Brain Research 883 2000 148–156 www.elsevier.com locate bres
Interactive report
Characterisation of vasopressin V , angiotensin AT and AT receptor
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distribution and density in normotensive and hypertensive rat brain
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stem and kidney: effects of restraint stress
Stuart J. McDougall, Carlie A. Roulston, Robert E. Widdop, Andrew J. Lawrence
Department of Pharmacology , Monash University, Wellington Rd., Clayton, Victoria, Australia 3168
Accepted 14 September 2000
Abstract
In the present study, we have examined neurochemical correlates that may be involved in the differential cardiovascular responses observed in normotensive and hypertensive rats during stress. Using a restraint stress paradigm, both normotensive Wistar Kyoto WKY
and Spontaneously Hypertensive rats SHR underwent acute 1 h restraint in a perspex tube, chronic 1 h restraint for ten consecutive days or no restraint control stress. Following cessation of restraint, rats were processed by incubating sections of brain stem and kidney
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with [ I]-HO-LVA 0.03 nM or [
I]Sar Ile -AngiotensinII 0.5 nM, in the presence of PD123319 10 mM or losartan 10 mM, to determine the distribution and density of vasopressin V , angiotensin AT and AT receptors, respectively. Analysis of autoradiograms
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indicated changes in the density of radioligand binding in acutely and chronically-stressed rats, as compared to controls. For example, V
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binding in the medial nucleus tractus solitarius SolM decreased in the WKY but increased in the SHR. AT binding in SolM did not
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significantly change in the WKY but decreased in the SHR with repeated restraint. In kidney slices, AT binding decreased with stress in
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the WKY 217 but increased in SHR 110–15. AT binding in the kidney showed a pattern similar to that of AT binding in SHR,
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but not WKY. Graded increases in V binding were measured in kidney medulla and cortex of both strains 150–60 with chronic
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restraint. These results suggest that physiological adaptation to restraint is associated with specific changes in V , AT and AT receptor
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density within brain nuclei and kidney.
2000 Elsevier Science B.V. All rights reserved.
Theme : Neurotransmitters, modulators, transporters, and receptors
Topic : Receptor modulation, up- and down-regulation
Keywords : Restraint; WKY; SHR; Angiotensin; Vasopressin; Receptor autoradiography
1. Introduction telemetry, that Spontaneously Hypertensive rats SHR
have an impaired capacity to cope with the stress-induced Acute stressors, such as restraint are well characterised
tachycardia compared to normotensive Wistar–Kyoto rats to cause immediate and profound tachycardia; however,
WKY [22]. Considering that coping adaptation or such responses may diminish with subsequent stress
habituation occurs due to changes within the central encounters [6]. Associated with this adaptation are also
nervous system CNS nuclei that regulate the stress alterations in the activity of the hypothalamo–pituitary–
response [15], it would appear that SHR may have a adrenal axis HPA and sympatho-adrenal system SAS
reduced plasticity within the CNS compared to WKY. [14,17]. We have recently demonstrated, using radio-
Central control of the cardiovascular system is achieved via a complex network of interconnected nuclei. While the
basic control is primarily mediated at the level of the
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Published on the World Wide Web on 2 October 2000.
medulla oblongata, significant supramedullary modulation
Corresponding author. Tel.: 161-3-9905-4855; fax: 161-3-9905-
also occurs [8]. For example, the locus coeruleus LC
5851.
makes up a large portion of the central noradrenergic
E-mail address : andrew.lawrencemed.monash.edu.au A.J. Law-
rence.
system, which is thought to play an important role in the
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 1 7 - 6
S .J. McDougall et al. Brain Research 883 2000 148 –156
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initiation of the stress response [15] and can regulate in diameter, Plastic Labs, Lansing, MI for 60 min between
sympathetic vasomotor outflow [24,41]. A number of 900 and 1200 h. This procedure of restraint stress was
neurotransmitters have been implicated in central car- performed once for the acute study and for 10 consecutive
diovascular control including arginine vasopressin AVP days in the chronic study, as previously described [37,22].
and angiotensin II Ang II [18]. Elevations in plasma AVP reset arterial baroreflex control of sympathetic nerve
2.2. Receptor autoradiography activity and heart rate HR to lower pressures, an effect
mediated by V receptors in rabbits [20]. Similarly in rats,
Frozen brainstems and kidneys were equilibrated from
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AVP is known to facilitate the baroreceptor heart-rate 2808C to 2178C and then sectioned in a cryostat Cryocut
reflex and sensitize low and high pressure baroreceptors 1800, Leica. Coronal sections 14 mm were collected
[13]. Conversely, Ang II inhibits baroreflex function at the from specific brainstem regions. In the kidney, sagittal
level of the NTS [5]. AVP and Ang II are also involved in sections incorporating both cortex and medulla were cut on
the control of the stress response. Thus AVP is known to a cryostat and sections were thaw-mounted onto gelatin-
become a major ACTH stimulator with repeated restraint chrome alum coated slides. All tissue slices were stored at
immobilisation stress [32], while it has been suggested that 2808C until autoradiography experiments. For the three
Ang II plays a role in the control of SAS activity due to autoradiography assays, tissue slices from all treatment
immobilisation-induced stress response [16]. groups day 0, 1 and 10 days of stress and from both
Since we have recently documented the differential strains WKY and SHR were processed simultaneously
effects of restraint on cardiovascular regulation between for any particular receptor assay, to ensure a valid com-
WKY and SHR [22], the present study was designed to parison between groups.
investigate whether this differential coping between WKY The vasopressin V
receptor autoradiography technique
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and SHR with chronic restraint was associated with using
[ I]HO-LVA
0.03 nM
sequence: 4-HO-
differential alterations in neuropeptide receptor binding. To Phenylacetyl-
D
-TyrMe-Phe-Gln-Asn-Arg-Pro-Arg-NH ;
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achieve this aim, quantitative in vitro autoradiography was specific activity: 2000 Ci mmol; Auspep, Parkville, Aus-
employed to assess the effect of restraint stress upon the tralia was adapted from a published protocol [2] by
density of V , AT and AT
receptors in selected increasing bovine serum albumin concentration in the
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brainstem regions and the kidneys of acutely and chroni- incubation medium from 0.1 to 1; the addition of a 15
cally stressed WKY and SHR. min pre-incubation of tissue in incubation medium at room
temperature; 1 mM AVP Sigma, St. Louis, USA was used to define non-specific binding. Following the assay incuba-
2. Methods tion, brain sections were washed for 735 min in ice-cold
