SUMMARY1. Single smooth muscle cells obtained by enzymic dispersion of the longitudinal muscle layer of guinea-pig ileum were used for recording membrane currents under whole-cell voltage clamp in response to carbachol (100 /bM, unless otherwise stated) or histamine (100 jam) applied extracellularly.2. At a holding potential of 0 mV, a transient outward current was evoked by carbachol and histamine. Responses to the two agonists were very similar in size and time course to the current response to caffeine (10 mM). The response to carbachol was virtually absent in the presence of histamine, and vice versa. Caffeine was without effect in the presence of either of these agonists. Inclusion of EGTA (10 or 20 mM) in the pipette abolished the responses to carbachol, histamine and caffeine. Thus, the outward current responses were considered to represent opening of Ca2+_ activated K+ channels in response to a massive release of Ca2+ from the same stores by these three agents.3. An inward current was evoked by carbachol and histamine, but not by caffeine at a holding potential of -40 mV, which was considered to represent opening of cationic channels. The carbachol-induced inward current was much longer in duration and larger in size than the histamine-induced inward current.4. Inclusion of GDPpJS (2 mm) in the pipette abolished the inward and outward current responses to histamine, but inhibited only part of those to carbachol.5. When the holding potential was held at 0 mV with inclusion of GTPyS (0'1-1 mM) in the pipette, spontaneous transient outward currents appeared immediately after break-through but disappeared a few minutes later. Under these conditions, caffeine (10 mM) was almost without effect, suggesting that GTPyS had released Ca2+ stores. When the holding potential was held at -40 mV and GTPyS (0-1 or 0-2 mm) was present in the pipette, an inward current developed a few minutes after break-through. During the GTPyS-induced inward current, application of carbachol or histamine produced no further inward current. However, when 0-01 mmGTPyS was included in the pipette solution, carbachol-and histamine-induced inward currents were potentiated.6. Pretreated with 2-5 gug/ml pertussis toxin (PTX) did not change noticeably the MS 9646 106 S. KOMORI, M. KA WAI, T. TAKEWAKI AND H. OHASHI outward current responses to carbachol and histamine, but abolished or markedly reduced the inward current responses.7. The results suggest that stimulation of muscarinic receptor or histamine receptor caused release of Ca2+ from storage sites and activation of cationic channels, and that regardless of the receptor type, calcium store release may be mediated via a PTX-insensitive G-protein, while the cation channels are activated via another G-protein which is sensitive to PTX.

1 Isometric contractile responses to carbachol were studied in ileal longitudinal smooth muscle strips from wild-type mice and mice genetically lacking M 2 or M 3 muscarinic receptors, in order to characterize the mechanisms involved in M 2 and M 3 receptor-mediated contractile responses. 2 Single applications of carbachol (0.1-100 mM) produced concentration-dependent contractions in preparations from M 2 -knockout (KO) and M 3 -KO mice, mediated via M 3 and M 2 receptors, respectively, as judged by the sensitivity of contractile responses to blockade by the M 2 -preferring antagonist methoctramine (300 nM) or the M 3 -preferring antagonist 4-DAMP (30 nM). 3 The M 2 -mediated contractions were mimicked in shape by submaximal stimulation with high K þ concentrations (up to 35 mM), almost abolished by voltage-dependent Ca 2 þ channel (VDCC) antagonists or depolarization with 140 mM K þ medium, and greatly reduced by pertussis toxin (PTX) treatment. 4 The M 3 -mediated contractions were only partially inhibited by VDCC antagonists or 140 mM K þ -depolarization medium, and remained unaffected by PTX treatment. The contractions observed during high K þ depolarization consisted of different components, either sensitive or insensitive to extracellular Ca 2 þ . 5 The carbachol contractions observed with wild-type preparations consisted of PTX-sensitive and -insensitive components. The PTX-sensitive component was functionally significant only at low carbachol concentrations. 6 The results suggest that the M 2 receptor, through PTX-sensitive mechanisms, induces ileal contractions that depend on voltage-dependent Ca 2 þ entry, especially associated with action potential discharge, and that the M 3 receptor, through PTX-insensitive mechanisms, induces contractions that depend on voltage-dependent and -independent Ca 2 þ entry and intracellular Ca 2 þ release. In intact tissues coexpressing M 2 and M 3 receptors, M 2 receptor activity appears functionally relevant only when fractional receptor occupation is relatively small.

SUMMARY1. Single smooth muscle cells obtained by enzymic dispersion of the longitudinal muscle layer of rabbit jejunum were held under voltage clamp using patch pipettes and membrane currents measured. The effects of carbachol or caffeine applied externally were examined in cells dialysed with normal pipette solutions or with a solution containing heparin (which blocks receptors for D-myo-inositol 1,4,5-trisphosphate, InsP3), guanosine 5-0-(y-thio)triphosphate (GTPyS) or guanosine 5-O-(/i-thio)diphosphate (GDPflS).2. Outward current in response to application of carbachol or caffeine was considered to represent the opening of calcium-activated potassium channels in response to a localized rise in the free ionized calcium concentration occasioned by the rapid discharge of stored calcium (Ca) by these agents.3. Heparin included in the pipette solution blocked outward current to muscarinic receptor activation by carbachol but not that to caffeine, suggesting that receptorevoked discharge of stored cellular Ca is caused by InsP3 action. However, heparin did not affect muscarinic-receptor inward current.4. After dialysis with 0-1-0{5 mM-GTPyS, carbachol inward current was evoked in two out of three of the cells; after dialysis with 0-1-0'2 mM-GTPyS for an average of 7-7 min it was 80 % of the normal response; after dialysis for an average of 8-6 min with 0 5 mM-GTPyS it was 31 % of the normal response. In contrast, 0 1 mM-GTPyS reduced caffeine outward current by 93 % after an average 4-5 min dialysis and spontaneous transient outward currents (STOCs) were abolished in 2-9 min on average.5. Carbachol inward current (at -40 or -50 mV) and carbachol outward current (at 0 mV) in responding cells were reduced only by half after 8-10 min dialysis with 1 mM-GDP,?S which has been shown in portal vein cells to antagonize the depletion of Ca stores by intracellular GTPyS (Komori & Bolton, 1989). After 8-10 min dialysis with 5 mM-GDP,8S outward current was 27 % of normal. However, if GDP/JS was present, outward current generally could not be evoked by a second application of carbachol.6. The discharge of Ca stores by dialysis with 01 mm-GTPyS was prevented completely by heparin included in the pipette solution, suggesting that activation of a G-protein associated with phospholipase C (PLC) enzyme accelerates PLC activity, MS 8003 S. KOMORI AND T. B. BOLTON InsP3 production and depletion of Ca stores. However, dialysis with GDP/?S or heparin did not affect Ca storage or discharge of STOCs suggesting that any tonic production of InsP3 is without effect on Ca storage and that InsP3 does not contribute to the cyclical process which gives rise to STOCs.7. The results suggest that a G-protein is associated with PLC and InsP3 production. Upon muscarinic receptor activation, InsP3 production is essential for Ca store release. The opening of cationic (inward current) channels in response to muscarinic receptor activation did not have properties typical of a G-proteinmediated system, and if a G-protein is involved it does not seem to...

In single cells isolated from guinea-pig ileal smooth muscle, held under voltage clamp at -40 mV or -50 mV by patch pipette in the whole-cell recording mode, carbachol (CCh) evoked an oscillatory inward cationic current. The frequency of current oscillations increased with increasing CCh concentration. CCh-evoked current oscillations were followed very closely by oscillations in intracellular free Ca2+ estimated from the Indo-1 signal, and were abolished by inclusion of EGTA in the pipette solution. Ryanodine and heparin, but not nifedipine, blocked the generation of current oscillations. CCh-evoked current oscillations were abolished upon withdrawal of extracellular calcium and restored upon its reintroduction. Inclusion of GTP[gamma S] in the pipette solution caused the generation of an oscillatory inward current, which was blocked by ryanodine. The present results are consistent with the hypothesis that CCh-evoked cationic current is gated by activation of a G protein and is steeply dependent on [Ca2+]i, fluctuations in the release of Ca2+ from stores during carbachol's action produce oscillations in [Ca2+]i which cause similar oscillations in the cationic current.

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