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in vimentin distribution in glial cells in culture revealed with an antibody against a phosphorylated epitope.

We have previously described that spatial and temporal changes in the organization of vimentin that are correlated with protein kinase C (PKC)-induced phosphorylation of vimentin can be detected with the mouse monoclonal antibody B3 in cultures of amoeboid microglia [Ciesielski-Treska et al. (1991) J. Neurosci. Res. 29, 362-378]. The antibodies were generated to native form of vimentin-containing filaments and antibody B3 reveals a restricted immunostaining of vimentin in glial cells from human, rat and mouse origin. In the present study we show the distribution of epitope B3 analyzed by immunofluorescence within defined populations of rat glial cells. Relatively high immunoreactivity was found in Type 1 astrocytes, Type 2 astrocytes and oligodendrocytes had low immunoreactivity. Although the results suggested that in Type 1 astrocytes the phosphorylated epitope is prominent, its phosphorylation was not found to be cell cycle-dependent, and appeared unrelated to the organizational changes of intermediate filaments associated with the morphological conversion of polygonal to stellate astrocytes. As expected, the immunofluorescence was increased by exposition of astrocyte cultures to an activator of PKC, confirming our previous conclusion that the immunoreactivity of the epitope B3 depends on PKC-mediated phosphorylation. In addition, the finding that the immunofluorescence of vimentin was more homogeneous in quiescent, serum-deprived astrocytes and also in astrocytes exposed to an inhibitor of protein synthesis, cycloheximide, may suggest that phosphorylation of the epitope B3 depends on a protein factor present in fetal calf serum. The immunofluorescence studies on cultures enriched in Type 2 astrocytes and in oligodendrocytes indicate that the epitope B3 is hypophosphorylated in glial cells of this lineage and becomes dephosphorylated after terminal differentiation of oligodendrocytes. These observations suggest that in Type 2 astrocytes and in oligodendrocytes the low level of phosphorylation of vimentin could be related to the down regulation in vimentin expression.

Phosphorylation of cellular proteins in response to treatment with Clostridium difficile toxin B and Clostridium sordellii toxin L.

Toxin B from Clostridium difficile induces typical morphological changes of cultured cells consisting of rounding up and arborization, which are associated with a dramatic disruption of microfilaments. In this study, we show that toxin L, a cytotoxin produced by bacterial strain Clostridium sordellii, has similar effects on cultured cells including the redistribution of F-actin and of the adhesion plaque protein vinculin. It has been assumed that the mechanisms involved in cytopathic effects of toxin B are related to the function of an unidentified component that regulates the organization of the actin cytoskeleton. We demonstrate that the treatment of cultured astrocytes with toxin B or toxin L alters the incorporation of inorganic phosphate into several proteins. Immunoblot analysis revealed that one of these proteins is tropomyosin. Since tropomyosin stabilizes microfilaments and inhibits the severing activity of gelsolin, the toxin-induced phosphorylation may counteract this inhibition resulting in severing of microfilaments and capping of short filaments. A decrease in the radioactivity associated with intermediate filament protein vimentin was also detected using a monoclonal antibody which specifically recognizes a phosphorylated epitope of vimentin. Since vimentin is an in vivo substrate for various protein kinases, these data are in favor of broad effects of these toxins. Direct measurement of protein kinase C in cells exposed to toxin B or to toxin L did not reveal a significant change in protein kinase C activity. Furthermore, treatments with toxins do not increase cAMP levels, suggesting that toxins do not activate protein kinase A. Although further studies are required to determine the primary target site for the clostridial cytotoxin B and L, our results show that they provoke the alteration in the phosphorylation of cellular proteins.

Protein kinase C-induced redistribution of the cytoskeleton and phosphorylation of vimentin in cultured brain macrophages.

The phorbol ester 12-O-tetradecanoyl-acetate (TPA) induced prominent and transient changes in the organization of the cytoskeleton in cultured amoeboid microglial cells including redistribution of actin toward the center of the cells and in the subplasmalemmal region, appearance of fine actin filaments, retraction of microtubules (MT), and rearrangement of intermediate filaments (IF) containing vimentin. The possible implication of protein kinase C (PKC) in mediating the effects of TPA was suggested by a parallel shift of PKC activity from the soluble to membrane fractions and phosphorylation of several microglial proteins. The rearrangement of IF closely correlated with increased vimentin phosphorylation, detected by pulse labeling of intact cells. Two monoclonal antivimentin antibodies, B3 and V9, showed different staining patterns. Immunoreactivity with the antibody B3 was more restricted and could be abolished by treatment of fixed, permeabilized cells with alkaline phosphatase, thus suggesting that the antibody reacts with a phosphorylated epitope. Using this antibody, rearrangement of IF involving vimentin phosphorylation was detected within 15 to 60 min of treatment with 50 nM TPA and consisted in the appearance of intense perinuclear fluorescent label. This perinuclear fluorescence persisted up to 24 hr after TPA removal and gradually diminished during the following 2 to 3 days. Immunochemical analysis of nonionic detergent-soluble and -insoluble extracts from untreated and TPA-treated cells revealed no differences in vimentin solubility suggesting that TPA induced vimentin phosphorylation does not result in notable vimentin filament disassembly. However the extent of vimentin degradation was more prominent in TPA-treated cultures indicating a higher sensitivity of vimentin to proteolytic degradation. The data show that PKC-mediated phosphorylation of vimentin results in precise spatial and temporal rearrangement of IF which are not associated with altered vimentin solubility, but possibly changes the mechanical properties and interactions of vimentin filaments.

Mechanism of action of Clostridium difficile toxin B: role of external medium and cytoskeletal organization in intoxicated cells.

Toxin B, an exotoxin produced by Clostridium difficile, induces the rounding-up and arborization of cultured mammalian cells, a typical effect which resembles that provoked by cytochalasins. In this study, the effect of toxin B was examined on astroglial cells grown in primary culture. A specific antiserum to toxin B was used to investigate its mechanisms of action. We found that the toxin exerts its effects on cell morphology after its incorporation into cells. The internalization of toxin B requires the presence of calcium ions in the extracellular medium. Replacement of NaCl with sucrose or with potassium glutamate prevents the internalization of the toxin. The direct introduction of calcium ions into cells by the calcium ionophore A23187 stimulates toxin-induced morphological changes. In contrast, toxin-induced morphological transformations were prevented in cells treated with tumor-promoting phorbol. esters or with dibutyryl-cAMP, although such treatment did not abolish the internalization of the toxin. As in the other cell types, the earliest effect of toxin B on astrocyte cytoskeleton is the disruption of actin filaments, without no visible alteration of intermediate filament nor microtubule networks. As astrocytes with toxin-induced stellate morphology survive toxin treatment, the progression of cell morphology and cytoskeleton organization were followed for several weeks. Twenty-six days after exposure to toxin B, stellate astrocytes have processes which were markedly longer and much more branched than those of cells freshly exposed to toxin. At that time, cells are still devoid of F-actin as assessed with rhodamine-conjugated phalloidin and only 70% contain vimentin while all astrocytes present in control cultures express vimentin. Some flat epithelioid astrocytes with prominent bundles of microfilaments reappear during the second week after toxin treatment. Our results show that Clostridium difficile toxin B is internalized into brain astrocytes in culture where it acts by modifying cytoskeletal elements. Its cytopathic effects are reversible. Although actin-related components of the cytoskeleton are the major target of toxin B, other cytoskeletal elements also seem to be affected.

Acquisition of vimentin in astrocytes cultured from postnatal rat brain.

Vimentin and glial fibrillary acidic protein (GFAP) represent the principal constituents of intermediate filaments found in astrocytes. In contrast to vimentin-GFAP transition which occurs during glial development in situ, vimentin coexists with GFAP in cortical astrocytes allowed to differentiate in culture. To examine whether culture conditions or proliferative activity of the cells is responsible for the expression of vimentin, we generated cultures of GFAP-positive, vimentin-negative astrocytes isolated from 26-day postnatal rat brain cortices. Isolated astrocytes are characterized by a very thin rim of perinuclear cytoplasm and by numerous processes. Antiserum to GFAP labelled major processes and cell somata of some astrocytes, especially those with relatively short and large processes. Within 3 days in culture, all astrocytes accumulated GFAP in hypertrophic cell bodies and many began to express vimentin. Vimentin appeared primarily close to nuclei, and filaments of vimentin extended into proximal segments of the cell processes. In some astrocytes, however, vimentin was always absent. Combined double immunolabelling and histoautoradiography experiments demonstrated that the acquisition of vimentin was independent of the ability of astrocytes to incorporate tritiated thymidine. The results indicate that astrocytes isolated from 26-day postnatal rat brain are heterogeneous with respect to their ability to express vimentin and that vimentin synthesis is not correlated with the growth state of the cells as had been previously suspected.

Immunocytochemistry and heterogeneity of rat brain vimentin.

In unfixed cryostat sections of the brains of early postnatal and adult rats, we screened for cells containing vimentin-positive intermediate filaments (VI+-IFs) by applying a panel of four monoclonal antibodies (Mabs VI-01, VI-02, VI-05 and VI-5B3) using indirect immunofluorescence. All of the Mabs stained VI+-IFs in the stromal part of the choroid plexus, in endothelial cells of blood vessels and in meninges in both adult and immature brains, although with varying strength (VI-5B3 and VI-01 stained more strongly than VI-05 and VI-02). In the brain parenchyma of adults, intense staining was mainly localized in ventricular ependymal cells (VI-5B3/VI-01 greater than VI-02/VI-05) and fibrous astrocyte-like cells (FAs). In the immature brain, the ependymal cells were activated in appearance, with evidence of cell enlargement, greater spreading of VI+-IFs within the cytoplasm and more pronounced VI+ cytoplasmic protrusions into the brain parenchyma. VI+-FAs were found near the ependymal and meningeal borders as well as in the white matter tracts of adult brain (VI-5B3/VI-01 greater than VI-05 greater than VI-02). In immature animals, VI+-FAs were less frequently encountered in the forebrain regions, except in and near the subepenydmal layer (in the adjacent parenchyma) as well as in submeningeal layers. Weaker staining was usually elicited by Mabs VI-02 and VI-05. In the cerebellum, Bergmann cell fibers were stained in both age groups. In adults, the most intense fluorescence usually occurred in segments close to the pia (VI-5B3/VI-01 greater than VI-05 greater than VI-02).(ABSTRACT TRUNCATED AT 250 WORDS)

Fibronectin and collagens modulate the proliferation and morphology of astroglial cells in culture.

The proliferation and morphology of astroglia derived from neonatal rat cortex and cultured in serum-free medium on either untreated, or fibronectin-, or collagen I-, or collagen IV-treated substrates were investigated using tritiated thymidine autoradiography and immunocytochemical staining of glial fibrillary acidic protein (GFAP) and actin. Modification of culture substratum with fibronectin enhanced the rate of proliferation of astroglial cells and increased the proportion of process-bearing astroglial cells. The distribution of actin and patterns of adhesion observed were typical for motile cells. Both types of collagen decreased the proportion of astroglial cells undergoing mitosis. Many of the astroglial cells exhibited a flat morphology and displayed prominent stress fibres in the cell body and processes. The data suggest that specific interactions with the substratum modulate the proliferation and morphological behaviour of astroglial cells.

Purkinje cell maturation in the light of RNA synthesis.

Wistar rats 1- to 90-day-old received an injection of 3H-uridine and were killed 20 min to 44 h later. Autoradiographic examination revealed the highest grain count densities in Purkinje cell nuclei around postnatal day (PD) 6 while the incidence of labelled nuclei stayed at the peak values till PD 15. Silver staining of Purkinje cell nuclei showed that the expression of nucleolar r-RNA coding genes is maximal at PD 15; in some cells it even slightly exceeds adult values. After PD 15, the percentage of labelled Purkinje cell nuclei declined; this was more pronounced in the nucleolar region than outside the nucleolus. The percentage of cells with cytoplasmic labelling culminated on PD 15. The highest grain counts were found in Purkinje cell cytoplasm on PD 6 at 44 h p.i. interval. Reversal in nuclear grain counts at 2 and 6 h p.i. intervals observed between PD 15 and PD 25 suggests faster degradation, or processing and export, of a newly synthesized nuclear RNA in these age groups. Frequency distribution analysis of grain count densities revealed a small group of Purkinje cells with higher incorporation of 3H-uridine both in the nucleolar region and the whole nucleus at PD 15. In situ hybridization of 3H-r-RNA revealed a slight binding excess to DNA of some Purkinje cell nuclei but not in granule cells of 1-month-old rats. These data, together with those published recently by Brodsky et al. (1985), indicate an uneven structural organization and partial overexpression of the genom coding r-RNA synthesis in the population of Purkinje cells.

The organization and solubility properties of intermediate filaments and microtubules of cortical astrocytes in culture.

The organization of intermediate filaments (IF) and microtubules (MT) and the solubility of intermediate filament proteins and tubulin in astrocytes which develop from cerebral hemispheres of neonatal rats in culture were examined using immunocytochemical and immunochemical approaches. Results of immunocytochemical studies demonstrated that in flat astrocytes which develop after 3 weeks of culturing in serum-supplemented medium, the IF containing vimentin and glial fibrillary acidic protein (GFAP) are concentrated around the nucleus and dispersed in an irregular fashion throughout the cytoplasm. Astrocytes which develop in serum-free hormonally-defined medium irrespective of whether they are bipolar, multipolar or flattened, have IF organized as a fibrous network of filaments distributed from the nuclear regions to the cell periphery. Under both culture conditions, vimentin and GFAP are resistant to extraction with low salt buffer containing nonionic detergent, indicating that the different cytoplasmic distribution of IF is unrelated to the solubility properties of vimentin and GFAP. Double immunolabelling experiments with polyclonal antibody to GFAP and monoclonal antibody to each alpha-tubulin or beta-tubulin reveal an extensive codistribution and parallel organization of IF and MT in all morphological types of astrocytes studied. Stabilization of MT with taxol, or depolymerization of MT with colchicine, cause dramatic changes in the distribution of IF and inhibit the extension of astrocyte processes in response to dibutyryl cyclic AMP (dBcAMP). In early stages of treatment with dBcAMP, renewal of culture medium without dBcAMP produces a rapid and permanent retraction of astrocyte processes, whereas in later stages the processes only retract partially and are then restored and maintained for several days in the absence of dBcAMP. The retraction of processes is accompanied by changes of immunocytochemical staining of IF with antibody to GFAP, which appears more intense and diffuse. However, electrophoretic and immunoblot analyses of detergent-extracted proteins from parallel cultures demonstrate that neither the amount nor the solubility of GFAP and vimentin are changed. Detergent extraction in MT stabilizing conditions shows that a substantial proportion of tubulin in astrocytes cultured in serum-containing and serum-free media is assembled into MT, most of which depolymerize on treatment with low temperature and Ca2+. Following long exposure to dBcAMP the proportion of cold/Ca2+-stable MT increases. The results suggest that the IF of astrocytes in culture are dependent on MT with respect to their cytoplasmic distribution.(ABSTRACT TRUNCATED AT 400 WORDS)

Cold-stable microtubules and microtubule-organizing centres in astrocytes in primary culture.

Immunocytochemical staining with anti-tubulin antibodies showed cold-stable microtubules in astrocytes in culture. On rewarming, microtubule-organizing centre (MTOC) could be visualized. MTOCs showed structural complexity, from 2-3 fibres to a mass of interweaving fibres. Only one MTOC per cell was found in cultures of both polygonal and stellate-bearing cells.

Proteolytic degradation of vimentin and glial fibrillary acidic protein in rat astrocytes in primary culture.

Vimentin and glial fibrillary acidic (GFA) protein in rat astrocytes in primary culture were identified with their respective specific antisera on electroblots of one-dimensional and two-dimensional polyacrylamide gels. Each antiserum revealed respectively vimentin (58 kDa, pI 5.3), GFA protein (50 kDa, pI 6-5.8) and several more acidic, lower molecular mass vimentin-immunoreactive and GFA protein-immunoreactive polypeptides. These were more apparent when astrocyte proteins were extracted in the absence of EGTA and in the presence of Ca2+ ions suggesting that the lower immunoreactive polypeptides result from a Ca2+-sensitive proteolytic degradation of vimentin and GFA protein. The extent of proteolysis of GFA protein was higher when the differentiation of astrocytes was induced with 1,N6-dibutyryl-adenosine 3',5'-monophosphate (Bt2cAMP). In contrast, the proteolysis of vimentin was not affected by the state of differentiation of astrocytes. The results indicate the existence of at least two distinct degradative pathways for intermediate filament proteins in astrocytes in primary culture and suggest that in differentiated astrocytes the activity of proteinase(s) involved in the degradation of GFA protein increases.

Electrophoretic pattern and distribution of cytoskeletal proteins in flat-epitheloid and stellate process-bearing astrocytes in primary culture.

One- and two-dimensional electrophoresis patterns and distribution of major cytoskeletal proteins were studied in primary astrocytes with either flat-epitheloid or stellate appearance. No major differences in the electrophoretic patterns of actin, tubulin, glial fibrillary acidic protein (GFAP) and vimentin were detected between flat-epitheloid and stellate process-bearing astrocytes produced by the exposure of cultures to dibutyryl cyclic AMP (dBcAMP). However the morphological changes of astrocytes were accompanied by marked changes in the quantitative distribution of cytoskeletal proteins. The most prominent change was a large and specific decrease in the amount of actin, detected by [(35)S]methionine incorporation, densitometric scanning of one-dimensional gels and DNase inhibition assay. In stellate astrocytes produced by a 4 day treatment with dibutyryl cyclic AMP, the amount of actin decreased by 50%. This decrease was not apparently related to the depolymerization of actin.

Activation of brain guanylate cyclase by phospholipase A2.

Various pure snake venom phospholipases A2 were used for studying their effect on guanylate cyclase activity. All the phospholipases A2 tested were found to activate guanylate cyclase from a rat brain homogenate. It was shown that particulate guanylate cyclase was especially affected. Intact glial cells incubated in presence of phospholipase A2 showed also an increased guanylate cyclase activity, demonstrating that the phospholipase effect, observed in disrupted cells, occurs also at the cellular level. These results suggest that in intact cells membrane-bound phospholipase A2 activity could be involved in the modulation of the cellular cyclic GMP content.