Abstract:
:Co-expression of EP2 and EP3 subtypes of prostaglandin E2 (PGE2) receptors (R) by U937 human monocytic cells permitted comparative studies of desensitization of each subtype. Specific binding of [3H]PGE2 to membranes of U937 cells showed a Kd of 2.9 +/- 0.3 nM (mean +/- SEM) and a Bmax of 40.5 +/- 1.0 fmol/mg protein, and was competitively inhibited by PGE2 > or = PGE1 > PGF2 alpha > PGD2 > PGI2. EP2 R and EP3 R mRNA were detected by reverse transcription-polymerase chain reaction and Northern blots. EP3 R expression was demonstrated by inhibition of [3H]PGE2 binding with the EP1/EP3 agonist sulprostone [50% inhibitory concentration (IC50 = 3.3 +/- 0.6 nM)] and the EP3/EP2 agonist M&B 28767 (IC50 = 2.1 +/- 0.3 nM), but not with the EP1 antagonist SC-19220. EP2 R protein was identified by Western blot analysis using specific rabbit IgG antibodies to an amino-terminal peptide of the EP2 R. EP2 R transduced PGE2 stimulation of significant increases in cellular [cAMP]i [50% effective concentration (EC50 = 20 +/- 2.5 nM)], and EP3 R mediated sulprostone inhibition of forskolin elevation of [cAMP]i (IC50 = 1.3 +/- 0.4 nM). Pretreatment of U937 cells with phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), for 1 hr reduced the total number, but not the affinity, of PGE2 R by down-regulating principally EP2 R. In contrast, a 24-hr exposure to PMA, which is known to down-regulate PKC, suppressed both the total number and affinity of PGE2 R on U937 cells with concurrent reductions in EP2 R and EP3 R. The down-regulation of EP2 R by PMA at 1 hr was blocked by staurosporine, an inhibitor of PKC, whereas the down-regulation of EP3 R by PMA at 24 hr was blocked by indomethacin. Pretreatment of U937 cells with PGE2 for 1 and 24 hr reduced both the binding affinity and the total number of PGE2 R, by co-ordinate suppression of the EP2 R and EP3 R. Desensitization of EP2 R and EP3 R for 1 hr with PGE2 suppressed subsequent PGE2-evoked chemokinetic responses to PGE2, whereas selective down-regulation of EP2 R alone by PMA for 1 hr had no effects on U937 cell migration. Thus expression of each subtype of PGE2 R is regulated independently and EP3 R, but not EP2 R, transduces PGE2 effects on migration of mononuclear phagocytes.
journal_name
Immunologyjournal_title
Immunologyauthors
Zeng L,An S,Goetzl EJsubject
Has Abstractpub_date
1995-12-01 00:00:00pages
620-8issue
4eissn
0019-2805issn
1365-2567journal_volume
86pub_type
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