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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA

Neutral Endopeptidase Up-Regulation in Isolated Human Umbilical Artery: Involvement in Desensitization of Bradykinin-Induced Vasoconstrictor Effects

Departamento de Farmacología, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina

Received September 3, 2006; accepted November 1, 2006.

	Abstract

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Previous reports show that bradykinin B₂ receptors mediate contractile responses induced by bradykinin (BK) in human umbilical artery (HUA). However, although it has been reported that BK-induced responses can desensitize in several inflammatory models, the effects of prolonged in vitro incubation on BK-induced vasoconstriction in HUA have not been studied. In isolated HUA rings, BK-induced responses after a 5-h in vitro incubation showed a marked desensitization compared with responses at 2 h. Inhibition of either angiotensin-converting enzyme (ACE) or neutral endopeptidase (NEP), both BK-inactivating enzymes, failed to modify responses to BK at 2 h. After 5 h, ACE inhibition produced only a slight potentiation of BK-induced responses. In contrast, BK-induced vasoconstriction at 5 h was markedly potentiated by NEP inhibition. Moreover, NEP activity, measured by hydrolysis of its synthetic substrate (Z-Ala-Ala-Leu-p-nitroanilide), showed a 2.4-fold increase in 5-h incubated versus 2-h incubated tissues, which was completely reversed by cycloheximide (CHX) treatment. Furthermore, CHX significantly potentiated BK-induced responses, suggesting that NEP-mediated kininase activity increase at 5 h depends on de novo protein synthesis. In addition, under NEP inhibition, CHX treatment failed to produce an additional potentiation of BK-induced vasoconstriction. Still, NEP up-regulation was confirmed by Western blot, showing a 2.1-fold increase in immunoreactive NEP in 5-h incubated versus 2-h incubated HUA. In summary, the present study provides strong pharmacological evidence that NEP is up-regulated and plays a key role in desensitization of BK-induced vasoconstriction after prolonged in vitro incubation in HUA. Our results provide new insights into the possible mechanisms involved in BK-induced response desensitization during sustained inflammatory conditions.

Whereas receptor expression is a major determinant of the action of kinins, agonist production and inactivation rates have been shown to play an important role in BKB₁ and BKB₂ receptor-mediated effects (Marceau et al., 1998). Vascular inactivation of BK can be mediated by several enzymes. Among them, angiotensin-converting enzyme (ACE) has been classically described as the main inactivator of BK in vascular tissues (Erdos, 1990). In addition, Gafford et al. (1983) have shown that neutral endopeptidase (NEP) is able to hydrolyze BK, yielding inactive fragments. Interestingly, we have recently shown that ACE and NEP are present in HUA smooth muscle after prolonged in vitro incubation (Pelorosso et al., 2005).

Taking into account the above-mentioned evidence, our objectives were 1) to evaluate changes in HUA contractile sensitivity to BK after prolonged in vitro incubation and 2) to elucidate the mechanisms involved and the role played, if any, by ACE and NEP in these phenomena.

	Materials and Methods

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Tissue Preparation. Human umbilical cords were obtained from normal full-term deliveries and excised midway between the child and the placenta. Cords were immediately placed in modified Krebs' solution (of the following composition: 119 mM NaCl, 4.7 mM KCl, 25 mM NaHCO₃, 1.2 mM KH₂PO₄, 2.5 mM CaCl₂, 1.0 mM MgSO₄, 0.004 mM EDTA, and 11 mM D-glucose) at 4°C. The use of umbilical samples was approved by the ethics committee of the School of Pharmacy and Biochemistry of the University of Buenos Aires, and written informed consent was obtained from each parturient.

Usually within 3 h after delivery, the samples were placed onto dissecting dishes containing Krebs' solution, and arteries (internal diameter 1 mm) were carefully dissected free from Wharton's jelly using microdissecting instruments and cut into rings of 3 mm width.

Functional Studies. Immediately after dissection, rings were suspended in 5-ml organ baths and stretched with an optimal resting tension of 2 to 4 g (Tufan et al., 2003). Changes in tension were measured with Grass isometric force transducers (FT-03C; Grass Instruments, Quincy, MA) and displayed on Grass polygraphs (model 7D). During incubation, the Krebs' solution was maintained at 37°C and pH 7.4 by constant bubbling with 95% O₂·5% CO₂. The bath solution was replaced every 15 min with fresh bubbled buffer. Concentration response curves (CRCs) to BKB₂ receptor subtype agonists were constructed after a 2- or 5-h in vitro incubation by cumulative addition in 0.25 log₁₀ increments. In other experiments, HUA rings were incubated for 2, 3, or 5 h and then sequentially challenged with 1 µM des-Arg¹⁰-kallidin, 0.1 µM BK, and 10 µM serotonin (5-HT). Whenever necessary, effective inhibitory doses of peptidase inhibitors were used: 1 µM captopril (ACE inhibitor, IC₅₀ 38 nM) (Miyamoto et al., 2002), 10 µM phosphoramidon (NEP inhibitor, IC₅₀ 10 nM) (Loffler, 2000), 10 µM thiorphan (NEP inhibitor, IC₅₀ 1.4 nM) (Miyamoto et al., 2002), and 10 µM amastatin [aminopeptidase M (APM) inhibitor, IC₅₀ 50 nM] (Proud et al., 1987) were applied 30 min before the addition of BKB₂ and BKB₁ receptor agonists. The concentrations of amastatin, captopril, and phosphoramidon used in this study were previously shown to produce the inhibition of des-Arg¹⁰-kallidin inactivation as assessed by functional contractility studies in HUA (Pelorosso et al., 2005). Phosphoramidon is also able to inhibit ACE (IC₅₀ 78 µM) (Kukkola et al., 1995) and endothelin-converting enzyme (ECE) (IC₅₀ 2.5 µM) (Hoang and Turner, 1997). However, we have previously shown that 10 µM phosphoramidon is unable to inhibit ACE kininase activity in functional contractility studies carried out in HUA (Pelorosso et al., 2005). Although partial inhibition of ECE could be expected at the phosphoramidon concentrations used in the present work, Hoang and Turner (1997) have shown that up to 100 µM phosphoramidon is necessary to completely inhibit human ECE. On the other hand, ECE is highly resistant to inhibition by thiorphan (IC₅₀ higher than 200 µM) (Hoang and Turner, 1997). Nevertheless, it is important to note that thiorphan is also able to inhibit ACE (IC₅₀ 295 nM) (Miyamoto et al., 2002). Therefore, all experiments involving thiorphan were carried out in presence of 1 µM captopril to avoid possible ACE interference. In addition, whenever necessary 10 µM cycloheximide (protein synthesis inhibitor) was added to the bath during the entire incubation. None of the inhibitors tested produced any significant change in the basal tone of the HUA rings when applied.

At the end of each CRC, 10 µM 5-HT was applied to determine the tissue maximal contractile response (Altura et al., 1972). All experiments were performed in parallel with rings from the same umbilical cord. Only one agonist CRC was performed in each ring.

NEP Assay. HUA rings were snap-frozen after a 2- or 5-h in vitro incubation or as fresh, nonincubated tissue. On the day of the experiment, tissues were ground to powder and then resuspended in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM benzamidine hydrochloride). Whole-cell extracts were prepared by centrifugation at 3000g for 15 min at 4°C. Supernatants were collected and assayed for protein content.

Whole-cell extracts (150 µg of protein) from fresh or incubated tissues were incubated with 500 µM Z-Ala-Ala-Leu-p-nitroanilide in 100 µl of 50 mM HEPES (pH 7.4) for 1 h at 37°C. The reaction was stopped by addition of 10 µM phosphoramidon. Leucine-aminopeptidase (5 mU) was then added, and the reaction mixture was further incubated for 30 min at 37°C. Reaction mixtures were taken to ice and then measured for absorbance at 405 nm. NEP activity was determined by the absorbance of the liberated p-nitroaniline and from the decrease in digestion rate caused by 10 µM phosphoramidon. Specific activity was calculated upon construction of a standard curve with known Leu-p-nitroanilide concentrations.

Western Blot. HUA rings were snap-frozen after a 2- or a 5-h in vitro incubation or as fresh, nonincubated tissue. On the day of the experiment, tissues were ground to powder and then resuspended in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM benzamidine hydrochloride). Whole-cell extracts were prepared by centrifugation at 3000g for 15 min at 4°C. Supernatants were collected and assayed for protein content.

Whole-cell extracts (70 µg of proteins) were separated by electrophoresis on 8% SDS-polyacrylamide gels, and resolved proteins were electrotransferred onto polyvinylidene difluoride membranes. Membranes were blocked in Tris-buffered saline containing 0.5% Tween 20 and 5% nonfat dried milk and then incubated overnight with either anti-NEP or anti-APM rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were washed three times in Tris-buffered saline containing 0.5% Tween 20 before incubation with alkaline phosphatase-conjugated goat anti-rabbit IgG for 1 h. Immunoreactive bands were detected by enhanced chemiluminescence, scanned, and quantified using Quantity One software (Bio-Rad, Hercules, CA). Equal gel loading was verified by Ponceau Red staining of the membrane. Changes in immunoreactive NEP levels were calculated on the basis of a standard curve constructed with protein extracts from rat kidney. Negative controls were carried out in the absence of the primary antibody.

Expression of Results and Statistical Analysis. All data are expressed as means ± S.E.M. The number of experiments (n) represents the number of rings from different cords tested. Contractile responses are expressed as a percentage of the tissue maximal response elicited by 10 µM 5-HT. The estimates of EC₅₀ (i.e., the agonist concentration that produces 50% of the maximal response), the maximal response (E_max), and the slope factor (n_H) were obtained using ALLFIT (DeLean et al., 1978). Briefly, responses obtained for each agonist concentration in each ring tested in the same group were averaged and then fitted to a four-parameter logistic model expressed as eq. 1: Y = a – E_max/1 + (X/EC₅₀)^nH + E_max. where Y is the response, X is the arithmetic dose, and a is the response when X = 0. EC₅₀ values were transformed into pEC₅₀ values (–log EC₅₀). Statistical analysis was performed by means of unpaired Student's t test or one-way analysis of variance followed by Tukey's post-test, when appropriate. P values < 0.05 were taken to indicate significant differences.

Reagents. The following compounds were used for functional studies: 5-hydroxytryptamine creatine sulfate complex from Sigma/RBI (Natick, MA); des-Arg¹⁰-kallidin, BK, and N-methyl-D-Phe⁷-BK from Bachem Biosciences (King of Prussia, PA); amastatin hydrochloride [(2S,3R)-3-amino-2-hydroxy-5-methylhexanoyl-Val-Val-Asp hydrochloride], captopril (N-[(S)-3-mercapto-2-methylpropionyl]-L-proline), cycloheximide (3-[2-(3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]glutarimide), and thiorphan [(±)-N-(3-mercapto-2-benzylpropionyl)glycine] from Sigma Chemical (St. Louis, MO); and phosphoramidon [N--L-rhamnopyranosyloxy(hydroxyphosphinyl)-L-leucyl-L-tryptophan] and Z-Ala-Ala-Leu-p-nitroanilide (benzyloxycarbonyl-L-alanyl-L-alanyl-L-leucine p-nitroanilide) from Peptides International Inc. (Louisville, KY). Preparation of all stock solutions and their subsequent dilutions were performed in glass-bidistilled water. Stock solutions were stored in frozen aliquots and thawed and diluted daily.

	Results

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Effects of in Vitro Incubation Time on BK-Induced Contractile Responses in HUA. Contractile responses induced by BK obtained after a 2-h in vitro incubation yielded a pEC₅₀ of 8.38 ± 0.05 and a maximal response of 82.9 ± 7.9% (n = 8) (Fig. 1). When tissues were incubated during 5 h, pEC₅₀ was significantly lower (7.98 ± 0.06, n = 8, P < 0.05), but the maximal response was not modified (E_max 69.5 ± 9.3%, n = 8) (Fig. 1).

View larger version (13K): [in this window] [in a new window]   Fig. 1. Responses elicited by BK after a 2 ()- or 5 ()-h in vitro incubation. *, significant differences between pEC50 (P < 0.05).

View larger version (16K): [in this window] [in a new window]   Fig. 2. A, responses elicited by BK after a 2-h in vitro incubation in the presence () or absence () of 1 µM captopril. B, responses elicited by BK after a 5-h in vitro incubation in the presence () or absence () of 1 µM captopril. *, significant differences between pEC50 (P < 0.05). Data for BK effects in the absence of peptidase inhibitors correspond to those shown in Fig. 1.

View larger version (17K): [in this window] [in a new window]   Fig. 3. A, responses elicited by BK after a 2-h in vitro incubation in the presence () or absence () of 10 µM phosphoramidon. B, responses elicited by BK after a 5-h in vitro incubation in the presence () or absence () of 10 µM phosphoramidon. *, significant differences between pEC50 (P < 0.05). Data for BK effects in the absence of peptidase inhibitors correspond to those shown in Fig. 1.

However, contractile responses induced by BK after a 5-h in vitro incubation in HUA rings (pEC₅₀ 7.98 ± 0.06, E_max 69.5 ± 9.3%, n = 8) were significantly potentiated by treatment with 10 µM phosphoramidon (pEC₅₀ 8.61 ± 0.10, P < 0.05, E_max 89.1 ± 5.9%, n = 8) (Fig. 3B).

Lack of Effects of Phosphoramidon on Responses Elicited by N-Methyl-D-Phe⁷-BK after a 5-h in Vitro Incubation. Contractile responses induced by N-methyl-D-Phe⁷-BK, a BKB₂ receptor agonist analog (Reissmann et al., 1996), after a 5-h in vitro incubation in HUA in the presence of 1 µM captopril (pEC₅₀ 6.23 ± 0.06, E_max 92.0 ± 3.5%, n = 6) failed to be modified by further addition of 10 µM phosphoramidon (pEC₅₀ 6.28 ± 0.09, E_max 85.7 ± 3.5%, n = 6) (Fig. 4A). As a positive control, we evaluated the effects of the addition of 10 µM phosphoramidon on BK-elicited responses obtained in presence of 1 µM captopril after a 5-h in vitro incubation (captopril: pEC₅₀ 8.26 ± 0.06, E_max 75.9 ± 7.9%, n = 8; phosphoramidon plus captopril: pEC₅₀ 8.60 ± 0.05, P < 0.05, E_max 91.8 ± 2.5%, n = 8) (Fig. 4B).

View larger version (18K): [in this window] [in a new window]   Fig. 4. A, responses elicited by N-methyl-D-Phe7-BK after a 5-h in vitro incubation in the presence of 1 µM captopril () or 1 µM captopril and 10 µM phosphoramidon (). B, responses elicited by BK after a 5-h in vitro incubation in the presence of 1 µM captopril () or 1 µM captopril and 10 µM phosphoramidon (). *, significant differences between pEC50 (P < 0.05).

View larger version (18K): [in this window] [in a new window]   Fig. 5. A, responses elicited by BK after a 2-h in vitro incubation in the presence of 1 µM captopril () or 1 µM captopril and 10 µM thiorphan (). B, responses elicited by BK after a 5-h in vitro incubation in the presence of 1 µM captopril () or 1 µM captopril and 10 µM thiorphan (). * represents significant differences between pEC50 (P < 0.05).

Role of de Novo Protein Synthesis in Changes in NEP Kininase Activity as a Function of in Vitro Incubation Time in HUA. Contractile responses induced by BK after a 5-h in vitro incubation in HUA rings in the presence of 1 µM captopril (pEC₅₀ 7.77 ± 0.05, E_max 83.6 ± 3.6%, n = 6) were significantly potentiated by treatment with 10 µM cycloheximide (pEC₅₀ 8.10 ± 0.04, P < 0.05, E_max 89.3 ± 2.4%, n = 6) (Fig. 6A). However, when rings were treated with 1 µM captopril plus 10 µM phosphoramidon, continuous exposure to 10 µM cycloheximide failed to modify BK-induced contractile responses at 5 h (captopril plus phosphoramidon: pEC₅₀ 8.48 ± 0.03, E_max 94.8 ± 2.2%, n = 6; captopril plus phosphoramidon plus cycloheximide: pEC₅₀ 8.42 ± 0.03, E_max 93.9 ± 2.2%, n = 6) (Fig. 6B). In addition, neither potency nor maximal responses to BK were modified when 10 µM cycloheximide was applied 15 min before the construction of the CRC (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 6. A, responses elicited by BK after a 5-h in vitro incubation in the presence of 1 µM captopril with () or without () continuous exposure to 10 µM cycloheximide. B, responses elicited by BK after a 5-h in vitro incubation in the presence of 1 µM captopril and 10 µM phosphoramidon with () or without () continuous exposure to 10 µM cycloheximide. *, significant differences between pEC50 (P < 0.05).

Role of in Vitro Incubation Time and de Novo Protein Synthesis in Changes in NEP Enzymatic Activity in HUA Whole-Cell Extracts. NEP activity was measured on HUA whole-cell extract preparations from fresh tissues and from rings incubated during 2 and 5 h (Fig. 7). Activity in fresh tissues was 0.20 ± 0.05 nmol/mg/min (n = 6). In addition, activity at 2 h was 0.29 ± 0.07 nmol/mg/min (n = 3), which was not higher than that in fresh tissues. However, activity measured after a 5-h incubation period was higher than those observed in fresh and 2-h incubated tissues (0.70 ± 0.08 nmol/mg/min, n = 7, P < 0.05). Treatment with 10 µM cycloheximide during 5 h produced a decrease in NEP activity compared with 5-h incubated tissues (0.27 ± 0.07 nmol/mg/min, n = 2, P < 0.05) (Fig. 7).

View larger version (25K): [in this window] [in a new window]   Fig. 7. NEP enzymatic activity measured in whole-cell extracts from fresh HUA (), tissues incubated for 2 h () tissues incubated for 5 h (), and tissues incubated for 5 h with 10 µM cycloheximide (CHX, ). *, significant differences between means (P < 0.05).

A single band of approximately 100 kDa was detected in extracts obtained from fresh and 2- and 5-h incubated HUA rings (Fig. 8A). NEP content after a 2-h incubation was 1.71 ± 0.40 (n = 3, Fig. 8B)-fold higher than that in fresh HUA. Moreover, NEP content after a 5-h incubation was higher than that observed at 2 h (3.62 ± 0.26, n = 6, P < 0.05) (Fig. 8B).

View larger version (32K): [in this window] [in a new window]   Fig. 8. A, representative blot showing a 100-kDa band corresponding to immunoreactive NEP protein in whole-cell extracts from fresh HUA, HUA incubated for 2 h, HUA incubated for 5 h, and fresh rat kidney (positive control). B, densitometric analysis of NEP protein content in membrane extracts obtained from fresh HUA (, n = 6), HUA incubated for 2 h (, n = 3), and HUA incubated for 5 h (, n = 6). Results are expressed as fold increase of NEP protein content in comparison with fresh tissues. *, significant differences between means (P < 0.05).

Role of in Vitro Incubation Time in Changes in Contractile Responses Mediated by BKB₁ Receptor Subtype in HUA. Maximal contractile responses in HUA evoked by a single exposure to 1 µM des-Arg¹⁰-kallidin in presence of 10 µM amastatin, 10 µM phosphoramidon, and 1 µM captopril were determined at different incubation times. Responses obtained after a 2-h in vitro incubation were 37.9 ± 11.8% (n = 9). Responses at 3 h were higher, although not significantly different (64.4 ± 12.1%, n = 9). However, when HUA rings were challenged with 1 µM des-Arg¹⁰-kallidin after a 5-h in vitro incubation, maximal responses were significantly higher than those obtained at 2 h (81.0 ± 11.4%, P < 0.05, n = 9). In contrast, maximal contractile responses evoked by a single exposure to 0.1 µM BK remained unchanged through the incubation times tested (2 h: 90.2 ± 3.0%, n = 9; 3 h: 94.1 ± 2.0%, n = 9; 5 h: 95.6 ± 1.1, n = 9) (Fig. 9).

View larger version (15K): [in this window] [in a new window]   Fig. 9. Maximal responses elicited by single exposure to 1 µM des-Arg10-kallidin () or 0.1 µM BK () in HUA rings incubated for 2, 3, and 5 h. All rings were exposed to 10 µM amastatin, 1 µM captopril, and 10 µM phosphoramidon. *, significant differences between Emax values.

	Discussion

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Yet, our report also shows that the selective BKB₂ receptor agonist, BK, displays a significant decrease in potency for contractile effects as a function of in vitro incubation time in isolated HUA. Similar findings have been reported in other models in which sensitization of responses mediated by BKB₁ receptor is associated with a clear reduction of BKB₂ receptor-mediated effects (Cruwys et al., 1994; Campos et al., 1996; El Sayah et al., 2006). For instance, Campos et al. (1996) have shown that LPS causes an increase in BKB₁ receptor-mediated rat paw edema, accompanied by a patent drop in the BKB₂ receptor-mediated edematogenic response. Recently, El Sayah et al. (2006) have reported that BK-induced contractile responses in isolated LPS-treated pig iris sphincter muscle are markedly reduced in a time-dependent process. Still, mechanisms underlying the functional desensitization of BKB₂ receptor-mediated responses are currently unknown. True BKB₂ receptor down-regulation is only observed in some forms of inflammation (acute renal transplant rejection) (Naidoo et al., 1996). Nevertheless, sustained, agonist-mediated stimulation does not lead to detectable degradation of rabbit BKB₂ receptor (Bachvarov et al., 2001). Moreover, clinical samples of chronic inflammatory tissues (psoriatic skin or nasal tissue samples from subjects with allergic rhinitis) show the up-regulation of BKB₁ receptor mRNA without the down-regulation of BKB₂ receptor mRNA (Schremmer-Danninger et al., 1999; Christiansen et al., 2002). In addition, whereas a patent up-regulation of BKB₁ receptor is observed in aortas obtained from LPS-treated pigs, neither autoradiographic visualization nor displacement studies using BKB₂ receptor ligands yield differences in BKB₂ receptor population characteristics between control and LPS-treated animals (Schremmer-Danninger et al., 1998). Interestingly, our group has recently demonstrated a relevant role for ACE and NEP in the functional inactivation of kinins after prolonged in vitro incubation of HUA (Pelorosso et al., 2005). This fact, in addition to poor evidence in favor of the possible down-regulation of BKB₂ receptor, led us to analyze the potential role of these enzymes in the BK desensitization phenomenon observed in isolated HUA.

In the present report we have shown that whereas BK-induced responses after a 2-h in vitro incubation failed to be modified by captopril treatment, a small, but significant, potentiation was observed by exposure to the ACE inhibitor after a 5-h incubation. There are several studies using recombinant BKB₂ receptor models that suggest a possible interaction of ACE inhibitors with BKB₂ receptors leading to the potentiation of BK-induced responses independently of inhibition of ACE activity (Tom et al., 2003). However, strong pharmacological evidence suggests that potentiation of the vasoactive effects of BK by ACE inhibitors in physiological models is due to inhibition of kinin metabolism (Dendorfer et al., 2001; Tom et al., 2002). Therefore, our results are compatible with a possible increase in ACE kininase activity after a 5 h-incubation period in isolated HUA.

Furthermore, although BK-elicited responses obtained after 2 h in vitro incubation were not modified by phosphoramidon treatment, contractile responses induced by BK in HUA at 5 h were markedly potentiated by NEP inhibition. These results suggest that the ability of NEP to perform functional inactivation of BK in biophase is significantly increased after a 5-h in vitro incubation in isolated HUA. Moreover, BK potency at 5 h in the presence of phosphoramidon (pEC₅₀ 8.61) was not significantly different from that observed at 2 h with (pEC₅₀ 8.48) or without (pEC₅₀ 8.38) phosphoramidon treatment. These results, in addition, suggest that the possible occurrence of BKB₂ receptor down-regulation in our model is unlikely.

Given the magnitude of the effects of NEP inhibition in comparison to those observed under ACE blockade, we decided to pursue a more profound characterization of NEP activity in HUA. Therefore, all subsequent CRCs were carried out in presence of 1 µM captopril to rule out any involvement of ACE in further observations.

In the following series of experiments, we tested the effects of phosphoramidon treatment on responses elicited by the BKB₂ analog, N-methyl-D-Phe⁷-BK (Reissmann et al., 1996) at 5 h. This compound behaved as a full, although less potent agonist, in comparison to BK. These observations are consistent with those reported for this analog in different BKB₂ receptor models (Dendorfer et al., 1999, 2001). In addition, 10 µM phosphoramidon failed to modify N-methyl-D-Phe⁷-BK-induced contractile responses in HUA. This result suggests that preservation of the original Pro⁷–Phe⁸ bond structure of BK, the main cleavage site of NEP, is necessary for phosphoramidon-potentiating effects. Previous reports indicate the possibility that NEP inhibitors may potentiate BK-induced responses by other mechanisms beside enzyme inhibition (Deddish et al., 2002). However, our results suggest that potentiation of BK-induced responses by NEP inhibition at 5 h in HUA is due to impairment of kinin biotransformation in biophase.

To confirm that potentiation of BK-induced contractile responses at 5 h in HUA is mediated by NEP inhibition, a chemically unrelated NEP inhibitor, thiorphan, was tested (Roques et al., 1995). Although thiorphan failed to modify BK-induced responses in HUA rings after a 2-h in vitro incubation, it significantly potentiated responses elicited by BK at 5 h. Moreover, potentiation of BK-elicited responses produced by thiorphan was quantitatively equivalent to that observed with phosphoramidon. These results provide further evidence of changes in NEP kininase activity as a function of in vitro incubation time in HUA.

To evaluate the possible correlation between de novo protein synthesis and the increase in NEP kininase activity in isolated HUA incubated for 5 h, responses to BK were evaluated in rings continuously treated with cycloheximide, a protein synthesis inhibitor. The significative potentiation of BK-induced responses after a 5-h incubation with cycloheximide suggests that NEP might be up-regulated during prolonged in vitro incubation in HUA. Furthermore, the lack of effect of cycloheximide on BK-induced responses in presence of phosphoramidon further substantiates our hypothesis that the effects of cycloheximide depend on NEP synthesis inhibition.

The increase in NEP activity as a function of in vitro incubation time in HUA was further evaluated by means of its biochemical analysis in HUA whole-cell extracts. The sharp increase in NEP activity after a 5-h incubation is consistent with up-regulation of the enzyme in HUA. Furthermore, the abolishment of this increase observed with cycloheximide treatment constitutes strong biochemical evidence suggesting that NEP is newly synthesized in HUA during prolonged in vitro incubation. In addition, the significant increment in NEP immunoreactive protein content observed in HUA whole-cell extracts after a 5-h incubation further suggests that NEP is up-regulated in this tissue.

Previous reports have provided evidence that NEP up-regulation might constitute a common response to tissue injury. For instance, Walther et al. (2002) showed that NEP activity in 6-day infarcted left ventricles from rats was 100% higher than in the control (sham-operated) group. Further evidence in support of this hypothesis has been provided by Olerud et al. (1999). This group has shown that whereas in normal skin NEP is strikingly localized to keratinocytes of the epidermal basal layer, incisional wounds produce an up-regulation of NEP already evident 6 h after wounding in these cells.

During sustained inflammatory insult, kinin-mediated responses adapt from a BKB₂ receptor type in the acute phase to a BKB₁ receptor type in the chronic phase (Dray and Perkins, 1993). This adaptation is explained in part by BKB₁ receptor induction from essentially a null level, which is triggered by several proinflammatory stimuli (Leeb-Lundberg et al., 2005). However, additional synchronous processes may take part in the shift leading to the prevalence of BKB₁ over BKB₂ receptor-mediated responses during continuous inflammatory stimuli. For instance, Schremmer-Danninger et al. (1998) have shown that LPS treatment is able to produce an increase of up to 200% in carboxypeptidase M activity in pig aortic samples. Carboxypeptidase M cleaves carboxyl terminal arginine in BK and kallidin, yielding the BKB₁ selective agonists des-Arg⁹-BK and des-Arg¹⁰-kallidin, respectively. In contrast, preincubation of rabbit aortic rings for 6 h, a procedure that sharply up-regulates BKB₁ receptor expression, produces a negligible increase in activity of APM, a major inactivating enzyme of des-Arg¹⁰-kallidin, the endogenous BKB₁ receptor agonist (Fortin et al., 2005). This result coincides with that found in our model in which the APM level as determined by Western blot remains unchanged in fresh versus 5-h incubated HUA tissue (data not shown). Thus, the above-mentioned evidence, in addition to that described in the present study, suggests that kinin-mediated responses may shift from a BKB₂ type to a BKB₁ type during sustained inflammatory insult not only by changes in relative receptor densities but also by changes in the expression of kininases, leading to increased BKB₁ agonist production and, at the same time, higher BKB₂ agonist inactivation rates.

In summary, our work clearly demonstrates that prolonged in vitro incubation of HUA rings leads to NEP up-regulation, evidenced by the increase in immunoreactive NEP content and its correlation with increased enzymatic activity. Furthermore, data presented here provide strong pharmacological evidence that NEP up-regulation produces an increase in BK biological inactivation, resulting in a marked reduction of kinin-induced contractile responses in HUA rings. Taking into account the fact that NEP constitutes a minor functional inactivator of des-Arg¹⁰-kallidin (Pelorosso et al., 2005) our results suggest that NEP up-regulation might play a role in the shift of kinin-mediated responses from a BKB₂ type in the acute phase to a BKB₁ type during sustained inflammatory processes.

	Acknowledgements

 
We thank the Instituto Médico de Obstetricia (Buenos Aires) for their efforts in providing umbilical tissues.

	Footnotes

 
This research was supported by grants from the University of Buenos Aires (Grant M-003). F.G.P. and W.N. are research fellows of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). A.E.E. is member of CONICET.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.113381.

ABBREVIATIONS: BK, bradykinin; HUA, human umbilical artery; ACE, angiotensin-converting enzyme; NEP, neural endopeptidase; CRC, concentration-response curve; 5-HT, 5-hydroxytryptamine, serotonin; APM, aminopeptidase M; ECE, endothelin-converting enzyme; LPS, lipopolysaccharide; IL, interleukin.

Address correspondence to: Dr. Rodolfo Pedro Rothlin, 2155 Paraguay St., 9th floor, Ciudad Autónoma de Buenos Aires (CP 1121), Argentina. E-mail: farmaco3{at}fmed.uba.ar

	References

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