Enhanced effectiveness of pulsatile 3',5'-cyclic adenosine monophosphate in stimulating prolactin and alpha-subunit gene expression

Abstract

cAMP is involved in the regulation of secretory activity in lactotrope, thyrotrope, and gonadotrope cells. The present study examined whether pulsatile or intermittent changes in cAMP are more effective than a continuous stimulation in increasing pituitary hormone gene expression. Pituitaries from adult female rats were dissociated, plated for 48 h (7-8 x 10(6) cells per well) to allow attachment to Matrigel-coated plastic coverslips, then inserted into perifusion chambers (five to eight chambers per group). After 24 h of treatment, the cells were recovered, RNA extracted, and messenger RNAs (mRNAs) determined by dot blot hybridization. Perfused cells were exposed to either hourly pulses of monobutyryl cAMP (Bt cAMP, 0.01, 0.1, or 1 mM; 1 mM butyrate pulses to controls), or continuously to forskolin (10 microM). Bt cAMP pulses increased both PRL and alpha-subunit mRNAs, maximal after the 0.1 mM dose for PRL (51% increase vs. butyrate controls) and after the 1 mM dose for alpha (60% increase). However, forskolin was ineffective in increasing PRL or alpha mRNA concentrations. TSH, LH, and FSH beta-subunit mRNAs were not altered by Bt cAMP pulses or forskolin. To confirm the different effects of pulsatile vs. continuous cAMP on PRL and alpha-subunit mRNAs, the response to pulsatile 8-bromo cAMP (1 mM) or Bt cAMP (0.5 mM) was compared to continuous Bt cAMP (0.5 mM). PRL and alpha-subunit mRNAs were increased by both cAMP analogs given in a pulsatile manner but not by continuous Bt cAMP. PRL and LH secretory responses (determined in perifusate samples after 2 h and 22 h of treatment) revealed that both PRL and LH release was increased by cAMP stimulation, given either in a pulsatile or continuous manner. These results show that PRL and alpha-subunit gene expression were sensitive to changes in cAMP stimulation, whereas that of TSH, LH, and FSH beta were unaltered. Only intermittent cAMP stimuli were effective in increasing PRL and alpha mRNAs. These data suggest that pulsatile fluctuations in intracellular cAMP may be essential for maximal expression of the PRL and alpha genes. Thus, intermittent changes in intracellular second messengers may be a necessary part of the pathway involved in the transduction of signals from the plasma membrane to the nucleus.