PGE₂ receptor EP4 plays an important role in mediating increased intracellular cAMP during the cardiomyocyte response to LPS.

<p>(<i>A</i>) The effects of <i>Mkp1</i> knockdown on COX-2 mRNA induction by LPS. H9c2 cardiomyocytes were transfected with either scrambled RNA (SC) or <i>Mkp1</i> siRNA. After 48 h, cells were stimulated with LPS (5 µg/mL) for 4 h or left unstimulated. Total RNA was harvested and COX-2 expression was assessed by PCR. COX-2 expression is presented as fold change relative to unstimulated SC controls. (<i>B</i>) The effects of LPS on COX-2 protein expression. H9c2 cells were stimulated with LPS (5 µg/mL) for the indicated periods of time. COX-2 protein in lysates was assessed by Western blot. The membrane was stripped and reblotted with a β-actin antibody to verify comparable protein loading. (<i>C</i>) Detection of the mRNA transcripts of different prostaglandin receptors in H9c2 cardiomyocytes. Total RNA was extracted from unstimulated H9c2 cells. The transcripts of the receptors of PGE₂ (EP1–4) and PGD₂ (DP) were amplified by RT-PCR. PCR products were separated by electrophoresis on a 2% agarose gel. (<i>D</i>) The differential effects of prostaglandin receptor antagonists on LPS-induced increases in cAMP. H9c2 cells were first pretreated with DMSO (as control (Con)), 10 µM AH23848 (an inhibitor of EP4), or 10 µM AH 6809 (inhibitor of EP1, EP2, EP3, and DP) for 60 min, and then stimulated with LPS for an additional 18 h. Cells were harvested and intracellular cAMP levels were determined using an EIA kit. Data represent means ± SE of at least 3 experiments. *, <i>P</i><0.05. Images shown in panels <i>B</i> and <i>C</i> are representative results.