Figure 3

UROtsa cell β-AR activation stimulates production of pro-inflammatory mediators. UROtsa cells were incubated with 100 nM isoproterenol or 300 nM lipopolysaccharide (LPS) in serum-free DMEM for the indicated times. Cells were lysed in modified RIPA and total cell lysates were processed as described under "Methods" for immunoblotting with anti-COX-2 or anti-iNOS antibody to determine β-AR mediated changes in protein expression. As reported by the antibody supplier, COX-2 is identified as a doublet band running at 72–74 kD, and iNOS is detected as a band at 130 kD. Peak expression of the pro-inflammatory mediators COX-2 and iNOS was observed 2 hrs after addition of isoproterenol. Semi-quantitative analysis revealed that there was a significant 1.8 ± 0.3 and 2.1 ± 0.3 fold increase in COX-2 and iNOS expression, respectively when compared to basal. Similarly, a 2 hr incubation with LPS led to increased expression of COX-2 and iNOS when compared to basal. Values are presented as the mean ± S.E. and the autoradiographs are representative immunoblots of n = 3–9 independent UROtsa cell treatments.