Vol. 273, Issue 4, R1400-R1406, October 1997
Role of guanylyl cyclase receptors for CNP in salt secretion
by shark rectal gland
Mark
Gunning1,2,
Richard J.
Solomon1,2,3,
Franklin H.
Epstein1,3, and
Patricio
Silva1,2,3
1 Department of Medicine, Beth
Israel Deaconess Medical Center,
2 Joslin Diabetes Center,
Harvard Medical School, Boston, Massachusetts 02215; and
3 The Mount Desert Island Biological
Laboratory, Salsbury Cove, Maine 04672
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ABSTRACT |
The role of
C-type natriuretic peptide (CNP) and its guanylyl cyclase-linked
receptors in mediating salt secretion by the rectal gland of the spiny
dogfish shark (Squalus acanthias)
was investigated using HS-142-1, a competitive inhibitor of the
binding of natriuretic peptides to their guanylyl cyclase receptors.
CNP binds to receptors and activates guanylyl cyclase in rectal gland membranes in a way that is inhibited by HS-142-1. Guanylyl cyclase activation in rectal gland membranes is far more sensitive to CNP than
to atrial natriuretic peptide, whereas the reverse is true for
membranes derived from mammalian (rabbit) renal collecting duct cells.
HS-142-1 inhibited the stimulatory effect of CNP on ouabain-inhibitable oxygen consumption by rectal gland tubules. In
explanted rectal glands continuously perfused with blood from intact
donor sharks, HS-142-1 inhibited the increase in salt secretion normally provoked by infusing isotonic saline solutions into the donor
animal. These results strongly support the view that CNP released into
the systemic circulation in response to volume expansion mediates the
secretion of chloride by the rectal gland via receptors linked to
guanylyl cyclase.
Squalus acanthias; HS-142-1; oxygen consumption
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INTRODUCTION |
THE RECTAL GLAND of the spiny dogfish
(Squalus acanthias) actively
secretes chloride when the shark's blood volume is expanded (2).
Control of secretion was thought to be neurogenic when it was found
that secretion could be elicited by vasoactive intestinal peptide
(VIP), a neurotransmitter present in nerves innervating the gland (25),
and by its second messenger adenosine 3',5'-cyclic monophosphate (cAMP) (26). The importance of humoral control was
established by Solomon et al. (22), who perfused an isolated rectal
gland with blood from an intact shark and demonstrated that secretion
was stimulated by infusing saline into the donor. A cardiac peptide
resembling mammalian atrial natriuretic peptide (ANP) appeared to be
the most likely candidate for the responsible hormone when it was
discovered that ANP released VIP from nerves within the gland and
initiated secretion by isolated perfused rectal glands (19, 21). The
cardiac natriuretic peptide native to the shark is the C-type
natriuretic peptide (CNP) (18, 27), which strongly stimulates chloride
secretion by isolated perfused rectal glands (20), as well as cultured
rectal gland cells, binds with high affinity to rectal gland plasma
membranes, and activates membrane-bound guanylyl cyclase (3). Thus
receptors for CNP on rectal gland cells have been presumed that may
entrain cell transduction pathways (like guanylyl cyclase) other than the adenylyl cyclase cascade that is activated in response to VIP.
At the level of the intact shark, evidence for the role of CNP in the
rectal gland's homeostatic response to volume expansion has thus far
been indirect, consisting chiefly of the facts that the mRNA for CNP is
present in the heart of the shark (18), the peptide has been isolated
from the heart (27), and that CNP stimulates isolated rectal gland
preparations (20). In the present experiments we attempted to clarify
the role of CNP by utilizing a newly discovered receptor antagonist of
cardiac peptides (HS-142-1) on the coupled guanylyl cyclase
receptors that are thought to mediate many of the cellular actions of
this class of hormones.
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METHODS |
Dogfish of either sex were taken by gill nets from Frenchman Bay,
Maine, and kept in marine live cars until used, usually within 3 days
of capture. The dogfish were pithed, and their rectal glands were
removed via an abdominal incision.
Explanted rectal gland.
The rectal gland was excised from a dogfish, and the duct and vein were
catheterized with PE-50 tubing. The artery of the gland was
catheterized with the free end of a section of PE-50 tubing, which had
previously been inserted (via percutaneous puncture through a
thin-walled needle) into the dorsal aorta of a second heparinized
perfusing fish that had been pithed and placed in a running-seawater
(15°C) tank where the head and gills were kept submerged, dorsal
side down, leaving the ventral surface of the abdomen and tail exposed
above the water level. This blood-perfused rectal gland had no neural
connection to the perfusing fish. The explanted gland was placed on a
glass chamber cooled to 15°C with running seawater and positioned
so as to maintain a hydrostatic perfusion pressure between 40 and 45 cmH2O (30-35 mmHg) at the level of the gland. In these experiments infusions were given to the
fish through a second aortic catheter located downstream from the
catheter supplying blood to the explanted gland. Infusions directly
into the rectal gland artery of the explanted gland were performed with
an infusion pump (Harvard Apparatus) connected to a T in the catheter
between the explanted gland and the perfusing fish. All solutions were
made up fresh before each experiment. The concentration of the infused
solution was adjusted so that infusion rates of <10% of the
simultaneously measured venous blood flow from the gland would deliver
the desired concentration of drugs to the rectal gland. During control
periods blood flow to the gland was maintained, but no additional
substances were infused directly into the gland. Rectal gland duct flow
and the secretion rate of chloride were measured for timed intervals
during a control period before infusions and for experimental periods
during the infusions. Blood was sampled from the dorsal aorta at the
midpoint of each collection.
Separated rectal gland tubules.
Two rectal glands were used for each tubule preparation. The rectal
glands were perfused in vitro as described previously with 100 ml of
shark Ringer. The glands were then perfused with 10 ml of shark Ringer
containing 0.2% collagenase and 10% fetal calf serum. The glands were
sectioned longitudinally and minced into 1-mm cubes with a razor blade.
The minced tissue was then incubated in shark Ringer containing
collagenase and fetal calf serum, in the same proportions given
previously, while constantly agitated for 45 min at room temperature.
The tissue digest was then centrifuged at 50 g for 1 min in a refrigerated
centrifuge to remove undigested tubules. The supernatant was then spun
at 500 g for 3 min to harvest the
tubules. The tubules were washed twice in shark Ringer and kept on ice
until used.
Oxygen consumption measurement.
Oxygen consumption was measured in a constant-temperature (25°C)
chamber using a Clark type polarographic electrode connected to a
recorder. The rate of oxygen consumption was calculated from the
tangent of the recorded slope of the oxygen consumption, the solubility
of oxygen in shark Ringer at 25°C, the barometric pressure, the
volume of incubation solution in the measuring chamber, generally 2 ml,
and the wet weight of the cells. The wet weight of the cells was
determined at the end of the experiment by removing a measured aliquot
of the cell suspension, spinning it down in a tared centrifuge tube,
removing the supernatant, and reweighing the tube. Exogenous substrates
used included (in mM) 5 glucose, 10 pyruvate, and 2.5 acetate. In the
experiments reported here oxygen consumption was measured under basal
conditions, and the rate of oxygen consumption was allowed to reach a
steady rate. A peptide was then added, and the rate of oxygen
consumption was allowed to reach a new steady state. Once this new
steady state was established, 10 mM ouabain was added and the rate of
oxygen consumption was determined again. In the experiments in which
HS-142-1 was used, it was added simultaneously with the peptide.
Ouabain-sensitive oxygen consumption was calculated as the difference
between the rate of oxygen consumption under basal or stimulated
conditions and the rate after 10 mM ouabain.
Membrane preparation.
Shark rectal glands were frozen in liquid nitrogen immediately on
removal and subsequently stored at 
80°C until use. Membrane preparations for binding studies and guanylyl cyclase assay were prepared in the same manner. Rectal glands were ground on ice and
homogenized three times using a polytron at setting 6, for 30 s in a
medium containing (in mM) 250 sucrose, 1 EDTA, and 1 dithiothreitol, pH
7.4. The resulting homogenate was centrifuged at 100 g to remove large unbroken cellular
debris, and the supernatant was centrifuged twice at 40,000 g for 40 min, being finally
resuspended in buffered solution specific for the type of assay to be
performed. For binding experiments the pellet was resuspended at a
concentration of ~1.5 mg protein/ml in buffered shark Ringer solution
consisting of (in mM) 10 tris(hydroxymethyl)aminomethane (Tris), 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 305 NaCl, 350 urea, 5 KCl, 3 MgCl2, 2.5 CaCl2, 1 KH2PO4,
and 0.5 Na2SO4,
pH 7.4, and frozen in aliquots at 80°C until use. For
guanylyl cyclase experiments membranes were used on the day of
preparation, the final pellet being resuspended at ~5 mg/ml in
guanylyl cyclase assay buffer consisting of (in mM) 100 HEPES and 50 NaCl, pH 7.4.
Equilibrium binding assay.
Binding studies were performed in a manner similar to that previously
used by us (3). Membranes were incubated to equilibrium at 4°C in
buffered shark Ringer with the following additions, 0.2% bovine serum
albumin, 0.15% bacitracin, 1 mM phenylmethylsulfonyl fluoride, and 2 µM phosphoramidon and in the presence of radiolabeled human CNP
(hCNP) and the indicated concentrations of the competing ligand.
HS-142-1 was added to a final concentration of 100 µg/ml in the
appropriate experiments. At the end of incubations bound radioactive
hormone was separated from free by suction filtration through glass
fiber filters (Whatman GF/C), pretreated with 1% polyethylenimine, and
the filters were washed with an additional 10 ml ice-cold 0.9% NaCl
containing 0.2% bovine serum albumin. The filters on which the intact
cells or membranes were retained were collected, and the associated
radioactivity was counted in a gamma counter (LKB 1272 Clinigamma).
Nonspecific binding was defined as that amount of
125I-CNP bound in the presence of
excess (10
6 M) unlabeled
hormone. Specific binding was the difference between total binding and
nonspecific binding. Nonspecific binding was always <3% of the total
added radioactivity.
Guanylyl cyclase assay.
Measurement of enzyme activity was performed by incubating 30-60
µg protein of the membrane preparation with the manganese salt of
guanosine triphosphate (Mn2GTP, 5 mM) as substrate in the presence of the phosphodiesterase inhibitor
3-isobutyl-1-methylxanthine, 1 mM as described in detail previously
(4). All incubations took place for 2 min in a water bath at 22°C
in a final volume of 200 or 250 µl. The reaction was stopped with
12% trichloroacetic acid. Samples were centrifuged at 2,300 g for 15 min, and the supernatant was
removed, extracted with 10 volumes of water-saturated ether four times,
dried, and reconstituted in 4 ml of 50 mM sodium acetate buffer (pH
6.2) for measurement of guanosine 3',5'-cyclic monophosphate (cGMP) content. cGMP concentration was measured by
radioimmunoassay. In experiments using natriuretic peptides, membrane
suspensions with and without added peptide were assayed simultaneously
from the same preparation. Protein concentration was measured by a
modification of the method of Bradford (1) (Bio-Rad).
Adenylyl cyclase assay.
The rectal glands were removed, and all connective tissue was dissected
out and discarded. The glands were diced into 2- to 3-mm cubes with a
razor blade, suspended in ten times the volume of homogenizing solution
containing (in mM) 50 Tris, 1 EDTA, 1 dithiothreitol, and 250 sucrose,
pH 8.0, and homogenized in a glass homogenizer with a Teflon pestle.
The homogenate was centrifuged at 500 g for 10 min. The pellet was
discarded, and the supernatant was diluted 1:5 in homogenizing solution
and stored on ice until used.
For the adenylyl cyclase assay, 50 µl of dilute supernatant were
added to 50 µl of assay solution containing (final concentrations) 50 mM Tris, pH 7.6, 15 mM phosphocreatine, 26.35 µg/ml creatine phosphokinase, and 0.1 mM theophylline. Peptides were
added and allowed to incubate for 5 min before starting the reaction.
The reaction was started by adding 10 µl of 10 mM ATP and 40 mM
MgCl2 at 10-s intervals. After 15 min of incubation the reaction was stopped by the addition of 900 µl
of 50 mM sodium acetate, pH 4.0, boiled for 3 min, centrifuged at 5,000 revolutions/min for 15 min, and the supernatant was stored frozen for
cAMP assay. cAMP generated was measured using a commercial
radioimmunoassay kit (Amersham). Protein concentration was measured by
a modification of the method of Bradford (1) (Bio-Rad). Results are
expressed as picomoles of cAMP generated per milligram of protein per
hour. Chloride was measured by amperometric titration using a
Buchler-Cotlove chloridometer.
VIP and procaine were purchased from Sigma Chemical. hCNP and human ANP
(hANP) were purchased from Peninsula Laboratories. Shark CNP (sCNP) was
provided by California Biotechnologies. HS-142-1 was a generous
gift of the Pharmaceutical Research Laboratories of Kyowa Hakko Kogyo
and Dr. Barry M. Brenner.
Chloride secretion is expressed as microequivalent per hour per gram
wet weight. Oxygen consumption is expressed as micromole of oxygen
consumed per hour per gram wet weight. All values are means ± SE.
Statistical significance was determined using Student's t-test, paired
t-test, and analysis of variance
wherever applicable.
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RESULTS |
Effect of HS-142-1 on binding of CNP to rectal gland plasma
membranes.
CNP binds with high affinity to abundant receptors in plasma membranes
from shark rectal gland (3). In mammalian tissues HS-142-1 is a
specific inhibitor of the natriuretic peptide receptors coupled to
guanylyl cyclase (9, 11, 16), but it does not bind to the
low-molecular-weight natriuretic peptide receptor, the so-called
clearance receptor (11). In isolated shark rectal gland membranes,
HS-142-1, at a concentration of 100 µg/ml, inhibited ~40% of
total specific binding of hCNP without an apparent change in the
association constant (Fig.
1).
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Fig. 1.
Effect of HS-142-1 on C-type natriuretic peptide (CNP) specific
binding to shark rectal gland plasma membranes. Membranes were
incubated as described in presence or absence of 100 µg/ml
HS-142-1. Data are expressed as ratio of specific bindings B/B0,
where B is specific binding of labeled CNP in presence of cold CNP and
or HS-142-1 and B0 is amount of labeled ligand specifically bound
in absence of competing unlabeled ligand and/or HS-142-1.
As shown in control curve, binding is of high affinity with association
constant (Ka) of ~0.1 nM. In presence of 100 µg/ml HS-142-1 specific binding is reduced by about 40%,
whereas Ka is unchanged. Each value represents
means ± SE of triplicate measurements in 3 different
plasma membrane preparations.
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Effect of natriuretic peptides and HS-142-1 on guanylyl cyclase
and adenylyl cyclase.
The activation of guanylyl cyclase by different classes of cardiac
peptides appears to be species specific. Figure
2A shows that both shark and human forms of CNP stimulate the production of cGMP
by shark rectal gland membranes in a similar and dose-dependent fashion. This effect was several orders of magnitude greater than that
of hANP, which produced only a minor stimulation in shark membranes
even at the highest concentration studied. Half-maximal stimulation
with sCNP or hCNP in shark rectal gland was 10 nM, similar to that
observed for hANP in rabbit kidney inner medullary collecting duct
cells (4). On the other hand, as shown in Fig. 2B, hANP stimulates guanylyl cyclase
in membranes derived from the inner medulla of the rabbit kidney,
whereas sCNP does not.
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Fig. 2.
Comparison of effects of natriuretic peptides on rectal gland and
rabbit inner medullary collecting duct guanylyl cyclase.
A: shark CNP (sCNP) and human CNP
(hCNP) stimulate production of cGMP. Their effect is several orders of
magnitude greater than that of human atrial natriuretic peptide (hANP).
B: effect of sCNP and hANP on guanylyl
cyclase from plasma membranes of rabbit inner medullary collecting
duct. sCNP does not stimulate guanylyl cyclase in these membranes,
whereas hANP does. Values are means ± SE. Number of observations
varied between 3 and 7 for each peptide concentration. For lower
peptide concentrations alternating concentrations were skipped.
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As illustrated in Fig. 3, 100 µg/ml
HS-142-1 reduced the activation of guanylyl cyclase induced by CNP
in shark rectal gland membranes by ~60% when CNP was present at 10 nM, close to the half-maximal concentration seen in Fig.
2A. At higher concentrations of CNP
(500 nM) this concentration of HS-142-1 was not sufficient to
inhibit the effect of CNP.
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Fig. 3.
Effect of HS-142-1 on guanylyl cyclase activity in plasma
membranes from shark rectal gland. Membranes were incubated as
described in METHODS for assay of
guanylyl cyclase activity in presence or absence of HS-142-1 (100 µg/ml). HS-142-1 reduced activation of guanylyl cyclase induced
by CNP by ~60% at concentration of CNP of
108 M. Each value
represents means ± SE of 3 measurements in 3 different plasma
membrane preparations.
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CNP had no effect on adenylyl cyclase of the shark rectal gland
membranes. In the presence of 100 µM theophylline, 1 nM VIP and 10 mM
aluminum fluoride, by contrast, briskly stimulated the production of
cAMP in the same preparation (Table
1).
Effect of CNP and HS-142-1 on ouabain-sensitive oxygen
consumption in rectal gland tubules.
sCNP was found to stimulate ouabain-inhibitable (transport-dependent)
oxygen consumption by separated rectal gland tubules in a
dose-dependent way (data not shown). The lowest concentration of sCNP
that had this effect was 10 nM, and maximal stimulation was reached
between 50 and 100 nM. At 50 nM, sCNP increased ouabain-sensitive oxygen consumption approximately 10-fold, by 137 ± 17 µM
oxygen · h1 · g
wet wt1
(n = 16, P < 0.01). The stimulatory effect of
sCNP was completely prevented by 500 µg/ml HS-142-1 (Fig.
4).
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Fig. 4.
Effect of HS-142-1 on ouabain-sensitive oxygen consumption
( O2) by separated rectal
gland tubules. In control experiments 50 nM sCNP increased
O2 from basal of 57 ± 5 to 182 ± 18 µM · h1 · g1
after CNP (n = 16, P < 0.001); subsequent addition of
10 mM ouabain reduced O2 to
44 ± 3, implying ouabain-sensitive
O2 of 137 ± 17 µM · h1 · g1.
In presence of 500 µg/ml HS-142-1, sCNP increased
O2 from basal of 49 ± 8 to only 76 ± 16 µM · h1 · g1
after CNP (n = 9;
P not significant), whereas subsequent
addition of ouabain reduced it to 62 ± 15 µM · h1 · g1.
Thus in freshly separated rectal gland tubules, sCNP stimulated
transport-dependent O2 in a
way that was completely inhibited by HS-142-1.
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HS-142 blocks effect of volume expansion on chloride secretion by
rectal gland.
Having demonstrated that HS-142-1 inhibits the binding and action
of C-type cardiac peptides in shark rectal gland cells, we next used
HS-142-1 to determine whether endogenous natriuretic peptides
mediate the effect of volume expansion on chloride secretion by the
rectal gland in intact sharks (Fig. 5).
After perfusion of explanted rectal glands was established, the donor
fish was infused with 50 ml/kg weight of shark Ringer solution given
over 30 min. Simultaneous with the volume load, a constant infusion of
either shark Ringer or shark Ringer with HS-142-1 (5 mg/ml final
concentration) was delivered into the arterial catheter connected to
the explanted rectal gland. The infusion rate was maintained at 10%
the rate of arterial blood flow to the gland for 150 min, so that the
effective concentration of HS-142-1 perfusing the gland was ~500
µg/ml. The usual response to volume expansion of the
donor fish is an increase in secretion of chloride by the explanted
gland associated with an increase in blood flow that starts
30-60 min after the infusion is delivered and lasts for 2-3 h
(22, 23). This pattern can be seen in the control experiments depicted
in Fig. 5. Perfusion of the explanted rectal gland with 500 µg/ml of HS-142-1 completely inhibited the increase in chloride secretion observed after volume expansion of the donor fish. After cessation of the HS-142-1 infusion, chloride secretion by the explanted gland increased toward the levels found in control glands that had received vehicle only. There was no significant difference between the two groups in the rate of blood flow to the explanted rectal gland (37 ± 4 vs. 39 ± 3 ml · h1 · g
peak flow1 in HS-142-1
group vs. shark Ringer, respectively) or in perfusion pressure (46.7 ± 2.7 vs. 43.5 ± 0.9 cmH2O
peak pressure) in the HS-142-1 group vs. shark Ringer,
respectively.
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Fig. 5.
Effect of HS-142-1 on effect of volume expansion on chloride
secretion by explanted glands. At time
0 a volume load (VL) of shark Ringer, 50 ml · kg1 · 30 min1, was started into
distal aorta of donor shark. Simultaneously, an infusion of either
shark Ringer (control experiments) or shark Ringer and HS-142-1
(500 µg/ml final concentration) was delivered via a T-tube to
explanted gland. In control experiments volume expansion of donor shark
doubled secretion of chloride. HS-142-1 completely prevented
effect of volume expansion. After infusion of HS-142-1 was
stopped, secretion of chloride started to rise toward values seen in
glands that did not receive HS-142-1. Values are means ± SE,
n = 6 for control and 5 for
experimental sharks. * P < 0.01.
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DISCUSSION |
HS-142-1, a polysaccharide produced by
Aureobasidium pullulans var.
melanigenum was shown by Morishita et al. (9) to
inhibit the binding of ANP to its receptor in rabbit kidney cortex and bovine adrenocortical membranes. In mammalian tissues it reduces or
suppresses the generation of cGMP evoked by ANP (5, 6, 9, 12,
15-17, 24, 28, 29), brain natriuretic peptide (BNP) (29), or CNP
(6, 7, 12, 29, 30) in a variety of preparations from isolated membranes
to live animals. It inhibits the binding of ANP, BNP, or
CNP (9, 10, 12, 29) and prevents or reverses the effect of endogenous
or exogenous natriuretic peptides (6, 10, 13, 17, 24, 29). It has been
used to study the distribution of guanylyl cyclase-linked natriuretic peptide receptors (14, 30) and to explore the possible role of
natriuretic peptides in a variety of pathophysiological conditions (8,
15, 28, 29). Because natriuretic peptides, including CNP, stimulate
guanylyl cyclase, their effect has been thought to be mediated by cGMP.
The present experiments establish the validity of HS-142-1 as a
competitive inhibitor of the action of natriuretic peptides in the
shark rectal gland and provide direct evidence for the importance of
CNPs in the native response of the rectal gland of intact sharks to
salt loading. HS-142-1 inhibited the binding of human and shark
CNP to plasma membranes derived from the rectal gland, inhibited its
activation of membrane-associated guanylyl cyclase, and prevented the
stimulation of transport-dependent oxygen consumption in separated
rectal gland tubules by CNP. In experiments with explanted glands
perfused with blood from intact donor sharks HS-142-1 prevented
the effect of volume expansion of the donor to increase chloride
secretion by the explanted gland. This inhibition was reversible, so
that chloride secretion by the explant increased when glands were no
longer exposed to HS-142-1. These results provide strong support
for the notion that CNP released in response to a volume stimulus
mediates the secretion of chloride by the shark rectal gland.
Marked species specificity was apparent when the action of various
natriuretic peptides was tested on guanylyl cyclase from elasmobranch
and mammalian tissues. Both sCNP and hCNP (which are 82% homologous,
differing in 4 of 22 amino acids, 3 in the aminoterminal end, those in
positions 2, 4, 5, and 1 in the loop, that in position 16) stimulated
guanylyl cyclase in plasma membranes of shark rectal gland and were
equipotent. Half-maximal stimulation was observed at 10 nM, within the
range of concentration at which chloride secretion is stimulated in
intact glands (20) and comparable to the Michaelis constant found for
ANP in guanylyl cyclase assays in membranes derived from rabbit kidney
collecting duct (4). The potency of hANP to stimulate
guanylyl cyclase in rectal gland membranes was almost negligible. On
the other hand, sCNP did not stimulate guanylyl cyclase in rabbit
collecting duct membranes, where hANP was highly active. These results
support the view that in the shark, CNPs and their receptors
predominate over ANP-type isoforms characteristic of
mammalian species.
In a recently published report we showed that CNP binds to plasma
membranes of shark rectal gland (3). In that report we demonstrated the
presence of two receptors with similar affinity for CNP. Kinetic
analysis of the data indicated that 50% of the receptors were highly
specific for CNP and 50% exhibited characteristics for
low-molecular-weight receptors. The experiments reported here support
that conclusion. In the present experiments HS-142-1 displaced ~40% of the CNP binding to rectal gland plasma membranes. Because HS-142-1 prevents the binding of natriuretic peptide to guanylate cyclase-linked receptors and has no effect on the low-molecular-weight receptor, displacement by HS-142-1 of 40% of the CNP binding
suggests that close to one-half of the receptors present in the rectal gland cell membrane are of the guanylate cyclase-linked type and the
remainder must be of the low-molecular-weight type. This represents a
second line of evidence indicating that there are two types of
natriuretic peptide receptors in the rectal gland: a guanylyl cyclase-linked receptor that by binding and functional characteristics appears to be a GC-B receptor and a low-molecular weight receptor, the
so-called clearance receptor.
Intravascular volume expansion of the spiny dogfish evokes an increase
in blood flow to the rectal gland of ~300% as well as an increase in
glandular secretion (22). In the explanted rectal gland preparation,
the changes seen in the donor shark are mirrored in the explanted
gland. The increased blood flow to the gland is not a result of an
increase in aortic perfusion pressure and must therefore represent a
decrease in local vascular resistance (23). A striking feature of the
present experiments is that HS-142-1 inhibited chloride secretion
without altering the increase in glandular blood flow that commonly
accompanies volume expansion. Bumetanide similarly inhibits the
secretory response of the explanted gland to volume expansion of the
donor shark, without preventing the increase in blood flow (23). It seems reasonable to hypothesize that the vasodilation stimulated in
explanted rectal glands by expansion of the donor blood stream is
produced by a hormonal substance that may not be a cardiac peptide and
is not an agonist of the guanylyl-cyclase-linked receptor inhibited by
HS-142-1.
Perspectives
Natriuretic peptides, secreted by the heart in response to volume
expansion, promote the excretion of salt by the mammalian kidney. This
mechanism, which prevents the retention of salt, also operates in other
vertebrates like the shark, where the target organ is the rectal gland
and not the kidney. This represents an evolutionary convergence in
vertebrates where a heart hormone has retained its specific action
while the target organ has changed. The present report documents for
the first time that CNP, the natriuretic peptide present in the shark
heart, is the mediator of the effect of volume expansion to stimulate
the secretion of salt by the rectal gland. Previous work in this area
suggested that the effect of volume expansion might involve the
sequential release of two hormones, a natriuretic peptide from the
heart and VIP from nerves within the gland. The previous experiments were done using rat or hANP that stimulated the secretion of salt by
the rectal gland by releasing VIP (19, 21). However, ANP is not present
in the shark heart (18) and, as described previously, does not
stimulate guanylyl cyclase in rectal gland plasma membranes. On the
other hand, CNP binds to a receptor, activates guanylyl cyclase, and
stimulates oxygen consumption, providing direct evidence that CNP
exerts a functional effect on rectal gland cells. More importantly, in
vivo, the effect of volume expansion is prevented by blockade of the
natriuretic peptide receptor indicating that CNP mediates the effect of
volume expansion.
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ACKNOWLEDGEMENTS |
The technical help of H. Brignull, S. Hornung, N. Katz, J. Landsberg, M. Silva, H. Solomon, K. Spokes, M. Taylor, and D. Wolff is
gratefully acknowledged.
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FOOTNOTES |
This research was supported by the National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-18078 and DK-16684, the
National Institute of Environmental Health Sciences ES-O3828, and the
American Heart Association, Maine Affiliate.
Address for reprint requests: P. Silva, Joslin Diabetes Center, 1 Joslin Place, Boston, MA 02215.
Received 3 March 1997; accepted in final form 18 June 1997.
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