Introduction

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Inhibitors of angiotensin I-converting enzyme (ACE or kininase II) are administered to millions of patients with strikingly beneficial effects in a variety of cardiovascular and other conditions, including hypertension, congestive heart failure, myocardial infarction, diabetic nephropathy (Pfeffer et al., 1992; Gavras, 1994; Ambrosioni et al., 1995; Lewis, 1996; Mancini et al., 1996; HOPE Study Investigators, 2000). Recently, it was reported after extensive clinical studies that an ACE inhibitor (ACEi) even reduced the occurrence of diabetes (HOPE Study Investigators, 2000). ACEis block both the release of angiotensin II and the inactivation of bradykinin (BK; Yang et al., 1971; Linz et al., 1995). The inhibitors significantly enhance the activity of BK and the release of mediators such as nitric oxide (NO) and prostaglandins by the peptide (Bhoola et al., 1992; Carretero and Scicli, 1995; Margolius, 1995). The interaction of the inhibitors with BK, however, cannot be attributed only to blocking the breakdown of the peptide (Skidgel and Erdös, 1998); for example, the potentiation of the effect of BK and its ACE resistant analogs on the B₂ receptor (Auch-Schwelk et al., 1993; Hecker et al., 1994) and the immediate resensitization of the receptor desensitized by the agonist BK (Minshall et al., 1997a,b; Marcic et al., 1999, 2000) have been documented. ACE and the B₂ receptor have to be sterically close to each other on the cell membrane to induce these effects, possibly by forming a heterodimer (Marcic et al., 2000).

The very favorable results obtained while treating a large number of patients with ACEi in a variety of clinical conditions (HOPE Study Investigators, 2000) made it even more interesting to study further the activities of ACEis at the cellular level on the phenomena induced by them, independent of inhibition of BK inactivation (Minshall et al., 1997b; Deddish et al., 1998; Erdös et al., 1999; Benzing et al., 1999; Marcic et al., 1999, 2000). The use of protein kinase C (PKC) or phosphatase inhibitors clearly distinguished the primary action of the ligand BK on the receptor from the reactivation of the desensitized B₂ receptor by BK, after it was resensitized by an ACEi. This tenet, based on determining arachidonic acid (AA) release and [Ca²⁺]_i equally applies to cultured cells that were either transfected to express human B₂ receptor and ACE [Chinese hamster ovary (CHO)/AB] or expressed both proteins constitutively, such as human pulmonary artery endothelial (HPAE) cells.

	Experimental Procedures

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Materials. BK, hippuryl-His-Leu (Hip-His-Leu), tissue culture medium, buffers, and reagents were from Sigma Chemical Co. (St. Louis, MO). [Phe⁸(CH₂NH)Arg⁹]BK, ACE-resistant BK analog (Drapeau et al., 1988; Marcic et al., 2000) was obtained from Novabiochem (San Diego, CA). Fetal bovine serum was from Atlanta Biologicals (Norcross, GA) and Z-Phe-His-Leu was from Bachem (Philadelphia, PA). Enalaprilat was provided by Merck, Sharpe & Dohme Research Division (Whitehouse, NJ), ramiprilat by Upjohn Laboratories (Kalamazoo, MI), and HOE 140 by Hoechst Co. (Frankfurt, Germany). [³H]BK (97 Ci/mmol) was from Amersham Pharmacia Biotech, Inc. (Piscataway, NJ). [5,6,8,9,11,12,14,15-³H(N)]AA ([³H]AA; 100 Ci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO). The original cDNA of wild-type ACE was kindly donated by Professor P. Corvol of College de France, Paris. B₂ receptor cDNA was provided by Dr. K. Jarnigan (Syntex, Palo Alto, CA). CHO cells were from the American Type Culture Collection (Rockville, MD). HPAE cells were purchased from Clonetics (San Diego, CA). Mammalian expression vectors pcDNA3 and pCEP4 were from Invitrogen (Carlsbad, CA). Anti-B₂ receptor antibodies were kindly provided by Professor W. Müller-Esterl of Gutenberg University, Mainz, Germany. Transfection reagent (Superfect) and geneticin (G418) were from Life Technologies (Grand Island, NY). Hygromycin B, staurosporine, calphostin C, calyculin A, and okadaic acid were purchased from Calbiochem (San Diego, CA)

Cell Culture. CHO cells were grown as described (Minshall et al., 1997b; Marcic et al., 1999). HPAE cells were cultured in Dulbecco's modified Eagle's medium with the same supplements. Cells were routinely subcultured with trypsin-EDTA to mobilize them (Minshall et al., 1997b). For transfection, CHO cells were plated at density 1 × 10⁵ cells/60-mm dish 1 day before transfection.

Transfection and Cloning. To establish cell lines with stable expression, CHO cells were transfected with human wild-type ACE cDNA in pCDNA3 expression vector, carrying neomycin resistance gene with Superfect method with serum-free Ham's F-12 medium without antibiotic. The Superfect-DNA complex was added into the dish over the entire surface and incubated with the cells for 3 h at 37°C. The cells were subcultured into medium containing 500 µg/ml geneticin G418. In 10 to 20 days, clones of transfected cells were formed (Marcic et al., 1999).

Screening of Clones for ACE Activity. Individual clones were evaluated both for cell-associated and -released ACE activity. Cells were incubated in serum-free medium for 24 h, medium was collected, the cells were washed and lysed in 3 ml of 8 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid in PBS, pH 7.4. Both the supernatants and the lysates were centrifuged (for 15 min at 900g) and their aliquots were mixed with 1 mM Hip-His-Leu or Z-Phe-His-Leu of substrate and incubated 4 h at 37°C. After the reaction was terminated, His-Leu released was determined by coupling with o-phthaldialdehyde at 365 nm excitation and 500 nm emission wavelengths (Deddish et al., 1998). Clones with highest levels of ACE expression were transfected with B₂ receptor cDNA (Marcic et al., 1999).

Transfection with Human B₂ Receptor cDNA. The selected clones were transfected with pCEP4 vector containing human B₂ BK receptor cDNA with the Superfect transfection method (Marcic et al., 1999). After transfection, cells were selected in Ham's F-12 medium containing 0.5 mg/ml Hygromycin B (pCEP4 vector with Hygromycin B resistant gene).

Radioligand Binding on Selected Clones. Clones with the highest expression of B₂ receptors were selected by [³H]BK saturation binding on whole-cell monolayers expressing both ACE and B₂ receptors. Equilibrium binding of 0.05 to 20 nM [³H]BK with or without 10 µM unlabeled BK was done in Ham's F-12 cell culture medium for 1 h at 37°C (Minshall et al., 1997). Bound radioactivity was separated from excess [³H]BK by washing and counted in liquid scintillation vials. Clones with the high expression of B₂ receptors on the cell surface were chosen and used further. Cells expressing both human B₂ receptor and wild-type ACE were designated as CHO/AB cells.

Measurement of Changes in [Ca²⁺]_i and [³H]AA. Free cytosolic calcium [Ca²⁺]_i was measured with a microspectrofluorometer (PTI Deltascan, Princeton, NJ), or Attofluor Ratiovision with fura-2/AM reagent. Cells were grown to confluence on glass coverslips, then incubated with 2 to 5 µM fura-2/AM for 1 h at 37°C, washed with buffer, and mounted in a Sykes-Moore chamber (Bellco, Vineland, NJ) at room temperature. Cellular fluorescence at 510 nm was measured after excitation at wavelengths of 340 and 380 nm (Erdös et al., 1999; Marcic et al., 2000). [³H]AA release was measured as described in Minshall et al. (1997b).

De- and Resensitization of B₂ Receptor. After desensitization of the receptor by initial exposure of cells to a kinin (Minshall et al., 1997b), the restoration of sensitivity to the agonist (resensitization) was measured either by [³H]AA release or by mobilization of [Ca²⁺]_i. For example, monolayers of transfected CHO cells loaded with [³H]AA were stimulated with 1 µM BK or its ACE-resistant analog for 30 min. Then, without removal of BK, cells were exposed to either enalaprilat (5 nM or 1 µM) or ramiprilat (5 nM or 1 µM) without adding more kinin or, as control, to a second dose of only kinin for an additional 5 min. (Minshall et al., 1997b). The amount of [³H]AA released was determined by taking AA released during the first 30 min as baseline, and normalizing to the amount released by buffer alone during the 5-min reactivation.

B₂ Receptor Phosphorylation. The phosphorylation of B₂ receptors by BK was measured in confluent monolayers of CHO/AB cells loaded with [³²P]orthophosphate (100 µCi/ml medium) for 4 h. Subsequently, the cells were either treated with buffer alone, or 1 µM BK, or 1 µM BK and 1 µM enalaprilat for 30 min. As control, 1 µM phorbol-12-myristate-13-acetate (PMA) was used for 30 min at 37°C. The cells in monolayers were solubilized with 1% Triton X-100, centrifuged at 100,000g for 15 min, and the supernatants were saved. Anti-B₂ receptor polyclonal antibodies were added at 1:1000 v/v dilution and samples were incubated overnight with shaking at 4°C. When insoluble protein A was added to each sample to 10%, incubation continued for 2 h at 4°C. Beads with immune complexes were sedimented by centrifugation at 1000g for 15 min. The presence of B₂ receptors was verified by [³H]BK binding and the receptors were subjected to 10% SDS-polyacrylamide gel electrophoresis. The bands labeled with ³²P were visualized by autoradiography and the densities of B₂ receptor bands were quantitated by scanning densitometry.

Kinase and Phosphatase Inhibition. Inhibitors of the PKC and phosphatases 1 and 2A were used. Cultured HPAE or CHO/AB cells were pretreated with 100 nM staurosporine, an inhibitor of all PKC isoenzymes except the atypical one (Tamaoki et al., 1986; Hofmann, 1997), for 15 min. Alternatively, the same cells were pretreated with 1 µM calphostin C, an inhibitor of all PKC isoenzymes (Hofmann, 1997), for 15 min.

Statistics. The data in the figures and text are expressed as mean ± S.E. when n = 3 or more. When some previously reported experiments were repeated with the same results, they were done once or twice in duplicate and not pursued further. [Ca²⁺]_i levels are represented as the percentage of mean fluorescence intensity increase relative to control levels, calculated as nanomolar concentrations. Statistical evaluation was performed by one-way ANOVA for matched values. Values of P less than .05 were considered statistically significant.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

CHO/AB and HPAE Cells

Transfected CHO/AB cells express 2 × 10⁵ B₂ receptors/cell as determined by [³H]BK saturation binding, in agreement with our previous results (Marcic et al., 1999; n = 5). Binding experiments were performed regularly during culture and the B₂ receptor expression did not change with passage. The very high expression of transfected ACE (10⁶ ACE molecules/cell) in CHO/AB cells and the inhibition of the enzyme by enalaprilat (72% at 5 nM) were previously described (Marcic et al., 1999). HPAE cells constitutively expressed 14,000 B₂ receptors/cell (n = 4). Expression of ACE in HPAE cells decreased with the number of passages (Johnson, 1980); after eight passages (the highest used), it was 7000 to 9000 molecules/cell (n = 3).

B₂ Receptor Phosphorylation

The effects of ACE inhibitors on the phosphorylation of the B₂ receptors were tested in transfected CHO/AB cells, expressing wild-type human ACE and human B₂ receptors, after treating them with ³²P. The results, calculated as changes in relative optical densities, were the following: no treatment = 1.0, enalaprilat = 1.1 ± 0.1, BK = 1.9 ± 0.3, and BK and enalaprilat = 1.3 ± 0.1 (P < .05). As control, PMA was added, which increased phosphorylation to 2.4 ± 0.3 (n = 5; P < .05). Thus, in these experiments, 1 µM enalaprilat significantly decreased the phosphorylation of the B₂ receptor induced by 1 µM BK (Fig. 1). Enalaprilat added alone without BK had no effect.

View larger version (45K): [in this window] [in a new window]   Fig. 1.   Phosphorylation of B2 receptors immunoprecipitated from CHO/AB cells was visualized by autoradiography and quantitated by scanning densitometry. Before solubilization, cell monolayers were treated with buffer alone (no treatment), 1 µM enalaprilat (EPT), 1 µM BK, 1 µM EPT and 1 µM BK together (BK + EPT), or with 1 µM PMA for 30 min. Ordinate, densitometric quantification of B2 receptor phosphorylation in relative units (n = 5; *P < .05).

Effects of PKC and Phosphatase Inhibition on [Ca²⁺]_i Increase

ACE inhibitors can resensitize the B₂ receptor to the agonist both in transfected cultured cells and in cells that constitutively express both ACE and B₂ receptors (Minshall et al., 1997b, Marcic et al., 1999). We wished to determine whether the first response to activation of the receptor by the agonist that immediately desensitizes it, and the one after reactivation of the B₂ receptor, resensitized by ACEi to the agonist present in the medium, may proceed through different signal transduction pathways. For this purpose, we tested inhibitors of PKC and phosphatases 1 and 2A on both transfected CHO/AB cells and on HPAE cells, which constitutively express the proteins.

CHO/AB Cells. ACE inhibitors resensitize the B₂ receptor to BK in the medium that previously desensitized the receptor in CHO/AB cells. The response was measured by the increase in [Ca²⁺]_i level to BK analog in the medium after adding an ACEi (n = 6; Fig. 2A). Pretreatment of cultured CHO/AB cells with PKC inhibitor 1 µM calphostin C (Hofmann, 1997) for 15 min did not affect the elevation of [Ca²⁺]_i by BK because 10 nM BK analog still transiently increased [Ca²⁺]_i. However, after calphostin C 1 µM ramiprilat did not resensitize the B₂ receptor to BK analog (n = 4; Fig. 2B). Pretreatment of CHO/AB cells with another PKC inhibitor, 100 nM staurosporine, also blocked the resensitization by ACEi (data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   A, [Ca2+]i was measured (ordinate) in CHO/AB cell monolayers over time (abscissa). The first increase was induced by administration of 10 nM ACE-resistant BK analog (BKan); the second increase was caused by addition of 1 µM ramiprilat (RAM), which resensitized B2 receptor to BKan present in the medium (typical experiment representing 1 of 6). B, as in A except CHO/AB cell monolayers were pretreated with 1 µM calphostin C for 15 min. Ramiprilat failed to resensitize the receptor to BK (n = 4), but the primary effect of BKan was not blocked. C, CHO/AB cell monolayers were pretreated with 100 nM calyculin A for 30 min. Ramiprilat failed to resensitize the receptor (n = 3), although the phosphatase inhibitor did not inhibit BK action.

HPAE Cells. Experiments with HPAE cells that constitutively express ACE and B₂ receptor yielded the same results as with the transfected cells. Enalaprilat (1 µM) resensitized the B₂ receptor to [Ca²⁺]_i mobilization induced by BK (n = 3; Fig. 3A). Exposing HPAE cells to 1 µM calphostin C for 15 min did not affect the primary action of BK, but blocked the effect of enalaprilat. It did not resensitize the receptor to the BK (10 nM) in the medium (n = 3; Fig. 3B). The results with staurosporine (100 nM) were identical (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   A, [Ca2+]i was measured (ordinate) in HPAE cell monolayers over time (abscissa). The first increase was induced by administration of 10 nM BK. Addition of 1 µM enalaprilat (EPT) resensitized the B2 receptor to BK in the medium (n = 3). B, HPAE cells pretreated with 1 µM calphostin C; enalaprilat failed to resensitize the receptor (n = 3). C, HPAE cells pretreated with 100 nM calyculin A; enalaprilat, again, did not resensitize the receptor to BK (n = 4). Inhibitors had no effect on the primary activation of the receptor by BK.

Okadaic Acid. Another inhibitor of the phosphatases 1 and 2A, okadaic acid (Gjertsen et al., 1994), also was tested in both cell types. When both HPAE or CHO/AB cells were pretreated with 500 nM okadaic acid for 30 min, the mobilization of [Ca²⁺]_i by BK (10 nM) was not affected (Fig. 4). However, just as with calcyculin, okadaic acid abolished resensitization of the receptor to BK by 1 µM enalaprilat both in HPAE cells (n = 5; Fig. 4A) and in CHO/AB cells (n = 4; Fig. 4B).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   A, HPAE cell monolayers were pretreated with 500 nM okadaic acid for 30 min. [Ca2+]i level was measured (ordinate) over time (abscissa). The first increase was induced by administration of 10 nM BK. Addition of 1 µM enalaprilat (EPT) failed to resensitize the receptor (n = 5). B, same experiment performed on CHO/AB cells.

Effects of Kinase and Phosphatase Inhibition on [³H]AA Release

ACE inhibitors also can resensitize the B₂ receptor to BK to release AA from cells expressing both B₂ receptors and ACE (Minshall et al., 1997b; Marcic et al., 1999). Because [Ca²⁺]_i elevation and AA leading to the synthesis of different mediators, prostaglandins and NO, involve the coupling of different G-proteins to the receptor (deWeerd and Leeb- Lundberg, 1997; Erdös et al., 1999), we tested the effects of kinase and phosphatase inhibitors on AA release from CHO/AB cells.

Kinase Inhibition. Enalaprilat (1 µM) resensitized the B₂ receptor, desensitized by the ligand (Marcic et al., 1999) in CHO/AB cells, and enhanced [³H]AA release 3.4 ± 0.6-fold compared with the addition of buffer alone (n = 4, P < .05). Calphostin C (1 µM; 15 min) pretreatment abolished 98 ± 7% (n = 4; P < .05) of this resensitization by enalaprilat (Fig. 5).

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Monolayers of CHO/AB cells loaded with [3H]AA were stimulated with 1 µM BK to desensitize the B2 receptor. Then, without removal of the agonist, cells were exposed to buffer alone (buffer), or 1 µM BK again, or 1 µM enalaprilat (EPT) for an additional 5 min. One group of cells was pretreated with 1 µM calphostin C (CalpC) for 15 min, followed by 1 µM BK for 30 min and then 1 µM EPT for 5 min. Note that CalpC blocked the resensitization of the receptor to BK by enalaprilat. The amount of [3H]AA released (ordinate) was determined by taking AA released during the first 30 min as baseline, and normalizing values to that obtained by adding buffer alone for 5 min (n = 4; *P < .05).

Phosphatase Inhibition. Effect of the phosphatase inhibitor calyculin A on the resensitization of B₂ receptor by enalaprilat was tested similarly by measuring [³H]AA release. Enalaprilat (1 µM) resensitized BK-induced [³H]AA release from CHO/AB cells 4.9 ± 0.7-fold compared with the addition of buffer alone (n = 3; P < .05). Calyculin A (100 nM; 30 min) pretreatment abolished 87 ± 16% (n = 3; P < .05) of the resensitization (Fig. 6).

View larger version (33K): [in this window] [in a new window]   Fig. 6.   Monolayers of CHO/AB cells loaded with [3H]AA were treated as in Fig. 5 except that one group of cells was pretreated with 100 nM calyculin A (CalA) for 30 min (n = 3; *P < .05). Ordinate as in Fig. 5. Resensitization of the receptor by EPT was blocked by phosphatase inhibitor.

Calcium-Free Medium

We studied a potential involvement of extracellular calcium influx in the resensitization of the receptor to release [³H]AA. This was done to establish whether blocking of the phosphorylation or dephosphorylation might block a putative opening of calcium channels by ACE inhibitors and thereby inhibit the resensitization of B₂ receptor. We measured the liberation of AA by BK in calcium-free medium. Even in the absence of extracellular calcium, ramiprilat both in high (1 µM) and low (5 nM) concentrations resensitized [³H]AA release induced by the ACE-resistant BK analog (n = 3; P < .05; Fig. 7), negating the possibility that a blockade of extracellular Ca²⁺ uptake would be responsible for the lack of ACEi effect in the above-mentioned experiments.

View larger version (36K): [in this window] [in a new window]   Fig. 7.   Monolayers of CHO/AB cells loaded with [3H]AA were stimulated with 1 µM ACE-resistant BK analog for 30 min in calcium-free medium. Then, without removal of the agonist, cells were exposed to buffer alone (Buffer), 1 µM ACE-resistant BK analog (1 µM BKan), 5 nM ramiprilat (5 nM RAM), or 1 µM ramiprilat (1 µM RAM; n = 3; *P < .05. Ordinate as in Fig. 5.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

BK as an agonist induces the phosphorylation of the B₂ receptor at serine residues, which leads to the desensitization of the receptor (Leeb-Lundberg et al., 1987; Roberts and Gullick, 1990; Blaukat et al., 1996; Pitcher et al., 1998). In the above-experiments, enalaprilat decreased the phosphorylation of the receptor stimulated by BK (Fig. 1), in good accord with the potentiation of BK actions by ACEis. Enalaprilat, also in agreement with previous experiments describing no direct effects of ACEis on the receptor (Minshall et al., 1997a,b; Marcic et al., 1999, 2000), did not induce phosphorylation in the absence of BK.

After using PKC and phosphatase inhibitors, we concluded that the activation of the receptor by BK and its reactivation by BK after resensitization by ACEi initiate different signal transduction pathways.

ACEis do not act directly on the B₂ receptor because they are inactive on cells where ACE is not expressed (Minshall et al., 1997a,b; Marcic et al., 1999). They can enhance the primary effects of BK and its ACE-resistant peptide analogs on the receptor, and, as also shown in Fig. 2, they resensitize the receptor desensitized by BK. This response to the peptides present in the medium is immediate without adding more BK. All of these experiments exclude the blocking of BK breakdown as the sole reason for the effects of ACEis in this system. ACEis enhance the effect of BK on the B₂ receptor indirectly, first by reacting with an active center of ACE (Marcic et al., 1999), resulting in an allosteric modification in the enzyme receptor complex. This, in turn, would lead to resensitization of the receptor desensitized to its ligand. In this study, we used PKC and phosphatase inhibitors that caused no harm to the primary effect of BK on the receptor or any good to the reactivation of the B₂ receptor. Or, in other words, they did not affect primary activation of the receptor, which is followed by desensitization (tachyphylaxis) to the agonist peptide. The elevation of [Ca²⁺]_i or the release of [³H]AA by BK was not blocked by PKC inhibitors, in line with the observations of others (Blaukat et al., 1996; Pizard et al., 1998), or, in our experiments, by two membrane phosphatase inhibitors. However, the application of either one of the two PKC or phosphatase inhibitors abolished the very well documented resensitization of the B₂ receptor by the ACEi to BK present in the medium (Minshall et al., 1997a,b; Benzing et al., 1999; Erdös et al., 1999; Marcic et al., 1999, 2000). We tested two different ACEis, enalaprilat and ramiprilat, to distinguish between specific and group-related structural effects of the compounds, and the results were identical with both inhibitors. Besides BK, an ACE-resistant peptide analog, a ligand to B₂ receptor not cleaved by ACE was used (Drapeau et al., 1988; Marcic et al., 2000). The activation of the B₂ receptor was assessed by measuring AA release and [Ca²⁺]_i elevation as parameters of the important indirect cardiovascular actions of BK, namely, the liberation of prostaglandins and NO (Cachofeiro and Nasjletti, 1991; Bhoola et al., 1992; Carretero and Scicli, 1995). In addition, in some tissues, AA is metabolized to endothelium-derived hyperpolarizing factors (Campbell and Harder, 1999).

The lack of effect of PKC inhibitors on the primary activation of the B₂ receptor by the ligand is understandable if the receptor is phosphorylated by another kinase. The -adrenergic receptor kinase can phosphorylate BK B₂ receptor in vitro followed by a secondary action of PKC (Blaukat et al., 1996). If -adrenergic receptor kinase is involved in BK-induced phosphorylation of B₂ receptors, ACEis can still lower phosphorylation directly or indirectly. Reducing B₂ receptor phosphorylation, thus, desensitization, renders it more reactive to BK already present in the medium.

Speculative explanations of roles of PKC and phosphatase inhibitors in blocking the resensitization of B₂ receptors by ACEi include the following. Dephosphorylation is the final step of receptor recycling. Inhibition of phosphatases that act on cell membranes may block removal of phosphate groups from B₂ receptors, which are recycled back to the surface and prevent signaling of receptors. In this model, rapid recycling would be promoted by ACE inhibitors. However, we found previously that B₂ receptor endocytosis in transfected CHO cells is a slow process (Minshall et al., 1997b).

Inhibition of PKC also could affect phosphorylation of an intermediate protein that is mediating the interaction between B₂ receptors and ACE induced by ACEi. This putative switch protein, which may be a regulator of B₂ receptor signaling, is phosphorylated by PKC and dephosphorylated on the membrane. Inhibiting either process blocks the rapid resensitization of the receptor. For example, PKC can affect a G-protein-coupled receptor kinase (Pitcher et al., 1998) that may be responsible for B₂ receptor desensitization, thus inhibiting PKC may block this secondary event. Such an enzyme may be the primary factor in receptor desensitization, thus PKC may destabilize it. Phosphatase inhibition may block the recycling of this intermediary protein kinase.

The cytosolic portion of ACE is unlikely to be phosphorylated because deletion of three of five potentially phosphorylated residues from the C-terminal end of recombinant ACE did not affect the actions of ACEis (Marcic et al., 2000).

The above-mentioned experiments showed that the transduction pathway mediating the action on the resensitized B₂ receptor differs from the initial pathway involved in primary response to BK. Dalemar et al. (1996) described that PKC and PKA can modulate expression of B₂ receptor affinity forms. Consequently, there is a possible conformational change in the receptor involved in the signaling, and after the application of ACEi, the reactivated receptor signals through alternate pathways. We reported that a physical proximity between B₂ receptors and ACE is required for the resensitization phenomenon to occur (Marcic et al., 2000), possibly the formation of an ACE-B₂ receptor heterodimer. It follows that in the absence of ACE expression, ACEis are ineffective (Minshall et al., 1997b; Marcic et al., 1999).

Judging from experiments based on AA release and [Ca²⁺]_i elevation, both G_i- and G_q-coupled B₂ receptors are involved in the reactivation process (Burch and Axelrod, 1987; de Weerd and Leeb-Lundberg, 1997; Marcic et al., 1999). The G_q-mediated activation of phospholipase C by BK is followed by inositol phosphate₃ release that mobilizes [Ca²⁺]_i from internal sources. Reactivation of the receptor to the BK in the medium elevates [Ca²⁺]_i level in CHO cells by promoting the entry of calcium from extracellular sources (Marcic et al., 1999). However, as shown above, resensitization by ACEis to a G_i-mediated, BK-induced [³H]AA release is possible even in the absence of extracellular calcium, suggesting an involvement of a calcium-insensitive phospholipase A₂ (Balsinde and Dennis, 1997). These findings also indicate that mechanisms of AA release and increase in [Ca²⁺]_i level induced by BK on the resensitized receptor are different.

In conclusion, the use of PKC and phosphatase inhibitors clearly distinguishes the primary activation of the B₂ receptor by agonist and subsequent action on the receptor reactivated by an ACEi. These observations point to possible additional beneficial consequences of ACEi application by amplification of BK effects.