Biphasic redistribution of muscarinic receptor and the altered receptor phosphorylation and gene transcription are underlying mechanisms in the rat heart during sepsis

Objective: The purpose of this study was to investigate intracellular redistribution of muscarinic cholinergic receptor (m2AChR) and the roles of receptor phosphorylation and gene transcription as underlying mechanisms in the rat heart during different phases of sepsis. Methods: Sepsis was induced by cecal ligation and puncture (CLP). The density of m2AChR in the sarcolemmal and Light vesicle fractions was studied using [H-3]-quinuclidinyl benzilate ([H-3]-QNB). Phosphorylation of m2AChR was studied by labeling of the myocardial ATP pool by perfusing isolated hearts with [P-32]H3PO4 followed by identification of the phosphorylated m2AChR with SDS-PAGE. The steady-state level of m2AChR mRNA was determined by RT-PCR and Southern blot analysis. Results: Septic rat hearts exhibit an initial hypercardiodynamic (9 h after CLP, early sepsis) and a subsequent hypocardiodynamic (18 h after CLP, late sepsis) state. During early sepsis, the B-max for [H-3]-QNB binding was increased in sarcolemma (+69%) but decreased in light vesicles (-22%), whereas during late sepsis, the B-max was decreased in sarcolemma (-20%) but increased in light vesicles (+32%). The sum of B-max for sarcolemmal and light vesicle fractions was increased during early sepsis (+43%) but decreased during late sepsis (-14%). The phosphorylation of m2AChR was decreased during early sepsis (-73%) but increased during late sepsis (+36% to +90%). The m2AChR mRNA abundance was increased during early sepsis (+52%) but decreased during late sepsis (-28%). Conclusions: The m2AChR in the rat heart was externalized from light vesicles to sarcolemma (overexpression) during early sepsis but internalized from surface membranes to intracellular sites (underexpression) during late sepsis. Furthermore, changes in the receptor phosphorylation and gene transcription are responsible for the biphasic redistribution and the altered expression of m2AChR in the rat heart during the progression of sepsis. (C) 2000 Elsevier Science B.V. All rights reserved.