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Department of Pharmacology, College of Medicine, University of California, Irvine, Irvine, California 92697-4625
Submitted 11 July 2003 ; accepted in final form 5 October 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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60 Torr, arteries from ORX+T developed greater tone compared with ORX arteries. This difference was abolished by removal of the endothelium but remained after treatment of intact arteries with indomethacin or L-NAME. In addition, testosterone treatment had no effect on cerebrovascular production of endothelin-1 or prostacyclin nor did it alter protein levels of endothelial NOS or COX-1. Endothelium removal after L-NAME/indomethacin exposure caused an additional increase in tone. Interestingly, the latter effect was smaller in arteries from ORX+T, suggesting testosterone affects endothelial vasodilators that are independent of NOS and COX. Apamin/CTX, in the presence of L-NAME/indomethacin, abolished the difference in tone between ORX and ORX+T and resulted in vessel diameters similar to those of endothelium-denuded preparations. In conclusion, testosterone may modulate vascular tone in cerebral arteries by suppressing EDHF.
endothelium; vascular tone; endothelium-derived hyperpolarizing factor
In the cerebral circulation, functional studies (10, 12) have demonstrated gender differences in vascular reactivity. Both chronic estrogen and testosterone treatment affect cerebral arteries but in opposite ways. Estrogen attenuates vascular tone by enhancing endothelium-dependent vasodilators derived from the endothelial nitric oxide (NO) synthase (eNOS) and cyclooxygenase-1 (COX-1) pathways (10, 24). In contrast, we found vascular tone of isolated middle cerebral arteries (MCA) is increased by prior in vivo testosterone treatment of castrated male rats (11). The effect of testosterone was endothelium dependent. Several studies (9, 15, 17) in other vascular beds also suggest that testosterone modulates tone via the endothelium; however, the factors involved have not been determined.
In the cerebral vasculature, healthy endothelium responds to intrinsic and extrinsic stimuli by releasing a variety of endothelium-derived factors that modulate the autoregulatory response, an important determinant of blood flow (8). The most studied endothelium-derived factors include the potent vasoconstrictor endothelin-1 (18) and vasodilators such as NO (10) and the COX-1-derived metabolite prostacyclin (PGI2) (25). In addition to NO and PGI2, a third unidentified vasodilatory substance, referred to as endothelium-derived hyperpolarizing factor (EDHF), also plays an important role in regulating cerebrovascular homeostasis (13, 28). EDHF is characterized as an endothelium-derived substance that elicits vascular smooth muscle hyperpolarization (1, 2, 13). Although the identity of EDHF is still in debate, EDHF-mediated vasodilatation has been shown to be synergistically blocked by apamin and charybdotoxin (CTX), suggesting the involvement of Ca2+-activated K+ channels (KCa) (1, 6, 28).
In the present study, we hypothesized that changes in one or more endothelial factor(s) are responsible for increased vascular tone after testosterone treatment. We first established a rat model for testosterone replacement in vivo that resulted in hormone levels comparable to those observed in intact males. Second, because the contribution of endothelial factors to vascular tone may be affected by vessel size (38), we compared the effects of testosterone treatment in isolated, pressurized large (second order) and small (third order) branches of the MCA. In our initial study of this vessel, we examined only the main first-order branch (300 µm diameter) (11). Third, we investigated the effects of testosterone on various endothelium-derived substances using functional and pharmacological approaches in isolated vessels of the cerebral circulation.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Animal model. Male Fischer-344 rats (3 mo old; Harlan) were anesthetized with a mixture of ketamine (91 mg/kg im) and xylazine (9 mg/kg im) for bilateral removal of the testes under aseptic surgical conditions. After orchiectomy (ORX), rats were divided into two groups. At the time of surgery, one group was implanted with testosterone-filled pellets (1.57-mm ID Silastic tubing) subcutaneously at the base of the neck (ORX+T) (11, 14). To establish an animal treatment model, the dose of testosterone was varied by changing the length of the Silastic implant (10 to 30 mm) (Table 1). Fifteen millimeters was chosen as the standard treatment length for all blood vessel experiments in this study. A second group of rats did not receive an implant and were used as testosterone-deficient controls (ORX). All rats received an injection of penicillin (30,000 U, penicillin G benzathine/penicillin G procain) at the time of surgery. After recovery from anesthesia, all animals were returned to animal housing (12:12-h light/dark cycle) and were given fresh water, food, and bedding.
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Four weeks after surgery, with or without testosterone treatment, animals were deeply anesthetized with pentobarbital sodium (50 mg/kg ip), and the thoracic cavity was exposed. Heparin (100 U/0.1 ml) was immediately injected into the right ventricle to prevent clotting, and a blood sample was drawn for plasma testosterone measurements using radioimmunoassay (DiSorin; Stillwater, MN).
Isolated, pressurized cerebral artery preparation. After exsanguination, the brain was removed and placed in ice-cold PSS and pinned in a Sylgard-coated dissection dish aerated with 21% O2-6% CO2-balance N2. Small segments of second- and third-order arteries (250 and 150 µm diameter, respectively) were carefully dissected and placed in a small vessel chamber (Living Systems; Burlington, VT) containing PSS. The proximal end of the vessel was mounted on a glass micropipette, secured with a nylon ligature, and the lumen was gently rinsed of any remaining red blood cells. The proximal cannula was connected to a pressure transducer and reservoir containing PSS equilibrated with 21% O2-6% CO2-balance N2. Next, the distal end was mounted on a second cannula and tied in place. A stopcock located distal to the vessel was closed, and the vessel was gradually pressurized to 60 Torr and then maintained at constant pressure with a pressure servo control unit (Living Systems). A constant-flow peristaltic pump continuously superfused (25 ml/min) the tissue with warmed PSS (37°C, pH 7.4) aerated with 21% O2-6% CO2-balance N2. The vessel preparation was viewed using an inverted microscope (Nikon) equipped with a video camera and monitor. A video-electronic dimension analyzer (Living Systems) was used to measure intraluminal diameter. Changes in intraluminal diameter were recorded on a Macintosh computer and digitized from an analog signal using a computer-based data-acquisition system (MacLab; Colorado Springs, CO).
Pressurized vessel experimental protocol. After 1 h of equilibration at 60 Torr and development of spontaneous tone, vessel diameter was recorded during increasing step pressure changes ranging from 20 to 100 Torr in the presence of PSS. After the last pressure recording, intraluminal pressure was returned to 60 Torr, and arteries were superfused with PSS in the presence of drug/inhibitor treatment for 30 min. To assess the contribution of basal NO production to vascular tone, vessel diameters with step pressure changes were recorded in the presence of PSS containing the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 10 and 100 µM). In separate experiments, the contribution of prostanoid production was assessed using the nonselective COX-1 and -2 inhibitor indomethacin (10 µM) in the presence of L-NAME. In some vessels, indomethacin was added before L-NAME to determine whether the order of drug administration influenced vessel diameter.
To assess the contribution of an EDHF component in modulating vascular tone, a combination of the K+ channel blockers apamin (50 nM) and CTX (11 nM) was added in the presence of L-NAME and indomethacin (1, 34). For experiments involving endothelium removal, an air bubble (1 to 2 ml) was intentionally introduced into the vessel lumen. Endothelium-denuded preparations were perfused for 10 min to wash out endothelium-derived factors and equilibrated at 60 Torr for an additional 20 min. The efficacy of endothelium removal was assessed by adding the vasodilator ADP (10 µM) via the lumen (11, 37). At the end of every experiment, passive responses to step pressure changes were determined in the presence of Ca2+-free PSS containing EDTA (3 mM). Tissues were incubated in Ca2+-free PSS for 20 min, and then intraluminal step pressure changes were applied to obtain passive blood vessel diameters.
Enzyme immunoassay measurements. The effect of in vivo testosterone treatment on endothelin and PGI2 production was determined in small segments of MCA isolated from ORX and ORX+T rats using an enzyme immunoassay (endothelin; Cayman Chemical, Ann Arbor, MI; and PGI2 measured as 6-keto PGF1
; Amersham Biosciences, Piscataway, NJ). Briefly, after deep anesthesia with pentobarbital sodium and heparin injection (described previously in Animal model), isolated brains were removed and immediately placed in ice-cold HEPES-buffered salt solution. With the use of a dissecting microscope, a 6-mm-long segment of MCA from each animal brain was carefully removed and placed into a 96-well plate containing ice-cold HEPES. After an initial equilibration period, each vessel was transferred to a well containing fresh HEPES equilibrated to 37°C and placed for 5 h in a 37°C incubator. After incubation, the vessel segment was removed, and the medium stored at –80°C until analyzed. Protocols and data analysis were performed according to the manufacturers' instructions.
Western blot analysis. Protein levels of eNOS and COX-1 were evaluated using standard immunoblotting methods. Whole brains from ORX and ORX+T animals were removed, rinsed in ice-cold PSS, rapidly frozen on dry ice, and stored at –80°C. Before Western blot analysis, the brains were thawed on ice, and cerebral vessels were isolated using a previously published procedure (19). In brief, the whole brain was gently homogenized with the use of a Dounce tissue grinder in cold PBS (pH 7.4) and centrifuged (720 g; 10 min at 4°C). The supernatant was then discarded and the pellets resuspended in cold PBS. The pellet was layered over a 15% dextran solution (35–45 kDa) and separated by centrifugation (1,300 g; 30–40 min at 4°C). Next, the pellet containing blood vessels was washed with ice-cold PBS over a 50 µm nylon mesh. Vessels were collected from the nylon mesh using fine-tip forceps and transferred to a small glass homogenizer containing ice-cold lysis buffer. The collected vessel fraction consisted of a mixture of arteries, arterioles, venules, veins, and capillaries as determined by light microscopy.
After incubation on ice (20 min), the homogenates were centrifuged (4,500 g for 10 min 4°C), the supernant was drawn off, and an aliquot was analyzed for protein concentration using a bicinchoninic acid protein assay (Pierce; Rockford, IL). Samples were dissolved in Tris-glycine SDS sample buffer and boiled for 4 min. Equal amounts of sample protein (15 µg/lane) were then loaded and separated in 8% polyacrylamide gels using SDS-PAGE. In addition to ORX and ORX+T samples, gels were loaded with a molecular weight marker and standards for eNOS (Transduction Labs; Lexington, KY) or COX-1 (Cayman). Separated proteins were transferred to polyvinylidene difluoride membranes and blocked overnight at 4°C in PBS (0.1% Tween and 6.5% nonfat dry milk). Membranes were probed with an antibody specific for either eNOS (1:500), COX-1 (1:600), or -smooth muscle actin (1:15,000). Enhanced chemiluminescence development in conjunction with a horseradish peroxidase-labeled secondary antibody (1:15,000) was used to visualize the proteins of interest. Densitometry of bands was analyzed with UN-SCAN-it software (Silk Scientific; Orem, UT).
Drugs and Chemicals. PSS bicarbonate-phosphate buffer stock solution containing (in mM) 122 NaCl, 5.1 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25.5 NaHCO3, and 0.03 EDTA was prepared weekly. Before each experiment, the superfusate buffer was prepared from the PSS stock solution by the addition of 11.5 mM glucose and 1.6 mM CaCl2. HEPES buffer was prepared each experimental day and consisted of (in mM) 130 NaCl, 4.0 KCl, 1.2 MgSO4, 1.2 KH2PO4, 4.0 NaHCO3, 10.0 HEPES, 6.0 glucose, and 0.03 EDTA. L-NAME, apamin, CTX, and ADP was dissolved in double-distilled H2O and diluted to a final concentration in PSS. Indomethacin was prepared in 8% NaHCO3 and diluted to a final concentration in buffer. A lysis buffer containing 50 mmol/l 
-glycerophosphate, 100 µmol/l sodium orthovanadate, 2 mmol/l MgCl, 1 mmol/l EGTA, and 0.5% Triton X-100 was prepared on the same day as tissues were homogenized. dl-Dithiolthreitol (1 mmol/l), 1 mmol/l PMSF, 20 µmol/l pepstatin, 20 µmol/l leupeptin, and 0.1 U/ml aprotinin were dissolved in solvent (double-distilled H2O or DMSO) and stored as stock solutions at –20°C. Unless noted otherwise, all drugs and chemicals were purchased from Sigma (St. Louis, MO).
Data analysis. Data are reported as means ± SE. Data were compared between ORX and ORX+T groups using Student's paired t-test or ANOVA for repeated measures as appropriate. Multiple comparisons were made with the Student-Newman-Keuls test when ANOVA indicated that differences existed. P 
0.05 was considered statistically significant for all comparisons.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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15 mm were significantly greater than the weight of ORX animals. There was no difference in body weight between ORX+T with 10-mm implants compared with ORX. Thus 15-mm-long tubing implants were used in all subsequent experiments. Effect of testosterone on intraluminal diameter. To determine the effects of chronic testosterone treatment on vascular diameter in pressurized arteries, MCA isolated from ORX and ORX+T were exposed to increasing pressures from 20 to 100 Torr in 20-Torr increments. Vessel diameters at an intraluminal pressure of 80 Torr in branches of MCA are illustrated in Fig. 1. Passive diameters in Ca2+-free PSS in second- (Fig. 1A) and third-order (Fig. 1B) arteries were not different between ORX and ORX+T, indicating that for each group, similarly sized vessels were obtained from ORX and ORX+T. In the presence of Ca2+-containing PSS, intraluminal diameters were significantly smaller compared with passive conditions demonstrating that all vessels developed vascular tone. However, at 80 Torr, the diameter of vessels from ORX+T was significantly smaller than that from ORX for both second- and third-order MCA. Removal of the endothelium resulted in a significant decrease in vessel diameter compared with diameters in either passive or Ca2+-containing PSS conditions. After endothelium removal, diameters were not different between the ORX and ORX+T groups. Smaller third-order arteries were used in all subsequent functional experiments.
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Effect of testosterone on vascular tone. The degree of vascular tone development with increasing intraluminal pressure is represented in Fig. 2A. Vascular tone was calculated as the difference between passive diameter (Ca2+-free PSS) and diameter in Ca2+-containing PSS at each intraluminal pressure and presented as the percentage of the passive diameter. Vascular tone was significantly greater in arteries isolated from ORX+T compared with ORX at intraluminal pressures between 60 and 100 Torr.
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Endothelium removal. The contribution of endothelial factors to modulate vessel diameter in arteries isolated from ORX and ORX+T was demonstrated by substantial arterial constriction after endothelium removal (Fig. 2B). The absence of ADP-induced vasodilation in pressurized MCA confirmed that the endothelial cell layer lining the lumen was removed or functionally impaired by the air bubble procedure. Removal of the endothelium abolished the differences between ORX and ORX+T seen at higher pressures (80–100 Torr) in intact vessels. This suggests that testosterone increased tone by altering endothelium-dependent modulators. Interestingly, denuded vessels from ORX+T showed less tone than ORX at lower pressures (20–60 Torr).
Effects of testosterone on endothelin production in MCA. Previous evidence suggests that testosterone may influence levels of the potent endothelium-derived vasoconstrictor endothelin (29). Because the modulation of vascular tone by testosterone appears to be endothelium dependent, we examined the effect of in vivo testosterone treatment on in vitro endothelin production in segments of MCA isolated from ORX and ORX+T rats. However, there was no difference in endothelin production in MCA from ORX versus ORX+T (28 ± 4 and 29 ± 4 pg/ml, respectively).
Effects of testosterone on prostanoid and NOS pathways. We next investigated the role of testosterone in modulating two other endothelium-dependent mechanisms: production of NO and prostanoids. Diameters isolated from ORX and ORX+T were first recorded in the presence of the nonselective COX inhibitor indomethacin. Figure 3 represents MCA diameters during superfusion with Ca2+-containing PSS and Ca2+-containing PSS with indomethacin. Data were collected at pressures from 20 to 100 Torr; Fig. 3 illustrates data at 100 Torr. Although diameters in arteries from ORX +T were significantly smaller than diameters in arteries from ORX animals, treatment with indomethacin did not affect vessel diameter in ORX or ORX+T at any pressure.
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Figure 4 illustrates the effect of treatment with two concentrations of L-NAME in the presence of indomethacin. The magnitude of change in diameter due to L-NAME (Ca2+-containing PSS and L-NAME) is shown for each pressure tested. Treatment with L-NAME, either 10 µM (Fig. 4A)or100 µM (Fig. 4B), caused a significant decrease in diameter in MCA from both ORX and ORX+T compared with diameters recorded in the presence of Ca2+-containing PSS. The decrease in diameter caused by 10 µM L-NAME was significantly greater in vessels from ORX+T compared with ORX at intraluminal pressures from 20 to 100 Torr (Fig. 4A). However, in the presence of 100 µM L-NAME, the differences in luminal diameters were abolished between ORX and ORX+T at pressures of 60 Torr and above (Fig. 4B). When a series of pressure steps were repeated in PSS without the addition of drugs (time control), there were no differences in diameters attained at each pressure.
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Complementing these functional studies, the effect of in vivo testosterone treatment on levels of eNOS and COX-1 proteins was examined. Western blot was performed on homogenates from freshly isolated cerebral vessels. Representative blots of eNOS (Fig. 5A) and COX-1 (Fig. 6A) demonstrate that cerebral blood vessels express these proteins. However, mean data from a series of experiments revealed no effect of testosterone treatment on eNOS (n = 6) or COX-1 (n = 9) protein levels (Figs. 5B and 6B). Additionally, PGI2 production in MCA segments isolated from ORX and ORX+T was also not different (ORX 1,741 ± 571 pg/ml; ORX+T 1,791 ± 374 pg/ml). On the basis of these results, enhanced vascular tone development after testosterone treatment does not appear to involve alterations in either COX or NOS in cerebral arteries.
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Effect of testosterone on non-NO, nonprostanoid vasodilators. Figure 7 illustrates vessel diameters in MCA from ORX and ORX+T in the presence of Ca2+-free PSS (passive), Ca2+-containing PSS, Ca2+-containing PSS after endothelial removal, and after addition of a cocktail of endothelial factor inhibitors (L-NAME-indomethacin-apamin-CTX). In the presence of "all blockers," diameters from MCA isolated from ORX and ORX+T were similar to those observed in denuded preparations; this was true at all intraluminal pressures studied (data shown at 100 Torr). Diameters in the absence of endothelium or in presence of all blockers were unaffected by testosterone. However, as seen before, there was a significant difference in diameter between ORX and ORX+T in PSS with intact endothelium.
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To evaluate a possible effect of testosterone treatment on a non-NO, nonprostanoid endothelium-dependent factor, the effect of K+ channel blockers in the presence of L-NAME and indomethacin was determined. In denuded preparations, addition of apamin-CTX did not alter vessel diameter, supporting an exclusive effect on the endothelium (data not shown). As demonstrated in Fig. 8, treatment with the K+ channel blockers apamin and CTX in vessels pretreated with L-NAME and indomethacin caused a significant decrease in vessel diameter. Furthermore, the decrease in diameter caused by the addition of the K+ channel blockers was greater in ORX compared with ORX+T. These findings suggest that testosterone may modulate vascular tone in small cerebral arteries primarily via modulation of an endothelial factor(s) resembling EDHF.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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To date, there is not a clear consensus as to the effects of testosterone on vascular reactivity (32, 35). The hormone has been reported to increase (3, 9, 11, 17, 31) as well as decrease (4, 5, 16, 23, 33, 39) vascular tone. Effects of testosterone are found to be both endothelium dependent (4, 5, 9, 11, 16, 17, 39) and independent (16, 31), and they may not always be mediated by androgen receptors (5, 31, 39). Differences among studies have been attributed to vascular bed specificity, mode of administration (acute vs. chronic), and in particular, the concentration of hormone used to elicit a response (nM vs. µM).
Previous studies (32, 35) have demonstrated that androgen receptors are present in vascular tissue, indicating that blood vessels have the capacity to respond to physiological levels of hormone. Thus it was important for us to establish a treatment model with appropriate concentrations of testosterone. In our previous study (11) on cerebral artery reactivity, testosterone replacement in ORX rats achieved 
35% of physiological levels. In the present study, we used 15-mm Silastic implants that resulted in plasma testosterone levels in the range of those reported previously for intact male rats (11, 14), although intact males in the present study had slightly lower levels. We also demonstrated that the 15-mm implant restored ORX body weight to that of intact males. Although we did not measure blood pressure, testosterone treatment has been shown to increase systemic blood pressure (21). However, a recent study using an SHR rat model, that has a substantially high blood pressure compared with WKY controls, demonstrated that the difference in systemic pressure did not significantly impact vascular tone in MCA pressurized to 100 Torr, an intraluminal pressure known to stimulate pressure-induced constriction. On the basis of these previous data, if chronic testosterone treatment altered blood pressure in our animal model, these changes in systemic pressure would not likely impact the effect of testosterone on cerebral vascular tone. Thus, using this model of testosterone replacement, we found vascular tone was increased in larger branches of the MCA (second order), similar to what we found previously in the main branch of the MCA (11). We extended this finding to smaller branches of the MCA, suggesting that circulating testosterone may influence tone throughout the cerebrovascular tree.
It is clear that endothelial factors modulate vascular tone in cerebral arteries (8, 10, 13, 25). In our study, a substantial contribution of the endothelium to the tone of small, pressurized segments of MCA was demonstrated by the considerable decrease in vessel diameter that occurred after endothelial removal. Importantly, endothelial denuding abolished the difference in vessel diameters between ORX and ORX+T, such that small branches of MCA from ORX+T rats no longer had greater tone; and in fact at lower pressures, lumen diameters became slightly smaller in denuded ORX vessels. In the larger, main branch of MCA isolated from ORX and ORX+T rats, differences in tone also relied on the presence of intact endothelium (11). A similar finding was made in pig coronary artery, where 2 wk of testosterone treatment enhanced vasoconstrictor responses in an endothelium-dependent manner (9). Together, these studies imply that testosterone treatment increases vascular tone by influencing production and/or function of endothelial factor(s). Because it is known that endothelium releases multiple factors that alter vascular smooth muscle tone, we next investigated the role of testosterone in modulating endothelium-dependent vasoconstrictors and vasodilators in cerebral vessels.
An important vasoconstrictor that contributes to vascular tone is the endothelium-derived peptide, endothelin (36). In cerebral arteries, the predominant isoform that produces constriction is endothelin-1 (18). Circulating endothelin levels have been shown to correlate with testosterone levels in vivo (29). In transsexual males receiving testosterone therapy, endothelin levels increased significantly during therapy. Given that endothelin is a potent vasoconstrictor, we tested whether chronic testosterone treatment alters endothelin production in isolated rat MCA. We did not detect any testosterone effect on endothelin production under nonpressurized conditions, suggesting that the ability of testosterone to enhance cerebral vascular tone is not dependent on changes in endothelin. However, our studies do not rule out the possibility of changes in endothelin receptor number or sensitivity after chronic testosterone treatment. Nevertheless, as discussed below, the difference in diameter between vessels of ORX and ORX+T is eliminated by KCa channel blockers in the presence of NOS/COX inhibition suggesting that endothelin is not a primary mediator of the testosterone effect in this isolated, pressurized vessel preparation.
Endothelial modulation of vascular tone also may involve COX-mediated pathways that produce vasoactive prostanoids, such as the potent vasodilator PGI2 (25). Studies in cultured aortic vascular smooth muscle cells demonstrate that testosterone treatment can decrease the production of PGI2 (22). In female rats, there is considerable regulation by endothelium-derived prostanoids in the smaller diameter branches of MCA (25). Therefore, we investigated the possibility that effects of circulating testosterone on vascular tone in small male cerebral arteries involved COX pathways. However, functional studies using the nonselective COX-1/COX-2 inhibitor, indomethacin, did not alter vascular tone in MCA from either ORX or ORX+T during changes in intraluminal pressure. Similarly, COX-1 protein levels were not affected by the long-term administration of testosterone. In addition, chronic testosterone treatment did not alter PGI2 production in MCA segments in vitro. Although testosterone treatment elicits an effect on prostanoid production in cultured cells in vitro (22), chronic in vivo testosterone treatment with physiological levels does not appear to influence COX-dependent endothelial modulation in male cerebral arteries.
NO is another important factor released from endothelial cells in many vascular beds. Therefore, we investigated the effect of testosterone on the contribution of NOS-dependent modulation of cerebral artery tone using the competitive NOS inhibitor L-NAME. In the presence of COX blockade, L-NAME caused a significant decrease in diameter of ORX and ORX+T vessels compared with diameters in PSS. In the presence of a low concentration of L-NAME (10 µM), NOS inhibition was significantly greater in vessels from ORX+T compared with ORX at intraluminal pressures from 20 to 100 Torr. Interestingly, a higher concentration of L-NAME (100 µM) further decreased diameter in ORX vessels eliminating the differences between groups at pressures great enough to elicit tone development (60–100 Torr). Differences in the concentration of inhibitor suggest that more L-NAME is required to inhibit NOS in ORX MCA. This may imply that production of NO is higher in ORX compared with ORX+T. However, Western blot analysis revealed that testosterone treatment had no effect on eNOS levels in cerebral vessel homogenates from ORX and ORX+T, similar to what was found using a lower dose of testosterone (19). The functional studies suggest, however, that testosterone may influence enzyme activity, perhaps at the level of phosphorylation or substrate availability. Nevertheless, differences in tone between ORX and ORX+T persist after complete blockade of NOS with 100 µM.
The most striking finding of our study was that endothelium removal after inhibition of both NO and prostanoids revealed an additional increase in tone that was greater in vessels from ORX compared with ORX+T. These data suggest that a greater contribution of a non-NO, nonprostanoid endothelium-dependent factor or mechanism is present in testosterone-deficient rats. Epoxyeicosatrienoic acids (EETs) (2), K+ (7), or other factors may act as endothelium-derived vasodilators that hyperpolarize vascular smooth muscle (1, 13). Regardless of their nature, it is commonly accepted that EDHF-like mechanisms are important modulators of vascular tone (1), particularly in cerebral arteries (13, 28, 38). Therefore, we tested the commonly accepted protocol for functionally defining EDHF, i.e., blockade by the combination of apamin, a specific inhibitor of small conductance KCa channels, and CTX, a nonselective inhibitor of large and intermediate conductance KCa channels (1) in vessels pretreated with L-NAME and indomethacin. This combination of drugs caused a significant decrease in vessel diameter, and the diameter achieved was similar to diameters seen in denuded preparations. This suggests that an EDHF-like factor accounts for the remainder of the endothelium-dependent modulation of vascular tone seen in this segment of male rat MCA. Moreover, the increase in diameter caused by addition of KCa channel blockers was greater in ORX compared with ORX+T. These findings strongly support the contention that testosterone primarily modulates vascular tone in small, pressurized, cerebral arteries by suppression of EDHF-mediated relaxation.
Future studies will be required to confirm that the effect of testosterone is due to a hyperpolarizing factor, the specific nature of the substance involved, and whether testosterone acts by suppressing the production or action of this factor. In addition, future investigation on EDHF and other endothelial vasoactive factors during stimulated conditions may provide a better understanding about the effects of testosterone on the modulation of cerebrovascular tone. Testosterone and its active metabolite 5-dihydrotestosterone act via androgen receptors (35); alternatively, testosterone can be converted by vascular aromatase to estrogen (20). Both mechanisms could contribute to effects of testosterone treatment on EDHF. Estrogen has been reported to decrease agonist-stimulated, EDHF-mediated dilation in female MCA (12). However, if significant amounts of estrogen were generated in the present study, it would be expected that this would also increase levels of cerebrovascular eNOS protein (19), but this was not seen. Interestingly, a recent study found that androgen treatment inhibits EET formation in rat kidney (21). Animals were injected for 2 wk with 5-dihydrotestosterone, which resulted in decreased epoxygenase conversion of arachidonic acid to EETs in isolated renal microsomes. These authors suggest that androgens regulate specific P-450 monooxygenases that are responsible for metabolism of arachidonic acid to either the vasorelaxant EETs or the vasoconstrictor HETEs. Because EETs are one of the leading candidates for EDHF activity (1, 2), it is possible that a similar mechanism occurs in cerebral arteries such that chronic testosterone exposure suppresses endothelial enzymes involved in EET formation.
Inhibition of EDHF may underlie several clinical observations that correlate circulating testosterone levels with diminished endothelial dilator function (15, 26). Little is known regarding androgen modulation of the cerebral circulation, but one clinical report (27) suggests chronic testosterone administration increases cerebral vascular resistance. An effect on cerebral blood flow could contribute to the increase in ischemic damage observed in testosterone-treated male rats subjected to experimental stroke (14). Clearly, a better understanding of the roles of EDHF and testosterone in cerebral arteries is needed to fully assess the impact of this hormone on the cerebral circulation.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by National Heart, Lung, and Blood Institute Grant HL-50775 and by a grant-in-aid from the American Heart Association.
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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estradiol increases rat cerebrovascular prostacyclin synthesis by elevating cyclooxygenase-1 and prostacyclin synthase. Stroke 33: 600–605, 2002.-Estradiol decreases vascular tone in cerebral arteries by shifting COX-dependent vasoconstriction to vasodilation. Am J Physiol Heart Circ Physiol 285: H241–H250, 2003.-estradiol and testosterone on the contractile responses of porcine coronary arteries. Br J Pharmacol 129: 1301–1308, 2000.[CrossRef][Web of Science][Medline]
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F. M. Faraci, C. Lynch, and K. G. Lamping Responses of cerebral arterioles to ADP: eNOS-dependent and eNOS-independent mechanisms Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2871 - H2876. [Abstract] [Full Text] [PDF]
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D. K. Bowles, K. K. Maddali, V. K. Ganjam, L. J. Rubin, D. L. Tharp, J. R. Turk, and C. L. Heaps Endogenous testosterone increases L-type Ca2+ channel expression in porcine coronary smooth muscle Am J Physiol Heart Circ Physiol, November 1, 2004; 287(5): H2091 - H2098. [Abstract] [Full Text] [PDF]
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