INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

METALLOCARBOXYPEPTIDASES specifically catalyze the hydrolysis of the COOH-terminal peptide bond of peptides and proteins (44). Although the release of a single COOH-terminal amino acid could appear to be of limited physiological importance, it can have profound effects on the biological activity of peptides and proteins (42, 44, 46). This was first established by the pioneering work of Erdös and Sloane (12, 16), who discovered a B-type carboxypeptidase (which cleave only COOH-terminal Arg or Lys) in plasma that inactivates bradykinin. It was initially named kininase I to distinguish it from another bradykinin inactivator named kininase II, which was later shown to be identical with angiotensin-converting enzyme (ACE) (12, 13). Although kininase I was later renamed carboxypeptidase N (CPN), the kininase I and II designation is still used to categorize enzymes that cleave either one (Arg⁹) or two (Phe⁸-Arg⁹) amino acids from the COOH-terminus of bradykinin (13, 47). Later discoveries of the first membrane-bound carboxypeptidase (CPM) (45, 48) and a secretory vesicle carboxypeptidase (CPE or CPH) involved in peptide hormone processing (17, 18) provided evidence for the existence of a subfamily of carboxypeptidases that have important functions in regulating peptide hormone activity (42). Cloning and sequencing confirmed this distinction and they came to be known as the "regulatory carboxypeptidases" (or more recently the CPN/E subfamily) as a way to distinguish them from the pancreatic carboxypeptidases (or CPA/B subfamily) (40, 44, 55). Interestingly, all catalytically active members of the regulatory or CPN/E subfamily are B-type carboxypeptidases, which cleave only COOH-terminal Arg or Lys residues. The regulatory carboxypeptidases play important roles in the maintenance of physiological homeostasis and the organism's response to pathological perturbations (40, 44, 46).

There are only two true membrane-bound regulatory carboxypeptidases: CPM and CPD. CPM is a widely distributed ectoenzyme anchored to plasma membranes via glycosylphosphatidylinositol (9, 46, 49). CPM cleaves only basic COOH-terminal residues, and arginine is cleaved faster than lysine in a variety of synthetic peptide substrates (45). The naturally occurring peptide substrates of CPM include bradykinin, Arg⁶- and Lys⁶-enkephalins, dynorphin A_1-13, epidermal growth factor (EGF), and hemoglobin (33, 35, 45). CPD was discovered as a unique B-type carboxypeptidase in rat and bovine tissues (51) as well as in human and mouse cells (34), with an acidic pH optimum of about 6.2. Subsequent cloning and sequencing of the human and rat cDNAs (54, 60) showed CPD to be the mammalian homolog of duck gp180, a protein originally identified as a duck hepatitis B-virus binding protein (26). CPD is a widely distributed 180-kDa glycoprotein containing three tandem homologous carboxypeptidase domains, of which the first two are active and the third domain is inactive, due to mutation of several critical active site residues (40, 46). CPD is a type I membrane protein with a single transmembrane sequence near the COOH-terminus and is primarily found in the trans-Golgi network in the cell, where it participates in protein processing in the constitutive secretory pathway (40, 46, 57). However, some CPD can be found on the plasma membrane where it can also function as a cell surface protein, for example, in binding hepatitis B-virus on duck liver cells (25, 26, 52) or by cleaving extracellular substrates and generating Arg to enhance nitric oxide (NO) production in macrophages (19).

Kinins are important peptide hormones that stimulate a variety of signal transduction pathways via two known G protein-coupled receptors named B₁ and B₂ (3). Activation of either the B₁ or B₂ receptor leads to stimulation of similar signal transduction pathways, causing increases in intracellular calcium and the release of mediators such as prostaglandins and NO (3, 31). However, there are several major differences between the two receptor types. B₁ receptor stimulation results in a more prolonged response because the receptor is resistant to desensitization and internalization in contrast to the B₂ receptor, which is rapidly sequestered and desensitized (2, 10, 27, 31). Another distinguishing feature is that the B₂ receptor is constitutively expressed, whereas the B₁ receptor is not normally expressed in most tissues, but injury or inflammatory mediators upregulate its transcription (3, 31). In human cells, interleukin-1 (IL-1) is especially effective in upregulating expression (31). Finally, bradykinin and Lys-bradykinin (kallidin) are specific ligands for the B₂ receptor, whereas the carboxypeptidase-derived products des-Arg⁹-bradykinin and des-Arg¹⁰-kallidin are ligands for the B₁ receptor (3, 39). Thus B-type carboxypeptidases not only inactivate B₂ agonists but act as required processing enzymes for the generation of B₁ agonists. In vitro, bradykinin is one of the best biological substrates for CPM, which cleaves only the COOH-terminal Arg⁹ to form des-Arg⁹-bradykinin (33, 45). The membrane localization of CPM and CPD makes them ideally situated to regulate bradykinin activity near cell surface receptors. However, their roles in generating B₁ agonists have not been directly addressed experimentally at the cellular level. The goal of this study was to investigate the ability of endothelial cell carboxypeptidases to affect NO production in stimulated endothelial cells through the generation of B₁ agonists.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials. Microvascular Endothelial Cell Growth Medium-2 Kit (EGM-2 BulletKit) was from Clonetics (San Diego, CA). Phenol red-free Dulbecco's modified Eagle's medium/Ham's nutrient mixture F-12 (DME/F-12) was from Sigma (St. Louis, MO). dl-2-Mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGTA) and IL-1 were from Calbiochem (La Jolla, CA). FBS was from Atlanta Biologicals (Norcross, GA). Human recombinant interferon- (IFN-) was from Life Technologies (Gaithersburg, MD). 5-Dimethylaminonaphthalene-1-sulfonyl-L-alanyl-L-arginine (dansyl-Ala-Arg) was synthesized and purified as described previously (53). Gelatin, PBS, N^G-monomethyl-L-arginine (L-NMMA), bradykinin, kallidin, des-Arg⁹-bradykinin, des-Arg¹⁰-kallidin, and des-Arg¹⁰-Leu⁹-kallidin were from Sigma. HOE 140 was a gift from Dr. B. A. Schölkens (Hoechst; Frankfurt, Germany). Common chemicals were from Fisher Scientific (Pittsburgh, PA).

Cell culture. Human lung microvascular endothelial cells (HLMVEC) were obtained from Clonetics, BioWhittaker (San Diego, CA). Cells were cultured in EGM-2 containing 10 ng/ml human epidermal growth factor, 5 ng/ml vascular endothelial growth factor, 2 ng/ml human fibroblast growth factor, 2 ng/ml insulin-like growth factor, 0.2 µg/ml ascorbic acid, 50 ng/ml gentamicin-amphotericin-B, and 10% FBS that was heat inactivated at 56°C for 30 min (to destroy any endogenous serum carboxypeptidase). For NO measurements, cells (from passages 4-8) were seeded at a density of 1 × 10⁶ cells/well on gelatin-coated 12-well plates. To upregulate B₁ receptor expression, 24 h after reaching confluence, HLMVEC were washed once with PBS and then incubated in fresh fully supplemented EGM-2 (as above except with 0.5% FBS) containing 5 ng/ml IL-1 and 200 U/ml IFN- at 37°C for an additional 16 h. Control cells were treated identically except the medium lacked IL-1 and IFN- during the 16-h incubation.

Measurement of NO production. NO production was determined by measurement of NO in real time with a porphyrinic microsensor (5, 30). The microsensor consisted of 5-7 carbon fibers (diameter 0.2 µm) that were electroplated with highly conductive polymeric porphyrin, which facilitates the electron transfer from NO to the sensor. This microsensor is supercoated with a negatively charged polymer (Nafion); negatively charged species such as nitrite or nitrate cannot gain access to the porphyrinic surface and therefore do not interfere with NO measurements. This microsensor is highly sensitive for NO (10⁹ mol/l detection limit), and its response time is rapid (on the order of 10⁴ s), providing the ability to make kinetic measurements of NO concentration. The porphyrinic sensor was positioned with the help of a micromanipulator close to the endothelial cell culture surface (20 ± 1 µm). A three-electrode system, which includes the porphyrinic NO-sensitive electrode, counter platinum electrode, and calomel reference electrode, was used with a constant potential of 700 mV. Before the experiment was started, the medium was changed to phenol red-free and serum-free DME/F-12 medium. In some experiments, cells were incubated with either the specific B-type carboxypeptidase inhibitor MGTA, B₂ receptor antagonist HOE 140, or the B₁ receptor antagonist des-Arg¹⁰-Leu⁹-kallidin for 10 min at 37°C before the addition of the agonist. To initiate NO generation, bradykinin, kallidin, or des-Arg¹⁰-kallidin was added and the responses (current versus time) were recorded continuously. Current generated was proportional to the NO released, and a computer-based Camry VP600 potentiostat was used to monitor NO concentration over time. Each electrode was calibrated with a NO standard.

Cell harvesting and membrane fractionation. HLMVEC grown in culture dishes were washed two times with cold PBS, scraped off into a small volume of PBS, and pelleted at 1,000 g for 10 min. Cell pellets were resuspended in 1 ml of 50 mM HEPES and 0.25 M sucrose, pH 7.4. The cell suspension was put on ice, and cells were then lysed by sonication three times for 15 s each. The high-speed membrane fraction was obtained by sequential differential centrifugation as described (37). Briefly, lysates were centrifuged at 1,000 g for 10 min, and the supernatant was centrifuged at 10,000 g for 25 min. The resulting supernatant was then centrifuged at 100,000 g for 1 h to obtain the final membrane (P₃) fraction, which was resuspended in the original fractionation buffer.

Carboxypeptidase activity assays. CPM and CPD enzyme activity was measured using dansyl-Ala-Arg substrate (34, 41, 53). Samples were incubated at 37°C for 2 h with 200 µM dansyl-Ala-Arg in a final volume of 250 µl in either 0.1 M HEPES with 0.2% Triton X-100 buffer at pH 7.5 for CPM or 0.2 M sodium acetate with 0.2% Triton X-100 at pH 5.5 for CPD. The assay was done at a slightly higher pH than the optimum for CPM (which is 7.0) and a slightly lower pH than the optimum for CPD (which is 6.2) to minimize any crossover activity of CPM in the CPD assay and vice versa. The reaction was stopped with 150 µl of 1 M citric acid, and the fluorescent product was extracted into chloroform and measured at 340-nm excitation/495-nm emission. Specific activity was calculated based on the protein concentration as determined by the method of Bradford.

Immunostaining. Confluent monolayers of HLMVEC on gelatin-coated glass coverslips were incubated in the absence or presence of 5 ng/ml IL-1 and 200 U/ml IFN- for 16 h at 37°C. Medium was changed to phenol red-free DME/F-12 for 1 h, and cells were then fixed in 4% paraformaldehyde for 20 min. Cells were washed with 100 mM glycine three times for 10 min to quench free aldehydes and followed by another three washes with Hank's balanced salt solution (HBSS). The cells were preincubated with blocking buffer (5% normal goat serum, 0.2% BSA, 0.1% Triton X-100 in HBSS, and 0.01% NaN₃) for 1 h at room temperature. The primary antibodies mouse monoclonal anti-CPM IgG (Novocastra Laboratories, diluted 1:50) or 1:500 anti-CPD antiserum (19, 34) were incubated at 4°C overnight. Normal mouse IgG or normal rabbit antiserum (for CPM or CPD, respectively) was used as controls. After three washes with HBSS, cell were preincubated for 1 h with blocking buffer and then incubated with secondary antibodies (goat anti-mouse Alexa fluor 488 or goat anti-rabbit Alexa fluor 546, diluted 1:750) for 2 h. Cells were washed and then mounted on glass slides using ProLong Antifade mounting medium. Confocal microscopy was performed on a Zeiss LSM 510 using a ×63 1.3 numerical aperature objective in optical sections <1 µm in thickness (pinhole set to achieve 1 Airy unit).

Data analysis. Data are presented as means ± SE. Statistical analysis was done with ANOVA using the Tukey-Kramer multiple-comparison test. Values of P < 0.05 were considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A porphyrinic microsensor was used to measure NO production in HLMVEC. This technique has the advantage of measuring nanomolar concentrations of authentic NO in real time (5, 30) as opposed to end-point measurements of accumulated breakdown products as in most other methods. When HLMVEC were exposed to increasing concentrations of bradykinin, a dose-dependent increase in NO production was seen that was transient and returned to baseline in about 5 min (Fig. 1A). The NO production was due to stimulation of the B₂ receptor as the B₂ receptor antagonist HOE 140 blocked the response (Fig. 1A). In the presence of medium alone, there was no apparent production of NO (Fig. 1B). Stimulation of control cells with the B₁-specific agonist 100 nM des-Arg¹⁰-kallidin (kallidin = Lys-bradykinin) also stimulated the production of NO but in a completely different pattern with a slow linear rise that was blocked by the B₁ receptor antagonist des-Arg¹⁰-Leu⁹-kallidin (Fig. 1B). These data indicate that HLMVEC constitutively express both B₁ and B₂ receptors.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Stimulation of nitric oxide (NO) production by bradykinin (BK) in control human lung microvascular endothelial cells (HLMVEC). Cells were stimulated with BK (A) at concentrations of 1 nM (n = 3), 10 nM (n = 3), or 100 nM (n = 6) or 100 nM des-Arg10-kallidin (DAKD; B, n = 3), and NO production was measured electrochemically with a porphyrinic electrode. In some cases, HLMVEC were preincubated for 10 min with the B2 antagonist HOE 140 (n = 5) or the carboxypeptidase inhibitor dl-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGTA, n = 4) before addition of 100 nM BK or with 1 µM des-Arg10-Leu9-kallidin (DALKD; n = 3) before addition of 100 nM des-Arg10-kallidin (DAKD). Bottom: typical real-time tracings of NO production. Vertical bar shows response to standard 200 nM concentration of NO. Length of horizontal arrow indicates 5 min (left) or 20 min (right). Top: mean values ± SE. NO release was measured as area under the response curve (A) or as NO concentration reached at 20 min (B). ** or ##Difference significant at P < 0.01 compared with 100 nM agonist alone.

To induce upregulation of the B₁ receptor, HLMVEC were stimulated with 5 ng/ml IL-1 and 200 U/ml IFN- for 16 h. In the presence of medium alone, there was no generation of NO in the stimulated HLMVEC (Fig. 2). The B₁ agonist des-Arg¹⁰-kallidin caused a dose-dependent progressively increasing and prolonged generation of NO that was almost linear and still increasing after 20 min (Fig. 2). The response to 100 nM des-Arg¹⁰-kallidin was about fourfold greater in the stimulated cells compared with that generated by the control cells (Figs. 1B and 2). Extension of the measurement time revealed a plateau in the increase in NO production between 20 and 30 min and then a slow decline, although NO concentration was still elevated over baseline at 60 min (not shown). Because the electrode directly measures NO, which is unstable and continually breaking down, a plateau in the response indicates achievement of a steady-state level of NO production that is balanced by NO degradation. The response to des-Arg¹⁰-kallidin in stimulated HLMVEC was blocked by 20 µM L-NMMA, the NO synthase inhibitor, and by 1 µM des-Arg¹⁰-Leu⁹-kallidin, the specific B₁ receptor antagonist, but not by the B₂ antagonist HOE-140 (Fig. 3).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Dose-dependent stimulation of NO production by DAKD in stimulated endothelial cells. HLMVEC were treated with 5 ng/ml interleukin-1 (IL-1) and 200 U/ml interferon- (IFN-) for 16 h and then stimulated with various concentrations DAKD, and NO production was measured electrochemically with a porphyrinic electrode. A: mean values from 3 separate experiments ± SE. NO release was calculated as NO concentration reached at 20 min. B: typical real-time tracing of NO production. Vertical bar shows response to standard 400 nM concentration of NO. Length of horizontal arrow indicates 20 min.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   DAKD stimulates prolonged NO production via B1 receptor activation in stimulated endothelial cells. HLMVEC were treated with 5 ng/ml IL-1 and 200 U/ml IFN- for 16 h and then stimulated with 100 nM DAKD without (n = 10) or with pretreatment with the B1 antagonist DALKD (n = 3), the B2 antagonist HOE 140 (n = 3), the NO synthase inhibitor NG-nitro-L-arginine (L-NMMA; n = 3), or the carboxypeptidase inhibitor MGTA (n = 3), and NO production was measured electrochemically with a porphyrinic electrode. A: mean values ± SE. *** Difference significant at P < 0.001 compared with 100 nM DAKD alone. B: typical real-time tracing of NO production. For further details, see Fig. 2.

Interestingly, addition of the B₂ agonists bradykinin or kallidin to HLMVEC that had been pretreated with IL-1 and IFN- also increased NO production but in a pattern similar to that seen with des-Arg¹⁰-kallidin (Fig. 4). The response was significantly inhibited by 20 µM MGTA, the specific kininase I-type carboxypeptidase inhibitor, indicating that bradykinin and kallidin were converted to the corresponding des-Arg B₁ agonists by endothelial carboxypeptidase activity. In control experiments, MGTA did not inhibit the generation of NO in response to bradykinin in control cells (Fig. 1) or to des-Arg¹⁰-kallidin in HLMVEC stimulated with IL-1 and IFN- (Fig. 3). Further proof of the generation of the B₁ agonists was obtained by use of the B₁ antagonist des-Arg¹⁰-Leu⁹-kallidin, which inhibited the response to bradykinin or kallidin to a similar extent as MGTA (Fig. 4). The B₂ receptor antagonist HOE 140 elicited a smaller inhibition that was nevertheless statistically significant, indicating that some of the response could be due to direct stimulation of the B₂ receptor and is consistent with the lack of complete inhibition by the B₁ antagonist (Fig. 4). Indeed, in the presence of both B₁ and B₂ receptor antagonists, the generation of NO in response to either bradykinin or kallidin was almost completely inhibited in stimulated HLMVEC. Because we had noticed a small B₁ receptor response in control HLMVEC (Fig. 1), we carried out additional experiments in control cells and continued the measurement of NO production in response to bradykinin beyond the 5-min peak due to B₂ receptor stimulation, and we observed a very small linear increase in NO production consistent with the B₁ pattern of generation we found in stimulated cells (not shown). The B₁ antagonist des-Arg¹⁰-Leu⁹-kallidin did not inhibit the initial response (peak at 5 min) to bradykinin but did block the delayed slow rise, indicating it is due to B₁ receptor stimulation.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   B2 receptor agonists stimulate prolonged NO production via B1 and B2 receptor activation in stimulated endothelial cells. HLMVEC were treated with 5 ng/ml IL-1 and 200 U/ml IFN- for 16 h and then stimulated with 100 nM BK or kallidin (KD) without or with preincubation with the B1 antagonist DALKD, B2 antagonist HOE 140, the B1 and B2 antagonists combined, NO synthase inhibitor L-NMMA, or the carboxypeptidase inhibitor MGTA, and NO production was measured electrochemically with a porphyrinic electrode. A: mean values from 3 to 8 separate experiments ± SE. Difference significant at P < 0.001 (*** or ###), P < 0.01 (** or ##), or P < 0.05 (* or #) compared with 100 nM BK (*) or 100 nM KD (#) alone. B: typical real-time tracing of NO production in response to BK (left) or KD (right). For further details, see Fig. 2.

To determine whether HLMVEC contained membrane-bound kininase I (or B-type) carboxypeptidase activity, we isolated a high-speed membrane fraction from the cells and assayed with the specific B-type carboxypeptidase substrate dansyl-Ala-Arg (41, 53). To differentiate between CPM, which has a neutral pH optimum, and CPD, which has an acidic pH optimum, assays were carried out at both pH 5.5 (for CPD) and pH 7.5 (for CPM). These conditions minimize overlap between the two enzymes because CPM is only 31% active at pH 5.5 and CPD is only 20-25% active at pH 7.5 (8, 34). There was significant carboxypeptidase activity at both pH values: 36.8 ± 5.2 nmol · h¹ · mg protein¹ (n = 4) at pH 5.5 and 18.7 ± 0.5 nmol · h¹ · mg protein¹ (n = 4) at pH 7.5, indicating that the cells contain both CPM and CPD. The specific B-type carboxypeptidase inhibitor MGTA (10 µM) inhibited 97 ± 3.5% of the CPM activity and 96.5 ± 4.7% of the CPD activity measured with dansyl-Ala-Arg (n = 4). To determine whether inflammatory cytokines that upregulate the B₁ receptor might also upregulate carboxypeptidase activity, HLMVEC that were treated with 5 ng/ml IL-1 and 200 U/ml IFN- for 16 h were fractionated and assayed for CPD and CPM activity as described above. After stimulation, there was about a twofold increase in both CPD and CPM activity (Fig. 5), indicating an upregulation of the enzymes that generate B₁ agonists under conditions that also upregulate B₁ receptors.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Upregulation of carboypeptidase M (CPM) and carboypeptidase D (CPD) activity in endothelial cells by inflammatory cytokines. HLMVEC were treated without (control) or with 5 ng/ml IL-1 and 200 U/ml IFN- for 16 h. Cells were lysed and membrane fractions were isolated and assayed for CPM or CPD activity with dansyl-Ala-Arg. Results are calculated as %control (untreated = 100%) and are means ± SE for n = 4.

To investigate the subcellular localization of CPM and CPD in endothelial cells, immunocytochemical labeling and laser scanning confocal microscopy were performed on control HLMVEC and cells stimulated with 5 ng/ml IL-1 and 200 U/ml IFN- for 16 h (Fig. 6). CPD was found primarily in a perinuclear region consistent with its localization in the Golgi, whereas CPM staining was primarily on the plasma membrane in a punctuate pattern that is commonly found with glycosylphosphatidylinositol-anchored proteins (Fig. 6). The intensity of CPM and CPD staining increased after cytokine treatment, which is consistent with the increases in membrane carboxypeptidase activity measured above.

View larger version (82K): [in this window] [in a new window]   Fig. 6.   Effect of cytokines on CPM and CPD expression and localization in endothelial cells. HLMVEC were exposed to vehicle or 5 ng/ml IL-1 and 200 U/ml IFN- for 16 h, fixed, and immunostained as described in MATERIALS AND METHODS. Both CPD (top, red) and CPM (bottom, green) immunostaining increased after cytokine treatment. CPM expression was mainly observed on the cell membrane, whereas CPD was localized primarily to the perinuclear/Golgi region. No membrane or Golgi staining was seen in cells stained with control normal mouse IgG or normal rabbit serum (not shown). Results are representative of 4 separate experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

These studies show that kinin-dependent NO production in endothelial cells can be enhanced by upregulation of the B₁ receptor, whose activation leads to a prolonged response. This phenomenon likely does not require exposure of cells to inflammatory cytokines. For example, bovine pulmonary artery endothelial cells, which constitutively express B₁, also generate a greater and more prolonged output of NO in response to direct stimulation of B₁ by either ACE inhibitors or peptide ligand compared with B₂ stimulation (21). However, B₁ agonists can only be generated physiologically by carboxypeptidase conversion of B₂ agonists to their des-Arg forms, which highlights the importance of membrane kininase-I type enzymes in regulating this response, as shown here. In this way, kininase I degradation of kinins is actually a processing step, analagous to ACE, which cleaves angiotensin I to generate angiotensin II, the agonist for the AT1 receptor.

We found significant CPD and CPM activity in membrane fractions of HLMVEC. However, subcellular fractionation does not separate plasma membranes from intracellular membranes; therefore, these assays cannot determine how much of the activity was on the plasma membrane. Immunohistochemical staining of HLMVEC revealed the same distribution for CPD and CPM found in other cell types; CPM is primarily on the plasma membrane and CPD is highly concentrated in the Golgi. Although CPD can be detected on the plasma membrane, the maximum reported is about 10% of the total CPD in the cell (19, 25, 57). It is likely in our studies that the primary enzyme involved in the conversion of kinins to des-Arg kinins was CPM based on the following considerations. First, although the total membrane CPD activity was about two times higher than CPM activity, assuming that 10% of the CPD is on the plasma membrane, the ratio would be about 5:1 CPM:CPD. Second, CPD has an acidic pH optimum of 6.2 and at pH 7.4 CPD has only about 20-25% of its activity remaining with dansyl-Ala-Arg, whereas CPM is fully active at pH 7.4. There are no specific inhibitors that will distinguish between CPM and CPD, therefore, a definitive resolution to this question will require the development of knockout animals or RNA interference approaches.

Another way in which membrane carboxypeptidases can enhance NO production is by cleaving substrates to generate free Arg to be utilized by NO synthase as was shown in a macrophage cell line (19). However, this is unlikely to play a significant role in the present experiments, which were carried out in the presence of medium containing 0.7 mM free Arg. The maximum amount of Arg that would be generated by carboxypeptidase cleavage of all the added kallidin or bradykinin would be only 100 nM, which is 7,000-fold lower than the Arg in the medium.

An important physiological indicator of B₁ receptor stimulation is NO (31). The ability to measure NO production in real time revealed a striking difference in the initial time course of NO production after addition of the agonist in control HLMVEC. Thus, the B₂ receptor-mediated response was relatively transient and short lived, whereas the B₁ receptor response resulted in a slowly rising, almost linear increase in the generation of NO. The presence of a B₁ response in control cells was unexpected because the receptor is usually absent in normal tissues or cells and requires injury or inflammatory mediators to upregulate its transcription (3, 31). Although it is possible that our culture conditions caused upregulation of B₁ receptors, we took great care to eliminate potential sources of endotoxin contamination in medium, buffers, etc. It is also possible that these cells constitutively express B₁ receptors at a low level as found, for example, in bovine pulmonary artery endothelial cells (21, 50). The difference in pattern of NO production in response to B₁ or B₂ agonists is consistent with the marked difference in the ability of the B₂ and B₁ receptors to be desensitized after stimulation; the B₂ receptor is rapidly sequestered and the response terminated, whereas the B₁ receptor is not, resulting in a more prolonged response (2, 10, 27, 31). However, in HLMVEC stimulated with IL-1 and IFN-, it is apparent that B₂ receptor stimulation can also result in prolonged generation of NO in a pattern similar to that found with B₁ stimulation. Thus, in the presence of a B₁ antagonist in stimulated cells, a B₂ agonist stimulated a slow linear increase in NO production that was inhibited by a B₂ antagonist (Fig. 4). The reason for the prolonged B₂ response in stimulated cells is unclear, but it is possible that sequestration/desensitization of B₂ is altered under these conditions. Alternatively, activation of NO synthase in stimulated cells might differ from that in control cells in the absence of a change in receptor desensitization. For example, B₂-stimulated phosphorylation of endothelial NO synthase might predominate in stimulated cells [which would generate a slower but more prolonged response (43)] compared with only calcium activation in control cells. Another possibility is that either B₁ or B₂ receptor activation in stimulated HLMVEC activates inducible NO synthase instead of endothelial NO synthase, which leads to a different pattern of NO production. These will be interesting areas to explore in future investigations.

The ability of endothelial cells to generate higher levels of NO under inflammatory conditions could have important physiological and pathological relevance. NO is a reactive radical gas that regulates cellular functions in both physiological and pathological conditions (20, 23, 29, 38). NO has a variety of activities in the cardiovascular system, including regulation of vascular tone and endothelial barrier function (24, 36). In physiological states, NO can serve a protective function, but, under conditions of high output, NO may contribute to tissue damage by reacting with superoxide to form peroxynitrite, a strong oxidant (22). NO not only participates in the inflammatory response via its physiological effects, but also by its ability to regulate the expression of inflammatory proteins (56, 59) via its regulation of the transcription factor, nuclear factor-kB (7). Thus it has been suggested that NO may exert both deleterious and protective effects in inflammatory conditions such as sepsis, depending on species, timing, the cell type, inflammatory stimulus, the NO concentration, and NO-related metabolites generated (58).

Kinins cause numerous biological effects, including regulation of smooth muscle tone or salt and water excretion in the kidney, and are important mediators of the inflammatory response (3, 31). Kinins also have beneficial effects in the heart and are now thought to be major mediators by which inhibitors of ACE have beneficial cardiovascular effects that go beyond lowering blood pressure (14, 28). The B₁ receptor may have protective roles in cardiovascular diseases (1, 4, 6), and ACE inhibitors were recently found to directly bind and activate the B₁ bradykinin receptor through a unique zinc-binding motif (21). ACE inhibitors also induce expression of the B₁ receptor in rodent kidney, heart, and vasculature, which was shown to play a role in the hypotensive effect of ACE inhibitors (32). Because ACE is a predominant peptidase for the degradation of bradykinin and kallidin in vivo (11, 12, 15), blockade with ACE inhibitors would increase the level of kinin substrates for conversion by cellular carboxypeptidases. Indeed, the generation of des-Arg kinins has been reported to increase in several studies in the presence of ACE inhibitors (31). Thus kininase I-type carboxypeptidases could potentially play a role in some of the therapeutic effects of ACE inhibitors through enhancement of signaling through the B₁ receptor.