© 2000 American Heart Association, Inc.
Integrative Physiology |
Biphasic Actions of Prostaglandin E2 on the Renal Afferent Arteriole
Role of EP3 and EP4 Receptors
From the Smooth Muscle Research Group, Department of Pharmacology and Therapeutics, The University of Calgary, Calgary, Alberta, Canada.
Correspondence to Rodger D. Loutzenhiser, PhD, Department of Pharmacology and Therapeutics, The University of Calgary, Health Sciences Centre, 3330 Hospital Dr NW, Calgary, Alberta T2N 4N1, Canada. E-mail rloutzen{at}ucalgary.ca
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Abstract |
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Abstract—Prostaglandin (PG) E2 is an important modulator of the actions of angiotensin (Ang) II. In the present study, we investigated the renal microvascular actions of PGE2 and the EP receptor subtypes involved. Ibuprofen potentiated Ang II–induced vasoconstriction in in vitro perfused normal rat kidneys and augmented afferent arteriolar, but not efferent arteriolar, responses in the hydronephrotic rat kidney model. This preglomerular effect of endogenous prostanoids was mimicked by exogenous PGE2, which reversed Ang II–induced afferent arteriolar vasoconstriction at concentrations of 0.1 to 10 nmol/L without affecting the efferent arteriole. The PGE2-induced vasodilation was potentiated by the phosphodiesterase inhibitor Ro 20-1724 and was mimicked by 11-deoxy-PGE1 (0.01 to 1 nmol/L). Butaprost, which acts preferentially at EP2 receptors, was relatively ineffective. Whereas 0.1 to 10 nmol/L PGE2 elicited vasodilation, higher concentrations (1 to 10 µmol/L) restored Ang II–induced afferent arteriolar vasoconstriction. This response was blocked by pertussis toxin (200 µg/mL) and was mimicked by the EP1/EP3 agonist sulprostone (1 to 300 nmol/L). Reverse transcription–polymerase chain reaction of individually isolated afferent arterioles revealed the presence of message for EP4 and all 3 EP3 splice variants (
, ß,
and 
) but not EP1 or EP2. Our findings thus
indicate that PGE2 elicits both vasodilatory and
vasoconstrictor actions on the afferent arteriole. The vasodilation is
mediated by EP4 receptors coupled to cAMP, presumably via
Gs. The vasoconstriction is mediated by an
EP3 receptor coupled to Gi and appears to
reflect a functional antagonism of the EP4-induced
vasodilation.
Key Words: receptors • microcirculation • arterioles • cyclooxygenase • angiotensin II
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Prostaglandin (PG) E2 is the major prostanoid produced by the kidney and plays an important role in modulating the renal microvascular response to vasoconstrictors.1 During conditions associated with low renal perfusion pressure, elevated renin activity, and increased renal sympathetic traffic, normal renal function may depend on the modulatory actions of PGE2 on angiotensin (Ang) II– and catecholamine-induced renal vasoconstriction. In clinical settings such as congestive heart failure, interference with this compensatory mechanism by the administration of nonsteroidal anti-inflammatory agents can lead to a deterioration of renal function and renal insufficiency.2 It has now been disclosed that the diverse biological actions of PGE2 are mediated by specific members of a group of G protein–linked (EP) receptors (reviewed in Reference 3 ). Thus, future therapies may allow a more selective intervention based on the development of selective receptor antagonists capable of blocking undesirable effects of the prostanoids while preserving their renal protective actions. This approach will require information on the renal microvascular actions of PGE2 and the identification of the specific EP receptor subtypes involved.
Although PGE2 is recognized to play a critical role in modulating renal vasoconstriction, the actions of PGE2 on the renal arterioles have not been thoroughly investigated. Only 2 studies to date have directly examined the renal microvascular actions of PGE2. In a study of isolated rabbit arterioles, Edwards4 reported that PGE2 elicited afferent arteriolar vasodilation but had no effect on the efferent arteriole. No constrictor responses to PGE2 were observed in this study. In contrast, Inscho et al,5 using the blood-perfused juxtamedullary nephron preparation, found 1.0 µmol/L PGE2 to evoke afferent arteriolar vasoconstriction in the rat. Only 1 concentration was used, and the effects of PGE2 on the efferent arteriole were not examined. Neither study examined the EP receptor subtypes or signaling pathways involved.
At least 4 distinct genes encoding for 4 separate subtypes of
PGE2 receptors, EP1 to
EP4 (reviewed in Reference 3 ),
have been identified. The mRNA for all 4 EP receptor subtypes has been
reported to be expressed in the kidney.6 7 8 9
EP2 and EP4 receptors are
generally associated with smooth muscle relaxation, whereas
EP1 and EP3 receptors are
reported to elicit smooth muscle contractile responses. However,
although the receptor subtypes involved in the tubular epithelial
actions of PGE2 have been partially characterized
(reviewed in Reference 10 ), we know little of the EP
receptor subtypes and signaling pathways involved in the renal
microvascular actions of PGE2. In the present
study, we used the in vitro perfused normal rat kidney and the in vitro
perfused hydronephrotic rat kidney models to investigate the actions of
PGE2 on the afferent and efferent arterioles. We
used reverse transcription–polymerase chain reaction (RT-PCR) assays
of microdissected vessels to determine EP receptor subtype expression
in the renal afferent arteriole. Our findings indicate that
PGE2 exerts a selective effect on the afferent
arteriole, eliciting both vasodilation and vasoconstriction of this
vessel. The vasodilation appears to be mediated by the
EP4 receptor coupled to a cAMP-dependent
mechanism. The vasoconstrictor actions of PGE2
are mediated by an EP3 receptor coupled to
Gi and appear to involve a functional
antagonism of the EP4-induced vasodilation.
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Materials and Methods |
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In Vitro Perfused Hydronephrotic Kidney
Male Sprague-Dawley rats were used in all experiments. The in vitro hydronephrotic rat kidney11 was used to examine the afferent and efferent arteriolar actions of PGE2. Unilateral hydronephrosis was induced by ligating the left ureter. Kidneys were harvested after 6 weeks, when tubular atrophy allows a direct visualization of the renal microvasculature. The renal artery was cannulated, and the kidney was excised with continuous perfusion. Kidneys were perfused at a renal arterial pressure of 80 mm Hg, with a modified Dulbecco’s medium containing 30 mmol/L bicarbonate, 5 mmol/L glucose, and 5 mmol/L HEPES and equilibrated with 95% air/5% CO2 (37°C, pH 7.4). Diameters were measured by online image processing. Afferent diameters were measured between the midpoint and origin. Efferent arteriolar diameters were measured within 50 µm of the glomerulus.
Initial studies were performed to determine the effects of basal prostaglandins. In all subsequent studies, kidneys were pretreated with 10 µmol/L ibuprofen to eliminate endogenous prostanoids. PGE2 was obtained from Calbiochem and prepared fresh daily. Ro 20-1724 was obtained from Research Biochemicals International and was administered at 0.5 µmol/L. Sulprostone and 11-deoxy-PGE1 were obtained from Cayman Chemical Co. Butaprost was provided by Dr Kluender of Bayer Pharmaceutical Division. SC-51322 was obtained from BIOMOL Research Laboratories.
In Vitro Perfused Normal Kidney
For the normal kidney studies, rats weighing from 220 to
275 g were anesthetized with methoxyflurane. The left
renal artery was cannulated in situ, and the kidney was excised with
continuous perfusion. Kidneys were perfused in vitro at 80 mm Hg
as described above. Perfusate flow was measured with a
transonic flowmeter (model T106, Transonic Systems, Inc).
EP mRNA Expression in Isolated Afferent Arterioles
Normal kidneys were perfused with 2% agarose (37°C in DMEM)
and chilled (4°C). Cortical slices (400 µm) were prepared and
treated with collagenase IV, hyaluronidase IV, dispase II,
and DNAse I. Six to 8 arterioles were individually isolated and washed
3 times. RT-PCR was run on the final wash to test for contamination.
Arteriolar total RNA was extracted, and cDNA was synthesized with
random primers. To eliminate genomic contamination, a separate sample
was treated identically, but without reverse transcriptase (Figure 8A
and 8B). Primers for RT-PCR assays were designed from the
reported EP receptor sequences12 13 14 15 16 17 (Table 1 online, see
http://www.circresaha.org). We assayed for a sequence common to all
EP3 receptors
(EP3ß) and sequences specific for
EP1, EP2,
EP4, and each EP3 splice
variant. A whole-kidney homogenate was used as a positive
control, and primers for GAPDH18 were used for an internal
control. After 35 cycles, PCR products were separated by
electrophoresis (2% agarose gel) and stained with ethidium bromide.
The identity of the products obtained with each set of primers was
confirmed by DNA sequencing. When negative results were obtained, fresh
polymerase was added after 35 cycles, and the PCR was continued to 70
cycles.
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Analysis
Data are expressed as the mean±SEM. Differences were evaluated
by ANOVA and Student’s t test (paired or unpaired). Values
of P<0.05 were considered significant. For multiple
comparisons, the Bonferroni correction was applied, and values of
P<0.05/n were considered significant.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
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Results |
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Initial studies were conducted to determine the influence of endogenous prostaglandins on Ang II responses of the in vitro perfused hydronephrotic kidney preparation and of in vitro perfused normal rat kidneys. The effects of 10 µmol/L ibuprofen on the afferent and efferent arteriolar actions of Ang II are presented in Figure 1A
. Basal
diameters were not affected by ibuprofen in either the afferent
(15.6±1.8 versus 15.4±1.6 µm, n=8, P=0.84) or
efferent (11.2±1.2 versus 11.7±1.2 µm, n=8, P=0.68)
arteriole. In the afferent arteriole, Ang II elicited significant
vasoconstriction at 0.1 nmol/L (11.4±2.2 µm,
P=0.003) in controls and at 0.03 nmol/L (11.2±1.4
µm, P=0.0016) in the presence of ibuprofen. As shown in
Figure 1A, ibuprofen pretreatment resulted in significant
potentiation of the afferent arteriolar response at Ang II
concentrations of 0.01 to 0.1 nmol/L (P<0.05). In contrast,
the efferent arteriolar response to Ang II was not significantly
altered by ibuprofen, and 0.03 nmol/L Ang II elicited significant
efferent arteriolar vasoconstriction in both settings.
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Ibuprofen also potentiated Ang II–induced vasoconstriction in normal
perfused rat kidneys (Figure 1B
). In the control group, basal
perfusate flows were 14.6±0.9 mL ·
min-1 · g kidney
wt-1 (n=5), and Ang II elicited significant
vasoconstriction at 0.1 nmol/L (9.5±1.0 mL ·
min-1 · g-1,
P<0.0001). In kidneys pretreated with 10 µmol/L
ibuprofen, basal perfusate flows were 13.4±0.6 mL ·
min-1 · g-1
(P=0.26 versus control, n=5) and Ang II elicited significant
vasoconstriction at 0.03 nmol/L (11.8±0.5 mL ·
min-1 · g-1,
P=0.0036). As presented in Figure 1B, ibuprofen significantly potentiated the vasoconstrictor response to Ang
II at concentrations of 0.03 and 0.1 nmol/L (P<0.05). In
concert, the findings presented in Figures 1A and 1B
illustrate that the threshold for Ang II–mediated vasoconstriction
occurs over similar concentrations in the in vitro perfused
hydronephrotic kidney and normal kidney preparation and that
cyclooxygenase inhibition potentiates reactivity to
Ang II in both models.
Because the major prostanoid produced by the kidney is
PGE2, we examined the effects of exogenous
PGE2 on the afferent and efferent arteriolar
response to Ang II. In these studies and those that follow, kidneys
were pretreated with 10 µmol/L ibuprofen to eliminate
endogenous prostanoid formation. As shown in Figure 2, 0.1 nmol/L Ang II reduced afferent
arteriolar diameter by 43±7% (n=8) and efferent arteriolar diameter
by 23±5% (n=7, P<0.001). PGE2
exerted a biphasic action on the afferent arteriole. Over
concentrations ranging from 1 to 10 nmol/L, PGE2
elicited afferent arteriolar vasodilation, completely reversing the Ang
II–induced vasoconstriction. At concentrations >100 nmol/L,
PGE2 elicited vasoconstriction. Thus, whereas
PGE2 completely reversed Ang II–induced
vasoconstriction at a concentration of 10 nmol/L (14.2±0.6, 8.2±1.2,*
and 13.4±0.7 µm for control, Ang II, and Ang II plus 10 nmol/L
PGE2, respectively; *P<0.05 versus
control), increasing the PGE2 concentration to
1.0 µmol/L fully restored the vasoconstriction (7.0±1.4
µm, P>0.05 versus Ang II alone). In the absence of Ang II
(Figure 2, top), PGE2 also elicited
a modest vasodilation and constriction at concentrations of 10 nmol/L
and 1 µmol/L (P<0.05). However, the constrictor
actions of PGE2 (1.0 µmol/L) were far
greater in the presence of Ang II (51±8% decrease in diameter) than
in its absence (11±3% decrease). Finally, in contrast to the afferent
arteriole, the efferent arteriolar actions of Ang II were not
significantly altered by PGE2 over concentrations
ranging from 1.0 nmol/L to 1.0 µmol/L (Figure 2, bottom).
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, n=8). Bottom, Lack of effect of
PGE2 on reactivity of the efferent arteriole to Ang II
(n=7).
To investigate the signaling pathway involved in the vasodilation, we
determined the effects of the specific type IV phosphodiesterase
inhibitor Ro 20-1724. A concentration of 0.5 µmol/L
Ro 20-1724 was selected for these studies because we found that this
concentration does not produce vasodilation by itself but rather
potentiates the vasodilatory actions of adenosine (not shown).
Two groups of hydronephrotic kidneys (controls and Ro 20-1724–treated,
n=5 for each) were preconstricted with 0.1 nmol/L Ang II, and the
afferent arteriolar vasodilatory actions of PGE2
were assessed. The inhibition of the Ang II–induced vasoconstriction
was calculated, and these data are summarized in Figure 3. Ro 20-1724 markedly potentiated the
actions of PGE2, shifting the
ED50 by 
1 log unit. These findings are
consistent with the currently held view that the vasodilatory
actions of PGE2 are mediated by cAMP.
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Both EP2 and EP4 have been
shown to be coupled to Gs and are thought to
produce vasodilation by stimulating the production of
cAMP.3 To characterize the receptor involved in the
afferent arteriolar vasodilation, we compared the potency of
11-deoxy-PGE1 and butaprost. These 2 agonists
have similar affinities for the EP2 receptor but
markedly different affinities for the EP4
receptor.13 19 Figure 4
summarizes the results of studies comparing the actions of these 2
agonists on afferent arterioles preconstricted with 0.1 nmol/L Ang II
(n=5 for both groups). As depicted, 11-deoxy-PGE1
fully reversed Ang II–induced afferent arteriolar vasoconstriction
(ED50 0.1 nmol/L). In contrast, butaprost was
a relatively weak vasodilator in this preparation. These results are
consistent with an EP4-mediated
vasodilation.3 13 19 20 21
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The biphasic response of the afferent arteriole to
PGE2 indicates actions at multiple receptor
subtypes, linked to both vasodilator and vasoconstrictor mechanisms. To
characterize the constrictor response, we examined the effects of
pretreatment with pertussis toxin (PTX). In these studies, kidneys were
pretreated with 200 µg/mL PTX for 1 hour with a recirculating
circuit. Control kidneys were treated in an identical manner with
vehicle alone. The kidneys were then returned to the single-pass
perfusion system and challenged with Ang II and
PGE2. As shown in Figure 5, PTX had no effect on the magnitude of
the Ang II–induced vasoconstriction and did not alter the vasodilatory
actions of 10 nmol/L PGE2. However, PTX treatment
completely abolished the vasoconstrictor actions of 1.0 µmol/L
PGE2.
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Of the known PGE2 receptors, the
EP3 receptor subtype has been shown to be coupled
to the PTX-sensitive G protein
Gi.3 We next examined the effects
of the EP3/EP1 receptor
agonist sulprostone.3 22 As shown in Figure 6A, sulprostone did not elicit afferent
arteriolar vasodilation in kidneys preconstricted with Ang II and, in
this setting, had no vasoconstrictor activity. However, as described
above (Figure 2), the vasoconstrictor actions of
PGE2 were much more evident when administered to
preconstricted vessels that were then dilated with low concentrations
of PGE2. We therefore designed studies to mimic
this condition, first inducing vasoconstriction with Ang II, then
eliciting full vasodilation with 10 nmol/L PGE2,
and then administering sulprostone (Figure 6B). Under these
conditions, sulprostone caused a dose-dependent vasoconstriction,
eliciting threshold contractions at concentrations of 1.0 to 10 nmol/L.
Because sulprostone can activate both EP1
and EP3, we examined the effects of SC-51322, an
EP1-selective antagonist
(pA2=8.1),23 on this response. As
shown in Figure 1 online (see http://www.circresaha.org), SC-51322 at
concentrations of 3 and 10 µmol/L had no effect on the
sulprostone-induced vasoconstriction, suggesting that this response is
mediated by EP3 receptors.
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The results of the above studies suggest that an
EP3 receptor coupled to a PTX-sensitive G protein
mediates the afferent arteriolar vasoconstrictor effects of
PGE2 in the hydronephrotic kidney model. To
determine whether a similar response occurs in the nonhydronephrotic
kidney, parallel studies were performed using isolated perfused normal
kidneys. Kidneys were treated with 10 µmol/L ibuprofen and
perfused at a constant arterial pressure of 80 mm Hg.
Vasoconstriction was established with 0.1 nmol/L Ang II, and then 10
nmol/L PGE2 was administered. As depicted in
Figure 7, PGE2 partially reversed the Ang II–induced
vasoconstriction, consistent with a selective afferent
arteriolar vasodilation. In this setting, sulprostone elicited a
similar vasoconstrictor response.
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Finally, to determine the expression of EP receptors in the renal
afferent arteriole, RT-PCR was used to assay EP receptor mRNA in
individually isolated afferent arterioles. As depicted in Figure 8A, the presence of afferent arteriolar
message for EP3 (EP3ß)
and EP4 receptors, but not for
EP1 or EP2 receptors, could
be demonstrated with this approach. To further identify the
EP3 receptors present, individual primers for
mRNA regions specific for the , ß, and splice variants were
used. As shown in Figure 8B, we found the mRNA for all 3 splice
variants to be expressed in the normal afferent arteriole.
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Discussion |
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The diverse actions of PGE2 on the kidney are mediated by a distinct group of G protein–coupled receptors. Four genes encoding for differing PGE2 receptors have been cloned (EP1 to EP4), and message for all 4 receptor subtypes is found in the kidney.6 7 8 9 10 24 Previous studies have focused on the EP receptors mediating the renal tubular actions of PGE2, but no information is currently available on the renal microvascular EP receptors. The present study indicates that the afferent arteriole of the rat expresses message for the EP4 receptor and for 3 known splice variants of the EP3 receptor (
, ß, and ). We found
PGE2 to elicit vasodilation at low concentrations
(ED50 <3 nmol/L) by activating
EP4 receptors coupled to cAMP. This vasodilation
is reversed at higher concentrations (>100 nmol/L). This
vasoconstrictor response is mediated by an EP3
receptor coupled to Gi and reflects a
functional antagonism of the EP4-mediated
vasodilation.
The present study used the hydronephrotic rat kidney model to
assess microvascular responses. Although this model is a widely used
method of studying the renal microvasculature, it was important to
characterize the role of endogenous prostanoids in this
preparation. During the onset of hydronephrosis, infiltrating
macrophages increase renal thromboxane
production, eliciting vasoconstriction.25 26 For
microvascular studies, a long-term preparation (6 to 8 weeks’
duration) is used. At this stage, tubular atrophy is complete, renal
thromboxane production returns to normal
levels,27 and SQ29548 has no effect on basal tone,
indicating a lack of thromboxane-dependent
vasoconstriction.28 The reactivity of our in vitro model
to Ang II was similar to that of the normal kidney, because threshold
vasoconstrictor responses were observed at concentrations of 0.03 to
0.1 nmol/L in both preparations. Moreover, the reactivity to Ang II was
similarly potentiated by cyclooxygenase inhibition,
suggesting a predominant influence of endogenous
vasodilatory prostanoids in both models (Figure 1A and 1B).
Cyclooxygenase inhibition potentiated the afferent arteriolar actions of Ang II but had no effect on the efferent arteriole, indicating a selective preglomerular action of endogenous prostanoids in our model. These findings are consistent with previous observations using in vivo preparations. For example, Heller and Horacek29 and Olsen et al30 found that cyclooxygenase inhibition potentiates the ability of Ang II to reduce glomerular filtration rate in the dog. These authors suggested a protective role of vasodilatory prostanoids in suppressing Ang II–mediated afferent arteriolar vasoconstriction. Our results are in agreement with this interpretation and suggest that the prostanoid involved in this action is PGE2, the major prostanoid produced by the normal kidney1 2 and by cortical interstitial cells of the hydronephrotic kidney.26 Like the endogenous prostanoid, exogenous PGE2 altered the afferent arteriolar effects of Ang II but had no effect on the efferent arteriole. This selective afferent arteriolar effect of PGE2 observed in our rat kidney model agrees with previous findings by Edwards,4 who used isolated afferent and efferent arterioles from the rabbit. Edwards reported PGE2 to block the afferent arteriolar response to norepinephrine but to have no effect on the efferent arteriolar actions of either norepinephrine or Ang II. Ang II does not constrict the afferent arteriole in Edwards’ preparation.31
At concentrations >100 nmol/L, we found PGE2 to promote afferent arteriolar vasoconstriction. A number of previous studies have documented both vasodilator and vasoconstrictor actions of PGE2 (reviewed in Reference 11 ). Edwards did not observe PGE2-induced vasoconstriction in isolated rabbit arterioles.4 However, in a study using the blood-perfused juxtamedullary nephron preparation of the rat, Inscho et al5 found PGE2 to elicit afferent arteriolar vasoconstriction. In the Inscho study, only a single concentration of PGE2 (1.0 µmol/L) was used, and efferent arteriolar responses were not reported. Our observation that 1.0 µmol/L PGE2 elicited afferent arteriolar vasoconstriction in the hydronephrotic kidney model is consistent with these findings. The fact that PGE2 elicits both constrictor and dilator responses clearly indicates an involvement of multiple EP receptor subtypes.
EP2 and EP4 receptors are
both linked to vasodilation (reviewed in Reference 3 ).
Our studies indicate that in the afferent arteriole,
PGE2 elicits vasodilation by activating
EP4 receptors. In this vessel, we found
11-deoxy-PGE1 to be a potent vasodilator, whereas
butaprost was relatively ineffective (Figure 4). Butaprost and
11-deoxy-PGE1 are both EP2
agonists and exhibit similar potencies at this receptor
subtype.13 19 However, 11-deoxy-PGE1
is also a potent EP4 agonist, whereas butaprost
has very weak activity at this receptor.3 13 19 20 21 Thus,
the rank order of potencies we observed
(11-deoxy-PGE1 >> butaprost) suggests that the
vasodilation involves the EP4 rather than the
EP2 receptor. This interpretation is
consistent with the results of our RT-PCR studies, in that we
found that message for EP4 but not
EP2 was expressed in the afferent arteriole. We
could not demonstrate the presence of EP2 mRNA
even under extreme conditions (70 cycles). Previous studies have
reported low expression levels of EP2 mRNA in the
kidney and an absence of this signal in the renal cortex.6
We also found low levels of EP2 mRNA in
whole-kidney homogenates (Figure 8A). In contrast,
EP4 receptor mRNA is highly expressed in the
kidney and has been localized to the glomerulus.6 7 Both
EP2 and EP4 receptors have
been shown to be coupled to Gs and stimulation
of adenylyl cyclase (reviewed in Reference 3 ). The
phosphodiesterase inhibitor Ro 20-1724 potentiated the
vasodilatory response to PGE2 in our study, a
finding in agreement with previous observations suggesting that the
renal microvascular actions of PGE2 are mediated
by an elevation in cAMP.1 32 Our observations are thus
consistent with a signaling pathway involving
PGE2 stimulation of EP4
receptors, leading to the activation of Gs,
adenylyl cyclase, and vasodilation via cAMP-dependent mechanisms.
Our studies suggest that the vasoconstrictor actions of
PGE2 involve an EP3
receptor. Sulprostone caused vasoconstriction at concentrations of 10
to 100 nmol/L, whereas constrictor responses to
PGE2 occurred only at much higher concentrations
(0.3 to 1.0 µmol/L). Sulprostone acts on both
EP1 and EP3 receptors but
is less potent than PGE2 on
EP1 receptors and more potent on
EP3 receptors.3 13 20 The relative
potency of sulprostone versus PGE2 is thus
consistent with an EP3 response. This
interpretation is supported by our observation that SC53122, an
EP1-selective antagonist, did not
alter the vasoconstrictor response to sulprostone. The RT-PCR studies
are also consistent with an involvement of
EP3 rather than EP1
receptors, because we found that the afferent arteriole expresses
message for all 3 splice variants of the rat EP3
receptor, but we could not demonstrate message for
EP1 in this vessel. Finally, the contractile
response to PGE2 was prevented by PTX, indicating
a coupling to Go or
Gi. EP1-mediated
responses are insensitive to PTX and generally linked to
Gq, whereas Gi
coupling is a common characteristic of EP3
receptors.3 33 In concert, these observations provide
compelling evidence that the EP3 receptor
mediates PGE2-induced afferent arteriolar
vasoconstriction.
Thus, our functional studies indicate that in the rat, the renal microvascular actions of PGE2 are mediated by EP4 and EP3 receptors. We found no evidence for the expression of EP1 or EP2 receptors in the rat afferent arteriole. In contrast, Morath et al24 recently reported that in the human kidney, the afferent arteriole stains positively for all 4 EP receptor subtypes. One obvious explanation of this difference is the possibility of species-dependent variability in EP receptor expression. Another possibility is that EP1 and EP2 mRNAs are present, but at levels that were below the detection limits in our assay. However, we found the pharmacological attributes of the responses we observed to be consistent with the results of the RT-PCR assays in that each approach suggested a primary role of EP4 and EP3 receptors.
We found message for all 3 known splice variants of the rat
EP3 receptor in the rat afferent arteriole. The
variable region of the EP3 receptor subtypes
is confined to the intracellular carboxylic tail and determines G
protein coupling.34 Because the sequence is identical from
the amino-terminus through the seventh transmembrane domain, including
the external portion of the receptor, the EP3
, ß, and isoforms are thought to have similar ligand-binding
characteristics.3 All 3 rat EP3
receptor isoforms can couple to Gi.
EP3 can also couple to
Gs and can stimulate adenylyl
cyclase.3 16 34 However, sulprostone had no vasodilatory
effect on the afferent arteriole (Figure 6), which argues
against a role of EP3 receptors in the
PGE2-induced afferent arteriolar vasodilation. A
fourth variant, EP3
, is coupled to a
PTX–insensitive Gq.3 34 However,
to the best of our knowledge, this splice variant has not yet been
found in the rat.
Although the role of the vasodepressant action of
PGE2 is widely appreciated, the significance of
the PGE2-induced vasoconstriction is not
understood. In our study, PGE2 elicited a
pronounced vasoconstriction in the presence of Ang II but only a modest
response when administered alone (Figure 2). Similarly, full
expression of the sulprostone-induced vasoconstriction required
pretreatment with both Ang II and PGE2. We
interpret these observations as suggesting that
EP3 receptor activation alone is not sufficient
to cause vasoconstriction but rather promotes vasoconstriction by
counteracting EP4 signaling and reversing the
PGE2-induced vasodilation. Thus, at low
concentrations, PGE2 stimulates
EP4 receptors, activates
Gs and adenylyl cyclase, and elevates cAMP. At
higher concentrations or under conditions in which the
EP3 receptor expression is upregulated,
PGE2 concurrently stimulates
EP3 receptors. EP3 receptor
activation inhibits adenylyl cyclase and decreases cAMP via
Gi, thereby attenuating the
EP4-mediated vasodilation. Activation of
EP3 receptors by PGE2 and
sulprostone have been shown to reduce cAMP-dependent water flux in the
cortical collecting duct by a similar mechanism.33 Thus,
the EP3 receptor pathway could provide a
functional antagonism of the vasodepressant actions of
PGE2. At the moment, one can only speculate on
the potential significance of this interaction. However, it is
important to note that although the preglomerular
vasodepressor actions of PGE2 are beneficial in
maintaining adequate PGC during conditions of low renal perfusion
pressure, this action may be detrimental when blood pressure is
elevated, because myogenic vasoconstriction is also attenuated by
PGE2 (eg, see Reference 35 ).
Future studies assessing the impact of the EP3
receptor on the reactivity of the afferent arteriole to
PGE2 and on the differential regulation of
EP3 and EP4 receptor
expression may provide insights into the
physiological or
pathophysiological importance of the afferent
arteriolar EP3 receptor.
In summary, the present study demonstrates that
endogenous prostanoids and exogenous
PGE2 preferentially modulate the reactivity of
the afferent arteriole. In this vessel, PGE2
elicits a biphasic response, eliciting vasodilation at low
concentrations (1 to 10 nmol/L) and reversing this effect at higher
concentrations. The vasodilation is mediated by
EP4 receptors and is dependent on cAMP. The
vasoconstriction is mediated by an EP3 receptor
and is blocked by PTX, implicating a mechanism dependent on
Gi and reduced cAMP formation. These findings
suggest a functional antagonism of these 2 receptor systems. We suggest
that in the renal microvasculature, EP3 receptors
may play a modulatory role on the vasodepressant and renal
hemodynamic actions of PGE2.
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Acknowledgments |
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This work was supported by an operating grant from the Heart and Stroke Foundation of Alberta and the Northwest Territories. R. Loutzenhiser is a Senior Medical Scholar of the Alberta Heritage Foundation for Medical Research. The authors wish to thank Dr Mike Walsh, Dr Neal Davies, Bob Winkfein, and Cindy Sutherland for their help with the RT-PCR studies.
Received August 30, 1999; accepted January 6, 2000.
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