Neurotrophic actions of PACAP-38 and LIF on human neuroblastoma SH-SY5Y cells.

The neurotrophic actions of pituitary adenylate cyclase-activating polypeptide (PACAP)-38 and leukemia inhibitory factor (LIF) were investigated in human neuroblastoma SH-SY5Y cells. Effects on differentiation were assessed through monitoring morphological changes and Western blot analysis of the expression of neuronal marker proteins. In contrast to PACAP-38, which induced a 5.5-fold increase in the number of neurite-bearing cells, LIF had no significant effect on cell morphology compared to control cells over the 4-day time course. Cells co-treated with PACAP-38+LIF showed a similar increase in neurite-bearing cells compared to those treated with PACAP-38 alone. Cell morphology was similar for PACAP-38-treated and PACAP-38+LIF-co-treated cells, with the formation of bipolar neuron-like cells with long thin neurites, topped by growth cone-like structures and varicosities. SH-SY5Y cells express tyrosine hydroxylase (TH) but only low levels of the neuronal marker proteins: Bcl-2, GAP-43 and choline acetyltransferase (ChAT). Treatment of cells with PACAP-38 induced the expression of Bcl-2, GAP-43, and ChAT but did not appear to alter the expression of TH. LIF failed to induce the expression of GAP-43 and had little effect on the expression of TH, but did induce the expression of Bcl-2 and upregulated the expression of ChAT. Co-treatment with LIF had no effect on PACAP-38-induced expression of Bcl-2, GAP-43, and ChAT. Cells differentiated for 4 days with PACAP-38 or treated with LIF also displayed increased resistance to hypoxic conditions and to treatment with H2O2 and TNFalpha. The increased resistance to hypoxic conditions for PACAP-differentiated cells was blocked by the p38 MAP kinase inhibitor, SB203580, but not by the MEK1 inhibitor, PD98059. Additionally, cell proliferation assays show that LIF, but not PACAP-38, stimulates proliferation of SH-SY5Y cells, and this observed increase by LIF is not attenuated by co-treatment with PACAP. Further investigation of the intracellular signaling pathways mediating the neurotrophic effects of PACAP on SH-SY5Y cells indicate that neither phospholipase C activation nor Ca2+/calmodulin-dependent kinase II (CAMKII) are involved.

PACAP-38 induces neuronal differentiation of human SH-SY5Y neuroblastoma cells via cAMP-mediated activation of ERK and p38 MAP kinases.

The intracellular signaling pathways mediating the neurotrophic actions of pituitary adenylate cyclase-activating polypeptide (PACAP) were investigated in human neuroblastoma SH-SY5Y cells. Previously, we showed that SH-SY5Y cells express the PAC(1) and VIP/PACAP receptor type 2 (VPAC(2)) receptors, and that the robust cAMP production in response to PACAP and vasoactive intestinal peptide (VIP) was mediated by PAC(1) receptors (Lutz et al. 2006). Here, we investigated the ability of PACAP-38 to differentiate SH-SY5Y cells by measuring morphological changes and the expression of neuronal markers. PACAP-38 caused a concentration-dependent increase in the number of neurite-bearing cells and an up-regulation in the expression of the neuronal proteins Bcl-2, growth-associated protein-43 (GAP-43) and choline acetyltransferase: VIP was less effective than PACAP-38 and the VPAC(2) receptor-specific agonist, Ro 25-1553, had no effect. The effects of PACAP-38 and VIP were blocked by the PAC(1) receptor antagonist, PACAP6-38. As observed with PACAP-38, the adenylyl cyclase activator, forskolin, also induced an increase in the number of neurite-bearing cells and an up-regulation in the expression of Bcl-2 and GAP-43. PACAP-induced differentiation was prevented by the adenylyl cyclase inhibitor, 2',5'-dideoxyadenosine (DDA), but not the protein kinase A (PKA) inhibitor, H89, or by siRNA-mediated knock-down of the PKA catalytic subunit. PACAP-38 and forskolin stimulated the activation of extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MAP; p38 MAP kinase) and c-Jun N-terminal kinase (JNK). PACAP-induced neuritogenesis was blocked by the MEK1 inhibitor PD98059 and partially by the p38 MAP kinase inhibitor SB203580. Activation of exchange protein directly activated by cAMP (Epac) partially mimicked the effects of PACAP-38, and led to the phosphorylation of ERK but not p38 MAP kinase. These results provide evidence that the neurotrophic effects of PACAP-38 on human SH-SY5Y neuroblastoma cells are mediated by the PAC(1) receptor through a cAMP-dependent but PKA-independent mechanism, and furthermore suggest that this involves Epac-dependent activation of ERK as well as activation of the p38 MAP kinase signaling pathway.

Characterisation of the mouse vasoactive intestinal peptide receptor type 2 gene, Vipr2, and identification of a polymorphic LINE-1-like sequence that confers altered promoter activity.

The VPAC(2) receptor is a seven transmembrane spanning G protein-coupled receptor for two neuropeptides, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). It has a distinct tissue-specific, developmental and inducible expression that underlies an important neuroendocrine role. Here, we report the characterisation of the gene that encodes the mouse VPAC(2) receptor (Vipr2), localisation of the transcriptional start site and functional analysis of the promoter region. The Vipr2 gene contains 12 introns within its protein-coding region and spans 68.6 kb. Comparison of the 5' untranslated region sequences for cloned 5'-RACE products amplified from different tissues showed they all were contained within the same exon, with the longest extending 111 bp upstream of the ATG start site. Functional analysis of the 3.2-kb 5'-flanking region using sequentially deleted sequences cloned into a luciferase gene reporter vector revealed that this region is active as a promoter in mouse AtT20 D16:16 and rat GH4C1 cell lines. The core promoter is located within a 180-bp GC-rich region proximal to the ATG start codon and contains potential binding sites for Sp1 and AP2, but no TATA-box. Further upstream, in two out of three mice strains examined, we have discovered a 496-bp polymorphic DNA sequence that bears a significant identity to mouse LINE-1 DNA. Comparison of the promoter activity between luciferase reporter gene constructs derived from the BALB/c (which contains this sequence) and C57BL/6J (which lacks this sequence) Vipr2 promoter regions has shown three-fold difference in luciferase gene activity when expressed in mouse AtT20 D16:16 and alphaT3-1 cells, but not when expressed in the rat GH4C1 cells or in COS 7 cells. Our results suggest that the mouse Vipr2 gene may be differentially active in different mouse strains, depending on the presence of this LINE-1-like sequence in the promoter region.

Characterization of novel splice variants of the PAC1 receptor in human neuroblastoma cells: consequences for signaling by VIP and PACAP.

Expression of VPAC and PAC1 receptor isoforms was determined in six neuroblastoma cell lines as well as in human embryonic and adult brain using reverse transcriptase PCR and quantitative PCR. PAC1 receptor splice variants missing a 21 amino acid sequence in the amino terminal domain were found to be the major receptor variants in the neuroblastoma cell lines and also were highly expressed in embryonic brain compared to adult brain. In four of the neuroblastoma cell lines, VIP and PACAP stimulated cyclic AMP production with different potencies and levels of maximal stimulation. High potency and greatest maximal stimulation of cyclic AMP for each peptide were recorded in SH-SY5Y cells, indicating the presence of high affinity VIP and PACAP receptors. Further characterization of specific VPAC and PAC1 receptor isoforms was carried out in the SH-SY5Y cell line, where along with known PAC1 receptor splice variants and the VPAC2 receptor, a number of novel PAC1 receptor splice variants were identified. The comparatively low level expression of the VPAC2 receptor along with the poor responsiveness of SH-SY5Y cells to the VPAC2 receptor-specific agonist Ro 25-1553 indicated that this receptor did not contribute significantly to the observed VIP responses. When the individual PAC1 receptor isoforms were expressed in COS 7 cells, the ability of VIP to activate cyclic AMP production was increased more than 50-fold at the majority of the PAC1 receptor variants lacking the 21 amino acid amino terminal domain sequence compared to those with the complete domain. Smaller changes were seen in the potency of PACAP-38. Similar trends were seen with inositol phosphate responses, where in each case agonist potencies were lower than for cyclic AMP production. The results of this study show that the combination of different amino terminal and intracellular loop 3 splicing variants in the PAC1 receptor dictates the ability of agonists, particularly VIP, to activate signaling pathways. VIP has considerably greater potency at most PAC1 receptors with the short amino terminal domain, and these therefore may mediate physiological effects of both VIP and PACAP. Furthermore, there may be a phenotypic switch in the expression of different PAC1 receptor amino terminal splice variants between embryonic and mature nervous system, indicating that regulation of this event may have an important role in VIP/PACAP function, particularly in the developing nervous system.

Specific interaction between the hop1 intracellular loop 3 domain of the human PAC(1) receptor and ARF.

The PAC(1), VPAC(1) and VPAC(2) receptors are members of the secretin (Group II) family of G protein-coupled receptors. All members of this family activate adenylate cyclase and several have also been shown to activate phospholipase C. We have recently reported that the rat VPAC(1), VPAC(2) and PAC(1) receptors activate phospholipase D and that distinct pathways are utilised by two intracellular loop 3 splice variants of PAC(1), one of which is ARF-dependent. Phospholipase D activation by the hop1, but not the null (short), form of the PAC(1) receptor is sensitive to brefeldin A, an inhibitor of GTP exchange at ARF. We have expressed the null and hop1 intracellular loop 3 domains of the human PAC(1) receptor in bacteria as GST-fusion proteins and used them as peptide affinity matrices to determine whether a functional interaction exists between these domains and ARF. Using this GST pull-down assay, we have shown binding of the small G protein ARF6 to the hop1 but not the null domain of this receptor.

Additional signals from VPAC/PAC family receptors.

The receptors for the neuropeptides vasoactive intestinal polypeptide and pituitary adenylate cyclase-activating polypeptide are strong activators of adenylate cyclase, but recent evidence suggests that they can elicit a number of additional intracellular signals. Some of these are likely to be downstream of the conventional adenylate cyclase pathway, but it is now clear that others reflect novel primary coupling events of the receptors.

ADP-ribosylation factor-dependent phospholipase D activation by VPAC receptors and a PAC(1) receptor splice variant.

The VPAC(1) and VPAC(2) receptors for vasoactive intestinal polypeptide and the PAC(1) receptor for pituitary adenylate cyclase-activating polypeptide are members of a subfamily of G protein-coupled receptors (GPCRs). We recently reported that phospholipase D (PLD) activation by members of the rhodopsin group of GPCRs occurs by at least two routes, one of which seems to involve the small G protein ADP-ribosylation factor (ARF) and its physical association with GPCRs. Here we report that rat VPAC and PAC(1) receptors can also stimulate PLD (albeit less potently than adenylate cyclase) in transfected cells and also in cells where they are natively expressed. PLD responses of the VPAC receptors and the hop1 spice variant of the PAC(1) receptor but not its null form are sensitive to brefeldin A (BFA), an inhibitor of GTP exchange at ARF. The presence of the hop1 cassette in the rat PAC(1) receptor facilitates PLD activation in the absence of marked changes in ligand binding, receptor internalization, and adenylate cyclase activation, with some reduction in phospholipase C activation. Both VPAC(2) and PAC(1-hop1) (but not PAC(1-null)) receptors were shown to associate with immunoprecipitates directed against native or epitope-tagged ARF. A chimeric construct of the VPAC(2) receptor body with intracellular loop 3 (i3) of the PAC(1-null) receptor mediated BFA-insensitive activation of PLD, whereas the response of the corresponding PAC(1-hop1) construct was BFA-sensitive. Motifs in i3 of the PAC(1-hop1) receptor may act as critical determinants of coupling to ARF-dependent PLD activation by contributing to the GPCR:ARF interface.

Mechanisms of phospholipase C activation by the vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide type 2 receptor.

The vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide type 2 (VPAC(2)) receptor was shown to induce both [(3)H]inositol phosphate ([(3)H]InsP)and cAMP production in transfected COS7 cells and in GH(3) cells where it is natively expressed. Neither cholera toxin nor forskolin could elicit an equivalent [(3)H]InsP response, suggesting independent coupling of the two pathways. The VPAC(2) receptor-mediated [(3)H]InsP response was partially inhibited by pertussis toxin (Ptx) and by the G beta gamma-sequestering C-terminal fragment of GRK2 (GRK2-ct) in COS7 and GH(3) cells, whereas responses of control receptors were unaffected. Blockers of receptor-activated Ca(2+) influx pathways (Co(2+) and SKF 96365) also partially inhibited VPAC(2) receptor-mediated [(3)H]InsP responses. This inhibition was not present in the component of the response remaining after Ptx treatment. A range of blockers of voltage-sensitive Ca(2+) channels were ineffective, consistent with the reported lack of these channels in COS7 cells. The data suggest that the VPAC(2) receptor may couple to phospholipase C through both Ptx-insensitive and Ptx-sensitive G proteins (G(q/11) and G(i/o), respectively) to generate [(3)H]InsP. In addition to G beta gamma, G(i/o) activation appears to require receptor-activated Ca(2+) entry. This is consistent with the possibility that not only G alpha(q/11)-responsive and G beta gamma-responsive isoforms of phospholipase C but also Ca(2+)-responsive forms may contribute to the overall [(3)H]InsP response.

Gonadotropin-releasing hormone receptor activation of extracellular signal-regulated kinase and tyrosine kinases in transfected GH3 cells and in alphaT3-1 cells.

GH3 cells were stably transfected with the wild-type murine GnRH receptor and a clonal cell line selected on the basis of inositol phosphate production and PRL/GH release in response to GnRH. This cell line (wt28) was characterized by [125I]GnRH analog binding, [3H]inositol phosphate response to GnRH, and hormone secretion. We examined the activation of the mitogen-activated protein kinase isoforms, extracellular signal-regulated kinase 1/2 (ERK1/2) and tyrosine kinases in wt28 cells and alphaT3-1 cells (which express a native GnRH) using specific phospho-ERK1/2 and phosphotyrosine antibodies. Concentration-response and time-course data revealed that a sustained ERK1/2 response was seen only in aT3-1 cells. Furthermore, GnRH-induced tyrosine phosphorylation was detectable in alphaT3-1 cells, but not in wt28 cells. Activators for several different signaling pathways revealed distinct differences between the cell types. Protein kinase C activation by phorbol 12,13-dibutyrate was very effective in alphaT3-1 cells at phosphorylation of both ERK1/2 and tyrosine, whereas raising cAMP levels using forskolin also strongly increased wt28 cell ERK1/2 phosphorylation. Only the tyrosine phosphatase inhibitor pervanadate increased tyrosine phosphorylation in wt28 cells. The lack of sustained ERK1/2 phosphorylation in wt28 cells could be the result of minimal tyrosine kinase activation by GnRH compounded by a different pathway profile for ERK1/2 activation. When pervanadate and GnRH were combined, ERK1/2 phosphorylation was synergistic and sustained in wt28 cells, whereas the response was additive in alphaT3-1 cells. In sum, the intracellular pathways leading to ERK1/2 and tyrosine phosphorylation in alphaT3-1 and wt28 cells are distinct; thus, activating GnRH receptors in each of the two cell types leads to different sequelae of events regarding ERK1/2 activation.

Differential activation of phospholipase D by VPAC and PAC1 receptors.

To investigate the phospholipase D (PLD) responses of the VIP/PACAP receptors, VPAC1 and VPAC2, and the PACAP-specific PAC1 receptors (short and "hop" intracellular loop 3 (i3) splice variants), stable CHO cell lines expressing similar levels of each wildtype receptor were generated (except for the VPAC1 receptor clone which showed considerably lower expression and lesser responses in signalling assays). All clones caused activation of PLD in response to agonists, as monitored by [3H]phosphatidylbutanol production. The PLD responses of the PAC1 "hop", but not the "null" receptor, were sensitive to the ARF inhibitor, brefeldin A (BFA) (as were VPAC1 and VPAC2 responses). Chimeric constructs of VPAC2 receptors containing i3 of either PAC1 hop or PAC1 null receptors were transiently expressed in COS 7 cells and PLD responses were measured. Only the PLD response of the hop construct was sensitive to BFA. This suggests that i3 motifs in certain Group II GPCRs may play a key role in determining their linkage to ARF-dependent PLD activation.

Domains determining agonist selectivity in chimaeric VIP2 (VPAC2)/PACAP (PAC1) receptors.

1 The VPAC2 and PAC1 receptors are closely related members of the Group II G protein-coupled receptor family. At the VPAC2 receptor, VIP is equipotent to PACAP-38 in stimulating cyclic AMP production, whereas at the PAC1 receptor PACAP-38 is many fold more potent than VIP. In this study, domains which confer this selectivity were investigated by constructing four chimaeric receptors in which segments of the VPAC2 receptor were exchanged with the corresponding segment from the PAC1 receptor. 2 When expressed in COS 7 cells all the chimaeric receptors bound the common ligand [125I]PACAP-27 and produced cyclic AMP in response to agonists. 3 Relative selectivity for agonists was determined primarily by the amino terminal extracellular domain of the PAC1 receptor and the VPAC2 receptor. The interchange of other domains had little effect on the potency of PACAP-38 or PACAP-27. 4 For chimaeric constructs with a PAC1 receptor amino terminal domain, the substitution of increasing portions of the VPAC2 receptor decreased the potency of VIP yet increased that of helodermin. 5 This suggests that the interaction of VIP/helodermin but not PACAP with the PAC1 receptor may be influenced (and differentially so) by additional receptor domains.

Structure of the human VIPR2 gene for vasoactive intestinal peptide receptor type 2.

The VPAC(2) (vasoactive intestinal peptide (VIP)(2)) receptor is a seven-transmembrane spanning G protein-coupled receptor which responds similarly to VIP and pituitary adenylate cyclase activating polypeptide (PACAP) in stimulating cAMP production. Recently, we reported the localisation of the human VPAC(2) receptor gene (VIPR2) to chromosome 7q36.3 (Mackay, M. et al. (1996) Genomics 37, 345-353). Here, we describe the characterisation of the VIPR2 gene structure and promoter region. The VIPR2 gene is encoded by 13 exons, the initiator codon of the 438 amino acid open reading frame is located in exon 1 and the termination signal and a poly-adenylation signal sequence are located in exon 13. The 5' untranslated region extends 187 bp upstream of the initiator codon and is extremely GC-rich (80%). The poly-adenylation signal is located 2416 bp downstream of the stop codon. Intron sizes range from 68 bp (intron 11) to 45 kb (intron 4) and the human gene spans 117 kb.

Expression of PACAP, and PACAP type 1 (PAC1) receptor mRNA during development of the mouse embryo.

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) have been reported to have a number of neurotrophic effects. We have examined the expression of mRNA for PACAP and PACAP type 1 (PAC1) receptor in the mouse embryo by in situ hybridization and the effects of PACAP and VIP on the growth of mouse embryos in vitro. Although we were unable to detect gross effects of either peptide on the growth rates of embryos maintained in culture, mRNAs for both PAC1 receptor and PACAP peptide were present in the nervous system from day 9.5 of embryonic development. PAC1 receptor mRNA was most abundant in the neural tube and the rhombencephalon and was present also in the dorsal root and trigeminal ganglia and the sympathetic chain. The distribution of mRNA for the PACAP peptide overlapped in part with that of the receptor, but was more extensively distributed in the rhombencephalon and in the developing hypothalamus. Within the neural tube, PAC1 receptor mRNA was located in the roof and floor plates, while the distribution of PACAP peptide mRNA was more complex, being located in two columns of cells in the ventromedial neural tube (consistent with the position of developing autonomic motor neurons) and in cells in the dorsolateral neural tube. These data are concordant with a role for PACAP or a related peptide in neural development.

Evidence for roles of vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) receptors in modulating the responses of rat dorsal horn neurons to sensory inputs.

The extracellularly recorded electrophysiological activity of single multireceptive dorsal horn neurons was markedly increased by ionophoretic administration of vasoactive intestinal polypeptide (VIP) or pituitary adenylate cyclase activating polypeptide (PACAP)-38. Some cells responded selectively to PACAP-38 (suggesting mediation by a PACAP receptor), whereas others responded to both VIP and PACAP-38 (suggesting a VIP1 and/or VIP2 receptor). Most non-nociceptive cells were unaffected by PACAP-38 and all were unaffected by VIP. The selectivity of VIP/PACAP receptor antagonists was established on cloned rat VIP1, VIP2 and PACAP receptors in vitro before their utilization to indicate the likely involvement of VIP1, and possibly PACAP receptors, in VIP- and PACAP-38-mediated responses of dorsal horn neurons. The VIP/PACAP receptor antagonists inhibited responses of multireceptive cells to sustained innocuous (brush) and noxious (mustard oil) stimuli, with a selectivity suggesting the involvement of VIP1 and PACAP receptors, although the participation by VIP2 receptors cannot be excluded. These data implicate both VIP and PACAP in regulating the basal responsiveness of multireceptive dorsal horn neurons to sensory stimuli.

The peptide pQFYRFamide is encoded on the FMRFamide precursor of the snail Helix aspersa but does not activate the FMRFamide-gated sodium current.

The complete sequence of the FMRFamide precursor cDNA from Helix aspersa is reported here. Since the 5' end of this cDNA is identical to that of the precursor that encodes the heptapeptide analogs of FLRFamide, the two transcripts are probably derived by alternative RNA splicing. A novel pentapeptide, Glp-Phe-Tyr-Arg-Phe-NH2 (pQFYRFamide), predicted from the cDNA sequence, was purified from extracts of H. aspersa ganglia and identified by mass spectroscopy. Partial gene sequences for the FMRFamide precursors of two closely related pulmonate species-Cepaea nemoralis and Polydontes acutangula-were also determined and compared with the cDNA sequence of H. aspersa and a partial gene sequence previously determined from H. pomatia. Not only are the FMRFamide-related sequences and proteolytic processing sites conserved, but the linear arrangement of these landmarks is also retained. Synthetic pQFYRFamide has some effects on the isolated heart and on neuronal potassium currents in H. aspersa that are similar to those of FMRFamide, but it does not activate the neuronal FMRFamide-gated sodium channel.

Expression of pituitary adenylate cyclase activating polypeptide receptors in the early mouse embryo as assessed by reverse transcription polymerase chain reaction and in situ hybridisation.

We have examined the expression of mRNA for the pituitary adenylate cyclase activating polypeptide (PACAP) receptor in the mouse from day 9.5 of embryonic development onwards by reverse transcription polymerase chain reaction (RT-PCR) and by in situ hybridisation. PACAP receptor mRNAs were detected by PCR in the earliest developmental stage examined and thereafter the levels of mRNA for all receptors increased during development. Wholemount in situ hybridisation first showed the expression of the PACAP receptor in the neural tube of 9.5 day old embryos, in later embryos heaviest expression of the PACAP receptor was found in the developing rhombencephalon (day 10.5 and 11.5). These results are consistent with a role for PACAP or a related peptide in the early development of the central nervous system.

The distribution of vasoactive intestinal peptide2 receptor messenger RNA in the rat brain and pituitary gland as assessed by in situ hybridization.

The distribution of rat vasoactive intestinal peptide2 (VIP2) receptor messenger RNA in the brain and the pituitary gland was examined by in situ hybridization and by ribonuclease protection assay. labelled cells were found chiefly in the suprachiasmatic nucleus, the central nucleus of the amygdala and the thalamus (the lateral geniculate nucleus, and the paraventricular, mediodorsal and ventral nuclei of the thalamus). The distribution of the VIP2 receptor overlaps only in part with that of the VIP1 receptor, for example in the hippocampus, where VIP2 receptor messenger RNA was found in the pyramidal cells of the CA1-CA3 subfields and in the granule cells of the dentate gyrus. Small numbers of neurons containing high concentrations of VIP2 receptor messenger RNA were present in the brainstem in the principal sensory trigeminal nucleus and in the substantia gelatinosa of the spinal cord, suggesting a role for the VIP2 receptor in the processing of sensory information. The presence of the VIP2 receptor in the suprachiasmatic nucleus suggests that it is this receptor subtype which is involved in the control of circadian rhythms.