Treatment with forskolin or PGE1 increases cAMP in K562-GPIb/V/IX cells without changing receptor surface expression.
(A) K562-GPIb/V/IX and K562-GPIb(βSer166Gly)/V/IX cells (1 × 106/mL) were treated (+Fsk) or not (-Fsk) with 50 μM forskolin. GPIb/V/IX cell surface expression was then analyzed by flow cytometry after incubation with 10 μg/mL of ALMA.12 against GPIbα (thick black line), RAM.1 against GPIbβ (gray line), ALMA.16 against GPIX (thin black line), V.1 against GPV (stippled line), or control MOPC21 (filled gray histogram). Histograms are representative of 2 separate experiments. (B) K562-GPIb/V/IX and K562-GPIb(βSer166Gly)/V/IX cells were treated with 10 μM (░) or 50 μM (▪) forskolin (Fsk) for one hour at 37°C, or with 10 μM PGE1 (PGE1) or 10 μM adrenaline (Adr) for 3 to 5 minutes at 37°C. Levels of cAMP were determined with a cAMP (125I) assay system and results are expressed as the mean ± SEM of 3 separate experiments. Surface expression of GPIb/V/IX was not affected by forskolin treatment, although forskolin and PGE1 increased cAMP levels in both cell lines.
