Gi-1/Gi-2-dependent signaling by single-transmembrane natriuretic peptide clearance receptor

K. S. Murthy, B.-Q. Teng, H. Zhou, J.-G. Jin, J. R. Grider, and G. M. Makhlouf

Departments of Physiology and Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0711


    ABSTRACT
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Single-transmembrane natriuretic peptide clearance receptor (NPR-C), which is devoid of a cytoplasmic guanylyl cyclase domain, interacts with pertussis toxin (PTx)-sensitive G proteins to activate endothelial nitric oxide synthase (eNOS) expressed in gastrointestinal smooth muscle cells. We examined the ability of NPR-C to activate other effector enzymes in eNOS-deficient tenia coli smooth muscle cells; these cells expressed NPR-C and NPR-B but not NPR-A. Atrial natriuretic peptide (ANP), the selective NPR-C ligand cANP-(4-23), and vasoactive intestinal peptide (VIP) inhibited 125I-ANP and 125I-VIP binding to muscle membranes in a pattern indicating high-affinity binding to NPR-C. Interaction of VIP with NPR-C was confirmed by its ability to inhibit 125I-ANP binding to membranes of NPR-C-transfected COS-1 cells. In tenia muscle cells, all ligands selectively activated Gi-1 and Gi-2; VIP also activated Gs via VIP2 receptors. All ligands stimulated phosphoinositide hydrolysis, which was inhibited by ANP-(1-11), PTx, and antibodies to phospholipase C-beta 3 (PLC-beta 3) and Gbeta . cANP-(4-23) contracted tenia muscle cells; contraction was blocked by U-73122 and PTx and by antibodies to PLC-beta 3 and Gbeta in intact and permeabilized muscle cells, respectively. VIP and ANP contracted muscle cells only after inhibition of cAMP- and cGMP-dependent protein kinases. ANP and cANP-(4-23) inhibited forskolin-stimulated cAMP in a PTx-sensitive fashion. We conclude that NPR-C is coupled to activation of PLC-beta 3 via beta gamma -subunits of Gi-1 and Gi-2 and to inhibition of adenylyl cyclase via alpha -subunits.

G protein; smooth muscle; vasoactive intestinal peptide; signal transduction; phosphoinositide metabolism; phospholipase C


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THREE MAMMALIAN single-transmembrane natriuretic peptide receptors (NPR-A, NPR-B, and NPR-C) have been identified (5, 8, 14). NPR-A and NPR-B are receptor guanylyl cyclases, with intracellular kinase and guanylyl cyclase domains corresponding to guanylyl cyclase-A (NPR-A) and guanylyl cyclase-B (NPR-B) (8, 11). The intracellular domain of NPR-C is devoid of kinase and guanylyl cyclase activities and consists of a 37-amino acid sequence, recently shown to bind and activate pertussis toxin (PTx)-sensitive, guanine nucleotide-binding proteins (G proteins) (4, 23). NPR-D, a recently cloned natriuretic peptide receptor, is a species homologue of NPR-C expressed in the tissues of the eel (13).

The high affinity of all natriuretic peptides for NPR-C and the ability of NPR-C to recycle rapidly in the presence or absence of natriuretic peptides suggests that it might act as a clearance receptor to internalize and degrade circulating natriuretic peptides (15, 24, 29). Its ability to couple to PTx-sensitive G proteins implies that it can also act to transduce external signals. Consistent with this notion, atrial natriuretic peptide (ANP) and the selective NPR-C agonist [des(Gln18,Ser19,Gln20,Leu21,Gly22)ANP-(4-23)-NH2; cANP-(4-23)] inhibited adenylyl cyclase activity in platelets that express only NPR-C and in membranes from vascular and cardiac muscle, the anterior pituitary, the adrenal cortex, and the brain (2, 3). A synthetic peptide consisting of the 37-amino acid intracellular sequence of NPR-C inhibited adenylyl cyclase activity in cardiac muscle membranes (4). cANP-(4-23) and/or ANP also stimulated phosphoinositide (PI) hydrolysis in aortic smooth muscle membranes (10) and isolated parotid acinar cells (6), suggesting G protein-dependent activation of other effector enzymes [e.g., phospholipase C-beta (PLC-beta )]. Our recent studies (23, 30) showed that both ANP and cANP-(4-23) activate nitric oxide synthase (NOS) and stimulate nitric oxide (NO) formation in gastric smooth muscle cells that express endothelial NOS (eNOS). The signaling cascade involves G protein-dependent stimulation of Ca2+ influx and activation of eNOS bound to calmodulin in the plasma membrane; in turn, NO activates soluble guanylyl cyclase, resulting in formation of cGMP and activation of cGMP-dependent protein kinase. The homologous neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) bind with high affinity to NPR-C and initiate an identical signaling cascade. The two neuropeptides interact also with their cognate VIP2/PACAP3 receptors (current nomenclature, VPAC2 receptors) coupled via Gs to adenylyl cyclase (23, 31).

The signaling cascade initiated by interaction of ANP, cANP-(4-23), VIP, and PACAP with NPR-C is mediated by Gi-1 and Gi-2 (23). We postulate that the same G proteins mediate activation of other effector enzymes. In this study, we have used freshly dispersed and cultured tenia coli smooth muscle cells that do not express eNOS (30) to examine the ability of NPR-C to activate other effector enzymes via Gi-1 and Gi-2. Our results show that ANP and cANP-(4-23) activated PLC-beta 3 via the beta gamma -subunits of Gi-1 and Gi-2, inducing PI hydrolysis and muscle contraction, and inhibited adenylyl cyclase activity. VIP also activated PLC-beta 3 via the beta gamma -subunits of Gi-1 and Gi-2. Inhibition of adenylyl cyclase was masked by a predominant activation via Gs mediated by VIP2 receptors.


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Preparation of dispersed and cultured muscle cells. Muscle cells were isolated from guinea pig tenia coli by sequential enzymatic digestion, filtration, and centrifugation as described previously (23, 30). In experiments with blocking antibodies, the cells were permeabilized with saponin (35 µg/ml) in a medium containing 0.34 mM CaCl2 and 1 mM EGTA and resuspended in 1.5 mM ATP and ATP-regenerating system (5 mM creatine phosphate and 10 U/ml creatine phosphokinase). The cells were cultured as described previously (7, 30) in DMEM containing 10% FCS. All experiments were done on cells in first passage.

Expression of natriuretic peptide receptors in smooth muscle cells. Expression of natriuretic peptide receptors (NPR-A, NPR-B, and NPR-C) was determined by RT-PCR and Northern blot analysis as described previously (23, 30, 31). Six micrograms of total RNA were reverse transcribed. The specific primers and conditions for RT-PCR were those used in studies (23) with cultured rabbit gastric smooth muscle cells. Cloned full-length cDNAs for rat NPR-A, NPR-B, and NPR-C were used as positive controls.

For Northern blot analysis, 20 µg of total RNA were fractionated by electrophoresis in 1.1% formaldehyde agarose gel. 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.

Transfection of NPR-C into COS-1 cells. The rat NPR-C cDNA was cloned into the mammalian expression vector pCDL-SRalpha . The plasmid DNA was purified using a Qiagen Midi kit. COS-1 cells were cultured in 90% DMEM supplemented with 10% fetal bovine serum at 37°C as previously described (23). Transfection of recombinant plasmid DNA was performed using Lipofectamine Plus reagent. COS-1 cells were cotransfected with 6 µg of pCDL-SRalpha -rat NPR-C DNA and 6 µg of pGreen Lantern-1 DNA for 72 h. Control cells were transfected with 6 µg of pCDL-SRalpha DNA and 6 µg of pGreen Lantern-1 DNA. Transfection efficiency was monitored microscopically by the expression of the green fluorescent protein using FITC filters.

Radioligand binding. Homogenates of cultured tenia coli muscle cells or NPR-C transfected COS-1 cells were centrifuged at 500 g for 5 min at 4°C. The supernatant was centrifuged at 30,000 g for 1 h at 4°C, and the pellet was suspended at a concentration of 3 mg protein/ml in 50 mM Tris · HCl medium containing 5 mM MgCl2, 0.5% BSA, 0.5% bacitracin, and 10 mg/ml aprotinin. Membranes (30 µg protein/sample) were incubated with 50 pM 125I-labeled ANP or 125I-VIP at 25°C for 15 min in the presence or absence of unlabeled ANP, cANP-(4-23), or VIP. The samples were centrifuged at 12,000 g for 4 min, and the pellet was washed with PBS. Nonspecific binding to membranes of muscle cells was ~30% of total binding.

Identification of receptor-activated G proteins. G proteins selectively activated by ANP, cANP-(4-23), and VIP were identified by an adaptation of the method of Okamoto et al. (25), as described previously (23). Muscle cells were homogenized in 20 mM HEPES medium, centrifuged at 25,000 g for 15 min, and solubilized in 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate at 4°C. The solubilized membranes were incubated for 20 min at 37°C with 60 nM 35S-labeled guanosine 5'-O-(gamma -thio)triphosphate ([35S]GTPgamma S) in 10 mM HEPES medium containing 100 µM EDTA and 10 mM MgCl2 in the presence or absence of various agonists. After the reaction was stopped, the membranes were incubated for 2 h on ice in wells precoated with specific antibodies to Galpha i-1, Galpha i-2, Galpha i-3, Gsalpha , and Galpha q/11. The wells were washed with phosphate buffer containing 0.05% Tween 20, and the radioactivity from each well was counted.

Assay of PLC-beta activity. PLC-beta activity was determined by a modification of the method of Uhing et al. (32) in plasma membranes of muscle cells prelabeled with myo-[3H]inositol, as described previously (17, 20). The assay was initiated by addition of 0.1 mg of membrane protein to 25 mM Tris · HCl medium containing 0.5 mM EGTA, 10 mM MgCl2, 300 nM free Ca2+, 1 µM GTPgamma S, 5 mM phosphocreatine, and 50 U/ml creatine phosphokinase in a total volume of 0.4 ml. After incubation at 31°C for 60 s, the supernatant was extracted with diethyl ether and the amount of labeled inositol phosphates in the aqueous phase was counted. PLC-beta activity was expressed as counts per minute per milligram of protein per minute.

Assay for cAMP and cGMP in dispersed muscle cells. cAMP and cGMP were measured in freshly dispersed muscle cells as described previously (23). Aliquots (0.5 ml) containing 106 cells/ml were incubated for 60 s with forskolin (10 µM) in the presence of 200 µM IBMX followed by addition of 1 µM ANP or 1 µM cANP-(4-23) for 60 s. cAMP and cGMP were measured in duplicate by RIA using 100-µl aliquots of reconstituted samples, and the results were expressed as picomoles per 106 cells.

Measurement of contraction in dispersed muscle cells. Contraction was measured in intact and permeabilized muscle cells by scanning micrometry as described previously (16, 20). The effects of specific antibodies for PLC-beta and G protein subunits were determined in saponin-permeabilized muscle cells after preincubation for 1 h with 10 µg/ml of each antibody (16, 20). Under each condition, the lengths of muscle cells treated with agonists were measured and compared with the lengths of untreated cells. Contraction was expressed in micrometers as the mean decrease in cell length from control.

Materials. ANP, cANP-(4-23), and VIP were obtained from Bachem (Torrance, CA). (8R,9S,11S)-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-trizadibenzo (a,g)cycloocta(c,d,e)-trinden-1-one (KT-5823) was from Kamiya Biomedical (Thousand Oaks, CA). N-2[(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride (H-89) and PTx were obtained from Calbiochem (San Diego, CA). 125I-ANP, 125I-VIP, 125I-cAMP, 125I-cGMP, [35S]GTPgamma S, and myo-[3H]inositol were from New England Nuclear (Boston, MA), and polyclonal antibodies to Galpha subunits, a common Gbeta subunit, and various PLC-beta isoforms were from Santa Cruz Biotechnology (Santa Cruz, CA). pGreen Lantern-1 and Lipofectamine Plus reagent were obtained from Life Technologies GIBCO BRL (Rockville, MD). NPR-A and NPR-B cDNA were gifts from Dr. David L. Garbers, University of Texas Southwestern Medical Center. NPR-C cDNA was a gift from Dr. David G. Lowe, Genentech.


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Selective expression of NPR-C and NPR-B in tenia coli smooth muscle cells. RT-PCR on RNA extracted from cultured tenia coli muscle cells yielded PCR products of the expected size with specific primers for NPR-B and NPR-C but not NPR-A (Fig. 1). Northern blot analysis detected a single mRNA transcript for NPR-B (3.5 kb) and a main transcript for NPR-C (7.5 kb) with some of smaller size (<3.5 kb) but none for NPR-A (Fig. 1). The absence of NPR-A expression was confirmed using primers based on the human NPR-A sequence (data not shown). A similar pattern of selective expression of NPR-B and NPR-C was previously obtained in rabbit gastric muscle cells (23). The size of the NPR-C transcripts was similar to that reported in other tissues (9, 27, 28).


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Fig. 1.   Expression of natriuretic peptide clearance receptor (NPR)-C and NPR-B in cultured tenia coli smooth muscle cells. Total RNA isolated from cultured (first passage) guinea pig tenia coli smooth muscle cells was reverse transcribed, and cDNA was amplified with specific rat NPR-C and NPR-A primers and human NPR-B primers. Experiments were done in the presence or absence of RT. A: PCR products were obtained with NPR-C and NPR-B but not NPR-A primers. B: transcripts corresponding to NPR-B (3.5 kb) and NPR-C (7.5 kb) but not NPR-A were detected by Northern blot analysis in cultured tenia coli smooth muscle cells. NPR-C transcripts of smaller size may represent splice variants. Lanes 1 and 2 represent different samples. MW, molecular weight. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Binding of 125I-ANP and 125I-VIP to muscle membranes. 125I-ANP binding to tenia coli muscle membranes was completely inhibited by ANP (dissociation constant 0.26 ± 0.03 nM; density of binding sites 9.3 ± 0.2 fmol/mg protein) but only partly inhibited by VIP (58 ± 4% at 10 µM) and the selective NPR-C ligand cANP-(4-23) (66 ± 2% at 10 µM) (Fig. 2). The results implied that VIP and cANP-(4-23) recognized a set of binding sites labeled by 125I-ANP. Competition binding with 125I-VIP as radioligand was examined at 1 nM and 10 µM. 125I-VIP binding was completely inhibited by 10 µM VIP but only partly inhibited by 10 µM ANP (58 ± 6%) and cANP-(4-23) (65 ± 3%) (Fig. 3). The results implied that ANP and cANP-(4-23) recognized a set of binding sites labeled by 125I-VIP. These binding sites were analyzed further by eliminating the component of VIP binding to VIP2 receptors. VIP2 receptors were desensitized by treating cultured muscle cells for 30 min with 1 µM VIP: 125I-VIP binding to membranes derived from these cells decreased by 57 ± 2%, and residual binding was abolished by 10 µM ANP, cANP-(4-23), and VIP. The pattern of binding was similar to that observed in rabbit gastric muscle cells (23) and implied that ANP and VIP bound to a receptor, i.e., NPR-C, that is selectively recognized by cANP-(4-23).


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Fig. 2.   125I-labeled atrial natriuretic peptide (125I-ANP) binding to tenia coli muscle membranes. Triplicate samples (30 µg membrane protein/sample) were incubated at 25°C for 15 min with 50 pM 125I-ANP in the presence or absence of unlabeled ANP, cANP-(4-23), or vasoactive intestinal peptide (VIP). Specific 125I-ANP binding was completely inhibited by ANP and partially inhibited by cANP-(4-23) and VIP. The extent of inhibition by cANP-(4-23) and VIP reflected the component of 125I-ANP binding to NPR-C. Values are means ± SE of 5 experiments.



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Fig. 3.   Inhibition of 125I-VIP binding to tenia coli muscle membranes by ANP and cANP-(4-23). 125I-VIP was completely inhibited by 10 µM VIP but only partially inhibited by 10 µM ANP or cANP-(4-23). The extent of inhibition by cANP-(4-23) and ANP reflected the component of 125I-VIP binding to NPR-C. Values are means ± SE of 4 experiments.

Binding of VIP to NPR-C was confirmed using COS-1 cells transfected with NPR-C. In these cells, 125I-ANP binding was completely inhibited by cANP-(4-23) (with an IC50 of 0.6 nM), confirming the absence of other natriuretic peptide receptors in COS-1 cells (Fig. 4). VIP inhibited 125I-ANP binding in a concentration-dependent fashion with an IC50 of 20 nM, providing conclusive evidence of the ability of VIP to bind to NPR-C.


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Fig. 4.   Specific binding of cANP-(4-23) and VIP to COS-1 cells transfected with NPR-C. Binding was determined in membranes from COS-1 cells transfected with NPR-C. Specific 125I-ANP binding was completely inhibited by cANP-(4-23), confirming absence of other natriuretic peptide receptors. Inhibition of binding by VIP provided conclusive evidence of interaction of VIP with NPR-C. Values are means ± SE of 4-6 experiments.

Identification of G proteins and PLC-beta isoform activated by natriuretic peptides and VIP. Both ANP and cANP-(4-23) selectively activated Gi-1 and Gi-2, causing a significant increase in the binding of [35S]GTPgamma S to Galpha i-1 and Galpha i-2 but not to Gsalpha , Galpha i-3, and Galpha q/11 (Table 1). VIP also caused a significant increase in the binding of [35S]GTPgamma S to Galpha i-1 and Galpha i-2, as well as to Galpha s (Table 1).

                              
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Table 1.   Binding of agonist-stimulated GTPgamma S.Galpha complexes in muscle membranes to G protein antibodies

PI-specific PLC-beta activity in muscle membranes was stimulated in a concentration-dependent fashion by cANP-(4-23) with an EC50 of 1.0 ± 0.1 nM and a maximal increase of 91 ± 9% above activity induced by 1 µM GTPgamma S (1,463 ± 105 cpm · mg protein-1 · min-1). ANP (1 µM) and VIP (1 µM) increased PLC-beta activity by 86 ± 10% and 43 ± 6%, respectively (Fig. 5). PLC-beta activity stimulated by all three agonists was inhibited by the NPR antagonist ANP-(1-11) (1, 4) and by pretreatment of the cells for 1 h with 400 ng/ml of PTx before membrane isolation (Fig. 5).


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Fig. 5.   Inhibition of agonist-stimulated phospholipase C-beta (PLC-beta ) activity by ANP-(1-11), pertussis toxin (PTx), and antibodies to PLC-beta 3 and Gbeta . PLC-beta activity induced by cANP-(4-23) (1 µM), ANP (1 µM), and VIP (1 µM) was determined as described in MATERIALS AND METHODS and expressed as the increase above activity induced by 1 µM guanosine 5'-O-(gamma -thio)triphosphate (1,688 ± 295 cpm · mg protein-1 · min-1). PLC-beta activity was inhibited by 10-min treatment with the antagonist ANP-(1-11) (10 µM) or 60-min treatment with antibodies to PLC-beta 3 (10 µg/ml) or Gbeta (10 µg/ml). For studies with PTx, cells were treated with 400 ng/ml for 1 h before membrane isolation. Antibodies to other isoforms of PLC-beta (PLC-beta 1, PLC-beta 2, and PLC-beta 4), or Galpha subunits (Galpha i-1, Galpha i-2, Galpha i-3, and Galpha q/11) had no effect (data not shown). Values are means ± SE of 4-6 experiments. All responses were significantly inhibited (P < 0.01).

A panel of isoform-specific PLC-beta antibodies was used to identify the PLC-beta isoform activated by cANP-(4-23). Pretreatment of muscle membranes for 1 h with a maximally effective concentration (10 µg/ml) of PLC-beta 3 antibody abolished PLC-beta activity stimulated by cANP-(4-23), ANP, and VIP (93 ± 2% to 99 ± 5% inhibition) (Fig. 5), whereas pretreatment with PLC-beta 1, PLC-beta 2, and PLC-beta 4 antibodies had no effect (3 ± 5% to 10 ± 8%). Pretreatment for 1 h with a maximally effective concentration (10 µg/ml) of a common Gbeta antibody also abolished PLC-beta activity stimulated by cANP-(4-23), ANP, and VIP (93 ± 5% to 97 ± 4% inhibition) (Fig. 5).

Inhibition of adenylyl cyclase by natriuretic peptides. ANP and cANP-(4-23) inhibited forskolin-stimulated cAMP formation (33.3 ± 4.9 pmol/106 cells) by 62 ± 4% and 64 ± 7%, respectively, in freshly dispersed tenia coli muscle cells (Fig. 6). The inhibition was reversed by pretreatment of the cells for 1 h with 400 ng/ml of PTx. The inhibition was consistent with activation of Gi-1 and Gi-2 by ANP and cANP-(4-23).


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Fig. 6.   Inhibition of forskolin-stimulated cAMP formation by cANP-(4-23) and ANP and reversal by PTx in freshly dispersed tenia coli muscle cells. Muscle cells were treated with 10 µM forskolin alone or in the presence of 1 µM cANP-(4-23) or ANP. Measurements were made with and without preincubation of cells for 1 h with 400 ng/ml PTx. Results are expressed as pmol cAMP/106 cells above level induced by 200 µM IBMX (7.8 ± 1.0 pmol/106 cells). Values are means ± SE of 3 experiments. ** P < 0.01, significant inhibition.

Stimulation of muscle cell contraction by NPR-C agonists. Activation of PLC-beta 3 by cANP-(4-23) in tenia coli muscle cells suggested that this ligand could act as a contractile agonist by stimulating inositol 1,4,5-trisphosphate-dependent Ca2+ release from sarcoplasmic stores. Consistent with this notion, cANP-(4-23) caused a concentration-dependent contraction of freshly dispersed tenia coli muscle cells (EC50 7.5 ± 3.4 nM) that was inhibited by U-73122 (86 ± 4%) and by 1-h treatment with 400 ng/ml of PTx (93 ± 5%) (Table 2). Contraction was not affected by 10-min treatment with nifedipine (1 µM).

                              
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Table 2.   Inhibition of contraction induced by cANP-(4-23), ANP, and VIP in tenia coli smooth muscle cells by U-73122, PTx, and PLC-beta 3 and Gbeta antibodies

Pretreatment of saponin-permeabilized muscle cells for 1 h with PLC-beta 3 antibody (10 µg/ml) inhibited maximal contraction induced by cANP-(4-23) by 70 ± 5% (P < 0.01) (Table 2), whereas pretreatment with PLC-beta 1, PLC-beta 2, and PLC-beta 4 antibodies had no effect (5 ± 5% to 12 ± 6% inhibition). Pretreatment for 1 h with a common Gbeta antibody (10 µg/ml) inhibited maximal contraction by 76 ± 4% (Table 2), whereas pretreatment with Galpha i-1, Galpha i-2, Galpha i-3, Galpha o, Gsalpha , or Galpha q/11 antibody had no effect (1 ± 7% to 6 ± 8% inhibition).

Although VIP and ANP interacted with NPR-C and activated PLC-beta 3, neither peptide caused contraction. The failure to cause contraction was attributed to concurrent stimulation of cGMP by ANP and of cAMP by VIP. ANP interacted with NPR-B to stimulate cGMP (30 ± 7% above basal with 10 nM ANP) and activate cGMP-dependent kinase, whereas VIP interacted with VIP2 receptors to stimulate cAMP and activate cAMP-dependent kinase. Activation of both protein kinases masked the contractile effect mediated by PLC-beta 3 by inhibiting PI-dependent Ca2+ mobilization (18). Consistent with this notion, both VIP and ANP induced muscle cell contraction in the presence of the cAMP-dependent kinase and cGMP-dependent kinase inhibitors H-89 and KT-5823. The contraction was inhibited 60 ± 6% to 67 ± 7% by U-73122 and 79 ± 5% to 82 ± 7% by PTx, implying that contraction resulted from activation of PLC-beta 3 by Gi-1 and Gi-2 (Table 2).


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This study shows that ANP, cANP-(4-23), and VIP bind with high affinity to NPR-C expressed in tenia coli smooth muscle cells and stimulate PI hydrolysis by activating PLC-beta 3 via the beta gamma -subunits of Gi-1 and Gi-2. ANP and cANP-(4-23) also inhibit adenylyl cyclase, presumably via the alpha -subunit of both G proteins. The results identify Gi-1 and Gi-2 as the G proteins mediating PTx-sensitive inhibition of adenylyl cyclase and stimulation of PI hydrolysis reported in previous studies (2-4, 10). Inhibition of adenylyl cyclase by VIP is probably masked by concurrent activation of adenylyl cyclase via the VIP2 receptor recently shown to be expressed in these muscle cells (12, 31). We base these conclusions on the following findings.

RT-PCR and Northern blot analysis demonstrated expression of NPR-C and NPR-B but not NPR-A, a pattern identical to that in gastric muscle cells (23). The absence of NPR-A was confirmed by primers derived from rat and human NPR-C.

125I-ANP binding was partially inhibited by the selective NPR-C ligand cANP-(4-23) and VIP, and this reflected the component of 125I-ANP binding to NPR-C. 125I-VIP binding was partially inhibited by ANP and cANP-(4-23), and this reflected the component of 125I-VIP binding to NPR-C. The pattern of binding to tenia coli muscle cells was similar to that observed in gastric muscle cells (23). In both muscle cell types, VIP bound to cognate VIP2 receptors and to NPR-C, whereas ANP bound to NPR-C and NPR-B, the only other natriuretic peptide receptor expressed in these cells. cANP-(4-23) bound exclusively to NPR-C. A previous study by Akiho et al. (1) in guinea pig cecal muscle cells showed that VIP partially inhibited ANP binding and ANP partially inhibited VIP binding, but that group did not identify NPR-C as the receptor recognized by VIP.

The ability of VIP to interact with NPR-C was demonstrated conclusively in COS-1 cells transfected with NPR-C. In these cells, both cANP-(4-23) and VIP inhibited 125I-ANP binding in a concentration-dependent fashion, providing direct evidence for interaction of VIP with NPR-C. This was further corroborated by recent functional studies (23) showing that COS-1 cells transfected with NPR-C could be activated by ANP and cANP-(4-23), as well as by VIP.

All three ligands selectively activated both Gi-1 and Gi-2 in tenia coli muscle cells. In addition, VIP activated Gs.

PI hydrolysis was stimulated by all three ligands and inhibited by the NPR antagonist ANP-(1-11) (1, 4), implying interaction of all three ligands with NPR-C. PI hydrolysis was also inhibited by PTx and by antibodies to PLC-beta 3 and Gbeta , consistent with activation of PLC-beta 3 by the beta gamma -subunits of Gi-1 and Gi-2. The results conformed to a pattern previously established for selective activation of PLC-beta 3 by the beta gamma -subunits of inhibitory G proteins in gastric and intestinal muscle cells. The specific G protein involved depended on the receptor (16, 19-21, 23).

Both ANP and cANP-(4-23) inhibited forskolin-stimulated cAMP. The inhibition was completely reversed by PTx.

cANP-(4-23) contracted dispersed tenia coli muscle cells, and the contraction was abolished by the PLC-beta inhibitor U-73122 and PTx. Contraction was also inhibited by antibodies to PLC-beta 3 and Gbeta . The effectiveness of Gbeta and PLC-beta 3 antibodies in inhibiting contraction paralleled their ability to block PI hydrolysis.

VIP and ANP stimulated PI hydrolysis but only caused contraction in the presence of cAMP-dependent kinase and cGMP-dependent kinase inhibitors. The contraction was inhibited by U-73122, PTx, and antibodies to PLC-beta 3 and Gbeta .

The results obtained in tenia coli muscle cells suggested that the inhibition of adenylyl cyclase or activation of PLC-beta by ANP or cANP-(4-23) observed in other cell types (2-4, 10) may have been mediated by the alpha -subunits (inhibition of adenylyl cyclase) and beta gamma -subunits (activation of PLC-beta ) of Gi-1 and Gi-2. Natriuretic peptides possess high affinity for their specific receptors, (i.e., the receptor guanylyl cyclases NPR-A and NPR-B), as well as for NPR-C. There is no evidence that either NPR-A or NPR-B is coupled to G proteins, whereas NPR-C, which is devoid of an intracellular guanylyl cyclase domain, is coupled to inhibitory G proteins, identified in this and a previous study (23) on gastric muscle as Gi-1 and Gi-2. G protein coupling is mediated by the 37-amino acid cytoplasmic domain of NPR-C. A synthetic peptide corresponding to this domain inhibited adenylyl cyclase activity in cardiac muscle membranes; the inhibition was reversed by PTx and by antibodies to the peptide (4). In recent studies (22), we have identified a fragment in the middle region of the cytoplasmic domain of NPR-C that selectively activated Gi-1 and Gi-2 and stimulated PI hydrolysis. A sequence with similar motif capable of binding to various G proteins has been identified in the cytoplasmic domain of the single-transmembrane receptor (insulin-like growth factor-II/mannose 6-phosphate receptor) (26).


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-15564 and 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 and other correspondence: G. M. Makhlouf, PO Box 980711, Medical College of Virginia, Virginia Commonwealth Univ., Richmond, VA 23298-0711.

Received 17 September 1999; accepted in final form 4 January 2000.


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ABSTRACT
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DISCUSSION
REFERENCES

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