Photodynamic treatment of melanoma cells using aza-dipyrromethenes as photosensitizers.

In this study, we report for the first time the use of four aza-dipyrromethenes (ADPMs) as photosensitizers for cancer PDT. The synthesis and characterization of the ADPMs and their photodynamic action against B16F10 melanoma cells were assessed. ADPM 2 is the best singlet oxygen generator and the most phototoxic (at 2.5 µM) towards B16F10 cells.

A glutathione-responsive photosensitizer with fluorescence resonance energy transfer characteristics for imaging-guided targeting photodynamic therapy.

Here, we have synthesized and characterized a novel activatable photosensitizer (PS) 8a in which two well-designed boron dipyrromethene (BODIPY) derivatives are utilized as the photosensitizing fluorophore and quencher respectively, which are connected by a disulfide linker via two successive Cu (І) catalyzed click reactions. The fluorescence emission and singlet oxygen production of 8a are suppressed via intramolecular fluorescence resonance energy transfer (FRET) from the excited BODIPY-based PS part to quencher unit, but both of them can be simultaneously switched on by cancer-related biothiol glutathione (GSH) in phosphate buffered saline (PBS) solution with 0.05% Tween 80 as a result of cleavage of disulfide. Also, 8a exhibits a bright fluorescence image and a substantial ROS production in A549 human lung adenocarcinoma, HeLa human cervical carcinoma and H22 mouse hepatoma cells having a relatively high concentration of GSH, thereby leading to a significant photocytotoxicity, with IC50 values as low as 0.44 µM, 0.67 µM and 0.48 µM, respectively. In addition, the photosensitizer can be effectively activated and imaged in H22 transplanted hepatoma tumors of mice and shows a strong inhibition on tumor growth. All these results suggest that such a GSH-responsive photosensitizer based on FRET mechanism may provide a new strategy for tumor-targeted and fluorescence imaging-guided cancer therapy.

Ruthenium(II) Conjugates of Boron-Dipyrromethene and Biotin for Targeted Photodynamic Therapy in Red Light.

The ruthenium(II) complexes [RuCl(L1)(L3)]Cl (1), [RuCl(L1)(L4)]Cl (2), [RuCl(L2)(L4)]Cl (3), [RuCl(L1)(L5)]Cl (4), and [RuCl(L2)(L5)]Cl (5) of NNN-donor dipicolylamine (dpa) bases (L4, L5) having BODIPY (boron-dipyrromethene) moieties, NN-donor phenanthroline derivatives (L1, L2), and benzyldipicolylamine (bzdpa, L3) were prepared and characterized by spectroscopic techniques and their cellular localization/uptake and photocytotoxicity studied. Complex 1, as its PF6 salt (1a), has been structurally characterized with help of a single-crystal X-ray diffraction technique. It has a RuN5Cl core with the Cl bonded trans to the amine nitrogen atom of bzdpa. The complexes showed intense absorption spectral bands near 500 nm (ε ≈ 58000 M-1 cm-1) in 2 and 3 and 654 nm (ε ≈ 80000 M-1 cm-1) in 4 and 5 in 1/1 DMSO/DPBS (v/v). Complex 5 having biotin and PEGylated-disteryl BODIPY gave a singlet oxygen quantum yield (ΦΔ) of ∼0.65 in DMSO. Complex 5 exhibited remarkable PDT (photodynamic therapy) activity (IC50 ≈ 0.02 µM) with a photocytotoxicity index (PI) value of >5000 in red light of 600-720 nm in A549 cancer cells. The biotin-conjugated complexes showed better photocytotoxicity in comparison to nonbiotinylated analogues in A549 cells. The complexes displayed less toxicity in HPL1D normal cells in comparison to A549 cancer cells. The emissive BODIPY complexes 3 and 5 (ΦF ≈ 0.07 in DMSO) showed significant mitochondrial localization.

Crystal structure, DNA crosslinking and photo-induced cytotoxicity of oxovanadium(IV) conjugates of boron-dipyrromethene.

Cis-dichloro-oxovanadium(IV) complexes [VO(L1/L2)Cl2], where L1 is N-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4ʎ4,5ʎ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)benzyl)-1-(pyridin-2-yl)-N-(pyridin-2-ylmethyl)methanamine in 1 and L2 is N-(4-(5,5-difluoro-2,8-diiodo-1,3,7,9-tetramethyl-5H-4ʎ4,5ʎ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)benzyl)-1-(pyridin-2-yl)-N-(pyridin-2-ylmethyl)methanamine in 2) having 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene as boron-dipyrromethene (BODIPY) appended dipicolylamine bases were prepared, characterized and their photocytotoxicity studied. X-ray crystal structure of 1 showed distorted octahedral geometry with a VIVON3Cl2 core having Cl-V-Cl angle of 91.93(4)°. The complexes showed variable solution conductivity properties. They were non-electrolytes in dry DMF at 25 °C but showed 1:1 electrolytic behavior in an aqueous medium due to dissociation of one chloride ligand as evidenced from the mass spectral study. Complexes 1 and 2 showed absorption bands at 500 and 535 nm, respectively. The calf thymus DNA melting study revealed their interaction through DNA crosslinking on exposure to light which was further confirmed from the alkaline agarose gel electrophoresis using plasmid supercoiled pUC19 DNA. Complex 2 showed disruption of the mitochondrial membrane potential in the JC-1 (1,1',3,3'-tetraethyl-5,5',6,6'-tetrachloroimidacarbocyanine iodide) assay. The complexes were photocytotoxic in visible light (400-700 nm, power: 10 J cm-2) in cervical cancer HeLa and breast cancer MCF-7 cells. Complex 2 having a photoactive diiodo­boron-dipyrromethene moiety gave a singlet oxygen quantum yield (ΦΔ) value of ~0.6. It showed singlet oxygen mediated apoptotic photodynamic therapy activity with remarkably low IC50 (half maximal inhibitory concentration) value of ~0.15 µM. The cis-disposition of chlorides gave a cis-divacant 4-coordinate intermediate structure from the density functional theory (DFT) study thus mimicking the DNA crosslinking property of cisplatin.

A novel distyryl boron dipyrromethene with two functional tags for site-specific bioorthogonal photosensitisation towards targeted photodynamic therapy.

A distyryl boron dipyrromethene based photosensitiser substituted with 1,2,4,5-tetrazine and alkyne moieties was prepared. Through site-specific bioorthogonal reactions with the complementary functional tags anchored on the membrane of A431 human epidermoid carcinoma cells, this versatile photosensitiser exhibited enhanced cellular uptake and photocytotoxicity. The bioorthogonal ligation was also demonstrated in tumour-bearing nude mice.

Construction of cathepsin B-responsive fluorescent probe and photosensitizer using a ferrocenyl boron dipyrromethene dark quencher.

A ferrocenyl boron dipyrromethene (BODIPY) has been developed and utilized as a dark quencher to construct a cathepsin B-responsive fluorescent probe and photosensitizer. The smart fluorescent probe and photosensitizer (Pc-FcQ) contains a zinc(II) phthalocyanine as the fluorescent and photosensitizing unit which is conjugated to the ferrocenyl BODIPY dark quencher via a cathepsin B-cleavable peptide substrate [Gly-Phe-Leu-Gly-Lys]. The photosensitizing properties of Pc-FcQ, including fluorescence and singlet oxygen generation, are significantly quenched through energy transfer to the BODIPY unit and subsequently by the photoinduced electron transfer from the nearby ferrocenyl moiety. Upon exposure of cathepsin B in human hepatocellular carcinoma HepG cells, the fluorescence emission of Pc-FcQ could be restored, indicating the cleavage of the peptide substrate and the separation of the phthalocyanine and ferrocenyl BODIPY unit. However, the intracellular fluorescence intensity of Pc-FcQ was largely diminished after the cells were pre-treated with cathepsin B inhibitor. Its intracellular fluorescence intensity was comparable to that of the control compound in which the peptide substrate was replaced by the non-cleavable one [Gly-Gly-Gly-Gly-Lys]. The singlet oxygen generation of Pc-FcQ was also examined in HepG2 cells as reflected by the cytotoxicity assay. The Pc-FcQ exhibited higher potency when compared with the non-cleavable analogue due to the cleavage of peptide substrate and the detachment of the BODIPY dark quencher from the phthalocyanine. The activation of the Pc-FcQ was also demonstrated in tumor-bearing nude mice. After intratumoral injection of Pc-FcQ, the fluorescence intensity at the tumor region increased gradually over 10 h as a result of the detachment of the dark quencher upon the action of cathepsin B. All the results suggest that this ferrocenyl BODIPY could serve as an efficient dark quencher and the resulting Pc-FcQ could act as the cathepsin B-responsive fluorescent probe and activatable photosensitizer.

Boron Dipyrromethene Nano-Photosensitizers for Anticancer Phototherapies.

As traditional phototherapy agents, boron dipyrromethene (BODIPY) photosensitizers have attracted increasing attention due to their high molar extinction coefficients, high phototherapy efficacy, and excellent photostability. After being formed into nanostructures, BODIPY-containing nano-photosensitizers show enhanced water solubility and biocompatibility as well as efficient tumor accumulation compared to BODIPY molecules. Hence, BODIPY nano-photosensitizers demonstrate a promising potential for fighting cancer. This review contains three sections, classifying photodynamic therapy (PDT), photothermal therapy (PTT), and the combination of PDT and PTT based on BODIPY nano-photosensitizers. It summarizes various BODIPY nano-photosensitizers, which are prepared via different approaches including molecular precipitation, supramolecular interactions, and polymer encapsulation. In each section, the design strategies and working principles of these BODIPY nano-photosensitizers are highlighted. In addition, the detailed in vitro and in vivo applications of these recently developed nano-photosensitizers are discussed together with future challenges in this field, highlighting the potential of these promising nanoagents for new tumor phototherapies.

Pyriplatin-Boron-Dipyrromethene Conjugates for Imaging and Mitochondria-Targeted Photodynamic Therapy.

Monofunctional pyriplatin analogues cis-[Pt(NH3)2(L)Cl](NO3) (1-3) having boron-dipyrromethene (BODIPY) pendants (L) with 1,3,5,7-tetramethyl-8-(4-pyridyl)-4,4'-difluoroboradiazaindacene moieties were designed and synthesized, and their photocytotoxic properties were studied. The Pt-BODIPY conjugates displayed an absorption band within 505-550 nm and a green emissive band near 535 nm in 1% DMSO/DMEM (Dulbecco's modified Eagle's medium) buffer. Complex cis-[Pt(NH3)2(4-Me-py)Cl](NO3) (4) was used as a control for determining the structural aspects by X-ray crystallography. The mono- and diiodinated BODIPY complexes 2 and 3 showed generation of singlet oxygen on light activation as evidenced from the 1,3-diphenylisobenzofuran (DPBF) titration experiments. The cytotoxicity of the BODIPY complexes was tested against A549 (human lung cancer), MCF-7 (human breast cancer), and HaCaT (human skin keratinocyte) cells in dark and visible light (400-700 nm, 10 J cm-2). While complexes 2 and 3 showed excellent photocytotoxicity (IC50 ≈ 0.05 µM), they remained essentially nontoxic in the dark (IC50 > 100 µM). The emissive bands of 1 and 2 were used for cellular imaging by confocal microscopy study, which showed their mitochondrial localization. This was further supported by platinum estimation from isolated mitochondria and mitochondrial depolarization through a JC-1 assay. The photomediated apoptotic cell death was evidenced from flow cytometric assays, annexin-V/FITC-PI (fluorescein isothiocyanate-propidium iodide) and cell cycle arrest in sub-G1 and G2/M phases. The complexes bind to 9-ethylguanine as a model nucleobase to form monoadducts. A mechanistic study on DNA photocleavage activity using pUC19 DNA showed singlet oxygen as the reactive oxygen species (ROS). The combination of photodynamic therapy with DNA cross-linking property enhanced the anticancer potential of the monofunctional BODIPY-conjugates of pyriplatins.

Effects of volatile anaesthetics on heme metabolism in a murine genetic model of Acute Intermittent Porphyria. A comparative study with other porphyrinogenic drugs.

BACKGROUND: Acute Intermittent Porphyria (AIP) is an inherited disease produced by a deficiency of Porphobilinogen deaminase (PBG-D). The aim of this work was to evaluate the effects of Isoflurane and Sevoflurane on heme metabolism in a mouse genetic model of AIP to further support our previous proposal for avoiding their use in porphyric patients. A comparative study was performed administering the porphyrinogenic drugs allylisopropylacetamide (AIA), barbital and ethanol, and also between sex and mutation using AIP (PBG-D activity 70% reduced) and T1 (PBG-D activity 50% diminished) mice. METHODS: The activities of 5-Aminolevulinic synthetase (ALA-S), PBG-D, Heme oxygenase (HO) and CYP2E1; the expression of ALA-S and the levels of 5-aminolevulinic acid (ALA) were measured in different tissues of mice treated with the drugs mentioned. RESULTS: Isoflurane increased liver, kidney and brain ALA-S activity of AIP females but only affected kidney AIP males. Sevoflurane induced ALA-S activity in kidney and brain of female AIP group. PBG-D activity was further reduced by Isoflurane in liver male T1; in AIP male mice activity remained in its low basal levels. Ethanol and barbital also caused biochemical alterations. Only AIA triggered neurological signs similar to those observed during human acute attacks in male AIP being the symptoms less pronounced in females although ALA-S induction was greater. Heme degradation was affected. DISCUSSION: Biochemical alterations caused by the porphyrinogenic drugs assayed were different in male and female mice and also between T1 and AIP being more affected the females of AIP group. GENERAL SIGNIFICANCE: This is the first study using volatile anaesthetics in an AIP genetic model confirming Isoflurane and Sevoflurane porphyrinogenicity.

A General Strategy Toward Highly Fluorogenic Bioprobes Emitting across the Visible Spectrum.

A general approach toward highly fluorogenic probes across the visible spectrum for various analytes offers significant potential for engineering a wide range of bioprobes with diverse sensing and imaging functions. Here we show a facile and general strategy that involves introducing a new fluorogenic mechanism in boron dipyrromethene (BODIPY) dyes, based on the principle of stimuli-triggered dramatic reduction in the electron-withdrawing capabilities of the meso-substituents of BODIPYs. The fluorogenic mechanism has been demonstrated to be applicable in various BODIPYs with emission maxima ranging from green to far red (509, 585, and 660 nm), and the synthetic strategy allows access to a panel of highly fluorogenic bioprobes for various biomolecules and enzymes (H2O2, H2S, and protease) via introducing specific triggering motifs. The potency of the general design strategy is exemplified by its application to develop a mitochondria-targeting far-red probe capable of imaging of endogenous H2O2 in living cells.

Oral administration of SR-110, a peroxynitrite decomposing catalyst, enhances glucose homeostasis, insulin signaling, and islet architecture in B6D2F1 mice fed a high fat diet.

Peroxynitrite has been implicated in type 2 diabetes and diabetic complications. As a follow-up study to our previous work on SR-135 (Arch Biochem Biophys 577-578: 49-59, 2015), we provide evidence that this series of compounds are effective when administered orally, and their mechanisms of actions extend to the peripheral tissues. A more soluble analogue of SR-135, SR-110 (from a new class of Mn(III) bis(hydroxyphenyl)-dipyrromethene complexes) was orally administered for 2 weeks to B6D2F1 mice fed a high fat-diet (HFD). Mice fed a HFD for 4 months gained significantly higher body weights compared to lean diet-fed mice (52 ± 1.5 g vs 34 ± 1.3 g). SR-110 (10 mg/kg daily) treatment significantly reduced fasting blood glucose and insulin levels, and enhanced glucose tolerance as compared to HFD control or vehicle (peanut butter) group. SR-110 treatment enhanced insulin signaling in the peripheral organs, liver, heart, and skeletal muscle, and reduced lipid accumulation in the liver. Furthermore, SR-110 increased insulin content, restored islet architecture, decreased islet size, and reduced tyrosine nitration. These results suggest that a peroxynitrite decomposing catalyst is effective in improving glucose homeostasis and restoring islet morphology and ß-cell insulin content under nutrient overload.

SR-135, a peroxynitrite decomposing catalyst, enhances ß-cell function and survival in B6D2F1 mice fed a high fat diet.

Peroxynitrite has been implicated in ß-cell dysfunction and insulin resistance in obesity. Chemical catalysts that destroy peroxynitrite, therefore, may have therapeutic value for treating type 2 diabetes. To this end, we have recently demonstrated that Mn(III) bis(hydroxyphenyl)-dipyrromethene complexes, SR-135 and its analogs, can effectively catalyze the decomposition of peroxynitrite in vitro and in vivo through a 2-electron mechanism (Rausaria et al., 2011). To study the effects of SR-135 on glucose homeostasis in obesity, B6D2F1 mice were fed with a high fat-diet (HFD) for 12 weeks and treated with vehicle, SR-135 (5mg/kg), or a control drug SRB for 2 weeks. SR-135 significantly reduced fasting blood glucose and insulin levels, and enhanced glucose tolerance as compared to HFD control, vehicle or SRB. SR-135 also enhanced glucose-stimulated insulin secretion based on ex vivo studies. Moreover, SR-135 increased insulin content, restored islet architecture, decreased islet size, and reduced tyrosine nitration and apoptosis. These results suggest that a peroxynitrite decomposing catalyst enhances ß-cell function and survival under nutrient overload.

High prevalence of and potential mechanisms for chronic kidney disease in patients with acute intermittent porphyria.

Acute intermittent porphyria (AIP) is a genetic disorder of the synthesis of heme caused by a deficiency in hydroxymethylbilane synthase (HMBS), leading to the overproduction of the porphyrin precursors δ-aminolevulinic acid and porphobilinogen. The aim of this study is to describe the clinical and biological characteristics, the renal pathology, and the cellular mechanisms of chronic kidney disease associated with AIP. A total of 415 patients with HMBS deficiency followed up in the French Porphyria Center were enrolled in 2003 in a population-based study. A follow-up study was conducted in 2013, assessing patients for clinical, biological, and histological parameters. In vitro models were used to determine whether porphyrin precursors promote tubular and endothelial cytotoxicity. Chronic kidney disease occurred in up to 59% of the symptomatic AIP patients, with a decline in the glomerular filtration rate of ~1 ml/min per 1.73 m(2) annually. Proteinuria was absent in the vast majority of the cases. The renal pathology was a chronic tubulointerstitial nephropathy, associated with a fibrous intimal hyperplasia and focal cortical atrophy. Our experimental data provide evidence that porphyrin precursors promote endoplasmic reticulum stress, apoptosis, and epithelial phenotypic changes in proximal tubular cells. In conclusion, the diagnosis of chronic kidney disease associated with AIP should be considered in cases of chronic tubulointerstitial nephropathy and/or focal cortical atrophy with severe proliferative arteriosclerosis.

Intracellular modulation of excited-state dynamics in a chromophore dyad: differential enhancement of photocytotoxicity targeting cancer cells.

The photosensitized generation of reactive oxygen species, and particularly of singlet oxygen [O2 (a(1) Δg )], is the essence of photodynamic action exploited in photodynamic therapy. The ability to switch singlet oxygen generation on/off would be highly valuable, especially when it is linked to a cancer-related cellular parameter. Building on recent findings related to intersystem crossing efficiency, we designed a dimeric BODIPY dye with reduced symmetry, which is ineffective as a photosensitizer unless it is activated by a reaction with intracellular glutathione (GSH). The reaction alters the properties of both the ground and excited states, consequently enabling the efficient generation of singlet oxygen. Remarkably, the designed photosensitizer can discriminate between different concentrations of GSH in normal and cancer cells and thus remains inefficient as a photosensitizer inside a normal cell while being transformed into a lethal singlet oxygen source in cancer cells. This is the first demonstration of such a difference in the intracellular activity of a photosensitizer.

Antimicrobial and anti-biofilm effect of a novel BODIPY photosensitizer against Pseudomonas aeruginosa PAO1.

Photodynamic therapy (PDT) combines the use of organic dyes (photosensitizers, PSs) and visible light in order to elicit a photo-oxidative stress which causes bacterial death. GD11, a recently synthesized PS belonging to the boron-dipyrromethene (BODIPY) class, was demonstrated to be efficient against planktonic cultures of Pseudomonas aeruginosa, causing a 7 log unit reduction of viable cells when administered at 2.5 µM. The effectiveness of GD11 against P. aeruginosa biofilms grown in flow-cells and microtiter trays was also demonstrated. Confocal laser scanning microscopy of flow-cell-grown biofilms suggests that the treatment has a biocidal effect against bacterial biofilm cells.

LDL cholesterol recycles to the plasma membrane via a Rab8a-Myosin5b-actin-dependent membrane transport route.

Mammalian cells acquire cholesterol, a major membrane constituent, via low-density lipoprotein (LDL) uptake. However, the mechanisms by which LDL cholesterol reaches the plasma membrane (PM) have remained obscure. Here, we applied LDL labeled with BODIPY cholesteryl linoleate to identify this pathway in living cells. The egress of BODIPY cholesterol (BC) from late endosomal (LE) organelles was dependent on acid lipase and Niemann-Pick C1 (NPC1) protein, as for natural cholesterol. We show that NPC1 was needed to recruit Rab8a to BC-containing LEs, and Rab8a enhanced the motility and segregation of BC- and CD63-positive organelles from lysosomes. The BC carriers docked to the cortical actin by a Rab8a- and Myosin5b (Myo5b)-dependent mechanism, typically in the proximity of focal adhesions (FAs). LDL increased the number and dynamics of FAs and stimulated cell migration in an acid lipase, NPC1, and Rab8a-dependent fashion, providing evidence that this cholesterol delivery route to the PM is important for cell movement.

DNA binding and anti-cancer activity of redox-active heteroleptic piano-stool Ru(II), Rh(III), and Ir(III) complexes containing 4-(2-methoxypyridyl)phenyldipyrromethene.

The synthesis of four novel heteroleptic dipyrrinato complexes [(η(6)-arene)RuCl(2-pcdpm)] (η(6)-arene = C6H6, 1; C10H14, 2) and [(η(5)-C5Me5)MCl(2-pcdpm)] (M = Rh, 3; Ir, 4) containing a new chelating ligand 4-(2-methoxypyridyl)-phenyldipyrromethene (2-pcdpm) have been described. The complexes 1-4 have been fully characterized by various physicochemical techniques, namely, elemental analyses, spectral (ESI-MS, IR, (1)H, (13)C NMR, UV/vis) and electrochemical studies (cyclic voltammetry (CV) and differential pulse voltammetry (DPV)). Structures of 3 and 4 have been determined crystallographically. In vitro antiproliferative and cytotoxic activity of these complexes has been evaluated by trypan blue exclusion assay, cell morphology, apoptosis, acridine orange/ethidium bromide (AO/EtBr) fluorescence staining, and DNA fragmentation assay in Dalton lymphoma (DL) cell lines. Interaction of 1-4 with calf thymus DNA (CT DNA) has also been supported by absorption titration and electrochemical studies. Our results suggest that in vitro antitumor activity of 1-4 lies in the order 2 > 1 > 4 > 3.

A reversible fluorescence probe based on Se-BODIPY for the redox cycle between HClO oxidative stress and H2S repair in living cells.

We have developed a new reversible fluorescence probe MPhSe-BOD for the redox cycle process between hypochlorous acid and hydrogen sulfide in solution and in living cells. Confocal microscopy imaging using RAW264.7 cell lines shows that the probe has good cell membrane permeability, and can monitor intracellular HClO/H(2)S redox cycles continuously.

In vitro and in vivo photocytotoxicity of boron dipyrromethene derivatives for photodynamic therapy.

To understand the effects of substitution patterns on photosensitizing the ability of boron dipyrromethene (BODIPY), two structural variations that either investigate the effectiveness of various iodinated derivatives to maximize the "heavy atom effect" or focus on the effect of extended conjugation at the 4-pyrrolic position to red-shift their activation wavelengths were investigated. Compounds with conjugation at the 4-pyrrolic position were less photocytotoxic than the parent unconjugated compound, while those with an iodinated BODIPY core presented better photocytotoxicity than compounds with iodoaryl groups at the meso-positions. The potency of the derivatives generally correlated well with their singlet oxygen generation level. Further studies of compound 5 on HSC-2 cells showed almost exclusive localization to mitochondria, induction of G(2)/M-phase cell cycle block, and onset of apoptosis. Compound 5 also extensively occluded the vasculature of the chick chorioallantoic membrane. Iodinated BODIPY structures such as compound 5 may have potential as new photodynamic therapy agents for cancer.

Suppression of the induction of long-term depression by carbon monoxide in rat cerebellar slices.

Carbon monoxide (CO) suppresses brain functions at doses lower than that suppressing oxygen (O(2)) supply to the brain, and the cerebellum is one of the sites most susceptible to the neurotoxic effects of CO. We investigated the effects of CO on the induction of cerebellar long-term depression (LTD) in the synapses between parallel fibres (PFs) and Purkinje cells. CO, at concentrations between 8 nM and 5 microM, exhibited almost no effect on synaptic responses in Purkinje cells, O(2) consumption and NO release from PFs in rat cerebellar slices. However, the induction of LTD was significantly suppressed by CO at concentrations between 40 and 200 nM. The suppressive effect of 40 nM CO was antagonized by 10 microM NOR3, an NO donor. In contrast, CO exhibited no clear effect on the induction of LTD at concentrations between 1 and 5 microM. The induction of LTD, suppressed by 10 microM N(G)-nitro-L-arginine, an inhibitor of NO synthase, was not restored by 5 microM CO. CO is not only a neurotoxic substance but also a candidate for an intercellular messenger. delta-Aminolevulinate (30 microM), a substance facilitating endogenous CO production, suppressed the induction of LTD, and the effect of delta-aminolevulinate was antagonized by 10 microM NOR3. These findings suggest that CO may have a suppressive effect on the induction of cerebellar LTD at nanomolar concentrations, probably via its effects on NO/cGMP signalling.