Mayra Paolillo

Pharmacological and molecular evidence for dopamine D1 receptor expression by striatal astrocytes in culture

The neurotransmitter dopamine (DA) at a 10 μM concentration elicited a stimulation of intracellular cyclic AMP (cAMP) accumulation in cultured astrocytes derived from embryonic rat striatum. This accumulation was partially blocked by the β‐adrenergic receptors antagonist propranolol, mimicked by the D1 agonist SKF 38393 and by the mixed D1/D2 agonist apomorphine. A regional heterogeneity in the magnitude of dopamine‐induced cAMP accumulation was observed in cultured astrocytes obtained from different brain areas. The maximum effect was observed in striatal astrocytes, a lower effect in cortical astrocytes, and no increase was detected in cerebellar astrocytes. Reverse transcription‐polymerase chain reaction (RT‐PCR) coupled to Southern blot hybridization demonstrated that striatal astrocytes express only D1 receptor mRNA and Western blot analysis confirmed the expression of the D1 receptor protein in striatal astrocytes. In contrast to what found in neurons, the D1‐dependent cAMP formation in striatal astrocytes is partially reduced by pertussis toxin (PTX) treatment. The stimulation of D1 receptors or the activation of adenylyl cyclase by forskolin led to an increase of cytosolic and nuclear protein kinase A (PKA) catalytic activity. The presence of dopamine D1 receptors in cultured striatal astrocytes suggests a role of dopamine in the regulation of cellular processes in striatal astrocytes. J. Neurosci. Res. 58:544–552, 1999.

Oxidative metabolism in cultured fibroblasts derived from sporadic Alzheimer's disease (AD) patients

Fibroblasts from Alzheimers disease (AD) patients displayed decreased cytochrome c oxidase (complex IV) activity (P < 0.05). The basal oxygen consumption rate (QO2) and the response to an uncoupler of oxidative phosphorylation did not differ between AD and control fibroblasts. The QO2 of AD fibroblasts was more susceptible (P < 0.05) to inhibition by azide in the range 0.5-5 mM. The basal intracellular pH (pHi) in AD fibroblasts was significantly more acidic than in control ones. The results support the hypothesis that subtle dysfunctions of oxidative energy-producing processes are present in fibroblasts from sporadic AD patients. The alterations observed scantly influence the fibroblasts functioning even in stressful conditions; however in tissues, such as the brain, that rely heavily on oxidative metabolism for their function, similar alterations may trigger molecular mechanisms leading to cell damage.

Opposing Actions of D1 and D2‐Dopamine Receptors on Arachidonic Acid Release and Cyclic AMP Production in Striatal Neurons

Abstract: D1‐and D2‐dopamine receptors exert important physiological actions on striatal neurons, but the intracellular second messenger pathways activated by these receptors are still incompletely understood. Using primary cultures of rat striatal cells, we have examined the effects of activating D1 or D2 receptors on arachidonic acid (AA) release and cyclic AMP accumulation. In striatal neurons labeled by incubation with [3H]AA, D2‐receptor stimulation enhanced release of [3H]AA produced by application of the Ca2+ ionophore A23187 or of the purinergic agonist ATP. By contrast, D1‐receptor stimulation inhibited [3H]AA release. This inhibitory effect of D1 receptors was accompanied by stimulation of adenylyl cyclase activity, measured as accumulation of cyclic AMP, and was mimicked by application of the adenylyl cyclase activator forskolin. The results indicate the existence of a novel signaling pathway for D2 and D1 receptors in striatum, potentiation and inhibition, respectively, of Ca2+‐evoked AA release.

The Differential Response of Protein Kinase A to Cyclic AMP in Discrete Brain Areas Correlates with the Abundance of Regulatory Subunit II

Abstract: We analyzed the expression and relative distribution of mRNA for the regulatory subunits (RIα, RIIα, and RIIβ) and of 150‐kDa RIIβ‐anchor proteins for cyclic AMP (cAMP)‐dependent protein kinase (PKA) into discrete brain regions. The subcellular distribution of both holoenzyme and free catalytic subunit was evaluated in the same CNS areas. In the neocortex and corpus striatum high levels of RIIβ paralleled the presence of specific RII‐anchoring proteins, high levels of membrane‐bound PKA holoenzyme, and low levels of cytosolic free catalytic activity (C‐PKA). Conversely, in brain areas showing low RIIβ levels (cerebellum, hypothalamus, and brainstem) we found an absence of RII‐anchoring proteins, low levels of membrane‐bound holoenzyme PKA, and high levels of cytosolic dissociated C‐PKA. Response to cAMP stimuli was specifically evaluated in the neocortex and cerebellum, prototypic areas of the two different patterns of PKA distribution. We found that cerebellar holoenzyme PKA was highly sensitive to cAMP‐induced dissociation, without, however, a consistent translocation of C‐PKA into the nucleus. In contrast, in the neocortex holoenzyme PKA was mainly in the undissociated state and poorly sensitive to cAMP. In nuclei of cortical cells cAMP stimulated the import of C‐PKA and phosphorylation of cAMP‐responsive element binding protein. Taken together, these data suggest that RIIβ (whose distribution is graded throughout the CNS, reaching maximal expression in the neocortex) may represent the molecular cue of the differential nuclear response to cAMP in different brain areas, by controlling cAMP‐induced holoenzyme PKA dissociation and nuclear accumulation of catalytic subunits.

Small Molecule Integrin Antagonists in Cancer Therapy

Integrins are a large family of dimeric receptors composed by alpha and beta subunits that, once bound to extra-cellular matrix (ECM) proteins, regulate a variety of cellular processes such as cell motility, migration, and proliferation. The integrins transduce signals from inside-out and outside-in the cell, thus representing the cellular link to the external environment. For these properties, integrin activation has been involved in pathological processes like tumor growth and metastasis formation. Recent advances in the elucidation of the crystallographic structures of the alphavbeta3 and alphaIIbeta3 integrins are promoting studies focused to the search of small molecule antagonists that can block the integrin binding to ECM and inhibit the biological effects exerted by these receptors. In this review we will focus on small molecule antagonists of alphavbeta3 and alphavbeta5 integrin as tools for cancer therapy while other integrins will only be briefly mentioned. Cilengitide (cyclic peptidic alphavbeta3 and alphavbeta5 antagonist) is currently in clinical trials for anti cancer therapy. Combination of integrin alphavbeta3 antagonists and other traditional therapeutic approaches may represent a future strategy to inhibit tumor growth and metastasis spreading.

The Type and the Localization of cAMP-dependent Protein Kinase Regulate Transmission of cAMP Signals to the Nucleus in Cortical and Cerebellar Granule Cells

cAMP signals are received and transmitted by multiple isoforms of cAMP-dependent protein kinases, typically determined by their specific regulatory subunits. In the brain the major regulatory isoform RIIβ and the RII-anchor protein, AKAP150 (rat) or 75 (bovine), are differentially expressed. Cortical neurons express RIIβ and AKAP75; conversely, granule cerebellar cells express predominantly RIα and RIIα. Cortical neurons accumulate PKA catalytic subunit and phosphorylated cAMP responsive element binding protein very efficiently into nuclei upon cAMP induction, whereas granule cerebellar cells fail to do so. Down-regulation of RIIβ synthesis by antisense oligonucleotides inhibited cAMP-induced nuclear signaling in cortical neurons. Expression in cerebellar granule cells of RIIβ and AKAP75 genes by microinjection of specific expression vectors, markedly stimulated cAMP-induced transcription of the lacZ gene driven by a cAMP-responsive element promoter. These data indicate that the composition of PKA in cortical and granule cells underlies the differential ability of these cells to transmit cAMP signals to the nucleus.

Potentiation of dopamine-induced cAMP formation by group I metabotropic glutamate receptors via protein kinase C in cultured striatal neurons

Metabotropic glutamate receptors have been shown to potentiate the cyclic adenosine monophosphate (cAMP) formation induced by activation of several receptors linked to adenylyl cyclase via GS‐protein. Here we show that, in primary cultures of striatal neurons, group I metabotropic receptors potentiate the cAMP formation induced by activation of D1‐like dopamine receptors. Reverse transcription associated with polymerase chain reaction revealed that mGluR5, mGluR3, mGluR4 and mGluR7 are present in striatal cell cultures. The potentiation of cAMP formation is induced by the selective group I mGluRs agonist (S)‐3,5‐dihydroxyphenylglycine and by other non‐selective mGluRs agonists with a typical group I‐like pharmacology (quisqualate > ibotenate > 1‐aminocyclopentane‐1,3‐dicarboxylic acid). The rank order potency of mGluRs agonists in potentiating cAMP formation correlates with their ability to induce inositol phosphates production; the potentiation of cAMP formation and the inositol phosphates production are blocked by the group I mGluRs antagonists (S)‐4‐carboxyphenylglycine and are not affected by group II antagonist 2S,3S,4S)‐2‐methyl‐2‐(carboxycyclopropyl)‐glycine or group III antagonist (S)‐2‐amino‐2‐methyl‐4‐phosphonobutanoic acid. The potentiating mechanism involves the activation of protein kinase C, being mimicked by phorbol‐12‐myristate‐13‐acetate and blocked by the specific protein kinase C inhibitors bisindolylmaleimide I and chelerythrine or by protein kinase C downregulation. Our results indicate that this interaction could have a functional importance in modulating the cAMP‐dependent transmission in the striatum.

Coexpression of phospholipase A2 isoforms in rat striatal astrocytes

The expression and activity of phospholipase A2 (PLA2) isoforms were investigated in primary cultures of striatal astrocytes. The calcium ionophore A23187 together with the protein kinase C activator phorbol ester was the most potent stimulus in eliciting [3H]arachidonic acid release in the extracellular medium. Reverse transcription coupled to polymerase chain reaction (RT-PCR) showed the presence of the 85 kDa cytosolic PLA2 mRNA and the 14 kDa secretory PLA2 mRNA in untreated astrocytes. Immunoblot experiments with isoform-specific antibodies showed the presence of the cytosolic PLA2 in untreated astrocytes, while the secretory PLA2 was detected only in lipopolysaccharide-treated astrocytes. These data suggest that the two PLA2 isoforms expressed in striatal astrocytes might play different roles in cellular processes mediated by astrocytes.

A small-molecule RGD-integrin antagonist inhibits cell adhesion, cell migration and induces anoikis in glioblastoma cells

In cancer cells integrins modulate important cellular events that regulate the metastasic cascade which involves detachment from the tumor mass, dissemination and attachment to the oncogenic niche. The α5β1, αvβ3 and αvβ5 integrins are widely expressed in different cancer types and recognize the tripeptide Arg-Gly-Asp (RGD) motif present in several extracellular matrix proteins. In human glioblastoma, αvβ3 integrin expression correlates with tumor grade, suggesting that this integrin may play a crucial role in the highly infiltrative behavior of high grade gliomas. However, few selective RGD-like antagonists have been developed and few studies have investigated their effects in in vitro models of human glioblastoma. In this study, we investigated several cellular effects and the underlying molecular mechanisms exerted by a new small-molecule RGD antagonist, 1a-RGD, in the U251 and U373 human glioblastoma cell lines. Treatment with 1a-RGD (20 μM) demonstrated a weak effect on cell viability and cell proliferation but strongly inhibited cell attachment and cell migration together with actin cytoskeleton disassembly. Prolonged 1a-RGD treatment (72 h) induced anoikis, assessed by Annexin staining and nucleosome assay, particularly in the detached cells. When integrin-linked transduction pathways were investigated, 1aRGD was found to exert a marked reduction in focal adhesion kinase (FAK) phosphorylation without affecting the AKT- and ERK-dependent pathways. Our data indicate that 1a-RGD, probably via modulation of the FAK-dependent pathway, inhibits cell migration and attachment and induces anoikis in glioblastoma cells. This novel finding suggests that the development of an RGD-like molecule may represent a promising tool for the pharmacological approach aimed at reducing the malignancy of glioblastoma cells.

Endothelin B receptor antagonists block proliferation and induce apoptosis in glioma cells.

The proliferative and antiapoptotic actions of endothelin (ET)-1 in cancer cells have been documented and ET receptor antagonists have been exploited as potential anticancer drugs. Glioblastoma cell lines express both ETA and ETB receptors and previous works have shown that ETB receptors are involved in the proliferation of different cancer cell types. In this study we have investigated the effects of two structurally unrelated ETB receptor antagonists, BQ788 and A192621, on cell survival, proliferation and apoptosis in 1321-N1, U87 and IPDDCA2 glioma cell lines. BQ788 and A192621 reduced glioma cells viability and proliferation assessed by BrdU incorporation and cell cycle analysis by flow cytometry, while in contrast the ETA receptor antagonist BQ123 had no effect on cell survival. TUNEL assay and immunocytochemical experiments showed that BQ788 and A192621 trigger apoptotic processes mainly via activation of the intrinsic mitochondrial pathway involving caspase-9 activation, AIF release and cytochrome c translocation. Furthermore, treatment with ETB antagonists downregulates ERK- and p38MAPK-dependent pathways but does not affect VEGF mRNA levels. Our findings support the hypothesis that ETB antagonists represent a new promising therapeutic strategy for the treatment of high grade gliomas.