Seasonality of the red blood cell stress response in rainbow trout (Oncorhynchus mykiss)
1 School of Biological Sciences, University of Liverpool, The Biosciences
Building, Crown Street, Liverpool L69 7ZB, UK
2 CEH Windermere, The Ferry House, Far Sawrey, Ambleside, Cumbria LA22 0LP,
UK
3 Department of Biology, University of Ottawa, Ottawa, Ontario, Canada K1N
6N5
* Author for correspondence (e-mail: piak{at}liv.ac.uk)
Accepted 9 October 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: adrenergic receptor, Na+/H+ exchanger, oestradiol, testosterone, cortisol, seasonal changes, rainbow trout, Oncorhynchus mykiss
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The seasonal loss of ßNHE activity is likely to impair the normal
respiratory response to stress and this might negatively affect stress
tolerance. Understanding the underlying mechanisms of this seasonally reduced
ß-adrenergic responsiveness and its physiological significance requires
an intimate understanding of how RBC biology varies over the seasonal
timescale and how this relates to circulating hormones. Thus, a reduction in
rainbow trout RBC adrenergic responsiveness and flounder (Platichthys
flesus) Na+/H+ exchanger activity has been
circumstantially linked to the reproductive cycle
(Lecklin and Nikinmaa, 1999;
Weaver et al., 1999, respectively). Furthermore, plasma cortisol levels also
show seasonal variation in salmonid fish
(Pickering and Christie,
1981
), and Reid and Perry
(1991
) have shown an
upregulation of RBC ß-AR numbers in response to highly elevated plasma
cortisol level. Also, Perry et al.
(1996
) observed an increased
RBC ßNHE responsiveness when cortisol was elevated through repeated
physical stress.
Thus, in order to throw light on the occurrence, duration and magnitude of
seasonal downregulation of the ß-adrenergic response and to identify the
underlying mechanisms, we have undertaken an extensive monthly monitoring
programme over 27 months. We have sought to relate changes in RBC adrenergic
responsiveness to the number and affinity of RBC ß-ARs and also to link
changes in both to a range of other physiological parameters, including
reproductive condition and circulating levels of cortisol and reproductive
hormones. In particular, we have sought measures of erythropoietic activity,
since Lecklin et al. (2000)
have shown that immature RBCs have a higher adrenergic responsiveness than
mature RBCs. Thus, we have determined the cellular amounts of transcripts for
both the ß-AR and the ßNHE through the seasons. Finally, we employed
known pharmacological activators of the ßNHE response to probe the
effectiveness during winter and summer of the transduction pathway linking
ß-AR with adrenergically activated NHE activity.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Red blood cell ß-adrenergic receptor determination
Freshly drawn blood was washed three times in isotonic saline (145 mmol
l-1 NaCl, 6 mmol l-1 KCl, 1 mmol l-1
MgSO4, 5 mmol l-1 CaCl2, 5 mmol
l-1 D-glucose and 10 mmol l-1 Hepes, pH 7.9 and an
osmolality of 319±1 mosmol kg-1; mean ±
S.E.M.; N=4) to remove the buffy coat. The red cells were
re-suspended in fresh saline at a haematocrit (Hct) of 30%. Plasma
removed after the first spin was frozen (-20°C) in aliquots (200 µl)
for subsequent hormone and metabolite assays. High-affinity ß-ARs were
measured using CGP 12177 as a ß-adrenergic agonist according to Marttila
and Nikinmaa (1988
). Briefly,
duplicate samples (50 µl) of the RBC suspension were added to a series of
tubes containing 450 µl saline with final concentrations of 0.3-4.3 nmol
l-1 of [3H]CGP 12177
{[(-)-4-(3-t-butylamino-2-hydroxypropoxy)-(5,7-[3H]benzimidazol-2-one)];
specific activity 1.85 TBq mmol-1; Amersham, Hertfordshire, UK}
either alone (total binding, Bt) or in the presence of
excess unlabelled CGP 12177 (gift from Novartis Pharma AG, Basel, Switzerland;
non-specific binding, Bns). The samples were mixed and
incubated for 6 h at 4°C, during which time they were regularly agitated.
The RBCs were counted using a haemocytometer (Weber Scientific International
Ltd, Teddington, UK). At the end of the incubation period, 400 µl of each
individual sample was filtered through glass microfibre filters (2.5 cm
Ø, retention
1.0 µm; Whatman, Maidstone, UK) held on a vacuum
filtration manifold (Model 1225; Millipore, Watford, UK) attached to a vacuum
pump. The filters were washed three times with 2 ml ice-cold saline to remove
unbound ligand. The filters were placed in vials containing 5 ml of
scintillation cocktail (Eco-Safe; Meridian, Epsom, UK), shaken vigorously and
stored in the dark at 4°C for 72 h before liquid scintillation counting
(Tri-Carb 2100 TR; Packard, Perkin Elmer, Seer Green, UK). The activity
present in the incubation media was determined by counting of the stock
standard solution activities. Counts for Bt and
Bns were multiplied by 1.25 to account for the fact that
only 80% of the total volume of the samples was counted. Values for
specifically bound ligand (Bs) were determined by
subtraction of Bns from Bt and
subsequently converted to fmol tube-1 by division by the specific
activity. Receptor density (Bmax) and affinity
(Kd) were determined by use of non-linear regression
analysis (Sigmaplot 4; Jandel Scientific, Woking, UK) of
Bs as a function of free [3H]CGP 12177 [total
([3H]-CGP)-Bt]. Bmax values
were then converted from fmol tube-1 to receptors cell-1
by multiplication by Avogadro's number fmol-1 cells
tube-1.
Cortisol may influence the number of RBC membrane-bound ß-ARs upon
exposure to a stressor (Reid and Perry,
1991). To establish whether seasonal fluctuations in plasma
cortisol concentrations influence the number of ß-ARs, we incubated
sub-samples of RBC suspensions under anoxic conditions at 5°C for 1 h
prior to incubation with labelled and unlabelled ligand (July 2000-March
2001). The subsequent procedures were as described above.
Red blood cell ßNHE activity measurements
Samples of 1.2 ml washed RBC suspension were placed in rotating Eschweiler
tonometers maintained thermostatically at 15°C and equilibrated with
humidified N2 (anoxic condition) for 60 min. After 50 min of
equilibration, ouabain (5x10-4 mol l-1 final
concentration) was added and duplicate samples of the suspension were taken
for Hct determination. The RBC suspension was transferred from the tonometer
flask into polypropylene test tubes containing saline with ouabain
(5x10-4 mol l-1 final concentration; 0.5% DMSO v/v
vehicle) and 22Na (0.05 MBq ml-1) equilibrated to
the same conditions as the blood cells, resulting in a 10-fold RBC dilution
(this was defined as time zero). The 22Na influx was measured in
unstimulated cells (basal flux) and in cells stimulated by 10-5 mol
l-1 (final concentration) isoproterenol added at 5 min.
Triplicate samples (300 µl) from control and treated RBC suspensions
were taken at 5 min and 10 min. The samples were centrifuged (Model 5410;
Eppendorf, Cambridge, UK) and the supernatant removed, whereupon the RBCs were
washed three times in ice-cold isotonic Hepes-containing MgCl2
solution (adjusted to pH 7.9). The remaining RBC pellet was lysed in 0.5 ml
0.05% Triton-X solution and deproteinised by subsequent addition of 0.5 ml 5%
trichloroacetic acid. The samples were centrifuged for 2 min and 0.5 ml of the
supernatant was counted (Tri-Carb 2100 TR, Packard) in 5 ml scintillation
cocktail (Eco-Safe, Meridian). In addition, triplicate samples of 200 µl
extracellular medium were counted for each experiment. The Na+
influx (mmol Na l-1 RBC h-1) was calculated as:
![]() |
Plasma cortisol, sex steroids and lactate
Plasma cortisol, testosterone and oestradiol-17ß concentrations were
measured by previously validated radioimmunoassays (cortisol:
Pickering et al., 1987;
testosterone: Pottinger and Pickering,
1985
; oestradiol-17ß:
Pottinger and Pickering,
1990
). Plasma lactate concentration was determined enzymatically
using lactate oxidase and peroxidase followed by spectrophometric analysis
(Roche, Basel, Switzerland).
Effects of repeated disturbance stress on adrenergic
responsiveness
Eighty rainbow trout from a stock population were divided evenly between
four 1000 litre outdoor holding tanks (conditions as above) and acclimated for
two weeks. At the onset of the experiment, the water level in two of the tanks
was dropped to 10 cm for
5 min once or twice daily for a two-week
period. The control tanks were left undisturbed. Upon sampling, three fish
from each tank (mean mass ± S.E.M., 1773±92 g;
N=12) were anaesthetised and blood was collected and prepared for
Na+ influx measurements exactly as described above. The abundance
of RBC ß-ARs was determined for RBC suspensions incubated under normoxic
and anoxic conditions as described above.
The effects of isoproterenol, forskolin, dibutyryl cAMP and calyculin
A on RBC Na+ influx
Six rainbow trout from the stock population (mean mass ±
S.E.M., 1847±83 g) were netted into anaesthetic and blood
was sampled, washed and stored overnight for Na+ influx
measurements as described above. The Na+ influx was measured as
described above 5 min after addition of (1) isoproterenol (10-5 mol
l-1; non-selective ß-AR agonist), (2) forskolin
(1.5x10-4 mol l-1; adenylate cyclase activator),
(3) dibutyryl cAMP (10-3 mol l-1; membrane-permeable
cAMP analogue) or (4) calyculin A (10-7 mol l-1;
phosphatase inhibitor). In addition, the Na+ influx was measured in
unstimulated RBCs.
ßNHE activity in RBCs separated according to density (age)
Blood was sampled, washed and stored as described above from rainbow trout
(Shasta strain; mean mass ± S.E.M., 937±68 g;
N=6) during October-November 2001. This treatment assured that the
RBC membrane transporters and cell volume were in an unstimulated steady-state
condition (Bourne and Cossins,
1982). After overnight storage, the RBCs were washed once and
re-suspended in saline containing 1% bovine serum albumin (BSA) at an Hct of
approximately 80%. The RBCs were separated into age fractions by fixed-angle
(30°) centrifugation (10 000 g, 4°C, 15 min) in narrow
tubes (diameter 4 mm, length 45 mm, volume 0.5 ml;
Speckner et al., 1989
;
optimized for trout by Phillips et al.,
2000
). The youngest, least dense, cells are located in the top
layer, whereas the older, more dense cells are located in the middle and
bottom of the tube. The tubes were cut into a top (24±2%), middle
(60±3%) and bottom (15±1%) fraction, containing RBCs of
increasing age, which were washed in isotonic saline three times to remove
BSA. The mean cellular haemoglobin concentration (MCHC), determined as
[Hb]/Hct, was used to verify that the cells were separated according to age.
MCHC is lower in younger cells than in older cells
(Speckner et al., 1989
;
Lund et al., 2000
).
Adrenergically stimulated Na+ influx was measured in each of the
different cell fractions and also in the original unseparated population of
RBCs.
RBC ßNHE and ß3b receptor mRNA determinations
Total RNA was isolated from frozen tissue by homogenisation in guanidinium
thiocyanate (Chomczynski and Sacchi,
1987) using Trizol Reagent (Invitrogen, Ontario, Canada). After
treatment with DNase I (5 units per µg RNA; Invitrogen) to remove any
remaining genomic DNA, the quality of the RNA was assessed by gel
electrophoresis. cDNA was synthesised from 1-2 µg RNA using random hexamer
primers and Superscript II reverse transcriptase (Invitrogen). Previous
studies (Nickerson, 2003) have demonstrated that the trout RBC ß-AR most
closely resembles the ß3-AR of mammals or the
ß4c-AR of turkey RBCs (Chen
et al., 1994
). Because the trout RBC ß-AR is found
exclusively in the blood, it has been termed ß3b-AR
(Nickerson et al., 2003
).
ß3b-AR or ßNHE mRNA levels were assessed by Q-PCR on
duplicate samples of cDNA (1 µl) using a Hot StarTaq Master Mix kit
(Qiagen, Ontario, Canada) and a Stratagene (West Cedar Creek, TX, USA) MX-4000
multiplex quantitative PCR system. CYBR green (Molecular Probes Inc., Boulder,
CO, USA) and ROX (Stratagene) were used as DNA and reference dyes,
respectively. The PCR conditions (final reaction volume=20 µl) were as
follows: cDNA template=1 µl; forward and reverse [primer]=150 pmol
l-1; [Mg2+]=2.0 mmol l-1; CYBR green=1:50 000
final dilution; ROX=1:30 000 final dilution; dNTP=200 µmol l-1.
The annealing and extension temperatures over 40 cycles were 58°C (45 s)
and 72°C (60 s), respectively. The following primer pairs were designed
using Primer3 software
(http://www-genome.wi.mit.edu/cgibin/primer/primer3_www.cgi):
actin forward 5'-CAC CAT GAA GAT CAA GAT CAT YGC-3'
actin reverse 5'-ATT TRC GGT GGA CGA TGG AG-3'
ß3b forward 5'-CTT GGG CTA TGG TGG CAG TA-3'
ß3b reverse 5'-CCA TGA TAA TGC CCA AGG TC-3'
ßNHE forward 5'-GGG TAA TGC GTC AGA CAA CC-3'
ßNHE reverse 5'-CCA TGA TAA TGC CCA AGG TC-3'
The specificity of the primers was verified by cloning (TOPO TA cloning
kit; Invitrogen) and sequencing of the amplified products. To ensure that CYBR
green was not being incorporated into primer dimers or non-specific amplicons
during the Q-PCR runs, PCR products were analysed by gel electrophoresis in
initial experiments; single bands of expected size were obtained in all
instances. Furthermore, the construction of CYBR green dissociation curves
after completion of 40 PCR cycles revealed the presence of single amplicons
for each primer pair. Relative expression of mRNA levels was determined (using
actin as an endogenous standard) by a modification of the delta-delta Ct
method (Pfaffl, 2001).
Amplification efficiencies were determined from standard curves generated by
serial dilution of plasmid DNA.
Data presentation and statistics
Throughout, the data are presented as means ± S.E.M. The
data sets were analysed using analysis of variance (ANOVA; Genstat 5, Lawes
Agricultural Trust, Harpenden, UK) with, for the seasonal study, individual
fish, tank and time as factors; for the implant studies, fish, tank and
treatment as factors; and for the separated cell study, fish and cell fraction
as factors. Significant differences between times, treatment groups or
fractions were determined using the estimated standard error of the
differences between means. Where mean and variance were found to be
interdependent, the data were log-transformed prior to analysis. Sigma Plot
4.0 was used to assess statistical significance of fitted linear and
non-linear regressions.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Seasonal variations in RBC ß-AR characteristics and ßNHE
activity
Fig. 1 presents the time
course over 27 months of ß-AR numbers together with the measured water
temperatures. The number of ß-ARs varied seasonally from a minimum of
668±112 receptors cell-1 to a maximum of 2654±882
receptors cell-1 (N=8;
Fig. 1). During late summer
2000, the ß-AR number increased, remained high until late autumn and then
decreased into the winter. A similar, though less pronounced, rhythm was
evident in 2001 (Fig. 1).
Throughout this period, the ß-AR number was positively related in a
linear fashion to the average ambient water temperature (P=0.002,
r2=0.417; Fig.
1 inset). The equilibrium dissociation constant
(KD) for CGP binding to the RBC ß-ARs varied between
0.31±0.08 nmol l-1 and 1.35±0.26 nmol l-1
(N=8; not shown) but did not correlate with the ambient water
temperature (linear regression: P=0.77).
|
Reid and Perry (1991)
showed that cortisol applied through an osmotic pump can pre-adapt the RBCs to
cope with a stressor by elevating the number of membrane-bound ß-ARs, and
this potentially increases adrenergic responsiveness. During our routine
procedures for measuring ß-adrenergic responsiveness we also stressed the
RBCs by incubating cells throughout under anoxic conditions. We therefore
examined whether seasonal fluctuations in cortisol, with elevated levels
during the spawning periods (Fig.
2), influenced the number or affinity of functional ß-ARs
when incubated under oxygenated or anoxic conditions. There was, however, no
statistical difference in ß-AR characteristics (number and affinity)
between oxygenated and anoxically treated RBC suspensions during the entire
period between July 2000 and March 2001 (data not shown).
The mean isoproterenol-stimulated Na+ influx was
110.4±2.3 mmol l-1 RBCs h-1 (N=128)
during the months where the ß-adrenergic response was not reduced
(Fig. 2). A downregulated
response was arbitrarily defined as any flux lower than 85 mmol l-1
RBCs h-1. In December 1999 and February 2001, the ßNHE
activity was reduced by 57% and 34%, respectively, compared with the average
value, whereas during the third winter of the study there was no statistically
significant reduction in the ßNHE activity
(Fig. 2). Surprisingly, there
was no obvious dependence of the magnitude of the Na+ influx on the
number (linear regression: P=0.83; N=163) or affinity
(linear regression: P=0.68; N=163) of the ß-ARs. These
data suggest that the seasonal changes in ßNHE activity do not reflect
changes in the binding characteristics of the hormone to the receptors. Also,
the Na+ influx did not correlate with ambient water temperature
(linear regression: P=0.73; non-linear regression: P=0.72),
i.e. the temperature at which the fish were acclimated, which agrees with the
results obtained by Cossins and Kilbey
(1989).
Effects of repeated disturbance stress on ß-adrenergic
responsiveness
RBC ß-AR density has been shown to decrease in response to chronically
elevated plasma catecholamine levels
(Gilmour et al., 1994). We
exposed fish to repeated daily stress over a 2-week period in January 2001,
when the ßNHE responsiveness was normal, to elevate plasma catecholamine
levels and test if unidentified stressors in the holding facilities would
affect the ß-adrenergic responsiveness. The ßNHE activity was not
affected by repeated physical stress, with Na+ influx values of
129.7±4.7 mmol l-1 RBCs h-1 and 128.8±7.4
mmol l-1 RBCs h-1 in control and disturbed fish,
respectively.
Comparison of the effects of isoproterenol, forskolin, dibutyryl cAMP
and calyculin A
Having established that the seasonal reduction in ßNHE activity was
not linked to seasonal changes in ß-AR number or affinity, we looked for
other causal factors. Binding of catecholamine to the membrane-bound ß-AR
triggers an adenylate cyclase-catalysed synthesis of cAMP
(Mahe et al., 1985), which in
turn stimulates protein kinase A to activate the ßNHE
(Guizouarn et al., 1993
).
Accordingly, we tested whether the cause of the seasonally reduced adrenergic
responsiveness lay in the transduction pathway linking the receptor with the
transporting effector using chemical compounds known to stimulate the
ßNHE by intervening at different points on a possibly long transduction
pathway. None of the compounds stimulated the Na+ influx to a
greater extent than isoproterenol (data not shown). More significantly in the
present context, the extent to which these compounds stimulated the ßNHE
relative to stimulation by isoproterenol was similar in RBCs with a seasonally
reduced response compared with RBCs with a normal response
(Table 1). Thus, the reduced
isoproterenol-induced ßNHE activity could not be rescued by any of the
transduction activators, indicating that the seasonal diminution of ßNHE
activity was not linked to any of the steps lying between the ß-AR and
the ßNHE. This together with the absence of any changes in the binding
affinity and number of ß-ARs, suggests that the winter loss of ßNHE
activity was linked to changes in the ßNHE itself.
|
Red blood cell ßNHE and ß3b mRNA levels
The seasonal variation in ß3b mRNA levels is shown in
Fig. 3. The relative quantity
of ß3b mRNA was more or less constant between May and October, decreased
markedly by 90% between October and December and remained at low levels
between December and May. The data showed that the level of ß3b mRNA,
like the receptor number per cell, was correlated in a linear fashion with the
ambient water temperature (P=0.01, r2=0.632) and
also that the number of receptors depended in a linear fashion
(P=0.005, r2=0.755) on the level of ß3b mRNA
present in the RBCs (Fig. 3
inset).
|
Fig. 4 illustrates the corresponding changes in ßNHE mRNA. The relative quantity increased between May and November, decreased markedly between November and December and increased again progressively between December and May (Fig. 4). Examination of the levels of ßNHE mRNA at different times of the year showed that the ßNHE activity had a one-sampling time delayed hyperbolic dependency on the mRNA level (P=0.038; r2=0.538) present in the cell (Fig. 4 inset).
|
ßNHE activity of age-separated RBCs
After separation, the top layer contained 23.9±2.9%, the middle
layer 61.5±3.0% and the bottom layer 14.7±1.1% of the cells
(N=6). The values for MCHC are presented in
Table 2. MCHC increased from
the top to the bottom fractions of RBCs, showing that the cells had been
separated according to age. Based on measurements for the individual
fractions, MCHC of the unseparated RBCs suspension was calculated to be 4.19
mmol l-1, which is within the 0.05 significance limits of the
measured value. The activity of the ßNHE of the top fraction RBCs was
23.6 mmol l-1 RBCs h-1 and 35.5 mmol l-1 RBCs
h-1 higher than that of the middle and bottom fractions,
respectively (P<0.01 and P<0.05, respectively;
Table 2).
|
Seasonal variations in plasma oestradiol, testosterone and
lactate
The seasonal fluctuations in plasma sex steroids in female fish within the
experimental population are illustrated together with the ßNHE activity
in Fig. 5. Plasma testosterone
levels in the female fish increased steeply just prior to or concomitantly
with the observed major changes in the RBC ßNHE activity
(Fig. 5). It is noticeable that
the maximal levels of plasma testosterone observed in successive years
decreased gradually and in a statistically significant manner; i.e. in the
third year of the study, the plasma testosterone level was reduced compared
with the level in the first (P<0.01) and second
(P<0.001) year of the study. Plasma oestradiol levels varied in a
very predictable manner, i.e. the maximal levels and the timing of the
increases and decreases were similar each year
(Fig. 5).
|
Plasma lactate varied seasonally throughout the study, with minimal values of 1.13±0.11 mmol l-1 (N=16) in February and maximal values of 2.54±0.24 mmol l-1 (N=16) in August. The response to ß-adrenergic stimulation, which lowers the risk of anoxic metabolism and lactic acid production, was also lowest in the winter (see above). The lactate data, therefore, indicate that, despite the reduction in ß-adrenergic responsiveness, the oxygen uptake from the water was entirely sufficient to prevent activation of anaerobic metabolism under conditions of routine activity in the well-aerated holding facilities.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In considering the magnitude of the winter reduction in ß-adrenergic
responsiveness, it is worth pointing out that the trout RBC ßNHE activity
is markedly temperature dependent, with a Q10 of 7.9 over the
temperature range 0-19°C (Cossins and
Kilbey, 1990). Thus, assay of ßNHE activity in vitro
at a standard temperature of 15°C irrespective of seasonally varying
environmental temperature must, therefore, profoundly underestimate the
seasonal fluctuation in the in vivo RBC ß-adrenergic response.
Thus, the flux of 72 mmol l-1 RBCs h-1 measured in
vitro in February 2001 at 15°C would be reduced in vivo to
9 mmol l-1 RBCs h-1 at the prevailing water
temperature at that time.
Repeated physical stress and confinement both elevate plasma catecholamine
and cortisol levels, with opposite effects on the ß-adrenergic response
(Perry et al., 1996). Thus,
exposure of rainbow trout to daily physical stress reduced the number of
membrane-bound ß-ARs determined both under normoxic and hypoxic
conditions but elevated adrenergic responsiveness due to increased ß-AR
affinity (Perry et al., 1996
).
We therefore considered the possibility that (unidentified) seasonal
differences in the degree of stress to which the experimental fish were
exposed could have influenced RBC adrenergic responsiveness and contributed to
the seasonal variation in RBC function observed during this study. However, at
a time of the year when the ß-adrenergic response was normal, exposure of
the fish to a regime of repeated daily disturbance stress did not change the
ßNHE responsiveness. The seasonal reductions in adrenergic
responsiveness, therefore, seem to occur independently of increased stress
levels.
An obvious factor that might underlie the seasonal variations in RBC
ß-adrenergic response is the effectiveness of the cellular ß-AR
system. Previous work has implicated altered ß-AR numbers in the enhanced
response to hypoxia (Reid and Perry,
1991; Marttila and Nikinmaa,
1988
); however, the fluctuations in ß-AR numbers or affinity
in the present study were not in any way related to the seasonal changes in
ßNHE activity. An alternative explanation is that there was some
impairment during winter in the effectiveness of the transduction pathway
linking the ß-AR with the ßNHE. We stimulated the ßNHE in a
number of receptor-independent ways, using forskolin and calyculin A, which
activate the ßNHE by effects on adenylate cyclase and protein
phosphatase, respectively (Seamon et al.,
1981
; Guizouarn et al.,
1995
), and the membrane-permeable cAMP analogue dibutyryl cAMP. We
compared the effect of these compounds on the ßNHE activity of winter
fish with reduced ß-adrenergic responsiveness with those in summer fish
with the normal high ß-adrenergic responsiveness. If winter suppression
were due to impairment at a specific step in this pathway we would expect a
larger effect of pharmacological activation at a downstream step, relative to
the effects of the ß-adrenergic agonist isoproterenol acting alone. We
found that all three compounds stimulated the ßNHE to the same extent in
RBCs with a reduction in ßNHE activity compared with RBCs with a normal
response. This indicates no rescue of ß-adrenergic response in RBCs of
winter fish by downstream activation; the red cells from animals collected at
different times of the year behaved identically with respect to transduction
manipulation when tested under common experimental conditions. Whilst we
cannot exclude the possibility that there is a critical step lying downstream
of the calyculin A-sensitive protein phosphatase, we suggest that the limiting
step during winter months in the ß-adrenergic response was not upstream
to the ßNHE. Given that it was not due to changes in ß-AR numbers,
we therefore conclude that it was due to variations in the number or affinity
of the exchanger itself or properties of its microenvironment.
The linkage between seasonal changes in ß-adrenergic responsiveness
and reduced expression of the ßNHE is supported by measurements of
ßNHE transcript expression. We found that transcript amounts declined in
the autumn and increased in the spring, the changes in the mRNA levels
preceding by one sampling period the spring increase in ßNHE activity.
The changes in ßNHE mRNA levels could be caused either by seasonal
effects on the transcriptional activity in the circulating population of RBCs
or by seasonality in erythropoietic activity coupled with age-dependent
changes in RBC transcriptional activity. Age-dependent reductions in mRNA
levels have been reported for carbonic anhydrase and the Band 3 anion
exchanger in rainbow trout RBCs (Lund et
al., 2000). At the physiological level, Lecklin et al.
(2000
) showed that the volume
increase following ß-adrenergic stimulation was considerably lower in
mature than in immature RBCs, suggesting lower ßNHE activity in the older
cells. Consistent with this observation, age-dependent reductions in enzyme
activity levels have been reported for citrate synthase, cytochrome oxidase,
lactate dehydrogenase and pyruvate kinase in rainbow trout RBCs
(Phillips et al., 2000
).
Age-dependent decreases in activity levels therefore seem to be widespread at
both the transcriptional and functional levels. Erythropoietic activity and
the release of newly synthesized RBCs into the circulation is reduced in
rainbow trout during the winter (Lane,
1979
), and in Baltic salmon (Salmo salar) the proportion
of immature cells in circulation decreases during the winter
(Härdig and Höglund,
1984
). Seasonal changes in the age profile of the circulating RBCs
make the age dependency of the RBC ß-adrenergic volume response and
enzyme activities of great interest in the interpretation of seasonal changes
in RBC function. We show with density-separated RBCs that the ßNHE
activity of the top (youngest RBCs) of the separated fractions was
significantly higher than that of the middle and bottom fractions, containing
older RBCs, which is consistent with the changing age profile of circulating
RBCs underpinning the seasonally reduced ß-adrenergic responsiveness. In
the present study, we do not have any measure of the erythropoietic activity.
However, previous work in the same fish-holding facilities at CEH Windermere
showed that the circulating RBC number decreased during autumn and winter in
mature females of the closely related brown trout Salmo trutta
(Pottinger and Pickering,
1987
). The critical factor might be influence of low water
temperatures during late autumn and winter upon erythropoietic activity
(Hevesy et al., 1964
),
progressively reducing the proportion of young RBCs with a high
ß-adrenergic responsiveness in circulation.
Linking circulating hormones and erythropoietic activity with the
ßNHE activity
Elevated levels of plasma oestradiol were observed prior to and concomitant
with the seasonally reduced ßNHE activity in December 1999 and
January-February 2001. It remains to be investigated whether seasonal
elevations of plasma oestradiol play any role in the ß-adrenergic
response of the circulating RBCs.
Testosterone is known to stimulate erythropoiesis in humans
(Barcelo et al., 1999;
Snyder et al., 2000
) and birds
(Jones and Johansen, 1972
;
Robinzon and Rogers, 1979
;
Thapliyal et al., 1982
) and is
suspected to have the same effect in fish
(Pottinger and Pickering,
1987
). Thus, we suggest that the increased testosterone levels
observed during the spawning period in the present work may, by stimulating
erythropoiesis, have promoted the rapid increase in adrenergic responsiveness
during winter or early spring, the increasing proportion of immature RBCs with
increased ßNHE activity explaining the enhanced ß-adrenergic
responsiveness. The increases in ß-adrenergic responsiveness were
observed 1-2 sampling periods after the increases in plasma testosterone and
decreases in ßNHE activity. This delay is consistent with the lengthy lag
time known to precede the entry of immature RBCs into the circulation
(Lecklin et al., 2000
).
Seasonality in the ß-adrenergic receptor characteristics
We provide the first evidence linking, in a linear fashion, the abundance
of a receptor, i.e. the ß-AR, and its cognate transcript with the ambient
water temperature. The spring increase in water temperature was closely
associated with an increase in ß3b-receptor mRNA and subsequently to an
increase in the number of functional receptors. This seems appropriate, as
oxygen consumption increases with a rise in water temperature, and stimulation
of the ß-AR optimizes oxygen transport
(Nikinmaa, 1992). It is
paradoxical, therefore, that the increase in receptor ß-AR numbers did
not elevate ßNHE activity and, consequently, in functional terms, does
not seem to improve oxygen transport. No other parameter monitored during the
present study co-varied with the ß3b mRNA levels and ß-AR numbers.
However, we cannot exclude the idea that the influence of temperature on
ß3b mRNA levels and ß-AR numbers was indirect. How would the lower
ambient temperatures routinely experienced by fish in Northern USA, Canada and
Scandinavia (0-2°C) affect seasonal fluctuations in ß-ARs?
Extrapolation of the linear relationship between ß-AR numbers and ambient
temperature to 1°C predicts 549 receptors cell-1. This might be
entirely sufficient to initiate events leading to ßNHE activation.
Indeed, the Windermere fish included specimens with very low ß-AR numbers
yet with powerfully expressed ßNHEs, suggesting that exceptionally low
winter temperatures would not necessarily incur greater reductions in
ßNHE activity.
Experimental elevation of plasma cortisol is able to increase ß-AR
density, probably by increasing transcription of the ß3b-receptor gene.
This enlarges the pool of cytosolic, physiologically inactive
ß-receptors, which can be mobilised to the plasma membrane on exposure to
stress (Reid and Perry, 1991).
Whilst we found considerable circannual fluctuations in plasma cortisol, with
high levels during February-March, we failed to observe changes in
membrane-bound ß-ARs in anoxically treated RBCs compared with control
RBCs. Cortisol therefore appears to play no role in the seasonal variation of
RBC adrenergic responsiveness.
Conclusions
We showed that the number of RBC ß-ARs is linearly related both to the
level of ß-AR transcript and the ambient water temperature. Naturally
occurring seasonal fluctuation in ß-AR numbers was not related to the
more inconsistent fluctuations in ßNHE activity. On the other hand,
ßNHE activity was positively related to the ßNHE transcript level
and depended on the age of the RBC, showing reduced activity in older RBCs.
The seasonally reduced ßNHE activity could not be rescued by
pharmacological intervention in the transduction cascade, suggesting that
fluctuations in ßNHE activity were a property of the transporter itself
rather than any other factor. The inconsistency in timing of the reductions in
ßNHE activity was matched by corresponding fluctuations in plasma
testosterone. Since testosterone stimulates erythropoietic activity in birds
and humans, we suggest that the seasonally occurring fluctuations in ßNHE
activity of trout red cells are caused by changes in the age profile of
circulating RBCs. We further hypothesise that this is due to the seasonal
influences of testosterone.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barcelo, A. C., Olivera, M. L., Bozzini, C., Alippi, R. M. and Bozzini, C. E. (1999). Androgens and erythropoiesis. Induction of erythropoietin-hypersecretory state and effect of finasteride on erythropoietin secretion. Comp. Haematol. Int. 9, 1-6.
Bourne, P. K. and Cossins, A. R. (1982). On the instability of K+ influx of erythrocytes of the rainbow trout, Salmo gairdneri, and the role of catecholamine hormones in maintaining in vivo influx activity. J. Exp. Biol. 101,93 -104.[Abstract]
Chen, X. H., Harden, T. K. and Nicholas, R. A.
(1994). Molecular-cloning and characterization of a novel
beta-adrenergic-receptor. J. Biol. Chem.
269,24810
-24819.
Chomczynski, P. and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate phenol chloroform extraction. Analyt. Biochem. 162,156 -159.[CrossRef][Medline]
Cossins, A. R. and Kilbey, R. V. (1989). The seasonal modulation of Na+/H+ exchanger activity in trout erythrocytes. J. Exp. Biol. 144,463 -478.
Cossins, A. R. and Kilbey, R. V. (1990). The temperature-dependence of the adrenergic Na+/H+ exchanger of trout erythrocytes. J. Exp. Biol. 148,303 -312.[Abstract]
Gilmour, K. M., Didyk, N. E., Reid, S. G. and Perry, S. F.
(1994). Down-regulation of red blood cell ß-adrenoreceptors
in response to chronic elevation of plasma catecholamine levels in the rainbow
trout. J. Exp. Biol.
186,309
-314.
Guizouarn, H., Borgese, F., Pellissier, B., Garcia-Romeu, F. and
Motais, R. (1993). Role of protein phosphorylation and
dephosphorylation in activation and desensitisation of the cyclic
AMP-dependent Na+/H+ antiport. J. Biol.
Chem. 268,8632
-8639.
Guizouarn, H., Borgese, F., Pellissier, B., Garcia-Romeu, F. and Motais, R. (1995). Regulation of Na+/H+ exchange activity by recruitment of new Na+/H+ antiporters: effect of calyculin A. Am. J. Physiol. 268,C434 -C441.[Medline]
Härdig, J. and Höglund, L. B. (1984). Seasonal variation in blood components of reared Baltic salmon, Salmo salar L. J. Fish. Biol. 24,565 -579.
Hevesy, G., Lockner, D. and Sletten, K. (1964). Iron metabolism and erythrocyte formation in fish. Acta. Physiol. Scand. 60,256 -266.[Medline]
Jones, D. R. and Johansen, K. (1972). The blood vascular system of birds. In Avian Biology, vol.II (ed. D. S. Farner and J. R. King), pp.157 -285. New York: Academic Press.
Lane, H. C. (1979). Progressive changes in haematology and tissue water of sexually mature trout, Salmo gairdneri Richardson during the autumn and winter. J. Fish Biol. 15,425 -436.
Lecklin, T. and Nikinmaa, M. (1999). Seasonal
and temperature effects on the adrenergic responses of Arctic charr
(Salvelinus alpinus) erythrocytes. J. Exp.
Biol. 202,2233
-2238.
Lecklin, T., Tuominen, A. and Nikinmaa, M.
(2000). The adrenergic volume changes of immature and mature
rainbow trout (Oncorhynchus mykiss) erythrocytes. J. Exp.
Biol. 203,3025
-3031.
Lund, S. G., Phillips, M. C. L., Moyes, C. D. and Tufts, B. L. (2000). The effects of cell ageing on protein synthesis in rainbow trout (Oncorhynchus mykiss) red blood cells. J. Exp. Biol. 203,2210 -2228.
Mahe, Y., Garcia-Romeu, F. and Motais, R. (1985). Inhibition by amiloride of both adenylate cyclase activity and the Na+/H+ antiporter in fish erythrocytes. Eur. J. Pharmacol. 116,199 -206.[CrossRef][Medline]
Marttila, O. N. T. and Nikinmaa, M. (1988). Binding of ß-adrenergic antagonists 3H-DNA and 3H-CGP 12177 to intact rainbow trout (Salmo gairdneri) and carp (Cyprinus carpio) red blood cells. Gen. Comp. Endocrinol. 70,429 -435.[Medline]
Nickerson, J. G., Dugan, S. G., Drouin, G., Perry, S. F. and Moon, T. W. (2003). Activity of the unique ß-adrenergic Na+/H+ exchanger in trout erythrocytes in controlled by a novel ß3-AR subtype. Am. J. Physiol. 285,R526 -R535.
Nikinmaa, M. (1992). Membrane transport and
control of hemoglobin-oxygen affinity and nucleated erythrocytes.
Physiol. Rev. 72,301
-321.
Nikinmaa, M. and Jensen, F. B. (1986). Blood oxygen transport and acid-base status of stressed trout (Salmo gairdnerii): pre- and postbranchial values in winter fish. Comp. Biochem. Physiol. A 84,391 -396.[CrossRef]
Perry, S. F., Reid, S. G. and Salama, A.
(1996). The effects of repeated stress on the ß-adrenergic
response of the rainbow trout red blood cell. J. Exp.
Biol. 199,549
-562.
Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29,2002 -2007.
Phillips, M. C. L., Moyes, C. D. and Tufts, B. L.
(2000). The effects of cell ageing on metabolism in rainbow trout
(Oncorhynchus mykiss) red blood cells. J. Exp.
Biol. 203,1039
-1045.
Pickering, A. D. and Christie, P. (1981). Changes in the concentrations of plasma cortisol and thyroxine during sexual maturation of the hatchery--reared brown trout, Salmo trutta L. Gen. Comp. Endocrinol. 44,487 -496.[Medline]
Pickering, A. D., Pottinger, T. G. and Sumpter, J. P. (1987). On the use of dexamethasone to block the pituitary-interrenal axis in the brown trout, Salmo trutta L. Gen. Comp. Endocrinol. 65,346 -353.[Medline]
Pottinger, T. G. and Pickering, A. D. (1985). Changes in skin structure associated with elevated androgen levels in maturing male brown trout, Salmo trutta L. J. Fish Biol. 26,745 -753.
Pottinger, T. G. and Pickering, A. D. (1987). Androgen levels and erythrocytosis in maturing brown trout, Salmo trutta L. Fish Physiol. Biochem. 3, 121-126.
Pottinger, T. G. and Pickering, A. D. (1990). The effect of cortisol administration on hepatic and plasma estradiol-binding capacity in immature female rainbow trout (Oncorhynchus mykiss). Gen. Comp. Endocrinol. 80,264 -273.[Medline]
Primmett, D. R. N., Randall, D. J., Mazeaud, M. and Boutilier, R. G. (1986). The role of catecholamines in erythrocyte pH regulation and oxygen transport in rainbow trout (Salmo gairdneri) during exercise. J. Exp. Biol. 122,139 -148.[Abstract]
Reid, S. D. and Perry, S. F. (1991). The effects and physiological consequences of raised levels of cortisol on rainbow trout (Oncorhynchus mykiss) erythrocyte ß-adrenoceptors. J. Exp. Biol. 158,217 -240.[Abstract]
Robinzon, B. and Rogers, J. G., Jr (1979). The effect of gonadal and thyroidal hormones on the regulation of food intake and adiposity, and on various endocrine glands, in the red-winged blackbird (Agelaius phoeniceus). Gen. Comp. Endocrinol. 38,135 -147.[Medline]
Seamon, K. B., Padgett, W. L. and Daly, J. W. (1981). Forskolin: unique diterpene activator of adenylate cyclase in membranes and in intact cells. Proc. Natl. Acad. Sci. USA 78,3363 -3367.[Abstract]
Snyder, P. J., Peachey, H., Berlin, J. A., Hannoush, P., Haddad,
G., Dlewati, A., Santanna, J., Loh, L., Lenrow, D. A., Holmes, J. H. et
al. (2000). Effects of testosterone replacement in
hypogonadal men. J. Clin. Endocrin. Metab.
85,2670
-2677.
Speckner, W., Schindler, J. F. and Albers, C. (1989). Age-dependent changes in volume and haemoglobin content of erythrocytes in the carp (Cyprinus carpio). J. Exp. Biol. 141,133 -149.[Abstract]
Tetens, V., Lykkeboe, G. and Christensen, N. J. (1988). Potency of adrenaline and noradrenaline for ß-adrenergic proton extrusion from red cell of rainbow trout, Salmo gairdneri. J. Exp. Biol. 134,267 -280.[Abstract]
Thapliyal, J. P., Pati, A. K. and Gupta, B. B. PD. (1982). The role of erythropoietin, testosterone, an L-thyroxine in the tissue oxygen consumption and erythropoiesis of spotted munia, Lonchura punctulata. Gen. Comp. Endocrinol. 48,84 -88.[Medline]