Department of Physiology and Groupe de Recherche sur le Système Nerveux Autonome, Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada H3C 3J7
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ABSTRACT |
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We have
recently reported enhanced levels of Gi proteins in
genetic and other experimentally induced models of hypertension, whereas the levels of Gs
were decreased in hypertensive
rats expressing cardiac hypertrophy. The present studies were
undertaken to investigate whether the decreased levels of
Gs
are associated with cardiac hypertrophy per se and
used an aortocaval fistula (AV shunt; volume overload) rat model that
exclusively expresses cardiac hypertrophy. Cardiac hypertrophy in
Sprague-Dawley rats (200-250 g) was induced under anesthesia, and,
after a period of 10 days, the hearts were used for adenylyl cyclase
activity determination, protein quantification, and mRNA level
determination. A temporal relationship between the expression of
Gs
proteins and cardiac hypertrophy was also examined on
days 2, 3, 7, and 10 after induction of AV shunt
in the rat. The heart-to-body-weight ratio (mg/g) was significantly
increased in AV shunt rats after 3, 7, and 10 days of induction of AV
shunt compared with sham-operated controls, whereas arterial blood
pressure was not different between the two groups. Guanosine
5'-O-(3-thiotriphosphate) (GTP
S) stimulated adenylyl
cyclase activity in a concentration-dependent manner in heart membranes
from both groups; however, the degree of stimulation was significantly
decreased in AV shunt rats. In addition, the stimulatory effects of
isoproterenol were also diminished in AV shunt rats compared with
control rats, whereas glucagon-stimulated adenylyl cyclase activity was
not different in the two groups. The inhibitory effects of oxotremorine
(receptor-dependent Gi functions) and low concentrations of
GTP
S on forskolin-stimulated adenylyl cyclase activity
(receptor-independent Gi functions) were not different in
the two groups. In addition forskolin and NaF also stimulated adenylyl
cyclase activity to a lesser degree in AV shunt rats compared with
control rats. The levels of Gi
-2 and Gi
-3
proteins and mRNA, as determined by immunoblotting and Northern
blotting, respectively, were not different in both groups; however, the
levels of Gs
45 and
Gs
47, and not of
Gs
52, proteins were significantly decreased
in AV shunt rats by days 7 and 10 compared with
control rats, whereas no change was observed on days 2 and
3 after induction of AV shunt. These results suggest that
the decreased expression of Gs
proteins may not be the
cause but the effect of hypertrophy and that the diminished
responsiveness of adenylyl cyclase to GTP
S, isoproterenol, NaF, and
forskolin in hearts from AV shunt rats may partly be due to the
decreased expression of Gs
. It can be concluded from
these studies that the decreased expression of Gs
may be
associated with cardiac hypertrophy and not with arterial hypertension.
G protein; adenylyl cyclase; aortocaval fistula; AV shunt
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INTRODUCTION |
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THE ADENYLYL
CYCLASE/cAMP signal transduction
system has been implicated in the regulation of various physiological
functions such as vascular tone and reactivity, cardiac functions,
platelet functions, etc. (49). The adenylyl cyclase system
consists of three distinct components: receptor, catalytic subunit, and
guanine nucleotide regulatory proteins (G proteins). The stimulatory
and inhibitory responses of hormones on adenylyl cyclase are mediated via the stimulatory (Gs) and inhibitory (Gi)
proteins, respectively (20, 41, 42). G proteins are
heterotrimeric proteins composed of -,
-, and
-subunits, and
the specificity of G proteins is attributed to the
-subunit
(42). Molecular cloning has revealed four different
isoforms of Gs
resulting from the differential splicing
of one gene (8, 34, 36) and three distinct isoforms of
Gi
: Gi
-1, Gi
-2, and
Gi
-3 encoded by three different genes (23-25). All three forms of Gi
have
been shown to be implicated in adenylyl cyclase inhibition
(51) and the activation of ACh-K+ channels
(9). Five different
-subunits of 35-36 kDa and
seven
-subunits of 8-10 kDa have been identified by molecular
cloning (10, 40). The G
-subunit has been shown to
regulate various effectors including adenylyl cyclase, phospholipase
C
, and K+ channels (40, 45, 50). Of the
eight types of adenylyl cyclase that have been cloned and expressed
(14), only two types, namely, types V and VI, have been
identified in the heart, aorta, and brain (26, 35).
Adenylyl cyclase types II and IV are activated by G
in the
presence of Gs
; type I is inhibited by G
; and types III, V, and VI do not appear to be directly regulated by G
(46, 48).
Alterations in G protein levels and adenylyl cyclase activity and its
responsiveness to various hormones have been documented in
cardiovascular tissues from genetic (spontaneously hypertensive rats,
SHR) and various experimental models of hypertension (1, 2-4, 7, 30). We have recently shown an increased expression of Gi-2 and Gi
-3 at protein and mRNA
levels and an altered hormonal inhibition and stimulation of
adenylyl cyclase in heart and aorta from SHR as well as in
DOCA-salt hypertensive rats (2, 4, 5, 7, 30), whereas an
unaltered expression of Gi
and Gs
proteins has also been reported in hearts from SHR and other models of
hypertension (32, 33). However, due to the expression of
cardiac hypertrophy with hypertension, it is not known whether these
changes are due to the expressed hypertrophy or hypertension. We have
recently shown that
N
-nitro-L-arginine methyl ester
(L-NAME) hypertensive rats that do not express
cardiac hypertrophy (6) exhibited an increased expression
of Gi levels with unaltered Gs levels
(15). Taken together, it may be possible that decreased
levels of Gs
may be associated with cardiac hypertrophy
and not with arterial hypertension. To investigate this possibility, we
have used volume-overload (VO) cardiac hypertrophied rats that express
cardiac hypertrophy but not arterial hypertension (18) and
examined the G protein expression (Gi
and
Gs
) and regulation of adenylyl cyclase by various
modulators in VO hypertrophied rat hearts. We have shown that
aortocaval fistula (AV shunt) rats exhibit decreased expression of
Gs
protein and mRNA, which occurs only after the
development of cardiac hypertrophy.
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MATERIALS AND METHODS |
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Materials.
Plasmids containing rat cDNAs encoding Gi-2,
Gi
-3, Gs
, and the adenylyl cyclase
catalytic subunit V were kindly obtained from Dr. Randall Reed from the
John Hopkins University and Dr. Hiroshi Itoh from the University of
Tokyo. The 32-mer oligonucleotide that recognizes a highly conserved
region of the 28S rRNA was kindly donated by Dr. Yoshihiro Ishiwaka
from Lederle Laboratory of New York. Chemicals necessary for total RNA
extraction and Northern blot analysis were obtained from Sigma Chemical
(St. Louis, MO), except guanidinium thiocyanate, which was from
Research Organics (St. Laurent, PQ, Canada). Enzymes used for
radiolabeling of cDNA probes were obtained from BRL (Burlington,
Ontario, Canada), and other chemicals were from Pharmacia (Baie
D'Urfee, PQ, Canada). Nylon filter (Hybond-N),
[
-32P]dCTP (3,000 Ci/mmol) and
[
-32P]ATP (3,000 Ci/mmol) were purchased from Amersham
(Oakville, Ontario, Canada).
Induction of VO hypertrophy by AV shunt. Male Sprague-Dawley rats (200-250 g) were purchased from Charles River Canada (St. Constant, Quebec, Canada). Cardiac hypertrophy was induced in rats by the method described by Garcia and Diebold (18). Briefly, each rat was anesthetized with pentobarbital sodium (60 mg/kg body wt), and an excision was made along the abdominal cavity and the visceral organs displaced. The descending aorta was isolated and ligated caudal to the renal artery and cephalic to the aortic bifurcation. The aorta was punctured through the adjacent wall and into the inferior vena cava with a 20-gauge needle. The needle was removed fully, and the initial aortic entry puncture point was sealed with a drop of cyanoacrylate glue. The ligation was removed after 10-20 s to insure proper drying, and the patency of the shunt was verified visually by mixing of arterial and venous blood. The entire surgical procedure took <10 min. The sham-operated control protocol was identical to AV shunt induction, except no aortic puncture was performed. After 10 days, the blood pressure was measured by the tail-cuff method, and the rats were killed by decapitation. Some rats were killed after 2, 3, and 7 days after the induction of AV shunt. The hearts were removed for adenylyl cyclase activity determination, mRNA levels, and G protein quantification.
This method of induction of VO cardiac hypertrophy has been shown to produce an "eccentric" form of cardiac hypertrophy that is characterized by normal wall thickness, a disproportionately large increase in heart chamber volume, and the serial addition of sarcomeres (19, 37).Preparation of heart particulate fraction.
Heart particulate fraction was prepared as described previously
(31). The dissected hearts were quickly frozen in liquid N2 and pulverized to a fine powder using a mortar and
pestle cooled in liquid N2. The powder was stored at
80°C until assayed. The powder was homogenized (12 stokes) in a
teflon glass homogenizer, in a buffer containing 10 mM
Tris · HCl, 1 mM EDTA (pH 7.5). The homogenate was centrifuged
at 1,000 g for 10 min. The supernatant was discarded, and
the pellet was finally suspended in the above buffer and used for
adenylyl cyclase activity determination and immunoblotting studies.
Immunoblotting. Immunoblotting of G proteins was performed as described previously (3). After SDS-PAGE, the separated proteins were electrophoretically transferred to a nitrocellulose sheet (Schleicher & Schuell) with a semidry transblot apparatus (Bio-Rad) at 15 V for 45 min. After transfer, the membranes were washed twice in phosphate-buffered saline (PBS) and were incubated in PBS containing 3% skim milk at room temperature for 2 h. The blots were then incubated with antibodies against G proteins in PBS containing 1.5% skim milk and 0.1% Tween-20 at room temperature overnight. The antigen-antibody complexes were detected by incubating the blots with goat anti-rabbit IgG (Bio-Rad) conjugated with horseradish peroxidase for 2 h at room temperature. The blots were then washed three times with PBS before reaction with enhanced chemiluminescence Western blotting detection reagents (Amersham).
Adenylyl cyclase activity determination.
Adenylyl cyclase activity was determined by measuring
[32P]cAMP formation from [-32P]ATP, as
described previously (2, 3). The assay medium containing
50 mM glycylglycine, pH 7.5, 0.5 mM MgATP, [
-32P]ATP
(1-1.5 × 106 counts/min), 5 mM
MgCl2, 100 mM NaCl, 0.5 mM cAMP, 1 mM IBMX, 0.1 mM EGTA, 10 µM guanosine 5'-O-(3-thiotriphosphate) (GTP
S; or
otherwise as indicated), and an ATP-regenerating system consisting of 2 mM phosphocreatine, 0.1 mg creatine kinase/ml, and 0.1 mg myokinase/ml
in a final volume of 200 µl. Incubations were initiated by addition
of the membrane preparation (50-100 µg) to the reaction mixture,
which had been thermally equilibrated for 2 min at 37°C. The
reactions, conducted in triplicate for 10 min at 37°C, were terminated by the addition of 0.6 ml of 120 mM zinc acetate. cAMP was
purified by coprecipitation of other nucleotides with
ZnCO3, by addition of 0.5 ml of 144 mM
Na2CO3, and subsequent chromatography by the
double-column system, as described by Salomon et al. (38). Under the assay conditions used, adenylyl cyclase activity was linear
with respect to protein concentration and time of incubation.
Na+-K+-ATPase activity determination. Na+-K+-ATPase activity was determined as described previously (43). Briefly, the reaction mixture containing 50 mM Tris-EDTA, 4 mM MgCl2, 100 mM NaCl, 20 mM KCl, and 4 mM Tris-ATP was preincubated at 37°C for 5 min. The reaction was initiated by addition of the membrane protein (50 µg) and was further incubated for 10 min. The reaction was terminated by the addition of 1 ml of cold 12% (TCA), and the Pi in the supernatant was determined by the method of Taussky and Shorr (47).
Northern analysis.
Total RNA was isolated by the guanidinium thiocyanate-phenol-chloroform
method described by Chomczynski and Sacchi (12). cDNA
inserts encoding for Gi -2, Gi
-3, and
Gs
were radiolabeled with [
-32P]dCTP by
random priming essentially described by Feinberg and Vogelstein
(16).
Data analysis. Results are expressed as means ± SE. Comparisons between groups (control and AV shunt rats) were made with Student's t-test for unpaired samples. Results were considered significant at a value of P < 0.05.
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RESULTS |
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Blood pressure and indexes of cardiac hypertrophy.
The heart-to-body-weight ratio in AV shunt rats was significantly
increased compared with the control sham-operated rats
(P < 0.001), whereas no change in arterial blood
pressure was detected (Table 1),
suggesting that the AV shunt rat model exhibits cardiac hypertrophy and
no hypertension. There was no significant difference in the
heart-to-body-weight ratio at 2 days of induction of AV shunt; however,
it was significantly increased by 15 ± 0.5% and 20 ± 2%
at 3 and 7 days, respectively, relative to sham-operated control rats
(P < 0.001, n = 4-6 for each
group). In addition, no significant difference in the activity of
Na+-K+-ATPase as a membrane marker was observed
in AV shunt rats compared with control rats (control rats; 18.7 ± 0.07 µmol Pi · mg
protein1 · h
1; n = 4), and AV shunt rats (19.4 ± 0.25 µmol
Pi · mg
protein
1 · h
1; n = 4). The data presented for adenylyl cyclase activity are related to
milligrams membrane protein and not to the amount of protein measured
in the hypertrophied myocardium, because the protein content per heart
was increased in AV shunt rats (305.0 ± 10 vs. 241.2 ± 12 mg/heart) due to an elevated mass of cardiac proteins; however, protein
content (mg/g heart) was not different in AV shunt rats compared with
control rats (control, 254.8 mg/g; AV shunt rats, 258.1 mg/g).
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G protein levels.
To investigate whether the altered levels of G proteins (enhanced
Gi and decreased Gs) observed in
hypertensive rats associated with hypertrophy (4) were
attributed to hypertension or to hypertrophy, the levels of
Gi and Gs proteins were determined in hearts
for AV shunt rats by immunoblotting techniques using specific
antibodies AS/7 against Gi
-1 and Gi
-2,
EC/2 against Gi
-3, RM/1 against Gs
, and
SW/1 against the common
-subunit of G
. The results depicted in
Fig. 1, show that the AS/7, EC/2, and SW/1 antibodies recognized a single protein of relative molecular mass 40 kDa referring to Gi
-2, [Gi
-1 has
been shown to be absent in heart (25)], 41 kDa referring
to Gi
-3, and 35 kDa referring to the common
-subunit
of G
, respectively, in both AV shunt and sham-operated rats;
however, no differences in the amounts of immunodetectable
Gi
-2 and Gi
-3 and G
were detected in
both groups. In addition, RM/1 antibody recognized three isoforms of Gs
: Gs
45,
Gs
47, and Gs
52 in
hearts from control and AV rats; however, the amounts of
immunodetectable Gs
45 and
Gs
47 and not of
Gs
52 were significantly decreased in AV
shunt rats compared with control rats.
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GTPS-stimulated adenylyl cyclase activity.
To investigate whether the decreased levels of Gs
in AV
shunt rats also resulted in decreased Gs
-mediated
functions, the effect of GTP
S, on adenylyl cyclase activity was
examined, and the results are shown in Fig.
4. GTP
S stimulated adenylyl cyclase in
a concentration-dependent manner in hearts from both groups; however,
the extent of stimulation was significantly decreased in AV shunt rats.
For example, GTP
S at 5 µM increased the adenylyl cyclase activity
by ~650% in the control rats and 580% in AV shunt rats.
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Hormonal regulation of adenylyl cyclase.
Because G proteins couple the hormone receptors to adenylyl cyclase and
mediate the stimulatory and inhibitory responses of hormones on
adenylyl cyclase, it was interesting to determine whether the altered
expression of Gs is reflected in the hormonal regulation
of adenylyl cyclase. For this reason, the effect of some hormones that
stimulate or inhibit adenylyl cyclase through Gs or
Gi, respectively, on adenylyl cyclase activity was examined in hearts from AV shunt rats. Figure 5
shows that isoproterenol and glucagon stimulated adenylyl cyclase
activity in hearts from control and AV shunt rats to various degrees;
however, the extent of isoproterenol-mediated stimulation was
significantly diminished in AV shunt rats compared with control rats
(580% vs. 410%), whereas the stimulatory effect of glucagon was not
different in the two groups of rats.
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Forskolin and NaF-stimulated adenylyl cyclase activity.
Forskolin stimulates adenylyl cyclase by interacting directly with the
catalytic subunit of adenylyl cyclase. To explore whether the catalytic
subunit of adenylyl cyclase is impaired in AV shunt rats, the effect of
forskolin on adenylyl cyclase was determined in hearts from control and
AV shunt rats. Figure 7 shows that forskolin stimulated adenylyl cyclase activity in hearts from both VO
and sham-operated controls; however, the extent of stimulation was
significantly decreased by ~55% in VO rats. Similarly, NaF, which
stimulates adenylyl cyclase by a receptor-independent mechanism but
requires Gs proteins, also stimulated adenylyl cyclase to a
lower extent in VO rats compared with sham-operated control rats.
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DISCUSSION |
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We have previously reported an increased expression of
Gi protein and mRNA in hearts and aorta from SHR and
DOCA-salt hypertensive rats compared with their respective controls
(2, 4). In addition, we have shown recently that
L-NAME hypertensive rats, which do not exhibit cardiac
hypertrophy, also exhibited an increased expression of
Gi
proteins (6). However, the levels of
Gs
were unaltered in L-NAME hypertensive
rats (6) and SHR (2) but decreased in
DOCA-salt hypertensive rats (4). These results suggest
that the decreased expression of Gs
may be associated with cardiac hypertrophy and not with arterial hypertension per se. In
the present studies, we report that the AV shunt rat model that
expressed eccentric cardiac hypertrophy and no arterial hypertension exhibited a decreased expression of Gs
protein and mRNA,
whereas the expression of Gi
-2 and Gi
-3
was not altered. Our results are in agreement with the studies reported
in pigs after chronic VO cardiac hypertrophy, where the amounts of
Gs
as measured by reconstitution assays were decreased
in hearts and the levels of pertussis toxin substrates were unaltered
(21). The decreased levels and functions of
Gs
protein have been shown in compensated left
ventricular hypertrophy (11), pressure overload left
ventricular failure (28), and myocardial ischemia
(44). The increased levels of catecholamines have been
reported in VO-induced cardiac hypertrophy in rats (13) as
well as in pigs (21), which may be responsible for
eliciting a decreased expression of Gs
proteins in the
current studies. In this regard, isoproterenol treatment of the rats
has been shown to decrease the levels of Gs
proteins without changing the levels of Gi
protein in ventricular
myocardium (27). The alteration in Gs
mRNA
in AV shunt rats may not be attributable to the variations in the
amounts of total RNA applied to the gels, because hybridization with a
32-mer oligonucleotide that recognizes a highly conserved region of 28S
rRNA showed a similar amount of 28S rRNA loaded from control and AV
shunt rats onto the gels.
We have also shown that the responsiveness of adenylyl cyclase to
GTPS stimulation was decreased in the hearts from AV shunt rats
compared with control rats, which may be due to the decreased levels of
Gs
protein or increased levels of Gi
protein. Because the levels of Gi
were not altered in AV
shunt rats, the decreased stimulation of adenylyl cyclase by GTP
S
can be explained by the decreased expression of Gs
protein in these rats. A relationship between decreased levels of
Gs
protein and a decreased stimulation of adenylyl
cyclase by guanine nucleotides in DOCA-salt hypertensive rats has been
shown previously (4). Furthermore, attenuated responsiveness of adenylyl cyclase to isoproterenol in AV shunt rats
compared with control rats may be due to the decreased levels of
Gs
and/or decreased number of
-adrenergic receptors
or impaired catalytic subunit, or a combination of all the three
components. In this regard, a downregulation of
-adrenergic
receptors due to increased levels of catecholamines and reduced
sensitivity to the chronotropic effect of isoproterenol has been
reported in pigs with VO hypertrophy (21). In addition,
since catalytic subunit of adenylyl cyclase was also impaired in AV
shunt rats as was demonstrated by decreased stimulation of adenylyl
cyclase by forskolin, it may be suggested that the decreased expression of Gs
, impaired catalytic subunit of adenylyl cyclase,
and downregulation of
-adrenergic receptors may all be responsible
for the decreased responsiveness of adenylyl cyclase to isoproterenol
stimulation in AV shunt rats. However, the reason for unaltered
glucagon-mediated stimulation of adenylyl cyclase in AV shunt rats is
not clear and needs to be investigated. It may be possible that
Gs
52 protein that is not altered in AV shunt
rats may be implicated in the coupling of glucagon receptors to
adenylyl cyclase.
The fact that GTPS inhibited forskolin-stimulated enzyme activity to
the same extent in hearts from control and AV shunt rats suggests that
Gi functions were not altered in volume hypertrophied rats.
These results correlate very well with the levels of Gi proteins, which were also not changed in these rats. Our results are in
agreement with the studies of Hammond et al. (21) who did
not observe any changes in the levels of pertussis toxin substrates in
pigs after chronic VO hypertrophy. Furthermore, unaltered
responsiveness of adenylyl cyclase to oxotremorine inhibition in hearts
from AV shunt rats may also be due to the unaltered levels and
functions of Gi proteins that couple these receptors to the
adenylyl cyclase system and/or may be due to the possibility that
muscarinic receptors are not downregulated in these rats.
The decreased sensitivity of adenylyl cyclase to forskolin stimulation
in AV shunt rats may be due to the defective catalytic subunit of
adenylyl cyclase and/or to the decreased levels of Gs
proteins. In this regard, the requirement of Gs
and
guanine nucleotides for forskolin activation has been reported
(22). In addition, a diminished stimulation of adenylyl
cyclase by NaF in AV shunt rats may also be attributed to the decreased
levels of Gs
proteins. Similar decreases in forskolin
and NaF stimulation of adenylyl cyclase have also been reported in
hearts from DOCA-salt hypertensive rats with established hypertrophy
(4, 30) and Syrian hamster with dilated cardiomyopathy
(17), myocardial ischemia (42), and VO
hypertrophy (21).
In conclusion, we have shown that the expression of
Gs45 and Gss
47
but not of Gs
52 was decreased in hearts of
VO-hypertrophied rats, whereas the levels of Gi
were
unaltered. The decreased expression of Gs
proteins was
observed only after the development of cardiac hypertrophy. The
attenuated responsiveness of adenylyl cyclase to isoproterenol,
forskolin, and NaF stimulation in AV shunt rats may partly be
attributed to the decreased levels of Gs
proteins in
this model of hypertrophy. From these results, it is suggested that the
decreased expression of Gs
in AV shunt rats may be
associated with cardiac hypertrophy and not with arterial hypertension.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Randall Reed and Hiroshi Itoh for their kind gift of cDNAs of G proteins. We thank Matteo Pagano for help in performing some experiments and Christiane Laurier for her valuable secretarial help.
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FOOTNOTES |
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This work was supported by grants from the Quebec Heart Foundation and the Medical Research Council of Canada.
M. B. Anand-Srivastava was a recipient of the Medical Research Council Scientist Award, from the Medical Research Council of Canada, during the course of these studies.
Address for reprint requests and other correspondence: M. B. Anand-Srivastava, Dept. of Physiology, Faculty of Medicine, Univ. of Montreal, C.P. 6128, Succ. Centre-ville, Montreal, Quebec, Canada H3C 3J7 (E-mail: anandsrm{at}physio.umontreal.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 August 1999; accepted in final form 6 April 2000.
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