Afferent arteriolar adenosine A2a receptors are
coupled to KATP in in vitro perfused hydronephrotic rat
kidney
Lilong
Tang1,
Michael
Parker2,
Qing
Fei1, and
Rodger
Loutzenhiser2
1 Smooth Muscle Research Group, Department of
Pharmacology and Therapeutics, University of Calgary, Calgary,
Alberta, Canada T2N 4N1, and 2 Department of Pharmacology,
University of Miami School of Medicine, Miami, Florida
33125
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ABSTRACT |
Adenosine is known to exert
dual actions on the afferent arteriole, eliciting vasoconstriction, by
activating A1 receptors, and vasodilation at higher
concentrations, by activating lower-affinity A2 receptors.
We could demonstrate both of these known adenosine responses in the in
vitro perfused hydronephrotic rat kidney. Thus, 1.0 µM adenosine
elicited a transient vasoconstriction blocked by
8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and 10-30 µM
adenosine reversed KCl-induced vasoconstriction. However, when we
examined the effects of adenosine on pressure-induced afferent
arteriolar vasoconstriction, we observed a third action. In this
setting, a high-affinity adenosine vasodilatory response was observed
at concentrations of 10-300 nM. This response was blocked by both 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM-241385) and glibenclamide and was mimicked
by 2-phenylaminoadenosine (CV-1808) (IC50 of 100 nM),
implicating adenosine A2a receptors coupled to
ATP-sensitive K channels (KATP). Like adenosine,
5'-N-ethylcarboxamidoadenosine (NECA) elicited both
glibenclamide-sensitive and glibenclamide-insensitive vasodilatory
responses. The order of potency for the glibenclamide-sensitive component was NECA > adenosine = CV-1808. Our findings suggest that,
in addition to the previously described adenosine A1 and low-affinity A2b receptors, the renal microvasculature is
also capable of expressing high-affinity adenosine A2a
receptors. This renal adenosine receptor elicits afferent arteriolar
vasodilation at submicromolar adenosine levels by activating
KATP.
ATP-sensitive potassium channels; glibenclamide; CV-1808; 5'-N-ethylcarboxamidoadenosine; ZM-241385; renal
microcirculation; afferent arteriole; hydronephrosis; myogenic
vasoconstriction; adenosine 3',5'-cyclic monophosphate; Ro-20-1724
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INTRODUCTION |
ADENOSINE IS AN IMPORTANT modulator of renal vascular
resistance. In the normal kidney, adenosine elicits both
vasoconstriction and vasodilation (reviewed in Refs. 16 and 28).
Physiological or pathophysiological elevations of adenosine are
generally associated with renal afferent arteriolar vasoconstriction
and a reduction in glomerular filtration rate (GFR). This
vasoconstrictor response is mediated by activation of the adenosine
A1 receptor subtype (24) and is purported to contribute to
a metabolic regulation of GFR (18, 25). Abnormal adenosine-induced
vasoconstriction has also been demonstrated to contribute to renal
pathophysiology in models of acute renal insufficiency (1, 7). Less is
known of the role of adenosine-induced renal vasodilation.
Experimentally, vasodilation is usually observed only at very high
adenosine concentrations and is believed to be mediated by the
activation of a low-affinity adenosine A2 receptor subtype
coupled to adenylate cyclase (16, 28). However, studies in conscious
animal models and anecdotal reports in humans subjects suggest
important additional renal vasodilatory actions of adenosine (1, 26).
In the present study, we examined the renal microvascular effects of
adenosine in the in vitro perfused hydronephrotic rat kidney (15).
Using this model, we could demonstrate both of the renal vascular
responses previously described (adenosine A1 receptor-mediated vasoconstriction and low-affinity adenosine A2 receptor-induced vasodilation). However, in addition to
these two actions of adenosine, we observed a third type of adenosine response. Submicromolar concentrations (10-300 nM) of adenosine were capable of reversing or inhibiting pressure-induced afferent arteriolar vasoconstriction. This novel vasodilatory response occurred
at concentrations well below that required to elicit adenosine A1-induced vasoconstriction, and was blocked by
both 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM-241385) and glibenclamide. Thus our findings indicate that the
renal microvasculature is capable of expressing high-affinity adenosine
A2a receptors and that the afferent arteriolar vasodilatory response mediated by the activation of this receptor type is linked to
the activation of ATP-sensitive K channels.
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METHODS |
Isolated perfused hydronephrotic kidney. The isolated
hydronephrotic rat kidney was used to examine the effects of adenosine on the renal afferent arteriole. Unilateral hydronephrosis was produced
by ligating the left ureter under methoxyflurane-induced anesthesia.
Hydronephrotic kidneys were harvested after 6 wk. Rats were
anesthetized with methoxyflurane, the renal artery of the
hydronephrotic kidney was cannulated, and the kidney was excised for in
vitro perfusion. During the initial cannulation and throughout the
isolation procedure, kidneys were continuously perfused with medium to
avoid a disruption of nutritive flow or exposure to hypoxia or ischemia.
The perfusing apparatus used in the present study employed a
single-pass presentation of medium to the kidney (13, 15, 23). The
medium was pumped on demand through a heat exchanger to a pressurized
reservoir, supplying the renal artery. Perfusion pressure was monitored
within the renal artery and altered by adjusting the pressure within
the reservoir. Kidneys were perfused with Dulbecco's modified Eagle's
medium (GIBCO; Life Technologies, Gaithersburg, MD)
containing 30 mM bicarbonate, 5 mM glucose, and 5 mM HEPES (GIBCO) and
passed through 0.2-µm filters before use. The perfusate was
equilibrated with 95% air-5% CO2
(PO2 = 150 Torr). Temperature and pH were
maintained at 37°C and 7.40, respectively.
Kidneys were allowed to equilibrate for at least 1 h before initiating
study protocols. To assess the effects of DPCPX and diltiazem on the
contractile responses to adenosine, initial responses to 1.0 µM
adenosine were assessed; the kidneys were then washed 10-20 min
with control media or media containing either blocker, and the
adenosine responses were reassessed. The effects of
4-[(3-butoxy-4-methoxyphenyl) methyl]-2-imidaxolidinone
(Ro-20-1724) were assessed using a similar experimental design, in
which initial responses were obtained, and then responses were
reassessed after 10-min pretreatment with 0.5 µM Ro-20-1724. In
studies examining the effects of adenosine and adenosine analogues on
the response of the afferent arteriole to stepped increases in
pressure, we first obtained two to three reproducible control pressure
responses. Renal arterial pressure was increased from 60 to 180 mmHg in
20-mmHg steps and held at each pressure for 1 min. Pressure was then
returned to 80 mmHg, kidneys were pretreated for 10 min with the
adenosine analogue at each concentration, and reactivity was
reassessed. We then sought to assess the impact of the KATP
channel blocker glibenclamide (10 µM) on the responses
to each adenosine analogue. In these studies, glibenclamide was added
at the onset of the study and was present throughout the experiment. To
cover the full concentration range for adenosine, 25 kidneys were used.
Of these, 11 were exposed to only 4 concentrations, and 14 were exposed
to each concentration. These data were combined. The single-pass
adaptation of the in vitro perfused hydronephrotic kidney model is very
stable, and reproducible constrictor responses to
angiotensin II and pressure ramps are obtained for at least 6-8 h
(e.g., see Refs. 15, 23). Moreover, KATP appears to be
quiescent in the basal state of this preparation, as we have previously
found glibenclamide to have no effect on the basal reactivity to either
angiotensin II or elevated pressure (15, 22, 23).
Materials. Adenosine, glibenclamide, DCPCX,
2-phenylaminoadenosine (CV-1808),
5'-N-ethylcarboxamidoadenosine (NECA), and
Ro-20-1724 were purchased from Research Biochemicals International
(Natick, MA). ZM-241385 was purchased from Tocris Cookson (Ballwin, MO).
Analysis of data. Video images were transmitted to computer
with a video acquisition board (model IVG-128; Datacube, Peabody, MA)
for online analysis. Custom software was used to measure vessel diameter from the digitized video image as described in detail in
previous publications (13). Vessel segments (20-30 µm in length)
were scanned automatically at 1- to 2-s intervals. The mean-diameter
measurements obtained during the plateau of the response were then
averaged. Typically, one diameter determination was derived from the
mean of 30-40 individual measurements, each in turn representing
the mean diameter over the length of arteriolar segment.
Throughout the text, data are expressed as the mean followed by the
standard error of the mean, as an index of dispersion. The number of
replicates refers to the number of afferent arterioles examined.
Differences between means were evaluated by analysis of variance
followed by Student's t-test (paired or unpaired). Probabilities (P) less than 0.05 were considered statistically significant.
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RESULTS |
Figures 1 and 2 demonstrate the classic
renal adenosine A1 and A2 responses observed in
the in vitro perfused hydronephrotic rat kidney preparation. As
depicted in Fig. 1, the administration of 1.0 µM adenosine elicited a
transient afferent arteriolar vasoconstriction that spontaneously
abates after 5-10 min. This vasoconstriction was blocked by 0.1 µM 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (Fig. 1, A and
C), an adenosine A1 antagonist. The
vasoconstriction was also blocked by diltiazem (Fig. 1D) and
thus exhibits characteristics quite similar to adenosine
A1-mediated responses reported using other renal
microvascular models (24).

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Fig. 1.
Adenosine-induced afferent arteriolar vasoconstriction. A:
tracing of transient afferent arteriolar vasoconstrictor response to
1.0 µM adenosine (ADO) and blockade of this response by the
A1 adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine
(DPCPX, 0.1 µM). B: mean time control data illustrating
sequential responses to adenosine (n = 5). C: blockade
of response by DPCPX (n = 5). D: blockade of the
response by 10 µM diltiazem (n = 5). ADO-1 and ADO-2 refer to
the first and second applications of adenosine (compare with
A). CTL-1 and CTL-2 refer to pre-adenosine (control)
diameters.
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Figure 2 illustrates the vasodilation seen
in response to stimulation of low-affinity adenosine A2
receptors. In these experiments, afferent arteriolar tone was increased
by elevating medium potassium concentration to 30 mM, and
adenosine-induced vasodilation was observed over a range of 3-30
µM. In these studies, KCl decreased afferent arteriolar diameter from
12.3 ± 0.5 to 3.2 ± 0.2 µm (n = 5). The addition of 1 µM adenosine did not cause a further decrease in afferent arteriolar
diameter in the presence of 30 mM KCl (3.4 ± 0.4 µm), indicating
either that adenosine has no further effect on depolarized vessels or
that the response to KCl was maximal. At 3, 10, and 30 µM adenosine,
diameters increased to 4.5 ± 0.6, 6.4 ± 0.6 (P < 0.01),
and 7.9 ± 0.9 µm (P < 0.01), respectively. It is
important to emphasize that we have previously shown that KATP-dependent vasodilation is abolished by the presence of
30 mM KCl in this preparation (15, 22). The vasodilation elicited by
1-30 µM adenosine was potentiated by 0.5 µM Ro-20-1724, a
selective phosphodiesterase inhibitor (11). In these experiments, KCl reduced diameters from 12.4 ± 0.8 to 3.8 ± 0.3 µm (n = 5). The addition of 0.3 µM Ro-20-1724 did not significantly alter
diameter (3.9 ± 0.3 µm) alone, but potentiated the vasodilatory
actions of adenosine. Thus, 1, 3, 10, and 30 µM adenosine increased
diameters to 5.7 ± 0.5 (P < 0.005), 7.7 ± 0.7 (P < 0.005), 8.6 ± 0.8 (P < 0.001), and 10.1 ± 0.5 µm
(P < 0.001), respectively. These data are plotted as the
percent inhibition of KCl-induced vasoconstriction in Fig. 2.
Ro-20-1724 potentiated the response at each concentration of adenosine.
These findings are consistent with the purported role of cAMP in renal
response to stimulation of low-affinity A2b receptors (28).

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Fig. 2.
Adenosine-induced vasodilation of arterioles preconstricted with 30 mM
KCl (to eliminate any contribution of altered K channel activity).
Under control conditions (open circles), vasodilation was initiated
only at adenosine concentrations above 1 µM. This vasodilation was
potentiated by 0.5 µM Ro-20-1724 (solid circles). *P < 0.05 vs. control; n = 5.
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Figure 3 illustrates the effects of a much
lower concentration of adenosine (100 nM) on the vasoconstrictor
response of the afferent arteriole to elevated renal perfusion
pressure. The tracing in Fig. 3A illustrates the protocol.
Pressure-dependent afferent arteriolar tone was established by
elevating perfusion pressure from 80 to 180 mmHg. In this setting, the
administration of 100 nM adenosine elicited a reversible vasodilation.
This response was abolished by pretreatment with the adenosine
A2a receptor antagonist ZM-241385 (100 nM). The mean data
obtained from five such experiments are summarized in Fig. 3B.
Additional studies were conducted in which sequential responses to 100 nM adenosine were assessed as controls. Afferent arteriolar diameters
for 80, 180, and 180 mmHg plus adenosine were 14.7 ± 1.5, 6.3 ± 1.0, and 13.4 ± 1.6 µm, respectively, for the first response and
14.2 ± 1.3, 6.6 ± 1.0, and 13.1 ± 1.5 µm for the second
response (n = 3, P > 0.80).

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Fig. 3.
A: tracings of the high-affinity vasodilatory response to
adenosine observed during pressure-induced (myogenic) afferent
arteriolar vasoconstriction. Adenosine (ADO) at 100 nM dilated vessels
preconstricted by elevated pressure. This response was prevented by
pretreatment with the adenosine A2a-selective antagonist
4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol
(ZM-241385). B: mean data (n = 5).
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This high-affinity vasodilatory response was also potentiated by 0.5 µM Ro-20-1724. Thus elevating perfusion pressure from 80 to 180 mmHg
decreased afferent arteriolar diameter from 14.0 ± 0.7 to 6.2 ± 0.6 µm (n = 5). At concentrations of 1, 3, 10, and 30 nM,
adenosine increased diameters to 6.3 ± 0.7, 6.7 ± 0.7, 7.8 ± 0.7 (P < 0.01), and 8.5 ± 0.9 µm (P < 0.01),
respectively. In the presence of Ro-20-1724, elevating perfusion
pressure reduced afferent arteriolar diameters from 13.0 ± 0.6 to 6.5 ± 0.5 µm (n = 5) and 1, 3, 10, and 30 nM adenosine
increased diameters to 7.7 ± 0.6 (P < 0.01), 9.2 ± 0.7 (P < 0.01), 10.2 ± 0.6 (P < 0.001), and
11.2 ± 0.7 µm (P < 0.001), respectively. These data are
plotted as the percent inhibition in Fig.
4.

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Fig. 4.
Potentiation of high-affinity adenosine-induced vasodilation (1-30
nM adenosine) by the phosphodiesterase inhibitor
4-[(3-butoxy-4-methoxyphenyl)methyl]-2-imidaxolidinone
(Ro-20-1724, 0.5 µM; solid circles). *P < 0.05 vs. control
(open circles); n = 5.
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The effects of adenosine pretreatment on the response of the afferent
arteriole to stepped increases in renal perfusion pressure are
illustrated in Figs. 5-7. In these
studies, the renal arterial pressure is reduced to 60 mmHg and then
increased in 20-mmHg steps to a final pressure of 180 mmHg. The
pressure at each step is held for 1 min, and constrictor responses are
assessed. Stable control responses were obtained and these data are
represented by the solid circles in Figs. 5 and 6. The
kidneys were then pretreated with adenosine and responses were
reassessed. Responses in the presence of increasing concentrations of
adenosine are depicted by the open symbols in Figs. 5 and
6. Note that over a concentration range of 1-100 nM,
adenosine exerted a concentration-dependent inhibition of myogenic
reactivity (Fig. 5, left). For reasons that are not presently
known, 100 nM adenosine appeared to be more effective in reversing
preestablished pressure-induced vasoconstriction (Fig. 3) than in
preventing the response. The myogenic response was further attenuated
when adenosine concentrations were increased to 10 µM and 30 µM.

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Fig. 5.
Effects of adenosine on myogenic reactivity of renal afferent
arterioles. Adenosine was added cumulatively to concentrations depicted
to right of each curve. To facilitate comparisons, the control
response is depicted by solid circles in both the right and
left. Note that adenosine inhibited myogenic reactivity at
1-100 nM. At higher concentrations, myogenic reactivity partially
recovered (1.0 µM). Increasing adenosine levels further (3-30
µM) resulted in further inhibition.
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The effects of KATP channel-blockade on this action of
adenosine are illustrated in Fig. 6. The
same protocol was repeated using a separate group of kidneys in the
presence of 10 µM glibenclamide. In the presence of glibenclamide,
1-100 nM adenosine had no effect on the myogenic
response (Fig. 6, left). However, the inhibitory effects
observed at the higher adenosine concentrations (10-30 µM) were
preserved. This effect of glibenclamide on the actions of adenosine are
summarized in Fig. 7, in which
concentration-response curves to adenosine were constructed in the
absence and presence of glibenclamide. To facilitate this comparison,
only data obtained at the highest pressure (180 mmHg) are plotted. In
the absence of glibenclamide (solid circles in Fig. 7),
adenosine elicited a biphasic response, characterized by vasodilatory
components at 10
8 to 10
7 M and at
10
5 to 10
4 M adenosine. Note that
in the kidneys pretreated with glibenclamide, only the
low-affinity component (10
5 to
10
4 M adenosine) was seen.

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Fig. 6.
Effects of adenosine on myogenic reactivity in presence of 10 µM
glibenclamide. Note that the inhibitory effects of 1-100 nM
adenosine normally seen in the absence of glibenclamide (Fig. 5) were
abolished by this treatment. In contrast, the vasodilatory effects of
adenosine at concentrations of 10-30 µM were preserved; ,
control.
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Fig. 7.
Summary of effects of glibenclamide on adenosine-induced inhibition of
myogenic vasoconstriction. Percent inhibition of vasoconstrictor
response to elevating perfusion pressure to 180 mmHg is plotted. During
control ( ), a biphasic vasodilatory response to adenosine was
observed, whereas only the low-affinity response was observed in the
presence of glibenclamide ( ). Data were taken from Figs. 5 and 6.
Number of replicates is indicated in parentheses as "(control,
glibenclamide)." *P < 0.05 vs. control.
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Figures 8 and 9 summarize studies examining
the effects of glibenclamide on the inhibition of myogenic reactivity
elicited by the adenosine agonists CV-1808 and NECA. In each case,
cumulative dose-response data were obtained, and separate kidneys were
used for the glibenclamide studies. As shown in Fig. 8, CV-1808
significantly inhibited myogenic vasoconstriction with an
IC50 of 100 nM (n = 5). Over this concentration
range, CV-1808 has been shown to activate adenosine
A2a receptors (9). These actions of CV-1808 were completely
prevented by 10 µM glibenclamide (n = 6). Figure 9 illustrates that NECA inhibited myogenic
responses over concentrations ranging from 3 to 30 nM (n = 5).
The dose response to NECA was right-shifted following pretreatment with
10 µM glibenclamide (n = 6). As seen with adenosine, the
actions of NECA appeared to be mediated by both glibenclamide-sensitive
and glibenclamide-insensitive mechanisms. Figure
10 compares the potencies of adenosine,
CV-1808, and NECA against myogenic afferent arteriolar
vasoconstriction. The order of potency for these vasodilatory actions
was NECA > CV-1808 = adenosine.

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Fig. 8.
Inhibition of myogenic vasoconstriction by 2-phenylaminoadenosine
(CV-1808). Glibenclamide completely prevented inhibitory effects of
this adenosine agonist; n = 6 and n = 5 for control
( ) and glibenclamide ( ), respectively. *P < 0.05 vs.
control.
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Fig. 9.
Inhibition of myogenic vasoconstriction by
5'-N-ethylcarboxamidoadenosine (NECA). Like adenosine,
NECA exhibited glibenclamide-sensitive and glibenclamide-insensitive
vasodilatory actions; n = 6 for both control ( )
and glibenclamide ( ). *P < 0.05 vs.
control.
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Fig. 10.
Comparison of the potencies of NECA (solid circles), CV-1808 (open
circles), and adenosine (gray-shaded circles) in inhibiting myogenic
vasoconstriction in renal afferent arteriole. Data are taken from Figs.
6, 8, and 9.
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DISCUSSION |
In this report, we partially characterize an unusual renal vasodilatory
response to adenosine that we have observed in the in vitro
perfused hydronephrotic rat kidney. In this preparation, adenosine
exerts a vasodilatory action on afferent arteriolar myogenic
vasoconstriction at submicromolar concentrations. This response was
mimicked by NECA and CV-1808, indicating that it is mediated by an
adenosine A2 receptor subtype. The fact that this response
was elicited at submicromolar levels of adenosine, the relatively high
sensitivity to CV-1808 (EC50 of approximately 100 nM, Fig.
8), and our observation that this response was blocked by ZM-241385 all
suggest an involvement of the high-affinity adenosine A2a
receptor subtype (9).
This novel adenosine response was potentiated by Ro-20-1724 and was
prevented by glibenclamide, implicating a role of cAMP and
ATP-sensitive K channels in the receptor-effector coupling mechanism.
Both A2a and A2b receptors are known to be
coupled through Gs
proteins to the activation of
adenylate cyclase (9). Furthermore, cAMP-dependent protein kinase has
been implicated in the activation of KATP in vascular
smooth muscle (12, 21), and adenosine responses have been shown to be
coupled to K channel activation in other vascular beds (7, 8, 10, 12).
The glibenclamide-sensitive afferent arteriolar vasodilation we
observed in our model was blocked by 100 nM ZM-241385. At this
concentration, ZM-241385 is highly selective against the adenosine
A2a receptor (19, 20). In the normal kidney, adenosine A2 receptors are generally considered to be low-affinity
A2b type and are activated only at adenosine concentrations
above that eliciting vasoconstriction (2, 28). We did observe a
comparable low-affinity adenosine-induced vasodilation in our model,
which we attribute to activation of A2b receptors. The
vasodilatory actions of adenosine observed during KCl-induced
vasoconstriction appear to be mediated exclusively by the low-affinity
A2b receptor. The lack of effect of the high-affinity
A2a receptor in this setting is consistent with a
vasodilatory mechanism requiring K channel activation. We have
previously demonstrated that KATP activation by hypoxia or
pinacidil does not induce afferent arteriolar vasodilation in the
presence of 30 mM KCl (15, 22). Low-affinity A2b receptors also likely account for inhibition of myogenic responses by high adenosine concentrations in the presence of glibenclamide. Adenosine concentrations of 10-30 µM were required for both responses
(compare Figs. 2 and 7).
Our findings thus indicate that both high-affinity A2a and
low-affinity A2b adenosine receptors are expressed in the
renal microvasculature of our model. It is not clear whether both
receptor subtypes are present on the same cell type (e.g., the afferent arteriolar myocyte). Both responses were potentiated by Ro-20-1724, suggesting each is coupled to cAMP. However only the
A2a-mediated response requires KATP. It is
difficult to imagine a signaling pathway in which cAMP mediates either
KATP-dependent or KATP-independent vasodilation
within a single cell, depending on which receptor (A2a or
A2b) is activated. Conceivably, this phenomenon could reflect a compartmentalization of cAMP-dependent responses within the
cell. Alternatively, differing cell types could be involved in each
response. For example, adenosine is reported to have
endothelial-dependent vasodilatory actions, and adenosine-induced
endothelial activation could conceivably induce hyperpolarization in
the underlying arteriolar smooth muscle cells (4, 17, 27).
We interpret our findings as indicating that the renal microcirculation
has the potential of expressing the high-affinity adenosine
A2a receptor and that activation of this receptor modulates myogenic afferent arteriolar vasoconstriction through KATP.
As discussed above, this type of adenosine response has not been reported in the normal kidney. Indeed, adenosine infusion or elevation of endogenous adenosine by uptake blockade normally elicits renal vasoconstriction via activation of A1 adenosine receptors
(2, 28). We consider it very likely that our ability to demonstrate the
A2a-mediated response is related to the hydronephrotic
state. The nonfiltering hydronephrotic kidney would be anticipated to have lower metabolic activity and reduced renal interstitial levels of
adenosine, which could induce an upregulation of the adenosine A2a receptor.
Alternatively, it is possible that the conditions of our study (e.g.,
low basal adenosine levels, elevated pressure-induced arteriolar tone,
low levels of endogenous vasoconstrictors) may have facilitated the
observation of this response. Microdialysis studies report cortical
renal interstitial adenosine concentrations to be approximately 200 nM
in the anesthetized rat (3), and the glibenclamide-sensitive adenosine
response we observed may not be readily demonstrated in the presence of
high basal levels of adenosine. Adenosine levels may be lower in
conscious animal preparations, but conscious animals generally have
very low basal renal vascular tone, and renal vasodilation is difficult
to demonstrate in the absence of significant renal vasoconstriction
(e.g., see Ref. 14). Certainly, the ability of the normal kidney to
generate myogenic tone in the anesthetized preparation, despite
presumably high basal levels of adenosine, would argue against the
presence of this receptor or, at least, its chronic activation in the
normal kidney.
The presence of circulating or intrarenal vasoconstrictors may also
impact on the ability to observe KATP-dependent responses in the normal kidney. We have recently reported that the
KATP component of the afferent arteriolar actions of
calcitonin gene-related peptide (CGRP), readily observed
during pressure-induced vasoconstriction, is eliminated during
angiotensin II-induced vasoconstriction, suggesting angiotensin II
inhibits KATP activation (23). Studies in isolated smooth
muscle cells indicate that vasoconstrictor agonists such as angiotensin
II are capable of inhibiting KATP by stimulating protein
kinase C (5). Accordingly, specific experimental conditions, including
low basal intrarenal adenosine levels, elevated pressure-induced renal
vascular tone and low levels of agonist-induced vasoconstriction, may
be required to observe this unusual adenosine response in the normal
kidney. Of interest, an anecdotal report by Smits et al. (26)
demonstrated that submicromolar levels of adenosine administered into
the renal artery of conscious hypertensive patients caused a twofold
increase in renal blood flow. Similarly, a study by Arakawa et al. (1) indirectly suggested the presence of a high-affinity adenosine vasodilatory mechanism in the normal, conscious dog. It is interesting to speculate that the high-affinity A2a receptor may play
some role in the regulation of normal renal microvascular reactivity or
contribute to abnormal myogenic reactivity in pathophysiological states. Further investigations will be required to address these issues.
In summary, in the present study we describe three distinct actions of
adenosine on the renal afferent arteriole of the in vitro perfused
hydronephrotic rat kidney. In addition to adenosine A1-induced vasoconstriction, we find two vasodilatory
responses. One response corresponds to that previously ascribed to
activation of low-affinity adenosine A2b receptors and is
elicited at adenosine concentrations of 10 µM and above. The second
response was elicited at 10-100 nM adenosine and was blocked by
glibenclamide, suggesting a mechanism coupled to activation of
KATP. We suggest that this high-affinity response is
mediated by renal microvascular adenosine A2a receptors.
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ACKNOWLEDGEMENTS |
This study was supported by grants from the Medical Research
Council of Canada and the Alberta Heritage Foundation for Medical Research.
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FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. D. Loutzenhiser, Department of Pharmacology and Therapeutics, University
of Calgary, Health Sciences Centre, 3330 Hospital Drive N.W., Calgary,
Alberta, Canada T2N 1N4 (E-mail:rloutzen{at}ucalgary.ca).
Received 10 February 1999; accepted in final form 17 August 1999.
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REFERENCES |
1.
Arakawa, K.,
H. Suzuki,
M. Naitoh,
A. Matsumoto,
K. Hayashi,
H. Matsuda,
A. Ichihara,
E. Kubota,
and
T. Saruta.
Role of adenosine in the renal responses to contrast medium.
Kidney Int.
4:
1199-1206,
1996.
2.
Arend, L. J.,
C. I. Thompson,
and
W. S. Spielman.
Dipyridamole decreases glomerular filtration in sodium-depleted dog: evidence for mediation by intrarenal adenosine.
Circ. Res.
56:
242-251,
1985[Abstract].
3.
Baranowski, R. L.,
and
C. Westenfelder.
Estimation of renal interstitial adenosine and purine metabolites by microdialysis.
Am. J. Physiol.
267 (Renal Fluid Electrolyte Physiol. 36):
F174-F182,
1994[Abstract/Free Full Text].
4.
Barrett, R. J.,
and
D. A. Dropplema.
Interactions of adenosine A1 receptor-mediated renal vasoconstriction with endogenous nitric oxide and ANG II.
Am. J. Physiol.
265 (Renal Fluid Electrolyte Physiol. 34):
F651-F659,
1993[Abstract/Free Full Text].
5.
Bonev, A. D.,
and
M. T. Nelson.
Vasoconstrictors inhibit ATP-sensitive K channels in arterial smooth muscle cells through activation of protein kinase C.
J. Gen. Physiol.
108:
315-323,
1996[Abstract].
6.
Churchill, P. C.,
and
A. K. Bidani.
Hypothesis: adenosine mediates hemodynamic changes in renal failure.
Med. Hypotheses
8:
275-285,
1982[Medline].
7.
Cornfield, L. J.,
S. Hu,
S. D. Hurt,
and
M. A. Sills.
[3H]2-phenylaminoadenosine ([3H]CV 1808) labels a novel adenosine receptor in rat brain.
J. Pharmacol. Exp. Ther.
263:
552-561,
1992[Abstract].
8.
Dart, C.,
and
N. B. Standen.
Adenosine-activated potassium current in smooth muscle cells isolated from the pig coronary artery.
J. Physiol. (Lond.)
471:
767-786,
1993[Abstract].
9.
Fredholm, B. B.,
M. P. Abbracchio,
G. Burnstock,
J. W. Daly,
T. K. Harden,
K. A. Jacobson,
P. Leff,
and
M. Williams.
Nomenclature and classification of purinoceptors.
Pharmacol. Rev.
46:
143-156,
1994[Medline].
10.
Gidday, J. M.,
R. G. Maceren,
A. R. Shah,
J. A. Meier,
and
Y. Zhu.
KATP channels mediate adenosine-induced hyperemia in retina.
Invest. Ophthalmol. Vis. Sci.
37:
2624-2633,
1996[Abstract].
11.
Jackson, E. K.,
Z. Mi,
J. A. Carcillo,
D. G. Billespie,
and
R. K. Dubey.
Phosphodiesterases in the rat renal vasculature.
J. Cardiovasc. Pharmacol.
30:
798-801,
1997[Medline].
12.
Kleppisch, T.,
and
M. T. Nelson.
Adenosine activates ATP-sensitive potassium channels in arterial myocytes via A2 receptors and cAMP-dependent protein kinase.
Proc. Natl. Acad. Sci. USA
92:
12441-12445,
1995[Abstract].
13.
Loutzenhiser, R.
In situ studies of renal arteriolar function using the in vitro-perfused hydronephrotic rat kidney.
Int. Rev. Exp. Pathol.
36:
145-160,
1996[Medline].
14.
Loutzenhiser, R.,
and
M. Epstein.
Effects of calcium antagonists on renal hemodynamics.
Am. J. Physiol.
249 (Renal Fluid Electrolyte Physiol. 18):
F619-F629,
1985[Medline].
15.
Loutzenhiser, R.,
and
M. Parker.
Hypoxia inhibits myogenic reactivity of renal afferent arterioles by activating ATP-sensitive K channels.
Circ. Res.
74:
861-869,
1994[Abstract].
16.
McCoy, D. E.,
S. Bhattacharya,
B. A. Olson,
D. G. Levier,
L. J. Arend,
and
W. S. Spielman.
The renal adenosine system: structure, function and regulation.
Semin. Nephrol.
13:
31-40,
1993[Medline].
17.
Olanrewaju, H. A.,
P. T. Hargittai,
E. A. Lieberman,
and
S. J. Mustafa.
Role of endothelium in hyperpolarization of coronary smooth muscle by adenosine and its analogues.
J. Cardiovasc. Pharmacol.
25:
234-239,
1995[Medline].
18.
Osswald, H.,
B. Mülbauer,
and
F. Schenk.
Adenosine mediates tubuloglomerular feedback response: an element of metabolic control of kidney function.
Kidney Int.
39, Suppl.32:
S128-S131,
1991.
19.
Palmer, T. M.,
S. M. Poucher,
K. A. Jacobson,
and
G. L. Stiles.
125I-4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]- triazin-5-ylamino]ethyl)phenol, a high affinity antagonist radioligand selective for the A2a adenosine receptor.
Mol. Pharmacol.
48:
970-974,
1995[Abstract].
20.
Poucher, S. M.,
J. R. Keddie,
P. Singh,
S. M. Stoggall,
P. W. R. Caulkett,
G. Jones,
and
M. G. Collis.
The in vitro pharmacology of ZM 241385, a potent, non-xanthine, A2a selective adenosine receptor antagonist.
Br. J. Pharmacol.
115:
1096-1102,
1995[Abstract].
21.
Quayle, J. M.,
A. D. Bonev,
J. E. Brayden,
and
M. T. Nelson.
Calcitonin gene-related peptide activated ATP-sensitive K currents in rabbit arterial smooth muscle via protein kinase A.
J. Physiol. Lond.
475:
9-13,
1994[Abstract].
22.
Reslerova, M.,
and
R. Loutzenhiser.
Divergent mechanisms of KATP-induced vasodilation in renal afferent and efferent arterioles: evidence for both L-type calcium channel-dependent and -independent actions of pinacidil.
Circ. Res.
77:
1114-1120,
1995[Abstract/Free Full Text].
23.
Reslerova, M.,
and
R. Loutzenhiser.
Renal microvascular actions of calcitonin gene-related peptide.
Am. J. Physiol.
274 (Renal Physiol. 43):
F1078-F1085,
1998[Abstract/Free Full Text].
24.
Rossi, N.,
P. C. Churchill,
and
B. Amore.
Mechanism of adenosine receptor induced renal vasoconstriction in the rat.
Am. J. Physiol.
255 (Heart Circ. Physiol. 24):
H885-H890,
1988[Abstract/Free Full Text].
25.
Schnermann, J.,
H. Weihprecht,
and
J. Briggs.
Inhibition of tubuloglomerular feedback during adenosine1 receptor blockade.
Am. J. Physiol.
258 (Renal Fluid Electrolyte Physiol. 27):
F553-F561,
1990[Abstract/Free Full Text].
26.
Smits, P.,
P. W. de Leeuw,
P. N. van Es,
C. T. Postma,
and
T. Thien.
Adenosine induces renal vasodilation in primary hypertensive patients.
J. Hypertens.
9, Suppl. 6:
S214-S215,
1991.
27.
Smits, P.,
S. B. Williams,
D. E. Lipson,
P. Banitt,
B. A. Rongen,
and
M. A. Creager.
Endothelial release of nitric oxide contributes to the vasodilator effects of adenosine in humans.
Circulation
92:
2135-1241,
1995[Abstract/Free Full Text].
28.
Spielman, W. S.,
and
L. J. Arend.
Adenosine receptors and signalling in the kidney.
Hypertension
17:
17-130,
1991.
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