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Vol. 286, Issue 2, 718-726, August 1998
Division of Pharmacology and Experimental Therapeutics, College of Pharmacy, University of Kentucky, Lexington, Kentucky (L.C., A.L., M.F., S.A., G.B., V.K., L.D.), and the Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, Washington (R.S.)
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Abstract |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The effect of cold exposure on the systemic renin-angiotensin system and on regulation of the angiotensin II (Ang II) receptor was examined in target organs for Ang II with cardiovascular relevance (left ventricle, kidney, lung) and metabolic relevance [interscapular brown adipose tissue (ISBAT), liver] to the functional consequences of cold exposure. In time course studies, the effects were examined of 4 hr or 1, 3 and 7 days of exposure to cold (4°C) on plasma Ang II concentration and Ang II receptor binding characteristics in rat liver. Plasma Ang II concentration increased 10-fold after 4 hr of cold exposure, returned to control levels at days 1 and 3 of cold exposure, and was again increased (2-fold) at 7 days of cold exposure. The affinity of [125I]Sar1,Ile8-Ang II binding in membranes prepared from rat liver was not altered in cold-exposed rats. The density (Bmax) of binding sites in liver from cold-exposed rats was increased by day 1 and remained elevated over time-matched controls. Alterations in Ang II receptor density did not parallel plasma Ang II concentration in their time course, suggesting that cold-induced regulation of the Ang II receptor was not substrate mediated. In rats from the 7-day time point of cold exposure, Ang II receptor binding characteristics were examined in ISBAT and lung. Increases in Ang II receptor density were evident in ISBAT but not lung. To determine whether cold-induced increases in food intake contributed to elevations in plasma Ang II concentration and/or Ang II receptor density, a group of cold-exposed rats (7 days) were pair-fed to food intake levels of control rats. Pair-feeding of cold-exposed rats eliminated increases in plasma Ang II and norepinephrine concentration but did not prevent increases in Ang II receptor density in liver, ISBAT, kidney and left ventricle. Moreover, increases in Ang II receptor density were augmented in kidney and left ventricle from cold-exposed rats that were pair-fed. Results from these studies demonstrate that cold exposure resulted in an increase in plasma Ang II concentration through mechanisms related to increased food intake. Elevations in food intake in cold-exposed rats contributed to tissue-specific increases in Ang II receptor density. Moreover, cold-induced increases in Ang II receptor density were not related to plasma Ang II concentration.
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Introduction |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Cold
exposure is a well-documented stimulus for activation of the
sympathetic nervous system (Saito, 1928
; Leduc, 1961). After prolonged
exposure of rats to cold, a sustained increase in systolic pressure
occurs, giving rise to a cold-induced model of hypertension (Fregly
et al., 1989). Activation of the sympathetic nervous system is thought to contribute to the development of hypertension in cold-exposed rats (Papanek et al., 1991). Sympathetic input
to the kidney increases the release of renin, which in turn contributes to the production of Ang II.
Several lines of evidence suggest activation of the
renin-angiotensin system in response to cold exposure. Experimental
hypertension in cold-exposed rats is associated with enhanced
responsiveness to the dipsogenic effect of Ang II (Fregly et
al., 1991). Moreover, pharmacological interference with the
renin-angiotensin system with angiotensin-converting enzyme inhibitors
(Shechtman et al., 1991) or AT1 receptor antagonists (Fregly
et al., 1993) has been demonstrated to prevent increases in
systolic blood pressure in cold-exposed rats. Previous studies in our
laboratory demonstrated that after 7 days of cold exposure, plasma
renin activity was not altered, whereas Ang II content was elevated in
ISBAT (Cassis, 1993). ISBAT has been shown to be activated by the
sympathetic nervous system after cold exposure (Young and Landsberg,
1982). Moreover, the presynaptic facilitatory effect of Ang II on
sympathetic neurotransmission in ISBAT from cold-exposed rats was
augmented, suggesting alterations in the functional responsiveness of
the angiotensin receptor (Cassis, 1993). Collectively, these results suggest coincident activation of the sympathetic nervous system and the
renin-angiotensin system in response to cold exposure.
We hypothesize that cold exposure represents a model of heightened activity of the systemic renin-angiotensin system resulting from increased sympathetic activation. Increases in the activity of the systemic renin-angiotensin system after cold exposure are hypothesized to result in regulation of the angiotensin receptor. In the present study, plasma Ang II concentration and Ang II receptor binding characteristics were examined in target organs for Ang II with cardiovascular relevance (left ventricle, kidney, lung) and metabolic relevance (ISBAT, liver) to the functional consequences of cold exposure. The following studies determined whether cold exposure regulates the systemic renin-angiotensin system, whether plasma Ang II concentration correlates to Ang II receptor density, whether cold exposure results in tissue-specific regulation of Ang II receptor density and whether cold-induced increases in food intake contribute to alterations in plasma Ang II concentration or Ang II receptor density.
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Methods |
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Abstract
Introduction
Methods
Results
Discussion
References
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General experimental design. In time course studies, alterations in Ang II receptor binding characteristics were examined in liver membranes from rats exposed to cold for different time periods (4 hr or 1, 3, 5 and 7 days; n = 5/group, cold-exposed and control rats at each time period). Comparisons were made between cold-exposed and control rats within a time period. The liver was chosen for study in these experiments due to its relative high density of Ang II receptor sites. From these studies, a time point (7 days) was chosen representing maximal cold-induced alterations in liver Ang II receptor density. Additional tissues (ISBAT and lung) then were examined for Ang II receptor binding characteristics in 7-day cold-exposed and control rats. In a separate study, cold-exposed rats were pair-fed to control rat food intake levels to determine the effect of increased food intake on Ang II receptor binding characteristics. Three groups of rats were studied for a period of 7 days, (1) control rats fed ad libitum (n = 6), (2) cold-exposed rats fed ad libitum (n = 6), and (3) cold-exposed rats that were pair-fed to the mean food intake level of control rats over the preceding 24-hr period (n = 6). Measurements of food and water consumption were obtained daily at 10:00 a.m.
Cold exposure. Male Sprague-Dawley rats (250-300 g, Harlan Laboratories, Indianapolis, IN) were used in all studies. All rats were housed two per cage in the animal quarters for 1 week on arrival. Preceding the experimental protocol, all rats were given free access to food and water, were maintained under a 12-hr light/dark cycle and were housed at ambient room temperature (23°C). For the cold exposure experimental protocols, rats were housed at 4°C in an animal hibernactim facility located in the Veteran's Administration Research facility at the University of Kentucky. Cold-exposed rats were housed individually in cages beginning at study day 1 (first day of cold exposure), with controlled humidity (79%) and a 12-hr light/dark cycle. Control rats were housed in individual cages for the duration corresponding to cold exposure.
For all studies, rats were fed standard rat chow. Measurements of food intake were obtained by providing 50 g of standard rat chow to the cage, followed by weighing the remaining food (food intake was corrected for spillage) 24 hr later. Water intake was measured by filling leak-proof water bottles with premeasured amounts of fresh water (500 ml/day), followed by measuring the water remaining in the bottle 24 hr later.Radioligand receptor binding assays. In the time course study, binding characteristics for the Ang II receptor were examined in membranes prepared from livers of cold-exposed and time-matched control rats. Ang II receptor binding characteristics were also determined in ISBAT and lung from 7-day cold-exposed and control rats. In the pair-feeding study, Ang II binding assays were performed using membranes prepared from liver, ISBAT, kidney and left ventricle from control rats fed ad libitum, cold-exposed rats fed ad libitum and cold-exposed pair-fed rats.
Tissues were removed, homogenized in membrane buffer (0.25 M sucrose/50 mM sodium phosphate, pH 7.2) and centrifuged for 30 min at 48,000 × g (4°C) (Lu et al., 1995). The pellet was
rehomogenized, washed and centrifuged additionally 3 times. The final
pellet was resuspended (1 mg protein/ml for each tissue) by
homogenization in 50 mM sodium phosphate (pH 7.2). The crude membranes
were frozen and stored at 
70°C until use. For Ang II receptor
binding saturation isotherms,
[125I]Sar1,Ile8-Ang
II (specific activity, 2,176 Ci/mmol; radiolabeled by Dr. Robert Speth
at Washington State University) was used as a nonselective Ang II
receptor antagonist to radiolabel the receptor sites. Initial experiments determined an amount of membrane protein representing the
linear portion of a membrane protein curve (liver, 20 µg of protein;
ISBAT, 150 µg; lung, 50 µg; kidney, 100 µg) for
[125I]Sar1,Ile8-Ang
II binding. Saturation binding isotherms in all tissues except ISBAT
were performed by incubating duplicate aliquots of membrane protein
with a fixed concentration of
[125I]Sar1,Ile8-Ang
II radioligand (0.5 nM) and increasing concentrations of unlabeled Ang
II (0.1 nM to 0.5 µM) in binding assay buffer (50 mM sodium
phosphate, 0.1 mM EDTA, 0.014% bacitracin, 0.2% fatty acid free
bovine serum albumin; pH 7.2) for 60 min at 26°C. For ISBAT,
saturation binding isotherms were performed with increasing concentrations of
[125I]Sar1,Ile8-Ang
II (1 pM to 10 nM). Nonspecific binding was determined in duplicate by
incubating membranes with
[125I]Sar1,Ile8-Ang
II and an excess (10 µM) of unlabeled Ang II. After incubation the
samples were filtered (Brandel Cell Harvester) through Whatman GF/B
glass fiber filters presoaked in a 0.2% bovine serum albumin solution.
The filters were washed three times, and the radioactivity was
determined by counting in a gamma counter (70% efficiency of counting
for [125I]). Kd
and Bmax (normalized to mg of protein)
values were derived using LIGAND software.
For competition studies, liver membranes were incubated with
[125I]Sar1,Ile8-Ang
II (0.5 nM) in the presence of increasing concentrations (0.1 nM to 10 µM) of the AT1 receptor antagonist losartan for 60 min at 26°C. The
inhibitory constant (Ki) was calculated
from the IC50 value using the equation of Cheng
and Prusoff (1973).
Measurement of plasma angiotensins.
Trunk blood was
collected into vacuum test tubes containing the following buffer:
pepstatin A, 0.15 mM; phenanthroline, 20 mM; EDTA, 125 mM; neomycin,
0.2%; ethanol, 2%; DMSO, 2%; and renin inhibitor kallikrein, 0.1 µM, pH 7.4. The inhibitors in this buffer were added to eliminate
breakdown of angiotensin peptides as well as further production of
peptides during sample handling (Campbell et al., 1995).
Plasma was obtained by centrifugation (3000 × g) of blood at
4°C for 30 min. Plasma samples were partially purified using SepPak
C-18 column chromatography (Waters, MA) with the columns
preequilibrated with 4 ml methanol, 4 ml water and 10 ml buffer.
Angiotensin peptides were eluted from the columns with 2 ml of
methanol/water/TFA (70:29:1). The eluate was evaporated overnight using
a SpeedVac (Savant). Plasma angiotensin peptides were measured in
preextracted samples that were reconstituted in 100 µl of Ang II RIA
buffer (0.1 M K2HPO4, 3.0 mM EDTA, 0.15 mM 8-hydroxyquinoline, 0.25% bovine serum albumin; pH
7.2), sonicated for 5 min and stored at 20°C. Angiotensin peptide
concentration in each sample was measured by Ang II RIA using a
polyclonal Ang II antibody (kindly supplied by Dr. A. Freedlender,
University of Virginia) exhibiting minimal cross reactivity to Ang I
(2%) and angiotensin5-8
(A5-8) (4%) but 100% cross reactivity to
angiotensin2-8 (Ang III),
angiotensin3-8 (A3-8 or
Ang IV) and angiotensin4-8
(A4-8). The sensitivity of the RIA for
angiotensin was 2 pg/ml.
Measurement of plasma leptin. Blood was obtained as described, and an aliquot (500 µl) of plasma was removed for measurement of plasma leptin levels. Plasma concentration of leptin was measured using a commercial RIA kit (Linco Research, MO) with a rat leptin antibody. The sensitivity of the kit for rat leptin was 0.5 ng/ml and required 100 µl of rat plasma for assay.
Measurement of plasma NE. The reverse-phase HPLC system consisted of a System Gold Module 116 pump (Beckman), a model 7725 injection valve fitted with a 50-µl sample loop (Rheodyne, Cotati, CA), a Coulochem model 5100A electrochemical detector (ESA, Bedford, MA) and model 5011 analytical cell (ESA) and a catecholamine HR-90 reverse-phase column (ESA) packed with 3-µm spherical silica bonded with octadecylsilane with a graphite guard filter. HPLC chromatograms were displayed on an Omniscribe chart recorder (Houston Instruments, TX).
The mobile phase (flow rate of 1.0 ml/min) consisted of 70 mM citric acid anhydrous, 0.16 mM EDTA, 100 mM l-octane sulfonic acetate trihydrate, 11 mM NaCl and 4.5% (v/v) methanol, pH 4.0. The HPLC column was equilibrated with mobile phase for 12 hr before use. Free catecholamines were extracted from plasma as follows: 250 µl plasma was added to 25 mg of activated alumina, 1 ml of 0.45 µm-filtered Tris·HCl (1.5 M Tris·HCl, 0.5 mM EDTA and 0.4 mM sodium metabisulfite, pH 8.7) and 400 pg of DHBA as internal standard. After the addition of alumina, the samples were vortexed for 10 min, interrupting every 2 min to allow the alumina to settle. The alumina mixture was washed three times with 3-ml portions of 50-fold dilutions of ice-cold Tris·HCl and water. The supernatant was removed, and the alumina slurry was transferred to microfilter tubes and centrifuged at 3500 rpm for 1 min. The supernatant from this centrifugation was discarded. Catecholamines were eluted from the alumina by the addition of 100 µl of 0.1 M perchloric acid, vortexing for 10 sec followed by centrifugation at 3500 rpm for 1 min. Extracts (50 µl) were injected onto the HPLC for catecholamine analysis. A set of catecholamine standards (NE, DHBA, Epi; 5-600 pg) were used to determine plasma catecholamine content with the DHBA content in extracts used to correct for recovery through the alumina extraction procedure.Statistical analysis. Statistical analysis of Ang II receptor density in livers from cold-exposed and control rats was performed at each time period by unpaired t test. Statistical analysis of the characteristics of cold-exposed rats (body weight, organ mass, plasma leptin levels) were also performed by unpaired t test. In the pair-feeding study, receptor binding data was analyzed by one-way ANOVA followed by Tukey-Kramer multiple comparisons test for post-hoc analysis. Food intake, water intake and body weight in the pair-feeding study were analyzed by one-way ANOVA with repeated measures, followed by Tukey-Kramer multiple comparisons test for post-hoc analysis.
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Results |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Characteristics of rats. In the time course study, at 7 days of cold exposure, body weight was significantly decreased in cold-exposed rats compared with control (control, 340 ± 5; cold-exposed, 323 ± 5 g; P < .05). In the pair-feeding study, body weights were significantly decreased in both groups of 7-day cold-exposed rats compared with control rats (control fed ad libitum, 305 ± 4; cold-exposed fed ad libitum, 273 ± 3; cold-exposed, pair-fed, 246 ± 2 g, P < .05). Moreover, body weights of cold-exposed rats that were pair-fed were significantly decreased from cold-exposed rats fed ad libitum. The time course for alterations in body weight and food intake were monitored throughout the experimental pair-feeding protocol and demonstrated a decrease in body weight in cold-exposed rats fed ad libitum and cold-exposed pair-fed rats compared with controls by day 5 (fig. 1A). Food intake was significantly increased in cold-exposed rats fed ad libitum by day 5 (fig. 1B). Food intake of cold-exposed rats that were pair-fed was maintained at control rat levels throughout the experimental protocol (fig. 1B). Water intake increased in cold-exposed rats fed ad libitum rats by 1 day of cold exposure and remained elevated (fig. 1C). Pair-feeding cold-exposed rats to food intake levels of control rats did not prevent cold-induced increases in water intake.
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Plasma angiotensin, NE and leptin concentration. In the time course study, angiotensin peptide concentration in plasma markedly increased (10-fold) in 4-hr cold-exposed rats compared with controls (table 1). Interestingly, at 1 and 3 days of cold exposure, plasma angiotensin peptide concentration was not significantly increased compared with controls; however, plasma angiotensin peptide concentration was again elevated (2-fold) above controls at day 7 (table 1).
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Characterization of Ang II receptor binding. In all tissues examined, [125I]Sar1,Ile8-Ang II bound to a single, high-affinity site. In the time course study, the affinity of binding was not significantly altered at any time period examined in livers from cold-exposed rats compared with controls (table 2). Nonspecific binding ranged from <5% (liver) to 40% (left ventricle) and was similar in membranes prepared from tissues of rats from each group. In the time course study, at 4 hr of cold exposure, Ang II receptor density in the liver was not altered (table 2, fig. 4A). After 1 day of cold exposure, Ang II receptor density in the liver increased (2-fold) compared with controls (table 2, fig. 4B). Increases in Ang II receptor density in livers from cold-exposed rats compared with controls were also evident at 3 (2-fold) and 7 (3-fold) days of cold exposure (table 2, fig. 4, C and D). In ISBAT, Ang II receptor binding density significantly increased (2-fold) in 7-day cold-exposed rats compared with control (table 3). In contrast, lung Ang II receptor binding density was not significantly altered after cold exposure (table 3).
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Discussion |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The present study clearly demonstrates that cold exposure
regulates the systemic renin-angiotensin system, resulting in an increase in plasma angiotensin peptide concentration. Concentrations of
angiotensin in the plasma of control rats from this study agree with
reported values in the literature, ranging from 8 to 30 pg/ml (Simon
et al., 1995). Moreover, characteristics of cold-exposed rats observed in the present study, including reductions in body weight, decreased plasma leptin concentration, increased plasma NE
concentration, increased food intake, and increases in the mass of
brown adipose tissue, are in agreement with previous reports in the
literature documenting the physiological effects of cold exposure
(Johnson et al., 1982; Giacobino, 1996).
The time course for cold-induced increases in plasma NE concentration
after cold exposure is well documented, demonstrating increases in the
plasma concentration of NE as early as 30 min (Picotti et
al., 1982) to 1 hr (Fukuhara et al., 1996a, 1996b) after cold exposure, which remain elevated as long as the exposure lasts. In contrast, angiotensin peptide concentration in plasma has not
been previously measured after cold exposure. Alterations in plasma
angiotensin peptide concentration in the present study included an
immediate marked increase in plasma angiotensin after acute cold
exposure that returned to base-line values within 1 day. The initial
increase in plasma angiotensin concentration after acute cold exposure
observed in the present study occurred in a time frame consistent with
previously reported stimulation of sympathetic nervous system activity,
suggesting that sympathetic activation may contribute to the acute
increase in plasma angiotensin peptide concentration.
Previous investigators have demonstrated that abrupt exposure to cold
resulted in a rapid increase in mean arterial pressure within 45 min
that was maintained over 24 hr (Fregly and Shechtman, 1994).
Thereafter, mean arterial pressure progressively increased above levels
observed in 24-hr cold-exposed rats to reach a level of 133 mm Hg after
7 days of cold exposure. The earliest time period of cold exposure
examined in the present study was 4 hr. Thus, it is unclear from this
study whether increases in plasma angiotensin peptide concentration are
related to previously reported abrupt rises in blood pressure in
cold-exposed rats. However, the marked increase in plasma angiotensin
peptide concentration after 4 hr of cold exposure may contribute to
early elevations in mean arterial pressure after cold exposure (Fregly
and Shechtman, 1994). The initial increase in plasma angiotensin
peptide concentration was not maintained and returned to normal within
24 hr despite continued cold exposure. Previous studies have
demonstrated that mean arterial pressure remains elevated from 45 min
to 7 days of cold exposure (Fregly and Shechtman, 1994). Thus, although increases in plasma angiotensin peptide concentration after cold exposure may contribute to an acute increase in mean arterial pressure,
results from this study do not support elevations in plasma angiotensin
peptides as the sole mechanism for initial elevations in blood pressure
after cold exposure.
In the feedback regulation loop for control of Ang II synthesis, the
end-product peptide (Ang II) of the system acts at kidney AT1 receptors
to inhibit further renin synthesis and release from the kidney (Peach,
1986). Thus, in the present study, plasma angiotensin peptide
concentration may have returned to normal values at days 1 and 3 of
cold exposure as a result of Ang II-mediated inhibition of subsequent
renin synthesis and release. Interestingly, in the present study,
plasma angiotensin peptide concentration again increased markedly after
7 days of cold exposure. Previous results from our laboratory suggested
that plasma renin activity was not altered after 7 days of cold
exposure (Cassis, 1993). In contrast, previous investigators reported
that plasma renin activity tended to increase slightly after 7 days of
cold exposure, followed by a decline to levels significantly lower than
controls at 4 weeks of cold exposure (Fregly et al., 1991).
These results suggest a dynamic interaction between plasma Ang II
concentration and feedback regulation of kidney renin synthesis and
release. Moreover, the resurgent increase in plasma angiotensin at 7 days of cold exposure in the present study suggests a potential role
for Ang II in the maintenance of cold-induced hypertension.
Interestingly, results from the present study demonstrate that increases in plasma angiotensin peptide concentration after cold exposure were primarily the result of increased concentrations of catabolic angiotensin peptide fragments. Of the angiotensin peptides resolved in the present study, Ang II, Ang III and Ang IV have known biological activity. Although plasma Ang II concentrations were not different between cold-exposed and control rats, concentrations of both Ang III and Ang IV were increased in plasma from cold-exposed rats. Thus, the physiological effects from increased plasma angiotensin peptide concentration after cold exposure may be mediated primarily by elevated concentrations of Ang III and/or Ang IV.
Results from the pair-feeding study demonstrate that pair-feeding
cold-exposed rats to food intake levels of control rats eliminated
increases in plasma angiotensin peptide concentration. Thus, activation
of the systemic renin-angiotensin system is dependent on cold-induced
increases in food intake. These results are the first to demonstrate
that food intake regulates plasma angiotensin peptide concentration.
Previous investigators have demonstrated that cold-exposed rats
increase their food intake to help maintain stimulated thermogenesis
(Johnson et al., 1982). In agreement with previous studies,
results from the present study demonstrate increases in food intake in
cold-exposed rats fed ad libitum. Plasma concentrations of
Ang II and NE measured in the present study were increased in parallel
after cold exposure. Moreover, increases in plasma Ang II and NE
concentration were eliminated in cold-exposed rats that were pair-fed
to control levels. These results demonstrate that elevations in food
intake in cold-exposed rats contribute to activation of the sympathetic
nervous system and the renin-angiotensin system.
Results from the present study clearly demonstrate that cold exposure
regulates Ang II receptor density. In time course studies examining the
effect of different periods of cold exposure on Ang II receptor binding
characteristics, increases in Ang II receptor density in liver were
evident within 24 hr of cold exposure. The time delay (4-24 hr) for
cold-induced increases in Ang II receptor density suggests mechanisms
related to de novo synthesis of Ang II receptor sites. The
dissimilarity in the time course for the observed increase in liver Ang
II receptor density and the time course for increased plasma
angiotensin concentration does not support interdependency of these two
variables. The sustained increase in liver Ang II receptor density in
the face of a return to normal plasma angiotensin concentration in
cold-exposed rats demonstrates regulation of Ang II receptor density
independent of angiotensin substrate concentration. Several
laboratories have examined substrate-mediated regulation of the Ang II
receptor. Using cell culture systems, chronic Ang II exposure decreased Ang II receptor density (Makita et al., 1992). In addition,
aortic coarctation and unilateral ureteral obstruction, both of which resulted in stimulated renin secretion and increased Ang II formation, inhibited AT1 mRNA expression in the affected kidney (Pimentel et
al., 1994; Tufro-McReddie et al., 1993). Moreover,
chronic infusion of Ang II in rats resulted in a decrease in Ang II
receptor density in heart and kidney (Sun and Weber, 1993). In the
present study, despite elevations in plasma angiotensin concentration, an increase in Ang II receptor density was observed in several tissues
from cold-exposed rats. Cold-induced increases in Ang II receptor
density in the present study may have resulted from substrate-mediated
up-regulation of Ang II receptor density or may be independent of
plasma angiotensin concentration. However, these results do not support
substrate-mediated down-regulation of Ang II receptor density in
response to elevated plasma angiotensin concentration.
Increases in Ang II receptor density were paralleled in ISBAT, a tissue
relevant to the maintenance of body temperature after cold exposure.
Previous investigators have demonstrated a marked increase in turnover
of NE in ISBAT after cold exposure, contributing to the maintenance of
nonshivering thermogenesis (Young and Landsberg, 1982). Previous
studies in our laboratory have demonstrated that after 7 days of cold
exposure, Ang II content in ISBAT is increased (Cassis, 1993). In
addition, the presynaptic effect of Ang II to facilitate NE release
from slices of ISBAT was enhanced in cold-exposed rats (Cassis, 1993).
Increases in Ang II receptor density in ISBAT from 7-day cold-exposed
rats in the present study may contribute to the enhanced presynaptic
effect of Ang II after cold exposure.
In the present study, cold exposure did not significantly increase Ang
II receptor density in lung. Thus, cold exposure regulates Ang II
receptor density through tissue-specific mechanisms. Previous investigators have demonstrated that the AT1 receptor is the
predominant angiotensin receptor subtype present in liver, lung and
ISBAT (Kitami et al., 1992; Cassis et al., 1996).
In kidney and left ventricle, both the AT1 and AT2 receptor are present
(Chang and Lotti, 1991; Kitami et al., 1992). In the present
study, losartan, used in a concentration range specific for AT1
blockade, displaced 100% of radioligand binding in livers from
cold-exposed and control rats. These results demonstrate that
cold-induced alterations in angiotensin receptor density in rat liver
are specific for the AT1 receptor subtype. Given that the lung also
contains predominately the AT1 receptor subtype, reasons for a lack of
cold-induced regulation of Ang II receptor density in this tissue
appear to be unrelated to the Ang II receptor subtype distribution.
Interestingly, cold exposure did not result in a significant increase in Ang II receptor density in kidney and left ventricle, the two tissues examined as Ang II target organs with cardiovascular relevance. However, Ang II receptor density increased in kidney and left ventricle when cold-exposed rats were pair-fed and were evident despite normalized plasma angiotensin concentrations in pair-fed rats. Mechanisms for tissue-specific regulation of Ang II receptor density by cold exposure and restricted food intake were not identified in the present study; however, results demonstrate that cold-induced regulation of Ang II receptor density in kidney and left ventricle occurred independent of plasma angiotensin concentration.
In summary, results from this study demonstrate that cold exposure regulates the systemic renin-angiotensin system, coincident with activation of systemic sympathetic nervous system activity. Moreover, cold exposure resulted in a tissue-specific increase in Ang II receptor density. The time course for cold-induced increases in plasma angiotensin concentration did not coincide with the time course for alterations in liver Ang II receptor density, suggesting regulation of Ang II receptor density was not substrate-mediated. Moreover, pair-feeding cold-exposed rats to food intake levels of control rats prevented increases in plasma angiotensin and NE concentration but did not prevent tissue-specific increases in Ang II receptor density. Results from these studies do not support substrate-mediated regulation of Ang II receptor density in response to cold exposure. Collectively, these results demonstrate that cold exposure is a model for systemic activation of the renin-angiotensin system and regulation of angiotensin receptor density.
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Acknowledgments |
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The authors acknowledge the University of Kentucky Veterans Administration research facility for use of the animal hibernactim for cold exposure studies.
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Footnotes |
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Accepted for publication April 20, 1998.
Received for publication September 23, 1997.
1 This work was supported by the National Heart, Lung and Blood Institute Grant HL52934.
Send reprint requests to: Lisa A. Cassis, Ph.D., Room 417, College of Pharmacy, Division of Pharmacology and Experimental Therapeutics, University of Kentucky, Lexington, KY 40536-0082.
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Abbreviations |
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NE, norepinephrine; Ang II, angiotensin II; Ang III, angiotensin III; Ang IV, angiotensin IV; ISBAT, interscapular brown adipose tissue; RIA, radioimmunoassay; AT1, angiotensin type 1 receptor; HPLC, high performance liquid chromatography; BSA, bovine serum albumin; EDTA, ethylenediamine tetraacetic acid; DMSO, dimethylsulfoxide; TFA, trifluoroacetic acid; DHBA, dihydroxybenzylamine.
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Abstract
Introduction
Methods
Results
Discussion
References
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 J. B. Chambers, T. D. Williams, A. Nakamura, R. P. Henderson, J. M. Overton, and M. E. Rashotte Cardiovascular and metabolic responses of hypertensive and normotensive rats to one week of cold exposure Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2000; 279(4): R1486 - R1494. [Abstract] [Full Text] [PDF]
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 S. Engeli, R. Negrel, and A. M. Sharma Physiology and Pathophysiology of the Adipose Tissue Renin-Angiotensin System Hypertension, June 1, 2000; 35(6): 1270 - 1277. [Abstract] [Full Text] [PDF]
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, Significantly
different from cold exposed.
