Posttranscriptional regulation of endothelial nitric oxide synthase during cell growth.

The expression of the endothelial NO synthase (eNOS) is dramatically influenced by the state of cell growth. In proliferating cells, mRNA levels are increased 4-fold compared with postconfluent, nonproliferating cells. Nuclear run-on analysis indicated that there is no difference in the transcriptional rate of eNOS in proliferating versus postconfluent cells. The half-life of eNOS mRNA, measured after actinomycin D transcriptional arrest, was 3-fold greater in preconfluent compared with confluent endothelial cells. Using UV-cross-linking analysis, a cytoplasmic protein with an apparent molecular mass of 51 kDa was found to bind to terminal 545-nt eNOS mRNA 3-fold more in confluent cells than in proliferating cells. Further characterization of the eNOS mRNA indicated that a 43-nt sequence at the origin of the 3'-untranslated region (UTR) is critical in binding of this protein. Endothelial cells were stably transfected with a chimeric cDNA plasmid containing chloramphenicol acetyl transferase (CAT) ligated to the eNOS coding region and either the wild-type 3'-UTR (pcDNACAT/eNOS((wtUTR))) or a mutant 3'-UTR lacking the 43 nt found to bind the 51-kDa protein (pcDNACAT/eNOS((DeltaUTR))). The CAT/eNOS mRNA half-life was dramatically stabilized in these latter cells as compared with cells transfected with pcDNACAT/eNOS((wtUTR))). Thus, this 43-nt region plays a critical role in destabilizing eNOS mRNA. These studies demonstrate a mechanism for modulation of eNOS expression during cell growth via interactions between the proximal 3'-UTR and a novel approximately 51-kDa cytosolic protein.

The effect of aluminum on markers for synaptic neurotransmission, cyclic AMP, and neurofilaments in a neuroblastoma x glioma hybridoma (NG108-15).

The effects of the neurotoxin aluminum on markers of synaptic neurotransmission, adenosine 3',5'-monophosphate, and neurofilaments have been evaluated in a neuroblastoma x glioma hybridoma (NG108-15). Cells were exposed for 4 days to 2 mM aluminum lactate, a concentration that did not suppress growth. Compared to controls, the activity of choline acetyltransferase was significantly increased by 37% associated with an up-regulation in enzyme activity (Vmax). Muscarinic receptors, measured by [3H]QNB binding, were reduced by 41%. In contrast, the activities of acetylcholinesterase and glutamate decarboxylase were not significantly changed. Aluminum raised the level of cyclic AMP by 20%, although adenylate cyclase activity was unchanged. Small amounts of both phosphorylated and non-phosphorylated neurofilaments were detected in NG108-15 cells. Aluminum intoxication, however, did not alter the quantity, ultrastructure, or immunoreactivity of neurofilaments. Our results demonstrate the capability of aluminum to produce selected changes in cholinergic markers and levels of cyclic AMP in a rapidly dividing cell line.

Effects of anticonvulsants on cholinergic and GABAergic properties in the neuronal cell clone NG108-15.

The effects of anticonvulsant drugs on growth, cholinergic, and GABAergic properties were examined in the neuronal cell clone NG108-15. Cells were exposed for 4 days to valproic acid, phenobarbital, phenytoin, or carbamazepine in concentrations equivalent to therapeutic free levels in human serum. Experiments were also performed with varying concentrations of a recently proposed antiepileptic, gamma-vinyl GABA. Of these five anticonvulsants, cell growth (total protein and cell counts) was decreased with valproic acid and phenytoin but only valproic acid and gamma-vinyl GABA altered neurotransmitter markers. Therapeutic concentrations of valproic acid increased choline acetyltransferase activity to 142% of control but had no effect on either the activity of glutamate decarboxylase or the level of GABA. The effects of a higher (toxic) concentration of valproic acid (200 micrograms/ml) were similar to those induced by the differentiating agent dibutyryl cyclic AMP: both decreased cell growth, enhanced the activity of choline acetyltransferase and reduced the activity of glutamate decarboxylase. Gamma-vinyl GABA had no effect on cholinergic markers but, at 1300 micrograms/ml, increased GABA levels to 135% of control despite the reduction of glutamate decarboxylase to 68% of control. In the NG108-15 cell clone, anticonvulsants have varying effects on cell growth, differentiation, and neurotransmitter systems. Our findings do not support the proposal that the mechanism of action for valproic acid, phenobarbital, phenytoin, and carbamazepine is via alteration of GABA levels.

The identification and characterization of a GABAergic system in the cholinergic neuroblastoma x glioma hybrid clone NG108-15.

gamma-Aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD) activity were identified and characterized in the cholinergic neuronal cell clone NG108-15. GAD activity is similar to form A in rat brain by being temperature sensitive and independent of pyridoxal 5'-phosphate in the assay mixture. Most of the NG108-15 GAD activity is inactivated by 1 mM amino-oxyacetic acid. In contrast to NG108-15 choline acetyltransferase, GAD activity is not enhanced when cells are grown with dibutyryl cyclic AMP. The GAD activity shows a cell-density dependent increase that does not correlate with changing level of endogenous GABA. The identification of GABAergic properties in the NG108-15 clone enhances its use as a neuronal model for studies of differentiation and neurotoxicity of drugs or chemicals.