Introduction

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Guanylyl cyclase (GC) (E.C. 4.6.1.2) catalyzes the conversion of guanosine triphosphate to guanosine 3',5'-cyclic monophosphate (cGMP), which is a ubiquitous second messenger in intracellular signaling cascades and is responsible for a wide variety of physiologic responses (1, 2). Two GC forms exist, a membrane-bound or particulate isoform (mGC) and a heme-containing, soluble (sGC) isoform. The latter is a cytosolic, heterodimeric enzyme formed by ₁ and ₁ subunits of relative molecular mass 82,000 and 70,000, respectively (3). Particulate GCs are single polypeptides consisting of an extracellular (ligand-binding) domain, a single membrane-spanning domain, a kinase-like domain, and a catalytic domain. In vertebrates, three major groups of mGCs are known: natriuretic peptide (NP) receptors, Escherichia coli heat-stable enterotoxin/guanylin receptors (known as GC-C) (4), and sensory organ-specific GCs. Binding of a ligand, such as an NP or enterotoxin, to the extracellular domain activates mGCs, whereas sensory organ-specific GCs are activated intracellularly (5).

Recently, the mGC receptor family activated by NPs has been subclassified because C-type NP 1-22 (CNP) and its extended N-terminal form, CNP-53, have been isolated from porcine brain (6). Even though these peptides are structurally related to other endogenous NPs, including atrial NP 1-28 (ANP) and brain NP 1-26 (BNP) (6), their receptor selectivity differs from other NPs. The designated GC-A (ANP-sensitive) receptor is activated by ANP and BNP, and exerts well-defined biologic functions via GC activation, whereas the GC-B (CNP-sensitive) receptor is selectively activated by CNP (2, 7). CNP, a local regulator, is functionally and evolutionarily distinct from ANP and BNP (8).

cGMP plays a significant role in the control of airway tone, exerting a relaxant effect by activating cGMP-dependent protein kinases (9). In bovine tracheal smooth muscle (BTSM), a muscarinic-sensitive (Mn⁺²-supported) mGC activity has been described (10). In these studies, muscarinic agonists were able to stimulate mGC activity in the presence of NaCl; the latter salt inhibits basal BTSM mGC activity (10). This chloride inhibition was abolished by GTP analogs such as GTPS and greatly reduced by pertussis toxin (PTX), suggesting that G proteins are involved in the regulation of this GC activity (11). This mGC appears to be regulated by two opposing muscarinic receptor subtypes, namely an M₃-mediated GC activation occurring at low agonist concentration through a PTX-insensitive G protein and an M₂-mediated inhibition taking place at high agonist concentrations through a G_i/o-like protein (12). Data presented in this report suggest that in bovine airways G protein-regulated mGC is predominantly a B-type guanylyl cyclase, i.e., a CNP-activated GC.

	Materials and Methods

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Compounds and Statistics

The following compounds were purchased from Sigma Chemicals (St. Louis, MO): adenosine triphosphate, creatine phosphokinase (rabbit muscle), bovine serum albumin (BSA) (fraction V), guanylin, NAD⁺, phenylmethylsulfonyl fluoride (PMSF), Trizma base, dithiothreitol (DTT), sucrose, 3-(N-morpholino)propanesulfonic acid, and thymidine. The kit for quantification of cGMP was obtained from the Radiochemical Centre (Amersham, UK). CNP (rat/porcine/human) and ANP (rat) were obtained from Peninsula Laboratories (San Carlos, CA). CNP-53 (rat/porcine) was purchased from Bachem Bioscience Inc. (King of Prussia, PA). Kits for complementary DNA (cDNA) synthesis were purchased from GIBCO BRL (Rockville, MD). TOPO TA Cloning kits were from Invitrogen (Carlsbad, CA). All other reagents were of analytical grade. Data are shown as mean ± standard error of the mean. Statistical analysis was performed using paired Student's t test to determine statistical significance.

Smooth Muscle

Bovine tracheas were obtained from a local slaughterhouse, and the thin layer of tracheal smooth muscle was immediately dissected from serosal, mucosal, and submucosal layers as described by Katsuki and Murad (13). Dissected tissue used for subcellular fractionations was rinsed with 20 mM Tris-HCl buffer (pH 7.2) containing 0.3 M sucrose, 0.5 mM DTT, and 0.1 mM PMSF (ISP buffer) before homogenization. Tissue used for RNA extractions was minced and snap-frozen in liquid nitrogen.

RNA Extraction

Total RNA was extracted from tracheal smooth muscle using Trizol (GIBCO BRL) as described (14), modified to include a LiCl precipitation step to eliminate glycogen (15). Total RNA was quantitated by ultraviolet spectrophotometry.

Degenerate Reverse Transcriptase/Polymerase Chain Reaction

Five micrograms of total RNA was suspended in 20 µl of reverse transcriptase (RT) buffer (containing 10 mM Tris [pH 8.3], 50 mM KCl, 5 mM MgCl₂, 1 mM each of deoxyadenosine triphosphate [dATP], deoxythymidine triphosphate [dTTP], deoxycytidine triphosphate [dCTP], and deoxyguanosine triphosphate [dGTP]), 20 U of ribonuclease inhibitor, 2.5 µM oligo(dT), and 150 U of Superscript H reverse transcriptase (GIBCO BRL), and incubated at 25°C for 10 min and at 42°C for 50 min. The reaction was stopped by heat inactivation at 90°C for 5 min and then chilled on ice. cDNA products were amplified by polymerase chain reaction (PCR) using the following degenerate oligonucleotide primers (Integrated DNA Technologies, Coralville, IA) designed to anneal to a catalytic domain-encoding region spanning 90 amino acids, highly conserved among all known GCs (Figure 1A): sense, 5'-GTI·TA (T/C)·AA(G/A)·GTI·GA(G/A)·ACI·ITI·GGI·GA(T/C)·III·TA (T/C)·ATG-3' (amino-acid sequence VYKVETIGDAY); antisense, 5'-CC·(G/A)AA·IA(G/A)·(G/A)CA·(G/A)TA-ICI·IGG-3' (amino-acid sequence MPRYCLF). Deoxyinosine (I) was included to reduce degeneracy (16). Appropriate amounts of cDNA were subjected to PCR in 50-µl reactions containing 10 mM Tris (pH 8.3), 50 mM KCl, 3 mM MgCl₂, 200 µM each of dATP, dTTP, dCTP, and dGTP, 0.5 mg/ml BSA, 2.5 U of native Taq DNA polymerase (MBI Fermentas, Hanover, MD), and 10 pmol each of the sense and antisense primers, in the absence or presence of 5% (vol/vol) dimethylsulfoxide (DMSO). The temperature profile of amplification consisted of one cycle at 94°C (5 min), 40 cycles consisting of three steps at 94°C (1 min), 40°C (10 min), and 60°C (2 min), respectively, and one last cycle at 72°C (7 min). PCR products were separated in 1.4% agarose gels, and DNA bands were visualized by staining with ethidium bromide.

View larger version (43K): [in this window] [in a new window]   Figure 1.   (A) Primary structure comparison between all known GCs around the amino-acid region amplified during RT-PCR experiments (alignment is modified from www.swmed.edu/ pharmacology). Bold, italicized sequences indicate the primer binding sites. (B) Agarose gel electrophoresis showing products of degenerate RT-PCR for BTSM GCs in the presence or absence of DMSO as described in MATERIALS AND METHODS. (C ) Agarose gel electrophoresis showing RT-PCR products for BTSM GC-A, GC-B, and GC-C using isoform-specific oligonucleotide primers as described in MATERIALS AND METHODS. M = DNA molecular weight markers.

Cloning and Characterization of RT-PCR Products

PCR fragments were isolated from agarose gels by binding to sodium borosilicate using the GeneClean kit (Bio101 Inc., Vista, CA) and ligated to the pCR2.1-TOPO (Invitrogen). E. coli TOP10 transformant cells were spread onto Luria-Bertani plates containing 40 mg/ml 5-bromo-4-chloro-3-indolyl--D-galactopyranoside. Insert-containing plasmids were checked by colony PCR for the presence of sGC sequences using the M13 oligonucleotide (5'-CAGGAAACAGCTATGAC-3') and a sGC-specific oligonucleotide (5'-TGGCCAGG-TCCAAGTAAATGG-3') as reverse and forward PCR primers, respectively. Plasmids were isolated by alkaline lysis from recombinant colonies that were negatives for colony PCR, and their inserts were characterized by dideoxy sequencing (17) using an ABI373 DNA sequencer.

RT-PCR for GC-A, GC-B, and GC-C

cDNA was synthesized from total RNA as described previously and subjected to amplification using the following primers according to Seebacher and coworkers (18): GC-A sense, 5'-AAGAGCCTGATAATACCTGAGTACT-3' and GC-A antisense, 5'-TTGCAGGCTGGGTCCTCATTGTCA-3' (product size, 414 bp); GC-B sense, 5'-AACGGGCGCATTGTGTATATCTGCGGC-3' and GC-B antisense, 5'-TTATCACAG-GATGGGTCGTCCAA-3' (product size, 562 bp); GC-C sense, 5'-ATAAGTCAG-GTGACTGCCGGA-3' and GC-C antisense, 5'-TGGCGTGCCACAC-ATAACAAT-3' (product size, 488 bp). PCR products were isolated from 1.4% agarose gels and cloned into pCR2.1-TOPO for DNA sequencing as described previously. Identity of PCR products was confirmed by comparison with GeneBank sequences using the National Center for Biotechnology Information BLAST server.

Subcellular Fractionation Procedure

Subcellular fractions were obtained from BTSM as described (10, 19). Briefly, tissue rinsed in ISP buffer was minced and homogenized twice with 3 vol of this buffer per gram of wet tissue in a Waring blender at full speed for 30 s. The dispersed material was centrifuged at 850 × g for 10 min and the supernatant fraction was retained. The sediment was re-extracted with 2 vol of ISP buffer (20 mM Tris-HCl [pH 7.2], 0.5 mM DTT, 0.3 M sucrose, and 0.25 mM PMSF) per gram wet tissue and filtered sequentially through two, four, and eight layers of cheesecloth. The filtrate and the previous extract were mixed. This pool (fraction E) was centrifuged at 1,000 × g for 10 min to remove nuclei (fraction N), the supernatant was centrifuged at 31,000 × g for 10 min to sediment mitochondria (fraction M), and the resulting supernatant was centrifuged at 150,000 × g for 1 h, yielding a microsomal fraction (fraction P) and the supernatant or cytosol (fraction S).

Plasma Membrane Preparation

Fraction P was dispersed in 50 ml ISP buffer and aliquots (15 ml) were fractionated on a discontinuous sucrose gradient (0.3/0.82/ 1.28 M) in a Beckman SW 28 rotor at 80,000 × g for 1 h. Three fractions, P1 (between 0.3 and 0.82 M sucrose), P2 (between 0.82 and 1.28 M sucrose), and P3 (at the bottom) were thus obtained. The P1 (light plasma membrane fraction) and P2 (heavy plasma membrane fraction) interfaces were collected and each one was diluted with 20 mM Tris-HCl (pH 7.2), 0.5 mM DTT (buffer I) and centrifuged at 150,000 × g for 30 min. The sediment was suspended in about 10 ml of 20 mM Tris-HCl (pH 7.2) buffer containing 0.3 M sucrose and 0.5 mM DTT (buffer IS), divided into small aliquots, frozen in liquid nitrogen, and stored at 80°C. Each particulate fraction was diluted at a ratio of 1:80 (vol/vol) with buffer I, and the sediment was collected at 150,000 × g for 30 min and suspended in a small volume of the same buffer. This washed material was employed in all assays.

GC Activity

GC activity was determined as described (10). Briefly, the reaction mixture, in a total volume of 125 µl, contained 50 mM Tris-HCl (pH 7.6), 3 mM MnCl₂, 50 to 100 µM GTP, and a GTP-regenerating system (5 mM creatine phosphate and 10 IU phosphocreatine kinase per assay in 0.1% defatted BSA). Incubations were initiated by adding the enzyme preparation (10 to 20 µg protein) and were performed for 5 min at 37°C. Reactions were terminated by addition of 10 µl of 167 mM ethylenediaminetetraacetic acid-Tris (pH 7.5), followed by heating for 3 min in a boiling water bath and cooling on ice. Samples incubated with heat-inactivated enzyme served as blanks. cGMP was determined by radioimmunoassay using the cGMP kit from Amersham in 50 to 100 µl supernatant of the reaction mixture obtained after centrifugation at 12,000 × g for 3 min at 4°C. Radioactivity was measured by liquid scintillation spectrometry. In the GC assays with plasma membranes, the P2 fraction was the most extensively used as described previously.

	Results

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RT-PCR

A degenerate RT-PCR was designed to allow amplification of all GCs expressed in BTSM, including the novel, muscarinic-sensitive isoform that should also contain the highly conserved GC catalytic domain (Figure 1A). The products of PCR from oligo(dT)-primed cDNA were approximately 270 bp in length, which corresponded to the predicted size for all seven mGC so far described (2) and the low molecular weight sGC 1 subunit (20) (Figure 1B). After cloning of the PCR products, a colony PCR assay was designed to distinguish those clones containing sGC inserts. From 500 clones processed, 380 (76%) were positive for the assay, thus coding for the sGC 1 subunit. The remaining colony PCR-negative clones (24%) yielded a nucleotide sequence identical to the bovine GC-B receptor (21). Amplification of cDNAs for GC-A, GC-B, and GC-C using specific oligonucleotides yielded fragments of the predicted size. Identity of fragments was confirmed by comparison of their nucleotide sequences to the cloned mammalian GC-A (22), GC-B (21), and GC-C (4) receptors.

Subcellular Distribution of GC Activity in BTSM

To investigate the distribution of total GC activity into soluble and particulate (membrane-bound) fractions, BTSM cell-free extracts were fractionated by differential centrifugation (10, 19) and GC activity was assayed per fraction (without osmotic shock) as indicated previously. For comparative purposes, GC-specific activity per fraction was expressed as the ratio of total activity to total protein content per fraction relative to fractions E + N (100%) as described elsewhere (23). Specific activities per subcellular BTSM fraction have already been reported (11). Table 1 shows the relative GC-specific activities per fraction (as percentages of E + N). GC activities in the soluble and particulate fractions amounted to 67 and 33%, respectively.

Effect of NPs and Guanylin on Plasma Membrane BTSM GC Activity

To determine whether BTSM mGC activity was sensitive to NPs and guanylin, increasing concentrations (10¹¹ to 10⁵ M) of CNP-53, ANP, or guanylin were tested on plasma membrane fractions (P2) prepared from BTSM (Figure 2). Each active peptide produced concentration-dependent cGMP formation. However, the maximal response elicited by CNP-53 (10⁵ M) was at least 1.6 times higher than that exerted by ANP, with a half-maximal response (EC₅₀) of ~ 10 nM. Although ANP also showed an EC₅₀ of ~ 10 nM, the CNP-53 effect was significantly higher (P < 0.001) than that exerted by ANP at all concentrations from 10⁷ M onward. Maximal CNP activation (at 10⁵ M) was 2.8-fold with respect to GC basal activity (Figure 2). Guanylin had negligible effects on cGMP production under our assay conditions. Similar GC activation was found using CNP-22 (data not shown).

View larger version (24K): [in this window] [in a new window]   Figure 2.   Effects of NP (CNP-53 and ANP) and guanylin on the plasma membrane bound GC. GC activities were measured in plasma membranes (P2 fraction) as described in MATERIALS AND METHODS in the presence of increasing amounts of CNP-53 (open squares), ANP-28 (open triangles), and guanylin (half-shaded squares). Each value is the mean ± standard error (SE) of three different membrane preparations assayed by triplicates. *CNP-53 effect significantly different (P < 0.05) versus ANP effect; **CNP-53 effect significantly different (P < 0.001) versus ANP effect.

Effect of ATP on the GC Activation by CNP

The effect of ATP on NP-GC-coupled receptors has been shown to be receptor- and cation-dependent, and cGMP production by GC-A and GC-B can be abolished when using ATP-Mg⁺² or ATP-Mn⁺² (24). To test whether this was the case for the GC-B receptor found in BTSM, a CNP concentration-effect experiment on cGMP production by the P2 fraction was conducted in the presence of 0.1 mM ATP-Mn⁺². It can be seen in Figure 3 that a significant decrement in the CNP-stimulated GC activity occurred in the presence of ATP.

View larger version (22K): [in this window] [in a new window]   Figure 3.   Effect of ATP on the CNP-22-stimulated GC activity from tracheal smooth muscle. GC activity was measured in plasma membranes (P2 fraction) as described in MATERIALS AND METHODS in the absence (open squares) or presence of 0.1 mM ATP (solid squares) and increasing amounts of CNP-22 (1 × 1010 to 1 × 105 M). Each value is the mean ± SE of three different plasma membrane preparations assayed by triplicates.

Effect of CNP-53 on NaCl Inhibition of mGC Activity

To test whether NaCl could influence BTSM mGC activation by CNP, as is the case for the muscarinic-sensitive mGC activity (10), cGMP production by P2 fractions after CNP-53 addition was determined in the presence of 0.2 M NaCl (Figure 3), a salt concentration known to produce about 50% inhibition of mGC activity in BTSM (10). It can be seen in Figure 4 that CNP-53 reverses the NaCl inhibition in a dose-dependent manner, suggesting that CNP-mediated receptor activation prevents the chloride inhibitory effect on mGC.

View larger version (22K): [in this window] [in a new window]   Figure 4.   Effect of NaCl on the CNP-53-stimulated GC activities from tracheal smooth muscle. GC activity was measured in plasma membranes (P2 fraction) as described in MATERIALS AND METHODS in the absence (open squares) or presence of 0.2 M NaCl (solid squares) and increasing amounts of CNP (1 × 10 10 to 1 × 105 M). Each value is the mean ± SE of three different plasma membrane preparations assayed by triplicates.

	Discussion

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There is ample evidence to imply that the G protein-coupled GC activity described by us in the plasma membrane fraction from BTSM is the same muscarinic-regulated, particulate GC found in this tissue (1). Given the potential role of mGC-produced cGMP in airway smooth muscle physiology (25), we undertook the cloning and biochemical characterization of this mGC to investigate its interaction with other components of the signaling cascade by protein engineering. The results presented in this report suggest that in bovine airways, G protein-regulated mGCs are predominantly of the B subtype. The signaling cascade proposed by us in this tissue (1) should at least involve this CNP-responsive mGC, given its abundance and NaCl sensitivity.

While searching for the gene coding for this potentially new tracheal mGC isoform, screening of degenerate RT-PCR products revealed that about 76% of all GC transcripts coded for sGC and 24% for mGCs. That this sGC: mGC messenger RNA (mRNA) ratio does indeed occur in BTSM is supported by biochemical evidence also presented here. After differential centrifugation of BTSM homogenates, GC activity was highest (67%) in the soluble (S) fraction (Table 1). It is important to point out that the percentage of sGC may be higher because particulate fractions may contain trapped hemeprotein isoenzymes. Nucleotide sequence of all mGC transcripts corresponded to the bovine GC-B isoform (18), indicating the predominance of this NP-sensitive receptor over GC-A and GC-C, which are also expressed in BTSM. To explore biochemically whether this was the case, particulate GC activity was measured in BTSM P2 fraction in the presence of ANP, CNP, and guanylin, which are classic activators of GC-A, GC-B, and GC-C receptors, respectively. Both ANP and CNP stimulated total mGC activity in a concentration- dependent manner, but the CNP effect (10⁸ to 10⁵ M) was significantly higher than that exerted by ANP, whereas guanylin showed no effect. Given that CNP specifically activates GC-B receptors and that it activates GC-A receptors only marginally (26), we take the significant CNP effect on BTSM cGMP production to indicate that GC-B is the most abundantly expressed tracheal mGC isoform, further confirming the RT-PCR results. Thus, our molecular and biochemical evidence suggest that although GC-A is also present, NP-sensitive receptors with GC activity in BTSM are predominantly of the GC-B subtype, resembling the smooth muscles from the rat penile corpus cavernosum (27) and from cultured vascular intima (28).

The inhibitory effect of ATP-Mn⁺² on NP-GC-coupled receptors is an important biologic feature of these GC subtypes (24, 29), which was the case for the CNP-sensitive mGC activity found in BTSM plasma membrane fractions. The mechanism of this inhibitory transduction process has been evaluated through deletion mutagenesis/expression studies of mGCs (29). Thus, the ATP-regulated inhibitory domain resides within the C-terminal segment of the cyclase. This domain is in a different location from the one representing the ATP-regulated stimulatory domain. Identification of the inhibitory domain in the C-terminal segment of the cyclase indicates that this segment is composed of two separate domains: one representing a catalytic cyclase domain and the other, an inhibitory ATP-regulated domain (ARMi) (29). Regarding the inhibition of BTSM GC-B by ATP, the latter may impair CNP activation by acting on the GC-B ARMi domain, inhibiting transmission to the catalytic site of the conformational changes induced extracellularly by CNP. ATP inhibition of the tracheal mGC activity described here supports the finding that it belongs to the NP-sensitive mGC family.

It is well known that NaCl promotes dissociation of heterotrimeric G proteins (30). The finding that the muscarinic-regulated mGC in BTSM is inhibited by NaCl through its effect on G proteins (11) prompted us to study the effect of CNP on the chloride mGC inhibition. In this sense, CNP was able to reverse the NaCl inhibition, suggesting that a NaCl-sensitive G protein is regulating tracheal GC-B, as has been described for the muscarinic-regulated mGC (1). There is evidence to suggest that in GC-B the conformational change elicited on CNP binding exposes previously inaccessible phosphoserine/phosphothreonine residues to a protein phosphatase activity (31). We propose that such conformational change also makes GC-B insensitive to regulation by NaCl-sensitive G proteins by preventing its binding to GC-B. Such a possibility and also the existence of a cross-talk between tracheal GC-B and the muscarinic signaling cascades in bovine airways are currently being studied in this laboratory. Although NP-sensitive GC activation is considered to be independent of GTP binding proteins, the existence of G protein-regulated NP-GCs has been suggested in Leydig tumor cells (32) and PC12 cells (33) for the GC-A subtype. Our data suggest the existence for the first time of a G protein-regulated GC-B.

The functional role of CNP and the CNP-sensitive GC in BTSM is presently unknown, but the high amounts of CNP and CNP-related peptides in trachea (34) and CNP mRNA during lung development (35) suggest that they may have a role to play in the cGMP-dependent control of smooth muscle contraction/relaxation and/or cell proliferation as noted in other systems. In contrast to ANP or BNP as a circulating hormone, a possible role for CNP is likely to be autocrine or paracrine because it is detectable at low levels in plasma (36). The data available suggest that CNP is a poor NP but a potent muscle relaxant. Thus, the diuretic/natriuretic and hypotensive effects of CNP were about 0.01 as potent as those of ANP and BNP in rat, whereas the chick rectum-relaxing activity was three to four times more potent than that of ANP (6). The relaxant effect of CNP on tracheal smooth muscle has already been noted (37), but it remains to be determined whether CNP is produced intracellularly by BTSM or is secreted elsewhere, e.g., in the airway epithelium, where GC-B is also the most abundant mGC isoform (38). Takagi and colleagues (37) have found CNP to induce a dose-dependent increase in guinea-pig tracheal smooth muscle cGMP levels, which reaches a peak after 1 min of incubation, paralleling CNP-induced muscle relaxation. In contrast, at ANP concentrations that cause a maximum increase in cGMP accumulation in BTSM, little relaxation was observed (39). Interestingly, muscarinic stimulation of BTSM mGC activity appears to be responsible for the production of an ODQ-insensitive cGMP peak produced 1 min after BTSM contraction has reached a plateau (25). ODQ is a classic inhibitor of sGCs (40). It has been suggested that cGMP produced through this muscarinic-regulated GC activity is likely to be involved in the onset of smooth muscle relaxation (25). It would be of interest to determine whether this ODQ-insensitive cGMP peak is also produced after CNP stimulation of intact BTSM and determine whether its appearance parallels smooth muscle relaxation. It also remains to be determined whether other tracheal CNP variants, which can be more active than CNP itself on cGMP production (29), are the actual natural ligands for this mGC with novel functions.