A Novel Role of VIP in Colonic Motility Function: Induction of Excitation-Transcription Coupling in Smooth Muscle Cells

Background & Aims: Vasoactive intestinal polypeptide (VIP) relaxes smooth muscle by generation of cAMP and activation of protein kinase A (PKA). However, PKA activation also phosphorylates the transcription factor CREB. The aim of this study was to investigate whether the phosphorylation of CREB induces gene expression of the pore-forming α_1C subunit of Ca_v1.2 channels (L-type calcium channels), whose promoter has 2 binding sites for CREB. Methods: The experiments were performed on primary cultures of human colonic circular smooth muscle cells and freshly obtained human and rat colonic circular muscle strips. Results: The incubation of human colonic circular smooth muscle cells or muscle strips with VIP for 24 hours enhanced the expression of α_1C protein and mRNA as well as the contractile response to acetylcholine and KCl. On the contrary, incubation of the muscle strips with VIP antagonist for 24 hours suppressed cell contractility. The incubation of the cells with VIP caused sustained generation of cAMP for 24 hours, but PKA activation and CREB phosphorylation were transient. The inhibition of PKA by H-89 or silencing of CREB gene with targeted RNAi blocked the transcription of α_1C. Progressive 5′ deletions of hα_1C1b promoter and site-directed mutations of the 2 CREB binding cis-elements indicated that most of α_1C transcription was mediated by the 5′ cAMP response element. Conclusions: The excitation-transcription coupling stimulated by VIP induces expression of the Ca_v1.2 channels. The influx of calcium through these channels is a critical step in excitation-contraction coupling in smooth muscle cells.