Force relaxes before the fall of cytosolic calcium in the photomechanical response of rat sphincter pupillae

2000 ◽  
Vol 279 (1) ◽  
pp. C274-C280 ◽  
Author(s):  
Andrew P. Krivoshik ◽  
Lloyd Barr
Keyword(s):  
Myosin Light Chain ◽  
Smooth Muscles ◽  
Cytosolic Calcium ◽  
Myosin Phosphorylation ◽  
Primary Signal ◽  
Intracellular Ca ◽  
Phosphorylation Cascade ◽  
Muscle Myosin ◽  
Light Flashes ◽  
Stimulation Of

In the rat sphincter pupillae, as in other smooth muscles, the primary signal transduction cascade for agonist activation is receptor → G protein → phospholipase C → inositol trisphosphate → intracellular Ca2+concentration ([Ca2+]i) → calmodulin → myosin light chain kinase → phosphorylated myosin → force development. Light stimulation of isolated sphincters pupillae can be very precisely controlled, and precise reproducible photomechanical responses (PMRs) result. This precision makes the PMR ideal for testing models of regulation of smooth muscle myosin phosphorylation. We measured force and [Ca2+]iconcurrently in sphincter pupillae following stimulation by light flashes of varying duration and intensity. We sampled at unusually short (0.01–0.02 s) intervals to adequately test a PMR model based on the myosin phosphorylation cascade. We found, surprisingly, contrary to the behavior of intestinal muscle and predictions of the phosphorylation model, that during PMRs force begins to decay while [Ca2+]iis still rising. We conclude that control of contraction in the sphincter pupillae probably involves an inhibitory process as well as activation by [Ca2+]i.

scholarly journals Embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.

1985 ◽  
Vol 100 (6) ◽  
pp. 2025-2030 ◽  
Author(s):  
H Takano-Ohmuro ◽  
T Obinata ◽  
M Kawashima ◽  
T Masaki ◽  
T Tanaka
Keyword(s):  
Light Chain ◽  
Gel Electrophoresis ◽  
Myosin Light Chain ◽  
Smooth Muscles ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Striated Muscles ◽  
Two Dimensional Gel Electrophoresis ◽  
Embryonic Chicken ◽  
Muscle Myosin

It has been demonstrated that embryonic chicken gizzard smooth muscle contains a unique embryonic myosin light chain of 23,000 mol wt, called L23 (Katoh, N., and S. Kubo, 1978, Biochem. Biophys. Acta, 535:401-411; Takano-Ohmuro, H., T. Obinata, T. Mikawa, and T. Masaki, 1983, J. Biochem. (Tokyo), 93:903-908). When we examined myosins in developing chicken ventricular and pectoralis muscles by two-dimensional gel electrophoresis, the myosin light chain (Le) that completely comigrates with L23 was detected in both striated muscles at early developmental stages. Two monoclonal antibodies, MT-53f and MT-185d, were applied to characterize the embryonic light chain Le of striated muscles. Both monoclonal antibodies were raised to fast skeletal muscle myosin light chains; the former antibody is specific to fast muscle myosin light chains 1 and 3, whereas the latter recognizes not only fast muscle myosin light chains but also the embryonic smooth muscle light chain L23. The immunoblots combined with both one- and two-dimensional gel electrophoresis showed that Le reacts with MT-185d but not with MT-53f. These results strongly indicate that Le is identical to L23 and that embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.

Isoproterenol attenuates myosin phosphorylation and contraction of tracheal muscle

1989 ◽  
Vol 66 (5) ◽  
pp. 2017-2022 ◽  
Author(s):  
K. Obara ◽  
P. de Lanerolle
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Isometric Force ◽  
Smooth Muscles ◽  
Inhibitory Effect ◽  
Isometric Tension ◽  
Myosin Light Chain Phosphorylation ◽  
Response Curves ◽  
Shortening Velocity ◽  
Myosin Phosphorylation

The effects of isoproterenol on isometric force, unloaded shortening velocity, and myosin phosphorylation were examined in thin muscle bundles (0.1–0.2 mm diam) dissected from lamb tracheal smooth muscle. Methacholine (10(-6) M) induced rapid increases in isometric force and in phosphorylation of the 20,000-Da myosin light chain. Myosin phosphorylation remained elevated during steady-state maintenance of isometric force. The shortening velocity peaked at 15 s after stimulation with methacholine and then declined to approximately 45% of the maximal value by 3 min. Isoproterenol pretreatment inhibited methacholine-stimulated myosin light chain phosphorylation, shortening velocity, and force during the early stages of force generation. However, the inhibitory effect of isoproterenol on force and myosin phosphorylation is proportionally greater than that on shortening velocity. Isoproterenol pretreatment also caused a rightward non-parallel shift in the methacholine dose-response curves for both isometric tension and myosin light chain phosphorylation. These data demonstrate that isoproterenol attenuates the contractile properties of airway smooth muscles by affecting the rate and extent of myosin light chain phosphorylation, perhaps through a mechanism that involves the synergistic interaction of myosin light chain kinase phosphorylation and Ca2+ metabolism.

Thrombin-Stimulated Myosin Phosphorylation in Intact Platelets and its Possible Involvement Secretion

1977 ◽  
Vol 38 (04) ◽  
pp. 0984-0989 ◽  
Author(s):  
James L. Daniel ◽  
Holm Holmsen ◽  
Robert S. Adelstein
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Adenine Nucleotide ◽  
Fold Increase ◽  
Acid Hydrolase ◽  
Myosin Phosphorylation ◽  
Stimulation Of ◽  
Platelet Secretion

SummaryA 20,000 dalton polypeptide, which is phosphorylated in intact platelets pre-incubated with 32P-P04, has been identified as a platelet myosin light chain. Stimulation of intact platelets with thrombin produced a 5-fold increase in the amount of radioactive phosphate incorporated into the light chain. Myosin phosphorylation preceeded acid hydrolase secretion and occurred concomitantly with adenine nucleotide secretion. These results are suggestive of participation of contractile mechanisms in platelet secretion.

Increased Ca2+ and myosin phosphorylation, but not calmodulin activity in sensitized airway smooth muscles

1995 ◽  
Vol 269 (1) ◽  
pp. L135-L135
Author(s):  
He Jiang ◽  
Kang Rao ◽  
Xueliang Liu ◽  
Gang Liu ◽  
Newman L. Stephans
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Smooth Muscles ◽  
Ragweed Pollen ◽  
Myosin Light Chain Phosphorylation ◽  
Myosin Phosphorylation

Page L739: He Jiang, Kang Rao, Xueliang Liu, Gang Liu, and Newman L. Stephans. “Increased Ca2+ and myosin phosphorylation, but not calmodulin activity in sensitized airway smooth muscles.” This title is incorrect and conveys the opposite meaning of the article. The correct title should read: “Intracellular Ca2+, myosin light chain phosphorylation, and calmodulin activity in ragweed pollen-sensitized airway smooth muscles.”

scholarly journals Cytosolic calcium as a second messenger for collagen-induced platelet responses

1992 ◽  
Vol 288 (3) ◽  
pp. 925-929 ◽  
Author(s):  
J B Smith ◽  
M A Selak ◽  
C Dangelmaier ◽  
J L Daniel
Keyword(s):  
Arachidonic Acid ◽  
Cytosolic Calcium ◽  
Dense Granule ◽  
Acetoxymethyl Ester ◽  
Myosin Phosphorylation ◽  
Kinase C ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Adhesion Of Platelets ◽  
Stimulation Of

We showed previously that direct platelet activation by collagen involves an increase in the platelet cytosolic free Ca2+ concentration ([Ca2+]i) but that this increase is not required for the adhesion of platelets to collagen. We now report that collagen-induced arachidonic acid liberation, myosin phosphorylation and 5-hydroxytryptamine secretion are dependent on increases in [Ca2+]i, as they were markedly inhibited in platelets loaded with the acetoxymethyl ester of the Ca2+ chelator BAPTA but not in cells loaded with the acetoxymethyl ester of the non-chelating diazo-3. BAPTA also partially inhibited the rate of collagen-induced phosphatidic acid (PtdA) formation but had little effect on increases in phosphorylation of pleckstrin (47 kDa protein; P47). From these results we infer that collagen-induced increases in [Ca2+]i are required for dense granule secretion and arachidonic acid liberation, but are not necessary for stimulation of the protein kinase C pathway.

Relation between free cytosolic calcium and amylase release by pancreatic acini

1985 ◽  
Vol 249 (3) ◽  
pp. G389-G398 ◽  
Author(s):  
D. L. Ochs ◽  
J. I. Korenbrot ◽  
J. A. Williams
Keyword(s):  
Acinar Cells ◽  
Cytosolic Calcium ◽  
Pancreatic Acinar Cells ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Intracellular Ca ◽  
Free Media ◽  
Induced Changes ◽  
Stoichiometric Relation ◽  
Stimulation Of

Pancreatic acini were loaded with the Ca-selective fluorescent indicator quin-2 by incubation with its acetyoxymethyl ester. Loading acini with 844 +/- 133 microM quin-2 altered neither their ultrastructure nor their viability. The rate of amylase release from quin-2-loaded acini in response to the secretagogue carbachol, however, was significantly smaller than that of control acini. Studies in which acini were loaded with both quin-2 and a similar Ca-chelating compound, BAPTA, indicated that this reduced amylase release was related to the Ca buffering properties of quin-2. The concentration of free intracellular Ca calculated from the fluorescence of quin-2 was 90 +/- 18 nM. Stimulation by carbachol of acini suspended in media containing 1.25 mM Ca caused a rapid, transient enhancement of this value. After stimulation amylase release, the onset of the rise in free cytosolic Ca levels was observed in 1.1 +/- 0.1 s following the addition of agonist, and peak Ca levels (545 +/- 112 nM) were obtained within 5.3 +/- 0.3 s. For concentrations of carbachol less than or equal to 10(-6) M, a stoichiometric relation was found between stimulated amylase release and the peak concentration of free cytosolic Ca achieved. At higher concentrations of carbachol, however, the peak free cytosolic Ca remained constant while amylase release declined. The latency of the rise in intracellular Ca following stimulation of acini suspended in Ca-free media was not different from that observed for acini suspended in normal media, but the rise time was significantly prolonged. In the presence of extracellular Ca, the intracellular level of Ca remained elevated 2.8-fold above basal levels for at least 15 min following stimulation with 10(-6) M carbachol, whereas it had returned to near resting levels by 15 min when either 3 X 10(-7) or 3 X 10(-5) M carbachol was the stimulus. The Ca ionophore ionomycin (10–6 M) induced changes in the level of free cytosolic Ca similar to those caused by 10(-6) M carbachol. Ionomycin, however, stimulated only approximately one-third as much amylase release. These data suggest that factors in addition to changes in free cytosolic Ca may be important in regulating enzyme secretion by pancreatic acinar cells.

scholarly journals A mathematical model of vasoreactivity in rat mesenteric arterioles. II. Conducted vasoreactivity

2010 ◽  
Vol 298 (1) ◽  
pp. H52-H65 ◽  
Author(s):  
Adam Kapela ◽  
Sridevi Nagaraja ◽  
Nikolaos M. Tsoukias
Keyword(s):  
Gap Junctions ◽  
Cytosolic Calcium ◽  
Resistance Arteries ◽  
Local Site ◽  
Agonist Stimulation ◽  
Physiological Relevance ◽  
Transduction Mechanisms ◽  
Intracellular Ca ◽  
Transmembrane Currents ◽  
Stimulation Of

This study presents a multicellular computational model of a rat mesenteric arteriole to investigate the signal transduction mechanisms involved in the generation of conducted vasoreactivity. The model comprises detailed descriptions of endothelial (ECs) and smooth muscle (SM) cells (SMCs), coupled by nonselective gap junctions. With strong myoendothelial coupling, local agonist stimulation of the EC or SM layer causes local changes in membrane potential ( Vm) that are conducted electrotonically, primarily through the endothelium. When myoendothelial coupling is weak, signals initiated in the SM conduct poorly, but the sensitivity of the SMCs to current injection and agonist stimulation increases. Thus physiological transmembrane currents can induce different levels of local Vm change, depending on cell's gap junction connectivity. The physiological relevance of current and voltage clamp stimulations in intact vessels is discussed. Focal agonist stimulation of the endothelium reduces cytosolic calcium (intracellular Ca2+ concentration) in the prestimulated SM layer. This SMC Ca2+ reduction is attributed to a spread of EC hyperpolarization via gap junctions. Inositol (1,4,5)-trisphosphate, but not Ca2+, diffusion through homocellular gap junctions can increase intracellular Ca2+ concentration in neighboring ECs. The small endothelial Ca2+ spread can amplify the total current generated at the local site by the ECs and through the nitric oxide pathway, by the SMCs, and thus reduces the number of stimulated cells required to induce distant responses. The distance of the electrotonic and Ca2+ spread depends on the magnitude of SM prestimulation and the number of SM layers. Model results are consistent with experimental data for vasoreactivity in rat mesenteric resistance arteries.

The role of THOA in calcium sensitization in human myometrial smooth muscles

2007 ◽  
Vol 30 (4) ◽  
pp. 70
Author(s):  
Hector Aguilar ◽  
B. F. (Peter) Mitchell
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Mammalian Cells ◽  
Myosin Light Chain ◽  
Smooth Muscles ◽  
Human Myometrium ◽  
Uterine Contractility ◽  
Western Blots ◽  
Intracellular Ca

Improved understanding of the regulation of contractions of uterine smooth muscle (myometrium) is essential to develop more successful strategies for prevention of premature birth, which remains the most common cause of infant death and disability. Oxytocin (OT) and prostaglandin (PG) F2a are potent myometrial stimulants which induce an increase in intracellular Ca++. This activates myosin light chain kinase (MLCK) and subsequently the cell’s contractile machinery. However, there is poor correlation between the rise in Ca++ and the strength of the myometrial contraction. Contractile strength greatly increases at the time of parturition partly due to Ca++-independent factors that sensitize the muscle to the rise in intracellular Ca++. In vascular smooth muscle, one key regulator in this process of Ca++ sensitization is the monomeric G-protein, RhoA which in turn activates Rho-associated kinase (ROK). Studies in the rat myometrium have shown that RhoA and ROK are involved in enhancing OT-induced contractions. The potential role of the RhoA/ROK system in uterine contractility at the time of parturition has not been investigated in the human myometrium or in human cell lines. We propose to investigate the role of RhoA/ROK in the human myometrium using molecular cloning techniques. We have constructed expression plasmids for wild type, dominant positive (constitutively active), and dominant negative (constitutively inactive) isoforms of RhoA and synthesized purified proteins using a bacterial (BL21) translation system. These constructs will be introduced into primary and immortalized human myocytes using the protein transduction domain (TAT) derived from the HIV virus, which is capable of introducing whole proteins into mammalian cells. RhoA activation/translocation to the plasma membrane will be visualized using real time confocal microscopy in experiments where the RhoA proteins have been tagged with green fluorescent protein (GFP). Following introduction of the normal and mutant G-proteins, the downstream targets of activated ROK will be assayed for phosphorylation status using near infrared (NIR) fluorescence imaging of western blots or in-cell westerns. These targets include the myosin binding subunit (MBS) of myosin light chain phosphatase (MLCP), and two peptide phosphatase inhibitors of MLCP, CPI-17 and PHI-1. In addition, we propose to measure ROK activity directly using a direct enzyme assay. We will monitor calcium transients using fluorescence microscopy to verify the calcium independence of our measurements. These experiments will determine the role of the RhoA/ROK system in the mechanisms that may determine human uterine contractility. This information may direct new strategies to prevent or treat preterm labour.

Chronic hypoxia modulates relations among calcium, myosin light chain phosphorylation, and force differently in fetal and adult ovine basilar arteries

2005 ◽  
Vol 99 (1) ◽  
pp. 120-127 ◽  
Author(s):  
Surya M. Nauli ◽  
James M. Williams ◽  
William T. Gerthoffer ◽  
William J. Pearce
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Cerebral Arteries ◽  
Cytosolic Calcium ◽  
Myosin Light Chain Phosphorylation ◽  
Postnatal Maturation ◽  
Myosin Phosphorylation ◽  
Age Related ◽  
Basilar Arteries ◽  
Nonpregnant Adult

The present study tests the hypothesis that age-related differences in contractility of cerebral arteries from hypoxic animals involve changes in myofilament Ca2+ sensitivity. Basilar arteries from term fetal and nonpregnant adult sheep maintained 110 days at 3,820 m were used in measurements of cytosolic calcium concentration ([Ca2+]i), myosin light chain phosphorylation, and contractile tensions induced by graded concentrations of K+ or serotonin (5-HT). Slopes relating [Ca2+]i to tension were similar in fetal (0.83 ± 0.07) and adult (1.02 ± 0.08) arteries for K+ contractions but were significantly greater for fetal (3.77 ± 0.64) than adult (2.00 ± 0.13) arteries for 5-HT contractions. For both K+ and 5-HT contractions, these relations were left shifted in fetal compared with adult arteries, indicating greater Ca2+ sensitivity in fetal arteries. In contrast, slopes relating [Ca2+]i and %myosin phosphorylation for K+ contractions were less in fetal (0.37 ± 0.08) than adult (0.81 ± 0.07) arteries, and the fetal curves were right shifted. For 5-HT contractions, the slope of the Ca2+-phosphorylation relation was similar in fetal (0.33 ± 0.09) and adult (0.33 ± 0.23) arteries, indicating that 5-HT depressed Ca2+-induced myosin phosphorylation in adult arteries. For slopes relating %myosin phosphorylation and tension, fetal values (K+: 1.52 ± 0.22, 5-HT: 7.66 ± 1.70) were less than adult values (K+: 2.13 ± 0.30, 5-HT: 8.29 ± 2.40) for both K+- and 5-HT-induced contractions, although again fetal curves were left shifted relative to the adult. Thus, in hypoxia-acclimatized basilar arteries, a downregulated ability of Ca2+ to promote myosin phosphorylation is offset by an upregulated ability of phosphorylated myosin to produce force yielding an increased fetal myofilament Ca2+ sensitivity. Postnatal maturation reprioritizes the mechanisms regulating hypoxic contractility through changes in the source of activator Ca2+, the pathways governing myosin light chain phosphorylation, and its interaction with actin.

Increased Ca2+ and myosin phosphorylation, but not calmodulin activity in sensitized airway smooth muscles

1995 ◽  
Vol 269 (6) ◽  
pp. 1-1
Author(s):  
He Jiang ◽  
Kang Rao ◽  
Xueliang Liu ◽  
Gang Liu ◽  
Newman L. Stephans
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Smooth Muscles ◽  
Ragweed Pollen ◽  
Myosin Light Chain Phosphorylation ◽  
Myosin Phosphorylation

Page L739: He Jiang, Kang Rao, Xueliang Liu, Gang Liu, and Newman L. Stephans. “Increased Ca2+ and myosin phosphorylation, but not calmodulin activity in sensitized airway smooth muscles.” This title is incorrect and conveys the opposite meaning of the article. The correct title should read: “Intracellular Ca2+, myosin light chain phosphorylation, and calmodulin activity in ragweed pollen-sensitized airway smooth muscles.”

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