Calcium regulation of the contractile state of isolated mammalian fibroblast cytoplasm

1975 ◽  
Vol 18 (2) ◽  
pp. 241-256
Author(s):  
C.S. Izzard ◽  
S.L. Izzard
Keyword(s):  
Plasma Membrane ◽  
Striated Muscle ◽  
Calcium Regulation ◽  
Threshold Concentration ◽  
Free Access ◽  
Contractile State ◽  
Relaxing Solution ◽  
Calcium Dependent ◽  
Rigor Solution

Calcium-dependent contractions have been induced in fresh, naked cytoplasm of L-929 fibroblasts using physiological solutions (rigor, relaxing and contracting) similar to those designed to control the contractile state of vertebrate striated muscle. Free access of solutions to the cytoplasm was achieved by popping and stripping the plasma membrane from cells using 7–10 strokes of a Dounce homogenizer. Contracting solution (free Ca2+ 7 X 10(−5) M; with added MgATP) applied locally from a micropipette to cells popped in rigor (free Ca2+ less than 10(−8) M) or relaxing (free Ca2+ less than 10(−8) M; with added MgATP) solutions induced symmetrical contractions of unstretched cytoplasm and directional shortening of stretched cytoplasm. The contractions produced 12–18% shortenings and were complete in 1–3 s. The cytoplasm could be cycled repeatedly through the contracted state from the relaxed state. Exogenous MgATP was required for the Ca2+-dependent contractions. At low free Ca2+ concentrations (less than 10(−8) M), MgATP had a marked plasticizing effect on the cytoplasm. Thus cytoplasm prepared in relaxing solution was less cohesive and more easily deformed than cytoplasm prepared in rigor solution. When induced to contract, relaxed cytoplasm showed a loss of plasticity. Using this criterion, the threshold concentration of free Ca2+ for contraction was determined to lie between 7 X 10(−8) M and 5 X 10(−7) M.

scholarly journals Myotubes from transgenic mdx mice expressing full-length dystrophin show normal calcium regulation.

1994 ◽  
Vol 5 (10) ◽  
pp. 1159-1167 ◽  
Author(s):  
W F Denetclaw ◽  
F W Hopf ◽  
G A Cox ◽  
J S Chamberlain ◽  
R A Steinhardt
Keyword(s):  
Single Channel ◽  
Calcium Regulation ◽  
Full Length ◽  
Mdx Mice ◽  
Free Calcium ◽  
Muscle Degeneration ◽  
Channel Activity ◽  
Mouse Muscle ◽  
Calcium Dependent ◽  
Normal Calcium

A lack of dystrophin results in muscle degeneration in Duchenne muscular dystrophy. Dystrophin-deficient human and mouse muscle cells have higher resting levels of intracellular free calcium ([Ca2+]i) and show a related increase in single-channel open probabilities of calcium leak channels. Elevated [Ca2+]i results in high levels of calcium-dependent proteolysis, which in turn increases calcium leak channel activity. This process could initiate muscle degeneration by further increasing [Ca2+]i and proteolysis in a positive feedback loop. Here, we tested the direct effect of restoration of dystrophin on [Ca2+]i and channel activity in primary myotubes from mdx mice made transgenic for full-length dystrophin. Transgenic mdx mice have been previously shown to have normal dystrophin localization and no muscle degeneration. Fura-2 calcium measurements and single-channel patch recordings showed that resting [Ca2+]i levels and open probabilities of calcium leak channels of transgenic mdx myotubes were similar to normal levels and significantly lower than mdx littermate controls (mdx) that lack dystrophin. Thus, restoration of normal calcium regulation in transgenic mdx mice may underlie the resulting absence of degeneration.

scholarly journals Calcium-dependent fusion of the plasma membrane fraction from human neutrophils with liposomes

1990 ◽  
Vol 1025 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Joseph W. Francis ◽  
James E. Smolen ◽  
Kenneth J. Balazovich ◽  
Rebecca R. Sandborg ◽  
Laurence A. Boxer
Keyword(s):  
Plasma Membrane ◽  
Membrane Fraction ◽  
Human Neutrophils ◽  
Plasma Membrane Fraction ◽  
Calcium Dependent

scholarly journals Calcium-Dependent Myosin from Insect Flight Muscles

1974 ◽  
Vol 63 (5) ◽  
pp. 553-563 ◽  
Author(s):  
William Lehman ◽  
Belinda Bullard ◽  
Kathleen Hammond
Keyword(s):  
Atpase Activity ◽  
Insect Flight ◽  
Calcium Regulation ◽  
Regulatory Proteins ◽  
Myosin Molecule ◽  
Thin Filaments ◽  
Actomyosin Atpase ◽  
Calcium Dependent ◽  
Regulatory Systems ◽  
Flight Muscles

Calcium regulation of the insect actomyosin ATPase is associated with the thin filaments as in vertebrate muscles, and also with the myosin molecule as in mollusks. This dual regulation is demonstrated using combinations of locust thin filaments with rabbit myosin and locust myosin with rabbit actin; in each case the ATPase of the hybrid actomyosin is calcium dependent. The two regulatory systems are synergistic, the calcium dependency of the locust actomyosin ATPase being at least 10 times that of the hybrid actomyosins described above. Likewise Lethocerus myosin also contains regulatory proteins. The ATPase activity of Lethocerus myosin is labile and is stabilized by the presence of rabbit actin. Tropomyosin activates the ATPase of insect actomyosin and the activation occurs irrespective of whether the myosin is calcium dependent or rendered independent of calcium.

Effects of Octopamine on Calcium Action Potentials in Insect Oocytes

1989 ◽  
Vol 142 (1) ◽  
pp. 115-124
Author(s):  
M. J. O'DONNELL ◽  
B. SINGH
Keyword(s):  
Action Potentials ◽  
Rank Order ◽  
Potassium Conductance ◽  
Membrane Resistance ◽  
Threshold Concentration ◽  
Resting Potential ◽  
Calcium Dependent ◽  
Octopamine Receptors ◽  
Voltage Dependent ◽  
Maximum Rate Of Rise

Our experiments show that octopamine receptors are present on the developing follicles of an insect, Rhodnius prolixus. Application of D,L-octopamine decreased the duration and overshoot of calcium-dependent action potentials (APs), and increased the intrafollicular concentration of cyclic AMP. The threshold concentration of D,L-octopamine for the reduction in electrical excitability was between 1 and 5×10−7moll−1, and maximal effects of a 40–50% reduction in AP overshoot and duration were apparent at 10−4moll−1. At concentrations above 10−5moll−1, a small (<10%) hyperpolarization of the resting potential was also apparent. Effects of D,L-octopamine on oocyte excitability were independent of these small shifts in resting potential. Current injection experiments, in which calcium entry was blocked by cobalt, demonstrated that D,L-octopamine reduced membrane resistance at both hyperpolarizing and depolarizing potentials. Octopamine did not affect the maximum rate of rise of the AP, dV/dtmax, which is an indicator of inward calcium current. It is suggested that octopamine may mediate its effects on excitability through an increase in a voltage-dependent potassium conductance. Application of other phenolamines indicated a rank order of potency of D, Loctopamine > D,L-synephrine > tyramine. The α-adrenergic agonists clonidine, naphazoline and tolazoline were without significant effect at 10−5-10−3moll−1. Reduction of excitability by D,L-octopamine was effectively blocked by phentolamine and metoclopramide. Yohimbine and gramine were less effective as antagonists. Possible functions of octopamine receptors in insect follicles are discussed.

Ultrastructural Evidence for Myosin of the Smooth Muscle Type at the Surface of Trypsin-Dissociated Embryonic Chick Cells

1974 ◽  
Vol 15 (2) ◽  
pp. 279-289
Author(s):  
I. AP GWYNN ◽  
R. B. KEMP ◽  
B. M. JONES ◽  
U. GRÖSCHEL-STEWART
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Striated Muscle ◽  
Smooth Muscle Myosin ◽  
Rabbit Serum ◽  
Embryonic Chick ◽  
Antibody Preparation ◽  
Rabbit Igg ◽  
Antibody Conjugate ◽  
Muscle Myosin

Cells dissociated from embryonic chick muscle tissue using trypsin were rotated in the presence of globulin-enriched rabbit antisera against both smooth and striated muscle actomyosins originating from chicken gizzard (GAM) and pectoralis (PAM) muscles respectively. The presence of the rabbit antibodies was demonstrated using peroxidase-labelled sheep anti-rabbit λ-globulins, the enzyme-antibody conjugate being located by electron-microscope histochemistry. Anti-GAM λ-globulins reacted strongly with the plasma membrane. Judging from the complete absence of staining, λ-globulins from non-immunized rabbit serum did not interact with the membrane.When λ-globulins of sheep anti-rabbit IgG serum were applied alone, that is in the absence of pretreatment with rabbit λ-globulin, there was an observable reaction with the cell surface. Preincubation of anti-GAM with the heavy meromyosin fraction from smooth-muscle myosin inhibited the interaction of the antibodies with the membrane, as evidenced by the absence of staining. A weak positive reaction obtained with anti-PAM was due to components of the antibody preparation which were reactive with actin and not with PAM. It was concluded that a smooth-muscle myosin-like protein is an integral part of the plasma membrane of embryonic chick muscle cells.

scholarly journals Cell Secretion: Current Structural and Biochemical Insights

2010 ◽  
Vol 10 ◽  
pp. 2054-2069 ◽  
Author(s):  
Saurabh Trikha ◽  
Elizabeth C. Lee ◽  
Aleksandar M. Jeremic
Keyword(s):  
Plasma Membrane ◽  
Secretory Vesicles ◽  
Intercellular Signaling ◽  
Cell Secretion ◽  
Membrane Structures ◽  
Calcium Dependent ◽  
Cell Plasma ◽  
Membrane Bound ◽  
And Control ◽  
Fusion Pores

Essential physiological functions in eukaryotic cells, such as release of hormones and digestive enzymes, neurotransmission, and intercellular signaling, are all achieved by cell secretion. In regulated (calcium-dependent) secretion, membrane-bound secretory vesicles dock and transiently fuse with specialized, permanent, plasma membrane structures, called porosomes or fusion pores. Porosomes are supramolecular, cup-shaped lipoprotein structures at the cell plasma membrane that mediate and control the release of vesicle cargo to the outside of the cell. The sizes of porosomes range from 150nm in diameter in acinar cells of the exocrine pancreas to 12nm in neurons. In recent years, significant progress has been made in our understanding of the porosome and the cellular activities required for cell secretion, such as membrane fusion and swelling of secretory vesicles. The discovery of the porosome complex and the molecular mechanism of cell secretion are summarized in this article.

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