The hypothalamus is involved in the regulation of homeostatic mechanisms and migraine-related trigeminal nociception and as such has been hypothesized to play a central role in the migraine syndrome ...from the earliest stages of the attack. The hypothalamus hosts many key neuropeptide systems that have been postulated to play a role in this pathophysiology. Such neuropeptides include but are not exclusive too orexins, oxytocin, neuropeptide Y, and pituitary adenylate cyclase activating protein, which will be the focus of this review. Each of these peptides has its own unique physiological role and as such many preclinical studies have been conducted targeting these peptide systems with evidence supporting their role in migraine pathophysiology. Preclinical studies have also begun to explore potential therapeutic compounds targeting these systems with some success in all cases. Clinical efficacy of dual orexin receptor antagonists and intranasal oxytocin have been tested; however, both have yet to demonstrate clinical effect. Despite this, there were limitations in these cases and strong arguments can be made for the further development of intranasal oxytocin for migraine prophylaxis. Regarding neuropeptide Y, work has yet to begun in a clinical setting, and clinical trials for pituitary adenylate cyclase activating protein are just beginning to be established with much optimism. Regardless, it is becoming increasingly clear the prominent role that the hypothalamus and its peptide systems have in migraine pathophysiology. Much work is required to better understand this system and the early stages of the attack to develop more targeted and effective therapies aimed at reducing attack susceptibility with the potential to prevent the attack all together.
Pituitary adenylate cyclase-activating polypeptide (PACAP-38) is a common neuropeptide exerting a wide spectrum of functions in many fields, including immunology. In the present study, 5-day ...post-fertilization (dpf) zebrafish larvae of three diverse genetic lines transgenic lines Tg(MPX:GFP) with GFP-labelled neutrophils and Tg(pou4f3:GAP-GFP) with GFP-labelled hair cells and the wild-type Tuebingen were used to investigate an inhibitory role of PACAP-38 in inflammation associated with damaged hair cells of the lateral line. Individuals of each genetic line were assigned to four groups: (1) control, and those consisting of larvae exposed to (2) 10 µM CuSO4, (3) 10 µM CuSO4+100 nM PACAP-38 and (4) 100 nM PACAP-38, respectively. Forty-minute exposure to CuSO4 solution was applied to evoke necrosis of hair cells and consequent inflammation. The inhibitory role of PACAP-38 was investigated in vivo under a confocal microscope by counting neutrophils migrating towards damaged hair cells in Tg(MPX:GFP) larvae. In CuSO4-treated individuals, the number of neutrophils associated with hair cells was dramatically increased, while PACAP-38 co-treatment resulted in its over 2-fold decrease. However, co-treatment with PACAP-38 did not prevent hair cells from extensive necrosis, which was found in Tg(pou4f3:GAP-GFP) individuals. Real-Time PCR analysis performed in wild-type larvae demonstrated differential expression pattern of stress and inflammation inducible markers. The most significant findings showed that CuSO4 exposure up-regulated the expression of IL-8, IL-1β, IL-6 and ATF3, while after PACAP-38 co-treatment expression levels of these genes were significantly decreased. The presence of transcripts for all PACAP receptors in neutrophils was also revealed. Adcyap1r1a and vipr1b appeared to be predominant forms. The present results suggest that PACAP-38 should be considered as a factor playing an important regulatory role in inflammatory response associated with pathological processes affecting zebrafish hair cells and it cannot be excluded that this interesting property has more universal significance.
The acylated pore-forming Repeats in ToXin (RTX) cytolysins α-hemolysin (HlyA) and adenylate cyclase toxin (CyaA) preferentially bind to β2 integrins of myeloid leukocytes but can also promiscuously ...bind and permeabilize cells lacking the β2 integrins. We constructed a HlyA1–563/CyaA860–1706 chimera that was acylated either by the toxin-activating acyltransferase CyaC, using sixteen carbon-long (C16) acyls, or by the HlyC acyltransferase using fourteen carbon-long (C14) acyls. Cytolysin assays with the C16- or C14-acylated HlyA/CyaA chimeric toxin revealed that the RTX domain of CyaA can functionally replace the RTX domain of HlyA only if it is modified by C16-acyls on the Lys983 residue of CyaA. The C16-monoacylated HlyA/CyaA chimera was as pore-forming and cytolytic as native HlyA, whereas the C14-acylated chimera exhibited very low pore-forming activity. Hence, the capacity of the RTX domain of CyaA to support the insertion of the N-terminal pore-forming domain into the target cell membrane, and promote formation of toxin pores, strictly depends on the modification of the Lys983 residue by an acyl chain of adapted length.
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•A palmitoylated HlyA/CyaA chimera is as cytolytic as myristoylated HlyA toxin.•Myristoylation (C14-acylation) fails to activate the HlyA/CyaA chimera.•Palmitoylated (C16-acylated) HlyA/CyaA chimera forms HlyA-like pores.•This HlyA/CyaA chimera binds the β2 integrin CR3 (CD11b/CD18).
PAC1 receptor is abundant in the CNS and plays an important role in neuronal survival. To identify the molecular determinants and the conformational components responsible for the activation of the ...PAC1 receptor, we performed a SAR study focusing on the N-terminal domain of its endogenous ligand, PACAP. This approach revealed that residues Asp3 and Phe6 are key elements of the pharmacophore of the PAC1 receptor. This study, supported by NMR structural analyses, suggests that the N-terminal tail of PACAP (residues 1 to 4) adopts a specific conformation similar to a turn when it activates the PAC1 receptor. Moreover, the integrity of the α-helix conformation observed at positions 5 to 7 appears crucial to allow the binding of PACAP. Characterization of analogues led to the identification of several superagonists, such as Bip6PACAP27, and of a new potent PAC1 receptor antagonist, Sar4PACAP38. The bioactive conformation inferred from this SAR study could constitute an appropriate molecular scaffold supporting the design of nonpeptidic PAC1 receptor agonists.
Obesity arises from disrupted energy balance and is caused by chronically higher energy intake compared to expenditure via basal metabolic rate, exercise, and thermogenesis. The brown adipose tissue ...(BAT), the primary thermogenic organ, has received considerable attention as a potential therapeutic target due to its ability to burn lipids in the production of heat. Pituitary adenylate cyclase-activating polypeptide (PACAP) has been identified as a key regulator of the physiological stress response both centrally and peripherally. While PACAP has been shown to increase thermogenesis by acting at the hypothalamus to increase sympathetic output to BAT, a peripheral role for PACAP-activated thermogenesis has not been studied. We identified PACAP receptor (PAC1, VPAC1/2) expression for the first time in murine BAT and confirmed their expression in white adipose tissues. PAC1 receptor expression was significantly altered in all three adipose tissues studied in response to 3.5-week cold acclimation, with expression patterns differing by depot type. In primary cell culture, VPAC1 was increased in differentiated compared to non-differentiated brown adipocytes, and the same trend was observed for the PACAP-specific receptor PAC1 in gonadal white fat primary cultures. The primary PAC1R mRNA splice variant in interscapular BAT was determined as isoform 2 by RNA-Seq. These results show that PACAP receptors are present in adipose tissues and may have important functional roles in adipocyte differentiation, lipid metabolism, or adipose sensitization to sympathetic signaling in response to thermogenic stimuli.
The airway epithelium restricts the penetration of inhaled pathogens into the underlying tissue and plays a crucial role in the innate immune defense against respiratory infections. The whooping ...cough agent,
, adheres to ciliated cells of the human airway epithelium and subverts its defense functions through the action of secreted toxins and other virulence factors. We examined the impact of
infection and of adenylate cyclase toxin-hemolysin (CyaA) action on the functional integrity of human bronchial epithelial cells cultured at the air-liquid interface (ALI).
adhesion to the apical surface of polarized pseudostratified VA10 cell layers provoked a disruption of tight junctions and caused a drop in transepithelial electrical resistance (TEER). The reduction of TEER depended on the capacity of the secreted CyaA toxin to elicit cAMP signaling in epithelial cells through its adenylyl cyclase enzyme activity. Both purified CyaA and cAMP-signaling drugs triggered a decrease in the TEER of VA10 cell layers. Toxin-produced cAMP signaling caused actin cytoskeleton rearrangement and induced mucin 5AC production and interleukin-6 (IL-6) secretion, while it inhibited the IL-17A-induced secretion of the IL-8 chemokine and of the antimicrobial peptide beta-defensin 2. These results indicate that CyaA toxin activity compromises the barrier and innate immune functions of
infected airway epithelia.
PACAP and its role in primary headaches Edvinsson, Lars; Tajti, János; Szalárdy, Levente ...
Journal of headache and pain,
03/2018, Letnik:
19, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide implicated in a wide range of functions, such as nociception and in primary headaches. Regarding its localization, PACAP has ...been observed in the sensory trigeminal ganglion (TG), in the parasympathetic sphenopalatine (SPG) and otic ganglia (OTG), and in the brainstem trigeminocervical complex. Immunohistochemistry has shown PACAP-38 in numerous cell bodies of SPG/OTG, co-stored with vasoactive intestinal peptide (VIP), nitric oxide synthase (NOS) and, to a minor degree, with choline acetyltransferase. PACAP has in addition been found in a subpopulation of calcitonin gene-related peptide (CGRP)-immunoreactive cells in the trigeminal system. The PACAP/VIP receptors (PAC
1
, VPAC
1
, and VPAC
2
) are present in sensory neurons and in vascular smooth muscle related to the trigeminovascular system. It is postulated that PACAP is involved in nociception. In support, abolishment of PACAP synthesis or reception leads to diminished pain responses, whereas systemic PACAP-38 infusion triggers pain behavior in animals and delayed migraine-like attacks in migraine patients without marked vasodilatory effects. In addition, increased plasma levels have been documented in acute migraine attacks and in cluster headache, in accordance with findings in experimental models of trigeminal activation. This suggest that the activation of the trigeminal system may result in elevated venous levels of PACAP, a change that can be reduced when headache is treated. The data presented in this review indicate that PACAP and its receptors may be promising targets for migraine therapeutics.
Migraine is the seventh most disabling disorder globally, with prevalence of 11.7% worldwide. One of the prevailing mechanisms is the activation of the trigeminovascular system, and calcitonin ...gene-related peptide (CGRP) is an important therapeutic target for migraine in this system. Recent studies suggested an emerging role of pituitary adenylate cyclase-activating peptide (PACAP) in migraine. However, the relation between CGRP and PACAP and the role of PACAP in migraine remain undefined. In this study, we established a novel repetitive (one, three, and seven days) electrical stimulation model by stimulating dura mater in conscious rats. Then, we determined expression patterns in the trigeminal ganglion and the trigeminal nucleus caudalis of the trigeminovascular system. Electrical stimulation decreased facial mechanical thresholds, and the order of sensitivity was as follows: vibrissal pad >inner canthus >outer canthus (P < 0.001). The electrical stimulation group exhibited head-turning and head-flicks (P < 0.05) nociceptive behaviors. Importantly, electrical stimulation increased the expressions of CGRP, PACAP, and the PACAP-preferring type 1 (PAC1) receptor in both trigeminal ganglion and trigeminal nucleus caudalis (P < 0.05). The expressions of two vasoactive intestinal peptide (VIP)-shared type 2 (VPAC1 and VPAC2) receptors were increased in the trigeminal ganglion, whereas in the trigeminal nucleus caudalis, their increases were peaked on Day 3 and then decreased by Day 7. PACAP was colocalized with NEUronal Nuclei (NeuN), PAC1, and CGRP in both trigeminal ganglion and the trigeminal nucleus caudalis. Our results demonstrate that the repetitive electrical stimulation model can simulate the allodynia during the migraine chronification, and PACAP plays a role in the pathogenesis of migraine potentially via PAC1 receptor.
Repeats-in-Toxin (RTX) proteins of Gram-negative bacteria are excreted through the type I secretion system (T1SS) that recognizes non-cleavable C-terminal secretion signals. These are preceded by ...arrays of glycine and aspartate-rich nonapeptide repeats grouped by four to eight β strands into blocks that fold into calcium-binding parallel β-roll structures. The β-rolls are interspersed by linkers of variable length and sequence and the organization of multiple RTX repeat blocks within large RTX domains remains unknown. Here we examined the structure and function of the RTX domain of Bordetella pertussis adenylate cyclase toxin (CyaA) that is composed of five β-roll RTX blocks. We show that the non-folded RTX repeats maintain the stability of the CyaA polypeptide in the Ca2+-depleted bacterial cytosol and thereby enable its efficient translocation through the T1SS apparatus. The efficacy of secretion of truncated CyaA constructs was dictated by the number of retained RTX repeat blocks and depended on the presence of extracellular Ca2+ ions. We further describe the crystal structure of the RTX blocks IV–V of CyaA (CyaA1372–1681) that consists of a contiguous assembly of two β-rolls that differs substantially from the arrangement of the RTX blocks observed in RTX lipases or other RTX proteins. These results provide a novel structural insight into the architecture of the RTX domains of large RTX proteins and support the “push-ratchet” mechanism of the T1SS-mediated secretion of very large RTX proteins.
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•Calcium-binding RTX repeats operate in a dual structure–function regime.•The non-folded RTX repeats maintain the stability of the T1SS substrates.•The number of RTX repeats dictates the secretion efficacy of large RTX substrates.•The RTX domain of the CyaA toxin forms an extended parallel β-roll structure.
Chronic pain and stress-related psychopathologies, such as depression and anxiety-associated abnormalities, are mutually reinforcing; however, the neuronal circuits and mechanisms that underlie this ...reinforcement are still not well understood. Pituitary adenylate cyclase-activating polypeptide (PACAP; Adcyap1) and its cognate PAC1 receptor (Adcyap1r1) are expressed in peripheral nociceptive pathways, participate in anxiety-related responses and have been have been linked to posttraumatic stress disorder and other mental health afflictions.
Using immunocytochemistry, pharmacological treatments and behavioral testing techniques, we have used a rodent partial sciatic nerve chronic constriction injury model (n = 5–8 per group per experiment) to evaluate PACAP plasticity and signaling in nociceptive and stress-related behaviors.
We show that chronic neuropathic pain increases PACAP expression at multiple tiers along the spinoparabrachioamygdaloid tract. Furthermore, chronic constriction injury bilaterally augments nociceptive amygdala (in the central nucleus of the amygdala CeA) PACAP immunoreactivity, extracellular signal–regulated kinase phosphorylation, and c-Fos activation, in parallel with heightened anxiety-like behavior and nociceptive hypersensitivity. Acute CeA infusions with the PACAP receptor antagonist PACAP(6-38) blocked chronic constriction injury–induced behavioral responses. Additionally, pretreatments with inhibitors of mitogen-activated protein kinase enzymes or endocytosis to block endosomal PACAP receptor extracellular signal–regulated kinase signaling attenuated PACAP-induced CeA neuronal activation and nociceptive responses.
Our data suggest that chronic pain-induced PACAP neuroplasticity and signaling in spinoparabrachioamygdaloid projections have an impact on CeA stress- and nociception-associated maladaptive responses, which can be ameliorated upon receptor antagonism even during injury progression. Thus, the PACAP pathway provides for an important mechanism underlying the intersection of stress and chronic pain pathways via the amygdala.
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