Rat Aortic Tissue Research Articles

Direct Stimulation of KATP Channels by Exogenous and Endogenous Hydrogen Sulfide in Vascular Smooth Muscle Cells

ATP-sensitive K+ (K(ATP)) channels in vascular smooth muscle cells (VSMC) are important targets for endogenous metabolic regulation and exogenous drug therapy. H2S, as a novel gasotransmitter, has been shown to relax rat aortic tissues via opening of K(ATP) channels. However, interaction of H2S, exogenous-applied or endogenous-produced, with K(ATP) channels in resistance artery VSMC has not been delineated. In the present study, using the whole-cell and single-channel patch-clamp technique, we demonstrated that exogenous H2S activated K(ATP) channels and hyperpolarized cell membrane in rat mesenteric artery VSMC. H2S enhanced the amplitude of whole-cell K(ATP) currents with an EC50 value of 116 +/- 8.3 microM and increased the open probability of single K(ATP) channels. H2S hyperpolarized membrane potentials by -12 mV in nystatin-perforated VSMC. Furthermore, inhibition of endogenous H2S production with D,L-propargylglycine (PPG) reduced whole-cell K(ATP) currents. PPG alone had no effect on unitary K(ATP) channel currents in cell-free membrane patches. In addition, effects of H2S on K(ATP) channels and membrane potentials were independent of cGMP-mediated phosphorylation. This study demonstrated modulation of K(ATP) channel activity by exogenous and endogenous H2S in resistance artery VSMC, thus helping elucidate cardiovascular functions of this endogenous gas.

Reactive Oxygen Species Induce Tyrosine Phosphorylation of and Src Kinase Recruitment to NO-sensitive Guanylyl Cyclase

Soluble guanylyl cyclase (sGC) is the major cytosolic receptor for nitric oxide (NO) that converts GTP into the second messenger cGMP in a NO-dependent manner. Other factors controlling this key enzyme are intracellular proteins such as Hsp90 and PSD95, which bind to sGC and modulate its activity, stability, and localization. To date little is known about the effects of posttranslational modifications of sGC, although circumstantial evidence suggests that reversible phosphorylation may contribute to sGC regulation. Here we demonstrate that inhibitors of protein-tyrosine phosphatases such as pervanadate and bisperoxo(1,10-phenanthroline)oxovanadate(V) as well as reactive oxygen species such as H2O2 induce specific tyrosine phosphorylation of the beta1 but not of the alpha1 subunit of sGC. Tyrosine phosphorylation of sGCbeta1 is also inducible by pervanadate and H2O2 in intact PC12 cells, rat aortic smooth muscle cells, and in rat aortic tissues, indicating that tyrosine phosphorylation of sGC may also occur in vivo. We have mapped the major tyrosine phosphorylation site to position 192 of beta1, where it forms part of a highly acidic phospho-acceptor site for Src-like kinases. In the phosphorylated state Tyr(P)-192 exposes a docking site for SH2 domains and efficiently recruits Src and Fyn to sGCbeta1, thereby promoting multiple phosphorylation of the enzyme. Our results demonstrate that sGC is subject to tyrosine phosphorylation and interaction with Src-like kinases, revealing an unexpected cross-talk between the NO/cGMP and tyrosine kinase signaling pathways at the level of sGC.

1H NMR Assessment of Safe Triton X-100 Levels in Decellularized Rat Aortic Valve Tissue

Decellularization of allograft heart valves has been proposed as a method to reduce the alloreactive immune response, which limits the durability of these tissues. Rat models are essential for prel...

Roles of nitric oxide and prostacyclin in triethyleneglycol dimethacrylate (TEGDMA)-induced vasorelaxation

Most dental resinous materials contain the diluent monomer triethyleneglycol dimethacrylate (TEGDMA), which has been reported to be bioactive. Previously, it was demonstrated that TEGDMA induces vasorelaxation. The present study examines the mechanism(s) of the TEGDMA-induced vasorelaxation by measuring vascular nitrite and prostacyclin levels. Nitrite and prostacyclin levels were assayed in rat aortic tissues in response to TEGDMA. The involvement of guanylyl and adenylyl cyclases in TEGDMA-induced aortic vasorelaxation was determined using the enzyme inhibitors 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536), respectively. TEGDMA enhanced the levels of nitrites in endothelium-intact and that of protacyclin in both endothelium-intact and denuded rat aortas. The increase in nitrites was associated with endothelium-dependent aortic relaxation mediated via the activation of guanylyl cyclase, while the increase in prostacyclin was associated with both endothelium-dependent and independent relaxation linked to adenylyl cyclase stimulation. Data from the present investigation can be relevant to dental practice employing materials containing TEGDMA by providing insights into the vasorelaxant effect of the monomer following placement of the materials in the oral cavity. Additional studies that are more relevant to the clinical situation are required to confirm these initial results and further explore their implications.

Wild blueberry ( Vaccinium angustifolium) consumption affects the composition and structure of glycosaminoglycans in Sprague-Dawley rat aorta

It has been documented that increased intake of polyphenols may provide protection against coronary heart disease and stroke. Blueberries ( Vaccinium angustifolium) are one of the richest sources of antioxidants among fruits and vegetables. Phenolic compounds from berry extracts inhibit human low density lipoprotein and liposome oxidation. Glycosaminoglycans (GAGs) and proteoglycans (PGs) are structural components of aortas with great structural diversity. Their interaction with compounds such as enzymes, cytokines, growth factors, proteins and lipoproteins and their subsequent role in degenerative diseases has been documented. We investigated the effects of a diet rich in blueberries on the content and structure of GAGs. Sprague-Dawley rats were fed either a control (C) or a blueberry (B) diet for 13 weeks. Aortic tissue GAGs were isolated with papain digestion, alkaline borohydride treatment and anion-exchange chromatography. Cellulose acetate electrophoresis and treatment of the fractions with specific lyases revealed the presence of three GAG populations, i.e. hyaluronan (HA), heparan sulfate (HS) and galactosaminoglycans (GalAGs). Disaccharide composition was determined by high-performance capillary electrophoresis following enzymatic degradation. A 13% higher amount of total GAGs in aortas of B-fed rats was attributed to a higher content of GalAGs (67%). Determination of the sulfated disaccharides showed an overall lower concentration of oversulfated disaccharides in both HS and GalAG populations in the aortas of the B group. Our results demonstrate for the first time that a diet rich in blueberries results in structural alterations in rat aortic tissue GAGs. These changes may affect cellular signal transduction pathways and could have major consequences for the biological function of GAG molecules within the vascular environment.

Reactive oxygen species induce tyrosine phosphorylation of and Src kinase recruitment to NO-sensitive guanylyl cyclase

Soluble guanylyl cyclase (sGC) is a cytosolic enzyme that converts GTP into the second messenger cGMP in a nitric oxide (NO)-dependent manner. Other factors controlling this key enzyme are intracellular proteins such as Hsp90 and PSD95, which bind to sGC and modulate its activity, stability and localization. To date little is known about the effects of posttranslational modifications of sGC though circumstantial evidence suggests that reversible phosphorylation may contribute to sGC regulation. Here we demonstrate that inhibitors of protein tyrosine phosphatases (PTP) such as pervanadate and bpV(phen) as well as reactive oxygen species such as H2O2 induce specific tyrosine phosphorylation of the β1 but not of the α1 subunit of sGC. Tyrosine phosphorylation of sGCβ1 is also inducible by pervanadate or H2O2 in intact PC12 cells, rat aortic smooth muscle cells and in rat aortic tissues indicating that tyrosine phosphorylation of sGC may also occur in vivo. We have mapped the major tyrosine phosphorylation site to position 192 of β1 where it forms part of a highly acidic phospho-acceptor site for Src-like kinases. In the phosphorylated state, pTyr192 exposes a docking site for SH2 domains and efficiently recruits Src and Fyn to sGCβ1 thereby promoting further phosphorylation of the enzyme. Our results demonstrate that sGC is subject to tyrosine phosphorylation and interaction with Src-like kinases, revealing an unexpected crosstalk between the NO/cGMP and tyrosine kinase signaling pathways at the level of sGC. from 2nd International Conference of cGMP Generators, Effectors and Therapeutic Implications Potsdam, Germany, 10–12 June, 2005

Expression pattern of fibroblast growth factors (FGFs), their receptors and antagonists in primary endothelial cells and vascular smooth muscle cells

Fibroblast growth factors (FGFs) are important angiogenic growth factors. While basic FGF (FGF2) is well established as a potent inducer of angiogenesis much less is known about other FGFs possibly expressed by EC. We investigated the expression of all known FGFs, their main tyrosine kinase receptors and antagonists by RT-PCR analysis in human umbilical vascular endothelial cells (HUVECs) to obtain a complete expression profile of this important growth factor system in model endothelial cells (EC). In addition to FGFR1IIIc, which is considered as the major FGF receptor in EC, HUVECs express similar levels of FGFR3IIIc, detectable amounts of FGFR2IIIc and a new FGF receptor without an intracellular kinase domain (FGFR5). HUVECs express several secreted FGFs, including FGF5, 7, 8, 16 and 18 and two members of the fibroblast growth factor homologous factors (FHFs), not yet reported to be expressed in EC. The expression panel was compared with that obtained from human vascular smooth muscle cells (VSMCs) and human aortic tissue. Human umbilical artery smooth muscle cells (HUASMCs) and HUVECs express the identical FGF receptor and ligand panel implicating that both cell types act, according the FGF signals more as an entity than as individual cell types. Expression of Fgf1, 2, 7, 16 and 18 and the antagonists Sprouty 2,3 and 4 was demonstrated for all analysed cDNAs. The IIIc isoforms of FGFR1 and 2 and the novel FGFR5 were expressed in the aorta, but expression of the FGF receptor 3 was not detected in cDNAs derived from aortic tissue. In the VSMC of rat aortic tissue and in HUASM cultured cells we could demonstrate FGF18 immunoreactivity in the nucleus of the cells. The expression of several secreted FGFs by EC may focus the view more on their paracrine effects on neighbouring cells during tissue regeneration or tumor formation.

Tyrosine phosphorylation of NO-sensitive guanylyl cyclase

NO-sensitive guanylyl cyclases (GC) are the principal receptors for nitric oxide (NO) and convert GTP into the second messenger cGMP. We showed that GC is prone to tyrosine phosphorylation in COS1 cells overexpressing the human holoenzyme. Similar results were obtained in PC12 cells and in rat aortic tissue slices. The major phosphorylation site was mapped to position 192 in the regulatory domain of the β1 subunit. Tyrosine phosphorylation of GC was reduced in the presence of the inhibitors PP1 and PP2 indicating that Src-like kinases are critically involved in phosphorylation. Moreover, coimmunoprecipitation experiments revealed an interaction between Src and GC. To further analyse the relevance of this posttranslational modification we generated a phospho-specific antibody raised against pTyr192. This antibody clearly distinguishes between phosphorylated and non-phosphorylated GC and may be a powerful tool to analyse the subcellular localisation of the phosphorylated enzyme. from 2nd International Conference of cGMP Generators, Effectors and Therapeutic Implications Potsdam, Germany, 10–12 June, 2005

626. Ultrasound-Triggered/Enhanced Delivery of Oligonucleotides into Aorta

Synthesized oligonucleotides (ODNs) are used in anti-sense and anti-gene technology to control gene expression. As tissues and cells do not easily take up ODNs, the development of inexpensive and effective approaches to transiently decrease gene expression in vivo would be useful clinically. These data demonstrate that ultrasound can release ODNs from acoustically-active cationic liposomes and increase the delivery of ODNs into cultured rat aorta tissue. Cationic liposomes were made using the component lipids 1, 2-Myristoyl-sn-glycero-3-ethylphosphocholine(EDMPC), phosphotidylcholine and cholesterol (CH). Liposomes were made acoustically-active by sonication in water, addition of mannitol, freezing, lyophilization, and rehydration. Acoustically-active lipoplexes were made by mixing ODNs with acoustically-active liposomes in a ratio of 1:2. Ultrasound triggered-ODN release was measured as follows: acoustically-active liposomes/FITC-ODNs complex were incorporated with cultured cells, ultrasound (1 MHz, 2W/cm2,10% duty cycle for 10 seconds) was applied after washing out all free ODNs and unattached lipoplexes. The release of ODNs into solution was measured before and after the ultrasound application. ODN release was calculated by subtracting the fluorescence of the background media from the cellular fluorescence. One application of ultrasound caused a 45% release of attached FITC-ODNs from cationic liposomes. Nuclear localization of FITC-ODNs can be seen immediately after ultrasound application in about 3% of the cells. This delivery method was also tested on arterial tissue using freshly-removed rat aorta. Acoustically-active lipoplexes were injected into the endothelium of the aorta in a perfusion system with 20 cm H2O pressure. Ultrasound (1 MHz, 2W/cm2,100% duty cycle) was applied to the aorta for 60 seconds. Aortas were then washed and frozen-sectioned. The distribution of FITC-ODNs was assessed by fluorescent microscopy. Ultrasound enhanced the transport of ODNs through endothelium and the FITC-ODNs was seen extending into the arterial media. These results suggest that ultrasound can both increase the release of nucleic acid from acoustically-active liposome complexes as well as increase the penetration of oligonucleotides into a complex tissue such as arterial muscle. These data indicate that therapeutic transfer using these methods is possible. (See Figure)

Blockade of Nuclear Factor of Activated T Cells Activation Signaling Suppresses Balloon Injury-induced Neointima Formation in a Rat Carotid Artery Model

We have previously reported that nuclear factor of activated T cells (NFATs) play an important role in the regulation of vascular smooth muscle cell migration and proliferation by receptor tyrosine kinase and G protein-coupled receptor agonists, platelet-derived growth factor-BB and thrombin, respectively. To understand the role of NFATs in vascular disease, we have now studied the involvement of these transcription factors in neointima formation in a rat carotid artery balloon injury model. The levels of NFATc1 in injured right common carotid arteries were increased at 72 h, 1 week, and 2 weeks after balloon injury compared with its levels in uninjured left common carotid arteries. Intraperitoneal injection of cyclosporine A (CsA), a pharmacological inhibitor of the calcineurin-NFAT activation pathway, suppressed balloon injury-induced neointima formation by 40%. Similarly, adenoviral-mediated expression of GFPVIVIT, a competent peptide inhibitor of the calcineurin-NFAT activation pathway, in injured arteries also reduced neointima formation by about 40%. Furthermore, CsA and GFPVIVIT attenuated balloon injury-induced neointimal smooth muscle cell proliferation as determined by bromodeoxyuridine staining. Platelet-derived growth factor-BB induced the expression of COX-2 in cultured VSMC in a time- and NFAT-dependent manner. COX-2 expression was also increased in the right common carotid artery in a time-dependent manner after balloon injury as compared with its levels in uninjured left common carotid artery and both CsA and GFPVIVIT negated this response. Together these results for the first time demonstrate that NFATs play a critical role in neointima formation via induction of expression of COX-2.

Decline in caveolin-1 expression and scaffolding of G protein receptor kinase-2 with age in Fischer 344 aortic vascular smooth muscle

Beta-adrenergic receptor (beta-AR)-mediated vasorelaxation declines with age in humans and animal models. This is not caused by changes in expression of beta-AR, G alpha s, adenylyl cyclase, or protein kinase A but is associated with decreased cAMP production. Expression and activity of G protein receptor kinase-2 (GRK-2), which phosphorylates and desensitizes the beta-AR, increases with age in rat aortic tissue. Caveolin scaffolds the beta-AR, GRK, and other proteins within "signaling pockets" and inhibits GRK activity when bound. We questioned the effect of age on caveolin-1 expression and interaction between caveolin-1 and GRK-2 in vascular smooth muscle (VSM) isolated from 2-, 6-, 12-, and 24-mo-old male Fischer 344 rat aorta. Western blot analysis found expression of caveolin-1 declined with age (6-, 12- and 24-mo-old rat aortas express 92, 50, and 42% of 2-mo-old rat aortas, respectively). Results from density-buoyancy analysis showed a lower percentage of GRK in caveolin-1-specific fractions with age (6-, 12- and 24-mo-old rat aortas express 95, 56, and 12% of 2-mo-old rat aortas, respectively). Coimmunoprecipitation confirmed this finding; density of GRK in caveolin-1 immunoprecipitates was 97, 30, and 21% of 2-mo-old aortas compared with 6-, 12- and 24-mo-old animals, respectively. Immunohistocytochemistry and confocal microscopy confirmed that GRK-2 and caveolin-1 colocalize in VSM. These results suggest that in nonoverexpressed, intact tissue, the decline in beta-AR-mediated vasorelaxation may be caused by both a reduction in caveolin-1 expression and a reduction in binding of GRK-2 by caveolin-1. This could lead to an increase in the fraction of free GRK-2, which could phosphorylate and desensitize the beta-AR.

Performance of diamino fluorophores for the localization of sources and targets of nitric oxide

An emergent approach to the detection of nitric oxide (NO) in tissues relies on the use of fluorescence probes that are activated by products of NO autoxidation. Here we explore the performance of the widely used NO probe 4,5-diaminofluorescein diacetate (DAF-2 DA) for the localization of sources of NO in rat aortic tissue, either from endogenous NO synthesis or from chemically or photolytically released NO from targets of nitrosation/nitrosylation. Of importance toward understanding the performance of this probe in tissues is the finding that, with incubation conditions commonly used in the literature (10 μM DAF-2 DA), intracellular DAF-2 accumulates to concentrations that approach the millimolar range. Whereas such high probe concentrations do not interfere with NO release or signaling, they help to clarify why DAF-2 nitrosation is possible in the presence of endogenous nitrosation scavengers (e.g., ascorbate and glutathione). The gain attained with such elevated concentrations is, however, mitigated by associated high levels of background autofluorescence from the probe. This, together with tissue autofluorescence, limits the sensitivity of the probe to low-micromolar levels of accumulated DAF-2 triazole (DAF-2 T), the activated form of the probe, which is higher than the concentrations of most endogenous nitrosation/nitrosylation products found in tissues. We further show that the compartmentalization of DAF-2 around elastic fibers further limits its potential to characterize the site of NO production at the subcellular level. Moreover, we find that reaction of DAF-2 with HgCl 2 and other commonly employed reagents is associated with spectral changes that may be misinterpreted as NO signals. Finally, UV illumination can lead to high levels of nitrosating species that interfere with NO detection from enzymatic sources. These findings indicate that while DAF-2 may still represent an important tool for the localization of NO synthesis, provided important pitfalls and limitations are taken into consideration, it is not suited for the detection of basally generated nitrosation/nitrosylation products.

Immunofluorescence revealed the presence of NHE-1 in the nuclear membranes of rat cardiomyocytes and isolated nuclei of human, rabbit, and rat aortic and liver tissues

Using immunofluorescence and 3-dimensional confocal microscopy techniques, the present study was designed to verify if NHE-1 is present at the level of the nuclear membrane in cells that are known to express this type of exchanger. Nuclei were isolated from aortic tissues of adult human, rabbit, and rats, as well as from liver tissues of human fetus, and adult rabbit and rat. In addition, cultured ventricular cardiomyocytes were isolated from 2-week-old rat. Our results showed the presence of NHE-1 in isolated nuclei of aortic vascular smooth muscle and liver of human, rabbit, and rat. NHE-1 seems to be distributed throughout the isolated nucleus and more particularly at the level of the nuclear membranes. The relative fluorescence density of NHE-1 was significantly higher (p < 0.05) in isolated liver nuclei of human, when compared with those of rabbit and rat. However, in isolated nuclei of aortic vascular smooth muscle, the relative fluorescence density of NHE-1 was significantly (p < 0.001) higher in the rabbit when compared with human and rat. In cultured rat ventricular cardiomyocytes, NHE-1 fluorescent labeling could be easily seen throughout the cell, including the nucleus, and more particularly at both the sarcolemma and the nuclear membranes. In rat cardiomyocytes, the relative fluorescence density of NHE-1 of the sarcolemma membrane, including the cytosol, was significantly lower than that of the whole nucleus (including the nuclear envelope membranes). In conclusion, our results showed that NHE-1 is present at the nuclear membranes and in the nucleoplasm and its distribution and density may depend on cell type and species used. These results suggest that nuclear membranes' NHE-1 may play a role in the modulation of intranuclear pH.

Hydrogen sulfide-induced relaxation of resistance mesenteric artery beds of rats.

Hydrogen sulfide (H2S) has been shown recently to function as an important gasotransmitter. The present study investigated the vascular effects of H2S, both exogenously applied and endogenously generated, on resistance mesenteric arteries of rats and the underlying mechanisms. Both H2S and NaHS evoked concentration-dependent relaxation of in vitro perfused rat mesenteric artery beds (MAB). The sensitivity of MAB to H2S (EC50, 25.2 +/- 3.6 microM) was about fivefold higher than that of rat aortic tissues. Removal of endothelium or coapplication of charybdotoxin and apamin to endothelium-intact MAB significantly reduced the vasorelaxation effects of H2S. The H2S-induced relaxation of MAB was partially mediated by ATP-sensitive K+ (KATP) channel activity in vascular smooth muscle cells. Pinacidil (EC50, 1.7 +/- 0.1 microM, n=6) mimicked, but glibenclamide (10 microM, n=6) suppressed, the vasorelaxant effect of H2S. KATP channel currents in isolated mesenteric artery smooth muscle cells were significantly augmented by H2S. L-cysteine, a substrate of cystathionine-gamma-lyase (CSE), at 1 mM increased endogenous H2S production by sixfold in rat mesenteric artery tissues and decreased contractility of MAB. DL-propargylglycine (a blocker of CSE) at 10 microM abolished L-cysteine-dependent increase in H2S production and relaxation of MAB. Our results demonstrated a tissue-specific relaxant response of resistance arteries to H2S. The stimulation of KATP channels in vascular smooth muscle cells and charybdotoxin/apamin-sensitive K+ channels in vascular endothelium by H2S represents important cellular mechanisms for H2S effect on MAB. Our study also demonstrated that endogenous CSE can generate sufficient H2S from exogenous L-cysteine to cause vasodilation. Future studies are merited to investigate direct contribution of endogenous H2S to regulation of vascular tone.

Alseis yucatanensis: a natural product from Belize that exhibits multiple mechanisms of vasorelaxation

An aqueous extract of the bark of Alseis yucatanensis was studied to determine its mechanism of action in the relaxation of endothelium-denuded rat aortic tissues. The extract relaxed both norepinephrine (NE) and KCl-contracted vessels, with ED50’s of 0.12 and 1.73 mg/mL, respectively. In NE-contracted vessels, two phases of relaxation were evident which were separated in both time and dose range. At high concentrations, a rapid relaxation was seen that was due to the blocking of internal (ED50=0.49 mg/mL) and external (ED50=2.34 mg/mL) calcium channels. A second, slowly developing (i.e., long-term) relaxation to baseline was seen at lower concentrations. The time to complete relaxation was dose-dependent. This long-term response was not seen in KCl-contracted vessels, was prolonged by TEA, and could be reversed by the addition of KCl to the bath. These data suggest that the long-term relaxation is due to the opening of potassium channels.

Alseis yucatanensis: a natural product from Belize that exhibits multiple mechanisms of vasorelaxation

An aqueous extract of the bark of Alseis yucatanensis was studied to determine its mechanism of action in the relaxation of endothelium-denuded rat aortic tissues. The extract relaxed both norepinephrine (NE) and KCl-contracted vessels, with ED 50’s of 0.12 and 1.73 mg/mL, respectively. In NE-contracted vessels, two phases of relaxation were evident which were separated in both time and dose range. At high concentrations, a rapid relaxation was seen that was due to the blocking of internal (ED 50=0.49 mg/mL) and external (ED 50=2.34 mg/mL) calcium channels. A second, slowly developing (i.e., long-term) relaxation to baseline was seen at lower concentrations. The time to complete relaxation was dose-dependent. This long-term response was not seen in KCl-contracted vessels, was prolonged by TEA, and could be reversed by the addition of KCl to the bath. These data suggest that the long-term relaxation is due to the opening of potassium channels.

Mechanisms of ADRF release from rat aortic adventitial adipose tissue.

Blood vessels are surrounded by variable amounts of adipose tissue. We showed earlier that adventitial adipose tissue inhibits rat aortic contraction by release of a transferable factor, adventitium-derived relaxing factor (ADRF), which activates smooth muscle K(+) channels. However, little is known about the mechanisms of ADRF release. Using isolated rat aortic rings and isometric contraction measurements, we show that ADRF release depends on extracellular [Ca(2+)] (EC(50) approximately 4.7 mM). ADRF effects do not involve neuronal presynaptic N-type Ca(2+) and Na(+) channels or vanilloid, cannabinoid, and CGRP receptors. ADRF release is strongly inhibited by the protein tyrosine kinase inhibitors genistein and tyrphostin A25. In contrast, daidzein, an inactive genistein analog, and the protein tyrosine kinase inhibitor ST638 had no effect. Protein kinase A inhibition by H89 also inhibited ADRF release, whereas the protein kinase G inhibitor KT-5823 had no effect. We propose that ADRF release is Ca(2+) dependent and is regulated by intracellular signaling pathways involving tyrosine kinase and protein kinase A. Furthermore, ADRF release does not depend on perivascular nerve endings.

Effect of vigabatrin on contractile response to arachidonic acid and prostaglandins in smooth muscle preparations and platelet aggregation in experimental laboratory animals

γ-Vinyl GABA (vigabatrin; VGB), an irreversible inhibitor of GABA-transaminase, was evaluated for its effect on the contractile responses to PGE 2 or its precursor arachidonic acid (AA) in guinea pig ileum (GPI) and non-pregnant rat uterus. Rationale behind this study was the rise in γ-aminobutyric acid (GABA) contents in the peripheral organs after treatment with GABAergic agents and extensive interconnections of neuromediators and their interactions. Indomethacin, the standard NSAID, at 6.1×10 −5 and 12.2×10 −5 M concentrations highly significantly ( P<0.001) inhibited contractile responses induced by AA (4.3×10 −5 M) in the isolated GPI. Incubation of the GPI segments with VGB at different concentrations (25, 100 and 200 mM) failed to inhibit AA-induced contractile responses in the same preparation. In fact, VGB at 25 mM significantly ( P<0.05) potentiated the contractile effect of AA 4.3×10 −5 M. Incubation of the same tissue with GABA at 13×10 −3 and 26×10 −3 M inhibited responses elicited by AA at 4.3×10 −5 M that was significant ( P<0.05) only with low concentration of GABA. Conversely, a higher concentration (64×10 −3 M) of GABA was needed to antagonise PGE 2 (9.4×10 −6 M)-induced contractions in the same tissue. Almost similar results (i.e. inhibition of contractile responses to AA and PGE 2) were obtained with isolated non-pregnant rat uterus. These findings suggested that GABA had the potential to inhibit prostaglandin (PG) synthesis and/or antagonise PGE 2 responses in isolated GPI and rat uterus. The addition of 35 or 70 μl of rat aorta incubation medium caused a dose-dependent inhibition of ADP-induced aggregation being 21.5 and 43.5%, respectively, an indication of prostacycline (PGI 2) contents. Pre-incubation of rat aortic tissues with VGB (96.8 mM) significantly ( P<0.05) reversed the anti-aggregatory activity of control aortic prostacycline on ADP-induced aggregation. These findings suggested that VGB might inhibit PGI 2 activity through the inhibition of its synthesis.

H(2)S-induced vasorelaxation and underlying cellular and molecular mechanisms.

H(2)S is endogenously generated in vascular smooth muscle cells. The signal transduction pathways involved in the vascular effects of H(2)S have been unclear and were investigated in the present study. H(2)S induced a concentration-dependent relaxation of rat aortic tissues that was not affected by vascular denervation. The vasorelaxant potency of H(2)S was attenuated by the removal of the endothelium. Similarly, the blockade of nitric oxide synthase or the coapplication of the Ca(2+)-dependent K(+) channel blockers apamin and charybdotoxin reduced the H(2)S-induced relaxation of the endothelium-intact aortic tissues. Sodium nitroprusside (SNP)-induced relaxation was completely abolished by either 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) or NS- 2028, two soluble guanylate cyclase inhibitors. Instead of inhibition, ODQ and NS-2028 potentiated the H(2)S-induced vasorelaxation, which was suppressed by superoxide dismutase. The vasorelaxant effect of H(2)S was also significantly attenuated when Ca(2+)-free bath solution was used. Finally, pretreatment of aortic tissues with H(2)S reduced the relaxant response of vascular tissues to SNP. Our results demonstrate that the vascular effect of H(2)S is partially mediated by a functional endothelium and dependent on the extracellular calcium entry but independent of the activation of the cGMP pathway.

Heat shock proteins, inflammation, and cardiovascular disease.

Atherosclerosis and its associated complications, such as coronary artery disease, peripheral vascular disease, and stroke, are the leading causes of morbidity and mortality in all racial groups of most westernized societies. A better understanding of the events that lead to the induction and progression of atherosclerosis and the development of strategies that control these processes would have a significant impact on human health. Atherosclerosis is a chronic inflammatory condition that usually begins at an early age in the absence of lipid accumulation, with fatty streaks composed of lipid-laden macrophages (foam cells) developing at later stages. T lymphocytes, predominantly of the CD4+ phenotype, are associated with the intima layer of the vessel. The inflammatory process at atherogenic sites leads to the production of cytokines and other inflammatory mediators, resulting in cell migration, proliferation, extracellular matrix production, and plaque development.1–3⇓⇓ Monocytes and macrophages appear intimately involved with the development of atherosclerosis; mice with the osteopetrotic ( op ) mutation in the macrophage colony-stimulating factor (M-CSF) gene, which results in a complete absence of M-CSF in the serum and tissues and a marked reduction in the number of circulating monocytes, exhibit significantly less atherosclerosis than control littermates when bred into an apolipoprotein (apo) E–deficient background,.4 The precise role of T and B cells in atherosclerosis remains unclear. Although activated T cells are a dominant feature of atherosclerotic lesions,5,6⇓ studies demonstrating that atherosclerosis can be induced by hypercholesterolemia in mice deficient in T and B cells suggest that these cells are not essential.7–9⇓⇓ However, the cholesterol levels in these studies far exceeded those present in human subjects without any genetic defect, and the findings should therefore be interpreted with caution. Indeed, other evidence indicates that T cells may attenuate atherogenesis by some means, because the elimination of …