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Introduction The sympathetic nervous system (SNS) has significant responsibility in the action of cardiac functions of amphibians such as the cane toad Bufo marinus ( Erlij et al. , 1965). Agonists and antagonists such as adrenaline and propranolol, respectively, are known to act on α- and β-adrenoc...

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Introduction The sympathetic nervous system (SNS) has significant responsibility in the action of cardiac functions of amphibians such as the cane toad Bufo marinus (Erlij et al., 1965). Agonists and antagonists such as adrenaline and propranolol, respectively, are known to act on α- and β-adrenoceptors found on cardiomyocytes which are G protein-coupled receptors (GPCR) (Morris et al., 1981). The SNS act on these β-adrenoceptors to mediate heart contractility through the modulation of ionic channels, particularly the L-type Ca2+ channel, which increases intracellular concentration of cyclic adenosine monophosphate (cAMP) (Chen et al., 1999). Increased cAMP concentration most importantly activates ryanodine receptors (RyR2), which release Ca2+ into the intracellular fluid (Chen et al., 1999). Ju & Allen (1998) have shown evidence with their study that this influx of Ca2+ affects the firing rate of pacemaker cells. The effects of adrenaline on α- and β-adrenoceptors are well known to increase intracellular Ca2+ concentration, ultimately having positive inotropic effects on the B. marinus heart (Morris et al. , 1981). Propranolol, on the other hand, is a non-selective antagonist and is known to diminish the effects of agonists such as adrenaline or isoproterenol on β-adrenoceptors (Cano-Martinez et al., 2004; Chiu & Chu, 1989). This is done by competitively blocking these receptors to produce negative inotropic effects (Lillo, 1979). However, Propranolol does not behave like adrenaline on α-adrenoceptors. This is due to the nature of adrenaline acting on both α- and β-adrenoceptors, thus harder to determine the effects of each type of receptor. Ju & Allen’s study (1999) conducted an experiment to observe if adrenaline and isoprenaline acted via the β-adrenoceptors and if the application of propranolol would be able to reverse the effects of the agonists on the B. marinus species. Their study confirmed the reversal of adrenaline and isoprenaline action through propranolol application by decreasing the transient Ca2+ concentration and firing rate of pacemaker cells,

which were previously enhanced by adrenaline. However, the study conducted by Ju & Allen (1999) did not research the effects of α-adrenoceptors in isolation, thus this current study has aimed to study the effects of α-adrenoceptors by isolating its action through the combination of adrenaline and propranolol. It was hypothesised that the topical application of adrenaline on the B. marinus heart would increase its heart rate (HR) and ventricular contractile force (VCF), application of propranolol would decrease HR and VCF, and the combination of propranolol with adrenaline on the B. marinus heart would also show an increase in HR and VCF, but not to the extent of adrenaline only. Methods The B. marinus species was dissected to expose the beating heart, and the heart was hooked through the apex of the ventricle on to the force transducer. The height of the force transducer was adjusted so that the string was taut to give a passive force reading of 6mN. A copper wire was placed through the side of the ventricle, which one end of it was wrapped around the positive ECG lead. The earth lead was clipped onto one of the hind limbs of the B. marinus, and the negative lead was clipped to the clavicle. The force transducer was connected to Lab Chart PowerLab (Version 8, GraphPad Software Inc.) and ECG and contractile force were recorded, while the temperature was kept constant at room temperature of 22oC. The force transducer was adjusted to have the baseline contractile force at 6mN and left to stabilise for 3 minutes to record baseline cardiac activity, before applying 100μl of 1mM propranolol over the heart of the B. marinus, for which data was recorded for 3 minutes. 100μl of 1 mM adrenaline was then applied immediately after the 3 minutes, and data was recorded again for another 3 minutes. Once data was obtained, the heart was lowered back down into the thoracic cavity and thoroughly rinsed with Ringer’s solution,

removing excess liquid with a paper towel. The heart was returned to its original baseline recording and a second baseline was recorded to standardise any discrepancies between the first and second baseline data. For the second condition, 100μl of 1mM adrenaline only was applied over the toad heart, and data was obtained over 3 minutes. Due to the nature of the experiment, only technical replicates were obtained by splitting the 3-minute intervals recorded in the first and second experiment into 1-minute intervals to act as the replicates. Results A paired t-test was used to analyse the difference between the two baselines and found the difference to be significant, hence data was transformed to show percentage change from baseline. Figure 1A shows a significant difference between the VCF of baseline and PROP group, PROP and PROP+ADR group, baseline and PROP+ADR group, baseline and ADR group, and PROP+ADR and ADR group, an 8.87% (±0.76%) decrease, 3.29% (±0.76%) decrease, 12.2% (±0.76%) decrease, 56.4% (±0.76%) increase, and a 68.6% (±0.76%) increase respectively. On the contrary, figure 1B shows no significance in the differences between the HR of baseline and PROP group, baseline and PROP+ADR group, and baseline and ADR group (ns, p>0.05). However, there was significance in the difference between the baseline and PROP+ADR group and between the PROP+ADR and ADR group, of 37.0% (±3.7%) and 17.1% (±1.9%) respectively.

Figure 1: A) Effects of antagonist and agonist on VCF (%) of B. marinus. Graph represents the VCF without any drug application (baseline) and after drug application of propranolol only (PROP), combination of propranolol and adrenaline (PROP+ADR), and adrenaline only (ADR). B) Effects on HR (%) without any drug application (baseline) and after drug application (PROP, PROP+ADR, ADR). Each drug was of 1mM and 100μl of each were applied in each group. Data in both A & B are means of triplicate measurements recorded and error bars represent 1 standard error of the mean (SEM). Statistical analyses were done by one-way ANOVA followed by Tukey’s multiple comparisons test for each parameter (ns= no significance, p>0.05; *=p...