Loading* of tissue with ^Rb and preparation of samples

Freshly dissected tissues were tied to frames made from stainless steel tubing, through which gas was passed to provide stirring.

The tissue was loaded with tracer by incubating it in a solution containing *^Rb at a specific activity of 3 pCi/ml for a period of 90 minutes. The concentration of Rb^ in the load solution did not exceed 100 |iM. At the end of the loading period the muscles were washed in a large volume of non-radioactive solution to remove excess tracer from the extracellular space. Thirty minutes after removal from the load the muscles were transferred through a series of test-tubes each containing 5 ml of solution. When a bicarbonate buffered solution was used the tubes were capped immediately after

preparation and pre-gassed for 4 minutes before the tissue was placed in them. Finally the tubes were emptied into scintillation vials and 5 ml of 5 mM KCl was used to wash any activity remaining in the tube into the vial.

At the end of the experiment the tissue was blotted dry and dissolved in 0.2 ml of

concentrated nitric acid. The extract was then diluted into 10 ml of bathing solution. The difference in quenching between the tissue extract and the other samples was taken into account by internal standardisation ('spiking'). Two 2 ml aliquots of tissue extract were diluted to 10 ml with 5 ml of 5 mM KCl and 3 ml of bathing solution. A known quantity of tracer (20 pi of load solution - the 'spike') was added to tissue extract and to the solution used for the other samples. The observed count was compared to the unspiked tissue sample and a correction factor calculated by the equation :-

where, m= cpm in tissue extract

ms~ cpm in tissue extract + spike

s = cpm in saline + spike

The activity in the samples was determined by counting Cerenkov radiation in a scintillation spectrophotometer (Beckman LS801).

A = ^

c_.At

where, k = the rate constant for tracer efflux Ac = cpm in sample

Cm = mean cpm remaining in tissue during collection period

At = duration of collection period in minutes

In some instances in which the response to drugs was small relative to variations in resting efflux between preparations the data were normalised by dividing each point by the mean of the 4 rate constants prior to exposure to the drug.

3.4 Design of experiments

3.4.1 Studies on the action ofpotassium channel openers on efflux from skeletal muscle

Fifteen two-minute collection periods were used in these experiments. The tissue was exposed to the potassium channel opener for a ten minute period beginning 40 minutes after removal from the load solution. In experiments which examined the effect of blocking agents, the blocker was present 20 minutes prior to, and during, exposure to channel opener. One muscle from each pair served as a control while the other was exposed to blocker.

The effect of cromakalim was calculated from the difference between average o f the 2nd, 3rd and 4th drug application periods and the average of the three periods immediately prior to drug application. This difference was expressed as a percentage of the pre-drug average. The inhibition by blocking agents was calculated from the percentage difference between control and test preparations. In the case of mouse skeletal muscle it was found that the response peaked during the first exposure period and tended to decline more rapidly than with frog muscle and so the increase was calculated from the mean rate constants for the first and second periods.

In some preliminary experiments the muscle was exposed to cromakalim twice. This approach was quickly abandoned since a marked tachyphylaxis was apparent (see Chapter

12).

3.4.2 Rubidium efflux from metabolically inhibited muscle

Twenty two-minute collection periods were used. For the first ten minutes o f the

experimental period the muscle was exposed to normal solution which was then replaced by 'poisoning' solution containing cyanide and iodoacetate for the remaining 30 minutes. Glibenclamide was added 20 minutes prior to poisoning; all other blockers were present 4 minutes before poisoning. As with the potassium channel opener experiments pairs of muscles were used, one acting as a control, the being exposed to blocker.

Percentage inhibition was calculated from the formula:

Vo Inhibition = , 1 — Z - Z

c - c .100

Here T„ and C„ are the means of the two rate constants before poisoning for the test and control muscles respectively. Ce is the mean of the three peak rate constants for the control during poisoning and Te is the mean of the three corresponding points for the test muscle.

3.4.3 Effects ofpotassium channel openers on ^^Rb efflux fro m smooth muscle Eleven four-minute collection periods were used. Potassium channel openers were present from between 16 and 28 minutes after the tissue was removed from the load solution. Blockers were present 8 minutes prior to exposure to the potassium channel opener.

The increase in * ^ b efflux stimulated by potassium channel openers was calculated from the difference between the peak rate during exposure and the mean rate for the two collection periods immediately before exposure. This was expressed as a percentage of

the pre-drug average. In each experiment at least two pieces of tissue were used as controls. Inhibition by test compounds was calculated from the difference between the response in the presence of test compound and the average of the control responses. This difference was expressed as a percent age of the average control response.