The goal of our study was to assess whether the human immunodeficiency virus (HIV) coat protein gpl20 induces functional alterations in astrocytes and microglia, known for their reactivity and involvement in most types of brain pathology. We hypothesized that gpl20-induced anomalies in glial functions, ifpresent, might be mediated by changes in the levels of intracellular messengers important for signal transduction, such as cAMP. Acute (10 min) exposure of cultured rat cortical astrocytes or microglia to 100 pM gpl20 caused only a modest (50-60%), though statistically significant, elevation in cAMP levels, which was antagonized by the (3-adrenergic receptor antagonist propranolol. More importantly, the protein substantially depressed [by 30% (astrocytes) and 50% (microglia)] the large increase in cAMP induced by the .3-adrenergic agonist isoproterenol (10 nM), without affecting that induced by direct adenylate cyclase stimulation by forskolin. Qualitatively similar results were obtained using a glial fibrillary acidic protein (GFAP)-positive human glioma cell line. The depression of the (3-adrenergic response had functional consequences in both astrocytes and microglia. In astrocytes we studied the phosphorylation of the two major cytoskeletal proteins, vimentin and GFAP, which is normally stimulated by isoproterenol, and found that gpl20 partially (40-50%) prevented such stimulation. In microglial cells, which are the major producers of inflammatory cytokines within the brain, gpl20 partially antagonized the negative ,3-adrenergic modulation of lipopolysaccharide (10 ng/ml)-induced production of tumor necrosis factor a. Our results suggest that, by interfering with the ,B-adrenergic regulation of astrocytes and microglia, gpl20 may alter astroglial "reactivity" and upset the delicate cytokine network responsible for the defense against viral and opportunistic infections.The pathogenesis of AIDS encephalopathy, a complication present in 50-80% of AIDS patients, is still largely unknown (1-4). Besides direct infection of brain cells by human immunodeficiency virus (HIV), which seems to occur mainly in microglia (2-6), the intrinsic brain macrophages, other factors could contribute to the development of brain damage, such as neurotoxic substances produced by brain cells and/or by invading hematic cells, or proteins encoded by the viral genome. Among the latter, the envelope glycoprotein gp120 was reported to cause neuronal death in cell cultures from the rodent central nervous system (reviewed in ref. 7; see also ref. 8) and to induce interleukin 1 (9) and to cause learning impairment (10) in the rat brain in vivo. In the present study we evaluated the possibility that the viral protein may cause functional alterations in glial cell types (astrocytes and microglia) known for their reactivity and involvement in most neurological diseases. We found that acute exposure to picomolar gp120 depressed the (3adrenergic agonist-induced formation of cAMP and altered important cAMP-regulated functions in both cel...

In order to study the voltage-dependent ion channels in microglia, and their possible modulation by pro-inflammatory substances like lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) we employed the patch-clamp technique on purified rat microglial cell subcultures grown for 1 - 5 days in control condition or after a 24 hour treatment with those agents. Regardless of the culture condition, almost 100% of the cells presented inward-rectifying (IR) K+ currents identified by the following features: (a) extracellular K(+)-dependence of Vrev and whole-cell conductance; (b) inward-rectifying property; (c) channel blocking mechanism by Cs+; and (d) single channel conductance of 27 pS. A 'n' type outward-rectifying (OR) K+ current was present in 30% of the cells during the first 2 days of subcultivation. Its occurrence was strongly dependent on the preparation, varying from 0% to almost 80%, and it decreased to 13% of the cells after three days in culture. It showed the following features: (i) threshold of activation close to -30 mV; (ii) sigmoid current onset; (iii) voltage-dependent kinetics; and (iv) sensitivity to 4-aminopyridine (4-AP) and tetraethylammonium (TEA). Furthermore, we detected two ion currents not previously described in microglia: (i) a slowly activating outward current which appeared at potentials more positive than +20 mV and with a reversal potential close to 0 mV, tentatively identified as a proton current; and (ii) a Cl- conductance identified in ion substitution experiments as the current sensitive to the Cl- channel blocker SITS. The two agents, LPS (20 - 2,000 ng/ml) and IFN-gamma (10 - 100 u/ml), shared the following effects: (a) enhancement of membrane capacitance, and (b) increase of OR current amplitude and frequency of occurrence. Moreover, IFN-gamma was also able to increase IR current density, especially in cells with ameboid morphology, while LPS was ineffective. We conclude that the voltage-dependent ion channel pattern of microglia is more complex than previously thought and that activating agents such as LPS and IFN-gamma share some electrophysiological effects, but differ in others.

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.

Kainate (KA), quisqualate (QA), and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) stimulated gamma-aminobutyric acid [3H]gamma-aminobutyric acid (GABA) release from cultured cerebellar type 2 astrocytes and from their bipotential precursors. The evoked release was prevented by the antagonist 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX). AMPA and QA applied together with KA at concentrations around or above their EC50S (20-50 microM) antagonized the stimulatory effect of KA on [3H]GABA release. On the other hand, the releasing action of KA was potentiated by concentrations of QA in the low micromolar range (2-5 microM), particularly when the concentration of KA was at the borderline of effectiveness (10 microM). KA and QA did not elevate intracellular cyclic GMP levels in astrocyte cultures, although guanylate cyclase was present in both type 2 and type 1 astrocytes. The inability of KA to elevate cyclic GMP levels in astrocytes was the only major difference in the behavior of this glutamate agonist between astroglial and neuronal cultures. The GABA transport inhibitor nipecotic acid or replacement of NaCl with LiCl abolished [3H]GABA uptake and also KA- and QA-induced release of preaccumulated [3H]GABA. Therefore, [3H]GABA was released from type 2 astrocytes and their progenitors through its Na(+)-dependent transport system, operating in an outward direction when the cells were depolarized by non-NMDA receptor agonists.

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 microM 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 microM kainate or 50 microM 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 greater than glutamate greater than aspartate. The effect was somewhat more pronounced in type-2 than in type-1 astrocytes. Veratridine (50 microM) 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.