Investigating the functional role of adenosine receptor agonists on the contractility of canine isolated left atria in vitro

Emma Coles, Andrew Gray, Sidath Katugampola, & Carolyn Napier, Candidate Research Group, Pfizer, Sandwich, Kent, CT13 9NJ, UK.

Activation of adenosine receptors in the dog is known to produce cardiovascular effects in vivo, including increases in heart rate and contractility (e.g. Zhao et al., 2003). However, there are no reported in vitro studies examining the direct effects of adenosine receptor agonists on the contractility of isolated cardiac tissue in the dog. In this study the effects of a range of adenosine receptor agonists were compared in the canine isolated, electrically field stimulated (EFS) left atria (LA) preparation, a tissue that has been widely used to determine the inotropic effects of compounds in vitro (Priola et al., 1994).

Hearts were removed from beagle dogs of either sex (10-20kg) euthanised under anesthesia (pentobarbitone 0.5ml/kg, animals were used in other pharmacological studies, protocols were ethically reviewed by Pfizer ethics committee). Strips of LA (2 x 8 mm) were mounted in 15 ml organ baths containing oxygenated 37˚C Krebs and caused to rhythmically contract by EFS (10V, 5msec, 1Hz). After an equilibration period (at isometric tension of 1g) cumulative concentration response curves (CCRC) were constructed to isoprenaline (1nM-100 μM) to determine tissue viability. Following wash-out, CCRC (1nM-100 μM) to NECA (non-selective), CCPA (A1/A3 preferring), CGS21680 (A2A selective) (Fredholm et al., 2001) or vehicle (0.000001%-1%DMSO) were constructed. In separate tissues, the effect of these agents on 10 μM isoprenaline stimulated tissues in the presence of EFS was examined. Subsequently, the effect of carbachol either in the absence or presence of 100nM CGS21680 was investigated. Where available mean pEC50/Emax± s.e.mean were expressed, n-values refer to the number of dogs. Data were compared using Student’s t-test, with significance set at P<0.05.

Table 1. Mean pEC 50 and Emax ± s.e.mean of adenosine receptor agonists in canine LA.

* EC 50 could not be derived; response was similar to DMSO and curve incomplete at 100 μM. (-) Emax values denotes negative inotropic effects.

Adenosine receptor agonists produced weak direct negative inotropic effects in the canine isolated LA (see table 1). These effects were apparent at concentrations more than 500-fold their binding affinity at their subtype specific human receptor (Fredholm et al., 2001). The potencies in the dog LA are more than 100-fold weaker compared with guinea pig (Ford & Broadley, 1997), and thus may highlight a species difference. The negative inotropic effect produced under basal tension by NECA and CCPA were significantly (P<0.05) more potent, compared to tissues stimulated with isoprenaline (used to mimic sympathetic tone). There was no evidence for A 2A-mediated functional cardiac responses on either basal tension, isoprenaline stimulated or on the carbachol CCRC (used to mimic parasympathetic inhibition). In conclusion, canine cardiac LA tissues may not be the most appropriate in vitro model to investigate functional cardiac activity of compounds that bind to adenosine receptor subtypes.

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