This study seeks to explore the cellular mechanisms underlying myogenic tone in the urethra and the means by which these are regulated by neurotransmitters that are known to modulate urethral tone. Our central hypothesis is that specialized pacemaker cells (interstitial cells or IC) produce rhythmic electrical activity similar to that of the interstitial cells of Cajal in the gastrointestinal tract. This in turn is conducted to neighboring smooth muscle cells (SMC) which are recruited to cause a sustained contraction. To test this model we will first need a detailed understanding of the properties of both cell types. We will use five main techniques: 1) Fast confocal imaging to measure intracellular calcium levels in pacemaking and smooth muscle cells and individual muscle bundles ; 2) the perforated patch-clamp technique to determine the ionic basis of the electrical activity; 3) RT-PCR, including real-time quantitative PCR to measure differences in channel expression in both cell types 4) Intracellular microelectrode recording to determine the source of pacemaking electrical activity in the whole tissue and 5) Isometric tension recordings to establish the contribution of IC and SMC to the generation of myogenic tone. We know from previous indirect evidence and from our preliminary data that the rhythmic electrical activity results from oscillatory calcium release within the IC. We will use fast confocal imaging of fluo-4 loaded IC to identify the initiating calcium event and the means by which it is amplified to a level sufficient to activate the ionic conductances underlying electrical activity. We know that frequency of oscillation is greatly influenced by external calcium levels so the next key task will be to determine the calcium influx pathway responsible. This will require an examination of the expression levels of channels known to mediate calcium entry using both RT-PCR and pharmacological approaches. Simultaneous imaging and patch clamp techniques will then be used to relate the intracellular calcium changes to membrane electrical events to determine how membrane potential changes affect intracellular calcium levels and vice-versa. Similar approaches will be used to elucidate the electrical and calcium-handling properties of SMC. When the fundamental properties of both IC and SMC have been elucidated we will turn our attention to the ways in which these are modified by excitatory and inhibitory neurotransmitters and then examine the contribution of IC to myogenic tone in isolated strips of urethra. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK068565-04
Application #
7434476
Study Section
Urologic and Kidney Development and Genitourinary Diseases Study Section (UKGD)
Program Officer
Mullins, Christopher V
Project Start
2005-08-01
Project End
2010-05-31
Budget Start
2008-06-01
Budget End
2009-05-31
Support Year
4
Fiscal Year
2008
Total Cost
$163,015
Indirect Cost
Name
Dundalk Institute of Technology
Department
Type
DUNS #
989044656
City
Dundalk
State
Country
Ireland
Zip Code
12345
Kyle, Barry D; Bradley, Eamonn; Large, Roddy et al. (2013) Mechanisms underlying activation of transient BK current in rabbit urethral smooth muscle cells and its modulation by IP3-generating agonists. Am J Physiol Cell Physiol 305:C609-22
Kyle, B; Bradley, E; Ohya, S et al. (2011) Contribution of Kv2.1 channels to the delayed rectifier current in freshly dispersed smooth muscle cells from rabbit urethra. Am J Physiol Cell Physiol 301:C1186-200
Bradley, Eamonn; Kadima, Sonia; Kyle, Barry et al. (2011) P2X receptor currents in smooth muscle cells contribute to nerve mediated contractions of rabbit urethral smooth muscle. J Urol 186:745-52
Bradley, Eamonn; Kadima, Sonia; Drumm, Bernard et al. (2010) Novel excitatory effects of adenosine triphosphate on contractile and pacemaker activity in rabbit urethral smooth muscle. J Urol 183:801-11
Sergeant, Gerard P; Bradley, Eamonn; Thornbury, Keith D et al. (2008) Role of mitochondria in modulation of spontaneous Ca2+ waves in freshly dispersed interstitial cells of Cajal from the rabbit urethra. J Physiol 586:4631-42
Sergeant, G P; Johnston, Louise; McHale, N G et al. (2006) Activation of the cGMP/PKG pathway inhibits electrical activity in rabbit urethral interstitial cells of Cajal by reducing the spatial spread of Ca2+ waves. J Physiol 574:167-81
Sergeant, Gerard P; Hollywood, M A; McHale, N G et al. (2006) Ca2+ signalling in urethral interstitial cells of Cajal. J Physiol 576:715-20
McHale, N G; Hollywood, M A; Sergeant, G P et al. (2006) Organization and function of ICC in the urinary tract. J Physiol 576:689-94
Sergeant, G P; Thornbury, K D; McHale, N G et al. (2006) Interstitial cells of Cajal in the urethra. J Cell Mol Med 10:280-91
Bradley, E; Hollywood, M A; Johnston, L et al. (2006) Contribution of reverse Na+-Ca2+ exchange to spontaneous activity in interstitial cells of Cajal in the rabbit urethra. J Physiol 574:651-61