Ethanol-mediated inhibition of mitogen-activated protein kinase phosphorylation in mouse brain

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Abstract

Ethanol (1.5–3.5 g/kg body weight) was administered intraperitoneally to mice and the phosphorylation of MAP (mitogen-activated protein) kinase in the cerebral cortex was determined using phospho-specific MAP kinase antibodies. Ethanol inhibited the phosphorylation of MAP kinase in a dose- and time-dependent manner. Developmental studies demonstrated that the levels of phospho-MAP kinase increased from fetal cortex (prenatal) to 16-day-old mice (postnatal) and remained constant up to 4 months of age. However, ethanol (3.5 g/kg) decreased the phospho-MAP kinase staining in all of the age groups studied. Subcellular fractionation studies demonstrated that ethanol inhibited the phosphorylation of MAP kinase in both the cytosol as well as nucleus, but did not alter the levels of MAP kinase. Likewise, MK-801 (0.4 mg/kg) or flurazepam (75 mg/kg) also decreased the phospho-MAP kinase content. These data indicate that ethanol may inhibit the phosphorylation of MAP kinase in vivo by either inhibiting NMDA receptors or activating GABA receptors.

Introduction

Ethanol intoxication is associated with deleterious effects on the brain that include the inhibition of the NMDA receptors (Lovinger et al., 1989), voltage-dependent Ca2+ channels (Skattebol and Rabin, 1987) and potentiation of GABA receptor function (Mehta and Ticku, 1999). A variety of studies have demonstrated the involvement of NMDA receptors in the generation of long-term potentiation. Loss of memory and learning abilities are the result of ethanol's action on the central nervous system. Understanding the biochemical mechanisms that underlie alcohol's ability to inhibit long-term potentiation may lead to new treatments for alcoholism. Ca2+ influx through either voltage sensitive Ca2+ channels (Rosen et al., 1994) or glutamate receptor (Sala et al., 2000) leads to the activation of Ras and MAP (mitogen-activated protein) kinase signaling cascade. MAP kinase(s) play an important role in transducing the extracellular signals into the nucleus following stimulation by neurotransmitters and/or growth factors. Several studies have provided evidence for a role of MAP kinases in the cell growth (Mii et al., 1996), differentiation (Sale et al., 1995) and long-term memory (Kornhauser and Greenberg, 1997). A typical MAP kinase signaling pathway consists of a MAP kinase, a MAP kinase kinase and an activator of MAP kinase kinase (MAP kinase kinase kinase) that transmit signals by sequential phosphorylation. An ethanol-induced inhibition of MAP kinase activity has been reported earlier (Hendrickson et al., 1998). Another study, however, demonstrated the potentiation of MAP kinase by ethanol (Chen et al., 1998a). We have previously shown that acute ethanol treatment can inhibit the phosphorylation of MAP kinase in cultured cortical neurons (Kalluri and Ticku, 2000). In the present study, we demonstrate the inhibition of MAP kinase phosphorylation by ethanol in vivo. We used MAP kinase/ERK1/2 (extracellular signal regulated kinase) interchangeably.

Section snippets

Animals and tissue preparation

C57BL/67CR mice were purchased from Harlan (Indianapolis, IN) and housed in a room maintained at a constant temperature of 22±1°C with free access to food and water and kept on a 12-h light/12-h dark cycle. The protocol used to administer ethanol was described earlier (Miyakawa et al., 1997). Briefly, absolute ethanol was diluted in saline (25% w/v) and injected (3.5 g/kg body weight) intraperitoneally into mice (age 3–4 months) unless otherwise stated. Likewise, MK-801 (0.4 mg/kg) and

Results

To determine the phosphorylation of MAP kinase (ERK 1/2), we used phospho-specific antibodies and normalized the data by probing with nonphosphorylated ERK 1/2. The results are summarized as the ratio of phospho-specific ERK to total ERK. In preliminary experiments, varying doses of ethanol were used to study the dose response relationship of ethanol on phospho-MAP staining. Intraperitoneal injection of ethanol (1.5, 2.5, 3.5 g/kg body weight) decreased the phosphorylation of MAP kinase (Fig. 1)

Discussion

In this study, we demonstrate that ethanol inhibits the phosphorylation of MAP kinase in mouse cerebral cortex. Our data show that ethanol at various concentrations (1.5, 2.5, 3.5 g/kg body weight) decreased the phosphorylation of MAP kinase in vivo. The ethanol-mediated inhibition of MAP kinase phosphorylation has been previously shown in cultured cortical neurons (Kalluri and Ticku, 2000). Several other studies have shown that ethanol can decrease Davis et al., 1999, Arabi et al., 1999 or

Acknowledgements

This research was supported by the National Institutes of Health—National Institute on Alcohol Abuse and Alcoholism Grant AA 12297 and AA 10552.

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