The Gastrointestinal Peptide Obestatin Induces Vascular Relaxation Via Specific Activation of Endothelium-Dependent Nitric Oxide Signalling.

Background and purpose: Obestatin is a recently-discovered gastrointestinal peptide with established metabolic actions, which is linked to diabetes and may exert cardiovascular benefits. Here we aimed to investigate the specific effects of obestatin on vascular relaxation. Experimental approach: Cumulative relaxation responses to obestatin peptides were assessed in isolated rat aorta and mesenteric artery (n=8) in the presence/absence of selective inhibitors. Complementary studies were performed in cultured bovine aortic endothelial cells (BAEC). Key results: Obestatin peptides elicited concentration-dependent relaxation in both aorta and mesenteric artery. Responses to full-length obestatin(1-23) were greater than those to obestatin(1-10) and obestatin(11-23). Obestatin(1-23)-induced relaxation was attenuated by endothelial denudation, L-NAME (NO synthase inhibitor), high extracellular K(+) , GDP-ß-S (G protein inhibitor), MDL-12,330A (adenylate cyclase inhibitor), wortmannin (PI3K inhibitor), KN-93 (CaMKII inhibitor), ODQ (guanylate cyclase inhibitor) and iberiotoxin (BK(Ca) blocker), suggesting that it is mediated by an endothelium-dependent NO signalling cascade involving an adenylate cyclase-linked G protein-coupled receptor, PI3K/Akt, Ca(2+) -dependent eNOS activation, soluble guanylate cyclase and modulation of vascular smooth muscle K(+) . Supporting data from BAEC indicated that nitrite production, intracellular Ca(2+) and Akt phosphorylation were increased after exposure to obestatin(1-23). Relaxations to obestatin(1-23) were unaltered by inhibitors of candidate endothelium-derived hyperpolarising factors (EDHFs) and combined SK(Ca) /IK(Ca) blockade, suggesting that EDHF-mediated pathways were not involved. Conclusions and Implications: Obestatin produces significant vascular relaxation via specific activation of endothelium-dependent NO signalling. These actions may be important in normal regulation of vascular function and are clearly relevant to diabetes, a condition characterised by endothelial dysfunction and cardiovascular complications.

Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels.

Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable nonselective cation channels implicated in diverse physiological functions, including smooth muscle contractility and synaptic transmission. However, lack of potent selective pharmacological inhibitors for TRPC channels has limited delineation of the roles of these channels in physiological systems. Here we report the identification and characterization of ML204 as a novel, potent, and selective TRPC4 channel inhibitor. A high throughput fluorescent screen of 305,000 compounds of the Molecular Libraries Small Molecule Repository was performed for inhibitors that blocked intracellular Ca(2+) rise in response to stimulation of mouse TRPC4ß by µ-opioid receptors. ML204 inhibited TRPC4ß-mediated intracellular Ca(2+) rise with an IC(50) value of 0.96 µm and exhibited 19-fold selectivity against muscarinic receptor-coupled TRPC6 channel activation. In whole-cell patch clamp recordings, ML204 blocked TRPC4ß currents activated through either µ-opioid receptor stimulation or intracellular dialysis of guanosine 5'-3-O-(thio)triphosphate (GTP?S), suggesting a direct interaction of ML204 with TRPC4 channels rather than any interference with the signal transduction pathways. Selectivity studies showed no appreciable block by 10-20 µm ML204 of TRPV1, TRPV3, TRPA1, and TRPM8, as well as KCNQ2 and native voltage-gated sodium, potassium, and calcium channels in mouse dorsal root ganglion neurons. In isolated guinea pig ileal myocytes, ML204 blocked muscarinic cation currents activated by bath application of carbachol or intracellular infusion of GTP?S, demonstrating its effectiveness on native TRPC4 currents. Therefore, ML204 represents an excellent novel tool for investigation of TRPC4 channel function and may facilitate the development of therapeutics targeted to TRPC4.

Hydrothermal synthesis of a continuous zeolite Beta layer by optimization of time, temperature and heating rate of the precursor mixture

Highly crystalline zeolite Beta coatings in a range of Si/Al ratios of 12-23 were synthesized on a surface-modified molybdenum substrate by hydrothermal synthesis. The average thickness of the coatings was ca. 2 mu m corresponding to a coverage of 2.5 gm(-2). The coatings were obtained from a viscous Na, K, and TEAOH containing aluminosilicate precursor mixture with silica sol as reactive silicon source. A mechanism for the in situ growth of zeolite Beta coatings is proposed. According to this mechanism, the deposition of an amorphous gel layer on the substrate surface in the initial stage of the synthesis is an important step for the crystallization of continuous zeolite Beta coatings. The heating rate of the precursor mixture and the synthesis temperature were optimized to control the level of supersaturation and to stimulate the initial formation of a gel layer. At a Si/Al ratio of 23, fast heating and a temperature of 150 degrees C are required to obtain high coverage, while at a Si/Al ratio of 15, hydrothermal synthesis has to be performed with a slow initial heating rate at 140 degrees C. (c) 2007 Elsevier Inc. All rights reserved.

The photo-oxidation of water by sodium persulfate, and other electron acceptors, sensitised by TiO2

A number of different electron acceptors are tested for efficacy in the oxidation of water to oxygen, photocatalysed by titanium dioxide. The highly UV-absorbing metal ion electron acceptors, Ce4+ and Fe3+, appear ineffective at high concentration (10(-2) M), due to UV-screening, but more effective at lower concentrations (10(-3) M). The metal-depositing electron acceptor, Ag+, is initially effective, but loses activity upon prolonged irradiation due to metal deposition which promotes electron-hole recombination as well as UV-screening the titania particles. Most striking of the electron acceptors tested is persulfate, particularly in alkaline solution (0.1 M NaOH). The kinetics of the photo-oxidation of water by persulfate, photocatalysed by titania are studied as a function of pH, [S2O82-] and incident light intensity (I). The initial rate of water oxidation increases with pH, is directly proportional to the concentration of persulfate present and depends upon I-0.6. The TiO2/alkaline persulfate photosystem is robust and shows very little evidence of photochemical wear upon repeated irradiation. The results of this work are discussed with regard to previous work in this area and current mechanistic thinking. The formal quantum efficiency of the TiO2/alkaline persulfate photosystem was estimated as ca. 2%. (C) 2004 Elsevier B.V. All rights reserved.

Thick titanium dioxide films for semiconductor photocatalysis

Thick paste TiO2 films are prepared and tested for photocatalytic and photoinduced superhydrophilic (PSH) activity. The films are effective photocatalysts for the destruction of stearic acid using near or far UV and all the sol-gel films tested exhibited a quantum yield for this process of typically 0.15 %. These quantum yields are significantly greater (4-8-fold) than those for titania films produced by an APCVD technique, including the commercial self-cleaning glass product Activ(TM). The films are mechanically robust and optically clear and, as photocatalysts for stearic acid removal, are photochemically stable and reproducible. The kinetics of stearic acid photomineralisation are zero order with an activation energy of ca. 2.5 Kj mol(-1). All titania films tested, including those produced by APCVD, exhibit PSH. The light-induced fall, and dark recovery, in the water droplet contact angle made with titania paste films are similar in profile shape to those described by others for thin titania films produced by a traditional sol-gel route. (C) 2003 Elsevier Science B.V. All rights reserved.

Heterogeneity in cytosolic calcium regulation among different microvascular smooth muscle cells of the rat retina

Rat retinae were dissociated to yield intact microvessels 7 to 42 microm in diameter. These were loaded with fura-2 AM and single fragments anchored down in a recording bath. Intracellular Ca(2+) levels from 20- to 30-microm sections of vessel were estimated by microfluorimetry. The vessels studied were identified as metarterioles and arterioles. Only the microvascular smooth muscle cells loaded with fura-2 AM and changes in the fluorescence signal were confined to these cells: Endothelial cells did not make any contribution to the fluorescence signal nor did they contribute to the actions of the drugs. Caffeine (10 mM) or elevated K(+) (100 mM) produced a transient rise in cell Ca(2+) in the larger vessels (diameters >18 microm) but had no effect on smaller vessels (diameters 30 min) on washing out the endothelin and the vessel failed to relax. These results demonstrate heterogeneity between smaller and larger retinal vessels with regard to Ca(2+) mobilisation and homogeneity with respect to the actions of vasoactive peptides.

Diabetes downregulates large-conductance Ca2+-activated potassium beta 1 channel subunit in retinal arteriolar smooth muscle

Retinal vasoconstriction and reduced retinal blood flow precede the onset of diabetic retinopathy. The pathophysiological mechanisms that underlie increased retinal arteriolar tone during diabetes remain unclear. Normally, local Ca(2+) release events (Ca(2+)-sparks), trigger the activation of large-conductance Ca(2+)-activated K(+)(BK)-channels which hyperpolarize and relax vascular smooth muscle cells, thereby causing vasodilatation. In the present study, we examined BK channel function in retinal vascular smooth muscle cells from streptozotocin-induced diabetic rats. The BK channel inhibitor, Penitrem A, constricted nondiabetic retinal arterioles (pressurized to 70mmHg) by 28%. The BK current evoked by caffeine was dramatically reduced in retinal arterioles from diabetic animals even though caffeine-evoked [Ca(2+)](i) release was unaffected. Spontaneous BK currents were smaller in diabetic cells, but the amplitude of Ca(2+)-sparks was larger. The amplitudes of BK currents elicited by depolarizing voltage steps were similar in control and diabetic arterioles and mRNA expression of the pore-forming BKalpha subunit was unchanged. The Ca(2+)-sensitivity of single BK channels from diabetic retinal vascular smooth muscle cells was markedly reduced. The BKbeta1 subunit confers Ca(2+)-sensitivity to BK channel complexes and both transcript and protein levels for BKbeta1 were appreciably lower in diabetic retinal arterioles. The mean open times and the sensitivity of BK channels to tamoxifen were decreased in diabetic cells, consistent with a downregulation of BKbeta1 subunits. The potency of blockade by Pen A was lower for BK channels from diabetic animals. Thus, changes in the molecular composition of BK channels could account for retinal hypoperfusion in early diabetes, an idea having wider implications for the pathogenesis of diabetic hypertension.

Role of basal extracellular Ca2+ entry during 5-HT-induced vasoconstriction of canine pulmonary arteries.

1. Measurements of artery contraction, cytosolic [Ca(2+)], and Ca(2+) permeability were made to examine contractile and cytosolic [Ca(2+)] responses of canine pulmonary arteries and isolated cells to 5-hydroxytryptamine (5-HT), and to determine the roles of intracellular Ca(2+) release and extracellular Ca(2+) entry in 5-HT responses. 2. The EC(50) for 5-HT-mediated contractions and cytosolic [Ca(2+)] increases was approximately 10(-7) M and responses were inhibited by ketanserin, a 5-HT(2A)-receptor antagonist. 3. 5-HT induced cytosolic [Ca(2+)] increases were blocked by 20 microM Xestospongin-C and by 2-APB (IC(50)=32 microM inhibitors of InsP(3) receptor activation. 4. 5-HT-mediated contractions were reliant on release of InsP(3) but not ryanodine-sensitive Ca(2+) stores. 5. 5-HT-mediated contractions and cytosolic [Ca(2+)] increases were partially inhibited by 10 microM nisoldipine, a voltage-dependent Ca(2+) channel blocker. 6. Extracellular Ca(2+) removal reduced 5-HT-mediated contractions further than nisoldipine and ablated cytosolic [Ca(2+)] increases and [Ca(2+)] oscillations. Similar to Ca(2+) removal, Ni(2+) reduced cytosolic [Ca(2+)] and [Ca(2+)] oscillations. 7. Mn(2+) quench of fura-2 and voltage-clamp experiments showed that 5-HT failed to activate any significant voltage-independent Ca(2+) entry pathways, including store-operated and receptor-activated nonselective cation channels. Ni(2+) but not nisoldipine or Gd(3+) blocked basal Mn(2+) entry. 8. Voltage-clamp experiments showed that simultaneous depletion of both InsP(3) and ryanodine-sensitive intracellular Ca(2+) stores activates a current with linear voltage dependence and a reversal potential consistent with it being a nonselective cation channel. 5-HT did not activate this current. 9. Basal Ca(2+) entry, rather than CCE, is important to maintain 5-HT-induced cytosolic [Ca(2+)] responses and contraction in canine pulmonary artery.

Comparative capacitative calcium entry mechanisms in canine pulmonary and renal arterial smooth muscle cells.

Experiments were performed to determine whether capacitative Ca(2+) entry (CCE) can be activated in canine pulmonary and renal arterial smooth muscle cells (ASMCs) and whether activation of CCE parallels the different functional structure of the sarcoplasmic reticulum (SR) in these two cell types. The cytosolic [Ca(2+)] was measured by imaging fura-2-loaded individual cells. Increases in the cytosolic [Ca(2+)] due to store depletion in pulmonary ASMCs required simultaneous depletion of both the inositol 1,4,5-trisphosphate (InsP(3))- and ryanodine (RY)-sensitive SR Ca(2+) stores. In contrast, the cytosolic [Ca(2+)] rises in renal ASMCs occurred when the SR stores were depleted through either the InsP(3) or RY pathways. The increase in the cytosolic [Ca(2+)] due to store depletion in both pulmonary and renal ASMCs was present in cells that were voltage clamped and was abolished when cells were perfused with a Ca(2+)-free bathing solution. Rapid quenching of the fura-2 signal by 100 microM Mn(2+) following SR store depletion indicated that extracellular Ca(2+) entry increased in both cell types and also verified that activation of CCE in pulmonary ASMCs required the simultaneous depletion of the InsP(3)- and RY-sensitive SR Ca(2+) stores, while CCE could be activated in renal ASMCs by the depletion of either of the InsP(3)- or RY-sensitive SR stores. Store depletion Ca(2+) entry in both pulmonary and renal ASMCs was strongly inhibited by Ni(2+) (0.1-10 mM), slightly inhibited by Cd(2+) (200-500 microM), but was not significantly affected by the voltage-gated Ca(2+) channel (VGCC) blocker nisoldipine (10 microM). The non-selective cation channel blocker Gd(3+) (100 microM) inhibited a portion of the Ca(2+) entry in 6 of 18 renal but not pulmonary ASMCs. These results provide evidence that SR Ca(2+) store depletion activates CCE in parallel with the organization of intracellular Ca(2+) stores in canine pulmonary and renal ASMCs.

Na+/H+ exchange inhibitor cariporide attenuates skeletal muscle infarction when administered before ischemia or reperfusion

Administration of Na(+)/H(+) exchange isoform-1 (NHE-1) inhibitors before ischemia has been shown to attenuate myocardial infarction in several animal models of ischemia-reperfusion injury. However, controversy still exists as to the efficacy of NHE-1 inhibitors in protection of myocardial infarction when administered at the onset of reperfusion. Furthermore, the efficacy of NHE-1 inhibition in protection of skeletal muscle from infarction (necrosis) has not been studied. This information has potential clinical applications in prevention or salvage of skeletal muscle from ischemia-reperfusion injury in elective and trauma reconstructive surgery. The objective of this research project is to test our hypothesis that the NHE-1 inhibitor cariporide is effective in protection of skeletal muscle from infarction when administered at the onset of sustained ischemia or reperfusion and to study the mechanism of action of cariporide. In our studies, we observed that intravenous administration of cariporide 10 min before ischemia (1 or 3 mg/kg) or reperfusion (3 mg/kg) significantly reduced infarction in pig latissimus dorsi muscle flaps compared with the control, when these muscle flaps were subjected to 4 h of ischemia and 48 h of reperfusion (P <0.05; n = 5 pigs/group). Both preischemic and postischemic cariporide treatment (3 mg/kg) induced a significant decrease in muscle myeloperoxidase activity and mitochondrial-free Ca(2+) content and a significant increase in muscle ATP content within 2 h of reperfusion (P <0.05; n = 4 pigs/group). Preischemic and postischemic cariporide treatment (3 mg/kg) also significantly inhibited muscle NHE-1 protein expression within 2 h of reperfusion after 4 h of ischemia, compared with the control (P <0.05; n = 3 pigs/group). These observations support our hypothesis that cariporide attenuates skeletal muscle infarction when administered at the onset of ischemia or reperfusion, and the mechanism involves attenuation of neutrophil accumulation and mitochondrial-free Ca(2+) overload and preservation of ATP synthesis in the early stage of reperfusion.

Pathogenic Yersinia enterocolitica strains increase the outer membrane permeability in response to environmental stimuli by modulating lipopolysaccharide fluidity and lipid A structure

Pathogenic biotypes of Yersinia enterocolitica (serotypes O:3, O:8, O:9, and O:13), but not environmental biotypes (serotypes O:5, O:6, O:7,8, and O:7,8,13,19), increased their permeability to hydrophobic probes when they were grown at pH 5.5 or in EGTA-supplemented (Ca(2+)-restricted) media at 37 degrees C. A similar observation was also made when representative strains of serotypes O:8 and O:5 were tested after brief contact with human monocytes. The increase in permeability was independent of the virulence plasmid. The role of lipopolysaccharide (LPS) in this phenomenon was examined by using Y. enterocolitica serotype O:8. LPS aggregates of bacteria grown in acidic or EGTA-supplemented broth took up more N-phenylnaphthylamine than LPS aggregates of bacteria grown in standard broth and also showed a marked increase in acyl chain fluidity which correlated with permeability, as determined by measurements obtained in the presence of hydrophobic dyes. No significant changes in O-antigen polymerization were observed, but lipid A acylation changed depending on the growth conditions. In standard medium at 37 degrees C, there were hexa-, penta-, and tetraacyl lipid A forms, and the pentaacyl form was dominant. The amount of tetraacyl lipid A increased in EGTA-supplemented and acidic media, and hexaacyl lipid A almost disappeared under the latter conditions. Our results suggest that pathogenic Y. enterocolitica strains modulate lipid A acylation coordinately with expression of virulence proteins, thus reducing LPS packing and increasing outer membrane permeability. The changes in permeability, LPS acyl chain fluidity, and lipid A acylation in pathogenic Y. enterocolitica strains approximate the characteristics in Yersinia pseudotuberculosis and Yersinia pestis and suggest that there is a common outer membrane pattern associated with pathogenicity.

Adsorption and aggregation properties of norovirus GI and GII virus-like particles demonstrate differing responses to solution chemistry

The transport properties (adsorption and aggregation behavior) of virus-like particles (VLPs) of two strains of norovirus ("Norwalk" GI.1 and "Houston" GII.4) were studied in a variety of solution chemistries. GI.1 and GII.4 VLPs were found to be stable against aggregation at pH 4.0-8.0. At pH 9.0, GI.1 VLPs rapidly disintegrated. The attachment efficiencies (a) of GI.1 and GII.4 VLPs to silica increased with increasing ionic strength in NaCl solutions at pH 8.0. The attachment efficiency of GI.1 VLPs decreased as pH was increased above the isoelectric point (pH 5.0), whereas at and below the isoelectric point, the attachment efficiency was erratic. Ca(2+) and Mg(2+) dramatically increased the attachment efficiencies of GI.1 and GII.4 VLPs, which may be due to specific interactions with the VLP capsids. Bicarbonate decreased attachment efficiencies for both GI.1 and GII.4 VLPs, whereas phosphate decreased the attachment efficiency of GI.1, while increasing GII.4 attachment efficiency. The observed differences in GI.1 and GII.4 VLP attachment efficiencies in response to solution chemistry may be attributed to differential responses of the unique arrangement of exposed amino acid residues on the capsid surface of each VLP strain.

Activation of MAPK by modified low-density lipoproteins in vascular smooth muscle cells

A high concentration of circulating low-density lipoproteins (LDL) is a major risk factor for atherosclerosis. Native LDL and LDL modified by glycation and/or oxidation are increased in diabetic individuals. LDL directly stimulate vascular smooth muscle cell (VSMC) proliferation; however, the mechanisms remain undefined. The extracellular signal-regulated kinase (ERK) pathway mediates changes in cell function and growth. Therefore, we examined the cellular effects of native and modified LDL on ERK phosphorylation in VSMC. Addition of native, mildly modified (oxidized, glycated, glycoxidized) and highly modified (highly oxidized, highly glycoxidized) LDL at 25 microg/ml to rat VSMC for 5 min induced a fivefold increase in ERK phosphorylation. To elucidate the signal transduction pathway by which LDL phosphorylate ERK, we examined the roles of the Ca(2+)/calmodulin pathway, protein kinase C (PKC), src kinase, and mitogen-activated protein kinase kinase (MEK). Treatment of VSMC with the intracellular Ca(2+) chelator EGTA-AM (50 micromol/l) significantly increased ERK phosphorylation induced by native and mildly modified LDL, whereas chelation of extracellular Ca(2+) by EGTA (3 mmol/l) significantly reduced LDL-induced ERK phosphorylation. The calmodulin inhibitor N-(6-aminohexyl)-1-naphthalenesulfonamide (40 micromol/l) significantly decreased ERK phosphorylation induced by all types of LDL. Downregulation of PKC with phorbol myristate acetate (5 micromol/l) markedly reduced LDL-induced ERK phosphorylation. Pretreatment of VSMC with a cell-permeable MEK inhibitor (PD-98059, 40 micromol/l) significantly decreased ERK phosphorylation in response to native and modified LDL. These findings indicate that native and mildly and highly modified LDL utilize similar signaling pathways to phosphorylate ERK and implicate a role for Ca(2+)/calmodulin, PKC, and MEK. These results suggest a potential link between modified LDL, vascular function, and the development of atherosclerosis in diabetes.

Activation of protease calpain by oxidized and glycated LDL increases the degradation of endothelial nitric oxide synthase

Oxidation and glycation of low-density lipoprotein (LDL) promote vascular injury in diabetes; however, the mechanisms underlying this effect remain poorly defined. The present study was conducted to determine the effects of 'heavily oxidized' glycated LDL (HOG-LDL) on endothelial nitric oxide synthase (eNOS) function. Exposure of bovine aortic endothelial cells with HOG-LDL reduced eNOS protein levels in a concentration- and time-dependent manner, without altering eNOS mRNA levels. Reduced eNOS protein levels were accompanied by an increase in intracellular Ca(2+), augmented production of reactive oxygen species (ROS) and induction of Ca(2+)-dependent calpain activity. Neither eNOS reduction nor any of these other effects were observed in cells exposed to native LDL. Reduction of intracellular Ca(2+) levels abolished eNOS reduction by HOG-LDL, as did pharmacological or genetic through calcium channel blockers or calcium chelator BAPTA or inhibition of NAD(P)H oxidase (with apocynin) or inhibition of calpain (calpain 1-specific siRNA). Consistent with these results, HOG-LDL impaired acetylcholine-induced endothelium-dependent vasorelaxation of isolated mouse aortas, and pharmacological inhibition of calpain prevented this effect. HOG-LDL may impair endothelial function by inducing calpain-mediated eNOS degradation in a ROS- and Ca(2+)-dependent manner.

Human dental pulp fibroblasts express the "cold-sensing" transient receptor potential channels TRPA1 and TRPM8.

Abstract
INTRODUCTION:
Transient receptor potential (TRP) channels comprise a group of nonselective calcium-permeable cationic channels, which are polymodal sensors of environmental stimuli such as thermal changes and chemicals. TRPM8 and TRPA1 are cold-sensing TRP channels activated by moderate cooling and noxious cold temperatures, respectively. Both receptors have been identified in trigeminal ganglion neurones, and their expression in nonneuronal cells is now the focus of much interest. The aim of this study was to investigate the molecular and functional expression of TRPA1 and TRPM8 in dental pulp fibroblasts.
METHODS:
Human dental pulp fibroblasts were derived from healthy molar teeth. Gene and protein expression was determined by polymerase chain reaction and Western blotting. Cellular localization was investigated by immunohistochemistry, and TRP functionality was determined by Ca(2+) microfluorimetry.
RESULTS:
Polymerase chain reaction and Western blotting showed gene and protein expression of both TRPA1 and TRPM8 in fibroblast cells in culture. Immunohistochemistry studies showed that TRPA1 and TRPM8 immunoreactivity co-localized with the human fibroblast surface protein. In Ca(2+) microfluorimetry studies designed to determine the functionality of TRPA1 and TRPM8 in pulp fibroblasts, we showed increased intracellular calcium ([Ca(2+)](i)) in response to the TRPM8 agonist menthol, the TRPA1 agonist cinnamaldehyde, and to cool and noxious cold stimuli, respectively. The responses to agonists and thermal stimuli were blocked in the presence of specific TRPA1 and TRPM8 antagonists.
CONCLUSIONS:
Human dental pulp fibroblasts express TRPA1 and TRPM8 at the molecular, protein, and functional levels, indicating a possible role for fibroblasts in mediating cold responses in human teeth.