The presence and distribution of pituitary adenylate cyclase activating polypeptide and its receptor in the snail Helix pomatia.

The aim of this study was to show the presence, distribution and function of the pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors in the CNS and peripheral nervous system of the mollusk, Helix pomatia. PACAP-like and pituitary adenylate cyclase activating polypeptide receptor (PAC1-R)-like immunoreactivity was abundant both in the CNS and the peripheral nervous system of the snail. In addition several non-neuronal cells also revealed PACAP-like immunoreactivity. In inactive animals labeled cell bodies were mainly found and in the neuropile of active animals dense immunostained fiber system was additionally detected suggesting that expression of PACAP-like peptide was affected by the behavioral state of the animal. RIA measurements revealed the existence of both forms of PACAP in the CNS where the 27 amino acid form was found to be dominant. The concentration of PACAP27 was significantly higher in samples from active animals supporting the data obtained by immunohistochemistry. In Western blot experiments PACAP27 and PACAP38 antibodies specifically labeled protein band at 4.5 kDa both in rat and snail brain homogenates, and additionally an approximately 14 kDa band in snail. The 4.5 kDa protein corresponds to PACAP38 and the 14 kDa protein corresponds to the preproPACAP or to a PACAP-like peptide having larger molecular weight than mammalian PACAP38. In matrix-assisted laser desorption ionization time of flight (MALDI TOF) measurements fragments of PACAP38 were identified in brain samples suggesting the presence of a large molecular weight peptide in the snail. Applying antibodies developed against the PACAP receptor PAC1-R, immunopositive stained neurons and a dense network of fibers were identified in each of the ganglia. In electrophysiological experiments, extracellular application of PACAP27 and PACAP38 transiently depolarized or increased postsynaptic activity of neurons expressing PAC1-R. In several neurons PACAP elicited a long lasting hyperpolarization which was eliminated after 1.5 h continuous washing. Taken together, these results indicate that PACAP may have significant role in a wide range of basic physiological functions in snail.

PAC1 receptor localization in a model nervous system: light and electron microscopic immunocytochemistry on the earthworm ventral nerve cord ganglia.

The presence and pattern of pituitary adenylate cyclase activating polypeptide (PACAP) type I (PAC1) receptors were identified by means of pre- and post-embedding immunocytochemical methods in the ventral nerve cord ganglia (VNC) of the earthworm Eisenia fetida. Light and electron microscopic observations revealed the exact anatomical positions of labeled structures suggesting that PACAP mediates the activity of some interneurons, a few small motoneurons and certain sensory fibers that are located in ventrolateral, ventromedial and intermediomedial sensory longitudinal axon bundles of the VNC ganglia. No labeling was located on large interneuronal systems such as dorsal medial and lateral giant axon systems and ventral giant axons. At the ultrastructural level labeling was mainly restricted to endo- and plasma membranes showing characteristic unequal distribution in various neuron parts. An increasing abundance of PAC1 receptors located on both rough endoplasmic reticulum and plasma membranes was seen from perikarya to neural processes, indicating that intracellular membrane traffic might play a crucial role in the transportation of PAC1 receptors. High number of PAC1 receptors was found in both pre- and postsynaptic membranes in addition to extrasynaptic sites suggesting that PACAP acts as neurotransmitter and neuromodulator in the earthworm nervous system.

PKA-Bad-14-3-3 and Akt-Bad-14-3-3 signaling pathways are involved in the protective effects of PACAP against ischemia/reperfusion-induced cardiomyocyte apoptosis.

The neuropeptide PACAP (pituitary adenylate cyclase activating polypeptide) and its receptors are widely expressed in the nervous system and various other tissues. PACAP has well-known anti-apoptotic effects in neuronal cell lines. Recent data suggest that PACAP exerts anti-apoptotic effects also in non-neuronal cells. The peptide is present in the cardiovascular system, and has various distinct effects. The aim of the present study was to investigate whether PACAP is protective against in vitro ischemia/reperfusion-induced apoptosis in cardiomyocytes. Cultured cardiomyocytes were exposed to 60 min ischemia followed by 120 min reperfusion. The addition of PACAP1-38 significantly increased cell viability and decreased the ratio of apoptotic cells as measured by MTT test and flow cytometry. PACAP induced the phosphorylation of Akt and protein kinase A. In the present study we also examined the possible involvement of Akt- and protein kinase A-induced phosphorylation and thus inactivation of Bad, a pro-apoptotic member of the Bcl-2 family. It was found that ischemia significantly decreased the levels of phosphorylated Bad, which was counteracted by PACAP. Furthermore, PACAP increased the levels of Bcl-xL and 14-3-3 protein, both of which promote cell survival, and decreased the apoptosis executor caspase-3 cleavage. All effects of PACAP1-38 were inhibited by the PACAP antagonist PACAP6-38. In summary, our results show that PACAP has protective effects against ischemia/reperfusion-induced cardiomyocyte apoptosis and provides new insights into the signaling mechanisms involved in the PACAP-mediated anti-apoptotic effects.

Protective effects of pituitary adenylate cyclase activating polypeptide against neurotoxic agents.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide highly expressed in the central and peripheral nervous system, where it exerts several neuromodulatory functions and is an important trophic and protective factor. PACAP has been shown to activate several protective pathways, mainly through its specific PAC1 receptor and protein kinase A, C and MAP kinases downstream. It has been shown to have very potent neuroprotective actions against different neurotoxic agents both in vitro and in vivo. The aim of the present review is to provide an overview on the neurotoxic injuries against which PACAP exerts protection, and to give an insight into its protective mechanism. We give a summary of the neuroprotective effects against the most commonly used neurotoxic agents, such as 6-OHDA, MPTP, glutamate and some less well-known neurotoxic compounds. Also endogenous PACAP has neuroprotective effects, known from studies in PACAP knockout mice or from blocking endogenous effects by antagonists. Altogether, the vast amount of data for the neuroprotective effects of PACAP give a firm background for its endogenous role as part of the neuroprotective machinery and its possible future therapeutic use as a neuroprotective factor.

Changes in open-field activity and novelty-seeking behavior in periadolescent rats neonatally treated with monosodium glutamate.

Monosodium glutamate (MSG) treatment of neonatal rodents leads to degeneration of the neurons in the arcuate nucleus, inner retinal layers and various other brain areas. It also causes various changes in the motor activity, sensory performance and learning abilities. We have previously shown that MSG treatment delays the appearance of some reflexes during neurobehavioral development and leads to temporary changes in reflex performance and motor coordination. Investigation of novelty-seeking behavior is of growing importance for its relationship with sensitivity to psychomotor stimulants. Perinatal administration of numerous toxic agents has been shown to influence novelty-seeking behavior in rats, but little is known about the influence of neonatal MSG treatment on the novelty-seeking behavior. The aim of the present study was to compare changes in locomotor, spontaneous exploratory and novelty-seeking behavior in periadolescent rats neonatally treated with MSG. Newborn rats were treated with 4 mg/g MSG subcutaneously on postnatal days 1, 3, 5, 7 and 9. Open-field behavior was tested at 2, 3, 4, 6 and 8 weeks of age. We found that MSG administration led to only temporary increases in locomotor behavior, which was more pronounced during the first few postnatal weeks, followed by a subtle hypoactivity at 2 months of age. Novelty-seeking was tested in four 5-min trials at 3 weeks of age. Trial 1 was in an empty open-field, two identical objects were placed in the arena during trial 2 and 3, and one of them was replaced to a novel object during trial 4. We found that the behavioral pattern of MSG-treated rats was the opposite in all tested signs in the novelty exploration test compared to control pups. In summary, our present study shows that neonatal MSG treatment leads to early temporary changes in the locomotor activity followed by hypoactivity at 2 months of age. Furthermore, MSG-treated rats show a markedly disturbed novelty-seeking behavior represented by altered activity when subjected to a novel object.

Protective effects of PACAP in excitotoxic striatal lesion.

The present article investigated the effects of pituitary adenylate cyclase-activating polypeptide (PACAP) treatment in a quinolinic acid (QA)-induced unilateral lesion of the striatum, a model of Huntington;s disease (HD). PACAP was given locally, preceding the lesion. Behavioral analysis was performed after 1, 10, and 30 days, when motor activity and asymmetrical signs were evaluated. Three weeks after the treatment, a catalepsy test was performed by haloperidol administration, and finally histological assessment of the striatum was done. Our results show that PACAP treatment attenuated the behavioral deficits and reduced the number of lesioned neurons in the striatum.

Comparative study of the effects of PACAP in young, aging, and castrated males in a rat model of Parkinson's disease.

We have previously shown that PACAP ameliorates the neurological symptoms and reduces the dopaminergic cell loss in young male rats, in a 6-hydroxydopamine (6-OHDA)-induced lesion of the substantia nigra, a model of Parkinson's disease. In the present study, we compared the effects of PACAP in young, aging, and castrated males. Our results show that PACAP significantly reduced the dopaminergic cell loss in young and aging males. In castrated males, 6-OHDA did not induce such a severe cell loss, and it was not altered by PACAP. However, PACAP effectively ameliorated behavioral symptoms in all groups, with a degree of recovery depending on age and endocrine status.

Effects of systemic PACAP treatment in monosodium glutamate-induced behavioral changes and retinal degeneration.

The present article investigated effects of systemic pituitary adenylate cyclase-activating polypeptide (PACAP) treatment in monosodium glutamate (MSG)-induced retinal degeneration and neurobehavioral alterations in neonatal rats. It was found that the dose of PACAP that effectively enhances neurobehavioral development in normal rats was able to counteract the retarding effect of MSG on righting, forelimb placing, and grasp reflexes and caused a significant amelioration of the righting and gait reflex performance and motor coordination at 2 weeks of age. In the retina, significant amelioration of neuronal loss in the inner retinal layers was achieved, but it was much less than that observed by local administration.

Pituitary adenylate cyclase activating polypeptide protects cardiomyocytes against oxidative stress-induced apoptosis.

Pituitary adenylate cyclase activating polypeptide (PACAP) has well-known neuroprotective effects, and one of the main factors leading to neuroprotection seems to be its anti-apoptotic effects. The peptide and its receptors are present also in the heart, but whether PACAP can be protective in cardiomyocytes, is not known. Therefore, the aim of the present study was to investigate the effects of PACAP on oxidative stress-induced apoptosis in cardiomyocytes. Our results show that PACAP increased cell viability by attenuating H2O2-induced apoptosis in a cardiac myocyte culture. PACAP also decreased caspase-3 activity and increased the expression of the anti-apoptotic markers Bcl-2 and phospho-Bad. These effects of PACAP were counteracted by the PACAP antagonist PACAP6-38. In summary, our results show that PACAP is able to attenuate oxidative stress-induced cardiomyocyte apoptosis.

Effect of PACAP in 6-OHDA-induced injury of the substantia nigra in intact young and ovariectomized female rats.

Pituitary adenylate cyclase activating polypeptide (PACAP) has neuroprotective effects in various neuronal cultures and in models of brain pathologies in vivo. Among others, it protects dopaminergic neurons in vitro, against 6-OHDA- and rotenone-induced injury. Recently, we have shown that PACAP reduces dopaminergic cell loss and ameliorates behavioral outcome following unilateral 6-OHDA-induced injury of the substantia nigra in male rats. However, after castration, PACAP led only to a slight amelioration of the behavioral symptoms. The aim of the present study was to investigate the degree of neuroprotection exerted by PACAP in female rats, using the same model. It was found that PACAP had no effect on the dopaminergic cell loss in intact female rats, only caused amelioration of certain acute behavioral signs. In contrast, PACAP effectively increased dopaminergic cell survival and decreased behavioral deficits in ovariectomized females. These results indicate that the neuroprotective effect of PACAP in a rat model of Parkinson's disease is gender-specific.

Development of neurological reflexes and motor coordination in rats neonatally treated with monosodium glutamate.

Monosodium glutamate (MSG) treatment of neonatal rats causes neuronal degeneration in various brain areas and leads to several neurochemical, endocrinological and behavioral alterations. However, relatively little is known about the development of neurological reflexes and motor coordination of these animals. Therefore, the aim of the present study was to examine the neurobehavioral development of newborn rats treated with MSG. Rats received MSG at postnatal days 1, 3, 5, 7, and 9. Appearance of neural reflexes and reflex performance as well as motor coordination were examined for 5 weeks after birth. The efficacy of MSG treatment was confirmed by histological examination of the arcuate nucleus. We found that MSG treatment delayed the appearance of forelimb placing, forelimb grasp and righting reflexes, besides the retarded somatic development. The treated pups performed surface righting in significantly longer times. Also, worse performance was observed in the foot-fault and rota-rod tests. However, MSG-treated rats reached control levels by the end of the fifth postnatal week. These results show that MSG treatment does not cause permanent alterations in the neurobehavioral development, only delays the appearance of some reflexes and leads to temporary changes in reflex performance and motor coordination signs.

Gastrin- and cholecystokinin-like immunoreactivities in the nervous system of the earthworm.

The distribution of cholecystokinin and gastrin-like immunoreactive cell bodies and fibers in the nervous system of 2 annelid worms, Lumbricus terrestris and Eisenia fetida, has been studied by means of immunohistochemistry. The cerebral ganglion contains 170-250, the subesophageal ganglion contains 120-150, and the ventral ganglia contain 50-75 cholecystokinin immunoreactive cells, that represent 8-12%, 8-10% and 4-5% of the total cell number, respectively. The anti-gastrin serum stained 330-360 nerve cells in the cerebral, 32-46 in the subesophageal and 7-25 in the ventral cord ganglia, representing 15-16%, 2-3% and 0.5-2% of the total cell number. Immunopositivity was found with both antisera in the enteric nervous system, where the stomatogastric ganglia and the enteric plexus contain immunoreactive cells and fibers. Immunopositive cells were found in the epithelial and subepithelial cells, as well as in nerve cells innervating the muscular layer of the gastrointestinal tube. Various epidermal sensory cells also displayed strong immunoreactivity. According to our findings and the results of several functional studies, it is suggested that in annelids cholecystokinin- and gastrin-like peptides may be involved in digestive regulation, sensory processes and central integrating processes.

Co-localization of serotonin and FMRFamide-like immunoreactivities in the central nervous system of the earthworm, Eisenia fetida.

In the present study co-localization of serotonin and FMRFamide-like immunoreactivities was studied in the central nervous system of the earthworm, Eisenia fetida. Alternate serial sections were stained with antisera raised against serotonin and FMRFamide, using the peroxidase-antiperoxidase method. The immunostained consecutive sections were compared by light-microscopy and on reconstructed images using NIH Image and Neurolucida programs. Although there was a large number of serotonin and FMRFamide-like immunoreactive neurons in the central nervous system of Eisenia, co-localization of immunostaining was relatively rare. In the cerebral ganglion no cells were found that stained for both antisera. In the subesophageal and ventral cord ganglia the neurons reacting for both antisera represented approximately 17-23% and 10-14% of the serotonin- and 7-11% and 9-17% of the FMRF-amide-like immunoreactive cells, respectively. It is suggested, that FMRFamide-related peptides act as neuromodulators and may also influence the activity of serotonergic neurons.

Serotonin immunoreactivity in the peripheral nervous system of oligochaeta.

The distribution of serotonin immunoreactive elements in the peripheral nervous system of three earthworm species (Lumbricus terrestris, Eisenia fetida and Tubifex tubifex) was studied using the peroxidase-antiperoxidase method. Most parts of the peripheral nervous system contain immunoreactive fibers. All segmental nerves as well as branches of the prostomial nerves show strong immunoreactivity. Muscles of the body wall contain serotonergic fibers, mostly between the circular and longitudinal layers. The best supplied area is the buccal musculature. All parts of the enteric nervous system contain serotonergic fibers, strongest staining was observed in the buccal and pharyngeal walls. Along the wall of the calciferous glands thin immunoreactive fibers were found. The excretory system, the metanephridial tubules also display immunoreactivity. Under the surface epithelium, a serotonergic subepidermal plexus was observed. No significant differences were observed between the species studied. On the basis of our morphological findings and functional studies performed by other authors it is suggested that serotonin plays an essential role in the function of the peripheral nervous system in Oligochaeta.

Proctolin immunoreactive elements in the nervous system of earthworm (Lumbricus terrestris).

Proctolin (H-Arg-Try-Leu-Pro-Thr-OH) immunoreactive elements in the nervous system of earthworm (Lumbricus terrestris, L.) were detected in paraffin sections using peroxidase-antiperoxidase method. Each part of the central nervous system contains proctolin immunoreactive cell bodies. The nerve cells, according to their locations are motoneurons, sensory neurons as well as interneurons. A part of the sensory epithelial cells of the body surface are proctolin immunoreactive, too. The body wall muscles receive a rich proctolin immunoreactive network. It is supposed that proctolin acts as a neuromodulator in the nervous system of Lumbricus terrestris.

Substance P immunoreactive elements in the nervous system of earthworm (Lumbricus terrestris).

Substance P immunoreactive elements were detected in the nervous system of the earthworm, Lumbricus terrestris using peroxidase-antiperoxidase method. Each part of the central nervous system contains immunoreactive perikarya. The central neuropil shows no stained fibers. Immunopositive nerve cells were found in the stomatogastric ganglia as well as nerve cells and fibers in the wall of the alimentary canal. In the body wall, epidermal sensory cells and fibers surrounding the chaetae display immunoreactivity. The results are compared with those described previously by other authors.

PACAP is an endogenous protective factor-insights from PACAP-deficient mice.

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. In vivo, PACAP-deficient mice have a negative histological outcome and altered cytokine response in kidney and small intestine ischemia/reperfusion injury. Large intestinal inflammation, toxic lesion of the pancreas, and doxorubicin-induced cardiomyopathy are also more severe with a lack of endogenous PACAP. Finally, an increased inflammatory response has been described in subacute endotoxin-induced airway inflammation and in an oxazolone-induced allergic contact dermatitis model. In summary, lack of endogenous PACAP leads to higher vulnerability in a number of injuries in the nervous system and peripheral organs, supporting the hypothesis that PACAP is part of the endogenous cytoprotective machinery.

Review on the protective effects of PACAP in models of neurodegenerative diseases in vitro and in vivo.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic and multifunctional peptide exerting its effects via 3 main receptors (PAC1, VPAC1 and VPAC2). PACAP is now considered to be a potent neurotrophic and neuroprotective peptide. It plays an important role during the embryonic development of the nervous system. PACAP also protects neurons against various toxic insults in neuronal cultures of diverse origins. In vivo, PACAP shows neuroprotection in models of ischemic and traumatic brain injuries, and those of neurodegenerative diseases. The present review summarizes the findings on the neuroprotective potential of PACAP in models of neurodegenerative diseases, with special focus on in vitro and in vivo models of Parkinson`s disease, Huntington chorea and Alzheimer`s disease. Based on these observations, both endogenous and exogenously administered PACAP or its novel analogs, fragments offer a novel therapeutic approach in treatment of neurodegenerative diseases.

Presence of pituitary adenylate cyclase activating polypeptide and its type I receptor in the rat kidney.

Pituitary adenylate cyclase activating polypeptide (PACAP), a multifunctional neuropeptide, has 2 active forms, PACAP38 and PACAP27. It is now well-established that PACAP has several actions also in peripheral organs, including renoprotective effects. The peptide itself has not been previously identified in the rat kidney. The first aim of our study was to identify PACAP in the rat kidney using mass spectrometry and radioimmunoassay (RIA). Receptor mRNA and binding studies revealed the existence of all 3 PACAP receptors (PAC1, VPAC1, and VPAC2) in the kidney, but their exact localization in histologic sections was not evident. Because most of the cytoprotective effects of PACAP relate to its specific PAC1 receptor, our second aim was to identify the cell types wherein the PAC1 receptor is expressed in the rat kidney. Mass spectrometry revealed the presence of PACAP38 in the kidney. RIA measurements showed both PACAP38- and PACAP27-like immunoreactivities in kidney homogenates, with PACAP38 being dominant. Immunohistochemistry revealed PAC1 receptor-like immunoreactivity in kidney sections, mainly expressed in cortical tubular epithelial cells. These results showed PACAP to be endogenously present in the kidney. The tubular localization of the PAC1 receptor provides the basis for the renal effects of the peptide under physiologic and pathologic conditions.

Investigation of pituitary adenylate cyclase activating polypeptide in human gynecological and other biological fluids by using MALDI TOF mass spectrometry.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional and pleiotropic neuropeptide. PACAP has diverse effects in the endocrine system, among others, it plays important roles in oogenesis, implantation and development of the nervous system. However, it is not known whether PACAP is present in the fluids of the human reproductive organs. The aim of the present study was to determine, by means of mass spectrometry and radioimmunoassay, whether PACAP is present in human amniotic fluid, ovarian follicular fluid and cervico-vaginal fluid. Samples were obtained from healthy adult volunteers. Our MALDI TOF and MALDI TOF/TOF spectrometry results show that PACAP38 is present in all of the follicular fluid samples, and PACAP-like immunoreactivity was also measured by radioimmunoassay. However, we did not find the characteristic peak representing the unmodified 38 amino acid form of the peptide in normal cervico-vaginal smear and amniotic fluid samples. Furthermore, we analyzed other body fluids for comparison, such as human nasal fluid, saliva and aqueous humor. PACAP was not found in these latter samples. In summary, the present study provides evidence for the presence of PACAP in human follicular fluid, suggesting a role in oocyte function, but determination of the exact physiological significance awaits further investigation.