EDITORIAL FOCUS
G protein-dependent activation of smooth muscle eNOS via
natriuretic peptide clearance receptor
K. S.
Murthy,
B.-Q.
Teng,
J.-G.
Jin, and
G. M.
Makhlouf
Departments of Physiology and Medicine, Medical College of
Virginia, Virginia Commonwealth University, Richmond, Virginia
23298-0711
 |
ABSTRACT |
In gastrointestinal smooth muscle, the neuropeptides vasoactive
intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) induce relaxation by interacting with
VIP2/PACAP3 receptors coupled via Gs to
adenylyl cyclase and with distinct receptors coupled via
Gi1 and/or
Gi2 to a smooth muscle endothelial nitric oxide synthase (eNOS). The present study identifies the receptor
as the single-transmembrane natriuretic peptide clearance receptor
(NPR-C). RT-PCR and Northern analysis demonstrated expression of the
natriuretic peptide receptors NPR-C and NPR-B but not NPR-A in rabbit
gastric muscle cells. In binding studies using
125I-labeled atrial natriuretic
peptide (125I-ANP) and
125I-VIP as radioligands, VIP,
ANP, and the selective NPR-C ligand cANP(4-23) bound with high
affinity to NPR-C. ANP, cANP-(4-23), and VIP initiated identical
signaling cascades consisting of
Ca2+ influx, activation of eNOS
via Gi1 and
Gi2, stimulation of cGMP formation, and muscle relaxation. NOS activity and cGMP formation were
abolished (93 ± 3 to 96 ± 2% inhibition) by nifedipine,
pertussis toxin, the NOS inhibitor,
NG-nitro-L-arginine,
and the antagonists ANP-(1-11) and VIP-(10-28). NOS activity
stimulated by all three ligands in muscle membranes was additively
inhibited by Gi1 and
Gi2 antibodies (82 ± 2 to 84 ± 1%). In reconstitution studies, VIP, cANP-(4-23), and guanosine 5'-O-(3-thiotriphosphate) stimulated NOS activity in
membranes of COS-1 cells cotransfected with NPR-C and eNOS. The
results establish a unique mechanism for G protein-dependent activation of a constitutive NOS expressed in gastrointestinal smooth muscle involving interaction of the relaxant neuropeptides VIP and PACAP with a single-transmembrane natriuretic peptide receptor, NPR-C.
endothelial nitric oxide synthase; nitric oxide; smooth muscle
relaxation; natriuretic peptide receptors; cyclic nucleotides; signal
transduction; vasoactive intestinal peptide; pituitary adenylate
cyclase-activating peptide
 |
INTRODUCTION |
THE HOMOLOGOUS PEPTIDE NEUROTRANSMITTERS, vasoactive
intestinal peptide (VIP) and pituitary adenylate cyclase-activating
peptide (PACAP), are potent relaxants of vascular and visceral smooth muscle (6, 32, 34). Both neuropeptides are colocalized with nitric
oxide synthase (NOS) in neurons of the enteric nervous system (11):
nitric oxide (NO) formed in nerve terminals regulates the release of
VIP and PACAP and participates in gastric and intestinal smooth muscle
relaxation (14, 18). In turn, VIP and PACAP regenerate NO in smooth
muscle cells by activating a constitutive smooth muscle NOS, recently
identified as endothelial NOS (eNOS) by in situ RT-PCR in single
dispersed gastric smooth muscle cells and by cloning and sequence
analysis (21, 29, 35).
The pathway involved in VIP/PACAP-stimulated NO formation in
gastrointestinal smooth muscle is initiated by G protein-dependent stimulation of Ca2+ influx and
activation of eNOS bound to calmodulin in the plasma membrane (27). In
turn, NO activates soluble guanylyl cyclase, resulting in formation of
cGMP and activation of cGMP-dependent protein kinase (cG-kinase) (29).
VIP- or PACAP-stimulated NO formation in smooth muscle membranes is
inhibited by pretreatment of muscle cells with pertussis toxin (PTx)
and by incubation of smooth muscle membranes with guanosine
5'-O-(2-thiodiphosphate) (GDP
S) or
G
i1-2 antibody, implying
involvement of inhibitory G proteins in VIP- or PACAP-mediated
activation of smooth muscle NOS (27).
Previous studies have shown that both VIP and PACAP interact with
distinct, G protein-coupled receptors (29). We have recently shown that
one of these receptors is the VIP2
receptor (also known as the PACAP3
receptor), which exhibits equally high affinity for VIP and PACAP and
is coupled via Gs to adenylyl
cyclase (1, 3, 36, 37). The identity of the receptor that mediates VIP/PACAP-dependent activation of eNOS in gastrointestinal smooth muscle is not known. Akiho et al. (2) have recently reported that VIP
and the atrial natriuretic peptide (ANP) compete for binding to cecal
muscle cells and that relaxation of these cells by ANP is blocked by
NOS inhibitors. Neither the receptor nor the pathway involved in
relaxation was identified. We have postulated that the natriuretic
peptide clearance receptor (NPR-C), which can couple to inhibitory G
proteins (3), could be the shared receptor with which VIP/PACAP and ANP
interact to activate smooth muscle NOS. NPR-C is widely expressed and
is the predominant natriuretic peptide receptor in vascular and
visceral smooth muscle (4, 17, 33). The receptor exhibits high affinity
for all natriuretic peptides (ANP, BNP, and CNP). The present studies
provide functional and molecular evidence that VIP interacts with
NPR-C, which is coupled via Gi1
and Gi2 to activation of eNOS in
gastric smooth muscle cells. Reconstitution experiments in COS-1 cells
cotransfected with NPR-C and eNOS confirmed the ability of VIP to
activate eNOS in a G protein-dependent fashion.
 |
MATERIALS AND METHODS |
Dispersion of gastric smooth muscle
cells. Muscle cells were isolated from the circular
muscle layer of rabbit stomach by sequential enzymatic digestion,
filtration, and centrifugation as described previously (25-29).
The cells were harvested by filtration through 500-µm Nitex followed
by two centrifugations at 350 g for 10 min.
Binding of 125I-labeled ANP and
125I-VIP to dispersed muscle cells.
Radioligand binding to dispersed muscle cells was done as described
previously (25, 29). Triplicate samples (0.3 ml) of cell suspension
(106 cells/ml) were incubated for
5 min with 50 pM radioligand
(125I-ANP or
125I-VIP) in the presence or
absence of unlabeled ligand. Bound and free radioligands were separated
by rapid filtration. Nonspecific binding was 22 ± 6% of total
binding for ANP and 36 ± 5% for VIP. In some experiments, binding
and functional assays were done in cells enriched with NPR-C, with the
use of the selective NPR-C ligand cANP-(4-23) as protective ligand
(3, 22). Muscle cells were incubated with 1 µM cANP-(4-23) for 2 min at 31°C and then for 20 min with 5 µM
N-ethylmaleimide (NEM) to inactivate
all residual receptors (20, 25, 26). The cells were centrifuged twice
for 10 min at 350 g to remove NEM and
protective ligand and were resuspended in HEPES medium. Binding and
relaxation were also measured after selective desensitization of VIP
receptors as previously described (25, 29). The cells were incubated at
31°C for 30 min with 1 µM VIP, centrifuged twice for 10 min at
350 g, and resuspended in HEPES medium.
Measurement of cAMP, cGMP, cytosolic
Ca2+, and
relaxation in dispersed smooth muscle cells.
cAMP and cGMP were measured by radioimmunoassay as described previously
(25, 29). Agonists were added to 0.5 ml of muscle cell suspension
(106 cells/ml) in the presence of
10 µM IBMX and the reaction terminated after 60 s; the results were
expressed as picomoles per 106
cells above basal level. Intracellular
Ca2+ concentration
([Ca2+]i)
was measured in muscle cells loaded with fura 2 as described previously, and an estimate of
[Ca2+]i
was obtained from observed, maximal, and minimal fluorescence (25, 29).
Relaxation was measured in muscle cells contracted with cholecystokinin
octapeptide (CCK-8) as previously described (6, 25, 29). Relaxant
agonists were added for 60 s to 0.5 ml of cell suspension
(104 cells/ml); CCK-8 (1 nM) was
then added for 30 s and the reaction was terminated with 1% acrolein.
Relaxation was expressed as the increase in length of CCK-contracted
muscle cells.
Measurement of NOS activity in dispersed muscle cells
and muscle membranes. NOS activity in dispersed muscle
cells was measured from the formation of
L-[3H]citrulline
in cells loaded with
L-[3H]arginine
as previously described (8, 25, 29).
L-[3H]arginine
(3 µCi/ml) was added to 1 ml of cell suspension for 10 min; the cells
were treated during the last minute with ANP, cANP-(4-23), or VIP
(1 µM).
L-[3H]citrulline
formation was expressed as counts per minute (cpm) per
106 cells above basal levels
measured in separate samples.
NOS activity was also measured by a modification of the method of Bush
et al. (9) in membrane fractions prepared from dispersed muscle cells
as previously described (27). Membrane protein (0.4 mg) was incubated
for 15 min at 31°C in 50 mM Tris · HCl buffer (pH
7.4) containing 50 µM
L-arginine and
~150,000 cpm of L-[3H]arginine
(sp act 58.7 Ci/mmol), 1 mM NADPH, 1 mM DTT, 4 µM FMN, 4 µM FAD, 10 µM tetrahydrobiopterin, 2 µg calmodulin (10 µg/ml), and
Ca2+ (0.1 mM) in a final volume of
200 µl. In some experiments, the medium contained 100 µM GTP, 5 mM
creatine phosphate, and 50 U/ml creatine phosphokinase.
L-[3H]citrulline
formation was expressed as picomoles
L-citrulline per milligram
protein per minute.
Identification of G proteins activated by VIP, ANP,
and cANP-(4-23). G proteins selectively activated
by ANP, cANP-(4-23), or VIP were identified by an adaptation of
the method of Okamoto et al. (30) as previously described (26). Muscle
membranes were solubilized in CHAPS and incubated at 37°C with 60 nM [35S]GTP
S in a
medium containing 10 mM HEPES (pH 7.4), 100 µM EDTA, and 10 mM
MgCl2. After the reaction was
stopped, the solubilized membranes were placed in wells precoated with
specific antibodies to G
i1,
G
i2,
G
i3,
G
s, and
G
q/11. After incubation for 2 h on ice, the wells were washed three times with phosphate buffer solution containing 0.05% Tween-20, and the radioactivity in each well
was counted.
Expression of natriuretic peptide receptor subtypes in
smooth muscle cells. Expression of natriuretic peptide
receptor subtypes was determined by RT-PCR and Northern blotting and
confirmed for NPR-C by cloning and cDNA sequencing of the PCR product.
Total RNA was isolated from freshly dispersed and cultured (first
passage) gastric smooth muscle cells, and 6 µg were reverse
transcribed in a reaction volume of 20 µl containing 50 mM
Tris · HCl (pH 8.3), 75 mM KCl, 3.0 mM
MgCl2, 10 mM dithiothreitol, 0.5 mM dNTP, 2.5 µM random hexamers, and 200 units of RT. Three
microliters of reverse transcribed cDNA were amplified by PCR (35 cycles) under standard conditions with specific primers for human
NPR-A [CAAGCGCTCATGCTCTACGCCTAC (sense),
GATGTTCTCCCCATCAGTAACAGTTC (antisense)] and NPR-B
[GTGGCCCGCTTTGCCTCCCACTGG (sense), GGTGAAGTAGTGAGGCCGGTC (antisense)] and for bovine NPR-C [CTTCTATGGAGATGGCT
(sense), TGCTTTGCAAGGAGAGC (antisense)] (10, 15). The
amplified PCR products were analyzed on 1% agarose gel containing 0.1 µg/ml ethidium bromide. Cloned cDNAs for rat NPR-A, NPR-B, and NPR-C were used as positive controls for PCR under the same conditions. The
PCR product obtained with NPR-C-specific primers was purified by
electrophoresis on 1% agarose gel and was cloned into pCR II vector
(Invitrogen). The nucleotide sequence was determined for cDNA inserts
on both strands by a DNA sequencer. For Northern analysis, 20 µg of
total RNA were fractionated by electrophoresis in 1.1% formaldehyde
agarose gel and transferred to a nylon membrane. cDNA inserts for NPR-A
and NPR-B using full-length rat cDNA, and for NPR-C using the cloned
541-bp RT-PCR product, were labeled with
32P using random hexamers as a
probe. Hybridization was carried out under standard conditions, and
autoradiography was performed at
80°C for 12 h.
Expression of NPR-C and eNOS in COS-1
cells. The 3.7 kb of bovine eNOS cDNA and the 1.7 kb of
rat NPR-C cDNA cloned at the EcoR I
site of pBluescript were digested with
EcoR I and purified by agarose gel.
The purified cDNA inserts were subcloned into the mammalian expression
vector pCDL-SR
at the EcoR I site
in the sense orientation. COS-1 cells (2-2.5 × 106) were transfected with 15 µg of eNOS cDNA or cotransfected with 15 µg each of eNOS and NPR-C
cDNA in pCDL-SR
using the calcium phosphate precipitation method.
Control COS-1 cells were transfected with equal amounts of pCDL-SR
vector without insert under the same conditions. The transfected cells
were maintained in culture for 72-96 h. Expression was confirmed
by RT-PCR and Northern blotting for NPR-C and eNOS. For RT-PCR, the
specific primers for eNOS were CAGAGCTACGCTCAGCAG (sense) and
CGGGGAGCTGTTGTAGGG (antisense) (21), and the specific primers for NPR-C
were TGGAGGTGAAAAGTTCTGTTG (sense) and GTCATGGCAACCACAGAGAA (antisense)
(14).
Materials. ANP, cANP-(4-23), VIP,
and CCK-8 were obtained from Bachem (Torrance, CA); KT-5823 was from
Kamiya Biomedical (Thousand Oaks, CA); LY-83583, calmidazolium, and
H-89 were from Calbiochem; fura 2-AM was from Molecular Probes;
L-[3H]arginine,
125I-VIP,
125I-ANP,
125I-cAMP, and
125I-cGMP were from New England
Nuclear;
NG-nitro-L-arginine
(L-NNA) and all other chemicals
were from Sigma Chemical. NPR-A and NPR-B cDNAs were kind gifts from
Dr. David L. Garbers (University of Texas Southwestern Medical Center); NPR-C cDNA was a kind gift from Dr. David G. Lowe (Genentech); and eNOS
cDNA was a kind gift from Dr. Thomas Michel (Harvard Medical School).
 |
RESULTS |
Selective expression of NPR-C and NPR-B in gastric
smooth muscle cells. RT-PCR on RNA extracted from
cultured gastric muscle cells in first passage using NPR-C- and
NPR-B-specific primers yielded products of the expected size (541 and
228 bp, respectively; Fig. 1).
No PCR product was obtained using NPR-A-specific primers. Northern
analysis on RNA from freshly dispersed and cultured smooth muscle cells
detected a single mRNA transcript for NPR-B (4.0 kb), a main transcript
for NPR-C (7.9 kb) with some of smaller size (<3 kb), but none for
NPR-A. Cloning and sequence analysis of the PCR product obtained with
NPR-C-specific primers showed close similarity of the predicted amino
acid sequences in rabbit to those in bovine (94%), human (93%), and
rat (92%) proteins.

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 1.
Expression of natriuretic peptide clearance receptor (NPR-C) and NPR-B
in freshly dispersed and cultured gastric smooth muscle cells. Total
RNA was isolated from freshly dispersed and cultured (first passage)
rabbit gastric smooth muscle cells and reverse transcribed. The cDNA
was amplified with specific bovine NPR-C primers and human NPR-A and
NPR-B primers. Experiments were done in the presence and absence of RT.
A: PCR products of expected size were
obtained with NPR-C and NPR-B primers but not NPR-A primers.
B: transcripts corresponding to NPR-B
(4.0 kb) and NPR-C (7.9 kb) but not NPR-A were detected by Northern
analysis in freshly dispersed (lane
1) and cultured gastric smooth muscle cells
(lane 2). MW, molecular weight.
|
|
Binding of 125I-VIP and
125I-ANP to dispersed smooth muscle cells.
Both 125I-VIP and
125I-ANP bound with high affinity
to dispersed muscle cells, with
IC50 values of 4 ± 1 and 24 ± 6 nM, respectively (Fig. 2). The
competition binding curves could be resolved into high-affinity and
low-affinity binding sites with dissociation constant
values of 0.16 ± 0.03 and 40.2 ± 6.2 nM for VIP and 0.23 ± 0.4 and 155 ± 65 nM for ANP.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 2.
Characteristics of 125I-labeled
atrial natriuretic peptide
(125I-ANP;
A) and vasoactive intestinal peptide
(125I-VIP;
B) binding to dispersed smooth
muscle cells. Specific 125I-ANP
binding was completely inhibited by ANP and partially by VIP and
cANP-(4-23). Specific
125I-VIP binding was completely
inhibited by VIP and partially by ANP and cANP-(4-23). Triplicate
samples (0.3 × 106 cells)
were incubated for 5 min with 50 pM
125I-ANP or
125I-VIP alone or in the presence
of unlabeled ligand. Bound and free radioligands were separated by
rapid filtration followed by repeated washing (4 times) with ice-cold
HEPES medium containing 0.2% BSA. Nonspecific
125I-VIP and
125I-ANP binding was 36 ± 5 and 22 ± 6%, respectively. Values are means ± SE of 5 experiments.
|
|
125I-ANP binding was partly
(47-55%) inhibited by VIP and the selective NPR-C ligand,
cANP-(4-23), whereas 125I-VIP
binding was partly (55-61%) inhibited by ANP and cANP-(4-23) (Fig. 2). The pattern implied that both ANP and VIP bound with high
affinity to NPR-C receptors selectively recognized by cANP-(4-23). VIP bound also to receptors (i.e.,
VIP2/PACAP3)
that were not recognized by ANP or cANP(4-23), whereas ANP
bound also to receptors (i.e., NPR-B) that were not recognized by VIP
or cANP(4-23).
The pattern of binding was corroborated by studies in which the
VIP2/PACAP3
receptors were selectively desensitized or inactivated. Previous
studies had shown that the
VIP2/PACAP3
receptors coupled to adenylyl cyclase in smooth muscle were readily
desensitized, whereas the unidentified receptors coupled to NOS were
more resistant to desensitization (25, 29). Selective desensitization
of VIP2/PACAP3
receptors by exposure to VIP for 30 min decreased 125I-VIP binding by 52 ± 2% but had no effect on
125I-ANP binding; residual
125I-VIP binding was completely
inhibited by ANP and cANP-(4-23). Exposure of the cells to ANP for
30 min had no effect on 125I-ANP
or 125I-VIP binding (data not
shown). In muscle cells treated for 2 min with cANP(4-23) so
as to protect NPR-C, and then for 20 min with NEM to inactivate all
unprotected receptors, levels of both 125I-VIP and
125I-ANP binding were decreased
(40 ± 5 and 42 ± 4%, respectively); residual
125I-VIP and
125I-ANP binding was abolished by
VIP, ANP, or cANP-(4-23), implying that VIP and ANP can bind to
NPR-C, from which they could be displaced by VIP, ANP, or the selective
NPR-C ligand, cANP-(4-23).
Signaling cascade initiated by activation of NPR-C
receptors. Both ANP and cANP-(4-23) increased
[Ca2+]i,
NOS activity, and cGMP formation in dispersed muscle cells. The
increases in
[Ca2+]i
induced by ANP and cANP-(4-23) (145 ± 34 and 169 ± 32 nM,
respectively, above a resting
[Ca2+]i
of 60 ± 5 nM), NOS activity (73 ± 11 and 61 ± 8%
above basal levels, respectively), and cGMP formation (82 ± 13 and
75 ± 4% above basal level, respectively) were abolished by
nifedipine (1 µM; Fig. 3).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 3.
Inhibition of nitric oxide synthase (NOS;
A) activity and cGMP formation
(B) in dispersed smooth muscle cells
by blockade of various steps in the signaling pathway. Smooth muscle
cells were stimulated with ANP (1 µM) or cANP-(4-23) (1 µM)
after treatment for 60 min with pertussis toxin (PTx; 800 ng/ml) or for
10 min with nifedipine (1 µM),
NG-nitro-L-arginine
(L-NNA; 100 µM), or
calmidazolium (Calmidaz; 1 µM). NOS activity was expressed as counts
per minute (cpm) of
L-[3H]citrulline/106
cells above basal level (1,064 ± 127 cpm
L-[3H]citrulline/106
cells), and cGMP formation was measured by radioimmunoassay and
expressed as pmol/106 cells above
basal level (0.6 ± 0.2 pmol/106 cells). Values are means ± SE of 3-5 experiments.
|
|
NOS activity and cGMP formation stimulated by ANP and cANP-(4-23)
were inhibited also by 1) PTx (Fig.
3), 2) the NOS inhibitor L-NNA (Fig. 3),
3) the calmodulin antagonist
calmidazolium (Fig. 3), and 4) VIP
and ANP receptor antagonists [VIP-(10-28) and
ANP-(1-11)] (Fig. 4).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 4.
Inhibition of NOS activity, cGMP formation, and muscle relaxation in
dispersed smooth muscle cells by ANP and VIP receptor antagonists.
Smooth muscle cells were stimulated with cANP-(4-23) (1 µM) in
the presence and absence of VIP-(10-28) (10 µM) or
ANP-(1-11) (10 µM). NOS activity and cGMP formation were
expressed as percent increase above basal level, and relaxation as
percent increase in the length of muscle cells precontracted with
cholecystokinin octapeptide. Identical results (not shown) were
obtained with ANP (1 µM). Values are means ± SE of 3-5
experiments.
|
|
Relaxation of dispersed smooth muscle cells induced by ANP and
cANP-(4-23) was also inhibited by PTx, nifedipine,
L-NNA, ANP-(1-11), and
VIP-(10-28) (range 93 ± 3 to 96 ± 2% inhibition). Both
cGMP formation and muscle relaxation were completely inhibited (95 ± 2 and 96 ± 3%) by the soluble guanylyl cyclase inhibitor
LY-83583 (1 µM). KT-5823, a selective inhibitor of cG-kinase when
used at a concentration of 1 µM (28), abolished muscle relaxation elicited by ANP and cANP-(4-23) but only partly inhibited muscle relaxation induced by VIP; residual muscle relaxation by VIP was abolished by H-89, a selective inhibitor of cA-kinase (data not shown).
Relaxation induced by VIP was also inhibited by VIP-(10-28) (78 ± 8% inhibition) and ANP-(1-11) (53 ± 5%).
The patterns of stimulation of
[Ca2+]i,
NOS activity, and cGMP formation by ANP and cANP-(4-23) and
inhibition of both responses by various agents were identical to those
previously reported for VIP and PACAP (25, 29). The signaling cascade
was triggered by agonist-induced
Ca2+ influx and mediated by a
Ca2+/calmodulin-dependent NOS. It
is noteworthy that cGMP formation and relaxation by ANP were abolished
by inhibitors of NOS and soluble guanylyl cyclase, implying that ANP
interacted preferentially with NPR-C, for which it has high affinity,
triggering a cascade involving NO-dependent activation of soluble
guanylyl cyclase. ANP has a much lower affinity (2 × 103 times less) for NPR-B, the
only natriuretic peptide receptor/guanylyl cyclase expressed in gastric
muscle cells (Fig. 1) (5, 33).
Receptor binding and signal transduction in muscle
cells enriched with NPR-C.
125I-VIP and
125I-ANP binding, cGMP and cAMP
formation, and muscle relaxation were also measured in smooth muscle
cells in which NPR-C was selectively preserved, and all other receptors
including NPR-B and
VIP2/PACAP3 receptors were inactivated. In these cells, control
125I-ANP and
125I-VIP binding decreased and the
residual binding was abolished by VIP, cANP-(4-23), and ANP. The
pattern of binding confirmed that VIP and ANP were capable of
interacting with NPR-C. cGMP formation stimulated by ANP,
cANP-(4-23), and VIP was not affected in these cells (control:
0.46 ± 0.08 to 0.56 ± 0.06 pmol/106 cells above basal level;
cells with NPR-C only: 0.42 ± 0.08 to 0.51 ± 0.10 pmol/106 cells), whereas cAMP
formation stimulated by VIP was abolished (control: 5.8 ± 1.1 pmol/106 cells above basal
levels; cells with NPR-C only: 0.2 ± 0.1 pmol/106 cells). Muscle cell
relaxation induced by ANP and cANP-(4-23) was not affected,
whereas relaxation induced by VIP was decreased by 43 ± 5%; the
decrease reflected suppression of the relaxant component mediated by
VIP2/PACAP3
receptors coupled to adenylyl cyclase. The relaxant responses to all
three agonists in cells where only NPR-C was preserved were abolished
by L-NNA.
Identification of G proteins coupled to
NPR-C. In membranes isolated from dispersed muscle
cells, ANP and cANP-(4-23) (in the presence of GTP) stimulated NOS
activity over and above maximal NOS activity stimulated by 100 µM
Ca2+; NOS activity stimulated by
Ca2+ and agonists was virtually
abolished by L-NNA (93 ± 2%
inhibition). Previous studies on gastric smooth muscle membranes (27)
had shown that VIP, PACAP, and GTP
S stimulated NOS activity in a concentration-dependent fashion and that pretreatment of the cells with
PTx before membrane isolation abolished agonist-stimulated NOS
activity, implying involvement of an inhibitory G protein in the
activation of gastric smooth muscle NOS; incubation of smooth muscle
membranes with a common antibody to
G
i1-2 inhibited VIP- and
PACAP-stimulated NOS activity. In the present study, incubation of
smooth muscle membranes for 60 min with
G
i1 antibody (10 µg/ml)
inhibited NOS activity stimulated by ANP and cANP-(4-23) (by 61 ± 2 to 63 ± 3%; P < 0.001),
whereas incubation with G
i2
antibody (10 µg/ml) inhibited NOS activity (by 30 ± 5 to 31 ± 10%; P < 0.05); incubation with
both antibodies elicited additive inhibition (82 ± 2 and 82 ± 1%; P < 0.001; Fig.
5). Although VIP-stimulated NOS activity
was higher, the percentage of inhibition by
G
i1 antibody (56 ± 2%;
P < 0.001),
G
i2 antibody (29 ± 2%; P < 0.001), or a combination of both
antibodies (84 ± 3%; P < 0.001)
was similar to that observed with ANP or cANP-(4-23) (Fig. 5).
Incubation with G
s,
G
o,
G
q/11, and
G
i3 antibodies had no
significant effect (1 ± 6 to 5 ± 10% inhibition).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 5.
Inhibition of NOS activity stimulated by cANP-(4-23), ANP, and VIP
in muscle membranes by antibodies to
G i1 and
G i2. NOS activity in membranes
was determined from the conversion of
L-[3H]arginine
to
L-[3H]citrulline
as previously described (Refs. 9 and 27; see MATERIALS
AND METHODS). The results represent the increase in
NOS activity (pmol citrulline · mg
protein 1 · min 1)
stimulated by ANP, cANP-(4-23), and VIP over and above maximal NOS
activity stimulated by 0.1 mM Ca2+
(28.3 ± 3.7 pmol
L-citrulline · mg
protein 1 · min 1).
Agonist-induced increase in NOS activity was partly inhibited by
incubation for 60 min with G i1
or G i2 antibody (Ab; 10 µg/ml
each) and abolished by the combination of both antibodies. Antibodies
were used at concentrations (10 µg/ml) previously shown to be
maximally effective in blocking the response mediated by the
corresponding G protein (26, 41). Values are means ± SE
of 4 experiments. Inhibition of NOS activity:
** P < 0.01.
|
|
The selective activation of Gi1
and Gi2 by ANP, cANP-(4-23),
and VIP revealed by blockade of NOS activity on neutralization with
G
i1 and
G
i2 antibodies was confirmed by
direct measurement of activation of both G proteins. In solubilized
smooth muscle membranes, both ANP and cANP-(4-23) caused a
significant increase in the binding of
[35S]GTP
S to
G
i1 and
G
i2 (determined from the
binding of a
[35S]GTP
S · G
complex to the corresponding G
antibody) but not to
G
s,
G
i3,
G
o, or
G
q/11 (Table
1). VIP and PACAP also caused a significant
increase in the binding of
[35S]GTP
S to
G
i1 and
G
i2, as well as to
G
s (Table 1); the binding to
G
s reflected activation of
VIP2/PACAP3
receptors coupled to adenylyl cyclase.
Activation of eNOS by VIP and cANP-(4-23) in
COS-1 cells cotransfected with NPR-C and eNOS. Decisive
evidence demonstrating the ability of VIP to interact with NPR-C and
activate eNOS was obtained in reconstitution experiments using COS-1
cells cotransfected with NPR-C and eNOS. Expression of NPR-C and eNOS
in COS-1 cells was identified by RT-PCR and Northern analysis (Fig.
6, A and B). In membranes from COS-1 cells
cotransfected with NPR-C and eNOS, but not in membranes from wild-type
COS-1 cells, VIP and cANP-(4-23) (in the presence of 100 µM GTP)
and GTP
S (100 µM) stimulated NOS activity [VIP: 3.4 ± 0.7, cANP-(4-23): 3.0 ± 0.6, and GTP
S: 3.8 ± 0.4 pmol
L-citrulline · mg
protein
1 · min
1]
above maximal Ca2+-induced
activity. NOS activity stimulated by these agents or Ca2+ was abolished by
L-NNA (Fig.
7B). In
contrast, in membranes from COS-1 cells transfected with eNOS only,
GTP
S stimulated NOS activity (4.3 ± 0.5 pmol
L-citrulline · mg
protein
1 · min
1)
above maximal Ca2+-induced
activity, whereas neither VIP nor cANP-(4-23) had any significant
effect (Fig. 7A).

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 6.
Transfection of COS-1 cells with endothelial NOS (eNOS) and NPR-C.
A: RT-PCR using rat NPR-C and bovine
eNOS specific primers on RNA derived from wild-type (WT) COS-1 cells
and COS-1 cells cotransfected with NPR-C and eNOS. PCR products of
expected size (NPR-C, 256 bp; eNOS, 210 bp) were detected. Full-length
rat NPR-C and bovine eNOS cDNA were used as controls.
B: Northern blots demonstrating NPR-C
and eNOS transcripts in COS-1 cells cotransfected with eNOS and
NPR-C.
|
|

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 7.
Stimulation of NOS activity in membranes from COS-1 cells transfected
with eNOS (A) or cotransfected with
eNOS and NPR-C (B). NOS activity in
membranes was determined as described in legend to Fig. 5.
A: in membranes from COS-1 cells
transfected with eNOS only, GTP S (100 µM) but not VIP (1 µM) or
cANP-(4-23) (1 µM) stimulated NOS activity over activity induced
by 100 µM Ca2+ (8.1 ± 0.4 pmol L-citrulline/mg protein);
NOS activity was abolished by
L-NNA. Values are means ± SE
of 4 experiments. B: in membranes from
COS-1 cells cotransfected with eNOS and NPR-C, VIP (1 µM) and
cANP-(4-23) (1 µM) (in the presence of GTP) and GTP S (100 µM) stimulated NOS activity over activity induced by 100 µM
Ca2+ (7.8 ± 0.5 pmol
L-citrulline/mg protein); NOS
activity was abolished by L-NNA.
Values are means ± SE of 5 experiments.
* P < 0.05;
** P < 0.01.
|
|
 |
DISCUSSION |
This study establishes the mechanism whereby the neurotransmitter
peptide VIP activates the constitutive NOS isoform (eNOS) expressed in
gastrointestinal smooth muscle cells (35). In this unique instance, NOS
acts as a membrane-bound effector enzyme directly activated by two G
proteins (Gi1 and
Gi2) that couple to the
cytoplasmic domain of a single-transmembrane receptor, the natriuretic
peptide clearance receptor (NPR-C). In other tissues, G protein-coupled
seven-transmembrane receptors transduce the Ca2+ signals required for
activation of constitutive neuronal NOS (7, 8, 23) or
eNOS (24, 31), whereas in gastrointestinal smooth muscle, a G
protein-coupled single-transmembrane receptor mediates both
Ca2+ influx and direct activation
of eNOS. As previously shown (27) and confirmed in this study, direct,
G protein-dependent activation of NOS is evident in smooth muscle
membranes and could be reproduced in membranes from COS-1 cells
transfected with eNOS or cotransfected with eNOS and NPR-C.
As depicted in Fig. 8, VIP and PACAP
interact with cognate seven-transmembrane receptors
(VIP2/PACAP3)
coupled via Gs to adenylyl cyclase
(27, 36) and with single-transmembrane receptors (NPR-C) coupled via
Gi1 and
Gi2 to smooth muscle eNOS.
Although the NPR-C is devoid of cytoplasmic guanylyl cyclase and kinase
domains and normally serves to internalize and degrade natriuretic
peptides, its truncated 37-amino acid carboxy terminal appears to
activate inhibitory G proteins coupled to various effector enzymes (3, 12, 16, 19). The NPR-C-mediated coupling of
Gi1 and
Gi2 results in activation of
smooth muscle eNOS.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 8.
Dual signaling cascades initiated by interaction of VIP or pituitary
adenylate cyclase-activating peptide (PACAP) with single-transmembrane
NPR-C and seven-transmembrane VIP2/PACAP3 receptors. The NPR-C cascade
involves G protein (Gi1 and
Gi2)-dependent stimulation of
Ca2+ influx and activation of a
membrane-bound eNOS. Generation of NO leads to sequential activation of
soluble guanylyl cyclase (GC) and cGMP-dependent protein kinase
(cG-kinase). cG-kinase (activated by cGMP and cross-activated by cAMP)
and cAMP-dependent protein kinase (cA-kinase) are jointly responsible
for smooth muscle relaxation.
|
|
Evidence for the involvement of NPR-C in mediating activation of eNOS
by VIP is based on 1) radioligand
binding studies demonstrating interaction of VIP with a complement of
receptors recognized by the selective NPR-C ligand, cANP-(4-23);
2) pharmacological evidence that
cANP-(4-23) and VIP initiate identical signaling cascades involving coupling to specific G proteins
(Gi1 and
Gi2);
3) blockade of the signaling
cascades with both VIP and ANP antagonists; and 4) reconstitution experiments in
which VIP was shown to activate eNOS in COS-1 cells cotransfected with
NPR-C and eNOS.
The steps in the cascade leading to NO formation initiated by the
selective NPR-C agonist, cANP-(4-23), as well as by ANP, which has
high affinity for NPR-C (3, 5, 22, 33), are identical to those
initiated by VIP (Fig. 8) (25, 27, 29). cANP-(4-23) interacted
exclusively with NPR-C, whereas ANP interacted with both NPR-C and
NPR-B (the only other natriuretic peptide expressed in gastric muscle),
and VIP interacted with both NPR-C and
VIP2/PACAP3
receptors. This conclusion was confirmed by functional and binding
studies in naive cells and in cells where only NPR-C was preserved by
selective receptor protection, or where
VIP2/PACAP3 receptors were selectively eliminated by desensitization.
NOS activity and cGMP formation stimulated by VIP/PACAP,
cANP-(4-23), and ANP (25, 29) were abolished by PTx, implying that
they were mediated by one or more inhibitory G proteins. Previous
studies had shown that NOS activity stimulated by VIP and PACAP in
muscle membranes was inhibited by a common antibody to
G
i1 and
G
i2 (27). In the present study,
specific antibodies to G
i1 and
G
i2 inhibited NOS activity
stimulated by VIP, cANP-(4-23), and ANP. The effects of both
antibodies when used at optimal concentrations were additive, causing
>80% inhibition of NOS activity. Selective activation of
Gi1 and
Gi2 by ANP and cANP-(4-23)
was corroborated by direct measurement of
G
i1 and
G
i2 binding to
[35S]GTP
S. VIP
activated both Gi1 and
Gi2 as well as
Gs, which couples VIP2/PACAP3
receptors to adenylyl cyclase.
Reconstitution experiments using membranes derived from COS-1 cells
cotransfected with eNOS and NPR-C confirmed that VIP and cANP-(4-23) stimulate NOS activity in a G protein-dependent
fashion, over and above NOS activity stimulated by a maximally
effective concentration of Ca2+
(27). Consistent with involvement of a G protein, GTP
S alone stimulated NOS activity in membranes from COS-1 cells transfected with
eNOS only (Fig. 7A) or cotransfected
with eNOS and NPR-C (Fig. 7B).
Previous studies (27) on membranes isolated from dispersed gastric
muscle cells showed that GTP
S, VIP, and PACAP stimulated NOS
activity in a concentration-dependent fashion and that NOS activity was
abolished by GDP
S.
In summary, the relaxant neuropeptides, VIP and PACAP, initiate dual
signaling cascades by interacting with seven-transmembrane VIP2/PACAP3
receptors coupled via Gs to
activation of adenylyl cyclase and single-transmembrane natriuretic
receptors (NPR-C) coupled via Gi1
and Gi2 to activation of
membrane-bound,
Ca2+/calmodulin-dependent eNOS.
The signaling cascades make optimal use of the cyclic nucleotide system
in smooth muscle and underlie the potency of VIP and PACAP as relaxant neurotransmitters.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-28300.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: G. M. Makhlouf, PO Box 980711, Medical
College of Virginia, Richmond, VA 23298-0711.
Received 13 July 1998; accepted in final form 1 September 1998.
 |
REFERENCES |
1.
Adamou, J. E.,
N. Aiyar,
S. Van Horn,
and
N. A. Elshourbagy.
Cloning and functional characterization of the human intestinal peptide (VIP2) receptor.
Biochem. Biophys. Res. Commun.
209:
385-392,
1995[Medline].
2.
Akiho, H.,
Y. Chijiiwa,
H. Okabe,
N. Harada,
and
H. Nawata.
Interaction between atrial natriuretic peptide and vasoactive intestinal peptide in guinea pig cecal smooth muscle.
Gastroenterology
109:
1105-1112,
1995[Medline].
3.
Anand-Srivastava, M. B.,
P. D. Sehl,
and
D. G. Lowe.
Cytoplasmic domain of natriuretic peptide receptor-C inhibits adenylyl cyclase: involvement of a pertussis toxin-sensitive G protein.
J. Biol. Chem.
271:
19324-19329,
1996[Abstract/Free Full Text].
4.
Anand-Srivastava, M. B.,
and
G. J. Trachte.
Atrial natriuretic factor receptors and signal transduction mechanisms.
Pharmacol. Rev.
45:
455-497,
1993[Medline].
5.
Bennett, B. D.,
G. L. Bennett,
R. V. Vitangcol,
J. R. S. Jewett,
J. Burnier,
W. Henzel,
and
D. G. Lowe.
Extracellular domains-IgG fusion for three human natriuretic peptide receptors.
J. Biol. Chem.
266:
23060-23067,
1991[Abstract/Free Full Text].
6.
Bitar, K. N.,
and
G. M. Makhlouf.
Relaxation of isolated gastric smooth muscle cells by vasoactive intestinal peptide.
Science
216:
531-533,
1982[Medline].
7.
Bredt, D. S.,
P. M. Hwang,
and
S. H. Snyder.
Localization of nitric oxide synthase indicating a neuronal role for nitric oxide.
Nature
347:
768-770,
1990[Medline].
8.
Bredt, D. S.,
and
S. H. Snyder.
Isolation of nitric oxide synthetase, a calmodulin requiring enzyme.
Proc. Natl. Acad. Sci. USA
87:
682-685,
1990[Abstract].
9.
Bush, P. A.,
N. E. Gonzalez,
and
L. J. Ignarro.
Nitric oxide synthase from cerebellum catalyzes the formation of equimolar quantities of nitric oxide and citrulline from L-arginine.
Biochem. Biophys. Res. Commun.
186:
308-314,
1992[Medline].
10.
Canaan-Kuhl, S.,
B. D. Jamison,
R. E. Myers,
and
R. Pratt.
Identification of "B" receptor for natriuretic peptide in human kidney.
Endocrinology
130:
550-552,
1992[Abstract].
11.
Costa, M.,
J. B. Furness,
S. Pompolo,
S. J. H. Brookes,
J. C. Bornstein,
D. S. Bredt,
and
S. H. Snyder.
Projections and chemical coding of neurons with immunoreactivity for nitric oxide synthase in guinea pig small intestine.
Neurosci. Lett.
148:
121-125,
1992[Medline].
12.
Drewett, J. G.,
and
D. L. Garbers.
The family of guanylyl cyclase receptors and their ligands.
Endocr. Rev.
15:
135-162,
1994[Medline].
13.
Engel, A. M.,
J. R. Schoenfeld,
and
D. G. Lowe.
A single residue determines the distinct pharmacology of rat and human natriuretic peptide receptor-C.
J. Biol. Chem.
269:
17005-17008,
1994[Abstract/Free Full Text].
14.
Grider, J. R.,
and
J.-G. Jin.
VIP release and L-citrulline production from isolated ganglia of the myenteric plexus: regulation of VIP release by nitric oxide.
Neuroscience
54:
521-526,
1993[Medline].
15.
Hagiwara, H.,
H. Sakaguchi,
K. M. Lodhi,
K. Suda,
and
S. Hirose.
Subtype switching of natriuretic peptide receptors in rat chondrocytes during in vitro culture.
J. Biochem.
116:
606-609,
1994[Abstract].
16.
Hirata, M.,
C.-H. Chang,
and
F. Murad.
Stimulatory effects of atrial natriuretic factor on phosphoinositide hydrolysis in cultured bovine aortic smooth muscle cells.
Biochim. Biophys. Acta
1010:
346-351,
1989[Medline].
17.
Jamison, R. L.,
S. Canaan-Kuhl,
and
R. Pratt.
Physiology and cell biology update: the natriuretic peptides and their receptors.
Am. J. Kidney Dis.
10:
519-530,
1992.
18.
Jin, J.-G.,
K. S. Murthy,
J. R. Grider,
and
G. M. Makhlouf.
Stoichiometry of neurally induced VIP release, NO formation, and relaxation in rabbit gastric muscle.
Am. J. Physiol.
271 (Gastrointest. Liver Physiol. 34):
G357-G369,
1996[Abstract/Free Full Text].
19.
Koller, K. J.,
and
D. V. Goeddel.
Molecular biology of the natriuretic peptides and their receptors.
Circulation
86:
1081-1088,
1992[Abstract].
20.
Kuemmerle, J. F.,
D. C. Martin,
K. S. Murthy,
J. M. Kellum,
J. R. Grider,
and
G. M. Makhlouf.
Co-existence of contractile and relaxant 5-HT receptors coupled to distinct signaling pathways in intestinal muscle cells: convergence of the pathways on Ca2+ mobilization.
Mol. Pharmacol.
42:
1090-1096,
1992[Abstract].
21.
Lamas, S.,
P. A. Marsden,
G. K. Li,
P. Tempst,
and
T. Michel.
Endothelial nitric oxide synthase: molecular cloning and characterization of a distinct constitutive enzyme isoform.
Proc. Natl. Acad. Sci. USA
89:
6348-6352,
1992[Abstract].
22.
Maack, T.,
M. Suzuki,
F. A. Almeida,
D. Nussenzveig,
R. M. Scarborough,
G. A. McEnroe,
and
J. A. Lewicki.
Physiological role of silent receptors of atrial natriuretic factor.
Science
238:
675-678,
1987[Medline].
23.
Moncada, S.,
R. M. J. Palmer,
and
E. A. Higgs.
Nitric oxide: physiology, pathophysiology, and pharmacology.
Pharmacol. Rev.
43:
109-142,
1991[Medline].
24.
Murad, F.
The nitric oxide-cyclic GMP signal transduction system for intracellular and intercellular communication.
Recent Prog. Horm. Res.
49:
239-248,
1994[Medline].
25.
Murthy, K. S.,
J.-G. Jin,
J. R. Grider,
and
G. M. Makhlouf.
Characterization of PACAP receptors and signaling pathways in rabbit gastric muscle cells.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G1391-G1399,
1997[Abstract/Free Full Text].
26.
Murthy, K. S.,
and
G. M. Makhlouf.
Differential coupling of muscarinic m2 and m3 receptors to adenylyl cyclases V/VI in smooth muscle. Concurrent m2-mediated inhibition via G
i3 and m3-mediated stimulation via G
q.
J. Biol. Chem.
272:
21317-21324,
1997[Abstract/Free Full Text].
27.
Murthy, K. S.,
and
G. M. Makhlouf.
VIP/PACAP-mediated activation of membrane-bound NO synthase in smooth muscle is mediated by pertussis toxin-sensitive Gi1-2.
J. Biol. Chem.
269:
15977-15980,
1994[Abstract/Free Full Text].
28.
Murthy, K. S.,
and
G. M. Makhlouf.
Interaction of cA-kinase and cG-kinase in mediating relaxation of dispersed smooth muscle cells.
Am. J. Physiol.
268 (Cell Physiol. 37):
C171-C180,
1995[Abstract/Free Full Text].
29.
Murthy, K. S.,
K.-M. Zhang,
J.-G. Jin,
J. R. Grider,
and
G. M. Makhlouf.
VIP-mediated and G protein-coupled Ca2+ influx activates a constitutive NOS in dispersed gastric muscle cells.
Am. J. Physiol.
265 (Gastrointest. Liver Physiol. 28):
G660-G671,
1993[Abstract/Free Full Text].
30.
Okamoto, T.,
T. Ikezu,
Y. Murayama,
E. Ogata,
and
I. Nishimoto.
Measurement of GTP
S binding to specific G proteins in membranes using G-protein antibodies.
FEBS Lett.
305:
125-128,
1992[Medline].
31.
Palmer, R. M. J.,
A. G. Ferrige,
and
S. Moncada.
Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor.
Nature
327:
524-526,
1987[Medline].
32.
Said, S. I.,
and
V. Mutt.
Isolation from porcine intestinal wall of an active octacosapeptide related to secretin and to glucagon.
Eur. J. Biochem.
28:
199-204,
1972[Medline].
33.
Suga, S.-I.,
K. Nakao,
K. Hosoda,
M. Mukoyama,
Y. Ogawa,
G. Shirakami,
Y. Saito,
Y. Kambayashi,
K. Inoue,
and
H. Imura.
Receptor selectivity of natriuretic peptide family, atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide.
Endocrinology
130:
229-239,
1992[Abstract].
34.
Sundler, F.,
E. Ekbald,
A. Absood,
A. Hakanson,
K. Koves,
and
A. Arimura.
Pituitary adenylate cyclase activating peptide: a novel intestinal peptide-like neuropeptide in the gut.
Neuroscience
46:
439-454,
1992[Medline].
35.
Teng, B.-Q.,
K. S. Murthy,
J. F. Kuemmerle,
J. R. Grider,
and
G. M. Makhlouf.
Expression of endothelial nitric oxide synthase in human and rabbit gastrointestinal smooth muscle cells.
Am. J. Physiol.
275 (Gastrointest. Liver Physiol. 38):
G342-G351,
1998[Abstract/Free Full Text].
36.
Teng, B.-Q.,
K. S. Murthy,
J. F. Kuemmerle,
J. R. Grider,
and
G. M. Makhlouf.
Selective expression of VIP2/PACAP3 receptors in rabbit and guinea pig gastric and tenia coli smooth muscle cells.
Regul. Pept.
77:
127-134,
1998[Medline].
37.
Usdin, T. B.,
T. I. Bonner,
and
E. Mezey.
Two receptors for vasoactive intestinal polypeptide with similar specificity and complementary distribution.
Endocrinology
135:
2662-2680,
1994[Abstract].
Am J Physiol Cell Physiol 275(6):C1409-C1416
0002-9513/98 $5.00
Copyright © 1998 the American Physiological Society