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1 Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas Universidade Federal de Minas Gerais, Belo Horizonte and 2 Departamento de Ciências Biológicas, Instituto de Ciências Exatas e Biológicas Universidade Federal de Ouro Preto, Brazil
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The
objective of the present study was to determine the contribution of the
autonomic nervous system and nitric oxide to the depressor effect
produced by unilateral microinjection of ANG-(1-7) and ANG II into the caudal ventrolateral medulla (CVLM). Unilateral microinjection of ANG-(1-7), ANG II (40 pmol), or
saline (100 nl) was made into the CVLM of male Wistar rats anesthetized
with urethane before and after intravenous injection of 1)
methyl-atropine, 2.5 mg/kg; 2) prazosin, 25 µg/kg;
3) the nitric oxide synthase (NOS) inhibitor,
NG-nitro-L-arginine methyl ester
(L-NAME), 5 mg/kg; or 4) the specific inhibitor
of neuronal NOS, 7-nitroindazole (7-NI), 45 mg/kg. Arterial pressure
and heart rate (HR) were continuously monitored. Microinjection of
ANG-(1-7) or ANG II into the CVLM produced a
significant decrease in mean arterial pressure (MAP; 
11 ± 1 mmHg, n = 12 and 10 ± 1 mmHg, n = 10, respectively) that was not accompanied by consistent changes in
HR or in cardiac output. The effect of ANG-(1-7) was abolished after treatment with methyl-atropine (3 ± 0.6 mmHg, n = 9) or L-NAME (2.3 ± 0.5 mmHg,
n = 8) or 7-NI (2.8 ± 0.6 mmHg,
n = 5). In contrast, these treatments did not
significantly interfere with the ANG II effect (10 ± 2.6 mmHg,
n = 8; 8 ± 1.5 mmHg, n = 8; and
12 ± 3.6 mmHg, n = 6; respectively). Peripheral treatment with prazosin abolished the hypotensive effect of
ANG-(1-7) and ANG II. Microinjection of saline did
not produce any significant change in MAP or in HR. These results
suggest that the hypotensive effect produced by ANG II at the CVLM
depends on changes in adrenergic vascular tonus and, more importantly,
the hypotensive effect produced by ANG-(1-7) also
involves a nitric oxide-related mechanism.
angiotensin II; angiotensin-(1-7); arterial pressure; NG-nitro-L-arginine methyl ester, nitric oxide, sympathetic nervous system
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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THE CAUDAL VENTROLATERAL MEDULLA (CVLM) plays an important role in the central control of blood pressure through a complex neuronal network involved in the modulation of the peripheral sympathetic activity (8, 13, 18). It is generally believed that the CVLM acts by inhibiting a more rostral excitatory region of the ventrolateral medulla, the RVLM, resulting in a widespread inhibition of the sympathetic vasomotor activity (8, 13). Several studies have demonstrated that ANG II can influence these regions increasing (RVLM) or decreasing (CVLM) sympathetic activity (2, 4, 13, 30, 42). ANG II receptors and ANG II-like immunoreactivity have been demonstrated in both the CVLM and RVLM in a number of species (1, 29, 45). Microinjection of the ANG peptides, ANG II, ANG III, and ANG-(1-7), into the RVLM elicits a pressor effect associated with an increase in renal nerve activity (4, 13, 40). CVLM depressor neurons have also been shown to be tonically excited by endogenous ANG peptides (38, 42). Silva et al. (41) showed that microinjection of ANG II or ANG-(1-7) into the CVLM produces a depressor response in anesthetized rats. However, the precise mechanism of the depressor response elicited by angiotensins at the CVLM has not yet been investigated.
It is now clear that ANG II and ANG-(1-7) act through different mechanisms in the periphery and that these two peptides, on most occasions, exert opposing effects (39). In the brain, ANG-(1-7) is devoid of the drinking and pressor effects elicited by intracerebroventricular injection of ANG II (11, 39). The cardiovascular effects produced by ANG-(1-7), centrally, are blocked by its selective antagonist, A-779 (39, 40), and are not affected by AT1 or AT2 receptor antagonists (20, 40), suggesting that ANG-(1-7) effects are mediated by a selective receptor different from the classical AT1 or AT2 receptor subtypes. In the nucleus of the solitary tract (NTS), both peptides produce hypotension and bradycardia (4, 10); however, whereas the effect of ANG II is predominantly mediated by a reduction in the sympathetic activity, the ANG-(1-7) effect involves an increase in vagal activity (4). Considering the key role of the CVLM in the neuronal circuitry controlling blood pressure, it is important to establish whether differential mechanisms are involved in the hypotensive effects elicited by microinjections of ANG II and ANG-(1-7). It is likely that the cardiovascular effects of these peptides in the CVLM are exclusively due to a withdrawal of the sympathetic drive to the periphery. However, recent studies by the Lewis group (14-17, 37) opened another possibility. These studies suggested the existence of an active sympathetic neurogenic vasodilator system using nitrosyl factors that mediate the direct or reflex activation of the lumbar chain to produce hindquarter vasodilation. Therefore, in the present study we used selective pharmacological adrenergic, cholinergic, and nitrergic blockade to evaluate the mechanism of the depressor response produced by ANG peptides at the CVLM. This information would provide important clues concerning the cardiovascular physiological role of the brain renin-angiotensin system.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Surgical Procedures
Experiments were performed in male Wistar rats (260-300 g) anesthetized with urethane (1.2 g/kg ip, Sigma). After rats underwent a tracheostomy, a catheter was inserted into the abdominal aorta through the femoral artery for arterial pressure measurement. Another catheter was inserted into the inferior cava vein through the femoral vein for injection of drugs. Next, the animals were placed in a stereotaxic frame (David Kopf Instruments) with the tooth bar 11 mm below the level of the interaural line. The dorsal surface of the brain stem was exposed by a limited occipital craniotomy and incision of the atlantooccipital membrane and meninges, as previously described (20, 41). The animals were kept on a heating pad to maintain a constant body temperature (37 ± 0.5°C).Arterial Pressure Measurements
Arterial pressure and heart rate (HR) were continuously monitored with a solid-state strain gauge transducer coupled to a Nihon-Kohden polygraph (Japan) or to a blood pressure signal amplifier (Stemtech, Quintron, WI), connected to a data-acquisition system (DATAQ Instruments, Akron, OH) with a 600-Hz sampling rate.Cardiac Output Measurements
Cardiac output (CO) was measured by the thermodilution method using a CARDIOTHERM 500 apparatus (Columbus Instrument, Columbus, OH). A thermistor (Fr. 1.5 microprobe, outer diameter of 0.64 mm, Columbus Instrument) consisting of a teflon-coated constantan and copper wire with 2-3 mm epoxy-coated tip, was inserted into the aortic arch through the left carotid artery for blood temperature measurement. A polyethylene catheter (PE-10) was placed into the right atrium via the jugular vein for injection of saline. CO was measured by rapidly injecting 0.1 ml of cold saline (18-20°C) with a pump (Hamilton, Microlab 500 series) into the right atrial catheter as a thermal tracer indicator. Three to four thermodilution curves were generated (minimum of 10-min interval) in the control period to assure the reproducibility of the measurement. The CO values obtained 5 min before and at the peak of the response elicited by CVLM microinjections were used to express the control and after peptide values, respectively. Total peripheral resistance was calculated from MAP and CO values (TPR = MAP/CO, mmHg · ml
1 · min).
Microinjections Procedures
Unilateral microinjections of ANG-(1-7), ANG II, or sterile saline (NaCl 0.9%) in a volume of 100 nl were made over a 20- to 30-s period into the CVLM [0.7 mm anterior, 1.8 mm lateral to the obex, and just above pia mater in the ventral surface (usually 3.2-3.6 mm from the dorsal surface)], as previously described (19, 41). Microinjections were made with a triple glass micropipette, fixed to the stereotaxic manipulator that was inserted in the brain tissue through the dorsal surface. Experiments were made only at sites where the positioning of the micropipette produced a transitory depressor response (usually 10-20 mmHg with 30-60 s of duration). For all experiments, only one site of the CVLM was tested per animal.Protocols and Experimental Groups
Evaluation of the cardiovascular effects of ANG-(1-7) or ANG II at the CVLM. Arterial pressure and HR changes produced by unilateral microinjection of ANG-(1-7) (40 pmol, n = 12), ANG II (40 pmol, n = 10), or saline (NaCl, 0.9%, 100 nl, n = 7) into the CVLM were recorded continuously over a 30-min period. In all animals, a minimum interval of 20 min was given after positioning of the micropipette and the beginning of the experiments (peripheral treatments or CVLM microinjections). In the majority of the experiments only one peptide and saline were tested and, in a few cases, two peptides were tested in the same animal. A minimum 30-min interval was given between CVLM injections.
CO measurements. In subgroups of these animals, control thermodilution curves for CO measurements were obtained immediately before and at the peak of the response produced by CVLM microinjection of ANG-(1-7) (40 pmol, n = 6) or ANG II (40 pmol, n = 5) or saline (NaCl, 0.9%, 100 nl; n = 2). In addition to direct measurements, TPR was calculated and correlated to the measured baseline values of MAP and HR at same time intervals.
Cardiovascular effects of repeated injections of ANG-(1-7) or ANG II at the CVLM. In other groups of animals, repeated injections were made to determine the reproducibility of the responses (time control). In these animals, arterial pressure and HR changes induced by two successive microinjections (minimum of 30 min apart) of ANG-(1-7) (40 pmol, n = 8), ANG II (40 pmol, n = 8), or saline (NaCl, 0.9%, 100 nl; n = 7) into the CVLM were continuously recorded. In these experiments, only one peptide was tested in each animal.
Cardiovascular effects of ANG-(1-7) or ANG II
at the CVLM after peripheral treatments.
Arterial pressure and HR effects produced by unilateral microinjection
of ANG-(1-7) (40 pmol), ANG II (40 pmol), or saline (100 nl) into the CVLM were evaluated in different groups of animals before and 20 min after intravenous injection of 1)
methyl-atropine, a muscarinic receptor antagonist (2.5 mg/kg,
n = 5-8); 2)
NG-nitro-L-arginine methyl ester
(L-NAME), a nitric oxide (NO) synthase (NOS) inhibitor (5 mg/kg, n = 4-8); 3) 7-nitroindazole
(7-NI), a neuronal NOS inhibitor (45 mg/kg, n = 5-6); or 4) prazosin, an 
1-adrenergic
receptor antagonist (25 µg/kg, n = 4-7). In
these experiments, only one peptide or saline was tested in each animal.
Drugs
ANG-(1-7), ANG II, methyl-atropine nitrate, prazosin, and L-NAME were dissolved in sterile isotonic saline (NaCl, 0.9%) immediately before use. 7-NI was dissolved in Na2CO3 (8% wt/vol), and the pH of the solution was adjusted to 7.4 by the addition of 1.0 M HCl.ANG-(1-7) and ANG II were purchased from Bachem (Torrance, CA) or Peninsula Laboratories (Belmont, CA). Methyl-atropine nitrate, prazosin, and L-NAME were from Sigma (St. Louis, MO), and 7-NI was purchased from Research Biochemicals International (RBI, Natick, MA). Purity of ANG-(1-7) and ANG II was confirmed by high-performance liquid chromatography (33) and by amino acid analysis performed by Dr. L. J. Greene at the Centro Interdepartamental de Química de Proteínas, Faculdade de Medicina de Ribeirão Preto, Universidade de Sao Paulo, SP, Brazil.
Histological Verification of Injection Sites
At the end of each experiment, 100 nl of Alcian blue dye (5%) was microinjected into the CVLM. The animals were then killed with excess of anesthetic (ether), and the brain was carefully removed and fixed in 10% phosphate-buffered formalin for later histological examination. Serial coronal sections (40-50 µm) of the medulla oblongata were made and stained with neutral red. Microinjection sites were identified by the deposition of Alcian blue dye with light microscopy and referred to standard anatomic structures of the brain stem according to the atlas of Paxinos and Watson (36).Statistical Analysis
The results are expressed as means ± SE. Comparisons between before and after injection in the same animal were evaluated by Student's t-test for paired observations. Comparisons among different groups were made by one-way ANOVA followed by Newman-Keuls. The criterion for statistical significance was set at P < 0.05.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Cardiovascular Effects Produced by CVLM Microinjections of ANG-(1-7) or ANG II
Figure 1 illustrates the MAP and HR effects produced by unilateral microinjection of ANG-(1-7) (40 pmol), ANG II (40 pmol), and saline (NaCl 0.9%, 100 nl) into the CVLM. As shown in Fig. 2A, microinjection of ANG-(1-7) produced significant decreases in MAP (11 ± 1 mmHg; baseline MAP = 89 ± 4 mmHg;
n = 12) similar to that of ANG II (10 ± 1 mmHg;
baseline MAP = 87 ± 4 mmHg, n = 10). The
changes in blood pressure were statistically different from that
produced by saline (1.6 ± 0.9 mmHg, n = 7, baseline MAP = 90 ± 8 mmHg; Fig. 2A). There were
no significant changes in HR (averaged baseline of HR = 317 ± 14 beats/min, n = 29; Fig. 2B) or CO
(averaged of baseline CO = 114 ± 4.9 ml/min,
n = 13; Fig. 2C). Accordingly, the depressor
effect produced by the CVLM microinjections was due to a decrease in
the TPR for both ANG-(1-7) (0.59 ± 0.06 vs.
0.76 ± 0.05 mmHg · ml1 · min,
control; n = 6; Fig. 2D) and ANG II
(0.65 ± 0.05 vs. 0.92 ± 0.10 mmHg · ml1 · min,
control, n = 5; Fig. 2D). In two animals,
saline microinjection at CVLM did not change MAP (1 ± 0.6 mmHg), HR (0.5 ± 0.5 beats/min), CO (105 ± 17.5 ml/min),
or TPR (0.78 ± 0.12 mmHg · ml1 · min)
(data not shown).
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We also compared the MAP and HR effects produced by successive
microinjections in the same animal (time and "tissue damage" control). Two successive microinjections into the CVLM (20 min apart)
produced similar changes in MAP for both 40 pmol of
ANG-(1-7) (12.5 ± 1.8 mmHg, first
microinjection and 10 ± 2.8 mmHg, second microinjection;
n = 8), 40 pmol of ANG II (10 ± 1.2 mmHg, first microinjection and 9 ± 0.9 mmHg, second microinjection;
n = 8), and saline (3 ± 1.2 mmHg, first
microinjection and 4 ± 0.7 mmHg, second microinjection;
n = 7).
Evaluation of the Peripheral Mechanism Induced by CVLM Microinjections of ANG-(1-7) or ANG II
Pretreatment with the muscarinic antagonist methyl-atropine produced an expected increase in baseline HR that was accompanied by a small but significant decrease in MAP (Tables 1 and 2). Surprisingly, the hypotensive effect of ANG-(1-7) was abolished after peripheral treatment with methyl-atropine (3 ± 0.6 mmHg compared with
12 ± 1.3 mmHg, before treatment; n = 9; Fig.
3). The hypotensive effect of ANG II at
the CVLM after methyl-atropine was not statistically different from
that observed before the antagonist (Fig. 3). No significant changes in
HR were induced by microinjection of angiotensins into the CVLM before
or after methyl-atropine treatment. In addition, methyl-atropine
abolished the hypotensive effect produced by intravenous injection of
acetylcholine tested at the end of experiments (0.3 ± 0.5 mmHg
compared with 25 ± 2.6 mmHg, before treatment;
n = 12).
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To evaluate whether the effect of methyl-atropine could be related to
interference with the release of NO, the effect of
ANG-(1-7) and ANG II was tested after peripheral
treatment with the NOS inhibitor L-NAME and the specific
inhibitor of neuronal NOS 7-NI. Pretreatment with L-NAME
produced a sustained increase in MAP without significant changes in HR,
whereas pretreatment with 7-NI did not produce significant changes in
MAP or in HR (Tables 1 and 2). As shown in Fig.
4, although the CVLM hypotensive effect of ANG II was not modified by any of these treatments, the depressor effect of ANG-(1-7) was abolished by both
L-NAME (2.3 ± 0.5 mmHg compared with 8 ± 1 mmHg before treatment, n = 8; Fig. 4A) or 7-NI (2.8 ± 0.6 mmHg compared with 11 ± 2.3 mmHg before
treatment, n = 5; Fig. 4B). No significant
changes in HR were induced by microinjection of angiotensins into the
CVLM before or after L-NAME or 7-NI treatments.
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Treatment with the 1-adrenergic receptor antagonist
prazosin produced a sustained decrease in MAP without significant
changes in HR (Tables 1 and 2). The MAP effect of ANG II was abolished by peripheral treatment with prazosin (2.2 ± 0.6 mmHg compared with 11.5 ± 1.0 mmHg, before treatment, n = 6;
Fig. 5). The hypotensive effect of
ANG-(1-7) was also abolished by prazosin (2.1 ± 0.7 mmHg compared with 10 ± 1 mmHg, before treatment,
n = 7; Fig. 5). As in the other groups, no significant
changes were observed in HR. In addition, prazosin treatment blocked
the pressor effect of intravenous injection of phenylephrine (5 ± 2 mmHg compared with 24 ± 3 mmHg, before treatment,
n = 12).
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The loss of the hypotensive effect of ANG-(1-7) and
ANG II after prazosin treatment cannot be attributed to a nonspecific influence of the decreased level of MAP, because intravenous injection of sodium nitroprusside (0.5 µg), given at the end of experiment to
some animals treated with 1-adrenergic receptor
antagonist, produced a significant fall in MAP (12 ± 2 mmHg,
baseline after prazosin MAP= 72 ± 7 mmHg, n = 5;
data not shown).
The changes in MAP produced by microinjection of saline into the CVLM
were not modified by any of the peripheral treatments (3.2 ± 0.8 mmHg, average of the effect after all peripheral treatments, compared with 2.6 ± 0.8 mmHg, before treatment;
n = 18).
Histological Examination
As it can be seen by the composite of the center of injection in all animals determined by the deposition of the Alcian blue dye (Fig. 6), the microinjections were confined to a small area ventral to the lateral reticular nucleus.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The major finding of the present study was the observation that the hypotensive effect produced by ANG II at the CVLM depends essentially on changes in adrenergic vascular tonus, whereas the ANG-(1-7) hypotensive effect involves a different mechanism. Our data showed that the hypotensive effect of ANG-(1-7) at CVLM was not accompanied by significant changes in CO but, contrasting with ANG II, it was completely abolished by peripheral treatment with methyl-atropine and the NOS inhibitors L-NAME and 7-NI. These data have opened a novel and intriguing possibility: the existence of a new pathway for the central control of arterial pressure, probably mediated NO release.
In the present study we confirmed and extended our previous
observations showing that ANG-(1-7) and ANG II
produce hypotensive effects when microinjected into the CVLM
(41). However, our present data indicate that the
peripheral mechanisms triggered by these related peptides are
strikingly different. Previous studies have shown that ANG II acts on
AT1 receptors of sympathoinhibitory neurons of the CVLM
causing decrease in MAP and renal sympathetic activity (27, 30,
38). In addition, Muratani et al. (31) showed that
the effect induced by ANG II on CVLM neurons are dependent on the
activity of the RVLM, which is well known to control the preganglionic
neurons that innervate the peripheral vascular beds (13).
Accordingly, our data showed that the hypotensive effect produced by
ANG II at the CVLM is completely abolished by peripheral 1-adrenergic blockade, indicating that this effect is
predominantly due to a decrease in sympathetic outflow. Methyl-atropine
or the NOS inhibitors did not significantly modify the cardiovascular effects of ANG II at the CVLM.
Contrasting to ANG II, the hypotensive effect of ANG-(1-7) could be completely blocked by methyl-atropine, L-NAME, and by the selective inhibitor of neuronal NOS, 7-NI, indicating that ANG-(1-7) is evoking a vasodilator NO-related mechanism at the CVLM. Because no significant changes in HR and CO were observed after microinjection of ANG-(1-7) at the CVLM, involvement of a cardiac component in this response is unlikely.
It is well known that an active neurogenic vasodilator system, of noncholinergic origin, exists in the skeletal vasculature of several species (5, 6, 23). Davisson et al. (14) described that centrally mediated activation of the lumbar sympathetic chain by air-jet stress produces a pronounced hindlimb vasodilation in conscious rats that is reduced by prior administration of L-NAME. In addition, these authors also showed that lumbar sympathetic cell bodies and the postganglionic fibers and varicosites within the iliac and femoral arteries stained intensely for NOS (14, 15). The presence of NOS in the varicosites provides important evidence that postganglionic sympathetic nerves may synthesize NO-related substances, such as NO itself or S-nitrosothiols or dinitrosyl iron(II)-cysteine complexes (32, 43). Furthermore, in this study Davisson et al. (15) showed that hindlimb vasodilation produced by low-intensity electrical stimulation of the lumbar sympathetic chain was markedly attenuated by 7-NI, suggesting that the vasodilation is mediated by the release of NO-related substances from nerve terminals. In addition, Possas and Lewis (37) showed that selective inhibition of neuronal NOS abolished the vasodilation of the hindlimb induced by the stimulation of the superior laryngeal nerve. These data provide evidence for a functionally nitroxidergic vascular system. Our present data add the possibility that this pathway could be modulated by ANG-(1-7) at the CVLM.
In keeping with our current data, we recently found that the pressor effect produced by microinjection of ANG-(1-7) into the RVLM is mediated by the combination of an increase in sympathetic outflow, an increase in vasopressin release, and also by a decrease in a sympathetic-cholinergic pathway (34). In addition, in a recent study by the Lewis group (12), it was shown that microinjection of L-glutamate (L-Glu) at the NTS elicited a pronounced decrease in hindquarter resistance once the initial pressor and vasoconstriction effects had subsided. This vasodilation was persistent in the presence of both prazosin and L-NAME. However, subsequent microinjections of L-Glu produced progressively and substantially smaller responses. The authors suggest that the hindquarter vasodilation may be mediated by the release of preformed pools of nitrosyl factors from the NOS-positive lumbar sympathetic nerves innervating this bed. The progressive decrease in the hindquarter vasodilator responses in L-NAME-treated rats would be consistent with the release and gradual depletion of these preformed pools of nitrosyl factors that cannot be regenerated in the absence of NO synthesis. Taking all these data together, the results of our present study add an important evidence of the existence of a previously unsuspected vasodilator tone, possibly originating from RVLM neurons and under the influence of CVLM neurons. It is also possible that ANG-(1-7) is acting through a vasodilator tone originating from the CVLM through neurons that are projecting directly to the spinal cord. Actually, a dense group of horseradish-peroxidase-positive cells have been described in the CVLM that project to both thoracic and lumbar segments at the spinal cord (7).
ANG-(1-7) hypotensive effect was also abolished by
prazosin treatment, suggesting that ANG-(1-7) action
at CVLM may also depend on the prevailing level of sympathetic vascular
tone. Whether this dependence is due to a CVLM-mediated presynaptic
release of NO at the sympathetic nerve endings remains to be
established. In addition, an interference with the
ANG-(1-7) effect (and perhaps ANG II) of the
unloading of the baroreceptors due to the decrease of blood pressure
produced by the 1-adrenergic blockade cannot be ruled out.
Another possibility is that ANG-(1-7) acting at the
CVLM may increase the activity of a neuronal nitroxidergic pathway that produces a presynaptic inhibition of the release of norepinephrine at
the sympathetic nerve endings. For instance, it has been described that
the vasoconstrictor response during sympathetic nerve stimulation in
gastrocnemius muscle is enhanced by L-NAME, suggesting that norepinephrine induced the release of NO to "buffer" the degree of
constriction in skeletal muscle vasculature (24). More
interestingly, Vials et al. (44) suggested a presynaptic
inhibitory action for NO, probably derived from identified perivascular
nitroxidergic nerves, on perivascular sympathetic vasoconstrictor
nerve-mediated responses of the rabbit renal artery. Thus it is also
possible that ANG-(1-7) at the CVLM could be
selectively increasing this pathway, which is supported by our findings
that the ANG-(1-7) hypotensive effect was blocked by
1-adrenergic antagonists and NOS inhibitors (especially
7-NI). Further studies will be necessary to explore these possibilities.
The fact that L-NAME and 7-NI are lipophilic agents with significant penetration into brain tissue after peripheral administration (3, 21) raises the possibility that the effect of ANG-(1-7) could be due to modulation of nitroxidergic neurons in the CVLM. Recently, Paton et al. (35), using the working heart-brain stem preparation of rats, showed that NTS microinjection of L-NAME or L-NG-monomethyl-L-arginine, but not a neuron-specific NOS inhibitor, prevented the ANG II-induced baroreflex attenuation. In addition, these authors showed that ANG II into the NTS failed to affect baroreflex in animals that expressed a dominant negative mutant of endothelial NOS bilaterally in the NTS. Although these data strongly suggest a participation of central NO in the effect of ANG II in the NTS, these data cannot be directly extended to other regions of the brain. Even though we cannot rule out the possibility of an involvement of central nitric oxidergic pathways in the CVLM, we believe that this possibility is unlikely. ANG-(1-7) effects were also completely abolished by the muscarinic antagonist methyl-atropine, with little or no diffusion capability into the brain tissue. In addition, we showed in a previous study (26) that microinjection of the NO precursor L-arginine into the CVLM produced an increase in MAP mediated by the release of vasopressin. Thus the hypotensive effect of ANG-(1-7) at CVLM through this pathway would first assume the existence of a tonic NO-mediated release of vasopressin by CVLM neurons and, second and more unlikely, an inhibitory action of ANG-(1-7) on the NO release in this region, which is contrary to all described effects of ANG-(1-7) in several preparations studied to date (39).
One concern that can be raised with the use of L-NAME is the possibility of interference of such NOS inhibitors with muscarinic receptors as reported by Buxton et al. (9) in in vitro preparations. However, other studies showed that L-NAME has a very weak affinity with M1 and M2 receptors and does not present antimuscarinic properties against M1, M2, and M3 receptor-mediated effects in rats in vivo (22, 25). In our study, even if part of the L-NAME effect had been mediated by blockade of muscarinic receptors it did not affect our main findings, showing that the mechanisms triggered by angiotensin peptides at the CVLM were completely different. Thus, whereas pretreatment with L-NAME or 7-NI did not alter the ANG II effect, these treatments abolished the ANG-(1-7) effect.
Another concern that is always imposed in studies in anesthetized animals is the possibility of an altered autonomic outflow interfering with the central responses. However, we previously showed that microinjection of ANG peptides at the RVLM induces similar pressor effects in anesthetized and conscious rats (19, 20). All the data in the literature evaluating the cardiovascular effects of microinjections into the CVLM were performed in anesthetized rats due to methodological difficulties, but even in the anesthetized condition it was possible to investigate arterial pressure and baroreflex effect of different substances at this site (27, 30, 31, 38). In addition, in our study the tachycardia that followed methyl-atropine administration was a good indication of the presence of a significant vagal tonus, which rules out the possibility of a completely blunted baroreflex activity.
Perspectives
It is well accepted that the depressor response evoked by stimulation of CVLM neurons is mainly due to a decrease in TPR resulting from a widespread inhibition of sympathetic vasomotor activity. The data presented in this study showed that CVLM microinjection of angiotensin peptides elicits a similar fall in blood pressure not accompanied by significant changes in CO. However, whereas the effect of ANG II depends mainly on the decrease in sympathetic outflow, that of ANG-(1-7) involves the activation of a nitroxidergic mechanism. These findings have uncovered a highly relevant physiological possibility: the existence of a new pathway in the central modulation of the peripheral vascular tone originating from the ventral medulla that can be modulated, at least in part, by angiotensin peptides. Although our data indicate that the effect of the angiotensin peptides in CVLM is due to an overall fall in peripheral resistance, one important question that still remains open is which peripheral vascular bed is involved in mediating ANG II and ANG-(1-7) effects at this site. An interesting possibility is that the differential effects of ANG II and ANG-(1-7) could be attributed to the modulation of distinct neuronal cells involved in separate vascular beds. It has been found, for instance, that glutamate injections into different RVLM regions could selectively activate particular sympathetic outflows (28). Although there are no data in the literature showing that the projections of CVLM neurons follow a regional distribution, evaluation of the effect of ANG-(1-7) microinjection into the CVLM on specific regional blood flow needs to be addressed in future studies.| |
ACKNOWLEDGEMENTS |
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We are thankful to J. R. Silva and S. S. Silva for skillful technical assistance.
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FOOTNOTES |
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This study was supported by Programa de Núcleos de Excelência-Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de Minas Gerais, CNPq, and Coordenadoria de Apoio ao Pessoal de Nível Superior (CAPES). A. C. Alzamora was a recipient of CAPES-Programa Institucional de Capacitação Docente e Técnica (PICDT) fellowship (Doctoral Degree) from the Federal University of Ouro Preto.
Address for reprint requests and other correspondence: M. J. Campagnole-Santos, Departamento de Fisiologia e Biofísica, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627-ICB, UFMG, 31270-901, Belo Horizonte, MG, Brasil (E-mail: mjcs{at}icb.ufmg.br).
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.
July 18, 2002;10.1152/ajpregu.00580.2001
Received 20 September 2001; accepted in final form 16 July 2002.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Allen, AM,
Chai SY,
Sexton PM,
Lewis SJ,
Verbene AJ,
Jarrot B,
Louis WJ,
Clevers J,
McKinley MJ,
Paxinos G,
and
Mendelsohn FAO
Angiotensin II receptors and angiotensin converting enzyme in the medulla oblongata.
Hypertension
9:
III198-III205,
1987[Medline].
2.
Andreatta, SH,
Averill DL,
Santos RAS,
and
Ferrario CM.
The ventrolateral medulla: a new site of action of the renin-angiotensin system.
Hypertension
11:
I163-I166,
1988[Medline].
3.
Anne, O,
Janos PA,
and
Christian T.
Increased blood pressure in rats after long-term inhibition of the neuronal isoform of nitric oxide synthase.
J Clin Invest
99:
2212-2218,
1997[ISI][Medline].
4.
Averill, DB,
and
Diz DI.
Angiotensin peptides and the baroreflex control of sympathetic outflow: pathways and mechanisms of the medulla oblongata.
Brain Res Bull
51:
119-128,
2000[ISI][Medline].
5.
Ballard, DR,
Abboud FM,
and
Mayer HE.
Release of a humoral vasodilator substance during neurogenic vasodilatation.
Am J Physiol
219:
123-128,
1970.
6.
Beck, L,
Pollard AA,
Kaaylp SO,
and
Weiner LM.
Sustained dilatation elicited by sympathetic nerve stimulation.
Fed Proc
25:
1596-1606,
1966[ISI][Medline].
7.
Blessing, WW,
Goodchild AK,
Dampney RAL,
and
Chalmers JP.
Cell groups in the lower brain stem of the rabbit projecting to the spinal cord, with special reference to catecholamine-containing cells.
Brain Res
221:
35-55,
1981[ISI][Medline].
8.
Blessing, WW,
and
Reis DJ.
Inhibitory cardiovascular function of neurons in the caudal ventrolateral medulla of the rabbit: relationship to the area containing A1 noradrenergic cells.
Brain Res
253:
161-171,
1982[ISI][Medline].
9.
Buxton, IL,
Cheek DJ,
Eckman D,
Westfall DP,
Sanders KM,
and
Keef KD.
NG-nitro-L-arginine methyl ester and other alkyl esters of arginine are muscarinic receptor antagonists.
Circ Res
72:
387-395,
1993
10.
Campagnole-Santos, MJ,
Diz DI,
Santos RAS,
Khosla MC,
Brosnihan KB,
and
Ferrario CM.
Cardiovascular effects of angiotensin-(1-7) microinjected into the dorsal medulla of rats.
Am J Physiol Heart Circ Physiol
257:
H324-H329,
1989
11.
Campagnole-Santos, MJ,
Heringer SB,
Batista EN,
Khosla MC,
and
Santos RAS
Differential baroreceptor reflex modulation by centrally infused angiotensin peptides.
Am J Physiol Regul Integr Comp Physiol
262:
R89-R94,
1992.
12.
Colombari, E,
Davisson RL,
Shaffer RA,
Talman WT,
and
Lewis SJ.
Hemodynamic effects of L-glutamate in NTS of conscious rats: a possible role of vascular nitrosyl factors.
Am J Physiol Heart Circ Physiol
274:
H1066-H1074,
1998
13.
Dampney, RAL
Functional organization of central pathways regulating the cardiovascular system.
Physiol Rev
74:
323-364,
1994
14.
Davisson, RL,
Johnson AK,
and
Lewis SJ.
Nitrosyl factors mediate active neurogenic hindquarter vasodilation in the conscious rat.
Hypertension
23:
962-966,
1994
15.
Davisson, RL,
Possas OS,
Murphy SP,
and
Lewis SJ.
Neurogenically derived nitrosyl factors mediate sympathetic vasodilation in the hindlimb of the rat.
Am J Physiol Heart Circ Physiol
272:
H2369-H2376,
1997
16.
Davisson, RL,
Shaffer RA,
Johnson AK,
and
Lewis SJ.
Stimulation of lumbar sympathetic nerves may produce hindlimb vasodilation via the release of pre-formed stores of nitrosyl factors.
Neuroscience
72:
881-887,
1996[ISI][Medline].
17.
Davisson, RL,
Shaffer RA,
Johnson AK,
and
Lewis SJ.
Use-dependent loss of active sympathetic neurogenic vasodilation after nitric oxide synthase inhibition in conscious rats. Evidence for the presence of preformed stores of nitric oxide-containing factors.
Hypertension
28:
347-353,
1996
18.
Feldberg, W,
and
Guertzenstein PG.
Vasodepressor effects obtained by drugs acting on the ventral surface of the brainstem.
J Physiol
258:
337-355,
1976
19.
Fontes, MAP,
Martins Pinge MC,
Naves V,
Campagnole-Santos MJ,
Lopes OU,
Khosla MC,
and
Santos RAS
Cardiovascular effects produced by microinjection of angiotensins and angiotensin antagonists into the ventrolateral medulla of freely moving rats.
Brain Res
750:
305-310,
1997[ISI][Medline].
20.
Fontes, MAP,
Silva LCS,
Campagnole-Santos MJ,
Khosla MC,
Guertzenstein PG,
and
Santos RAS
Evidence that angiotensin-(1-7) plays a role in the central control of blood pressure at the ventrolateral medulla acting through specific receptors.
Brain Res
665:
175-180,
1994[ISI][Medline].
21.
Gillian, MM,
Sarah R,
Philip ABW,
Phillip KM,
Peter J,
and
David CM.
Time course of inhibition of brain nitric oxide synthase by 7-nitroindazole.
Neuroreport
5:
1993-1996,
1994[ISI][Medline].
22.
Hellmich, B,
and
Gyermek L.
NG-nitro-L-arginine methyl ester: a muscarinic receptor antagonist?
Fundam Clin Pharmacol
11:
305-314,
1997[ISI][Medline].
23.
Jones, RD,
and
Berne RM.
Vasodilatation in skeletal muscle.
Am J Physiol
204:
461-466,
1963[Medline].
24.
King-VanVlack, CE,
Curtis SE,
Mewburn JD,
Cain SM,
and
Chapler CK.
Endothelial modulation of neural sympathetic vascular tone in canine skeletal muscle.
J Appl Physiol
85:
1362-1367,
1998
25.
Koss, MC.
Effect of NG-nitro-L-arginine methyl ester on functionally characterized muscarinic receptors in anesthetized cats.
Eur J Pharmacol
335:
199-204,
1997[ISI][Medline].
26.
Lage, RC,
Campagnole-Santos MJ,
Fontes MAP,
and
Santos RAS
Cardiovascular effects produced by nitric oxide-related drugs in the caudal ventrolateral medulla.
Neuroreport
10:
731-735,
1999[ISI][Medline].
27.
Li, Y,
Polson JW,
and
Dampney RAL
Angiotensin II excites vasomotor neurons but not respiratory neurons in the rostral and caudal ventrolateral medulla.
Brain Res
577:
161-164,
1992[ISI][Medline].
28.
McAllen, RM,
and
May CN.
Differential drives from rostral ventrolateral medullary neurons to three identified sympathetic outflows.
Am J Physiol Regul Integr Comp Physiol
267:
R935-R944,
1994
29.
Mendelsohn, FAO,
Allen AM,
Clevers J,
Denton DA,
Tarjan E,
and
McKinley MJ.
Localization of angiotensin II receptor binding in rabbit brain by in vitro autoradiography.
J Comp Neurol
270:
372-384,
1988[ISI][Medline].
30.
Muratani, H,
Averill DB,
and
Ferrario CM.
Effect of angiotensin II in the ventrolateral medulla of spontaneously hypertensive rats.
Am J Physiol Regul Integr Comp Physiol
260:
R977-R984,
1991
31.
Muratani, H,
Ferrario CM,
and
Averill DB.
Ventrolateral medulla of spontaneously hypertensive rats: role of angiotensin II.
Am J Physiol Regul Integr Comp Physiol
264:
R388-R395,
1993
32.
Myers, PR,
Minor RL, Jr,
Guerra R, Jr,
Bates JN,
and
Harisson DG.
Vasorelaxant properties of the endothelium-derived relaxing factor more closely resemble S-nitrosocysteine than nitric oxide.
Nature
345:
161-163,
1990[Medline].
33.
Neves, LAA,
Almeida AP,
Khosla MC,
and
Santos RAS
Metabolism of angiotensin I in isolated rat hearts: effect of angiotensin converting inhibitors.
Biochem Pharmacol
50:
1451-1459,
1995[ISI][Medline].
34.
Oliveira, RC,
Campagnole-Santos MJ,
and
Santos RAS
The pressor effect of angiotensin-(1-7) at the rostral ventrolateral medulla is multimediated (Abstract).
J Hypertens
16, Suppl2:
S129,
1998.
35.
Paton, JF,
Deuchars J,
Ahmad Z,
Wong LF,
Murphy D,
and
Kasparov S.
Adenoviral vector demonstrates that angiotensin II-induced depression of the cardiac baroreflex is mediated by endothelial nitric oxide synthase in the nucleus tractus solitarii of the rat.
J Physiol
531:
445-458,
2001
36.
Paxinos, G,
and
Watson C.
The Rat Brain in Stereotaxic Coordinates. New York: Academic, 1986.
37.
Possas, OS,
and
Lewis SJ.
NO-containing factors mediate hindlimb vasodilation produced by superior laryngeal nerve stimulation.
Am J Physiol Heart Circ Physiol
273:
H234-H243,
1997
38.
Sasaki, S,
and
Dampney RAL
Tonic cardiovascular effects of angiotensin II in the ventrolateral medulla.
Hypertension
15:
274-283,
1990
39.
Santos, RAS,
Campagnole-Santos MJ,
and
Andrade SP.
Angiotensin-(1-7): an update.
Regul Pept
91:
45-62,
2000[ISI][Medline].
40.
Santos, RAS,
Campagnole-Santos MJ,
Baracho NCV,
Fontes MAP,
Silva CLS,
Neves LAA,
Oliveira DR,
Caligiorne SM,
Rodrigues ARV,
Geopenc Jr,
Carvalho WS,
Simões e Silva AC,
and
Khosla MC.
Characterization of a new angiotensin antagonist selective for angiotensin-(1-7) evidence that the actions of angiotensin-(1-7) are mediated by specific angiotensin receptors.
Brain Res Bull
35:
293-298,
1994[ISI][Medline].
41.
Silva, LC,
Fontes MAP,
Campagnole-Santos MJ,
Khosla MC,
Campos RR, Jr,
Guertzenstein PG,
and
Santos RAS
Cardiovascular effects produced by microinjection of angiotensin-(1-7) on vasopressor and vasodepressor sites of the ventrolateral medulla.
Brain Res
613:
321-325,
1993[ISI][Medline].
42.
Sesoko, S,
Muratani H,
Takishita S,
Teruya H,
Kawazoe N,
and
Fukiyama K.
Modulation of the baroreflex function by angiotensin II endogenous to the caudal ventrolateral medulla.
Brain Res
671:
38-44,
1995[ISI][Medline].
43.
Vedernikov, YP,
Mordvintcev PI,
Malenkova IV,
and
Vanin AF.
Similarity between the vasorelaxing activity of dinitrosyl iron cysteine complexes and endothelium-derived relaxing factor.
Eur J Pharmacol
211:
311-317,
1992.
44.
Vials, AJ,
Crowe R,
and
Burnstock G.
A neuromodulatory role for neuronal nitric oxide in the rabbit renal artery.
Br J Pharmacol
121:
213-220,
1997[ISI][Medline].
45.
Yang, SN,
Lippoldt A,
Janson A,
Phillips MI,
Ganten D,
and
Fuxe K.
Localization of angiotensin II AT1 receptor-like immunoreactivity in catecholaminergic neurons of the rat medulla oblongata.
Neuroscience
81:
503-515,
1997[ISI][Medline].
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P < 0.05 compared with before
treatment (Student's t-test for paired observations).
§P < 0.05 compared with ANG II (ANOVA followed by
Newman-Keuls).
