Circulating PACAP levels are associated with altered imaging measures of entorhinal cortex neurite density in posttraumatic stress disorder.

Introduction: Pituitary adenylate cyclase-activating polypeptide (PACAP) regulates plasticity in brain systems underlying arousal and memory and is associated with posttraumatic stress disorder (PTSD). Research in animal models suggests that PACAP modulates entorhinal cortex (EC) input to the hippocampus, contributing to impaired contextual fear conditioning. In PTSD, PACAP is associated with higher activity of the amygdala to threat stimuli and lower functional connectivity of the amygdala and hippocampus. However, PACAP-affiliated structural alterations of these regions have not been investigated in PTSD. Here, we examined whether peripheral PACAP levels were associated with neuronal morphology of the amygdala and hippocampus (primary analyses), and EC (secondary) using Neurite Orientation Dispersion and Density Imaging.Methods: Sixty-four (44 female) adults (19 to 54 years old) with DSM-5 Criterion A trauma exposure completed the Clinician-Administered PTSD Scale (CAPS-5), a blood draw, and magnetic resonance imaging. PACAP38 radioimmunoassay was performed and T1-weighted and multi-shell diffusion-weighted images were acquired. Neurite Density Index (NDI) and Orientation Dispersion Index (ODI) were quantified in the amygdala, hippocampus, and EC. CAPS-5 total score and anxious arousal score were used to test for clinical associations with brain structure.Results: Higher PACAP levels were associated with greater EC NDI (ß = 0.0099, q = 0.032) and lower EC ODI (ß = -0.0073, q = 0.047), and not hippocampal or amygdala measures. Neither EC NDI nor ODI was associated with clinical measures.Conclusions: Circulating PACAP levels were associated with altered neuronal density of the EC but not the hippocampus or amygdala. These findings strengthen evidence that PACAP may impact arousal-associated memory circuits in PTSD.

PACAP was associated with altered entorhinal cortex neurite density in PTSD.PACAP was not associated with altered neurite density in amygdala or hippocampus.PACAP may impact arousal-associated memory circuits.

Circulating PACAP levels are associated with altered imaging measures of entorhinal cortex neurite density in posttraumatic stress disorder.

Background: Pituitary adenylate cyclase-activating polypeptide (PACAP) regulates plasticity in brain systems underlying arousal and memory and is associated with posttraumatic stress disorder (PTSD). Research in animal models suggests that PACAP modulates entorhinal cortex (EC) input to the hippocampus, contributing to impaired contextual fear conditioning. In PTSD, PACAP is associated with higher activity of the amygdala to threat stimuli and lower functional connectivity of the amygdala and hippocampus. However, PACAP-affiliated structural alterations of these regions have not been reported. Here, we examined whether peripheral PACAP levels were associated with neuronal morphology of the amygdala and hippocampus (primary analysis), and EC (secondary analysis) using Neurite Orientation Dispersion and Density Imaging. Methods: Sixty-four (44 female) adults (19 to 54 years old) with DSM-5 Criterion A trauma exposure completed the Clinician-Administered PTSD Scale (CAPS-5), a blood draw, and magnetic resonance imaging. PACAP38 radioimmunoassay was performed and T1-weighted and multi-shell diffusion- weighted images were acquired. Neurite Density Index (NDI) and Orientation Dispersion Index (ODI) were quantified in the amygdala, hippocampus, and EC. CAPS-5 total score and anxious arousal score were used to test for clinical associations with brain structure. Results: Higher PACAP levels in blood were associated with greater EC NDI (ß=0.31, q=0.034) and lower EC ODI (ß=-0.30, q=0.042) and not hippocampal or amygdala measures. Neither EC NDI nor ODI was associated with clinical measures. Conclusions: Circulating PACAP levels were associated with altered neuronal density of the EC but not hippocampus or amygdala. These findings strengthen evidence that PACAP may impact arousal- associated memory circuits.

Circulating PACAP levels are associated with increased amygdala-default mode network resting-state connectivity in posttraumatic stress disorder.

The pituitary adenylate cyclase-activating polypeptide (PACAP) system is implicated in posttraumatic stress disorder (PTSD) and related amygdala-mediated arousal and threat reactivity. PTSD is characterized by increased amygdala reactivity to threat and, more recently, aberrant intrinsic connectivity of the amygdala with large-scale resting state networks, specifically the default mode network (DMN). While the influence of PACAP on amygdala reactivity has been described, its association with intrinsic amygdala connectivity remains unknown. To fill this gap, we examined functional connectivity of resting-state functional magnetic resonance imaging (fMRI) in eighty-nine trauma-exposed adults (69 female) screened for PTSD symptoms to examine the association between blood-borne (circulating) PACAP levels and amygdala-DMN connectivity. Higher circulating PACAP levels were associated with increased amygdala connectivity with posterior DMN regions, including the posterior cingulate cortex/precuneus (PCC/Precun) and left angular gyrus (lANG). Consistent with prior work, this effect was seen in female, but not male, participants and the centromedial, but not basolateral, subregions of the amygdala. Clinical association analyses linked amygdala-PCC/Precun connectivity to anxious arousal symptoms, specifically exaggerated startle response. Taken together, our findings converge with previously demonstrated effects of PACAP on amygdala activity in PTSD-related processes and offer novel evidence for an association between PACAP and intrinsic amygdala connectivity patterns in PTSD. Moreover, these data provide preliminary evidence to motivate future work ascertaining the sex- and subregion-specificity of these effects. Such findings may enable novel mechanistic insights into neural circuit dysfunction in PTSD and how the PACAP system confers risk through a disruption of intrinsic resting-state network dynamics.

GPCR Intracellular Loop Regulation of Beta-Arrestin-Mediated Endosomal Signaling Dynamics.

G protein-coupled receptors (GPCRs) are currently appreciated to be routed to diverse cellular platforms to generate both G protein-dependent and -independent signals. The latter has been best studied with respect to ß-arrestin-associated receptor internalization and trafficking to signaling endosomes for extracellular signal-regulated kinase (ERK) activation. However, how GPCR structural and conformational variants regulate endosomal ERK signaling dynamics, which can be central in neural development, plasticity, and disease processes, is not well understood. Among class B GPCRs, the PACAP-selective PAC1 receptor is unique in the expression of variants that can contain intracellular loop 3 (ICL3) cassette inserts. The nervous system expresses preferentially the PAC1Null (no insert) and PAC1Hop (28-amino acid Hop insert) receptor variants. Our molecular modeling and signaling studies revealed that the PAC1Null and PAC1Hop receptor variants can associate with ß-arrestin differentially, resulting in enhanced receptor internalization and ERK activation for the PAC1Hop variant. The study amplifies our understandings of GPCR intracellular loop structure/function relationships with the first example of how the duration of endosomal ERK activation can be guided by ICL3. The results provide a framework for how changes in GPCR variant expression can impact developmental and homeostatic processes and may be contributory to maladaptive neuroplasticity underlying chronic pain and stress-related disorders.

Activation of Lateral Parabrachial Nucleus (LPBn) PACAP-Expressing Projection Neurons to the Bed Nucleus of the Stria Terminalis (BNST) Enhances Anxiety-like Behavior.

Anxiety disorders are among the most common psychiatric disorders, and understanding the underlying neurocircuitry of anxiety- and stress-related behaviors may be important for treatment. The bed nucleus of the stria terminalis (BNST) has been studied for its role in many stress-related pathologies, such as anxiety, pain, depression, and addiction. Our prior work has demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) receptor activation in the BNST mediates many of the behavioral consequences of chronic stress. While the BNST contains local PACAP-expressing neurons, a major source of afferent PACAP is the lateral parabrachial nucleus (LPBn), and excitotoxic lesions of the LPBn substantially decreasess PACAP immunostaining in the BNST. Here, we first assessed Cre-dependent reporter expression by injecting AAV2-hSyn-DIO-mCherry into the LPBn of PACAP-IRES-Cre mice for circuit mapping studies and identified PACAP projections to the BNST, lateral capsular central nucleus of the amygdala (CeLC), and ventromedial hypothalamus (VMH). In a second study, we assessed the effects of chemogenetically activating LPBn PACAP afferents in the BNST by injecting AAV2-hSyn-DIO-hM3D(Gq)-mCherry into the LPBn of PACAP-IRES-Cre mice for Cre-dependent expression of excitatory designer receptors exclusively activated by designer drugs (DREADDs). Before behavioral testing, clozapine-N-oxide (CNO), the selective agonist of our DREADD, was infused directly into the BNST. We found that after specific activation of LPBn PACAP afferents in the BNST, mice had increased anxiety-like behavior compared with controls, while total locomotor activity was unaffected. These results indicate that activation of PACAPergic LPBn projections to the BNST may play an important role in producing anxiety-like behavior.

Chemoarchitecture of the bed nucleus of the stria terminalis: Neurophenotypic diversity and function.

The bed nucleus of the stria terminalis (BNST) is a compact but neurophenotypically complex structure in the ventral forebrain that is structurally and functionally linked to other limbic structures, including the amygdala nuclear complex, hypothalamic nuclei, hippocampus, and related midbrain structures, to participate in a wide range of functions, especially emotion, emotional learning, stress-related responses, and sexual behaviors. From a variety of sensory inputs, the BNST acts as a node for signal integration and coordination for information relay to downstream central neuroendocrine and autonomic centers for appropriate homeostatic physiological and behavioral responses. In contrast to the role of the amygdala in fear, the BNST has gained wide interest from work suggesting that it has main roles in mediating sustained responses to diffuse, unpredictable and/or long-duration threats that are typically associated with anxiety-related responses. Further, some BNST subregions are highly sexually dimorphic which appear contributory to the differential stress and social interactive behaviors, including reproductive responses, between males and females. Notably, maladaptive BNST neuroplasticity and function have been implicated in chronic pain, depression, anxiety-related abnormalities, and other psychopathologies including posttraumatic stress disorders. The BNST circuits are predominantly GABAergic-the glutaminergic neurons represent a minor population-but the complexity of the system results from an overlay of diverse neuropeptide coexpression in these neurons. More than a dozen neuropeptides may be differentially coexpressed in BNST neurons, and from variable G protein-coupled receptor signaling, may inhibit or activate downstream circuit activities. The mechanisms and roles of these peptides in modulating intrinsic BNST neurocircuit signaling and BNST long-distance target cell projections are still not well understood. Nevertheless, an understanding of some of the principal players may allow assembly of the circuit interactions.

Molecular Basis of Class B GPCR Selectivity for the Neuropeptides PACAP and VIP.

The related neuropeptides PACAP and VIP, and their shared PAC1, VPAC1 and VPAC2 receptors, regulate a large array of physiological activities in the central and peripheral nervous systems. However, the lack of comparative and molecular mechanistic investigations hinder further understanding of their preferred binding selectivity and function. PACAP and VIP have comparable affinity at the VPAC1 and VPAC2 receptor, but PACAP is 400-1,000 fold more potent than VIP at the PAC1 receptor. A molecular understanding of the differing neuropeptide-receptor interactions and the details underlying the receptor transitions leading to receptor activation are much needed for the rational design of selective ligands. To these ends, we have combined structural information and advanced simulation techniques to study PACAP/VIP binding selectivity, full-length receptor conformation ensembles and transitions of the PACAP/VIP receptor variants and subtypes, and a few key interactions in the orthosteric-binding pocket. Our results reveal differential peptide-receptor interactions (at the atomistic detail) important for PAC1, VPAC1 and VPAC2 receptor ligand selectivity. Using microsecond-long molecular dynamics simulations and the Markov State Models, we have also identified diverse receptor conformational ensembles and microstate transition paths for each receptor, the potential mechanisms underlying receptor open and closed states, and the interactions and dynamics at the transmembrane orthosteric pocket for receptor activation. These analyses reveal important features in class B GPCR structure-dynamics-function relationships, which provide novel insights for structure-based drug discovery.

PACAP orchestration of stress-related responses in neural circuits.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic polypeptide that can activate G protein-coupled PAC1, VPAC1, and VPAC2 receptors, and has been implicated in stress signaling. PACAP and its receptors are widely distributed throughout the nervous system and other tissues and can have a multitude of effects. Human and animal studies suggest that PACAP plays a role responding to a variety of threats and stressors. Here we review the roles of PACAP in several regions of the central nervous system (CNS) as they relate to several behavioral functions. For example, in the bed nucleus of the stria terminalis (BNST), PACAP is upregulated following chronic stress and may drive anxiety-like behavior. PACAP can also influence both the consolidation and expression of fear memories, as demonstrated by studies in several fear-related areas, such as the amygdala, hippocampus, and prefrontal cortex. PACAP can also mediate the emotional component of pain, as PACAP in the central nucleus of the amygdala (CeA) is able to decrease pain sensitivity thresholds. Outside of the central nervous system, PACAP may drive glucocorticoid release via enhanced hypothalamic-pituitary-adrenal axis activity and may participate in infection-induced stress responses. Together, this suggests that PACAP exerts effects on many stress-related systems and may be an important driver of emotional behavior.

PAC1 Receptor Internalization and Endosomal MEK/ERK Activation Is Essential for PACAP-Mediated Neuronal Excitability.

Pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) activation of PAC1 receptors (Adcyap1r1) can significantly increase the excitability of diverse neurons through differential mechanisms. For guinea pig cardiac neurons, the modulation of excitability can be mediated in part by PAC1 receptor plasma membrane G protein-dependent activation of adenylyl cyclase and downstream signaling cascades. By contrast, PAC1 receptor-mediated excitability of hippocampal dentate gyrus granule cells appears independent of membrane-delimited AC/cAMP/PKA and PLC/PKC signaling. For both neuronal types, there is mechanistic convergence demonstrating that endosomal PAC1 receptor signaling has prominent roles. In these models, neuronal exposure to Pitstop2 to inhibit ß-arrestin/clathrin-mediated PAC1 receptor internalization eliminates PACAP modulation of excitability. ß-arrestin is a scaffold for a number of effectors especially MEK/ERK and notably, paradigms that inhibit PAC1 receptor endosome formation and ERK signaling also blunt the PACAP-induced increase in excitability. Detailed PAC1 receptor internalization and endosomal ERK signaling mechanisms have been confirmed in HEK PAC1R-EGFP cells and shown to be long lasting which appear to recapitulate the sustained electrophysiological responses. Thus, PAC1 receptor internalization/endosomal recruitment efficiently and efficaciously activates MEK/ERK signaling and appears to represent a singular and critical common denominator in regulating neuronal excitability by PACAP.

Intrabladder PAC1 Receptor Antagonist, PACAP(6-38), Reduces Urinary Bladder Frequency and Pelvic Sensitivity in Mice Exposed to Repeated Variate Stress (RVS).

Stress causes symptom exacerbation in functional disorders of the urinary bladder. However, the potential mediators and underlying mechanisms of stress effects on micturition reflex function are unknown. We have characterized PACAP (Adcyap1) and PAC1 receptor (Adcyap1r1) signaling in stress-induced urinary bladder dysfunction in mice. We determined PACAP and PAC1 transcripts and protein expressions in the urinary bladder and lumbosacral dorsal root ganglia (DRG) and spinal cord in repeated variate stress (RVS) or control mouse (handling only) groups. RVS in mice significantly (p ≤ 0.01) increased serum corticosterone and urinary bladder NGF content and decreased weight gain. PACAP and PAC1 mRNA and protein were differentially regulated in lower urinary tract tissues with changes observed in lumbosacral DRG and spinal cord but not in urinary bladder. RVS exposure in mice significantly (p ≤ 0.01) increased (2.5-fold) voiding frequency as determined using conscious cystometry. Intrabladder administration of the PAC1 receptor antagonist, PACAP(6-38) (300 nM), significantly (p ≤ 0.01) increased infused volume (1.5-2.7-fold) to elicit a micturition event and increased the intercontraction interval (i.e., decreased voiding frequency) in mice exposed to RVS and in control mice, but changes were smaller in magnitude in control mice. We also evaluated the effect of PAC1 blockade at the level of the urinary bladder on pelvic sensitivity in RVS or control mouse groups using von Frey filament testing. Intrabladder administration of PACAP(6-38) (300 nM) significantly (p ≤ 0.01) reduced pelvic sensitivity following RVS. PACAP/receptor signaling in the CNS and PNS contributes to increased voiding frequency and pelvic sensitivity following RVS and may represent a potential target for therapeutic intervention.

The Role of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Signaling in the Hippocampal Dentate Gyrus.

Pituitary adenylate cyclase-activating polypeptide (PACAP, ADCYAP1) dysregulation has been associated with multiple stress-related psychopathologies that may be related to altered hippocampal function. In coherence, PACAP- and PAC1 receptor (ADCYAP1R1)-null mice demonstrate changes in hippocampal-dependent behavioral responses, implicating the PACAPergic system function in this structure. Within the hippocampus, the dentate gyrus (DG) may play an important role in discerning the differences between similar contexts, and DG granule cells appear to both highly express PAC1 receptors and receive inputs from PACAP-expressing terminals. Here, we review the evidence from our laboratories and others that PACAP is an important regulator of activity within hippocampal circuits, particularly within the DG. These data are consistent with an increasing literature implicating PACAP circuits in stress-related pathologies such as post-traumatic stress disorder (PTSD) and implicate the hippocampus, and in particular the DG, as a critical site in which PACAP dysregulation can alter stress-related behaviors.

Pituitary adenylate cyclase-activating polypeptide-induced PAC1 receptor internalization and recruitment of MEK/ERK signaling enhance excitability of dentate gyrus granule cells.

Pituitary adenylate cyclase activating polypeptide (PACAP; ADCYAP1) is a pleiotropic neuropeptide widely distributed in both the peripheral and central nervous systems. PACAP and its specific cognate PAC1 receptor (ADCYAP1R1) play critical roles in the homeostatic maintenance of multiple physiological and behavioral systems. Notably, maladaptations in the PACAPergic system have been associated with several psychopathologies related to fear and anxiety. PAC1 receptor transcripts are highly expressed in granule cells of the dentate gyrus (DG). Here, we examined the direct effects of PACAP on DG granule cells in brain slices using whole cell patch recordings in current clamp mode. PACAP significantly increased the intrinsic excitability of DG granule cells via PAC1 receptor activation. This increased excitability was not mediated by adenylyl cyclase/cAMP or phospholipase C/PKC activation, but instead via activation of an extracellular signal-regulated kinase (ERK) signaling pathway initiated through PAC1 receptor endocytosis/endosomal signaling. PACAP failed to increase excitability in DG granule cells pretreated with the persistent sodium current blocker riluzole, suggesting that the observed PACAP effects required this component of the inward sodium current.

Circulating PACAP peptide and PAC1R genotype as possible transdiagnostic biomarkers for anxiety disorders in women: a preliminary study.

Pituitary adenylate cyclase activating polypeptide (PACAP, gene Adcyap1) is a neuropeptide and hormone thought to play a critical role in stress response (Stroth et al., Ann NY Acad Sci 1220:49-59, 2011; Hashimoto et al., Curr Pharm Des 17:985-989, 2011). Research in humans implicates PACAP as a useful biomarker for the severity of psychiatric symptoms in response to psychological stressors, and work in rodent models suggests that PACAP manipulation exerts downstream effects on peripheral hormones and behaviors linked to the stress response, providing a potential therapeutic target. Prior work has also suggested a potential sex difference in PACAP effects due to differential estrogen regulation of this pathway. Therefore, we examined serum PACAP and associated PAC1R genotype in a cohort of males and females with a primary diagnosis of generalized anxiety disorder (GAD) and nonpsychiatric controls. We found that, while circulating hormone levels were not associated with a GAD diagnosis overall (p = 0.19, g = 0.25), PACAP may be associated with GAD in females (p = 0.04, g = 0.33). Additionally, among patients with GAD, the risk genotype identified in the PTSD literature (rs2267735, CC genotype) was associated with higher somatic anxiety symptom severity in females but lower somatic anxiety symptom severity in males (-3.27, 95%CI [-5.76, -0.77], adjusted p = 0.03). Taken together, the associations between the risk genotype, circulating PACAP, and somatic anxiety severity were stronger among females than males. These results indicate a potential underlying biological etiology for sex differences in stress-related anxiety disorders that warrants further study.

Targeting the PAC1 Receptor for Neurological and Metabolic Disorders.

The pituitary adenylate cyclase-activating polypeptide (PACAP)-selective PAC1 receptor (PAC1R, ADCYAP1R1) is a member of the vasoactive intestinal peptide (VIP)/secretin/glucagon family of G protein-coupled receptors (GPCRs). PAC1R has been shown to play crucial roles in the central and peripheral nervous systems. The activation of PAC1R initiates diverse downstream signal transduction pathways, including adenylyl cyclase, phospholipase C, MEK/ERK, and Akt pathways that regulate a number of physiological systems to maintain functional homeostasis. Accordingly, at times of tissue injury or insult, PACAP/PAC1R activation of these pathways can be trophic to blunt or delay apoptotic events and enhance cell survival. Enhancing PAC1R signaling under these conditions has the potential to mitigate cellular damages associated with cerebrovascular trauma (including stroke), neurodegeneration (such as Parkinson's and Alzheimer's disease), or peripheral organ insults. Conversely, maladaptive PACAP/PAC1R signaling has been implicated in a number of disorders, including stressrelated psychopathologies (i.e., depression, posttraumatic stress disorder, and related abnormalities), chronic pain and migraine, and metabolic diseases; abrogating PAC1R signaling under these pathological conditions represent opportunities for therapeutic intervention. Given the diverse PAC1R-mediated biological activities, the receptor has emerged as a relevant pharmaceutical target. In this review, we first describe the current knowledge regarding the molecular structure, dynamics, and function of PAC1R. Then, we discuss the roles of PACAP and PAC1R in the activation of a variety of signaling cascades related to the physiology and diseases of the nervous system. Lastly, we examine current drug design and development of peptides and small molecules targeting PAC1R based on a number of structure- activity relationship studies and key pharmacophore elements. At present, the rational design of PAC1R-selective peptide or small-molecule therapeutics is largely hindered by the lack of structural information regarding PAC1R activation mechanisms, the PACAP-PAC1R interface, and the core segments involved in receptor activation. Understanding the molecular basis governing the PACAP interactions with its different cognate receptors will undoubtedly provide a basis for the development and/or refinement of receptor-selective therapeutics.

Parallel signaling pathways of pituitary adenylate cyclase activating polypeptide (PACAP) regulate several intrinsic ion channels.

Pituitary adenylate cyclase activating polypeptide (PACAP), acting through its cognate receptors PAC1, VPAC1, and VPAC2, is a pleiotropic signaling neuropeptide of the vasoactive intestinal peptide/secretin/glucagon family. PACAP has known functions in neuronal growth, development, and repair, and central PACAP signaling has acute behavioral consequences. One of the ways in which PACAP may affect neuronal function is through the modulation of intrinsic membrane currents to control neuronal excitability. Here, we review the evidence of PACAP-dependent modulation of calcium- and voltage-gated potassium currents, hyperpolarization-activated cation currents, calcium currents, and voltage-gated sodium currents. Interestingly, PACAP signaling pathways diverge into parallel pathways to target different ionic currents for modulation, though single pathways are not limited to modulating just one target ionic current. Despite the various targets of modulation, the weight of the evidence suggests that PACAP signaling most commonly leads to a net increase in neuronal excitability. We discuss possible mechanisms by which PACAP signaling leads to the modulation of intrinsic membrane currents that may contribute to changes in behavior.

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 establish four 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-Induced PAC1 Receptor Internalization and Recruitment of Endosomal Signaling Regulate Cardiac Neuron Excitability.

Pituitary adenylate cyclase-activating polypeptide (PACAP, Adcyap1) activation of PAC1 receptors (Adcyap1r1) significantly increases excitability of guinea pig cardiac neurons. This modulation of excitability is mediated in part by plasma membrane G protein-dependent activation of adenylyl cyclase and downstream signaling cascades, as well as by endosomal signaling mechanisms. PACAP/PAC1 receptor-mediated activation of plasma membrane adenylyl cyclase (AC) and the resulting increase in cellular cAMP enhances a hyperpolarization-induced nonselective cationic current Ih, which contributes to the PACAP-induced increase in cardiac neuron excitability. Further, PACAP-mediated AC/cAMP/PKA downstream signaling also appears to enhance cardiac neuron IT to facilitate the excitatory responses. PACAP activation of PAC1 receptors rapidly stimulates receptor internalization, and reducing ambient temperature or treatments with the clathrin inhibitor Pitstop2 or the dynamin I/II inhibitor dynasore to block endocytic events can suppress PACAP-enhanced neuronal excitability. Thus, endocytosis inhibitors essentially eliminate PACAP-enhanced excitability suggesting that endosomal platforms represent a primary signaling mechanism. Endosomal signaling is associated canonically with ERK activation and in accord, PACAP-enhanced cardiac neuron excitability is reduced by MEK inhibitor pretreatments. PACAP activation of MEK/ERK signaling can enhance currents through voltage-dependent Nav1.7 channels. Hence, PACAP-induced PAC1 receptor internalization/endosomal signaling, recruitment of MEK/ERK signaling, and modulation of Nav1.7 are implicated as key mechanisms contributing to the PACAP-enhanced neuronal excitability. PACAP/PAC1 receptor-mediated endosomal ERK signaling in central circuits can play key roles in development of chronic pain and anxiety-related responses; thus, PAC1 endosomal signaling likely participates in a variety of homeostatic responses within neuronal circuits in the CNS.

PACAP38-Mediated Bladder Afferent Nerve Activity Hyperexcitability and Ca2+ Activity in Urothelial Cells from Mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP; Adcyap1) and its cognate PAC1 receptor (Adcyap1r1) have tissue-specific distributions in the lower urinary tract (LUT). The afferent limb of the micturition reflex is often compromised following bladder injury, disease, and inflammatory conditions. We have previously demonstrated that PACAP signaling contributes to increased voiding frequency and decreased bladder capacity with cystitis. Thus, the present studies investigated the sensory components (e.g., urothelial cells, bladder afferent nerves) of the urinary bladder that may underlie the pathophysiology of aberrant PACAP activation. We utilized bladder-pelvic nerve preparations and urothelial sheet preparations to characterize PACAP-induced bladder afferent nerve discharge with distention and PACAP-induced Ca2+ activity, respectively. We determined that PACAP38 (100 nM) significantly (p ≤ 0.01) increased bladder afferent nerve activity with distention that was blocked with a PAC1/VPAC2 receptor antagonist PACAP6-38 (300 nM). PACAP38 (100 nM) also increased Ca2+ activity in urothelial cells over that observed in control preparations. Taken together, these results establish a role for PACAP signaling in bladder sensory components (e.g., urothelial cells, bladder afferent nerves) that may ultimately facilitate increased voiding frequency.

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.

Pituitary Adenylate Cyclase-Activating Peptide (PACAP) Signaling and the Dark Side of Addiction.

While addiction to drugs of abuse represents a significant health problem worldwide, the behavioral and neural mechanisms that underlie addiction and relapse are largely unclear. The concept of the dark side of addiction, developed and explored by George Koob and colleagues, describes a systematic decrease in reward-related processing following drug self-administration and subsequent recruitment of anti-reward (i.e., stress) systems. Indeed, the activation of central nervous system (CNS) stress-response systems by drugs of abuse is contributory not only to mood and anxiety-related disorders but critical to both the maintenance of addiction and relapse following abstinence. In both human and animal studies, compounds that activate the bed nucleus of the stria terminalis (BNST) have roles in stress-related behaviors and addiction processes. The activation of pituitary adenylate cyclase-activating peptide (PACAP) systems in the BNST mediates many consequences of chronic stressor exposure that may engage in part downstream corticotropin-releasing hormone (CRH) signaling. Similar to footshock stress, the BNST administration of PACAP or the PAC1 receptor-specific agonist maxadilan can facilitate relapse following extinction of cocaine-seeking behavior. Further, in the same paradigm, the footshock-induced relapse could be attenuated following BNST pretreatment with PAC1 receptor antagonist PACAP6-38, implicating PACAP systems as critical components underlying stress-induced reinstatement. In congruence with previous work, the PAC1 receptor internalization and endosomal MEK/ERK signaling appear contributory mechanisms to the addiction processes. The studies offer new insights and approaches to addiction and relapse therapeutics.