Cyclic AMP is a major regulator of corticotrophin releasing hormone (CRH) transcription. However, receptors activating CRH neurons (alpha-adrenergic and glutamaergic) do not signal through cAMP, suggesting that calcium-phospholipid-dependent signaling synergizes with small elevations of intracellular cAMP. To test this hypothesis, we examined the relationship between activation of CRH transcription, cAMP production and CREB phosphorylation in neuronal cultures treated with the adenylyl cyclase stimulator, forskolin, the phorbol ester, PMA, or their combination. Forskolin, at threshold concentrations for cAMP production and CREB phosphorylation, induced CRH promoter-driven luciferase activity in 4B cells (EC50=0.7microM), and CRH primary transcript in hypothalamic neurons (EC50=0.6microM). PMA alone failed to activate CRH transcription in spite of being as effective as forskolin in phosphorylating CREB (Ser133 and Ser121). Although PMA potentiated the effect of low forskolin concentrations on CRH transcription and CREB phosphorylation, there was no correlation between phospho-CREB levels and activation of CRH transcription. Similarly, the calcium/calmodulin dependent kinase inhibitor, KN-93, enhanced PMA plus forskolin-stimulated CREB phosphorylation, while inhibiting CRH transcription. Suppression of CREB phosphorylation by the PKA inhibitor, H89, or the CREB dominant negative, A-CREB, did not affect basal but blocked forskolin-stimulated transcription. This study shows that calcium phospholipid-dependent pathways potentiate the ability of small elevations of intracellular cAMP to activate CRH transcription, providing a mechanism by which non-cAMP dependent regulators induce CRH gene expression. In addition, the data indicate that phospho-CREB is essential but not sufficient for activation of CRH transcription, suggesting that full promoter stimulation requires the interaction of phospho-CREB with a co-activator.