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HEART

Endothelin-1 Mobilizes Profilin-1–Bound PIP₂ in Cardiac Muscle

Department of Physiology, University of Wisconsin–Madison, Madison, Wisconsin 53706

To whom requests for reprints should be addressed at ¹ Department of Physiology, University of Wisconsin, 1300 University Avenue, Madison, WI 53706. E-mail: jwalker{at}physiology.wisc.edu

Abstract

Phosphatidylinositol 4,5-bisphosphate (PIP₂) is a key down-stream substrate of the endothelin signaling pathway and plays a role in regulating protein function at the membrane-cytoskel-etal interface. However, the dynamic properties of distinct pools of PIP₂ are poorly understood, especially for PIP₂ that is bound to cytoskeletal proteins. We investigated the effects of endothelin-1 (ET-1) stimulation on protein-bound PIP₂ in cardiac muscle. Isolated rat myocytes and homogenized mouse ventricles were exposed to 10 nM ET-1 for varying time periods and protein-bound PIP₂ was analyzed using an anti-PIP₂ antibody and Western blotting. Several cytoskeletal proteins were found to contain tightly bound PIP₂, including profilin-1 (~15 kDa), capZ (~32 kDa), gCap39, (~39 kDa) and -actinin (~106 kDa). Interestingly, ET-1 pretreatment reduced the amount of PIP₂ bound to profilin-1 by 46% after 15 mins, followed by a recovery to near basal levels after 60 mins. ET-1 had no effect on capZ-, gCap39-, or -actinin–bound PIP₂ levels. To further explore the dynamics of PIP₂ binding, brefeldin-A (BFA) was used to disrupt PIP₂ binding to ADP-ribosylation factors and to impair receptor internalization. Pretreatment with 1 µM BFA increased the PIP₂ signal on profilin-1 x54% after 15 mins, followed by a decline to subbasal levels after 60 mins. Like ET-1, BFA had no effect on levels of PIP₂ bound to capZ or to -actinin. Taken together, the data indicate that profilin-1 binds PIP₂ dynamically and may serve as a key regulator of the balance between cytoskeletal integrity and PIP₂ availability for Ca²⁺/PKC signaling in the heart.

Key Words: brefeldin-A • -actinin • phosphoinositides

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