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Cardiovascular Research 2005 67(4):736-744; doi:10.1016/j.cardiores.2005.04.006
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Copyright © 2005, European Society of Cardiology

Increased counteracting effect of eNOS and nNOS on an {alpha}1-adrenergic rise in total peripheral vascular resistance in spontaneous hypertensive rats

Torill Berg*

Department of Physiology, Institute of Basic Medical Sciences, University of Oslo, P.O. Box 1103, Blindern, 0317 Oslo, Norway

* Tel.: +47 22851090, fax: +47 22851502. Email address: torill.berg{at}basalmed.uio.no

Received 9 February 2005; revised 20 March 2005; accepted 12 April 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: The hypertension in spontaneous hypertensive rats (SHR) may result from a hyperactive sympathetic nervous system or from insufficient bioactive nitric oxide (NO) due to increased oxidative stress. The present investigation aimed to elucidate the balance between these two systems by studying the ability of NO to oppose an adrenergic rise in total peripheral vascular resistance (TPVR).

Methods: In anesthetized, open-chest SHR and normotensive controls (WKY) on a respirator, blood pressure was recorded in the femoral artery and cardiac output measured by ascending aorta flow. Tyramine infusion (15 min, intravenously) was used to stimulate neuronal noradrenaline release.

Results: Tyramine induced an immediate but transient increase in TPVR, which was 4.5 times greater in SHR. After the non-selective NO synthase (NOS) inhibitor (L-NAME: N{varpi}-nitro-L-arginine methyl ester), {Delta}TPVRimm was 8.6 and 5.3 times increased in SHR and WKY, respectively, and TPVR remained elevated throughout the infusion period. Addition of {alpha}1-adrenoceptor antagonist (prazosin+L-NAME) abolished the TPVR response to tyramine. Neuronal NOS inhibitor (7-introindazole) increased {Delta}TPVRimm only in SHR (2.1 times), and TPVR remained elevated. Inducible NOS inhibitor (1400W), free radical scavenger (tempol), NAD(P)H oxidase inhibitor (apocynin), angiotensin AT1 receptor antagonist (losartan), and ganglion blocker (hexamethonium) had no effect on the tyramine TPVR response in either strain. {Delta}TPVR to hexamethonium, prazosin, and L-NAME were greater in SHR than WKY, and hexamethonium reduced {Delta}TPVR to L-NAME in SHR only.

Conclusions: The {alpha}1-adrenoceptor TPVR response to endogenous noradrenaline release was increased in SHR. This was not due to reduced bioavailable NO; on the contrary, NO counteraction was greatly increased, derived from endothelial NOS, with an additional role of neuronal NOS not seen in WKY. An influence of oxidative stress on these responses was not detected in either strain. In addition, a central eNOS sympathoinhibitory component appeared to influence baseline TPVR in SHR.

KEYWORDS Autonomic nervous system; Hypertension; Oxygen radicals; Nitric oxide; Vasoconstriction/dilatation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
A normal blood pressure (BP) is maintained by a homeostatic balance between hypertensive and hypotensive control mechanisms. The sympathetic nervous system plays an important role in maintaining vascular tension, and an adrenergic hyperactivity has been suggested to be the reason for the high BP in essential hypertension in man and in the spontaneous hypertensive rat (SHR) [1–3]. However, hypertension may also result from a deficiency on the hypotensive side of the homeostatic balance. Nitric oxide (NO) is a potent vasodilator, and plays an important antihypertensive role in the BP homeostasis. This is demonstrated by the rise in BP after pharmacological inhibition of NO synthesis [4] and by the high BP in endothelial NO synthase (eNOS) knock-out mice [5]. Reduced endothelial NO synthesis [6,7] and lower levels of eNOS [8,9] have been demonstrated in SHR. However, several other studies have reported on an increased NO synthesis in SHR as well as elevated levels of neuronal (nNOS) and inducible NOS (iNOS) and some studies also of eNOS [10–14]. The functional implication of NOS expression is difficult to predict. The caveolar membrane proteins caveolin-1 and -3 inhibit eNOS activity [15,16], and caveolin-3 also nNOS [17]. Aorta caveolin-1 and -3 have been found to be reduced in SHR [18]. Furthermore, the bioavailability and function of NO may be reduced by other free radicals [19,20]. Vascular superoxide anion (O2.–) will rapidly scavenge NO to form the cytotoxic metabolite peroxynitrate (ONOO–) [21]. Increased oxidative stress and degradation of NO from eNOS has been described in SHR [22–28], and reduction of the concentration of superoxide has been shown to lower BP [29–31]. NAD(P)H oxidase is the main contributor to oxidative stress in the vessel wall and for the exaggerated O2.– production in the SHR [32]. The angiotensin AT1 receptor seems responsible for activation of NAD(P)H oxidase [33] also in the SHR [32,34]. Therefore, due to the complexity of the NO system, a functional approach may be preferable to determine its contribution to the control of BP.

The NO system contributes to the BP homeostasis not only in the basal condition, but also counteracts an adrenergic pressor response, activated for instance by an acute fall in BP [35] or by 4-aminopyridine [36]. The efficiency of the NO component was therefore in the present study investigated by its ability to counteract an adrenergically evoked rise in total peripheral vascular resistance (TPVR), induced by tyramine-activated noradrenaline release from peripheral sympathetic nerve endings. The biological effect of NO synthesis was visualized by pre-treatment with NOS inhibitors. Through this functional approach, the present study aimed to determine if the BP homeostasis in the SHR was dominated by a deficiency in the function of the NO system or an excessive adrenergic component. The results will show an enhanced effect of eNOS and also nNOS in SHR, counteracting an augmented adrenergic tension response. Superoxide anions, NAD(P)H oxidase and angiotensin AT1 receptor did not influence this response.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Preparation of animals
Male WKY (Wistar Kyoto, 311 ± 3 g and 12.8 ± 0.1 weeks, n = 91) and age-matched SHR rats (Okamoto, SHR/NHsd strain, 299 ± 2 g and 12.7 ± 0.1 weeks, n = 129) were included in the experiments as previously described [36]. In short, the rats, fed on conventional rat chow diet (0.7% NaCl), were anaesthetized (65 mg/kg Nembutal, intraperitoneally) and artificially ventilated with air. Cardiac output (CO=minus coronary flow) was measured with a 2SB perivascular flowprobe on the ascending aorta and a T206 Ultrasonic Transit-Time Flowmeter (Transonic Systems Inc., Ithaca, NY, USA), and systolic BP (SBP) and diastolic BP (DBP) through a heparinized catheter in the femoral artery. Tyramine was infused (217 µl/min/kg) through a catheter in the left femoral vein. Other drugs were administered as bolus injections (0.6–1.3 ml/kg) unless otherwise indicated through a catheter in the right femoral vein. All drugs were dissolved in PBS (0.01 M Na-phosphate, pH 7.4, 0.14 M NaCl). Arterial blood (100 µl) was sampled from the arterial catheter at the start of the experiment and at the end of the tyramine-infusion, and PCO2, PO2, pH and acid/base excess (ABE) were measured in an ABL 500 Radiometer (Radiometer Medical, Copenhagen, Denmark). Body temperature was sustained by external heating, guided by a thermosensor inserted inguinally. All experiments conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication NO. 85-23, revised 1996).

2.2. Experimental protocols
WKY and SHR controls were pre-treated with a sham-injection containing PBS (–10 min). They were subsequently infused for 15 min with tyramine (1.26 µmol/min/kg), which has been shown to activate catecholamine release exclusively from peripheral sympathetic nerves and not from the adrenals [37]. To evaluate if tyramine influenced central sympathetic output or if the response to tyramine was influenced by baroreceptor activation following the tyramine-induced rise in BP, rats were pre-treated with nicotinic receptor antagonist (37 µmol/kg hexamethonium chloride, –10 min) to block ganglion transmission. This concentration of hexamethonium abolished the tachycardia recorded 10 min after injection of phentolamine (6.3 µmol/kg), i.e., {Delta}HR=101 ± 10 and –15 ± 11 beats/min in control and hexamethonium-treated conscious WKY, respectively, and 74 ± 9 and –35 ± 11 beats/min in SHR (P<0.001). To determine the influence of NO on the response to tyramine, the pre-treatment with PBS was substituted with either a supramaximal dose [38] of the non-selective NO synthase inhibitor N{varpi}-nitro-L-arginine methyl ester (L-NAME, 1.1 mmol/kg, –30 min), the selective neuronal NOS (nNOS) inhibitor 7-nitroindazole (184 µmol/kg, intraperitoneally, –20 min [39]), or the inducible NOS (iNOS) inhibitor 1400W (N[[3-(aminomethyl)phenyl]methyl]-ethanimidamide dihydrochloride, 40 µmol/kg [40]). L-NAME was also given 10 min after administration of hexamethonium or prazosin (0.24 µmol/kg [41], tested in SHR only) injected 10 min prior to L-NAME. L-NAME was also administered to WKY and SHR after pre-treatment with phentolamine (6.3 µmol/kg, –10 min [41]), but without subsequent administration of tyramine. The response to prazosin was also recorded in WKY without subsequent drug injections. To evaluate the role of O2.– in the response to tyramine, rats were infused with the low molecular weight superoxide dismutase mimetic tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl, 3.7 µmol/min/kg [30]) first for 10 min alone, then dissolved in the tyramine solution. The influence of NAD(P)H oxidase on the tyramine response was evaluated by pre-treatment with the effective and selective NAD(P)H oxidase inhibitor apocynin (acetovanillone; 4-hydroxy-3-methoxy-acetophenone, 200 µmol/kg per os, –30 min [42]). In a separate SHR group, apocynin was administered before L-NAME. The role of angiotensin AT1 receptor activation was studied by pre-treatment with losartan (79 µmol/kg, –10 min). This concentration abolished the pressor response to 48 nmol/kg angiotensin II in anesthetized WKY ({Delta}MBP=89 ± 4 and 7 ± 1 mm Hg before and after losartan, respectively, P = 0.0001).

2.3. Drugs
7-nitroindazole was from Tocris Cookson Ltd., Avonmouth,UK., while the remaining drugs were from Sigma Chemical Co., St. Louis, MO, USA. Losartan was a kind gift from MSD Norge a/s, Drammen, Norway.

2.4. Statistical analysis
Each group consisted of 6–7 rats, except for the SHR tyramine control group which comprised 12 rats. The results are expressed as mean values ± s.e.m. The recorded data were averaged every minute throughout all the experiments. The response to tyramine was evaluated using the maximum response, i.e., after 3–4 min for TPVR and after 15 min for HR, SV and CO. The late tension response was also analyzed using the {Delta}TPVR and {Delta}DBP after 15 min. The response at a particular time was analyzed by one-way ANOVA, and when the presence of significant responses and differences between groups was indicated, these were located by one- and two-sample Student's t-tests, respectively. Similar analyses were used to evaluate the response to pre-treatment. Correlations were determined by the Pearson correlation test. P<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. The effect of pre-treatment on cardiovascular baselines
In the controls, baseline DBP, HR and TPVR were greater in SHR than in WKY whereas CO was less (P ≥ 0.005) (Table 1). The sham pre-treatment with PBS in the controls had little or no effect on these baselines (Table 1). The non-selective NOS inhibitor L-NAME increased baseline TPVR (Fig. 1) and DBP and decreased CO in both strains (P ≥ 0.005 compared to the controls) (Table 1). The increase in TPVR baseline was 4.7 times greater in SHR than in WKY (P = 0.002). Pre-treatment with the ganglion blocker hexamethonium or the adrenoceptor antagonists phentolamine ({alpha}1+2) and prazosin ({alpha}1) lowered baseline DBP and TPVR, all three more in SHR than in WKY (Fig. 1 legend). After hexamethonium, phentolamine and prazosin, the L-NAME-induced increase in TPVR was clearly reduced in SHR (Fig. 1). Hexamethonium had no effect on the TPVR-response to L-NAME in WKY, whereas a minor reduction was seen after phentolamine (Fig. 1). 7-nitroindazole (nNOS inhibitor) and 1400W (iNOS inhibitor) did not alter baselines (P = NS compared to the changes in WKY and SHR controls), whereas the small molecular scavenger tempol caused some reduction in baseline TPVR in SHR (Table 1). The angiotensin AT1 receptor antagonist losartan decreased (P ≥ 0.046) DBP in both strains, and in SHR also HR, CO (data not shown) and TPVR (–1.15 ± 0.36 mm Hg/ml/min). The NAD(P)H oxidase inhibitor apocynin had no significant effect on baselines in either strain (data not shown), and did not significantly alter the response to L-NAME (7.45 ± 1.00 and 4.90 ± 0.96 mm Hg/ml/min in the SHR L-NAME and apocynin+L-NAME groups, respectively, P = NS).


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  		Table 1 DBP, HR, CO and TPVR after pre-treatment, i.e., baselines prior to tyramine, in WKY and SHR

 

Figure 1
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  		Fig. 1 The TPVR-response to L-NAME in WKY and SHR controls and after ganglion blockade by hexamethonium or pre-treatment with the {alpha}-adrenoceptor antagonists phentolamine ({alpha}1+2) or prazosin ({alpha}1). Hexamethonium lowered baseline DBP and TPVR in both strains (Table 1). Phentolamine lowered baseline DBP with –13 ± 2 and –31 ± 4 mm Hg in WKY and SHR, respectively, and TPVR with –0.29 ± 0.09 and –0.81 ± 0.15 mm Hg/ml/min. Prazosin lowered baseline DBP with –12 ± 2 and –27 ± 5 mm Hg and TPVR with –0.45 ± 0.08 and –1.48 ± 0.18 mm Hg/ml/min. The fall in DBP and TPVR was for all drugs greater in SHR than WKY (P ≥ 0.01). The response to L-NAME was not tested in prazosin-treated WKY. {Delta}TPVR to L-NAME was statistically significant in all groups (P ≥ 0.002, one Student's t-tests). *–P<0.05.

 
3.2. The response to tyramine
The tyramine-infusion induced a sharp rise in DBP, followed by a subsequent DBP-plateau phase (Fig. 2). The DBP-response did not differ in the two strains. There was an immediate, but transient rise in TPVR ({Delta}TPVRimm), and a steady rise in SV (not shown), HR and CO, reaching maximum values after 15 min (Fig. 2). {Delta}TPVRimm was 4.5 times greater in SHR (Fig. 3), whereas {Delta}HR and {Delta}CO (after 15 min) were greater in WKY (P ≥ 0.002) (Fig. 4). Ganglion blockade with hexamethonium had no effect on TPVR-response to tyramine in either strain (Figs. 3 and 4Go).


Figure 2
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  		Fig. 2 The changes in DBP, HR, CO and TPVR during the 15-min infusion with tyramine in the WKY and SHR control groups. Strain-related differences in the maximum responses were detected as indicated (*).*–P<0.05.

 

Figure 3
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  		Fig. 3 The maximum change in TPVR during the immediate response to tyramine ({Delta}TPVRimm) and {Delta}DBP recorded at the same time in WKY and SHR. The rats were pre-treated as indicated by the symbol legends. Group comparisons (two-sample Student's t-tests) were made between the WKY and SHR controls, between the control and the experimental groups within each strain, and between the SHR L-NAME and hexamethonium+L-NAME, prazosin+L-NAME or 7-nitroindazole groups. Significant changes were detected as indicated (brackets). {Delta}TPVR and {Delta}DBP were statistically significant in all groups (P ≥ 0.05, one Student's t-tests) except for in the prazosin+L-NAME-treated SHR group (P = NS). *–P<0.05.

 

Figure 4
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  		Fig. 4 {Delta}DBP, {Delta}TPVR, {Delta}HR and {Delta}CO recorded at the end of the tyramine-infusion (after 15 min) in WKY and SHR. The rats were pre-treated as indicated by the symbol legends. Group comparisons (two-sample Student's t-tests) were made between the WKY and SHR controls, between the control and the experimental groups within each strain, and between the SHR L-NAME and hexamethonium+L-NAME, prazosin+L-NAME or 7-nitroindazole groups. Significant changes were detected as indicated (brackets). The response was significant (P<0.05, one-sample Student's t-tests) for {Delta}DBP in all but the SHR prazosin+L-NAME-group, for {Delta}HR in all groups, and also {Delta}CO except for the WKY hexamethonium+L-NAME group, and for {Delta}TPVR in the WKY control, hexamethonium+L-NAME and 1400 W groups, and in the SHR groups given hexamethonium, 7-nitroindazole, or L-NAME as part of the pre-treatment. *–P<0.05.

 
L-NAME increased {Delta}TPVRimm by 5.3 times in WKY (P = 0.016) and 8.6 times in SHR (P = 0.004), respectively (Figs. 3 and 5Go). The increase in {Delta}TPVRimm was followed by an increase in {Delta}DBP in WKY (P = 0.018), whereas there was no difference in SHR (Fig. 3). After L-NAME, TPVR remained elevated throughout the tyramine-infusion in both strains (P = 0.031 and 0.003 in WKY and SHR, respectively, Figs. 4 and 5Go). The TPVR-response to tyramine after L-NAME was not significantly different from that after hexamethonium+L-NAME in either strain (Figs. 3 and 4Go). The immediate rise in DBP and TPVR in SHR were totally abolished when prazosin was given prior to L-NAME (P ≥ 0.018 and 0.003 compared to the control and L-NAME groups, respectively) (Figs. 3 and 5Go). Prazosin also eliminated the L-NAME-induced late increase in TPVR (Figs. 4 and 5Go).


Figure 5
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  		Fig. 5 The changes in DBP and TPVR during the 15-min infusion with tyramine in WKY and SHR pre-treated as indicated by symbol legends.

 
The nNOS inhibitor 7-nitroindazole increased both the early (Figs. 3 and 5Go) and late (Figs. 4 and 5Go) TPVR-response to tyramine in SHR (P ≥ 0.003 compared to the controls) but had no effect on the response in WKY (Figs. 3–5GoGo). The increases in the immediate and late {Delta}TPVR in SHR after 7-nitroindazole were less than that after L-NAME (P ≥ 0.009). The concomitant rise in DBP was not statistically different from that in the SHR controls (Figs. 3 and 4Go).

Neither the iNOS inhibitor 1400W nor the free radical scavenger tempol altered the immediate (Fig. 3) or late (Fig. 4) TPVR-response to tyramine in either strain. Also the angiotensin AT1 receptor antagonist losartan and the NADP oxidase inhibitor apocynin had no effect on the response to tyramine, and the response after pre-treatment with apocynin+L-NAME (tested in SHR only) was not different from that after L-NAME alone (data not shown).

The tyramine-induced tachycardia was in both strains increased after hexamethonium and tempol (P ≥ 0.05) (Fig. 4). L-NAME lowered {Delta}HR in SHR, but hexamethonium still increased {Delta}HR in the presence of L-NAME (P ≥ 0.004) (Fig. 4). {Delta}CO in response to tyramine was in both strains totally eliminated in the groups including L-NAME in the pre-treatment, and in SHR also after the nNOS inhibitor 7-nitroindazole (P ≥ 0.006 compared to the controls) (Fig. 4). Significant correlations between {Delta}HR and {Delta}TPVR at 15 min were not detected in any of the groups.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
The main conclusion from the present results was that the biological activity of NO from eNOS was enhanced in SHR and strongly opposed an excessive {alpha}1-adrenergic TPVR-response during endogenous noradrenaline release. A counter-acting effect due to nNOS activity was also detected in the SHR. The hypotensive effect of NO was not influenced by oxidative stress in either strain. A similar shift in the BP homeostatic balance was also seen to control resting TPVR in the SHR, with the possible addition of a central sympathoinhibitory action of eNOS.

The pressor response to endogenous noradrenaline release during the tyramine-infusion comprised an immediate but transient increase in TPVR. HR, SV and CO increased more slowly and reached its maximum towards the end of the infusion period. The TPVR-response was apparently mediated by peripheral release of neuronal catecholamines and did not involve changes in central sympathetic output, since it was not reduced after acute removal of the adrenals (data not shown) and was not altered by ganglion blockade. This is in agreement with that observed by others [37]. The adrenergic TPVR-response was clearly opposed by NO synthesis in both strains. This was demonstrated by that the immediate, peak TPVR-response was clearly elevated after pre-treatment with the non-selective NOS inhibitor L-NAME. In addition, after L-NAME, the TPVR-response to tyramine was no longer transient but remained elevated throughout the 15-min-infusion period.

The immediate TPVR-response to tyramine in SHR differed from that in WKY by being 4.5 times greater, in spite of the 2.7 times higher TPVR baseline in SHR. This difference could not be explained by a decreased NO-function since L-NAME increased {Delta}TPVRimm by 5 and 8 times in WKY and SHR, respectively. In addition, the increase in TPVR at the end of the 15-min-tyramine infusion after L-NAME was 6.6 times greater in SHR than in WKY. The TPR-response to tyramine after L-NAME represented an {alpha}1-adrenergic vasoconstriction, since administration of prazosin prior to L-NAME totally abolished both the immediate and late TPVR-response to tyramine (tested in SHR only). The NO opposing the adrenergic tension response was exclusively of endothelial origin in WKY, since selective nNOS and iNOS inhibitors had no effect on the tyramine TPVR-response. However, both eNOS and nNOS were apparently involved in SHR, since also nNOS inhibitor but not iNOS inhibitor increased both the immediate and late TPVR-response to tyramine, although much less than L-NAME. The augmented peripheral vasoconstriction throughout the tyramine-infusion after L-NAME in both strains and also nNOS inhibitor in SHR was further reflected by a greater base deficit at the end of the experiment in these groups (data not shown). From these results, it was concluded that the vasodilatory action of eNOS was not impaired in the SHR, on the contrary, it was increased and strongly opposed the {alpha}1-adrenergic vasoconstriction both during the immediate response and during the subsequent return of TPVR to baseline. In addition, nNOS activity was present in SHR and contributed to ameliorate the adrenergic vasoconstriction. The activation of nNOS may have a direct effect on vascular smooth muscles, but may also function to counteract an excessive adrenergic vasoconstriction by hampering the release of noradrenaline, as demonstrated for sympathetic nerves in the rat heart [43]. The augmented TPVR-response to tyramine after NOS inhibitor was not likely to involve an effect of NO on central sympathetic output. This was concluded by that the tyramine TPVR-response was not different after losartan, and the L-NAME-induced changes in the tyramine-response were not altered by ganglion blockade with hexamethonium. Intravenous administration of losartan in a concentration 3 times less than that used in the present study, as well as ganglion blocker, has been shown to inhibit the pressor response to L-NAME injected into the cerebral ventricle [44]. However, the present observations were compatible with previous studies on isolated aortic rings from stroke-prone SHR, where the ability of basal NO production to oppose {alpha}1-adrenergic vasoconstriction was reduced in spite of that the NO-dependant vasodilatory response to acetycholine was increased in SHR [45].

Only L-NAME and not nNOS or iNOS inhibitor was observed to increase baseline DBP and TPVR, and the effect was much greater in SHR than in WKY. Also the fall in TPVR in response to ganglion blockade by hexamethonium and phentolamine ({alpha}1+2) and prazosin ({alpha}1) was much greater in SHR, suggesting an augmented neural, {alpha}1-adrenergic control of vascular tension in the SHR. This conclusion was in agreement with that prazosin enhanced voltage-sensitive K+ channel vasorelaxation in SHR but not WKY [41]. Furthermore, the rise in TPVR baseline in response to L-NAME was markedly reduced after hexamethonium, phentolamine and prazosin in SHR but not at all or only marginally in WKY. The response in SHR remained greater than in the WKY controls. On the other hand, hexamethonium did not influence the effect of L-NAME on the TPVR-response to tyramine, which activated peripheral noradrenaline release. The data therefore suggested that the reduced response to L-NAME after hexamethonium reflected an increased sympathoinhibitory effect of central NOS in SHR, apparently eNOS, since nNOS or iNOS inhibitor did not increase baseline TPVR. This is in agreement with that in stroke prone SHR, overexpression of eNOS in the rostral ventrolateral medulla caused sympathoinhibition [46], and the hypotensive effect of the cholesterol-lowering drug atorvastatin was mediated through an upregulation of central eNOS [47]. The present data therefore appeared to suggest that the function of peripheral eNOS and nNOS and central eNOS activity was upregulated in the SHR, and opposed an excessive, basal {alpha}1-adrenergic control of resting vascular tension and BP.

Current thinking suggests hypertension to result from reduced bioavailable NO due to its reaction with O2.–, resulting in peroxynitrite and reduced vasodilatation [48,49]. The production of O2.– may be stimulated by angiotensin II through activation of the angiotensin AT1 receptor and NAD(P)H oxidase [50,51]. NAD(P)H oxidase-driven oxidative stress has been demonstrated to scavenge endothelial NO and hamper vasorelaxation also in SHR [23,28,32,52]. In addition, administration of heparin-binding superoxide dismutase, xanthine oxidase inhibitor [29] and the low molecular weight, membrane-penetrable superoxide dismutase mimetic tempol [30] lowered BP in SHR. In the present study, although tempol lowered TPVR baseline in SHR but not WKY, it did not alter the TPVR-response to tyramine. The latter was also not influenced by angiotensin AT1 receptor antagonist (losartan) or NAD(P)H oxidase inhibitor (apocynin). Although bioactive NO may be reduced with age [12], the up-regulated biological effect of NO in the early hypertensive stage as in the present 12–14 weeks-old SHR, argued against that a failing vascular vasodilatation, due to a reduced NO bioavailability, was the underlying cause of the high BP and TPVR in these animals. This conclusion was in agreement with previous studies showing increased urinary excretion and plasma concentration of NO metabolites as well as kidney and aorta eNOS and iNOS proteins in prehypertensive (3 weeks) and early hypertensive (8–12 weeks) SHR [11]. However, both angiotensin II and oxidative stress may influence BP through other mechanisms than by lowering NO bioavailability. Presynaptic angiotensin AT1 receptors may stimulate the release of noradrenaline [53], and tempol, but not apocynin, has been shown to have a direct inhibitory effect on renal sympathetic nerve activity [42,54]. Angiotensin II, NAD(P)H oxidase, oxidative stress and increased sympathetic nerve activity may also increase TPVR by their stimulatory effect on vascular smooth muscle hypertrophy, deposition of collagen and vascular remodeling [55,56]. Such structural changes have been described also in the pre-hypertensive SHR [57], and these changes, as well as the development of hypertension, were totally prevented by neonatal sympathectomy [58]. NAD(P)H oxidase-derived O2.– was observed to be stimulated during chronic angiotensin II-, but not noradrenaline-infusion [33]. The role of O2.– and excessive sympathetic activity may therefore be different in different types of hypertension, and possibly also in different stages of the disease.

Baseline HR was higher in SHR than in WKY. However, this may be due to an elevated basal sympathetic tone in the SHR, since hexamethonium lowered baseline HR in SHR but not WKY. The elevated baseline may also be the reason for the lower HR-response to tyramine in the SHR. Both eNOS and nNOS contribute to cardiac function and the cardiac response to sympathetic nerve stimulation [59]. The present results detected an effect of eNOS in the HR-response to tyramine only in the SHR. However, nNOS and possibly also eNOS appeared to be involved in the tyramine-induced rise in CO in SHR. This was concluded since L-NAME and nNOS inhibitor completely eliminated {Delta}CO in SHR. In WKY, {Delta}CO was eliminated after L-NAME but not the other NOS inhibitors, suggesting a role of eNOS only. These differences in {Delta}CO were not paralleled by changes in {Delta}TPVR, and are therefore not likely to be due to deficiencies in cardiac blood flow.

In conclusion, the biological activity of NO from eNOS and nNOS in opposing the {alpha}1-adrenergic TPVR-response during endogenous noradrenaline release was greatly increased in SHR. Furthermore, neither tempol, apocynin nor losartan influenced the TPVR-response to tyramine in either strain. These results indicated that angiotensin II-NAD(P)H oxidase induced superoxide anion production did not hamper the ability of NO to oppose the rise in TPVR during endogenous noradrenaline release in SHR. The augmented TPVR-response to tyramine in SHR was therefore likely to reflect an excessive response to adrenergic stimulation rather than a reduction in the bioavailability of NO and NO-dependent vasorelaxation. A similar pattern was seen for the baseline TPVR, an increased role of {alpha}1-adrenergic control and an augmented eNOS counteraction in SHR. In addition, the data suggested an upregulated sympathoinhibitory action of central eNOS in SHR. An augmented {alpha}1-adrenergic response and not a reduced NO vasodilatory function may therefore be responsible for the high TPVR in SHR.


   Acknowledgement
 
I am grateful for the support from The Norwegian Council on Cardiovascular Diseases.


   Notes
 
Time for primary review 33 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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