The pharmacological properties of glutamate agonists were compared in astrocyte-rich and astrocyte-poor cultures derived from embryonic rat cerebral cortex. The object of this investigation was to determine the extent to which glutamate uptake might influence the receptor-mediated neurotoxic actions of these compounds. In astrocyte-rich cultures, using 30 min exposures, we observed that the potencies of the poorly transported agonists NMDA (35 microM) and D-glutamate (89 microM) were higher than that of L-glutamate (205 microM). In astrocyte-poor cultures, L-glutamate was much more potent, with an EC50 of 5 +/- 4 microM (3-12 microM), for a 30 min exposure, whereas the potencies of NMDA and D-glutamate were essentially unchanged. L- and D-aspartate were also more effective in astrocyte-poor cultures, again with EC50 values of approximately 6-10 microM, as compared with 130 and 108 microM, respectively, in astrocyte-rich cultures. In other experiments, blocking sodium-dependent glutamate uptake in astrocyte-rich cultures, by using a sodium-free medium, made glutamate as potent an agonist as in astrocyte-poor cultures. Finally, we directly assessed the glutamate uptake system in astrocyte-rich and astrocyte-poor cultures and found that uptake was reduced approximately 25-fold in the astrocyte-poor cultures. These results show that in the presence of abundant astrocytes the neurotoxic potencies of L-glutamate, L-aspartate, and D-aspartate are substantially under-estimated.
Identification of endogenous toxins and characterization of the mechanisms by which toxins produce cell injury and death may help understand both normal modeling of cell populations and connections in the CNS as well as abnormal cell loss. The toxicity of catecholamines intrinsic to the CNS was investigated using the model system of rat cerebral cortex in dissociated cell culture. All catecholamines tested, including norepinephrine (NE), dopamine, and epinephrine, were toxic to neurons as well as glia at a concentration of 25 microM when added to cultures 24 hr after plating. Toxicity was evident after 48 hr exposure to NE, as monitored by loss of cells from the cultures. Toxicity did not seem to be mediated by adrenergic receptors because, although the beta-adrenergic agonist isoproterenol (but not the alpha-adrenergic agonist phenylephrine) was similar in its toxic effect to NE, the beta-adrenergic antagonist atenolol did not block the toxic effect of NE. Toxicity could be mimicked by hydrogen peroxide, a product of the oxidative degradation of catecholamines. Toxicity of NE was blocked by catalase. The neurotoxin 6-hydroxydopamine (6-OHDA), supposedly selective for catecholaminergic neurons, was found to be toxic over the same concentration range as NE. These results suggest that endogenous catecholamines may play a role in normal and abnormal cell death, and suggest that caution be used in relying on the specificity of 6-OHDA and other supposedly selective neurotoxins.
Adenosine is an important inhibitory neuromodulator in the CNS, yet the sources of extracellular adenosine have yet to be well characterized. In this study we show that beta-adrenergic stimulation of cortical cultures results in the extracellular accumulation of cAMP as well as adenosine, and that the extracellular adenosine derives from extracellular cAMP. The concentration dependence of isoproterenol in evoking cAMP secretion was determined by radioimmunoassay, and the EC50 for this effect was found to be approximately 100 nM. In order to investigate the effect of beta-adrenergic stimulation on the regulation of extracellular adenosine, the effect of isoproterenol in stimulating the extracellular accumulation of adenine-containing compounds was examined by HPLC. Isoproterenol stimulated cAMP secretion in both astrocyte cultures and astrocyte-rich mixed cultures of astrocytes and neurons. However, no extracellular cAMP was detectable in neuron- enriched astrocyte-poor cultures. Extracellular adenosine increased in response to isoproterenol in the astrocyte-rich mixed cultures, but not in the neuron-enriched astrocyte-poor cultures. After 30 min exposure to isoproterenol, the concentration of adenosine in the extracellular medium increased by 47% in 56 experiments in the mixed astrocyte-rich cultures. In order to establish whether the adenosine that accumulates in response to isoproterenol stimulation actually derives from extracellular cAMP, phosphodiesterase inhibitors were tested for their ability to block isoproterenol-stimulated adenosine accumulation. Isobutylmethylxanthine (IBMX; 100 microM), RO 20–1724 (180 microM), carbazeran (10 microM), dipyridamole (10 microM), and trifluoperazine (10 microM) had no inhibitory effect on the isoproterenol-stimulated accumulation of extracellular adenosine. However 100 microM IBMX plus 180 microM RO 20–1724 effectively blocked isoproterenol-stimulated adenosine accumulation and, as expected, increased extracellular cAMP. As a further test of the origin of isoproterenol-stimulated adenosine accumulation, we attempted to block this phenomenon by blocking cAMP secretion itself. For this purpose probenecid, a known inhibitor of cAMP secretion in many different cell types, was used. We found that probenecid at 1 mM blocked isoproterenol-stimulated adenosine accumulation. These studies suggest that one potentially important source of extracellular adenosine in the cerebral cortex is endogenous extracellular cAMP, secreted from astrocytes in response to beta- adrenergic receptor stimulation. Since the receptors of neuromodulators other than norepinephrine may also be coupled to adenylyl cyclase in the cerebral cortex, there may be several neuromodulatory systems that regulate extracellular adenosine levels by this mechanism.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.