Mechanisms for PACAP-induced prolactin gene expression in grass carp pituitary cells.

Chengyuan Lin,Ling Zhao,Mulan He,Xue Jiang,Zhaoxiang Bian,Anderson O L Wong,Tao Huang

doi:10.1530/joe-16-0433

Abstract

In mammals, pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic hormone with diverse functions but its role in prolactin (PRL) regulation is highly controversial. To shed light on Prl regulation by PACAP in fish model, grass carp pituitary cells was used as a model to examine the receptor specificity and signal transduction for PACAP modulation of prl gene expression in the carp pituitary. Using RT-PCR, PACAP-selective PAC1 receptor was detected in carp lactotrophs. In carp pituitary cells, nanomolar doses of PACAP, but not VIP, could elevate Prl secretion and protein production with concurrent rise in prl mRNA and these stimulatory effects were blocked by PACAP antagonist but not VIP antagonist. PACAP-induced prl mRNA expression could be mimicked by activating adenylate cyclase (AC), increasing cAMP level by cAMP analog, or increasing intracellular Ca2+ ([Ca2+]i) by Ca2+ ionophore/voltage-sensitive Ca2+ channel (VSCC) activator. PACAP-induced prl gene expression, however, was attenuated/abolished by suppressing cAMP production, inhibiting PKA activity, blocking [Ca2+]i mobilization and VSCC activation, calmodulin (CaM) antagonism, and inactivation of JNK and CaM Kinase II (CaMK-II). Similar sensitivity to CaM, JNK, and CaMK-II blockade was also noted by substituting cAMP analog for PACAP as the stimulant for prl mRNA expression. These results, as a whole, provide evidence for the first time that (i) PACAP activation of PAC1 receptor expressed in carp lactotrophs could induce Prl synthesis and secretion, and (ii) Prl production induced by PACAP was mediated by upregulation of prl gene expression, presumably via functional coupling of cAMP/PKA-, Ca2+/CaM-, and MAPK-dependent cascades.

Highlights

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/glucagon family, is a 38-amino acid (a.a.) peptide isolated from ovine hypothalamus with stimulatory activity on cAMP production in rat pituitary cells (Miyata et al 1989)
To test if the effect of PACAP on Prl production was caused by stimulation of prl gene expression, pituitary cells were treated with increasing doses of PACAP (0.1–1000 nM) and total RNA harvested was used for prl mRNA measurement (Fig. 2A and B)
Within the CNS, PACAP can act as a neurotransmitter (Fahrenkrug 2006) and neurotrophic factor (Vallejo & Vallejo 2002, Botia et al 2007), and serve as a hypophysiotropic factor with pituitary functions (Wong et al 2000). This idea is supported by the findings that (1) PACAP cell bodies are located within the hypothalamus (Hannibal 2002, Durr et al 2007); (2) PACAP nerve fibers can be identified in the external zone of the median eminence (Piggins et al 1996); (3) PACAP immunoreactivity can be detected in the hypophyseal portal blood (Dow et al 1994); and (4) under certain conditions, PACAP can induce pituitary hormone secretion, for example, Lh, Gh and Prl (Murakami et al 2001, Kanasaki et al 2013)

Summary

Introduction

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/glucagon family, is a 38-amino acid (a.a.) peptide isolated from ovine hypothalamus with stimulatory activity on cAMP production in rat pituitary cells (Miyata et al 1989). Two forms of PACAP have been reported, namely PACAP38 and PACAP27 (Miyata et al 1989, 1990). The biological functions of PACAP are mediated by three subtypes of PACAP receptors, including PAC1, VPAC1 and VPAC2 receptors, functionally coupled to cAMP/PKA-, PLC/PKC-, and Ca2+-dependent cascades (Harmar et al 2012). The PAC1 receptor is specific for PACAP and with high affinity for PACAP but not VIP, whereas the VPAC receptors can bind PACAP and VIP with similar affinity (Vaudry et al 2009)

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Results

Conclusion