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Department of Biochemistry (S.K.), Vanderbilt University Medical School, Nashville, Tennessee 37212; Cardiovascular Research Department (D.G.L.), Genentech, Inc., South San Francisco, California 94080; and Department of Pharmacology (G.J.T.), University of Minnesota-Duluth, School of Medicine, Duluth, Minnesota 55812
Address all correspondence and requests for reprints to: George J. Trachte, Department of Pharmacology, University of Minnesota-Duluth, School of Medicine, 10 University Drive, Duluth, Minnesota 55812. E-mail: gtracht1{at}d.umn.edu
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Abstract |
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Introduction |
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The objective of this report is to test definitively for an involvement of the NPR-C in mediating the neuromodulatory effects of natriuretic peptides. The recent cloning of the rat NPR-C (11) has allowed the deduction of the amino acid sequence of this receptor. In this report, we examine the intracellular regions of this receptor for biological activity in permeabilized adrenergic tissue. We then further test for a role of this receptor by generating antibodies to the receptor fragments and testing whether these antibodies prevent neuromodulatory effects of natriuretic peptides. This study extends recent work showing that the 37 amino acid cytosolic portion of the NPR-C suppresses adenylyl cyclase activity in plasma membranes (12). Two critical tests of the hypothesis that the NPR-C mediates neuromodulatory effects of natriuretic peptides include the following: 1) a cytoplasmic fragment of this receptor should mimic effects of natriuretic peptides to attenuate evoked dopamine efflux in permeabilized cells; and 2) antibodies generated against the active portion of the receptor should ablate this effect of natriuretic peptides in permeabilized cells. The results are relevant because the natriuretic peptide system functions as endocrine regulators of numerous systems including cardiac hypertrophy (13) and skeletal growth (14). Elucidation of the relevant receptors mediating these effects is essential to understanding the natriuretic peptide system and to developing therapeutics to influence the system.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell permeabilization
Cells were permeabilized by exposure to digitonin (10
µM) for 4 min. The digitonin was dissolved in a buffer
consisting of potassium glutamate (157 mM), HEPES (10
mM), magnesium sulfate (5 mM) and ATP (5
mM). The digitonin was washed out and cells were exposed to
either a buffer containing 0 or 10 µM calcium. The 0
µM calcium solution consisted of the permeabilizing
buffer except that digitonin was replaced by 4 mM EGTA. The
10 µM calcium solution contained the same ingredients as
the permeabilizing buffer except that both 4 mM EGTA and 4
mM calcium chloride were included and digitonin was
excluded. This mixture of calcium chloride and EGTA yields a free
calcium concentration of approximately 10 µM (15). The
cell permeabilization was confirmed with ethidium homodimer, a nuclear
dye that fluoresces 100- to 200-fold more intensely when it interacts
with double stranded nucleic acids (16).
Catecholamine measurements
Dopamine efflux from cells was measured in the presence of both
the 0 and 10 µM calcium solutions. The difference in
dopamine efflux under the two conditions was designated as evoked
release. Catecholamines were extracted from the 0 or 10
µM calcium solutions after cells were exposed to the
solutions for 5 min. Catecholamines remaining in the cells also were
extracted after scraping the cells and disrupting cell membranes by
sonication for 30 sec. Cellular debris was pelleted by a 15,000 x
g centrifugation lasting 10 min. Catecholamine contents of
both the bathing medium and the cells were eluted onto 60 mg alumina by
incubation for 10 min in 3 M Tris buffer. The alumina
subsequently was washed with 6 mM Tris buffer followed by
water. Catecholamines were eluted with 0.5 ml of 0.2 N
perchloric acid. Recoveries of catecholamines were assessed by the
recovery of 10 ng dihydroxybenzylamine, which was added to each sample.
Recoveries averaged 50 to 70%. Catecholamine contents of samples were
assessed by HPLC with electrochemical detection. The assay was
accomplished with a Spectraphysics P100 pump attached to a RP18 column
with a 4.6-mm diameter and a 22-cm length. A Bioanalytical Systems, Inc. LC-4B electronic controller and a LC-17A oxidative
flow cell measured oxidation. The signals were integrated by a
Spectraphysics Datajet integrator. The mobile phase consisted of 0.1
M potassium phosphate monobasic, 0.1 mM
ethylenediamine-tetraacetic acid, 1.0 mM octyl sodium
sulfate and 2.5% methanol by volume. Injection volumes were 100 µl.
Dopamine contents averaged 113 ± 15 ng per culture, and no
treatment significantly altered the dopamine content of the treated
culture. The maximal and minimal values for dopamine contents were
151 ± 39 and 49 ± 17 ng per culture.
Peptides and antibodies
The cytosolic portion of the NPR-C contains 37 amino acids (17).
This 37 amino acid peptide was divided approximately in half in an
attempt to locate the transducing regions of the receptor. The amino
terminal pentadecapeptide of this portion of the receptor and the
nonadecapeptide carboxy terminal fragment of the receptor were
purchased from Chiron Mimitopes (Raleigh, NC). The sequences of these
two peptides were the following (amino to carboxy terminals):
R-K-K-Y-R-I-T-I-E-R-R-N-H-Q-E and
V-G-K-H-R-E-L-R-E-D-S-I-R-S-H-R-S-V-A. A scrambled peptide also was
generated for the amino terminal pentadecapeptide of the following
composition: T-Y-N-H-E-R-R-I-R-K-I-Q-E-K-R. The entire 37 amino acid
peptide, corresponding to the bovine sequence, was synthesized at
Genentech, Inc. and also was tested for biological
activity. The sequence was:
R-K-K-Y-R-I-T-I-E-R-R-N-Q-Q-E-E-S-N-V-G-K-H-R-E-L-R-E-D-S-I-R-S-H-F-S-V-A.
Its scrambled counterpart consisted of the following:
K-E-R-F-S-E-R-H-K-D-I-Y-S-R-I-T-N-Q-G-L-R-A-Q-N-R-H-R-K-S-I-R-V-E-S-V-E-E.
After testing these peptides for neuromodulatory activity, polyclonal
antibodies to the amino terminal pentadecapeptide were purchased from
Research Genetics, Inc. (Huntsville, AL). The antibodies
were purified over a protein A superose column. Preimmune and immune
sera were then matched for protein concentration by measuring
absorbency at 280 nm. A native peptide, C-type natriuretic peptide, was
purchased from Peninsula Laboratories, Inc. (Belmont, CA).
Prostaglandin E2 was purchased from Sigma Chemical Co. (St. Louis, MO).
Antibody specificity
The rabbit antibody generated against the amino terminal
pentadecapeptide of the NPR-C cytosolic region was tested for
specificity by testing its reactivity with the various peptides used in
this report. Dot blots were performed by adding the various peptides to
Protran BA75 nitrocellulose membranes (Schleicher & Schuell, Inc., Keene, NH) using a Bio-Dot apparatus from Bio-Rad Laboratories, Inc. (Hercules, CA). Peptides were spotted at 0.25
to 2 nmol. The nitrocellulose membrane was incubated overnight with a
blocking buffer consisting of 1% milk in the reaction buffer. The
blocking buffer was rinsed away and the nitrocellulose membrane was
incubated with the NPR-C antibody at a dilution of 1:100, for 4 h.
The antibody was washed away with numerous rinsings with the reaction
buffer, consisting of 50 mM Tris-HCl, 150 mM
NaCl and 0.1% Tween (vol/vol) with the pH adjusted to 8.0. The
membrane then was incubated with a secondary antirabbit antibody
conjugated with horseradish peroxidase (Pierce Chemical Co., Rockford, IL) for 2 h. Enhanced chemiluminescence was
used to assess the amount of antibody bound to the nitrocellulose
membrane using the ECL Detection kit RPN29 of Amersham Life Sciences (Buckinghamshire, UK). After the reaction, the
nitrocellulose membrane was placed adjacent to photographic film
(Hyperfilm, Amersham Life Sciences) in a film cassette.
The resulting exposure of the film was developed by a Konica QX-70
medical film processor.
Statistics
Curves were compared by ANOVA for repeated measures. Individual
points were compared with control values by Student’s paired
t test with Dunnett’s correction for multiple comparisons.
Values of P 
0.05 were considered statistically
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. All
permeabilized cells fluoresced when exposed to ethidium homodimer (4
µM) whereas none of the intact cells fluoresced
detectably when exposed to the nuclear dye. The difference in
accumulation of the dye between the two groups of treated cells
demonstrates that the digitonin treatment effected a successful
disruption of the plasma membrane. Representative bright fields of
either intact or permeabilized cells also are shown.
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. These inhibitory effects were
statistically significant (P < 0.05). A scrambled
peptide with the same amino acid composition, but with the amino acids
arranged in a random order, failed to attenuate evoked dopamine efflux,
as also is shown in Fig. 2. The two curves differed statistically
(P < 0.05) when compared by ANOVA. Lower
concentrations of the 37 amino acid intracellular fragment of the NPR-C
were tested for biological activity because no concentration response
relationship was discerned at concentrations of 0.1 and 1
pM. A concentration-response curve was observed with
maximal effects occurring at 40 fM and a half-maximal
effect occurring at 31 ± 7 fM (Fig. 3). These effects were statistically
significant at concentrations of 40 fM and higher. The
concentration-response curve exhibited a linear relationship with the
correlation coefficient equaling 0.98 (P < 0.01) in
the linear portion of the curve (i.e. up to 40
fM).
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. The two
curves differed statistically when compared by ANOVA (P
< 0.05). The absence of a concentration-response relationship induced
us to examine lower concentrations of the active peptide to test if a
concentration dependence existed. As seen in Fig. 5, a linear relationship existed between
concentrations of this agent and evoked dopamine efflux. The linear
regression for the line has a correlation coefficient of 0.92 and is
statistically significant (P < 0.01). The peptide
produced statistically significant effects at concentrations of 600
fM and greater (P < 0.05) and a
half-maximal effect occurred at a concentration of 457 ± 97
fM. These data are consistent with the hypothesis that the
NPR-C suppresses adrenergic neurotransmission and they identify the
amino terminus of the cytosolic region of the receptor as the
transducer of neuromodulatory activity.
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The effect of polyclonal antibodies generated against the amino
terminal fragment of the cytosolic region of the NPR-C was explored.
The specificity of the antibody was tested with dot blots probing its
reactivity with the peptides used in this study. The antibody reacted
with its pentadecapeptide antigen, as indicated by progressively darker
staining at antigen concentrations of 0.25 to 2.0 nmol, as shown in
Fig. 6. The antibody failed to react with
other peptides, including the carboxy terminal 19 amino acids of the
NPR-C or a scrambled control peptide for the amino terminal 15 amino
acids of the NPR-C cytosolic region. These dot blot results indicate
that the antibody is specific and reacts in a dose-dependent manner
with the antigenic peptide.
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. The pentadecapeptide
receptor fragment significantly suppressed evoked dopamine efflux at
concentrations of 1 and 10 pM in cultures exposed to the
preimmune serum (P < 0.05), but no statistically
significant changes were observed in cultures exposed to the immune
serum. The two curves were statistically different when compared by
ANOVA (P < 0.01). These data indicate that antibodies
to the pentadecapeptide fragment of the NPR-C cytosolic region blocked
its activity, as would be expected.
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. C-type natriuretic peptide attenuated
evoked dopamine efflux from permeabilized PC12 cells in a
concentration-dependent manner with an EC50 of 0.042
± 0.016 nM in the presence of the preimmune serum. The
maximal inhibitory effect averaged 38 ± 9% at a concentration of
1 nM. All concentrations exceeding 0.01 nM
significantly suppressed evoked dopamine efflux in cultures exposed to
the preimmune serum (P < 0.05). In stark contrast,
C-type natriuretic peptide lacked neuromodulatory effects in the
presence of a 1:2500 dilution of the antibody to the amino terminal 15
amino acids of the cytosolic region of the NPR-C. This attenuation of
natriuretic peptide effects was statistically significant
(P < 0.01). Furthermore, the antibody was inactive in
intact cells (data not shown), suggesting that it must penetrate into
cells to reach its active site. These data strongly support the
hypothesis that the NPR-C mediates a suppression of evoked dopamine
efflux.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The inhibitory effect of the pentadecapeptide corresponding to the NPR-C cytosolic region closest to the membrane was anticipated because this region contains a consensus sequence for coupling to GTP-binding proteins (17). The potency of either the pentadecapeptide receptor fragment or the peptide corresponding to the entire intracellular region of the NPR-C was far greater than that of any receptor fragment reported thus far. In fact, the peptide comprising the intracellular region of the receptor manifested a potency only 31-fold less than the affinity of avidin for biotin (i.e. 1 fM) (20), the most potent known attraction between two naturally occurring substances. In contrast, Okamoto et al. (21) typically observe EC50 in the µM range for active fragments of either the insulin or amyloid precursor protein receptor (22). Other investigators also require µM concentrations of receptor fragments to activate GTP-binding proteins (23, 24). Anand-Srivastava et al. (12) found the entire intracellular fragment of the NPR-C to suppress adenylyl cyclase activity with an EC50 of 1 nM. The potency of the amino terminal 15 amino acids of this fragment was at least 1000-fold more potent in the dopamine efflux assay, producing a maximal effect at concentrations as low as 1 pM. These data predict that maximal effects of NPR-C activation would be achieved when 600-1200 receptors are activated per cell if activation of native receptors equates with biological activity of these fragments. We have observed PC12 cells to possess 2500 NPR-C’s on their plasma membrane (9), a number exceeding the predicted requirement for maximal activity. Thus, the potency of this peptide is consistent with this fragment being the active transducing portion of the receptor.
The curves for the receptor fragments were exceedingly steep such that the entire curves were complete in less than an order of magnitude increase in concentration. In contrast, receptor agonists typically require 3 orders of magnitude for a complete concentration-response curve. The lesser slope of receptor agonists is attributed to the progressively greater difficulty in saturating receptor sites as one increases agonist concentrations. In the absence of positive or negative cooperativity or spare receptors, responses are predicted to correlate directly with receptor saturation with agonist. Thus, the steeper slope of the concentration-response curve for these receptor fragments is consistent with their predicted behavior if they represent activated receptors. Furthermore, linear regression analysis indicates a direct correlation between receptor fragment concentrations and submaximal biological responses, as would be predicted if the fragments represent activated receptors. This exquisite sensitivity of dopamine efflux to these receptor fragments or activated receptors would be required for biological activity of a receptor that is present in low concentrations and is consistent with the fragments behaving as activated receptors.
Prior work with receptor fragments typically revealed a stoichiometric interaction between receptor fragments and signal-transducing systems. This scenario results in the receptor fragments being equipotent to receptor agonists in stimulating biological systems (12, 22). Our results demonstrate a 10- to 1,000-fold greater potency of the receptor fragments relative to an endogenous ligand, C-type natriuretic peptide. This potency difference should be observed for active fragments of the receptor because only a fraction of receptor agonists actually binds to the receptor; most of the agonist remains free in solution. Therefore, receptor concentrations producing biological effects should be far less than agonist concentrations producing equivalent responses if agonist occupation of receptors is required for receptor activation. Our data are consistent with 10%, 3%, and 0.1% of agonist occupying the NPR-C at C-type natriuretic peptide concentrations producing 10%, 50% and 100% of maximal effects if the potency of the NPR-C fragment is identical to that of the activated receptor. This reduction in efficiency of receptor occupation with higher agonist concentration is the basis for the Scatchard analysis (25) and is totally consistent with receptor theory.
Antibodies to the active pentadecapeptide fragment prevented the suppression of dopamine efflux caused by both the pentadecapeptide receptor fragment and a natriuretic peptide, C-type natriuretic peptide. These data support the hypothesis that this receptor mediates the neuromodulatory effects of natriuretic peptides. They also represent a significant advancement in support for a signaling role for the NPR-C because they indicate that blockade of the cytosolic region of the NPR-C eliminates natriuretic peptide effects on neurotransmitter release. Prior data indicating signal-transducing activities of the NPR-C had relied on the pharmacological selectivity of peptides for the extracellular region of the receptor. The inhibitory effect of the antibody also appeared to be specific because prostaglandin effects to reduce dopamine efflux persisted in the presence of the antibody. The specificity of the antibody was confirmed further by dot blot analyses. Moreover, previous investigations indicated that the digitonin permeabilization employed allows entry of antibodies into adrenergic cells (26). Thus, these data are consistent with the NPR-C mediating neuromodulatory actions of natriuretic peptides because the antibody to a specific region of the NPR-C prevented the inhibition of dopamine efflux caused by natriuretic peptides.
The carboxy terminal 19 amino acids of the NPR-C failed to influence evoked dopamine efflux, indicating that it is not involved in mediating neuromodulatory effects of natriuretic peptides. The absence of neuromodulatory activity attributable to the cytosolic nonadecapeptide of the NPR-C provides a negative control indicating that not all peptides exhibit this activity. Furthermore, the content of basic and acidic amino acids in the two fractions is nearly identical, with the carboxy terminal fragment containing one more acidic amino acid and one less basic amino acid than the pentadecapeptide fragment. These data indicate that the activity of the pentadecapeptide fragment is not attributable to a random content of basic or acidic amino acids but requires a specific sequence of amino acids such as that described by Nishimoto (22). The specificity of the amino acid sequence of the pentadecapeptide is underscored further by the inability of scrambled peptides with the same amino acid content to mimic its activity.
Our conclusions regarding a signaling role for the NPR-C are consistent with those of many other investigators, although many of these known signal-transducing functions of the NPR-C would be expected to potentiate, not inhibit, evoked catecholamine efflux. For instance, the NPR-C has been implicated as an activator of both protein kinase C (27, 28) and calcium channels (29, 30) and a suppressor of both adenylyl cyclase (7, 8) and tyrosine kinase activity (31). The NPR-C also is thought to mediate antimitogenic effects of natriuretic peptides in both vascular smooth muscle (32) and glial cells (33). Thus this study adds to the evidence that the NPR-C is a signaling entity. The major advantage of the current work over these prior studies involves the demonstration that a portion of the NPR-C actually can mimic the biological activity of endogenous natriuretic peptides and that an antibody to the active portion of the NPR-C eliminates neuromodulatory activity of the natriuretic peptides.
Collectively, these data are consistent with the NPR-C mediating inhibitory neuromodulatory effects of natriuretic peptides on evoked dopamine efflux. Specifically, a 15-amino acid peptide, representing the juxtamembrane cytosolic region of the receptor, accounted for the majority of the neuromodulatory activity of the receptor. The greater potency of the entire cytosolic region of the receptor relative to the amino terminal pentadecapeptide could indicate that extensions of this receptor region enhance potency. The remarkable simplicity of this receptor provides an exceptional opportunity to investigate basic mechanisms accounting for both regulatory and signal-transducing mechanisms within receptors. Most receptors are far more complex, containing hundreds of amino acids in their cytosolic regions with the heptahelical receptors having the added complication of numerous peptide loops obfuscating potential interactions with signal-transduction systems. The NPR-C receptor only contains 37 amino acids in its cytosolic region and this study suggests that this short cytoplasmic segment has signal-transducing functions.
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Acknowledgments |
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Footnotes |
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Received July 15, 1998.
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References |
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2 adrenergic receptor i3c region. Mol Pharmacol 50:351–358[Abstract]
-opioid receptor involved in G protein
coupling using site-specific synthetic peptides. Mol Pharmacol 50:985–993[Abstract]
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