Contribution of prostaglandin EP2 receptors to renal microvascular reactivity in mice

John D. Imig1, Matthew D. Breyer2, and Richard M. Breyer3

1 Vascular Biology Center, Department of Physiology, Medical College of Georgia, Augusta, Georgia 30912; and Departments of 2 Medicine (Nephrology) and Molecular Biology and Biophysics and of 3 Medicine (Nephrology) and Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present studies were performed to determine the contribution of EP2 receptors to renal hemodynamics by examining afferent arteriolar responses to PGE2, butaprost, sulprostone, and endothelin-1 in EP2 receptor-deficient male mice (EP2-/-). Afferent arteriolar diameters averaged 17.8 ± 0.8 µm in wild-type (EP2+/+) mice and 16.7 ± 0.7 µm in EP2-/- mice at a renal perfusion pressure of 100 mmHg. Vessels from both groups of mice responded to norepinephrine (0.5 µM) with similar 17-19% decreases in diameter. Diameters of norepinephrine-preconstricted afferent arterioles increased by 7 ± 2 and 20 ± 6% in EP2+/+ mice in response to 1 µM PGE2 and 1 µM butaprost, respectively. In contrast, afferent arteriolar diameter of EP2-/- mice decreased by 13 ± 3 and 16 ± 6% in response to PGE2 and butaprost. The afferent arteriolar vasoconstriction to butaprost in EP2-/- mice was eliminated by angiotensin-converting enzyme inhibition. Sulprostone, an EP1 and EP3 receptor ligand, decreased afferent arteriolar diameter in both groups; however, the vasoconstriction in the EP2-/- mice was greater than in the EP2+/+ mice. Endothelin-1-mediated afferent arteriolar diameter responses were enhanced in EP2-/- mice. Afferent arteriolar diameter decreased by 29 ± 7% in EP2-/- and 12 ± 7% in EP2+/+ mice after administration of 1 nM endothelin-1. These results demonstrate that the EP2 receptor mediates a portion of the PGE2 afferent arteriolar vasodilation and buffers the renal vasoconstrictor responses elicited by EP1 and EP3 receptor activation as well as endothelin-1.

prostaglandins; endothelin; kidney; microcirculation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE REGULATION OF WATER and electrolyte homeostasis is dependent on the renal hemodynamic and tubular transport actions of PGE2 (5, 14, 25, 42). PGE2 is the major renal cyclooxygenase (COX)-derived metabolite in the kidney and the PGE2 receptors (EP) are abundantly expressed in the kidney (5, 7, 8). Four seven-transmembrane-spanning domain, G protein-coupled EP receptors have been identified (5, 7, 8). The intracellular signaling mechanisms for the EP receptors have been characterized and activate mechanisms that would either relax or contract smooth muscle (5, 14). Overall, PGE2 has been demonstrated to increase renal blood flow and glomerular filtration rate but the contribution of EP receptors to the control of renal hemodynamics remains unresolved.

An important role for the EP2 receptors in regulating fluid and electrolyte homeostasis has been suggested by studies in mice with targeted disruption of these receptors (21, 38, 43). Disruption of the EP2 receptor in mice does not alter renal blood flow but does unmask a systemic vasoconstriction in response to PGE2 (3, 43). These mice lacking EP2 receptors develop salt-sensitive hypertension (20). Thus further investigation of the renal microvascular actions of PGE2 is of extreme interest in these mice. The purpose of the present study was to determine the contribution of EP2 receptors to renal hemodynamics by examining afferent arteriolar responses to PGE2, selective EP receptor agonists, and endothelin-1 in mice lacking EP2 receptors.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemical reagents. Sulprostone, PGE2, and butaprost were purchased from Cayman Chemical. Norepinephrine (Levophed) was obtained from Winthrop Pharmaceuticals. Endothelin-1 was purchased from Phoenix Pharmaceuticals. Enalaprilat was a gift from Merck Sharp and Dohme. Indomethacin and all other reagents were purchased from Sigma.

Animal preparation. EP2 receptor-deficient mice were generated at Vanderbilt University as previously described (21). F2 wild-type (EP2+/+) and EP2-null (EP2-/-) mice were littermates produced from intercrossing F1 heterozygous (EP2+/-) mice. All mice were weaned at 3 wk of age and fed a standard chow diet. Genotypes of the mice were routinely determined by Southern analysis of genomic tail DNA. The wild-type (4.3 kb) and recombinant (7.5 kb) XbaI fragments were identified by using a 3' XbaI/SacI fragment as a probe. Animals were housed for at least 2 wk at the Tulane University School of Medicine vivarium. The Vanderbilt University and Tulane Advisory Committee for Animal Resources approved all experiments, and the procedures followed were in accordance with institutional guidelines.

Vascular preparation. Experiments were performed in male EP2+/+ and EP2-/- mice weighing an average of 33 ± 1 and 32 ± 1 g, respectively. Mice were anesthetized with a combination of thiobutabarbital (Inactin; 100 mg/kg ip) and ketamine (Ketaset; 10 mg/kg ip), and a midline abdominal incision was made. The right renal artery was cannulated via the superior mesenteric artery, and the kidney was immediately perfused with Tyrode solution containing 6% albumin and a mixture of L-amino acids (15). All protocols were conducted in the juxtamedullary microvascular preparation perfused with the cell-free Tyrode solution containing 6% albumin. We previously demonstrated that the main difference between a cell-free and red blood cell-containing solution is that nitric oxide levels are elevated in a cell-free perfusate (16). The Tyrode solution was stirred continuously in a closed reservoir that was pressurized by a 95% O2-5% CO2 tank. The kidney was removed from the mouse and maintained in an organ chamber at room temperature throughout the isolation and dissection procedure. The juxtamedullary microvasculature was isolated for study as previously described (15). The organ chamber was then warmed, and the tissue surface was continuously superfused with Tyrode solution containing 1% albumin at 37°C. Renal artery perfusion pressure, measured at the tip of the cannula, was set to 100 mmHg.

Determination of afferent arteriolar diameter was accomplished using transillumination videomicroscopy as previously described (15). The tissue was transilluminated, and the focused image was converted to a video signal by a high-resolution Newvicon camera. This video signal was electronically enhanced and recorded on videotape for later analysis. Afferent arteriolar inside diameters were measured at 15-s intervals using a digital image-shearing monitor. The image-shearing device is accurate to within 0.2% of the screen width or 0.2 µm, and measurement reproducibility is within 0.5 µm. The average diameter during the final 2 min of each 5-min treatment period was used for statistical analysis of steady-state responses.

Afferent arteriolar diameter response to PGE2. After a 20-min equilibration period, baseline diameter measurements of the afferent arteriole were made. Norepinephrine (0.5 µM) was added to the perfusate to elevate basal vascular tone. The endogenous ligand PGE2 (1 µM) was added to the perfusate, and vessel diameter changes were monitored for 5 min. In additional experiments, the influence of the renin-angiotensin system on the afferent arteriolar diameter response was evaluated. For these studies, the angiotensin- converting enzyme inhibitor enalaprilat (1 mg ip) was administered to the mice (17). One hour after the injection, the kidney was harvested and the afferent arteriolar response to PGE2 was determined as described above.

Afferent arteriolar response to the EP receptor activation with butaprost or sulprostone. After a 20-min equilibration period and baseline diameter measurements, the afferent arteriole was preconstricted with norepinephrine (0.5 µM). The arteriole was subsequently exposed to increasing concentrations of an EP2 receptor-selective ligand, butaprost (0.01-1 µM) (7, 8, 14, 25), and diameter change was monitored for 5 min at each concentration. In a separate series, the afferent arteriolar diameter response to butaprost was determined in enalaprilat-treated EP2+/+ and EP2-/- mice.

The afferent arteriole diameter response to an EP1 and EP3 receptor ligand, sulprostone (7, 8, 14, 25), was determined in EP2+/+ and EP2-/- mice. Administration of norepinephrine (0.5 µM) to the perfusate resulted in an elevated vascular tone. Sulprostone (0.01-1 µM) was superfused, and the afferent arteriolar diameter changes were monitored.

Involvement of the EP2 receptor in the afferent arteriolar vasoconstrictor response to endothelin-1. After a 20-min equilibration period, baseline diameter measurements of the afferent arteriole were made. Endothelin-1 (0.1-10 nM) was then administered in increasing concentrations, and diameter changes were monitored. In a separate series, the concentration-response profile to endothelin-1 was determined in the presence of the nonselective COX inhibitor indomethacin (10 µM) (15). Indomethacin was added to the perfusate and superfusate for 20 min to ensure complete tissue blockade (15).

Statistical analysis. In all experiments, steady-state diameter was attained by the end of the second minute, and the average diameter of the third to fifth minute of each treatment period was used for graphical representation. Data are presented as means ± SE. The basic design for each treatment protocol is a prospective randomized controlled trial with repeated measures over time for the independent groups. Standard parametric change-from-baseline analyses within each group were conducted for each of the outcome measures. Change scores were computed and used in between-group hypothesis testing (ANOVA). Post hoc multiple comparisons were made using standard statistical Student-Newman-Keuls methods to adjust the "comparison-wise" error rate. A value of P < 0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Afferent arteriolar diameter response to PGE2 in EP2+/+ and EP2-/- mice. Consistent with previous reports, body weight was similar between the groups and averaged 33 ± 1 g in EP2+/+ and 32 ± 1 g in EP2-/- mice. Afferent arteriolar diameter at a renal perfusion pressure of 100 mmHg was unaltered by the absence of EP2 receptors. Diameter of the afferent arteriole averaged 16.7 ± 0.7 µm (n = 39) in EP2-/- compared with 17.8 ± 0.8 µm (n = 40) in EP2+/+ mice. Norepinephrine decreased preglomerular diameter to the same extent in EP2+/+ and EP2-/- mice. Afferent arteriolar diameter decreased by 17 ± 3% (n = 29) in EP2+/+ and 18 ± 4% (n = 27) in EP2-/- mice in response to perfusion of 0.5 µM norepinephrine.

In the first series of experiments, the response of the afferent arteriole to the endogenous EP2 receptor ligand PGE2 was determined. Afferent arteriolar diameter decreased from 17.6 ± 0.6 to 14.6 ± 0.8 µm (n = 12) in EP2+/+ and from 16.5 ± 0.8 to 13.4 ± 0.7 µm (n = 11) in EP2-/- mice after norepinephrine administration. In EP2 +/+ mice, diameter of the afferent arteriole increased in response to superfusion of 1 µM PGE2 in five of the six vessels studied. In contrast, 1 µM PGE2 decreased preglomerular vessel caliber by 13 ± 4% (n = 5) in mice lacking EP2 receptors (Fig. 1). The afferent arteriolar response to PGE2 was determined in additional experiments to evaluate the involvement of the renin-angiotensin system. Enalaprilat treatment did not alter baseline values, and afferent arteriolar diameter averaged 18.4 ± 1.9 (n = 6) and 17.4 ± 1.2 µm (n = 6) in EP2+/+ and EP2-/- mice, respectively. PGE2 increased afferent arteriolar diameter in six of the six vessels studied, and the diameter response averaged 10 ± 2% in EP2+/+ mice. Interestingly, the afferent arteriolar vasoconstrictor response to PGE2 reversed to a 6 ± 3% increase in vessel diameter after angiotensin-converting enzyme inhibition in mice lacking EP2 receptors (Fig. 2). PGE2 significantly increased diameter from control in four afferent arterioles, and diameter did not change in the other two afferent arterioles taken from enalaprilat-treated EP2-/- mice.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 1.   Afferent arteriolar diameter responses to PGE2 in EP2+/+ and EP2-/- mice. The afferent arteriolar PGE2 responses in EP2+/+ mice are shown in A, and PGE2 responses in EP2-/- mice are shown in B. Diameter measurements at 15-s intervals are depicted under control conditions (first 5 min) and after addition of PGE2 (second 5 min).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2.   Afferent arteriolar diameter response to PGE2 in EP2+/+ and EP2-/- mice. Percent control diameter response is depicted under control conditions (left) and in enalaparilat [angiotensin-converting enzyme inhibitor (ACEI)]-treated mice (right). *Significant difference from response in EP2-/- mice; dagger significant difference from response to PGE2 under control conditions in EP2-/- mice.

Afferent arteriolar diameter response to butaprost in EP2+/+ and EP2-/- mice. Figure 3 depicts the preglomerular vascular response to the selective EP2 receptor ligand butaprost in EP2+/+ and EP2-/- mice. The diameter of norepinephrine-precontracted afferent arterioles increased by 20 ± 6% (n = 6) in response to 1 µM butaprost in EP2+/+ mice. Similar to the response to PGE2, superfusion of 1 µM butaprost constricted the preglomerular vessel caliber by 16 ± 6% (n = 6) in mice lacking EP2 receptors. Additional experiments were performed to determine the involvement of the renin-angiotensin system to the butaprost-mediated afferent arteriolar vasoconstriction in EP2-/- mice. After enalaprilat treatment, afferent arteriolar diameters were not different from untreated mice and averaged 18 ± 2 µm (n = 5) in EP2+/+ and 17 ± 1 µm (n = 5) in EP2-/- mice. Angiotensin-converting enzyme inhibition eliminated the preglomerular vasoconstrictor response in EP2-/- mice but did not significantly alter the vasodilatory response to butaprost in EP2+/+ mice (Fig. 4).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 3.   Afferent arteriolar diameter response to superfusion of butaprost in EP2+/+ and EP2-/- mice. Diameter response is depicted in A, and %control diameter is depicted in B. *Significant difference from control diameter; dagger significant difference from response to the same dose of butaprost between EP2+/+ and EP2-/- mice.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of enalaprilat on the afferent arteriolar diameter response to superfusion of butaprost in EP2+/+ and EP2-/- mice. *Significant difference from control diameter; dagger significant difference from response to the same dose of butaprost between EP2+/+ and EP2-/- mice.

Afferent arteriolar diameter response to sulprostone in EP2+/+ and EP2-/- mice. The preglomerular vascular response to EP1 and EP3 receptor activation with sulprostone is depicted in Fig. 5. The afferent arteriolar diameter response to sulprostone was significantly greater in EP2-/- mice compared with that of EP2+/+ mice. Sulprostone (1 µM) decreased afferent arteriolar diameter by 7 ± 2% (n = 6) in EP2+/+ mice and by 17 ± 3% (n = 5) in mice lacking EP2 receptors.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 5.   Afferent arteriolar diameter response to superfusion of sulprostone in EP2+/+ and EP2-/- mice. Diameter response is depicted in A, and %control diameter is depicted in B. *Significant difference from control diameter; dagger significant difference from response to the same dose of sulprostone between EP2+/+ and EP2-/- mice.

Enhanced afferent arteriolar reactivity to endothelin-1 in EP2-/- mice. Figure 6 depicts the afferent arteriolar vasoconstrictor response to endothelin-1 in EP2+/+ and EP2-/- mice. Afferent arteriolar diameter decreased after superfusion of endothelin-1 and reached a steady-state diameter by the end of the second minute. The preglomerular vascular response to endothelin-1 was significantly enhanced in mice lacking EP2 receptors. Afferent arteriolar diameter decreased by 12 ± 7% (n = 6) in EP2+/+ and 29 ± 7% (n = 5) in EP2-/- mice after administration of 1 nM endothelin-1.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 6.   Afferent arteriolar diameter response to superfusion of endothelin-1 in EP2+/+ and EP2-/- mice. Diameter response is depicted in A, and %control diameter is depicted in B. Dotted lines depict the effect of the cyclooxygenase inhibitor indomethacin on the afferent arteriolar response to endothelin-1 in EP2+/+ and EP2-/- mice. *Significant difference from control diameter; dagger significant difference between EP2-/- group and EP2+/+, EP2+/+ indomethacin-treated, and EP2-/- indomethacin-treated groups; Dagger significant difference between EP2-/- and EP2+/+ groups compared with EP2+/+ indomethacin-treated and EP2-/- indomethacin-treated groups.

The effects of the COX inhibition on endothelin-1 afferent arteriolar vasoconstriction were evaluated to determine whether the generation of endogenous COX metabolites was responsible for the difference between EP2+/+ and EP2-/- mice. In the presence of indomethacin, afferent arteriolar diameter averaged 18.3 ± 3 µm (n = 5) in EP2+/+ and 17.0 ± 0.9 µm (n = 6) in EP2-/- mice. Afferent arteriolar diameter in EP2+/+ mice decreased by 4 ± 1, 11 ± 3, and 24 ± 4% in response to 0.1, 1, and 10 nM endothelin-1. The preglomerular vascular response to endothelin-1 during COX inhibition was attenuated in EP2-/- mice and became similar to that observed in EP2+/+ mice. In mice lacking EP2 receptors, afferent arteriolar diameter decreased by 7 ± 4, 13 ± 4, and 21 ± 3% in response to 0.1, 1, and 10 nM endothelin-1 (Fig. 6).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Studies in mice lacking EP2 receptors point to a critical role for these receptors in the maintenance of renal blood flow and water homeostasis (21, 38, 43). The present study focused on the contribution of the EP2 receptor to the control of renal microvascular function. We found that PGE2 or the EP2 receptor ligand butaprost when administered to wild-type mice resulted in an increase in afferent arteriolar diameter. In contrast, PGE2 and butaprost decreased afferent arteriolar vessel caliber in EP2-/- mice. The vasoconstriction in response to PGE2 and butaprost in mice lacking EP2 receptors was eliminated by angiotensin-converting enzyme inhibition. These findings suggest that PGE2-mediated stimulation of the renin-angiotensin system in EP2-/- mice was responsible for the afferent arteriolar vasoconstriction observed in these mice. In addition to the afferent arteriolar vasoconstrictor response to the EP1 and EP3 receptor agonists, sulprostone was enhanced in mice lacking EP2 receptors. Endothelin-1 also resulted in a greater decrease in preglomerular diameter in EP2-/- mice. The enhanced vasoconstrictor response to endothelin-1 in mice lacking EP2 receptors appears to be COX-dependent because indomethacin eliminated this difference between EP2+/+ and EP2-/- mice. Overall, the results of these studies suggest that EP2 receptors help sustain renal blood flow.

The biological actions of PGE2 are mediated via activation of one of four EP receptors (5). EP receptors are abundant throughout the kidney and are expressed in the renal microcirculation (5, 31, 37, 43). Molecular and pharmacological characterization of four different EP receptors, designated EP1-4, have been completed (5, 39). Activation of vascular EP1 and EP3 receptors would be expected to contract smooth muscle cells. EP1 receptors act via the inositol trisphosphate (IP3), diacylglycerol (DAG), and protein kinase C (PKC) pathway (4, 12, 40), and EP3 receptors decrease cAMP and increase rho (1, 2, 24). Activation of either EP2 or EP4 receptors results in an increase in rabbit and rat preglomerular vessel cAMP levels and results in relaxation of vascular smooth muscle (5, 10, 17, 33). In many studies, PGE2 has been demonstrated to increase renal blood flow (5, 14, 25); however, under certain experimental conditions renal vasoconstriction has been observed during administration of PGE2 (19). These opposing results suggest that renal microvessels contain multiple EP receptors. Although all four EP receptors appear to be expressed in the renal microvasculature (23, 31, 37, 43), there is still controversy on this point, because half of these studies failed to find mRNA expression for all four EP receptors (31, 37). The fact that we observed an increase in afferent arteriolar diameter in EP2+/+ mice but a decrease in vessel caliber in EP2-/- mice in response to PGE2 supports the concept that the renal microcirculation is modulated by multiple PG receptor subtypes.

There is controversy regarding the EP receptor subtype that is responsible for the PGE2-mediated increase in renal blood flow. Recent studies have provided experimental evidence that the EP4 receptor is responsible for the dilator response to PGE2 (31, 37); however, these studies did not directly determine the actions of butaprost on afferent arteriolar diameter or renal blood flow. Interestingly, one of these studies did demonstrate that butaprost opposed the afferent arteriolar constrictor actions of angiotensin by 40% and attributed this response to EP4 receptor stimulation because the study failed to find EP2 mRNA expression in isolated renal microvessels (37). However, this interpretation is at variance with the pharmacological characterization of cloned receptors that suggests that butaprost does not stimulate EP4 receptor-evoked responses at concentrations up to 10 µM (26). Experimental studies in gene-disrupted mice performed by Audoly et al. (3) found that baseline renal blood flow was not different between EP2+/+ and EP2-/- mice and that EP2-/- mice had a vasodilator response to a single dose of PGE2 similar to mice with EP2 receptors. In agreement with these findings, we did not observe a difference in baseline afferent arteriolar diameter between EP2+/+ and EP2-/- mice. On the other hand, we observed a vasoconstriction to PGE2 and butaprost in mice lacking an EP2 receptor. The reason for this difference is unknown. One possible explanation is that the present study investigates afferent arterioles of the juxtamedullary area that give rise to the vasa recta in the medullary circulation. Previous studies have demonstrated that COX inhibition has a greater effect on medullary compared with outer cortical blood flow (11, 14, 32). Other studies also noted differences in responses to PGE2 between superficial and juxtamedullary afferent arterioles (14, 29, 35). The renal vascular distribution of the EP2 receptor and other EP receptors is presently not known. Our studies provide evidence that the EP2 receptor does participate in the renal hemodynamic response to PGE2 and butaprost (Fig. 7).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 7.   Afferent arteriolar diagram depicting potential mechanisms for the effects of PGE2, butaprost, and sulprostone. PGE2 vasodilates by activating the EP2 and EP4 receptors in EP2+/+ mice. PGE2 can activate the EP4 receptor to release renin from the granular cells that leads to the local formation of ANG II, and PGE2 can activate EP3 and EP1 receptors to constrict the afferent arteriole in EP2 gene-disrupted mice. Butaprost activates EP2 and possibly EP4 and prostaglandin I2 (IP) receptors to dilate afferent arteriole in EP2+/+ mice, whereas activation of the EP4 and IP receptors in EP2-/- mice will release renin and ultimately result in ANG II-mediated constriction. Last, sulprostone acts on the EP1 and EP3 receptor to cause afferent arteriolar constriction that is enhanced in mice lacking EP2 receptors. Ao, angiotensinogen; ACE, angiotensin-converting enzyme; AT1, angiotensin II type 1 receptor.

One very interesting finding of the present study is that afferent arterioles constricted in response to PGE2 and butaprost in mice lacking EP2 receptors. We further demonstrated that angiotensin-converting enzyme inhibition eliminated the afferent arteriolar constrictor response to PGE2 and butaprost in EP2-/- mice. Butaprost-mediated stimulation of the renin-angiotensin system may be due to prostaglandin I2 (IP) receptor activation because butaprost activates IP receptors at micromolar concentrations (22). Butaprost activation of IP receptors is consistent with the observation that prostacyclin is a mediator of COX-dependent renin release (41). These findings do not preclude the possibility that EP4 receptors participate in PGE2-evoked renin release as well. In contrast to the effects of butaprost, Tang et al. (37) demonstrated that the EP4 receptor agonist 11-deoxy-PGE1 completely reversed the afferent arteriolar vasoconstriction to angiotensin II, suggesting EP4 receptor activity opposes the renin-angiotensin system (37). It is important to note that these experiments were conducted in the hydronephrotic kidney that lacks tubules and interactions among the macula densa, juxtaglomerular apparatus, and vascular smooth muscle. Therefore, prostaglandin activation of the renin-angiotensin system would not occur under this experimental setting. Nevertheless, we did observe preglomerular vasodilation in response to PGE2 during angiotensin- converting enzyme inhibition in EP2-/- mice. This finding supports the concept that EP4 receptors on preglomerular vessels participate in maintaining renal blood flow.

As mentioned above, butaprost is reported to be an EP2-selective agonist and should not have influenced renal microvessel caliber in mice lacking EP2 receptors. IP receptor activation and renin release could mediate the butaprost-mediated vasoconstriction. Although butaprost has a much lower affinity for the EP4 receptor (5), butaprost actions on the EP4 receptor may be unmasked in mice lacking EP2 receptors. PGE2 also stimulates renal renin release (13, 20), and the EP4 receptor is presently the best candidate for mediating this response (5). PGE2 stimulates cAMP and renin release from juxtaglomerular cells, and intrarenal renin mRNA is not different between wild-type and EP2-/- mice (38). In addition, EP4 but not EP2 receptors are abundantly expressed in glomeruli (6, 9, 36). Thus the results of the present study support the concept that PGE2 activation of the renin-angiotensin system opposes the PGE2-mediated increase in renal blood flow and is not EP2 receptor mediated and may be mediated by EP4 and/or IP receptor activation (Fig. 7). We cannot rule out the possibility that activation of other vasoactive pathways might participate in the PGE2-mediated afferent arteriolar vasoconstriction observed in EP2-/- mice.

The afferent arteriolar response to sulprostone was assessed in mice lacking EP2 receptors to determine whether EP2 receptors opposed EP1 and EP3 receptor-mediated vasoconstriction. Sulprostone decreased afferent arteriolar diameter to a greater extent in mice lacking EP2 receptors compared with EP2+/+ mice. This finding confirms that EP1 or EP3 receptor activation results in an increase in renal vascular resistance (Fig. 7) (5, 31, 37). There is still controversy regarding which EP receptor is responsible for the vasoconstrictor response to PGE2. A recent study demonstrated afferent arteriolar constriction in response to the EP1/3-selective agonist sulprostone, but this response was not blocked by the EP1 antagonist SC-51322 (37). These findings suggest that the EP3 receptor is primarily responsible for the renal vasoconstrictor response to PGE2. In contrast, evidence for EP1 but not EP3 receptors in rat preglomerular vessels has recently been demonstrated (31). Purdy and Arendshorst (31) did not observe inhibition of isoproterenol elevation of cAMP levels in renal microvessels by the EP3 agonist M&B28767. Interestingly, this same group has data that suggest that EP3 receptors are important to the control of renal hemodynamics in the mouse (3). Renal blood flow was elevated and the vasodilatory response to PGE2 was enhanced in mice that lack the EP3 receptor. Additionally, systemic administration of the EP3 agonist SC-46275 resulted in a prolonged elevation of arterial blood pressure in mice lacking EP2 receptors (43). The results of the present study demonstrate that EP2 receptors oppose the preglomerular vasoconstrictor response to sulprostone.

The contribution of endothelin-1 to the development of salt-sensitive hypertension is well established (30, 34). Interestingly, mice that lack EP2 receptors develop hypertension when fed a high-salt diet (21). Therefore, we investigated the contribution of EP2 receptors to oppose the afferent arteriolar vasoconstrictor response to endothelin-1. Afferent arterioles from EP2 -/- mice were more responsive to endothelin-1 compared with EP2+/+ mice. Along these lines, Oyekan and McGiff (28) demonstrated that the endothelin-1-evoked decreases in renal blood flow and glomerular filtration were enhanced by COX inhibition. In contrast, indomethacin attenuated the increase in renal vascular resistance, the afferent arteriolar decrease in diameter, and renal microvascular smooth muscle cell calcium response to endothelin-1 (18, 27). The results of the present study also suggest involvement of COX-derived vasodilator and vasoconstrictor metabolites in the afferent arteriolar response to endothelin-1. The enhanced response to endothelin-1 observed in mice lacking EP2 receptors was eliminated by COX inhibition. Thus PGE2 activation of EP2 receptors and the resultant vasorelaxation oppose COX-mediated renal vasoconstrictor mechanisms in response to endothelin-1.

In summary, afferent arteriolar diameter of EP2+/+ increased in response to PGE2 and butaprost, whereas PGE2 and butaprost decreased the diameter of afferent arterioles in EP2-/- mice. The renal vasoconstriction to butaprost in EP2-/- mice was eliminated by enalapril. This observation supports the concept that the renin-angiotensin system contributed to the PGE2-mediated vasoconstriction in EP2-/- mice. Mice lacking EP2 receptors also exhibited a greater vasoconstriction to the EP1 and EP3 agonist sulprostone. Endothelin-1 elicited a greater afferent arteriolar vasoconstrictor response in mice lacking EP2 receptors. COX inhibition ameliorated this enhanced endothelin-1 response in EP2-/- mice. Overall, these studies support the concept that EP2 receptors participate in the maintenance of afferent arteriolar function.


    ACKNOWLEDGEMENTS

The work presented in this manuscript was conducted at the Tulane University School of Medicine in Dr. Imig's laboratory. The authors thank P. Diechmann and S. Brandon for technical assistance with the experimental studies. Assistance with the statistical analysis was provided by Dr. J. Dias at the Medical College of Georgia.


    FOOTNOTES

This work was supported by National Institutes of Health Grants DK-38226 (to J. D. Imig), HL-59699 (to J. D. Imig), GM-15431 (to R. M. Breyer), DK-46205 (to R. M. Breyer), and DK-37097 (to M. D. Breyer).

Address for reprint requests and other correspondence: J. D. Imig, Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912-2500 (E-mail: jdimig{at}mail.mcg.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.

March 19, 2002;10.1152/ajprenal.00351.2001

Received 27 November 2001; accepted in final form 14 March 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aoki, J, Katoh H, Yasui H, Yamaguchi Y, Nakamura K, Hasegawa H, Ichikawa A, and Negishi M. Signal transduction pathway regulating prostaglandin EP3 receptor-induced neurite retraction: requirement for two different tyrosine kinases. Biochem J 340: 365-369, 1999[ISI][Medline].

2.   Audoly, L, Ma L, Feoktistov I, Breyer M, and Breyer R. EP3 receptor activation of cAMP response element mediated gene transcription. J Pharmacol Exp Ther 289: 140-148, 1999[Abstract/Free Full Text].

3.   Audoly, LP, Ruan X, Wagner VA, Goulet JL, Tilley SL, Koller BH, Coffman TM, and Arendshorst WJ. Role of EP2 and EP3 PGE2 receptors in control of murine renal hemodynamics. Am J Physiol Heart Circ Physiol 280: H327-H333, 2001[Abstract/Free Full Text].

4.   Båtshake, B, Nilsson C, and Sundelin J. Molecular characterization of the mouse prostanoid EP1 receptor gene. Eur J Biochem 231: 809-814, 1995[Abstract].

5.   Breyer, MD, and Breyer RM. Prostaglandin E receptors and the kidney. Am J Physiol Renal Physiol 279: F12-F23, 2000[Abstract/Free Full Text].

6.   Breyer, MD, Davis L, Jacobson HR, and Breyer RM. Differential localization of prostaglandin E receptor subtypes in human kidney. Am J Physiol Renal Fluid Electrolyte Physiol 270: F912-F918, 1996[Abstract/Free Full Text].

7.   Breyer, MD, Jacobson HR, and Breyer RM. Functional and molecular aspects of renal prostaglandin receptors. J Am Soc Nephrol 7: 8-17, 1996[Abstract].

8.   Breyer, MD, Zhang YH, Guan YF, Hao CM, Hebert RL, and Breyer RM. Regulation of renal function by prostaglandin E receptors. Kidney Int 54: S88-S94, 1998[ISI].

9.   Breyer, RM, Davis LS, Nian C, Redha R, Stillman B, Jacobson HR, and Breyer MD. Cloning and expression of the rabbit prostaglandin EP4 receptor. Am J Physiol Renal Fluid Electrolyte Physiol 270: F485-F493, 1996[Abstract/Free Full Text].

10.   Chaudhari, A, Gupta S, and Kirschenbaum MA. Biochemical evidence for PGI2 and PGE2 receptors in the rabbit renal preglomerular vasculature. Biochim Biophys Acta 1053: 156-161, 1990[ISI][Medline].

11.   Cupples, WA, Sakai T, and Marsh DJ. Angiotensin II and prostaglandins in control of vasa recta blood flow. Am J Physiol Renal Fluid Electrolyte Physiol 254: F417-F424, 1988[Abstract/Free Full Text].

12.   Funk, C, Furchi L, Fitzgerald G, Grygorczyk R, Rochette C, Bayne MA, Abramovitz M, Adam M, and Metters KM. Cloning and expression of a cDNA for the human prostaglandin E receptor EP1 subtype. J Biol Chem 268: 26767-26772, 1993[Abstract/Free Full Text].

13.   Hockel, G, and Cowley A. Prostaglandin E2-induced hypertension I conscious dogs. Am J Physiol Heart Circ Physiol 237: H449-H454, 1979[Abstract/Free Full Text].

14.   Imig, JD. Eicosanoid regulation of the renal vasculature. Am J Physiol Renal Physiol 279: F965-F981, 2000[Abstract/Free Full Text].

15.   Imig, JD, and Deichmann PC. Afferent arteriolar responses to ANG II involve activation of PLA2 and modulation by lipoxygenase and P-450 pathways. Am J Physiol Renal Physiol 273: F274-F282, 1997[Abstract/Free Full Text].

16.   Imig, JD, Gebremedhin D, Harder DR, and Roman RJ. Modulation of vascular tone in renal microcirculation by erythrocytes: role of EDRF. Am J Physiol Heart Circ Physiol 264: H190-H195, 1993[Abstract/Free Full Text].

17.   Imig, JD, Navar GL, Zou LX, O'Reilly KC, Allen PL, Kaysen JH, Hammond TG, and Navar LG. Renal endosomes contain angiotensin peptides, converting enzyme, and AT1A receptors. Am J Physiol Renal Physiol 277: F303-F311, 1999[Abstract/Free Full Text].

18.   Imig, JD, Pham BT, LeBlanc EA, Reddy KM, Falck JR, and Inscho EW. Cytochrome P450 and cyclooxygenase metabolites contribute to the endothelin-1 afferent arteriolar vasoconstrictor and calcium responses. Hypertension 35: 307-312, 2000[Abstract/Free Full Text].

19.   Inscho, EW, Carmines PK, and Navar LG. Prostaglandin influences on afferent arteriolar responses to vasoconstrictor agonists. Am J Physiol Renal Fluid Electrolyte Physiol 259: F157-F163, 1990[Abstract/Free Full Text].

20.   Jensen, BL, Schmid C, and Kurtz A. Prostaglandins stimulate renin secretion and renin mRNA in mouse renal juxtaglomerular cells. Am J Physiol Renal Fluid Electrolyte Physiol 271: F569-F669, 1996.

21.   Kennedy, CRJ, Zhang Y, Brandon S, Guan Y, Coffee K, Funk CD, Magnuson MA, Oates JA, Breyer MD, and Breyer RM. Salt-sensitive hypertension and reduced fertility in mice lacking the prostaglandin EP2 receptor. Nat Med 5: 217-220, 1999[ISI][Medline].

22.   Kiriyama, M, Ushikubi F, Kobayashi T, Hirata M, Sugimoto Y, and Narumiya S. Ligand binding specificities of the eight types and subtypes of the mouse prostanoid receptors expressed in Chinese hamster ovary cells. Br J Pharmacol 122: 217-224, 1997[Abstract].

23.   Morath, R, Klein T, Seyberth HW, and Nusing R. Immunolocalization of the four prostaglandin E2 receptor proteins EP1, EP2, EP3, and EP4 in human kidney. J Am Soc Nephrol 10: 1851-1860, 1999[Abstract/Free Full Text].

24.   Namba, T, Sugimoto Y, Negishi M, Irie A, Ushikubi F, Kakizuka A, Ito S, Ichikawa A, and Narumiya A. Alternative splicing of C-terminal tail of prostaglandin E receptor subtype EP3 determines G-protein specificity. Nature 365: 166-170, 1993[ISI][Medline].

25.   Navar, LG, Inscho EW, Majid DSA, Imig JD, Harrison-Bernard LM, and Mitchell KD. Paracrine regulation of the renal microcirculation. Physiol Rev 76: 425-536, 1996[Abstract/Free Full Text].

26.   Nishigaki, N, Negishi M, Honda A, Sugimoto Y, Namba T, Narumiya S, and Ichikawa A. Identification of prostaglandin E receptor "EP2" cloned from mastocytoma cells as EP4 subtype. FEBS Lett 364: 339-341, 1995[ISI][Medline].

27.   Oyeken, A, Balazy M, and McGiff JC. Renal oxygenases: differential contribution to vasoconstriction induced by ET-1 and ANG II. Am J Physiol Regul Integr Comp Physiol 273: R293-R300, 1997[Abstract/Free Full Text].

28.   Oyekan, AO, and McGiff JC. Cytochrome P-450 derived eicosanoids participate in the renal functional effects of ET-1 in the anesthetized rat. Am J Physiol Regul Integr Comp Physiol 274: R52-R61, 1998[Abstract/Free Full Text].

29.   Parekh, N, Zou AP, Jungling I, Endlich K, Sadowski J, and Steinhausen M. Sex differences in control of renal outer medullary circulation in rats: role of prostaglandins. Am J Physiol Renal Fluid Electrolyte Physiol 264: F629-F636, 1993[Abstract/Free Full Text].

30.   Pollock, DM. Renal endothelin in hypertension. Curr Opin Nephrol Hypertens 9: 157-164, 2000[ISI][Medline].

31.   Purdy, KE, and Arendshorst WJ. EP1 and EP4 receptors mediate prostaglandin E2 actions in the microcirculation of rat kidney. Am J Physiol Renal Physiol 279: F755-F764, 2000[Abstract/Free Full Text].

32.   Roman, RJ, and Lianos EA. Influence of prostaglandins on papillary blood flow and pressure-natriuresis response. Hypertension 15: 29-35, 1990[Abstract].

33.   Ruan, X, Chatziantoniou C, and Arendshorst WJ. Impaired prostaglandin E2/prostaglandin I2 receptor-Gs protein interactions in isolated renal resistance arterioles of spontaneously hypertensive rats. Hypertension 34: 1134-1141, 1999[Abstract/Free Full Text].

34.   Schiffrin, EL. Role of endothelin-1 in hypertension. Hypertension 34: 876-881, 1999[Abstract/Free Full Text].

35.   Silldorff, EP, Yang S, and Pallone TL. Prostaglandin E2 abrogates endothelin-induced vasoconstriction in renal outer medullary descending vasa recta of rat. J Clin Invest 95: 2734-2740, 1995[ISI][Medline].

36.   Sugimoto, Y, Namba T, Shigemoto R, Negishi M, Ichikawa A, and Narumiya S. Distinct cellular localization of mRNAs for three subtypes of prostaglandin E receptor in kidney. Am J Physiol Renal Fluid Electrolyte Physiol 266: F823-F828, 1994[Abstract/Free Full Text].

37.   Tang, L, Loutzenhiser K, and Loutzenhiser R. Biphasic actions of prostaglandin E2 on the renal afferent arteriole: role of EP3 and EP4 receptors. Circ Res 86: 663-670, 2000[Abstract/Free Full Text].

38.   Tilley, SL, Audoly LP, Hicks EH, Kim HS, Flannery PJ, Coffman TM, and Koller BH. Reproductive failure and reduced blood pressure in mice lacking EP2 prostaglandin E2 receptor. J Clin Invest 103: 1539-1545, 1999[Abstract/Free Full Text].

39.   Toh, H, Ichikawa A, and Narumiya S. Molecular evolution of receptors for eicosanoids. FEBS Lett 361: 17-21, 1995[ISI][Medline].

40.   Watabe, A, Sugimoto Y, Irie A, Namba T, Negishi M, Ito S, Narumiya S, and Ichikawa A. Cloning and expression of cDNA for a mouse EP1 subtype of prostaglandin E receptor. J Biol Chem 268: 20175-20178, 1993[Abstract/Free Full Text].

41.   Whorton, AR, Misono K, Hollifield J, Frolich JC, Inagami T, and Oates JA. Prostaglandins and renin release: I. Stimulation of renin release from rabbit renal cortical slices by PGI2. Prostaglandins 14: 1095-1104, 1977[Medline].

42.   Yared, A, Kon V, and Ichikawa I. Mechanism of preservation of glomerular perfusion and filtration during acute extracellular volume depletion: importance of intrarenal vasopressin-prostaglandin interaction for protecting kidneys from constrictor action of vasopressin. J Clin Invest 75: 1477-1487, 1985[ISI][Medline].

43.   Zhang, Y, Guan Y, Schneider A, Brandon S, Breyer RM, and Breyer MD. Characterization of murine vasopressor and vasodepressor prostaglandin E2 receptors. Hypertension 35: 1129-1134, 2000[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 283(3):F415-F422
0363-6127/02 $5.00 Copyright © 2002 the American Physiological Society