Skip to main content
SpringerLink
Log in

Real-Time Monitoring the Spatiotemporal Dynamics of Intracellular cGMP in Vascular Smooth Muscle Cells

  • Protocol
  • First Online:
Guanylate Cyclase and Cyclic GMP

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1020))

Abstract

Real-time and noninvasive imaging of intracellular second messengers in mammalian cells, while ­preserving their in vivo phenotype, requires biosensors of exquisite constitution. Here we provide the methodology for utilizing the single wavelength cGMP-biosensor δ-FlincG in aortic vascular smooth muscle cells.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Friebe A, Koesling D (2003) Regulation of nitric oxide-sensitive guanylyl cyclase. Circ Res 93(2):96–105

    Article  PubMed  CAS  Google Scholar 

  2. Kuhn M (2003) Structure, regulation, and function of mammalian membrane guanylyl cyclase receptors, with a focus on guanylyl cyclase-A. Circ Res 93(8):700–709

    Article  PubMed  CAS  Google Scholar 

  3. Conti M, Beavo J (2007) Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic ­nucleotide signaling. Annu Rev Biochem 76:481–511

    Article  PubMed  CAS  Google Scholar 

  4. Francis SH, Blount MA, Zoraghi R et al (2005) Molecular properties of mammalian proteins that interact with cGMP: protein kinases, cation channels, phosphodiesterases, and multi-drug anion transporters. Front Biosci 10:2097–2117

    Article  PubMed  CAS  Google Scholar 

  5. Kemp-Harper B, Schmidt HH (2009) cGMP in the vasculature. Handb Exp Pharmacol 191:447–467

    Article  PubMed  CAS  Google Scholar 

  6. Alverdi V, Mazon H, Versluis C et al (2008) cGMP-binding prepares PKG for substrate binding by disclosing the C-terminal domain. J Mol Biol 375(5):1380–1393

    Article  PubMed  CAS  Google Scholar 

  7. Scholten A, Fuss H, Heck AJ et al (2007) The hinge region operates as a stability switch in cGMP-dependent protein kinase I alpha. FEBS J 274(9):2274–2286

    Article  PubMed  CAS  Google Scholar 

  8. Surks HK, Mochizuki N, Kasai Y et al (1999) Regulation of myosin phosphatase by a specific interaction with cGMP-dependent protein kinase Ialpha. Science 286(5444):1583–1587

    Article  PubMed  CAS  Google Scholar 

  9. Tang KM, Wang GR, Lu P et al (2003) Regulator of G-protein signaling-2 mediates vascular smooth muscle relaxation and blood pressure. Nat Med 9(12):1506–1512

    Article  PubMed  CAS  Google Scholar 

  10. Wooldridge AA, MacDonald JA, Erdodi F et al (2004) Smooth muscle phosphatase is regulated in vivo by exclusion of phosphorylation of threonine 696 of MYPT1 by phosphorylation of serine 695 in response to cyclic nucleotides. J Biol Chem 279(33):34496–34504

    Article  PubMed  CAS  Google Scholar 

  11. Sun X, Kaltenbronn KM, Steinberg TH et al (2005) RGS2 is a mediator of nitric oxide action on blood pressure and vasoconstrictor signaling. Mol Pharmacol 67(3):631–639

    Article  PubMed  CAS  Google Scholar 

  12. Schlossmann J, Ammendola A, Ashman K et al (2000) Regulation of intracellular calcium by a signalling complex of IRAG, IP3 receptor and cGMP kinase Ibeta. Nature 404(6774):197–201

    Article  PubMed  CAS  Google Scholar 

  13. Geiselhoringer A, Werner M, Sigl K et al (2004) IRAG is essential for relaxation of receptor-triggered smooth muscle contraction by cGMP kinase. EMBO J 23(21):4222–4231

    Article  PubMed  Google Scholar 

  14. Lalli MJ, Shimizu S, Sutliff RL et al (1999) [Ca2+]i homeostasis and cyclic nucleotide relaxation in aorta of phospholamban-deficient mice. Am J Physiol 277(3 Pt 2):H963–H970

    PubMed  CAS  Google Scholar 

  15. Sausbier M, Schubert R, Voigt V et al (2000) Mechanisms of NO/cGMP-dependent vasorelaxation. Circ Res 87(9):825–830

    Article  PubMed  CAS  Google Scholar 

  16. Ellerbroek SM, Wennerberg K, Burridge K (2003) Serine phosphorylation negatively regulates RhoA in vivo. J Biol Chem 278(21):19023–19031

    Article  PubMed  CAS  Google Scholar 

  17. Weber S, Bernhard D, Lukowski R et al (2007) Rescue of cGMP kinase I knockout mice by smooth muscle specific expression of either isozyme. Circ Res 101(11):1096–1103

    Article  PubMed  CAS  Google Scholar 

  18. Rybalkin SD, Bornfeldt KE, Sonnenburg WK et al (1997) Calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1C) is induced in human arterial smooth muscle cells of the synthetic, proliferative phenotype. J Clin Invest 100(10):2611–2621

    Article  PubMed  CAS  Google Scholar 

  19. Rybalkin SD, Rybalkina IG, Feil R et al (2002) Regulation of cGMP-specific phosphodiesterase (PDE5) phosphorylation in smooth muscle cells. J Biol Chem 277(5):3310–3317

    Article  PubMed  CAS  Google Scholar 

  20. Thomas MK, Francis SH, Corbin JD (1990) Substrate- and kinase-directed regulation of phosphorylation of a cGMP-binding phosphodiesterase by cGMP. J Biol Chem 265(25):14971–14978

    PubMed  CAS  Google Scholar 

  21. Corbin JD, Turko IV, Beasley A et al (2000) Phosphorylation of phosphodiesterase-5 by cyclic nucleotide-dependent protein kinase alters its catalytic and allosteric cGMP-binding activities. Eur J Biochem 267(9):2760–2767

    Article  PubMed  CAS  Google Scholar 

  22. Francis SH, Poteet-Smith C, Busch JL et al (2002) Mechanisms of autoinhibition in cyclic nucleotide-dependent protein kinases. Front Biosci 7:d580–d592

    Article  PubMed  CAS  Google Scholar 

  23. Rybalkin SD, Rybalkina IG, Shimizu-Albergine M et al (2003) PDE5 is converted to an activated state upon cGMP binding to the GAF A domain. EMBO J 22(3):469–478

    Article  PubMed  CAS  Google Scholar 

  24. Zoraghi R, Bessay EP, Corbin JD et al (2005) Structural and functional features in human PDE5A1 regulatory domain that provide for allosteric cGMP binding, dimerization, and regulation. J Biol Chem 280(12):12051–12063

    Article  PubMed  CAS  Google Scholar 

  25. Omori K, Kotera J (2007) Overview of PDEs and their regulation. Circ Res 100(3):309–327

    Article  PubMed  CAS  Google Scholar 

  26. Held KF, Dostmann WR (2012) Sub-nanomolar sensitivity of nitric oxide mediated regulation of cGMP and vasomotor reactivity in vascular smooth muscle. Front Pharmacol 3:130

    Article  PubMed  CAS  Google Scholar 

  27. Halvey EJ, Vernon J, Roy B et al (2009) Mechanisms of activity-dependent plasticity in cellular no-cGMP signaling. J Biol Chem 284(38):25630–25641

    Article  PubMed  CAS  Google Scholar 

  28. Batchelor AM, Bartus K, Reynell C et al (2010) Exquisite sensitivity to subsecond, picomolar nitric oxide transients conferred on cells by guanylyl cyclase-coupled receptors. Proc Natl Acad Sci USA107(51):22060–22065

    Article  PubMed  CAS  Google Scholar 

  29. Bellamy TC, Wood J, Goodwin DA et al (2000) Rapid desensitization of the nitric oxide receptor, soluble guanylyl cyclase, underlies diversity of cellular cGMP responses. Proc Natl Acad Sci USA97(6):2928–2933

    Article  PubMed  CAS  Google Scholar 

  30. Steiner AL, Parker CW, Kipnis DM (1972) Radioimmunoassay for cyclic nucleotides. I. Preparation of antibodies and iodinated cyclic nucleotides. J Biol Chem 247(4):1106–1113

    PubMed  CAS  Google Scholar 

  31. Wehmann RE, Blonde L, Steiner AL (1972) Simultaneous radioimmunoassay for the measurement of adenosine 3′,5′-monophosphate and guanosine 3′,5′-monophosphate. Endocrinology 90(1):330–335

    Article  PubMed  CAS  Google Scholar 

  32. Lakowicz JR, Gryczynski I, Gryczynski Z et al (1999) Anisotropy-based sensing with reference fluorophores. Anal Biochem 267(2):397–405

    Article  PubMed  CAS  Google Scholar 

  33. Heim R, Prasher DC, Tsien RY (1994) Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci USA91(26):12501–12504

    Article  PubMed  CAS  Google Scholar 

  34. Zukin RS, Hartig PR, Koshland DE Jr (1977) Use of a distant reporter group as evidence for a conformational change in a sensory receptor. Proc Natl Acad Sci USA74(5):1932–1936

    Article  PubMed  CAS  Google Scholar 

  35. Hahn LH, Hammes GG (1978) Structural mapping of aspartate transcarbamoylase by fluorescence energy-transfer measurements: determination of the distance between catalytic sites of different subunits. Biochemistry 17(12):2423–2429

    Article  PubMed  CAS  Google Scholar 

  36. Honda A, Adams SR, Sawyer CL et al (2001) Spatiotemporal dynamics of guanosine 3′,5′-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator. Proc Natl Acad Sci USA98(5):2437–2442

    Article  PubMed  CAS  Google Scholar 

  37. Sawyer CL, Honda A, Dostmann WR (2003) Cygnets: spatial and temporal analysis of intracellular cGMP. Proc West Pharmacol Soc 46:28–31

    PubMed  CAS  Google Scholar 

  38. Cawley SM, Sawyer CL, Brunelle KF et al (2007) Nitric oxide-evoked transient kinetics of cyclic GMP in vascular smooth muscle cells. Cell Signal 19(5):1023–1033

    Article  PubMed  CAS  Google Scholar 

  39. Nikolaev VO, Gambaryan S, Lohse MJ (2006) Fluorescent sensors for rapid monitoring of intracellular cGMP. Nat Methods 3(1):23–25

    Article  PubMed  CAS  Google Scholar 

  40. Baird GS, Zacharias DA, Tsien RY (1999) Circular permutation and receptor insertion within green fluorescent proteins. Proc Natl Acad Sci USA96(20):11241–11246

    Article  PubMed  CAS  Google Scholar 

  41. Nakai J, Ohkura M, Imoto K (2001) A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein. Nat Biotechnol 19(2):137–141

    Article  PubMed  CAS  Google Scholar 

  42. Nausch LW, Ledoux J, Bonev AD et al (2008) Differential patterning of cGMP in vascular smooth muscle cells revealed by single GFP-linked biosensors. Proc Natl Acad Sci USA105(1):365–370

    Article  PubMed  CAS  Google Scholar 

  43. Isner JC, Maathuis FJ (2011) Measurement of cellular cGMP in plant cells and tissues using the endogenous fluorescent reporter FlincG. Plant J 65(2):329–334

    Article  PubMed  CAS  Google Scholar 

  44. Wood KC, Batchelor AM, Bartus K et al (2011) Picomolar nitric oxide signals from central neurons recorded using ultrasensitive detector cells. J Biol Chem 286(50):43172–43181

    Article  PubMed  CAS  Google Scholar 

  45. Miller CL, Cai Y, Oikawa M et al (2011) Cyclic nucleotide phosphodiesterase 1A: a key regulator of cardiac fibroblast activation and extracellular matrix remodeling in the heart. Basic Res Cardiol 106(6):1023–1039

    Article  PubMed  CAS  Google Scholar 

  46. Chao YC, Cheng CJ, Hsieh HT et al (2010) Guanylate cyclase-G, expressed in the Grueneberg ganglion olfactory subsystem, is activated by bicarbonate. Biochem J 432(2):267–273

    Article  PubMed  CAS  Google Scholar 

  47. Tsai EJ, Kass DA (2009) Cyclic GMP signaling in cardiovascular pathophysiology and therapeutics. Pharmacol Ther 122(3):216–238

    Article  PubMed  CAS  Google Scholar 

  48. Cocks TM, Angus JA, Campbell JH et al (1985) Release and properties of endothelium-derived relaxing factor (EDRF) from endothelial cells in culture. J Cell Physiol 123(3):310–320

    Article  PubMed  CAS  Google Scholar 

  49. Griffith TM, Edwards DH, Lewis MJ et al (1984) The nature of endothelium-derived vascular relaxant factor. Nature 308(5960):645–647

    Article  PubMed  CAS  Google Scholar 

  50. Hakim TS, Sugimori K, Camporesi EM et al (1996) Half-life of nitric oxide in aqueous solutions with and without haemoglobin. Physiol Meas 17(4):267–277

    Article  PubMed  CAS  Google Scholar 

  51. Keefer LK, Nims RW, Davies KM et al (1996) “NONOates” (1-substituted diazen-1-ium-1,2-diolates) as nitric oxide donors: convenient nitric oxide dosage forms. Methods Enzymol 268:281–293

    Article  PubMed  CAS  Google Scholar 

  52. Griffiths C, Wykes V, Bellamy TC et al (2003) A new and simple method for delivering clamped nitric oxide concentrations in the physiological range: application to activation of guanylyl cyclase-coupled nitric oxide ­receptors. Mol Pharmacol 64(6):1349–1356

    Article  PubMed  CAS  Google Scholar 

  53. Bellamy TC, Griffiths C, Garthwaite J (2002) Differential sensitivity of guanylyl cyclase and mitochondrial respiration to nitric oxide measured using clamped concentrations. J Biol Chem 277(35):31801–31807

    Article  PubMed  CAS  Google Scholar 

  54. Garthwaite J, Southam E, Boulton CL et al (1995) Potent and selective inhibition of nitric oxide-sensitive guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Mol Pharmacol 48(2):184–188

    PubMed  CAS  Google Scholar 

  55. Schrammel A, Behrends S, Schmidt K et al (1996) Characterization of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one as a heme-site inhibitor of nitric oxide-sensitive guanylyl cyclase. Mol Pharmacol 50(1):1–5

    PubMed  CAS  Google Scholar 

  56. Evgenov OV, Pacher P, Schmidt PM et al (2006) NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potential. Nat Rev Drug Discov 5(9):755–768

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Drs. Carolyn Sawyer, Sharon Cawley, and Lydia Nausch for their help in perfecting this technique. Support was provided by NIH grants HL68991 (W.R.D.) and T323 HL07944 (K.F.H.), and the Totman Trust for Biomedical Research.

Author information

Authors and Affiliations

  1. Department of Pharmacology, Yale University, New Haven, USA

    Kara F. Held

  2. Department of Pharmacology, University of Vermont, Burlington, USA

    Wolfgang R. Dostmann

Authors
  1. Kara F. Held
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wolfgang R. Dostmann
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Addenbrooke’s Hospital, Department of Medicine, University of Cambridge, Hills Road, Cambridge, CB2 2QQ, United Kingdom

    Thomas Krieg

  2. Toxicology and Clinical Pharmacy, Department of Pharmacology, Universität Tübingen, Auf der Morgenstelle 8, Tübingen, 72076, Germany

    Robert Lukowski

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Held, K.F., Dostmann, W.R. (2013). Real-Time Monitoring the Spatiotemporal Dynamics of Intracellular cGMP in Vascular Smooth Muscle Cells. In: Krieg, T., Lukowski, R. (eds) Guanylate Cyclase and Cyclic GMP. Methods in Molecular Biology, vol 1020. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-459-3_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-459-3_8

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-458-6

  • Online ISBN: 978-1-62703-459-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation