In summary, I have demonstrated that mouse gut smooth muscle cells are endowed with three distinct pathways mediating mICat generation. Two of these pathways are initiated by activation of either of M2 and M3 receptors and target 70-pS channels or both 70-pS and 120-pS channels, respectively. The third pathway requires the presence of both M2 and M3 receptors to be effective and targets 70-pS channels. This M2/M3 pathway most strongly activates the 70-pS channel resulting in a considerably longer open state and a substantial rise in the opening probability. Thus, the M2/M3 pathway is the major mediator of whole-cell mICat and most potently depolarizes the membrane. The
delineation of the M2/M3 pathway is consistent with the existence of a signaling complex involving the M2-Go system, the M3-PLC system and cationic channels predicted to mediate mICat. These findings provide novel insights into the signal transduction mechanisms underlying the muscarinic regulation of electrical and mechanical activity of intestinal smooth muscle. Further studies are needed to identify the molecular basis of the muscarinic cationic channels and to elucidate their activation mechanisms in more detail.
Ⅵ. Summary
1. The present study aimed to elucidate activation mechanisms for the muscarinic receptor-operated cationic channel in gut myocyte from mice lacking M2 and/or M3
muscarinic receptor and their WT strains. Whole-cell musucarinic cationic currents (mICat) were recorded from single myocytes using whole-cell patch clamp techniques, and single cationic channel activities also done in excised membrane patches using outside-out patch clamp techiniques.
2. Carbachol (1-300 μM)-induced mICat was pharmacologically characterized in the myocytes from conventional mouse of ddY strain. The mICat was inhibited competitively by M2 preferring antagonist and non-competitively by M3 preferring one. It was severely inhibited by PTX treatment or PLC inhibitor application and had a U-shape I-V relationship. The mICat was sensitive to Ca2+ influx via voltage dependent Ca2+ channels. These results show that muscarinic signaling mechanisms for the mICat generation were almost the same as those for guinea-pig intestinal mICat. 3. To elucidate the role of M2 and M3 receptors for leading to mICat generation, M2 or
M3 single knockout (KO) and M2/M3-double KO mice were used as experimental tools. Carbachol (100 μM) evoked sustained mICat in myocytes from WT strains, but evoked only a small inward current in myocytes from M2-KO or M3-KO strains. In M2/M3 double-KO strain, carbachol could not evoke any appreciable current. All mutant type cells preserved normal G protein-cationic channel coupling, judged from their ability to generate cationic currents in response to the direct G protein activator, GTPγS. Measurements of Ca2+-activated K+ current and intracellular cAMP responses to carbachol indicated that the M2 and M3 receptor subtypes were able to couple normally to their respective G protein/effector systems (i.e. the Gi/adenylyl cyclase and the Gq/PLCβ respectively) in M3-KO and M2-KO cells, respectively.
These results suggested that mICat in WT cells is not simple mixture of M2 and M3
responses and that three distinct pathways leading to mICat generation exist. Two of which involved either M2 and M3 receptors and the third requires the presence of both receptor subtypes. The latter pathway may mediate the major part of mICat. 4. Voltage dependency and Ca2+ sensitivity of mICat were investigated in M2-KO,
M3-KO and WT cells. I-V curves were U-shaped in WT and M3KO cells, but roughly linear in M2KO cells. These results suggested that cationic channels activated via M2- and M2/M3 pathways are similar in their intrinsic voltage dependency. Ca2+
injection via voltage dependent Ca2+ channels activated by depolarizing pulse potentiated WT mICat, but could not potentiate mICat in M2- or M3-KO cells. These observations suggested that Ca2+ has no potentiating role in channel activation in M2
and M3 pathways.
5. To assess the physiological relevance of the three mICat-inducing pathways, I observed changes in membrane potential produced by carbachol in cells from the four different mouse strains, using nystatin-perforated patch clamp techniques. In WT cells, carbachol at 0.1-0.3 μM produced a moderate depolarization of 10-20 mV and generation or acceleration of spike activity. Higher concentrations of carbachol (> 1 μM) evoked a full depolarization. In M2KO cells, 0.1 μM carbachol was almost without effect, but at higher concentration of 30 or 100 μM, a full depolarization was evoked. In M3KO cells, lower concentrations of 1-3 μM were usually ineffective. At 100 μM, a significant depolarization of about 10 mV was evoked, but full response was not evoked. These observations suggest that the three muscarinic pathways are relevant in depolarizing the membrane with a rank order of M2/M3 > M3 > M2, similar to that for mICat generation.
6. To identify target channels for the muscarinic pathways, single channel activity was
observed in outside-out patches that were excised from M2-KO, M3-KO or WT cells.
Channel analysis in outside-out patches recognized 70-pS and 120-pS channels as the major muscarinic cationic channels. Active patches of M2-KO cells exhibited both 70-pS and 120-pS channel activity usually together, either of which consisted of brief openings (the respective mean open times Oτ = 0.55 and 0.23 msec). In contrast, active M3-KO patches showed only 70-pS channel activity, which had three open states (Oτ = 0.55, 3.1 and 17.4 msec). In WT patches, besides the M2-KO and M3-KO types, another type of channel activity was also observed that consisted of 70-pS channel openings with four open states (Oτ = 0.62, 2.7, 16.9 and 121.1 msec), and patch current of this channel activity showed a U-shaped I-V curve similar to the WT mICat. These finding suggested that the major part of whole-cell mICat depends on 70-pS channel activated by M2/M3 pathway.
7. In conclusion, intestinal myocytes are endowed with three distinct muscarinic pathways mediating cationic channel activation. The M2/M3 pathway targeting 70-pS channels, serves as the major contributor to mICat generation. The delineation of this pathway is consistent with the formation of a functional unit by the M2/Go protein and the M3/PLC systems predicted to control cationic channels.
Ⅶ. Acknowledgements
Firstly, I am greatly indebted to Professor Seiichi Komori, Department of Pathogenic Veterinary Science, United Graduate School of Veterinary Science, Gifu University, for excellent guidance and invaluable suggestions and criticisms trough the experiments and in the preparation of this thesis.
I am grateful to Associate Professor Toshihiro Unno, Department of Pathogenic Veterinary Science, United Graduate School of Veterinary Science, Gifu University, for helpful suggestions and discussions on my research.
I am also grateful to Professor Masakazu Nishimura and Professor Minoru Shimoda, Department of Pathogenic Veterinary Science, United Graduate School of Veterinary Science, Gifu University, Professor Yoshio Yamamoto and Professor Tadashi Takewaki, Department of Basic Veterinary Science, United Graduate School of Veterinary Science, Gifu University, for their critical suggestions and careful reading of this thesis.
I wish to thank Research Associate Hayato Matsuyama, Laboratory of Pharmacology, Department of Veterinary Medicine, Faculty of Applied Biological Science, Gifu University for kind suggestions on my research and colleagues in Laboratory of Pharmacology, Department of Veterinary Medicine, Faculty of Applied Biological Science, Gifu University, for the assistance in the experiments.
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