Ya Li

Intracellular Redox State Determines Whether Nitric Oxide Is Toxic or Protective to Rat Oligodendrocytes in Culture

Abstract : We found that several nitric oxide donors had similar potency in killing mature and immature forms of oligodendrocytes (OLs). Because of the possibility of interaction of nitric oxide with intracellular thiols, we tested the effect of the nitrosonium ion donor S‐nitrosylglutathione (SNOG) in OL cultures in the setting of cystine deprivation, which has been shown to cause intracellular glutathione depletion. Surprisingly, the presence of 200 μM SNOG completely protected OLs against the toxicity of cystine depletion. This protection appeared to be due to nitric oxide, because it could be blocked by hemoglobin and potentiated by inclusion of superoxide dismutase. We tested the effect of three additional NO• donors and found that protection was not seen with diethylamine NONOate, a donor with a half‐life measured in minutes, but was seen with dipropylenetriamine NONOate and diethylaminetriamine NONOate, donors with half‐lives measured in hours. This need for donors with longer half‐lives for the protective effect suggested that NO• was required when intracellular thiol concentrations were falling, a process evolving over hours in medium depleted of cystine. These studies suggest a novel protective role for nitric oxide in oxidative stress injury and raise the possibility that intracerebral nitric oxide production might be a mechanism of defense against oxidative stress injury in OLs.

Adenylyl cyclase activation underlies intracellular cyclic AMP accumulation, cyclic AMP transport, and extracellular adenosine accumulation evoked by β-adrenergic receptor stimulation in mixed cultures of neurons and astrocytes derived from rat cerebral cortex

We have previously shown that stimulation of cortical cultures containing both neurons and astrocytes with the beta-adrenergic agonist isoproterenol (ISO) results in transport of cAMP from astrocytes followed by extracellular hydrolysis to adenosine [Rosenberg et al. J. Neurosci. 14 (1994) 2953-2965]. In this study we found that the endogenous catecholamines epinephrine (EPI) and norepinephrine (NE), but not dopamine, serotonin, or histamine, all at 10 microM, significantly stimulated intracellular cAMP accumulation, cAMP transport, and extracellular adenosine accumulation in cortical cultures. Detailed dose-response experiments were performed for NE and EPI, as well as ISO. For each catecholamine, the potencies in evoking intracellular cAMP accumulation, cAMP transport, and extracellular adenosine accumulation were similar. These data provide additional evidence that a single common mechanism, namely beta-adrenergic mediated activation of adenylyl cyclase, underlies intracellular cAMP accumulation, cAMP transport, and extracellular adenosine accumulation. It appears that regulation of extracellular adenosine levels via cAMP transport and extracellular hydrolysis to adenosine may be a final common pathway of neuromodulation in cerebral cortex for catecholamines, and, indeed, any substance whose receptors are coupled to adenylyl cyclase.

Forskolin evokes extracellular adenosine accumulation in rat cortical cultures

In this study, the effect on extracellular adenosine concentration of direct activation of adenylyl cyclase by forskolin was investigated using rat cortical cultures. Forskolin evoked intracellular and extracellular cAMP accumulation as well as extracellular adenosine accumulation. The accumulation of adenosine in response to forskolin could be blocked by the cyclic nucleotide phosphodiesterase inhibitor 4-[(3-butoxy-4-methoxyphenyl)methyl]-2-imidazolidinone (RO 20-1724; 180 microM), but not isobutylmethylxanthine (100 microM). The accumulation of adenosine in response to forskolin could be blocked by the 5-ectonucleotidase inhibitor guanosine 5-monophosphate. These results demonstrate that forskolin can increase extracellular adenosine levels, and that this adenosine is ultimately derived from cAMP.

Vasoactive intestinal peptide regulates extracellular adenosine levels in rat cortical cultures.

Adenosine is an important inhibitory neuromodulator in the cerebral cortex, yet it remains unclear how extracellular adenosine concentrations are regulated. Recently, it has been shown that beta-adrenergic receptor stimulation in rat cortical cultures causes the accumulation of extracellular adenosine derived by enzymatic hydrolysis from adenosine cyclic monophosphate (cAMP) transported into the extracellular space. In this study we show that vasoactive intestinal peptide (VIP), in addition to activating adenylyl cyclase and promoting the accumulation of intracellular cAMP in rat cortical cultures, also causes transport of cAMP and accumulation of extracellular adenosine. We further show that the extracellular accumulation of adenosine in response to VIP can be blocked by inhibition of cAMP transport, cyclic nucleotide phosphodiesterase activity, and 5-nucleotidase, indicating that extracellular cAMP is the source of the adenosine. Cyclic AMP transport may be a general mechanism by which a variety of neuromodulators that act upon receptors coupled to adenylyl cyclase might regulate extracellular adenosine levels and thereby inhibitory tone in the cerebral cortex.

NMDA receptor-mediated extracellular adenosine accumulation in rat forebrain neurons in culture is associated with inhibition of adenosine kinase

The effect of N‐methyl‐d‐aspartate (NMDA) on regulation of extracellular adenosine was investigated in rat forebrain neurons in culture. NMDA evoked accumulation of extracellular adenosine with an EC50 value of 4.8u2003±u20031.2u2003µm. The effect of NMDA was blocked by (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo [a, d] cyclohepten‐5,10‐imine hydrogen maleate indicating that NMDA receptor activation was involved. The NMDA effect was also blocked by chelation of extracellular Ca2+ indicating that influx of calcium was required. The nitric oxide–cyclic GMP signalling pathway was not involved, as nitric oxide synthase inhibitors were unable to block, and cGMP analogs were unable to mimic, the effect of NMDA. The source for extracellular adenosine was likely to be intracellular adenosine as the ecto‐5′‐nucleotidase inhibitor αβ‐methylene‐ADP was unable to block the effect of NMDA. One possible cause of intracellular adenosine accumulation might be NMDA receptor‐mediated inhibition of mitochondrial function and ATP hydrolysis. We found that NMDA caused a concentration dependent depletion of intracellular ATP with an EC50 value of 21u2003±u20038u2003µm. NMDA also caused a significant decrease in adenosine kinase activity, assayed by two different methods. Consistent with the hypothesis that inhibition of adenosine kinase is sufficient to cause an increase in extracellular adenosine, inhibition of adenosine kinase by 5′‐iodotubercidin resulted in elevation of extracellular adenosine. However, in the presence of a concentration of 5′‐iodotubercidin that inhibited over 90% of adenosine kinase activity, exposure to NMDA still caused adenosine accumulation. These studies suggest that several possible mechanisms are likely to be involved in NMDA‐evoked extracellular adenosine accumulation.

Elevation of intracellular cAMP evokes activity-dependent release of adenosine in cultured rat forebrain neurons

Adenosine is an important regulator of neuronal excitability. Zaprinast is a cyclic nucleotide phosphodiesterase inhibitor, and has been shown in the hippocampal slice to suppress excitation. This action can be blocked by an adenosine receptor antagonist, and therefore is presumably due to adenosine release stimulated by exposure to zaprinast. To explore the mechanism of this phenomenon further, we examined the effect of zaprinast on adenosine release itself in cultured rat forebrain neurons. Zaprinast significantly stimulated extracellular adenosine accumulation. The effect of zaprinast on adenosine appeared to be mediated by increasing intracellular cyclic adenosine monophosphate (cAMP) and activation of protein kinase A (PKA): (i) zaprinast stimulated intracellular cAMP accumulation; (ii) a cAMP antagonist (Rp‐8‐Br‐cAMP) significantly reduced the zaprinast effect on adenosine; (iii) an inhibitor of phosphodiesterase (PDE)1 (vinpocetine) and an activator of adenylate cyclase (forskolin) mimicked the effect of zaprinast on adenosine. We also found that zaprinast had no effect on adenosine in astrocyte cultures, and tetrodotoxin completely blocked zaprinast‐evoked adenosine accumulation in neuronal cultures, suggesting that neuronal activity was likely to be involved. Consistent with a dependence on neuronal activity, NMDA receptor antagonists (MK‐801 and D‐APV) and removal of extracellular glutamate by glutamate–pyruvate transaminase blocked the effect of zaprinast. In addition, zaprinast was shown to stimulate glutamate release. Thus, our data suggest that zaprinast‐evoked adenosine accumulation is likely to be mediated by stimulation of glutamate release by a cAMP‐ and PKA‐dependent mechanism, most likely by inhibition of PDE1 in neurons. Furthermore, regulation of cAMP, either by inhibiting cAMP–PDE activity or by stimulating adenylate cyclase activity, may play an important role in modulating neuronal excitability. These data suggest the existence of a homeostatic negative feedback loop in which increases in neuronal activity are damped by release of adenosine following activation of glutamate receptors.

Zaprinast stimulates extracellular adenosine accumulation in rat pontine slices

Adenosine appears to be an endogenous somnogen. The lateral dorsal tegmental/pedunculopontine nucleus (LDT/PPT) located in the mesopontine tegmentum is important in the regulation of arousal. Neurons in this nucleus are strongly hyperpolarized by adenosine and express neuronal nitric oxide synthase. Zaprinast is a cyclic nucleotide phosphodiesterase inhibitor, and has been shown in the hippocampal slice to inhibit the field excitatory postsynaptic potential. This action could be blocked by an adenosine receptor antagonist, and therefore is presumably due to adenosine release stimulated by zaprinast. In the present study we tested the effect of zaprinast on extracellular adenosine accumulation in pontine slices containing the LDT. Zaprinast at 10 microM evoked an increase in extracellular adenosine concentration. This effect was blocked by impermeant inhibitors of 5-nucleotidase, indicating that the extracellular adenosine was derived from extracellular AMP. However, inhibitors of cAMP degradation had little or no effect on zaprinast-evoked adenosine accumulation, suggesting that extracellular cAMP was not the source. Removal of extracellular calcium inhibited the effect of zaprinast. These results demonstrate that a pathway exists by which zaprinast stimulates extracellular adenosine accumulation, and the presence of this pathway in the pontine slice suggests the possibility that it may be relevant for the regulation of behavioral state.