Vascular smooth muscle cell contraction and endothelium-dependent relaxation was evaluated in aortic rings isolated from weaned, 5-mo-old Sprague-Dawley rats fed a normal (NS; 0.8% NaCl) or high (HS; 8% NaCl) sodium diet. Arterial pressure was 140 ± 6 (NS) and 145 ± 6 mmHg (HS). In endothelium-denuded rings, the response to phenylephrine (PE) was not modified by the sodium diet, while that of depolarizing agent KCl and intracellular calcium releasing agent caffeine increased in the HS group. When endothelium was preserved, PE-evoked contraction was reduced in both NS and HS groups, the contraction being yet lower in the HS group. This effect was partially obliterated by addition of NG-nitro-l-arginine methyl ester (l-NAME), independently of the sodium diet. Relaxation to ACh in intact rings and to sodium nitroprusside (SNP) and 8-bromoadenosine 3′5′ cyclic guanosine monophosphate (8-BrcGMP) in the absence of endothelium was enhanced in rings isolated from HS rats. In addition, the dose-response curve to 8-BrcGMP was shifted to the right in the presence of iberiotoxin, an inhibitor of large conductance, voltage-dependent, and calcium-sensitive potassium channel (BKCa). However, shift was more marked in rings from HS rats. Present results provide evidence that response of vascular smooth muscle cell to nitric oxide/cGMP-related compounds is increased in HS rings and is associated with a greater activation of the repolarizing BKCa channels. Such changes might counterbalance enhanced contractile response to membrane depolarization and thus participate in maintenance of arterial pressure in the present model of early and long-term HS feeding in rats.
- vascular smooth muscle cell
- endothelial cell
- sodium nitroprusside
excessive consumption of sodium is reported to induce cardiac, vascular, and renal alterations in normotensive and hypertensive humans and animals (9, 30). In various strains of rat, a high salt (HS) diet is associated with changes in the reactivity of aortic rings. Many studies conducted in salt-loaded models have shown an increased contraction of vascular smooth muscle cells in response to agonists (1, 14). In addition, relaxation evoked by acetylcholine (ACh) or sodium nitroprusside (SNP) was obliterated in aortic rings isolated from adult spontaneously hypertensive and Dalh-sensitive rats fed a 8% NaCl diet for 4–8 wk (12, 14, 16). These results suggest that concomitant increase in vascular smooth muscle cell contraction and impaired endothelium-dependent relaxation both participate in the development of hypertension in the salt-sensitive strains. In contrast, an enhanced influence of nitric oxide (NO) may have a major role in the lack of rise in blood pressure of sodium-resistant rats when fed a HS diet. In the Dahl-resistant strain (16) and more clearly in the Sprague-Dawley rats (18), NG-nitro-l-arginine methyl ester (l-NAME) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a guanylate cyclase inhibitor, enhanced phenylephrine (PE) contraction, and to a greater extent in rats fed an HS than a normal salt (NS) diet. Taken together, these results favor an adaptive response of both the endothelium and the vascular smooth muscle cells to chronic HS intake in adult rats of resistant strains.
Among the mechanisms implicated in endothelial and vascular smooth muscle cell adaptation to HS, the NO pathway might play a key role, as suggested by the enhanced expression of endothelial NO synthase (eNOS) in glomeruli and aorta of Sprague-Dawley rats fed 8% NaCl diet for 4–7 days (28, 29). NO induces activation of the guanylate cyclase and production of cyclic guanosine monophosphate (cGMP), which elicits relaxation through various mechanisms. The stable cGMP analog, 8-bromoadenosine 3′5′ cGMP (8-BrcGMP) was reported to activate the myosin light chain phosphatase of smooth muscle strips isolated from the rabbit femoral artery (13, 27). In feline aorta strips, the relaxant effect of nitroprusside and 8-BrcGMP partly occurred via calcium uptake by the endoplasmic reticulum and involves sarcoplasmic reticulum calcium-ATPase (SERCA) (15). cGMP relaxes vascular smooth muscle cells also via hyperpolarization, resulting from activation of cGMP-dependent protein kinase (PKG) and the large-conductance voltage-gated, calcium-sensitive potassium channel (BKCa) (2). In addition, the BKCa channel is highly expressed in the vascular smooth muscle cell (26) and appears to be involved in the vasorelaxing response to ACh in rat aortic rings (19).
In a previous study, feeding weanling rats with an 8% NaCl diet for 4 mo was devoid of major effect on arterial pressure, and it was accompanied by a cardiac and carotid artery hypertrophy (7). Facing the salt loading, change in vascular reactivity may be one of the factors involved in the lack of hypertension observed in the latter model. Therefore, the current investigation was designed to assess vascular smooth muscle cell reactivity to agonists as well as to NO/cGMP related compounds during HS intake. Among the targets of cGMP that can participate in the putative greater influence of the NO pathway in HS-fed rats, the contribution of BKCa channel was evaluated acutely using its pharmacological blockade with iberiotoxin.
Fifty weanling male Sprague-Dawley rats were randomly assigned to two groups of 25 animals and fed NS or HS diet containing 0.8 or 8% NaCl, respectively, as previously described (7). After 18–20 wk i.e., at 5 mo of age, arterial pressure was measured in the carotid artery of the anesthetized animal (pentobarbital sodium, 50 mg/kg body wt ip; Sanofi), and rats were prepared for the aortic reactivity study.
Aortic Rings Preparation
The thoracic aorta was carefully excised and placed in a physiological solution of the following composition (in mM): 122 NaCl, 5 KCl, 1 MgCl2, 0.5 KH2PO4, 0.5 NaH2PO4, 10 HEPES, 1 NaHCO3, 1.8 CaCl2, and 11 glucose; pH was adjusted to 7.4. The aorta was cleaned of superficial fat and connective tissue and rings of ∼3 mm in length were mounted between two triangular stainless steel hooks and placed into an organ bath containing 20 ml of the medium described above. The medium was maintained at 37°C and continuously gassed with oxygen. Isometric tension was measured by an isometric force transducer (EMKA Instruments), connected to a computer system, and recorded by IOX 7.0 software. After an equilibration period of 1 h at a resting tension of 2 g, functional integrity of the rings was tested by the response to a single dose of PE and ACh (10−5 and 10−6 M, respectively). At this dose of PE, the contraction level was similar in the two diet groups. At least 6–8 rings from each rat of the two diet groups were isolated and kept intact or mechanically deendothelialized. In six rats per regimen group, one ring was immediately fixed in 4% formaldehyde for the histological study.
All procedures were in accordance with French law and conform to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (publication No. 85-23, revised 1996).
Aortic Rings Reactivity
VASCULAR SMOOTH MUSCLE CELL RESPONSE.
The contractile capacity of vascular smooth muscle cells was evaluated through the dose response to three different agonists of deendothelialized rings. In a first set of rings, the α-adrenergic pathway was assessed via the contraction evoked by PE (10−10 to 10−5 M). In the second set of rings, the response to calcium channels activation was determined by direct depolarization of the cell membrane with KCl (10−3 to 10−1 M), while mobilization of intracellular calcium store was evaluated using caffeine (10−4 to 10−2 M).
INFLUENCE OF ENDOTHELIUM ON CONTRACTILE RESPONSE.
The dose response to PE was also determined in aortic rings with preserved endothelium, as well as in intact rings preincubated (30 min) with the NO synthase inhibitor l-NAME (10−4 M).
RINGS WITH A PRESERVED ENDOTHELIUM.
The dose response to ACh (10−9 to 10−6 M) was determined in rings precontracted by 10−5 M of PE. Both PE and ACh responses were repeated 30 min after addition of l-NAME (10−4 M) in the bath.
RINGS WITHOUT ENDOTHELIUM.
The vascular smooth muscle cell sensitivity to NO pathway was assessed by the dose response to the NO donor SNP (10−10 to 10−6 M) and the cGMP mimetic 8-BrcGMP (10−6 to 10−4 M) in two different sets of endothelium-denuded rings precontracted with 10−5 M PE. In the last set of experiments, the dose response to 8-BrcGMP was determined in the presence of 10−7 M iberiotoxin, a BKCa channel inhibitor, added to the incubation bath.
Rings fixed in 4% formaldehyde solution were embedded in paraffin. Three 6-μm-thick slices per ring were stained with Sirius Red and Fast Green, and the cross-sectional area (CSA; in mm2) of the media was measured on each section using Image J software (National Institutes of Health, Bethesda, MD). The three measurements were averaged to give a single value of CSA per rat.
Drugs and Data Analysis
All compounds were purchased from Sigma. The doses used in the present study are in the range of those reported by others in the evaluation of vascular reactivity (3, 8, 11, 25). The dose of iberiotoxin chosen was reported to reduce the maximal relaxation induced by an NO-producing drug in aortic rings of normotensive Wistar rats (4).
Data are means ± SE of at least 10 measurements for each dose. The response to PE, KCl, and caffeine was expressed as a change in the basal tension (contraction in g). To minimize the impact of variations in the precontraction level to PE due to the presence or not of endothelium, the response to ACh, SNP, and 8-BrcGMP was expressed as the percentage of the contraction to PE achieved for each ring.
The concentration of drug causing EC50 was determined from the concentration response curve for each ring by using the best fit to a logistic sigmoid function and was averaged to give one value per experimental group.
Data were analyzed with one- or two-way analysis of variance when required. Difference between groups was then assessed with the Fisher least significant difference test. P values <0.05 were considered significant.
Rats fed the NS or HS diet had similar final body weight (513 ± 10 and 499 ± 8 g body wt, respectively) and mean arterial pressure (140 ± 6 and 145 ± 6 mmHg, respectively). No statistical difference between aortic CSA of the NS and HS groups was observed (CSA value of 0.989 ± 0.058 and 0.897 ± 0.058 mm2, respectively).
Vascular smooth muscle cell response.
The influence of sodium diet on the response of deendothelialized rings to various constrictor agents is depicted in Fig. 1. The maximal isometric tension developed in response to PE was similar in the HS and NS rings (Fig. 1A). A clear and significant increase in the response to each dose of KCl (Fig. 1B) or caffeine (Fig. 1C) was observed in rings from the HS group. Of note, the EC50 for PE, KCl, and caffeine in endothelium-denuded rings was not modified by the sodium diet (Table 1).
Influence of endothelium on contractile response.
Isometric tension developed in response to PE was greatly reduced in both sodium regimens when the endothelium was preserved (Fig. 2). Although significance was not achieved, the maximal isometric tension was lower, and the EC50 of PE was higher in intact rings isolated from HS than NS rats (Table 1). When intact rings were preincubated with l-NAME, the isometric tension developed in response to the higher doses of PE was restored to that observed in the absence of endothelium in both regimens (Fig. 2).
Rings with a preserved endothelium.
Intact rings from HS diet rats presented a greater relaxation and sensitivity to ACh characterized by a significantly lower remaining contraction to PE and a smaller EC50. Pretreatment with l-NAME totally abolished the ACh-induced relaxation in the two groups of diet (Fig. 3, Table 1).
Rings without endothelium.
On deendothelialized rings, relaxation induced by SNP was more marked in the HS than in the NS group, and the EC50 was lowered in rings isolated from HS rats (Fig. 3, Table 1). Similarly, the contraction remaining after addition of 8-BrcGMP and the EC50 were significantly reduced in denuded rings of the HS group (Fig. 4, Table 1). Addition of iberiotoxin to the incubation bath obliterated the relaxation induced by 8-BrcGMP in the HS and the NS group (Fig. 4). However, the rise in EC50 was more marked in the HS group with approximately a threefold increase compared with the EC50 obtained in the NS group (Table 1).
The main results of the present study are as follows: 1) the vascular reactivity to PE is lower in intact rings of HS rats and the difference vanishes in the absence of endothelium; 2) the vascular smooth muscle cell reactivity to KCl and caffeine is greater in HS rings; and 3) NO-cGMP dependent relaxation is enhanced in HS rings with a greater activation of the repolarizing BKCa channels.
The present study indicates that vascular reactivity to the α-agonist PE equated with the EC50 was reduced in endothelium-intact rings isolated from rats fed the HS diet. This finding is in agreement with the reduced precontraction induced by 10−7 M of noradrenaline in aortic rings (17) and the lack of difference in PE-evoked contraction of aortic strips from control and salt-loaded rats (10). Various factors may be involved in the decrease responsiveness to PE in HS rings, including change in arterial pressure and vascular hypertrophy. In the current study, HS diet caused no significant increase in blood pressure. This observation confirms our previous report (7) and is in agreement with observations made with similar (22) or a shorter (10) period of HS feeding. In addition, no aortic hypertrophy was detected in the present HS rats, as previously shown on the carotid artery of a 4-mo-old rat on 2% NaCl diet for 6 mo (25). It is therefore suggested that the reduction of PE response observed in intact rings from HS rats is not related to changes in hemodynamics or structure of the aorta.
Interestingly, PE-evoked contraction was largely increased in the two groups of regimen when the endothelium was removed, and the difference between normal and HS groups vanished as reported by Payne et al. (18). In addition, the response of denuded rings to KCl, which induces voltage-activated calcium entry from the extracellular space, and to caffeine, which mobilizes calcium from the sarcoplasmic reticulum, was markedly higher in the HS than in NS group. An increased sensitivity of the vascular smooth muscle cell to vasoconstrictors, such as noradrenaline, was also previously described in rats fed a HS diet (1). It was also reported that vascular reactivity to PE and KCl is greater in endothelin-infused rats, particularly when fed a 8% NaCl diet during 9 days, and this was associated with increases in calcium entry from the extracellular space (23). Altogether, these results are in favor of an enhanced contractile response of the vascular smooth muscle cell, partly through an increase in calcium stores and activity of voltage-activated calcium channels. The reduced response to PE observed in the present intact HS rings is therefore probably related to functional modification of the endothelium rather than alteration of vascular smooth muscle cell reactivity to constrictors.
In the present experiment, blockade of NO generation by l-NAME in intact rings was associated with a marked enhancement of PE contraction to a level comparable to that observed in the absence of endothelium. In addition, ACh-induced relaxation of intact rings precontracted by PE was higher in the HS than the NS group and was totally abolished by l-NAME in both groups. Although other factor(s), including the endothelium-derived hyperpolarizing factor (24), may participate in the endothelial response of HS rings, the NO/cGMP pathway seems to have a pivotal role in the reduced response to PE of intact HS rings. The higher mRNA and protein levels of eNOS in the arterial wall and the greater basal nitrite/nitrate production by aortic strips reported in rats fed an 8% NaCl diet for 7 days (29) or 9 wk (18) is consistent with such a role. At variance with the present observation, unaffected ACh-induced relaxation (10) and lesser response to methacholine (31) was reported in aortic rings of adult rats fed an HS diet for a short period of time (7 and 3 days, respectively). In the latter study, it was suggested that an HS diet impairs endothelium-dependent relaxation via reduced NO levels. Although changes in NO production cannot be excluded, the greater response to ACh of the present HS rings suggests that compensatory mechanisms developed when the HS diet is proposed since weaning and for a longer period (22 wk) of time.
Besides involvement of the endothelium, an enhanced sensitivity of the vascular smooth muscle cell to NO-related compounds may participate in both the lower response to PE and the higher relaxation evoked by ACh in the present HS- intact rings. The greater relaxation induced by SNP and 8-BrcGMP, as well as their lower EC50 in the denuded HS rings, favors a higher sensitivity of the vascular smooth muscle cells of HS rats to NO-related compounds and suggests that events occurring after cGMP formation are involved. In addition to SERCA activation (15) and actin-myosin complex dissociation (13, 27) by cGMP, the enhanced relaxation to 8-BrcGMP observed in the current HS rings may be also related to changes in repolarization through the large-conductance, voltage-dependent, and BKCa. Highly expressed in the vascular smooth muscle cell (26), BKCa is involved in the relaxation to ACh and NO donor (4, 5, 6, 19), and cGMP has a role in its modulation (2, 20, 21). We show in the present study that iberiotoxin induced a shift in the dose-response curve to 8-BrcGMP in the two diets, confirming the participation of BKCa in the vascular smooth muscle cell relaxation to cGMP in aortic rings. However, the shift was larger in the HS than in the NS group, indicating a more important contribution of BKCa in the 8-BrcGMP-induced relaxation of aorta in animals fed the HS diet. Therefore, it is suggested that the increased activity of the BKCa participates in the enhanced response to 8-BrcGMP, and counteracts the hypersensitivity to membrane depolarization in our model.
In conclusion, the concomitant increase in the vascular smooth muscle cell response to membrane depolarization and NO/cGMP-related compounds is consistent with a local adaptation contributing to maintain normal blood pressure in response to an early and long-lasting HS intake. However, an alteration of the endothelium function might therefore favor imbalance between agonists and endothelial relaxing factors, and lead to a deleterious effect on arterial pressure in this model of HS intake.
M. Cordaillat was supported by a fellowship from the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche. A. Fort was supported by a fellowship from the Servier Laboratories.
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