Gabriel Schreiber

Hyperfunctional G proteins in mononuclear leukocytes of patients with mania

In a recent study, we found that lithium inhibits the function of guanine nucleotide-binding proteins, implicating G proteins as the common site for both the antimanic and antidepressant therapeutic effects of lithium. These findings may also suggest that an altered G protein function is of pathophysiological importance in bipolar affective disorder. In the present study, the coupling of both muscarinic-cholinergic receptors and beta-adrenergic receptors to pertussis toxin-sensitive G proteins or cholera toxin-sensitive G proteins was compared among untreated manic patients, lithium-treated euthymic bipolar patients, and healthy volunteers using mononuclear leukocyte (MNL) membrane preparations. Hyperactive function of G proteins was detected in untreated manic patients. Both isoproterenol-induced and carbamylcholine-induced increases in Gpp(NH)p binding capacity were twofold to threefold higher than the increases observed in healthy volunteers. On the other hand, lithium-treated euthymic bipolar patients showed G protein responses to agonist activation that were no different from the healthy volunteers. Altered G protein function may be of pathophysiological importance in bipolar affective disorder.

Magnesium reversal of lithium inhibition of β-adrenergic and muscarinic receptor coupling to G proteins

Recently, lithium was found to inhibit the coupling of both muscarinic cholinergic and beta-adrenergic receptors to pertussis toxin-sensitive and cholera toxin-sensitive G proteins respectively. These findings suggest that G proteins are the common site for both the antimanic and antidepressant therapeutic effects of lithium. Magnesium ions are crucial to the function of G proteins and interact with them at multiple sites. In the present study using rat cerebral cortex, we determined that magnesium can reverse the ability of lithium to inhibit isoprenaline- and carbamylcholine-induced increases in guanosine triphosphate (GTP) binding to G proteins. Lithium concentrations effective in attenuating G protein function were found to be hyperbolically dependent on free Mg2+ concentrations, suggesting multiple sites of competition between lithium and magnesium on G proteins. Free intracellular Mg2+ concentrations in rat cerebral cortex in vivo are known to be less than 1 mM. At such Mg2+ concentrations, therapeutically efficacious lithium concentrations (1 to 1.5 mM) were still able to alter G protein function, which supports the physiological and clinical relevance of lithium action on G proteins.

Interaction of the Antiarrhythmic Drug Amiodarone with the Muscarinic Receptor in Rat Heart and Brain

The possible interaction between the muscarinic receptor and the antiarrythmic drug amiodarone was studied physiologically in the guinea pig ileum, as well as by competition binding experiments in rat brain and cardiac tissues, using the highly specific tritiated muscarinic antagonist N-methyl-4-piperidyl benzilate. In these studies, amiodarone was found to affect both antagonist and agonist binding to the muscarinic receptor. The drugs inhibitory effect on the binding of antagonist to cerebral cortex muscarinic receptors was consistent with mutually exclusive binding of the compounds [KI = (1.0 +/- 0.2)10(-5) M]. On the other hand, in the brain stem and in cardiac tissues (atrium and ventricle) the inhibitory effect on the binding of muscarinic antagonist could not be fitted to a simple model of competitive inhibition. The possible mode of interaction is discussed. Compared with its activity in the cerebral cortex, amiodarone was a more potent inhibitor of muscarinic antagonist binding in the brain stem and in the atrium and ventricle of the heart [apparent KI values were (6.5 +/- 0.1)10(-6), (4.0 +/- 0.1)10(-6), and (4.0 +/- 0.1)10(-6) M, respectively]. In view of the KI values and the serum concentration of amiodarone observed therapeutically (10(-6) M), the effect of amiodarone on the muscarinic system may have clinical relevance. In both the brain stem and the cardiac preparations, amiodarone converted sites that bind agonist with high affinity into low-affinity sites. Agonist binding in the cerebral cortex was not affected.

Lithium-selective alteration of the function of brain versus cardiac Gs protein.

Lithium was recently demonstrated to inhibit the coupling of both muscarinic cholinergic receptors and beta-adrenergic receptors to pertussis toxin-sensitive and cholera toxin-sensitive G proteins, respectively, thus suggesting alteration of the function of G protein by lithium, as the single site for both the antimanic and antidepressant effects of this drug. One of the most puzzling aspects of the ability of lithium to ameliorate the manic-depressive condition, is its relatively selective action upon the central nervous system (CNS). In the present study, it was shown that lithium selectively attenuated the function of Gs proteins in the CNS, assessed through isoproterenol-induced increases in the binding of guanosine triphosphate (GTP) to these proteins. Therapeutic concentrations of lithium (1-1.5 mM in vitro) inhibited the function of Gs protein in the cerebral cortex of the rat, while 4- to 6-fold larger concentrations of lithium were required to alter the function of Gs protein equivalently in the cardiac ventricles of the rat. Chronic administration of lithium via rat chow, containing lithium carbonate, to rats totally abolished the effect of isoproterenol on the binding of GTP in the CNS but did not affect the function of peripheral cardiac Gs protein. The lithium-selective action on the function of Gs protein in the CNS may stem from the heterogeneity of the alpha s subunit proteins: in the heart, the major species is a 45 kDa molecule, while in the brain, a 52 kDa molecular weight species predominates. The heterogeneity in alpha s subunits may thus be the biochemical basis for the selective action of lithium on the CNS and for the scarcity of peripheral side effects.

Antidepressants, β-Arrestins and GRKs: From Regulation of Signal Desensitization to Intracellular Multifunctional Adaptor Functions

G protein-coupled receptors (GPCR) have generated considerable interest in the pharmaceutical industry as drug targets. Theories concerning antidepressant targets of action suggested pre-synaptic monoamine reuptake mechanisms regulating GPCR activities including delayed receptor desensitization and down-regulation. GRKs and beta-arrestins translocate to the cell membrane and bind to agonist-occupied receptors. This uncouples these receptors from G proteins and promotes their internalization, leading to desensitization and down-regulation. Thus, GRKs and beta-arrestins serve as negative regulators of GPCR signaling. Recently, GPCR have been demonstrated to elicit signals through interaction with beta-arrestin as scaffolding proteins, independent of heterotrimeric G-protein coupling. beta-arrestins function as scaffold proteins that interact with several cytoplasmic proteins and link GPCR to intracellular signaling pathways such as MAPK cascades. Recent work has also revealed that beta-arrestins translocate from the cytoplasm to the nucleus and associate with transcription cofactors such as p300 and CREB. They also interact with regulators of transcription factors. We review findings concerning effects of antidepressants on GRKs and beta-arrestins and the plethora of antidepressants effects on signal transduction elements in which GRKs and beta-arrestins serve as signaling scaffold proteins, and on transcription factors and cofactors in which beta-arrestins mediate regulation of transcription. The emergence of G-protein-independent signaling pathways, through beta-arrestins, changes the way in which GPCR signaling is evaluated, from a cell biological to a pharmaceutical perspective and raises the possibility for the development of pathway specific therapeutics e.g., antidepressant medications targeting GRKs and beta-arrestin regulatory and signaling proteins.

Antidepressants increase β-arrestin2 ubiquitinylation and degradation by the proteasomal pathway in C6 rat glioma cells

β-Arrestins, regulators of G protein-coupled receptor-G protein coupling and receptor desensitization and internalization, function also as scaffolding proteins mediating cellular signaling events. β-Arrestin1 was previously implicated by us in the pathophysiology of depression and in the mechanism of action of antidepressants (ADs). The ubiquitously expressed β-arrestins1 and 2 are structurally highly homologous. There has been extensive investigation of these two proteins to determine whether they serve different roles in receptor signaling. In this study, we show that treatment of C6 rat glioma cells with ADs of various types for 3 days resulted in decreased β-arrestin2 levels. In contrast, β-arrestin2 mRNA expression was found to be up-regulated by ADs. To unravel the mechanism for these opposite effects several possible β-arrestin2 post-transcriptional events and modifications were examined. C6 rat glioma cells transfected with β-arrestin1-targeted short hairpin RNA showed similar effects of ADs on β-arrestin2 levels. AD-induced decreases in β-arrestin2 protein levels were not due to cytosolic membrane translocation. Immunoprecipitation experiments showed that ADs were able to increase coimmunoprecipitation of ubiquitin with β-arrestin2. AD-induced increases in β-arrestin2 ubiquitinylation led to its degradation by the proteasomal pathway, as the proteasome inhibitor N-[(phenylmethoxy)carbonyl]-l-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-l-leucinamide (MG-132) prevented antidepressant-induced decreases in β-arrestin2 protein levels.

Application of G-proteins in the molecular diagnosis of psychiatric disorders

Mental disorders are highly prevalent and often difficult to diagnose. Although significant advances have been achieved in medication for mental disorders, the diagnosis and treatment monitoring of these disorders remain in a static situation. The absence of objective diagnostic ‘gold standards’, derives from the special complexity of diagnosis in psychiatry. Heterotrimeric G-proteins are crucial elements in post-receptor information transduction. These proteins have been implicated in the biochemical mechanism of action of drugs used to treat psychiatric disorders. G-protein measurements have unravelled a differential pattern characteristic of each of the major mental disorders. The accumulated data supports the potential use of G-protein measures as state-dependent markers for the biochemical diagnosis of mental disorders and as aid in the biochemical monitoring of the response to a specific treatment.

Disopyramide and quinidine bind with inverse selectivity to muscarinic receptors in cardiac and extracardiac rat tissues

We investigated the interactions of disopyramide and quinidine with the muscarinic receptor in tissue homogenates from rat atrium, ventricle, cortex, submandibular gland, and urinary bladder by means of competition binding experiments, using the tritium-labeled antagonist N-methyl-4-piperidyl benzilate. The drugs displayed heterogeneous characteristics of binding to the muscarinic receptors in the different tissues. The binding affinity of quinidine to the muscarinic receptor in atrial tissue was five to 10 times greater than in the other tissues studied, whereas the affinity of disopyramide to the muscarinic receptor in the heart was five times lower than in the other tissues. This inverse selectivity shown by the two drugs in their binding to cardiac and to non-cardiac tissues may explain the extracardiac antimuscarinic side effects of treatment with disopyramide and their absence with quinidine.

Photoperiodicity and annual rhythms of wars and violent crimes

The seasonal variations of individual violent crimes, i.e. sexual offenses and aggravated assaults, and non-violent offenses, i.e. burglary, in Israel, the USA, Denmark and New South Wales, Australia, representing four continents, were analyzed. Seasonal variations in the opening dates of wars were similarly analyzed. In northern hemisphere countries, although non-violent offenses are distributed equally throughout the year, individual violent crimes and collective acts of hostility are characterized by an annual rhythm of incidence, with a peak in the months of July-August and a nadir in December-February. Inverse rhythms were obtained in southern hemisphere countries. These rhythms were found to be correlated in a statistically significant manner with the duration of the daily photoperiod. The existence of similar patterns of annual variations in violent crimes and in the opening dates of wars indicate similarities between individual and collective aggressiveness with respect to the underlying mechanisms and probably also to the means of their prevention.

Carbamylcholine inhibits β-adrenergic receptor-coupled Gs protein function proximal to adenylate cyclase

The specific mechanism by which the inhibitory guanine nucleotide binding protein (Gi) mediates the inhibition of adenylate cyclase activity is still unclear. The subunit dissociation model, based on studies in purified or reconstituted systems, suggests that the βγ subunit, which is dissociated with activation of Gi, inhibits the function of the stimulatory guanine nucleotide binding protein (Gs) by reducing the concentration of the free αs subunit. In the present study, Gs, protein function is determined by measuring cholera toxin‐blockable, isoproterenol‐induced increases in guanosine triphosphate (GTP) binding capacity to rat cardiac ventricle membrane preparations. Carbamylcholine totally inhibited this β‐adrenergic receptor‐coupled Gs protein function. Pretreatment of the cardiac ventricle membrane preparation with pertussis toxin prevented this muscarinic agonist effect. These results confirm the possibility of an inhibitory agonist‐receptor coupled effect through Gi on Gs protein function proximal to the catalytic unit of adenylate cyclase in an intact membrane preparation.