Relaxation Of Cerebral Arteries Research Articles

Apelin inhibits an endothelium-derived hyperpolarizing factor-like pathway in rat cerebral arteries

Apelin has complex vasomotor actions inasmuch as the peptide may cause either vasodilation or vasoconstriction depending on the vascular bed and experimental conditions. In cerebral arteries, apelin inhibits endothelium-dependent relaxations mediated by nitric oxide (NO); however, its effects on relaxation to other endothelium-derived substances (e.g. prostacyclin, endothelium-derived hyperpolarizing factors(s) (EDHF)) are unknown. The present study was designed to determine effects of apelin on endothelium-dependent relaxations that are independent of NO in rat cerebral arteries. In arterial rings contracted with 5-HT, A23187 caused endothelium-dependent relaxation that was unaffected by inhibitors of eNOS, guanylyl cyclase or cyclooxygenase, but was attenuated by MS-PPOH, a selective inhibitor of cytochrome P450 catalyzed synthesis of epoxyeicosatrienoic acids (EETs) and by 14,15-EE(Z)E, an EET-receptor antagonist. Apelin inhibited A23187-induced relaxation, as well as relaxations evoked by exogenous 11,12- and 14,15-EET. These effects of apelin were mimicked by the selective BKCa channel blocker, iberiotoxin. The APJ receptor antagonist, F13A abolished the effects of apelin on A23187-induced relaxations. Both 11,12- and 14,15-EET also increased BKCa channel current density in isolated cerebral artery smooth muscle cells, effects that were inhibited in a similar manner by apelin and iberiotoxin. These findings provide evidence that apelin impairs endothelium-dependent relaxation of cerebral arteries by inhibiting an NO-independent pathway (i.e. “EDHF-like”) involving activation of smooth muscle cell BKCa channels by endothelium-derived EETs. Inhibition of such pathway may create an environment favoring vasoconstriction in cerebral arteries.

Abstract WP543: Apelin Impairs Endothelium-Derived Hyperpolarizing Factor (EDHF)-Induced Relaxation of Cerebral Arteries by Inhibiting Activation of Large Conductance, Calcium-Activated K (BK Ca ) Channels in Vascular Smooth Muscle

Introduction: Apelin is a peptide found in fat cells, endothelial cells and CNS neurons. Apelin levels are increased in conditions linked to cardiovascular disease (e.g. obesity, diabetes). Apelin inhibits NO-induced relaxation of cerebral arteries (CA) but its effect on relaxation to EDHF, which often compensates for impaired NO responses in diseased arteries, is unknown. Methods and Results: Immunoblot analysis identified apelin receptor expression in human and rat CA. In isolated rat CA, A23187 (10 -9 – 10 -6 M) caused endothelium-dependent relaxation that was unaffected by inhibitors of eNOS (nitro-l-arginine (3 x 10 -5 M), guanylyl cyclase (ODQ (10 -5 M), cyclooxygenase (indomethacin (10 -5 M) and the H 2 O 2 -scavenger, PEG-catalase (500 U/ml) (n=6). A23187-induced relaxation was attenuated by MS-PPOH (10 -5 M), a selective inhibitor of cyp450-catalyzed synthesis of epoxyeicosatrienoic acids (EETs) (pD 2 =6.86 ± 0.3 vs 5.90 ± 0.1, without and with MS-PPOH; n=6; p<0.05), and abolished by 14,15-EE(Z)E (10 -5 M), a selective EET-receptor antagonist (n=7; p<0.05). Apelin (10 -7 M) inhibited A23187-induced relaxation (pD 2 =7.08 ± 0.2 vs 6.44 ± 0.1, without and with apelin; n=7; p<0.05) as well as relaxation to 11,12-EET, 14,15-EET and NS1619 (BK Ca channel opener), but had no effect on relaxation evoked by levcromakalim (K ATP channel opener). In whole cell patch clamp studies (10mv steps from -60mv to +80mv, 200ms duration) in CA smooth muscle cells, 11,12-EET (10 -5 M), 14,15-EET (10 -5 M), and NS1619 (10 -5 M) each caused an approximately 2-fold increase in BK Ca peak current density (at +80 mV; n=6; p<0.05), which was completely abrogated in the presence of apelin (10 -7 M). The effects of apelin on EET-induced relaxation and activation of BK Ca currents were mimicked by iberiotoxin (10 -7 M) (selective BK Ca channel blocker) and abolished by F13A (10 -7 M), an apelin receptor antagonist. Conclusion: Apelin regulates CA vascular tone by inhibiting endothelium-dependent relaxation evoked by an EDHF-like mediator (possibly an EET) through a mechanism involving activation of apelin receptors and inhibition of BK Ca channels in CA smooth muscle cells. These effects could increase the risk of cerebrovascular dysfunction under conditions where apelin levels are elevated.

Apelin Reduces Nitric Oxide-Induced Relaxation of Cerebral Arteries by Inhibiting Activation of Large-Conductance, Calcium-Activated K Channels.

Activation of the apelin/APJ receptor signaling system causes endothelium-dependent and nitric oxide (NO)-dependent relaxation in several peripheral arteries. The effects of apelin in cerebral arteries are unknown; however, apelin inhibits voltage-dependent increases in large-conductance, calcium-activated K channel (BKCa) currents in cerebral artery smooth muscle cells. Because NO-induced relaxation of cerebral arteries is mediated, in part, by activation of BKCa channels, the goals of this study were to determine the net effect of apelin in cerebral arteries, as well as test the hypothesis that the actions of apelin in cerebral arteries are secondary to stimulation of APJ receptors. Immunoblot and quantitative reverse transcription polymerase chain reaction analyses detected APJ receptors in cerebral arteries of male Sprague-Dawley rats, and immunofluorescence studies using confocal microscopy confirmed APJ receptor localization in smooth muscle cells. In myograph studies, apelin itself had no direct vasomotor effect but inhibited relaxations to the NO-donor, diethylamine NONOate, and to the endothelium-dependent vasodilator, bradykinin. These effects of apelin were mimicked by the selective BKCa-channel blocker, iberiotoxin, and suppressed by the APJ receptor antagonist, F13A. Apelin also inhibited relaxations evoked by the BKCa-channel openers, NS1619 and BMS 191011, but had no effect on relaxation to levcromakalim, a selective KATP-channel opener. Apelin had no effect on diethylamine NONOate-induced or bradykinin-induced increases in cyclic guanosine monophosphate levels. Patch clamp recordings demonstrated that apelin and iberiotoxin each suppressed the increase in BKCa currents induced by DEA and NS1619 in freshly isolated cerebral artery smooth muscle cells. The results demonstrate that apelin inhibits NO-induced relaxation of cerebral arteries through a mechanism involving activation of APJ receptors and inhibition of BKCa channels in cerebral arterial smooth muscle cells.

Relaxant Effects of the Selective Estrogen Receptor Modulator, Bazedoxifene, and Estrogen Receptor Agonists in Isolated Rabbit Basilar Artery.

We have previously shown that the selective estrogen receptor modulator, bazedoxifene, improves the consequences of ischemic stroke. Now we aimed to characterize the effects and mechanisms of action of bazedoxifene in cerebral arteries. Male rabbit isolated basilar arteries were used for isometric tension recording and quantitative polymerase chain reaction. Bazedoxifene relaxed cerebral arteries, as 17-β-estradiol, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol [estrogen receptor (ER) α agonist], and G1 [G protein-coupled ER (GPER) agonist] did it (4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol > bazedoxifene = G1 > 17-β-estradiol). 2,3-Bis(4-hydroxyphenyl)-propionitrile (ERβ agonist) had no effect. Expression profile of genes encoding for ERα (ESR1), ERβ (ESR2), and GPER was GPER > ESR1 > ESR2. As to the endothelial mechanisms, endothelium removal, N-nitro-L-arginine methyl ester, and indomethacin, did not modify the relaxant responses to bazedoxifene. As to the K channels, both a high-K medium and the Kv blocker, 4-aminopyridine, inhibited the bazedoxifene-induced relaxations, whereas tetraethylammonium (nonselective K channel blocker), glibenclamide (selective KATP blocker) or iberiotoxin (selective KCa blocker) were without effect. Bazedoxifene also inhibited both Ca- and Bay K8644-elicited contractions. Therefore, bazedoxifene induces endothelium-independent relaxations of cerebral arteries through (1) activation of GPER and ERα receptors; (2) increase of K conductance through Kv channels; and (3) inhibition of Ca entry through L-type Ca channels. Such a profile is compatible with the beneficial effects of estrogenic compounds (eg, SERMs) on vascular function and, specifically, that concerning the brain. Therefore, bazedoxifene could be useful in the treatment of cerebral disorders in which the cerebrovascular function is compromised (eg, stroke).

Role of Mitochondria in Cerebral Vascular Function: Energy Production, Cellular Protection, and Regulation of Vascular Tone.

Mitochondria not only produce energy in the form of ATP to support the activities of cells comprising the neurovascular unit, but mitochondrial events, such as depolarization and/or ROS release, also initiate signaling events which protect the endothelium and neurons against lethal stresses via pre-/postconditioning as well as promote changes in cerebral vascular tone. Mitochondrial depolarization in vascular smooth muscle (VSM), via pharmacological activation of the ATP-dependent potassium channels on the inner mitochondrial membrane (mitoKATP channels), leads to vasorelaxation through generation of calcium sparks by the sarcoplasmic reticulum and subsequent downstream signaling mechanisms. Increased release of ROS by mitochondria has similar effects. Relaxation of VSM can also be indirectly achieved via actions of nitric oxide (NO) and other vasoactive agents produced by endothelium, perivascular and parenchymal nerves, and astroglia following mitochondrial activation. Additionally, NO production following mitochondrial activation is involved in neuronal preconditioning. Cerebral arteries from female rats have greater mitochondrial mass and respiration and enhanced cerebral arterial dilation to mitochondrial activators. Preexisting chronic conditions such as insulin resistance and/or diabetes impair mitoKATP channel relaxation of cerebral arteries and preconditioning. Surprisingly, mitoKATP channel function after transient ischemia appears to be retained in the endothelium of large cerebral arteries despite generalized cerebral vascular dysfunction. Thus, mitochondrial mechanisms may represent the elusive signaling link between metabolic rate and blood flow as well as mediators of vascular change according to physiological status. Mitochondrial mechanisms are an important, but underutilized target for improving vascular function and decreasing brain injury in stroke patients. © 2016 American Physiological Society. Compr Physiol 6:1529-1548, 2016.

Effect of natriuretic peptides on cerebral artery blood flow in healthy volunteers

The natriuretic peptides (NPs), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), have vasoactive functions that concern humans and most animals, but their specific effects on cerebral circulation are poorly understood. We therefore examined the responsiveness of cerebral arteries to different doses of the natriuretic peptides in animals and humans. We conducted a dose-response experiment in guinea pigs (in vitro) and a double-blind, three-way cross-over study in healthy volunteers (in vivo). In the animal experiment, we administered cumulative doses of NPs to pre-contracted segments of cerebral arteries. In the main study, six healthy volunteers were randomly allocated to receive two intravenous doses of ANP, BNP or CNP, respectively, over 20min on three separate study days. We recorded blood flow velocity in the middle cerebral artery (VMCA) by transcranial Doppler. In addition, we measured temporal and radial artery diameters, headache response and plasma concentrations of the NPs. In guinea pigs, ANP and BNP but not CNP showed significant dose-dependent relaxation of cerebral arteries. In healthy humans, NP infusion had no effect on mean VMCA, and we found no difference in hemodynamic responses between the NPs. Furthermore, natriuretic peptides did not affect temporal and radial artery diameters or induce headache. In conclusion, natriuretic peptides in physiological and pharmacological doses do not affect blood flow velocity in the middle cerebral artery or dilate extracerebral arteries in healthy volunteers.

Pituitary adenylate cyclase-activating polypeptide (PACAP) induces relaxations of peripheral and cerebral arteries, which are differentially impaired by aging.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a well-known neuropeptide, which also has vasomotor effects. However, little is known regarding its age-related and organ-specific vasomotor effects. We hypothesized that the vasomotor effects of PACAP depend on the tissue origin of the vessels and aging substantially modulates its actions. Thus, carotid (CA) and basilar arteries (BA) were isolated from young (2months old), middle age (12months old), and old (30months old) rats. Their vasomotor responses were measured with an isometric myograph (DMT610M) in response to cumulative concentrations of PACAP1-38 (10(-9)-10(-6)M). PACAP1-38 induced (1) significantly greater concentration-dependent relaxations in CA compared to that of BA of young, middle age, and old rats; (2) relaxations of CA significantly decreased, whereas they did not change substantially in BA, as a function of age; (3) sodium nitroprusside (SNP)-induced relaxation did not change after PACAP1-38 administration in any conditions; and (4) inhibition of PAC1 receptors by selective PAC1 receptor blocker (PACAP6-38) completely diminished the responses to PACAP in all age groups of BA and CA. In conclusion, these findings suggest that PACAP1-38 has greater vasomotor effect in CA than that in BA, whereas aging has less effect on PACAP-induced relaxation of cerebral arteries and BA than that in peripheral arteries and CA suggesting that the relaxation to PACAP is maintained in cerebral arteries even in old age.

Dahl Salt-Sensitive Rats Are Protected Against Vascular Defects Related to Diet-Induced Obesity

Obesity increases plasma renin activity and angiotensin II levels, leading to vascular damage, elevated blood pressure, diabetes mellitus, and renal damage. Because genetic deletion of crucial parts of the renin-angiotensin system protect against obesity-related cardiovascular defects, we hypothesized that Dahl salt-sensitive (SS) rats, a model of chronically low plasma renin activity and angiotensin II levels, would be protected against vascular defects during diet-induced obesity compared with SS.13(BN) consomic rats showing normal renin-angiotensin system regulation. We evaluated vascular function in middle cerebral arteries of SS or SS.13(BN) rats fed high-fat (45% kcal from fat) versus normal-fat diet for 15 to 20 weeks from weaning. Endothelium-dependent relaxation in response to acetylcholine (10(-8) to 10(-4) mol/L) was restored in middle cerebral arteries of high-fat SS rats versus normal-fat diet controls, whereas vasodilation to acetylcholine was dramatically reduced in high-fat SS 13(BN) rats versus normal-fat diet controls. These findings support the hypothesis that physiological levels of angiotensin II play an important role in maintaining normal vascular relaxation in cerebral arteries and suggest that the cerebral vasculature of the SS rat model is genetically protected against endothelial dysfunction in diet-induced obesity.

NaHS relaxes rat cerebral artery in vitro via inhibition of l-type voltage-sensitive Ca2+ channel

H2S, a gaseous signalling molecule, relaxes blood vessels partly through activation of ATP-sensitive K+ channels. It is however unclear whether H2S or its donors could affect other ion transporting proteins. The present study examined the hypothesis that NaHS, a H2S donor inhibits voltage-sensitive Ca2+ channels and thus relaxes vascular smooth muscle cells (VSMC) in the cerebral arteries. NaHS dilated cerebral arteries from Sprague–Dawley rats with the same potency against pre-contraction by 5-HT and 60mmol/L KCl, which were unaffected by several K+ channel blockers, NG-nitro-l-arginine methyl ester or indomethacin, as assessed in wire myograph under an isometric condition. Likewise, NaHS also dilated cerebral arteries against myogenic constriction in pressurized myograph under an isobaric condition. NaHS concentration-dependently inhibited CaCl2-induced contraction in Ca2+-free, 60mM K+-containing Krebs solution. Patch clamp recordings showed that NaHS reduced the amplitude of l-type Ca2+ currents in single myocytes isolated enzymatically from the cerebral artery. Calcium fluorescent imaging using fluo-4 showed a reduced [Ca2+]i in 60mmol/L KCl-stimulated rat cerebral arteries in response to NaHS. H2S precursor l-cysteine-induced relaxation in cerebral arteries was inhibited by cystathionine γ–lyase (CSE) inhibitor dl-propargylglycine. CSE was expressed in cerebral arteries. In summary, NaHS dilates rat cerebral arteries by reducing l-type Ca2+ currents and suppressing [Ca2+]i of arterial myocyte, indicating that NaHS relaxes cerebral arteries primarily through inhibiting Ca2+ influx via Ca2+ channels

Introgression of the Brown Norway Renin Allele Onto the Dahl Salt-Sensitive Genetic Background Increases Cu/Zn SOD Expression in Cerebral Arteries

Nitric oxide (NO)-dependent vasodilation is impaired in middle cerebral arteries (MCAs) from Dahl salt-sensitive (SS) rats that are fed normal salt (NS) diet, due to low plasma renin activity and chronic exposure to low plasma angiotensin II (ANG II) levels. NO-dependent vasodilator responses are rescued in MCAs from Ren1-BN congenic rats, which have a 2.0 Mbp portion of Brown Norway (BN) chromosome 13 containing the renin gene introgressed onto the Dahl SS genetic background. Vascular superoxide levels were measured with dihydroethidium (DHE) fluorescence in basilar arteries from 10- to 14-week-old, male Dahl SS and Ren1-BN congenic rats that fed NS diet. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and xanthine oxidase (XO) activity were also measured in cerebral artery tissue homogenates. Expression of the superoxide dismutase (SOD) enzymes was evaluated via western blotting in cerebral arteries from the two rat strains. Superoxide levels were significantly higher in basilar arteries from Dahl SS rats compared to Ren1-BN congenic rats. NADPH oxidase and XO activity were similar between the two rat strains. Cu/Zn SOD expression was significantly higher in cerebral arteries from Ren1-BN congenic rats vs. those from Dahl SS rats. The expression of Mn-SOD was similar in cerebral arteries from both strains. These findings suggest that introgressing the BN renin allele onto the Dahl SS genetic background to restore normal activity of the renin-angiotensin system (RAS) protects NO-dependent vascular relaxation in cerebral arteries by increasing the expression of Cu/Zn SOD and lowering vascular superoxide levels.

Impaired relaxation of cerebral arteries in the absence of elevated salt intake in normotensive congenic rats carrying the Dahl salt-sensitive renin gene

This study evaluated endothelium-dependent vascular relaxation in response to acetylcholine (ACh) in isolated middle cerebral arteries (MCA) from Dahl salt-sensitive (Dahl SS) rats and three different congenic strains that contain a portion of Brown Norway (BN) chromosome 13 introgressed onto the Dahl SS genetic background through marker-assisted breeding. Two of the congenic strains carry a 3.5-Mbp portion and a 2.6-Mbp portion of chromosome 13 that lie on opposite sides of the renin locus, while the third contains a 2.0-Mbp overlapping region that includes the BN renin allele. While maintained on a normal salt (0.4% NaCl) diet, MCAs from Dahl SS rats and the congenic strains retaining the Dahl SS renin allele failed to dilate in response to ACh, whereas MCAs from the congenic strain carrying the BN renin allele exhibited normal vascular relaxation. In congenic rats receiving the BN renin allele, vasodilator responses to ACh were eliminated by nitric oxide synthase inhibition with N(G)-nitro-l-arginine methyl ester, angiotensin-converting enzyme inhibition with captopril, and AT(1) receptor blockade with losartan. N(G)-nitro-l-arginine methyl ester-sensitive vasodilation in response to ACh was restored in MCAs of Dahl SS rats that received either a 3-day infusion of a subpressor dose of angiotensin II (3 ng·kg(-1)·min(-1) iv), or chronic treatment with the superoxide dismutase mimetic tempol (15 mg·kg(-1)·day(-1)). These findings indicate that the presence of the Dahl SS renin allele plays a crucial role in endothelial dysfunction present in the cerebral circulation of the Dahl SS rat, even in the absence of elevated dietary salt intake, and that introgression of the BN renin allele rescues endothelium-dependent vasodilator responses by restoring normal activation of the renin-angiotensin system.

Restoration of Vascular Relaxation in Cerebral Arteries of Congenic Dahl Rats Receiving the Brown Norway (BN) Renin Gene

The Dahl Salt Sensitive (SS) rat exhibits impaired regulation of the renin‐angiotensin system (RAS), chronically low levels of angiotensin II, and impaired vascular relaxation in response to vasodilator stimuli. This impaired vascular relaxation is likely due to chronic exposure of the animals to low levels of ANG II. The present study investigates responses to vasodilator stimuli in two different congenic strains in which a portion of the BN chromosome 13 was inserted onto the SS genetic background. Congenic strain SS.BN‐(D13rat88‐D13rat105) contains a 2.9 Mbp portion of chromosome 13 which excludes the BN renin gene, and would not be expected to restore normal regulation of the RAS. Congenic strain SS.BN‐(D13hmgc41‐D13hmgc23) contains a 2.0 Mbp adjacent region that includes the BN renin gene, which should restore normal ANG II levels in animals fed low salt (LS) diet. Males from each congenic strain were fed a LS diet (0.4% NaCl) after weaning. At the age of 10–14 weeks, middle cerebral arteries (MCA) were isolated and cannulated and dilator responses to acetylcholine (10−10M–10−5M) and reduced PO2 were assessed using television microscopy. MCA from the congenic strain containing the BN allele for renin dilated in response to acetylcholine and reduced PO2 while the MCA from the congenic strain containing the SS renin allele did not. These results suggest that the presence of a normally functioning renin gene from the BN rat in the SS genetic background restores normal vasodilator responses in the MCA. [NIH #HL‐29587; #HL‐65289, and #HL‐72920].

Effects of chronic in vivo administration of nitroglycerine on ACh‐induced endothelium‐dependent relaxation in rabbit cerebral arteries

In the setting of nitrate tolerance, endothelium-dependent relaxation is reduced in several types of peripheral vessels. However, it is unknown whether chronic in vivo administration of nitroglycerine modulates such relaxation in cerebral arteries. Isometric force and smooth muscle cell membrane potential were measured in endothelium-intact strips from rabbit middle cerebral artery (MCA) and posterior cerebral artery (PCA). ACh (0.1-10 microM) concentration-dependently induced endothelium-dependent relaxation during the contraction induced by histamine in both MCA and PCA. Chronic (10 days) in vivo administration of nitroglycerine reduced the ACh-induced relaxation in PCA but not in MCA, in the presence of the cyclooxygenase inhibitor diclofenac (3 microM). In the presence of the NO-synthase inhibitor N (omega)-nitro-L-arginine (L-NNA, 0.1 mM) plus diclofenac, in MCA from both nitroglycerine-untreated control and -treated rabbits, ACh (0.1-10 microM) induced a smooth muscle cell hyperpolarization and relaxation, and these were blocked by the small-conductance Ca(2+)-activated K(+)-channel inhibitor apamin (0.1 microM), but not by the large- and intermediate-conductance Ca(2+)-activated K(+)-channel inhibitor charybdotoxin (0.1 microM). In contrast, in PCA, ACh (<3 microM) induced neither hyperpolarization nor relaxation under these conditions, suggesting that the endothelium-derived relaxing factor is NO in PCA, whereas endothelium-derived hyperpolarizing factor (EDHF) plays a significant role in MCA. It is suggested that in rabbit cerebral arteries, the function of the endothelium-derived relaxing factor NO and that of EDHF may be modulated differently by chronic in vivo administration of nitroglycerine.

Endothelial nitric oxide synthase uncoupling: Is it a physiological mechanism of endothelium-dependent relaxation in cerebral artery?

See article by Drouin et al. [8] (pages 73–81) in this issue. Nitric oxide (NO) is generated from the conversion of l -arginine to l-citrulline by endothelial nitric oxide synthase (eNOS), which requires Ca2+/calmodulin, FAD, FMN, and tetrahydrobiopterin (BH4) as cofactors. Chemical studies in vitro demonstrated that the catalytic mechanisms of NOS involve flavin-mediated electron transport from a flavin-containing reductase domain to a heme-containing oxygenase domain. Here, oxygen is reduced and incorporated into the guanidine group of l-arginine, giving rise to NO and l-citrulline. Calcium/calmodulin binding to NOS increases the rate of reduction of both flavins and the heme iron. Reduction of iron (III) to iron (II) facilitates oxygen binding to the heme group to form a transient ferrous–dioxygen complex. NOS can also produce superoxide under certain conditions. Superoxide is generated from the oxygenase domain by dissociation of the ferrous–dioxygen complex [1,2]. This state is referred to as the “uncoupled state of eNOS” (eNOS uncoupling). BH4 couples l-arginine oxidation to NADPH consumption and prevents dissociation of the ferrous–dioxygen complex. Although in vitro data using purified eNOS demonstrate that modulation of the BH4 concentration may regulate the ratio of superoxide to NO … *Corresponding author. Tel.: +81 78 382 5840; fax: +81 78 382 5858. Email address: yokoyama{at}med.kobe-u.ac.jp

Evidence for Involvement of Both IK Ca and SK Ca Channels in Hyperpolarizing Responses of the Rat Middle Cerebral Artery

Endothelium-derived hyperpolarizing factor responses in the rat middle cerebral artery are blocked by inhibiting IKCa channels alone, contrasting with peripheral vessels where block of both IKCa and SKCa is required. As the contribution of IKCa and SKCa to endothelium-dependent hyperpolarization differs in peripheral arteries, depending on the level of arterial constriction, we investigated the possibility that SKCa might contribute to equivalent hyperpolarization in cerebral arteries under certain conditions. Rat middle cerebral arteries (approximately 175 microm) were mounted in a wire myograph. The effect of KCa channel blockers on endothelium-dependent responses to the protease-activated receptor 2 agonist, SLIGRL (20 micromol/L), were then assessed as simultaneous changes in tension and membrane potential. These data were correlated with the distribution of arterial KCa channels revealed with immunohistochemistry. SLIGRL hyperpolarized and relaxed cerebral arteries undergoing variable levels of stretch-induced tone. The relaxation was unaffected by specific inhibitors of IKCa (TRAM-34, 1 micromol/L) or SKCa (apamin, 50 nmol/L) alone or in combination. In contrast, the associated smooth-muscle hyperpolarization was inhibited, but only with these blockers in combination. Blocking nitric oxide synthase (NOS) or guanylyl cyclase evoked smooth-muscle depolarization and constriction, with both hyperpolarization and relaxation to SLIGRL being abolished by TRAM-34 alone, whereas apamin had no effect. Immunolabeling showed SKCa and IKCa within the endothelium. In the absence of NO, IKCa underpins endothelium-dependent hyperpolarization and relaxation in cerebral arteries. However, when NOS is active SKCa contributes to hyperpolarization, whatever the extent of background contraction. These changes may have relevance in vascular disease states where NO release is compromised and when the levels of SKCa expression may be altered.

KMUP‐1 activates BKCa channels in basilar artery myocytes via cyclic nucleotide‐dependent protein kinases

This study investigated whether KMUP-1, a synthetic xanthine-based derivative, augments the delayed-rectifier potassium (K(DR))- or large-conductance Ca2+-activated potassium (BKCa) channel activity in rat basilar arteries through protein kinase-dependent and -independent mechanisms. Cerebral smooth muscle cells were enzymatically dissociated from rat basilar arteries. Conventional whole cell, perforated and inside-out patch-clamp electrophysiology was used to monitor K+- and Ca2+ channel activities. KMUP-1 (1 microM) had no effect on the K(DR) current but dramatically enhanced BKCa channel activity. This increased BKCa current activity was abolished by charybdotoxin (100 nM) and iberiotoxin (100 nM). Like KMUP-1, the membrane-permeable analogs of cGMP (8-Br-cGMP) and cAMP (8-Br-cAMP) enhanced the BKCa current. BKCa current activation by KMUP-1 was markedly inhibited by a soluble guanylate cyclase inhibitor (ODQ 10 microM), an adenylate cyclase inhibitor (SQ 22536 10 microM), competitive antagonists of cGMP and cAMP (Rp-cGMP, 100 microM and Rp-cAMP, 100 microM), and cGMP- and cAMP-dependent protein kinase inhibitors (KT5823, 300 nM and KT5720, 300 nM). Voltage-dependent L-type Ca2+ current was significantly suppressed by KMUP-1 (1 microM), and nearly abolished by a calcium channel blocker (nifedipine, 1 microM). In conclusion, KMUP-1 stimulates BKCa currents by enhancing the activity of cGMP-dependent protein kinase, and in part this is due to increasing cAMP-dependent protein kinase. Physiologically, this activation would result in the closure of voltage-dependent calcium channels and the relaxation of cerebral arteries.

Nitrergic Neurodegeneration in Cerebral Arteries of Streptozotocin-Induced Diabetic Rats

Although autonomic neuropathy is recognized as an independent risk factor for stroke in diabetes, the mechanism by which autonomic nerves are involved in this pathology is unknown. Parasympathetic (cholinergic) nerves of the autonomic nervous system are known to innervate and to cause relaxation of cerebral arteries by releasing nitric oxide (NO); hence, they are called nitrergic nerves. However, the effect of diabetes on nitrergic nerves is unknown. Here, we show that perivascular nitrergic nerves around the cerebral arteries degenerate in two phases in streptozotocin-induced diabetic rats. In the first phase, perivascular nitrergic nerve fibers remain intact while they lose their neuronal NO synthase content. This phase is reversible with insulin treatment. In the second phase, nitrergic cell bodies in the ganglia are lost via apoptosis in an irreversible manner. Throughout the two phases, irreversible thickening of the smooth muscle layer of cerebral arteries is observed. This is the first demonstration of nitrergic degeneration in diabetic cerebral arteries, which could elucidate the link between diabetic autonomic neuropathy and stroke.

Vasoconstrictor mechanisms in the cerebral circulation are unaffected by insulin resistance.

Insulin-resistance (IR) impairs agonist-induced relaxation in cerebral arteries, but little is known about its effect on constrictor mechanisms. We examined the vascular responses of the basilar artery (BA) and its side branches in anesthetized Zucker lean (ZL) and IR Zucker obese (ZO) rats using a cranial window technique. Endothelin-1 (ET-1) constricted the BAs in both the ZL and ZO rats, but there was no significant difference between the two groups (ZL: 36 +/- 8%; ZO: 33 +/- 3% at 10(-8) M). Inhibition of the ET(A) receptors by BQ-123 slightly increased the diameters of the BAs, with no difference shown between the ZL (6 +/- 1%) and ZO (5 +/- 3%) rats. Expressions of the ET(A) receptors and ET-1 mRNA examined by immunoblot analysis and RT-PCR, respectively, were also similar in the ZL and ZO groups. Phorbol 12,13-dibutyrate (PDBu), an activator of protein kinase C (PKC), and the thromboxane A(2) (TxA(2)) mimetic U-46619 constricted the BAs, but similarly to ET-1, there was no significant difference between the ZL and ZO groups (10(-6) M PDBu: ZL: 33 +/- 2%; ZO: 32 +/- 4%; and 10(-7) M U-46619: ZL: 23 +/- 1%; ZO: 19 +/- 2%). Inhibition of Rho-kinase with Y-27632 induced dilation of the BAs, and these responses were also comparable in the ZL and ZO rats (ZL: 39 +/- 4%; ZO: 38 +/- 2% at 10(-5) M). In contrast, nitric oxide-dependent relaxation to bradykinin was significantly reduced in the ZO rats (10(-6) M: 10 +/- 3%) compared with ZLs (29 +/- 7%, P < 0.01). These findings indicate that vasoconstrictor responses of the BA mediated by ET-1, TxA(2), PKC, and Rho-kinase are not affected by IR.

Maturation Alters Cyclic Nucleotide and Relaxation Responses to Nitric Oxide Donors in Ovine Cerebral Arteries

To examine the hypothesis that maturation modulates nitric oxide (NO)-induced relaxation in cerebral arteries, we quantified concentration-relaxation relations and the corresponding dynamic responses of guanosine 3′:5′-cyclic monophosphate (cGMP) and adenosine 3′:5′-cyclic monophosphate (cAMP) levels following administration of nitroglycerin and S-nitroso-N-acetyl-penicilamine (SNAP), an NO donor, in posterior communicating and middle cerebral arteries from newborn (3–7 days) and adult sheep. The results offer 5 main observations: (1) the efficacy and potency of NO donors were generally greater in newborn than in adult cerebral arteries; (2) rates of relaxation, and presumably rates of NO release, were faster for equimolar concentrations of SNAP than for nitroglycerin in both newborn and adult arteries; (3) basal concentrations were greater for cAMP than for cGMP, and both were greater in newborn than adult cerebral arteries; (4) in adult cerebral arteries, NO-induced increases in cGMP occurred faster but relaxation developed more slowly than in newborn cerebral arteries, and (5) responses to NO donors involved significant cross-reactivity between cGMP and cAMP, the characteristics of which were age, artery, and agent specific. From these results, we conclude that postnatal changes in reactivity to NO reflect corresponding changes in soluble guanylate cyclase activity and possible decreases in NO half-life. We also conclude that maturation slows the mechanisms mediating NO-induced relaxation, and that this effect is more pronounced in distal than in proximal cerebral arteries. The data also suggest that the rate-limiting step governing rates of response to NO is probably downstream from cGMP synthesis. From the basal cyclic nucleotide levels, we conclude that basal ratios of synthesis to hydrolysis were greater in fetal than adult arteries. Because NO increased both cGMP and cAMP, we speculate that Type III phosphodiesterase has a possible influence upon cerebrovascular responses to NO, and that this influence varies with postnatal age and artery type. Together, these findings emphasize that the cerebrovascular effects of NO are highly age dependent and artery specific, and should be carefully considered when administering NO therapeutically in the neonate.

High-salt diet impairs vascular relaxation mechanisms in rat middle cerebral arteries.

Male Sprague-Dawley rats were maintained on a low-salt (LS) diet (0.4% NaCl) or a high-salt (HS) diet (4% NaCl) for 3 days or 4 wk. PO(2) reduction to 40-45 mmHg, the stable prostacyclin analog iloprost (10 pg/ml), and stimulatory G protein activation with cholera toxin (1 ng/ml) caused vascular smooth muscle (VSM) hyperpolarization, increased cAMP production, and dilation in cerebral arteries from rats on a LS diet. Arteries from rats on a HS diet exhibited VSM depolarization and constriction in response to hypoxia and iloprost, failed to dilate or hyperpolarize in response to cholera toxin, and cAMP production did not increase in response to hypoxia, iloprost, or cholera toxin. Low-dose angiotensin II infusion (5 ng x kg(-1) x min(-1) i.v.) restored normal responses to reduced PO(2) and iloprost in arteries from animals on a HS diet. These observations suggest that angiotensin II suppression with a HS diet leads to impaired relaxation of cerebral arteries in response to vasodilator stimuli acting at the cell membrane.