(Hypertension. 2000;35:1248.)
© 2000 American Heart Association, Inc.
Scientific Contributions |
From Instituto de Química y Fisicoquímica Biológicas (M.M.G., C.P.) and Cátedras de Fisiología (M.V.) y Fisiopatología (M.R.-F., B.E.F.), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina.
Correspondence to Dr Clara Peña, Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113-Buenos Aires, Argentina. E-mail clara{at}qb.ffyb.uba.ar
| Abstract |
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-nitro-L-arginine
methylester, an inhibitor of nitric oxide synthase
activity, and was restored by 100 µmol/L L-arginine,
precursor of nitric oxide synthesis. Methylene blue (10 µmol/L),
an inhibitor of guanylate cyclase considered as
the target of nitric oxide action, as well as Hoe 140 (10
µmol/L), a bradykinin B2-receptor antagonist,
prevented the inhibitory effect of the heptapeptide on
neuronal norepinephrine release, whereas no modification
was observed in the presence of 0.1 to 10 µmol/L
indomethacin, a cyclooxygenase
inhibitor. Our results indicate that Ang-(1-7) has a
tissue-specific neuromodulatory effect on noradrenergic
neurotransmission, being inhibitory at the central nervous
system by a nitric oxidedependent mechanism that involves
angiotensin type 2 receptors and local bradykinin
production.
Key Words: angiotensin norepinephrine nitric oxide angiotensin antagonist bradykinin prostaglandins
| Introduction |
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Ferrario and coworkers2 demonstrated the presence of Ang-(1-7) in several regions of the rat brain such as the hypothalamus, amygdala, and medulla oblongata but not in the cerebral cortex and cerebellum. In addition, type 1 (AT1) and type 2 (AT2) Ang receptors were described in several regions and nucleus of the central nervous system, including the hypothalamus.3 4
Although Ang-(1-7) is not an agonist in terms of activating vasoconstriction,5 stimulating thirst,6 or promoting aldosterone secretion,5 the heptapeptide causes neuronal excitation in the paraventricular nucleus of hypothalamus and dorsal vagal complex of the medulla oblongata,7 facilitates the noradrenergic neurotransmission,8 and stimulates prostaglandin9 10 11 and vasopressin release12 with potency comparable to that of Ang II. Conversely, some of the effects of Ang-(1-7) are opposite to those elicited by Ang II, that is, it displays an antiproliferative action on vascular smooth muscle cells,13 produces natriuresis14 and diuresis15 as well as vasodilation,16 17 and facilitates the baroreflex activity.7 18
Several studies indicated that Ang-(1-7) effects are tissue-specific, that is, the heptapeptide activates Na+, K+-ATPase in rat brain synaptosomal membranes, whereas a biphasic effect on this enzymatic system is observed in rat renal membranes.19 Furthermore, Gironacci et al8 have reported a facilitatory effect of the heptapeptide on the sympathetic neurotransmission in rat atria. Conversely, this stimulatory action was not detected in the rabbit vas deferens.11
Because of the suggested tissue-specific activity and the presynaptic effect of Ang-(1-7) on peripheral noradrenergic neurotransmission,8 the present study was performed to assess the effect of Ang-(1-7) on K+-evoked neuronal release of norepinephrine (NE) in rat hypothalamus.
| Methods |
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-nitro-L-arginine
methylester were from Sigma Chemical Co. Ang-(1-7) and
[D-Ala7]Ang-(1-7) were
synthesized in our laboratory by the Merrifield solid-phase procedure,
as previously described.8
Experimental Protocol
[3H]NE release was measured according to
the technique described by Vatta et al,20 with slight
modification. Briefly, minced rat hypothalami were incubated at 37°C
for 30 minutes in 2 mL of standard Krebs solution. Monoamine-oxidase
activity and extraneuronal NE uptake were inhibited by the addition of
0.1 mmol/L pargyline and 0.1 mmol/L hydrocortisone,
respectively. NE stores were labeled with 2.5 µCi/mL
[3H]NE (100 nmol/L) during a 30-minute
incubation period. After 8 consecutive 5-minute washes with Krebs
solution, tissues were then incubated during a period of 4 minutes in
high-potassium Krebs solution containing 0.1 mmol/L cocaine to
inhibit neuronal NE uptake, 0.1 mmol/L hydrocortisone, and
0.1 mmol/L pargyline. Two consecutive samples of incubation medium
were collected every 2 minutes (S1 and
S2), and [3H]NE release
was measured in each period as the amount of radioactivity present
in the incubation medium. Ang-(1-7) and losartan were
present during the second 2-minute period. PD 123319 and
[D-Ala7]Ang-(1-7) were added during
S1 and S2. Methylene blue
was present in the medium 15 minutes before sample collection.
Results are expressed as the ratio of the radioactivity released by the
second and first potassium-stimulation periods
(S2/S1).
Statistical Analysis
All values are mean±SEM. Data were submitted to 1-way ANOVA
followed by the Bonferroni test. Probability values <0.05 were
considered statistically significant.
| Results |
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To study the Ang-receptor subtypes coupled to the inhibitory activity of Ang-(1-7) on neuronal NE release, the effects of selective antagonists for AT1- and AT2-receptor subtypes were assessed. Results showed that losartan (10 nmol/L to 1 µmol/L), a selective AT1-receptor antagonist, diminished neuronal NE release evoked by 25 mmol/L KCl (data not shown). In addition, the reduction of evoked NE release produced by 100 nmol/L Ang-(1-7) was not blocked by losartan (10 nmol/L to 1 µmol/L) (Figure 2). On the other hand, the role of AT2 receptor on Ang-(1-7) reduction of evoked NE release in hypothalamus was studied in the presence of PD 123319, a selective AT2-receptor blocker, which, at levels >1 µmol/L, abolished Ang-(1-7)inhibitory effects (Figure 2). Simultaneous addition of losartan and PD 123319 did not modify the stimulated NE release (data not shown).
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The selective Ang-(1-7) antagonist [D-Ala7]Ang-(1-7) (1 µmol/L) partially blocked the effect of Ang-(1-7) on NE release (Figure 2). PD 123319 and D-Ala7-Ang-(1-7) each had no effect by itself on NE release (data not shown).
It has been demonstrated that Ang-(1-7) exerts several effects through
the NO pathway.16 21 22 23 To assess the role of NO as
possible mediator of Ang-(1-7) effect on neuronal NE release evoked by
K+ in rat hypothalamus, we investigated the
effect of Ang-(1-7) in the presence of
N
-nitro-L-arginine
methylester (L-NAME), an inhibitor of NO synthase activity.
The reduction of evoked NE release produced by 100 nmol/L Ang-(1-7) was
prevented by the addition of 10 µmol/L L-NAME and was restored
when L-arginine (100 µmol/L), the
precursor of NO synthesis, was simultaneously present
(Figure 3). Moreover, 10 µmol/L
methylene blue, an inhibitor of guanylate
cyclase considered as the target of NO action, prevented the
inhibitory effect of Ang-(1-7) on NE release (Figure 3). L-NAME and methylene blue did not alter
K+-evoked neuronal NE output by itself (data not
shown).
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It has been reported that NO formation after AT2-receptor stimulation is due to the activation of local bradykinin (BK) production.24 25 Therefore, we investigated the effect of Hoe 140, a kinin B2-receptor antagonist, on the inhibitory effect of Ang-(1-7) on NE release. As shown in Figure 4, 10 µmol/L Hoe 140 completely blocked the induced reduction of K+-evoked neuronal NE release caused by the heptapeptide in rat hypothalamus. The antagonist had no direct effect.
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To assess if the prostaglandin pathway is involved in Ang-(1-7) effects, experiments were performed in the presence of 0.1 to 10 µmol/L indomethacin, a cyclooxygenase inhibitor. No differences in the effect elicited by 100 nmol/L Ang-(1-7) were observed (Figure 5). Indomethacin (0.1 to 10 µmol/L) failed to modify by itself the K+-evoked neuronal NE release (data not shown).
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| Discussion |
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It has been reported that Ang-(1-7) elicits a tissue-specific neuromodulatory action. In this regard, a facilitatory effect on noradrenergic neurotransmission in rat atria was observed,8 but no effect in rabbit vas deferens was described.11
Biological activity of Ang-(1-7) is distinguishable from that of Ang II, and frequently contrasting effects were observed27 : For example, Ang-(1-7) facilitates the baroreflex7 18 as well as induces vasodilation,16 17 21 effects not produced by Ang II. There is evidence that the renin-angiotensin system regulates several functions through multiple-level feedback mechanisms.28 29 Contrasting activities of Ang-(1-7) and Ang II would further confirm the hypothesis that the renin-angiotensin system may limit Ang II effects through generation of the heptapeptide.27 In agreement, the present results show that Ang-(1-7) opposes the enhancement on K+-induced [3H]-NE release from rat hypothalamus caused by Ang II.30 31
The decreased NE release caused by Ang-(1-7) was blocked by the AT2-receptor antagonist PD 123319 and not by the AT1-receptor antagonist losartan, suggesting that AT2 receptors are involved in such response. In fact, central responses of Ang-(1-7) appear to be more sensitive to inhibition by AT2-receptor antagonists. For example, prostaglandin synthesis in human astrocytes9 and substance P release in rat hypothalamus32 as well as neuronal excitation in the paraventricular nucleus7 induced by Ang-(1-7) were blocked by AT2-receptor antagonists.
Interestingly, both AT1 and AT2 receptors appear to have antagonistic roles on central noradrenergic neurotransmission, since Ang IIfacilitated NE release is mediated by AT1 receptors4 whereas Ang-(1-7) inhibitory action on NE release is coupled to AT2 receptors (present results). Several reports have suggested that these receptors mediate opposite physiological effects.24
Furthermore, the inhibitory action of Ang-(1-7) on NE release was partially prevented by [D-Ala7]Ang-(1-7), suggesting that Ang-(1-7) receptors are also involved. Likewise, the stimulation of specific Ang-(1-7) receptors accounts for the Ang-(1-7)induced excitation of paraventricular neurones in rat33 and [3H]arachidonic acid release in rabbit aortic smooth muscle cells.34 In this latter case, both PD 123319 and [D-Ala7]Ang-(1-7) were required to fully block the response.
Because Ang II increases NE release through AT1-receptor activation and not through AT2 receptors, the opposite response induced by losartan itself may result from blockade of endogenous Ang II binding to AT1 receptors, as it was previously suggested.35 36 We disregarded this possibility in our results because the addition of losartan plus PD 123319 did not modify the stimulated NE release, suggesting that losartan may unmask the binding of endogenous either Ang-(1-7) or Ang II to AT2 sites.
Ang-(1-7) is a potent stimulator of prostaglandin release in neural and vascular cells.9 10 11 Furthermore, vasodilation of cerebral arteries as well as the natriuresis and depressor activities produced by Ang-(1-7) could be abolished by indomethacin, suggesting that these effects are mediated by prostaglandins.37 Despite their inhibitory role on NE release, the attenuation on sympathetic neurotransmission caused by Ang-(1-7) in the rat hypothalamus (present results) was not prevented by indomethacin, excluding prostaglandin involvement.
Because the neuromodulatory effect of Ang-(1-7) on NE release was blocked by L-NAME and methylene blue, an NO-mediated mechanism is suggested in accordance with previous reports that demonstrated NO dependence in various Ang-(1-7) effects.16 21 22 23 Moreover, NO formation induced by Ang-(1-7) (present data) after AT2-receptor stimulation appears to be due to the activation of local BK production, since the inhibitory effect of the heptapeptide on NE release disappeared in the presence of Hoe 140, a B2-receptor antagonist. Accordingly, Seyedi et al38 have shown that the increased aortic NO production induced by Ang-(1-7) resulted from the activation of AT2 receptors and also involved local BK production. In fact, it recently has been shown that mice lacking AT2 receptors have low renal BK and NO production,25 suggesting that these receptors mediate BK and NO formation. It should be pointed out that Ang-(1-7) interaction with kinins is a receptor-mediated event and not simply attributable to angiotensin-converting enzyme inhibition, which may possibly prevent BK degradation39 or downregulation of the B2 receptor.40
In conclusion, Ang-(1-7) has a tissue-specific neuromodulatory effect on noradrenergic neurotransmission, being inhibitory at the central level by a NO-dependent mechanism that involves AT2 receptors and local BK generation.
| Acknowledgments |
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Received July 28, 1999; first decision August 24, 1999; accepted January 17, 2000.
| References |
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