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PubMed1998

Modulation of the in situ activity of tissue transglutaminase by calcium and GTP.

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes the posttranslational modification of proteins by transamidation of specific polypeptide-bound glutamine residues. Previous in vitro studies have demonstrated that the transamidating activity of tTG requires calcium and is inhibited by GTP. To investigate the endogenous regulation of tTG, a quantitative in situ transglutaminase (TG) activity assay was developed. Treatment of human neuroblastoma SH-SY5Y cells with retinoic acid (RA) resulted in a significant increase in tTG levels and in vitro TG activity. In contrast, basal in situ TG activity did not increase concurrently with RA-induced increased tTG levels. However, stimulation of cells with the calcium-mobilizing drug maitotoxin (MTX) resulted in increases in in situ TG activity that correlated (r2 = 0.76) with increased tTG levels. To examine the effects of GTP on in situ TG activity, tiazofurin, a drug that selectively decreases GTP levels, was used. Depletion of GTP resulted in a significant increase in in situ TG activity; however, treatment of SH-SY5Y cells with a combination of MTX and tiazofurin resulted in significantly less in situ TG activity compared with treatment with MTX alone. This raised the possibility of calcium-dependent proteolysis due to the effects of tiazofurin, because in vitro GTP protects tTG against proteolysis by trypsin. Studies with a selective membrane permeable calpain inhibitor indicated that tTG is likely to be an endogenous substrate of calpain, and that depletion of GTP increases tTG degradation after elevation of intracellular calcium levels. TG activity was also increased in response to activation of muscarinic cholinergic receptors, which increases intracellular calcium through inositol 1,4,5-trisphosphate generation. The results of these experiments demonstrate that selective changes in calcium and GTP regulate the activity and levels of tTG in situ.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

Neuroexcitatory actions of ciguatoxin on brain regions associated with thermoregulation.

We have investigated the action of ciguatoxin (CTX) in the mouse following i.p. administration. CTX (0.5 mouse units) induced a rapid (10-20 min) decrease in body temperature that persisted for several hours. This corresponded closely with a neuroexcitatory action of ciguatoxin on c-fos mRNA in the brain. We identified the neuronal pathways activated by CTX action in the mouse brain by immunostaining of c-fos translational product, a biomarker for neuroexcitability. c-fos-like immunoreactivity was prominent in the hypothalamus, including the medial preoptic and supraoptic nuclei. Immunostaining was also evident in certain regions of the brain stem, including the locus coeruleus, dorsolateral parabranchial nucleus, area postrema and the nucleus of the solitary tract. These studies indicate that CTX has neuroexcitatory actions on brain stem regions receiving vagal afferents and ascending pathways associated with visceral and thermoregulatory responses.

Animals↗
PubMed1999

Maitotoxin and P2Z/P2X(7) purinergic receptor stimulation activate a common cytolytic pore.

The effects of maitotoxin (MTX) on plasmalemma permeability are similar to those caused by stimulation of P2Z/P2X(7) ionotropic receptors, suggesting that 1) MTX directly activates P2Z/P2X(7) receptors or 2) MTX and P2Z/P2X(7) receptor stimulation activate a common cytolytic pore. To distinguish between these two possibilities, the effect of MTX was examined in 1) THP-1 monocytic cells before and after treatment with lipopolysaccharide and interferon-gamma, a maneuver known to upregulate P2Z/P2X(7) receptor, 2) wild-type HEK cells and HEK cells stably expressing the P2Z/P2X(7) receptor, and 3) BW5147.3 lymphoma cells, a cell line that expresses functional P2Z/P2X(7) channels that are poorly linked to pore formation. In control THP-1 monocytes, addition of MTX produced a biphasic increase in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)); the initial increase reflects MTX-induced Ca(2+) influx, whereas the second phase correlates in time with the appearance of large pores and the uptake of ethidium. MTX produced comparable increases in [Ca(2+)](i) and ethidium uptake in THP-1 monocytes overexpressing the P2Z/P2X(7) receptor. In both wild-type HEK and HEK cells stably expressing the P2Z/P2X(7) receptor, MTX-induced increases in [Ca(2+)](i) and ethidium uptake were virtually identical. The response of BW5147.3 cells to concentrations of MTX that produced large increases in [Ca(2+)](i) had no effect on ethidium uptake. In both THP-1 and HEK cells, MTX- and Bz-ATP-induced pores activate with similar kinetics and exhibit similar size exclusion. Last, MTX-induced pore formation, but not channel activation, is greatly attenuated by reducing the temperature to 22 degrees C, a characteristic shared by the P2Z/P2X(7)-induced pore. Together, the results demonstrate that, although MTX activates channels that are distinct from those activated by P2Z/P2X(7) receptor stimulation, the cytolytic/oncotic pores activated by MTX- and Bz-ATP are indistinguishable.

Adenosine Triphosphate↗
PubMed2001

The mechanism of maitotoxin-induced elevation of the cytosolic free calcium level in rat cerebrocortical synaptosomes.

The present study was conducted to elucidate the mechanism of the maitotoxin (MTX)-induced increase in intrasynaptosomal free calcium level ([Ca2+]i). The MTX (1 ng/ml)-induced increase in [Ca2+]i was partially inhibited by the omission of extracellular Ca2+ (Ca2+e) or the addition of verapamil, but not by adding nifedipine, omega-agatoxin IVA, omega-conotoxin GVIA and omega-conotoxin MVIIC. An increase in [Ca2+]i in the absence of Ca2+e was sensitive to procaine, TMB-8, genistein and verapamil, but not to ryanodine and U-73122. These results may suggest that MTX increases [Ca2+]i by stimulating Ca2+ entry through voltage-independent nonselective cation channels and Ca2+ release from stores through a phospholipase C-gamma1-mediated pathway in rat cerebrocortical synaptosomes.

Animals↗
PubMedDate not supplied

Determination of brevetoxins in shellfish by the neuroblastoma assay.

A neuroblastoma assay for determination of brevetoxins in shellfish was developed together with a method for sample cleanup that allows separation of brevetoxins from most of the components that cause matrix interference in the assay. This improved assay method was applied to a range of shellfish samples with different characteristics. Extracts of naturally contaminated and nontoxic shellfish together with extracts spiked with known amounts of toxin were tested. The results demonstrated that brevetoxins could be reliably detected in shellfish extracts at concentrations below the regulatory limit. Brevetoxin activity was detected in 15 of 23 samples from 5 separate toxicity incidents in which shellfish tested positive in the neurotoxic shellfish poisoning (NSP) mouse bioassay. Twelve of these positive NSP results came from 2 toxicity incidents. Yessotoxin was the major contributor to toxicity in 2 other incidents, although some samples contained both yessotoxin and brevetoxin. The sample from the remaining incident contained an unidentified toxin bioactivity, together with gymnodimine. In contrast to earlier versions of the neuroblastoma assay, gymnodimine was not detected by this modified method.

Animals↗
PubMed2000

Processing of cdk5 activator p35 to its truncated form (p25) by calpain in acutely injured neuronal cells.

Recently, it was shown that conversion of cdk5 activator protein p35 to a C-terminal fragment p25 promotes a deregulation of cdk5 activity, which may contribute to neurodegeneration in Alzheimer's disease. In this study, we present evidence that calpain is a protease involved in the conversion of p35 to p25. To activate calpain, rat cerebellar granule neurons were treated with maitotoxin (MTX). A C-terminus-directed anti-p35 antibody detected that p35 conversion to p25 paralleled the formation of calpain-generated alpha-spectrin (alpha-fodrin) breakdown products (SBDP's) in a maitotoxin-dose-dependent manner. Two calpain inhibitors (MDl28170 and SJA6017) reduced p35 processing but were unchanged when exposed to the caspase inhibitor carbobenzoxy-Asp-CH(2)OC(=O)-2, 6-dichlorobenzene or the proteasome inhibitors (lactacystin and Z-Ile-Glu(OtBu)Ala-Leu-CHO). p35 protein was also degraded to p25 when rat brain lysate was subjected to in vitro digestion with purified mu- and m-calpains. Additionally, in a rat temporary middle cerebral artery occlusion model, p35 processing to p25 again paralleled SBDP formation in the ischemic core. Lastly, in malonate-injured rat brains, the ipsilateral side showed a striking correlation of SBDP formation with p35 to p25 conversion and tau phosphorylation (at Ser202 and Thr205) increase. These data suggest that calpain is a major neuronal protease capable of converting p35 to p25 and might play a pathological role of activating cdk5 and its phosphorylation of tau in Alzheimer's disease.

Animals↗
PubMed1985

Calcium mobilization potentiates prolactin release induced by protein kinase C activators.

The in vitro effect of synthetic diacylglycerol (DG) and phorbol myristate acetate (PMA), potent stimulators of protein kinase C, was studied on prolactin release. These substances increased, in a concentration-dependent manner, prolactin release from primary cultures of anterior pituitary cells. Similarly, exposure of pituitary cells to phospholipase C, which liberates endogenous DG from various substrates, also enhanced prolactin release. The effect of Ca2+ mobilization on PMA-, synthetic DG- or phospholipase C-induced prolactin release was examined. A23187 at 400 nM or 2 ng/ml maitotoxin, a Ca2+ channel activator, did not affect prolactin release by themselves, but enhanced the release of prolactin induced by DG, PMA or phospholipase C. The stimulatory effects of DG, PMA and phospholipase C on prolactin release were reduced by co-incubation with dopamine. These results suggest that the presumed activation of protein kinase C by DG and mobilization of Ca2+ may be synergistically involved in the regulation of prolactin release. Dopamine appears to inhibit prolactin release at a point distal to the DG-enhanced stimulation of the process.

Animals↗
PubMed1999

Maitotoxin-induced nerve growth factor production accompanied by the activation of a voltage-insensitive Ca2+ channel in C6-BU-1 glioma cells.

1. The aim of the present study was to determine the effects of maitotoxin on nerve growth factor production and the Ca2+ influx in clonal rat glioma cells (C6-BU-1). 2. Maitotoxin (1 - 10 ng ml-1) induced a profound increase in 45Ca2+ influx in an extracellular Ca2+-dependent manner. However, high KCl had no effect at all. These effects were supported by the results from the analysis of intracellular Ca2+ concentration using fura 2. 3. The maitotoxin-induced 45Ca2+ influx was inhibited by inorganic Ca2+ antagonists, such as Mg2+, Mn2+ and Co2+. The inhibitory effect of Co2+ was antagonized by increasing the extracellular Ca2+ concentrations. 4. Maitotoxin (3 ng ml-1) as well as A-23187 (1microM) and dibutyryl cyclic AMP (0.5 mM) caused an acceleration of nerve growth factor (NGF) production in C6-BU-1 cells, as determined by NGF enzyme immunoassay. 5. Reverse transcription polymerase chain reaction (RT - PCR) analysis showed that maitotoxin (10 ng ml-1) enhanced the expression of NGF mRNA, which was abolished by the removal of extracellular Ca2+. A-23187 also accelerated its expression. 6. These results suggest that maitotoxin activates a voltage-insensitive Ca2+ channel and accelerates NGF production mediated through a Ca2+ signalling pathway in C6-BU-1 glioma cells.

Animals↗
PubMed1994

Appearance of depolarization- and maitotoxin-induced [Ca2+]i elevation in single LAN-1 human neuroblastoma cells on exposure to retinoic acid.

LAN-1 is a human neuroblastoma cell line that, in the undifferentiated state, does not respond to membrane depolarization with an elevation of [Ca2+]i, monitored by fura-2 single-cell microfluorimetry. The exposure of LAN-1 cells to the differentiating agent retinoic acid induced the appearance of [Ca2+]i elevation elicited by 55 mM K+. Maitotoxin, a putative activator of voltage-sensitive Ca2+ channels, did not evoke an elevation of [Ca2+]i in undifferentiated LAN-1 cells, but produced a marked and sustained increase in [Ca2+]i when superfused in retinoic acid-treated cells. Both high K(+)- and maitotoxin-induced [Ca2+]i elevation in retinoic acid-differentiated LAN-1 cells was reversed by the lanthanide Gd3+, an inorganic Ca(2+)-entry blocker, and by the snail toxin omega-conotoxin GVIA, which interacts with the N subtype of voltage-sensitive Ca2+ channels. In contrast, both Bay K 8644 and nimodipine, dihydropyridines that selectively activate or block, respectively, the L-channel subtype, were completely ineffective. The tumor promoter phorbol 12-myristate 13-acetate (100 nM), a protein kinase C activator, inhibited the elevation of [Ca2+]i due to Ca2+ influx elicited by membrane depolarization. K(+)-induced [Ca2+]i elevation appeared 24 h after the addition of retinoic acid and reached the highest magnitude after 72 h. Furthermore, 8 days after the removal of the differentiating agent from the culture medium, the high K(+)-induced increase of [Ca2+]i was still present.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1997

Up-regulation of sodium channel subunit mRNAs and their cell surface expression by antiepileptic valproic acid: activation of calcium channel and catecholamine secretion in adrenal chromaffin cells.

Treatment of cultured bovine adrenal chromaffin cells with a therapeutic concentration (0.6 mM) of valproic acid (VPA) for > 24 h caused a time-dependent (t1/2 = 74 h) increase in [3H]saxitoxin binding up to 1.4-fold without altering the KD value; it was prevented by the simultaneous treatment with cycloheximide (an inhibitor of protein synthesis). VPA also raised Na+ channel alpha- and beta 1-subunit mRNA levels 1.4- and 1.7-fold at 24 h, and 1.6- and 1.8-fold at 72 h, respectively. Chronic (but not acute) exposure to VPA enhanced 22Na+ influx caused by various concentrations of veratridine 1.4-2.1-fold, even when assayed in the presence of Na+,K(+)-ATPase inhibitor, but did not change the EC50 value of veratridine. Ptychodiscus brevis toxin-3 allosterically potentiated veratridine-induced 22Na+ influx by approximately 2-fold in VPA-treated cells as in nontreated cells. Long-term treatment with VPA augmented veratridine-induced 45Ca2+ influx via voltage-dependent Ca2+ channels and catecholamine secretion, but had no effect on 45Ca2+ influx and catecholamine secretion caused by high K+ (a direct activation of voltage-dependent Ca2+ channels). Chronic treatment with VPA also enhanced nicotine-induced 22Na+ influx via the nicotinic receptor-ion channel complex 1.2-1.4-fold with little change in the EC50 value of nicotine, thereby increasing the nicotine-induced 45Ca2+ influx via voltage-dependent Ca2+ channels and catecholamine secretion. These results suggest that chronic treatment with VPA up-regulates cell surface expression of Na+ channels via the transcription/translation-dependent mechanisms, and probably of nicotinic receptors, thereby resulting in the enhancement of Ca2+ channel gating and catecholamine secretion.

Animals↗
PubMed2000

Brevetoxin-induced autocrine excitotoxicity is associated with manifold routes of Ca2+ influx.

Real-time alterations in intracellular Ca2+ ([Ca2+]i) were monitored in fluo-3-loaded cerebellar granule neurons (CGNs) exposed to the brevetoxin PbTx-1. [Ca2+]i was measured using a fluorescent plate reader (FLIPR), which measures simultaneously the mean intracellular Ca2+ change in a population of cultured cells in each well of a 96-well plate. PbTx-1 produced rapid and concentration-dependent increases in neuronal [Ca2+]i with a potency nearly identical to that determined previously for PbTx-1-induced neurotoxicity. The NMDA receptor antagonists MK-801, dextrorphan, and D(-)-2-amino-5-phosphonopentanoic acid, and tetanus toxin, an inhibitor of Ca2+-dependent exocytotic neurotransmitter release, effected significant reductions in both the integrated fluo-3 fluorescence response and excitatory amino acid release and protected CGNs against PbTx-1 neurotoxicity. The L-type Ca2+ channel antagonist nifedipine produced a modest reduction in the fluo-3 response but reduced substantially the plateau phase of the PbTx-1 increment in [Ca2+]i when combined with MK-801. When nifedipine and MK-801 were combined with the Na+/Ca2+ exchanger (reversed mode) inhibitor KB-R7943, the PbTx-1 increment in [Ca2+]i was nearly completely attenuated. These data show that Ca2+ entry into PbTx-1-exposed CGNs occurs through three primary routes: NMDA receptor ion channels, L-type Ca2+ channels, and reversal of the Na+/Ca2+ exchanger. There was a close correlation between reduction of the integrated fluo-3 fluorescence response and the level of neuroprotection afforded by blockers of each Ca2+ entry pathway; however, simultaneous blockade of L-type Ca2+ channels and the Na+/Ca2+ exchanger, although reducing the integrated [Ca2+]i response to a level below that provided by NMDA receptor blockade alone, failed to completely attenuate PbTx-1 neurotoxicity. This finding suggests that in addition to total [Ca2+]i load, neuronal vulnerability is governed principally by the NMDA receptor Ca2+ influx pathway.

2-Amino-5-phosphonovalerate↗
PubMed1996

Site of covalent labeling by a photoreactive batrachotoxin derivative near transmembrane segment IS6 of the sodium channel alpha subunit.

The binding site for batrachotoxin, a lipid-soluble neurotoxin acting at Na+ channel receptor site 2, was localized using a photoreactive radiolabeled batrachotoxin derivative to covalently label purified and reconstituted rat brain Na+ channels. In the presence of the brevetoxin 1 from Ptychodiscus brevis and the pyrethroid RU51049, positive allosteric enhancers of batrachotoxin binding, a protein with an apparent molecular mass of 240 kDa corresponding to the Na+ channel alpha subunit was specifically covalently labeled. The region of the alpha subunit specifically photolabeled by the photoreactive batrachotoxin derivative was identified by antibody mapping of proteolytic fragments. Even after extensive trypsinization, and anti-peptide antibody recognizing an amino acid sequence adjacent to Na+ channel transmembrane segment IS6 was able to immunoprecipitate up to 70% of the labeled peptides. Analysis of a more complete digestion with trypsin or V8 protease indicated that the batrachotoxin receptor site is formed in part by a portion of domain I. The identification of a specifically immunoprecipitated photolabeled 7.3-kDa peptide containing transmembrane segment S6 from domain I restricted the site of labeling to residues Asn-388 to Glu-429 if V8 protease digestion was complete or Leu-380 to Glu-429 if digestion was incomplete. These results implicate the S6 transmembrane region of domain I of the Na+ channel alpha subunit as an important component of the batrachotoxin receptor site.

Allosteric Regulation↗
PubMed1986

Protein kinase C activators and calcium-mobilizing agents synergistically increase GH, LH, and TSH secretion from anterior pituitary cells.

A series of studies was designed to determine the effects of protein kinase C activators on TSH, LH, and GH release from anterior pituitary cells. A 15-min incubation of cultured pituitary cells with synthetic diacylglycerol or phorbol myristate acetate, stimulators of protein kinase C, increased GH, LH, and TSH release. Similarly phospholipase C, which liberates endogenous diacylglycerol, stimulated GH, LH, and TSH secretion. The potentiation of the effects of protein kinase C activators is achieved by calcium mobilization in various cell types. The results of the present studies show that calcium ionophore A23187 or calcium channel activator maitotoxin potentiate diacylglycerol-, phorbol ester-, or phospholipase C-induced GH, LH, or TSH release. These findings suggest that activation of protein kinase C by diacylglycerol and mobilization of calcium may be synergistically involved in the regulation of GH, LH, and TSH release.

Animals↗
PubMed1990

Calcitonin decreases thyrotropin-releasing hormone-stimulated prolactin release through a mechanism that involves inhibition of inositol phosphate production.

Calcitonin is present in both the hypothalamus and pituitary of the rat, and normal rat anterior pituitary cells express calcitonin receptors. Calcitonin has been reported to inhibit or to stimulate PRL release from rat anterior pituitary cells. We have investigated the effects of salmon calcitonin on basal and stimulated PRL release from rat anterior pituitary cells and have studied the effects of this peptide on the intracellular biochemical pathways involved in PRL release. Salmon calcitonin had no significant effect on basal PRL release, but inhibited (P less than 0.01) TRH-stimulated PRL release without affecting PRL release promoted by angiotensin II, neurotensin, phorbol myristate acetate (a protein kinase C activator), or maitotoxin (a calcium channel activator). Salmon calcitonin had no effect on the increase in PRL release and intracellular cAMP concentration after exposure of pituitary cells to vasoactive intestinal peptide or forskolin. Salmon calcitonin significantly decreased (P less than 0.01) the TRH-stimulated rise in inositol phosphates without affecting the angiotensin II-stimulated increase in inositol phosphates. Similarly, salmon calcitonin decreased the TRH-stimulated increase in cytosolic calcium and arachidonate liberation by pituitary cells. We conclude that salmon calcitonin selectively decreases TRH-stimulated PRL release by a mechanism that involves a decrease in inositol phosphate production, as well as a subsequent reduction in cytosolic calcium levels and in arachidonate liberation.

Angiotensin II↗
PubMed1991

Photoaffinity labeling of the brevetoxin receptor on sodium channels in rat brain synaptosomes.

Brevetoxin, a neurotoxin isolated from the marine dinoflagellate Ptychodiscus brevis, has been derivatized into a photoaffinity probe by carbodiimide linkage to p-azidobenzoic acid. Rosenthal analysis of a tritiated p-azidobenzoate brevetoxin derivative indicates that specific binding of the toxin occurs at two distinct and separate sites, with Kd and Bmax values of 0.21 nM and 2.12 pmol/mg of protein for the high affinity site and 50.7 nM and 91.5 pmol/mg of protein for the low affinity site, respectively. Binding of tritiated photoaffinity probe to the high affinity/low capacity site can be displaced in a competitive manner by native brevetoxin (Kd = 1.9 nM), demonstrating a specific competitive interaction with the receptor site. Rat brain synaptosomes, covalently labeled with the brevetoxin photoaffinity probe, were subjected to detergent solubilization. The covalently labeled membrane protein was estimated to have a Stokes radius of 55 +/- 3 A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed specific labeling of a 260-kDa protein. Treatment with 2-mercaptoethanol and neuraminidase resulted in retention of brevetoxin binding to this high molecular weight protein. The affinity-purified membrane protein-brevetoxin photoaffinity probe complex was specifically recognized by a sodium channel antibody directed against the intracellular side of transmembrane segment IS6. The sodium channel alpha subunit is implicated as the specific site of brevetoxin interaction.

Affinity Labels↗
PubMed1992

Agents that stimulate phosphoinositide turnover also elevate cAMP in SK-N-SH human neuroblastoma cells.

Stimulation of m1 and of m3 muscarinic receptors has previously been shown to increase intracellular cAMP levels in a variety of cells. Although the mechanism underlying this response is not fully understood, it has been hypothesized to be secondary to the IP3-mediated rise in intracellular calcium. In order to determine whether other means of elevating intracellular calcium also raise cAMP levels, we stimulated SK-N-SH human neuroblastoma cells with bradykinin or with maitotoxin. Both of these agents stimulated phospholipase C, stimulated inositol phosphate release and elevated cAMP levels, thus demonstrating that this cAMP response is not unique to muscarinic receptor stimulation.

Bradykinin↗
PubMed2006

Identification of pectenotoxin-11 as 34S-hydroxypectenotoxin-2, a new pectenotoxin analogue in the toxic dinoflagellate Dinophysis acuta from New Zealand.

A new pectenotoxin, which has been named pectenotoxin-11 (PTX11), was isolated from the dinoflagellate Dinophysis acuta collected from the west coast of New Zealand. The structure of PTX11 was determined as 34S-hydroxypectenotoxin-2 by tandem mass spectrometry and UV and NMR spectroscopy. PTX11 appears to be only the third pectenotoxin identified as a natural biosynthetic product from algae after pectenotoxin-2 and pectenotoxin-12. The LD50 of PTX11 determined by mouse intraperitoneal injection was 244 microg/kg. The LD(min) of PTX11 in these experiments was 250 microg/kg. No signs of toxicity were recorded in mice following an oral dose of PTX11 at 5000 microg/kg. No diarrhea was observed in any of the animals administered with the test substance by either route of administration. Unlike pectenotoxin-2 (PTX2), PTX11 was not readily hydrolyzed to its corresponding seco acid by enzymes from homogenized green-lipped mussel (Perna canaliculus) hepatopancreas.

Animals↗
PubMed2000

Impaired mitochondrial function results in increased tissue transglutaminase activity in situ.

Tissue transglutaminase (tTG) is a transamidating enzyme that is elevated in Huntington's disease (HD) brain and may be involved in the etiology of the disease. Further, there is evidence of impaired mitochondrial function in HD. Therefore, in this study, we examined the effects of mitochondrial dysfunction on the transamidating activity of tTG. Neuroblastoma SH-SY5Y cells stably overexpressing human tTG or mutated inactive tTG were treated with 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase. 3-NP treatment of tTG-expressing cells resulted in a significant increase of TG activity in situ. In vitro measurements demonstrated that 3-NP had no direct effect on tTG activity. However, 3-NP treatment resulted in a significant decrease of the levels of GTP and ATP, two potent inhibitors of the transamidating activity of tTG. No significant changes in the intracellular levels of calcium were observed in 3-NP-treated cells. Treatment with 3-NP in combination with antioxidants significantly reduced the 3-NP-induced increase in in situ TG activity, demonstrating that oxidative stress is a contributing factor to the increase of TG activity. This study demonstrates for the first time that impairment of mitochondrial function significantly increases TG activity in situ, a finding that may have important relevance to the etiology of HD.

Adenosine Triphosphate↗