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An experimental model of coronary microembolization
J Mol Cell Cardiol23 (Supplement III) (1991)
P-2-38 CHARACTERISTICS
AND Ca” SENSITIVITY OF FOLYPEOSPHOINOSITIDE SYNTHESIS IN RAT HEART SARCOLXMMA Nasrin Mesaeli, Ios MI Lamers‘, Vincenzo Panagia. Divisionof Cardi~~a~~ul~ Sciences,St BonifaceGH ResearchCentre,University of Manitoba,Winnipeg,CANADA and %ept. of Biochemistry,ErasmusUniversity, Rotterdam,THE NETHERLANDS. In vitro [?]ATP (0.25 mM)-induced phosphorylation of phosphoinositides was examined in rat heart sarcolemma (SL) after preincubation with alamethicin for 30 min at 30°C. The reaction was linear for 1 min. The optimal requirement for Mg” was 2.5 mM. The reaction was observed over a broad pH range (6-9) with an optimum at 7.5. Under these conditions the synthesis of phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5bisphosphate (PtdIns(4,5)PJ was 1101.0 f 78.8 and 115.3 f 13.3 pmol/mg/min, respectively. Exogenous PtdIns4P (lo-200 PM) progressively increased PtdIns(4,5)P, synthesis to a maximum of 3.7 fold. Furthermore, neomycin (an inhibitor of phosphoinositide hydrolysis) increased the formation of both PtdIns4P (2 fold) and PtdIns(4,5)P, (1.7 fold). l-100 I.IM free Ca” induced a progressive decline in both PtdIns4P and PtdIns(4,5)PZ formation with IDS, of 20 and 6.3 PM Ca”, respectively. However, neomycin did not change the inhibitory effect of Ca”‘, thus excluding the possibility of a Ca”-activated breakdown of phosphoinositides by phospholipase C. These results indicate the occurrence of a Ca” sensitive polyphosphoinositide-generating kinase system for the phosphatidylinositol signalling pathway in rat heart SL. (Supportedby MRC, CanadaandNATO, Belgium).
p-2-39a,-ADRENERGIC STIMULATION AND PROTEIN PHOSPHORYLATIONIN BEATING HEARTS Laszlo Talosi, Evangelia G Kranias. Department of Pharmacology and Cell Biophysics, University of Cincinnati, Cincinnati, OH 45267-0575 USA The present study examines the hypothesis that activation of protein kinase C and phosphorylation of key regulatory proteins may mediate the inotropic response of the heart to cl-adrenergie agonists. Rabbit hearts were perfused with modified Krebs-buffer containing 32P-orthophosphate and upon introduction of phenylephrine (IO uM) there was a 1.54-fold increase in +dP/dt. Examination of the protein kinase C activity levels in these hearts revealed a redistribution of the enzymatic activity from the cytosolic to the membranous fraction. Examination of the phosphate incorporation into cardiac proteins indicated: a) increases in a 15 kDa sarcolemmal and a 28 kDa cytosolic protein; b) no changes in phospholamban in sarcoplasmic reticulum and troponin I, troponin T and C-protein in the myofibrills, although these proteins were found to be substrates of protein kinase C in vitro. Prazosin, an al-adrenergic antagonist, prevented the changes in inotropy,pmn These kinase C activity and protein phosphorylation, mediated by phenylephrine. findings provide evidence that protein kinase C is activated in response to ul-adrenergic stimulation and activation is associated with increased phosphorylation of a 15 kDa sarcolemmal protein and a 28 kDa cytosolic protein in the myocar(Supported by NIH HL26057, HL22619.1 dium.
P-3-1
AN EXPERIMENTAL MODEL OF CORONARYMICROEMBOLIZATION M Pauliina R;imij, Michael A Lauer, Marjorie Gabel, Ronald W Millard, Department of University of Cincinnati, Cincinnati, OH USA. Pharmacology and Cell Biophysics, in coronary embolization is associated with Thrombus fragmentation resulting A porcine model is described with features salient atherosclerotic plaque evolution. to clinical observations. Regional left ventricular microembolization was induced by injecting 50 urn polycarbonate microspheres (MS) into LAD coronary artery of domestic pigs (12i-0.7 kg) either under fluoroscopy guidance (n=6) with light ketamine sedation (20 mg/kg) or during alpha-chloralose (80 mg/kg iv) anesthesia in open chest pigs (n=8). In all animals microembolization (IO4 MS/kg) produced transient, 1.5 to 3 mm often combined with intermittent venST-segment changes, without Q wave appearance, In anesthetized pigs with constant coronary blood flow (CBF) tricular arrhythmias. this MS dose increased coronary perfusion pressure 37+5$ and decreased systolic shorHeart rate (126+2 beats/min) and mean tening fraction (SF) from 16+3 to lOi2$. arterial pressure (73?6 mmHg) remained constant while dP/dtmax decreased 13*2%. One pig developed reversible ventricular fibrillation immediately after MS injection. SF a MS dose of decreased as a function of the injected MS dose. All animals receiving 104/kg survived a 10 day follow-up period. In conclusion, a MS dose of 104/kg injected into the LAD leads to a hemodynamically stab-le coronary microembolization in pigs which resembles clinical findings typically observed during unstable angina. S.66
P-2-38 CHARACTERISTICS
AND Ca” SENSITIVITY OF FOLYPEOSPHOINOSITIDE SYNTHESIS IN RAT HEART SARCOLXMMA Nasrin Mesaeli, Ios MI Lamers‘, Vincenzo Panagia. Divisionof Cardi~~a~~ul~ Sciences,St BonifaceGH ResearchCentre,University of Manitoba,Winnipeg,CANADA and %ept. of Biochemistry,ErasmusUniversity, Rotterdam,THE NETHERLANDS. In vitro [?]ATP (0.25 mM)-induced phosphorylation of phosphoinositides was examined in rat heart sarcolemma (SL) after preincubation with alamethicin for 30 min at 30°C. The reaction was linear for 1 min. The optimal requirement for Mg” was 2.5 mM. The reaction was observed over a broad pH range (6-9) with an optimum at 7.5. Under these conditions the synthesis of phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5bisphosphate (PtdIns(4,5)PJ was 1101.0 f 78.8 and 115.3 f 13.3 pmol/mg/min, respectively. Exogenous PtdIns4P (lo-200 PM) progressively increased PtdIns(4,5)P, synthesis to a maximum of 3.7 fold. Furthermore, neomycin (an inhibitor of phosphoinositide hydrolysis) increased the formation of both PtdIns4P (2 fold) and PtdIns(4,5)P, (1.7 fold). l-100 I.IM free Ca” induced a progressive decline in both PtdIns4P and PtdIns(4,5)PZ formation with IDS, of 20 and 6.3 PM Ca”, respectively. However, neomycin did not change the inhibitory effect of Ca”‘, thus excluding the possibility of a Ca”-activated breakdown of phosphoinositides by phospholipase C. These results indicate the occurrence of a Ca” sensitive polyphosphoinositide-generating kinase system for the phosphatidylinositol signalling pathway in rat heart SL. (Supportedby MRC, CanadaandNATO, Belgium).
p-2-39a,-ADRENERGIC STIMULATION AND PROTEIN PHOSPHORYLATIONIN BEATING HEARTS Laszlo Talosi, Evangelia G Kranias. Department of Pharmacology and Cell Biophysics, University of Cincinnati, Cincinnati, OH 45267-0575 USA The present study examines the hypothesis that activation of protein kinase C and phosphorylation of key regulatory proteins may mediate the inotropic response of the heart to cl-adrenergie agonists. Rabbit hearts were perfused with modified Krebs-buffer containing 32P-orthophosphate and upon introduction of phenylephrine (IO uM) there was a 1.54-fold increase in +dP/dt. Examination of the protein kinase C activity levels in these hearts revealed a redistribution of the enzymatic activity from the cytosolic to the membranous fraction. Examination of the phosphate incorporation into cardiac proteins indicated: a) increases in a 15 kDa sarcolemmal and a 28 kDa cytosolic protein; b) no changes in phospholamban in sarcoplasmic reticulum and troponin I, troponin T and C-protein in the myofibrills, although these proteins were found to be substrates of protein kinase C in vitro. Prazosin, an al-adrenergic antagonist, prevented the changes in inotropy,pmn These kinase C activity and protein phosphorylation, mediated by phenylephrine. findings provide evidence that protein kinase C is activated in response to ul-adrenergic stimulation and activation is associated with increased phosphorylation of a 15 kDa sarcolemmal protein and a 28 kDa cytosolic protein in the myocar(Supported by NIH HL26057, HL22619.1 dium.
P-3-1
AN EXPERIMENTAL MODEL OF CORONARYMICROEMBOLIZATION M Pauliina R;imij, Michael A Lauer, Marjorie Gabel, Ronald W Millard, Department of University of Cincinnati, Cincinnati, OH USA. Pharmacology and Cell Biophysics, in coronary embolization is associated with Thrombus fragmentation resulting A porcine model is described with features salient atherosclerotic plaque evolution. to clinical observations. Regional left ventricular microembolization was induced by injecting 50 urn polycarbonate microspheres (MS) into LAD coronary artery of domestic pigs (12i-0.7 kg) either under fluoroscopy guidance (n=6) with light ketamine sedation (20 mg/kg) or during alpha-chloralose (80 mg/kg iv) anesthesia in open chest pigs (n=8). In all animals microembolization (IO4 MS/kg) produced transient, 1.5 to 3 mm often combined with intermittent venST-segment changes, without Q wave appearance, In anesthetized pigs with constant coronary blood flow (CBF) tricular arrhythmias. this MS dose increased coronary perfusion pressure 37+5$ and decreased systolic shorHeart rate (126+2 beats/min) and mean tening fraction (SF) from 16+3 to lOi2$. arterial pressure (73?6 mmHg) remained constant while dP/dtmax decreased 13*2%. One pig developed reversible ventricular fibrillation immediately after MS injection. SF a MS dose of decreased as a function of the injected MS dose. All animals receiving 104/kg survived a 10 day follow-up period. In conclusion, a MS dose of 104/kg injected into the LAD leads to a hemodynamically stab-le coronary microembolization in pigs which resembles clinical findings typically observed during unstable angina. S.66