Requirement of Ryanodine Receptor Subtypes 1 and 2 for Ca2+-induced Ca2+ Release in Vascular Myocytes
2000; Elsevier BV; Volume: 275; Issue: 13 Linguagem: Inglês
10.1074/jbc.275.13.9596
ISSN1083-351X
AutoresFrédéric Coussin, Nathalie Macrez, Jean‐Luc Morel, Jean Mironneau,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoWhile the roles of subtypes 1 and 2 of the ryanodine receptors (RYRs) have been studied in cellular systems expressing specifically one or the other of these subtypes (i.e. skeletal and cardiac muscle), the function of these receptors has not been evaluated in smooth muscles. We have previously reported RYR-mediated elementary (Ca2+ sparks) and global Ca2+ responses in rat portal vein myocytes. Here, we investigated the respective roles of all three RYR subtypes expressed in these cells as revealed by reverse transcriptase-polymerase chain reaction. Antisense oligonucleotides targeting each one of the three RYR subtypes were shown to specifically inhibit the expression of the corresponding mRNA and protein without affecting the other RYR subtypes. Confocal Ca2+ measurements revealed that depolarization-induced Ca2+ sparks and global Ca2+ responses were blocked when either RYR1 or RYR2 expression was suppressed. Caffeine-induced Ca2+ responses were partly inhibited by the same antisense oligonucleotides. Neither the corresponding scrambled oligonucleotides nor the antisense oligonucleotides targeting RYR3 affected depolarization- or caffeine-induced Ca2+ responses. Our results show that, in vascular myocytes, the two RYR1 and RYR2 subtypes are required for Ca2+ release during Ca2+ sparks and global Ca2+ responses, evoked by activation of voltage-gated Ca2+ channels. While the roles of subtypes 1 and 2 of the ryanodine receptors (RYRs) have been studied in cellular systems expressing specifically one or the other of these subtypes (i.e. skeletal and cardiac muscle), the function of these receptors has not been evaluated in smooth muscles. We have previously reported RYR-mediated elementary (Ca2+ sparks) and global Ca2+ responses in rat portal vein myocytes. Here, we investigated the respective roles of all three RYR subtypes expressed in these cells as revealed by reverse transcriptase-polymerase chain reaction. Antisense oligonucleotides targeting each one of the three RYR subtypes were shown to specifically inhibit the expression of the corresponding mRNA and protein without affecting the other RYR subtypes. Confocal Ca2+ measurements revealed that depolarization-induced Ca2+ sparks and global Ca2+ responses were blocked when either RYR1 or RYR2 expression was suppressed. Caffeine-induced Ca2+ responses were partly inhibited by the same antisense oligonucleotides. Neither the corresponding scrambled oligonucleotides nor the antisense oligonucleotides targeting RYR3 affected depolarization- or caffeine-induced Ca2+ responses. Our results show that, in vascular myocytes, the two RYR1 and RYR2 subtypes are required for Ca2+ release during Ca2+ sparks and global Ca2+ responses, evoked by activation of voltage-gated Ca2+ channels. ryanodine receptor Ca2+-induced Ca2+ release polymerase chain reaction reverse transcription Ca2+ signaling is a common step for activation of all muscle cell types and Ca2+ release from the sarcoplasmic reticulum plays an essential role in the regulation of the cytosolic Ca2+ concentration. Two families of intracellular Ca2+ release channels are known; the inositol 1,4,5-trisphosphate receptor family is activated by the second messenger inositol 1,4,5-trisphosphate, while the ryanodine receptor (RYR)1 family is activated by cytosolic Ca2+ and is able to bind the plant alkaloid ryanodine (1.Berridge M.J. Nature. 1993; 365: 388-389Crossref PubMed Scopus (296) Google Scholar, 2.Berridge M.J. J. Physiol. (Lond.). 1997; 499: 291-306Crossref Scopus (924) Google Scholar). Since the description of a Ca2+-induced Ca2+ release (CICR) mechanism in skeletal muscle (3.Endo M. Tanaka M. Ogawa Y. Nature. 1970; 228: 34-36Crossref PubMed Scopus (588) Google Scholar), the function of RYR as a Ca2+-activated Ca2+release channel has been widely studied in both skeletal and cardiac muscles, where the mechanisms of RYR activation by plasma membrane depolarization are different. In skeletal muscle, RYR is thought to be operated by a mechanical coupling with the dihydropyridine receptor Ca2+ channels without requirement of Ca2+influx (4.Schneider M.F. Chandler W.K. Nature. 1973; 242: 244-246Crossref PubMed Scopus (672) Google Scholar, 5.Marks A.R. Tempst P. Hwang K.S. Taubman M.B. Inui M. Chadwick C. Fleischer S. Nadal-Ginard B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8683-8687Crossref PubMed Scopus (183) Google Scholar, 6.Rios E. Pizarro G. Physiol. Rev. 1991; 71: 849-908Crossref PubMed Scopus (500) Google Scholar). In contrast, the cardiac type of excitation-contraction coupling involves a depolarization-dependent Ca2+ influx via dihydropyridine receptor Ca2+channels, which triggers Ca2+ release through RYR (7.Fabiato A. J. Gen. Physiol. 1985; 85: 247-289Crossref PubMed Scopus (709) Google Scholar, 8.Nabauer M. Callewaert G. Cleemann L. Morad M. Science. 1989; 244: 800-803Crossref PubMed Scopus (349) Google Scholar). In smooth muscle, depolarization-induced Ca2+ release via RYR has been described and requires Ca2+ influx (9.Ganitkevich V.Y. Isenberg G. J. Physiol. (Lond.). 1995; 484: 287-306Crossref Scopus (62) Google Scholar, 10.Kamishima T. McCarron J.G. J. Physiol. (Lond.). 1997; 501: 497-508Crossref Scopus (50) Google Scholar). Localized Ca2+ release events, called Ca2+sparks, have been shown to be produced by RYRs in smooth muscle as well as in other muscles (11.Cheng H. Lederer W.J. Cannell M.B. Science. 1993; 262: 740-744Crossref PubMed Scopus (1637) Google Scholar, 12.Nelson M.T. Cheng H. Rubart M. Santana L.F. Bonev A.D. Knot H.J. Lederer W.J. Science. 1995; 270: 633-637Crossref PubMed Scopus (1221) Google Scholar, 13.Arnaudeau S. Macrez-Leprêtre N. Mironneau J. Biochem. Biophys. Res. Commun. 1996; 222: 809-815Crossref PubMed Scopus (56) Google Scholar, 14.Arnaudeau S. Boittin F-X. Macrez N. Lavie J-L. Mironneau C. Mironneau J. Cell Calcium. 1997; 22: 399-411Crossref PubMed Scopus (38) Google Scholar). It has been proposed that global Ca2+ signals result from the spatiotemporal summation of individual calcium release events, giving rise to Ca2+waves (14.Arnaudeau S. Boittin F-X. Macrez N. Lavie J-L. Mironneau C. Mironneau J. Cell Calcium. 1997; 22: 399-411Crossref PubMed Scopus (38) Google Scholar, 15.Cannell M.B. Cheng H. Lederer W.J. Biophys. J. 1994; 67: 1942-1956Abstract Full Text PDF PubMed Scopus (333) Google Scholar, 16.Cannell M.B. Cheng H. Lederer W.J. Science. 1995; 268: 1045-1050Crossref PubMed Scopus (516) Google Scholar, 17.Lipp P. Niggli E. Prog. Biophys. Mol. Biol. 1996; 65: 265-296Crossref PubMed Scopus (73) Google Scholar). In the last few years, the cloning and sequencing of three genes encoding different RYR subtypes and 10 genes encoding dihydropyridine receptor Ca2+ channel subtypes has provided a structural basis for the understanding of the different types of RYR activation by membrane depolarization. Both RYR and dihydropyridine receptor Ca2+ channel subtypes involved in depolarization-induced Ca2+ release are different between cardiac and skeletal muscle. The skeletal Ca2+ release depends on ryanodine receptor subtype 1 (RYR1) activation (18.Takeshima H. Iino M. Takekura H. Nishi M. Kun J. Minowa O. Takano H. Noda T. Nature. 1994; 369: 556-559Crossref PubMed Scopus (330) Google Scholar). Despite a recent report showing that in embryonic cardiac myocytes isolated from mutant mice lacking RYR2, the CICR mechanism is not significantly affected (19.Takeshima H. Komazaki S. Hirose K. Nishi M. Noda T. Iino M. EMBO J. 1998; 17: 3309-3316Crossref PubMed Scopus (190) Google Scholar), the mature cardiac CICR is thought to occur through subtype 2 (RYR2). Since smooth and cardiac muscles share the expression of RYR2 subtype and highly homologous α1C L-type Ca2+ channel subclones (20.Neylon C.B. Richards S.M. Larsen M.A. Agrotis A. Bobik A. Biochem. Biophys. Res. Commun. 1995; 215: 814-821Crossref PubMed Scopus (95) Google Scholar, 21.Sutko J.L. Airey J.A. Physiol. Rev. 1996; 76: 1027-1071Crossref PubMed Scopus (367) Google Scholar, 22.Catterall W.A. Cell. 1991; 64: 871-874Abstract Full Text PDF PubMed Scopus (164) Google Scholar, 23.Bosse E. Bottlender R. Kleppisch T. Hescheler J. Welling A. Hofmann F. Flockerzi V. EMBO J. 1992; 11: 2033-2038Crossref PubMed Scopus (46) Google Scholar, 24.Takimoto K. Li D. Nerbonne J.M. Levitan E.S. J. Mol. Cell. Cardiol. 1997; 29: 3035-3042Abstract Full Text PDF PubMed Scopus (110) Google Scholar), a cardiac-like mechanism is generally proposed to underlie the vascular or visceral Ca2+ channel activation-induced Ca2+ release. However, some studies have shown that both RYR1 and RYR3 may also be activated by Ca2+ influx upon L-type Ca2+ channel activation (18.Takeshima H. Iino M. Takekura H. Nishi M. Kun J. Minowa O. Takano H. Noda T. Nature. 1994; 369: 556-559Crossref PubMed Scopus (330) Google Scholar, 25.Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 344: 451-453Crossref PubMed Scopus (208) Google Scholar). In the present study, we performed a series of experiments to determine which RYR subtypes are responsible for Ca2+ sparks and global Ca2+ responses in rat portal vein myocytes. Subtypes of RYR potentially expressed in these cells were first identified by RT-PCR, and then we designed antisense oligonucleotides that specifically targeted each RYR subtype. The efficiency of these antisense oligonucleotides was checked by studying their ability to specifically inhibit RYR subtype expression on one hand and to inhibit Ca2+ sparks or global Ca2+ responses induced by membrane depolarization or caffeine application on the other. We show for the first time that both RYR1 and RYR2 subtypes are required for Ca2+ sparks and global Ca2+ responses induced by activation of voltage-gated Ca2+ channels. These results suggest the existence of mixed functional Ca2+ release channel units composed of RYR1 and RYR2 subtypes in vascular myocytes. 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 (Hanks' balanced salt solution), and then 0.8 mg/ml collagenase (EC 3.4.24.3), 0.2 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 an enzyme-free solution and triturated using a fire-polished Pasteur pipette to release cells. Cells were seeded at a density of 103 cells/mm2 on 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 mmpyruvate, 20 units/ml penicillin, and 20 μg/ml streptomycin; they were kept in an incubator gassed with 95% air and 5% CO2at 37 °C. The myocytes were cultured in this medium for 4 days. Total RNA was extracted from portal vein media, heart, or skeletal muscle (Fig. 1 A) and from about 100 portal vein myocytes (Fig. 1, C and D) using the RNeasy minikit (Qiagen, Hilden, Germany), following the instructions of the supplier. The reverse transcription reaction was performed using Sensiscript RT kit (Qiagen). Total RNA was incubated with random primers (Promega, Lyon, France) at 65 °C for 5 min. After a cooling time of 15 min at 25 °C, RT mix was added, and the total mixture was incubated for 60 min at 37 °C. The resulting cDNA was stored at −20 °C. PCR was performed with 2 μl of cDNA (in RT-PCR mix), 1.25 units of HotStartTaq DNA polymerase (Qiagen), a 1 μmconcentration of each primer, and a 200 μm concentration of each deoxynucleotide triphosphate, in a final volume of 50 μl. The PCR conditions were 95 °C for 15 min and then 35 cycles at 94 °C for 1 min, 60 °C (RYR1 and RYR2) or 56 °C (RYR3) for 1 min, and 72 °C for 1 min, and at the end of PCR, samples were kept at 72 °C for 10 min for final extension and then stored at 4 °C. Reverse transcription and PCR were performed with a thermal cycler (Techne, Cambridge, UK). Amplification products were separated by electrophoresis (2% agarose gel) and visualized by ethidium bromide staining. Gels were photographed with EDAS 120 and analyzed with KDS1D 2.0 software (Kodak Digital Science, Paris, France). Sense (s) and antisense (as) primer pairs specific for RYR1, RYR2, and RYR3 were designed on the known cloned receptor sequences deposited in the GenBankTM sequence data base (accession nos. X83932,X83933, and X83934, respectively) with Lasergene software (DNASTAR, Madison, WI). The nucleotide sequence and the length of the expected PCR products (in parentheses) for each primer pair were, respectively, as follows: RYR1(s), GAAGGTTCTGGACAAACACGGG; RYR1(as), TCGCTCTTGTTGTAGAATTTGCGG (435 base pairs); RYR2(s), GAATCAGTGAGTTACTGGGCATGG; RYR2(as), CTGGTCTCTGAGTTCTCCAAAAGC (635 base pairs); RYR3(s), AGAAGAGGCCAAAGCAGAGG; RYR3(as), GGAGGCCAACGGTCAGA (269 bp). After electrophoresis, the amplified DNA fragments were cleaned and purified with the Qiaquick gel extraction kit (Qiagen). PCR fragments were sequenced by the Qiagen sequencing service. The deduced DNA sequences of RYR1, RYR2, and RYR3 fragments were 98, 99, and 97% identical, respectively, to the published sequences. Sequences of phosphorothioate antisense oligonucleotides used in the present study were determined by sequence comparison with Lasergene software. Sequence of all three RYR cDNAs were aligned with each other, and specific antisense oligonucleotide sequences were chosen in the 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 GenBankTM 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 described previously (26.Macrez-Leprêtre N. Kalkbrenner F. Schultz G. Mironneau J. J. Biol. Chem. 1997; 272: 5261-5268Abstract Full Text Full Text PDF PubMed Scopus (71) 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 no. X83932); those of as1RYR2 and as2RYR2 are GTGTCCTCACAGAAGTT and TGAAATCTAGTGCAGCCT, corresponding to nucleotides 137–153 and 1587–1604, respectively, of RYR2 cDNA (accession no. X83933); and those of as1RYR3 and as2RYR3 are AAGTCAAGGGCATTTTTG and ACTTAGCCATGACACCAG, corresponding to nucleotides 502–519 and 557–574, respectively, of RYR3 cDNA (accession no. 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). Voltage clamp and membrane current recordings were made with a standard patch clamp technique using a List EPC7 patch clamp amplifier (Darmstadt, Eberstadt, Germany). The whole-cell-recording mode was performed with patch clamp pipettes of 2–5-megaohm resistance. Membrane potential and current records were stored and analyzed using pCLAMP software (Axon Instruments, Forster City, CA). Peak current density (picoamps/picofarads) was calculated by dividing the peak inward current at 0 mV by the cell capacitance. The normal physiological solution contained 130 mm NaCl, 5.6 mm KCl, 1 mm MgCl2, 1.7 mm CaCl2, 11 mm glucose, and 10 mm HEPES (pH 7.4, with NaOH). The basic pipette solution contained 130 mm CsCl and 10 mm HEPES (pH 7.3 with CsOH). For experiments that used membrane depolarization, fluo 3 (60 μm) was dialyzed into the cells through the patch clamp pipette. In the other experiments, cells were loaded by incubation in physiological solution containing 4 μm fluo 3-acetoxymethylester for 1 h at room temperature. These cells were washed and allowed to cleave the dye to the active fluo 3 compound for 1 h. Images were acquired using the line-scan mode of a confocal Bio-Rad MRC1000 (Bio-Rad, Paris, France) 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 the position along the scanned line was given by vertical displacement. Fluorescence signals are expressed as pixel per pixel fluorescence ratios (F/F 0), where F is the fluorescence during a response andF 0 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). BayK 8644, angiotensin II, and caffeine 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. Three days after injection, myocytes were washed in physiological solution and incubated with BODIPY® FL-X ryanodine (1 μm) for 60 min at 37 °C. After incubation, cells were washed and maintained in physiological solution during fluorescence measurements. 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). Nonspecific fluorescence was estimated by incubating cells with both 1 μmBODIPY® FL-X ryanodine and 10 μm ryanodine. 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, fluo 3-acetoxymethylester, and BODIPY® FL-X ryanodine were from Molecular Probes, Inc. (Leiden, The Netherlands). Caffeine was from Merck (Nogent sur Marne, France). BayK 8644 was from Bayer (Puteaux, France). Angiotensin II was from Neosystem laboratories (Strasbourg, France). Ryanodine was from Calbiochem (Meudon, France). 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. (Cergy Pontoise, France). All primers and phosphorothioate antisense oligonucleotides were synthesized and purchased from Eurogentec (Seraing, Belgium). All other chemicals were from Sigma. 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. Depending on tissues and species, various RYR subtype expression and Ca2+ responses via RYRs have been reported in smooth muscle. This variability prompted us to identify the RYR subtypes expressed in rat portal vein myocytes by RT-PCR. Since RYR subtype complete cDNA sequences were not known in the rat, we designed primer pairs amplifying fragments of each subtype displaying a common sequence in mouse and rabbit or pig and included in the partial sequences of rat RYR1 and RYR2, recently submitted to GenBankTM. RT-PCR performed on RNA extracts prepared from portal vein myocytes maintained 4 days in primary culture gave the same results as the one performed on RNA extracts prepared from freshly isolated myocytes or from portal vein media (Fig. 1 A). The expression of all three RYR subtypes in portal vein myocytes contrasts with the specific expression of RYR2 and RYR3 in cardiac myocytes or RYR1 and RYR3 in skeletal myocytes (Fig. 1 A). PCR experiments performed directly on RNA extracts (by omitting the reverse transcription) attested that genomic DNA was not amplified (not shown). Since our PCR results were in agreement with those of Neylon et al. (20.Neylon C.B. Richards S.M. Larsen M.A. Agrotis A. Bobik A. Biochem. Biophys. Res. Commun. 1995; 215: 814-821Crossref PubMed Scopus (95) Google Scholar), reporting that all three RYR subtypes are expressed in vascular myocytes, 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 by PCR (named as2RYR) and the other one (named as1RYR) designed to hybridize the mRNA outside the amplified fragment but close to the start codon. We determined the time course of antisense oligonucleotide efficiency by checking the ability of a mixture of as1RYR1 plus as1RYR2 plus as1RYR3 (10 μm each; named as1RYR1+2+3) to inhibit caffeine-induced Ca2+ wave in isolated myocytes (Fig.1 B). The Ca2+ responses were dramatically decreased 3 days after injection of the antisense oligonucleotides, and recovery began the fourth day. We noted nonspecific effects of antisense oligonucleotides only at concentrations higher than 50 μm (for example, inhibition of Gαq protein expression by 50 μm asRYRs and vice versa;n = 15). Based on this time scale, we verified that mRNA encoding each RYR subtype was specifically decreased by the corresponding as2RYR 3 days after injection. Fig. 1, C and D, illustrate RT-PCR experiments performed on RNA preparations from injected cells located in a marked area of glass slides. The noninjected cells located outside the marked area of each glass slide were used as control and displayed in all cases all three subtypes of RYR (not shown). Each antisense oligonucleotide (Fig. 1 C) or combinations of two or three antisense oligonucleotides (Fig. 1 D) inhibited the amplification of the targeted RYR subtype mRNAs, while the nontargeted subtypes were still amplified, thus showing the efficiency and specificity of our antisense strategy. Looking at the protein expression level, we used BODIPY®-labeled ryanodine to detect RYRs, since this has been previously reported on several cell types (27.Holz G.G. Leech C.A. Heller R.S. Castonguay M. Habener J.F. J. Biol. Chem. 1999; 274: 14147-14156Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 28.Zhang J.J. Williams A.J. Sitsapesan R. Br. J. Pharmacol. 1999; 126: 1066-1074Crossref PubMed Scopus (12) Google Scholar) and validated by the ability of BODIPY®-labeled ryanodine to inhibit [3H]ryanodine binding on skeletal muscle microsomes (29.Zhang X. Wen J. Bidasee K.R. Besch Jr., H.R. Rubin R.P. Am. J. Physiol. 1997; 273: C1306-C1314Crossref PubMed Google Scholar). BODIPY®-labeled ryanodine staining in noninjected portal vein myocytes after 3 days of primary culture revealed a network of RYRs throughout the cytoplasm (Fig.2 A) with some spots of fluorescence in discrete areas probably expressing more RYRs (Fig.2 B). In cells injected with all three as1RYRs, RYR fluorescent staining by binding of BODIPY®-labeled ryanodine was abolished (Fig. 2, C, D, andG). Similar results were obtained with injection of all three as2RYRs. The weak fluorescence detected in as1RYR1+2+3-injected cells is thought to correspond to the nonspecific fluorescence, since it is not different from the background fluorescence measured in cells incubated with 1 μm BODIPY® FL-X ryanodine plus 10 μm ryanodine (Fig. 2 G). Although the fluorescence was largely decreased in cells injected with as1RYR1+2 (Fig. 2, E, F, and G), when compared with control cells, a specific fluorescence (about 30% of the fluorescence measured in control cells) could be distinguished from the background fluorescence observed in as1RYR1+2+3-injected cells, attesting that the expression of RYR3 specifically observed in asRYR1+2-injected cells was inhibited by asRYR3 antisense oligonucleotides. Similar results were obtained for the other RYR subtypes, and statistical analysis of BODIPY® FL-X ryanodine fluorescence revealed that each one of the RYR subtypes represented approximately one-third of the total amount of RYR expressed per cell, as shown by the remaining fluorescence in cells injected with asRYR1+2, asRYR1+3, or asRYR2+3 (Fig. 2 G). Taken together, these results indicate that the antisense oligonucleotides were efficient and specific in inhibiting expression of RYR subtypes 3 days after injection in the nucleus and that they could be used to identify the role of each RYR subtype in Ca2+ responses of vascular myocytes. We have previously shown that in venous myocytes, Ca2+ sparks or global Ca2+ responses can be generated by controlling membrane depolarization (14.Arnaudeau S. Boittin F-X. Macrez N. Lavie J-L. Mironneau C. Mironneau J. Cell Calcium. 1997; 22: 399-411Crossref PubMed Scopus (38) Google Scholar). Electrophysiological protocols eliciting Ca2+ sparks (depolarization from −60 to −20 mV) or global Ca2+responses (depolarization from −60 to 0 mV) were applied on cells injected with the various asRYRs in order to determine which RYR subtypes were required for transducing depolarization-induced Ca2+ events. Cells injected with as1RYR1 or as1RYR2 were not able to produce any Ca2+sparks and Ca2+ responses in response to depolarizations eliciting normal Ca2+ currents (Figs. 3 and 4). In contrast, the Ca2+ events activated by membrane depolarizations in as1RYR3-injected cells (Figs. 3 D and 4 D) were not different from those elicited in control cells (Figs. 3 A and4 A). Scrambled RYR1 and scrambled RYR2 antisense oligonucleotides did not affect the Ca2+ events activated by depolarization or caffeine (not shown). The properties of Ca2+ sparks in as1RYR3-injected cells were similar to those of the Ca2+ sparks obtained in control cells (Fig. 3). The mean amplitude of Ca2+ sparks was estimated to be 1.04 ± 0.08 ratio units (ΔF/F 0) in control cells (n = 63) versus 0.91 ± 0.12 ratio units in as1RYR3-injected cells (n = 25). The mean time to reach the peak Ca2+ spark, the mean half-time of decay, and the mean full width at half-maximal amplitude were, respectively, 22.1 ± 0.4 ms, 25.5 ± 0.6 ms, and 1.5 ± 0.1 μm in control noninjected cells (n = 63) and 22.2 ± 0.6 ms, 25.2 ± 0.5 ms, and 1.5 ± 0.1 μm in asRYR3-injected cells (n = 25). It should be noted that spontaneous Ca2+ sparks have never been detected in either as1RYR1- or as1RYR2-injected cells (n = 48), whereas they were observed in as1RYR3-injected and control cells. The mean amplitude of global Ca2+ responses in as1RYR3-injected cells was similar to that observed in control noninjected cells, while the responses were almost abolished in as1RYR1- or as1RYR2-injected cells (Fig. 5 A). The maximal rate of Ca2+ increase (56 ± 6 ΔF/F 0·s−1,n = 63) in control cells was not significantly affected in as1RYR3-injected cells (48 ± 8 ΔF/F 0·s−1,n = 25).Figure 4Effects of asRYR1, asRYR2, and asRYR3 antisense oligonucleotides on global Ca2+ responses induced by membrane depolarizations. Typical Ca2+ responses induced by depolarizing steps from −60 to 0 mV in a noninjected control cell (A) and in cells injected with 10 μm as1RYR1 (B), as1RYR2 (C), and as1RYR3 (D) antisense oligonucleotides.Traces show (from top to bottom) membrane potential, line-scan fluorescence image, and averaged fluorescence from a 2-μm region of the line-scan image. Myocytes were loaded with fluo 3 through the patch pipette.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Effects of asRYR antisense oligonucleotides on amplitude of global Ca2+ responses (A) and maximal Ca2+ current densities (B) induced by depolarizing steps from −60 to 0 mV in noninjected control cells and in cells injected with 10 μm as1RYR1, as1RYR2, and as1RYR3 antisense oligonucleotides. Data are means ± S.E., with the number of cells tested indicated in parentheses. Cells were obtained from four different batches. ★, values significantly different from those obtained in noninjected cells. Myocytes were loaded with fluo 3 through the patch pipette.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The Ca2+ channel agonist BayK 8644 has been used to increase the frequency of Ca2+ sparks in ventricular myocytes (30.Satoh H. Hayashi H. Blatter L.A. Bers D.M. Heart Vessels. 1997; 12: 58-61PubMed Google Scholar). In vascular myocytes, the application of 1 nm BayK 8644 similarly increased the number of Ca2+ sparks in noninjected cells and in cells injected with as1RYR3 (Table I). In contrast, in as1RYR1- or as1RYR2-injected cells, BayK 8644 failed to induced any Ca2+ sparks (Table I), suggesting that whatever the mode of activation of voltage-gated Ca2+ channels (membrane depolarization or Ca2+ channel agonist), both RYR1 and RYR2 are required for Ca2+ release during Ca2+sparks.Table IEffects of antisense oligonucleotides directed against the mRNAs of RYRs on the number of Ca 2+ sparks per line-scan image evoked by Bay K 8644 and on the mean amplitude of angiotensin II-mediated Ca 2+ responsesNoninjected cellsnas1RYR1-injected cellsnas1RYR2-injected cellsnas1RYR3-injected cellsnBay K 8644 (1 nm) Ca2+ spark/line sc
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