Cyclic GMP specifically suppresses Type-Iα cGMP-dependent protein kinase expression by ubiquitination

Type I cGMP-dependent protein kinase (PKG-I) mediates nitric oxide (NO) and hormone dependent smooth muscle relaxation and stimulates smooth muscle cell-specific gene expression. Expression of PKG-I in cultured smooth muscle cells depends on culture conditions and is inhibited by inflammatory cytokines such as interleukin-I and tumor necrosis factor-α, which are known to stimulate Type II NO synthase (iNOS) expression. We report here that the suppression of PKG-I protein levels in smooth muscle cells is triggered by the ubiquitin/26S proteasome pathway. Incubation of vascular smooth muscle cells with phosphodiesterase-resistant cyclic GMP analogs (e.g., 8-bromo-cGMP) decreases PKG-I protein level in a time- and concentration-dependent manner. To study this process, we tested the effects of 8-Br-cGMP on PKG-I protein level in Cos7 cells, which do not express endogenous type I PKG mRNA. 8-Br-cGMP induced the ubiquitination and down-regulation of PKG-Iα, but not PKG-Iβ. Treatment of cells with the 26S proteasome inhibitor, MG-132, increased ubiquitination of PKG. Blocking PKG-I catalytic activity using the cell-permeant specific PKG-I inhibitor, DT-2, inhibited cGMP-induced PKG-I ubiquitination and down-regulation, suggesting that PKG catalytic activity and autophosphorylation were required for suppression of PKG-I level. Mutation of the known autophosphorylation sites of PKG-Iα to alanine uncovered a specific role for autophosphorylation of serine-64 in cGMP-dependent ubiquitination and suppression of PKG-I level. The results suggest that chronic elevation of cGMP, as seen in inflammatory conditions, triggers ubiquitination and degradation of PKG-Iα in smooth muscle.     

Role of Potassium Channels in the Effects of Hydrogen Sulfide on Contractility of Gastric Smooth Muscle Cells in Rats

The effect of sodium hydrosulfide (NaHS), a hydrogen sulfide (H₂S) donor, on spontaneous contractile activity of rat gastric smooth muscle cells was analyzed. Experiments were conducted on gastric stripes under conditions of isometric contraction. It was shown that NaHS has a biphasic effect on spontaneous contractile activity, increasing tonic tension and the amplitude of phasic contractions within the first minutes since application. This initial phase is followed by a decrease in amplitude, basal tone, and frequency of spontaneous contractions. The inhibitory effect of NaHS was dose-dependent at concentrations from 10 to 600 μM. Preliminary application of tetraethylammonium and 4-aminopirydine, inhibitors of voltage-gated and calciumactivated potassium channels, prevented a NaHS-induced initial increase in basal tone and phasic contraction amplitude. Activation of ATP-dependent potassium channels (KATP-channels) by diazoxide prevented in part a NaHS-induced decrease in basal tone and amplitude of spontaneous contractions. Glibenclamide, an inhibitor of K_ATP-channels, decreased the inhibitory effect of NaHS on amplitude, basal tone and frequency of spontaneous contractions. It was concluded that in rat gastric smooth muscles the excitatory effect of H₂S is mediated by the inhibition of voltagegated and calcium-activated potassium channels, while its inhibitory effect involves the activation of K_ATP-channels.     

环鸟苷酸依赖的蛋白激酶对心血管功能的调节作用

环鸟苷酸(cGMP)依赖的蛋白激酶(PKG)是一氧化氮-cGMP的主要细胞内受体,在哺乳动物细胞中分为PKG-I和PKG-II两型。在PKG介导的血管平滑肌舒张作用中,其主要通过活化细胞膜上的钙活化的钾通道(BK通道),磷酸化肌质网上的受磷蛋白(phospholamban,PLB)和三磷酸肌醇受体相关的PKG-I底物(IP3receptor-associated PKG-I substrate,IRAG),降低细胞内Ca2 浓度。PKG还可通过活化肌球蛋白轻链磷酸酶及抑制Rho激酶降低肌球蛋白对Ca2 敏感性。PKG调节血管平滑肌细胞的基因表达和表型调变,调节细胞增生。PKG活化以后还具有抑制血小板聚集,抑制心肌细胞肥大等功能。最近的研究证明,PKG的表达水平和活性改变与动脉粥样硬化和再狭窄、高血压、糖尿病心血管病变以及硝酸盐耐受等的发病机制有密切关系。     

Activation of cGMP-dependent protein kinase stimulates cardiac ATP-sensitive potassium channels via a ROS/calmodulin/CaMKII signaling cascade

Background

Cyclic GMP (cGMP)-dependent protein kinase (PKG) is recognized as an important signaling component in diverse cell types. PKG may influence the function of cardiac ATP-sensitive potassium (K_ATP) channels, an ion channel critical for stress adaptation in the heart; however, the underlying mechanism remains largely unknown. The present study was designed to address this issue.

Methods and Findings

Single-channel recordings of cardiac K_ATP channels were performed in both cell-attached and inside-out patch configurations using transfected human embryonic kidney (HEK)293 cells and rabbit ventricular cardiomyocytes. We found that Kir6.2/SUR2A (the cardiac-type K_ATP) channels were activated by cGMP-selective phosphodiesterase inhibitor zaprinast in a concentration-dependent manner in cell-attached patches obtained from HEK293 cells, an effect mimicked by the membrane-permeable cGMP analog 8-bromo-cGMP whereas abolished by selective PKG inhibitors. Intriguingly, direct application of PKG moderately reduced rather than augmented Kir6.2/SUR2A single-channel currents in excised, inside-out patches. Moreover, PKG stimulation of Kir6.2/SUR2A channels in intact cells was abrogated by ROS/H₂O₂ scavenging, antagonism of calmodulin, and blockade of calcium/calmodulin-dependent protein kinase II (CaMKII), respectively. Exogenous H₂O₂ also concentration-dependently stimulated Kir6.2/SUR2A channels in intact cells, and its effect was prevented by inhibition of calmodulin or CaMKII. PKG stimulation of K_ATP channels was confirmed in intact ventricular cardiomyocytes, which was ROS- and CaMKII-dependent. Kinetically, PKG appeared to stimulate these channels by destabilizing the longest closed state while stabilizing the long open state and facilitating opening transitions.

Conclusion

The present study provides novel evidence that PKG exerts dual regulation of cardiac K_ATP channels, including marked stimulation resulting from intracellular signaling mediated by ROS (H₂O₂ in particular), calmodulin and CaMKII, alongside of moderate channel suppression likely mediated by direct PKG phosphorylation of the channel or some closely associated proteins. The novel cGMP/PKG/ROS/calmodulin/CaMKII signaling pathway may regulate cardiomyocyte excitability by opening K_ATP channels and contribute to cardiac protection against ischemia-reperfusion injury.     

cGMP/Protein Kinase G Signaling Suppresses Inositol 1,4,5-Trisphosphate Receptor Phosphorylation and Promotes Endoplasmic Reticulum Stress in Photoreceptors of Cyclic Nucleotide-gated Channel-deficient Mice

Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. We have shown endoplasmic reticulum (ER) stress-associated apoptotic cone death and increased phosphorylation of the ER Ca²⁺ channel inositol 1,4,5-trisphosphate receptor 1 (IP₃R1) in CNG channel-deficient mice. We also presented a remarkable elevation of cGMP and an increased activity of the cGMP-dependent protein kinase (protein kinase G, PKG) in CNG channel deficiency. This work investigated whether cGMP/PKG signaling regulates ER stress and IP₃R1 phosphorylation in CNG channel-deficient cones. Treatment with PKG inhibitor and deletion of guanylate cyclase-1 (GC1), the enzyme producing cGMP in cones, were used to suppress cGMP/PKG signaling in cone-dominant Cnga3^−/−/Nrl^−/− mice. We found that treatment with PKG inhibitor or deletion of GC1 effectively reduced apoptotic cone death, increased expression levels of cone proteins, and decreased activation of Müller glial cells. Furthermore, we observed significantly increased phosphorylation of IP₃R1 and reduced ER stress. Our findings demonstrate a role of cGMP/PKG signaling in ER stress and ER Ca²⁺ channel regulation and provide insights into the mechanism of cone degeneration in CNG channel deficiency.     

Effect of pretreatment with hydrogen sulfide donor sodium hydrosulfide on heat tolerance in relation to antioxidant system in maize (Zea mays) seedlings

Hydrogen sulfide (H₂S) is a signal molecule that is involved in plant growth, development and the acquisition of stress tolerance including heat tolerance, but the mechanism of H₂S-induced heat tolerance is not completely clear. In present study, the effect of sodium hydrosulfide (NaHS), a H₂S donor, treatment on heat tolerance of maize seedlings in relation to antioxidant system was investigated. The results showed that NaHS treatment improved survival percentage of maize seedlings under heat stress in a concentration-dependent manner, indicating that H₂S treatment could improve heat tolerance of maize seedlings. To further study mechanism of NaHS-induced heat tolerance, catalase (CAT), guaiacol peroxidase (GPX), superoxide dismutase (SOD), glutathione reductase (GR) and ascorbate peroxidase (APX) activities, and glutathione (GSH) and ascorbic acid (AsA) contents in maize seedlings were determined. The results showed that NaHS treatment increased the activities of CAT, GPX, SOD and GR, and GSH and AsA contents as well as the ratio of reduced antioxidants to total antioxidants [AsA/(AsA+DHA) and GSH/(GSH +GSSG)] in maize seedlings under normal culture conditions compared with the control. Under heat stress, antioxidant enzymes activities, antioxidants contents and the ratio of the reduced antioxidants to total antioxidants in control and treated seedlings all decreased, but NaHS-treated seedlings maintained higher antioxidant enzymes activities and antioxidants levels as well as the ratio of reduced antioxidants to total antioxidants. All of above-mentioned results suggested that NaHS treatment could improve heat tolerance of maize seedlings, and the acquisition of this heat tolerance may be relation to enhanced antioxidant system activity.     

Essential roles of the nitric oxide (no)/cGMP/protein kinase G type-Iα (PKG-Iα) signaling pathway and the atrial natriuretic peptide (ANP)/cGMP/PKG-Iα autocrine loop in promoting proliferation and cell survival of OP9 bone marrow stromal cells

Inappropriate signaling conditions within bone marrow stromal cells (BMSCs) can lead to loss of BMSC survival, contributing to the loss of a proper micro-environmental niche for hematopoietic stem cells (HSCs), ultimately causing bone marrow failure. In the present study, we investigated the novel role of endogenous atrial natriuretic peptide (ANP) and the nitric oxide (NO)/cGMP/protein kinase G type-Iα (PKG-Iα) signaling pathway in regulating BMSC survival and proliferation, using the OP9 BMSC cell line commonly used for facilitating the differentiation of HSCs. Using an ANP-receptor blocker, endogenously produced ANP was found to promote cell proliferation and prevent apoptosis. NO donor SNAP (S-nitroso-N-acetylpenicillamine) at low concentrations (10 and 50 μM), which would moderately stimulate PKG activity, protected these BMSCs against spontaneous apoptosis. YC-1, a soluble guanylyl cyclase (sGC) activator, decreased the levels of apoptosis, similar to the cytoprotective effects of low-level NO. ODQ (1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one), which blocks endogenous NO-induced activation of sGC and thus lowers endogenous cGMP/PKG activity, significantly elevated apoptotic levels by 2.5- and three-fold. Pre-incubation with 8-Bromo-cGMP or ANP, which bypass the ODQ block, almost completely prevented the ODQ-induced apoptosis. A highly-specific PKG inhibitor, DT-3, at 20, and 30 μM, caused 1.5- and two-fold increases in apoptosis, respectively. ODQ and DT-3 also decreased BMSCs proliferation and colony formation. Small Interfering RNA gene knockdown of PKG-Iα increased apoptosis and decreased proliferation in BMSCs. The data suggest that basal NO/cGMP/PKG-Iα activity and autocrine ANP/cGMP/PKG-Iα are necessary for preserving OP9 cell survival and promoting cell proliferation and migration.     

Quantitative automated microscopy (QuAM) elucidates growth factor specific signalling in pain sensitization

Background

Hydrogen sulphide (H₂S) is a gaseous neuro-mediator that exerts analgesic effects in rodent models of visceral pain by activating K_ATP channels. A body of evidence support the notion that K_ATP channels interact with endogenous opioids. Whether H₂S-induced analgesia involves opioid receptors is unknown.

Methods

The perception of painful sensation induced by colorectal distension (CRD) in conscious rats was measured by assessing the abdominal withdrawal reflex. The contribution of opioid receptors to H₂S-induced analgesia was investigated by administering rats with selective μ, κ and δ opioid receptor antagonists and antisenses. To investigate whether H₂S causes μ opioid receptor (MOR) transactivation, the neuronal like cells SKNMCs were challenged with H₂S in the presence of MOR agonist (DAMGO) or antagonist (CTAP). MOR activation and phosphorylation, its association to β arrestin and internalization were measured.

Results

H₂S exerted a potent analgesic effects on CRD-induced pain. H₂S-induced analgesia required the activation of the opioid system. By pharmacological and molecular analyses, a robust inhibition of H₂S-induced analgesia was observed in response to central administration of CTAP and MOR antisense, while κ and δ receptors were less involved. H₂S caused MOR transactivation and internalization in SKNMCs by a mechanism that required AKT phosphorylation. MOR transactivation was inhibited by LY294002, a PI3K inhibitor, and glibenclamide, a K_ATP channels blocker.

Conclusions

This study provides pharmacological and molecular evidence that antinociception exerted by H₂S in a rodent model of visceral pain is modulated by the transactivation of MOR. This observation provides support for development of new pharmacological approaches to visceral pain.     

Vasodilatation of sulfur dioxide derivatives and signal transduction

The vasodilatation of sulfur dioxide (SO₂) derivatives on the rat thoracic aortic rings and its cell signal transduction pathway were studied. The levels of cAMP, cGMP, PGI₂, TXA₂ and activities of PKA and adenylyl cyclase (AC) in the rings exposed to SO₂ derivatives were determined. The results indicated that SO₂ derivatives could dose-dependently relax the isolated rat aorta rings with or without endothelium precontracted by NE, no difference was found between the rings with and without endothelium; Levels of cAMP, PGI₂, AC activity and PKA activity in the aortic rings were significantly increased by the derivatives in a dose-related way; No change of cGMP and TXA₂ levels in rings was observed; cAMP/cGMP and PGI₂/TXA₂ ratios were increased significantly by the SO₂ derivatives. These results led to the conclusions that SO₂ derivatives might cause the endothelium-independent vasorelaxation effect, and the vasorelaxation was mediated in partly by the signal transduction pathway of PGI₂-AC-cAMP-PKA.     

Hydrogen sulphide triggers VEGF-induced intracellular Ca signals in human endothelial cells but not in their immature progenitors

Hydrogen sulphide (H₂S) is a newly discovered gasotransmitter that regulates multiple steps in VEGF-induced angiogenesis. An increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) is central to endothelial proliferation and may be triggered by both VEGF and H₂S. Albeit VEGFR-2 might serve as H₂S receptor, the mechanistic relationship between VEGF- and H₂S-induced Ca²⁺ signals in endothelial cells is unclear. The present study aimed at assessing whether and how NaHS, a widely employed H₂S donor, stimulates pro-angiogenic Ca²⁺ signals in Ea.hy926 cells, a suitable surrogate for mature endothelial cells, and human endothelial progenitor cells (EPCs). We found that NaHS induced a dose-dependent increase in [Ca²⁺]_i in Ea.hy926 cells. NaHS-induced Ca²⁺ signals in Ea.hy926 cells did not require extracellular Ca²⁺ entry, while they were inhibited upon pharmacological blockade of the phospholipase C/inositol-1,4,5-trisphosphate (InsP₃) signalling pathway. Moreover, the Ca²⁺ response to NaHS was prevented by genistein, but not by SU5416, which selectively inhibits VEGFR-2. However, VEGF-induced Ca²⁺ signals were suppressed by dl-propargylglycine (PAG), which blocks the H₂S-producing enzyme, cystathionine γ-lyase. Consistent with these data, VEGF-induced proliferation and migration were inhibited by PAG in Ea.hy926 cells, albeit NaHS alone did not influence these processes. Conversely, NaHS elevated [Ca²⁺]_i only in a modest fraction of circulating EPCs, whereas neither VEGF-induced Ca²⁺ oscillations nor VEGF-dependent proliferation were affected by PAG. Therefore, H₂S-evoked elevation in [Ca²⁺]_i is essential to trigger the pro-angiogenic Ca²⁺ response to VEGF in mature endothelial cells, but not in their immature progenitors.     

Cofilin phosphorylation is involved in nitric oxide/cGMP-mediated nociception

There is convincing evidence that nitric oxide (NO), cGMP and cGMP-dependent protein kinase I (PKG-I) are involved in the development of hyperalgesia in response to noxious stimuli. However, downstream target proteins contributing to nociception have not been completely identified so far. Several reports indicate a role of the NO/cGMP/PKG cascade in the regulation of neurite outgrowth which is suggested to be involved in specific mechanisms of nociception. Since neurite outgrowth is strongly dependent on modulation of cytoskeleton proteins we were interested in the impact of PKG-I activation on the actin cytoskeleton and its role in inflammatory hyperalgesia. Therefore we investigated the actin-destabilising protein cofilin and its NO-dependent effects in vitro in primary neuronal cultures as well as in vivo in the zymosan-induced paw inflammation model in rats. In primary neurons from rats, treatment with the PKG-I activator 8-Br-cGMP induced a time-dependent phosphorylation of cofilin and significantly increased neurite outgrowth. Further functional analysis revealed that the underlying signal transduction pathways involve activation of the Rho-GTPases RhoA, Rac1 and Cdc42 and their corresponding downstream targets Rho-kinase (ROCK) and p21-activated kinase (PAK). In vivo, treatment of rats with the NO-synthase inhibitor l-NAME and the ROCK-inhibitor Y-27632, respectively, led to a significant decrease of cofilin phosphorylation in the spinal cord and resulted in antinociceptive effects in a model of inflammatory hyperalgesia. Our results suggest that cofilin represents a downstream target of NO/cGMP/PKG signal transduction in neurons thus indicating that it is involved in NO-mediated nociception.     

Vasoconstrictive effect of hydrogen sulfide involves downregulation of cAMP in vascular smooth muscle cells

Hydrogen sulfide (H(2)S), a new endogenous mediator, produces both vasorelaxation and vasoconstriction. This study was designed to examine whether cAMP mediates the vasoconstrictive effect of H(2)S. We found that NaHS at a concentration range of 10-100 microM (yields approximately 3-30 microM H(2)S) concentration-dependently reversed the vasodilation caused by isoprenaline and salbutamol, two beta-adrenoceptor agonists, and forskolin, a selective adenylyl cyclase activator, in phenylephrine-precontracted rat aortic rings. Pretreatment with NaHS (10-100 microM) for 5 min also significantly attenuated the vasorelaxant effect of salbutamol and forskolin. More importantly, NaHS (5-100 microM) significantly reversed forskolin-induced cAMP accumulation in vascular smooth muscle cells. However, NaHS produced significant, but weaker, vasoconstriction in the presence of N(G)-nitro-l-arginine methyl ester (100 microM), a nitric oxide synthase inhibitor, or in endothelium-denuded aortic rings. Blockade of ATP-sensitive potassium channels with glibenclamide (10 microM) failed to attenuate the vasoconstriction induced by H(2)S. Taken together, we demonstrated for the first time that the vasoconstrictive effect of H(2)S involves the adenyly cyclase/cAMP pathway.     

TRPA1 Has a Key Role in the Somatic Pro-Nociceptive Actions of Hydrogen Sulfide

Hydrogen sulfide (H₂S), which is produced endogenously from L-cysteine, is an irritant with pro-nociceptive actions. We have used measurements of intracellular calcium concentration, electrophysiology and behavioral measurements to show that the somatic pronociceptive actions of H₂S require TRPA1. A H₂S donor, NaHS, activated TRPA1 expressed in CHO cells and stimulated DRG neurons isolated from Trpa1^+/+ but not Trpa1^−/− mice. TRPA1 activation by NaHS was pH dependent with increased activity at acidic pH. The midpoint of the relationship between NaHS EC₅₀ values and external pH was pH 7.21, close to the expected dissociation constant for H₂S (pK_a 7.04). NaHS evoked single channel currents in inside-out and cell-attached membrane patches consistent with an intracellular site of action. In behavioral experiments, intraplantar administration of NaHS and L-cysteine evoked mechanical and cold hypersensitivities in Trpa1^+/+ but not in Trpa1^−/− mice. The sensitizing effects of L-cysteine in wild-type mice were inhibited by a cystathionine β-synthase inhibitor, D,L-propargylglycine (PAG), which inhibits H₂S formation. Mechanical hypersensitivity evoked by intraplantar injections of LPS was prevented by PAG and the TRPA1 antagonist AP-18 and was absent in Trpa1^−/− mice, indicating that H₂S mediated stimulation of TRPA1 is necessary for the local pronociceptive effects of LPS. The pro-nociceptive effects of intraplantar NaHS were retained in Trpv1^−/− mice ruling out TRPV1 as a molecular target. In behavioral studies, NaHS mediated sensitization was also inhibited by a T-type calcium channel inhibitor, mibefradil. In contrast to the effects of NaHS on somatic sensitivity, intracolonic NaHS administration evoked similar nociceptive effects in Trpa1^+/+ and Trpa1^−/− mice, suggesting that the visceral pro-nociceptive effects of H₂S are independent of TRPA1. In electrophysiological studies, the depolarizing actions of H₂S on isolated DRG neurons were inhibited by AP-18, but not by mibefradil indicating that the primary excitatory effect of H₂S on DRG neurons is TRPA1 mediated depolarization.     

Inhibition of cyclic guanosine 5'-monophosphate-dependent protein kinase I (PKG-I) in lumbar spinal cord reduces formalin-induced hyperalgesia and PKG upregulation.

Nitric oxide-mediated nociception has been suggested to involve formation of cyclic guanosine 5'-monophosphate (cGMP) and activation of cGMP-dependent protein kinase (PKG). To further evaluate this pathway we assessed the effects of the PKG-inhibiting cGMP analog Rp-8-Br-cGMPS in the rat formalin assay and analyzed the regulation of PKG expression in rat lumbar spinal cord. Spinally delivered Rp-8-Br-cGMPS (0.1-0.5 micro mol i.t.) reduced the nociceptive behavior in a dose-dependent manner. Similar effects were achieved with Rp-8-Br-PET-cGMPS (0.5 micro mol i.t.), another PKG-inhibitory cGMP analog. In contrast, Rp-8-Br-cAMPS (0.5 micro mol i.t.), an inhibitor of protein kinase A, had no effect in this model. Formalin treatment resulted in a rapid (within 1h), long-lasting (up to 96h) upregulation of PKG-I protein expression. This increase was prevented in animals pretreated with Rp-8-Br-cGMPS (0.5 micro mol i.t.) or morphine (2.5-5mg/kg i.p.) 10min prior to formalin injection. Spinal delivery of 8-Br-cGMP, a PKG-activating cGMP analog, without subsequent formalin treatment also caused an increase of PKG-I protein expression. Hence, the upregulation of PKG-I might possibly be mediated by cGMP itself. Our data suggest that PKG-I activation is involved in the synaptic transmission of nociceptive stimuli in the spinal cord and that PKG-I inhibitors might be interesting novel drugs for pain treatment.     

Actions of Hydrogen Sulfide and ATP-Sensitive Potassium Channels on Colonic Hypermotility in a Rat Model of Chronic Stress

Objective

To investigate the potential role of hydrogen sulphide (H₂S) and ATP-sensitive potassium (K_ATP) channels in chronic stress-induced colonic hypermotility.

Methods

Male Wistar rats were submitted daily to 1 h of water avoidance stress (WAS) or sham WAS (SWAS) for 10 consecutive days. Organ bath recordings, H₂S production, immunohistochemistry and western blotting were performed on rat colonic samples to investigate the role of endogenous H₂S in repeated WAS-induced hypermotility. Organ bath recordings and western blotting were used to detect the role of K_ATP channels in repeated WAS.

Results

Repeated WAS increased the number of fecal pellets per hour and the area under the curve of the spontaneous contractions of colonic strips, and decreased the endogenous production of H₂S and the expression of H₂S-producing enzymes in the colon devoid of mucosa and submucosa. Inhibitors of H₂S-producing enzymes increased the contractile activity of colonic strips in the SWAS rats. NaHS concentration-dependently inhibited the spontaneous contractions of the strips and the NaHS IC₅₀ for the WAS rats was significantly lower than that for the SWAS rats. The inhibitory effect of NaHS was significantly reduced by glybenclamide. Repeated WAS treatment resulted in up-regulation of Kir6.1 and SUR2B of K_ATP channels in the colon devoid of mucosa and submucosa.

Conclusion

The colonic hypermotility induced by repeated WAS may be associated with the decreased production of endogenous H₂S. The increased expression of the subunits of K_ATP channels in colonic smooth muscle cells may be a defensive response to repeated WAS. H₂S donor may have potential clinical utility in treating chronic stress- induced colonic hypermotility.     

Cadmium-Induced Hydrogen Sulfide Synthesis Is Involved in Cadmium Tolerance in Medicago sativa by Reestablishment of Reduced (Homo)glutathione and Reactive Oxygen Species Homeostases

Until now, physiological mechanisms and downstream targets responsible for the cadmium (Cd) tolerance mediated by endogenous hydrogen sulfide (H₂S) have been elusive. To address this gap, a combination of pharmacological, histochemical, biochemical and molecular approaches was applied. The perturbation of reduced (homo)glutathione homeostasis and increased H₂S production as well as the activation of two H₂S-synthetic enzymes activities, including _L-cysteine desulfhydrase (LCD) and _D-cysteine desulfhydrase (DCD), in alfalfa seedling roots were early responses to the exposure of Cd. The application of H₂S donor sodium hydrosulfide (NaHS), not only mimicked intracellular H₂S production triggered by Cd, but also alleviated Cd toxicity in a H₂S-dependent fashion. By contrast, the inhibition of H₂S production caused by the application of its synthetic inhibitor blocked NaHS-induced Cd tolerance, and destroyed reduced (homo)glutathione and reactive oxygen species (ROS) homeostases. Above mentioned inhibitory responses were further rescued by exogenously applied glutathione (GSH). Meanwhile, NaHS responses were sensitive to a (homo)glutathione synthetic inhibitor, but reversed by the cotreatment with GSH. The possible involvement of cyclic AMP (cAMP) signaling in NaHS responses was also suggested. In summary, LCD/DCD-mediated H₂S might be an important signaling molecule in the enhancement of Cd toxicity in alfalfa seedlings mainly by governing reduced (homo)glutathione and ROS homeostases.     

A New Slow Releasing,H2S Generating Compound,GYY4137 Relaxes Spontaneous and Oxytocin-Stimulated Contractions of Human and Rat Pregnant Myometrium

Better tocolytics are required to help prevent preterm labour. The gaseotransmitter Hydrogen sulphide (H₂S) has been shown to reduce myometrial contractility and thus is of potential interest. However previous studies used NaHS, which is toxic and releases H₂S as a non-physiological bolus and thus alternative H₂S donors are sought. GYY4137 has been developed to slowly release H₂S and hence better reflect endogenous physiological release. We have examined its effects on spontaneous and oxytocin-stimulated contractility and compared them to NaHS, in human and rat myometrium, throughout gestation. The effects on contractility in response to GYY4137 (1 nM–1 mM) and NaHS (1 mM) were examined on myometrial strips from, biopsies of women undergoing elective caesarean section or hysterectomy, and from non-pregnant, 14, 18, 22 day (term) gestation or labouring rats. In pregnant rat and human myometrium dose-dependent and significant decreases in spontaneous contractions were seen with increasing concentrations of GYY4137, which also reduced underlying Ca transients. GYY4137 and NaHS significantly reduced oxytocin-stimulated and high-K depolarised contractions as well as spontaneous activity. Their inhibitory effects increased as gestation advanced, but were abruptly reversed in labour. Glibenclamide, an inhibitor of ATP-sensitive potassium (K_ATP) channels, abolished the inhibitory effect of GYY4137. These data suggest (i) H₂S contributes to uterine quiescence from mid-gestation until labor, (ii) that H₂S affects L-type calcium channels and K_ATP channels reducing Ca entry and thereby myometrial contractions, (iii) add to the evidence that H₂S plays a physiological role in relaxing myometrium, and thus (iv) H₂S is an attractive target for therapeutic manipulation of human myometrial contractility.     

Peptide blockers of PKG inhibit ROS generation by acetylcholine and bradykinin in cardiomyocytes but fail to block protection in the whole heart

Bradykinin and acetylcholine (ACh) trigger preconditioning by ATP-sensitive K(+) (K(ATP)) channel-dependent production of reactive oxygen species (ROS). Recent evidence suggests that ROS production may in turn be influenced by cGMP-dependent protein kinase (PKG). This study utilized DT-2 and DT-3 peptides, highly specific membrane-permeable blockers of PKG. Rabbit cardiomyocytes were incubated for 15 min in reduced MitoTracker red, which becomes fluorescent only after exposure to ROS. Bradykinin (400 nM) and ACh (250 microM) caused a 49.9 +/- 5.9% and 46.8 +/- 1.7% increase in ROS production, respectively (P < 0.005 vs. untreated cells). Coincubation with DT-3 (250 nM) abolished both the ACh- and bradykinin-induced ROS signal, whereas a nonpermeable form of the peptide (W45) had no effect on ACh-induced ROS production. DT-3 was unable to block ROS production from diazoxide (100 microM), a selective opener of mitochondrial K(ATP) channels, suggesting that these channels are downstream of PKG. DT-2 (125 nM) also prevented ACh from triggering ROS production. 8-(4-Chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (100 microM), a cGMP analog and potent direct activator of PKG, increased ROS production of cardiomyocytes by 44.7 +/- 7.1% (P < 0.001 vs. untreated cells). This increase was blocked by DT-2. Neither DT-2 nor DT-3 could block the anti-infarct effect of bradykinin in isolated rabbit hearts. Studies with fluorescent-tagged DT-3 revealed that it was confined to endothelial cells and never reached the myocytes. We conclude that both bradykinin and ACh trigger ROS generation by a pathway that includes PKG. Although the peptides may be inappropriate for a whole heart model, they are likely to become important tool drugs for elucidation of signal transduction pathways in cell preparations.