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 |
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
-monooxygenase; endothelial cells; peptidylamidoglycolate lyase; nitric oxide synthase
 |
INTRODUCTION |
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
-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
-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
-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 |
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
-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 |
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, ) and glycine-extended SP (SP-Gly, ) 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
-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.
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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
(
A340 · ml
1 · min
1,
where
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
(
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
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|
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 -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- -hydroxy-Gly.
Lyase activity was determined by using
TNP-D-Tyr-Val- -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.
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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).
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|
 |
DISCUSSION |
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 |
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Behzadian, M. A.,
X. Wang,
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