Journal of Molecular Neuroscience

, Volume 36, Issue 1–3, pp 299–309 | Cite as

Expression of Phosphorylated cAMP Response Element Binding Protein (p-CREB) in Bladder Afferent Pathways in VIP−/− Mice with Cyclophosphamide (CYP)-Induced Cystitis

  • Dorthe G. Jensen
  • Simon Studeny
  • Victor May
  • James Waschek
  • Margaret A. Vizzard


The expression of phosphorylated cAMP response element binding protein (p-CREB) in dorsal root ganglia (DRG) with and without cyclophosphamide (CYP)-induced cystitis (150 mg/kg, i.p; 48 h) was determined in VIP−/− and wild-type (WT) mice. p-CREB immunoreactivity (IR) was determined in bladder (Fast blue) afferent cells. Nerve growth factor (NGF) bladder content was determined by enzyme-linked immunosorbent assays. Basal expression of p-CREB-IR in DRG of VIP−/− mice was (p ≤ 0.01) greater in L1, L2, L5-S1 DRG compared to WT mice. CYP treatment in WT mice increased (p ≤ 0.05) p-CREB-IR in L1, L2, L5-S1 DRG. CYP treatment in VIP−/− mice (p ≤ 0.01) increased (p ≤ 0.01) p-CREB-IR in L6-S1 DRG compared to WT with CYP. In WT mice, bladder afferent cells (20–38%) in DRG expressed p-CREB-IR under basal conditions. With CYP, p-CREB-IR increased in bladder afferent cells (60–65%; L6-S1 DRG) in WT mice. In VIP−/− mice, bladder afferent cells (12–58%) expressed p-CREB-IR under basal conditions, and CYP increased p-CREB expression (78–84%) in L6-S1 DRG. Urinary bladder NGF expression in VIP−/− mice under basal conditions or after cystitis was significantly greater than WT. Detrusor smooth muscle thickness was significantly increased in VIP−/− mice. Bladder NGF expression may contribute to differences in p-CREB expression.


Inflammation Urinary bladder Afferent neurons Growth factors Cytokines 



The authors acknowledge the technical support of Susan E. Malley. This work was funded by NIH grants DK051369, DK060481, and DK065989. D.G. Jensen was supported by Drug Research Academy at Copenhagen University and Ferring Pharmaceutical, Copenhagen.


  1. Abad, C., Martinez, C., Juarranz, M. G., et al. (2003). Therapeutic effects of vasoactive intestinal peptide in the trinitrobenzene sulfonic acid mice model of Crohn’s disease. Gastroenterology, 124, 961–971.PubMedCrossRefGoogle Scholar
  2. Amir, R., & Devor, M. (1996). Chemically mediated cross-excitation in rat dorsal root ganglia. Journal of Neuroscience, 16, 4733–4741.PubMedGoogle Scholar
  3. Arthur, J. S., Fong, A. L., Dwyer, J. M., et al. (2004). Mitogen- and stress-activated protein kinase 1 mediates cAMP response element-binding protein phosphorylation and activation by neurotrophins. Journal of Neuroscience, 24, 4324–4332.PubMedCrossRefGoogle Scholar
  4. Avelino, A., Cruz, C., & Cruz, F. (2002). Nerve growth factor regulates galanin and c-jun overexpression occurring in dorsal root ganglion cells after intravesical resiniferatoxin application. Brain Research, 951, 264–269.PubMedCrossRefGoogle Scholar
  5. Bancroft, J. D., & Stevens, A. (1990). Theory and practice of histological techniques. New York: Churchill Livingstone.Google Scholar
  6. Bik, W., Wolinska-Witort, E., Chmielowska, M., Baranowska-Bik, A., Rusiecka-Kuczalek, E., & Baranowska, B. (2004). Vasoactive intestinal peptide can modulate immune and endocrine responses during lipopolysaccharide-induced acute inflammation. Neuroimmunomodulation, 11, 358–364.PubMedCrossRefGoogle Scholar
  7. Braas, K. M., May, V., Zvara, P., et al. (2006). Role for pituitary adenylate cyclase activating polypeptide in cystitis-induced plasticity of micturition reflexes. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology, 290, R951–R962.PubMedGoogle Scholar
  8. Chapple, C. R., Milner, P., Moss, H. E., & Burnstock, G. (1992). Loss of sensory neuropeptides in the obstructed human bladder. British Journal of Urology, 70, 373–381.PubMedGoogle Scholar
  9. Chorny, A., Gonzalez-Rey, E., Varela, N., Robledo, G., & Delgado, M. (2006). Signaling mechanisms of vasoactive intestinal peptide in inflammatory conditions. Regulatory Peptide, 137, 67–74.CrossRefGoogle Scholar
  10. Colwell, C. S., Michel, S., Itri, J., Rodriguez, W., Tam, J., Lelievre, V., et al. (2003). Disrupted circadian rhythms in VIP- and PHI-deficient mice. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology, 285, R939–949.PubMedGoogle Scholar
  11. Cox, P. J. (1979). Cyclophosphamide cystitis—identification of acrolein as the causative agent. Biochemical Pharmacology, 28, 2045.PubMedCrossRefGoogle Scholar
  12. Delgado, M., Gomariz, R. P., Martinez, C., Abad, C., & Leceta, J. (2000). Anti-inflammatory properties of the type 1 and type 2 vasoactive intestinal peptide receptors: Role in lethal endotoxic shock. European Journal of Immunology, 30, 3236–3246.PubMedCrossRefGoogle Scholar
  13. Delgado, M., Munoz-Elias, E. J., Gomariz, R. P., & Ganea, D. (1999). Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide enhance IL-10 production by murine macrophages: In vitro and in vivo studies. Journal of Immunology, 162, 1707–1716.Google Scholar
  14. Dickinson, T., Mitchell, R., Robberecht, P., & Fleetwood-Walker, S. M. (1999). The role of VIP/PACAP receptor subtypes in spinal somatosensory processing in rats with experimental peripheral mononeuropathy. Neuropharmacology, 38, 167–180.PubMedCrossRefGoogle Scholar
  15. Dmitrieva, N., Shelton, D., Rice, A. S. C., & McMahon, S. B. (1997). The role of nerve growth factor in a model of visceral inflammation. Neuroscience, 78, 449–459.PubMedCrossRefGoogle Scholar
  16. Donovan, M. K., Winternitz, S. R., & Wyss, J. M. (1983). An analysis of the sensory innervation of the urinary system of the rat. Brain Research Bulletin, 11, 321–324.PubMedCrossRefGoogle Scholar
  17. Driscoll, A., & Teichman, J. M. H. (2001). How do patients with interstitial cystitis present? Journal of Urology, 166, 2118–2120.PubMedCrossRefGoogle Scholar
  18. Erol, K., Ulak, G., Donmez, T., Cingi, M. I., Alpan, R. S., & Ozdemir, M. (1992). Effects of vasoactive intestinal polypeptide on isolated rat urinary bladder smooth muscle. Urology International, 49, 151–153.CrossRefGoogle Scholar
  19. Girard, B. A., Lelievre, V., Braas, K. M., et al. (2006). Noncompensation in peptide/receptor gene expression and distinct behavioral phenotypes in VIP- and PACAP-deficient mice. Journal of Neurochemistry, 99, 499–513.PubMedCrossRefGoogle Scholar
  20. Gold, B. G., Storm-Dickerson, T., & Austin, D. R. (1993). Regulation of the transcription factor c-JUN by nerve growth factor in adult sensory neurons. Neuroscience Letters, 154, 129–133.PubMedCrossRefGoogle Scholar
  21. Gonzalez-Rey, E., & Delgado, M. (2006). Therapeutic treatment of experimental colitis with regulatory dendritic cells generated with vasoactive intestinal peptide. Gastroenterology, 131, 1799–1811.PubMedCrossRefGoogle Scholar
  22. Harmar, A. J., Arimura, A., Gozes, I., et al. (1998). International Union of Pharmacology. XVIII. Nomenclature of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. Pharmacological Reviews, 50, 265–270.PubMedGoogle Scholar
  23. Hawley, R. J., Scheibe, R. J., & Wagner, J. A. (1992). NGF induces the expression of the VGF gene through a cAMP response element. Journal of Neuroscience, 12, 2573–2581.PubMedGoogle Scholar
  24. Hernandez, M., Barahona, M. V., Recio, P., et al. (2006). Neuronal and smooth muscle receptors involved in the PACAP- and VIP-induced relaxations of the pig urinary bladder neck. British Journal of Urology, 149, 100–109.Google Scholar
  25. Ho, N., Koziol, J. A., & Parsons, C. L. (1997). Epidemiology of interstitial cystitis. In G. R. Sant (Ed.) Interstitial cystitis (pp. 9–16). Philadelphia: Lippincott-Raven Publishers.Google Scholar
  26. Hu, V. Y., Zvara, P., Dattilio, A., et al. (2005). Decrease in bladder overactivity with REN1820 in rats with cyclophosphamide induced cystitis. Journal of Urology, 173, 1016–1021.PubMedCrossRefGoogle Scholar
  27. Igawa, Y., Persson, K., Andersson, K. E., Uvelius, B., & Mattiasson, A. (1993). Facilitatory effect of vasoactive intestinal polypeptide on spinal and peripheral micturition reflex pathways in conscious rats with and without detrusor instability. Journal of Urology, 149, 884–889.PubMedGoogle Scholar
  28. Ishihara, T., Shigemoto, R., Mori, K., Takahashi, K., & Nagata, S. (1992). Functional expression and tissue distribution of a novel receptor for vasoactive intestinal polypeptide. Neuron, 8, 811–819.PubMedCrossRefGoogle Scholar
  29. Jaggar, S. I., Scott, H. C., & Rice, A. S. (1999). Inflammation of the rat urinary bladder is associated with a referred thermal hyperalgesia which is nerve growth factor dependent. British Journal of Anaesthesia, 83, 442–448.PubMedGoogle Scholar
  30. Jennings, L. J., & Vizzard, M. A. (1999). Cyclophosphamide-induced inflammation of the urinary bladder alters electrical properties of small diameter afferent neurons from dorsal root ganglia. FASEB Journal, 13, A57.Google Scholar
  31. Ji, R. R., & Rupp, F. (1997). Phosphorylation of transcription factor CREB in rat spinal cord after formalin-induced hyperalgesia: Relationship to c-fos induction. Journal of Neuroscience, 17, 1776–1785.PubMedGoogle Scholar
  32. Johansson, S. L., Ogawa, K., & Fall, M. (1997). The pathology of interstitial cystitis. In G. R. Sant (Ed.) Interstitial cystitis (pp. 143–152). Lippincott-Raven Publishers: Philadelphia.Google Scholar
  33. Juarranz, Y., Abad, C., Martinez, C., et al. (2005). Protective effect of vasoactive intestinal peptide on bone destruction in the collagen-induced arthritis model of rheumatoid arthritis. Arthritis Research & Therapy, 7, R1034–R1045.CrossRefGoogle Scholar
  34. Karin, M., Liu, Z., & Zandi, E. (1997). AP-1 function and regulation. Current Opinion in Cell Biology, 9, 240–246.PubMedCrossRefGoogle Scholar
  35. Keast, J. R., & de Groat, W. C. (1992). Segmental distribution and peptide content of primary afferent neurons innervating the urogenital organs and colon of male rats. Journal of Comparative Neurology, 319, 615–623.PubMedCrossRefGoogle Scholar
  36. Lantéri-Minet, M., Bon, K., de Pommery, J., Michiels, J. F., & Menétrey, D. (1995). Cyclophosphamide cystitis as a model of visceral pain in rats: Model elaboration and spinal structures involves as revealed by the expression of c-Fos and Krox-24 proteins. Experimental Brain Research, 105, 220–232.CrossRefGoogle Scholar
  37. Lasanen, L. T., Tammela, T. L., Liesi, P., Waris, T., & Polak, J. M. (1992). The effect of acute distension on vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and substance P (SP) immunoreactive nerves in the female rat urinary bladder. Urological Research, 20, 259–263.PubMedCrossRefGoogle Scholar
  38. Lelievre, V., Favrais, G., Abad, C., et al. (1997). Gastrointestinal dysfunction in mice with a targeted mutation in the gene encoding vasoactive intestinal polypeptide: A model for the study of intestinal ileus and Hirschsprung’s disease. Peptides, 28, 1688–1699.CrossRefGoogle Scholar
  39. Maggi, C. A., Lecci, A., Santiciolo, P., Del Biance, E., & Giuliani, S. (1992). Cyclophosphamide cystitis in rats: involvement of capsaicin-sensitive primary afferents. Journal of the Autonomic Nervous System, 38, 201–208.PubMedCrossRefGoogle Scholar
  40. Martinez, C., Juarranz, Y., Abad, C., et al. (2005). Analysis of the role of the PAC1 receptor in neutrophil recruitment, acute-phase response, and nitric oxide production in septic shock. Journal of Leukocyte Biology, 77, 729–738.PubMedCrossRefGoogle Scholar
  41. Maruno, K., Absood, A., & Said, S. I. (1995). VIP inhibits basal and histamine-stimulated proliferation of human airway smooth muscle cells. American Journal of Physiology, 268, L1047–L1051.PubMedGoogle Scholar
  42. Mayr, B., & Montminy, M. (2001). Transcriptional regulation by the phosphorylation-dependent factor CREB. Nature Reviews. Molecular Cell Biology, 2, 599–609.PubMedCrossRefGoogle Scholar
  43. Messersmith, D. J., Kim, D. J., & Iadarola, M. J. (1998). Transcription factor regulation of prodynorphin gene expression following rat hindpaw inflammation. Molecular Brain Research, 53, 259–269.CrossRefGoogle Scholar
  44. Morgan, C. W., Ohara, P. T., & Scott, D. E. (1999). Vasoactive intestinal polypeptide in sacral primary sensory pathways in the cat. Journal of Comparative Neurology, 407, 381–394.PubMedCrossRefGoogle Scholar
  45. Murray, E., Malley, S. E., Qiao, L. Y., Hu, V. Y., & Vizzard, M. A. (2004). Cyclophosphamide induced cystitis alters neurotrophin and receptor tyrosine kinase expression in pelvic ganglia and bladder. Journal of Urology, 172, 2434–2439.PubMedCrossRefGoogle Scholar
  46. Nadelhaft, I., & Vera, P. L. (1995). Central nervous system neurons infected by pseudorabies virus injected into the rat urinary bladder following unilateral transection of the pelvic nerve. Journal of Comparative Neurology, 359, 443–456.PubMedCrossRefGoogle Scholar
  47. Newma, R., Cuan, N., Hampartzoumian, T., Connor, S. J., Lloyd, A. R., & Grimm, M. C. (2005). Vasoactive intestinal peptide impairs leucocyte migration but fails to modify experimental murine colitis. Clinical and Experimental Immunology, 139, 411–420.CrossRefGoogle Scholar
  48. Petrone, R. L., Agha, A. H., Roy, J. B., & Hurst, R. E. (1995). Urodynamic findings in patients with interstitial cystitis. Journal of Urology, 153, 290A.Google Scholar
  49. Qiao, L. Y., & Vizzard, M. A. (2002a). Cystitis-induced upregulation of tyrosine kinase (TrkA, TrkB) receptor expression and phosphorylation in rat micturition pathways. Journal of Comparative Neurology, 454, 200–211.PubMedCrossRefGoogle Scholar
  50. Qiao, L. Y., & Vizzard, M. A. (2002b). Up-regulation of tyrosine kinase (TrkA, TrkB) receptor expression and phosphorylation in lumbosacral dorsal root ganglia after chronic spinal cord (T8-T10) injury. Journal of Comparative Neurology, 449, 217–230.PubMedCrossRefGoogle Scholar
  51. Qiao, L. Y., & Vizzard, M. A. (2004). Up-regulation of phosphorylated CREB but not c-Jun in bladder afferent neurons in dorsal root ganglia after cystitis. Journal of Comparative Neurology, 469, 262–274.PubMedCrossRefGoogle Scholar
  52. Riccio, A., Ahn, S., Davenport, C. M., Blendy, J. A., & Ginty, D. D. (1999). Mediation by a CREB family transcription factor of NGF-dependent survival of sympathetic neurons. Science, 286, 2358–2361.PubMedCrossRefGoogle Scholar
  53. Said, S. I. (1991). Vasoactive intestinal polypeptide (VIP) in asthma. Annals of the New York Academy of Sciences, 629, 305–318.PubMedCrossRefGoogle Scholar
  54. Sant, G., & Hanno, P. M. (2001). Interstitial cystitis: current issues and controversies in diagnosis. Urology, 57, 82.PubMedCrossRefGoogle Scholar
  55. Singh, S., Natarajan, K., & Aggarwal, B. B. (1996). Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is a potent inhibitor of nuclear transcription factor-kappa B activation by diverse agents. Journal of Immunology, 157, 4412–4420.Google Scholar
  56. Smet, P. J., Moore, K. H., & Jonavicius, J. (1997). Distribution and colocalization of calcitonin gene-related peptide, tachykinins, and vasoactive intestinal peptide in normal and idiopathic unstable human urinary bladder. Laboratory Investigation, 77, 37–49.PubMedGoogle Scholar
  57. Steers, W. D., Creedon, D. J., & Tuttle, J. B. (1996). Immunity to nerve growth factor prevents afferent plasticity following urinary bladder hypertrophy. Journal of Urology, 155, 379–385.PubMedCrossRefGoogle Scholar
  58. Steers, W. D., & de Groat, W. C. (1988). Effect of bladder outlet obstruction on micturition reflex pathways in the rat. Journal of Urology, 140, 864–871.PubMedGoogle Scholar
  59. Steers, W. D., Kolbeck, S., Creedon, D., & Tuttle, J. B. (1991). Nerve growth factor in the urinary bladder of the adult regulates neuronal form and function. Journal of Clinical Investigation, 88, 1709–1715.PubMedCrossRefGoogle Scholar
  60. Steers, W. D., & Tuttle, J. B. (2006). Mechanisms of Disease: the role of nerve growth factor in the pathophysiology of bladder disorders. Natural Clinical Practice in Urology, 3, 101–110.CrossRefGoogle Scholar
  61. Szema, A. M., Hamidi, S. A., Lyubsky, S., et al. (2006). Mice lacking the VIP gene show airway hyperresponsiveness and airway inflammation, partially reversible by VIP. American Journal of Physiology. Lung Cellular and Molecular Physiology, 291, L880–886.PubMedCrossRefGoogle Scholar
  62. Tuttle, J. B., Steers, W. D., Albo, M., & Nataluk, E. (1994). Neural input regulates tissue NGF and growth of the adult rat urinary bladder. Journal of the Autonomic Nervous System, 49, 147–158.PubMedCrossRefGoogle Scholar
  63. Uckert, S., Stief, C. G., Lietz, B., Burmester, M., Jonas, U., & Machtens, S. A. (2002). Possible role of bioactive peptides in the regulation of human detrusor smooth muscle—Functional effects in vitro and immunohistochemical presence. World Journal of Urology, 20, 244–249.PubMedGoogle Scholar
  64. Verge, V. M. K., Richardson, P. M., Wiesenfeld-Hallin, Z., & Hokfelt, T. (1995). Differential influence of nerve growth factor on neuropeptide expression in vivo: a novel role in peptide suppression in adult sensory neurons. Journal of Neuroscience, 15(3), 2081–2096.PubMedGoogle Scholar
  65. Vizzard, M. A. (1997). Increased expression of neuronal nitric oxide synthase in bladder afferent and spinal neurons following spinal cord injury. Developments in Neuroscience, 19, 232–246.CrossRefGoogle Scholar
  66. Vizzard, M. A. (2000a). Alterations in spinal cord Fos protein expression induced by bladder stimulation following cystitis. American Journal of Physiology. I Regulatory, Integrative and Comparative Physiology, 278, R1027–1039.Google Scholar
  67. Vizzard, M. A. (2000b). Changes in urinary bladder neurotrophic factor mRNA and NGF protein following urinary bladder dysfunction. Experimental Neurology, 161, 273–284.PubMedCrossRefGoogle Scholar
  68. Vizzard, M. A. (2000c). Up-regulation of pituitary adenylate cyclase-activating polypeptide in urinary bladder pathways after chronic cystitis. Journal of Comparative Neurology, 420, 335–348.PubMedCrossRefGoogle Scholar
  69. Vizzard, M. A. (2001). Alterations in neuropeptide expression in lumbosacral bladder pathways following chronic cystitis. Journal of Chemical Neuroanatomy, 21, 125–138.PubMedCrossRefGoogle Scholar
  70. Vizzard, M. A., & Boyle, M. M. (1999). Increased expression of growth-associated protein (GAP-43) in lower urinary tract pathways following cyclophosphamide (CYP)-induced cystitis. Brain Research, 844, 174–187.PubMedCrossRefGoogle Scholar
  71. Vizzard, M. A., Braas, K. M., Studeny, S., et al. (2007a). Vasoactive intestinal polypeptide knockout (VIP−/−) mice exhibit altered bladder function and somatic sensitivity with cyclophosphamide (CYP)-induced cystitis. Journal of Molecular Neuroscience, 33, 311.Google Scholar
  72. Vizzard, M. A., Malley, S., Braas, K. M., Waschek, J. A., & May, V. (2007b). Exaggerated cytokine and chemokine expression in vasoactive intestinal polypeptide knockout (VIP−/−) mice with cyclophosphamide (CYP)-induced cystitis. Journal of Molecular Neuroscience, 33, 338–339.Google Scholar
  73. Voice, J. K., Dorsam, G., Chan, R. C., Grinninger, C., Kong, Y., & Goetzl, E. J. (2002). Immunoeffector and immunoregulatory activities of vasoactive intestinal peptide. Regulatory Peptide, 109, 199–208.CrossRefGoogle Scholar
  74. Wanigasekara, Y., Kepper, M. E., & Keast, J. R. (2003). Immunohistochemical characterisation of pelvic autonomic ganglia in male mice. Cell and Tissue Research, 311, 175–185.PubMedGoogle Scholar
  75. Yoshimura, N., & de Groat, W. C. (1999). Increased excitability of afferent neurons innervating rat urinary bladder after chronic bladder inflammation. Journal of Neuroscience, 19, 4644–4653.PubMedGoogle Scholar
  76. Zvara, P., & Vizzard, M. A. (2007). Exogenous overexpression of nerve growth factor in the urinary bladder produces bladder overactivity and altered micturition circuitry in the lumbosacral spinal cord. BMC Physiology, 7, 9.PubMedCrossRefGoogle Scholar
  77. Zvarova, K., & Vizzard, M. A. (2006). Changes in galanin-immunoreactivity in rat micturition reflex pathways after cyclophosphamide (CYP)-induced cystitis. Cell and Tissue Research, 324, 213–224.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press 2008

Authors and Affiliations

  • Dorthe G. Jensen
    • 1
  • Simon Studeny
    • 2
  • Victor May
    • 3
  • James Waschek
    • 4
  • Margaret A. Vizzard
    • 2
    • 3
  1. 1.Departments of Pharmacology and Pharmacotherapy, Faculty of Pharmaceutical SciencesUniversity of CopenhagenCopenhagenDenmark
  2. 2.Department of NeurologyUniversity of Vermont College of MedicineBurlingtonUSA
  3. 3.Department of Anatomy/NeurobiologyUniversity of Vermont College of MedicineBurlingtonUSA
  4. 4.University of CaliforniaLos AngelesUSA

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