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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Vasodilator prostaglandin PGE2 protects the kidney from excessive vasoconstriction during contraction of extracellular fluid volume and pathophysiological states. However, it is not yet clear which of the four known E-prostanoid (EP) receptors is localized to resistance vessels and mediates net vasodilation. In the present study, we assessed the presence, signal transduction, and actions of EP receptor subtypes in preglomerular arterioles of Sprague-Dawley rat kidneys. RNA encoding EP1, an EP1-variant, and EP4 receptors was identified by RT-PCR in freshly isolated preglomerular microvessels; cultured preglomerular vascular smooth muscle cells (VSMC) had EP1-variant and EP4 RNA but lacked EP1. EP2 and EP3 receptors were undetectable in both vascular preparations. In studies of cell signaling, stimulation of cAMP by various receptor agonists is consistent with primary actions of PGE2 on the EP4 receptor, with no inhibition of cAMP by EP1 receptors. Studies of cytosolic calcium concentration in cultured renal VSMC support an inhibitory role of EP4 during ANG II stimulation. In vivo renal blood flow (RBF) studies indicate that the EP4 receptor is the primary receptor mediating sustained renal vasodilation produced by PGE2, whereas the EP1 receptor elicits transient vasoconstriction. The EP1-variant receptor does not appear to possess any cAMP or cytosolic calcium signaling capable of affecting RBF. Collectively, these studies demonstrate that the EP4 receptor is the major receptor in preglomerular VSMC. EP4 mediates PGE2-induced vasodilation in the rat kidney and signals through Gs proteins to stimulate cAMP and inhibit cytosolic calcium concentration.
kidney; renal circulation; afferent arteriole; vascular smooth muscle cells; prostanoids; hemodynamics; hypertension; receptor subtypes; cyclic adenosine 3,5,-monophosphate; cytosolic calcium
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INTRODUCTION |
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THE PARACRINE AGENT PGE2 is the predominant cyclooxygenase metabolite of arachidonic acid in the kidney (9). PGE2 plays an important role in tubular reabsorption of salt and water as well as in the control of renal vascular resistance and the maintenance of glomerular hemodynamics. Vasoconstrictor hormones such as ANG II and norepinephrine, which are released during periods of effective volume depletion, stimulate PGE2 production in many vascular beds, including the kidney. Despite several reports of PGE2-induced vasoconstriction (5, 20), there is convincing evidence that the primary action of PGE2 is to counteract the effects of these pressor hormones and protect the kidney from excessive vasoconstriction. Injection of small amounts of PGE2 directly into the renal artery causes vasodilation in vivo (12, 22), and infusion of PGE2 into the renal artery effectively attenuates hormone-induced vasoconstriction (11, 25). Conversely, blockade of prostaglandin production enhances vasoconstrictor activity (1, 17). PGE2 appears to act primarily on the preglomerular vasculature to decrease vascular resistance (2, 18). It has been shown to buffer ANG II-induced constriction in isolated renal arterioles (16, 38) and buffer ANG II-induced increases in cytosolic calcium in vascular smooth muscle cells (VSMC) isolated from the preglomerular vasculature (32). Little is known about the receptor subtype initiating and cellular signaling events underlying PGE2 action in the renal vasculature.
In general, the actions of PGE2 are mediated by a family of G protein-coupled receptors. Four E-prostanoid (EP) receptors, termed EP1 through EP4, were originally identified pharmacologically and then further characterized by cloning (14, 30, 31, 37). Transfection of recombinant receptors into host cells, such as Chinese hamster ovary or human embryonic kidney cells, has revealed that each receptor couples to a different signal transduction pathway. In these noncontractile cells, the EP1 receptor activates Gq proteins to elevate intracellular calcium (31); EP2 and EP4 stimulate cAMP production via Gs (8, 30); and EP3 inhibits cAMP formation via Gi (8). Alternatively, spliced isoforms have been identified for both EP1 and EP3 that may couple to various signaling pathways. Whether these same second messenger systems are coupled to their respective EP receptor subtypes in natural cells remains to be established.
Although the role of individual EP receptors in the renal vasculature is not known, expression and function of EP receptor subtypes in the distal nephron have been characterized. The EP1 receptor, detected in the collecting duct of humans (10, 28) and mice (35), is reported to mediate PGE2 inhibition of sodium reabsorption (19). EP3 is present in the thick ascending limb and collecting duct of humans (10, 28), mice (29), and rats (36) and also appears to inhibit salt and water reabsorption.
Recent studies of EP receptor distribution in rat renal cells other than epithelial have focused on mRNA expression in the glomerulus, more than on renal arteries and arterioles. Ribonuclease protection assays demonstrated predominant expression of EP4 in rat glomeruli with lower levels of EP1 and EP3 (23). In cell culture models, gene expression of EP1 and EP4 receptors is noted in rat mesangial cells (21) and podocytes (7). Little is known about the function and cellular actions of EP receptor types in resistance vessels in general and the renal microcirculation in particular. Whether findings for glomeruli and cultured mesangial cells correlate with message, protein expression, and functional effects in the preglomerular arterioles is not known.
The purpose of the present study was to determine which EP receptors are expressed in preglomerular resistance vessels and, of these, which subtypes act to mediate vascular actions of PGE2. RT-PCR was used to identify the presence of receptor mRNA in both freshly isolated preglomerular microvessels and cultured preglomerular VSMC. Cell signaling via EP receptors was assessed in vitro by measuring cAMP production using radioimmunoassay and by measuring changes in cytosolic calcium concentration using the fluorescent dye fura 2. In vivo renal blood flow (RBF) studies evaluated renal vascular reactivity to PGE2 injected into the renal artery. The contribution of the different EP receptors in renal resistance vessels was further defined by using pharmacological receptor agonists and antagonists in cell signaling and RBF experiments.
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METHODS |
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Isolation of preglomerular resistance vessels. Renal preglomerular arterioles were isolated from kidneys of 6-wk-old male Sprague-Dawley rats from our Chapel Hill breeding colony (originally derived from Harlan, Indianapolis, IN) by use of previously established methodology (32, 40). Approximately three rats were used for each preparation of isolated VSMC. Sterile solutions and equipment were used throughout. Briefly, the kidneys were infused with a magnetized iron oxide suspension (1% Fe3O4 in phosphate-buffered saline), and the preglomerular vessels were separated from the rest of the cortex with the aid of a magnet, sequential sieving, and collagenase treatment. All animal protocols were performed in accordance with the University of North Carolina at Chapel Hill institutional guidelines (IACUC approval nos. 96-07-0 and 99-030-0).
Culture of VSMC. The method used to culture renal arteriolar VSMC has been described by our laboratory previously (32, 40). Cells of the digested microvessels were collected after brief centrifugation and suspended in 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 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.
RT-PCR of EP receptor subtypes. Total RNA was isolated from cultured preglomerular VSMC, freshly isolated preglomerular microvessels, whole kidney, and lung using RNA STAT-60 (Tel-Test) and treated with DNase (Life Technologies) to eliminate genomic DNA contamination. cDNA was synthesized by Superscript II RT (Life Technologies) with either random hexamer or oligo(dT) primers. PCR was performed by using Taq DNA polymerase (Boehringer Mannheim) on both cDNA and RNA with subtype-specific gene primers. The specificity of the primers was verified by using positive control clones for each subtype. EP1 and EP4 full-length clones were gifts from Emiko Okuda Ashitaka (Kansai Medical Univ.) and William Smith (Michigan State Univ.), respectively. EP2 and EP3 clones were obtained by designing flanking primers, performing PCR on cDNA from lung and kidney, cloning the amplified fragments by using the pGEM-T Easy Vector System (Promega), and sequencing the ligated products. Primers for the housekeeping gene cyclophilin were used to verify the quality of the cDNA samples (15). To ensure the removal of genomic DNA, a separate RNA sample was treated in the same way, except for the addition of RT. PCR assays were run in triplicate, and all PCR products were found to be the predicted size and directly sequenced to ensure their expected identity.
Determination of cAMP content. Freshly isolated preglomerular arterioles were prepared as described above, and cAMP generation was determined by using standard methodology. Briefly, a portion of the cell suspension was incubated with an equal volume of buffer A [(in mM) 50 HEPES, 8 MgCl2, IBMX, 2 ATP, 4 GTP, and 60 phosphocreatine as well as 800 µg/ml creatine phosphokinase] and buffer B [(in mM) 50 HEPES, 1 EDTA, 1 dithiothreitol, and 0.25 sucrose as well as 1 mg/ml BSA] to give a final concentration of 50 µg protein/ml. This mixture was incubated for 15 min at 37°C with ANG II (Sigma), PGE2 (Cayman Chemical), isoproterenol (Sigma), or various EP receptor agonists [sulprostone (Cayman Chemical), 17-phenyl-PGE2 (Cayman Chemical), butaprost (Bayer), misoprostol (Biomol), and M&B28767 (Rhone-Poulenc Rorer)]. In another set of experiments, fresh VSMC were pretreated with EP receptor antagonists [SC-19220 (Searle), AH-23848 (Glaxo-Wellcome), or AH-6809 (Biomol)] for 5 min before PGE2 stimulation for 15 min. The reaction was stopped by adding TCA to produce a 6% final concentration. The samples were immediately put on ice and sonicated for 1 min. The cell lysate was extracted four times with 1 ml of water-saturated ether and evaporated. Aliquots were acetylated, and cAMP was measured by radioimmunoassay (Biomedical Technologies).
Measurement of cytosolic free calcium concentration. Cytosolic free calcium concentration ([Ca2+]i ) was measured in cultured VSMC of preglomerular arterioles by using the calcium-sensitive dye fura 2-acetoxymethyl ester (AM) as described previously (32, 40). Confluent VSMC grown on glass coverslips were subjected to serum-free medium 24 h before an experiment. Before the study, monolayers of VSMC were washed twice with Hanks' balanced salt solution [HBSS; (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 fura loading, the VSMC were washed three times with HBSS and allowed to stabilize for 20 min. Cells were excited alternately with light of 340- and 380-nm wavelength from dual monochrometers of a Photon Technology International dual-excitation wavelength Deltascan (model RMD) interfaced with an inverted microscope (Olympus IX-70). After passing through a barrier filter (510 nm), fluorescence was detected by a photometer. Fluorescence signal intensity was acquired, stored, and processed by a Pentium II computer and Felix software (Photon Technology International). Each preparation was tested only once, to avoid possible receptor desensitization or tachyphylaxis.
RBF studies. Experiments were performed on 5- to 6-wk-old Sprague-Dawley rats derived from our Chapel Hill breeding colony by using standard methods for our laboratory (13). Anesthesia was induced by an intraperitoneal injection of pentobarbital sodium (65 mg/kg body wt), and the animals were placed on a servo-controlled heating table that maintained body temperature at 37°C. A tracheostomy was performed to facilitate free breathing. Carotid arterial pressure was monitored via an indwelling PE-50 catheter connected to a Statham P23 Db transducer and a Hewlett-Packard oscillographic recorder interfaced to an IBM-clone Pentium II computer. Maintenance infusions of isooncotic albumin were made via a jugular vein. To measure RBF, a noncannulating electromagnetic flow probe (1.5-mm circumference, Carolina Medical) was placed around the left renal artery. A catheter inserted in a femoral artery was advanced into the abdominal aorta, and the tip was positioned at the origin of the renal artery for continuous infusion of heparinized isotonic saline (5 µl/min) and intrarenal injection of vasoactive agents. Before injection of EP agonists, the rate of saline infusion was increased to 120 µl/min; 60 s later a 10-µl bolus of the particular agent was injected into the catheter positioned in the renal artery. In some experiments, the EP1 blocker SC-19220 was infused into the artery at 120 µl/min for 2 min before injection of the PGE2 bolus. After completion of the surgical preparation, the animals were allowed to stabilize for 30 min before the measurements were started. The RBF and mean arterial pressure values were normalized and expressed as a percentage of baseline values, determined separately for each injection. Mean arterial pressure was constant during RBF responses to each injection. Plots of normalized RBF as a function of time were prepared by using the SigmaPlot software package (SPSS, Chicago, IL).
Statistics. Data are presented as means ± SE. Data sets concerning signal transduction were analyzed by analysis of variance followed by post hoc testing according to Student-Newman-Keuls. Maximum RBF responses were analyzed by Student's t-test. P values <0.05 are considered statistically significant.
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RESULTS |
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RT-PCR was used to evaluate gene expression of EP receptor
subtypes in freshly isolated preglomerular arterioles and cultured preglomerular VSMC preparations. As is shown in Fig.
1A, signals indicating the
presence of mRNA encoding EP1 and EP4 were
detected in both fresh and cultured preparations by identity with the
predicted product sizes of 415 and 318 bp, respectively. It should be
noted that the primer used to identify EP1 mRNA does not
differentiate between the two known splice variants of the
EP1 receptor. PCR products were directly sequenced and
found to be identical to reported cDNA sequences for EP1
and EP4. Although we were able to amplify cyclophilin
fragments from the same cDNA, expression of EP2 or
EP3 message was undetectable in both fresh and cultured renal VSMC (Fig. 1B). It is unlikely that this finding of
receptor absence is due to an error in primer design because
subtype-specific primers produced fragments of the predicted size using
cDNA samples from positive control clones as well as from tissues known
to strongly express each subtype, lung for EP2 and kidney
for EP3 (30, 36). The absence of bands in the
RNA lanes indicates the elimination of genomic DNA contamination.
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To assess the expression of the vasoconstrictor EP1 receptor in more detail, another set of PCR primers was designed to distinguish between the EP1 receptor and its variant, nonsignaling form. The EP1-variant consists of the first 951 bp of EP1, with an alternative sequence in place of the seventh transmembrane section containing a stop codon; the remaining sequence is common to both receptors. We designed the 5' primer to anneal within the first common region and the 3' primer to anneal to the terminal common sequence so that amplification of EP1-variant cDNA would produce a longer PCR fragment (730 bp) than native EP1 (360 bp). Figure 1C shows detection of amplification fragments indicative of the presence of the EP1-variant in both cultured and fresh preparations, as well as in whole kidney samples. However, the presence of signal for EP1 receptor mRNA differed in the two preparations. Whereas EP1 expression was evident as a faint band in freshly isolated preglomerular microvessels, no signal was detected in cultured VSMC. A stronger band for EP1 was clearly observed in cDNA samples derived from whole kidney tissue. As before, the identity of these amplification fragments was confirmed by direct sequencing.
To examine the functional coupling of EP1 and
EP4 receptors to Gi and or Gs
proteins in freshly isolated preglomerular microvessels, cAMP levels
were measured after stimulation of EP receptors by relatively
subtype-specific agonists. Baseline production of cAMP by renal
microvessels of Sprague-Dawley rats was 6.8 ± 2.5 pmol cAMP · 50 mg
protein1 · ml
1 · 15 min
1, consistent with an earlier report in preglomerular
VSMC isolated from Wistar-Kyoto and spontaneously hypertensive rat
strains (33). All EP receptor agonists were tested at
three concentrations; results for the highest concentration
(10
5 M) are presented in Fig.
2. Sulprostone and
17-phenyl-PGE2, both putative EP1 and
EP3 agonists, had no effect on cAMP. Because the
EP1 receptor is thought to signal via inositol
1,4,5-triphosphate and
[Ca2+]i, the lack of a change
in cAMP after EP1 stimulation is not unexpected.
Interpretation of results with EP3 stimulation is difficult
because this receptor subtype can act via either activating or
inhibiting adenylate cyclase. Butaprost, which is predicted to
stimulate the EP2 receptor and presumably increase cAMP
formation, had no effect on cAMP in our VSMC, suggesting a paucity of
functional EP2 receptor. These data confirm our RT-PCR
findings of the absence of EP2 mRNA transcripts. Addition
of the nonselective EP2-4 stimulator misoprostol enhanced
cAMP production by 4.3 ± 0.8 pmol cAMP · 50 mg
protein
1 · ml
1 · 15 min
1 (P < 0.05), a stimulatory effect
consistent with our earlier observation of EP4 mRNA
expression. The EP3 agonist M&B28767 had no effect on cAMP.
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In another set of experiments on the renal microvessels, cAMP
production was initially stimulated by isoproterenol in an attempt to
unmask any possible inhibition mediated by EP3 receptors.
Isoproterenol elevated cAMP levels to 20.1 ± 1.8 pmol
cAMP · 50 mg
protein1 · ml
1 · 15 min
1 (P < 0.01). Coadministration of
misoprostol further stimulated cAMP production by 8.5 ± 2.8 pmol
cAMP · 50 mg
protein
1 · ml
1 · 15 min
1 (P < 0.05), whereas M&B28767 had no
effect (Fig. 2B). These results are consistent with the
notion that there is no EP3 receptor coupling to
Gi proteins in our VSMC.
To evaluate the effect of available subtype-specific antagonists on
PGE2-induced increases in cAMP, cells were pretreated with
blockers for 5 min before PGE2 stimulation. As is shown in Fig. 2C, PGE2 alone produced 23.9 ± 1.2 pmol cAMP · 50 mg
protein1 · ml
1 · 15 min
1 (P < 0.001). The EP4
antagonist AH-23848 dose dependently inhibited the ability of
PGE2 to stimulate cAMP production. The highest AH-23848
dose tested reduced cAMP formation by 9.2 ± 1.1 pmol cAMP · 50 mg
protein
1 · ml
1 · 15 min
1 (P < 0.02). The EP1
antagonists were predicted to have no effect on
PGE2-induced cAMP production due to presumed coupling to
Gq proteins. Although this was clearly the case for
SC-19220, AH-6809 (10
5 M) attenuated
PGE2-induced cAMP levels by 7.1 ± 2.9 pmol
cAMP · 50 mg
protein
1 · ml
1 · 15 min
1 (P < 0.03), suggesting AH-6809 may
not be as specific for EP1 as SC-19220.
A separate series of experiments evaluated the influence of EP receptor
activation on another second messenger system,
[Ca2+]i, in cultured preglomerular VSMC.
Pharmacological agents were initially administered alone to test for
possible effects on baseline values. Subsequently, ANG II was added to
determine the effect of these EP agents on vasoconstrictor-mediated
calcium signaling. In an earlier study, we found that PGE2
itself has no effect on basal or unstimulated
[Ca2+]i (32). Misoprostol did
not alter baseline [Ca2+]i but did dose
dependently inhibit the [Ca2+]i response to
ANG II (Fig. 3A). Maximal
inhibition was reached at 105 M misoprostol, attenuating
the peak [Ca2+]i response to ANG II from
245 ± 14.6 to 129 ± 8.8 nM (P < 0.002). Neither sulprostone nor 17-phenyl-PGE2 altered baseline
[Ca2+]i or the ANG II-induced
[Ca2+]i response (Fig. 3B), as
would be expected with the lack of expression of EP1 mRNA
in cultured VSMC. Butaprost and M&B28767 were also without effect
on calcium signaling (data not shown). These calcium data confirm the
absence of EP2 and EP3 receptors in renal VSMC. Without EP2 and EP3 present, it is reasonable
to conclude that misoprostol acts through the EP4 receptor
to antagonize the actions of ANG II.
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In other studies we assessed the effect of EP-receptor antagonists on
ANG II-induced calcium responses. Figure
4A shows that the
EP4-receptor antagonist AH-23848 dose dependently enhanced the peak level of calcium stimulation by ANG II. At the highest concentration of the EP4-receptor antagonist
(105 M), the response nearly doubled to 462 ± 30.5 nM (P < 0.002). AH-23848 did not alter baseline
[Ca2+]i when administered in the absence of
ANG II, indicating low levels of EP4 receptor stimulation
during basal conditions. Figure 4B reveals that the
EP1 antagonists AH-6809 and SC-19220 affected neither
baseline [Ca2+]i nor ANG II-induced calcium
signaling, findings that are consistent with the lack of PCR evidence
for EP1 in cultured preglomerular VSMC.
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To verify the functional expression of EP receptor subtypes in renal
resistance arterioles in vivo, we examined vascular reactivity by
measuring changes in RBF after administration of PGE2 and
subtype-specific agents into the renal artery. Basal values for RBF and
mean arterial pressure averaged 5.8 ± 0.6 ml/min and 112 ± 5 mmHg (n = 15), respectively. A bolus of the
EP1-receptor agonist sulprostone (200 ng) caused transient
renal vasoconstriction, reducing RBF to 91.9 ± 1.2% of control
flow (P < 0.001) (Fig.
5A), without affecting
systemic arterial pressure. In contrast, misoprostol (200 ng) elicited
a longer lasting increase in RBF to 106 ± 0.8% of control flow
(P < 0.001), indicating renal vasodilation in the
absence of a change in arterial pressure (Fig. 5B). To
determine whether the EP1 subtype expressed in fresh renal
preglomerular VSMC is functional in vivo, rats were challenged with
PGE2. Bolus injection of PGE2 (200 ng) into the
renal artery produced a biphasic response, characterized by immediate,
short-lived constriction followed by a more sustained phase of dilation
(Fig. 5C). During the immediate transient constriction, RBF
fell to 85.3 ± 4.2% of control (P < 0.02),
followed by a longer lasting dilatory phase in which RBF rose to a peak
of 110.8 ± 0.9% of baseline (P < 0.001). The
constrictor period of ~15 s, contrasted with the prolonged dilatory
phase, returned to control levels after >5 min. It is noteworthy that
the initial constrictor response was completely abolished by 2-min
pretreatment with the EP1 blocker SC-19220. Selectivity was
indicated by the lack of change in the sustained, elevated phase during
SC-19220 administration.
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DISCUSSION |
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We present new evidence that preglomerular resistance vessels of the rat kidney express two of the four known EP receptors, with EP1 causing transient vasoconstriction and EP4 eliciting long-lasting vasodilation. As PGE2 produces net vasodilation, EP4 is the functionally predominant receptor. Detection of the EP4 receptor provides a mechanism for the long-standing observation that prostaglandins can effectively buffer the actions of vasoconstrictors in the renal microvasculature and thereby protect kidney function (11, 25). EP1 receptor expression is evident pharmacologically at the whole kidney level in vivo, and message is localized by RT-PCR to the preglomerular resistance vessels. Also present in freshly isolated microvessels is mRNA encoding a nonsignaling isoform, termed the EP1-variant. Interestingly, EP1 receptors are absent in cell culture, whereas the EP1-variant and EP4 receptors are retained. These observations provide a cellular explanation for previous observations of PGE2-induced renal vasodilation as well as highlight the complexities of prostaglandin signaling in a critical vascular bed.
In previous studies, PGE2 receptors have been identified in preglomerular vessels by radioligand binding, and PGE2 produces an increase in cAMP in freshly isolated VSMC, suggesting a dominance of vasodilator EP receptors coupled to Gs proteins (33). The present study identified a biphasic RBF response to injection of PGE2 into the renal artery: an initial, transient vasoconstriction, followed by a much more pronounced, sustained vasodilation. On the basis of earlier work in nonvascular cell types and transfection studies in host cells, one might predict that vasodilatory signals are transduced by either EP2 or EP4 receptors, both of which are thought to stimulate adenylate cyclase.
Along these lines, our studies of second messenger signal transduction indicate the presence of EP4 and absence of EP2 receptors in preglomerular vessels. The EP2 agonist butaprost had no effect on basal cAMP levels, baseline [Ca2+]i, or ANG II-induced calcium signaling. On the other hand, the EP2,3,4 agonist misoprostol significantly increased cAMP, both from baseline and when previously stimulated by isoproterenol. With regard to the limited specificity of misoprostol, it is important to note that activation of an EP3 receptor, if present, might be predicted to decrease, rather than increase, cAMP levels. In other signaling experiments, misoprostol inhibited PGE2-elicited cAMP production and attenuated ANG II-induced increases in [Ca2+]i . ANG II is known to enhance prostanoid production by preglomerular VSMC, and cAMP has been shown to counteract ANG II-elicited increases in [Ca2+]i (32). In this regard, we found in the present study that the EP4 antagonist AH-23848 markedly enhanced the calcium response to ANG II. These findings are consistent with ANG II stimulation of PGE2 production and PGE2 action on EP4 receptors to inhibit [Ca2+]i via cAMP.
Further support for PGE2-induced renal vasodilation mediated by EP4 derives from the demonstration that misoprostol increased RBF by reducing renal arteriolar resistance in acute animal experiments. In other RBF studies we tested the efficacy of AH-23848 to block PGE2 actions on EP4 receptors. The preliminary results, however, were inconclusive, in large part because of the very high concentration of drug required to antagonize PGE2 binding to EP4 receptors (inhibition constant difference of 10,000-fold) (8) and limited availability of the compound. One can speculate at this juncture that the weak affinity of AH-23848 has less of an influence in vitro because of the drug's longer access time in isolated microvessels and VSMC. Nevertheless, our RT-PCR experiments confirm the predominance of EP4 in PGE2-induced vasodilation by indicating the presence of RNA for EP4 and the absence of message for EP2 in both fresh and cultured preglomerular preparations. Taken together, these comprehensive data extend to resistance arterioles the suggestive pharmacological evidence of a dilator effect of EP4 in isolated large vessels of other vascular beds (4, 27).
The presence and functional expression of EP4 in rat arterioles elucidate the mechanisms underlying the well-known vasodilatory actions of PGE2. In the renal microcirculation, it has been clear for many years that PGE2 can oppose constriction in preglomerular vessels (6, 11, 22, 25). The activation of EP4 by PGE2 is crucial to maintain RBF when vasoconstrictor systems are active as, for example, during extracellular fluid volume contraction and in disease states. In regard to experimental models, previous investigations have established that an imbalance between vasodilatory and vasoconstrictor systems may contribute to the development of hypertension. In young spontaneously hypertensive rats, the dysfunction appears to arise in the interaction between a vasodilatory prostaglandin receptor and the Gs protein that activates adenylate cyclase (32). On the basis of the present results, it is reasonable to predict that this defect lies in the coupling of the EP4 receptor to Gs proteins and the cAMP pathway.
The expression of vasoconstrictor EP1 receptors was unexpected in view of the noted predominant vasodilatory actions of PGE2. Nevertheless, message for this subtype has been reported in isolated glomeruli (23) and cultured mesangial cells (21). Our RBF studies establish functional expression of the vasoconstrictor EP1 subtype in the microcirculation in vivo. The immediate transient decrease in RBF caused by PGE2 injection into the renal artery is fully abolished by the EP1 antagonist SC-19220 while having little, if any, effect on the more pronounced, sustained vasodilation. Moreover, the EP1,3 agonist sulprostone elicits renal vasoconstriction in our Sprague-Dawley rats. The fact that sulprostone does not alter cAMP in our VSMC preparation supports the notion that its actions are mediated by EP1, rather than EP3, in vivo. Interestingly, previous studies of the effect of PGE2 on RBF in our laboratory demonstrated a vasodilator effect only, suggesting there may be strain differences in EP receptor subtype expression (12).
Of particular interest are the changes in EP1 expression in renal VSMC maintained in cell culture. Sulprostone as well as the EP1 antagonists AH-6809 and SC-19220 had no effect on the putative EP1 signaling pathway, cytosolic calcium, in cultured VSMC. This loss of expression during primary cell culture is most clearly evidenced by the detection of EP1 message in freshly isolated cells and its absence in cultured cells. A similar loss of EP receptors in culture has been reported in rabbit cortical collecting tubule cells, evidenced by a decrease in PGE2 binding and a loss of inhibition of cAMP formation (34). The altered distribution of EP receptor expression in cultured VSMC may impact future studies of prostaglandin action. Although global PGE2 function cannot be assessed in cultured preparations, the cultured VSMC could facilitate the study of EP4 actions without confounding interactions with EP1 signaling.
The overall modulatory influence of PGE2-induced vasoconstriction via EP receptors is not clear. Audoly and colleagues (3) found that, compared with male wild-type control animals, systemic administration of PGE2 lowered arterial pressure less in mice in which the coding sequence common to EP1 and EP1-variant receptors was disrupted by gene targeting. Thus the enhanced depressor response in wild-type mice presumably reflects an unexpected contribution of EP1 receptors to apparent vasodilation, although cardiac effects cannot be excluded. Our RBF data clearly demonstrate that the EP1 receptor mediates vasoconstriction in the renal vasculature of Sprague-Dawley rats. This divergence in EP1 function may be explained by heterogeneous effects of EP receptor activation in various vascular beds or species differences in receptor signaling. Nevertheless, it is important to appreciate that renal vasoconstriction mediated by EP1 is transient and plays a weaker role compared with that of the prolonged vasodilation mediated by EP4. Perhaps the expression of this vasoconstrictor receptor provides a means of further modulating the prostaglandin effect in the microcirculation. It is also possible that EP1 is expressed preferentially in a subpopulation of minority nephrons with EP4 as the predominant receptor in the more numerous population of cortical nephrons. Such a model is consistent with a previous study demonstrating solely vasoconstrictive effects of PGE2 in the juxtamedullary nephron preparation (20).
The only other study to differentiate between EP1 and EP1-variant expression is the original work in which the EP1 receptor variant was cloned (31). Our results document for the first time mRNA expression for the EP1-variant in both cultured and freshly isolated preglomerular preparations. The EP1-variant receptor displays the same ligand binding specificity as EP1 but lacks the COOH-terminal signaling domain (31). Thus the function, or lack thereof, of this subtype is difficult to establish because the portion of the receptor that couples to the G protein is truncated, and the receptor does not appear to generate signals along traditional transduction pathways. One can speculate that the expression of EP1-variant protein at the cell surface is regulated to finely tune the response to PGE2. This variant receptor may serve as a type of clearance receptor, removing excess PGE2 from the circulation and guarding against the sudden onset of dangerously low blood pressure. It may bind PGE2 and release it slowly to VSMC in a manner similar to that postulated for the similarly truncated form of the atrial natriuretic peptide receptor (24). Also, the possibility that the EP1-variant couples to an alternative signaling pathway, such as growth regulation or metabolism, cannot be ruled out.
Species comparisons of EP renal vascular expression are complicated by conflicting results. Although ribonuclease protection assays detect primarily transcripts for EP4 with lower levels of EP1 and EP3 in freshly isolated rat glomerular tissue (23), cultured mesangial cells are reported to express EP1 and EP4 (21). In mice, only EP2 transcripts are detected in glomeruli (35). Similarly, in situ hybridization experiments using human tissue support solely EP4 expression (10), whereas immunohistochemistry studies detect both EP3 and EP4 in human glomeruli (28). The latter study examined expression in the preglomerular vasculature, and reactivity for EP1-3, but not EP4, was reported (28). It is possible that the differences in arteriolar expression may also reflect species variation. Nevertheless, it should be emphasized that our findings concerning EP receptors in rat preglomerular VSMC are based on multiple techniques examining gene expression, cellular signaling ability, and in vivo responses, in contrast to a human study that is confined to antibody recognition of protein.
Our comprehensive findings in the preglomerular vasculature extend the several studies that have examined EP receptor expression in cultured rat glomerular mesangial cells to physiological control of renal vascular resistance and glomerular hemodynamics. Measurements of changes in cross-sectional area suggested both constrictor and dilatory actions of PGE2 in cultured mesangial cells (26). Furthermore, PGE2 has been shown to evoke an increase in cytosolic calcium that can be blocked by EP1 antagonists. The receptor subtype responsible for the increase in cAMP was not identified. In addition, mRNA message for both EP1 and EP4 was detected by Northern blotting in cultured rat mesangial cells (21). A comparison of results provide evidence for phenotypic differences between cultured cells derived from preglomerular resistance arterioles and glomeruli on the basis of EP1 receptor expression. It is not clear whether mesangial cells express the EP1-variant because the riboprobe used was nonselective.
Of particular interest is the potential therapeutic utility of EP subtype-specific drugs. Nonsteroidal anti-inflammatory drugs, which inhibit the cyclooxygenase enzyme that produces PGE2, are presently among the most widely prescribed drugs. In volume-compromised conditions, administration of nonsteroidal anti-inflammatory drugs can severely reduce RBF and lead to reversible acute renal failure (39). These undesirable side effects arise from inhibiting renal PGE2 production and actions, which lead to tubular as well as vascular disturbances. With the high probability that EP4 tranduces the protective vasodilator signal in the renal microcirculation, it is tempting to speculate about the development of EP receptor-specific agents that would be more selective for desired vascular effects and free of the undesirable side effects of present medications. Therapeutic drugs specific to EP receptors may allow better treatment of conditions of extracellular fluid volume contraction and hyperdynamic vasoconstrictor status in pathophysiological conditions.
In summary, the present study provides important new information about gene expression and functional characterization of EP1 and EP4 receptors localized to preglomerular resistance arterioles of Sprague-Dawley rats. In contrast, message for EP2 and EP3 was absent and no functional effect of these receptor subtypes was observed. Preglomerular VSMC express an EP1-variant receptor that does not appear to possess any cAMP or cytosolic calcium signaling capable of affecting RBF. In vivo RBF studies indicate that the EP4 receptor is the primary receptor mediating sustained renal vasodilation produced by PGE2, whereas the EP1 receptor can produce transient vasoconstriction. We conclude that the EP4 receptor is responsible for PGE2-induced vasodilation in the rat kidney, signaling through Gs proteins and the cAMP pathway to reduce [Ca2+]i when stimulated by a vasoconstrictor such as ANG II.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Sharon Milgram (Univ. of North Carolina at Chapel Hill) for scientific advice and helpful discussions regarding PCR experiments. We also thank Dr. Emiko Okuda Ashitaka (Kansai Medical Univ.) and Dr. William Smith (Michigan State Univ.) for the generous gifts of EP1 and EP4 subtype clones, respectively. SC-19220 was donated by Searle Pharmaceuticals (Skokie, IL), and AH-23848 was provided by Glaxo-Wellcome Research and Development, Ltd. (Stevenage, Hertfordshire, UK). Butaprost was a gift of Bayer (West Haven, CT), and M&B28767 was donated by Rhone-Poulenc Rorer (Dagenham, Essex, UK).
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FOOTNOTES |
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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.
Address for reprint requests and other correspondence: W. J. Arendshorst, Dept. of Cell and Molecular Physiology, Rm. 152, Medical Sciences Research Bldg., CB #7545, School of Medicine, Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545 (E-mail: arends{at}med.unc.edu).
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. Section 1734 solely to indicate this fact.
Received 2 March 2000; accepted in final form 20 June 2000.
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