Homogeneous Ca2+ stores in rat adrenal chromaffin cells

Inoue, Masumi; Sakamoto, Youichi; Fujishiro, Naoji; Imanaga, Issei; Ozaki, Shoichiro; Prestwich, Glenn D.; Warashina, Akira

doi:10.1016/s0143-4160(02)00178-1

Cited by 34 publications

(23 citation statements)

References 25 publications

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“…In our experimental conditions, there was no significant cross-talk between FITC-and rhodamine-like fluorescence. To identify organelles stained with the anti-InsP 3 R2 Abs, cells with immunoreactions were immersed in PBS containing 30 µM BODIPY-FL-InsP 3 [16], 1 µM ER Tracker (Molecular Probe), or 4.4 µM 3,3´-dihexyloxacarbocyanine iodide (DIO) (Molecular Probe) whereas to study the relation between the ER and Ca 2+ store sites, the cells treated with the anti-calnexin Ab were placed in 0.5 µM BODIPY-FLthapsigargin (Molecular Probe). The immunoreactivity was observed using a confocal laser scanning microscope (Zeiss LSM 410) with X63 objective lenses (a numerical aperture of 1.25 or 1.4).…”

Section: Methodsmentioning

confidence: 99%

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Organelles Containing Inositol Trisphosphate Receptor Type 2 in Adrenal Medullary Cells

et al. 2006

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Abstract:To identify which organelles contained inositol trisphosphate (InsP 3 ) receptor type 2 (InsP 3 R2) in adrenal medullary (AM) cells, immunocytochemical and biochemical studies were performed on AM cells of several species. InsP 3 R2-like immunoreactive materials produced by two different anti-InsP 3 R2 antibodies (Abs) (Chemicon and Sigma) were distributed in rat AM cells in agreement with BODIPY-FL-InsP 3 binding sites. For two other Abs (KM1083 and Santa Cruz), some of the antiInsP 3 R2 immunoreactive materials were stained with an antidopamine-β-hydroxylase Ab, but not by BODIPY-FL-InsP 3 . BO-DIPY-FL-thapsigargin binding sites were consistent with a distribution of the endoplasmic reticulum (ER) identified by an anticalnexin Ab, and a prior application of thapsigargin significantly eliminated BODIPY-FL-thapsigargin bindings, suggesting that BODIPY-FL-thapsigargin bindings were mediated by thapsigargin, but not the fluorescence molecule. The anti-InsP 3 R2 Ab that produced stainings consistent with BODIPY-FL-InsP 3 bindings recognized a protein with about 250 kDa. A fractional analysis of bovine adrenal medullae revealed that the 250 kDa InsP 3 R2 was detected in a crude membrane fraction, but not in a secretory granule fraction. The results suggest that the InsP 3 R2 was present in the ER, but not in secretory granules in AM cells.

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Section: Methodsmentioning

confidence: 99%

“…Therefore, InsP 3 Rs below the plasma membrane are not necessarily located in secretory granules, but they are possibly present in the peripheral ER [16]. In pancreatic acinar cells, InsP 3 Rs in the apical pole were reported to be present in secretory granules [17,18].…”

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confidence: 99%

Organelles Containing Inositol Trisphosphate Receptor Type 2 in Adrenal Medullary Cells

et al. 2006

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“…78 Cell-permeable BODIPY FL-X-and BODIPY TR-X-conjugates of thapsigargin, ryanodine, and InsP 3 proved useful to study the spatial distribution and density of SR/ER ryanodine receptors, 79 -81 InsP 3 receptors, 82 and Ca 2ϩ -ATPases. 79,81,82 FRAP experiments revealed remarkable changes in the mobility of proteins in the ER lumen under conditions of cell stress, 83 thus proving ER to be a very dynamic structure responding to cell stress.…”

Section: Sarcoplasmic/endoplasmic Reticulummentioning

confidence: 99%

Examining Intracellular Organelle Function Using Fluorescent Probes

Zorov¹,

Kobrinsky²,

Juhaszova³

et al. 2004

Circulation Research

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Abstract-Fluorescence microscopy imaging has become one of the most useful techniques to assess the activity of individual cells, subcellular trafficking of signals to and between organelles, and to appreciate how organelle function is regulated. The past 2 decades have seen a tremendous advance in the rational design and development in the nature and selectivity of probes to serve as reporters of the intracellular environment in live cells. These probes range from small organic fluorescent molecules to fluorescent biomolecules and photoproteins ingeniously engineered to follow signaling traffic, sense ionic and nonionic second messengers, and report various kinase activities. These probes, together with recent advances in imaging technology, have enabled significantly enhanced spatial and temporal resolution. This review summarizes some of these developments and their applications to assess intracellular organelle function. Key Words: microscopy Ⅲ calcium Ⅲ redox Ⅲ mitochondria Ⅲ fluorescent proteins R esponses of single cells undergoing physiological activation processes or pathological stresses can be quite heterogeneous in time and space. Measurements of these parameters in bulk (such as in tissue or cellular suspension), using conventional physiological and biochemical methods, can often fail to resolve discrete differences in the kinetics and magnitudes of the responses between cells, as well as complex intracellular and subcellular dynamical processes, critical to understanding how cells actually work. For example, during embryonic development or periods of environmental stress, the decision for a certain cell to undergo apoptosis may be triggered in a moment and distinct from its neighbors, the resulting intracellular processes may proceed along a more or less protracted time frame, and these events may occur heterogeneously among (and inside) other cells. Yet, bulk measures of the progression of apoptosis, such as by DNA-laddering, although undeniably valuable, would show only the overall slow progression with time. All-ornone phenomenon occurring in discrete organelles, such as the mitochondrial permeability transition (MPT), appears as a graded response when examined in bulk suspensions of cells or mitochondria. Similar arguments can be made for resolving questions of Ca 2ϩ signaling and gene expression. Ca and, unless they are synchronized (such as in the beating heart), their occurrence or nature (eg, frequency, amplitude, etc) might not be apparent from ensemble measurements. Significant information about these processes would obviously need to use a single cells studies. Currently, one of the most useful techniques to assess the activities of individual cells, subcellular trafficking of signals to and between organelles, and to appreciate how organelle function is regulated is based on fluorescence microscopy imaging. Each of the various imaging techniques requires that the signaling molecule(s), compartment(s), or organelle(s) be labeled in a specific fashion such that they can be tracked in time...

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“…Epinephrine is produced by neurosecretory cells called chromaffin cells located in the medulla (middle) of the adrenal gland. A study that employed laser microscopy and amperometry showed that chromaffin cells contain both IP 3 -and ryanodine-sensitive ER Ca 2ϩ pools; agonist coupled to IP 3 production released approximately twice the amount of Ca 2ϩ released in response to caffeine (Inoue et al, 2003). Muscarine-induced Ca 2ϩ responses lasted for 10 to 20 s, whereas caffeine-induced Ca 2ϩ responses lasted only 3 to 6 s.…”

Section: Exocrine and Endocrine Systemsmentioning

confidence: 99%