Novel technique to determine the pKA of clonidine at prejunctional α2-adrenoceptors in sympathetic transmission
Professor James Angus
+61 3 834 45896
For many years it has been known that clonidine, a powerful and potent antihypertensive drug, could inhibit sympathetic transmission centrally and peripherally by inhibiting transmitter noradrenaline release. In effect, clonidine acting as an agonist at prejunctional α2-adrenoceptors results in the decrease of noradrenaline concentrations at post-junctional β-adrenoceptors in the heart, for example. But, normal kinetics of equilibrium do not apply here because the agonist action only results in an effect when the nerve varicosity is depolarised leading to a transient release of transmitter that can be measured by an increase in atrial rate or contraction of an artery.
We have used the robust isolated right atrial assay of mouse, rat and guinea pig and increase in rate response with low (1-4) numbers of electrical field pulses to measure the effect of clonidine that shifts the response lines to the right. Low numbers of field pulses prevent autoinhibitory feedback. By equating the peak response to the field pulses with the response to cumulative additions of exogenous noradrenaline, we determined the pKA for clonidine using global fitting analysis and Clark plot. The pKA for clonidine varied from 8.95 in mouse, 7.87 in rat and 8.3 in guinea pig atria. To calibrate this assay and technique for nerve-released transmitter, we tested propranolol which had a pKB of 8.87 in mouse and 8.91 in rat atria; very similar values as for equilibrium agonist/antagonist assays. In mesenteric small arteries mounted in a myograph, the electrical field stimulation was inhibited by clonidine with a pKA of 7.1 in mouse arteries, but clonidine was inactive in rat arteries due to competing autoinhibitory feedback.
In summary, we have developed a novel approach to estimate the pKA of clonidine under varying physiological conditions and sites in the sympathetic nervous system. It just goes to show that revisiting scientific questions started nearly 37 years ago with fresh thinking and approach can unravel scientific beauty in the most elegant of functional, calibrated tissue assays. The search for mechanism is dependent on rigorous assays with sound analytics.
Associate Professor Christine E. Wright, BSc Hons, PhD, Laboratory Head,
Mr Mark Ross-Smith, BSc Hons, Senior Research Assistant
- Angus JA, Rajasekaran P, Wright CE. Novel technique to determine the pKA of clonidine at prejunctional α2-adrenoceptors in cardiac and vascular sympathetic transmission. Eur J Pharmacol 2017; 800: 81-95.
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