Morris James B, Huynh Huy, Vasilevski Oliver, Woodcock Elizabeth A
Cellular Biochemistry Laboratory, Baker Heart Research Institute, PO Box 6492 St. Kilda Road Central, Melbourne, 8008 Victoria, Australia.
J Mol Cell Cardiol. 2006 Jul;41(1):17-25. doi: 10.1016/j.yjmcc.2006.03.011. Epub 2006 May 30.
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 alpha1-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 alpha1-adrenergic receptor agonist phenylephrine (50 microM) caused reductions in PIP2, but only in caveolae. PIP2 in other fractions was unaffected. In agreement with this, PLCbeta1 and, to a lesser extent, Galphaq were concentrated in this fraction. PLCbeta3 was primarily observed in heavier membranes. We conclude that PIP2 in cardiomyocyte sarcolemma is compartmentalized and that alpha1-adrenergic receptor signaling is localized to caveolae.
在新生大鼠心肌细胞中，磷脂酰肌醇-4,5-二磷酸（PIP2）是第二信使的前体、离子通道和交换体的稳定剂、细胞骨架的锚定点，此外，其本身还可作为一种信号分子。我们研究了肌膜PIP2是否存在于不同的池，以及其中只有一个池为α1-肾上腺素能受体激活的磷脂酶C（PLC）提供底物的可能性。根据浮力密度对膜进行分离，使用免疫沉淀法将轻脂筏部分进一步分离为小窝和非小窝脂筏。PIP2主要位于轻脂筏部分，且在小窝和非小窝膜之间均匀分布。较重的膜部分也含有一些PIP2。添加α1-肾上腺素能受体激动剂去氧肾上腺素（50微摩尔）会导致PIP2减少，但仅在小窝中。其他部分的PIP2不受影响。与此一致的是，PLCβ1以及程度较轻的Gαq集中在该部分。PLCβ3主要在较重的膜中观察到。我们得出结论，心肌细胞膜中的PIP2是分隔存在的，并且α1-肾上腺素能受体信号传导定位于小窝。
