Intracellular Ca^^ fluorescent indicator.

In principle, an ion-specific fluorescent indicator was loaded into isolated myocytes and the fluorescent output of a single cell monitored. The fluorescent output was obtained fi*om two exciting wavelengths, chosen so that at one exciting wavelength the output changed little with variation of intracellular Ca^^ concentration whereas at the other the change was large.

The fluorescent indicator Fura-2 (Grynkiewicz et al, 1985) was used as an index of the intracellular flree Ca^^ concentration, ([Ca^^Ji). Fura-2 has a high affinity and selectivity for Ca^^ and the measured Kd (224 nM) lies within the physiological range o f intracellular [Ca^^]. During recording Fura-2 was excited at wavelengths of 340 ± 1 0 nm and 380 ± 10 nm and the emitted light at wavelengths between 400-510 nm was collected. The fluorescence excitation spectrum o f Fura-2 shifts progressively to a shorter wavelength as [Ca^"^]i increases and as a result the recorded emission intensity at 340 nm excitation increases while the emission intensity at 380 nm decreases. The isofluoresence wavelength is approximately 360 nm. The emission spectrum is unaltered by Ca^^ binding and has a maximum at about 510 nm (Grynkiewicz et al 1985). Measuring the ratio of the emission intensity at the two exciting wavelengths ensures that the signal is independent of fluorochrome concentration variation that may arise from loading or leakage from the cell or an uneven distribution of the dye throughout the cytosol.

2.4,4 Intracellular loading o f indicator

The commonest approach for the loading o f fluorescent indicators is as their acetoxymethyl (AM) esters (Thomas & Delaville 1991). These AM esters are lipophilic and therefore freely pass through the lipid bilayer cell membrane. Once within the cell the ester is cleaved by intracellular esterases releasing the hydrophilic acid, which is then trapped within the cell.

Fura-2 AM (Molecular Probes Inc, Eugene, Oregon, USA) was dissolved in the organic solvent dimethyl sulphoxide (DMSO; Sigma Chemical Co. Ltd) to a concentration of 1 mM and stored at -20°C in 50pl aliquots.

Isolated detrusor myocytes were loaded at room temperature by incubation for 30 minutes in Ca^^-free HEPES- Tyrode’s containing 3jaM of Fura-2 AM.

2,4.5 Experimental set-up,

A Perspex superfusion chamber with a heated water jacket was situated on an inverted stage microscope (Diaphot-TMD, Nikon Corporation, Tokyo, Japan) which was mounted on an air table (Ealing optics Ltd., Watford, UK) and enclosed in a Faraday cage covered with a black blanket. Tyrode’s superfusate solutions were continually gassed with 95% O2; 5% CO2, warmed in a water tank at 37°C

and delivered to the preparation in the superfusion trough via a gravity-fed, water- jacketed system via tubing o f 2mm diameter (Thermoflow, MEDIC 471, Coniar Churchill Scientific supplies Ltd, Middlesex, UK). The superfusate had a flow rate of approximately 3 ml/minute, a solution change taking 20 seconds to reach the

preparation. The total chamber volume was 0.1 ml and therefore the time for exchange of solution in the trough would take approximately 2 seconds. The solution in the chamber was drawn to waste by suction to ensure a constant level o f fluid.

The epifluorescence system was supplied by Cairn Research Ltd., (Sittingboume, Kent, UK). A Xenon short arc light (75W XBO; Osram Ltd., Berlin, Germany) was used to provide a light source, which provided a focused, high intensity, wide band-width, white light. This was directed via six band-width (± 10 nm) interference filters of differing wavelength (340, 360, 380, 400, 420 and 500 nm) housed in a spinning wheel, to provide light of specific wavelengths. The rate of rotation of the wheel was variable and was typically 20-30rps. For excitation at the two wavelengths of 340 and 380 nm, the other four filters were redundant and were blacked out. The excitation light was transmitted via a quartz fibre optic cable into the substage of the microscope and was reflected onto the cells in the superfusion trough by a dichroic mirror (400 nm). Emitted light was focused in the light tube using a X 40 objective magnification oil immersion lens before a variable rectangular diaphragm which was closed around the cell chosen for experiment to limit the extracellular light signal, and passed down through a dichroic mirror. The microscope light source (halogen 40 W, with an optional red filter) was used to position the cell and adjust the diaphragm before the experiment. A second dichroic mirror (510 nm) acted as a beam splitter. The higher wavelengths, obtained mainly from the microscope light source, were directed to a CCD camera (Heiman CCD: Alrad Instruments Ltd., Newbury, Berks, UK.) and the image displayed on a monitor to facilitate adjustment o f the diaphragm prior to recording. The lower

wavelengths 400-510 nm were transmitted to the photomultiplier tube (PMT) as the fluorescence signal from Fura-2.

The intensities of the emitted light at the two exciting wavelengths were recorded by two sample-and-hold amplifiers incorporated into the spectrophotometer system. The frequency of switch between these two amplifiers was synchronised to the rotation of the spinning filter wheel by an internal high frequency time clock so that sampling occurred only during the passage o f the specific filter. The magnitude of the output could be adjusted by changing the voltage applied to the PMT. An analogue division circuit was also incorporated into the spectrophotometer system to produce a simultaneous ratio o f the two signals which was displayed onto an oscilloscope (Model DSO 1604; Gould Inc., Essex, UK). This was facilitated by signals o f similar size and therefore a ten-fold gain was applied to the signal at 340nm excitation. The emitted light intensities and ratio from the two excitation wavelengths were recorded onto a chart recorder (EasyGraf TA240: Gould Electronics Ltd., Hainault, Essex, UK) or displayed and printed out on a storage oscilloscope for subsequent analysis.

Signal 1 Signal 2 Ratio 1/2 Microscope light Red filter Sample & hold Superfused single cell Microscope objective (x40) High pass filter Dichroic Xenon Light source mirror 400 nm CCD camera Filter wheel

Microscope Adjustable Dichroic

mirror diaphragm mirror

510 nm

Figure 2,7; A s c h e m a t ic d ia g r a m o f t h e e p if lu o r e s c e n c e m ic r o s c o p y s y s t e m u s e d fo r th e m e a s u r e m e n t o f in tr a c e llu la r [Ca^^j w it h F u r a -2 .