Figure 4. G protein, adenylate cyclase, and cAMP generation in orbital fibroblasts and fibrocytes.

(A) Confluent cultures were treated with bTSH (5 mIU/mL), PGE₂ (1 µM), forskolin (20 µM) or nothing (control) for 16 h. Cell layers were analyzed for cAMP content and protein determination. (B) Adenylate cyclase mRNA levels were determined in orbital fibroblasts and fibrocytes from healthy donors (n = 5) and those with GD (n = 5). (C) RNA from orbital fibroblasts, fibrocytes, and thyroid tissue was subjected to RT-PCR for Gq and Gs using the C_T method. Data are expressed as mean±SD of triplicate determinations from three different strains of each. (D) Cultures indicated were treated with nothing, bTSH, PKA inhibitors H89 (10 µM), KT5720 (10 µM) or Rp-cAMP (1 mM) alone or in the combinations indicated for 6 h. RNA was extracted and subjected to RT-PCR for IL-6. Signals were normalized to GAPDH. Data are expressed as the mean ± SD of fold-change in three independent determinations from a single experiment, representative of three experiments performed. In 3 separate experiments, H89 failed to inhibit TSH-dependent IL-6 expression (1.62±0.23-fold and 1.06±0.3-fold increase in fibroblasts and fibrocytes, respectively vs TSH alone).