In cultured pituitary cells of tilapia, gonadotropin-releasing hormone (GnRH; IO11M 4-24 h), elevation of cyclic AMP (by 10 μM forskolin or 0.2 mM3-isobutyl-1-methylxanthine: IBMX 0.5-36 h) or activation of protein kinase C (PKC; by 12.5 nM tetradecanoyl phorbol-13-acetate: TPA, 0.5–24 h) all increased gonadotropin (GtH) IIβ steady state mRNA levels by three- to fourfold. The involvement of PKA and PKC in the GnRH stimulatory effect on both GtH release and GtH IIβ mRNA levels was corroborated by use of the PKA andPKC inhibitors, H89 and GF109203X, respectively (100 nM) which attenuated the GnRH effect. Incubation with actinomycin D (8 μM, 4–21 h) after preexposure for 24 h to either forskolin (10 μM) or TPA (12.5 nM), revealed that rates of transcript degradation were slower in forskolin-treated cells (T½ = 14.1 h) than in control or TPA-treated cells (T½ = 8.47 or 8.38 h), suggesting a stabilizing effect on the mRNA. Dopamine (DA; 10 μM, 4–36 h) had no apparent effect onsteady state mRNA levels of GtH Ilβ, but reduced GtH release by as much as 75%. Steady state levels of growth hormone (GH) mRNA were not affected by exposure to GnRH (10 nM, 4–24 h), although GH release was more than doubled. Similarly, activation of PKC (by TPA 12.5 nM, 1.5–36 h), which was shownto be essential for the GnRH-stimulatory effect on GH release, did not alter levels of the GH transcript, but increased GHrelease by more than fivefold. DA (10 μM, 4–24 h) moderately increased GH transcript levels (160%) with similar kinetics but lower potency than direct elevation of cAMP (by 10 μM forskolin or 0.2 mM f IBMX, 0.5-36 h) which increased transcript levels by more than fourfold. The involvement of PKA in the DA effect was confirmed when the PKA inhibitor H89 (100 nM, 15 min prior to DA exposure) attenuated the DA effect on GH mRNA levels. Exposure of cells to actinomycin D (8μM, 2–16 h) after treatment with forskolin (10 μM, 24 h) led to a slower rate of transcript degradation than in control cells (T½ = 6.5 h vs. T½ = 4.36 h), suggesting that cAMPalso elicits a stabilizing effect on GH mRNA. Somatostatin (100 nM, 0.5-36 h) had no clear effect on GH transcript levels, but reduced GH release by as much as 90%. These results suggest that activation of either cAMP-PKA or PKC pathways can, possibly by different mechanisms, stimulate mRNA levels of the GtH IIβ gene, but that only the cAMP-PKA pathway stimulates GH mRNA levels. It would appear therefore that GnRH, although stimulating GH release, does not regulate GH transcription in this fish.
- Cyclic AMP
- Gonadotropin-releasing hormone
- Growth hormone
- Protein kinases