Cultured myometrial cells establish communicating gap junctions

Loch‐Caruso, Rita; Juberg, Daland R.; Caldwell, V.; Corcos, Isabel A.

doi:10.1016/0309-1651(90)91160-6

Cited by 26 publications

(15 citation statements)

References 21 publications

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“…After 8 -1 0 days, cells had formed a confluent monolayer. The cells were positively identified as smooth muscle by indirect immunofluorescence staining for a-actin [13].…”

Section: Isolation O F Mesenteric Arterial Smooth Muscle Cellsmentioning

confidence: 99%

Gap Junctional Communication and Vascular Smooth Muscle Reactivity: Use of Tetraethylammonium Chloride

et al. 1994

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Oscillatory contractions in uterine smooth muscle are mechanistically related to gap junction complex formation. We have tested the hypothesis that agonist-induced oscillations in vascular smooth muscle are also mediated by gap junctions and that gap junctions are important for vascular smooth muscle cell communication. Total RNA from cultured Wistar-Kyoto rat (WKY) mesenteric arterial cells hybridized strongly with a cDNA probe for the message for connexin43, a monomer of the gap junction. In these same cells, the quaternary ion tetraethylammonium (TEA) (10 mM) increased Lucifer yellow dye transfer between contiguous cells, a measure of cell-to-cell communication via gap junctions, approximately 35% above basal levels. Heptanol, an established inhibitor of gap junction communication, completely blocked both basal- and TEA-stimulated dye transfer between neighboring cells. In other experiments, helical strips of superior mesenteric and tail arteries from WKY rats were mounted in tissue baths for measurement of isometric contractile force. TEA (10^–3–10^–1M) induced oscillatory contractions (1–5 cycle/min) in both mesenteric and tail arteries. Removal of endothelium did not affect the pattern of TEA-stimulated oscillations. Oscillations to TEA were blocked in a concentration-dependent manner in both arteries by heptanol (10^–7–10^–3M). Heptanol (10^–3M) also significantly reduced (40%) acetylcholine-induced relaxation in the mesenteric artery (contracted with phenylephrine). Thus, these data document that: (1) the message for the gap junction protein connexin43 is located in cultured arterial cells; (2) TEA induces oscillatory contractions in vascular smooth muscle and stimulates intercellular communication in cultured cells, and (3) oscillatory contractions, vascular relaxation and intercellular communication are blocked by the gap junction inhibitor heptanol. Collectively, these data strongly support the importance of gap junctional communication in vascular smooth muscle reactivity, including both oscillatory contractions and vascular relaxation.

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“…After 8 -1 0 days, cells had formed a confluent monolayer. The cells were positively identified as smooth muscle by indirect immunofluorescence staining for a-actin [13].…”

Section: Isolation O F Mesenteric Arterial Smooth Muscle Cellsmentioning

confidence: 99%

Gap Junctional Communication and Vascular Smooth Muscle Reactivity: Use of Tetraethylammonium Chloride

et al. 1994

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“…Since gap junctions were absent in endometrial epithelial cells of anestrus mares but detected when cells were isolated and placed into culture and since similar upregulation of gap junctions in myometrial cells occurs upon introduction of cells into primary culture (Loch-Caruso et al, 1990;Dookwah et al, 1992), it was hypothesized that porcine endometrial epithelial cells might develop gap junctions in vitro. However, neither nonpolarized nor polarized epithelial monolayers expressed gap junctions or functional communication under conditions evaluated in the present study.…”

Section: Discussionmentioning

confidence: 99%

“…The rationale for performing this analysis was based on evidence that suppression of gap junction expression in uterine myometrial cells does not occur when cells are placed into culture (Loch-Caruso et al, 1990;Dookwah et al, 1992). Similarly, equine endometrial epithelial cells obtained from anestrus mares lacked gap junctional contacts in intact tissues but developed Cx43-containing gap junctions that were functionally coupled after 3 days in culture (data not shown).…”

Section: Gap Junctional Intercellular Communicationmentioning

confidence: 99%

Endometrial connexin expression in the mare and pig: Evidence for the suppression of cell-cell communication in uterine luminal epithelium

et al. 1998

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This investigation examines the relationship between implantation strategy and gap junction protein expression in uterine endometrium. The pattern of gap junction and connexin protein expression was analyzed in porcine and equine endometrium from cycling and pregnant animals using electron microscopy and immunocytochemistry. Functional analysis of cellcell communication was also monitored by laser cytometry in primary cultures of endometrial epithelial cells. Gap junctions were detected in endometrial stroma of cycling and pregnant animals, which was correlated with immunoreactive Cx43 within stromal fibroblasts and vascular elements. No Cx26, Cx32, or Cx43 immunostaining was detected in luminal endometrial epithelium in either the mare or the pig at any stage of the estrous cycle or pregnancy. In contrast, endometrial glands of the mare exhibited a spatiotemporal pattern of Cx43 expression in the apicolateral plasma membrane which, when present, colocalized with the tight junction-associated protein, ZO-1. Uterine glandular Cx43 expression in mares was present from day 3 postovulation through day 14 of diestrus and until day 23 of pregnancy, whereas Cx43 was absent within uterine glands during seasonal anestrus, estrus, and after day 30 of pregnancy. Primary cultures of equine endometrial epithelial cells expressed both immunoreactive Cx43 and significant gap junction-mediated intercellular communication (GJIC) which was rapidly upregulated by 1.0 mM 8-bromo-cAMP or blocked with 1.0 mM octanol. No GJIC or connexin protein was detected in cultured porcine epithelial cells despite incubation with a variety of agents, including 8-bromo-cAMP, steroid hormones, retinoic acid, and/or prolactin.

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“…Myometrial smooth muscle cells were isolated from midgestation (Day 10) Sprague-Dawley rats by methods previously described (Caruso et al, 1990) with a modified digestion enzyme solution containing 100 μg/ml deoxyribonuclease I, 150 μg/ml type II collagenase, and 150 μg/ml crude trypsin. Cells were seeded into flasks containing RPMI 1640 medium supplemented with 10% bovine calf serum (BCS) and maintained at 37°C in a 5% CO 2 /95% air atmosphere.…”

Section: Myometrial Cell Isolation and Culturementioning

confidence: 99%

“…All cells were used between passages three and five. To verify the smooth muscle character of the cultured cells, indirect immunofluorescence labeling with mouse anti-α-smooth muscle actin monoclonal antibody was performed as previously described (Caruso et al, 1990), and 99-100% of cultured cells were labeled by the antibody. …”

Section: Myometrial Cell Isolation and Culturementioning

confidence: 99%

Phospholipase-Mediated Inhibition of Spontaneous Oscillatory Uterine Contractions by Lindane in Vitro,

Wang

Loch‐Caruso

2002

Toxicology and Applied Pharmacology

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Although regulation of uterine contractility is fundamental for parturition, mechanisms by which toxicants modify uterine muscle contractions remain poorly understood. In a previous cumulative concentration-response study, 10 μM lindane (γ-hexachlorocyclohexane) reduced contraction force and 30 μM lindane abolished contractions in Gestation Day 10 rat uterine strips when lindane was added to muscle baths at 10-min intervals. Other studies showed that brief (<10 min) exposures to 10-100 μM lindane inhibit gap junctions and activate phospholipase pathways in rat myometrial cells in culture. Consequently, lindane was used as a prototype toxicant with known uterine activity to investigate the hypothesis that activation of a specific phospholipase pathway provides a mechanistic link between inhibition of uterine contraction and inhibition of myometrial gap junctions. Uterine tissue and cells were pretreated with phospholipase pathway inhibitors to evaluate the role of phospholipase pathways in lindane's actions in the uterus. Concentrations of inhibitors were selected based on previous reports of effective concentrations for the enzyme activity and on pilot toxicity studies of the inhibitors on uterine contraction and gap junction communication. To monitor uterine contractions, longitudinal uterine strips were excised from Gestation Day 10 rats and suspended in isometric muscle baths, consistent with previous experiments. Exposure in vitro for 60 min to 10-50 μM lindane, an effective concentration range for the uterine responses of interest, revealed that 30 μM lindane rapidly abolished contractions. Subsequently, uterine strips were pretreated with phospholipase pathway inhibitors and then challenged with 30 μM lindane, the lindane concentration that elicited maximal inhibition of uterine contraction. Pretreatment with 20-50 μM of the phosphatidylinositol-specific phospholipase C inhibitor 1-O-octadecyl-2-O-methyl-sn -glycerol-3-phosphorylcholine (ET-18-OCH 3 ) reversed lindane-induced inhibition of spontaneous uterine contractions. Gap junction intercellular communication was monitored by injecting the fluorescent dye Lucifer yellow into rat myometrial cells grown in culture and assessing dye transfer to adjacent cells using epifluorescence microscopy. Similar to uterine contraction, pretreatment of cell cultures with phospholipase C inhibitors (30 μM ET-18-OCH 3 , 50 μM tricyclodecan-p-yl-xanthogenate · K [D609] or 50 μM tricyclodecan-p-yl-xanthogenate · K or 2-nitro-4-carboxyphenyl-N,Ndophenylcarbamate [NCDC]) partially reversed inhibition of dye transfer by 100 μM lindane, a 4To whom correspondence and reprint requests should be addressed at 1420 Washington Heights, Department of Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor, MI 48109-2029. Fax: (734) lindane concentration previously shown to abolish myometrial Lucifer yellow dye transfer under similar culture conditions. In contrast, pretreatment with 20 μM of bromoenol lactone (BEL) to inhibit the calcium-i...

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Cultured myometrial cells establish communicating gap junctions

Cited by 26 publications

References 21 publications

Gap Junctional Communication and Vascular Smooth Muscle Reactivity: Use of Tetraethylammonium Chloride

Gap Junctional Communication and Vascular Smooth Muscle Reactivity: Use of Tetraethylammonium Chloride

Endometrial connexin expression in the mare and pig: Evidence for the suppression of cell-cell communication in uterine luminal epithelium

Phospholipase-Mediated Inhibition of Spontaneous Oscillatory Uterine Contractions by Lindane in Vitro,

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