Vol.
174,
No.
February
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
14, 1991
COMMUNICATIONS
1248-1254
Pages
REDUCTION IN THE Ca'*-INDUCED Ca2+ RELEASE FROM CANINE CARDIAC SARCOPLASMIC RETICULUM FOLLOWING ENDOTOXIN ADMINISTRATION
Maw-Shung Department St.
Received
December
Liu
of Pharmacological Louis University St. Louis, 19,
and Li-Ling
Wu*
and Physiological School of Medicine MO 63104
Science
1990
SUMMARY:Effects of endotoxin administration on the Ca"-induced Ca2+ release from canine cardiac sarcoplasmic reticulum (SR) were studied. Results show that the Ca2+-induced Ca2+ release from either passively or actively loaded SR vesicles was decreased by 28 to 46% (pcO.05) 4 h after endotoxin administration. Kinetic analysis reveals that the Vmax for Ca2+ was decreased significantly without changing the S,, and the Hill coefficient values. The binding of [3H]ryanodine to cardiac SR was reduced by 25.3% (pcO.01) following endotoxin administration. These data demonstrate that the Ca'+induced Ca2' release via the ryanodine-sensitive Ca2+ channel in canine cardiac SR was reduced during endotoxin shock. A reduction in the SR Ca'+-induced Ca2+ release may have a pathophysiological significance in contributing to the development of myocardial G 1991AcadfmlcPress, Inc. depression during endotoxin shock. In cardiac muscle cell, contraction and relaxation are regulated by the myoplasmic free Ca2+ ion concentration which in turn is regulated by various membrane systems (1,2). Extracellular Ca2+ must first enter the myocardial cell via the slow inward Ca" channel following depolarization of sarcolemmal membrane. The Ca2+ ions which enter the cell then trigger the release of Ca2' from the sarcoplasmic reticulum (SR) compartment, a process known as IICa2+induced Ca2' release" Contraction is then initiated by the (3) release of Ca2+ from the SR while relaxation occurs when myoplasmic Ca2+ is reaccumulated within the lumen of the SR and by extrusion through sarcolemma (4-9) . Because of its role in regulating the contraction and relaxation of cardiac muscle, any derangement in the SR Ca2+ release channel would affect myocardial function as a Since myocardial contractility has been reported to depress pump. *Present University,
address: Department Beijing, People's
of Pathophysiology, Republic of China.
Beijing
Medical
Vol.
174,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
the present study dealing during the progression of shock (lo-13), with the effect of endotoxin administration on the Ca2+-induced Ca2+ release from canine cardiac SR was undertaken in an attempt to understand the pathogenesis of myocardial depression in endotoxin shock. MATERIALS AND METHODS Experiments were carried out on adult male mongrel dogs. Endotoxin shock was induced by a single I.V. injection of 0.5 mg/kg endotoxin (lipopolysaccharide W from g. u; Difco Laboratories) and the endotoxin-injected dogs were sacrificed 4 h after endotoxin administration (14). Control dogs received no treatment. Cardiac SR vesicles were prepared as described previously (14) except that the sucrose gradient was modified to consist of 0.6 M, 0.8 M, 1.0 and that the sample (suspended in 0.25 M M, and 1.2 M sucrose sucrose) was layered on top of the sucrose gradient. Fractions obtained at 0.8 M:l.O M and 1.0 M:1.2 M interfaces were combined and used as a SR preparation. Preliminary experiments indicate that the SR vesicles used for the released study is "heavy" junctional derived membrane fraction because the preparation exhibited a comparable number of specific high-affinity ryanodine binding sites (Bmax was 8 pmoles/mg protein with a Kd of 13.7 nM) as reported by others (15-17). The Ca"-induced Ca2+ release from passively and actively loaded SR vesicles was determined by Millipore filtration as described by Meissner and Henderson (15) with modification. For passively loaded experiments, SR vesicles (2 mg protein/ml) were loaded with 4SCa2+ for 90 min at 4°C in a medium containing 1 mM "CaCl 120 mM KCl, 1 mM dithiothreitol and 20 mM Hepes (pH 7.2). 45Ca22'release behavior of the vesicles was measured by diluting the vesicles 200fold into an unlabeled release medium (120 mM KCl, 20 mM Hepes, pH 7.2, and 5 FM of free Ca" after the addition of vesicles). After incubating at 25°C for varied time intervals (O-40 set), 45Ca2+ release was inhibited by the addition of an inhibiting medium (120 mM KCl, 10 mM MgC$, 0.1 mM EGTA, 2 mM LaC12, 15 PM ruthenium red and 10 mM Hepes, pH 7.2). Extravesicular "Ca2+ was removed by placing the vesicles on 0.45-p Millipore filters followed by rapid rinsing with inhibiting medium. The radioactivity retained by the vesicles on the filters was determined by liquid scintillation counting. For actively loaded experiments, SR vesicles (0.1 mg/ml) were incubated at 37°C for various time intervals (O-10 min) in an uptake medium containing 120 mM KCl, 20 mM Hepes (pH 7.2), 5 mM At the end of each NaN, , 3 mM MgC12 0.1 mM 45CaC12 and 3 mM ATP. time interval, the vesicles were diluted into either inhibiting medium (for uptake determination) or release medium (for release determination) followed by Millipore filtration as described above. [3H]Ryanodine binding was carried out by the method of Inui et al with modification. The binding was measured at 200 nM (18) [3H]ryanodine in the presence of 120 mM KC1 and 20 PM CaCl, for 30 min at 37°C.
Figure Ca2+-induced
RESULTS AND DISCUSSION 1 depicts the effect of endotoxin administration Ca2+ release from passively loaded SR vesicles 1249
on the as a
Vol.
174,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
I (W 2-
p
. p < 0.05
25
;; $ 20 E : x0
l5
l
l
2 e
‘0
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5
rp
174,
No.
February
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
14, 1991
COMMUNICATIONS
1248-1254
Pages
REDUCTION IN THE Ca'*-INDUCED Ca2+ RELEASE FROM CANINE CARDIAC SARCOPLASMIC RETICULUM FOLLOWING ENDOTOXIN ADMINISTRATION
Maw-Shung Department St.
Received
December
Liu
of Pharmacological Louis University St. Louis, 19,
and Li-Ling
Wu*
and Physiological School of Medicine MO 63104
Science
1990
SUMMARY:Effects of endotoxin administration on the Ca"-induced Ca2+ release from canine cardiac sarcoplasmic reticulum (SR) were studied. Results show that the Ca2+-induced Ca2+ release from either passively or actively loaded SR vesicles was decreased by 28 to 46% (pcO.05) 4 h after endotoxin administration. Kinetic analysis reveals that the Vmax for Ca2+ was decreased significantly without changing the S,, and the Hill coefficient values. The binding of [3H]ryanodine to cardiac SR was reduced by 25.3% (pcO.01) following endotoxin administration. These data demonstrate that the Ca'+induced Ca2' release via the ryanodine-sensitive Ca2+ channel in canine cardiac SR was reduced during endotoxin shock. A reduction in the SR Ca'+-induced Ca2+ release may have a pathophysiological significance in contributing to the development of myocardial G 1991AcadfmlcPress, Inc. depression during endotoxin shock. In cardiac muscle cell, contraction and relaxation are regulated by the myoplasmic free Ca2+ ion concentration which in turn is regulated by various membrane systems (1,2). Extracellular Ca2+ must first enter the myocardial cell via the slow inward Ca" channel following depolarization of sarcolemmal membrane. The Ca2+ ions which enter the cell then trigger the release of Ca2' from the sarcoplasmic reticulum (SR) compartment, a process known as IICa2+induced Ca2' release" Contraction is then initiated by the (3) release of Ca2+ from the SR while relaxation occurs when myoplasmic Ca2+ is reaccumulated within the lumen of the SR and by extrusion through sarcolemma (4-9) . Because of its role in regulating the contraction and relaxation of cardiac muscle, any derangement in the SR Ca2+ release channel would affect myocardial function as a Since myocardial contractility has been reported to depress pump. *Present University,
address: Department Beijing, People's
of Pathophysiology, Republic of China.
Beijing
Medical
Vol.
174,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
the present study dealing during the progression of shock (lo-13), with the effect of endotoxin administration on the Ca2+-induced Ca2+ release from canine cardiac SR was undertaken in an attempt to understand the pathogenesis of myocardial depression in endotoxin shock. MATERIALS AND METHODS Experiments were carried out on adult male mongrel dogs. Endotoxin shock was induced by a single I.V. injection of 0.5 mg/kg endotoxin (lipopolysaccharide W from g. u; Difco Laboratories) and the endotoxin-injected dogs were sacrificed 4 h after endotoxin administration (14). Control dogs received no treatment. Cardiac SR vesicles were prepared as described previously (14) except that the sucrose gradient was modified to consist of 0.6 M, 0.8 M, 1.0 and that the sample (suspended in 0.25 M M, and 1.2 M sucrose sucrose) was layered on top of the sucrose gradient. Fractions obtained at 0.8 M:l.O M and 1.0 M:1.2 M interfaces were combined and used as a SR preparation. Preliminary experiments indicate that the SR vesicles used for the released study is "heavy" junctional derived membrane fraction because the preparation exhibited a comparable number of specific high-affinity ryanodine binding sites (Bmax was 8 pmoles/mg protein with a Kd of 13.7 nM) as reported by others (15-17). The Ca"-induced Ca2+ release from passively and actively loaded SR vesicles was determined by Millipore filtration as described by Meissner and Henderson (15) with modification. For passively loaded experiments, SR vesicles (2 mg protein/ml) were loaded with 4SCa2+ for 90 min at 4°C in a medium containing 1 mM "CaCl 120 mM KCl, 1 mM dithiothreitol and 20 mM Hepes (pH 7.2). 45Ca22'release behavior of the vesicles was measured by diluting the vesicles 200fold into an unlabeled release medium (120 mM KCl, 20 mM Hepes, pH 7.2, and 5 FM of free Ca" after the addition of vesicles). After incubating at 25°C for varied time intervals (O-40 set), 45Ca2+ release was inhibited by the addition of an inhibiting medium (120 mM KCl, 10 mM MgC$, 0.1 mM EGTA, 2 mM LaC12, 15 PM ruthenium red and 10 mM Hepes, pH 7.2). Extravesicular "Ca2+ was removed by placing the vesicles on 0.45-p Millipore filters followed by rapid rinsing with inhibiting medium. The radioactivity retained by the vesicles on the filters was determined by liquid scintillation counting. For actively loaded experiments, SR vesicles (0.1 mg/ml) were incubated at 37°C for various time intervals (O-10 min) in an uptake medium containing 120 mM KCl, 20 mM Hepes (pH 7.2), 5 mM At the end of each NaN, , 3 mM MgC12 0.1 mM 45CaC12 and 3 mM ATP. time interval, the vesicles were diluted into either inhibiting medium (for uptake determination) or release medium (for release determination) followed by Millipore filtration as described above. [3H]Ryanodine binding was carried out by the method of Inui et al with modification. The binding was measured at 200 nM (18) [3H]ryanodine in the presence of 120 mM KC1 and 20 PM CaCl, for 30 min at 37°C.
Figure Ca2+-induced
RESULTS AND DISCUSSION 1 depicts the effect of endotoxin administration Ca2+ release from passively loaded SR vesicles 1249
on the as a
Vol.
174,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
I (W 2-
p
. p < 0.05
25
;; $ 20 E : x0
l5
l
l
2 e
‘0
t 8
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rp
