Aequorin luminescence, myosin phosphorylation, and active stress in tracheal smooth muscle

W. T. Gerthoffer, K. A. Murphey, S. J. Gunst

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

During muscarinic activation of canine tracheal smooth muscle with carbachol, myosin phosphorylation is significantly more sensitive than stress to the external Ca2+ concentration ([Ca2+](o)) [W.T. Gerthoffer. Am. J. Physiol. 250 (Cell Physiol. 19): C597-C604, 1986]. To determine whether the intracellular Ca2+ concentration ([Ca2+](i)) correlated more closely with changes in phosphorylation or force, we measured isometric force and light emitted by the luminescent intracellular Ca2+ indicator aequorin as [Ca2+](o) was increased in the presence of 1 μM carbachol or 60 mM K+. Myosin phosphorylation was measured using an immunoblot assay in a second set of muscle strips treated identically. Stimulation with carbachol increased aequorin luminescence slightly in strips incubated in Ca2+-free solution. Active stress and aequorin luminescence subsequently increased in parallel as [Ca2+](o) was increased. Myosin phosphorylation at 0.05 mM [Ca2+](o) (0.30 ± 0.04 mol P(i)/mol light chain) was significantly higher than phosphorylation in Ca2+-free solution with no carbachol (0.12 ± 0.048 mol P(i)/mol light chain) and increased to a maximum of 0.56 ± 0.03 mol P(i)/mol light chain at 1.6 mM [Ca2+](o). In contrast, active stress and aequorin luminescence remained low at low [Ca2+](o) and reached a maximum at 2.4 mM [Ca2+](o). Stimulation with carbachol produced greater increases in myosin phosphorylation and active stress for a given change in aequorin luminescence than did K+ depolarization. Stimulation with carbachol also produced a different phosphorylation-stress relationship than did K+ depolarization. These observations are consistent with the possibility that carbachol induces increases in the Ca2+ sensitivity of contractile proteins in tracheal smooth muscle.

Original languageEnglish (US)
Pages (from-to)26/6
JournalAmerican Journal of Physiology - Cell Physiology
Volume257
Issue number6
StatePublished - Dec 1 1989

Fingerprint

Aequorin
Phosphorylation
Carbachol
Myosins
Luminescence
Smooth Muscle
Muscle
Light
Depolarization
Contractile Proteins
Cholinergic Agents
Canidae
Assays
Chemical activation
Muscles

Keywords

  • calcium
  • carbachol
  • potassium
  • trachea

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

Cite this

Aequorin luminescence, myosin phosphorylation, and active stress in tracheal smooth muscle. / Gerthoffer, W. T.; Murphey, K. A.; Gunst, S. J.

In: American Journal of Physiology - Cell Physiology, Vol. 257, No. 6, 01.12.1989, p. 26/6.

Research output: Contribution to journalArticle

@article{e3f8ee0036854c25ade40e0ca38f0bf7,
title = "Aequorin luminescence, myosin phosphorylation, and active stress in tracheal smooth muscle",
abstract = "During muscarinic activation of canine tracheal smooth muscle with carbachol, myosin phosphorylation is significantly more sensitive than stress to the external Ca2+ concentration ([Ca2+](o)) [W.T. Gerthoffer. Am. J. Physiol. 250 (Cell Physiol. 19): C597-C604, 1986]. To determine whether the intracellular Ca2+ concentration ([Ca2+](i)) correlated more closely with changes in phosphorylation or force, we measured isometric force and light emitted by the luminescent intracellular Ca2+ indicator aequorin as [Ca2+](o) was increased in the presence of 1 μM carbachol or 60 mM K+. Myosin phosphorylation was measured using an immunoblot assay in a second set of muscle strips treated identically. Stimulation with carbachol increased aequorin luminescence slightly in strips incubated in Ca2+-free solution. Active stress and aequorin luminescence subsequently increased in parallel as [Ca2+](o) was increased. Myosin phosphorylation at 0.05 mM [Ca2+](o) (0.30 ± 0.04 mol P(i)/mol light chain) was significantly higher than phosphorylation in Ca2+-free solution with no carbachol (0.12 ± 0.048 mol P(i)/mol light chain) and increased to a maximum of 0.56 ± 0.03 mol P(i)/mol light chain at 1.6 mM [Ca2+](o). In contrast, active stress and aequorin luminescence remained low at low [Ca2+](o) and reached a maximum at 2.4 mM [Ca2+](o). Stimulation with carbachol produced greater increases in myosin phosphorylation and active stress for a given change in aequorin luminescence than did K+ depolarization. Stimulation with carbachol also produced a different phosphorylation-stress relationship than did K+ depolarization. These observations are consistent with the possibility that carbachol induces increases in the Ca2+ sensitivity of contractile proteins in tracheal smooth muscle.",
keywords = "calcium, carbachol, potassium, trachea",
author = "Gerthoffer, {W. T.} and Murphey, {K. A.} and Gunst, {S. J.}",
year = "1989",
month = "12",
day = "1",
language = "English (US)",
volume = "257",
pages = "26/6",
journal = "American Journal of Physiology",
issn = "0363-6143",
publisher = "American Physiological Society",
number = "6",

}

TY - JOUR

T1 - Aequorin luminescence, myosin phosphorylation, and active stress in tracheal smooth muscle

AU - Gerthoffer, W. T.

AU - Murphey, K. A.

AU - Gunst, S. J.

PY - 1989/12/1

Y1 - 1989/12/1

N2 - During muscarinic activation of canine tracheal smooth muscle with carbachol, myosin phosphorylation is significantly more sensitive than stress to the external Ca2+ concentration ([Ca2+](o)) [W.T. Gerthoffer. Am. J. Physiol. 250 (Cell Physiol. 19): C597-C604, 1986]. To determine whether the intracellular Ca2+ concentration ([Ca2+](i)) correlated more closely with changes in phosphorylation or force, we measured isometric force and light emitted by the luminescent intracellular Ca2+ indicator aequorin as [Ca2+](o) was increased in the presence of 1 μM carbachol or 60 mM K+. Myosin phosphorylation was measured using an immunoblot assay in a second set of muscle strips treated identically. Stimulation with carbachol increased aequorin luminescence slightly in strips incubated in Ca2+-free solution. Active stress and aequorin luminescence subsequently increased in parallel as [Ca2+](o) was increased. Myosin phosphorylation at 0.05 mM [Ca2+](o) (0.30 ± 0.04 mol P(i)/mol light chain) was significantly higher than phosphorylation in Ca2+-free solution with no carbachol (0.12 ± 0.048 mol P(i)/mol light chain) and increased to a maximum of 0.56 ± 0.03 mol P(i)/mol light chain at 1.6 mM [Ca2+](o). In contrast, active stress and aequorin luminescence remained low at low [Ca2+](o) and reached a maximum at 2.4 mM [Ca2+](o). Stimulation with carbachol produced greater increases in myosin phosphorylation and active stress for a given change in aequorin luminescence than did K+ depolarization. Stimulation with carbachol also produced a different phosphorylation-stress relationship than did K+ depolarization. These observations are consistent with the possibility that carbachol induces increases in the Ca2+ sensitivity of contractile proteins in tracheal smooth muscle.

AB - During muscarinic activation of canine tracheal smooth muscle with carbachol, myosin phosphorylation is significantly more sensitive than stress to the external Ca2+ concentration ([Ca2+](o)) [W.T. Gerthoffer. Am. J. Physiol. 250 (Cell Physiol. 19): C597-C604, 1986]. To determine whether the intracellular Ca2+ concentration ([Ca2+](i)) correlated more closely with changes in phosphorylation or force, we measured isometric force and light emitted by the luminescent intracellular Ca2+ indicator aequorin as [Ca2+](o) was increased in the presence of 1 μM carbachol or 60 mM K+. Myosin phosphorylation was measured using an immunoblot assay in a second set of muscle strips treated identically. Stimulation with carbachol increased aequorin luminescence slightly in strips incubated in Ca2+-free solution. Active stress and aequorin luminescence subsequently increased in parallel as [Ca2+](o) was increased. Myosin phosphorylation at 0.05 mM [Ca2+](o) (0.30 ± 0.04 mol P(i)/mol light chain) was significantly higher than phosphorylation in Ca2+-free solution with no carbachol (0.12 ± 0.048 mol P(i)/mol light chain) and increased to a maximum of 0.56 ± 0.03 mol P(i)/mol light chain at 1.6 mM [Ca2+](o). In contrast, active stress and aequorin luminescence remained low at low [Ca2+](o) and reached a maximum at 2.4 mM [Ca2+](o). Stimulation with carbachol produced greater increases in myosin phosphorylation and active stress for a given change in aequorin luminescence than did K+ depolarization. Stimulation with carbachol also produced a different phosphorylation-stress relationship than did K+ depolarization. These observations are consistent with the possibility that carbachol induces increases in the Ca2+ sensitivity of contractile proteins in tracheal smooth muscle.

KW - calcium

KW - carbachol

KW - potassium

KW - trachea

UR - http://www.scopus.com/inward/record.url?scp=0024826080&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024826080&partnerID=8YFLogxK

M3 - Article

C2 - 2610246

AN - SCOPUS:0024826080

VL - 257

SP - 26/6

JO - American Journal of Physiology

JF - American Journal of Physiology

SN - 0363-6143

IS - 6

ER -