α1-Adrenergic receptor signaling is localized to caveolae in neonatal rat cardiomyocytes

In neonatal rat cardiomyocytes, phosphatidylinositol(4,5)bisphosphate (PIP2) is a precursor of second messengers, a stabilizer of ion channels and exchangers, an anchor point for the cytoskeleton and, in addition, can serve as a signaling molecule in its own right. We examined the possibility that sarcolemmal PIP2 exists in different pools and that only one of these provides the substrate for alpha(1)-adrenergic receptor activated phospholipase C (PLC). Membranes were separated on the basis of buoyant density, and the light lipid raft fractions were further separated into caveolae and non-caveolar rafts using immunoprecipitation. PIP2 was principally located in the light lipid raft fractions and was equally distributed between caveolae and non-caveolar membranes. Heavier membrane fractions also contained some PIP2. Addition of the alpha(1)-adrenergic receptor agonist phenylephrine (50 mu M) caused reductions in PIP2, but only in caveolae. PIP2 in other fractions was unaffected. In agreement with this, PLC beta 1 and, to a lesser extent, G alpha q were concentrated in this fraction. PLC beta 3 was primarily observed in heavier membranes. We conclude that PIP2 in cardiomyocyte sarcolemma is compartmentalized and that alpha(1)-adrenergic receptor signaling is localized to caveolac. (c) 2006 Elsevier Inc. All rights reserved.