Volume 5 Supplement 1

2nd International Conference of cGMP Generators, Effectors and Therapeutic Implications

Open Access

Effects of stimulating the cGMP pathway on spontaneous activity in pacemaker cells isolated from the rabbit urethra

  • Gerard Sergeant1,
  • Lovise Johnston2,
  • Noel McHale1,
  • Mark Hollywood1 and
  • Keith Thornbury1Email author
BMC Pharmacology20055(Suppl 1):S34

DOI: 10.1186/1471-2210-5-S1-S34

Published: 16 June 2005

Nitric oxide (NO) is the main inhibitory transmitter in the urethra [1] but little is known about how it mediates relaxation in this tissue. One possibility is that NO inhibits the Ca2+ oscillations underlying spontaneous electrical activity in the urethra and consequently reduces tone. We have recently demonstrated the presence of specialized, spontaneously active cells in the rabbit urethra that resemble interstitial cells of Cajal (ICC) in the gut. We hypothesized that urethral interstitial cells (IC) serve to drive the surrounding bulk smooth muscle and thus may contribute significantly to the generation of urethral tone and its modulation by neurotransmitters [24]. The purpose of the present study was to examine if spontaneous Ca2+ oscillations in isolated urethral IC were altered by NO donors and other activators of the cGMP pathway. Rabbits were humanely killed with pentobarbitone (I.V.) and their urethras removed. Cells were isolated as previously described [2] and incubated for 15 minutes at room temperature with the fluorescent Ca2+-sensitive indicator Fluo-4AM (2 μM). Cells were then plated onto glass-bottomed petri dishes for 30 minutes before being perfused with Hanks solution. Single cell imaging was performed at 5–15 frames sec-1 using a Nipkow spinning disk laser confocal microscope. When cells were maintained at 37°C calcium waves usually initiated at one or more discrete sites and then propagated throughout the cell as recently reported [5]. Wave amplitudes were calculated as a ratio (ΔF/F0) by dividing the mean intensity of the ROI during the peak of the wave by the mean intensity during quiescent periods. The waves occurred at a mean frequency of 5.5 ± 0.4 min-1 and had peak amplitudes of 1.6 ± 0.2 (ΔF/F0, n = 42). These were abolished in the presence of ryanodine (30 μM, n = 12) or tetracaine (100 μM n = 12), suggesting that calcium waves were dependant on functional RyR. In contrast, application of the IP3R blocker 2APB (100 μM) failed to reduce wave frequency (4.8 ± 0.5 compared to 4.1 ± 0.4 min-1) but reduced the spatial spread of the Ca2+ wave suggesting that IP3R were necessary for propagation of the waves. Application of the NO donor DEA-NO (30 μM), also failed to reduce the frequency of the Ca2+ waves but reduced their spatial spread from 83.25 ± 2.5 to 34.9 ± 5.6 μm (n = 5, p < 0.05), as did 8Br cGMP (1 mM; from 83.54 ± 17.23 to 28.26 ± 9.1 μm, n = 4, p < 0.05). A reduction in spatial spread also occurred on application of the protein kinase GI activator SP-8-BrcGMPs (25 μM). In 4 cells, Ca2+ waves propagated 103 ± 19.25 μm under control conditions compared to 39.2 ± 9.2 μm in the presence of SP-8-BrcGMPs (n = 4, p < 0.05).

Given the similarity between the effects of the IP3R blocker 2-APB and the activators of cGMP/GK pathway, these data suggest that NO may mediate its effects by interfering with IP3R and thus prevent propagation of the Ca2+ signal in rabbit IC.

Declarations

Acknowledgements

This work was supported by the Wellcome Trust (grant no 064212) and a Department of Education and Learning (N.I.) studentship to Louise Johnston

Authors’ Affiliations

(1)
Dundalk Institute of Technology
(2)
Queen's University

References

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  3. Sergeant GP, Hollywood MA, McCloskey KD, McHale NG, Thornbury KD: Role of IP(3) in modulation of spontaneous activity in pacemaker cells of rabbit urethra. Am J Physiol Cell Physiol. 2001, 280: C1349-56.PubMedGoogle Scholar
  4. Sergeant GP, Thornbury KD, McHale NG, Hollywood MA: Characterization of norepinephrine-evoked inward currents in interstitial cells isolated from the rabbit urethra. Am J Physiol Cell Physiol. 2002, 283: C885-94.View ArticlePubMedGoogle Scholar
  5. Johnston L, Sergeant GP, Hollywood MA, Thornbury KD, McHale NG: Calcium oscillations in interstitial cells of the rabbit urethra. J Physiol. 2005Google Scholar

Copyright

© BioMed Central Ltd 2005

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