title: Novel technique to determine the pKAof clonidine at prejunctional α2-adrenoceptors in cardiac and vascular sympathetic transmission.
abstract: Analytical pharmacology draws heavily on the concept of equilibrium of agonist and silent antagonist concentrations competing at a specific receptor site. This condition breaks down in nerve transmission when transmitter release is inhibited by prejunctional α2-adrenoceptors activated by an agonist such as clonidine. We have developed a method that allows the agonist dissociation constant KAof clonidine to be determined in a robust isolated right atrial assay of mouse, rat and guinea pig. By applying low numbers of field pulses 1-4 to prevent autoinhibitory feedback, clonidine shifted the nerve pulse stimulation-tachycardia curves to the right. These peak responses to field pulses were equated to responses to exogenous noradrenaline and the pKAdetermined by global fitting and display in the Clark plot. The pKAfor clonidine ranged from 8.95 in the mouse, 7.8 in rat and 8.3 in guinea pig. The propranolol pKBwas 8.87 in mouse and 8.91 in rat atria, reading very similarly to those values from β-adrenoceptor agonist assays under equilibrium conditions. In mesenteric resistance arteries mounted in a myograph for electrical field stimulation, clonidine again inhibited contractions to field pulses in mouse arteries with a pKAof 7.12, but was inactive in rat arteries due to competing autoinhibitory feedback from nerve-released noradrenaline. In both species, prazosin inhibited the field pulses with a pKBof 9.08 in rat and 9.03 in mouse arteries. We conclude that pKBfor antagonists and pKAfor the prejunctional inhibitors of nerve transmission can be determined with this novel analytical approach.
authors.affiliation: Cardiovascular Therapeutics Unit, Department of Pharmacology and Therapeutics, University of Melbourne, Victoria 3010, Australia. Electronic address: jamesaa@unimelb.edu.au.
authors.forename: James A
authors.initials: JA
authors.lastname: Angus
authors.affiliation: Cardiovascular Therapeutics Unit, Department of Pharmacology and Therapeutics, University of Melbourne, Victoria 3010, Australia. Electronic address: rajasekaran@wehi.edu.au.
authors.forename: Pravin
authors.initials: P
authors.lastname: Rajasekaran
authors.affiliation: Cardiovascular Therapeutics Unit, Department of Pharmacology and Therapeutics, University of Melbourne, Victoria 3010, Australia. Electronic address: cewright@unimelb.edu.au.
authors.forename: Christine E
authors.initials: CE
authors.lastname: Wright
mesh_terms: D000818:Animals; D003000:Clonidine / Q000494:pharmacology*; D003538:Cystamine / Q000031:analogs & derivatives / Q000494:pharmacology; D003891:Desipramine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006168:Guinea Pigs; D006325:Heart Atria / Q000187:drug effects / Q000294:innervation*; D008297:Male; D008638:Mesenteric Arteries / Q000187:drug effects / Q000294:innervation*; D051379:Mice; D009638:Norepinephrine / Q000494:pharmacology; D051381:Rats; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism*; D013564:Sympathetic Nervous System / Q000187:drug effects* / Q000502:physiology*; D009435:Synaptic Transmission / Q000187:drug effects*; D015016:Yohimbine / Q000494:pharmacology
chemical_list: D018341:Receptors, Adrenergic, alpha-2; D015016:Yohimbine; C029998:benextramine; D003000:Clonidine; D003538:Cystamine; D003891:Desipramine; D009638:Norepinephrine
keywords: (-)-noradrenaline bitartrate (PubChem CID: 18530234); (±)-propranolol hydrochloride (PubChem CID: 62882); Benextramine; Clonidine; Desipramine; Mesenteric artery; Prazosin; Right atria; Yohimbine; and yohimbine hydrochloride (PubChem CID: 6169); benextramine tetrahydrochloride (PubChem CID: 2317); clonidine hydrochloride (PubChem CID: 20179); desipramine hydrochloride (PubChem CID: 65327); prazosin hydrochloride (PubChem CID: 68546); α(2)-adrenoceptors