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【摘要】 Evidence indicates that prostaglandin E 2 (PGE 2 ) preferentially affects preglomerular renal vessels. However, whether this is limited to small-caliber arterioles or whether larger vessels farther upstream also respond to PGE 2 is currently unclear. In the present study, we first investigated the effects of PGE 2 along the preglomerular vascular tree and subsequently focused on proximal interlobular arteries (ILAs). Proximal ILAs in hydronephrotic rat kidneys as well as isolated vessels from normal kidneys constricted in response to PGE 2, both under basal conditions and after the induction of vascular tone. By contrast, smaller vessels, i.e., distal ILAs and afferent arterioles, exhibited PGE 2 -induced vasodilation. Endothelium removal and pretreatment of single, isolated proximal ILAs with an EP1 receptor blocker (SC51322, 1 µmol/l) or a thromboxane A 2 receptor blocker (SQ29548, 1 µmol/l) did not prevent vasoconstriction to PGE 2. Furthermore, in the presence of SC51322, responses of these vessels to PGE 2 and the EP1/EP3 agonist sulprostone were superimposable, indicating that PGE 2 -induced vasoconstriction is mediated by EP3 receptors on smooth muscle cells. Immunohistochemical staining of proximal ILAs confirmed the presence of EP3 receptor protein on these cells and the endothelium. Adding PGE 2 to normal isolated kidneys induced a biphasic flow response, i.e., an initial flow increase at PGE 2 concentrations 0.1 µmol/l followed by a flow decrease at 1 µmol/l PGE 2. Thus our results demonstrate that PGE 2 affects multiple segments of the preglomerular vascular tree in a different way. At the level of the proximal ILAs, PGE 2 had a direct vasoconstrictor action mediated by EP3 receptors.
【关键词】 interlobular arteries vasoconstriction
PGE 2 IS ONE OF the major cyclooxygenase-derived products produced by the kidney ( 17 ). The actions of PGE 2 intrarenally include both tubular and vascular effects ( 2 ). The latter are important for regulating renal function under various circumstances. So far, the effects of PGE 2 on smaller pre- and postglomerular arterioles have mainly received attention. The postglomerular efferent arterioles were found to be insensitive to PGE 2 ( 4, 23 ), while for the preglomerular afferent arterioles (AAs) and distal interlobular arteries (ILAs) most studies indicated vasodilation ( 4, 8, 23 ), although vasoconstriction in certain instances has been reported as well ( 9, 23 ). Whether larger vessels further upstream of the glomerulus are also sensitive to PGE 2 is currently unknown. Previous studies have demonstrated that larger preglomerular vessels constrict in response to inflammatory mediators, such as serotonin and leukotrienes ( 5, 19 ), suggesting a similar mode of action for PGE 2.
In general, the actions of PGE 2 are mediated by a family of four G protein-coupled receptors, designated EP1-EP4 ( 3, 16 ). Of these receptors, EP2 and EP4 have been shown to stimulate intracellular processes leading to smooth muscle cell relaxation and, hence, vasodilation. EP1 and EP3 receptors, on the other hand, stimulate processes leading to vasoconstriction. For example, in cell cultures, EP1 receptor activation has been shown to increase intracellular Ca 2+ concentration ( 26 ). In addition, EP3 receptors have been found to inhibit intracellular cAMP production ( 21, 22 ) and are thereby able to reduce PKA activity.
The purpose of the present study was to determine the effects of PGE 2 on intermediate and proximal ILAs. Initially, experiments were performed using the isolated, perfused hydronephrotic rat kidney model. In this preparation, nearly the entire microvasculature can be visualized, allowing a direct comparison of responses of different vessel segments. Using this model, we found that, in contrast to distal ILAs and AAs, larger preglomerular arterioles manifested a direct vasoconstrictor response to PGE 2. Thereupon, we used normal kidneys to determine in isolated proximal ILAs the receptor subtype involved, both pharmacologically and immunohistochemically. In addition, we assessed the effects of PGE 2 on renal hemodynamics.
MATERIALS AND METHODS
All experiments were performed using 12- to 15-wk-old male Sprague-Dawley rats (Harlan, Horst, The Netherlands). The animals were housed in macrolon cages with free access to tap water and a standard rat chow (Hope Farm, Woerden, The Netherlands). All their handling was reviewed and approved by the Institutional Animal Care and Use Committee.
Drugs. PGE 2 was obtained from Calbiochem. The EP1 receptor blocker SC51322, the thromboxane A 2 (TXA 2 ) receptor antagonist SQ29548, and the EP1/EP3 agonist sulprostone were obtained from Biomol and Cayman Chemical, respectively. ANG II, norepinephrine, and acetylcholine were purchased from Sigma.
Isolated, perfused hydronephrotic and normal kidneys. To obtain hydronephrotic kidneys, rats were anesthetized with isoflurane (3% in O 2, 0.7 l/min and N 2 O, 1.2 l/min, Abbott Laboratory, Queensborough, Kent, UK) at a younger age, i.e., 8 wk before the in vitro experiments. Subsequently, the left ureter was located and tied off with a suture. Ureter ligation is known to induce almost complete tubular atrophy ( 20 ), allowing direct microscopic visualization of nearly the entire renal microvascular bed.
For in vitro perfusion, both hydronephrotic and normal kidneys were isolated in a similar manner (see Ref. 25 for an extensive description). Briefly, the rat was anesthetized with a combination of pentobarbital sodium (50 mg/kg ip, Sanofi Sante, Maassluis, The Netherlands) and ketamine (25 mg/kg im, Kombivet, Etten-Leur, The Netherlands). The kidney was exposed by opening the abdominal cavity, and the renal artery was cannulated via the abdominal aorta. Perfusion was started in situ with preheated DMEM (Sigma) supplemented with (in mmol/l) 23.8 NaHCO 3, 5.6 HEPES, 5.5 D -glucose, and 1 sodium pyruvate (all Sigma). DMEM was equilibrated with 95% air-5% CO 2 at 37°C, resulting in a pH of 7.4. Then, the kidney was excised and moved to a heated chamber on the stage of an inverted microscope (Axiovert 100, Zeiss) without disruption of renal flow. For all studies, a single-pass perfusion was employed. The perfusion apparatus consisted of a small pressurized reservoir ( 25 ml) connected to the renal arterial cannula, that was filled on demand from a larger preheated and oxygenated reservoir ( 600 ml). Agents were added in a cumulative manner to this larger reservoir. Both hydronephrotic and normal kidneys were perfused at a constant pressure of 80 mmHg, monitored at the level of the renal artery. Pressure changes were eliminated by adjusting the outflow of gas (95% air-5% CO 2 ) from the small reservoir. In normal kidneys, renal perfusate flow was measured using an electromagnetic flow probe (TS410, Transonic Systems) mounted in the perfusion line proximal to the kidney.
To visualize different vessel segments in hydronephrotic kidneys, a small hole was made in the cortex through which a fiber optic probe was inserted, transilluminating a portion of the membranous cortex. Vessel images were generated using a x 40 objective lens (numerical aperture 0.6, Zeiss) and a CCD camera (7020/20, Philips, Eindhoven, The Netherlands). The images were recorded on a VCR for offline analysis using a custom designed vessel wall tracking system ( 12 ). Changes in afferent arteriolar diameters were analyzed just after branching from ILAs. ILAs were divided into four different groups based on their location and basal diameters: distal (connected to AAs), intermediate (30-50 and 50-70 70 µm). ILA diameters were measured near their midpoint.
Isolated cannulated proximal interlobular arteries. Proximal ILAs were isolated from normal dissected kidneys and kept in ice-cold dissection DMEM in which NaHCO 3 was lowered to 4.2 mmol/l by replacing NaHCO 3 with NaCl. This medium was equilibrated with atmospheric CO 2, and pH was set at 7.4 using NaOH. The isolated ILAs were mounted with sutures between two glass micropipettes in a water-jacketed chamber of a pressure myograph. The chamber was moved to the stage of a microscope, filled with dissection DMEM, and sealed with a glass cover. One of the micropipettes was connected to a pressure column, used to gradually pressurize the ILA to 75 mmHg. The other pipette was clamped off and connected to a micrometer to stretch the vessel to its in vivo length. The time period from dissection to mounting the vessel did not exceed 2 h. Superfusion with normal DMEM was subsequently started, and the temperature was raised to 37°C. Superfusion rate was set at 2 ml/min, with refreshing of the myograph chamber volume every minute. Agents were added directly to the superfusion medium in a cumulative manner. Changes in vessel diameter were measured online using a custom designed measurement system. Only those vessels exhibiting a clear endothelium-dependent vasodilator response to 0.3 µmol/l 50% reversal of PGE 2 -induced vasoconstriction) were used for determining mean responses. In one series of experiments, the endothelium was removed deliberately by perfusing the vessels with a bubble of air. When ILAs were subjected to this treatment, acetylcholine-induced vasodilatation was completely absent. To exclude the possibility that PGE 2 -induced vasoconstriction of ILAs was mediated by TXA 2 receptors, isolated ILAs were treated in another series of experiments with the specific TXA 2 receptor blocker SQ29548 (1 µmol/l, 10 min) after which PGE 2 concentration-response curves were constructed.
Equilibration and elimination of endogenous prostanoids. All in vitro preparations described above were allowed to equilibrate for at least 30 min before experiments began. All were pretreated with 10 µmol/l ibuprofen (Sigma) to eliminate the influence of endogenous prostanoids. Sufficient cyclooxygenase (COX)-2 inhibition by this ibuprofen concentration is indicated by our observation that treatment with a selective COX-2 inhibitor (NS-398, 10 µmol/l, Sigma) in hydronephrotic kidneys did not change basal diameters of pre- or postglomerular vessels. Changes in vessel diameter or perfusion flow caused by a drug were evaluated during the plateau of the response, usually 10 min after its addition.
Immunohistochemical staining of EP3 and EP1 receptors. Cannulated proximal ILAs were fixated with 2% ice-cold paraformaldehyde in dissection DMEM, embedded in 15% gelatin, and frozen in liquid nitrogen. Cut 10-µm sections were permeabilized with 0.3% Triton X-100, blocked with 10% goat serum in PBS [1 h, at room temperature (RT)], and incubated with a rabbit polyclonal antibody against EP3 or EP1 receptors (1:500 in PBS containing 3% goat serum, 48 h, 4°C, both Cayman Chemical). In the negative background control, the primary antibody was omitted from the incubation medium. The sections of both types were then washed (PBS, 3 x 10 min, RT) and incubated with the secondary antibody consisting of 1:100 diluted Alexa Fluor 488-conjugated donkey anti-rabbit (1 h, RT, Molecular Probes). This fluorochrome allows short illumination times, thereby precluding autofluorescence. After a final wash (3 x 30 min, RT), sections were mounted in medium (Vectashield from Vector) containing 4',6-diamino-2-phenylindole (DAPI) as a nuclear counterstain. To distinguish smooth muscle cells from endothelial cells, F-actin was labeled in additional sections using Cy3-conjungated-rhodamine phalloidin (1:60, Molecular Probes), incubated together with the secondary antibody. The vascular localization of EP3 and EP1 receptor protein was studied using an inverted fluorescence microscope (Axiovert 200 Marianas, Zeiss). Images were generated with a x 10 air and x 40 oil-immersion objective (numerical aperture 0.50 and 1.30, respectively; Zeiss) and recorded using a cooled CCD camera (1,280 x 1,024 pixels, Cooke Sensicam, Cooke, Tonawanda, NY). This microscope and camera, as well as the data viewing and processing, including deconvolution, were conducted and controlled by Slidebook software (Slidebook version 4.0, Intelligent Images Innovations, Denver, CO).
Statistical analyses. All values are presented as means ± SE; n refers to the number of animals studied. Statistical analyses were performed using Prism 4 (GraphPad Software, San Diego, CA). Differences within concentration-response curves were assessed using one-way ANOVA for repeated measurements followed by a Newman-Keuls post hoc test. P < 0.05 was considered statistically significant. Differences between groups were assessed using ANOVA followed by Student's t -test. For multiple comparisons, the Bonferroni correction was applied.
RESULTS
Effects of PGE 2 on different segments of the preglomerular vascular tree in isolated hydronephrotic rat kidneys. As shown in Fig. 1, marked differences in the response to PGE 2 were seen along the preglomerular vascular tree. Under basal conditions ( Fig. 1 A ), proximal and intermediate ILAs (see Table 1 for basal values) manifested a concentration-dependent constriction to PGE 2, which appeared to increase in magnitude with their diameter. Thus, at the highest PGE 2 concentration, i.e., 1 µmol/l, mean diameters were reduced by 15.4 ± 3.7% in proximal 70 µm, n = 5) and by 12.0 ± 3.4 and 8.0 ± 3.3% in intermediate ILAs (50-70 and 30-50 µm, respectively; n = 6 and 5). By contrast, the smallest caliber ILAs (i.e., distal) and AAs were hardly or not affected under basal conditions by increasing concentrations of PGE 2 ( Fig. 1 A ).
Fig. 1. Effects of PGE 2 on successive segments of the preglomerular vascular tree in isolated, perfused hydronephrotic rat kidneys. A : under basal conditions, PGE 2 30 µm. B : in contrast, during treatment with the vasoconstrictor ANG II (0.1 nmol/l), PGE 2 responses displayed a gradual change from vasodilation at the small-caliber sites to again vasoconstriction in the largest ILAs, with a biphasic response in between; n = 5-6. * P < 0.05 vs. basal condition.
Table 1. Diameters of successive segments of the preglomerular vascular tree in isolated hydronephrotic rat kidneys under basal conditions and after treatment with PGE 2 (1 µmol/l) or ANG II (0.1 nmol/l)
The vascular bed of isolated hydronephrotic rat kidneys is almost maximally vasodilated. Thus PGE 2 effects were also assessed after the induction of vascular tone with ANG II to observe possible vasodilator responses. As shown in Fig. 1 B, ANG II-induced vasoconstriction was inversely related to the basal ILA diameters. Proximal ILAs ( n = 5) failed to respond to ANG II, while subsequent segments (see Table 1 ) were reduced in diameter by 7.8 ± 6.2 ( n = 6), 21.2 ± 6.3 ( n = 6), and 51.1 ± 7.8% ( n = 6), respectively. AAs exhibited a similar level of vasoconstriction to ANG II to distal ILAs (48.0 ± 3.9%, n = 4). In all vessels exhibiting tone, PGE 2 completely reversed at lower concentrations ( 10 nmol/l) the ANG II-induced vasoconstriction. In distal ILAs and AAs, higher PGE 2 10 nmol/l) had no further effect, with mean vessel diameters remaining near basal values (24.2 ± 1.2 and 20.7 ± 0.3 µm at 1 µmol/l PGE 2, respectively). In intermediate ILAs, however, the diameter increase at lower PGE 2 concentrations was followed by a subsequent diameter decrease. At the level of the proximal ILAs, no vasodilation to PGE 2 was observed. Instead, also in the presence of ANG II, PGE 2 decreased their diameters in a concentration-dependent manner similar to that observed under basal conditions (to 79.3 ± 4.5 µm at 1 µmol/l).
Effects of PGE 2 on proximal ILAs obtained from normal rat kidneys. Because in the experiments reported above proximal ILAs exhibited the largest vasoconstrictor response, proximal ILAs isolated from normal kidneys were studied as well. As shown in Fig. 2 A, these isolated ILAs also displayed a concentration-dependent diameter reduction in response to PGE 2. Significant vasoconstriction occurred from 10 nmol/l PGE 2 onward (14.8 ± 3.0%), up to 34.9 ± 2.6% at the highest concentration of PGE 2 (1 µmol/l). Removal of the endothelium did not prevent but enhanced the vasoconstrictor response of isolated ILAs to PGE 2 ( Fig. 2 A ). At 1 µmol/l PGE 2, diameters of endothelium-denuded ILAs were decreased by 62.0 ± 6.9% ( n = 4 ). Also after induction of a moderate amount of vascular tone ( 15%) using norepinephrine, isolated ILAs only displayed PGE 2 -induced vasoconstriction ( Fig. 2 B ). Thus, in the presence of norepinephrine, PGE 2 at concentrations of 0.01, 0.1, and 1 µmol/l decreased mean diameters of ILAs by 18.4 ± 3.6, 33.3 ± 5.6, and 45.0 ± 5.7% ( n = 6), respectively.
Fig. 2. PGE 2 -induced vasoconstriction of proximal ILAs obtained from normal kidneys. A : note enhanced response of endothelium-denuded vessels (solid symbols). B : furthermore, also after the induction of vascular tone using norepinephrine (NE; 30-70 nmol/l), proximal ILAs only displayed concentration-dependent vasoconstriction; n = 4-7. EC, endothelium. * P < 0.05 vs. basal condition and NE-induced tone, respectively. # P < 0.05 vs. control vessels.
Characterization of the receptor subtype mediating vasoconstriction of ILAs to PGE 2. Initially, the role of the EP1 receptor in the vasoconstriction of ILAs to PGE 2 was assessed. As shown in Fig. 3 A, a selective EP1 blocker had no effect on basal diameters of isolated proximal ILAs ( n = 7). Furthermore, PGE 2 -induced vasoconstriction of isolated ILAs was not inhibited. Instead, the blocker significantly increased their reactivity to PGE 2, as depicted in Fig. 3 B. During EP1 receptor blockade, the PGE 2 -induced vasoconstriction of ILAs increased from 14.8 ± 3.0 to 23.8 ± 1.9% ( P = 0.03 vs. PGE 2 alone), from 26.0 ± 2.4 to 38.3 ± 4.6% ( P = 0.05), and from 34.9 ± 2.6 to 46.1 ± 4.1% ( P = 0.04) at 0.01, 0.1, and 1 µmol/l PGE 2, respectively.
Fig. 3. Failure of the selective EP1 receptor antagonist SC512322 (1 µmol/l) to prevent PGE 2 -induced vasoconstriction of isolated proximal ILAs from normal kidneys. Shown are absolute data ( A ) and %change from pre-PGE 2 values ( B ) for both the blocked and control condition; n = 7. * P < 0.05 vs. pre-PGE 2 value. # P < 0.05 vs. PGE 2 alone.
Because a commercially available antagonist for the other constrictive PGE 2 receptor, i.e., EP3, is lacking, its potential role in ILA vasoconstriction was investigated using the EP1/EP3 agonist sulprostone. As shown in Fig. 4 A, sulprostone reduced diameters of isolated ILAs in a concentration-dependent manner. The highest concentration of sulprostone, i.e., 1 µmol/l, reduced diameters by 42.1 ± 5.1% (to 49.0 ± 3.8 µm, n = 7). Pretreatment with the selective EP1 antagonist did not affect sulprostone-induced vasoconstriction ( Fig. 4, B and C, n = 6). Moreover, when the potencies of sulprostone and PGE 2 in the presence of the EP1 blocker were compared, the concentration-response curves to both agonists were superimposable ( Fig. 4 D ), indicating that the vasoconstrictor actions of PGE 2 on ILAs are mediated by the EP3 receptor.
Fig. 4. Vasoconstrictor effects of the EP1/EP3 receptor agonist sulprostone on isolated proximal ILAs from normal kidneys. Shown are absolute data under control conditions ( n = 7, A ) and after pretreatment with the selective EP1 receptor antagonist SC512322 (1 µmol/l, n = 6, B ), respectively. C : comparison of %change from presulprostone values. D : comparison of effects of sulprostone and PGE 2 during EP1 receptor blockade. Note that the concentration-response curves for both agonists are superimposable, indicating that PGE 2 -induced vasoconstriction is mediated by EP3 receptor activation. * P < 0.05 vs. presulprostone values.
To exclude a potential involvement of TXA 2 receptors in the PGE 2 response, PGE 2 concentration-response curves were also constructed in the presence of a specific TXA 2 receptor blocker (SQ29548; 1 µmol/l). As shown in Fig. 5, TXA 2 receptor blockade did not prevent PGE 2 -induced vasoconstriction, supporting our conclusion as mentioned above.
Fig. 5. Failure of selective thromboxane A 2 (TXA 2 ) receptor antagonist SQ29548 (1 µmol/l) to prevent PGE 2 -induced vasoconstriction of isolated proximal ILAs from normal kidneys; n = 5. * P < 0.05 vs. pre-PGE 2 values.
Expression of EP3 receptor protein in proximal ILAs. As shown in Fig. 6, EP3 receptor protein was present in smooth muscle cells of isolated proximal ILAs. Near the nucleus, labeling was more dense ( Fig. 6 G, white arrows), probably reflecting receptor protein present in the perinuclear Golgi. In addition, EP3 receptor protein was also present on endothelial cells, showing a similar distribution as in smooth muscle cells. In contrast to EP3, labeling of smooth muscle cells was relatively weak when an anti-EP1 antibody was used ( Fig. 7, F - J ). Immunofluorescence of endothelial cells, on the other hand, was very high. Dense labeling near the nucleus was not observed when the anti-EP1 receptor antibody was used.
Fig. 6. Presence of EP3 receptor protein in vascular smooth muscle cells and endothelial cells of isolated proximal ILAs of normal kidneys. Shown are EP3 receptor protein (green), F-actin (red), and nuclei (blue). A - C : x 10 objective. D-F : x 40 oil-immersion objective. G : deconvoluted image using x 40 objective. White arrows, examples of dense EP3 receptor labeling near the nucleus of smooth muscle and endothelial cells.
Fig. 7. Comparison of EP3 ( A - E ) and EP1 ( F - J ) receptor expression in consecutive proximal ILA slides. Shown are EP3 or EP1 receptor protein (green), F-actin (red), and nuclei (blue). A - C and F-H : x 10 objective. D and I : x 40 oil-immersion objective. E and J : deconvoluted image using x 40 objective. Note that in contrast to EP3, EP1 receptor staining is most dense in the endothelium. Also note that in I, the elastin membrane is visible as a black line adjacent to the fluorescent endothelium; with the short illumination times used, the elastin membrane did not show autofluorescence.
Effects of PGE 2 on renal hemodynamics in isolated normal kidneys. As shown in Fig. 8, PGE 2 predominantly increased renal perfusion in isolated normal kidneys. Thus, at concentrations 0.1 µmol/l, PGE 2 increased renal perfusate flow both under basal conditions ( Fig. 8 A; from 19.3 ± 2.3 to 20.6 ± 2.5 ml·min -1 ·g -1; n = 6) and after pretreatment with ANG II ( Fig. 8 B; from 9.1 ± 0.9 to 16.8 ± 1.3 ml·min -1 ·g -1; n = 6). In both cases, this initial flow increase was partially reversed at the highest PGE 2 concentration of 1 µmol/l (basal: to 18.1 ± 2.5 ml·min -1 ·g -1; ANG II to 15.8 ± 1.4 ml·min -1 ·g -1; for both P < 0.05 vs. 0.1 µmol/l PGE 2 ).
Fig. 8. Effects of PGE 2 on renal perfusate flow in normal isolated kidneys during control conditions ( A ) and after preconstriction with ANG II (0.1 nmol/l; B ); n = 6. * P < 0.05 vs. pre-PGE 2 values. P < 0.05 vs. 0.1 µmol/l PGE 2.
DISCUSSION
We found that PGE 2 induced exclusive vasoconstriction of proximal ILAs both in hydronephrotic rat kidneys and in those isolated from normal kidneys. Smaller vessels, on the other hand, i.e., distal ILAs and AAs, were found to dilate to PGE 2. Treatment of isolated proximal ILAs with drugs interacting with the different PGE 2 receptor subtypes indicated a role for EP3 receptors in PGE 2 -induced vasoconstriction. The presence of this receptor subtype on smooth muscle cells and also on endothelial cells of proximal ILAs was confirmed using immunohistochemical staining. Adding PGE 2 to normal isolated kidneys induced a biphasic flow response; i.e., flow increased at PGE 2 concentrations 0.1 µmol/l and subsequently decreased at 1 µmol/l PGE 2.
Our study is the first to demonstrate that the vasoconstrictor action of PGE 2 in the kidney is confined to the larger preglomerular vessels. In hydronephrotic kidneys, we mapped the PGE 2 response along the preglomerular tree, finding vasoconstriction of proximal ILAs, while distal ILAs and AAs only manifested PGE 2 -induced vasodilation. This latter observation is consistent with the response of AAs observed in most other studies ( 4, 8, 24 ), although PGE 2 -induced vasoconstriction of these vessels has also occasionally been reported ( 9, 24 ). In addition, we found that intermediate ILAs displayed a biphasic response to increasing concentrations of PGE 2, i.e., vasodilation followed by vasoconstriction. Our data indicate that the actions of PGE 2 along the preglomerular vascular tree gradually change from exclusively vasoconstriction upstream to vasodilation downstream. A similar response pattern has been reported for serotonin ( 5 ), suggesting heterogeneity in receptor expression of the different preglomerular segments.
Renal prostaglandin production has been reported to alter during hydronephrosis ( 14, 15 ), which could have influenced reactivity of the larger ILAs to PGE 2. Therefore, we also performed single-vessel experiments using tissue from normal kidneys. We found that PGE 2 elicited vasoconstriction of isolated proximal ILAs, both under basal conditions and after the induction of vascular tone. Thus responses were similar to those observed in hydronephrotic rat kidneys, indicating a general action of PGE 2 on proximal ILAs. In addition, we found in normal isolated kidneys that with increasing concentrations of PGE 2 only the highest PGE 2 concentration reversed the increase to a decrease in renal perfusate flow, indicating that, also in this preparation, not all vessels but probably only the larger ones constrict in response to PGE 2.
Of the different PGE 2 receptors, EP1 and EP3 have been shown to activate signal transduction pathways leading to contraction of smooth muscle cells ( 3, 16 ). In proximal ILAs, our findings indicate that the vasoconstrictor actions of PGE 2 involve EP3 receptors. First, we found that PGE 2 -induced vasoconstriction of isolated vessels could not be abolished by selective TXA 2 or EP1 receptor blockade. Furthermore, during EP1 receptor antagonism, concentration-response curves to PGE 2 and to the EP1/EP3 agonist sulprostone were superimposable. Removal of the endothelium did not prevent the vasoconstriction to PGE 2, indicating that EP3 receptors are activated directly on smooth muscle cells. The presence of EP3 receptor protein on smooth muscle cells was confirmed by immunohistochemical staining. By contrast, EP1 receptor labeling was predominantly found at the level of the endothelium. Tang et al. ( 24 ), who in contrast to our and various other studies ( 4, 8 ), observed afferent arteriolar constriction to high PGE 2 concentrations, also found that this response was mediated via EP3 receptors.
We observed that when isolated proximal ILAs were pretreated with a selective EP1 receptor antagonist, PGE 2 -induced vasoconstriction was not inhibited but rather potentiated, indicating that EP1 receptor activation stimulates a vasodilator process. This observation seems consistent with the study of Audoly et al. ( 1 ) showing that PGE 2 lowered arterial pressure to a lesser extent in male EP1 receptor knockout mice than in wild-type mice. Our immunohistochemical data indicate that EP1 receptors are predominantly expressed on endothelial cells. This observation extends the findings of previous studies demonstrating the presence of EP1 receptors in renal vascular structures ( 11, 13, 18 ). Thus EP1 receptor activation may stimulate endothelial cells to produce and release vasodilatory factors modulating EP3-mediated constriction. Indeed, we found that after deliberate damage of endothelial cells vasoconstrictor responses to PGE 2 were enhanced. Furthermore, in cultured cells, EP1 receptor activation has been shown to increase intracellular Ca 2+ concentration ( 26 ), which, in endothelial cells, is known to stimulate nitric oxide production ( 6 ).
Our immunohistochemical staining revealed that EP3 receptors were present on both smooth muscle and endothelial cells. Thus, besides by directly activating smooth muscle cells, PGE 2 might also induce vasoconstriction of ILAs by stimulating endothelial cells to release vasoconstrictor agents. Using endothelium-denuded proximal ILAs, we investigated the possible contribution of endothelial cells to the vasoconstrictor actions of PGE 2. We found in these vessels that PGE 2 was still able to induce vasoconstriction, suggesting that EP3 as well as EP1 receptors on endothelial cells fulfill a different role.
A final comment should be made regarding our flow data. The influence of PGE 2 on flow in normal kidneys was biphasic. At concentrations 0.1 µmol/l, flow increased both under basal conditions and after preconstriction with ANG II. This finding is consistent with previous data obtained in this model ( 7, 10 ) and likely reflects the vasodilatory response of the small preglomerular vessels to PGE 2. However, at a concentration of 1 µmol/l, flow decreased in both situations. At this concentration, the arterioles and small arteries, which are the most important resistance regulators, are completely dilated ( Fig. 1 B ) and, hence, vasoconstriction of the larger arteries becomes visible in the flow signal.
In summary, the present study demonstrates that PGE 2 elicits vasoconstriction of proximal ILAs, mediated via EP3 receptor activation. Toward the glomerulus, the vasoconstrictor effect of PGE 2 gradually changes to PGE 2 -induced vasodilation.
GRANTS
This study was supported by Grant C 00.1903 from the Dutch Kidney Foundation.
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作者单位：1 Laboratory for Physiology and 2 Department of Nephrology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands