Publications by authors named "Jens Schlossmann"

54 Publications

Asymmetric dimethylarginine in psychiatric disorders.

Psychiatry Res 2021 Mar 23;300:113901. Epub 2021 Mar 23.

Clinical Pharmacology, Department of Pharmacology and Toxicology, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany.

Serum concentrations of asymmetric dimethylarginine (ADMA) in patients with schizophrenia, schizoaffective disorder, bipolar disorder, and depression were determined and compared to serum concentrations in healthy individuals. In all psychiatric diseases investigated, the ADMA concentration was elevated compared to the control group. Patients with recurrent depressive disorder had higher ADMA levels than patients with only one depressive episode. No differences between women and men were found. The elevated ADMA levels suggest that ADMA is involved in the pathophysiology of psychiatric diseases.
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http://dx.doi.org/10.1016/j.psychres.2021.113901DOI Listing
March 2021

Targeted Delivery of Soluble Guanylate Cyclase (sGC) Activator Cinaciguat to Renal Mesangial Cells via Virus-Mimetic Nanoparticles Potentiates Anti-Fibrotic Effects by cGMP-Mediated Suppression of the TGF-β Pathway.

Int J Mol Sci 2021 Mar 4;22(5). Epub 2021 Mar 4.

Department of Pharmaceutical Technology, University of Regensburg, 93053 Regensburg, Germany.

Diabetic nephropathy (DN) ranks among the most detrimental long-term effects of diabetes, affecting more than 30% of all patients. Within the diseased kidney, intraglomerular mesangial cells play a key role in facilitating the pro-fibrotic turnover of extracellular matrix components and a progredient glomerular hyperproliferation. These pathological effects are in part caused by an impaired functionality of soluble guanylate cyclase (sGC) and a consequentially reduced synthesis of anti-fibrotic messenger 3',5'-cyclic guanosine monophosphate (cGMP). Bay 58-2667 (cinaciguat) is able to re-activate defective sGC; however, the drug suffers from poor bioavailability and its systemic administration is linked to adverse events such as severe hypotension, which can hamper the therapeutic effect. In this study, cinaciguat was therefore efficiently encapsulated into virus-mimetic nanoparticles (NPs) that are able to specifically target renal mesangial cells and therefore increase the intracellular drug accumulation. NP-assisted drug delivery thereby increased in vitro potency of cinaciguat-induced sGC stabilization and activation, as well as the related downstream signaling 4- to 5-fold. Additionally, administration of drug-loaded NPs provided a considerable suppression of the non-canonical transforming growth factor β (TGF-β) signaling pathway and the resulting pro-fibrotic remodeling by 50-100%, making the system a promising tool for a more refined therapy of DN and other related kidney pathologies.
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http://dx.doi.org/10.3390/ijms22052557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961750PMC
March 2021

Activation of soluble guanylyl cyclase signalling with cinaciguat improves impaired kidney function in diabetic mice.

Br J Pharmacol 2021 Mar 2. Epub 2021 Mar 2.

Institute of Pharmacy, Department of Pharmacology and Toxicology, University of Regensburg, Regensburg, Germany.

Background And Purpose: Diabetic nephropathy is the leading cause for end-stage renal disease worldwide. Until now, there is no specific therapy available. Standard treatment with inhibitors of the renin-angiotensin system just slows down progression. However, targeting the NO/sGC/cGMP pathway using sGC activators does prevent kidney damage. Thus, we investigated if the sGC activator cinaciguat was beneficial in a mouse model of diabetic nephropathy, and we analysed how mesangial cells (MCs) were affected by related conditions in cell culture.

Experimental Approach: Type 1 diabetes was induced with streptozotocin in wild-type and endothelial NOS knockout (eNOS KO) mice for 8 or 12 weeks.. Half of these mice received cinaciguat in their chow for the last 4 weeks. Kidneys from the diabetic mice were analysed with histochemical assays and by RT-PCR and western blotting. . Additionally, primary murine MCs under diabetic conditions were stimulated with 8-Br-cGMP or cinaciguat to activate the sGC/cGMP pathway.

Key Results: The diabetic eNOS KO mice developed most characteristics of diabetic nephropathy, most marked at 12 weeks. Treatment with cinaciguat markedly improved GFR, serum creatinine, mesangial expansion and kidney fibrosis in these animals. We determined expression levels of related signalling proteins. Thrombospondin 1, a key mediator in kidney diseases, was strongly up-regulated under diabetic conditions and this increase was suppressed by activation of sGC/cGMP signalling.

Conclusion And Implications: Activation of the NO/sGC/PKG pathway with cinaciguat was beneficial in a model of diabetic nephropathy. Activators of sGC might be an appropriate therapy option in patients with Type 1 diabetes.
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http://dx.doi.org/10.1111/bph.15425DOI Listing
March 2021

IRAG1 Deficient Mice Develop PKG1β Dependent Pulmonary Hypertension.

Cells 2020 10 13;9(10). Epub 2020 Oct 13.

Universities of Giessen and Marburg Lung Centre, German Center for Lung Research (DZL), 35392 Giessen, Germany.

PKGs are serine/threonine kinases. PKG1 has two isoforms-PKG1α and β. Inositol trisphosphate receptor (IPR)-associated cGMP-kinase substrate 1 (IRAG1) is a substrate for PKG1β. IRAG1 is also known to further interact with IPRI, which mediates intracellular Ca release. However, the role of IRAG1 in PH is not known. Herein, WT and IRAG1 KO mice were kept under normoxic or hypoxic (10% O) conditions for five weeks. Animals were evaluated for echocardiographic variables and went through right heart catheterization. Animals were further sacrificed to prepare lungs and right ventricular (RV) for immunostaining, western blotting, and pulmonary artery smooth muscle cell (PASMC) isolation. IRAG1 is expressed in PASMCs and downregulated under hypoxic conditions. Genetic deletion of IRAG1 leads to RV hypertrophy, increase in RV systolic pressure, and RV dysfunction in mice. Absence of IRAG1 in lung and RV have direct impacts on PKG1β expression. Attenuated PKG1β expression in IRAG1 KO mice further dysregulates other downstream candidates of PKG1β in RV. IRAG1 KO mice develop PH spontaneously. Our results indicate that PKG1β signaling via IRAG1 is essential for the homeostasis of PASMCs and RV. Disturbing this signaling complex by deleting IRAG1 can lead to RV dysfunction and development of PH in mice.
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http://dx.doi.org/10.3390/cells9102280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601978PMC
October 2020

Homozygous mutation in murine retrovirus integration site 1 gene associated with a non-syndromic form of isolated familial achalasia.

Neurogastroenterol Motil 2020 12 22;32(12):e13923. Epub 2020 Jun 22.

Medizinische Fakultät Carl Gustav Carus, Children's Hospital, Technische Universität Dresden, Dresden, Germany.

Background: Achalasia is a condition characterized by impaired function of esophageal motility and incomplete relaxation of the lower esophagus sphincter, causing dysphagia and regurgitation. Rare cases of early-onset achalasia appear often in combination with further symptoms in a syndromic form as an inherited disease.

Methods: Whole genome sequencing was used to investigate the genetic basis of isolated achalasia in a family of Tunisian origin. We analyzed the function of the affected protein with immunofluorescence and affinity chromatography study.

Key Results: A homozygous nonsense mutation was detected in murine retrovirus integration site 1 (MRVI1) gene (Human Genome Organisation Gene Nomenclature Committee (HGNC) approved gene symbol: IRAG1) encoding the inositol 1,4,5-trisphosphate receptor 1 (IP R1)-associated cyclic guanosine monophosphate (cGMP) kinase substrate (IRAG). Sanger sequencing confirmed co-segregation of the mutation with the disease. Sequencing of the entire MRVI1 gene in 35 additional patients with a syndromic form of achalasia did not uncover further cases with MRVI1 mutations. Immunofluorescence analysis of transfected COS7 cells revealed GFP-IRAG with the truncating mutation p.Arg112* (transcript variant 1) or p.Arg121* (transcript variant 2) to be mislocalized in the cytoplasm and the nucleus. Co-transfection with cGMP-dependent protein kinase 1 isoform β (cGK1β) depicted a partial mislocalization of cGK1β due to mislocalized truncated IRAG. Isolation of protein complexes revealed that the truncation of this protein causes the loss of the interaction domain of IRAG with cGK1β.

Conclusions & Inferences: In individuals with an early onset of achalasia without further accompanying symptoms, MRVI1 mutations should be considered as the disease-causing defect.
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http://dx.doi.org/10.1111/nmo.13923DOI Listing
December 2020

Protein Kinase G Is Involved in Acute but Not in Long-Term Regulation of Renin Secretion.

Front Pharmacol 2019 18;10:800. Epub 2019 Jul 18.

Institute of Pharmacy, Department of Pharmacology and Toxicology, University of Regensburg, Regensburg, Germany.

Pharmacological inhibition of the renin-angiotensin-aldosterone system (RAAS) is, in combination with diuretics, the first-choice treatment for hypertension, although 10-20% of patients do not respond adequately. Next to the RAAS, the nitric oxide/cGMP/protein kinase G (PKG) system is the second fundamental blood pressure regulator. Whether both systems influence each other is not well-studied. It has been shown that nitric oxide (NO) supports renin recruitment activation of soluble guanylate cyclase (sGC) and subsequent generation of cGMP. Whether this leads to an ensuing activation of PKGs in this context is not known. PKGIα, as well as PKGII, is expressed in renin-producing cells. Hence, we analyzed whether these enzymes play a role regarding renin synthesis, secretion, or recruitment. We generated renin-cell-specific PKGI-knockout mice and either stimulated or inhibited the renin system in these mice by salt diets. To exclude the possibility that one kinase isoform can compensate the lack of the other, we also studied double-knockout animals with a conditional knockout of PKGI in juxtaglomerular cells (JG cells) and a ubiquitous knockout of PKGII. We analyzed blood pressure, renin mRNA and renal renin protein content as well as plasma renin concentration. Furthermore, we stimulated the cGMP system in these mice using BAY 41-8543, an sGC stimulator, and examined renin regulation either after acute administration or after 7 days (application once daily). We did not reveal any striking differences regarding long-term renin regulation in the studied mouse models. Yet, when we studied the acute effect of BAY 41-8543 on renin secretion in isolated perfused kidneys as well as in living animals, we found that the administration of the substance led to a significant increase in plasma renin concentration in control animals. This effect was completely abolished in double-knockout animals. However, after 7 days of once daily application, we did not detect a persistent increase in renin mRNA or protein in any studied genotype. Therefore, we conclude that in mice, cGMP and PKG are involved in the acute regulation of renin release but have no influence on long-term renin adjustment.
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http://dx.doi.org/10.3389/fphar.2019.00800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6657341PMC
July 2019

Relaxin and extracellular matrix remodeling: Mechanisms and signaling pathways.

Mol Cell Endocrinol 2019 05 17;487:59-65. Epub 2019 Jan 17.

Research Service, VA Nebraska-Western Iowa Health Care System, Departments of Internal Medicine and Biochemistry & Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA. Electronic address:

Fibrosis is associated with accumulation of excess fibrillar collagen, leading to tissue dysfunction. Numerous processes, including inflammation, myofibroblast activation, and endothelial-to-mesenchymal transition, play a role in the establishment and progression of fibrosis. Relaxin is a peptide hormone with well-known antifibrotic properties that result from its action on numerous cellular targets to reduce fibrosis. Relaxin activates multiple signal transduction pathways as a mechanism to suppress inflammation and myofibroblast activation in fibrosis. In this review, the general mechanisms underlying fibrotic diseases are described, along with the current state of knowledge regarding cellular targets of relaxin. Finally, an overview is presented summarizing the signaling pathways activated by relaxin and other relaxin family peptide receptor agonists to suppress fibrosis.
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http://dx.doi.org/10.1016/j.mce.2019.01.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7384500PMC
May 2019

Determination of total and free ceftolozane and tazobactam in human plasma and interstitial fluid by HPLC-UV.

J Pharm Biomed Anal 2019 Jan 24;163:34-38. Epub 2018 Sep 24.

Institute of Pharmacy, University of Regensburg, Universitätsstraße 31, 93053, Regensburg, Germany. Electronic address:

Ceftolozane/tazobactam is a new cephalosporin/beta-lactamase inhibitor combination. An HPLC-UV method is described for the determination of total and free ceftolozane and tazobactam in human plasma and in microdialysate of subcutaneous tissue, respectively. Separation was performed using a reversed-phase column with phosphate buffer/acetonitrile as eluent and photometric detection at 260 nm (ceftolozane) or 220 nm (tazobactam). Linearity has been shown down to ceftolozane/tazobactam 0.1/0.05 mg/L in plasma and 0.03/0.015 mg/L in saline, respectively. The plasma protein binding of both drugs as determined by ultrafiltration was less than 10%. Temperature, pH or relative centrifugation force (up to 3000 x g) had no significant impact on the protein binding. The method was applied to the determination of ceftolozane and tazobactam in plasma and interstitial fluid of healthy volunteers following intravenous infusion of ceftolozane/tazobactam 1.0/0.5 g.
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http://dx.doi.org/10.1016/j.jpba.2018.09.044DOI Listing
January 2019

Two isoforms of cyclic GMP-dependent kinase-I exhibit distinct expression patterns in the adult mouse dorsal root ganglion.

Mol Pain 2018 Jan-Dec;14:1744806918796409

1 Department of Medical Chemistry, Kansai Medical University, Japan.

cGMP-dependent kinase-I (cGKI) is known to regulate spinal pain processing. This enzyme consists of two isoforms (cGKIα and cGKIβ) that show distinct substrate specificity and tissue distribution. It has long been believed that the α isoform is exclusively expressed in the adult dorsal root ganglion. The aim of the present study was to reexamine the expression of cGKI isoforms in the adult mouse dorsal root ganglion using isoform-specific cGKI antibodies whose specificities had been validated in the previous studies. Immunoblot and immunohistochemical analyses revealed the presence of both isoforms in the dorsal root ganglion. Moreover, cGKIα was found to be mainly expressed within the cytoplasm of small- to medium-sized peptidergic and nonpeptidegic C-fibers, whereas cGKIβ was located within the nuclei of a wide range of dorsal root ganglion neurons. In addition, glutamine synthetase-positive satellite glial cells expressed both isoforms to varying degrees. Finally, using an experimental model for neuropathic pain produced by L5 spinal nerve transection, we found that cGKIα expression was downregulated in the injured, but not in the uninjured, dorsal root ganglion. In contrast, cGKIβ expression was upregulated in both the injured and uninjured dorsal root ganglions. Also, injury-induced cGKIβ upregulation was found to occur in small-to-medium-diameter dorsal root ganglion neurons. These data thus demonstrate the existence of two differently distributed cGKI isoforms in the dorsal root ganglion, and may provide insight into the cellular and molecular mechanisms of pain.
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http://dx.doi.org/10.1177/1744806918796409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113733PMC
December 2018

Real-Time Imaging Reveals Augmentation of Glutamate-Induced Ca Transients by the NO-cGMP Pathway in Cerebellar Granule Neurons.

Int J Mol Sci 2018 Jul 26;19(8). Epub 2018 Jul 26.

Interfaculty Institute of Biochemistry, University of Tübingen, 72076 Tübingen, Germany.

Dysfunctions of NO-cGMP signaling have been implicated in various neurological disorders. We have studied the potential crosstalk of cGMP and Ca signaling in cerebellar granule neurons (CGNs) by simultaneous real-time imaging of these second messengers in living cells. The NO donor DEA/NO evoked cGMP signals in the granule cell layer of acute cerebellar slices from transgenic mice expressing a cGMP sensor protein. cGMP and Ca dynamics were visualized in individual CGNs in primary cultures prepared from 7-day-old cGMP sensor mice. DEA/NO increased the intracellular cGMP concentration and augmented glutamate-induced Ca transients. These effects of DEA/NO were absent in CGNs isolated from knockout mice lacking NO-sensitive guanylyl cyclase. Furthermore, application of the cGMP analogues 8-Br-cGMP and 8-pCPT-cGMP, which activate cGMP effector proteins such as cyclic nucleotide-gated cation channels and cGMP-dependent protein kinases (cGKs), also potentiated glutamate-induced Ca transients. Western blot analysis failed to detect cGK type I or II in our primary CGNs. The addition of phosphodiesterase (PDE) inhibitors during cGMP imaging showed that CGNs degrade cGMP mainly via Zaprinast-sensitive PDEs, most likely PDE5 and/or PDE10, but not via PDE1, 2, or 3. In sum, these data delineate a cGK-independent NO-cGMP signaling cascade that increases glutamate-induced Ca signaling in CGNs. This cGMP⁻Ca crosstalk likely affects neurotransmitter-stimulated functions of CGNs.
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http://dx.doi.org/10.3390/ijms19082185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6121606PMC
July 2018

Establishing a Split Luciferase Assay for Proteinkinase G (PKG) Interaction Studies.

Int J Mol Sci 2018 Apr 12;19(4). Epub 2018 Apr 12.

Department of Pharmacology and Toxicology, University of Regensburg, 93053 Regensburg, Germany.

Nitric oxide (NO/cyclic guanosine monophosphate (cGMP)-regulated cellular mechanisms are involved in a variety of (patho-) physiological processes. One of the main effector molecules in this system, proteinkinase G (PKG), serves as a molecular switch by phosphorylating different target proteins and thereby turning them on or off. To date, only a few interaction partners of PKG have been described although the identification of protein-protein interactions (PPI) is indispensable for the understanding of cellular processes and diseases. Conventionally used methods to detect PPIs exhibit several disadvantages, e.g., co-immunoprecipitations, which depend on suitable high-affinity antibodies. Therefore, we established a cell-based protein-fragment complementation assay (PCA) for the identification of PKG target proteins. Here, a reporter protein ( luciferase) is split into two fragments and fused to two different possible interaction partners. If interaction occurs, the reporter protein is functionally complemented and the catalyzed reaction can then be quantitatively measured. By using this technique, we confirmed the regulator of G-Protein signaling 2 (RGS2) as an interaction partner of PKGIα (a PKG-isoform) following stimulation with 8-Br-cGMP and 8-pCPT-cGMP. Hence, our results support the conclusion that the established approach could serve as a novel tool for the rapid, easy and cost-efficient detection of novel PKG target proteins.
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http://dx.doi.org/10.3390/ijms19041180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5979328PMC
April 2018

Impact of Experimental Variables on the Protein Binding of Tigecycline in Human Plasma as Determined by Ultrafiltration.

J Pharm Sci 2018 02 18;107(2):739-744. Epub 2017 Sep 18.

Department of Anesthesiology, University Hospital Regensburg, Regensburg, Germany.

Tigecycline, a tetracycline derivative, shows atypical plasma protein binding behavior. The unbound fraction decreases with increasing concentration at therapeutic concentrations. Moreover, uncertainty exists about the magnitude of tigecyline's protein binding in man. Unbound fractions between 2.5% and 35% have been reported in plasma from healthy volunteers, and between 25% and 100% in patients, respectively. In the present study, the protein binding of tigecycline has been investigated by ultrafiltration using different experimental conditions. Whereas temperature had only a marginal influence, the unbound fraction at 0.3/3.0 mg/L was low at pH 8.2 (9.4%/1.9%) or in unbuffered pooled plasma (6.3%/1.2%), compared with plasma buffered with HEPES to pH 7.4 (65.9%/39.7%). In experiments with phosphate buffer and EDTA, the concentration dependency was markedly attenuated or abolished, which is compatible with a cooperative binding mechanism involving divalent cations such as calcium. The unbound fraction in clinical plasma samples from patients treated with tigecycline was determined to 66.3 ± 13.7% at concentrations <0.3 mg/L compared with 41.3 ± 16.0% at >1 to <5 mg/L. To summarize, tigecycline appears to be only moderately bound to plasma proteins as determined by ultrafiltration, when a physiological pH is maintained.
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http://dx.doi.org/10.1016/j.xphs.2017.09.006DOI Listing
February 2018

Differences in the renal antifibrotic cGMP/cGKI-dependent signaling of serelaxin, zaprinast, and their combination.

Naunyn Schmiedebergs Arch Pharmacol 2017 Sep 28;390(9):939-948. Epub 2017 Jun 28.

Department of Pharmacology and Toxicology, University of Regensburg, Regensburg, Germany.

Renal fibrosis is an important factor for end-stage renal failure. However, only few therapeutic options for its treatment are established. Zaprinast, a phosphodiesterase 5 inhibitor, and serelaxin, the recombinant form of the naturally occurring hormone relaxin, are differently acting modulators of cyclic guanosine monophosphate (cGMP) signaling. Both agents enhance cGMP availability in kidney tissue. These substances alone or in combination might interfere with the development of kidney fibrosis. Therefore, we compared the effects of combination therapy with the effects of monotherapy on renal fibrosis. Renal fibrosis was induced by unilateral ureteral obstruction (UUO) for 7 days in wild-type (WT) and cGKI knockout (KO) mice. Renal antifibrotic effects were assessed after 7 days. In WT, zaprinast and the combination of zaprinast and serelaxin significantly reduced renal interstitial fibrosis assessed by α-SMA, fibronectin, collagen1A1, and gelatinases (MMP2 and MMP9). Intriguingly in cGKI-KO, mRNA and protein expression of fibronectin and collagen1A1 were reduced by zaprinast, in contrast to serelaxin. Gelatinases are not regulated by zaprinast. Although both substances showed similar antifibrotic properties in WT, they distinguished in their effect mechanisms. In contrast to serelaxin which acts both on Smad2 and Erk1, zaprinast did not significantly diminish Erk1/2 phosphorylation. Interestingly, the combination of serelaxin/zaprinast achieved no additive antifibrotic effects compared to the monotherapy. Due to antifibrotic effects of zaprinast in cGKI-KO, we hypothesize that additional cGKI-independent mechanisms are supposed for antifibrotic signaling of zaprinast.
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http://dx.doi.org/10.1007/s00210-017-1394-zDOI Listing
September 2017

Inhibition of the TGFβ signalling pathway by cGMP and cGMP-dependent kinase I in renal fibrosis.

FEBS Open Bio 2017 04 1;7(4):550-561. Epub 2017 Mar 1.

Department of Pharmacology and Toxicology University of Regensburg Germany.

Agents that enhance production of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) ameliorate the progression of renal fibrosis. However, the molecular mechanism of this process is not fully understood. We hypothesize that the antifibrotic effects of cGMP and cGMP-dependent kinase I (cGKI) are mediated via regulation of the TGFβ signalling pathway, both via ERK and the Smad-dependent route. Kidney fibrosis was induced by unilateral ureter obstruction (UUO) in wild-type and cGKI-deficient (cGKI-KO) mice. The cGMP/cGKI signalling pathway was activated by application of the soluble guanylate cyclase (sGC) stimulator BAY 41-8543 (BAY), beginning 1 day after UUO. After 7 days, the antifibrotic effects of BAY were analysed by measuring mRNA and protein expression of characteristic fibrotic biomarkers. The effects of cGMP/TGFβ on cultured fibroblasts were also analysed . BAY application influenced the activity of the extracellular matrix (ECM)-degrading matrix metalloproteases (MMP2 and MMP9) and their inhibitor tissue inhibitors of metalloproteinase-1, the secretion of cytokines (e.g. IL-6) and the expression pattern of ECM proteins (e.g. collagen, fibronectin) and profibrotic mediators (e.g. connective tissue growth factors and plasminogen-activator inhibitor-1). Activation of the cGMP/cGKI signalling pathway showed protective effects against fibrosis which were mediated by inhibition of P-Erk1/2 and translocation of P-smad3. The elucidation of these signalling mechanisms might support the development of new therapeutic options regarding cGMP/cGKI-mediated antifibrotic actions.
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http://dx.doi.org/10.1002/2211-5463.12202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5377407PMC
April 2017

Antimicrobial and Hemolytic Studies of a Series of Polycations Bearing Quaternary Ammonium Moieties: Structural and Topological Effects.

Int J Mol Sci 2017 Jan 30;18(2). Epub 2017 Jan 30.

Institut für Organische Chemie, Universität Regensburg, Universitätsstr. 31, Regensburg 93053, Germany.

A series of polycations bearing quaternary ammonium moieties have shown antimicrobial activity against the Gram-negative bacterium Escherichia coli. Different polymer topologies governed by a disubstituted aromatic core as well as different diamine-based linkers were found to influence the antimicrobial properties. Moreover, the hemolytic activity against human red blood cells was measured and demonstrated good biocompatibility and selectivity of these polycations for bacteria over mammalian cells.
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http://dx.doi.org/10.3390/ijms18020303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5343839PMC
January 2017

Involvement of Cyclic Guanosine Monophosphate-Dependent Protein Kinase I in Renal Antifibrotic Effects of Serelaxin.

Front Pharmacol 2016 12;7:195. Epub 2016 Jul 12.

Department of Pharmacology and Toxicology, University of Regensburg Regensburg, Germany.

Introduction: Kidney fibrosis has shown to be ameliorated through the involvement of cyclic guanosine monophosphate (cGMP) and its dependent protein kinase I (cGKI). Serelaxin, the recombinant form of human relaxin-II, increases cGMP levels and has shown beneficial effects on kidney function in acute heart failure patients. Antifibrotic properties of serelaxin are supposed to be mediated via relaxin family peptide receptor 1 and subsequently enhanced nitric oxide/cGMP to inhibit transforming growth factor-β (TGF-β) signaling. This study examines the involvement of cGKI in the antifibrotic signaling of serelaxin.

Methods And Results: Kidney fibrosis was induced by unilateral ureteral obstruction in wildtype (WT) and cGKI knock-out (KO) mice. After 7 days, renal antifibrotic effects of serelaxin were assessed. Serelaxin treatment for 7 days significantly increased cGMP in the kidney of WT and cGKI-KO. In WT, renal fibrosis was reduced through decreased accumulation of collagen1A1, total collagen, and fibronectin. The profibrotic connective tissue growth factor as well as myofibroblast differentiation were reduced and matrix metalloproteinases-2 and -9 were positively modulated after treatment. Moreover, Smad2 as well as extracellular signal-regulated kinase 1 (ERK1) phosphorylation were decreased, whereas phosphodiesterase (PDE) 5a phosphorylation was increased. However, these effects were not observed in cGKI-KO.

Conclusion: Antifibrotic renal effects of serelaxin are mediated via cGMP/cGKI to inhibit Smad2- and ERK1-dependent TGF-β signaling and increased PDE5a phosphorylation.
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http://dx.doi.org/10.3389/fphar.2016.00195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4940422PMC
July 2016

Identification of cCMP and cUMP Substrate Proteins and Cross Talk Between cNMPs.

Handb Exp Pharmacol 2017 ;238:149-167

Pharmacology and Toxicology, Institute of Pharmacy, University Regensburg, Universitätsstr. 31, D-93040, Regensburg, Germany.

cCMP and cUMP are pyrimidine cyclic nucleotides which are present in several types of cells. These molecules could exert diverse cellular functions and might act as second messengers. In the last years, diverse approaches were performed to analyze possible cellular substrates and signaling pathways of cCMP and cUMP. In this review these approaches are summarized, and probable cross talk of these signaling molecules is described. These analyses might lead to the (patho)physiological and pharmacological relevance of these noncanonical cyclic nucleotides.
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http://dx.doi.org/10.1007/164_2015_38DOI Listing
October 2017

Regulation of the Na(+)-K(+)-2Cl(-) cotransporter by cGMP/cGMP-dependent protein kinase I after furosemide administration.

FEBS J 2015 Oct 30;282(19):3786-98. Epub 2015 Jul 30.

Pharmacology and Toxicology, Institute of Pharmacy, University of Regensburg, Germany.

Sodium chloride reabsorption in the thick ascending limb of the loop of Henle is mediated by the Na(+)-K(+)-2Cl(-) cotransporter (NKCC2). The loop diuretic furosemide is a potent inhibitor of NKCC2. However, less is known about the mechanism regulating the electrolyte transporter. Considering the well-established effects of nitric oxide on NKCC2 activity, cGMP is likely involved in this regulation. cGMP-dependent protein kinase I (cGKI; PKGI) is a cGMP target protein that phosphorylates different substrates after activation through cGMP. We investigated the potential correlation between the cGMP/cGKI pathway and NKCC2 regulation. We treated wild-type (wt) and cGKIα-rescue mice with furosemide. cGKIα-rescue mice expressed cGKIα only under the control of the smooth muscle-specific transgelin (SM22) promoter in a cGKI deficient background. Furosemide treatment increased the urine excretion of sodium and chloride in cGKIα-rescue mice compared to that in wt mice. We analyzed the phosphorylation of NKCC2 by western blotting and immunostaining using the phosphospecific antibody R5. The administration of furosemide significantly increased the phosphorylated NKCC2 signal in wt but not in cGKIα-rescue mice. NKCC2 activation led to its phosphorylation and membrane translocation. To examine whether cGKI was involved in this process, we analyzed vasodilator-stimulated phosphoprotein, which is phosphorylated by cGKI. Furosemide injection resulted in increased vasodilator-stimulated phosphoprotein phosphorylation in wt mice. We hypothesize that furosemide administration activated cGKI, leading to NKCC2 phosphorylation and membrane translocation. This cGKI-mediated pathway could be a mechanism to compensate for the inhibitory effect of furosemide on NKCC2.
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http://dx.doi.org/10.1111/febs.13376DOI Listing
October 2015

IL-3 contributes to development of lupus nephritis in MRL/lpr mice.

Kidney Int 2015 Nov 1;88(5):1088-98. Epub 2015 Jul 1.

Department of Internal Medicine II, University Hospital Regensburg, Regensburg, Germany.

MRL/lpr mice develop a spontaneous autoimmune disease that closely resembles human systemic lupus erythematosus (SLE) with DNA autoantibodies, hypergammaglobulinemia, immune complex glomerulonephritis, and systemic vasculitis. Little is known about the role of IL-3 in SLE. In order to study this we analyzed the expression of IL-3 in murine lupus and determined whether blockade of IL-3 with a monoclonal antibody or injection of recombinant IL-3 affects lupus nephritis in MRL/lpr mice. During disease progression IL-3 levels were increased in the plasma and in the supernatant of cultured splenocytes from MRL/lpr mice. Administration of IL-3 aggravated the disease with significantly higher renal activity scores, more renal fibrosis, and more glomerular leukocyte infiltration and IgG deposition. Blockade of IL-3 significantly improved acute and chronic kidney damage, reduced the glomerular infiltration of leukocytes and the glomerular deposition of IgG, and decreased the development of renal fibrosis. Furthermore, DNA autoantibody production, proteinuria, and serum creatinine levels were significantly lower in the anti-IL-3 group. Thus, IL-3 plays an important role in the pathogenesis of SLE and the progression of lupus nephritis. Hence, blockade of IL-3 may represent a new strategy for treatment of lupus nephritis.
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http://dx.doi.org/10.1038/ki.2015.196DOI Listing
November 2015

Interaction of cCMP with the cGK, cAK and MAPK Kinases in Murine Tissues.

PLoS One 2015 15;10(5):e0126057. Epub 2015 May 15.

Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Regensburg, Regensburg, Germany.

cAMP and cGMP are well established second messengers that are essential for numerous (patho)physiological processes. These purine cyclic nucleotides activate cAK and cGK, respectively. Recently, the existence of cCMP was described, and a possible function for this cyclic nucleotide was investigated. It was postulated that cCMP plays a role as a second messenger. However, the functions regulated by cCMP are mostly unknown. To elucidate probable functions, cCMP-binding and -activated proteins were identified using different methods. We investigated the effect of cCMP on purified cyclic nucleotide-dependent protein kinases and lung and jejunum tissues of wild type (WT), cGKI-knockout (cGKI KO) and cGKII-knockout (cGKII KO) mice. The catalytic activity of protein kinases was measured by a (γ-32P) ATP kinase assay. Cyclic nucleotide-dependent protein kinases (cAK, cGKI and cGKII) in WT tissue lysates were stimulated by cCMP. In contrast, there was no stimulation of phosphorylation in KO tissue lysates. Competitive binding assays identified cAK, cGKI, and cGKII as cCMP-binding proteins. An interaction between cCMP/MAPK and a protein-protein complex of MAPK/cGK were detected via cCMP affinity chromatography and co-immunoprecipitation, respectively. These complexes were abolished or reduced in jejunum tissues from cGKI KO or cGKII KO mice. In contrast, these complexes were observed in the lung tissues from WT, cGKI KO and cGKII KO mice. Moreover, cCMP was also able to stimulate the phosphorylation of MAPK. These results suggest that MAPK signaling is regulated by cGMP-dependent protein kinases upon activation by cCMP. Based on these results, we propose that additional cCMP-dependent protein kinases that are capable of modulating MAPK signaling could exist. Hence, cCMP could potentially act as a second messenger in the cAK/cGK and MAPK signaling pathways and play an important role in physiological processes of the jejunum and lung.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126057PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4433244PMC
April 2016

Renal effects of soluble guanylate cyclase stimulators and activators: a review of the preclinical evidence.

Curr Opin Pharmacol 2015 Apr 31;21:95-104. Epub 2015 Jan 31.

University of Potsdam, Potsdam, Germany.

Direct stimulation of soluble guanylate cyclase (sGC) is emerging as a potential new approach for the treatment of renal disorders. sGC catalyzes the formation of cyclic guanosine monophosphate (cGMP), deficiency of which is implicated in the pathogenesis of chronic kidney disease (CKD). Therefore, new classes of drugs - sGC stimulators and activators - are being investigated in preclinical models under conditions where nitric oxide is deficient. In preclinical models with different etiologies of CKD, the sGC stimulators BAY 41-2272, BAY 41-8543, BAY 60-4552, riociguat and vericiguat and the sGC activators cinaciguat, ataciguat, BI 703704 and GSK2181236A have shown consistently renoprotective effects. Clinical trials are required to confirm these findings in humans, and to ascertain whether these agents could provide a future alternative to guideline-recommended treatments.
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http://dx.doi.org/10.1016/j.coph.2014.12.014DOI Listing
April 2015

Cyclic nucleotide signalling in kidney fibrosis.

Int J Mol Sci 2015 Jan 22;16(2):2320-51. Epub 2015 Jan 22.

Pharmacology and Toxicology, University Regensburg, Regensburg 93053, Germany.

Kidney fibrosis is an important factor for the progression of kidney diseases, e.g., diabetes mellitus induced kidney failure, glomerulosclerosis and nephritis resulting in chronic kidney disease or end-stage renal disease. Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were implicated to suppress several of the above mentioned renal diseases. In this review article, identified effects and mechanisms of cGMP and cAMP regarding renal fibrosis are summarized. These mechanisms include several signalling pathways of nitric oxide/ANP/guanylyl cyclases/cGMP-dependent protein kinase and cAMP/Epac/adenylyl cyclases/cAMP-dependent protein kinase. Furthermore, diverse possible drugs activating these pathways are discussed. From these diverse mechanisms it is expected that new pharmacological treatments will evolve for the therapy or even prevention of kidney failure.
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http://dx.doi.org/10.3390/ijms16022320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4346839PMC
January 2015

Roles of cGMP-dependent protein kinase I (cGKI) and PDE5 in the regulation of Ang II-induced cardiac hypertrophy and fibrosis.

Proc Natl Acad Sci U S A 2014 Sep 19;111(35):12925-9. Epub 2014 Aug 19.

Forschergruppe 923, Institut für Pharmakologie und Toxikologie, Technische Universität München, 80802 Munich, Germany;

Conflicting results have been reported for the roles of cGMP and cGMP-dependent protein kinase I (cGKI) in various pathological conditions leading to cardiac hypertrophy and fibrosis. A cardioprotective effect of cGMP/cGKI has been reported in whole animals and isolated cardiomyocytes, but recent evidence from a mouse model expressing cGKIβ only in smooth muscle (βRM) but not in cardiomyocytes, endothelial cells, or fibroblasts has forced a reevaluation of the requirement for cGKI activity in the cardiomyocyte antihypertrophic effects of cGMP. In particular, βRM mice developed the same hypertrophy as WT controls when subjected to thoracic aortic constriction or isoproterenol infusion. Here, we challenged βRM and WT (Ctr) littermate control mice with angiotensin II (AII) infusion (7 d; 2 mg ⋅ kg(-1) ⋅ d(-1)) to induce hypertrophy. Both genotypes developed cardiac hypertrophy, which was more pronounced in Ctr animals. Cardiomyocyte size and interstitial fibrosis were increased equally in both genotypes. Addition of sildenafil, a phosphodiesterase 5 (PDE5) inhibitor, in the drinking water had a small effect in reducing myocyte hypertrophy in WT mice and no effect in βRM mice. However, sildenafil substantially blocked the increase in collagen I, fibronectin 1, TGFβ, and CTGF mRNA in Ctr but not in βRM hearts. These data indicate that, for the initial phase of AII-induced cardiac hypertrophy, lack of cardiomyocyte cGKI activity does not worsen hypertrophic growth. However, expression of cGKI in one or more cell types other than smooth muscle is necessary to allow the antifibrotic effect of sildenafil.
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http://dx.doi.org/10.1073/pnas.1414364111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4156763PMC
September 2014

Function of cGMP-dependent protein kinase II in volume load-induced diuresis.

Pflugers Arch 2014 Oct 18;466(10):2009-18. Epub 2014 Jan 18.

Pharmacology and Toxicology, Institute of Pharmacy, University of Regensburg, Universitätsstr 31, 93053, Regensburg, Germany.

Atrial natriuretic peptide (ANP)/cGMPs cause diuresis and natriuresis. Their downstream effectors beyond cGMP remain unclear. To elucidate a probable function of cGMP-dependent protein kinase II (cGKII), we investigated renal parameters in different conditions (basal, salt diets, starving, water load) using a genetically modified mouse model (cGKII-KO), but did not detect any striking differences between WT and cGKII-KO. Thus, cGKII is proposed to play only a marginal role in the adjustment of renal concentration ability to varying salt loads without water restriction or starving conditions. When WT mice were subjected to a volume load (performed by application of a 10-mM glucose solution (3% of BW) via feeding needle), they exhibited a potent diuresis. In contrast, urine volume was decreased significantly in cGKII-KO. We showed that AQP2 plasma membrane (PM) abundance was reduced for about 50% in WT upon volume load, therefore, this might be a main cause for the enhanced diuresis. In contrast, cGKII-KO mice almost completely failed to decrease AQP2-PM distribution. This significant difference between both genotypes is not induced by an altered p-Ser256-AQP2 phosphorylation, as phosphorylation at this site decreases similarly in WT and KO. Furthermore, sodium excretion was lowered in cGKII-KO mice during volume load. In summary, cGKII is only involved to a minor extent in the regulation of basal renal concentration ability. By contrast, cGKII-KO mice are not able to handle an acute volume load. Our results suggest that membrane insertion of AQP2 is inhibited by cGMP/cGKII.
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http://dx.doi.org/10.1007/s00424-014-1445-yDOI Listing
October 2014

cGMP-Dependent Protein Kinase Inhibitors in Health and Disease.

Pharmaceuticals (Basel) 2013 Feb 7;6(2):269-86. Epub 2013 Feb 7.

Department of Pharmacology and Toxicology, Institute of Pharmacy, University Regensburg, Universitätsstr. 31, 93053 Regensburg, Germany.

cGMP-dependent protein kinases (PKG) exhibit diverse physiological functions in the mammalian system e.g., in vascular and gastrointestinal smooth muscles, in platelets, in kidney, in bone growth, nociception and in the central nervous system. Furthermore, PKG were found in insects and in the malaria parasite Plasmodium falciparum. Two different genes of PKG exist: a) the PKG-I gene that is expressed as cytosolic PKG-Iα or PKG-Iβ isoform, and b) the PKG-II gene, which expresses the membrane associated PKG-II protein. The enzyme kinetics, the localization and the substrates of these PKG enzymes differ utilizing different physiological functions. Various inhibitors of PKG were developed directed against diverse functional regions of the kinase. These inhibitors of PKG have been used to analyse the specific functions of these enzymes. The review article will summarize these different inhibitors regarding their specificity and their present applications in vitro and in vivo. Furthermore, it will be discussed that the distinct inhibition of the PKG enzymes could be used as a valuable pharmacological target e.g., in the treatment of cardiovascular diseases, diarrhea, cancer or malaria.
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http://dx.doi.org/10.3390/ph6020269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3816681PMC
February 2013

Atrial natriuretic peptide-mediated inhibition of microcirculatory endothelial Ca2+ and permeability response to histamine involves cGMP-dependent protein kinase I and TRPC6 channels.

Arterioscler Thromb Vasc Biol 2013 Sep 27;33(9):2121-9. Epub 2013 Jun 27.

Institute of Physiology, University of Würzburg, Würzburg, Germany.

Objective: Histamine increases microvascular endothelial leakage by activation of complex calcium-dependent and -independent signaling pathways. Atrial natriuretic peptide (ANP) via its cGMP-forming guanylyl cyclase-A (GC-A) receptor counteracts this response. Here, we characterized the molecular mechanisms underlying this interaction, especially the role of cGMP-dependent protein kinase I (cGKI).

Approach And Results: We combined intravital microscopy studies of the mouse cremaster microcirculation with experiments in cultured microvascular human dermal endothelial cells. In wild-type mice, ANP had no direct effect on the extravasation of fluorescent dextran from postcapillary venules, but strongly reduced the histamine-provoked vascular leakage. This anti-inflammatory effect of ANP was abolished in mice with endothelial-restricted inactivation of GC-A or cGKI. Histamine-induced increases in endothelial [Ca(2+)]i in vitro and of vascular leakage in vivo were markedly attenuated by the Ca(2+)-entry inhibitor SKF96365 and in mice with ablated transient receptor potential canonical (TRPC) 6 channels. Conversely, direct activation of TRPC6 with hyperforin replicated the hyperpermeability responses to histamine. ANP, via cGKI, stimulated the inhibitory phosphorylation of TRPC6 at position Thr69 and prevented the hyperpermeability responses to hyperforin. Moreover, inhibition of cGMP degradation by the phosphodiesterase 5 inhibitor sildenafil prevented the edematic actions of histamine in wild types but not in mice with endothelial GC-A or cGKI deletion.

Conclusions: ANP attenuates the inflammatory actions of histamine via endothelial GC-A/cGMP/cGKI signaling and inhibitory phosphorylation of TRPC6 channels. The therapeutic potential of this novel regulatory pathway is indicated by the observation that sildenafil improves systemic endothelial barrier functions by enhancing the endothelial effects of endogenous ANP.
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http://dx.doi.org/10.1161/ATVBAHA.113.001974DOI Listing
September 2013

The cyclic GMP-dependent protein kinase Iα suppresses kidney fibrosis.

Kidney Int 2013 Dec 12;84(6):1198-206. Epub 2013 Jun 12.

Lehrstuhl für Pharmakologie und Toxikologie, Institut für Pharmazie, Universität Regensburg, Regensburg, Germany.

Cyclic guanosine monophosphate (cGMP) is synthesized by nitric oxide or natriuretic peptide-stimulated guanylyl cyclases and exhibits pleiotropic regulatory functions in the kidney. Hence, integration of cGMP signaling by cGMP-dependent protein kinases (cGKs) might play a critical role in renal physiology; however, detailed renal localization of cGKs is still lacking. Here, we performed an immunohistochemical analysis of cGKIα and cGKIβ isozymes in the mouse kidney and found both in arterioles, the mesangium, and within the cortical interstitium. In contrast to cGKIα, the β-isoform was not detected in the juxtaglomerular apparatus or medullary fibroblasts. Since interstitial fibroblasts play a prominent role in interstitial fibrosis, we focused our study on cGKI function in the interstitium, emphasizing a functional differentiation of both isoforms, and determined whether cGKIs influence renal fibrosis induced by unilateral ureter obstruction. Treatment with the guanylyl cyclase activators YC1 or isosorbide dinitrate showed stronger antifibrotic effects in wild-type than in cGKI-knockout or in smooth muscle-cGKIα-rescue mice, which are cGKI deficient in the kidney except in the renal vasculature. Moreover, fibrosis influenced the mRNA and protein expression levels of cGKIα more strongly than cGKIβ. Thus, our results indicate that cGMP, acting primarily through cGKIα, is an important suppressor of kidney fibrosis.
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http://dx.doi.org/10.1038/ki.2013.219DOI Listing
December 2013

Editorial of the special issue: signaling molecules and signal transduction in cells.

Authors:
Jens Schlossmann

Int J Mol Sci 2013 May 29;14(6):11438-43. Epub 2013 May 29.

Pharmacology and Toxicology, Institute of Pharmacy, University Regensburg, Universitätsstr, 31, D-93040 Regensburg, Germany.

In the special issue "Signaling Molecules and Signal Transduction in Cells" authors were invited to submit papers regarding important and novel aspects of extra- and intracellular signaling which have implications on physiological and pathophysiological processes. These aspects included compounds which are involved in these processes, elucidation of signaling pathways, as well as novel techniques for the analysis of signaling pathways. In response, various novel and important topics are elucidated in this special issue.
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http://dx.doi.org/10.3390/ijms140611438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3709741PMC
May 2013

Methods for identification of cGKI substrates.

Methods Mol Biol 2013 ;1020:147-62

Pharmakologie und Toxikologie, Institut für Pharmazie, Universität Regensburg, Regensburg, Germany.

The cGMP-dependent protein kinases (cGK), which belong to the family of serine/threonine kinases, exhibit their diverse functions in cells through interaction with a variety of substrate proteins. Several substrates were identified and the interactions studied using different methods inter alia co-immunoprecipitation (Co-IP) and cGMP-agarose affinity purification. In the following chapter, we will describe the preparation of cell or tissue lysates, the procedures of cGMP-agarose affinity purification and co-immunoprecipitation, and finally the separation and analysis of the protein complexes by SDS-PAGE or mass spectrometry.
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http://dx.doi.org/10.1007/978-1-62703-459-3_9DOI Listing
December 2013