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and neurogenic control of blood pressure in
normal rats and rats with chronic renal failure
Division of Nephrology, Department of Medicine, University of Southern California, Los Angeles, California 90033
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Increased sympathetic
nervous system (SNS) activity plays a role in the genesis of
hypertension in rats with chronic renal failure (CRF). The rise in
central SNS activity is mitigated by increased local expression of
neuronal nitric oxide synthase (NOS) mRNA and
NO2/NO3 production. Because interleukin
(IL)-1
may activate nitric oxide in the brain, we have tested the
hypothesis that IL-1 may modulate the activity of the SNS via
regulation of the local expression of neuronal NOS (nNOS) in the brain
of CRF and control rats. To this end, we first found that
administration of IL-1 in the lateral ventricle of control and CRF
rats decreased blood pressure and norepinephrine (NE) secretion from
the posterior hypothalamus (PH) and increased NOS mRNA expression.
Second, we observed that an acute or chronic injection of an
IL-1-specific antibody in the lateral ventricle raised blood
pressure and NE secretion from the PH and decreased NOS mRNA abundance
in the PH of control and CRF rats. Finally, we measured the IL-1
mRNA abundance in the PH, locus coeruleus, and paraventricular nuclei of CRF and control rats by RT-PCR and found it to be greater in CRF
rats than in control rats. In conclusion, these studies have shown that
IL-1 modulates the activity of the SNS in the central nervous system
and that this modulation is mediated by increased local expression of
nNOS mRNA.
sympathetic nerve activity; nitric oxide; nitric oxide synthase; posterior hypothalamus
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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RECENT STUDIES PROVIDED evidence that nitric oxide (NO) synthase (NOS) is present in specific areas of the brain involved in the neurogenic control of blood pressure (6,44). The neuronal isoform of NOS (nNOS) is an important component of the transduction pathways that tonically inhibit the sympathetic outflow from the brain stem (42). In normal rats, the basal activity of the central sympathetic nervous system (SNS) is inhibited by local NO production (47), and the hypertensive response to NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, is greatly attenuated by sympathectomy or renal denervation (22). In the brain of 5/6 nephrectomized rats, nNOS mRNA abundance and NO2/NO3 content were greater than in control rats. Administration of L-NAME increased norepinephrine (NE) turnover rate in the posterior hypothalamic (PH) nuclei and blood pressure in these rats (47). This suggests that increased local expression of NO in certain brain nuclei partially mitigates the increase in SNS activity and hypertension that occurs in this model of hypertension. The mechanisms that mediate the increase in NO and nNOS mRNA expression in the brain of chronic renal failure (CRF) rats remain to be elucidated.
Because of the complex relationships existing between cytokines, NO,
and SNS activity (18, 25, 32-33, 41), one possible mediator for the increase in NO expression is interleukin (IL)-1. Proinflammatory cytokines increase the expression of an inducible form
of NOS (iNOS) in rat microvascular brain endothelial cells (5) as well as in airway epithelial cells (3,
30), smooth muscle cells, mesangial cells (20), and
endothelial cells (39, 40). Bacterial lipopolysaccharide
induction of iNOS activity in brain cells is mediated in part by
IL-1 (31, 35). However, nNOS expression in the brain
was not increased after administration of endotoxin, despite a
significant rise in IL-1. Some evidence however, suggests that NO is
involved in the IL-1-induced central activation of SNS outflow in
rats (6). Murakami et al. (23) showed that
IL-1 induces a prostaglandin-mediated central excitation of the SNS
and that NO is also involved in this activation. Other studies,
however, indicate that although IL-1 may mediate the stimulation of
rat hypothalamic-pituitary axis induced by endotoxin, NO may be
involved in the counterregulation of this response (35). Thus the physiological interactions among IL-1, nNOS, and the noradrenergic regulation of blood pressure require further
clarification. In addition, the role of these factors in the
pathophysiology of hypertension in CRF remains to be elucidated.
To address these issues, we first studied the effects of an
intracerebroventricular infusion of IL-1 on blood pressure, NE secretion from the PH nuclei, and brain nNOS mRNA abundance in control
and CRF rats. Second, we examined the effects of a specific anti-IL-1 antibody on blood pressure and NE secretion from the PH
nuclei of control and CRF rats. Third, we examined the expression of
IL-1 in several brain nuclei of Sprague-Dawley rats subjected to 5/6
nephrectomy (CRF) and sham nephrectomy.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animal Preparation
Male Sprague-Dawley rats weighing 250-300 g were used for these studies. Rats received normal rat chow (ICN, Nutritional Biochemical, Cleveland, OH) and tap water. Rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (35 mg/kg). For the studies in rats with chronic renal insufficiency, we performed 5/6 nephrectomy (CRF group) or sham nephrectomy (control group). CRF rats underwent 2/3 nephrectomy of the right kidney and total nephrectomy of the left kidney as previously described (4). Rats were studied 4-5 wk after nephrectomy or sham operation. Blood pressure measurements were performed before surgery and weekly thereafter by the tail cuff method. Blood samples were drawn from the tail of the animals before and 4 wk after 5/6 nephrectomy for measurement of serum creatinine by autoanalyzer.Measurements of Blood Pressure
For chronic studies, blood pressure was measured weekly by the tail cuff method using an electrosphygmomanometer and physiograph recorder (MK-III, Narco Bio-Systems, Houston, TX). Each blood pressure recorded was the average of six to eight readings over 30-40 min.For acute studies, polyethylene catheters (PE-10) were implanted in a femoral artery and vein for subsequent measurements of mean arterial pressure and for administration of drugs. The femoral catheters were connected to a pressure transducer (P-1000B, Narco Bio-Systems) and strain-gauge amplifier and recorder (7-A, Grass Instruments).
NE Secretion from PH Nuclei
Four weeks after nephrectomy or sham surgery, rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (35 mg/kg). Subsequently, the rat's head was accurately placed in a stereotaxic apparatus and a 2-mm-long Teflon 22-gauge guide cannula (IV Catheter Placement Unit; Critikon, Tampa, FL) was implanted in the PH using coordinates anteroposterior
4.0 mm, lateral ± 0.4 mm, and vertical 8 mm. The guide was secured in place with dental
cement. A 28-gauge stainless steel stylus was lowered through the guide
cannula to a depth 1.5 mm dorsal to the dorsoventral coordinate for the
PH, namely, 8.5 mm from the skull surface.
Microdialysis probes were constructed from 25-gauge stainless tubing (Critikon) and 1-mm lengths of cuproammonium rayon dialysis tubes as previously described (47). One end of the dialysis tube was sealed with epoxy resin (Rapid Araldite; Ciba-Geigy, Summit, NJ). Two lengths of fused silica capillary tubing (outside diameter × inside diameter = 150 × 75 µm) were inserted into the 25-gauge tubing and the longer length, which formed the inlet, was inserted into the dialysis tube with the tip 200 µm from the sealed end. The short capillary formed the outlet of the probe. The inlet and outlet fused silica tubes were covered with 10 mm of 27-gauge stainless steel tubing for connection of polyethylene tubing.
The stylus was removed from the guide cannula and replaced with the
dialysis probe, which was secured to the guide with sticky wax. The
inlet tubing of the dialysis probe was connected by polyethylene (PE-20) tubing to a 1-ml disposable syringe driven by a microinfusion pump (model A-99, Razel), and an infusion of artificial cerebrospinal fluid (aCSF) prepared by us (in mM: 150 Na+, 3.0 K+, 1.4 Ca2+, 0.8 Mg2+, 1.0 P, 155 Cl
, pH 7.2) was initiated at the rate of 1.7 µl/min. PE-10 tubing was attached to the outlet side of the
probe, and the free end led to a 0.5-ml vial set in a small box of ice.
The vial contained 2 µl of 0.1 N HCl for preservation of NE. After
120 min of dialysis equilibration, dialysate samples were collected for
5 min each. All samples were immediately frozen and stored at 70°C
until the time of assay.
Effects of Intracerebroventricular Infusion of
IL-1 Injection on Blood Pressure and
NE Secretion from PH
(R&D Systems, Minneapolis, MN) was then
infused in the lateral ventricle of control rats (in doses of 0, 5, and
10 ng in 50 µl of aCSF over a period of 30 min). Because the response
to IL-1 in CRF was found to be reduced compared with that of control
rats, CRF rats received IL-1 in doses of 10 and 100 ng in 50 µl of
aCSF. Blood pressure was measured continuously, and dialysate from the
PH was collected every 5 min for measurement of NE concentration three
times before IL-1 infusion and for 90-120 min thereafter.
Control rats received 50 µl of aCSF in the lateral ventricle. The
infusion in the lateral ventricle was performed with the cannula
connected by a polyethylene tube to a 100-µl microsyringe. To measure
the effects of IL-1 on nNOS mRNA abundance, groups of control rats
were killed at 90 min (time of maximum effect of drug) or 120 min after
initiation of the IL-1 infusion (time when blood pressure and NE
secretion had returned to baseline levels), and the brains were
immediately separated and frozen in dry ice and stored at 70°C
until measurements of NOS mRNA gene expression were made. CRF rats were
killed at 60 or 90 min after initiation of IL-1 infusion.
Effects of IL-1 Antibody on Blood Pressure and
NE Secretion From PH
antibody (15 µg/150 µl dissolved in PBS buffer solution; R&D Systems, Minneapolis, MN) or vehicle was infused in the
lateral ventricle via a micropump for a period of 30 min. Blood
pressure was continuously recorded for 15 min before the injection of
IL-1 antibody and for 120 min thereafter. Samples for determination
of NE concentration in the dialysate from the PH were collected every 5 min, starting 15 min before the infusion of IL-1 antibody and for
120 min thereafter. At the end of the experiment, rats were killed by
decapitation, and the brain was isolated, immediately frozen in dry
ice, and stored at 70°C for 
3 wk for determination of nNOS mRNA
abundance in the PH, locus coeruleus (LC), and paraventricular nuclei (PVN).
In two separate group of CRF rats, a cannula (23 gauge) was placed in
the right lateral ventricle as described, and IL-1 antibody (15 µg/150 µl dissolved in PBS buffer solution; R&D Systems) or vehicle
was injected in the lateral ventricle for a period of 1 h for 3 consecutive days. After 3 days, blood pressure was measured by the tail
cuff method while rats were not anesthetized. The rats were then killed
by decapitation, and the brain was immediately separated, frozen in dry
ice, and stored at 70°C for 3 wk for measurements of NE content
and NOS mRNA abundance in the PH, LC, and PVN.
Effects of Phentolamine on Blood Pressure, NE Secretion From PH, and nNOS mRNA Expression in PH, PVN, and LC of CRF Rats
In a separate group of five anesthetized rats, phentolamine (0.15 mg in 0.2 ml of normal saline iv) was injected 3 wk after nephrectomy or sham operation and arterial pressure and NE secretion from the PH were measured as described above. At the end of the experiments, brains were isolated for measurement of nNOS and IL-1
mRNA expression in the PH, LC, and PVN.
Effects of Angiotensin II on Blood Pressure and NE Secretion From PH and nNOS mRNA Expression in PH, PVN, and LC of Control Rats
In five normal rats, angiotensin II (8-16 ng/min in 30 µl of saline iv) was infused and arterial pressure and NE secretion from the PH were measured as described above. At the end of the experiments, brains were isolated for measurement of nNOS and IL-1 mRNA
expression in the PH, LC, and PVN.
Isolation of Brain Nuclei
Brains were cut into consecutive 200-µm sections in a cryostat at20°C, and bilateral micropunches 0.5 mm in diameter were obtained from several brain nuclei using the following coordinates. For
the anterior hypothalamic nuclei, the coordinates were anteroposterior from bregma 1.1 mm to 1.9 mm, lateral ±0.9, and vertical 8.6 mm
from skull surface, according to the Paxinos and Watson rat atlas (Ref.
28; see also Refs. 13,
26). The coordinates for the PH were
anteroposterior from 3.5 to 4.1 mm, lateral ±0.4 mm, and vertical
8 mm; for the PVN anteroposterior from 1.4 to 2.0 mm, lateral ± 0.3 mm, and vertical 7.9 mm; and for the LC anteroposterior from
9.8 mm to 10.2 mm, lateral ±1.4 mm, and vertical 7.2 mm. The
nuclei so isolated were used to measure NE content and IL-1 and nNOS
mRNA expression. In CRF rats, IL-1 mRNA abundance was also measured
in other brain nuclei using the following coordinates: for the nucleus
tractus solitarii anteroposterior from 11.6 mm to 12.6 mm, lateral
±1.4 mm, and vertical 8.3 mm; for the rostral ventrolateral medulla
(C1) the coordinates were anteroposterior from 11.8 mm to
12.8 mm, lateral ±2.3 mm, and vertical 10 mm. For the ventral
ventrolateral medulla (A1), we performed micropunches at
two different levels with coordinates anteroposterior from 13.6 to
14.3 mm, lateral ± 2.0 mm, and vertical 9.9 mm, and
anteroposterior from 14.31 to 14.60 mm, lateral ±2.4, and vertical
10.0 mm, respectively.
NE Microassay
All brain samples were first sonicated in 0.03 N perchloric acid and then centrifuged (10,000 g for 30 s). The supernatants were assayed for NE by a highly sensitive microradioenzymatic assay (48). Dialysate (10 µl) was added to 5 µl of reaction mixture containing 1 µl of 3.7 M Tris base (with 0.37 M EGTA and 1.8 M MgCl2, pH 8.2), 0.06 µl of 36 mM benzoxylamine, 1.5 µl of S-[methyl-3H]adenosyl-L-methionine, and 2.4 µl of partially purified catechol-o-methyltransferase and incubated for 60 min at 37°C. The sensitivity of this method is 0.5 pg.Reverse Transcription-Polymerase Chain Reaction
Total RNA was extracted from the tissues with the TRIzol Reagent (Life Technologies), which is an improvement of the single-step RNA isolation method described by Chomczynski and Sacchi (10). The quantity and purity of total RNA for each sample was measured by optical density at 260 nm and at 280 nm (Du-64 Sprectrophotometer, Beckman Instruments, Fullerton, CA). Total RNA measurement of all samples ranged between 0.2 and 0.8 µg. All samples were stored at70°C for the next part of the experiments.
For reverse transcription, total RNA (0.2-0.8 µg) was mixed with
3 µl of random hexamer primers (0.5 ng/µl), incubated at 70°C for
10 min, and then transferred on ice for 5 min. Nine microliters of RT
reaction mixture [containing 4 µl of 5× reaction buffer, 2 µl of
25 mM MgCl2, 1 µl of 10 mM deoxynucleotide mixture
(dNTP), and 2 µl of 0.1 dithiothreitol (DTT)] were added to each
sample tube. The mixture was incubated at 25°C for 5 min. Thereafter, 1 µl (200 units) of SuperScript II RT was added, and samples were incubated at 25°C for 10 min and at 42°C for 50 min. Subsequently, the reaction mixture was heated to 70°C for 15 min to inactivate the
RT and then chilled on ice for 5 min. Four microliters of cDNA template
were used for each PCR reaction. PCR was performed on the resulting RT
product using specific oligonucleotide primers for either nNOS or
IL-1 derived from cDNAs cloned from rat brain (6) or
rat liver (36) (Table 1). A
master mix of PCR reagents was made for duplex reactions containing
primers for the "housekeeping" gene -actin (Genbank accession
no. J00691) and primers for either nNOS (Genbank accession no. X59949)
or IL-1 (Genbank accession no. M98820). The PCR reaction mixture
contained 10 µl of 10× PCR buffer, 5 units Taq DNA
polymerase, 4 µl of cDNA, 2 mM MgCl2, 0.2 mM dNTP, and
0.1 µM of each primer set. The final volume of each PCR was 100 µl.
Each reaction mixture tube was overlaid with 50 µl of mineral
oil. The PCR was performed with a DNA Thermal Cycler 480 (Perkin-Elmer,
Branchburg, NJ). The cycling programs were as follows: denaturation for
1 min at 94°C, annealing for 1.5 min at 58°C, and extension for 1.5 min at 72°C. After completion of PCR (25 cycles for -actin, 28 cycles for IL-1, and 28 cycles for NOS), the thermal cycler was
stopped in the course of an extension and 80 µl of the reaction
volume were removed through the mineral oil from each vial to be used
for quantification of RT-PCR. To ensure that the PCR reaction was
appropriate, the remaining 20 µl of the PCR mixture were subjected to
an additional 15 cycles of amplification. Later, PCR products were
separated by 1.5% agarose gel electrophoresis with ethidium bromide
staining. Only PCR products with a distinct target band corresponding
to the appropriate product on the electrophoresis gel were used for further analysis.
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The RT-PCR products were quantified by a method based on that of
Higuchi and Dollinger (16). Fluorescence was measured in a
fluorescence spectrofluorometer (F-2000, Hitachi, Tokyo, Japan). Excitation was at 280 nm, and emitted light was selected at 590 nm.
Results were expressed as a ratio of the resultant optical densities
for the specific gene to -actin.
Random hexamers, DTT, SuperScript II RT with reaction buffer (5×; 20 mM Tris · HCl, 10 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.01% NP-40, and 50% glycerol), Taq DNA polymerase with reaction buffer (10×; 50 mM Tris · HCl, 10 mM NaCl, 0.1 mM EDTA, 5 mM DTT, 50% glycerol, and 1.0% Triton X-100), dNTP, and MgCl2 were purchased from GIBCO-BRL (Gaithersburg, MD).
Measurements of IL-1 Expression in Brain of
CRF and Control Rats
70°F until assay (3 wk). Later, the brains were cut into consecutive 200-µm sections in a cryostat at
20°C and bilateral micropunches 0.5 mm in diameter from several brain nuclei were obtained for determination of IL-1 mRNA abundance.
Location of Probes
At the end of the experiments, while rats were still anesthetized, the dialysis probes were removed and rats were killed by decapitation. The brains were immediately removed, frozen in dry ice, and stored at70°C. Later, brains were sliced in 200-µm sections
and the proper location of the lesion in the PH nuclei was identified.
Only rats with probes properly implanted in the PH nuclei were
considered for further analysis.
Statistical Analyses
Data were analyzed by one-way analysis of variance and by Fisher's test for comparisons among groups using the computer program Statview and Graphics 4.01 (Labacus Concepts). When indicated, repeated-measures ANOVA was performed. Simple regression analyses were performed to determine correlations among different parameters. Results are expressed as means ± SE. The accepted level of significance was P < 0.05.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Blood Pressure and Serum Creatinine
Data on body weight, blood pressure, and serum creatinine are summarized in Table 2. Serum creatinine concentration was significantly greater in CRF compared with control rats, whereas body weight was significantly lower in CRF than control rats.
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Effect of IL-1 on Arterial Blood Pressure and NE Secretion
From PH Nuclei
Control rats.
The infusion of IL-1 in the lateral ventricle (in doses of 0, 5, and
10 ng in 50 µl of aCSF, over a period of 30 min) caused a
dose-dependent decrease in blood pressure (Figs.
1A and
2A). The
hypotensive response was not immediate, but it became significant only
50 min after the initiation of the infusion, it reached its nadir after
60-70 min, and it reverted to baseline values after 120 min.
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in the lateral ventricle also caused a
dose-dependent decrease in NE secretion from the PH (Figs.
1B and 2B). The changes in NE release from the PH
became significant 40 min after initiation of IL-1 infusion,
therefore, preceding the changes in blood pressure. This suggests that
the decrease in blood pressure may be a consequence rather than a cause
of the fall in NE secretion. There was a highly significant
relationship between the levels of blood pressure and NE secretion from
the PH measured throughout the 120 min of observation (r =
0.82; P < 0.0001).
The administration of IL-1 in the lateral ventricle increased NOS
mRNA abundance in the PH, LC, and PVN. The difference was evident when
rats were killed 90 min after initiation of the IL-1 infusion, but
it was not present when rats were killed 120 min after initiation of
IL-1 infusion, at the time when the hypotensive action and the
inhibition of NE secretion from the PH had subsided (Figs.
3 and 4).
The effect of IL-1- on NOS mRNA abundance was dose
dependent (Fig. 5).
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Rats with CRF.
In CRF rats, the infusion of IL-1 in the lateral ventricle (10 and
100 ng in 50 µl of perfusate, over a period of 30 min) caused a
dose-dependent decrease in blood pressure (Fig.
6A). The hypotensive response
was not immediate, but it became significant 50 min after the
initiation of the infusion, it reached its nadir after 55 min, and it
reverted to baseline values after 90 min. The decrease in blood
pressure caused by the intracerebroventricular injection of IL-1 (10 ng) was significantly less (P < 0.01) in CRF rats
(15 ± 2.0 mmHg) than it was in control animals (35.7 ± 2.04).
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(10 and 100 ng/50 µl of aCSF)
in the lateral ventricle caused a significant decrease in NE secretion
from the PH (Fig. 6B), which became significant 40 min after
the beginning of the infusion, reached a nadir after 65-70 min,
and reverted to baseline values after 90 min. Quantitatively, the
decrease in NE secretion from the PH caused by the
intracerebroventricular injection of IL-1 (10 ng) was less
pronounced (P < 0.01) in CRF (31 ± 6.0 pg/ml)
than that observed in control rats (73 ± 3.7 pg/ml).
Qualitatively, the decrease in NE secretion subsided faster in CRF rats
than it did in control rats (90 as opposed to 120 min). In CRF rats, as
in control rats, the changes in NE release from the PH preceded the
changes in blood pressure and there was a highly significant
relationship between the levels of blood pressure and NE secretion from
the PH (r = 0.75; P < 0.0001). The
administration of IL-1 in the lateral ventricle increased NOS mRNA
abundance in the PH nuclei, LC, and PVN. The difference was evident
when rats were killed 60 min after initiation of the IL-1 infusion,
but it was not present when rats were killed 90 after initiation of
IL-1 infusion, when the hypotensive action and the inhibition of NE
secretion from the PH had subsided (Fig. 7).
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Effects of IL-1 Antibody on Blood
Pressure, NE Content, and NOS
mRNA Abundance in Brains of CRF and
Control Rats
mRNA in the brain
modulates the abundance of nNOS mRNA, SNS activity, and blood pressure
in control as well as CRF rats, we studied the effects of an acute and
a subacute (3 days) infusion of a specific anti-rat IL-1 antibody in
the lateral ventricle.
An acute infusion of IL-1 antibody (15 µg/150 µl in PBS buffer
solution) in the lateral ventricle raised blood pressure and NE
secretion from the PH both in control and in anesthetized CRF rats
(Fig. 8, A and B)
and decreased nNOS mRNA abundance in the PH, LC, and PVN of both
control and CRF rats (Fig. 9).
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The infusion of IL-1 antibody (15 µg/150 µl in PBS buffer
solution) in the lateral ventricle of awake CRF rats for 3 consecutive days raised blood pressure from 160 ± 1.6 to 179 ± 1.9 mmHg
(P < 0.01), whereas no changes occurred in CRF rats
infused with vehicle only (161 ± 1.9 vs. 158 ± 2.6 mmHg)
(Fig. 10). IL-1 antibody also caused
a significant (P < 0.001) decrease in NOS mRNA
abundance in the PH (from 80.8 ± 2.1 to 47.4 ± 2.6), PVN
(from 69.4 ± 3.2 to 38.0 ± 2.2), and LC (from 114.8 ± 6.6 to 36.8 ± 2.2) and increased (P < 0.001) NE
content in the PH (from 23,639 ± 625 to 31,393 ± 1,142 pg/mg of tissue), PVN (from 15,096 ± 179 to 19,984 ± 304 pg/mg of tissue), and LC (from 11,414 ± 541 to 20,172 pg/mg of tissue).
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In control rats, infusion of IL-1 antibody or vehicle in the lateral
ventricle for 3 consecutive days caused a significant (P < 0.05) rise in blood pressure (from 112 ± 1.25 to 119.0 ± 1.25 mmHg), whereas blood pressure did not change
in rats that received vehicle (113 ± 1.4 and 111.3 ± 1.25 mmHg, respectively). After 3 days of infusion of anti-IL-1 in the
lateral ventricle of control rats, NE secretion from the PH increased
compared with that of rats injected with vehicle (171 ± 1.56 vs.
160 ± 2.7 pg/ml, P < 0.01). After infusion of
anti-IL-1, the expression of IL-1 mRNA did not change in the PH
(26.7 ± 0.5 vs. 25.1 ± 0.5), PVN (25.9 ± 0.7 vs.
26.2 ± 0.5), and LC (22.2 ± 0.7 vs. 21.6 ± 0.6) of
control rats. On the other hand, the infusion of anti-IL-1 in the
lateral ventricle decreased nNOS expression in the PH (from 32.3 ± 0.7 to 29.8 ± 0.5, P < 0.05), PVN (from
24.5 ± 0.4 to 21.4 ± 0.5, P < 0.005), and
LC (from 31.4 ± 0.7 to 28.1 ± 0.4, P < 0.01) of control rats.
Effect of Phentolamine and Angiotensin II on Blood Pressure, NE Secretion From PH, and NOS mRNA Abundance in Brain of CRF Rats
Infusion of phentolamine (0.15 mg iv) in five anesthetized CRF rats caused a marked decrease in blood pressure from 168.8 ± 4.3 to 112.5 ± 1.4 mmHg. This was accompanied by a significant decrease in nNOS and IL-1 mRNA expression in the PH, PVN, and LC
(Fig. 11). NE secretion from the PH, on
the other hand, increased significantly from 312.5 ± 8.4 to
349 ± 3.0 pg/ml (P < 0.01).
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Infusion of angiotensin II (8-16 ng/min iv) in five control rats
to achieve a rise in blood pressure of ~180 mmHg, levels similar to
those of CRF rats, increased nNOS mRNA gene expression in the PH, PVN,
and LC but decreased NE secretion from the PH (see Refs.
47 and 48). Angiotensin II also increased IL-1 mRNA
expression in the PH from 35 ± 0.9 to 47 ± 1.6 (P < 0.0002).
Expression of IL-1-mRNA in Brain of
CRF and Control Rats
-mRNA was greater in several brain nuclei
of CRF compared with control rats (Fig.
12).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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These studies have shown that IL-1 exerts a modulatory action
on SNS activity both in control and in CRF rats. This action of IL-1
is mediated by increased expression of nNOS mRNA in the brain. Several
lines of evidence support this conclusion. First, administration of
IL-1 in the lateral ventricle of control and CRF rats caused a
dose-dependent decrease in blood pressure and NE secretion from the PH
and an increase in nNOS mRNA abundance in several brain nuclei. Second,
infusion of a specific anti-rat IL-1 antibody in the lateral
ventricle led to an increase in blood pressure and NE secretion from
the PH of control rats and to a further rise in blood pressure and NE
secretion from the PH of CRF rats. Third, the administration of an
anti-rat IL-1 antibody decreased NOS mRNA expression in the PH, PVN,
and LC of both control and CRF rats. Finally, in CRF rats we observed an increase in the abundance of IL-1 mRNA in all brain nuclei tested. In all, these findings suggest that IL-1 modulates the activity of the SNS via activation of nNOS and partially mitigates the
rise in blood pressure and in SNS activity in CRF as well as in control rats.
To determine whether the increased expression of IL-1 is modulated
specifically by the uremic state or is a nonspecific consequence of
changes in blood pressure, we administered phentolamine, an 
-blocking agent, to CRF rats. This caused a marked decrease in blood
pressure, a significant decrease in nNOS and IL-1 mRNA expression in
the PH, PVN, and LC, and an increase in NE secretion from the PH. In
contrast, in control rats, a rise in blood pressure caused by the
infusion of angiotensin II increased nNOS mRNA abundance in the PH,
PVN, and LC but decreased NE secretion from the PH. The studies with
phentolamine and angiotensin II suggest that changes in blood pressure,
per se, may alter the expression of IL-1 and nNOS in the brain. The
data also confirm that upregulation of IL-1 and nNOS is associated
with suppression of SNS activity, whereas downregulation of IL-1 and
nNOS is associated with increased SNS activity. In all, these studies
confirm that IL-1 and nNOS exert a modulatory action on central SNS
activity and blood pressure.
Previous studies on the relationship between IL-1 and central or
peripheral SNS activity have provided conflicting results. In the
myenteric plexus of noninflamed intestine, IL-1 suppressed NE
release (33), an action mediated by products of the
cyclooxygenase pathway (32). In contrast, Ichijo et al.
(17) observed that an infusion of recombinant human
IL-1 in the third ventricle of anesthetized rats elicited a
dose-dependent increase in the electrical activity of the splenic
sympathetic nerves. This action was prostaglandin dependent and
sensitive to -melanocyte-stimulating hormone. Terao et al.
(41) observed that an intracerebroventricular injection of
IL-1 caused a dose-dependent increase in NE turnover in the spleen,
lung, diaphragm, and pancreas but not in the heart, kidney, liver,
adrenal glands, and brown adipose tissue. Niijima et al.
(25) showed that, when injected intravenously, IL-1 increased splenic sympathetic activity but suppressed renal sympathetic activity. Murakami et al. (23) showed that
intracerebroventricular administration of IL-1 induced a gradual
elevation of plasma NE levels that was abolished by pretreatment with
chemical sympathectomy, indomethacin, and a NOS inhibitor.
Unfortunately, measurements of plasma catecholamines in anesthetized
animals cannot be used as a reliable marker of the effects of IL-1
on SNS activity. Moreover, Murakami et al. (23) did not
report the effects of IL-1 on blood pressure. Thus one cannot rule
out the possibility that the elevation of plasma catecholamine or of
regional SNS activity might be secondary to hypotension rather than primary.
Our study has demonstrated for the first time that, when injected in
the lateral ventricle, IL-1 lowers blood pressure in control and CRF
rats and that this action is associated with a decrease in NE secretion
from the PH. The decrease in NE secretion from the PH precedes the fall
in blood pressure, suggesting a cause-effect relationship.
The temporal pattern of the responses to IL-1 given
intracerebroventricularly seems inconsistent with a neural response
only in appearance. The slow onset of response could be caused by the time needed for IL-1 to induce the increase in expression of nNOS,
which may be the ultimate mediator of the action of this cytokine. The
mechanisms for the increased IL-1 gene expression are not apparent
at this time. It is possible that the increased sheer stress related to
hypertension may be responsible, but we cannot rule out the possibility
of hormonal or humoral mechanism.
Also, the reasons for the differences in blood pressure and NE
secretion in response to IL-1 between control and CRF rats remain to
be established. One could speculate that this may be the result of
differences in receptor binding or metabolism of this cytokine, but
further studies are needed to address these possibilities.
NE secretion from the PH is considered a marker of increased SNS
activity. An increase in noradrenergic activity in the PH is associated
with increased peripheral SNS activity and blood pressure. Electrical
stimulation (27) or perfusion with phenylephrine (24) of the PH areas increases blood pressure, and
destruction of these areas decreases blood pressure in rats
(7). One could speculate that the decrease in NE secretion
from the PH might be the consequence rather than the cause of
hypotension. This, however, is unlikely because NE turnover in this
region increases when arterial pressure falls and decreases when
arterial pressure rises (9, 28). Moreover, administration
of angiotensin II in doses that raised blood pressure up to 180 mmHg
caused a significant decrease in NE secretion from the PH nuclei
(48) and an increase in NOS mRNA abundance. In contrast,
the decrease in blood pressure caused by phentolamine was associated
with an increase, not a decrease, in NE secretion. In all, these
studies support the notion that the hypotension caused by
intracerebroventricular infusion of IL-1 is the consequence of
decreased noradrenergic outputs from the PH rather than the cause.
A specific neuronal isoform of NO synthase (nNOS) has been described as an independent gene product that has been implicated in neuronal signaling in the central and peripheral autonomic nervous systems (6, 47). nNOS is an important component of the transduction pathways that tonically inhibit SNS outflow from the brain stem (2, 14, 38, 43-44). Sakuma and colleagues (34) showed that administration of NG-methyl-L-arginine to male Wistar rats increased renal sympathetic nerve activity and blood pressure. We showed previously (47) that the basal activity of the central SNS in normal rats is inhibited by local NO production. In CRF rats, increased expression of nNOS mRNA and NO2/NO3 content in the PH mitigates the rise in blood pressure and in SNS activity (47). Nitric oxide in the PVN has also been shown to have an inhibitory effect on renal sympathetic outflow, and this action is mediated by GABA (49, 50).
Complex relationships also exist between IL-1 and NO. Most studies
have evaluated the effects of IL-1 on iNOS rather than on nNOS.
Bacterial lipopolysaccharide induced iNOS activity in brain cells, and
this action was mediated in part by IL-1 (31, 35).
However, nNOS expression in the brain was not increased after
administration of endotoxin, despite a significant rise in IL-1.
Moreover, the changes in release of hypothalamic peptides induced by
cytokines in response to infections are mediated by NO (29,
35). Some evidence suggests that NO is involved in the
IL-1-induced central activation of sympathetic outflow in rats
(6). Our current studies lend strong support to the
hypothesis that IL-1 may stimulate the neuronal form of NOS mRNA in
the brain of CRF rats, and through this mechanism it may partially modulate SNS activity.
Several factors have been implicated in the pathogenesis of
hypertension associated with renal disease and/or failure. These include sodium retention, volume expansion, and increased activity of
the renin-angiotensin system (21, 37) or the SNS (1, 12, 15, 18, 20). We have shown a greater NE turnover rate in the
PH and the LC of CRF rats than in control rats (4) and greater secretion of NE from the PH of CRF rats than in control rats
(48). Bilateral dorsal rhizotomy prevented the development of hypertension and the increase in NE turnover rate in the PH and LC
of CRF rats (8). The decrease in arterial pressure
observed in uremic patients after bilateral nephrectomy was associated with lower sympathetic nerve firing and lower regional vascular resistance (11). In all, these findings suggest that
afferent impulses from the kidney of rats and human subjects with renal diseases may activate areas of the brain involved in the noradrenergic regulation of blood pressure and largely contribute to the development and maintenance of hypertension associated with CRF. In the CRF model,
the primary increase in SNS activity may raise blood pressure, which
may then activate IL-1 and NO production. The latter may partially
mitigate the increase in SNS activity and blood pressure.
Previous studies using in situ hybridization have shown that kainic
acid or transient ischemia can induce IL-1 mRNA in several regions
of the rat brain, and the expression may vary in different areas of the
brain (45, 46). With the PCR technique, we have been able
to identify the presence of IL-1 mRNA in several brain nuclei, even
during nonstimulated conditions. Quantitative comparisons of the
abundance of IL-1 mRNA in different regions of the brains using
these different techniques are not possible.
In conclusion, these studies have shown that IL-1 modulates central
SNS activity and that this modulation is mediated by increased local
expression of nNOS mRNA abundance. Our studies have also shown
increased IL-1 expression in the brain of CRF rats. Moreover,
administration of a specific antibody to IL-1 caused a further rise
in blood pressure and in SNS activity in CRF rats. Although the
mechanisms for the increase in IL-1 expression in the brain of CRF
rats remain to be elucidated, our data suggest that activation of
IL-1 may be responsible for the upregulation of NO and for the
partial attenuation of the increased SNS activity in CRF rats.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant 1-RO1-HL-47881 and by an Extramural Grant from Baxter Healthcare Corp.
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FOOTNOTES |
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Address for reprint requests and other correspondence: V. M. Campese, Div. of Nephrology, Keck School of Medicine, Univ. of Southern California, 1200 North State St., Los Angeles, CA 90033 (E-mail: campese{at}hsc.usc.edu).
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.
Received 29 July 1999; accepted in final form 20 June 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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