Characteristics of cultured subepithelial fibroblasts in the rat small intestine. II. Localization and functional analysis of endothelin receptors and cell-shape-independent gap junction permeability

Abstract

Subepithelial fibroblasts form a cellular network with gap junctions under the epithelium of the gastrointestinal tract. Previously, we have reported their unique characteristics, such as reversible rapid cell-shape changes from a flat to a stellate configuration induced by dBcAMP and endothelins (ETs), and Ca2+ responses to, for example, ETs, ATP, and substance-P. We have now investigated the subtypes of ET receptors both in the rat small intestine and in primary cultured subepithelial fibroblasts isolated from rat duodenal villi. Their properties were compared between wild-type and endothelin-B-receptor-mutant sl/sl rats. Light- and electron-microscopic immunohistochemistry showed intense ETA immunoreactivity in the subepithelial fibroblasts from the small intestine and colon of both wild-type and sl/sl rats. In culture, immunocytochemistry, reverse transcription/polymerase chain reaction analysis, Ca2+ response measurements, and cell-shape change analysis indicated functional ETA and ETB receptors in the wild-type cells, but only ETA in the sl/sl cells. However, wild-type cells were more sensitive to ET-1 than to ET-3 by about one order of magnitude. ETA seemed to be dominant both in vivo and in vitro. The relationship between cell-shape change and gap junction permeability was examined by fluorescence recovery after photobleaching; the gap junctions were usually open but were blocked by carbenoxolone. Permeability did not change significantly with cell-shape change. This network of differentiated subepithelial fibroblasts may maintain intercellular communication via gap junctions to transduce signals evoked in the local network to the whole network. The cell-shape change of the cells through ETA activation may play an important role as a barrier and for intercellular signaling in the intestinal villi.

Characteristics of subepithelial fibroblasts as a mechano-sensor in the intestine: cell-shape-dependent ATP release and P2Y1 signaling

“…4C). In addition, as discussed in later, in dBcAMP treated stellate-shaped cells, touchinduced Ca 2+ waves were suppressed, although gap junction permeability was still open (Furuya et al, 2005). This also confirmed that mechanically induced Ca 2+ waves were propagated via released ATP but not via gap junctions.…”

“…Interestingly, unlike the mechano-sensitivity, gap junction permeability did not change with cell shape, and usually stayed open (Furuya et al, 2005). So, subepithelial fibroblast networks always form a syncytium in villi.…”

“…We found that subepithelial fibroblasts of rat small intestinal villi are rich in endothelin (ET) receptors by electron microscopic autoradiography and immunohistochemistry (Furuya et al, 1990;Furuya et al, 1991;Furuya et al, 2005). By the establishment of a primary culture system from rat duodenum villi (Furuya and Furuya, 1993), we found rapid cell-shape changes from flat morphology with broad cell processes (flat-shape) to round cell body with thin cell processes (stellate-shape) by dibutyryl cyclic AMP (dBcAMP), forskolin and cholera toxin, and reversed morphological changes from stellate to flat upon the addition of serum and ETs, just as reported in cultured astrocytes (Goldman and Abramson, 1990).…”

“…In contrast with cultured astrocytes, subepithelial fibroblasts are rich in endothelin A (ET A ) receptors, and gap junctions are commonly opened in a cAMP-independent manner, i.e. independent of cell-shape changes (Furuya et al, 2005). We have preliminarily reported that subepithelial fibroblasts are sensitive to mechanical stimulation and communicate not only via gap junctions, but also via humoral factors, especially ATP (Furuya and Furuya, 2002).…”

Isolation and Characterization of Bovine Intestinal Subepithelial Myofibroblasts

“…Indeed previous reports showed that ATP, serotonin, endothelin-1, and bradykinin, but not norepinephrine or angiotensin II, elevated [Ca 2+ ] i in ISMFs isolated from rat duodenum (7,18). To characterize cellular reactivity, we measured [Ca 2+ ] i in bovine ISMFs at passage 3 -7.…”