Adam Bielawski

α1-Adrenergic receptor subtypes in the central nervous system: insights from genetically engineered mouse models

α1-Adrenergic receptors (α1-ARs) are important players in peripheral and central nervous system (CNS) regulation and function and in mediating various behavioral responses. The α1-AR family consists of three subtypes, α1A, α1B and α1D, which differ in their subcellular distribution, efficacy in evoking intracellular signals and transcriptional profiles. All three α1-AR subtypes are present at relatively high densities throughout the CNS, but the contributions of the individual subtypes to various central functions are currently unclear. Because of the lack of specific ligands, functionally characterizing the α1-ARs and discriminating between the three subtypes are difficult. To date, studies using genetically engineered mice have provided some information on subtype-related functions of the CNS α1-ARs. In this mini-review, we discuss several CNS processes where the α1-ARs role has been delineated with pharmacological tools and by studies using mutated mice strains that infer specific α1-AR subtype functions through evaluation of behavioral phenotypes.

Effects of the noradrenergic neurotoxin DSP-4 on the expression of α1-adrenoceptor subtypes after antidepressant treatment

We have previously reported that chronic imipramine and electroconvulsive treatments increase the α(1A)-adrenoceptor (but not the α(1B) subtype) mRNA level and the receptor density in the rat cerebral cortex. Furthermore, we have also shown that chronic treatment with citalopram does not affect the expression of either the α(1A)- or the α(1B)-adrenoceptor, indicating that the previously observed up-regulation of α(1A)-adrenoceptor may depend on the noradrenergic component of the pharmacological mechanism of action of these antidepressants. Here, we report that previous noradrenergic depletion with DSP-4 (50 mg/kg) (a neurotoxin selective for the noradrenergic nerve terminals) significantly attenuated the increase of α(1A)-adrenoceptor mRNA induced by a 14-day treatment with imipramine (IMI, 20 mg/kg, ip) and abolished the effect of electroconvulsive shock (ECS, 150 mA, 0.5 s) in the prefrontal cortex of the rat brain. The changes in the receptor protein expression (as reflected by its density) that were induced by IMI and ECS treatments were differently modulated by DSP-4 lesioning, and only the ECS-induced increase in α(1A)-adrenoceptor level was abolished. This study provides further evidence corroborating our initial hypothesis that the noradrenergic component of the action of antidepressant agents plays an essential role in the modulation of α(1A)-adrenoceptor in the rat cerebral cortex.

Effects of morphine and methadone treatment on mRNA expression of Gα(i) subunits in rat brains.

Methadone is clinically effective as substitution therapy in patients with opioid dependence. The diversity of methadone and morphine in their intracellular activity is postulated. We compared the effects of repeated daily treatment of Sprague-Dawley rats with morphine (10 mg/kg) and methadone (1 mg/kg) on the expression of the Gα(i1-i3) mRNAs in several rat brain areas using RT-qPCR. We found that both opioid receptor agonists decreased Gα(i3) mRNA in only the nucleus accumbens. Although there was no difference in the influence of morphine and methadone on Gα(i), our results indicate that among Gα(i) subunits, the Gα(i3) is specifically involved in the mechanism of action of both drugs.

Cryptic peptide derived from the rat neuropeptide FF precursor affects G-proteins linked to opioid receptors in the rat brain.

Recently, we reported the discovery of a novel amino acid sequence derived from the NPFF precursor NAWGPWSKEQLSPQA, which blocked the expression of conditioned place preference induced by morphine and reversed the antinociceptive activity of morphine (5mg/kg, s.c.) in the tail-immersion test in rats. Here, we name it as NPNA (Neuropeptide NA from its flanking amino acid residues). The synthetic peptide influenced the expression of mRNA coding for Galpha(i1), (i2), and (i3) subunits. The results provide further evidence that yet another bioactive sequence might be present within the NPFF precursor.

Imipramine administration induces changes in the phosphorylation of FAK and PYK2 and modulates signaling pathways related to their activity.

BACKGROUND Antidepressants can modify neuronal functioning by affecting many levels of signal transduction pathways that are involved in neuroplasticity. We investigated whether the phosphorylation status of focal adhesion kinase (FAK/PTK2) and its homolog, PYK2/PTK2B, and their complex with the downstream effectors (Src kinase, p130Cas, and paxillin) are affected by administration of the antidepressant drug, imipramine. The treatment influence on the levels of ERK1/2 kinases and their phosphorylated forms (pERK1/2) or the Gαq, Gα11 and Gα12 proteins were also assessed. METHODS Rats were injected with imipramine (10 mg/kg, twice daily) for 21 days. The levels of proteins investigated in their prefrontal cortices were measured by Western blotting. RESULTS Imipramine induced contrasting changes in the phosphorylation of FAK and PYK2 at Tyr397 and Tyr402, respectively. The decreased FAK phosphorylation and increased PYK2 phosphorylation were reflected by changes in the levels of their complex with Src and p130Cas, which was observed predominantly after chronic imipramine treatment. Similarly only chronic imipramine decreased the Gαq expression while Gα11 and Gα12 proteins were untouched. Acute and chronic treatment with imipramine elevated ERK1 and ERK2 total protein levels, whereas only the pERK1 was significantly affected by the drug. CONCLUSION The enhanced activation of PYK2 observed here could function as compensation for FAK inhibition. GENERAL SIGNIFICANCE These data demonstrate that treatment with imipramine, which is a routine in counteracting depressive disorders, enhances the phosphorylation of PYK2, a non-receptor kinase instrumental in promoting synaptic plasticity. This effect documents as yet not considered target in the mechanism of imipramine action.

Morphine-induced place preference affects mRNA expression of G protein α subunits in rat brain

BACKGROUND The conditioned place preference (CPP) test is an animal model serving to assess addictive potential of drugs in which environmental cues become associated with the subjective effects of drugs of abuse. Morphine, a known addictive drug, is an agonist of opioid receptors that couple to the G(i/o) family of guanine nucleotide-binding proteins (GP). We have recently found that chronic treatment with morphine affects mRNA levels of GPs that are not coupled to opioid receptors (OR). Therefore, in this study, we investigated the influence of morphine-induced CPP on mRNA expression of the Gα subunits, G(i/o), G(s), G(q/11), and G(12), in the rat prefrontal cortex (PFC) and nucleus accumbens (NAc) using standard PCR techniques. METHODS CPP and NO-CPP experiments were conducted; Wistar rats were either subjected to the standard CPP procedure or were injected with morphine (or saline) in their home cage. All rats were decapitated 24 h after the last injection. RESULTS We found that mRNA levels of Gα(q), Gα(11) and Gα(12) were increased after morphine in non-conditioned treatment in the PFC but remained unchanged in the NAc. In rats showing conditioned place preference to morphine, levels of Gα(i2) in the PFC and levels of Gα(oA) in the NAc were diminished by ≈58% and ≈30%, respectively (p < 0.05 vs. saline), but levels of Gα(s-l) in NAc were increased (≈60%, p = 0.05). CONCLUSION Our data indicate that only G(i/o) and G(s) were specifically changed in animals after morphine-induced CPP, thus suggesting that the effect was related to learning environmental cues associated with morphine.