Local oestrogenic/androgenic balance in the cerebral vasculature.

Reproductive effects of sex steroids are well-known; however it is increasingly apparent that these hormones have important actions on non-reproductive tissues such as the vasculature. The latter effects can be relevant throughout the lifespan, not just limited to reproductive years, and are not necessarily restricted to one gender or the other. Our work has established that cerebral blood vessels are a non-reproductive target tissue for sex steroids. We have found that oestrogen and androgens alter vascular tone, endothelial function, oxidative stress and inflammatory responses in cerebral vessels. Often the actions of oestrogen and androgens oppose each other. Moreover, it is clear that cerebral vessels are directly targeted by sex steroids, as they express specific receptors for these hormones. Interestingly, cerebral blood vessels also express enzymes that metabolize sex steroids. These findings suggest that local synthesis of 17ß-estradiol and dihydrotestosterone can occur within the vessel wall. One of the enzymes present, aromatase, converts testosterone to 17ß-estradiol, which would alter the local balance of androgenic and oestrogenic influences. Thus cerebral vessels are affected by circulating sex hormones as well as locally synthesized sex steroids. The presence of vascular endocrine effector mechanisms has important implications for male-female differences in cerebrovascular function and disease. Moreover, the cerebral circulation is a target for gonadal hormones as well as anabolic steroids and therapeutic drugs used to manipulate sex steroid actions. The long-term consequences of these influences are yet to be determined.

Mechanisms of cerebrovascular protection: oestrogen, inflammation and mitochondria.

Investigation of oestrogen action reveals a multitude of diverse effects. This brief review focuses on the impact of oestrogen on the vasculature, with particular emphasis on the cerebral circulation. Three major actions of oestrogen are discussed: enhancement of vasodilator capacity, suppression of vascular inflammation and increase in mitochondrial efficiency. In both humans and animals, oestrogen increases vasodilator tone, an effect dependent on a functional endothelium. Two distinct mechanisms are involved: increase in endothelial nitric oxide synthase (eNOS) mRNA and protein and phosphorylation of eNOS via the PI-3 kinase/Akt pathway. Both effects are mediated by oestrogen receptors (ER), but through two pathways, ER-mediated nuclear gene transcription and cell membrane-associated ERs respectively. Oestrogen also increases function of other endothelium-dependent vasodilators. Oestrogen suppresses vascular inflammation through an NF-κB-dependent effect. The inflammatory response has also been shown to vary significantly during the oestrous cycle of rodents. Emerging information shows that oestrogen increases mitochondrial biogenesis and decreases superoxide production. Suppression of mitochondrial superoxide production by 17ß-estradiol in cerebral blood vessels is mediated by the ER-alpha receptor and not dependent on increased Mn superoxide dismutase activity. Oestrogen treatment also increases protein levels for a number of components of the electron transfer chain, as well as levels of transcription factors that regulate mitochondrial function. All of these actions of oestrogen could be important in mediating vascular protection, especially in the cerebral circulation. Furthermore, given the potential of mitochondrial DNA damage to contribute to pathophysiology and ageing, mitochondrial protective effects of oestrogen might contribute to the longer average lifespan of women.

Selected contribution: cerebrovascular nos and cyclooxygenase are unaffected by estrogen in mice lacking estrogen receptor-alpha.

Estrogen alters reactivity of cerebral arteries by modifying production of endothelium-dependent vasodilators. Estrogen receptors (ER) are thought to be involved, but the responsible ER subtype is unknown. ER-alpha knockout (alphaERKO) mice were used to test whether estrogen acts via ER-alpha. Mice were ovariectomized, with or without estrogen replacement, and cerebral blood vessels were isolated 1 mo later. Estrogen increased levels of endothelial nitric oxide synthase and cyclooxygenase-1 in vessels from wild-type mice but was ineffective in alphaERKO mice. Endothelium-denuded middle cerebral artery segments from all animals constricted when pressurized. In denuded arteries from alphaERKO but not wild-type mice, estrogen treatment enhanced constriction. In endothelium-intact, pressurized arteries from wild-type estrogen-treated mice, diameters were larger compared with arteries from untreated wild-type mice. In addition, contractile responses to indomethacin were greater in arteries from wild-type estrogen-treated mice compared with arteries from untreated wild-type mice. In contrast, estrogen treatment of alphaERKO mice had no effect on diameter or indomethacin responses of endothelium-intact arteries. Thus ER-alpha regulation of endothelial nitric oxide synthase and cyclooxygenase-1 pathways appears to contribute to effects of estrogen on cerebral artery reactivity.

Human urotensin II mediates vasoconstriction via an increase in inositol phosphates.

The cyclic peptide urotensin II has recently been cloned from human and reported to potently constrict primate blood vessels. To elucidate the cellular signalling mechanisms of this peptide, we investigated a possible relationship of vasomotor effects of human urotensin II and phosphoinositide turnover in isolated rabbit thoracic aorta. Human urotensin II produced a slowly developing increase in isometric contractile force (pEC(50)=9.0) that was endothelium-independent. The contractile effect of urotensin II was significantly inhibited by the phospholipase C inhibitor, 2-nitro-4-carboxyphenyl-N,N,-diphenylcarbamate (NCDC), but not by the cyclooxygenase inhibitor, indomethacin. In slices of rabbit thoracic aorta, human urotensin II increased phosphoinositide hydrolysis, and this effect was also inhibited by NCDC. The potency of urotensin II (pEC(50)=8.6) was similar to that found in the contractile studies. Thus, vasoconstrictor effects of human urotensin II appear to be mediated by a phospholipase C-dependent increase in inositol phosphates, suggesting that the peptide acts via a G(q) protein-coupled receptor.

Estrogen reduces mouse cerebral artery tone through endothelial NOS- and cyclooxygenase-dependent mechanisms.

Gender and estrogen status are known to influence the incidence and severity of cerebrovascular disease. The vasoprotective effects of estrogen are thought to include both nitric oxide-dependent and independent mechanisms. Therefore, using small, resistance-sized arteries pressurized in vitro, the present study determined the effect of gender and estrogen status on myogenic reactivity of mouse cerebral arteries. Luminal diameter was measured in middle cerebral artery segments from males and from females that were either untreated, ovariectomized (OVX), or OVX with estrogen replacement (OVX + EST). The maximal passive diameters of arteries from all four groups were similar. In response to increases in transmural pressure, diameters of arteries from males and OVX females were smaller compared with diameters of arteries from either untreated or OVX + EST females. In the presence of N(G)-nitro-L-arginine methyl ester, artery diameters decreased in all groups, but diameters remained significantly smaller in arteries from males and OVX females compared with untreated and OVX + EST females. After endothelium removal or when inhibition of nitric oxide synthase and cyclooxygenase were combined, differences in diameters of arteries from OVX and OVX + EST were abolished. These data suggest that chronic estrogen treatment modulates myogenic reactivity of mouse cerebral arteries through both endothelium-derived cyclooxygenase- and nitric oxide synthase-dependent mechanisms.

Gonadal hormones affect diameter of male rat cerebral arteries through endothelium-dependent mechanisms.

Gender is known to influence the incidence and severity of cerebrovascular disease. In the present study, luminal diameter was measured in vitro in pressurized middle cerebral artery segments from male rats that were either untreated, orchiectomized (ORX), ORX with testosterone treatment (ORX+TEST), or ORX with estrogen treatment (ORX+EST). The maximal passive diameters (0 Ca(2+) + 3 mM EDTA) of arteries from all four groups were similar. In endothelium-intact arteries, myogenic tone was significantly greater in arteries from untreated and ORX+TEST compared with arteries from either ORX or ORX+EST. During exposure to N(G)-nitro-L-arginine-methyl ester (L-NAME), an NO synthase (NOS) inhibitor, myogenic tone significantly increased in all groups. The effect of L-NAME was significantly greater in arteries from untreated and ORX+EST compared with arteries from ORX and ORX+TEST rats. Differences in myogenic tone between ORX and ORX+TEST persisted after inhibition of NOS. After endothelium removal or inhibition of the cyclooxygenase pathway combined with K(+) channel blockers, myogenic tone differences between ORX and ORX+TEST were abolished. Wall thickness and forced dilation were not significantly different between arteries from ORX and ORX+TEST. Our data show that gonadal hormones affect myogenic tone in male rat cerebral arteries through NOS- and/or endothelium-dependent mechanisms.

Chronic estrogen treatment increases levels of endothelial nitric oxide synthase protein in rat cerebral microvessels.

BACKGROUND AND PURPOSE: A number of studies indicate that the female gonadal hormone, estrogen, confers protection against cerebrovascular disorders such as stroke. One postulated mechanism for these effects of estrogen is an action on the enzyme endothelial nitric oxide synthase (eNOS), which produces the vasodilatory molecule NO. We have investigated the hypothesis that estrogen increases expression of eNOS in cerebral microvessels of male and female rats. METHODS: We measured levels of eNOS protein by Western blot in cerebral microvessels isolated from 7 groups of animals: females, ovariectomized females, ovariectomized females treated with estrogen, males, castrated males, castrated males treated with estrogen, and castrated males treated with testosterone. RESULTS: Ovariectomized female rats treated with estrogen had 17. 4-fold greater levels of eNOS protein in cerebral microvessels than ovariectomized females, and intact females had 16.6-fold greater levels than ovariectomized females (P<0.01). In intact females, cerebral microvessel eNOS protein levels were 9.2-fold higher than those of intact males (P<0.05). Levels of eNOS protein in castrated males, castrated males treated with testosterone, and males were not different from each other. Estrogen treatment of castrated animals resulted in an 18.8-fold increase in cerebral microvessel eNOS protein (P<0.05). CONCLUSIONS: Chronic estrogen treatment increases levels of eNOS protein in cerebral microvessels of male and female rats. This increase in eNOS protein correlates with our previous functional findings indicating that estrogen exposure increases NO modulation of cerebrovascular reactivity in both male and female animals. Upregulation of eNOS expression may contribute to the neuroprotective effect of estrogen.

Postjunctional alpha2-adrenoceptors in the rat tail artery: effect of sex and castration.

To investigate sex-related differences in vasoconstrictor responses to postjunctional alpha2-adrenoceptor activation, isolated ring segments of tail arteries from Fischer-344 rats were studied. Addition of the alpha2-adrenoceptor agonist, UK-14304 [5-bromo-6-(2-imidazoline-2yl)-aminol-quinoxaline], enhanced vasoconstriction to the selective alpha1-adrenoceptor agonist, methoxamine, in arteries from both males and females. The response to UK-14304 was significantly greater in arteries from males as compared to female arteries. Addition of alpha2-adrenoceptor antagonist, idazoxan or rauwolscine, shifted norepinephrine concentration response curves to the right. Antagonist effects also tended to be greater in arteries from males as compared to females. After gonadectomy, male-female differences persisted; thus, removal of sex hormones in either males or females did not alter responses to either agonists or antagonists of alpha2-adrenoceptors. These findings suggest that sex differences in alpha2-adrenoceptor function are not maintained by either male or female gonadal steroid hormones but may be developmentally regulated.

Estradiol modulates vascular response to melatonin in rat caudal artery.

The purpose of this study was to determine whether estrogen modulates the function of vascular melatonin receptors. We used the rat caudal artery and found that the contractile effects of melatonin were influenced by the estrous cycle, ovariectomy, and estrogen replacement. In arterial ring segments isolated from female rats, melatonin potentiated, in a concentration-dependent manner, contractions produced either by adrenergic nerve stimulation or by phenylephrine. Constrictor responses to melatonin were smaller in arteries from female rats in proestrus compared with other stages of the estrous cycle and after ovariectomy. Administration of 17beta-estradiol to ovariectomized female rats also resulted in decreased constriction of isolated arteries to melatonin; however, in vitro addition of 17beta-estradiol (10(-7) M) had no effect. In the caudal artery, melatonin appears to act on two receptor subtypes that mediate contraction and relaxation, respectively. The selective melatonin MT2-receptor antagonist 4-phenyl-2-propionamidotetraline (4P-PDOT) enhanced constrictor responses to melatonin in arterial segments from intact female rats, consistent with the inhibition of MT2 receptor-mediated relaxation. In contrast, 4P-PDOT had no significant effect in arteries from ovariectomized female rats. However, when estradiol was replaced in vivo, the effect of 4P-PDOT on melatonin responses was restored. Thus circulating estradiol appears to enhance MT2 melatonin-receptor function in the thermoregulatory caudal artery of the female rat resulting in increased vasodilatation in response to melatonin.

Gender difference in levels of alpha2-adrenoceptor mRNA in the rat tail artery.

To investigate the hypothesis that differing mRNA levels underlie gender differences in the contractile response of the rat tail artery, alpha2-adrenoceptor mRNA was measured using in situ hybridization. Messenger RNA for the alpha2A- and alpha2C-adrenoceptor subtypes was found localized to the smooth muscle layer. There was no detectable mRNA present for the alpha2B-adrenoceptor subtype. Levels of alpha2C-adrenoceptor mRNA were greater in female compared to male tail arteries (417 +/- 35 vs. 263 +/- 38 dpm/mg, P = 0.01), while levels of alpha2A-adrenoceptor mRNA were the same in both sexes. Levels of alpha2-adrenoceptor mRNA may parallel levels of functioning protein present in the rat tail artery.

Simulated microgravity increases myogenic tone in rat cerebral arteries.

Adaptation of the cerebral circulation to microgravity was investigated in rat middle cerebral arteries after 20 days of hindlimb unweighting (HU). Myogenic responses were measured in isolated, pressurized arteries from HU and control animals. Maximal passive lumen diameters, obtained in the absence of extracellular Ca2+ plus EDTA, were not significantly different between groups (249 vs. 258 micrometer). In physiological salt solution, arteries from both HU and control animals maintained a constant lumen diameter when subjected to incremental increases in transmural pressure (20-80 mmHg). However, the diameter of arteries from HU animals was significantly smaller than that of arteries from control animals at all pressures; this difference could be eliminated by exposure to the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester. After HU treatment, transient distensibility of the artery wall in response to pressure was also significantly decreased, whereas the frequency and amplitude of vasomotion were increased. The latter changes were not affected by NG-nitro-L-arginine methyl ester. Thus simulated microgravity increases cerebral artery myogenic tone through both nitric oxide synthase-dependent and -independent mechanisms.

Effect of simulated microgravity on vascular contractility.

Microgravity was simulated in Sprague-Dawley (SD) and Wistar (W) rats by using a tail harness to elevate the hindquarters, producing hindlimb unweighting (HU). After 20 days of HU treatment, blood vessels from both HU and control rats were cut into 3-mm rings and mounted in tissue baths for the measurement of isometric contraction. HU treatment decreased the contractile response to 68 mM K+ in abdominal aorta from W rats. HU treatment also decreased the contraction to 68 mM K+ in carotid arteries from both rat strains and in femoral arteries from W but not SD rats. HU treatment reduced the maximal response to norepinephrine in all arteries except the femoral from SD rats. HU treatment reduced the maximal response of jugular vein from W rats to 68 mM K+ but had no effect on that response in femoral vein from either rat strain. HU treatment also had no significant effect on the maximal response to norepinephrine in veins. These results demonstrate that HU treatment caused a nearly universal reduction of contractility in arteries, but generally had no effect in veins.

Estrogen reduces myogenic tone through a nitric oxide-dependent mechanism in rat cerebral arteries.

Gender differences in the incidence of stroke and migraine appear to be related to circulating levels of estrogen; however, the underlying mechanisms are not yet understood. Using resistance-sized arteries pressurized in vitro, we have found that myogenic tone of rat cerebral arteries differs between males and females. This difference appears to result from estrogen enhancement of endothelial nitric oxide (NO) production. Luminal diameter was measured in middle cerebral artery segments from males and from females that were either untreated, ovariectomized (Ovx), or ovariectomized with estrogen replacement (Ovx + Est). The maximal passive diameters (0 Ca2+ + 1 mM EDTA) of arteries from all four groups were identical. In response to a series of 10-mmHg step increases in transmural pressure (20-80 mmHg), myogenic tone was greater and vascular distensibility less in arteries from males and Ovx females compared with arteries from either untreated or Ovx + Est females. In the presence of NG-nitro-L-arginine methyl ester (L-NAME; 1 microM), an NO synthase inhibitor, myogenic tone was increased in all arteries, but the differences among arteries from the various groups were abolished. Addition of L-arginine (1 mM) in the presence of L-NAME restored the differences in myogenic tone, suggesting that estrogen works through an NO-dependent mechanism in cerebral arteries. To determine the target of NO-dependent modulation of myogenic tone, we used tetraethylammonium (TEA; 1 mM) to inhibit large-conductance, calcium-activated K+ (BKCa) channels. In the presence of TEA, the myogenic tone of arteries from all groups increased significantly; however, myogenic tone in arteries from males and Ovx females remained significantly greater than in arteries from either untreated or Ovx + Est females. This suggests that activity of BKCa channels influences myogenic tone but does not directly mediate the effects of estrogen. Estrogen appears to alter myogenic tone by increasing cerebrovascular NO production and/or action.

Effect of melatonin in the rat tail artery: role of K+ channels and endothelial factors.

1. The role of endothelial factors and potassium channels in the action of the pineal hormone melatonin to potentiate vasoconstrictor responses was investigated in the isolated perfused tail artery of the rat. 2. Melatonin (100 nM) potentiated contractile responses to both adrenergic nerve stimulation and alpha1-adrenoceptor stimulation by phenylephrine. After removal of the endothelium, melatonin no longer caused potentiation. 3. The potentiating effect of melatonin was also lost when nitric oxide synthase was inhibited with L-NAME (10 nM). Thus potentiating effects depend on the presence of nitric oxide released by the endothelium. However, melatonin did not affect relaxation responses to acetylcholine in endothelium-intact arteries, nor did melatonin modulate relaxing responses to sodium nitroprusside in endothelium-denuded arteries. While melatonin does not appear to modulate agonist-induced release of nitric oxide nor its effect, melatonin may modulate nitric oxide production induced by flow and shear stress. 4. When the Ca2+-activated K+ channel opener, NS 1619 (10 microM), was present, potentiating effects of melatonin were restored in endothelium-denuded vessels. However, addition of the opener of ATP-sensitive K+ channels, cromakalim (3 microM), did not have the same restorative effect. Furthermore, addition of a blocker of Ca2+-activated K+ channels, tetraethylammonium (1 mM), significantly attenuated potentiating effects of melatonin. These findings support the hypothesis that melatonin inhibits the activity of large conductance Ca2+-activated K+ channels to produce its potentiating effects. 5. Thus in the rat perfused tail artery, potentiation of constriction by melatonin depends on the activity of both endothelial factors and Ca2+-activated K+ channels. Our findings suggest that melatonin inhibits endothelial K+ channels to decrease flow-induced release of nitric oxide as well as block smooth muscle K+ channels to enhance vascular tone.

Melatonin mediates two distinct responses in vascular smooth muscle.

The pineal hormone melatonin was found to produce two distinct contractile responses in vascular smooth muscle. In isolated rat caudal artery segments, denuded of endothelium, melatonin (10(-10)-10(-7) M) potentiated phenylephrine-induced contractions in a concentration-dependent manner. At higher melatonin concentrations (10(-7)-10(-5) M), however, the potentiating effect was attenuated. In the presence of the melatonin MT2 receptor antagonist, 4-phenyl-2-acetamidotetraline (4P-ADOT), the attenuated constrictor responses were selectively enhanced. These results are consistent with the hypothesis that melatonin activates two receptor subtypes in vascular smooth muscle; MT2 receptors may induce relaxation, while a second receptor subtype mediates vasoconstriction.

Melatonin directly constricts rat cerebral arteries through modulation of potassium channels.

The pineal hormone melatonin was found to decrease luminal diameter of rat middle cerebral artery segments, pressurized in vitro, in a concentration-dependent manner (concentration that produced a half-maximal effect = 2.7 nM). Contractile responses to melatonin were inhibited by luzindole, a melatonin receptor antagonist, but not by the serotonin receptor antagonist ketanserin. Pertussis toxin abolished the effect of melatonin, which is consistent with involvement of Gi or G(o) protein-coupled receptors. The maximal effect of melatonin was increased by elevating transmural pressure. When compared at the same pressure, contractions elicited by melatonin were smaller than those elicited by serotonin but similar in magnitude to those produced by tetraethylammonium or charybdotoxin, blockers of Ca(2+)-dependent, large-conductance K+ (BKCa) channels. The effect of melatonin was significantly attenuated in the presence of BKCa channel blockers, but not by apamin, a blocker of Ca(2+)-dependent, small-conductance K+ channels. Melatonin, like tetraethylammonium, significantly reduced vasodilation produced by NS-1619, an opener of BKCa channels. Contractile responses to melatonin were diminished in the presence of elevated extracellular K+ (16 mM), but they were not significantly affected by NG-nitro-L-arginine methyl ester. The results suggest that activation of melatonin receptors on rat cerebral arteries increases vascular tone through Gi or G(o) protein-mediated inhibition of BKCa channels. Thus melatonin, which is secreted during the night, can directly influence the contractile state of cerebral arteries.

Ovariectomy eliminates sex differences in rat tail artery response to adrenergic nerve stimulation.

The influence of gonadal hormones on vasoconstrictor responses to adrenergic nerve stimulation was investigated by comparing tail arteries from intact and gonadectomized male and female Fisher 344 rats. Arterial ring segments from females were significantly less responsive to transmural nerve stimulation (1-8 Hz) than arteries from age-matched males. Significant male-female differences persisted after correcting the contractile responses for sex-related differences in arterial mass, optimal resting tension, and maximal contractile force. Arteries were taken from cycling, intact females in either proestrus, estrus, metestrus, or diestrus, but no significant differences were found among the four stages for vasoconstrictor responses to either adrenergic nerve stimulation or exogenous norepinephrine. These data suggest adrenergic function in the artery is not affected by hormonal variations during the estrous cycle. After bilateral ovariectomy, however, contractile responses of female arteries to adrenergic nerve stimulation were increased to levels similar to those observed in male arteries. Orchidectomy of males, in contrast, had no effect on neural-evoked contraction. Low concentrations of norepinephrine also produced greater contractile responses in male compared with female arteries; however, this sex-related difference was eliminated by orchidectomy but not ovariectomy. Taken together, the results indicate that circulating gonadal hormones contribute to gender differences observed in rat tail artery. Vasoconstrictor responses to exogenous norepinephrine appear to be enhanced by testicular hormones. In contrast, vasoconstriction induced by adrenergic nerve stimulation appears to be influenced by chronic exposure to circulating ovarian hormones, resulting in a smaller vascular response in female arteries.

Noradrenaline content and release in male and female rat tail arteries.

In tail-artery segments isolated from male and female control and gonadectomized rats, noradrenaline content and noradrenaline released by electrical stimulation were measured by high-pressure liquid chromatography. Noradrenaline content, expressed as a function of tissue wet weight, was higher in tail arteries from female than from male rats, but there were no significant differences between control and orchiectomized males or control and ovariectomized females. Electrical stimulation of vascular segments in the presence of cocaine (10(-5) M) and deoxycorticosterone (10(-5) M) induced release of noradrenaline that was increased in the presence of the alpha 2-adrenergic antagonist idaxozan (10(-6) M). However, no differences were found in either basal or stimulation-evoked fractional noradrenaline release between male or female, control or gonadectomized animals. These results indicate that control of noradrenaline release at the nerve ending does not appear to be different between genders. There may be differences in adrenergic density, but noradrenaline content does not appear to be modified by circulating gonadal hormones.

Vascular responses to neuropeptide Y are greater in female than male rats.

Sex differences in vascular effects of neuropeptide Y (NPY) were investigated in isolated tail artery ring segments from male and female F344 rats. Both pre- and postjunctional effects of NPY appeared to be greater in females. NPY potentiated contractions elicited by transmural adrenergic nerve stimulation (TNS), consistent with an effect on smooth muscle Y1 receptors. The degree of potentiation was significantly greater in arteries from females than males. Ovariectomy of the females resulted in a decrease in potentiation, while orchiectomy of the males resulted in an enhanced NPY effect. When NPY potentiation was measured in the presence of peptidase inhibitors, the effect of exogenous NPY was enhanced; however the enhancement was greater in arteries from females than either males or ovariectomized females. Possible male-female differences in inhibitory prejunctional Y2 receptor function were addressed using the selective agonist NPY13-36. At a low frequency of stimulation (0.5 Hz), NPY13-36 inhibited the response to TNS in arteries from females and castrated males; however, NPY13-36 had no significant effect on responses to TNS in arteries from males or ovariectomized females. Thus gonadal hormones appear to modulate several components involved in NPY neurotransmission, including tissue peptidase activity, postjunctional Y1 and prejunctional Y2 receptors. These effects may contribute to significant differences observed in vascular reactivity between females and males.