PACAP system evolution and its role in melanophore function in teleost fish skin

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP) administered to tilapia melanophores ex-vivo causes significant pigment aggregation and this is a newly identified function for this peptide in fish. The G-protein coupled receptors (GPCRs), adcyap1r1a (encoding Pac1a) and vipr2a (encoding Vpac2a), are the only receptors in melanophores with appreciable levels of expression and are significantly (p < 0.05) down-regulated in the absence of light. Vpac2a is activated exclusively by peptide histidine isoleucine (PHI), which suggests that Pac1a mediates the melanin aggregating effect of PACAP on melanophores. Paradoxically activation of Pac1a with PACAP caused a rise in cAMP, which in fish melanophores is associated with melanin dispersion. We hypothesise that the duplicate adcyap1ra and vipr2a genes in teleosts have acquired a specific role in skin and that the melanin aggregating effect of PACAP results from the interaction of Pac1a with Ramp that attenuates cAMP-dependent PKA activity and favours the Ca(2+)/Calmodulin dependent pathway.

The calcitonin-like system is an ancient regulatory system of biomineralization

“…Mammalian cell cultures and transfections followed the methodology described by 89,90 . Mammalian HEK293 cells (ECACC, UK) were used and they were maintained in complete Dulbecco's modified Eagle's medium (DMEM, 4.5 g/l glucose, 110 mg/l sodium pyruvate and L-glutamine, SIGMA-ALDRICH, Spain) supplemented with 10% sterile foetal bovine serum (FBS), 0.1% penicillin: streptomycin antibiotic mix (10,000 U:10 mg/ml, Sigma) and 250 μg /ml sterile filtered 1:100 amphotericin B solution (SIGMA-ALDRICH) in a humid 5% CO 2 incubator (HERAEUS, Portugal) at 37 °C.…”

“…cAMp assay. The amount of cAMP produced was determined using a competitive immunoassay with a cryptate labelled anti-cAMP antibody (CISBIO, France) and followed the methodology describe by 89,90 . Briefly, approximately 15,000 stably transfected mammalian HEK293 cells were assayed per well and cell-peptide incubations were performed for a final reaction volume of 20 μl in white 384 well small Volume HiBase Polystyrene microplates (GREINER, Germany).…”

Tracing the Origins of the Pituitary Adenylate-Cyclase Activating Polypeptide (PACAP)

“…Six putative PACAP receptor genes (two Pac 1 , adcyap1r1a and adcyap1r1b; two Vpac 1 , vipr1a and vipr1b and two Vpac 2 , vipr2a, and vipr2b) have been characterized and receptor activation triggers similar signaling pathways to those in mammals, birds, and amphibians. The teleost PACAP receptor gene paralogs stimulate similar functions to those of other vertebrates but also acquired specialized functions during the teleost radiation (Cardoso et al, 2004(Cardoso et al, , 2007b(Cardoso et al, , 2015Roch et al, 2009). In common with the human receptors, interaction of teleost family B1 GPCRs with receptor activity-modifying proteins (RAMPs) (Christopoulos et al, 2003;Archbold et al, 2011;Couvineau and Laburthe, 2011), a class of membrane accessory proteins, can modulate their activity by changing receptor pharmacology (Cardoso et al, 2015).…”

“…In fish a single gene encoding the PACAP peptide precursor that shares high sequence similarity and organization with the tetrapod homolog was identified in the genomes of lamprey, elephant shark, spotted gar, and coelacanth. In contrast, in the teleosts, two PACAP precursor genes adcyap1a (protein; Pacapa) and adcyap1b (protein; Pacapb), have been isolated and arose from the teleost specific genome duplication event and four mature PACAP peptides (two PACAP-38 and two PACAP-27) are considered to be produced (Cardoso et al, 2007a(Cardoso et al, , 2015Ng et al, 2012). Analysis across the vertebrates of the genes flanking ADCYAP1 reveals syntenic genome regions in fish and tetrapods and supports a common evolutionary origin for PACAP (Cardoso et al, 2007b(Cardoso et al, , 2015.…”

“…Although, the distribution of PACAP is relatively well-characterized in fish, few studies have characterized the function of the teleost duplicate PACAPs. The outcome of the studies that exist suggest that the duplicate PACAPs possess similar functions to the mammalian homolog although teleost specific functions are proposed to have also been acquired (Cardoso et al, 2010(Cardoso et al, , 2015.…”