Flow-induced dilation of gracilis muscle arterioles was examined in both genders of control rats and rats chronically treated withN ω-nitro-l-arginine methyl ester (l-NAME). After l-NAME treatment (4 wk), systolic blood pressure was significantly increased compared with control, whereas the plasma concentration of nitrate/nitrite was significantly reduced. Isolated and pressurized arterioles dilated significantly in response to increases in flow (0–25 μl/min). Flow-induced dilation was comparable in arterioles of control andl-NAME-treated rats but was significantly greater in female than in male rats. l-NAME + indomethacin, which abolished flow-induced dilation in arterioles of male control rats, inhibited the dilation by only ∼75% in female control rats. The residual portion of the response was eliminated by additional administration of miconazole, an inhibitor of cytochromeP-450. Indomethacin did not affect the dilation in femalel-NAME-treated rats but completely inhibited the response in male l-NAME-treated rats. The indomethacin-insensitive, flow-induced dilation in female l-NAME-treated arterioles was abolished by miconazole, 6-(2-proparglyoxyphenyl)hexanoic acid, or charybdotoxin. Thus an augmented release of endothelial prostaglandins accounts for the preserved flow-induced dilation in arterioles of male rats, whereas a metabolite of cytochrome P-450 is responsible for the maintenance of flow-induced dilation in female rats, suggesting important differences in the adaptation of the endothelium of arterioles from male and female rats to the lack of nitric oxide (NO) synthesis.
- NO synthesis
- hyperpolarizing factor
- cytochrome P-450 metabolites
- potassium channels
the endothelium plays an important role in the regulation of vascular tone via release of dilator mediators, including nitric oxide (NO), prostaglandins, and endothelium-derived hyperpolarizing factor (EDHF), the latter generally being characterized as a metabolite of arachidonic acid via cytochromeP-450 (CYP) epoxygenase (6, 13, 18, 19). One of the important local factors governing arteriolar tone is wall shear stress, which is the primary stimulus for release of endothelial NO, as well as prostaglandins, in vivo (14, 15).
There is but scant evidence regarding the release of EDHF in response to increases in shear stress elicited by flow, especially because its synthesis is believed to be inhibited by the other two endothelial mediators released to flow (1, 24). Indeed, there is increasing evidence suggesting a feedback inhibition on EDHF production by NO and/or prostaglandins (2, 20, 24), as well as potential interactions among these three endothelial mediators (16). Upregulation of one system in response to a suppression or deficiency of the others has also been demonstrated (3, 4, 20). Our previous studies have shown that, in skeletal muscle arterioles, flow-induced dilation in male wild-type (WT) mice is mediated by endothelial NO and prostaglandins, whereas it is mediated exclusively by prostaglandins in arterioles of male endothelial NO synthase gene-deficient (eNOS-KO) mice (22). In the same vessels isolated from female WT mice, EDHF, together with prostaglandins, participates in the mediation of flow-induced dilation when NO is acutely inhibited; however, EDHF is solely responsible for the maintenance of this response in female eNOS-KO mice (9).
The question, therefore, arose as to whether the gender-specific compensation involving flow-induced dilation in response to NO deficiency is a universal phenomenon or simply a species-specific response. In addition, whether the effects of a short-term adaptation to the absence of NO synthesis are any different from those caused by a genetic lack of eNOS is of interest. Accordingly, we designed experiments to be conducted on arterioles of male and female rats that were chronically treated withN ω-nitro-l-arginine methyl ester (l-NAME), an inhibitor of the synthesis of NO.
Twelve-week-old Wistar rats (Charles River Laboratories, Wilmington, MA) were divided into four groups: control untreated male,l-NAME-treated male, untreated female, andl-NAME-treated female rats. Rats received l-NAME in the drinking water (50 mg/100 ml) for 4 wk. Age-matched normal male and female rats drinking tap water alone were studied concomitantly. All protocols were approved by the Institutional Animal Care and Use Committee of New York Medical College and conform to the guidelines of the National Institutes of Health and the American Physiological Society for the use and care of laboratory animals.
Systolic blood pressure (SBP) and heart rate were monitored every 3 days after the start of treatment by using the tail-cuff method. Rats were anesthetized with pentobarbital sodium (Nembutal, 50 mg/kg ip). Experiments were conducted on isolated gracilis muscle arterioles of all rats. The dissection of muscle and isolation of vessels have been described previously (10, 14). Changes in the diameter of arterioles in response to increases in flow were studied at 80 mmHg perfusion pressure. Perfusate flow was increased from 0 to 25 μl/min in 5 μl/min steps. After control experiments, inhibitors were administered in the perfusate for 30 min before and during the experimental protocols.
In the first series of experiments, the role of NO or prostaglandins in the mediation of flow-induced dilation was assessed by usingl-NAME (10−4 M) and indomethacin (Indo, 10−5 M), inhibitors of NO synthase and cyclooxygenase, respectively. After control flow-diameter curves were obtained, an inhibitor was administered alone or in combination with the other before the flow-diameter relationships were once more assessed.
In the second series of experiments, the role and interaction of metabolites of cytochrome P-450 (CYP) and eNOS on flow-induced dilations were assessed by using miconazole (MCZ, 2 × 10−6 M), an inhibitor of CYP epoxygenase, andl-NAME. After control experiments, MCZ orl-NAME was administered before the experiments were repeated. Then flow-induced responses were studied once more in the additional presence of the other inhibitor. In a separate group of experiments, the effect of metabolites of CYP on flow-induced dilation in arterioles of l-NAME-treated female rats was confirmed further by using 6-(2-proparglyoxyphenyl)hexanoic acid (PPOH, 10−5 M), which is thought to be a more specific inhibitor of CYP epoxygenase (23).
In the third series of experiments, the contribution of EDHF to flow-induced responses was again evaluated by performing the experiments before and after abluminal administration of charybdotoxin (ChTX, 2 × 10−8 M), a blocker of Ca2+-dependent K+ channels. These channels are thought to be activated by EDHF, causing hyperpolarization of vascular smooth muscle and vessel dilation.
At the conclusion of each experiment, the suffusion solution was changed to a Ca2+-free solution containing 1 mM EGTA. Vessels were incubated for 10 min to reach maximal diameter at 80 mmHg perfusion pressure.
Measurement of plasma nitrate/nitrite.
After the muscle had been excised, blood (5 ml) was withdrawn from the abdominal aorta with a 10-ml syringe containing 0.1 ml of heparin sodium (1,000 U/ml). The blood sample was centrifuged immediately (3,000 rpm at 4°C for 20 min) to obtain the plasma, which was then filtered through a 10,000-mol wt cutoff filter (NANOSEP, Fisher) to remove Hb. The plasma was kept at −80°C for later measurement of plasma nitrite/nitrate (NO2/NO3) concentration (25) by fluorometric assay.
All chemicals were obtained from Sigma (St. Louis, MO). PPOH was dissolved in ethanol at 10−2 M and further diluted with physiological salt solution (PSS). l-NAME and ChTX were dissolved in saline. Indo and MCZ were dissolved in DMSO at 10−1 and 10−2 M for Indo and MCZ, respectively, and further diluted with PSS. The highest concentration of DMSO in the chamber was 0.1% (vol/vol), which had no effect on vessel tone.
Calculations and statistics.
Passive diameter (PD) was used to assess the active tone (%PD) generated by arterioles in response to intravascular pressure and to normalize the changes in diameter in response to increases in flow in each vessel. Values are means ± SE; n is the number of rats. Statistical significance was calculated by repeated-measures of ANOVA followed by the Tukey-Kramer multiple-comparison test. Student'st-test was also used, as appropriate. Significance level was taken at P < 0.05.
Table 1 shows the changes in SBP, heart rate, and plasma concentrations of NO2/NO3 in four groups of rats. SBP was significantly increased and heart rate was correspondingly reduced inl-NAME-treated rats compared with untreated rats (P < 0.05). Also, plasma concentration of NO2/NO3 was significantly reduced afterl-NAME treatment (P < 0.05).
Characteristics of arterioles of gracilis muscle from the four groups of rats studied are summarized in Table2. PDs were significantly smaller in arterioles of l-NAME-treated than untreated rats, whereas active diameters and basal tone, expressed as percentage of PD, were comparable in vessels from rats of the same gender. On the other hand, basal arteriolar tone in female rats was significantly attenuated compared with that in male rats (P < 0.05).
Increasing flow from 0 to 25 μl/min elicited significant increases in diameter of arterioles from all four groups of rats (Fig.1). The magnitude of flow-induced dilation of arterioles was comparable in untreated and treated rats of the same gender but was significantly greater in female than in male rats (∼89.8 ± 1.3 vs. 73.9 ± 1.3% at 25 μl/min), confirming our previous findings (11).
Arterioles of male rats.
The endothelial mediators responsible for the mediation of flow-induced dilation in arterioles of male rats are summarized in Fig.2, showing that in untreated rats (A), Indo or l-NAME alone inhibited flow-induced dilation by ∼50%. Combined administration of both inhibitors abolished the responses. In rats treated chronically withl-NAME (Fig. 2 B), Indo eliminated the dilator responses to flow, revealing a solely prostaglandin-mediated flow-induced dilation as a consequence of l-NAME treatment.
Arterioles of female rats.
The endothelial mediators responsible for the mediation of flow-induced dilation in arterioles of female rats are summarized in Figs.3 and 4. A prostaglandin-mediated portion of flow-induced dilation in arterioles of untreated female rats was demonstrated by the fact that Indo inhibited the responses by ∼50% whether Indo was administered first or last among the inhibitors used (Fig. 3). The roles of NO and metabolites of CYP in the mediation of flow-induced dilation in vessels of untreated female rats were also assessed by using l-NAME and MCZ. The Indo-resistant portion of the response was partially inhibited by l-NAME or MCZ after l-NAME (Fig.3, top and middle). On the other hand, unlikel-NAME, MCZ alone did not affect the Indo-resistant portion of the response, which, however, was eliminated by additional administration of l-NAME (Fig. 3, bottom), suggesting an interaction between NO and metabolites of CYP.
Figure 4 shows that, in female rats treated chronically withl-NAME, flow-induced dilation was independent of prostaglandins, since Indo had no effect on the response. Dilation, however, was eliminated by MCZ or PPOH (Fig. 4 A), revealing the involvement of the CYP pathway in the mediation of the responses, as a consequence of chronic l-NAME treatment. When these arterioles were treated with ChTX, flow-induced dilation was abolished (Fig. 4 B).
The present study demonstrates a gender-specific adaptation to the lack of NO in the mediation of endothelium-dependent flow-induced dilations in rat gracilis muscle arterioles. It also shows that although NO and prostaglandins participate equally in the mediation of flow-dependent responses in control rats of both genders, after chronic treatment with l-NAME, prostaglandins account solely for this response in vessels of male rats and EDHF in vessels of female rats. Also, EDHF contributes partially to the mediation of flow-induced dilation when NO synthesis is acutely inhibited in female control rats. These findings are consistent with our previous studies in WT and eNOS-KO mice (9, 22). The congruence between the present and our previous studies reveals that a deficiency of eNOS-derived NO activates gender-specific signal transduction pathways.
It was reported previously that the cardiovascular system adapts to an acute inhibition of NO synthesis in a manner that is different from that observed with a chronic lack of NO (5). Also our previous studies demonstrated that, in skeletal muscle arterioles of eNOS-KO mice, endothelial cells adapt to the chronic lack of NO and maintain a normal or close-to-normal response to shear stress by upregulation of the synthesis of other mediators, which, however, operate in a heterogeneous fashion dependent on gender (9,22). Given that compensatory mechanisms may play an important role in the maintenance of cardiovascular function and that sex hormones may participate in the control of this compensation, it was of interest to define the nature of the mechanisms by which vessels are capable of responding normally to flow/shear stress in the absence of NO. To this end, flow-induced dilation and the nature of the endothelial factors mediating this response were investigated in gracilis muscle arterioles of rats of both genders treated chronically with l-NAME.
SBP was significantly enhanced, resulting in a reflex attenuation of heart rate in l-NAME-treated rats. Also, plasma concentrations of NO2/NO3 were significantly reduced after chronic l-NAME treatment (Table 1). The PD of arterioles of l-NAME-treated rats was significantly smaller than that of untreated littermates, a finding similar to that in eNOS-KO mice (9, 22), whereas the active diameters and the basal tone of vessels were comparable in the vessels of the two groups (Table 2). In addition, the results showing attenuated basal tone (Table 2) and enhanced flow-induced dilation (Fig. 1) in arterioles of female compared with male rats are consistent with our previous findings (10-12) showing that the greater basal and stimulated release of endothelial NO, triggered by the presence of estrogen, is responsible for these differences. Interestingly, in the present study, the reduced basal tone and greater flow-induced dilation in female than in male rats seem not to be purely NO dependent, since they are also present in l-NAME-treated littermates. On the other hand, the similar magnitude of flow-induced dilation in treated and untreated rats of the same gender (Fig. 1) further supports our hypothesis that arterioles of skeletal muscle are able to compensate for the absence of NO to maintain dilator responses to shear stress.
Adaptation of arterioles of male rats.
Our present findings are similar to those we reported previously (14) showing that endothelium-derived NO and prostaglandins are coreleased in gracilis muscle arterioles of untreated male rats in response to increases in flow, that endothelium-derived NO and prostaglandins are responsible for the ensuing vasodilation, and that inhibition of NO or prostaglandin synthesis reduces the dilation by ∼50%. Combination of both inhibitors eliminated the responses (Fig. 2 A). In contrast, in arterioles of male rats treated chronically with l-NAME, Indo completely eliminated flow-induced dilation, indicating that the response is solely mediated by enhanced release of dilator prostaglandins (Fig. 2 B), a finding that corresponds to that observed in the same vessels of male eNOS-KO mice (22). Recently, we demonstrated that this compensatory upregulation of prostaglandin synthesis most likely involves inducible cyclooxygenase (unpublished observations). In keeping with the present findings, an upregulation of cyclooxygenase activity, as a consequence of chronic NO deficiency, has also been demonstrated in the dog coronary (3,21) and rat mesenteric circulations (8).
Adaptation of arterioles of female rats.
As for the endothelial factors responsible for the mediation of flow-induced responses in arterioles of female mice, results shown in Fig. 3 indicate that in untreated rats, apart from the coparticipation of NO and prostaglandins, CYP metabolites contribute, in part, to the mediation of the responses, as indicated by the inhibitory effect of MCZ on the dilations. However, such a role for CYP metabolites was observed only in the presence of l-NAME, since MCZ alone did not affect the responses (Fig. 3, bottom). Unlike MCZ, either l-NAME or Indo alone significantly inhibited flow-induced dilation (Fig. 3, top and middle), indicating that NO and prostaglandins are the primary mediators of this response in normal conditions. The absence of NO after the acute administration of l-NAME activates CYP, eliciting EDHF formation, which then contributes to the mediation of flow-dependent dilation. A similar effect of a NO donor on agonist-induced EDHF release from porcine (1, 2) and canine coronary arteries (20) was also reported.
In female rats treated chronically with l-NAME (Fig. 4), MCZ or PPOH alone abolished flow-induced dilation, indicating a solely CYP metabolite-dependent response, unaffected by inhibition of cyclooxygenase (Fig. 4 A). Furthermore, the dilation is completely inhibited by ChTX (Fig. 4 B), suggesting further that the CYP-mediated dilation to flow is indeed dependent on hyperpolarization of vascular smooth muscle, via activation of Ca2+-sensitive K+ channels (6).
These results, together with those observed in eNOS-KO mice, may well form the basis of the gender-dependent mechanisms by which compensation for the lack of endothelial NO occurs in resistance vessels. Regarding the relationships between EDHF and female hormones, previous studies have provided some evidence suggesting that estrogen enhances the contribution of EDHF in the mediation of agonist-induced vasodilation (17) and smooth muscle membrane hyperpolarization (7). Further studies are necessary to establish the possible role(s) of specific hormones that are responsible for the activation of endothelial pathways leading to the synthesis of NO, prostaglandins, and EDHF in the two genders.
In conclusion, we demonstrated that, in skeletal muscle arterioles of male and female rats, NO and prostaglandins are the primary mediators of flow-induced dilation in control conditions. When NO synthesis is blocked acutely, EDHF participates in the responses of vessels of female rats. In arterioles of male rats chronically treated withl-NAME, prostaglandins are solely responsible for the preserved flow-induced dilation, whereas in vessels ofl-NAME-treated female rats, this response is exclusively mediated by EDHF. These data, together with our previous findings in eNOS-KO mice, support our hypothesis that compensatory mechanisms in arterioles evoked by the absence of NO are indeed gender dependent in nature, through which enhanced contribution of endothelial mediators other than NO contribute to the maintenance of shear stress-sensitive regulation of skeletal muscle arterioles and, consequently, peripheral resistance.
We appreciate the excellent secretarial assistance of Miriam Nunez and Dana M. Spencer.
This study was supported by National Heart, Lung, and Blood Institute Grants HL-43023 and HL-46813, and American Heart Association Grant 9930244N.
Address for reprint requests and other correspondence: G. Kaley, Dept. of Physiology, New York Medical College, Valhalla, NY 10595 (E-mail:).
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- Copyright © 2001 the American Physiological Society