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

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.

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Characterization and confocal imaging of calponin in gastrointestinal smooth muscle

AJP Cell Physiology ◽

10.1152/ajpcell.1993.265.5.c1371 ◽

1993 ◽

Vol 265 (5) ◽

pp. C1371-C1378 ◽

Cited By ~ 30

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.

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Characterization of colonic circular smooth muscle cells in culture

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1992.263.3.g365 ◽

1992 ◽

Vol 263 (3) ◽

pp. G365-G370 ◽

Cited By ~ 2

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)

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243. In vitro culture significantly reduces differences in gene expression profiles between myometrial and fibroid smooth muscle cells

Reproduction Fertility and Development ◽

10.1071/srb05abs243 ◽

2005 ◽

Vol 17 (9) ◽

pp. 96

Author(s):

M. Zaitseva ◽

P. A. W. Rogers

Keyword(s):

Gene Expression ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Cultured Cells ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Muscle Cells ◽

Compare Gene Expression

Fibroids are benign neoplasms of the smooth muscle cells of the uterus. Cultured myometrial (M) and fibroid (F) smooth muscle cells (SMC) have been widely used as a model for the study of fibroid growth. Although it has been shown that FSMC can behave differently in culture to MSMC, it is not clear how relevant the cultured cells and their responses are to the in-vivo situation. The aim of the present study was to compare gene expression profiles of M and F tissue to cells isolated from the same tissue and cultured for up to 3 passages. M and F were collected from hysterectomy specimens (n = 6), part was snap frozen for RNA and the rest used to isolate SMC, which were cultured for 3 passages and RNA was collected at passage 0 (P0) and 3 (P3). 36 microarrays were performed on 8K human cDNA slides, 6 per each specimen (3 for M and 3 for F: tissue, cell at P0 and P3) against reference RNA. Analysis revealed significant differences between tissues and cultured cells. Independent clustering assigned tissues versus cells into two distinct groups based on their expression profiles. Parametric ANOVA with Benjamini-Hochberg correction and post-hoc testing was used to determine similarities and differences between tissues and cells. 128 genes were found to be statistically different between M and F tissue, 66 between MSMC and FSMC at P0, and only 9 at P3. More than 1100 genes were significantly changed between tissues and cultured cells, with 648 genes common between both M and F cells at P0 and P3. Similar numbers of genes were up regulated as were down regulated. Expression profiles of genes of interest including estrogen receptor α and progesterone receptor were also validated using real-time PCR. This is the first study to compare gene expression of in vivo and in vitro fibroid and myometrial SMC. The results demonstrate that large changes occur in SMC gene expression in culture, reducing differences between myometrial and fibroid cells. This study indicates that results of in vitro studies should be interpreted with caution as many genes have an altered gene expression profile in culture.

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Use of cultured airway myocytes for study of airway smooth muscle

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1995.268.1.l1 ◽

1995 ◽

Vol 268 (1) ◽

pp. L1-L11 ◽

Cited By ~ 17

Author(s):

I. P. Hall ◽

M. Kotlikoff

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Airway Smooth Muscle ◽

Cultured Cells ◽

Muscle Cells ◽

Cellular Level ◽

Receptor Expression ◽

Airway Smooth Muscle Cells ◽

Wide Range ◽

Airway Responses

Cultured airway smooth muscle cells provide a convenient model system for studying the regulation of a wide range of airway responses at the cellular level. This review describes the characteristics of cultured airway smooth muscle cells and differences that exist between cultured cells and acutely dissociated cells or muscle strips. Receptor and ion-channel expression and control of coupling in cultured airway smooth muscle are reviewed. The methodology for airway smooth muscle culture is discussed. The main advantage of using cultured airway smooth muscle cells is that studies can be performed to examine long-term control of cell responses. Studies of the regulation of receptor expression and coupling, desensitization of receptor or channel-mediated responses, or regulation of the expression of important enzymes or muscle proteins can be readily performed in cell culture. In addition, cultured airway myocytes provide a useful secondary screening system for the development of novel therapeutic agents targeted at airway receptors that are expressed upon these cells.

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3P-0688 An HMG-CoA reductase inhibitor inhibits the migration of smooth muscle cells through the LR11-mediated urokinase receptor expression system in vitro

Atherosclerosis Supplements ◽

10.1016/s1567-5688(03)90907-x ◽

2003 ◽

Vol 4 (2) ◽

pp. 212-213

Author(s):

J. Meizi ◽

H. Bujo ◽

H. Yamazaki ◽

M. Shibasaki ◽

K. Takahashi ◽

...

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Reductase Inhibitor ◽

Expression System ◽

Muscle Cells ◽

Urokinase Receptor ◽

Receptor Expression ◽

Hmg Coa Reductase Inhibitor ◽

Coa Reductase

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

AJP Cell Physiology ◽

10.1152/ajpcell.1998.274.5.c1206 ◽

1998 ◽

Vol 274 (5) ◽

pp. C1206-C1214 ◽

Cited By ~ 88

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.

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