Rates of onset of contraction of rat aorta to octopamine for calculation of receptor affinities (Km) and to distinguish responses mediated via α 1 - and trace amine-associated receptors

KJ Broadley, C Richards. Cardiff University, Cardiff, UK

The “Occupation Theory” of drug-receptor interaction states that response is proportional to the number of receptors occupied. The “Rate Theory” of Paton (1) argued that the magnitude of response is proportional to rate of drug-receptor interaction, but is now generally discounted. However, in a related approach, we have used the rate of contraction and borrowed from enzyme kinetics to apply the Lineweaver-Burk plot to drug-receptor interactions. The rates of onset of contraction of rat aorta to octopamine were determined in the absence and presence of the α1-adrenoceptor antagonist prazosin. Octopamine acts directly on α1-adrenoceptors (2) and trace amine-associated receptors-1 (TAAR-1) in rat aorta (3) and this study attempts to distinguish these mechanisms by calculating the rate of reaction (Km) which is equivalent to receptor affinity.

Aortic rings from male Sprague–Dawley rats were passed onto fixed and mobile hangers and isometric contraction measured using a computerized Power Lab, Chart 5 data acquisition system (ADInstruments). Endothelium was removed and the rings immersed in Kreb’s solution gassed with 5% CO2 in O2, and 1.5g resting tension applied. Tissues were incubated with 10µM cocaine HCl, 10µM pargyline HCl and 1µM ICI 118,551 HCl to block indirect sympathomimetic actions, monoamine oxidase and β2-adrenoceptors, respectively. Cumulative or non-cumulative octopamine concentration-response curves (CRC) were obtained in the absence and presence of α1-antagonist (prazosin 1, 10 and 30µM). Geometric mean -log EC50 values ± SEM (n=4) were calculated and compared by ANOVA + Bonferroni post hoc tests. Contractions were measured by subtracting baseline tension from plateau responses. Rates of onset and offset of non-cumulative contractions were found from the gradient of the line for responses measured every 15s over the first 1-2 mins.

Cumulative CRCs for octopamine were shifted to the right by prazosin 1µM (EC50 from 3.49±0.09 to 2.56±0.06). There was no further shift on increasing concentration to 10 (EC50 2.31±0.03) and 30µM (2.30±0.001). There was no shift with the prazosin vehicle, 0.3ml DMSO. Thus, α1-adrenoceptors were no longer involved in the vasoconstriction in the presence of 1µM prazosin and this concentration was used to study TAAR-1-mediated responses. Rates of onset of contractions to 100µM octopamine alone and 3000µM octopamine with prazosin were compared as they caused similar ~70% maximum contractions. Rates of onset of contraction in the presence of 1µM (0.52±0.07mg/s) and 10µM (0.41±0.05mg/s) prazosin were significantly slower than in its absence (1.49±0.23mg/s). Lineweaver-Burk plots of 1/rate vs 1/concentration for each non-cumulative response yielded straight lines from which the rate of reaction, Km (equivalent to receptor affinity) = gradient x Vmax (1/yintercept). –logKm in the presence of 1µM (2.74±0.14) and 10µM (2.39±0.23) prazosin were significantly less than in its absence (3.92±0.67). Therefore affinity for the receptor in presence of α1-blockade was significantly less than for α1-adrenoceptors. Rates of offset of contraction after washout of octopamine were, however, not significantly different in the absence (-2.14±0.65) and presence of prazosin (1µM, -1.47±0.68; 10µM, -2.08±0.96).

Octopamine contracts rat aorta via α1-adrenoceptors and by a non-adrenergic response in the presence of prazosin, probably via TAAR-1, which has a significantly slower onset rate.

1. Paton WDM. (1961). Proc Royal Soc B 154: 21-69.

2. Brown CM et al. (1988). Br J Pharmacol 93: 417-429.

3. Broadley KJ (2010). Pharmacol Ther 125: 363–375.