Angelika Schobert

Release of ATP from cultured rat astrocytes elicited by glutamate receptor activation

The release of ATP was studied in cultures of astrocytes derived from the brain hemispheres of newborn rats. There was a basal efflux of ATP, which was increased up to 19-fold by glutamate (300-1000 microM). N-methyl-D-aspartate (20-500 microM), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA; 30-100 microM) and kainate (20 microM). The N-methyl-D-aspartate receptor-selective antagonist 2-amino-5-phosphonopentanoate (100 microM) blocked the effect of N-methyl-D-aspartate but not the effects of AMPA, kainate and glutamate. The AMPA receptor-selective antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (30 microM) blocked the effect of AMPA and also of glutamate and N-methyl-D-aspartate, but not the effect of kainate. The kainate receptor-selective antagonist D-glutamyl-amino-methanesulfonate (30 microM) blocked the effect of kainate but not of glutamate. Glutamate (1000 microM) did not increase the release of lactate dehydrogenase from astrocytes. Excitatory amino acids are known to release adenyl compounds in the brain. The present results identify one adenyl compound thus released, namely ATP, and identify astrocytes as one source. The release is brought about by activation of any of the three ionotropic glutamate receptor types-N-methyl-D-aspartate, AMPA and kainate receptors. AMPA receptors seem to mediate at least a part of the effect of glutamate itself, but the involvement of other receptors cannot be ruled out. ATP and its degradation products, such as adenosine, once released, may exert acute as well as trophic effects on neurons and glial cells.

Co-release of noradrenaline and ATP from cultured sympathetic neurons

The vesicles of postganglionic sympathetic axons store both noradrenaline and ATP. The theory of noradrenaline-ATP co-transmission implies that both compounds are released by nerve action potentials and elicit postjunctional effects. Many properties of postjunctional responses support the theory. However, neural release of ATP has been difficult to detect biochemically: by far the major part of the overflow of ATP from intact tissues upon sympathetic nerve stimulation comes from non-neural elements, especially smooth muscle and endothelial cells. Here we describe a parallel electrically evoked overflow of [3H]noradrenaline and endogenous ATP from cultured chick sympathetic neurons. The overflow was abolished by tetrodotoxin, omega-conotoxin and withdrawal of Ca2+, was increased by tetraethylammonium and 4-aminopyridine, and was not changed by prazosin or suramin. The results demonstrate directly the action potential-evoked, Ca(2+)-dependent and presumably vesicular and exocytotic release of ATP from postganglionic sympathetic neurons. They support the co-transmitter theory and suggest that cultured sympathetic neurons are a preparation in which noradrenaline-ATP co-release can be examined free from postjunctional components.

Rat microglial interleukin-3

Interleukin-3 (IL-3, multi-CSF) is a growth factor for a variety of hematopoietic progenitor cells. Recently, microglial cells, the resident macrophages of the central nervous system (CNS) have been shown to proliferate in the presence of IL-3 both in vivo and in culture. Data obtained from cultured astrocytes gave rise to the hypothesis that astrocytes synthesize the microglial growth factor. This is the first report identifying rat microglial cells themselves as a source of IL-3. Culture media conditioned by isolated microglia enhanced microglial proliferation above fresh media controls. IL-3 polypeptide was detected in both conditioned media (CM) and in microglial cells by Western blotting and immunoprecipitation. Furthermore, anti-IL-3 antibodies were able to inhibit microglial proliferation induced by conditioned media. mRNAIL-3 was present in single microglial cells as revealed by in situ hybridization. Total RNA prepared from purified microglia yielded a single PCR amplification product. Identity of the PCR product was confirmed by Southern blot hybridization using a cDNAIL-3 probe and by DNA sequencing. Expression of mRNAIL-3 was observed in both absence and presence of lipopolysaccharide, a bacterial endotoxin, that commonly induces expression of inflammatory cytokines and inhibits microglial proliferation. It is concluded that IL-3 expression in ensuring the recruitment of enhanced numbers of immunocompetent cells at sites of lesion. In the light of weak immune reactions in the brain, it is hypothesized that the expression of a characteristic T cell feature in monocyte-derived microglia may be a partial compensation of T cell functions in brain lesions.

P2-purinoceptor induced prostaglandin synthesis in primary rat astrocyte cultures

SummaryAdenosine triphosphate (ATP) is one of the cotransmitters that are commonly released at catecholaminergic and cholinergic nerve terminals. The glial cell type most closely associated with the synapse is the astrocyte and, thus, is the next cellular element beside the postsynaptic neuron to face the transmitters released. This report gives evidence of P2-purinoceptors on cultured astroglial cells. Upon stimulation with nucleoside triphosphates and nucleoside diphosphates, the cells respond with synthesis of prostaglandins of the D2 type, which is the predominant prostaglandin made in rat brain. Nucleoside triphosphate analogues, such as 5′-adenylyl-imido diphosphate, β,γ-methylene, or α,β-methylene ATP were less effective than ATP or its non-hydrolysable analogue ATP [γ S]. The receptor was desensitized by ATP [γ S] within 15 min, whereas desensitization by α,β-methylene ATP was significantly delayed. 8-phenyl-theophylline (10−4 M) had no influence on ATP-stimulated prostaglandin synthesis. Adenosine 5′-monophosphate (AMP) and adenosine were unable to stimulate prostaglandin D2 formation. According to the common nomenclature for purinoceptors, the described astroglial receptor would fulfill the characteristics of a P2-purinoceptor. Furthermore, it is shown that pertussis toxin sensitive G-proteins influence some early step in prostaglandin synthesis. The inactivation of these proteins results in reduced prostaglandin formation. It is assumed that ATP serves as an important mediator in the cross-talk between neurons and astroglial cells at the synaptic cleft.

Cultured chick sympathetic neurons: modulation of electrically evoked noradrenaline release by P2-purinoceptors

The present study investigates the pharmacological profile of P2-purinoceptors modulating noradrenaline release from cultured chick sympathetic neurons. ATP (30 μM-3 mM) and 2-methylthio-ATP (3–100 μM), but not α,β-methylene-ATP (up to 100 μM), caused a significant facilitation of electrically evoked [3H]-noradrenaline release when added 2 min before depolarization. The facilitation declined with time of exposure suggesting receptor desensitization. The facilitatory effect was markedly diminished by the P2-purinoceptor antagonists reactive blue 2 (3 μM) and suramin (300 μM), but not changed by mecamylamine (10 μM), a nicotinic receptor antagonist. At 1 mM and higher concentrations, ATP added for 12 min, inhibited noradrenaline release; release was virtually abolished by 6 mM ATP. The inhibitory effect of ATP was slightly diminished by suramin but not affected by reactive blue 2. Electrically evoked [3H]-noradrenaline release remained unaffected in the presence of the adenosine (P1)-receptor agonists R(−)N6-(2-phenylisopropyl)adenosine (R-PIA), 2-[p-(2-carboxyethyl)phenylethylamino]5′-N-ethylcarboxamidoadenosine (CGS-21680), 5′-N-ethylcarboxamidoadenosine (NECA), and N6-2-(4-aminophenyl)ethyladenosine (APNEA), used up to 1 μM.The present results confirm the existence of two P2-purinoceptors affecting noradrenaline release: 1) a facilitatory receptor which is activated by 2-methyl thio-ATP as well as ATP, and blocked by suramin as well as reactive blue 2, and 2) an inhibitory receptor which is activated by ATP, only slightly affected by suramin but not at all by reactive blue 2 and does not belong to the established P2-purinoceptor subtypes.

Regulation and Glucocorticoid-Independent Induction of Lipocortin I in Cultured Astrocytes

Abstract: Stimulation of prostaglandin (PG) release in rat astroglial cultures by various substances, including phorbol esters, melittin, or extracellular ATP, has been reported recently. It is shown here that glucocorticoids (GCs) reduced both basal and stimulated PGD2 release. Hydrocortisone, however, did not inhibit ATP‐, calcium ionophore A23187‐, or tetradecanoyl phorbol acetate (TPA)‐stimulated arachidonic acid release, and only TPA stimulations were affected by dexamethasone. GC‐mediated inhibition of PGD2 release thus appeared to exclude regulation at the phospholipase A2(PLA2) level. Therefore, the effects of GCs on the synthesis of lipocortin I (LC I), a potent, physiological inhibitor of PLA2, were studied in more detail. Dexamethasone was not able to enhance de novo synthesis of LC I in freshly seeded cultures and failed to increase LC I synthesis in 2–3‐week‐old cultures. It is surprising that LC I was the major LC synthesized in those cultures, and marked amounts accumulated with culture time, reaching plateau levels at approximately day 10. In contrast, LC I was barely detectable in vivo. This tonic inhibition of PLA2 is the most likely explanation for unsuccessful attempts to evoke PG release in astrocyte cultures by various physiological stimuli. GC receptor antagonists (progesterone and RU 38486) given throughout culture time reduced LC I accumulation and simultaneously increased PGD2 release. Nonetheless, a substantial production of LC I persisted in the presence of antagonists. Therefore, LC I induction did not seem to involve GC receptor activation. This was confirmed in serum‐and GC‐free brain cell aggregate cultures. Here also a marked accumulation of LC I was observed. The data raise the hypothesis that enriched astrocyte cultures synthesize steroid‐like compounds (neurosteroids).

Cultured chick sympathetic neurons: ATP-induced noradrenaline release and its blockade by nicotinic receptor antagonists.

The ATP-induced increase in tritium outflow from cultured chick sympathetic neurons prelabelled with [3H]-noradrenaline was investigated.Seven days-old dissociated cell cultures of embryonic paravertebral ganglia, loaded with [3H]-noradrenaline (0.05 μM), were superfused in the presence of (+)-oxaprotiline and exposed to ATP, ATP-analogues, or 1,1-dimethyl-4-piperazinium (DMPP) for 2 min. ATP (3 μLM-3 mM), 2-methylthio-ATP (3–100 μM), as well as DMPP (10 and 100 μM) induced a significant overflow of tritium. The EC50-value of ATP was 20 μM. Both the ATP-induced and the DMPP-induced tritium overflow was Ca2+-dependent and sensitive to tetrodotoxin (0.3 μM) and ω-conotoxin (0.1 μM); in addition, it was inhibited by the α2-adrenoceptor agonist 5-bromo-6-(2-imidazoline-2-ylamino)-quinoxaline (UK-14,304; 1 μM). The effects of ATP and DMPP were not additive. The ATP-induced as well as the DMPP-induced overflow of tritium was diminished by the P2-purinoceptor antagonists suramin (300 μM) and reactive blue 2 (3 μM); in all 4 cases, the inhibition amouted to approximately 40%. The tritium overflow induced by ATP or DMPP was almost abolished by the nicotinic receptor antagonist mecamylamine (10 μM) and markedly inhibited by hexamethonium (100 μM). Neither ATP nor electrical stimulation caused an overflow of tritium from cultures loaded with [3H]-choline.The results suggest that ATP at μmolar concentrations induces noradrenaline release from cultured chick sympathetic neurons via an action on a subclass of the nicotinic cholinoceptor.

P2 purinoceptors modulating noradrenaline release from sympathetic neurons in culture

ATP (1 mM) inhibited, whereas 2-methylthio-ATP (30 microM), a P2Y-selective purinoceptor agonist, increased electrically evoked release of [3H]noradrenaline from chick sympathetic neurons. The P2X-selective purinoceptor agonist alpha,beta-methylene-ATP (30 microM) had no effect. The ATP-induced inhibition of release as well as the facilitation caused by 2-methylthio-ATP was not affected by the selective adenosine (P1) receptor antagonist 8-(p-sulfophenyl)-theophylline (8-PST; 100 microM), but completely prevented by the non-selective P2 antagonist suramin (300 microM). The present data reveal a dual regulation of noradrenaline release from sympathetic neurons. Facilitation seems to be mediated by a P2Y purinoceptor, whereas inhibition is caused by a P2 purinoceptor which needs further subtype characterization.

Secretogranin II Is Synthesized and Secreted in Astrocyte Cultures

Abstract: Astrocyte cultures from rat brain were analyzed for their ability to synthesize and secrete secretogranin II (chromogranin C). Northern blot analysis of polyA‐selected RNA established the presence of secretogranin II mRNA in these cells. By radioimmunoassay, 11.6 fmol/106 astrocytes of secretogranin II was found in these cells. About twice the amount was released into the medium within 3 days. Secretogranin II within the astrocytes was practically unprocessed, as shown by HPLC. These results establish for the first time that astrocytes in vitro synthesize and sec rete a protein of the acidic chromogranin family.

Characterization of cysteinyl-leukotriene formation in primary astroglial cell cultures.

Abstract— The formation and composition of cysteinyl‐leukotrienes (LT) in primary astroglial cell cultures prepared from newborn rat brain has been studied. Small amounts of cysteinyl‐LT determined in terms of LTC4‐like material in the supernatants of the cultures, became detectable after stimulation of the cells with 10−5 M ionophore A23187. Cysteinyl‐LT formation increased with time, reaching about 600 pg (mg protein)−1 after 60 min incubation. In contrast, considerable thromboxane (TX) B2 synthesis was found at 5 min following A23187‐stimulation (about 30 ng TXB2 (mg protein)−1). The synthesis of cysteinyl‐LT was abolished by 5 × 10−5 M nordihydroguaiaretic acid (NDGA). Irrespective of the duration of incubation, blockage of prostanoid synthesis by 10−6 M indomethacin did not result in increased cysteinyl‐LT production. Reversed phase HPLC combined with radioimmunological detection showed that, after 60 min incubation in the presence of A23187, LTC4 and LTD4 accounted for practically all the LTC4‐like immunoreactive material in the supernatants of cell cultures. No significant amounts of LTE4 could be detected. The results show that astrocytes may contribute to brain LTC4 and LTD4 synthesis. However, the cellular site of cerebral LTE4 formation seems to be other than the astroglia.