Responses Of Cerebral Arterioles Research Articles

Prenatal exposure to alcohol impairs responses of cerebral arterioles to activation of potassium channels: Role of oxidative stress.

Potassium channels play an important role in the basal tone and dilation of cerebral resistance arterioles in response to many stimuli. However, the effect of prenatal alcohol exposure (PAE) on specific potassium channel function remains unknown. The first goal of this study was to determine the influence of PAE on the reactivity of cerebral arterioles to activation of ATP-sensitive potassium (KATP ) and BK channels. Our second goal was to determine whether oxidative stress contributed to potassium channel dysfunction of cerebral arterioles following PAE. We fed Sprague-Dawley dams a liquid diet with or without alcohol (3% EtOH) for the duration of their pregnancy (21 to 23 days). We examined in vivo responses of cerebral arterioles in control and PAE male and female offspring (14 to 16 weeks after birth) to activators of potassium channels (Iloprost [BK channels] and pinacidil [KATP channels]), before and following inhibition of oxidative stress with apocynin. We found that PAE impaired dilation of cerebral arterioles in response to activation of potassium channels with iloprost and pinacidil, and this impairment was similar in male and female rats. In addition, treatment with apocynin reversed the impaired vasodilation to iloprost and pinacidil in PAE rats to levels observed in control rats. This effect of apocynin also was similar in male and female rats. PAE induces dysfunction in the ability of specific potassium channels to dilate cerebral arterioles which appears to be mediated by an increase in oxidative stress. We suggest that these alterations in potassium channel function may contribute to the pathogenesis of cerebral vascular abnormalities and/or behavioral/cognitive deficits observed in fetal alcohol spectrum disorders.

Sex-related differences in reactivity of cerebral arterioles during moderate exercise training.

Our goals were to determine the influence of sex on reactivity of cerebral arterioles and whether MExT could influence sex-related differences in reactivity of cerebral arterioles. Responses of cerebral arterioles were measured in Sed and MExT adult male and female Sprague-Dawley rats to eNOS-dependent (ADP), nNOS-dependent (NMDA), and NOS-independent (nitroglycerin) agonists before and following L-NMMA. In addition, protein expression for eNOS and nNOS was determined. NOS-dependent vasodilation was enhanced in Sed and MExT female rats compared to their male counterparts. L-NMMA produced a greater decrease in baseline diameter of arterioles in females compared to males, and produced less inhibition of NOS-dependent vasodilation in females. Expression of eNOS protein was significantly increased in Sed female when compared to Sed male rats; nNOS protein was similar in Sed males and females, but increased in MExT females. The findings from this study indicate that while NOS-dependent vascular reactivity is increased in females, MExT does not alter vasodilation in males or females. These studies provide insights into the influence of sex and MExT on the cerebral microcirculation and may have implications regarding mechanisms that protect the brain in females compared to males.

Influence of type 1 diabetes on basal and agonist-induced permeability of the blood-brain barrier.

Type 1 diabetes mellitus (T1D) impairs endothelial nitric oxide synthase (eNOS)-dependent responses of cerebral arterioles. However, the influence of T1D on another critical aspect of endothelial cell function in the cerebral microcirculation, i.e., regulation of permeability of the blood-brain barrier (BBB), remains largely unknown. Our goal was to examine basal and agonist-induced changes in permeability of the BBB in nondiabetic and type 1 diabetic (streptozotocin; 50mg/kg IP) rats. On the day of the experiment (2-3months after streptozotocin), a craniotomy was made over the parietal cortex in nondiabetic and diabetic rats. We measured the permeability of the BBB (FITC-dextran-10K) under basal conditions and during application of histamine. We also measured diameter of cerebral arterioles in response to histamine in the absence and presence of NG-monomethyl-L-arginine (L-NMMA). We found that basal permeability of the BBB was elevated in T1D and application of histamine did not produce a further increase in permeability. In contrast, basal permeability of the BBB was minimal in nondiabetics and histamine produced an increase in permeability. In addition, histamine-induced arteriolar dilation was less in diabetics than in nondiabetics, and vasodilation to histamine was inhibited by L-NMMA. Our findings suggest that T1D-induced endothelial dysfunction leads to an increase in basal permeability of the BBB, but decreases the ability of the endothelium of the BBB to respond to an important inflammatory mediator. Thus, T1D impairs two critical aspects of endothelial cell function in the cerebral microcirculation, i.e., basal and agonist-induced changes in permeability of the BBB and arteriolar dilation.

Abstract 72: Vascular and Cardiac Effects of Inducible Nitric Oxide Synthase in Hyperhomocysteinemic Mice

Background Diet-induced hyperhomocysteinemia (HHcy) produces endothelial and cardiac dysfunction and promotes thrombosis through a mechanism proposed to involve oxidative stress. Inducible nitric oxide synthase (iNOS) is upregulated in HHcy and can generate oxidants such as superoxide. We tested the hypothesis that iNOS mediates the adverse oxidative vascular, thrombotic, and cardiac effects of HHcy. Methods Mice deficient in iNOS ( Nos2-/ -) and their wild-type (WT) littermates were fed either a control (C) diet or a high methionine/low folate (HM/LF) diet for 3-6 months. Vasomotor function was assessed in cerebral arterioles in response to the endothelium-dependent dilator acetylcholine or the endothelium-independent dilator nitroprusside. Thrombotic occlusion of the carotid artery was measured in response to photochemical injury (rose bengal). Regional myocardial injury was measured after 30 minutes of ischemia and 2 hours of reperfusion of the left coronary artery. Results Nos2 -/- and WT mice fed the HM/LF diet developed similar levels of HHcy, with a 2-fold increase in plasma total homocysteine (P<0.001 vs. C diet). As expected, WT mice with HHcy exhibited endothelial dysfunction, with impaired dilatation to acetylcholine but not nitroprusside, and enhanced susceptibility to thrombosis, with shortened times to occlusion of the carotid artery (P<0.05 vs. C diet). Interestingly, deficiency of iNOS resulted in decreased, rather than increased dilatation responses to acetylcholine (P<0.05 vs. WT mice). Nos2 -/- mice also exhibited shortened times to thrombotic occlusion in mice fed the C diet (P<0.05 vs. WT mice) and failed to protect from endothelial dysfunction or accelerated thrombosis in mice fed the HM/LF diet. Deficiency of iNOS did not alter myocardial infarct size in mice fed the C diet but significantly increased infarct size and cardiac superoxide production in mice fed the HM/LF diet (P<0.05 vs. WT mice). Conclusions These findings suggest that endogenous iNOS protects from, rather than exacerbates, endothelial dysfunction, thrombosis, and myocardial ischemia-reperfusion injury. In the setting of HHcy, iNOS appears to function to blunt cardiac oxidative stress rather than as a source of superoxide.

Glutathione Improves Impaired Responses of Cerebral Arterioles in Type 1 Diabetes

Our goal was to examine whether glutathione improves impaired nitric oxide synthase‐dependent responses of cerebral arterioles during Type 1 diabetes. We measured diameter of cerebral arterioles in nondiabetic and diabetic (streptozotocin; 50 mg/kg) rats to nitric oxide synthase‐dependent, acetylcholine and adenosine 5’‐diphosphate (ADP) and a nitric oxide synthase‐independent agonists, nitroglycerin. We measured responses before and during topical application of glutathione (5 mM). In addition, we measured superoxide production by brain tissue in nondiabetic and diabetic rats. In nondiabetic rats, acetylcholine and ADP produced dilatation of cerebral arterioles. However, there was significant impairment in reactivity of arterioles to acetylcholine and ADP in diabetic rats. Vasodilatation to nitroglycerin was similar in nondiabetic and diabetic rats. Glutathione did not alter baseline diameter of cerebral arterioles, but significantly improved impaired nitric oxide synthase‐dependent vasodilatation in diabetic rats. Superoxide production was increased under basal states in diabetic rats and glutathione restored superoxide levels to that observed in nondiabetic rats. Our findings suggest that glutathione selectively improves endothelial dysfunction of cerebral arterioles during Type 1 diabetes.

Long-term oral vitamin C administration improves cerebral microvascular vasodilatory impairment in diabetes: in vivo evidence using diabetic rats

Background: Many clinical reports have indicated that ascorbic acid (vitamin C) improves vasodilatory impairments in patients with diabetes mellitus, but there is very little in vivo evidence to demonstrate its effectiveness on the brain. Objective: To investigate long-term effects of oral vitamin C administration on the cerebral microvascular vasodilation in diabetes, using streptozotocin (STZ)-induced diabetic rats. Materials and methods: Diabetes was induced in male Wistar Furth rats by a single intravenous injection of STZ (55 mg/kg b.w). Ascorbic acid (vitamin C) was administered in drinking water (1g/l). The rats were divided into control and diabetic groups with or without administration of vitamin C. The cerebral microcirculation was observed at different times (12, 24 and 36 weeks) after vitamin C supplementation, using fluorescence videomicroscopy. Responses of cerebral arterioles to acetylcholine (ACh), adenosine-5 diphosphate (ADP) and nitroglycerine (NTG) were studied by measuring diameters of cerebral arterioles before and after topical application on the cortical surface. Results: The vasodilatory responses of cerebral arterioles to ACh and ADP were significantly decreased in diabetic rats, compared with non-diabetic (control) rats. The response to NTG was not altered in diabetic rats, indicating that the vasodilatory impairment involves at the endothelium. The impaired endothelium-dependent vasodilation was prevented by long-term vitamin C administration. Conclusion: Long-term oral vitamin C administration might be of clinical relevance in improving cerebral microvascular vasodilatory impairment in diabetes.

Hypertrophy of Cerebral Arterioles in Mice Deficient in Expression of the Gene for CuZn Superoxide Dismutase

Reactive oxygen species are believed to be an important determinant of vascular growth. We examined effects of genetic deficiency of copper-zinc superoxide dismutase (CuZnSOD; SOD1) on structure and function of cerebral arterioles. Systemic arterial pressure (SAP) and cross-sectional area of the vessel wall (CSA) and superoxide (O2-) levels (relative fluorescence of ethidium [ETH]) were examined in maximally dilated cerebral arterioles in mice with targeted disruption of one (+/-) or both (-/-) genes encoding CuZnSOD. Wild-type littermates served as controls. Vasodilator responses were tested in separate groups of mice. CSA and ETH were significantly increased (P<0.05) in both CuZnSOD+/- and CuZnSOD-/- mice (CSA=435+/-24 and 541+/-48 microm2; ETH=18+/-1 and 34+/-2%) compared with wild-type mice (CSA=327+/-28 microm2; ETH=6%). Furthermore, the increases in CSA and ETH relative to wild-type mice were significantly greater (P<0.05) in CuZnSOD-/- mice than in CuZnSOD+/- mice (CSA=108 versus 214 microm2; ETH=12 versus 28%). In addition, dilatation of cerebral arterioles in response to acetylcholine, but not nitroprusside, was reduced by approximately 25% in CuZnSOD+/- (P<0.075) and 50% in CuZnSOD-/- mice (P<0.05) compared with wild-type mice. Cerebral arterioles in CuZnSOD+/- and CuZnSOD-/- mice undergo marked hypertrophy. These findings provide the first direct evidence in any blood vessel that CuZnSOD normally inhibits vascular hypertrophy suggesting that CuZnSOD plays a major role in regulation of cerebral vascular growth. The findings also suggest a gene dosing effect of CuZnSOD for increases in O2-, induction of cerebral vascular hypertrophy and impaired endothelium-dependent dilatation.

Impaired Endothelium-Dependent Responses and Enhanced Influence of Rho-Kinase in Cerebral Arterioles in Type II Diabetes

Although the incidence of type II diabetes is increasing, very little is known regarding vascular responses in the cerebral circulation in this disease. The goals of this study were to examine the role of superoxide in impaired endothelium-dependent responses and to examine the influence of Rho-kinase on vascular tone in the cerebral microcirculation in type II diabetes. Diameter of cerebral arterioles (29+/-1 microm; mean+/-SE) was measured in vivo using a cranial window in anesthetized db/db and control mice. Dilatation of cerebral arterioles in response to acetylcholine (ACh; 1 and 10 micromol/L), but not to nitroprusside, was markedly reduced in db/db mice (eg, 10 micromol/L ACh produced 29+/-1% and 9+/-1% in control and db/db mice, respectively). Superoxide levels were increased (P<0.05) in cerebral arterioles from db/db mice (n=6) compared with controls (n=6). Vasodilatation to ACh in db/db mice was restored to normal by polyethylene glycol-superoxide dismutase (100 U/mL). Y-27632 (1 to 100 micromol/L; a Rho-kinase inhibitor) produced modest vasodilatation in control mice but much greater responses in db/db mice. N(G)-nitro-L-arginine (100 micromol/L; an inhibitor of NO synthase) significantly enhanced Y-27632-induced dilatation in control mice to similar levels as observed in db/db mice. These findings provide the first evidence for superoxide-mediated impairment of endothelium-dependent responses of cerebral vessels in any model of type II diabetes. In addition, the influence of Rho-kinase on resting tone appears to be selectively enhanced in the cerebral microcirculation in this genetic model of type II diabetes.

Cerebral Vascular Dysfunction in Methionine Synthase-Deficient Mice.

Methionine synthase (MS) is a key enzyme involved in remethylation of homocysteine to methionine. We tested the hypothesis that deficiency of MS influences endothelial function. Mice heterozygous for disruption of the Ms gene (Ms+/−), and wild type littermates (Ms+/+), were fed either a control diet or a low folate (LF) diet. Plasma total homocysteine was similar in Ms+/+ and Ms+/− mice fed the control diet (4.5±0.3 and 5.3±0.4 μmol/L, respectively), and was mildly elevated in Ms+/+ or Ms+/− mice fed the LF diet (7.5±0.7 and 9.6±1.2 μmol/L, respectively; p<0.001 vs. control diet). Dilatation of cerebral arterioles (~30 μm diameter) to the endothelium-dependent dilator, acetylcholine (10 μM), was blunted in Ms+/− mice compared with Ms+/+ mice fed the control diet (21±4 vs. 32±4%; p<0.05). Both Ms+/+ and Ms+/− mice exhibited impaired dilatation of cerebral arterioles to acetylcholine when they were fed the LF diet (12±2 and 17±3%, respectively; p<0.01 vs. Ms+/+ mice fed the control diet). Dilatation of cerebral arterioles in response to the endothelium-independent dilator, nitroprusside, was similar in all groups of mice. Relaxation of aortic rings to acetylcholine was not influenced by Ms genotype or diet. Elevated levels of superoxide and hydrogen peroxide were detected by dihydroethidium (DHE) and dichlorodihydrofluorescein (DCF), respectively, in cerebral arterioles of Ms+/− mice fed the control diet and in both Ms+/+ and Ms+/− mice fed the LF diet. These findings demonstrate that defective homocysteine remethylation caused by deficiency of either MS or folate produces endothelial dysfunction and increased oxidative stress in the cerebral microcirculation of mice. Interestingly, impairment of cerebral vascular function was independent of plasma tHcy concentration.

Altered regulation of endothelin A receptor subtype in the cerebral arterioles in response to a Japanese-style diet, in stroke-prone hypertensive rats.

To investigate the expression of endothelin (ET)-1 and its receptors in the cerebral arterioles of stroke-prone (spSHR) and control spontaneously hypertensive rats (SHRs) and the changes in endothelin receptor subtypes A and B density elicited by a stroke-permissive diet, before the development of stroke. Six-week-old SHRs (n=11) and spSHRs (n=11) were assigned to either a regular or a "Japanese"-style diet, in addition to 1% NaCl in the drinking water, for 4 weeks. Cryosections (10 microm thick) of rat brain were assessed for endothelin receptor distribution and density by autoradiography with [125I]ET-1 (10(-10) mol/l) in the presence of cold ET-1 (10(-6) mol/l) or the peptide antagonists BQ-123 (10(-6) mol/l) or BQ-788 (10(-6) mol/l). Reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry were used to detect specific mRNAs and localize immunoreactive ET-1 and ET(A) and ET(B). In both strains, immunoreactive ET-1 was detected in the endothelium of cerebral arterioles, and RT-PCR and autoradiography demonstrated the coexistence of both receptor subtypes in brain homogenates and the cerebral arteriole walls, respectively. With the regular diet, the ET(A) receptor density was lower in SHRs than in spSHRs (P = 0.007), whereas the ET(B) receptor density was similar (P = NS). The Japanese-style diet increased the density of ET(A) receptors (P = 0.006) in SHRs, but decreased it (P = 0.019) in spSHRs. No effect was seen on ET(B) receptor density. ET(A) and ET(B) receptor subtypes are expressed in the wall of cerebral arterioles of SHRs and spSHRs. The latter strain showed a marked increase in ET(A) receptor density under a regular diet, in addition to an altered regulation in response to a stroke-permissive diet.

Arachidonate dilates basilar artery by lipoxygenase-dependent mechanism and activation of K(+) channels.

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 micrometer) (means +/- SE) was measured using a cranial window in anesthetized rats. Arachidonic acid (10 and 100 microM) produced concentration-dependent vasodilatation that was not inhibited by indomethacin (10 mg/kg iv) or N(G)-nitro-L-arginine (100 microM) but was inhibited markedly by baicalein (10 micrometerM) or nordihydroguaiaretic acid (NDGA; 10 microM), 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 microM 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 [(3)H]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.

COX-2-dependent delayed dilatation of cerebral arterioles in response to bradykinin.

Bradykinin (BK) is released in the brain during injury and inflammation. Activation of endothelial BK receptors produces acute dilatation of cerebral arterioles that is mediated by reactive oxygen species (ROS). ROS can also modulate gene expression, including expression of the inducible isoform of cyclooxygenase (COX-2). We hypothesized that exposure of the brain to BK would produce acute dilatation, which would be followed by a delayed dilatation mediated by COX-2. To test this hypothesis in anesthetized rats, BK was placed twice in cranial windows for 7 min, after which the windows were flushed to remove residual BK. The two BK exposures were separated by 30 min. Each BK exposure produced acute dilatation of cerebral arterioles, after which diameter rapidly returned to baseline. Over the subsequent 4.5 h after the second BK exposure, arterioles dilated 48 +/- 8%. Treatment of the cranial window with NS-398, a selective COX-2 inhibitor, or dexamethasone, significantly attenuated the delayed dilatation. Aminoguanidine, a selective inhibitor of inducible nitric oxide synthase, did not alter the delayed dilatation. Cotreatment of cranial windows with BK, superoxide dismutase, and catalase also prevented the delayed dilatation. In separate experiments, exposure of the cortical surface to BK upregulated leptomeningeal expression of COX-2 mRNA. Our results suggest that acute, time-limited exposure of the brain to BK produces delayed dilatation of cerebral arterioles dependent on expression and activity of COX-2.

Expression and vascular effects of cyclooxygenase-2 in brain.

Cyclooxygenase-2 (COX-2) is an inducible isoform of cyclooxygenase. Several types of brain cells in culture can express COX-2 when treated with lipopolysaccharide (LPS) or some cytokines. LPS produces dilatation of cerebral arterioles in vivo through a mechanism that is partially inhibited by indomethacin. In the present study we examined the hypothesis that LPS causes increased expression of COX-2 in brain as well as COX-2-dependent dilatation of cerebral arterioles. Cranial windows were implanted in anesthetized rats and used to measure diameter of cerebral arterioles under control conditions and during topical application of various agonists and antagonists. Windows were flushed every 30 minutes for 4 hours with vehicle (artificial cerebrospinal fluid; n=5), LPS (100 ng/mL; n=8), LPS and NS-398 (100 micromol/L; n=8), a selective inhibitor of COX-2, or LPS and dexamethasone (1 micromol/L; n=5), which attenuates expression of COX-2. To examine expression of COX-2 protein in vivo, other animals were injected intracisternally with artificial cerebrospinal fluid (n=3) or LPS (40 ng; n=4). Four hours after injection, the leptomeninges were harvested and analyzed by Western blot for expression of COX-2 protein. In a third group of experiments, COX-2 expression and prostaglandin E2 (PGE2) production were determined in leptomeningeal tissue treated for 4 hours ex vivo with vehicle (n=4), LPS (100 ng/mL; n=4), LPS and NS-398 (100 micromol/L; n=4), or LPS and dexamethasone (1 micromol/L; n=4). LPS caused marked, progressive dilatation of cerebral arterioles, with a maximum increase in diameter of 55+/-9% (mean+/-SEM) at 4 hours. Coapplication of either NS-398 or dexamethasone with LPS reduced dilatation of cerebral arterioles at hours 2 through 4 (P<0.05). In contrast, NS-398 did not inhibit dilatation of cerebral arterioles in response to bradykinin or ADP. In animals injected intracisternally with vehicle, COX-2 protein was expressed at a very low level in leptomeningeal tissue. Intracisternal injection of LPS increased COX-2 protein expression by approximately 20-fold (P<0.05). In leptomeningeal tissue treated ex vivo with LPS, there was also expression of COX-2. Both dexamethasone and NS-398 markedly reduced COX-2 protein expression in ex vivo LPS-treated tissue. PGE2 production was detectable under control conditions in leptomeningeal tissue incubated in vehicle ex vivo for 4 hours (6.5+/-1.1 pmol/mg protein). LPS treatment significantly increased PGE2 production to 12.8+/-1.1 pmol/mg protein (P<0.05). Both dexamethasone and NS-398 significantly attenuated LPS-induced PGE2 production (P<0.05). LPS increased expression of COX-2 protein in leptomeningeal tissue and caused COX-2-dependent dilatation of cerebral arterioles in vivo. Ex vivo, both NS-398 and dexamethasone suppressed LPS-induced PGE2 production and COX-2 expression in leptomeningeal tissue. Inhibition of LPS-induced dilatation of cerebral arterioles in vivo by NS-398 and dexamethasone suggests that the dilatation was dependent on expression and activity of COX-2. These findings support the concept that exposure of brain to LPS causes cerebral vasodilatation that is dependent in part on expression and activity of COX-2.

Potassium channels mediate dilatation of cerebral arterioles in response to arachidonate.

We tested the hypothesis that cerebral vasodilatation in response to arachidonate is dependent on activation of cyclooxygenase and cytochrome P-450 pathways and formation of endogenous reactive oxygen species and is mediated by activation of potassium channels. The diameter of cerebral arterioles was measured using cranial windows in anesthetized rats. Under control conditions [baseline diameter = 45 +/- 1 micrometer (mean +/- SE)], arachidonate (1-100 microM) and papaverine (10-50 microM) produced concentration-dependent vasodilatation. Cerebral vasodilator responses to arachidonate, but not papaverine, were abolished during topical application of indomethacin (10 microM, an inhibitor of cyclooxygenase) or catalase (100 U/ml, which inactivates hydrogen peroxide). In contrast, clotrimazole (10 microM) and 17-ODYA (20 microM), inhibitors of cytochrome P-450 activity, had no effect on dilator responses of cerebral arterioles to arachidonate. Superoxide dismutase (SOD, 100 U/ml) had no effect on vasodilator responses to papaverine or lower concentrations of arachidonate, whereas dilator responses to 100 microM arachidonate were inhibited modestly (by 22%) by SOD. Similarly, deferoxamine (1 mM) partly inhibited dilator responses to 10 and 100 microM arachidonate (by approximately 30% at each concentration). Tetraethylammonium ion (1 mM) or iberiotoxin (50 nM), inhibitors of calcium-activated potassium channels, markedly inhibited vasodilatation in response to arachidonate (by 70-90%) but not papaverine. These findings suggest that dilatation of cerebral arterioles in response to arachidonate is mediated largely by endogenously formed reactive oxygen species, which are generated from cyclooxygenase activity, and activation of calcium-activated potassium channels. Thus activation of potassium channels appears to be a major mechanism of cerebral vasodilatation in response to reactive oxygen species produced endogenously.

Tumor necrosis factor-alpha-induced dilatation of cerebral arterioles.

In brain, several cell types produce tumor necrosis factor-alpha (TNFalpha) after injury or exposure to endotoxin. TNFalpha alone or in combination with endotoxin or other cytokines can cause expression of inducible nitric oxide (NO) synthase. We have previously demonstrated that endotoxin caused NO-dependent dilatation of cerebral arterioles in vivo. In the present study we examined the hypothesis that TNFalpha causes NO-mediated dilatation of cerebral arterioles in vivo. Cranial windows were implanted in anesthetized rats and used to measure the diameter of cerebral arterioles. Windows were flushed every 30 minutes for 4 hours with artificial cerebrospinal fluid (aCSF) (n=6); aCSF with TNFalpha (100 ng/mL; n=10); aCSF with TNFalpha and aminoguanidine (0.3 mmol/L; n=5), an inhibitor of inducible NO synthase; or aCSF with TNFalpha and dexamethasone (1 micromol/L; n=6), which attenuates expression of inducible NO synthase. In some animals, brain from beneath the cranial window was examined by immunocytochemistry for inducible NO synthase expression. Application of TNFalpha caused marked, progressive dilatation of cerebral arterioles, with a maximum increase in diameter of 46+/-9% (mean+/-SEM) at 4 hours. Coapplication of either aminoguanidine or dexamethasone with TNFalpha prevented dilatation of cerebral arterioles compared with TNFalpha alone (4+/-2% and 1+/-1% dilatation at 4 hours, respectively; P<.05). Dexamethasone did not inhibit dilatation of cerebral arterioles in response to adenosine diphosphate. However, 2 hours of aminoguanidine treatment produced moderate inhibition of adenosine diphosphate-induced dilatation of cerebral arterioles. After treatment with TNFalpha, immunocytochemistry for inducible NO synthase demonstrated expression in perivascular and arachnoid cells but not brain cells. There was no detectable expression of inducible NO synthase after treatment with aCSF. The present study indicates that TNFalpha causes cerebral vasodilatation and expression of inducible NO synthase in perivascular and arachnoid cells. Inhibition of TNFalpha-induced dilatation by aminoguanidine and dexamethasone suggests that the vasodilatation was due predominantly to expression of inducible NO synthase. These findings support the concept that cerebral vasodilatation that occurs during pathophysiological conditions associated with increased TNFalpha production in brain is mediated by expression of inducible NO synthase.

Influence of hypoxia/ischemia on cerebrovascular responses to oxytocin in piglets.

We examined the effects of hypoxic/ischemic stress on cerebral arteriolar responses to oxytocin in anesthetized piglets. Pial arteriolar diameters were measured using a cranial window and intravital microscopy. First, we evaluated arteriolar responses to topical application of oxytocin during normoxic conditions. We then determined whether 5-10 min of arterial hypoxia, ischemia, or asphyxia alters oxytocin-induced responses. Arterial hypoxia was produced by inhalation of 7.5% O2-92.5% N2 for 10 min. Ischemia was achieved by increasing intracranial pressure for 10 min. Asphyxia was achieved by turning off the ventilator for 5 min. During normoxic conditions, oxytocin dilated pial arterioles by 9 +/- 1% at 10(-8) and by 16 +/- 1% at 10(-6) mol/l (n = 47, p < 0.05). Arteriolar responses to oxytocin did not change with repeated applications (n = 10). Following hypoxia, dilator effect of oxytocin was not changed at 10(-8) (8 +/- 2%) but it was reduced at 10(-6) mol/l (7 +/- 2%; p < 0.05, n = 8). After asphyxia or ischemia, oxytocin did not dilate arterioles at 10(-8) mol/l, whereas 10(-6) mol/l resulted in a mild vasoconstriction (-4 +/- 3 to -6 +/- 4%, n = 6 and 8). Topically applied superoxide dismutase did not preserve arteriolar responses to oxytocin after asphyxia although the arterioles did not constrict to 10(-6) mol/l oxytocin (n = 5). Dilatation of cerebral arterioles in response to oxytocin was reversed to constriction by N(omega)-nitro-L-arginine methyl ester (L-NAME) (15 mg/kg, i.v.; n = 5) and by endothelial impairment by intra-arterial infusion of phorbol ester (10[-5] mol/l; n = 5). We conclude that the absence of pial arteriolar dilation to oxytocin after ischemia and asphyxia indicates endothelial dysfunction which may be involved in the pathology of perinatal brain injury.

Role of Ca(2+)-dependent K+ channels in cerebral vasodilatation induced by increases in cyclic GMP and cyclic AMP in the rat.

The mechanisms by which cAMP and cGMP produce vasorelaxation are not entirely clear. In this study we examined the hypothesis that relaxation of cerebral arterioles in response to receptor-mediated activation of adenylate cyclase (increase in cAMP) is mediated through Ca(2+)-dependent K+ channels. We measured the diameter of cerebral arterioles (basal diameter, 47 +/- 1 microns) using an open cranial window in anesthetized rats. Agonists and antagonists were applied locally in the cranial window. Topical application of adenosine (0.1 and 1 mmol/L), a receptor-mediated activator of adenylate cyclase, and dibutyryl cAMP (60 and 200 mumol/L), a cell-permeable analogue of cAMP, dilated cerebral arterioles. Iberiotoxin (50 nmol/L), a selective inhibitor of Ca(2+)-dependent K+ channels, reduced vasodilatation in response to 0.1 and 1 mmol/L adenosine by 66% and 28%, respectively. Tetraethylammonium (TEA) (1 mmol/L), another inhibitor of Ca(2+)-dependent K+ channels, reduced vasodilatation to 0.1 and 1 mmol/L adenosine by 58% and 42%, respectively, and reduced vasodilatation in response to 60 and 200 mumol/L dibutyryl cAMP by 75% and 66%, respectively. Topical application of sodium nitroprusside (0.1 and 1 mumol/L), a direct activator of guanylate cyclase, and 8-bromo cGMP (200 and 600 mumol/L), a cell-permeable analogue, produced dilatation of cerebral arterioles that was inhibited by iberiotoxin (50 nmol/L) and TEA (1 and 3 mmol/L). In contrast, dilatation of cerebral arterioles in response to papaverine (which produces vasodilatation in large part by inhibition of Ca2+ channels) and aprikalim (which produces vasodilation by activation of ATP-sensitive K+ channels) was not inhibited by iberiotoxin or TEA. These findings suggest that dilatation of cerebral arterioles by receptor-mediated activation of adenylate cyclase and by direct activation of guanylate cyclase in the rat is mediated in large part by activation of Ca(2+)-dependent K+ channels.

Effect of chronic alcohol consumption on responses of cerebral arterioles.

The goal of this study was to determine whether chronic ingestion of alcohol alters dilatation of cerebral arterioles in response to agonists that produce activation of adenylate cyclase and activation of ATP-sensitive potassium channels. Rats were fed liquid diets with or without ethanol for 2 to 2.5 months. In vivo diameter of pial arterioles was measured in alcohol-fed and nonalcohol-fed rats during superfusion with isoproterenol, forskolin, cromakalim, and nitroglycerin. Dilatation of pial arterioles in response to activation of adenylate cyclase via stimulation of beta-adrenergic receptors using isoproterenol was impaired in alcohol-fed rats. Isoproterenol (1.0 microM) dilated cerebral arterioles by 15 +/- 3% in nonalcohol-fed rats, but by only 7 +/- 2% in alcohol-fed rats. In contrast, dilatation of pial arterioles in response to forskolin was similar in nonalcohol-fed and alcohol-fed rats. Dilatation of pial arterioles in response to activation of ATP-sensitive potassium channels was impaired in alcohol-fed compared with nonalcohol-fed rats. Cromakalim (1.0 microM) dilated pial arterioles by 22 +/- 5% in nonalcohol-fed rats, but by only 5 +/- 2% in alcohol-fed rats (p < 0.05). In contrast, dilatation of pial arterioles in response to nitroglycerin was similar in alcohol and nonalcohol-fed rats. The findings of the present study suggest that chronic alcohol ingestion impairs dilatation of cerebral resistance arterioles in response to activation of adenylate cyclase via stimulation of beta-adrenergic receptors and in response to activation of ATP-sensitive potassium channels. We suggest that impaired vasodilator mechanisms during chronic alcohol consumption may have important implications for the pathogenesis of cerebrovascular abnormalities observed during chronic alcoholism.

7-Nitroindazole inhibits brain nitric oxide synthase and cerebral vasodilatation in response to N-methyl-D-aspartate.

N-Methyl-D-aspartate (NMDA) produces dilatation of cerebral arterioles that is dependent on production of nitric oxide (NO). In these experiments we examined the hypothesis that cerebral vasodilatation in response to NMDA is mediated by the neuronal isoform of NO synthase. We measured diameters of cerebral arterioles (baseline diameter, 89 +/- 7 microns) using a closed cranial window in anesthetized rabbits that received either vehicle (10 mL/kg IP peanut oil) or 7-nitroindazole (7-NI; 50 mg/kg IP). 7-NI is reported to be a selective inhibitor of neuronal NO synthase. Two hours after administration of 7-NI, activity of brain NO synthase (measured by conversion of L-arginine to L-citrulline) was reduced by 33% compared with vehicle (24 +/- 1 versus 16 +/- 3 pmol/min per milligram protein; n = 7; P < .05). Dilatation of cerebral arterioles in response to NMDA (100 and 300 mumol/L) was inhibited by 30% to 40% by 7-NI compared with responses in the presence of vehicle (23 +/- 6% versus 14 +/- 5% and 30 +/- 4% versus 21 +/- 5%, respectively; P < .05 for both concentrations; n = 10). In contrast, vasodilatation in response to acetylcholine (1 mumol/L) was similar in vehicle- and 7-NI-treated animals (17 +/- 5% versus 21 +/- 4%; P > .05). These findings suggest that vasodilatation in response to NMDA is mediated by neuronally derived NO. 7-NI appears to produce selective inhibition of brain NO synthase but not endothelial NO synthase.

Acute effects of glucose on reactivity of cerebral microcirculation: role of activation of protein kinase C.

Our first goal was to determine whether acute hyperglycemia alters endothelium-dependent reactivity of rat cerebral arterioles. Our second goal was to investigate a possible mechanism for impaired reactivity during acute hyperglycemia. Diameter of pial arterioles was measured during suffusion with ADP, acetylcholine, histamine, N-methyl-D-aspartate (NMDA), and nitroglycerin before and during application of a suffusate containing D-glucose (5, 10, 20, and 25 mM). ADP, acetylcholine, histamine, NMDA, and nitroglycerin produced dose-related vasodilation before application of D-glucose. Vasodilatation in response to the agonists was not altered by 5 and 10 mM D-glucose. In contrast, vasodilatation in response to ADP, acetylcholine, histamine, and NMDA was impaired during application of 20 and 25 mM D-glucose. Dilatation in response to nitroglycerin was not altered. Application of the protein kinase C inhibitor calphostin C (1.0 nM) or chelerythrine (10 nM) restored endothelium-dependent vasodilatation during application of 25 mM D-glucose. Thus acute hyperglycemia impairs endothelium-dependent responses of cerebral arterioles via the activation of protein kinase C.