Pituitary Adenylate Cyclase Research Articles

Role of the Bed Nucleus of the Stria Terminalis in PTSD: Insights From Preclinical Models.

Post-traumatic stress disorder (PTSD) afflicts approximately 8% of the United States population and represents a significant public health burden, but the underlying neural mechanisms of this and other anxiety- and stressor-related disorders are largely unknown. Within the last few decades, several preclinical models of PSTD have been developed to help elucidate the mechanisms underlying dysregulated fear states. One brain area that has emerged as a critical mediator of stress-related behavioral processing in both clinical and laboratory settings is the bed nucleus of the stria terminalis (BNST). The BNST is interconnected with essential emotional processing regions, including prefrontal cortex, hippocampus and amygdala. It is activated by stressor exposure and undergoes neurochemical and morphological alterations as a result of stressor exposure. Stress-related neuro-peptides including corticotropin-releasing factor (CRF) and pituitary adenylate cyclase activating peptide (PACAP) are also abundant in the BNST, further implicating an involvement of BNST in stress responses. Behaviorally, the BNST is critical for acquisition and expression of fear and is well positioned to regulate fear relapse after periods of extinction. Here, we consider the role of the BNST in stress and memory processes in the context of preclinical models of PTSD.

Regulatory networks upon neurogenesis induction in PC12 cell line by small molecules.

Alteration in the normal regulatory pathway ofdifferentiation can lead to the induction of programmed cell death. Accordingly, some chemicals like staurosporine, nerve growth factor, pituitary adenylate cyclase activating peptide, and trimethyltin are shown to be able to induce differentiation in vitro, via different mechanisms in the PC12 cell line. Hence, understanding the details of the molecular mechanisms of differentiation induction by these small molecules are important for further application of these molecules in neurogenesis. Therefore, we sought to determine these signaling pathways, using gene regulatory networks analysis. Then, we have conducted a comparative analysis of the alterations in the gene expression pattern of the PC12 cell lines in response to these chemicals at the early stages. Based on the comparative analysis and previous knowledge, we have proposed the affected pathways during differentiation and apoptosis. Our findings could be useful in the development of protocols to reprogramming of neurons by such small molecules with high efficiency.

PACAP-38 and PACAP(6-38) Degranulate Rat Meningeal Mast Cells via the Orphan MrgB3-Receptor.

Infusion of pituitary adenylate cyclase activating peptide-38 (PACAP-38) provokes migraine attacks in migraineurs and headache in non-migraineurs. Adverse events like long-lasting flushing and heat sensation can be terminated with oral antihistamine treatment, indicating the involvement of mast cell activation after PACAP-infusion. Degranulation of rat peritoneal mast cells was provoked by several isoforms of PACAP via previously unknown receptor pharmacology. The effect might thus be mediated either via specific splice variants of the PAC1-receptor or via an unknown receptor for PACAP-38. In the present study, we characterize degranulation of rat meningeal mast cells in response to PACAP-receptor ligands. Furthermore, we investigate if PACAP-38-induced mast cell degranulation is mediated via PAC1-receptor splice variants and/or via the orphan Mas-related G-protein coupled member B3 (MrgB3)-receptor. To address this, the pharmacological effect of different PACAP isoforms on meningeal mast cell degranulation was investigated in the hemisected skull model after toluidine blue staining followed by microscopic quantification. Presence of mRNA encoding PAC1-receptor splice variants and the MrgB3-receptor in rat mast cells was investigated by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis. The effect of PACAP isoforms on PAC1- and MrgB3-receptor-expressing Xenopus laevis oocytes were performed by two-electrode voltage-clamp (TEVC) electrophysiology. PACAP-38 is a more potent mast cell degranulating agent than Pituitary Adenylate Cyclase Activating Peptide-27 (PACAP-27) in the meninges. Presence of mRNA encoding the PAC1-receptor and its different splice variants could not be detected in peritoneal mast cells by RT-PCR, whereas the orphan MrgB3-receptor, recently suggested to be a mediator of basic secretagogues-induced mast cell degranulation, was widely present. In PAC1-receptor-expressing Xenopus laevis oocytes both PACAP-38, PACAP-27 and the specific PAC1-receptor agonist maxadilan were equipotent, however, only PACAP-38 showed a significant degranulatory effect on mast cells. We confirmed Pituitary Adenylate Cyclase Activating Peptide(6–38) [PACAP(6–38)] to be a PAC1-receptor antagonist, and we demonstrated that it is a potent mast cell degranulator and have an agonistic effect on MrgB3-receptors expressed in oocytes. The present study provides evidence that PACAP-induced mast cell degranulation in rat is mediated through a putative new PACAP-receptor with the order of potency being: PACAP-38 = PACAP(6–38) > > PACAP-27 = maxadilan. The results suggest that the observed responses are mediated via the orphan MrgB3-receptor.

Mitochondrial and calcium perturbations in rat CNS neurons induce calpain-cleavage of Parkin: Phosphatase inhibition stabilizes pSer65Parkin reducing its calpain-cleavage

Mitochondrial impairment and calcium (Ca++) dyshomeostasis are associated with Parkinson's disease (PD). When intracellular ATP levels are lowered, Ca++-ATPase pumps are impaired causing cytoplasmic Ca++ to be elevated and calpain activation. Little is known about the effect of calpain activation on Parkin integrity. To address this gap, we examined the effects of mitochondrial inhibitors [oligomycin (Oligo), antimycin and rotenone] on endogenous Parkin integrity in rat midbrain and cerebral cortical cultures. All drugs induced calpain-cleavage of Parkin to ~36.9/43.6 kDa fragments. In contrast, treatment with the proinflammatory prostaglandin J2 (PGJ2) and the proteasome inhibitor epoxomicin induced caspase-cleavage of Parkin to fragments of a different size, previously shown by others to be triggered by apoptosis. Calpain-cleaved Parkin was enriched in neuronal mitochondrial fractions. Pre-treatment with the phosphatase inhibitor okadaic acid prior to Oligo-treatment, stabilized full-length Parkin phosphorylated at Ser65, and reduced calpain-cleavage of Parkin. Treatment with the Ca++ ionophore A23187, which facilitates Ca++ transport across the plasma membrane, mimicked the effect of Oligo by inducing calpain-cleavage of Parkin. Removing extracellular Ca++ from the media prevented oligomycin- and ionophore-induced calpain-cleavage of Parkin. Computational analysis predicted that calpain-cleavage of Parkin liberates its UbL domain. The phosphagen cyclocreatine moderately mitigated Parkin cleavage by calpain. Moreover, the pituitary adenylate cyclase activating peptide (PACAP27), which stimulates cAMP production, prevented caspase but not calpain-cleavage of Parkin. Overall, our data support a link between Parkin phosphorylation and its cleavage by calpain. This mechanism reflects the impact of mitochondrial impairment and Ca++-dyshomeostasis on Parkin integrity and could influence PD pathogenesis.

Alternative Routes of Administration of the Neuroprotective Pituitary Adenylate Cyclase Activating Polypeptide.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with diverse actions, including strong neurotrophic and neuroprotective effects. The aim of our present review is to provide a summary of the different approaches how in vivo neuroprotective effects can be achieved, emphasizing the potential translational values for future therapeutic applications. In the central nervous system, PACAP has been shown to have in vivo protective effects in models of cerebral ischemia, Parkinson's and Alzheimer's disease, Huntington chorea, traumatic brain and spinal cord injury and different retinal pathologies. PACAP passes through the bloodbrain barrier and therefore, systemic administration can affect the nervous system and lead to neuroprotective effects. This review summarizes results obtained in neuronal injury studies via local, such as intracerebral, intrathecal, intracerebroventricular, intravitreal and systemic treatments, such as intravenous, intraperitoneal and subcutaneous administration of PACAP. A few other options are summarized, like intranasal and eye drops treatments, as well as difficulties and side effects of different treatments are also discussed.

PET Imaging of PARP Expression Using 18F-Olaparib.

502 Objectives: Urothelial Bladder Cancer (UBC) affects >80,000 new patients annually. Treatment is stage dependent, thus accurate staging with conventional imaging and biopsy are crucial, but are often inaccurate. To improve visualization of primary disease, the extent of loco-regional disease and the presence or absence of metastatic lesions, a large number of PET biomarkers have been developed. However serious limitations, including urinary excretion of radioactivity, compromises their ability to accurately assess the bladder lumen and invasive tumors. This ongoing investigation is aimed to validate a hypothesis that VPAC (combined for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP)) receptors expressed in high density on UBC cells can be targeted to PET image UBC, its loco-regional disease and metastatic lesions. Materials and Methods:64Cu-TP3805, shown in our laboratory to have high affinity (3.1 x 10-9 M) for VPAC was the imaging agent. Patients (M, N=15, 44-80 yrs.) diagnosed with invasive UBC scheduled for radical cystectomy were enrolled. Chemotherapy eligible patients were treated with standard of care cisplatin regimens. All subjects were administered 148±10% MBq 64Cu-TP3805 (20 µg) intravenously. Urine and blood samples were collected at predetermined time points. Patients then underwent PET/CT imaging 90 minutes later. Images were read independently by two nuclear medicine physicians. Radical cystoprostatectomy and lymphadenectomy was performed 1 to 4 weeks later, and VPAC PET images were correlated with final surgical pathology. Results: There were no adverse events. Blood clearance, t½ (∝), was 22.3 ± 2.7 min (> 85%) and t½ (β) was 118.2 ± 4.9 min (< 15%). Urinary excretion of radioactivity was negligible. As confirmed by post cystectomy histology, PET images were true positive in the bladder for UBC in 10 patients and true negative in 4. There was one false negative image. When examining the prostate, there was PET uptake in 12 patients. Of these, 5 patients had incidental prostate cancer or HGPIN; 2 patients had UCB involving the prostate; and 5 patients had negative histology in the prostate. PET image was true positive for one lymph node, true negative for 11 and false negative in 3. In one patient several bone lesions in iliac crest and spine were seen by PET imaging. Biopsy of iliac crest confirmed metastasis. These pilot data demonstrate 64Cu-TP3805 VPAC targeting for imaging UBC has 91% sensitivity (95% CI 59%-100%), 100% specificity (95% CI 40%-100%) , and has positive predictive and negative predictive values 100% (95% CI 69%-100) and 80% (95% CI 38%-96%), respectively. Conclusions: The results of this first-in-human study to image UBC by targeting VPAC receptor are encouraging. Further investigation continues. Acknowledgement: Supported in part by NIH, NCI R01 157372 (MLT) Dean’s Translational Science Award (MLT) and Sidney Kimmel Cancer Center Award (MLT).

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Reduces Oxidative and Mechanical Stress-Evoked Matrix Degradation in Chondrifying Cell Cultures.

Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide also secreted by non-neural cells, including chondrocytes. PACAP signaling is involved in the regulation of chondrogenesis, but little is known about its connection to matrix turnover during cartilage formation and under cellular stress in developing cartilage. We found that the expression and activity of hyaluronidases (Hyals), matrix metalloproteinases (MMP), and aggrecanase were permanent during the course of chondrogenesis in primary chicken micromass cell cultures, although protein levels changed daily, along with moderate and relatively constant enzymatic activity. Next, we investigated whether PACAP influences matrix destructing enzyme activity during oxidative and mechanical stress in chondrogenic cells. Exogenous PACAP lowered Hyals and aggrecanase expression and activity during cellular stress. Expression and activation of the majority of cartilage matrix specific MMPs such as MMP1, MMP7, MMP8, and MMP13, were also decreased by PACAP addition upon oxidative and mechanical stress, while the activity of MMP9 seemed not to be influenced by the neuropeptide. These results suggest that application of PACAP can help to preserve the integrity of the newly synthetized cartilage matrix via signaling mechanisms, which ultimately inhibit the activity of matrix destroying enzymes under cellular stress. It implies the prospect that application of PACAP can ameliorate articular cartilage destruction in joint diseases.

The PACAP-derived peptide MPAPO facilitates corneal wound healing by promoting corneal epithelial cell proliferation and trigeminal ganglion cell axon regeneration.

It is well known that the cornea plays an important role in providing protection to the eye, but it is fragile and vulnerable. To clarify the biological effects and molecular mechanisms of the pituitary adenylate cyclase activating polypeptide (PACAP)-derived peptide MPAPO (named MPAPO) to promote corneal wound healing, we applied a mechanical method to establish a corneal injury model and analyzed the repair effects of MPAPO on corneal injury. MPAPO significantly promoted corneal wound repair in C57BL/6 mice. In addition, we established injury models of epithelial cells and trigeminal ganglion cells with H2O2. The results show that when the concentration of MPAPO is 1 μM, it can significantly promote the repair of injured corneal epithelial cells and the regeneration of trigeminal ganglion cell axons. MPAPO repairs epithelial cells through the promotion of GSK3β phosphorylation by binding to PAC1 and activating AKT. β-catenin escapes the phosphorylation of GSK3β and enters the nucleus to promote the expression of cyclin D1, accelerate cell cycle progression and promote cell proliferation. MPAPO promotes axonal regeneration by binding to the PAC1 receptor and activating adenylate cyclase activity, followed by the cAMP activation of protein kinase A activity and the promotion of CREB phosphorylation. Phosphorylated CREB promotes Bcl2 expression and axonal regeneration. In conclusion, our data support the role of MPAPO to facilitate corneal wound healing by promoting corneal epithelial cell proliferation and trigeminal ganglion cell axon regeneration.

Assessment of Conformational State Transitions of Class B GPCRs Using Molecular Dynamics.

Class B G protein-coupled receptors (GPCRs) comprise a family of 15 peptide-binding members, which are crucial targets for endocrine, metabolic, and stress-related disorders. While their protein structures and dynamics remain largely unclear, computer modeling and simulations represent a promising means to help solve such puzzles. Herein, we present a basic introduction to the methodology of molecular dynamics (MD) simulations and two analytical methods to assess the conformational ensembles and transitions of Class B GPCRs, using our recent studies of the human pituitary adenylate cyclase activating polypeptide (PAC1) receptor as an example. From long MD simulations, conformational ensembles with different roles in ligand binding and receptor activation are sampled to establishfour states identified as either "open" or "closed" for the PAC1 receptor. Next, the dynamical network can be applied to analyze the simulations and identify key features within each conformational ensemble, which help distinguish the ligand-bound states of the PAC1 receptor from the ligand-free one. Further, the Markov State Model has emerged as a key approach to construct the transition network and connect the GPCR ensembles, providing detailed information for the transition pathways and kinetics. For the ligand-free PAC1 receptor, the transitions within the closed states are near 10-30 times faster than the open-closed transitions, which is likely related to the activation mechanism of the receptor. Overall, long MD simulations and analyses are useful to assess conformational transitions for the Class B GPCRs and to gain mechanistic insight, which is difficult to obtain using other methods.

PACAP and VIP expression in the periaqueductal grey of the rat following sciatic nerve constriction injury

Nerve injuries often result in neuropathic pain with co-morbid changes in social behaviours, motivation, sleep-wake cycles and neuroendocrine function. In an animal model of neuropathic injury (CCI) similar co-morbid changes are evoked in a subpopulation (~30%) of injured rats. In addition to anatomical evidence of altered neuronal and glial function, the periaqueductal grey (PAG) of these rats shows evidence of cell death. These changes in the PAG may play a role in the disruption of the normal emotional coping responses triggered by nerve injury. Cell death can occur via a number of mechanisms, including the disruption of neuroprotective mechanisms. Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two endogenous neuropeptides whose activities are tightly regulated by two receptors subtypes, namely the PAC1 and VPAC receptors. These peptides and their receptors exert robust neuroprotective roles. In these studies, we hypothesized that rats expressing disabilities following CCI showed altered expression of PACAP and VIP in the PAG. Rats were categorized as having either Pain alone, Transient or Persistent disability, based on changes in social behaviours pre- and post-CCI. Social interaction behavioural tested (BT), sham-injured and naïve untested rats were also included. For measurements of mRNA and protein expression we utilised micro-dissected PAGs blocks taken from each group. At the mRNA level, VIP was downregulated and PAC1 was upregulated in BT animals, whilst VPAC1 mRNA was specifically increased in the Pain alone group. Interestingly, protein levels of both PACAP and VIP were remarkably increased in the Persistent Disability group. Taken together, sciatic nerve CCI that triggers neuropathic pain and persistent disability results in abnormally increased VIP and PACAP expression in the PAG. Our data also suggest that these effects are likely to be governed by post-transcriptional mechanisms.

Retinoprotective Effects of TAT-Bound Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase Activating Polypeptide

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) belong to the same peptide family and exert a variety of biological functions. Both PACAP and VIP have protective effects in several tissues. While PACAP is known to be a stronger retinoprotective peptide, VIP has very potent anti-inflammatory effects. The need for a non-invasive therapeutic approach has emerged and PACAP has been shown to be retinoprotective when administered in the form of eye drops as well. The cell penetrating peptide TAT is composed of 11 amino acids and tagging of TAT at the C-terminus of neuropeptides PACAP/VIP can enhance the traversing ability of the peptides through the biological barriers. We hypothesized that TAT-bound PACAP and VIP could be more effective in exerting retinoprotective effects when given in eye drops, by increasing the traversing efficacy and enhancing the activation of the PAC1 receptor. Rats were subjected to bilateral carotid artery occlusion (BCCAO), and retinas were processed for histological analysis 14 days later. The efficiency of the TAT-bound peptides to reach the retina was assessed as well as their cAMP increasing ability. Our present study provides evidence, for the first time, that topically administered PACAP and VIP derivatives (PACAP-TAT and VIP-TAT) attenuate ischemic retinal degeneration via the PAC1 receptor presumably due to a multifactorial protective mechanism.

Activation of Serotonin 5-HT7 Receptors Modulates Hippocampal Synaptic Plasticity by Stimulation of Adenylate Cyclases and Rescues Learning and Behavior in a Mouse Model of Fragile X Syndrome.

We have previously demonstrated that activation of serotonin 5-HT7 receptors (5-HT7R) reverses metabotropic glutamate receptor-mediated long term depression (mGluR-LTD) in the hippocampus of wild-type (WT) and Fmr1 Knockout (KO) mice, a model of Fragile X Syndrome (FXS) in which mGluR-LTD is abnormally enhanced. Here, we have investigated intracellular mechanisms underlying the effect of 5-HT7R activation using patch clamp on hippocampal slices. Furthermore, we have tested whether in vivo administration of LP-211, a selective 5-HT7R agonist, can rescue learning and behavior in Fmr1 KO mice. In the presence of an adenylate cyclase blocker, mGluR-LTD was slightly enhanced in WT and therefore the difference between mGluR-LTD in WT and Fmr1 KO slices was no longer present. Conversely, activation of adenylate cyclase by either forskolin or Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) completely reversed mGluR-LTD in WT and Fmr1 KO. 5-HT7R activation reversed mGluR-LTD in WT and corrected exaggerated mGluR-LTD in Fmr1 KO; this effect was abolished by blockade of either adenylate cyclase or protein kinase A (PKA). Exposure of hippocampal slices to LP-211 caused an increased phosphorylation of extracellular signal regulated kinase (ERK), an intracellular effector involved in mGluR-LTD, in WT mice. Conversely, this effect was barely detectable in Fmr1 KO mice, suggesting that 5-HT7R-mediated reversal of mGluR-LTD does not require ERK stimulation. Finally, an acute in vivo administration of LP-211 improved novel object recognition (NOR) performance in WT and Fmr1 KO mice and reduced stereotyped behavior in Fmr1 KO mice. Our results indicate that mGluR-LTD in WT and Fmr1 KO slices is bidirectionally modulated in conditions of either reduced or enhanced cAMP formation. Activation of 5-HT7 receptors reverses mGluR-LTD by activation of the cAMP/PKA intracellular pathway. Importantly, a systemic administration of a 5-HT7R agonist to Fmr1 KO mice corrected learning deficits and repetitive behavior. We suggest that selective 5-HT7R agonists might become novel pharmacological tools for FXS therapy.

A transcriptomic investigation of appetite‐regulation and digestive processes in giant grouper Epinephelus lanceolatus during early larval development

The giant grouper Epinephelus lanceolatus is an ecologically vulnerable species with high market demand. However, efforts to improve larval husbandry are hindered by a lack of knowledge surrounding larval developmental physiology. To address this shortfall, a transcriptomic approach was applied to larvae between 1 and 14 days post hatch (dph) to characterise the molecular ontogenesis of genes that influence appetite and digestion. Appetite regulating factors were detected from 1 dph, including neuropeptide Y, nesfatin-1, cocaine and amphetamine regulated transcript, cholecystokinin and pituitary adenylate cyclase activating peptide and the expression level of several genes changed sharply with the onset of exogenous feeding. The level of expression for proteases, chitinases, lipases and amylases typically followed one of two expression patterns, a general increase as development progressed, or an inverted U-shape with maximal expression at c. 6 dph. Similarly, the tendency among both expression patterns was for the level of expression to increase around the time of mouth-opening. There was also evidence to suggest the presence of putative isoforms for several digestion-related genes. We have provided an insight into appetite-regulation and digestive processes in groupers during early larval development and have developed a transcriptomic database that will aid future efforts to rear this species in an aquaculture setting.

PAC1 regulates receptor tyrosine kinase transactivation in a reactive oxygen species-dependent manner

Pituitary adenylate cyclase activating polypeptide (PACAP) is a growth factor for lung cancer cells. PACAP-27 or PACAP-38 binds with high affinity to non-small cell lung cancer (NSCLC) cells, causing elevated cytosolic Ca2+, increased proliferation and increased phosphorylation of extracellular regulated kinase (ERK) and the epidermal growth factor receptor (EGFR). The role of reactive oxygen species (ROS) was investigated in these processes. Addition of PACAP-38 to NCI-H838 or A549 cells increased the tyrosine phosphorylation of the EGFR, HER2 and ERK significantly by 4-, 3-, and 2-fold, respectively. The transactivation of the EGFR and HER2 was inhibited by gefitinib or lapatinib (tyrosine kinase inhibitors), PACAP (6–38) (PAC1 antagonist), N-acetylcysteine (NAC is an anti-oxidant) or dipheyleneiodonium (DPI is an inhibitor of Nox and Duox enzymes). PACAP-38 addition to NSCLC cells increased ROS which was inhibited by PACAP (6–38), NAC or DPI. Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2 mRNA was present in many NSCLC cell lines. PACAP-38 stimulated the growth of NSCLC cells whereas PACAP (6–38), gefitinib or DPI inhibited proliferation. The results show that ROS are essential for PAC1 to regulate EGFR and HER2 transactivation as well as proliferation of NSCLC cells.

Effect of the H1-antihistamine clemastine on PACAP38 induced migraine.

To investigate the effect of the H1-antihistamine clemastine on the migraine-inducing abilities of pituitary adenylate cyclase activating peptide-38. We conducted a double-blind, randomized, placebo controlled two-way cross-over study. Twenty migraine without aura patients were randomly allocated to receive bolus clemastine 2 mg (1 mg/ml) or bolus saline 2 ml intravenously over 2 min on two study days. Following each bolus injection, 10 pmol/kg/min of pituitary adenylate cyclase activating peptide-38 was administered intravenously over 20 min. We recorded migraine/headache characteristics every 10 min until 90 min after the start of infusion, and collected blood to investigate mast cell degranulation and the inflammation markers tryptase and tumor necrosis factor-alpha before and after infusion of pituitary adenylate cyclase activating peptide-38. After clemastine pretreatment, five out of 20 participants developed a migraine-like attack in response to a pituitary adenylate cyclase activating peptide-38 infusion compared to nine out of 20 after placebo pretreatment ( p = 0.288). Following clemastine pretreatment, 15 out of 20 participants reported headache in response to a pituitary adenylate cyclase activating peptide-38 infusion, whereas 19 out of 20 participants did so following placebo pretreatment ( p = 0.221). We found no difference in area under the curve 12 h for headache intensity between the two experimental days ( p = 0.481). We found no difference in area under the curve 180 min for tryptase ( p = 0.525) or tumor necrosis factor-alpha ( p = 0.487) between clemastine and placebo pretreatment days. H1-antihistamine, clemastine, failed to prevent migraine or headache after pituitary adenylate cyclase activating peptide-38 infusion, thus making a role for histamine release or mast cell degranulation in pituitary adenylate cyclase activating peptide-38-induced migraine less likely.

Signalling Alterations in Bones of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Gene Deficient Mice.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with diverse developmental roles, including differentiation of skeletal elements. It is a positive regulatory factor of chondrogenesis and osteogenic differentiation in vitro, but little is known about its in vivo role in bone formation. In our experiments, diaphyses of long bones from hind limbs of PACAP gene-deficient mice showed changes in thickness and increased staining intensity. Our main goal was to perform a detailed morphological and molecular biological analysis of femurs from PACAP knockout (KO) and wild type (WT) mice. Transverse diameter and anterior cortical bone thickness of KO femurs showed significant alterations with disturbed Ca2+ accumulation and collagen type I expression. Higher expression and activity of alkaline phosphatase were also observed, accompanied by increased fragility PACAP KO femurs. Increased expression of the elements of bone morphogenic protein (BMP) and hedgehog signalling was also observed, and are possibly responsible for the compensation mechanism accounting for the slight morphological changes. In summary, our results show that lack of PACAP influences molecular and biomechanical properties of bone matrix, activating various signalling cascade changes in a compensatory fashion. The increased fragility of PACAP KO femur further supports the role of endogenous PACAP in in vivo bone formation.

Effect of PACAP on Bacterial Adherence and Cytokine Expression in Intestinal Cell Cultures

Bacterial adhesion is a crucial event of intestinal pathological conditions evoked by bacterial infections. Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide having a widespread distribution throughout the entire body including the digestive tract. It has diverse physiological functions in the gastrointestinal system, including protective effects in several models of intestinal inflammatory conditions. However, its effects on bacterial adherence and the inflammatory reactions as a result of that have not been elucidated yet. The aim of our study was therefore to investigate the effect of PACAP on bacterial adherence and cytokine expression upon lipopolysaccharide (LPS) exposure. Small intestinal INT407 and colonic Caco-2 cells were treated with PACAP prior to exposure to bacteria (Escherichia coli, Salmonella Typhimurium, Klebsiella pneumoniae, Enterococcus faecalis) and colonies were counted. PACAP had no significant influence on bacterial adhesion, as it did not change the number of colonies of investigated bacteria. However, PACAP was able to counteract the LPS-induced increases in the expression of the cytokines IL-8 and CXCL-1 in INT407 cells, as assessed by cytokine array. These results indicate that while PACAP has no direct effect on bacterial adherence, it can influence the cytokine expression of intestinal cells upon endotoxin-induced exposure, possibly contributing to the known anti-inflammatory actions of PACAP in the intestinal system.

PACAP38 and PAC1 receptor blockade: a new target for headache?

Pituitary adenylate cyclase activating polypeptide-38 (PACAP38) is a widely distributed neuropeptide involved in neuroprotection, neurodevelopment, nociception and inflammation. Moreover, PACAP38 is a potent inducer of migraine-like attacks, but the mechanism behind this has not been fully elucidated.Migraine is a neurovascular disorder, recognized as the second most disabling disease. Nevertheless, the antibodies targeting calcitonin gene-related peptide (CGRP) or its receptor are the only prophylactic treatment developed specifically for migraine. These antibodies have displayed positive results in clinical trials, but are not effective for all patients; therefore, new pharmacological targets need to be identified.Due to the ability of PACAP38 to induce migraine-like attacks, its location in structures previously associated with migraine pathophysiology and the 100-fold selectivity for the PAC1 receptor when compared to VIP, new attention has been drawn to this pathway and its potential role as a novel target for migraine treatment. In accordance with this, antibodies against PACAP38 (ALD 1910) and PAC1 receptor (AMG 301) are being developed, with AMG 301 already in Phase II clinical trials. No results have been published so far, but in preclinical studies, AMG 301 has shown responses comparable to those observed with triptans. If these antibodies prove to be effective for the treatment of migraine, several considerations should be addressed, for instance, the potential side effects of long-term blockade of the PACAP (receptor) pathway. Moreover, it is important to investigate whether these antibodies will indeed represent a therapeutic advantage for the patients that do not respond the CGRP (receptor)-antibodies.In conclusion, the data presented in this review indicate that PACAP38 and PAC1 receptor blockade are promising antimigraine therapies, but results from clinical trials are needed in order to confirm their efficacy and side effect profile.

PAC1 Receptors: Shapeshifters in Motion.

Shapeshifters, in common mythology, are entities that can undergo multiple physical transformations. As our understanding of G protein-coupled receptors (GPCRs) has accelerated and been refined over the last two decades, we now understand that GPCRs are not static proteins, but rather dynamic structures capable of moving from one posture to the next, and adopting unique functional characteristics at each transition. This model of GPCR dynamics underlies our current understanding of biased agonism-how different ligands to the same receptor can generate different intracellular signals-and constitutive receptor activity, or the level of unbound basal receptor signaling that can be attenuated by inverse agonists. From information derived from related class B receptors, we have recently modeled the structure and molecular dynamics of the full-length pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1)-selective PAC1 receptor (PAC1R, Adcyap1r1). The class B receptors are different from the class A GPCRs in part from the presence of a large extracellular domain (ECD); the transitions of the ECD along with the dynamics of the transmembrane domains (TMD or 7TM) of the PAC1R describes a series of open- and closed-state conformations that appear to identify the mechanisms for receptor activation. The PAC1R shapeshifts also have the ability of delineating the mechanisms and the design of reagents that may direct biased agonism (or antagonism) for potential therapeutics.

PACAP neurons in the ventral premammillary nucleus regulate reproductive function in the female mouse.

Pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) is a neuromodulator implicated in anxiety, metabolism and reproductive behavior. PACAP global knockout mice have decreased fertility and PACAP modulates LH release. However, its source and role at the hypothalamic level remain unknown. We demonstrate that PACAP-expressing neurons of the ventral premamillary nucleus of the hypothalamus (PMVPACAP) project to, and make direct contact with, kisspeptin neurons in the arcuate and AVPV/PeN nuclei and a subset of these neurons respond to PACAP exposure. Targeted deletion of PACAP from the PMV through stereotaxic virally mediated cre- injection or genetic cross to LepR-i-cre mice with Adcyap1fl/fl mice led to delayed puberty onset and impaired reproductive function in female, but not male, mice. We propose a new role for PACAP-expressing neurons in the PMV in the relay of nutritional state information to regulate GnRH release by modulating the activity of kisspeptin neurons, thereby regulating reproduction in female mice.