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Anxiety and depression are a major health burden. Angiotensin II, via activation of angiotensin II type 1 receptor (AT1R)-mediated brain oxidative stress and inflammation may contribute to these emotional abnormalities. In this study, we investigated the role of a regulator of G-protein signaling 5 (RGS5) protein, which regulates AT1R activity, in angiotensin II-induced brain oxidative stress, inflammation and anxiety-, and depression-like behavior. We hypothesized that deletion of the RGS5 protein would worsen angiotensin II-induced anxiety- and depression-like behavior, cerebral vascular oxidative stress, and brain inflammation. Adult male wild-type and RGS5-deficient mice were implanted with osmotic minipumps delivering either vehicle (saline) or angiotensin II (1 mg/kg/d) for three weeks. Subsequently, mice were tested for locomotor activity, anxiety-like behavior (using the elevated plus maze), and depression-like behavior (using the tail suspension test). After behavioral testing, brain tissue was collected to assess oxidative stress and inflammatory proteins. RGS5 deletion resulted in anxiety-like but not depression-like behavior when compared to wild-type mice. Combined deletion of RGS5 and angiotensin II treatment did not further worsen anxiety-like behavior observed in RGS5-deficient mice. In contrast, depression-like behavior was worsened in RGS5-deficient mice treated with angiotensin II. Interestingly, RGS5 deficiency and angiotensin II treatment had no effect on cerebral vascular oxidative stress, or on expression of the inflammatory marker vascular cell adhesion molecule-1 in the brain. RGS5 deficiency was also associated with decreased blood pressure and an enhanced pressor response to angiotensin II. These data suggest that RGS5 protects against anxiety-like behavior and against angiotensin II-induced depression-like behavior.

Aging is commonly associated with chronic low-grade inflammation and hypertension but it is unknown whether a cause-effect relationship exists between them. We compared the sensitivity of young adult (8-12 w) and aged (23-31 mo) male C57Bl6J mice to develop hypertension in response to a slow-pressor dose of angiotensin II (Ang II; 0.28 mg/kg/d; 28 d). In young mice, the pressor response to Ang II was gradual and increased to 142±8 mmHg over 28 d. However, in aged mice, Ang II promptly increased SBP and reached 155±12 mmHg by 28 d. Aging increased renal but not brain expression of Ang II receptors (At1ar and At2r) and elevated AT1R:AT2R expression ratio in mesenteric artery. Maximal contractile responses of mesenteric arteries to Ang II were enhanced in aged mice and were not affected by L-NAME, indomethacin or tempol. Mesenteric arteries and thoracic aortae from aged mice exhibited higher Nox2-dependent superoxide production. Despite having higher renal expression of Nlrp3, Casp-1 and Il-1β, Ang II-induced hypertension (SBP: 139±7 mmHg) was unaffected by co-infusion of the NLRP3 inflammasome inhibitor, MCC950 (10 mg/kg/d; SBP: 145±10 mmHg). Thus, increased vascular AT1R:AT2R expression, rather than NLRP3 inflammasome activation, may contribute to enhanced responses to Ang II in aging.

Aims The G protein-coupled estrogen receptor 1 (GPER) may modulate some effects of aldosterone. In addition, G-1 (a GPER agonist) can lower blood pressure (BP) and promote T cell-mediated anti-inflammatory responses. This study aimed to test the effects of G-1 and G-15 (a GPER antagonist) on aldosterone-induced hypertension in mice and to examine the cellular mechanisms involved. Methods and results C57Bl/6 (wild-type, WT), RAG1-deficient and GPER-deficient mice were infused with vehicle, aldosterone (0.72 mg/kg/day S.C. plus 0.9% NaCl for drinking) ± G-1 (0.03 mg/kg/day S.C.) ± G-15 (0.3 mg/kg/day S.C.) for 14 days. G-1 attenuated aldosterone-induced hypertension in male WT but not male GPER-deficient mice. G-15 alone did not alter hypertension but it prevented the anti-hypertensive effect of G-1. In intact female WT mice, aldosterone-induced hypertension was markedly delayed and suppressed compared with responses in males, with BP remaining unchanged until after Day 7. In contrast, co-administration of aldosterone and G-15 fully increased BP within 7 days in WT females. Similarly, aldosterone robustly increased BP by Day 7 in ovariectomized WT females, and in both sexes of GPER-deficient mice. Whereas aldosterone had virtually no effect on BP in RAG1-deficient mice, adoptive transfer of T cells from male WT or male GPER-deficient mice into male RAG1-deficient mice restored the pressor response to aldosterone. This pressor effect could be attenuated by G-1 in RAG1-deficient mice that were reconstituted with either WT or GPER-deficient T cells, suggesting that G-1 does not act via T cells to lower BP. Conclusion Our findings indicate that although aldosterone-induced hypertension is largely mediated by T cells, it can be attenuated by activation of GPER on non-T cells, which accounts for the sex difference in sensitivity to the pressor effect.

Although hypertension may involve underlying inflammation, it is unknown whether advanced atherosclerosis − a chronic inflammatory condition − can by itself promote hypertension. We thus tested if advanced atherosclerosis in chronically hypercholesterolemic mice is associated with systemic and end-organ inflammation, vascular dysfunction and oxidative stress, and whether blood pressure is higher than in control mice. Male ApoE −/− and wild-type (C57Bl6J) mice were placed on a high fat or chow diet, respectively, from 5 to 61 weeks of age. Expression of several cytokines (including IL-6, TNF-α, IFN-γ and/or IL-1β) was elevated in plasma, brain, and aorta of ApoE −/− mice. Aortic superoxide production was ∼3.5-fold greater, and endothelium-dependent relaxation was markedly reduced in aorta and mesenteric artery of ApoE −/− versus wild-type mice. There was no difference in blood pressure of aged ApoE −/− (104 ± 3 mmHg, n = 13) and wild-type mice (113 ± 1 mmHg, n = 18). To clarify any effects of aging alone, findings from 61 week-old wild-type mice were compared with those from young (8–12 weeks old) chow-fed wild-type mice. The data indicate that aging alone increased renal and aortic expression of numerous cytokines (including CCL2, CCL7 and IL-1β). Aging had no effect on blood pressure, systemic inflammation, oxidative stress or endothelial function. Despite systemic and end-organ inflammation, oxidative stress and endothelial dysfunction, advanced atherosclerosis does not necessarily result in elevated blood pressure.

Aldosterone binds to mineralocorticoid receptors (MRs) on renal epithelial cells to regulate sodium and water reabsorption, and therefore blood pressure. Recently, the actions of aldosterone outside the kidney have been extensively investigated, with numerous reports of aldosterone having detrimental actions, including in the vasculature. Notably, elevated aldosterone levels are an independent cardiovascular risk factor, and in addition to causing an increase in blood pressure, aldosterone can have blood pressure-dependent and -independent effects commonly manifested in the vasculature in cardiovascular diseases, including oxidative stress, endothelial dysfunction, inflammation, remodeling, stiffening, and plaque formation. Receptor-dependent mechanisms mediating these actions include the MR expressed on vascular endothelial and smooth muscle cells, but also include the angiotensin II type 1 receptor, epidermal growth factor receptor and vascular endothelial growth factor receptor 1, with downstream mechanisms including NADPH oxidase, cyclooxygenase, glucose-6-phosphate dehydrogenase, poly-(ADP ribose) polymerase and placental growth factor. The beneficial actions of MR antagonism in experimental hypertension include improved endothelial function, reduced hypertrophy and remodeling, and in atherosclerosis beneficial actions include reduced plaque area, inflammation, oxidative stress and endothelial dysfunction. Aldosterone excess is detrimental and MR antagonism is beneficial in humans also. The emerging concept of the contribution of aldosterone/MR-induced immunity to vascular pathology will also be discussed.

Macrophage mineralocorticoid receptor (MR) signaling is an important mediator of cardiac tissue inflammation and fibrosis. The goal of the present study was to determine the cellular mechanisms of MR signaling in macrophages that promote cardiac tissue injury and remodeling. We sought to identify specific markers of MR signaling in isolated tissue macrophages (cardiac, aortic) vs splenic mononuclear cells from wild-type and myeloid MR-null mice given vehicle/salt or deoxycorticosterone (DOC)/salt for 8 weeks. Cardiac tissue fibrosis in response to 8 weeks of DOC/salt treatment was found in the hearts from wild-type but not myeloid MR-null mice. This was associated with an increased expression of the profibrotic markers TGF-β1 and matrix metalloproteinase-12 and type 1 inflammatory markers TNFα and chemokine (C-X-C motif) ligand-9 in cardiac macrophages. Differential expression of immunomodulatory M2-like markers (eg, arginase-1, macrophage scavenger receptor 1) was dependent on the tissue location of wild-type and MR-null macrophages. Finally, intact MR signaling is required for the phosphorylation of c-Jun NH2-terminal kinase in response to a proinflammatory stimulus in bone marrow monocytes/macrophages in culture. These data suggest that the activation of the c-Jun NH2-terminal kinase pathway in macrophages after a tissue injury and inflammatory stimuli in the DOC/salt model is MR dependent and regulates the transcription of downstream profibrotic factors, which may represent potential therapeutic targets in heart failure patients.

Angiotensin II (Ang II) promotes vascular disease through several mechanisms including by producing oxidative stress and endothelial dysfunction. Although multiple potential sources of reactive oxygen species exist, the relative importance of each is unclear, particularly in individual vascular beds. In these experiments, we examined the role of NADPH oxidase (Nox1 and Nox2) in Ang II-induced endothelial dysfunction in the cerebral circulation. Treatment with Ang II (1.4 mg·kg−1·day−1 for 7 days), but not vehicle, increased blood pressure in all groups. In wild-type (WT; C57Bl/6) mice, Ang II reduced dilation of the basilar artery to the endothelium-dependent agonist acetylcholine compared with vehicle but had no effect on responses in Nox2-deficient (Nox2−/y) mice. Ang II impaired responses to acetylcholine in Nox1 WT (Nox1+/y) and caused a small reduction in responses to acetylcholine in Nox1-deficient (Nox1−/y) mice. Ang II did not impair responses to the endothelium-independent agonists nitroprusside or papaverine in either group. In WT mice, Ang II increased basal and phorbol-dibutyrate-stimulated superoxide production in the cerebrovasculature, and these increases were abolished in Nox2−/y mice. Overall, these data suggest that Nox2 plays a relatively prominent role in mediating Ang II-induced oxidative stress and cerebral endothelial dysfunction, with a minor role for Nox1.

Angiotensin II (Ang II) produces oxidative stress and endothelial dysfunction in blood vessels. The vasculature from females may be protected against deleterious effects of Ang II. We tested the hypothesis that manganese superoxide dismutase (MnSOD) protects against Ang II–induced endothelial dysfunction. Experiments were performed in C57Bl/6, wild-type (MnSOD +/+ ), and MnSOD-deficient (MnSOD +/− ) mice treated systemically with vehicle or Ang II. Basilar arteries were isolated from mice treated for 1 week with a nonpressor dose of Ang II (0.28 mg/kg per day). Ang II treatment produced superoxide-mediated impairment of responses to the endothelium-dependent vasodilator acetylcholine ( P +/+ mice, Ang II modestly inhibited responses to acetylcholine ( P +/− mice ( P P +/− female mice. Vascular superoxide levels after treatment with an inhibitor of CuZn and extracellular superoxide dismutase were higher in Ang II–treated versus vehicle-treated MnSOD +/− mice. Thus, a nonpressor dose of Ang II produces endothelial dysfunction in male mice only, suggesting that the female vasculature is protected from Ang II. In male but not female mice, MnSOD deficiency enhanced endothelial dysfunction, suggesting that MnSOD normally protects the vasculature during disease states in which Ang II contributes to vascular dysfunction.

Levels of reactive oxygen species, including hydrogen peroxide , increase in blood vessels during hypertension and in response to angiotensin II (Ang II). Although glutathione peroxidases are known to metabolize hydrogen peroxide, the role of glutathione peroxidase during hypertension is poorly defined. We tested the hypothesis that glutathione peroxidase-1 protects against Ang II–induced endothelial dysfunction. Responses of carotid arteries from Gpx1 -deficient ( Gpx1 +/− and Gpx1 −/− ) and Gpx1 transgenic mice, and their respective littermate controls, were examined in vitro after overnight incubation with either vehicle or Ang II. Under control conditions, relaxation to acetylcholine (ACh; an endothelium-dependent agonist) was similar in control, Gpx1 +/− , and Gpx1 transgenic mice, whereas in Gpx1 −/− mice, responses to ACh were impaired. In control mice, ACh-induced vasorelaxation was not affected by 1 nmol/L of Ang II. In contrast, relaxation to ACh in arteries from Gpx1 +/− mice was inhibited by ≈60% after treatment with 1 nmol/L of Ang II, indicating that Gpx1 haploinsufficiency markedly enhances Ang II–induced endothelial dysfunction. A higher concentration of Ang II (10 nmol/L) selectively impaired relaxation to ACh in arteries from control mice, and this effect was prevented in arteries from Gpx1 transgenic mice or in arteries from control mice treated with polyethylene glycol-catalase (which degrades hydrogen peroxide). Thus, genetic and pharmacological evidence suggests a major role for glutathione peroxidase-1 and hydrogen peroxide in Ang II–induced effects on vascular function.

Background and purpose Equol is the main active intestinal metabolite of the isoflavone daidzein and is postulated to be responsible for the cardiovascular benefits of soy. Cerebral vascular effects of equol are unknown. We compared the vasorelaxant and antioxidant effects of equol and daidzein in carotid and basilar artery of normal and hypertensive rats. Experimental approach Relaxant responses to equol and daidzein were measured in the isolated carotid artery and in the basilar artery in vivo. Effects of nitric oxide synthase (NOS) inhibition, high extracellular K+, endothelial removal and gender on responses to equol were investigated in carotid arteries. Antioxidant activity was assessed as the reduction of NADPH-induced superoxide levels. Hypertension was induced using angiotensin II (0.7 mg/kg per day for 14 days). Key results In normotensive rats, equol displayed vasorelaxant activity similar to daidzein. The relaxant effect of equol was independent of an intact endothelium, NOS activity, K+ channels and gender. In the basilar artery, where superoxide levels are higher, equol exerted weak antioxidant effects, whereas effects of daidzein were insignificant. During hypertension, equol-induced vasorelaxation was preserved, whereas relaxant responses to daidzein were impaired. Conclusions and implications Equol possesses substantial vasodilator and weak antioxidant activity in cerebral arteries, with similar activity to daidzein, whereas in hypertension the vasorelaxant response to equol, but not daidzein, is preserved. However, daidzein possesses comparable direct vascular effects with equol, without the need for intestinal conversion to equol. Nevertheless, equol may represent a more useful therapeutic agent during cerebral vascular disease.

Elevated aldosterone levels, which promote cerebral vascular oxidative stress, inflammation, and endothelial dysfunction, may increase stroke risk, independent of blood pressure and other risk factors. The main target receptor of aldosterone, the mineralocorticoid receptor (MR), is expressed in many cell types, including endothelial cells. Endothelial cell dysfunction is thought to be an initiating step contributing to cardiovascular disease and stroke; however the importance of MR expressed on endothelial cells in the brain is unknown. Here we have examined whether endothelial cell MR mediates cerebral vascular oxidative stress and brain inflammation during aldosterone excess. In male mice, aldosterone (0.72 mg/kg/day, 14 days) caused a small increase (~14 mmHg) in blood pressure. The MR blocker spironolactone (25 mg/kg/d, ip) abolished this increase, whereas endothelial cell MR-deficiency had no effect. Aldosterone increased superoxide production capacity in cerebral arteries, and also mRNA expression of the pro-inflammatory cytokines chemokine (C–C motif) ligand 7 (CCL7), CCL8 and interleukin (IL)-1β in the brain. These increases were prevented by both spironolactone treatment and endothelial cell MR-deficiency; whereas IL-1β expression was blocked by spironolactone only. Endothelial cell MR mediates aldosterone-induced increases in cerebrovascular superoxide levels and chemokine expression in the brain, but not blood pressure or brain IL-1β. Endothelial cell-targeted MR antagonism may represent a novel approach to treat cerebrovascular disease and stroke, particularly during conditions of aldosterone excess.

Background and Purpose— Premenopausal women are less susceptible to cardiovascular diseases than men or postmenopausal women. Such disease states are often associated with increased vascular RhoA/Rho-kinase activity and decreased activity of nitric oxide (NO). This study tested whether female gender is associated with lower Rho-kinase activity or higher NO activity in cerebral arteries in vivo and whether estrogen contributes to any such gender differences. Methods— Changes in basilar artery diameter were measured with the use of a cranial window preparation in anesthetized Sprague-Dawley rats. Some female rats were ovariectomized (OVX) and treated subcutaneously daily for 14 days with vehicle (dimethyl sulfoxide) or 17β-estradiol. Vascular expression of RhoA or Rho-kinase was assessed by Western blotting. Results— The Rho-kinase inhibitor Y-27632 was selectively ≈3-fold more potent as a cerebral vasodilator in males versus females. Expression of total RhoA or Rho-kinase did not differ between males and females. In OVX rats, vasodilator responses to Y-27632 resembled responses in males. Treatment of OVX rats with 17β-estradiol normalized the vasodilator effects of Y-27632 to be equivalent to responses in intact female controls. The NO synthase inhibitor N -nitro- l -arginine methyl ester caused ≈50% greater constriction of the basilar artery in females versus males, but responses in OVX rats treated with either vehicle or 17β-estradiol did not differ from those recorded in intact females. Conclusions— These data indicate that vascular Rho-kinase function is suppressed in females because of the effects of estrogen, whereas the higher NO activity in females is estrogen independent.

An elevated plasma aldosterone level is an independent cardiovascular risk factor. Although excess aldosterone promotes cardiovascular disease, no studies have examined the effect of increased plasma aldosterone on the cerebral circulation. A major source of vascular reactive oxygen species (ROS) during cardiovascular disease is the NADPH oxidases. Because Nox2-containing NADPH oxidase (Nox2 oxidase) is highly expressed in the cerebral endothelium, we postulated that it might contribute to ROS generation and vascular dysfunction in response to aldosterone. Here, we examined the effect of aldosterone and Nox2 oxidase on ROS production and endothelial dysfunction in the cerebral circulation, and whether the effects of aldosterone are exacerbated in aged mice.In adult (average age ∼24-25 weeks) wild-type and Nox2-deficient (Nox2(/y)) mice, neither vehicle nor aldosterone (0.28 mg/kg per day for 14 days) affected blood pressure (measured using tail-cuff). By contrast, aldosterone treatment reduced dilation of the basilar artery (measured using myography) to the endothelium-dependent agonist acetylcholine in wild-type mice (P 0.05), but had no such effect in Nox2(/y) mice (P 0.05). Aldosterone increased basal and phorbol dibutyrate-stimulated superoxide production (measured using L-012-enhanced chemiluminesence) in cerebral arteries from wild-type but not from Nox2(/y) mice. In aged wild-type mice (average age ∼70 weeks), aldosterone treatment increased blood pressure, but had a similar effect on cerebral artery superoxide levels as in adult wild-type mice.These data indicate that Nox2 oxidase mediates aldosterone-induced increases in ROS production and endothelial dysfunction in cerebral arteries from adult mice independently of blood pressure changes. Aldosterone-induced hypertension is augmented during aging.

Abstract —The small G protein Rho and its target Rho-kinase may participate in the mechanisms underlying vascular contractile tone via inhibition of myosin light chain phosphatase. The present study has tested the hypothesis that Rho-kinase activity normally contributes to cerebral vascular tone in vivo, and that this effect is augmented during chronic hypertension. Comparative studies also examined the role of protein kinase C (PKC) in regulation of cerebral artery tone. Two Rho-kinase inhibitors, Y-27632 (0.1 to 100 μmol/L) and HA1077 (1 to 10 μmol/L), caused marked concentration-dependent increases in basilar artery diameter of anesthetized normotensive rats (Sprague-Dawley and Wistar-Kyoto [WKY] strains), as measured using a cranial window approach. By comparison, the selective PKC inhibitors calphostin C (0.01 to 0.5 μmol/L) and Ro 31-8220 (5 μmol/L) had little or no effect on basilar artery diameter. Vasodilator responses to Y-27632 were unaffected by PKC inhibition or activation. In two models of chronic hypertension (spontaneously hypertensive rats and WKY rats treated with N -nitro- l -arginine methyl ester for 4 weeks), Y-27632 elicited cerebral vasodilator responses that were significantly greater than in control WKY rats ( P

In the normal physiologic state, reactive oxygen species (ROS) generation is intentional and important for the functioning of cerebral and systemic circulations. Furthermore, emerging evidence indicates that cerebral arteries generate higher levels of ROS than arteries outside of the brain in the normal physiologic state. As such, it has been proposed that ROS may play a more prominent role in the physiologic regulation of cerebral arteries. There are numerous potential enzymatic sources of ROS in the cerebral vasculature; however, increasing evidence indicates that the family of NADPH oxidases is a major source. Aberrant redox signaling or oxidative stress in the cerebral circulation, usually as a result of excessive production of ROS and reactive nitrogen species (RNS), is a common feature in diverse models of cardiovascular risk factors (e.g., hypertension, hypercholesterolemia) and cerebrovascular disease. Furthermore, oxidative stress is now believed to be an underlying cause of cerebrovascular dysfunction and damage associated with these disease states. In this chapter, we summarize the effects and potential roles of ROS/RNS in modulating cerebral artery function in the normal physiologic state, with a particular focus on their roles in modulating cerebrovascular tone. Furthermore, we will highlight current evidence for the involvement of ROS/RNS in cerebrovascular dysfunction associated with cardiovascular risk factors, stroke, and Alzheimer's disease.

Background and Purpose— Receptors for calcitonin gene-related peptide (CGRP) are composed of the calcitonin-like receptor in association with receptor activity-modifying protein-1 (RAMP1). CGRP is an extremely potent vasodilator and may protect against vascular disease through other mechanisms. Methods— We tested the hypothesis that overexpression of RAMP1 enhances vascular effects of CGRP using transgenic mice with ubiquitous expression of human RAMP1. Because angiotensin II (Ang II) is a key mediator of vascular disease, we also tested the hypothesis that RAMP1 protects against Ang II-induced vascular dysfunction. Results— Responses to CGRP in carotid and basilar arteries in vitro as well as cerebral arterioles in vivo were selectively enhanced in human RAMP1 transgenic mice compared to littermate controls ( P P P Conclusions— RAMP1 overexpression increases CGRP-induced vasodilation and protects against Ang II-induced endothelial dysfunction. These findings suggest that RAMP1 may be a new therapeutic target to regulate CGRP-mediated effects during disease including pathophysiological states in which Ang II plays a major role.

Oxidative stress refers to increases in reactive metabolites of molecular oxygen that occur as a result of increases in formation and/or reductions in scavenging or degradation. Oxidative stress plays an important role in the pathogenesis of hypertension. In this chapter, we examine the relationship between oxidative stress in the development and maintenance of hypertension and changes in vascular structure and function. We focus primarily on enzymatic systems that contribute to oxidative stress, such as NAD(P)H oxidase, and those that limit oxidative stress, including superoxide dismutase, glutathione peroxidase, and catalase.

The mineralocorticoid receptor (MR), well known to be expressed in renal epithelial cells where it is important in fluid and electrolyte homeostasis, has aldosterone as one of its main agonists. Much research in the last 10–15 years indicates that MRs are also expressed outside of the kidney, including in the brain, vasculature and heart, where they contribute to the pathophysiology of disease (Dinh et al., 2012; Jaisser and Farman, 2016). Excess aldosterone is a cardiovascular risk factor, and MR antagonism is beneficial in the setting of cardiovascular disease (both clinically and experimentally), including in experimental stroke, whereby MR antagonism is beneficial in reducing both cerebral infarct size (Iwanami et al., 2007; Oyamada et al., 2008) and cerebral vascular remodeling (reviewed in Dinh et al., 2012) following cerebral ischemia. MR antagonism also reverses remodeling, both during aldosterone/mineralocorticoid excess and following cerebral ischemia. The advent of technology to generate mice which lack specific genes in specific cell types has allowed investigation into the contribution of MR in cell types such as vascular endothelial and myeloid cells to the pathophysiology of cerebrovascular disease and stroke (Frieler et al., 2011, 2012; Dinh et al., 2016). Endothelial cell MRs mediate cerebrovascular oxidative stress and brain inflammation in response to excess aldosterone (Dinh et al., 2016), and myeloid MR contribute to the ischemic damage, inflammation and neurological impairment following cerebral ischemia/reperfusion (Frieler et al., 2011, 2012). These and further investigations into the contribution of cell-specific MRs to cerebrovascular disease and stroke can help guide the future design of therapeutic strategies for stroke treatment.

Background and Purpose— It is not known whether cerebral vasoprotective mechanisms in females include increased function of arterial K + channels. We hypothesized that vasodilator responses mediated by activation of inwardly rectifying K + (K IR ) channels are greater in cerebral arteries of female versus male rats and that this is due to the effects of estrogen. Methods— Changes in basilar artery diameter were measured with a cranial window preparation in anesthetized Sprague-Dawley rats. Results— K + (5 and 10 mmol/L) caused greater vasodilatation in females (percent maximum, 21±3% and 58±7%, respectively) versus males (11±1% and 37±4%, respectively; P IR channel inhibitor barium ion (30 μmol/L) decreased basilar artery diameter in males but not females (−7±1% versus −2±1%, P + -induced vasodilatation by ≈50% in both groups. Ovariectomy of female rats resulted in smaller vasodilator effects of K + , and chronic treatment of these rats with 17β-estradiol (0.01 mg/kg per day for 7 days) normalized K+-induced vasodilatation. Furthermore, the selective M2 muscarinic ACh receptor antagonist methoctramine (1 μmol/L) increased responses to K + in males to levels equivalent to responses in females but had no effect on responses to K + in females. Conclusions— K + is a more powerful vasodilator in the female versus male cerebral circulation. This difference is estrogen dependent and could be due to a lack of M2 muscarinic ACh receptor–induced inhibition of K IR channel activation by K + in female cerebral arteries.

Background and Purpose— We examined the importance of NADPH-oxidase in reactive oxygen species production in cerebral arteries and its effect on vascular tone in vivo. Furthermore, we investigated whether chronic hypertension affects function or expression of this enzyme in cerebral vessels. Methods— Superoxide generation was detected in isolated rat basilar arteries with the use of lucigenin-enhanced chemiluminescence. mRNA expression of NADPH-oxidase subunits was assessed by real-time polymerase chain reaction. Basilar artery diameter was measured with the use of a cranial window preparation in anesthetized rats. Results— NADPH-stimulated superoxide production was 2.3-fold higher in arteries from spontaneously hypertensive rats (SHR) versus normotensive Wistar-Kyoto rats (WKY) and could be blocked by the NADPH-oxidase inhibitor diphenyleneiodonium. Higher NADPH-oxidase activity was also reflected at the molecular level as mRNA expression of the NADPH-oxidase subunit Nox4 was 4.1-fold higher in basilar arteries from SHR versus WKY. In contrast, expression of Nox1, gp91phox, p22phox, and p47phox did not differ between strains. Application of NADPH to basilar arteries caused larger vasodilatation in SHR than WKY. Vasodilatation to NADPH could be attenuated by diphenyleneiodonium, as well as diethyldithiocarbamate (Cu 2+ /Zn 2+ –superoxide dismutase inhibitor), catalase (H 2 O 2 scavenger), or tetraethylammonium (BK Ca channel inhibitor). Conclusions— Activation of NADPH-oxidase in cerebral arteries generates superoxide, which is dismutated by Cu 2+ /Zn 2+ –superoxide dismutase to H 2 O 2 . H 2 O 2 then elicits vasodilatation via activation of BK Ca channels. Upregulation of Nox4 during chronic hypertension is associated with elevated cerebral artery NADPH-oxidase activity.

Background and Purpose — The structurally related, inwardly rectifying K + (K IR ) channel and the ATP-sensitive K + (K ATP ) channel are important modulators of cerebral artery tone. Although protein kinase C (PKC) activators have been shown to inhibit these channels with the use of patch-clamp electrophysiology, effects of PKC on K + channel function in intact cerebral blood vessels are unknown. We therefore tested whether pharmacological alteration of PKC activity affects cerebral vasodilator responses to K IR and/or K ATP channel activators in vivo. Methods — We measured changes in basilar artery diameter using a cranial window preparation in anesthetized rats. In addition, intracellular recordings of smooth muscle membrane potential were made in isolated basilar arteries. Results — K + (5 to 15 mmol/L) and aprikalim (1 to 10 μmol/L) each elicited reproducible vasodilatation. The PKC activator phorbol-12,13-dibutyrate (PdBu) (50 nmol/L) inhibited responses to K + (by 40% to 55%) and aprikalim (by 40% to 70%), whereas responses to papaverine were unaffected. The PKC inhibitor calphostin C (0.1 μmol/L) augmented responses to K + (by 2- to 3-fold) and aprikalim (2-fold) but not papaverine. In addition, K + (5 mmol/L) and aprikalim (3 μmol/L) each hyperpolarized the basilar artery. PdBu inhibited these responses to aprikalim by 45% but had no effect on K + -induced hyperpolarization. Conclusions — These data suggest that both basal and stimulated PKC activity inhibit K IR and K ATP channel–mediated cerebral vasodilatation in vivo. The inhibitory effect on K ATP channel–mediated vasodilatation occurs at least partly by inhibition of hyperpolarization mediated by K ATP channels. PKC inhibits K + -induced vasodilatation without affecting hyperpolarization, suggesting that the inhibitory effect of PKC on vasodilator responses to K + does not involve altered K IR channel function.

Potassium ion (K + ) normally causes cerebral vasodilatation by activating inwardly rectifying K + (K IR ) channels. We tested whether chronic hypertension affects the magnitude and/or mechanism of K + -induced cerebral vasodilatation in vivo. Basilar artery responses were examined in anesthetized Wistar-Kyoto (WKY; mean arterial pressure, 114±4 mm Hg) and spontaneously hypertensive (SHR; 176±3 mm Hg) rats. In WKY, elevating cerebrospinal fluid K + concentration from 3 mmol/L to 5 and 10 mmol/L caused vasodilatation (percent maximum, 12±1 and 48±7, respectively). The response to 5 mmol/L K + was greater in SHR (percent maximum, 17±2 [ P IR channel inhibitor, barium ion (Ba 2+ , 100 μmol/L) selectively inhibited dilator responses to 5 and 10 mmol/L K + by ≈75% in WKY. In SHR, Ba 2+ had no effect on the response to 5 mmol/L K + , and only partially inhibited (by ≈40%) the response to 10 mmol/L K + . The nonselective NO synthase (NOS) inhibitor Nω-nitro- l -arginine methyl ester, the neuronal NOS (nNOS) inhibitor 1-(2-trifluromethyl-phenyl)imidazole, and the N -type calcium channel inhibitor ω-conotoxin GVIA, were all without effect in WKY, but markedly inhibited the response to 5 mmol/L K + in SHR. When applied together with Ba 2+ , each of these inhibitors also profoundly reduced responses to 10 mmol/L K + in SHR. Immunostaining of basilar arteries revealed that the perivascular nNOS-containing nerve plexus was denser in SHR. Thus, K + dilates the normotensive basilar artery predominantly via K IR channel activation. During chronic hypertension, small physiological elevations in K + dilate the basilar artery by an nNOS-dependent mechanism that appears to be upregulated in a compensatory manner.

Dilatation of cerebral arterioles in response to arachidonic acid is dependent on activity of cyclooxygenase. In this study, we examined mechanisms that mediate dilatation of the basilar artery in response to arachidonate. Diameter of the basilar artery (baseline diameter = 216 ± 7 μm) (means ± SE) was measured using a cranial window in anesthetized rats. Arachidonic acid (10 and 100 μM) produced concentration-dependent vasodilatation that was not inhibited by indomethacin (10 mg/kg iv) or NG-nitro-l-arginine (100 μM) but was inhibited markedly by baicalein (10 μM) or nordihydroguaiaretic acid (NDGA; 10 μM), inhibitors of the lipoxygenase pathway. Dilatation of the basilar artery was also inhibited markedly by tetraethylammonium ion (TEA; 1 mM) or iberiotoxin (50 nM), inhibitors of calcium-dependent potassium channels. For example, 10 μM arachidonate dilated the basilar artery by 19 ± 7 and 1 ± 1% in the absence and presence of iberiotoxin, respectively. Measurements of membrane potential indicated that arachidonate produced hyperpolarization of the basilar artery that was blocked completely by TEA. Incubation with [3H]arachidonic acid followed by reverse-phase and chiral HPLC indicated that the basilar artery produces relatively small quantities of prostanoids but large quantities of 12(S)-hydroxyeicosatetraenoic acid (12-S-HETE), a lipoxygenase product. Moreover, the production of 12-HETE was inhibited by baicalein or NDGA. These findings suggest that dilatation of the basilar artery in response to arachidonate is mediated by a product(s) of the lipoxygenase pathway, with activation of calcium-dependent potassium channels and hyperpolarization of vascular muscle.

Maintenance of vascular tone by the endothelium involves the production of endothelium-derived nitric oxide (NO). NO, produced from endothelial nitric oxide synthase diffuses to the underlying smooth muscle to stimulate soluble guanylate cyclase, resulting in increased cyclic GMP levels, and subsequent smooth muscle relaxation and blood vessel dilatation. Endothelial dysfunction, manifested as diminished NO bioavailability, is a common feature of a number of vascular-related diseases.. Oxidative stress can be defined as an imbalance between reactive oxygen species (ROS) production and/or impaired ROS metabolism that favours them being present in excess of physiological levels. Oxidative stress can negatively impact many cell types, including in the vasculature. There is now a wealth of evidence suggesting that oxidative stress is a major cause of endothelial dysfunction in the cerebral circulation. This review will summarize disease models in which both oxidative stress and endothelial dysfunction occur in the cerebral circulation, namely hypertension involving angiotensin II (Ang II), diabetes, subarachnoid hemorrhage, stroke and Alzheimer's disease. Molecular mechanisms by which oxidative stress occurs, (eg increased NADPH-oxidase activity) will also be discussed.