181
Pharmacological Research Communications, Vol. 7, No. 2, 1975
STIMULUS-COUPLED 3H-NOREPINEPHRINE
Maurizio
RAITERI
(") Istituto
("), Giulio LEVI
dello Shock
Sacchetti
Cellu!are,
CNR,
OF U N M E T A B O L I Z E D
FROM RAT BRAIN
di F a r m a c o l 0 g i a
Fisiopatoiogia Pineta
RELEASE
("") and Rodolfo
and Centro (CNR),
FEDERICO
per lo Studio della
Universit~
644, Roma and ("") Roma,
SYNAPTOSOMES
Cattolica,
Laboratorio
Via
di Biologia
Italy.
Received 12 November 1974
SUMMARY.
-
, . • The calcium-triggered release •of 3~ s-norepznepnrzne from
prelabelled rat brain synaptosomes was studied by a superfusion tech nique. It was found that the radioactivity released by the depolarizing stimulus, in the presence of calcium, consisted of unmetabolized 3Hi norepinephrine. In contrast, the increased release caused by reserpine was totally accounted for by 3H-deaminated metabolites. The results suggest that calcium triggers a release of the amine directly from synaptic vesicles.
INTRODUCTION. - The m e c h a n i s m s by w h i c h n e u r o t r a n s m i t t e r s are released from nerve terminals are not yet c o m p l e t e l y understood.
It is w i d e l y
key role in t r i g g e r i n g in conditions
a c c e p t e d that c a l c i u m plays
the release
of m e m b r a n e
of n e u r o t r a n ~ i t t e r s
depoiarization.
evidence was o b t a i n e d
in studies
(Katz,
and Misu, ~ 1967;
1969; ~ K i r k e p a r
some investigations calcium
Most of the
on p e r i p h e r a l
have d e m 0 n s t r a t e d
ions for the r e l e a s e
a
nerves
D o U g l a s , 1968i , the i m p o r t a n c e
of several
but of
neurotransmitters
(")
Pharmacological Research Communications, Vol. 7, No. 2, 1975
182
also in preparations'from central nervous tissues (Baldessarini and K0pin, 1967; Blaustein et al., 1972; Bradford et al., 1973). In the case of norepinephrine •(NE), the data obtained with adrenal medulla and sYmPathetic nerves have given str0ng support:to the/hyF0thesis that a mechanism of exbcytosis is operiting' in the release 0f the amine from the storage granules (Douglas, I•968; weinshilboum et al., •
,
,
,
, ,
'
.,
,
,
"
1971). •However, our knoWledg e about••the mechanisms of stimulated release of NE in nerve terminals of the central nervous system is~n0t as advanced. Evidence has•been presented suggesting that the mechanisms Of storage and release/of ME in synaptic vesicles from cen£ral nervous system may be different from those described for adrenal medulla (MaYnert~/ 1973). In the present investigation we utilized a new superfusion technique• (Raiteri•et al•., 1974) tO analyze the reiease of NE from isolated•PresynaPtiC nerve endings. The/data obtained show that a depolarizing stimulus, in the presence of calcium ions, triggers a release of unme tabolized NE from central nerve terminals. •
METHODS.
-
',
.
,
Synaptosomes prepared from adult (250 g) Wistar
rat cerebrum according to Gray and whittaker (1962) were resuspended in 0.32M g l u c o s e a t a protein concentration of about 5 mg/ml. The suspension was diluted 1:i0 in a calciunt-free Krebs-Ringer medium (.128 ~M NaCI, r5mM KCI, 1.2 m M MgSO 4 , 5mM Na2HP04, 10 mM Tris-HCl buffer at pH 7.4) ~nd equilibrate d for 15 min at 37"in a~rotary water-bath, in an:air athm0sPhere. Then a small voiume (I/50 of the final volume) of a solution containing 3H-norepinephrine (Amersham Radiochemicai C e n t r e ) w a s added to the incubation flask, • to give a fina! concentration of 0-2 ~ M. After 10 min incubati0n, 0.4 ml aliquots of the suspension were placed on 0.65 ~m Millipore f i l t e r s l a y i n g at the bot£on of several parallel superfusi0n chambers, thermostated at 37 ° , as already described (Raiteriret al. 1974). The fil-
Pharmacological Research Communications, Vol. 7, No. 2, 1975
183
ters were washed with g l u c o s e - c o n t a i n i n g , c a l c i u m - f r e e , o x y genated m e d i u m , a t
37°,and then superfusion was started with
the same medium at a rate of 0.45 ml/min. After 7 min,
the
superfusion m e d i u m was substituted with new m e d i u m contai~ hinge56 mM KCI NaCI)
and,
(replacing an equimolar concentration of
in some cases,
2.7 m M C a C I 2. T h e : s u p e r f u s i o n
was continued for an other 11 min and the r a d i o a c t i v i t y was counted in aliqu0ts of the s u P e r f u s a t e P f r a c t i o n s in the filter.
and
The amount of 3H-deaminated m e t a b o l i t e s was
measured as described by Baldessarini and Kopin
(1967).
In some e x P e r i m e n t s synaptosomes were prelabelled with 3H-NE in a m e d i u m containing 2.7 mM CaCI 2. Aliquots were superfused either with standard,
calcium-containing,medium
or with m e d i u m containing also I0-7M reserpine.
The super-
fusates were analyzed for t o t a l radioactivity and 3H-deami nated metabolites
RESULTS
AND
as mentioned above. Blaustein et al.
DISCUSSION..
(1972)
showed
~hat depolarized synaFtosomes release a larger amount of NE in the presence of calcium than in its absence. study NE was determined
fluorimetrically in the medium,
after 10 min incubation;
the p r o b l e m of the origin of the
NE released by the stimulation was not approached experiments,
In that
in these
which were performed in the presence of
monoaminoloxydase
inhibitor.
a
A determination of the ratio
between NE and its deaminated metabolites,
during s p o n t a -
neous and stimulated release, Can provide some i n f o r m a t i o n as to the origin of the amine liberated. in vivo Philippu et al.
In experiments
(1970-) showed that, under different
conditions of stimulation,
the ratio between unmetabolized
radioactive NE and deaminated metab~lites was the same as during spontaneous ni and Kopin
release.
(1967)found
On the other hand,
Baldessari-
t h a t electrical s t i m u l a t i o n of
brain s l i c e s caused an increase in the release of 3H-NE and of deaminated metabolites,
and also in the ratio bet-
ween 3 H - N E and 3H-deaminated metaboiites. in experiments
The situation
in vivo and also with relatively intact
tissue preparations,
such as brain slices,
is certainly
Pharmaco/ogical Research Communications, Vol. 7, No. 2, 1975
184
13 12 t
,-
0
f~ i \\
11
¢J
,,--A H o--o ,-----
~
~
m 0.
.~
9 8'
&J
Total 3H.no calcium Total I.I.2.7mM CaCi2 3H- deaminated meta~01ites, no calcium ~H-deaminated metabolites, Z.7mM CaCi2 3
"
'
7 "0
m
6
c
5
0 ..,. c
4-
~
3
&~
--
\
21
f ..
•
1
I
2
.
.
J
3
.
.i
4
[
5
6
.
L
7
_
J
JL
8
9
__.
Fraction number (1 fraction= 2 rnin = 0.9 ml) Fig, I - C a l c i u m - d e p e n d e n t release of 3H-norepinephrine from depolarized synaptosomes. Purified synaptosomes from rat cerebrum were prelabelled with 0;2 p M 3H'NE for 10 min and then SuPerfused with standard medium, ~ in the absence of calcium./~fter 7 min the superfuSion m e d i u m was substituted with:,new m e d i u m containing 56.mM KCI (with or without 2.7 mM CaCI~). The total radioactivity of the superfusate fractions-and that present in the form Of deaminated m e t a b o l i t e s were measured as indicated ±n the text. T h e results are expressed as percentages of total r a d i o a c t i v i t y recovered (that is, total fractions plus r a d i o a c t i v i t y remaining in the filters). The curves were taken f r o m experiments run in duplicate, using four parallel superfusion chambers. The pattern shown was reproduced in:for:experiments run in different days.
Pharmacological Research Communications, Vol. 7, No. 2, 1975 complex,
185
and the NE released does not necessarily
only and directly
from nerve
In the p r e s e n t s t u d y perimental
conditions
the kinetics without ~he
terminals.
we approached
the p r o b l e m
that were appropriate
of NE release interference
derive in ex-
to follow
from p u r i f i e d nerve herminals,
of r e - u p t a k e of the amine re-
i
leased
(Raiteri et al.,
res other
1974)
than nerve endings.
depolarizing
stimulus,
13 12 0
~
of calcium
a ions,
A--,~ Control Total =H =~'A lO"MReserpine } o-.-o vC i ~H-deaminated 10"7-MReserpine metabolites
9 8
~
7
0
i
from structu-
In these conditions,
in the presence
/\ ~i\
•~
0
and of release
~
•- -
6
~
5
~
4
%%%,
3--
"~.Q
2 1
"
1
3
4
5
D--___~O__._O_____..
6
7
O
8
Fraction number (1 fraction= 2min = 0.9 ml) Fig. 2 - Effect o f reserpine on the release of 3H-norepinephrine and 3H-deaminated m e t a b o l i t e s from synaptosomes. Expe:rimental details as in the legend for Fig. 1, except that calcium (2.7 mM) was present in incubation and superfusion media. Reserpine was added at the b e g i n n i n g of superfusion. This pattern of release was typical of four experiments run in duplicate in different days.
Pharmacological Research Communications, Vol. 7, No. 2, 1975
1 86
released unmctabolized
3H-NE from prelabelled
synapto-
somes, but did not alter appreciably the efflux of 3Hdeaminated metabolites
(Fig. 1).
Most of the synaptosomal NE seems to be localized in synaptic vesicles, (Glowinski,
and Very little in the cytoplasm
1970; Raiteri and Levi,
1973). Any increase
in the level of the free amine in the c y t o p l a s m would result in an increase of deaminated metabolites.
This
is confirmed by the experiments reported in Fig.
2, which
shows the eff!ux of radioactivity
from synaptosomes pre-
labelled with 3H-NE and superfused with standard medium, or with m e d i u m c o n t a i n i n g I0-7M r e s e r p i n e . The larger amount of radioactivity released/in the Presence of the drug
(which is known to release N E
the cytoplasm)
from the vesicles
into
was totally accounted for by 3H-deaminated
metabolites. It seems therefore likely that the NE released by-a depolarizing stimulus in the presence of calcium derives directly from synaptic vesicles, pin order to establish whether this release occurs through a•process of exocytosis it would be necessary to have more information on the composition of synaptic vesicles from central noradrenergic terminals,
and to s t u d y the release of some vesicular com-
f
ponents together w i t h that of NE. ACKNOWLEDGMENT.
- This study was partly supported by Grant
n. 73.00.741.O4 of the Italian National Research Council to one of us
(M.R.).
REFERENCES.
Baldessarini, R . J . and I.J.Kopin (1967), J.Pha~macol.exp. Ther. 156, 31. Blaustein, M.P., E.M, J o h s o n ,and P. N e e d l e m a n (1972), Proc. Nat.Acad. S c i . U S A 69, 2237. Bradford, H.F., G.W. Bennett and A.J.Thomas (I 973) , J.Neurochem. 21, 495. Douglas, W.W. (1968), Br. ~. Pharmacol. 34, 451. Glowinski, J. (1 970) S t o r a g e and release"of monoamines in the central nervous system, in: Handbook of Neurochemistry, e¢%, A. Lajtha, (Plenum Press, New York and London) p. 91.
Pharmacological Research Communications, Vol. 7, No. 2, 1975 Gray, E.G. and V.P. Whittaker (1962), J. Anat. (Lond.) __96,79Katz, B. (1969) The release of neural transmitter substances, in: The S h e r r i n g t o n Lectures, No.X (University Press, Liverpool) . Kirkepar, S.M. and J. MIsu (1967), J. Physiol. (Lond.)
18__8, 219. Maynert,
EoW.
(1973) Tissue storage of catecholamines, in: Chemical m o d u l a t i o n of brain function, ed. H.C. Sabelli (Raven Press, ]New York) p. 31. Philippu, A., G. Heyd and A. Burger (1970), Eur. J.Pharmacol. 9, 52. Raiteri, M. and G. Levi (1973), Nature New Biol. 245, 89. Raiteri, M., F. Angelini /nd G. Levi (1974), Eur. J. Pharmacol. 25, 411. Weinshilboum, R.M., N,B. ThDa, D.G. Johnson, I.J. Kopin and J. Axelrod (I 971) , Science 174, 1349.
187
Pharmacological Research Communications, Vol. 7, No. 2, 1975
STIMULUS-COUPLED 3H-NOREPINEPHRINE
Maurizio
RAITERI
(") Istituto
("), Giulio LEVI
dello Shock
Sacchetti
Cellu!are,
CNR,
OF U N M E T A B O L I Z E D
FROM RAT BRAIN
di F a r m a c o l 0 g i a
Fisiopatoiogia Pineta
RELEASE
("") and Rodolfo
and Centro (CNR),
FEDERICO
per lo Studio della
Universit~
644, Roma and ("") Roma,
SYNAPTOSOMES
Cattolica,
Laboratorio
Via
di Biologia
Italy.
Received 12 November 1974
SUMMARY.
-
, . • The calcium-triggered release •of 3~ s-norepznepnrzne from
prelabelled rat brain synaptosomes was studied by a superfusion tech nique. It was found that the radioactivity released by the depolarizing stimulus, in the presence of calcium, consisted of unmetabolized 3Hi norepinephrine. In contrast, the increased release caused by reserpine was totally accounted for by 3H-deaminated metabolites. The results suggest that calcium triggers a release of the amine directly from synaptic vesicles.
INTRODUCTION. - The m e c h a n i s m s by w h i c h n e u r o t r a n s m i t t e r s are released from nerve terminals are not yet c o m p l e t e l y understood.
It is w i d e l y
key role in t r i g g e r i n g in conditions
a c c e p t e d that c a l c i u m plays
the release
of m e m b r a n e
of n e u r o t r a n ~ i t t e r s
depoiarization.
evidence was o b t a i n e d
in studies
(Katz,
and Misu, ~ 1967;
1969; ~ K i r k e p a r
some investigations calcium
Most of the
on p e r i p h e r a l
have d e m 0 n s t r a t e d
ions for the r e l e a s e
a
nerves
D o U g l a s , 1968i , the i m p o r t a n c e
of several
but of
neurotransmitters
(")
Pharmacological Research Communications, Vol. 7, No. 2, 1975
182
also in preparations'from central nervous tissues (Baldessarini and K0pin, 1967; Blaustein et al., 1972; Bradford et al., 1973). In the case of norepinephrine •(NE), the data obtained with adrenal medulla and sYmPathetic nerves have given str0ng support:to the/hyF0thesis that a mechanism of exbcytosis is operiting' in the release 0f the amine from the storage granules (Douglas, I•968; weinshilboum et al., •
,
,
,
, ,
'
.,
,
,
"
1971). •However, our knoWledg e about••the mechanisms of stimulated release of NE in nerve terminals of the central nervous system is~n0t as advanced. Evidence has•been presented suggesting that the mechanisms Of storage and release/of ME in synaptic vesicles from cen£ral nervous system may be different from those described for adrenal medulla (MaYnert~/ 1973). In the present investigation we utilized a new superfusion technique• (Raiteri•et al•., 1974) tO analyze the reiease of NE from isolated•PresynaPtiC nerve endings. The/data obtained show that a depolarizing stimulus, in the presence of calcium ions, triggers a release of unme tabolized NE from central nerve terminals. •
METHODS.
-
',
.
,
Synaptosomes prepared from adult (250 g) Wistar
rat cerebrum according to Gray and whittaker (1962) were resuspended in 0.32M g l u c o s e a t a protein concentration of about 5 mg/ml. The suspension was diluted 1:i0 in a calciunt-free Krebs-Ringer medium (.128 ~M NaCI, r5mM KCI, 1.2 m M MgSO 4 , 5mM Na2HP04, 10 mM Tris-HCl buffer at pH 7.4) ~nd equilibrate d for 15 min at 37"in a~rotary water-bath, in an:air athm0sPhere. Then a small voiume (I/50 of the final volume) of a solution containing 3H-norepinephrine (Amersham Radiochemicai C e n t r e ) w a s added to the incubation flask, • to give a fina! concentration of 0-2 ~ M. After 10 min incubati0n, 0.4 ml aliquots of the suspension were placed on 0.65 ~m Millipore f i l t e r s l a y i n g at the bot£on of several parallel superfusi0n chambers, thermostated at 37 ° , as already described (Raiteriret al. 1974). The fil-
Pharmacological Research Communications, Vol. 7, No. 2, 1975
183
ters were washed with g l u c o s e - c o n t a i n i n g , c a l c i u m - f r e e , o x y genated m e d i u m , a t
37°,and then superfusion was started with
the same medium at a rate of 0.45 ml/min. After 7 min,
the
superfusion m e d i u m was substituted with new m e d i u m contai~ hinge56 mM KCI NaCI)
and,
(replacing an equimolar concentration of
in some cases,
2.7 m M C a C I 2. T h e : s u p e r f u s i o n
was continued for an other 11 min and the r a d i o a c t i v i t y was counted in aliqu0ts of the s u P e r f u s a t e P f r a c t i o n s in the filter.
and
The amount of 3H-deaminated m e t a b o l i t e s was
measured as described by Baldessarini and Kopin
(1967).
In some e x P e r i m e n t s synaptosomes were prelabelled with 3H-NE in a m e d i u m containing 2.7 mM CaCI 2. Aliquots were superfused either with standard,
calcium-containing,medium
or with m e d i u m containing also I0-7M reserpine.
The super-
fusates were analyzed for t o t a l radioactivity and 3H-deami nated metabolites
RESULTS
AND
as mentioned above. Blaustein et al.
DISCUSSION..
(1972)
showed
~hat depolarized synaFtosomes release a larger amount of NE in the presence of calcium than in its absence. study NE was determined
fluorimetrically in the medium,
after 10 min incubation;
the p r o b l e m of the origin of the
NE released by the stimulation was not approached experiments,
In that
in these
which were performed in the presence of
monoaminoloxydase
inhibitor.
a
A determination of the ratio
between NE and its deaminated metabolites,
during s p o n t a -
neous and stimulated release, Can provide some i n f o r m a t i o n as to the origin of the amine liberated. in vivo Philippu et al.
In experiments
(1970-) showed that, under different
conditions of stimulation,
the ratio between unmetabolized
radioactive NE and deaminated metab~lites was the same as during spontaneous ni and Kopin
release.
(1967)found
On the other hand,
Baldessari-
t h a t electrical s t i m u l a t i o n of
brain s l i c e s caused an increase in the release of 3H-NE and of deaminated metabolites,
and also in the ratio bet-
ween 3 H - N E and 3H-deaminated metaboiites. in experiments
The situation
in vivo and also with relatively intact
tissue preparations,
such as brain slices,
is certainly
Pharmaco/ogical Research Communications, Vol. 7, No. 2, 1975
184
13 12 t
,-
0
f~ i \\
11
¢J
,,--A H o--o ,-----
~
~
m 0.
.~
9 8'
&J
Total 3H.no calcium Total I.I.2.7mM CaCi2 3H- deaminated meta~01ites, no calcium ~H-deaminated metabolites, Z.7mM CaCi2 3
"
'
7 "0
m
6
c
5
0 ..,. c
4-
~
3
&~
--
\
21
f ..
•
1
I
2
.
.
J
3
.
.i
4
[
5
6
.
L
7
_
J
JL
8
9
__.
Fraction number (1 fraction= 2 rnin = 0.9 ml) Fig, I - C a l c i u m - d e p e n d e n t release of 3H-norepinephrine from depolarized synaptosomes. Purified synaptosomes from rat cerebrum were prelabelled with 0;2 p M 3H'NE for 10 min and then SuPerfused with standard medium, ~ in the absence of calcium./~fter 7 min the superfuSion m e d i u m was substituted with:,new m e d i u m containing 56.mM KCI (with or without 2.7 mM CaCI~). The total radioactivity of the superfusate fractions-and that present in the form Of deaminated m e t a b o l i t e s were measured as indicated ±n the text. T h e results are expressed as percentages of total r a d i o a c t i v i t y recovered (that is, total fractions plus r a d i o a c t i v i t y remaining in the filters). The curves were taken f r o m experiments run in duplicate, using four parallel superfusion chambers. The pattern shown was reproduced in:for:experiments run in different days.
Pharmacological Research Communications, Vol. 7, No. 2, 1975 complex,
185
and the NE released does not necessarily
only and directly
from nerve
In the p r e s e n t s t u d y perimental
conditions
the kinetics without ~he
terminals.
we approached
the p r o b l e m
that were appropriate
of NE release interference
derive in ex-
to follow
from p u r i f i e d nerve herminals,
of r e - u p t a k e of the amine re-
i
leased
(Raiteri et al.,
res other
1974)
than nerve endings.
depolarizing
stimulus,
13 12 0
~
of calcium
a ions,
A--,~ Control Total =H =~'A lO"MReserpine } o-.-o vC i ~H-deaminated 10"7-MReserpine metabolites
9 8
~
7
0
i
from structu-
In these conditions,
in the presence
/\ ~i\
•~
0
and of release
~
•- -
6
~
5
~
4
%%%,
3--
"~.Q
2 1
"
1
3
4
5
D--___~O__._O_____..
6
7
O
8
Fraction number (1 fraction= 2min = 0.9 ml) Fig. 2 - Effect o f reserpine on the release of 3H-norepinephrine and 3H-deaminated m e t a b o l i t e s from synaptosomes. Expe:rimental details as in the legend for Fig. 1, except that calcium (2.7 mM) was present in incubation and superfusion media. Reserpine was added at the b e g i n n i n g of superfusion. This pattern of release was typical of four experiments run in duplicate in different days.
Pharmacological Research Communications, Vol. 7, No. 2, 1975
1 86
released unmctabolized
3H-NE from prelabelled
synapto-
somes, but did not alter appreciably the efflux of 3Hdeaminated metabolites
(Fig. 1).
Most of the synaptosomal NE seems to be localized in synaptic vesicles, (Glowinski,
and Very little in the cytoplasm
1970; Raiteri and Levi,
1973). Any increase
in the level of the free amine in the c y t o p l a s m would result in an increase of deaminated metabolites.
This
is confirmed by the experiments reported in Fig.
2, which
shows the eff!ux of radioactivity
from synaptosomes pre-
labelled with 3H-NE and superfused with standard medium, or with m e d i u m c o n t a i n i n g I0-7M r e s e r p i n e . The larger amount of radioactivity released/in the Presence of the drug
(which is known to release N E
the cytoplasm)
from the vesicles
into
was totally accounted for by 3H-deaminated
metabolites. It seems therefore likely that the NE released by-a depolarizing stimulus in the presence of calcium derives directly from synaptic vesicles, pin order to establish whether this release occurs through a•process of exocytosis it would be necessary to have more information on the composition of synaptic vesicles from central noradrenergic terminals,
and to s t u d y the release of some vesicular com-
f
ponents together w i t h that of NE. ACKNOWLEDGMENT.
- This study was partly supported by Grant
n. 73.00.741.O4 of the Italian National Research Council to one of us
(M.R.).
REFERENCES.
Baldessarini, R . J . and I.J.Kopin (1967), J.Pha~macol.exp. Ther. 156, 31. Blaustein, M.P., E.M, J o h s o n ,and P. N e e d l e m a n (1972), Proc. Nat.Acad. S c i . U S A 69, 2237. Bradford, H.F., G.W. Bennett and A.J.Thomas (I 973) , J.Neurochem. 21, 495. Douglas, W.W. (1968), Br. ~. Pharmacol. 34, 451. Glowinski, J. (1 970) S t o r a g e and release"of monoamines in the central nervous system, in: Handbook of Neurochemistry, e¢%, A. Lajtha, (Plenum Press, New York and London) p. 91.
Pharmacological Research Communications, Vol. 7, No. 2, 1975 Gray, E.G. and V.P. Whittaker (1962), J. Anat. (Lond.) __96,79Katz, B. (1969) The release of neural transmitter substances, in: The S h e r r i n g t o n Lectures, No.X (University Press, Liverpool) . Kirkepar, S.M. and J. MIsu (1967), J. Physiol. (Lond.)
18__8, 219. Maynert,
EoW.
(1973) Tissue storage of catecholamines, in: Chemical m o d u l a t i o n of brain function, ed. H.C. Sabelli (Raven Press, ]New York) p. 31. Philippu, A., G. Heyd and A. Burger (1970), Eur. J.Pharmacol. 9, 52. Raiteri, M. and G. Levi (1973), Nature New Biol. 245, 89. Raiteri, M., F. Angelini /nd G. Levi (1974), Eur. J. Pharmacol. 25, 411. Weinshilboum, R.M., N,B. ThDa, D.G. Johnson, I.J. Kopin and J. Axelrod (I 971) , Science 174, 1349.
187
