Extracellular Adenosine Triphosphate Triggers Arrhythmias and Elemental Redistribution in Electrically Stimulated Rat Cardiac Myocytes.

An increase in extracellular adenosine triphosphate (ATP) is arrhythmogenic in rat cardiac myocytes and extracellular ATP levels are elevated during cardiac ischemia. To gain insight into the mechanism by which the arrhythmic contractions are generated, we investigated changes in subcellular elemental content by electron probe microanalysis (EPMA) in isolated adult rat cardiac myocytes stimulated by the ATP analog, 2-methylthio-ATP (2-M-S-ATP). We also measured the effects of 2-M-S-ATP stimulation on myocyte cell shortening. In electrically stimulated myocytes, 2-M-S-ATP stimulation generated arrhythmic contractions and also increased the amplitude of cell shortening. However, only the arrhythmic contractions were reversed by 2-M-S-ATP washout. EPMA of freeze-dried cryosections of rapidly frozen 2-M-S-ATP-stimulated myocytes showed increased cytosolic Na and Cl, decreased K, but no significant change in mitochondrial Ca upon 2-M-S-ATP stimulation. The arrhythmias were abolished upon 2-M-S-ATP washout, and the observed changes in cytosolic elemental content also reversed upon agonist washout, thus suggesting that the increased Na and Cl, and decreased K, are specifically associated with the ATP-dependent spontaneous contractile activity. The observed increase in intracellular Na upon 2-M-S-ATP stimulation may explain our observation of prolonged relaxation time of 2-M-S-ATP-stimulated contractions. This may be due to inhibition of Ca(2+) efflux via the Na(+) Ca(2+) exchanger.

AT1 receptor induced alterations in histone H2A reveal novel insights into GPCR control of chromatin remodeling.

Chronic activation of angiotensin II (AngII) type 1 receptor (AT(1)R), a prototypical G protein-coupled receptor (GPCR) induces gene regulatory stress which is responsible for phenotypic modulation of target cells. The AT(1)R-selective drugs reverse the gene regulatory stress in various cardiovascular diseases. However, the molecular mechanisms are not clear. We speculate that activation states of AT(1)R modify the composition of histone isoforms and post-translational modifications (PTM), thereby alter the structure-function dynamics of chromatin. We combined total histone isolation, FPLC separation, and mass spectrometry techniques to analyze histone H2A in HEK293 cells with and without AT(1)R activation. We have identified eight isoforms: H2AA, H2AG, H2AM, H2AO, H2AQ, Q96QV6, H2AC and H2AL. The isoforms, H2AA, H2AC and H2AQ were methylated and H2AC was phosphorylated. The relative abundance of specific H2A isoforms and PTMs were further analyzed in relationship to the activation states of AT(1)R by immunochemical studies. Within 2 hr, the isoforms, H2AA/O exchanged with H2AM. The monomethylated H2AC increased rapidly and the phosphorylated H2AC decreased, thus suggesting that enhanced H2AC methylation is coupled to Ser1p dephosphorylation. We show that H2A125Kme1 promotes interaction with the heterochromatin associated protein, HP1α. These specific changes in H2A are reversed by treatment with the AT(1)R specific inhibitor losartan. Our analysis provides a first step towards an awareness of histone code regulation by GPCRs.

Site-specific cleavage of G protein-coupled receptor-engaged beta-arrestin. Influence of the AT1 receptor conformation on scissile site selection.

The discovery of beta-arrestin-related approximately 46-kDa polypeptide in transfected cells and mouse hearts led us to examine angiotensin II type 1 receptor (AT(1)R)-dependent proteolytic cleavage of beta-arrestin(s). Receptor-ligand induced proteolysis of beta-arrestin(s) is novel, especially in the endocrine system, since proteolytic and/or splice variants of nonvisual arrestins are unknown. We used a strategy to retrieve AT(1)R-engaged isoforms of beta-arrestin 1 to confirm direct interaction of fragments with this G protein-coupled receptor and determine cleavage sites. Here we show that the angiotensin II-AT(1)R complex is associated with full-length and approximately 46-kDa beta-arrestin forms. Mass spectrometric analysis of the AT(1)R-associated short form suggested a scissile site located within the Arg(363)-Arg(393) region in the bovine beta-arrestin 1. Edman degradation analysis of a beta-arrestin 1 C-terminal fragment fused to enhanced green fluorescent protein confirmed the major cleavage to be after Phe(388) and a minor cleavage after Asn(375). Rather unexpectedly, the inverse agonist EXP3174-bound AT(1)R generated different fragmentation of bovine beta-arrestin 1, at Pro(276). The angiotensin II-induced cleavage is independent of inositol 1,4,5-trisphosphate- and Ca(2+)-mediated signaling pathways. The proteolysis of beta-arrestin 2 occurs, but the pattern is more complex. Our findings suggest that beta-arrestin cleavage upon AT(1)R stimulation is a part of the unraveling beta-arrestin-mediated G protein-coupled receptor signaling diversity.