Amidative peptide processing and vascular function

Charlie D. Oldham1, Cuizhen Li1, Jun Feng1, Robert O. Scott2, Wen Z. Wang2, Allison B. Moore1, Peggy R. Girard2, Jianzhong Huang3, Ruth B. Caldwell3, R. William Caldwell3, and Sheldon W. May1

1 School of Chemistry and Biochemistry and 2 School of Biology, Georgia Institute of Technology, Atlanta 30332; and 3 School of Medicine, Medical College of Georgia, Augusta, Georgia 30912

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Substance P (SP), an amidated peptide present in many sensory nerves, is known to affect cardiovascular function, and exogenously supplied SP has been shown to activate nitric oxide synthase (NOS) in endothelial cells. We now report that SP-Gly, the glycine-extended biosynthetic precursor of SP (which is enzymatically processed to the mature amidated SP), causes relaxation of rat aortic strips with an efficacy and potency comparable to that of SP itself. Pretreatment of the aortic strips with 4-phenyl-3-butenoic acid (PBA), an irreversible amidating enzyme inactivator, results in marked inhibition of the vasodilation activity induced by SP-Gly but not of that induced by SP itself. Isolated endothelial cell basal NOS activity is also decreased by pretreatment with PBA, with no evidence of cell death or direct action of PBA on NOS activity. Both bifunctional and monofunctional forms of amidating enzymes are present in endothelial cells, as evidenced by affinity chromatography and Western blot analysis. These results provide evidence for a link between amidative peptide processing, NOS activation in endothelial cells, and vasodilation and suggest that a product of amidative processing provides intrinsic basal activation of NOS in endothelial cells.

peptidylglycine alpha -monooxygenase; endothelial cells; peptidylamidoglycolate lyase; nitric oxide synthase

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

BIOACTIVE PEPTIDES, NOW recognized to be vitally involved in many cellular functions, are generated biosynthetically from larger precursors via a variety of posttranslational modifications. One such processing event is carboxy-terminal amidation. Amidation is a prevalent process that occurs in a variety of tissues, including sensory and sympathetic nerves (3) as well as in chromaffin granules (10). We and others have demonstrated that formation of peptide amides from their glycine-extended precursors is a two-step process, entailing sequential enzymatic action by peptidylglycine alpha -monooxygenase (PAM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PGL, EC 4.3.2.5) (9, 12, 13, 18, 21, 22). The monooxygenase, PAM, first catalyzes formation of the alpha -hydroxyglycine derivative of the glycine-extended precursor, in a process dependent on ascorbate, copper, and molecular oxygen (12, 21, 24). The lyase, PGL, then catalyzes the breakdown of this alpha -hydroxyglycine derivative to produce the amidated peptide plus glyoxylate (12, 13).

Endothelial cells, which play a critical role in vascular circulation, are known to produce and release a variety of vasoactive substances, including a number of vasoactive peptides (6). However, none of the peptides heretofore considered to be "endothelially derived" is amidated, and the same is true of atrial natriuretic peptides, the family of peptide hormones produced in cardiac atrial cells (19). Recently, we reported (17) that the two enzymes essential for amidation, PAM and PGL, are present in cultured bovine aortic endothelial cells and that they exhibit enzymological characteristics that correspond to those of PAM and PGL from other tissues. This finding is consistent with a role for amidative peptide processing in vascular function.

Substance P (SP), an amidated peptide present in many sensory nerves, is known to affect cardiovascular function (2). Exogenously supplied SP has been shown to activate nitric oxide synthase (NOS) in endothelial and other cells (5, 27) and to produce nitric oxide (NO), an extremely important effector of cardiac and vascular function. Although SP has also been detected in vascular endothelial cell extracts (14-16), it has not been known whether this SP had been passively accumulated or actively processed within these endothelial cells.

We now report that SP-Gly, the glycine-extended biosynthetic precursor of SP (which is enzymatically processed to the mature, amidated SP), causes vascular tissue relaxation with an efficacy and potency comparable to that of SP itself. Moreover, we demonstrate that pretreatment of vessels with the amidating enzyme inactivator, 4-phenyl-3-butenoic acid (PBA), results in marked inhibition of the vasodilation activity induced by SP-Gly but not of that induced by SP itself. In addition, we demonstrate that PBA causes reduction of the basal NOS activity in endothelial cells, and we characterize the molecular forms of the PAM and PGL present in endothelial cells. Together, these results provide evidence for a link between amidative peptide processing, NOS activation in endothelial cells, and vasodilation. We believe that some product of amidation in endothelial cells is capable of providing intrinsic basal activation of the NOS system within these cells.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Vascular tone. Male Sprague-Dawley rats were killed by decapitation. The thoracic aorta was rapidly removed, cleaned from the adjacent tissue, and cut into four rings of ~3-5 mm in length. Two metal hooks were carefully passed through the lumen of each ring and then mounted in 25-ml organ baths containing Krebs solution under 2 g of tension. Krebs solution had the following composition (10-3 M): 118 NaCl, 6.75 KCl, 2.54 CaCl2, 1.2 MgSO4 · 7H2O, 1.19 KH2PO4, 23 NaHCO3, and 11 dextrose; the pH of the solution was 7.35-7.45. Rings were equilibrated for 90 min with solution changes every 15 min. The bathing solution was kept at 37°C and was continuously aerated with a mixture of 5% CO2 and 95% O2. After equilibrium was established, a submaximal dose of phenylephrine, 3 × 10-7 M, was added to the organ bath, and the contraction was allowed to develop fully. Vessels with good integrity of the endothelial cells, as assessed by ~80% relaxation to acetylcholine (10-7 M), were used. Increasing concentrations of either SP or SP-Gly, from 10-9 to 10-5 M, were added to the organ baths every 10 min, and the degree of relaxation was monitored. A cumulative dose-response curve (DRC) to either SP or SP-Gly and control was constructed over a period of 1 h. A full relaxation occurred with addition of acetylcholine (10-5 M). The bathing solution was drained and replaced with fresh solution. The rings were allowed to return to baseline, and another DRC was built using the same process as described above. Rings were pretreated with PBA, 10-5 M, during the last 30 min of the 90-min equilibrium period and subsequently washed out. Vascular tension was not altered by this treatment. After reconstriction of the rings, DRCs to SP or SP-Gly and control were constructed.

Endothelial cell NOS activity. NOS activity was measured by monitoring the conversion of [3H]arginine to [3H]citrulline (5). Bovine aortic endothelial cells (106 cells per dish) were incubated in 2 ml of 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer (pH 7.4) containing L-[3H]arginine (10 mCi/ml, DuPont NEN, Boston, MA), 1 mM NADPH, and 1.25 mM CaCl2 for 30 min in the absence and presence of 10-6 or 10-5 M PBA. After marked acidification with HEPES buffer (pH 5.5, 2 mM EDTA), cells were disrupted by freezing and thawing (two times) and the supernatant was applied to 2-ml columns containing Dowex 50 W resin (Na+ form) to separate L-[3H]arginine from L-[3H]citrulline, formation of which is a measure of NO production. L-[3H]citrulline elutes through the resin and was counted by liquid scintillation. Following this same method, we determined the action of SP (10-6 M) on NOS activity ([3H]citrulline production) by adding this agent to control cells and cells pretreated for 30 min with PBA (10-5 M). The incubation time with SP was also 30 min. The difference in the amount of citrulline formed in the presence and absence of SP was considered to be the effect of SP.

Enzyme assays. Assays for PAM and PGL activity were performed as previously described (12, 13, 17).

Fumarase assays (8) and lactate dehydrogenase (LDH) assays (20) were performed as described.

Endothelial cell procedures. Bovine aortic endothelial cells (passages 8-12) were purchased from Cell Systems (Kirkland, WA). Cells were seeded onto 100-mm tissue culture plates and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal bovine serum, penicillin (100,000 U/l), and streptomycin sulfate (100 mg/l). The cultures were fed every 48 h until the cells were confluent and kept at 37°C, with 97% humidity and 5% CO2. Fifteen dishes of confluent endothelial cells were rinsed four times with DMEM, and each dish was cultured in 10 ml of serum and protein-free DMEM at 37°C. Endothelial cells were fed every 24 h, for a period of 72 h, and the conditioned medium was collected into a 250-ml centrifuge tube containing 0.8 g of KI, 0.03 mg of phenylmethylsulfonyl fluoride (PMSF), and 1.4 mg of benzamide and centrifuged at 1,000 g for 25 min. The supernatant was kept at -70°C. Cultures of bovine retinal endothelial (BRE) cells were prepared and amplified in serum-defined endothelial cell growth medium (Clonetics, San Diego, CA) as described (1). The cultures, which were 98% pure as demonstrated by immunoreactivity for von Willebrand's factor and by their ability to take up acetylated low-density lipoprotein, were used between passages 4 and 6.

For preparation of cell extracts, cells from 10 to 20 plates were harvested, suspended in pH 7.4 buffer containing 0.3 mg/ml PMSF, 16 µg/ml benzamidine, and 2 µg/ml leupeptin, and homogenized using a Dounce homogenizer. The supernatant obtained after centrifugation was used as the soluble fraction. The pellet was resuspended and washed with Na2CO3, pH 11.5, to remove surface proteins. The pellet was then resuspended in 300 µl of 20 mM N-tris(hydroxymethyl) methyl-2-aminoethanesulfonic acid, pH 7.4, containing 1% Triton X-100 and allowed to stand for 60 min at 4°C before centrifuging to remove debris. This supernatant was used as the detergent-soluble fraction of the cell extract.

In the secretion experiments, confluent endothelial cells in 100-mm culture dishes were washed four times with serum-free DMEM and cultured in 10 ml of DMEM containing glutamine (0.584 mg/ml), bovine serum albumin (1 mg/ml), transferrin (5 µg/ml), sodium selenite (30 nM), penicillin, and streptomycin at 37°C. Aliquots of 150 µl were withdrawn from the incubation medium at the appropriate time and kept at -70°C until assay. An equal volume of fresh medium was added to the incubation solution whenever aliquots were withdrawn.

Solid phase peptide synthesis. PGL peptide (PGL 561-579), NH2-V-I-D-P-N-N-A-A-V-L-Q-S-S-G-K-N-L-F-Y-COOH, and PAM peptide (PAM 288-310), NH2-C-V-F-T-G-E-G-R-T-E-V-T-H-I-G-G-T-S-S-D-E-M-C-COOH (7), were synthesized using a PS3 peptide synthesizer (Rainin Instrument, Woburn, MA) with p-benzyloxybenzyl alcohol resin as solid support. The peptides were cleaved from the resin with trifluoroacetic acid and purified by high-performance liquid chromatography (HPLC) on a C8 reverse phase column, and the sequences of both purified peptides were analyzed by the Edman degradation method on a Porton 1090 sequencer. The molecular weights of both peptides were analyzed by fast-atom bombardment mass spectrometry.

Two other peptides, a modified PAM peptide (PAM 289-309, missing both terminal cysteines of the PAM 288-310 peptide) and glycine-extended SP (NH2-R-P-K-P-Q-Q-F-F-G-L-M-G-COOH), were also synthesized by the same method. As expected, SP-Gly was found to be a substrate for the PAM-PGL enzyme system.

Affinity purification of antibodies. Affinity columns were prepared by coupling either 1 mg of PGL peptide (PGL 561-579) or 1 mg of PAM peptide (PAM 289-309) with 0.4 mg of dried activated CH Sepharose 4B according to company directions (Pharmacia, Piscataway, NJ). One milliliter of New Zealand White rabbit antiserum raised against either peptide was loaded onto an affinity column prepared from the corresponding peptide equilibrated with 20 mM HEPES-100 mM NaCl, pH 7.4. The column was washed with the same buffer to remove unbound material and then eluted with 4.5 M MgCl2. The eluted material was used as the primary antibody for Western blot analyses.

Western blot analysis. Endothelial cell extracts were separated on 10% sodium dodecyl sulfate (SDS) polyacrylamide gels. Kaleidoscope prestained molecular weight standards (Bio-Rad Laboratories, Hercules, CA) were used. After electrophoresis, proteins in the gel were transferred to a nitrocellulose membrane. The blots were incubated for 1 h at room temperature in 25 mM tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 7.4), 150 mM NaCl, and 0.05% Tween 20 (TBST) containing 5% nonfat milk to block the nonspecific binding sites. The blots were then incubated with primary antibody diluted with TBST containing 5% nonfat milk for 2 h at room temperature and washed five times for 10 min each time with TBST. Secondary antibody (horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin) diluted with TBST containing 5% nonfat milk was added for 1 h, and immunoreactive bands were visualized by enhanced chemiluminescence using the kit from Amersham.

For blots from BRE cells, the blots were stripped in stripping buffer (62.5 mM Tris · HCl, pH 6.8, 2% SDS, and 100 mM beta -mercaptoethanol) at 50°C for 30 min to remove previous antibody. Stripped blots were washed in TBST for 2 × 10 min and reblocked in 5% nonfat milk for 1 h at room temperature. The blots were then reprobed with a different primary antibody, and immunodetection was performed as described above.

Analysis of data. The data in Figs. 1 and 2 are expressed as mean absolute or percent change in values ± SE. Statistical evaluation for differences among experimental groups was performed by the Student-Newman-Keuls test following analysis of variance. The differences were considered significant when P < 0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Vascular tone. Both SP and SP-Gly were essentially equipotent in producing concentration-relaxation curves; these curves were superimposable in the control state (Fig. 1A). The mean percent relaxations to SP and SP-Gly at 10-5 M were 48 ± 5 and 49 ± 4, respectively, compared with the maximum response observed for acetylcholine (10-5 M). Relaxation responses to the two peptides occurred with the same latency (~5 s).


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Fig. 1.   A: effect of graded concentrations of substance P (SP, bullet ) and glycine-extended SP (SP-Gly, open circle ) on the tension in aortic vascular strips preconstricted with phenylephrine. Relaxation is represented as percent of the maximum relaxation induced in these tissues by acetylcholine (10-5 M). B: effect of pretreatment with 4-phenyl-3-butenoic acid (PBA, 10-5 M) on vasodilation responses of rat aortic strips to SP and SP-Gly. Bar labeled control represents the full relaxation response (100%) to concentrations of 10-7-10-5 M of either SP or SP-Gly. Second and third bar represent the mean percent of control responses to SP and SP-Gly, respectively, after PBA treatment and washout. Each bar represents 10-13 observations. * Response is different from control, P < 0.05.

To examine the effect of PBA, we compared the pooled maximum relaxation responses to SP or SP-Gly (10-7-10-5 M) without PBA treatment (100% of control response for each) to those obtained after PBA pretreatment (10-5 M) (Fig. 1B). Tissues were washed after the PBA pretreatment period (30 min) and before the application of SP or SP-Gly. Pretreatment with PBA reduced the vasorelaxing response to SP to 75% of the control. In contrast to this minor effect on the responses to SP, PBA markedly reduced the vasodilation responses to SP-Gly to only 26% of that for control vessels.

These data demonstrate that intact blood vessels contain the functional enzyme system to effectively convert SP-Gly to the vasoactive SP. Furthermore, PBA appears to inhibit this conversion. The 25% reduction in responses to SP itself by PBA pretreatment (10-5 M) may be due to nonspecific actions on vascular contractile processes.

Endothelial cell NOS activity. Treatment of isolated endothelial cells for 30 min with PBA (10-6 M and 10-5 M) reduced basal NOS activity (L-arginine to L-citrulline conversion) by 17% and 28%, respectively (Fig. 2). There was no evidence of cell death with these PBA treatments. Moreover, SP was fully capable of activating endothelial cell NOS in these cells treated with PBA (10-5 M). SP (10-6 M) raised citrulline formation by 541 ± 38 and 605 ± 42 pmol/mg protein, respectively, before and after treatment with PBA.


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Fig. 2.   Effect of PBA (10-6-10-5 M) on the conversion of L-[3H]arginine to L-[3H]citrulline, a measure of nitric oxide synthase activity, in bovine aortic endothelial cells. Each bar represents mean of 5 or 6 observations. * Response is different from control, P < 0.05.

These data suggest that an alpha -amidated peptide(s), possibly SP, is produced by endothelial cells and exerts some tonic stimulation of NOS function in these cells.

Secretion of amidation activity. Initial experiments confirmed that both PAM and PGL are secreted into the culture medium by endothelial cells. When conditioned medium from a 24-h culture of confluent endothelial cells was incubated with trinitrophenyl (TNP)-D-Tyr-Val-Gly, formation of the amide product, TNP-D-Tyr-Val-NH2 is readily detectable using our standard HPLC assay procedure (10). Amide formation in this assay entails sequential action of both PAM and PGL. Amidation activity, which was typically at the level of 100 mU/l of conditioned medium from a 24-h culture, increased continuously over several days of cell culture, and no amidation activity was detectable in control medium in which no endothelial cells had been grown (Fig. 3).


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Fig. 3.   Time course of endothelial amidation activity secretion. Amidation activity in the medium conditioned by endothelial cells for various periods of time was assayed with 60 µM TNP-D-Tyr-Val-Gly as the substrate in an assay mixture containing 4 µM Cu(II), 1 mg/ml catalase, and 4.5 mM L-ascorbate. Amidation activity is represented by the production of TNP-D-Tyr-Val-NH2 determined by high-performance liquid chromatography. Data shown are mean values for duplicate experimental samples; error range is less than the size of the data points.

To test whether this amidation activity could be due to cell lysis rather than secretion, the conditioned medium was also assayed for both fumarase (mitochondrial marker) (8) and LDH (cytosol marker) (20) activities. The LDH activity (Delta A340 · ml-1 · min-1, where Delta A is the change in absorbance at 340 nm) of the cell lysate was -27.4, and the LDH activity of the conditioned medium was +0.01. The fumarase activity (Delta A240 · ml-1 · min-1) of the cell lysate was 0.6 and that of the conditioned medium was 0.04. Clearly, neither of these activities was present in the conditioned medium, whereas high levels of both fumarase and LDH were found in endothelial cell lysate.

It has been reported that dexamethasone treatment decreases amidation activity in cultured mouse pituitary tumor cells (AtT-20), whereas dexamethasone increases amidation activity of cultured rat atrial cells (23). We find that incubation of endothelial cells with dexamethasone for 24 h results in no increase of the amidation activity in the conditioned medium compared with controls.

Chromatographic characterization of secreted amidation activity. Initial characterization of the amidating enzymes secreted by cultured endothelial cells was carried out using an affinity chromatography gel that specifically binds to PAM and not to PGL. Concentrated conditioned medium was first partially purified by gel filtration (Superose 12, Pharmacia), and fractions containing either PAM or PGL activities were then analyzed using an affinity column consisting of activated CH Sepharose 4B linked to the PAM substrate, D-Tyr-Trp-Gly. As shown in Table 1, only PGL activity was present in the nonadsorbed fraction from the affinity column, whereas both PAM and PGL activities were present in the adsorbed fraction. If only monofunctional PAM and PGL are present in the conditioned medium, the nonadsorbed fraction from the PAM affinity column should contain only PGL activity, whereas the adsorbed fraction of this column should contain only PAM activity. On the other hand, if only bifunctional proteins possessing both PAM and PGL domains are present, all the PAM and PGL activity should be retained on the affinity column. Finally, if both monofunctional and bifunctional enzymes are present, all species possessing the PAM domain should be adsorbed onto the column, whereas only PGL activity should be evident in the nonadsorbed fraction. Our results are clearly in accord with this latter possibility, thus indicating that both monofunctional and bifunctional forms of amidating enzymes are secreted by cultured endothelial cells.

                              
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Table 1.   Analysis of amidating enzyme proteins in conditioned medium from endothelial cells using PAM substrate affinity column

The adsorbed and nonadsorbed fractions from the PAM affinity column were further analyzed using Superose 12 gel filtration chromatography. Figure 4, A and B, shows the Superose 12 elution profiles of PAM and PGL activities present in the adsorbed fraction from the affinity column. The profiles confirm that this fraction contains mainly bifunctional enzyme, since the major peak of PAM activity coelutes with the peak of PGL activity. In addition, a minor lower-molecular-weight peak exhibiting only PAM activity, thus corresponding to monofunctional PAM, is also present. In contrast, the elution profile of the nonadsorbed fraction from the affinity column (Fig. 5C) confirms the presence of only monofunctional PGL, with an elution position in the 40,000 molecular weight region.


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Fig. 4.   Gel filtration profiles of peptidylglycine alpha -monooxygenase (PAM) and peptidylamidoglycolate lyase (PGL) activities in the adsorbed and nonadsorbed fractions from the PAM substrate affinity column. Monooxygenase activity in the fractions was assayed using TNP-D-Tyr-Val-Gly as substrate and monitoring production of TNP-D-Tyr-Val-alpha -hydroxy-Gly. Lyase activity was determined by using TNP-D-Tyr-Val-alpha -hydroxy-Gly as the substrate and monitoring production of TNP-D-Tyr-Val-NH2. Adsorbed material clearly shows 2 peaks of monooxygenase activity (A) and only a single peak of lyase activity (B). Thus the early peak is bifunctional, and the latter peak is monofunctional monooxygenase. Nonadsorbed material shows only lyase activity (C) and is therefore monofunctional.

Western blot analysis of endothelial cell extracts. The affinity conjugate used in the above analysis clearly binds only to either monofunctional PAM or to the PAM domain of the bifunctional enzyme. Therefore, we proceeded to analyze the amidating enzymes from endothelial cell extracts using Western blot analysis with both affinity-purified anti-PAM and anti-PGL antibodies. The resulting Western blots are shown in Fig. 5. It is apparent that the major immunoreactive band that migrates at ~80 kDa reacts with both anti-PAM and anti-PGL antibodies and is thus a bifunctional enzyme. Additional minor immunoreactive bands are also evident and likely are processed forms of PAM or PGL, proteolytic fragments formed during sample preparation, or proteins bound nonspecifically. Thus, together, these Western blots and the PAM-affinity chromatography elution profiles confirm the presence of both bifunctional and monofunctional amidating enzymes in endothelial cell extracts and conditioned medium.


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Fig. 5.   Western blot analysis of PAM and PGL in endothelial cells. Thirty micrograms of endothelial cell extract (soluble fraction or detergent-soluble fraction prepared as described under MATERIALS AND METHODS) were loaded onto a 10% polyacrylamide-SDS gel and subjected to electrophoresis. Proteins were transferred to a nitrocellulose membrane, and membrane was then incubated with polyclonal antibodies against either PAM or PGL. Blot was subsequently incubated with horseradish peroxidase-conjugated donkey anti-rabbit antibody (secondary antibody) and developed using enhanced chemiluminescence. Extracts from bovine aortic endothelial cells were run in lanes 1 and 3 (soluble fraction) and lanes 2 and 4 (detergent-soluble fraction). Polyclonal antibodies were used to probe lanes 1 and 2 (anti-PAM) and lanes 3 and 4 (anti-PGL). Extracts from bovine retinal epithelial cells were run in lanes 5 and 7 (soluble fraction) and lanes 6 and 8 (detergent-soluble fraction). Polyclonal antibodies were used to probe lanes 5 and 6 (anti-PAM) and lanes 7 and 8 (anti-PGL). In all cases, a single major immunoreactive band migrating at ~80 kDa was seen. Controls probed with secondary antibody alone showed no immunoreactive bands (data not shown).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Previous work has clearly established that SP exerts significant effects on cardiovascular function (2, 27), and there is evidence that at least some of these effects are mediated by NO (27). The data in Fig. 1 now demonstrate for the first time that SP-Gly, the immediate biosynthetic precursor of SP, also exhibits vasodilation activity in rat aortic strips. Moreover, the potency of SP-Gly is similar to that of SP itself.

Conversion of SP-Gly to SP entails sequential enzymatic action by the two amidating enzymes PAM and PGL. Because we recently found that both PAM and PGL are present in endothelial cells (17), it was of great interest to determine whether the vasodilation activity observed for SP-Gly is a consequence of its amidative processing to SP within the endothelial cells of these aortic strips. Accordingly, the effect of the mechanism-based amidation inhibitor, PBA, on the vasodilation activities of SP and SP-Gly was determined. As is evident from Fig. 1, PBA pretreatment reduces the vasodilation activity of SP-Gly to 26% of its control response; in contrast, the activity of SP itself is only reduced to 75% of its control response. Because we have previously shown that PBA is a potent inactivator for endothelial cell PAM (11, 17), these results support the view that amidative processing of SP-Gly contributes significantly to the vasodilation activity of this peptide. Indeed, the fact that SP is still capable of increasing NO production in cells pretreated with PBA to the same extent as in control cells confirms that PBA does not exert direct action on NOS activity in endothelial cells or on cell viability.

Previous studies on the link between SP and endothelial cell NOS activity have utilized exogenously supplied SP, which presumably acts only on endothelial cell surface receptors to activate NOS (27). In contrast, it has never been determined whether SP processed within an endothelial cell can activate the cell's own NOS. The data in Fig. 2 now demonstrate that PBA causes a concentration-dependent reduction of basal NOS activity, with no evidence of cell death or direct action of PBA on NOS itself. These findings provide evidence for a link between amidated peptide production and the spontaneous basal production of NO by endothelial cells. In this regard, we certainly recognize that an amidated peptide, so produced, could exit the cell and subsequently affect cell surface receptors to activate endothelial NOS in that or adjacent cells in autocrine and paracrine fashions.

The PAM-PGL system in endothelial cells may work on a variety of glycine-extended peptide substrates. Whether such peptides are produced by endothelial cells is not known. It is possible that glycine-extended precursors for amidated peptides could be released from sensory nerves (SP, calcitonin gene-related peptide), sympathetic autonomic nerves (neuropeptide Y), or the neurohypophysis (vasopressin), carried humorally and processed by the vascular endothelium. Moreover, amidating enzymes secreted by endothelial cells could react with glycine-extended substrates in the blood to produce mature, amidated peptides. Such secretory action in endothelial cells would parallel that which occurs when atrial granules fuse with the cell membrane, release atrial natriuretic peptide, and possibly expose the blood to the PAM-PGL enzyme system (4). Our finding that amidating enzymes are present in microvascular (retinal) endothelial cells (Fig. 5) as well as aorta suggests that amidation may also play a role in processes that more selectively affect microvessels, such as angiogenesis and vascular permeability, both of which are processes shown to be induced by SP (25, 26).

    ACKNOWLEDGEMENTS

We gratefully acknowledge partial support of this work by the Georgia Institute of Technology/Medical College of Georgia Biomedical Research and Education Program.

    FOOTNOTES

Address for reprint requests: S. W. May, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332.

Received 4 March 1997; accepted in final form 7 August 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Cell Physiol 273(6):C1908-C1914
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