(Hypertension. 1997;29:53.)
© 1997 American Heart Association, Inc.
Research Articles (Issue 1, Part 1) |
Amplification of Kinin-Induced Hypotension by Nitric Oxide Synthesis in Spontaneously Hypertensive Rats
the Department of Physiology, Institute of Basic Medical Sciences, University of Oslo (Norway).
Correspondence to Torill Berg, Institute of Physiology, Box 1103, Blindern, 0317 Oslo 3, Norway.
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Abstract |
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We studied the role of nitric oxide and adrenergic activation in the blood pressure (BP) response to exogenous bradykinin in spontaneously hypertensive rats (SHR) compared with normotensive Wistar-Kyoto rats (WKY). Rats were pretreated with the nitric oxide synthase inhibitor N
-nitro-L-arginine methyl ester (L-NAME), the 
-adrenergic receptor antagonist phentolamine together with L-NAME, or phentolamine alone. Sham-injected rats were used as controls. All rats subsequently received bradykinin (3, 6, and 30 µg/kg IV). Bradykinin induced a concentration-dependent fall in BP in both WKY and SHR (P<.0005). The change in BP was greater in SHR than WKY (P<.0001). BP before bradykinin administration was elevated in the L-NAME group in both strains. In WKY, L-NAME or L-NAME plus phentolamine did not alter the 
BP concentration-response curve to bradykinin (P=NS), whereas in SHR, the BP concentration-response curve was attenuated (P<.0048). The attenuation was observed for the two lower bradykinin doses (P<.0005) but not the highest. In SHR, phentolamine alone reduced BP before bradykinin to the same level as in WKY controls, and its BP concentration-response curve was not different from that of the normotensive controls or L-NAME and L-NAME plus phentolamine SHR groups. No difference was observed in the duration of the hypotensive response in SHR compared with WKY. The present results confirm that in normotensive rats, the hypotensive effect of bradykinin was mediated by an unknown mechanism other than through the release of nitric oxide. However, in SHR, this mechanism was amplified by additional activation of nitric oxide synthesis. This bradykinin-activated nitric oxide production may be a pressure-induced mechanism to counteract the hypertensive condition.
Key Words: bradykinin • endothelium-derived relaxing factor • nitric oxide • blood pressure • rats, inbred SHR
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Introduction |
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Bradykinin is a potent vasodilator peptide that induces hypotension when injected intravenously. A role for bradykinin in BP control has been suggested from studies in normotensive men1 2 and hypertensive rats.3 4 5 NO has been suggested to act as a mediator in bradykinin-induced hypotension. Several in vitro studies document a role of NO as a mediator for bradykinin,6 7 and a shortened hypotensive response has been shown in normotensive rats after inhibition of NO synthase.8 However, we have recently shown that this shortened response could be explained by the fact that NO synthase inhibition eliminates the continuous hypotensive effect of the NO system and thus no longer hampers the compensatory adrenergic response after the acute fall in BP, resulting in a shorter duration of the hypotension.9 We therefore concluded that NO did not play a role in the hypotensive effect of bradykinin in normotensive rats and that the effect of bradykinin is mediated through another, yet unknown, system.
However, in hypertensive rats, an attenuation in the hypotensive response to converting enzyme (kininase II) inhibition was observed after NO synthase inhibition as well as after bradykinin antagonist.5 Although a similar reduction was seen also after angiotensin II receptor blockade, the possibility cannot be excluded that the response to bradykinin in hypertension may differ from that in normotensive animals. The vasodilator response to bradykinin depends on endothelial cell activity.10 Endothelial cells are of major importance in determining the balance between hypertensive and hypotensive systems, and in hypertension, endothelial cell function may be altered or impaired.11 12 The mediators normally mediating a response to vasoactive agents may therefore be altered, and the balance between systems working in opposite directions may be changed. In the present study, we therefore wanted to study the role of NO synthesis and adrenergic activation in the hypotensive effect of bradykinin in SHR compared with normotensive rats (WKY). The results show that in SHR, unlike in WKY, part of the hypotensive effect of bradykinin was mediated through the NO system.
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Methods |
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Male WKY and age-matched SHR (13 to 15 weeks) were anesthetized with pentobarbital (70 mg/kg IP) and tracheotomized. All drugs were dissolved in PBS (0.01 mol/L sodium phosphate [pH 7.4], 0.14 mol/L NaCl) and administered as bolus injections through a catheter in the femoral vein (0.6 mL/kg, 20 seconds). The catheter was flushed with 0.1 mL PBS after each injection. Arterial BP was recorded throughout the experiment by a strain-gauge transducer (Statham) and recorder (Hewlett-Packard 7402 A) connected to a heparin-treated catheter in the femoral artery. As a measure of the duration of the hypotensive response to bradykinin, the time when BP had recovered three quarters of the maximum fall was recorded.
The rats were divided into four groups (n=6 per group) of WKY and SHR and given the following treatments: The control groups, WKY1 and SHR1, received two sham injections of PBS 5 minutes apart; WKY2 and SHR2 received PBS and L-NAME (0.3 g/kg) at the same time intervals; WKY3 and SHR3 received the -adrenergic receptor antagonist phentolamine (2 mg/kg) followed by L-NAME (0.3 g/kg); and WKY4 and SHR4 received phentolamine and PBS. Fifteen minutes later, all rats were given bradykinin (3, 6, and 30 µg/kg) as consecutive injections administered 5 minutes apart.
Bradykinin acetate salt and L-NAME were obtained from Sigma Chemical Co. Phentolamine (Regitin) was from CIBA-Geigy and heparin from Nycomed. Pentobarbital sodium was purchased from The National Hospital, Oslo, Norway.
Results are expressed as mean±SE. A concentration-response relationship was tested by two-way ANOVA. For BP and BP concentration-response curves, group differences and interactions between groups over dose were tested by one-way ANOVA and ANCOVA with repeated measures (BMDP) first as an overall test including all groups of WKY or SHR, respectively (P value limit, .05), and subsequently, when differences or interactions were observed within the groups, between all groups to locate differences (P value limit, .0083 after Bonferroni adjustment). When differences or an interaction between the control group and an experimental group was observed, two-sample Student's t tests were used to locate differences at a particular dose (P value limit, .017 after Bonferroni adjustment). Group differences between the control and the three experimental groups in the duration of the hypotensive response and differences between the response in WKY compared with SHR were tested similarly. To determine differences in BP at the start of the experiment and at the time before bradykinin injection, we used two-sample Student's t tests for intergroup comparisons within the WKY and SHR, respectively (P value limit, .017 after Bonferroni adjustment). For intragroup comparisons of the changes in BP from before pretreatment to BP after bradykinin administration, one-sample t tests were applied (P value limit, .05).
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Results |
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At the start of the experiment, basal BP of WKY (all rats) was 83±3 mm Hg, which was significantly lower than in SHR (148±4 mm Hg, P<.0001). Basal BP within the four groups of SHR or WKY did not differ significantly (Table). Phentolamine administration reduced basal BP, whereas L-NAME induced an increase in BP in both strains. The resulting BP, ie, BP before bradykinin injection, is shown in the Table. BP before bradykinin administration was higher in the corresponding SHR than in the WKY (P<.0007), except for the phentolamine group (P=NS) (Table, Fig 1). In this group (SHR4), BP before bradykinin was not significantly different from that of the WKY control group (WKY1) (P=NS).
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Bradykinin administration induced an acute dose-dependent fall in BP in both WKY and SHR (WKY1 and SHR1, P<.0005) (Fig 1). The fall in BP was greater in the SHR1 than the WKY1 (P<.0001) (Fig 1).
In WKY, a small but significant group difference (P<.0012) and interaction (P<.0001) were observed for the absolute BP dose-response curve between the control (WKY1) and phentolamine+L-NAME (WKY3) groups as well as the phentolamine (WKY4) group but not the L-NAME group (WKY2) (P=NS) (Fig 1). However, BP in response to bradykinin did not differ between the control group and the three experimental WKY groups (P=NS) (Fig 1).
The duration of the hypotensive response in WKY (Fig 2) decreased in L-NAME–treated rats (P<.0015), but this shorter response was abolished by addition of phentolamine (P=NS, WKY1 compared with WKY3). Phentolamine alone (WKY4) resulted in a prolongation of the hypotensive response compared with the control group (P<.0001).
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In SHR, on the other hand, differences (P<.0005) as well as significant interactions (P<.0005) were observed in the concentration-response curves to bradykinin between the control group and all three experimental groups, with a higher BP in the L-NAME (SHR2) and L-NAME+phentolamine (SHR3) groups and a lower BP in the phentolamine group (SHR4) (Fig 1). The difference between SHR1 and SHR3 was evident for the two lowest bradykinin concentrations (P<.0005), whereas no difference was seen for BP before bradykinin injection or for the highest bradykinin concentration (P=NS). Furthermore, the BP concentration-response curve in the L-NAME group (SHR2) paralleled that of the phentolamine+L-NAME group (SHR3) (no interaction, P=NS) but at a higher BP level (P<.0001). Thus, BP in response to bradykinin was found to be the same for the L-NAME and phentolamine+L-NAME groups but was clearly less than that in the control group (P<.011 and P<.0016, respectively) for the two low bradykinin concentrations (P<.010) but not for the highest bradykinin concentration (P=NS) (Fig 1). The BP concentration-response curve of the phentolamine group (SHR4) paralleled (no interaction, P=NS) that of the control group (SHR1) but with smaller changes in BP (P<.0036). For the BP concentration-response curve in the SHR4 group, group differences were not observed compared with the L-NAME (SHR2) or L-NAME+phentolamine (SHR3) groups (P=NS). Although an interaction was detected, the differences when tested with two-sample t tests for the three bradykinin doses were not significant. Furthermore, no difference or interaction was detected when the SHR4 group was compared with the WKY control group (WKY1) (P=NS), whereas between SHR2 (SHR L-NAME–treated group) and WKY1, an interaction (P<.0001) but no group difference (P=NS) was observed. For the latter comparison, the difference was evident only for the highest dose of bradykinin (P<.0066).
The pattern for the duration of the hypotensive response among the four SHR groups was found to be the same as for the WKY groups (Fig 2). No differences were observed between corresponding WKY and SHR groups (P=NS).
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Discussion |
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NO has been suggested as a mediator for bradykinin-induced hypotension. Studies on cultured cells6 13 14 15 or isolated organs7 clearly show the ability of bradykinin to activate NO synthesis. However, in a previous study,9 we found that NO synthase inhibition did not attenuate the acute fall in BP after intravenous administration of bradykinin in normotensive rats. Although we, like others,8 16 observed the hypotensive response to be shortened, this shortened response was found to be adrenergically dependent and thus did not indicate a role of NO as a mediator for bradykinin. Since arteriole wall tension, and thus arterial BP, is the result of several vectors working in the hypertensive or hypotensive direction, removal of one system will cause BP to change as a result of the remaining forces. When the continuous production of NO is blocked, the counteracting effect of NO on both an acute compensatory and a continuous adrenergic hypertensive force will be prevented, and the return in BP after acute hypotension will be faster than when NO production is present. In the present study, the same results were obtained with normotensive rats. However, in SHR, NO was found to partly mediate the hypotensive response since L-NAME administration reduced the acute hypotensive response to bradykinin. The pattern for the duration of the hypotensive response between the SHR groups was similar to that of the WKY groups, as described above, with no differences between corresponding WKY and SHR groups, thus further confirming that the difference in the acute fall in BP between the WKY and SHR groups was a result of an altered response to bradykinin and not of alterations in mechanisms compensating for the acute fall in BP. The NO-dependent bradykinin response in SHR could also not be explained by the elevated BP after L-NAME, because after reduction of BP by additional administration of the
1+2-adrenergic receptor blocker phentolamine, the same BP dose-response curve as for the L-NAME group was observed. Although BP in the L-NAME group was higher than that of the phentolamine+L-NAME group for all doses, no interaction was observed between these two groups, whereas an interaction was seen between these two groups and the control group. Therefore, it seems that in normotensive rats, the hypotensive response to bradykinin was exclusively mediated through a mechanism other than NO release. On the other hand, in hypertensive rats, this mechanism as well as NO appeared to mediate the response for the two lower bradykinin doses. The "normotensive" mechanism appeared to function at the same level as that in normotensive rats, since BP in L-NAME–treated SHR was for these two doses not different from that of WKY controls. In phentolamine-treated SHR, BP before bradykinin was not different from that of normotensive controls, and their BP concentration-response curves were not different and no interaction was observed. Moreover, the fall in BP in the SHR phentolamine group was not significantly different from that of the SHR L-NAME or L-NAME+phentolamine groups. These results may indicate that the activated NO responsiveness in SHR was induced by the high BP. A pressure-induced activation of NO in addition to the "normotensive" mechanism may therefore represent a way to amplify the response to bradykinin of high BP and may also explain the increased sensitivity to bradykinin in SHR compared with WKY. This seems physiologically favorable as an attempt to oppose a hypertensive condition. After the highest dose of bradykinin, NO did not participate significantly in the hypotensive response. Still, the fall in BP was greater than in normotensive controls. It is therefore possible that after a dose that may have reached pharmacological levels, yet other mediator mechanisms may contribute to the hypotensive response.
The nature of the mechanism mediating bradykinin-induced hypotension in normotensive rats is not yet known. Prostaglandins do not appear to be a likely candidate, at least in normotensive rats, because the hypotensive response to bradykinin in normotensive rats as observed by Rees et al8 in the presence of indomethacin was similar to that observed by us. However, the possibility cannot be excluded that prostaglandins were a contributor after the high dose of bradykinin in hypertensive rats. Another candidate for the "normotensive" mechanism may be endothelium-derived hyperpolarizing factor because a mediator role for endothelium-derived hyperpolarizing factor in the response to bradykinin has been suggested.17
NO synthase inhibitors have been shown to attenuate the vasodilator action of bradykinin.18 19 20 Although the present results clearly opposed the concept of NO as a mediator in a bradykinin-induced overall hypotension in normotensive rats, this does not contradict a role of NO in mediating local hemodynamic effects induced by bradykinin since vasodilatation locally may not necessarily result in changes in BP.
Endogenous formation of bradykinin has been suggested to play a role in BP homeostasis, particularly in the hypertensive condition. Kinins have been shown to participate in the hypotensive response to converting enzyme inhibition in both normotensive humans1 and hypertensive rats,3 4 5 21 and bradykinin antagonists potentiated hypertension induced by mineralocorticoids and salt in uninephrectomized rats.22 Moreover, several studies on cultured endothelial cells indicate that endogenous bradykinin production is present in these cells.15 23 We have recently shown by the use of converting enzyme inhibition in nephrectomized rats21 that endogenous production of bradykinin is present in SHR but not WKY. It therefore seems that bradykinin production and mechanisms to amplify its response are present in SHR. However, bradykinin antagonists did not have any effect on basal BP in either awake or anesthetized nephrectomized SHR.21 Still, the possibility cannot be excluded that these amplifications of the bradykinin system play a role in counteracting the hypertensive condition.
In nephrectomized SHR, we have previously shown21 that L-NAME did not significantly attenuate the hypotensive response to converting enzyme inhibition, although a tendency was observed. This observation may seem to contradict the present results but is nonetheless in accordance with a pressure-dependent activation of the bradykinin-NO amplification, since basal BP in anesthetized, nephrectomized SHR is not different from that of anesthetized, non-nephrectomized WKY.21
The present study may also offer some explanation regarding the discrepancies between the frequently observed bradykinin-induced NO production in in vitro studies6 7 13 14 15 and our previous9 and present in vivo studies showing a failure of L-NAME to attenuate bradykinin-induced hypotension in normotensive rats. The vasodilator response to bradykinin depends on endothelial cell activity,10 and endothelial function is altered in hypertension.11 12 It is possible that endothelial cells in in vitro systems may have undergone changes similar to that occurring in hypertension.
Measuring BP changes in anesthetized rats should perhaps be interpreted with some caution because anesthesia is known to influence sympathetic nervous activity. In the present study, we observed a higher level of sympathetic activity in SHR than in WKY as assessed by differences in response to phentolamine. However, blockade of basal sympathetic or NO synthase activity has in normotensive rats been shown to alter the duration of the hypotensive response through differences in compensatory activity but not the acute fall in BP.9 Since the pattern for the duration of the hypotensive response to bradykinin was found to be similar in the two strains, it does not seem likely that the anesthesia had any influence on the presently observed differences in BP in response to bradykinin between the two strains. This would most probably be true also for other vasoactive systems that may be altered by anesthesia, such as the renin-angiotensin system.
In conclusion, the present study confirms that in normotensive rats, the hypotensive effect of bradykinin was mediated by an unknown mechanism other than through the release of NO. However, in SHR, this mechanism was amplified by additional activation of NO synthesis. This bradykinin-activated NO production may be a pressure-induced mechanism to counteract the hypertensive condition.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by The Norwegian Research Council and The Norwegian Council on Cardiovascular Diseases.
Received March 11, 1996; first decision July 23, 1996; accepted July 23, 1996.
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