Protein Kinase G1α Research Articles

Regulation of vascular tone and blood pressure by singlet molecular oxygen in inflammation.

The principle aim of this review is to prompt vascular researchers interested in vascular inflammation and oxidative stress to consider singlet molecular oxygen (1O2) as a potentially relevant contributor. A secondary goal is to propose novel treatment strategies to address haemodynamic complications associated with septic shock. Increased inflammation and oxidative stress are hallmarks of a range of vascular diseases. We recently showed that in systemic inflammation and oxidative stress associated with models of inflammation including sepsis, the tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase-1 (Ido1) contributes to hypotension and decreased blood pressure through production of singlet molecular oxygen (1O2). Once formed, 1O2 converts tryptophan bound to Ido1 to a vasoactive hydroperoxide which decreases arterial tone and blood pressure via oxidation of a specific cysteine residue of protein kinase G1α. These works show, for the first time, that 1O2 contributes to arterial redox signalling and that Ido1 contributes to the regulation of blood pressure through production of a novel tryptophan-derived hydroperoxide, thus presenting a new signalling pathway as novel target in the treatment of blood pressure disorders such as sepsis.

Singlet molecular oxygen regulates vascular tone and blood pressure in inflammation.

Singlet molecular oxygen (1O2) has well-established roles in photosynthetic plants, bacteria and fungi1-3, but not in mammals. Chemically generated 1O2 oxidizes the amino acid tryptophan to precursors of a key metabolite called N-formylkynurenine4, whereas enzymatic oxidation of tryptophan to N-formylkynurenine is catalysed by a family of dioxygenases, including indoleamine 2,3-dioxygenase 15. Under inflammatory conditions, this haem-containing enzyme is expressed in arterial endothelial cells, where it contributes to the regulation of blood pressure6. However, whether indoleamine 2,3-dioxygenase 1 forms 1O2 and whether this contributes to blood pressure control have remained unknown. Here we show that arterial indoleamine 2,3-dioxygenase 1 regulates blood pressure via formation of 1O2. We observed that in the presence of hydrogen peroxide, the enzyme generates 1O2 and that this is associated with the stereoselective oxidation of L-tryptophan to a tricyclic hydroperoxide via a previously unrecognized oxidative activation of the dioxygenase activity. The tryptophan-derived hydroperoxide acts in vivo as a signalling molecule, inducing arterial relaxation and decreasing blood pressure; this activity is dependent on Cys42 of protein kinase G1α. Our findings demonstrate a pathophysiological role for 1O2 in mammals through formation of an aminoacid-derived hydroperoxide that regulates vascular tone and blood pressure under inflammatory conditions.

Synthesis and Characterization of 8-Nitroguanosine 3',5'-Cyclic Monophosphorothioate Rp-Isomer as a Potent Inhibitor of Protein Kinase G1α.

Guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinases (PKG) are kinases regulating diverse physiological functions including vascular smooth muscle relaxation, neuronal synaptic plasticity, and platelet activities. Certain PKG inhibitors, such as Rp-diastereomers of derivatives of guanosine 3',5'-cyclic monophosphorothioate (Rp-cGMPS), have been designed and used to study PKG-regulated cell signaling. 8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is an endogenous cGMP derivative formed as a result of excess production of reactive oxygen species and nitric oxide. 8-Nitro-cGMP causes persistent activation of PKG1α through covalent attachment of cGMP moieties to cysteine residues of the enzyme (i.e., the process called protein S-guanylation). In this study, we synthesized a nitrated analogue of Rp-cGMPS, 8-nitroguanosine 3',5'-cyclic monophosphorothioate Rp-isomer (Rp-8-nitro-cGMPS), and investigated its effects on PKG1α activity. We synthesized Rp-8-nitro-cGMPS by reacting Rp-8-bromoguanosine 3',5'-cyclic monophosphorothioate (Rp-8-bromo-cGMPS) with sodium nitrite. Rp-8-Nitro-cGMPS reacted with the thiol compounds cysteine and glutathione to form Rp-8-thioalkoxy-cGMPS adducts to a similar extent as did 8-nitro-cGMP. As an important finding, a protein S-guanylation-like modification was clearly observed, by using Western blotting, in the reaction between recombinant PKG1α and Rp-8-nitro-cGMPS. Rp-8-Nitro-cGMPS inhibited PKG1α activity with an inhibitory constant of 22 µM in a competitive manner. An organ bath assay with mouse aorta demonstrated that Rp-8-nitro-cGMPS inhibited vascular relaxation induced by acetylcholine or 8-bromo-cGMP more than Rp-8-bromo-cGMPS did. These findings suggest that Rp-8-nitro-cGMPS inhibits PKG through induction of an S-guanylation-like modification by attaching the Rp-cGMPS moiety to the enzyme. Additional study is warranted to explore the potential application of Rp-8-nitro-cGMPS to biochemical and therapeutic research involving PKG1α activation.

81 - Regulation of Vascular Tone and Blood Pressure by a Tryptophan-Derived Tricyclic Hydroperoxide

Vascular inflammation is associated with increased arterial O2●–/H2O2 that affect vascular tone and disease. We reported previously that in inflammation, vascular endothelial cells express indoleamine 2,3-dioxygenase 1 (IDO1, a heme-containing enzyme that oxidizes Trp to N-formyl-kynurenine) and that IDO1-mediated metabolism of Trp regulates vascular tone and blood pressure (Nat Med 16:279). Here we show that in the presence of H2O2 purified IDO1 forms singlet oxygen (1O2), as assessed by the conversion of specific probes to their endoperoxides, and light emission at 1270 nm. 1O2 formed by isolated IDO1/H2O2 stereospecifically converts Trp to a tricyclic hydroperoxide (cis-WOOH) that decays to N-formyl-kynurenine. Formation of cis-WOOH is via an oxidative dioxygenase reaction, distinct from the known reductive dioxygenase and peroxidase activities of IDO1. In vitro, cis-WOOH but not trans-WOOH oxidizes protein kinase G1α (PKG1α) to a dimer in a reaction dependent on Cys42. Similarly, exposure of endothelium-denuded arteries to cis-WOOH dimerizes PKG1α. Such cis-WOOH-induced PKG1α dimerization is associated with arterial relaxation, whereas arteries from “redox dead” PKG1α-C42S knock-in mice are refractory to relaxation by cis-WOOH. Moreover, Trp-induced relaxation of IDO1-expressing and endothelium-intact arteries depends on PKG1α-Cys42 as corresponding segments from PKG1α-C42S knock-in mice do not relax, and it is attenuated by pretreatment with PEG-catalase. Together, our data indicate that in ‘inflamed arteries’, endothelial IDO1/H2O2 generate light (i.e., 1O2) in a dark reaction. This results in formation of a tricyclic Trp-derived hydroperoxide that signals relaxation via oxidative activation of PKG1α in vascular smooth muscle cells.

20-HETE-induced mitochondrial superoxide production and inflammatory phenotype in vascular smooth muscle is prevented by glucose-6-phosphate dehydrogenase inhibition.

20-Hydroxyeicosatetraeonic acid (20-HETE) produced by cytochrome P-450 monooxygenases in NADPH-dependent manner is proinflammatory, and it contributes to the pathogenesis of systemic and pulmonary hypertension. In this study, we tested the hypothesis that inhibition of glucose-6-phosphate dehydrogenase (G6PD), a major source of NADPH in the cell, prevents 20-HETE synthesis and 20-HETE-induced proinflammatory signaling that promotes secretory phenotype of vascular smooth muscle cells. Lipidomic analysis indicated that G6PD inhibition and knockdown decreased 20-HETE levels in pulmonary arteries as well as 20-HETE-induced 1) mitochondrial superoxide production, 2) activation of mitogen-activated protein kinase 1 and 3, 3) phosphorylation of ETS domain-containing protein Elk-1 that activate transcription of tumor necrosis factor-α gene (Tnfa), and 4) expression of tumor necrosis factor-α (TNF-α). Moreover, inhibition of G6PD increased protein kinase G1α activity, which, at least partially, mitigated superoxide production and Elk-1 and TNF-α expression. Additionally, we report here for the first time that 20-HETE repressed miR-143, which suppresses Elk-1 expression, and miR-133a, which is known to suppress synthetic/secretory phenotype of vascular smooth muscle cells. In summary, our findings indicate that 20-HETE elicited mitochondrial superoxide production and promoted secretory phenotype of vascular smooth muscle cells by activating MAPK1-Elk-1, all of which are blocked by inhibition of G6PD.

Hyperglycemia down-regulates cGMP-dependent protein kinase I expression in first trimester cytotrophoblast cells.

Diabetes in pregnancy is associated with microvascular complications and a higher incidence of preeclampsia. The regulatory signaling pathways involving nitric oxide, cGMP, and cGMP-dependent protein kinase (PKG) have been shown to be down-regulated under diabetic conditions and contribute to the pathogenesis of vascular complications in diabetes. The present study was undertaken to investigate how high glucose concentrations regulate PKG expression in cytotrophoblast cells (CTBs). Human CTBs (Sw. 71) were treated with 45, 135, 225, 495, or 945 mg/dL glucose for 48 h. Some cells were pretreated with a p38 inhibitor (10 μM SB203580) or 10 μM rosiglitazone. After treatment, the cell lysates were subjected to measure the expression of protein kinase G1α (PKG1α), protein kinase G1β (PKG1β), soluble guanylate cyclase 1α (sGC1α), and soluble guanylate cyclase 1 β (sGC1β) by Western blot. Statistical comparisons were performed using analysis of variance with Duncan's post hoc test. The expressions of PKG1α, PKG1β, sGC1α, and sGC1β were significantly down-regulated (p < 0.05) in CTBs treated with >135 mg/dL glucose compared to basal (45 mg/dL). The hyperglycemia-induced down-regulation of cGMP and cGMP-dependent PKG were attenuated by the SB203580 or rosiglitazone pretreatment. Exposure of CTBs to excess glucose down-regulates cGMP and cGMP-dependent PKG, contributing to the development of vascular complications in diabetic mothers during pregnancy. The attenuation of hyperglycemia-induced down-regulation of PKG proteins by SB203580 or rosiglitazone pretreatment further suggests the involvement of stress signaling mechanisms in this process.

The stimulating effects of nitric oxide on intermediate conductance Ca²⁺-activated K⁺ channels in human dermal fibroblasts through PKG pathways but not the PKA pathways.

Nitric oxide (NO) is produced by nitric oxide synthase (NOS) in dermal fibroblasts and is important during wound healing. Intermediate conductance Ca²⁺-activated K+ (IK; IK1; KCa3.1; IKCa; SK4; KCNN4) channels contribute to NOS upregulation, NO production, and various NO-mediated essential functions in many kinds of cells. To determine if the action of NO is linked to IK channel regulation in human dermal fibroblasts, we investigated the expression of IK channels in the cells and the effects and mechanisms of NO on the channels using RT-PCR, western blot analysis, immunocytochemistry and whole-cell and single-channel patch-clamp techniques. The presence of functional IK channels at the RNA, protein and membrane levels was demonstrated and S-nitroso-N-acetylpenicillamine (SNAP) was shown to significantly increase IK currents. The effects of NO were abolished by pretreatment with KT5823 or 1H-[1,2,4]-oxadiazolo [4,3-a]quinoxalin-1-one (ODQ) but not with KT5720. In addition, IK currents were increased by protein kinase G1α or 8-bromo-cGMP but not by forskolin, 8-bromo-cAMP, or catalytic subunits of protein kinase A (PKAcs). On the other hand, PKAcs with cGMP did not increase IK currents, and pretreatment with KT5720 did not block the stimulating effects of 8-Br-cGMP on the IK channels. These data suggest that NO activates IK channels through the PKG but not the PKA pathways, and it seems there is no cross activation between PKG and PKA pathways in human dermal fibroblasts.

Dehydroepiandrosterone promotes pulmonary artery relaxation by NADPH oxidation‐elicited subunit dimerization of protein kinase G1α (1175.9)

The activity of glucose‐6‐phosphate dehydrogenase (G6PD) controls a vascular smooth muscle relaxing mechanism promoted by the oxidation of cytosolic NADPH, which has been associated with activation of the 1α form of protein kinase G (PKG1α) by a thiol oxidation‐elicited subunit dimerization. This PKG1α‐activation mechanism appears to contribute to responses of isolated endothelium‐removed bovine pulmonary arteries (BPA) elicited by peroxide, cytosolic NADPH oxidation resulting from G6PD inhibition and hypoxia. Dehydroepiandrosterone (DHEA) is a steroid hormone with pulmonary vasodilator activity, which has beneficial effects in treating pulmonary hypertension. Since one of the known actions of DHEA is inhibiting G6PD, we investigated if it promoted relaxation associated with NADPH oxidation, PKG1α dimerization and PKG activation detected by increased vasodilator‐stimulated phosphoprotein (VASP) phosphorylation. Relaxation of BPA to DHEA under aerobic or hypoxic conditions was associated with NADPH oxidation, PKG1α dimerization and increased VASP phosphorylation. The vasodilator activity of DHEA was markedly attenuated in pulmonary arteries and aorta from a PKG knockin mouse containing a serine in place of a cysteine involved in PKG dimerization. DHEA promoted increased PKG dimerization in lungs from wild type mice, which was not detected in the PKG knockin mouse model. Thus, PKG1α dimerization is a major contributing factor to the vasodilator actions of DHEA, and perhaps its beneficial effects in treating pulmonary hypertension.Grant Funding Source: Supported by NIH Grant R01HL115124

Hyperglycemia down‐regulates the cgmp‐dependent protein kinase i expression in cytotrophoblasts (LB241)

Objective: The signaling pathway of nitric oxide (NO), cGMP, and cGMP‐dependent protein kinase (PKG) down‐regulates under diabetic conditions. High glucose exposure reduces PKG activity in smooth muscle cells; however, there is no data in cytotrophoblasts (CTBs). The present study evaluates the effect of high glucose on PKG‐I expression in CTBs. Study Design: Human CTBs (Sw. 71) were treated with 100, 150, 200, 300 or 400 mg/dL glucose for 72h. Some cells were pretreated with a p38 inhibitor (10 μM SB203580). After the treatment, the expression of protein kinase G1α (PKG1α), protein kinase G1β (PKG1β), soluble guanylate cyclase 1α (sGC1α), and soluble guanylate cyclase 1 β (sGC1β) were measured in the cell lysates by western blot analysis. The p38 MAPK phosphorylation was evaluated by western blot. Statistical comparisons were performed using analysis of variance with Duncan’s post hoc test. Results: The expression of PKG1α, PKG1β, sGC1α, and sGC1β were significantly down‐regulated (p&lt;0.05) in CTBs treated with 蠅150 mg/dL glucose compared to basal (100 mg/dL) that were attenuated by SB203580. The p38 MAPK phosphorylation was up‐regulated (p&lt;0.05) in excess glucose‐treated CTBs. Conclusions: Exposure of CTBs to excess glucose down‐regulates the PKG. The attenuation of hyperglycemia‐induced down‐regulation of PKG proteins by SB203580 pretreatment suggests the involvement of stress signaling in this process.Grant Funding Source: Mohammad Uddin

Roles for cytosolic NADPH redox in regulating pulmonary artery relaxation by thiol oxidation-elicited subunit dimerization of protein kinase G1α

The activity of glucose-6-phosphate dehydrogenase (G6PD) appears to control a vascular smooth muscle relaxing mechanism regulated through cytosolic NADPH oxidation. Since our recent studies suggest that thiol oxidation-elicited dimerization of the 1α form of protein kinase G (PKG1α) contributes to the relaxation of isolated endothelium-removed bovine pulmonary arteries (BPA) to peroxide and responses to hypoxia, we investigated whether cytosolic NADPH oxidation promoted relaxation by PKG1α dimerization. Relaxation of BPA to G6PD inhibitors 6-aminonicotinamide (6-AN) and epiandrosterone (studied under hypoxia to minimize basal levels of NADPH oxidation and PKG1α dimerization) was associated with increased PKG1α dimerization and PKG-mediated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Depletion of PKG1α by small inhibitory RNA (siRNA) inhibited relaxation of BPA to 6-AN and attenuated the increase in VASP phosphorylation. Relaxation to 6-AN did not appear to be altered by depletion of soluble guanylate cyclase (sGC). Depletion of G6PD, thioredoxin-1 (Trx-1), and Trx reductase-1 (TrxR-1) in BPA with siRNA increased PKG1α dimerization and VASP phosphorylation and inhibited force generation under aerobic and hypoxic conditions. Depletion of TrxR-1 with siRNA inhibited the effects of 6-AN and enhanced similar responses to peroxide. Peroxiredoxin-1 depletion by siRNA inhibited PKG dimerization to peroxide, but it did not alter PKG dimerization under hypoxia or the stimulation of dimerization by 6-AN. Thus regulation of cytosolic NADPH redox by G6PD appears to control PKG1α dimerization in BPA through its influence on Trx-1 redox regulation by the NADPH dependence of TrxR-1. NADPH regulation of PKG dimerization may contribute to vascular responses to hypoxia that are associated with changes in NADPH redox.

EXOGENOUS NITRIC OXIDE (NO) MODULATES THE G‐PROTEIN COUPLED SIGNALING PROTEINS IN CULTURED LYMPHATIC SMOOTH MUSCLE CELLS

Lymphatics use both phasic and tonic contractions of lymphatic muscle. The cellular and molecular mechanisms by which lymphatic muscle performs these dual tasks are still unknown. Recently, we demonstrated that cGMP and PKG (protein kinase G) are involved in the NO modulation of rat thoracic duct contractility. We aim to study the NO dependent signaling mechanisms in cultured lymphatic muscle cells. Rat mesenteric lymphatic muscle cells (RMLMC) were used in this study. Cells were maintained in DMEM media supplemented with 10% fetal bovine serum and were incubated at 37°C and 5% CO2. Cells were treated with two NO donors SNAP (S‐Nitroso‐N‐Acetyl‐D,L‐Penicillamine) and DETA NONOate (Diethylenetriamine NONOate) at 10, 25, 50, 100 or 200 μM for 4 and 8 hours in different wells (n=6 in each treatment) separately. The sGC (soluble guanylate cyclase) isoforms α and β, PKG 1α, and PKG 1β were measured by Western blotting using anti‐sGC α, anti‐sGC β, anti‐PKG 1α, or anti‐PKG 1β antibodies. The expression of sGC α, sGC β, PKG 1α, and PKG 1β proteins were significantly upregulated in 25–200 μM SNAP and DETA NONOate treated RMLMC. Both dose and time dependent effects of SNAP and DETA NONOate on these proteins were observed. These findings demonstrate the usefulness of using cultured lymphatic muscle cell lines in the study of the involvement of sGC and PKG on NO dependent signaling. Supported by NIH HL70308

Mechanisms for Enhanced Endothelium-Derived Hyperpolarizing Factor-Mediated Responses in Microvessels in Mice

Endothelium-derived relaxing factors play an important role in cardiovascular homeostasis. Among them, endothelium-derived hyperpolarizing factor (EDHF) is important especially in microcirculation. It has previously been demonstrated that endothelium-derived hydrogen peroxide (H(2)O(2)) is an EDHF in animals and humans and that endothelial nitric oxide synthase (eNOS) plays diverse roles as a nitric oxide (NO) generating system in conduit arteries and as an EDHF/H(2)O(2) generating system in microvessels. As compared with NO-mediated responses, those by EDHF are resistant to atherosclerosis, contributing to the maintenance of cardiovascular homeostasis. The aim of this study is to elucidate the molecular mechanisms for enhanced EDHF-mediated responses in microvessels. This study used male wild-type mice and caveolin-1-deficient mice (caveolin-1(-/-) mice). In the endothelium, eNOS was functionally suppressed in mesenteric arteries (microvessels) compared with the aorta (conduit arteries), for which Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ) and caveolin-1 are involved, as EDHF-mediated responses were inhibited by STO-609 (an inhibitor of CaMKKβ) and in caveolin-1(-/-) mice, respectively. In vascular smooth muscle, relaxation responses to H(2)O(2) were enhanced through a protein kinase G1α (PKG1α)-mediated mechanism in mesenteric arteries compared with the aorta, as they were inhibited by Rp-8-Br-cGMPS (an inhibitor of PKG1α). These results indicate that CaMKKβ, caveolin-1, and PKG1α are substantially involved in the mechanisms for the enhanced EDHF-mediated responses in microvessels in mice.

Pregnancy modifies the large conductance Ca2+-activated K+ channel and cGMP-dependent signaling pathway in uterine vascular smooth muscle

Regulation of uteroplacental blood flow (UPBF) during pregnancy remains unclear. Large conductance, Ca(2+)-activated K(+) channels (BK(Ca)), consisting of alpha- and regulatory beta-subunits, are expressed in uterine vascular smooth muscle (UVSM) and contribute to the maintenance of UPBF in the last third of ovine pregnancy, but their expression pattern and activation pathways are unclear. We examined BK(Ca) subunit expression, the cGMP-dependent signaling pathway, and the functional role of BK(Ca) in uterine arteries (UA) from nonpregnant (n = 7), pregnant (n = 38; 56-145 days gestation; term, approximately 150 days), and postpartum (n = 15; 2-56 days) sheep. The alpha-subunit protein switched from 83-87 and 105 kDa forms in nonpregnant UVSM to 100 kDa throughout pregnancy, reversal occurring >30 days postpartum. The 39-kDa beta(1)-subunit was the primary regulatory subunit. Levels of 100-kDa alpha-subunit rose approximately 70% during placentation (P < 0.05) and were unchanged in the last two-thirds of pregnancy; in contrast, beta(1)-protein rose throughout pregnancy (R(2) = 0.996; P < 0.001; n = 13), increasing 50% during placentation and approximately twofold in the remainder of gestation. Although UVSM soluble guanylyl cyclase was unchanged, cGMP and protein kinase G(1alpha) increased (P < 0.02), paralleling the rise and fall in beta(1)-protein during pregnancy and the puerperium. BK(Ca) inhibition not only decreased UA nitric oxide (NO)-induced relaxation but also enhanced alpha-agonist-induced vasoconstriction. UVSM BK(Ca) modify relaxation-contraction responses in the last two-thirds of ovine pregnancy, and this is associated with alterations in alpha-subunit composition, alpha:beta(1)-subunit stoichiometry, and upregulation of the cGMP-dependent pathway, suggesting that BK(Ca) activation via NO-cGMP and beta(1) augmentation may contribute to the regulation of UPBF.