PKGIα Activation Research Articles

Naringenin alleviates the excessive lipid deposition of polycystic ovary syndrome rats and insulin-resistant adipocytes by promoting PKGIα.

Naringenin (NGEN) has anti-inflammatory and anti-diabetic effects. On this basis, this study aims to determine whether NGEN affects insulin resistance (IR) in polycystic ovary syndrome (PCOS). CCK-8 assay and oil red O staining were used to detect the cytotoxicity of NGEN and lipid production in cells or tissues, respectively. The differentiated mature SW872 cells were treated with palmitic acid (PA) to mimic IR cell model. Through detecting glucose consumption, the changes of inflammation and glycolipid metabolism can be observed with the assessment on expression levels of the inflammatory factors as well as lipid synthesis- (ACC, SREBP1c, PPARγ), glucose metabolism- and thermogenesis (ATGL, GLUT4, UCP1)-related genes. Insulin sensitivity was determined by changes in glucose consumption and PKGIα pathway. PKGIα was silenced to verify the protective mechanism of NGEN. PCOS rat model was constructed to confirm the results of cell experiments in vivo. NGEN generated no effect on SW872 cell viability. SW872 cells were differentiated and mature, as evidenced by lipid droplet formation, lipid synthesis gene activation, sugar metabolism and inhibition of thermogenesis-related genes. PA induction promoted lipid synthesis in mature adipocytes, and inhibited glucose metabolism and cell insulin sensitivity. NGEN pretreatment effectively alleviated the above-mentioned abnormalities. The protective mechanism of NGEN was achieved through promoting PKGIα activation. NGEN also mitigated the abnormal glucose and lipid metabolism in PCOS rats. NGEN inhibits the expression of PKGIα to alleviate IR that occurs in PCOS.

PKGIα is activated by metal-dependent oxidation in vitro but not in intact cells

Type I cGMP-dependent protein kinases (PKGIs) are important components of various signaling pathways and are canonically activated by nitric oxide– and natriuretic peptide–induced cGMP generation. However, some reports have shown that PKGIα can also be activated in vitro by oxidizing agents. Using in vitro kinase assays, here, we found that purified PKGIα stored in PBS with Flag peptide became oxidized and activated even in the absence of oxidizing agent; furthermore, once established, this activation could not be reversed by reduction with DTT. We demonstrate that activation was enhanced by addition of Cu2+ before storage, indicating it was driven by oxidation and mediated by trace metals present during storage. Previous reports suggested that PKGIα Cys43, Cys118, and Cys196 play key roles in oxidation-induced kinase activation; we show that activation was reduced by C118A or C196V mutations, although C43S PKGIα activation was not reduced. In contrast, under the same conditions, purified PKGIβ activity only slightly increased with storage. Using PKGIα/PKGIβ chimeras, we found that residues throughout the PKGIα-specific autoinhibitory loop were responsible for this activation. To explore whether oxidants activate PKGIα in H9c2 and C2C12 cells, we monitored vasodilator-stimulated phosphoprotein phosphorylation downstream of PKGIα. While we observed PKGIα Cys43 crosslinking in response to H2O2 (indicating an oxidizing environment in the cells), we were unable to detect increased vasodilator-stimulated phosphoprotein phosphorylation under these conditions. Taken together, we conclude that while PKGIα can be readily activated by oxidation in vitro, there is currently no direct evidence of oxidation-induced PKGIα activation in vivo.

Important Roles of Endothelium-Dependent Hyperpolarization in Coronary Microcirculation and Cardiac Diastolic Function in Mice.

Endothelium-dependent hyperpolarization (EDH) factor is one of endothelium-derived relaxing factors and plays important roles especially in microvessels. We have previously demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an EDH factor produced by all types of nitric oxide synthases (NOSs), including endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS. Recent studies have suggested the association between coronary microvascular dysfunction and cardiac diastolic dysfunction. However, the role of EDH in this issue remains to be fully elucidated. We thus examined whether EDH plays an important role in coronary microcirculation and if so, whether endothelial dysfunction, especially impaired EDH, is involved in the pathogenesis of cardiac diastolic dysfunction in mice. Using a Langendorff-perfused heart experiment, we examined the increase in coronary flow in response to bradykinin in the presence of indomethacin and N-nitro-L-arginine (EDH condition) in wild-type, eNOS-knockout (KO), and nNOS/eNOS-double-KO mice. Compared with wild-type mice, EDH-mediated relaxations were increased in eNOS-KO mice but were significantly reduced in n/eNOS-KO mice. Catalase, a specific H2O2 scavenger, markedly inhibited EDH-mediated relaxations in all 3 genotypes, indicating compensatory roles of nNOS-derived H2O2 as an EDH factor in coronary microcirculation. Although both eNOS-KO and n/eNOS-KO mice exhibited similar extents of cardiac morphological changes, only n/eNOS-KO mice exhibited cardiac diastolic dysfunction. The expression of oxidized protein kinase G I-α (PKGIα) in the heart was significantly increased in eNOS-KO mice compared with n/eNOS-KO mice. These results indicate that EDH/H2O2 plays important roles in maintaining coronary microcirculation and cardiac diastolic function through oxidative PKGIα activation.

Oxidation of cysteine 117 stimulates constitutive activation of the type Iα cGMP-dependent protein kinase

The type I cGMP-dependent protein kinase (PKG I) is an essential regulator of vascular tone. It has been demonstrated that the type Iα isoform can be constitutively activated by oxidizing conditions. However, the amino acid residues implicated in this phenomenon are not fully elucidated. To investigate the molecular basis for this mechanism, we studied the effects of oxidation using recombinant WT, truncated, and mutant constructs of PKG I. Using an in vitro assay, we observed that oxidation with hydrogen peroxide (H2O2) resulted in constitutive, cGMP-independent activation of PKG Iα. PKG Iα C42S and a truncation construct that does not contain Cys-42 (Δ53) were both constitutively activated by H2O2 In contrast, oxidation of PKG Iα C117S maintained its cGMP-dependent activation characteristics, although oxidized PKG Iα C195S did not. To corroborate these results, we also tested the effects of our constructs on the PKG Iα-specific substrate, the large conductance potassium channel (KCa 1.1). Application of WT PKG Iα activated by either cGMP or H2O2 increased the open probabilities of the channel. Neither cGMP nor H2O2 activation of PKG Iα C42S significantly increased channel open probabilities. Moreover, cGMP-stimulated PKG Iα C117S increased KCa 1.1 activity, but this effect was not observed under oxidizing conditions. Finally, we observed that PKG Iα C42S caused channel flickers, indicating dramatically altered KCa 1.1 channel characteristics compared with channels exposed to WT PKG Iα. Cumulatively, these results indicate that constitutive activation of PKG Iα proceeds through oxidation of Cys-117 and further suggest that the formation of a sulfur acid is necessary for this phenotype.

An N-terminally truncated form of cyclic GMP–dependent protein kinase Iα (PKG Iα) is monomeric and autoinhibited and provides a model for activation

The type I cGMP-dependent protein kinases (PKG I) serve essential physiological functions, including smooth muscle relaxation, cardiac remodeling, and platelet aggregation. These enzymes form homodimers through their N-terminal dimerization domains, a feature implicated in regulating their cooperative activation. Previous investigations into the activation mechanisms of PKG I isoforms have been largely influenced by structures of the cAMP-dependent protein kinase (PKA). Here, we examined PKG Iα activation by cGMP and cAMP by engineering a monomeric form that lacks N-terminal residues 1-53 (Δ53). We found that the construct exists as a monomer as assessed by whole-protein MS, size-exclusion chromatography, and small-angle X-ray scattering (SAXS). Reconstruction of the SAXS 3D envelope indicates that Δ53 has a similar shape to the heterodimeric RIα-C complex of PKA. Moreover, we found that the Δ53 construct is autoinhibited in its cGMP-free state and can bind to and be activated by cGMP in a manner similar to full-length PKG Iα as assessed by surface plasmon resonance (SPR) spectroscopy. However, we found that the Δ53 variant does not exhibit cooperative activation, and its cyclic nucleotide selectivity is diminished. These findings support a model in which, despite structural similarities, PKG Iα activation is distinct from that of PKA, and its cooperativity is driven by in trans interactions between protomers.

Oxidant sensor in the cGMP-binding pocket of PKGI\u03b1 regulates nitroxyl-mediated kinase activity

Despite the mechanisms for endogenous nitroxyl (HNO) production and action being incompletely understood, pharmacological donors show broad therapeutic promise and are in clinical trials. Mass spectrometry and site-directed mutagenesis showed that chemically distinct HNO donors 1-nitrosocyclohexyl acetate or Angeli’s salt induced disulfides within cGMP-dependent protein kinase I-alpha (PKGIα), an interdisulfide between Cys42 of the two identical subunits of the kinase and a previously unobserved intradisulfide between Cys117 and Cys195 in the high affinity cGMP-binding site. Kinase activity was monitored in cells transfected with wildtype (WT), Cys42Ser or Cys117/195Ser PKGIα that cannot form the inter- or intradisulfide, respectively. HNO enhanced WT kinase activity, an effect significantly attenuated in inter- or intradisulfide-deficient PKGIα. To investigate whether the intradisulfide modulates cGMP binding, real-time imaging was performed in vascular smooth muscle cells expressing a FRET-biosensor comprising the cGMP-binding sites of PKGIα. HNO induced FRET changes similar to those elicited by an increase of cGMP, suggesting that intradisulfide formation is associated with activation of PKGIα. Intradisulfide formation in PKGIα correlated with enhanced HNO-mediated vasorelaxation in mesenteric arteries in vitro and arteriolar dilation in vivo in mice. HNO induces intradisulfide formation in PKGIα, inducing the same effect as cGMP binding, namely kinase activation and thus vasorelaxation.

Proof of Principle for a Novel Class of Antihypertensives That Target the Oxidative Activation of PKG Iα (Protein Kinase G Iα).

Arterial hypertension continues to be a major health burden. Development of new antihypertensive drugs that engage vasodilatory mechanisms not harnessed by available therapies offer therapeutic potential. Oxidants induce an interprotein disulfide in PKG Iα (protein kinase G Iα) at C42, which is associated with its targeting and activation, resulting in vasodilation and blood pressure lowering. Consequently, we developed an assay and screened for electrophilic drugs that activate PKG Iα by selectively targeting C42, as such compounds have potential as novel antihypertensives with a mechanism of action that differs from current therapies. In this way, a drug that we termed G1 was identified, which targets C42 of PKG Iα to induce vasodilation of isolated resistance blood vessels and blood pressure lowering in a mouse model of angiotensin II-induced hypertension. In contrast, these antihypertensive effects were deficient in angiotensin II-induced hypertensive C42S PKG Iα knockin mice. These transgenic mice were engineered to have the reactive cysteinyl thiol replaced with a hydroxyl so that it cannot react with endogenous vasodilatory oxidants or electrophiles such as drug G1. These studies, therefore, provide validation of PKG Iα C42 as the target of G1, as well as proof-of-principle for a new class of antihypertensive drugs that have potential for further development for clinical use in humans.

The activity of cGMP-dependent protein kinase Iα is not directly regulated by oxidation-induced disulfide formation at cysteine 43

The type I cGMP-dependent protein kinases (PKGs) are key regulators of smooth muscle tone, cardiac hypertrophy, and other physiological processes. The two isoforms PKGIα and PKGIβ are thought to have unique functions because of their tissue-specific expression, different cGMP affinities, and isoform-specific protein-protein interactions. Recently, a non-canonical pathway of PKGIα activation has been proposed, in which PKGIα is activated in a cGMP-independent fashion via oxidation of Cys43, resulting in disulfide formation within the PKGIα N-terminal dimerization domain. A "redox-dead" knock-in mouse containing a C43S mutation exhibits phenotypes consistent with decreased PKGIα signaling, but the detailed mechanism of oxidation-induced PKGIα activation is unknown. Therefore, we examined oxidation-induced activation of PKGIα, and in contrast to previous findings, we observed that disulfide formation at Cys43 does not directly activate PKGIα in vitro or in intact cells. In transfected cells, phosphorylation of Ras homolog gene family member A (RhoA) and vasodilator-stimulated phosphoprotein was increased in response to 8-CPT-cGMP treatment, but not when disulfide formation in PKGIα was induced by H2O2 Using purified enzymes, we found that the Cys43 oxidation had no effect on basal kinase activity or Km and Vmax values; however, PKGIα containing the C43S mutation was less responsive to cGMP-induced activation. This reduction in cGMP affinity may in part explain the PKGIα loss-of-function phenotype of the C43S knock-in mouse. In conclusion, disulfide formation at Cys43 does not directly activate PKGIα, and the C43S-mutant PKGIα has a higher Ka for cGMP. Our results highlight that mutant enzymes should be carefully biochemically characterized before making in vivo inferences.

Insulin increases filtration barrier permeability via TRPC6-dependent activation of PKGIα signaling pathways

Podocytes are dynamic polarized cells on the surface of glomerular capillaries and an essential component of the glomerular filtration barrier. Insulin increases the activation of protein kinase G type Iα (PKGIα) subunits, leading to podocyte dysfunction. In addition, accumulating evidence suggests that TRPC6 channels are crucial mediators of podocyte calcium handling and involved in the regulation of glomerular filtration. Therefore, we investigated whether TRPC6 is involved in the regulation of filtration barrier permeability by insulin via the PKGIα-dependent manner.TRPC channel inhibitor SKF96365 abolished insulin-dependent glomerular albumin permeability and transepithelial albumin flux in cultured rat podocytes. Insulin-evoked albumin permeability across podocyte monolayers was also blocked using TRPC6 siRNA. The effect of insulin on albumin permeability was mimicked by treating podocytes with TRPC channel activator (oleolyl-2-acetyl-sn-glycerol, OAG). Insulin or OAG treatment rapidly increased the superoxide generation through activation of NADH oxidase. TRPC inhibitor SKF96365 or siRNA knockdown of TRPC6 attenuated insulin-dependent increase of ROS production. Furthermore, TRPC inhibitor or downregulation of TRPC6 blocked insulin-induced rearrangement of the actin cytoskeleton and attenuated oxidative activation of PKGIα and changes in the phosphorylation of PKG target proteins MYPT1 and MLC. Moreover insulin regulated the PKGIα interaction with TRPC6 in cultured rat podocytes.Taken together, our data suggest a key role of TRPC6 channels in the mediation of insulin-dependent activation of PKGIα signaling pathways. Overall, we have identified a potentially important mechanism that may explain disturbances in filtration barrier permeability in many diseases with increased expression of TRPC6 and chronic Ca2+ overload.

348 - Redox Control of Protein Kinase G Iα Fine-Tunes the Frank-Starling Law of the Heart in Vivo by Regulating Diastolic Relaxation

The Frank-Starling law describes a regulatory mechanism that allows the amount of blood that enters the heart to be precisely matched with the amount that is pumped out to the arterial circulation, independently of external regulation. Mechanisms that contribute to the Frank-Starling response include stretch-induced increases in myofilament overlap, myofilament Ca2+ sensitivity and actin-myosin cross-bridge formation. Recently, it was reported that there is also an increased production of oxidants during diastolic stretch, which are involved in regulation of cardiac Ca2+ cycling and contractile performance. Here we show that myocardial stretch mediates oxidation of cysteine 42 in protein kinase G Iα (PKGIα), causing it to form a disulfide dimer which is catalytically active. An unbiased phosphoproteomic study revealed that this disulfide-activated PKGIα selectively phosphorylates phospholamban (PLN) Ser16. Isothermal titration calorimetry data suggest that the interaction between PLN and the reduced form of PKGIα is markedly weaker than when the kinase is oxidised to the disulfide. Hearts isolated from C42S PKGIα knock-in (KI) mice, in which the kinase is resistant to oxidant-induced activation, were deficient in PLN Ser16 phosphorylation and displayed impaired end-diastolic relaxation and Frank-Starling responses compared to wild-type (WT). Ventricular myocytes isolated from KI mice were significantly deficient in their systolic Ca2+ transient and SR Ca2+ content and had ~50 % slower rate of Ca2+ decay compared to WT cells. In vivo analysis of cardiac function by echocardiography and using pressure-volume catheter showed KI hearts had a slower diastolic relaxation leading to an elevated end-diastolic pressure and an impaired Frank-Starling mechanism, in agreement with isolated heart data. Furthermore, in the absence of this redox control mechanism as is the case in the KI, the peak force the heart generated in vivo from beat-to-beat was erratic. We conclude that myocardial stretch-induced reactive oxygen species (ROS) production causes oxidative activation of PKGIα, fine-tuning the Frank-Starling response.

Role of Protein Kinase G and Reactive Oxygen Species in the Regulation of Podocyte Function in Health and Disease.

Podocytes and their foot processes form an important cellular layer of the glomerular barrier involved in the regulation of glomerular permeability. Disturbing the function of podocytes plays a central role in the development of proteinuria in diabetic nephropathy. Retraction of the podocyte foot processes that form slit diaphragms is a common feature of proteinuria; although, the correlation between these events in not well understood. Notably, it is unclear whether podocyte foot processes are able to regulate slit diaphragm permeability and glomerular ultrafiltration. The occurrence of reactive oxygen species generation, insulin resistance, and hyperglycemia characterizes early stages of type 2 diabetes. Protein kinase G type I alpha (PKGIα) is an intracellular target for vasorelaxant factors. It is activated in both cGMP-dependent and cGMP-independent manners. Recently, we demonstrated a relationship between oxidative stress, PKGIα activation, actin reorganization, and changes in the permeability of the filtration barrier. This review discusses how redox imbalance affects both the activity of PKGIα and PKGI-dependent signaling pathways in podocytes. J. Cell. Physiol. 232: 691-697, 2017. © 2016 Wiley Periodicals, Inc.

Synthetic Peptides as cGMP-Independent Activators of cGMP-Dependent Protein Kinase Iα

PKG is a multifaceted signaling molecule and potential pharmaceutical target due to its role in smooth muscle function. A helix identified in the structure of the regulatory domain of PKG Iα suggests a novel architecture of the holoenzyme. In this study, a set of synthetic peptides (S-tides), derived from this helix, was found to bind to and activate PKG Iα in a cyclic guanosine monophosphate (cGMP)-independent manner. The most potent S-tide derivative (S1.5) increased the open probability of the potassium channel KCa1.1 to levels equivalent to saturating cGMP. Introduction of S1.5 to smooth muscle cells in isolated, endothelium-denuded cerebral arteries through a modified reversible permeabilization procedure inhibited myogenic constriction. In contrast, in endothelium-intact vessels S1.5 had no effect on myogenic tone. This suggests that PKG Iα activation by S1.5 in vascular smooth muscle would be sufficient to inhibit augmented arterial contractility that frequently occurs following endothelial damage associated with cardiovascular disease.

Insulin Increases Glomerular Barrier Permeability: Role of Intracellular Calcium and PKGIα‐Dependent Signaling

Podocytes are dynamic polarized cells that lie on the surface of glomerular capillaries and comprise an essential component of the glomerular filtration barrier. We demonstrated recently that insulin increasesactivation of protein kinase G type Ia (PKGIa) subunits, leading to podocyte dysfunction. Here we investigated whether intracellular calciumis involved in insulin-dependent regulation of filtration barrier permeability in PKGIα-dependent manner. The RT-PCR showed the presence of mRNAs encoding SERCA isoforms 1-3, PMCA isoforms 1,3,4 and TRPC6 channel in podocyte. The thapsigargin (TG, SERCA inhibitor) and SKF96365 (TRPC inhibitor) abolished insulin-dependent glomerular albumin permeability in Wistar rats and PKGI-dependent transmembrane albumin flux in cultured podocytes. The same result we observed after preincubation cells with PKGI inhibitor Rp-8-cGMPS. Our data showed that insulin evoked a sustained increase of [Ca2+]in in podocytes, about 69%. This [Ca2+]in increase was blocked by TG and SKF96365. Furthermore, Rp-8-cGMPS strongly reduced the insulin-induced [Ca2+]in increase, about 71%. Moreover, the TG and SKF96365 abolished insulin induce changes in the phosphorylation of the PKG target proteins MYPT1 and RhoA. The results indicate that insulin increases filtration barrier permeability through increase level of intracellular Ca2+ concentration and via PKGIα activation in podocytes. This work was supported by grant from the FNP (POMOST/2011-4/6) and from the NSC (2012/05/B/NZ4/02587).

Abstract B01: Phosphorylation of pro-oncogenic c-Src at serine-17 by protein kinase G-Iα promotes chemoresistance/cell proliferation in human non-small cell lung cancer cells.

Abstract We previously showed that PKG-Iα promotes cell survival in neural cells (N1E-115 neuroblastoma and NG108-15 neuroblastoma-glioma hybrid cells) and significantly contributes to serine-155 phosphorylation of BAD, an apoptosis-regulating protein (Johlfs and Fiscus 2010). We also found, by using both gene knockdown and pharmacological inhibitors (ODQ, DT-2 and DT-3) to decrease PKG-Iα kinase activity, that PKG-Iα promotes cell proliferation and chemoresistance in human ovarian cancer cells, involving a novel interaction between PKG-Iα and the oncogenic protein c-Src (Fiscus et al 2012, Leung et al 2010). PKG-Iα activity was dependent on c-Src-catalyzed tyrosine phosphorylation of PKG-Iα; and, reciprocally, c-Src activation was dependent on expression and kinase activity of PKG-Iα;. We hypothesized that activation of c-Src involves PKG-Iα-catalyzed phosphorylation at serine-17 of c-Src, because amino acids around serine-17 provide consensus sequence for PKG-I, and tαhat this plays an important role in proliferation and chemoresistance. As c-Src has been proposed to promote cell proliferation and chemoresistance in NSCLC cells, the present study aimed to: 1) determine if c-Src increases PKG-Iα kinase activity in NSCLC cells using our newly-developed near-infrared-fluorescence (NIRF)-kinase assay, 2) determine if serine-17-phosphorylation of c-Src, in intact cancer cells, is dependent on PKG-Iα expression and kinase activity (using siRNA gene knockdown and pharmacological inhibitors), and 3) determine if inhibition/knockdown of c-Src and PKG-Iα alters chemoresistance and proliferation of NSCLC cells. Protein expression of c-Src and PKG-Iα was determined by conventional Western blot analysis and new ultrasensitive capillary-electrophoresis-based NanoPro100/1000 (ProteinSimple, Santa Clara, CA, USA). NanoPro100/1000 allows clear separation and identification of different c-Src and PKG-Iαphospho-forms (a novel way of identifying the activation of c-Src and PKG-Iα). We found PKG-Iα was able to directly catalyze phosphorylation of serine-17 of c-Src, increasing autophosphorylation of c-Src at tyrosine-419, the activation site of c-Src. Inhibition of PKG-Iα kinase activity using DT-2 or silencing of PKG-Iα expression using siRNA dramatically reduced the intracellular phosphorylation of c-Src at serine-17. Both kinase-activity inhibition and knockdown of PKG-Iα caused significant increases in apoptosis, synergistically enhancing apoptosis induced by the chemotherapeutic agent cisplatin, and dramatically decreased cell proliferation/colony formation in NSCLC cells. This novel reciprocal relationship between c-Src and PKG-Iα provides a new target for the development of new anti-cancer therapeutic agents. Fiscus RR, Leung EL, Wong JC, Johlfs MG (2012). Nitric oxide/protein kinase G-Ialpha promote c-Src activation, proliferation and chemoresistance in ovarian cancer. In: Farghaly S (ed). Ovarian Cancer - Basic Science Perspective. Intech Open Access Publisher. pp 315-334. Johlfs MG, Fiscus RR (2010). Protein kinase G type-Ialpha phosphorylates the apoptosis-regulating protein Bad at serine 155 and protects against apoptosis in N1E-115 cells. Neurochem Int 56: 546-553. Leung EL, Wong JC, Johlfs MG, Tsang BK, Fiscus RR (2010). Protein kinase G type Ialpha activity in human ovarian cancer cells significantly contributes to enhanced Src activation and DNA synthesis/cell proliferation. Mol Cancer Res 8: 578-591. Citation Format: Mary G. Johlfs, Ronald R. Fiscus, Priyatham Gorjala, Renee Coffman, Harry Rosenberg. Phosphorylation of pro-oncogenic c-Src at serine-17 by protein kinase G-Iα promotes chemoresistance/cell proliferation in human non-small cell lung cancer cells. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B01.

High glucose increases glomerular filtration barrier permeability by activating protein kinase G type Iα subunits in a Nox4-dependent manner

Hyperglycemia is a primary factor that disturbs podocyte function in the glomerular filtration process; this disturbance leads to the development of diabetic nephropathy, and ultimately, renal failure. Podocyte function may also be altered by biological agents that modify protein kinase activity, including the cGMP-activated protein kinase type Iα (PKGIα). We hypothesized that hyperglycemia-induced podocyte protein hyperpermeability was dependent on PKGIα activation, and that PKGIα was activated via dimerization induced by reactive oxygen species. This hypothesis was investigated in rat podocytes cultured in high glucose (HG, 30mM). Protein expression was measured with Western blot and immunofluorescence. Podocyte permeability was measured with a transmembrane albumin flux assay. We found that HG increased podocyte permeability in long-term incubations (1, 3, and 5 days); permeability was increased by 66% on day 5. This effect was abolished with apocynin, a NAD(P)H inhibitor, and Rp-8-Br-cGMPS, a PKG inhibitor. It was also abolished by introducing small interfering RNAs (siRNAs) against Nox4 and PKGIα into cultured podocytes. Furthermore, HG increased PKGIα dimerization by 138% (0.23±0.04 vs. 0.54±0.09; P<0.05); this effect was abolished with a siRNA against Nox4. Our observations suggested that HG could increase albumin permeability across the podocyte filtration barrier via Nox4-dependent PKGIα dimerization.

Protein kinase G oxidation is a major cause of injury during sepsis

Sepsis is a common life-threatening clinical syndrome involving complications as a result of severe infection. A cardinal feature of sepsis is inflammation that results in oxidative stress. Sepsis in wild-type mice induced oxidative activation of cGMP-dependent protein kinase 1 alpha (PKG Iα), which increased blood vessel dilation and permeability, and also lowered cardiac output. These responses are typical features of sepsis and their combined effect is a lowering of blood pressure. This hypotension, a hallmark of sepsis, resulted in underperfusion of end organs, resulting in their damage. A central role for PKG Iα oxidative activation in injury is supported by oxidation-resistant Cys42Ser PKG Iα knock-in mice being markedly protected from these clinical indices of injury during sepsis. We conclude that oxidative activation of PKG Iα is a key mediator of hypotension and consequential organ injury during sepsis.

H 2 O 2 -Induced Dilation in Human Coronary Arterioles: Role of Protein Kinase G Dimerization and Large-Conductance Ca 2+ -Activated K + Channel Activation

Hydrogen peroxide (H(2)O(2)) serves as a key endothelium-derived hyperpolarizing factor mediating flow-induced dilation in human coronary arterioles (HCAs). The precise mechanisms by which H(2)O(2) elicits smooth muscle hyperpolarization are not well understood. An important mode of action of H(2)O(2) involves the oxidation of cysteine residues in its target proteins, including protein kinase G (PKG)-Iα, thereby modulating their activities. Here we hypothesize that H(2)O(2) dilates HCAs through direct oxidation and activation of PKG-Iα leading to the opening of the large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel and subsequent smooth muscle hyperpolarization. Flow and H(2)O(2) induced pressure gradient/concentration-dependent vasodilation in isolated endothelium-intact and -denuded HCAs, respectively. The dilation was largely abolished by iberiotoxin, a BK(Ca) channel blocker. The PKG inhibitor Rp-8-Br-PET-cGMP also markedly inhibited flow- and H(2)O(2)-induced dilation, whereas the soluble guanylate cyclase inhibitor ODQ had no effect. Treatment of coronary smooth muscle cells (SMCs) with H(2)O(2) elicited dose-dependent, reversible dimerization of PKG-Iα, and induced its translocation to the plasma membrane. Patch-clamp analysis identified a paxilline-sensitive single-channel K(+) current with a unitary conductance of 246-pS in freshly isolated coronary SMCs. Addition of H(2)O(2) into the bath solution significantly increased the probability of BK(Ca) single-channel openings recorded from cell-attached patches, an effect that was blocked by the PKG-Iα inhibitor DT-2. H(2)O(2) exhibited an attenuated stimulatory effect on BK(Ca) channel open probability in inside-out membrane patches. H(2)O(2) dilates HCAs through a novel mechanism involving protein dimerization and activation of PKG-Iα and subsequent opening of smooth muscle BK(Ca) channels.

Abstract 334: Cyclic GMP-dependent Protein Kinase Iα Dependent Regulator of G-protein Signaling 2 Activation Is Required for Early Cardiac Compensation to Pressure-overload

In response to pressure-overload, the heart initially develops adaptive hypertrophy with enhanced contractility, but later decompensates with chamber dilation. Such maladaptation can be induced by activation of G αq -coupled signaling, whereas enhancing cyclic GMP-dependent protein kinase I-α (PKGI-α) by phosphodiesterase 5A inhibition (PDE5A-I) ameliorates pressure-overload pathobiology. A potential coupler of both pathways is RGS2 (regulator of G-protein signaling 2), which is activated by PKGI-α to suppress G αq -stimulation in vascular smooth muscle cells. Here we tested this signaling in hearts exposed to pressure-overload by transverse aortic constriction (TAC). RGS2 is up-regulated in the particulate fraction of control heart lysate within 48 hours of TAC. Unlike controls, RGS2 −/− hearts display no early compensation to TAC, developing accelerated dilation and severe hypertrophy within 1week, with high mortality (Table ). Downstream signaling of G αq stimulation including ERK1/2, CaM kinase II and calcineurin is markedly activated or up-regulated in RGS2 −/− -TAC versus RGS2 +/+ -TAC. In normal myocytes, RGS2 displays weak diffuse subcellular localization by confocal immunofluorescent histochemistry, whereas PKGI-α has a striated banding pattern. Stimulation of G αq (angiotensin II, 1μM; endothelin 1, 0.5μM) induces translocation of both RGS2 and PKGI-α to the sarcolemmal membrane, which is dependent on PKG activation. Similar translocation is observed in 48hours-TAC myocytes, but it declines by 1 week. Concomitant PDE5A-I (oral sildenafil; 200mg/kg/day) sustains PKGI-α and RGS2 at the membrane in RGS2 +/+ -TAC 1week, ameliorating hypertrophy, whereas PDE5A-I has no effect on RGS2 −/− -TAC 1week (Table ). Thus RGS2 is an essential regulator of early compensation to pressure-overload, is regulated by PKGI-α activation, and also plays a key mechanistic role in the anti-hypertrophic effects from PDE5A inhibitors. Results TAC 1 week (n = 4–6 for hemodynamics in each group)

Myosin Phosphatase Regulatory Pathways

In many ways, the smooth muscle cell (SMC) has been at the forefront in defining cellular signaling pathways. For example, the initial description of agonist-dependent intracellular calcium ([Ca2+]i) transients was made on vertebrate vascular SMC using aqueorin as the indicator.1 Delineation of nitric oxide (NO) and cGMP signaling was first accomplished in smooth muscle.2,3 And more recently, insights into the general mechanisms of gene transcription that dictate phenotypic traits of cells are being defined by smooth muscle specific gene expression.4,5 It should come as no surprise then that the interactions among many signaling pathways in cells should be explained first in the SMC. Perhaps the reason for the central position of the SMC in defining paradigms in cellular biology is that there are well-defined physiologic end points amenable to measurement (ie, contractile force) and even better described and easily quantified biochemical pathways that underlie said physiologic end points (ie, myosin regulatory light chain phosphorylation). From a historical perspective, the role of protein phosphorylation in smooth muscle contraction as a paradigm for cell signaling follows only that for the control of glycogen metabolism (see 6,7 for reviews). Increases in the levels of cytosolic [Ca2+]i initiate smooth muscle contraction by binding to the universal intracellular Ca2+ receptor protein, calmodulin (CaM), which in turn binds to and activates smooth muscle myosin light chain kinase (MLCK). Activated MLCK catalyzes the phosphorylation serine-19 of the regulatory myosin light chain (MLC) thereby increasing cross-bridge cycling and the rate of tension development. Dephosphorylation of MLC is catalyzed by smooth muscle specific myosin light chain phosphatase (MLCP). Decreases in cytosolic [Ca2+]i and MLC dephosphorylation are considered salient events in smooth muscle relaxation. What has become apparent only within the last decade is the fact that …

Differential relaxing responses to particulate or soluble guanylyl cyclase activation on endothelial cells: a mechanism dependent on PKG-I alpha activation by NO/cGMP.

cGMP is generated in endothelial cells after stimulation of soluble guanylyl cyclase (sGC) by nitric oxide (NO) or of particulate guanylyl cyclase (pGC) by natriuretic peptides (NP). We examined whether localized increases in cytosolic cGMP have distinct regulatory roles on the contraction induced by H2O2 treatment in human umbilical vein endothelial cells. cGMP concentrations and temporal dynamics were different upon NO stimulation of sGC or C-type NP (CNP) activation of pGC and did not correlate with their relaxing effects measured as planar cell surface area after H2O2 challenge. cGMP production due to sGC stimulation was always smaller and more brief than that induced by pGC stimulation with CNP, which was greater and remained elevated longer. The NO effects on cell relaxation were cGMP dependent because they were blocked by sGC inhibition with 1H-(1,2,4)Oxadiazolo(4,3-a)quinoxaline-1-one and mimicked by 8-Br-cGMP. An antagonist of the cGMP-dependent protein kinase type-I (PKG-I) also inhibited the NO-induced effects. The cell contraction induced by H2O2 produces myosin light chain (MLC) phosphorylation and NO prevented it completely, whereas CNP only produced a partial inhibition. Transfection with a dominant negative form of PKG type-I alpha completely reversed the NO-induced effects on MLC phosphorylation, whereas it only partially inhibited the effects due to CNP. Taken together, these results demonstrate that the NO/sGC/cGMP pathway induces endothelial cell relaxation in a more efficient manner than does CNP/pGC/cGMP pathway, an effect that might be related to a selective stimulation of PKG-1 alpha by NO-derived cGMP. Consequently, stimulated PKG-I alpha may phosphorylate important protein targets that are necessary to inhibit the endothelial contractile machinery activated by oxidative stress.