Limits...
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: The function of the two enzymes is additive.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.

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

Calcium oscillations in single intestines isolated from an (A) egl-8(n488) loss-of-function or a (B) plc-3(RNAi) worm. Images were acquired at 10-s intervals.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2266627&req=5

fig7: Calcium oscillations in single intestines isolated from an (A) egl-8(n488) loss-of-function or a (B) plc-3(RNAi) worm. Images were acquired at 10-s intervals.

Mentions: Fig. 7 shows examples of Ca2+ oscillations observed in intestines isolated from egl-8(n488) or plc-3(RNAi) worms. Like pBoc (Figs. 5 and 6), loss of EGL-8 or PLC-3 activity gave rise to Ca2+ oscillation periods that were highly arrhythmic. Calcium oscillations in intestines from egl-8(RNAi) worms showed similar arrhythmia (unpublished data). Mean ± SEM Ca2+ oscillation coefficients of variance were 48 ± 11% (n = 7) (coefficients of variance for Ca2+ oscillations in egl-8(n488) and egl-8(RNAi) worms were not significantly different and were therefore averaged together) and 54 ± 11% (n = 6) for EGL-8 and PLC-3 loss-of-function animals, respectively. These coefficients of variance were significantly (P < 0.01) different from the mean CV of 16% for Ca2+ oscillations observed in intestines of control worms (Table I). The mean ± SEM Ca2+ oscillation CV observed in plc-3(tm1340) worms was 36 ± 4% (n = 4) and was not significantly (P > 0.2) different from that of plc-3(RNAi) animals. Consistent with the severe disruption of pBoc induced by combined loss of EGL-8 and PLC-3 function (Fig. 6, C and D), we were unable to detect Ca2+ oscillations in 40 intestines isolated from egl-8(n488);plc-3(RNAi) worms.


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)

Calcium oscillations in single intestines isolated from an (A) egl-8(n488) loss-of-function or a (B) plc-3(RNAi) worm. Images were acquired at 10-s intervals.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2266627&req=5

fig7: Calcium oscillations in single intestines isolated from an (A) egl-8(n488) loss-of-function or a (B) plc-3(RNAi) worm. Images were acquired at 10-s intervals.
Mentions: Fig. 7 shows examples of Ca2+ oscillations observed in intestines isolated from egl-8(n488) or plc-3(RNAi) worms. Like pBoc (Figs. 5 and 6), loss of EGL-8 or PLC-3 activity gave rise to Ca2+ oscillation periods that were highly arrhythmic. Calcium oscillations in intestines from egl-8(RNAi) worms showed similar arrhythmia (unpublished data). Mean ± SEM Ca2+ oscillation coefficients of variance were 48 ± 11% (n = 7) (coefficients of variance for Ca2+ oscillations in egl-8(n488) and egl-8(RNAi) worms were not significantly different and were therefore averaged together) and 54 ± 11% (n = 6) for EGL-8 and PLC-3 loss-of-function animals, respectively. These coefficients of variance were significantly (P < 0.01) different from the mean CV of 16% for Ca2+ oscillations observed in intestines of control worms (Table I). The mean ± SEM Ca2+ oscillation CV observed in plc-3(tm1340) worms was 36 ± 4% (n = 4) and was not significantly (P > 0.2) different from that of plc-3(RNAi) animals. Consistent with the severe disruption of pBoc induced by combined loss of EGL-8 and PLC-3 function (Fig. 6, C and D), we were unable to detect Ca2+ oscillations in 40 intestines isolated from egl-8(n488);plc-3(RNAi) worms.

Bottom Line: The function of the two enzymes is additive.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.

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