A Ca2+-sensing receptor modulates shark rectal gland function
1 Department of Cell and Molecular Physiology, University of North Carolina
at Chapel Hill, Chapel Hill, NC 27599, USA
2 Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672,
USA
* Author for correspondence (e-mail: sfellner{at}med.unc.edu )
Accepted 17 April 2002
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Summary |
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Key words: dogfish, Squalus acanthias, norepinephrine, procaine, cyclic AMP, ryanodine, rectal gland
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Introduction |
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Less well studied is the relationship between blood flow to the rectal
gland and secretory activity. Blood flow to the artery of the rectal gland
(RGA) in conscious sharks varies from less than 1 % in some animals to 2-7 %
of the cardiac output in others, suggesting a pattern of intermittent blood
flow (Kent and Olson, 1982).
Such variability may reflect changes in extracellular fluid volume and the
effect of hormonal influence on vascular tone during feeding cycles.
Shuttleworth has shown that
-adrenergic catecholamines cause
vasoconstriction of the RGA and that agents known to stimulate gland secretion
of salt, such as cyclic AMP (cAMP) and vasoactive intestinal peptide,
abolished the vasoconstriction in Squalus acanthias
(Shuttleworth, 1983
).
Subsequent studies in the shark, Scyliorhinus canicula L., perfused
at in vivo pressures, showed that reducing perfusion to one third of
control values caused a marked decline in Na+ excretion by the
gland (Shuttleworth and Thompson,
1986
).
To better delineate the relationships between catecholamines and substances
that stimulate secretion in the rectal gland tubule (RGT) but potentially
dilate the RGA [e.g. vasoactive intestinal peptide, cardiac naturiuretic
peptide (CNP)], we undertook an examination of calcium signaling pathways in
both tissues. Whereas cytosolic calcium ([Ca2+]i) of
mammalian vascular smooth muscle cells is relatively insensitive to changes in
extracellular Ca2+ levels ([Ca2+]e)
(Fellner and Arendshorst,
2000; Champigneulle et al.,
1997
), we found that both RGA and RGT responded in a
concentration-dependent fashion to changes in [Ca2+]e.
Our preliminary studies appeared to exclude voltage-gated L-type channels or
Na+/Ca2+ exchange operating in the reverse direction as
pathways for the dependency of [Ca2+]i on
[Ca2+]e (Fellner and Parker,
2001a
,
b
).
Prior studies in isolated perfused rectal gland tubules showed that
carbachol and agents that stimulate cAMP production increased
[Ca2+]i by both Ca2+ mobilization and entry,
by enhancing the basolateral K+ conductance. Blockade of
voltage-gated L-type channels with nifedipine did not inhibit cAMP-mediated
Ca2+ influx (Warth et al.,
1998).
A calcium sensing receptor (CaSR) has been described in mammalian kidney
tubules, parathyroid and bone cells, dorsal root ganglion cells and other
neural tissues (Bukoski et al.,
1997; Brown et al.,
2001
). Only one example of a CaSR in vascular smooth muscle cells
has been reported, in the spiral modiolar artery of the cochlea of the gerbil
(Wonneberger et al., 2000
). We
postulated that a CaSR might be responsible for the calcium sensitivity we
observed in RGA and RGT of Squalus acanthias.
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Materials and Methods |
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These protocols were approved by the IACUC at Mount Desert Island Biological Laboratory.
RGA and RGT were loaded with the calcium-sensitive fluorescent dye
fura-2AM, and [Ca2+]i was measured as previously
described (Fellner and Arendshorst,
1999,
2000
). RGA or RGT were placed
in an open static chamber and examined in a small window of the optical field
of a x40 oil-immersion fluorescence objective of an inverted microscope
(Olympus IX70). The tissue was excited alternately with light of 340 and 380
nm wavelengths from a dual-excitation wavelength Delta-Scan equipped with dual
monochronometers and a chopper [Photon Technology International (PTI), NJ,
USA]. After passing signals through a barrier filter (510 nm), fluorescence
was detected by a photomultiplier tube. The calibration of
[Ca2+]i was based on the signal ratio at 340/380 nm and
known concentrations of calcium
(Grynkiewicz et al.,
1985
).
Reagents
Nifedipine, GdCl3, spermine, 3,4,5-trimethoxybenzoic
acid-8-(diethylamino)octyl ester (TMB-8), TMAO and 2-amino-ethoxydiphenyl
borane (2-APB) were purchased from Sigma (St Louis, MO, USA), thapsigargin,
VIP, CNP and ryanodine from Cal Biochem (La Jolla, CA, USA) and fura-2-AM from
Teflab (Austin, TX, USA).
Statistics
The data are presented as means ± S.E.M. Each data set is derived
from tissue originating from at least three separate experimental days. Paired
data sets were tested with Student's paired t-test. Multiple
comparisons were analyzed using one-way analysis of variance (ANOVA) for
repeated measures followed by StudentNeumanKeuls post
hoc test. P<0.05 was considered statistically
significant.
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Results |
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RGA response to extracellular calcium
To test the response of RGA (in nominally calcium-free buffer) to graded
concentrations of extracellular Ca2+, we added CaCl2 in
shark Ringer's solution to the chamber. The concentrationresponse curve
(Fig. 1) shows that there is a
stepwise increase in [Ca2+]i with each increment of
extracellular Ca2+ from 0.8 to 5.3 mmol l-1. We chose
this range of extracellular Ca2+ to be within approximately
±2 mmol l-1 of the physiological
[Ca2+]e of S. acanthias (2.5 mmol
l-1). Every data point is part of a pair, that is, at least two
concentrations of calcium were tested on any one RGA sample. Within this
concentration range, there is a linear correlation of
[Ca2+]i with changes in [Ca2+]e
(r2=0.51, P<0.01).
|
To assess calcium entry via voltage-gated L-type channels, we
employed the dihydropyridine, nifedipine. Utilizing concentrations of
nifedipine known to block L-channels
(Fellner and Arendshorst,
1999), we tested the response of RGA to external calcium (2.5 mmol
l-1) either with pre- or post-treatment with nifedipine
(3x10-6 mol l-1).
Fig. 2 shows that L-channel
blockade did not have statistically significant effects on the response to the
addition of calcium (2.5 mmol l-1) (N=8 pre; N=9,
post).
|
Data to support presence of CaSR in RGA
Agonist stimulation
A variety of cations act as agonists for the CaSR. Gadolinium, which is an
inhibitor of store-operated (or capacitative) calcium entry (SOC) at
concentrations of 1-3 µmol l-1
(Broad et al., 1999),
stimulates the mammalian CaSR by 50 % (EC50) at 35 µmol
l-1 (Brown et al.,
1993
). We conducted studies of RGA in nominally calcium-free
buffer to which we sequentially added Ca2+ (3 mmol l-1)
and Gd3+ (333 µmol l-1). The baseline
[Ca2+]i of 165±26 nmol l-1 increased
to 263±27 nmol l-1 following addition of Ca2+ to
the buffer (P<0.01) and to 314±23 nmol l-1 after
subsequent addition of Gd3+ (P<0.05) (N=10;
Fig. 3A). These data suggest
that Gd3+ stimulates additional mobilization of Ca2+
from the sarcoplasmic reticulum (vide infra).
|
Spermine and other polyamines may also serve as agonists for the CaSR
(Quinn et al., 1997).
Fig. 3B is a representative
response of RGA in Ca2+-free buffer to the addition of spermine
(333 µmol l-1). The mean increase in
[Ca2+]i following the addition of spermine was
133±21 nmol l-1 (N=5, P<0.01).
Calcium signaling mechanisms
Agonist activation of a CaSR stimulates mobilization of Ca2+
from the endoplasmic (ER) or sarcoplasmic reticulum (SR) followed by
Ca2+ entry through non-voltage-gated calcium channels
(Nemeth and Scarpa, 1987). The
majority of CaSRs identified currently are linked to G-protein-coupled
activation of phospholipase C (Brown et
al., 1993
; McNeil et al.,
1998
), with the generation of inositol trisphosphate
(IP3) and diacylglycerol. IP3 activation of the
IP3 receptor on the ER causes release of Ca2+ into the
cytosol. The resulting depletion of ER Ca2+ then stimulates SOC
(Putney, 1990
). It follows,
therefore, that any agent which depletes the ER of Ca2+ should
diminish the response achieved by activation of the CaSR. Accordingly, we
chose two pathways of depleting SR/ER calcium that are independent of the
IP3 pathway, namely inhibition of the SR/ER Ca2+-ATPase
with thapsigargin and stimulation of the ryanodine-sensitive receptor
(Fellner and Arendshorst,
2000
).
Stimulation of RGA in Ca2+-free buffer with ryanodine (3 µmol
l-1), a concentration known to activate the ryanodine receptor
(Fellner and Arendshorst,
2000), mobilized Ca2+ from the SR and increased
[Ca2+]i by 64±15 nmol l-1
(N=7), demonstrating that the shark RGA has a ryanodine-sensitive
receptor that, when stimulated, promotes the release of Ca2+ from
the SR into the cytosol. Subsequent addition of external calcium (3.3 mmol
l-1) further increased [Ca2+]i by 55±9
nmol l-1 (N=8), which is approximately half the response
we measured in the absence of ryanodine (N=11, P<0.01).
Similar experiments were performed using thapsigargin (10-6 mol
l-1). Inhibition of Ca2+ reentry into the SR with
thapsigargin caused an increase in [Ca2+]i of
69±8 nmol l-1; subsequent addition of calcium to the buffer
further increased [Ca2+]i by 44±8 nmol
l-1 (N=8, P<0.01 versus response to
Ca2+ without thapsigargin). Thus, using two separate methods of
depleting SR Ca2+, the response to external calcium was blunted.
That the response to external calcium was not completely obliterated suggests
that ryanodine and thapsigargin did not totally empty the calcium stores of
RGA SR during the 100 s time period. Fig.
4 compares representative responses of RGA in calcium-free buffer
to calcium alone or to calcium in the presence of ryanodine or
thapsigargin.
|
The compound 2-APB has been shown to inhibit the IP3 receptor
and, as well, the SOC channel independent of IP3
(Missiaen et al., 2001;
Ma et al., 2001
;
Gregory et al., 2001
). It has
no known effect on Na2+/Ca2+ exchange or calcium entry
via L-channels. Because the CaSR stimulates the generation of
IP3 with subsequent mobilization of Ca2+ from the SR/ER
and then activation of SOC, 2-APB should inhibit its activity. In experiments
in which RGA was stimulated with Ca2+ with or without ryanodine,
thapsigargin or Gd3+, 2-APB (100 µmol l-1)
consistently diminished the response by approximately 50 % (N=8,
P<0.01). A representative example of the effect of 2-APB on the
response of RGA to the addition of external Ca2+ is illustrated in
Fig. 5.
|
RGA and Ni2+
Because Ni2+ (1 µmol l-1) constricts ventral
aortic rings of the dogfish shark (Evans
et al., 1993) and has been shown to be an agonist for the CaSR in
some mammalian tissues at concentrations of 0.5-5.0 mmol l-1 or higher
(Adebanjo et al., 1998
), we did
experiments to test the hypothesis that a CaSR sensitive to Ni2+ is
present in RGA. Ni2+ (2-3 mmol l-1) is an inhibitor of
SOC and of Na2+/Ca2+ exchange. Thus, the choice of the
concentration of Ni2+ employed is important. RGA in nominally
calcium-free medium responded to Ni2+ (1.0 mmol l-1),
increasing the baseline [Ca2+]i of 83±14
nmoll-1 to 109±20 nmoll-1, suggesting that
Ni2+ promoted mobilization of Ca2+ from the SR.
Subsequent addition of external calcium (in the presence of 0.7
mmoll-1 Ni2+) further elevated
[Ca2+]i to 253±43 nmoll-1
(N=6, P<0.01 for both comparisons;
Fig. 6A). Ni2+ given
after Ca2+ to RGA previously maintained in calcium-free buffer
could either further stimulate Ca2+ mobilization or could inhibit
SOC. When Ni2+ (1.5 mmoll-1) was added after stimulation
with calcium, there was a fall in [Ca2+]i, suggesting
that inhibition of SOC blunted the calcium entry component of the rise in
[Ca2+]i. Baseline [Ca2+]i of
94±11 nmoll-1 rose to 298±29 nmoll-1
following the addition of calcium; subsequent addition of Ni2+
caused a fall in [Ca2+]i to 158±14
nmoll-1 (N=9, P<0.01) A representative example
is illustrated in Fig. 6B.
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RGA and procaine
Studies of mesenteric arteries of the rat have suggested that activation of
a perivascular sensory nerve CaSR causes Ca2+-induced relaxation of
the vessel (Bukoski et al.,
1997). Accordingly, we performed experiments in which procaine (5
mmoll-1) was added to the bath in order to inhibit sensory nerve
function. There was no difference in the [Ca2+]i
response of RGA in calcium-free Ringer's solution to 2.5 mmoll-1
external calcium (136±27 nmoll-1, N=6) in the
presence of procaine compared to the response without it (110±13
nmoll-1), suggesting that the CaSR was not located in the
perivascular nerve network.
RGT and the CaSR
Agonists
Experiments similar to those conducted with RGA were performed with RGT to
investigate the possibility that the rectal gland might respond to agonists of
the CaSR as well. Tubules in nominally calcium-free Ringer responded to the
addition of calcium (5 mmoll-1) to the medium with a rapid increase
in [Ca2+]i of 250±53 nmoll-1
(N=7). Addition of spermine (333 µmoll-1), a known
agonist of the CaSR (Quinn et al.,
1997), caused a further increase in [Ca2+]i
of 235±94 nmoll-1 (P<0.01 for both comparisons,
Fig. 7A).
|
Fig. 7B is a representative example of the effect of spermine added after the response of RGT to external calcium. As shown in Fig. 7C, RGT, like RGA, responded to incremental increases in external Ca2+ with a stepwise increase in [Ca2+]i and responded to Gd3+ with a further rise in [Ca2+]i. Group data are illustrated in Fig. 7A. When calcium (5 mmoll-1) was added to tubules in normal calcium Ringer (2.5 mmoll-1), [Ca2+]i increased by 162 nmoll-1 (N=10, P<0.01). Gd3+ further increased [Ca2+]i by 60±21 nmoll-1 (P<0.05). The increment in [Ca2+]i produced by the addition of Gd3+ is considerably less than that achieved by spermine (235±94 nmoll-1, P<0.01), which may be a consequence of the inhibitory effect of Gd3+ on calcium entry via SOC.
RGT and TMB-8
To assess the participation of IP3-mediated calcium release from
the ER of RGT, we used the drug TMB-8 (10-6 moll-1),
which inhibits the IP3 receptor
(Palade et al., 1989;
Salomonsson and Arendshorst,
1999
). Tubules were prepared in calcium-free buffer with or
without the addition of TMB-8. The increment in [Ca2+]i
when Ca2+ (5 mmoll-1) was added was 47±10
nmoll-1 (N=7) in the TMB-8 group and 118±24
nmoll-1 in the control group (N=18, P<0.01),
demonstrating that inhibition of the IP3 receptor blunted the
response of RGT to added external calcium (data not shown).
RGT and thapsigargin
In a manner similar to that performed in RGA, we prepared tubules in
calcium-free Ringer's solution, and added thapsigargin (10-6
mmoll-1) for 100 s before adding external Ca2+ (3.3
mmoll-1). The increment in [Ca2+]i after
thapsigargin was 108±23 (N=5, P<0.01).
Fig. 8 illustrates a typical
experiment in which calcium was added after thapsigargin. In contrast, in the
absence of thapsigargin, the addition of Ca2+ to cells in
calcium-free buffer increased [Ca2+]i by 203±10
nmoll-1 (N=17) (not shown). This nearly 50 % reduction in
response is similar to our findings in RGA with thapsigargin and ryanodine.
Because cyclic ADP-ribose, which activates ryanodine receptors, has not been
found in renal tubules (Chini et al.,
1997), we chose not to study ryanodine in RGT.
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Discussion |
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The RGA arises from the posterior mesenteric artery of S.
acanthias. Studies of the anterior mesenteric artery show that
acetylcholine, endothelin-1 (ETB) and nitric oxide constrict and
that cardiac naturiuretic peptide, prostaglandin E and carbaprostacyclin
dilate the artery (Evans,
2001). A band of smooth muscle in the periphery of the rectal
gland constricts in response to endothelin and nitric oxide and dilates
following treatment with cardiac naturiuretic peptide, suggesting that the
dimensions of the entire rectal gland may play a role in its secretory
function (Evans and Piemarini,
2001
). Because we dissected the capsule and outer layer of tubules
from the gland prior to analysis and inspected the tubules microscopically
before measuring [Ca2+]i, we are confident that smooth muscle
tissue was not present in our samples.
Our initial studies explored pathways of calcium signaling in RGA and RGT
(Fellner and Parker,
2001a,b
).
Whereas [Ca2+]e of mammalian vascular smooth muscle cells, and also
many epithelial cells, is relatively resistant to changes in
[Ca2+]e (Fellner and
Arendshorst, 2000
;
Champigneulle et al., 1997
), we
were surprised to find that both RGA and RGT responded to changes in
[Ca2+]e with an increase in [Ca2+]i. Furthermore, these
studies appeared to exclude voltage-gated L-type channels or
Na+/Ca2+ exchange operating in the reverse direction as
pathways for the dependency of [Ca2+]i on [Ca2+]e. Thus,
exploration of the possibility of the presence of a CaSR on RGA and RGT was
the aim of the present study.
Cloning and characterization of a CaSR from bovine parathyroid cells was
achieved in 1993 (Brown et al.,
1993); subsequently the CaSR was identified on cell membranes of
tissues involved in the regulation of [Ca2+]e such as kidney, bone
and intestine. The receptor is also involved in the activation of some ion
channels, control of hormonal secretion, apoptosis and cell proliferation
(Brown et al., 2001
). A single
study in vascular smooth muscle suggests that the CaSR functions as a
modulator of vasoconstriction in the spiral modiolar artery of the cochlea of
the gerbil (Wonneberger et al.,
2000
). A number of cations (Gd3+, Ni2+,
Pb2+) are agonists for the CaSR
(Wonneberger et al., 2000
;
Chattopadhyay, 2000
;
Handlogten et al., 2001
; Quinn
et al., 1998), as are polyamines (Quinn et
al., 1997
) and aromatic and L-amino acids
(Conigrave et al., 2000
).
There is an inverse relationship between ionic strength and activity of the
CaSR. Reduction in ionic strength enhances sensitivity of the receptor and an
increase in ionic strength inhibits its activity (Quinn et al., 1998). This
latter characteristic of the CaSR may be its major function in Squalus
acanthias.
Our studies of both the RGA and RGT of the dogfish shark are entirely
compatible with these unique characteristics of the CaSR. (1) We have shown
that there is a concentrationresponse relationship between changes in
[Ca2+]e and [Ca2+]i. (2) Spermine activated the receptor
of RGT and RGA, increasing [Ca2+]i. (3) Gadolinium, which is an
irreversible inhibitor of SOC at concentrations of 1-3 µmol l-1
(Broad et al., 1999), modestly
increased [Ca2+]i in both RGA and RGT. The response to
Gd3+ was considerably less than that achieved by spermine, which
suggests that although Gd3+ was able to initiate
IP3-directed mobilization of Ca2+ from the SR/ER, SOC
that would have occurred as a consequence of depletion of SR/ER calcium stores
was inhibited by the presence of Gd3+. (4) Nickel, an inhibitor of
Na2+/Ca2+ exchange in millimolar concentrations and also
an inhibitor of SOC (Sarosi et al.,
1998
), is an agonist for the CaSR (at concentrations of 0.5-5.0
mmol l-1) (Adebanjo et al.,
1998
). We found that RGA in nominally calcium-free Ringer's
solution responded to Ni2+ (1.0 mmol l-1) with
mobilization of [Ca2+]i from the SR. Subsequent addition of
external calcium (in the presence of 0.7 mmol l-1 Ni2+)
further increased [Ca2+]i. When Ni2+ was added after
calcium, there was inhibition of the [Ca2+]i response, presumably
because calcium entry via SOC was blocked.
The CaSR can operate via pertussis toxin-sensitive or -
insensitive G proteins to activate phospholipase C, A or D, depending on the
milieu of the cell in which it is expressed
(Brown et al., 2001). Thus,
both Gi and Gq proteins may be involved in the signaling pathways of the CaSR.
Agonist activation of the CaSR stimulates mobilization of Ca2+ from
the SR/ER followed by Ca2+ entry through non-voltage-gated calcium
channels (Nemeth and Scarpa,
1987
; Wonneberger et al.,
2000
). We did not find evidence for CaSR-mediated L-channel
calcium entry in the present study.
The majority of CaSRs identified currently are linked to G-protein coupled
activation of phospholipase C (Brown et
al., 1993; McNeil et al.,
1998
) with the generation of IP3 and diacylglycerol.
IP3 activation of the IP3 receptor on the SR/ER causes
release of Ca2+ into the cytosol. To assess the participation of
IP3 generation on the response of RGA and RGT to stimulation with
[Ca2+]e, we utilized the compound TMB-8, which inhibits the
IP3 receptor (Palade et al.,
1989
, Salomosson and
Arendshorst, 1999
), or 2-APB. The cell-permeant compound 2-APB
blocks the IP3 receptor in a variety of cell types
(Missiaen et al., 2001
;
Ma et al., 2001
) and also
inhibits the SOC channel, independent of IP3
(Gregory et al., 2001
). Both
TMB-8 (RGT) and 2-APB (RGA) diminished the [Ca2+]i response to
external calcium by approximately 50%.
In addition, depletion of SR/ER calcium via non-IP3
mechanisms should diminish the global response of RGT or RGA to external
calcium because of inhibition of SOC. We employed ryanodine to stimulate the
SR ryanodine-sensitive receptor and thapsigargin to inhibit reuptake of
calcium into the SR by blocking the Ca2+-ATPase (Fellner and
Arendshorst, 1999,
2000
). Both ryanodine and
thapsigargin diminished the cytosolic response of RGA to external calcium by
approximately 50%. Thus, independently of the mechanism utilized to deplete
the SR/ER of Ca2+, the response to activation of the CaSR is
diminished because of reduction in Ca2+ entry via SOC.
Further confirmation of the presence of a CaSR in the dogfish shark has been obtained in preliminary studies (Dr Marlies Betka, Marical, Portland, ME, USA, personal communication) where positive immunocytochemical staining for the CaSR was seen in both RGA and RGT of Squalus acanthias captured in Frenchman's Bay, ME, USA.
The mechanism by which a CaSR may influence the function of RGT is related
to its effect on cAMP generation and degradation. The co-expression of a CaSR
and a Ca2+-inhibitable adenyl cyclase has been studied in cells of
the thick ascending limb of the rat kidney
(Ferreira et al., 1998).
Changes in extracellular Ca2+, coupled to a pertussis
toxin-insensitive G-protein-activated phospholipase C, caused a dose-dependent
inhibition of cAMP content both from inhibition of cAMP synthesis and from
stimulation of cAMP hydrolysis (Ferreira
et al., 1998
). As salt secretion in RGT is dependent upon the
generation of cAMP (Warth et al.,
1998
), agonist stimulation of the CaSR should inhibit NaCl
secretion. Stimulation of salt secretion by RGT involves a complex sequence of
events in which cardiac naturiuretic peptide, released from the shark heart in
response to volume expansion, stimulates the release of vasoactive intestinal
peptide from the neural network of the rectal gland. Vasoactive intestinal
peptide then stimulates the accumulation of cAMP and promotes salt secretion
(Silva et al., 1996
). Cardiac
naturiuretic peptide has also been shown to have a direct effect on RGT cells
maintained in tissue culture (Karnaky et
al., 1993
).
Because the extracellular domain of the CaSR interacts with polyvalent
cations and polycationic molecules such as polyamines, it was hypothesized
that activation of the receptor might occur through the screening of charged
side chains of acidic or basic amino acids. Furthermore, if ionic strength
were increased by the addition of salts to the extracellular environment, the
ability of polycations to trigger the CaSR should be decreased (Quinn et al.,
1998). Such a property of the CaSR suggests that, rather than sensing changes
in calcium levels exclusively, the receptor might serve as a `salt sensor' in
some cells or species. Non-mammalian vertebrates have developed specialized
salt-excretory organs to maintain osmolar and volume homeostasis, such as the
salt gland of birds (Shuttleworth and
Hildebrandt, 1999) and the rectal gland of the shark
(Burger, 1960
). Only one study
has examined the effect of changes in ionic strength on secretory function of
the shark RG. In the isolated perfused RGT, cAMP causes a biphasic response,
the second of which arises from stimulation of the
Na+/K+/2Cl- co-transporter. Hypotonic
solutions (reduction in NaCl concentration of 150 mmol l-1) caused
RGT cell swelling and abolished this second phase, whereas hypertonic
solutions (addition of NaCl, 150 mmol l-1) resulted in cell
shrinkage and an increase in relative cell chloride concentration
(Greger et al., 1999
). These
data support a role for the effect of changes in ionic strength on secretion
of salt by the RGT. Our future studies will be directed at testing the effect
of changes in ionic strength on salt secretion in the isolated, perfused shark
rectal gland and on calcium signaling in RGA and RGT cells.
We propose that the function of a CaSR in RGA and RGT of Squalus acanthias is to inhibit tonically blood flow to the gland and to inhibit salt secretion (via a reduction in cAMP levels) by the RGT during non-feeding periods. When the shark ingests fish and sea water during feeding, the resultant increase in salt concentration (ionic strength) of the blood and interstitial spaces would then inhibit the CaSR (Quinn et al., 1998), resulting in disinhibition of RGA vascular contraction and reversal of inhibition of salt secretion. Such a control mechanism would permit the animal to efficiently recruit the function of the rectal gland only during periods of feeding.
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Acknowledgments |
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