HT4 Cells Research Articles

HT22 cell differentiation reduces insulin receptor levels

Purpose: The brain is an insülin-sensitive organ and has widespread insulin receptor (IR) expression. IR signaling in the brain is essential for neuronal development, feeding behavior, body weight, and cognitive processes such as attention, learning, and memory. HT22 cells, which are derived from parent HT4 cells that are immortalized from primary mouse hippocampal neuronal cells are used in research related to insulin signaling. However, the role of these cells in insulin signaling is not known. In this study, we aimed to examine IR levels in cells differentiated using neurobasal medium. Material and methods: For the study, briefly, the cells were seeded in 6-well plates at 2x105 cells/well for 24 h. After the cells reached 80% confluence, the normal growth medium was replaced with a differentiation medium and the cells were incubated for 72 hours at 37 0C in 5% CO2. Western blot procedure was used to determine the expression of the IR. Result: Our results show that differentiation of HT22 cells stimulates neurite outgrowth. Furthermore, IR protein levels were significantly downregulated in differentiated HT22 cells. Conclusion: This finding may require careful consideration of the use of neurobasal medium in conditions where IR signaling is important.

Differentiated HT22 cells as a novel model for in vitro screening of serotonin reuptake inhibitors.

The mouse hippocampal neuronal cell line HT22 is frequently used as an in vitro model to investigate the role of hippocampal cholinergic neurons in cognitive functions. HT22 cells are derived from hippocampal neuronal HT4 cells. However, whether these cells exhibit the serotonergic neuronal phenotype observed in mature hippocampal neurons has not been determined yet. In this present study, we examined whether the differentiation of HT22 cells enhances the serotonergic neuronal phenotype, and if so, whether it can be used for antidepressant screening. Our results show that differentiation of HT22 cells promoted neurite outgrowth and upregulation of N-methyl-D-aspartate receptor and choline acetyltransferase, which is similar to that observed in primary cultured hippocampal neurons. Furthermore, proteins required for serotonergic neurotransmission, such as tryptophan hydroxylase 2, serotonin (5-hydroxytryptamine, 5-HT)1a receptor, and serotonin transporter (SERT), were significantly upregulated in differentiated HT22 cells. The transcription factor Pet-1 was upregulated during HT22 differentiation and was responsible for the regulation of the serotonergic neuronal phenotype. Differentiation also enhanced the functional serotonergic properties of HT22 cells, as evidenced by increase in intracellular 5-HT levels, serotonin transporter SERT glycosylation, and 5-HT reuptake activity. The sensitivity of 5-HT reuptake inhibition by venlafaxine in differentiated HT22 cells (IC50, 27.21nM) was comparable to that in HEK293 cells overexpressing serotonin transporter SERT (IC50, 30.65nM). These findings suggest that the differentiation of HT22 cells enhances their functional serotonergic properties, and these cells could be a potential in vitro system for assessing the efficacy of antidepressant 5-HT reuptake inhibitors.

HT22 hippocampal neuronal cell line possesses functional cholinergic properties

Aims Hippocampal cholinergic hypofunction is known to be involved in the cognitive deficits of Alzheimer's disease, but the detailed mechanisms remain to be elucidated. In order to establish an in vitro hippocampal cholinergic neuronal model for the relevant mechanistic studies, we have characterized a widely used hippocampal neuronal cell line, HT22, a sub-line derived from parent HT4 cells that were originally immortalized from primary mouse hippocampal neuronal culture. Main methods Western blot and immunocytochemistry were used to examine expression of cholinergic markers in HT22 cells. High potassium-evoked [ 3H]ACh release was used to evaluate the cholinergic functional properties of the cells. Key findings We found that HT22 cells express essential cholinergic markers, such as the high affinity choline transporter, choline acetyltransferase, vesicular acetylcholine transporter, and muscarinic acetylcholine receptors. Exposure of HT22 cells to high potassium evoked [ 3H]ACh release in a dose-dependent manner. In addition, the [ 3H]ACh release was significantly potentiated when presynaptic autoreceptors were blocked. Significance Our results suggest that HT22 cells possess functional cholinergic properties, and can be used for an in vitro model for defining the mechanisms in cognitive deficits of Alzheimer's disease.

Neuronal AKAP150 coordinates PKA and Epac-mediated PKB/Akt phosphorylation

In diverse neuronal processes ranging from neuronal survival to synaptic plasticity cyclic adenosine monophosphate (cAMP)-dependent signaling is tightly connected with the protein kinase B (PKB)/Akt pathway but the precise nature of this connection remains unknown. In the current study we investigated the effect of two mainstream pathways initiated by cAMP, cAMP-dependent protein kinase (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) on PKB/Akt phosphorylation in primary cortical neurons and HT-4 cells. We demonstrate that PKA activation leads to a reduction of PKB/Akt phosphorylation, whereas activation of Epac has the opposite effect. This effect of Epac on PKB/Akt phosphorylation was mediated by Rap activation. The increase in PKB/Akt phosphorylation after Epac activation could be blocked by pretreatment with Epac2 siRNA and to a somewhat smaller extent by Epac1 siRNA. PKA, PKB/Akt and Epac were all shown to establish complexes with neuronal A-kinase anchoring protein150 (AKAP150). Interestingly, activation of Epac increased phosphorylation of PKB/Akt complexed to AKAP150. From experiments using PKA-binding deficient AKAP150 and peptides disrupting PKA anchoring to AKAPs, we conclude that AKAP150 acts as a key regulator in the two cAMP pathways to control PKB/Akt phosphorylation.

Inhibition of ATP-induced calcium influx in HT4 cells by glucocorticoids: involvement of protein kinase A1

In our previous observations, adenosine triphosphate (ATP) was found to evoke immediate elevations in intracellular free calcium concentration ([Ca2+]i) in HT4 neuroblastoma cells of mice. We tried to see if a brief pretreatment of glucocorticoids could inhibit the Ca2+ response and reveal the underlying signaling mechanism. Measurement of [Ca2+]i was carried out using the dual-wavelength fluorescence method with Fura-2 as the indicator. Pre-incubation of HT4 cells for 5 min with corticosterone (B) or bovine serum albumin conjugated corticosterone (B-BSA) inhibited the peak [Ca2+]i increments in a concentration-dependent manner. Cortisol and dexamethasone had a similar action, while deoxycorticosterone and cholesterol were ineffective. Both extracellular Ca2+ influx and internal Ca2+ release contributed to ATP-induced [Ca2+]i elevation. The brief treatment with only B attenuated Ca2+ influx. Furthermore, the [Ca2+]i elevation induced by the P2X receptor agonist adenosine 5'-(beta, gamma-methylene) triphosphate (beta, gamma-meATP) was also suppressed. The rapid inhibitory effect of B can be reproduced by forskolin 1 mmol/L and blocked by H89 20 mmol/L. Neither nuclear glucocorticoid receptor antagonist mifepristone nor protein kinase C inhibitors influenced the rapid action of B. Our results suggest that glucocorticoids modulate P2X receptor-medicated Ca2+ influx through a membrane-initiated, non-genomic and PKA-dependent pathway in HT4 cells.

Tocotrienol: The Natural Vitamin E to Defend the Nervous System?

Vitamin E is essential for normal neurological function. It is the major lipid-soluble, chain-breaking antioxidant in the body, protecting the integrity of membranes by inhibiting lipid peroxidation. Mostly on the basis of symptoms of primary vitamin E deficiency, it has been demonstrated that vitamin E has a central role in maintaining neurological structure and function. Orally supplemented vitamin E reaches the cerebrospinal fluid and brain. Vitamin E is a generic term for all tocopherols and their derivatives having the biological activity of RRR-alpha-tocopherol, the naturally occurring stereoisomer compounds with vitamin E activity. In nature, eight substances have been found to have vitamin E activity: alpha-, beta-, gamma- and delta-tocopherol; and alpha-, beta-, gamma- and delta-tocotrienol. Often, the term vitamin E is synonymously used with alpha-tocopherol. Tocotrienols, formerly known as zeta, , or eta-tocopherols, are similar to tocopherols except that they have an isoprenoid tail with three unsaturation points instead of a saturated phytyl tail. Although tocopherols are predominantly found in corn, soybean, and olive oils, tocotrienols are particularly rich in palm, rice bran, and barley oils. Tocotrienols possess powerful antioxidant, anticancer, and cholesterol-lowering properties. Recently, we have observed that alpha-tocotrienol is multi-fold more potent than alpha-tocopherol in protecting HT4 and primary neuronal cells against toxicity induced by glutamate as well as by a number of other toxins. At nanomolar concentration, tocotrienol, but not tocopherol, completely protected neurons by an antioxidant-independent mechanism. Our current work identifies two major targets of tocotrienol in the neuron: c-Src kinase and 12-lipoxygenase. Dietary supplementation studies have established that tocotrienol, fed orally, does reach the brain. The current findings point towards tocotrienol as a potent neuroprotective form of natural vitamin E.

The Organellular Chloride Channel Protein CLIC4/mtCLIC Translocates to the Nucleus in Response to Cellular Stress and Accelerates Apoptosis

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to the mitochondria and cytoplasm of keratinocytes and participates in the apoptotic response to stress. We now show that multiple stress inducers cause the translocation of cytoplasmic CLIC4 to the nucleus. Immunogold electron microscopy and confocal analyses indicate that nuclear CLIC4 is detected prior to the apoptotic phenotype. CLIC4 associates with the Ran, NTF2, and Importin-alpha nuclear import complexes in immunoprecipitates of lysates from cells treated with apoptotic/stress-inducing agents. Deletion or mutation of the nuclear localization signal in the C terminus of CLIC4 eliminates nuclear translocation, whereas N terminus deletion enhances nuclear localization. Targeting CLIC4 to the nucleus via adenoviral transduction accelerates apoptosis when compared with cytoplasmic CLIC4, and only nuclear-targeted CLIC4 causes apoptosis in Apaf null mouse fibroblasts or in Bcl-2-overexpressing keratinocytes. These results indicate that CLIC4 nuclear translocation is an integral part of the cellular response to stress and may contribute to the initiation of nuclear alterations that are associated with apoptosis.

N-Acetylcysteine and Celecoxib Lessen Cadmium Cytotoxicity Which Is Associated with Cyclooxygenase-2 Up-regulation in Mouse Neuronal Cells

In many neurodegenerative disorders, aggregates of ubiquitinated proteins are detected in neuronal inclusions, but their role in neurodegeneration remains to be defined. To identify intracellular mechanisms associated with the appearance of ubiquitin-protein aggregates, mouse neuronal HT4 cells were treated with cadmium. This heavy metal is a potent cell poison that mediates oxidative stress and disrupts the ubiquitin/proteasome pathway. In the current studies, the following intracellular events were found to be also induced by cadmium: (i) a specific rise in cyclooxygenase-2 (COX-2) gene expression but not COX-1; (ii) an increase in the extracellular levels of the proinflammatory prostaglandin E2, a product of COX-2; and (iii) production of 4-hydroxy-2-nonenal-protein adducts, which result from lipid peroxidation. In addition, cadmium treatment led to the accumulation of high molecular weight ubiquitin-COX-2 conjugates and perturbed COX-2 glycosylation. The thiol-reducing antioxidant N-acetylcysteine, and, to a lesser extent, the COX-2 inhibitor celecoxib, attenuated the loss of cell viability induced by cadmium demonstrating that oxidative stress and COX-2 activation contribute to cadmium cytotoxicity. These findings establish that disruption of the ubiquitin/proteasome pathway is not the only event triggered by cadmium. This oxidative stressor also activates COX-2 function. Both events could be triggered by formation of 4-hydroxy-2-nonenal as a result of cadmium-induced lipid peroxidation. Proinflammatory responses stimulated by oxidative stressors that mimic the cadmium effects may, therefore, be important initiators of the neurodegenerative process and exacerbate its progress.

Evoked intracellular Ca2+ elevations in HT4 neuroblastoma cells.

In this study, membrane depolarization and multiple neurotransmitters (5-HT, acetylcholine, histamine, norepinephrine, epinephrine, glutamate, and ATP) were tested for the ability to elevate the intracellular free Ca2+ concentration ([Ca2+]i) in mouse HT4 neuroblastoma cells. Apart from ATP, none of the treatments gave rise to a detectable Ca2+ response, no matter whether the cells were subjected to temperature-induced neuronal differentiation. Our results provide pharmacological evidence for the co-existence in HT4 cells of both P2X and P2Y receptors, the activation of which by ATP led to Ca2+ influx and Ca2+ release, respectively. The P2Y receptor was found to couple to more than one type of G protein in the signaling pathway, causing the activation of phospholipase C (PLC) and Ca2+ mobilization from intracellular stores. cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) attenuated ATP-evoked [Ca2+]i elevations in different ways. However, no correlation was identified between neuronal differentiation and the ATP-evoked Ca2+ responses in HT4 cells. This work indicates that HT4 cells can serve as a good model to study P2 purinoceptor-associated signaling pathways.

Cellular and mitochondrial changes in glutamate-induced HT4 neuronal cell death

Elevated levels of extracellular glutamate are neurotoxic. The cytotoxic property of extracellular glutamate is known to mediate two primary mechanisms, excitotoxicity and excitotoxicity-independent processes. The excitotoxicity-independent pathway was investigated in the current study in a mouse hippocampal-derived HT4 cell line. Exposure of HT4 cells to glutamate for 12 h induced loss of cell viability preceded by rapid loss of intracellular reduced glutathione followed by accumulation of intracellular reactive oxygen species, elevation of intracellular Ca 2+, progressive loss of mitochondrial membrane potential swelling and loss of mitochondrial outer membrane integrity. Glutamate-induced loss of DNA integrity has been detected. The antioxidants α-tocopherol and trolox, mitochondrial calcium uniporter inhibitor Ruthenium Red and protein synthesis inhibitor cycloheximide all showed protection against glutamate-induced toxicity. None of the protective agents except for α-tocopherol controlled the glutamate-induced reactive oxygen species build-up. However, these cell death regulators prevented the glutamate-induced mitochondrial damage and regulated glutamate-induced increase in intracellular Ca 2+. Carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone, a mitochondrial uncoupler, partially protected against glutamate-induced cell death and mitochondrial damage, while the mitochondrial ribosomal inhibitor chloramphenicol and extracellular Ca 2+ chelator ethylene glycol-bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid did not protect the cells against glutamate treatment. The results of this study demonstrated that mitochondrial dysfunction was a key event in the excitotoxicity-independent component of neuronal cell death. Reactive oxygen species accumulation and glutathione depletion were prominent in glutamate-treated cells; however, these events were not direct mediators of cell death.

Antioxidants and herbal extracts protect HT-4 neuronal cells against glutamate-induced cytotoxicity

Antioxidant therapy has been shown to be beneficial in neurological disorders including Alzheimer's disease and cerebral ischemia. Glutamate-induced cytotoxicity in HT-4 neuronal cells has been previously demonstrated to be due to oxidative stress caused by depletion of cellular glutathione (GSH). The present study demonstrates that a wide variety of antioxidants inhibit glutamate-induced cytotoxicity in HT-4 neuronal cells. Low concentrations of α-tocopherol and its analogs were highly effective in protecting neuronal cells against cytotoxicity. Purified flavonoids and herbal extracts of Gingko biloba (EGb 761) and French maritime pine bark (Pycnogenol®) were also effective. We have previously shown that pro-glutathione agents can spare GSH and protect cells from glutamate insult in a C6 glial cell model. The protective effects of nonthiol-based antioxidants tested in the HT-4 line were not mediated via GSH level modulation. In contrast, protective effects of thiol-based pro-glutathione agents α-lipoic acid (LA) and N-acetyl cysteine (NAC) corresponded with a sparing effect on GSH levels in glutamate-treated HT-4 cells. Glutamate-induced cytotoxicity in HT-4 cells is a useful model system for testing compounds or mixtures for antioxidant activity.

Regulation of the murine NMDA-receptor-subunit NR2C promoter by Sp1 and fushi tarazu factor1 (FTZ-F1) homologues.

We have cloned the 5'-region of the murine N-methyl-d-aspartate (NMDA) receptor channel subunit NR2C (GluRepsilon3) gene and characterized the cis- and trans-activating regulatory elements responsible for its tissue specific activity. By using a native epsilon3-promoter/lacZ-construct & various 5'-deletion constructs, we compared beta-galactosidase expression in non-neuronal NIH3T3 cells and in neuronal epsilon3-gene-expressing HT-4 cells and show that large parts of the epsilon3 promoter are responsible for the repression of the epsilon3 gene in non-neuronal cells. Deletion of exon 1 sequences led to an enhancement of epsilon3 transcription, suggesting a role of the 5'-untranslated region in epsilon3 gene regulation. Sequence analysis of the promoter region revealed potential binding sites for the transcription factor Sp1, the murine fushi tarazu factor1 (FTZ-F1) homologues, embryonic LTR binding proteins (ELP1,2,3) and steroidogenic factor (SF-1), as well as for the chicken ovalbumin upstream promoter transcription-factor (COUP-TF). Electrophoretic mobility shift assays confirmed specific binding of Sp1, SF-1 and COUP-TFI. Whereas point mutation studies indicate that, in neuronal HT-4 cells, Sp1 is apparently not critically involved in basal epsilon3 gene transcription, SF1 is a positive regulator. This was evident from a selective enhancement of epsilon3-promoter-driven reporter gene expression upon cotransfection of an SF1-expression vector, which was reverted by deletion and point mutation of the SF1 binding site.

Intracellular Localization of SNAP-23 to Endosomal Compartments

We have reexamined the intracellular localization of the ubiquitously expressed target membrane SNAP receptor (t-SNARE), SNAP-23. While SNAP-23 appears on the plasma membrane, in the cell types examined there is a significant pool associated with endosomal compartments. Immuno-staining and expression of green fluorescent protein-tagged SNAP-23, show that it has a punctate, perinuclear localization in HepG2 and HT4 cells. This distribution overlaps significantly with transferrin receptor and slightly with the late endosome/lysosomal protein LAMP-1. The localization of SNAP-23 changes as HepG2 cells polarize. Initially it is concentrated at sites of cell-cell contact and then almost exclusively to the apical (or bile canalicular) domain of the cell. These data are consistent with a role for SNAP-23 in both endosome-plasma membrane trafficking as well as endosome-endosome transport.

The central role of lipid peroxidation in glutamate neurotoxicity in HT-4 cells: [formula omitted] and Enrique Cadenas School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033

The central role of lipid peroxidation in glutamate neurotoxicity in HT-4 cells: [formula omitted] and Enrique Cadenas School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033

The ubiquitin/proteasome pathway: friend or foe in zinc-, cadmium-, and H2O2-induced neuronal oxidative stress.

One of the hallmarks of neurodegeneration is the accumulation of ubiquitinated proteins in intraneuronal inclusions in the cytosol, endosomes/lysosomes and nuclei of affected cells. The relationship between inclusion production and cell viability is not well understood. On the one hand inclusions may be beneficial and result from an attempt of the cell to isolate a subclass of ubiquitinated proteins that are not effectively degraded. On the other hand, the inclusions may impede normal cell function contributing to cell death. To address this issue we treated mouse neuronal HT4 cells with three toxic agents cadmium, zinc and H2O2, and investigated their effects on glutathione homeostasis, on accumulation of ubiquitinated proteins and on cell viability. The three treatments induce oxidative stress manifested by decreases in glutathione (GSH) and/or increases in protein mixed disulfides (PrSSG). After an overnight recovery period in the absence of treatment, GSH and PrSSG were restored to almost normal levels. However, the levels of ubiquitinated proteins were significantly increased, and cell viability was sharply reduced. These results suggest that the ubiquitin-proteasome pathway is recruited for removal of proteins that are oxidatively modified. However, if the ubiquitinated proteins are not efficiently degraded, they accumulate in the cell and contribute to a decrease in cell viability.

Disruption of the Intracellular Sulfhydryl Homeostasis by Cadmium-induced Oxidative Stress Leads to Protein Thiolation and Ubiquitination in Neuronal Cells

Cadmium is a potent cell poison known to cause oxidative stress by increasing lipid peroxidation and/or by changing intracellular glutathione levels and to affect the ubiquitin/ATP-dependent proteolytic pathway. However, the cellular mechanisms involved in cadmium toxicity are still not well understood, especially in neuronal cells. To investigate the relationship between cadmium-induced oxidative stress and the ubiquitin/ATP-dependent pathway, we treated cultures of neuronal cells with different concentrations of the metal ion. In addition to decreases in glutathione levels, we observed marked increases in protein-mixed disulfides (Pr-SSGs) after exposure of HT4 cells (a mouse neuronal cell line) or rat primary mesencephalic cultures to Cd2+. The increases in intracellular levels of Pr-SSGs were concurrent with increases in the levels of ubiquitinated proteins (Ub proteins) when the HT4 cells were subjected to lower (25 microM or less) concentrations of cadmium. However, higher concentrations of cadmium (50 microM), which were toxic, led to increases in Pr-SSGs but inhibited ubiquitination, probably reflecting inhibition of ubiquitinating enzymes. The cadmium-induced changes in Pr-SSGs and Ub proteins were not affected when more than 85% of intracellular glutathione was removed from the cells by the glutathione synthetase inhibitor L-buthionine-(S,R)-sulfoximine. However, the reducing agent dithiothreitol, which prevented the build up of Pr-SSGs in the cell, also blocked the accumulation of Ub proteins induced by cadmium. In addition, dithiothreitol blocked the effects of the higher toxic (50 microM) concentrations of cadmium on cytotoxicity and on glutathione, Pr-SSGs, and Ub proteins. Together, these results strongly suggest that changes in the levels of intracellular Pr-SSGs and ubiquitin-protein conjugates in neuronal cells are responses closely associated with the disruption of intracellular sulfhydryl homeostasis caused by cadmium-mediated oxidative stress.

Rat Brain p64H1, Expression of a New Member of the p64 Chloride Channel Protein Family in Endoplasmic Reticulum

Many plasma membrane Cl- channels have been cloned, including the cystic fibrosis transmembrane conductance regulator and several members of the voltage-gated ClC family. In contrast, very little is known about the molecular identity of intracellular Cl- channels. We used a polymerase chain reaction-based approach to identify candidate genes in mammalian brain and cloned the cDNA corresponding to rat brain p64H1. This encoded a microsomal membrane protein of predicted Mr 28,635 homologous to the putative intracellular bovine kidney Cl- channel p64. In situ mRNA hybridization histochemistry showed marked expression in hippocampus and cerebellum, and in vitro expression revealed a large cytoplasmic domain, one membrane-spanning segment, and a small nonglycosylated N-terminal luminal domain. The predicted protein contained consensus phosphorylation sites for protein kinase C and protein kinase A, and protein kinase C-mediated phosphorylation increased the Mr of p64H1 to approximately 43,000, characteristic of the native protein in Western blots. Recombinant p64H1 was immunolocalized to the endoplasmic reticulum of human embryonic kidney 293 and HT-4 cells, and incorporation of human embryonic kidney 293 endoplasmic reticulum vesicles into planar lipid bilayers gave rise to intermediate conductance, outwardly rectifying anion channels. Although p64H1 is the first intracellular Cl- channel component or regulator to be identified in brain, Northern blotting revealed transcripts in many other rat tissues. This suggests that p64H1 may contribute widely to intracellular Cl- transport.

Accumulation of ubiquitinated proteins in mouse neuronal cells induced by oxidative stress.

Ubiquitin protein conjugates are commonly detected in neuronal brain inclusions of patients with neurodegenerative disorders. The failure to eliminate the ubiquitin-protein deposits in the degenerating neurons may result from changes in the activity of the ubiquitin/ATP-dependent proteolytic pathway. This proteolytic pathway plays a major role in the degradation of short lived, abnormal and denatured proteins. Cadmium is a potent cell poison and is known to affect the ubiquitin pathway and to cause oxidative stress. Increases in protein mixed-disulfides (Pr-SSG) and decreases in glutathione (GSH) are often used as markers of oxidative stress. To investigate the relationship between the ubiquitin pathway and cellular glutathione (GSH), we treated HT4 cells (a mouse neuronal cell line) and rat mesencephalic primary cultures with different concentrations of the heavy metal. We observed marked increases in Pr-SSG as well as decreases in GSH, after exposure of HT4 cells or primary mesencephalic cultures to Cd2+. Furthermore, our results show that Cd2+ induced the accumulation of ubiquitinated proteins. Detection was by Western blotting of total cell extracts probed with antibodies that recognize ubiquitin-protein conjugates. These results suggest that the ubiquitin-pathway is closely involved in the cell response to cadmium-mediated oxidative stress.

A New Inhibitor of the Chymotrypsin‐Like Activity of the Multicatalytic Proteinase Complex (20S Proteasome) Induces Accumulation of Ubiquitin‐Protein Conjugates in a Neuronal Cell

Exposure of HT4 cells (a mouse neuronal cell line) to a new potent permeable peptidyl aldehyde inhibitor of the chymotrypsin-like activity of the multicatalytic proteinase complex (MPC) causes accumulation of ubiquitinylated proteins. In contrast, inhibition of calpain or treatment with a lysosomotropic agent failed to produce detectable ubiquitin-protein conjugates. The appearance of such conjugates is not a nonspecific phenomenon because incubation with the peptidyl alcohol analogue of the inhibitor does not produce accumulation of ubiquitinylated proteins. The MPC inhibitor may therefore be a useful tool for identification and study of physiological pathways involving MPC. Furthermore, the inhibitor may help develop a model for the study of neurodegeneration where accumulation of ubiquitin-protein conjugates is commonly detected in abnormal brain inclusions.

Conditional activation of cAMP signal transduction by protein kinase C. The effect of phorbol esters on adenylyl cyclase in permeabilized and intact cells.

To understand the convergence of cAMP and protein kinase C signal transduction, adenylyl cyclase isozyme identification and biochemical studies were performed on the HT4 neural cell line. In HT4 cells, basal cAMP production by adenylyl cyclase types I and VI were unaffected by phorbol esters, nor did phorbol esters have any effect on forskolin-induced cAMP production. However, phorbol esters synergistically increased cAMP production when adrenaline receptors were simultaneously activated, indicating a conditional activation of cAMP production by phorbol esters. A permeabilized cell preparation was used to analyze the mechanism by which phorbol esters were affecting cAMP production. This preparation enables G-proteins to be activated directly by GTP gamma S or bacterial toxins. In the permeabilized cell preparation, phorbol esters enhanced cAMP produced by GTP gamma S-activated G-protein. A stimulatory G-protein pathway may be involved since phorbol esters synergistically increased cAMP production by cholera toxin, yet had no effect on that produced by pertussis toxin. In this cell culture model, phorbol esters stimulate cAMP production only when some component of the cAMP cascade is simultaneously activated. Moreover, the pattern of modulation suggests that protein kinase C acts on an activated component of the second messenger system, such as the G-protein or the coupling of the G-protein with adenylyl cyclase, and not on the resting state of the protein components.