Characterization of colonic circular smooth muscle cells in culture

1992 ◽  
Vol 263 (3) ◽  
pp. G365-G370 ◽  
Author(s):  
H. S. Ennes ◽  
J. A. McRoberts ◽  
P. E. Hyman ◽  
W. J. Snape
Keyword(s):  
Smooth Muscle ◽  
Adenylate Cyclase ◽  
Smooth Muscle Cells ◽  
Primary Culture ◽  
Cultured Cells ◽  
Muscle Cells ◽  
Intracellular Camp ◽  
Isolated Cells ◽  
Vip Receptors ◽  
Circular Smooth Muscle

The receptor-binding properties of isolated rabbit colonic circular smooth muscle cells in primary culture have been investigated. In intact smooth muscle, acetylcholine, acting through M2 muscarinic receptors, and vasoactive intestinal polypeptide (VIP), acting through VIP receptors, are two of the principal neurotransmitters mediating contraction and relaxation, respectively. The muscarinic receptor was present in very high levels (600,000 receptors/cell) on freshly isolated colonic smooth muscle cells as shown by binding of the muscarinic receptor antagonist N-methylscopolamine (NMS). However, NMS binding sites decreased rapidly when the cells were placed in primary culture. After 21 h in culture, specific binding of [3H]NMS decreased to 20%, and after 48 h to less than 10% that of preculture values. This loss was not associated with a change in receptor affinity, since Kd was unchanged for the receptors still present. In contrast, high-affinity VIP receptors were expressed on cultured smooth muscle cells but could not be detected on freshly isolated cells. Cultured cells responded to VIP with an increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP), indicating that the VIP receptors were functionally coupled to adenylate cyclase. Cultured cells also responded to calcitonin gene-related peptide (CGRP) and forskolin with increased production of intracellular cAMP. In contrast, neither VIP nor CGRP elicited an increase in intracellular cAMP when added to freshly isolated cells. Furthermore, freshly isolated cells had a greatly diminished response to forskolin, suggesting that the isolation procedure not only destroyed cell surface receptors for VIP and CGRP, but also damaged the cells sufficiently to decrease cellular adenylate cyclase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum deprivation induces a unique hypercontractile phenotype of cultured smooth muscle cells

1998 ◽  
Vol 274 (5) ◽  
pp. C1206-C1214 ◽  
Author(s):  
Xuefei Ma ◽  
Ying Wang ◽  
Newman L. Stephens
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Cultured Cells ◽  
Muscle Cells ◽  
Serum Deprivation ◽  
Airway Smooth Muscle Cells ◽  
Shortening Velocity ◽  
Isolated Cells

Chronic asthma is characterized by hypertrophy and hyperplasia of airway smooth muscle cells (SMC) that limit airflow by a geometric effect. Whether contractility of airway SMC is altered is not clear. Cultured cells were used as a model of hyperplasia. Phenotypic changes seen indicated conversion to a synthetic, weakly contractile type. At confluence, although limited reversal of protein changes was seen, no restoration in contractility occurred. Phenotypic modulation of postconfluent cultured airway SMC under prolonged serum deprivation (arrested cells) is reported here. Two phenotypically distinct groups of cells were identified in primary airway SMC cultures: 1) elongated spindle-shaped cells, which expressed large amounts of smooth muscle contractile and regulatory proteins, and 2) flat and stellate cells, which expressed very little. The first group showed a surprising shortening capacity and a velocity that was even greater than that of the freshly isolated cells, whereas the second group became spherical and noncontractile. Even more surprising was that the myosin heavy chain (MHC) isoform (SM-B) generally said to be associated with the higher shortening velocity disappeared from the cell, while the content of the key rate-limiting regulating enzyme, myosin light chain kinase (MLCK), increased 30-fold. We conclude that a functional, contractile phenotype of airway SMC can be obtained by prolonged serum deprivation. We speculate that the increased contractility could be the result of increased phosphorylation of the 20-kDa myosin light chain resulting from increased content of smooth muscle MLCK rather than any increase in endogenous MHC ATPase activity. This model may be useful for study of SMC differentiation and contraction.

scholarly journals 269.Estrogen and progesterone receptor expression is significantly reduced in cultured myometrial and fibroid smooth muscle cells

2004 ◽  
Vol 16 (9) ◽  
pp. 269
Author(s):  
M. Zaitseva ◽  
P. A. W. Rogers
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Internal Control ◽  
Cultured Cells ◽  
Progesterone Receptors ◽  
Muscle Cells ◽  
Receptor Expression ◽  
Isolated Cells ◽  
Large Variability

Fibroids are benign neoplasms of the smooth muscle cells of the uterus. Cultured myometrial and fibroid smooth muscle cells (MSMC & FSMC) have been widely used as a model for the study of fibroid growth. However, there is ongoing controversy regarding expression levels of estrogen and progesterone receptors (ER and PR) in cultured cells vs tissue. The aim of the present study was to measure levels of mRNA for ER� and PR in myometrium and fibroid, and in cultured MSMC & FSMC. Myometrium and fibroids were collected from hysterectomy specimens (n = 8). Part of the tissue was snap frozen and the rest was used to isolate SMC, which were cultured for 3 passages and collected for RNA at P0 (2 weeks in culture) and P3 (5 weeks in culture). ERα and PR levels were quantified using real-time PCR and normalized using 18S rRNA as an internal control. Both ERα and PR were detected in all samples. Large variability in receptor levels between different isolates was detected. Surprisingly, despite large differences between the means, none of comparisons of tissue v. P0 cells were significant by non-parametric tests. However, there was a statistically significant reduction in both ERα and PR expression between whole tissue and isolated cells at P3 (Table 1, see PDF file). This is the first study to provide objective data to support a significant decline in ERα and PR expression in cultured MSMC and FSMC. Despite this decline, detectable levels of ERα and PR mRNA were present at both P0 and P3, potentially explaining why some published studies have been able to demonstrate in vitro response to steroids in these cells.

Characterization and confocal imaging of calponin in gastrointestinal smooth muscle

1993 ◽  
Vol 265 (5) ◽  
pp. C1371-C1378 ◽  
Author(s):  
M. P. Walsh ◽  
J. D. Carmichael ◽  
G. J. Kargacin
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Smooth Muscle Cells ◽  
Thin Filament ◽  
Muscle Cells ◽  
Biochemical Properties ◽  
Confocal Imaging ◽  
Isolated Cells ◽  
Independent Manner

Calponin isolated from chicken gizzard smooth muscle binds in vitro to actin in a Ca(2+)-independent manner and thereby inhibits the actin-activated Mg(2+)-adenosinetriphosphatase of smooth muscle myosin. This inhibition is relieved when calponin is phosphorylated by protein kinase C or Ca2+/calmodulin-dependent protein kinase II, suggesting that calponin is involved in thin filament-associated regulation of smooth muscle contraction. To further examine this possibility, calponin was isolated from toad stomach smooth muscle, characterized biochemically, and localized in intact isolated cells. Toad stomach calponin had the same basic biochemical properties as calponin from other sources. Confocal immunofluorescence microscopy revealed that calponin in intact smooth muscle cells was localized to long filamentous structures that were colabeled by antibodies to actin or tropomyosin. Preservation of the basic biochemical properties of calponin from species to species suggests that these properties are relevant for its in vivo function. Its colocalization with actin and tropomyosin indicates that calponin is associated with the thin filament in intact smooth muscle cells.

NUMERICAL SIMULATION OF THE ROLE OF CO-TRANSMISSION BY ACETYLCHOLINE AND SEROTONIN ON MOTILITY OF THE GUT

2006 ◽  
Vol 06 (04) ◽  
pp. 399-428
Author(s):  
R. MIFTAHOF
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Transit Time ◽  
Receptor Agonist ◽  
Muscle Cells ◽  
Receptor Antagonists ◽  
One Dimensional ◽  
Circular Smooth Muscle ◽  
Spatio Temporal ◽  
Stimulation Of

Electrophysiological mechanisms of co-transmission by serotonin (5-HT) and acetylcholine (ACh), co-expression of their receptor types, i.e., 5-HT type 3 and 4, nicotinic cholinerginc (nACh) and muscarinic cholinergic (μACh), and effects of selective and non-selective 5-HT3 and 5-HT4 receptor agonists/antagonists, on electromechanical activity of the gut were studied numerically. Two series of numerical experiments were performed. First, the dynamics of the generation and propagation of electrical signals interconnected with the primary sensory (AH) neurons, motor (S) neurons and smooth muscle cells were studied in a one-dimensional model. Simulations showed that stimulation of the 5-HT3 receptors reduced the threshold of activation of the mechanoreceptors by 17.6%. Conjoint excitation of the 5-HT3 and 5-HT4 receptors by endogenous serotonin converted the regular firing pattern of electrical discharges of the AH and S neurons to a beating mode. Activation confined to 5-HT3 receptors, located on the somas of the adjacent AH and S type neurons, could not sustain normal signal transduction between them. It required ACh as a co-transmitter and co-activation of the nACh receptors. Application of selective 5-HT3 receptor antagonists inhibited dose-dependently the production of action potentials at the level of mechanoreceptors and the soma of the primary sensory neuron and increased the threshold activation of the mechanoreceptors. Normal mechanical contractile activity depended on co-stimulation of the 5-HT4 and μACh receptors on the membrane of smooth muscle cells. In the second series of simulations, which involved a spatio-temporal model of the functional unit, effects of co-transmission by ACh and 5-HT on the electromechanical response in a segment of the gut were analyzed. Results indicated that propagation of the wave of excitation between the AH and S neurons within the myenteric nervous plexus in the presence of 5-HT3 receptor antagonists was supported by co-release of ACh. Co-stimulation of 5-HT3, nACh and μACh receptors impaired propulsive activity of the gut. The bolus showed uncoordinated movements. In an ACh-free environment Lotronex (GlaxoSmithKline), a 5-HT3 receptor antagonist, significantly increased the transit time of the pellet along the gut. In the presence of ACh, Lotronex produced intensive tonic-type contractions in the longitudinal and circular smooth muscle layers and eliminated propulsive activity. The 5HT4 receptor agonist, Zelnorm (Novartis), preserved the reciprocal electromechanical relationships between the longitudinal and circular smooth muscle layers. The drug changed the normal propulsive pattern of activity to an expulsive (non-mixing) type. Treatment of the gut with selective 5HT4 receptor antagonists increased the transit time by disrupting the migrating myoelectrical complex. Cisapride (Janssen), a mixed 5HT3 and 5HT4 receptor agonist, increased excitability of the AH and S neurons and the frequency of slow waves. Longitudinal and circular smooth muscle syncytia responded with the generation of long-lasting tonic contractions, resulting in a "squeezing" type of pellet movement. Comparison of the theoretical results obtained on one-dimensional and spatio-temporal models to in vivo and in vitro experimental data indicated satisfactory qualitative, and where available, quantitative agreement.

SCN5A is expressed in human jejunal circular smooth muscle cells

2002 ◽  
Vol 14 (5) ◽  
pp. 477-486 ◽  
Author(s):  
Y. Ou ◽  
S. J. Gibbons ◽  
S. M. Miller ◽  
P. R. Strege ◽  
A. Rich ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Circular Smooth Muscle
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