Activation of the newly discovered cyclostome reninangiotensin system in the river lamprey Lampetra fluviatilis
1 School of Biological and Chemical Sciences, Hatherly Laboratories,
University of Exeter, Prince of Wales Road, Exeter, EX4 4PS, UK
2 Aquatic Biology Research Centre, Institute of Biology, University of
Southern Denmark Odense University, Hindsholmvej 11, DK-5300
Kerteminde, Denmark
* Author for correspondence (e-mail: J.A.Brown{at}exeter.ac.uk)
Accepted 27 October 2004
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Summary |
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Key words: reninangiotensin system, river lamprey, Lampetra fluviatilis, plasma angiotensin, salinity adaptation, volume regulation
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Introduction |
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In teleost fish there is evidence that the RAS plays important roles in
regulating blood pressure, controlling renal and cardiovascular function,
stimulating interrenal steroidogenesis and in determining drinking rates
(Brown et al., 1980;
Arnold-Reed and Balment, 1994
;
Tierney et al.,
1995a
,b
;
Bernier et al., 1999
;
Bernier and Perry, 1999
;
Butler and Zhang, 2001
).
For decades, the RAS was believed to have first evolved in bony fishes and
to be absent from both elasmobranchs and cyclostomes
(Nishimura et al., 1970;
Nishimura and Ogawa, 1973
;
Nishimura, 1985
;
Henderson et al., 1993
).
However, Ang I has now been isolated and sequenced in the elasmobranch
Triakis scyllia (Takei et al.,
1993
) and more recently in two cyclostomes: the sea lamprey
Petromyzon marinus (Takei et al.,
2004
) and the river lamprey Lampetra fluviatilis
(Rankin et al., 2004
). The Ang
II component in these lamprey species was identified as
Asn1,Val5-Ang II, as is found in most, but not all
teleosts (Hasegawa et al.,
1984
; Khosla et al.,
1985
; Conlon et al.,
1996
; Balment et al.,
2003
). Recognition of this similarity enabled our first
measurements of circulating angiotensin in cyclostomes, using commercial
antisera with high cross-reactivity to Asn1,Val5-Ang II
and Val5-Ang III (Rankin et
al., 2001
). This study showed higher plasma Ang II and Ang III
concentrations in river lampreys acclimated to seawater (SW) than in
freshwater (FW)-acclimated lampreys
(Rankin et al., 2001
). From
this it can be suggested that for anadromous lamprey species, migrating
between rivers and the marine environment during their life cycle
(Hardisty, 1979
), the RAS
could be involved in body fluid homeostasis as has been identified in teleost
fish (Olson, 1992
;
Kobayashi and Takei,
1996
).
There are similarities in the processes responsible for regulating body
fluid volume and composition of teleosts and lampreys with, for example,
hyper-osmoregulation in FW requiring excretion of large amounts of dilute
urine to eliminate the high osmotic water influx
(Brown et al., 1980;
Evans, 1993
). Also, in both
lampreys and teleosts, hypo-osmoregulation in more saline environments is
associated with an osmotic water loss that is balanced by drinking the
surrounding water, with fluid absorption in the gut, together with a drastic
reduction in urine output and renal adjustments to excrete high concentrations
of divalent ions (Logan et al.,
1980
; Evans, 1993
;
Rankin, 1997
,
2002
;
Brown and Rankin 1999
).
In teleosts and elasmobranchs there is evidence that the RAS is controlled
by volume receptors with, for example, release of renin and elevated
circulating angiotensin concentrations during hypovolaemia and/or reduction in
blood pressure (Nishimura et al.,
1979; Bernier et al.,
1999
; Anderson et al.,
2001
). However, the physiological signals responsible for
activation of the lamprey RAS are as yet unclear. We therefore undertook a
range of experimental manipulations of river lampreys followed by measurement
of circulating angiotensin levels to investigate the activation of the lamprey
RAS and to facilitate the development of hypotheses relating to its
physiological role. Specifically, we have examined: (1) whether blood removal
activates the lamprey RAS and hence increases circulating angiotensin
concentration; for these studies we used lampreys held in a hyperosmotic
environment to ensure that volume depletion could not be rapidly compensated
for by the natural osmotic influx of water; (2) the impact of extracellular
fluid expansion after intraperitoneal (i.p.) injection of a nominally
isosmotic saline; (3) whether changes in extracellular fluid osmolality
accompanying volume expansion influence the lamprey RAS; (4) how circulating
angiotensin concentration is affected after rapid changes in external salinity
from FW to a hyperosmotic medium (605 mOsm kg1) and from a
hyperosmotic medium (758 mOsm kg1) to FW.
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Materials and methods |
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Anaesthesia and blood sampling
River lampreys were anaesthetised by immersion in MS222 (3-amino-benzoic
acid ester methanesulfonate salt; Sigma, Poole, UK; 0.065 g
l1, as previously, Brown
and Rankin, 1999). Once anaesthetised, lampreys were weighed and
placed on their backs in individual Perspex troughs with their heads immersed
in aerated water containing anaesthetic and maintained at 10°C; the rest
of the body was covered with damp tissue. Respiratory movements were
monitored, and anaesthesia adjusted to maintain strong ventilation and stable
blood pressure and renal function (McVicar
and Rankin, 1983
; Brown and
Rankin, 1999
; Rankin et al.,
2004
). Blood samples were collected by needle puncture of the
caudal vein. Blood for measurement of circulating angiotensin concentrations
was collected into 100 µl of inhibitor solution (0.225 mol
l1 EDTA, 50 Kallikrein IU aprotinin and 0.05 mol
l1 1,10-phenanthroline; Sigma, UK) in chilled syringes
containing air-dried ammonium heparin (Sigma, UK). Samples were centrifuged
(20 000 g, 2 min at 4°C, Microcentrifuge, Ole Dich,
Hvidovre, Denmark), plasma removed and transferred into a fresh tube, and
tubes frozen in liquid nitrogen. Haematocrit (with inhibitor dilution) was
also determined in heparinised microhaematocrit tubes (Mikro 12-24 Hettich
Zentrifugen, Berlin, Germany; 13 000 g, 2 min) to enable
correction for the dilution of measured plasma angiotensin concentration by
the inhibitor solution; the dilution factor was determined from haematocrit
and total mass of blood sample. Immediately after collecting the first blood
sample, a further small sample (0.2 ml blood) was collected into a 1 ml
syringe coated with ammonium heparin (Sigma, UK). This sample was used for
determination of haematocrit (without inhibitor) as before, and plasma
osmolality (model 5520 Vapor Pressure Osmometer, Westcor Inc.). Plasma samples
for angiotensin analysis were held at 80°C until transport on
dry-ice to the University of Exeter, UK, where they were held at
80°C until extraction and radioimmunoassay of angiotensins.
Experimental series
Blood volume depletion
River lampreys (4291 g; N=30) acclimated to Kerteminde SW
(576 mOsm kg1; 21 p.p.t.) were anaesthetised in MS222 as
described above and an initial blood sample of 3.2% of body mass
(1.32.9 ml, calculated to reduce blood volume by 40%) was collected
from the caudal vein for analysis of plasma angiotensin and determination of
sample haematocrit (with inhibitor). A further 0.2 ml was taken immediately
for measurement of blood haematocrit (without inhibitor) and plasma
osmolality. For each lamprey, blood samples (approximately 1 ml) were
subsequently collected into inhibitor at 30 min (N=10), 60 min
(N=11) or 90 min (N=7) after the initial blood volume
depletion (one time point per lamprey) and used to measure plasma angiotensin
and haematocrit (in presence of inhibitor). Immediately after the allocated
sampling point a further 0.2 ml blood sample was collected to determine
haematocrit (without inhibitor) and plasma osmolality.
Isosmotic and hyperosmotic injections
In order to determine the effects of volume expansion on the RAS, with and
without salt loading, FW-acclimated river lampreys were lightly anaesthetised
and injected i.p. with 1% body mass by volume of either nominally isosmotic
saline or hyperosmotic saline, with control, non-injected lampreys run in
parallel.
FW-acclimated river lampreys (43101 g) were lightly anaesthetised as outlined above and weighed. Experimental lampreys were i.p.-injected at 1% by volume of body mass with either nominally isosmotic saline (120 mmol l1 NaCl; 233 mOsm kg1) or hyperosmotic saline (4 mol l1 NaCl). Blood samples were collected from each lamprey as described earlier for determination of plasma angiotensin, blood haematocrit and plasma osmolality. Samples were collected from separate lampreys 15 min after i.p. injection of hyperosmotic saline (N=8) or isosmotic saline (N=5) and 30 min after i.p. injection of hyperosmotic saline or isosmotic saline (N=5 in each group). Control non-injected lampreys from the same stock tanks were held under light anaesthesia until removal of a single blood sample after 15 min (N=8) or 30 min (N=5).
Acute changes in external salinity
The aquarium system used allowed changes in environmental salinity to be
rapidly achieved, avoiding the stress of removing the lampreys from the water.
This allowed investigations to determine how the lamprey RAS is affected
during the initial period after rapid shifts in external salinity. In the
first experiment, the initial FW (14 mOsm kg1) was altered
to a hyperosmotic medium (605 mOsm kg1); in a second
experiment hyperosmotic Kerteminde SW (758 mOsm kg1) was
rapidly replaced by FW to reach 22 mOsm kg1.
For the experiment involving transfer from FW to a hyperosmotic environment, river lampreys (4587 g) were held in a flow-through FW system (approx. 500 litres) for 2 weeks. At the start of the experiment (time zero), the tank FW volume was reduced to a depth of 10 cm (approx. 80 litres) and switched to a closed system with filtration for the 24 h prior to the start of the experiment. An initial group of lampreys (N=8) was removed, anaesthetised and blood samples collected. Kerteminde SW (salinity 25 p.p.t.) was pumped into the tank to increase the salinity from 0.5 p.p.t. to 20 p.p.t. within 6 min and achieve a peak salinity of 21 p.p.t. (605 mOsm kg1) within 20 min. Sub-groups of river lampreys (N=8 per group) were blood sampled at 1, 2, 4, 8 or 24 h from the start of SW addition in order to determine plasma osmolality, haematocrit and plasma angiotensin concentration, as described in the blood volume depletion experiments.
For the experiment involving transfer from a hyperosmotic environment to FW, river lampreys (4487 g) were held in a closed and filtered system of aerated Kerteminde SW (26 p.p.t., 758 mOsm kg1 at 10°C) for 3 weeks. 24 h before starting the experiment, the SW was reduced to a depth of approximately 10 cm. At the start of the experiment a group of lampreys (N=10) was removed from the experimental tank, anaesthetised, and blood samples collected as in other experiments. Salinity was lowered by rapid addition of FW to reach 1 p.p.t. (23 mOsm kg1) within 30 min and 0.6 p.p.t. at 1 h. Sub-groups of lampreys (N=8 per group) were blood sampled at 2 h, 4 h, 8 h and 24 h after the start of FW addition.
Extraction and radioimmunoassay of plasma angiotensin
Plasma angiotensin was extracted according to the method described by
Bernier et al. (1999). Briefly,
100 µl samples of river lamprey plasma held on ice at 4°C were each
mixed with 100 µl acidic acetone (acetone:water:1 mol l1
HCl, ratio 40:5:1) and vortexed vigorously for 1 min. The mixture was
centrifuged at 10 000 g for 10 min at 4°C. The supernatant
was collected and the pellet re-solubilised and re-extracted, as before.
Supernatants were combined, freeze dried under vacuum at 45°C
(Edwards EF4 Modulyo freeze dryer, Edwards High Vacuum, Crawley, UK) and
stored at 80°C until radioimmunoassay (RIA).
Prior to RIA, the extracted residues were resuspended in phosphate-buffered
saline (PBS, 400 µl at 0.01 mol l1, pH 7.4, containing
0.25% (w/v) bovine serum albumin fraction V RIA grade and 0.25% (v/v) Triton
X-100; Sigma, UK). Triplicate samples of these plasma extracts and
[Asn1,Val5]-Ang II standards (100 µl) were incubated
overnight at 4°C with 100 µl (8000 c.p.m.)
125I-[Ile5]-Ang II (74 Tbq mmol1,
2000 Ci mmol1; Amersham, UK) and 100 µl of
angiotensin antiserum (100 000x diluted in PBS). The heterologous
antiserum was initially raised against mammalian
[Asp1,Ile5]-Ang II
(Yamaguchi, 1981
). Serially
diluted extracted lamprey plasma ran parallel to the standard curve of
[Asn1,Val5]-Ang II, the native Ang II sequence in
lampreys (Fig. 1). The
antiserum used shows <0.5% cross-reactivity with Ang I from mammals,
teleosts and elasmobranchs but high cross-reactivity with Ang II from mammals
(100%), teleosts (6385%), elasmobranchs (74%) and lampreys (63%)
(Bernier et al., 1999
; Gary
Anderson, Gatty Marine Laboratory, University of St Andrews, UK, personal
communication). However, the antiserum, in common with most commercial Ang II
antisera, also shows high cross-reactivity (
90%) with mammalian Ang III
and Ang IV (G. Anderson, personal communication). Therefore, our RIA
measurements of angiotensin levels would incorporate Ang II, Ang III and Ang
IV.
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The following day, separation of free and bound angiotensins was achieved by addition of a 100 µl of a solid phase second antibody-coated cellulose suspension (anti-rabbit IgG serum; Sac-Cel IDS, Boldon, UK) and incubation at room temperature for 30 min. Distilled water (1 ml) was then added and tubes centrifuged (2500 g, 4 min, 4°C). Supernatants were aspirated and radioactivity of the pellets containing bound Ang II was determined (Packard Cobra Auto-gamma, B5002, Reading, UK). The angiotensin content of samples of river lamprey plasma was determined using a software package (RIASMART, Biosoft, Cambridge, UK). Final values of plasma angiotensin concentration were corrected for the calculated dilution of plasma samples by the inhibitor mix. This dilution was determined by calculation of the plasma volume in each sample based on the measured haematocrit and the gravimetric determination of the blood volume collected.
Estimated recovery of angiotensin through the extraction procedure and RIA was 78.5±5.7% for [Asn1,Val5]-Ang II and 105.7±3.7% for [Val5]-Ang III (N=10 in both cases). Intra-assay variability was 10.1% and 7.2% for Ang II at 9.8 pmol l1 and 78.1 pmol l1, respectively, and 7.3% and 10.6% for Ang III at identical concentrations (N=10 in all cases). The minimum detectable level of plasma angiotensin concentration was 11.7 pmol l1.
Statistical analyses
All data are presented as means or percentages of means ± standard
error (S.E.M.). All statistical analyses used SPSS version 10.0 for
Windows. All data were initially tested by the KolmogorovSmirnov test
to determine whether they were normally distributed. When data were normally
distributed or normality was achieved by transformations, further analyses
used analysis of variance (ANOVA), followed by post-hoc multiple
comparison tests (Tukey's HSD when data showed homogeneous variances after
Levene's tests, and Games Howell when variances differed significantly). When
transformations failed to achieve normality, data were analysed by a
non-parametric ANOVA for independent samples and MannWhitney
U-tests. Plasma angiotensin data after transfer of lampreys from FW
to higher environmental salinities showed significant differences between
groups (ANOVA) that could not be located by post-hoc multiple
comparison tests; data were therefore analysed by linear contrasts comparing
angiotensin concentrations at each time point after exposure to the higher
environmental salinity with angiotensin concentrations in FW-acclimated
lampreys at time 0 h. Significant differences between pre-blood volume
depletion and post-blood volume depletion data for plasma angiotensins,
osmolarity and haematocrit were established using paired t-tests.
Statistical differences between means were considered significant at
P<0.05.
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Results |
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A time course investigation of the changes in plasma angiotensin concentration after blood volume depletion showed that the plasma concentration of angiotensin had more than doubled after 30 min (Fig. 2C; P<0.01). Plasma angiotensin concentration remained significantly elevated in lampreys sampled 60 min after blood volume depletion (P<0.001) with a 62% increase compared to basal levels (Fig. 2C). After 90 min, there was no significant difference in plasma angiotensin concentration compared to that of basal samples taken to achieve the imposed blood volume depletion (Fig. 2C).
Isosmotic and hyperosmotic saline injection
The haematocrit of non-injected lampreys remained stable under the light
anaesthesia (Fig. 3A).
Injection with either of the saline solutions resulted in a pronounced
reduction in haematocrit with the mean values in non-injected controls of
38.7% and 41.8% at the two time points declining to mean values of
2026% in the four injected groups
(Fig. 3A). The reduction in
haematocrit was similar after injection of isoosmotic and hyperosmotic saline
and not significantly affected over the two sampling time points.
|
Injection of hyperosmotic saline significantly increased plasma osmolality (Fig. 3B). The nominally isosmotic saline (233 mOsm kg1) proved to be slightly hypo-osmotic in these lampreys, but not sufficiently so as to result in any change in plasma osmolality (Fig. 3B). The two experimental groups showed no differences over the two time points, but non-injected lampreys showed a slight (3.8%) decrease in plasma osmolality between the 15 min and 30 min sampling points.
Injection with the nominally isoosmotic saline resulted in a significant depression in the concentration of plasma angiotensin at 15 min (P<0.01; Fig. 3C), but plasma angiotensin concentration of lampreys injected with hyperosmotic saline was not significantly different from that of the non-injected controls both after 15 min and 30 min (Fig. 3C).
Acute changes in external salinity
The rapid increase in environmental salinity, rising from 14 to 605 mOsm
kg1 within 20 min, resulted in a significant increase in
plasma osmolality within 1 h (Fig.
4A). Blood haematocrit was not significantly altered by exposure
to high salinities (Fig. 4B).
Plasma angiotensin concentrations followed a visibly similar pattern to the
rising plasma osmolality (Fig.
4C) but there was a large variability in plasma angiotensin
concentration at all time points, particularly at time 0 h. Statistical
analysis by ANOVA indicated a significant change after exposure to higher
environmental salinity, but multiple comparison procedures did not locate a
significant difference between groups. However, linear contrasts of
angiotensin concentrations after exposure to higher salinity, compared to
control values in FW-acclimated lampreys, indicated a significant rise after 4
h (P=0.022), 8 h (P=0.005) and 24 h (P=0.004).
|
A rapid decrease in environmental salinity, from 758 to 22 mOsm kg1, significantly lowered the blood haematocrit and plasma osmolality in river lampreys within 4 h (Fig. 5A,B; P<0.05 and P<0.001, respectively). Plasma osmolality remained depressed at 8 h and 24 h but blood haematocrit was restored. Plasma angiotensin concentrations dropped steadily after the acute change in environmental salinity, but only reached a significantly lower value after 24 h (Fig. 5C).
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Discussion |
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In previous studies, transfer of river lampreys from FW to 503 mOsm
kg1 suggested a possible link between the circulating Ang II
concentration and rising plasma osmolality
(Rankin et al., 2001). The
present studies therefore examined more fully the changes in plasma osmolality
and plasma angiotensin concentrations after rapidly increasing external
salinity from FW to 605 mOsm kg1 and also after the reverse
manoeuvre, rapidly decreasing salinity from 758 mOsm kg1 to
FW. Lampreys exposed to these rapid changes in environmental salinity showed
good osmoregulation over the next 24 h, as shown previously
(Rankin, 2002
). After an acute
rise in external salinity, there was no significant change in blood
haematocrit, indicating limited, if any, volume depletion, but there was a
small though significant rise in plasma osmolality that was associated with a
similar pattern of change in plasma angiotensin concentration, with a
significant rise after 4 h, 8 h and 24 h suggested by the linear contrast
analyses of data at each time point compared to the initial level in FW. In
agreement with these trends, transfer from a hyperosmotic saline environment
to FW, resulted in a steady decline in plasma angiotensin concentration
alongside declining plasma osmolality. However, our data indicate that
relationships are complex since the largest (and significant) decline in
plasma angiotensin concentration occurred between 8 h and 24 h when plasma
osmolality was stable.
Movements between different environmental salinities are highly likely to
affect extracellular fluid volume as well as plasma osmolality, as in teleosts
(Olson, 1992). We therefore
examined the impact of both hypovolaemia and volume expansion. A severe
haemorrhage of 40% of a nominal blood volume of 8% of body mass was chosen to
test for stimulation of the RAS in SW-acclimated lampreys. Another lamprey
species (Petromyzon marinus) was estimated to have a blood volume of
8.5% of body mass, based on the volume of distribution of Evan's Blue
(Thorson, 1959
). This
technique is inaccurate in teleost fish
(Olson, 1992
), but in two
Petromyzon marinus obtained from Ringkøbing Fjord, Denmark,
the more accurate technique involving re-injection of red blood cells labelled
with 51Cr (Olson,
1992
), gave values of 8.3% and 8.1% of body mass (J. C. Rankin,
unpublished observations).
SW-acclimated lampreys are faced with the need to continually drink their
environmental medium in order to regulate body fluid volume, but anaesthesia
has been found to block drinking (Rankin,
1997). Therefore, in the hypovolaemia experiments the lampreys
would not have been able to increase drinking in order to restore blood
volume. Based on expected drinking rates in SW-acclimated lampreys, an
inhibition of drinking would have resulted in a further volume loss of 4.8 ml
h1 kg1 body mass that could not be
absorbed from the gut to replace branchial and renal losses
(Rankin, 2002
). Within the 90
min experimental period after the imposed blood volume depletion this would
have resulted in a further 4.5% hypovolaemia.
Hypovolaemia, induced by removal of an estimated 40% of the blood volume,
in river lampreys held in hyperosmotic media, would potentially cause an
immediate drop in blood pressure, as in teleost fish (e.g.
Nishimura et al., 1979). This
could cause a decrease in kidney perfusion pressure, releasing renin. However,
as yet, there is no evidence for renin-releasing granular epithelioid cells in
the lamprey kidney or measurement of plasma renin concentration in cyclostomes
(Henderson et al., 1993
;
Kobayashi and Takei, 1996
).
Nevertheless, the more than twofold increase in plasma angiotensin
concentration observed 30 min after hypovolaemia suggests that pressure/volume
receptors play an important role in activating the lamprey RAS, as has been
reported in both teleosts and elasmobranchs
(Bailey and Randall, 1981
;
Nishimura et al., 1979
;
Nishimura and Bailey, 1982
;
Galli and Phillips, 1996
;
Bernier et al., 1999
).
Hypotension following haemorrhage will result in fluid influx to the blood
from the interstitial fluid due to the oncotic pressure of plasma proteins. In
lampreys, which lack a spleen (and a secondary circulation, as is found in
teleost fish), reductions in haematocrit may quantitatively reflect such
haemodilution. If so, the resultant falls in haematocrit (by 36.7%, 36.3% and
40.3% after 30, 60 and 90 min, respectively) indicate that the intention of
reducing blood volume by 40%, had indeed been achieved. The initial transient
reduction in blood volume (prior to compensatory fluid influx) can be
predicted to have triggered a rapid activation of the RAS and plasma
angiotensin concentration could well have peaked before the 30 min sample was
taken.
The demonstration of pressure/volume-sensitive regulation in the lamprey
RAS is perhaps indicative of the most ancient role of the RAS in blood
pressure regulation, which has remained a fundamental feature throughout
vertebrate evolution. The most immediate effect of elevated levels of
circulating angiotensin after blood volume depletion is likely to be
vasoconstriction, which would serve to restore blood pressure. The mean
circulating concentration of angiotensin that we measured at 30 min after
hypovolaemia was ca. 1 nmol l1 (1054.5±203.5 pmol
l1, N=10). Injection of 1 nmol
kg1 body mass angiotensin II produced a significant (6 mmHg)
increase in dorsal aortic blood pressure in the river lamprey
(Rankin et al., 2004).
Although this would have rapidly produced a concentration of about 12 nmol
l1 if distributed only in the blood volume, pressure effects
were observed with lower doses and initial angiotensin concentrations
following blood volume depletion could well have been much higher than 1 nmol
l1.
In the present study, plasma angiotensin concentrations recovered within
1.5 h of blood volume depletion. Our measurements of the declining
concentrations of plasma angiotensin after an infusion of Ang II have shown
that the half-life of angiotensin in lampreys is approximately 16 min (J. A.
Brown, S. C. Frankling, C. S. Cobb and J. C. Rankin, unpublished data) and
thus if RAS activation after volume depletion is a short-lived event, basal
levels could be achieved within 90 min of volume depletion. Pressure effects
would be very rapidly achieved (Rankin et
al., 2004
) and a restoration of blood pressure would be likely to
inhibit further angiotensin formation. A further part of the recovery, at
least in FW-acclimated lampreys, could involve a reduction in urine flow rates
initiated by angiotensin, as has been shown to occur in teleosts
(Brown and Balment, 1997
;
Brown et al., 2000
). Studies
are required to describe fully the renal actions of the lamprey RAS, but
Asn1,Val5-Ang II at 1010 or
109 mol min1 kg1 body
mass has been shown to reduce urine flow rates of FW-acclimated Lampetra
fluviatilis and Petromyzon marinus (J. A. Brown, C. S. Cobb and
J. C. Rankin, unpublished data). However, in the blood volume depletion
experiments, lampreys were acclimated to hyperosmotic SW and urine flows would
already have been minimal (McVicar and
Rankin, 1983
; Brown and
Rankin, 1999
).
In addition to examining the effects of blood volume depletion, we explored
the effect of i.p. injection of nominally isosmotic saline on plasma
angiotensin concentrations with the aim of inducing extracellular fluid
expansion, with non-injected FW lampreys as controls. Anaesthesia in the
control lampreys resulted in a slight reduction in plasma osmolality between
the 15 and 30 min sample times, perhaps reflecting some water retention. This
could be the result of a slight decrease in blood pressure that would
influence individual nephron filtration rates
(McVicar and Rankin, 1985) and
hence reduce urine flow (Brown and Rankin,
1999
). This argument is questionable, however, as lampreys given
approximately isosmotic saline (actually slightly hyposmotic), showed no
evidence of any change in plasma osmolality. The i.p. injection of isosmotic
saline resulted in a significant decline in plasma angiotensins compared to
the level in non-injected FW-acclimated river lampreys at 15 min after the
injection, and supports our hypothesis that volume/pressure receptors are
important in regulating the lamprey RAS. Similar findings were reported in the
Australian lungfish Neoceratodus forsteri, in which plasma renin
activity was significantly lower after injection of isosmotic saline, although
plasma angiotensin concentration was not measured in this study
(Blair-West et al., 1977
). Our
results suggest that the i.p. injection of approximately isosmotic saline led
to expansion of the extracellular fluid volume, lowering blood haematocrit by
35% (at 15 min), and hence inhibited the RAS, whereas stimulation of the
lamprey RAS occurred after volume depletion.
Although a significant reduction in plasma angiotensin concentration was induced by i.p. injection of nominally isosmotic saline, this did not occur after injection of lampreys with a similar volume of hyperosmotic saline. It is arguable that i.p. injection of hyperosmotic saline might stimulate initial withdrawal of extracellular fluid into the peritoneal cavity and that reduced plasma volume may account for the absence of any reduction in plasma angiotensin concentration. However, there was no evidence of any increase in haematocrit that would have accompanied fluid redistribution into the peritoneal cavity. After both isosmotic and hyperosmotic saline injections, haematocrit was dramatically reduced compared to the haematocrit of non-injected lampreys and did not differ significantly between lampreys injected with isosmotic saline and hyperosmotic saline. Therefore, it would appear that both groups of lampreys were exposed to a volume expansion. However, declining plasma angiotensin concentration was not seen in lampreys i.p.-injected with hyperosmotic saline. The injection of hyperosmotic saline significantly raised plasma osmolality within 15 min and this suggests that osmoreceptors may either directly activate the lamprey RAS, or inhibit the impact of volume/pressure receptors. These results are in agreement with the longer-term increase in plasma angiotensin concentration that accompanied the rise in plasma osmolality after exposure to increased environmental salinity.
In mammals, renin has been suggested to be the major rate-limiting
component of the RAS and a complex array of mechanisms involving renal nerve
stimulation, baroreceptors and the macula densa interact to control renin
release (Kobayashi and Takei,
1996; Nishimura,
2004
; Peti-Peterdi et al.,
2004
). In non-mammalian vertebrates, the control of renin release
is still poorly understood (Henderson et
al., 1993
; Kobayashi and
Takei, 1996
; Nishimura,
2004
) and few studies have investigated the impact of
extracellular osmolality on renin release. In the fowl, infusion of hypertonic
saline into the kidney (via the renal portal system) and intravenous
injection of hypertonic saline depress plasma renin release and plasma
angiotensin, respectively (Nishimura and
Bailey, 1982
; Kobayashi and
Takei, 1996
). This inhibition of renin release by high osmolality
agrees with the results in mammalian studies, suggesting an inverse
relationship between plasma sodium/osmolality and the number of renin cells,
renin release and plasma renin activity
(Kobayashi and Takei, 1996
;
Peti-Peterdi et al., 2004
).
Our results show an opposite pattern in lampreys, since high plasma
osmolalities were associated with high plasma angiotensin concentrations in
both experiments involving changes in external osmolality. Furthermore, while
plasma angiotensin concentration declined after injection of lampreys with
nominally isosmotic saline, similar changes did not occur after the
hyperosmotic saline injection. Stimulation of the RAS after injection of
hyperosmotic saline was also reported for the eel (reviewed by
Kobayashi and Takei, 1996
), so
this may be a feature of lower vertebrates. However, we cannot assume that
renin was released in either the study of eels or in the present study of
lampreys, since plasma renin activity was not determined in either study. Even
if plasma renin activity was known, this may not be the only rate-limiting
step in the lamprey RAS. In mammals, secretion of angiotensinogen has a
rate-limiting effect on maximal formation of plasma Ang I and ultimately, the
production of Ang II (Klett and Granger,
2001
). Furthermore, our recent studies of the rainbow trout
exposed to an osmotic stress have shown increased hepatic angiotensinogen mRNA
(R. K. Paley, J. G. Aust, S. J. Aves and J. A. Brown, unpublished
observations; Aust, 2002
). This
suggests that regulation of hepatic angiotensinogen secretion plays a
significant role in producing the reported elevations in circulating
angiotensin levels and adaptation of teleost fish to hyperosmotic media.
However, as yet we have no information on the regulation of lamprey
angiotensinogen secretion.
In conclusion, our results indicate that volume receptors exert control on
the lamprey RAS, but these receptors may be modulated by
sodium/chloride/osmo-sensitive receptors and circulating angiotensin levels
will be determined by the interaction of the putative volume and osmo/salt
receptors and their relative sensitivities. However, the predictable changes
in plasma volume and electrolytes when anadromous lampreys migrate between FW
and SW would act as complementary signals in activating the RAS in
hyperosmotic environments and inhibition of the RAS in FW. While feeding on
fish during the marine phase of their life cycle, lampreys may face imposed
changes on extracellular fluid volume and osmolarity that impact on the
functioning of the RAS. For example, river lampreys caught in
Ringkøbing Fjord, Denmark, just as they begin their upstream migration,
have been found with intestines distended with blood representing up to 17% of
their body mass (Rankin,
1997). This gut distension when feeding on teleosts may result in
a rapid isosmotic volume load, a potential inhibitory signal to the RAS. In
contrast, sea lampreys (Petromyzon marinus) may meet simultaneous
volume and hyperosmotic challenges when feeding on marine sharks that have
body fluids roughly isosmotic to seawater
(Wilkie et al., 2004
).
Although undoubtedly difficult to achieve, investigations exploring the
potential regulatory role of endocrine systems such as the RAS during the
parasitic feeding of lampreys, would be extremely valuable.
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Acknowledgments |
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Footnotes |
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Present address: Department of Chemical and Biological Sciences, University
of Huddersfield, Queensgate, Huddersfield, HD1 3DH, UK
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References |
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