Pituitary Adenylate Cyclase-Activating Polypeptide Excites Proopiomelanocortin Neurons: Implications for the Regulation of Energy Homeostasis.

OBJECTIVE: We examined whether pituitary adenylate cyclase-activating polypeptide (PACAP) excites proopiomelanocortin (POMC) neurons via PAC1 receptor mediation and transient receptor potential cation (TRPC) channel activation. METHODS: Electrophysiological recordings were done in slices from both intact male and ovariectomized (OVX) female PACAP-Cre mice and eGFP-POMC mice. RESULTS: In recordings from POMC neurons in eGFP-POMC mice, PACAP induced a robust inward current and increase in conductance in voltage clamp, and a depolarization and increase in firing in current clamp. These postsynaptic actions were abolished by inhibitors of the PAC1 receptor, TRPC channels, phospholipase C, phosphatidylinositol-3-kinase, and protein kinase C. Estradiol augmented the PACAP-induced inward current, depolarization, and increased firing, which was abrogated by estrogen receptor (ER) antagonists. In optogenetic recordings from POMC neurons in PACAP-Cre mice, high-frequency photostimulation induced inward currents, depolarizations, and increased firing that were significantly enhanced by Gq-coupled membrane ER signaling in an ER antagonist-sensitive manner. Importantly, the PACAP-induced excitation of POMC neurons was notably reduced in obese, high-fat (HFD)-fed males. In vivo experiments revealed that intra-arcuate nucleus (ARC) PACAP as well as chemogenetic and optogenetic stimulation of ventromedial nucleus (VMN) PACAP neurons produced a significant decrease in energy intake accompanied by an increase in energy expenditure, effects blunted by HFD in males and partially potentiated by estradiol in OVX females. CONCLUSIONS: These findings reveal that the PACAP-induced activation of PAC1 receptor and TRPC5 channels at VMN PACAP/ARC POMC synapses is potentiated by estradiol and attenuated under conditions of diet-induced obesity/insulin resistance. As such, they advance our understanding of how PACAP regulates the homeostatic energy balance circuitry under normal and pathophysiological circumstances.

Discovery of PACAP and its receptors in the brain.

Pituitary adenylate-cyclase-activating polypeptide (PACAP) is a 27- or 38-amino acid neuropeptide, which belongs to the vasoactive intestinal polypeptide (VIP)/glucagon/secretin family. PACAP shows particularly high homology (~ 68%) to VIP. Because of the high homology of the amino acid sequences of PACAP and VIP, these peptides share three class B-G-protein coupled receptors: the PAC1-Receptor (PAC1-R), the VPAC1-Receptor (VPAC1-R) and VPAC2-Receptor (VPAC2-R). These receptors have high homology to each other, and their high homology is utilized for these discoveries. This review provides mainly an overview of the history of the discovery of PACAP and its three receptors.

Pituitary adenylate cyclase-activating polypeptide promotes eccrine gland sweat secretion.

BACKGROUND: Sweat secretion is the major function of eccrine sweat glands; when this process is disturbed (paridrosis), serious skin problems can arise. To elucidate the causes of paridrosis, an improved understanding of the regulation, mechanisms and factors underlying sweat production is required. Pituitary adenylate cyclase-activating polypeptide (PACAP) exhibits pleiotropic functions that are mediated via its receptors [PACAP-specific receptor (PAC1R), vasoactive intestinal peptide (VIP) receptor type 1 (VPAC1R) and VPAC2R]. Although some studies have suggested a role for PACAP in the skin and several exocrine glands, the effects of PACAP on the process of eccrine sweat secretion have not been examined. OBJECTIVES: To investigate the effect of PACAP on eccrine sweat secretion. METHODS: Reverse transcriptase-polymerase chain reaction and immunostaining were used to determine the expression and localization of PACAP and its receptors in mouse and human eccrine sweat glands. We injected PACAP subcutaneously into the footpads of mice and used the starch-iodine test to visualize sweat-secreting glands. RESULTS: Immunostaining showed PACAP and PAC1R expression by secretory cells from mouse and human sweat glands. PACAP immunoreactivity was also localized in nerve fibres around eccrine sweat glands. PACAP significantly promoted sweat secretion at the injection site, and this could be blocked by the PAC1R-antagonist PACAP6-38. VIP, an agonist of VPAC1R and VPAC2R, failed to induce sweat secretion. CONCLUSIONS: This is the first report demonstrating that PACAP may play a crucial role in sweat secretion via its action on PAC1R located in eccrine sweat glands. The mechanisms underlying the role of PACAP in sweat secretion may provide new therapeutic options to combat sweating disorders.

Zac1 Regulates the Differentiation and Migration of Neocortical Neurons via Pac1.

Imprinted genes are dosage sensitive, and their dysregulated expression is linked to disorders of growth and proliferation, including fetal and postnatal growth restriction. Common sequelae of growth disorders include neurodevelopmental defects, some of which are indirectly related to placental insufficiency. However, several growth-associated imprinted genes are also expressed in the embryonic CNS, in which their aberrant expression may more directly affect neurodevelopment. To test whether growth-associated genes influence neural lineage progression, we focused on the maternally imprinted gene Zac1. In humans, either loss or gain of ZAC1 expression is associated with reduced growth rates and intellectual disability. To test whether increased Zac1 expression directly perturbs neurodevelopment, we misexpressed Zac1 in murine neocortical progenitors. The effects were striking: Zac1 delayed the transition of apical radial glial cells to basal intermediate neuronal progenitors and postponed their subsequent differentiation into neurons. Zac1 misexpression also blocked neuronal migration, with Zac1-overexpressing neurons pausing more frequently and forming fewer neurite branches during the period when locomoting neurons undergo dynamic morphological transitions. Similar, albeit less striking, neuronal migration and morphological defects were observed on Zac1 knockdown, indicating that Zac1 levels must be regulated precisely. Finally, Zac1 controlled neuronal migration by regulating Pac1 transcription, a receptor for the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP). Pac1 and Zac1 loss- and gain-of-function presented as phenocopies, and overexpression of Pac1 rescued the Zac1 knockdown neuronal migration phenotype. Thus, dysregulated Zac1 expression has striking consequences on neocortical development, suggesting that misexpression of this transcription factor in the brain in certain growth disorders may contribute to neurocognitive deficits. Significance statement: Altered expression of imprinted genes is linked to cognitive dysfunction and neuropsychological disorders, such as Angelman and Prader-Willi syndromes, and autism spectrum disorder. Mouse models have also revealed the importance of imprinting for brain development, with chimeras generated with parthenogenetic (two maternal chromosomes) or androgenetic (two paternal chromosomes) cells displaying altered brain sizes and cellular defects. Despite these striking phenotypes, only a handful of imprinted genes are known or suspected to regulate brain development (e.g., Dlk1, Peg3, Ube3a, necdin, and Grb10). Herein we show that the maternally imprinted gene Zac1 is a critical regulator of neocortical development. Our studies are relevant because loss of 6q24 maternal imprinting in humans results in elevated ZAC1 expression, which has been associated with neurocognitive defects.

Pituitary adenylate cyclase-activating polypeptide type 1 receptor signaling evokes long-lasting nociceptive behaviors through the activation of spinal astrocytes in mice.

Intrathecal (i.t.) administration of pituitary adenylate cyclase-activating polypeptide (PACAP) induces long-lasting nociceptive behaviors for more than 60 min in mice, while the involvement of PACAP type1 receptor (PAC1-R) has not been clarified yet. The present study investigated signaling mechanisms of the PACAP-induced prolonged nociceptive behaviors. Single i.t. injection of a selective PAC1-R agonist, maxadilan (Max), mimicked nociceptive behaviors in a dose-dependent manner similar to PACAP. Pre- or post-treatment of a selective PAC1-R antagonist, max.d.4, significantly inhibited the nociceptive behaviors by PACAP or Max. Coadministration of a protein kinase A inhibitor, Rp-8-Br-cAMPS, a mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase inhibitor, PD98059 or a c-Jun N-terminal kinase (JNK) inhibitor, SP600125, significantly inhibited the nociceptive behaviors by Max. Immunohistochemistry and immunoblotting analysis revealed that spinal administration of Max-induced ERK phosphorylation and JNK phosphorylation, and also augmented an astrocyte marker, glial fibrillary acidic protein in mouse spinal cord. Furthermore, an astroglial toxin, l-α-aminoadipate, significantly attenuated the development of the nociceptive behaviors and ERK phosphorylation by Max. These results suggest that the activation of spinal PAC1-R induces long-lasting nociception through the interaction of neurons and astrocytes.

PACAP and VIP increase the expression of myelin-related proteins in rat schwannoma cells: involvement of PAC1/VPAC2 receptor-mediated activation of PI3K/Akt signaling pathways.

PACAP and its cognate peptide VIP participate in various biological functions, including myelin maturation and synthesis. However, defining whether these peptides affect peripheral expression of myelin proteins still remains unanswered. To address this issue, we assessed whether PACAP or VIP contribute to regulate the expression of three myelin proteins (MAG, MBP and MPZ, respectively) using the rat schwannoma cell line (RT4-P6D2T), a well-established model to study myelin gene expression. In addition, we endeavored to partly unravel the underlying molecular mechanisms involved. Expression of myelin-specific proteins was assessed in cells grown either in normal serum (10% FBS) or serum starved and treated with or without 100 nM PACAP or VIP. Furthermore, through pharmacological approach using the PACAP/VIP receptor antagonist (PACAP6-38) or specific pathway (MAPK or PI3K) inhibitors we defined the relative contribution of receptors and/or signaling pathways on the expression of myelin proteins. Our data show that serum starvation (24h) significantly increased both MAG, MBP and MPZ expression. Concurrently, we observed increased expression of endogenous PACAP and related receptors. Treatment with PACAP or VIP further exacerbated starvation-induced expression of myelin markers, suggesting that serum withdrawal might sensitize cells to peptide activity. Stimulation with either peptides increased phosphorylation of Akt at Ser473 residue but had no effect on phosphorylated Erk-1/2. PACAP6-38 (10 µM) impeded starvation- or peptide-induced expression of myelin markers. Similar effects were obtained after pretreatment with the PI3K inhibitor (wortmannin, 10 µM) but not the MAPKK inhibitor (PD98059, 50 µM). Together, the present finding corroborate the hypothesis that PACAP and VIP might contribute to the myelinating process preferentially via the canonical PI3K/Akt signaling pathway, providing the basis for future studies on the role of these peptides in demyelinating diseases.

Possible role of PACAP and its PAC1 receptor in the differential regulation of pituitary LHbeta- and FSHbeta-subunit gene expression by pulsatile GnRH stimulation.

The pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are mainly under the control of hypothalamic gonadotropin-releasing hormone (GnRH), which regulates male and female gonadal function. GnRH is released in a pulsatile manner from the hypothalamus, and the frequency of GnRH pulses determines the dominance of output of LH and FSH from pituitary gonadotrophs. That is, more rapid pulses of GnRH preferentially increase synthesis and secretion of LH, whereas FSH is preferentially stimulated by slower GnRH pulses. The detailed mechanisms underlying this phenomenon remain unknown. Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally identified as a hypothalamic activator of cAMP production in pituitary cells. PACAP is produced within the pituitary gonadotroph as well as in the central nervous system. PACAP stimulates gonadotropin alpha-, LHbeta-, and FSHbeta-subunits as well as receptors for GnRH in the pituitary gonadotropin-secreting cells. In addition, its own receptor, PACAP type I receptor (PAC1R), is also regulated by PACAP in gonadotrophs. GnRH stimulates expression of PACAP as well as PAC1R, and lower frequencies of GnRH pulses preferentially increase PACAP and PAC1R expression in gonadotrophs. Increasing concentrations of PACAP further increase the levels of gonadotropin subunit and that increasing amounts of PAC1R in gonadotrophs potentiates the effects of PACAP or GnRH on gonadotropin subunit expression. In addition, we have observed that GnRH-increased FSHbeta-subunit expression was prevented in the presence of PAC1R antagonist. These observations suggest the involvement of locally produced PACAP and its PAC1R in the differential regulation of specific gonadotropin subunit expression by pulsatile GnRH stimulation. Here, we review the possible involvement of PACAP and its PAC1R in gonadotropin control on the basis of our observations with gonadotroph cell lines.

Upregulation of pituitary adenylate cyclase activating polypeptide and its receptor expression in the rat carotid body in chronic and intermittent hypoxia.

The carotid body (CB) plays important roles in cardiorespiratory changes in chronic and intermittent hypoxia. Pituitary adenylate cyclase activating polypeptide (PACAP) is involved in the regulation of respiratory chemoresponse. We hypothesized an upregulation of the expressions of PACAP and its receptor (PAC1) in the rat CB in chronic and intermittent hypoxia. The CB expressions of PACAP and PAC1 were examined in rats breathing 10% O(2) (in isobaric chamber for chronic hypoxia, 24 h/day) or in intermittent hypoxia (cyclic between air and 5% O(2) per minute, 8 h/day) for 7 days. Immunohistochemical studies showed that the PACAP and PAC1 proteins were localized in CB glomic clusters containing tyrosine hydroxylase. The proportional amount of cells with positive staining of PACAP and PAC1 was significantly increased in both hypoxic groups when compared with the normoxic control. In addition, the mRNA level of PAC1 expression was markedly elevated in the hypoxic groups, despite no changes in the PACAP expression. These results suggest an upregulation of PACAP and its receptor expression in the rat CB under chronic and intermittent hypoxic conditions. The PACAP binding to its receptor could activate the PKA signaling pathway leading to an increased CB excitability under hypoxic conditions.

Pituitary adenylate cyclase-activating polypeptide and its receptors: 20 years after the discovery.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid C-terminally alpha-amidated peptide that was first isolated 20 years ago from an ovine hypothalamic extract on the basis of its ability to stimulate cAMP formation in anterior pituitary cells (Miyata et al., 1989. PACAP belongs to the vasoactive intestinal polypeptide (VIP)-secretin-growth hormone-releasing hormone-glucagon superfamily. The sequence of PACAP has been remarkably well conserved during evolution from protochordates to mammals, suggesting that PACAP is involved in the regulation of important biological functions. PACAP is widely distributed in the brain and peripheral organs, notably in the endocrine pancreas, gonads, respiratory and urogenital tracts. Characterization of the PACAP precursor has revealed the existence of a PACAP-related peptide, the activity of which remains unknown. Two types of PACAP binding sites have been characterized: type I binding sites exhibit a high affinity for PACAP and a much lower affinity for VIP, whereas type II binding sites have similar affinity for PACAP and VIP. Molecular cloning of PACAP receptors has shown the existence of three distinct receptor subtypes: the PACAP-specific PAC1-R, which is coupled to several transduction systems, and the PACAP/VIP-indifferent VPAC1-R and VPAC2-R, which are primarily coupled to adenylyl cyclase. PAC1-Rs are particularly abundant in the brain, the pituitary and the adrenal gland, whereas VPAC receptors are expressed mainly in lung, liver, and testis. The development of transgenic animal models and specific PACAP receptor ligands has strongly contributed to deciphering the various actions of PACAP. Consistent with the wide distribution of PACAP and its receptors, the peptide has now been shown to exert a large array of pharmacological effects and biological functions. The present report reviews the current knowledge concerning the pleiotropic actions of PACAP and discusses its possible use for future therapeutic applications.

Regulation of spinal dynorphin 1-17 release by endogenous pituitary adenylyl cyclase-activating polypeptide in the male rat: relevance of excitation via disinhibition.

Opioids inhibit release of primary afferent transmitters but it is unclear whether the converse occurs. To test the hypothesis that primary afferent transmitters influence opioid-ergic tone, we studied the functional and anatomical relationships between pituitary adenylyl cyclase-activating polypeptide (PACAP) and dynorphin 1-17 (Dyn) in spinal cord. We found that activation of the PACAP-specific receptor PAC(1) (PAC(1)R) inhibited, whereas PAC(1)R blockade augmented, spinal release of Dyn. It is noteworthy that in the formalin-induced pain model PAC(1)R blockade (via PACAP6-38) also resulted in antinociception that was abolished by spinal κ-opioid receptor blockade. These findings indicate that Dyn release is tonically inhibited by PACAP and that blocking this inhibition, which increases the spinal release of Dyn, results in antinociception. Consistent with this conclusion, we found in the spinal dorsal horn that Dyn-immunoreactive neurons 1) expressed PAC(1)R and 2) were apposed by PACAP terminals. Present results, in combination with the previous demonstration that the release of spinal Dyn is tonically inhibited by opioid- and nociceptin/orphanin FQ-coupled pathways (J Pharmacol Exp Ther 298:1213-1220, 2001), indicate that spinal Dyn-ergic neurons integrate multiple inhibitory inputs, the interruption of any one of which (i.e., disinhibition) is sufficient to enhance spinal Dyn release and generate antinociception. Gaining a better understanding of the role of primary afferent neurotransmitters in negatively modulating the spinal release of Dyn and the physiological use of disinhibition to increase spinal Dyn activity could suggest novel clinically useful approaches for harnessing endogenous Dyn for pain control.

PACAP-PAC1 Signaling Regulates Serotonin 2A Receptor Internalization.

Mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP) display psychomotor abnormalities, most of which are ameliorated by atypical antipsychotics with serotonin (5-HT) 2A receptor (5-HT2A) antagonism. Heterozygous Pacap mutant mice show a significantly higher hallucinogenic response than wild-type mice to a 5-HT2A agonist. Endogenous PACAP may, therefore, affect 5-HT2A signaling; however, the underlying neurobiological mechanism for this remains unclear. Here, we examined whether PACAP modulates 5-HT2A signaling by addressing cellular protein localization. PACAP induced an increase in internalization of 5-HT2A but not 5-HT1A, 5-HT2C, dopamine D2 receptors or metabotropic glutamate receptor 2 in HEK293T cells. This PACAP action was inhibited by protein kinase C inhibitors, ß-arrestin2 silencing, the PACAP receptor PAC1 antagonist PACAP6-38, and PAC1 silencing. In addition, the levels of endogenous 5-HT2A were decreased on the cell surface of primary cultured cortical neurons after PACAP stimulation and were increased in frontal cortex cell membranes of Pacap-/- mice. Finally, intracerebroventricular PACAP administration suppressed 5-HT2A agonist-induced head twitch responses in mice. These results suggest that PACAP-PAC1 signaling increases 5-HT2A internalization resulting in attenuation of 5-HT2A-mediated signaling, although further study is necessary to determine the relationship between behavioral abnormalities in Pacap-/- mice and PACAP-induced 5-HT2A internalization.

VPAC and PAC receptors: From ligands to function.

Vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase activating polypeptides (PACAPs) share 68% identity at the amino acid level and belong to the secretin peptide family. Following the initial discovery of VIP almost four decades ago a substantial amount of knowledge has been presented describing the mechanisms of action, distribution and pleiotropic functions of these related peptides. It is now known that the physiological actions of these widely distributed peptides are produced through activation of three common G-protein coupled receptors (VPAC(1), VPAC(2) and PAC(1)R) which preferentially stimulate adenylate cyclase and increase intracellular cAMP, although stimulation of other intracellular messengers, including calcium and phospholipase D, has been reported. Using a range of in vitro and in vivo approaches, including cell-based functional assays, transgenic animals and rodent models of disease, VPAC/PAC receptor activation has been associated with numerous physiological processes (e.g. control of circadian rhythms) and clinical conditions (e.g. pulmonary hypertension), which underlies on-going research efforts and makes these peptides and their cognate receptors attractive targets for the pharmaceutical industry. However, despite the considerable interest in VPAC/PAC receptors and the processes which they mediate, there is still a paucity of selective and available, non-peptide ligands, which has hindered further advances in this field both at the basic research and clinical level. This review summarises the current knowledge of VIP/PACAP and the VPAC/PAC receptors with regard to their distribution, pharmacology, signalling pathways, splice variants and finally, the utility of animal models in exploring their physiological roles.

Epac mediates PACAP-dependent long-term depression in the hippocampus.

Extensive work has shown that activation of the cAMP-dependent protein kinase A (PKA) is crucial for long-term depression (LTD) of synaptic transmission in the hippocampus, a phenomenon that is thought to be involved in memory formation. Here we studied the role of an alternative target of cAMP, the exchange protein factor directly activated by cyclic AMP (Epac). We show that pharmacological activation of Epac by the selective agonist 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT) induces LTD in the CA1 region. Paired-pulse facilitation of synaptic responses remained unchanged after induction of this LTD, suggesting that it depended on postsynaptic mechanisms. The 8-pCPT-induced LTD was blocked by the Epac signalling inhibitor brefeldin-A (BFA), Rap-1 antagonist geranylgeranyltransferase inhibitor (GGTI) and p38 mitogen activated protein kinase (P38-MAPK) inhibitor SB203580. This indicated a direct involvement of Epac in this form of LTD. As for other forms of LTD, a mimetic peptide of the PSD-95/Disc-large/ZO-1 homology (PDZ) ligand motif of the AMPA receptor subunit GluR2 blocked the Epac-LTD, suggesting involvement of PDZ protein interaction. The Epac-LTD also depended on mobilization of intracellular Ca(2+), proteasome activity and mRNA translation, but not transcription, as it was inhibited by thapsigargin, lactacystin and anisomycin, but not actinomycin-D, respectively. Finally, we found that the pituitary adenylate cyclase activating polypeptide (PACAP) can induce an LTD that was mutually occluded by the Epac-LTD and blocked by BFA or SB203580, suggesting that the Epac-LTD could be mobilized by stimulation of PACAP receptors. Altogether these results provided evidence for a new form of hippocampal LTD.

Differential activation of guinea pig intrinsic cardiac neurons by the PAC1 agonists maxadilan and pituitary adenylate cyclase-activating polypeptide 27 (PACAP27).

Pituitary adenylate cyclase-activating polypeptide (PACAP) evokes tachycardia followed by a larger cholinergic bradycardia in isolated guinea pig hearts. We used the selective PAC1 receptor agonist maxadilan and vasoactive intestinal polypeptide (VIP) to test the hypothesis that PACAP27-evoked tachycardia and bradycardia are mediated by VPAC and PAC1 receptors, respectively. Chronotropic actions of these peptides were evaluated in isolated perfused hearts. Direct neuronal actions were determined by intracellular voltage recordings from cholinergic neurons in atrial ganglion whole mounts. Administration of 1 nmol of PACAP27 to isolated hearts evoked typical biphasic rate responses, whereas 1 nmol of maxadilan caused only a minor rate decrease. Desensitization with VIP eliminated the positive chronotropic effect of PACAP27 selectively. Local application of PACAP27 to cardiac neurons frequently evoked slow depolarization and caused prolonged increase of neuronal excitability. Maxadilan rarely affected membrane potential but consistently increased excitability. VIP had no effect on excitability and evoked depolarization in only a few neurons. Because maxadilan increased neuronal excitability but did not trigger action potentials as PACAP often does, we evaluated the interaction of maxadilan with substance P (SP) in isolated hearts. SP depolarizes cardiac neurons more consistently than PACAP, often triggers neuronal action potentials, and causes bradycardia but does not increase neuronal excitability. Maxadilan had a persistent effect to augment negative chronotropic responses to SP. These findings support our hypothesis that PACAP evokes tachycardia and bradycardia through VPAC and PAC1 receptors, respectively. They also suggest that maxadilan and PACAP27 differ in activating PAC1 receptors on cardiac neurons and/or stimulating downstream signaling mechanisms.

G protein-coupled receptors control NMDARs and metaplasticity in the hippocampus.

Long-term potentiation (LTP) and long-term depression (LTD) are the major forms of functional synaptic plasticity observed at CA1 synapses of the hippocampus. The balance between LTP and LTD or "metaplasticity" is controlled by G-protein coupled receptors (GPCRs) whose signal pathways target the N-methyl-D-asparate (NMDA) subtype of excitatory glutamate receptor. We discuss the protein kinase signal cascades stimulated by Galphaq and Galphas coupled GPCRs and describe how control of NMDAR activity shifts the threshold for the induction of LTP.

Characterization, expression, and functional activity of pituitary adenylate cyclase-activating polypeptide and its receptors in human granulosa-luteal cells.

CONTEXT: Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are found in the ovary of mammalian species, although nothing is known about the possible role of PACAP and VIP in the human ovary. OBJECTIVE: We investigated the expression of PACAP and PACAP/VIP receptors in human granulosa-luteal (GL) cells obtained from consenting in vitro fertilization patients attending a private fertility clinic and assessed a possible antiapoptotic effect of these molecules. MAIN OUTCOME MEASURES: We measured the expression of PACAP and PACAP/VIP receptor mRNAs in GL cells in response to FSH or LH, as well as the effects of PACAP and VIP on apoptosis. We also evaluated the levels of procaspase-3 in GL cells cultured in the absence of serum. RESULTS: After 7 d in culture, GL cells displayed increased responsiveness to FSH and LH (100 ng/ml). FSH and LH promoted PACAP expression, LH doing so in a time-dependent fashion. VIP receptor (VPAC1-R and VPAC2-R) mRNAs were also induced by gonadotropin stimulation. Although PACAP receptor (PAC1-R) mRNA was barely detectable, Western blot analysis revealed its presence. The apoptotic effect of serum withdrawal from the culture environment was reverted by both PACAP and VIP. Both peptides showed the ability to reverse a decrease in procaspase-3 levels induced by culture in the absence of serum. CONCLUSIONS: PACAP and VIP appear to play a role in maintenance of follicle viability as a consequence of the antiapoptotic effect. Further studies are warranted to evaluate the respective roles of PACAP and VIP in ovarian physiology and to identify their mechanism of action.

GPCR-jacking: from a new route in RTK signalling to a new concept in GPCR activation.

A large body of evidence indicates that agonists of some G protein-coupled receptors (GPCRs) can activate growth factor receptor tyrosine kinases (RTKs) in the absence of added growth factor. This phenomenon, called transactivation, is an important pathway that contributes to growth-promoting activity of many GPCR ligands. Reciprocally, recent advances indicate that RTKs utilize GPCR signalling molecules to transduce signals and that RTK ligands themselves can transactivate GPCRs. This novel transactivation process, which places GPCR signalling downstream of RTKs, either requires the production of a GPCR ligand of the transactivated GPCR or occurs in a ligand independent manner within an integrated signalling network. Here, we provide an overview of the molecular mechanisms involved in this novel cross-communication between GPCRs and RTKs and discuss its relevance in the specification of growth factor signalling and functions.

Characterization of signaling pathway for the translocation of neuronal nitric oxide synthase to the plasma membrane by PACAP.

In the central nervous system, the activation of neuronal nitric oxide synthase (nNOS) is closely associated with activation of NMDA receptor, and trafficking of nNOS may be a prerequisite for efficient NO production at synapses. We recently demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) and NMDA synergistically caused the translocation of nNOS to the membrane and stimulated NO production in PC12 (pheochromocytoma) cells. However, the mechanisms responsible for trafficking and activation of nNOS are largely unknown. To address these issues, here we constructed a yellow fluorescent protein (YFP)-tagged nNOS N-terminal (1-299 a.a.) mutant, nNOSNT-YFP, and visualized its translocation in PC12 cells stably expressing it. PACAP enhanced the translocation synergistically with NMDA in a time- and concentration-dependent manner. The translocation was blocked by inhibitors of protein kinase A (PKA), protein kinase C (PKC), and Src kinase; and the effect of PACAP could be replaced with PKA and PKC activators. The beta-finger region in the PSD-95/disc large/zonula occludens-1 domain of nNOS was required for the translocation of nNOS and its interaction with post-synaptic density-95 (PSD-95), and NO formation was attenuated by dominant negative nNOSNT-YFP. These results demonstrate that PACAP stimulated nNOS translocation mediated by PKA and PKC via PAC(1)-receptor (a PACAP receptor) and suggest cross-talk between PACAP and NMDA for nNOS activation by Src-dependent phosphorylation of NMDA receptors.

Long-term administration of maxadilan improves glucose tolerance and insulin sensitivity in mice.

Maxadilan and its truncated variant, M65, are agonist and antagonist specific, respectively, for the PAC1 receptor. PAC1 is the specific receptor for the pituitary adenylate cyclase-activating peptide (PACAP), which is not shared by vasoactive intestinal peptide (VIP). PACAP is a ubiquitous peptide of the glucagon superfamily that is involved in glucose homeostasis and regulation of insulin secretion. This study employed the recombinant maxadilan and M65 to evaluate the PAC1 receptor-mediated effects on energy metabolism using NIH mice. First, the acute effect of maxadilan-induced hyperglycemia was blocked by M65. In long-term studies, NIH mice were given daily intraperitoneal injections with maxadilan, M65, or vehicle for 21 days. Maxadilan suppressed feeding and enhanced water intake significantly for the first several days. After that period, maxadilan treatment continued to promote food and water intake. Long-term administration of maxadilan led to an increase in body weight (P<0.01), decrease in body fat (P<0.01), down-regulation of basal plasma glucose (P<0.01), upregulation of basal plasma insulin (P<0.01) and improved glucose tolerance (P<0.01) and insulin sensitivity (P<0.01). An elevation in plasma LDL (P<0.01) was also observed in the maxadilan group. However, M65 displayed no significant adverse effects on the aforementioned parameters except basal plasma glucose (P<0.05). The significant changes induced by maxadilan indicate that the PAC1 receptor plays multiple key roles in carbohydrate metabolism, lipid metabolism and energy homeostasis in mice.