FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2000245366> ?p ?o ?g. }

• W2000245366 endingPage "22661" @default.
• W2000245366 startingPage "22654" @default.
• W2000245366 abstract "In oligodendrocyte processes, methacholine-evoked Ca2+ waves propagate via regions of specialized Ca2+ release kinetics (wave amplification sites) at which the amplitude and rate of rise of local Ca2+ signals are markedly higher than in surrounding areas (Simpson, P. B., and Russell, J. T. (1996) J. Biol. Chem. 271, 33493–33501). In the present study we have examined the effects of other phosphoinositide-coupled agonists on Ca2+ in these cells, and the structural specializations underlying regenerative wave amplification sites. Both bradykinin and norepinephrine evoke Ca2+ waves, which initiate at the same loci and propagate through the cell body and multiple processes via identical wave amplification sites. Antibodies against type 2 inositol 1,4,5-trisphosphate receptors (InsP3R2) and calreticulin identify expression of these proteins in oligodendrocyte membranes in Western blots. Immunocytochemistry followed by high resolution fluorescence microscopy revealed that both InsP3R2 and calreticulin are expressed in high intensity patches along processes. Cross-correlation analysis of the profiles of local Ca2+release kinetics during a Ca2+ wave and immunofluorescence for these proteins along cellular processes showed that the domains of high endoplasmic reticulum protein expression correspond closely to wave amplification sites. Staining cells with the mitochondrial dye, MitoTracker®, showed that mitochondria are only found in intimate association with these sites possessing high density endoplasmic reticulum proteins, and they remain in the same locations over relatively long periods of time. It appears, therefore, that multiple specializations are found at domains of elevated Ca2+ release in oligodendrocyte processes, including high levels of calreticulin, InsP3R2 Ca2+ release channels, and mitochondria. In oligodendrocyte processes, methacholine-evoked Ca2+ waves propagate via regions of specialized Ca2+ release kinetics (wave amplification sites) at which the amplitude and rate of rise of local Ca2+ signals are markedly higher than in surrounding areas (Simpson, P. B., and Russell, J. T. (1996) J. Biol. Chem. 271, 33493–33501). In the present study we have examined the effects of other phosphoinositide-coupled agonists on Ca2+ in these cells, and the structural specializations underlying regenerative wave amplification sites. Both bradykinin and norepinephrine evoke Ca2+ waves, which initiate at the same loci and propagate through the cell body and multiple processes via identical wave amplification sites. Antibodies against type 2 inositol 1,4,5-trisphosphate receptors (InsP3R2) and calreticulin identify expression of these proteins in oligodendrocyte membranes in Western blots. Immunocytochemistry followed by high resolution fluorescence microscopy revealed that both InsP3R2 and calreticulin are expressed in high intensity patches along processes. Cross-correlation analysis of the profiles of local Ca2+release kinetics during a Ca2+ wave and immunofluorescence for these proteins along cellular processes showed that the domains of high endoplasmic reticulum protein expression correspond closely to wave amplification sites. Staining cells with the mitochondrial dye, MitoTracker®, showed that mitochondria are only found in intimate association with these sites possessing high density endoplasmic reticulum proteins, and they remain in the same locations over relatively long periods of time. It appears, therefore, that multiple specializations are found at domains of elevated Ca2+ release in oligodendrocyte processes, including high levels of calreticulin, InsP3R2 Ca2+ release channels, and mitochondria. Endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; InsP3, inositol 1,4,5-trisphosphate; InsP3R, inositol 1,4,5-trisphosphate receptor; EPR, extensive photon reassignment; PBS, phosphate-buffered saline; MCh, methacholine.Ca2+-binding proteins provide a high capacity buffering mechanism which results in the lowering of [Ca2+]free in the ER, and thus a reduction in the gradient against which pumps must transport cytoplasmic Ca2+ into the store. They are also thought to be important in localizing Ca2+ to sites of release, and in modulating release activity, via protein-protein interactions with release channels (1Ikemoto N. Ronjat M. Meszaros L.G. Koshita M. Biochemistry. 1989; 28: 6764-6771Crossref PubMed Scopus (188) Google Scholar, 2Milner R.E. Famulski K.S. Michalak M. Mol. Cell. Biochem. 1992; 112: 1-13Crossref PubMed Scopus (99) Google Scholar, 3Jaffe L.F. Cell Calcium. 1993; 14: 736-745Crossref PubMed Scopus (143) Google Scholar, 4Camacho P. Lechleiter J.D. Cell. 1995; 82: 765-771Abstract Full Text PDF PubMed Scopus (200) Google Scholar). The best described of these Ca2+-binding proteins are calsequestrin and calreticulin. In many cells, calreticulin is the major calcium-binding protein of the ER lumen (2Milner R.E. Famulski K.S. Michalak M. Mol. Cell. Biochem. 1992; 112: 1-13Crossref PubMed Scopus (99) Google Scholar,5Nori A. Villa A. Podini P. Witcher D.R. Volpe P. Biochem. J. 1993; 291: 199-204Crossref PubMed Scopus (42) Google Scholar). Three subtypes of inositol 1,4,5-trisphosphate receptor (InsP3R) are now known (see Ref. 6Simpson P.B. Challiss R.A.J. Nahorski S.R. Trends Neurosci. 1995; 18: 299-306Abstract Full Text PDF PubMed Scopus (279) Google Scholar for review), which can have different modulatory properties and discrete functions even when expressed together in the same cell (7Simpson P.B. Challiss R.A.J. Nahorski S.R. J. Neurochem. 1994; 63: 2369-2372Crossref PubMed Scopus (30) Google Scholar, 8Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 9DeLisle S. Blondel O. Longo F.J. Schnabel W.E. Bell G.I. Welsh M.J. Am. J. Physiol. 1996; 270: C1255-C1261Crossref PubMed Google Scholar). Localization of calreticulin to InsP3R-containing membrane vesicles has been reported in some cell types using density gradient techniques (2Milner R.E. Famulski K.S. Michalak M. Mol. Cell. Biochem. 1992; 112: 1-13Crossref PubMed Scopus (99) Google Scholar,10Enyedi P. Szabadkai G. Krause K.-H. Lew D.P. Spat A. Cell Calcium. 1993; 14: 485-492Crossref PubMed Scopus (22) Google Scholar). The function of this coexpression, however, has remained controversial. Recent reports have indicated that calreticulin may play a role in regulating Ca2+ signals, including perhaps serving as a luminal sensor for Ca2+ store depletion (4Camacho P. Lechleiter J.D. Cell. 1995; 82: 765-771Abstract Full Text PDF PubMed Scopus (200) Google Scholar,11Bastianutto C. Clementi E. Codazzi F. Podini P. De Giorgi F. Rizzuto R. Meldolesi J. Pozzan T. J. Cell Biol. 1995; 130: 847-855Crossref PubMed Scopus (169) Google Scholar). InsP3-mediated Ca2+ waves in several cell types propagate over long distances by regenerative Ca2+ release at specialized cellular domains (12Yagodin S.V. Holtzclaw L. Sheppard C.A. Russell J.T. J. Neurobiol. 1994; 25: 265-280Crossref PubMed Scopus (65) Google Scholar, 13Yagodin S. Holtzclaw L.A. Russell J.T. Mol Cell Biochem. 1995; 149/150: 137-144Crossref Scopus (29) Google Scholar, 14Simpson P.B. Russell J.T. J. Biol. Chem. 1996; 271: 33493-33501Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 15Bootman M.D. Berridge M.J. Curr. Biol. 1996; 6: 855-865Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In glial cells, these specialized wave amplification sites are characterized by significantly higher amplitude in local Ca2+ signals and steeper rate of rise of the signals (12Yagodin S.V. Holtzclaw L. Sheppard C.A. Russell J.T. J. Neurobiol. 1994; 25: 265-280Crossref PubMed Scopus (65) Google Scholar, 14Simpson P.B. Russell J.T. J. Biol. Chem. 1996; 271: 33493-33501Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 16Roth B.J. Yagodin S.V. Holtzclaw L.A. Russell J.T. Cell Calcium. 1995; 17: 53-64Crossref PubMed Scopus (41) Google Scholar). Ca2+ waves typically travel in complex nonlinear paths through three-dimensional space (12Yagodin S.V. Holtzclaw L. Sheppard C.A. Russell J.T. J. Neurobiol. 1994; 25: 265-280Crossref PubMed Scopus (65) Google Scholar,15Bootman M.D. Berridge M.J. Curr. Biol. 1996; 6: 855-865Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 17Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6188) Google Scholar, 18Hajnoczky G. Robb-Gaspers L.D. Seitz M.B. Thomas A.P. Cell. 1995; 82: 415-424Abstract Full Text PDF PubMed Scopus (955) Google Scholar, 19Jouaville L.S. Ichas F. Holmuhamedov E.L. Camacho P. Lechleiter J.D. Nature. 1995; 377: 438-441Crossref PubMed Scopus (374) Google Scholar), making analysis of their mechanisms of propagation problematical. In recent studies, however, we have investigated Ca2+ waves in cultured cortical oligodendrocytes which because of their long, thin, relatively linear processes, allow for the analysis of Ca2+ waves as one dimensional propagatory entities. Oligodendrocytes express a variety of receptors coupled to the hydrolysis of phosphoinositides and consequent mobilization of Ca2+ from InsP3R-expressing intracellular stores. These include α1A adrenoreceptors, M1 muscarinic cholinoceptors, and bradykinin receptors (20Kastritsis C.H. McCarthy K.D. Glia. 1993; 8: 106-113Crossref PubMed Scopus (81) Google Scholar, 21Marriott D.R. Wilkin G.P. J. Neurochem. 1993; 61: 826-834Crossref PubMed Scopus (32) Google Scholar, 22Cohen R.I. Almazan G. NeuroReport. 1993; 4: 1115-1118PubMed Google Scholar, 23Cohen R.I. Almazan G. Eur. J. Neurosci. 1994; 6: 1213-1224Crossref PubMed Scopus (76) Google Scholar). We have previously demonstrated that oligodendrocytes respond to the muscarinic receptor agonist methacholine (MCh) by the induction of Ca2+ waves initiating in several distinct regions of oligodendrocyte processes. These waves travel along each process and into the cell body via multiple amplification sites (14Simpson P.B. Russell J.T. J. Biol. Chem. 1996; 271: 33493-33501Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). One or more mitochondria are closely associated with each of these specialized Ca2+ wave amplification sites in oligodendrocytes, and inhibition of mitochondrial activity markedly affects methacholine-evoked Ca2+ responses (14Simpson P.B. Russell J.T. J. Biol. Chem. 1996; 271: 33493-33501Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The present study was undertaken to investigate whether specializations in ER protein distribution as well as mitochondrial location would underlie the specialized Ca2+ release sites found during InsP3-evoked Ca2+ waves. Our results indicate that type 2 InsP3Rs (InsP3R2), and calreticulin, are expressed in much higher intensity at Ca2+ wave amplification sites along oligodendrocyte processes compared with other regions. Furthermore, in the processes stationary mitochondria were found only at these specialized Ca2+ release sites in close association with high density of ER proteins. These findings suggest that wave propagation in glia may be modulated by specialized microdomains of Ca2+release involving both mitochondria and ER proteins. (±)-Norepinephrine hydrochloride and acetyl-β-methylcholine chloride were obtained from Sigma. Bradykinin, fura 2-AM and fluo 3-AM were obtained from Research Biochemicals International. MitoTracker CMXRos was from Molecular Probes. PA3-900 was from Affinity Bioreagents Inc. AP42 was a gift from Dr. A. Sharp (Johns Hopkins University, Baltimore, MD). Oligodendrocytes were prepared from 2-day-old rat pups as described previously (14Simpson P.B. Russell J.T. J. Biol. Chem. 1996; 271: 33493-33501Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 24Patneau D.K. Wright P.W. Winters C. Mayer M.L. Gallo V. Neuron. 1994; 12: 357-371Abstract Full Text PDF PubMed Scopus (284) Google Scholar). Briefly, cortices were removed and manually dissociated, and cells cultured in plastic flasks (25McCarthy K.D. de Vellis J. J. Cell Biol. 1980; 85: 890-898Crossref PubMed Scopus (3398) Google Scholar). After 8 days in vitro, the flasks were vigorously shaken overnight. The supernatant was repeatedly plated onto plastic dishes, to which endothelial cells, microglia, and fibroblasts quickly attach. Non-adherent cells were then replated onto glass coverslips coated with 0.1 mg/ml polyornithine. Cells were cultured in DME-N1 containing 0.5% fetal bovine serum for 24 h, and thereafter in DME-N1 containing 2% fetal bovine serum, and maintained in 10% CO2, 90% air, under which conditions the bipotential cells developed into oligodendrocytes. Cells were >85% positive for the oligodendrocyte marker galactocerebroside. Culture medium was replaced every 3 days, and all cells were used 4–8 days after replating. For the study of Ca2+ wave propagation in glial cell processes, cells were incubated with 5 μm fluo 3-AM for 20 min at room temperature as described previously (12Yagodin S.V. Holtzclaw L. Sheppard C.A. Russell J.T. J. Neurobiol. 1994; 25: 265-280Crossref PubMed Scopus (65) Google Scholar, 26Fatatis A. Russell J.T. Glia. 1992; 5: 95-104Crossref PubMed Scopus (75) Google Scholar). Experiments were performed in a Leiden coverslip chamber continuously perfused with balanced salt solution. The perfusion chamber was positioned on the stage of an inverted microscope, and fluorescence images acquired at 495 nm excitation (510 nm emission) wavelength through a microchannel plate intensifier with a CCD camera (12Yagodin S.V. Holtzclaw L. Sheppard C.A. Russell J.T. J. Neurobiol. 1994; 25: 265-280Crossref PubMed Scopus (65) Google Scholar). Images were digitized and averaged (2 frames at each wavelength) in a Trapix 55/4256 image processor. Cells were divided for analysis into 0.8–2.0-μm-wide regions sequentially along the longitudinal axis of the cell (14Simpson P.B. Russell J.T. J. Biol. Chem. 1996; 271: 33493-33501Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Fluorescence intensity values in the nonzero pixels within each slice were averaged (F) and plotted as normalized fluorescence intensities (ΔF/F) against time, where ΔF was calculated as the difference between the average value of the first 20 data points prior to stimulation of the cell and F. Oligodendrocyte membranes were prepared and Western blots performed according to previously published methods (7Simpson P.B. Challiss R.A.J. Nahorski S.R. J. Neurochem. 1994; 63: 2369-2372Crossref PubMed Scopus (30) Google Scholar). Membrane proteins were separated in 7.5% SDS-polyacrylamide gels using the Phastgel system (Pharmacia Biotech Inc.). Proteins were electrophoretically transferred to nitrocellulose and blots were incubated overnight at 4 °C in Tris-buffered saline-Tween 20 containing 5% nonfat dried milk, followed by incubation in primary antibodies (1:1000 dilution). After incubation in peroxidase-coupled secondary antibodies (Amersham Corp.), blots were developed using enhanced chemiluminescence reagents. Rabbit polyclonal antibody AP42, raised against a peptide sequence corresponding to the C-terminal region of mouse InsP3R subtype 2 (GSNTPHENHHMPPH) (27Ross C.A. Danoff S.K. Schell M.J. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4265-4269Crossref PubMed Scopus (220) Google Scholar), was a gift from Dr. A. Sharp. This sequence is unconserved, i.e.it is not present in the other known subtypes of InsP3R or in any other known oligodendrocyte protein. PA3-900, a rabbit polyclonal antibody raised against recombinant human calreticulin produced in the baculovirus insect cell system (28Stendahl O. Krause K.H. Krischer J. Jerstrom P. Theler J.M. Clark R.A. Carpentier J.L. Lew D.P. Science. 1994; 265: 1439-1441Crossref PubMed Scopus (127) Google Scholar), was from Affinity Bioreagents. Immunocytochemistry on either naive cells or cells in which Ca2+ waves were first measured was performed as described previously (14Simpson P.B. Russell J.T. J. Biol. Chem. 1996; 271: 33493-33501Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Cy3- or fluorescein isothiocyanate-labeled anti-rabbit secondary antibodies (Jackson Immunocytochemical Laboratories) were used as appropriate. Briefly, cells were fixed in 100% methanol at −20 °C for 3 min, washed three times in phosphate-buffered saline (PBS) (pH 7.1), then incubated in primary antibody (1:200 for anti-InsP3R antibodies, 1:300 for anti-calreticulin) overnight at 4 °C. After further PBS washes, cell were incubated for 1–2 h in secondary antibody (1:400 dilution in 10% goat serum). Cells were then washed three times, and the coverslips were mounted on a glass microscope slide using Mowiol. Controls consisted of substitution of the primary antibody with normal serum from the same host species, used at a dilution equal to that of the primary antibody, and consistently showed negligible fluorescence levels. For the study of mitochondrial distribution, living cells were incubated with MitoTracker Red CMXRos (500 nm, 30 min, Molecular Probes) at 37 °C, then washed in prewarmed PBS. Cells were then imaged using a Cy3 filter set in a fluorescence microscope. To examine the spatial relationship between mitochondria and ER markers, MitoTracker-loaded cells were fixed in 2% paraformaldehyde (4 min, 4 °C) and 100% methanol (−20 °C, 3 min), washed, and developed for immunocytochemistry as above. Use of fluorescein isothiocyanate-labeled secondary antibodies enabled good resolution and separation of antibody fluorescence from that of MitoTracker. Cells previously subjected to [Ca2+] i measurements were processed for immunocytochemistry or organelle labeling on the microscope stage following the same procedures described for naive cells. For comparison of immunofluorescence or mitochondrial staining with Ca2+ release kinetics, cells were imaged in the Ca2+ imaging system using a Cy3 filter set (Chroma Technologies, Inc., Brattleboro, VT). For analysis of subcellular immunofluorescence with high resolution, a digital confocal microscopy technique was employed (14Simpson P.B. Russell J.T. J. Biol. Chem. 1996; 271: 33493-33501Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). For this, cells were imaged with a cooled CCD camera (Photometrics, Inc., Tucson, AZ) using the Cellscan software environment (Scanalytics, Inc., Billerica, MA). The Cellscan environment allows for acquisition of wide angle fluorescence microscope images at all the focal planes through cells (z-series). Images are then restored using a deconvolution procedure (extensive photon reassignment protocol, EPR) into confocal images by removing out-of-focus light. The software system is based on the algorithm developed by Dr. Fay and co-workers (14Simpson P.B. Russell J.T. J. Biol. Chem. 1996; 271: 33493-33501Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 29Carrington W.A. Fogarty K.E. Fay F.S. Foster K. Non-invasive Techniques in Cell Biology. Wiley-Liss, New York1990: 53-72Google Scholar). The algorithm used the point-spread function of the microscope obtained by acquiring a z-series images of a subresolution polystyrene bead 200 nm in diameter filled with fluorophore. This data set was used by the algorithm to reassign out-of-focus plane light, which causes blurring of confocal images (29Carrington W.A. Fogarty K.E. Fay F.S. Foster K. Non-invasive Techniques in Cell Biology. Wiley-Liss, New York1990: 53-72Google Scholar). Under our measurement conditions, thez-resolution by our optics was 0.48 μm (measured as full width at half-maximum intensity). The spatial patterns of local Ca2+ release kinetics measured in 0.83-μm-wide subregions of oligodendrocyte processes and of mitochondrial distribution measured by fluorescently tagging mitochondria in the same cells were compared using a cross-correlation function as a quantitative test for similarity (14Simpson P.B. Russell J.T. J. Biol. Chem. 1996; 271: 33493-33501Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 30Press W.H. Flannery B.P. Teukolsky S.A. Vetterling W.T. Numerical Recipes in Pascal. Cambridge University Press, Cambridge1989: 120-129Google Scholar). Cross-correlation function is derived from the fast Fourier transform of the two data sets being compared. For this analysis the mean values of the amplitudes in the patterns were subtracted out and the resulting zero mean waves were embedded in surrounding zeros, to eliminate circular correlations, i.e.between the beginning and end of the data sets, due to the periodic nature of Fourier series (30Press W.H. Flannery B.P. Teukolsky S.A. Vetterling W.T. Numerical Recipes in Pascal. Cambridge University Press, Cambridge1989: 120-129Google Scholar). The data were then analyzed via a Fast Fourier Transform algorithm, using standard functions in Mathematica (Wolfram Research Inc.). Cross-spectra were formed as a product of one data set with the complex conjugate transform of a second data set. The cross-correlation function was produced by inverse Fourier transformation of the cross-spectrum. Performing these operations on a single data set produced the power density spectrum and the auto-correlation function. Results are presented as mean ± S.D. The validity of this quantitative analysis technique was evaluated in two different control experiments. In one, we compared synthesized noisy sine waves of identical frequency but slightly out of phase with each other (Fig. 4 A, bottom traces). The cross-correlation of these waveforms (Fig. 4 B, solid circles) is a wave pattern of the same frequency as the original waves, with peak correlation shifted from phase to a position equivalent to the delay between the two signals. The noise experimentally inserted into the two sine waves (see figure legend) has minimal effect on the outcome of the cross-correlation. In the second control experiment, a real data set was compared with a scrambled data set (Fig. 4 A, top traces) to determine if chance alone would cause high cross-correlation. The method used was a random permutation in the order of the data points, generated by the program Random Permutation in the Discrete package of Mathematica. By this method non-correlational statistics (mean, variance, and all other parameters of the distribution) were kept constant, but spatial correlations were deleted via a random permutation in the order of the data points. The bottom patterns in Fig. 4 A are equivalent to the data presented in Fig. 4 C (see below), except that one of the patterns has been scrambled with respect to position. The resultant cross-correlation (Fig. 4 B,open circles) is a somewhat noisy, relatively flat function that does not greatly deviate from zero correlation at any delay, unlike the cross-correlation function of the original data sets shown in Fig. 4 D. These results are consistent with cross-correlation analysis conservatively detecting genuine but not coincidental correlations within related data sets. Initial experiments characterized Ca2+ waves in oligodendrocytes evoked by activation of different phosphoinositide-coupled receptors. Oligodendrocytes stimulated with norepinephrine (200 nm) or bradykinin (200 μm) responded with propagating Ca2+ wavefronts. High resolution spatiotemporal analysis revealed that these waves initiated at discrete cellular regions and propagated along the processes and the cell body. In Fig.1 (A and B), offset plots show typical time course of the local changes in [Ca2+] observed in successive 2.0-μm-wide sections of the same cell receiving the two stimuli. Ca2+ waves initiated at four distinct sites where the wave reaches 50% of maximum amplitude sooner than surrounding regions. These wave initiation sites (marked with asterisks) are identifiable as the local “minima” in Fig. 1 C. Three are located in cellular processes and one in the cell body. In contrast wave amplification occurs at other more numerous loci. At these wave amplification sites (marked with arrows), the magnitude and rate of Ca2+ rise were substantially higher than in surrounding regions (Fig. 1, D and E). We have previously described similar distinct initiation and propagation domains in oligodendrocytes responding to methacholine (14Simpson P.B. Russell J.T. J. Biol. Chem. 1996; 271: 33493-33501Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) and in astrocytes responding to norepinephrine (12Yagodin S.V. Holtzclaw L. Sheppard C.A. Russell J.T. J. Neurobiol. 1994; 25: 265-280Crossref PubMed Scopus (65) Google Scholar, 13Yagodin S. Holtzclaw L.A. Russell J.T. Mol Cell Biochem. 1995; 149/150: 137-144Crossref Scopus (29) Google Scholar). Applying the cross-correlation analysis also used in the previous work, we have now examined the distribution of sites associated with responses to norepinephrine and bradykinin. Local Ca2+ peak amplitudes had a maximal cross-correlation coefficient of 0.81 at 0.0 μm from phase. Similarly, the comparison between the half-rise time and the rates of Ca2+ rise for norepinephrine and bradykinin responses gave peak coefficients of 0.79 and 0.75 at 2.0 μm from phase. Such analysis also confirms that wave initiation and amplification sites are indeed distinct. For example, comparison of half-rise time for bradykinin with norepinephrine local Ca2+ peak amplitudes had a maximal coefficient of 0.08 at 0.0 μm. Other control analyses (see “Experimental Procedures”) established that this analytical protocol does not readily identify false positive relationships. The expression of InsP3R2 and calreticulin in oligodendrocytes was investigated using Western blotting and immunocytochemical analyses. The antibody AP42, raised against an unique sequence in the C-terminal region of InsP3R2, detected a single band of approximately 250 kDa in oligodendrocyte membranes (Fig.2 A), consistent with the expected size of this protein from previous reports (8Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 27Ross C.A. Danoff S.K. Schell M.J. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4265-4269Crossref PubMed Scopus (220) Google Scholar). Previous experiments clearly showed that this antibody does not cross-react with either InsP3R1 or InsP3R3. Antibody PA3-900, raised against recombinant human calreticulin, reacted against a single band at approximately 60 kDa (Fig. 2 B) (28Stendahl O. Krause K.H. Krischer J. Jerstrom P. Theler J.M. Clark R.A. Carpentier J.L. Lew D.P. Science. 1994; 265: 1439-1441Crossref PubMed Scopus (127) Google Scholar), indicating the expression of calreticulin in or associated with oligodendrocyte membranes. These antibodies were then used to investigate the distribution of InsP3R2 and calreticulin in oligodendrocytes using standard immunocytochemical techniques and high resolution fluorescence microscopy (see “Experimental Procedures”) (Fig. 3). InsP3R2 immunofluorescence (Fig. 3 A) was found distributed in a variegated manner throughout the cell body, except the nucleus, and along the length of the cell processes. Similarly, calreticulin immunofluorescence was also found in the cell body and in a punctate pattern along oligodendrocyte processes (Fig. 3 B). The size of these clusters of immunofluorescence varied between different cells, being typically longer and more graded in thick processes, but small and highly punctate in thin processes (see also Fig. 5).Figure 3Distribution of InsP3R2, calreticulin, and mitochondria in oligodendrocytes. A andB, digital images of oligodendrocytes reacted with AP42 (A) and PA3-900 (B). Image in A is a single plane digital confocal image through the nucleus of a cell (see “Experimental Procedures”). While the nucleus shows no staining with AP42, InsP3R2 immunofluorescence is detectable in most of the cell body and in a variegated pattern along the processes. The image in B is a single digital confocal image from another cell incubated with the anti-calreticulin antibody, PA3-900. This optical section is from a region just above the nucleus of the cell. Note that calreticulin immunofluorescence also extends throughout the cell and, in processes, the staining appears in patches. Like InsP3R2, calreticulin immunofluorescence also does not extend into the nucleus in other optical sections (data not shown).Scale bars correspond to 30 μm in A andB. C, time-lapse images of mitochondria in an oligodendrocyte process. Images shown were acquired at 10-s intervals for 15 min. Note that mitochondria are stationary during the first 130 s of this experiment. No movement was observed over the rest of the 15 min acquisition period (data not shown). Rapid image acquisition at 2-s intervals in other experiments (n = 5) revealed no rapid mitochondrial motion (data not shown).Vertical scale bar in the first panel corresponds to 15 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Comparison of MitoTracker staining with calreticulin immunofluorescence in an oligodendrocyte process.Live cells were incubated with 500 nm MitoTracker in PBS for 30 min at 37 °C, fixed, and processed for immunocytochemistry as described under “Experimental Procedures.” 42 serial optical sections (every 0.09 μm in the z-dimension) obtained by digital EPR restoration were rendered into a three-dimensional voxel as shown in A and B. Scale bar = 20 μm. A, an oligodendrocyte process reconstructed from az-series through the cell is depicted, labeled with PA3-900 to detect localization of calreticulin (green). Calreticulin immunofluorescence in this process is predominantly concentrated in multiple high intensity patches. Mitochondria labeled wi" @default.
• W2000245366 created "2016-06-24" @default.
• W2000245366 creator A5031890627 @default.
• W2000245366 creator A5041145233 @default.
• W2000245366 creator A5041313514 @default.
• W2000245366 creator A5074277478 @default.
• W2000245366 date "1997-09-01" @default.
• W2000245366 modified "2023-10-16" @default.
• W2000245366 title "High Density Distribution of Endoplasmic Reticulum Proteins and Mitochondria at Specialized Ca2+ Release Sites in Oligodendrocyte Processes" @default.
• W2000245366 cites W1498235130 @default.
• W2000245366 cites W1592841312 @default.
• W2000245366 cites W1817999602 @default.
• W2000245366 cites W1963791094 @default.
• W2000245366 cites W1975551314 @default.
• W2000245366 cites W1976746355 @default.
• W2000245366 cites W1980166477 @default.
• W2000245366 cites W1980235848 @default.
• W2000245366 cites W1980756989 @default.
• W2000245366 cites W1992347253 @default.
• W2000245366 cites W1992482300 @default.
• W2000245366 cites W1996426136 @default.
• W2000245366 cites W2000363488 @default.
• W2000245366 cites W2000626734 @default.
• W2000245366 cites W2001574683 @default.
• W2000245366 cites W2015426747 @default.
• W2000245366 cites W2015967728 @default.
• W2000245366 cites W2018702981 @default.
• W2000245366 cites W2035058927 @default.
• W2000245366 cites W2036031534 @default.
• W2000245366 cites W2038404316 @default.
• W2000245366 cites W2052351195 @default.
• W2000245366 cites W2057665893 @default.
• W2000245366 cites W2058759144 @default.
• W2000245366 cites W2059994807 @default.
• W2000245366 cites W2061115935 @default.
• W2000245366 cites W2067254948 @default.
• W2000245366 cites W2073706613 @default.
• W2000245366 cites W2076502472 @default.
• W2000245366 cites W2078844809 @default.
• W2000245366 cites W2085741168 @default.
• W2000245366 cites W2103299031 @default.
• W2000245366 cites W2152691057 @default.
• W2000245366 cites W2153609006 @default.
• W2000245366 cites W2189607915 @default.
• W2000245366 cites W2397904797 @default.
• W2000245366 cites W4235927881 @default.
• W2000245366 doi "https://doi.org/10.1074/jbc.272.36.22654" @default.
• W2000245366 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9278423" @default.
• W2000245366 hasPublicationYear "1997" @default.
• W2000245366 type Work @default.
• W2000245366 sameAs 2000245366 @default.
• W2000245366 citedByCount "162" @default.
• W2000245366 countsByYear W20002453662012 @default.
• W2000245366 countsByYear W20002453662013 @default.
• W2000245366 countsByYear W20002453662014 @default.
• W2000245366 countsByYear W20002453662015 @default.
• W2000245366 countsByYear W20002453662016 @default.
• W2000245366 countsByYear W20002453662017 @default.
• W2000245366 countsByYear W20002453662018 @default.
• W2000245366 countsByYear W20002453662019 @default.
• W2000245366 countsByYear W20002453662020 @default.
• W2000245366 countsByYear W20002453662021 @default.
• W2000245366 countsByYear W20002453662022 @default.
• W2000245366 countsByYear W20002453662023 @default.
• W2000245366 crossrefType "journal-article" @default.
• W2000245366 hasAuthorship W2000245366A5031890627 @default.
• W2000245366 hasAuthorship W2000245366A5041145233 @default.
• W2000245366 hasAuthorship W2000245366A5041313514 @default.
• W2000245366 hasAuthorship W2000245366A5074277478 @default.
• W2000245366 hasBestOaLocation W20002453661 @default.
• W2000245366 hasConcept C110121322 @default.
• W2000245366 hasConcept C134306372 @default.
• W2000245366 hasConcept C158617107 @default.
• W2000245366 hasConcept C169760540 @default.
• W2000245366 hasConcept C185592680 @default.
• W2000245366 hasConcept C2776985911 @default.
• W2000245366 hasConcept C2778609137 @default.
• W2000245366 hasConcept C28859421 @default.
• W2000245366 hasConcept C33923547 @default.
• W2000245366 hasConcept C529278444 @default.
• W2000245366 hasConcept C55493867 @default.
• W2000245366 hasConcept C86803240 @default.
• W2000245366 hasConcept C95444343 @default.
• W2000245366 hasConceptScore W2000245366C110121322 @default.
• W2000245366 hasConceptScore W2000245366C134306372 @default.
• W2000245366 hasConceptScore W2000245366C158617107 @default.
• W2000245366 hasConceptScore W2000245366C169760540 @default.
• W2000245366 hasConceptScore W2000245366C185592680 @default.
• W2000245366 hasConceptScore W2000245366C2776985911 @default.
• W2000245366 hasConceptScore W2000245366C2778609137 @default.
• W2000245366 hasConceptScore W2000245366C28859421 @default.
• W2000245366 hasConceptScore W2000245366C33923547 @default.
• W2000245366 hasConceptScore W2000245366C529278444 @default.
• W2000245366 hasConceptScore W2000245366C55493867 @default.
• W2000245366 hasConceptScore W2000245366C86803240 @default.
• W2000245366 hasConceptScore W2000245366C95444343 @default.
• W2000245366 hasIssue "36" @default.
• W2000245366 hasLocation W20002453661 @default.

• next