Search

Maize ( Zea mays ) is moderately sensitive to salt stress. Therefore, increasing salinity in soil causes the arrestment of physiological processes and retention of growth and development, consequently leading to yield loss. Although many strategies have been launched to improve salt stress tolerance, plant growth-promoting rhizobacteria (PGPR) are considered the most promising approach due to being more environmentally friendly and agronomically sustainable than chemicals. Therefore, this study aims to investigate the potential of Bacillus spp. and the role of microRNA-mediated genetic regulation in maize subjected to seed bio-priming application to mitigate salt stress effects. To this end, maize seeds were bio-primed with the vegetative form of B. pumilus , B. licheniformis, and B. coagulans both individually or combined, subsequently treated to NaCl, and the seedlings were screened morphologically, physiologically, and transcriptionally. The study revealed that seed bio-priming with B. licheniformis reduced the stress effects of maize seedlings by increasing catalase (CAT) and ascorbate peroxidase (APX) activities by 2.5- and 3-fold, respectively, tolerating the decrease in chlorophyll content (CC), upregulating miR160d expression which led to a 36% increase in root fresh weight (RFW) and a 39% increase in shoot fresh weight (SFW). In conclusion, Bacillus spp. successfully alleviated salt stress effects on maize by modulating antioxidant enzymes and miRNA expression., Competing Interests: Conflict of interestThe authors declare they have no conflict of interest in the publication., (© King Abdulaziz City for Science and Technology 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Background: Fusarium verticillioides is among the most devastating fungal pathogen of cereals. Therefore, it is crucial to employ effective and long-term strategies for managing F. verticillioides for sustainable agriculture. RNA interference (RNAi) being a natural defense mechanism of plants via regulation of gene expression, has emerged as a promising tool for eradicating pathogens. RNAi also operates between the host and pathogen through RNA exchange. RNAi interacts with competitive endogenous RNAs (ceRNAs) including long non-coding RNA (lncRNA), microRNA (miRNA), and mRNA. Due to the lack of an elaborate model to investigate all these mechanisms, this study aimed to establish a Nicotiana benthamiana (Nb)-F. verticillioides (Fv) phyto-pathosystem as an experimental model., Methods and Results: Nb seedlings were inoculated with Fv, and the pathogenicity was monitored morphologically, microscopically, biochemically, and transcriptionally. To observe the role of RNAi and RNA-exchange in pathogenicity, Nb-miR172 and Nb-miR399 targeting Nb-lncRNA-IPS (Induced by Phosphate Starvation1) and Nb-AP2 (Apetala2) and Nb-PHO2 (phosphate over-accumulator) ceRNA network and Fv-V-ATPase (Vesicle-fusing ATPase) targeted by Nb-miR172 were investigated. As a result, epidermal cell density, leaf area, petiole length, and chlorophyll content were reduced while the density of stomata and trichome and phenolic content and the activity of ascorbate peroxidase (APX) and glutathione reductase (GR) were increased in response to Fv infection in Nb. The expression of AP2 and PHO2 were downregulated against Fv but no significant changes were detected in IPS, miR172, and miR399 expression., Conclusion: These findings suggested the Fv-Nb phyto-pathosystem as a useful experimental model to reveal genetic regulations., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

We investigated the role of RhoA/Rho-kinase (ROCK) and PKC in the inhibitory effect of L-cysteine/hydrogen sulfide (H 2 S) pathway on the carbachol-mediated contraction of mouse bladder smooth muscle. Carbachol (10 -8 -10 -4  M) induced a concentration-dependent contraction in bladder tissues. L-cysteine (H 2 S precursor; 10 -2  M) and exogenous H 2 S (NaHS; 10 -3  M) reduced the contractions evoked by carbachol by ~ 49 and ~ 53%, respectively, relative to control. The inhibitory effect of L-cysteine on contractions to carbachol was reversed by 10 -2  M PAG (~ 40%) and 10 -3  M AOAA (~ 55%), cystathionine-gamma-lyase (CSE) and cystathionine-β-synthase (CBS) inhibitor, respectively. Y-27632 (10 -6  M) and GF 109203X (10 -6  M), a specific ROCK and PKC inhibitor, respectively, reduced contractions evoked by carbachol (~ 18 and ~ 24% respectively), and the inhibitory effect of Y-27632 and GF 109203X on contractions was reversed by PAG (~ 29 and ~ 19%, respectively) but not by AOAA. Also, Y-27632 and GF 109203X reduced the inhibitory responses of L-cysteine on the carbachol-induced contractions (~ 38 and ~ 52% respectively), and PAG abolished the inhibitory effect of L-cysteine on the contractions in the presence of Y-27632 (~ 38%). Also, the protein expressions of CSE, CBS, and 3-MST enzymes responsible for endogenous H 2 S synthesis were detected by Western blot method. H 2 S level was increased by L-cysteine, Y-27632, and GF 109203X (from 0.12 ± 0.02 to 0.47 ± 0.13, 0.26 ± 0.03, and 0.23 ± 0.06 nmol/mg respectively), and this augmentation in H 2 S level decreased with PAG (0.17 ± 0.02, 0.15 ± 0.03, and 0.07 ± 0.04 nmol/mg respectively). Furthermore, L-cysteine and NaHS reduced carbachol-induced ROCK-1, pMYPT1, and pMLC20 levels. Inhibitory effects of L-cysteine on ROCK-1, pMYPT1, and pMLC20 levels, but not of NaHS, were reversed by PAG. These results suggest that there is an interaction between L-cysteine/H 2 S and RhoA/ROCK pathway via inhibition of ROCK-1, pMYPT1, and pMLC20, and the inhibition of RhoA/ROCK and/or PKC signal pathway may be mediated by the CSE-generated H 2 S in mouse bladder., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In this study, we investigated the possible role of the l-cysteine/hydrogen sulfide pathway in β 3 -adrenoceptors-mediated relaxation in isolated mouse gastric fundus tissue. l-cysteine (endogenous H 2 S; 10 -6 -10 -2  M), sodium hydrogen sulfide (NaHS; exogenous H 2 S; 10 -6 -10 -3  M), selective β 3 -adrenoceptors agonist BRL 37344 (10 -9 -10 -4  M) and non-selective β-adrenoceptor agonist isoprenaline (10 -9 -10 -4  M) produced concentration-dependent relaxation in mouse gastric fundus. The non-selective β-adrenoceptors antagonist propranolol (10 -6  M) inhibited the relaxant response to isoprenaline but not to BRL 37344. On the other hand, the selective β 3 -adrenoceptors antagonist SR 59230A (10 -5  M) inhibited the relaxant responses to BRL 37344. In addition, cystathionine-gamma-lyase (CSE) inhibitor D,L-propargylglycine (PAG, 10 -2  M), cystathionine-beta-synthase inhibitor (CBS) aminooxyacetic acid (AOAA, 10 -2  M), and the combination of these inhibitors significantly reduced the relaxant responses induced by l-cysteine and BRL 37344. Pre-incubation of gastric fundal strips with propranolol (10 -6  M) and SR 59230A (10 -5  M) did not affect relaxations to l-cysteine and NaHS. Also, the existence of CSE, CBS, 3-mercaptopurivate sulfur transferase (3-MST) enzymes and β 3 -adrenoceptors were detected in gastric fundal tissue. Furthermore, basal H 2 S release was detected in the measurements. H 2 S level increased in the presence of l-cysteine, NaHS, and BRL 37344. The increase in H 2 S level by l-cysteine and BRL 37344 decreased significantly with PAG and AOAA enzyme inhibitors. These results suggest that endogenous H 2 S is synthesized from l-cysteine at least by CBS and CSE enzymes. Also, β 3 -adrenoceptors are found in the mouse stomach fundus and mediate BRL 37344-induced relaxations, and l-cysteine/hydrogen sulfide pathway plays a partial role in β 3 -adrenoceptors-mediated relaxation in mouse gastric fundus tissue., (Copyright © 2021. Published by Elsevier Inc.)

MAIN CONCLUSION: miRNAs control leaf size of maize crop during chilling stress tolerance by regulating developmentally important transcriptional factors and sustaining redox homeostasis of cells. Chilling temperature (0-15 °C) is a major constraint for the cultivation of maize (Zea mays) which inhibits the early growth of maize leading to reduction in leaf size. Growth and development take place in meristem, elongation, and mature zones that are linearly located along the leaf base to tip. To prevent shortening of leaf caused by chilling, this study aims to elucidate the regulatory roles of microRNA (miRNA) genes in the controlling process switching between growth and developmental stages. In this respect, hybrid maize ADA313 seedlings were treated to the chilling temperature which caused 26% and 29% reduction in the final leaf length and a decline in cell production of the fourth leaf. The flow cytometry data integrated with the expression analysis of cell cycle genes indicated that the reason for the decline was a failure proceeding from G2/M rather than G1/S. Through an miRNome analysis of 321 known maize miRNAs, 24, 6, and 20 miRNAs were assigned to putative meristem, elongation, and mature zones, respectively according to their chilling response. To gain deeper insight into decreased cell production, in silico, target prediction analysis was performed for meristem specific miRNAs. Among the miRNAs, miR160, miR319, miR395, miR396, miR408, miR528, and miR1432 were selected for confirming the potential of negative regulation with their predicted targets by qRT-PCR. These findings indicated evidence for improvement of growth and yield under chilling stress of the maize.

Rho-kinase activity is a key regulator in the maintenance of corporal vasoconstriction and penile detumescense. Also, importance of l-cysteine/H 2 S pathway in erectile tissue has been shown; however it is currently unknown the role RhoA/Rho-kinase pathway in H 2 S-induced inhibition in cavernosal tissue. We investigated the role of RhoA/Rho-kinase pathway in the inhibitory effect of l-cysteine and NaHS, as endogenous and exogenous H 2 S, respectively, on phenylephrine-induced contractions of mouse cavernosal strips. Phenylephrine, α 1 receptor agonist, (10 nM-100 μM) induced a concentration-dependent contraction in CC. l-cysteine (endogenous H 2 S substrate; 10 mM) and exogenous H 2 S (NaHS; 1 mM) significantly inhibited the contractile response to phenylephrine (P < 0.05). Inhibition of CSE and CBS enzymes by PAG (10 mM) and AOAA (1 mM), respectively, significantly reversed the inhibitory effects of l-cysteine on phenylephrine-induced contraction (P < 0.05). Y-27632 (1 μM), a specific Rho-kinase inhibitor, significantly augmented the inhibitory effect of l-cysteine and NaHS on phenylephrine-induced contraction, and this inhibition was reversed by PAG and AOAA (P < 0.05). In addition, the formation of H 2 S was increased by approximately 1.8 fold over basal values after incubation of tissue homogenates with l-cysteine. Y-27632 significantly increased both basal and l-cysteine-induced H 2 S formation and this augmentation diminished by PAG and AOAA (P < 0.05). Furthermore, the pMYPT-1 expression was significantly decreased by l-cysteine, NaHS or Y-27632 alone. Also, pMYPT-1 expression was completely abolished by the l-cysteine/NaHS plus Y-27632 combination, and this inhibition was reversed by PAG and AOAA (P < 0.05). These results suggest that there is an interaction between Rho-kinase and H 2 S pathways. Rho-kinase may be, at least in part, inhibits CSE/CBS enzymes in mouse corpus cavernosal tissue; however, it is not excluded the other kinases such as PKC and Zip-kinase., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Throughout the plant life cycle, growth of new leaves is governed by cell division and cell expansion. During steady-state growth of the maize leaf, these processes are spatially separated between the meristem zone, consisting of dividing cells at the leaf base, the elongation zone, consisting of expanding cells moving upwards from the meristem, and the mature zone containing differentiated mature cells. Increased leaf size can be achieved through increasing cell number or cell size, for example by manipulating the genes controlling the transition between those zones. In this study, microRNA (miRNA) genes, which are a class of endogenous small, non-coding gene regulatory RNAs, were investigated in the growth zones, to gain insight into their role in the transition between cell division and cell expansion. A genome-wide survey was conducted using a miRNA-microarray and 59 miRNA genes were detected to be differentially expressed between the growth zones. miR160, miR166, miR168, miR172, miR319 and miR390 families were significantly up-regulated in the meristem relative to the elongation and mature zones. In contrast, expression of the miR167 and miR396 families was lower in the meristem and higher in the mature zone. Therefore, these were considered to be candidate growth-regulated miRNAs that control cell division processes indirectly by repressing target genes. The miR156, miR166, miR167, miR399, miR408 and miR2275 families were expressed most highly in the elongation zone, and so were classified as elongation-specific, with possible roles in switching from cell division to cell elongation during leaf differentiation. In silico target prediction analysis showed that these miRNAs target several transcription factors and metabolic genes, and a reciprocal relationship between the expression levels of miR319 and miR396 and their targets was confirmed by qRT-PCR. Furthermore, 12 candidate novel miRNAs were identified from the microarray data and computationally verified. Three out of twelve were also validated by qRT-PCR. These findings provide important information regarding the regulatory functions of miRNAs in controlling progression of growth mechanisms., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In several animal and human studies, the contribution of the endothelium, nitric oxide/soluble guanosine monophosphate (NO/cGMP) pathway, adenylyl cyclase, phosphodiesterase (PDE), potassium (K + ) channels, L-type calcium channels, Na + -K + -ATPase, muscarinic acetylcholine receptors, RhoA/Rho-kinase pathway, and cyclooxygenase (COX)/arachidonic acid cascade on the relaxant mechanism of L-cysteine/H 2 S pathway in corpus cavernosum has been investigated. In this chapter the relaxant mechanisms of H 2 S in corpus cavernosum is discussed with data available in the current relevant literature. Also, in vitro experimental procedure for mice corpus cavernosum which used to investigate the relaxant effect of H 2 S is given in detail.

One of the important challenges in electron beam lithography is nanofabrication on nonflat or irregular surfaces. Although spin coating is the most popular technique for resist coating, it is not suitable for nonflat, irregular substrates because a uniform film cannot be achieved on those surfaces. Here, it is demonstrated that single layer surface-grafted PMMA can be used as a negative-tone e-beam resist, and it can be applied to nonflat, irregular surfaces as well as flat, conventional surfaces. Although it is well known that heavily exposed PMMA undergoes cross-linking and works as a negative-tone e-beam resist when developed by solvent, solvent does not work as a developer for negative-tone single-layer surface-grafted PMMA. Instead, thermal treatment at 360 °C for 1 min is used to develop PMMA.

The aim of this study was to investigate the possible interaction of l-cysteine/H 2 S pathway and muscarinic acetylcholine receptors (mAChRs) in the mouse corpus cavernosum (CC). l-cysteine (endogenous H 2 S substrate; 10 -6 -10 -3  M), sodium hydrogen sulfide (NaHS; exogenous H 2 S; 10 -6 -10 -3  M) and acetylcholine (10 -9 -10 -4  M) produced concentration-dependent relaxation in isolated mouse CC tissues. Relaxations to endogenous and exogenous H 2 S were reduced by non-selective mAChR antagonist atropine (5 × 10 -5  M), selective M 1 mAChR antagonist pirenzepine (5 × 10 -5  M) and selective M 3 mAChR antagonist 4-DAMP (10 -7  M) but not by selective M 2 mAChR antagonist AF-DX 116 (10 -6  M). Also, acetylcholine-induced relaxations were reduced by atropine, pirenzepine, 4-DAMP and AF-DX 116, confirming the selective effects of mAChR antagonists. Furthermore, acetylcholine-induced relaxations were attenuated by cystathionine-gamma-lyase (CSE) inhibitor d,l-propargylglycine (PAG, 10 -2  M) and cystathionine-β-synthase inhibitor (CBS) aminooxyacetic acid (AOAA, 10 -3  M). l-nitroarginine, nitric oxide synthase inhibitor, augmented the inhibitory effects of mAChR antagonists and H 2 S enzyme inhibitors on acetylcholine-induced relaxations. In addition, the existence and localization of CSE, CBS and 3-MST were demonstrated in mouse CC. Furthermore, tissue acetylcholine release was significantly increased by l-cysteine but not by exogenous H 2 S. The increase in acetylcholine level was completely inhibited by AOAA and PAG. These results suggest that M 1 and M 3 mAChRs contributes to relaxant effect mediated by endogenous H 2 S but at same time l-cysteine triggers acetylcholine release from cavernosal tissue. Also, the role of NO in the interaction of l-cysteine/H 2 S pathway and muscarinic acetylcholine receptors (mAChRs) could not be excluded., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Penile corpus cavernosum is an extremely vascularized tissue and cavernosal smooth muscle tone is regulated by the balance between contractile and relaxant factor. We investigated the possible role of arachidonic acid/cyclooxygenase cascade, phosphodiesterase IV (PDEIV) and Rho-kinase in exogenous hydrogen sulfide (H 2 S)-induced relaxation in mouse corpus cavernosum., Methods: The relaxant response to H 2 S (NaHS as exogenous H 2 S; 1-1000μM) were obtained in isolated mouse corpus cavernosum tissues which pre-contracted by phenylephrine (5μM). The effects of 4-(4-octadecylphenyl)-4-oxobutenoic acid (OBAA; 10μM), a selective phospholipase A 2 (PLA 2 ) inhibitor, indomethacin (1μM), a non-selective cyclooxygenase (COX) inhibitor, baicalein (10μM), a lipoxygenase (LOX) inhibitor, and proadifen (10μM), cytochrome P450 inhibitor, on the relaxant responses to H 2 S were investigated. Furthermore, the effects of theophylline (500μM) and rolipram (1μM), a non-selective and selective PDEIV inhibitor, and fasudil (3μM), a specific Rho-kinase inhibitor, were studied on H 2 S-induced relaxation., Results: H 2 S-induced relaxations were significantly reduced by OBAA, indomethacin and proadifen but not baicalein. Furthermore, theophylline, rolipram and fasudil reduced H 2 S-induced relaxations., Conclusion: These results suggest that PLA 2 , COX, cytochrome P450, PDEIV and Rho-kinase pathway may involve in H 2 S-induced relaxation in mouse corpus cavernosum tissues., (Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Although spin coating is the most widely used electron-beam resist coating technique in nanolithography, it cannot typically be applied for nonflat or irregular surfaces. Here, we demonstrate that monolayer polystyrene brush can be grafted on substrates and used as both positive and negative electron-beam resist, which can be applied for such unconventional surfaces. Polystyrene is a popular negative resist when using solvent developer but solvent cannot be used for grafted polystyrene brush that is firmly bonded to the substrate. Instead, we employed two unconventional development methods to lead polystyrene brush to positive or negative tone behavior. Negative tone was achieved by thermal development at 300 °C because exposed thus cross-linked polystyrene brush is more thermally stable against vaporization than unexposed linear one. Surprisingly, positive tone behavior occurred when the brush was grafted onto an aluminum (Al) layer and the film stack was developed using diluted hydrofluoric acid (HF) that etched the underlying Al layer. By transferring the patterns into the silicon (Si) substrates using the thin Al layer as a sacrificial hard mask for dry etch, well-defined structures in Si were obtained in two different electron-beam resist tones as well as in nonflat surfaces.

The aim of this study was to investigate the mechanism of H2 S-induced relaxation in mouse corpus cavernosal tissue. l-cysteine (10(-6) × 10(-3) mol/L) and exogenous H2 S (NaHS; 10(-6) to 10(-3) mol/L) induced concentration-dependent relaxation. l-cysteine-induced relaxations was reduced by d,l-propargylglycine, a cystathionine gamma lyase (CSE) inhibitor but not influenced by aminooxyacetic acid, a cystathionine beta synthase (CBS) inhibitor. l-cysteine induced relaxations, but not of those of H2 S diminished in endothelium-denuded tissues. N(ω) -nitro-l-arginine (l-NA; 10(-4) mol/L), a nitric oxide synthase inhibitor, and ODQ (10(-4) mol/L), a guanylyl cyclase inhibitor, increased the H2 S-induced relaxation. Zaprinast (5 × 10(-6) mol/L) and sildenafil (10(-6) mol/L), phosphodiesterase inhibitors, inhibited H2 S-induced relaxation. Adenylyl cyclase inhibitors N-ethylmaleimide (2.5 × 10(-5) mol/L) and SQ22536 (10(-4) mol/L) reduced relaxation to H2 S. Also, H2 S-induced relaxation was reduced by KCl (50 mmol/L), 4-aminopyridine (10(-3) mol/L), a Kv inhibitor, glibenclamide (10(-5) mol/L), a KATP inhibitor or barium chloride (10(-5) mol/L), a KIR inhibitor. However, H2 S-induced relaxation was not influenced by apamin (10(-6) mol/L), a SKC a (2+) inhibitor, charybdotoxin (10(-7) mol/L), an IKC a (2+) and BKC a (2+) inhibitor or combination of apamin and charybdotoxin. Nifedipine (10(-6) mol/L), an L-type calcium channel blocker and atropine (10(-6) mol/L), a muscarinic receptor blocker, inhibited H2 S-induced relaxation. However, H2 S-induced relaxation was not influenced by ouabain (10(-4) mol/L), a Na(+) /K(+) -ATPase inhibitor. This study suggests that H2 S endogenously synthesizes from l-cysteine by CSE endothelium-dependent in mouse corpus cavernosum tissue, and exogenous H2 S may cause endothelium-independent relaxations via activation of K channels (KATP channel, KV channels, KIR channels), L-type voltage-gated Ca(2+) channels, adenylyl cyclase/cAMP pathway and muscarinic receptor, and there is the interaction between H2 S and NO/cGMP., (© 2016 John Wiley & Sons Australia, Ltd.)

The aim of this study was to evaluate whether the sub-chronic systemic ethanol exposure has direct effect on cavernosal smooth muscle contractions induced by KCl (depolarizing) and phenylephrine (α1-receptor agonist), and the possible involvement of RhoA/Rho-kinase pathway. Sub-chronic systemic ethanol was applied to mice with inhalation route for 14 days. The blood levels in ethanol-treated mice averaged 121.2 ± 9.1 mg/dl. KCl (80 mM) and phenylephrine (10 nM-100 μM) induced sustained contractions in corpus corporal strips from sham-treated mice. Sub-chronic ethanol treatment reduced the contractions to KCl. However, phenylephrine-induced contractions were not affected by ethanol treatment. Rho-kinase inhibitor fasudil (50 μM) and Y-27632 (50 μM) inhibited contractions to KCl and phenylephrine in sham-treated mice. Ethanol treatment increased the inhibitory effect of Rho-kinase inhibitors on contractions to phenylephrine. The relaxations induced by fasudil (100 μM) and Y-27632 (500 μM) did not change in ethanol treatment group. In ethanol-treated group, the expression of RhoA decreased compared to sham-treated group. Also, ROCK1 expression was reduced by ethanol but not statically significant to sham-treated group; however, the expression of ROCK2 increased in ethanol group. From these findings, it seems that phenylephrine and KCl-induced contractions depends on RhoA/Rho-kinase-mediated Ca(2+) sensitization. Also, these results suggest that the ethanol treatment decreased the expression of RhoA, and the inhibitory effect of ethanol on KCl-induced contractions may be due to, at least in part, the inhibition of a RhoA/Rho-kinase in mouse corpus cavernosum.

Calcium sensitization by the RhoA/Rho-kinase (ROCK) pathway contributes to the contraction in smooth muscle. Contractile stimuli can sensitize myosin to Ca(2+) by activating RhoA/Rho-kinase that inhibits myosin light chain phosphatase activity. The present study was aimed at investigating the possible involvement of RhoA/Rho-kinase pathway in contractile responses to agonist (phenylephrine) and depolarizing (KCl) of mouse lung parenchymal tissues. Also, we investigated the effect of ethanol on RhoA/Rho-kinase pathway. Phenylephrine (10(-8)-10(-4) M) and KCl (10-80 mM) induced sustained contractions in parenchymal strips. Ethanol significantly attenuated the contractions to phenylephrine and KCl. The Rho-kinase inhibitors fasudil (5×10(-5) M) and Y-27632 (5×10(-5) M) inhibited contractions to in both control and ethanol-treated parenchymal strips. In addition, the relaxations induced by fasudil (10(-4) M) and Y-27632 (5×10(-4) M) on parenchymal strips contracted by phenylephrine but not KCl was decreased in ethanol-treatment group. Also, RhoA, ROCK1 and ROCK2 expressions were detected in mouse lung parenchymal tissue. In ethanol-treated group, expression of RhoA and ROCK1 but not ROCK2 decreased compared to control. Furthermore, ethanol causes apoptotic changes in alveolar type I epithelial cells of parenchymal tissue. These results suggest that RhoA/Rho-kinase signaling pathway plays an important role in phenylephrine- and KCl-induced Ca(2)(+) sensitization in mouse lung parenchymal tissue. Also, ethanol may be decrease phenylephrine- and KCl-induced contraction due to lowering the RhoA/Rho-kinase-mediated Ca(2+)-sensitizing by inhibiting RhoA/Rho-kinase pathway in parenchymal tissue. These results may be lead to important insights into the mechanisms of lung diseases due to alcohol consumption., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background and Purpose: NAD(P)H oxidase and COX-1 participate in vascular damage induced by angiotensin II. We investigated the effect of rosuvastatin on endothelial dysfunction, vascular remodelling, changes in extracellular matrix components and mechanical properties of small mesenteric arteries from angiotensin II-infused rats., Experimental Approach: Male rats received angiotensin II (120 ng·kg⁻¹ ·min⁻¹ , subcutaneously) for 14 days with or without rosuvastatin (10 mg·kg⁻¹ ·day⁻¹ , oral gavage) or vehicle. Vascular functions and morphological parameters were assessed by pressurized myography., Key Results: In angiotensin II-infused rats, ACh-induced relaxation was attenuated compared with controls, less sensitive to L-NAME, enhanced by SC-560 (COX-1 inhibitor) or SQ-29548 (prostanoid TP receptor antagonist), and normalized by the antioxidant ascorbic acid or NAD(P)H oxidase inhibitors. After rosuvastatin, relaxations to ACh were normalized, fully sensitive to L-NAME, and no longer affected by SC-560, SQ-29548 or NAD(P)H oxidase inhibitors. Angiotensin II enhanced intravascular superoxide generation, eutrophic remodelling, collagen and fibronectin depositions, and decreased elastin content, resulting in increased vessel stiffness. All these changes were prevented by rosuvastatin. Angiotensin II increased phosphorylation of NAD(P)H oxidase subunit p47phox and its binding to subunit p67phox, effects inhibited by rosuvastatin. Rosuvastatin down-regulated vascular Nox4/NAD(P)H isoform and COX-1 expression, attenuated the vascular release of 6-keto-PGF1α , and enhanced copper/zinc-superoxide dismutase expression., Conclusion and Implications: Rosuvastatin prevents angiotensin II-induced alterations in resistance arteries in terms of function, structure, mechanics and composition. These effects depend on restoration of NO availability, prevention of NAD(P)H oxidase-derived oxidant excess, reversal of COX-1 induction and its prostanoid production, and stimulation of endogenous vascular antioxidant defences., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

The aim of this work was to compare the preventing capacity of quercetin with Cu/Zn superoxide dismutase (Cu/Zn SOD), ascorbic acid and glutathione on nitric oxide (NO)-induced relaxation in mouse gastric fundus. Furthermore, the effects of the quercetin on the tissue level of total oxidant and antioxidant was investigated. Nitrergic stimulation (4Hz, 25V, 0.1 ms, 10s-train) and exogenous NO (10 μM) induced relaxation. Pyrogallol (10 μM), hydroquinone (100 μM) and LY83583 (6-Anilino-quinolin-5,8-quinone, 5 μM) inhibited nitrergic relaxations. The inhibition observed with pyrogallol, hydroquinone and LY83583 was prevented by quercetin (0.1 μM). Also, ascorbic acid (500 μM), glutathione (100 μM) and Cu/Zn SOD (100 U/ml) prevented the inhibitory effect of superoxide anion generators on the relaxation to nitrergic stimulation and NO. Diethyldithiocarbamic acid (DETCA; 8mM) inhibited nitrergic relaxations. DETCA-induced inhibition on nitrergic stimulation and NO-induced relaxation was prevented by quercetin, ascorbic acid, glutathione or Cu/Zn SOD. DETCA plus pyrogallol, hydroquinone or LY83583 strengthened the inhibition on the relaxations. Also, pre-treatment with quercetin, ascorbic acid and glutathione prevented the inhibitory effect of DETCA plus LY-83583 on the relaxation to nitrergic stimulation and NO but Cu/Zn SOD did not prevent this inhibition. Also, quercetin increased tissue total antioxidant capacity and decreased tissue oxidant level and oxidative stress index in DETCA-treatment group. These results indicate that quercetin has antioxidant effect and protects NO from endogenous superoxide anion-driven inactivation and enhances its biological activity, suggesting that quercetin may scavenge superoxide anion in a Cu/Zn SOD, glutathione or ascorbic acid-inhibitable manner., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: The aim of this study was to investigate whether thiols are involved in the nitrergic neurotransmission in mouse duodenum., Methods: The effects of thiol-modulating agents, ethacrynic acid (100 μM), a non-specific sulfhydryl alkylator, and diamide (100 μM), an alkylating agent that oxidizes protein sulfhydryl groups and depletes intracellular glutathione, on relaxations to nitrergic stimulation (electrical field stimulation, EFS;10 Hz, 25 V, 1 ms, 15 s-train), S-nitrosoglutathione (GSNO; 5 μM), S-nitroso-acetylpenicillamine (SNAP; 5 μM), and S-nitrosocysteine (CysNO; 10 μM) were investigated. Moreover, the effects of buthionine sulfoximine (100 μM), an inhibitor of γ-glutamylcysteine synthetase, and sulfobromophthalein (100 μM), an inhibitor of glutathione-S-transferase, were studied on relaxant responses to EFS and S-nitrosothiols in mouse duodenum., Results: Exogenous free thiol, glutathione (GSH, 100 μM) did not influence relaxation to EFS, GSNO, SNAP, and CysNO. Ethacrynic acid and diamide significantly decreased relaxation of duodenum to EFS, GSNO, SNAP, and CysNO. This inhibition was prevented by addition of GSH. Buthionine sulfoximine and sulfobromophthalein significantly decreased relaxation to EFS and GSNO but did not influence relaxation to SNAP and CysNO. The inhibitory effect of buthionine sulfoximine and sulfobromophthalein on the relaxant response to EFS and GSNO was prevented by addition of GSH., Conclusions: These results suggest that relaxation to nitrergic stimulation is thiol-dependent, and nitrosothiols, possibly S-nitrosoglutathione may play a role, as an intermediate compound in nitrergic neurotransmission in mouse duodenum.

Aims: The aim of this study was to investigate the role of cyclooxygenase (COX)-1 on vascular alterations in structure, mechanics, and extracellular matrix (ECM) components induced by angiotensin (Ang) II in mesenteric arteries from wild-type (WT) and COX-1 knockout (COX-1(-/-)) mice., Methods and Results: Animals were infused with vehicle or Ang II (400 ng/kg/min, s.c.) ± SC-560 (COX-1 inhibitor), DFU (COX-2 inhibitor), or SQ-29548 (TP receptor antagonist). After 2 weeks, vessels were isolated and exposed to intraluminal pressures (3-140 mmHg, pressurized myograph) to determine mechanical properties. Angiotensin II-induced vascular hypertrophic remodelling in WT was reversed by SC-560 or SQ-29548, but unaffected by DFU. Angiotensin II increased vessel stiffness (P< 0.01), this effect being ameliorated by SC-560 or SQ-29548, but unmodified by DFU. Angiotensin II failed to modify vessel elasticity in COX-1(-/-) mice. In WT vessels, Ang II enhanced COX-1 immunostaining, induced collagen and fibronectin depositions and decreased elastin content (P< 0.01). These effects were reversed by SC-560 or SQ-29548, but unaffected by DFU. In COX-1(-/-) mice, Ang II did not affect ECM contents. In WT, Ang II increased COX-1 and decreased COX-2 expression, and enhanced the vascular release of 6-keto-PGF1α which was prevented by COX-1 blockade. Human coronary artery smooth muscle cells, incubated with Ang II, showed an increased expression of procollagen I, which was abrogated by SC-560 or SQ-29548., Conclusion: Angiotensin II-induced alterations of resistance arteries in structure, mechanics, and ECM composition were prevented by COX-1 inhibition and TP receptor antagonism, indicating that Ang II-mediated vascular damage is mediated by COX-1-derived prostanoid prostacyclin, activating TP receptors.

The relaxation of cavernous smooth muscle is critical for inducing and maintaining a penile erection. The neurogenic- and endothelium-dependent relaxation of corpus cavernosum smooth muscle and the degenerative effect of subacute ethanol treatment on the endothelial cells of corpus cavernosum was investigated in mice. In the cavernous strips contracted with phenylephrine, electrical field stimulation (EFS), acetylcholine and exogenous nitric oxide (NO) induced relaxations in the control group. Ethanol treatment abolished the endothelium-dependent relaxations induced by acetylcholine but failed to alter the relaxation to EFS and NO. L-nitroarginine, a NO synthase inhibitor, reduced relaxations induced by EFS and acetylcholine, but not those induced by NO in control and ethanol-treated mice. L-arginine prevented the response inhibited by L-nitroarginine. ODQ, a guanylyl cyclase inhibitor, inhibited relaxations in response to EFS, NO and acetylcholine in control and ethanol-treated mice. Corpus cavernosum tissues were investigated using electron microscopy and endothelial damage was observed in ethanol-treated mice. These results suggest that ethanol impairs the endothelial function of corpus cavernosum in mouse, and it may lead to erectile dysfunction through a reduced NO release via endothelial impairment.