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First published online 19 July 2005
doi: 10.1242/jcs.02453

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

¹ Cell Mechano-Sensing Project, ICORP and SORST, Japan Science and Technology Agency, Nagoya, 466-8550, Japan
² Department of Physiology, Nagoya University School of Medicine, Nagoya, 466-8550, Japan
³ Section of Brain Structure, Center for Brain Experiment, Okazaki 444-8585, Japan
⁴ The Graduate University for Advanced Studies, National Institute for Physiological Sciences, Okazaki 444-8585, Japan

View larger version (65K): [in a new window]   Fig. 1. Localization of P2Y1 in rat duodenum and in cultured subepithelial fibroblasts. Cryosections of 6W Wistar rat duodenum were incubated with (A) rabbit anti-P2Y1 antibody and (B) anti-P2Y1 antibody pre-absorbed with 2 µg ml-1 antigenic peptide, followed with anti-rabbit Envision+ and visualized with DAB-H2O2 reactions. (C) Subepithelial fibroblasts isolated from rat duodenal villi in primary cultures were incubated with rabbit anti-P2Y1 antibody, then biotinylated anti-rabbit IgG, followed with streptavidin-Texas red. Bar in A and B is 100 µm, and in C is 10 µm.

View larger version (34K): [in a new window]   Fig. 2. Expression of P2Y1 mRNA and P2Y1 protein. RT-PCR analysis of subtypes of ATP receptors (A) in the primary culture of subepithelial fibroblasts and (B) in 18Co cells. (C) Western blotting analysis of P2Y1 receptors. Samples were loaded as follows. Lane A: subepithelial fibroblasts cultured in medium containing 10% FCS (control) (20 µg per lane). Lane B: subepithelial fibroblasts incubated in medium with 1 mM dBcAMP without FCS for 2 hours (20 µg per lane). Lane C: 18Co cells (50 µg per lane). Lane D: 3-week-old rat cerebrum (100 µg per lane). Samples were run on 10% SDS-polyacrylamide gels, transferred onto blotting membranes and detected with anti-P2Y1 antibody.

View larger version (52K): [in a new window]   Fig. 3. The effect of agonists and antagonists of ATP receptors on Ca2+ responses. Intracellular Ca2+ responses shown by indo-1 fluorescence ratios (F405/F480), were measured in subepithelial fibroblasts cultured for 3 days using a laser confocal microscope. Color traces show responses in each cell and black traces with filled circles indicate the average response. (A) Ca2+ responses to UTP, ATP, UDP and ADP. The nucleotide concentrations were 10 µM. ATP and ADP are equally potent to induce Ca2+ increase, UTP is less effective, and UDP is not effective at 10 µM. (B) In Ca2+-free solution, a similar Ca2+ response to ATP was initially observed, but later was suppressed. (C) 2MeSATP, a P2Y1 agonist, is more effective than ATP. The responses to each agonist at concentration 0.1 µM are compared. In the same cells, carbachol, a cholinergic agonist, is not effective even at 100 µM. (D) MRS2179 (30 µM), a P2Y1 antagonist, potently blocks the Ca2+ response to 2MeSATP (1 µM). The response is recovered after 10 minutes washout of MRS2179. Conversely, MRS2159 (100 µM), a P2X1 antagonist, is not effective on the Ca2+ response.

View larger version (99K): [in a new window]   Fig. 4. Mechanical stimulation, `touch', induces intercellular Ca2+ waves. Two images of indo-1 fluorescence (F405 and F480) were superimposed onto a Nomarski image where red represented F405 and green represented F480. So, cells in which Ca2+ increased turned red, as shown in color scale bar for Ca2+-increases. Arrows and asterisks show touched cells. Magnification scales are 20 µm (A-C are the same magnification). (A) A Slight touch with a blunted thin glass rod induced intercellular Ca2+-waves in subepithelial fibroblasts. See also supplementary material Movie 1. (B) MRS2179 (100 µM) inhibited touch-induced Ca2+ waves. The washing out of MRS2179 caused the recovery of Ca2+ waves. (C) CBX (100 µM), a blocker of gap junctions, is not effective on the initiation and propagation of Ca2+ waves. (D) Touch-induced Ca2+ waves could propagate to physically non-contacted cells. (E) Ca2+ waves were induced and propagated even in Ca2+-free solution similarly under normal conditions. (F) Time courses of Ca2+ changes in individual cells are shown by increases in fluorescence ratios (F405/F480) after touch stimulation in normal Ringer (left) and in Ca2+-free solutions (right). Cells touched for a second time also induced Ca2+ waves, although Ca2+ increases in touched cells (shown by dark-blue rhomboids) were suppressed in Ca2+-free solution.

View larger version (98K): [in a new window]   Fig. 5. Touch-induced cellular contractions propagate like a wave. Touching stimulations of a cell induced a propagation of contraction to the surrounding cells. The contraction seemed to spread with Ca2+ wave propagations. To show the contraction clearly, high magnification Nomarski images after stimulation were arranged with time. Arrowheads at 15.8 seconds indicate transient bleb formation in the cell membrane, sometimes accompanied with Ca2+ waves. Rows (a) and (b) show images magnified x2 of two areas marked `a' and `b' in the top row (see image labeled 0 seconds). Arrows indicate corresponding regions to show the contractions. Supplementary material Movies 1, 2 facilitate the understanding of the contraction.

View larger version (58K): [in a new window]   Fig. 6. Mechanical stimulation, `stretch', induces Ca2+ responses and Ca2+ waves. Subepithelial fibroblasts were cultured on elastic chambers made by thin silicone elastomer membrane coated with collagen, and stretched using a stretch machine. Stretch stimulation (12% for 3 seconds) was applied to vertical direction in the figures between 0 seconds and 5 seconds. Ca2+ increases in the cells, measured by indo-1 fluorescence, occurred under the presence (A) and the absence (B) of extracellular Ca2+. Wave-like Ca2+ responses were also observed in the process (see supplementary material Movie 3). Here, each image was a merged RGB color image of F405 (as red), F480 (as green) and F405/F480 (as blue). The scale for Ca2+ increase is shown in the right color bar.

View larger version (16K): [in a new window]   Fig. 7. Stretch induces ATP release. During the stretch stimulation, external solutions were perfused continuously at a speed of about 300 µl per minute and the perfusate was collected every minute. The ATP content in each elute was measured using a luciferin-luciferase bio-luminescence assay. ATP was released by stretching in a dose-dependent manner and repetitive stretching induced repetitive ATP release.

View larger version (146K): [in a new window]   Fig. 8. Suppression of Ca2+ responses to touch and stretch in the stellate-shape cells. Stellate-shaped cells treated with 1 mM dBcAMP for 1.5 hours did not respond to touch (A1) and stretch (B1), although an extremely strong stimulation that punctured cells induced Ca2+ waves (A2). After 10 minutes treatment with 10 nM ET1, cells changed to flat, and sensitivity to touch and stretch was recovered (A3,B2). Arrows indicate touched cells. Ca2+ increases are shown by superimposed images: (A) F405 is red, F480 is green, Nomarski is gray; and (B) F405 is red, F480 is green, ratio of F405/F480 is blue. Similar results were obtained in over 10 dishes in several different cultures in both A and B. Bars, 100 µm.

View larger version (16K): [in a new window]   Fig. 9. Suppression of stretch-induced ATP-release in stellate-shaped cells. Subepithelial fibroblasts cultured on elastic chambers were treated with 1 mM dBcAMP for about 1 hour to induce the cells to change shape to stellate. During stretching, ATP contents in perfusates were measured every minute. (A) A typical example involving a strong stretch (32%, 40%) induces a small ATP release even in stellate-shaped cells. After ET1 (but not substance-P treatment) ATP release is obviously enhanced. (B) The average data for the effects of dBcAMP treatment and ET1 application on ATP release are shown, which were normalized by controls. n=5, bars show s.e.m.

View larger version (15K): [in a new window]   Fig. 10. Suppression of ATP sensitivity in stellate-shaped cells. Ca2+ responses to 10 µM ATP were measured in control (flat-shape), 1 mM dBcAMP-treated stellate-shaped cells and ET1-treated flat-shaped cells. Average data and s.e.m. (bars) were obtained from more than 70 cells in three or four experiments.

View larger version (75K): [in a new window]   Fig. 11. Propagation of Ca2+ waves from subepithelial fibroblasts to neuronal cells. (A) Differentiated NG108-15 cells were seeded onto the culture of subepithelial fibroblasts and co-cultured for 1 day (upper left, Nomarski image). A NG108-15 cell is indicated by `N' in the figure. Ca2+ waves were generated by touching a subepithelial fibroblast (3.7-18.6 seconds). Each image is superimposed onto the ratio (F405:F480) image (yellow) with Nomarski images. Yellow parts indicate the cells in which Ca2+ increased. Co-cultured NG108-15 cell (indicated by an asterisk) was activated with the Ca2+ waves. (B) Time courses of Ca2+ wave propagation in subepithelial fibroblasts (colored lines) and Ca2+ increases in NG108-15 cells (black line with filled circles) are shown by the ratio (F405/F480) in each cell.

View larger version (64K): [in a new window]   Fig. 12. A model for subepithelial fibroblasts working as a mechano-sensor and other functions in the intestinal villi. Food and water intake deform villi and cause mechanical stress to the network of subepithelial fibroblasts (dark-blue cells). ATP is released from subepithelial fibroblasts by the mechanical stimulation and activates P2Y1 on surrounding cells as an autocrine mediator. The released ATP also activates the nerve endings of mucosal sensory neurons via P2X receptors. This represents a mechanosensing process in the villi and may contribute to the peristaltic reflex. Subepithelial fibroblast networks communicate with -SMA-negative lamina propria fibroblasts-like cells (gray cells) and they form three-dimensional networks. They closely contact capillary networks, sensory and motor neuronal networks, smooth muscles, and epithelium in the villi. Subepithelial fibroblasts communicate among these cell systems via released ATP and other humoral factors, such as TGF-ß, TNF-. From these views, we consider subepithelial fibroblasts work as barrier or sieve, mechano-sensor, mechanical frame, and signal transduction machinery in the villi. These functions are most likely locally and dynamically regulated in the villi by the rapid changes in cell-shape and mechano-sensitivity.

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