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...

Mycobacterium ulcerans, the causative agent of Buruli ulcer, produces a macrolide toxin, mycolactone A/B, which is thought to play a major role in virulence. A disease similar to Buruli ulcer recently appeared in United States frog colonies following importation of the West African frog, Xenopus tropicalis. The taxonomic position of the frog pathogen has not been fully elucidated, but this organism, tentatively designated Mycobacterium liflandii, is closely related to M. ulcerans and Mycobacterium marinum, and as further evidence is gathered, it will most likely be considered a subspecies of one of these species. In this paper we show that M. liflandii produces a novel plasmid-encoded mycolactone, mycolactone E. M. liflandii contains all of the genes in the mycolactone cluster with the exception of that encoding CYP140A2, a putative p450 monooxygenase. Although the core lactone structure is conserved in mycolactone E, the fatty acid side chain differs from that of mycolactone A/B in the number of hydroxyl groups and double bonds. The cytopathic phenotype of mycolactone E is identical to that of mycolactone A/B, although it is less potent. To further characterize the relationship between M. liflandii and M. ulcerans, strains were analyzed for the presence of the RD1 region genes, esxA (ESAT-6) and esxB (CFP-10). The M. ulcerans genome strain has a deletion in RD1 and lacks these genes. The results of these studies show that M. liflandii contains both esxA and esxB.

drogen sulfide (H2S) is a gaseous signaling molecule that appears to be involved in numerous biological processes, including regulation of blood pressure and vascular tone. The present study is designed to address the hypothesis that H2S is a functionally significant, endogenous dilator in the newborn cerebrovascular circulation. In vivo experiments were conducted using newborn pigs with surgically implanted, closed, cranial windows. Topical application of H2S concentration-dependently (10 Ϫ6 to 2 ϫ 10 Ϫ4 M) dilated pial arterioles. This dilation was blocked by glibenclamide (10 Ϫ6 M). L-Cysteine, the substrate of the H2S-producing enzymes cystathionine ␥-lyase (CSE) and cystathionine ␤-synthase (CBS), also dilated pial arterioles. The dilation to L-cysteine was blocked by the CSE inhibitor D,L-propargylglycine (PPG, 10 mM) but was unaffected by the CBS inhibitor amino-oxyacetate (AOA, 1 mM). Western blots detected CSE, but not CBS, in cerebral microvessels, whereas CBS is detected in brain parenchyma. Immunohistological CSE expression is predominantly vascular while CBS is expressed mainly in neurons and astrocytes. L-Cysteine (5 mM) increased H2S concentration in cerebrospinal fluid (CSF), measured by GC-MS, from 561 Ϯ 205 to 2,783 Ϯ 818 nM before but not during treatment with PPG (1,030 Ϯ 70 to 622 Ϯ 78 nM). Dilation to hypercapnia was inhibited by PPG but not AOA. Hypercapnia increased CSF H2S concentration from 763 Ϯ 243 to 4,337 Ϯ 1789 nM before but not during PPG treatment (357 Ϯ 178 vs. 425 Ϯ 217 nM). These data show that H2S is a dilator of the newborn cerebral circulation and that endogenous CSE can produce sufficient H2S to decrease vascular tone. H2S appears to be a physiologically significant dilator in the cerebral circulation.

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.

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