Jonathan H. Jaggar

Heme Is a Carbon Monoxide Receptor for Large-Conductance Ca2+-Activated K+ Channels

Carbon monoxide (CO) is an endogenous paracrine and autocrine gaseous messenger that regulates physiological functions in a wide variety of tissues. CO induces vasodilation by activating arterial smooth muscle large-conductance Ca2+-activated potassium (BKCa) channels. However, the mechanism by which CO activates BKCa channels remains unclear. Here, we tested the hypothesis that CO activates BKCa channels by binding to channel-bound heme, a BKCa channel inhibitor, and altering the interaction between heme and the conserved heme-binding domain (HBD) of the channel α subunit C terminus. Data obtained using thin-layer chromatography, spectrophotometry, mass spectrometry (MS), and MS-MS indicate that CO modifies the binding of reduced heme to the α subunit HBD. In contrast, CO does not alter the interaction between the HBD and oxidized heme (hemin), to which CO cannot bind. Consistent with these findings, electrophysiological measurements of native and cloned (cbv) cerebral artery smooth muscle BKCa channels show that CO reverses BKCa channel inhibition by heme but not by hemin. Site-directed mutagenesis of the cbv HBD from CKACH to CKASR abolished both heme-induced channel inhibition and CO-induced activation. Furthermore, on binding CO, heme switches from being a channel inhibitor to an activator. These findings indicate that reduced heme is a functional CO receptor for BKCa channels, introduce a unique mechanism by which CO regulates the activity of a target protein, and reveal a novel process by which a gaseous messenger regulates ion channel activity.

Carbon Monoxide Dilates Cerebral Arterioles by Enhancing the Coupling of Ca2+ Sparks to Ca2+-Activated K+ Channels

Abstract— Carbon monoxide (CO) is generated endogenously by the enzyme heme oxygenase. Although CO is a known vasodilator, cellular signaling mechanisms are poorly understood and are a source of controversy. The goal of the present study was to investigate mechanisms of CO dilation in porcine cerebral arterioles. Data indicate that exogenous or endogenously produced CO is a potent activator of large-conductance Ca2+-activated K+ (KCa) channels and Ca2+ spark–induced transient KCa currents in arteriole smooth muscle cells. In contrast, CO is a relatively poor activator of Ca2+ sparks. To understand the apparent discrepancy between potent effects on transient KCa currents and weak effects on Ca2+ sparks, regulation of the coupling relationship between these events by CO was investigated. CO increased the percentage of Ca2+ sparks that activated a transient KCa current (ie, the coupling ratio) from ≈62% in the control condition to 100% and elevated the slope of the amplitude correlation between these events ≈2.6-fold, indicating that Ca2+ sparks induced larger amplitude transient KCa currents in the presence of CO. This signaling pathway for CO is physiologically relevant because ryanodine, a ryanodine-sensitive Ca2+ release channel blocker that inhibits Ca2+ sparks, abolished CO dilation of pial arterioles in vivo. Thus, CO dilates cerebral arterioles by priming KCa channels for activation by Ca2+ sparks. This study presents a novel dilatory signaling pathway for CO in the cerebral circulation and appears to be the first presents of a vasodilator that acts by increasing the effective coupling of Ca2+ sparks to KCa channels.

Mitochondria-Derived Reactive Oxygen Species Dilate Cerebral Arteries by Activating Ca2+ Sparks

Mitochondria regulate intracellular calcium (Ca2+) signals in smooth muscle cells, but mechanisms mediating these effects, and the functional relevance, are poorly understood. Similarly, antihypertensive ATP-sensitive potassium (KATP) channel openers (KCOs) activate plasma membrane KATP channels and depolarize mitochondria in several cell types, but the contribution of each of these mechanisms to vasodilation is unclear. Here, we show that cerebral artery smooth muscle cell mitochondria are most effectively depolarized by diazoxide (−15%, tetramethylrhodamine [TMRM]), less so by levcromakalim, and not depolarized by pinacidil. KCO-induced mitochondrial depolarization increased the generation of mitochondria-derived reactive oxygen species (ROS) that stimulated Ca2+ sparks and large-conductance Ca2+-activated potassium (KCa) channels, leading to transient KCa current activation. KCO-induced mitochondrial depolarization and transient KCa current activation were attenuated by 5-HD and glibenclamide, KATP channel blockers. MnTMPyP, an antioxidant, and Ca2+ spark and KCa channel blockers reduced diazoxide-induced vasodilations by >60%, but did not alter dilations induced by pinacidil, which did not elevate ROS. Data suggest diazoxide drives ROS generation by inducing a small mitochondrial depolarization, because nanomolar CCCP, a protonophore, similarly depolarized mitochondria, elevated ROS, and activated transient KCa currents. In contrast, micromolar CCCP, or rotenone, an electron transport chain blocker, induced a large mitochondrial depolarization (−84%, TMRM), reduced ROS, and inhibited transient KCa currents. In summary, data demonstrate that mitochondria-derived ROS dilate cerebral arteries by activating Ca2+ sparks, that some antihypertensive KCOs dilate by stimulating this pathway, and that small and large mitochondrial depolarizations lead to differential regulation of ROS and Ca2+ sparks.

Carbon monoxide as an endogenous vascular modulator

Carbon monoxide (CO) is produced by heme oxygenase (HO)-catalyzed heme degradation to CO, iron, and biliverdin. HO has two active isoforms, HO-1 (inducible) and HO-2 (constitutive). HO-2, but not HO-1, is highly expressed in endothelial and smooth muscle cells and in adjacent astrocytes in the brain. HO-1 is expressed basally only in the spleen and liver but can be induced to a varying extent in most tissues. Elevating heme, protein phosphorylation, Ca(2+) influx, and Ca(2+)/calmodulin-dependent processes increase HO-2 activity. CO dilates cerebral arterioles and may constrict or dilate skeletal muscle and renal arterioles. Selected vasodilatory stimuli, including seizures, glutamatergic stimulation, hypoxia, hypotension, and ADP, increase CO, and the inhibition of HO attenuates the dilation to these stimuli. Astrocytic HO-2-derived CO causes glutamatergic dilation of pial arterioles. CO dilates by activating smooth muscle cell large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels. CO binds to BK(Ca) channel-bound heme, leading to an increase in Ca(2+) sparks-to-BK(Ca) channel coupling. Also, CO may bind directly to the BK(Ca) channel at several locations. Endothelial nitric oxide and prostacyclin interact with HO/CO in circulatory regulation. In cerebral arterioles in vivo, in contrast to dilation to acute CO, a prolonged exposure of cerebral arterioles to elevated CO produces progressive constriction by inhibiting nitric oxide synthase. The HO/CO system is highly protective to the vasculature. CO suppresses apoptosis and inhibits components of endogenous oxidant-generating pathways. Bilirubin is a potent reactive oxygen species scavenger. Still many questions remain about the physiology and biochemistry of HO/CO in the circulatory system and about the function and dysfunction of this gaseous mediator system.

Mitochondrial modulation of Ca2+ sparks and transient KCa currents in smooth muscle cells of rat cerebral arteries

Mitochondria sequester and release calcium (Ca2+) and regulate intracellular Ca2+ concentration ([Ca2+]i) in eukaryotic cells. However, the regulation of different Ca2+ signalling modalities by mitochondria in smooth muscle cells is poorly understood. Here, we investigated the regulation of Ca2+ sparks, Ca2+ waves and global [Ca2+]i by mitochondria in cerebral artery smooth muscle cells. CCCP (a protonophore; 1 μm) and rotenone (an electron transport chain complex I inhibitor; 10 μm) depolarized mitochondria, reduced Ca2+ spark and wave frequency, and elevated global [Ca2+]i in smooth muscle cells of intact arteries. In voltage‐clamped (−40 mV) cells, mitochondrial depolarization elevated global [Ca2+]i, reduced Ca2+ spark amplitude, spatial spread and the effective coupling of sparks to large‐conductance Ca2+‐activated potassium (KCa) channels, and decreased transient KCa current frequency and amplitude. Inhibition of Ca2+ sparks and transient KCa currents by mitochondrial depolarization could not be explained by a decrease in intracellular ATP or a reduction in sarcoplasmic reticulum Ca2+ load, and occurred in the presence of diltiazem, a voltage‐dependent Ca2+ channel blocker. Ru360 (10 μm), a mitochondrial Ca2+ uptake blocker, and lonidamine (100 μm), a permeability transition pore (PTP) opener, inhibited transient KCa currents similarly to mitochondrial depolarization. In contrast, CGP37157 (10 μm), a mitochondrial Na+–Ca2+ exchange blocker, activated these events. The PTP blockers bongkrekic acid and cyclosporin A both reduced inhibition of transient KCa currents by mitochondrial depolarization. These results indicate that mitochondrial depolarization leads to a voltage‐independent elevation in global [Ca2+]i and Ca2+ spark and transient KCa current inhibition. Data also suggest that mitochondrial depolarization inhibits Ca2+ sparks and transient KCa currents via PTP opening and a decrease in intramitochondrial [Ca2+].

Photolysis of intracellular caged sphingosine-1-phosphate causes Ca2+ mobilization independently of G-protein-coupled receptors.

Sphingosine‐1‐phosphate (S1P), the product of sphingosine kinase, activates several widely expressed G‐protein‐coupled receptors (GPCR). S1P might also play a role as second messenger, but this hypothesis has been challenged by recent findings. Here we demonstrate that intracellular S1P can mobilize Ca2+ in intact cells independently of S1P‐GPCR. Within seconds, S1P generated by the photolysis of caged S1P raised the intracellular free Ca2+ concentration in HEK‐293, SKNMC and HepG2 cells, in which the response to extracellularly applied S1P was either blocked or absent. Ca2+ transients induced by photolysis of caged S1P were caused by Ca2+ mobilization from thapsigargin‐sensitive stores. These results provide direct evidence for a true intracellular action of S1P.

IP3 Constricts Cerebral Arteries via IP3 Receptor–Mediated TRPC3 Channel Activation and Independently of Sarcoplasmic Reticulum Ca2+ Release

Vasoconstrictors that bind to phospholipase C–coupled receptors elevate inositol-1,4,5-trisphosphate (IP3). IP3 is generally considered to elevate intracellular Ca2+ concentration ([Ca2+]i) in arterial myocytes and induce vasoconstriction via a single mechanism: by activating sarcoplasmic reticulum (SR)-localized IP3 receptors, leading to intracellular Ca2+ release. We show that IP3 also stimulates vasoconstriction via a SR Ca2+ release–independent mechanism. In isolated cerebral artery myocytes and arteries in which SR Ca2+ was depleted to abolish Ca2+ release (measured using D1ER, a fluorescence resonance energy transfer–based SR Ca2+ indicator), IP3 activated 15 pS sarcolemmal cation channels, generated a whole-cell cation current (ICat) caused by Na+ influx, induced membrane depolarization, elevated [Ca2+]i, and stimulated vasoconstriction. The IP3-induced ICat and [Ca2+]i elevation were attenuated by cation channel (Gd3+, 2-APB) and IP3 receptor (xestospongin C, heparin, 2-APB) blockers. TRPC3 (canonical transient receptor potential 3) channel knockdown with short hairpin RNA and diltiazem and nimodipine, voltage-dependent Ca2+ channel blockers, reduced the SR Ca2+ release–independent, IP3-induced [Ca2+]i elevation and vasoconstriction. In pressurized arteries, SR Ca2+ depletion did not alter IP3-induced constriction at 20 mm Hg but reduced IP3-induced constriction by ≈39% at 60 mm Hg. [Ca2+]i elevations and constrictions induced by endothelin-1, a phospholipase C–coupled receptor agonist, were both attenuated by TRPC3 knockdown and xestospongin C in SR Ca2+-depleted arteries. In summary, we describe a novel mechanism of IP3-induced vasoconstriction that does not occur as a result of SR Ca2+ release but because of IP3 receptor–dependent ICat activation that requires TRPC3 channels. The resulting membrane depolarization activates voltage-dependent Ca2+ channels, leading to a myocyte [Ca2+]i elevation, and vasoconstriction.

TMEM16A/ANO1 Channels Contribute to the Myogenic Response in Cerebral Arteries

Rationale: Pressure-induced arterial depolarization and constriction (the myogenic response) is a smooth muscle cell (myocyte)-specific mechanism that controls regional organ blood flow and systemic blood pressure. Several different nonselective cation channels contribute to pressure-induced depolarization, but signaling mechanisms involved are unclear. Similarly uncertain is the contribution of anion channels to the myogenic response and physiological functions and mechanisms of regulation of recently discovered transmembrane 16A (TMEM16A), also termed Anoctamin 1, chloride (Cl−) channels in arterial myocytes. Objective: To investigate the hypothesis that myocyte TMEM16A channels control membrane potential and contractility and contribute to the myogenic response in cerebral arteries. Methods and Results: Cell swelling induced by hyposmotic bath solution stimulated Cl− currents in arterial myocytes that were blocked by TMEM16A channel inhibitory antibodies, RNAi-mediated selective TMEM16A channel knockdown, removal of extracellular calcium (Ca2+), replacement of intracellular EGTA with BAPTA, a fast Ca2+ chelator, and Gd3+ and SKF-96365, nonselective cation channel blockers. In contrast, nimodipine, a voltage-dependent Ca2+ channel inhibitor, or thapsigargin, which depletes intracellular Ca2+ stores, did not alter swelling-activated TMEM16A currents. Pressure-induced (−40 mm Hg) membrane stretch activated ion channels in arterial myocyte cell–attached patches that were inhibited by TMEM16A antibodies and were of similar amplitude to recombinant TMEM16A channels. TMEM16A knockdown reduced intravascular pressure-induced depolarization and vasoconstriction but did not alter depolarization-induced (60 mmol/L K+) vasoconstriction. Conclusions: Membrane stretch activates arterial myocyte TMEM16A channels, leading to membrane depolarization and vasoconstriction. Data also provide a mechanism by which a local Ca2+ signal generated by nonselective cation channels stimulates TMEM16A channels to induce myogenic constriction.

Isoform-Selective Physical Coupling of TRPC3 Channels to IP3 Receptors in Smooth Muscle Cells Regulates Arterial Contractility

Rationale: Inositol 1,4,5-trisphosphate (IP3)-induced vasoconstriction can occur independently of intracellular Ca2+ release and via IP3 receptor (IP3R) and canonical transient receptor potential (TRPC) channel activation, but functional signaling mechanisms mediating this effect are unclear. Objectives: Study mechanisms by which IP3Rs stimulate TRPC channels in myocytes of resistance-size cerebral arteries. Methods and Results: Immunofluorescence resonance energy transfer (immuno-FRET) microscopy using isoform-selective antibodies indicated that endogenous type 1 IP3Rs (IP3R1) are in close spatial proximity to TRPC3, but distant from TRPC6 or TRPM4 channels in arterial myocytes. Endothelin-1 (ET-1), a phospholipase C–coupled receptor agonist, elevated immuno-FRET between IP3R1 and TRPC3, but not between IP3R1 and TRPC6 or TRPM4. TRPC3, but not TRPC6, coimmunoprecipitated with IP3R1. TRPC3 and TRPC6 antibodies selectively inhibited recombinant channels, but only the TRPC3 antibody blocked IP3-induced nonselective cation current (ICat) in myocytes. TRPC3 knockdown attenuated immuno-FRET between IP3R1 and TRPC3, IP3-induced ICat activation, and ET-1 and IP3-induced vasoconstriction, whereas TRPC6 channel knockdown had no effect. ET-1 did not alter total or plasma membrane-localized TRPC3, as determined using surface biotinylation. RT-PCR demonstrated that C-terminal calmodulin and IP3R binding (CIRB) domains are present in myocyte TRPC3 and TRPC6 channels. A peptide corresponding to the IP3R N-terminal region that can interact with TRPC channels activated ICat. A TRPC3 CIRB domain peptide attenuated IP3- and ET-1–induced ICat activation and vasoconstriction. Conclusions: IP3 stimulates direct coupling between IP3R1 and membrane-resident TRPC3 channels in arterial myocytes, leading to ICat activation and vasoconstriction. Close spatial proximity between IP3R1 and TRPC3 establishes this isoform-selective functional interaction.

Essential role for smooth muscle BK channels in alcohol-induced cerebrovascular constriction

Binge drinking is associated with increased risk for cerebrovascular spasm and stroke. Acute exposure to ethanol at concentrations obtained during binge drinking constricts cerebral arteries in several species, including humans, but the mechanisms underlying this action are largely unknown. In a rodent model, we used fluorescence microscopy, patch-clamp electrophysiology, and pharmacological studies in intact cerebral arteries to pinpoint the molecular effectors of ethanol cerebrovascular constriction. Clinically relevant concentrations of ethanol elevated wall intracellular Ca2+ concentration and caused a reversible constriction of cerebral arteries (EC50 = 27 mM; Emax = 100 mM) that depended on voltage-gated Ca2+ entry into myocytes. However, ethanol did not directly increase voltage-dependent Ca2+ currents in isolated myocytes. Constriction occurred because of an ethanol reduction in the frequency (–53%) and amplitude (–32%) of transient Ca2+-activated K+ (BK) currents. Ethanol inhibition of BK transients was caused by a reduction in Ca2+ spark frequency (–49%), a subsarcolemmal Ca2+ signal that evokes the BK transients, and a direct inhibition of BK channel steady-state activity (–44%). In contrast, ethanol failed to modify Ca2+ waves, a major vasoconstrictor mechanism. Selective block of BK channels largely prevented ethanol constriction in pressurized arteries. This study pinpoints the Ca2+ spark/BK channel negative-feedback mechanism as the primary effector of ethanol vasoconstriction.