PACAP key interactions with PAC1, VPAC1, and VPAC2 identified by molecular dynamics simulations.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) belongs to the glucagon/secretin family. PACAP interacts with the pituitary adenylate cyclase-activating polypeptide receptor type 1 (PAC1) and vasoactive intestinal peptide receptors 1 and 2 (VPAC1 and VPAC2), exhibiting functions in the immune, endocrine, and nervous systems. This peptide is upregulated in numerous instances of brain injury, acting as a neuroprotective agent. It can also suppress HIV-1 and SARS-CoV-2 viral replication in vitro. This work aimed to identify, in each peptide-receptor system, the most relevant residues for complex stability and interaction energy communication via Molecular Dynamics (MD), Free Energy calculations, and Protein-energy networks, thus revealing in detail the underlying mechanisms of activation of these receptors. Hydrogen bond formation, interaction energies, and computational alanine scanning between PACAP and its receptors showed that His1, Asp3, Arg12, Arg14, and Lys15 are crucial to the peptide's stability. Furthermore, several PACAP interactions with structurally conserved positions deemed necessary in GPCR B1 activation, including Arg2.60, Lys2.67, and Glu7.42, were significant for the peptide's stability within the receptors. According to the protein-energy network, the connection between Asp3 of PACAP and the receptors' conserved Arg2.60 represents a critical energy communication hub in all complexes. Additionally, the ECDs of the receptors were also found to function as energy communication hubs for PACAP. Although the overall binding mode of PACAP in the three receptors was found to be highly conserved, Arg12 and Tyr13 of PACAP were more prominent in complex with PAC1, while Ser2 of PACAP was with VPAC2. The detailed analyses performed in this work pave the way for using PACAP and its receptors as therapeutic targets.Communicated by Ramaswamy H. Sarma.

Role of VPAC1 and VPAC2 receptors in the etiology of pregnancy rhinitis: an experimental study in rats / O papel dos receptores VPAC1 e VPAC2 na etiologia da rinite gestacional: um estudo experimental em ratos

Abstract Introduction: Pregnancy rhinitis is a common sex hormone-related otorhinolaryngological disorder. There are some epidemiological and physiological studies on pregnancy rhinitis, but histopathological and biomolecular changes have not been studied thoroughly. Objectives: The receptors VPAC1 and VPAC2 are known for their roles in allergic rhinitis. On the other hand, activation of subclinical allergy has been suggested in the pathophysiology of pregnancy rhinitis. Therefore, we aimed to compare the physiological and gestational pattern of VPAC1 and VPAC2 expression in rat nasal mucosa. Methods: Twenty adult Wister albino female rats were enrolled into the study. Two groups constituted as 10 control (group A) and 10 pregnant (group B) rats. They were fed ad libitum and sheltered at room temperature (22°±2°C). The rats were sacrificed at the 20th day of gestation by intraperitoneal injection of 400mg/kg Na-pentobarbitone. Then, 10 - 15 mL of blood was taken, and samples were reserved for the detection of serum estradiol and progesterone levels by ELISA test. The nasal septum was resected and divided in half for immunohistochemical analyses and real time polymerase chain reaction testing of VPAC1 and VPAC2. Results: VPAC1 and VPAC2 were found to be in all layers of septal specimens, but the immunostaining of surface epithelium was more distinct in specimens of both groups. We demonstrated higher overall staining intensity in the pregnant group. PCR revealed significant increase in expression of VPAC1 (p = 0.023) and VPAC2 (p = 0.021) in pregnant group when compared with control group. In addition, we demonstrated upregulatory effect of estradiol and progesterone on the vasoactive intestinal peptide receptor expression. Conclusions: Gestational up-regulation of nasal VPAC1 and VPAC2 was shown both by PCR and immunohistochemical analysis. These findings support the hypothesis that PR is caused by the activation of subclinical allergy that is present before pregnancy.

Resumo Introdução: A rinite gestacional é um distúrbio comum da otorrinolaringologia relacionado a hormônios sexuais. Existem alguns estudos epidemiológicos e fisiológicos sobre rinite gestacional, mas as alterações histopatológicas e biomoleculares ainda não foram estudadas completamente. Objetivo: Os receptores VPAC1 e VPAC2 são conhecidos por seu papel na rinite alérgica. Por outro lado, a ativação da alergia subclínica tem sido sugerida na fisiopatologia da rinite gestacional. Portanto, objetivamos comparar o padrão fisiológico e gestacional da expressão de VPAC1 e VPAC2 na mucosa nasal de ratos. Método: Vinte ratas fêmeas Wistar albinas adultas foram incluídas no estudo. Os dois grupos foram divididos em 10 ratas; controle (grupo A) e 10 ratas prenhes (grupo B). Elas foram alimentadas ad libitum e abrigadas em temperatura ambiente (22° ±2° C). Sacrificamos as ratas no 20° dia de gestação por injeção intraperitoneal de 400 mg/kg de sódio-pentobarbital. Em seguida, foram coletados 10 a 15 mL de sangue e as amostras foram reservadas para a detecção dos níveis séricos de estradiol e progesterona pelo método Elisa. O septo nasal foi ressecado e dividido em 2 para análises imuno-histoquímicas e testes de reação em cadeia da polimerase em tempo real, RT-PCR, de VPAC1 e VPAC2. Resultados: VPAC1 e VPAC2 foram encontrados em todas as camadas da amostra septal, mas a imunocoloração do epitélio de superfície foi mais distinta nas amostras de ambos os grupos. Demonstramos maior intensidade geral de coloração no grupo gestante. A reação de polimerase em cadeia revelou aumento significante na expressão de VPAC1 (p = 0,023) e VPAC2 (p = 0,021) no grupo gestante quando comparado ao grupo controle. Além disso, demonstramos um efeito up-regulador do estradiol e progesterona na expressão do receptor peptídeo intestinal vasoativo. Conclusão: A up-regulação gestacional dos receptores VPAC1 e VPAC2 nasais foi demonstrada tanto por reação de polimerase em cadeia quanto por análise imuno-histoquímica. Esses achados corroboram a hipótese de que a rinite gestacional é causada pela ativação de alergia subclínica presente antes da gestação.

Role of VPAC1 and VPAC2 receptors in the etiology of pregnancy rhinitis: an experimental study in rats.

INTRODUCTION: Pregnancy rhinitis is a common sex hormone-related otorhinolaryngological disorder. There are some epidemiological and physiological studies on pregnancy rhinitis, but histopathological and biomolecular changes have not been studied thoroughly. OBJECTIVES: The receptors VPAC1 and VPAC2 are known for their roles in allergic rhinitis. On the other hand, activation of subclinical allergy has been suggested in the pathophysiology of pregnancy rhinitis. Therefore, we aimed to compare the physiological and gestational pattern of VPAC1 and VPAC2 expression in rat nasal mucosa. METHODS: Twenty adult Wister albino female rats were enrolled into the study. Two groups constituted as 10 control (group A) and 10 pregnant (group B) rats. They were fed ad libitum and sheltered at room temperature (22°±2°C). The rats were sacrificed at the 20th day of gestation by intraperitoneal injection of 400mg/kg Na-pentobarbitone. Then, 10-15mL of blood was taken, and samples were reserved for the detection of serum estradiol and progesterone levels by ELISA test. The nasal septum was resected and divided in half for immunohistochemical analyses and real time polymerase chain reaction testing of VPAC1 and VPAC2. RESULTS: VPAC1 and VPAC2 were found to be in all layers of septal specimens, but the immunostaining of surface epithelium was more distinct in specimens of both groups. We demonstrated higher overall staining intensity in the pregnant group. PCR revealed significant increase in expression of VPAC1 (p=0.023) and VPAC2 (p=0.021) in pregnant group when compared with control group. In addition, we demonstrated upregulatory effect of estradiol and progesterone on the vasoactive intestinal peptide receptor expression. CONCLUSIONS: Gestational up-regulation of nasal VPAC1 and VPAC2 was shown both by PCR and immunohistochemical analysis. These findings support the hypothesis that PR is caused by the activation of subclinical allergy that is present before pregnancy.