Modulation of Muscarinic Cholinergic Receptor Affinity for Antagonists in Rat Heart (original) (raw)
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Cardiac cholinergic muscarinic receptors: changes in multiple affinity forms with down-regulation
The Journal of pharmacology and experimental therapeutics, 1985
The isolated working rat heart exhibits dynamic changes in the cholinergic muscarinic receptor in response to perfusion with the acetylcholine congener methacholine. For example, perfusion with 4 microM methacholine for 2.5 hr mediates a statistically significant and reversible 10 to 15% decrease in receptor content in right atrium and left atrium and ventricle. The muscarinic receptor exhibits a single affinity state for antagonists but multiple affinity states for receptor agonists. When agonist 3H-antagonist competition experiments are performed, the concentration dependence of displacement by agonists is flattened and extends over more than 2 log units. From such curves, it is difficult to extract values for the relative proportions of the multiple receptors or their KD values by inspection. We have developed a procedure for plotting the competition curves so that the KD values and proportion of multiple receptor forms can be estimated graphically. We determined these more accur...
Partial functional reconstitution of the cardiac muscarinic cholinergic receptor
Journal of Biological Chemistry
~ Digitonin-solubilized cardiac muscarinic receptors were reconstituted by dialysis into human erythrocyte acceptor membranes which lack high-affinity muscarinic receptors. The number of receptors reconstituted was proportional to the quantity of soluble receptors added to the reconstitution system.
Cooperativity and Oligomeric Status of Cardiac Muscarinic Cholinergic Receptors †
Biochemistry, 2002
Muscarinic cholinergic receptors can appear to be more numerous when labeled by [ 3 H]quinuclidinylbenzilate (QNB) than by N-[ 3 H]methylscopolamine (NMS). The nature of the implied heterogeneity has been studied with M 2 receptors in detergent-solubilized extracts of porcine atria. The relative capacity for [ 3 H]NMS and [ 3 H]QNB was about 1 in digitonin-cholate, 0.56 in cholate-NaCl, and 0.44 in Lubrol-PX. Adding digitonin to extracts in cholate-NaCl increased the absolute capacity for both radioligands, and the relative capacity increased to near 1. The latency cannot be attributed to a chemically impure radioligand, instability of the receptor, an irreversible effect of NMS, or a failure to reach equilibrium. Binding at near-saturating concentrations of [ 3 H]QNB in cholate-NaCl or Lubrol-PX was blocked fully by unlabeled NMS, which therefore appeared to inhibit noncompetitively at sites inaccessible to radiolabeled NMS. Such an effect is inconsistent with the notion of functionally distinct, noninterconverting, and mutually independent sites. Both the noncompetitive effect of NMS on [ 3 H]QNB and the shortfall in capacity for [ 3 H]NMS can be described quantitatively in terms of cooperative interactions within a receptor that is at least tetravalent; no comparable agreement is possible with a receptor that is only di-or trivalent. The M 2 muscarinic receptor therefore appears to comprise at least four interacting sites, presumably within a tetramer or larger array, and ligands appear to bind in a cooperative manner under at least some conditions.
Functional M3 muscarinic acetylcholine receptors in mammalian hearts
British Journal of Pharmacology, 2004
In contrast to most peripheral tissues where multiple subtypes of muscarinic acetylcholine receptor (mAChR) coexist, with each of them playing its part in the orchestra of parasympathetic innervation, the myocardium has been traditionally considered to possess a single mAChR subtype. Although there is much evidence to support the notion that one receptor subtype (M2) orchestrates myocardial muscarinic transduction, there is emerging evidence that M1 and M3 receptors are also expressed and are of potential physiological, pathophysiological and pharmacological relevance. Clarifying this issue has a profound impact on our thinking about the cholinergic control of the heart function and disease and approaches to new drug development for the treatment of heart disease associated with parasympathetic dysfunction. This review article presents evidence for the presence of the M3 receptor subtype in the heart, and analyzes the controversial data from published pharmacological, functional and...
Biochemical Pharmacology, 1983
The affinities of atropine, scopolamine, 3-quinuclidinyl benzilate and twelve analogues of 3-quinuclidinyl benzilate were determined for the muscarinic acetylcholine receptor (m-AChR) using membrane preparations from caudate/putamen. The affinity constants thus obtained were compared with affinities previously reported for the m-AChR obtained from ventricular muscle. The affinities differed significantly for six of the compounds, the largest difference being 1Bfold. Neither solubilization nor variation of physiologically significant salts led to a significant change in the affinity of that compound. These results are interpreted as supporting the subclassification of the muscarinic acetylcholine receptor.
Assessment of mechanistic proposals for the binding of agonists to cardiac muscarinic receptors
Biochemistry, 1986
Methylscopolamine has been used to characterize muscarinic receptors in crude homogenates prepared from hearts of Syrian golden hamsters. The Hill coefficient is one for specific binding of the radioligand itself and for its inhibition by muscarinic antagonists; markedly lower values are obtained for its inhibition by muscarinic agonists. The binding patterns of agonists have been analyzed in terms of a mixture of sites differing in affinity for the drug and reveal the following. (1) All agonists discern at least two classes of receptor in atrial and ventricular homogenates. (2) The number of classes and the relative size of each differ for different agonists in the same region and for the same agonist in different regions. ' Abbreviations: GMP-PNP, guanylyl imidodiphosphate; HEPES, N-(2-hydroxyethyl)piperazine-N'2-ethanesulfonic acid. C A R D I A C M U S C A R I N I C R E C E P T O R S V O L . 2 5 , N O . 2 2 , 1 9 8 6 6997 P carbachol LV(5), n = 2 RV(5), n = 2 K , , K2 F2 K I , K2, F2 arecoline AT(2), n = 2 LV(3), n = 2 K , , K2 F2 carbachol AT(5), n = 3 LV(5), n = 2 K
Cardiac muscarinic receptor binding is regulated by Na+ and guanine nucleotides
Journal of Biological Chemistry
To define cardiac muscarinic cholinergic receptor regulation, we have investigated the influences of sodium chloride and the guanyl nucleotides, GTP, GDP, and guanyl5'-yl-imidodiphosphate (Gpp(NH)p), on the rat cardiac muscarinic cholinergic receptor. In competition experiments, the radiolabeled muscarinic antagonist, [3H]quinuclidinyl benzilate (QNB), sodium chloride (0 to 140 nm) and/or the guanyl nucleotides (1 PM) were added to a 50 nm Tris-HC1 buffer to detect changes of binding affinity for both the muscarinic cholinergic agonist, oxotremorine sesquifumarate, and the muscarinic antagonist, atropine sulfate. In the absence of Na+, the ICs0 value for oxotremorine was 0.12 p. However, in the presence of Na+ at concentrations of 50,90, and 140 n m , the ICso values increased to 0.81, 0.90, and 1.0 PM, respectively. The IC50 value for atropine increased from 5.0 mu in the absence of Na+ to 9.3 mu in the presence of 140 n m Na+. The monovalent ions Cs' and Rb' were without effect; however, 140 nm K+ shifted the agonist affinity approximately 2-fold but had no effect on antagonist affinity. GTP, GDP, and Gpp(NH)p (1 PM) selectively decreased the affinity of the muscarinic cholinergic receptor for oxotremorine. Atropine competition was not changed by these guanine nucleotides. The order of potency for the nucleotides was Gpp(NH)p > GTP and GDP. Guanosine and 5'GMP were without effect on agonist and antagonist affinity at concentrations up to 0.1 nm. The affinity of oxotremorine was further decreased by 5fold in the presence of both 140 m Na+ and 1 PM guanyl nucleotides. Thus, NaCl and guanyl nucleotides appear to interact in a synergistic fashion to decrease the affinity of the cardiac muscarinic cholinergic receptor for agonists.