Low volume perfusion-superfusion system for measurement of transmitter release from blood vessels in vitro.

An in vitro technique has been developed for the simultaneous recording of vasoconstriction and transmitter release from small perfused blood vessels. To avoid excessive dilution of the released substances, 6-12-mg segments of rabbit ear arteries or rat tail arteries were mounted in 0.5-mL tissue chambers and perfused-superfused in a closed system. Drug administration and removal were performed by timer-controlled solenoid valves. Oscillation in the baseline generated by the roller pump was dampened by delivering Kreb's solution through a 4-mL buffer vial filled partly with air. Vasoconstriction was elicited by electrical stimulation and detected by pressure sensitive transducers. Transducer-generated electrical signals were amplified, digitalized, and recorded by a computerized system. Release of norepinephrine was measured after the tissue was preloaded with [3H]norepinephrine. This system allowed us to measure minimum vasoconstrictions of 1-2 mm Hg and [3H]norepinephrine efflux evoked by 4-8 electrical pulses. There was a good linear correlation (r = 0.98) between the contractile responses and tritiated norepinephrine release. Endogenous norepinephrine released by electrical stimulation into the perfusion media was determined by high-performance liquid chromatography (HPLC) in the low picogram range (80-100 pg). Our novel experimental setup permits computerized data acquisition and automated in vitro pharmacological experiments on isolated blood vessels.