Abstract-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 largeconductance Ca 2ϩ -activated potassium (BK Ca ) channels. However, the mechanism by which CO activates BK Ca channels remains unclear. Here, we tested the hypothesis that CO activates BK Ca channels by binding to channel-bound heme, a BK Ca channel inhibitor, and altering the interaction between heme and the conserved heme-binding domain (HBD) of the channel ␣ 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 ␣ 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 BK Ca channels show that CO reverses BK Ca 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 BK Ca 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. Key Words: vascular smooth muscle Ⅲ vasodilation Ⅲ potassium channels Ⅲ signal transduction L arge-conductance Ca 2ϩ -activated potassium (BK Ca ) channels regulate the physiological functions of many tissues, including smooth muscle, neuronal and endocrine cells. 1 BK Ca channels are typically composed of pore-forming ␣ subunits that are encoded by the Slo1 (or KCNMA1) gene, and accessory ␤ subunits that modulate channel gating. 2 In smooth muscle cells, BK Ca channels regulate cellular membrane potential and, thus, Ca 2ϩ entry through voltage-gated Ca 2ϩ channels, providing a mechanism to control contractility. 3 BK Ca channel activity is regulated by a variety of signaling molecules, including intracellular Ca 2ϩ ([Ca 2ϩ ] i ), protein kinases, 4 -6 tyrosine kinases, 7 cytochrome P-450 metabolites of arachidonic acid, 8 and heme. 9 BK Ca channels are also activated by physiologically relevant gases, including O 2 , CO, and NO. Although these gases can use cellular signaling pathways, O 2 , CO, and NO also activate BK Ca channels in cell-free membrane patches isolated from the intracellular milieu. 10 -12 Carbon monoxide is a physiological paracrine and autocrine messenger and neurotransmitter produced by heme oxygenase (HO) catalyzed metabolism of heme. [13][14][15] Heme is found in virtually all cell types, and many cell types contain HO-2, including arterial smooth muscle cells, endothelial cells, and neurons. CO regulates a variety o...

Astrocyte signals can modulate arteriolar tone, contributing to regulation of cerebral blood flow, but specific intercellular communication mechanisms are unclear. Here we used isolated cerebral arteriole myocytes, astrocytes, and brain slices to investigate whether carbon monoxide (CO) generated by the enzyme heme oxygenase (HO) acts as an astrocyte-to-myocyte gasotransmitter in the brain. Glutamate stimulated CO production by astrocytes with intact HO-2, but not those genetically deficient in HO-2. Glutamate activated transient K(Ca) currents and single K(Ca) channels in myocytes that were in contact with astrocytes, but did not affect K(Ca) channel activity in myocytes that were alone. Pretreatment of astrocytes with chromium mesoporphyrin (CrMP), a HO inhibitor, or genetic ablation of HO-2 prevented glutamate-induced activation of myocyte transient K(Ca) currents and K(Ca) channels. Glutamate decreased arteriole myocyte intracellular Ca2+ concentration and dilated brain slice arterioles and this decrease and dilation were blocked by CrMP. Brain slice arteriole dilation to glutamate was also blocked by L-2-alpha aminoadipic acid, a selective astrocyte toxin, and paxilline, a K(Ca) channel blocker. These data indicate that an astrocytic signal, notably HO-2-derived CO, is used by glutamate to stimulate arteriole myocyte K(Ca) channels and dilate cerebral arterioles. Our study explains the astrocyte and HO dependence of glutamatergic functional hyperemia observed in the newborn cerebrovascular circulation in vivo.

BackgroundKATP channels, assembled from pore‐forming (Kir6.1 or Kir6.2) and regulatory (SUR1 or SUR2) subunits, link metabolism to excitability. Loss of Kir6.2 results in hypoglycemia and hyperinsulinemia, whereas loss of Kir6.1 causes Prinzmetal angina–like symptoms in mice. Conversely, overactivity of Kir6.2 induces neonatal diabetes in mice and humans, but consequences of Kir6.1 overactivity are unknown.Methods and ResultsWe generated transgenic mice expressing wild‐type (WT), ATP‐insensitive Kir6.1 [Gly343Asp] (GD), and ATP‐insensitive Kir6.1 [Gly343Asp,Gln53Arg] (GD‐QR) subunits, under Cre‐recombinase control. Expression was induced in smooth muscle cells by crossing with smooth muscle myosin heavy chain promoter–driven tamoxifen‐inducible Cre‐recombinase (SMMHC‐Cre‐ER) mice. Three weeks after tamoxifen induction, we assessed blood pressure in anesthetized and conscious animals, as well as contractility of mesenteric artery smooth muscle and KATP currents in isolated mesenteric artery myocytes. Both systolic and diastolic blood pressures were significantly reduced in GD and GD‐QR mice but normal in mice expressing the WT transgene and elevated in Kir6.1 knockout mice as well as in mice expressing dominant‐negative Kir6.1 [AAA] in smooth muscle. Contractile response of isolated GD‐QR mesenteric arteries was blunted relative to WT controls, but nitroprusside relaxation was unaffected. Basal KATP conductance and pinacidil‐activated conductance were elevated in GD but not in WT myocytes.ConclusionsKATP overactivity in vascular muscle can lead directly to reduced vascular contractility and lower blood pressure. We predict that gain of vascular KATP function in humans would lead to a chronic vasodilatory phenotype, as indeed has recently been demonstrated in Cantu syndrome.

Existing pressure sensitive adhesives (PSAs) are mainly derived from petrochemicals. This study describes a novel approach for development of biobased PSAs. Epoxidized soybean oil was polymerized and cross-linked with a dicarboxylic acid to generate superior PSAs. The dicarboxylic acids used in this study included dimer acid (DA), sebacic acid, adipic acid, and a difunctional polymeric carboxylic acid that was prepared from polymerization of bisphenol A diglycidyl ether (BPAGE) and an excess of DA. AMC-2, a chromium-(III)-based organometallic compound, was found to be the most effective catalyst for the polymerization/cross-linking. The PSAs had a peel strength of 1.4−5.0 N/cm, a loop tack of 7.1−12.6 N, a shear strength of 34 min to more than 168 h, and a good aging resistance. The adhesive properties of the PSAs can be tailored for specific applications such as PSA tapes and labels through the selection of the dicarboxylic acid and its usage. Incorporation of a small amount of phenylene-containing monomer BPAGE into the PSAs significantly increases the peel and shear strengths of the resulting PSAs. This new class of PSAs can be fully based on renewable materials. The preparation of the PSAs does not use any organic solvent or toxic chemicals, thus being environmentally friendly.

A new aldehyde-functionalized glycomonomer, 1,2:3,4-di-O-isopropylidene-6-O-(2′-formyl-4′vinylphenyl)-D-galactopyranose (IVDG), was designed and prepared. The "living"/controlled radical polymerization of IVDG was successfully achieved using 2,2′-azobis(isobutyronitrile) as the initiator and 1-phenylethyl dithiobenzoate as the reversible addition-fragmentation chain transfer (RAFT) agent at 60 °C in tetrahydrofuran. The polymerization followed first-order kinetics, the number-average molecular weight of the obtained polymers increased in direct proportion to the monomer conversion, and the molecular weight distribution was narrow (polydispersity index <1.1). Removal of protective isopropylidene groups from the sugar residue in polyIVDG was carried out quantitatively using 88% formic acid at room temperature, yielding a novel amphiphilic polymer containing both galactopyranose and aldehyde functionalities. These amphiphilic polymers self-assembled into well-defined aldehyde-bearing polymeric micelles in aqueous solution without recourse to any surfactant. The size of the micelles increased almost linearly with the molecular weight of polyIVDG precursor, which could be controlled directly via the aforementioned RAFT polymerization process. Protein-bioconjugated nanoparticles were also successfully prepared by the immobilization of bovine serum albumin (as a model protein) onto the aldehyde-functionalized micelles.

A new monomer, 2-formal-4-vinylphenyl ferrocenecarboxylate (FVFC), containing both aldehyde and ferrocene functional groups was designed and prepared by the reaction of ferrocenecarboxylic acid chloride with 2-hydroxy-5-vinylbenzaldehyde. The controlled radical polymerization of FVFC was achieved using 2,2′azobis(isobutyronitrile) (AIBN) as the initiator and 2-cyanopropyl-2-yl dithiobenzoate (CPDB) as the reversible addition-fragmentation chain transfer (RAFT) agent at 60 °C in tetrahydrofuran (THF). The polymerization nearly followed first-order kinetics, the number-average molecular weight of the obtained polymers increased almost in direct proportion to the monomer conversion, and the molecular weight distribution was narrow (polydispersity index < 1.2). The chain and terminal structures of the obtained polyFVFC were confirmed by 1 H NMR and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry analysis. The obtained polyFVFC can be employed as a macro-RAFT agent for styrene polymerization, resulting in polyFVFC-bpolystyrene.

A new class of renewable pressure-sensitive adhesive (PSA) designed and developed from soybean oil was reported in this study. Soybean oil was epoxidized and hydrolysed selectively on the ester groups to afford a mixture of epoxidized fatty acids (EFAs) which were characterized by FTIR and 1 H NMR spectroscopy. The EFA mixture without further purification was then polymerized directly in the presence and absence of a small amount of dicarboxylic acid compounds to afford hydroxyl-functionalized polymers. The peel strength, loop tack, shear strength and viscoelastic properties of the resulting (co)polymers revealed that the (co)polymers were suitable for PSA applications. The new PSAs could be fully bio-based and potentially biodegradable, and their preparation and application did not require the use of an organic solvent or a toxic chemical, thus being environmentally friendly.

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