Threshold Change in Muscarinic Receptor-operated All-or-none Response by Desensitization in Isolated Single Smooth Muscle Cells from Taenia Caecum

2015 ◽  
pp. 21-46
Author(s):  
Mitsuo Mita ◽  
Kazuhiko Oishi ◽  
Takao Hashimoto ◽  
Masaatsu K. Uchida
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscarinic Receptor ◽  
Muscle Cells

Human esophageal smooth muscle cells express muscarinic receptor subtypes M1 through M5

2000 ◽  
Vol 279 (5) ◽  
pp. G1059-G1069 ◽  
Author(s):  
Jian Wang ◽  
Pawel S. Krysiak ◽  
Lisanne G. Laurier ◽  
Stephen M. Sims ◽  
Harold G. Preiksaitis
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscarinic Receptor ◽  
Muscle Cells ◽  
Receptor Subtypes ◽  
Muscarinic Receptor Subtypes ◽  
Functional Responses ◽  
Selective Antagonist ◽  
Esophageal Physiology ◽  
Esophageal Smooth Muscle Cells

Receptor characterization in human esophageal smooth muscle is limited by tissue availability. We used human esophageal smooth muscle cells in culture to examine the expression and function of muscarinic receptors. Primary cultures were established using cells isolated by enzymatic digestion of longitudinal muscle (LM) and circular muscle (CM) obtained from patients undergoing esophagectomy for cancer. Cultured cells grew to confluence after 10–14 days in medium containing 10% fetal bovine serum and stained positively for anti-smooth muscle specific α-actin. mRNA encoding muscarinic receptor subtypes M1–M5 was identified by RT-PCR. The expression of corresponding protein for all five subtypes was confirmed by immunoblotting and immunocytochemistry. Functional responses were assessed by measuring free intracellular Ca2+ concentration ([Ca2+]i) using fura 2 fluorescence. Basal [Ca2+]i, which was 135 ± 22 nM, increased transiently to 543 ± 29 nM in response to 10 μM ACh in CM cells ( n = 8). This response was decreased <95% by 0.01 μM 4-diphenylacetoxy- N-methylpiperidine, a M1/M3-selective antagonist, whereas 0.1 μM methoctramine, a M2/M4-selective antagonist, and 0.1 μM pirenzepine, a M1-selective antagonist, had more modest effects. LM and CM cells showed similar results. We conclude that human smooth muscle cells in primary culture express five muscarinic receptor subtypes and respond to ACh with a rise in [Ca2+]i mediated primarily by the M3 receptor and involving release of Ca2+ from intracellular stores. This culture model provides a useful tool for further study of esophageal physiology.

Muscarinic receptor size on smooth muscle cells and membranes

1986 ◽  
Vol 251 (2) ◽  
pp. G195-G200
Author(s):  
S. M. Collins ◽  
C. Y. Jung ◽  
A. K. Grover
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscarinic Receptor ◽  
Plasma Membranes ◽  
Muscle Cells ◽  
High Energy ◽  
Membrane Preparation ◽  
Galactose Oxidase ◽  
Radiation Inactivation ◽  
Gastric Smooth Muscle

The loss of [3H]quinuclidinyl benzilate ([3H]QNB) binding following high-energy radiation was used to compare the muscarinic receptor size on single smooth muscle cells isolated by collagenase digestion from the canine stomach and on plasma membranes derived from intact gastric smooth muscle without exposure to exogenous proteolysis. Radiation inactivation of galactose oxidase (68 kdaltons), yeast alcohol dehydrogenase (160 kdaltons), and pyruvate kinase (224 kdaltons) activities were used as molecular-weight standards. Radiation inactivation of [3H]QNB binding to rat brain membranes, which gave a target size of 86 kdaltons, served as an additional control. In isolated smooth muscle cells, the calculated size of the muscarinic receptor was 80 +/- 8 kdaltons. In contrast, in a smooth muscle enriched plasma membrane preparation, muscarinic receptor size was significantly smaller at 45 +/- 3 kdaltons. Larger molecular sizes were obtained either in the presence of protease inhibitors (62 +/- 4 kdaltons) or by using a crude membrane preparation of gastric smooth muscle 86 +/- 7 kdaltons).

Expression and muscarinic receptor coupling of Lyn kinase in cultured human airway smooth muscle cells

2006 ◽  
Vol 290 (3) ◽  
pp. L492-L500 ◽  
Author(s):  
Thomas Pertel ◽  
Defen Zhu ◽  
Reynold A. Panettieri ◽  
Naoto Yamaguchi ◽  
Charles W. Emala ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscarinic Receptor ◽  
Airway Smooth Muscle ◽  
Tyrosine Kinases ◽  
Muscle Cells ◽  
Airway Smooth Muscle Cells ◽  
Human Airway Smooth Muscle ◽  
Lyn Kinase ◽  
Src Family Tyrosine Kinases

Src family tyrosine kinases are signaling intermediates in a diverse array of cellular events including cell differentiation, motility, proliferation, and survival. In nonairway smooth muscle cells, muscarinic receptors directly interact with Src family tyrosine kinases. As little is known about the expression and signaling of these Src family tyrosine kinases in human airway smooth muscle cells, we determined the expression of Src family members and characterized the muscarinic receptor-mediated activation of Lyn kinase in these cells. RT-PCR revealed mRNA transcripts for FYN, c- SRC, YES, FRK, and LYN. Fyn, c-Src, Yes, and Lyn were identified in cultured airway smooth muscle cells by immunoblot analysis. In both nontransformed human cultured airway smooth muscle cells and cells transduced with wild-type human Lyn kinase, carbachol increased Lyn kinase activity. Pertussis toxin pretreatment failed to block carbachol activation of Lyn kinase but did attenuate the carbachol-induced increase in ERK/MAPK phosphorylation. Moreover, carbachol inhibited adenylyl cyclase but failed to increase total inositol phosphate synthesis in these cells. The present study shows that Lyn kinase is expressed in human cultured airway smooth muscle cells at both the mRNA and protein levels and that carbachol, an M2 muscarinic receptor agonist in these cells, activates Lyn kinase by a pertussis toxin-insensitive signaling pathway.

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