Evidence for the binding of β-adrenoceptor blockers to microsomal Na+/K+-ATPase in guinea pig heart preparations

2001 ◽  
Vol 79 (1) ◽  
pp. 8-12
Author(s):  
Abdulrahman A Almotrefi ◽  
Chona Basco ◽  
Azadali Moorji ◽  
Nduna Dzimiri
Keyword(s):  
Guinea Pig ◽  
Receptor Occupancy ◽  
Antiarrhythmic Agents ◽  
Binding Assays ◽  
Binding Properties ◽  
Guinea Pig Heart ◽  
Receptor Sites ◽  
Series Of Experiments ◽  
Β Blockers ◽  
Arrhythmogenic Effects

We reported in a previous study that β-adrenoceptor blockers inhibit the Mg2+-dependent ATP-hydrolytic function of Na+/K+-ATPase. To determine if this action is a result of binding of β-blockers to the receptor sites that bind the digitalis glycosides, we performed displacement binding assays of eight β-blockers with [3H]-ouabain (OUA) in guinea pig myocardial microsomal preparations. In the first series of experiments, 10-200 µM of the β-blockers were displaced with 250 nM OUA. In the second set of experiments, 10-500 nM of OUA was displaced using 200 µM of the β-blockers. The drugs showed concentration-dependent receptor occupancy at the different OUA levels. Propranolol (PPN), metoprolol (MTP), and sotalol (STL) showed the strongest binding; nadolol (NDL), indenolol (IDN), and atenolol (ATN) had intermediate binding; carazolol (CRZ) and celiprolol (CLP) had the weakest binding properties. The results suggest that β-blockers may compete for the same binding sites with ouabain in their inhibition of the Na+/K+-ATPase. These actions may contribute to the mechanism for some of their cardiac effects, especially their proarrhythmic and arrhythmogenic actions.Key words: β-adrenoceptor blockers, antiarrhythmic agents, arrhythmogenic effects, Na+/K+-ATPase, ouabain binding.

Class I antiarrhythmic drug effects on ouabain binding to guinea pig cardiac Na+-K+ATPase

1999 ◽  
Vol 77 (11) ◽  
pp. 866-870 ◽  
Author(s):  
Abdulrahman A Almotrefi ◽  
Chona Basco ◽  
Azadali Moorji ◽  
Nduna Dzimiri
Keyword(s):  
Guinea Pig ◽  
Drug Effects ◽  
Receptor Occupancy ◽  
Class I ◽  
Antiarrhythmic Agents ◽  
Binding Assays ◽  
Ouabain Binding ◽  
Guinea Pig Heart ◽  
Receptor Sites ◽  
Displacement Assays

The notion that the inhibition of the Mg2+-dependent ATP-hydrolytic function of the myocardial Na+-K+ATPase by class I antiarrhythmic agents occurs as a result of their binding to the same receptor sites as the digitalis glycosides was tested by performing competitive binding assays of [3H]ouabain (OUA) with eight drugs: disopyramide, encainide, lidocaine, lorcainide, phenytoin, procainamide, quinidine, and tocainide in guinea pig heart microsomal preparations. In the first set of experiments, 10-200 µM concentrations of the drugs were preincubated with the enzyme and displacement assays performed with 250 nM OUA. The drugs showed receptor occupancy of 19-32% at 50 µM, 25-44% at 100 µM, and 37-56% at 200 µM. Then, 10-500 nM concentrations of OUA were preincubated with the enzyme, and competitive assays were performed using 200 µM concentrations of the drugs. OUA occupied 39-51% of the receptor sites at 100 nM, 44-67% at 250 nM, and 62-82% at 500 nM, displacing the drugs in a concentration-dependent fashion. The results show that antiarrhythmic drugs interact with the same or similar receptor sites as ouabain on the Na+-K+ATPase, pointing to a possible contribution of these interactions to the mechanism for their inhibitory actions on the enzyme, and perhaps their arrhythmogenic effects.Key words: class I antiarrhythmic agents, proarrhythmias, Na+-K+ATPase, ouabain binding.

scholarly journals Visualizing Pharmacological Activities of Antidepressants: A Novel Approach

2008 ◽  
Vol 2 (1) ◽  
pp. 54-62 ◽  
Author(s):  
Hieronymus J. Derijks ◽  
Eibert R. Heerdink ◽  
Rob Janknegt ◽  
Fred H.P. De Koning ◽  
Berend Olivier ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Hierarchical Cluster ◽  
Receptor Occupancy ◽  
Pharmacological Properties ◽  
Therapeutic Action ◽  
Pharmacological Activities ◽  
Binding Properties ◽  
Novel Approach ◽  
Receptor Sites ◽  
Serotonin Reuptake Transporter

Antidepressants have different receptor binding profiles, which are related to therapeutic action and adverse drug reactions. We constructed a model to classify antidepressants on the basis of their binding properties of most common transporter- and receptor sites. Receptor binding was quantified by calculating receptor occupancy for the 5-HT (serotonin) reuptake transporter, norepinephrinic reuptake transporter, 5-HT2C-receptor, M3-receptor, H1-receptor and 􀀁1- receptor. To identify groups of antidepressants that show similar patterns of receptor occupancy for different receptors, hierarchical cluster analysis (HCA) and principle component analysis (PCA) were used. In addition, to visualize (a)symmetry between binding profiles of antidepressants, radar plots were constructed. On the basis of both analyses, four clusters of antidepressants which exert similar pharmacological properties were identified. Potentially, this model could be a helpful tool in medical practice and may be used as a prediction model for adverse effects of drugs entering the market.

Mitochondrial Oxidation of p-N, N-Dimethylamino-β-Phenethylamine (DAPA)

1975 ◽  
Vol 33 ◽  
pp. 458-459
Author(s):  
W. Allen Shannon ◽  
Hannah L. Wasserkrug ◽  
andArnold M. Seligman
Keyword(s):  
Guinea Pig ◽  
Oxidase Activity ◽  
Amine Oxidase ◽  
Histochemical Demonstration ◽  
Guinea Pig Heart ◽  
Pig Kidney ◽  
Cytoplasmic Vacuoles ◽  
Liver And Kidney ◽  
Mitochondrial Oxidation ◽  
Amine Oxidase Activity

The synthesis of a new substrate, p-N,N-dimethylamino-β-phenethylamine (DAPA)3 (Fig. 1) (1,2), and the testing of it as a possible substrate for tissue amine oxidase activity have resulted in the ultracytochemical localization of enzyme oxidase activity referred to as DAPA oxidase (DAPAO). DAPA was designed with the goal of providing an amine that would yield on oxidation a stronger reducing aldehyde than does tryptamine in the histochemical demonstration of monoamine oxidase (MAO) with tetrazolium salts.Ultracytochemical preparations of guinea pig heart, liver and kidney and rat heart and liver were studied. Guinea pig kidney, known to exhibit high levels of MAO, appeared the most reactive of the tissues studied. DAPAO reaction product appears primarily in mitochondrial outer compartments and cristae (Figs. 2-4). Reaction product is also localized in endoplasmic reticulum, cytoplasmic vacuoles and nuclear envelopes (Figs. 2 and 3) and in the sarcoplasmic reticulum of heart.

059 Daridorexant: A dual, equipotent, and insurmountable antagonist of both orexin-1 and orexin-2 receptors

SLEEP ◽  
2021 ◽  
Vol 44 (Supplement_2) ◽  
pp. A25-A25
Author(s):  
Celia Mueller Grandjean ◽  
Manon Kiry ◽  
Catherine Vaillant ◽  
Oliver Nayler ◽  
John Gatfield
Keyword(s):  
Calcium Release ◽  
Brain Activity ◽  
Specific Treatment ◽  
Receptor Occupancy ◽  
Kinetic Properties ◽  
Receptor Subtypes ◽  
Residence Times ◽  
Binding Assays ◽  
Receptor System ◽  
Highly Effective

Abstract Introduction The orexin neuropeptide–receptor system is a central sleep and wake regulator in the brain. The two orexin receptor subtypes, OX1R and OX2R, are expressed either alone or together in all major wake-promoting brain areas. OX1R and OX2R activation by orexins causes elevation of intracellular calcium, which enhances synaptic transmission in secondary, monoaminergic wake- and arousal-promoting neurotransmitter circuits. Orexin receptor antagonists represent a novel and specific treatment of insomnia, which is different from classical therapy that more broadly inhibits brain activity via GABAA activation. Here we describe the molecular pharmacology of daridorexant, an orexin receptor antagonist which has proven highly effective in improving sleep and daytime functioning in insomnia patients. Methods Orexin-A(OxA)-induced calcium release assays in OX1R- and OX2R-expressing recombinant cell lines were applied to measure the antagonistic potency and kinetic properties of daridorexant in functional assays. Whole-cell competitive binding assays, using an orthosteric tracer were employed to determine the Ki of daridorexant. Comparisons were made with suvorexant and lemborexant. Results In OxA-induced calcium release assays with 2-h pre-incubation time, daridorexant displayed apparent Kb values of 0.5 nM (OX1R) and 0.8 nM (OX2R) with insurmountable antagonism on both receptors, demonstrating equipotent and highly effective functional inhibition of both receptor subtypes. On-target residence times of daridorexant (37oC) expressed as receptor occupancy half-lives (ROt1/2) were 4 min (OX1R) and 8 min (OX2R). In binding assays, daridorexant behaved as highly potent orthosteric antagonist. Also suvorexant behaved as dual insurmountable antagonist at OX1R/OX2R (appKb=0.7nM/1.0nM; ROt1/2=9 min/6 min) and as potent orthosteric antagonist in binding assays. Interestingly, lemborexant displayed a different interaction profile at OX1R/OX2R (appKb=13nM/0.4nM, ROt1/2<2min/<2min), i.e. it behaved as preferential OX2R antagonist with a very short on-target residence time and little insurmountability. Conclusion Daridorexant displays the desired target interaction profile of a dual, equipotent, and insurmountable antagonist of both OX1R and OX2R, which ensures equally efficient inhibition of both arousal-/wake-promoting receptor subtypes. Daridorexant′s on-target residence times are long enough to cause insurmountable inhibition, but short enough to avoid pharmacodynamic effects after drug elimination. Support (if any) Funded by Idorsia Pharmaceuticals Ltd.

Functional and autoradiographic characterization of dopamine D2-like receptors in the guinea pig heart

2002 ◽  
Vol 80 (6) ◽  
pp. 578-587 ◽  
Author(s):  
María de Jesús Gómez ◽  
Guy Rousseau ◽  
Réginald Nadeau ◽  
Roberto Berra ◽  
Gonzalo Flores ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Western Blot ◽  
Dopamine Receptors ◽  
Negative Inotropic Effect ◽  
Cyclase Activity ◽  
Inotropic Effect ◽  
Receptor Subtypes ◽  
Guinea Pig Heart ◽  
Additional Information ◽  
The Right

Dopamine receptors include the D1- (D1 and D5 subtypes) and D2-like (D2, D3, and D4 subtypes) families. D1-like receptors are positively and D2-like receptors negatively coupled to the adenylyl cyclase. Dopamine D2-like (D4 subtype) receptors have been identified in human and rat hearts. However the presence of D2 and D3 receptor subtypes is unclear. Furthermore, their role in cardiac functions is unknown. By autoradiographic studies of guinea pig hearts, we identified D3 and D4 receptors, using the selective radioligands [3H]-7-OH-DPAT and [3H]emonapride (YM-09151-2 plus raclopride). Western blot analysis confirmed D3 and D4 receptors in the right and left ventricle of the same species. Selective agonists of D3 and D4 receptors (±)-7-OH-DPAT and PD 168 077 (10–9 to 10–5 M, respectively) induced a significant negative chronotropic and inotropic effect in the isolated guinea pig heart preparation. Negative inotropic effect induced by PD 168 077 was associated with an inhibition in cyclase activity. No changes in cyclase activity were found with (±)-7-OH-DPAT. The aim of this study is to support the presence of D3 and D4 receptors in the heart. Although our results suggest that D3 and D4 receptors are functionally active in the heart, we need additional information with an antagonist and an agonist of improved potency and selectivity to understand the respective roles of D3 and D4 receptors in the cardiac functions.Key words: Dopamine receptors (D2, D3, D4 subtypes), autoradiography, Western blot, cAMP, heart.

scholarly journals The C2A-C2B Linker Defines the High Affinity Ca2+ Binding Mode of Rabphilin-3A

2006 ◽  
Vol 282 (7) ◽  
pp. 5015-5025 ◽  
Author(s):  
Pierre Montaville ◽  
Christine Schlicker ◽  
Andrei Leonov ◽  
Markus Zweckstetter ◽  
George M. Sheldrick ◽  
...  
Keyword(s):  
Binding Mode ◽  
Bound State ◽  
C2 Domain ◽  
Binding Assays ◽  
Host Proteins ◽  
Binding Properties ◽  
C2 Domains ◽  
Phospholipid Binding ◽  
Acidic Residues ◽  
Structural Base

The Ca2+ binding properties of C2 domains are essential for the function of their host proteins. We present here the first crystal structures showing an unexpected Ca2+ binding mode of the C2B domain of rabphilin-3A in atomic detail. Acidic residues from the linker region between the C2A and C2B domains of rabphilin-3A interact with the Ca2+-binding region of the C2B domain. Because of these interactions, the coordination sphere of the two bound Ca2+ ions is almost complete. Mutation of these acidic residues to alanine resulted in a 10-fold decrease in the intrinsic Ca2+ binding affinity of the C2B domain. Using NMR spectroscopy, we show that this interaction occurred only in the Ca2+-bound state of the C2B domain. In addition, this Ca2+ binding mode was maintained in the C2 domain tandem fragment. In NMR-based liposome binding assays, the linker was not released upon phospholipid binding. Therefore, this unprecedented Ca2+ binding mode not only shows how a C2 domain increases its intrinsic Ca2+ affinity, but also provides the structural base for an atypical protein-Ca2+-phospholipid binding mode of rabphilin-3A.

scholarly journals Involvement of A pertussis Toxin Sensitive G-Protein in the Inhibition of Inwardly Rectifying K+Currents by Platelet-Activating Factor in Guinea-Pig Atrial Cardiomyocytes

1994 ◽  
Vol 3 (1) ◽  
pp. 45-51
Author(s):  
M. Gollasch ◽  
T. Kleppisch ◽  
D. Krautwurst ◽  
D. Lewinsohn ◽  
J. Hescheler
Keyword(s):  
Cyclic Amp ◽  
Guinea Pig ◽  
G Protein ◽  
Pertussis Toxin ◽  
Platelet Activating Factor ◽  
Resting Membrane Potential ◽  
Patch Clamp Technique ◽  
Guinea Pig Heart ◽  
Ventricular Cells ◽  
Inwardly Rectifying

Platelet-activating factor (PAF) inhibits single inwardly rectifying K+channels in guinea-pig ventricular cells. There is currently little information as to the mechanism by which these channels are modulated. The effect of PAF on quasi steady-state inwardly rectifying K+currents (presumably of the IK1type) of auricular, atrial and ventricular cardiomyocytes from guinea-pig were studied. Applying the patch-clamp technique in the whole-cell configuration, PAF (10 nM) reduced the K+currents in all three cell types. The inhibitory effect of PAF occurred within seconds and was reversible upon wash-out. It was almost completely abolished by the PAF receptor antagonist BN 50730. Intracellular infusion of atrial cells with guanine 5′-(β-thio)diphosphate (GDPS) or pretreatment of cells with pertussis toxin abolished the PAF dependent reduction of the currents. Neither extracellularly applied isoproterenol nor intracellularly applied adenosine 3′,5′-cyclic monophosphate (cyclic AMP) attenuated the PAF effect. In multicellular preparations of auricles, PAF (10 nM) induced arrhythmias. The arrhythmogenic activity was also reduced by BN 50730. The data indicate that activated PAF receptors inhibit inwardly rectifying K+currents via a pertussis toxin sensitive G-protein without involvement of a cyclic AMP-dependent step. Since IK1is a major component in stabilizing the resting membrane potential, the observed inhibition of this type of channel could play an important role in PAF dependent arrhythmogenesis in guinea-pig heart.

Sign in / Sign up

Export Citation Format

Share Document