Localisation and functional significance of ryanodine receptors during beta-adrenoceptor stimulation in the guinea-pig sino-atrial node.

OBJECTIVE Recent evidence shows that calcium released from the sarcoplasmic reticulum (SR) plays an important role in the regulation of heart rate. The aim of this study was to investigate the subcellular distribution of ryanodine receptors in the guinea-pig sino-atrial (SA) node and to determine their functional role in the regulation of pacemaker frequency in response to beta-adrenoceptor stimulation. METHODS Monoclonal antibodies raised against the cardiac ryanodine receptor were used with confocal microscopy to investigate ryanodine receptor distribution in single guinea-pig SA node cells. The functional role of ryanodine receptors was investigated in both multicellular SA node/atrial preparations and in single SA node cells. RESULTS Ryanodine receptor labelling was observed in all SA node cells studied and showed both subsarcolemmal and intracellular staining. In the latter, labelling appeared as transverse bands with a regular periodicity of approximately 2 microm. This interval resembled that of the expected sarcomere spacing but did not, however, depend on the presence of transverse tubules. The bands of ryanodine receptors appeared to be located in the region of the Z lines, based on co-distribution studies with antibodies to alpha-actinin, myomesin and binding sites for phalloidin. Functional studies on single SA node cells showed that application of ryanodine (2 micromol/l) reduced the rate of firing of spontaneous action potentials (measured using the perforated patch clamp technique) and this was associated with changes in action potential characteristics. Ryanodine also significantly decreased the positive chronotropic actions of isoprenaline in both multicellular and single cell preparations. In single cells exposed to 100 nmol/l isoprenaline, ryanodine caused a decrease in the rate of firing and this was associated with a decrease in the amplitude of the measured calcium transients. CONCLUSIONS These findings are the first to show immunocytochemical evidence for the presence and organisation of ryanodine receptor calcium release channels in mammalian SA node cells. This study also provides evidence of a role for ryanodine sensitive sites in the beta-adrenergic modulation of heart rate in this species.