Download Aminobutyric Acid (GABA)-A Function and Binding in

␥-Aminobutyric Acid (GABA)-A Function and Binding in
the Paraventricular Nucleus of the Hypothalamus in
Chronic Renal-Wrap Hypertension
Joseph R. Haywood, Steven W. Mifflin, Teresa Craig, Alfred Calderon,
Julie G. Hensler, Carmen Hinojosa-Laborde
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Abstract—The goal of this study was to determine whether ␥-aminobutyric acid (GABA)ergic transmission and GABA
binding are altered in chronic renal-wrap hypertension. Three groups of hypertensive and sham-operated rats were
prepared for separate protocols. Four weeks later, the animals were prepared with femoral artery catheters for the
measurement of mean arterial pressure. In all groups, blood pressure was significantly higher in the renal-wrapped
animals. In the first study, bilateral microinjection of the GABA-A antagonist, bicuculline (50 pmol/site), into the
paraventricular nucleus of the hypothalamus (PVN) caused a greater increase in arterial pressure (21.9⫾1.4 versus
16.7⫾1.8 mm Hg, P⬍0.05) and heart rate (135⫾15 versus 98⫾12 bpm, P⫽0.064) in hypertensive rats. [3H]Flunitrazepam was used to measure binding to the GABA-A receptor. Magnocellular neurons and the adjacent medial
parvicellular neurons had more intense binding compared with the remainder of the PVN. Bmax was greater for the higher
density binding area; the Kd value was less in the high-density region. There were no differences in these parameters
between normotensive and hypertensive animals. Competitive reverse transcription–polymerase chain reaction was used
to measure the expression of mRNA for the ␣1 subunit of the GABA-A receptor. No difference was observed in the
mRNA between renal-wrapped and sham-operated rats. In summary, inhibition of GABA-A receptors in the PVN is
augmented in the chronic phase of hypertension and is unrelated to a change in the expression of the number or affinity
to the receptor. These findings suggest that the greater GABAergic activity is the result of an increase in GABA release
in the PVN in chronic renal-wrap hypertension. (Hypertension. 2001;37[part 2]:614-618.)
Key Words: [3H]flunitrazepam 䡲 sympathetic nervous system 䡲 bicuculline 䡲 receptors
angiotensin.14 Inhibition of ␥-aminobutyric acid (GABA)-A
receptors also elicits an increase in sympathetic outflow.11,12,15 During the onset of sodium-dependent hypertension, the tonic inhibition by GABA appears to be diminished,
permitting excitatory neurotransmission to prevail.16
The goals of the present study were 2-fold. First, a
functional assessment of GABA-A was performed to determine whether the actions of GABA were altered in chronic
hypertension. Then, benzodiazepine binding and mRNA for
the ␣1 subunit of the GABA-A receptor were measured to
ascertain whether physiological dysfunction was related to
changes in the postsynaptic receptor.
T
here is considerable evidence supporting a role for
increased sympathetic nervous system function in
sodium-dependent hypertension.1– 4 Enhanced neural function
is further supported by ablation studies of central nervous
system structures that interfere with the hypertensive process.
Pathways from the sodium-sensitive anteroventral third ventricle (AV3V) area to the paraventricular nucleus of the
hypothalamus (PVN) appear to be a major link in sympathoadrenal nervous system activation in hypertensive animals.5–7
In turn, descending pathways from the PVN through the
rostral ventrolateral medulla and spinal cord have been shown
to mediate efferent neural function to increase blood pressure.8,9 Additionally, projections to the nucleus tractus solitarii inhibit baroreceptor input, which can contribute to an
elevated sympathetic outflow.10
Little is known about the neurotransmitters in the PVN that
are responsible for the activation of the sympathoadrenal
nervous system. Glutamate stimulation in the conscious
animal increases arterial pressure, heart rate, efferent renal
nerve activity, and circulating norepinephrine and epinephrine.11–13 Similar cardiovascular responses are observed with
Methods
Animal Maintenance and Surgery
Male Sprague-Dawley rats (Harlan, Indianapolis, Ind) weighing 275
to 325 g were used in the present study. Animals were maintained on
a 14-hour light/10-hour dark cycle in temperature-controlled quarters. Rats were offered an ad libitum water intake and normal sodium
diet (100 ␮Eq/g chow, Teklad). All animals were subjected to renal
wrap or sham operation. The rats were anesthetized with methoxyflurane, and a figure-8 renal wrap was performed on one kidney
Received October 25, 2000; first decision November 27, 2000; revision accepted December 14, 2000.
From the Department of Pharmacology, the University of Texas Health Science Center, San Antonio.
Correspondence to Joseph R. Haywood, PhD, Department of Pharmacology, the University of Texas Health Science Center, 7703 Floyd Curl Dr, San
Antonio, TX 78229-3900. E-mail [email protected]
© 2001 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
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Haywood et al
PVN GABA in Sodium-Dependent Hypertension
615
period and at the peak pressor response after the second microinjection, blood samples were taken for measurement of plasma catecholamines. Blood samples were kept on ice during the experiment. After
centrifugation, the plasma was frozen at ⫺80°C for later analysis of
norepinephrine and epinephrine by radioenzymatic assay.
At the conclusion of the experiments, the animals were deeply
anesthetized with pentobarbital (Abbott Laboratories) and perfused
transcardially with 0.9% saline, followed by 10% buffered formalin
solution. Frozen sections (60 ␮m) were cut through the region of the
PVN and stained with cresyl violet for identification of the injector
placement. Criteria for the appropriate placement of the injection
were for at least 1 cannula to be 0.6 mm from the PVN, with the
contralateral cannula 1.0 mm from the PVN.17 Hemodynamic data
are presented as mean⫾SEM. Changes in arterial pressure, heart
rate, and plasma catecholamines were compared by t test. A value of
P⬍0.05 was taken to be statistically significant.
Receptor Autoradiography
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Figure 1. A, Increase in mean arterial pressure (MAP) and heart
rate (HR) are shown in response at bilateral microinjection of
bicuculline (50 pmol/site) into the PVN. Blood pressure rose
more in the hypertensive rats than in the sham-operated rats.
Significance was taken at P⬍0.05. B, Plasma norepinephrine
(NE) and epinephrine (EPI) responses are shown during bicuculline administration into the PVN. Plasma NE and EPI increased
similarly in both normotensive and hypertensive animals.
while the contralateral kidney was removed.1 Sham-operated animals
underwent a unilateral nephrectomy. Aseptic technique was used for
all surgical procedures according to the Guide for the Care and Use
of Laboratory Animals. Studies were performed according to the
Guiding Principals for the Use of Animals in Research and Teaching
of the American Physiological Society and the Statement of Principles on the Use of Animals in Research and Teaching of the
Federation of American Societies for Experimental Biology. The
University of Texas Health Science Center, San Antonio, is fully
accredited by the Association for the Assessment and Accreditation
of Laboratory Animal Care, International.
Microinjection Study
Eight renal-wrapped and 8 sham-operated animals were used for
microinjection of the GABA-A antagonist, bicuculline, into the
PVN. Approximately 3 weeks after the induction of hypertension,
the rats were anesthetized with rat cocktail (ketamine/xylazine/
acepromazine) and prepared with bilateral guide cannulas directed at
the PVN. After 1 week of recovery, the animals were subjected to
catheterization of the femoral artery and vein while they were
anesthetized with methoxyflurane. One to 3 days later, the animals
were brought to the laboratory. The arterial line was attached to a
pressure transducer, and the animal was permitted to acclimate for 1
hour. Mean arterial pressure and heart rate were continuously
monitored at a rate of 0.1 Hz with use of a MacLab data acquisition
system. After measurements were taken during a control period,
bicuculline (50 pmol/site, 50 nL) was injected bilaterally into the
PVN. Blood pressure and heart rate were followed for an additional
20 minutes. Hemodynamic data were analyzed by averaging
1-minute bins of data for the control period and 15-second bins after
bicuculline injection. Two microinjections were performed in each
animal, with a recovery period between the injections. Peak mean
arterial pressure and heart rate responses were taken from the
15-second bin averages during the first injection. During the control
Twelve renal-wrapped and 12 sham-operated rats were used to
measure [3H]flunitrazepam binding. After baseline arterial pressure
and heart rate were measured, the animals were anesthetized with
methoxyflurane and perfused with PBS before the brain was removed and frozen. Coronal sections (20 ␮m) were cut through the
PVN with use of a cryostat and thaw-mounted onto gelatin-coated
microscope slides. Slides with mounted sections were desiccated and
stored at ⫺80°C. The method of Olsen et al18 was modified to
measure [3H]flunitrazepam binding. The sections were thawed and
preincubated in ice-cold buffer (pH 7.4). The tissue was incubated in
0.3 to 12 nmol/L [3H]flunitrazepam (85 Ci/mmol, New England
Nuclear) in Tris buffer with or without 10 ␮mol/L flurazepam to
obtain nonspecific and total ligand binding. After it was washed, the
tissue was exposed to Hyperfilm for 2.5 weeks. After film development, sections were analyzed by use of autoradiographic standards
(ART-123, American Radiochemicals), which had been calibrated to
brain mash according to the methods of Geary et al.19 The area
analyzed was determined by definition of the PVN after cresyl violet
staining. Observation of the autoradiographs revealed an area in the
region of the magnocellular neurons and adjacent medial parvicellular area just below the magnocellular neurons that contained a
higher density of silver grains relative to the remainder of the PVN.
The high-density and light-density areas were analyzed by converting optical density measurements to femtomoles per milligram of
protein. Bmax and Kd were analyzed by using nonlinear regression
analysis of total binding, taking into account the linear increase in
nonspecific binding. Differences in binding parameters were determined by comparing the overall results of the nonlinear regression by
ANOVA.
Molecular Analyses
Six renal-wrapped and 3 sham-operated rats were used to measure
mRNA of the ␣1 subunit of the GABA-A receptor. After measurement of baseline arterial pressure and heart rate, animals were
anesthetized with methoxyflurane. The brains were rapidly removed
and frozen in isopentane on ice. By use of structural landmarks on
the ventral surface of the brain, a 1-mm-thick section was cut,
capturing the PVN. From the frozen section, bilateral micropunches
of the PVN were obtained by using a blunt-end, 20-gauge, stainlesssteel piece of hypodermic tubing. Quantitative competitive reverse
transcription (RT)–polymerase chain reaction (PCR) was used to
measure mRNA of the ␣1 subunit of the GABA-A receptor.20 Total
RNA was isolated from the homogenized micropunches. Approximately 100 ng of the RNA in a 20-␮L volume was used for the RT
reaction. PCR amplification was performed with 5 ␮L of the reaction
product. The PCR cycle consisted of the following: initial denaturation at 95°C for 3 minutes, 35 cycles at 95°C for 1 minute, 60°C for
1 minute, and 72°C for 2 minutes, and a final elongation step at 72°C
for 7 minutes. The PCR products, a target message band of 304 bp,
and the internal band of 192 bp were separated on a 2% agarose gel
and stained with ethidium bromide and photographed. The image
was scanned, and the density of the bands was measured by using an
imaging system. The mRNA levels were measured as the mass ratio
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February 2001 Part II
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Figure 2. Top, Total binding of [3H]flunitrazepam in a coronal section through the
rat brain at the level of the hypothalamus.
The PVN is defined by the solid line determined from staining the histological section with cresyl violet. Within the PVN, the
lateral magnocellular area of the nucleus
has a higher density of binding than does
the medial aspect of the nucleus. Bottom,
Nonspecific binding observed with coincubation of the [3H]flunitrazepam with 10
␮mol/L flurazepam.
of the target message to the internal standard. Mass ratios were
compared by t tests.
Results
Resting arterial pressure was significantly higher in the
chronically renal-wrapped hypertensive rats (139⫾3 versus
116⫾3 mm Hg). Microinjection of bicuculline into the PVN
caused an increase in arterial pressure in normotensive
animals that was similar to previous observations by this
laboratory. In hypertensive animals, there was a 31% greater
pressor response to bicuculline (Figure 1A). Resting heart
rate was not different between the sham-operated and renalwrapped animals (376⫾8 versus 362⫾12 bpm). After bicuculline administration, the cardiac rate rose in both groups of
animals, but the increase was not significantly different
between the groups (P⫽0.064). Resting plasma norepinephrine was not different between the renal-wrapped and normotensive animals (309⫾45 versus 259⫾27 pg/mL), nor was
plasma epinephrine (57⫾5 versus 65⫾5 pg/mL). After bicuculline, plasma norepinephrine increased ⬇50%, and epi-
nephrine rose nearly 5-fold. The elevation was similar in both
normotensive and hypertensive rats (Figure 1B).
Renal-wrapped animals included in the receptor binding
studies had significantly higher mean arterial pressure compared with that in the normotensive rats (140⫾4 versus
111⫾1 mm Hg), whereas heart rate was not different between
the groups (343⫾10 versus 360⫾7 bpm). Saturation binding
experiments were performed with the use of 9 concentrations
(0.3 to 12 nmol/L) of [3H]flunitrazepam. Figure 2 shows the
total and nonspecific binding of [3H]flunitrazepam in coronal
sections of the hypothalamus. High binding occurred in the
area of the PVN that contained the magnocellular neurons
and extended ventrally to the medial parvicellular region of
the nucleus. There were more binding sites in the highintensity region of the PVN (Table). In addition, the affinity
of the ligand to the receptor was greater in this portion of the
PVN. These differences between high- and light-intensity
binding regions were similar in normotensive and hypertensive animals Figure 3. [3H]Flunitrazepam binding was measured in the cerebral cortex as a reference. No differences
Haywood et al
PVN GABA in Sodium-Dependent Hypertension
617
Binding Parameters of [3H]Flunitrazepam for the Cerebral Cortex and PVN of the Hypothalamus
Cerebral Cortex
Parameter
Wrap
High-Density PVN
Sham
Wrap
Sham
Light-Density PVN
Wrap
Sham
Bmax
2400⫾65
2611⫾53
1699⫾67
1884⫾111
1579⫾91
1596⫾86
Kd
1.64⫾0.10
1.81⫾0.08
1.77⫾0.15
1.77⫾0.24
2.41⫾0.26
2.24⫾0.25
Values are mean⫾SEM.
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were observed between normotensive and hypertensive rats;
however, binding was more intense than in the PVN.
Hypertensive animals included in the present study for the
measurement of the GABA-A ␣1 subunit mRNA in the PVN
had significantly elevated arterial pressure (132⫾4 versus
117⫾2 mm Hg). The expression of the mRNA was not
different between the normotensive and hypertensive animals. The mass ratio of the target mRNA to the internal
standard was 3.5⫾1.1 in the sham-operated rats and 2.2⫾0.2
in the hypertensive animals.
Discussion
The major finding of the present study was that the tonic
activity of GABAergic function in the PVN is increased in
chronic renal-wrap hypertension. Autoradiographic analysis
of benzodiazepine receptor binding and examination of the
mRNA of the GABA-A receptor ␣1 subunit in the PVN
indicated that the augmented function was not the result of
increased postsynaptic expression of GABA-A receptors.
Together, these observations suggest that GABA release is
increased in the PVN of chronically hypertensive animals.
Evidence supports a role for central GABA in blood
pressure regulation. Intraventricular administration of a
GABA agonist causes a decrease in arterial pressure by
reducing sympathetic nervous system activity.21–23 Most of
the response is the result of a hindbrain action, inasmuch as
administration of GABA on the ventral surface of the medulla
or into the rostral ventrolateral medulla causes profound
depressor responses.24,25 However, a hypothalamic site of
action is suggested by GABAergic inhibition of the central
angiotensin response26 and baroreceptor-mediated vasopressin release.27 In addition, the administration of a GABA-A
Figure 3. Total binding and nonspecific binding of [3H]flunitrazepam in the PVN are shown for sham-operated (left) and renalwrapped (right) animals. High-density (circles) and light-density
(diamonds) areas were measured in the same histological sections. Nonspecific binding (squares) was performed on adjacent
sections. Linear (nonspecific) and nonlinear (total binding) analysis was performed by using all data points at all concentrations
of ligand. The best fit lines are represented.
antagonist localized to the forebrain increases arterial pressure.28 More recently, local direct administration of agonists
and antagonists into hypothalamic nuclei indicate that there is
a tonic GABAergic inhibition on sympathoadrenal function
from several forebrain sites.11,29
A role for GABA has been strongly implicated in hypertension.
Administration of GABA agonists intraventricularly causes a
greater fall in blood pressure in hypertensive animals.21,22,30 Muscimol microinjection into the dorsomedial hypothalamus causes an
augmented fall in blood pressure in spontaneously hypertensive rats
(SHR) compared with normotensive control rats.31 In addition, the
synthetic enzyme of GABA, glutamic acid decarboxylase, and its
mRNA are reduced in the posterior hypothalamus.32 A reduced
formation of GABA in the posterior hypothalamus of the SHR is
supported by an attenuated pressor response to a GABA synthesis
inhibitor.33 There is also evidence for a reduced number of GABA
receptors in the hypothalamus of the SHR.34–36 Thus, in the SHR,
a reduction in presynaptic and postsynaptic GABAergic function is
present in the hypothalamus, which leads to an activation of the
sympathetic nervous system.
In sodium-dependent hypertension, the PVN apparently
receives excitatory input from sodium-sensitive areas of the
AV3V.5 Descending pathways to the rostral ventrolateral
medulla, nucleus tractus solitarii, and spinal cord are then
activated to increase sympathoadrenal function.8 –10 Although
GABA function has been studied in the PVN, the neurochemical changes during the hypertensive process are not fully
delineated. During the onset of renal-wrap hypertension, the
response to bicuculline in the PVN is significantly attenuated.16 However, no change in benzodiazepine binding was
observed in the PVN or other hypothalamic nuclei (authors’
unpublished data, 2000). In the present study, no difference in
binding to the GABA receptor was found between normotensive and hypertensive animals, whereas GABA function was
enhanced in chronic hypertension. These differences suggest
that in sodium-dependent hypertension, GABA function
changes at the presynaptic level in the PVN. There appears to
be a decreased release of GABA during the early stage of the
hypertension either from a greater excitation from the AV3V
or reduced inhibitory input from the hindbrain. The increase
in PVN GABA function in chronic hypertension may occur
as an adaptation to the elevated pressure. In chronic hypertension, when baroreceptor input to the nucleus tractus
solitarii is elevated at resting levels of blood pressure,37 the
increased baroreceptor afferent input to the hindbrain could
lead to an activation of GABAergic inputs to the PVN that
counters the forebrain sympathoexcitatory pathways. The
ultimate outflow from the PVN will reflect the balance
between excitation and inhibition within this nucleus, and the
adaptive changes described in the present study could con-
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February 2001 Part II
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tribute to this balance. Final proof of this hypothesis rests
with further experiments.
The GABA-A receptor is made up of 5 subunits, usually in
(␣)2(␤)(␥)2 configuration. Benzodiazepines bind to the ␣1 and ␥2
subunits to facilitate neurotransmitter action.38 Distribution of
subunits in the PVN conform to the common configuration;
however, the specific ␣ and ␤ subunits appear to vary within the
nucleus.39 This variation may account for the differences in Kd
observed between the magnocellular neurons and the parvicellular portion of the nucleus. The increase in binding sites in the
magnocellular part of the PVN may be related to the configuration of subunits. A greater proportion of ␣1 and ␥2 in the
magnocellular neurons may account for greater benzodiazepine
binding. Alternatively, there may be more GABA receptors in
this area of the nucleus. An altered configuration of subunits
could also account for an augmented physiological response
independent of increased binding.
In summary, GABA function in the PVN changes during
the course of the hypertension. During the onset of hypertension, there is a reduced GABAergic inhibition, permitting an
increase in sympathetic nervous system activity to increase
arterial pressure. Chronically, there is an increase in GABA
activity in the PVN that competes with the sympathoexcitatory stimuli. In both phases of the hypertension, it appears
that the change in GABA function is related to altered GABA
neuronal function rather than changes in the GABA receptor.
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γ-Aminobutyric Acid (GABA)-A Function and Binding in the Paraventricular Nucleus of
the Hypothalamus in Chronic Renal-Wrap Hypertension
Joseph R. Haywood, Steven W. Mifflin, Teresa Craig, Alfred Calderon, Julie G. Hensler and
Carmen Hinojosa-Laborde
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Hypertension. 2001;37:614-618
doi: 10.1161/01.HYP.37.2.614
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2001 American Heart Association, Inc. All rights reserved.
Print ISSN: 0194-911X. Online ISSN: 1524-4563
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