We determined whether high fatty acid oxidation rates during aerobic reperfusion of ischemic hearts could be explained by a decrease in malonyl-CoA levels, which would relieve inhibition of carnitine palmitoyl-transferase 1, the rate-limiting enzyme involved in mitochondrial uptake of fatty acids. Isolated working rat hearts perfused with 1.2 mM palmitate were subjected to 30 min of global ischemia, followed by 60 min of aerobic reperfusion. Fatty acid oxidation rates during reperfusion were 136% higher than rates seen in aerobically perfused control hearts, despite the fact that cardiac work recovered to only 16% of pre-ischemic values. Neither the activity of carnitine palmitoyltransferase 1, or the IC50 value of malonyl-CoA for carnitine palmitoyl-transferase 1 were altered in mitochondria isolated from aerobic, ischemic, or reperfused ischemic hearts. Levels of malonyl-CoA were extremely low at the end of reperfusion compared to levels seen in aerobic controls, as was the activity of acetyl-CoA carboxylase, the enzyme which produces malonyl-CoA. The activity of 5'-AMP-activated protein kinase, which has been shown to phosphorylate and inactivate acetyl-CoA carboxylase in other tissues, was significantly increased at the end of ischemia, and remained elevated throughout reperfusion. These results suggest that accumulation of 5'-AMP during ischemia results in an activation of AMP-activated protein kinase, which phosphorylates and inactivates ACC during reperfusion. The subsequent decrease in malonyl-CoA levels wil result in accelerated fatty acid oxidation rates during reperfusion of ischemic hearts.

Prostaglandin E 2 (PGE 2 ) couples to stimulation of adenylyl cyclase through two distinct G protein-coupled receptors designated EP2 and EP4. Although they have similar affinities for PGE 2 , the EP 2 and EP4 receptors have distinct structural characteristics. EP2 is a 358-amino-acid protein with short third intracellular loop and C-terminal domains, whereas EP4 consists of 488 amino acids with a long third intracellular loop and a long cytoplasmic tail. The ability of the HA epitope-tagged receptors to undergo PGE 2 -induced internalization was examined by enzyme-linked immunosorbent assay and immunofluorescence microscopy after expression in human embryonic kidney 293 cells. The EP2 receptor did not internalize, whereas the EP4 receptor underwent rapid internalization. Truncation of the EP4 receptor after amino acid 350, which removes 138 residues, abolished internalization. Truncation after amino acid 369 markedly attenuated internalization, whereas truncation after amino acid 383 had little effect. Serine and threonine residues in the region 350 to 383 were mutated to determine their role in internalization. The mutants S370-382A, a fulllength receptor containing six serine-to-alanine mutations in the region 370 to 382, and S354-369A, containing four serine mutations and one threonine mutation in the region 350 to 370, both internalized to the same extent as the wild-type. A further mutant, designated S354-382A, containing amino acid substitutions S354A, S359A, S364A, S366G, T369A, S370A, S371A, S374A, S377A, S379A, and S382A, also internalized to the same extent as the wild-type. We conclude that the C terminus of the EP4 receptor is involved in internalization; however, serine and threonine residues do not seem to be involved. PGE 2 is an important autocrine mediator in the cardiovascular and other systems (Campbell and Halushka, 1996). PGE 2 is a potent vasodilator in the microvasculature, although it may also cause constriction at selected sites. It causes a fall in systemic blood pressure and an increase in blood flow to the heart; it increases blood flow to the kidneys, leading to increased diuresis, natriuresis, and kaluresis, and causes secretion of renin from the renal cortex. PGE 2 exerts its actions through four distinct G protein-coupled receptors, designated EP1, EP2, EP3, and EP4, that are encoded by different genes (for a review, see Negishi et al., 1995). The EP1 receptor couples to phospholipase C, the EP2 and EP4 receptors couple to stimulation of adenylyl cyclase, and the EP3 receptor couples to inhibition of adenylyl cyclase.EP2 and EP4 receptors are widely distributed; both receptors are found in uterus, spleen, and lung; the EP4 receptor is also present in small intestine, thymus, pancreas, leukocytes, and kidney (Regan et al., 1994;Katsuyama et al., 1995). There are no selective EP receptor antagonists that can be used in studies to functionally distinguish the two receptors. However, the selective EP2 receptor agonist butaprost is not active at the EP4 receptor (Regan et al.,...

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We examined the pathway of prostaglandin E 2 (PGE 2 )-induced internalization of the prostaglandin EP4 receptor in HEK 293 cells. Co-expression of dominant negative L Larrestin (319^418) or dynamin I (K44A) with the EP4 receptor reduced internalization. The activated receptor co-localized with GFP-arrestin 2 and GFP-arrestin 3, confirming the requirement for L L-arrestins in internalization. Inhibition of clathrin-coated vesicle-mediated internalization resulted in inhibition of sequestration, whereas inhibition of caveola-mediated internalization had no effect. PGE 2 stimulation of the EP4 receptor resulted in rapid mitogen-activated protein (MAP) kinase activation. Examination of an internalization-resistant mutant and coexpression of mutant accessory proteins with EP4 revealed that MAP kinase activation proceeds independently of internalization. ß

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