Calcium transients evoked by electrical stimulation of smooth muscle from guinea-pig ileum recorded by the use of Fura-2.

1. Intracellular free calcium levels were recorded in strips of longitudinal smooth muscle from guinea-pig ileum, by the use of the fluorescent calcium indicator Fura-2. 2. The resting intracellular free calcium concentration was estimated to be 210 nM. Many muscle strips showed spontaneous bursts of contractions, accompanied by bursts of calcium transients. Following these the calcium level often fell transiently below the resting level. The spontaneous transients were unaffected by tetrodotoxin (TTX) and atropine. 3. Field electrical stimulation of muscle strips evoked a series of calcium transients comprising: (i) an initial rise in free calcium, reaching a peak within 20-30 ms of stimulation, (ii) a second rise in calcium, beginning after a few hundred milliseconds, and finally (iii) a decline in calcium to below the resting level, persisting for a few seconds. The mean peak increase in free calcium above the resting level during components (i) and (ii) was, respectively, 130 and 200 nM. The mean decrease in free calcium during the third component was to 20 nM below the resting level. 4. The short-latency calcium transient required relatively long stimuli for activation, and was not blocked by TTX and atropine. The long-latency transient was selectively activated by brief stimuli, and was abolished by TTX and atropine. Thus, the short-latency component probably arose because of direct electrical stimulation of muscle fibres, while the long-latency component was due to stimulation of muscarinic nerves. 5. The first detectable increase in tension began about 100 ms after the peak of the initial calcium transient. Contractions associated with the long-latency calcium transient were much larger than those associated with the short-latency transient, even in muscle strips where the calcium levels were similar for both transients. 6. Removal of calcium in the bathing solution caused the resting intracellular calcium level to fall, following an initial rise accompanied by increased spontaneous transients. Electrically evoked contractions and calcium transients were abolished in calcium-free solution, and by the addition of verapamil or diltiazem to normal Krebs solution.