(Hypertension. 1997;30:1128-1134.)
© 1997 American Heart Association, Inc.
Articles |
Insulin Enhances Endothelial 
2-Adrenergic Vasorelaxation by a Pertussis Toxin Mechanism
From the IRCCS "Neuromed," Pozzilli (IS) (G.L., C.V., C.M., F.M., B.T.), and the Department of Internal Medicine, School of Medicine, "Federico II" University, Naples (G.I., E.B., L.P., B.T.), Italy.
Correspondence to Bruno Trimarco, MD, Department of Internal Medicine, "Federico II" University, Via Pansini 5, 80131 Naples, Italy. E-mail trimarco{at}ds.cised.unina.it
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Abstract |
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Abstract To investigate whether insulin effect on endothelium is related to a specific signal transduction pathway or reflects a more generalized action of the hormone, we studied in aortic rings of Wistar-Kyoto (WKY) rats the effects of the hormone on endothelium-dependent relaxations generated by acetylcholine, adenosine diphosphate, the selective
2-adrenergic agonist UK 14,304, and the
calcium ionophore ionomycin. The responses were evaluated both in
control conditions and after 30 minutes of exposure to three different
levels of insulin (30, 100, and 500 µU/mL). Insulin failed to modify
the phenylephrine aortic contractions and the relaxations
induced by acetylcholine, adenosine diphosphate, and ionomycin.
In contrast, both 100 and 500 µU/mL insulin were able to potentiate
the UK 14,304–induced vasorelaxation (+96±19% and +91±12%,
respectively). Pertussis toxin, which causes
2-adrenergic receptor Gi uncoupling, reduced
the 2-adrenergic vasorelaxation and prevented the
insulin potentiation of the response to UK 14,304. Furthermore, in
primary cultured aortic endothelial cells from WKY, we
evaluated the conversion of [3H]arginine to
[3H]citrulline in response to acetylcholine, ionomycin,
and UK 14,304, both in control conditions and during insulin exposure.
Again, insulin did not affect basal citrulline production or
the increase induced by acetylcholine and ionomycin, whereas it
potentiated the response to UK 14,304. Finally, in aortic rings of
spontaneously hypertensive rats, insulin treatment (100 and 500
µU/mL) was unable to enhance the 2-adrenergic
vasodilator response; in vascular endothelial cells
from spontaneously hypertensive rats, insulin did not potentiate the
increase in citrulline production evoked by UK 14,304. In
conclusion, insulin selectively enhances 2-adrenergic
endothelial vasorelaxation through a pertussis
toxin–sensitive mechanism, by potentiating endothelial
nitric oxide production. This vasorelaxant mechanism is altered
in spontaneously hypertensive rats.
Key Words: aortic ring • endothelium • nitric oxide • Gi protein • insulin resistance
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Insulin is a pleiotropic signal for the target tissues. In recent years, much attention has been paid to the finding that insulin, independently from its effects on intermediary metabolism, may have relevant effects on vascular tone, both directly through a vasorelaxant action1-3 and indirectly through an effect on the autonomic nervous system.4-7
In humans in particular, insulin at physiologically reached postprandial levels evokes a net reflex increase in sympathetic outflow5-7; coincidentally, it is able to blunt the vasoconstrictive effect resulting from this reflex sympathetic activation.5,7-10 Clinical studies have clarified that insulin effects on the sympathetic nervous system are likely evoked through a central neural mechanism.5,7,11 On the contrary, the precise mechanisms underlying insulin modulating action on sympathetic-mediated vasoconstriction are not completely elucidated. A growing body of evidence suggests that the vasorelaxant effect of insulin may be mediated by interference of the endothelial function, although other mechanisms have been demonstrated.12 Recent data obtained in humans indicate that the insulin-mediated vasorelaxant effect may depend on endothelium NO release.10,13,14 However, so far, it is still unclear whether the link between insulin and NO production is localized to a specific intracellular signal transduction pathway or, conversely, all endothelium-mediated responses are influenced by insulin.
Recently, the ability of insulin to modulate the vasoconstriction induced by norepinephrine, the major sympathetic neurotransmitter, has been demonstrated in rat aortic strips.1,15,16 Also, in this experimental model the vasorelaxant action of insulin is endothelium dependent because the endothelium denudation of the aortic vasculature abolishes the effect of the hormone.15-17 Thus, such an experimental approach seems to represent a reliable way to more accurately study the mechanisms underlying insulin influence on endothelial function.
We planned the present study to investigate in aortic rings of WKY whether the effect of physiological levels of insulin on endothelium-mediated responses is related to a specific intracellular signal transduction pathway or reflects a more generalized amplification of the endothelium-mediated responses. Second, to clarify whether the functional data obtained on aortic rings could be explained by an insulin effect on NO release, we evaluated the conversion of [3H]arginine to [3H]citrulline, which is stoichiometric with NO production, in primary cultured aortic endothelial cells from WKY.18 Third, because previous studies from several laboratories have shown that SHR are resistant to insulin action both at metabolic19,20 and vascular16,17 levels, we decided to extend to SHR our analysis to investigate whether vascular insulin resistance also implies a defect of insulin action on endothelial function.
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Methods |
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Experimental Animals
Most studies were conducted in 93 male WKY (Charles River Laboratories) aged 10 to 12 weeks. A group of 13 age- and sex-matched SHR (Charles River Laboratories) was also used. The animals were housed two to a cage and kept in a temperature-controlled room (between 23°C and 25°C) with a 12-hour light/dark cycle. Food and water were provided ad libitum. The experiments were performed after the rats were acclimatized to the housing condition for at least 1 week.
Systolic blood pressure was measured in conscious rats with tail-cuff plethysmography (PE-300, Narco Biosystems Inc) and recorded on a multichannel polygraph (Universal Oscillograph, Harvard Instruments). The systolic blood pressure values reported are the average of three to four consecutive determinations.
The experimental protocol was in accordance with institutional guidelines of the University of Naples School of Medicine for research in animals.
Preparation of Aortic Rings
On the day of contractile experiments, the rats were weighed and
then decapitated. The thoracic aorta was dissected out from each rat
and placed in cold Krebs-Henseleit bicarbonate buffer solution with the
following composition (mmol/L): NaCl 118.3, KCl 4.7,
CaCl2 2.5, MgSO4 · 7H2O 1.2,
KH2PO4 1.2, NaHCO3 25, and glucose
5.6. The aorta was cleansed of the adhering perivascular tissue and cut
into rings 3 mm long. Aortic rings were suspended in isolated
tissue baths filled with 20 mL Krebs' solution continuously bubbled
with a mixture of 5% CO2/95% O2 (pH 7.37 to
7.42) at 37°C. One end of the aortic ring was connected to a tissue
holder and the other to an isometric force transducer. The signal was
passed to a Gould pressure processor and then acquired in a
computerized system by Gould's Data Acquisition and Signal
Analysis. The analysis of the generated curves was
performed with View II software (Gould Instruments), and the
sensitivity of the system was 5±1 mg of tension generated. The rings
were equilibrated for 90 minutes in the unstretched condition, and the
buffer was replaced every 20 minutes. The length of the smooth muscle
was increased stepwise in the equilibration period to adjust passive
wall tension to 2 g. This tension was found to be optimal for
contractions of aorta from WKY and SHR rats by testing the contractions
to norepinephrine (10-3 mol/L). Once
basal tension was established, the length of the rings was not altered
thereafter. Care was taken to avoid endothelial damage,
and the functional integrity of this structure was reflected by the
response to 10-7 mol/L acetylcholine (WKY 37±1%
and SHR 33±1%).
Studies on Aortic Rings
The following drugs were used: acetylcholine, adenosine
diphosphate, ionomycin, pertussis toxin, phenylephrine
(Sigma Chemical Co), BHT-933 (a gift of Boehringer Ingelheim,
Biberach, Germany), and UK 14,304 (Research Biochemicals
International). Drugs were prepared daily in distilled water, except
ionomycin which was dissolved in DMSO (Sigma). Concentrations of the
drugs are reported as the final molar concentration in the organ
bath.
After the equilibration period, a cumulative concentration-response
curve to phenylephrine (10-9 to
10-5 mol/L) was obtained. To study
endothelium-dependent relaxations, the vessels were
contracted with phenylephrine (10-6
mol/L). Relaxations were then studied both in the absence of and
after 30 minutes of preincubation with human regular insulin. Three
different insulin doses were tested: 30, 100, and 500 µU/mL; each one
was examined in separate aortic rings. The
endothelium-mediated responses evoked by acetylcholine
(10-8 to 10-5 mol/L),
adenosine diphosphate (10-8 to 10-5
mol/L), the two different selective 2-agonists UK
14,304 (10-9 to 10-6 mol/L) and
BHT-933 (10-8 to 10-5 mol/L), and a
calcium ionophore ionomycin (10-9 to 10-7
mol/L) were each performed in a distinct aortic ring of WKY.
Full dose-response curves were obtained for each agent using the
maximal dose, allowing preservation of the specificity of the
pharmacological stimulus. In control experiments, acetylcholine,
adenosine, ionomycin, and 2-adrenergic agonists
were not able to induce concentration-dependent relaxations in aortic
rings in which endothelium was removed.
To explore the mechanism by which insulin enhances
endothelium-dependent relaxation to
2-adrenergic agonists in aortic rings, relaxations to UK
14,304 were tested in control conditions, after incubation with
pertussis toxin (100 ng/mL, 120 minutes), and finally during
insulin (100 and 500 µU/mL) plus pertussis toxin.
To explore whether the effect of insulin on 2-adrenergic
vasorelaxation was NO dependent, the vascular response to increasing
doses of UK 14,304 was tested in control conditions, after incubation
with the NO synthase competitive inhibitor L-NMMA (3
· 10-4 mol/L for 15 minutes), and finally during
insulin (100 and 500 µU/mL) plus L-NMMA.
To verify whether insulin has a similar effect on
endothelium-dependent relaxation to
2-adrenergic agonists in aortic rings of SHR, we studied
the endothelium-dependent relaxations to UK 14,304 in
the absence of and after 30 minutes of insulin exposure (100 and 500
µU/mL) on aortic rings of the genetically hypertensive rat strain
SHR. The doses of insulin tested were those that had shown an effect in
the WKY rats.
Preparation of Primary Isolated Aortic Endothelial Cells
Vascular endothelial cells were isolated by
outgrowth from rat aorta as previously described.20 WKY
rats of 10 weeks of age were heparinized and killed by decapitation. A
median thoracotomy was then performed, and the thoracic aorta was
removed and rinsed in PBS. The vessel was cleaned of periadventitial
fat and connective tissue and cut into flat pieces of about 4
mm2 surface area. The aortic pieces were then placed on the
top of Matrigel-coated, 35-mm Petri dishes (Becton-Dickinson) and
incubated with 1 mL DMEM (Bio-Whittaker) with 5% FBS (Bio-Whittaker),
1% ECGF, 100 U/mL penicillin, and 100 µg/mL streptomycin
(Sigma) in a humidified incubator at 37°C, 95% air and 5%
CO2. After 6 to 9 days, depending on the degree of
outgrowth, the aortic explants were removed. At confluence, cells on
Matrigel were detached using 50 U/mL dispase dissolved in Hank's
balanced salt solution and replated in 100-mm culture plastic dishes in
DMEM with 5% FBS and 1% ECGF. Cells were subcultured for up to 6
passages, and removal from culture dishes was performed using 0.1%
trypsin/0.02% EGTA. Cells were characterized by the expression of
endothelial constitutive NO synthase (Transduction
Laboratories).
Estimation of NO Production Through
[3H]Arginine to [3H]Citrulline
Conversion
Because direct measurement of NO is difficult because of its
short half-life, citrulline production (assayed by a radiometer
technique17) was used to monitor NO production. NO
generation is catalyzed by a class of NADPH-dependent NO synthases,
which favor the conversion of L-arginine into
L-citrulline and NO with a 1:1
stoichiometry. At the 4 to 6 passage, 10 000 cells per well were
plated in 6-well plates. At confluence, the medium was removed and
wells were washed three times; DMEM plus 1% BSA, 1% ECGF, and 2.5
µCi L-[3H]arginine was added to each well
and allowed to incubate for 24 hours. The medium was then removed, and
wells were washed once with HEPES buffer (mmol/L: 145 NaCl, 5
KCl, 1 MgSO4, 10 HEPES sodium salt, 10 glucose, and 1
CaCl2; pH 7.4) and then incubated at 37°C with 1 mL of
the same buffer, with or without 100 µU/mL insulin. After 20 minutes,
acetylcholine (10-4 mol/L, in DMSO), UK 14,304
(10-4 mol/L, in DMSO), ionomycin (2 ·
10-3 mol/L, in DMSO), or vehicle was added to each
well, and plates were incubated at 37°C for 30 minutes. The
agonist-induced stimulations were then stopped by washing cells with
PBS plus 4 mmol/L EDTA. Supernatant was then collected,
applied to 2-mL columns of Dowex AG50WX-8 (Na+ form), and
eluted with 4 mL H2O. Cell protein content in wells was
determined by the modified Lowry protein assessment (Bio-Rad). The
[3H]citrulline content in the eluate (6 mL) was assayed
by liquid scintillation. Citrulline production was expressed as
picomoles per minute per milligram protein.
Statistical Analysis
The results are expressed as mean±SEM. Student's t
test for paired observation was used to compare the insulin effect on
phenylephrine-induced contractions. Repeated measures
ANOVA with grouping factors was performed for evaluation of the
interaction between agonist-induced endothelium
responses and insulin. Post hoc simultaneous multiple
comparisons were done by Bonferroni analysis.22
Student's t test for paired observations was performed to
compare citrulline production in response to different
endothelial stimulations in control conditions and
during insulin exposure.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In WKY rats, systolic blood pressure was 121±2 mm Hg. Cumulative addition of phenylephrine to the muscle bath caused contractions in all aortic segments, revealing good general responsiveness of the vessels used for further investigations. The tension developed by phenylephrine (10-6 mol/L) was not affected by insulin (30 µU/mL: 1489±45 versus 1449±14, n=25, NS; 100 µU/mL: 1261±54 versus 1243±52, n=33, NS; 500 µU/mL: 1376±33 versus 1358±33, n=25, NS).
Endothelium-Dependent Responses
As shown in the Table, in all arteries, acetylcholine,
adenosine diphosphate, and the calcium ionophore ionomycin
produced concentration-dependent relaxations of rings with
endothelium (Fig 1
).
Insulin treatment was not able to significantly alter the relaxation of
aortic rings evoked by all these agonists (Table and Fig 1).
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) and during exposure to 100 µU/mL of regular
insulin (
).
UK 14,304 produced concentration-dependent relaxations of aortic rings
(Fig 2). However, insulin exposure at
both 100 and 500 µU/mL increased the vasorelaxant response evoked by
UK 14,304 to an extent similar to that of control conditions (Fig 2).
To better characterize the nature of insulin facilitation of UK
14,304–induced relaxations, we decided to evaluate the insulin effect
on the vasorelaxation induced by a different
2-adrenergic agonist, ie, BHT-933. Accordingly, this
2-adrenergic agonist was also able to induce
dose-dependent relaxations, and during insulin exposure this relaxant
response was significantly potentiated (Table).
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Effects of Pertussis Toxin on 2-Adrenergic
Vasorelaxation During Insulin
In this series of experiments, the ability of UK 14,304 to induce
dose-dependent vasorelaxations was confirmed (Fig 3). The addition of pertussis toxin,
while not altering the resting tension, enabled the significant
blunting of the response elicited by UK 14,304 (
% of maximal
response from 31±4% to 13±1%, P<.01; Fig 3). However,
in the presence of pertussis toxin, the facilitation of
2-adrenergic–evoked relaxations by insulin was
completely abolished both at 100 µU/mL (Fig 3) and 500 µU/mL (%
of maximal response 14±1% versus 15±2%, n=5, NS).
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), and during exposure to pertussis toxin plus insulin (100
µU/mL) (
Effects of L-NMMA on 2-Adrenergic Vasorelaxation
During Insulin
As expected, UK 14,304 induced dose-dependent relaxations (from
7±1% to 33±4%, n=5). L-NMMA exposure blunted the UK 14,304–evoked
relaxations (% of maximal response from 33±4% to 5±1%,
P<.01), and in this condition levels of both 100 and 500
µU/mL insulin were unable to exert their facilitating effect on
2-adrenergic–evoked relaxations (% of maximal
response: 100 µU/mL, from 5±1% to 6±2%, NS; 500 µU/mL, from
5±1% to 7±1%, NS).
2-Adrenergic Vasorelaxation During Insulin
in SHR
In SHR, systolic blood pressure was 175±4 mm Hg,
significantly higher than in WKY. The tension developed by
phenylephrine on aortic rings (1237±59% versus 1347±27%
mg) and the UK 14,304–evoked relaxations (36±2% versus 31±2%) were
comparable with those observed in WKY. However, unlike the findings in
WKY, the insulin exposure was not able to enhance UK 14,304–evoked
relaxations, even when higher levels of the hormone were used (Fig 4).
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Citrulline Production in Primary Isolated Aortic
Endothelial Cells From WKY and SHR
In supranatant of cultured aortic
endothelial cells from WKY, there was a significant
increase in citrulline production after exposure to
acetylcholine (from 1.79±0.39 to 2.46±0.48 pmol ·
min-1 · mg-1, n=6, P<.05),
UK 14,304 (from 1.41±0.07 to 2.11±0.33 pmol ·
min-1 · mg-1, n=6, P<.05),
and ionomycin (from 1.30±0.12 to 11.51±1.44 pmol ·
min-1 · mg-1, n=7, P<.01).
Insulin did not affect basal citrulline production (1.41±0.18
versus 1.44±0.18 pmol · min-1 ·
mg-1, NS) or the responses induced by acetylcholine
(+34±8 versus +36±12%, NS) and ionomycin (+969±210 versus
+872±210%, NS). In contrast, the hormone significantly enhanced
citrulline response to UK 14,304 (Fig 5).
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) and during insulin exposure
(
). *P<.05 compared with control conditions.
When aortic endothelial cells from SHR were exposed for
30 minutes to UK 14,304, there was a significant increase in citrulline
production (from 1.67±.14 to 3.31±.34 pmol ·
min-1 · mg-1, P<.05).
However, in these experiments, insulin did not affect either basal
citrulline production (1.77±.17 versus 1.67±.11 pmol ·
min-1 · mg-1, NS) or the response to
UK 14,304 (Fig 5
).
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Discussion |
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The major finding of this study is that physiological insulin levels affect heterogeneous endothelium-dependent relaxations. Specifically, the hormone amplifies the
2-adrenergic pathway, whereas it does not interfere with
other endothelium-mediated responses. Moreover, the
enhancement of endothelial 2-adrenergic
vasodilation is mediated through a pertussis toxin–sensitive pathway.
These findings from the aortic ring model are also supported by the
studies on primary cultured vascular endothelial cells.
In addition, in these latter experimental conditions, insulin is able
to potentiate exclusively the enhancement of citrulline
production induced by 2-adrenergic agonists. Several studies have demonstrated that insulin is able to modulate the vascular responses to some vasoactive agents, including norepinephrine, in normotensive1-3,23 but not in spontaneously hypertensive strains.16,17,23–25 Such an action is complex and poorly understood. Elucidation of this issue may be particularly relevant because insulin, at levels comparable with those normally achieved postprandially, evokes a net reflex increase in sympathetic nervous activity,4-7 and the lack of an vascular modulating effect of insulin could abnormally amplify the sympathetic effects on peripheral vascular tone. Thus, resistance to the vascular action of insulin may have a permissive role in the setting of hypertension.
Recent observations show that the endothelium plays a
major role in the vascular effect of insulin because in humans L-NMMA,
a specific inhibitor of NO synthase, is able to abolish the
vasorelaxant effect of insulin,13,14 and more convincingly,
in aortic rings the vascular effect of the hormone is suppressed by
endothelium removal.15-17 Therefore, the
purpose of this study was to determine whether this effect is due to
insulin interaction with a specific signal transduction pathway or it
is a consequence of a more generalized action of the hormone. The
finding that insulin does not modify the vascular response to
ionomycin, which increases endothelial cytosolic
calcium independent of receptors, indicates that the insulin action on
the endothelium-dependent NO release involves a
receptor-mediated mechanism. Furthermore, because acetylcholine,
adenosine, and 2-adrenergic agonists exert their
action through different initial signal transduction pathways, our
results seem to support the concept that insulin interaction with NO
production involves a specific receptor-mediated signal
transduction pathway.
The functional data obtained in the aortic rings allow us only to
postulate the effect of insulin on endothelium-mediated
NO relaxation. However, the parallel observation obtained in a more
elementary model, primary cultured aortic endothelial
cells, gives us more direct biochemical evidence of the ability of
insulin to induce NO production through a specific pathway.
Furthermore, to better compare the effects of insulin on the cellular
system to those obtained in aortic rings, we decided to use an insulin
exposure of comparable duration in both experimental models, since
different time courses of the two experiments may have explored
distinct phenomena. Moreover, the choice of that time was derived from
previous studies, which have clearly demonstrated that 30 minutes of
insulin exposure is enough to attenuate
norepinephrine-induced vasoconstriction.2,15,16
To study acute insulin-induced effects, we used a sensitive approach on
endothelial cells to evaluate the short-term NO
production by monitoring the conversion of
[3H]arginine to [3H]citrulline, which is
stoichiometric with NO production. The biochemical data on
endothelial cells confirmed the functional observations
obtained from the aortic rings. Actually, insulin did not affect basal
citrulline production, and its sensitizing effect was evident
only during 2-adrenergic stimulation. Our data could
appear to be in conflict with a recent observation showing that insulin
is able to exert a direct effect on the production of NO from
endothelial cells.26 Actually, a careful
perusal of those data reveals that more
physiological insulin concentrations, similar to
those used in the our study, are absolutely unable to stimulate NO
production. The conclusions of the study of Zeng and
Quon26 are mainly derived from the use of high
pharmacological insulin levels, from 100 to 1000 times more than used
in our study. Thus, the sensitizing effect on the
endothelial 2-adrenergic pathway is
realized by levels of the hormone that have no direct effect on the
release of NO.
Therefore, our results suggest a specific cross talk between insulin
and the 2-adrenergic pathway at the
endothelial level. Actually,
endothelium contains 2-adrenergic
receptors27,28 and the sympathetic vascular response
represents the balance of contrasting effects of
norepinephrine both on vascular smooth muscle and
endothelium.29-32 Thus, the common clinical
observation that insulin significantly blunts the sympathetic
vasoconstriction8,10,23 is in keeping with a sensitizing
effect of the hormone on endothelial
2-adrenergic pathway.
2-Adrenergic receptors are closely coupled to
Gi proteins to transduce their signal.33 In
particular, it has been reported that Gi2 subunits are
specifically involved in the vasorelaxant effect of
2-adrenergic receptor activation.34
Pertussis toxin uncouples Gi from the receptor through ADP
ribosylation of the carboxyl terminus of Gi proteins,
disrupting the signal transduction.35 However, pertussis
toxin abolishes only a part (
80%) of
endothelium-dependent relaxations induced by
2-adrenergic activation, indicating that a small amount
of NO is still released in response to 2-adrenergic
receptor stimulation via a non–pertussis-toxin–sensitive
pathway.34,36 In our study, during exposure to pertussis
toxin, insulin was no longer able to enhance the
2-adrenergic vasorelaxation, suggesting that a
Gi-sensitive mechanism is involved in vascular insulin
action. Our findings are also in keeping with several experimental
findings indicating that insulin can modify responsiveness to agents
that operate via Gi proteins.37-39 On the other
hand, it has been reported that Gi proteins can be
implicated in some action of insulin.40-42 In this regard,
recent data obtained in genetically engineered mice with a defect in
Gi protein clearly demonstrate resistance to insulin
action.43
Finally, to investigate whether insulin resistance depicted in
essential hypertension at both metabolic19,20
and vascular16,17 levels may be broadened to this novel
insulin action on endothelial
2-adrenergic stimulation, we decided to extend our
analysis to SHR. In particular, we observed that in aortic
rings of these animals, insulin was unable to potentiate the
vasorelaxant effect of 2-adrenergic receptor stimulation
even when a high dose of insulin was used. Simultaneously,
in aortic endothelial cells from SHR, the hormone did
not potentiate the 2-adrenergic–evoked citrulline
production. These findings suggest that in hypertension, the
defect in insulin vascular action may be due to the impairment of the
insulin sensitizing effect on endothelial
2-adrenergic–evoked NO production.
Taken together, our results demonstrate that
physiological levels of insulin selectively enhance
2-adrenergic endothelial vasorelaxation
through a pertussis toxin–sensitive mechanism by potentiating
endothelial NO production. The lack of this
action of insulin may represent the basis of the insulin
vascular resistance associated with arterial
hypertension.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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The present study was supported by grants from the Italian Ministry of Public Health and from the Italian Society of Hypertension (Dr Iaccarino). The authors are indebted to Dr A. Zimmer of the Boehringer Ingelheim Co for providing BHT-933.
Received March 3, 1997; first decision April 4, 1997; accepted April 4, 1997.
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