Monitoring of Ca2+Release from Intracellular Stores in Permeabilized Rat Parotid Acinar Cells Using the Fluorescent Indicators Mag-fura-2 and Calcium Green C18

Tojyo, Yosuke; Tanimura, Akihiko; Matsumoto, Yoshito

doi:10.1006/bbrc.1997.7584

Cited by 18 publications

(8 citation statements)

References 29 publications

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“…4); a similar observation was made by Tojyo et at. (17), albeit with saponin-treated cells (parotid acinar). Further studies will be needed for a more detailed exploration of conditions for Ca 2 + depletion.…”

Section: Resultsmentioning

confidence: 99%

A Mechanism for Both Capacitative Ca²⁺Entry and Excitation-Contraction Coupled Ca²⁺Release by the Sarcoplasmic Reticulum of Skeletal Muscle Cells

Islam

Narayanan

Ochs

2002

Exp Biol Med (Maywood)

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We have previously established that L6 skeletal muscle cell cultures display capacitative calcium entry (CCE), a phenomenon established with other cells in which Ca2+ uptake from outside cells increases when the endoplasmic reticulum (sarcoplasmic reticulum in muscle, or SR) store is decreased. Evidence for CCE rested on the use of thapsigargin (Tg), an inhibitor of the SR CaATPase and consequently transport of Ca2+ from cytosol to SR, and measurements of cytosolic Ca2+. When Ca2+ is added to Ca2+-free cells in the presence of Tg, the measured cytosolic Ca2+ rises. This has been universally interpreted to mean that as SR Ca2+ is depleted, exogenous Ca2+ crosses the plasma membrane, but accumulates in the cytosol due to CaATPase inhibition. Our goal in the present study was to examine CCE in more detail by measuring Ca2+ in both the SR lumen and the cytosol using established fluorescent dye techniques for both. Surprisingly, direct measurement of SR Ca2+ in the presence of Tg showed an increase in luminal Ca2+ concentration in response to added exogenous Ca2+. While we were able to reproduce the conventional demonstration of CCE—an increase of Ca2+ in the cytosol in the presence of thapsigargin—we found that this process was inhibited by the prior addition of ryanodine (Ry), which inhibits the SR Ca2+ release channel, the ryanodine receptor (RyR). This was also unexpected if Ca2+ enters the cytosol first. When Ca2+ was added prior to Ry, the later was unable to exert any inhibition. This implies a competitive interaction between Ca2+ and Ry at the RyR. In addition, we found a further paradox: we had previously found Ry to be an uncompetitive inhibitor of Ca2+ transport through the RyR during excitation-contraction coupling. We also found here that high concentrations of Ca2+ inhibited its own uptake, a known feature of the RyR. We confirmed that Ca2+ enters the cells through the dihydropyridine receptor (DHPR, also known as the L-channel) by demonstrating inhibition by diltiazem. A previous suggestion to the contrary had used Mn2+ in place of direct Ca2+ measurements; we showed that Mn2+ was not inhibited by diltiazem and was not capacitative, and thus not an appropriate probe of Ca2+ flow in muscle cells. Our findings are entirely explained by a new model whereby Ca2+ enters the SR from the extracellular space directly through a combined channel formed from the DHPR and the RyR. These are known to be in close proximity in skeletal muscle. Ca2+ subsequently appears in the cytosol by egress through a separate, unoccupied RyR, explaining Ry inhibition. We suggest that upon excitation, the DHPR, in response to the electrical field of the plasma membrane, shifts to an erstwhile-unoccupied receptor, and Ca2+ is released from the now open RyR to trigger contraction. We discuss how this model also resolves existing paradoxes in the literature, and its implications for other cell types.

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Section: Resultsmentioning

confidence: 99%

A Mechanism for Both Capacitative Ca²⁺Entry and Excitation-Contraction Coupled Ca²⁺Release by the Sarcoplasmic Reticulum of Skeletal Muscle Cells

Islam

Narayanan

Ochs

2002

Exp Biol Med (Maywood)

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“…[167] Therefore,i ti si mportant to develop membrane-targeting imaging probes to monitor Ca 2+ activities near the plasma membrane.I nt he early 1990s,s everal membrane-associated fluorescent probes (e.g.C18-Fura-2 (72)and Calcium Green-C18 (73), Scheme 28) were developed by conjugating al ipophilic alkyl chain to the Ca 2+ probes Fura-2 or Calcium Green-1. [168,169] To bypass the penetration issue of one-photon microscopy and to conduct deep tissue monitoring of Ca 2+ ,t he Cho research group developed in 2009 at wo-photon fluorescent probe (ACaL (74), Scheme 28) to visualize near-membrane Ca 2+ in live cells and live tissue samples. [168,169] To bypass the penetration issue of one-photon microscopy and to conduct deep tissue monitoring of Ca 2+ ,t he Cho research group developed in 2009 at wo-photon fluorescent probe (ACaL (74), Scheme 28) to visualize near-membrane Ca 2+ in live cells and live tissue samples.…”

Section: Memtafps For Metal Ionsmentioning

confidence: 99%

“…[168] Cell imaging experiments indicated these probes are located at the plasma membrane and are sensitive to Ca 2+ fluctuations. [168,169] To bypass the penetration issue of one-photon microscopy and to conduct deep tissue monitoring of Ca 2+ ,t he Cho research group developed in 2009 at wo-photon fluorescent probe (ACaL (74), Scheme 28) to visualize near-membrane Ca 2+ in live cells and live tissue samples. [170] This probe consists of BAPTA-5-Me as aC a 2+ chelator, 2-dimethylamino-6-laurylnaphthalene (laurdan) as the two-photon chromophore,a nd dodecanoyl group as the membrane anchor, with BAPTA-5-Me and the dodecanoyl group located separately at the two ends of laurdan to avoid crossinteraction.…”

Section: Memtafps For Metal Ionsmentioning

confidence: 99%

Discerning the Chemistry in Individual Organelles with Small‐Molecule Fluorescent Probes

et al. 2016

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Principle has it that even the most advanced super-resolution microscope would be futile in providing biological insight into subcellular matrices without well-designed fluorescent tags/probes. Developments in biology have increasingly been boosted by advances of chemistry, with one prominent example being small-molecule fluorescent probes that not only allow cellular-level imaging, but also subcellular imaging. A majority, if not all, of the chemical/biological events take place inside cellular organelles, and researchers have been shifting their attention towards these substructures with the help of fluorescence techniques. This Review summarizes the existing fluorescent probes that target chemical/biological events within a single organelle. More importantly, organelle-anchoring strategies are described and emphasized to inspire the design of new generations of fluorescent probes, before concluding with future prospects on the possible further development of chemical biology.

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“…In addition to IP $ , isolated pancreatic zymogen granules have been reported to respond to cADPr [13]. However, this is inconsistent with our previous result that cADPr did not evoke Ca# + release from intracellular Ca# + stores in saponin-permeabilized rat parotid acinar cells [30]. Since cADPr is thought to activate RyRs [18], we have assessed whether RyRs exist in rat parotid acinar cells.…”

Section: Discussionmentioning

confidence: 68%

Evidence that zymogen granules do not function as an intracellular Ca2+ store for the generation of the Ca2+ signal in rat parotid acinar cells

Nezu¹,

Tanimura²,

Mitsui³

et al. 2002

Biochem. J.

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Rat parotid acinar cells lacking zymogen granules were obtained by inducing granule discharge with the beta-adrenoceptor agonist isoproterenol. To assess whether zymogen granules are involved in the regulation of Ca(2+) signalling as intracellular Ca(2+) stores, changes in cytosolic free Ca(2+) ion concentration ([Ca(2+)](i)) were studied with imaging microscopy in fura-2-loaded parotid acinar cells lacking zymogen granules. The increase in [Ca(2+)](i) induced by muscarinic receptor stimulation was initiated at the apical pole of the acinar cells, and rapidly spread as a Ca(2+) wave towards the basolateral region. The magnitude of the [Ca(2+)](i) response and the speed of the Ca(2+) wave were essentially similar to those in control acinar cells containing zymogen granules. Western blot analysis of the inositol 1,4,5-trisphosphate receptor (IP(3)R) was performed on zymogen granule membranes and microsomes using anti-IP(3)R antibodies. The immunoreactivity of all three IP(3)Rs was clearly observed in the microsomal preparations. Although a weak band of IP(3)R type-2 was detected in the zymogen granule membranes, this band probably resulted from contamination by the endoplasmic reticulum (ER), because calnexin, a marker protein of the ER, was also detected in the same preparation. Furthermore, Western blotting and reverse transcriptase-PCR analysis failed to provide evidence for the expression of ryanodine receptors in rat parotid acinar cells, whereas expression was clearly detectable in rat skeletal muscle, heart and brain. These results suggest that zymogen granules do not have a critical role in Ca(2+) signalling in rat parotid acinar cells.

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Monitoring of Ca2+Release from Intracellular Stores in Permeabilized Rat Parotid Acinar Cells Using the Fluorescent Indicators Mag-fura-2 and Calcium Green C18

Cited by 18 publications

References 29 publications

A Mechanism for Both Capacitative Ca²⁺Entry and Excitation-Contraction Coupled Ca²⁺Release by the Sarcoplasmic Reticulum of Skeletal Muscle Cells

A Mechanism for Both Capacitative Ca²⁺Entry and Excitation-Contraction Coupled Ca²⁺Release by the Sarcoplasmic Reticulum of Skeletal Muscle Cells

Discerning the Chemistry in Individual Organelles with Small‐Molecule Fluorescent Probes

Evidence that zymogen granules do not function as an intracellular Ca2+ store for the generation of the Ca2+ signal in rat parotid acinar cells

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Monitoring of Ca2+Release from Intracellular Stores in Permeabilized Rat Parotid Acinar Cells Using the Fluorescent Indicators Mag-fura-2 and Calcium Green C18

Cited by 18 publications

References 29 publications

A Mechanism for Both Capacitative Ca2+Entry and Excitation-Contraction Coupled Ca2+Release by the Sarcoplasmic Reticulum of Skeletal Muscle Cells

A Mechanism for Both Capacitative Ca2+Entry and Excitation-Contraction Coupled Ca2+Release by the Sarcoplasmic Reticulum of Skeletal Muscle Cells

Discerning the Chemistry in Individual Organelles with Small‐Molecule Fluorescent Probes

Evidence that zymogen granules do not function as an intracellular Ca2+ store for the generation of the Ca2+ signal in rat parotid acinar cells

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A Mechanism for Both Capacitative Ca²⁺Entry and Excitation-Contraction Coupled Ca²⁺Release by the Sarcoplasmic Reticulum of Skeletal Muscle Cells

A Mechanism for Both Capacitative Ca²⁺Entry and Excitation-Contraction Coupled Ca²⁺Release by the Sarcoplasmic Reticulum of Skeletal Muscle Cells