Michel P. Rathbone

A randomized trial to assess the efficacy of surgery in addition to radiotherapy in patients with a single cerebral metastasis

Cerebral metastasis is a common oncologic problem that occurs in 15–30% of cancer patients; approximately half such metastases are single. Previous retrospective studies and two randomized trials reported that the addition of surgical extirpation prior to radiation therapy increased survival, neurologic function, and quality of life compared with radiation alone in patients with a single brain metastasis.

Trophic actions of extracellular nucleotides and nucleosides on glial and neuronal cells

In addition to their well-established roles as neurotransmitters and neuromodulators, growing evidence suggests that nucleotides and nucleosides might also act as trophic factors in both the central and peripheral nervous systems. Specific extracellular receptor subtypes for these compounds are expressed on neurons, glial and endothelial cells, where they mediate strikingly different effects. These range from induction of cell differentiation and apoptosis, mitogenesis and morphogenetic changes, to stimulation of synthesis or release, or both, of cytokines and neurotrophic factors, both under physiological and pathological conditions. Nucleotides and nucleosides might be involved in the regulation of development and plasticity of the nervous system, and in the pathophysiology of neurodegenerative disorders. Receptors for nucleotides and nucleosides could represent a novel target for the development of therapeutic strategies to treat incurable diseases of the nervous system, including trauma- and ischemia-associated neurodegeneration, demyelinating and aging-associated cognitive disorders.

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.

Nitric oxide donors enhance neurotrophin‐induced neurite outgrowth through a cGMP‐dependent mechanism

Nitric oxide (NO), a diffusible and unstable gas, has been implicated in inter‐ and intra‐cellular communication in the nervous system. NO also plays a role in neural development, plasticity and alterations of synaptic function such as long‐term potentiation and long‐term depression (Gally et al.: Proc NY Acad Sci, 87:354–355, 1990; Zhuo et al.: Science 260:1946–1950, 1993; Schuman and Madison.: Science 254:1503–1506, 1991; Bruhwyler et al.: Neurosci Biobehav Rev 17:373–384, 1993) some of which likely involve growth and remodelling of neurites. Some actions of NO are mediated directly by protein modification (e.g., nitrosylation) and others by activation of soluble guanylyl cyclase (soluble GC), which increases intracellular levels of guanosine 3′,5′‐cyclic monophosphate (cGMP). NO is synthesized by the enzyme nitric oxide synthase (NOS), which is induced by treatment of CNS neurons (Holtzman et al.: Neurobiol Disease 1:51–60, 1994) or pheochromocytoma PC12 cells (Hirsch et al.: Curr Biol 3:749–754, 1993) with NGF. NO has been proposed to mediate some of the effects of NGF on PC12 cells by inhibiting cell division (Peunova and Enikolopov: Nature 374:68–73, 1995). In addition, NO can substitute for NGF by delaying the death of trophic factor‐deprived PC12 cells through a mechanism that does not involve a cytostatic action (Farinelli et al.: J Neurosci 16:2325–2334, 1996). We investigated whether NO stimulated neurite outgrowth from hippocampal neurons and PC12 cells. Primary cultures of E17 mouse hippocampal neurons co‐cultured with neopallial astrocytes were exposed to the NO donors sodium nitrite (100 μM) or sodium nitroprusside (100 nM). After 48 hr, NO donor‐treated cultures contained a greater proportion of cells bearing neurites and neurites that were much longer than those found in control cultures. In cultures of PC12 cells, NO donors also enhanced the neuritogenic effects of NGF. The proportion of PC12 cells with neurites 48 hr after exposure to NO donors sodium nitrite (100 μM–10 mM) or sodium nitroprusside (100 nM–1 μM) plus 2.5S nerve growth factor (NGF) was approximately twice the proportion of cells with neurites in sister cultures grown in NGF alone. Neither of the NO donors elicited neurites from the PC12 cells in the absence of NGF. The effects of the NO donors were likely mediated by release of NO since their effects were antagonized by addition of hemoglobin, which avidly binds NO, to the culture medium. The enhancement by NO of NGF‐mediated neurite outgrowth in PC12 cells appeared to occur through a cGMP‐dependent mechanism. The NO donors stimulated a prompt increase in intracellular cGMP in PC12 cells. Moreover their action was mimicked by addition of the membrane‐permeant cGMP analogs 8‐Bromo‐cGMP (8‐Br‐cGMP) and para (chlorophenylthio)‐cGMP (pCPT‐cGMP) to the culture medium and by atrial natriuretic factor which stimulates particulate guanylyl cyclase. The neuritogenic activity of the NO donors was inhibited by LY83583 and methylene blue, inhibitors of guanylyl cyclase. These data imply that NO may act alone or with other growth factors to regulate synapse formation and maintenance by stimulating neurite outgrowth. J. Neurosci. Res. 47:427–439, 1997.

Rat cultured astrocytes release guanine-based purines in basal conditions and after hypoxia/hypoglycemia

Brain ischemia stimulates release from astrocytes of adenine‐based purines, particularly adenosine, which is neuroprotective. Guanosine, which has trophic properties that may aid recovery following neurological damage, is present in high local concentrations for several days after focal cerebral ischemia. We investigated whether guanine‐based purines, like their adenine‐based counterparts, were released from astrocytes and whether their release increased following hypoxia/hypoglycemia. HPLC analysis of culture medium of rat astrocytes showed spontaneous release of endogenous guanine‐based purines at a higher rate than their adenine‐based counterparts. The concentration of guanosine (≈120 nM) and adenosine (≈43 nM) in the culture medium remained constant, whereas concentrations of adenine and guanine nucleotides, particularly GMP, and their metabolites increased with time. Exposure of the cultures to hypoxia/hypoglycemia for 30 min increased the extracellular concentration of adenine‐based purines by 2.5‐fold and of guanine‐based purines by 3.5‐fold. Following hypoxia/hypoglycemia extracellular adenine nucleotide levels increased further. Adenosine concentration increased, but not proportionally to nucleotide levels. Accumulation of adenosine metabolites indicated it was rapidly metabolized. Conversely, the concentrations of extracellular guanine‐based nucleotides remained elevated and the concentration of guanosine continued to increase. These data indicate that astrocytes are a major source of guanine‐based purines, the release of which is markedly increased following hypoxia/hypoglycemia, permitting them to exert neurotrophic effects. GLIA 25:93–98, 1999.

Glial cells express multiple ATP binding cassette proteins which are involved in ATP release

Rat brain astrocyte and microglia cultures express different members of ATP-binding-cassette (ABC) proteins. RT-PCR analysis showed that astrocytes are equipped with P-glycoprotein (mdr1a, mdr1b), multidrug resistance-associated-protein (mrp1, mrp4, mrp5) and cystic fibrosis transmembrane conductance regulator (CFTR). No transcripts for mrp5 and CFTR were detected in microglia. The ABC protein functional activities are shown by the following results: (i) cyclosporin A (50 μM), verapamil (50 μM), probenecid (1 mM) or sulfinpyrazone (2 mM) enhanced [3H]vincristine accumulation; (ii) cyclosporin A or verapamil but not probenecid or sulfinpyrazone enhanced [3H]digoxin accumulation; (iii) glibenclamide (100 μM) inhibited 36Clefflux from astrocytes. ATP release from glial cells was inhibited by the pretreatment with ABC protein inhibitors indicating that ABC proteins are involved in nucleotide efflux from glial cells which represent the main source of cerebral extracellular purines.

Mechanisms of apoptosis induced by purine nucleosides in astrocytes

Astrocytes release adenine‐based and guanine‐based purines under physiological and, particularly, pathological conditions. Thus, the aim of this study was to determine if adenosine induced apoptosis in cultured rat astrocytes. Further, if guanosine, which increases the extracellular concentration of adenosine, also induced apoptosis determined using the TUNEL and Annexin V assays. Adenosine induced apoptosis in a concentration‐dependent manner up to 100 μM. Inosine, hypoxanthine, guanine, and guanosine did not. Guanosine or adenosine (100 μM) added to the culture medium was metabolized, with 35% or 15%, respectively, remaining after 2–3 h. Guanosine evoked the extracellular accumulation of adenosine, and particularly of adenine‐based nucleotides. Cotreatment with EHNA and guanosine increased the extracellular accumulation of adenosine and induced apoptosis. Inhibition of the nucleoside transporters using NBTI (100 μM) or propentophylline (100 μM) significantly decreased but did not abolish the apoptosis induced by guanosine + EHNA or adenosine + EHNA, respectively. Apoptosis produced by either guanosine + EHNA or adenosine + EHNA was unaffected by A1 or A2 adenosine receptor antagonists, but was significantly reduced by MRS 1523, a selective A3 adenosine receptor antagonist. Adenosine + EHNA, not guanosine + EHNA, significantly increased the intracellular concentration of S‐adenosyl‐L‐homocysteine (SAH) and greatly reduced the ratio of S‐adenosyl‐L‐methioine to SAH, which is associated with apoptosis. These data demonstrate that adenosine mediates apoptosis of astrocytes both, via activation of A3 adenosine receptors and by modulating SAH hydrolase activity. Guanosine induces apoptosis by accumulating extracellular adenosine, which then acts solely via A3 adenosine receptors. GLIA 38:179–190, 2002.

Specific [3H]‐guanosine binding sites in rat brain membranes

Extracellular guanosine has diverse effects on many cellular components of the central nervous system, some of which may be related to its uptake into cells and others to its ability to release adenine‐based purines from cells. Yet other effects of extracellular guanosine are compatible with an action on G‐protein linked cell membrane receptors. Specific binding sites for [3H]‐guanosine were detected on membrane preparations from rat brain. The kinetics of [3H]‐guanosine binding to membranes was described by rate constants of association and dissociation of 2.6122×107 M−1 min−1 and 1.69 min−1, respectively. A single high affinity binding site for [3H]‐guanosine with a KD of 95.4±11.9 nM and Bmax of 0.57±0.03 pmol mg−1 protein was shown. This site was specific for guanosine, and the order of potency in displacing 50 nM [3H]‐guanosine was: guanosine=6‐thio‐guanosine>inosine>6‐thio‐guanine>guanine. Other naturally occurring purines, such as adenosine, hypoxanthine, xanthine caffeine, theophylline, GDP, GMP and ATP were unable to significantly displace the radiolabelled guanosine. Thus, this binding site is distinct from the well‐characterized receptors for adenosine and purines. The addition of GTP produced a small concentration‐dependent decrease in guanosine binding, suggesting this guanosine binding site was linked to a G‐protein. Our results therefore are consistent with the existence of a novel cell membrane receptor site, specific for guanosine.

Cultured astrocyte proliferation induced by extracellular guanosine involves endogenous adenosine and is raised by the co‐presence of microglia

Extracellular adenosine (Ado) and ATP stimulate astrocyte proliferation through activation of P1 and P2 purinoceptors. Extracellular GTP and guanosine (Guo), however, that do not bind strongly to these receptors, are more effective mitogens than ATP and Ado. Exogenous Guo, like GTP and 5′‐guanosine‐βγ‐imidotriphosphate (GMP‐PNP), dose‐dependently stimulated proliferation of rat cultured astrocytes; potency order GMP‐PNP > GTP ≥ Guo. The mitogenic effect of Guo was independent of the extracellular breakdown of GTP to Guo, because GMP‐PNP, a GTP analogue resistant to hydrolysis, was the most mitogenic. In addition to a direct effect on astrocytes, Guo exerts its proliferative activity involving Ado. Exogenous Guo, indeed, enhanced the extracellular levels of endogenous Ado assayed by HPLC in the medium of cultured astrocytes. Culture pretreatment with Ado deaminase (ADA), that converts Ado into inosine, reduced but did not abolish Guo‐induced astrocyte proliferation whereas erythro‐9‐(2‐hydroxy‐3‐nonyl)adenine (EHNA), that inhibits ADA activity, amplified Guo effect. Moreover, the mitogenic activity of Guo was partly inhibited by 8‐cyclopentyl‐1,3‐dipropylxanthine and alloxazine, antagonists of Ado A1 and A2B receptors, respectively. Also microglia seem to be a target for the action of Guo. Indeed, the mitogenic effect of Guo on astrocytes was: i) increased proportionally to the number of microglial cells present in the astrocyte cultures; ii) amplified when purified cultures of astrocytes were supplemented with conditioned medium deriving from Guo‐pretreated microglial cultures. These data indicate that the mitogenic effects exerted by exogenous Guo on rat astrocytes are mediated via complex mechanisms involving extracellular Ado and microglia‐derived soluble factors. GLIA 29:202–211, 2000.

Purine nucleosides and nucleotides stimulate proliferation of a wide range of cell types

SummaryPresumptive astrocytes isolated from 10-day white Leghorn chick embryos, Factor VIII-positive human brain capillary endothelial cells, meningeal fibroblasts from 10-day chick embryos, Swiss mouse 3T3 cells, and human astrocytoma cell lines, SKMG-1 and U373, were rendered quiescent when placed in culture medium that contained 0 or 0.2% serum for 48 h; their proliferation was markedly reduced and they incorporated [3H]thymidine at a low rate. [3H]Thymidine incorporation and cell proliferation were induced in all types of cells by addition of guanosine, GMP, GDP, GTP, and to a lesser extent, adenosine, AMP, ADP or ATP to the culture medium. The stimulation of proliferation by adenosine and guanosine was abolished by 1,3-dipropyl-7-methylxanthine (DPMX), an adenosine A2 receptor antagonist, but not by 1,3,-dipropyl-8-(2-amino-4-chorophenyl)xanthine (PACPX), an A1 antagonist. Stimulation of proliferation by the nucleotides was not abolished by either DPMX or PACPX. The P2 receptor agonists,α,β-methyleneATP and 2-methylthioATP, also stimulated [3H]thymidine incorporation into the cells with peak activity at approximately 3.5 and 0.03 nM, respectively. These data imply that adenosine and guanosine stimulate proliferation of these cell types through activation of an adenosine A2 receptor, and the stimulation of cell proliferation by the nucleotides may be due to the activation of purinergic P2y receptors. As the primary cultures grew older their growth rate slowed. The capacity of the purine nucleosides and nucleotides to stimulate their growth diminished concomitantly. The 3T3 cells showed neither decreased growth with increased passages nor reduced response to the purines. In contrast, although the doubling time of the immortalized human astrocytoma cell lines SKMG-1 and U373 remained constant, the responsiveness to purinergic stimulation of the U373 cells decreased but that of the SKMG-1 did not. These data are compatible with a decrease in the number, or the ligand-binding affinity of the purinergic receptors, or a decreased coupling of purinergic receptors to intracellular mediators in primary cells aged in tissue culture.