Pituitary adenylate cyclase-activating polypeptide plays an anti-inflammatory role in endotoxin-induced airway inflammation: In vivo study with gene-deleted mice

Krisztian Elekes, Katalin Sandor, Andras Moricz, Laszlo Kereskai, Agnes Kemeny, Eva Szoke, Aniko Perkecz, Dora Reglodi, Hitoshi Hashimoto, Erika Pinter, Janos Szolcsanyi, Zsuzsanna Helyes

Peptides 2011

The presence of pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors in capsaicin sensitive peptidergic sensory nerves, inflammatory and immune cells suggest its involvement in inflammation. However, data on its role in different inflammatory processes are contradictory and there is little known about its functions in the airways. Therefore, our aim was to examine intranasal endotoxininduced subacute airway inflammation in PACAP gene-deficient (PACAP−/−) and wild-type (PACAP+/+) mice. Airway responsiveness to inhaled carbachol was determined in unrestrained mice with whole body plethysmography 6 h and 24 h after LPS. Myeloperoxidase (MPO) activity referring to the number of accumulated neutrophils and macrophages was measured with spectrophotometry and interleukin-1
(IL-1) concentration with ELISA from the lung homogenates. Histological evaluation and semiquantitative scoring were also performed. Bronchial responsiveness, as well as IL-1 concentration and MPO activity markedly increased at both timepoints. Perivascular edema dominated the histological picture at 6 h, while remarkable peribronchial granulocyte accumulation, macrophage infiltration and goblet cell hyperplasia were seen at 24 h. In PACAP−/− mice, airway hyperreactivity was significantly higher 24 h after LPS and inflammatory histopathological changes were more severe at both timepoints.
MPO increase was almost double in PACAP−/− mice compared to the wild-types at 6 h. In contrast, there was no diffrence between the IL-1 concentrations of the PACAP+/+ and PACAP−/− mice. These results provide evidence for a protective role for PACAP in endotoxin-induced airway inflammation and hyperreactivity.

Pituitary adenylate cyclase-activating polypeptide plays a key role in nitroglycerol-induced trigeminovascular activation in mice

Adrienn Markovics, Viktoria Kormos, Balazs Gaszner, Arvin Lashgarara, Eva Szoke, Katalin Sandor, Krisztina Szabadfi, Bernadett Tuka, Janos Tajti, Janos Szolcsanyi, Erika Pinter, Hitoshi Hashimoto, Jozsef Kun, Dora Reglodi, Zsuzsanna Helyes

Neurobiology of Diseases 2011

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors (PAC1, VPAC) are present in sensory neurons and vascular smooth muscle. PACAP infusionwas found to trigger migraine-like headache in humans and we showed its central pro-nociceptive function in several mouse pain models. Nitroglycerol (NTG)-induced pathophysiological changes were investigated in this study in PACAP gene-deleted (PACAP−/−) and wildtype (PACAP+/+) mice. Chemical activation of the trigeminovascular system was induced by 10 mg/kg i.p. NTG.
Light-aversive behavior was determined in a light–dark box, meningeal microcirculation by laser Doppler blood perfusion scanning and the early neuronal activation marker c-Fos with immunohistochemistry. NTG induced photophobia both in the early (0–30 min) and late phases (90–120 min) due to direct vasodilation
and trigeminal sensitization, respectively,was significantly reduced in PACAP−/− mice. Meningeal blood flow increased by 30–35% during 4 h in PACAP+/+ mice, but only a 5–10% elevation occurred from the second hour in PACAP−/− ones. The number of c-Fos expressing cells referring to neuronal activation in the trigeminal ganglia and nucleus caudalis significantly increased 4 h after NTG in PACAP+/+, but not in PACAP−/− animals. Similar PAC1 receptor immunostaining was detected in both groups, which did not change 4 h after NTG treatment.
PACAP-38 (300 μg/kg, i.p.) produced photophobia similarly to NTG and 30% meningeal vasodilatation for 30 min in PACAP+/+, but not in PACAP−/− mice. It significantly increased neural activation 4 h later in the trigeminal ganglia of both groups, but in the nucleus caudalis of only the PACAP+/+ mice.
We provide the first experimental results that PACAP is a pivotal mediator of trigeminovascular activation/sensitization and meningeal vasodilation related to migraine.

Comparative Examination of Inner Ear in Wild Type and Pituitary Adenylate Cyclase Activating Polypeptide (PACAP)-Deficient Mice

A. Tamas • K. Szabadfi • A. Nemeth • B. Fulop • P. Kiss • T. Atlasz • R. Gabriel • H. Hashimoto • A. Baba • N. Shintani • Zs. Helyes • D. Reglodi

Neurotoxicity Research 2011

Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional neuropeptide with wellknown neuroprotective and neurotrophic effects. The involvement of PACAP in sensory processing has also been documented, but little is known about its effects in the auditory system. PACAP and its specific receptor (PAC1) are present in the cochlea and in brain structures involved in auditory pathways. Recently, we have shown that PACAP protects cochlear cells against oxidative stressinduced apoptosis. The endolymphatic Ca2+ concentration controlled by Ca2+ buffers of the hair cells is essential for the normal hearing processes. In this study we examined the localization of PAC1 receptor and Ca2+ buffering proteins (parvalbumin, calretinin, calbindin) in the inner ear of 5-day-old PACAP-deficient mice compared with wild-type mice in order to get a closer insight into the effect of endogenous PACAP in the cochlear function. We did not find differences in the distribution pattern of PAC1 receptors between the two groups, but wild-type animals showed significantly higher PAC1 receptor expression. In contrast, inner and outer hair cells of PACAP-deficient mice showed more pronounced parvalbumin, calbindin, and calretinin immunopositivity compared with wild-type mice. Elevated endolymphatic Ca2+ is deleterious for cochlear function, while the high concentration of Ca2? buffers in hair cells may offer protection. The increased immunoreactivity of Ca2+ binding proteins in the absence of PACAP provide further evidence the important role of PACAP in the hearing processes.

publiCATIONS 2012

Effects of Pituitary Adenylate Cyclase Activating Polypeptide on Human Sperm Motility

R. Brubel, P. Kiss, A. Vincze, A. Varga, A. Varnagy, J. Bodis, L. Mark, E. Jambor, G. Maasz, H. Hashimoto, Zs. Helyes, G. Toth, A. Tamas, M. Koppan, D. Reglodi

Jurnal of Molecular Neuroscience 2012

Pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide with diverse effects, was originally isolated as a hypothalamo-hypophyseal peptide. Subsequent studies showed highest levels of PACAP in the testis after the brain, suggesting that it influences the development and functioning of spermatozoa. Indeed, it has been proven that PACAP has an effect on spermatogenesis, both locally and via influencing the hypothalamo-hypophyseal–gonadal axis. The aim of the present study was to determine whether PACAP has an effect on human sperm motility and whether it is present in the human seminal fluid. Furthermore, the sperm head morphology was studied in mice lacking endogenous PACAP. Human samples were obtained from healthy adult volunteers and andrological patients. The effects of PACAP on the motility of human sperm cells were investigated using a computer aided sperm analysis system. In cases where the motility was lower, addition of PACAP to the samples increased the motility and the ratio of rapid progressive and medium progressive sperm motility groups. The presence of PACAP could not be detected in human seminal fluid samples by means of mass spectrometry. Investigating sperm head morphology with routine histology in PACAP deficient mice revealed that both the longitudinal and transverse diameters were significantly lower in PACAP deficient mice, without marked difference in the shape, as revealed by scanning electron microscopy.

Effects of PACAP on Intracellular Signaling Pathways in Human Retinal Pigment Epithelial Cells Exposed to Oxidative Stress

E. Fabian, D. Reglodi, L. Mester, A. Szabo, K. Szabadfi, A. Tamas, G. Toth, K. Kovacs

Journal of Molecular Neuroscience 2012

The integrity of retinal pigment epithelial cells is critical for photoreceptor survival and vision. Pituitary adenylate cyclase activating polypeptide (PACAP) exerts retinoprotective effects against several types of injuries in vivo, including optic nerve transection, retinal ischemia, excitotoxic injuries, UVA-induced lesion, and diabetic retinopathy. In a recent study, we have proven that PACAP is also protective in oxidative stress-induced injury in human pigment epithelial cells (ARPE-19 cells). The aim of the present study was to investigate the possible mechanisms of this protection. ARPE cells were exposed to a 24-h hydrogen peroxide treatment. Expressions of kinases and apoptotic markers were studied by complex array kits and Western blot. Oxidative stress induced the activation of several apoptotic markers, including Bad, Bax, HIF-1α, several heat shock proteins, TNF-related apoptosis-inducing ligand, and Fas-associated protein with death domain, while PACAP treatment decreased them. The changes in the expression of MAP kinases showed that PACAP activated the protective ERK1/2 and downstream CREB, and decreased the activation of the pro-apoptotic p38MAPK and c-Jun N-terminal kinase, an effect opposite to that observed with only oxidative stress. Furthermore, PACAP increased the activation of the protective Akt pathway. In addition, the effects of oxidative stress on several other signaling molecules were counteracted by PACAP treatment (Chk2, Yes, Lyn, paxillin, p53, PLC, STAT4, RSK). These play a role in cell death, cell cycle, inflammation, adhesion, differentiation and proliferation. In summary, PACAP, acting at several levels, influences the balance between pro- and antiapoptotic factors in favor of anti-apoptosis, thereby providing protection in oxidative stress-induced injury of human retinal pigment epithelial cells.

The behavioral phenotype of Pituitary Adenylatecyclase Activating Polypeptide-deficient mice in anxiety and deprsession tests is accompanied by blunted c-Fos expression in the bed nucleus of the stria terminalis, central projecting Edinger-Westphal nucleus, ventral lateral septum and dorsal raphe nucleus


Neuroscience 2012

Pituitary adenylate-cyclase activating polypeptide (PACAP) has been implicated in the (patho)physiology of stress-adaptation. PACAP deficient (PACAP -/-) mice show altered anxiety levels and depression-like behavior, but little is known about the underlying mechanisms in stress-related brain areas. Therefore, we aimed at investigating PACAP/mice in light–dark box, marble burying, open field, and forced swim paradigms. We also analyzed whether the forced swim test-induced c-Fos expression would be affected by PACAP deficiency in the following stress-related brain areas: magnoand parvocellular paraventricular nucleus of the hypothalamus (PVN); basolateral (BLA), medial (MeA), and central (CeA) amygdaloid nuclei; ventral (BSTv), dorsolateral (BSTdl), dorsomedial (BSTdm), and oval (BSTov) nuclei of the bed nucleus of stria terminalis; dorsal (dLS) and ventral parts (vLS) of lateral septal nucleus, central projecting Edinger–Westphal nucleus (EWcp), dorsal (dPAG) and lateral (lPAG) periaqueductal gray matter, dorsal raphe nucleus (DR). Our results revealed that PACAP-/- mice showed greatly reduced anxiety and increased locomotor activity compared with wildtypes. In forced swim test PACAP/ mice showed increased depression-like behavior. Forced swim exposure increased c-Fos expression in all examined brain areas in wildtypes, whereas this was markedly blunted in the DR, EWcp, BSTov, BSTdl, BSTv, PVN, vLS, dPAG, and in the lPAG of PACAP-/- mice vs. wildtypes, strongly suggesting their involvement in the behavioral phenotype of PACAP-/- mice. PACAP deficiency did not influence the c-Fos response in the CeA, MeA, BSTdm, and dLS. Therefore, we propose that PACAP exerts a brain area-specific effect on stressinduced neuronal activation and it might contribute to stressrelated mood disorders. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Correlation Between Oocyte Number and Follicular Fluid Concentration of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) in Women After Superovulation Treatment

M. Koppan, A. Varnagy, D. Reglodi, R. Brubel, J. Nemeth, A. Tamas, L. Mark, J. Bodis

Journal of Molecular Neuroscience 2012

Follicular growth, ovulation, and luteinization are influenced by interactions of peptide and steroid hormonesignaling cascades in the ovary. Pituitary adenylate cyclaseactivating polypeptide (PACAP) plays an important role in the regulation of several endocrine processes and is present in ovarian follicular fluid (FF). However, little is known about PACAP in FF with regard to maturation, ovulation, fertilization, and successful pregnancy. The aim of this pilot study was to investigate whether there is a correlation between PACAP concentration in FF and ovarian response to superovulation treatment in infertile women, performed in volunteers (n0 132; aged between 20 and 35). After treatment, the number of harvested oocytes was recorded and PACAP immunoreactivity in FF was measured by radioimmunoassay. All the corresponding PACAP concentrations were below 290 fmol/ml in cases when the number of harvested oocytes exceeded 14 per patient, while in all cases above 290 fmol/ml, the number of oocytes was below 14. Using these cutoff values, we determined three study groups: high-PACAP concentration, highoocyte number, and low-PACAP concentration–low-oocyte number groups. Median values of PACAP concentration in these groups were 411.2, 106.5, and 101.0 fmol/ml, respectively, while themedian values of harvested oocyteswere 5.5, 19.0, and 5.0, respectively. Differences were significant, indicating a correlation between concentration of PACAP in FF and the number of recruited oocytes. Higher concentrations of PACAP in FF might be associated with lower number of developing oocytes, while low concentrations of PACAP might correlate with a markedly higher number of ova retrieved, thus predicting a higher chance for ovarian hyperstimulation. Our present study is among the first few human clinical studies with direct conclusions drawn for possible clinical impact of PACAP.

Intestinal Autotransplantation Using PACAP-38-Containing 5 Preservation Solution

Klara Nedvig, Gyorgy Weber, Jozsef Nemeth, Krisztina Kovacs, Dora Reglodi, Agnes Kemeny, Andrea Ferencz

Journal of Molecular Neuroscience 2012

Small bowel is one of the most sensitive organs to ischemia–reperfusion injury, which is a significant problem during transplantation. Pituitary adenylate cyclase-activating polypeptide (PACAP) has cytoprotective effect in ischemic injuries of various tissues. The aim of our study was to measure changes of PACAP-38 and PACAP-27 immunoreactivities and cytokine levels in intestinal grafts stored in PACAP-38-containing preservation solution. Small bowel autotransplantation was performed on male Wistar rats. Grafts were stored in University of Wisconsin (UW) solution at 4 °C for 1 h (group (G)I), for 3 h (GII), and for 6 h (GIII) and in PACAP-38-containing UW solution for 1 h (GIV), for 3 h (GV), and for 6 h (GVI). After preservation, performing vessel anastomosis reperfusion began, which lasted 3 h in each group. Tissue biopsies were collected after laparotomy (control) and at the end of the reperfusion periods. Intestinal PACAP-38 and PACAP-27 immunoreactivities were measured by radioimmunoassay. To measure cytokine array from tissue homogenates, we used rat cytokine array and Luminex Multiplex Immunoassay. Levels of PACAP-38 and PACAP-27 immunoreactivity decreased after 1 and 3 h preservation compared to control levels. This decrease was significant following 6 h cold storage (p<0.05). Values remained significantly higher in grafts stored in PACAP-38-containing UW. Cytokine array revealed that expression of the soluble intercellular adhesion 37molecule-1 (CD54) and L-selectin (CD62L/LECAM-1) was increased in GIII. Both 6 h cold storage in PACAP-38 - containing UW solution and 3 h reperfusion caused strong reduction in these cytokines activation in GVI. RANTES (CCL5) levels were increased in all groups. Strong activation of the tissue inhibitor of metalloproteinase-1 was in GIII. However, PACAP-38-containing cold storage could decrease its activation in GVI. Furthermore, strong activation of the tissue inhibitor of metalloproteinase-1 was 46
detected in 6 h preserved grafts without PACAP-38 (GIII). PACAP-38-containing cold storage could decrease its activation in GVI. Our present study showed that PACAP-38 and PACAP-27 immunoreactivities decreased in a timedependent manner during intestinal cold preservation, which could be ameliorated by administration of exogenous PACAP-38 to the preservation solution. Moreover, PACAP-38 could attenuate tissue cold ischemic injury-induced 54 changes in cytokine expression.

Role of PACAP in female fertility and reproduction at gonadal level – recent advances

Dora Reglodi, Andrea Tamas, Miklos Koppan, Donat Szogyi and Laura Welke

Frontiers in Endocrinology 2012

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide, first isolated from hypothalamic extracts, but later shown in peripheral organs, such as endocrine glands, gastrointestinal system, cardiovascular system, and reproductive organs. PACAP plays a role in fertility and reproduction. Numerous studies report on the gonadal regulatory effects of PACAP at hypothalamo-hypophyseal levels. However, the local effects of PACAP at gonadal levels are also important.The present review summarizes the effects of PACAP in the ovary. PACAP and its receptors are present in the ovary, and PACAP plays a role in germ cell migration, meiotic division, follicular development, and atresia. The autocrineparacrine hormonal effects seem to play a regulatory role in ovulation, luteinization, and follicular atrophy. Altogether, PACAP belongs to the ovarian regulatory peptides.

PACAP is an Endogenous Protective Factor - Insights from PACAP-Deficient Mice

D. Reglodi, P. Kiss, K. Szabadfi, T. Atlasz, R. Gabriel, G. Horvath, P. Szakaly, B. Sandor, A. Lubics, E. Laszlo, J.Farkas, A.Matkovits, R. Brubel, H. Hashimoto, A. Ferencz, A. Vincze, Z. Helyes, L. Welke, A. Lakatos, A. Tamas

Journal of Molecular Neuroscience 2012

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a widespread neuropeptide with a diverse array of biological functions. Not surprisingly, the lack of endogenous PACAP therefore results in a variety of abnormalities. One of the important effects of PACAP is its neuroprotective and general cytoprotective role. PACAP protects neurons and other tissues against ischemic, toxic, and traumatic lesions. Data obtained from PACAP-deficient mice provide evidence that endogenous PACAP also has protective functions. Mice lacking PACAP are more vulnerable to different in vitro and in vivo insults. The present review summarizes data on the increased sensitivity of PACAP-deficient mice against harmful stimuli. Mice lacking PACAP respond with a higher degree of injury in cerebral ischemia, autoimmune encephalomyelitis, and axonal lesion. Retinal ischemic and excitotoxic injuries also produce increased cell loss in PACAP-deficient mice. In peripheral organs, kidney cell cultures from PACAP-deficient mice are more sensitive to oxidative stress and in vitro hypoxia.

Protective effects of the neuropeptide PACAP in diabetic retinopathy

Krisztina Szabadfi, Tamas Atlasz, Peter Kiss, Dora Reglodi, Aliz Szabo, Krisztina Kovacs, Balint Szalontai, Gyorgy Setalo Jr., Eszter Banki, Katalin Csanaky, Andrea Tamas, Robert Gabriel

Cell Tissue Research 2012

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with highly potent neurotrophic and neuroprotective effects. PACAP and its receptors occur in the retina and PACAP has been applied in animal models of metabolic retinal disorders to reduce structural and functional damage. Furthermore, PACAP has been implicated as a potential anti-diabetic peptide. Our aim has been to investigate, by using a complex morphological, immunochemical and molecular biological approach, whether PACAP attenuates diabetic retinopathy. Diabetes was induced in rats with a single streptozotocin injection. PACAP was injected intravitreally into one eye (100 pmol) three times during the last week of a 3-week survival period. Retinas were processed for the following procedures: routine histology, immunohistochemistry (single and double labeling, whole-mount), quantitative reverse transcription with the polymerase chain reaction and Western blotting. Cone photoreceptors and dopaminergic amacrine and ganglion cells degenerated in diabetic retinas and glial fibrillary acidic protein were upregulated in Müller glial cells. The number of cones, the length of their outer segments and the cell number in the ganglion cell layer were decreased. PACAP ameliorated these structural changes. Moreover, PACAP increased the levels of PAC1-receptor and tyrosine-hydroxylase as detected by molecular biological methods. Thus, PACAP has significant protective effects in the diabetic retina. PACAP treatment attenuates neuronal cell loss in diabetic retinopathy, the protective effects of PACAP probably being mediated through the activation of PAC1-
receptor. These results suggest that PACAP has a therapeutic potential in diabetic retinopathy.

Preconditioning with volatile anaesthetic sevoflurane in ischemic retinal lesion in rats

Krisztina Szabadfi, Bese Danyadi, Peter Kiss, Sridharan Manavalan, Robert Gabriel, Dora Reglodi, Andrea Tamas, Domonkos Trasy, Istvan Batai

Journal of Molecular Histology 2012

Volatile anaesthetic agents have been recognized for their neuroprotective properties since the 1960s. However, little is known regarding the potential retinoprotective effects of preconditioning by anaesthetic drugs. Retinal ischemia can be modeled by permanent bilateral common carotid artery occlusion (BCCAO). Here we studied the degree of ischemic injury with preconditioning by sevoflurane in the rat retina. During the BCCAO operation and preconditioning Wistar rats were anaesthetized with 1 MAC of sevoflurane. The oxygen, carbon dioxide, and anaesthetic vapor concentration in the anaesthetizing box was monitored with a gas analyzer. We examined 4 groups: non- and preconditioning groups in control and BCCAO animals. The duration of preconditioning period was 1 h and it was performed 1 day before BCCAO. The
retinas were processed for histological evaluation after 2 weeks survival to determine the cell number in the ganglion cell layer and the thickness of the whole retina and that of all retinal layers. BCCAO-induced retinal ischemic injury was ameliorated by sevoflurane preconditioning. Retinal thickness and the cell number in the ganglion cell layer were more retained in preconditioned animals after BCCAO compared to non-preconditioned group. These results suggest that preconditioning using sevoflurane could provide a new perspective in retinoprotective strategies.

Protective Effects of Vasoactive Intestinal Peptide (VIP) 5 in Ischemic Retinal Degeneration

K. Szabadfi, B. Danyadi, P. Kiss, A. Tamas, E. Fabian, R. Gabriel, D. Reglodi

Journal of Molecular Neuroscience 2012

Vasoactive intestinal peptide (VIP) is a pleiotropic neuropeptide, acting as a neuromodulator and neuroprotective peptide in the CNS after injuries. We have previously described that pituitary adenylate cyclase-activating polypeptide, another member of the same peptide family, is retinoprotective in ischemic lesions. The aim of this study was to investigate the protective potential of VIP in bilateral common carotid artery occlusion (BCCAO)-induced ischemic retinal lesion. Two-month-old rats were subjected to BCCAO and treated with intravitreal VIP injection. Their retinas were processed for histology after 2 weeks of survival. We measured the number of the cells/100 μm of the ganglion cell layer and the thickness of each layer such as the outer nuclear, outer plexiform, inner nuclear, and inner plexiform layers as well as that of the whole retina. We found that treatment with 1,000 pmol VIP, but not 100 pmol VIP, had significant protective effects in BCCAO-injured retina, as Q229 shown by the morphometric analysis. Comparing the neuroprotective effects of VIP and pituitary adenylate cyclase-activating polypeptide (PACAP) in BCCAO-operated retinas, PACAP was more effective, already protective at 100-pmol doses. Similar to other studies, we found that VIP must be given at least in 10 times more concentration than PACAP to achieve a similar degree of neuroprotection in the retina.

Mice Deficient in Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) are More Susceptible to Retinal Ischemic Injury In Vivo

K. Szabadfi, T. Atlasz, P. Kiss, B. Danyadi, A. Tamas, Zs. Helyes, H. Hashimoto, N. Shintani, A. Baba, G. Toth, R. Gabriel, D. Reglodi

Neurotoxicity Research 2012

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuroprotective peptide exerting protective effects in neuronal injuries. We have provided evidence that PACAP is neuroprotective in several models of retinal degeneration in vivo. Our previous studies showed that PACAP treatment ameliorated the damaging effects of chronic hypoperfusion modeled by permanent bilateral carotid artery occlusion. We have also demonstrated in earlier studies that treatment with PACAP antagonists further aggravates retinal lesions. It has been shown that PACAP deficient mice have larger infarct size in cerebral ischemia. The aim of this study was to compare the degree of retinal damage in wild type and PACAP deficient mice in ischemic retinal insult. Mice underwent 10 min of bilateral carotid artery occlusion followed by 2-week reperfusion period. Retinas were then processed for histological analysis. It was found that PACAP deficient mice had significantly greater retinal damage, as shown by the thickness of the whole retina, the morphometric analysis of the individual retinal layers, and the cell numbers in the inner nuclear and ganglion cell layers. Exogenous PACAP administration could partially protect against retinal degeneration in PACAP deficient mice. These results clearly show that endogenous PACAP reacts as a stressresponse peptide that is necessary for endogenous protection against different retinal insults.

Effect of PACAP on MAP kinases, Akt and cytokine expressions in rat retinal hypoperfusion

Aliz Szabo, Bese Danyadi, Eszter Bognar, Krisztina Szabadfi, Eszter Fabian, Peter Kiss, Laszlo Mester, Sridharan Manavalan, Tamas Atlasz, Robert Gabriel, Gabor Toth,Andrea Tamas, Dora Reglodi, Krisztina Kovacsa

Neuroscience letters 2012

Pituitary adenylate cyclase activating polypeptide (PACAP) is known for its potent neuroprotective effects, including the retinoprotective actions in several types of retinal injuries. We have shown earlier that PACAP treatment causes activation of protective pathways and inhibition of pro-apoptotic signaling in excitotoxic retinal lesions. The aim of the present study was to gain insight into the in vivo protective mechanism of PACAP in retinal hypoperfusion injury induced by bilateral common carotid artery occlusion (BCCAO). Rats underwent BCCAO and received intravitreal PACAP (PACAP38) treatment. We investigated the activation level of the protective Akt pathway as well as the different mitogen activated protein kinases (MAPKs) by Western blot analysis and the expression of cytokines using a cytokine array kit. We found that PACAP treatment alone did not influence the phosphorylation of Akt or the MAPKs, but decreased the hypoperfusion-induced activation of both p38MAPK and JNK and increased the activation of the protective Akt and ERK1/2 in hypoperfused retinas. The cytokine profile was dramatically changed after BCCAO, with most cytokines and chemokines showing an increase, which was attenuated by PACAP (such as CINC, CNTF, fractalkine, sICAM, IL-1, LIX, Selectin, MIP-1, RANTES and TIMP-1). In addition, PACAP increased the expression of VEGF and thymus chemokine. The present results provide further insight into the neuroprotective mechanism induced by PACAP in ischemic retinal injuries, showing that PACAP ameliorates hypoperfusion injury involving Akt, MAPK pathways and anti-inflammatory actions.

PACAP Immunoreactivity in Human Malignant Tumor Samples and Cardiac Diseases

Z. Szanto, Zs. Sarszegi, D. Reglodi, J. Nemeth, K. Szabadfi, P. Kiss, A. Varga, E. Banki, K. Csanaky, B. Gaszner, O. Pinter, Zs. Szalai , A. Tamas

Journal of Molecular Neuroscience 2012

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic and multifunctional neuropeptide having important roles in various physiological processes. Recent trends in PACAP research point to the clinical introduction of PACAP or its analogs/fragments possibly in the near future. Recently, we have shown the presence of PACAP in human plasma, milk, placenta, and follicular fluid samples. However, relatively few data are available on PACAP in human tissues from patients with different disorders. The aim of the present study was to determine, by radioimmunoassay, the tissue level of PACAP38-like immunoreactivity (LI) and PACAP27-LI in different primary nonsmall cell lung cancer, colon tumor samples, and in cardiac muscle samples from patients suffering from ischemic heart disease and valvular disorders. We also labeled the PAC1 receptors in human cardiac cells. All samples showed significantly higher PACAP38-LI compared with PACAP27-LI. We found significantly lower levels of PACAP38-LI and PACAP27-LI in tumoral and peripheral samples compared with normal healthy tissue in both lung and colon cancers. Further investigations are necessary to describe the exact function of PACAP in oncogenesis. We showed that PACAP38-LI and PACAP27-LI are significantly higher in ischemic heart diseases compared with valvular abnormalities, suggesting that PACAP might play a role in ischemic heart disorders.

Effect of PACAP in Central and Peripheral Nerve Injuries

Andrea Tamas, Dora Reglodi, Orsolya Farkas, Erzsebet Kovesdi, Jozsef Pal, John T. Povlishock, Attila Schwarcz, Endre Czeiter, Zalan Szanto, Tamas Doczi, Andras Buki and Peter Bukovics

International Journal of Molecular Sciences 2012

Pituitary adenylate cyclase activating polypeptide (PACAP) is a bioactive peptide with diverse effects in the nervous system. In addition to its more classic role as a neuromodulator, PACAP functions as a neurotrophic factor. Several neurotrophic factors have been shown to play an important role in the endogenous response following both cerebral ischemia and traumatic brain injury and to be effective when given exogenously. A number of studies have shown the neuroprotective effect of PACAP in different models of ischemia, neurodegenerative diseases and retinal degeneration. The aim of this review is to summarize the findings on the neuroprotective potential of PACAP in models of different traumatic nerve injuries. Expression of endogenous PACAP and its specific PAC1 receptor is elevated in different parts of the central and peripheral nervous system after traumatic injuries. Some experiments demonstrate the protective effect of exogenous PACAP treatment in different traumatic brain injury models, in facial nerve and optic nerve trauma. The upregulation of endogenous PACAP and its receptors and the protective effect of exogenous PACAP after different central and peripheral nerve injuries show the important function of PACAP in neuronal regeneration indicating that PACAP may also be a promising therapeutic agent in injuries of the nervous system.

Peripheral and central alterations of pituitary adenylate cyclase activating polypeptide-like immunoreactivity in the rat in response to activation of the trigeminovascular system

Bernadett Tuka, Zsuzsanna Helyes, Adrienn Markovics, Teréz Bagoly, József Németh, László Márk, Réka Brubel, Dóra Reglodi, Árpád Párdutz, János Szolcsányi, László Vécsei, János Tajti

Peptides 2012

Pituitary adenylate cyclase activating polypeptide (PACAP) is present in the cranial arteries and trigeminal sensory neurons. We therefore examined the alterations in PACAP-like immunoreactivity (PACAP-LI) in a time-dependent manner in two rat models of trigeminovascular system (TS) activation. In one group chemical stimulation (CS) was performed with i.p. nitroglycerol (NTG), and in the other one the trigeminal ganglia (TRG) were subjected to electrical stimulation (ES). The two biologically active forms, PACAP-38 and PACAP-27, were determined by means of radioimmunoassay (RIA) and mass spectrometry (MS) in the plasma, the cerebrospinal fluid (CSF), the trigeminal nucleus caudalis (TNC), the spinal cord (SC) and the TRG. The tissue concentrations of PACAP-27 were 10 times lower than those of PACAP-38 in the TNC and SC, but about half in the TRG. PACAP-38, but not PACAP-27, was present in the plasma. Neither form could be identified in the CSF. PACAP-38-LI in the plasma, SC and TRG remained unchanged after CS, but it was increased significantly in the TNC 90 and 180 min after NTG injection. In response to ES of the TRG, the level of PACAP-38 in the plasma and the TNC was significantly elevated 90 and 180 min later, but not in the SC or the TRG. The alterations in the levels of PACAP-27 in the tissue homogenates in response to both forms of stimulation were identical to those of PACAP-38. The selective increases in both forms of PACAP in the TNC suggest its important role in the central sensitization involved in migraine-like headache.

publiCATIONS 2013

Effect of PACAP treatment on kidney morphology and cytokine expression in rat diabetic nephropathy

E. Banki, P. Degrell, P. Kiss, K. Kovacs, A. Kemeny, K. Csanaky, A. Duh, D. Nagy, G. Toth, A. Tamas, D. Reglodi

Peptides 2013

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide, exerting diverse effects. One of its frequently examined functions is cell protection, which is achieved mainly via inhibiting apoptotic, inflammatory and oxidative processes. All its three receptors (PAC1, VPAC1, VPAC2) are expressed in the kidney and PACAP has been shown to have protective effects against different renal pathologies. Diabetic nephropathy is the leading cause of end stage renal disease. The aim of the present study was to investigate the possible ameliorative effect of PACAP in streptozotocin-induced diabetic nephropathy and to evaluate its anti-inflammatory effect in this model. Diabetes was induced by a single intravenous injection of streptozotocin (65 mg/kg) in male Wistar rats. PACAP-treated animals were administered ip. 20 g PACAP every second day, while untreated animals were given vehicle. Kidneys were removed after 8-weeks survival. Besides the complex histological analysis (glomerular PAS positive area/glomerulus area, tubular damage, arteriolar hyalinosis), expression of several cytokines was evaluated by cytokine array and Luminex assay. Histological analysis revealed severe diabetic changes in kidneys of control diabetic animals (glomerular PAS-positive area expansion, tubular damage, Armanni-Ebstein phenomenon). PACAP treatment significantly diminished the damage. Diabetic kidneys showed significant cytokine activation compared to their healthy controls. PACAP was effective in downregulation of several cytokines including CINC-1, TIMP-1, LIX, MIG, s-ICAM. To conclude, PACAP is effective in ameliorating diabetic nephropathy at least partly through its well-known anti-inflammatory effect. These results raise the opportunity for the use of PACAP as a possible therapeutic or preventive method in treating the complications of diabetes.

Role of Pituitary Adenylate-Cyclase Activating Polypeptide and Tac1 gene derived tachykinins in sensory, motor and vascular functions under normal and neuropathic conditions

Balint Botza, Andras Imreh, Katalin Sandor, Krisztian Elekes, Janos Szolcsanyi, Dora Reglodi, John P. Quinn, James Stewart, Andreas Zimmer, Hitoshi Hashimoto, Zsuzsanna Helyes

Peptides 2013

Pituitary Adenylate-Cyclase Activating Polypeptide (PACAP) and Tac1 gene-encoded tachykinins (substance P: SP, neurokinin A: NKA) are expressed in capsaicin-sensitive nerves, but their role in nociception, inflammation and vasoregulation is unclear. Therefore, we investigated the function of these neuropeptides and the NK1 tachykinin receptor (from Tacr1 gene) in the partial sciatic nerve ligationinduced traumatic mononeuropathy model using gene deficient (PACAP−/−, Tac1−/−, and Tacr1−/−) mice.
Mechanonociceptive threshold of the paw was measured with dynamic plantar aesthesiometry, motor coordination with Rota-Rod and cutaneous microcirculation with laser Doppler imaging. Neurogenic vasodilation was evoked by mustard oil stimulating sensory nerves. In wildtype mice 30–40% mechanical hyperalgesia developed one week after nerve ligation, which was not altered in Tac1−/− and Tacr1−/− mice, but was absent in PACAP−/− animals. Motor coordination of the PACAP−/− and Tac1−/− groups was significantly worse both before and after nerve ligation compared to their wildtypes, but it did not change in Tacr1−/− mice. Basal postoperative microcirculation on the plantar skin of PACAP−/− mice did not differ from the wildtypes, but was significantly lower in Tac1−/− and Tacr1−/− ones. In contrast, mustard oil-induced neurogenic vasodilation was significantly smaller in PACAP−/− mice, but not in Tacr1−/− and Tac1−/− animals. Both PACAP and SP/NKA, but not NK1 receptors participate in normal motor coordination. Tachykinins maintain basal cutaneous microcirculation. PACAP is a crucial mediator of neuropathic mechanical hyperalgesia and neurogenic vasodilation. Therefore identifying its target and developing selective, potent antagonists, might open promising new perspectives for the treatment of neuropathic pain and vascular complications.

PACAP Protects Against Salsolinol-Induced Toxicity in Dopaminergic SH-SY5Y Cells: Implication for Parkinson’s Disease

Dwayne Brown, Andrea Tamas, Dora Reglodi, Yousef Tizabi

Journal of Molecular Neuroscience 2013

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an endogenous 38 amino acid containing neuropeptide with various cytoprotective functions including neuroprotection. Administration of PACAP has been shown to reduce damage induced by ischemia, trauma, or exogenous toxic substances. Moreover, mice deficient in PACAP are more vulnerable to damaging insults. In this study, we sought to determine whether PACAP may also be protective against salsolinol-induced toxicity in SH-SY5Y cells and, if so, elucidate its mechanism(s) of action. Salsolinol (SALS) is an endogenous dopamine metabolite with selective toxicity to nigral dopaminergic neurons, which are directly implicated in Parkinson’s disease (PD). SH-SY5Y cells, derived from human neuroblastoma cells, express high levels of dopaminergic activity and are used extensively as a model to study these neurons. Exposure of SH-SY5Y cells to 400 μM SALS for 24 h resulted in approximately 50 % cell death that was mediated by apoptosis as determined by cell flow cytometry and increases in caspase-3 levels. Cellular toxicity was also associated with reductions in brainderived neurotrophic factor and phosphorylated cyclic AMPresponse element-binding protein. Pretreatment with PACAP dose-dependently attenuated SALS-induced toxicity and the associated apoptosis and the chemical changes. PACAP receptor antagonist PACAP6-38, in turn, dosedependently blocked the effects of PACAP. Neither PACAP
nor PACAP antagonist had any effect of its own on cellular viability. These results suggest the protective effects of PACAP in a cellular model of PD. Hence, PACAP or its agonists could be of therapeutic benefit in PD.

Examination of PACAP38-like immunoreactivity in different milk and infant formula samples

K Csanaky, D Reglődi, E Bánki, I Tarcai, L Márk, Zs Helyes, T Ertl, J Gyarmati, K Horváth, L Sántik, A Tamás

Acta Physiologica Hungarica 2013

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with special importance in reproductive and developmental processes. PACAP is found in two bioactive forms: PACAP27 and PACAP38. Recently, we have described that PACAP38 is present in high levels in the milk of human and ruminant animals. Breastfeeding is of utmost importance in proper nutrition of the newborn, but artificial nursing with infant formulas is necessary when breastfeeding is not available. Composition of the breast milk varies during the whole period of nursing and it shows
differences at the beginning (foremilk) and the end of an actual suckling (hindmilk). The aim of this study was to investigate PACAP38-like immunoreactivity (PACAP38-LI) in different milk and infant formula samples by radioimmunoassay and to prove the presence of PACAP38 in the infant formula by mass spectrometry. We found similar PACAP38-LI in human mature foremilk and hindmilk samples, in the fresh and pasteurized cow milk and also in formulas. However, we found significantly higher PACAP38-LI in the hypoantigenic formula undergoing extensive hydrolysis compared to the non-hypoantigenic ones. Our results suggest that PACAP38 is relatively stable
in the milk and it can withstand the manufacturing processes.

Gender-Dependent Effects of Enriched Environment and Social Isolation in Ischemic Retinal Lesion in Adult Rats

Peter Kiss, Krisztina Szabadfi, Gabor Horvath, Andrea Tamas, Jozsef Farkas, Robert Gabriel and Dora Reglodi

International Journal of Molecular Sciences 2013

Exposure to an enriched environment has been shown to have many positive effects on brain structure and function. Numerous studies have proven that enriched environment can reduce the lesion induced by toxic and traumatic injuries. Impoverished environment, on the other hand, can have deleterious effects on the outcome of neuronal injuries. We have previously shown that enriched conditions have protective effects in retinal injury in newborn rats. It is well-known that the efficacy of neuroprotective strategies can depend on age and gender. The aim of the present study, therefore, was to examine the effects of environmental enrichment and social isolation in retinal ischemia. We used bilateral common carotid artery occlusion to induce retinal hypoperfusion in adult Wistar rats of both genders. Groups were housed in standard, enriched or impoverished conditions. Impoverished environment was induced by social isolation. Retinas were processed for histological analysis after two weeks of survival. In the present study, we show that (1) enriched environment has protective effects in adult ischemic retinal lesion, while (2) impoverished environment further increases the degree of ischemic injury, and (3) that these environmental effects are gender-dependent: females are less responsive to the positive effects of environmental enrichment and more vulnerable to retinal ischemia in social isolation. In summary, our present study shows that the effects of both positive and negative environmental stimuli are gender-dependent in ischemic retinal lesions.