Differential distribution and functional impact of BK channel beta1 subunits across mesenteric, coronary, and different cerebral arteries of the rat.

Large conductance, Ca2+i- and voltage-gated K+ (BK) channels regulate myogenic tone and, thus, arterial diameter. In smooth muscle (SM), BK channels include channel-forming α and auxiliary ß1 subunits. BK ß1 increases the channel's Ca2+ sensitivity, allowing BK channels to negatively feedback on depolarization-induced Ca2+ entry, oppose SM contraction and favor vasodilation. Thus, endothelial-independent vasodilation can be evoked though targeting of SM BK ß1 by endogenous ligands, including lithocholate (LCA). Here, we investigated the expression of BK ß1 across arteries of the cerebral and peripheral circulations, and the contribution of such expression to channel function and BK ß1-mediated vasodilation. Data demonstrate that endothelium-independent, BK ß1-mediated vasodilation by LCA is larger in coronary (CA) and basilar (BA) arteries than in anterior cerebral (ACA), middle cerebral (MCA), posterior cerebral (PCA), and mesenteric (MA) arteries, all arterial segments having a similar diameter. Thus, differential dilation occurs in extracranial arteries which are subjected to similar vascular pressure (CA vs. MA) and in arteries that irrigate different brain regions (BA vs. ACA, MCA, and PCA). SM BK channels from BA and CA displayed increased basal activity and LCA responses, indicating increased BK ß1 functional presence. Indeed, in the absence of detectable changes in BK α, BA and CA myocytes showed an increased location of BK ß1 in the plasmalemma/subplasmalemma. Moreover, these myocytes distinctly showed increased BK ß1 messenger RNA (mRNA) levels. Supporting a major role of enhanced BK ß1 transcripts in artery dilation, LCA-induced dilation of MCA transfected with BK ß1 complementary DNA (cDNA) was as high as LCA-induced dilation of untransfected BA or CA.

[INFLUENCE OF NATURAL COMPOUNDS WITH POLYPHENOL STRUCTURE ON THE VASODILATION FUNCTION OF CEREBROVASCULAR ENDOTHELIUM IN RATS UNDER FOCAL ISCHEMIA CONDITIONS.]

It was studied the effect of two natural compounds with polyphenol structure, green tea polyphenols (Sigma-Aldrich) and catechol hydrate (Sigma-Aldrich) in a dose of 100 mg/kg, on the vasodilating function of endothelium in brain vessels of rats with model ischemic damage. The focal ischemia was modeled by occlusion of the right middle cerebral artery. The vasodilating function of endothelium was estimated from NO synthesis modification determined by means of the Doppler sonography technique. It was established that at the focal brain ischemia leads to the pronounced endothelial dysfunction that is confirmed by the suppressed reactivity of brain vessels to the action of a vasodilating factor (acetylcholine, Ach) and by development of the L-arginine paradox phenomenon. Upon the treatment with polyphenolic compounds studied, the ability of brain vessels to vasodilatation upon ACh introduction is retained. The observed blood flow enhancement (relative to negative control group) in response to the introduction of L-arginine significantly decreases upon the administration of green tea polyphenols and catechol hydrate (by a factor of 3.15 and 2.29, respectively). This result, in turn, gives grounds to assume that the polyphenolic compounds studied possess endothelium protecting properties.

Neuroprotective effect of ethyl acetate extract from gastrodia elata against transient focal cerebral ischemia in rats induced by middle cerebral artery occlusion.

OBJECTIVE: To investigate the protective effect of neuroprotection against transient focal cerebral ischemia of the extract from Tianma (Rhizoma Gastrodiae) and the possible mechanisms underlying the action. METHODS: Cerebral ischemia-reperfusion injury was induced through middle cerebral artery occlusion (MCAO). Adult male Sprague-Dawley rats were randomly divided into four groups: sham-operated, ischemia-reperfusion model, 102.6 mg/kg extract treated and 11.4 mg/kg extract treated groups. The extract was prepared from gastrodia elata with ethyl acetate. The effect of the extract tested on rat neurological deficits and Cerebral index, cerebral infarct volume, brain injury, terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and B-cell lymphoma-2 (Bcl-2) positive cells. RESULTS: The extract was able to reduce neurological scores, cerebral index and cerebral infarction rate. The brain injury was also relieved by the extract. The results of immunofluorescence staining analysis indicated that the extract increased the expression of Bcl-2 and reduced TUNEL-positive cells significantly in the extract treated groups. CONCLUSION: These results suggested that the extract relieved ischemic injury induced by transient focal cerebral ischemia in rats, and this neuroprotective effect might be partially due to the attenuated apoptosis pathway.

Eugenol dilates rat cerebral arteries by inhibiting smooth muscle cell voltage-dependent calcium channels.

Plants high in eugenol, a phenylpropanoid compound, are used as folk medicines to alleviate diseases including hypertension. Eugenol has been demonstrated to relax conduit and ear arteries and reduce systemic blood pressure, but mechanisms involved are unclear. Here, we studied eugenol regulation of resistance-size cerebral arteries that control regional brain blood pressure and flow and investigated mechanisms involved. We demonstrate that eugenol dilates arteries constricted by either pressure or membrane depolarization (60 mM K) in a concentration-dependent manner. Experiments performed using patch-clamp electrophysiology demonstrated that eugenol inhibited voltage-dependent calcium (Ca) currents, when using Ba as a charge carrier, in isolated cerebral artery smooth muscle cells. Eugenol inhibition of voltage-dependent Ca currents involved pore block, a hyperpolarizing shift (∼-10 mV) in voltage-dependent inactivation, an increase in the proportion of steady-state inactivating current, and acceleration of inactivation rate. In summary, our data indicate that eugenol dilates cerebral arteries by means of multimodal inhibition of voltage-dependent Ca channels.

Effects of Krill-derived phospholipid-enriched n-3 fatty acids on Ca(2+) regulation system in cerebral arteries from ovariectomized rats.

AIMS: To investigate the effects of n-3 polyunsaturated fatty acids on cerebral circulation, ovariectomized (OVX) rats were administered with phospholipids in krill oil (KPL) or triglycerides in fish oil (FTG); effects on the Ca(2+) regulating system in their basilar artery (BA) were then analyzed. MAIN METHODS: The rats were divided into 4 groups: control, OVX, OVX given KPL (OVXP), and OVX given FTG (OVXT) orally, daily for 2weeks. Time dependent relaxation (TDR) of contractile response to 5HT in BA was determined myographically, Na(+)/Ca(2+) exchanger (NCX) 1 mRNA expression was determined by real time PCR, and nucleotides were analyzed by HPLC. KEY FINDINGS: The level of TDR in OVX that was significantly lower in the control was inhibited by l-NAME and indomethacin; TEA inhibited TDR totally in the control but only partly in OVXP and OVXT. Relaxation induced by the addition of 5mM KCl to the BA pre-contracted with 5-HT was inhibited by TEA in the controls, OVXP and OVXT, but not in OVX. Overexpression of NCX1 mRNA in the BA from OVX was significantly inhibited by FTG. The ratio of ADP/ATP in cerebral arteries from OVX was significantly inhibited by KPL and FTG. Levels of triglyceride and arachidonic acid in the plasma of OVX increased, but were significantly inhibited by KPL and FTG. SIGNIFICANCE: Ovarian dysfunction affects Ca(2+) activated-, ATP-sensitive-K(+) channels and NCX1, which play crucial roles in the autoregulation of cerebral blood flow. Also, KPL may become as good a supplement as FTG for postmenopausal women.

Chronic supplementation of paeonol combined with danshensu for the improvement of vascular reactivity in the cerebral basilar artery of diabetic rats.

One of the leading causes of death in the world is cerebrovascular disease. Numerous Chinese traditional medicines, such as Cortex Moutan (root bark of Paeonia suffruticosa Andrew) and Radix Salviae miltiorrhizae (root and rhizome of Salvia miltiorrhiza Bunge), protect against cerebrovascular diseases and exhibit anti-atherosclerotic effects. Traditional medicines have been routinely used for a long time in China. In addition, these two herbs are prescribed together in clinical practice. Therefore, the pharmacodynamic interactions between the active constituents of these two herbs, which are paeonol (Pae) and danshensu (DSS), should be particularly studied. The study of Pae and DSS can provide substantial foundations in understanding their mechanisms and empirical evidence to support clinical practice. This study investigated the effects and possible mechanisms of the pharmacodynamic interaction between Pae and DSS on cerebrovascular malfunctioning in diabetes. Experimental diabetes was induced in rats, which was then treated with Pae, DSS, and Pae + DSS for eight weeks. Afterward, cerebral arteries from all groups were isolated and equilibrated in an organ bath with Krebs buffer and ring tension. Effects of Pae, DSS, and Pae + DSS were observed on vessel relaxation with or without endothelium as well as on the basal tonus of vessels from normal and diabetic rats. Indexes about oxidative stress were also determined. We report that the cerebral arteries from diabetic rats show decreased vascular reactivity to acetylcholine (ACh) which was corrected in Pae, DSS, and Pae + DSS treated groups. Furthermore, phenylephrine (PE)-induced contraction response decreased in the treated groups. Phenylephrine and CaCl(2)-induced vasoconstrictions are partially inhibited in the three treated groups under Ca2+-free medium. Pre-incubated with tetraethylammonium, a non-selective K+ channel blocker, the antagonized relaxation responses increased in DSS and Pae + DSS treated diabetic groups compared with those in diabetic and Pae-treated diabetic groups. In addition, superoxide dismutase activity and thiobarbituric acid reactive substances content significantly changed in the presence of Pae + DSS. We therefore conclude that both Pae and DSS treatments prevent diabetes-induced vascular damage. Furthermore, Pae + DSS prove to be the most efficient treatment regimen. The combination of Pae and DSS produce significant protective effects through the reduction of oxidative stress and through intracellular Ca2+ regulatory mechanisms.

Insulin-induced generation of reactive oxygen species and uncoupling of nitric oxide synthase underlie the cerebrovascular insulin resistance in obese rats.

Hyperinsulinemia accompanying insulin resistance (IR) is an independent risk factor for stroke. The objective is to examine the cerebrovascular actions of insulin in Zucker obese (ZO) rats with IR and Zucker lean (ZL) control rats. Diameter measurements of cerebral arteries showed diminished insulin-induced vasodilation in ZO compared with ZL. Endothelial denudation revealed vasoconstriction to insulin that was greater in ZO compared with ZL. Nonspecific inhibition of nitric oxide synthase (NOS) paradoxically improved vasodilation in ZO. Scavenging of reactive oxygen species (ROS), supplementation of tetrahydrobiopterin (BH(4)) precursor, and inhibition of neuronal NOS or NADPH oxidase or cyclooxygenase (COX) improved insulin-induced vasodilation in ZO. Immunoblot experiments revealed that insulin-induced phosphorylation of Akt, endothelial NOS, and expression of GTP cyclohydrolase-I (GTP-CH) were diminished, but phosphorylation of PKC and ERK was enhanced in ZO arteries. Fluorescence studies showed increased ROS in ZO arteries in response to insulin that was sensitive to NOS inhibition and BH(4) supplementation. Thus, a vicious cycle of abnormal insulin-induced ROS generation instigating NOS uncoupling leading to further ROS production underlies the cerebrovascular IR in ZO rats. In addition, decreased bioavailability and impaired synthesis of BH(4) by GTP-CH induced by insulin promoted NOS uncoupling.

Daidzein relaxes rat cerebral basilar artery via activation of large-conductance Ca2+-activated K+ channels in vascular smooth muscle cells.

Daidzein, a phytoestrogen, has been reported to produce vasodilation via inhibition of Ca(2+) inflow. However, the involvement of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in the effect of daidzein is debated. Therefore, the present study was designed to investigate the effect of daidzein on the rat cerebral basilar artery and the underlying molecular mechanisms. Isolated cerebral basilar artery rings and single vascular smooth muscle cells (VSMCs) were used for vascular reactivity and electrophysiology measurements, to investigate the effect of daidzein on BK(Ca) channels in cerebral basilar artery smooth muscle. In addition, the human BK(Ca) channel alpha-subunit gene (hslo) was transfected into HEK293 cells, to directly assess whether daidzein activates BK(Ca) channels. The results showed that daidzein produced a concentration-dependent but endothelium-independent relaxation in rat cerebral basilar arteries. Paxilline, a selective BK(Ca) channel blocker, significantly inhibited the daidzein-induced vasodilation, whereas NS1619, a selective BK(Ca) channel opener, enhanced the vasodilation. In the whole-cell configuration, daidzein increased noisy oscillation currents in cerebral basilar artery VSMCs in a concentration-dependent manner, and washout of daidzein or blockade of BK(Ca) channels with paxilline fully reversed the increase. However, daidzein did not substantially affect hSlo currents in HEK293 cells when applied to the outside of the cell membrane. In conclusion, these results indicate that the activation of BK(Ca) channels in VSMCs at least partly contributes to the daidzein-induced vasodilation of the rat cerebral basilar artery. The beta1-subunit of BK(Ca) channels plays a critical role in the activation of BK(Ca) currents by daidzein.

The central analgesia induced by antimigraine drugs is independent from Gi proteins: superiority of a fixed combination of indomethacin, prochlorperazine and caffeine, compared to sumatriptan, in an in vivo model.

A hypofunctionality of Gi proteins has been found in migraine patients. The fixed combination of indomethacin, prochlorperazine and caffeine (Indoprocaf) is a drug of well-established use in the acute treatment of migraine and tension-type headache. The aim of this study was to investigate if Indoprocaf was able to exert its central antinociceptive action when Gi proteins activity is abolished by pertussis toxin (PTX), compared to its single active ingredients and to sumatriptan. The mice model of abdominal constriction test induced by an i.p. injection of a 0.6% solution of acetic acid was used. The study showed that Indoprocaf (a fixed combination of indomethacin 1 mg/kg, prochlorperazine 1 mg/kg and caffeine 3 mg/kg, s.c.) and sumatriptan (20 mg/kg, s.c.) exert their central antinociceptive action independently from the Gi proteins. In addition, the antinociceptive efficacy of Indoprocaf in this study was statistically superior to that of sumatriptan. This study also showed that the single active ingredients of Indoprocaf, indomethacin (1 mg/kg, s.c.), prochlorperazine (1 mg/kg, s.c.) and caffeine (3 mg/kg, s.c.), were able to exert their central antinociceptive action independently from the Gi proteins. However, Indoprocaf at analgesic doses was able to abolish almost completely the abdominal constrictions, with a statistically higher efficacy compared to the single active ingredients, showing an important synergic effect of Indoprocaf. This synergic effect was evident not only when Gi proteins activity was abolished by PTX, but also under control condition, when Gi proteins were active. This study suggests that the central antinociceptive action induced by antimigraine drugs is independent from Gi proteins.

Rotenone partially reverses decreased BK Ca currents in cerebral artery smooth muscle cells from streptozotocin-induced diabetic mice.

1. Reactive oxygen species (ROS) cause vascular complications and impair vasodilation in diabetes mellitus. Large-conductance Ca(2+)-activated potassium channels (BK(Ca)) modulate vascular tone and play an important negative feedback role in vasoconstriction. In the present study, we tested the hypothesis that ROS regulate the function of BK(Ca) in diabetic cerebral artery smooth muscle cells. 2. Diabetes was induced in male BALB/c mice by injection of streptozotocin (STZ; 180 mg/kg, i.p., dissolved in sterile saline). Control and diabetic mice were treated with 12.7 micromol/L rotenone, an inhibitor of the mitochondrial electron transport chain complex I, or placebo every other day for 5 weeks. The whole-cell patch clamp-technique and functional vasomotor methods were used to record BK(Ca) currents and myogenic tone of cerebral artery smooth muscle cells. 3. In the diabetic group, there was a significant decrease in spontaneous transient outward currents in cerebral artery smooth muscle cells compared with control. Although the currents were only moderately increased in rotenone-treated diabetic mice, they remained significantly lower than in the control group. Furthermore, the macroscopic BK(Ca) currents that were decreased in diabetic mice were partially recovered in rotenone-treated diabetic mice (P < 0.05 vs untreated diabetic group). 4. The posterior cerebral artery from diabetic mice had a significantly higher myogenic tone than the control group, but this impaired contraction was partially reversed in the rotenone-treated diabetic group (P < 0.05 vs untreated diabetic group). 5. The H(2)O(2) concentration was significantly increased in cerebral arteries from diabetic mice compared with control. This increase in H(2)O(2) was significantly blunted by rotenone treatment. 6. In conclusion, rotenone partially reverses the decreased macroscopic BK(Ca) currents in STZ-induced Type 1 diabetic mice and this reversal of BK(Ca) currents may be related to the inhibitory effects of rotenone on H(2)O(2) production. Reactive oxygen species, particularly H(2)O(2), are important regulators of BK(Ca) channels and myogenic tone in diabetic cerebral artery.

[Vasopressin and angiogenesis]. / Vasopressine et angiogenèse.

In adult mammals, the CNS vasculature remains essentially quiescent, excepted for specific pathologies. In the seventies, it was reported that proliferation of astrocytes and endothelial cells occurs within the hypothalamic magnocellular nuclei when strong metabolic activation of the vasopressinergic and oxytocinergic neurons was induced by prolonged hyperosmotic stimulation. Using more appropriate techniques, we first demonstrated that in these nuclei, the proliferative response to osmotic stimulus is essentially associated with local angiogenesis. We then showed that hypothalamic magnocellular neurons express vascular endothelial growth factor (VEGF), a potent angiogenic factor, that plays a major rôle in the angiogenesis induced by osmotic stimuli. We then demonstrated a correlation between increased VEGF secretion and local hypoxia. In AVP-deficient Brattleboro rats, the dramatic activation of magnocellular hypothalamic neurons failed to induce hypoxia, VEGF expression or angiogenesis suggesting a major role of hypothalamic AVP. Lastly we showed that 1) hypoxia and angiogenesis were not observed in non-osmotically stimulated Wistar rats in which circulating AVP was increased by the prolonged infusion of exogenous AVP, 2) contractile arterioles afferent to the magnocellular nuclei were strongly constricted by the perivascular application of AVP via V1a receptors (V1a-R) stimulation, and 3) following the intracerebral administration of selective V1a-R antagonist to osmotically stimulated rats, hypothalamic hypoxia and angiogenesis were inhibited. Together, these data strongly suggest that the angiogenesis induced by osmotic stimulation relates to tissue hypoxia resulting from the constriction of local arterioles, via the stimulation of perivascular V1a-R by AVP locally released from dendrites.

Low vitamin d levels predict stroke in patients referred to coronary angiography.

BACKGROUND AND PURPOSE: Vitamin D deficiency is common among the elderly and may contribute to cerebrovascular diseases. We aimed to elucidate whether low vitamin D levels are predictive for fatal stroke. METHODS: The LUdwigshafen RIsk and Cardiovascular Health (LURIC) study includes 3316 patients who were referred to coronary angiography at baseline between 1997 and 2000. 25-Hydroxyvitamin D [25(OH)D] and 1,25-dihydroxyvitamin D [1,25(OH)2D] were measured in 3299 and 3315 study participants, respectively. To account for the seasonal variation of vitamin D metabolites, we calculated z values for the 25(OH)D and 1,25(OH)2D concentrations within each month of blood draw. RESULTS: During a median follow-up time of 7.75 years, 769 patients died, including 42 fatal (ischemic and hemorrhagic) strokes. When compared with survivors in binary logistic-regression analyses, the odds ratios (with 95% CIs) for fatal stroke were 0.58 (0.43 to 0.78; P<0.001) per z value of 25(OH)D and 0.62 (0.47 to 0.81; P<0.001) per z value of 1,25(OH)2D. After adjustment for several possible confounders, these odds ratios remained significant for 25(OH)D at 0.67 (0.46 to 0.97; P=0.032) and for 1,25(OH)2D at 0.72 (0.52 to 0.99; P=0.047). Z values of 25(OH)D and 1,25(OH)2D were also reduced in the 274 patients who had a history of previous cerebrovascular disease events at baseline. CONCLUSIONS: Low levels of 25(OH)D and 1,25(OH)2D are independently predictive for fatal strokes, suggesting that vitamin D supplementation is a promising approach in the prevention of strokes.

Quantitative analysis of brain perfusion SPECT in Alzheimer's disease using a fully automated regional cerebral blood flow quantification software, 3DSRT.

PURPOSE: The clinical symptoms of Alzheimer's disease (AD) show great diversity depending on the clinical stage. We investigated the correlation of regional cerebral blood flow (rCBF) changes and the clinical severity of AD patients. METHODS: Thirty-nine AD patients and 16 normal subjects participated in this study. AD patients were divided into three subgroups by clinical severity. Quantitative brain perfusion SPECT analyses were performed using a rCBF quantification software, 3DSRT. RESULTS: In mild AD, significant decreases of rCBF were detected in the bilateral parietal, angular gyrus, pericallosal, thalamus, right temporal and left hippocampal regions. Moderate AD patients showed significantly lower blood flow than those with mild AD only to the right hippocampus. Analysis of the severe AD group revealed a nearly diffuse decrease of rCBF throughout the cerebral cortex except for part of the frontal lobe compared with moderate patients. CONCLUSIONS: These results were consistent with previous findings demonstrated by qualitative analysis of CBF. The decreased thalamic blood flow was noteworthy as this finding has rarely been reported. In consideration of the structure and function of the Papez circuit, which connects the medial temporal lobe and thalamus, a remote metabolic effect might be the cause of lower rCBF in the thalamus.

Brain microvascular and intracranial artery resistance to atherosclerosis is associated with heme oxygenase and ferritin in Japanese quail.

Oxidative stress and increased oxidation of low-density lipoprotein (oxLDL) through free radical-mediated tissue injury may be important factors in the development of extracranial atherosclerotic lesions. However, the roles of oxidative stress and hypercholesterolemia in intracranial atherosclerosis is less established. The induction of heme oxygenase (HO) is a cellular response to oxidative stress, and inducible HO (HO-1) may protect against oxidized lipids such as those produced by oxidative stress. We investigated the effects of oxLDL on cell and tissue viability, HO-1 and ferritin expression in extracranial and intracranial endothelial cells, and the arteries of cholesterol-induced atherosclerosis (CIA) Japanese quail. We report that cultured microvascular endothelial cells from the brain (QBMEC) and carotid (QCEC) differ in their response to oxidative stress. The QCECs are less responsive than QBMECs to oxidative stress induced by oxLDL, as evident by lower expression of HO-1 mRNA, HO activity, and ferritin levels. Furthermore, the higher levels of catalytic iron, thiobarbituric acid reactive substances, and lactate dehydrogenase released in QCECs indicated that these cells are more susceptible to oxidative stress than QBMECs. We also investigated the relationship between extent of atherosclerotic plaque deposition and the extracranial and intracranial arterial expression of HO-1 in quail. The common carotid and vertebral (extracranial) arteries had higher tissue cholesterol levels (starting at 2 weeks of cholesterol-supplementation) and a greater atherosclerotic plaque score (starting at 4 weeks of cholesterol-supplementation) compared with middle cerebral and basilar (intracranial) arteries, and this may be relevant to the effect of aging on the process of atherogenesis. The extracranial arteries also had early and greater levels of lipid peroxidation and catalytic iron coupled with lower expression of HO-1 protein, HO activity, and ferritin compared to the intracranial vessels. These observations suggest that the extracranial and intracranial arterial walls respond differently to oxidation of lipoproteins, and support the feasibility of increased HO-1 expression as a means of protection against oxidant injury.

Developmental changes of leptin receptors in cerebral microvessels: unexpected relation to leptin transport.

The adipokine leptin participates not only in the regulation of feeding and obesity in adults but also in neonatal development. It crosses the blood-brain barrier (BBB) by receptor-mediated transport. Leptin concentrations in blood differ between neonates and adults. We determined the developmental changes of leptin receptor subtypes in the cerebral microvessels composing the BBB and examined their expected correlation with leptin transport across the BBB. Total RNA was extracted from enriched cerebral microvessels of mice 1, 7, 14, and 60 d of age for real-time RT-PCR analysis of leptin receptor subtypes. In cerebral microvessels from neonates, ObRa, ObRb, ObRc, and ObRe mRNA were all higher than in adults, but ObRd was not detectable. Hypothalamus showed similar age-related changes except for ObRb, which was higher in adults. The homologous receptor gp130 did not show significant age-related changes in either region. Despite the increase of leptin receptors, leptin permeation across the BBB after iv injection was less in the neonates. In situ brain perfusion with blood-free buffer showed no significant difference in the brain uptake of leptin between neonates and adults, indicating an antagonistic role of leptin-binding proteins in the circulation, especially the soluble receptor ObRe. The results are consistent with our previous finding that ObRe antagonizes leptin endocytosis in cultured endothelia and transport from blood to brain in mice. Overall, the developmental changes observed for leptin receptors unexpectedly failed to correlate with the entry of leptin into brain, and this may indicate different functions of the receptors in neonates and adults.

[Effect of electroacupuncture on inositol triphosphate and diacylglycerol levels in cerebral arteries of cerebral ischemia rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on inositol triphosphate (IP3) and diacylglycerol (DAG) contents in cerebral arteries of the cerebral cortex in rats with cerebral ischemia (CI), so as to explore its effect on intracellular signal transduction pathway. METHODS: Fifty-six Wistar rats wererandomly divided into control (n=8), CI (n=24) and EA (n=24) groups, and the later 2 groups were further separately divided into 1 h, 3 h and 6 h subgroups (CI-1 h, CI-3 h, CI-6 h, EA-CI-1 h, EA-CI-3 h and EA-CI-6 h), with 8 cases in each. CI model was made by occlusion of the middle cerebral artery. EA (15 Hz, 0.1 mA) was applied to "Shuigou" (GV 26) for 20 min. The cerebral arteries in the cerebral shallow layer were collected for assaying intracellular DAG content by thin-layer chromatography (TLC) and IP3 content by competitive protein binding assay (CPBA). RESULTS: Compared with control group,the levels of IP3 and DAG in cerebral vascular smooth muscle were significantly increased in model 1 h, 3 h and 6 h subgroups (P<0.01). Compared with the corresponding model subgroups,the levels of IP3 and DAG were significantly decreased in EA-CI-1 h, EA-CI-3 h and EA-CI-6 h subgroups (P<0.01). CONCLUSION: EA of GV 26 can effectively inhibit CI-induced increase of intracellular IP3 and DAG levels in rat cerebral arteries, which may contribute to its effect in modulating vascular motor to ameliorate cerebral microcirculation.

Role of BK(Ca) channels in cephalic vasodilation induced by CGRP, NO and transcranial electrical stimulation in the rat.

Both calcitonin gene-related peptide (CGRP) and nitric oxide (NO) are potent vasodilators that have been shown to induce headache in migraine patients. Their antagonists are effective in the treatment of migraine attacks. In the present study, we hypothesize that vasodilation induced by the NO donor glyceryltrinitrate (GTN) or by CGRP is partially mediated via large conductance calcium-activated potassium (BK(Ca)) channels. The effects of the BK(Ca) channel selective inhibitor iberiotoxin on dural and pial vasodilation induced by CGRP, GTN and endogenously released CGRP by transcranial electrical stimulation (TES) were examined. Iberiotoxin significantly attenuated GTN-induced dural and pial artery dilation in vivo and in vitro, but had no effect on vasodilation induced by CGRP and TES. Our results show that GTN- but not CGRP-induced dural and pial vasodilation involves opening of BK(Ca) channels in rat.

Cholinergic neuronal deficits in CADASIL.

BACKGROUND AND PURPOSE: Previous evidence from MRI and acetylcholinesterase histochemistry suggests cholinergic fibers are affected in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). METHODS: As a measure of cholinergic function, we assessed choline acetyltransferase (ChAT) activities in the frontal and temporal neocortices and the immunocytochemical distribution of ChAT and p75 neurotrophin receptor (P75(NTR)) by in vitro imaging in the nucleus basalis of Meynert of CADASIL subjects. RESULTS: ChAT activities were significantly reduced by 60% to 70% in frontal and temporal cortices of CADASIL cases, as were ChAT and P75(NTR) immunoreactivities in the nucleus basalis. CONCLUSIONS: Our findings suggest cholinergic neuronal impairment in CADASIL and implicate cholinomimetic therapy for subcortical vascular dementias.

Altered gene expression in cerebral capillaries of stroke-prone spontaneously hypertensive rats.

Stroke-prone spontaneously hypertensive rats (SHRSP) are a well-characterized, genetic model for stroke. We showed earlier that the structure and function of the tight junctions in SHRSP blood-brain barrier endothelial cells is disturbed prior to stroke. To investigate the molecular events leading to endothelial dysfunction in SHRSP cerebral capillaries, we carried out suppression subtractive hybridization (SSH) in combination with a cDNA filter screening step. We identified two cDNA fragments that were upregulated in SHRSP, compared to stroke-resistant spontaneously hypertensive rats (SHR), and found open reading frames of 133 and 138 amino acids, respectively. These peptides did not match any known proteins in public databases. A third upregulated SHRSP cDNA fragment was identified as the rat sulfonylurea receptor 2B (SUR2B). We also isolated and cloned the cDNA of the rat homologue for the mouse G-protein signaling 5 (RGS5) regulator. This regulator was downregulated in SHRSP. We used in situ hybridization to show that rat RGS5 is expressed in the brain capillary endothelium and in the choroid plexus. Our findings may lead to the identification of new stroke-related genes.

Sex steroid hormones exert biphasic effects on cytosolic magnesium ions in cerebral vascular smooth muscle cells: possible relationships to migraine frequency in premenstrual syndromes and stroke incidence.

Clinically, it is known that: (1) magnesium (Mg) supplementation relieves premenstrual problems (e.g., migraine, bloating and edema) occurring in the late luteal phase of the menstrual cycle; and (2) migraine syndromes, particularly in women, are associated with deficits in brain and serum ionized Mg levels. We investigated whether concentrations of sex steroid hormones, found in the serum during the menstrual cycle of women, are associated with changes in the levels of cytosolic free magnesium ions ([Mg2+]i in single cultured canine cerebral vascular smooth muscle cells. The resting level of [Mg2+]i in these cells was 645 +/- 89 microM before exposure to sex steroid hormones. Exposure of these vascular cells to a low concentration of estrogen (10 pg/ml) failed to interfere with the levels of [Mg2+]i. However, exposure to estrogen, at concentrations ranging from 40 to 200 pg/ml, induced significant loss of [Mg2+]i in a concentration-dependent manner. At a concentration of 200 pg/ml estrogen, the level of [Mg2+]i decreased approximately 30% in comparison with controls. Progesterone produced biphasic effects on the levels of [Mg2+]i, depending on its concentration. Exposure of the cultured cells to a low concentration of progesterone (0.5 ng/ml) resulted in an increased level of [Mg2+]i (from 690 +/- 50 microM to 753 +/- 56 microM, p < 0.05). However, when these cells were exposed to higher concentrations of progesterone (i.e., from 5.0 to 20 ng/ml), the cellular levels of [Mg2+]i were decreased significantly. The higher the estrogen or progesterone concentration, the lower the levels of [Mg2+]i. In contrast, testosterone, a male hormone, didn't produce any significant alteration in [Mg2+]i levels in these cerebral vascular smooth muscle cells. These data indicate that low, physiological concentrations of female sex hormones, estrogen and progesterone, help cerebral vascular smooth cells sustain normal concentrations of [Mg2+]i, which are beneficial to vascular function, whereas high levels of estrogen and progesterone deplete, significantly, [Mg2+]i in cerebral vascular smooth muscle cells, possibly resulting in cerebrovasospasms and reduced cerebral blood flows related to premenstrual syndromes, migraine and stroke risk. Our findings could provide new insight into the mechanism whereby migraine occurs frequently in the late luteal phase in the premenstrual syndrome. In addition, our results demonstrate that female sex steroids but not testosterone (in physiologic concentrations) can exert direct effects on [Mg2+]i in cerebral vascular cells.