Vol. 186, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
July 15, 1992
Pages 595-500
VOLATILE ANESTHETICS SELECTIVELY ALTER ['H]RYANODINE BINDING TO SKELETAL AND CARDIAC RYANODINE RECEPTORS*
Timothy J. Connelly a, Roque-El H a y e k a'b, B e n F. Rusy a a n d Roberto Coronado b D e p a r t m e n t s of aAnesthesiology and bphysiology, University of Wisconsin-Madison, Madison, Wisconsin 5 3 7 9 2
Received
June
i, 1992
SUMMARY. The effect of clinical concentrations of volatile a n e s t h e t i c s on r y a n o d i n e receptors of cardiac a n d skeletal m u s c l e sarcoplasmic reticulum w a s evaluated u s i n g [3H]ryanodine binding. At 2 volume percent, halothane a n d enflurane stimulated binding to cardiac SR b y 238% a n d 204%, respectively, while isoflurane h a d no effect. In contrast, h a l o t h a n e a n d enflurane h a d no effect on [3H]ryanodine binding to skeletal ryanodine receptors, while isoflurane p r o d u c e d a significant stimulation. These results s u g g e s t t h a t volatile a n e s t h e t i c s interact in a site-specific m a n n e r with r y a n o d i n e receptors of cardiac or skeletal m u s c l e to effect Ca 2÷ releasec h a n n e l gating. ®1992AcademicP.........
Halothane, anesthetics
that
evidence s u g g e s t s
isoflurane
and
enflurane
are
halogenated
inhallational
d e p r e s s myocardial contractility (1). A p r e p o n d e r a n c e that
excitation-contraction
these
coupling
agents
restrict the
(2). This
alteration
of
availability of Ca 2÷ for may
result
from
an
i m p a i r m e n t of the Ca 2. u p t a k e a n d release b y the SIR, although a specific target h a s n o t b e e n identified. Ca 2÷ release from the SR of cardiac and skeletal m u s c l e is controlled b y t h e ryanodine receptor, a 550 k D a protein t h a t b i n d s the p l a n t alkaloid r y a n o d i n e at n a n o m o l a r concentrations
(3).
Since the r y a n o d i n e binding site is formed w h e n the c h a n n e l is in the open conformational state, [3H]ryanodine binding activity h a s b e e n widely u s e d as 1 S u p p o r t e d b y International A n e s t h e s i a R e s e a r c h Society, NIH (GM 36852), American H e a r t Association, a n d M u s c u l a r D y s t r o p h y Association. Abbreviations: SIR, sarcoplasmic reticulum; EGTA, ethyleneglycol-bis-(Jlaminoethyl ether)N,N,N',N-tetraacetic acid; Pipes, piperazine-N,N'-bis-(2e t h a n e s u l f o n i c acid). 0006-291X/92 $4.00 595
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
V o l . 186, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
a powerful index of c h a n n e l activity (4,5). The p u r p o s e of the p r e s e n t s t u d y was to u s e [SHlryanodine binding to investigate the effect of the volatile a n e s t h e t i c s on the conformational s t a t e s of the SR Ca 2+ release c h a n n e l of cardiac a n d skeletal SR.
EXPERIMENTAL PROCEDURES I s o l a t i o n o f r y a n o d t n e receptors. With institutional approval, a d u l t swine were a n e s t h e t i z e d with s u b l e t h a l doses of t h i o b a r b i t u r a t e a n d e u t h a n i z e d by e x s a n g u i n a t i o n . Skeletal m u s c l e was harvested from the v a s t u s a n d trapezius m u s c l e s a n d i m m e d i a t e l y frozen in liquid nitrogen. The entire h e a r t was excised, rapidly dissected free of connective tissue a n d frozen in liquid nitrogen. Skeletal a n d cardiac muscle was t h e n stored at -80°C for later use. Heavy SR was p r e p a r e d from skeletal m u s c l e a n d ventricular free wall by a differential a n d s u c r o s e g r a d i e n t m e t h o d (6) in the presence of protease inhibitors. [nil] ryanodtne binding. All a s s a y s were performed in 800 111 Teflon-capped vials (Kimax, Owens-Illinois) for 120 m i n u t e s at 37°C u s i n g 50-75 lag of SR protein in the presence of 7 nM [3Hlryanodine (60 m C i / m m o l e - D u P o n t New E n g l a n d Nuclear, Boston, MA). Incubation m e d i u m consisted of 750 lal of buffer (Na Pipes 10raM; KCI 0.2 M, pH 7.2), equilibrated with N2 or a N2a n e s t h e t i c m i x t u r e delivered to a reservoir by a calibrated vaporizer t h r o u g h scinterred glass. Isoflurane a n d enflurane were obtained commercially (Anaquest, Madison, WI) a n d thymol-free h a l o t h a n e was generously provided by Halocarbon Laboratories (Augusta, NC). Free Ca 2÷ was 10 .5 M in all experiments, except in t h a t a d d r e s s i n g the calcium d e p e n d e n c e of ryanodine binding. The stability c o n s t a n t s of Fabiato (7) were u s e d to prepare CaC12EGTA (1 raM) buffers to set free [Ca 2÷] at the desired value. All a s s a y s were completed in quadruplicate. At the end of the i n c u b a t i o n period, 24 samples were filtered s i m u l t a n o u s l y t h r o u g h W h a t m a n G F / C filters u s i n g a Brandel Cell Harvester (Brandel, Gaithersberg, MD) a n d c o u n t e d by liquid scintillation. The specific binding was calculated as the difference between the b i n d i n g in the abscence (total binding) and the presence of (nonspecific binding) 100 pM unlabeHed ryanodine.
M e a s u r e m e n t o f a n e s t h e t i c c o n c e n t r a t i o n s . At the end of the i n c u b a t i o n period, b u t before filtering, 25 pl was aspirated from the h e a d space gas (approximately 50 lai) in each vial with a gas-tight syringe. The concentration was m e a s u r e d b y gas c h r o m a t o g r a p h y (Varian Model 3100, Sugarland, TX) with a flame ionization detector a n d referenced to calibration gases commercially p r e p a r e d in N2 (Scott Medical Products, Plumsteadville, PAL The m e a n a n e s t h e t i c c o n c e n t r a t i o n for q u a d r u p l i c a t e s a m p l e s was u s e d for t h a t set. The coefficient of variation for gas c h r o m a t o g r a p h i c m e a s u r e m e n t s of s t a n d a r d gases w a s 10% over the course of this s t u d y .
RESULTS Anesthetics were delivered as a gas u s i n g a calibrated vaporizer into the
[aH]ryanodine binding reaction mixture. The 596
protocol
(see Methods)
Vol. 186, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
~0
300 •
~
r..)
•
•
•
mm
200
!i ,,° 100
i
i
1
2
[HALOTHANE], vol%
FiRure I. Dose-response effect of halothane on cardiac (squares) and skeletal (circles) [3H]ryanodine binding, plotted as percent of control binding (no halothane). The solid line represents a linear regression of the data shown (r=0.92). Each data point the mean of four separate assays.
ensured
that
anesthetics
would
be
equilibrated with
the
SR-cont ai ni ng
a q u e o u s p h a s e in t he s a m e w a y as would o c c u r w h e n t i s s u e is exposed to a n a n e s t h e t i c /n s/tu. Fig. 1 (filled s q u a r e s ) s h o w s t h a t h a l o t h a n e c a u s e d a dose-dependent
stimulation
of
[3Hlryanodine
binding
to
cardi ac
SR.
S t i m u l a t i o n w a s linear over t he clinical c o n c e n t r a t i o n r a n g e of 0.5 to 2 v o l u m e p e r c e n t , w h i c h c o r r e s p o n d s to a n a q u e o u s p h a s e c o n c e n t r a t i o n of 0.25 to 1 mM h a l o t h a n e at r o o m t e m p e r a t u r e . Actual c o n c e n t r a t i o n s at the end
of t h e i n c u b a t i o n period were verified b y gas c h r o m a t o g r a p h y . The
stimulatory
effect of h a l o t h a n e
receptor inasmuch
was
specific for t h e
as h a l o t h a n e h a d
cardi ac
ryanodine
little or no effect on [3H]ryanodine
b i n d i n g to r y a n o d i n e r e c e p t o r s of skeletal m u s c l e
a s s a y e d in a similar
m a n n e r (open circles). In s e p a r a t e e x p e r i m e n t s (not shown), h a l o t h a n e also h a d n o effect on [3H]ryanodine b i n d i n g to b r a i n r y a n o d i n e r e c e p t o r s a s s a y e d in r a b b i t b r a i n m i c r o s o m e s (8). T h u s t he effect was specific for t he cardiac i s o f o r m in spite of t he fact t h a t car diac a n d b r a i n r y a n o d i n e r e c e p t o r s are s t r u c t u r a l l y h o m o l o g o u s (9). Fig.
2
compares
the
effect of h a l o t h a n e
with
that
of two
other
c o m m o n l y u s e d a n e s t h e t i c s , t he s t e r e o i s o m e r s isoflurane a n d enflurane. T he three
anesthetics
separately on
were
cardiac
tested
at
a
c o n c e n t r a t i o n of 2
volume
(left) or skeletal r y a n o d i n e r e c e p t o r s
percent
(right). B o t h
h a l o t h a n e a n d e n f l u r a n e c a u s e d a significant s t i m u l a t i o n of [3Hlryanodine b i n d i n g to th e car di a c r e c e p t o r while isoflurane h a d no effect. On t he o t h e r h a n d , isoflurane, b u t n o t h a l o t h a n e or enflurane, s t i m u l a t e d [3H]ryanodine b i n d i n g to t h e skeletal r y a n o d i n e receptor. T h u s t h e r e w a s a r e m a r k a b l e 597
Vol. 186, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
~
~o o ~
ISOFLURANE ENFLURANE
Ill
300
.OTHANE
250
.ge ~
150
ii o° ta
50 CARDIAC
SKELETAL
Figure 2. Effect of isoflurane, enflurane and halothane (2 vol%) on [3Hlryanodine binding to cardiac (left) and skeletal (right) SR vesicles, plotted as a percent of control binding (no anesthetic). Each bar represents the mean of two experiments, each completed in quadruplicate assays. Error bars represent the standard deviations of duplicate experiments for each anesthetic.
selectivity of receptors.
each
Since
anesthetic
[3Hlryanodine
for
either
binding
cardiac
varies
or
with
skeletal free
ryanodine
Ca 2÷ (10),
we
investigated t h e Ca2*-dependence of t h e s t i m u l a t i o n p r o d u c e d b y h a l o t h a n e in c a r d i a c SR. Fig. 3 shows the Ca2*-dependence of [3H]ryanodine b i n d i n g in t h e a b s c e n c e (triangles) or p r e s e n c e (circles) of 2 v o l u m e p e r c e n t a n e s t h e t i c . In
controls,
the
Ca2+-dependence w a s
bell-shaped
with
a
maximum
at
a p p r o x i m a t e l y 10 pM. H a l o t h a n e s t i m u l a t e d binding over t h e entire r a n g e of free Ca 2÷ w i t h o u t p r o d u c i n g a change in the overall s h a p e of the curve.
•
CONTROL
•
HALOTHANE 2%
125
/
/
o"'-,
///
~S
50
•
, "'',
/
25
"~
"
"'O
m~ -9
-8
-7
-6
-5
-4
-3
-2
log [Ca], M Figure 3. Calcium dependence of [3H]ryanodine binding in the abscence (triangles) and presence (circles) of 2% halothane. Each data point represents a single experiment performed with quadruplicate assays. 598
Vol. 186, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DISCUSSION
C o n t r a c t i o n s t u d i e s in isolated myocardial t i s s u e myofibrfls
(14),
halothane,
the
and
isolated
most
widely
myocytes studied
(15-17), anesthetic,
(Ii-13),
have
skinned
suggested
decreases
the
that net
s e q u e s t r a t i o n of Ca 2÷ by the SIL Exposure of cardiac t i s s u e to h a l o t h a n e leads to myocardial depression a n d negative inotropy (11-13). Our results are c o n s i s t e n t with a direct effect of h a l o t h a n e on t h e cardiac Ca 2÷ release c h a n n e l since a d o s e - d e p e n d e n t stimulation of [3H]ryanodine binding was evident in the clinical range of anesthetic delivered to SR in a g a s e o u s form. The s t i m u l a t i o n of binding suggested t h a t h a l o t h a n e increases
C a 2÷
release
c h a n n e l activity. This conclusion is based on the generalized finding t h a t ligands t h a t e n h a n c e
C a 2÷
release from the SR also e n h a n c e [3H] ryanodine
binding, w h e r e a s ligands t h a t inhibit Ca 2+ release also inhibit [3H]ryanodine b i n d i n g (10). Hence, the open conformational state of the receptor a p p e a r s to r e p r e s e n t the high affinity binding site for ryanodine a n d binding site d e n s i t y therefore serves as a n indicator of the fraction of receptors t h a t are present, a t a given ligand concentration, in an open conformation. At this point however, we do n o t k n o w w h e t h e r h a l o t h a n e only increases [3H]ryanodine b i n d i n g site density, as would be required by the interpretation above, or if m a y also increase [3H]ryanodine binding site aff'mity. In the latter case, a n e s t h e t i c effects on
[3H]ryanodine binding activity m a y not be directly
related to c h a n g e s in c h a n n e l gating. Nevertheless, the increase of cardiac [3H]ryanodine b i n d i n g produced by h a l o t h a n e a n d enflurane, b u t n o t by isoflurane, is c o n s i s t e n t with a n alteration of gating b e c a u s e isoflurane c a u s e s m u c h less myocardial depression t h a n the other a n e s t h e t i c s (11-13). The idea t h a t anesthetics m a y affect Ca 2÷ release c h a n n e l gating is also s u p p o r t e d by the Ca2*-dependence of h a l o t h a n e stimulation. The Ca z÷ d e p e n d e n c e of [3H]ryanodine binding h a s been interpreted to reflect the b i n d i n g of Ca 2÷ to a high affinity a n d a low affinity site controlling c h a n n e l gating (10). T h u s , Ca 2÷ binding to a high affinity site would increase the open probability of the channel, leading to a n increase in the d e n s i t y of sites available for [3H]ryanodine binding. Conversely, Ca z÷ b i n d i n g to a low-affinity site would close or inactivate the c h a n n e l a n d t h u s decrease the n u m b e r of binding sites. Within this scheme, the major effect of h a l o t h a n e a p p e a r e d to be a n increase in the Ca 2÷ sensitivity to b o t h the high a n d low-affinity Ca 2÷ binding sites controlling c h a n n e l gating. The
selective interaction
of enflurane with
cardiac
receptors
and
isoflurane with skeletal receptors is significant since t h e s e two a n e s t h e t i c s 599
Vol. 186, No. 1, 1992
are
methyl-ethyl
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ether
isomers.
These
results
suggest
site-specifc
interactions of the a n e s t h e t i c s with each receptor type. Skeletal a n d cardiac r y a n o d i n e receptor isoforms are different
pharmacologic
responses
only =60% homologous (17), to
various
ligands
was
not
so t h a t totally
unexpected. Yet volatile a n e s t h e t i c s are simple h y d r o c a r b o n s w h i c h have been proposed to act on ion c h a n n e l s t h r o u g h a n o n - p r o t e i n m e d i a t e d interaction. Specifically, volatile a n e s t h e t i c s are classically t h o u g h t to alter b u l k m e m b r a n e lipid properties in the vicinity of protein c h a n n e l s (18). This explanation s e e m s less plausible, at least for the Ca 2÷ release c h a n n e l of SR, since enflurane a n d isoflurane are chemical isomers with similar physical properties.
The
results
presented
here
may
have
implications
for the
m e c h a n i s m of action of a n e s t h e t i c s in other t i s s u e s as well.
REFERENCES
1. 2. 3. 4. 5.
Brown BR, C r o u t JR. (1971) Anesthesiology 34:236-245. R u s y BF, Komai, H. (1987) Anesthesiology 67:745-766. P e s s a h IN a n d Zimanyi I. (1991) Mol P h a r m a c o l 39:679-689. Meissner G. (1986) J Biol Chem 261:6300-6306. Mickelson JR, Gallant EM, Litterer LA, J o h n s o n KM, Rempel WE, Louis CL. (1988) J Biol C h e m 263:9310-93158. 6. M e i s s n e r G. (1983) J Biol Chem 259:2365-2374. 7. Fabiato A. (1983) Am J Physiology 245:C1-C14. 8. McPherson PS, Kim Y-K, Valdivia H, K n u d s o n CM, T a k e k u r a H, FranziniA r m s t r o n g C, Coronado R, Campbell tiP. (1991) Neuron 7:17-25. 9. O t s u IC H u n t i n g t o n WF, K h a n n a VK, Zorzato F, Green NM McLerman DH. (1990) J Biol C h e m 265(23):13472-13483, 10. Valdivia HH, Hogan K, Coronado R. (1991) Am J Physiol 261:C237C245. 11. L y n c h C, Frazer MJ. (1989) Anesthesiology 70:511-522. 12. Komai H, R u s y BF. (1990) Anesthesiology 72:694-698. 13. H o u s m a n s PR, Murat. (1988) Anesthesiology 69:451-463. 14. S u JY, Kerrick WGL. (1978) Pflugers Arch 375:111-117. 15. Wheeler DM, Rice RT, Hansford RG, L a k a t t a EG. (1988) Anesthesiology 69:578-583. 16. Wilde DW, Knight PR, S h e t h N, Williams BS. (1991) Anesthesiology 74:1075-1086. 17. K a t s u o k a M, Kobayashi BL Ohnishi ST. (1989) Anesthesiology 70:954-960. 18. F r a n k s WP, Lieb WR. (1991) Science 254:427-436.
600
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
July 15, 1992
Pages 595-500
VOLATILE ANESTHETICS SELECTIVELY ALTER ['H]RYANODINE BINDING TO SKELETAL AND CARDIAC RYANODINE RECEPTORS*
Timothy J. Connelly a, Roque-El H a y e k a'b, B e n F. Rusy a a n d Roberto Coronado b D e p a r t m e n t s of aAnesthesiology and bphysiology, University of Wisconsin-Madison, Madison, Wisconsin 5 3 7 9 2
Received
June
i, 1992
SUMMARY. The effect of clinical concentrations of volatile a n e s t h e t i c s on r y a n o d i n e receptors of cardiac a n d skeletal m u s c l e sarcoplasmic reticulum w a s evaluated u s i n g [3H]ryanodine binding. At 2 volume percent, halothane a n d enflurane stimulated binding to cardiac SR b y 238% a n d 204%, respectively, while isoflurane h a d no effect. In contrast, h a l o t h a n e a n d enflurane h a d no effect on [3H]ryanodine binding to skeletal ryanodine receptors, while isoflurane p r o d u c e d a significant stimulation. These results s u g g e s t t h a t volatile a n e s t h e t i c s interact in a site-specific m a n n e r with r y a n o d i n e receptors of cardiac or skeletal m u s c l e to effect Ca 2÷ releasec h a n n e l gating. ®1992AcademicP.........
Halothane, anesthetics
that
evidence s u g g e s t s
isoflurane
and
enflurane
are
halogenated
inhallational
d e p r e s s myocardial contractility (1). A p r e p o n d e r a n c e that
excitation-contraction
these
coupling
agents
restrict the
(2). This
alteration
of
availability of Ca 2÷ for may
result
from
an
i m p a i r m e n t of the Ca 2. u p t a k e a n d release b y the SIR, although a specific target h a s n o t b e e n identified. Ca 2÷ release from the SR of cardiac and skeletal m u s c l e is controlled b y t h e ryanodine receptor, a 550 k D a protein t h a t b i n d s the p l a n t alkaloid r y a n o d i n e at n a n o m o l a r concentrations
(3).
Since the r y a n o d i n e binding site is formed w h e n the c h a n n e l is in the open conformational state, [3H]ryanodine binding activity h a s b e e n widely u s e d as 1 S u p p o r t e d b y International A n e s t h e s i a R e s e a r c h Society, NIH (GM 36852), American H e a r t Association, a n d M u s c u l a r D y s t r o p h y Association. Abbreviations: SIR, sarcoplasmic reticulum; EGTA, ethyleneglycol-bis-(Jlaminoethyl ether)N,N,N',N-tetraacetic acid; Pipes, piperazine-N,N'-bis-(2e t h a n e s u l f o n i c acid). 0006-291X/92 $4.00 595
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
V o l . 186, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
a powerful index of c h a n n e l activity (4,5). The p u r p o s e of the p r e s e n t s t u d y was to u s e [SHlryanodine binding to investigate the effect of the volatile a n e s t h e t i c s on the conformational s t a t e s of the SR Ca 2+ release c h a n n e l of cardiac a n d skeletal SR.
EXPERIMENTAL PROCEDURES I s o l a t i o n o f r y a n o d t n e receptors. With institutional approval, a d u l t swine were a n e s t h e t i z e d with s u b l e t h a l doses of t h i o b a r b i t u r a t e a n d e u t h a n i z e d by e x s a n g u i n a t i o n . Skeletal m u s c l e was harvested from the v a s t u s a n d trapezius m u s c l e s a n d i m m e d i a t e l y frozen in liquid nitrogen. The entire h e a r t was excised, rapidly dissected free of connective tissue a n d frozen in liquid nitrogen. Skeletal a n d cardiac muscle was t h e n stored at -80°C for later use. Heavy SR was p r e p a r e d from skeletal m u s c l e a n d ventricular free wall by a differential a n d s u c r o s e g r a d i e n t m e t h o d (6) in the presence of protease inhibitors. [nil] ryanodtne binding. All a s s a y s were performed in 800 111 Teflon-capped vials (Kimax, Owens-Illinois) for 120 m i n u t e s at 37°C u s i n g 50-75 lag of SR protein in the presence of 7 nM [3Hlryanodine (60 m C i / m m o l e - D u P o n t New E n g l a n d Nuclear, Boston, MA). Incubation m e d i u m consisted of 750 lal of buffer (Na Pipes 10raM; KCI 0.2 M, pH 7.2), equilibrated with N2 or a N2a n e s t h e t i c m i x t u r e delivered to a reservoir by a calibrated vaporizer t h r o u g h scinterred glass. Isoflurane a n d enflurane were obtained commercially (Anaquest, Madison, WI) a n d thymol-free h a l o t h a n e was generously provided by Halocarbon Laboratories (Augusta, NC). Free Ca 2÷ was 10 .5 M in all experiments, except in t h a t a d d r e s s i n g the calcium d e p e n d e n c e of ryanodine binding. The stability c o n s t a n t s of Fabiato (7) were u s e d to prepare CaC12EGTA (1 raM) buffers to set free [Ca 2÷] at the desired value. All a s s a y s were completed in quadruplicate. At the end of the i n c u b a t i o n period, 24 samples were filtered s i m u l t a n o u s l y t h r o u g h W h a t m a n G F / C filters u s i n g a Brandel Cell Harvester (Brandel, Gaithersberg, MD) a n d c o u n t e d by liquid scintillation. The specific binding was calculated as the difference between the b i n d i n g in the abscence (total binding) and the presence of (nonspecific binding) 100 pM unlabeHed ryanodine.
M e a s u r e m e n t o f a n e s t h e t i c c o n c e n t r a t i o n s . At the end of the i n c u b a t i o n period, b u t before filtering, 25 pl was aspirated from the h e a d space gas (approximately 50 lai) in each vial with a gas-tight syringe. The concentration was m e a s u r e d b y gas c h r o m a t o g r a p h y (Varian Model 3100, Sugarland, TX) with a flame ionization detector a n d referenced to calibration gases commercially p r e p a r e d in N2 (Scott Medical Products, Plumsteadville, PAL The m e a n a n e s t h e t i c c o n c e n t r a t i o n for q u a d r u p l i c a t e s a m p l e s was u s e d for t h a t set. The coefficient of variation for gas c h r o m a t o g r a p h i c m e a s u r e m e n t s of s t a n d a r d gases w a s 10% over the course of this s t u d y .
RESULTS Anesthetics were delivered as a gas u s i n g a calibrated vaporizer into the
[aH]ryanodine binding reaction mixture. The 596
protocol
(see Methods)
Vol. 186, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
~0
300 •
~
r..)
•
•
•
mm
200
!i ,,° 100
i
i
1
2
[HALOTHANE], vol%
FiRure I. Dose-response effect of halothane on cardiac (squares) and skeletal (circles) [3H]ryanodine binding, plotted as percent of control binding (no halothane). The solid line represents a linear regression of the data shown (r=0.92). Each data point the mean of four separate assays.
ensured
that
anesthetics
would
be
equilibrated with
the
SR-cont ai ni ng
a q u e o u s p h a s e in t he s a m e w a y as would o c c u r w h e n t i s s u e is exposed to a n a n e s t h e t i c /n s/tu. Fig. 1 (filled s q u a r e s ) s h o w s t h a t h a l o t h a n e c a u s e d a dose-dependent
stimulation
of
[3Hlryanodine
binding
to
cardi ac
SR.
S t i m u l a t i o n w a s linear over t he clinical c o n c e n t r a t i o n r a n g e of 0.5 to 2 v o l u m e p e r c e n t , w h i c h c o r r e s p o n d s to a n a q u e o u s p h a s e c o n c e n t r a t i o n of 0.25 to 1 mM h a l o t h a n e at r o o m t e m p e r a t u r e . Actual c o n c e n t r a t i o n s at the end
of t h e i n c u b a t i o n period were verified b y gas c h r o m a t o g r a p h y . The
stimulatory
effect of h a l o t h a n e
receptor inasmuch
was
specific for t h e
as h a l o t h a n e h a d
cardi ac
ryanodine
little or no effect on [3H]ryanodine
b i n d i n g to r y a n o d i n e r e c e p t o r s of skeletal m u s c l e
a s s a y e d in a similar
m a n n e r (open circles). In s e p a r a t e e x p e r i m e n t s (not shown), h a l o t h a n e also h a d n o effect on [3H]ryanodine b i n d i n g to b r a i n r y a n o d i n e r e c e p t o r s a s s a y e d in r a b b i t b r a i n m i c r o s o m e s (8). T h u s t he effect was specific for t he cardiac i s o f o r m in spite of t he fact t h a t car diac a n d b r a i n r y a n o d i n e r e c e p t o r s are s t r u c t u r a l l y h o m o l o g o u s (9). Fig.
2
compares
the
effect of h a l o t h a n e
with
that
of two
other
c o m m o n l y u s e d a n e s t h e t i c s , t he s t e r e o i s o m e r s isoflurane a n d enflurane. T he three
anesthetics
separately on
were
cardiac
tested
at
a
c o n c e n t r a t i o n of 2
volume
(left) or skeletal r y a n o d i n e r e c e p t o r s
percent
(right). B o t h
h a l o t h a n e a n d e n f l u r a n e c a u s e d a significant s t i m u l a t i o n of [3Hlryanodine b i n d i n g to th e car di a c r e c e p t o r while isoflurane h a d no effect. On t he o t h e r h a n d , isoflurane, b u t n o t h a l o t h a n e or enflurane, s t i m u l a t e d [3H]ryanodine b i n d i n g to t h e skeletal r y a n o d i n e receptor. T h u s t h e r e w a s a r e m a r k a b l e 597
Vol. 186, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
~
~o o ~
ISOFLURANE ENFLURANE
Ill
300
.OTHANE
250
.ge ~
150
ii o° ta
50 CARDIAC
SKELETAL
Figure 2. Effect of isoflurane, enflurane and halothane (2 vol%) on [3Hlryanodine binding to cardiac (left) and skeletal (right) SR vesicles, plotted as a percent of control binding (no anesthetic). Each bar represents the mean of two experiments, each completed in quadruplicate assays. Error bars represent the standard deviations of duplicate experiments for each anesthetic.
selectivity of receptors.
each
Since
anesthetic
[3Hlryanodine
for
either
binding
cardiac
varies
or
with
skeletal free
ryanodine
Ca 2÷ (10),
we
investigated t h e Ca2*-dependence of t h e s t i m u l a t i o n p r o d u c e d b y h a l o t h a n e in c a r d i a c SR. Fig. 3 shows the Ca2*-dependence of [3H]ryanodine b i n d i n g in t h e a b s c e n c e (triangles) or p r e s e n c e (circles) of 2 v o l u m e p e r c e n t a n e s t h e t i c . In
controls,
the
Ca2+-dependence w a s
bell-shaped
with
a
maximum
at
a p p r o x i m a t e l y 10 pM. H a l o t h a n e s t i m u l a t e d binding over t h e entire r a n g e of free Ca 2÷ w i t h o u t p r o d u c i n g a change in the overall s h a p e of the curve.
•
CONTROL
•
HALOTHANE 2%
125
/
/
o"'-,
///
~S
50
•
, "'',
/
25
"~
"
"'O
m~ -9
-8
-7
-6
-5
-4
-3
-2
log [Ca], M Figure 3. Calcium dependence of [3H]ryanodine binding in the abscence (triangles) and presence (circles) of 2% halothane. Each data point represents a single experiment performed with quadruplicate assays. 598
Vol. 186, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DISCUSSION
C o n t r a c t i o n s t u d i e s in isolated myocardial t i s s u e myofibrfls
(14),
halothane,
the
and
isolated
most
widely
myocytes studied
(15-17), anesthetic,
(Ii-13),
have
skinned
suggested
decreases
the
that net
s e q u e s t r a t i o n of Ca 2÷ by the SIL Exposure of cardiac t i s s u e to h a l o t h a n e leads to myocardial depression a n d negative inotropy (11-13). Our results are c o n s i s t e n t with a direct effect of h a l o t h a n e on t h e cardiac Ca 2÷ release c h a n n e l since a d o s e - d e p e n d e n t stimulation of [3H]ryanodine binding was evident in the clinical range of anesthetic delivered to SR in a g a s e o u s form. The s t i m u l a t i o n of binding suggested t h a t h a l o t h a n e increases
C a 2÷
release
c h a n n e l activity. This conclusion is based on the generalized finding t h a t ligands t h a t e n h a n c e
C a 2÷
release from the SR also e n h a n c e [3H] ryanodine
binding, w h e r e a s ligands t h a t inhibit Ca 2+ release also inhibit [3H]ryanodine b i n d i n g (10). Hence, the open conformational state of the receptor a p p e a r s to r e p r e s e n t the high affinity binding site for ryanodine a n d binding site d e n s i t y therefore serves as a n indicator of the fraction of receptors t h a t are present, a t a given ligand concentration, in an open conformation. At this point however, we do n o t k n o w w h e t h e r h a l o t h a n e only increases [3H]ryanodine b i n d i n g site density, as would be required by the interpretation above, or if m a y also increase [3H]ryanodine binding site aff'mity. In the latter case, a n e s t h e t i c effects on
[3H]ryanodine binding activity m a y not be directly
related to c h a n g e s in c h a n n e l gating. Nevertheless, the increase of cardiac [3H]ryanodine b i n d i n g produced by h a l o t h a n e a n d enflurane, b u t n o t by isoflurane, is c o n s i s t e n t with a n alteration of gating b e c a u s e isoflurane c a u s e s m u c h less myocardial depression t h a n the other a n e s t h e t i c s (11-13). The idea t h a t anesthetics m a y affect Ca 2÷ release c h a n n e l gating is also s u p p o r t e d by the Ca2*-dependence of h a l o t h a n e stimulation. The Ca z÷ d e p e n d e n c e of [3H]ryanodine binding h a s been interpreted to reflect the b i n d i n g of Ca 2÷ to a high affinity a n d a low affinity site controlling c h a n n e l gating (10). T h u s , Ca 2÷ binding to a high affinity site would increase the open probability of the channel, leading to a n increase in the d e n s i t y of sites available for [3H]ryanodine binding. Conversely, Ca z÷ b i n d i n g to a low-affinity site would close or inactivate the c h a n n e l a n d t h u s decrease the n u m b e r of binding sites. Within this scheme, the major effect of h a l o t h a n e a p p e a r e d to be a n increase in the Ca 2÷ sensitivity to b o t h the high a n d low-affinity Ca 2÷ binding sites controlling c h a n n e l gating. The
selective interaction
of enflurane with
cardiac
receptors
and
isoflurane with skeletal receptors is significant since t h e s e two a n e s t h e t i c s 599
Vol. 186, No. 1, 1992
are
methyl-ethyl
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ether
isomers.
These
results
suggest
site-specifc
interactions of the a n e s t h e t i c s with each receptor type. Skeletal a n d cardiac r y a n o d i n e receptor isoforms are different
pharmacologic
responses
only =60% homologous (17), to
various
ligands
was
not
so t h a t totally
unexpected. Yet volatile a n e s t h e t i c s are simple h y d r o c a r b o n s w h i c h have been proposed to act on ion c h a n n e l s t h r o u g h a n o n - p r o t e i n m e d i a t e d interaction. Specifically, volatile a n e s t h e t i c s are classically t h o u g h t to alter b u l k m e m b r a n e lipid properties in the vicinity of protein c h a n n e l s (18). This explanation s e e m s less plausible, at least for the Ca 2÷ release c h a n n e l of SR, since enflurane a n d isoflurane are chemical isomers with similar physical properties.
The
results
presented
here
may
have
implications
for the
m e c h a n i s m of action of a n e s t h e t i c s in other t i s s u e s as well.
REFERENCES
1. 2. 3. 4. 5.
Brown BR, C r o u t JR. (1971) Anesthesiology 34:236-245. R u s y BF, Komai, H. (1987) Anesthesiology 67:745-766. P e s s a h IN a n d Zimanyi I. (1991) Mol P h a r m a c o l 39:679-689. Meissner G. (1986) J Biol Chem 261:6300-6306. Mickelson JR, Gallant EM, Litterer LA, J o h n s o n KM, Rempel WE, Louis CL. (1988) J Biol C h e m 263:9310-93158. 6. M e i s s n e r G. (1983) J Biol Chem 259:2365-2374. 7. Fabiato A. (1983) Am J Physiology 245:C1-C14. 8. McPherson PS, Kim Y-K, Valdivia H, K n u d s o n CM, T a k e k u r a H, FranziniA r m s t r o n g C, Coronado R, Campbell tiP. (1991) Neuron 7:17-25. 9. O t s u IC H u n t i n g t o n WF, K h a n n a VK, Zorzato F, Green NM McLerman DH. (1990) J Biol C h e m 265(23):13472-13483, 10. Valdivia HH, Hogan K, Coronado R. (1991) Am J Physiol 261:C237C245. 11. L y n c h C, Frazer MJ. (1989) Anesthesiology 70:511-522. 12. Komai H, R u s y BF. (1990) Anesthesiology 72:694-698. 13. H o u s m a n s PR, Murat. (1988) Anesthesiology 69:451-463. 14. S u JY, Kerrick WGL. (1978) Pflugers Arch 375:111-117. 15. Wheeler DM, Rice RT, Hansford RG, L a k a t t a EG. (1988) Anesthesiology 69:578-583. 16. Wilde DW, Knight PR, S h e t h N, Williams BS. (1991) Anesthesiology 74:1075-1086. 17. K a t s u o k a M, Kobayashi BL Ohnishi ST. (1989) Anesthesiology 70:954-960. 18. F r a n k s WP, Lieb WR. (1991) Science 254:427-436.
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