Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC247
Calcium stimulated adenylyl cyclase modulates ion channel currents in the guinea-pig atrioventricular node
K. Yuill1,2, D. Terrar2
1. Biomedical and Biological Sciences, University of Plymouth, Plymouth, United Kingdom. 2. Department of Pharmacology, University of Oxford, Oxford, United Kingdom.
The atrioventricular node (AVN) is essential to cardiac function, forming the only pathway between the atria and ventricles, crucially timing ventricular filling and contraction. There is currently little information available regarding ion channel regulation by intracellular second messengers, and in particular the contribution of cyclical changes in intracellular calcium to ion channel function. cAMP is fundamental to cellular function, and its levels are maintained in dynamic equilibrium via breakdown by phosphodiesterases (PDE) and production by adenylyl cyclase (AC). Calcium stimulated ACs, which have been shown to play an important role in neuronal function, have been identified in sinoatrial nodal tissue, but are absent from ventricular tissue (Mattick et al., 2007). Adult male guinea-pigs (~400g) were killed by cervical dislocation. The heart was rapidly removed and retrogradely perfused with a collagenase solution using a modified langendorff apparatus. The AVN region was excised and cells isolated using previously described methods (Hancox et al., 1993; Yuill et al., 2010). Here we present evidence of calcium dependent AC activity in individual atrioventricular nodal myocytes from the guinea-pig, using perforated patch clamp electrophysiology to record ionic currents and action potentials, and confocal line scan imaging with Fluo-5 to record spontaneous calcium transients. Experiments were only conducted on healthy AVN myocytes exhibiting regular spontaneous activity. All recordings were carried out at 37οC, using standard physiological potassium-based solutions. Statistical analysis was performed using a paired t-test, with Bonferroni post-hoc correction. Inhibition of sarcoplasmic reticulum activity with cyclopiazonic acid (10μM) or ryanodine (1μM) induced a cessation in action potential firing, highlighting the importance of spontaneous calcium release to the generation of spontaneous electrical activity in AVN myocytes. We demonstrated the functional importance of calcium dependent AC activity on cellular electrical activity and calcium release, by addition of inhibitors of AC (MDL 12330A) and PDE (IBMX) activity. Application of 10μM MDL 12330A significantly attenuated spontaneous action potential firing rate, induced a negative shift in the activation of a hyperpolarisation activated non-selective cation current (‘funny current’, If,), inhibited L type calcium channel (ICaL) amplitude (65.3 ± 2.3%, S.E.M, n=5), and reduced the frequency of spontaneous calcium release (P=0.05, n>5). Conversely, IBMX caused a positive shift in the voltage dependence of If activation and an increase in the frequency of spontaneous calcium release. These effects are consistent with the presence of calcium dependent AC in AVN myocytes, which is integral to the generation of spontaneous electrical activity in these cells.
Where applicable, experiments conform with Society ethical requirements