Angiotensin Converting Enzyme in Brush-Border Membranes of Avian Small Intestine

Angiotensin converting enzyme (ACE, kininase II, peptidyldipeptidase; E.C. 3.4.15.1) converts angiotensin I to angiotensin II, and hydrolyses a variety of bioactive peptides such as bradykinin and enkephalins. Historically ACE has been associated with pulmonary endothelial tissue and, more recently, with epithelial and neuroepithelial tissue in mammals (Defendini et al. 1983). Angiotensin converting enzyme’s presence in the blood and lung has established it as an important physiological regulator of blood pressure via its control of vasoactive peptides (Yang, Erdos & Levin, 1971). Recently, however, ACE has been immunohistochemically identified as a major membrane-bound protein of the microvillus membrane of epithelial cells of mammalian small intestine and renal proximal tubule (Bruneval et al. 1986; Naim, Sterchi, Hauri & Lentze, 1985; Ward, Sheridan, Hammon & Erdos, 1980; Ward & Sheridan, 1983). It is notable that many of ACE’s substrate peptides originate locally in the intestine (Tapper, 1983), and regulate the physiological events of electrolyte and fluid transport (Crocker & Munday, 1970; Davies, Munday & Parsons, 1970; Levens, 1986; Turnberg, 1983). Avian intestine is a critical site for absorption/resorption of ions and water (Crocker & Holmes, 1971), and thus ACE activity of avian enterocytes may play an important role in mediating the physiological status of the avian gastrointestinal tract. An alternative role for intestinal lumen ACE may involve oligopeptide digestion for the processing of nutrient nitrogen. In this study we have identified and partially characterized ACE activity in brush-border membranes purified from the small intestine of the grackle, Quiscalus quiscula. Furthermore, membrane ACE activity was probed using Ramipril, a potent and selective inhibitor of angiotensin converting enzyme (Becker, Scholkens, Metzger & Schultz, 1984; Bunning, 1984; Scholkens, Becker & Kaiser, 1984; Unger et al. 1986).

Small intestinal membrane vesicles were purified from intestinal mucosal scrapings obtained from adult common grackles Quiscalus quiscula of mean mass 90±6g (;V = 6). Birds were killed using halothane. The small intestines were immediately excised, chilled in ice-cold 1·02% (w/v) saline, and then scraped of mucosa. One gram samples of mucosa from each bird were stored in cryotubes in liquid nitrogen, and used later to prepare brush-border membranes based on a Ca²⁺ aggregation technique (Stevens, Ross & Wright, 1982; Stevens, Kaunitz & Wright, 1984). Briefly, each gram of mucosa was homogenized for 20 s, using a Polytron homogenizer (Brinkman, Westbury, NY) at setting no. 10, with 8 ml of buffer containing 350mmoll⁻¹ mannitol and Immoll⁻¹ Hepes/Tris, pH7·5. CaC1₂ was added to the crude homogenate (H₁) to make a 10 mmol I⁻¹ solution, which was stirred for 20 min at 5 °C. This was centrifuged for 5 min at 1500g-, the supernatant was collected and centrifuged again in the same manner. The second supernatant was centrifuged at 45 000g for 30 min. The resulting pellet was homogenized (glass/Teflon) in buffer containing 350 mmol I⁻¹ mannitol and 10 mmol I⁻¹ Hepes/Tris, pH 7·5. This was then centrifuged at 45 000g for 30 min. The final pellet containing brush-border membranes vesicles (BBMV) was homogenized in the 350 mmol I⁻¹ mannitol buffer to give a final protein concentration adjusted to 9·5 μg μl⁻¹. Protein concentration was determined using the Bio-Rad reagent (Bio-Rad, Richmond, CA). Samples of membranes were stored frozen in cryotubes in liquid nitrogen.

The brush-border marker enzymes, alkaline phosphatase and aminopeptidase N were assayed by previously published methods (Stevens, Kempner & Wright, 1986; Hanna, Mircheff & Wright, 1979).

Measurement of angiotensin converting enzyme activity was based on the hydrolysis of N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPGG). FAPGG is a well-established definitive substrate for angiotensin converting enzyme (Bunning, Holmquist & Riordan, 1983; Holmquist, Bunning & Riordan, 1979).

Brush-border membranes were thawed in the cryotubes at 22°C, and a 95μg sample of membrane protein was preincubated for 30 min in a plastic cuvette containing 50mmoll⁻¹ Hepes/Tris, pH7μ5, 300mmoll⁻¹ NaCl, 10μmoll⁻¹ ZnCl₂ and 0μ2% (w/v) polyoxyethylene-9-lauryl ether detergent. The reaction was started at time zero by adding the same buffer also containing (final reaction concentration, pH 7·5): FAPGG (0·01–1·5 mmol I⁻¹), Ramipril-diacid (0μ40μmol I⁻¹) and NaOH (19μmoll⁻¹). The Ramipril-diacid was added from a stock dissolved in 0·1 moll⁻¹ NaOH; control reactions contained the same amount of NaOH lacking Ramiprildiacid.

Ramipril (Hoe-498) manufactured by Hoechst AG, Frankfurt, FRG, was kindly supplied by M. I. Phillips of the Department of Physiology, University of Florida. Ramipril was always used in the diacid form. All other reagents were obtained from Sigma Chemical Co. (St Louis, MO).

The activity of angiotensin converting enzyme of grackle small intestinal brushborder membranes is illustrated in Fig. 1. The time-dependent decrease in absorbance indicates that first-order hydrolysis of FAPGG substrate occurred during the measurement period. In this example 50μmoll⁻¹ FAPGG was hydrolysed at 280μmol min⁻¹ g⁻¹. Fig. 1 also demonstrates that ACE activity was arrested when Ramipril-diacid was added to the buffer at time zero. Subsequent experiments were based on measurements made in this manner.

The grackle intestinal ACE activity was 20 times higher in the brush-border membrane preparation than in the corresponding mucosal/enterocyte scrapes, as demonstrated in Table 1. Also shown in Table 1 are the activities and enrichments for the brush-border membrane marker enzymes, alkaline phosphatase and aminopeptidase N. The coenrichment of ACE and marker enzymes in the final BBMV pellet indicated that ACE was bound to the brush-border membrane.

The enzyme hydrolysis kinetic parameters were estimated by ^a Gauss-Newton nonlinear regression, giving a maximal velocity of the reaction, V_max, of 907 ± 41 units g⁻¹, and an apparent Michaelis-Menten constant, K_m, of 55 ± 6μmol I⁻¹ FAPGG. The nonlinear regression analysis also indicated that only one enzyme species existed in our system. When the data were.plotted in an Eadie—Hofstee plot (Fig. 2), the resulting single linear relationship indicated that one species of ACE enzyme was responsible for the hydrolysis of FAPGG (Segel, 1975). Linear regression of the Eadie-Hofstee data (Fig. 2) yielded the same values as the nonlinear analysis.

Ramipril served as a highly potent and selective pharmacological probe of ACE activity (Becker et al. 1984; Bunning, 1984; Scholkens et al. 1984; Unger et al. 1986). Ramipril-diacid strongly inhibited grackle intestine brush-border ACE activity in a log dose-dependent manner (Fig. 3), with a median inhibitory concentration (IC₅₀) of 1 nmoll⁻¹; the activity was completely inhibited by 40μmoll⁻¹ Ramipril (Figs 1, 3).

Angiotensin converting enzyme is a multifunctional enzyme which classically has been studied for its vasoactive properties. However, recent immunohistochemical studies have confirmed the prominence of ACE on epithelial brush-border membranes of mammalian kidney, choroid plexus and small intestine (Bruneval et al. 1986; Naim et al. 1985 ; Ward et al. 1980; Ward & Sheridan, 1983). The results of the present study using an avian intestinal preparation of enterocyte brush borders corroborate the immunohistochemical work conducted on mammalian tissues.

ACE predominately converts angiotensin I to angiotensin II, inactivates bradykinin and hydrolyses enkephalins (Yang et al. 1971). These peptides originate locally in these tissues, where they modulate salt and water transport (Bolton, Munday, Parsons & York, 1975 ; Crocker & Munday, 1970; Gaginella, 1984). Avian intestine is an important site for ion and water transport (Crocker & Holmes, 1971; Holmes & Phillips, 1985; Duke, 1986), and therefore epithelial membrane-bound ACE may play an important role in controlling the bioactive peptides which affect the physiological status of the gut. The present study indicates that the avian small intestinal epithelial brush-border membranes possess prominent ACE activity which is strongly inhibited by the ACE inhibitor, Ramipril.

Avian intestinal membrane-bound ACE displays efficient kinetic properties, compared with its mammalian lung counterpart. The lung has long been recognized as a source of ACE (Erdos & Skidgel, 1987). Utilizing FAPGG as a substrate, the avian intestinal K_m value of 55 μmol I⁻¹ (Fig. 2) is an order of magnitude lower than the K_m of 300 μmoll⁻¹ measured for rabbit lung ACE (Bunning et al. 1983; Holmquist et al. 1979; Bunning, 1984). This suggests that ACE in avian intestine may be better adapted than in mammalian lung to control bioactive peptides. We utilized FAPGG as the definitive synthetic substrate for ACE, because FAPGG demonstrates a greater selectivity and turnover efficiency than hippuryl-His-Leu, angiotensin I or bradykinin (Bunning et al. 1983).

The avian intestinal BBMV ACE was strongly inhibited by Ramipril (Fig. 1), a selective and potent ACE inhibitor originally developed as an orally administered antihypertensive drug (Becker et al. 1984; Bunning, 1984; Scholkens et al. 1984; Unger et al. 1986). Ramipril in the present study served as a definitive probe for ACE activity in the membranes. The IC₅₀ value of 1 nmol I⁻¹ (Fig. 3) is similar to the IC₅₀ of 3 nmol I⁻¹ found for Ramipril inhibition of plasma ACE (Unger et al. 1986), and similar to the competitive binding K_x of 10 nmol I⁻¹ for rabbit lung ACE (Bunning, 1984).

In conclusion, we have shown that purified avian small intestinal brush-border membranes possessed considerable angiotensin converting enzyme activity, as demonstrated by hydrolysis of the ACE synthetic substrate FAPGG. The activity was strongly inhibited by nanomolar concentrations of Ramipril. The results suggest that bird intestinal ACE has some properties in common with its mammalian pulmonary vascular counterpart. Intestinal ACE may serve the avian intestine by controlling endogenous bioactive peptides which regulate electrolyte and water fluxes, or it may serve a digestive role in hydrolysing luminal nutrient oligopeptides which possess penultimate N-terminal prolyl residues.

This study was supported in part by research grant DK-38715 from the National Institutes of Health.