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LOCAL CONTROL OF CIRCULATION
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892
Submitted 4 December 2002 ; accepted in final form 20 February 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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renal blood flow; ultrasonic flowmeter; renal vascular resistance; glomerular filtration rate; perfused arterioles
The converse relationship, i.e., a dependence of ANG II vasoconstriction on simultaneous activation of A1AR, is somewhat more controversial. For example, in the split hydronephrotic kidney, acute blockade of A1AR with the A1AR antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) had no effect on the
constrictor effect of ANG II
(4). On the other hand, DPCPX
caused an 
50% reduction in the constrictor response to ANG II in isolated
and perfused afferent arterioles from rabbit kidneys
(17). The availability of mice
with a targeted deletion of A1AR permits the study of renal vascular responses
to ANG II during chronic and complete A1AR deficiency
(15). Accordingly, the present
experiments were performed to determine whether the response of renal vascular
resistance to ANG II can be shown to be altered in A1AR knockout compared with
wild-type mice. These experiments were done as a first step to further
attempts of understanding the nature of the interaction between ANG II and
adenosine. Our experiments show that both the vasoconstriction and the fall in
glomerular filtration rate (GFR) in response to an acute change in angiotensin
plasma concentration are significantly blunted in the absence of functional
A1AR and that this reflects at least in part a reduced responsiveness of
afferent arterioles.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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To determine GFR, mice were infused with [125I]iothalamate (Glofil, Questcor Pharmaceutical, Hayward, CA) at 5 µCi/h. After 30–45 min of equilibration, three 10-min urine collection periods were made bracketed by two blood collections of 4 µl each collected in
heparinized 5-µl microcaps (Drummond). An ANG II infusion was then started
at a rate of 1.5 ng/min (ANG II concentration 1 ng/µl) with a flow of 1.5
µl/min without interrupting the iothalamate infusion. Three additional
10-min urine collections and a terminal blood collection were made.
[125I]iothalamate radioactivity was measured in duplicate 0.5-µl
aliquots of plasma and urine in a gamma counter.
Measurements of renal blood flow (RBF) were performed in a separate group of A1AR +/+ and A1AR -/- mice. The left renal artery was approached from a flank incision and carefully dissected free to permit placement of a 0.5 mm V-type ultrasonic flow probe (Transonic Systems, Ithaca, NY). The probe was held in place with a micromanipulator. The flow signal was digitized and analyzed using MacLab software. In each animal, RBF was determined during the intravenous injection of 1, 10, and 100 ng of ANG II. After each injection a waiting period of 10 min was allowed in which RBF and blood pressure returned to baseline.
Perfused afferent arterioles. To determine the effect of A1AR deficiency on ANG II-induced constriction at the arteriolar level, afferent arterioles from A1AR +/+ and -/- mice were isolated and microperfused. Mice (18–26 g) of either sex were used in the study. The method of isolation and perfusion was a modification of that used by Weihprecht et al. (18) and Jensen et al. (9). Afferent arterioles were microdissected at 4°C from slices of mouse kidneys. The dissection was performed in DMEM/Nutrient F12 (DMEM/F12) with 0.5% BSA under a stereomicroscope. The specimen was transferred to a thermoregulated chamber, containing oxygenated DMEM/F12 + 0.1% BSA, mounted on an inverted microscope (Olympus IMT-2), and perfused with concentric glass pipettes mounted in a moveable track system. The arteriole was aspirated into a holding pipette (tip diameter 20 µm) and cannulated with a perfusion pipette (tip diameter 5–6 µm). Once the arteriole was cannulated, the driving pressure was increased until the vessel opened, and perfusion was established. Perfusion was performed close to the minimum opening pressure because it was our impression that the perfused arterioles from the mouse are rather sensitive to pressure-induced damage. The perfusate consisting of physiological salt solution (PSS) + 1% BSA was driven from a reservoir pressurized to 60–110 mmHg. The temperature was increased to 37°C, and the vessel was allowed to recover for 25 min. In each experiment a test stimulus of high potassium (100 mmol/l K+) or a gentle increase in pressure was applied initially to ensure viability of the vessel. Sequences of interest were recorded with a digital camera (CoolSNAP-Pro, Media Cybernetics). Images were transferred to a computer, and intraluminal vessel diameters were assessed using imaging software (Image Pro-plus, Media Cybernetics). PSS had the following composition (mmol/l): 115 NaCl, 25 NaHCO3, 2.5 K2HPO4, 1.3 CaCl2, 1.2 MgSO4, 10 HEPES, and 5.5 glucose. High-potassium solution contained (mmol/l) 25 NaHCO3, 20 NaCl, 95 KCl, 1.2 MgSO4, 2.5 K2HPO4, 1.3 CaCl2, 10 HEPES, and 5.5 glucose. All solutions were equilibrated with 5% CO2 in O2 for 30 min before use, BSA was added, and pH was adjusted to 7.35. To study the vasoconstrictor response to ANG II in arterioles from A1AR wild-type and knockout mice, ANG II was added to the bath for 3 min at increasing concentrations (10-12, 10-11, 10-10, 10-9, and 10-8 mol/l) using a step-up protocol. Furthermore, in a small series of experiments in afferent arterioles from A1AR +/+ mice, we tested the effect of the A1AR-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (10-5 mol/l) on the vascular response to ANG II (10-10 mol/l).
Quantitative real-time PCR. The expression of ANG II receptors (AT1) in the kidney cortex of 5 A1AR +/+ and 5 A1AR -/- mice was investigated using real-time PCR analysis. In addition, we determined the expression levels of A2aAR and A2bAR in A1AR -/- compared with A1AR +/+ mice. RNA was isolated
using Trizol-reagent and reverse transcribed using Superscript (Invitrogen)
and oligo(dT) (Pharmacia). Quantitative PCR analysis was performed using an
ABI prism 7900 HT Sequence Detection System (Applied Biosystems). SyBR Green,
a double-stranded DNA binding dye, was used for the fluorescent detection of
DNA generated during the PCR. The PCR reaction was performed in a total volume
of 20 µl with 0.4 pmol/µl of each primer, and 2x SyBR Green PCR
master mix (Applied Biosystems); 1 µl cDNA corresponding to 100 ng of total
RNA was used as template. Negative controls included water instead of cDNA in
the PCR reaction and addition of RNA instead of cDNA. Published sequences for
mouse AT1a (S37484
[GenBank]
), A2aAR (Y13346
[GenBank]
), and A2bAR (NM_007413
[GenBank]
) were used to design
primers for PCR amplification
(13). The AT1a primers might
also amplify the AT1b isoform but not the AT2 receptor. Primer sequences were
AT1 sense 5'-CCA GAT CAA GTG ATT TTG AAC AGT G-3' and antisense
5'-GCT GTA GAG AGT AGG GAT CAT GAC AA-3'; A2aAR sense 5'-TCC
TGG TCC TCA CGC AGA GT-3' and antisense 5'-GGG TCA GGC CGA TGG
C-3'; A2bAR sense 5'-TGG CTG TCG ACC GAT ATC TG-3' and
antisense 5'-GTC AAT CCA ATG CCA AAG GC-3'; 
-actin sense
5'-GCT CTG GCT CCT AGC ACC AT-3' and antisense 5'-GCC ACC
GAT CCA CAC AGA GT-3'.
Statistics. Data are expressed as means ± SE. Statistical analysis was performed using the t-test of paired and non-paired data. Arteriolar diameters were compared by two-way ANOVA followed by Bonferroni test. Student's t-test on paired data in the individual arterioles was performed when comparing EC50 values. P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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RBF studies. Measurements of RBF were performed in eight wild-type and nine A1AR knockout mice. In response to intravenous bolus injections of 1, 10, and 100 ng ANG II, RBF of the left kidney fell in the wild-type mice from 930 ± 12 to 790 ± 9 µl/min, from 940 ± 15 to 490 ± 9 µl/min, and from 960 ± 18 to 220 ± 7 µl/min. In the A1AR knockout mice, the same injections caused RBF to fall from 1,030 ± 14 to 940 ± 10 µl/min, from 1,070 ± 15 to 640 ± 13 µl/min, and from 1,080 ± 20 to 400 ± 14 µl/min. All changes in both wild-type and A1AR knockout mice were significant at P < 0.01.
While levels of RBF were not significantly different before the ANG II injections, RBF was higher during the ANG II administration in knockout compared with wild-type mice (P = 0.019 at 1 ng, P = 0.018 at 10 ng, and P = 0.008 at 100 ng ANG II).
Arterial blood pressure levels were not significantly different between A1AR +/+ and A1AR -/- mice even though they tended to be lower in the latter group (103 ± 3.5 and 97 ± 2.9 mmHg, respectively; P = 0.17). Furthermore, blood pressure changes in response to ANG II at 1, 10, and 100 ng were nearly identical, increasing by 3.3 ± 0.2, 17.3 ± 0.4, and 47.3 ± 1 mmHg in wild-type and by 3.3 ± 0.15, 18.4 ± 0.4, and 45.5 ± 0.8 mmHg in the A1AR knockout mice. Because blood pressures tended to be lower and RBF higher in the A1AR knockout animals, renal vascular resistance under control conditions was lower in A1AR knockout than wild-type mice (112.3 ± 1.7 vs. 96.3 ± 1.5 mmHg min/ml; P = 0.05). Increases in renal vascular resistance caused by the injection of 1, 10, and 100 ng of ANG II are shown in Fig. 3. It can be seen that renal vascular resistance increased significantly less in the knockout mice during all three levels of angiotensin injection (paired t-test).
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Studies in perfused afferent arterioles. The vasoconstrictor response to ANG II was studied in perfused afferent arterioles from A1AR +/+ and -/- mice. Basal diameters averaged 10.1 ± 0.4 µm in A1AR wild-type mice (n = 6) and 9.0 ± 0.5 µm in knockout mice (n = 7). Administration of 10-12 and 10-11 mol/l ANG II had no measurable effect on the inner arteriolar diameter in either wild-type or knockout mice. The addition of 10-10 mol/l ANG II to afferent arterioles from wild-type mice induced a significant vasoconstriction (P < 0.05, ANOVA), whereas in knockout mice no significant vasoconstrictor effect was seen at this ANG II concentration. However, increasing the ANG II concentration to 10-9 mol/l was associated with significant constriction in both wild-type and knockout animals (Fig. 4A). The ANG II concentration causing half-maximal vasoconstriction was 1.5 ± 0.4 x 10-10 mol/l in wild-type mice and 7.3 ± 1.2 x 10-10 mol/l in A1AR knockout mice (P < 0.05, t-test, Fig. 4B). Figure 4C shows representative examples of perfused afferent arterioles from wild-type and knockout mice under control conditions and during exposure to ANG II at 10-10 mol/l.
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In three A1AR +/+ mice, the addition of DPCPX to the bath reduced the vasoconstrictor effect of a submaximal dose of ANG II. ANG II (10-10 mol/l) reduced the diameter of the afferent arterioles by 4.0 ± 0.6 µm before DPCPX and by 1.5 ± 1.2 µm after DPCPX administration.
Expression of AT1 and A2AR receptors. To test if the decreased sensitivity to ANG II in A1AR -/- mice was due to a downregulation of AT1 receptors, we determined the expression of ANG II receptor mRNA in kidneys of A1AR +/+ and -/- mice. The expression levels of AT1 mRNA normalized to
-actin were found to be not significantly different in A1AR +/+ (1.0
± 0.2) compared with A1AR -/- mice (1.1 ± 0.3). To test whether
an increase in A2AR expression in the A1AR -/- mice may cause attenuation of
the ANG II-induced constriction, we determined the mRNA expression levels of
the A2aAR and A2bAR genes. A2aAR mRNA levels ranged from 1.1 ± 0.2 in
A1AR +/+ to 1.0 ± 0.4 in -/- mice, and A2bAR ranged from 1.2 ±
0.3 in +/+ to 0.8 ± 0.2 in -/- mice (n = 4 in each group).
Even though the level of A2bAR tended to be lower in the -/- mice, the
differences were not significant.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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In view of the observation that the expression levels of ANG II receptor mRNA were comparable between A1AR +/+ and -/- mice, we consider it unlikely that differences in AT1 receptor density are responsible for the reduced angiotensin sensitivity in the A1AR knockout mice. Furthermore, the unchanged
expression levels of both A2a and A2b adenosine receptors between A1AR -/- and
A1AR +/+ mice do not support the possibility that an increase in A2AR
abundance and activation may be responsible for the attenuation of the ANG
II-induced response in the A1AR -/- animals. While there could be a number of
other reasons for the lower renal vascular responsiveness to ANG II in A1AR
knockout mice, it would seem most likely that it is an expression of the
previously noted relation between angiotensin-induced constriction and A1AR
activation. Studies from our laboratory have shown that the constrictor effect
of ANG II in isolated rabbit afferent arterioles was blocked by 50% when
the A1AR inhibitor DPCPX was present in the bath
(17), a finding that was
reproduced in the present studies in mouse afferent arterioles. In
confirmation of an earlier observation, a recent study in dogs showed that the
reduction in RBF caused by ANG II was augmented by intrarenal infusion of
adenosine and attenuated by the A1AR antagonist KW-3902
(1,
6). The present study is the
first to demonstrate that the vasoconstricting effects of acute changes in
plasma ANG II levels are attenuated in animals with a life-long absence of
A1AR. Thus the fundamental mechanism by which adenosine affects angiotensin
actions in renal blood vessels does not seem to adapt to chronic A1AR
deficiency.
It seems reasonable to predict that the effect of adenosine blockade on the ANG II-induced vasoconstriction may depend on the level of expression of A1AR in a given vascular bed. Thus in a vascular bed with low levels of A1AR expression, the effect of A1AR inhibition on angiotensin responses should be low. We believe that this notion may be the explanation for some of the apparently discrepant results that can be found in the literature. For example, blockade of A1AR did not measurably affect ANG II-induced vasoconstriction in afferent arterioles of juxtamedullary nephrons (3). Functional evidence mainly based on the vasoconstrictor efficacy of adenosine would indicate that the nucleoside causes no more than a 10% reduction in afferent arteriolar diameter and that even when A2AR receptors are blocked, the maximum vasoconstriction is only 15% (3, 10). In contrast, A1AR activation has previously been observed to cause a 45–50% diameter reduction in more superficial afferent arterioles of the rabbit and in the hydronephrotic kidney preparation (7, 19). We believe that this dataset is most likely a reflection of a markedly lower abundance of A1AR in deep nephrons. Thus any interaction between AT1 and A1AR activation would be expected to be less obvious in juxtamedullary afferent arterioles, an expectation directly supported by the observation that converting enzyme blockade reduced the vasoconstriction caused by N6-cyclohexyladenosine in superficial arterioles but enhanced it in juxtamedullary arterioles (5). A second important variable may be the rate of production of adenosine and its appearance in and around the A1AR expressing vessels. In the hydronephrotic kidney preparation, the selective A1AR antagonist DPCPX did not alter the vasoconstrictor effect of ANG II (4). If one assumes that the source of adenosine or its precursor ATP is predominantly the renal epithelial cells, one would not expect A1AR to be highly activated in a preparation that is devoid of epithelial structures.
The mechanisms responsible for the effect of A1AR activation on ANG II responsiveness have not been addressed in this study. However, there is substantial evidence for a synergistic interaction between Gq- and Gi-coupled receptors using a variety of different agonists in a number of different
tissues. Overall, these studies show that activation of Gi-coupled receptors
such as A1AR often synergistically enhances the accumulation of inositol
trisphosphate and subsequent increases in cytosolic calcium caused by
activation of Gq-coupled receptors. The underlying reason appears to be an
enhanced activation of phospholipase C, resulting from simultaneous exposure
to G
q and to the G
dimers dissociated from Gi
(21). There is also evidence
that this synergy is especially effective in cells with a high level of A1AR
expression, and this may explain why afferent arterioles are a site where the
angiotensin-adenosine interactions are detectable most clearly
(2). Further studies are needed
to test the validity of this concept in the renal vasculature.
ANG II has been found in the present experiments to constrict isolated afferent arterioles from wild-type mice with an EC50 of 10-10 M and to cause maximum constriction consisting of essentially complete vessel closure at
10-9 M. In previous studies in perfused afferent
arterioles from rabbits and rats, EC50 values for ANG II
vasoconstriction of 10-9 M and maximum effects at
10-7 M have been reported, suggesting that mouse
arterioles may be somewhat more sensitive to ANG II
(18,
20). This conclusion is
slightly at variance with a study in which vasoconstriction of isolated
perfused afferent arterioles from the mouse required higher threshold
concentrations of ANG II and in which the maximum effect consisted of an about
30–40% reduction in diameter
(12). Effects comparable to
those seen in our study were only observed when vessels were treated with the
NOS blocker L-NAME, suggesting that the effect of ANG II was
counteracted by the release of nitric oxide. Whether differences in
endothelial function are responsible for the enhanced response to ANG II in
our studies remains to be determined. Compared with vessels isolated from
wild-type mice, arterioles originating from A1AR knockout mice had a
significantly lower response to ANG II in the threshold concentration range
around 10-10 M, resulting in a significant right shift
of the dose-response relationship and a significantly higher EC50
concentration. This observation indicates that vessels from wild-type mice
generate adenosine that via activation of A1AR enhances the effect of ANG
II.
In conclusion, the reduction in GFR and the increase in renal vascular resistance caused by acute ANG II administration is significantly reduced in A1AR knockout mice. Furthermore, the relationship between bath ANG II and vasoconstriction of isolated perfused afferent arterioles is shifted to the right in vessels from A1AR knockout compared with vessels from wild-type mice. Thus chronic absence of A1AR diminishes the effectiveness of ANG II to constrict renal resistance vessels.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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(16) and
evidence for a synergic interaction between G and the
subunit of a receptor activated G protein. Proc Natl Acad Sci
USA 93:
2827–2831, 1996.This article has been cited by other articles:
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