Our understanding of renin-angiotensin system (RAS) has experienced remarkable change over the past two decades. The new functional components [e.g. Ang-(1-7), Ang IV, Ang-(1-12)] and pathways [e.g. angiotensin converting enzyme 2, (ACE2)] have been described (1-4). It has also became clear that all bioactive angiotensin peptides can be generated not only in systemic circulation, but also in several tissues and organs. Thus, local renin-angiotensin systems as well as various biological functions of multiple angiotensin peptides acting via autocrine and/or paracrine manner have been described in the kidney, heart, liver, pancreas, adrenal gland, brain, ovaries and testes (5-8).

There is relatively scarce information about formation and action of angiotensin peptides in gastrointestinal tract, especially in stomach wall. Previously ACE mRNA and protein and recently ACE2 have been demonstrated to be present in gastric mucosa (9-11). There are also several indirect evidences, coming from the use of ACE inhibitors and angiotensin receptor blockers, that angiotensin II may regulate mucosal blood flow and play role in mucosal damage (12-16). However, studies directly showing the metabolism and action of angiotensin peptides in stomach wall are still lacking. This is partly due to methodological challenges with comprehensive, accurate and reliable measurements of tissue angiotensin levels.

Only recently, through coupling of liquid chromatography and electrospray ionization - mass spectrometry (LC-ESI-MS), we developed an accurate, reproducible and comprehensive method of quantitation of formation of angiotensin peptides in organ bath of tissue fragments exposed to exogenous Ang I (17, 18). Here, using LC-ESI-MS method we assessed the metabolism of Ang I in organ bath of rat stomach wall.


MATERIALS AND METHODS
Isolation and treatment of rat stomach wall

Before experiment, animals were fasting (with free access to water) for 48 hrs. Male Wistar-Kyoto rats 7 months of age and 350-420 g of weight were administered fraxiparine (2850 IU, i.p.) and anaesthetized with 50 mg of thiopentone (50 mg/ml, i.p.). Fragments of stomach wall (3mm x 3mm, mid-portion of the corpus) were excised through abdominal incision, washed with cold, standard Krebs-Henseleit solution (glucose 10 mM, pyruvate 2 mM, HEPES 10 mM, EDTA 0.03 mM, NaCl 118 mM, KCl 4.7 mM, CaCl2 2.5 mM, KH2PO4 1.2 mM, MgSO4 1.2 mM, NaHCO3 15 mM) and cleaned of thrombi and tissue remnants. Tissue fragments were incubated for 30 minutes at 37°C in Eppendorf tubes in 450 µl of Krebs-Henseleit solution and continuously bubbled with 95%O2/5%CO2. The pH of incubation buffer was 7.3 and remained stable throughout experiment.

Sample of 50 µl of buffer was removed to provide information on background production of angiotensin metabolites. After 5 min of incubation, angiotensin I was added (final concentration 1 µM). Samples of 50 µl of buffer were removed after another 15 min of incubation. Each sample was promptly frozen at -70°C until further analysis with mass spectrometry. Tissue pieces were dried overnight at 60°C to allow estimation of angiotensin metabolites’ production per mg of dry tissue.

All procedures were approved by an Ethical Committee of the Jagiellonian University, School of Medicine.

Liquid chromatography – electrospray ionization - mass spectrometry (LC-ESI-MS)

Separation of angiotensins was performed on a reversed-phase, high performance liquid chromatography (HPLC) system using a PepMap (LC Packings, CA, USA) reverse-phased C18 column (150mm x 1mm ID, 5µm particle size). The mobile phase solvents were: 5% acetonitrile in a buffer of 4mM ammonium formate with 4 mM formic acid (phase A) and 95% acetonitrile in a buffer of 4mM ammonium formate with 4 mM formic acid (phase B). The angiotensins were separated at a flow rate of 45 µl/min with a linear gradient. The mobile phase gradient was started with linear increase from 0 to 25% phase B within 35 min, then increasing to 40% B in 40 min, next rapidly increasing to 80% B within 12 sec, followed by 80% B isocratic for 5 min. Finally B phase was rapidly decreased to 0% and left for isocratic 100% A column equilibration for 30 min.

Mass spectrometric detection was performed using a LCQ ion- trap mass spectrometer (Finnigan, San Jose, USA), equipped with an ESI source (electrospray). All experiments were carried out in the positive ion mode. The main working parameters were as follows: nitrogen (sheath gas) flow rate 60psi, ion spray voltage 4.5kV, capillary temperature 200°C. For detection, selected ion monitoring (SIM) mode was used. LCQ data were analyzed by using the Xcalibur Software (Finnigan, San Jose, USA). Concentrations of angiotensins (Ang I, II, III, IV, 1-9, 1-7, 1-5) were calculated using the standard calibration curves, constructed by linear regression analysis by plotting of peak area vs. angiotensin concentration and calculated as ng/mg dry tissue.

Assessment of mRNA expression

Frozen fragments of stomach , kidney artery, and aorta were grounded in liquid nitrogen and homogenized in Trizol buffer (TRIzol® Reagent, Gibco-BRL, USA), and total RNA was isolated by single-step, guanidium thiocyanate-phenol-chloroform extraction according to the manufacturer’s instructions (Gibco-BRL, USA).

1 µg of total RNA from each sample was reverse-transcribed to complementary DNA (cDNA) using oligo(dT)12-18 primer and MMLV reverse transcriptase (Gibco BRL, USA). The final RT reaction volume was 20 µl. The reaction was performed in T3 Thermocycler (Biometra, Germany) at 42°C during 2 h, and the enzyme was then denatured at 99°C for 5 min.

The ACE, ACE2, NEP, and ß-actin cDNA fragments (478, 425, 432, and 308 base pair long, respectively) were amplified using specific primer pairs for rat: ACE (5’- aga agg cca agg agc tgt atg -3’ and 5’- gac aaa ggc atg gag gtt cag -3’) (19), ACE2 (5’- gtg cac aaa ggt gac aat gg -3’ and 5’- atg cgg ggt cac agt atg tt -3’) (20), NEP (5’- ggc aac ctc tgc tca cac tgt tac -3’ and 5’- gca ttg ggt cat ttc ggt ctt c -3’) (21), and for b-actin (5’-agc ggg aaa tcg tgc gtg-3’ and 5’- cag ggt aca tgg tgg tgc c-3’) (22). The polymerase chain reactions were performed with 1 µl RT product (cDNA) in a 25- µl reaction volume containing 1.5 mM MgCl2, 0.2 mM of each dNTPs, 1 U HotStarTaq DNA Polymerase (Invitrogen, Brasil) and 1 µM of each primer. After an initial enzyme activation step for 15 min at 95°C, PCR was carried out for: ACE (30 s at 94°C, 30 s at 64°C and 60 s at 72°C), ACE2 (60 s at 94°C, 60 s at 60°C and 60 s at 72°C), NEP (30 s at 94°C, 30 s at 57°C and 45 s at 72°C), and ß-actin (1 min at 94°C, 30 s at 59°C and 30 s at 72°C), followed by a 10 min extension at 72°C. DNA fragments separated in 2% agarose gel were visualized by staining with ethidium bromide.

Chemicals

Standards of Ang I, II, III, IV, 1-9, 1-7, 1-5 were purchased from Sigma (USA). Formic acid (99%) (Riedel de Haen, Germany), acetonitrile (Baker, USA), ammonium formate (Fluka, Germany) were HPLC grade. Deionized water was obtained using a MillQ system (MilliPore, USA).

Statistics

Concentrations of angiotensins were expressed as in ng/mg dry tissue. All values in the figures and text are expressed as mean ± s.e.of n observations. A one way analysis of variance (ANOVA) followed, if appropriate, by a Bonferroni`s test for multiple comparisons was used to compare means between the groups. A P value less than 0,05 was considered to be statistically significant.


RESULTS
In general, the absolute amounts of main Ang I metabolites produced by stomach wall were much lower than that produced by aorta or renal artery [e.g. for Ang-(1-7): 10,6 ±1,6 vs. 489,5 ±47 and 181,6 ±35 ng/mg tissue, respectively]. Importantly, the pattern of angiotensin I metabolites in stomach wall was also different: opposite to aorta and renal artery, incubation of Ang I with stomach wall fragments resulted in predominant formation of Ang-(1-7) and relatively lower production of Ang II (Fig. 1). Both, ACE inhibitor – perindoprilat, and NEP inhibitor – tiorphan, decreased Ang II production by stomach wall furthermore, to not detectable levels (Table 1). Interestingly, tiorphan decreased significantly the production of Ang-(1-9), but has no influence on formation of Ang-(1-7) (Table 1).

Fig. 1. Representative (for n=6 experiments) chromatograms of products of Ang I conversion by the rat stomach wall, renal artery and aorta, all incubated for 15 minutes with Ang I (1µM). Peaks represent relative abundance (Ang I = 100%). (Inserts: magnifications of chromatogram fragments).

Table 1. Production of Ang I (1µM) metabolites by fragments of rat stomach wall (ng/mg dry tissue) incubated with or without perindoprilat (10µM) or tiorphan (10µM). All values are mean±SEM;

* p<0,05 vs. control value; n=6; ND: not detectable

The levels of mRNA for ACE were similar in stomach wall, aorta and renal artery (Fig. 2). The highest levels of ACE2 mRNA were present in renal artery (Fig. 2); slightly lower levels of ACE2 mRNA were detected in stomach wall and aorta (Fig. 2). Interestingly, the levels of NEP mRNA in stomach wall were much lower compared to aorta and renal artery (Fig. 2).

Fig. 2. Expression mRNA for ACE2, ACE, NEP and ß-actin in stomach wall, renal artery and aorta. The pattern is representative for n=3 experiments.

DISCUSSION
Here, using LC-ESI-MS method we identified Ang-(1-7) as the main product of Ang I conversion in rat stomach wall. The metabolism of Ang I in stomach wall has never been studied, partly due to methodological problems with accurate and comprehensive assessment of unstable peptides in tissues. Recently, we have shown that LC-ESI-MS method is reliable for comprehensive quantitation of angiotensin metabolites in organ bath of tissue fragments and in medium of cultured cells, exposed to Ang I for relatively short period of time (17, 18). Combination of this method with measurements of tissue mRNA levels of ACE, ACE2 and NEP revealed several surprising discrepancies between Ang I conversion pathways in stomach, aorta and renal artery. Despite similar levels of expression of mRNA for ACE and ACE2 in stomach and aorta the overall rate of generation of Ang I metabolites was much lower in stomach wall. Moreover, the pattern of Ang I metabolites differed significantly between these two organs – there was prevalence of Ang II formation in aorta, but Ang-(1-7) in stomach wall. Interestingly, in renal artery the expression of ACE2 seems to be higher than ACE, yet the main product of Ang I conversion is Ang II. These results may suggest that posttranscriptional regulation may be crucial for the function of enzymes involved in angiotensin metabolism in tissues. Clearly, the measurements of protein levels of ACE, NEP and ACE2 could shed a light on this question.

There is also a question about the involvement of other proteolytic enzymes in Ang I metabolism in our model. There is a high content of pepsin and chymase in the stomach wall (23, 24). Although both, theoretically may contribute to Ang I degradation (4), it seems not to be a case in our setting. Pepsin is inactivated in pH above 4,0. Chymase is responsible for Ang II formation and its activity is preserved in the presence of perindoprilat (6). Importantly, in rat stomach wall organ-bath perindoprilat decreased generation of Ang II to non-detectable levels.

Use of perindoprilat and tiorphan in our model brought some surprising findings. Unexpectedly, tiorphan inhibited formation of Ang II and Ang-(1-9), what could depend on the lack of selectivity of this compound in concentrations higher than 1 µM (25). Importantly, in our setting both perindoprilat and tiorphan left untouched formation of Ang-(1-7). Apparently, formation of Ang-(1-7) in rat stomach wall seems to not depend on activity of ACE and NEP. Typically, Ang-(1-7) could originate from Ang I directly (by NEP), as well as indirectly via Ang II (sequential action of ACE then ACE2) or via Ang-(1-9) (ACE2 then ACE) (Fig. 3) (26). The exact pathway of Ang I to Ang-(1-7) conversion in stomach wall require further investigation.

Fig. 3. The main pathways of conversion of Ang I and action of inhibitors. ACE, ACE2 – Angiotensin converting enzymes; NEP – neutral endopeptidase, neprilysin;

We can only speculate about the physiological significance of Ang-(1-7) formation in stomach wall. Recent studies suggest that Ang-(1-7), acting via MAS receptor directly antagonizes many actions of Ang II (27). According to recent view, RAS can be envisioned as a dual function system in which the vasoconstrictor/proliferative/damaging or vasodilatatory/antiproliferative/protective actions are primarily driven by the balance of ACE/Ang II/AT1 receptor and ACE2/Ang-(1-7)/MAS receptor pathways, respectively (26;28). It has been recently suggested that part of the beneficial effects of drugs like ACE inhibitors or AT1 receptor blockers may depend on the shifting the balance toward ACE-2/Ang-(1-7)/MAS axis (29). Indeed, in rat stomach wall organ-bath perindoprilat strongly inhibited generation of Ang II, but did not decrease Ang-(1-7) formation. It has been shown that ACE inhibitors and AT1 receptor blockers may prevent stress-induced gastric injury (12, 30, 31). We are tempted to speculate that this action may be mediated by Ang-(1-7).

Here, using LC-ESI-MS method we identified Ang-(1-7) as the main product of Ang I conversion in rat stomach wall. The role of Ang-(1-7) in rat stomach, as well as potential significance of our finding for human pathophysiology require further investigation.

Acknowledgment: This study was supported by MNiSW grants No: 2 P05F 040 29, 2 P05F 011 30 and N N401 3560 33. We acknowledge generosity of Servier sp. z.o.o in providing us perindoprilat. The authors are grateful to Ms Jolanta Reyman and Ms Alicja Starosciak for technical help with animal experiments.


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