The involvement of autonomic responses in nitrovasodilator action and vascular tolerance is controversial. 1 Nitric oxide (NO) has been reported to play a role in the regulation of the sympathetic nerve activity. 2–4 For example, NO inhibits sympathetic vasoconstrictor activity either by decreasing norepinephrine release or by attenuating the neuronal sympathetic excitability in the brain. 2,5,6 Zanzinger et al 4 suggested that impaired modulation of NO on sympathetic excitability may contribute to nitrate tolerance. These authors found that nitrate tolerance induced by 4 weeks of isosorbide dinitrate (4 mg/kg) treatment was accompanied by a 50% reduction in nitric oxide synthase (NOS)-positive neurons. Moreover, reflex sympathetic activation in response to S-nitroso-N-acetylpenicillamine (SNAP, 40 μg/kg) was much more pronounced in nitroglycerin (NTG) tolerant pigs pretreated with the NOS inhibitor, nitro-L-arginine. 4 Parker et al 7 also suggested that nitrate tolerance may be in part due to sympathetic activation resulting from neurohormonal compensation.
Recently, Ma et al 8 suggested that central sympathetic function may also play a role in acute NTG hemodynamic tolerance in rats. The hypotensive effects of NTG bolus doses were significantly attenuated after acute treatment of rats with subcutaneous (s.c.) injection of NTG (3 mg/kg). This vascular tolerance, however, was apparently reversed by bolus administration of either prazosin (300 μg/kg, an α1-adrenoceptor antagonist) or chlorisondamine (10 mg/kg, a sympathetic ganglion blocking agent). 8 While these results are suggestive of a role of the sympathetic nervous system in vascular nitrate tolerance, no control studies were performed to examine the effects of sympathetic blockade on the hypotensive effects of NTG in the absence of tolerance induction. It is therefore not known whether the apparent reversal of NTG tolerance by prazosin was simply due to the hypotensive effects of this α1-adrenoceptor blocker itself.
We recently showed that animals with congestive heart failure (CHF) exhibited similar NTG hemodynamic tolerance behavior when compared with normal rats. 9 These results would suggest that sympathetic activation may not be critically involved in nitrate tolerance in a conscious animal model, since CHF is associated with increased neurohormonal compensation such as increased plasma levels of norepinephrine. 10,11 However, direct evidence of the involvement, or lack thereof, of sympathetic activation in acute nitrate tolerance has not been examined. The goal of our present study, therefore, was to investigate the effects of sympathetic blockade with prazosin on the acute hemodynamic effects and tolerance development induced by NTG, using a conscious rat model of vascular nitrate tolerance. 12,13
Nitroglycerin solution (1 mg/mL in 5% dextrose, D5W) was obtained from Schwarz Pharma (Germany), and diluted with D5W when appropriate. Prazosin was obtained from Sigma Chemical Co. (St. Louis, MO), and its solution was prepared in distilled water.
All surgical procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee, University at Buffalo. Male Sprague-Dawley rats were obtained from Harlan (Indianapolis, IN). Two days before initiating the hemodynamic study, animals were cannulated at several sites, namely, the left femoral artery for blood pressure measurements, the left femoral vein for bolus drug administration, and the right jugular vein for drug infusion. 14
Effects of Prazosin on the Dose–Response Curve of Acute NTG
On the day of the study, systolic and diastolic blood pressures were recorded continuously via the left femoral artery using a Statham pressure transducer (Ohmeda Inc., Murray Hill, NJ) and a Gould RS3400 recorder (Gould Inc., Cleveland, OH). Mean arterial pressure (MAP) was calculated as [diastolic pressure + 1/3 (systolic pressure – diastolic pressure)]. Baseline blood pressure was recorded for at least 15–30 minutes before starting the experiment. To examine the effect of prazosin on NTG hemodynamics after acute dosing, intravenous (i.v.) bolus doses of NTG (5, 15, and 30 μg) were given in random order at 30-minute intervals (n = 5–6 animals for each dose). The hemodynamic response of each NTG bolus dose was recorded by the change in maximal MAP versus baseline MAP just prior to the specific dose. The apparent duration of NTG-induced MAP response was also measured after NTG dosing. The duration of NTG action was defined as the time it took for the MAP to return to 90% of MAP just prior to NTG dosing. This criterion was chosen because previous data 13 showed that the variability in MAP was approximately 10%. At 1 hour after the completion of NTG dose–response curve (DRC), animals received either prazosin (300 μg/kg) or vehicle by i.v. bolus, and the NTG DRC was then repeated in the same animal 30 minutes later. The injection volume for all NTG bolus doses was kept at 0.5 mL, using D5W as a diluent.
Effects of Prazosin on NTG-induced Hemodynamic Tolerance
In a separate study, rats were infused continuously with either 10 μg/min NTG or vehicle (D5W) for 5 hours (n = 4–6). A 30-μg NTG i.v. bolus challenge dose (CD) was given hourly, and the MAP response produced by the hourly CD was compared with that produced before NTG or vehicle infusion. To examine the effect of prazosin on NTG tolerance development, both NTG-infused and control animals were predosed with a single prazosin bolus dose (300 μg/kg) before NTG or vehicle infusion. The duration of NTG-induced hypotension by NTG CD was also measured at 1 hour after NTG infusion.
Data are presented as mean ± SD. Statistical analysis was performed, where appropriate, using the Student t test, or one-way ANOVA, followed by the Student-Newman-Keuls post-hoc test. Differences with P < 0.05 were considered statistically significant.
Effects of Prazosin on the Extent and Duration of the MAP Response Induced by Acute NTG
Figure 1 shows the representative blood pressure tracings obtained in a rat after NTG and prazosin bolus dosing. Consistent with our previous findings, 12 after a stable baseline has been established (region 1 in Fig. 1), a 30-μg NTG i.v. bolus dose caused an immediate but transient decrease in blood pressure (region 2 in Fig. 1), lasting only for a few seconds. When a prazosin bolus dose (300 μg/kg) was given 1 hour later, it produced an immediate but sustained decrease in blood pressure, which lasted throughout the 2-hour study period after its administration (panel B in Fig. 1). Since the baseline values before NTG and prazosin dosing were similar (regions 1 in panels B and A of Fig. 1), the hypotensive effect of prazosin was calculated by subtracting the values in region 3 from those in region 1. The effect of prazosin on the MAP response of acute NTG was then tested 30 minutes after the prazosin dose. The net MAP depressing effect of the NTG dose was calculated using region 3 as baseline, to correct for changes in baseline blood pressure prior to NTG injection but after prazosin dosing. It was noted that the duration of NTG effect was much longer in the presence of prazosin (panel C) compared with control (panel A).
The effects of prazosin on the maximum hypotensive effects produced by acute NTG bolus doses are shown in Figure 2. In the absence of prazosin, NTG dose-dependently decreased maximal MAP over the dose range examined. The maximal MAP depressions were 20.8 ± 5.8, 26.1 ± 5.0, and 30.6 ± 5.7 mm Hg for the 5, 15, and 30 μg NTG doses, respectively (differences between regions 2 and 1 in Fig. 1). From the differences between values observed in regions 3 and 1 in Figure 1, the prazosin dose (300 μg/kg) was shown to produce a significant reduction in MAP of 16.5 ± 1.7 mm Hg, (P < 0.01, versus baseline MAP before prazosin, Student t test). After prazosin treatment, the maximal MAP responses of the 5-, 15-, and 30-μg NTG doses were slightly enhanced in a dose-independent manner; the MAP was further depressed by an average of 8.9 ± 0.4 mm Hg for the 3 NTG doses. While these values were not statistically different from the corresponding dose before prazosin injection (P > 0.05, ANOVA), there appeared a trend that prazosin might potentiate the hypotensive effects of acute NTG slightly. Control vehicle injections had no apparent effect on the hypotensive effects of NTG; the maximal MAP responses of the three NTG doses before and after vehicle treatment were similar to that reported in Figure 2 (bars labeled NTG).
Figure 3 shows the apparent duration of NTG-induced MAP response after acute bolus dosing. The apparent duration of NTG-induced vasodilation was similar for the 5-, 15-, and 30-μg NTG doses, which lasted for 6 ± 2, 8 ± 1, and 9 ± 3 seconds, respectively (P > 0.05, ANOVA). Sympathetic blockade with prazosin significantly extended the hypotensive effects of all NTG doses examined, resulting in durations of action of 22 ± 7, 29 ± 8, 33 ± 13 seconds, for the 5-, 15-, and 30-μg NTG dose, respectively (P < 0.001 versus corresponding dose without prazosin, ANOVA). The magnitude of this apparent increase in the duration of NTG-induced MAP response was statistically independent (P > 0.05, ANOVA) of the NTG dose, with an average increase of approximately 4-fold.
Effects of Prazosin on NTG-Induced Hemodynamic Tolerance
Figure 4 shows the effects of prazosin bolus (300 μg/kg) administration on the hypotensive effects of the 30-μg NTG CD in rats that were infused with either vehicle (Fig. 4A) or NTG (10 μg/min, Fig. 4B). In control animals with or without prazosin pretreatment, the hourly NTG CD consistently produced a drop of 27–36 mm Hg in maximal MAP. ANOVA indicated that none of the responses produced by the hourly CD was significantly different from that observed prior to control infusion (P > 0.05 versus 0 time). Consistent with the results obtained from the acute hemodynamic study, a trend for a slightly greater hypotensive effect of the NTG CD was observed in the prazosin-treated and vehicle-infused group, but the difference did not reach statistical significance (P > 0.05 versus untreated, ANOVA, Fig. 4A).
In both prazosin-treated and untreated groups, the maximal MAP response of the NTG CD was significantly attenuated within 1 hour following NTG infusion (P < 0.001 versus 0 hours response, ANOVA, Fig. 4B), indicating the development of NTG hemodynamic tolerance. Consistent with results shown in Figure 2 and Figure 4A, while prazosin pretreatment slightly potentiated the hypotensive effects of the hourly NTG CD in NTG-infused animals, no statistical significance was reached between prazosin-treated and untreated groups (P > 0.05, ANOVA). At 5 hours after the start of NTG infusion, the beneficial effect of prazosin, if any, appeared to have been completely lost, and the MAP response of the NTG CD was essentially the same in animals with or without prazosin treatment. These results indicate that pretreatment of animals with prazosin did not prevent or attenuate the development of NTG hemodynamic tolerance.
The effects of prazosin on the apparent duration of NTG-induced hypotension in NTG-infused animals were also examined. At 1 hour after the start of NTG infusion, the apparent duration of NTG-induced MAP response by the 30 μg NTG CD was similar between prazosin-treated and untreated groups, lasting for 10 ± 2 and 7 ± 2 seconds, respectively (P > 0.05, ANOVA). The 1-hour time point was chosen as an example since results in Figure 4B showed that NTG hemodynamic tolerance was first observed at 1 hour following NTG infusion, and that this effect was similar at other time points. These results indicate that, during NTG tolerance development, prazosin no longer prolonged the duration of NTG-induced hypotension.
Ma et al 8 recently reported that NTG tolerance in rats was reversed by sympathetic blockade with either prazosin or chlorisondamine, and concluded that sympathetic activation contributes to NTG acute tolerance. This conclusion, if proven correct, would help to define a role of the sympathetic nervous system in vascular nitrate tolerance. However, the reported study was somewhat incomplete in that no control experiment was performed to examine the effects of prazosin or chlorisondamine, by themselves, in nontolerant animals. It is therefore possible that the pharmacological interaction between NTG and prazosin may occur independent of the phenomenon of vascular nitrate tolerance. The present study was designed to address this question.
Results from the acute NTG hemodynamic study (Figs. 1 and 3) showed that the MAP effects of a NTG bolus dose lasted for about 10 seconds after acute dosing. This duration of action was much shorter than the reported 4-minute half-life of NTG in rats. 15 These results suggest that even in the absence of tolerance, mechanisms other than NO or cyclic guanosine monophosphate degradation may be involved to counteract the hypotensive effects of NTG. One such possible mechanism is the activation of the sympathetic reflex after NTG dosing. Indeed, we observed that prazosin preadministration substantially prolonged the duration of NTG-induced MAP depression, and this observed enhancement was by and large independent of the NTG dose that was given in this study (Fig. 3). In preliminary studies not reported here, we found that this prazosin-induced effect on the duration of hypotensive response was also observed for S-nitroso-N-acetylpenicillamine (SNAP), which is a NO donor that does not cause vascular tolerance. 13 Thus, this observation may be interpreted as an abolishment of the sympathetic reflex, by prazosin, secondary to the acute hypotensive effects caused by NTG and SNAP, and this effect is not specific for organic nitrates.
In contrast to the observed results in the acute study (Fig. 3), prazosin was not able to prolong the duration of the acute MAP effects of NTG in NTG-tolerant animals. These results therefore suggested that NTG tolerance development might possibly attenuate the sympathetic compensation associated with the acute hypotensive action produced by the CD of NTG. In previous studies, Tseng et al had observed that 10 μg/min NTG infusion for 90 minutes led to ∼2-fold decrease in the sensitivity of the baroreceptor reflex in rats. 16 In addition, Gori et al 17 recently reported decreased baroreceptor reflex sensitivity and standard deviations of RR intervals (RRSD, an index of tonic overall short-term heart rate variability) in human subjects following 6 days of chronic NTG treatment. Thus, it is possible that NTG tolerance development may indeed alter the sympathetic reflex activity of the central nervous system in response to an acute dose of NTG.
In Figures 1 and 2, we showed that the maximal MAP responses of NTG were only slightly potentiated after prazosin treatment, after correcting for the MAP depression induced by prazosin itself. These results indicate that the pharmacological interaction between NTG and prazosin on the extent of NTG-induced MAP response was not substantial at the chosen doses. Consistent with these data, we also found that in control and NTG-infused animals, prazosin also slightly augmented the hypotensive responses of the hourly NTG CD (Fig. 4). In both prazosin-treated and untreated controls, NTG hemodynamic tolerance was observed within 1 hour of NTG infusion (Fig. 4B), suggesting that prazosin neither prevented nor attenuated NTG tolerance in our animal model. Consistent with the present study, we showed that NTG tolerance was not altered in CHF rats, 9 which are known to possess increased neurohormonal compensation when compared with normal rats.
Our findings (as shown in Fig. 4B), therefore, are in contrast to an earlier report by Ma et al, 8 which showed that prazosin (also at 300 μg/kg) completely reversed NTG hemodynamic tolerance in rats. The apparent discrepancy between the 2 studies may possibly be due to the differences in the protocol of NTG tolerance induction and the extent of tolerance induced. Ma et al 8 used a single s.c. injection of NTG (3 mg/kg) or 4 hourly bolus doses of 1 mg/kg, while we induced tolerance via continuous NTG infusion (3 mg NTG infused over 5 hours per rat). We believe that our dosing protocol is more relevant to the clinical practice of NTG administration in the treatment of chronic stable angina, which employs transdermal NTG patches, or controlled-release oral dosage forms, for continuous drug input, rather than a single large bolus dose. Our infusion rate of NTG was 10 μg/min, and tolerance was observed over the entire 1–5-hour period. At 1 hour, the total NTG dose was 0.6 mg/300 g rat (= 2 mg/kg), while at 5 hours, the total dose was 3 mg/300 g rat (= 10 mg/kg). In comparison, Ma et al 8 gave tolerance doses of 3–4 mg/kg. Thus, our tolerance regimen was slightly wider in range and duplicated the doses given in the study of Ma et al. 8 In addition, it was unclear in the study by Ma et al 8 whether the MAP decrease induced by prazosin was subtracted from NTG-induced MAP response after prazosin treatment. Our results showed that it is imperative to determine the net NTG response after prazosin treatment, since prazosin produced a substantial hypotensive effect of its own at the chosen dose.
In this study, we showed that prazosin exerted a slight potentiating effect on NTG-induced MAP response both in the absence and presence of NTG-induced hemodynamic tolerance. Thus, the “protective effect” of prazosin on the hypotensive effect of NTG, as described by Ma et al 8, probably occurs via a mechanism independent of NTG tolerance development. Our results therefore suggest that sympathetic activation may not be a major underlying cause of tolerance induction in our acute model of NTG dosing. This conclusion, however, could well be dependent on the methodology used, including the duration of NTG administration. For example, Gori et al 17 indicated that 6 days of chronic NTG dosing in humans reduced tonic and reflex vagal heart rate modulation, resulting in greater sympathetic influence. Thus, the relative influence of sympathetic control on NTG-induced response could be quite complex.
The findings reported here are in part consistent with our recent observation, 13 using gene microarray techniques, that extensive alterations in vascular (i.e., local) gene regulation accompany the development of nitrate tolerance. This finding was subsequently supported by another study. 18 The cause for such extensive changes in vascular gene expression is currently unknown, but is consistent with the prevailing theory that oxidative stress may be a dominant mechanism for vascular nitrate tolerance. 19,20 The source of free radical generation in nitrate tolerance has been thought to arise from the uncoupling of the endothelial nitric oxide synthase (eNOS) enzyme, 21 but our studies with eNOS knockout mice revealed that these animals exhibited similar vascular nitrate tolerance when compared with their wildtype controls. 22 A recent study indicated that inactivation of mitochondrial aldehyde dehydrogenase may be a critical mechanism for the development of nitrate tolerance. 23 However, questions have been raised about the exclusiveness of this mechanism. 24 The century-old mystery concerning the mechanisms of vascular nitrate tolerance 25 is therefore not quite completely resolved.
The authors thank Mr. David M. Soda for extensive technical support. This work was supported in part by funds from the University at Buffalo Foundation.
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