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Immunohistochemistry using an antibody to unphosphorylated connexin 43 to identify human myometrial interstitial cells.

Hutchings G, Gevaert T, Deprest J, Roskams T, Van Lommel A, Nilius B, De Ridder D - Reprod. Biol. Endocrinol. (2008)

Bottom Line: Immunohistochemistry using an antibody directed against connexin 43 unphosphorylated at serine 368 showed that it is this isoform that is expressed continually by these cells.No particular arrangement of cells as plexuses was observed.This antibody specificity may aid future study of this potentially important cell type.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics and Gynaecology, University Hospital Gasthuisberg, Katholieke Universiteit Leuven, Leuven, Belgium. graham.hutchings@uclouvain.be

ABSTRACT

Background: Myometrial smooth myocytes contract as a result of electrical signalling via a process called excitation-contraction coupling. This process is understood in great detail at the cellular level but the generation and coordination of electrical signals throughout the myometrium are incompletely understood. Recent evidence concerning the vital role of interstitial cells of Cajal in tissue-level signalling in gastrointestinal tract, and the presence of similar cells in urinary tract smooth muscle may be relevant for future research into myometrial contractility but there remains a lack of evidence regarding these cells in the myometrium.

Methods: Single stain immunohistochemical and double stain immunofluorescence techniques visualised antibodies directed against total connexin 43, unphosphorylated connexin 43, KIT, alpha-SMA and prolyl 4-hydroxylase in myometrial biopsies from 26 women representing all stages of reproductive life.

Results: Myometrial smooth myocytes from term uterine biopsies expressed connexin 43 in a punctate pattern typical of gap junctions. However, on the boundaries of the smooth muscle bundles, cells were present with a more uniform staining pattern. These cells continued to possess the same staining characteristics in non-pregnant biopsies whereas the smooth myocytes no longer expressed connexin 43. Immunohistochemistry using an antibody directed against connexin 43 unphosphorylated at serine 368 showed that it is this isoform that is expressed continually by these cells. Double-stain immunofluorescence for unphosphorylated connexin 43 and KIT, an established marker for interstitial cells, revealed a complete match indicating these cells are myometrial interstitial cells (MICs). MICs had elongated cell processes and were located mainly on the surface of the smooth muscle bundles and within the fibromuscular septum. No particular arrangement of cells as plexuses was observed. Antibody to prolyl 4-hydroxylase identified fibroblasts as separate from MICs.

Conclusion: MICs are identified consistently on the boundaries of smooth muscle bundles in both the pregnant and non-pregnant uterus and are distinct from fibroblasts. The uniform distribution of connexin 43 on the cell membrane of MICs, rather than localisation in gap junction plaques, may represent the presence of connexin hemichannels. This antibody specificity may aid future study of this potentially important cell type.

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Unphosphorylated connexin 43 distribution in myometrium during term labour. (a) Antibody for unphosphorylated (serine 368) connexin 43 binds only to cells on the boundaries of the smooth muscle bundles (arrowhead) and to vascular endothelial cells (arrows) but not to smooth myocytes (Magnification ×10, Bar 100 micrometres). (b) At higher magnification, these cells show homogenous staining throughout the cell membrane, an oval shaped nucleus, elongated cell body and extended dendritic processes. (Magnification ×100, Bar 10 micrometres).
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Figure 2: Unphosphorylated connexin 43 distribution in myometrium during term labour. (a) Antibody for unphosphorylated (serine 368) connexin 43 binds only to cells on the boundaries of the smooth muscle bundles (arrowhead) and to vascular endothelial cells (arrows) but not to smooth myocytes (Magnification ×10, Bar 100 micrometres). (b) At higher magnification, these cells show homogenous staining throughout the cell membrane, an oval shaped nucleus, elongated cell body and extended dendritic processes. (Magnification ×100, Bar 10 micrometres).

Mentions: In contrast to total connexin 43, unphosphorylated connexin 43 was confined to the cells described above, located mainly on the boundary of the smooth muscle bundles and in the fibromuscular septum in all of the biopsies examined (Figure 2a). In all biopsies from pregnant women in labour or prior to labour, the antibody for unphosphorylated connexin 43 did not bind to the smooth myocytes. Under higher-powered magnification the positive cells had oval shaped nuclei and little perinuclear cytoplasm. The cell body was elongated and possessed dendritic processes (Figure 2b). Again the antibody was bound to the entire cell surface in contrast to the punctate staining seen on smooth myocytes with antibody to total connexin 43. Endothelial cells lining small blood vessels also expressed unphosphorylated connexin 43 but were distinguishable by their continuity with other similar cells to form vessels. Examination of the single biopsy taken from the upper segment of the uterus revealed an identical pattern. In biopsies taken from non-pregnant pre-menopausal women the smooth muscle bundles showed less hypertrophy making rows of unphosphorylated connexin 43 expressing cells more easily visible (data not shown). Although this architecture was less apparent in samples from postmenopausal women, unphosphorylated connexin 43 expressing cells were still identifiable as separate from smooth muscle cells that did not express connexin 43 (data not shown). Thus all biopsies demonstrated the permanent presence of unphosphorylated connexin 43-expressing cells on the boundaries of the smooth muscle bundles.


Immunohistochemistry using an antibody to unphosphorylated connexin 43 to identify human myometrial interstitial cells.

Hutchings G, Gevaert T, Deprest J, Roskams T, Van Lommel A, Nilius B, De Ridder D - Reprod. Biol. Endocrinol. (2008)

Unphosphorylated connexin 43 distribution in myometrium during term labour. (a) Antibody for unphosphorylated (serine 368) connexin 43 binds only to cells on the boundaries of the smooth muscle bundles (arrowhead) and to vascular endothelial cells (arrows) but not to smooth myocytes (Magnification ×10, Bar 100 micrometres). (b) At higher magnification, these cells show homogenous staining throughout the cell membrane, an oval shaped nucleus, elongated cell body and extended dendritic processes. (Magnification ×100, Bar 10 micrometres).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553078&req=5

Figure 2: Unphosphorylated connexin 43 distribution in myometrium during term labour. (a) Antibody for unphosphorylated (serine 368) connexin 43 binds only to cells on the boundaries of the smooth muscle bundles (arrowhead) and to vascular endothelial cells (arrows) but not to smooth myocytes (Magnification ×10, Bar 100 micrometres). (b) At higher magnification, these cells show homogenous staining throughout the cell membrane, an oval shaped nucleus, elongated cell body and extended dendritic processes. (Magnification ×100, Bar 10 micrometres).
Mentions: In contrast to total connexin 43, unphosphorylated connexin 43 was confined to the cells described above, located mainly on the boundary of the smooth muscle bundles and in the fibromuscular septum in all of the biopsies examined (Figure 2a). In all biopsies from pregnant women in labour or prior to labour, the antibody for unphosphorylated connexin 43 did not bind to the smooth myocytes. Under higher-powered magnification the positive cells had oval shaped nuclei and little perinuclear cytoplasm. The cell body was elongated and possessed dendritic processes (Figure 2b). Again the antibody was bound to the entire cell surface in contrast to the punctate staining seen on smooth myocytes with antibody to total connexin 43. Endothelial cells lining small blood vessels also expressed unphosphorylated connexin 43 but were distinguishable by their continuity with other similar cells to form vessels. Examination of the single biopsy taken from the upper segment of the uterus revealed an identical pattern. In biopsies taken from non-pregnant pre-menopausal women the smooth muscle bundles showed less hypertrophy making rows of unphosphorylated connexin 43 expressing cells more easily visible (data not shown). Although this architecture was less apparent in samples from postmenopausal women, unphosphorylated connexin 43 expressing cells were still identifiable as separate from smooth muscle cells that did not express connexin 43 (data not shown). Thus all biopsies demonstrated the permanent presence of unphosphorylated connexin 43-expressing cells on the boundaries of the smooth muscle bundles.

Bottom Line: Immunohistochemistry using an antibody directed against connexin 43 unphosphorylated at serine 368 showed that it is this isoform that is expressed continually by these cells.No particular arrangement of cells as plexuses was observed.This antibody specificity may aid future study of this potentially important cell type.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics and Gynaecology, University Hospital Gasthuisberg, Katholieke Universiteit Leuven, Leuven, Belgium. graham.hutchings@uclouvain.be

ABSTRACT

Background: Myometrial smooth myocytes contract as a result of electrical signalling via a process called excitation-contraction coupling. This process is understood in great detail at the cellular level but the generation and coordination of electrical signals throughout the myometrium are incompletely understood. Recent evidence concerning the vital role of interstitial cells of Cajal in tissue-level signalling in gastrointestinal tract, and the presence of similar cells in urinary tract smooth muscle may be relevant for future research into myometrial contractility but there remains a lack of evidence regarding these cells in the myometrium.

Methods: Single stain immunohistochemical and double stain immunofluorescence techniques visualised antibodies directed against total connexin 43, unphosphorylated connexin 43, KIT, alpha-SMA and prolyl 4-hydroxylase in myometrial biopsies from 26 women representing all stages of reproductive life.

Results: Myometrial smooth myocytes from term uterine biopsies expressed connexin 43 in a punctate pattern typical of gap junctions. However, on the boundaries of the smooth muscle bundles, cells were present with a more uniform staining pattern. These cells continued to possess the same staining characteristics in non-pregnant biopsies whereas the smooth myocytes no longer expressed connexin 43. Immunohistochemistry using an antibody directed against connexin 43 unphosphorylated at serine 368 showed that it is this isoform that is expressed continually by these cells. Double-stain immunofluorescence for unphosphorylated connexin 43 and KIT, an established marker for interstitial cells, revealed a complete match indicating these cells are myometrial interstitial cells (MICs). MICs had elongated cell processes and were located mainly on the surface of the smooth muscle bundles and within the fibromuscular septum. No particular arrangement of cells as plexuses was observed. Antibody to prolyl 4-hydroxylase identified fibroblasts as separate from MICs.

Conclusion: MICs are identified consistently on the boundaries of smooth muscle bundles in both the pregnant and non-pregnant uterus and are distinct from fibroblasts. The uniform distribution of connexin 43 on the cell membrane of MICs, rather than localisation in gap junction plaques, may represent the presence of connexin hemichannels. This antibody specificity may aid future study of this potentially important cell type.

Show MeSH
Related in: MedlinePlus