- Email: [email protected]
Calcium wave propagation between isolated ventricular cell pairs — Confocal laser scanning microscopic analysis
J Mol Cell Cardiol 24 (Supplement I) (1992)
O-32-8
GLYCOLVGIS,
CATECHOIAMINE
RELEA6E
AND ENERGY YElABGlJ6Y
WRING
RECURRENT
CARDIAC
i6CHMlA
‘Jan W. de Jong, Anna Cargnonl, Salvatom Cur&lo, Eva&o Passlnl, Rob&o Ferrarl, Thoraxcenter, Rotterdam, The Netherlands; Department of CanSdogy, Unhrerslty of Brescla, Italy.
Erasmus University
Whyfunction and metabolism of lschemlc heart are better ofl wkh intem~htentrepetfusku~is poorly understood. We compared rabbil Langendorfl hearts (190 beats/mln, flow 22 ml/mln), made Lschemlc at 37% for 24’ or 36’ followed by 36’ repffilsbn, with a model of 12 x 2’ or 6 x 2’ + 6 x 4’ of Ischemla,with 3’ reperfuskma(thus total rep&&on timealso 36’). In addition to lactate and noradrenaline (norA) release, we measured adenoslne, lnosine and hypoxanthlne production during reperfusion. Repeffusionfunction and cumllathre metabolite release were: Variable
Control
24’
IntermIttent
Dev. P (mmHg) Xpurlne @md) Zlaetste (mmd ) XnorA (nmd) Mean
f SE (n=57),
57
f
2
0.56 f 0.06 0.30 t 0.03 0.06
f
0.01
52 1.71
zt f
36’ lschemia
isdlernia
Contlnuous 4 0.12
37
*
10.9 f
0.45 * 0.04
0.34 f
0.12
2.5
*P
+
0.01
f
control, #P
5’
1.3-
Intermittent
Continuous
42 3.2
21 12.5
f f
3 0.2’
* f
5y 15.r
0.06
0.65 f
0.06’
0.34 * 0.w
0.6
0.10
0.01
5.6
vs
f
f
1.4-
htermitient ischemia.
Thus intermittent repetfusion suppresses ATP breakdown and norA release to a large extent. In contrast, lactate production was higher il we rep&used the heatts repeatedly. In line with earlier data [JMCC 20 (1988) 2471, we speculate that inhibited glycdysls during continuous ischemla, with lack of anaerobic ATP generation, is responsible for damage to nerve endings, resulting in noradrenaline release.
O-33-1
CALCIUM WAVE PROPAGATION BETWEEN ISOLATED VENTRICULAR CELL PAIRS - CONFOCAL LASER SCANNING MICROSCOPIC ANALYSIS Hideyuki Kawachi, Tetsuro Takamatsu, Tetsuhiro Minamikawa, Setsuya Fujita. Department of Pathology, Kyoto Prefectural University of Medicine, Kyoto 602, Japan By using confocal laser scanning microscope (CLSM), we analyzed calcium wave propagation between isolated ventricular cell pairs. Ventricular cell pairs were obtained by enzymatic dispersion of young adult guinea-pig hearts and were loaded with fluorescent calcium indicator, fluo-3, by internal perfusion with a micropipette electrode. Calcium waves were induced by whole-cell-clamping of the cell membrane potential at a depolarized level. The CLAM provided tomographic images of calcium wave at SO-90 msec intervals. Most calcium waves propagated from one of the cell pairs to the other. When the cell pairs were perfused with Tyrode solution containing enflurane, calcium waves did not propagate over the cell-to-cell junction. The results suggest that oscillations of the [Ca’+]i propagate through the gap junctions between ventricular cell pairs, and that the mechanism of intercellular calcium wave propagation is calcium-induced calcium release.
O-33-2
THAPSIGARGIN IS A POTENT INHIBITOR OF INTRACELLULAR CALCIUM PUMPS OF HEART, SKELETAL MUSCLE, AND BRAIN Yoshivuki Kiiima. Eunice Oaunbunmi. Sidnev Fleischer. Department of Molecular Biology, Vanderbilt University, Nashville, TN 37238 USA. The effect of thapsigargin (TG) to inhibit intracellular Ca’+ pump protein (CPP), was studied in cardiac (C) sarcoplasmic reticulum (SR), skeletal muscle (Sk) SR, and brain microsomes (BMc). The molar ratio of TG/CPP for complete inhibition of Ca2+ loading (MF+& is 3.8 for CSR, and -1.0 for SkSR and BMc. TG also inhibited Ca2+-ATPase and formation of phosphoenzyme (EP). The mechanism of inhibition by TG was studied in Sk%. Bindings of ATP and Ca2+ to CPP were both completely inhibited, when CPP was pretreated with TG in EGTA (E2 state). Pretreatment with Ca + in the absence of ATP (E, state) protects the CPP from inactivation by TG with respect to Ca2’ binding and EP formation. Phosphorylation with protein kinase A (P-PKA) of CSR increased the sensitivity to TG (MR,, = 2.8). In summary, we find: 1) TG is a potent inhibitor of intracellular CPP; 2) CSR is less sensitive to TG than SkSR and BMc; 3) the decreased sensitivity of CSR is enhanced by PPKA; 4) TG appears to inactivate preferentially the E, form of CPP. [Muscular Dystrophy Assoc. (MDA) Fellowship to Y.K., and MDA grant and NIH-HL32711 to S.F.] s.131
O-32-8
GLYCOLVGIS,
CATECHOIAMINE
RELEA6E
AND ENERGY YElABGlJ6Y
WRING
RECURRENT
CARDIAC
i6CHMlA
‘Jan W. de Jong, Anna Cargnonl, Salvatom Cur&lo, Eva&o Passlnl, Rob&o Ferrarl, Thoraxcenter, Rotterdam, The Netherlands; Department of CanSdogy, Unhrerslty of Brescla, Italy.
Erasmus University
Whyfunction and metabolism of lschemlc heart are better ofl wkh intem~htentrepetfusku~is poorly understood. We compared rabbil Langendorfl hearts (190 beats/mln, flow 22 ml/mln), made Lschemlc at 37% for 24’ or 36’ followed by 36’ repffilsbn, with a model of 12 x 2’ or 6 x 2’ + 6 x 4’ of Ischemla,with 3’ reperfuskma(thus total rep&&on timealso 36’). In addition to lactate and noradrenaline (norA) release, we measured adenoslne, lnosine and hypoxanthlne production during reperfusion. Repeffusionfunction and cumllathre metabolite release were: Variable
Control
24’
IntermIttent
Dev. P (mmHg) Xpurlne @md) Zlaetste (mmd ) XnorA (nmd) Mean
f SE (n=57),
57
f
2
0.56 f 0.06 0.30 t 0.03 0.06
f
0.01
52 1.71
zt f
36’ lschemia
isdlernia
Contlnuous 4 0.12
37
*
10.9 f
0.45 * 0.04
0.34 f
0.12
2.5
*P
+
0.01
f
control, #P
5’
1.3-
Intermittent
Continuous
42 3.2
21 12.5
f f
3 0.2’
* f
5y 15.r
0.06
0.65 f
0.06’
0.34 * 0.w
0.6
0.10
0.01
5.6
vs
f
f
1.4-
htermitient ischemia.
Thus intermittent repetfusion suppresses ATP breakdown and norA release to a large extent. In contrast, lactate production was higher il we rep&used the heatts repeatedly. In line with earlier data [JMCC 20 (1988) 2471, we speculate that inhibited glycdysls during continuous ischemla, with lack of anaerobic ATP generation, is responsible for damage to nerve endings, resulting in noradrenaline release.
O-33-1
CALCIUM WAVE PROPAGATION BETWEEN ISOLATED VENTRICULAR CELL PAIRS - CONFOCAL LASER SCANNING MICROSCOPIC ANALYSIS Hideyuki Kawachi, Tetsuro Takamatsu, Tetsuhiro Minamikawa, Setsuya Fujita. Department of Pathology, Kyoto Prefectural University of Medicine, Kyoto 602, Japan By using confocal laser scanning microscope (CLSM), we analyzed calcium wave propagation between isolated ventricular cell pairs. Ventricular cell pairs were obtained by enzymatic dispersion of young adult guinea-pig hearts and were loaded with fluorescent calcium indicator, fluo-3, by internal perfusion with a micropipette electrode. Calcium waves were induced by whole-cell-clamping of the cell membrane potential at a depolarized level. The CLAM provided tomographic images of calcium wave at SO-90 msec intervals. Most calcium waves propagated from one of the cell pairs to the other. When the cell pairs were perfused with Tyrode solution containing enflurane, calcium waves did not propagate over the cell-to-cell junction. The results suggest that oscillations of the [Ca’+]i propagate through the gap junctions between ventricular cell pairs, and that the mechanism of intercellular calcium wave propagation is calcium-induced calcium release.
O-33-2
THAPSIGARGIN IS A POTENT INHIBITOR OF INTRACELLULAR CALCIUM PUMPS OF HEART, SKELETAL MUSCLE, AND BRAIN Yoshivuki Kiiima. Eunice Oaunbunmi. Sidnev Fleischer. Department of Molecular Biology, Vanderbilt University, Nashville, TN 37238 USA. The effect of thapsigargin (TG) to inhibit intracellular Ca’+ pump protein (CPP), was studied in cardiac (C) sarcoplasmic reticulum (SR), skeletal muscle (Sk) SR, and brain microsomes (BMc). The molar ratio of TG/CPP for complete inhibition of Ca2+ loading (MF+& is 3.8 for CSR, and -1.0 for SkSR and BMc. TG also inhibited Ca2+-ATPase and formation of phosphoenzyme (EP). The mechanism of inhibition by TG was studied in Sk%. Bindings of ATP and Ca2+ to CPP were both completely inhibited, when CPP was pretreated with TG in EGTA (E2 state). Pretreatment with Ca + in the absence of ATP (E, state) protects the CPP from inactivation by TG with respect to Ca2’ binding and EP formation. Phosphorylation with protein kinase A (P-PKA) of CSR increased the sensitivity to TG (MR,, = 2.8). In summary, we find: 1) TG is a potent inhibitor of intracellular CPP; 2) CSR is less sensitive to TG than SkSR and BMc; 3) the decreased sensitivity of CSR is enhanced by PPKA; 4) TG appears to inactivate preferentially the E, form of CPP. [Muscular Dystrophy Assoc. (MDA) Fellowship to Y.K., and MDA grant and NIH-HL32711 to S.F.] s.131