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Type I nitric oxide synthase (NOS) is the predominant NOS in rat small intestine. Regulation by platelet-activating factor
Biochimica et Biophysica Acta 1451 (1999) 211^217 www.elsevier.com/locate/bba
Type I nitric oxide synthase (NOS) is the predominant NOS in rat small intestine. Regulation by platelet-activating factor Xiao-Wu Qu a , Hao Wang a , Ranna A. Rozenfeld b , Wei Huang b , Wei Hsueh a b
a;
*
Department of Pathology, Children's Memorial Hospital, Northwestern University Medical School, Chicago, IL 60614, USA Department of Pediatrics, Children's Memorial Hospital, Northwestern University Medical School, Chicago, IL 60614, USA Received 8 December 1998; received in revised form 31 March 1999; accepted 2 June 1999
Abstract Constitutive nitric oxide synthase (cNOS) may play an important protective role in the intestine, since our previous study has shown that the degree of bowel injury induced by platelet-activating factor (PAF), a potent inflammatory mediator, is inversely related to the cNOS content of the intestine. This study aims to examine the composition of the cNOS system in rat small intestine, and its regulation by PAF. We found that an approximately 120 kDa NOS I (neuronal NOS) is the predominant NOS in rat intestine, as evidenced by the following: (a) immunoblotting with specific antibodies detected a NOS I of approximately 120 kDa, but little NOS III; (b) the Ca2 -dependent, constitutive NOS (cNOS) activity of the rat intestine was removed by immunoprecipitation with the anti-NOS I, but not anti-NOS II or anti-NOS III antibodies; (c) RT^PCR revealed constitutive expression of NOS I in the intestinal tissue, but only a minute amount of NOS III. Immunofluorescent staining with anti-NOS I located NOS in the Auerbach plexus and nerve fibers in the muscle layer. We also found that this 120 kDa NOS I is rapidly (within 1 h) down-regulated in response to PAF administration. The protein level, enzyme activity as well as mRNA of nNOS were all decreased in the intestine. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Nitric oxide; Nitric oxide synthase; Platelet-activating factor; (Rat); (Intestine)
1. Introduction Nitric oxide (NO), a molecule with extremely versatile function, is produced in the body by the three members of the nitric oxide synthase (NOS) family: the constitutive neuronal (type I) nNOS and endothelial (type III) eNOS, and inducible (type II) iNOS
* Corresponding author. Fax: +1-773-880-8127; E-mail: [email protected]
(reviewed in [1]). Previous reports demonstrated that in the intestine, endogenous NO produced by constitutive NOS (cNOS) plays a pivotal role in maintaining normal physiological functions, such as regulation of intestinal motility [2], and protection of the mucosal barrier [3]. Blockage of physiological NO production has many adverse e¡ects, such as loss of epithelial integrity [4], breakdown of the mucosal barrier, microvascular leakage [5], and leukocyte adhesion in postcapillary venules of rat mesentery [6]. Administering NO has been shown to protect against ischemia/reperfusion injury in the small intestine [7]. Our previous work also showed that endogenous NO [8] as well as NO donors [9]
0167-4889 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 8 9 ( 9 9 ) 0 0 0 7 6 - 2
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protect against platelet-activating factor (PAF)-induced intestinal injury. PAF is a pro-in£ammatory phospholipid mediator central to endotoxin shock [10] and intestinal injury [11]. Systemic injection of PAF [12] causes intestinal necrosis, and lipopolysaccharide (LPS)-induced bowel injury can be prevented by pretreatment with PAF antagonists [11]. Our previous studies [9] have shown that: (a) in normal intestinal tissue, the predominant NOS activity comes from cNOS, whereas iNOS, which is localized in the epithelium, comprises only 5^10% of the total NOS activity; (b) PAF downregulates intestinal cNOS activity; and the development of intestinal injury after PAF injection inversely correlates with cNOS activity in the intestine. These ¢ndings call for further characterization of the regulation of NOS isoforms in the small intestine by in£ammatory stimuli such as PAF. In the ¢rst part of the present study we identi¢ed nNOS as the predominant cNOS in the intestine. In the second part, we examined the e¡ect of PAF on nNOS enzyme activity, protein levels, and gene expression in the intestine. 2. Materials and methods 2.1. Animal experiment Male Sprague^Dawley rats (80^120 g) were anesthetized with Nembutal and tracheotomized. The carotid artery and jugular vein were cannulated for continuous blood pressure monitoring, blood sampling, and drug administration. The animals were either sham-operated, or injected with 2.5 Wg/kg (i.v.) of PAF (1-O-hexadecyl-2-acetyl-sn-glycero-3phosphocholine, Sigma, St. Louis, MO). One hour after PAF, the animals were euthanized, the small intestine (and in some animals, also the heart and brain) was removed immediately, washed with icecold phosphate-bu¡ered saline, and stored at 370³C. For iNOS assay, intestinal epithelial cells were isolated and assayed as previously described [13]. Epithelial cells rather than whole tissue homogenate was used because our previous study showed that iNOS is localized mainly in the epithelial cells and comprises less than 10% of the total intestinal NOS activity [9].
2.2. Assay of the NOS activity of Ca2+-dependent (cNOS) and Ca2+-independent (iNOS) isoforms, and the inhibition of speci¢c NOS isoform The activity of NOS isoforms was determined by the L-[14 C]arginine conversion method as previously published [14]. In some experiments, immunoprecipitation was performed to remove a speci¢c NOS isoform before the enzyme assay. 10 Wl of anti-NOS I, anti-NOS II, or anti-NOS III antibody (Santa Cruz Biotechnology, Santa Cruz, CA) were mixed with 0.5 ml tissue homogenate (protein concentration 400 Wg/ml), shaken at 4³C for 2 h, and the antigen^antibody complex was removed by incubating with protein-A agarose beads (Santa Cruz) for 1 h followed by centrifugation. Control samples contained no antibody. 2.3. Detection of NOS isoforms by immunoprecipitation and Western blotting Intestinal tissue lysate was prepared as described previously [15]. The protein concentration of the tissue lysate was determined using a Sigma total protein kit (Sigma), and was adjusted to 0.5 mg/ml. After precleaning with protein-A agarose, 1.0 ml tissue lysate (0.5 mg protein) was incubated with 30 Wl anti-NOS antibody at 4³C with gentle shaking for 3 h, followed by incubation with 40 Wl protein-A agarose beads for 2 h at 4³C. The immunoprecipitate was collected and the bound immune complex was eluted by the addition of 200 Wl Laemmli bu¡er and boiling. After centrifugation, 150 Wl supernatant was loaded on a 7.5% sodium dodecyl sulfate^polyacrylamide gel for electrophoresis resolution. The resolved protein was transferred to a nitrocellulose membrane, and the blot was detected subsequently with anti-NOS antibody, horseradish peroxidase-labeled goat anti-rabbit IgG antibody, and visualized with an ECL system from Amersham. 2.4. RNA extraction and RT^PCR Total RNA was extracted from intestinal tissue by homogenizing the samples in a RNA-extracting agent, STAT-60 (TEL-TEST, Friendswood, TX), together with chloroform treatment and isopropanol precipitation following the manufacturer's instruc-
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tions. The total RNA was reversely transcribed (RT) at 37³C by using random hexamer (pdN6), and Moloney murine leukemia virus (M-MLV) reverse transcriptase (Gibco-BRL). An eNOS- or nNOS- sequence-speci¢c cDNA segment and a speci¢c sequence of L-actin (housekeeping gene) were ampli¢ed by polymerase chain reaction (PCR) with AmpliTaq DNA polymerase and speci¢c primers £anking the corresponding gene segments. The sequence of the primers and the PCR thermocycle pro¢les used are as previously reported [16]. The size of the corresponding PCR products for nNOS, eNOS, and L-actin are 280 bp, 345 bp, and 764 bp, respectively. The PCR products were resolved by agarose gel electrophoresis and visualized by staining with SYBR Green-1 (Molecular Probes, Eugene, OR). nNOS transcription was analyzed by semiquantitative RT^ PCR. The £uorescence of the PCR products of nNOS and L-actin ampli¢ed from the same reversetranscription reaction was analyzed using a STORM860 Phosphor-imager, and the nNOS/L-actin ratio between sham-operated and PAF-treated groups was compared. 2.5. Immuno£uorescent stains for nNOS Five-Wm frozen tissue sections were mounted on polylysine-coated slides and post-¢xed with ice-cold acetone. After blocking with goat serum, the slides were incubated with rabbit anti-nNOS antibody, followed by incubation with biotinylated goat anti-rabbit IgG antibody, and £uorescein isothiocyanate (FITC)-conjugated streptavidin. 2.6. Statistical analysis Results are expressed as mean þ S.E.M. Comparison between means are made by unpaired Student's t-test. P 6 0.05 is considered signi¢cant. 3. Results and discussion 3.1. An approximately 120 kDa nNOS is the predominant cNOS in rat small intestine By using the speci¢c anti-nNOS antibody R-20, which recognizes the amino acids 1400^1419 at the
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Fig. 1. Immune-blotting of nNOS and eNOS in brain, heart, and ileal tissue. (A) Western blotting of nNOS (after immunoprecipitation) with anti-NOS I Ab R-20. A major band of approximately 120 kDa was detected in ileal tissue. The molecular mass of nNOS from rat brain homogenate is 160 kDa. In addition, there were a few smaller and fainter bands with lower molecular mass. These may represent either sample degradation or low-level expression of other nNOS isoform in the brain. (B) Western blotting of eNOS (after immunoprecipitation) with anti-NOS III Ab C-20.
C-terminus of the rat nNOS, a band of approximately 120 kDa was precipitated from ileal tissue. The molecular mass of this protein is less than that of nNOS (160 kDa) from rat brain homogenate, which was precipitated with the same antibody (Fig. 1A). The 160 kDa nNOS is also detected, albeit weakly, in heart tissue, but not in the rat ileum. In contrast to nNOS, eNOS (135 kDa) is almost undetectable in the small intestine (Fig. 1B), as we failed to detect eNOS protein using a speci¢c anti-NOS III antibody. The validity of the antibody was con¢rmed by its strong reaction with the 135 kDa eNOS in the heart homogenate for immunoblotting (Fig. 1B). The PCR results support the ¢ndings of Western blotting. RT^PCR revealed a large amount of nNOS mRNA constitutively present in the rat ileum, whereas little eNOS mRNA was detected (Fig. 2). In contrast, RT^PCR detected abundant eNOS mRNA in heart tissue, but a relatively low level of nNOS mRNA (Fig. 2). These ¢ndings suggest that nNOS is the predominant cNOS in the intestine. Immuno£uorescent stains using anti-type I NOS antibody R-20 showed predominant localization of the protein in the myenteric plexus and in nerve ¢bers within the muscle layer (Fig. 3). This is identical
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to previous reports on the localization of nNOS in the intestine [17], suggesting that the 120 kDa protein is nNOS. In order to functionally verify that the 120 kDa protein is a NOS isoform, we compared the cNOS (Ca2 -dependent) activity of the ileal homogenate before and after the removal of type I, II, and III NOS isoform by immunoprecipitation with the speci¢c antibody. Fig. 4 (upper) shows that immunoprecipitation with anti-NOS I antibody removed 80 þ 5% of the cNOS (Ca2 -dependent) activity, whereas immunoprecipitation with anti-NOS II or anti-NOS III antibody did not signi¢cantly a¡ect the cNOS activity. The e¡ectiveness of the latter two antibodies to inhibit iNOS and eNOS, respectively, is demonstrated by the almost 100% inhibition of iNOS in isolated ileal epithelial cells (Fig. 4, lower), and approximately 70% inhibition of cNOS activity in heart homogenate (data not shown). These results strongly suggest that the 120 kDa protein is responsible for most of the Ca2 -dependent NOS activity in the intestine. Based on these observations and our previous ¢nding that more than 90% of the total NOS activity in normal intestine is composed of cNOS [9], we conclude that nNOS is the predominant NOS in the small intestine of normal rats. The remaining (usually 2^5%) NOS activity, as measured by L-[14 C]arginine conversion, is Ca2 -independent, and thus is probably accounted for by iNOS in in-
Fig. 2. Gene expression of nNOS and eNOS in the ileum analyzed by semiquantitative RT^PCR. The numbers of the thermocycles used for PCR ampli¢cation for nNOS, eNOS, and Lactin were 30, 30, and 20, respectively. The sizes of the corresponding PCR products are: nNOS, 280 bp; eNOS, 345 bp; Lactin (control), 764 bp.
Fig. 3. Immuno£uorescent staining of the small intestine using anti-nNOS Ab. Frozen ileal tissue section was incubated with R-20 (rabbit anti-rat NOS I, polyclonal, 40 Wg/ml), followed by biotinylated goat anti-rabbit IgG Ab and FITC-conjugated streptavidin.
testinal epithelium [9]. (Although iNOS is supposed to be only inducible, a low level of iNOS is constitutively present in normal rat intestine, mainly in the epithelial cells [9,18].) The nNOS mRNA and mature protein are structurally diverse as a consequence of alternative promoters and alternate mRNA splicing [19]. In addition to the originally cloned nNOS from a brain cDNA library [20], several di¡erentially spliced variants have been described in the rat [21]. Some of them have a molecular mass close to 120 kDa, such as nNOS-Q found in the skeletal muscle of mutant mice [22]; also described is a novel NOS mRNA transcript with a N-terminal truncation in the human testis which, when translated in CHO-K1 cells through transfection with a construct carrying the NOS sequence, turned out to have a molecular mass of 125 kDa [23]. Whether the 120 kDa nNOS we found in rat intestine is related to these nNOS isoforms awaits its cloning. It has been suggested that the expression of the alternative transcripts occurs with a high degree of tissue speci¢city [24]. Thus, it is possible that the diversity in alternative splicing re£ects a means of tight and speci¢c regulation of nNOS in di¡erent organs.
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dilators did not improve intestinal tissue perfusion. Furthermore, since most of the cNOS activity in the small intestine is accounted for by nNOS, it is possible that nNOS in the small intestine functions like eNOS in other organs, i.e., regulating perfusion and antagonizing leukocyte adhesion, directly or indirectly. This is a plausible hypothesis, in view of the reported localization of nNOS in the perivascular nerve ¢bers and the adventitia of blood vessels in many organs [34^36]. Although in this study we did not observe nNOS in the small intestinal vessels, other studies using more sensitive methods have revealed nNOS localizing in the endothelium [37]. 3.2. In vivo administration of PAF suppresses the activity and decreases the protein content of the 120 kDa nNOS, as well as nNOS mRNA in the intestine
Fig. 4. NOS activity after the removal of certain NOS isoforms by speci¢c antibodies. (Upper) Ca2 -dependent NOS activity in the ileal tissue homogenate after immunoprecipitation with antiNOS I Ab (R-20), anti-NOS II Ab (M-19), or anti-NOS III Ab (C-20), and agarose protein-A beads. *Inhibition level is signi¢cantly higher than 0 (n = 5, P 6 0.05). (Lower) Ca2 -independent NOS activity in isolated ileal epithelial cell homogenate after immunoprecipitation with anti-NOS I Ab (R-20), antiNOS II Ab (M-19), or anti-NOS III Ab (C-20), and agarose protein-A beads. *Inhibition level is signi¢cantly higher than 0 (n = 5, P 6 0.05).
It is surprising to ¢nd an almost complete absence of eNOS in the small intestine, since it is generally assumed (reviewed in [1]) that eNOS exerts an important protective function by maintaining adequate blood £ow and perfusion [25^27], protecting the mucosal barrier [28], and inhibiting leukocyte-endothelial adhesion [6]. This assumption probably derives from the vast amount of data from studies of isolated mesenteric vessels where eNOS is abundantly expressed [29^32]. However, the regulating mechanism of the larger mesenteric vessels and their smaller intramural branches (within the bowel wall) may be di¡erent. This is supported by our previous study [33] which showed that reversal of PAF-induced vasoconstriction of superior mesenteric artery by vaso-
To examine the regulation of the activity, protein content and gene transcription of this 120 kDa nNOS, we administered PAF to induce intestinal injury. At the dose used (2.5 Wg/kg, i.v.), all rats develop various degree of bowel injury in 1 h. As shown in Fig. 5 (upper), 1 h after PAF injection the cNOS activity in the intestine of PAF-treated rats is decreased to half of that in the sham-operated group. This decreased enzyme activity correlates with a marked reduction of intestinal nNOS protein content, as shown by Western blot (Fig. 5, lower), suggesting that PAF down-regulates nNOS protein synthesis. There is also a signi¢cant reduction in the nNOS mRNA after PAF injection, as revealed by RT^PCR (Fig. 5, middle), suggesting that PAF administration also causes a decline of nNOS mRNA, either due to a down-regulation of nNOS gene transcription, or to a decrease in mRNA stability. Previously, we demonstrated [9] that the activity of cNOS (or more accurately, nNOS) slowly increased with time in sham-operated rats, suggesting that mild stress (which may compromise intestinal perfusion) such as general anesthesia and surgery, could up-regulate nNOS activity. However, when a potent in£ammatory stimulus such as PAF is administered, the protective nNOS is rapidly down-regulated, and injury develops as a consequence [9]. The severity of the injury is inversely correlated with the tissue cNOS activity [9]. Further, this injury may be allevi-
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Acknowledgements This study was supported by NIH Grants DK34574 and HD31840.
References
Fig. 5. PAF (2.5 Wg/kg) down-regulates the enzyme activity (upper), protein content (lower), and gene transcription (middle) of nNOS in the small intestine. (upper) Ca2 -dependent NOS activity in the ileal tissue homogenate of sham-operated (n = 6) and PAF-treated (n = 7) rats. *Signi¢cantly di¡erent from shamoperated group (P 6 0.05). (lower) Western blot showing reduction of nNOS in the ileum after PAF injection. A typical experiment is shown. (middle) nNOS mRNA (expressed as the ratio of nNOS/L-actin mRNA by RT^PCR) in the ileum of sham-operated (n = 6) and PAF-treated (n = 7) rats. *Signi¢cantly di¡erent from sham-operated group (P 6 0.05).
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Type I nitric oxide synthase (NOS) is the predominant NOS in rat small intestine. Regulation by platelet-activating factor Xiao-Wu Qu a , Hao Wang a , Ranna A. Rozenfeld b , Wei Huang b , Wei Hsueh a b
a;
*
Department of Pathology, Children's Memorial Hospital, Northwestern University Medical School, Chicago, IL 60614, USA Department of Pediatrics, Children's Memorial Hospital, Northwestern University Medical School, Chicago, IL 60614, USA Received 8 December 1998; received in revised form 31 March 1999; accepted 2 June 1999
Abstract Constitutive nitric oxide synthase (cNOS) may play an important protective role in the intestine, since our previous study has shown that the degree of bowel injury induced by platelet-activating factor (PAF), a potent inflammatory mediator, is inversely related to the cNOS content of the intestine. This study aims to examine the composition of the cNOS system in rat small intestine, and its regulation by PAF. We found that an approximately 120 kDa NOS I (neuronal NOS) is the predominant NOS in rat intestine, as evidenced by the following: (a) immunoblotting with specific antibodies detected a NOS I of approximately 120 kDa, but little NOS III; (b) the Ca2 -dependent, constitutive NOS (cNOS) activity of the rat intestine was removed by immunoprecipitation with the anti-NOS I, but not anti-NOS II or anti-NOS III antibodies; (c) RT^PCR revealed constitutive expression of NOS I in the intestinal tissue, but only a minute amount of NOS III. Immunofluorescent staining with anti-NOS I located NOS in the Auerbach plexus and nerve fibers in the muscle layer. We also found that this 120 kDa NOS I is rapidly (within 1 h) down-regulated in response to PAF administration. The protein level, enzyme activity as well as mRNA of nNOS were all decreased in the intestine. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Nitric oxide; Nitric oxide synthase; Platelet-activating factor; (Rat); (Intestine)
1. Introduction Nitric oxide (NO), a molecule with extremely versatile function, is produced in the body by the three members of the nitric oxide synthase (NOS) family: the constitutive neuronal (type I) nNOS and endothelial (type III) eNOS, and inducible (type II) iNOS
* Corresponding author. Fax: +1-773-880-8127; E-mail: [email protected]
(reviewed in [1]). Previous reports demonstrated that in the intestine, endogenous NO produced by constitutive NOS (cNOS) plays a pivotal role in maintaining normal physiological functions, such as regulation of intestinal motility [2], and protection of the mucosal barrier [3]. Blockage of physiological NO production has many adverse e¡ects, such as loss of epithelial integrity [4], breakdown of the mucosal barrier, microvascular leakage [5], and leukocyte adhesion in postcapillary venules of rat mesentery [6]. Administering NO has been shown to protect against ischemia/reperfusion injury in the small intestine [7]. Our previous work also showed that endogenous NO [8] as well as NO donors [9]
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protect against platelet-activating factor (PAF)-induced intestinal injury. PAF is a pro-in£ammatory phospholipid mediator central to endotoxin shock [10] and intestinal injury [11]. Systemic injection of PAF [12] causes intestinal necrosis, and lipopolysaccharide (LPS)-induced bowel injury can be prevented by pretreatment with PAF antagonists [11]. Our previous studies [9] have shown that: (a) in normal intestinal tissue, the predominant NOS activity comes from cNOS, whereas iNOS, which is localized in the epithelium, comprises only 5^10% of the total NOS activity; (b) PAF downregulates intestinal cNOS activity; and the development of intestinal injury after PAF injection inversely correlates with cNOS activity in the intestine. These ¢ndings call for further characterization of the regulation of NOS isoforms in the small intestine by in£ammatory stimuli such as PAF. In the ¢rst part of the present study we identi¢ed nNOS as the predominant cNOS in the intestine. In the second part, we examined the e¡ect of PAF on nNOS enzyme activity, protein levels, and gene expression in the intestine. 2. Materials and methods 2.1. Animal experiment Male Sprague^Dawley rats (80^120 g) were anesthetized with Nembutal and tracheotomized. The carotid artery and jugular vein were cannulated for continuous blood pressure monitoring, blood sampling, and drug administration. The animals were either sham-operated, or injected with 2.5 Wg/kg (i.v.) of PAF (1-O-hexadecyl-2-acetyl-sn-glycero-3phosphocholine, Sigma, St. Louis, MO). One hour after PAF, the animals were euthanized, the small intestine (and in some animals, also the heart and brain) was removed immediately, washed with icecold phosphate-bu¡ered saline, and stored at 370³C. For iNOS assay, intestinal epithelial cells were isolated and assayed as previously described [13]. Epithelial cells rather than whole tissue homogenate was used because our previous study showed that iNOS is localized mainly in the epithelial cells and comprises less than 10% of the total intestinal NOS activity [9].
2.2. Assay of the NOS activity of Ca2+-dependent (cNOS) and Ca2+-independent (iNOS) isoforms, and the inhibition of speci¢c NOS isoform The activity of NOS isoforms was determined by the L-[14 C]arginine conversion method as previously published [14]. In some experiments, immunoprecipitation was performed to remove a speci¢c NOS isoform before the enzyme assay. 10 Wl of anti-NOS I, anti-NOS II, or anti-NOS III antibody (Santa Cruz Biotechnology, Santa Cruz, CA) were mixed with 0.5 ml tissue homogenate (protein concentration 400 Wg/ml), shaken at 4³C for 2 h, and the antigen^antibody complex was removed by incubating with protein-A agarose beads (Santa Cruz) for 1 h followed by centrifugation. Control samples contained no antibody. 2.3. Detection of NOS isoforms by immunoprecipitation and Western blotting Intestinal tissue lysate was prepared as described previously [15]. The protein concentration of the tissue lysate was determined using a Sigma total protein kit (Sigma), and was adjusted to 0.5 mg/ml. After precleaning with protein-A agarose, 1.0 ml tissue lysate (0.5 mg protein) was incubated with 30 Wl anti-NOS antibody at 4³C with gentle shaking for 3 h, followed by incubation with 40 Wl protein-A agarose beads for 2 h at 4³C. The immunoprecipitate was collected and the bound immune complex was eluted by the addition of 200 Wl Laemmli bu¡er and boiling. After centrifugation, 150 Wl supernatant was loaded on a 7.5% sodium dodecyl sulfate^polyacrylamide gel for electrophoresis resolution. The resolved protein was transferred to a nitrocellulose membrane, and the blot was detected subsequently with anti-NOS antibody, horseradish peroxidase-labeled goat anti-rabbit IgG antibody, and visualized with an ECL system from Amersham. 2.4. RNA extraction and RT^PCR Total RNA was extracted from intestinal tissue by homogenizing the samples in a RNA-extracting agent, STAT-60 (TEL-TEST, Friendswood, TX), together with chloroform treatment and isopropanol precipitation following the manufacturer's instruc-
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tions. The total RNA was reversely transcribed (RT) at 37³C by using random hexamer (pdN6), and Moloney murine leukemia virus (M-MLV) reverse transcriptase (Gibco-BRL). An eNOS- or nNOS- sequence-speci¢c cDNA segment and a speci¢c sequence of L-actin (housekeeping gene) were ampli¢ed by polymerase chain reaction (PCR) with AmpliTaq DNA polymerase and speci¢c primers £anking the corresponding gene segments. The sequence of the primers and the PCR thermocycle pro¢les used are as previously reported [16]. The size of the corresponding PCR products for nNOS, eNOS, and L-actin are 280 bp, 345 bp, and 764 bp, respectively. The PCR products were resolved by agarose gel electrophoresis and visualized by staining with SYBR Green-1 (Molecular Probes, Eugene, OR). nNOS transcription was analyzed by semiquantitative RT^ PCR. The £uorescence of the PCR products of nNOS and L-actin ampli¢ed from the same reversetranscription reaction was analyzed using a STORM860 Phosphor-imager, and the nNOS/L-actin ratio between sham-operated and PAF-treated groups was compared. 2.5. Immuno£uorescent stains for nNOS Five-Wm frozen tissue sections were mounted on polylysine-coated slides and post-¢xed with ice-cold acetone. After blocking with goat serum, the slides were incubated with rabbit anti-nNOS antibody, followed by incubation with biotinylated goat anti-rabbit IgG antibody, and £uorescein isothiocyanate (FITC)-conjugated streptavidin. 2.6. Statistical analysis Results are expressed as mean þ S.E.M. Comparison between means are made by unpaired Student's t-test. P 6 0.05 is considered signi¢cant. 3. Results and discussion 3.1. An approximately 120 kDa nNOS is the predominant cNOS in rat small intestine By using the speci¢c anti-nNOS antibody R-20, which recognizes the amino acids 1400^1419 at the
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Fig. 1. Immune-blotting of nNOS and eNOS in brain, heart, and ileal tissue. (A) Western blotting of nNOS (after immunoprecipitation) with anti-NOS I Ab R-20. A major band of approximately 120 kDa was detected in ileal tissue. The molecular mass of nNOS from rat brain homogenate is 160 kDa. In addition, there were a few smaller and fainter bands with lower molecular mass. These may represent either sample degradation or low-level expression of other nNOS isoform in the brain. (B) Western blotting of eNOS (after immunoprecipitation) with anti-NOS III Ab C-20.
C-terminus of the rat nNOS, a band of approximately 120 kDa was precipitated from ileal tissue. The molecular mass of this protein is less than that of nNOS (160 kDa) from rat brain homogenate, which was precipitated with the same antibody (Fig. 1A). The 160 kDa nNOS is also detected, albeit weakly, in heart tissue, but not in the rat ileum. In contrast to nNOS, eNOS (135 kDa) is almost undetectable in the small intestine (Fig. 1B), as we failed to detect eNOS protein using a speci¢c anti-NOS III antibody. The validity of the antibody was con¢rmed by its strong reaction with the 135 kDa eNOS in the heart homogenate for immunoblotting (Fig. 1B). The PCR results support the ¢ndings of Western blotting. RT^PCR revealed a large amount of nNOS mRNA constitutively present in the rat ileum, whereas little eNOS mRNA was detected (Fig. 2). In contrast, RT^PCR detected abundant eNOS mRNA in heart tissue, but a relatively low level of nNOS mRNA (Fig. 2). These ¢ndings suggest that nNOS is the predominant cNOS in the intestine. Immuno£uorescent stains using anti-type I NOS antibody R-20 showed predominant localization of the protein in the myenteric plexus and in nerve ¢bers within the muscle layer (Fig. 3). This is identical
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to previous reports on the localization of nNOS in the intestine [17], suggesting that the 120 kDa protein is nNOS. In order to functionally verify that the 120 kDa protein is a NOS isoform, we compared the cNOS (Ca2 -dependent) activity of the ileal homogenate before and after the removal of type I, II, and III NOS isoform by immunoprecipitation with the speci¢c antibody. Fig. 4 (upper) shows that immunoprecipitation with anti-NOS I antibody removed 80 þ 5% of the cNOS (Ca2 -dependent) activity, whereas immunoprecipitation with anti-NOS II or anti-NOS III antibody did not signi¢cantly a¡ect the cNOS activity. The e¡ectiveness of the latter two antibodies to inhibit iNOS and eNOS, respectively, is demonstrated by the almost 100% inhibition of iNOS in isolated ileal epithelial cells (Fig. 4, lower), and approximately 70% inhibition of cNOS activity in heart homogenate (data not shown). These results strongly suggest that the 120 kDa protein is responsible for most of the Ca2 -dependent NOS activity in the intestine. Based on these observations and our previous ¢nding that more than 90% of the total NOS activity in normal intestine is composed of cNOS [9], we conclude that nNOS is the predominant NOS in the small intestine of normal rats. The remaining (usually 2^5%) NOS activity, as measured by L-[14 C]arginine conversion, is Ca2 -independent, and thus is probably accounted for by iNOS in in-
Fig. 2. Gene expression of nNOS and eNOS in the ileum analyzed by semiquantitative RT^PCR. The numbers of the thermocycles used for PCR ampli¢cation for nNOS, eNOS, and Lactin were 30, 30, and 20, respectively. The sizes of the corresponding PCR products are: nNOS, 280 bp; eNOS, 345 bp; Lactin (control), 764 bp.
Fig. 3. Immuno£uorescent staining of the small intestine using anti-nNOS Ab. Frozen ileal tissue section was incubated with R-20 (rabbit anti-rat NOS I, polyclonal, 40 Wg/ml), followed by biotinylated goat anti-rabbit IgG Ab and FITC-conjugated streptavidin.
testinal epithelium [9]. (Although iNOS is supposed to be only inducible, a low level of iNOS is constitutively present in normal rat intestine, mainly in the epithelial cells [9,18].) The nNOS mRNA and mature protein are structurally diverse as a consequence of alternative promoters and alternate mRNA splicing [19]. In addition to the originally cloned nNOS from a brain cDNA library [20], several di¡erentially spliced variants have been described in the rat [21]. Some of them have a molecular mass close to 120 kDa, such as nNOS-Q found in the skeletal muscle of mutant mice [22]; also described is a novel NOS mRNA transcript with a N-terminal truncation in the human testis which, when translated in CHO-K1 cells through transfection with a construct carrying the NOS sequence, turned out to have a molecular mass of 125 kDa [23]. Whether the 120 kDa nNOS we found in rat intestine is related to these nNOS isoforms awaits its cloning. It has been suggested that the expression of the alternative transcripts occurs with a high degree of tissue speci¢city [24]. Thus, it is possible that the diversity in alternative splicing re£ects a means of tight and speci¢c regulation of nNOS in di¡erent organs.
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dilators did not improve intestinal tissue perfusion. Furthermore, since most of the cNOS activity in the small intestine is accounted for by nNOS, it is possible that nNOS in the small intestine functions like eNOS in other organs, i.e., regulating perfusion and antagonizing leukocyte adhesion, directly or indirectly. This is a plausible hypothesis, in view of the reported localization of nNOS in the perivascular nerve ¢bers and the adventitia of blood vessels in many organs [34^36]. Although in this study we did not observe nNOS in the small intestinal vessels, other studies using more sensitive methods have revealed nNOS localizing in the endothelium [37]. 3.2. In vivo administration of PAF suppresses the activity and decreases the protein content of the 120 kDa nNOS, as well as nNOS mRNA in the intestine
Fig. 4. NOS activity after the removal of certain NOS isoforms by speci¢c antibodies. (Upper) Ca2 -dependent NOS activity in the ileal tissue homogenate after immunoprecipitation with antiNOS I Ab (R-20), anti-NOS II Ab (M-19), or anti-NOS III Ab (C-20), and agarose protein-A beads. *Inhibition level is signi¢cantly higher than 0 (n = 5, P 6 0.05). (Lower) Ca2 -independent NOS activity in isolated ileal epithelial cell homogenate after immunoprecipitation with anti-NOS I Ab (R-20), antiNOS II Ab (M-19), or anti-NOS III Ab (C-20), and agarose protein-A beads. *Inhibition level is signi¢cantly higher than 0 (n = 5, P 6 0.05).
It is surprising to ¢nd an almost complete absence of eNOS in the small intestine, since it is generally assumed (reviewed in [1]) that eNOS exerts an important protective function by maintaining adequate blood £ow and perfusion [25^27], protecting the mucosal barrier [28], and inhibiting leukocyte-endothelial adhesion [6]. This assumption probably derives from the vast amount of data from studies of isolated mesenteric vessels where eNOS is abundantly expressed [29^32]. However, the regulating mechanism of the larger mesenteric vessels and their smaller intramural branches (within the bowel wall) may be di¡erent. This is supported by our previous study [33] which showed that reversal of PAF-induced vasoconstriction of superior mesenteric artery by vaso-
To examine the regulation of the activity, protein content and gene transcription of this 120 kDa nNOS, we administered PAF to induce intestinal injury. At the dose used (2.5 Wg/kg, i.v.), all rats develop various degree of bowel injury in 1 h. As shown in Fig. 5 (upper), 1 h after PAF injection the cNOS activity in the intestine of PAF-treated rats is decreased to half of that in the sham-operated group. This decreased enzyme activity correlates with a marked reduction of intestinal nNOS protein content, as shown by Western blot (Fig. 5, lower), suggesting that PAF down-regulates nNOS protein synthesis. There is also a signi¢cant reduction in the nNOS mRNA after PAF injection, as revealed by RT^PCR (Fig. 5, middle), suggesting that PAF administration also causes a decline of nNOS mRNA, either due to a down-regulation of nNOS gene transcription, or to a decrease in mRNA stability. Previously, we demonstrated [9] that the activity of cNOS (or more accurately, nNOS) slowly increased with time in sham-operated rats, suggesting that mild stress (which may compromise intestinal perfusion) such as general anesthesia and surgery, could up-regulate nNOS activity. However, when a potent in£ammatory stimulus such as PAF is administered, the protective nNOS is rapidly down-regulated, and injury develops as a consequence [9]. The severity of the injury is inversely correlated with the tissue cNOS activity [9]. Further, this injury may be allevi-
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Acknowledgements This study was supported by NIH Grants DK34574 and HD31840.
References
Fig. 5. PAF (2.5 Wg/kg) down-regulates the enzyme activity (upper), protein content (lower), and gene transcription (middle) of nNOS in the small intestine. (upper) Ca2 -dependent NOS activity in the ileal tissue homogenate of sham-operated (n = 6) and PAF-treated (n = 7) rats. *Signi¢cantly di¡erent from shamoperated group (P 6 0.05). (lower) Western blot showing reduction of nNOS in the ileum after PAF injection. A typical experiment is shown. (middle) nNOS mRNA (expressed as the ratio of nNOS/L-actin mRNA by RT^PCR) in the ileum of sham-operated (n = 6) and PAF-treated (n = 7) rats. *Signi¢cantly di¡erent from sham-operated group (P 6 0.05).
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