RESUMEN
RATIONALE: Recent data suggest that passive smoking has a risk comparable to active smoking. Passive smoking is considered dangerous in children and is suspected as a cause of asthma. However, some reports are opposing such claims, indicating the need for solid results and large-scale studies. This scientific work aims to develop a method for the determination of nicotine (NCOT) and major nicotine's metabolite cotinine (COT) in urine samples, using gas chromatography-mass spectrometry (GC-MS). METHODS: Analysis was performed using a gas chromatograph Agilent Technologies 7890A with an MS 5975C inert XL, EI/CI MSD with Triple-Axis detector. For sample preparation, liquid-liquid extraction was applied after an optimization study with different extraction media. Eventually, 1 mL of dichloromethane was selected for the extraction of 0.5 mL of urine. Suitable chromatographic conditions were found for the rapid and accurate determination of NCOT and COT. Injection of 2 µL was performed using GC-MS, and selected ion monitoring (SIM) analysis was performed with the following ions (m/z): 162 (quantifier ion) and 84, 133, 161 qualifier ions for NCOT, and 176 (quantifier ion) and 98, 118, 119, 147 qualifier ions for COT. Nicotine-D4 (NCOT-D4) and cotinine-D3 (COT-D3) were used as internal standards with quantifier ions 101 and 166, respectively. The retention time (Rt) for NCOT was 7.557 min and 9.743 min for COT. RESULTS: The method was validated following international principles, assessing characteristics such as absolute recovery, carryover, linearity, specificity, selectivity, accuracy, precision, and stability. The method showed a linear dynamic range from 0.5 to 50 ng/mL, and the limits of detection and quantification were for both NCOT and COT 0.2 and 0.5 ng/mL, respectively. Validation results were found satisfactory. Finally, the method was applied to the analysis of 60 clinical pediatric samples obtained from Aristotle University's pediatric clinic to check for possible exposure to smoke. Concentration levels ranged between 0.5 and 16.2 ng/mL for NCOT and between 1.0 and 25.1 ng/mL for COT. CONCLUSIONS: A rapid, sensitive, accurate, and simple method was developed and used as a tool for the confirmation of passive smoking in children. It is the first method applied to the analysis of such samples belonging to nonsmokers of young age. The total runtime of the GC-MS analysis was short (20 min), and the pretreatment protocol was simple, giving the ability for analysis of a large number of samples on a daily routine basis.
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Cotinina , Cromatografía de Gases y Espectrometría de Masas , Nicotina , Contaminación por Humo de Tabaco , Cotinina/orina , Cromatografía de Gases y Espectrometría de Masas/métodos , Humanos , Contaminación por Humo de Tabaco/análisis , Nicotina/orina , Nicotina/análisis , Reproducibilidad de los Resultados , Límite de Detección , NiñoRESUMEN
PURPOSE: In the present study, a method for the detection of 25 psychoactive substances in cerumen was developed and validated. This method targets opiates, cocaine, antidepressants, benzodiazepines, antipsychotics and antiparkinsons. METHODS: Analysis was performed on a SCIEX Triple Quad 6500+ system after liquid-liquid extraction. Methanol with 1% acetic acid was chosen as the extraction solvent. After the addition of the solvent, samples were vortexed, sonicated, centrifuged and directly injected into the liquid chromatography-tandem mass spectrometry system. RESULTS: The method was found to be selective and sensitive (limit of detection: 0.017 ng-0.33 ng/mg), the assay was linear for all analytes with linear regression coefficient ranging 0.9911-1.00. The values for intra-assay precision was between 4.34 and 14.6% and inter-assay precision between 5.81 and 17.7%, with accuracy within the acceptable criteria for all analytes. All analytes in cerumen specimens were stable for 48 h at 4 °C and 72 h at - 20 °C, whilst no significant matrix effect or carryover was observed. Applicability was proven by analyzing cerumen samples from 25 deceased with a history of drug abuse. All analytes were detected in real samples, thus confirming the sensitivity of the developed method. CONCLUSIONS: According to our knowledge, it is the first time that a method for the simultaneous detection of 25 psychoactive drugs in cerumen was developed, fully validated and finally applied to 25 postmortem samples.
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Cerumen , Espectrometría de Masas en Tándem , Cerumen/química , Espectrometría de Masas en Tándem/métodos , Reproducibilidad de los Resultados , Cromatografía Liquida/métodos , Psicotrópicos/análisis , SolventesRESUMEN
Cerumen is an emerging alternative biological matrix in the field of forensic toxicology. An ultra-high-pressure liquid chromatography-mass spectrometry/mass spectrometry [UHPLC-MS/MS] method for the determination of fentanyl and norfentanyl in cerumen was developed and applied in a mixed drug toxicity fatal case. The method was found to be selective and sensitive (LOQ: 0.05 ng/mg for fentanyl and 0.02 ng/mg for norfentanyl), while validation included recovery, carryover, short-term stability, matrix effect, accuracy, and precision (RSD%). Accuracy ranged from 83.1% to 103.5%, while intra- and inter-day precision ranged from 8.6% to 13.1% and from 8.3% to 15.8%, respectively. Matrix effect experiments showed that matrix did not significantly affect signal intensity (82.3%-96.8%). Short-term stability concerning sample extracts was found satisfactory. Fentanyl and norfentanyl were detected in cerumen at a concentration of 1.17 and 0.36 ng/mg respectively. The findings in cerumen corroborate the cause of death and suggest that cerumen is a potential specimen for detecting drugs of abuse in forensic cases.
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Cerumen , Espectrometría de Masas en Tándem , Analgésicos Opioides/análisis , Cerumen/química , Fentanilo/análogos & derivados , Fentanilo/análisis , Toxicología Forense/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
RATIONALE: N,N-Dimethyldodecylamine is produced from lauryl alcohol and dimethylamine. C12-C16 alkyldimethylamines are used as intermediates for the manufacture of amineoxides and quaternary amino compounds. In the present study a gas chromatography-mass spectrometry (GC/MS) method for the determination of C12-C16 alkyldimethylamines in blood was developed and validated. The reason for this study was the detection of the above compounds in the postmortem blood sample of a fatal suicide case. METHODS: Analysis of amines was performed using a gas chromatograph (Agilent Technologies 7890A) with an MS 5975C inrXL, EI/CI MSD with triple-axis detector in selected ion monitoring mode, after liquid-liquid extraction. Four different organic solvents (butyl acetate, ethyl acetate, n-hexane and n-heptane) were used for the optimization of the extraction procedure, resulting in ethyl acetate being the solvent of choice for the extraction procedure. A QuEChERS step was applied (20 mg of MgSO4 , 5 mg of NaCl) to 1 mL of blood and pH was adjusted at 12 (K2 CO3 buffer solution). After the addition of the extraction solvent, samples were vortexed, centrifuged and directly injected into the GC/MS system. RESULTS: In validation, the method was found to be selective and sensitive (limit of detection from 0.3 to 0.5 ng/mL, limit of quantitation from 10.0 to 20.0 ng/mL), whilst validation included recovery, stability, accuracy and precision (relative standard deviation). Validation results were found satisfactory: intra- and interday precision ranged from 0.4% to 2% and from 0.6% to 1.9% respectively, while intra- and interday accuracy ranged from 87% to 109% and from 86% to112.8%. C12-C16 alkyldimethylamines were detected in blood samples at a concentration of 8.39 µg/mL (C12), 3.01 µg/mL (C14) and 0.42 µg/mL (C16). CONCLUSIONS: A rapid, sensitive and reliable method was developed for the determination of C12-C16 alkyldimethylamines in postmortem blood, after optimization of the sample preparation procedure, and finally successfully applied to a real postmortem blood sample from a fatal case involving these compounds.
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Carbono/química , Extracción Líquido-Líquido , Metilaminas/química , Cromatografía de Gases y Espectrometría de Masas/métodos , Humanos , Espectrometría de Masas , SolventesRESUMEN
Food contact materials (FCM) polyethylene terephthalate (PET) and polybutylene terephthalate (PBT) used extensively in food packaging may contain cyclic oligomers which may migrate into food and thus cause toxic effects on human health. A simple, fast, and sensitive ultra-high-performance liquid chromatography method quadrupole time-of-flight mass spectrometer was developed for the analysis of 7 cyclic oligomers in post-mortem blood samples. The targeted analytes were separated on a Waters BEH C18 (150 × 2.1 mm, 1.7 µm) analytical column by gradient elution. Calibration curves were constructed based on standard solutions and blood samples and Student's t-test was applied to evaluate the matrix effect. The LODs ranged from 1.7 to 16.7 µg mL-1, while the method accuracy was assessed by recovery experiments and resulting within the range 84.2-114.6%. Such an analytical method for the determination of PET and PBT cyclic oligomers in biological samples is reported for the first time. The developed methodology allows the determination of these oligomers in blood providing a useful analytical tool to assess the exposure and thus the potential hazard and health risks associated with these non-intentionally added substances (NIAS) from PET and PBT FCM through food consumption. The method was validated and successfully applied to the analysis of 34 post-mortem whole blood samples. Polyethylene terephthalate trimer was detected in four of them, for the first time in literature.
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Análisis Químico de la Sangre/métodos , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , Poliésteres/análisis , Tereftalatos Polietilenos/análisis , Anciano , Embalaje de Alimentos , Humanos , Límite de Detección , Extracción Líquido-Líquido , Poliésteres/química , Tereftalatos Polietilenos/química , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The analysis of blood samples for forensic or clinical intoxication cases is a daily routine in an analytical laboratory. The list of 'suspect' drugs of abuse and pharmaceuticals that should be ideally screened is large, so multi-targeted methods for comprehensive detection and quantification are a useful tool in the hands of a toxicologist. In this study, the development of an ultra-high performance liquid chromatography (LC)-tandem mass spectrometry (MS-MS) method is described for the detection and quantification of 84 drugs and pharmaceuticals in postmortem blood. The target compounds comprise pharmaceutical drugs (antipsychotics, antidepressants, etc.), some of the most important groups of drugs of abuse: opiates, cocaine, cannabinoids, amphetamines, benzodiazepines and new psychoactive substances. Sample pretreatment was studied applying a modified Mini-QuEChERS single step, and the best results were obtained after adding a mixture of 20 mg MgSO4, 5 mg K2CO3 and 5 mg NaCl together with 600 µL of cold acetonitrile in 200 µL of sample. After centrifugation, the supernatant was collected for direct injection. LC-MS analysis took place on a C18 column with a gradient elution over 17 min. The method was found to be selective and sensitive, offering limits of detection ranging from 0.01 to 9.07 ng/mL. Validation included evaluation of limit of quantification, recovery, carryover, matrix effect, accuracy and precision of the method. The method performed satisfactorily in relation to established bioanalytical criteria and was therefore applied to the analysis of blood obtained postmortem from chronic drug abusers, offering unambiguous identification and quantitative determination of drugs in postmortem blood.
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Toxicología Forense , Preparaciones Farmacéuticas/análisis , Detección de Abuso de Sustancias/métodos , Acetonitrilos , Anfetaminas , Analgésicos Opioides , Benzodiazepinas , Cromatografía Líquida de Alta Presión , Cocaína , Medicina Legal , Humanos , Límite de Detección , Alcaloides Opiáceos , Reproducibilidad de los Resultados , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Fatal drowning is one of the leading causes of unintentional injury mortality worldwide and a persistent public health concern in Greece. While several pathologic and sociodemographic contributing factors have been previously identified, these have not been extensively investigated in conjunction with the effects of psychoactive substances. METHODS: A retrospective case-control study of drowning deaths was conducted in the Greek regions of Northern Greece and Thessaly during a 10-year period. A regression model was constructed examining differences in detected substances, autopsy findings and sociodemographic characteristics between 240 victims of unintentional fatal submersion and 480 victims of other causes of sudden or violent death. RESULTS: The majority of victims were males (69.4%) and foreign nationality was associated with increased odds of drowning. Cardiomegaly and coronary bypass grafts were significantly more likely to have been recorded among drowning victims, while the frequency of other circulatory system disorders was also elevated. Several of these findings were potential arrhythmogenic substrates which could adversely interact with the diving reflex. Selective serotonin reuptake inhibitors (SSRIs) were the most commonly detected pharmacological group (9.0%), and along with tramadol, there was an increased likelihood of exposure to them. These drugs have been previously associated with QT prolongation and other adverse effects which may contribute to fatal outcomes in a seawater environment. In contrast, there was a decreased risk of exposure to dependence-inducing drugs and paracetamol. CONCLUSIONS: Male sex, older age, foreign nationality and cardiovascular disease predisposed individuals to an elevated risk of fatal submersion. SSRI antidepressants and tramadol may contribute to this outcome.
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Ahogamiento , Anciano , Estudios de Casos y Controles , Grecia/epidemiología , Humanos , Masculino , Estudios Retrospectivos , Factores de RiesgoRESUMEN
Analysis of post-mortem liver for toxicological reasons is a considerable option when blood is unavailable. The development of analytical methods for tissue specimens is not as straightforward as for biological fluids as tissue presents challenges to the analytical chemist. The present study reports the development of a UHPLC-MS/MS method for the detection and quantification of 84 drugs and pharmaceuticals in human liver. The selected target drugs include pharmaceutical drugs and drugs of abuse. Sample preparation was studied using QuEChERS and different ratios of solvent volume and sample mass. Best results were attained by homogenizing 1 g of liver with acetonitrile K2CO3 buffer (pH = 10), QuEChERS salts MgSO4/ NaCl (1st purification step) and PSA/ 150 mg MgSO4 (2nd purification step). The extracted sample was analysed on UHPLC-MS/MS in multiple reaction monitoring mode (MRM) on a reversed-phase (Acquity BEH C18) column. Elution was accomplished by gradient program of mobile phase A: water, 0.1% formic acid and B: methanol, 0.1% formic acid that lasted 17 min. The method was specific, without interferences from the complex matrix. Sensitivity was satisfactory with limit of detection (LOD) ranging from 0.01 ng/g to 4.94 ng/g. Validation study was based on the guidelines of international bodies and included evaluation of recovery, carry-over, matrix effect, accuracy, stability, and precision of the method. The method performed satisfactory in relation to established bioanalytical criteria and was therefore applied to the analysis of liver tissue obtained post-mortem from chronic drug abusers, offering unambiguous identification and quantitative determination of drugs in postmortem blood.
RESUMEN
Lacosamide is a functionalized amino acid with antiepileptic function. Therapeutic drug monitoring (TDM) in patients for lacosamide is critical as it allows clinicians to control epileptic seizures. A single liquid-liquid extraction step was applied for the extraction of lacosamide from whole blood samples which were thereafter analyzed by GC-MS. Optimum extraction conditions were selected on the basis of experiments with various solvents at different pHs, indicating ethyl acetate at pH 12 as the most efficient parameters for the extraction of lacosamide. Method exhibited linearity from 2 to 100 µg/mL with R2 = 0.998. Accuracy and precision were evaluated at three concentrations and found to be within acceptable limits. LOD and LOQ were determined at 0.1 and 0.5 µg/mL, respectively. Lacosamide was found to be stable at storage conditions. The developed method was applied successfully in clinical samples and postmortem blood sample from an overdose case.
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Anticonvulsivantes/sangre , Cromatografía de Gases y Espectrometría de Masas , Lacosamida/sangre , Anticonvulsivantes/envenenamiento , Monitoreo de Drogas , Toxicología Forense , Humanos , Lacosamida/envenenamiento , Límite de Detección , Modelos Lineales , Extracción Líquido-Líquido , Intoxicación/diagnósticoRESUMEN
In several medico-legal cases, bone samples analysis may provide the only source of toxicological information. This case study reports the analysis of a human bone specimen, belonging to a 46-year-old man, found 3 months after his death due to cervical-thoracic injuries in a motorcycle accident. Bone specimen was the only available material for toxicological analysis, among few skull hair and rotten skin. Analysis was performed by a newly developed and validated ultra-high-pressure liquid chromatography-mass spectrometry/mass spectrometry (UHPLC-MS/MS) method, following simple and efficient sample pretreatment. The results were in accordance with the man's medical record: Alprazolam and zolpidem were found at 2.2 and 5.4 ng/g of bone, respectively. Both these drugs were prescribed to the deceased.
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Alprazolam/análisis , Restos Mortales , Cambios Post Mortem , Detección de Abuso de Sustancias , Zolpidem/análisis , Accidentes de Tránsito , Cromatografía Líquida de Alta Presión , Fémur/química , Cabello/química , Humanos , Hipnóticos y Sedantes/análisis , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Motocicletas , Piel/química , Trastornos Relacionados con Sustancias/diagnósticoRESUMEN
In several medico legal cases bone analysis may provide the only source of toxicological information. The present study reports the development of an UHPLC-MS/MS method for the detection and quantification of 27 drugs and pharmaceuticals in human bones. The target compounds comprise pharmaceuticals (antipsychotics and antidepressants) and some of the most important groups of drugs of abuse: opiates, cocaine, cannabinoids, amphetamines and benzodiazepines. Sample pretreatment was studied and the best results were obtained after extraction with methanol, stirring and ultra-sonication. The extract, after filtration, evaporation and reconstitution was analysed on a reversed-phase column (C18) in gradient elution over 17min. The method was found to be selective, and sensitive offering limits of detection (LOD) from 0.03 to 1.35ng/g of bone. Validation included evaluation of limit of quantification (LOQ), recovery, carry-over, matrix effect, accuracy and precision (RSD%) of the method. The method performed satisfactory in relation to established bioanalytical criteria and was therefore applied to the analysis of bone and bone marrow obtained post-mortem from chronic drug abusers, offering unambiguous identification and quantitative determination of drugs in bones from legal cases where the analysis of blood was not feasible.
Asunto(s)
Cromatografía Líquida de Alta Presión , Fémur/química , Drogas Ilícitas/análisis , Preparaciones Farmacéuticas/análisis , Espectrometría de Masas en Tándem , Toxicología Forense/métodos , Humanos , Límite de Detección , MasculinoRESUMEN
Serotonin-specific reuptake inhibitors (SSRIs) are generally considered safe drugs but fatal adverse effects do sometimes occur, often as a consequence of interactions with other serotonin active drugs. Polypharmacy is usually a problem that the elderly encounter, but it can also have dire consequences for young people, especially when an underlying heart condition is present. Thus, failure to diagnose heart disease and the use of contraindicated medications can be a lethal combination, irrespective of age. Here we present a case of a young adult suffering from bipolar disorder who used a combination of two SSRIs (citalopram and fluoxetine) and a monoamine oxidase inhibitor (MAO; moclobemide) with tragic consequences. The deceased also suffered from undiagnosed hypertrophic cardiomyopathy and was carrier of a genotype that may have predisposed him to increased sensitivity to SSRIs. The apparent difficulty in establishing the manner of death in this case is also discussed.
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Cardiomiopatía Hipertrófica/diagnóstico , Citalopram/envenenamiento , Fluoxetina/envenenamiento , Variantes Farmacogenómicas , Inhibidores Selectivos de la Recaptación de Serotonina/envenenamiento , Adulto , Trastorno Bipolar/tratamiento farmacológico , Citalopram/análisis , Fluoxetina/análisis , Genotipo , Heterocigoto , Humanos , Masculino , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Inhibidores Selectivos de la Recaptación de Serotonina/análisisRESUMEN
Cytochrome P450 2B6 (CYP2B6) is responsible for the initial biotransformation of profol, an extensively metabolized intravenous anesthetic. In this study we examined the effect of the apparently functional CYP2B6 c.516G>T polymorphism on the distribution of propofol concentrations, quantified by GC/MS analysis following a single bolus dose, in the blood of 44 Greek women undergoing oocyte retrieval. Univariate analysis using age, height, weight and smoking status as covariates, as well as the Mann-Whitney non-parametric test, revealed a strong trend of association of the T allele with high propofol concentrations determined in whole blood, shortly after a single bolus dose. Propofol concentrations which were higher than one standard deviation of the mean were almost invariably associated with carriage of the T allele.
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Anestésicos Intravenosos/sangre , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2B6/metabolismo , Polimorfismo de Nucleótido Simple , Propofol/sangre , Adulto , Anestésicos Intravenosos/administración & dosificación , Anestésicos Intravenosos/farmacocinética , Biotransformación , Femenino , Cromatografía de Gases y Espectrometría de Masas , Genotipo , Grecia , Humanos , Inyecciones Intravenosas , Recuperación del Oocito , Fenotipo , Propofol/administración & dosificación , Propofol/farmacocinética , Factores SexualesAsunto(s)
Antipsicóticos/envenenamiento , Muerte Súbita Cardíaca/etiología , Dibenzotiazepinas/envenenamiento , Sobredosis de Droga/fisiopatología , Antipsicóticos/sangre , Antipsicóticos/uso terapéutico , Dibenzotiazepinas/sangre , Dibenzotiazepinas/uso terapéutico , Resultado Fatal , Cromatografía de Gases y Espectrometría de Masas , Humanos , Masculino , Persona de Mediana Edad , Fumarato de Quetiapina , Esquizofrenia/complicaciones , Esquizofrenia/tratamiento farmacológicoRESUMEN
On incubation with epinephrine, PC12 cells stably expressing alpha2-adrenergic receptor (alpha2-AR) undergo morphological and biochemical changes characteristic of neuron-like differentiation. The present study shows that alpha2-AR stimulation increases the phosphorylation of the transcription factor cAMP-response element-binding protein (CREB), the activity of a CRE-reporter plasmid and the expression of cyclin D1 with subtype-dependent efficiency (alpha2A approximately alpha2C >> alpha2B). The effects of epinephrine were mimicked by cell exposure to forskolin or to exogenous arachidonic acid (AA) and they were abrogated by prior treatment with the inhibitor of phospholipase C (PLC) (U73122) or the inhibitor of cytochrome P450-dependent epoxygenase, ketoconazole. On the other hand, treatment of the cells with epinephrine caused activation of protein kinase A (PKA), which was fully abolished by ketoconazole. Inhibition of PKA activity with H89 or ketoconazole abolished the effects of epinephrine on CREB, suggesting that activation of the cAMP/PKA pathway by AA epoxy-derivatives is responsible for CREB activation by alpha2-ARs. The effects of epinephrine were unaffected by LY294002. Furthermore, treatment with staurosporine, tyrphostin AG1478, PP1 or PD98059 did not change the extent of CREB phosphorylation but enhanced its transcriptional activity. Altogether, our results demonstrate that, in PC12 cells, the alpha2-AR subtypes cause phosphorylation and activation of CREB through a pathway involving stimulation of PLC, AA release, generation of epoxygenase derivative and increase of PKA activity. They also suggest attenuation of CREB transcriptional activity by mitogen-activated protein kinase, protein kinase C and Src kinases.
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Ácido Araquidónico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Neuronas/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2 , Agonistas alfa-Adrenérgicos/farmacología , Animales , Ácido Araquidónico/farmacología , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Ciclina D1/genética , Ciclina D1/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Epinefrina/metabolismo , Epinefrina/farmacología , Genes Reporteros/efectos de los fármacos , Genes Reporteros/genética , Neuronas/efectos de los fármacos , Células PC12 , Fosforilación/efectos de los fármacos , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Previous study carried out on PC12 cells expressing each alpha(2)-adrenergic receptor subtype individually (PC12/alpha(2A), /alpha(2B) or /alpha(2C)) have shown that epinephrine causes activation of PI3K and phosphorylation of Erk 1/2. The signal transduction mechanisms whereby each alpha(2)-AR subtype triggers these actions were investigated in the present study. In all three clones, epinephrine-induced phosphorylation of MAPK or Akt was abolished by prior treatment with ketoconazole, but not with indomethacin or nordihydroguaiaretic acid. On the other hand, treatment of the clones with epinephrine caused a rapid increase of AA release, which was fully abolished by the PLC inhibitor U73122, but was unaffected by the PLA(2) inhibitor quinacrine. The effects of epinephrine on MAPK and Akt were mimicked by cell exposure to exogenous AA. Furthermore, whereas U73122 abolished the effects of epinephrine, quinacrine only prevented the effects of epinephrine, suggesting that AA release through PLC and its metabolites are responsible for MAPK and Akt activation by alpha(2)-ARs. Treatment with 1,10-phenanthroline, CRM197, or tyrphostin AG1478 suppressed MAPK and Akt phosphorylation by epinephrine or AA, in a subtype-specific manner. Furthermore, conditioned culture medium from epinephrine-treated PC12/alpha(2) induced MAPK and Akt phosphorylation in wild-type PC12. Inhibition of NGFR tyrosine phosphorylation had no effect but the src inhibitor PP1 abolished MAPK and Akt phosphorylation in all three clones. Our results provide evidence for a putative pathway by which alpha(2)-ARs activate MAPK and Akt in PC12 cells, involving stimulation of PLC, AA release, AA metabolism by cytochrome P450-dependent epoxygenase, stimulation of matrix metalloproteinases and subtype-specific transactivation of EGFR through src activation and heparin-binding EGF-like growth factor release.