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,
*
Johannes-Müller-Institute of Physiology,
Humboldt-University of Berlin, Berlin, Germany.
Department of Nephrology, University-Hospital
Charité, Humboldt-University of Berlin,
Berlin, Germany.
Institute of Molecular Biology and Biochemistry, Free University of
Berlin, Berlin, Germany.
Correspondence to Dr. A. Patzak, Johannes-Müller-Institut für Physiologie, Humboldt-Universität zu Berlin, Universitätsklinikum Charité, Tucholskystrasse 2, 10117 Berlin, Germany. Phone: +49-30-2802-6683; Fax: +49-30-2802-6662; E-mail: andreas.patzak{at}charite.de
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Abstract |
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 Top Abstract IntroductionMaterial and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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Enhanced Af vasoconstrictor responses to NOS blockade were observed in angiotensin II (Ang II)—induced hypertensive rats in comparison with responses in normotensive controls (3). These results suggest an Ang II—induced NO production that is mediated by either subtype 1 (AT1) or subtype 2 (AT2) angiotensin receptors (4,5). Experiments in isolated perfused rabbit arterioles underscore an important interaction between Ang II and NO: Ang II—induced arteriolar constriction in this preparation is transient only because of activation of local NO (6).
It remains unclear whether this effect is due to endothelial-derived NO or whether the neuronal NOS isoform located in the macula densa cells is responsible for counteracting Ang II vasoconstriction. The availability of mice that are homozygous (-/-) for disruption of the endothelial NOS (eNOS) gene allows the study of selectively inhibited NO production by eNOS. However, to perfuse isolated mouse Af, a perfusion setup must be miniaturized. In the present study, this was put into effect.
Recent studies demonstrated significantly higher BP in eNOS knockout mice (7,8). Furthermore, overexpression of eNOS causes hypotension and reduces NO-elicited vasorelaxation in mice (9). Thus, in this animal model, an important contribution of endothelial-derived NO on vascular resistance was apparent. However, the role of NO in adjustment of renal arterioles in the mice still is not understood fully. Therefore, in the present study, we examined to what extent NO modulates the constriction of Af because of Ang II. Reduction of NO production was achieved by application of the unspecific NO inhibitor NG-nitro-L-arginine methyl ester (L-NAME). In addition, eNOS knockout mice were studied. Experiments were carried out on isolated perfused Af, which are not under control of the sympathetic nervous system and circulating vasoactive substances.
We tested further whether the Ang II-NO interaction is gender dependent. There are some indications that sex hormones interact with the renin-Ang and NO system. For example, it has been found that estrogen reduces Ang II plasma levels (10) and downregulates the AT1 receptor expression in ovariectomized female rats (11). Moreover, a significant association between the number of estrogen receptors and basal release of NO in mouse aorta has been described elsewhere (12).
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Material and Methods |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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Animals and Microperfusion Procedure
A total of 57 mice (20 to 30 g) of both genders were used in this study.
The mice were wild types from the C57BL/6 strain and eNOS mutants
(8) (The Jackson Laboratory,
Bar Harbor, ME). Animals were fed with standard mouse chow and allowed free
access to tap water. All animal procedures conformed with the guidelines for
care and handling of animals established by the U.S. Department of Health and
Public Services and published by the National Institutes of Health.
After each animal was killed, the kidneys were removed immediately and sliced along the corticomedullary axis. The Af were prepared in DMEM at 4°C. The dissection of individual arterioles was performed under a stereoscopic microscope at magnifications up to 250x and with the help of sharpened forceps (No. 5; Dumont, La Chaux-de-Fonds, Switzerland). The position of Af in the slice was indicated by the glomerulus that belonged to it. Tubuli were removed, and the arterioles were identified by preparation of the arterial tree, including the interlobular artery. Normally, Af used were located at the end of the arterial tree. The arteriole with its glomerulus intact was cut with miniblades and transferred into a thermoregulated chamber (vol 1.5 ml; VETEC, Rostock-Warnemünde, Germany) on a stage of an inverted microscope. The perfusion system allowed movement and adjustment of concentric, holding, and perfusion pipettes (Luigs & Neumann, Ratingen, Germany). Pipettes were produced with the help of a self-manufactured apparatus and with the use of custom glass tubes (Drummond Scientific Company, Broomall, PA). The holding pipette (outer diameter, 0.084''; inner diameter, 0.064'') into which the proximal end of the arteriole was aspirated had an aperture of approximately 26 µm at the tip and a constriction of approximately 20 µm after customization. The inner perfusion pipette (outer diameter, 0.047''; inner diameter, 0.040'') with an aperture of 5 µm was advanced into the lumen of the arteriole. It was connected to a reservoir that contained the perfusion solution and to a manometer. Af of wild-type and eNOS knockout mice were perfused with similar pressures between 60 and 80 mmHg at 37°C. Although the arterial BP in eNOS knockout mice is approximately 20 mmHg higher than that of wild types (8), pressure was unchanged in all series to exclude the influence of this factor. If perfusion was not achieved within 120 min after the mouse was killed, then the experiment was stopped. The time of adaptation after the start of perfusion and heating of the bath chamber was 20 min. Only arterioles with remaining basal tone were used. Hypoxic or otherwise injured vessels were identified readily by pronounced vasodilatation and failed constriction to K+ -rich solution, which was used to test the viability of the arterioles at the beginning of the experiment. Thereafter, a recovery of 10 min was allowed. In all series, the last 10 s of the treatment period were used for statistical analysis of steady-state responses.
Experimental Protocols
Series 1. The effect of the NO precursor L-arginine on arteriolar
diameter was determined, L-arginine was added to the bath in concentrations of
10-6, 10-5, 10-4, and 10-3 mol/L.
A 3-min treatment period was used. Af diameters were measured under control
conditions and during exposure of L-arginine.
Series 2. To determine whether an endogenous, tonic NO release plays a role for the arteriolar diameter in this preparation, we added the NO synthase inhibitor L-NAME to the bath solution. The L-NAME concentration was increased from 10-6 to 10-3 mol/L with treatment periods of 3 min. The diameter was determined before and during the exposure of L-NAME.
Series 3. To exclude that the lack of the NO precursor L-arginine prevents a tonic production and release of NO as tested in series 2, all solutions were enriched with L-arginine (10-4 mol/L). The protocol corresponds to that in series 2.
Series 4. In this series, the dose response to Ang II was determined at concentrations of 10-12, 10-10, 10-8, and 10-6 mol/L. The treatment period was 2 min. Af diameters were measured before and during the addition of Ang II. Female as well as male mice were used, to assess any gender-specific effects of Ang II on arteriolar diameter.
Series 5. Experiments were performed to determine whether NO modulates the arteriolar reactivity to Ang II. The bath solution was enriched with L-NAME at a concentration of 10-4 mol/L. The doses of Ang II were the same as those in series 3. Female and male mice were included, to test for gender-specific effects.
Series 6. Mice that were homozygous (-/-) for disruption of the eNOS gene were used to test the effect of total blockade of endothelial NO production by eNOS. The reactivity of the vessel to Ang II was determined by addition of Ang II in concentrations of 10-12, 10-10, 10-8, and 10-6 mol/L.
Data Analyses
Experiments were recorded on SVHS videotapes (video recorder AG-MD 830;
Panasonic, Yokohama, Japan). The magnification results from an objective
(x40; Zeiss, Oberkochen, Germany) and projection (x1) on a
0.3'' chip digital camera (CB-3803S; GKB, Tai Chung, Taiwan). Video
sequences were digitized by use of a frame grabber card (UDT 55-LC-EZ-50; Data
Translation, Marlboro, MA). The vessel diameter was determined by use of
customized software (Dr. H. Siegmund, Institute of Physiology,
Humboldt-University of Berlin, Berlin, Germany). The equipment allowed a
resolution of 0.2 µm of the vessel structures.
Statistical Analyses
The luminal diameter of the arterioles was determined. In all series, the
last 10 s of the treatment and control period were used for statistical
analysis of steady-state responses. For each 10-s period, five measurements
were averaged. To test diameter dose responses, we performed a nonparametric
Friedman two-way ANOVA. If significance was found, then the Mann-Whitney
U test was applied. The same test was used for the comparison of
independent measures. The confidence level, 
, was set to 0.05. Data are
presented as mean ± SEM.
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Results |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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Effect of Ang II
Ang II reduced the diameter significantly, from 8.1 ± 0.5 µm
(control) to 5.6 ± 0.8 µm (66.5 ± 7.0%) and to 5.2 ±
0.8 µm (62.2 ± 8.0%) at doses of 10-8 and 10-6
mol/L, respectively. There were no differences between female (n = 9)
and male (n = 8) mice, either for the absolute diameter or for
percentage changes (Figure
3).
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Effect of L-NAME on Responses to Ang II
Ang II significantly reduced arteriolar diameter in vessels that were
pretreated with L-NAME (male mice, n = 7). The diameter averaged 8.9
± 1.0 µm during control, 1.8 ± 1.3 µm (19.5 ±
12.6%; P < 0.05) at 10-8 mol/L Ang II, and 2.4 ±
1.1 µm (25.5 ± 10.2%; P < 0.05) at 10-6 mol/L
Ang II. As shown in Figure 3,
Ang II + L-NAME led to further constriction, in comparison with Ang II without
L-NAME pretreatment (1.8 ± 1.3 versus 5.6 ± 0.8 µm
[P < 0.05] and 19.5 ± 12.6% versus 66.5 ±
7.0% [P < 0.05] at 10-8 mol/L Ang II; 2.3 ± 1.1
µm versus 5.2 ± 0.8 µm [P = 0.054], 25.5
± 10.2% versus 62.2 ± 8.0% [P < 0.05] at
10-6 mol/L Ang II, male mice). Female (n = 11) and male
(n = 7) mice did not differ in regard to their response to Ang II
during the exposure to L-NAME (Figure
3).
Ang II Effect in eNOS Knockout Mice
In mice that were homozygous (-/-) for disruption of the eNOS gene (female
mice, n = 8), Ang II reduced diameters from 8.2 ± 0.9 µm
(control) to 0.7 ± 0.5 µm (P < 0.05) and 9.1 ±
6.0% (P < 0.05), respectively, at 10-8 mol/L Ang II and
to 0.8 ± 0.6 µm (P < 0.05) and 11.2 ± 8.2%
(P < 0.05), respectively, at 10-6 mol/L Ang II
(Figure 4). The values at
10-8 and 10-6 mol/L were significantly less in
comparison with arteriolar diameters of wild-type mice that were treated with
the same Ang II dose (female mice, n = 9). No significant differences
in Ang II reactivity were found between eNOS knockout and wild-type mice that
were pretreated with L-NAME (female mice, n = 11;
Figure 4). In both groups,
complete constriction of the vessels usually occurred at 10-8 and
10-6 mol/L Ang II (for example, see
Figure 5).
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Discussion |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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In vivo, L-arginine causes an increased plasma flow and GFR in rats (13). Accordingly, pharmacologic blockade of NO production leads to an increase in renal vascular resistance (1, 13,14,15) and a decrease in renal plasma flow in the rat (13,16). A bulk of evidence indicates that the L-arginine-mediated vasodilatation depends on NO (for overview, see reference 17). As was mentioned above, in the present study, L-arginine did not significantly change afferent vessel diameter. This may be explained, in part, by experimental conditions, e.g., the absence of circulating vasoactive substances. Notably, the inhibition with L-NAME did not change the arteriolar diameter either. In isolated perfused vessels, there is no supply of NO precursors during the experiment, which could explain the lacking L-NAME vasoconstriction. However, replenishing L-arginine to the bath solution failed to elicit an L-NAME response. These results differ from studies on isolated Af of the rabbit (6,18). In those studies, a clear reduction of the vessel diameter in response to L-NAME pretreatment was observed. Methods and drug concentration used in these studies match with our experimental protocols. Thus, our results suggest that, in mice, vasoconstriction caused by Ang II (or possibly other vasoconstrictors) is a prerequisite for a functional significance of the NO system in the control of vascular diameter.
In our study, Ang II constricted the Af significantly in a dose-dependent manner. The contraction reached significance at 10-8 and 10-6 mol/L Ang II. The results agree well with investigations in vivo and in vitro that demonstrated the vasoconstrictor properties of Ang II in Af in rabbit and rat (6,19,20,21). In contrast to previous experiments in isolated Af in the rabbit (6), we observed a prolonged contraction. This can be explained by the proposed low eNOS activity in mice. Accordingly, pretreatment with L-NAME augmented the vasoconstrictor action of Ang II at 10-8 and 10-6 mol/L.
The advantage of miniaturizing the experimental setup to allow Af perfusion of the mouse is that knockout models can be investigated. In eNOS knockout mice, which have a very low endothelial NO production, the response to Ang II was increased (Figure 4). An antagonistic action of Ang II and NO on Af in normotensive rats (19,21) and in isolated perfused Af in rabbits has been reported elsewhere (6,18). From these studies, as well as from studies in Ang II—hypertensive rats (22), it is assumed that Ang II induces NO production in renal vessels. Thus, blocking NO withdraws the counterbalance to Ang II vasoconstriction. Remarkably, in the present study, the entire effect of NO relied on Ang II induction. It is not clear whether the activation of AT1 or AT2 receptors stimulates the release of NO from Af. Both AT1 receptors of endothelial cells and AT2 receptors have been discussed elsewhere (4,18,23,24).
In the kidney, all isoforms of NOS have been discovered (25,26). Neuronal NOS and eNOS are found in the renal vasculature and in the glomerulus (27,28). The question arises as to which of the isoforms are involved in the Ang II—NO interaction. It could be shown that Ang II acts dose dependently and that the vasoconstrictive effect is enhanced in eNOS knockout mice. Because both the L-NAME treatment and knockout of the eNOS produces comparable increases in Ang II vasoconstriction, it is concluded that the mainly endothelial-derived NO counteracts the Ang II effect in our preparation. The clear enhancement of Ang II reactivity in the eNOS knockout mice is a little surprising, because compensating mechanisms can be assumed in such mice with congenital defect of NO.
Af vasoconstriction in response to Ang II did not differ between male and female mice. Moreover, there were no differences after pretreatment with L-NAME in the Ang II dose response in either of these groups. Our perfusion model is free of circulating hormonal influences and does not contain estrogen. Hormones were not added, and so, in the present model, it is assumed that gender does not change the response to Ang II.
In conclusion, the present study shows that it is feasible to microdissect and microperfuse mouse Af, thereby allowing studies on genetically modified mice. From the data obtained in Af of eNOS knockout mice and Af pretreated with L-NAME, it seems that the Ang II vasoconstriction elicits a counterbalancing NO release. The endothelial NOS isoform is responsible for this action. Furthermore, the NO system of isolated perfused Af in mice differs from other species in regard to its dependence on Ang II. Finally, this study also shows that there are no gender differences in Ang II—NO interaction in Af.
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Acknowledgments |
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) and after pretreatment with (
) L-arginine
(10-4 mol/L, male mice, n = 5).
and 
for female and male mice, respectively.
