Human alpha6 AChR subtypes: subunit composition, assembly, and pharmacological responses

Many nicotinic acetylcholine receptor (AChR) subunits are known to be co-expressed with the alpha6 subunit in neurons. Because alpha6beta4 AChRs assemble inefficiently and alpha6beta2 AChRs not at all, more complex mixtures of human subunit cDNAs were tested for their abilities to form functional AChRs when expressed in Xenopus oocytes. alpha6beta4beta3 AChRs produced the largest and most consistent responses. alpha6alpha3beta2 AChRs exhibited reduced potency for ACh and increased potency and efficacy for nicotine compared to alpha3beta2 AChRs, but similar resistance to functional inactivation after prolonged exposure to nicotine. alpha6alpha4beta2 AChRs differed little in potency or efficacy for ACh or nicotine compared to alpha4beta2 AChRs, and had similarly high sensitivity to inactivation by prolonged exposure to nicotine. Co-expression of alpha6 and beta2 cRNAs resulted in large numbers of (3)H-epibatidine binding sites in the form of large aggregates but not in functional pentameric AChRs. Co-expression of alpha6, beta2, and alpha5 resulted in assembly of some functional pentameric AChRs. Chimeras with the large extracellular domain of alpha6 and the rest from either alpha3 or alpha4 efficiently formed functional AChRs. Thus, the extracellular domain of alpha6 efficiently assembles with beta2 to form ACh binding sites, but more C-terminal domains cause difficulties in forming pentameric AChRs. Chimeric alpha6/alpha3 and alpha6/alpha4 AChRs containing either beta2 or beta4 subunits were blocked by alpha-conotoxin MII which had previously been reported to be specific for alpha3beta2 AChRs.     

Biotransformation of post-clozapine antipsychotics: pharmacological implications

The need to develop new antipsychotics that have fewer motor adverse effects and offer better treatment of negative symptoms has led to a new generation of drugs. Most of these drugs undergo extensive first-pass metabolism and are cleared almost exclusively by metabolism, except for amisulpride whose clearance is largely due to urinary excretion. Risperidone has metabolic routes in common with ziprasidone but shows differences in regard to other main pathways: the benzisoxazole moiety of risperidone is oxidised by cytochrome P450 (CYP) 2D6 to the active 9-hydroxyrisperidone, whereas the benzisothiazole of ziprasidone is primarily oxidised by CYP3A4, yielding sulfoxide and sulfone derivatives with low affinity for target receptors in vitro. Olanzapine, quetiapine and zotepine also have some common metabolic features. However, for the thienobenzodiazepine olanzapine a main metabolic route is direct conjugation at the benzodiazepine nucleus, whereas for the dibenzothiazepine quetiapine and the dibenzothiepine zotepine it is CYP3A4-mediated oxidation, leading to sulfoxidation, hydroxylation and dealkylation for quetiapine, but N-demethylation to the active nor-derivative for zotepine. Although the promising benzisoxazole (iloperidone) and benzisothiazole (perospirone) antipsychotics share some metabolic routes with the structurally related available drugs, they too have pharmacologically relevant compound-specific pathways. For some of the new antipsychotics we know the isoenzymes involved in their main metabolic pathways and the endogenous and exogenous factors that, by affecting enzyme activity, can potentially modify steady-state concentrations of the parent drug or its metabolite(s), but we know very little about others (e.g. amisulpride isomers, nemonapride). For yet others, information is scarce about the activity of the main metabolites and whether and how these contribute to the effect of the parent drug. Aging reduces the clearance of most antipsychotics, except amisulpride (which requires further evaluation) and ziprasidone. Liver impairment has little or no effect on the pharmacokinetics of olanzapine, quetiapine, risperidone (and 9-hydroxy-risperidone) and ziprasidone, but information is lacking for amisulpride. Renal impairment significantly reduces the clearance and prolongs the elimination half-life of amisulpride and risperidone. Again, studies are still not available for some drugs (zotepine) and have focused on the parent drug for others (olanzapine, quetiapine, ziprasidone) despite the fact that renal impairment would be expected to lower the clearance of more polar metabolites. Addressing these issues may assist clinicians in the design of safe and effective regimens for this group of drugs, and in selecting the best agent for each specific population.     

Regulation of liver cytochrome P450 by activation of brain dopaminergic system: physiological and pharmacological implications

The aim of the present study was to investigate the influence of activation of brain dopaminergic system by different dopaminomimetics on the level and activity of liver cytochrome P450 (CYP) isoforms. Studies into the identification of hormones and cytokines which are known to mediate liver CYP expression were also simultaneously carried out.Stimulation of dopaminergic receptors in the pituitary, a target for the tuberoinfundibular pathway, by dopamine (a D₁/D₂ receptor agonist) administered intraperitoneally caused a significant increase in the activities and protein levels of CYP2B, CYP2C11 and CYP3A, a substantial increase in the blood plasma level of growth hormone (GH) and a significant decrease in triiodothyronine (T₃) level. Local stimulation of dopaminergic receptors in the nucleus accumbens, a target for the mesolimbic pathway, by apomorphine (a D₁/D₂ receptor agonist), amphetamine (an indirect D₁/D₂ dopaminemimetic) and quinpirole (a D₂ receptor agonist) produced a substantial rise in CYP3A activity and protein level, caused a large increase in corticosterone concentration and a moderate drop in T₃ level. SKF82958 (a D₁ receptor agonist) did not significantly affect the CYP isoforms or hormones studied. In both cases (activation of the tuberoinfundibular or mesolimbic pathway), the activity and the protein level of CYP1A considerably decreased. Plasma levels of thyroxine, testosterone, interleukin-2 and interleukin-6 were not changed after activation of the two pathways.The obtained results establish the brain dopaminergic system as a physiological centre regulating cytochrome P450 (engaging D₂ receptors and pituitary hormones) and demonstrate new pharmacological aspects of neuroactive drugs that affect this system.     

Bopindolol: pharmacological basis and clinical implications

Bopindolol, a non-selective antagonist of beta 1- and beta 2-adrenoceptors (ARs), has been found by pharmacological, molecular biological techniques and molecular modeling to have several unique properties. Bopindolol produces sustained blockade of beta 1- and beta 2-ARs, has intrinsic sympathomimetic as well as membrane stabilizing actions, inhibits renin secretion, and interacts with 5-HT receptors. Also, our recent molecular modeling studies identified possible interaction sites between bopindolol and beta-AR subtypes. The reviewed studies support our findings that bopindolol is non-selective for beta 1- and beta 2-ARs, has low affinity for beta 3-AR subtype and has pharmacological properties that are likely to be beneficial in the treatment of cardiovascular diseases.     

Apoptosis in atherosclerosis: pathological and pharmacological implications.

Normal embryonic development, tissue differentiation and repair in the eukaryote requires a tightly regulated apoptosis, or programmed cell death. Apoptosis also plays an essential role in different pathological processes including atherosclerosis, in which it affects all cell types in the atherosclerotic lesion, including endothelial cells, vascular smooth muscle cells, and macrophages. During atherosclerosis progression, pro- and anti-apoptotic signals abound in the evolving lesion. Apoptosis limits the number of a particular cell type that accumulates in the lesion and slows down the overall progression of the lesion. On the other hand, it contributes to the production of unstable plaques. Many pharmacological agents used to treat cardiovascular and lipid disorders have pro- or/and anti-apoptotic effects. Pharmaceuticals that modulate apoptosis in specific types of cell can potentially serve as anti-atherogenic agents. However, to develop agents for clinical use requires a thorough knowledge of the pathophysiology of apoptosis in atheromatous lesions, a highly cell-specific process. Here we review our current understanding of the process to provide a background for future pharmacological research in the area.     

Regulation of G protein-coupled receptor signalling: focus on the cardiovascular system and regulator of G protein signalling proteins

G protein-coupled receptors (GPCRs) are involved in many biological processes. Therefore, GPCR function is tightly controlled both at receptor level and at the level of signalling components. Well-known mechanisms by which GPCR function can be regulated comprise desensitization/resensitization processes and GPCR up- and downregulation. GPCR function can also be regulated by several proteins that directly interact with the receptor and thereby modulate receptor activity. An additional mechanism by which receptor signalling is regulated involves an emerging class of proteins, the so-called regulators of G protein signalling (RGS). In this review we will describe some of these control mechanisms in more detail with some specific examples in the cardiovascular system. In addition, we will provide an overview on RGS proteins and the involvement of RGS proteins in cardiovascular function.     

Central nervous system control of cough: pharmacological implications

For many years the idea of a cough center in the brain dominated discussions in the field without any substantial progress in defining what this cough center is or how it functions. Substantial progress has now been made and many of the central neural elements involved in coughing are being described. Furthermore, hypothesis driven research into the function of these neural elements is providing exciting new leads for possible therapeutic targets. The concept of a specific, centrally acting drug for cough suppression is fast becoming a reality. This review summarizes the key findings from the past few years and provides a perspective on future directions for the development of novel antitussives.     

Regions in the G protein gamma subunit important for interaction with receptors and effectors

G betagamma dimers containing the gamma11 or gamma1 subunits are often less potent and effective in their ability to regulate effectors compared with dimers containing the gamma2 subunit. To explore the regions of the gamma subunit that affect the activity of the betagamma dimer, we constructed eight chimeric gamma subunits from the gamma1 and gamma2 subunits. Two chimeras were made in which the N-terminal regions of gamma1 and gamma2 were exchanged and two in which the C-terminal regions were transposed. Another set of chimeras was made in which the CAAX motifs of the chimeras were altered to direct modification with different prenyl groups. All eight gamma chimeras were expressed in Sf9 cells with the beta1 subunit, G betagamma dimers were purified, and then they were assayed in vitro for their ability to bind to the G alpha(i1) subunit, to couple G alpha(i1) to the A1 adenosine receptor, to stimulate phospholipase C-beta, and to regulate type I or type II adenyl cyclases. Dimers containing the C-terminal sequence of the gamma2 subunit modified with the geranylgeranyl lipid had the highest affinity for G(i1)alpha (range, 0.5-1.2 nM) and were most effective at coupling the G(i1)alpha subunit to receptor. These dimers were most effective at stimulating the phosphatidylinositol-specific phospholipase C-beta isoform and inhibiting type I adenyl cyclase. In contrast, betagamma dimers containing the N-terminal sequence of the gamma2 subunit and a geranylgeranyl group are most effective at activating type II adenyl cyclase. The results indicate that both the N- and C-terminal regions of the gamma subunit impart specificity to receptor and effector interactions.     

Arsenic trioxide-induced apoptosis in H9c2 cardiomyocytes: implications in cardiotoxicity

Arsenic trioxide (As(2)O(3)) achieved dramatic remissions in patients with acute promyelocytic leukaemia. Clinical reports have shown that treatment was associated with cardiotoxicity. We investigated the toxic mechanisms of As(2)O(3) in H9c2 cardiomyocytes. Clinically relevant concentrations of As(2)O(3) (2-10 microM) reduced the viability of H9c2 cells in a concentration-dependent manner. The decreased cell viability was because As(2)O(3) induced cell apoptosis (cell shrinkage, nuclear alterations and caspase-3 activation), or even necrosis at higher concentrations. Inhibition of caspase-3 with a specific inhibitor, Ac-DEVD-CHO, suppressed apoptosis induced by As(2)O(3). In addition, reactive oxygen species formation and cellular Ca(2+) overload were observed in H9c2 cells exposed to As(2)O(3), which was partly inhibited by vitamin E and verapamil. These results suggest that As(2)O(3)-induced cardiotoxicity is mediated, at least in part, by activation of caspase-3 pathway, which may be triggered by reactive oxygen species formation and intracellular Ca(2+) overload.     

The role of the G protein gamma(2) subunit in opioid antinociception in mice

We examined the role of the gamma(2) subunit of G proteins (Ggamma(2)) in the antinociception produced by c[D-Pen(2), D-Pen(5)]enkephalin (DPDPE) in mice. DPDPE produced 84.0+/-9.0% antinociception in vehicle-treated mice. After intracerebroventricular (i.c.v.) treatment with an antisense phosphorothioate oligodeoxynucleotide to the Ggamma(2) subunit, DPDPE-mediated antinociception decreased to 24.4+/-7.4%. The mismatch phosphorothioate oligodeoxynucleotide-treated mice showed 65.1+/-10.3% antinociception, while the missense phosphorothioate oligodeoxynucleotide-treated mice showed 76.4+/-23.6% antinociception by DPDPE. The reduction of analgesia in antisense phosphorothioate oligodeoxynucleotide-treated mice was significant in comparison with vehicle-treated (P<0.001), mismatch phosphorothioate oligodeoxynucleotide-treated (P<0.01) and missense phosphorothioate oligodeoxynucleotide-treated (P<0.05) mice. These results suggest that the G protein gamma(2) subunit is involved in the transduction pathway leading to antinociception by DPDPE.     

Regulation of NMDA receptor subunit expression and its implications for LTD, LTP, and metaplasticity

NMDA-type glutamate receptors (NMDARs) mediate many forms of synaptic plasticity. These tetrameric receptors consist of two obligatory NR1 subunits and two regulatory subunits, usually a combination of NR2A and NR2B. In the neonatal neocortex NR2B-containing NMDARs predominate, and sensory experience facilitates a developmental switch in which NR2A levels increase relative to NR2B. In this review, we clarify the roles of NR2 subunits in synaptic plasticity, and argue that a primary role of this shift is to control the threshold, rather than determining the direction, for modifying synaptic strength. We also discuss recent studies that illuminate the mechanisms regulating NR2 subunits, and suggest that the NR2A/NR2B ratio is regulated by multiple means, which may control the ratio both locally at individual synapses and globally in a cell-wide manner. Finally, we use the visual cortex as a model system to illustrate how activity-dependent modifications in the NR2A/NR2B ratio may contribute to the development of cortical functions.     

Allosteric modulation of G protein-coupled receptors: A pharmacological perspective

G protein-coupled receptor (GPCR)-based drug discovery has traditionally focused on targeting the orthosteric site for the endogenous agonist. However, many GPCRs possess allosteric sites that offer enormous potential for greater selectivity in drug action. The complex behaviors ascribed to allosteric ligands also present challenges to those interested in preclinical lead discovery. These challenges include the need to detect and quantify various phenomena when screening for allosteric ligands, such as saturability of effect, probe dependence, differential effects on orthosteric ligand affinity vs. efficacy, system-dependent allosteric agonism, stimulus-bias (functional selectivity), and the potential existence of bitopic (hybrid orthosteric/allosteric) ligands. These issues are also critical when interpreting structure-function studies of allosteric GPCR modulators because mutations in receptor structure, either engineered or naturally occurring, can differentially affect not only modulator affinity, but also the nature, magnitude and direction of the allosteric effect on orthosteric ligand function. The ever-expanding array of allosteric modulators arising from both academic and industrial research also highlights the need for the development of a uniform approach to nomenclature of such compounds.     

Central nervous system control of the airways: pharmacological implications

Autonomic innervation of the airways is derived primarily from the parasympathetic nervous system. Preganglionic fibers originating in the brainstem project to parasympathetic ganglion neurons, which regulate airway smooth-muscle tone, glandular secretion and blood-vessel diameter. Airway preganglionic nerve activity is regulated by subsets of pulmonary and extrapulmonary afferent nerve fibers, which continuously provide polysynaptic input to brainstem preganglionic nuclei. Each of these synapses in the central nervous system is a potential site for therapeutic intervention. Potential targets include increasing opioid, GABAergic and serotonergic controls on central neurons, and blockade of tachykinin and glutamate receptors. Unfortunately, much is still unknown about the control of airway nerves at the level of the central nervous system. Recently, however, interaction between vagal afferent nerve subtypes regulating airway function has been described. This interaction, made possible by their convergence at key sites of integration in the brainstem, may lead to central sensitization analogous to that described in somatic pathways regulating pain sensation. Improved understanding of the central pharmacology of airway reflexes may provide novel therapeutics for treating symptoms associated with respiratory disorders such as chronic obstructive pulmonary disease, asthma and sleep-disordered breathing.     

Genomics and phenotypic profiles in dementia: implications for pharmacological treatment

Constitutive genomics are probably determinant for the onset of dementia in conjunction with cerebrovascular and environmental factors. Furthermore, pharmacogenomic studies predict that the therapeutic response in Alzheimer's disease (AD) is genotype-specific, and that the expression of genes involved in the regulation of drug metabolism can influence efficacy and safety issues in pharmacotherapy. AD and dementia with a vascular component (DVC = VD + MXD) are the most prevalent forms of dementia. These clinical entities share many similarities, but they differ in major phenotypic and genotypic profiles, as revealed by structural and functional genomics studies. Comparative phenotypic studies have identified significant differences in 25% of more than 100 parametric variables, including anthropometry, cardiovascular function, aortic atherosclerosis, brain atrophy, blood pressure, blood biochemistry, hematology, thyroid function, folic acid and vitamin B(12) levels, brain hemodynamics and lymphocyte markers. The phenotypic profile of patients with DVC differs from that of AD patients in the following: (a) anthropometric values, (b) cardiovascular function, (c) blood pressure, (d) lipid metabolism, (e) uric acid levels, (f) peripheral calcium levels, (g) liver function (GOT, GPT, GGT), (h) alkaline phosphatase, (i) lactate dehydrogenase, (j) red and white blood cells, (k) regional brain atrophy (left temporal region, inter-hippocampal distance) and (l) brain blood flow velocity. Functional genomics studies incorporating APOE-related changes in biological markers extended the difference between AD and DVC up to 57%. Structural genomics studies with AD-related genes, including APP, MAPT, APOE, PS1, PS2, A2M, ACE, AGT, cFOS and PRNP genes, demonstrate different genetic profiles in AD and DVC, with an absolute genetic variation rate ranging from 30 to 80%, depending upon genes and genetic clusters. Single gene analysis identifies relative genetic variations ranging from 0 to 5%. The relative polymorphic variation in genetic clusters integrated by 2, 3 or 4 genes associated with AD ranges from 1 to 3%. The main phenotypic differences between AD and DVC are genotype-dependent, especially in AD, probably indicating that different genomic factors are essential for the expression of dementia symptoms that might be accelerated or induced by environmental and/or cerebrovascular factors.     

Cytokines: abnormalities in major depression and implications for pharmacological treatment

The role of cytokines in depression was first considered when the cytokine interferon resulted in "sickness behaviour", the symptoms of which are similar to those of major depression. The latter is associated with an increase in pro-inflammatory cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF-alpha). These cytokines are potent modulators of corticotropin-releasing hormone (CRH) which produces heightened hypothalamic-pituitary-adrenal axis (HPA) activity characterized by increases in ACTH and cortisol, both of which are reported elevated in major depression. Antidepressant treatment has immunomodulatory effects with increases in the production of IL-10, which is an anti-inflammatory cytokine. This review based on a Medline search from 1980-2003, focuses on the evidence available of cytokine changes in acute stress, chronic stress and major depression. It examines the effects of antidepressant treatment on immune parameters in both animal models and clinical trials. We suggest that future antidepressants may target the immune system by either blocking the actions of pro-inflammatory cytokines or increasing the production of anti-inflammatory cytokines.     

Dopamine-sensitive adenylyl cyclases in neuronal development: physiopathological and pharmacological implications

Pharmacological studies of molecular mechanisms leading to the differentiation of neurons with retained dopaminergic fate and function suggest that such differentiation could be a form of treatment of neurodegenerative disorders, such as Parkinson's disease (PD) and schizophrenia. This goal could be achieved by neuronal replacement therapies based upon the manipulation of endogenous precursors in situ or by transplantation-based approaches. Signals conveyed by the adenylyl cyclase (AC) pathway appear to be crucial for the suitable differentiation of neurons. Here, we discuss dopamine (DA)-sensitive isoforms of AC as key cues for dopaminergic neuronal patterning and as interesting therapeutic targets for the induction of regenerative processes or to drive correct neuronal development.     

Interaction of G protein beta subunit with inward rectifier K(+) channel Kir3

G protein betagamma subunits bind and activate G protein-coupled inward rectifier K+ (GIRK) channels. This protein-protein interaction is crucial for slow hyperpolarizations of cardiac myocytes and neurons. The crystal structure of Gbeta shows a seven-bladed propeller with four beta strands in each blade. The Gbeta/Galpha interacting surface contains sites for activating GIRK channels. Furthermore, our recent investigation using chimeras between Gbeta1 and yeast beta (STE4) suggested that the outer strands of blades 1 and 2 of Gbeta1 could be an interaction area between Gbeta1 and GIRK. In this study, we made point mutations on suspected residues on these outer strands and investigated their ability to activate GIRK1/GIRK2 channels. Mutations at Thr-86, Thr-87, and Gly-131, all located on the loops between beta-strands, substantially reduced GIRK channel activation, suggesting that these residues are Gbeta/GIRK interaction sites. These mutations did not affect the expression of Gbeta1 or its ability to stimulate PLCbeta2. These residues are surface-accessible and located outside Gbeta/Galpha interaction sites. These results suggest that the residues on the outer surface of blades 1 and 2 are involved in the interaction of Gbetagamma with GIRK channels. Our study suggests a mechanism by which different effectors use different blades to achieve divergence of signaling. We also observed that substitution of alanine for Trp-332 of Gbeta1 impaired the functional interaction of Gbeta1 with GIRK, in agreement with the data on native neuronal GIRK channels. Trp-332 plays a critical role in the interaction of Gbeta1 with Galpha as well as all effectors so far tested.     

The role of the endocannabinoid system in eating disorders: pharmacological implications

The endocannabinoid (eCB) system is a widespread intercellular signalling mechanism that plays a critical role in body homoeostasis. It is located in key points involved in food intake and energy expenditure, coordinating all the players involved in energy balance. As such, it has come to be seen as an interesting target for the management of diseases characterized by an imbalanced energy homoeostasis, such as obesity and eating disorders. The aetiology of eating disorders and the molecular systems involved are still largely a mystery. Research has focused on brain circuits where the eCB system plays an important role, such as those related to feeding behaviour and the rewarding properties of food. Accordingly, recent findings have suggested a deregulation of the eCB system in eating disorders. At present, cannabinoid agonists are safe and effective tools in the management of diseases in which weight gain is needed, for example cachexia in AIDS patients. However, studies on the potential therapeutic validity of cannabinoids in eating disorders are scarce and inconclusive. Taken together, all these considerations warrant more preclinical and clinical investigations in the role of the eCB system in eating disorders. Eventually, they may provide novel pharmacological approaches for the treatment of these diseases.