Role of Angiotensin-(1-7) in the Modulation of the Baroreflex in Renovascular Hypertensive Rats
Abstract In this study, we evaluated the effect produced by lateral ventricle (intracerebroventricular, ICV) infusion of the selective angiotensin (Ang)-(1-7) antagonist, d-Ala7-Ang-(1-7) (A-779), in the modulation of the baroreflex control of heart rate in two-kidney, one clip renovascular hypertensive rats (2K1C) treated with the angiotensin-converting enzyme (ACE) inhibitor enalapril. Twenty days after the surgery to produce renovascular hypertension, ICV cannulas were implanted in the rats with blood pressure (BP) greater than 145 mm Hg (n=33) and in sham-operated rats (n=32). Five days later, the rats were treated with enalapril (10 mg · kg−1 · d−1; 6 days, in the drinking water) or vehicle (tap water). On the sixth day of treatment, direct continuous BP recording and measurement of reflex changes in heart rate elicited by phenylephrine were made in conscious rats before and at 1 hour of ICV infusion of saline (8 μL/h) or A-779 (4 μg/h). To evaluate the degree of ACE blockade produced by enalapril treatment, the pressor effect of Ang I (50 ng, IV, and 100 ng, ICV) and plasma ACE activity was determined. As expected, enalapril treatment in 2K1C produced a significant fall in BP, significant attenuation in the pressor response of Ang I (IV), and a reduction in plasma ACE activity. In addition, enalapril treatment increased the baroreflex sensitivity (0.76±0.04 versus 0.43±0.04 ms/mm Hg in 2K1C untreated rats). ICV infusion of A-779 reverted the improvement in baroreflex sensitivity produced by enalapril treatment in 2K1C (from 0.80±0.07 to 0.42±0.08 ms/mm Hg) and also attenuated the baroreflex sensitivity in untreated 2K1C (0.36±0.05 versus 0.48±0.06 ms/mm Hg) and untreated sham-operated rats (1.21±0.05 versus 0.78±0.17 ms/mm Hg). These results suggest that central endogenous Ang-(1-7) is involved at least in part in the improvement of baroreflex sensitivity observed in 2K1C after peripheral chronic ACE inhibition.
Angiotensin converting enzyme inhibitors, which lower BP in several models of experimental hypertension, have been largely used in the treatment of human hypertension.1 The mechanism of action of ACE inhibitors has been attributed to the inhibition of Ang II formation and/or BK inactivation.1 2 Recently it was shown that ACE inhibition produces an increase in plasma levels of another biologically active angiotensin peptide, Ang-(1-7), in SHR3 and in humans.4
Ang-(1-7) is a biologically active end product of the renin-angiotensin system that presents selective actions.5 6 Peripherally, Ang-(1-7) produces a potent antidiuretic effect on water-loaded rats7 8 and potentiates the hypotensive effect of BK after intravenous injection in freely moving rats.9 Centrally, Ang-(1-7) causes cardiovascular responses when microinjected into the rat dorsomedial10 11 or ventrolateral medulla12 13 and produces a significant facilitation of the baroreflex control of HR in normotensive rats after ICV infusion.14 More recently we have shown that ICV infusion of the Ang-(1-7) antagonist A-779 [d-Ala7-Ang-(1-7)] produces a severe impairment of baroreflex control of HR,15 suggesting an endogenous role for this peptide in the modulation of the baroreceptor control of BP. In addition, Ang-(1-7) is a major Ang I metabolite formed by a pathway independent of ACE,16 17 18 and for this reason it is likely that the beneficial effects of ACE inhibitors may at least in part depend on Ang-(1-7) formation in plasma or tissue.
In the present study, we attempted to evaluate the contribution of central Ang-(1-7) to the improvement of the baroreflex sensitivity observed in hypertensive rats treated orally with the ACE inhibitor enalapril. For this purpose we have used the recently characterized Ang-(1-7) antagonist A-779 that was shown to be potent in inhibiting Ang-(1-7) actions in several preparations in vitro and in vivo, without intrinsic agonistic properties and with no interference with the action of other related peptides.19
Production of Renal Hypertension
Male Wistar rats weighing 180 to 200 g, bred at the animal facility of the Biological Sciences Institute (CEBIO, UFMG, Brazil), were anesthetized with ether, and a silver clip (0.25 mm ID) was placed around the left renal artery through a midline incision to produce the Goldblatt 2K1C renovascular hypertension. Other rats were submitted to similar procedures (sham operation) but without the renal artery clip placement (sham). Presurgery and postsurgery, the animals were kept in a temperature-controlled room on a 14/10-hour light/dark cycle with free access to standard chow and tap water.
Indirect Measurement of BP
The development of hypertension was evaluated by indirect measurement of BP by the tail-cuff method. Briefly, the artery of the tail was occluded with a small cuff connected to a mercury column, and the pulse was detected by a water column. In our laboratory, the BP values obtained with this method were similar to the MAP obtained by direct measurement in a polygraph (correlation, 87%; n=42).
ICV Cannula Implantation
Twenty days after the surgery to produce 2K1C hypertension, cannulas were implanted into the lateral ventricle of rats with BP greater than 145 mm Hg (n=33) and in sham (n=32), according to a procedure described previously.14 Briefly, the rats were anesthetized with ether and placed in a stereotaxic frame (David Kopf Instruments) with the head in the horizontal position. A metallic siliconized cannula (25-gauge butterfly needle) bent at a right angle was inserted into the lateral ventricle (1.5 mm lateral and 1.0 mm caudal to the bregma and 4.5 mm below the skull) through a small hole drilled in the skull and fixed with dental cement and a jeweler’s screw. The external end of the cannula was connected to polyethylene tubing (PE-10) fixed to the interscapular region. The total dead space of the cannula was 2.5 to 3.0 μL, calibrated before cannula implantation was filled with sterile isotonic saline. At the end of each experiment, 5 μL of Evans blue dye was injected ICV to verify the cannula positioning.
Twenty-five days after the surgery to produce renal hypertension, subgroups of the hypertensive 2K1C (n=16) and sham (n=16) rats were treated with enalapril, 10 mg · kg−1 · d−1 (Merck), in the drinking water during 6 days. The other animals, 2K1C (n=17) and sham (n=16) received normal tap water. One hour before the ICV infusion, the animals received 5 mg/kg of enalaprilat intravenously. All the animals were kept in metabolic cages from day 18 to day 26 after surgery.
Arterial Pressure Measurement
On the fifth day of treatment, ie, 24 hours before the ICV infusion, catheters were inserted under ether anesthesia into the femoral artery and vein for BP measurement and intravenous injections, respectively. The catheters were filled with saline, closed by metallic pins, and tunneled subcutaneously to the back of the neck. On the next day, arterial pressure was monitored in conscious freely moving rats by a solid state strain-gauge transducer (model TP-200T, Nihon Kohden), and HR was determined with an HR counter (model AT-601G, Nihon Kohden) triggered by the arterial pressure wave. All variables were recorded continuously on a direct-writing polygraph (model CP-640G, Nihon Kohden).
ICV Infusion Procedures
ICV infusion of sterile saline (vehicle, 8 μL/h) or the Ang-(1-7) antagonist A-779 [d-Ala7-Ang-(1-7), 4 μL/h, synthesized by one of us (M.C. Khosla) at the Cleveland Clinic Foundation] was carried out with a Hamilton syringe (10 μL, Hamilton Co) as follows: untreated sham rats (saline, n=8; A-779, n=8), enalapril-treated sham rats (saline, n=7; A-779, n=9), untreated 2K1C rats (saline, n=9; A-779, n=8), and enalapril-treated 2K1C rats (saline, n=8; A-779, n=8).
Baroreceptor Reflex Test
Baroreflex control of HR was evaluated in conscious rats before and at 1 hour of ICV infusion by the reflex changes in HR in response to MAP changes produced by repeated bolus injections of grade doses of phenylephrine (0.2 to 40 μg/kg, IV). Phenylephrine doses were injected 1 to 2 minutes apart into the femoral vein in 0.1 mL isotonic NaCl. Peak changes in HR occurring during the initial 5 seconds of the corresponding maximum change in MAP produced with phenylephrine were recorded. The HR changes were converted to PI (ms) by the formula 60 000/HR. Baroreceptor reflex sensitivity was estimated by the ratio between changes in HR (as PI) and changes in MAP (ΔPI/ΔMAP, ms/mm Hg) in each rat before and at 1 hour of infusion. The data were also illustrated graphically by the best-fit regression line drawn from the mean±SE of pressure and HR changes for each dose of phenylephrine.
Evaluation of ACE Inhibition
To evaluate the degree of ACE blockade produced by enalapril treatment, we determined (1) the pressor response produced by intravenous (50 ng) and ICV (100 ng) Ang I injection in all animals at the end of the experiment and (2) plasma ACE activity. The plasma ACE activity was determined by a fluorimetric enzymatic assay using the synthetic substrate Hip-His-Leu (Sigma) as described previously by Santos et al.20
Comparisons among different groups or different time points were made by one-way ANOVA followed by the least significant difference test. Differences between two groups and between before and after ICV infusion were made by Student’s t test or Student’s t test for paired observations, respectively. A value of P<.05 was considered statistically significant. Numerical values are given as mean±SE.
Baseline Changes in Arterial Pressure
As shown in Table 1⇓, BP was significantly high on the 15th day after surgery (average of groups 1 and 2, 145±3 mm Hg compared with sham rats, average of groups 3 and 4, 100±2 mm Hg). BP of 2K1C rats continued to increase through the end of the experiment in 2K1C untreated rats (vehicle). As expected, enalapril treatment produced a significant fall in BP in 2K1C hypertensive rats (134±5 mm Hg on the 6th day of treatment compared with 169±3 mm Hg before treatment or with 182±5 mm Hg in untreated 2K1C rats; Table 1⇓). No changes in BP were observed during enalapril treatment in sham rats (Table 1⇓).
Degree of ACE Inhibition
As shown in Fig 1⇓, enalapril treatment produced a significant attenuation of the pressor response of intravenous injection of Ang I in 2K1C hypertensive rats (change in MAP, 16±3 mm Hg compared with 37±4 mm Hg in untreated 2K1C; Fig 1A⇓) as well as in sham rats (change in MAP, 13±2 mm Hg compared with 45±4 mm Hg in untreated sham rats; Fig 1A⇓). Treatment with enalapril also produced a significant reduction in plasma ACE activity in 2K1C hypertensive rats (40.9±7.5 nmol His-Leu per milliliter per minute compared with 92.9±14.4 nmol His-Leu per milliliter per minute in untreated 2K1C rats; Fig 1B⇓) and in sham rats (30.3±3 nmol His-Leu per milliliter per minute compared with 94.8±10.1 nmol His-Leu per milliliter per minute in untreated sham rats; Fig 1B⇓). In contrast, enalapril treatment did not affect the pressor and drinking responses to ICV injection of Ang I in 2K1C hypertensive rats (44±4 mm Hg and 12±1 mL, respectively) compared with untreated 2K1C rats (35±3 mm Hg and 12±1 mm Hg, respectively). Similarly, enalapril treatment did not affect the pressor and drinking responses to ICV injection of Ang I in sham rats (35±3 mm Hg and 11±2 mL, respectively) compared with untreated sham rats (33±2 mm Hg and 14±1 mL, respectively).
Baroreflex Control of HR in 2K1C Rats
As expected, renal hypertensive rats presented a marked attenuation of the reflex bradycardia. The sensitivity of the baroreflex bradycardia taken as the mean ratio ΔPI/ΔMAP, for each animal, was significantly attenuated in 2K1C rats (0.43±0.04 ms/mm Hg, n=17; Table 2⇓) compared with sham rats (1.15±0.07 ms/mm Hg, n=16; Table 2⇓).
Effect of Enalapril on Baroreflex Control of HR
Enalapril treatment did not affect the baroreflex bradycardia of sham rats (averaged ΔPI/ΔMAP: 0.87±0.08 ms/mm Hg, n=16, compared with 1.15±0.07 ms/mm Hg in untreated rats, n=16; Table 2⇑). In contrast, enalapril treatment produced a significant increase in the baroreflex bradycardia of 2K1C (averaged ΔPI/ΔMAP: 0.76±0.04 ms/mm Hg, n=16, compared with 0.43±0.04 ms/mm Hg in untreated 2K1C, n=17; Table 2⇑).
Effect of ICV Infusion on Baseline MAP and HR
As shown in Table 3⇓, ICV infusion of the Ang-(1-7) antagonist (A-779) or saline did not significantly change baseline values of MAP and HR in 2K1C hypertensive rats or in sham rats untreated or treated with enalapril.
Effect of A-779 ICV Infusion on Baroreflex Control of HR
Normotensive Sham Rats
ICV infusion of A-779 significantly decreased the baroreflex bradycardia of untreated sham rats (ΔPI/ΔMAP: 0.78±0.17 ms/mm Hg versus 1.21±0.05 ms/mm Hg before infusion, n=8; inset Fig 2A⇓). As shown in Fig 2A⇓, A-779 infusion produced a shift to the right of the line that correlates reflex changes in HR (as PI) and changes in MAP in untreated sham rats. In contrast, ICV infusion of A-779 did not change the sensitivity of the baroreflex bradycardia of sham rats treated with enalapril (ΔPI/ΔMAP: 0.96±0.13 ms/mm Hg versus 1.00±0.13 ms/mm Hg before infusion, n=7; inset Fig 2C⇓). ICV infusion of saline did not modify baroreflex sensitivity of untreated or enalapril-treated sham rats (Fig 2B⇓ and 2D⇓).
Hypertensive 2K1C Rats
ICV infusion of A-779 further decreased the already low sensitivity of the baroreflex bradycardia of untreated 2K1C hypertensive rats, as shown by the small but significant decrease in the ratio ΔPI/ΔMAP (0.36±0.05 ms/mm Hg versus 0.48±0.06 ms/mm Hg before infusion, n=9; inset Fig 3A⇓) or by the shift to the right in the line that correlates reflex changes in HR (as PI) and changes in MAP (Fig 3A⇓). More interestingly, ICV infusion of A-779 reverted the improvement in baroreflex bradycardia produced by enalapril treatment in 2K1C hypertensive rats. After A-779 infusion, there was a significant decrease in the ratio ΔPI/ΔMAP (0.42±0.08 ms/mm Hg versus 0.80±0.07 ms/mm Hg before infusion, n=8; inset Fig 3C⇓) and a shift to the right of the line that correlates reflex changes in HR (as PI) and changes in MAP (Fig 3C⇓). The sensitivity of the baroreflex bradycardia after A-779 infusion in 2K1C treated rats (0.42±0.08 ms/mm Hg) was not different from that of untreated 2K1C rats (0.48±0.06 ms/mm Hg before infusion) or 2K1C treated rats infused with saline (0.37±0.05 ms/mm Hg). As observed for the other groups, ICV infusion of saline did not modify baroreflex sensitivity of untreated or enalapril-treated rats (Fig 3B⇓ and 3D⇓).
The major finding of the present study was that central infusion of the Ang-(1-7) selective antagonist A-779 produces a complete reversal of the improvement of the sensitivity of the baroreflex control of HR observed in renal hypertensive rats by chronic oral treatment with enalapril. This observation suggests that Ang-(1-7) may play a central role in the beneficial cardiovascular effects of peripheral ACE inhibition.
In our study, ACE inhibition with enalapril produced in 2K1C hypertensive rats, as expected, a significant decrease in BP. This effect was associated with a 50% decrease in plasma ACE activity and 50% decrease in the pressor effect produced by intravenous injection of Ang I. In addition, enalapril treatment enhanced by 70% the baroreflex bradycardia induced by increases in MAP produced by phenylephrine. These results are in accordance with the well-known beneficial effects of ACE inhibitors in the treatment of hypertension1 2 that have been ascribed to the decrease in Ang II formation.21
One may argue that the effect of the ACE inhibitor treatment on baroreflex sensitivity could be due only to the decrease in MAP. However, Brooks22 has shown that angiotensin-induced chronic baroreflex resetting is partially reversed soon after Ang II infusion is stopped, despite maintenance of the hypertensive state by another vasoconstrictor. In addition, Moreira et al23 have shown that rats with high-renin renal hypertension have impairment of the baroreflex control of HR independently of the severity of hypertension, while these animals had normal bradycardia elicited by electrical stimulation of the vagus nerve and normal reflex bradycardia produced by ether stimulation. These data suggest that inhibition of the baroreflex-mediated bradycardia in renal hypertension is most probably due to impairment of the central integration of the vagal component of the baroreceptor reflex, which is modulated by angiotensin peptides.10 11 23 24
It is well established that the reflex bradycardia elicited by pressor stimulus results mainly from increase in cardiac vagal activity and to a minor extent from the decrease in sympathetic drive to the heart.25 Additionally, it has been extensively reported that the vagal component is most affected by Ang II.26 27 28 It should be pointed out, however, that enalapril treatment prevents the action of ACE on other peptides capable of influencing baroreflex sensitivity, such as bradykinin,2 4 and produces an increase in Ang-(1-7) levels.3 Thus, as discussed below, the overall effect of enalapril treatment on baroreflex cannot be ascribed solely to the interference with Ang II formation.
Effect of ICV Infusion of A-779 in Normotensive Rats
In the present study, ICV infusion of A-779 in normotensive untreated sham rats produced a significant attenuation of the bradycardic component of the baroreflex, similar to that previously observed in our laboratory,15 suggesting that endogenous Ang-(1-7) is importantly involved in the central modulation of the baroreceptor control of HR. This finding is in accordance with previous studies in which we have shown the involvement of Ang-(1-7) in the modulation of the bradycardic component of the baroreflex either with central infusion (ICV) or microinjection into the nTS of Ang-(1-7)10 11 14 or A-779.11 15 19 In the present study, we have evaluated the reflex bradycardia at time intervals (3 to 5 seconds of the peak change in MAP) corresponding mainly to an increase in vagus nerve activity.29 Thus, it is likely that the effects observed after A-779 infusion are related to the blockade of the effect of endogenous Ang-(1-7) on sites within the central nervous system involved in regulating baroreceptor reflex control of parasympathetic outflow to the heart. However, further studies are obviously needed to confirm this possibility.
The effect produced by the Ang-(1-7) analog A-779 is attributable to the antagonism of Ang-(1-7), based on the studies performed previously by us13 15 19 and others.30 31 We have shown that A-779 is potent to block the antidiuretic effect of Ang-(1-7) in water-loaded rats and to antagonize the BP changes produced by Ang-(1-7) at medullary nuclei (nTS and ventrolateral medulla).19 In addition, A-779 produced a selective blockade of the Ang-(1-7) stimulatory effect on the neuronal activity at the paraventricular nucleus of the hypothalamus.30 In contrast, A-779 did not alter the dipsogenic, pressor, or myotropic effects of Ang II or the effects of other related peptides, such as Ang III, vasopressin, bradykinin, or substance P in rats. Additionally, A-779 action does not seem to be related to AT1 and AT2 receptor subtypes, since A-779 did not compete significantly for the binding of 125I-Ang II to adrenal cortical or medullary membranes19 but produced a complete inhibition of the 125I-Ang-(1-7) binding to bovine aortic endothelial cells.31
Interestingly, A-779 did not change baroreflex sensitivity in sham rats treated with enalapril. Although the reason for this unexpected finding is not clear, the absence of A-779 effect on these animals indicates that the changes produced by this Ang-(1-7) analogue in the other group of rats, or in previous studies,15 were not due to an unspecific effect. Kohara et al3 have observed a 26-fold increase in plasma levels of Ang-(1-7) in normotensive Wistar-Kyoto rats after ACE inhibitor treatment. Thus, one possible explanation could be that the rate or duration of ICV infusion of A-779 was not sufficient to compete with the increased endogenous levels of Ang-(1-7) induced by enalapril treatment in sham rats. Another possibility would be that enalapril treatment could produce differential enzymatic or neuropeptide expression (as BK or vasopressin) in sham animals that could account for the lack of A-779 effect. In this regard, it has been shown that the Ang I ICV pressor effect after long-term treatment (3 months) with an ACE inhibitor was not affected in stroke-prone SHR but was attenuated in normotensive Wistar-Kyoto rats,32 suggesting that ACE inhibitors could cause differential effects depending on the strain or BP levels. A less unlikely possibility is that enalapril treatment in sham rats may impair the activity of neuronal pathways in which Ang-(1-7) plays a role in modulating the bradycardic component of the baroreflex control of HR.
Effect of ICV Infusion of A-779 in Hypertensive Rats
ICV infusion of A-779 produced a small but significant attenuation of the already low baroreflex sensitivity in 2K1C untreated rats. This effect was not accompanied by changes in the high level of BP presented by these animals. This finding is in contrast with our previous observation in another model of hypertension, SHR, in which ICV infusion of A-779 was not able to modify baroreflex sensitivity.15 Differences in the degree of activation of the central renin-angiotensin system in SHR and 2K1C hypertensive rats probably account for these observations.
In the present study, we showed that the improvement of baroreflex sensitivity produced by oral treatment with enalapril was reversed by short-term ICV infusion with the Ang-(1-7)–selective antagonist A-779. ACE inhibitors may gain access to circumventricular organs of the brain and bind to neural elements that contain ACE.33 34 In this regard, it has been shown that a single dose of enalaprilat results in a selective inhibition of ACE activity in the nTS,16 a key region for baroreflex modulation. Therefore, enalapril could be expressing its activity at brain sites where angiotensins are modulating baroreflex transmission. The presumable accumulation of Ang-(1-7) and/or other peptides, such as BK, in association with the decrease in Ang II formation in these areas could account for the effect of ACE inhibition. Thus, the Ang-(1-7) antagonist infused ICV could be directly or indirectly interfering with Ang-(1-7) action in these sites. One could argue that A-779 was not acting at the same sites where ACE inhibition was occurring and/or that its effect was not related to ACE inhibition. However, this hypothesis seems unlikely because we were unable to show an effect of A-779 on baroreflex modulation in SHR.15 In addition, in sham rats treated with enalapril, A-779 infusion did not affect baroreflex. Importantly, the effect of A-779 was unrelated to changes in baseline MAP and HR.
Another possibility would be that the effect of A-779 could be due to its leakage to the periphery. However, this possibility seems unlikely. Our previous finding that 3 hours of intravenous infusion of Ang-(1-7) did not affect baroreflex control of HR14 and our present data showing significant changes in baroreflex sensitivity by ICV administration of its selective antagonist indicate that enalapril treatment and A-779 infusion are modulating baroreflex-mediating bradycardia by changing local angiotensin metabolism and action in the brain. The involvement of the local renin-angiotensin system in the cardiovascular effects of ACE inhibitors was also suggested by the observation that the reduction in BP caused by ACE inhibitors is not directly related to high plasma levels of renin activity or the lowering in Ang II plasma levels in patients and experimental animals.34 In addition, ICV administration of saralasin or captopril, at doses that were either not effective or much less efficacious when given intravenously, markedly lowered BP and altered baroreflex sensitivity in adult SHR or attenuated the development of hypertension in young SHR.35 36
The area postrema–solitarii–vagal neuronal complex in the dorsomedial medulla is a potential candidate for a central site where enalapril and A-779 could be acting to interfere with the bradycardia mediated by baroreflex. Dense concentrations of specific high-affinity Ang II binding sites exist in the nTS and dorsal motor nucleus of the vagus, with lower concentrations in area postrema. In addition, Diz et al37 have shown that Ang II receptors in both rats and dogs are associated with vagal afferent fibers in the nTS and vagal motor neurons. Although the characteristics and location of Ang-(1-7) receptors within the neuronal elements of the brain have not been determined yet, we have previously shown that Ang-(1-7) elicited cardiovascular effects when microinjected into the nTS and dorsal motor nucleus of the vagus. Additionally, we have shown that microinjection of Ang-(1-7) into the nTS produces a significant increase in the bradycardia induced by baroreflex stimulation, while microinjection of its selective antagonist, A-779, produces a significant attenuation.11 The cardiovascular actions of Ang-(1-7) at the nTS appear not to be dependent on presynaptic elements, since they were potentiated rather than impaired after unilateral sinoaortic denervation.38 Thus, an increase in Ang-(1-7) formation at this site induced by enalapril treatment may directly affect central integration of baroreflex. Alternatively, changes in the angiotensin profile at the circumventricular region could be influencing the neuronal activity at the postrema-solitarii-vagal complex in integrating baroreflex.
In summary, our data provide new evidence for an important role of Ang-(1-7) in the central modulation of baroreflex control of HR. More importantly, we have obtained evidence that changes in Ang-(1-7) formation and/or action in the brain may contribute to the improvement of baroreflex sensitivity produced by peripheral administration of ACE inhibitors.
Selected Abbreviations and Acronyms
|2K1C||=||two-kidney, one clip renovascular hypertensive rats|
|MAP||=||mean arterial pressure|
|nTS||=||nucleus tractus solitarii|
|SHR||=||spontaneously hypertensive rat(s)|
This work was supported by FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico). We are thankful to Jose R. Silva and Soraia S. Silva for technical assistance.
- Received March 15, 1997.
- Revision received April 17, 1997.
- Accepted May 6, 1997.
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