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1.
J Mol Neurosci ; 71(8): 1567-1574, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32323126

RESUMO

PACAP is a neuropeptide with widespread distribution and diverse biological functions. It has strong cytoprotective effects mediated mainly through specific PAC1 receptors. Experimental data show protective effects of PACAP in the retina and cornea in several pathological conditions. Although intravitreal injections are a common practice in some ocular diseases, delivery of therapeutic agents in the form of eye drops would be more convenient and would lead to fewer side effects. We have previously shown that PACAP, in the form of eye drops, is able to pass through the ocular barriers and can exert retinoprotective effects. As eye drops represent a promising form of administration of PACAP in ocular diseases, it is important to investigate the stability of PACAP in solutions used in eye drops. In this study, the stability of PACAP1-27 and PACAP1-38 in eye drops was measured in four common media and a commercially available artificial tear solution at both room temperature and +4 °C. Mass spectrometry results show that the highest stability was gained with PACAP1-38 in water and 0.9% saline solution at +4 °C, representing 80-90% drug persistence after 2 weeks. PACAP1-38 in the artificial tear showed very fast degradation at room temperature, but was stable at +4 °C. In summary, PACAP1-38 has higher stability than PACAP1-27, with highest stability at +4 °C in water solution, but both peptides in each medium can be stored for relatively longer periods without significant degradation. These data can provide reference for future therapeutic use of PACAP in eye drops.


Assuntos
Soluções Oftálmicas/química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Espectrometria de Massas/métodos , Soluções Oftálmicas/normas , Estabilidade Proteica , Lágrimas/química , Temperatura
2.
Nat Commun ; 11(1): 2399, 2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32404879

RESUMO

The ability to monitor molecules volumetrically throughout the body could provide valuable biomarkers for studies of healthy function and disease, but noninvasive detection of molecular targets in living subjects often suffers from poor sensitivity or selectivity. Here we describe a family of potent imaging probes that can be activated by molecules of interest in deep tissue, providing a basis for mapping nanomolar-scale analytes without the radiation or heavy metal content associated with traditional molecular imaging agents. The probes are reversibly caged vasodilators that induce responses detectable by hemodynamic imaging; they are constructed by combining vasoactive peptides with synthetic chemical appendages and protein blocking domains. We use this architecture to create ultrasensitive biotin-responsive imaging agents, which we apply for wide-field mapping of targets in rat brains using functional magnetic resonance imaging. We also adapt the sensor design for detecting the neurotransmitter dopamine, illustrating versatility of this approach for addressing biologically important molecules.


Assuntos
Imagem Molecular/métodos , Sondas Moleculares/metabolismo , Peptídeos/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Vasodilatadores/metabolismo , Animais , Biotina/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Células CHO , Cricetinae , Cricetulus , Dopamina/metabolismo , Células HEK293 , Humanos , Imageamento por Ressonância Magnética/métodos , Sondas Moleculares/química , Neurotransmissores/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Ratos , Reprodutibilidade dos Testes , Vasodilatadores/química
3.
Brain Res ; 1739: 146858, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32348776

RESUMO

PACAP-38 (P38) is a pleiotropic peptide that exerts multiple peripheral and central actions, including neurotrophic, neuroprotective and anti-inflammatory actions. Previous studies have suggested an improvement of memory in rats that have received a single systemic injection of P38. In a therapeutic perspective, we used an analog, acetyl-[Ala15, Ala20]PACAP-38-propylamide (ALG), to improve both stability and affinity for PAC1 receptors vs. endogen PACAP. We investigated the effect of P38 and ALG on memory consolidation using a spatial novelty detection (SND) task in which rats had to memorize a configuration of objects to identify that, during a test session, a familiar object has been moved to a new location. Rats received an intravenous injection of P38 or ALG after the last training session. In Experiment 1, P38 (30 µg/kg) improved spatial memory consolidation allowing detection of novelty vs. saline injection. In Experiment 2, we confirmed this effect and showed that P38 restored the performance similar to what was found using non-injected rats. This suggests that, contrary to ALG, P38 exerted a promesiant rather than an anxiety-related effect whereas ALG did not show similar action. We also examined whether P38 effect involved an interaction with NR2B-containing NMDA receptors (NMDARs) by administrating ifenprodil (IFE; a selective NR2B-containing NMDAR antagonist) alone or in combination with P38 or ALG. The results suggested that P38 action on memory involved NR2B-containing NMDARs. Lastly, brain-derived neutrophic factor (BDNF) modulation appeared to be not related to the behavioral performance in the SND task. Overall, the results indicate that P38 exerted a beneficial effect on memory consolidation in a non-associative task, whereas ALG did not have this action.


Assuntos
Consolidação da Memória/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Memória Espacial/efeitos dos fármacos , Animais , Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Hipocampo/metabolismo , Aprendizagem/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Consolidação da Memória/fisiologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/síntese química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Ratos , Ratos Wistar , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/efeitos dos fármacos , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo
4.
Nat Struct Mol Biol ; 27(3): 274-280, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32157248

RESUMO

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide hormone. The PACAP receptor PAC1R, which belongs to the class B G-protein-coupled receptors (GPCRs), is a drug target for mental disorders and dry eye syndrome. Here, we present a cryo-EM structure of human PAC1R bound to PACAP and an engineered Gs heterotrimer. The structure revealed that transmembrane helix TM1 plays an essential role in PACAP recognition. The extracellular domain (ECD) of PAC1R tilts by ~40° compared with that of the glucagon-like peptide-1 receptor (GLP-1R) and thus does not cover the peptide ligand. A functional analysis demonstrated that the PAC1R ECD functions as an affinity trap and is not required for receptor activation, whereas the GLP-1R ECD plays an indispensable role in receptor activation, illuminating the functional diversity of the ECDs in class B GPCRs. Our structural information will facilitate the design and improvement of better PAC1R agonists for clinical applications.


Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/química , Peptídeos/química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Humanos , Modelos Moleculares , Peptídeos/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Homologia Estrutural de Proteína
5.
Int J Biol Sci ; 15(12): 2676-2691, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31754339

RESUMO

It is well known that the cornea plays an important role in providing protection to the eye, but it is fragile and vulnerable. To clarify the biological effects and molecular mechanisms of the pituitary adenylate cyclase activating polypeptide (PACAP)-derived peptide MPAPO (named MPAPO) to promote corneal wound healing, we applied a mechanical method to establish a corneal injury model and analyzed the repair effects of MPAPO on corneal injury. MPAPO significantly promoted corneal wound repair in C57BL/6 mice. In addition, we established injury models of epithelial cells and trigeminal ganglion cells with H2O2. The results show that when the concentration of MPAPO is 1 µM, it can significantly promote the repair of injured corneal epithelial cells and the regeneration of trigeminal ganglion cell axons. MPAPO repairs epithelial cells through the promotion of GSK3ß phosphorylation by binding to PAC1 and activating AKT. ß-catenin escapes the phosphorylation of GSK3ß and enters the nucleus to promote the expression of cyclin D1, accelerate cell cycle progression and promote cell proliferation. MPAPO promotes axonal regeneration by binding to the PAC1 receptor and activating adenylate cyclase activity, followed by the cAMP activation of protein kinase A activity and the promotion of CREB phosphorylation. Phosphorylated CREB promotes Bcl2 expression and axonal regeneration. In conclusion, our data support the role of MPAPO to facilitate corneal wound healing by promoting corneal epithelial cell proliferation and trigeminal ganglion cell axon regeneration.


Assuntos
Proliferação de Células/efeitos dos fármacos , Lesões da Córnea/tratamento farmacológico , Células Epiteliais/efeitos dos fármacos , Peptídeos/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Gânglio Trigeminal/citologia , Animais , Axônios/efeitos dos fármacos , Axônios/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Córnea , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regeneração Nervosa/efeitos dos fármacos , Peptídeos/química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Distribuição Aleatória , Gânglio Trigeminal/efeitos dos fármacos
6.
Biochim Biophys Acta Gen Subj ; 1863(11): 129410, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31401178

RESUMO

BACKGROUND: Neurodegenerative disorders, such as Parkinson's disease (PD), are characterized by neuronal death involving, among other events, mitochondrial dysfunction and excitotoxicity. Along these lines, several attempts have been made to slow this pathology but none have been yet discovered. Based on its capacity to cross the blood-brain barrier and provide neuronal protection in vitro and in vivo, the pituitary adenylate cyclase-activating polypeptide (PACAP) represents a promising lead molecule. Pharmacological studies showed that PACAP interacts with three different G protein-coupled receptors, i.e. PAC1, VPAC1 and VPAC2. However, only PAC1 is associated with neuronal anti-apoptotic actions, whilst VPAC activation might cause adverse effects. In the context of the development of PAC1-selective agonists, PACAP(1-23) (PACAP23) appears as the shortest known PACAP bioactive fragment. METHODS: Hence, the capacity of this peptide to bind PACAP receptors and protect neuroblastoma cells was evaluated under conditions of mitochondrial dysfunction and glutamate excitotoxicity. In addition, its ability to activate downstream signaling events involving G proteins (Gαs and Gαq), EPAC, and calcium was also assessed. RESULTS: Compared to the endogenous peptide, PACAP23 showed a reduced affinity towards PAC1, although this fragment exerted potent neuroprotection. However, surprisingly, some disparities were observed for PACAP23 signaling compared to full length PACAP, suggesting that downstream signaling related to neuroprotection is distinctly regulated following subtle differences in their PAC1 interactions. CONCLUSIONS: Altogether, this study demonstrates the potent neuroprotective action of amidated PACAP23. GENERAL SIGNIFICANCE: PACAP23 represents an attractive template for development of shorter PACAP-derived neuroprotective molecules.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Fármacos Neuroprotetores , Peptídeos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Animais , Células CHO , Cricetulus , Humanos , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Receptores Tipo II de Peptídeo Intestinal Vasoativo/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo
7.
Biochemistry ; 58(9): 1214-1225, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30720275

RESUMO

Amyloids are polypeptide supramolecular assemblies that have been historically associated with numerous pathologies. Nonetheless, recent studies have identified many amyloid structures that accomplish vital physiological functions. Interestingly, amyloid fibrils, either pathological or functional, have been reported to be consistently associated with other biomolecules such as RNA and glycosaminoglycans (GAGs). These linear polyanions, RNA and GAGs, have also demonstrated an inherent ability to accelerate and/or promote amyloid formation. GAGs, including heparan sulfate, are highly charged polysaccharides that may have essential roles in the storage of peptide hormones in the form of amyloids. In this study, we evaluated the ability of sulfated GAGs to promote the self-assembly of the peptide (neuro)hormone PACAP27 and investigated the secondary and quaternary conformational transitions associated with the amyloidogenic process. PACAP27 readily self-assembled into insoluble, α-helix-rich globular particulates in the presence of sulfated GAGs, which gradually condensed and disappeared as nontoxic ß-sheet-rich amyloid fibrils were formed. By designing a PACAP27 derivative for which helical folding was hindered, we observed that the α-helix-to-ß-sheet conformational transition within the amorphous particulates constitutes the rate-limiting step of primary nucleation events. The proposed mechanism of GAG-induced self-assembly within insoluble particulates appears to be fundamentally different from usual amyloidogenic systems, which commonly implicates the formation of soluble prefibrillar proteospecies. Overall, this study provides new insights into the mechanistic details involved in the formation of functional amyloids catalyzed by polyanions, such as the assembly of nuclear amyloid bodies and the storage of peptide hormones.


Assuntos
Amiloide/metabolismo , Glicosaminoglicanos/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Amiloide/química , Animais , Linhagem Celular , Dicroísmo Circular , Difusão Dinâmica da Luz , Glicosaminoglicanos/química , Microscopia de Força Atômica , Miócitos Cardíacos , Concentração Osmolar , Conformação Proteica , Dobramento de Proteína , Ratos , Espectrometria de Fluorescência
8.
J Mol Neurosci ; 68(3): 397-407, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30542799

RESUMO

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) belong to the same peptide family and exert a variety of biological functions. Both PACAP and VIP have protective effects in several tissues. While PACAP is known to be a stronger retinoprotective peptide, VIP has very potent anti-inflammatory effects. The need for a non-invasive therapeutic approach has emerged and PACAP has been shown to be retinoprotective when administered in the form of eye drops as well. The cell penetrating peptide TAT is composed of 11 amino acids and tagging of TAT at the C-terminus of neuropeptides PACAP/VIP can enhance the traversing ability of the peptides through the biological barriers. We hypothesized that TAT-bound PACAP and VIP could be more effective in exerting retinoprotective effects when given in eye drops, by increasing the traversing efficacy and enhancing the activation of the PAC1 receptor. Rats were subjected to bilateral carotid artery occlusion (BCCAO), and retinas were processed for histological analysis 14 days later. The efficiency of the TAT-bound peptides to reach the retina was assessed as well as their cAMP increasing ability. Our present study provides evidence, for the first time, that topically administered PACAP and VIP derivatives (PACAP-TAT and VIP-TAT) attenuate ischemic retinal degeneration via the PAC1 receptor presumably due to a multifactorial protective mechanism.


Assuntos
Anti-Inflamatórios/farmacologia , Fármacos Neuroprotetores/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Retina/efeitos dos fármacos , Degeneração Retiniana/tratamento farmacológico , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/uso terapêutico , Células CHO , Cricetinae , Cricetulus , Masculino , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/uso terapêutico , Soluções Oftálmicas , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Retina/metabolismo , Peptídeo Intestinal Vasoativo/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética
9.
PLoS One ; 13(11): e0207366, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30462698

RESUMO

The Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP), a polycationic, amphiphilic and helical neuropeptide, is well known for its neuroprotective actions and cell penetrating properties. In the present study, we evaluated the potent antibacterial property of PACAP38 and related analogs against various bacterial strains. Interestingly, PACAP38 and related analogs can inhibit the growth of various bacteria including Escherichia coli (JM109), Bacillus subtilis (PY79), and the pathogenic Burkholderia cenocepacia (J2315). Investigation of the mechanism of action suggested that a PACAP metabolite, identified as PACAP(9-38), might indeed be responsible for the observed PACAP38 antibacterial action. Surprisingly, PACAP(9-38), which does not induce haemolysis, exhibits an increased specificity toward Burkholderia cenocepacia J2315 compared to other tested bacteria. Finally, the predisposition of PACAP(9-38) to adopt a π-helix conformation rather than an α-helical conformation like PACAP38 could explain this gain in specificity. Overall, this study has revealed a new function for PACAP38 and related derivatives that can be added to its pleiotropic biological activities. This innovative study could therefore pave the way toward the development of new therapeutic agents against multiresistant bacteria, and more specifically the Burkholderia cenocepacia complex.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Complexo Burkholderia cepacia/crescimento & desenvolvimento , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Animais , Células CHO , Cricetulus , Humanos , Estrutura Secundária de Proteína
10.
Biochem Biophys Res Commun ; 503(3): 1973-1979, 2018 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-30077368

RESUMO

Vasoactive intestinal peptide receptor 2 (VIPR2, also known as VPAC2) is a class B G-protein coupled receptor (GPCR) and plays important roles in the physiology of central nervous system (CNS) by interaction with natural ligands; vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Because it has been reported that high-expression and/or overactivation of VIPR2 link to schizophrenic symptoms, VIPR2 antagonists could be good drug candidates for schizophrenia therapeutics. In this study, we discovered several artificial peptides that antagonize both human and rodent VIPR2 with selectivities against receptor subtypes VIPR1 (also known as VPAC1) and pituitary adenylate cyclase-activating polypeptide type-1 receptor (PAC1). Of them, the representative 16-mer cyclic peptide VIpep-3 (Ac-CPPYLPRRLCTLLLRS-OH) exhibited strong binding affinity with KD value of 41 nM to extracellular domain of human VIPR2 in SPR analysis and showed potent antagonist activity with IC50 values of 47 nM (human), 180 nM (mouse), and 44 nM (rat) against VIP-VIPR2 signal in cell-based Ca influx assay. This is not only the first report on artificial VIPR2-selective antagonist peptides but also good example of the effective approach to discover novel antagonist against class B GPCR. Our peptides will contribute to study and development of the novel CNS drugs targeting to VIPR2.


Assuntos
Descoberta de Drogas , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Receptores Tipo II de Peptídeo Intestinal Vasoativo/antagonistas & inibidores , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Técnicas Biossensoriais , Células CHO , Cricetulus , Humanos , Ligantes , Camundongos , Biblioteca de Peptídeos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/síntese química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Ratos , Receptores Tipo II de Peptídeo Intestinal Vasoativo/metabolismo , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Peptídeo Intestinal Vasoativo/síntese química , Peptídeo Intestinal Vasoativo/química
11.
Peptides ; 104: 35-40, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29654809

RESUMO

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a naturally occurring cationic peptide with potent immunosuppressant and cytoprotective activities. We now show that full length PACAP38 and to a lesser extent, the truncated form PACAP27, and the closely related vasoactive intestinal peptide (VIP) and secretin had antimicrobial activity against the Gram-negative bacteria Escherichia coli in the radial diffusion assay. PACAP38 was more potent than either the bovine neutrophil antimicrobial peptide indolicidin or the synthetic antimicrobial peptide ARVA against E. coli. PACAP38 also had activity against the Gram-positive bacteria Staphylococcus aureus in the same assay with comparable potency to indolicidin and ARVA. In the more stringent broth dilution assay, PACAP38 had moderate sterilizing activity against E. coli, and potent sterilizing activity against the Gram-negative bacteria Pseudomonas aeruginosa. PACAP27, VIP and secretin were much less active than PACAP38 in this assay. PACAP38 also had some activity against the Gram-positive bacteria Bacillus cereus in the broth dilution assay. Many exopeptidase-resistant analogs of PACAP38, including both receptor agonists and antagonists, had antimicrobial activities equal to, or better than PACAP38, in both assays. PACAP38 made the membranes of E. coli permeable to SYTOX Green, suggesting a classical membrane lytic mechanism. These data suggest that analogs of PACPAP38 with a wide range of useful biological activities can be made by judicious substitutions in the sequence.


Assuntos
Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Membrana Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Hemólise/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-Atividade , Peptídeo Intestinal Vasoativo/química , Peptídeo Intestinal Vasoativo/farmacologia
12.
Clin Lab ; 64(1): 113-122, 2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-29479877

RESUMO

BACKGROUND: The self-developed portable surface plasmon resonance (SPR) biosensor was used in the quantitative detection and kinetic study of pituitary adenylate cyclase-activating polypeptide (PACAP), as the existing detection methods were complicated, with a long detection period, high-cost instrument, and sample needing to be labeled. METHODS: After preparing SPR biochip, the direct detection proceeded in an immune reaction detection between the PACAP samples with concentrations of 0.5 mg/L, 1 mg/L, 2 mg/L, 5 mg/L, 8 mg/L, 10 mg/L, and PACAP type 1 receptor (PAC1R). The standard curve of PACAP direct detection was established. According to the 1:1 Langumair model, the immune responses dynamic characteristic parameters of PAC1R with PACAP-38, and their reconstructive PN37R, PK38W were calculated, respectively. RESULTS: The direct detection limit of PACAP could be 0.5 mg/L. The absolute deviation and relative deviation of the detected value and the true value are both low. The magnitude orders of kinetic parameters of immune response between PAC1R and PACAP-38, PK38W, PN37R are basically the same. However, the specific values of the binding rate constant and dissociation rate constant of PK38W and PN37R are slightly larger than that of PACAP-38. CONCLUSIONS: The experimental results show that the SPR biochip detection system can be used for the effective quantitative detection of PACAP and can be used for kinetic study. The developed device could provide a labelfree, simple, quick, and low-cost method for the concentration detection of PACAP in the samples and the immune response study between PACAP and its receptors. It could promote PACAP to play an important role in the pharmaceutical industry, clinical treatment, and other industries.


Assuntos
Técnicas Biossensoriais/métodos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Algoritmos , Humanos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/imunologia , Ligação Proteica , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/imunologia , Reprodutibilidade dos Testes
13.
J Pharmacol Exp Ther ; 365(1): 1-8, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29363578

RESUMO

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are present in the spinal dorsal horn and dorsal root ganglia, suggesting an important role of PACAP signaling systems in the modulation of spinal nociceptive transmission. Previously, we found that intrathecal injection of PACAP or maxadilan, a selective PACAP type I (PAC1) receptor agonist, induced transient aversive responses followed by a long-lasting mechanical allodynia in mice, suggesting that PACAP-PAC1 receptor systems are involved in chronic pain and that selective PAC1 antagonists may become a new class of analgesics. Although several PAC1 antagonists, such as PACAP 6-38, have been reported, all of them are peptide compounds. In the present study, we identified new small-molecule antagonists of the PAC1 receptor using in silico screening and in vitro/vivo pharmacological assays. The identified small-molecule compounds, named PA-8 and PA-9, dose dependently inhibited the phosphorylation of CREB induced by PACAP in PAC1-, but not VPAC1- or VPAC2-receptor-expressing CHO cells. PA-8 and PA-9 also dose dependently inhibited PACAP-induced cAMP elevation with an IC50 of 2.0 and 5.6 nM, respectively. In vivo pharmacological assays showed that intrathecal injection of these compounds blocked the induction of PACAP-induced aversive responses and mechanical allodynia in mice. In contrast, the compounds when administered alone exerted neither agonistic nor algesic actions in the in vitro/vivo assays. The compounds identified in the present study are new and the first small-molecule antagonists of the PAC1 receptor; they may become seed compounds for developing novel analgesics.


Assuntos
Simulação por Computador , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/antagonistas & inibidores , Animais , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Avaliação Pré-Clínica de Medicamentos , Hiperalgesia/tratamento farmacológico , Masculino , Camundongos , Simulação de Acoplamento Molecular , Nociceptividade/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/uso terapêutico , Domínios Proteicos , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo
14.
Gen Comp Endocrinol ; 255: 90-101, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-28974369

RESUMO

The somatotropic axis (SA) regulates numerous aspects of vertebrate physiology such as development, growth, and metabolism and has influence on several tissues including neural, immune, reproductive and gastric tract. Growth hormone (GH) is a key component of SA, it is synthesized and released mainly by pituitary somatotrophs, although now it is known that virtually all tissues can express GH, which, in addition to its well-described endocrine roles, also has autocrine/paracrine/intracrine actions. In the pituitary, GH expression is regulated by several hypothalamic neuropeptides including GHRH, PACAP, TRH and SST. GH, in turn, regulates IGF1 synthesis in several target tissues, adding complexity to the system since GH effects can be exerted either directly or mediated by IGF1. In reptiles, little is known about the SA components and their functional interactions. The aim of this work was to characterize the mRNAs of the principal SA components in the green iguana and to develop the tools that allow the study of the structural and functional evolution of this system in reptiles. By employing RT-PCR and RACE, the cDNAs encoding for GHRH, PACAP, TRH, SST and IGF1 were amplified and sequenced. Results showed that these cDNAs coded for the corresponding protein precursors of 154, 170, 243, 113, and 131 amino acids, respectively. Of these, GHRH, PACAP, SST and IGF1 precursors exhibited a high structural conservation with respect to its counterparts in other vertebrates. On the other hand, iguana's TRH precursor showed 7 functional copies of mature TRH (pyr-QHP-NH2), as compared to 4 and 6 copies of TRH in avian and mammalian proTRH sequences, respectively. It was found that in addition to its primary production site (brain for GHRH, PACAP, TRH and SST, and liver for IGF1), they were also expressed in other peripheral tissues, i.e. testes and ovaries expressed all the studied mRNAs, whereas TRH and IGF1 mRNAs were observed ubiquitously in all tissues considered. These results show that the main SA components in reptiles of the Squamata Order maintain a good structural conservation among vertebrate phylogeny, and suggest important physiological interactions (endocrine, autocrine and/or paracrine) between them due to their wide peripheral tissue expression.


Assuntos
Hormônio Liberador de Hormônio do Crescimento/genética , Iguanas/genética , Fator de Crescimento Insulin-Like I/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Somatostatina/genética , Hormônio Liberador de Tireotropina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Hormônio Liberador de Hormônio do Crescimento/química , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Fator de Crescimento Insulin-Like I/química , Fator de Crescimento Insulin-Like I/metabolismo , Filogenia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Somatostatina/química , Somatostatina/metabolismo , Hormônio Liberador de Tireotropina/química , Hormônio Liberador de Tireotropina/metabolismo
15.
PLoS One ; 12(7): e0182100, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28759637

RESUMO

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an inhibitor of megakaryopoiesis and platelet function. Recently, PACAP deficiency was observed in children with nephrotic syndrome (NS), associated with increased platelet count and aggregability and increased risk of thrombosis. To further study PACAP deficiency in NS, we used transgenic Tg(cd41:EGFP) zebrafish with GFP-labeled thrombocytes. We generated two models for congenital NS, a morpholino injected model targeting nphs1 (nephrin), which is mutated in the Finnish-type congenital NS. The second model was induced by exposure to the nephrotoxic compound adriamycin. Nephrin RNA expression was quantified and zebrafish embryos were live-screened for proteinuria and pericardial edema as evidence of renal impairment. Protein levels of PACAP and its binding-protein ceruloplasmin were measured and GFP-labeled thrombocytes were quantified. We also evaluated the effects of PACAP morpholino injection and the rescue effects of PACAP-38 peptide in both congenital NS models. Nephrin downregulation and pericardial edema were observed in both nephrin morpholino injected and adriamycin exposed congenital NS models. However, PACAP deficiency was demonstrated only in the adriamycin exposed condition. Ceruloplasmin levels and the number of GFP-labeled thrombocytes remained unchanged in both models. PACAP morpholino injections worsened survival rates and the edema phenotype in both congenital NS models while injection with human PACAP-38 could only rescue the adriamycin exposed model. We hereby report, for the first time, PACAP deficiency in a NS zebrafish model as a consequence of adriamycin exposure. However, distinct from the human congenital NS, both zebrafish models retained normal levels of ceruloplasmin and thrombocytes. We further extend the renoprotective effects of the PACAP-38 peptide against adriamycin toxicity in zebrafish.


Assuntos
Proteínas de Membrana/metabolismo , Síndrome Nefrótica/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Plaquetas/metabolismo , Ceruloplasmina/metabolismo , Doxorrubicina/toxicidade , Proteínas de Membrana/genética , Síndrome Nefrótica/etiologia , Síndrome Nefrótica/genética , Fragmentos de Peptídeos/farmacologia , Pericárdio/efeitos dos fármacos , Pericárdio/metabolismo , Pericárdio/patologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
16.
Curr Med Chem ; 24(13): 1332-1349, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28245765

RESUMO

BACKGROUND: Migraine is a highly disabling neurovascular primary headache disorder, with its exact pathomechanism being still unrevealed. The current leading hypotheses are based on the sensitization and activation of the trigeminovascular system. OBJECTIVE: To review the literature with focus on the effects of kynurenines (L-kynurenine and kynurenic acid) and pituitary adenylate cyclase-activating polypeptide on the regulation of the trigeminovascular system. METHOD: A literature search was conducted to identify preclinical and clinical publications (198 references) by using the keywords 'kynurenines', 'pituitary adenylate cyclase-activating polypeptide', and 'migraine' in the database of MEDLINE/PubMed up to 10 September 2016 for topical review. Additional filters used included 'review', 'systematic review', 'original article', and 'English language'. RESULTS: L-kynurenine and kynurenic acid act on the glutamatergic system at the level of the second-order nociceptive neurons in the trigeminal nucleus caudalis. Pituitary adenylate cyclase- activating polypeptide is released from the peripheral nerve endings of the trigeminal pseudounipolar neurons and causes vasodilation and mast cell degranulation, leading to consequent peripheral sensitization of the dural nociceptors. Centrally released pituitary adenylate cyclase-activating polypeptide in the trigeminal nucleus caudalis results in the central sensitization of the second-order neurons. The sensitization process leads to the characteristic features of migraine. CONCLUSION: L-kynurenine, kynurenic acid, and pituitary adenylate cyclase-activating polypeptide may have fundamental roles in the initiation of migraine headache attacks.


Assuntos
Cinurenina/metabolismo , Transtornos de Enxaqueca/patologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Animais , Química Farmacêutica , Ácido Glutâmico/metabolismo , Humanos , Cinurenina/química , Cinurenina/uso terapêutico , Transtornos de Enxaqueca/tratamento farmacológico , Transtornos de Enxaqueca/metabolismo , Neurônios/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo
17.
Cancer Biother Radiopharm ; 31(1): 29-36, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26844850

RESUMO

OBJECTIVE: The authors have conjugated chelating agents (DOTA and NODAGA) with a peptide (pituitary adenylate cyclase-activating peptide [PACAP] analogue) that has a high affinity for VPAC1 receptors expressed on cancer cells. To determine a suitable chelating agent for labeling with (68)Ga, they have compared the labeling kinetics and stability of these peptide conjugates. METHODS: For labeling, (68)GaCl3 was eluted in 0.1 M HCl from a [(68)Ge-(68)Ga] generator. The influences of peptide concentration, pH, and temperature on the radiolabeling efficiency were studied. The stability was evaluated in saline, human serum, DTPA, transferrin, and metallic ions (FeCl3, CaCl2, and ZnCl2). Cell binding assay was performed using human breast cancer cells (T47D). Tissue biodistribution was studied in normal athymic nude mice. RESULTS: Optimal radiolabeling (>95.0%) of the DOTA-peptide conjugates required a higher (50°C-90°C) temperature and 10 minutes of incubation at pH 2-5. The NODAGA-peptide conjugate needed incubation only at 25°C for 10 minutes. Both radiocomplexes were stable in saline, serum, as well as against transchelation and transmetallation. Cell binding at 37°C for 15 minutes of incubation with (68)Ga-NODAGA-peptide was 34.0% compared to 24.5% for (68)Ga-DOTA-peptide. Tissue biodistribution at 1 hour postinjection of both (68)Ga-labeled peptide conjugates showed clearance through the kidneys. CONCLUSIONS: NODAGA-peptide showed more convenient radiolabeling features than that of DOTA-peptide.


Assuntos
Quelantes/farmacocinética , Radioisótopos de Gálio/farmacocinética , Neurotransmissores/fisiologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacocinética , Animais , Quelantes/química , Feminino , Radioisótopos de Gálio/química , Humanos , Camundongos , Camundongos Nus , Neurotransmissores/química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Tomografia por Emissão de Pósitrons , Distribuição Tecidual
18.
Artigo em Inglês | MEDLINE | ID: mdl-26393433

RESUMO

The role of PACAP in spermatogenesis and steroidogenesis has been largely investigated in last years in mammals; conversely, a few studies have been performed in non mammalian vertebrates. In this paper we investigated the sequence, expression and localization of PACAP and its PAC1 receptor in the testis of the benthic elasmobranch Torpedo marmorata, the marbled electric ray. Cloning a partial PACAP cDNA, we demonstrated for the first time in elasmobranches that PACAP shows a highly conserved sequence, compared with the PACAP of other chordates (tunicates and vertebrates). Moreover, the phylogenetic analysis revealed that PACAP has been well preserved during evolution and that a negative selection acts on PACAP sequence, leading to the conservation of the coding sites. The phylogenetic consensus tree showed also that Torpedo PACAP is more related with the amphibian PACAP than with the teleost one. Finally, we demonstrated that in T. marmorata PACAP and its PAC1 receptor are synthesized directly in the testis, where they show a wider localization than mammals, suggesting that this neuropeptide is involved in the control of Torpedo spermatogenesis.


Assuntos
Filogenia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Testículo/metabolismo , Torpedo/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Humanos , Masculino , Dados de Sequência Molecular , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Transporte Proteico , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Espermatogênese
19.
Neuropharmacology ; 108: 440-50, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-26006268

RESUMO

Parkinson's disease (PD) is characterized by a steady loss of dopamine neurons through apoptotic, inflammatory and oxidative stress processes. In that line of view, the pituitary adenylate cyclase-activating polypeptide (PACAP), with its ability to cross the blood-brain barrier and its anti-apoptotic, anti-inflammatory and anti-oxidative properties, has proven to offer potent neuroprotection in various PD models. Nonetheless, its peripheral actions, paired with low metabolic stability, hampered its clinical use. We have developed Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27) as an improved PACAP-derived neuroprotective compound. In vitro, this analog stimulated cAMP production, maintained mitochondrial potential and protected SH-SY5Y neuroblastoma cells from 1-methyl-4-phenylpyridinium (MPP(+)) toxicity, as potently as PACAP. Furthermore, contrasting with PACAP, it is stable in human plasma and against dipeptidyl peptidase IV activity. When injected intravenously to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, PACAP and Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27) restored tyrosine hydoxylase expression into the substantia nigra and modulated the inflammatory response. Albeit falls of mean arterial pressure (MAP) were observed with both PACAP- and Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27)-treated mice, the intensity of the decrease as well as its duration were significantly less marked after iv injections of the analog than after those of the native polypeptide. Moreover, no significant changes in heart rate were measured with the animals for both compounds. Thus, Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27) appears as a promising lead molecule for the development of PACAP-derived drugs potentially useful for the treatment of PD or other neurodegenerative diseases.


Assuntos
Doenças Cardiovasculares/prevenção & controle , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/uso terapêutico , Transtornos Parkinsonianos/tratamento farmacológico , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/uso terapêutico , Animais , Células CHO , Doenças Cardiovasculares/induzido quimicamente , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores/farmacologia , Transtornos Parkinsonianos/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia
20.
J Biomed Nanotechnol ; 11(12): 2111-23, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26510306

RESUMO

For the first time the efficiency of gene knockdown of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor 1 (PAC1R) is demonstrated by employing gold nanocomplexes. This gene knockdown subsequently affects the action of PACAP on neurite outgrowth in PC12 cells. These nanocomplexes comprise cholera toxin B (CTB)-gold nanoparticle conjugates loaded with double-stranded morpholinos (MOs) (photo MO and antisense MO). Nanocomplexes are introduced into cells via lipid raft-dependent endocytosis. After UV light exposure, the photolinker in the photo MO is cleaved, bisecting the photo MO and releasing the antisense MO from the conjugate. The antisense MO then binds the PAC1R mRNA and decreases gene expression. The maximal efficiency of gene knockdown is observed after 24 hours, resulting in a 65% ± 12 reduction of the protein level. This reduction in PAC1R impairs the responsiveness of cells to PACAP exposure. Following PAC1R gene knockdown, only 10% ± 8 and 11%± 9 of cells exhibit neurite outgrowth after 4-day exposure to PACAP-38 and PACAP-27, respectively. These results demonstrate an efficient PAC1R gene knockdown and noticeable difference in response to PACAP action on neural cell differentiation, adding an extra dimension to determine the involvement of PACAP and its PAC1R in the neurotropic effect to PC12 cells.


Assuntos
Técnicas de Silenciamento de Genes , Ouro/química , Nanopartículas Metálicas , Morfolinos/química , Morfolinos/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/deficiência , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Animais , Sequência de Bases , Transporte Biológico , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Toxina da Cólera/metabolismo , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Ouro/metabolismo , Modelos Moleculares , Fator de Crescimento Neural/farmacologia , Neurônios/citologia , Conformação de Ácido Nucleico , Células PC12 , Fragmentos de Peptídeos/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Ratos , Raios Ultravioleta
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