Protein kinase G expression in the small intestine and functional importance for smooth muscle relaxation

Andrea Huber1, Peter Trudrung1, Martin Storr1, Hartmut Franck1, Volker Schusdziarra1, Peter Ruth2, and Hans-Dieter Allescher1

1 Department of Internal Medicine II and 2 Department of Pharmacology and Toxicology, Technical University of Munich, 81675 Munich, Germany

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In functional experiments, the nitric oxide (NO) donor N-morpholino-N-nitroso-aminoacetonitrile or the cGMP analog 8-(4-chlorophenylthio)-cGMP caused a concentration-dependent, tetrodotoxin-resistant relaxation of precontracted strips from rat small intestine. The inhibitory effect of both substances was completely blocked at lower concentrations and was significantly attenuated at higher concentrations by the selective cGMP-dependent protein kinase (cGK) antagonist KT-5823 (1 µM). cGK-I was identified by immunohistochemistry in circular and longitudinal muscle, lamina muscularis mucosae, and smooth muscle cells of the villi and in fibroblast-like cells of the small intestine. Additionally, there was staining of a subpopulation of myenteric and submucous plexus neurons. Double staining for neuronal NO synthase (nNOS) and cGK-I demonstrated a colocalization of these two enzymes. Western blot analysis of smooth muscle preparations and isolated nerve terminals demonstrated that these structures predominantly contain the cGK-Ibeta isoenzyme, whereas the cGK-Ialpha expression is about threefold less. The isoform cGK-II was entirely confined to mucosal epithelial cells. These results show that cGK-I is expressed in different muscular structures of the small intestine and participates in the NO-induced relaxation of gastrointestinal smooth muscle. The presence of cGK-I in NOS-positive enteric neurons further suggests a possible neuronal action site.

gastrointestinal tract; nitric oxide; signal transduction; KT-5823; rat; guinea pig; immunohistochemistry; cGMP-dependent protein kinase

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

RELAXATION OF GASTROINTESTINAL smooth muscle is mainly mediated by nonadrenergic, noncholinergic inhibitory mechanisms. ATP, vasoactive intestinal polypeptide (VIP), and nitric oxide (NO) have been suggested as putative mediators (7, 10, 18). In various systems, NO acts on smooth muscle cells to induce hyperpolarization and relaxation (13, 38, 41). The mechanisms through which these inhibitory cellular effects are mediated include either direct effects of NO on cellular structures, such as stimulation of ion channels (6) or activation of protein kinase C (8), or effects mediated via stimulation of soluble guanylyl cyclase (3, 24, 45). NO binds to the heme ring of soluble guanylyl cyclase, causing activation of this enzyme (39) and leading to the synthesis and intracellular rise of cGMP. Analogs of cGMP can mimic the inhibitory effects of NO on isolated smooth muscle strips (26) and cause transmitter release in isolated nerve terminals (NT) (2), similar to NO. cGMP may act on ion channels either directly or via modulation of phosphodiesterases or cGMP-dependent protein kinases (cGK) (39, 44). Three different isoforms of cGK have been identified so far (17). The isoforms Ialpha and Ibeta are present in vascular smooth muscle, lung, cerebellum, platelets, and heart (17, 27). The isoform II is expressed in secretory epithelium (22, 32) as well as in various regions of the brain (31, 42).

The purpose of the present study was, first, to demonstrate in functional studies that cGK is involved in the NO-induced relaxation of small intestinal smooth muscle; second, to investigate which isoforms of cGK are present in various layers and different structures of the small intestine; and third, to localize these forms by immunohistochemistry.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Functional studies. The experimental model and protocol for investigation of inhibitory response used has been previously described (1). In brief, male Wistar rats (400-500 g) were killed by intraperitoneal injection of phenobarbital sodium (100 mg/kg). The terminal ileum was immediately removed and kept in oxygenated Krebs-Ringer bicarbonate solution (KRS). Six segments of full-thickness strips were prepared from the terminal ileum (length 1.5-2 cm) and fixed to a hook on a holder. Holders with tissue were placed in a jacketed organ bath containing 3 ml of KRS [(in mM) 115.5 NaCl, 1.16 MgSO4, 1.16 NaH2PO4, 11.1 glucose, 24.9 NaHCO3, 2.5 CaCl2, and 4.16 KCl] gassed with 95% O2-5% CO2 and maintained at 37°C by circulating water through the jackets. The free end of one segment was connected with a thread to an isometric force transducer (Swegma force displacement transducer SG 4-500); 1 g of tension was applied to the muscle, and the preparation was allowed to equilibrate for at least 30 min. Changes in the tension were amplified by Hellige couplers and recorded on a Rikadenki chart recorder. The segment was precontracted by addition of the muscarinic receptor agonist carbachol (CCH, 1 µM).

The effect of the NO donor N-morpholino-N-nitroso-aminoacetonitrile (SIN-1; 10 µM-1 mM) or the cGMP analog 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP; 10 µM-1 mM) was tested when the CCH-induced response had reached a plateau, which was usually 30 s after addition of CCH. After each concentration of SIN-1 and 8-pCPT-cGMP, the strips were washed and stimulated again with CCH (1 µM). KT-5823 (1 µM) and KT-5720 (1 µM) were applied 10 min before application of CCH. These concentrations of both blockers have been used in other systems and have been shown to be effective and selective (33, 34). In additional experiments, the inhibitory effect of the guanylyl cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) against the SIN-1-induced relaxation was tested. After a concentration response curve, ODQ (1 µM) (16) was added 15 min before the concentration response curve of the SIN-1-induced relaxation was repeated. In separate experiments, the inhibitory effect of SIN-1 or 8-pCPT-cGMP on CCH-induced contraction was tested for stability by repeating this protocol several times. The inhibitory action of both agents remained stable for >3 h with this protocol.

Isolation of proteins of LM and CM layers. Protein isolation was performed from muscularis externa of rat and guinea pig small intestine [longitudinal muscle (LM)/circular muscle (CM), whole mount preparation]. The muscle layers were further subdivided in LM with attached myenteric plexus (LM/MP) and CM layer (after removal of mucosa). The tissue was minced into small pieces with scissors and homogenized with a fourfold volume of buffer A [(in mM) 12.2 K2HPO4, 7.8 KH2PO4, 2 EDTA, and 2 benzamidine, pH 7, 4°C] in a Polytron PT20 homogenizer at ~1,500 rpm for 25 s (5 × 5 s, cooled on ice in between homogenizations). The homogenate was centrifuged at 25,000 g for 30 min, and the pellet was rehomogenized with buffer B (buffer A + 1% Triton X-100). After a second centrifugation step, the supernatants were combined and stored at -20°C.

Isolation of smooth muscle cells and NT. Smooth muscle cells were isolated from the LM/MP of guinea pig small intestine as described previously (4). Segments of ~5 cm were slipped over a 3-ml glass pipette, the mesenteric attachment was gently scraped off with forceps, and the LM/MP layer was carefully pulled off the CM layer. The tissue was cut into small pieces in a medium containing (in mM) 120 NaCl, 2.6 KH2PO4, 4.0 KCl, 2.0 CaCl2, 0.6 MgCl2, 25 HEPES, and 14 glucose and 2% essential amino acid mixture, pH 7.4. After two periods of incubation (20 min at 31°C) in a medium containing 0.05% collagenase (Worthington CLS type II) and 0.01 soybean trypsin inhibitor, the tissue was filtered through a 224-µm mesh, washed several times with collagenase-free medium, and incubated for spontaneous cell dispersion in collagenase-free medium gently bubbled with 95% O2-5% CO2. The dispersed cells were harvested by filtration through a 500-µm mesh.

Rat enteric NT were isolated by differential centrifugation as described previously (29) from the LM/MP layer isolated from the serosal side as described above. The tissue was minced with scissors and homogenized with a Polytron PT20 homogenizer at ~1,500 rpm for 15 s. The tissue homogenate was centrifuged at 800 g for 10 min, and the supernatant was collected (postnuclear supernatant) and then subjected to various differential centrifugation steps (3,500 g for 10 min, 120,000 g for 60 min, and 10,000 g for 10 min). The resulting pellet of the last centrifugation step (P2) was the enriched NT. Protein concentrations were determined with a commercial kit using bovine plasma gamma -globulin as a standard (Bio-Rad, Munich, Germany).

Western blot analysis. For Western blot analysis, the cells and the isolated NT were treated with ultrasound (4 strokes of 15 s, cooled on ice between treatments). Isolated cells were transferred to sample buffer 1 (62.5 mM Tris · HCl, 2% SDS, 10% sucrose, 10% beta -mercaptoethanol, and 0.02% bromphenol blue, pH 6.8), and isolated NT as well as protein extracts were transferred to sample buffer 2 (62.5 mM Tris · HCl, 2% SDS, 10% glycerol, 10% beta -mercaptoethanol, and 0.02% bromphenol blue, pH 6.8). The proteins were separated by SDS-PAGE on 7.5% slab gels using a 3% stacking gel in a Bio-Rad minigel apparatus according to the procedure of Laemmli (30). Protein bands were visualized by Coomassie blue staining or blotted onto a nitrocellulose membrane (Bio-Rad) using Tris-glycine-SDS buffer (50 mM Tris, 380 mM glycine, 0.05% SDS, and 20% methanol). After blocking the membrane with 5% dry milk, we probed blots with selective antibodies for cGK-Ialpha , cGK-Ibeta , and cGK-II. The cGK-Ialpha and -Ibeta antibodies were raised against the NH2-terminal domain of the corresponding enzyme (27). The peptide antibody for cGK-II was raised against the COOH-terminal domain of the kinase (P. Ruth, unpublished results). The detection was performed either with alkaline phosphatase-labeled second antibody (Bio-Rad) or horseradish peroxidase-labeled second antibody and enhanced chemiluminescence (ECL system; Amersham, Braunschweig, Germany).

To assess the amounts of cGK-Ialpha and -Ibeta in intestinal smooth muscle homogenate, we performed Western blot analysis with the extracted proteins and with different amounts (1, 3, 10, and 30 ng) of pure recombinant cGK-Ialpha and -Ibeta (37). The intensity of the bands was measured by an imaging analyzer.

RNA isolation and PCR amplification. RNA from rat small intestine was isolated from the LM/MP layer and from the mucosa, based on the method of Chirgwin et al. (12) and using a commercially available RNA isolation kit (Stratagene, Heidelberg, Germany). We placed 0.5 g of tissue in 5 ml of denaturation solution and subsequently homogenized it with a Polytron PT20 homogenizer at ~1,500 rpm for 20 s (4 × 5 s) on ice. Total yield from 0.5 g of starting tissue was 60 µg of RNA from the LM/MP layer and 1,500 µg from the mucosa. One microgram of RNA from each tissue was subjected to DNase treatment (15 min at room temperature, 1 U DNase I; GIBCO BRL, Eggenstein, Germany) and transcribed in complementary DNA by using avian myeloblastosis virus RT and oligo-p(dT)15 primer (Boehringer Mannheim, Mannheim, Germany) at 25°C over 10 min and 42°C over 60 min. The synthesized cDNA was stored at -20°C until PCR experiments were performed.

PCR amplification of cGK-II was carried out using a PrimeZyme polymerase kit (Biometra, Göttingen, Germany) and primers specific for the known cGK-II sequence in rat (22). Primers were selected by software analysis (DNAstar) using the enzyme sequences available on GeneWorks (Intelligenetics, San Francisco, CA). Primers for the cGK-II were 5'-GTGGCCAGATTCTCAACCTC-3' for the sense primer and 5'-ACCTCGGGGGCCACATACTCT-3' for the antisense primer. The calculated product length was 542 bp for the cGK-II amplification. Annealing temperature was 58°C, and PCR amplification was carried out over 35 cycles. Primer products were sequenced (Medigene, Martinsried, Germany) and revealed homology to the known enzyme sequence.

Immunohistochemistry. Segments of the proximal rat small intestine were removed, opened along the mesenteric border, and fixed in 4% freshly depolymerized paraformaldehyde in 0.1 M Tris-buffered saline (TBS, pH 7.4) at 4°C overnight. After several washes in TBS, the tissue was placed in TBS supplemented with 30% sucrose at 4°C overnight. Frozen sections 10 - 30 µm thick were cut on a cryostat and melted onto glass slides. Activity of endogenous peroxidase was blocked with 0.3% methanol and 3% hydrogen peroxide in TBS. After three rinses in TBS, 10% normal goat serum (NGS) and 1% L-lysine were applied for 90 min. Afterwards, sections were incubated with primary antisera specific for cGK-I or -II (36) diluted 1:1,000 in TBS supplemented with 5% NGS overnight in a humid chamber at room temperature. After the sections were washed, they were incubated with biotinylated goat anti-rabbit serum (Vectastain; Vector, Burlingame, CA) followed by horseradish peroxidase-labeled avidin-biotin complex (Vectastain) diluted in carbonate buffer (11) to prevent nonspecific staining of mast cells. After several additional rinses, peroxidase was revealed by a 3,3'-diaminobenzidine substrate kit (Vectastain). Negative controls were performed without primary antiserum.

For double staining with neuronal NO synthase (nNOS) and cGK-I antibodies, the sections were blocked with 10% NGS and 1% L-lysine and incubated with anti-nNOS (Transduction Laboratories, Lexington, KY) diluted 1:200 in TBS supplemented with 5% NGS overnight at room temperature in a humid chamber. After the sections were washed, positive reactions were visualized by goat anti-mouse IgG antibody coupled with FITC immunofluorescence (Vectastain). This incubation was followed by photography and subsequently by staining for cGK-I as described.

Drugs. CCH was obtained from Boehringer Mannheim, SIN-1 was obtained from Cassella-Riedel (Frankfurt, Germany), 8-pCPT-cGMP was obtained from Biolog Life Science Institute (Bremen, Germany), ODQ was obtained from Biotrend (Cologne, Germany), KT-5823 and KT-5720 were from Calbiochem (Bad Soden, Germany), essential amino acid mixture was from GIBCO BRL, collagenase CLS type II was from Worthington Biochemicals (Freehold, NJ), and soybean trypsin inhibitor, EDTA, benzamidine, and Triton X-100 were from Sigma (Munich, Germany). All other chemicals were purchased from Merck (Darmstadt, Germany).

Statistics. For analysis of data, the contraction level of CCH before addition of SIN-1 or 8-pCPT-cGMP was determined. Relaxation induced by SIN-1 or 8-pCPT-cGMP in the presence or absence of the respective blockers (KT-5823, KT-5720, and ODQ) was expressed relative to the contraction level before the addition of SIN-1 or 8-pCPT-cGMP. Data of the functional experiments are given as means ± SE; n indicates the number of independent observations in different muscle strips. Each protocol was repeated with ileal segments of at least three different animal preparations. ANOVA for repeated measures with a subsequent post hoc test (Newman-Keuls) was used for comparison of the functional data, and values of P < 0.05 were considered significant.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Influence of cGK on smooth muscle relaxation. After prestimulation of LM strips from rat small intestine with the muscarinic agonist CCH (1 µM), addition of the membrane-permeable cGMP analog 8-pCPT-cGMP (10 µM-1 mM) caused a concentration-dependent relaxation, which reached 65.0 ± 2.8% at 1 mM (n = 8). In the presence of the specific cGK antagonist KT-5823 (1 µM), the inhibitory effect of the cGMP analog 8-pCPT-cGMP was significantly attenuated (P < 0.05). At the highest concentration tested, the inhibitory effect of 8-pCPT-cGMP was further reduced by a combination of KT-5823 (1 µM) and the selective cAMP-dependent protein kinase (cAK) blocker KT-5720 (1 µM) (Fig. 1A).


View larger version (16K):
[in this window]
[in a new window]
 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1.   Concentration-dependent effect of membrane-permeable cGMP analog 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) (A) and NO donor N-morpholino-N-nitroso-aminoacetonitrile (SIN-1, B) on cholinergic prestimulated (1 µM carbachol) segments of rat ileum. Inhibitory response was abolished at lower concentrations and significantly reduced in higher concentrations in both cases by cGMP-dependent protein kinase (cGK) antagonist KT-5823 (1 µM; n = 8). Combination of KT-5823 with the selective cAMP-dependent protein kinase inhibitor KT-5720 blocked residual response of 8-pCPT-cGMP and further reduced inhibitory effect on SIN-1 at higher concentrations. ANOVA for multiple measures with Newman-Keuls post hoc test, n = 8; * P < 0.05, ** P < 0.01 vs. control; # P < 0.05 vs. KT-5823.

The NO donor SIN-1 (10 µM-1 mM) caused a similar concentration-dependent relaxation of the precontracted smooth muscle strips, which reached 73.8 ± 7.6% at 1 mM (Fig. 1B, n = 8). The SIN-1-induced inhibitory effect up to a concentration of 300 µM was blocked by the guanylyl cyclase inhibitor ODQ (300 µM SIN-1, 42.9 ± 4.1%; 300 µM SIN-1 + 1 µM ODQ, 8.8 ± 6.3%; P < 0.01, n = 10), indicating that this relaxation is mediated by an activation of guanylyl cyclase. However, at higher concentrations of SIN-1, an ODQ-resistant inhibitory effect was present (1 mM SIN-1, 61.8 ± 3.2%; 1 mM SIN-1 + 1 µM ODQ, 29.2 ± 3.1%; P < 0.05, n = 10).

In the presence of the specific cGK antagonist KT-5823 (1 µM), the inhibitory effect of SIN-1 was completely blocked at low concentrations of SIN-1 (30-100 µM) and significantly attenuated at higher concentrations (300 µM-1 mM) (Fig. 1B). A combination of the cGK antagonists KT-5823 (1 µM) and the selective cAK blocker KT-5720 reduced the response to the highest concentration of SIN-1 tested further (Fig. 1B; n = 8). However, even in the presence of both protein kinase inhibitors (KT-5832 and KT-5720), a small inhibitory effect of SIN-1 (14.0 ± 4.2%, n = 8) was still present. The inhibitory effects of both SIN-1 and 8-pCPT-cGMP were resistant to neural blockade with TTX (data not shown).

Identification of specific isoforms of cGK by Western blot analysis and RT-PCR. For demonstration of the presence of different isoforms of cGK, protein extracts of rat and guinea pig small intestine muscle and isolated myenteric NT of rat small intestine were analyzed with specific antibodies. The isoform Ibeta could be detected in all muscle preparations of both species studied. It was present in the full-thickness preparations (LM/CM) and in preparations of the LM/MP and CM layers (Fig. 2, A and B). cGK-Ibeta was also detected in isolated longitudinal smooth muscle cells of the guinea pig (Fig. 2A). Additionally, the isoform Ibeta was shown to occur in isolated NT of rat small intestine (Fig. 2A). Probing of protein extracts of LM and CM layers with an antibody directed against cGK-Ialpha also resulted in a positive signal (Fig. 2C), but this signal was about threefold less intense compared with the isoform Ibeta [cGK-Ialpha : ~44 ng/mg protein in LM/CM, 62 ng/mg protein in LM/MP, and below the detection limit (<35 ng/mg protein) in CM; cGK-Ibeta : ~134 ng/mg protein in LM/CM, 196 ng/mg protein in LM/MP, and 112 ng/mg protein in CM (Fig. 2, B and C; see MATERIALS AND METHODS for calculation)]. The molecular mass of the detected isoforms Ialpha and Ibeta corresponded, at ~75 kDa, to the pure recombinant cGK-Ialpha and -Ibeta , respectively (Fig. 2).


View larger version (46K):
[in this window]
[in a new window]
 


View larger version (16K):
[in this window]
[in a new window]
 


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2.   Expression of cGK-I in small intestine. A: Western blot analysis of proteins isolated from muscular and neuronal elements of guinea pig and rat small intestine with an antibody specific for cGK-Ibeta . cGK-Ibeta is expressed in both muscular and neuronal structures of both species. LM, longitudinal muscle; MP, myenteric plexus; LM/C, isolated smooth muscle cells of LM layer; CM, circular muscle; NT, nerve terminals. B and C: semiquantitative Western blot analysis with selective antibodies for cGK-Ialpha and -Ibeta (27). cGK-Ibeta is expressed about threefold higher than cGK-Ialpha . Standard curve was created with different amounts of pure recombinant cGK-Ibeta and -Ialpha , respectively (1, 3, 10, and 30 ng). Muscularis externa (LM/CM in A) and isolated NT were from rat; muscularis externa (LM/CM in B and C), LM/MP, LM/C, and CM were from guinea pig.

The membrane-associated isoform cGK-II could not be demonstrated in the LM/MP or CM layer (Fig. 3A) of guinea pig small intestine. Because cGK-II is present in various regions of the brain (31, 42), we chose two different approaches to investigate a possible localization of cGK-II in the enteric nervous system. By immunoblotting, we analyzed proteins of NT isolated from rat small intestine, strictly avoiding mucosal contamination. Probing the proteins with an antibody specific for cGK-II showed no positive signal (Fig. 3A); however, cGK-Ibeta could be detected (Fig. 2A). As a second approach, RT-PCR was carried out using RNA isolated from the LM/MP layer and the mucosal layer of rat small intestine. mRNA coding for cGK-II could be detected in the mucosa but was absent in the LM/MP layer. However, mRNA coding for beta -actin, a ubiquitous protein, could be shown to the same extent in both preparations (Fig. 3B). The different distribution of cGK-I and -II in the analyzed tissues suggests specific functions of these isoforms in various layers.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3.   Western blot analysis and RT-PCR for cGK-II. A: protein extracts of LM/MP and CM of guinea pig small intestine and of isolated NT of rat small intestine incubated with a selective antibody for cGK-II. Positive control (50 ng pure recombinant cGK-II) gave a signal at ~86 kDa, whereas in samples, cGK-II could not be detected. B: RT-PCR for beta -actin and cGK-II with RNA isolated from LM/MP layer and mucosa (M) of rat small intestine. beta -Actin was used to demonstrate that equal amounts of mRNA were used.

Immunohistochemical localization of cGK-I and -II in rat small intestine. The localization of the two enzymes was further investigated immunohistochemically with antibodies specific for cGK-I or -II in segments of rat small intestine. Additionally, to detect colocalization of the two enzymes, the segments were probed with an antibody for nNOS before incubation with the antibody for cGK-I. The antibody specific for cGK-I showed positive staining with different types of smooth muscle cells within the entire wall of rat small intestine, e.g., smooth muscle cells in the CM and LM layer (Fig. 4A), in the lamina muscularis mucosae (Fig. 4, A and C), and running with the longitudinal axis of the villi (Fig. 4B). There was no staining of mucosal epithelial or submucosal cells (Fig. 4, A and C). However, two additional types of cell which showed a positive reaction with the cGK-I antibody were subepithelial and pericryptal myofibroblast-like cells (Fig. 4, D and E) and subepithelial stellate, fibroblast-like cells (Fig. 4B). In the submucosal plexus and MP, there was positive staining for cGK-I of ~15% of the neurons present (Fig. 4, A and C). When double staining for nNOS and cGK-I was performed, neurons as well as nerve processes showed positive staining for both enzymes (Fig. 5, A and B), demonstrating a colocalization of nNOS and cGK-I. The coexpression of both enzymes, nNOS and cGK-I, might suggest a neuromodulatory action of NO via cGK-I. These data demonstrate for the first time that cGK-I is expressed not only in intestinal smooth muscle but also in fibroblast-like cells and in enteric neurons.


View larger version (156K):
[in this window]
[in a new window]
 
Fig. 4.   Immunohistochemical localization of cGK-I in rat small intestine. cGK-I was expressed in LM and CM layers, in muscularis mucosa (MM) (A, C), and in smooth muscle cells (SMC) running with longitudinal axis of the villi (B). Subepithelial stellate fibroblast-like cells (SF) (B), subepithelial myofibroblast-like cells (MF) (E), and pericryptal myofibroblast-like cells (PCMF) (D) were also positive for cGK-I. About 15% of all neurons present in myenteric and submucous plexus were positive for cGK-I (arrows in C). SM, submucosa; CR, crypts. Bars = 50 µm in A and 25 µm in B-E.


View larger version (99K):
[in this window]
[in a new window]
 
Fig. 5.   Immunohistochemical double staining for cGK-I and neuronal NO synthase (nNOS). Within MP, nNOS was present in neurons (long arrows) and nerve processes (short arrows in A) and cGK-I was localized in same neurons (long arrows) and nerve processes (short arrows in B), indicating a colocalization of both enzymes. Bar = 25 µm.

cGK-II was entirely confined to mucosal epithelial cells, and there was an increasing staining intensity from crypt bases toward the top of the villi (Fig. 6, A and B). There was no positive staining in either the muscle layers or in the region of the submucosal or myenteric nerve plexus.


View larger version (156K):
[in this window]
[in a new window]
 
Fig. 6.   Immunohistochemical localization of cGK-II in rat small intestine. cGK-II was entirely confined to mucosal cells (Mu), with an increase in staining intensity from crypts (CR) toward top of villi. There was no staining within submucosa (SM) or muscularis externa (ME). Bar = 500 µm in A and 100 µm in B.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We demonstrated that both the cGMP analog 8-pCPT-cGMP and the NO donor SIN-1 produced a concentration-dependent relaxation of rat ileal longitudinal smooth muscle. At lower concentrations of SIN-1 and 8-pCPT-cGMP, this inhibitory effect was abolished by the specific cGK blocker KT-5823 at a concentration that was demonstrated in various smooth muscle cell preparations, including gastrointestinal smooth muscle, to interact specifically with cGK (34, 35, 47). This indicates that the inhibitory effects of the NO donor SIN-1 and the cGMP analog were mediated by an activation of the cGK. This is also consistent with the finding that the SIN-1-induced inhibitory effect at low concentration is dependent on an activation of the guanylyl cyclase. The inhibitory effect of SIN and 8-pCPT-cGMP at high concentrations was significantly attenuated by KT-5823 and further reduced by a combination of KT-5823 and the specific cAK blocker KT-5720 at a concentration that was shown to selectively interact with cAK in smooth muscle cells (33).

It has been demonstrated that NO- and cGMP-dependent mechanisms can release VIP, another inhibitory neurotransmitter (2, 19). This NO-induced release of VIP also occurred in isolated enteric synaptosomes, suggesting a presynaptic site of actions that is resistant to TTX (2). Thus this release of VIP and subsequent activation of cAK could contribute to the inhibition observed. However, because this release was abolished by KT-5823 (19), it is unlikely that this effect contributes to the residual relaxation in the presence of the cGK blocker. Our results suggest that high concentrations of both SIN-1 and 8-pCPT-cGMP could lead to a cross-activation of the cAK, as postulated in other systems (23), which is partially responsible for the inhibitory effect observed at these concentrations. Whether these high concentrations reflect physiological conditions remains speculative and cannot be answered from our study.

In reference to immunohistochemistry, positive staining for cGK-I was detected in the contractile elements within rat small intestine, supporting the important role in the relaxation of these cells as shown by the functional experiments. cGK-I was detected in the CM and LM layers of the muscularis externa, in the lamina muscularis mucosae, and, according to Markert et al. (32), in smooth muscle cells in the lamina propria mucosae. This latter type of cells is thought to act as a motility mechanism for the villi ("villi pump") that might be of importance for mixing the chyme and intensifying contact with the digestible and resorbable substances. Western blot analysis of proteins isolated from whole thickness muscle layer preparations or from LM and CM layers of guinea pig and rat small intestine probed with selective antibodies for cGK-Ialpha and -Ibeta further corroborates this distribution of cGK-I. A semiquantitative Western blot analysis of cGK-Ialpha and -Ibeta showed that the signal of cGK-Ialpha was about threefold less compared with cGK-Ibeta , suggesting a predominant role of cGK-Ibeta in gastrointestinal smooth muscle.

Additionally, cGK-I was localized with immunohistochemistry in fibroblast-like cells, which form an interconnected contractile network in the lamina propria mucosae (21, 25). We could demonstrate different types of these cells, which have been characterized as subepithelial stellate, fibroblast-like cells, pericryptal myofibroblast-like cells, and subepithelial myofibroblast-like cells (5, 28). The exact function of these cells remains speculative, but the positive staining with the cGK-I antibody might indicate that these cells could also play a role in motility of the villi (14).

There was also a positive staining with the cGK-I antibody in a subset of ~15% of the neurons in the submucous plexus and MP, which was further confirmed by Western blot analysis of proteins isolated from NT of rat small intestine with the cGK-Ibeta antibody. Immunohistochemically, cGK-I was colocalized with the neuronal isoform of NO synthase. On the other hand, NO-synthesizing neurons identified by immunostaining showed a colocalization with VIP in the rat small intestine (15). There is evidence for an NO-dependent VIP release in the rat and guinea pig small intestine that can be mimicked by cGMP analogs (2) and is mediated by activation of cGK, because it was abolished by KT-5823 (19). Thus our structural and Western blot data are in good agreement with these functional findings.

However, in species like the dog and guinea pig, cGMP immunoreactivity and NOS or NADPH diaphorase immunoreactivity are not colocalized within the same neuron, whereas cGMP and VIP immunoreactivity are colocalized (40, 46). This suggests that NO might act as a neuromodulator not only within the same neuron but also between different neurons. Additionally, other sources of NO such as interstitial cells of Cajal and smooth muscle cells have to be considered (9, 20, 35).

cGK-II is suggested to be involved in the regulation of intestinal ion transport and fluid secretion (43). In accordance with this function and with other groups (22, 32), the immunohistochemical experiments showed that the enzyme was entirely confined to mucosal cells, with an increase in staining intensity from the crypts toward the top of the villi. There was no evidence for the presence of cGK-II in muscular elements of the gut wall. Because cGK-II is expressed in high concentrations in the CNS (31, 42), we were especially interested as to whether this isoform would also be present in the enteric nervous system. However, there was no evidence for cGK-II in enteric neurons either by immunohistochemistry, Western blot analysis of proteins from enriched enteric NT, or RT-PCR of RNA isolated from the LM/MP layer containing the MP. Thus, in contrast to the CNS, there was no evidence for the presence of cGK-II in the enteric nervous system.

In conclusion, in the present study, we demonstrated an important functional role of the activation of cGK for intestinal smooth muscle relaxation in response to the NO donor SIN-1 and the cGMP analog 8-pCPT-cGMP. We characterized the presence and distribution of the cGK isoforms in the small intestine. cGK-I was present in all contractile elements, and the isoenzyme cGK-Ibeta was the predominant form. Because of its neuronal localization, this cGK isoform could have an additional neuromodulatory role. In contrast, the cGK-II isoform was confined to mucosal epithelium, and, in contrast to the brain, there was no evidence for its presence in neuronal elements.

    ACKNOWLEDGEMENTS

We thank I. Eiglmeier and S. Kerling for expert technical assistance concerning the immunohistochemical experiments.

    FOOTNOTES

This study was supported by Deutsche Forschungsgesellschaft Sonderforschungsbereich 391 C5.

Preliminary results of this study were presented at the 1997 annual meeting of the American Gastroenterological Association (Washington, DC).

Address for reprint requests: H.-D. Allescher, Dept. of Internal Medicine II, Technical Univ. of Munich, Ismaningerstr. 22, 81675 Munich, Germany.

Received 19 May 1997; accepted in final form 29 May 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Allescher, H. D., H. Fick, V. Schusdziarra, and M. Classen. Mechanisms of neurotensin-induced inhibition in rat ileal smooth muscle. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G767-G774, 1992[Abstract/Free Full Text].

2.   Allescher, H. D., M. Kurjak, A. Huber, P. Trudrung, and V. Schusdziarra. Regulation of VIP release from rat enteric nerve terminals: evidence for a stimulatory effect of NO. Am. J. Physiol. 271 (Gastrointest. Liver Physiol. 34): G568-G574, 1996[Abstract/Free Full Text].

3.   Bayguinov, O., and K. M. Sanders. Role of nitric oxide as an inhibitory neurotransmitter in the canine pyloric sphincter. Am. J. Physiol. 264 (Gastrointest. Liver Physiol. 27): G975-G983, 1993[Abstract/Free Full Text].

4.   Bitar, K. N., and G. M. Makhlouf. Receptors on smooth muscle cells. Characterization by contraction and specific antagonists. Am. J. Physiol. 242 (Gastrointest. Liver Physiol. 5): G400-G407, 1982[Abstract/Free Full Text].

5.   Bloom, W., and D. W. Fawcett. Intestines: A Textbook of Histology. New York: Chapman and Hall, 1994, p. 617-651.

6.   Bolotina, V. M., S. Najibi, J. J. Palacino, P. Pagano, and R. A. Cohen. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature 368: 850-853, 1994[Medline].

7.   Bult, H., G. E. Boeckxstaens, P. A. Pelckmans, F. H. Jordeans, Y. M. Van Maercke, and A. G. Herman. Nitric oxide as an inhibitory non-adrenergic non-cholinergic neurotransmitter. Nature 345: 346-347, 1990[Medline].

8.   Burgstahler, A. D., and M. H. Nathanson. NO modulates the apicolateral cytoskeleton of isolated hepatocytes by a PKC-dependent, cGMP-independent mechanism. Am. J. Physiol. 269 (Gastrointest. Liver Physiol. 32): G789-G799, 1995[Abstract/Free Full Text].

9.   Burns, A. J., A. E. Lomax, S. Torihashi, K. M. Sanders, and S. M. Ward. Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach. Proc. Natl. Acad. Sci. USA 93: 12008-12013, 1996[Abstract/Free Full Text].

10.   Burnstock, G., G. Campell, D. G. Satchell, and A. Smythe. Evidence that adenosine triphosphate or a related nucleotide is the transmitter substance released by non-adrenergic inhibitory nerves in the gut. Br. J. Pharmacol. 40: 668-688, 1970[Medline].

11.   Bussolati, G., and P. Gugliotta. Nonspecific staining of mast cells by avidin-biotin-peroxidase complexes (ABC). J. Histochem. Cytochem. 31: 1419-1421, 1983[Abstract].

12.   Chirgwin, J. M., A. E. Przybyla, R. J. Macdonald, and W. J. Rutter. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294-5299, 1979[Medline].

13.   Christinck, F., J. Jury, F. Cayabyab, and E. E. Daniel. Nitric oxide may be the final mediator of nonadrenergic, noncholinergic inhibitory junction potentials in the gut. Can. J. Physiol. Pharmacol. 69: 1448-1458, 1991[Medline].

14.   Desaki, J., T. Fujiwara, and T. Komuro. A cellular reticulum of fibroblast-like cells in the rat intestine: scanning and transmission electron microscopy. Arch. Histol. Jpn. 47: 179-186, 1984[Medline].

15.   Ekblad, E., H. Mulder, R. Uddman, and F. Sundler. NOS-containing neurons in the rat gut and coeliac ganglia. Neuropharmacology 33: 1323-1331, 1994[Medline].

16.   Ellis, J. L. Role of soluble guanylyl cyclase in the relaxations to a nitric oxide donor and to nonadrenergic nerve stimulation in guinea pig trachea and human bronchus. J. Pharmacol. Exp. Ther. 280: 1215-1218, 1997[Abstract/Free Full Text].

17.   Francis, S. H., and J. D. Corbin. Structure and function of cyclic nucleotide-dependent protein kinases. Annu. Rev. Physiol. 56: 237-272, 1994[Medline].

18.   Goyal, R. K., S. Rattan, and S. I. Said. VIP as a possible neurotransmitter of non-cholinergic non-adrenergic inhibitory neurons. Nature 288: 378-380, 1980[Medline].

19.   Grider, J. R., and J. G. Jin. Vasoactive intestinal peptide release and L-citrulline production from isolated ganglia of the myenteric plexus: evidence for regulation of vasoactive intestinal peptide release by nitric oxide. Neuroscience 54: 521-526, 1993[Medline].

20.   Grider, J. R., K. S. Murthy, J. G. Jin, and G. M. Makhlouf. Stimulation of nitric oxide from muscle cells by VIP: prejunctional enhancement of VIP release. Am. J. Physiol. 262 (Gastrointest. Liver Physiol. 25): G774-G778, 1992[Abstract/Free Full Text].

21.   Güldner, F. H., J. R. Wolff, and D. Graf Keyserlingk. Fibroblasts as a part of the contractile system in duodenal villi of rat. Z. Zellforsch. Mikrosk. Anat. 135: 349-360, 1972[Medline].

22.   Jarchau, T., C. Häusler, T. Markert, D. Pöhler, J. Vandekerckhove, H. R. De Jonge, S. M. Lohmann, and U. Walter. Cloning, expression, and in situ localization of rat intestinal cGMP-dependent protein kinase II. Proc. Natl. Acad. Sci. USA 91: 9426-9430, 1994[Abstract/Free Full Text].

23.   Jiang, H., J. B. Shabb, and J. D. Corbin. Cross-activation: overriding cAMP/cGMP selectivities of protein kinases in tissues. Biochem. Cell Biol. 70: 1283-1289, 1992[Medline].

24.   Jin, J. G., K. S. Murthy, J. R. Grider, and G. M. Makhlouf. Activation of distinct cAMP- and cGMP-dependent pathways by relaxant agents in isolated gastric muscle cells. Am. J. Physiol. 264 (Gastrointest. Liver Physiol. 27): G470-G477, 1993[Abstract/Free Full Text].

25.   Joyce, N. C., M. F. Haire, and G. E. Palade. Morphologic and biochemical evidence for a contractile cell network within the rat intestinal mucosa. Gastroenterology 92: 68-81, 1987[Medline].

26.   Kanada, A., F. Hata, N. Suthamnatpong, T. Maehara, T. Ishii, T. Takeuchi, and O. Yagasaki. Key roles of nitric oxide and cyclic GMP in nonadrenergic and noncholinergic inhibition in rat ileum. Eur. J. Pharmacol. 216: 287-292, 1992[Medline].

27.   Keilbach, A., P. Ruth, and F. Hofmann. Detection of cGMP dependent protein kinase isoenzymes by specific antibodies. Eur. J. Biochem. 208: 467-473, 1992[Abstract].

28.   Komuro, T. Re-evaluation of fibroblast and fibroblast-like cells. Anat. Embryol. (Berl.) 182: 103-112, 1990[Medline].

29.   Kurjak, M., H. D. Allescher, V. Schusdziarra, and M. Classen. Release of bombesin-like immunoreactivity from synaptosomes isolated from the rat ileum. Eur. J. Pharmacol. 257: 169-179, 1994[Medline].

30.   Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[Medline].

31.   Lohmann, S. M., U. Walter, P. E. Miller, P. Greengard, and P. De Camilli. Immunohistochemical localization of cyclic GMP-dependent protein kinase in mammalian brain. Proc. Natl. Acad. Sci. USA 78: 653-657, 1981[Abstract].

32.   Markert, T., A. B. Vaandrager, S. Gambaryan, D. Pöhler, C. Häusler, U. Walter, H. R. De Jonge, T. Jarchau, and S. M. Lohmann. Endogenous expression of type II cGMP-dependent protein kinase mRNA and protein in rat intestine. J. Clin. Invest. 96: 822-830, 1995[Medline].

33.   Maruno, K., A. Absood, and S. I. Said. VIP inhibits basal and histamine-stimulated proliferation of human airway smooth muscle cells. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L1047-L1051, 1995[Abstract/Free Full Text].

34.   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].

35.   Murthy, K. S., K. M. Zhang, J. G. Jin, J. R. Grider, and G. M. Makhlouf. VIP-mediated 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].

36.   Pfeifer, A., A. Aszodi, U. Seidler, P. Ruth, F. Hofmann, and R. Fassler. Intestinal secretory defects and dwarfism in mice lacking cGMP dependent protein kinase II. Science 274: 2082-2086, 1996[Abstract/Free Full Text].

37.   Ruth, P., A. Pfeifer, S. Kamm, P. Klatt, W. R. G. Dostmann, and F. Hofmann. Identification of the amino acid sequences responsible for high affinity activation of cGMP kinase Ialpha . J. Biol. Chem. 272: 10522-10528, 1997[Abstract/Free Full Text].

38.   Sanders, K. M., and S. M. Ward. Nitric oxide as a mediator of non-adrenergic noncholinergic neurotransmission. Am. J. Physiol. 262 (Gastrointest. Liver Physiol. 25): G379-G392, 1992[Abstract/Free Full Text].

39.   Schmidt, H. H. W., S. M. Lohmann, and U. Walter. The nitric oxide and cGMP signal transduction system: regulation and mechanism of action. Biochim. Biophys. Acta 1178: 153-175, 1993[Medline].

40.   Shuttleworth, C. W., C. Xue, S. M. Ward, J. De-Vente, and K. M. Sanders. Immunohistochemical localization of 3',5'-cyclic guanosine monophosphate in the canine proximal colon: responses to nitric oxide and electrical stimulation of enteric inhibitory neurons. Neuroscience 56: 513-522, 1993[Medline].

41.   Stark, M. E., A. J. Bauer, and J. H. Szurszewski. Effect of nitric oxide on circular muscle of the canine small intestine. J. Physiol. (Lond.) 444: 743-761, 1991[Abstract].

42.   Uhler, M. D. Cloning and expression of a novel cyclic GMP-dependent protein kinase from mouse brain. J. Biol. Chem. 268: 13586-13591, 1993[Abstract/Free Full Text].

43.   Vaandrager, A. B., A. G. M. Bot, and H. R. Dejonge. Guanosine 3',5'cyclic monophosphate dependent protein kinase II mediates heat stable enterotoxin provoked chloride secretion in rat intestine. Gastroenterology 112: 437-443, 1997[Medline].

44.   Wahler, G. M., and S. J. Dollinger. Nitric oxide donor Sin-1 inhibits mammalian cardiac calcium current through cGMP-dependent protein kinase. Am. J. Physiol. 268 (Cell Physiol. 37): C45-C54, 1995[Abstract/Free Full Text].

45.   Ward, S. M., H. H. Dalziel, M. E. Bradley, I. L. Buxton, K. Keef, D. P. Westfall, and K. M. Sanders. Involvement of cyclic GMP in non-adrenergic, non-cholinergic inhibitory neurotransmission in dog proximal colon. Br. J. Pharmacol. 107: 1075-1082, 1992[Abstract].

46.   Young, H. M., K. McConalogue, J. B. Furness, and J. De-Vente. Nitric oxide targets in the guinea-pig intestine identified by induction of cyclic GMP immunoreactivity. Neuroscience 55: 583-596, 1993[Medline].

47.   Zhou, X. B., P. Ruth, J. Schlossmann, F. Hofmann, and M. Korth. Protein phosphatase 2A is essential for the activation of Ca2+-activated K+currents by cGMP-dependent protein kinase in tracheal smooth muscle and Chinese hamster ovary cells. J. Biol. Chem. 271: 19760-19767, 1996[Abstract/Free Full Text].


Am J Physiol Gastroint Liver Physiol 275(4):G629-G637
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society