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• W2000551229 abstract "Using an antisense strategy, we have previously shown that in vascular myocytes, subtypes 1 and 2 of ryanodine receptors (RYRs) are required for Ca2+ release during Ca2+ sparks and global Ca2+ responses, evoked by activation of voltage-gated Ca2+ channels, whereas RYR subtype 3 (RYR3) has no contribution. Here, we investigated the effects of increased Ca2+ loading of the sarcoplasmic reticulum (SR) on the RYR-mediated Ca2+ responses and the role of the RYR3 by injecting antisense oligonucleotides targeting the RYR subtypes. RYR3 expression was demonstrated by immunodetection in both freshly dissociated and cultured rat portal vein myocytes. Confocal Ca2+ measurements revealed that the number of cells showing spontaneous Ca2+ sparks was strongly increased by superfusing the vascular myocytes in 10 mmCa2+-containing solution. These Ca2+ sparks were blocked after inhibition of RYR1 or RYR2 by treatment with antisense oligolucleotides but not after inhibition of RYR3. In contrast, inhibition of RYR3 reduced the global Ca2+responses induced by caffeine and phenylephrine, indicating that RYR3 participated together with RYR1 and RYR2 to these Ca2+responses in Ca2+-overloaded myocytes. Ca2+transients evoked by photolysis of caged Ca2+ with increasing flash intensities were also reduced after inhibition of RYR3 and revealed that the [Ca2+]i sensitivity of RYR3 would be similar to that of RYR1 and RYR2. Our results show that, under conditions of increased SR Ca2+ loading, the RYR3 becomes activable by caffeine and local increases in [Ca2+]i. Using an antisense strategy, we have previously shown that in vascular myocytes, subtypes 1 and 2 of ryanodine receptors (RYRs) are required for Ca2+ release during Ca2+ sparks and global Ca2+ responses, evoked by activation of voltage-gated Ca2+ channels, whereas RYR subtype 3 (RYR3) has no contribution. Here, we investigated the effects of increased Ca2+ loading of the sarcoplasmic reticulum (SR) on the RYR-mediated Ca2+ responses and the role of the RYR3 by injecting antisense oligonucleotides targeting the RYR subtypes. RYR3 expression was demonstrated by immunodetection in both freshly dissociated and cultured rat portal vein myocytes. Confocal Ca2+ measurements revealed that the number of cells showing spontaneous Ca2+ sparks was strongly increased by superfusing the vascular myocytes in 10 mmCa2+-containing solution. These Ca2+ sparks were blocked after inhibition of RYR1 or RYR2 by treatment with antisense oligolucleotides but not after inhibition of RYR3. In contrast, inhibition of RYR3 reduced the global Ca2+responses induced by caffeine and phenylephrine, indicating that RYR3 participated together with RYR1 and RYR2 to these Ca2+responses in Ca2+-overloaded myocytes. Ca2+transients evoked by photolysis of caged Ca2+ with increasing flash intensities were also reduced after inhibition of RYR3 and revealed that the [Ca2+]i sensitivity of RYR3 would be similar to that of RYR1 and RYR2. Our results show that, under conditions of increased SR Ca2+ loading, the RYR3 becomes activable by caffeine and local increases in [Ca2+]i. ryanodine receptor sarcoplasmic reticulum AM, 1-(4,5-dimethoxy-2-nitrophenyl)-EDTA, tetra(acetoxymethylester) fluo 3- acetoxymethylester Since the description of a Ca2+-induced Ca2+ release mechanism in skeletal muscle (1Endo M. Tanaka M. Ogawa Y. Nature. 1970; 228: 34-36Crossref PubMed Scopus (577) Google Scholar), the function of ryanodine receptor channels (RYRs)1 have been widely studied in both skeletal and cardiac muscles (2Fleischer S. Inui M. Annu. Rev. Biophys. Biophys. Chem. 1989; 18: 333-364Crossref PubMed Scopus (445) Google Scholar, 3Rios E. Pizarro G. Physiol. Rev. 1991; 71: 849-908Crossref PubMed Scopus (499) Google Scholar, 4Cheng H. Lederer W.J. Cannell M.B. Science. 1993; 262: 740-744Crossref PubMed Scopus (1623) Google Scholar, 5Tsugorka A. Rios E. Blatter L.A. Science. 1995; 269: 1723-1726Crossref PubMed Scopus (252) Google Scholar, 6Berridge M.J. J. Physiol. ( Lond. ). 1997; 499: 291-306Crossref PubMed Scopus (918) Google Scholar, 7Niggli E. Annu. Rev. Physiol. 1999; 61: 311-335Crossref PubMed Scopus (113) Google Scholar) and more recently in smooth muscle (8Nelson M.T. Cheng H. Rubart M. Santana M.F. Bonev A.D. Knot H.J. Lederer W.J. Science. 1995; 270: 633-637Crossref PubMed Scopus (1208) Google Scholar, 9Arnaudeau S. Macrez-Leprêtre N. Mironneau J. Biochem. Biophys. Res. Commun. 1996; 222: 809-815Crossref PubMed Scopus (56) Google Scholar). After the cloning and sequencing of three genes encoding different RYR subtypes, the localization and role of each RYR subtype in Ca2+ signaling have begun to be studied. Of the three RYRs, RYR subtype 3 (RYR3) is the most widely expressed (10Hakamata Y. Nakai J. Takeshima H. Imoto K. FEBS Lett. 1992; 312: 229-235Crossref PubMed Scopus (353) Google Scholar), whereas RYR1 and RYR2 are mainly found in skeletal and cardiac muscles, respectively (11Takeshima H. Iino M. Takekura H. Nishi M. Kuno J. Minowa O. Takano H. Noda T. Nature. 1994; 369: 556-559Crossref PubMed Scopus (326) Google Scholar, 12Takeshima H. Komazaki S. Hirose K. Nishi M. Noda T. Iino M. EMBO J. 1998; 17: 3309-3316Crossref PubMed Scopus (187) Google Scholar). However, in arterial and venous smooth muscles, the three RYR subtypes have been identified (13Neylon C.B. Richards S.M. Larsen M.A. Agrotis A. Bobik A. Biochem. Biophys. Res. Commun. 1995; 215: 814-821Crossref PubMed Scopus (93) Google Scholar, 14Coussin F. Macrez N. Morel J.L. Mironneau J. J. Biol. Chem. 2000; 275: 9596-9603Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), but their role in Ca2+ release is still unclear. The regulation of the different RYR subtypes has been extensively studied using single channel recordings in lipid bilayers. The single channel properties of RYR subtypes are rather similar, i.e.they form a large conductance channel permeable to monovalent and divalent cations, which can be activated by ATP, caffeine, and submicromolar concentrations of Ca2+; inhibited by Mg2+, ruthenium red, and millimolar concentrations of Ca2+; and modulated by ryanodine (15Chu A. Fill M. Stefani E. Entman M.L. J. Membr. Biol. 1993; 135: 49-59Crossref PubMed Scopus (93) Google Scholar, 16Yamazawa T. Takeshima H. Sakurai T. Endo M. Iino M. EMBO J. 1996; 15: 6172-6177Crossref PubMed Scopus (52) Google Scholar, 17Murayama T. Oba T. Katayama E. Oyamada H. Oguchi K. Kobayashi M. Otsuka K. Ogawa Y. J. Biol. Chem. 1999; 274: 17297-17308Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). However, differences in responses to cyclic ADP-ribose and caffeine as well as in sensitivities to Ca2+ activation and Ca2+inactivation have been reported between RYR1 and RYR3 and between RYR3s cloned from skeletal and smooth muscles (17Murayama T. Oba T. Katayama E. Oyamada H. Oguchi K. Kobayashi M. Otsuka K. Ogawa Y. J. Biol. Chem. 1999; 274: 17297-17308Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 18Chen S.R.W. Li X. Ebisawa K. Zhang L. J. Biol. Chem. 1997; 272: 24234-24246Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 19Sonnleitner A. Conti A. Bertocchini F. Schindler H. Sorrentino V. EMBO J. 1998; 17: 2790-2798Crossref PubMed Scopus (100) Google Scholar, 20Jeyakumar L.H. Copello J.A. O'Malley A.M. Wu G.M. Grassucci R. Wagenknecht T. Fleischer S. J. Biol. Chem. 1998; 273: 16011-16020Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). It has been proposed that these differences may result from the expression of different splice variants of RYR3 (21Miyatake R. Furukawa A. Matsushita M. Iwahashi K. Nakamura K. Ichikawa Y. Suwaki H. FEBS Lett. 1996; 395: 123-126Crossref PubMed Scopus (33) Google Scholar). The physiological contribution of the different RYR subtypes to Ca2+ signaling has been addressed first by using either RYR1 and RYR2 knockout mice (12Takeshima H. Komazaki S. Hirose K. Nishi M. Noda T. Iino M. EMBO J. 1998; 17: 3309-3316Crossref PubMed Scopus (187) Google Scholar, 22Takeshima H. Yamazawa T. Ikemoto T. Takekura H. Nishi M. Noda T. Iino M. EMBO J. 1995; 14: 2999-3006Crossref PubMed Scopus (129) Google Scholar) or RYR3 knockout mice (23Takeshima H. Ikemoto T. Nishi M. Nishiyama N. Shimuta M. Sugitani Y. Kuno J. Saito I. Saito H. Endo M. Iino M. Noda T. J. Biol. Chem. 1996; 271: 19649-19652Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 24Bertocchini F. Ovitt C.E. Conti A. Barone V. Scholer H.R. Bottinelli R. Reggiani C. Sorrentino V. EMBO J. 1997; 16: 6956-6963Crossref PubMed Scopus (123) Google Scholar, 25Conklin M.W. Barone V. Sorrentino V. Coronado R. Biophys. J. 1999; 77: 1394-1403Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). In RYR1-null myotubes in culture, the Ca2+ release from the sarcoplasmic reticulum (SR) in response to increases in cytosolic Ca2+ concentration ([Ca2+]i) or caffeine is strongly reduced, but a similar decrease in caffeine sensitivity is also observed in RYR3-null neonatal myocytes, suggesting a possible co-contribution of each RYR subtype to Ca2+signaling, at least at some stages of myogenesis. Accordingly, it has been recently proposed that, in embryonic skeletal muscle, both RYR1 and RYR3 may co-contribute to Ca2+ release during Ca2+ sparks (25Conklin M.W. Barone V. Sorrentino V. Coronado R. Biophys. J. 1999; 77: 1394-1403Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). In addition, Ca2+ sparks produced independently in RYR1- or RYR3-null cells reveal similar spatio-temporal parameters (26Conklin M.W. Ahern C.A. Vallejo P. Sorrentino V. Takeshima H. Coronado R. Biophys. J. 2000; 78: 1777-17785Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Another approach using antisense oligonucleotides, that specifically targeted each one of the RYR subtypes, has been used to determine which RYR subtypes are responsible for Ca2+ sparks and global Ca2+ responses in smooth muscle cells (14Coussin F. Macrez N. Morel J.L. Mironneau J. J. Biol. Chem. 2000; 275: 9596-9603Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). It appears that both RYR1 and RYR2 are required for Ca2+ release during Ca2+ sparks and Ca2+ waves induced by activation of L-type Ca2+ currents and that RYR3 does not contribute to these Ca2+ signals. In smooth muscle cells, Ca2+ sparks are observed spontaneously or in response to Ca2+ influx through L-type Ca2+ channels (8Nelson M.T. Cheng H. Rubart M. Santana M.F. Bonev A.D. Knot H.J. Lederer W.J. Science. 1995; 270: 633-637Crossref PubMed Scopus (1208) Google Scholar, 9Arnaudeau S. Macrez-Leprêtre N. Mironneau J. Biochem. Biophys. Res. Commun. 1996; 222: 809-815Crossref PubMed Scopus (56) Google Scholar, 27Mironneau J. Arnaudeau S. Macrez-Leprêtre N. Boittin F.X. Cell Calcium. 1996; 20: 153-160Crossref PubMed Scopus (118) Google Scholar, 28Arnaudeau S. Boittin F.X. Macrez N. Lavie J.L. Mironneau C. Mironneau J. Cell Calcium. 1997; 22: 399-411Crossref PubMed Scopus (38) Google Scholar), and their localization corresponds to coupling areas between the plasma membrane and the SR (28Arnaudeau S. Boittin F.X. Macrez N. Lavie J.L. Mironneau C. Mironneau J. Cell Calcium. 1997; 22: 399-411Crossref PubMed Scopus (38) Google Scholar, 29Lesh R.E. Nixon G.F. Fleischer S. Airey J.A. Somlyo A.P. Somlyo A.V. Circ. Res. 1998; 82: 175-185Crossref PubMed Scopus (80) Google Scholar). In rat portal vein myocytes, spatial and temporal recruitment of Ca2+ sparks results in propagating Ca2+waves, which trigger cell contraction (30Boittin F.X. Macrez N. Halet G. Mironneau J. Am. J. Physiol. 1999; 277: C139-C151Crossref PubMed Google Scholar). In the present study, we investigated the effects of elevating the extracellular Ca2+ concentration ([Ca2+]o) on both Ca2+ sparks and global Ca2+ responses induced by Ca2+, caffeine, and phenylephrine. Under conditions of increased SR Ca2+ loading, we provide the first evidence that the RYR3, which becomes activable by caffeine and localized increases in [Ca2+]i, is responsible for the increased global Ca2+ responses. We also found that spontaneous Ca2+ sparks are highly abundant but remain dependent on activation of only RYR1 and RYR2. Rats (160–180 g) were killed by cervical dislocation. The portal vein was cut into several pieces and incubated for 10 min in low Ca2+ (40 μm) physiological solution, and then 0.8 mg/ml collagenase (EC 3.4.24.3), 0.20 mg/ml Pronase E (EC 3.4.24.31), and 1 mg/ml bovine serum albumin were added at 37 °C for 20 min. After this time, the solution was removed, and pieces of portal vein were incubated again in a fresh enzyme solution at 37 °C for 20 min. Tissues were placed in a enzyme-free solution and triturated using fire-polished Pasteur pipette to release cells. Cells were seeded at a density of 103 cells/mm2on glass slides imprinted with squares for localization of injected cells. Cells were maintained in short term primary culture in medium M199 containing 2% fetal calf serum, 2 mm glutamine, 1 mm pyruvate, 20 units/ml penicillin, and 20 μg/ml streptomycin; they were kept in an incubator gassed with 95% air and 5% CO2 at 37 °C. The myocytes were cultured in this medium for 4 days. The normal physiological solution contained 130 mm NaCl, 5.6 mm KCl, 1 mmMgCl2, 1.7 mm CaCl2, 11 mm glucose, and 10 mm HEPES (pH 7.4, with NaOH). Phosphorothioate antisense oligonucleotides (denoted with the prefix “as”) used in the present study were designed on the known cloned RYR sequences deposited in the GenBank™ sequence data base with Lasergene software (DNASTAR, Madison, WI). Sequences of all three RYR cDNAs were aligned with each other, and specific antisense oligonucleotide sequences were chosen in region of the cDNA of interest, completely different from the sequences of the two other RYR subtypes. Then antisense and scrambled sequences displaying putative binding to any other mammalian sequences deposited in GenBank™ were discarded. Oligonucleotides were injected into the nuclei of myocytes by a manual injection system (Eppendorf, Hamburg, Germany). Intranuclear oligonucleotide injection with Femtotips II (Eppendorf) was performed as previously described (14Coussin F. Macrez N. Morel J.L. Mironneau J. J. Biol. Chem. 2000; 275: 9596-9603Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The myocytes were then cultured for 3–4 days in culture medium, and the glass slides were transferred into the perfusion chamber for physiological experiments. The sequences of as1RYR1 and as2RYR1 are AGCGTGTGCAGCAGGCTCA and GCAATCCGCTCCCGCCCA, corresponding to nucleotides 325–343 and 584–601, respectively, of RYR1 cDNA deposited in GenBankTM (accession numberX83932); those of as1RYR2 and as2RYR2 are GTGTCCTCACAGAAGTT and TGAAATCTAGTGCAGCCT, corresponding to nucleotides 137–153 and 1587–1604, respectively, of RYR2 cDNA (accession number X83933); and those of as1RYR3 and as2RYR3 are AAGTCAAGGGCATTTTTG and ACTTAGCCATGACACCAG, corresponding to nucleotides 502–519 and 557–574, respectively, of RYR3 cDNA (accession number X83934). In some control experiments, myocytes were injected with the following scrambled oligonucleotides: CACGCCTACGCACCTCCG, corresponding to a scrambled sequence of as2RYR1 (nucleotides 584–601 of RYR1 cDNA); AGTCGTACATGACTCGTA, corresponding to a scrambled sequence of as2RYR2 (nucleotides 1587–1604 of RYR2 cDNA); and CAGCACTATCAGTACGAC, corresponding to a scrambled sequence of as2RYR3 (nucleotides 557–574 of RYR3 cDNA). In most experiments, cells were loaded by incubation in physiological solution containing 4 μm fluo 3-acetoxymethylester (fluo 3-AM) for 1 h at room temperature. These cells were washed and allowed to cleave the dye to the active fluo 3 compound for at least 30 min. Images were acquired using the line scan mode of a confocal Bio-Rad MRC1000 microscope connected to a Nikon Diaphot microscope. Excitation light was delivered by a 25-milliwatt argon ion laser (Ion Laser Technology, Salt Lake City, UT) through a Nikon Plan Apo × 60, 1.4 NA objective lens. Fluo 3 was excited at 488 nm, and emitted fluorescence was filtered and measured at 540 ± 30 nm. At the setting used to detect fluo 3 fluorescence, the resolution of the microscope was near 0.4 × 0.4 × 1.5 μm (x,y, and z axis). Images were acquired in the line scan mode at a rate of 6 ms/scan. Scanned lines were plotted vertically, and each line was added to the right of the preceding line to form the line scan image. In these images, time increased from the left to the right, and position along the scanned line was given by vertical displacement. Fluorescence signals are expressed as pixel per pixel fluorescence ratios (F/Fo), whereF is the fluorescence during a response andFo is the rest level fluorescence of the same pixel. Image processing and analysis were performed by using COMOS, TCSM, and MPL 1000 software (Bio-Rad). In other experiments, cells were loaded by incubation in physiological solution containing 1 μm indo 1-AM for 30 min. [Ca2+]i measurements were estimated from the 405-/480-nm fluorescence ratio, as previously reported (31Morel J.L. Macrez-Leprêtre N. Mironneau J. Br. J. Pharmacol. 1996; 118: 73-78Crossref PubMed Scopus (44) Google Scholar). The minimum and maximum fluorescence (Rmin andRmax, respectively) values were determinedin vivo, in the absence of Ca2+ and at saturating Ca2+, in cells superfused in 1.7 and 10 mm [Ca2+]o. Caffeine and phenylephrine were applied by pressure ejection from a glass pipette for the period indicated on the records. All experiments were carried out at 26 ± 1 °C. Caged Ca2+, 1-(4,5-dimethoxy-2-nitrophenyl)-EDTA, tetra(acetoxymethylester) (DMNP-EDTA, AM) at 15 μm, was added to the bathing solution and maintained in the presence of cells for 1 h in an incubator at 37 °C. Photolysis was produced by a 1-ms pulse from a xenon flash lamp (Hi-Tech Scientific, Salisbury, United Kingdom) focused to a ∼2-mm diameter spot around the cell. Light was band pass-filtered with a UG11 glass between 300 and 350 mm. Flash intensity could be adjusted by varying the capacitor-charging voltage between 0 and 380 V, which corresponded to a change in the energy input into the flash lamp from 0 to 240 J. On flash photolysis, Ca2+ was released within 2 ms, and the small percentage of conversion of the caged compound (∼10%) allowed us to apply repetitive pulses without altering the Ca2+ responses and the reserve of caged Ca2+. Freshly dissociated and cultured myocytes (3 days after injection) were immunostained as previously described (30Boittin F.X. Macrez N. Halet G. Mironneau J. Am. J. Physiol. 1999; 277: C139-C151Crossref PubMed Google Scholar). Briefly, myocytes were incubated in the presence of anti-RYR3-specific antibody (20Jeyakumar L.H. Copello J.A. O'Malley A.M. Wu G.M. Grassucci R. Wagenknecht T. Fleischer S. J. Biol. Chem. 1998; 273: 16011-16020Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) (at 1:100 dilution) for 20 h at 4 °C and with the secondary antibody (donkey anti-rabbit IgG conjugated to fluorescein isothiocyanate, diluted at 1:200) for 3 h at 20 °C. Thereafter, cells were mounted in Vectashield. Images of the stained cells were obtained with the Bio-Rad confocal microscope. Control cells and injected cells on the same glass slide were compared with each other by keeping acquisition parameters constant (gray values, exposure time, aperture). Fluorescent labeling was estimated by gray level analysis using MPL software and expressed in arbitrary units of fluorescence. Collagenase was obtained from Worthington. Fluo 3-AM and DMNP-EDTA, AM were from Molecular Probes (Leiden, The Netherlands). Caffeine was from Merck. Indo 1-AM, ryanodine, and cyclopiazonic acid were from Calbiochem. Medium M199 was from ICN (Costa Mesa, CA). Fetal calf serum was from Bio Media (Boussens, France). Streptomycin, penicillin, glutamine, and pyruvate were from Life Technologies, Inc. All primers and phosphorothioate antisense oligonucleotides were synthesized and purchased from Eurogentec (Seraing, Belgium). All other chemicals were from Sigma. The rabbit anti-RYR3-specific antibody was directed against the deduced amino acid sequence, 4326–4336 (11 amino acids), of rabbit RYR3 (20Jeyakumar L.H. Copello J.A. O'Malley A.M. Wu G.M. Grassucci R. Wagenknecht T. Fleischer S. J. Biol. Chem. 1998; 273: 16011-16020Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Data are expressed as means ± S.E.;n represents the number of tested cells. Significance was tested by means of Student's test. p values < 0.05 were considered as significant. The effects of increasing the external Ca2+concentration ([Ca2+]o) in rat portal vein myocytes were studied in a series of experiments, in which caffeine (10 mm) was applied through a micropipette positioned near the surface of the cells and the resulting [Ca2+]ichanges were measured either in the entire cytosol (indo 1 experiments) or in a single line repeatedly scanned through the confocal cell section (fluo 3 experiments). In indo 1-loaded cells, the peak fluorescence (Δ ratio 405/480 nm) as well as the total fluorescence measured during 10-s applications of caffeine (Δ ratio × 10 s) increased in a time-dependent manner and reached a steady-state value within 1 h in 10 mmCa2+-containing solution. In 1.7 mm[Ca2+]o, the values for the peak and total fluorescence were 0.69 ± 0.03 and 119.2 ± 7.6, respectively (n = 46). In 10 mm[Ca2+]o, these values increased to 0.98 ± 0.03 (n = 79) and 272.2 ± 26.1 (n= 46), respectively, indicating a 40% increase in caffeine response amplitude and a 230% increase in SR Ca2+ release. In contrast, the basal [Ca2+]i was not significantly increased (from 55 ± 8 nm in 1.7 mm[Ca2+]o to 64 ± 10 nm in 10 mm [Ca2+]o, n = 46). In fluo 3-loaded cells (Fig. 1), the amplitude of the caffeine-induced Ca2+ waves, measured from a 2-μm region of the line scan image, also increased from 2.01 ± 0.10 (ΔF/Fo, n = 32) in 1.7 mm [Ca2+]o to 2.66 ± 0.08 (n = 45) in 10 mm[Ca2+]o, indicating a 30% increase in Ca2+ response amplitude (Fig. 1 B). Furthermore, the upstroke velocity of the caffeine-induced Ca2+response, corresponding to the initiation site of the response, was enhanced from 8.57 ± 0.71 (ΔF/Fo·s−1,n = 32) in 1.7 mm[Ca2+]o to 33.57 ± 4.27 (n= 45) in 10 mm [Ca2+]o, indicating a 350% increase in Ca2+ release velocity (Fig.1 B). Taken together, these results suggest that the SR Ca2+ content of vascular myocytes is increased by sustained elevation in extracellular [Ca2+] and that both amplitude and upstroke velocity of the Ca2+ responses can be used as significant parameters to study the cellular mechanisms involved during increased SR Ca2+ loading. To assess the role of the SR Ca2+ loading in the generation of large and fast Ca2+ responses to caffeine, the effects of 10 μm cyclopiazonic acid were first investigated on the caffeine-induced Ca2+ responses. Inhibition of the Ca2+ uptake capacity of the intracellular store by cyclopiazonic acid resulted in a small elevation of the basal [Ca2+]i and the suppression of the caffeine-induced Ca2+ response in the continuous presence of cyclopiazonic acid for 5 min (n = 6). In a second set of experiments, caffeine (10 mm) was applied in Ca2+-free, 0.5 mm EGTA-containing solution for 10 s (a time sufficient to remove voltage-dependent Ca2+ current) on myocytes superfused either in 1.7 mm [Ca2+]o or 10 mm[Ca2+]o. After 10 s in Ca2+-free solution, the amplitude of the caffeine-induced Ca2+responses (measured with indo 1) was 0.55 ± 0.03 (Δ ratio,n = 26) in myocytes pretreated with 1.7 mm[Ca2+]o and 0.79 ± 0.07 (n= 26) in myocytes pretreated with 10 mm[Ca2+]o, indicating an increase in Ca2+ response amplitude similar to that obtained in Ca2+-containing solutions (about 40%). Taken together, these results suggest that the increased accumulation of Ca2+ in the SR is responsible for the large and fast caffeine-induced Ca2+ responses under conditions of increased [Ca2+]o. As recently published (14Coussin F. Macrez N. Morel J.L. Mironneau J. J. Biol. Chem. 2000; 275: 9596-9603Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), we designed antisense oligonucleotides specifically targeting each RYR subtype mRNA. For each RYR subtype, two antisense sequences were chosen, one targeting the region of the mRNA amplified in PCR experiments (named as2RYR) and the other one (named as1RYR) designed to hybridize the mRNA outside the amplified fragment but close to the start codon. The time course of antisense oligonucleotide efficiency was determined by checking the ability of a mixture of as1RYR1 + as1RYR2 + as1RYR3 (10 μm each) to inhibit the Ca2+ waves induced by 10 mmcaffeine in isolated myocytes superfused in 10 mm[Ca2+]o for 1 h. The Ca2+responses were strongly inhibited 3 days after nuclear injection of the antisense oligonucleotides (83 ± 5%, n = 30); recovery began the fourth day with an inhibition of 48 ± 5% (n = 25). Nonspecific effects of antisense oligonucleotides were detected only at concentrations higher than 50 μm (for example, inhibition of RYR2 protein expression by 50 μm anti-Gαo antisense oligonucleotide). Immunodetection of RYR3 with an anti-RYR3-specific antibody (20Jeyakumar L.H. Copello J.A. O'Malley A.M. Wu G.M. Grassucci R. Wagenknecht T. Fleischer S. J. Biol. Chem. 1998; 273: 16011-16020Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) revealed a homogeneous distribution of fluorescence in cell sections from freshly isolated and cultured myocytes (Fig.2, A and B). In cells injected with as1RYR3, the immunostaining was very weak (Fig.2 C), whereas it was not significantly changed in cells injected with as1RYR1 + 2 (Fig. 2, E and F). These results indicate that RYR3 is expressed in rat portal vein myocytes and can be selectively inhibited by asRYR3 without variation in the expression of the other RYR subtypes; they are in good agreement with previous data using BODIPY®-labeled ryanodine staining, which showed that inhibition of each one of the three RYR subtypes decreased by approximately one-third the specific fluorescence (14Coussin F. Macrez N. Morel J.L. Mironneau J. J. Biol. Chem. 2000; 275: 9596-9603Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). We have previously reported that, in 1.7 mm [Ca2+]o, both RYR1 and RYR2 are required for Ca2+ release during Ca2+ sparks evoked by activation of voltage-gated Ca2+ channels (14Coussin F. Macrez N. Morel J.L. Mironneau J. J. Biol. Chem. 2000; 275: 9596-9603Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Since elevation of luminal [Ca2+] has been suggested to increase the activity of RYRs (32Gyorke I. Gyorke S. Biophys. J. 1998; 75: 2801-2810Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar), we studied the parameters of spontaneous Ca2+ sparks in Ca2+-overloaded cells. In control cells, superfused in 1.7 mm[Ca2+]o, spontaneous Ca2+ sparks were rarely detected, in less than 25% of cells tested (7/32 cells), and the number of initiation sites per line scan image was 1.1 ± 0.1 (n = 7). In 10 mm[Ca2+]o, spontaneous Ca2+ sparks were detected in about 80% of cells tested (116/140 cells; Fig.3 A), and the number of initiation sites per line scan image was 2.17 ± 0.21 (n = 44, Fig. 3 C). In contrast, the spatio-temporal parameters of Ca2+ sparks were not significantly different in 1.7 mm[Ca2+]o and 10 mm[Ca2+]o (TableI).Table IEffects of RYR3 antisense oligonucleotides on the parameters of Ca2+ sparksAmplitudeTime to peakFTHMFWHMnΔ (F/Fo)msmsμm1.7 mm [Ca2+]oNoninjected cells1.01 ± 0.0922.1 ± 0.936.1 ± 1.81.6 ± 0.23210 mm [Ca2+]oNoninjected cells1.02 ± 0.0721.4 ± 0.736.3 ± 1.31.9 ± 0.144asRYR3-injected cells0.88 ± 0.0622.1 ± 0.735.3 ± 1.81.8 ± 0.238Data are means ± S.E., with n indicating the number of cells tested in each conditions. FTHM, full time at half-maximal amplitude; FWHM, full width at half-maximal amplitude. Open table in a new tab Data are means ± S.E., with n indicating the number of cells tested in each conditions. FTHM, full time at half-maximal amplitude; FWHM, full width at half-maximal amplitude. When cells were injected with asRYR1, asRYR2, or asRYR1 + 2, the number of cells with spontaneous Ca2+ sparks was strongly decreased (Fig. 3 A). In contrast, the number of cells with spontaneous Ca2+ sparks and the number of initiation sites per line scan image were not significantly affected in cells injected with asRYR3 (Fig. 3, B and C). These results suggest that under both normal (14Coussin F. Macrez N. Morel J.L. Mironneau J. J. Biol. Chem. 2000; 275: 9596-9603Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) and increased SR Ca2+content conditions, Ca2+ sparks are due to activation of both RYR1 and RYR2 and that RYR3 does not contribute to triggering Ca2+ sparks. In addition, the spatio-temporal parameters of Ca2+ sparks in 10 mm[Ca2+]o were not significantly different in noninjected cells and in cells injected with asRYR3 (Table I). Taken together, these results suggest that increased SR Ca2+loading potentiates the activity of Ca2+ release units formed by RYR1 and RYR2, leading to an increase in Ca2+spark frequency without alterations of the spatio-temporal parameters. We have previously shown that in 1.7 mm[Ca2+]o, caffeine triggers Ca2+ waves by activating both RYR1 and RYR2 (14Coussin F. Macrez N. Morel J.L. Mironneau J. J. Biol. Chem. 2000; 275: 9596-9603Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Consequently, inhibition of RYR1 or RYR2 by treatment with antisense oligonucleotides partly inhibited the caffeine-induced Ca2+ responses, whereas inhibition of RYR3 was ineffective (14Coussin F. Macrez N. Morel J.L. Mironneau J. J. Biol. Chem. 2000; 275: 9596-9603Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Using the same an" @default.
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