Calpain Inhibitors Alter the Excitable Membrane Properties of Cultured Aplysia Neurons

2008 ◽  
Vol 100 (5) ◽  
pp. 2784-2793 ◽  
Author(s):  
Arkady Khoutorsky ◽  
Micha E. Spira
Keyword(s):  
Calcium Influx ◽  
Potassium Conductance ◽  
Cysteine Proteases ◽  
Excitable Membrane ◽  
Membrane Properties ◽  
Calpain Inhibitor ◽  
Calcium Dependent ◽  
Aplysia Neurons ◽  
The One ◽  
Calpain Inhibitors

The calpain superfamily of calcium-dependent papain-like cysteine proteases constitutes highly conserved proteases that function to posttranslationally modify substrates by partial proteolysis. Calpains are known to proteolyze >100 substrates that lack strong sequence homology. Consequently, the calpain superfamily has been implicated in playing a central role in diverse physiological and pathological processes. Investigation of the physiological functions of calpains, on the one hand, and the need to develop pharmacological reagents to inhibit calpain-mediated pathological processes, on the other hand, led to the development of numerous calpain inhibitors. Using cultured Aplysia neurons and voltage-clamp analysis, we report here that the calpain inhibitors calpeptin, MG132, and the calpain inhibitor XII inhibit voltage-gated potassium conductance and moderately reduce the sodium conductance. These consequently lead to spike broadening and increased calcium influx. Such alterations of the excitable membrane properties may alter the normal patterns of neuronal and muscle electrical activities and thus should be taken into account when evaluating the effects of calpain inhibitors as protective/therapeutic drugs and as research tools.

scholarly journals Calpain 1 and 2 Are Required for RNA Replication of Echovirus 1

2007 ◽  
Vol 82 (3) ◽  
pp. 1581-1590 ◽  
Author(s):  
Paula Upla ◽  
Varpu Marjomäki ◽  
Liisa Nissinen ◽  
Camilla Nylund ◽  
Matti Waris ◽  
...  
Keyword(s):  
Cysteine Proteases ◽  
Host Cells ◽  
Calpain Inhibitor ◽  
Calpain Activity ◽  
Caveolin 1 ◽  
Calcium Dependent ◽  
Cytoplasmic Proteins ◽  
Calpain 2 ◽  
Calpain Inhibitors

ABSTRACT Calpains are calcium-dependent cysteine proteases that degrade cytoskeletal and cytoplasmic proteins. We have studied the role of calpains in the life cycle of human echovirus 1 (EV1). The calpain inhibitors, including calpeptin, calpain inhibitor 1, and calpain inhibitor 2 as well as calpain 1 and calpain 2 short interfering RNAs, completely blocked EV1 infection in the host cells. The effect of the inhibitors was not specific for EV1, because they also inhibited infection by other picornaviruses, namely, human parechovirus 1 and coxsackievirus B3. The importance of the calpains in EV1 infection also was supported by the fact that EV1 increased calpain activity 3 h postinfection. Confocal microscopy and immunoelectron microscopy showed that the EV1/caveolin-1-positive vesicles also contain calpain 1 and 2. Our results indicate that calpains are not required for virus entry but that they are important at a later stage of infection. Calpain inhibitors blocked the production of EV1 particles after microinjection of EV1 RNA into the cells, and they effectively inhibited the synthesis of viral RNA in the host cells. Thus, both calpain 1 and calpain 2 are essential for the replication of EV1 RNA.

Isoproterenol enhances a calcium-independent potassium current in mouse anterior pituitary tumor cells

1992 ◽  
Vol 68 (1) ◽  
pp. 117-123
Author(s):  
R. Weik ◽  
J. Spiess
Keyword(s):  
Tumor Cells ◽  
Pituitary Tumor ◽  
Anterior Pituitary ◽  
Potassium Current ◽  
Calcium Influx ◽  
Potassium Conductance ◽  
Patch Clamp Technique ◽  
Calcium Dependent ◽  
Gs Protein ◽  
Current Reduction

1. The patch-clamp technique was used to study the action of the beta-adrenergic agonist (-)-isoproterenol in anterior pituitary tumor cells of the mouse. 2. (-)-Isoproterenol induced an inward-rectifying potassium conductance with half-maximal stimulation at a concentration of approximately 67 nM. The isomer (+)-isoproterenol was less effective in stimulating the current. 3. The effect of (-)-isoproterenol was abolished by cholera toxin treatment, indicating the involvement of a Gs protein, whereas pertussis toxin treatment did not exhibit a current reduction. 4. We blocked or stimulated phosphorylation pathways in cells to test the involvement of adenosine 3',5'-cyclic monophosphate (cAMP). It was concluded that the current stimulation probably was not exclusively mediated by cAMP. 5. Activation of calcium-dependent potassium channels by an isoproterenol-induced calcium influx into the cell could be excluded. 6. Therefore it is suggested that the observed activation of a potassium current by isoproterenol could be directly mediated by a Gs protein

In vitro selection of Phytomonas serpens cells resistant to the calpain inhibitor MDL28170: alterations in fitness and expression of the major peptidases and efflux pumps

Parasitology ◽  
2017 ◽  
Vol 145 (3) ◽  
pp. 355-370 ◽  
Author(s):  
SIMONE S. C. OLIVEIRA ◽  
INÊS C. GONÇALVES ◽  
VITOR ENNES-VIDAL ◽  
ANGELA H. C. S. LOPES ◽  
RUBEM F. S. MENNA-BARRETO ◽  
...  
Keyword(s):  
In Vitro Selection ◽  
Oncopeltus Fasciatus ◽  
Calpain Inhibitor ◽  
Insect Vector ◽  
Peptidase Activity ◽  
Wild Type ◽  
Calcium Dependent ◽  
Calpain Inhibitors ◽  
Selection Of

SUMMARYThe species Phytomonas serpens is known to express some molecules displaying similarity to those described in trypanosomatids pathogenic to humans, such as peptidases from Trypanosoma cruzi (cruzipain) and Leishmania spp. (gp63). In this work, a population of P. serpens resistant to the calpain inhibitor MDL28170 at 70 µm (MDLR population) was selected by culturing promastigotes in increasing concentrations of the drug. The only relevant ultrastructural difference between wild-type (WT) and MDLR promastigotes was the presence of microvesicles within the flagellar pocket of the latter. MDLR population also showed an increased reactivity to anti-cruzipain antibody as well as a higher papain-like proteolytic activity, while the expression of calpain-like molecules cross-reactive to anti-Dm-calpain (from Drosophila melanogaster) antibody and calcium-dependent cysteine peptidase activity were decreased. Gp63-like molecules also presented a diminished expression in MDLR population, which is probably correlated to the reduction in the parasite adhesion to the salivary glands of the insect vector Oncopeltus fasciatus. A lower accumulation of Rhodamine 123 was detected in MDLR cells when compared with the WT population, a phenotype that was reversed when MDLR cells were treated with cyclosporin A and verapamil. Collectively, our results may help in the understanding of the roles of calpain inhibitors in trypanosomatids.

scholarly journals The Calcium-Dependent Protease Calpain-1 Links TRPC6 Activity to Podocyte Injury

2018 ◽  
Vol 29 (8) ◽  
pp. 2099-2109 ◽  
Author(s):  
Kim A.T. Verheijden ◽  
Ramon Sonneveld ◽  
Marinka Bakker-van Bebber ◽  
Jack F.M. Wetzels ◽  
Johan van der Vlag ◽  
...  
Keyword(s):  
Transient Receptor Potential ◽  
Calcium Influx ◽  
Receptor Potential ◽  
Transient Receptor Potential Channel ◽  
Slit Diaphragm ◽  
Calpain Inhibitor ◽  
Podocyte Injury ◽  
Calcium Dependent ◽  
Transient Receptor ◽  
Calcineurin Activity

BackgroundThe hallmark of podocytopathies, such as FSGS, is podocyte injury resulting in proteinuria. Transient receptor potential channel C6 (TRPC6) is a calcium-conducting ion channel expressed at the slit diaphragm. TRPC6 gain-of-function mutations and glomerular TRPC6 overexpression are associated with proteinuria. However, the pathways linking TRPC6 to podocyte injury, which is characterized by loss of the slit diaphragm protein nephrin, activation of several intracellular pathways (including calcineurin-NFAT signaling), and cytoskeletal rearrangement, remain elusive.MethodsWe tested whether the calcium-dependent protease calpain-1 mediates TRPC6-dependent podocyte injury in human and experimental FSGS and cultured podocytes.ResultsCompared with kidneys of healthy controls, kidneys of patients with FSGS had increased TRPC6 expression, increased calpain and calcineurin activity, and reduced expression of the calpain target Talin-1, which links the actin cytoskeleton to integrins and is critical for podocyte cytoskeletal stability. In a rat model of human FSGS, increased glomerular and urinary calpain activity associated with reduced Talin-1 abundance, enhanced calcineurin activity, and increased proteinuria. Treatment with the calpain inhibitor calpeptin prevented these effects. In cultured podocytes, pharmacologic stimulation of TRPC6-dependent calcium influx increased calpain-1 and calcineurin activity and reduced Talin-1 expression, and knockdown of TRPC6 or calpain-1 prevented these effects.ConclusionsWe elucidated a novel mechanism that links TRPC6 activity to calpain-1 activation and through Talin-1 loss and possibly, calcineurin activation, the podocyte injury characterizing FSGS. Therefore, calpain-1 and/or TRPC6 inhibition could be future therapeutic options to treat patients with FSGS or other podocytopathies.

scholarly journals Rab coupling protein is selectively degraded by calpain in a Ca2+-dependent manner

2005 ◽  
Vol 389 (1) ◽  
pp. 223-231 ◽  
Author(s):  
Nicolas MARIE ◽  
Andrew J. LINDSAY ◽  
Mary W. McCAFFREY
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Calpain Inhibitor ◽  
Dependent Manner ◽  
Interacting Proteins ◽  
Wild Type ◽  
A431 Cells ◽  
Calcium Dependent ◽  
Coupling Protein ◽  
Calpain Inhibitors

RCP (Rab coupling protein) belongs to the recently identified Rab11-FIPs (Rab11 family of interacting proteins). All the Rab-FIP members have the ability to bind Rab11 tightly via a Rab-binding domain located near their C-termini. RCP belongs to the class I Rab11-FIP subfamily, characterized by the presence of a conserved C2 domain near its N-terminus. The function of this protein in Rab11-dependent membrane trafficking remains to be fully understood. In the present study, we have identified three putative PEST (Pro, Glu, Ser/Thr-rich) sequences in RCP. PEST motifs play a role in targeting a protein for proteolytic degradation. We have demonstrated that RCP undergoes calcium-dependent degradation which can be prevented by specific calpain inhibitors. Using a mutant, lacking the three PEST sequences, RCPΔPEST, we demonstrated that they are necessary for the cleavage of RCP by calpains. When expressed in A431 cells, RCPΔPEST displays significantly greater localization to the plasma membrane, compared with the wild-type protein. Similarly, treatment with the calpain inhibitor, calpeptin, results in the redistribution of endogenous RCP to the periphery of the cell. We propose that once the Rab11/RCP-regulated cargo has been delivered from the endocytic recycling compartment to the plasma membrane, RCP is inactivated by calpain-mediated proteolysis.

scholarly journals Listeriolysin O-Dependent Bacterial Entry into the Cytoplasm Is Required for Calpain Activation and Interleukin-1α Secretion in Macrophages Infected with Listeria monocytogenes

2010 ◽  
Vol 78 (5) ◽  
pp. 1884-1894 ◽  
Author(s):  
Sita R. Dewamitta ◽  
Takamasa Nomura ◽  
Ikuo Kawamura ◽  
Hideki Hara ◽  
Kohsuke Tsuchiya ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Intracellular Calcium ◽  
Mutant Strain ◽  
Calcium Influx ◽  
Cytolytic Activity ◽  
Calpain Inhibitor ◽  
Listeriolysin O ◽  
Wild Type ◽  
Calcium Dependent ◽  
Interleukin 1Α

ABSTRACT Listeriolysin O (LLO), an hly-encoded cytolysin of Listeria monocytogenes, plays an essential role in the entry of L. monocytogenes into the host cell cytoplasm. L. monocytogenes-infected macrophages produce various proinflammatory cytokines, including interleukin-1α (IL-1α), that contribute to the host immune response. In this study, we have examined IL-1α production in macrophages infected with wild-type L. monocytogenes or a nonescaping mutant strain deficient for LLO (Δhly). Expression of IL-1α mRNA and accumulation of pro-IL-1α in the cytoplasm were induced by both strains. In contrast, the secretion of the mature form of IL-1α from infected macrophages was observed in infection with wild-type L. monocytogenes but not with the Δhly mutant. A recovery of the ability to induce IL-1α secretion was shown in a mutant strain complemented with the hly gene. The Toll-like receptor (TLR)/MyD88 signaling pathway was exclusively required for the expression of pro-IL-1α, independently of LLO-mediated cytoplasmic entry of L. monocytogenes. The LLO-dependent secretion of mature IL-1α was abolished by addition of calcium chelators, and only LLO-producing L. monocytogenes strains were able to induce elevation of the intracellular calcium level in infected macrophages. A calcium-dependent protease, calpain, was implicated in the maturation and secretion of IL-1α induced by LLO-producing L. monocytogenes strains based on the effect of calpain inhibitor. Functional activation of calpain was detected in macrophages infected with LLO-producing L. monocytogenes strains but not with a mutant strain lacking LLO. These results clearly indicated that LLO-mediated cytoplasmic entry of bacteria could induce the activation of intracellular calcium signaling, which is essential for maturation and secretion of IL-1α in macrophages during L. monocytogenes infection through activation of a calcium-dependent calpain protease. In addition, recombinant LLO, when added to macrophages infected with the Δhly strain, could induce calcium influx and IL-1α secretion at doses exhibiting cytolytic activity, suggesting that LLO produced by intracellular L. monocytogenes may be implicated in induction of calcium influx through pore formation.

scholarly journals The expression of calpain 1 and calpain 2 in spermatogenic cells and spermatozoa of the mouse

Reproduction ◽  
2005 ◽  
Vol 129 (4) ◽  
pp. 435-442 ◽  
Author(s):  
Irit Ben-Aharon ◽  
Paula R Brown ◽  
Nir Etkovitz ◽  
Edward M Eddy ◽  
Ruth Shalgi
Keyword(s):  
Acrosome Reaction ◽  
Cysteine Proteases ◽  
Calpain Inhibitor ◽  
Dependent Manner ◽  
Spermatogenic Cells ◽  
Calcium Dependent ◽  
Protein Levels ◽  
Complex Process ◽  
And Function ◽  
Mitosis And Meiosis

There is some evidence suggesting that Ca2+is involved in processes that occur during the development and function of spermatozoa. Calcium-dependent proteins, such as calmodulin, are expressed during mammalian spermatogenesis further suggesting that Ca2+takes part in its regulation. However, the precise roles of Ca2+in spermatogenesis remain to be elucidated. Calpains are a family of Ca2+-dependent cysteine proteases whose members are expressed ubiquitously or in a tissue-specific manner. Calpain has been demonstrated to mediate specific Ca2+-dependent processes including cell fusion, mitosis and meiosis. We herein followed the expression pattern of calpain’s ubiquitous isoforms, 1 and 2, throughout spermatogenesis at the RNA and protein levels by RT-PCR and Western blotting analysis. Both RNA and protein studies revealed that these isoforms are expressed in all spermatogenic cells. The expression of calpain 1 levels is slightly higher in spermatocytes entering the meiotic phase. Both calpain isoforms are also expressed in mouse spermatozoa and are localized to the acrosomal cap. Inducing capacitated spermatozoa to undergo the acrosome reaction in the presence of a selective calpain inhibitor significantly reduced the acrosome reaction rate in a dose-dependent manner. Thus, calpain, a pluripotential protease with numerous substrates, may serve as an effector in more than one pathway in the complex process of spermatogenesis and in the events preceding fertilization, such as the acrosome reaction.

scholarly journals Pneumolysin-Dependent Calpain Activation and Interleukin-1α Secretion in Macrophages Infected with Streptococcus pneumoniae

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Rendong Fang ◽  
Rui Wu ◽  
Huihui Du ◽  
Meilan Jin ◽  
Yajing Liu ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Calcium Influx ◽  
Critical Role ◽  
Pneumococcal Infections ◽  
Content Type ◽  
Calpain Activation ◽  
Calcium Dependent ◽  
Interleukin 1Α ◽  
Calpain Inhibitors

ABSTRACT Pneumolysin (PLY), a major virulence factor of Streptococcus pneumoniae, is a pore-forming cytolysin that modulates host innate responses contributing to host defense against and pathogenesis of pneumococcal infections. Interleukin-1α (IL-1α) has been shown to be involved in tissue damage in a pneumococcal pneumonia model; however, the mechanism by which this cytokine is produced during S. pneumoniae infection remains unclear. In this study, we examined the role of PLY in IL-1α production. Although the strains induced similar levels of pro-IL-1α expression, wild-type S. pneumoniae D39, but not a deletion mutant of the ply gene (Δply), induced the secretion of mature IL-1α from host macrophages, suggesting that PLY is critical for the maturation and secretion of IL-1α during S. pneumoniae infection. Further experiments with calcium chelators and calpain inhibitors indicated that extracellular calcium ions and calpains (calcium-dependent proteases) facilitated the maturation and secretion of IL-1α from D39-infected macrophages. Moreover, we found that PLY plays a critical role in calcium influx and calpain activation, as elevated intracellular calcium levels and the degradation of the calpain substrate α-fodrin were detected in macrophages infected with D39 but not the Δply strain. These results suggested that PLY induces the influx of calcium in S. pneumoniae-infected macrophages, followed by calpain activation and subsequent IL-1α maturation and secretion.

scholarly journals The Diverse Calpain Family in Trypanosomatidae: Functional Proteins Devoid of Proteolytic Activity?

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 299
Author(s):  
Vítor Ennes-Vidal ◽  
Marta Helena Branquinha ◽  
André Luis Souza dos Santos ◽  
Claudia Masini d’Avila-Levy
Keyword(s):  
Proteolytic Activity ◽  
Current Knowledge ◽  
Current Therapy ◽  
Calcium Dependent ◽  
Administration Routes ◽  
Life Threatening ◽  
Living Organisms ◽  
Almost All ◽  
Open Question ◽  
Calpain Inhibitors

Calpains are calcium-dependent cysteine peptidases that were originally described in mammals and, thereafter, their homologues were identified in almost all known living organisms. The deregulated activity of these peptidases is associated with several pathologies and, consequently, huge efforts have been made to identify selective inhibitors. Trypanosomatids, responsible for life-threatening human diseases, possess a large and diverse family of calpain sequences in their genomes. Considering that the current therapy to treat trypanosomatid diseases is limited to a handful of drugs that suffer from unacceptable toxicity, tough administration routes, like parenteral, and increasing treatment failures, a repurposed approach with calpain inhibitors could be a shortcut to successful chemotherapy. However, there is a general lack of knowledge about calpain functions in these parasites and, currently, the proteolytic activity of these proteins is still an open question. Here, we highlight the current research and perspectives on trypanosomatid calpains, overview calpain description in these organisms, and explore the potential of targeting the calpain system as a therapeutic strategy. This review gathers the current knowledge about this fascinating family of peptidases as well as insights into the puzzle: are we unable to measure calpain activity in trypanosomatids, or are the functions of these proteins devoid of proteolytic activity in these parasites?

scholarly journals Intracellular versus extracellular inhibition of calpain I causes differential effects on pain in a rat model of joint inflammation

2021 ◽  
Vol 17 ◽  
pp. 174480692110161
Author(s):  
Jason J McDougall ◽  
Miranda McConnell ◽  
Allison R Reid
Keyword(s):  
Joint Pain ◽  
Acute Inflammation ◽  
Weight Bearing ◽  
Joint Inflammation ◽  
Local Administration ◽  
Calpain Inhibitor ◽  
Mast Cell Tryptase ◽  
Calcium Dependent ◽  
Protease Activated Receptor 2 ◽  
Arthritic Joints

Calpain I is a calcium-dependent cysteine protease which has dual effects on tissue inflammation depending on its cellular location. Intracellularly, calpain I has pro-inflammatory properties but becomes anti-inflammatory when exteriorised into the extracellular space. In this study, the effect of calpain I on joint pain was investigated using the kaolin/carrageenan model of acute synovitis. Evoked pain behaviour was determined by von Frey hair algesiometry and non-evoked pain was measured using dynamic hindlimb weight bearing. Local administration of calpain I reduced secondary allodynia in the acute inflammation model and this effect was blocked by the cell impermeable calpain inhibitor E-64c. Calpain I also blocked the algesic effect of the protease activated receptor-2 (PAR-2) cleaving enzyme mast cell tryptase. The cell permeable calpain blocker E-64d also produced analgesia in arthritic joints. These data suggest that calpain I produces disparate effects on joint pain viz. analgesia when present extracellularly by disarming PAR-2, and pro-algesic when the enzyme is inside the cell.

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