cAMP production mediated through P2Y₁₁-like receptors in rat striatum due to severe, but not moderate, carbon monoxide poisoning

Abstract

We examined the effect of carbon monoxide (CO) poisoning on the production of cAMP, an intracellular second messenger, in rat striatum in terms of extracellular cAMP, which is highly correlated with intracellular cAMP, by using microdialysis. Severe poisoning due to 3000 ppm CO, but not moderate poisoning due to 1000 ppm CO, caused an increase in cAMP, which was susceptible to a voltage-dependent Na⁺ channel blocker, tetrodotoxin, and more profound than that under comparable hypoxia caused by 5% O₂. These results were similar to our previous findings on the production of hydroxyl radical (^•OH), suggesting a close relationship between cAMP and ^•OH production. The increase in cAMP was suppressed by a non-selective purine P2 receptor antagonist, suramin. However, other non-selective P2 receptor antagonists, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid and reactive blue 2, exhibited no effect and weak non-significant suppression, respectively. A P2Y₁₁ receptor antagonist, NF157, dose-dependently suppressed the increase in cAMP, although rats lack the P2Y₁₁ receptor. These results suggest that a threshold for cAMP production mediated through P2Y₁₁-like receptors following depolarization triggered by Na⁺ influx exists in rat striatum during CO poisoning, and that the threshold is reached only in cases of severe CO poisoning. It is also likely that the threshold is related to the generation of ^•OH, contributing to the toxicity of CO in the brain.

Introduction

Carbon monoxide (CO) is well known as a toxic gas, which binds to hemoglobin more strongly than does O₂, forming carboxyhemoglobin (COHb) and interfering with the supply of O₂ to tissues. Therefore, the brain, which strongly demands O₂ to function, is vulnerable to CO poisoning. Injuries in the brain are salient in the cerebral cortex, globus pallidus, hippocampus, and caudate putamen, accompanying neuropsychological disorders, such as parkinsonism and amnesia, in humans (Choi and Cheon, 1999, Gale et al., 1999, Ginsberg, 1980, O’Donnell et al., 2000) and experimental animals (Ishimaru et al., 1992, Nabeshima et al., 1991). We found that CO poisoning stimulated generation of hydroxyl radical (^•OH), the most toxic of the reactive oxygen species (ROS), in rat striatum (Hara et al., 2004), which could play a role in brain injury due to CO poisoning as well as brain ischemia and trauma (Gilgun-Sherki et al., 2002, Leker and Shohami, 2002, Lewén et al., 2000). Interestingly, ^•OH production was evident in rats with over 70% COHb due to 3000 ppm CO, but not those with approximately 50% COHb due to 1000 ppm CO, suggesting that severe, though not moderate, CO poisoning stimulates the generation of ^•OH (Hara et al., 2011). Therefore, a threshold is likely to exist for CO-induced ^•OH production.

An increase in an intracellular second messenger, cAMP, in the brain is a well-recognized event in response to brain ischemia and hypoxia/anoxia (Palmer, 1985). The increase could be attributed to activation of adenylate cyclase mediated through a stimulatory guanine nucleotide-binding protein (Gs) coupled with receptors of various neurotransmitters and neuromodulators, including dopamine, noradrenaline, serotonin (5-HT), histamine, adenosine and prostaglandins (Tanaka, 2001), which are increased in the extracellular fluid in response to brain insults (Adachi, 2005, Cheng et al., 2000, Hagberg et al., 1987, Stevens and Yaksh, 1988, Thaminy et al., 1997, Yang et al., 2007). Prado et al. (1992) reported that the cAMP increase in rat striatum due to brain ischemia was attenuated by SCH23390, an antagonist of the DA receptors coupled to Gs (Missale et al., 1998). The antagonist also attenuated striatal damage following brain ischemia in newborn piglets (Yang et al., 2007). Therefore, activation of the cAMP signaling pathways mediated through the dopamine system might at least in part play a deleterious role in the brain damage (Prado et al., 1992, Yang et al., 2007). In addition, Tsukada et al. (2004) suggested that the cAMP signaling pathways mediated through 5-HT_1A receptors as well as the dopamine receptors are involved in ischemic brain damage in monkeys. On the other hand, studies have proposed that activation of the cAMP pathway protects against ischemic brain damage (Tanaka, 2001). This is supported by the protective effect of rolipram and cilostazol, which increase intracellular cAMP levels by inhibiting phosphodiesterases (Block et al., 1997, Choi et al., 2002). Furthermore, pretreatment with a cell-permeable cAMP analogue, dibutyryl cAMP (db-cAMP) (Qiu et al., 2002), as well as phosphodiesterase inhibitors (Nikulina et al., 2004), promotes axonal regeneration in animal models of spinal cord lesions. However, direct administration of db-cAMP to the brain elicits seizures (Kuriyama and Kakita, 1980), which could lead to neural injury (Berg et al., 1993). In fact, Amadio et al. (2005) reported cell death induced by forskolin (a direct activator of adenylate cyclase), 3-isobutyl-1-methyl-xanthine (a phosphodiesterase inhibitor) and (4-chloro phenylthio)-cAMP (a cAMP analogue) in cultured cerebellar granule neurons. In these neurons, stimulation of purine receptors by an ATP analogue, ATPγS, enhanced both cAMP and ROS production (Amadio et al., 2005). In addition, direct administration of ATP to rat striatum was injurious (Ryu et al., 2002). Thus, it is likely that the cAMP signaling system has paradoxical functions in the brain.

It is known that intracellular production of cAMP is highly correlated with the efflux of cAMP to the extracellular space (Egawa et al., 1988, Rosenberg and Li, 1995), which can be monitored in the brain in vivo by microdialysis (Egawa et al., 1988, Hashimoto and Kuriyama, 1997, Klamer et al., 2005, Wade et al., 2004). In the present study, we found that the extracellular cAMP level was increased in the striatum of rats exposed to CO at 3000 ppm, but not 1000 ppm, as in the case of ^•OH production. It is likely that cAMP is a factor associated with the threshold of CO-induced ^•OH generation. Therefore, we explored the mechanism of increase in cAMP.