Novel technique to determine the pKA of clonidine at prejunctional α2-adrenoceptors in cardiac and vascular sympathetic transmission

James A Angus; Pravin Rajasekaran; Christine E Wright

doi:10.1016/j.ejphar.2017.02.030

Novel technique to determine the pK_A of clonidine at prejunctional α₂-adrenoceptors in cardiac and vascular sympathetic transmission

Eur J Pharmacol. 2017 Apr 5:800:81-95. doi: 10.1016/j.ejphar.2017.02.030. Epub 2017 Feb 17.

Authors

James A Angus¹, Pravin Rajasekaran², Christine E Wright³

Affiliations

¹ Cardiovascular Therapeutics Unit, Department of Pharmacology and Therapeutics, University of Melbourne, Victoria 3010, Australia. Electronic address: jamesaa@unimelb.edu.au.
² Cardiovascular Therapeutics Unit, Department of Pharmacology and Therapeutics, University of Melbourne, Victoria 3010, Australia. Electronic address: rajasekaran@wehi.edu.au.
³ Cardiovascular Therapeutics Unit, Department of Pharmacology and Therapeutics, University of Melbourne, Victoria 3010, Australia. Electronic address: cewright@unimelb.edu.au.

PMID: 28219708
DOI: 10.1016/j.ejphar.2017.02.030

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 K_A of 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 pK_A determined by global fitting and display in the Clark plot. The pK_A for clonidine ranged from 8.95 in the mouse, 7.8 in rat and 8.3 in guinea pig. The propranolol pK_B was 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 pK_A of 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 pK_B of 9.08 in rat and 9.03 in mouse arteries. We conclude that pK_B for antagonists and pK_A for the prejunctional inhibitors of nerve transmission can be determined with this novel analytical approach.

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.

MeSH terms

Animals
Clonidine / pharmacology*
Cystamine / analogs & derivatives
Cystamine / pharmacology
Desipramine / pharmacology
Dose-Response Relationship, Drug
Guinea Pigs
Heart Atria / drug effects
Heart Atria / innervation*
Male
Mesenteric Arteries / drug effects
Mesenteric Arteries / innervation*
Mice
Norepinephrine / pharmacology
Rats
Receptors, Adrenergic, alpha-2 / metabolism*
Sympathetic Nervous System / drug effects*
Sympathetic Nervous System / physiology*
Synaptic Transmission / drug effects*
Yohimbine / pharmacology

Substances

Receptors, Adrenergic, alpha-2
Yohimbine
benextramine
Clonidine
Cystamine
Desipramine
Norepinephrine