Changes of PACAP levels in the brain show gender differences following short-term water and food deprivation.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide exerting diverse actions in the central and peripheral nervous systems. A few studies indicate that PACAP is involved in the regulation of feeding and water homeostasis. The aim of the present study was to investigate changes in PACAP38 concentrations in different brain areas following food or water deprivation in male and female rats. Rats were sacrificed 12, 36 and 84h after water or food removal. PACAP levels were determined by radioimmunoassay. Our results show that levels of PACAP decreased in the hypothalamus in both sexes after water deprivation, with a more marked, significant decrease in females at 12h. A decrease was observed also in the telencephalon, with a similar pattern in both genders: levels were lowest after 12h, and showed a gradual increase at the other two time-points. PACAP levels increased in the brainstem of male rats, while females had a decrease 12h after water deprivation. The pattern of changes in PACAP levels was very different after food deprivation. In male rats, PACAP levels showed a significant increase in the hypothalamus, telencephalon and brainstem 12h after the beginning of starvation. In females, a less marked increase was observed only in the hypothalamus while no changes were found in the other brain areas. Our results show a sensitive reaction in changes of endogenous PACAP levels to water and food deprivation in most brain areas, but they are differentially regulated in male and female rats.

Protective effects of pituitary adenylate cyclase activating polypeptide in endothelial cells against oxidative stress-induced apoptosis.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a widely distributed neuropeptide that has various different functions in the nervous system and in non-neural tissues. Little is known about the effects of PACAP in endothelial cells. The aim of the present study was to investigate the effects of PACAP on endothelial cell survival and apoptotic signaling pathways under oxidative stress. Mouse hemangioendothelioma (EOMA) cells were exposed to 0.5mM H(2)O(2) which resulted in a marked reduction of cell viability and a parallel increase of apoptotic cells assessed by MTT test and flow cytometry. Co-incubation with 20nM PACAP1-38 increased cell viability and reduced the percentage of apoptotic cells. Flow cytometry analysis showed that oxidative stress reduced the phosphorylation of the anti-apoptotic ERK and increased the phosphorylation of the pro-apoptotic JNK and p38 MAP kinases. PACAP1-38 treatment ameliorated these changes: levels of phospho-ERK were elevated and those of phospho-JNK and p38 were decreased. All these effects were abolished by simultaneous treatment with the PACAP antagonist PACAP6-38. In summary, our results show that PACAP effectively protects endothelial cells against the apoptosis-inducing effects of oxidative stress.

Effects of pretreatment with PACAP on the infarct size and functional outcome in rat permanent focal cerebral ischemia.

PACAP exerts neuroprotective effects under various neurotoxic conditions in vitro. In vivo, it reduces brain damage after global and transient focal ischemia. The present study investigated whether PACAP has neuroprotective effects when applied before the onset of permanent ischemia. Rats were given bolus injections of PACAP38 intracerebroventricularly, and then underwent permanent middle cerebral artery occlusion. The results show that 2 microg of PACAP significantly reduced the infarct size measured 12 and 24h after the onset of ischemia. No further reduction was obtained by a 7-day pretreatment. PACAP also ameliorated certain sensorimotor deficits. Our present study provides further evidence for the neuroprotective effects of PACAP, and implies that it might be a promising preventive therapeutic agent in ameliorating ischemic brain damage.

Distribution and daily variations of PACAP in the chicken brain.

Levels of PACAP38 were measured in different areas of the chicken brain under various lighting conditions by radioimmunoassay (RIA). Selected groups of animals were maintained under light for 14 h alternating with 10 h of darkness (LD), reversed lighting conditions (DL) and constant light (LL) or constant dark (DD). Daily variations of PACAP levels were observed in the brainstem, diencephalon, telencephalon and retina. In the brainstem and diencephalon, levels of PACAP increased during subjective nighttime, except in the DL group where levels were elevated between 15-21 h. In the telencephalon, the lowest level of PACAP was measured between 12-21 h except in the DL group where two peaks occurred at 18 and 03 h. In the retina, all 4 groups showed a similar level and pattern, with lowest levels during midday hours. No daily variation was observed in the pineal gland. According to the present observations, it is suggested that PACAP levels differ in several areas of the chicken brain under various lighting conditions and photic stimuli do not appear to be the main regulators of the circadian variations of PACAP.

Pituitary adenylate cyclase activating polypeptide is highly abundant in the nervous system of anoxia-tolerant turtle, Pseudemys scripta elegans.

The levels of the pituitary adenylate cyclase activating polypeptide (PACAP) were measured in the central nervous system and in peripheral organs of the anoxia-tolerant freshwater turtle, Pseudemys scripta elegans by radioimmunoassay. The concentration of PACAP38 was strikingly high in the central nervous system and lower but considerable immunoreactivity was detected in the peripheral organs. Levels of PACAP38 in the turtle brain exceed those measured in rat and human brain areas by 10-100-fold. Based on these exceptionally high levels of PACAP and the known neuroprotective role of the peptide, it can be suggested that PACAP38 plays a role in the extraordinary resistance of the turtle brain from anoxia-induced neuronal damage.

GH mRNA levels are elevated by forskolin but not GH releasing hormone in GHRH receptor-expressing MtT/S somatotroph cell line.

The MtT/S somatotroph cell line should be a growth hormone-releasing hormone (GHRH)-responsive model system for the study of physiological control of growth hormone (GH) transcription because GH secretion from these cells is stimulated by GHRH. To examine the GH transcriptional activity of these cells, endogenous GH mRNA levels were measured using a ribonuclease protection assay following treatment under a variety of hormonal conditions. While omission of serum led to reduction of GH mRNA to 22% of control levels by 2 days and to 8% by 5 days (P<0.05 for both), GH mRNA levels were maintained at control values in serum-free medium containing 5 nM dexamethasone and 30 pM triiodothyronine (TDM). However, the addition of 10 nM GHRH under any treatment condition did not significantly alter GH mRNA levels. Characterization of the MtT/S cells showed that GHRH-receptor (GHRH-R) mRNA was detectable by reverse transcription-polymerase chain reaction (RT-PCR) amplification. Measurement of extracellular cAMP showed that the MtT/S cells have basal levels of > or =20 nmol/10(6) cells per h in both serum-containing and serum-free media, and that GHRH had no effect on cAMP levels, suggesting constitutive activation. To rule out the possibility of autocrine stimulation by GHRH produced endogenously, GHRH mRNA was not detectable in MtT/S cells using RT-PCR amplification. The stimulatory G-protein alpha subunit, mutations of which are known to activate adenylate cyclase constitutively in acromegaly, was sequenced but found not to differ from normal pituitary in the regions most commonly mutated. Finally, treatment with 10 microM forskolin, to directly activate adenylate cyclase, increased GH mRNA to 140% of controls in TDM, and to 163% in serum-free medium after 2 days, and to 166% in TDM-treated cells and 174% in serum-free culture after 5 days (all P<0.05). Taken together, these data indicate that although MtT/S cells express the GHRH-R, GHRH cannot stimulate adenylate cyclase to increase GH transcription due to constitutive elevation of cAMP levels, by a means that may be similar to that in cases of acromegaly not caused by oncogenic gsp mutations.

Tissue distribution of PACAP27 and -38 in oligochaeta: PACAP27 is the predominant form in the nervous system of Lumbricus polyphemus.

The levels of pituitary adenylate cyclase-activating polypeptide (PACAP)27 and -38 were measured in the nervous, intestinal, excretory, and reproductive systems of Lumbricus polyphemus by radioimmunoassay. Although both PACAP27 and -38 were significantly detectable in all of the examined tissues, the distribution of the peptides was very heterogeneous. Their highest concentrations were found in the cerebral ganglia and the ventral cord, followed by the alimentary tract and the nephridial system, respectively. Moreover, the reproductive system also contained a substantial amount of PACAP. The dominant form of the peptide discovered in the majority of tissues was PACAP27. Interestingly, about 10 times more PACAP27 than PACAP38 was found, with the latter representing only a fraction of PACAP-like immunoreactivity in the tissues of Lumbricus polyphemus.

Effect of middle cerebral artery occlusion on the passage of pituitary adenylate cyclase activating polypeptide across the blood-brain barrier in the rat.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to be a potent neuroprotective agent in global and focal ischemia. We demonstrated that PACAP could cross the blood-brain barrier (BBB) by a saturable transport system, and a systemic administration of PACAP reduced the infarct induced by unilateral middle cerebral artery occlusion (MCAO). Therefore, we studied whether this transport system is affected by MCAO in the rat. The entry of PACAP38 into the brain was compared in five groups: control, 4, 6, 24, and 48 h after MCAO. [(125)I]PACAP38 was injected intravenously and serum and various brain regions were collected 3 min later. The rate of entry into the brain of PACAP38 was also determined. We showed that PACAP entered the rat brain via a rapid transport system when the BBB is intact. After transient (2 h) unilateral MCAO, all regions of the brain, showed a selective increase in the passage of PACAP38 across the BBB after 4 h after the occlusion, which was not related to any generalized change in the permeability of the BBB, as measured with albumin. A significant decrease in the amount of PACAP38 entering the brain was observed in the 6- and 24-h groups, but it returned to the baseline level in the 48-h group. These results suggest that focal cerebral ischemia can selectively modify the passage of PACAP38 across the BBB, in both damaged and undamaged sides of the brain, and that these changes in influx are not solely due to the disruption of BBB. These findings imply the necessity of adjusting the dose of intravenously administered PACAP38 in order to maximize its therapeutic effect on the brain damage resulting from focal ischemia

Suckling-induced increase in cyclic AMP exclusively in the central region of the rat adenohypophysis.

Investigating the cellular events in the pituitary gland, the intracellular cyclic AMP (cAMP) of the neural lobe (NL), intermediate lobe (IL), the inner (IZ-AL) and outer zone (OZ-AL) of the anterior lobe (AL) have been measured during the suckling stimulus. Ten-minutes suckling, parallel to the elevation of plasma PRL, induced a significant increase of cAMP concentration in the IZ-AL. In contrast, 10- and 30-min suckling resulted in a decrease of cAMP level in the NL. Changes in cAMP of the OZ-AL and the IL as well as in the plasma level of alpha-MSH could not be detected. These region-specific changes of cAMP in the pituitary gland during suckling stimulus seems to be related to interacting neuroendocrine signals delivered concomitantly from the hypothalamus and from the NIL to the IZ-AL.

Postischemic spontaneous hyperthermia and its effects in middle cerebral artery occlusion in the rat.

This study examined the time course and effects of postischemic spontaneous hyperthermia after transient and permanent focal ischemia. Rats underwent a 90-min, 120-min, or permanent middle cerebral artery occlusion (MCAO). Body temperatures started rising 15-20 min after MCAO and reached 39-40.5 degrees C during the first hour. Sustained hyperthermia was observed during the rest of the first 24 h. In another experiment, rats were subjected to the same interventions, but a normothermic body temperature was maintained. Spontaneous hyperthermia significantly increased the infarct volumes measured 48 h after MCAO in all groups. Reperfusion 2 h after the onset of ischemia was not beneficial in the hyperthermic animals in contrast to the normothermic group. We also examined the effect of spontaneous hyperthermia on the temporal progression of infarcted and penumbral areas 4, 12, or 48 h after MCAO. During spontaneous hyperthermia, penumbral areas became infarcted areas more rapidly, which was most expressed at 4 h. These findings demonstrate that severe spontaneous hyperthermia can occur in rats after MCAO and that it not only increases the infarct volumes in both transient and permanent ischemia, but also accelerates the incorporation of penumbral areas into necrotic areas, which significantly decreases the window of opportunity for therapeutic interventions.

Delayed systemic administration of PACAP38 is neuroprotective in transient middle cerebral artery occlusion in the rat.

BACKGROUND AND PURPOSE: Many substances have been shown to reduce brain damage in models of stroke, but mainly when given either before or shortly after the onset of ischemia. Delayed systemic administration of pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to attenuate the neuronal damage in the hippocampus in a model of global ischemia in rats. The present study examined the neuroprotective action of delayed systemic administration of PACAP38 in a model of transient focal ischemia produced by middle cerebral artery occlusion (MCAO) in rats. METHODS: We administered PACAP38 as an intravenous bolus (20 nmol/kg body wt) followed by an intravenous infusion for 48 hours using a micro-osmotic pump at a rate of 160 pmol/microL per hour, beginning 4, 8, or 12 hours after a 2-hour transient MCAO using a filament model. The size of the infarct was determined by examining 2-mm-thick brain sections stained with triphenyltetrazolium chloride, followed by image analysis. Control animals received intravenously 0.1% bovine serum albumin in 0.9% saline as a bolus and infusion at the same time intervals. RESULTS: The administration of PACAP38 beginning 4 hours after MCAO significantly reduced the infarct size by 50.88%. Treatment with PACAP38 starting 8 or 12 hours after the onset of ischemia did not result in a significant reduction of the infarct size, although infarct volumes tended to be smaller than in the control groups. CONCLUSIONS: Systemic administration of PACAP38 should be clinically useful for reducing brain damage resulting from stroke even when administration is delayed for several hours.

Neuroprotective effects of PACAP38 in a rat model of transient focal ischemia under various experimental conditions.

In the study reported here, we investigated the neuroprotective effect of PACAP38 in a rat model of middle cerebral artery occlusion (MCAO) under various experimental conditions. Stroke patients often develop mild hyperthermia that increases the infarct size and worsens the outcome. We compared the neuroprotective effect of PACAP38 in normothermic and moderately hyperthermic animals. Brain damage was more extensive in the hyperthermic animals, but PACAP38 significantly reduced the infarct size, measured 48 h after MCAO, in both the normothermic and the moderately hyperthermic animals, by more than 50%. However, a significant neuroprotection with PACAP38 could only be demonstrated when animals with no definite circling behavior and/or convulsions were excluded from the evaluations. In conclusion, moderate hyperthermia did not influence the effect of PACAP38, but animals with questionable brain damage or convulsions must be excluded from the sample in order to demonstrate the neuroprotection with PACAP38.

Prohormone convertases 1 and 2 process ProPACAP and generate matured, bioactive PACAP38 and PACAP27 in transfected rat pituitary GH4C1 cells.

Pituitary adenylate cyclase-activating polypetide (PACAP) exists in two amidated forms, PACAP38 and PACAP27, which are expressed in the magnocellular and parvocellular neurons of the paraventricular nucleus (PVN) and the magnocellular neurons of the supraoptic nucleus (SON) of the hypothalamus. The prohormone convertases PC1 and PC2, subtilisin-like PCs of the Kex2 family, are expressed in neuroendocrine cells. Immunocytochemistry and in situ hybridization of PC1 and PC2 in the hypothalamus have shown that PC1 and PC2 are also present in the PVN and SON. Therefore, it is possible that the precursor of PACAP is processed by PC1 and/or PC2 in the hypothalamic nuclei and then converted to its mature forms. To test this hypothesis, rat pituitary GH4C1 cells were supertransfected with human PACAP cDNA and either rat PC1 or PC2 cDNA. The acid extracts of these cells were analyzed by reversed-phase HPLC for proPACAP, PACAP38 and/or PACAP27 radioimmunoassays using three antibodies with different recognition sites, and then bioassayed for the ability to stimulate adenylate cyclase. The cells transfected with PACAP cDNA alone yielded PACAP-like immunoreactivity (PACAP-li) corresponding to molecular weights between 15 and 20 kDa without PACAP bioactivity. Cotransfection of these cells with PC1 or PC2 generated PACAP-li, which coeluted with synthetic PACAP38 and PACAP27, respectively. Western blot also revealed 4.5- and 3.0-kDa PACAP-li bands, which correspond to the molecular weights of PACAP38 and PACAP27, respectively. The HPLC fractions containing PACAP-li, which were coeluted with synthetic PACAP38 and PACAP27, showed marked bioactivities. These findings suggest that the precursor of PACAP expressed in the PVN and SON of the hypothalamus could be efficiently processed by PC1 and PC2, and then converted to mature, bioactive PACAP38 and PACAP27.

Testis-specific prohormone convertase PC4 processes the precursor of pituitary adenylate cyclase-activating polypeptide (PACAP).

The physiological substrate for proprotein convertase (PC) 4, which is expressed only in the testis, has remained unknown. Pituitary adenylate cyclase activating polypeptide (PACAP), originally isolated from the hypothalamus, exists as two amidated forms with 38 (PACAP38) and 27 (PACAP27) residues. PACAP-like immunoreactivity (PACAP-li) is found not only in the brain, but also in the peripheral tissues, and is especially abundant in the testis. Immunohistochemistry of the rat testis demonstrated strong PACAP-li in spermatids in the cap and acrosome phases. The nearly simultaneous expression of PC4 transcripts and PACAP-li in spermatids during spermatogenesis led to the hypothesis that PACAP precursor is processed by PC4. To investigate this possibility, rat pituitary GH4C1 cells were stably transfected with human PACAP cDNA, and some of these cells were co-transfected with mouse PC4 cDNA. The acid extracts of the cells were fractionated by reversed-phase HPLC. Each fraction was examined for PACAP-li using three antisera which recognize PACAP precursor, PACAP38 and/or PACAP27. Negligible PACAP-li that eluted with synthetic PACAP38 or PACAP27 was detected from cells transfected with PACAP cDNA; however, PC4 co-transfected cells showed marked PACAP-li peaks with the retention times for both PACAP38 and PACAP27. Moreover, Western blot analysis revealed immunostained bands, corresponding to the Mr for PACAP38 and PACAP27, in the PC4 co-transfected cells. Bioactivity, as indicated by stimulation of cAMP production in pituitary cell cultures, was found only in the extracts of PC4 co-transfected cells. These results provide evidence that PACAP precursor in the testis is a substrate for PC4. The processing of PACAP precursor by PC4 at a critical time in spermatogenesis suggests an important regulatory role of PC4 and PACAP in the maturation of germ cells in the testis.

Immunohistochemical demonstration of the intracellular localization of pituitary adenylate cyclase activating polypeptide-like immunoreactivity in the rat testis using the stamp preparation.

Radioimmunoassay has revealed an abundance of PACAP in the rat testis. In the present study, a novel stamp preparation together with light microscopic immunohistochemistry were used to investigate in detail the intracellular localization of PACAP-like immunoreactivity (PACAP-LI) in rat germ cells. Samples were obtained by pressing the freshly cut surfaces of the testes against glass slides. PACAP-LI was clearly identified in developing acrosomes using five antisera which recognize PACAP 38, or both PACAP 27 and PACAP 38. Immunoreactivity with the antisera specific to PACAP38 was strictly localized in the developing acrosomes of the spermatids but vanished in the mature spermatids. Using an antiserum which detects PACAP 27 specifically, little staining was observed. Based on the specificities of antisera used, it was suggested that the PACAP-LI in the acrosomes represents mainly PACAP 38-LI. In addition, the present results supported the usefulness of the stamp preparation for immunohistochemical study of testicular tissues.

Localization and gene expression of the receptor for pituitary adenylate cyclase-activating polypeptide in the rat brain.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a recently identified member of the secretin/vasoactive intestinal polypeptide (VIP) family. There are at least two types of receptor for PACAP: type I (PACAPR), which specifically binds PACAP; and type II (VIP/PACAPR), which binds both PACAP and VIP. The localization of PACAPR in the rat brain was determined by in situ hybridization and immunocytochemistry. We raised antisera against a synthetic peptide that corresponds to the carboxy-terminal cytoplasmic domain which is found in all subtypes of PACAPR in order to localize PACAPR-like immunoreactivity (PACAPR-LI) in the rat brain. In general, the distribution of PACAPR-LI correlated well with the distribution of PACAPR transcripts. Particularly strong PACAPR mRNA expression was detected in the olfactory bulb, hippocampus, cerebellum and hypothalamus and moderate labeling was detected in other scattered regions. At the cellular level, PACAPR-LI appeared to be concentrated predominantly in neuronal perikarya and dendrites. At the ultrastructural level, strong immunostaining for the PACAPR was found in plasma membranes, rough endoplasmic reticulum, cytoplasmic matrix, and at synapses. This study provides the basis for a better understanding of the functions of PACAP in the rat brain.

Effect of the lack of light impulses on the hypothalamic PACAP and C-fos immunoreactivities in rats.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the secretin family. It is widely distributed in the central and peripheral nervous systems. The highest concentration of PACAP was found in the hypothalamus. In the present work it has been studied whether PACAP is involved in the mediation of photic stimuli to the anterior pituitary gland. We have examined the effect of the lack of light impulses on the hypothalamic PACAP and C-fos immunoreactivities. In adult rats 10 days after the removal of the eyes (surgical enucleation) and in those received monosodium glutamate treatment neonatally (chemical enucleation). The PACAP immunostaining enhanced in the hypothalamic magnocellular nuclei and in the extemal zone of the median eminence. C-fos immunoreactivity also enhanced in a few hypothalamic nuclei 2 hours after the surgical enucleation indicating that the lack of light impulses activated hypothalamic neurons which, in turn, might stimulate the release of PACAP into the portal circulation. It has been concluded that PACAP may be involved in photoendocrine regulations.

Specific antibody recognition of rat pituitary adenylate cyclase activating polypeptide receptors.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a new member of the secretin/VIP family of peptides. The specific receptor for PACAP has been cloned in rat, human, and bovine tissues. The distribution of the transcripts of PACAP receptor genes has been studied in various tissues using in situ hybridization. However, the unavailability of a specific antibody against the PACAP receptor has hampered further study of the expression of receptor proteins. In the present study, rabbit antisera were generated against a synthetic 25-residue peptide corresponding to the C-terminal intracellular domain of the rat PACAP receptor. To validate the specificity of the antisera, CHO cells and cells stably transfected with rat PACAP receptor cDNA were prepared. Using one of these antisera, the membrane and soluble fractions of the transformants were examined by Western blot analysis. Three bands were observed in subcellular fractions from the transfected CHO cells, but no bands were found in similar preparations from the nontransfected cells. A distinct 57-kDa band, which corresponds to the size of cloned rat PACAP receptor, was detected. In addition, a less intense band, larger than 57 kDa, and a very weakly stained band, smaller than 57 kDa, were demonstrated. All of these bands disappeared or were considerably diminished when the antiserum was preabsorbed with the synthetic immunogen peptide. This suggests that these bands are PACAP receptor-related proteins. The membranes from the transfected CHO cells bound to [125I]PACAP27. The size of the ligand/protein crosslinked product approximated 60 kDa, corresponding to the combined size of the PACAP receptor and PACAP27. No additional bands were observed, indicating that the immunopositive proteins larger or smaller than 57 kDa do not bind to the ligand and are not functional. Unlabeled PACAP27 and PACAP38, but not VIP, displaced the binding, suggesting that the receptors expressed in CHO cells are specific for PACAP. Solubilized membrane fractions prepared from rat brains were used for an immunoprecipitation study with [125I]PACAP27 and [125I]VIP. The PACAP receptor antiserum recognized [125I]PACAP-, but not [125I]VIP-bound proteins in the solubilized brain membrane fractions. Immunohistochemistry using this antiserum showed a distribution of PACAP receptor-like immunoreactivities similar to the distribution of the mRNA of PACAP receptor in the rat brain. Thus, the PACAP receptor antiserum is sufficiently specific to be used as a tool for studying the expression of PACAP receptors and related proteins.