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Oscillatory Ca2+ signaling in the isolated Caenorhabditis elegans intestine: role of the inositol-1,4,5-trisphosphate receptor and phospholipases C beta and gamma.

Espelt MV, Estevez AY, Yin X, Strange K - J. Gen. Physiol. (2005)

Bottom Line: Loss-of-function mutations in the inositol-1,4,5-trisphosphate (IP(3)) receptor ITR-1 reduce pBoc and Ca(2+) oscillation frequency and intercellular Ca(2+) wave velocity.In contrast, gain-of-function mutations in the IP(3) binding and regulatory domains of ITR-1 have no effect on pBoc or Ca(2+) oscillation frequency but dramatically increase the speed of the intercellular Ca(2+) wave.Our findings provide new insights into intestinal cell Ca(2+) signaling mechanisms and establish C. elegans as a powerful model system for defining the gene networks and molecular mechanisms that underlie the generation and regulation of Ca(2+) oscillations and intercellular Ca(2+) waves in nonexcitable cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.

ABSTRACT
Defecation in the nematode Caenorhabditis elegans is a readily observable ultradian behavioral rhythm that occurs once every 45-50 s and is mediated in part by posterior body wall muscle contraction (pBoc). pBoc is not regulated by neural input but instead is likely controlled by rhythmic Ca(2+) oscillations in the intestinal epithelium. We developed an isolated nematode intestine preparation that allows combined physiological, genetic, and molecular characterization of oscillatory Ca(2+) signaling. Isolated intestines loaded with fluo-4 AM exhibit spontaneous rhythmic Ca(2+) oscillations with a period of approximately 50 s. Oscillations were only detected in the apical cell pole of the intestinal epithelium and occur as a posterior-to-anterior moving intercellular Ca(2+) wave. Loss-of-function mutations in the inositol-1,4,5-trisphosphate (IP(3)) receptor ITR-1 reduce pBoc and Ca(2+) oscillation frequency and intercellular Ca(2+) wave velocity. In contrast, gain-of-function mutations in the IP(3) binding and regulatory domains of ITR-1 have no effect on pBoc or Ca(2+) oscillation frequency but dramatically increase the speed of the intercellular Ca(2+) wave. Systemic RNA interference (RNAi) screening of the six C. elegans phospholipase C (PLC)-encoding genes demonstrated that pBoc and Ca(2+) oscillations require the combined function of PLC-gamma and PLC-beta homologues. Disruption of PLC-gamma and PLC-beta activity by mutation or RNAi induced arrhythmia in pBoc and intestinal Ca(2+) oscillations. The function of the two enzymes is additive. Epistasis analysis suggests that PLC-gamma functions primarily to generate IP(3) that controls ITR-1 activity. In contrast, IP(3) generated by PLC-beta appears to play little or no direct role in ITR-1 regulation. PLC-beta may function instead to control PIP(2) levels and/or G protein signaling events. Our findings provide new insights into intestinal cell Ca(2+) signaling mechanisms and establish C. elegans as a powerful model system for defining the gene networks and molecular mechanisms that underlie the generation and regulation of Ca(2+) oscillations and intercellular Ca(2+) waves in nonexcitable cells.

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Confocal imaging of Ca2+ oscillations in isolated glo-1 intestines. (A, left) Confocal micrograph of a glo-1 intestine loaded with fluo-4 AM. Focal plane is located at the apical pole on the bottom of the intestine. Fluo-4 intensity was quantified in regions of interest outlined in red. One region is located over the apical pole of the intestine. A second region is located in the basal pole adjacent to the apical region. Bar, 10 μm. (A, right) Changes in apical and basal pole fluo-4 intensity. Calcium oscillations are detected only in the apical pole of the epithelium. Removal of extracellular Ca2+ induces similar reductions in fluo-4 intensity in both apical and basal poles. Similar results were obtained in two additional intestines. (B) Effect of elevation of bath Ca2+ on fluo-4 intensity in the basal cell pole. A region of the basal cell pole only was imaged by laser scanning. Elevation of bath Ca2+ to 10 mM induced a rapid rise in basal cell pole fluo-4 fluorescence intensity. Similar results were obtained in two additional intestines.
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fig3: Confocal imaging of Ca2+ oscillations in isolated glo-1 intestines. (A, left) Confocal micrograph of a glo-1 intestine loaded with fluo-4 AM. Focal plane is located at the apical pole on the bottom of the intestine. Fluo-4 intensity was quantified in regions of interest outlined in red. One region is located over the apical pole of the intestine. A second region is located in the basal pole adjacent to the apical region. Bar, 10 μm. (A, right) Changes in apical and basal pole fluo-4 intensity. Calcium oscillations are detected only in the apical pole of the epithelium. Removal of extracellular Ca2+ induces similar reductions in fluo-4 intensity in both apical and basal poles. Similar results were obtained in two additional intestines. (B) Effect of elevation of bath Ca2+ on fluo-4 intensity in the basal cell pole. A region of the basal cell pole only was imaged by laser scanning. Elevation of bath Ca2+ to 10 mM induced a rapid rise in basal cell pole fluo-4 fluorescence intensity. Similar results were obtained in two additional intestines.

Mentions: We used confocal microscopy to determine if Ca2+ oscillations were polarized to apical or basal poles of intestinal cells. A confocal fluorescence micrograph of a fluo-4–loaded intestine is shown in the left panel of Fig. 3 A. The focal plane of the image is located above the apical cell pole at the bottom of the intestine. Two regions of interest are outlined in this image. One region is located in the lumen over the apical membrane and the other in the basal cell pole adjacent to the brush border. Relative changes in fluo-4 intensity are shown in the right panel of Fig. 3 A. Calcium oscillations were detected in the apical pole only.


Oscillatory Ca2+ signaling in the isolated Caenorhabditis elegans intestine: role of the inositol-1,4,5-trisphosphate receptor and phospholipases C beta and gamma.

Espelt MV, Estevez AY, Yin X, Strange K - J. Gen. Physiol. (2005)

Confocal imaging of Ca2+ oscillations in isolated glo-1 intestines. (A, left) Confocal micrograph of a glo-1 intestine loaded with fluo-4 AM. Focal plane is located at the apical pole on the bottom of the intestine. Fluo-4 intensity was quantified in regions of interest outlined in red. One region is located over the apical pole of the intestine. A second region is located in the basal pole adjacent to the apical region. Bar, 10 μm. (A, right) Changes in apical and basal pole fluo-4 intensity. Calcium oscillations are detected only in the apical pole of the epithelium. Removal of extracellular Ca2+ induces similar reductions in fluo-4 intensity in both apical and basal poles. Similar results were obtained in two additional intestines. (B) Effect of elevation of bath Ca2+ on fluo-4 intensity in the basal cell pole. A region of the basal cell pole only was imaged by laser scanning. Elevation of bath Ca2+ to 10 mM induced a rapid rise in basal cell pole fluo-4 fluorescence intensity. Similar results were obtained in two additional intestines.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2266627&req=5

fig3: Confocal imaging of Ca2+ oscillations in isolated glo-1 intestines. (A, left) Confocal micrograph of a glo-1 intestine loaded with fluo-4 AM. Focal plane is located at the apical pole on the bottom of the intestine. Fluo-4 intensity was quantified in regions of interest outlined in red. One region is located over the apical pole of the intestine. A second region is located in the basal pole adjacent to the apical region. Bar, 10 μm. (A, right) Changes in apical and basal pole fluo-4 intensity. Calcium oscillations are detected only in the apical pole of the epithelium. Removal of extracellular Ca2+ induces similar reductions in fluo-4 intensity in both apical and basal poles. Similar results were obtained in two additional intestines. (B) Effect of elevation of bath Ca2+ on fluo-4 intensity in the basal cell pole. A region of the basal cell pole only was imaged by laser scanning. Elevation of bath Ca2+ to 10 mM induced a rapid rise in basal cell pole fluo-4 fluorescence intensity. Similar results were obtained in two additional intestines.
Mentions: We used confocal microscopy to determine if Ca2+ oscillations were polarized to apical or basal poles of intestinal cells. A confocal fluorescence micrograph of a fluo-4–loaded intestine is shown in the left panel of Fig. 3 A. The focal plane of the image is located above the apical cell pole at the bottom of the intestine. Two regions of interest are outlined in this image. One region is located in the lumen over the apical membrane and the other in the basal cell pole adjacent to the brush border. Relative changes in fluo-4 intensity are shown in the right panel of Fig. 3 A. Calcium oscillations were detected in the apical pole only.

Bottom Line: Loss-of-function mutations in the inositol-1,4,5-trisphosphate (IP(3)) receptor ITR-1 reduce pBoc and Ca(2+) oscillation frequency and intercellular Ca(2+) wave velocity.In contrast, gain-of-function mutations in the IP(3) binding and regulatory domains of ITR-1 have no effect on pBoc or Ca(2+) oscillation frequency but dramatically increase the speed of the intercellular Ca(2+) wave.Our findings provide new insights into intestinal cell Ca(2+) signaling mechanisms and establish C. elegans as a powerful model system for defining the gene networks and molecular mechanisms that underlie the generation and regulation of Ca(2+) oscillations and intercellular Ca(2+) waves in nonexcitable cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.

ABSTRACT
Defecation in the nematode Caenorhabditis elegans is a readily observable ultradian behavioral rhythm that occurs once every 45-50 s and is mediated in part by posterior body wall muscle contraction (pBoc). pBoc is not regulated by neural input but instead is likely controlled by rhythmic Ca(2+) oscillations in the intestinal epithelium. We developed an isolated nematode intestine preparation that allows combined physiological, genetic, and molecular characterization of oscillatory Ca(2+) signaling. Isolated intestines loaded with fluo-4 AM exhibit spontaneous rhythmic Ca(2+) oscillations with a period of approximately 50 s. Oscillations were only detected in the apical cell pole of the intestinal epithelium and occur as a posterior-to-anterior moving intercellular Ca(2+) wave. Loss-of-function mutations in the inositol-1,4,5-trisphosphate (IP(3)) receptor ITR-1 reduce pBoc and Ca(2+) oscillation frequency and intercellular Ca(2+) wave velocity. In contrast, gain-of-function mutations in the IP(3) binding and regulatory domains of ITR-1 have no effect on pBoc or Ca(2+) oscillation frequency but dramatically increase the speed of the intercellular Ca(2+) wave. Systemic RNA interference (RNAi) screening of the six C. elegans phospholipase C (PLC)-encoding genes demonstrated that pBoc and Ca(2+) oscillations require the combined function of PLC-gamma and PLC-beta homologues. Disruption of PLC-gamma and PLC-beta activity by mutation or RNAi induced arrhythmia in pBoc and intestinal Ca(2+) oscillations. The function of the two enzymes is additive. Epistasis analysis suggests that PLC-gamma functions primarily to generate IP(3) that controls ITR-1 activity. In contrast, IP(3) generated by PLC-beta appears to play little or no direct role in ITR-1 regulation. PLC-beta may function instead to control PIP(2) levels and/or G protein signaling events. Our findings provide new insights into intestinal cell Ca(2+) signaling mechanisms and establish C. elegans as a powerful model system for defining the gene networks and molecular mechanisms that underlie the generation and regulation of Ca(2+) oscillations and intercellular Ca(2+) waves in nonexcitable cells.

Show MeSH
Related in: MedlinePlus