Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7545
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ABSTRACT |
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In order to exert an
appropriate biological effect, the action of the vasoconstrictive
hormone angiotensin II (ANG II) is modulated by vasoactive factors such
as prostaglandins PGE2 and PGI2. The present
study investigates whether prostaglandins alter ANG II-mediated
increases in cytosolic calcium concentration
([Ca2+]i) in vascular smooth muscle
cells (VSMC) isolated from rat renal preglomerular arterioles.
[Ca2+]i was assessed using the
calcium-sensitive dye fura 2 and a microscope-based photometer system.
ANG II (107 M) caused a biphasic, time-dependent
[Ca2+]i response: an initial peak
increase from 52 ± 7 to 264 ± 25 nM, followed by a
sustained plateau of 95 ± 9 nM in cultured VSMC. Coadministration of PGE2 or PGI2 or synthetic
mimetics caused dose-dependent decreases in the peak
[Ca2+]i response to ANG II, with
attenuation of 40-50%. This degree of inhibition was even more
pronounced in individual freshly isolated preglomerular VSMC.
Increasing cAMP levels in cultured VSMC, by using either a
cell-permeable analog or inhibiting phosphodiesterase activity,
mirrored the antagonistic effects of prostaglandins on ANG
II-stimulated increases in [Ca2+]i.
Radioimmunoassays demonstrate that ANG II (10
7 M)
stimulates production of PGI2 and PGE2; the
stable prostacyclin metabolite 6-keto-PGF1
was released in 10-fold greater concentrations than PGE2.
Indomethacin blockade of prostaglandin production potentiated both the
peak (264 to 337 ± 26 nM) and sustained
[Ca2+]i responses (95 to 181 ± 22 nM) to ANG II. When prostaglandin analogs were added
during indomethacin treatment, the ANG II response was restored to the
typical pattern. In conclusion, we demonstrate that modulation of
intracellular calcium levels is one mechanism by which prostaglandins
can buffer ANG II-mediated constriction in renal preglomerular VSMC.
PGI2 is more potent than PGE2 in this regard.
renal circulation; afferent arteriole; vascular smooth muscle cells; fura 2; angiotensin II; prostaglandin I2; prostaglandin E2; iloprost; 15-(S)-15-methyl-prostaglandin E2; indomethacin; cyclooxygenase; adenosine 3',5'-cyclic monophosphate; phosphodiesterase
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INTRODUCTION |
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RESISTANCE IN THE RENAL PREGLOMERULAR vasculature influences glomerular filtration rate, sodium excretion, and renin release, which in turn affect blood volume and blood pressure. Angiotensin II (ANG II) is one of the primary mediators of vascular smooth muscle cell (VSMC) tone, with increases in cytosolic calcium concentration ([Ca2+]i) triggering contraction. The actions of ANG II are modulated by a variety of paracrine agents such as the prostaglandins PGE2 and PGI2, which are produced via cyclooxygenase metabolism of arachidonic acid. Prostaglandins are thought to be primarily derived from the neighboring endothelium (35), but there is growing evidence that VSMC can produce prostaglandins themselves (4, 34, 39).
In vivo studies have shown that these prostaglandins play a critical role in buffering ANG II-mediated constriction (19). Previous work from our laboratory examining renal blood flow in anesthetized rats demonstrated that cyclooxygenase blockade enhances reactivity to ANG II (1). Similar results have also been reported in microdissected afferent arterioles (19, 36). Cyclooxygenase blockade is reported to have no effect on afferent arteriolar diameter in the in vitro juxtamedullary nephron preparation (15). This variation may be due to differences in phenotype between superficial and juxtamedullary populations of nephrons or the ex vivo preparation. However, intrarenal infusion of prostaglandin analogs reduced the whole kidney vasoconstrictor effect of ANG II (6). We (8) and others (18) have shown that a defect in this prostanoid buffering system is responsible for the exaggerated renal vascular reactivity associated with development of hypertension in genetically susceptible rats.
The interaction of signaling cascades initiated by prostaglandin receptor activation and that by ANG II which result in this buffering effect have yet to be fully elucidated, particularly in VSMC of renal resistance arterioles. Earlier studies in this area have found that vasodilatory prostaglandins can affect cAMP levels in freshly isolated preglomerular vessels from the rabbit kidney as both PGE2 and PGI2 dose-dependently stimulate adenylate cyclase activity (9). Our laboratory has recently shown similar prostaglandin effects in freshly isolated preglomerular arterioles from rat kidneys, although strain differences were noted between spontaneously hypertensive rats and Wistar-Kyoto rats (27). Increased levels of cAMP are known to activate protein kinase A (PKA) and a number of downstream effector sites. It has been proposed that PKA can cause vasodilation via at least one of several mechanisms that range from changes in contractile sensitivity to interactions with calcium entry and mobilization pathways (10, 23, 24, 31). It is unclear which of these possible actions plays a role in vasodilation caused by prostaglandins in VSMC in general and resistance arterioles in particular.
The purpose of the present study was to determine whether altering [Ca2+]i is one mechanism by which PGE2 and PGI2 can modulate ANG II activity in renal preglomerular VSMC. The interaction of ANG II with exogenous and endogenous prostaglandins was assessed using the calcium-sensitive dye fura 2 and a microscope-based fluorescence photometer system. In addition, the effect of increasing intracellular levels of cAMP independent of receptor activation on ANG II stimulation was examined. Radioimmunoassays (RIAs) were used to quantify prostaglandin release from these arteriolar cells before and after ANG II stimulation.
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METHODS |
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Isolation of preglomerular resistance vessels. Experiments were performed on 200- to 300-g male Sprague-Dawley rats from our Chapel Hill breeding colony. To isolate VSMC from renal resistance vessels, we used a technique previously described by Zhu and Arendshorst (38) for the rat kidney. Sterile solutions and equipment were used throughout the procedure. Briefly, for each culture, three rats were anesthetized with pentobarbital sodium (60 mg/kg ip), and the abdominal aorta was cannulated below the renal arteries through a midline abdominal incision. The proximal aorta was compressed to halt blood flow, the left renal vein was cut, and the kidneys were perfused with ice-cold PBS (in mM: 17 K2HPO4, 3 Na2H2PO4, 125 NaCl, and 5 MgCl2, pH 7.4) until renal venous effluent was blood free. Thereafter, the kidneys were perfused with approximately 5 ml of a magnetized iron oxide suspension (1% Fe3O4 in PBS), excised, and placed in fresh cold PBS. Thereafter, the isolation procedure was carried out on ice and in a sterile tissue culture hood, unless otherwise noted. After decapsulation, the cortex was dissected from the medulla. The cortical tissue was placed on a glass petri dish, gently minced with a razor blade for 3 min, and then transferred to a beaker with 5 ml cold PBS. Renal vessels containing iron oxide as well as surrounding connective tissue were removed from the solution with a magnet. The crude homogenate was then resuspended in PBS, passed through needles of decreasing size (22- and 23-gauge), and filtered through a 120-µm sieve. The microvessels were recovered from the top of the sieve and then purified once more by magnetic separation. The final preparation was digested with collagenase (8 mg/10 ml, type 1A; Worthington Biochemical, Lakewood, NJ) for 30 min with constant shaking at 37°C. After collagenase digestion, the tube was shaken vigorously to disperse the cells and iron oxide. The remaining solution consisted of isolated VSMC and short pieces of vessels (17).
Culture of VSMC. The method used to culture renal arteriolar VSMC has been described by Zhu and Arendshorst (38). Cells of the digested microvessels were collected by brief centrifugation and washed once with PBS to remove collagenase. Next, the cells were suspended in 36 ml culture medium [RPMI 1640, supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 0.6 mM L-glutamine, and 10% fetal calf serum (Hyclone Laboratories, Salt Lake City, UT)]. The microvascular suspension was aliquoted into twelve 60-mm culture dishes and incubated at 37°C in 5% CO2-95% air at 98% humidity. The next day, the medium was changed and thereafter every 2 or 3 days until the cells became confluent. After approximately 3 wk in primary culture, the cells were passaged by harvesting with 0.05% trypsin and subpassaged every 7-10 days thereafter. The cells were seeded at a density of 3-5 × 103 cells/cm2. Monolayers were studied between passages 5 and 9.
Measurement of cytosolic free calcium concentration.
Measurements of [Ca2+]i in cultured
VSMC were performed using the acetoxymethyl ester of the
calcium-sensitive dye, fura 2 (fura 2-AM), as described previously for our laboratory (38). A monolayer of VSMC was grown on
22-mm2 glass coverslips in the same conditions as described
above. Confluent cells were subjected to serum-free medium 24 h before
an experiment. Prior to the study, the VSMC were washed twice with
physiological saline solution [PSS, containing (in mM) 135 NaCl,
5 KCl, 1 CaCl2, 1 MgCl2, 5 D-glucose, and 10 HEPES, pH 7.4] and incubated in the dark at room temperature with 2 µM fura 2-AM for 60 min. After loading, the cells were washed three times with PSS and allowed to sit
for 20 min. Immediately before testing, a coverslip was mounted in a
plastic chamber, creating a well for drug addition directly over the
center of the coverslip. The chamber was then centered in the field of
a ×40 oil-immersion fluorescence objective of an inverted
microscope (Olympus IX-70). Then cells were excited alternately with
light of 340- and 380-nm wavelength from dual monochrometers of a
Photon Technology International (PTI) dual-excitation wavelength
Deltascan (model RMD). Fluorescence was detected by a photometer after
passing through a barrier filter (510 nm). Fluorescence signal
intensity was acquired, stored, and processed by a Dell computer with
Felix software (PTI). After subtracting background readings,
[Ca2+]i was calculated based on the
ratio of 340/380 nm, according to the formula
[Ca2+]i = [(R Rmin)/(Rmax
R) × (Sf2/Sb2) × Kd], described by Grynkiewicz et al. (13),
using external calibration.
The effect of ANG II and its interactions with prostaglandin analogs
and antagonists on renal VSMC were evaluated in terms of changes in
[Ca2+]i. After a 50-s control
period, ANG II (107 M) was added, and the response
was measured for 200 s. Coadministration of stable synthetic analogs of
PGE2 [15-(S)-15-methyl-PGE2;
Cayman Chemical, Ann Arbor, MI] or PGI2 (iloprost;
Berlex Laboratories, Cedar Knolls, NJ) and ANG II was used to determine
the effects of exogenous prostaglandins on the calcium response to ANG
II. These mimetics were tested in varying concentrations:
15-(S)-15-methyl-PGE2 (10
11 to
10
5 M) and iloprost (10
11 to
10
7 M), all dissolved in PSS. The actions of
15-(S)-15-methyl-PGE2 were confirmed by
coadministration of native PGE2 (10
7 and
10
5 M). Conversely, the effect of endogenous
prostaglandins was assessed by pretreating cells for 15 min with the
cyclooxygenase inhibitor indomethacin (10
5 M). The
effect of exogenous prostaglandins, without interference from
endogenous compounds, was determined by coadministering the prostaglandin mimetics and ANG II on the background of indomethacin pretreatment. The effect of increasing intracellular levels of cAMP
during ANG II stimulation was assessed by coadministration of two
different compounds: 10
5 M dibutyryl-cAMP, a
cell-permeable cAMP analog, or 10
6 M milrinone, an
inhibitor of cAMP phosphodiesterase PDE-3B (Sigma Chemical, St. Louis,
MO). Each preparation was tested only once, to avoid possible receptor
desensitization or tachyphylaxis.
Preliminary studies were conducted on freshly isolated cells from preglomerular arterioles according to methodology previously described (12, 17) to determine whether the PGI2 analog iloprost influenced ANG II-induced increases in [Ca2+]i.
RIAs for PGE2 and 6-keto-PGF1. Cells
were grown to confluence in 12-well plates and rendered quiescent by
maintenance in serum-free medium 24 h before an experiment. On the day
of the study, cells were washed twice with PSS and incubated at
25°C with various drugs. Cells were pretreated with indomethacin or vehicle for 15 min and then exposed to varying concentrations of ANG II
for an additional 15 min. The culture supernatant was then removed and
stored at
80°C until analysis. Cells were assayed for
PGE2 and 6-keto-PGF1
(a stable metabolite of
PGI2) using RIA kits purchased from PerSeptive Biosystems
(Framingham, MA). Protein levels were determined using the Bradford
method after digestion of cells with 1 M NaOH, as described previously
(7).
Statistical analysis. Data are presented as means ± SE. Data sets were analyzed statistically with analysis of variance followed by post hoc testing according to Student-Newman-Keuls. P < 0.05 was considered statistically significant.
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RESULTS |
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The stimulatory effect of ANG II was assessed in preglomerular VSMC
using the calcium-sensitive dye fura 2. A representative tracing of the
ANG II effect is shown in Fig. 1. After
recording basal control levels for 50 s, ANG II (107
M) was added to the chamber enclosing the coverslip of cultured VSMC.
The raw data were recorded as photons emitted at 510 nm after
excitation from dual monochrometers at 340 and 380 nm.
These counts changed in an antiparallel fashion, as expected for
typical behavior of fura-2 in response to a change in
[Ca2+]i. The ratio of these counts
is shown in Fig. 1B, and the calculated [Ca2+]i is shown in Fig.
1C. ANG II stimulation resulted in an immediate peak increase
in [Ca2+]i lasting approximately
100 s, followed by a smaller, but sustained increase in
[Ca2+]i.
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To determine the effect of exogenous prostaglandins on ANG II
stimulation of [Ca2+]i,
biologically stable analogs of PGI2 (iloprost) and
PGE2
[15-(S)-15-methyl-PGE2] were used to
avoid the complications of rapid degradation of the native compounds.
The prostaglandin mimetics alone had an insignificant effect on the
baseline [Ca2+]i;
107 M iloprost caused an increase of 11 ± 4 nM
(P > 0.4), and 10
5 M
15-(S)-15-methyl-PGE2 caused an increase of 15 ± 8 nM (P > 0.6). On average, ANG II alone caused
[Ca2+]i to increase from a baseline
of 52 ± 7 nM to a peak of 264 ± 25 nM and a sustained level of 95 ± 9 nM at 240 s (Fig. 2A). When given concurrently with ANG II at 50 s, iloprost
(10
9 and 10
7 M) significantly
reduced the immediate peak calcium response. Blockade by
10
7 M iloprost is evidenced by an ANG II-induced
increase from a baseline of 59 ± 6 nM to a suppressed peak of 175 ± 19 nM (P < 0.001), with essentially no effect on the
sustained phase. Figure 2B shows the change in
[Ca2+]i calculated from the
difference between the initial baseline readings and the immediate peak
response. Iloprost dose-dependently attenuated the calcium response to
ANG II; the highest concentration of iloprost tested
(10
7 M) blocked ANG II stimulation by 50%. We did
not test iloprost at higher concentrations because of limitations in
the amount of the drug available.
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15-(S)-15-methyl-PGE2 was less potent than iloprost
when given simultaneously with ANG II at 50 s (Fig.
3A), although the qualitative
effects on the ANG II calcium response were similar to those observed
with iloprost. At 107 M,
15-(S)-15-methyl-PGE2 did not significantly affect
the peak response to ANG II. However, this PGE2 analog at
10
5 M effectively attenuated the peak ANG II
response to 191 ± 18 nM, a 42% change (P < 0.01).
15-(S)-15-methyl-PGE2 also decreased the peak
response to ANG II in a dose-dependent fashion while having little
effect on the flat plateau phase of the calcium response (Fig.
3B). To ensure that the PGE2 analog retains the specificity and potency of the native compound, the above experiments were repeated using native PGE2. The results obtained were
almost identical to those using the analog. At 10
7
M, native PGE2 did not significantly affect the peak
response to ANG II, whereas 10
5 M PGE2
attenuated the peak response to 176 ± 12 nM (P < 0.005). Thus, both native and synthetic PGE2 blunt the increase in
[Ca2+]i elicited by ANG II.
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Preliminary studies were conducted to verify that iloprost would
attenuate the calcium response to ANG II in freshly isolated preglomerular arteriolar VSMC in a manner similar to that observed in
the cultured preglomerular VSMC. In the individual fresh cells, ANG II
(107 M) caused an immediate peak increase in
[Ca2+]i from a baseline of 111 ± 9 to 202 ± 22 nM (n = 7). Iloprost (10
7 M)
attenuated this response by 83% (P < 0.01, n = 7).
Because prostaglandins are thought to exert their effects on VSMC via
adenylate cyclase stimulation, the effect of increasing intracellular
levels of cAMP was assessed during ANG II stimulation (Fig.
4). Two different approaches were used to
increase cytosolic cAMP: 1) 105 M
dibutyryl-cAMP, a cell-permeable cAMP analog, and 2)
10
6 M milrinone, an inhibitor of cAMP
phosphodiesterase PDE-3B. This enzyme has been shown to be the primary
PDE present in freshly isolated renal preglomerular VSMC (29).
Coadministration of dibutyryl-cAMP with ANG II resulted in a
significant attenuation of the peak
[Ca2+]i response, from a baseline
of 53 ± 4 to a peak of 177 ± 19 nM (P < 0.001). Similar
results were observed with coadministration of milrinone, increasing
from a similar baseline to a peak of 180 ± 9 nM
(P < 0.001). Both maneuvers reduced the peak response to ANG
II by ~50%. These results are not statistically different (P > 0.5) from those obtained by maximum attenuation with the exogenous
prostaglandin analogs noted earlier.
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To determine whether renal preglomerular VMSC, in the absence of
endothelial cells, could produce endogenous prostaglandins in response
to ANG II, we measured release of PGE2 and
6-keto-PGF1 (a stable metabolite of PGI2)
into the cell culture supernatant using RIAs. In the absence of ANG II,
VSMC produced 40 ± 10 pg 6-keto-PGF1
· 100 µg
protein
1 · 15 min
1 (Fig. 5A).
We could not detect basal release of PGE2 above the lower
limit of detection of the assay (10 pg · 100 µg
protein
1 · 15 min
1) (Fig. 5B). However, the addition of
increasing concentrations of ANG II (10
11 to
10
7 M) stimulated the cells dose dependently to
increase the synthesis of both prostaglandins. It is clear that
6-keto-PGF1
was produced at much greater amounts than
PGE2 during both basal conditions and in response to ANG II
challenge. At 10
9 M ANG II, cells released 92 ± 13 pg 6-keto-PGF1
· 100 µg protein
1 · 15 min
1 (P < 0.01), whereas PGE2
production remained at nondetectable levels. At 10
7
M ANG II, the concentration used in fura 2 studies, both prostaglandins were released in significantly greater amounts than baseline levels: 459 ± 40 pg · 100 µg
protein
1 · 15 min
1 for 6-keto-PGF1
(P < 0.001) and approximately 10 times less, 42 ± 8 pg · 100 µg
protein
1 · 15 min
1, for PGE2 (P < 0.01). To
ensure that the RIA was specific for cyclooxygenase products, the cells
were pretreated with the cyclooxygenase blocker indomethacin for 15 min
and then subjected to the same treatment. Indomethacin decreased
release of both prostaglandins to undetectable levels, even at the
highest concentration of ANG II.
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To determine the effect of endogenous prostaglandins on the calcium
response to ANG II, cultured VSMC were pretreated with indomethacin for
15 min before calcium determinations. Cyclooxygenase blockade resulted
in elevated baseline levels of
[Ca2+]i from 52 ± 7 to 85 ± 7 nM (Fig. 6), indicating that prostaglandins are produced and exert effects under basal conditions. Moreover, indomethacin potentiated ANG II effects on
[Ca2+]i, both in terms of the
immediate peak (from 264 ± 25 to 337 ± 26 nM) and the
sustained phase (from 95 ± 9 to 181 ± 22 nM). These observations
indicate that prostanoids synthesized by the cyclooxygenase pathway
blunt both the immediate and sustained phases of the calcium response
to ANG II stimulation.
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Other experiments were conducted to determine the effects of exogenous
prostanoids during cyclooxygenase inhibition. In these studies, VSMC
were pretreated with indomethacin for 15 min, and then exogenous
prostaglandins were administered at the maximal concentrations used
previously to block ANG II effects. When basal production of
prostaglandins was blocked, all groups exhibited an elevated baseline
[Ca2+]i of ~80 nM, an increase of
~25 nM above that in untreated VSMC (Fig.
7A). When added concurrently with
ANG II in the presence of indomethacin, iloprost
(107 M) caused a small decrease in the peak response
from 337 ± 26 to 296 ± 46 nM, which was not statistically
significant (P > 0.4), and a larger decrease in the sustained
response, from 181 ± 22 nM to 98 ± 10 nM (P < 0.005). Thus, iloprost treatment during indomethacin blockade results
in a calcium response similar to that observed during challenge with
ANG II alone in the presence of endogenous prostaglandins (Fig.
2A). Concurrent application of
15-(S)-15-methyl-PGE2 (10
5 M)
with ANG II at 50 s during indomethacin pretreatment produced similar
results (Fig. 7B). The peak calcium response was largely unaffected (352 ± 48 vs. 337 ± 26 nM; P > 0.7),
consistent with the less potent effects of the PGE2 analog
observed earlier. The sustained phase was significantly reduced by the
PGE2 mimetic from 181 ± 22 to 103 ± 16 nM (P < 0.01) at 240 s.
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DISCUSSION |
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We demonstrate for the first time that PGE2 and PGI2 and/or their synthetic analogs can modify the effect of ANG II on [Ca2+]i in cultured renal arteriolar VSMC. Preliminary data confirm these results for the PGI2 mimetic in freshly isolated renal arteriolar VSMC as well. These findings reveal a mechanism consistent with the long-standing observation that prostaglandins can buffer ANG II-mediated constriction of resistance vessels (19, 36). Addition of PGE2 or PGI2 analogs dose-dependently decrease the peak [Ca2+]i increase during ANG II stimulation. This was also the case when cytosolic cAMP was increased by dibutyryl-cAMP or the phosphodiesterase inhibitor milrinone. We also demonstrate that blockade of cyclooxygenase activity, and thus endogenous PGE2 and PGI2 production, potentiates the ANG II-induced calcium response. Furthermore, the typical response to ANG II was restored when exogenous prostaglandins were coadministered with ANG II during cyclooxygenase blockade. The present study also reveals that cultured rat preglomerular VSMC, in the absence of endothelial cells, can release PGE2 and a stable derivative of PGI2 in response to ANG II stimulation in a dose-dependent fashion.
A novel finding of our studies is the demonstration that PGE2 and PGI2 can significantly attenuate the calcium response to ANG II in renal VSMC. Animal studies indicate that prostaglandins can buffer ANG II-mediated constriction in many vascular beds and in many preparations, but the mechanisms by which this occurs at the cellular level have never been fully elucidated (2, 3, 6). To our knowledge, only one other study has specifically examined the effects of an arachidonic acid derivative on ANG II-mediated changes in intracellular calcium, and it dealt with lipoxygenase metabolites (28).
The physiological importance of the modulatory effect of prostaglandins on the microvasculature of the kidney is well known. For instance, patients with elevated ANG II activity experience acute renal failure when given nonsteroidal anti-inflammatory drugs that block cyclooxygenase, because the unopposed ANG II-mediated vasoconstriction severely reduces renal blood flow and glomerular filtration rate (25, 27). We (8) and others (18) have shown that a defect in this buffering system is associated with the development of hypertension in genetically susceptible rats.
Our observations indicate that several mechanisms are involved in receptor-mediated increases in [Ca2+]i and that prostaglandins appear to interact with them differently. ANG II receptor stimulation in the preglomerular VSMC results in a biphasic response consisting of an immediate peak increase in [Ca2+]i followed by a sustained increase of lesser magnitude. Prostaglandin analogs given in the presence of endogenous prostanoids predominantly attenuate the peak (Figs. 2A and 3A), whereas blockade of endogenous prostanoids potentiates both peak and plateau portions of the response (Fig. 6). This difference can be explained if distinct mechanisms are responsible for the increases in [Ca2+]i observed during peak and sustained phases. Exogenous prostaglandins can act additively with endogenous compounds to further suppress the peak response to ANG II. However, endogenous prostaglandins appear to be saturating the mechanism responsible for the sustained phase so that no additional buffering is observed when exogenous mimetics are added. This view is supported by evidence indicating that both calcium mobilization from intracellular stores and calcium entry participate in ANG II-mediated contraction in renal resistance vessels (26) and, more specifically, preglomerular VSMC (10, 16).
It has been generally thought that prostaglandins are primarily if not
exclusively synthesized by endothelial cells, with subsequent diffusion
to adjacent VSMC to exert their effect (22, 35). However, several
reports utilizing fresh cell preparations in various vascular beds
(e.g., pulmonary, coronary, and aortic vessels) suggest a synthetic
role for VSMC in addition to the endothelium (4, 34, 39). A recent
immunological study reports that cyclooxygenase-1 is expressed in
preglomerular vessels of human kidneys (22). A previous study of
cultured rabbit renal preglomerular VSMC reported synthesis of the
prostacyclin (PGI2) metabolite 6-keto-PGF1
(2 ng · mg
protein
1 · 15 min
1) and PGE2 (11 ng · mg
1 · 15 min
1) in the basal state 1(9). These results, however,
contrast with our data indicating that unstimulated cultured VSMC
produce 6-keto-PGF1
(0.4 ng · mg
1 · 15 min
1), whereas PGE2 production is below
the detection limits of the assay. A novel observation in the present
report is that ANG II stimulation significantly increases production of
both PGI2 (to 5 ng · mg
1 · 15 min
1) and PGE2 (to 0.4 ng · mg
1 · 15 min
1) in renal preglomerular VSMC. This is
consistent with earlier work showing that a freshly isolated membrane
preparation from rabbit preglomerular microvessels with an intact
endothelium produce three times as much prostacyclin metabolite
6-keto-PGF1
as PGE2 (20).
Previous studies on cultured VSMC from nonrenal vessels indicate
greater production of 6-keto-PGF1
compared with PGE2 during ANG II stimulation (5, 14). Thus, our data,
together with those of other investigators, suggest that
PGI2 is produced in greater abundance than PGE2
in renal preglomerular VSMC. One possible caveat is that RIA antibodies
may bind to metabolites from other arachidonic acid pathways. However,
because indomethacin completely abolished release of both prostanoid
compounds as measured by RIA, it is reasonable to conclude that the
antibodies are specific to cyclooxygenase products.
To minimize the potential influence of basal prostanoid activity in some of our calcium studies, indomethacin treatment was employed and exogenous prostaglandins were coadministered with ANG II. These experiments demonstrate that the indomethacin effect is reversed by coadministration of either prostanoid. This confirms that cyclooxygenase blockade with indomethacin is specific, in agreement with the elimination of prostaglandin release by indomethacin treatment in the RIAs. These data argue against the possibility that the observed effects are due to indomethacin rerouting arachidonic acid metabolism to alternative enzymatic pathways, thereby increasing the levels of an eicosanoid vasoconstrictor rather than decreasing levels of cyclooxygenase-derived vasodilators. Furthermore, these results exclude the involvement of the vasoconstrictor thromboxane, another cyclooxygenase product. Previous studies have shown that thromboxane analogs can increase [Ca2+]i and constrict aortic VSMC (11, 33).
The effects of endogenous prostaglandins and PGE2 and
PGI2 analogs on [Ca2+]i
during ANG II stimulation can be compared by subtracting the experimental from control curves obtained in our experiments (Figs. 6
and 7). This analysis, shown in Fig. 8,
effectively cancels the effect of ANG II on
[Ca2+]i, showing the change in
[Ca2+]i specifically elicited by
prostaglandins. The curve labeled endogenous prostaglandins (Fig.
8A) shows the difference between the data in Fig. 6 for ANG II
alone and ANG II with indomethacin. The remaining curves (Fig. 8,
B and C) represent the subtraction of exogenous
prostaglandin treatment from ANG II alone, both during cyclooxygenase
blockade (Fig. 7, A and B). Endogenous prostaglandins lower the baseline [Ca2+]i by ~40
nM (Fig. 8A). The baseline change is approximately zero in Fig.
8, B and C, however, because both curves within a group were treated with indomethacin, and thus the two curves
showed approximately the same baseline values for
[Ca2+]i. Both prostaglandin analogs
effectively restore the sustained [Ca2+]i response observed with
endogenous prostaglandin action. With all interventions,
[Ca2+]i during the sustained phase
is reduced by approximately 80 nM from basal levels. However, the
kinetics of the response differ. The effect of iloprost is similar to
that of endogenous prostaglandins, although a bit slower; it takes
about 50 s longer for iloprost to exert a maximum effect in reducing
[Ca2+]i.
15-(S)-15-methyl-PGE2 actually shows some
vasoconstrictor-like activity by initially increasing
[Ca2+]i and then reducing it to an
apparent maximum of 80 nM. This phenomena could be explained by the
expression of multiple PGE2 receptor subtypes in the renal
preglomerular VSMC. Some subtypes (EP1, EP3)
are known to mediate vasoconstriction, whereas others (EP2,
EP4) are thought to elicit vasodilation (32). Only one receptor subtype has been identified for PGI2, the IP
receptor. The calcium data discussed above, together with greater
release of 6-keto-PGF1 and greater potency
of iloprost in the earlier calcium experiments, suggest that
prostacyclin is the primary vasodilator cyclooxygenase
product involved in the ANG II response in renal preglomerular VSMC.
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The mechanisms by which prostaglandins affect [Ca2+]i are incompletely understood. PGE2 and PGI2 are known to activate adenylate cyclase and to increase intracellular cAMP levels in several different vascular and epithelial cells, including renal preglomerular VSMC preparations (9, 27). In the current preparation, we have shown that increasing intracellular cAMP levels attenuates the peak [Ca2+]i response during ANG II stimulation (Fig. 4). The magnitude of this effect is similar to that observed with addition of exogenous prostaglandins (Fig. 2). These results are consistent with the hypothesis that prostaglandins can exert their vasodilatory actions via adenylate cyclase stimulation.
cAMP is thought to activate PKA to carry out the intracellular effects of prostaglandins. However, the relationship between increased PKA activity and changes in [Ca2+]i remains imprecise. One view is that PKA phosphorylates and thereby increases the activity of calcium ATPases that remove calcium from the cytoplasm. Accumulating evidence suggests that the primary effect of PKA is on a calcium-ATPase in the sarcoplasmic reticulum to increase calcium reuptake (23). PKA also has been reported to phosphorylate the IP3 receptor, thereby reducing its efficiency to release calcium from intracellular stores (31). It is also possible that PKA can affect calcium entry and/or the sensitivity of the contractile apparatus. Indirect support for this view comes from studies in which iloprost inhibits KCl-induced contraction to a greater degree than it reduces [Ca2+]i (24). Several reports indicate that prostaglandins can act through PKA to modulate potassium channel activity (10, 30).
In conclusion, both PGI2 and PGE2 effectively attenuate the stimulation of [Ca2+]i by ANG II. This was the case for cultured VSMC from rat preglomerular arterioles before and during inhibition of cyclooxygenase and production of endogenous prostanoids. Prostacyclin also blocks ANG II-induced stimulation of [Ca2+]i in individual freshly isolated renal arteriolar VSMC. Our calcium results demonstrate that the buffering effect of prostacyclin is closely mimicked by endogenous prostanoids. The counteracting effects of endogenous cyclooxygenase products as well as PGI2 and PGE2 analogs are most likely mediated by cAMP. Cell-permeable cAMP and increases in endogenous cAMP associated with phosphodiesterase inhibition by milrinone produced effects similar to those of the prostanoid analogs. The RIA data indicate that cultured preglomerular VSMC release more PGI2 than PGE2 in response to ANG II. Thus, it is tempting to speculate that prostaglandins may act in an autacrine/paracrine manner in the afferent arteriole. This work demonstrates that modulation of [Ca2+]i is one mechanism by which prostaglandins may attenuate ANG II-mediated constriction in the renal microcirculation.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Susan K. Fellner for conducting the preliminary studies on freshly isolated renal arteriolar VSMC. Iloprost was a gracious gift of Berlex Laboratories.
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FOOTNOTES |
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We appreciate the assistance of Julie Vorobiov on prostaglandin assays performed at a core facility supported by National Institutes of Health Grant P30-DK-34987. This research was supported by National Heart, Lung, and Blood Institute Grant HL-02334. K. E. Purdy was supported by a Howard Hughes Predoctoral Fellowship.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: W. J. Arendshorst, Department of Cell and Molecular Physiology, Room 152, Medical Sciences Research Building, CB 7545, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545 (E-mail: arends{at}med.unc.edu).
Received 21 December 1998; accepted in final form 9 July 1999.
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