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 sarcoplasmi c reticulum (SR) plays an important role in the regulation of heart rate. T he aim of this study was to investigate the subcellular distribution of rya nodine receptors in the guinea-pig sino-otrial (SA) node and to determine t heir functional role in the regulation of pacemaker frequency in response t o beta -adrenoceptor stimulation. Methods: Monoclonal antibodies raised aga inst the cardiac ryanodine receptor were used with confocal microscopy to i nvestigate ryanodine receptor distribution in single guinea-pig SA node cel ls. The functional role of ryanodine receptors was investigated in both mul ticellular SA node/atrial preparations and in single SA node cells. Results : Ryanodine receptor labelling was observed in all SA node cells studied an d showed both subsarcolemmal and intracellular staining. In the latter, lab elling appeared as transverse bands with a regular periodicity of similar t o2 mum. This interval resembled that of the expected sarcomere spacing but did not, however, depend on the presence of transverse tubules. The bands o f ryanodine receptors appeared to be located in the region of the Z lines, based on co-distribution studies with antibodies to alpha -actinin. myomesi n and binding sites for phalloidin. Functional studies on single SA node ce lls showed that application of ryanodine (2 mu mol/l) reduced the rate of f iring 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 chrono tropic actions of isoprenaline in both multicellular and single cell prepar ations. In single cells exposed to 100 nmol/l isoprenaline, ryanodine cause d a decrease in the rate of firing and this was associated with a decrease in the amplitude of the measured calcium transients. Conclusions: These fin dings are the first to show immunocytochemical evidence for the presence an d organisation of ryanodine receptor calcium release channels in mammalian SA node cells. This study also provides evidence of a role for ryanodine se nsitive sites in the beta -adrenergic modulation of heart rate in this spec ies. (C) 2000 Elsevier Science B.V. All rights reserved.