A method for measurement of muscarinic receptor-mediated responses in dissociated single colon longitudinal smooth muscle cells.
We previously reported a simple method of acutely preparing dissociated smooth muscle cells from urinary bladder tissue, but the feasibility of this method has not been well ascertained. In the present study, we assessed whether this method is applicable for measuring muscarinic receptor function in intestinal smooth muscle cells. Single smooth muscle cells were prepared from the longitudinal muscle tissue of guinea pig colon by the enzymatic dissociation with papain and hyaluronidase, followed by collagenase digestion. Muscarinic responses in the isolated smooth muscle cells were measured by intracellular Ca(2+) mobilization and extracellular acidification through Fura-2 fluorometry and Cytosensor microphysiometry, respectively. A single, viable population of colon longitudinal smooth muscle cells (approximately 6 x 10(6) cells/animal) was obtained. In these cells, carbachol (muscarinic agonist) induced Ca(2+) mobilization and extracellular acidification over the concentration range similar to that previously reported to produce contraction of the intact colon muscle strips. Atropine (nonselective muscarinic antagonist) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, M(3)-selective antagonist) inhibited the Ca(2+) mobilization with potencies approximately 3 log units greater than that for methoctramine (M(2)-selective antagonist). For extracellular acidification, the potency differences between these antagonists was approximately 2 log units. In addition, the carbachol-induced extracellular acidification was inhibited by 5-[N-ethyl-N-isopropyl]-amiloride, a selective inhibitor of the Na(+)/H(+) exchanger. These findings indicate that in isolated colonic smooth muscle cells, M(3) receptors are predominantly involved in Ca(2+) mobilization, while a mixed population of M(2) and M(3) receptors seems to contribute to extracellular acidification. Our results further suggest the role of the Na(+)/H(+) exchanger in muscarinic-mediated extracellular acidification. Consequently, our method produces viable isolated colonic smooth muscle cells that display physiologically appropriate responses to muscarinic receptor activation, and the method may be applicable for several types of nonvascular smooth muscle tissues.
Kobayashi
S
S
Pharmacology Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan. kobayashi.seiji@yamanouchi.co.jp
Ikeda
K
K
Miyata
K
K
Yamada
T
T
Honda
K
K
D000584:Amiloride / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001285:Atropine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002469:Cell Separation; D018679:Cholinergic Agonists / Q000494:pharmacology; D003106:Colon; D003959:Diamines / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D016257:Fura-2 / Q000493:pharmacokinetics; D006168:Guinea Pigs; D008297:Male; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D010880:Piperidines / Q000494:pharmacology; D011976:Receptors, Muscarinic / Q000145:classification / Q000502:physiology*
D018679:Cholinergic Agonists; D003959:Diamines; D010880:Piperidines; D011976:Receptors, Muscarinic; D001285:Atropine; D000584:Amiloride; C042375:4-diphenylacetoxy-1,1-dimethylpiperidinium; D002217:Carbachol; C054938:methoctramine; D002118:Calcium; D016257:Fura-2; C039614:ethylisopropylamiloride