Skip to main content

Advertisement

SpringerLink
Log in

Activation of MEK/ERK Signaling by PACAP in Guinea Pig Cardiac Neurons

  • Published:
Journal of Molecular Neuroscience Aims and scope Submit manuscript

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP) signaling can increase guinea pig cardiac neuron excitability in part through extracellular signal-regulated kinase (ERK) activation. The present study examined the PACAP receptors and signaling cascades that stimulate guinea pig cardiac neuron ERK signaling using confocal microscopy to quantify PACAP-induced neuronal phosphorylated ERK (pERK) immunoreactivity. PACAP and maxadilan, but not vasoactive intestinal polypeptide (VIP), increased cardiac neuron pERK, implicating primary roles for PACAP-selective PAC1 receptor (Adcyap1r1) signaling rather than VPAC receptors (Vipr1 and Vipr2) in the generation of cardiac neuron pERK. The adenylyl cyclase (AC) activator forskolin, but not the protein kinase C (PKC) activator phorbol myristate acetate (PMA), increased pERK. Also, Bim1 did not blunt PACAP activation of pERK. Together, the results suggest PAC1 receptor signal transduction via Gs/adenylyl cyclase (AC)/cAMP rather than Gq/phospholipase C (PLC) generated neuronal pERK. Activator and inhibitor studies suggested that the PACAP-mediated pERK activation was PKA-dependent rather than an exchange protein directly activated by a cAMP (EPAC), PKA-independent mechanism. The PACAP-induced pERK was inhibited by the clathrin inhibitor Pitstop2 to block receptor internalization and endosomal signaling. We propose that the PACAP-mediated MEK/ERK activation in cardiac neurons involves both AC/cAMP/PKA signaling and PAC1 receptor internalization/activation of signaling endosomes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Barrie AP, Clohessy AM, Buensuceso CS, Rogers MV, Allen JM (1997) Pituitary adenylyl cyclase-activating peptide stimulates extracellular signal-regulated kinase 1 or 2 (ERK1/2) activity in a Ras-independent, mitogen-activated protein kinase/ERK kinase 1 or 2-dependent manner in PC12 cells. J Biol Chem 272:19666–19671

    Article  CAS  PubMed  Google Scholar 

  • Botia B, Basille M, Allais A, Raoult E, Falluel-Morel A, Galas L, Jolivel V, Wurtz O, Komuro H, Fournier A, Vaudry H, Burel D, Gonzalez BJ, Vaudry D (2007) Neurotrophic effects of PACAP in the cerebellar cortex. Peptides 28:746–752

    Article  Google Scholar 

  • Bouschet T, Perez V, Fernandez C, Bockaert J, Eychene A, Journot L (2003) Stimulation of the ERK pathway by GTP-loaded Rap1 requires the concomitant activation of Ras, protein kinase C, and protein kinase A in neuronal cells. J Biol Chem 278:4778–4785

    Article  CAS  PubMed  Google Scholar 

  • Braas KM, May V (1999) Pituitary adenylate cyclase-activating polypeptides directly stimulate sympathetic neuron neuropeptide Y release through PAC(1) receptor isoform activation of specific intracellular signaling pathways. J Biol Chem 274:27702–27710

    Article  CAS  PubMed  Google Scholar 

  • Braas KM, May V, Harakall SA, Hardwick JC, Parsons RL (1998) Pituitary adenylate cyclase-activating polypeptide expression and modulation of neuronal excitability in guinea pig cardiac ganglia. J Neurosci 18:9766–9779

    CAS  PubMed  Google Scholar 

  • Broca C, Quoyer J, Costes S, Linck N, Varrault A, Deffayet PM, Bockaert J, Dalle S, Bertrand G (2009) Beta-arrestin 1 is required for PAC1 receptor-mediated potentiation of long-lasting ERK1/2 activation by glucose in pancreatic beta-cells. J Biol Chem 284:4332–4342

    Article  CAS  PubMed  Google Scholar 

  • Calebiro D, Nikolaev VO, Persani L, Lohse MJ (2010) Signaling by internalized G-protein-coupled receptors. Trends Pharmacol Sci 3:221–228

    Article  Google Scholar 

  • Calupca MA, Vizzard MA, Parsons RL (2000) Origin of pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive fibers innervating guinea pig parasympathetic cardiac ganglia. J Comp Neurol 423:26–39

    Article  CAS  PubMed  Google Scholar 

  • Emery AC, Eiden LE (2012) Signaling through the neuropeptide GPCR PAC1 induces neuritogenesis via a single linear cAMP- and ERK-dependent pathway using a novel cAMP sensor. FASEB J 26:3199–3211

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Emery A.C., Eiden M.V., Mustafa T., and Eiden L.E. (2013) Rapgef2 connects GPCR-mediated cAMP signals to ERK activation in neuronal and endocrine cells. Sci Signal. 6, ra51

  • Goldsmith ZG, Dhanasekaran DN (2007) G protein regulation of MAPK networks. Oncogene 26(22): 3122–3142

  • Gupte RP, Kadunganattil S, Shepherd AJ, Merrill R, Planer W, Bruchas MR, Strack S, Mohapatra DP (2016) Convergent phosphomodulation of the major neuronal dendritic potassium channel Kv4.2 by pituitary adenylate cyclase-activating polypeptide. Neuropharmacology. 101:291–308

    Article  CAS  PubMed  Google Scholar 

  • Harmar AJ, Fahrenkrug J, Gozes I, Laburthe M, May V, Pisegna JR, Vaudry D, Vaudry H, Waschek JA, Said SI (2012) Pharmacology and functions of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide: IUPHAR review 1. Br J Pharmacol 166:4–17

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hoover DB, Tompkins JD, Parsons RL (2009) Differential activation of guinea pig intrinsic cardiac neurons by the PAC1 agonists maxadilan and pituitary adenylate cyclase-activating polypeptide 27 (PACAP27). J Pharmacol Exp Ther 331:197–203

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Irannejad R, von Zastrow M (2014) GPCR signaling along the endocytic pathway. Curr Opin Cell Biol 27:109–116

    Article  CAS  PubMed  Google Scholar 

  • Irannejad R, Tomshine JC, Tomshine JR, Chevalier M, Mahoney JP, Steyaert J, Rasmussen SG, Sunahara RK, El-Samad H, Huang B, von Zastrow M (2013) Conformational biosensors reveal GPCR signalling from endosomes. Nature 495:534–538

    Article  CAS  PubMed  Google Scholar 

  • Kao S, Jaiswal RK, Kolch W, Landreth GE (2001) Identification of the mechanisms regulating the differential activation of the mapk cascade by epidermal growth factor and nerve growth factor in PC12 cells. J Biol Chem 276:18169–18177

    Article  CAS  PubMed  Google Scholar 

  • Lazarovici P, Jiang H, Fink D Jr (1998) The 38-amino-acid form of pituitary adenylate cyclase-activating polypeptide induces neurite outgrowth in PC12 cells that is dependent on protein kinase C and extracellular signal-regulated kinase but not on protein kinase A, nerve growth factor receptor tyrosine kinase, p21(ras) G protein, and pp60(c-src) cytoplasmic tyrosine kinase. Mol Pharmacol 54:547–558

  • Liu C, Takahashi M, Li Y, Song S, Dillon TJ, Shinde U, Stork PJ (2008) Ras is required for the cyclic AMP-dependent activation of Rap1 via Epac2. Mol Cell Biol 28:7109–7125

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Marshall CJ (1995) Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80:179–185

    Article  CAS  PubMed  Google Scholar 

  • May V, Lutz E, MacKenzie C, Schutz KC, Dozark K, Braas KM (2010) Pituitary adenylate cyclase-activating polypeptide (PACAP)/PAC1HOP1 receptor activation coordinates multiple neurotrophic signaling pathways: Akt activation through phosphatidylinositol 3-kinase gamma and vesicle endocytosis for neuronal survival. J Biol Chem 285:9749–9761

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • May V, Buttolph TR, Girard BM, Clason TA, Parsons RL (2014) PACAP-induced ERK activation in HEK cells expressing PAC1 receptors involves both receptor internalization and PKC signaling. Am J Physiol Cell Physiol 306:C1068–C1079

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Merriam LA, Baran CN, Girard BM, Hardwick JC, May V, Parsons RL (2013) Pituitary adenylate cyclase 1 receptor internalization and endosomal signaling mediate the pituitary adenylate cyclase activating polypeptide-induced increase in guinea pig cardiac neuron excitability. J Neurosci 33:4614–4622

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Monaghan TK, Mackenzie CJ, Plevin R, Lutz EM (2008) PACAP-38 induces neuronal differentiation of human SH-SY5Y neuroblastoma cells via cAMP-mediated activation of ERK and p38 MAP kinases. J Neurochem 104:74–88

    CAS  PubMed  PubMed Central  Google Scholar 

  • Moro O, Lerner EA (1997) Maxidilan the vasodilator from sand flies, is a specific pituitary adenlyate cyclase activating peptide type I receptor agonist. J Biol Chem 272:966–970

    Article  CAS  PubMed  Google Scholar 

  • Murphy JE, Padilla BE, Hasdemir B, Cottrell GS, Bunnett NW (2009) Endosomes: a legitimate platform for the signaling train. Proc Natl Acad Sci U S A 106:17615–17622

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Oakley RH, Laporte SA, Holt JA, Barak LS, Caron MG (1999) Association of beta-arrestin with G protein-coupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization. J Biol Chem 274:32248–32257

    Article  CAS  PubMed  Google Scholar 

  • Oakley RH, Laporte SA, Holt JA, Caron MG, Barak LS (2000) Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G protein-coupled receptors delineate two major classes of receptors. J Biol Chem 275:17201–17210

    Article  CAS  PubMed  Google Scholar 

  • Obara Y, Horgan AM, Stork PJ (2007) The requirement of Ras and Rap1 for the activation of ERKs by cAMP, PACAP, and KCl in cerebellar granule cells. J Neurochem 101:470–482

    Article  CAS  PubMed  Google Scholar 

  • Pellegrino MJ, Stork PJ (2006) Sustained activation of extracellular signal-regulated kinase by nerve growth factor regulates c-fos protein stabilization and transactivation in PC12 cells. J Neurochem 99:1480–1493

    Article  CAS  PubMed  Google Scholar 

  • Shenoy SK, Lefkowitz RJ (2003) Multifaceted roles of beta-arrestins in the regulation of seven-membrane-spanning receptor trafficking and signalling. Biochem J 375:503–515

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shi GX, Rehmann H, Andres DA (2006) A novel cyclic AMP-dependent Epac-Rit signaling pathway contributes to PACAP38-mediated neuronal differentiation. Mol Cell Biol 26:9136–9147

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Spengler D, Waeber C, Pantaloni C, Holsboer F, Bockaert J, Seeburg PH, Journot L (1993) Differential signal transduction by five splice variants of the PACAP receptor. Nature 365:170–175

    Article  CAS  PubMed  Google Scholar 

  • Tompkins JD, Parsons RL (2008) Identification of intracellular signaling cascades mediating the PACAP-induced increase in guinea pig cardiac neuron excitability. J Mol Neurosci 36:292–298

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tompkins JD, Hardwick JC, Locknar SA, Merriam LA, Parsons RL (2006) Ca2+ influx, but not Ca2+ release from internal stores, is required for the PACAP-induced increase in excitability in guinea pig intracardiac neurons. J Neurophysiol 95:2134–2142

    Article  CAS  PubMed  Google Scholar 

  • Tompkins JD, Ardell JL, Hoover DB, Parsons RL (2007) Neurally released pituitary adenylate cyclase-activating polypeptide enhances guinea pig intrinsic cardiac neurone excitability. J Physiol 582:87–93

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tsvetanova NG, Irannejad R, Zastrow M (2015) G protein-coupled receptor (GPCR) signaling via heterotrimeric G proteins from endosomes. J Biol Chem 290:6689–6696

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Villalba M, Bockaert J, Journot L (1997) Pituitary adenylate cyclase-activating polypeptide (PACAP-38) protects cerebellar granule neurons from apoptosis by activating the mitogen-activated protein kinase (MAP kinase) pathway. J Neurosci 1:83–90

    Google Scholar 

  • von Kriegsheim A, Baiocchi D, Birtwistle M, Sumpton D, Bienvenut W, Morrice N, Yamada K, Lamond A, Kalna G, Orton R, Gilbert D, Kolch W (2009) Cell fate decisions are specified by the dynamic ERK interactome. Nat Cell Biol 11:1458–1464

    Article  Google Scholar 

  • Wang Z, Dillon TJ, Pokala V, Mishra S, Labudda K, Hunter B, Stork PJ (2006) Rap1-mediated activation of extracellular signal-regulated kinases by cyclic AMP is dependent on the mode of Rap1 activation. Mol Cell Biol 26:2130–2145

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wortzel I, Seger R (2011) The ERK cascade: distinct functions within various subcellular organelles. Genes Cancer 2:195–209

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yoon S, Seger R (2006) The extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions. Growth Factors 24:21–44

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported in part by NIH grant NIGMS P30 GM103498/NCRR P30 RR032135 (RLP) and S10 OD017969-01 (RLP).

Author Contributions

RLP and VM designed the experiments, TAC and BMG collected and analyzed the data, RLP and VM interpreted the data, RLP and VM wrote the paper with contributions from TAC and BMG. All authors approved the final version.

Author information

Authors and Affiliations

  1. Department of Neurological Sciences, College of Medicine, University of Vermont, Burlington, VT, 05405, USA

    Todd A. Clason, Beatrice M. Girard, Victor May & Rodney L. Parsons

Authors
  1. Todd A. Clason
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Beatrice M. Girard
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Victor May
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rodney L. Parsons
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Rodney L. Parsons.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Clason, T.A., Girard, B.M., May, V. et al. Activation of MEK/ERK Signaling by PACAP in Guinea Pig Cardiac Neurons. J Mol Neurosci 59, 309–316 (2016). https://doi.org/10.1007/s12031-016-0766-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12031-016-0766-z

Keywords

Search

Navigation