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A quantitative assay for atrial natriuretic factor (ANF)-mRNA
J Mol
Cell
Cardiol
24
(Supplement
I) (1992)
0-18-8 ISCHAEMIC
PRECONDITIONING CAUSES RAPID ELEVATION OF 65kDa STRESS PROTEIN IN RABBIT HEART. Richard J. Heads. Vanlata C. Patel, David S. Latchman and &mk M. Yellon. The Hatter hpartnmt of Aca&mic Cardiology, University College Hospital, London, UK. Plwious shldies have demonstrated that tk promtim affaded by ischaemlc pmconditiolling (PC) is appatently indepetxlent of & novo ptotein synthesis. In a& to investigate the possible role of individual smss proteins we have urasuted levels of bath the 70 kD (hsp 70) and 60 kD (hsp 60) families of stnss proteins by SDS-PAGE and Western blotting. Whereas hsp 70i is induced by many stresses (including heat) and is pFtdaninantly cytoplasmic, hsp 65 is mildly heat inducible but has a constitutive function in the n-folding of mitocho&ially impor& pmteins and as such, may implicate the mitochondria as a possible intracellular site for pmtection. Levels of the inducible hsp 70i and hsp 65 were measured in frozzn biopsies of LV ischaemic and RV normal myocardium following: (1) control untrcahcd, (2) control sham ligated, (3) 5 mins occlusion+ 10 minr tqerfusion (PC)? (4) 30 min ischaemic insult (I) and (5) 2 hrs tepetfusion (R), in anaesthetised rabbits. A rapid and transient inuease in LV and RV hsp 65 occurs to high levels compared to that in control (untrtated) hearts following PC. Hsp 65 appears to dccnase during subsequent I. ln contrast, levels of hsp 70i remained constant during PC and I. High levels of hsp 65 wen also detected in 2 of 4 control sham ligated heatts which had a coronary ligaaue placed but not occlu&d. Hsp 65 therefore, may be induced by dimct myocardiel injury as well as during ischaemia/xepcfision. It is at present unknown whether changes in hsp 65 turnover/expression an involved in the preconditioning mechanism.
O-19-1
A QUANTITATIVE ASSAY FOR ATRIAL NATRIURETIC FACTOR (ANF) -mRNA Laura Comini, Gianni Mantero, AnnaFranca Pan& Roberto Ferrari. Chair of Cardiology and Chemistry, University of Brescia, Brescia, Italy. Evaluation of ANF-mRNA expression in cardiac or other tissues has proved to be an important research tool. The available methods, however, suffers from the limit that quantitation is accomplished in “relative units”. No methods for absolute quantitation of ANF-mRNA have been so far published. We designed a dot blot assay which allows absolute quantitation of ANF-mRNA. The assay is based on the use of “in vitro” trabscribed RNA from a 429 bp insert of ANF gene in Bluescript vector. cRNA was prepared in batch, purified by ion-exchange chromatography and accurately quantified by spectrophotometric reading at 260/280 nm. We then evaluated ANF-mRNA expression in the four cardiac chambers from normal Sprague Dowley rats by dot blot analysis with s*P-end labelled 4Omer oligonucleotide probe or with 32P RNA probe obtained by “in vitro” transcription. The amout of bound probe was evaluated by liquid scintillation and compared to the standard curve of cRNA(from 20 ng to 10 pg) loaded on to the same membrane. The filters were stripped and rehybridized with an oligo dT probe. The sensitivity of the assay is 100 pg. This assay is now being applied to the evaluation of ANFmRNA expression in congestive cardiac failure and in hypertrophic Sprague Dowley rats.
o-19-2
ISUiAEMA4EFERF!JSION STIRAATED ISCLATEDlXRFUXD RATtEPRT
ATRIAL NATRILETIC
RPTIU
Rwanda Centre
(ANP)fiELEA?X
BY THE
Lochner, Sonia Genade, R&lle Routon. Department of medical Physiology and Biochemistry, MRC for Nolecular and Cellular Biology, Medical Faculty, University of Stellenbosch, Tygerberg, Republic of South Africa. Recent studies reported the stimulatory effects of Ca 2+ (1) c h anneQnd CX,-receptor agonists (2) on ANP release by atria1 tissue. In view of the increased cytosolic Ca (3) and release of endogenous catecholamines (4) cmurring during ischaemia-reperfusion, the Possibility of ANP release under these conditions was investigated. ANP release in the coronary effluent of the isolated perfused rat heart was monitored before and after induction of ischamia. Reperfusion after both 15 or 25 min ischaemia, caused a lo-fold irmrediate increase in ANP release which was maintained for at least 20 min. Normothermic cardioplegic arrest for 35 min resulted in similar high rates of ANQ release both into the cardioplegic solution and the reperfusion fluid. Hypothermic cardioplegic arrest significantly attenuated ANP release during arrest and reperfusion. Exposure to ischaemia also caused loss of stretch-induced ANP release: whereas atria1 stretch (induced by switching from retrograde to working heart perfusion) caused a 2-3 fold increase in ANP release during the control period, it had no additional effect on ANP release during reperfusion. 1. Ruskoaho, H. et al., 6.6. Aes cm. 138, 266, 1966. 2. Shields, P.P., U&&ski, L.C. J. Biol. Chem. 264, 9322, 1989. 3. Bourdillon P.D.V., Poole-Wilson, P.A. Cardiovasc. Res. 15, 121, 1981. 4. c;rc. Res. 55, 669, 1964. Schtimig, A. et al., 5.94
Cell
Cardiol
24
(Supplement
I) (1992)
0-18-8 ISCHAEMIC
PRECONDITIONING CAUSES RAPID ELEVATION OF 65kDa STRESS PROTEIN IN RABBIT HEART. Richard J. Heads. Vanlata C. Patel, David S. Latchman and &mk M. Yellon. The Hatter hpartnmt of Aca&mic Cardiology, University College Hospital, London, UK. Plwious shldies have demonstrated that tk promtim affaded by ischaemlc pmconditiolling (PC) is appatently indepetxlent of & novo ptotein synthesis. In a& to investigate the possible role of individual smss proteins we have urasuted levels of bath the 70 kD (hsp 70) and 60 kD (hsp 60) families of stnss proteins by SDS-PAGE and Western blotting. Whereas hsp 70i is induced by many stresses (including heat) and is pFtdaninantly cytoplasmic, hsp 65 is mildly heat inducible but has a constitutive function in the n-folding of mitocho&ially impor& pmteins and as such, may implicate the mitochondria as a possible intracellular site for pmtection. Levels of the inducible hsp 70i and hsp 65 were measured in frozzn biopsies of LV ischaemic and RV normal myocardium following: (1) control untrcahcd, (2) control sham ligated, (3) 5 mins occlusion+ 10 minr tqerfusion (PC)? (4) 30 min ischaemic insult (I) and (5) 2 hrs tepetfusion (R), in anaesthetised rabbits. A rapid and transient inuease in LV and RV hsp 65 occurs to high levels compared to that in control (untrtated) hearts following PC. Hsp 65 appears to dccnase during subsequent I. ln contrast, levels of hsp 70i remained constant during PC and I. High levels of hsp 65 wen also detected in 2 of 4 control sham ligated heatts which had a coronary ligaaue placed but not occlu&d. Hsp 65 therefore, may be induced by dimct myocardiel injury as well as during ischaemia/xepcfision. It is at present unknown whether changes in hsp 65 turnover/expression an involved in the preconditioning mechanism.
O-19-1
A QUANTITATIVE ASSAY FOR ATRIAL NATRIURETIC FACTOR (ANF) -mRNA Laura Comini, Gianni Mantero, AnnaFranca Pan& Roberto Ferrari. Chair of Cardiology and Chemistry, University of Brescia, Brescia, Italy. Evaluation of ANF-mRNA expression in cardiac or other tissues has proved to be an important research tool. The available methods, however, suffers from the limit that quantitation is accomplished in “relative units”. No methods for absolute quantitation of ANF-mRNA have been so far published. We designed a dot blot assay which allows absolute quantitation of ANF-mRNA. The assay is based on the use of “in vitro” trabscribed RNA from a 429 bp insert of ANF gene in Bluescript vector. cRNA was prepared in batch, purified by ion-exchange chromatography and accurately quantified by spectrophotometric reading at 260/280 nm. We then evaluated ANF-mRNA expression in the four cardiac chambers from normal Sprague Dowley rats by dot blot analysis with s*P-end labelled 4Omer oligonucleotide probe or with 32P RNA probe obtained by “in vitro” transcription. The amout of bound probe was evaluated by liquid scintillation and compared to the standard curve of cRNA(from 20 ng to 10 pg) loaded on to the same membrane. The filters were stripped and rehybridized with an oligo dT probe. The sensitivity of the assay is 100 pg. This assay is now being applied to the evaluation of ANFmRNA expression in congestive cardiac failure and in hypertrophic Sprague Dowley rats.
o-19-2
ISUiAEMA4EFERF!JSION STIRAATED ISCLATEDlXRFUXD RATtEPRT
ATRIAL NATRILETIC
RPTIU
Rwanda Centre
(ANP)fiELEA?X
BY THE
Lochner, Sonia Genade, R&lle Routon. Department of medical Physiology and Biochemistry, MRC for Nolecular and Cellular Biology, Medical Faculty, University of Stellenbosch, Tygerberg, Republic of South Africa. Recent studies reported the stimulatory effects of Ca 2+ (1) c h anneQnd CX,-receptor agonists (2) on ANP release by atria1 tissue. In view of the increased cytosolic Ca (3) and release of endogenous catecholamines (4) cmurring during ischaemia-reperfusion, the Possibility of ANP release under these conditions was investigated. ANP release in the coronary effluent of the isolated perfused rat heart was monitored before and after induction of ischamia. Reperfusion after both 15 or 25 min ischaemia, caused a lo-fold irmrediate increase in ANP release which was maintained for at least 20 min. Normothermic cardioplegic arrest for 35 min resulted in similar high rates of ANQ release both into the cardioplegic solution and the reperfusion fluid. Hypothermic cardioplegic arrest significantly attenuated ANP release during arrest and reperfusion. Exposure to ischaemia also caused loss of stretch-induced ANP release: whereas atria1 stretch (induced by switching from retrograde to working heart perfusion) caused a 2-3 fold increase in ANP release during the control period, it had no additional effect on ANP release during reperfusion. 1. Ruskoaho, H. et al., 6.6. Aes cm. 138, 266, 1966. 2. Shields, P.P., U&&ski, L.C. J. Biol. Chem. 264, 9322, 1989. 3. Bourdillon P.D.V., Poole-Wilson, P.A. Cardiovasc. Res. 15, 121, 1981. 4. c;rc. Res. 55, 669, 1964. Schtimig, A. et al., 5.94