Contribution of Ryanodine Receptor Subtype 3 to Ca2+Responses in Ca2+-overloaded Cultured Rat Portal Vein Myocytes

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. EMBO J. 1998; 17: PubMed Scopus Google Scholar, T. S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). has been that these differences from the expression of different of RYR3 A. M. K. K. Y. H. FEBS Lett. 1996; PubMed Scopus Google Scholar). The of the different RYR subtypes to Ca2+ signaling has been by using RYR1 and RYR2 H. Komazaki S. Hirose K. Nishi M. Noda T. Iino M. EMBO J. 1998; 17: 3309-3316Crossref PubMed Scopus (187) Google Scholar, H. T. T. Takekura H. Nishi M. Noda T. Iino M. EMBO J. 1995; PubMed Scopus Google Scholar) or RYR3 H. T. Nishi M. N. M. Y. Kuno J. H. Endo M. Iino M. Noda T. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, F. Conti A. EMBO J. 1997; PubMed Scopus Google Scholar, Biophys. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In in the Ca2+ release from the sarcoplasmic reticulum (SR) in to increases in Ca2+ or caffeine is strongly but a similar in caffeine sensitivity is also in myocytes, a of each RYR subtype to of has been recently that, in skeletal both RYR1 and RYR3 to Ca2+ release during Ca2+ sparks Biophys. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In Ca2+ sparks in or cells similar Takeshima H. Biophys. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). using antisense that each of the RYR subtypes, has been to which RYR subtypes are for Ca2+ sparks and global Ca2+ responses in smooth muscle cells 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). that both RYR1 and RYR2 are required for Ca2+ release during Ca2+ sparks and Ca2+ induced by activation of Ca2+ and that RYR3 not to these Ca2+ In smooth muscle Ca2+ sparks are or in to Ca2+ 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, J. S. Macrez-Leprêtre N. 1996; PubMed Scopus Google Scholar, S. Macrez N. J.L. Mironneau Mironneau J. 1997; PubMed Scopus Google Scholar), and their localization to between the and the SR S. Macrez N. J.L. Mironneau Mironneau J. 1997; PubMed Scopus Google Scholar, S. Res. 1998; PubMed Scopus Google Scholar). In rat portal vein myocytes, and of Ca2+ sparks results in which Macrez N. G. Mironneau J. J. Physiol. 1999; PubMed Google Scholar). In the we investigated the effects of the Ca2+ on both Ca2+ sparks and global Ca2+ responses induced by caffeine, and conditions of increased SR Ca2+ loading, we the that the which becomes activable by caffeine and increases in is for the increased global Ca2+ also found that spontaneous Ca2+ sparks are but on activation of RYR1 and RYR2. were by The portal vein was and for 10 in Ca2+ and and 1 were for After the was and of portal vein were in a for were in a and using to release were a of with for localization of were in in 2 1 and were in an with and The myocytes were cultured in for The 1 and 10 with antisense oligonucleotides with the in the were on the cloned RYR in the with of three RYR were with each and antisense were in of the of different from the of the RYR subtypes. antisense and to in were were the of myocytes by a with was as previously 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 cultured for in and the were the for The of and are and to and of RYR1 in of and are and to and of RYR2 number and of and are and to and of RYR3 number In myocytes were with the to a of of RYR1 to a of of RYR2 and to a of of RYR3 In most cells were by in fluo for 1 These cells were and to the to the fluo 3 for were using the of a to a was by a a 3 was and was and the to fluo 3 the of the was and were in the a of were and each was to the of the to form the In these increased from the to the and the was by are expressed as is the during a is the of the and were by using and In cells were by in 1 for [Ca2+]i measurements were from the as previously reported J.L. Macrez-Leprêtre N. Mironneau J. J. 1996; PubMed Scopus Google Scholar). The and were in the of Ca2+ and in cells in and 10 and were by from a for the on the were 1 1-(4,5-dimethoxy-2-nitrophenyl)-EDTA, tetra(acetoxymethylester) was to the and in the of cells for 1 in an was by a from a flash to a the was with a between and be by the between and which to a in the the flash from to J. flash Ca2+ was 2 and the of of the caged to the Ca2+ responses and the of caged dissociated and cultured myocytes after were as previously Macrez N. G. Mironneau J. J. Physiol. 1999; PubMed Google Scholar). myocytes were in the of T. S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) for and with the to for 3 cells were in of the cells were with the cells and cells on the were with each by was by using and expressed in of was from and were from The was from and were from was from was from and were from and antisense oligonucleotides were and from were from The was the of RYR3 T. S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). are expressed as the number of was by of were as The effects of increasing the in rat portal vein myocytes were studied in a of in which caffeine was a the of the cells and the were in the 1 or in a single the 3 In the as well as the during of caffeine 10 increased in a and a 1 in 10 mmCa2+-containing solution. In the for the and were and respectively In 10 these increased to and indicating a in caffeine and a in SR Ca2+ In contrast, the [Ca2+]i was not increased in to 10 in 10 In fluo cells the of the Ca2+ from a of the also increased from in to in 10 indicating a in Ca2+ 1 the of the to the of the was from in to in 10 indicating a in Ca2+ release these results that the SR Ca2+ of vascular myocytes is increased by in and that both and of the Ca2+ responses can be as to the during increased SR Ca2+ the role of the SR Ca2+ loading in the of large and Ca2+ responses to caffeine, the effects of 10 were investigated on the Ca2+ of the Ca2+ of the by in a of the [Ca2+]i and the of the Ca2+ in the of for In a of caffeine was in for 10 to Ca2+ on myocytes in or 10 After 10 in the of the Ca2+responses with was in myocytes with and in myocytes with 10 indicating an in Ca2+ similar to that in these results that the increased of Ca2+ in the SR is for the large and Ca2+ responses under conditions of increased recently 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 antisense oligonucleotides targeting each RYR subtype each RYR antisense were targeting the of the in and the to the the but to the The of antisense was by the of a of to the Ca2+ induced by 10 in myocytes in 10 for 1 The Ca2+responses were strongly inhibited 3 after of the antisense oligonucleotides the with an inhibition of effects of antisense oligonucleotides were concentrations inhibition of RYR2 expression by antisense of RYR3 with an T. S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) revealed a of in from freshly and cultured myocytes and In cells with the was whereas was not in cells with 2 and These results that RYR3 is expressed in rat portal vein myocytes and can be inhibited by in the expression of the RYR are in with using ryanodine which that inhibition of each of the three RYR subtypes by the 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). have previously reported that, in both RYR1 and RYR2 are required for Ca2+ release during Ca2+ sparks evoked by activation of voltage-gated Ca2+ channels 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 of has been to the of S. Biophys. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), we studied the of spontaneous Ca2+ sparks in Ca2+-overloaded In in spontaneous Ca2+ sparks were in of cells and the number of was In 10 spontaneous Ca2+ sparks were in of cells and the number of was 3 In contrast, the of Ca2+ sparks were not different in and 10 of RYR3 antisense oligonucleotides on the of Ca2+ to are with indicating the number of cells in each in a are with indicating the number of cells in each cells were with or the number of cells with spontaneous Ca2+ sparks was strongly 3 In contrast, the number of cells with spontaneous Ca2+ sparks and the number of were not in cells with and These results that under both 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+ sparks are to activation of both RYR1 and RYR2 and that RYR3 not to Ca2+ In the of Ca2+ sparks in 10 were not different in cells and in cells with these results that increased SR the of Ca2+ release by RYR1 and to an in of the have previously shown that in caffeine Ca2+ by both RYR1 and RYR2 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). inhibition of RYR1 or RYR2 by treatment with antisense oligonucleotides inhibited the Ca2+ responses, whereas inhibition of RYR3 was 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 antisense we the role of each RYR subtype in the of the Ca2+ responses under conditions of increased SR Ca2+ with or evoked Ca2+ responses to 10 caffeine that were similar to in cells in 10 However, of both and of the Ca2+ responses by and respectively and inhibition was in cells with a of 2 3 RYR3 antisense oligonucleotides not the Ca2+responses evoked by caffeine is that the of the Ca2+ responses in and cells were not different from that in whereas that in cells was by of 2 the of the Ca2+ inhibition was that in cells These results that the RYR3 becomes activable by caffeine in cells with Since inhibition of RYR1 or RYR2 can be by activation of these results that the properties of the RYR3 be different from of RYR1 and RYR2. the effects of a in [Ca2+]i on activation of the RYR subtypes, flash photolysis of was to the as previously reported S. Macrez N. J.L. Mironneau Mironneau J. 1997; PubMed Scopus Google Scholar). in and and both and of the Ca2+ responses evoked by a flash of were increased in 10 cells with cells in The increased from in to in 10 The in [Ca2+]i to Ca2+ release from the caged in to a flash was in cells with ryanodine for to of the In cells with the of the Ca2+responses evoked by flash photolysis of caged Ca2+ in 10 was and and similar to that in cells in and In contrast, inhibition of RYR1 or RYR2 by treatment with antisense oligonucleotides no effects on the Ca2+responses evoked by flash photolysis of caged Ca2+ in 10 However, of 2 the Ca2+-induced Ca2+ responses the Ca2+ sensitivity of the RYR subtypes to the of the Ca2+ from the was as a function of flash in cells and after inhibition of the RYR subtypes in 10 the Ca2+-induced in [Ca2+]i was reduced by treatment with and 2 of the flash intensities inhibition was in cells with ryanodine for was that the in was similar to that in 10 after inhibition of RYR3s The Ca2+ sensitivity of RYR subtypes was by the between the Ca2+transients and the Ca2+ different flash intensities in cells in 10 and after inhibition of RYR1 2 or RYR3 The were that the Ca2+ sensitivities of the three RYR subtypes to [Ca2+]i be These results show that in Ca2+-overloaded the RYR3 is also activable by local increases in showing the of RYR antisense oligonucleotides on the of Ca2+responses evoked by flash photolysis of caged show in and in 10 in cells in the or in the of ryanodine show in 10 in cells and in cells with 10 and a of 2 were from different in 10 different from that in different from in cells in 10 were from different were with fluo and caged Ca2+ sensitivity of RYR subtypes in 10 Ca2+ induced by Ca2+ were from the and of increasing in cells in the or in the of ryanodine for and in cells with or 2 in in are for cells from different by flash in cells and in cells with or 2 were with the were with fluo and have reported previously that Ca2+ release Ca2+ channels Macrez N. G. Mironneau J. J. Physiol. 1999; PubMed Google Scholar). In Ca2+-overloaded both and of the Ca2+ induced by 10 were increased The and of the Ca2+responses increased from to and from to in and in 10 In inhibition of the RYR3 subtype by treatment with antisense oligonucleotides no on the and of Ca2+ In contrast, in 10 inhibition of RYR3 reduced both the and the of the Ca2+ the and of the Ca2+ responses in cells in 10 were similar to in cells in these results that the RYR3 is for the of the Ca2+ in Ca2+-overloaded that the of of the SR can be by Ca2+ S. Biophys. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Membr. Biol. 1994; PubMed Scopus Google Scholar, S. S. J. Physiol. ( Lond. ). 1999; PubMed Scopus Google Scholar), but the of the different RYR subtypes in increased Ca2+ release has been on the antisense strategy, the that, in rat portal vein myocytes, the RYR3 becomes activable under conditions of increased SR Ca2+ loading and is for the in Ca2+ release during and global Ca2+ the of both RYR1 and RYR2 but not of RYR3 to in Ca2+-overloaded myocytes. of RYR3s revealed that these receptors were in freshly dissociated and cultured portal vein myocytes. Since the of RYR3 was in the the that RYR3s not Ca2+ release of the conditions for an increased SR Ca2+ loading, as previously reported in cardiac myocytes Cannell M.B. E. Lederer W.J. Science. PubMed Scopus Google Scholar, L.A. J. E. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus (78) Google Scholar), that a of 1 in 10 was for vascular myocytes to a found that inhibition of Ca2+ the SR by the Ca2+ responses, whereas of Ca2+ for 10 no on the increased Ca2+ responses in 10 indicating that were on Ca2+ release from the the conditions of increased SR Ca2+ loading, the of spontaneous Ca2+ sparks was The number of vascular myocytes showing spontaneous was from of cells to more and the number of was similar in Ca2+ has been reported after a SR Ca2+ in myocytes H. Blatter L.A. J. Physiol. 1997; 272: PubMed Google Scholar). in smooth muscle cells have shown that the Ca2+ is on the Ca2+ K. J. Physiol. 1999; PubMed Scopus Google Scholar), but the RYR subtypes in have not been of RYR1 or RYR2 by treatment with antisense oligonucleotides strongly reduced the number of vascular myocytes with spontaneous Ca2+ whereas inhibition of RYR3 was inhibition of both RYR1 and RYR2 was not with inhibition of RYR in with showing that both RYR1 and RYR2 are required for activation of Ca2+ sparks under conditions of Ca2+ loading 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 that of were not in cells with an T. S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) after inhibition of both RYR1 and RYR2 by treatment with antisense oligonucleotides the that RYR3s not form not to Ca2+ is in with showing that with properties were in both and skeletal cells Takeshima H. Biophys. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). the of Ca2+ sparks the and in cells and in cells with were not the that RYR1 and RYR2 are in Ca2+ in vascular myocytes. can be to increased Ca2+ and the number of spontaneous Ca2+ increased an of a as previously in cardiac myocytes S. Biophys. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the conductance as well as the of RYR1 and RYR2. Our that the Ca2+ were not in 10 and the from single channel showing that increased the number of rather their S. Biophys. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) not Ca2+ release would be with that increased activated by as J. Membr. Biol. 1994; PubMed Scopus Google Scholar). the these three be The sensitivity of RYR subtypes to [Ca2+]i was by Ca2+ by flash photolysis of caged The of for the in 10 in cells and in or that the three RYR subtypes have a similar sensitivity to activation by local increases in [Ca2+]i. have previously that the Ca2+ for activation of Ca2+ sparks was a that is not different from the for activation of the RYR1 from skeletal muscle and of the RYR3 from in cells 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). However, found a sensitivity for Ca2+ of the RYR3 from skeletal muscle (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, 19Sonnleitner A. Conti A. Bertocchini F. Schindler H. EMBO J. 1998; 17: PubMed Scopus Google Scholar, T. S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). be to of rat portal vein myocytes expressed L. and J. in to skeletal which expressed A. M. K. K. Y. H. FEBS Lett. 1996; PubMed Scopus Google Scholar). activation of RYR3s not to in vascular myocytes, results with flash photolysis of caged Ca2+ and that local increases in [Ca2+]i induced or in to activation of channels are to RYR3s under conditions of increased SR Ca2+ results the that RYR3 a in to be activable by local increases in [Ca2+]i. cells were with or both and Ca2+-induced Ca2+ responses were not that activation of RYR3s for the inhibition of or is in with the that the single channel properties of the RYR3 from of the of the RYR3 activated by Ca2+ is to whereas that of the RYR1 is and the RYR3 is 10 to by the RYR1 (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). have that the expressed in is to in the of 1 and not M. E. Schindler H. FEBS Lett. 2000; PubMed Scopus Google Scholar). Our that the of the Ca2+ in 2 is that in is in with the of RYR3 with that of the RYR subtypes. cells their Ca2+ in by Ca2+ the SR and Ca2+ to the that and Ca2+ of the SR be of of in cardiac myocytes has been with an in SR Ca2+ Biophys. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Since both RYR1 and RYR2 activated are from as a of RYR Lederer W.J. Science. 1995; PubMed Scopus Google Scholar) or by Ca2+ Y. Cheng H. S. A. 1998; PubMed Scopus Google Scholar), can be that the which has been reported to be to Ca2+ concentrations (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, M. E. Schindler H. FEBS Lett. 2000; PubMed Scopus Google Scholar), Ca2+ release the RYR subtypes are In these results show that, in vascular myocytes, RYR3 can be activated by caffeine and local increases in under conditions of increased SR Ca2+ N. for