GH mRNA levels are elevated by forskolin but not GH releasing hormone in GHRH receptor-expressing MtT/S somatotroph cell line

The MtT/S somatotroph cell line should be a growth hormone-releasing hormon e (GHRH)-responsive model system for the study of physiological control of growth hormone (GH) transcription because GH secretion from these cells is stimulated by GHRH. To examine the GH transcriptional activity of these cel ls, endogenous GH mRNA levers were measured using a ribonuclease protection assay following treatment under a variety of hormonal conditions. While om ission of serum led to reduction of GK mRNA to 22% of control levels by 2 d ays and to 8% by 5 days (P < 0.05 for both), GH mRNA levels were maintained at control values in serum-free medium containing 5 nM dexamethasone and 3 0 pM triiodothyronine (TDM). However, the addition of 10 nM GHRH under any treatment condition did not significantly alter GH mRNA levels. Characteriz ation of the MtT/S cells showed that GHRH-receptor (GHRH-R) mRNA was detect able by reverse transcription-polymerase chain reaction (RT-PCR) amplificat ion. Measurement of extracellular cAMP showed that the MtT/S cells have bas al levels of <greater than or equal to>20 nmol/10(6) cells per h in both se rum-containing and serum-free media, and that GHRH had no effect on cAMP le vels, suggesting constitutive activation. To rule out the possibility of au tocrine stimulation by GHRH produced endogenously, GHRH mRNA was not detect able in MtT/S cells using RT-PCR amplification. The stimulatory G-protein a subunit, mutations of which are known to activate adenylate cyclase consti tutively in acromegaly, was sequenced but found not to differ from normal p ituitary in the regions most commonly mutated. Finally, treatment with 10 p M forskolin, to directly activate adenylate cyclase, increased GH mRNA to 1 40% of controls in TDM, and to 163% in serum-free medium after 2 days, and to 166% in TDM-treated cells and 174% in serum-free culture after 5 days ta ll P < 0.05). Taken together, these data indicate that although MtT/S cells express the GHRH-R, GHRH cannot stimulate adenylate cyclase to increase GH transcription due to constitutive elevation of cAMP levels, by a means tha t may be similar to that in cases of acromegaly not caused by oncogenic gap mutations. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.