In vivo regulation of Na/Ca exchanger expression by adrenergic effectors

The Na/Ca exchanger encoded by the NCX1gene plays an important role in calcium homeostasis in cardiac muscle. We previously identified three in vitro signaling pathways that are of major importance in the regulation of Na/Ca exchanger gene expression in neonatal cardiac myocytes, the protein kinase A (PKA) and protein kinase C (PKC) pathways, and intracellular Ca2+. To determine whether these pathways are important in vivo, we stimulated the PKA and PKC pathways and examined functional expression of the Na/Ca exchanger in adult rat heart. After a 3- and 7-day treatment, norepinephrine (200 μg · kg−1 · h−1), isoproterenol (150 μg · kg−1 · h−1), and phenylephrine (200 μg · kg−1 · h−1) each stimulated a significant increase in NCX1 mRNA levels (35–85%, P < 0.05). Norepinephrine also stimulated a 35% increase in protein abundance ( P < 0.05), a 20% decrease in relaxation duration ( P < 0.05), and a 25% reduction in the fluorescence decay constant ( P < 0.05) after a 7-day treatment. We conclude that a 7-day treatment of α- and β-adrenergic agonists increases the expression of functional Na/Ca exchangers in adult rat heart.

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Anti-echinococcal effect of verapamil involving the regulation of the calcium/calmodulin-dependent protein kinase II response in vitro and in a murine infection model

Parasites & Vectors ◽

10.1186/s13071-021-04618-4 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Hai-Jun Gao ◽

Xu-Dong Sun ◽

Yan-Ping Luo ◽

Hua-Sheng Pang ◽

Xing-Ming Ma ◽

...

Keyword(s):

Protein Kinase ◽

Mrna Expression ◽

Echinococcus Granulosus ◽

Calcium Content ◽

Mrna Levels ◽

Dependent Protein Kinase ◽

Calcium Calmodulin Dependent ◽

Dependent Protein

Abstract Background Echinococcosis, which is caused by the larvae of cestodes of the genus Echinococcus, is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and associated side effects after its long-term use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil (Vepm) can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of Vepm on Echinococcus with a particular focus on the regulatory effect of Vepm on calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-CaMKII) in infected mice. Methods The anti-echinococcal effects of Vepm on Echinococcus granulosus protoscoleces (PSC) in vitro and Echinococcus multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by Vepm were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CaMKII in infected mice, and in the mRNA levels of CaMKII in E. granulosus PSC, were evaluated after treatment with Vepm by immunohistochemistry and/or real-time quantitative polymerase chain reaction. Results In vitro, E. granulosus PSC could be killed by Vepm at a concentration of 0.5 μg/ml or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied by obvious calcium loss and downregulation of CaMKII mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg Vepm, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CaMKII protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with Vepm. Conclusions Vepm exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca2+/CaM-CaMKII, which was over-activated by parasitic infection. The results suggest that Ca2+/CaM-CaMKII may be a novel drug target, and that Vepm is a potential anti-echinococcal drug for the future control of echinococcosis.

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5′-AMP-activated protein kinase is inactivated by adrenergic signalling in adult cardiac myocytes

Bioscience Reports ◽

10.1042/bsr20110076 ◽

2011 ◽

Vol 32 (2) ◽

pp. 197-209 ◽

Cited By ~ 10

Author(s):

Yugo Tsuchiya ◽

Fiona C. Denison ◽

Richard B. Heath ◽

David Carling ◽

David Saggerson

Keyword(s):

Protein Kinase ◽

Cardiac Myocytes ◽

Calcium Signalling ◽

Metabolic Stress ◽

Protein Kinase Activity ◽

Adult Rat ◽

Protein Kinase C Inhibitor ◽

Amp Activated Protein Kinase

In adult rat cardiac myocytes adrenaline decreased AMPK (AMP-activated protein kinase) activity with a half-time of approximately 4 min, decreased phosphorylation of AMPK (α-Thr172) and decreased phosphorylation of ACC (acetyl-CoA carboxylase). Inactivation of AMPK by adrenaline was through both α1- and β-ARs (adrenergic receptors), but did not involve cAMP or calcium signalling, was not blocked by the PKC (protein kinase C) inhibitor BIM I (bisindoylmaleimide I), by the ERK (extracellular-signal-regulated kinase) cascade inhibitor U0126 or by PTX (pertussis toxin). Adrenaline caused no measurable change in LKB1 activity. Adrenaline decreased AMPK activity through a process that was distinct from AMPK inactivation in response to insulin or PMA. Neither adrenaline nor PMA altered the myocyte AMP:ATP ratio although the adrenaline effect was attenuated by oligomycin and by AICAR (5-amino-4-imidazolecarboxamide-1-β-D-ribofuranoside), agents that mimic ‘metabolic stress’. Inactivation of AMPK by adrenaline was abolished by 1 μM okadaic acid suggesting that activation of PP2A (phosphoprotein phosphatase 2A) might mediate the adrenaline effect. However, no change in PP2A activity was detected in myocyte extracts. Adrenaline increased phosphorylation of the AMPK β-subunit in vitro but there was no detectable change in vivo in phosphorylation of previously identified AMPK sites (β-Ser24, β-Ser108 or β-Ser182) suggesting that another site(s) is targeted.

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LmjF.22.0810 from Leishmania major Modulates the Th2-Type Immune Response and Is Involved in Leishmaniasis Outcome

Biomedicines ◽

10.3390/biomedicines8110452 ◽

2020 ◽

Vol 8 (11) ◽

pp. 452

Author(s):

Andrés Vacas ◽

Celia Fernández-Rubio ◽

Esther Larrea ◽

José Peña-Guerrero ◽

Paul A. Nguewa

Keyword(s):

Immune Response ◽

Protein Kinase ◽

Leishmania Major ◽

Parasite Burden ◽

Mrna Levels ◽

Expression Change ◽

Arginase 1 ◽

Th2 Immune Response

A novel serine/threonine protein kinase, LmjF.22.0810, was recently described in Leishmania major. After generating an L. major cell line overexpressing LmjF.22.0810 (named LmJ3OE), the ability of this novel protein to modulate the Th2-type immune response was analyzed. Our results suggest that the protein kinase LmjF.22.0810 might be involved in leishmaniasis outcomes. Indeed, our study outlined the LmJ3OE parasites infectivity in vitro and in vivo. Transgenic parasites displayed lower phagocytosis rates in vitro, and their promastigote forms exhibited lower expression levels of virulence factors compared to their counterparts in control parasites. In addition, LmJ3OE parasites developed significantly smaller footpad swelling in susceptible BALB/c mice. Hematoxylin–eosin staining allowed the observation of a lower inflammatory infiltrate in the footpad from LmJ3OE-infected mice compared to animals inoculated with control parasites. Gene expression of Th2-associated cytokines and effectors revealed a dramatically lower induction in interleukin (IL)-4, IL-10, and arginase 1 (ARG1) mRNA levels at the beginning of the swelling; no expression change was found in Th1-associated cytokines except for IL-12. Accordingly, such results were validated by immunohistochemistry studies, illustrating a weaker expression of ARG1 and a similar induction for inducible NO synthase (iNOS) in footpads from LmJ3OE-infected mice compared to control L. major infected animals. Furthermore, the parasite burden was lower in footpads from LmJ3OE-infected mice. Our analysis indicated that such significant smaller footpad swellings might be due to an impairment of the Th2 immune response that subsequently benefits Th1 prevalence. Altogether, these studies depict LmjF.22.0810 as a potential modulator of host immune responses to Leishmania. Finally, this promising target might be involved in the modulation of infection outcome.

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BRF1 Protein Turnover and mRNA Decay Activity Are Regulated by Protein Kinase B at the Same Phosphorylation Sites

Molecular and Cellular Biology ◽

10.1128/mcb.01099-06 ◽

2006 ◽

Vol 26 (24) ◽

pp. 9497-9507 ◽

Cited By ~ 62

Author(s):

Don Benjamin ◽

Martin Schmidlin ◽

Lu Min ◽

Brigitte Gross ◽

Christoph Moroni

Keyword(s):

Protein Kinase ◽

Mrna Decay ◽

Protein Kinase B ◽

Proteasomal Degradation ◽

Mrna Levels ◽

Cell Compartment ◽

Protein Levels ◽

Dependent Protein

ABSTRACT BRF1 posttranscriptionally regulates mRNA levels by targeting ARE-bearing transcripts to the decay machinery. We previously showed that protein kinase B (PKB) phosphorylates BRF1 at Ser92, resulting in binding to 14-3-3 and impairment of mRNA decay activity. Here we identify an additional regulatory site at Ser203 that cooperates in vivo with Ser92. In vitro kinase labeling and wortmannin sensitivity indicate that Ser203 phosphorylation is also performed by PKB. Mutation of both serines to alanine uncouples BRF1 from PKB regulation, leading to constitutive mRNA decay even in the presence of stabilizing signals. BRF1 protein is labile because of proteasomal degradation (half-life, <3 h) but becomes stabilized upon phosphorylation and is less stable in PKBα−/− cells. Surprisingly, phosphorylation-dependent protein stability is also regulated by Ser92 and Ser203, with parallel phosphorylation required at these sites. Phosphorylation-dependent binding to 14-3-3 is abolished only when both sites are mutated. Cell compartment fractionation experiments support a model in which binding to 14-3-3 sequesters BRF1 through relocalization and prevents it from executing its mRNA decay activity, as well as from proteasomal degradation, thereby maintaining high BRF1 protein levels that are required to reinstate decay upon dissipation of the stabilizing signal.

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Stanniocalcin 1 and 2 are secreted as phosphoproteins from human fibrosarcoma cells

Biochemical Journal ◽

10.1042/bj3500453 ◽

2000 ◽

Vol 350 (2) ◽

pp. 453-461 ◽

Cited By ~ 34

Author(s):

Derek A. JELLINEK ◽

Andy C. CHANG ◽

Martin R. LARSEN ◽

Xin WANG ◽

Phillip J. ROBINSON ◽

...

Keyword(s):

Protein Kinase ◽

Mammalian Cells ◽

Phosphorylation Site ◽

Mrna Levels ◽

Intact Cells ◽

Fibrosarcoma Cells ◽

Ht1080 Cells ◽

Human Fibrosarcoma Cells

Stanniocalcin 1 (STC1) and stanniocalcin 2 (STC2) are two recently identified mammalian peptide hormones. STC1 plays a role in calcium and phosphate homoeostasis, while the role of STC2 is unknown. We examined a human fibrosarcoma cell line, HT1080, that has high steady-state STC1 and STC2 mRNA levels, to determine whether these proteins are secreted. Following incubation of HT1080 cells with 32P, labelled STC1 and STC2 were found to be secreted into the medium. STC1 was phosphorylated in vitro by protein kinase C (PKC). In vitro and in vivo phosphorylation both occurred exclusively on serine and the phosphopeptide maps were similar, suggesting that PKC might be the in vivo kinase. STC2 was phosphorylated in vitro by casein kinase II (CK2), in vitro and in vivo phosphorylation were exclusively on serine and the phosphopeptide maps were indistinguishable. Phosphorylation of STC2 in intact cells resulted from the action of an ecto-protein kinase, since exogenous STC2 was phosphorylated by HT1080 cells and no phosphorylated STC2 was detectable inside the cells. The ectokinase activity was abolished by heparin and GTP could substitute for ATP as the phosphate donor, indicative of an ecto-CK2-like activity. The in vitro CK2 phosphorylation site was shown by matrix-assisted laser-desorption ionization–time-of-flight MS to be a single serine located between Ser-285 and Ser-298 in the C-terminal region of STC2. This is the first report of the secretion of STC1 or STC2 from mammalian cells. We conclude that these human fibrosarcoma cells express both STC1 and STC2 as secreted phosphoproteins in vivo, with STC2 being phosphorylated by an ecto-CK2-like enzyme.

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Regulation of mitogen-activated protein kinase cascade in adult rat heart preparations in vitro.

Circulation Research ◽

10.1161/01.res.75.5.932 ◽

1994 ◽

Vol 75 (5) ◽

pp. 932-941 ◽

Cited By ~ 58

Author(s):

A Lazou ◽

M A Bogoyevitch ◽

A Clerk ◽

S J Fuller ◽

J Marshall C ◽

...

Keyword(s):

Protein Kinase ◽

Rat Heart ◽

Mitogen Activated Protein Kinase ◽

Adult Rat ◽

Mitogen Activated Protein ◽

Kinase Cascade ◽

Protein Kinase Cascade

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Insulin increases mRNA levels of protein kinase C-α and -β in rat adipocytes and protein kinase C-α, -β and -θ in rat skeletal muscle

Biochemical Journal ◽

10.1042/bj3080181 ◽

1995 ◽

Vol 308 (1) ◽

pp. 181-187 ◽

Cited By ~ 33

Author(s):

A Avignon ◽

M L Standaert ◽

K Yamada ◽

H Mischak ◽

B Spencer ◽

...

Keyword(s):

Adipose Tissue ◽

Protein Kinase C ◽

Protein Kinase ◽

Mrna Levels ◽

Rat Adipocytes ◽

Kinase C ◽

Pkc Epsilon ◽

Pkc Alpha

Effects of insulin of levels of mRNA encoding protein kinase C (PKC)-alpha, PKC-beta, PKC-epsilon and PKC-theta were examined by ribonuclease protection assay in primary cultures of rat adipocytes in vitro, and in rat adipose tissue and gastrocnemius muscle in vivo. In all cases, insulin increased the levels of PKC-alpha mRNA and PKC-beta mRNA, and, in muscle, insulin also increased the level of PKC-theta mRNA. PKC-epsilon mRNA levels, on the other hand, were not altered significantly. Insulin also stimulated the apparent translocation of PKC-alpha, -beta, -epsilon and -theta, to the membrane fractions of adipocytes, adipose tissue and gastrocnemius muscles, and, in some instances, total PKC levels were diminished, e.g. PKC-alpha and PKC-beta in cultured adipocytes in vitro and/or whole adipose tissue in vivo, and PKC-alpha and PKC-theta in the gastrocnemius muscle. Thus, insulin-induced increases in PKC mRNA may have been partly compensatory in nature to restore PKC levels following translocation and proteolytic losses. However, much more severe depletion of PKC-alpha and PKC-beta by phorbol ester treatment in cultured rat adipocytes in vitro resulted in, if anything, smaller increases in PKC-alpha mRNA and PKC-beta mRNA, and it therefore appears that insulin effects on PKC mRNA levels were not simply due to decreases in respective PKC levels. In addition, effects of insulin, particularly on PKC-beta mRNA, could not be attributed to increased glucose metabolism, which alone decreased PKC-beta mRNA in cultured adipocytes in vitro. We conclude that insulin-induced translocation and degradation of PKC-alpha, PKC-beta and PKC-theta are attended by selective increases in their mRNAs. This mechanism of increasing mRNA may be important in maintaining PKC levels during the continued action of insulin.

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Anti-echinococcal effect of verapamil involving the regulation of the calcium/calmodulin-dependent protein kinase Ⅱ response in vitro and in a murine infection model

10.21203/rs.3.rs-107151/v2 ◽

2021 ◽

Author(s):

Hai-Jun Gao ◽

Xu-Dong Sun ◽

Yan-Ping Luo ◽

Hua-Sheng Pang ◽

Xing-Ming Ma ◽

...

Keyword(s):

Protein Kinase ◽

Mrna Expression ◽

Calcium Content ◽

Mouse Serum ◽

Mrna Levels ◽

Dependent Protein Kinase ◽

Calcium Calmodulin Dependent ◽

Dependent Protein

Abstract Background: Echinococcosis caused by the larvae of cestodes of the genus Echinococcus is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and the assocaiated side effects after long-time use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of verapamil on Echinococcus with a particular focus on the regulatory effect of verapamil on calcium/calmodulin-dependent protein kinase Ⅱ (Ca2+/CaM-CamKⅡ) in infected mice.Methods: The anti-echinococcal effects of verapamil on E. granulosus protoscoleces (PSC) in vitro and E. multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by verapamil were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and Alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CamKⅡ in infected mice, and in the mRNA levels of CamKⅡ in E. granulosus PSC were evaluated after treatment with verapamil by immunohistochemistry and/or real-time quantitative polymerase chain reaction.Results: In vitro, E. granulosus PSC could be killed by verapamil at a concentration of 0.5 μg/mL or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied obvious calcium loss and downregulation of CamKⅡ mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg verapamil, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CamKⅡ protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with verapamil.Conclusions: Verapamil exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca2+/CaM-CamKⅡ, which were over-activated by parasitic infection. The results suggested that Ca2+/CaM-CamKⅡ may be a novel drug-target, and verapamil was shown to be a potential anti-echinococcal drug for controlling echinococcosis in the future.

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Anti-echinococcal effect of verapamil involving regulating calcium/calmodulin-dependent protein kinase Ⅱ response in vitro and in a murine infection model

10.21203/rs.3.rs-107151/v1 ◽

2020 ◽

Author(s):

Hai-Jun Gao ◽

Xu-Dong Sun ◽

Yan-Ping Luo ◽

Hua-Sheng Pang ◽

Xing-Ming Ma ◽

...

Keyword(s):

Protein Kinase ◽

Calcium Content ◽

Morphological Alteration ◽

Mrna Levels ◽

Down Regulation ◽

Dependent Protein Kinase ◽

Calcium Calmodulin Dependent ◽

Dependent Protein

Abstract Background: Echinococcosis caused by the larval stage of cestode of the genus Echinococcus is a parasitic zoonosis, imposing serious threat on the health of humans and animals globally. Albendazole is the drug of choice for treatment of echinococcosis, but it is difficult to meet the clinical challenge in chemotherapy due to its low curative rate and severe side effects. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. Previous study has showed that verapamil can suppress the growth of Echinococcus granulosus (E. granulosus) larva, but the mechanism remains unclear. The aim of the present study is to gain insight into the anti-echinococcal effect of verapamil on Echinococcus with particular focus on the regulatory role of verapamil to calcium/calmodulin-dependent protein kinase Ⅱ (Ca2+/CaM-CamK Ⅱ) in infected mice.Methods: The anti-echinococcal effect of verapamil on E. granulosus protoscolex (PSCx) in vitro and Echinococcus multilocularis (E. multilocularis) metacestodes in infected mice were assessed. The morphological alteration of Echinococcus spp. induced by verapamil was observed by scanning electron microscope (SEM), and the changes of calcium content in both parasite and mice sera and livers were measured by SEM-energy dispersive spectrometer, inductively coupled plasma mass spectrometry and alizarin red staining accordingly. Additionally, the changes on the protein and mRNA levels of CaM and CamK Ⅱ in infected mice, and on the mRNA levels of CamK Ⅱ in E. granulosus PSCx after treatment of verapamil were evaluated by immunohistochemistry or/and real-time quantitative polymerase chain reaction (RT-qPCR).Results: In vitro, E. granulosus PSCx could be killed by verapamil at 0.5 μg/mL or more within 8 days. Under these conditions, the ultrastructure of PSCx was damaged and accompanied with obvious calcium loss and down-regulation of CamK Ⅱ mRNA. In vivo, the weight and the calcium content of E. multilocularis metacestodes from the mice were reduced after treatment with verapamil at 40 mg/kg, meanwhile, an elevation of calcium content in the serum and liver of infected mice was observed. In addition, down-regulation of both the over-expressed CaM and CamK Ⅱ proteins and mRNAs in the liver of mice infected with E. multilocularis metacestodes were found after treatment of verapamil.Conclusions: Verapamil exerted a parasiticidal activity on Echinococcus both in vitro and in vivo through down-regulation of Ca2+/CaM-CamK Ⅱ that was over-activated by parasitic infection. It has been speculated that the Ca2+/CaM-CamK Ⅱ can be a novel drug-target and verapamil is shown to be a potential anti-echinococcal drug for controlling echinococcosis in the future.

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