RESUMO
OBJECTIVE: The phytohormone abscisic acid (ABA) as a signaling molecule exists in various types of organisms from early multicellular to animal cells and tissues. It has been demonstrated that ABA has an antinociceptive effect in rodents. The present study was designed to assess the possible role of PKA and phosphorylated ERK (p-ERK) on the antinociceptive effects of intrathecal (i.t.) ABA in male Wistar rats. METHODS: The animals were cannulated intrathecally and divided into different experimental groups (n=6â7): Control (no surgery), vehicle (received ABA vehicle), ABA-treated groups (received ABA in doses of 10 or 20 µg/rat), ABA plus H.89 (PKA inhibitor)-treated group which received the inhibitor 15 min prior to the ABA injection. Tail-flick and hot-plate tests were used as acute nociceptive stimulators to assess ABA analgesic effects. p-ERK was evaluated in the dorsal portion of the spinal cord using immunoblotting. RESULTS: Data showed that a microinjection of ABA (10 and 20 µg/rat, i.t.) significantly increased the nociceptive threshold in tail flick and hot plate tests. The application of PKA inhibitor (H.89, 100 nM/rat) significantly inhibited ABA-induced analgesic effects. Expression of p-ERK was significantly decreased in ABA-injected animals, which were not observed in the ABA+H.89-treated group. CONCLUSIONS: Overall, i.t. administration of ABA (10 µg/rat) induced analgesia and p-ERK down-expression likely by involving the PKA-dependent mechanism.
Assuntos
Ácido Abscísico/farmacologia , Analgésicos/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , Reguladores de Crescimento de Plantas/farmacologia , Medula Espinal/metabolismo , Animais , Western Blotting , Proteínas Quinases Dependentes de AMP Cíclico/análise , MAP Quinases Reguladas por Sinal Extracelular/análise , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Masculino , Ratos Wistar , Valores de Referência , Reprodutibilidade dos Testes , Medula Espinal/efeitos dos fármacos , Fatores de TempoRESUMO
Abstract Objective: The phytohormone abscisic acid (ABA) as a signaling molecule exists in various types of organisms from early multicellular to animal cells and tissues. It has been demonstrated that ABA has an antinociceptive effect in rodents. The present study was designed to assess the possible role of PKA and phosphorylated ERK (p-ERK) on the antinociceptive effects of intrathecal (i.t.) ABA in male Wistar rats. Methods: The animals were cannulated intrathecally and divided into different experimental groups (n=6‒7): Control (no surgery), vehicle (received ABA vehicle), ABA-treated groups (received ABA in doses of 10 or 20 µg/rat), ABA plus H.89 (PKA inhibitor)-treated group which received the inhibitor 15 min prior to the ABA injection. Tail-flick and hot-plate tests were used as acute nociceptive stimulators to assess ABA analgesic effects. p-ERK was evaluated in the dorsal portion of the spinal cord using immunoblotting. Results: Data showed that a microinjection of ABA (10 and 20 µg/rat, i.t.) significantly increased the nociceptive threshold in tail flick and hot plate tests. The application of PKA inhibitor (H.89, 100 nM/rat) significantly inhibited ABA-induced analgesic effects. Expression of p-ERK was significantly decreased in ABA-injected animals, which were not observed in the ABA+H.89-treated group. Conclusions: Overall, i.t. administration of ABA (10 µg/rat) induced analgesia and p-ERK down-expression likely by involving the PKA-dependent mechanism.
Resumo Objetivo: O ácido fito-hormônio abscísico (ABA) existe como molécula sinalizadora em vários tipos de organismos, de multicelulares a células e tecidos animais. Foi demonstrado que o ABA tem efeito antinociceptivo em roedores. O presente estudo foi desenhado para avaliar o possível papel da PKA e da ERK fosforilada (p-ERK) nos efeitos antinociceptivos do ABA intratecal (i.t.) em ratos Wistar machos. Métodos: Os animais foram canulados por via i.t. e divididos em diferentes grupos experimentais (n=6‒7): controle (sem cirurgia), veículo (veículo ABA recebido), grupos tratados com ABA (recebeu ABA em doses de 10 ou 20 µg/rato), grupo tratado com ABA mais H.89 (inibidor de PKA) que recebeu o inibidor 15 minutos antes da injeção de ABA. Os testes de movimento da cauda e placa quente foram utilizados como estimuladores nociceptivos agudos para avaliar os efeitos analgésicos da ABA. A p-ERK foi avaliada na porção dorsal da medula espinhal por imunotransferência. Resultados: A microinjeção de ABA (10 e 20 µg/rato, i.t.) aumentou significativamente o limiar nociceptivo nos testes de movimento da cauda e placa quente. A aplicação de inibidor de PKA (H.89, 100 nM/rato) inibiu significativamente os efeitos analgésicos induzidos por ABA. A expressão de p-ERK diminuiu significativamente em animais injetados com ABA que não foram observados no grupo tratado com ABA+H.89. Conclusões: No geral, a administração i.t. de ABA (10 µg/rato) induziu a analgesia e expressão negativa de p-ERK provavelmente envolvendo mecanismo dependente de PKA.
Assuntos
Animais , Masculino , Reguladores de Crescimento de Plantas/farmacologia , Medula Espinal/metabolismo , Ácido Abscísico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , Analgésicos/farmacologia , Valores de Referência , Medula Espinal/efeitos dos fármacos , Fatores de Tempo , Western Blotting , Reprodutibilidade dos Testes , Ratos Wistar , Proteínas Quinases Dependentes de AMP Cíclico/análise , MAP Quinases Reguladas por Sinal Extracelular/análise , Peptídeos e Proteínas de Sinalização Intracelular/farmacologiaRESUMO
We previously reported that elevated extracellular calcium (Ca2+) levels increase bone morphogenetic protein 2 expression in human dental pulp (hDP) cells. However, it is unknown whether extracellular Ca2+ affects the expression of other growth factors such as fibroblast growth factor 2 (FGF2). The present study aimed to examine the effect of extracellular Ca2+ on FGF2 gene expression in hDP and immortalized mouse dental papilla (mDP) cells. Cells were stimulated with 10 mM CaCl2 in the presence or absence of cell signaling inhibitors. FGF2 gene expression was assessed using real-time polymerase chain reaction. The phosphorylation status of signaling molecules was examined by Western blotting. Extracellular Ca2+ increased FGF2 gene expression in mDP and hDP cells. Gene expression of the calcium-sensing receptor and G protein-coupled receptor family C group 6 member A, both of which are extracellular Ca2+ sensors, was not detected. Ca2+-mediated Fgf2 expression was reduced by pretreatment with the protein kinase A (PKA) inhibitor H-89 or extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 but not by pretreatment with the protein kinase C inhibitor GF-109203X or p38 inhibitor SB203580. Extracellular Ca2+ increased PKA activity and ERK1/2 phosphorylation. Ca2+-induced PKA activity decreased by pretreatment with PD98059. These findings indicate that elevated extracellular Ca2+ levels led to increased Fgf2 expression through ERK1/2 and PKA in mDP cells and that this mechanism may be useful for designing regenerative therapies for dentin.
Assuntos
Cálcio/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Papila Dentária/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/efeitos dos fármacos , Animais , Western Blotting , Cloreto de Cálcio/farmacologia , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/análise , Ensaio de Imunoadsorção Enzimática , Fator 2 de Crescimento de Fibroblastos/análise , Fator 2 de Crescimento de Fibroblastos/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/análise , Proteína Quinase 3 Ativada por Mitógeno/análise , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Abstract We previously reported that elevated extracellular calcium (Ca2+) levels increase bone morphogenetic protein 2 expression in human dental pulp (hDP) cells. However, it is unknown whether extracellular Ca2+ affects the expression of other growth factors such as fibroblast growth factor 2 (FGF2). Objective: The present study aimed to examine the effect of extracellular Ca2+ on FGF2 gene expression in hDP and immortalized mouse dental papilla (mDP) cells. Materials and Methods: Cells were stimulated with 10 mM CaCl2 in the presence or absence of cell signaling inhibitors. FGF2 gene expression was assessed using real-time polymerase chain reaction. The phosphorylation status of signaling molecules was examined by Western blotting. Results: Extracellular Ca2+ increased FGF2 gene expression in mDP and hDP cells. Gene expression of the calcium-sensing receptor and G protein-coupled receptor family C group 6 member A, both of which are extracellular Ca2+ sensors, was not detected. Ca2+-mediated Fgf2 expression was reduced by pretreatment with the protein kinase A (PKA) inhibitor H-89 or extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 but not by pretreatment with the protein kinase C inhibitor GF-109203X or p38 inhibitor SB203580. Extracellular Ca2+ increased PKA activity and ERK1/2 phosphorylation. Ca2+-induced PKA activity decreased by pretreatment with PD98059. Conclusions: These findings indicate that elevated extracellular Ca2+ levels led to increased Fgf2 expression through ERK1/2 and PKA in mDP cells and that this mechanism may be useful for designing regenerative therapies for dentin.
Assuntos
Animais , Camundongos , Expressão Gênica/efeitos dos fármacos , Cálcio/farmacologia , Fator 2 de Crescimento de Fibroblastos/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Papila Dentária/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/efeitos dos fármacos , Fatores de Tempo , Cloreto de Cálcio/farmacologia , Ensaio de Imunoadsorção Enzimática , Células Cultivadas , Western Blotting , Reprodutibilidade dos Testes , Fator 2 de Crescimento de Fibroblastos/análise , Fator 2 de Crescimento de Fibroblastos/genética , Proteínas Quinases Dependentes de AMP Cíclico/análise , Proteína Quinase 1 Ativada por Mitógeno/análise , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/análise , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Tityus serrulatus sting causes thousands of deaths annually worldwide. T. serrulatus-envenomed victims exhibit local or systemic reaction that culminates in pulmonary oedema, potentially leading to death. However, the molecular mechanisms underlying T. serrulatus venom (TsV) activity remain unknown. Here we show that TsV triggers NLRP3 inflammasome activation via K(+) efflux. Mechanistically, TsV triggers lung-resident cells to release PGE2, which induces IL-1ß production via E prostanoid receptor 2/4-cAMP-PKA-NFκB-dependent mechanisms. IL-1ß/IL-1R actions account for oedema and neutrophil recruitment to the lungs, leading to TsV-induced mortality. Inflammasome activation triggers LTB4 production and further PGE2 via IL-1ß/IL-1R signalling. Activation of LTB4-BLT1/2 pathway decreases cAMP generation, controlling TsV-induced inflammation. Exogenous administration confirms LTB4 anti-inflammatory activity and abrogates TsV-induced mortality. These results suggest that the balance between LTB4 and PGE2 determines the amount of IL-1ß inflammasome-dependent release and the outcome of envenomation. We suggest COX1/2 inhibition as an effective therapeutic intervention for scorpion envenomation.
Assuntos
Proteínas de Transporte/genética , Dinoprostona/farmacologia , Interleucina-1beta/efeitos dos fármacos , Leucotrieno B4/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Picadas de Escorpião/imunologia , Venenos de Escorpião/farmacologia , Animais , Araquidonato 5-Lipoxigenase/genética , Western Blotting , Proteínas de Transporte/imunologia , Celecoxib/farmacologia , AMP Cíclico/imunologia , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/imunologia , Inibidores de Ciclo-Oxigenase/farmacologia , Dinoprostona/imunologia , Técnicas In Vitro , Indóis/farmacologia , Indometacina/farmacologia , Inflamassomos/imunologia , Interleucina-1beta/imunologia , Leucotrieno B4/imunologia , Inibidores de Lipoxigenase/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Knockout , NF-kappa B/efeitos dos fármacos , NF-kappa B/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Fosfoproteínas , Antagonistas de Prostaglandina/farmacologia , Receptores de Prostaglandina E Subtipo EP2/efeitos dos fármacos , Receptores de Prostaglandina E Subtipo EP2/imunologia , Receptores de Prostaglandina E Subtipo EP4/efeitos dos fármacos , Receptores de Prostaglandina E Subtipo EP4/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Picadas de Escorpião/mortalidade , Escorpiões , Xantonas/farmacologiaRESUMO
Ethanol exposure to offspring during pregnancy and lactation leads to developmental disorders, including central nervous system dysfunction. In the present work, we have studied the effect of chronic ethanol exposure during pregnancy and lactation on the phosphorylating system associated with the astrocytic and neuronal intermediate filament (IF) proteins: glial fibrillary acidic protein (GFAP), and neurofilament (NF) subunits of low, medium, and high molecular weight (NFL, NFM, and NFH, respectively) in 9- and 21-day-old pups. Female rats were fed with 20% ethanol in their drinking water during pregnancy and lactation. The homeostasis of the IF phosphorylation was not altered in the cerebral cortex, cerebellum, or hippocampus of 9-day-old pups. However, GFAP, NFL, and NFM were hyperphosphorylated in the hippocampus of 21-day-old pups. PKA had been activated in the hippocampus, and Ser55 in the N-terminal region of NFL was hyperphosphorylated. In addition, JNK/MAPK was activated and KSP repeats in the C-terminal region of NFM were hyperphosphorylated in the hippocampus of 21-day-old pups. Decreased NFH immunocontent but an unaltered total NFH/phosphoNFH ratio suggested altered stoichiometry of NFs in the hippocampus of ethanol-exposed 21-day-old pups. In contrast to the high susceptibility of hippocampal cytoskeleton in developing rats, the homeostasis of the cytoskeleton of ethanol-fed adult females was not altered. Disruption of the cytoskeletal homeostasis in neural cells supports the view that regions of the brain are differentially vulnerable to alcohol insult during pregnancy and lactation, suggesting that modulation of JNK/MAPK and PKA signaling cascades target the hippocampal cytoskeleton in a window of vulnerability in 21-day-old pups. Our findings are relevant, since disruption of the cytoskeleton in immature hippocampus could contribute to later hippocampal damage associated with ethanol toxicity.
Assuntos
Depressores do Sistema Nervoso Central/toxicidade , Citoesqueleto/efeitos dos fármacos , Etanol/toxicidade , Hipocampo/efeitos dos fármacos , Lactação , Animais , Animais Recém-Nascidos , Peso Corporal/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ingestão de Energia/efeitos dos fármacos , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/embriologia , Homeostase , Filamentos Intermediários/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas de Neurofilamentos/metabolismo , Fosforilação , Gravidez , Ratos , Ratos WistarRESUMO
The establishment of extinction of one-trial avoidance involves the dorsal hippocampus (DH) and basolateral amygdala (BLA), two areas that participate in its original consolidation. The posterior parietal (PARIE) and posterior cingulate (CING) cortices also participate in consolidation of this task but their role in extinction has not been explored. Here we study the effect on the extinction of one-trial avoidance in rats of three different drugs infused bilaterally into DH, BLA, PARIE or CING 5min before the first of four daily unreinforced test sessions: The glutamate NMDA receptor antagonist, AP5 (5.0microg/side),and the inhibitors of calcium-calmodulin dependent kinase II (CaMKII), KN-93 (0.3microg/side), or of the cAMP-dependent protein kinase (PKA), Rp-cAMPs (0.5microg/side) hindered extinction when given into DH or BLA. Levels of pPKA and pCaMKII increased in DH after the first extinction trial; in BLA only the CaMKII increase was seen. Thus, this pathway appears to participate in extinction in BLA at the "basal" levels, and at enhanced levels in DH. None of the treatments affected extinction when given into PARIE or CING. The present findings indicate that: (1) the DH and BLA are important for the initiation of extinction at the time of the first unreinforced retrieval session; (2) both the CaMKII and the PKA signaling pathway are necessary for the development of extinction in the two regions; (3) PARIE and CING are probably unrelated to extinction.
Assuntos
Tonsila do Cerebelo/enzimologia , Aprendizagem da Esquiva/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Extinção Psicológica/fisiologia , Hipocampo/enzimologia , 2-Amino-5-fosfonovalerato/farmacologia , Tonsila do Cerebelo/efeitos dos fármacos , Animais , Benzilaminas/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/efeitos dos fármacos , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Extinção Psicológica/efeitos dos fármacos , Giro do Cíngulo/efeitos dos fármacos , Giro do Cíngulo/enzimologia , Hipocampo/efeitos dos fármacos , Masculino , Microinjeções , Lobo Parietal/efeitos dos fármacos , Lobo Parietal/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Sistemas do Segundo Mensageiro/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sulfonamidas/farmacologia , Tionucleotídeos/farmacologiaRESUMO
Vanadium is a trace element widely distributed in the environment. In vertebrates it is mainly stored in bone tissue. The unique cellular environment in the bone and the variety of interactions that mediate cancer metastasis determine that certain types of cancer, such as breast and prostate cancer, preferentially metastize in the skeleton. Since this effect usually signifies serious morbidity and grave prognosis there is an increasing interest in the development of new treatments for this pathology. The present work shows that vanadium complexes can inhibit some parameters related to cancer metastasis such as cell adhesion, migration and clonogenicity. We have also investigated the role of protein kinase A in these processes.
Assuntos
Metástase Neoplásica/prevenção & controle , Osteossarcoma/tratamento farmacológico , Oligoelementos/farmacologia , Vanádio/farmacologia , Animais , Aspirina/química , Aspirina/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Estabilidade de Medicamentos , Glucose/química , Glucose/farmacologia , Ratos , Oligoelementos/química , Trealose/química , Trealose/farmacologia , Vanádio/químicaRESUMO
The present study investigated the antinociceptive effect of p-methoxy-diphenyl diselenide (MeOPhSe)(2), a simple organochalcogenide, in chemical and thermal behavioural models of nociception in mice, without accompanying changes in ambulation when assessed in an open field. This compound given by oral route (p.o.) produced antinociception when assessed on acetic acid-induced visceral nociception, with mean ID(50) value of 9.64 (3.28-28.35) mg/kg. In addition, the per oral administration of (MeOPhSe)(2) exhibited significant inhibition of the neurogenic nociception induced by intraplantar (i.pl.) injection of capsaicin, with mean ID(50) value of 16.29 (11.43-23.22) mg/kg. (MeOPhSe)(2) showed an antinociceptive effect when measured by the tail-immersion and hot-plate tests. Likewise, compound inhibited both neurogenic and inflammatory phases of the overt nociception caused by i.pl injection of formalin, with mean ID(50) values of 22.32 (17.84-27.92) and 19.65 (13.67-28.24) mg/kg, respectively. (MeOPhSe)(2) reduced the nociception produced by i.pl. injection of glutamate and 8-bromo-cAMP (8-Br-cAMP, a protein kinase A [PKA] activator), with mean ID(50) values of 11.05 (7.12-17.15) and 8.72 (5.42-14.02) mg/kg, respectively. (MeOPhSe)(2) also reduced formalin-, glutamate-, induced paw oedema formation. A marked inhibition of the biting behaviour induced by intrathecal (i.t.) injection of glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and (+/-)-1 aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) was caused by (MeOPhSe)(2). However, (MeOPhSe)(2) completely failed to affect the nociception induced by i.t. injection of N-methyl-D-aspartate (NMDA; 450 pmol/site) and kainate (110 pmol /site). The antinociceptive effect caused by (MeOPhSe)(2) was blocked by picrotoxin (a chloride ion channel blocker) and bicucculine (a specific GABA(A) receptor antagonist) but not by phaclofen (a specific GABA(B) receptor antagonist) in the hot-plate test. Together, these results indicate that (MeOPhSe)(2) produces antinociception in several models of nociception through mechanisms that involve an interaction with glutamatergic and GABAergic systems, as well as the inhibition of protein kinase A pathway.
Assuntos
Analgésicos , Derivados de Benzeno/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ácido Glutâmico/fisiologia , Compostos Organosselênicos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ácido gama-Aminobutírico/fisiologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Ácido Acético , Animais , Capsaicina , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Edema/induzido quimicamente , Edema/prevenção & controle , Ativadores de Enzimas/farmacologia , Aminoácidos Excitatórios/fisiologia , Feminino , Formaldeído , Temperatura Alta , Imersão/fisiopatologia , Injeções Espinhais , Camundongos , Atividade Motora/efeitos dos fármacos , Dor/induzido quimicamente , Dor/psicologia , Tempo de ReaçãoRESUMO
Inositol is the precursor for most Trypanosoma cruzi surface molecules, including phosphoinositides, glycosylinositolphospholipids and glycosylphosphatidylinositol anchors. As the parasite is an inositol auxotroph, the inositol transport system might be a potential target for new trypanocide drugs, as some of its properties are different from its mammalian counterpart. Here, we investigated the modulation exerted by effectors of PKA and PKC on this transport system to comply with the parasite physiology. Pre-incubation of the cells with either dibutyryl-cyclic AMP (25 microM) or forskolin (30 microM) decreased the myo-inositol uptake by half, this effect being reversed by KT5720 (PKA inhibitor). Conversely, pre-incubation of the cells with PMA (2.8 microg/ml) or serum (5%) had a approximately 50% stimulation in myo-inositol uptake, being this effect reversed by staurosporine (0.5 microM) or sphingosine (10 microM). These results allow us to conclude that the myo-inositol transport system in T. cruzi epimastigotes is inhibited by PKA and stimulated by PKC effectors.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inositol/metabolismo , Proteína Quinase C/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Bucladesina/farmacologia , Carbazóis/farmacologia , Colforsina/farmacologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Indóis/farmacologia , Cinética , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/efeitos dos fármacos , Pirróis/farmacologia , Transdução de Sinais/fisiologia , Esfingosina/farmacologia , Estaurosporina/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologiaRESUMO
Cellular prion protein (PrPc) has a pivotal role in prion diseases. PrPc is a specific receptor for laminin (LN) gamma1 peptide and several lines of evidence indicate that it is also involved in neural plasticity. Here we investigated whether the interaction between PrPc and LN plays a role in rat memory formation. We found that post-training intrahippocampal infusion of PrPc-derived peptides that contain the LN binding site (PrPc163-182 and PrPc173-192) or of anti-PrPc or anti-LN antibodies that inhibit PrPc-LN interaction impaired inhibitory avoidance memory retention. The amnesic effect of anti-PrPc antibodies and PrPc173-192 peptide was reversed by co-infusion of a LN gamma1 chain-derived peptide containing the PrPc-binding site, suggesting that PrPc-LN interaction is indeed crucial for memory consolidation. In addition, PrPc173-192 peptide and anti-PrPc or anti-LN antibodies also inhibited the activation of hippocampal cAMP-dependent protein kinase A (PKA) and extracellular regulated kinase (ERK1/2), two kinases that mediate the up-regulation of signaling pathways needed for consolidation of inhibitory avoidance memory. Our findings show that, through its interaction with LN, hippocampal PrPc plays a critical role in memory processing and suggest that this role is mediated by activation of both PKA and ERK1/2 signaling pathways.
Assuntos
Hipocampo/metabolismo , Laminina/metabolismo , Aprendizagem/fisiologia , Memória/fisiologia , Proteínas PrPC/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Sítios de Ligação/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Laminina/antagonistas & inibidores , Laminina/imunologia , Masculino , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Proteínas PrPC/química , Estrutura Terciária de Proteína/fisiologia , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Because of the importance of cell signalling processes in proliferation and differentiation, the adenylate cyclase pathway was studied, specifically the protein kinase A (PKA) in Leishmania amazonensis. The PKAs of soluble (SF) and enriched membrane fractions (MF) from infective/non-infective promastigotes and axenic amastigotes were assayed. In order to purify the PKA molecule, fractions were chromatographed on DEAE-cellulose columns and the phosphorylative activity was evaluated using [gamma(32)P]-ATP as the phosphate source. These experiments were performed in the presence of cyclic adenosine monophosphate (cAMP) and an inhibitor of PKA. Our data demonstrated that the PKA activity was significantly higher (about two times) in SF from promastigotes with a high concentration of metacyclic forms, when compared with the non-infective promastigotes, suggesting an association of this activity and the metacyclogenesis process. A discrete phosphorylative activity in axenic amastigotes was observed. As the adenylate cyclase/cAMP pathway would be involved in the parasite-host interiorization, the PKA activity may constitute a good intracellular target for studies of leishmanicidal drugs.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Leishmania mexicana/enzimologia , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , GMP Cíclico/metabolismo , Leishmania mexicana/crescimento & desenvolvimento , Inibidores de Proteínas Quinases/farmacologia , Proteínas de Protozoários/análiseRESUMO
The contribution of Ca2+ entry through different voltage-activated Ca2+ channel (VACC) subtypes to the phosphorylation of extracellular signal regulated kinase (ERK) was examined in bovine adrenal-medullary chromaffin cells. High K+ depolarization (40 mM, 3 min) induced ERK phosphorylation, an effect that was inhibited by specific mitogen-activated protein kinase kinase inhibitors. By using selective inhibitors, we observed that depolarization-induced ERK phosphorylation completely depended on protein kinase C-alpha (PKC-alpha), but not on Ca2+/calmodulin-dependent protein kinase nor cyclic AMP-dependent protein kinase. Blockade of L-type Ca2+ channels by 3 microm furnidipine, or blockade of N channels by 1 micromomega-conotoxin GVIA reduced ERK phosphorylation by 70%, while the inhibition of P/Q channels by 1 micromomega-agatoxin IVA only caused a 40% reduction. The simultaneous blockade of L and N, or P/Q and N channels completely abolished this response, yet 23% ERK phosphorylation remained when L and P/Q channels were simultaneously blocked. Confocal imaging of cytosolic Ca2+ elevations elicited by 40 mm K+, showed that Ca2+ levels increased throughout the entire cytosol, both in the presence and the absence of Ca2+ channel blockers. Fifty-eight percent of the fluorescence rise depended on Ca2+ entering through N channels. Thus, ERK phosphorylation seems to depend on a critical level of Ca2+ in the cytosol rather than on activation of a given Ca2+ channel subtype.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Células Cromafins/metabolismo , Citosol/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Bovinos , Polaridade Celular/efeitos dos fármacos , Células Cultivadas , Células Cromafins/citologia , Células Cromafins/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativadores de Enzimas/farmacologia , Inibidores Enzimáticos/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Potássio/farmacologia , Proteína Quinase C/efeitos dos fármacos , Proteína Quinase C/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
The effect of several second messengers on the functional expression of gap junctions was investigated in primary cultures of newborn rat microglia. As previously reported, microglia cultured under resting conditions expressed low levels of the gap junction protein connexin 43, and exhibited little dye coupling. After treatment with 4bromo-A23187, a Ca(2+) ionophore, the incidence of dye coupling between microglia increased progressively over a 12-h period. Dye coupling was markedly reduced by gap junction blockers. Induction of dye coupling by 4bromo-A23187 was prevented by the addition of a synthetic peptide with the same sequence as a region of the extracellular loop 1 of connexin 43 (residues 53-66). The increase in dye coupling induced by 4bromo-A23187 was associated with increased connexin 43 mRNA and protein levels. Treatment of microglia with phorbol 12-myristate 13-acetate, an activator of protein kinase C, did not promote gap junctional communication in untreated microglia and reversed 4bromo-A23187-induced dye coupling. Thus, gap junctional communication between microglia can be regulated oppositely by calcium- and protein kinase C-dependent pathways. Activators of cGMP-dependent protein kinase (8bromo-cGMP) or protein kinase A (8bromo-cAMP) had no effect on untreated microglia or on 4bromo-A23187-induced dye coupling. Differential regulation of gap junctions by intracellular calcium concentration and protein kinase C activity may help to explain how various stimuli evoke differences in microglia responses, such as synthesis and secretion of cytokines and proteases.
Assuntos
Sistema Nervoso Central/imunologia , GMP Cíclico/análogos & derivados , Proteína GAP-43/metabolismo , Junções Comunicantes/metabolismo , Gliose/imunologia , Microglia/imunologia , Proteína Quinase C/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Animais Recém-Nascidos , Calcimicina/farmacologia , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Células Cultivadas , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , GMP Cíclico/farmacologia , Corantes Fluorescentes , Proteína GAP-43/efeitos dos fármacos , Proteína GAP-43/genética , Junções Comunicantes/efeitos dos fármacos , Gliose/patologia , Gliose/fisiopatologia , Ionóforos/farmacologia , Isoquinolinas , Microglia/citologia , Microglia/metabolismo , Proteína Quinase C/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
To test for a role for the cellular prion protein (PrP(c)) in cell death, we used a PrP(c)-binding peptide. Retinal explants from neonatal rats or mice were kept in vitro for 24 h, and anisomycin (ANI) was used to induce apoptosis. The peptide activated both cAMP/protein kinase A (PKA) and Erk pathways, and partially prevented cell death induced by ANI in explants from wild-type rodents, but not from PrP(c)-null mice. Neuroprotection was abolished by treatment with phosphatidylinositol-specific phospholipase C, with human peptide 106-126, with certain antibodies to PrP(c) or with a PKA inhibitor, but not with a MEK/Erk inhibitor. In contrast, antibodies to PrP(c) that increased cAMP also induced neuroprotection. Thus, engagement of PrP(c) transduces neuroprotective signals through a cAMP/PKA-dependent pathway. PrP(c) may function as a trophic receptor, the activation of which leads to a neuroprotective state.
Assuntos
Anisomicina/farmacologia , Apoptose/efeitos dos fármacos , AMP Cíclico/análogos & derivados , AMP Cíclico/fisiologia , Proteínas do Olho/fisiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas PrPC/metabolismo , Retina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Anisomicina/antagonistas & inibidores , Anticorpos Monoclonais/farmacologia , Apoptose/fisiologia , Colforsina/farmacologia , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/biossíntese , Proteínas do Olho/imunologia , Flavonoides/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Soros Imunes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Cultura de Órgãos , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , Fosforilação , Proteínas PrPC/química , Processamento de Proteína Pós-Traducional , Ratos , Ratos Endogâmicos , Retina/metabolismo , Tionucleotídeos/farmacologia , Fosfolipases Tipo C/farmacologiaRESUMO
Permeabilized germlings from the dimorphic fungus Mucor rouxii were used for in situ measurement of protein kinase A (PKA) activation, to compare the results with those obtained in vitro at low or high (nonlinear) enzyme concentrations. The apparent total activity per cell when measured in situ is 5- to 10-fold lower than the in vitro measured activity in crude extracts from those cells. Polyamines and NaCl stimulate the activity in situ. The apparent relative specific activity of the in situ measured PKA toward four peptide substrates is similar to the results obtained in vitro at high holoenzyme concentration and not to those obtained with the free catalytic subunit. Saturation in the activation of PKA by cAMP in situ is attained at low concentrations (2 to 10 microM), while in vitro, at high holoenzyme concentration, no saturation was attained up to 1 mM cAMP (V. Zaremberg et al. Arch. Biochem. Biophys. 381, 74-82, 2000). Activation of PKA by site-selective cAMP analogs is assayed in situ and in vitro at two enzyme concentrations. Site B-selective cAMP analogs are good activators of PKA at low enzyme concentration in vitro but poor activators either at high enzyme concentration in vitro or in permeabilized cells. A physiological correlation with the behavior of site-selective analogs in situ is demonstrated in vivo when assaying the effect of increasing concentrations of site-selective cAMP analogs on the impairment of polarized growth of M. rouxii spores.
Assuntos
Permeabilidade da Membrana Celular/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Líquido Intracelular/enzimologia , Mucor/enzimologia , Transdução de Sinais/fisiologia , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Bioensaio , Catálise/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Líquido Intracelular/efeitos dos fármacos , Modelos Biológicos , Mucor/citologia , Mucor/efeitos dos fármacos , Peptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Rats implanted bilaterally with cannulae in the CA1 region of the dorsal hippocampus or in the amygdala were trained in one-trial step-down inhibitory (passive) avoidance using a 0.4 mA footshock. At various times after training (0, 1.5, 3, 6 or 9 h for animals implanted in the hippocampus; 0 or 3 h for those implanted in the amygdala), they received infusions of 8-Br-cAMP (cyclic adenosine monophosphate) (1.25 micrograms/side), SKF38393 (7.5 micrograms/side), SCH23390 (0.5 microgram/side), norepinephrine ClH (0.3 microgram/side), timolol ClH (0.3 microgram/side), 8-HO-DPAT (2.5 micrograms/side), NAN-190 (2.5 micrograms/side), forskolin (0.5 microgram/side) or KT5720 (0.5 microgram/side). Rats were tested for retention 24 h after training. SKF38393 is an agonist and SCH23390 an antagonist at dopamine D1 receptors, timolol is a beta-adrenoceptor antagonist, 8-HO-DPAT is an agonist and NAN-190 an antagonist at 5HT1A receptors, forskolin enhances adenylyl cyclase, and KT5720 inhibits protein kinase A. When given into the hippocampus 0 h post-training, norepinephrine enhanced memory and KT5720 was amnestic. When given 1.5 h after training, all treatments were ineffective. When given 3 or 6 h post-training, 8-Br-cAMP, forskolin, SKF 38393, noradrenaline and NAN-190 caused memory facilitation, and KT5720, SCH23390, timolol and 8-HO-DPAT caused retrograde amnesia. At 9 h from training, all treatments were again ineffective. When given into the amygdala 0 or 3 h post-training all treatments were ineffective, except for noradrenaline at 0 h, which caused retrograde facilitation. The data agree with the suggestion that in the hippocampus, but not the amygdala, a cAMP/protein kinase A pathway is involved in memory consolidation at 3 and 6 h from training, and that this is regulated by D1, beta, and 5HT1A receptors. This correlates with a previous report of increased cAMP levels, protein kinase A activity and P-CREB levels at 3-6 h from training in rat hippocampus in this task. This may be taken to suggest that the hippocampus, but not the amygdala, is involved in the long-term storage of step-down inhibitory avoidance in the rat.
Assuntos
Carbazóis , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Memória/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/farmacologia , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Agonistas alfa-Adrenérgicos/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Tonsila do Cerebelo , Animais , Aprendizagem da Esquiva , Benzazepinas/farmacologia , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Hipocampo , Indóis/farmacologia , Masculino , Norepinefrina/farmacologia , Piperazinas/farmacologia , Pirróis/farmacologia , Ratos , Ratos Wistar , Receptores Adrenérgicos beta/efeitos dos fármacos , Receptores de Dopamina D1/efeitos dos fármacos , Receptores de Serotonina/efeitos dos fármacos , Antagonistas da Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologia , Fatores de Tempo , Timolol/farmacologiaRESUMO
Male Wistar rats were trained in one-trial step-down inhibitory avoidance using a 0.4-mA footshock. At various times after training (0, 1.5, 3, 6 and 9 h for the animals implanted into the CA1 region of the hippocampus; 0 and 3 h for those implanted into the amygdala), these animals received microinfusions of SKF38393 (7.5 micrograms/side), SCH23390 (0.5 microgram/side), norepinephrine (0.3 microgram/side), timolol (0.3 microgram/side), 8-OH-DPAT (2.5 micrograms/side), NAN-190 (2.5 micrograms/side), forskolin (0.5 microgram/side), KT5720 (0.5 microgram/side) or 8-Br-cAMP (1.25 micrograms/side). Rats were tested for retention 24 h after training. When given into the hippocampus 0 h post-training, norepinephrine enhanced memory whereas KT5720 was amnestic. When given 1.5 h after training, all treatments were ineffective. When given 3 or 6 h post-training, 8-Br-cAMP, forskolin, SKF38393, norepinephrine and NAN-190 caused memory facilitation, while KT5720, SCH23390, timolol and 8-OH-DPAT caused retrograde amnesia. Again, at 9 h after training, all treatments were ineffective. When given into the amygdala, norepinephrine caused retrograde facilitation at 0 h after training. The other drugs infused into the amygdala did not cause any significant effect. These data suggest that in the hippocampus, but not in the amygdala, a cAMP/protein kinase A pathway is involved in memory consolidation at 3 and 6 h after training, which is regulated by D1, beta, and 5HT1A receptors. This correlates with data on increased post-training cAMP levels and a dual peak of protein kinase A activity and CREB-P levels (at 0 and 3-6 h) in rat hippocampus after training in this task. These results suggest that the hippocampus, but not the amygdala, is involved in long-term storage of step-down inhibitory avoidance in the rat.
Assuntos
Tonsila do Cerebelo/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , AMP Cíclico/análise , Hipocampo/efeitos dos fármacos , Memória/fisiologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Animais , Benzazepinas/farmacologia , Colforsina/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/análise , Masculino , Norepinefrina/farmacologia , Ratos , Ratos Wistar , Transdução de SinaisRESUMO
Male Wistar rats were trained in one-trial step-down inhibitory avoidance using a 0.4-mA footshock. At various times after training (0, 1.5, 3,6 and 9 h for the animals implanted into the CA1 region of the hippocampus; 0 and 3 h for those implanted into the amygdala), these animals received microinfusions of SKF38393 (7.5 mug/side), SCH23390 (0.5 mug/side), norepinephrine (0.3 mug/side), timolol (0.3 mug/side), 8-OH-DPAT (2.5 mug/side), NAN-190 (2.5 mug/side), forskolin (0.5 mug/side), KT5720 (0.5 mug/side) or 8-Br-cAMP (1.25 mug/side). Rats were tested for retention 24 h after training. When given into the hippocampus 0 h post-training, norepinephrine enhanced memory whereas KT5720 was amnestic. When given 1.5 h after training, all treatments were ineffective. When given 3 or 6 h post-training, 8-Br-cAMP, forskolin, SKF38393, norepinephrine and NAN-190 caused memory facilitation, while KT5720, SCH23390, timolol and 8-OH-DPAT caused retrograde amnesia. Again, at 9 h after training, all treatments were inffective. When given into the amygdala, norepinephrine caused retrograde facilitation at 0 h after training. The other drugs infused into the amygdala did not cause any significant effect. These data suggest that in the hippocampus, but not in the amygdala, a cAMP/protein kinase A pathway is involved in memory cosolidation at 3 and 6 h after training, which is regulated by D1, Beta, and 5HT1A receptors. This correlates with data on increased post-training cAMP levels and a dual peak of protein kinase A activity and CREB-P levels (at 0 and 3-6 h) in rat hippocampus after training in this task. These results suggest that the hippocampus, but not the amygdala, is involved in long-term storage of step-down inhibitory avoidance in the rat.
Assuntos
Ratos , Animais , Masculino , Tonsila do Cerebelo/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , AMP Cíclico/análise , Hipocampo/efeitos dos fármacos , Memória/fisiologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Benzazepinas/farmacologia , Colforsina/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/análise , Norepinefrina/farmacologia , Ratos Wistar , Transdução de SinaisRESUMO
Miniature end-plate currents (MEPCS), from synaptic spots on the caudal muscles of Bufo marinus tadpoles, were analyzed in both pre- and postsynaptic domains, when protein kinase A (PKA) activity modificators were used. Sp-cAMPS diasteromer induced an increase in MEPC frequency, which was completely reversed by Rp-cAMPS. However, changes in the decay time of MEPCS were not detected. Dibutyryl-cAMP produced a similar presynaptic action, but its postsynaptic action was similar to butyrate. Presynaptic effect of forskolin (FSK), if any, is masked by the increase of MEPC frequency produced by dimethylsulfoxide (DMSO), the solvent used.