β₁ adrenoceptors (ARs) couple to G_s proteins whereas β₂ ARs couple to both G_s and G_i. It is G_i protein activation that confines the cAMP-dependent β₂ AR signal to the membrane compartment (Chen-Izu et al. 2000). We have recently shown that disrupting caveolae (invaginated lipid raft domains) in rat ventricular myocytes enhances the inotropic response to β₂ AR stimulation, and that this effect can be mimicked by disabling G_i signalling with pertussis toxin (Calaghan & White, 2006). If G_i signalling requires caveolae, disrupting caveolae should convert the membrane-localised β₂ signal to a more diffuse signal that reaches intracellular targets such as the sarcoplasmic reticulum and the myofilaments. This hypothesis has been tested by looking at the effect of caveolar disruption on the phosphorylation of phospholamban (PLB) and one of its functional correlates (the rate of relaxation) in response to β₂ AR stimulation. Rat ventricular myocytes were treated with methyl-β-cyclodextrin (MβC) to disrupt caveolae (see Calaghan & White, 2006). β₂ AR stimulation was achieved with 50 μM salbutamol in the presence of 1 μM atenolol. All myocytes were field-stimulated (0.5 Hz) and maintained at room temperature (22-24°C). Populations of cells were fixed under basal conditions, or at 5 min after β₂ AR stimulation. Phosphorylation of PLB was measured following SDS-PAGE and Western blotting using an antibody specific for Ser¹⁶-phosphorylated PLB (Calaghan et al., 1998). Change in phosphorylation status was assessed with densitometry. Some cells were perfused with salbutamol and atenolol through a rapid-switching device and the lusitropic response indexed by t_0.5 relaxation at steady state (≈5 min). A reduction in t_0.5 relaxation in response to β₂ AR was observed in both populations of cells, but the effect was much greater (P<0.05; Student’s t test) in myocytes in which caveolae were disrupted (-25.9 ± 5.1%; mean ± S.E.M; n=19 cells) than in controls (-13.8 ± 2.9%; n=19). Preliminary data showed a 42.6 ± 10.5% increase in phosphorylation of PLB at Ser¹⁶ in response to β₂ AR stimulation in MβC-treated cells which bordered on significance (P=0.056; n=3 hearts; t test). However, no change in Ser¹⁶-phosphorylated PLB was seen following β₂ AR stimulation in controls (P>0.2; n=3). These data suggest that disruption of caveolae in the adult ventricular myocyte converts the more membrane-confined β₂ AR cAMP-dependent signal to a global β₁-like response, and provide further support for the hypothesis that coupling of G_i proteins to the β₂ AR receptor requires the spatial confinement offered by caveolae.