Differential Effects of Haloperidol and Clozapine on [H]cAMP Binding, Protein Kinase A (PKA) Activity, and mRNA and Protein Expression of Selective Regulatory and Catalytic Subunit Isoforms of PKA in Rat Brain

The present study was undertaken to examine whether the mechanism of action of typical and atypical antipsychotics is related in their ability to regulate key phosphorylating enzyme of adenylyl cyclase-cAMP pathway, i.e., protein kinase A (PKA). For this purpose, regulatory (R) and catalytic (Cat) activities of PKA and expression of various isoforms of regulatory and catalytic subunits were examined in rat brain after single or chronic (21-day) treatment with haloperidol (HAL, 1 mg/kg) or clozapine (CLOZ, 20 mg/kg). It was observed that chronic but not acute treatment of CLOZ significantly decreased [H]cAMP binding to the regulatory subunit of PKA as well as catalytic activity of PKA in particulate and cytosol fractions of the rat cortex, hippocampus, and striatum. In these fractions, CLOZ significantly decreased protein levels of selective RII -, RII -, and Cat -subunit isoforms of PKA. These decreases were accompanied by decreases in their respective mRNA expression. In contrast, chronic but not acute treatment of HAL significantly increased [H]cAMP binding and the catalytic activity of PKA in particulate and cytosol fractions of only the striatum brain area. In addition, chronic treatment of HAL significantly increased mRNA and protein levels of RII and RII -subunit isoforms in the striatum. None of the antipsychotics caused any change in the expression of the Cat -, RI -, or RI -subunit isoform. These results, thus, suggest that HAL and CLOZ differentially regulate PKA catalytic and regulatory activities and the expression of selective catalytic and regulatory subunit isoforms of PKA, which may be associated with their mechanisms of action. Haloperidol (HAL) and clozapine (CLOZ), the two most commonly used antipsychotic agents, share the common property of blocking dopamine D2 receptors (Deutsch et al., 1991; Dixon et al., 1995). Despite this common feature, the clinical and behavioral profiles of these drugs differ. For example, although effectively blocking psychoses, HAL causes extrapyramidal side effects (EPS), including a Parkinson’s-like syndrome, and Tardive Dyskinesia (TD). On the other hand, CLOZ is associated with low incidence of EPS and TD. Some studies even suggest that CLOZ reduces symptoms of TD (Safferman et al., 1991), which is effective at ameliorating motor dysfunction in patients with idiopathic Parkinson’s disease (Pakkenberg and Pakkenberg, 1986; Arevalo and Gershanik, 1993) and in the treatment-resistant negative symptoms of schizophrenia (Kane et al., 1988). The mechanisms of action of antipsychotics in alleviating the symptoms associated with psychoses and the mechanisms responsible for their differential effects on EPS are not clear. Whereas one hypothesis is that different affinities of these two drugs toward dopamine D2 receptors may be responsible for their different clinical efficacy and incidence of EPS, several other biological factors may also be associated with their actions. This is based upon reports that suggest that CLOZ binds to numerous neurotransmitter receptors besides dopamine D2, including 5HT2A, 5HT2C, 5HT1A, 5HT6, 1and 2-adrenergic, and muscarinic receptors (Bolden et al., 1991; Baldessarini et al., 1992; Kuoppamaki et al., 1994; Millan, 2000; Zhukovskaya and Neumaier, 2000). In search for the mechanisms of action of antipsychotic drugs, several studies have been performed at the postreceptor sites, particularly of their effects on receptor-mediated phosphoinositide hydrolysis and various components of this signaling transduction system such as PKC and phospholipase C (Hokin-Neaverson, 1980; Li et al., 1991; KuoppaThis study was supported by Grant KO1 MH01836 from the National Institute of Mental Health and Young Investigator Award from the American Foundation of Suicide Prevention (to Y.D.) and Grant RO1-MH56528 from the National Institute of Mental Health (to G.N.P.). ABBREVIATIONS: Cat, catalytic; CLOZ, clozapine; HAL, haloperidol; PKA, protein kinase A; PKC, protein kinase C; R, regulatory; ECL, enhanced chemiluminescence; EPS, extrapyramidal side effects; TD, tardive dyskinesia; RT-PCR, reverse transcriptase-polymerase chain reaction; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride; bp, base pair(s). 0022-3565/02/3011-197–209$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 301, No. 1 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 4565/973881 JPET 301:197–209, 2002 Printed in U.S.A. 197 at A PE T Jornals on N ovem er 4, 2017 jpet.asjournals.org D ow nladed from maki et al., 1994; Dwivedi and Pandey, 1999). On the other hand, in adenylyl cyclase-cAMP signaling pathway, most of the studies of the effects of antipsychotic drugs are confined either to receptor-stimulated adenylyl cyclase activity or to the levels of G proteins, such as Gs and Gi, linked to this signaling system via stimulatory and inhibitory fashion, respectively. For example, HAL, which antagonizes dopamine D2 receptor, leads to an increase in cAMP (Kaneko et al., 1992). More recent studies suggest that CLOZ decreases 5HT1A-mediated (Assie et al., 1997) and muscarinic M4-mediated (Zeng et al., 1997) cAMP formation. On the other hand, Kaplan et al. (1999) showed that whereas HAL increases GTP -S-stimulated adenylyl cyclase activity in rat cortex, olanzapine decreases it. But in the striatum, olanzapine produces effects opposite those of the cortex, and HAL has no effects on GTP -S-stimulated adenylyl cyclase activity. Other significant observations are that whereas HAL decreases Gi and Gs in rat striatum, CLOZ increases them (Gupta and Mishra, 1992; Shin et al., 1995). In contrast, Kaplan et al. (1999) reported no change in the striatum but a decrease in Gs levels in the cortex. No change in these two subunits after HAL (See et al., 1993; Meller and Bohmaker, 1996) or olanzapine (Kaplan et al., 1999) treatment has also been reported. In light of these observations, it is quite possible that the mechanism of action of typical and atypical antipsychotic drugs may lie in their ability to differentially regulate adenylyl cyclase-cAMP pathway at the level of functional response. In the adenylyl cyclase-cAMP pathway, this functional response is mediated by phosphorylating enzyme protein kinase A (PKA), which is activated by cAMP generated by the conversion of ATP in response to the activation of adenylyl cyclase by receptor-activated Gs or Gi proteins. PKA then phosphorylates various substrate proteins in cells, thereby mediating a variety of hormonal and physiological responses (Nestler and Greengard, 1994). In a native state, PKA exists as a tetramer holoenzyme that consists of two regulatory and two catalytic subunits. In the holoenzyme state, PKA exists in an inactive form. After an increase in intracellular cAMP, the regulatory subunits bind to cAMP, which results in the dissociation of the holoenzyme into a regulatory dimer and two monomers of catalytic subunits. The free catalytic subunits can then phosphorylate various substrates. Thus, both catalytic and regulatory subunits are important in facilitating PKA-mediated functions (Skálhegg and Taskén, 1997) In the present study, we tested whether different clinical and behavioral profiles of HAL and CLOZ are associated with their ability to differentially regulate adenylyl cyclase pathway at the level of PKA. For this purpose, we examined [H]cAMP binding, catalytic activity, and expression of various regulatory and catalytic subunits of PKA after acute and chronic treatment of HAL and CLOZ to rats. Experimental Procedures