Dexmedetomidine-Induced ERK Phosphorylation In Brain Tissue By Transactivation Of EGF Receptors

IntroductionDexmedetomidine is a potent and highly specific α₂ - adrenergic agonist, which in receptor binding experiments has an α₂ /α₁ selectivity ratio of 1600 or several times higher than clonidine. Besides hypnotic/sedative and analgesic effects low concentrations of dexmedetomidine have, with one exception, consistently been found to have neuroprotective properties in experimental cerebral ischemia and excitotoxic neuronal injury. It potently activates each of the three subtypes of the α₂ — adrenergic receptor (theα_2A/D, the α_{2 B}, and the α_2C receptor) , which all are coupled to pertussis toxin - sensitive Gi/o - coupled receptors.Based upon inhibition of the phosphorylation by tyrphostin AG 1478, an inhibitor of receptor tyrosine - kinases ( RTKs) , and by heparin, an antagonist of heparin - binding epithelial growth factor ( HB - EGF) we suggested that this effect is a result of transactivation of the epithelial growth factor receptor ( EG-FR) . The demonstration of dexmedetomidine - induced EGFR transactivation in cultured astrocytes raises two questions: (i) do astrocytes in the brain behave similarly;and (ii) do neurons respond in the same manner?Materials and methodsMale adult CD - 1 mice were injected intraperitoneally with dexmedetomidine. After 7, 15 min they were decapitated. The tissues were homogenized in ice - cold lysis buffer. Brain slices pre - incubated for stabilization for 2 hrs. One of the inhibitors tyrphostin AG 1478 , GM 6001, atipamezole or heparin wasadded to the slices, and the incubation was continued for another 15 min. Subsequently dexmedetomidine to a final concentration of 50 nM was added to the slices in both the group treated with an inhibitor and the group to which no inhibitor had been added, and the slices were incubated for another 20 min. The slices were homogenized in ice - cold lysis buffer. The primary cerebellar granule cells were incubated by a 20 min incubation in similar medium in the presence or absence of 50 nM dexmedetomidine. The cells were homogenized in ice - cold lysis buffer. Then sigals of ERK1/2 phosphorylation of all the samples were messured by Western blotting.Results1. In vivo -7 min after intraperitoneal administration of 3 |xg/kg dexmedetomidine, but the effect was not statistically significant(P >0. 05) until 15 min after drug administration ( P < 0. 05 ) .2. Brain slicesThe effect, if any, by 25 nM dexmedetomidine is marginal, whereas both 50 nM and 100 nM dexmedetomidine caused a significant increase in ERK phoshorylation ( P < 0. 05 ). This increase was inhibited by treatment with 10 |xM of the metalloproteinase inhibitor GM 6001 ( P <0. 05 ) . TRK inhibitor tyr-phostin AG 1478 also inhibited ERK1/2 phosphorylation by 50 nM dexmedetomidine (P<0. 05). Inhibition of ERK} phosphorylation by heparin (1 mg/ml) , which inhibits specifically HB - EGF was statistically significant (P <0. 05);atipamezole, a specific inhibitor of the a2 — adrenergic receptor abolished the stimulation of ERK1/2 phosphorylation by dexmedetomidine ( P < 0. 05 ). This finding shows that dexmedetomidine owes its action exclusively to the effect on a2 — adrenergic receptors, not to stimulation of any of the other receptors, where supranormal concentrations of dexmedetomidine may act.3. Cerebellar granule neuronsIn contrast to the observations in brain slices there was no significant stimulation of ERK1/2 phosphorylation in primary cultures of cerebellar granule cells ( P>0.05) .DiscussionThe pronounced inhibition of dexmedetomidine - stimulated ERK phospho-rylation by GM 6001, tyrphostin AG 1478, and heparin indicates that the phos-phorylation of ERK in cultured astrocytes and in brain slices mainly occurred as a result of HB - EGF * shedding' and EGFR activation. The present observations seem to represent the first demonstration of transactivation of EGFR in non — cultured brain tissue.The results with heparin indicates that one of the released EGFR agonists is HB - EGF, but they do not allow the conclusison that HB - EGF is the only agonist released. EGFRs are activated by at least six different agonists, epithelium growth factor ( EGF) , HB - EGF, transforming growth factor - a (TGF - a) , amphiregulin, betacellulin, and epiregulin. HB - EGF is expressed at high level in brain in vivo at early developmental stages, and declines thereafter. However , some HB - EGF remains in the cerebellum, and more recently it has also been demonstrated in other regions, including cerebral cortex. TGF — a is found both in neurons and astrocytes of the adult brain, and amphiregulin has also been demonstrated in brain. EGF expression in postnatal brain is rare, and betacellulin and epiregulin have not been found to be expressed in the central nervous system tissue so far.The absence of dexmedetomidine - promoted ERK phosphorylation in cultured neurons suggests that a2 - adrenergic stimulation does not lead to EGFR transactivation in neurons. This probably indicates an absence of neuronal posts-ynaptic a2 - adrenergic receptors, as reflected by the observation that cultured cortical intemeurons show no increase in free cytosolic Ca2+ ([Ca2 + ];in response to dexmedetomidine. In contrast [ Ca + ];is increased in cultured or freshly dissociated astrocytes by the 2 a - adrenergic agonists clonidine and dexmedetomidine, suggesting that the dexmedetomidine effect on ERK phosphorylation in brain slices represents ERK phoshorylation in astrocytes.ConclusionIn the brain in vivo, shedded EGFR - ligands in response to a2 - adrener-gic stimulation of astrocytes are likely to exert a paracrine stimulation not only of surrounding astrocytes but also of adjacent neurons, expressing EGFR. Since stimulation of EGF receptors in the CNS by EGF, HB - EGF or transforming growth factor - a ( TGF - a ) provides neuroprotection during ischemia, such a activation may be causally related to the neuroprotective effect of dexmedetomi-dine.