PACAP-38

Evidence has been accumulating that elements of the vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) system are missing in non-chordate genomes, which is at odds with the partial sequence-, immunohistochemical-, and physiological data in the literature. Multilevel experiments were performed on the great pond snail (Lymnaea stagnalis) to explore the role of PACAP in invertebrates. Screening of neuronal transcriptome and genome data did not reveal homologs to the elements of vertebrate PACAP system. Despite this, immunohistochemical investigations with an anti-human PAC1 receptor antibody yielded a positive signal in the neuronal elements in the heart. Although Western blotting of proteins extracted from the nervous system found a relevant band for PACAP-38, immunoprecipitation and mass spectrometric analyses revealed no corresponding peptide fragments. Similarly to the effects reported in vertebrates, PACAP-38 significantly increased cAMP synthesis in the heart and had a positive ionotropic effect on heart preparations. Moreover, it significantly modulated the effects of serotonin and acetylcholine. Homologs to members of Cluster B receptors, which have shared common evolutionary origin with the vertebrate PACAP receptors, PTHRs, and GCGRs, were identified and shown not to be expressed in the heart, which does not support a potential role in the mediation of PACAP-induced effects. Our findings support the notion that the PACAP system emerged after the protostome-deuterostome divergence. Using antibodies against vertebrate proteins is again highlighted to have little/no value in invertebrate studies. The physiological effects of vertebrate PACAP peptides in protostomes, no matter how similar they are to those in vertebrates, should be considered non-specific.

Retinal ischemia is an ophthalmic emergency often caused by cardiovascular diseases, leading to irreversible vision loss and even blindness. Innovative retinal ischemia treatments are needed due to limited options. The pathological mechanisms involve retinal cell apoptosis and microglial activation. The pituitary adenylate cyclase-activating polypeptide (PACAP) is a well distributed neuropeptide found in both central nervous system and peripheral organs. Though it shows great anti-apoptosis and anti-microglia activation properties, it is rapidly cleared by intravitreal injection. Herein, we established a novel poly(ethylene glycol) (PEG) hydrogel system by cross-linking 4arm-PEG-NHS and 4arm-PEG-NH₂ to load PACAP (PACAP@Gel-PEG), which exhibited great fluidity, injectability, structural recovery ability, moderate swelling ratio and drug release ability that were appropriate for drug delivery. Then the safety and effectiveness of the PACAP@Gel-PEG were evaluated in vitro in three retinal cell lines (ARPE-19, 661 W and rRMC) and in vivo using the unilateral common carotid artery occlusion (UCCAO) mice model. The CCK-8 test and live/dead staining demonstrated that PACAP@Gel-PEG exhibited excellent biocompatibility in three retinal cell lines. Furthermore, after PACAP@Gel-PEG treatment, a great anti-apoptotic effect was observed in cells treated by CoCl₂. Application of PACAP@Gel-PEG greatly improved the therapeutic efficacy of PACAP in restoring retinal function, maintaining retinal integrity, and suppressing apoptosis and microglia activation in retinal tissues. Moreover, in mice, the biosafety of PACAP@Gel-PEG was confirmed by H&E staining of systemic organs. Taken together, our results demonstrated PACAP@Gel-PEG as a promising therapeutic option for retinal ischemia, providing new strategies for vision restoration.