R. Ciccarelli

Involvement of astrocytes in purine-mediated reparative processes in the brain

Astrocytes are involved in multiple brain functions in physiological conditions, participating in neuronal development, synaptic activity and homeostatic control of the extracellular environment. They also actively participate in the processes triggered by brain injuries, aimed at limiting and repairing brain damages. Purines may play a significant role in the pathophysiology of numerous acute and chronic disorders of the central nervous system (CNS). Astrocytes are the main source of cerebral purines. They release either adenine‐based purines, e.g. adenosine and adenosine triphosphate, or guanine‐based purines, e.g. guanosine and guanosine triphosphate, in physiological conditions and release even more of these purines in pathological conditions. Astrocytes express several receptor subtypes of P1 and P2 types for adenine‐based purines. Receptors for guanine‐based purines are being characterised. Specific ecto‐enzymes such as nucleotidases, adenosine deaminase and, likely, purine nucleoside phosphorylase, metabolise both adenine‐ and guanine‐based purines after release from astrocytes. This regulates the effects of nucleotides and nucleosides by reducing their interaction with specific membrane binding sites. Adenine‐based nucleotides stimulate astrocyte proliferation by a P2‐mediated increase in intracellular [Ca2+] and isoprenylated proteins. Adenosine also, via A2 receptors, may stimulate astrocyte proliferation, but mostly, via A1 and/or A3 receptors, inhibits astrocyte proliferation, thus controlling the excessive reactive astrogliosis triggered by P2 receptors. The activation of A1 receptors also stimulates astrocytes to produce trophic factors, such as nerve growth factor, S100β protein and transforming growth factor β, which contribute to protect neurons against injuries. Guanosine stimulates the output of adenine‐based purines from astrocytes and in addition it directly triggers these cells to proliferate and to produce large amount of neuroprotective factors. These data indicate that adenine‐ and guanine‐based purines released in large amounts from injured or dying cells of CNS may act as signals to initiate brain repair mechanisms widely involving astrocytes.

Activation of A1 adenosine or mGlu3 metabotropic glutamate receptors enhances the release of nerve growth factor and S-100β protein from cultured astrocytes

Pharmacological activation of A1 adenosine receptor with 2‐chloro‐N6‐cyclopentyladenosine (CCPA) or mGlu3 metabotropic glutamate receptors with (2S,2′R,3′R)‐2‐(2′,3′‐dicarboxycyclopropyl)glycine (DCG‐IV) or aminopyrrolidine‐2R,4R‐dicarboxylate (2R,4R‐APDC) enhanced the release of nerve growth factor (NGF) or S‐100β protein from rat cultured astrocytes. Stimulation of release by CCPA and DCG‐IV or 2R,4R‐APDC was inhibited by the A1 adenosine receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine and by the mGlu2/3 receptor antagonist (2S,1′S,2′S,3′R)‐2‐(2′‐carboxy‐3′‐phenylcyclopropyl)glycine (PCCG‐4), respectively. Time‐course studies revealed a profound difference between the release of S‐100β protein and the release of NGF in response to extracellular signals. Stimulation of S‐100β protein exhibited rapid kinetics, peaking after 1 h of drug treatment, whereas the enhancement of NGF release was much slower, requiring at least 6 h of A1 adenosine or mGlu3 receptor activation. In addition, stimulation of NGF but not S‐100β release was substantially reduced in cultures treated with the protein synthesis inhibitor cycloheximide. In addition, a 6–8 h treatment of cultured astrocytes with A1 or mGlu3 receptor agonists increased the levels of both NGF mRNA and NGF‐like immunoreactive proteins, including NGF prohormone. We conclude that activation of A1 adenosine or mGlu3 receptors produces pleiotropic effects in astrocytes, stimulating the synthesis and/or the release of protein factors. Astrocytes may therefore become targets for drugs that stimulate the local production of neurotrophic factors in the CNS, and this may provide the basis for a novel therapeutic strategy in chronic neurodegenerative disorders. GLIA 27:275–281, 1999.

Physiology and pharmacology of natural and synthetic nonadenine-based purines in the nervous system

Like their adenine‐based counterparts, increasing evidence implicates extracellular nonadenine‐based purines such as guanosine and GTP as trophic effector molecules, affecting the growth and differentiation of cells in the nervous system. The extracellular concentration of guanosine is higher than that of adenosine, both in physiological and pathological conditions. Extracellular guanosine and GTP stimulate the astrocyte cell division, apparently through enhancing release of their adenine‐based counterparts, which act in an autocrine fashion. Guanosine and GTP also stimulate the synthesis by astrocytes of several neurotrophic factors, e.g., NGF, and bFGF, and the release of NGF and S100β. As well, guanosine and GTP enhance the differentiation of PC12 cells and hippocampal neurons in vitro. Their action on PC12 cells is associated with the early synthesis of adenotin‐1, a chaperone protein. A hypoxanthine analog, AIT‐082, has similar activity on PC12 cells and neurons to guanosine, but is not metabolized. It enhances memory in both old memory‐deficient mice and in young mice, stimulates neurotrophic factor synthesis in astrocytes in vitro and in brain in vivo, and protects hippocampal neurons in vitro from a “dying‐back” neuropathy caused by brief exposure to high concentrations of glutamate. Given systemically, it protects neurons from NMDA‐induced toxicity. Its neuroprotective effects may partly be related to its ability to stimulate release of NGF from astrocytes. Although AIT‐082 enhances NGF synthesis, unlike exogenously administered NGF it does not produce hyperalgesia. AIT‐082 may prove useful in the treatment of Alzheimers disease, for which it is in Phase II trials, and also in the treatment of acute neuronal injuries due, e.g., to trauma and stroke. Drug Dev. Res. 45:356–372, 1998.

AIT-082 as a potential neuroprotective and regenerative agent in stroke and central nervous system injury

The synthetic purine 4-[[3-(1,6 dihydro-6-oxo-9-purin-9-yl)-1-oxypropyl] amino] benzoic acid (AIT-082, Neotrofin, leteprinim potassium) possesses several biological properties of note: it stimulates outgrowth of neurites from PC12 cells and neurones, stimulates synthesis and/or release of neurotrophic factors from astrocytes, enhances nerve fibre regeneration in vivo and enhances of memory in animals and humans. AIT-082 also protects against glutamate neurotoxicity in vitro and in vivo, which has led to successful tests of AIT-082 in animal models of acute central nervous system injury. In such cases, AIT-082 probably functions by both acutely reducing glutamate excitotoxicity and, over a longer period, by enhancing neuronal sprouting and functional recovery.

Influence of PLA2-PG system on purine release and cAMP content in dissociated primary glial cultures from rat striatum

Purine release and prostaglandin (PG) outflow were simultaneously evaluated from untreated glial primary cultures of rat striatum, at rest and under field electrical stimulation. Purine release was also assayed from sister cultured cells in which a suitable pharmacological treatment with 1 x 10(-6) M dexamethasone or 1 x 10(-4) M indomethacin had produced a complete inhibition of the phospholipase A2-prostaglandin (PLA2-PG) system. Purine release from untreated cells seems to be regulated by specific receptor sites for released adenosine (Ado); A1 receptors exert an inhibitory control on purine release while A2 receptors facilitate it. PG release appears to be related to A1-mediated Ado activity, since culture treatment with 1 x 10(-10) M 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or 1 x 10(-4) M N-ethylmaleimide (NEM), A1 receptor inhibitory agents able to increase purine release, induced a significant reduction of the evoked PG outflow. Purine amount, released from glial cells with inhibited PLA2-PG system, was remarkably greater than that one assayed from control cultured cells. In so treated cultures, no additive effect, NEM-induced, was detected, while the addition of a mixture of PGs partially reduced the increased purine outflow. An electrically evoked cAMP accumulation, significantly greater than that found in controls, was even detected in cultured cells with inhibited PLA2-PG system. Since 10 micrograms/ml adenosine deaminase (ADA) reduced while DPCPX enhanced the evoked cAMP accumulation, it seems partially due to released Ado and accounts for a prevalent A2-stimulating rather than an A1-inhibitory control on adenylate cyclase activity. Thus, in cultured glial cells, the PLA2-PG system, likely linked to A1 receptor sites, concurs to control purine release and seems to affect less directly cAMP accumulation.

Influence of Ca2+ channel modulators on [3H]purine release from rat cultured glial cells

Abstract[3H]Purine release from rat striatum astrocyte cultures was studied at 14 days in vitro (DIV). Superfusion of cultures with a Ca2+-free medium +0.5 mM ethylene glycol-bis(β-aminoethylether)N,N,N′,N′-tetracetic acid (EGTA) reduced the electrically evoked [3H]purine release. Nimodipine only at the concentration of 10 μM modified [3H]purine outflow whereas 0.1 μM ω-conotoxin and 0.03–0.1 μM nitrendipine reduced the evoked one. Superfusion of cultures with 0.1 μM ω-conotoxin +0.1 μM nitrendipine antagonized the evoked [3H]purine release similarly to each drug given alone. Neither nitrendipine nor ω-conotoxin influenced the uptake of45Ca2+ by the cultures. The treatment of cells with the Ca2+ agonist Bay K 8644 did not affect [3H]purine release or the45Ca2+ uptake. The drug did not either alter [Ca2+]i, evaluated by loading the cells with 3 μM Fura-2/AM. 10–30 μM 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), a blocker of intracellular Ca2+ discharge, significantly reduced the evoked [3H]purine release. On the other hand, 2 μM thapsigargin, an inhibitor of the ion store Ca2+ ATPase, was able to increase either the culture [3H]purine release or the [Ca2+]i. Together, the findings indicate that voltage-sensitive calcium channels (VSCCs) of the neuronal N and L-types are not involved in the modulation of [3H]purine release from rat cultured astrocytes whereas Ca2+ coming from intracytoplasmic stores seems to play a prevailing role. Moreover, agents which block VSCCs seem to be able to affect [3H]purine outflow with mechanisms other than VSCC gating.

A1 and A2 Adenosine Receptors Involvement in Controlling Purine and Acetylcholine Release From Rat Hippocampal Slices

Abstract The different role of presynaptic A1 and A2 adenosine receptor subtypes on a possible autoregulation of endogenous purine outflow as well as on the ACh release, simultaneously assayed, was evaluated in rat hippocampal slices, at rest and under a field electrical stimulation.

Influence of Phospholipase A2-Prostaglawdin System Linked to A1 Adexosine Becepmb on Protein Kinase C Activity of Cultubed Glial Cells

Abstract In cultured astrocytes phospholipase A2 seems to be functionally linked to G1 protein of A1 adenosine receptors. Phospholipase A2 inhibition and A1-linked G1 protein inactivation resulted in purine release increase due to protein kinase C activation.

Influence of n-isopropylamino-2-pyrimidine and its derivatives on neuronal growth and adenosine release

In contrast to mouse and chicken astrocytes the ganglioside pattern of cultured rat astrocytes is usually less differentiated. We have obtained apparently a line of rat astrocytes probably through a slight spontaneous transformation. The doubling-time of this cultures is 24 hours while that of primary cultures of rat astrocytes is 36 hours in similar conditions. After 40 generations the specific glial fibrillar acidic protein is still present in our astrocyte cultures as demonstrated by immunocytochemistry. A ganglioside pattern of differentiated cells with the presence of diand tetra-sialogangliosides was obtained. (Isolation and analysis of gangliosides were performed according to R.W. Ledeen & R.K. Yu, Methods in enzymology, 1982, 83, PP 139).

Adenosine-peptides interactions during the development of the rai hypothalamus

Douglas W. Hoffman, Departments of Psychiatry and Pharmacology, Southern Illinois University School of Medicine, P. 0. Box 3!126, Springfield, Illinois 62708 There is currently great interest in the role of endogenous and exogenous opioid peptides and opiates both in neurogenesis and in modulation of neurotransmitter function. Research in this field must take into account the great diversity of molecular form which is becoming apparent in both the proenkephalin A and proenkephalin B derived neuropeptides. Interactions between these two classes of neuropeptides also should not be disregarded. Yhe differing receptor specificities of the many endogenous opioid peptides indicate that they may be serving different roles and interacting with different systems within -,;he nervous system. The guinea pig and rat hippocampus have been studied extensively with the use of high performance liquid chromatography, radioimnunoassays, and rad,ioreceptor assays to identify the endogenous opioid peptides of this brain structure. This evidence will be presented to demonstrate the multiplicity of possible molecular forms which must be taken into account in the design of experiments and interpretation of data derived from experiments involving opioid peptides.