Manjunatha B. Bhat

Cytochrome c is released from mitochondria into the cytosol in cells undergoing apoptosis. The temporal relationship between cytochrome c release and loss of mitochondrial membrane potential was monitored by laser-scanning confocal microscopy in single living pheochromocytoma-6 cells undergoing apoptosis induced by staurosporine. Mitochondrial membrane potential monitored by tetramethylrhodamine methyl ester decreased abruptly in individual cells from 2 to 7 h after treatment with staurosporine. Depolarization was accompanied by cytochrome c release documented by release of transfected green fluorescent protein-tagged cytochrome c in these cells. The results show that mitochondrial depolarization accompanies cytochrome c release in pheochromocytoma-6 cells undergoing apoptosis.

Dantrolene is a drug that suppresses intracellular Ca2+ release from sarcoplasmic reticulum (SR) in skeletal muscle and is used as a therapeutic agent in individuals susceptible to malignant hyperthermia. Although its precise mechanism of action has not been elucidated, we have identified the N-terminal region (amino acids 1–1400) of the skeletal muscle isoform of the ryanodine receptor (RyR1), the primary Ca2+ release channel in SR, as a molecular target for dantrolene using the photoaffinity analog [3H]azidodantrolene. Here, we demonstrate that heterologously expressed RyR1 retains its capacity to be specifically labeled with [3H]azidodantrolene, indicating that muscle specific factors are not required for this ligand-receptor interaction. Synthetic domain peptides of RyR1 previously shown to affect RyR1 function in vitro andin vivo were exploited as potential drug binding site mimics and used in photoaffinity labeling experiments. Only DP1 and DP1–2s, peptides containing the amino acid sequence corresponding to RyR1 residues 590–609, were specifically labeled by [3H]azidodantrolene. A monoclonal anti-RyR1 antibody that recognizes RyR1 and its 1400-amino acid N-terminal fragment recognizes DP1 and DP1–2s in both Western blots and immunoprecipitation assays and specifically inhibits [3H]azidodantrolene photolabeling of RyR1 and its N-terminal fragment in SR. Our results indicate that synthetic domain peptides can mimic a native, ligand-binding conformation in vitro and that the dantrolene-binding site and the epitope for the monoclonal antibody on RyR1 are equivalent and composed of amino acids 590–609. Dantrolene is a drug that suppresses intracellular Ca2+ release from sarcoplasmic reticulum (SR) in skeletal muscle and is used as a therapeutic agent in individuals susceptible to malignant hyperthermia. Although its precise mechanism of action has not been elucidated, we have identified the N-terminal region (amino acids 1–1400) of the skeletal muscle isoform of the ryanodine receptor (RyR1), the primary Ca2+ release channel in SR, as a molecular target for dantrolene using the photoaffinity analog [3H]azidodantrolene. Here, we demonstrate that heterologously expressed RyR1 retains its capacity to be specifically labeled with [3H]azidodantrolene, indicating that muscle specific factors are not required for this ligand-receptor interaction. Synthetic domain peptides of RyR1 previously shown to affect RyR1 function in vitro andin vivo were exploited as potential drug binding site mimics and used in photoaffinity labeling experiments. Only DP1 and DP1–2s, peptides containing the amino acid sequence corresponding to RyR1 residues 590–609, were specifically labeled by [3H]azidodantrolene. A monoclonal anti-RyR1 antibody that recognizes RyR1 and its 1400-amino acid N-terminal fragment recognizes DP1 and DP1–2s in both Western blots and immunoprecipitation assays and specifically inhibits [3H]azidodantrolene photolabeling of RyR1 and its N-terminal fragment in SR. Our results indicate that synthetic domain peptides can mimic a native, ligand-binding conformation in vitro and that the dantrolene-binding site and the epitope for the monoclonal antibody on RyR1 are equivalent and composed of amino acids 590–609. Dantrolene is a hydantoin derivative used to treat malignant hyperthermia (MH), 1The abbreviations used are: MH, malignant hyperthermia; AMP-PCP, β,γ-methyleneadenosine 5′-triphosphate; CHO, Chinese hamster ovary; DHPR, dihydropyridine receptor; DP, domain peptide; mAb, monoclonal antibody; PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid); PVDF, polyvinyl difluoride; RyR, ryanodine receptor; SCR, scrambled peptide; SR, sarcoplasmic reticulum. a rare, pharmacogenetic disorder of skeletal muscle characterized by uncontrolled Ca2+ release from sarcoplasmic reticulum (SR) stores in response to volatile anesthetics. Triggering of MH results in hypercontracture, hyperthermia, and eventually death. Therapeusis with dantrolene results from its effective suppression of skeletal muscle SR Ca2+ release, presumably by modulating the activity of the ryanodine receptor (RyR1), the primary Ca2+ release channel in skeletal muscle via that Ca2+ stored in the SR is released into the myoplasm to initiate muscle contraction in response to membrane depolarization (1Fruen B.R. Mickelson J.R. Louis C.F. J. Biol. Chem. 1997; 272: 26965-26971Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). The genetic defect that causes MH in humans has been linked to point mutations in RyR1 in about 50% of patients. To date, some 26 MH-linked mutations in RyR1 have been identified: 9 in the extreme N-terminal region, 16 in the central region, and 1 in the extreme C-terminal region (2McCarthy T.V. Quane K.A. Lynch P.J. Hum. Mutat. 2000; 15: 410-417Crossref PubMed Scopus (307) Google Scholar, 3Sambuughin N. Nelson T.E. Jankovic J. Xin C. Meissner G. Mullakandov M., Ji, J. Rosenberg H. Sivakumar K. Goldfarb L.G. Neuromuscul. Disord. 2001; 11: 530-537Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 4Froemming G.R. Ohlendieck K. Front. Biosci. 2001; 6: D65-D74Crossref PubMed Scopus (26) Google Scholar, 5MacLennan D.H. Eur. J. Biochem. 2000; 267: 5291-5297Crossref PubMed Scopus (188) Google Scholar, 6Jurkat-Rott K. McCarthy T. Lehmann-Horn F. Muscle Nerve. 2000; 23: 4-17Crossref PubMed Scopus (280) Google Scholar, 7Denborough M. Lancet. 1998; 352: 1131-1136Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Interestingly, a single amino acid deletion, rather than mutation, in the central region of RyR1 (Glu2347) has also been found to be associated with MH susceptibility (8Sambuughin N. McWilliams S. de Bantel A. Sivakumar K. Nelson T.E. Am. J. Hum. Genet. 2001; 69: 204-208Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Whether dantrolene suppression of Ca2+ release occurs via direct interaction with RyR1 is not entirely clear. Some have found evidence suggesting that RyR1 is not the target (9Palnitkar S.S. Mickelson J.R. Louis C.F. Parness J. Biochem. J. 1997; 326: 847-852Crossref PubMed Scopus (41) Google Scholar, 10Szentesi P. Collet C. Sarkozi S. Szegedi C. Jona I. Jacquemond V. Kovacs L. Csernoch L. J. Gen. Physiol. 2001; 118: 355-375Crossref PubMed Scopus (81) Google Scholar), and others have found evidence that it is (1Fruen B.R. Mickelson J.R. Louis C.F. J. Biol. Chem. 1997; 272: 26965-26971Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 11Nelson T.E. Lin M. Zapata-Sudo G. Sudo R.T. Anesthesiology. 1996; 84: 1368-1379Crossref PubMed Scopus (76) Google Scholar, 12Zhao F., Li, P. Chen S.R. Louis C.F. Fruen B.R. J. Biol. Chem. 2001; 276: 13810-13816Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Given the controversy as to the mechanism and, hence, the targets of dantrolene action, we have embarked on a project to directly identify the molecular target(s) of dantrolene by photoaffinity labeling. Knowing the target(s) of dantrolene binding would allow genetic and physiological manipulation of these molecular entities to not only elucidate the mechanism of action of this drug but also provide insights into the in vivo mechanisms controlling the RYR1 Ca2+ release channel in skeletal muscle excitation-contraction coupling and the pathophysiology of MH. Recently, we have demonstrated that [3H]azidodantrolene, a pharmacologically active, photoaffinity analog of dantrolene, specifically labels the N-terminal, 1400-amino acid residue fragment of RyR1 cleaved by n-calpain, a tissue-specific isoform of this Ca2+- and thiol-activated protease (13Paul-Pletzer K. Palnitkar S.S. Jimenez L.S. Morimoto H. Parness J. Biochemistry. 2001; 40: 531-542Crossref PubMed Scopus (41) Google Scholar). Several studies have demonstrated that this portion of the RyR plays a significant role in the regulation of channel function (14Wu Y. Aghdasi B. Dou S.J. Zhang J.Z. Liu S.Q. Hamilton S.L. J. Biol. Chem. 1997; 272: 25051-25061Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 15Aghdasi B. Zhang J.Z., Wu, Y. Reid M.B. Hamilton S.L. J. Biol. Chem. 1997; 272: 3739-3748Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 16Yamazawa T. Takeshima H. Shimuta M. Iino M. J. Biol. Chem. 1997; 272: 8161-8164Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 17Leong P. MacLennan D.H. J. Biol. Chem. 1998; 273: 29958-29964Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 18Leong P. MacLennan D.H. J. Biol. Chem. 1998; 273: 7791-7794Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Additionally, the nine, N-terminal mutations in RyR1 linked to MH alluded to above are localized within this region of the channel. The DHPR and RyR1 are intimate physiological partners during skeletal muscle excitation-contraction coupling. The evidence indicates that the DHPR is the voltage sensor in the T-tubule membrane that upon sensing depolarization undergoes a conformational change associated with intramolecular charge movement. This is believed to result in the movement of the intracellular loop between transmembrane domains II and III (II–III loop) of the DHPR α-1 subunit, which physically contacts the RyR1, inducing its opening and resultant Ca2+ release from the SR (for reviews see Refs. 19Protasi F. Front. Biosci. 2002; 7: D650-D658Crossref PubMed Google Scholar and 20Shoshan-Barmatz V. Ashley R.H. Int. Rev. Cytol. 1998; 183: 185-270Crossref PubMed Google Scholar). Previous studies have demonstrated the experimental utility of using synthetic domain peptides derived from the DHPR and RyR1 to define physiologically significant domains within the parent protein. In particular, peptides A and C of the DHPR II–III loop and domain peptides DP1 and DP4 of the RyR1 have been shown to be active in in vitro studies of excitation-contraction coupling (21El Hayek R. Saiki Y. Yamamoto T. Ikemoto N. J. Biol. Chem. 1999; 274: 33341-33347Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 22Yamamoto T. Ikemoto N. J. Biol. Chem. 2002; 277: 984-992Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar, 23Yamamoto T. Rodriguez J. Ikemoto N. J. Biol. Chem. 2002; 277: 993-1001Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 24Ikemoto N. Yamamoto T. Front. Biosci. 2002; 7: D671-D683Crossref PubMed Google Scholar). Interestingly, DP1 and DP4 are from the N-terminal and central regions of the RyR1 that are “hot spots” for mutations conferring sensitivity to MH and/or the rare myopathy, central core disease (24Ikemoto N. Yamamoto T. Front. Biosci. 2002; 7: D671-D683Crossref PubMed Google Scholar). We therefore used these peptides as potential in vitro targets for [3H]azidodantrolene photoaffinity labeling, as well as others from RyR1 not demonstrated to have physiological activity as negative controls, in our attempt to define the dantrolene-binding sequence(s) in these proteins. In the present study, we demonstrate specific [3H]azidodantrolene photolabeling of two domain peptides containing the core sequence corresponding to amino acid residues 590–609 on RyR1-DP1 (amino acids 590–609) and DP1–2s, an elongated version of the skeletal sequence present in DP1 (590–628). A monoclonal anti-RyR1 antibody raised against rabbit terminal cisternae (25Campbell K.P. Knudson C.M. Imagawa T. Leung A.T. Sutko J.L. Kahl S.D. Raab C.R. Madson L. J. Biol. Chem. 1987; 262: 6460-6463Abstract Full Text PDF PubMed Google Scholar) that recognizes both the intact rabbit RyR1 and the 172 kDa, n-calpain-cleaved, N-terminal fragment of this channel (13Paul-Pletzer K. Palnitkar S.S. Jimenez L.S. Morimoto H. Parness J. Biochemistry. 2001; 40: 531-542Crossref PubMed Scopus (41) Google Scholar), also recognizes these synthetic RyR1 peptides. This antibody specifically inhibited [3H]azidodantrolene photolabeling of RyR1 in SR in a concentration-dependent manner. These results indicate, therefore, that the dantrolene-binding site on RyR1 is comprised of amino acids 590–509. Dantrolene sodium·3.5H2O and azumolene sodium·2H2O were generous gifts of Proctor & Gamble (Norwich, NY). Polyclonal sheep anti-rabbit RyR1, monoclonal mouse (IgM) anti-rabbit RyR1 XA7 and rabbit anti-rabbit RyR1 C-terminal against a synthetic C-terminal RyR1 corresponding to amino acids were generous gifts of K. P. of skeletal muscle by H. and characterized as S.S. B. Jimenez L.S. Morimoto H. K. Parness J. J. Chem. 1999; PubMed Scopus Google Scholar), and the specific activity to be RyR1 domain peptides DP1–2s, and DP1 scrambled and rabbit skeletal muscle DHPR peptides and were on an A and by as (21El Hayek R. Saiki Y. Yamamoto T. Ikemoto N. J. Biol. Chem. 1999; 274: 33341-33347Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, Hayek R. Ikemoto N. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). DP1 scrambled by DP1 were by and and were for The of RyR1 into the the of the into using the as previously H. T. J. S. A. PubMed Scopus Google Scholar, M. C. 1996; PubMed Scopus Google Scholar). of with RyR1 were using M.B. Hayek J. Takeshima H. J. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, M.B. J. Takeshima H. J. Biochem. J. 2000; PubMed Scopus Google Scholar). SR were from rabbit skeletal muscle in the of protease and as in M. Hamilton S.L. Biochem. PubMed Scopus (32) Google from were by of of as M.B. Hayek J. Takeshima H. J. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). specifically from used in membrane to specifically allow for of RyR1 and of its N-terminal present (13Paul-Pletzer K. Palnitkar S.S. Jimenez L.S. Morimoto H. Parness J. Biochemistry. 2001; 40: 531-542Crossref PubMed Scopus (41) Google Scholar, S. M. V. J. Biol. 1998; PubMed Scopus Google Scholar). SR and were with [3H]azidodantrolene in binding PIPES, containing in the of azumolene as previously (13Paul-Pletzer K. Palnitkar S.S. Jimenez L.S. Morimoto H. Parness J. Biochemistry. 2001; 40: 531-542Crossref PubMed Scopus (41) Google Scholar). of photolabeling by mouse were to the binding of and in the of protease with the of 1 Synthetic peptides were with in binding containing of in the of the were by for SR and for synthetic PubMed Scopus Google Scholar). and Western the were H. T. J. S. A. PubMed Scopus Google Scholar). as (13Paul-Pletzer K. Palnitkar S.S. Jimenez L.S. Morimoto H. Parness J. Biochemistry. 2001; 40: 531-542Crossref PubMed Scopus (41) Google Scholar). Western the were with primary antibody mAb, by were with using The using and the as a The using for and and the were The were for and binding and expressed as of the amino acids that the RyR1 we have shown that a dantrolene-binding site between amino acids 1 and of the N-terminal region of the channel (13Paul-Pletzer K. Palnitkar S.S. Jimenez L.S. Morimoto H. Parness J. Biochemistry. 2001; 40: 531-542Crossref PubMed Scopus (41) Google Scholar). In an attempt to a molecular to define the amino acids that the dantrolene-binding we RyR1 is of specifically with dantrolene in the of a muscle We that it this would our that the RyR1 is a target of dantrolene and we attempt to a region of the N-terminal portion of the channel that would its to dantrolene and in of the We used RyR1, expressed in as a target for our photoaffinity labeling experiments. from these with rabbit skeletal muscle SR as were with [3H]azidodantrolene in the of and as shown in 1 specific photolabeling of a corresponding to the RyR1 in SR in This photolabeling on the of This is to the for photolabeling of RyR1 and its fragment in SR previously (13Paul-Pletzer K. Palnitkar S.S. Jimenez L.S. Morimoto H. Parness J. Biochemistry. 2001; 40: 531-542Crossref PubMed Scopus (41) Google Scholar). The Western of the membrane used for with a anti-RyR1 equivalent in the experimental and that the in is specific photolabeling anti-RyR1 were in from These results demonstrate that dantrolene can to heterologously expressed RyR1 in the of and this binding is pharmacologically specific and on a of the channel protein. To the amino acids on RyR1 that the binding site for dantrolene, we to specifically a heterologously fragment containing amino acids This fragment not presumably of not We to synthetic domain peptides of RyR1 as targets for the dantrolene-binding We peptides with amino acid residues from within the N-terminal, 1400-amino acid region of RyR1 DP1–2s, and as well as peptides from the central region of RyR1 and from the II–III loop of the and These peptides were with [3H]azidodantrolene in the the of and the peptides were for specific photolabeling by The in specific photolabeling of DP1 (amino acid residues 590–609) and an elongated version of DP1–2s (amino acid residues (amino acid residues the peptides were not labeled The of [3H]azidodantrolene labeling of DP1 demonstrated by the of dantrolene and but not the to this interaction In of the DP1 sequence and only to labeling of These results that DP1 an in of the dantrolene-binding site on photolabeling of synthetic peptides. synthetic peptides were with [3H]azidodantrolene in the the of azumolene on and to of photolabeling of DP1 in the of dantrolene an drug as negative photolabeling of DP1 and scrambled peptides of and sequence of DP1 and scrambled Our has shown that an anti-RyR1 RyR1 and its fragment from SR (13Paul-Pletzer K. Palnitkar S.S. Jimenez L.S. Morimoto H. Parness J. Biochemistry. 2001; 40: 531-542Crossref PubMed Scopus (41) Google Scholar). Given the epitope of monoclonal and that the epitope of this monoclonal to be on the we that it that of our domain peptides corresponding to from the N-terminal region of RyR1 with this it it would demonstrate the of using synthetic peptides to mimic a epitope in this channel. to our results we demonstrated that only intact RyR1 and its N-terminal fragment were with the monoclonal antibody by photolabeling SR with [3H]azidodantrolene, these and with anti-RyR1 and the with anti-RyR1 C-terminal A that the recognizes RyR1 and its fragment but not the portion of the channel. The anti-RyR1 C-terminal on the recognizes the RyR1 and its C-terminal fragment but not its N-terminal fragment This to that the epitope of mAb, within the residues of the N-terminal region of To of our RyR1 domain peptides be by this we the peptides on a and the with Only DP1 and DP1–2s, both of which the corresponding core of amino acid residues of RyR1, were by the The scrambled DP1 peptides and were not by the antibody These results indicate that the epitope for the is within the DP1 of DP1 peptides by RyR1 domain DP1–2s, and DHPR II–III loop peptide; and and scrambled DP1 peptides and were on a and membrane and with Only the DP1 peptides are the results above demonstrate a between and specific [3H]azidodantrolene photolabeling of a sequence in the N-terminal of The of these two of that anti-RyR1 [3H]azidodantrolene labeling of RyR1 in SR. To this we SR in the of of mouse The a concentration-dependent of specific photolabeling of both the RyR1 as well as its this equivalent anti-RyR1 in of the of photolabeling the for the fragment The of of photolabeling of intact RyR1 by anti-RyR1 is in this of its and the of photolabeling to the N-terminal for the of photolabeling of the fragment results in of the sensitivity of the for the labeling of the RyR1 not a the two are not entirely This not the of specific of [3H]azidodantrolene photolabeling of both of the channel and of labeling is not to of mouse the used in mouse not affect photolabeling of the intact channel its fragment is our that the molecular mechanism of action of dantrolene to a of the of MH, the mechanism of excitation-contraction and the regulation of intracellular Ca2+ release from SR. A for molecular mechanism the of molecular The of amino acids in drug binding for the of to in drug Here, we have evidence that the amino acid sequence of the dantrolene-binding site on RyR1 to amino acids 590–609, corresponding to the synthetic domain The evidence for the DP1 sequence the dantrolene-binding sequence in RyR1 from our with the monoclonal In K.P. Knudson C.M. Imagawa T. Leung A.T. Sutko J.L. Kahl S.D. Raab C.R. Madson L. J. Biol. Chem. 1987; 262: 6460-6463Abstract Full Text PDF PubMed Google Scholar) an anti-RyR1 mAb, of that specifically Our present studies demonstrate that this antibody recognizes its epitope not only on the RyR1 and its N-terminal, fragment but also on the synthetic DP1 and DP1–2s, containing the core RyR1 sequence 590–609. this antibody specifically inhibits [3H]azidodantrolene photolabeling of RyR1 and its fragment in SR in a concentration-dependent manner. This result is with our the of a dantrolene-binding site on RyR1 C-terminal to the site between amino acids (13Paul-Pletzer K. Palnitkar S.S. Jimenez L.S. Morimoto H. Parness J. Biochemistry. 2001; 40: 531-542Crossref PubMed Scopus (41) Google Scholar). The that we were to demonstrate of [3H]azidodantrolene photolabeling of the fragment with a monoclonal antibody is a that the epitope by this amino acids 590–609, the only dantrolene-binding site on The of mouse required to about of specific photolabeling is to the of which are of the response of the to M. C. 1996; PubMed Scopus Google Scholar). These also the experimental evidence in synthetic domain peptides are of the of domains present in the parent RyR1 in this J. R.H. M. 2002; PubMed Scopus Google Scholar, I. I. Biochemistry. 2001; 40: PubMed Scopus Google Scholar, B. A. J. J. Biol. 2001; PubMed Scopus Google Scholar). Previous studies have shown that heterologously expressed RyR1 in M.B. J. Takeshima H. J. J. 1997; Full Text PDF PubMed Scopus Google Scholar) and to the of dantrolene binding to RyR1 the of factors in heterologously expressed Here, we have demonstrated that RyR1 can directly with dantrolene in the of a this interaction is by AMP-PCP, as it is in SR (13Paul-Pletzer K. Palnitkar S.S. Jimenez L.S. Morimoto H. Parness J. Biochemistry. 2001; 40: 531-542Crossref PubMed Scopus (41) Google Scholar), our that this interaction is pharmacologically indicates that this analog RyR1 directly in the of this channel to photolabeling of the synthetic on the be of specific of RyR1 of the on RyR1 would be to be associated with this acid our demonstrate that [3H]azidodantrolene photolabeling of the two DP1 peptides is of not Although we have evidence that the dantrolene-binding site on RyR1 is by this it is not the activity of this A of the physiological of dantrolene action in skeletal muscle has shown that dantrolene suppresses but intracellular Ca2+ release in and muscle from SR but has on RyR1 into P. Collet C. Sarkozi S. Szegedi C. Jona I. Jacquemond V. Kovacs L. Csernoch L. J. Gen. Physiol. 2001; 118: 355-375Crossref PubMed Scopus (81) Google Scholar). These results to that RyR1 is not the site of action of This result is in to our results above and of Nelson and T.E. Lin M. Zapata-Sudo G. Sudo R.T. Anesthesiology. 1996; 84: 1368-1379Crossref PubMed Scopus (76) Google Scholar). of these results are to be and the the dantrolene-binding site on RyR1 is to during the it dantrolene as the physiological of dantrolene binding to RyR1 the interaction of RyR1 factors that be during are not well in the the DP1 sequence in RyR1 with the rabbit that is an sequence in and a in with a amino acid to H. N. Y. PubMed Scopus Google Scholar). this sequence is the dantrolene-binding be to this evidence indicates that only the RyR1 and are to We have shown by [3H]azidodantrolene photolabeling of SR from skeletal muscle that only RyR1 is a target for dantrolene, not (13Paul-Pletzer K. Palnitkar S.S. Jimenez L.S. Morimoto H. Parness J. Biochemistry. 2001; 40: 531-542Crossref PubMed Scopus (41) Google Scholar). have shown that dantrolene can binding to RyR1 and but not (1Fruen B.R. Mickelson J.R. Louis C.F. J. Biol. Chem. 1997; 272: 26965-26971Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 12Zhao F., Li, P. Chen S.R. Louis C.F. Fruen B.R. J. Biol. Chem. 2001; 276: 13810-13816Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). the DP1 sequence is the dantrolene-binding is the isoform to this drug it the The our present of is that the within the conformation this sequence that dantrolene is to with to this of the channel that to the The for the in dantrolene sensitivity of to RyR1 are active in our the DP1 sequence of RyR1 is in Whether this isoform is to dantrolene in the in which this isoform is expressed to be studies that dantrolene has on muscle C. PubMed Scopus Google Scholar), studies that T. H. H. Gen. 1996; PubMed Scopus Google Scholar). the studies are by the that heterologously expressed has been shown to be to dantrolene F., Li, P. Chen S.R. Louis C.F. Fruen B.R. J. Biol. Chem. 2001; 276: 13810-13816Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). is a target of dantrolene, this result the of dantrolene in that and H. 2002; PubMed Scopus Google Scholar, C. Y. Y. J. 2001; PubMed Scopus (76) Google Scholar). Recently, Ikemoto and (21El Hayek R. Saiki Y. Yamamoto T. Ikemoto N. J. Biol. Chem. 1999; 274: 33341-33347Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, Hayek R. Ikemoto N. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) demonstrated that DP1 to SR RyR1, as by These have that DP1 be the conformation of the sequence in the channel with this domain for its domain on RyR1, in of the RyR1 domain (24Ikemoto N. Yamamoto T. Front. Biosci. 2002; 7: D671-D683Crossref PubMed Google N. Yamamoto T. 2000; PubMed Scopus Google Scholar, T. Ikemoto N. Biochemistry. 2002; PubMed Scopus Google Scholar). domain would the channel in a these result in of the channel. that the of DP1 into the domain to domain of RyR1, would result in of the conformational by these domains and to channel the above is the of the results to is that dantrolene inhibits Ca2+ release by the dantrolene of these result in of within RyR1 with the of channel and Ca2+ of the of the and of the dantrolene-binding site in the channel would to our of the regulation of RyR channel We the of the as a and the of this to our in and in

Retinal capillary pericytes undergo premature death, possibly by apoptosis, during the early stages of diabetic retinopathy. The alpha-oxoaldehyde, methylglyoxal (MGO), has been implicated as a cause of cell damage in diabetes. We have investigated the role of MGO and its metabolizing enzyme, glyoxalase I, in high glucose-induced apoptosis (annexin V binding) of human retinal pericyte (HRP). HRP incubated with high glucose (30 mm d-glucose) for 7 days did not undergo apoptosis despite accumulation of MGO. However, treatment with a combination of high glucose and S-p-bromobenzylglutathione cyclopentyl diester, a competitive inhibitor of glyoxalase I, resulted in apoptosis along with a dramatic increase in MGO. Overexpression of glyoxalase I in HRP protected against S-p-bromobenzylglutathione cyclopentyl diester-induced apoptosis under high glucose conditions. Incubation of HRP with high concentrations of MGO resulted in an increase of apoptosis relative to untreated controls. We found an elevation of nitric oxide (NO.) in HRP that was incubated with high glucose when compared with those incubated with either the l-glucose or untreated controls. When HRP were incubated with an NO. donor, DETANONOATE ((Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate), we observed both decreased glyoxalase I expression and activity relative to untreated control cells. Further studies showed that HRP underwent apoptosis when incubated with DETANONOATE and that apoptosis increased further on co-incubation with high glucose. Our findings indicate that glyoxalase I is critical for pericyte survival under hyperglycemic conditions, and its inactivation and/or down-regulation by NO. may contribute to pericyte death by apoptosis during the early stages of diabetic retinopathy.