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. ? ? ?
