Balazs Opper

Mice deficient in pituitary adenylate cyclase activating polypeptide (PACAP) show increased susceptibility to in vivo renal ischemia/reperfusion injury

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with well-known cytoprotective effects. We have reported earlier that PACAP decreases mortality and the degree of tubular atrophy in a rat model of renal ischemia/reperfusion injury. Recently, we have shown that kidney cultures isolated from PACAP deficient mice show increased susceptibility to renal oxidative stress. Based on these previous studies, we raised the question whether PACAP deficient mice display increased sensitivity to in vivo kidney ischemia/reperfusion. PACAP⁻/⁻ mice underwent 45 or 60 min of renal ischemia followed by 2 weeks reperfusion. Kidneys were processed for histological analysis. Sections stained with PAS-haematoxylin were graded for the following parameters: degree of tubular dilation, Bowmanns capsule dilation, lymphocyte and macrophage infiltration, thyroidization and the disappearance of the PAS-positive glycocalyx from under the brush border. In other sets of experiments, tissue cytokine expression and the level of the endogenous antioxidant superoxide dismutase (SOD) were also determined after 60 min ischemia/reperfusion. Our results show that while intact kidneys were not different between wild-type and PACAP deficient mice, marked differences were observed in the histological structures in groups that underwent ischemia/reperfusion. PACAP deficient mice had a worse histological outcome, with significantly higher histological scores for all tested parameters. Cytokine expression was also markedly different between wild-type and PACAP deficient mice. In addition, the level of SOD was significantly lower in PACAP⁻/⁻ animals after ischemia/reperfusion. In conclusion, the lack of endogenous PACAP leads to higher susceptibility to in vivo renal ischemia/reperfusion, suggesting that PACAP has an endogenous renoprotective effect.

Effects of PACAP on Oxidative Stress-Induced Cell Death in Rat Kidney and Human Hepatocyte Cells

Oxidative stress plays an important role in various renal and hepatic pathologies, and reduction of oxidative stress-induced processes is an important protective strategy in tissues of diverse origins against harmful stimuli. Pituitary adenylate cyclase activating polypeptide (PACAP) is a well-known cytotrophic and cytoprotective peptide. PACAP promotes cell survival in numerous cells and tissues exposed to various stimuli. Protective effects of PACAP have been shown in the kidney, but it is not known whether PACAP is protective against oxidative stress in renal cells. Little is known about the effects of PACAP in the liver. The aim of the present study was to investigate whether PACAP is protective against oxidative stress in primary rat kidney cell culture and whether PACAP has any effect on cell survival in human WRL-68 hepatocytes and HEP-G2 hepatocellular carcinoma cells subjected to oxidative stress. Cells were exposed to various concentrations of H2O2 with or without PACAP co-treatment and cell viability was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test (MTT). We found that oxidative stress induced a significant decrease in cell viability in both cell lines. PACAP could dose-dependently increase the percentage of living cells in kidney cells, but it failed to do so in hepatocytes. Given the survival-promoting effects of PACAP against oxidative stress in rat kidney, we conducted a further experiment to determine whether PACAP influences the markers of oxidative stress in vivo. We have proven earlier that PACAP was effective in kidney ischemia/reperfusion injury in vivo. In the present study, we determined the levels of the oxidative stress marker malondialdehyde and the activity of the scavenger molecules glutathione (GSH) and superoxide dismutase (SOD) following kidney ischemia/reperfusion in rats. We found that PACAP significantly increased the level of GSH and counteracted the marked reduction of SOD activity after ischemia/reperfusion in vivo. In summary, the present study showed that while PACAP was able to significantly increase the cell survival in primary kidney cell cultures exposed to oxidative stress, possibly involving interaction with the endogenous scavenger system, it failed to influence the viability of normal or cancerous hepatocytes.

Revising lysenin expression of earthworm coelomocytes

Lysenin is a species-specific bioactive molecule of Eisenia andrei earthworms. This protein is a potent antimicrobial factor; however its cellular expression and induction against pathogens are still not fully understood. We developed a novel monoclonal antibody against lysenin and applied this molecular tool to characterize its production and antimicrobial function. We demonstrated by flow cytometry and immunocytochemistry that one subgroup of earthworm immune cells (so called coelomocytes), the chloragocytes expressed the highest amount of lysenin. Then, we compared lysenin expression with earlier established coelomocyte (EFCC) markers. In addition, we determined by immunohistology of earthworm tissues that lysenin production is only restricted to free-floating chloragocytes. Moreover, we observed that upon in vitro Staphylococcus aureus but not Escherichia coli challenged coelomocytes over-expressed and then secreted lysenin. These results indicate that among subpopulations of coelomocytes, lysenin is mainly produced by chloragocytes and its expression can be modulated by Gram-positive bacterial exposure.

Calcium is required for coelomocyte activation in earthworms

The role of calcium signaling in activation of both innate and adaptive immunity is basically important, however, the evolutionary aspects are not clarified yet. Currently limited data are available about calcium levels of coelomocytes, cellular mediators of earthworm immunity. We aimed to observe basal and induced Ca(2+) levels of coelomocyte subgroups after various stimulations in Eisenia fetida and Allolobophora caliginosa using a Ca(2+)-sensitive dye. E. fetida chloragocytes had the highest basal Ca(2+) levels among subpopulations; however there was no detectable Ca(2+) influx after any stimuli, while coelomocytes showed strong Ca(2+) increase after ionomycin treatment, which could be attenuated using phorbol ester. A. caliginosa coelomocytes showed a weak response to ionophore, while chloragocytes, similar to those in E. fetida, exhibited no changes after this stimulation. Intracellular calcium is mainly stored in the endoplasmic reticulum of coelomocytes as proved by thapsigargin treatments. Among several mitogens only phytohemagglutinin caused increased Ca(2+) level in E. fetida coelomocytes, but not in A. caliginosa coelomocytes. Moreover, the chemoattractant fMLP revealed calcium influx of Eisenia coelomocytes. For the first time we observed various basal Ca(2+) levels and sensibility to Ca(2+) influx inducers (including mitogens and chemoattractant) of coelomocyte subgroups using flow cytometry. These observations suggest that Ca(2+) influx and signal transduction may play crucial roles in the innate immunity of the earthworm.

Earthworm Innate Immune System

Nonself recognition is the basis of innate and adaptive immune responses and it depends on immune receptors. Caenorhabditis elegans and Drosophila animal models have added ample information to define molecular homology especially at the level of signal transduction. Yet, it is unwise to generalize about the immune system/signaling mechanisms of all invertebrates based upon two species. Other invertebrate species (mollusks and annelids) have also evolved effective innate immune responses, although those might have more unique pathways than other more popular and better studied species. We analyzed cellular immune responses in earthworms, and began to identify conserved signal molecules relative to MAP kinase pathways. These pathways appear at every level of evolution suggesting ubiquitous roles in various biological processes (differentiation, adaptation, stress response, apoptosis) including immunity. Analysis of seemingly less complex invertebrate immune systems can provide useful information with putative applications to more complex vertebrate immune system and to the evolution of immune response, including regulation of signaling systems in innate immunity. This knowledge can also help to identify and characterize new bioactive molecules with possible therapeutic use both in human and veterinary medicine.

Effects of PACAP on the oxidative stress-induced cell death in chicken pinealocytes is influenced by the phase of the circadian clock

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with highly potent neuro- and general cytoprotective actions. PACAP is also an important modulator of circadian rhythmic functions, including time-dependent effects in the pineal gland. It is not known whether PACAP influences the survival of pinealocytes. The present study had two aims. First, we tested whether the cytoprotective effects of PACAP are present also in the pineal cells. As the pineal gland is the main circadian master clock in birds, we also tested whether this effect depends on the time of day. Using flow cytometry, we detected a significant decrease of cell viability after hydrogen peroxide-induced oxidative stress in chicken pinealocytes. PACAP alone did not influence cell survival. Co-incubation with PACAP in the dark phase (9 PM) was able to attenuate the toxic effect of H2O2. The survival-promoting effect could be counteracted by simultaneously applied PACAP antagonist, PACAP6-38. However, co-treatment with PACAP during the light phase (9 AM) did not result in significant differences in the percentage of living cells. In summary, our results show that PACAP has a protective effect against the oxidative stress-induced cell death in chicken pinealocytes, but this effect is dependent on the phase of the circadian biological clock.

Induction of apoptosis-like cell death by coelomocyte extracts from Eisenia andrei earthworms.

Earthworms innate immunity is maintained by cellular and humoral components. Our objective was to characterize the cytotoxicity leading to target cell death caused by earthworm coelomocytes. Coelomocyte lysates induced strong cytotoxicity in tumor cell lines. Transmission electron microscopy revealed cell membrane and intracellular damage in cells treated with coelomocyte lysates. Using TUNEL-assay, within 5 min of incubation we detected DNA fragmentation. Moreover, we found phosphatidylserine translocation in target cell-membranes. Furthermore, we detected dose-dependent Ca(2+) influx and decrease of mitochondrial membrane potential in coelomocyte lysate-treated cells. Interestingly, caspase 3/8 activation was undetectable in exposed tumor cells. One such cytotoxic molecule, lysenin identified in earthworms binds to sphingomyelin and causes target cell lysis in vertebrates. Pretreatment with our anti-lysenin monoclonal antibody rescued the majority but not all target cells from coelomocyte induced death. These data suggest that, not only lysenin but also other factors participate in the caspase-independent apoptosis induced by coelomocytes.

Investigation of the Possible Functions of PACAP in Human Trophoblast 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 female reproductive system. Both the peptide and its receptors have been shown in the placenta but its role in placental growth, especially its human aspects, remains unknown. The aim of the present study was to investigate the effects of PACAP on invasion, proliferation, cell survival, and angiogenesis of trophoblast cells. Furthermore, cytokine production was investigated in human decidual and peripheral blood mononuclear cells. For in vitro studies, human invasive proliferative extravillous cytotrophoblast (HIPEC) cells and HTR-8/SVneo human trophoblast cells were used. Both cell types were used for testing the effects of PACAP on invasion and cell survival in order to investigate whether the effects of PACAP in trophoblasts depend on the examined cell type. Invasion was studied by standardized invasion assay. PACAP increased proliferation in HIPEC cells, but not in HTR-8 cells. Cell viability was examined using MTT test, WST-1 assay, and annexin V/propidium iodide flow cytometry assay. Survival of HTR-8/SVneo cells was studied under oxidative stress conditions induced by hydrogen peroxide. PACAP as pretreatment, but not as co-treatment, significantly increased the number of surviving HTR-8 cells. Viability of HIPEC cells was investigated using methotrexate (MTX) toxicity, but PACAP1-38 could not counteract its toxic effect. Angiogenic molecules were determined both in the supernatant and the cell lysate by angiogenesis array. In the supernatant, we found that PACAP decreased the secretion of various angiogenic markers, such as angiopoietin, angiogenin, activin, endoglin, ADAMTS-1, and VEGF. For the cytokine assay, human decidual and peripheral blood lymphocytes were separated and treated with PACAP1-38. Th1 and Th2 cytokines were analyzed with CBA assay and the results showed that there were no significant differences in control and PACAP-treated cells. In summary, PACAP seems to play various roles in human trophoblast cells, depending on the cell type and microenvironmental influences.

Protective Intestinal Effects of Pituitary Adenylate Cyclase Activating Polypeptide

Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide widely distributed throughout the body, including the gastrointestinal tract. Several effects have been described in human and animal intestines. Among others, PACAP influences secretion of intestinal glands, blood flow, and smooth muscle contraction. PACAP is a well-known cytoprotective peptide with strong anti-apoptotic, anti-inflammatory, and antioxidant effects. The present review gives an overview of the intestinal protective actions of this neuropeptide. Exogenous PACAP treatment was protective in a rat model of small bowel autotransplantation. Radioimmunoassay (RIA) analysis of the intestinal tissue showed that endogenous PACAP levels gradually decreased with longer-lasting ischemic periods, prevented by PACAP addition. PACAP counteracted deleterious effects of ischemia on oxidative stress markers and cytokines. Another series of experiments investigated the role of endogenous PACAP in intestines in PACAP knockout (KO) mice. Warm ischemia–reperfusion injury and cold preservation models showed that the lack of PACAP caused a higher vulnerability against ischemic periods. Changes were more severe in PACAP KO mice at all examined time points. This finding was supported by increased levels of oxidative stress markers and decreased expression of antioxidant molecules. PACAP was proven to be protective not only in ischemic but also in inflammatory bowel diseases. A recent study showed that PACAP treatment prolonged survival of Toxoplasma gondii infected mice suffering from acute ileitis and was able to reduce the ileal expression of proinflammatory cytokines. We completed the present review with recent clinical results obtained in patients suffering from inflammatory bowel diseases. It was found that PACAP levels were altered depending on the activity, type of the disease, and antibiotic therapy, suggesting its probable role in inflammatory events of the intestine.

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.