Therapeutic targeting of g-protein coupled receptor-mediated epidermal growth factor receptor transactivation in human glioma brain tumors.

The epidermal growth factor receptor (EGFR) is the main tyrosine kinase receptor dysregulated or overexpressed in brain cancer types and its expression is directly correlated with tumor malignancy and unfavorable prognosis. Recently, the availability of endogenous EGFR ligands has been reported to be also regulated indirectly by the activation of several G-protein-coupled receptors (GPCRs) in many cancer cell types. This EGFR transactivation mechanism requires the initial activation of a GPCR that in turn induces the cleavage of membrane-bound EGFR ligands precursors via the involvement of the family of disintegrin and metalloproteases (ADAMs). The discovery of ADAMs in this transactivation mechanism led to the development of small molecule inhibitors. In this minireview we describe the expression of GPCR, ADAMs and EGFR ligands in human glioma brain tumors and the characteristics of small molecule ADAMs inhibitors. The addition of ADAM inhibitors to our pharmacological arsenal could enhance the outcome of combination therapies when using EGFR inhibitors against human brain tumors.

Stimulation of endothelin B receptors in astrocytes induces cAMP response element-binding protein phosphorylation and c-fos expression via multiple mitogen-activated protein kinase signaling pathways.

The vasoconstrictor peptide endothelin (ET-1) exerts its physiological and pathological effects via activation of ET(A) and ET(B) receptor (ET-R) subtypes. In this study, we demonstrate that both ET-R subtypes are highly expressed in rat astrocytes in vivo, indicating that these cells are potential targets of the biological effects of ET-1 in the brain. In cultured cortical astrocytes, both ET-R subtypes are expressed, and selective stimulation of ET(B)-R with ET-1 induces phosphorylation of cAMP response element-binding protein (CREB). The signal transduction pathway activated by ET-1 includes the Rap1/B-Raf and the Ras/Raf-1 complexes, protein kinase C (PKC) together with extracellular signal-regulated kinases (ERK), and the ribosomal S6 kinase (RSK) isoforms RSK2 and RSK3, two kinases that lie immediately downstream of ERK and are able to phosphorylate CREB. Moreover, ET-1 activates the p38 mitogen-activated protein kinase (MAPK)-dependent, but not the c-jun N-terminal kinase (JNK)-dependent pathway. By using selective protein kinase inhibitors and expression of dominant-negative Rap1 protein, we also found that the Rap1/PKC/ERK-dependent pathway induces the phosphorylation of activating transcription factor-1, CREB, and Elk-1, whereas the p38MAPK-dependent pathway only causes CREB phosphorylation. ET-1-induced transcription of the immediate early gene c-fos requires the concomitant activation of both the PKC/ERK- and p38MAPK-dependent pathways, because inhibitors of either pathway block the ET-1-induced increase of c-fos mRNA. Our findings indicate that changes in the expression of cAMP response element-dependent immediate and delayed response genes could play a pivotal role in the physiological effects elicited by ET-1 in astrocytes.

Modulation of dopamine-induced cAMP production in rat striatal cultures by the calcium ionophore A23187 and by phorbol-12-myristate-13-acetate.

The modulation of cAMP formation by protein kinase C (PKC), activated by phorbol-12-myristate-13-acetate, and by Ca2+ entry, using the ionophore A23187, was investigated in rat striatal neurons grown in primary dissociated cell culture. Phorbol-12-myristate-13-acetate (PMA) potentiated forskolin-induced and dopamine-induced cAMP formation in a concentration-dependent manner. In contrast, the calcium ionophore A23187 inhibited dopamine-induced cAMP formation. When PMA and A23187 were tested simultaneously, the levels of cAMP were not statistically different from those found in the presence of dopamine alone. Furthermore, the decreasing effect of A23187 on cAMP formation was enhanced when PKC was desensitized by pretreating the neurons with 1 microM PMA for 18 h. These data indicate that in striatal neurons Ca2+ entry and PKC activation exert opposing effect on cAMP production.

Aspects of amitriptyline and nortriptyline plasma levels monitoring in depression.

Fifty-nine depressed female inpatients were treated with 100 mg amitriptyline (AMT) IM for 4 weeks. Depression ratings and determinations of the parent drug and nortriptyline (NT) were performed weekly. No direct relationship between plasma AMT + NT concentrations and therapeutic response was apparent, but beneficial therapeutic responses and significantly lower side-effect scores were more frequently noted in subjects with concentrations in the 100-200 ng/ml range. AMT + NT concentrations were significantly correlated with age. No significant difference was found in the number of responders between younger and older subjects with two clinical improvement criteria; however, a significant difference emerged when a third more restrictive clinical outcome criterion was adopted. The implications of the present findings for patient treatment and for the interpretation of previous studies are discussed. The data collected point to a possible usefulness of monitoring AMT and NT plasma levels, even if further investigations are needed.

Differential effects of CGRP on adenylyl cyclase in adult and embryonic rat brain.

The presence of functional receptors for calcitonin gene-related peptide (CGRP) in the brain of adult rats and on nerve cell cultures was investigated. Neuronal and glial cultures were obtained from mesencephalons of embryos at gestational day 16. The response to CGRP was tested by measuring the adenylyl cyclase (AC) activity on isolated membranes. CGRP binding in adult rat brains was ineffective in activating AC, whereas a dose-dependent stimulation of AC activity was induced by the peptide both in neuronal and glial cultures. This effect was more pronounced in the glial cells where high affinity binding sites for CGRP were detected. The presence of functional CGRP receptors in embryonic mesencephalic cells, suggests a role for CGRP in the development of rat mesencephalon.

MPTP treatment combined with ethanol or acetaldehyde selectively destroys dopaminergic neurons in mouse substantia nigra.

We have previously reported that ethanol and acetaldehyde potentiate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in mice, enhancing dopamine (DA) depletion in the striatum. The present study was designed to determine whether such enhancement of neurotoxicity was specific for the nigro-striatal DA pathway. In 5-week-old mice acetaldehyde treatment did not enhance DA depletion seen 7 days after MPTP treatment. In 8-week-old animals, however, acetaldehyde or ethanol given with MPTP decreased striatal DA content to about 10% of controls, whereas the depletion was to 43% of controls when MPTP was given alone. In acetaldehyde or ethanol and MPTP-treated mice, changes in DA levels were observed only in the striatum. DA contents in the hypothalamus, olfactory bulb and frontal cortex were similar to that in controls. Contents of norepinephrine and serotonin in striatum, hypothalamus, olfactory bulb and cerebral cortex were not affected by any of the treatments. Three months after MPTP alone, striatal DA recovered to 74% of controls in 8-week-old mice, whereas no recovery occurred in acetaldehyde and MPTP-treated mice. Moreover, both tyrosine hydroxylase (TH) immunocytochemistry and Cresyl violet staining showed an extensive and selective cell loss in the pars compacta of the substantia nigra (SNc) of the mice treated with acetaldehyde or ethanol and MPTP, whereas MPTP alone caused only a limited cell degeneration.

Acetaldehyde directly enhances MPP+ neurotoxicity and delays its elimination from the striatum.

We have previously shown that ethanol and acetaldehyde (ACE) potentiate MPTP toxicity in mice, selectively enhancing dopamine (DA) depletion in the striatum and markedly increasing loss of DA neurons in the substantia nigra. Several months after these combined treatments there is no evidence of any recovery. In the present study, we measured the accumulation of the MPTP toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) in both striatum and whole brain, after MPTP alone or after combined treatments with ethanol or acetaldehyde, in order to determine whether this enhancement of toxicity is caused by changes in the MPTP metabolism. We also investigated whether acetaldehyde interfered with the conversion of MPTP to MPP+ by glial cells in vitro and studied its effects on the MPP+ uptake and spontaneous release from mesencephalic DA neurons or striatal astrocytes in primary cell cultures from E13 mouse embryos. The results from the in vivo experiments indicated that relatively low doses of ethanol or acetaldehyde potentiate directly MPP+ toxicity, apparently without interfering with its pharmacokinetics. However when higher doses of these drugs were administered, they also decreased MPP+ clearance from the striatum. ACE also increased initial MPTP accumulation in the whole brain but failed to enhance MPP+ levels, thus indicating that ACE effect is not related to MPTP metabolism. In vitro studies confirmed that ACE does not modify MPTP metabolism in striatal or mesencephalic astrocytes in culture. In mesencephalic neuronal cultures ACE does not change the levels of MPP+ uptake (MPP+ is accumulated in putative DA neurons in vitro with a mechanism similar to that of the DA high affinity uptake) nor its spontaneous release. These results indicate that the slower MPP+ clearance from the stratum after ACE is not related to a direct effect of ACE on DA neurons or astrocytes.

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.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in mice is enhanced by ethanol or acetaldehyde.

Persistent neurochemical changes consistent with parkinsonism have been reported in brains of mice treated with repeated high doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We now report that ethanol or acetaldehyde potentiate MPTP-induced damage to mouse striatum. One hour after the combined treatments (ethanol and MPTP or acetaldehyde and MPTP), the animals exhibited a marked and long-lasting catatonic posture and then returned gradually to apparently normal locomotion. Seven days after MPTP administration, depletion of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in mouse striatum were further potentiated in the group of animals treated with ethanol. This effect was more evident when the treatment was repeated twice and was dose-dependent. Acetaldehyde was more potent than ethanol in enhancing MPTP neurotoxicity. A single exposure to acetaldehyde before and during MPTP treatment produced a very consistent fall of DA, DOPAC and HVA but not serotonin (5HT) or 5-hydroxyindoleacetic acid (5HIAA) in the striatum. This suggests that ethanol effects on MPTP neurotoxicity might be related to acetaldehyde formation.

Membrane localization of N-acylphosphatidylethanolamine in central neurons: studies with exogenous phospholipases.

We studied the localization of N-acyl phosphatidylethanolamine (NAPE), a putative cannabinoid precursor, in primary cultures of striatal and cortical neurons from the rat brain. We probed intact neurons with various exogenous phospholipases, including S. chromofuscus phospholipase D (PLD). S. chromofuscus PLD does not penetrate into neurons (as demonstrated by a lack of internalization of 125I-labeled PLD), and does not cause gross damage to the neuronal membrane (as demonstrated by a lack of effect of PLD on [3H]gamma-aminobutyric acid release). When neurons, labeled to isotopic equilibrium with [3H]ethanolamine, were incubated for 10 min with S. chromofuscus PLD, approximately 50% of neuronal NAPE was hydrolysed. This hydrolysis was accompanied by the release of a family of N-acyl ethanolamines (NAE) (assessed by high performance liquid chromatography), which included the cannabinoid receptor agonist, anandamide. Exogenous phospholipase A2 (PLA2) (Apis mellifera) and PLC (B. cereus) mobilized [3H]arachidonate and [3H]diacylglycerol, respectively, but had no effect on NAE formation under these conditions. These experiments indicate that approximately 50% of neuronal NAPE is localized in a compartment that is easily accessible to extracellular PLD, possibly the plasmalemma, where it would also be easily hydrolyzed upon stimulation to produce NAE.

Dopamine synthesis, uptake and metabolism in embryonic rat mesencephalic cultures.

We have developed an HPLC method using electrochemical detection (ED) to study the synthesis, uptake and metabolism of dopamine (DA) in primary cell cultures dissociated from rat embryonic mesencephalon. The method is rapid and simple and is also able to detect, after 7 days in vitro incubation (DIV), intracellular levels of L-3,4-dihydroxyphenylalanine (L-DOPA) and 3,4-dihydroxyphenylacetic acid (DOPAC). The amount of DA synthesized and taken up from the cells is directly proportional to in vitro development time; the contents of endogenous DA is related to the number of mesencephalic neurons originally plated. When the dopa decarboxylase inhibitor alpha-methyldopa is added to the incubation medium, it reduces DA levels and conversely increases the amount of L-DOPA in a dose-dependent manner. In mesencephalic-striatal cocultures a statistically significant increase in the amount of DA is observed. This is not observed when either cerebellar or cortical cells are used in the cocultures which confirms the importance of target striatal cells in the maturation of dopaminergic mesencephalic neurons.

Measurement of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in cultured rat mesencephalic neurons by high-performance liquid chromatography with electrochemical detection.

An HPLC method with electrochemical detection for the simultaneous measurement of serotonin (5-hydroxytryptamine) and 5-hydroxyindoleacetic acid in primary mesencephalic cell culture is described. The serotonin and 5-hydroxyindoleacetic acid cell content was measured on different days of growth in vitro; after twelve days in culture the amounts of serotonin and 5-hydroxyindoleacetic acid detected were 916.0 +/- 70.2 and 215.8 +/- 15.5 pg per well, respectively. The heterogeneity of neurons in our cultures and their capacity to take up serotonin were assessed by measuring the amounts of exogenous serotonin taken up in the presence of different monoamine uptake inhibitors. This method, sensitive and reliable, can represent a valid alternative to the use of labelled compounds.

Opposing actions of D1- and D2-dopamine receptors on arachidonic acid release and cyclic AMP production in striatal neurons.

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 Ca(2+)-evoked AA release.

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-13acetate 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.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine metabolism and 1-methyl-4-phenylpyridinium uptake in dissociated cell cultures from the embryonic mesencephalon.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a contaminant found in a synthetic illicit drug, can elicit in humans and monkeys a severe extrapyramidal syndrome similar to Parkinson's disease. It also induces alterations of the dopamine (DA) pathways in rodents. MPTP neurotoxicity requires its enzymatic transformation into 1-methyl-4-phenylpyridinium (MPP+) by monoamine oxidase followed by its concentration into target cells, the DA neurons. Here, we show that mesencephalic glial cells from the mouse embryo can take up MPTP in vitro, transform it into MPP+, and release it into the culture medium. MPTP is not taken up by neurons from either the mesencephalon or the striatum in vitro (8 days in serum-free conditions). However, mesencephalic neurons in culture revealed a high-affinity uptake mechanism for the metabolite MPP+, similar to that for DA. The affinity (Km) for DA uptake is fivefold higher than that for MPP+ (0.2 and 1.1 microM, respectively), whereas the number of uptake sites for MPP+ is double (Vmax = 25 and 55 pmol/mg of protein/min for DA and MPP+, respectively). Mazindol, a DA uptake inhibitor, blocks the uptake of DA and MPP+ equally well under these conditions. Moreover, by competition experiments, the two molecules appear to use the same carrier(s) to enter DA neurons. Small concentrations of MPP+ are also taken up by striatal neurons in vitro. The amount taken up represented less than 10% of the MPP+ uptake in mesencephalic neurons. Depolarization induced by veratridine released comparable proportions of labeled DA and MPP+ from mesencephalic cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

cAMP-dependent protein kinase induces cAMP-response element-binding protein phosphorylation via an intracellular calcium release/ERK-dependent pathway in striatal neurons.

Activation of the cAMP-dependent protein kinase A (PKA) pathway may induce cAMP-response element-binding protein (CREB) phosphorylation either directly or via cross-talk mechanisms with other signal transduction pathways. In this study, we have investigated in striatal primary cultures the mechanism by which activation of the cAMP/PKA-dependent pathway leads to CREB phosphorylation via the extracellular signal-regulated kinase (ERK)-dependent pathway. We have found that PKA-induced CREB phosphorylation and CREB-dependent transcription are mediated by calcium (Ca(2+)) release from intracellular stores and are blocked by inhibitors of the protein kinase C and ERK pathways. This mechanism appears to be mediated by the small G-protein Rap1, whose activation appears to be primed by PKA-induced Ca(2+) release but not further induced by direct or indirect PKA- or protein kinase C-dependent phosphorylation. These results suggest that, in striatal neurons, intracellular Ca(2+) release, Rap1, and ERK pathway play a crucial role in the PKA-induced CREB phosphorylation and CREB-dependent transcription.

Pharmacological and molecular evidence for dopamine D(1) receptor expression by striatal astrocytes in culture.

The neurotransmitter dopamine (DA) at a 10 microM 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 beta-adrenergic receptors antagonist propranolol, mimicked by the D(1) agonist SKF 38393 and by the mixed D(1)/D(2) 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 D(1) receptor mRNA and Western blot analysis confirmed the expression of the D(1) receptor protein in striatal astrocytes. In contrast to what found in neurons, the D(1)-dependent cAMP formation in striatal astrocytes is partially reduced by pertussis toxin (PTX) treatment. The stimulation of D(1) 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 D(1) receptors in cultured striatal astrocytes suggests a role of dopamine in the regulation of cellular processes in striatal astrocytes.

Assay of urinary vanilmandelic, homovanillic, and 5-hydroxyindole acetic acids by liquid chromatography with electrochemical detection.

A reversed-phase liquid chromatographic method with amperometric detection has been developed for the simultaneous determination of the noradrenaline metabolite, vanilmandelic acid, the dopamine metabolite, homovanillic acid, and the serotonin metabolite, 5-hydroxyindole acetic acid, in human urine. After purification by an extraction procedure, the metabolites were rapidly separated under isocratic conditions. Detection and quantification were performed with an electrochemical detector using a carbon paste electrode. The present method is sensitive, selective and, achieves a high degree of precision by the use of isovanilmandelic acid as an internal standard. This provides a suitable tool for clinical and research applications.

Urinary 3-methoxy-4-hydroxyphenylglycol determination using reversed-phase chromatography with amperometric detection.

A reversed-phase liquid chromatographic method with amperometric detection has been developed for the determination of urinary 3-methoxy-4-hydroxyphenylglycol. Before and after enzymatic deconjugation, it was purified by an extraction procedure and rapidly quantified under isocratic conditions. The 24-h excretion profile in normal human subjects (eight males and seven females) was determined; our results are consistent with those arrived at in a number of other studies. The present method is highly sensitive and selective; in addition, a good degree of precision is assured by use of 4-methoxy-3-hydroxyphenylglycol as internal standard.