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VASCULAR BIOLOGY

TNF- dilates cerebral arteries via NAD(P)H oxidase-dependent Ca²⁺ spark activation

Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee

Submitted 3 October 2005 ; accepted in final form 31 October 2005

	ABSTRACT

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Expression of TNF-, a pleiotropic cytokine, is elevated during stroke and cerebral ischemia. TNF- regulates arterial diameter, although mechanisms mediating this effect are unclear. In the present study, we tested the hypothesis that TNF- regulates the diameter of resistance-sized (150-µm diameter) cerebral arteries by modulating local and global intracellular Ca²⁺ signals in smooth muscle cells. Laser-scanning confocal imaging revealed that TNF- increased Ca²⁺ spark and Ca²⁺ wave frequency but reduced global intracellular Ca²⁺ concentration ([Ca²⁺]_i) in smooth muscle cells of intact arteries. TNF- elevated reactive oxygen species (ROS) in smooth muscle cells of intact arteries, and this increase was prevented by apocynin or diphenyleneiodonium (DPI), both of which are NAD(P)H oxidase blockers, but was unaffected by inhibitors of other ROS-generating enzymes. In voltage-clamped (–40 mV) cells, TNF- increased the frequency and amplitude of Ca²⁺ spark-induced, large-conductance, Ca²⁺-activated K⁺ (K_Ca) channel transients 1.7- and 1.4-fold, respectively. TNF--induced transient K_Ca current activation was reversed by apocynin or by Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a membrane-permeant antioxidant, and was prevented by intracellular dialysis of catalase. TNF- induced reversible and similar amplitude dilations in either endothelium-intact or endothelium-denuded pressurized (60 mmHg) cerebral arteries. MnTMPyP, thapsigargin, a sarcoplasmic reticulum Ca²⁺-ATPase blocker that inhibits Ca²⁺ sparks, and iberiotoxin, a K_Ca channel blocker, reduced TNF--induced vasodilations to between 15 and 33% of control. In summary, our data indicate that TNF- activates NAD(P)H oxidase, resulting in an increase in intracellular H₂O₂ that stimulates Ca²⁺ sparks and transient K_Ca currents, leading to a reduction in global [Ca²⁺]_i, and vasodilation.

cerebrovascular circulation; ryanodine-sensitive Ca²⁺ release channel; Ca²⁺-activated K⁺ channel; reactive oxygen species; vasodilation

TNF- acts in a paracrine manner by binding to two membrane receptors, p55 and p75. Besides its actions on neurons, astrocytes, and microglia, TNF- also regulates arterial contractility (15), an effect that may modulate injury by altering local blood supply. However, the regulation of arterial diameter and blood flow by TNF- is complex. Intracranial injection of TNF- in vivo constricted pial arterioles and reduced cerebral blood flow (29, 38, 44). TNF- also induced procontractile effects in coronary arteries (23, 51). In contrast, TNF- dilated cerebral, mesenteric, and cremaster muscle arterioles and relaxed endothelium-denuded aortas (4, 7, 16, 22, 32, 37). In bronchial arteries, TNF- initially induced dilation, followed by constriction 2 h later (45). TNF--mediated vasoregulation can also occur through both endothelium-dependent and endothelium-independent mechanisms (4, 7, 16, 22, 23, 29, 32, 37, 38, 44, 45, 51). Collectively, data suggest that TNF- can induce both vasoconstriction and vasodilation and that its effects may be time dependent. Regardless of these data, the mechanisms that mediate vasoregulation by TNF- are poorly understood.

A major regulator of arterial diameter is the smooth muscle cell global intracellular Ca²⁺ concentration ([Ca²⁺]_i). In smooth muscle cells, global [Ca²⁺]_i is regulated by extracellular Ca²⁺ influx and intracellular Ca²⁺ release (19). An elevation in global [Ca²⁺]_i induces constriction, whereas a decrease in global [Ca²⁺]_i leads to vasodilation. Another Ca²⁺ signal, termed a Ca²⁺ spark, is a rapid, localized intracellular Ca²⁺ transient that occurs as a result of the activation of several ryanodine-sensitive Ca²⁺ release (RyR) channels on the sarcoplasmic reticulum (SR) (19, 33). In arterial smooth muscle cells, Ca²⁺ sparks occur close to the plasma membrane and activate several large-conductance Ca²⁺-activated K⁺ (K_Ca) channels, resulting in a transient K_Ca current. The resulting membrane hyperpolarization reduces voltage-dependent Ca²⁺ channel activity, leading to a decrease in Ca²⁺ entry, a reduction in global [Ca²⁺]_i and vasodilation (19, 33). In contrast, Ca²⁺ spark inhibition leads to vasoconstriction due to depolarization-induced voltage-dependent Ca²⁺ channel activation (19). Alterations in Ca²⁺ sparks and effective coupling to K_Ca channels have been implicated in hypertension and diabetes (1, 6, 19, 31). Another intracellular Ca²⁺ signal, termed a Ca²⁺ wave, is a propagating [Ca²⁺]_i elevation that occurs as a result of the activation of SR RyR channels and/or inositol 1,4,5-trisphosphate-gated Ca²⁺ channels. Although the physiological functions of Ca²⁺ waves have been less well described, Ca²⁺ waves, when stimulated, contribute Ca²⁺ for contraction (35).

The goal of the present study was to investigate whether TNF- regulates cerebral artery diameter by modulating smooth muscle Ca²⁺ signaling and, if so, to determine the underlying mechanisms. Such information would improve the understanding of cytokine-mediated changes in arterial diameter during vascular pathologies, including stroke.

	METHODS

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Tissue Preparation and Single-Cell Isolation

Sprague-Dawley rats (250 g) of either sex were euthanized by administering an intraperitoneal injection of a pentobarbital sodium overdose (150 mg/kg body wt) in accordance with the policies and procedures of the Animal Care and Use Committee at the University of Tennessee. The brains were removed and placed into ice-cold (4°C) oxygenated (95% O₂-5% CO₂) physiological salt solution (PSS) containing (in mM) 119 NaCl, 4.7 KCl, 24 NaHCO₃, 1.2 KH₂PO₄, 1.6 CaCl₂, 1.2 MgSO₄, 0.023 EDTA, and 11 glucose. The posterior cerebral, middle cerebral, and cerebellar arteries (50–200 µm in diameter) were removed, cleaned of connective tissue, and maintained in ice-cold PSS. When appropriate, the endothelium was removed by introducing an air bubble into the arterial lumen for 2 min, followed by a 30-s wash with H₂O. Individual smooth muscle cells were dissociated from cerebral arteries using enzymes and an isolation solution composed of (in mM) 55 NaCl, 80 sodium glutamate, 5.6 KCl, 2 MgCl₂, 10 HEPES, and 10 glucose (pH 7.3 with NaOH) as previously described (18). Cells were stored on ice and used for experiments between 1 and 6 h after isolation.

Laser-Scanning Confocal Ca²⁺ Imaging

Artery segments (1–2 mm in length) were placed into HEPES-buffered PSS composed of (in mM) 134 NaCl, 6 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, and 10 glucose (pH 7.4, NaOH) containing fluo-4 AM (10 µM) and Pluronic F-127 (0.05%) for 60 min at 22°C. To allow for indicator deesterification, the arteries were subsequently placed into HEPES-buffered PSS for 30 min at 22°C. Smooth muscle cells within the arterial wall were scanned using an OZ laser-scanning confocal microscope (Noran Instruments, Middleton, WI) and a x60 magnification water-immersion lens objective (numerical aperture 1.2) attached to a TE300 microscope (Nikon). Cells were illuminated using a krypton-argon laser at 488 nm, and emitted light >500 nm was collected. Planar images (56.3 x 52.8 µm) were recorded every 16.7 ms (i.e., 60 images s^–1). To compare laser-scanning confocal Ca²⁺ imaging data with electrophysiological recordings obtained in this study, Ca²⁺ sparks in smooth muscle cells of arterial segments were measured in an extracellular solution containing 30 mM K⁺, which depolarizes smooth muscle cells to approximately –40 mV. This membrane potential is similar to that of cerebral arteries pressurized to 60 mmHg (see Ref. 20 for similar procedure). The 30 mM K⁺ bath solution contained (in mM) 110 NaCl, 30 KCl, 10 HEPES, 2 CaCl₂, 1 MgCl₂, and 10 glucose (pH 7.4 maintained using NaOH). At least two different representative areas of each artery were scanned for at least 10 s for each condition. The same area of each artery was scanned only once to avoid any laser-induced changes in Ca²⁺ signaling, and the effects of drugs were measured in paired experiments. Ca²⁺ sparks were detected in smooth muscle cells using custom analysis software written in IDL 5.2 (Research Systems, Boulder, CO), kindly provided by Drs. M. T. Nelson and A. D. Bonev (University of Vermont, Burlington, VT). Automated and manual detection of Ca²⁺ sparks were performed by dividing an area of 1.54 µm (7 pixels) x 1.54 µm (7 pixels) (i.e., 2.37 µm²) in each image (F) by a baseline level (F₀) that was determined by averaging 10 images without Ca²⁺ spark activity. The entire area of each image was analyzed to detect Ca²⁺ sparks. A Ca²⁺ spark was defined as a localized increase in F/F₀ >1.2 (10). Arterial Ca²⁺ spark frequency (measured in Hz) was calculated by averaging values from at least two different areas. Ca²⁺ waves were defined as an elevation in F/F₀ >1.2 that propagated for at least 20 µm. Global Ca²⁺ fluorescence was calculated from the same images used for Ca²⁺ spark analysis and was the mean pixel value of 100 different images acquired during a 10-s period. To calculate whether TNF- altered global [Ca²⁺]_i, global Ca²⁺ fluorescence in TNF- was divided by the corresponding control value.

Patch-Clamp Electrophysiology

K⁺ currents were measured using either the conventional whole cell or perforated configuration of the patch-clamp technique with an Axopatch 200B amplifier (Axon Instruments, Union City, CA). Bath solution was 6 mM K⁺ HEPES-buffered PSS (composition described above). For perforated patch-clamp experiments, the pipette solution contained (in mM) 110 potassium aspartate, 30 KCl, 10 NaCl, 1 MgCl₂, 10 HEPES, and 0.05 EGTA (pH 7.2 maintained using KOH). For conventional whole cell experiments, the pipette solution contained (in mM) 140 KCl, 1.9 MgCl₂, 0.037 CaCl₂, 0.1 EGTA, 10 HEPES, and 2 Na₂ATP (pH 7.2 maintained using KOH); the calculated free Ca²⁺ and free Mg²⁺ concentrations of this solution were 100 nM and 1 mM, respectively (WEBMAXC; Stanford University, Palo Alto, CA). In some experiments, 200 U/ml catalase was included in the whole cell pipette solution. When appropriate, catalase was heat inactivated by incubation at 92°C for 25 min. TNF- was applied to the bath solution 10 min after formation of the whole cell configuration to ensure exchange of the pipette solution with the cytosol. All experiments were performed using a holding potential of –40 mV. Membrane currents were recorded using a sample rate of 2.5 kHz and filtered at 1 kHz. Transient K_Ca current analysis was performed offline. A transient K_Ca current was defined as the simultaneous opening of three or more K_Ca channels.

Reactive Oxygen Species Measurements

Dichlorofluorescein imaging. Reactive oxygen species (ROS) were measured in isolated intact arterial segments using 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA). Isolated endothelium-denuded cerebral artery segments (1–2 mm) were incubated for 45 min at room temperature in 10 µM H₂DCFDA. H₂DCFDA is hydrolyzed by intracellular esterases to the nonfluorescent derivative dichlorodihydrofluorescein (H₂DCF). ROS oxidize H₂DCF to fluorescent dichlorofluorescein (DCF). DCF fluorescence was excited with 488 nm of light, and emitted light >525 nm was collected. Planar images were acquired using a Zeiss LSM5 laser-scanning confocal microscope. Images were recorded 10 min after TNF- or H₂O₂ application. Time controls were also performed in which agents were not applied. Because DCF fluorescence increases spontaneously upon exposure to excitation light (12), arterial regions were scanned only once. Images were background corrected. ImageJ software (National Institutes of Health, Bethesda, MD) was used for analysis.

Dihydroethidium imaging. Cerebral arterial segments were incubated for 20 min in HEPES-buffered PSS without (time-matched control) or with TNF-. Arteries were then embedded in OTC and snap-frozen in liquid N₂. Transverse sections 30 µm thick were cut, mounted onto glass slides, and incubated with 10 µM dihydroethidium (DHE) in a 100% N₂ atmosphere in the dark for 30 min at 37°C. Arterial sections were excited with 488 nm light, and emitted light >590 nm was collected using an OZ laser-scanning confocal microscope. Custom-written software was used to measure DHE fluorescence intensity. Fluorescence intensity in TNF- was divided by that in time-matched controls to determine the effects of TNF- on DHE intensity. Tissue processing did not elevate ROS, because the DHE fluorescence intensity of arteries preincubated with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) was 1.04 ± 0.06-fold of those that were left untreated (n = 5; P > 0.05).

Pressurized Artery Diameter Measurements

Cerebral arterial segments (100–200 µm diameter) were placed in a temperature-controlled perfusion chamber (Living Systems Instrumentation, Burlington, VT) and cannulated with glass pipettes at each end. The chamber was perfused continuously with PSS equilibrated with 5% CO₂, 21% O₂, and 74% N₂, pH 7.4, and maintained at 37°C. Arteries were observed using a charge-coupled device camera attached to an inverted microscope (TS100F; Nikon). Arterial diameter was monitored using edge detection software (IonWizard; IonOptix, Milton, MA). Arterial intraluminal pressure was elevated to 60 mmHg, which resulted in the development of myogenic tone. After diameter stabilization, tested agents were applied using chamber perfusion. Arterial diameter was digitized at 1 Hz using the edge detection function of IonWizard software. Endothelial denudation was confirmed by the absence of a dilatory response to carbachol (10^–5 M), an endothelium-dependent cerebral vasodilator. At the end of each experiment, passive arterial diameter was determined using a Ca²⁺-free bath solution containing 1 mM EGTA.

Data Analysis

Data are expressed as means ± SE. Student's t-test was used to compare paired and unpaired data from two populations, and ANOVA and the Student-Newman-Keuls test were used for multiple-group comparisons. Evaluation of whether distributions were Gaussian using the method of Kolmogorov and Smirnov. P < 0.05 was considered significant.

Chemicals

Unless stated otherwise, all chemicals used in this study were obtained from Sigma (St. Louis, MO) or Calbiochem (San Diego, CA). Papain was purchased from Worthington Biochemical (Lakewood, NJ), fluo-4 AM and H₂DCFDA were obtained from Molecular Probes (Eugene, OR), and rat recombinant TNF- was purchased from BD Pharmingen (San Diego, CA).

	RESULTS

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TNF- Activates Ca²⁺ Sparks and Ca²⁺ Waves and Reduces Global [Ca²⁺]_i in Smooth Muscle Cells of Intact Cerebral Arteries

The regulation of local and global Ca²⁺ signals in smooth muscle cells by TNF- was measured in cerebral artery segments. TNF- (50 ng/ml) increased mean Ca²⁺ spark frequency from 1.53 ± 0.16 Hz to 2.6 ± 0.51 Hz, or 1.7-fold (Fig. 1, A and B). TNF- did not change Ca²⁺ spark amplitude (control F/F₀, 1.36 ± 0.01, n = 184 sparks, and TNF- F/F₀, 1.34 ± 0.01, n = 329 sparks; P > 0.05). In the same cells, TNF- also elevated mean Ca²⁺ wave frequency from 0.32 ± 0.02 Hz to 0.48 ± 0.03 Hz, or 1.5-fold (Fig. 1C). In contrast, TNF- decreased mean global [Ca²⁺]_i to 0.85 ± 0.02 of control (n = 6 arteries, P < 0.05). These experiments suggest TNF- elevates Ca²⁺ spark and wave frequency and reduces global [Ca²⁺]_i in cerebral arterial smooth muscle cells.

View larger version (39K): [in this window] [in a new window]   Fig. 1. TNF- activates Ca2+ sparks and Ca2+ waves in smooth muscle cells of intact cerebral arteries. A: average fluorescence (100 of 600 images) during 10-s period in 2 different areas of the same cerebral artery in control and TNF-. Locations of Ca2+ sparks that occurred during 10-s period are indicated by white boxes (1.54 x 1.54 µm), and representative localized F/F0 changes with time are shown below respective images and labeled accordingly. In control, 8 sparks were identified in traces a and b. In TNF-, 11 sparks and 2 waves were detected in traces a–d. B: mean effects of TNF- (50 ng/ml) on Ca2+ spark frequency (n = 6 arteries). C: mean effects on Ca2+ wave frequency. *P < 0.05 vs. control.

TNF- Activates NAD(P)H Oxidase Leading to a ROS Elevation in Smooth Muscle Cells of Intact Cerebral Arteries

TNF- activates ROS generation in smooth muscle cells (13). Because RyR channels are redox sensitive (42), we tested the hypothesis that TNF- regulates local Ca²⁺ signaling by elevating intracellular ROS. Several enzymes generate ROS including NAD(P)H oxidase, xanthine oxidase, cytochrome P450, and mitochondrial electron transport chain complexes (13, 28, 41, 49). The DCF and DHE fluorescence methods were used to determine the pathways through which TNF- elevates ROS in the smooth muscle cells of small cerebral arteries. TNF- increased DCF fluorescence intensity 2.7 ± 0.32-fold and DHE intensity 1.51 ± 0.03-fold in the smooth muscle cells of intact arteries (Fig. 2, A–D). During the same period, H₂O₂ elevated DCF fluorescence 4.7 ± 0.68-fold, whereas DCF fluorescence did not change in controls (absence of applied agents) (Fig. 2B). In the presence of apocynin or diphenyleneiodonium (DPI), which are NAD(P)H oxidase blockers, TNF- did not elevate DCF fluorescence (Fig. 2B). MnTMPyP, a membrane-permeant O₂^–· and catalase mimetic, also prevented the TNF--induced DCF fluorescence elevation (Fig. 2B). Apocynin (1.07 ± 0.23-fold vs. control; n = 5), DPI (0.85 ± 0.1-fold vs. control; n = 4), and MnTMPyP (1.14 ± 0.17-fold vs. control; n = 4) did not change ROS when applied alone (P > 0.05 for each). TNF--induced DCF fluorescence elevations were not reduced by oxypurinol, a xanthine oxidase inhibitor; 17-octadecanoic acid, a cytochrome P450 blocker; or lonidamine, a mitochondrial permeability transition pore (PTP) opener (Fig. 2B). Oxypurinol (0.98 ± 0.13-fold vs. control; n = 4), 17-octadecanoic acid (1.09 ± 0.12-fold vs. control; n = 5), and lonidamine (0.86 ± 0.13-fold vs. control; n = 4) did not change DCF fluorescence when applied alone (P > 0.05 for each). These data indicate that TNF- activates NAD(P)H oxidase, leading to ROS elevation in cerebral arterial smooth muscle cells.

View larger version (35K): [in this window] [in a new window]   Fig. 2. TNF- elevates reactive oxygen species (ROS) in smooth muscle cells of intact cerebral arteries via NAD(P)H oxidase activation. A: representative confocal images of dichlorofluorescein (DCF) fluorescence in 2 different areas of the same cerebral artery in control (left) and after TNF- (50 ng/ml) (right). B: mean effects of TNF- on DCF fluorescence. TNF- (50 ng/ml; n = 10 arteries), H2O2 (100 µM; n = 6), TNF- + apocynin (Apo, 25 µM; n = 10), TNF- + diphenyleneiodonium (DPI, 10 µM; n = 7), TNF- + Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) (MnT, 10 µM; n = 10), TNF- + oxypurinol (Oxy, 10 µM; n = 6), TNF- + 17-octadecanoic acid (Oda, 10 µM; n = 10), TNF- + lonidamine (Lon, 100 µM; n = 5), time control (Cntrl; n = 5). C: TNF- (50 ng/ml) elevated dihydroethidium (DHE) fluorescence in intact cerebral arteries. D: average effects of TNF- (50 ng/ml) on DHE fluorescence (n = 5). *P < 0.05 vs. control. #P < 0.05 vs. TNF-.

TNF- Activates Transient K_Ca Currents in Isolated Arterial Smooth Muscle Cells

In arterial smooth muscle cells, a Ca²⁺ spark activates several K_Ca channels, resulting in a transient K_Ca current that induces membrane hyperpolarization (19). To investigate the effects of TNF--induced changes in Ca²⁺ signaling, transient K_Ca current regulation by TNF- was measured in isolated, voltage-clamped (–40 mV) arterial smooth muscle cells. When using the perforated patch-clamp configuration, TNF- evoked a gradual increase in transient K_Ca current frequency and amplitude that reached a steady-state elevation of 1.7- and 1.4-fold, respectively, in 10 min (Fig. 3, A, C, and D). Similar observations were made when using the conventional whole cell configuration (Fig. 3, B–D). Apocynin did not alter transient K_Ca current frequency (0.89 ± 0.12-fold vs. control; n = 6) or amplitude (1.0 ± 0.06-fold vs. control; n = 6) when applied alone (P > 0.05 for each) but reversed the TNF--induced increase in transient K_Ca current frequency and amplitude (Fig. 3, B–D). These data suggest that TNF- stimulates transient K_Ca currents by activating NAD(P)H oxidase.

View larger version (45K): [in this window] [in a new window]   Fig. 3. TNF- stimulates transient Ca2+-activated K+ (KCa) channel currents in isolated cerebral artery smooth muscle cells due to NAD(P)H oxidase activation. A and B: original traces showing transient KCa currents in cells voltage clamped at –40 mV. A: TNF--activated transient KCa currents using the perforated-patch-clamp configuration. B: TNF- activated transient KCa currents using conventional whole cell configurations, and this activation was reversed by addition of apocynin. C and D: mean effects on transient KCa current frequency and amplitude of TNF- (50 ng/ml) using perforated patch clamp (p-p; n = 6), TNF- (50 ng/ml) whole cell (w-c, n = 6), TNF- + apocynin (Apo, 25 µM; n = 6), TNF- washout (Wash; n = 6), ceramide (Cer, 10 µM; n = 6), and atractyloside (Atr, 100 µM; n = 4). *P < 0.05 vs. control. #P < 0.05 vs. TNF-.

To determine whether TNF--induced transient K_Ca current activation occurs as a result of a ROS elevation, we measured transient K_Ca current regulation by MnTMPyP as well as by catalase that converts H₂O₂ to H₂O. MnTMPyP reversed TNF--induced elevations in transient K_Ca current frequency and amplitude (Fig. 4, A–C). In contrast, MnTMPyP did not change transient K_Ca current frequency (1.0 ± 0.1-fold vs. control; n = 5) or amplitude (1.0 ± 0.07-fold vs. control) when applied alone (P > 0.05 for each). Similarly, when the conventional whole cell configuration was used, inclusion of catalase in the pipette solution (to allow intracellular access) prevented TNF--induced transient K_Ca current activation (Fig. 4, B and C). In contrast, when the pipette solution was supplemented with heat-inactivated catalase, TNF- increased transient K_Ca current frequency and amplitude 1.8- and 1.4-fold, respectively (Fig. 4, B and C). In summary, these data indicate that TNF- activates Ca²⁺ sparks and transient K_Ca currents by inducing an NAD(P)H oxidase-mediated ROS elevation.

View larger version (49K): [in this window] [in a new window]   Fig. 4. TNF--induced transient KCa current activation occurs as a result of ROS elevation. A: 50 ng/ml TNF--induced transient KCa current activation was reversed by addition of MnTMPyP. B and C: mean transient KCa current frequency and amplitude. TNF- (50 ng/ml; n = 12), TNF- + MnTMPyP (10 µM; n = 6), TNF- with catalase (Cat, 200 U/ml; n = 6), TNF- with boiled catalase (Boil. catalase, 200 U/ml; n = 6). *P < 0.05 vs. control. #P < 0.05 vs. TNF-.

TNF- Dilates Pressurized Cerebral Arteries by Elevating ROS, SR Ca²⁺-ATPase Release, and K_Ca Channel Activity

TNF- has been demonstrated to induce both vasoconstriction and vasodilation by endothelium-dependent and endothelium-independent pathways (4, 7, 16, 22, 23, 29, 32, 37, 38, 44, 51). The data reported herein suggest that TNF- activates a dilatory Ca²⁺ signaling pathway in the smooth muscle cells of small, resistance-sized cerebral arteries. Therefore, vasoregulation by TNF- was investigated using resistance-sized, pressurized (60 mmHg) cerebral arteries. In endothelium-intact arteries, from a mean active diameter of 142 ± 3 µm (0.64 ± 0.01-fold vs. passive diameter), TNF- induced a mean dilation of 22 ± 1 µm (Fig. 5, A–C). In endothelium-denuded arteries, TNF- increased mean diameter by 18 ± 3 µm from 130 ± 9 µm (0.57 ± 0.02-fold vs. passive diameter) (Fig. 5C). TNF--induced dilations were not significantly different in endothelium-intact or endothelium-denuded arteries (P > 0.05). TNF--induced dilations were fully reversible (Fig. 5A), and consecutive TNF- applications resulted in dilations of similar magnitude (first application, 19 ± 2 µm; second application, 21 ± 2 µm) (n = 2).

View larger version (23K): [in this window] [in a new window]   Fig. 5. TNF- dilates pressurized (60 mmHg) cerebral arteries by elevating ROS and activating sarcoplasmic reticulum (SR) Ca2+ release and KCa channels. A: TNF- reversibly dilated a pressurized (60 mmHg) endothelium-intact cerebral artery. B: 10 ng/ml TNF--induced vasodilation was inhibited by 100 nM iberiotoxin (IBTX). C: mean 10 ng/ml TNF--induced dilation in endothelium-intact arteries in control (Cntrl; n = 29), MnTMPyP (MnT, 10 µM; n = 4), thapsigargin (+Tg, 100 nM; n = 7), iberiotoxin (+IBTX, 100 nM; n = 9), and endothelium-denuded arteries (Denud; n = 6). MnTMPyP reduced arterial diameter by 4 ± 1 µm (n = 4; P < 0.05). Thapsigargin and IBTX reduced mean arterial diameter by 9 ± 3 µm and 9 ± 1 µm, respectively (P < 0.05 for each), consistent with Ca2+ spark and KCa channel inhibition (19). *P < 0.05 vs. control. #P < 0.05 vs. TNF- in endothelium-intact arteries.

	DISCUSSION

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In the present study, we investigated the mechanisms by which TNF-, a pleiotropic cytokine, regulates the diameter of small cerebral arteries. Our data indicate that TNF- activates smooth muscle cell NAD(P)H oxidase, leading to the generation of ROS that activate Ca²⁺ sparks and thus transient K_Ca currents. The ensuing decrease in global [Ca²⁺]_i leads to vasodilation. These findings show for the first time that an inflammatory mediator that is central to a variety of vascular diseases induces vasodilation through Ca²⁺ spark activation.

TNF- is produced in the brain during ischemia and is implicated in the etiology of several cardiovascular diseases, including stroke, cardiac failure, coronary artery disease, and atherosclerosis (15, 46). TNF- is reported to induce both vasoconstriction and vasodilation. Topical application of TNF- dilated cerebral arterioles (7, 37), whereas intracranial injection of TNF- induced cerebrovascular constriction and reduced blood flow (29, 38, 44). TNF- also dilated mesenteric and cremaster muscle arterioles (4, 32, 45). Earlier studies suggested that TNF- promotes vasodilation by elevating nitric oxide (NO) and prostaglandin production in the arterial wall and in perivascular tissues (4, 7, 37). In contrast, in pulmonary arterial rings, a hypoxia-induced increase in TNF- mRNA was associated with contraction (43). TNF- also reduced bradykinin- and ACh-induced vasodilation in rat mesenteric arteries (48) and bradykinin- and A23187 [GenBank] -induced vasodilation in bovine coronary arteries (51), blunted ACh-induced vasodilation in cat carotid arteries (2), and evoked endothelium-dependent vasoconstriction in human and rat coronary arteries (17, 23). In humans, intrabrachial TNF- injection impaired bradykinin- and ACh-induced vasodilation (11). Data also suggest that TNF- inhibits vasodilation by triggering ROS that scavenge endothelial NO (51). It is unclear what underlies differential regulation of arterial contractility by TNF-, but several possibilities exist. Vasoregulatory actions of TNF- may be vascular bed specific. Different experimental protocols used may also explain the diversity of observations reported in various studies. In addition, the effects of TNF- on arterial contractility may be time dependent. For example, in sheep bronchial arteries, TNF- initially induced dilation, followed by constriction 2 h later (45). Data also suggest that differential effects on contractility may occur when TNF- acts primarily on either endothelial or smooth muscle cells. Conceivably, TNF--induced vasoconstriction might occur primarily as a result of an endothelium-dependent mechanism, whereas vasodilation may be mediated by a direct effect on smooth muscle cells. Here, we have shown that in isolated, pressurized, myogenic rat cerebral arteries, acute TNF- application (<30 min) induced vasodilation that was endothelium independent and was markedly attenuated by an antioxidant and inhibitors of SR Ca²⁺ release and K_Ca channels.

TNF- activates several signaling pathways in vascular smooth muscle cells. TNF- stimulated NAD(P)H oxidase, leading to ROS production in rat aortic smooth muscle cells (13). TNF- relaxed endothelium-denuded, phenylephrine-constricted aortic rings via PLA₂ activation, implicating ceramide as a mediator (22). In cardiac and endothelial cells, signaling mechanisms for TNF- involve mitochondria (20, 23–25, 35, 36). TNF- disrupted the mitochondrial electron transport chain, caused release of cytochrome c, induced mitochondrial ROS generation, and triggered apoptosis (14, 28, 30, 41, 52). Although the downstream intramitochondrial target for TNF- is unclear, PTP involvement was proposed in endothelial cells (52). PTP is also a downstream target for TNF--induced signaling in other cell types, and PTP opening may be triggered by electron transport chain inhibition and mitochondrial depolarization (5, 10, 39). Ceramide had no effect on transient K_Ca currents, and PTP openers inhibited these events, consistent with the findings reported in a previous study of arterial smooth muscle cells (10). Therefore, TNF- is unlikely to activate Ca²⁺ sparks and transient K_Ca currents by elevating ceramide or by inducing PTP opening. In some cell types, including neutrophils, preincubation with apocynin is required to block association of NAD(P)H oxidase subunits (40). Here, we have shown that in cerebral artery smooth muscle cells, apocynin reverses TNF--induced transient K_Ca current activation within 10 min. This effect may reflect rapid subunit turnover in the presence of TNF- in these cells. Our present data indicate that TNF- stimulates NAD(P)H oxidase, generating ROS that activate Ca²⁺ sparks and transient K_Ca currents in cerebral artery smooth muscle cells. These findings are consistent with those described in a recent study that reported mitochondria-derived ROS activate Ca²⁺ sparks and transient K_Ca currents in arterial smooth muscle cells (50). Thus, ROS derived from different sources induce vasodilation through a common pathway, namely, Ca²⁺ spark activation.

TNF--induced ROS may activate Ca²⁺ sparks and transient K_Ca currents as a result of a direct or indirect effect on RyR and K_Ca channels. Cardiac and skeletal muscle RyR channels are redox sensitive and are activated by oxidizing agents, including H₂O₂ (42). ROS also activate a number of other signal transduction pathways that may regulate RyR channel activity (42, 49). Although O₂^–· is the initial product of NAD(P)H oxidase, O₂^–· has a short half-life and rapidly dismutates to H₂O₂, a relatively stable oxidant. O₂^–· is unlikely to mediate the effects of TNF- on Ca²⁺ sparks, particularly because catalase blocked TNF--induced transient K_Ca current activation. Data suggest that H₂O₂ formed from NAD(P)H oxidase-generated O₂^–· activates RyR channels, leading to a Ca²⁺ spark frequency elevation. Indeed, exogenous H₂O₂ activates transient K_Ca currents in cerebral artery smooth muscle cells (10). Another potential ROS mediating Ca²⁺ spark activation in this study is HO·, a highly reactive species produced in a transition metal-catalyzed reaction (the Fenton reaction) between H₂O₂ and O₂^–·. In conventional whole cell experiments, we used a pipette solution absent in transition metals, and dialysis of the pipette solution within the cell interior should have washed out endogenous cytosolic transition metals. Thus HO· generation in these experiments should have been minimal, arguing against a significant role for HO· in TNF--induced Ca²⁺ spark and K_Ca channel activation.

ROS production or inhibition of ROS degradation leads to vasoconstriction, high blood pressure, and vascular hypertrophy (24). However, ROS also stimulate vasodilation. For example, flow-mediated vasodilation occurs because of the generation of mitochondria-derived H₂O₂ (27). Furthermore, topical application of ROS dilates cerebral arteries in vivo (47). Thus ROS induce both vasoconstriction and vasodilation. Conceivably, there might be a delicate balance between ROS concentrations that regulate physiological functions and higher ROS concentrations that lead to pathologies (24). The data reported herein indicate that TNF- regulates Ca²⁺ sparks by generating ROS from a single source, NAD(P)H oxidase. During disease, vasoconstriction may result from ROS produced in higher concentrations, from additional enzymes, from exogenous sources, and for prolonged periods. For example, oxyhemoglobin, which may contribute to vasoconstriction after subarachnoid hemorrhage, inhibits Ca²⁺ sparks in cerebral artery smooth muscle cells, and this effect can be blocked by antioxidants (21). TNF- receptors were recently proposed to confine NAD(P)H oxidase-mediated redox signaling spatially in human microvascular endothelial cells (25). TNF- may also activate Ca²⁺ sparks by elevating ROS in the local vicinity of Ca²⁺ spark sites. Through restriction, the global effects of ROS could be lessened and dilatory signaling could be maintained. Because Ca²⁺ spark sites are close to sarcolemmal K_Ca channels, local NAD(P)H oxidase-derived ROS should also regulate K_Ca channel activity. TNF- did not alter Ca²⁺ spark amplitude but increased transient K_Ca current amplitude. NAD(P)H oxidase inhibitors and antioxidants reduced the TNF--induced transient K_Ca current amplitude elevation, indicating that the effect was mediated by NAD(P)H oxidase-derived ROS. Thus TNF- also enhances the effective coupling of Ca²⁺ sparks to K_Ca channels, presumably by elevating K_Ca channel Ca²⁺ sensitivity within the micromolar concentration range generated by these Ca²⁺ transients (34). The combined effects of elevated Ca²⁺ spark frequency and transient K_Ca current amplitude could significantly increase K_Ca channel activity, leading to vasodilation (19, 33).

TNF- elevated Ca²⁺ wave frequency 1.5-fold but reduced global [Ca²⁺]_i. In the absence of receptor agonists, Ca²⁺ wave frequency in arterial smooth muscle cells is low, and Ca²⁺ waves contribute little to the global [Ca²⁺]_i (18). When stimulated to high frequencies, Ca²⁺ waves can contribute to the global [Ca²⁺]_i and thus induce smooth muscle contraction (35). Although TNF- stimulated Ca²⁺ waves, the frequency elevation was insufficient to contribute significantly to the global [Ca²⁺]_i. In contrast, TNF--induced Ca²⁺ spark activation significantly elevated K_Ca channel activity. The resulting membrane hyperpolarization would reduce voltage-dependent Ca²⁺ channel activity, leading to the observed decrease in global [Ca²⁺]_i (19). Therefore, the net effect of TNF--induced Ca²⁺ spark and Ca²⁺ wave activation is a reduction in the global [Ca²⁺]_i, leading to vasodilation.

In summary, in this study, we have defined a novel vasodilatory signaling pathway for an inflammatory cytokine mediated by Ca²⁺ spark and K_Ca channel activation in smooth muscle cells. After ischemia and stroke, TNF--induced Ca²⁺ spark activation and cerebrovascular dilation would be important in regulating local blood supply in the affected brain region.

	GRANTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This project was supported by grants from the National Institutes of Health and the American Heart Association National Center (to J. H. Jaggar). S. Y. Cheranov is a recipient of a postdoctoral fellowship from the American Heart Association-Southeast Affiliate.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. H. Jaggar, Dept. of Physiology, College of Medicine, Univ. of Tennessee Health Science Center, 426 Nash Research Bldg., Memphis, TN 38163 (e-mail: jjaggar{at}physio1.utmem.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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