Figure 3, Panel is a representative immunoblot (= 3) of these studies showing IL-1 production changes in response to these individual or combined LPS and Iso treatments. generated Iso concentration-response curves in the presence of fixed subtype-selective -AR antagonist concentrations to demonstrate that 1-AR activation was exclusively linked with the generation of cAMP in THP-1 cells. Furthermore, use of a selective kinase inhibitor demonstrated that Iso potentiated the expression of soluble IL-1 through activation of cAMP-dependent protein kinase A. Finally, discriminating concentrations of subtype-selective -AR antagonists revealed that 1-AR stimulation alone accounted for the synergistic production of IL-1 in LPS stimulated monocytes co-incubated with Iso. These results demonstrate a unique synergistic pro-inflammatory response mediated through a 1-AR cAMP-dependent mechanism in LPS challenged monocytic cells. preparation of human mononuclear leukocytes (MNL), which comprise a mixed population of monocytes, lymphocytes WNT3 (adaptive immunocompetent cells) and platelets (Motulsky, et al., 1986). A subsequent investigation utilizing oral administration of subtype-selective -AR antagonists to block isoproterenol (Iso) induced changes in -AR density and generation of cAMP documented a 2-AR population in this same MNL preparation (van Tits, et al., 1990). Although an elegant analysis, this investigation did not quantify direct receptor interactions for subtype-selective -AR competitive antagonists and thus heterogeneous expression of -AR subtypes cannot be ruled out in this mixed population of immunocompetent cells. Nonetheless, and preparations of human monocytes are considered to solely express the 2-AR subtype, whose activation has further been shown to have anti-inflammatory effects resulting in dampening of the Rocuronium innate immune response to infection or injury (Farmer and Pugin, 2000; Mizuno, et al., 2005; van der Poll, et al., 1994). However, complicating the literature are reports of 1-AR subtype expression in preparations of human monocytes or pro-inflammatory responses attributed to 2-AR activation, suggesting Rocuronium a pluripotent 2-AR effect in these same cells (Kavelaars, et al., 1997; Szelenyi, et al., 2006; Talmadge, et al., 1993). In this study we tested the hypothesis that pro-inflammatory outcomes of -AR activation in monocytes are not due to the bi-functional ability of a single receptor, but instead are related to the signaling capacity for a specific -AR subtype expressed in a heterogeneous receptor population. We explored the dichotomous -AR inflammatory response in human monocytes that had been simultaneously incubated with the bacterial endotoxin, lipopolysaccharide (LPS). We characterize the expression of both 1- and 2-AR subtypes on human monocytes, which when stimulated concomitantly with LPS and Iso generated a unique synergistic increase in IL-1 production. Using subtype-selective receptor antagonists, we observed that this novel pro-inflammatory response is associated with exclusive activation of the 1-AR subtype and was functionally correlated to the generation of cAMP along with subsequent activation of protein kinase A (PKA). Our results are the first to demonstrate, using classical pharmacological techniques, a pro-inflammatory effect of 1-AR activation in human monocytes that have been pathogenically challenged to initiate an inflammatory Rocuronium response. EXPERIMENTAL Materials and Methods Cell Culture A human monocytic cell line, THP-1 (ATCC, Manassas, VA) was propagated using Rocuronium standard cell culture conditions (37 C/5% CO2) in RPMI 1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose and 10 mM HEPES (complete media), supplemented with 10% heat inactivated fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA). Confluent THP-1 cells (106 cells/mL) were washed in serum-free complete medium, allowed to become quiescent for 30 min before pre-incubating with or without AR antagonists or inhibitors of PKA for 60 min prior to addition of the selective -AR agonist, Iso (Sigma-Aldrich, St. Louis, MO) and/or.