GTP‐binding protein involvement in membrane currents evoked by carbachol and histamine in guinea‐pig ileal muscle.

Abstract

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.

Effects of G‐protein‐specific antibodies and Gβγ subunits on the muscarinic receptor‐operated cation current in guinea‐pig ileal smooth muscle cells

“…This was supported by the failure of Ga i1 , Ga i1 /Ga i2 , Ga q /Ga 11 and Ga s antibodies to reduce the cation current response. These results are also consistent with the block of the cation current response to M 2 receptor activation by PTX (Inoue & Isenberg, 1990;Komori et al, 1992;Unno et al, 1995;Pucovsky et al, 1998). The failure of Ga q /Ga 11 antibodies to inhibit mIcat is significant because Wang et al (1997) recently showed that dialysis of the same antibody inhibited muscarinic-receptor I Cl(Ca) in tracheal smooth muscle cells, although it had no effect on mIcat.…”

“…In rabbit portal vein myocytes, the cation current evoked by noradrenaline shows similar properties to mIcat in guinea-pig small intestine at negative potentials but, unlike the latter, passes little current in the positive range (Helliwell & Large, 1996); it can be evoked by 1-oleoyl-2-acetyl-sn-glycerol (OAG) (Helliwell & Large, 1997), which is completely ineffective in evoking mIcat in the guinea-pig intestine (our unpublished observation). mIcat in guinea-pig small intestine is pertussis toxin (PTX)-sensitive (Inoue & Isenberg, 1990;Komori et al, 1992), as it is in equine tracheal smooth muscle cells (Wang et al, 1997). In the latter, the current was blocked by Ga i1 /Ga i2 antibodies and by Ga i3 /Ga o antibodies, but not by Ga q /Ga 11 antibodies, suggesting that Ga i /Ga o was the link between muscarinic receptor and cation channel.…”

“…Single smooth muscle myocytes from the longitudinal muscle layer of the ileum were obtained after collagenase and papain treatment as previously described (Komori et al, 1992;Zholos & Bolton, 1996a).…”

Modulation of carbachol‐induced [Ca2+]i oscillations by Ca2+ influx in single intestinal smooth muscle cells

“…The longitudinal muscle layer of the segments was peeled from the underlying circular muscle and washed in physiological salt solution (PSS; composition given below). Smooth muscle cells were dispersed by a combination of collagenase (0.2-0.6 mg ml-') and papain (0.3-0.6 mg ml-'), as previously described (Komori et al, 1992 (Pacaud & Bolton, 1991;Komori et al, 1993) (Komori et al, 1992;1993; (Komori et al, 1993;Kohda et al, 1996).…”

“…Carbachol (CCh) is capable of inducing InsP3-dependent [Ca2+], oscillations in single smooth muscle cells of guinea-pig ileum (Komori et al, 1993;Kohda et al, 1996). The present study aimed to investigate potential path- (Benham et al, 1985;Inoue & Isenberg, 1990;Komori et al, 1992) and can change its intensity in a manner closely correlated with [Ca2+]i (Pacaud & Bolton, 1991; Brifish Journal of Phamacology (1996Phamacology ( ) 119, 245 252 1993 …”