Structural and functional properties of adult rat heart myocytes lysed with digitonin.

Altschuld, Ruth A.; Wenger, William C.; Lamka, Karla G.; Kindig, O.; Capen, Charles C.; Mizuhira, Vinci; Heide, Richard S. Vander; Brierley, Gerald P.

doi:10.1016/s0021-9258(17)38721-5

Cited by 100 publications

(13 citation statements)

References 37 publications

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“…39 Melittin was able to induce an alteration in the mitochondria (Figure S4B). In addition, a low concentration of digitonin, which allows the analysis of mitochondrial function in situ, 40,41 was able to induce YoPro-1 uptake with a parallel increase in MitoSOX fluorescence (Figure S4C). Furthermore, plasma membrane permeabilization with digitonin induced a similar mitochondrial network fragmentation to the one found during pyroptosis (Figure S4D) and mitochondrial DNA release (Figure S4E).…”

Section: Gsdmd Nt Plasma Membrane Pore Activity Is Required For Mitochondrial Damagementioning

confidence: 99%

Gasdermins mediate cellular release of mitochondrial DNA during pyroptosis and apoptosis

et al. 2021

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Section: Gsdmd Nt Plasma Membrane Pore Activity Is Required For Mitochondrial Damagementioning

confidence: 99%

Gasdermins mediate cellular release of mitochondrial DNA during pyroptosis and apoptosis

et al. 2021

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“…The initial rate of Ca2+ uptake in these experiments progressively increased as the initial Ca2+ concentration was raised to about 0.1 mM, which is a greater range than that noted by other workers using permeabilized myocytes (Altschuld et al 1985;Chiesi et al 1981). This difference may be due to the presence of oxalate which precipitates accumulated calcium, or procaine to inhibit calcium efflux in these other studies.…”

Section: Comparison With Isolated Organellesmentioning

confidence: 45%

“…(1984a) who found that, in the same preparation, mitochondrial Ca2+ uptake could only be demonstrated in the rod-shaped fraction. However, an ATP-dependent, nonmitochondrial accumulation of Ca2+ has also been demonstrated by Altschuld et al (1985) in a preparation of rounded cells from the rat heart. These authors suggested that the shape of the myocyte does not affect the ability of this fraction to accumulate Ca2+ but that the tendency of cells to change from a rod to round shape is strongly dependent upon the concentration of ATP and Ca2+ in the medium.…”

Section: Nature Of the Non-mitochondrial Ca2+ Uptakementioning

confidence: 89%

“…CP (15 mM) was also present to enable ATP to be regenerated as it was consumed. Both digitonin and saponin have been widely used to render the sarcolemma permeable while leaving intracellular organelles intact and functional (Endo & Kitazawa 1978;Williamson et al 1983 ;Fry et al 1984a;Altschuld et al 1985 ;Fry & Miller 1985). Shown in figure 1 are results with both agents, although in all further experiments digitonin has been used out of preference as it acts much faster than saponin (see also Endo & lino 1980) and therefore allows a more accurate measurement of the initial rate of Ca2+ uptake (Fry et al 1984a).…”

Section: Characteristics Of the Non-mitochondrial Ca2+ Exchangementioning

confidence: 99%

“…Altschuld et al (1981) have demonstrated that exposure of a suspension of rat myocytes to digitonin caused release of a sarcoplasmic enzyme, lactate dehydro genase, but not a mitochondrial enzyme, aspartate transferase. Several groups have established that digitonin or saponin does not impair the activity of either the sarcoplasmic reticulum (Endo & Kitazawa 1978;Altschuld et al 1985) or mitochondria (Kessar & Crompton 1983 ;Fry et al 1984a).…”

Section: Nature Of the Non-mitochondrial Ca2+ Uptakementioning

confidence: 99%

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Non-mitochondrial calcium ion regulation in rat ventricular myocytes

Fry

Harding

Miller

1989

Proc. R. Soc. Lond. B

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Ca 2+ exchange has been measured in a suspension of rat ventricular myocytes treated with digitonin or saponin to render the sarcolemma permeable to small molecules and ions. Two fractions of exchange were identified, one that was attributed to the mitochondrial component of the cell and the other to a non-mitochondrial fraction. Mitochondrial Ca 2+ uptake was blocked by sodium azide and depended on respiratory substrates whereas non-mitochondrial uptake occurred independently of these molecules but was dependent on ATP and creatine phosphate. Non-mitochondrial Ca 2+ uptake could be induced at a Ca 2+ concentration below 1 um and the initial rate increased with concentration up to 100 um Uptake could be reversed by sulmazole (a caffeine-like substance) and this reversal in turn inhibited by ryanodine. These properties suggest that the major locus for non-mitochondrial Ca 2+ exchange is at the sarcoplasmic reticulum. Ca 2+ exchange could be modulated by a number of agents, including carnosine, but was unaffected by others, including Na + , inositol trisphosphate and cyclic AMP. A kinetic model of the data is presented, which incorporates similar data of Ca 2+ uptake into the mitochondrial fraction. The rates of Ca 2+ exchange measured in these experiments suggest that these two components of the cell can reduce the sarcoplasmic Ca 2+ concentration rapidly enough to account for the observed transient nature of the isometric twitch. Furthermore, it is suggested that both non-mitochondrial and mitochondrial fractions of the cell could significantly contribute to tension relaxation in rat cardiac muscle.

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Permeabilization activated reduction in fluorescence: A novel method to measure kinetics of protein interactions with intracellular structures

et al. 2016

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Understanding kinetic information is fundamental in understanding biological function. Advanced imaging technologies have fostered the development of kinetic analyses in cells. We have developed Permeabilization Activated Reduction in Fluorescence (PARF) analysis for determination of apparent t1/2 and immobile fraction, describing the dissociation of a protein of interest from intracellular structures. To create conditions where dissociation events are observable, cells expressing a fluorescently-tagged protein are permeabilized with digitonin, diluting the unbound protein into the extracellular media. As the media volume is much larger than the cytosolic volume, the concentration of the unbound pool decreases drastically, shifting the system out of equilibrium--favoring dissociation events. Loss of bound protein is observed as loss of fluorescence from intracellular structures and can be fit to an exponential decay. We compared PARF dissociation kinetics with previously published equilibrium kinetics as determined by FRAP. PARF dissociation rates agreed with the equilibrium-based FRAP analysis predictions of the magnitude of those rates. When used to investigate binding kinetics of a panel of cytoskeletal proteins, PARF analysis revealed that filament stabilization resulted in slower fluorescence loss. Additionally, commonly used “general” F-actin labels display differences in kinetic properties, suggesting that not all fluorescently-tagged actin labels interact with the actin network in the same way. We also observed differential dissociation kinetics for GFP-VASP depending on which cellular structure was being labeled. These results demonstrate that PARF analysis of non-equilibrium systems reveals kinetic information without the infrastructure investment required for other quantitative approaches such as FRAP, photoactivation, or in vitro reconstitution assays.

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Structural and functional properties of adult rat heart myocytes lysed with digitonin.

Cited by 100 publications

References 37 publications

Gasdermins mediate cellular release of mitochondrial DNA during pyroptosis and apoptosis

Gasdermins mediate cellular release of mitochondrial DNA during pyroptosis and apoptosis

Non-mitochondrial calcium ion regulation in rat ventricular myocytes

Permeabilization activated reduction in fluorescence: A novel method to measure kinetics of protein interactions with intracellular structures

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