Limits...
Acetylcholine-induced calcium signaling and contraction of airway smooth muscle cells in lung slices.

Bergner A, Sanderson MJ - J. Gen. Physiol. (2002)

Bottom Line: Incubation with thapsigargin, xestospongin, or ryanodine inhibited the ACH-induced Ca(2+) signaling.Cessation of the Ca(2+) oscillations, induced by ACH-esterase, halothane, or the absence of extracellular Ca(2+) resulted in a relaxation of the airway.These results indicate that Ca(2+) oscillations, induced by ACH in murine bronchial SMCs, are generated by Ca(2+) release from the SR involving IP(3)- and ryanodine receptors, and are required to maintain airway contraction.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01655, USA.

ABSTRACT
The Ca(2+) signaling and contractility of airway smooth muscle cells (SMCs) were investigated with confocal microscopy in murine lung slices (approximately 75-microm thick) that maintained the in situ organization of the airways and the contractility of the SMCs for at least 5 d. 10--500 nM acetylcholine (ACH) induced a contraction of the airway lumen and a transient increase in [Ca(2+)](i) in individual SMCs that subsequently declined to initiate multiple intracellular Ca(2+) oscillations. These Ca(2+) oscillations spread as Ca(2+) waves through the SMCs at approximately 48 microm/s. The magnitude of the airway contraction, the initial Ca(2+) transient, and the frequency of the subsequent Ca(2+) oscillations were all concentration-dependent. In a Ca(2+)-free solution, ACH induced a similar Ca(2+) response, except that the Ca(2+) oscillations ceased after 1--1.5 min. Incubation with thapsigargin, xestospongin, or ryanodine inhibited the ACH-induced Ca(2+) signaling. A comparison of airway contraction with the ACH-induced Ca(2+) response of the SMCs revealed that the onset of airway contraction correlated with the initial Ca(2+) transient, and that sustained airway contraction correlated with the occurrence of the Ca(2+) oscillations. Buffering intracellular Ca(2+) with BAPTA prohibited Ca(2+) signaling and airway contraction, indicating a Ca(2+)-dependent pathway. Cessation of the Ca(2+) oscillations, induced by ACH-esterase, halothane, or the absence of extracellular Ca(2+) resulted in a relaxation of the airway. The concentration dependence of the airway contraction matched the concentration dependence of the increased frequency of the Ca(2+) oscillations. These results indicate that Ca(2+) oscillations, induced by ACH in murine bronchial SMCs, are generated by Ca(2+) release from the SR involving IP(3)- and ryanodine receptors, and are required to maintain airway contraction.

Show MeSH

Related in: MedlinePlus

Confocal microscopy images of immunocytochemical stainings of airways in lung slices using FITC-conjugated secondary antibodies. (A) The wall of an airway, cut in cross-section, after staining with anti–α-actin antibodies. Several smooth muscle cells (SMC) stained brightly, show an elongated fusiform cell shape, and are in close spatial proximity to epithelial cells (EC) and airway lumen (AL). (B) A slice stained with anti–pan cytokeratin antibodies showing the typical cobblestone-like appearance of epithelial cells. In this case, the plane of the section has passed obliquely through the airway to provide near longitudinal section at the top left. Bars: 15 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2233801&req=5

fig2: Confocal microscopy images of immunocytochemical stainings of airways in lung slices using FITC-conjugated secondary antibodies. (A) The wall of an airway, cut in cross-section, after staining with anti–α-actin antibodies. Several smooth muscle cells (SMC) stained brightly, show an elongated fusiform cell shape, and are in close spatial proximity to epithelial cells (EC) and airway lumen (AL). (B) A slice stained with anti–pan cytokeratin antibodies showing the typical cobblestone-like appearance of epithelial cells. In this case, the plane of the section has passed obliquely through the airway to provide near longitudinal section at the top left. Bars: 15 μm.

Mentions: To verify the morphological observations and to assess the spatial distribution of ECs and SMCs in the airways, we performed immunocytochemistry with antibodies specific for cytokeratin and α-smooth muscle actin, respectively. SMCs could be identified by their fusiform shape and were in many cases found around the whole circumference of the airway. However, in some airways, only parts of the circumference contained SMCs. SMCs were found to be closely associated with ECs, and were often separated from the airway lumen by just one layer of ECs (Fig. 2 A). By contrast, ECs showed a typical cobblestone-like appearance and completely lined the airway lumen (Fig. 2 B).


Acetylcholine-induced calcium signaling and contraction of airway smooth muscle cells in lung slices.

Bergner A, Sanderson MJ - J. Gen. Physiol. (2002)

Confocal microscopy images of immunocytochemical stainings of airways in lung slices using FITC-conjugated secondary antibodies. (A) The wall of an airway, cut in cross-section, after staining with anti–α-actin antibodies. Several smooth muscle cells (SMC) stained brightly, show an elongated fusiform cell shape, and are in close spatial proximity to epithelial cells (EC) and airway lumen (AL). (B) A slice stained with anti–pan cytokeratin antibodies showing the typical cobblestone-like appearance of epithelial cells. In this case, the plane of the section has passed obliquely through the airway to provide near longitudinal section at the top left. Bars: 15 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233801&req=5

fig2: Confocal microscopy images of immunocytochemical stainings of airways in lung slices using FITC-conjugated secondary antibodies. (A) The wall of an airway, cut in cross-section, after staining with anti–α-actin antibodies. Several smooth muscle cells (SMC) stained brightly, show an elongated fusiform cell shape, and are in close spatial proximity to epithelial cells (EC) and airway lumen (AL). (B) A slice stained with anti–pan cytokeratin antibodies showing the typical cobblestone-like appearance of epithelial cells. In this case, the plane of the section has passed obliquely through the airway to provide near longitudinal section at the top left. Bars: 15 μm.
Mentions: To verify the morphological observations and to assess the spatial distribution of ECs and SMCs in the airways, we performed immunocytochemistry with antibodies specific for cytokeratin and α-smooth muscle actin, respectively. SMCs could be identified by their fusiform shape and were in many cases found around the whole circumference of the airway. However, in some airways, only parts of the circumference contained SMCs. SMCs were found to be closely associated with ECs, and were often separated from the airway lumen by just one layer of ECs (Fig. 2 A). By contrast, ECs showed a typical cobblestone-like appearance and completely lined the airway lumen (Fig. 2 B).

Bottom Line: Incubation with thapsigargin, xestospongin, or ryanodine inhibited the ACH-induced Ca(2+) signaling.Cessation of the Ca(2+) oscillations, induced by ACH-esterase, halothane, or the absence of extracellular Ca(2+) resulted in a relaxation of the airway.These results indicate that Ca(2+) oscillations, induced by ACH in murine bronchial SMCs, are generated by Ca(2+) release from the SR involving IP(3)- and ryanodine receptors, and are required to maintain airway contraction.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01655, USA.

ABSTRACT
The Ca(2+) signaling and contractility of airway smooth muscle cells (SMCs) were investigated with confocal microscopy in murine lung slices (approximately 75-microm thick) that maintained the in situ organization of the airways and the contractility of the SMCs for at least 5 d. 10--500 nM acetylcholine (ACH) induced a contraction of the airway lumen and a transient increase in [Ca(2+)](i) in individual SMCs that subsequently declined to initiate multiple intracellular Ca(2+) oscillations. These Ca(2+) oscillations spread as Ca(2+) waves through the SMCs at approximately 48 microm/s. The magnitude of the airway contraction, the initial Ca(2+) transient, and the frequency of the subsequent Ca(2+) oscillations were all concentration-dependent. In a Ca(2+)-free solution, ACH induced a similar Ca(2+) response, except that the Ca(2+) oscillations ceased after 1--1.5 min. Incubation with thapsigargin, xestospongin, or ryanodine inhibited the ACH-induced Ca(2+) signaling. A comparison of airway contraction with the ACH-induced Ca(2+) response of the SMCs revealed that the onset of airway contraction correlated with the initial Ca(2+) transient, and that sustained airway contraction correlated with the occurrence of the Ca(2+) oscillations. Buffering intracellular Ca(2+) with BAPTA prohibited Ca(2+) signaling and airway contraction, indicating a Ca(2+)-dependent pathway. Cessation of the Ca(2+) oscillations, induced by ACH-esterase, halothane, or the absence of extracellular Ca(2+) resulted in a relaxation of the airway. The concentration dependence of the airway contraction matched the concentration dependence of the increased frequency of the Ca(2+) oscillations. These results indicate that Ca(2+) oscillations, induced by ACH in murine bronchial SMCs, are generated by Ca(2+) release from the SR involving IP(3)- and ryanodine receptors, and are required to maintain airway contraction.

Show MeSH
Related in: MedlinePlus