Nitric oxide (NO) is mainly generated by endothelial NO synthase (eNOS) or neuronal NOS (nNOS). Recent studies indicate that angiotensin II generates NO release, which modulates renal vascular resistance and sympathetic neurotransmission. Experiments in wild-type [eNOS(+/+) and nNOS(+/+)], eNOS-deficient [eNOS(−/−)], and nNOS-deficient [nNOS(−/−)] mice were performed to determine which NOS isoform is involved. Isolated mice kidneys were perfused with Krebs-Henseleit solution. Endogenous norepinephrine release was measured by HPLC. Angiotensin II dose dependently increased renal vascular resistance in all mice species. EC50 and maximal pressor responses to angiotensin II were greater in eNOS(−/−) than in nNOS(−/−) and smaller in wild-type mice. The nonselective NOS inhibitor Nω-nitro-l-arginine methyl ester (l-NAME; 0.3 mM) enhanced angiotensin II-induced pressor responses in nNOS(−/−) and wild-type mice but not in eNOS(−/−) mice. In nNOS(+/+) mice, 7-nitroindazole monosodium salt (7-NINA; 0.3 mM), a selective nNOS inhibitor, enhanced angiotensin II-induced pressor responses slightly. Angiotensin II-enhanced renal nerve stimulation induced norepinephrine release in all species. l-NAME (0.3 mM) reduced angiotensin II-mediated facilitation of norepinephrine release in nNOS(−/−) and wild-type mice but not in eNOS(−/−) mice. 7-NINA failed to modulate norepinephrine release in nNOS(+/+) mice. (4-Chlorophrnylthio)guanosine-3′, 5′-cyclic monophosphate (0.1 nM) increased norepinephrine release. mRNA expression of eNOS, nNOS, and inducible NOS did not differ between mice strains. In conclusion, angiotensin II-mediated effects on renal vascular resistance and sympathetic neurotransmission are modulated by NO in mice. These effects are mediated by eNOS and nNOS, but NO derived from eNOS dominates. Only NO derived from eNOS seems to modulate angiotensin II-mediated renal norepinephrine release.
- eNOS-deficient mice
- nNOS-deficient mice
- pressor response
the renin-angiotensin-aldosterone system (RAAS) and the nitric oxide (NO) system play a superior role in regulation of cardiovascular physiology and pathology (36). Hemodynamic effects of angiotensin II, the most important peptide of the RAAS, are mediated by increased sodium and water retention, vasoconstriction, and enhanced sympathetic neurotransmitter release. In mice and rat kidney, angiotensin II regulates vascular resistance and enhances neurotransmitter release solely by activation of post- and presynaptic AT1-receptors, respectively (27, 30). On the other hand, NO serves as a potent vasodilator, has an important role in the maintenance of vascular tone, and seems to counteract angiotensin II-mediated hemodynamic effects (4). In general, in the kidney, NO released from endothelial macula densa cells and sympathetic neurons is generated by endothelial NO synthase (eNOS) or neuronal NOS (nNOS) (2, 14, 15, 17, 35). The inducible NOS (iNOS) does not seem to play any important role in the kidney (32). It has been demonstrated from work in isolated, perfused mice kidneys and afferent arterioles that the increased vasoconstriction induced by angiotensin II is counteracted by locally produced NO (11, 23, 28). Furthermore, studies have shown a pivotal role of eNOS in generation of NO in arterioles of mice kidneys (24, 25). However, not much is known about the role of nNOS localized in macula densa cells in the regulation of renal blood flow. New data have indicated that nNOS also contributes to the regulation of renal vascular resistance, by influencing the tubuloglomerular feedback sensitivity (1, 26) and counteracting pre- and postglomerular vasoconstriction (6, 12). Moreover, chronic infusion of a selective nNOS inhibitor significantly increased blood pressure in rats (20).
In contrast to the important impact of NO in regulation of renal blood flow, not much is known about its role in modulating presynaptic neurotransmitter release (9). New studies suggested an important role of NO in modulating presynaptic sympathetic neurotransmitter release, whereas NO generated by angiotensin II increased norepinephrine release in mice kidneys and rat atrium (7, 28, 31). However, it is still unclear which isoform of NOS is generating presynaptic active NO. Thus, the aims of our study were 1) to assess the predominant NOS responsible for generating NO and counteracting angiotensin II-induced changes in pressor response in mice kidney; and 2) to confirm the important role of NO in modulating renal neurotransmitter release and investigate the NO synthase responsible for generating the presynaptic active NO. To perform experiments studying effects assigned to either eNOS or nNOS, we investigated kidneys of mice that are homozygous (−/−) for disruption of the eNOS or nNOS gene.
A total of 128 mice (20–30 g) of eNOS(−/−), nNOS(−/−), and wild-type mice were used in this study. Adult male wild-type [nNOS(+/+)] and nNOS-deficient mice [nNOS(−/−)] were used from the breeding colony of the Institute of Medical Neurobiology, Otto-von-Guericke University Magdeburg. The colony was originally established with breeders from the Cardiovascular Research Center, General Hospital, Boston, MA. Mutant mice show >95% loss of nNOS production in the brain due to the disruption of the α-isoform of the nNOS enzyme (10). Their genetic background is derived from multiple backcrossings with C57BL/6J mice.
The eNOS(−/−)-deficient mice were backcrossed with C57Bl/6J [eNOS(+/+)] all from Jackson Laboratory, Bar Harbor, ME, and kept as a homozygous inbreeding. To assess the genetic status of the breeding, PCR was done continuously. Animals were fed standard mouse chow and allowed free access to tap water. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Our experiments were approved by the federal government of North Rhine-Westphalia, Germany, which is responsible for experiments performed in animals (licence no. 50.8735.1 Nr. 109/6).
Mice were anesthetized with ketamine 0.168 mg/g mouse wt ip and xylazine 8 μg/g mouse wt. Kidneys were isolated microscopically (Olympus CO11) and perfused with Krebs-Henseleit solution according to the method described previously (28). An abdominal midline incision was made, the lower aorta cannulated with polyethylene tubing, and the animal given 250 IU heparin ia. Left and right suprarenal and spermatic vessels were ligated and cut. Both kidneys were flushed with warm Krebs-Henseleit solution through the aortic cannula. The right renal artery was cannulated via the upper aorta with polyethylene tubing (Portex; 0.28 mm ID) tubing and perfused in situ at a constant rate of 7.2 ml·min−1·g kidney−1. The left renal artery was cannulated via the lower aorta with polyethylene tubing and perfused with the same constant perfusion rate of 7.2 ml·min−1·g kidney−1. All connected tissue including the capsule of both kidneys was carefully removed before the renal veins and ureters were cut. Bipolar platinum electrodes were placed around the renal arteries to stimulate the renal sympathetic nerves. The kidneys were transferred into a jacketed glass chamber maintained at a temperature of 37°C. The perfusion medium was gassed continuously with a mixture of 95% O2-5% CO2 and passed through a 0.45-μm filter before it reached the kidney. The perfusate was allowed to drip out of the cut end of the renal vein and ureter and was then collected. Perfusion pressure was monitored continuously with a Statham P23 Db pressure transducer (Gould, Oxnard, CA) coupled to a Watanabe pen recorder (Graphtec, Tokyo, Japan).
Experimental Protocol I: Effects of Angiotensin II, 7-Nitroindazole Monosodium Salt, and Nω-nitro-l-arginine methyl ester on Renal Pressor Response
Kidney wet weights were calculated according to data obtained from control mice, showing that the wet weight of mice kidneys correspond to ∼0.5% of whole body wt. This is in accordance with studies previously performed in rat isolated kidneys (29). Immediately after preparation, a bolus injection of 60 mM KCl was delivered to test the viability of the preparation followed by a stabilization period of 30 min. The agonist angiotensin II was added to the perfusion solution in a cumulative manner (3-min time interval) expressed in a dose-response curve. The perfusion of drugs were stopped when the pressor responses had reached a maximum or when no effects were observed, respectively. When dose-response curves for angiotensin II were determined in the presence of the nonselective NOS inhibitor Nω-nitro-l-arginine methyl ester (l-NAME) or the selective nNOS inhibitor 7-nitroindazole monosodium salt (7-NINA), both were added to the perfusion solution 20 min before determination of the second dose-response curve.
Because flow was maintained at a constant rate, changes in perfusion pressure were used as an index of changes in renal vascular resistance, an increase indicating vasoconstriction. The pressor responses of angiotensin II were measured as the maximum increase of perfusion pressure above basal perfusion pressure (ΔPmax = Pmax − Pbasal). This increase was expressed in millimeters of mercury. The maximum increase of perfusion pressure above basal pressure by angiotensin II in the presence of l-NAME and 7-NINA was calculated in millimeters of mercury as well. It is generally known that in the preparation of the isolated perfused kidney, renal basal vascular resistance is compared with in vivo experiments. This fact had to be taken into consideration when interpreting the results.
Experimental Protocol II: Renal Nerve Stimulation and Sympathetic Neurotransmission
The kidneys were perfused as described in Experimental Protocol I: Effects of Angiotensin II, 7-Nitroindazole Monosodium Salt, and l-NAME on Renal Pressor Response. Immediately after preparation, a priming stimulation of 5 Hz for 30 s (1 ms pulse width, 40 mA) was delivered to test the viability of the preparation. After a stabilization period of 30 min, cocaine (10 μM) and corticosterone (20 μM) were added to the perfusion solution to prevent neuronal and extraneuronal uptake of released norepinephrine, respectively. After another 20 min, 3-min fractions of the effluent were collected by a fraction collector (LKB Bromma) into vials containing 167 μl of 1 M HCl, 13.3 μl of 0.067 M EDTA, and 3.3 μl of 1M Na2SO3. Five electrical renal nerve stimulations (RNS) (S0–S4), each at 5 Hz for 30 s (1 ms pulse width, 40 mA), were applied 6, 22, 38, 54, and 70 min after the start of fraction collection. Cumulative concentration response curves for angiotensin II were determined. Substances were infused into the perfusion line by a perfusion apparatus (Braun, Melsungen, Germany) at a constant flow rate of 0.158 μl/min in a cumulative manner starting 5 min before S1, S2, S3, and S4.
When l-NAME or (4- chlorophrnylthio)guanosine-3′, 5′- cyclic monophosphate (8-pCTP-cGMP) was present throughout the experiment, drugs were added to the perfusion solution 20 min before the start of fraction collection. When the influence of l-NAME and 7-NINA alone on RNS-induced norepinephrine release was examined, l-NAME and 7-NINA, respectively, were added to the perfusion solution 20 min before S1. When influence of 8-pCTP-cGMP on RNS-induced norepinephrine release was examined, 8-pCPT-cGMP was added to the perfusion solution 16 min before the stimulation.
Determination of endogenous norepinephrine.
Norepinephrine in the collected samples was extracted (adsorption onto alumina, elution with HCLO4). Norepinephrine content was determined by reversed-phase HPLC detection (27). The amount of norepinephrine present in each samples was corrected for recoveries (average %recovery of norepinephrine-HCl was 52.6% ± 9.1; n = 63).
Calculation of data.
RNS-induced outflow of norepinephrine was determined as the difference between the content of norepinephrine present in two 3-min samples collected immediately after onset of stimulation and spontaneous norepinephrine outflow. Spontaneous norepinephrine outflow was estimated from the norepinephrine content present in the 3-min sample collected immediately before RNS. Difference between RNS-induced norepinephrine outflow and the spontaneous norepinephrine outflow was taken as an index of norepinephrine release from sympathetic nerve endings. S0 served as reference stimulation. RNS-induced norepinephrine release in S1, S2, S3, and S4 was expressed as a percentage of S0 (Sn as %S0). For further evaluations of effects of angiotensin II in the absence or presence of l-NAME, the Sn/S0 ratios were calculated as a percentage of values, which were determined in corresponding control experiments (Sn/S0 as %control). For evaluation of effects of l-NAME or 8-pCTP-cGMP on RNS-induced norepinephrine release, the Sn-to-S0 ratios were calculated as a percentage of values, which were determined in corresponding control experiments, whereas S0 serves as the control stimulation without any drug (Sn/S0 as %control).
Animals of all groups, eNOS(+/+) (n = 6), eNOS(−/−) (n = 6), nNOS(+/+) (n = 6), and nNOS(−/−) (n = 5) were killed by cervical translocation. The kidneys were removed and immediately frozen in liquid nitrogen. To investigate the expression of eNOS, iNOS, or nNOS, whole kidney RNA was isolated using an RNA isolation kit (Peqlab, Erlangen, Germany). The tissue samples were homogenized in a mortar in liquid nitrogen. For the isolation of total RNA, 20 mg of homogenized tissue was used in accordance with the manufacturer's instructions.
The estimation of the eNOS, iNOS, and nNOS mRNA amounts were performed by real-time RT-PCR analyses. RNA was isolated with Trizol reagent and reverse transcribed with Superscript and random hexamers (Invitrogen) according to the manufacturer's protocol. Quantitative PCR analysis was performed with GeneAmp 5700 (Applied Biosystems). SYBR Green was used for the fluorescent detection of DNA generated during PCR. The PCR reaction was performed in a total volume of 25 μl with 0.4 pmol/μl of each primer, and 2× SYBR Green master mix (Applied Biosystems); 2 μl cDNA corresponding to 20 ng RNA were used as template. Primers, which bridge at least one intron, were designed for PCR amplification on the base of published sequences for mouse eNOS (NM_008713), eNOSfw: 5′-GTT TGT CTG CGG CGA TGT C-3′, eNOSrv: 5′-CAT GCC GCC CTC TGT TG-3′, iNOS (NM_010927), iNOSfw: 5′-GGC AGC CTG TGA GAC CTT TG-3′, iNOSrv: 5′-CAT TGG AAG TGA AGC GTT TCG-3′, nNOS (NM_008712), nNOSfw: 5′-TCG GCT GTG CTT TGAT GGA-3′, nNOSrv: 5′-TTG AAT CGG ACC TTG TAG CTC TTC-3′. Experiments were performed in triplicate with similar results. The expression levels of receptor mRNA were normalized to β-actin by the ΔCt-method. Parallelism of standard curves of the test and control was confirmed.
Data obtained in isolated perfused kidneys were expressed as means ± SE. Differences between dose-response curves were analyzed by two-factorial ANOVA for repeated measures followed by unpaired Student's t-test. For comparison of RNA expression, the nonparametric Wilcoxon test was used for comparison of two samples. Probability levels of P < 0.05 were considered statistically significant. The number of experiments indicates the number of individual kidneys.
Drugs and Vehicles
Krebs-Henseleit solution had the following composition (in mM): 118 NaCl, 4.7 KCl, 2.5 CaCl2, 0.45 MgSO4, 25 NAHCO3, 1.03 KH2PO4, 11.1 d-(+)-glucose, 0.067 Na2EDTA, and 0.07 ascorbic acid. The following drugs were purchased: corticosterone and angiotensin II (Sigma-Aldrich), cocaine HCl (Merk); l-arginine and l-NAME (Sigma-Aldrich), 7-NINA (BioTrend), and 8-pCTP-cGMP (BioLog). Drugs were dissolved in distilled water before being diluted with Krebs-Henseleit solution, except corticosterone (in absolute ethanol), 7-NINA (in DMSO) and 8-pCTP-cGMP (in DMSO).
The effects of angiotensin II on renal vascular resistance alone, in the presence of l-NAME, a nonselective inhibitor of NOS, or 7-NINA, a selective neuronal NOS inhibitor, were analyzed. Renal basal perfusion pressure did not differ significantly between the mice strains [eNOS(+/+), 45 ± 17; eNOS(−/−), 62 ± 25; nNOS(+/+), 47 ± 22; nNOS(−/−) 58 ± 16 mmHg].
l-NAME (0.3 mM), 7-NINA (0.3 mM), and l-Arginine (1 mM), a precursor of NO, by themselves did not alter basal renal perfusion pressure in kidneys of eNOS(−/−)- and nNOS(−/−)-deficient as well as of eNOS(+/+) and nNOS(+/+) mice (data not shown).
Effects of Angiotensin II in eNOS and nNOS Knockout Mice
Angiotensin II (3 pM–10 nM) dose dependently increased renal vascular resistance in kidneys of eNOS(−/−) and nNOS(−/−)-deficient mice as well as in kidneys of eNOS(+/+) and nNOS(+/+) wild-type mice. EC50 values were significantly smaller, and maximal pressor responses to angiotensin II were significantly greater in eNOS(−/−)-deficient mice than in wild-type mice [EC50 values: eNOS(−/−), 0.5 ± 0.1 nM; eNOS(+/+), 5 ± 0.3 nM; maximal pressor response to angiotensin II: eNOS(−/−), 176 ± 10 mmHg; eNOS(+/+), 116 ± 7] (Fig. 1). No difference was seen in EC50 values between nNOS(−/−)-deficient and nNOS(+/+) wild-type mice [EC50 values: nNOS(−/−), 5 ± 0.1 nM; nNOS(+/+), 6 ± 0.2 nM]. However, pressor responses to the highest angiotensin II concentrations (3 nM and 10 nM) were significantly greater in kidneys of nNOS(−/−) (angiotensin II: 3 nM, 124 ± 9 mmHg; 10 nM, 138 ± 5 mmHg) than in nNOS(+/+) (angiotensin II: 3 nM, 105 ± 6 mmHg; 10 nM, 111 ± 6 mmHg) (Fig. 2).
Effects of l-NAME and 7-NINA on Angiotensin II-Induced Pressor Responses in Kidneys of eNOS(−/−) and nNOS(−/−) Mice
To test the effect of l-NAME on angiotensin II-induced pressor responses, l-NAME (0.3 mM) was given throughout. l-NAME (0.3 mM)-enhanced angiotensin II-induced pressor responses in kidneys of nNOS(−/−) and wild-type mice [nNOS(+/+) and eNOS(+/+)] mice. However, l-NAME (0.3 mM) failed to augment angiotensin II-induced pressor responses in kidneys of eNOS(−/−)-deficient mice. No significant difference in angiotensin II-induced pressor responses were found in isolated perfused kidneys of untreated eNOS(−/−) knockout mice and eNOS(+/+) mice pretreated with l-NAME (0.3 mM) (Fig. 1). Furthermore, no difference was observed between angiotensin II-induced pressor responses in kidneys of nNOS(−/−) and nNOS(+/+) mice both pretreated with l-NAME (0.3 mM) (Fig. 2). 7-NINA (0.3 mM) significantly increased angiotensin II-induced pressor responses in wild-type mice (Fig. 3).
Effects of l-NAME on Angiotensin II-Mediated Facilitation of RNS Induced Norepinephrine Release in Kidneys of eNOS(−/−), nNOS(−/−), and Wild-Type Mice
Angiotensin II increased RNS-induced facilitation of norepinephrine release in eNOS(+/+) and nNOS(+/+) wild-type mice as well as in eNOS(−/−) and nNOS(−/−)-deficient mice (Figs. 4 and 5). To test the effect of l-NAME (0.3 mM) on angiotensin II-induced norepinephrine release, l-NAME was given throughout. l-NAME (0.3 mM)-reduced angiotensin II-induced norepinephrine release in nNOS(−/−) and nNOS(+/+) wild-type mice (Fig. 4). In contrast therefore, l-NAME (0.3 mM) had no effect on angiotensin II-induced norepinephrine release in eNOS(−/−) mice compared with eNOS(+/+) mice (Fig. 5). Moreover, absolute RNS-induced norepinephrine release facilitated by angiotensin II (10 nM) was significantly higher in eNOS(+/+) mice compared with wild-type mice [angiotensin II 10 nM: 8,151.3 ± 2,978.9 eNOS(−/−); angiotensin II 10 nM: 14,017.0 ± 3,999.2 eNOS(+/+)]. Moreover, 7-NINA (0.3 mM) has no significant effect on angiotensin II-induced norepinephrine release in nNOS(+/+) mice (Fig. 4A). In all mice species, l-NAME (0.3 mM) by itself had no effect on RNS-induced norepinephrine release (Fig. 6). However, 8-cPTC-cGMP (0.1 mM), a membrane-permeable cGMP analog increased RNS-induced norepinephrine release compared with control (Fig. 6).
eNOS, nNOS, and iNOS mRNA Expression in Renal Tissue of Wild-Type, and eNOS(−/−)- and nNOS(−/−)-Deficient Mice
Expression of mRNA for eNOS, nNOS, and iNOS was determined by quantitative PCR analysis in renal tissue of eNOS(+/+), eNOS(−/−), nNOS(+/+), and nNOS(−/−) mice. Expression of eNOS, nNOS, and iNOS revealed no significant difference in eNOS(−/−) and nNOS(−/−)-deficient mice and their respective control mice (Fig. 7).
In the cardiovascular system, the RAAS interacts with NO. Thus, angiotensin II induces NO generation, which modulates renal vascular resistance and sympathetic neurotransmission. NO is generated by three isoforms: eNOS, nNOS, and iNOS. All isoforms are expressed in the kidney; however, iNOS seems not to play a significant role under physiological conditions (21, 32). Aside from the predominant role of eNOS in regulating renal vascular resistance, there is some controversy about the function of nNOS located in the macula densa and in renal sympathetic neurons. These renal structures generate NO, but it is unclear whether this modulates renal vascular tone. Some studies showed a considerable role of nNOS in regulating renal blood flow by influencing pre- and postglomerular microcirculation, whereas other studies failed to show that nNOS modulates renal vascular resistance (5, 20, 33). For instance, Ollerstam et al. (20) observed an increase in blood pressure after long-term inhibition of nNOS in rats, whereas in another study, inhibition of nNOS had no effect on renal function in rats fed with low-salt diet (33). Therefore, the first part of the present study was designed to investigate the influence of eNOS and nNOS on renal vascular resistance in isolated perfused kidneys. To identify the responsible NOS isoforms, experiments were performed in either eNOS(−/−) or nNOS(−/−)-deficient mice.
Role of NO on Angiotensin II-Induced Pressor Responses in eNOS and nNOS Mice Kidneys
In all mice species, angiotensin II increased pressor responses in a cumulative manner. Pressor responses to angiotensin II were significantly greater in kidneys of eNOS-deficient mice than in kidneys of eNOS wild-type mice. l-NAME, a nonselective NOS inhibitor significantly increased angiotensin II-induced pressor response in kidneys of wild-type mice. In contrast, l-NAME had no effect on angiotensin II-induced pressor responses in eNOS(−/−)-deficient mice. Moreover, pressor responses to angiotensin II were comparable in eNOS(−/−)-deficient and eNOS(+/+) wild-type mice pretreated with l-NAME. These results, as well as studies performed by others (22, 24), emphasize the predominate role of endothelial-derived NO in counteracting angiotensin II-induced pressor responses. This angiotensin II-induced NO release is mediated by AT1-receptors as shown in perfused kidneys of AT2 receptor-deficient mice (28), isolated renal afferent arterioles (22), and macula densa cells (17).
In nNOS-deficient and nNOS wild-type mice, angiotensin II increases renal perfusion pressure. These pressor responses to angiotensin II were significantly greater in nNOS-deficient than in wild-type mice for the highest two concentrations of angiotensin II (3 and 10 nM). Moreover, 7-NINA, a selective nNOS inhibitor, increased angiotensin II-induced pressor responses in kidneys of wild-type mice. Thus, our results from nNOS(−/−) and wild-type mice indicate that nNOS plays, to some extent, a role in regulating pressor responses to angiotensin II in isolated perfused kidneys. This observation is in accordance with several studies that demonstrated, in vivo as well as in vitro, that nNOS might play a role in regulating renal vascular tone and blood pressure (5, 11, 18) by sensitization of the tubuloglomerular feedback, which leads to vasoconstriction and volume retention (3). Our study suggests that neuronal-derived NO has an influence on angiotensin II-regulated vascular tone in isolated kidneys; however, it does not play an as important role as endothelial-derived NO(−/−). On the basis of RT-PCR there was no evidence for adaptive changes in eNOS, nNOS, and iNOS in the respective knockout mice.
Effects of NO on Angiotensin II-Mediated Facilitation of Norepinephrine Release in eNOS and nNOS Mice Kidneys
Not much is known about the role of NO in modulating sympathetic neurotransmitter release. Recent studies have demonstrated that endogenously produced NO increases norepinephrine release in isolated perfused kidneys (28, 31) and other cardiovascular tissues (7) of mice and rats. In contrast, others have described inhibitory effects of NO on norepinephrine release in rat mesenteric resistance arteries (8). It is not known which NOS is responsible for generating presynaptic active NO. Thus, the second aim of the present study was to clarify whether NO is able to modulate neuronal norepinephrine release and to identify the responsible NOS in mice kidneys.
In kidneys of eNOS(−/−), nNOS(−/−), and wild-type mice, activation of presynaptic angiotensin II receptors facilitates RNS-induced neurotransmitter release. l-NAME alone had no effect on RNS-induced neurotransmitter release. This is in contrast to findings in rat kidney, in which l-NAME decreased norepinephrine release (31) but is in accordance with findings performed in vivo by measuring renal nerve activity in conscious rabbits, where NO modulates sympathetic outflow only in the presence of angiotensin II (16). However, l-NAME decreased angiotensin II facilitation of norepinephrine release in wild-type mice in the present study. This suggests that only angiotensin II and not neuronally released sympathetic neurotransmitters, such as norepinephrine or ATP (34), induce NO generation in mice kidneys to influence presynaptic norepinephrine release. Although presynaptic effects of angiotensin II mediated by NO have been demonstrated in different species and organs, up to now nothing is known about the responsible NOS generating presynaptic active NO. As described in results, maximal angiotensin II-induced facilitation of norepinephrine release was reduced in eNOS(−/−) mice compared with wild-type mice. This observation led us to the assumption that NO generated by eNOS might play a role in modulation of angiotensin II-induced facilitation of neurotransmitter release. In addition, in accordance with this observation, l-NAME did not affect angiotensin II-induced facilitation of norepinephrine release in eNOS(−/−)-deficient mice. In contrast, l-NAME decreased angiotensin II-induced facilitation of norepinephrine release in nNOS(−/−) mice in the same manner as in wild-type mice. Furthermore, 7-NINA failed to reduce angiotensin II-facilitated RNS-induced norepinephrine release in nNOS(+/+) mice. These results suggest that angiotensin II-induced formation of presynaptic active NO is generated by eNOS, which facilitates renal norepinephrine release in this mouse model. In line with this, a membrane-permeable analog cGMP (8-pCTP-cGMP) increased norepinephrine release by itself. This is in accordance with observations done in the iris-ciliary body of rabbits (19). However, further studies are needed to clarify the underlying mechanism and the location of NO generation modulating angiotensin II-induced neurotransmitter release. Recently, one explanation described in the brain suggested that NO generated at the postsynaptic site diffuses retrograde through the synaptic gap and then activates presynaptic cGMP, which modulates norepinephrine release (13).
All experiments were performed in the presence of the uptake1/2 blocker cocaine and corticosterone. Therefore, the observed effects of angiotensin II and l-NAME are independent of an influence on synthesis, reuptake, or renal clearance of norepinephrine.
In conclusion, the present study shows a significant role of both eNOS and nNOS in modulating angiotensin II-induced pressor responses in isolated perfused kidney of mice. Thus, postsynaptically, NO attenuates angiotensin II-mediated vasoconstriction and the presented data establish the predominant role of eNOS in this setting. Furthermore, our experiments demonstrate that NO influences angiotensin II-mediated facilitation of neurotransmitter release in mice kidneys. This presynaptic active NO seems to be exclusively generated by endothelial NOS. Thus, NO has pre- and postsynaptic divergent effects, and up to now it not possible to say what is the net effect on renal vascular resistance in vivo.
This study was supported by the Deutsche Forschungsgemeinschaft Grant RU-401/5-7.
We thank Ulrike Neumann for her excellent technical assistance.
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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