RESUMEN
Post-translational modification of neural cell adhesion molecule (NCAM) with alpha2,8-linked polysialic acid, which regulates homophilic adhesion and/or signal transduction events, is crucial to synaptic plasticity in the developing and adult brain. Evidence from in vitro models has implicated polysialylation in the regulation of cell growth, migration, and differentiation. Here, using two in vitro models, we demonstrate that polysialylation is downregulated by cell-cell contact and correlated with a state of neuronal differentiation. Furthermore, we report a role for protein kinase C delta (PKCdelta) in the regulation of NCAM polysialylation. Pharmacological studies using the PKC activator, phorbol myristate acetate, and inhibitors, calphostin-C, and staurosporine, demonstrated PKC activity to be inversely related to NCAM polysialylation in the mouse neuro-2A cell line. Isoform-specific immunoblot studies indicated this effect to be mediated by the calcium-independent PKCdelta isozyme, as its expression was inversely related to NCAM polysialylation state in both neuro-2A and rat PC-12 cell lines. Isoform specificity was further confirmed using the PKCdelta-selective inhibitor rottlerin, which produced a marked increase in PSA expression (36.9+/-5.25 a.u. vs. 24.7+/-0.80 arbitrary units control) coupled with a neuritogenic response. Likewise, decreased expression of PKCdelta was seen in nerve growth factor (NGF)-differentiated PC-12 cells. These findings suggest that the neuronal differentiation process may involve inhibition of PKCdelta, resulting in enhanced morphological plasticity, as evidenced by activation of NCAM polysialylation.
Asunto(s)
Comunicación Celular/fisiología , Isoenzimas/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuronas/citología , Neuronas/enzimología , Proteína Quinasa C/metabolismo , Ácidos Siálicos/metabolismo , Acetofenonas/farmacología , Animales , Benzopiranos/farmacología , Sitios de Unión/fisiología , Unión Competitiva/fisiología , Comunicación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Isoenzimas/antagonistas & inhibidores , Ratones , Moléculas de Adhesión de Célula Nerviosa/química , Neuroblastoma , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Células PC12 , Polisacáridos/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C-delta , Ratas , Ácidos Siálicos/química , Sialiltransferasas/metabolismo , Células Tumorales CultivadasRESUMEN
The antiproliferative potential of the volatile anesthetics isoflurane, enflurane and sevoflurane was determined and compared to the valproate teratogen. The in vitro system employed, a G1 phase proliferative arrest endpoint in C6 glioma, has served previously to discriminate agents with known teratogenic potential in vivo. Based on estimated IC(50) values that were within twice the estimated minimum aveolar concentration value, the rank antiproliferative potency of the inhalational anesthetics employed was isoflurane=enflurane>>sevoflurane. Flow cytometric analysis of growth-arrested cell populations failed to reveal specific accumulation in any cell cycle phase and the lack of a G1 phase-specific effect was confirmed by the absence of a transient, time-dependent sialylation event in synchronized cells. The antiproliferative mechanism of volatile anesthetics, and valproate, was mediated at hydrophobic binding sites, as increasing the hydration sphere of the drug-micelle complex, using the hygroscopic qualities of the dimethylsulfoxide vehicle, completely reversed this effect. Our findings suggest inhalational anesthetics lack the specific in vitro characteristics of the valproate teratogen.
Asunto(s)
Anestésicos por Inhalación/farmacología , Glioma , Isoflurano/farmacología , Neuronas/citología , Teratógenos/farmacología , Ácido Valproico/farmacología , Animales , Western Blotting , División Celular/efectos de los fármacos , Dimetilsulfóxido/farmacología , Enflurano/farmacología , Citometría de Flujo , Fase G1/efectos de los fármacos , L-Lactato Deshidrogenasa/metabolismo , Éteres Metílicos/farmacología , Micelas , Ácido N-Acetilneuramínico/metabolismo , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/metabolismo , Neuronas/efectos de los fármacos , Neuronas/enzimología , Fitohemaglutininas , Sevoflurano , Solventes/farmacología , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/enzimologíaRESUMEN
Previously, the ability of co-administered nefiracetam to reverse scopolamine-induced learning deficits has been attributed to the preservation of a transient increase in neural cell adhesion molecule (NCAM) polysialylation state during a late phase of memory consolidation (Doyle et al., J. Neurosci. Res., 31 (1992) 513-523). Using the PC-12 pheochromocytoma cell model, we now demonstrate nefiracetam pre-exposure to significantly enhance nerve growth factor-induced neuritogenesis and NCAM polysialylation, but not prevalence, in a dose-dependent manner with maximal effects being observed at the lowest dose (0.1 microM) examined. As the memory-associated increase in NCAM polysialylation in vivo is associated with a defined group of neurons at the dentate hilar/granule cell layer border (Regan and Fox, Neurochem. Res., 20 (1995) 521-526), the effect of chronic nefiracetam exposure in vivo was evaluated. Once-daily, intraperitoneal administration of either 3 or 9 mg/kg nefiracetam to adult male Wistar rats for 40 days significantly increased the number of hippocampal dentate polysialylated neurons only at the highest dose evaluated, suggesting it to prevent their age-dependent decline. These results are consistent with nefiracetam facilitating early induction events of long-term memory consolidation processes involving NCAM polysialylation state.