Billy K. C. Chow

Pituitary adenylate cyclase-activating polypeptide and its receptors: 20 years after the discovery.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid C-terminally α-amidated peptide that was first isolated 20 years ago from an ovine hypothalamic extract on the basis of its ability to stimulate cAMP formation in anterior pituitary cells (Miyata et al., 1989. PACAP belongs to the vasoactive intestinal polypeptide (VIP)-secretin-growth hormone-releasing hormone-glucagon superfamily. The sequence of PACAP has been remarkably well conserved during evolution from protochordates to mammals, suggesting that PACAP is involved in the regulation of important biological functions. PACAP is widely distributed in the brain and peripheral organs, notably in the endocrine pancreas, gonads, respiratory and urogenital tracts. Characterization of the PACAP precursor has revealed the existence of a PACAP-related peptide, the activity of which remains unknown. Two types of PACAP binding sites have been characterized: type I binding sites exhibit a high affinity for PACAP and a much lower affinity for VIP, whereas type II binding sites have similar affinity for PACAP and VIP. Molecular cloning of PACAP receptors has shown the existence of three distinct receptor subtypes: the PACAP-specific PAC1-R, which is coupled to several transduction systems, and the PACAP/VIP-indifferent VPAC1-R and VPAC2-R, which are primarily coupled to adenylyl cyclase. PAC1-Rs are particularly abundant in the brain, the pituitary and the adrenal gland, whereas VPAC receptors are expressed mainly in lung, liver, and testis. The development of transgenic animal models and specific PACAP receptor ligands has strongly contributed to deciphering the various actions of PACAP. Consistent with the wide distribution of PACAP and its receptors, the peptide has now been shown to exert a large array of pharmacological effects and biological functions. The present report reviews the current knowledge concerning the pleiotropic actions of PACAP and discusses its possible use for future therapeutic applications.

Discovery of growth hormone-releasing hormones and receptors in nonmammalian vertebrates

In mammals, growth hormone-releasing hormone (GHRH) is the most important neuroendocrine factor that stimulates the release of growth hormone (GH) from the anterior pituitary. In nonmammalian vertebrates, however, the previously named GHRH-like peptides were unable to demonstrate robust GH-releasing activities. In this article, we provide evidence that these GHRH-like peptides are homologues of mammalian PACAP-related peptides (PRP). Instead, GHRH peptides encoded in cDNAs isolated from goldfish, zebrafish, and African clawed frog were identified. Moreover, receptors specific for these GHRHs were characterized from goldfish and zebrafish. These GHRHs and GHRH receptors (GHRH-Rs) are phylogenetically and structurally more similar to their mammalian counterparts than the previously named GHRH-like peptides and GHRH-like receptors. Information regarding their chromosomal locations and organization of neighboring genes confirmed that they share the same origins as the mammalian genes. Functionally, the goldfish GHRH dose-dependently activates cAMP production in receptor-transfected CHO cells as well as GH release from goldfish pituitary cells. Tissue distribution studies showed that the goldfish GHRH is expressed almost exclusively in the brain, whereas the goldfish GHRH-R is actively expressed in brain and pituitary. Taken together, these results provide evidence for a previously uncharacterized GHRH-GHRH-R axis in nonmammalian vertebrates. Based on these data, a comprehensive evolutionary scheme for GHRH, PRP-PACAP, and PHI-VIP genes in relation to three rounds of genome duplication early on in vertebrate evolution is proposed. These GHRHs, also found in flounder, Fugu, medaka, stickleback, Tetraodon, and rainbow trout, provide research directions regarding the neuroendocrine control of growth in vertebrates.

The gene transfection efficiency of a folate–PEI600–cyclodextrin nanopolymer

The success of gene therapy relies on a safe and effective gene delivery system. In this communication, we describe the use of folate grafted PEI(600)-CyD (H(1)) as an effective polyplex-forming plasmid delivery agent with low toxicity. The structures of the polymer and polyplex were characterized, and the in vitro transfection efficiency, cytotoxicity, and in vivo transfection of H(1) were examined. We found that folate molecules were successfully grafted to PEI(600)-CyD. At N/P ratios between 5 and 30, the resulting H(1)/DNA polyplexes had diameters less than 120 nm and zeta potentials less than 10 mV. In various tumor cell lines examined (U138, U87, B16, and Lovo), the in vitro transfection efficiency of H(1) was more than 50%, which could be improved by the presence of fetal bovine serum or albumin. The cytotoxicity of H(1) was significantly less than high molecular weight PEI-25 kDa. Importantly, in vivo optical imaging showed that the efficiency of H(1)-mediated transfection (50 microg luciferase plasmid (pLuc), N/P ratio=20/1) was comparable to that of adenovirus-mediated luciferase transduction (1 x 10(9) pfu) in melanoma-bearing mice, and it did not induce any toxicity in the tumor tissue. These results clearly show that H(1) is a safe and effective polyplex-forming agent for both in vitro and in vivo transfection of plasmid DNA and its application warrants further investigation.

Characterization of an additional molt inhibiting hormone-like neuropeptide from the shrimp Metapenaeus ensis

We have identified a second form of the type-II neuropeptide encoding a molt inhibiting hormone-like (MeeMIH-B) neuropeptide. MeeMIH-B showed only a 70% amino acid identity to the MIH-A (formerly MIH) isolated from the same species, suggesting a possible different function of the deduced neuropeptide. Like other neuropeptide members of the CHH family, the MIH-B gene consists of three exons separated by two introns. The levels of MIH-B mRNA transcript in the eyestalk decrease in the initial phase of gonad maturation and increase towards the end of maturation. The drop in MIH-B level suggests an inhibitory role for this neuropeptide in the initiation of vitellogenesis. MIH-B transcripts can also be detected in the brain, thoracic ganglion and ventral nerve cord. Together with the CHH-B peptide that we have previously described, this is the second peptide of the CHH family that can also be identified in the ventral nerve cord and in the XOSG complex. A recombinant MIH-B was produced and a polyclonal antibody against rMIH-B was subsequently generated. Specific anti-rMIH-B antiserum recognized the presence of MIH-B in the sinus gland, X-organs, as well as a giant neuron of the ventral nerve cord. Injection of rMIH-B delayed the molting cycle of the maturing female. Taken together, the results of this study suggest that a drop in MIH-B level may be required for the delay in the molting of the maturing females.

Identification and Characterization of a Receptor from Goldfish Specific for a Teleost Growth Hormone-Releasing Hormone-Like Peptide

In mammals, growth hormone-releasing hormone (GHRH), acting via the GHRH receptor, plays an important role in the regulation of growth hormone (GH) synthesis and secretion as well as the proliferation and differentiation of somatotropes in the pituitary. In fishes, information concerning the functional role of the characterized GHRHs is limited. For that reason, a putative goldfish GHRH receptor cDNA was characterized in this study. The receptor cDNA is 2,243 bp in length, encoding a 438-amino-acid-long polypeptide with 7 putative transmembrane-spanning regions, which is a characteristic of G-protein-coupled receptors. The receptor, when expressed in COS-7 cells, showed minimal responses (2-fold cAMP responses) when stimulated with 100 nM of human GHRH, pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP). However, this receptor was found to be specific for a carp GHRH-like peptide isolated from the brain of common carp (Cyprinus carpio); there was a significant and dose-dependent increase in intracellular cAMP (a maximum response of 22-fold increase with an EC50 of 0.1 nM) when the transfected cells were stimulated with this peptide. As a preliminary study to investigate the functional role of this receptor, the tissue distribution of the mRNA was analyzed by reverse-transcription-polymerase chain reaction. The receptor mRNA was found to be present in the brain, pituitary, gut, gill, heart, liver, skeletal muscle, spleen, ovary and testis. Together with a goldfish PACAP type 1 receptor and a VIP1 receptor recently isolated in our laboratory, characterization of this putative GHRH receptor provides the molecular basis for the future understanding of the neuroendocrine control of growth and reproduction by these neuropeptides in goldfish as well as other teleosts.

Molecular cloning and expression studies of a prolactin receptor in goldfish (Carassius auratus).

A full-length cDNA clone, of a size of 4.6 kb, for the goldfish prolactin receptor has been isolated. This cDNA clone encodes a protein of 600 amino acids homologous to prolactin receptors of other species. A Kyte-Doolittle hydropathy analysis of the receptor indicates that the translated protein consists of a signal peptide of 22 amino acids, an extracellular domain of 228 amino acids, a single transmembrane domain of 24 amino acids, and an intracellular domain of 346 amino acids. Several characteristic landmarks of prolactin receptor could be identified in this clone. These include the four conserved cysteine residues and the WS motif within the extracellular domain, and the box 1 and box 2 regions of the intracellular domain. Among all the prolactin receptor sequences known to date, this clone bears the closest resemblance to the tilapia prolactin receptor, although homology between these two fish prolactin receptors is rather low. There are only 57.4% of nucleotide and 48.3% of amino acid sequence identities between these two fish receptors. This receptor cDNA was transfected into CHO-K1 cells for functional analysis. RT-PCR analysis with a pair of gene specific primers indicate that the receptor was transcribed in the transfected cells. Using a cell proliferation assay based on the reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, the receptor transfected CHO-K1 cells can be stimulated to proliferate upon the addition of ovine prolactin in the culture medium. The tissue distribution of the prolactin receptor in goldfish was studied by RT-PCR/Southern analysis and by Northern analysis. The results indicated that the receptor is expressed mostly in the kidney, the gill and the intestine of goldfish, corroborating with the osmoregulatory role of prolactin in fish. In addition, an appreciable level of the receptor is also found in the brain and gonads of goldfish. Northern analysis showed that there are two transcript sizes, a major 4.6 kb and a minor 3.5 kb mRNAs, in the kidney, gill and intestine.

Knockout of secretin receptor reduces large cholangiocyte hyperplasia in mice with extrahepatic cholestasis induced by bile duct ligation

During bile duct ligation (BDL), the growth of large cholangiocytes is regulated by the cyclic adenosine monophosphate (cAMP)/extracellular signal‐regulated kinase 1/2 (ERK1/2) pathway and is closely associated with increased secretin receptor (SR) expression. Although it has been suggested that SR modulates cholangiocyte growth, direct evidence for secretin‐dependent proliferation is lacking. SR wild‐type (WT) (SR+/+) or SR knockout (SR−/−) mice underwent sham surgery or BDL for 3 or 7 days. We evaluated SR expression, cholangiocyte proliferation, and apoptosis in liver sections and proliferating cell nuclear antigen (PCNA) protein expression and ERK1/2 phosphorylation in purified large cholangiocytes from WT and SR−/− BDL mice. Normal WT mice were treated with secretin (2.5 nmoles/kg/day by way of osmotic minipumps for 1 week), and biliary mass was evaluated. Small and large cholangiocytes were used to evaluate the in vitro effect of secretin (100 nM) on proliferation, protein kinase A (PKA) activity, and ERK1/2 phosphorylation. SR expression was also stably knocked down by short hairpin RNA, and basal and secretin‐stimulated cAMP levels (a functional index of biliary growth) and proliferation were determined. SR was expressed by large cholangiocytes. Knockout of SR significantly decreased large cholangiocyte growth induced by BDL, which was associated with enhanced apoptosis. PCNA expression and ERK1/2 phosphorylation were decreased in large cholangiocytes from SR−/− BDL compared with WT BDL mice. In vivo administration of secretin to normal WT mice increased ductal mass. In vitro, secretin increased proliferation, PKA activity, and ERK1/2 phosphorylation of large cholangiocytes that was blocked by PKA and mitogen‐activated protein kinase kinase inhibitors. Stable knockdown of SR expression reduced basal cholangiocyte proliferation. SR is an important trophic regulator sustaining biliary growth. Conclusion: The current study provides strong support for the potential use of secretin as a therapy for ductopenic liver diseases. HEPATOLOGY 2010

Phenotypes developed in secretin receptor-null mice indicated a role for secretin in regulating renal water reabsorption.

ABSTRACT Aquaporin 2 (AQP2) is responsible for regulating the concentration of urine in the collecting tubules of the kidney under the control of vasopressin (Vp). Studies using Vp-deficient Brattleboro rats, however, indicated the existence of substantial Vp-independent mechanisms for membrane insertion, as well as transcriptional regulation, of this water channel. The Vp-independent mechanism(s) is clinically relevant to patients with X-linked nephrogenic diabetes insipidus (NDI) by therapeutically bypassing the dysfunctional Vp receptor. On the basis of studies with secretin receptor-null (SCTR−/−) mice, we report here for the first time that mutation of the SCTR gene could lead to mild polydipsia and polyuria. Additionally, SCTR−/− mice were shown to have reduced renal expression of AQP2 and AQP4, as well as altered glomerular and tubular morphology, suggesting possible disturbances in the filtration and/or water reabsorption process in these animals. By using SCTR−/− mice as controls and comparing them with wild-type animals, we performed both in vivo and in vitro studies that demonstrated a role for secretin in stimulating (i) AQP2 translocation from intracellular vesicles to the plasma membrane in renal medullary tubules and (ii) expression of this water channel under hyperosmotic conditions. The present study therefore provides information for at least one of the Vp-independent mechanisms that modulate the process of renal water reabsorption. Future investigations in this direction should be important in developing therapeutic means for treating NDI patients.

Pituitary Growth Hormone Secretion in the Turbot, a Phylogenetically Recent Teleost, Is Regulated by a Species-Specific Pattern of Neuropeptides

In mammals, growth hormone (GH) is under a dual hypothalamic control exerted by growth hormone-releasing hormone (GHRH) and somatostatin (SRIH). We investigated GH release in a pleuronectiform teleost, the turbot (Psetta maxima), using a serum-free primary culture of dispersed pituitary cells. Cells released GH for up to 12 days in culture, indicating that turbot somatotropes do not require releasing hormone for their regulation. SRIH dose-dependently inhibited GH release up to a maximal inhibitory effect of 95%. None of the potential stimulators tested induced any change in basal GH release. Also, neither forskolin, an activator of adenylate cyclase, nor phorbol ester (TPA), an activator of protein kinase C, were able to modify GH release, suggesting that spontaneous basal release already represents the maximal secretory capacity of turbot somatotropes. In contrast, forskolin and TPA were able to increase GH release in the presence of SRIH. In this condition (coincubation with SRIH), pituitary adenylate cyclase-activating polypeptide (PACAP) stimulated GH release, whereas none of the other neuropeptides tested (GHRHs; sea bream or salmon or chicken II GnRHs; TRH; CRH) had any significant effect. These data indicate that inhibitory control by SRIH may be the basic control of GH production in teleosts and lower vertebrates, while PACAP may represent the ancestral growth hormone-releasing factor in teleosts, a role taken over in higher vertebrates by GHRH.

Orexins and their receptors from fish to mammals: A comparative approach

Although recently discovered, orexins have been rapidly established as important neuropeptides in regulating physiological processes including food intake, sleep/wake cycles and reproduction through binding to two class B G protein-coupled receptors (OX1R and OX2R). To date, a handful of sequences for orexins and their receptors ranging from fish to mammalian species have been identified, allowing a glimpse into their evolution. Structurally, the genetic and molecular organization of the peptides and receptors amongst vertebrates are highly similar, underlining the strong evolutionary pressure that has been exerted to preserve structure and ultimately function. Furthermore, the absence of invertebrate orexin-like sequences suggests early vertebrates as the origin from which orexins evolved. With respect to the receptors, OX2R is probably evolutionary more ancient whilst OX1R is specific to mammalian species and evolved only during this later lineage. In common to all vertebrates studied, the hypothalamus remains to be the key brain region in which orexinergic neurons and fibers are localized in, establishing orexin to be an important player in regulating physiological processes especially those related to food intake and energy metabolism. To allow better understanding of the evolution of orexins and their receptors, this review will provide a comparative approach to their structures and functions in vertebrates.