CALCIUM RELEASE INDUCED BY INOSITOL 1,4,5-TRISPHOSPHATE IN SINGLE-RABBIT INTESTINAL SMOOTH-MUSCLE CELLS

1. Single smooth muscle cells were isolated by enzymic digestion from the longitudinal muscle layer of rabbit jejunum, and the response of the cells to calcium (Ca2+) release by InsP3 (D-myo-inositol 1,4,5-trisphosphate) was studied. Changes in internal Ca2+ concentration were monitored by measuring Ca2+ -activated K+ currents (outward currents) using the whole-cell voltage-clamp technique. 2. At break-through from cell-attached patch to whole-cell recording mode using a 100-mu-M-InsP3-filled pipette, cells exhibited a brief outward current which reached its peak in 1.1 s and terminated within 10 s. Following this the generation of spontaneous transient outward currents (STOCs) was inhibited. (STOCs are considered to represent bursts of openings of Ca2+ -activated K+ channels in response to spontaneous discharges of Ca2+ from the stores.) When a pipette filled with 20-mu-M-InsP3 was used, similar current responses were also evoked, but some cells failed to respond. 3. The InsP3-induced outward current at membrane break-through was similar in size and time course to the outward current response of normal cells to bath-applied carbachol (CCh, 100-mu-M) or caffeine (20 mM). 4. Dialysis with InsP3-containing solution inhibited the caffeine-induced outward current, depending on the pipette InsP3 concentration. Inclusion of heparin (5 mg/ml) in the pipette completely prevented inhibition by InsP3 of the caffeine response and of STOC discharge. However, the InsP3-induced current at break-through remained unchanged, probably because of the slower rate of diffusion of heparin. 5. In cells dialysed with pipette solution containing 30 or 100-mu-M-caged InsP3, flash photolysis (producing up to 1.5-mu-M-InsP3) induced an outward current response after a latency of 31.0 +/- 1.8 ms (n = 15), which was followed by inhibition of STOCs. The reversal potential of the current to flash-release of InsP3 followed closely the Nernst potential for K+ ions (E(K)), suggesting negligible contributions from channels other than Ca2+ -activated K+ channels. 6. Photolysis of caged InsP3 (30 or 100-mu-M) still produced a current response after 3-6 min in Ca2+ -free (3 mM-EGTA added) bathing solution, but no response occurred if the cell was exposed to either caffeine (20 mM) or CCh (100-mu-M) to deplete Ca stores. Inclusion of heparin (0.5 or 5 mg/ml) in the pipette resulted in blockade of the current response to photolysis of caged InsP3 but no inhibition of the caffeine (20 mM) response. 7. The results show that InsP3 can extremely rapidly induce Ca2+ release from storage sites from which Ca2+ can also be release by CCh or caffeine. Heparin blocks the Ca2+ -releasing action of InsP3. The similarity in the Ca2+ -releasing actions of InsP3 and CCh provides evidence for the previous view (Komori & Bolton, 1990) that InsP3 production is essential for Ca2+ release following activation of the muscarinic receptor.