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Am J Physiol Heart Circ Physiol 293: H1673-H1681, 2007. First published June 8, 2007; doi:10.1152/ajpheart.00289.2007
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Increased spontaneous tone in renal arteries of spontaneously hypertensive rats

Frédéric S. Michel, Ricky Y. K. Man, and Paul M. Vanhoutte

Department of Pharmacology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong

Submitted 8 March 2007 ; accepted in final form 1 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The spontaneous tone of vascular smooth muscle is augmented in hypertension. The present study examined the role of nitric oxide (NO), cyclooxygenase (COX), thromboxane A2/prostanoid (TP) and PGE2/prostanoid (EP-1) receptors, reactive oxygen species, and large-conductance Ca2+-activated K+ (BKCa) channels in the regulation of spontaneous tone in renal arteries of young and mature Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Rings of arteries, with and without endothelium, were suspended in a myograph for isometric force recording. Spontaneous tone (increase above initial tension) was observed only in arteries of mature SHR and was greater in arteries without endothelium. N{omega}-nitro-L-arginine methyl ester (L-NAME, an inhibitor of NO synthases) induced larger contractions in arteries of SHR than WKY. Indomethacin (a COX inhibitor), SC-19220 (an EP-1 receptor antagonist), and terutroban (a TP receptor antagonist) reduced the L-NAME-evoked contractions. Tiron (a superoxide anion scavenger), catalase (an enzyme that degrades H2O2), and deferoxamine (a hydroxyl radical scavenger) augmented the L-NAME-induced contractions in arteries of mature SHR. Charybdotoxin (a BKCa channel blocker) caused contractions in arteries of mature SHR without endothelium and in arteries with endothelium incubated with L-NAME. A decreased protein level of endothelial NO synthase, an increased release of prostacyclin, and an increased expression of EP-1 receptors were observed in arteries of mature SHR. The present study suggests that spontaneous tone is precipitated by age and hypertension. The reduced production of NO, leading to decreased activation of BKCa channels, may leave the actions of endogenous vasoconstrictors unopposed. COX products that activate EP-1 and TP receptors are involved in the development of spontaneous tone.

nitric oxide; cyclooxygenase; large-conductance calcium-activated potassium channels

VASCULAR TONE REFERS IN VIVO to the ability of vascular smooth muscle to be in a state of permanent partial constriction, which depends on the level of intraluminal pressure (4). In vitro, stretching, in the absence of other exogenous stimuli, increases tension in the canine basilar artery (22) and in the isolated aorta of hypertensive rats (15, 47). Contractions in response to increases in intravascular pressure are mediated by changes in the activation state of K+ and/or Ca2+ channels, resulting in depolarization of the vascular smooth muscle and influx of Ca2+ (9, 18). Metabolites of arachidonic acid participate in the stretch-induced contraction of the canine basilar artery and in the pressure-induced constriction of renal arterioles in vitro (21, 22). Similarly, active tone is potentiated by a cyclooxygenase (COX) product in the aorta of the spontaneously hypertensive rat (SHR) (39). Consistent with a role of COX in determining spontaneous tone, thromboxane A2/prostanoid (TP) receptor antagonists reduce resting tone in aortas from deoxycorticosterone-salt hypertensive rats (15) and SHR (1).

The endothelium plays an important role in modulating vascular tone (13, 14). It contributes to the development of spontaneous tone (26). Nitric oxide (NO) is the primary endothelium-derived relaxing factor, mainly in large-conductance arteries (12). In vitro, the inhibition of NO synthase (NOS) increases tension in the aorta of the rabbit (33) and the rat (28). In the isolated coronary artery of the rat, inhibition of NOS, as well as removal of the endothelium, increases intrinsic tone (30). The removal of NO induces the depolarization of the smooth muscles cells, which opens voltage-dependent Ca2+ channels and, thereby, causes contraction (17). It also augments the production of endothelium-derived vasoconstrictor prostanoids (3, 45, 49). Increased endogenous reactive oxygen species are involved in the development of vascular tone (23, 46). Reactive oxygen species probably inactivate endothelium-derived NO and favor this process (15, 47).

The renal vasculature autoregulates to stabilize the glomerular filtration rate (10). The renal vascular tone is increased in patients with essential hypertension (20). The aim of the present study was to determine whether spontaneous tone exists in the renal arteries of young and mature normotensive Wistar-Kyoto (WKY) and hypertensive rats and whether age and hypertension have an impact on its potential occurrence. When this appeared to be the case, the underlying mechanism was examined.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and Tissue Preparation

Three- to 4-mo-old (young) and 8- to 9-mo-old (mature) male WKY rats and male SHR were purchased from the Chinese University of Hong Kong and raised at the University of Hong Kong. On the day of the experiments, the rats were anesthetized with an injection of pentobarbitone sodium (50 mg/kg ip) and exsanguinated. The present study was approved by the Committee on the Use of Live Animals for Teaching and Research of the University of Hong Kong.

The kidneys, including extrarenal arteries, were excised and placed in a cold modified Krebs-Ringer bicarbonate (control) solution (mmol/l: 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 11.1 glucose). The main branch of the renal arteries was isolated. Rings (2 mm long) were cleaned of fat and connective tissue and suspended in a Halpern-Mulvany myograph (model 610M, Danish Myo Technology, Aahrus, Denmark). In some preparations, the endothelium was removed by perfusion of the artery, before preparation of the rings, with 1 ml of a saponin solution (500 µg/ml) for 20 s (40). The rings were suspended between two 40-µm-diameter stainless steel wires in an organ chamber filled with control solution kept at 37°C and aerated with 95% O2-5% CO2. One of the wires was connected to a movable holder supporting a tension transducer (model FT03, Grass Instrument, Quincy, MA), so that the isometric force measurement could be collected by a data acquisition system (PowerLab 4SP, ADInstruments, Colorado Springs, CO). An optimal load of 5 mN (determined in preliminary experiments) was applied to the rings, which were allowed to equilibrate for 1 h. After stabilization, segments were exposed twice to KCl at 60 mmol/l, which was the concentration that produced a maximal response (Table 1) and was used as reference contraction.


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  		Table 1. Maximal response to KCl in young and mature WKY and SHR renal arteries with and without endothelium

 
Experimental Protocols

Rings of renal arteries from young and mature WKY and SHR, with and without endothelium, were suspended in the myograph. Some of the rings were incubated for 30 min with 1H-[1,2,4]oxalodiazolo[4,3-a]quinoxalin-1-one (ODQ, a soluble guanylate cyclase inhibitor, 0.1 mmol/l) (45) or the NOS inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME, 10 µmol/l) (40) in the absence or presence of indomethacin (a nonselective COX inhibitor, 10 µmol/l) (40), SC-19220 [a PGE2/prostanoid (EP-1) receptor antagonist, 0.1 mmol/l] (29), or terutroban [a thromboxane A2/prostanoid (TP) receptor antagonist, 0.1 µmol/l] (40, 41).

A group of rings from mature WKY and SHR, with or without endothelium, was incubated for 30 min with charybdotoxin [ChTx, a large-conductance Ca2+-activated K+ (BKCa) channel blocker, 0.1 µmol/l] (40) in the absence or presence of L-NAME. Another group of rings from mature SHR, with or without endothelium, was incubated for 30 min with L-NAME + 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron, an anion superoxide scavenger, 10 mmol/l) (48), L-NAME + deferoxamine mesylate salt (a hydroxyl radical scavenger, 0.1 mmol/l) (48), or L-NAME + catalase (an enzyme that reduces H2O2 to H2O and O2, 1,000 U/ml) (15, 48).

Protein Expression

Renal arteries were dissected free and homogenized in 70 µl of buffer solution [1% lauryl sulfate (SDS), 1 mmol/l sodium orthovanadate, and 10 mmol/l Tris·HCl (pH 7.4) containing protease inhibitors (2.5 mg/l leupeptin, 5 mg/l aprotinin, and 10 mg/l PMSF)]. The homogenate was centrifuged (13,000 rpm) for 15 min. The protein content in the supernatant was determined by the Bradford assay (8). Proteins (30 µg) were separated in denaturing SDS-10% polyacrylamide gels for measurement of the protein expression of BKCa channels and TP and EP-1 receptors and in denaturing SDS-10% polyacrylamide gels for the endothelial, neuronal, and inducible NOS protein levels. The proteins were blotted on a nitrocellulose sheet [Hybond enhanced chemiluminescence (ECL), Amersham, Piscataway, NJ]. Blots were blocked for 2 h at room temperature with 5% nonfat dry milk in Tris-buffered saline + Tween 20 (20 mmol/l Tris·HCl, 137 mmol/l NaCl, and 0.1% Tween 20). Some of the membranes were incubated overnight at 4°C with rabbit polyclonal antibodies against BKCa channels (Alomone, Jerusalem, Israel), TP receptors (Cayman Chemicals, Ann Arbor, MI), or EP-1 receptors (Cayman Chemicals) at a dilution of 1:500. Some membranes were incubated overnight at 4°C with mouse polyclonal antibodies against endothelial NOS (BD Biosciences, San Jose, CA; 1:7,500 dilution). Some membranes were incubated for 2 h at room temperature with rabbit polyclonal antibodies against neuronal NOS (BD Biosciences) or inducible NOS (Santa Cruz Biotech., Santa Cruz, CA) at a dilution of 1:1,500 dilution. The membranes were then incubated for 1 h at room temperature with the respective anti-rabbit or anti-mouse IgG conjugated with horseradish peroxidase (Amersham; 1:4,000 dilution). Specific proteins were detected by a chemiluminescence reaction (ECL+ kit, Amersham) followed by exposure of the membranes to Hyperfilm ECL (Amersham). Quantifications were performed by densitometric analysis after scanning using Gel Doc 1000 (Bio-Rad, Hercules, CA). Proteins were stained with Ponceau red [2% (wt/vol) Ponceau S in 30% trichloroacetic acid and 30% sulfosalicylic acid] to verify that comparable amounts of proteins were loaded.

Measurement of Thromboxane B2, PGE2, and 6-Keto-PGF1{alpha}

Rings were placed in minichambers containing 1 ml of control solution kept at 37°C and aerated with 95% O2-5% CO2 (16) and allowed 1 h for equilibration. The control solution was changed every 20 min. The renal artery rings were incubated with or without L-NAME (10 µmol/l) for 30 min and then removed from the incubation solutions, which were collected and stored at –80°C until analysis. Thromboxane B2, PGE2, and 6-keto-PGF1{alpha} concentrations were measured using a commercially available enzyme immunoassay kit (Cayman Chemical). The samples were undiluted for thromboxane B2 measurements, diluted 3 times for PGE2 measurements, and diluted 10 times for 6-keto-PGF1{alpha} measurements.

Chemicals

Catalase (from bovine liver), ChTx, deferoxamine mesylate salt, tiron, indomethacin, L-NAME, ODQ, Ponceau red, and SC-19220 were purchased from Sigma-Aldrich (St. Louis, MO); 3-((6R)6{[(4-chlorophenyl)sulfonyl]amino}-2-methyl-5,6,7,8 tetrahydro-1-naphthalenyl)propanoic acid sodium salt (S-18886, terutroban) was a kind gift from the Institut de Recherches Servier (Suresnes, France). Indomethacin was dissolved in 5 mM sodium bicarbonate and SC-19220 in DMSO; all other drugs were dissolved in water. Water was used for further dilutions. Concentrations are expressed as final molar concentrations in the bath solution.

Analysis of Results

Increases in force (means ± SE) are expressed in terms of area under the curve (percentage of the reference contraction to 60 mmol/l KCl). Western blot values are pixels (arbitrary units), and results (means ± SE) are expressed as the ratio of the protein of interest to the Ponceau red staining. Student's t-test for unpaired observations was used for statistical analysis. P < 0.05 was considered to indicate statistically significant differences.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
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Spontaneous Tone

Basal tension. The resting tension in control rings with endothelium was not statistically different in young and mature WKY and young SHR but was significantly greater in arteries from mature SHR (Fig. 1). The resting tension was significantly larger SHR arteries without endothelium than in those with endothelium (Fig. 1), but not in arteries of young WKY (data not shown).


Figure 1
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  		Fig. 1. Spontaneous tone (increase above initial tension) in arteries of young [3- to 4-mo-old (3m)] or mature [8- to 9-mo-old (8m)] Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) without and with endothelium (EC). Values, represented as area under the curve (AUC, percentage of reference contraction to 60 mmol/l KCl), are means ± SE (n = 5–10). *P < 0.05 vs. WKY(3m) with EC (Student's t-test for unpaired observations). {dagger}P < 0.05 vs. SHR(3m) with EC (Student's t-test for unpaired observations). $P < 0.05 vs. SHR(8m) (Student's t-test for unpaired observations).

 
NO pathway. In arteries of young WKY and SHR, L-NAME (10 µmol/l) caused an increase in tension. The response developed slowly and was characterized by the appearance of oscillations (Fig. 2). In both strains, the response was greater in rings without than in those with endothelium (Figs. 2 and 3). The L-NAME-induced contractions in artery rings with endothelium were significantly larger in young and mature SHR than in young WKY. The L-NAME-induced contractions in arteries without endothelium were significantly larger in SHR than in WKY.


Figure 2
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  		Fig. 2. Increases in isometric tension induced by N{omega}-nitro-L-arginine methyl ester (L-NAME, 10 µmol/l) in renal arteries with and without endothelium from young WKY (A) and SHR (B).

 

Figure 3
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  		Fig. 3. Increases in tension caused by L-NAME (10 µmol/l) in arteries of young and mature WKY and SHR. Values are means ± SE (n = 5–10). Rings with endothelium were incubated with L-NAME in the absence or presence of indomethacin (Indo, 10 µmol/l); rings without endothelium were incubated with L-NAME, L-NAME + SC-19220 (0.1 mmol/l), or L-NAME + terutroban (0.1 µmol/l). *P < 0.05 vs. WKY(3m) with EC (Student's t-test for unpaired observations). {dagger}P < 0.05 vs. respective rings with EC (Student's t-test for unpaired observations). $P < 0.05 vs. age-matched WKY without EC (Student's t-test for unpaired observations). {Psi}P < 0.05 vs. respective rings without EC incubated with L-NAME (Student's t-test for unpaired observations).

 
The effects of ODQ (0.1 mmol/l) were similar to those of L-NAME in young WKY and SHR arteries with and without endothelium of (Fig. 4).


Figure 4
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  		Fig. 4. Increases in tension caused by 1H-[1,2,4]oxalodiazolo[4,3-a]quinoxalin-1-one (ODQ, 0.1 mmol/l) in arteries of young WKY and SHR. Values are means ± SE (n = 5–7). *P < 0.05 vs. respective rings with EC (Student's t-test for unpaired observations).

 
COX and prostanoid receptors. In arteries of mature SHR, indomethacin (10 µmol/l) prevented the spontaneous increase above initial tension (data not shown).

In young and mature WKY and SHR arteries with endothelium, indomethacin abolished the L-NAME-induced contractions (Fig. 3). In mature WKY and SHR arteries without endothelium, the L-NAME-evoked contractions were decreased significantly by SC-19220 and terutroban (Fig. 3).

Oxygen-derived free radicals. In rings with endothelium from arteries of mature SHR, the L-NAME-induced contractions were significantly greater in the presence of tiron (10 mmol/l), deferoxamine (0.1 mmol/l), and catalase (1,000 U/ml; Fig. 4). The tiron-evoked increase of L-NAME-induced contractions was significantly greater in rings without than in those with endothelium (Fig. 5).


Figure 5
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  		Fig. 5. Effect of tiron (10 mmol/l), catalase (1,000 U/ml), and deferoxamine (0.1 mmol/l) on increases in tension induced by L-NAME (10 µmol/l) in arteries of mature SHR. Values are means ± SE (n = 5–8). *P < 0.05 vs. endothelium incubated with L-NAME + tiron (Student's t-test for unpaired observations).

 
BKCa channels. These experiments were performed only in arteries of mature rats. In SHR arteries, ChTx alone significantly increased resting tension without, but not with, endothelium (Fig. 6B). The L-NAME-induced contractions were significantly increased by ChTx (0.1 µmol/l; Fig. 6A). The ChTx-evoked increase of L-NAME-induced contractions was significantly greater in WKY arteries without than in those with endothelium (Fig. 6B). This difference was not observed in SHR.


Figure 6
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  		Fig. 6. A: increases in isometric tension induced by L-NAME (10 µmol/l) and L-NAME + charybdotoxin [ChTx, a large-conductance Ca2+-activated K+ (BKCa) channel blocker, 0.1 µmol/l] in renal arteries with endothelium from mature SHR. B: increases in resting tension induced by ChTx (0.1 µmol/l) and ChTx + L-NAME (10 µmol/l) in arteries of mature WKY and SHR. Values are means ± SE (n = 5–8). *P < 0.05 vs. rings with endothelium incubated with ChTx. {dagger}P < 0.05 vs. rings without endothelium incubated with L-NAME + ChTx (Student's t-test for unpaired observations).

 
Release of prostaglandins. Significantly less thromboxane A2 was released by arteries of SHR than WKY (Fig. 7A). The release of PGE2 was similar in the arteries of the different experimental groups (Fig. 7B). The release of prostacyclin was significantly greater in rings of mature than young rats and in rings of SHR than young WKY (Fig. 7C). Thromboxane B2 (a stable metabolite of thromboxane A2) and PGE2 and 6-keto-PGF1{alpha} (stable metabolites of prostacyclin) concentrations were not significantly affected by L-NAME (10 µmol/l; data not shown).


Figure 7
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  		Fig. 7. Thromboxane A2 (TXA2, A) PGE2 (B), and 6-keto-PGF1{alpha} (C) released in bath solution by renal arteries of young adult and mature WKY and SHR. Values are means ± SE (n = 5–7). *Significant difference between groups: P < 0.05 (Student's t-test for unpaired observations).

 
Molecular Biology Studies

NOS. The three different isoforms of NOS were present in the renal arteries of young adult and mature WKY and SHR (Fig. 8). The protein level of endothelial NOS was significantly lower in arteries from mature than young WKY and SHR (Fig. 8A). The protein level of neuronal NOS was significantly higher in arteries from adult SHR than adult WKY (Fig. 8B). The protein expression of inducible NOS was comparable in the arteries of the different experimental groups (Fig. 8C).


Figure 8
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  		Fig. 8. Representative Western blots and quantitative determination of protein levels of endothelial nitric oxide (NO) synthase (eNOS, A), neuronal NO synthase (nNOS, B), and inducible NO synthase (iNOS, C) in renal arteries from young and mature WKY and SHR. Values are means ± SE (n = 5–8). *Significant difference between groups: P < 0.05 (Student's t-test for unpaired observations).

 
TP and EP-1 receptors. The protein level of TP receptors was comparable in the arteries of the different experimental groups (Fig. 9A). The protein level of EP-1 receptors was significantly higher in arteries from mature SHR than young adult SHR or WKY (Fig. 9B).


Figure 9
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  		Fig. 9. Representative Western blots and quantitative measurement of protein levels of thromboxane A2/prostanoid receptor (TP, A), PGE2/prostanoid receptor isoform type 1 (EP-1, B), and large-conductance Ca2+-activated K+ (BKCa) channel (C) in renal arteries from young and mature WKY and SHR. Values are means ± SE (n = 5–7). *Significant difference between groups: P < 0.05 (Student's t-test for unpaired observations).

 
BKCa channels. The protein level of BKCa channels was comparable in the arteries of the different experimental groups (Fig. 9C).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrates in vitro that, in renal arteries from the rat, inhibition of NOS unmasks spontaneous tone mediated by vasoconstrictor prostaglandins. Age and hypertension augment the action of endogenous vasoconstrictors, thereby increasing spontaneous contractions in these arteries.

The term "spontaneous tone" refers to the presence of an increased contraction without agonist stimulation in large conduit arteries. Conductance arteries from healthy animals do not exhibit spontaneous tone, whereas hypertension induces the development of such tone (15, 44, 47). In the present in vitro study, renal arteries from mature SHR spontaneously developed tone. Removal of the endothelium in arteries from young hypertensive rats unmasked spontaneous contractions, suggesting that endothelium-derived relaxing factors counteract the development of spontaneous tone in the underlying vascular smooth muscle. Certain blood vessels can endogenously produce enough NO in the absence of neurohumoral stimuli or flow to decrease spontaneous tone (17, 32, 33, 47). Thus results obtained with L-NAME in the arteries of young WKY and SHR suggest that inhibition of NOS(s) unmasks the development of spontaneous contractions. In the present study, the effect of ODQ, an inhibitor of soluble guanylate cyclase (24), was comparable to that of L-NAME, suggesting that the local endothelial production of NO, with the resulting activation of soluble guanylate cyclase in the vascular smooth muscle, curtails contractions that can be attributed to spontaneous tone.

In the absence of the endothelium, NO inhibitors do not increase spontaneous tone in the aorta of deoxycorticosterone-salt hypertensive rats and SHR (1, 15). By contrast, the present experiments demonstrate that, in the renal artery of the rat, the endothelium is not the only source of the NO curtailing spontaneous contractions. This conclusion is prompted by the augmenting effect of L-NAME on spontaneous tone in the absence of endothelial cells. The protein levels suggest that endothelial NOS is to be the most abundant enzyme producing NO in the vascular wall of renal arteries of WKY and SHR. The development of spontaneous tone in arteries of mature SHR may be explained by the downregulation of endothelial NOS, demonstrated by the reduced protein presence of this isoform of the enzyme. However, a contribution to augmented L-NAME-induced contractions in arteries from hypertensive rats of the other isoforms of NOS in vascular smooth muscle cannot be excluded. Endothelium-independent relaxations of the rat aorta have been observed with the superperfusate of interleukin-1beta-treated cultured smooth muscle cells and have been attributed to the production of NO following activation of inducible NOS (38). However, in the present study in rat renal arteries, the protein level of the inducible NOS was unchanged with age and hypertension. Another contributor could be neuronal NOS. In cultured human aortic smooth muscle cells, flow stimulates NO production as a result of activation of this isoform of the enzyme (34). Neuronal NOS expression is upregulated in the carotid smooth muscles cells of adult hypertensive, but not normotensive, rats (7). This is consistent with the present findings in renal arteries of young SHR, in which this isoform of the enzyme is upregulated.

The basal production of NO suppresses the release of thromboxane A2 and regulates the tone in cerebral arteries (5). The present study suggests that, in renal arteries of WKY and SHR, inhibition of NO production unmasks the action of COX-derived metabolites of arachidonic acid. This conclusion is prompted by the inhibitory effect of indomethacin on the response to L-NAME. The effect of terutroban and SC-19220 on the artery of the mature SHR suggests that these metabolites activate TP and EP-1 receptors, respectively, of the vascular smooth muscle. Since arteries of adult hypertensive rats exhibit spontaneous tone and the greatest L-NAME-induced contractions associated with an upregulated expression of EP-1 receptors, the prolonged hypertensive process must favor this mechanism. The lack of NO may leave the actions of endogenous vasoconstrictors unopposed or may induce the production of constrictors in the vascular wall (3, 45, 49). NO increases (31, 37), inhibits (27), or does not change (16) the release of prostaglandins in the vascular wall. The present findings confirm the latter observations, since the release by renal arteries of thromboxane A2, PGE2, and prostacyclin was not affected by L-NAME. At high concentrations, prostacyclin is a TP receptor agonist, qualified as one of the endothelium-derived contractile factors in the aorta of the SHR (16). In the present study, the amount of prostacyclin released was quantitatively greater than that of the other metabolites measured in the studied preparation. The present study demonstrates that the release of prostacyclin also increases with age and hypertension, whereas release of the most potent agonist of the TP receptor, thromboxane A2, is reduced. Hence, prostacyclin is the likely mediator of the development of spontaneous tone in renal arteries of mature SHR, as it is for endothelium-dependent contractions of the aorta of the same strain (16).

The present study demonstrates that tiron (a superoxide anion scavenger), catalase (which degrades H2O2), and deferoxamine (a hydroxyl radical scavenger) augment the L-NAME-induced contractions in arteries of mature SHR. These observations suggest that, in the renal artery of mature SHR, oxygen-derived free radicals, such as NO, curtail the development of spontaneous tone. In rat cerebral arterioles, endogenous H2O2 mediates the dilatation caused by arachidonate and bradykinin (42, 43). H2O2 has been suggested to be an endothelium-derived hyperpolarizing factor (11, 25) and activates BKCa channels through stimulation of soluble guanylate cyclase (19). The present experiments do not permit further speculation on the mechanism by which reactive oxygen species modulate spontaneous tone in the renal artery of the rat.

Blockade of BKCa channels, in vitro, by ChTx or iberiotoxin results in the depolarization of the vascular smooth muscle cells, which unmasks spontaneous vascular tone (2, 36, 44). In the present study, the ChTx-evoked increase of the L-NAME-induced contractions in arteries from mature SHR and WKY suggests that the blockade of BKCa channels favors spontaneous tone. NO activates BKCa channels directly or through a cGMP-dependent protein kinase (6, 35). Since contractions upon administration of the toxin occurred only in the presence of L-NAME or in the absence of the endothelium in arteries of mature SHR, NO may act against the depolarization of the smooth muscle cells because of the blockade of these BKCa channels.

In summary, the vascular smooth muscle of rat renal artery can develop spontaneous tone (Fig. 10). The spontaneous tone is modulated by NO (produced by the endothelium and the vascular smooth muscle cells) and oxygen free radicals, which may activate BKCa channels. These spontaneous contractions are induced by COX products, which bind to EP-1 and TP receptors. Spontaneous tone in vitro may be caused by the increased action of metabolites of arachidonic acid derived from COX, unopposed by the low bioavailability of NO because of the endothelial dysfunction characteristic of age and hypertension.


Figure 10
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  		Fig. 10. Proposed mechanism underlying increased spontaneous tone in mature SHR. sGC, soluble guanylyl cyclase.

 

   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by Fondation pour la Recherche Médicale, France, Grant SPE20050303429 (to F. S. Michel), University of Hong Kong Small Project Funding Grant 200607176137, and the Research Centre of Heart, Brain, Hormone, and Healthy Aging of the University of Hong Kong.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. M. Vanhoutte, Dept. of Pharmacology, 2/F, Laboratory Block 1, Faculty of Medicine Bldg., 21 Sassoon Rd., Pokfulam, Univ. of Hong Kong, Hong Kong (e-mail: vanhoutt{at}hkucc.hku.hk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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