Effects of pituitary adenylate cyclase activating polypeptide on small intestinal INT 407 cells.

Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide having a widespread distribution both in the nervous system and peripheral organs including the gastrointestinal tract. It has been shown to exert actions on intestinal functions, mainly affecting glandular secretion and motility. PACAP has several different effects on cell survival depending on the cell type and the applied stimulus. Its influences on small intestinal epithelial cells are not yet elucidated, therefore the aim of the present study was to investigate the effects of PACAP on intestinal epithelial cells having high turnover (INT 407) against different harmful stimuli, such as oxidative stress, in vitro hypoxia and gamma radiation. We tested the effect of PACAP on proliferation and cell survival using MTT assay. Moreover, various cancer-related factors were evaluated by oncology array. PACAP did not influence the proliferation rate of INT 407 cells. Its cell survival-enhancing effect could be detected against oxidative stress, but not against in vitro hypoxia or gamma irradiation. Clonogenic survival assay was performed to analyze the effect of PACAP on clonogenic potential of cells exposed to gamma radiation. Surprisingly, PACAP enhanced the clone-forming ability decrease induced by irradiation. Western blot analysis of ERK1/2 phosphorylation was performed in order to obtain further information on the molecular background. Our data showed phospho-ERK1/2 suppression of PACAP in irradiated cells. Furthermore, the role of endogenous PACAP against oxidative stress was also investigated performing ADCYAP1 small interfering RNA transfection. We found significant difference in the cell vulnerability between cells undergoing silencing and cells without transfection suggesting the protective role of the endogenously present PACAP against oxidative stress in INT 407 cells. In summary, PACAP seems to be able to exert contradictory effects in INT 407 cells depending on the applied stressor, suggesting its regulatory role in the cellular household.

Changes in the expression of pituitary adenylate cyclase-activating polypeptide in the human placenta during pregnancy and its effects on the survival of JAR choriocarcinoma cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide with survival-promoting actions, has been observed in endocrine organs and is thought to play a role in reproductive functions, including pregnancy. PACAP occurs in two forms, 27 and 38 amino acid residues, with PACAP38 being the predominant form in human tissues. In the present study, we determined the concentrations of PACAP38 and PACAP27 in first-trimester and full-term human placentas using radioimmunoassay. We found high levels of PACAP38 and lower levels of PACAP27 in different parts of the full-term human placenta. PACAP38 content increased in the placenta during pregnancy, both on the maternal side and on the fetal side. The effects of PACAP on the survival of JAR human choriocarcinoma cells were investigated using flow cytometry and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) cell viability assay in cells exposed to the widely used chemotherapeutic agent methotrexate (MTX). It was found that PACAP neither influenced the survival of JAR cytotrophoblast cells nor affected cellular response to the death-inducing effect of the chemotherapeutic agent MTX. The present observations further support the significance of PACAP in the human placenta. The observation that PACAP did not influence the effects of MTX may have future clinical importance, showing that PACAP does not decrease the effects of certain chemotherapeutic agents.

Effects of pituitary adenylate cyclase activating polypeptide on the survival and signal transduction pathways in human choriocarcinoma cells.

The pituitary adenylate cyclase activating polypeptide (PACAP) has several effects in endocrine and reproductive organs, including the placenta. PACAP is generally known as a survival-promoting peptide acting on divergent signal transduction pathways. However, its effects on the survival and signaling mechanisms of trophoblast cells are not known. In the present study we found that 1-h pretreatment with PACAP38 did not significantly influence the survival of JAR cytotrophoblast cells. However, the survival rate of cells exposed to oxidative stress or CoCl(2)-induced in vitro hypoxia showed a significant further decrease in PACAP-treated cells, implying that PACAP sensitizes the cells to these stressors. This was not observed in the case of lipopolysaccharide or ethanol treatment. Western blot data revealed that, in cells exposed to oxidative stress, PACAP treatment decreased phosphorylation of all extracellular signal-regulated kinase (ERK), phospho-jun N-terminal kinase (JNK), protein kinase B, p38 mitogen-activated protein kinase (MAPK), and phospho-glycogen synthase kinase (GSK) and the expression of bax. The overall effect seems to be a sensitizing effect in almost all examined pathways when oxidative stress was applied, which may explain the enhancing effect of PACAP on cell death in contrast to most other cell types examined so far. Our data show that the signaling mechanism of PACAP may be different in trophoblast cells to that observed in other cell lines.

Agonistic behavior of PACAP6-38 on sensory nerve terminals and cytotrophoblast cells.

The effects of pituitary adenylate cyclase activating polypeptide (PACAP) are mediated through G-protein-coupled receptors, the specific PAC1 receptor and VPAC1 and VPAC2 receptors which bind vasoactive intestinal peptide with similar affinity. Based on binding affinity studies, PACAP6-38 was discovered as a potent antagonist of PAC1 and it has been used by hundreds of studies as a PACAP antagonist. Recently, we have found that in certain cells/tissues, PACAP6-38 does not antagonize PACAP-induced effects, but surprisingly, it exerts similar actions to PACAP1-38, behaving as an agonist. In the present study, we report on the agonistic behavior of PACAP6-38 on neuropeptide release from sensory nerves of the isolated rat trachea and on the MAPK signaling pathways in cytotrophoblast cells. In isolated rat tracheae, PACAP6-38, similarly to PACAP1-38, induced significant inhibitory effects on the release of three simultaneously measured sensory neuropeptides, substance P, calcitonin gene-related peptide, and somatostatin evoked by both chemical excitation and electrical field stimulation of capsaicin-sensitive afferents. Effects of PACAP6-38 were the same as those of PACAP1-38 on MAPK signaling in human cytotrophoblast cells. Western blot analysis showed that both peptide forms stimulated ERK1/2 and JNK phosphorylation, while they both inhibited p38 MAPK phosphorylation. The most pronounced effects were observed when both peptides were present. In summary, our results show that PACAP6-38, which is a PACAP receptor antagonist in most cells/tissues, can behave as an agonist in other systems. The increasing interest in the effects of PACAP requires further studies on the pharmacological properties of the peptide and its analogues.

Cloning, sequencing, structural and molecular biological characterization of placental protein 20 (PP20)/human thiamin pyrophosphokinase (hTPK).

Full-length cDNAs of placental protein 20 (PP20) were cloned by screening a human placental cDNA library, which encode a 243 amino acid protein, identical to human thiamin pyrophosphokinase (hTPK) as confirmed by protein sequence analysis. Genomic alignment showed that the PP20/hTPK gene contains 9 exons. It is abundantly expressed in placenta, as numerous EST clones were identified. As thiamine metabolism deficiencies have been seen in placental infarcts previously, these indicate that PP20/hTPK may have a role in placental diseases. Analysis of the 1kb promoter region showed numerous putative transcription factor binding sites, which might be responsible for the ubiquitous PP20/hTPK expression. This may also be in accordance with the presence of the protein in tissues responsible for the regulation of the exquisite balance between cell division, differentiation and survival. TPK activity of the purified and recombinant protein was proved by mass spectrometry with electrospray ionization. By Western blot, PP20/hTPK was found in all human normal and tumorous adult and fetal tissues in nearly equal amounts, but not in sera. By immunohistochemical and immunofluorescent confocal imaging methods, diffuse labelling in the cytoplasm of the syncytiotrophoblasts and weak staining of the trophoblasts were observed, and the amount of PP20/hTPK decreased from the first trimester to the end of gestation. A 3D model of PP20/hTPK was computed (PDB No.: 1OLY) by homology modelling. A high degree of structural homology showed that the thiamin binding site was highly similar to that of the mouse enzyme, but highly different from the bacterial ones. Comparison of the catalytic centre sequences revealed differences, raising the possibility of designing new drugs which specifically inhibit bacterial and fungal enzymes without affecting PP20/hTPK and offering the possibility for safe antimicrobial therapy during pregnancy.

Extremely high maternal alkaline phosphatase serum concentration with syncytiotrophoblastic origin.

An extremely high alkaline phosphatase (AP) concentration (3609 IU/litre) was found in a 20 year old primigravida at 37 week's gestation, prompting an examination of its histological and cellular origin. Immunohistochemistry and western blots using antibodies against AP, Ki-67, phospho-protein kinase B (Akt), phospho-p44/42 mitogen activated protein kinase/extracellular signal regulated kinase 1/2 (MAPK/Erk1/2), phospho-glycogen synthase kinase-3beta (GSK-3beta), phospho-stress activated protein kinase/c-Jun N-terminal kinase, total-Akt, total-GSK-3beta, and phospho-p38-MAPK were carried out on index and control placental samples of the same gestational age. Compared with controls, staining of the index placenta showed minimal AP labelling of the brush border and remarkable positivity of the intervillous space. Cytotrophoblastic proliferation was 8-10% in the index placenta compared with 1-2% in controls. The index placenta also had raised concentrations of protein kinases with important roles in cell differentiation. The proliferation and differentiation rates of the cytotrophoblasts were found to be five times higher in index samples than in controls. It is hypothesised that loss of syncytial membranes in immature villi led to increased AP concentrations in the maternal circulation and decreased AP staining of the placenta. Loss of the syncytium might also stimulate increased proliferation of villous cytotrophoblasts, which would then fuse and maintain the syncytium.