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␥-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 Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017 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 614 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 Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017 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 616 Hypertension February 2001 Part II Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017 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. Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017 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- 618 Hypertension February 2001 Part II Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017 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. References 1. Haywood JR, Williams SFD, Ball NA. Contribution of sodium to the mechanism of one-kidney, renal-wrap hypertension. Am J Physiol. 1984; 247:H797–H803. 2. Gordon FJ, Matsuguchi H, Mark AL. Abnormal baroreflex control of heart rate in prehypertensive and hypertensive Dahl genetically saltsensitive rats. Hypertension. 1981;3(suppl 1):135–141. 3. Hinojosa-Laborde C, Guerra P, Haywood JR. Sympathetic nervous system in high sodium one-kidney, figure-8 renal hypertension. Hypertension. 1992;20:96 –102. 4. 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Mifflin, Teresa Craig, Alfred Calderon, Julie G. Hensler and Carmen Hinojosa-Laborde Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017 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 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://hyper.ahajournals.org/content/37/2/614 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. 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