INTRODUCTION

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RATS CHRONICALLY TREATED WITH the orally active nitric oxide synthase (NOS) inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) develop a conspicuous hypertension (10, 29). Most of the data available suggest that NOS inhibition in the central nervous system may increase the sympathetic activity, which could be responsible, at least in part, for the increase in arterial pressure occurring in the L-NAME hypertensive model (7, 38). A previous study using double pharmacological blockade of the autonomic influences on the heart with atropine and propranolol associated with the ganglionic blocker trimethaphan suggested that, in chronic L-NAME-treated rats, the hypertensive levels may be maintained by central sympathoexcitation and by vagal suppression as well (7). However, a more recent observation (32) using pharmacological blockade with methylatropine and methoprolol demonstrated that moderate NOS inhibition by L-NAME does not seem to influence the cardiac sympathetic tonus.

The analysis of heart rate variability (HRV) in the frequency domain (spectral analysis) has been used to evaluate the autonomic modulation of the cardiovascular system (1, 14, 20, 37), and, despite the autonomic alterations found in the chronic L-NAME hypertensive model (7, 31, 32), there are only few reports addressing changes in the HRV in this model (4, 21), as well as the role of nitric oxide (NO) in cardiovascular variability (15, 26, 27).

Additionally, only few works have evaluated the baroreflex function during acute or chronic hypertension induced by L-NAME, and the results have revealed some disagreement. Studies on humans (4) and rats (25) did not show a deficit in baroreflex sensitivity after acute treatment with L-NAME. On the other hand, a decrease in baroreflex sensitivity has been described in the hypertensive model elicited by chronic NOS inhibition with L-NAME (17, 32, 32), contrasting with the only report of an increased sensitivity of the baroreflex control of heart rate (HR) in rats (42). These contradictory findings may be due to different methodological approaches such as different time elapsed after recovery from anesthesia for catheterization and dosage of L-NAME.

One of the difficulties in bringing together the findings with L-NAME has been that each of the studies has used only one or two methods of autonomic assessment. There has not been a comprehensive or systematic investigation in the L-NAME hypertensive model of the tonic and phasic autonomic influences on the heart.

There is evidence in the literature that, after 7 days of treatment with L-NAME, the sympathetic nervous system markedly contributes to the pathogenesis of this model of short-duration hypertension in rats (7, 31). Therefore, the aim of the present study was to investigate, in conscious rats, the possible alterations of the autonomic control of the heart using the following different approaches: 1) double blockade with methylatropine and propranolol, 2) the reflex control of HR, and 3) the autonomic modulation of HRV in the time domain by means of SD and in the frequency domain by means of spectral analysis using fast-Fourier transformation (FFT).

	METHODS

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Animals. Male Wistar rats (250-300 g) were divided into three groups and kept in individual cages with controlled temperature (21°C) and a 12:12-h dark-light cycle. The animals were allowed food and water ad libitum for 3 days before the experiment. The experimental protocol was then started with a 7-day treatment with ~60 mg/kg L-NAME, ~60 mg/kg N^G-nitro-D-arginine methyl ester (D-NAME group), or water ad libitum (control group). On the 6th day, under tribromoethanol anesthesia (250 mg/kg ip), the animals were instrumented with femoral venous and arterial catheters (PE-50 soldered to PE-10) filled with heparinized saline (500 IU/ml) and exteriorized through the animal's back. After the surgical procedures, the rats were allowed to drink L-NAME or D-NAME solution or tap water until the beginning of the experiment on the next day. The animals that did not drink a minimum amount of L-NAME or D-NAME solution to provide the proper daily intake of L-NAME or D-NAME were not used in the experiments.

Arterial pressure measurement. Twenty hours after the surgical procedures, the recording was performed with a pressure transducer (Statham P23 Gb), and the amplified (Hewlett-Packard 8805-A) signal was fed to a computer acquisition board (analog-to-digital converter CAD-12/36, software Aqdados; Lynx Tecnologia Eletrônica, São Paulo, Brazil). The animals were kept in individual cages during the experiment. Mean arterial pressure (MAP) and HR were calculated from the arterial pulse pressure.

Sympathovagal tonus and intrinsic rate of the cardiac pacemaker. Methylatropine (4 mg/kg) and propranolol (5 mg/kg) were used to block the parasympathetic and sympathetic influences on HR, respectively, in control (n = 24), D-NAME (n = 12), and L-NAME (n = 24) groups. After the basal period (15 min), methylatropine was injected in one-half of the rats, and the HR was recorded during the next 15 min to evaluate the parasympathetic effect on HR. Propranolol was then injected, and HR was recorded for another 12 min to determine the intrinsic heart rate (IHR). In the other half of the rats, the methylatropine/propranolol sequence was reversed to propranolol/methylatropine, observing the same time period (12/15 min) for each drug as in the previous sequence used to determine IHR. The data from the methylatropine/propranolol (control, n = 12; D-NAME, n = 06; L-NAME, n = 12) and propranolol/methylatropine (control, n = 12; D-NAME, n = 06; L-NAME; n = 12) sequence were pooled to provide the basal HR (before any blockade) and the IHR. In addition, HRV in the frequency domain (see below) was assessed in these animals before and after propranolol or methylatropine.

HRV. The method used to examine HRV in the frequency domain (control, n = 12; L-NAME, n = 12) has been described elsewhere (9). Briefly, the spectral density power of the various frequency components of HR was calculated using FFT. Time series (30 min) of beat-to-beat HR data were converted to data points every 100 ms using a cubic spline interpolation. The interpolated series were divided into half-overlapping sequential sets of 1,024 data points (102.4 s). Nonstationary data were removed visually. A Hanning window in the time domain was used to attenuate side effects, and the spectrum computation was performed using the FFT algorithm for discrete time series. The averaged spectra were integrated in three frequency bands defined as very low (VLF = 0.015-0.199 Hz; see Ref. 14), low (LF = 0.20-0.59 Hz; see Refs. 14 and 26), and high (HF = 0.60-2.5 Hz) frequency. This HF range was used because our animals always present respiratory frequencies ranging within this band. The mean ± SD of the beat-to-beat HR was calculated as an index of the overall variability of this parameter in the time domain.

Baroreflex sensitivity. The rats (control, n = 12; D-NAME, n = 06; L-NAME, n = 12) were brought to a quiet room and allowed to acclimatize for at least 30 min. Baroreflex sensitivity was determined by the method of Head and McCarty (12). Changes in MAP were elicited by alternating bolus injections of phenylephrine (0.1-16.0 µg/kg) and nitroprusside (0.1-16.0 µg/kg). MAP and HR were measured before and immediately after injection of phenylephrine (or sodium nitroprusside) when the arterial pressure achieved a new steady-state level. The two parameters were then allowed to return to baseline, after which the next injection was given. A total of at least seven increases and seven decreases in MAP of different degrees was elicited in each rat. Baseline and response values for MAP and HR were analyzed by fitting a sigmoidal curve to the following logistic equation (22)
where P₁ is the lower plateau (maximum reflex bradycardia), P₂ is the HR range, P₃ is the slope coefficient (a curvature parameter), and P₄ is the half-maximal MAP (i.e., the MAP at half-P₂). The maximum gain (G_max) of the baroreflex was obtained mathematically from the slope of the curve and is given by G_max = (P₂ × P₃)/4.

Statistical analysis. The data are presented as means ± SE. The results of sympathovagal control, spectral analysis, SD, and baroreflex sensitivity of HR were analyzed by one-way ANOVA followed by the post hoc Tukey test. The results of spectral analysis of HRV were analyzed by nonpaired Student's t-test. Significant differences were considered at P < 0.05.

	RESULTS

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Table 1 shows the basal hemodynamic parameters after 7 days of treatment for all groups. L-NAME-treated rats exhibited a significantly higher MAP and tachycardia compared with normotensive control and D-NAME-treated rats.

Despite the conspicuous tachycardia observed in the L-NAME hypertensive rats, the IHR obtained after double blockade of the autonomic influences on HR with methylatropine and propranolol was similar among groups (L-NAME = 385 ± 3 beats/min vs. control = 380 ± 2 beats/min, and D-NAME = 383 ± 3 beats/min). Figure 1 shows basal HR, IHR and percent change in HR elicited by methylatropine and propranolol. After parasympathetic blockade with methylatropine, the increase in HR (11 ± 0.6%) in L-NAME hypertensive rats was significantly smaller than in normotensive control (27 ± 2.7%) and D-NAME (21 ± 2.1%) rats. On the other hand, after the sympathetic blockade with propranolol, the decrease in HR (14 ± 0.8%) in L-NAME hypertensive rats was higher than in normotensive control (6 ± 0.9%) and D-NAME (7 ± 0.7%) rats.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Bar graphs showing basal heart rate (HR; broken line), intrinsic heart rate (IHR; hatched bars), and percent changes in HR (HR; arrows) after the injection of methylatropine and propranolol in control (vehicle), NG-nitro-D-arginine methyl ester (D-NAME), and NG-nitro-L-arginine methyl ester (L-NAME) groups. *P < 0.05 compared with normotensive control and D-NAME group. bpm, Beats/min.

The HRV measured by the SD in the time domain in L-NAME hypertensive rats (13 ± 3 beats/min) was significantly smaller than in normotensive control rats (22 ± 4 beats/min).

Figure 2 shows a typical power density spectrum of the HR in a control and L-NAME-treated rat. Chronic L-NAME-induced hypertension was associated with a lower power density in the LF band (1.35 ± 0.10 vs. 2.15 ± 0.19 beats · min¹ · Hz¹) but not the VLF (34.73 ± 1.51 vs. 31.05 ± 0.79 beats · min¹ · Hz¹) and HF (9.04 ± 0.49 vs. 10.17 ± 0.44 beats · min¹ · Hz¹) bands. Furthermore, as shown in Fig. 2, inset, the LF-to-HF ratio power density of the L-NAME (0.14 ± 0.01) group was significantly smaller than that of the control (0.21 ± 0.016) group. In control rats, parasympathetic blockade reduced both LF and HF bands of HR spectra, whereas cardiac sympathetic blockade reduced only the LF band. In contrast, the reduced LF band of HR spectra observed in L-NAME-treated rats was reduced further only by methylatropine (Fig. 3).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Spectrum power of the HR of a typical rat in the control and L-NAME groups. Dashed lines show the limits of the three frequency bands (very low, low, and high). Bar graphs (inset) show the ratio of the power in the low (LF) and high (HF) frequency bands for the control and L-NAME group. *P< 0.05 compared with normotensive control group.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Bar graph of spectral power density of HR in LF and HF bands in control (A) and L-NAME (B) groups before (open bars) and after propranolol (filled bars) or methylatropine (hatched bars). *P < 0.05 compared with normotensive control.

As shown in Fig. 4, assessment of the blood pressure-HR reflex by means of the barocurve demonstrated that there was no difference between the normotensive control and D-NAME rats, whereas the barocurve of the L-NAME hypertensive rats was shifted to the right, as indicated by the displacement of the half-maximal MAP to hypertensive levels (Table 1). As shown in Table 1, the upper and lower plateau and the HR range of the different groups did not differ. Figure 4 and Table 1 show that the gain of the baroreflex of L-NAME hypertensive rats was smaller than the gain of the normotensive control and D-NAME rats.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   A: average baroreflex sigmoidal curves obtained from mean arterial pressure (MAP)-HR. Solid lines, control (vehicle); dashed lines, D-NAME; dotted lines, L-NAME. Half-maximal MAP of control (), D-NAME (), and L-NAME () groups are shown. Inset: parameters characterizing the sigmoidal logistic equation of the cardiac baroreflex. P1, lower plateau; P2, HR range; P4, MAP location parameter half-way between the HR range (MAP50). B: slopes of the baroreflex control of HR with changes of MAP in the control, D-NAME, and L-NAME groups. *P < 0.05 compared with normotensive control and D-NAME groups.

	DISCUSSION

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The present study shows that the hypertension induced by chronic administration of the NOS inhibitor L-NAME is associated with tachycardia, increased sympathetic drive to the heart, decrease of HRV, and an attenuation of the baroreflex control of HR.

Because the administration of D-NAME failed to cause any alteration in blood pressure, HR, or baroreflex control of HR and taking into account that the main action of L-NAME is to inhibit NOS, the observed effects can be attributed to the lack of NO production.

It is unlikely that the observed tachycardia resulted from changes of IHR since no alterations were observed in this parameter, as determined by means of the double blockade with methylatropine and propranolol. Moreover, these experiments revealed an increased sympathetic and a decreased parasympathetic influence on HR. It has been reported that the elevated blood pressure after 7-10 days of L-NAME is associated with a decrease in basal HR reflecting an increase in parasympathetic and a decrease in sympathetic tone (32). It is most likely that this opposite result was related to a more intense inhibition of NO production in our study, since we used a dosage of L-NAME approximately six times higher than that used in Ref. 32.

The presence of abnormalities in the autonomic regulation of HR in rats with hypertension induced by L-NAME was further evaluated by the analysis of HRV. The results showed a decreased HRV in the chronic hypertensive rats, measured by the SD in the time domain that was also confirmed in the frequency domain by spectral analysis. This latter approach showed a reduction of power spectral density of the LF band and of the LF-to-HF ratio. Oscillations of HR in the LF band in awake rats have been associated with sympathetic and parasympathetic modulation, whereas oscillations in the HF band have been associated only with parasympathetic modulation (5, 6, 14). Our results with autonomic blockade in control rats confirm the participation of both components of the autonomic nervous system in the genesis of the LF band of HRV, whereas only the parasympathetic component contributes to the HF band. Interestingly, the already reduced LF band of HRV in L-NAME-treated rats was reduced further only by the parasympathetic blockade, indicating that the sympathetic component does not contribute to the genesis of the LF band of HRV in L-NAME rats. These observations indicate that the chronic deficit in NO production leads to alterations in the autonomic control of the heart.

Although it appears contradictory that an increased sympathetic activity promoting remarkable tachycardia in L-NAME hypertensive rats is associated with a decreased LF variability in HR and a decreased LF-to-HF ratio, this paradox was also observed in others situations, which presented a sharp increase in sympathetic activity, e.g., heavy physical exercise (2) or severe heart failure (40). In these situations, when several physiological mechanisms are mobilized to the maximum to keep homeostasis, the cardiovascular system has no reserve to maintain its variability. Accordingly, the cause of a reduced LF band in HRV in this model of hypertension is not known. Even though Van de Borne et al. (40) have suggested abnormalities in central autonomic modulation, impairment in arterial baroreflex regulation or changes in neurotransmitter sensitivity in the target organ might be responsible for the reduced LF band in HRV.

Sympathetic influence on HRV at the LF band has been related to baroreflex function, since simultaneous analysis of arterial pressure and HR has been applied to obtain an index of baroreflex sensitivity (5, 8) derived from cross-spectral analysis. Although we used only pharmacological stimulation, the present results showing a decreased baroreflex sensitivity agree with previous observations in less-prolonged L-NAME hypertension, i.e., 1 wk (32), and in more-prolonged L-NAME hypertension, i.e., 4-5 wk (17, 33). However, there is a report that rats treated with L-NAME for 6 days exhibited increased baroreflex sensitivity (42), and another report shows that rats treated for 2.5-3 wk did not show any baroreflex dysfunction (3). Compared with our results, these contradictory findings may have been due to different methodological approaches, such as the short time (6 h) elapsed after recovery from anesthesia for catheterization (42) or the more prolonged (4-5 wk) hypertension (3) as well.

A derangement of the baroreflex arch or development of left ventricular hypertrophy, as seen in spontaneously hypertensive rats (11), are among the mechanisms that might explain the deficit in baroreflex sensitivity observed in L-NAME hypertensive rats. Nevertheless, it is well documented that chronic (2.5-4 wk) L-NAME hypertensive rats lack ventricular hypertrophy (3, 17). On the other hand, the baroreflex may be altered by changes in the afferent, central, and/or efferent component. Baroreceptor afferents may have their activity modified by the chronic lack of NO under L-NAME treatment, since there is no evidence that baroreceptor function can be affected by NO (23, 41).

Results from different species have suggested that NO may play a role in the central processing of baroreceptor afferents, mainly in the nucleus tractus solitarius (NTS) or rostral ventrolateral medulla (RVLM). In anesthetized rats, intravenous L-NAME infusion reduced the NTS neuronal discharge (19), and unilateral microinjection of L-arginine into the NTS produced remarkable dose-related depressor and bradycardic effects and reduced renal sympathetic nerve activity (39). Additionally, the NO in the NTS increased the neuronal activity of adjacent neurons in the NTS through an increase in cGMP (36). Other authors (18) have suggested that the fall in MAP and HR after activation of N-methyl-D-arginine receptors by excitatory amino acids is mediated by NO in the NTS of rats. On the other hand, other studies also suggest that NO acts at the NTS level to increase the sympathetic outflow in normotensive rats, leading to an increase in arterial pressure and HR (24).

It has been demonstrated that microinjections of L-NAME or N^G-monomethyl-L-arginine into the RVLM of rats or cats increased arterial pressure (16, 35). On the other hand, a study on anesthetized rabbits with denervated baroreceptors (13) showed that NO has a pressor and sympathoexcitatory action in the RVLM. This opposite result may be ascribed to the effects of anesthesia or to the species studied. In addition, another study on anesthetized rats demonstrated a small pressor response and a marked increase in renal sympathetic nerve activity after intracisternal infusion of N^G-monomethyl-L-arginine; the effects were abolished by medullar transection at the C₁-C₂ levels or by intracisternal injection of L-arginine (38).

Furthermore, NO also appears to play a role in spinal cord preganglionic sympathetic neuron function, since neurons in the intermediolateral cell column showed the presence of NOS (30). It has also been suggested that NO could facilitate the release of excitatory transmitters, possibly through a presynaptic cGMP-dependent mechanism (43). In addition, sympathetic right stellate ganglion stimulation in rabbits demonstrated that endogenous NO plays an inhibitory role in cardiac sympathetic neurotransmission (34).

Most studies examining a role of NO on reflex cardiovascular regulation used the acute administration of NOS inhibitors, making a comparison with our findings obtained after chronic (7 days) treatment with L-NAME difficult. Nevertheless, a recent study (28) using chronic intracerebroventricular injection of L-NAME in normotensive Wistar-Kyoto rats demonstrated an increase of systemic arterial pressure associated with a decrease in baroreflex sensitivity. Therefore, considering the high dosage of systemic L-NAME used in the present study, we may suggest that the cardiac sympathetic overactivity associated with baroreflex impairment in the chronic L-NAME hypertensive model might be due at least in part to an effect of the NOS inhibitor on the central nervous system.

In conclusion, our findings indicate that the hypertension induced by L-NAME is associated with an altered cardiac autonomic control, characterized by a predominance of the sympathetic over the parasympathetic drive in the control of HR and associated with a decreased baroreflex sensitivity. In addition, a decrease in HRV in the LF band was observed similar to that present in physiological or pathophysiological conditions, causing sharp increases of sympathetic activity, e.g., heavy physical exercise and severe heart failure (2, 40). The precise site of the reflex arch where NO acts requires further study.