Tonic regulation of middle meningeal artery diameter by ATP-sensitive potassium channels

Animal models

Animal housing and all experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory animals (eighth edition, 2011), ARRIVE (Animals in Research: Reporting In Vivo Experiments) guidelines and followed protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Vermont. Sprague–Dawley rats (males, 300–350 g; Charles River Laboratories, Saint Constant, QC, Canada) were euthanized by decapitation under deep pentobarbital anesthesia (60 mg/kg). The brain and calvaria were carefully dissected and placed in ice-cold artificial cerebral spinal fluid (aCSF) of the following composition (in mM): 125 NaCl, 3 KCl, 18 NaHCO₃, 1.25 NaH₂PO₄, 1 MgCl₂, 2 CaCl₂, and 5 glucose (pH 7.4). The dura mater was removed from the calvaria and the MMA dissected out using iris scissors. Superior cerebellar (cerebral) arteries were dissected from the brain in a similar fashion. Eight-week-old smooth muscle reporter (SMMHC-tdTomato) mice were used for in vivo imaging of meningeal arterial diameter. These mice, expressing the red fluorescent protein, tdTomato, under control of the smooth muscle myosin heavy chain (SMMHC) gene promoter were obtained by cross-breeding B6.FVB-Tg(Mhg11-cre/ERT2)1Soff/J (Jackson Lab, Stock No: 019079) mice and B6.Cg-Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}/J (Jackson Lab, Stock No: 007914) mice. SMMHC-tdTomato mice were administrated tamoxifen (40–80 mg/kg/day, Harlan Laboratories) in their food for 7 days starting at the age of 21 days.

Ex vivo measurements of arterial diameter

Middle meningeal and cerebral arteries were cannulated by glass micropipettes mounted in a 5 ml myograph chamber perfused with aCSF at 37℃ that was aerated with 5% CO₂, 20% O₂, 75% N₂ (pH 7.35–7.40). Intravascular pressure was adjusted using a pressure servo-controller and a peristaltic pump as previously described. ²⁹ Changes in arterial diameter were measured by video edge detection (Living Systems Instrumentation, St Albans, VT) using WinDaq data acquisition software (Dataq Instruments; Akron, OH). After allowing the arteries to equilibrate for 1 h, viability of the artery was determined by brief superfusion with aCSF containing 60 mM KCl (aCSF isosmotic replacement of NaCl with KCl). Arteries that exhibited a constriction representing less than a 50% decrease in diameter were discarded. Unless noted, isolated MMA were studied at an intravascular pressure of 40 mmHg. In aCSF, arterial diameter was 54.3 ± 2.4 µm in aCSF compared to a fully dilated (passive) diameter of 90.4 ± 2.5 µm obtained at the end of experimental protocol using Ca²⁺-free aCSF containing 100 µM diltiazem and 1 µM forskolin. This pressure-induced constriction or “myogenic tone” represented a 39.9 ± 2.4% decrease in lumen diameter (n = 30 arteries; N = 30 animals), similar to our previous findings that examined MMA responses over a range of intravascular pressures. ³⁰ Constriction to pharmacological agents is expressed as a percent decrease in arterial diameter relative to the arterial diameter obtained prior to the addition of the test compound. Percent vasodilation to cromakalim was normalized to maximal dilation and calculated from the equation: ((D_cromakalim-D_start)÷(DP-D_start)) × 100, where D_cromakalim is the lumen diameter at a specific concentration of cromakalim, D_start is the diameter prior to giving the first concentration of cromakalim and DP (passive diameter) is the lumen diameter of the artery in Ca²⁺-free aCSF containing the vasodilators diltiazem (100 μM) and forskolin (1 μM). ³¹

Membrane potential measurements

Arteries were cannulated as described above and pressurized to 40 mmHg. Smooth muscle membrane potential was measured by insertion of a sharp glass microelectrode (∼100 MΩ) containing 0.5 M KCl into smooth muscle cells from the adventitial side of the arterial wall. The criteria for successful impalement were (1) an abrupt negative potential deflection upon entry, (2) a stable membrane potential for ≥30 s, and (3) an abrupt positive potential deflection upon microelectrode removal. ²⁹ Membrane potential measurements were made with an electrometer (World Precision Instruments, Sarasota, FL) and recorded via computer with Axotape software (Molecular Devices, Sunnyvale, CA).

Ratiometric measurements of arterial smooth muscle intracellular Ca²⁺ concentration ([Ca²⁺]_i)

Freshly isolated MMA were cannulated on glass micropipettes mounted in a 5 ml myograph chamber, as described above. After cannulation, arteries were loaded with the ratiometric Ca²⁺-sensitive dye fura-2 acetoxymethyl ester (fura-2 AM, 5 µM, Invitrogen, Carlsbad, CA) for 1 h in aCSF containing pluronic acid (0.1%) at room temperature (∼ 22℃). Next, the myograph chamber was placed on the stage of a Nikon TE2000-S inverted fluorescence microscope and superfused with aerated aCSF (37℃, 30 min) to allow de-esterification of intracellular fura-2 AM, and then pressurized to 40 mmHg for study. Fluorescence ratio was obtained from background corrected ratio of the 510 nm emission from arterioles alternately excited at 340 and 380 nm with hardware and software developed by IonOptix (Milton, MA). ³²

In vivo two-photon imaging of meningeal blood vessels

SMMHC-tdTomato mice were initially anesthetized with isoflurane (5% induction and 2% maintenance), and a dura-intact cranial window superfused with aCSF (37℃) aerated with 5% CO₂, 20% O₂, 75%N₂ placed over the MMA. Anesthesia was then switched to a combination of urethane (750 mg/kg, i.p.) and α-chloralose (50 mg/kg, i.p.), a change necessitated by the fact that isoflurane causes vasodilation and impaired cerebral blood flow autoregulation.^33,34 Animals were head-fixed to a stereotactic frame and meningeal blood vessels were visualized by intravenous injection of FITC-dextran (∼500 kDa). Fluorescent images (512 × 512 pixels, 0.5-1.0 µm/pixels) were acquired using a Zeiss LSM-7 multiphoton imaging system (excitation, 820 nm; fluorescent bandpass filter, 525/50 nm and 675/70 nm). Intraluminal diameter (i.e. the width of intravascular FITC green fluorescence line) was measured using custom software SparkAn (written by Dr. Adrian D. Bonev, University of Vermont). ³⁵ Meningeal arteries and veins were differentiated based on the presence and absence of vascular smooth muscle cells (identified by tdTomato red fluorescence), respectively. Drugs were applied by superfusion of the cranial window. Upon completion of the experimental protocol, animals were euthanized by decapitation while under a surgical plane of anesthesia.

Statistical analysis

Data are expressed as mean ± SEM (n: the number of observations, N: the number of animals). Analysis was performed by investigators that perform the studies; no blinding was done. Student’s two-tailed paired t test was used for comparisons between two groups.

Reagents

Fura-2 AM and pluronic acid were obtained from Invitrogen (Life Technologies, Eugene, OR). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Inhibition of basal K_ATP channel activity causes vasoconstriction via membrane potential depolarization and elevated [Ca²⁺]_i in MMA smooth muscle

Glibenclamide caused a concentration-dependent constriction of isolated MMA with an EC₅₀ of ∼11 nM, consistent with an action mediated through inhibition of K_ATP channels.^18,20 In marked contrast, glibenclamide (1 nM to 10 µM) had no effect on the diameter of isolated cerebral arteries (Figure 1(a) to (c)). PNU37883A (10 µM), a purported vascular-selective K_ATP channel inhibitor structurally distinct from sulfonylurea compounds, ³⁶ induced an ex vivo constriction of MMA similar in magnitude to that observed with glibenclamide (i.e. ∼20% decrease in diameter), but did not alter cerebral artery diameter (Figure 1(d)). Using two-photon imaging on dura-intact cranial windows, we also observed glibenclamide-induced MMA constriction in vivo (Figure 1(e) and (f)). These results suggest that basal K_ATP channel activity in MMA impacts arterial function by providing a tonic vasodilator influence. OPEN IN VIEWER

Activation of arterial smooth muscle K⁺ channels promotes membrane potential hyperpolarization, decreased VDCC-mediated Ca²⁺ influx and vasodilation. ¹⁷ Evidence provided above (Figure 1) suggests that inhibition of K_ATP channels in MMA causes the inverse response, i.e. membrane potential depolarization, an increase in VDCC open-state probability, and vasoconstriction. To directly examine this pathway, intracellular microelectrodes were used to measure arterial smooth muscle membrane potential in the absence and presence of K_ATP channel inhibition (Figure 2(a) to (c)). In MMAs, glibenclamide (10 µM) caused a membrane potential depolarization of ∼14 mV, from −45.4 ± 1.4 mV to −31.3 ± 2.7 mV. In contrast, cerebral artery smooth muscle membrane potential was not significantly different in the absence (−37.1 ± 0.9 mV) and presence (−36.8 ± 0.8 mV) of glibenclamide. Dimethyl sulfoxide (DMSO, 1:1000 dilution in aCSF), the vehicle used to dissolve glibenclamide, did not affect the membrane potential of MMA myocytes (46.0 ± 1.2 mV, n = 5; N = 5 animals). To examine the impact of K_ATP inhibition on [Ca²⁺]_i, MMA was loaded with the ratiometric Ca²⁺ indicator dye, fura-2. Treatment with 10 µM glibenclamide evoked a vasoconstriction of 23.4 ± 1.0% and an increase in the fura-2 340:380 fluorescence ratio from 1.4 ± 0.1 to 1.8 ± 0.2, consistent with an increase in [Ca²⁺]_i (n = 4; N = 4 animals). We further postulated that this glibenclamide-induced increase in [Ca²⁺]_i was the result of smooth muscle V_M depolarization and enhanced VDCC gating. Thus, VDCC inhibition with diltiazem should prevent glibenclamide-induced MMA constriction. As VDCCs are the predominant Ca²⁺ entry pathway underlying pressure-induced myogenic tone,^32,37 this experimental series was done using arteries held at 10 mmHg to minimize the confounding effects of elevated intravascular pressure on VDCC activity. In the absence of diltiazem, glibenclamide (10 µM) caused a constriction of 14.7 ± 2.1 µm representing a 21.2 ± 2.6% decrease in diameter, a response similar in magnitude to that observed at 40 mmHg. As predicted, this glibenclamide-induced vasoconstriction was abolished by diltiazem (100 µM) (Figure 2(d) to (f)). In sum, these data are consistent with basal K_ATP channel activity exerting a tonic hyperpolarizing/vasodilating influence on MMA, but not cerebral artery diameter. OPEN IN VIEWER

Cyclic AMP-dependent protein kinase (PKA) underlies tonic K_ATP channel activity in MMA

K_ATP channel activity is enhanced by PKA-mediated channel phosphorylation. ²³ We therefore hypothesized that ongoing PKA activity may underlie basal K_ATP channel activity in MMA. To explore this concept, we examined if KT5720, a PKA inhibitor, ³⁸ could mimic the actions of glibenclamide to decrease K_ATP channel activity promoting enhanced MMA constriction. Indeed, KT5720 (1 µM) caused constriction (25.2 ± 5.2%) of MMA comparable to that induced by glibenclamide. Further, in the presence of KT5720, glibenclamide was without effect, consistent with the two drugs acting through a common signaling pathway (Figure 3(a) to (c)). As with glibenclamide, KT5720 did not alter cerebral artery diameter (Figure 3(d)). In addition, we used a second PKA inhibitor, H89 ³⁹ (1 µM), to examine the impact of PKA inhibition on the membrane potential of MMA smooth muscle. Similar to the actions of glibenclamide, H-89 caused a membrane potential depolarization of ∼13 mV, from −46.8 ± 0.5 mV to −33.5 ± 0.6 mV. In the presence of H-89, glibenclamide no longer caused membrane potential depolarization or vasoconstriction (Figure 3(e) and (f)). These results indicate that basal PKA activity contributes to the activation of K_ATP channels in MMA. OPEN IN VIEWER

Ongoing PKA activity underlies enhanced sensitivity of MMA to the K_ATP channel opener cromakalim

In addition to marked differences in responsiveness to K_ATP channel inhibitors, MMAs and cerebral arteries exhibit a striking disparity in sensitivity to the synthetic K_ATP channel opener, cromakalim. As illustrated by the cumulative concentration response curves shown in Figure 4, MMA exhibited a 10-fold greater sensitivity to cromakalim (EC₅₀ ∼ 100 nM) compared to cerebral arteries (EC₅₀ ∼ 1 µM). Despite the difference in potency, the efficacy of cromakalim was similar between artery types with 10 µM cromaklim causing a dilation of 96.9 ± 3.1% and 89.1 ± 3.5% of tissue maximum in MMA and cerebral arteries, respectively. To examine whether basal PKA activity contributes to the greater potency of cromakalim in MMA, concentration response curves to this K_ATP opener were repeated in the presence of KT5720 (1 µM). As shown in Figure 4, KT5720 caused a rightward shift in the cromakalim concentration response curve in MMA and a three-fold increase in the EC₅₀ value to ∼300 nM. These findings indicate that ongoing PKA activity not only increases basal K_ATP activity in MMA, but also increases the sensitivity of this tissue to K_ATP channel activators. OPEN IN VIEWER