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Oxidation of methionine in proteins of isolated rat heart myocytes and tissue slices by neutrophil-generated oxygen free radicals
J Mol Cell Cardiol 18 (Supplement 3) (1986)
~ INO
ACID SEQUENCE OF CALSEQt~STRIN DEDUCED FROM cDNA AND PEPTIDE SEQDEI~CING. L. Fliegel, 2M. Ohnishi, IV.K. Khanna, 2R.A.F. Reithmeier and ID.H. MacLennan. iBanting and Best Department of Medical Research, University of Toronto, Toronto, Ontario and 2Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada. Calsequestrin is a major protein of the sarcoplasmic reticulum of both skeletal and cardiac muscle. It i~ located in the l~nen of the terminal cisternae where it binds and concentrates Ca Z+. Over half of the protein, including the amino and carboxyl-termini, has been sequenced by conventional methods. Synthetic oligon%~leotides (17 mers) encoding peptide sequences near the carboxyl-terminus were synthesized and the 3' synthetic sequence was used as a primer for reverse transcription of isolated from 14 day old rabbit skeletal muscle. A cDNA library of 3000 clones was made in the vector pBR322 and screened with a second oligomer 5' to the primer to obtain calsequestrin clones. Clones isolated from this and other full length libraries were sequenced to obtain the deduced amino acid sequence of calsequestrin and to study the molecular genetics of the protein. ('Supported by the MRC of Canada, the Muscular Dystrophy Association of Canada and the Heart and Stroke Foundation of Ontario).
OXIDATION OF METHIONINE IN PROTEINS OF ISOLATEn RAT HEART MYOCYTESAND TISSUE SLICES BY NEUTROPHIL-GENERATED OXYGENFREE RADICALS. H. Fliss, G. %cherty. Department of Physiology, School of Medicine, University of Ottawa, Ottawa, Canada, KIH ~M5. Much of the cellular damage observed durinQ reperfusion o~ ischemic myocardial tissues is apparently attributable to the formation of oxyqen free radicals, A large portion of these oxidants may be produced by neutrophils. We have examined the effects of neutrophil-generated oxidants on the proteins of isolated rat heart myocytes, as well as tissue slices, by determining the level of methionine oxidation. Myocyt~s and tissue slices were exposed to neutroDhils (rat peritoneal or human, 5 x 10~/ml) which had been activated with phorbol myristate acetate. Samples were then assayed for the presence of oxidized methionine using a recently developed assay for methionine sulfoxide !PNAS, 80, 7160, 1983). We found that in both myocytes and tissue slices a substantial portion (approximately 8%) of a l l the methionine residues was in the sulfoxide form prior to exposure to neutrophils. In samples exposed to neutrophil oxidants for 60 minutes the concentration of methionine sulfoxide approximately doubled. This level of oxidation was comoarab!e to that obtained with 1-2 mM hypochlorous acid, one of the more potent oxidants produced by neutrophils. Methionine was effectively protected from neutrophil-, and hypochlorous acid-mediated oxidation by thiol reQents I d i t h i o t h r e i t o l , Qlutathione). These data suggest that neutrophils may damaQereperfused myocardial tissue throuqh the oxidation of proteins.
INTERACTION OF CALCIUM ENTRY BLOCKERS AND ADRENERGIC SYSTEM. F.M. Fouad, R. Pedrinelli, R.C. Tarazi. Heart and Hypertension Department, Research Division, Cleveland Clinic Foundation, Cleveland, Ohio, U.S.A. Calcium influx plays a role in the response to receptor-mediated stimuli. In vivo experiments have shown that Ca++ influxes selectively influence alpha-2 adrenergic respooses with a preferential antagonism of alpha-2 adrenergic mediated vasoconstriction to calcium entry bioclede (CEB). However, studies in isolated vascular beds (rat hind quarter preparation demonstrated that both alpha-1 and alpha-2 adrenergic mediated vasoconstriction were equally sensitive to CEB. There was no clear evidence that beta-receptors were spared since increases in heart rate during treatment with CEB were only partly inhibited by beta-blockade indicating that a non-adrenergic mechanism (e.g. suppressed vaga] tone) is also involved in CEB-induced tachycardia. Baroreceptor sensitivity was reported to be normal during acute treatment with CEB but the baroceptor reflex was reset to a lower presssure after chronic therapy. Also, CEB did not seem to alter central peurones of the BR arc since they did not abolish the bradycardia induced by electrical stimulation of the central end of the carotid sinus nerve. Finally, release of neurotransmitters was not suppressed by CEB. Therefore, CEB may affect peripheral sensory baroreceptor function, alpha and p a r t i a l l y beta receptor responses but not vagal efferent responses nor release of neurotransmitters.
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~ INO
ACID SEQUENCE OF CALSEQt~STRIN DEDUCED FROM cDNA AND PEPTIDE SEQDEI~CING. L. Fliegel, 2M. Ohnishi, IV.K. Khanna, 2R.A.F. Reithmeier and ID.H. MacLennan. iBanting and Best Department of Medical Research, University of Toronto, Toronto, Ontario and 2Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada. Calsequestrin is a major protein of the sarcoplasmic reticulum of both skeletal and cardiac muscle. It i~ located in the l~nen of the terminal cisternae where it binds and concentrates Ca Z+. Over half of the protein, including the amino and carboxyl-termini, has been sequenced by conventional methods. Synthetic oligon%~leotides (17 mers) encoding peptide sequences near the carboxyl-terminus were synthesized and the 3' synthetic sequence was used as a primer for reverse transcription of isolated from 14 day old rabbit skeletal muscle. A cDNA library of 3000 clones was made in the vector pBR322 and screened with a second oligomer 5' to the primer to obtain calsequestrin clones. Clones isolated from this and other full length libraries were sequenced to obtain the deduced amino acid sequence of calsequestrin and to study the molecular genetics of the protein. ('Supported by the MRC of Canada, the Muscular Dystrophy Association of Canada and the Heart and Stroke Foundation of Ontario).
OXIDATION OF METHIONINE IN PROTEINS OF ISOLATEn RAT HEART MYOCYTESAND TISSUE SLICES BY NEUTROPHIL-GENERATED OXYGENFREE RADICALS. H. Fliss, G. %cherty. Department of Physiology, School of Medicine, University of Ottawa, Ottawa, Canada, KIH ~M5. Much of the cellular damage observed durinQ reperfusion o~ ischemic myocardial tissues is apparently attributable to the formation of oxyqen free radicals, A large portion of these oxidants may be produced by neutrophils. We have examined the effects of neutrophil-generated oxidants on the proteins of isolated rat heart myocytes, as well as tissue slices, by determining the level of methionine oxidation. Myocyt~s and tissue slices were exposed to neutroDhils (rat peritoneal or human, 5 x 10~/ml) which had been activated with phorbol myristate acetate. Samples were then assayed for the presence of oxidized methionine using a recently developed assay for methionine sulfoxide !PNAS, 80, 7160, 1983). We found that in both myocytes and tissue slices a substantial portion (approximately 8%) of a l l the methionine residues was in the sulfoxide form prior to exposure to neutrophils. In samples exposed to neutrophil oxidants for 60 minutes the concentration of methionine sulfoxide approximately doubled. This level of oxidation was comoarab!e to that obtained with 1-2 mM hypochlorous acid, one of the more potent oxidants produced by neutrophils. Methionine was effectively protected from neutrophil-, and hypochlorous acid-mediated oxidation by thiol reQents I d i t h i o t h r e i t o l , Qlutathione). These data suggest that neutrophils may damaQereperfused myocardial tissue throuqh the oxidation of proteins.
INTERACTION OF CALCIUM ENTRY BLOCKERS AND ADRENERGIC SYSTEM. F.M. Fouad, R. Pedrinelli, R.C. Tarazi. Heart and Hypertension Department, Research Division, Cleveland Clinic Foundation, Cleveland, Ohio, U.S.A. Calcium influx plays a role in the response to receptor-mediated stimuli. In vivo experiments have shown that Ca++ influxes selectively influence alpha-2 adrenergic respooses with a preferential antagonism of alpha-2 adrenergic mediated vasoconstriction to calcium entry bioclede (CEB). However, studies in isolated vascular beds (rat hind quarter preparation demonstrated that both alpha-1 and alpha-2 adrenergic mediated vasoconstriction were equally sensitive to CEB. There was no clear evidence that beta-receptors were spared since increases in heart rate during treatment with CEB were only partly inhibited by beta-blockade indicating that a non-adrenergic mechanism (e.g. suppressed vaga] tone) is also involved in CEB-induced tachycardia. Baroreceptor sensitivity was reported to be normal during acute treatment with CEB but the baroceptor reflex was reset to a lower presssure after chronic therapy. Also, CEB did not seem to alter central peurones of the BR arc since they did not abolish the bradycardia induced by electrical stimulation of the central end of the carotid sinus nerve. Finally, release of neurotransmitters was not suppressed by CEB. Therefore, CEB may affect peripheral sensory baroreceptor function, alpha and p a r t i a l l y beta receptor responses but not vagal efferent responses nor release of neurotransmitters.
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