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Agonist-induced phasic and tonic responses in smooth muscle are mediated by InsP₃

Neuroscience and Biomedical Systems, Institute of Biomedical and Life Sciences, West Medical Building, University of Glasgow, Glasgow G12 8QQ, UK

View larger version (20K): [in a new window]   Fig. 1. The effects of removing external Ca2+ or addition of the InsP3 receptor blocker 2-APB on the contractile response of the guinea-pig colon to carbachol (CCh). (A) The contraction produced by CCh (0.5 µM) alone comprised an initial fast phasic component then declined to a lower maintained level (tonic component). Ca2+ withdrawal for 3 minutes significantly reduced (P<0.05, n=17) the amplitude of the tonic component. (B) In the presence of 2-APB (100 µM, 15 minutes) both phasic and tonic components of the CCh response were reduced significantly. The contractions were largely restored on washout of 2-APB.

View larger version (32K): [in a new window]   Fig. 2. The effects of TEA and ryanodine on periodic outward currents. (A) Depolarisation to -20 mV (v) from a membrane potential (VM) of -69 mV activated periodic outward currents (ii) that increased in frequency and amplitude (mean amplitude 116±30 pA) even as [Ca2+]i (iv) declined. The current amplitude varied widely (i,iii). (B) TEA (20 mM, which blocks K+ channels) inhibited periodic outward currents (i) evoked by depolarisation to -30 mV (iii). (C) Ryanodine (50 µM), which places RyR in a subconductance state, inhibited periodic currents (i) evoked by depolarisation to 0 mV (iii) but failed to increase [Ca2+]i significantly. The small increase in [Ca2+]i (ii) represent a partial inhibition of conductance at the RyR. Together A, B and C indicate that the periodic outward currents are indeed STOCs.

View larger version (32K): [in a new window]   Fig. 3. The effects of InsP3, caffeine and carbachol (CCh) on STOCs. Depolarisation from -69 mV to -10 mV (iv) elevated [Ca2+]i (iii) and activated STOCs (ii). InsP3 (), caffeine (Caff, 10 mM, v) and CCh (50 µM) each increased [Ca2+]i (iii) and reversibly inhibited STOCs (ii and expanded time scale i).

View larger version (20K): [in a new window]   Fig. 4. The effects of ryanodine on InsP3-evoked [Ca2+]i transients and STOCs. At a holding potential of -20 mV (iv) InsP3 () increased [Ca2+]i (ii); ryanodine (50 µM) significantly reduced the InsP3-evoked Ca2+ transients (i,ii). Activation of RyR by caffeine (Caff, 10 mM, iii) increased [Ca2+]i (ii). A second application of Caff some 60 seconds later almost abolished both the [Ca2+]i transient, presumably by depleting the SR store and the InsP3 response () leaving only the artefact (ii). Because the InsP3-evoked Ca2+ transient was blocked after caffeine in the presence of ryanodine, InsP3 receptors and RyR may share a common Ca2+ store. i is a summary of eight experiments.

View larger version (23K): [in a new window]   Fig. 5. Effects of withdrawal of extracellular Ca2+ on the rate of decline of STOCs and on the response to InsP3. Depolarisation to -10 mV from a holding potential of -70 mV (iii) induced STOCs (i) and raised [Ca2+]i (ii). Removal of extracellular Ca2+ for the duration indicated by the bar abolished STOCs and lowered [Ca2+]i to pre-depolarisation levels. The times on the bars indicate the period in Ca2+-free/1mM EGTA solution prior to the release of InsP3 (ii). A 4 minute period separated the traces as indicated by the gap. At -10 mV the responses to InsP3 (, iii) were elicited 0.5, 1, 2, 4 and 8 minutes after Ca2+ withdrawal and compared with control responses to InsP3 obtained before removal of extracellular Ca2+. Ca2+ withdrawal reduced the amplitude of both STOCs and the InsP3 responses, the rate of decline of the former exceeded that of the latter (iv). These results suggest that the Ca2+ store content required to support STOCs is greater that that to maintain InsP3 responses.

View larger version (24K): [in a new window]   Fig. 6. The effects of tetracaine on InsP3-evoked [Ca2+]i transients and STOCs. (A) At a holding potential of -10 mV (iii) InsP3 () increased [Ca2+]i; tetracaine, an inhibitor of RyR did not reduce the InsP3-evoked Ca2+ transient (Ai,ii), although it inhibited STOCs (B).

View larger version (23K): [in a new window]   Fig. 7. The effects of InsP3 receptor blockade on the ability of carbachol (CCh) to suppress STOCs. Depolarisation to -20 mV from a holding potential of -70 mV (iv) induced STOCs (ii) and increased [Ca2+]i (iii). (A) 2-APB (50 µM), a membrane-permeable InsP3 receptor inhibitor, introduced by perfusion, inhibited the ability of InsP3 and CCh to affect STOCs (ii and i expanded time base). (i) represents some 30 second excerpts from ii as indicated by the dotted lines. The increased perfusion per se temporarily increased STOC amplitude and was unrelated to the presence of a particular drug. (B) Heparin (2.5 mg/ml), a membrane-impermeable InsP3 receptor inhibitor, introduced via the patch pipette, was present throughout the entire experiment. Other experiments (not shown) under identical conditions with no heparin present served as controls. In the presence of heparin, neither InsP3 () nor CCh (10 mM, v) significantly altered the amplitude or frequency (ii and expanded time base i) of STOCs. CCh activated a transient inward current causing the resting level of membrane current to fall (ii).

View larger version (30K): [in a new window]   Fig. 8. The effects of protein kinase C modulation on Ca2+ transients and STOCs. (A) Following depolarisation from a holding potential of -70 mV to -20 mV (iii), H-7 (10 µM), a protein kinase C inhibitor, had no significant effect on STOC frequency or amplitude (i) or on the ability of carbachol (CCh; iv) to inhibit these responses (i). (B) Following depolarisation from a holding potential of -70 mV to -18 mV, PKC19-36 (3 mM), an impermeant PKC inhibitor added to the patch pipette, failed to affect the ability of either InsP3 () or CCh (iv) to inhibit STOC frequency or amplitude (i). (C) Following depolarisation from a holding potential of -70 mV to -20 mV (iv) indolactam (10 µM), which activates protein kinase C, slightly decreased [Ca2+]i (ii), did not alter the InsP3-evoked Ca2+ transient (; ii) but significantly reduced STOC frequency (i), see text.

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