Mario Patrizio

Astrocyte Heterogeneity: Endogenous Amino Acid Levels and Release Evoked by Non‐N‐Methyl‐D‐Aspartate Receptor Agonists and by Potassium‐Induced Swelling in Type‐1 and Type‐2 Astrocytes

Abstract: The aim of the present study was to determine whether endogenous amino acids are released from type‐1 and type‐2 astrocytes following non‐N‐methyl‐D‐aspartate (NMDA) receptor activation and whether such release is related to cell swelling. Amino acid levels and release were measured by HPLC in secondary cultures from neonatal rat cortex, highly enriched in type‐1 or type‐2 astrocytes. The following observations were made. (a) The endogenous level of several amino acids (glutamate, alanine, glutamine, asparagine, taurine, serine, and threonine) was substantially higher in type‐1 than in type‐2 astrocytes. (b) The spontaneous release of glutamine and taurine was higher in type‐1 than in type‐2 astrocytes; that of other amino acids was similar. (c) Exposure of type‐2 astrocyte cultures to 50 μM kainate or quisqualate doubled the release of glutamate and caused a lower, but significant increase in that of aspartate, glycine, taurine, alanine, serine (only in the case of kainate), and glutamine (only in the case of quisqualate). These effects were reversed by the antagonist CNQX. (d) Exposure of type‐1 astrocyte cultures to 50–200 μM kainate or 50 μM quisqualate did not affect endogenous amino acid release, even after treating the cultures with dibutyryl cyclic AMP. (e) Exposure of type‐1 or type‐2 astrocyte cultures to 50 mM KCl (replacing an equimolar concentration of NaCl) enhanced the release of taurine > glutamate > aspartate. The effect was somewhat more pronounced in type‐2 than in type‐1 astrocytes. Veratridine (50 μM) did not cause any increase in amino acid release. (f) The release of amino acids induced by high [K+] appeared to be related to cell swelling, in both type‐1 and type‐2 astrocytes. Swelling and K+‐induced release were somewhat higher in type‐2 than in type‐1 astrocytes. In contrast, neither kainate nor quisqualate caused any appreciable increase in cell volume. It is concluded that non‐NMDA receptor agonists stimulate the release of several endogenous amino acids (some of which are neuroactive) from type‐2 but not from type‐1 astrocytes. The effect does not seem to be related to cell swelling, which causes a different release profile in both type‐1 and type‐2 astrocytes. The absence of kainate‐ and quisqualate‐evoked release in type‐1 astrocytes suggests that the density of non‐NMDA receptors in this cell type is very low.

Release of endogenous and newly synthesized glutamate and of other amino acids induced by non-N-methyl-D-aspartate receptor activation in cerebellar granule cell cultures.

Abstract: Amino acid release studies were performed by an HPLC procedure using differentiated rat cerebellar granule cell cultures. Kainic acid (KA; 50 μM) caused an increase (about threefold) in the release of endogenous glutamate and a lesser, but statistically significant, increase in the release of glutamine, glycine, threonine, taurine, and alanine. Quisqualic acid (QA) and, to a lesser degree, α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) (both 50 μM) enhanced the release of the following amino acids in the order glutamate > aspartate ≥ taurine, whereas the release of other amino acids was either unaffected or affected in a statistically nonsignificant way. The release of glutamate induced by KA was partially (43%) Ca2+ dependent. The other release‐inducing effects of KA and QA were not Ca2+ dependent. In all cases, the evoked release could be prevented by the non‐N‐methyl‐D‐aspartate (non‐NMDA) receptor antagonist 6‐cyano‐2,3‐hydroxy‐7‐nitroquinoxaline, and thus appeared to be receptor mediated. NMDA (5 and 50 μM) had no releaseinducing activity. The KA‐, QA‐, and AMPA‐evoked release of newly synthesized [3H]glutamate and [3H]aspartate (formed in the cells exposed to [3H]glutamine) was very similar to the evoked release of endogenous glutamate and aspartate. On the other hand, the release of preloaded D‐[3H]aspartate (purified by HPLC in the various fractions analyzed, before radioactivity determination) induced by 50 μM KA was twice as high as that of endogenous glutamate. In the case of high [K+] depolarization, in contrast, the release of preloaded D‐[3H]aspartate was ∼30% lower than that of endogenous glutamate. The reasons for the above differences in the susceptibility of the various glutamate pools to being released may be the following: Non‐NMDA receptor agonists cause essentially a carrier‐mediated efflux of glutamate from a cytoplasmic pool that is preferentially labeled by exogenous D‐[3H]aspartate, whereas depolarization with high [K+] causes mainly an exocytotic‐like release from a vesicular pool to which exogenous D‐[3H]aspartate has a more limited access.

Expression and translocation of protein kinase C isoforms in rat microglial and astroglial cultures

Cellular distribution and activation by phorbol myristate acetate (PMA) of classical (α, βI, βII,γ), novel (δ, ϵ, θ, η), and atypical (ζ, ι) protein kinase C (PKC) isoforms were studied in cultured rat neonatal microglial and astroglial cells by Western blot analysis. Among the classical isoforms, only βII was expressed in microglia and astrocytes in the same abundance. The expression of βI in microglia was less abundant, while PKCα was not detectable in this cell type. PKCγ was absent in both cell populations. A different pattern of expression was also found for novel and atypical isoenzymes: Both cell types expressed δ, θ, η, ζ, and ι isoforms, but PKCϵ was absent in microglia and the expression of PKCζ and PKCι in these cells was low compared to astrocytes. The pattern of PKC distribution in cytosolic and particulate fractions as well as activation by short (10 min) and prolonged (4 hr) PMA treatment in both cell types were similar. On the whole, in comparison with astrocytes, PKC in microglial cells was less expressed, both in terms of number of isoforms and level of expression. The microglial profile of PKC isoforms differed from that of rat peritoneal macrophages, which did express PKCα. Preliminary evidence suggests that the ability of PMA to enhance cyclic AMP responses in astrocytes, but not in microglia, is related to the different pattern of expression of PKCα and PKCϵ in the two cell types. J. Neurosci. Res. 57:33–38, 1999.

Glutamate production by cultured microglia : differences between rat and mouse, enhancement by lipopolysaccharide and lack effect of HIV coat protein gp120 and depolarizing agents

Glutamate release from rat and mouse microglia subcultures grown in a serum-free medium was substantially greater in the presence than in the absence of a physiological concentration of glutamine (0.5 mM). Mouse microglia produced and released more glutamate than rat microglia. Glutamate accumulation in the medium increased with time and cell density, which is consistent with the virtual absence of glutamate reuptake. Lipopolysaccharide (LPS; 10-100 ng/ml), HIV coat protein gp120 (0.1-10 nM), high K+ (35 mM) or ATP (150 microM), did not affect glutamate release from cells maintained in serum-free medium. In the presence of 1% dialyzed serum, however, LPS induced a dose- and time-dependent increase in the accumulation of glutamate in the medium, suggesting that, as in other cell types, serum factors are required for LPS binding to its receptors.

Multiple Actions of the Human Immunodeficiency Virus Type-1 Tat Protein on Microglial Cell Functions

The human immunodeficiency virus type-1 (HIV-1) regulatory protein Tat is produced in the early phase of infection and is essential for virus replication. Together with other viral products, Tat has been implicated in the pathogenesis of HIV-1–associated dementia (HAD). As HIV-1 infection in the brain is very limited and macrophage/microglial cells are the only cellular type productively infected by the virus, it has been proposed that many of the viral neurotoxic effects are mediated by microglial products. We and others have shown that Tat affects the functional state of microglial cells, supporting the hypothesis that activated microglia play a role in the neuropathology associated with HIV-1 infection. This review describes the experimental evidence indicating that Tat stimulates microglia to synthesize potentially neurotoxic molecules, including proinflammatory cytokines and free radicals, and interferes with molecular mechanisms controlling cAMP levels, intracellular [Ca2+], and ion channel expression.

Human CD4+ T lymphocytes with increased intracellular cAMP levels exert regulatory functions by releasing extracellular cAMP

We have previously shown that cholera toxin (CT) and other cAMP‐elevating agents induce up‐regulation of the inhibitory molecule CTLA‐4 on human resting T lymphocytes. In this study, we evaluated the function of these cells. We found that purified human CD4+ T lymphocytes pretreated with CT were able to inhibit proliferation of autologous PBMC in a dose‐dependent manner. It is interesting that this phenomenon was not mediated by inhibitory cytokines such as IL‐10, IL‐4, or TGF‐β but was in part caused by the release of extracellular cAMP by the CD4+ T lymphocytes. Purified CD4+ T cells pretreated with forskolin, a transient cAMP inducer, or with dibutyryl cAMP, an analog of cAMP, did not exert suppressive functions, suggesting that a sustained production of cAMP, such as that induced by CT, was required to identify a novel regulatory function mediated by CD4+ T cells. Our results show that CD4+ T lymphocytes can exert regulatory functions through the release of extracellular cAMP and that the cyclic nucleotide acts as a primary messenger, which could play a biological role in the modulation of immune responses.

Opposite Regulation of Adenylyl Cyclase by Protein Kinase C in Astrocyte and Microglia Cultures

Abstract: We studied the regulation of cyclic AMP responses by protein kinase C (PKC) in purified astrocyte and microglia cultures obtained from the neonatal rat brain. In astrocytes, a 10‐min treatment with the phorbol esters phorbol 12‐myristate 13‐acetate (PMA) and 4β‐phorbol 12,13‐didecanoate (4β‐PDD) (but not with 4α‐PDD) or with diacylglycerol, which activate PKC, dose‐dependently enhanced cyclic AMP accumulation induced by the β‐adrenergic agonist isoproterenol and the adenylyl cyclase activator forskolin. Such enhancement was prevented by the PKC inhibitors staurosporine and calphostin‐C and by down‐regulation of PKC and was not related to activation of membrane receptors or Gs proteins or to inhibition of Gi proteins or phosphodiesterases. Instead, the activity of adenylyl cyclase doubled in PMA‐treated astrocytes. In microglia, a 10‐min treatment with PMA or PKC inhibitors did not affect cyclic AMP accumulation, whereas longer treatments with PMA or 4β‐PDD (but not 4α‐PDD) inhibited the cyclic AMP response in a time‐ and dose‐dependent manner. Such inhibition was mimicked by staurosporine and calphostin‐C. Also, in the case of microglia, the modulation of cyclic AMP responses appeared to occur at the level of adenylyl cyclase, and not elsewhere in the cyclic AMP cascade. The inhibition of microglial adenylyl cyclase was apparently not due to aspecific cytotoxicity. A differential regulation of adenylyl cyclase by PKC in astrocytes and microglia may help to explain qualitative and quantitative differences in the response of these cells to various physiological and pathological stimuli.

Human monocytes respond to extracellular cAMP through A2A and A2B adenosine receptors

In this study, we test the hypothesis that cAMP, acting as an extracellular mediator, affects the physiology and function of human myeloid cells. The cAMP is a second messenger recognized as a universal regulator of several cellular functions in different organisms. Many studies have shown that extracellular cAMP exerts regulatory functions, acting as first mediator in multiple tissues. However, the impact of extracellular cAMP on cells of the immune system has not been fully investigated. We found that human monocytes exposed to extracellular cAMP exhibit higher expression of CD14 and lower amount of MHC class I and class II molecules. When cAMP‐treated monocytes are exposed to proinflammatory stimuli, they exhibit an increased production of IL‐6 and IL‐10 and a lower amount of TNF‐α and IL‐12 compared with control cells, resembling the features of the alternative‐activated macrophages or M2 macrophages. In addition, we show that extracellular cAMP affects monocyte differentiation into DCs, promoting the induction of cells displaying an activated, macrophage‐like phenotype with reduced capacity of polarized, naive CD4+ T cells into IFN‐γ‐producing lymphocytes compared with control cells. The effects of extracellular cAMP on monocytes are mediated by CD73 ecto‐5′‐nucleotidase and A2A and A2B adenosine receptors, as selective antagonists could reverse its effects. Of note, the expression of CD73 molecules has been found on the membrane of a small population of CD14+CD16+ monocytes. These findings suggest that an extracellular cAMP‐adenosine pathway is active in cells of the immune systems.

Low expression of estrogen receptor β in T lymphocytes and high serum levels of anti-estrogen receptor α antibodies impact disease activity in female patients with systemic lupus erythematosus

BackgroundCurrent evidence indicates that estrogens, in particular 17β-estradiol (E2), play a crucial role in the gender bias of autoimmune diseases although the underlying molecular mechanisms have not yet been fully elucidated. Immune cells have estrogen receptors (ERs), i.e., ERα and ERβ, that play pro- and anti-inflammatory functions, respectively, and the presence of one estrogen receptor (ER) subtype over the other might change estrogen effects, promoting or dampening inflammation. In this study, we contributed to define the influences of E2 on T cells from female patients with systemic lupus erythematosus (SLE), a representative autoimmune disease characterized by a higher prevalence in women than in men (female/male ratio 9:1). Particularly, our aim was to evaluate whether alterations of ERα and ERβ expression in T cells from female SLE patients may impact lymphocyte sensitivity to E2 and anti-ERα antibody (anti-ERα Ab) stimulation interfering with cell signaling and display a direct clinical effect.MethodsSixty-one premenopausal female patients with SLE and 40 age-matched healthy donors were recruited. Patients were divided into two groups based on the SLE Disease Activity Index 2000 (SLEDAI-2K) (i.e., <6 and ≥6). ER expression was evaluated in T lymphocytes by flow cytometry, immunofluorescence, and Western blot analyses. Serum anti-ERα Ab levels were analyzed by enzyme-linked immunosorbent assay (ELISA). ER-dependent signaling pathways were measured by a phosphoprotein detection kit.ResultsIntracellular ERβ expression was significantly lower in T cells from patients with SLEDAI-2K ≥6 as compared with healthy donors and patients with SLEDAI-2K <6 and negatively correlated with disease activity. The expression of intracellular and membrane-associated-ERα was similar in SLE and control T cells. ER-dependent signaling pathways were activated in T cells from SLE patients with SLEDAI-2K ≥6, but not with SLEDAI-2K <6, when both membrane and intracellular ERs were stimulated by co-treatment with E2 and anti-ERα Abs.ConclusionsOur results demonstrate an altered ER profile in SLE patients, possibly contributing to SLE pathogenesis and interfering with clinical activity, and highlight the potential exploitation of T cell-associated ERβ as a biomarker of disease activity.

Human immunodeficiency virus protein gp120 interferes with beta-adrenergic receptor-mediated protein phosphorylation in cultured rat cortical astrocytes.

Summary1. We have previously shown that acute exposure to the HIV coat protein gp120 interferes with the β-adrenergic regulation of astroglial and microglial cells (Leviet al., 1993). In particular, exposure to 100 pM gp120 for 30 min depressed the phosphorylation of vimentin and glial fibrillary acidic protein (GFAP) induced by isoproterenol in rat cortical astrocyte cultures. In the present study we have extended our analysis on the effects of gp120 on astroglial protein phosphorylation.2. We found that chronic (3-day) treatment of the cells with 100 pM gp120 before exposure to isoproterenol was substantially more effective than acute treatment in depressing the stimulatory effect of the β-adrenergic agonist on vimentin and GFAP phosphorylation.3. Even after chronic treatment with gp120, no differences were found in the levels and solubility of these proteins.4. Besides stimulating the phosphorylation of intermediate filament proteins, isoproterenol inhibited the incorporation of32P into a soluble acidic protein of 80,000Mr, which was only minimally present in Triton X-100-insoluble extracts.5. Treatment of astrocytes with a phorbol ester or exposure to3H-myristic acid indicated that the acidic 80,000Mr protein is a substrate for protein kinase C (PKC) and is myristoylated, thus suggesting that it is related to the MARCKS family of PKC substrates.6. Acute (30-min) treatment with 100 pM gp120 totally prevented the inhibitory effect of isoproterenol on the phorphorylation of the 80,000Mr MARCKS-like protein.7. Our studies corroborate the hypothesis that viral components may contribute to the neuropathological changes observed in AIDS through the alteration of signal transduction systems in glial cells.