Vol. 88, Issue 4, 1316-1320, April 2000
Renal vasopressin receptor expression and function in rats
following spaceflight
David P.
Brooks1,
Ponnal
Nambi1,
Nicholas J.
Laping1,
Barbara A.
Olson1,
Mark
Pullen1, and
Charles E.
Wade2
1 Department of Renal Pharmacology, SmithKline
Beecham Pharmaceuticals, King of Prussia, Pennsylvania
19406-0939; and 2 Life Sciences
Division, NASA Ames Research Center, Moffett Field, California
94035
 |
ABSTRACT |
It has been suggested
there is a decreased renal responsiveness to vasopressin following
spaceflight and that this may be the mechanism for the increased urine
flow that is observed following return to normal gravity. In the
present study, we have therefore measured vasopressin receptor
expression and activity in kidneys taken from rats 1 and 14 days
following spaceflight of 15 days duration. Measurements of renal
vasopressin V2 and V1a receptor mRNA expression
by quantitative RT-PCR demonstrated little difference at either 1 day
or at 14 days following return from space. Evaluation of
3H-labeled arginine vasopressin binding to membranes
prepared from kidneys indicated that the majority of the vasopressin
receptors were V2 receptors. Furthermore, the data
suggested that binding to vasopressin V2 or V1a
receptors was unaltered at 1 day and 14 days following spaceflight.
Similarly, the ability of vasopressin to stimulate adenylate cyclase
suggested no change in vasopressin V2 receptor activity in
these animals. These data suggest that, whatever changes in fluid and
electrolyte metabolism are observed following spaceflight, they are not
mediated by changes in vasopressin receptor number or
vasopressin-induced stimulation of adenylate cyclase.
microgravity; fluid balance; water metabolism; antidiuretic
hormone
| |
INTRODUCTION |
A NUMBER OF STUDIES HAVE DEMONSTRATED that renal
function is altered during and immediately following spaceflight (2, 9, 11, 14, 15). In particular, it has been noted that humans demonstrate a
diminished ability to concentrate urine on return to normal gravity.
Thus, in a study of renal function in cosmonauts, postflight urine
osmolality was always lower than preflight levels for any given urine
flow rate (12). In addition, the ability to excrete a fluid load
appears to be impaired following spaceflight (2, 14). It has been
suggested that this reduction in concentrating ability involves a
decreased responsiveness to vasopressin, since there appears to be an
inappropriate urine osmolality for any given vasopressin concentration
following spaceflight (6, 14). Findings similar to those of humans have
been noted in rats following spaceflight (1); however, more recent
research has suggested that the increased urine flow observed in rats
following spaceflight involves changes in solute excretion rather than
free water clearance (20). Because a decreased renal responsiveness to
vasopressin would involve either decreased density of vasopressin
receptors or decreased receptor activity, we evaluated these parameters in rats immediately (1 day) and 14 days after return to normal gravity.
We proposed to determine if there was indeed a decrease in the
expression and density of renal vasopressin receptors or in the
activity of the receptors, which could contribute to the reported
differences in renal handling of water following spaceflight.
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METHODS |
Studies were conducted on a total of 36, male Fischer 344 rats (Taconic
Farms). All animal protocols were approved by the appropriate National
Aeronautics and Space Administration (NASA) Ames Animal Care Committees
and adhered to the National Institutes of Health guidelines for the
humane care and use of animals.
Experimental procedures.
Animals were assigned to three groups (n = 11-12/group) so
that each group had the same initial mean body weight. The groups were
designated as flight, vivarium control (VIV-C), and flight simulation
control (SIM-C). Flight animals were individually housed and flown in a
research animal holding facility (RAHF), and SIM-C animals were individually housed in flight-simulation cages (~4 in.
high by 4 in. wide by 12 in. deep; the height of these cages does not
allow the animals to rear on their hind legs). The SIM-C group was
included as a control for the cage size and environmental parameters
(temperature and humidity) experienced by the flight group. The VIV-C
group was included to evaluate whether changes in caging and/or
environment had any effects. All animals were maintained on the same
12:12-h light-dark cycle, to which they were entrained before selection
for the study. Both groups were fed food bars [Teklad (Madison,
WI), NASA experimental rodent diet no. TD 88179 extruded into food
bars, dipped in 15% sorbate to retard mold growth, radiation
sterilized, sealed in polyethylene bags, and stored at 4°C until
use] and provided water ad libitum. VIV-C animals were housed two
animals per cage, in standard shoe box cages (8 in. high by 10.5 in.
wide by 19 in. deep), with a Plexiglas divider running parallel to the
long axis of the cage to separate the two rats. VIV-C subjects were fed
pieces of flight food bars and provided water ad libitum. Flight
animals were flown aboard the Space Shuttle Columbia (STS-90, Neurolab)
for 15 days. Flight animals were killed within 5 h of landing.
V2 and V1a receptor mRNA expression.
Real-time quantitative RT-PCR was used to determine mRNA levels for the
V2 and V1a receptors.
Two micrograms of total RNA, extracted by CsCl ultracentrifugation,
were reverse transcribed using First-Strand beads (Pharmacia Biotech)
with 0.2 µg of random hexamer oligonucleotides in a 30-µl volume at
37°C for 1 h to synthesize cDNA. The cDNA was diluted 20-fold and
used in the subsequent PCR reaction. The 50-µl PCR reaction contained
3 µl of the diluted cDNA, 1× Taqman universal PCR master mix
(Perkin-Elmer Applied Biosystems), 200 nM of the gene-specific forward
and reverse primers [V1a forward:
CGCCT-ACGTG-ACCTG-GATG, V1a reverse:
AGCAT-GTACC-CAAGA-CGACC-A, V2 forward:
TGCTG-CCTGT-CAGGT-TCTTA-TC, V2 reverse: TCGGA-TGGCC-CTGGC,
ribosomal protein L32 (rpL32) forward: GAAAC-TGGCG-GAAAC-CCA, rpL32
reverse: GGATC-TGGCC-CTTGA-ATCTT-C], and 200 nM of gene-specific
fluorescent oligonucleotide probes (V1a:
6fam-CCAGC-GGTGT-CTTCG-TGGCA-CC-tamra, V2:
6fam-TCCGG-GAGAT-ACACG-CCAGT-CTGG-tamra, rpL32:
6fam-AGGCA-TCGAC-AACAG-GGTGC-GG-tamra). The PCR reaction was performed
in the ABI Prism 7700 sequence detector (Perkin-Elmer Applied
Biosystems) with 40 cycles at 95°C for 20 s and 60°C for 30 s.
The standard curves were determined by making serial dilutions of
control cDNA from 5-fold to 160-fold dilution. Quantity is calculated
by comparing the cycle time of the unknown (at which the signal is 3 times above threshold) to the cycle times measured for the standards.
The quantity of the 20-fold dilution of the standard was arbitrarily
set at 1. All data are represented as relative to the signal obtained
by the rpL32 probe.
Vasopressin receptor binding.
Membranes were prepared as follows. Tissue from one-half of one kidney
(0.8-1.2 g) was homogenized in 10 ml of buffer containing 0.25 M
sucrose, 20 mM Tris, pH 7.5, 5 mM EDTA, 1 µg/ml leupeptin, 0.01 trypsin inhibitor unit/ml aprotinin, and 50 µg/ml
phenylmethylsulfonyl fluoride (PMSF) using a motor-driven, Teflon and
glass tissue homogenizer. The homogenate was centrifuged at 1,000 g for 10 min at 4°C. The supernatant was filtered through
two layers of cheesecloth and centrifuged at 40,000 g for 30 min at 4°C. The pellet was suspended in 1.5 ml of 50 mM Tris base,
pH 7.5, and 10 mM MgC12 and kept frozen at 
70°C
until use. Protein was measured using a Bio-Rad protein assay reagent
and BSA as a standard.
Binding assays were performed following the procedure of Wang et al.
(21), with some modifications. Saturation binding experiments were
conducted to determine the dissociation constant
(Kd) for 3H-labeled arginine
vasopressin ([3H]AVP) and the proportion of
V1a and V2 receptors by measuring binding in
the presence and absence of 10 nM of the V1a antagonist Pmp1-Tyr(Ome)2-[Arg8]vasopressin
(PYAVP). Nonspecific binding was measured in the presence of 3 µM AVP; 50 µl of increasing amounts of
[3H]AVP were added to give a final range of
concentrations of 0.1-2.3 nM in a final assay volume of 200 µl.
Whole kidney membrane protein (0.15-0.4 mg) was added to each
tube. The peptides and membranes were diluted in 50 mM Tris base, pH
7.5, and 10 mM MgC12 with 1 µM phosphoramidon and 0.1%
BSA. Initial experiments to quantitate the vasopressin receptors were
done using the Kd (0.2 nM) and saturating
concentrations (1.5 nM) of [3H]AVP. A follow-up
experiment was done in the presence and absence of 3 nM
deamino-[Arg8]vasopressin (DAVP) or 10 nM PYAVP
(to quantitate V1a and V2 receptors, respectively) and saturating concentration of
[3H]AVP.
Adenylate cyclase activity.
Adenylate cyclase activity was determined in membrane preparations
following the procedure of Salomon et al. (17). Various concentrations
of vasopressin or forskolin (10 µl of 10× concentration) were added
to membranes (30-40 µg protein) in a total volume of 50 µl.
Adenylate cyclase reactions were initiated by the addition of 50 µl
of substrate mixture containing (final concentrations) 50 mM
Tris · HCl, pH 7.4, 10 mM MgCl2, 1.2 mM
ATP, 1.0 µCi [
-32P]ATP, 0.1 mM cAMP, 0.1 mM GTP, 2.8 mM phospho(enol)pyruvate, and 5.2 µg/ml
myokinase. The reaction was carried out for 20 min at 30°C.
[32P]cAMP formed was isolated by sequential
chromatography using Dowex 50 cation exchanger and neutral alumina.
Recovery of cAMP was monitored by the addition of
[2,8-3H]cAMP to each tube. The cAMP eluted from
the alumina column was quantitated by liquid scintillation spectroscopy.
Materials.
Phenylalanyl-3,4,5-3H-8-arginine vasopressin, (68 Ci/mmol;
[3H]AVP) was obtained from New England Nuclear
(Boston, MA).
5-Pmp1-Tyr(Ome)2-[Arg8]vasopressin
(PYAVP), deamino-[Arg8]vasopressin (DAVP), and
[Arg8]vasopressin (AVP) were purchased from
American Peptide (Sunnyvale, CA).
Data analysis.
Data are reported as means ± SE. Data were evaluated statistically
using an ANOVA and subsequently Scheffé's F test, with the exception of some of the body weight data, which were analyzed using a repeated-measures ANOVA (super ANOVA).
| |
RESULTS |
The body weight of animals before flight was similar among the entire
group (Table 1). On return from space,
flight animals had lost between 12 and 20 g of body weight, whereas the
SIM-C and VIV-C groups had gained weight. The body weight of animals 14 days after return from space increased to preflight values (Table 1).
Body weights of the animals in the VIV-C and SIM-C groups increased
over the course of the experiment; however, there was some variation
between the groups. The reason for this variability was unclear.
Measurement of vasopressin V2 and V1a receptor
mRNA by quantitative RT-PCR demonstrated no statistically significant
difference in kidney vasopressin receptor expression either 1 day
(P = 0.360) or 14 days (P = 0.685) after return from
space (Fig. 1). The data are expressed as a
ratio of receptor mRNA and ribosomal protein L32 (rpL32) mRNA.
Confirmation of no changes in vasopressin receptor expression was
obtained by evaluating [3H]AVP binding to
membranes prepared from the kidneys. [3H]AVP
binding to rat kidney tissue was saturable and specific. The
nonspecific binding, as measured in the presence of high concentrations of AVP (3 µM), was between 5 and 20% of total binding (Fig.
2A). Saturation binding experiments
performed in the presence and absence of 10 nM of the V1a
antagonist PYAVP (Fig. 2B) indicated the majority of receptors
in the kidneys were vasopressin V2 receptors. Furthermore, the data indicate that binding to either the vasopressin V2
receptor or the V1a receptor was unaltered at 1 day and 14 days following spaceflight (Fig. 3). There
was a trend for the vasopressin V2 receptor binding to be
lower in the control groups than in the flight group; however, this did
not reach statistical significance (P = 0.204 vs. VIV-C,
P = 0.056 vs. SIM-C).
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Fig. 1.
Vasopressin V2 receptor mRNA (A) and
V1a receptor mRNA (B) as measured by RT-PCR in rats
1 day and 14 days after spaceflight and in control rats housed at
ground level in vivarium (VIV-C) or flight-simulation (SIM-C) cages.
Data are expressed as a ratio of receptor mRNA and ribosomal protein
L32 (rpL32) mRNA (n = 4-6
observations/group).
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Fig. 2.
Saturation binding of 3H-labeled arginine vasopressin
([3H]AVP) to membranes prepared from rat kidney
(A), and Scatchard transformation of the specific binding
(B) in the presence and absence of the V1a receptor
antagonist
Pmp1-Tyr(Ome)2-[Arg8]vasopressin
(PYAVP) (10 nM). B/F, bound counts/free counts; ns, nonspecific.
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Fig. 3.
Vasopressin V1a and V2 receptor binding to
renal membranes prepared from rats 1 day and 14 days following
spaceflight and in control rats housed at ground level in vivarium
(VIV-C) or flight-simulation (SIM-C) cages (n = 4-6
observations/group).
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The ability of AVP to stimulate adenylate cyclase was used as a measure
of the functional activity of the vasopressin V2 receptors. Baseline adenylate cyclase activity ranged between 4.4 and 6.3 pmol/mg
protein on day 1 postflight and between 4.9 and 5.4 pmol/mg protein on day 14 postflight. There were no differences between groups. Vasopressin increased adenylate cyclase activity between 50 and
75%; however, the increase was similar in all groups of animals
(P = 0.674). Furthermore, direct stimulation of adenylate cyclase with forskolin was not altered by space travel (P = 0.200).
| |
DISCUSSION |
In normal individuals, total body water and osmolality are exquisitely
controlled. Day-to-day fluctuations in total body water and osmolality
are on the order of 0.2%/24 h. On exposure to microgravity, there are
shifts in fluids among the various body compartments. Exposure of
humans to a weightless state results in a headward shift in fluids,
thereby expanding thoracic blood volume by as much as 0.5 liter (22).
The body establishes a new fluid and electrolyte homeostasis through
regulatory compensatory mechanisms. These compensatory mechanisms
include functional changes of the cardiovascular, renal, nervous, and
endocrine systems, leading to alterations in fluid and electrolyte metabolism.
A number of investigators have suggested that the posterior pituitary
peptide vasopressin shows compensatory changes in regulation and
function following microgravity exposure, leading to alterations in
fluid and electrolyte homeostasis (7, 14, 19). Circulating levels of
vasopressin have been measured on numerous spaceflights, with
inconclusive findings (15). Interpretation of the data has been
complicated by the timing of the samples, the duration of the
spaceflights, and the occurrence of space motion sickness resulting in
vomiting, a powerful stimulus to vasopressin release (16). Nonetheless, the majority of the data obtained to
date do not suggest a decrease in circulating vasopressin (3-5,
8-11, 18). It has therefore been suggested that increased urine
output following spaceflight may involve a diminished sensitivity of the kidneys to vasopressin. This suggestion is derived from earlier work in which an inappropriate urine osmolality for a given vasopressin concentration was noted (6, 14). On the morning after flight, plasma
vasopressin was increased from a preflight level of 2.7 to 8.7 ng/ml,
accompanied by a decrease in urine osmolality from 1040 mosmol/kg
preflight to 872 mosmol/kg postflight. In normal subjects, an increase
in plasma vasopressin is associated with an increase in the
concentrating ability of the kidney, producing an elevation in urine
osmolality (23). The administration of a water load 36-40 h postflight
also demonstrated differences attributable to altered sensitivity of
the kidney to vasopressin (2, 6, 13, 18). These data suggest variable
responses of vasopressin to microgravity and in end-organ responsiveness.
As stated before, a similarity has been shown between the findings in
humans after spaceflight and those of rats following spaceflight. The excretion of a water load by rats 1 day after spaceflight was faster than that of control animals (1).
Recently, urine output of flight animals was reported to be almost
twice that of control animals for 3 days following flight
(20). There was no significant difference in water
intake between groups. Although the increase in urine output was
predominately due to an increase in osmotic clearance, a change in free
water clearance could not be ruled out. Thus the present study was
proposed to assess whether a decrease in the density and expression of
the kidney vasopressin receptors, which may contribute to the reported differences in the renal handling of water, does exist. Our data, however, clearly demonstrate no change in vasopressin receptor density
following exposure to microgravity for 15 days. Determinations of
vasopressin V2 receptor gene expression, by quantitative
RT-PCR, and measurements of vasopressin binding indicated a similar
receptor density in all groups of animals. Furthermore, there appeared to be no change in responsiveness to vasopressin, since the ability of
vasopressin to stimulate adenylate cyclase activity was unaltered. Increased cAMP production, resulting from vasopressin-stimulated adenylate cyclase, mediates the changes in water permeability in the
collecting ducts. The absence of a change in the ability of vasopressin
to stimulate adenylate cyclase suggests no change in the activity of
kidney vasopressin receptors due to spaceflight.
If there is indeed a reduced ability to concentrate
urine following spaceflight, it is possible that this involves altered responsiveness to cAMP or, perhaps, changes in renal hemodynamics. However, it should be noted that the question of whether any change in
urine concentrating ability occurs following spaceflight has been
questioned by recent observations that indicate the increased urine
flow observed during some spaceflight involves changes in solute
excretion rather than free water clearance (20). Free water clearance
was decreased, being indicative of increased vasopressin activity in
the kidney. Our study was limited, as we were unable to measure plasma
vasopressin levels, data that might have provided information useful to
the present study.
In summary, we found no changes in either vasopressin receptor
expression, density, or activity in the kidneys of rats following 15 days in microgravity. These data suggest that the changes in fluid and
electrolyte metabolism observed following spaceflight are not mediated
by changes in renal vasopressin receptor expression or activity.
Therefore, we speculate that increased fluid excretion following
spaceflight involves increased solute excretion rather than changes in
free water homeostasis.
| |
ACKNOWLEDGEMENTS |
We are most grateful to Kim Webster, Mami Shao, Dr. David
Liskowsky, Dr. Alvins Mooreland, and the Biospecimen Sharing Program Team in the Bionetics Life Sciences Support Contract at Kennedy Space
Center, Florida, for animal handling and tissue preparation, and to
Maria McDevitt in the Department of Renal Pharmacology, SmithKline
Beecham Pharmaceuticals, for preparing the manuscript.
| |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. P. Brooks,
SmithKline Beecham Pharmaceuticals, Dept. of Renal Pharmacology, UW2521
709 Swedeland Road, Box 1539, King of Prussia, PA 19406-0939.
Received 14 June 1999; accepted in final form 1 December 1999.
| |
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ABSTRACT
INTRODUCTION
