Human alternative Klotho mRNA is a nonsense-mediated mRNA decay target inefficiently spliced in renal disease.

Klotho is a renal protein involved in phosphate homeostasis, which is downregulated in renal disease. It has long been considered an antiaging factor. Two Klotho gene transcripts are thought to encode membrane-bound and secreted Klotho. Indeed, soluble Klotho is detectable in bodily fluids, but the relative contributions of Klotho secretion and of membrane-bound Klotho shedding are unknown. Recent advances in RNA surveillance reveal that premature termination codons, as present in alternative Klotho mRNA (for secreted Klotho), prime mRNAs for degradation by nonsense-mediated mRNA decay (NMD). Disruption of NMD led to accumulation of alternative Klotho mRNA, indicative of normally continuous degradation. RNA IP for NMD core factor UPF1 resulted in enrichment for alternative Klotho mRNA, which was also not associated with polysomes, indicating no active protein translation. Alternative Klotho mRNA transcripts colocalized with some P bodies, where NMD transcripts are degraded. Moreover, we could not detect secreted Klotho in vitro. These results suggest that soluble Klotho is likely cleaved membrane-bound Klotho only. Furthermore, we found that, especially in acute kidney injury, splicing of the 2 mRNA transcripts is dysregulated, which was recapitulated by various noxious stimuli in vitro. This likely constitutes a novel mechanism resulting in the downregulation of membrane-bound Klotho.

Poligapolide, a PI3K/Akt inhibitor in immunodeficiency virus type 1 TAT-transduced CHME5 cells, isolated from the rhizome of Polygala tenuifolia.

The rhizome of Polygala tenuifolia WILLD (PT, family Polygalaceae) has been used in traditional Chinese medicine for inflammation, dementia, amnesia, neurasthenia and cancer. The phosphoinositide 3-kinase (PI3K)/Akt inhibitor(s) was isolated from PT by using the cytoprotective phenotype of human immunodeficiency virus type 1 (HIV-1) Tat-transduced CHME5 cells against lipopolysaccharide/cycloheximide. We isolated 9 constituents (1)-(9) from ethyl acetate fraction of PT, which potently showed anti-cytoprotective effect against HIV-1 TAT-transduced cells. Of them, (9R)-(-)-9-peptandecanolide (2), a new compound named poligapolide, most potently abolished the cytoprotective effect of HIV-1 Tat-transduced CHME5 cells. The compound (2) inhibited the phosphorylation of Akt and its downstream molecule, glycogen synthase kinase-3 beta (GSK3ß) in PI3K/Akt cell survival signaling pathway, but did not suppress the phosphorylation of PI3K and pyruvate dehydrogenase lipoamide kinase isozyme 1. Based on these finding, poligapolide may abolish the cytoprotective phenotype of HIV-1 Tat-transduced CHME5 cells by inhibiting Akt phosphorylation in PI3K/Akt pathway.

Hydroxylation of DHEA, androstenediol and epiandrosterone by Mortierella isabellina AM212. Evidence indicating that both constitutive and inducible hydroxylases catalyze 7α- as well as 7ß-hydroxylations of 5-ene substrates.

The course of transformation of DHEA, androstenediol and epiandrosterone in Mortierella isabellina AM212 culture was investigated. The mentioned substrates underwent effective hydroxylation; 5-ene substrates--DHEA and androstenediol--were transformed into a mixture of 7α- and 7ß- allyl alcohols, while epiandrosterone was converted into 7α- (mainly), 11α- and 9α- monohydroxy derivatives. Ketoconazole and cycloheximide inhibition studies suggest the presence of constitutive and substrate-induced hydroxylases in M. isabellina. On the basis of time course analysis of the hydroxylation of DHEA and androstenediol, the oxidation of allyl C(7)-H(α) and C(7)-H(ß) bonds by the same enzyme is a reasonable assumption.

Transforming growth factor-beta1 regulation of laminin gamma1 and fibronectin expression and survival of mouse mesangial cells.

The transforming growth factor-beta (TGF-beta) 1 is a mediator of extracellular matrix (ECM) gene expression in mesangial cells and the development of diabetic glomerulopathy. Here, we investigate the effects of TGF-beta1 on laminin gamma1 and fibronectin polypeptide expression and cell survival in mouse mesangial cells (MES-13). TGF-beta1 (10 ng/ml) stimulates laminin-gamma1 and fibronectin expression approximately two-fold in a time-dependent manner (0-48 h). TGF-beta1 treatment also retards laminin-gamma1 mobility on SDS-gels, and tunicamycin, an inhibitor of the N-linked glycosylation, blocks the mobility shift. TGF-beta1 increases the binding of laminin gamma1 to WGA-agarose and the binding is abolished by tunicamycin suggesting that laminin gamma1 is modified by N-linked glycosylation. TGF-beta1 also elevates fibronectin glycosylation but its mobility is not altered. The degradation of laminin gamma1 and fibronectin proteins is reduced by their glycosylation. In addition, TGF-beta1 enhances mesangial cell viability and metabolic activities initially (0-24 h); however, eventually leads to cell death (24-48 h). TGF-beta1 elevates pro-apoptotic caspase-3 activity and decrease cell cycle progression factor cyclin D1 expression, which parallels cell death. These results indicate that TGF-beta1 plays an important role in ECM expression, protein glycosylation and demise of mesangial cells in the diabetic glomerular mesangium.

IL-4 and IL-13 induce protection of porcine endothelial cells from killing by human complement and from apoptosis through activation of a phosphatidylinositide 3-kinase/Akt pathway.

Vascular endothelial cells (EC) perform critical functions that require a balance of cell survival and cell death. EC death by apoptosis and EC activation and injury by the membrane attack complex of complement are important mechanisms in atherosclerosis and organ graft rejection. Although the effects of various cytokines on EC apoptosis have been studied, little is known about their effects on complement-mediated EC injury. Therefore, we studied the abilities of various cytokines to induce protection of porcine aortic EC against apoptosis and killing by human complement, a model of pig-to-human xenotransplantation. We found that porcine EC incubated with IL-4 or IL-13, but not with IL-10 or IL-11, became protected from killing by complement and apoptosis induced by TNF-alpha plus cycloheximide. Maximal protection required 10 ng/ml IL-4 or IL-13, developed progressively from 12 to 72 h of incubation, and lasted 48-72 h after cytokine removal. Protection from complement was not associated with reduced complement activation, C9 binding, or changes in CD59 expression. Inhibition of PI3K prevented development of protection; however, inhibition of p38 MAPK or p42/44 MAPK had no effect. IL-4 and IL-13 induced rapid phosphorylation of Akt. Although protection was inhibited by an Akt inhibitor and a dominant negative Akt mutant transduced into EC, it was induced by transduction of EC with the constitutively active Akt variant, myristylated Akt. We conclude that IL-4 and IL-13 can induce protection of porcine EC against killing by apoptosis and human complement through activation of the PI3K/Akt signaling pathway.

Distinct IkappaB kinase regulation in adult T cell leukemia and HTLV-I-transformed cells.

We have recently shown constitutive IkappaB kinase (IKK) activation and aberrant p52 expression in adult T cell leukemia (ATL) cells that do not express human T cell leukemia virus type I (HTLV-I) Tax, but the mechanism of IKK activation in these cells has remained unknown. Here, we demonstrate distinct regulation of IKK activity in ATL and HTLV-I-transformed T cells in response to protein synthesis inhibition or arsenite treatment. Protein synthesis inhibition for 4 h by cycloheximide (CHX) barely affects IKK activity in Tax-positive HTLV-I-transformed cells, while it diminishes IKK activity in Tax-negative ATL cells. Treatment of ATL cells with a proteasome inhibitor MG132 prior to protein synthesis inhibition reverses the inhibitory effect of CHX, and MG132 alone greatly enhances IKK activity. In addition, treatment of HTLV-I-transformed cells with arsenite for 1 h results in down-regulation of IKK activity without affecting Tax expression, while 8 h of arsenite treatment does not impair IKK activity in ATL cells. These results indicate that a labile protein sensitive to proteasome-dependent degradation governs IKK activation in ATL cells, and suggest a molecular mechanism of IKK activation in ATL cells distinct from that in HTLV-I-transformed T cells.

Local regulation of the axonal phenotype, a case of merotrophism

In this essay, we show that several anatomical features of the axon, namely, microtubular content, caliber and extension of sprouts, correlate on a local basis with the particular condition of the glial cell, i.e., the anatomy of axons is dynamic, although it is seen usually in its `normal' state. The occurrence of ribosomes and messenger RNAs in the axon suggests that axoplasmic proteins are most likely synthesized locally, at variance with the accepted notion that they are supplied by the cell body. We propose that the supporting cell (oligodendrocyte or Schwann cell) regulates the axonal phenotype by fine-tuning the ongoing axonal protein synthesis.

Reversal of cycloheximide-induced memory disruption by AIT-082 (Neotrofin) is modulated by, but not dependent on, adrenal hormones.

RATIONALE: AIT-082 (Neotrofin), a hypoxanthine derivative, has been shown to improve memory in both animals and humans. In animals, adrenal hormones modulate the efficacy of many memory-enhancing compounds, including piracetam and tacrine (Cognex). OBJECTIVE: To investigate the role of adrenal hormones in the memory-enhancing action of AIT-082. METHODS: Plasma levels of adrenal hormones (corticosterone and aldosterone) in mice were significantly reduced by surgical or chemical (aminoglutethimide) adrenalectomy or significantly elevated by oral administration of corticosterone. The effects of these hormone level manipulations on the memory-enhancing activity of AIT-082 and piracetam were evaluated using a cycloheximide-induced amnesia/passive avoidance model. RESULTS: As previously reported by others, the memory enhancing action of piracetam was abolished by adrenalectomy. In contrast, the memory enhancement by 60 mg/kg AIT-082 (IP) was unaffected. However, a sub-threshold dose of AIT-082 (0.1 mg/kg, IP) that did not improve memory in control animals did improve memory in adrenalectomized animals. These data suggested that, similar to piracetam and tacrine, the memory enhancing action of AIT-082 might be inhibited by high levels of adrenal hormones. As expected, corticosterone (30 and 100 mg/kg) inhibited the action of piracetam, however no dose up to 100 mg/kg corticosterone inhibited the activity of AIT-082. CONCLUSIONS: These data suggest that while AIT-082 function is not dependent on adrenal hormones, it is modulated by them. That memory enhancement by AIT-082 was not inhibited by high plasma corticosterone levels may have positive implications for its clinical utility, given that many Alzheimer's disease patients have elevated plasma cortisol levels.

Detection of drug-induced, superoxide-mediated cell damage and its prevention by antioxidants.

The mode of the cytotoxic activity of three benzo(c)fluorene derivatives was characterized. The observed morphological changes of lysosomes or variations of mitochondrial activity are assumed to be the consequence of cell protection against oxidative damage and/or the part of the damage process. To establish the relationship between the quantity of superoxide (O2*-) generated and the degree of damage resulting from O2*-, a simple system based on measurement of 3-(4-iodophenyl)-2-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) reductase activity in the presence of superoxide dismutase (SOD) was used. The functionality of the chosen battery of in vitro tests was proved using several known superoxide inducers: cyclosporin A (CsA) and benzo(a)pyrene (BP), as well as noninducers: citrinin (CT) and cycloheximide (CH). From the results followed that the cell growth tests are much better indices of toxicity than the other tests. The model system for the evaluation of the protective capacity of antioxidants against superoxide-induced cytotoxicity included simultaneous exposure of HeLa cells to cytotoxic drugs and to quercetin (Qe), an antioxidant of plant origin. The complete abolishment of the inhibition of cell proliferation and clonogenic survival was concluded to be due to the protective effect of the antioxidant. These observations correlated with the decrease of superoxide content as estimated by the INT-reductase assay in the presence of SOD using the same model system, as well as with the increase of intracellular SOD content and its activity.

Prevention of cycloheximide-induced apoptosis in hepatocytes by adenosine and by caspase inhibitors.

The mechanism by which cycloheximide induces apoptosis in isolated rat hepatocytes was studied. Cycloheximide (1-300 microM) induced apoptosis within 3-4 hr in the hepatocytes. Specific apoptotic characteristics such as blebbing, phosphatidyl serine (PS) exposure, chromatin condensation, and nuclear fragmentation were induced. Cycloheximide (CHX) dose dependently activated the caspase-3-like proteases, but not the caspase-1-like proteases. Pretreatment of the hepatocytes with 100 microM of the caspase inhibitors z-Val-Ala-DL-Asp-fluoromethylketone or Ac-Asp-Glu-Val-Asp-aldehyde completely abrogated the caspase activation and the apoptosis. Addition of adenosine (100 microM) reduced phosphatidyl serine exposure and other morphological characteristics of apoptosis by 50%; however, it did not prevent the activation of the caspases, suggesting that adenosine inhibited downstream of caspase activation. The adenosine receptor antagonist 8-[4-[[[[(2-aminoethyl)amino]-carbonyl]methyl]oxy]phenyl]-1,3-dipropylxa nthine abolished the capacity of adenosine to prevent apoptosis, indicating that prevention was receptor-mediated. During apoptosis, the mitochondrial membrane potential in apoptotic cells (cells with PS exposition) was decreased to 50-60% of the control value; in the population viable cells, however, the mitochondrial membrane potential remained stable. Prevention of apoptosis by the caspase inhibitor z-Val-Ala-DL-Asp-fluoromethylketone or adenosine prevented the decrease in mitochondrial membrane potential. In conclusion, CHX rapidly induces apoptosis in isolated rat hepatocytes, which is inhibited by adenosine at a relatively late step.

Cyclooxygenase inhibitors decrease apoptosis initiated by actinomycin D, cycloheximide, and staurosporine in amnion-derived WISH cells.

Apoptosis is a process by which external or developmental factors induce a specific series of events leading to cell death. Recently, apoptotic cells have been described in rat amnion membrane at late gestation, suggesting apoptosis may be involved in membrane rupture. Mechanisms controlling amnion cell apoptosis are unknown. The objective of this study was to investigate whether cyclooxygenase and prostaglandins are integral to apoptosis in amnion, as reported in intestinal epithelial cells and renal mesangial cells. Amnion-derived WISH cells underwent apoptosis in a dose- and time-dependent manner after incubation with actinomycin D, cycloheximide, or staurosporine, as determined by cell viability, DNA fragmentation analysis, and fluorescent in situ fragmentation analysis. Cells cultured with increasing doses of these agents also demonstrated concomitant increases in prostaglandin E2 output. WISH cell coincubation with these agents and the cyclooxygenase inhibitors indomethacin or piroxicam resulted in dose-dependent decreases in both prostaglandin E2 and apoptosis. Cultures incubated with 0.5 microgram/mL actinomycin D showed 80.7% cell apoptosis after 12 hours compared with 1.1% in untreated cultures. After 24 hours incubation with actinomycin D, 0.8% of the original cell number remained attached to the plate. In cultures coincubated with 0.5 microgram/mL actinomycin D and 100 mumol/L indomethacin, only 19.2%, 24.7%, and 39.3% of the cells were found to be apoptotic after 12, 24, and 48 hours in culture, respectively. Similar trends were observed after the use of cycloheximide or staurosporine in combination with indomethacin or prioxicam. These data suggest that cyclooxygenase and/or prostaglandins play a role in programmed cell death of amnion-derived WISH cells in culture.

Ameliorating effect of p-hydroxybenzyl alcohol on cycloheximide-induced impairment of passive avoidance response in rats: interactions with compounds acting at 5-HT1A and 5-HT2 receptors.

The effect of p-hydroxybenzyl alcohol (HBA) on cycloheximide (CXM)-induced impairment in the step-through passive avoidance task was investigated in rats and compared to the effect of the nootropic piracetam. HBA and piracetam significantly counteracted the CXM-induced shortening of retention latencies. The effect of HBA was a bell-shaped dose-response curve with a maximal effect of 5 mg/kg. The counteractive effect of HBA was not depressed by either scopolamine or mecamylamine. The serotonin (5-HT) releaser, p-chloroamphetamine, and presursor, 5-hydroxytryptophan, significantly antagonized the counteractive effect of HBA on the CXM-induced shortening of retention latencies. Furthermore, the counteractive effect was also inhibited by the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and the 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2 aminopropane [(+/-)-DOI], but potentiated by the 5-HT1 receptor antagonist (+/-)-pindolol and the 5-HT2 receptor antagonist ritanserin. There results suggest that the beneficial effect of HBA on CXM-induced impairment is amplified by treatment with serotonergic receptor antagonists but reduced by serotonergic 5-HT1A and 5-HT2 receptor agonists, and insensitive to cholinergic manipulations.

No. 302, a newly synthesized [pGlu4,Cyt6]AVP(4-9) analogue, prevents the disruption of avoidance behavior.

We investigated the effects of [pGlu4,Cyt6]AVP(4-9) fragments and its analogues on cycloheximide (CHX)-induced learning impairment in rats using the step-through-type passive avoidance test in rats. CHX (2.8 mg/kg, s.c.) significantly shortened the step-through latency in the retention trial. pGlu-Asn-Cys(Cys)-Pro-Arg-Gly-NH2 ([pGlu4, Cyt6]AVP(4-9); 10 ng/kg, s.c.), a major metabolite of arginine vasopressin, improved the CHX-induced learning impairment. Asn-Cys-Pro-Arg-OH ([Cys6]AVP(5-8); 1 ng/kg) corrected avoidance learning in the CHX-treated group, whereas neither Cys(Cys)-Pro-Arg-OH nor pGlu-Asn-Cys(Cys)-Pro-OH had any effect (1, 10 and 100 ng/kg, s.c.). pGlu-Asn-Ser-Pro-Arg-Gly-NH2 (No. 302), a newly synthesized [pGlu4,Cyt6]AVP(4-9) analogue, significantly prolonged the latency shortened by CHX at doses of 0.1, 1 and 10 ng/kg (s.c.). Asn-Ser-Pro-Arg-OH also improved the learning disruption induced by CHX, although the effective dose was 100 times higher than that of No. 302. The half-life of No. 302 in rat blood was about 5.5, 22 and 25 times longer than that of [pGlu4,Cyt6]AVP(4-9), [Cys6]AVP(5-8) and Asn-Ser-Pro-Arg-OH, respectively. These results suggest that [Cys6]AVP(5-8) is the minimal effective amino acid sequence in [pGlu4,Cyt6]AVP(4-9), and show that No. 302 is a potent, pharmacologically active peptide with high stability in the blood.

Systemic administration of dynorphin A-(1-13) markedly improves cycloheximide-induced amnesia in mice.

The effects of systemic or intracerebroventricular injection of dynorphin A-(1-13), a kappa-selective opioid receptor agonist, on cycloheximide-induced amnesia were investigated by using a step-down-type passive avoidance task in mice. The intracerebroventricular injection of dynorphin A-(1-13) (0.3-3 micrograms) before training significantly prolonged step-down latency. The systemic administration of dynorphin A-(1-13) (1, 3 and/or 10 mg/kg, i.p.) before training or retention tests markedly inhibited the cycloheximide (30 mg/kg, s.c.)-induced shortening of step-down latency, indicating antiamnesic effects of dynorphin A-(1-13). One and 3 mg/kg doses of ((+/-)trans-3, 4-dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, methanesulfonate hydrate (U-50,488H), another kappa-selective opioid receptor agonist, significantly inhibited the shortening. The anti-amnesic effects of dynorphin A-(1-13) (3 and 10 mg/kg, i.p.) were almost completely antagonized by intracerebroventricular administration of the quaternary derivative of the opioid receptor antagonist naltrexone methobromide (0.3 microgram), but not by systemic administration of the opioid receptor antagonist (1 mg/kg, s.c.), demonstrating central mediation of the anti-amnesic effects of dynorphin A-(1-13). Furthermore, the kappa-selective opioid receptor antagonist, nor-binaltorphimine (2 mg/kg, s.c.), almost completely antagonized the effects of dynorphin A-(1-13) (3 and 10 mg/kg, i.p.). These results suggest that dynorphin A-(1-13) produces anti-amnesic effects through the blood-brain barrier.

NS-3 (CG3703), an analog of thyrotropin-releasing hormone, ameliorates cognitive impairment in rats.

The effects of thyrotropin-releasing hormone (TRH) and its analog, N-[[(3R,6R)-6-methyl-5-oxo-thiomorpholinyl] carbonyl]-L-histidyl-L- prolinamide tetrahydrate (NS-3, CG3703) on disturbance of memory of a passive avoidance response (PAR) and an escape response in rats were investigated. NS-3 improved amnesia caused by scopolamine, electroconvulsive shock (ECS), and cycloheximide (CXM), but TRH improved only the ECS-induced amnesia. NS-3 reversed learning deficits caused by hypercapnia, but TRH had no effect. These differences in the effect between NS-3 and TRH may be due to their biological half-life in rat plasma. These results suggest that NS-3 possesses more potent antiamnestic effects than TRH in rats.

Interleukin-3 inhibits cycloheximide induction of C-jun mRNA in human monocytes: possible role for a serine/threonine phosphatase.

Cycloheximide is a strong inducer of the c-jun protooncogene mRNA at concentrations (< or = 50 ng/ml) that do not inhibit protein synthesis in human monocytes. This induction is transient lasting 30-60 min in contrast to the sustained induction obtained with concentrations that inhibit protein synthesis. The pluripotent colony stimulating factor interleukin-3 (IL-3) (10 ng/ml) is also a modest inducer of the c-jun gene in these cells; however, in combination with cycloheximide, IL-3 dramatically reduces the c-jun induction below levels induced by cycloheximide alone. This is a true inhibition and is not due to a change in temporal kinetics of induction because the suppression in the presence of IL-3 is observed at both 30 and 60 min after simultaneous addition of both IL-3 and cycloheximide. Preincubation of monocytes with 12.5 nM okadaic acid (a potent inhibitor of protein phosphatases 1 and 2A) and cycloheximide prior to addition of IL-3 restored the level of c-jun induction to that mediated by cycloheximide alone. This concentration of okadaic acid inhibited almost 70% of the phosphorylase phosphatase activity in monocyte lysates. These observations suggest that activation of protein serine/threonine phosphatase(s) underlies the ability of IL-3 to inhibit cycloheximide induction of c-jun in monocytes.

Okadaic acid accelerates germinal vesicle breakdown and overcomes cycloheximide- and 6-dimethylaminopurine block in cattle and pig oocytes.

Pig and cattle oocytes, when released from the follicle, spontaneously resume first meiotic division within 20 or 8 hr, respectively. In oocytes of both species, the activity of histone H1 kinase increases during maturation, exhibiting a maximum in metaphase I. Treatment of these oocytes with okadaic acid results in acceleration of germinal vesicle breakdown (GVBD) and of histone H1 kinase activation. This effect is more important in pig oocytes, in which the acceleration rises for 6 hr, as compared to 2 hr in cattle. Moreover, under these conditions, H1 kinase activity measured after 12 hr of culture appears higher than that observed in control metaphase I oocytes. When added to prophase oocytes, both cycloheximide and 6-DMAP (6-dimethylaminopurine) block GVBD and histone H1 kinase activation. Okadaic acid, at a concentration of 2.5 microM, is able to release the inhibitory effect exerted by cycloheximide on histone H1 kinase activity; however, GVBD occurred only in two-thirds of pig and one-quarter of cattle oocytes after 20 hr of culture. In addition, okadaic acid fully reverses the effect of 6-DMAP on H1 kinase activity and on GVBD in both species. The opposite effects of 6-DMAP and okadaic acid on MPF activation are discussed, as well as the nature of the protein, which has to be synthesized during the first meiotic division and may be involved in the MPF activation cascade.

Epidermal growth factor and insulin-like growth factor-1 preserve cell viability in the absence of protein synthesis.

Prolonged exposure of cells to the potent protein synthesis inhibitor cycloheximide (CHX) terminates in cell death. In the present study we investigated the effect of epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and insulin on cell death induced by CHX in the human cancerous cell lines MDA-231 and MCF-7 (breast), KB (oral epidermoid), HEP-2 (larynx epidermoid), and SW-480 (colon), and correlated this effect to the inhibition rate of protein synthesis. Cell death was evaluated by measuring either dead cells by trypan blue dye exclusion test or by the release of lactic dehydrogenase into the culture medium. CHX was shown to induce cell death in a concentration (1 to 60 micrograms/ml) and time (24 to 72 h)-dependent manner in each of the five cell lines. EGF at physiologic concentrations (2 to 40 ng/ml) reduced cell death close to control level (without CHX) in the cell lines HEP-2, KB, MDA-231, and SW-480, but had almost no effect on cell death in the MCF-7 cells. IGF-1 at physiologic concentrations (2 to 40 ng/ml) reduced cell death nearly to control level in the MCF-7 cells, but had only a partial effect in the other four cell lines. Insulin at supraphysiologic concentration (10,000 ng/ml) mimicked the effect of IGF-1 in each of the cell lines. CHX at concentrations that induced about 60% cell death, inhibited about 90% of protein synthesis as measured by [3H]leucine incorporation. Protein synthesis remained inhibited although cell viability was preserved by EGF or IGF-1. These results indicated that the mechanism by which EGF or IGF-1 preserve cell viability does not require new protein synthesis and may be mediated via a posttranslational modification effect.

Involvement of serotonergic neuronal systems in the anti-amnesic action of naftidrofuryl oxalate.

The effects of naftidrofuryl oxalate on cycloheximide- and 5-hydroxytryptophan (5-HTP)-induced amnesia were investigated using a passive avoidance task in mice. Naftidrofuryl oxalate significantly improved the cycloheximide-induced amnesia. This effect of naftidrofuryl oxalate was antagonized by 5-HTP, a serotonin (5-HT) precursor, and by p-chloroamphetamine (PCA), a 5-HT releaser. Single administration of 5-HTP in combination with pargyline, a monoamine oxidase inhibitor, induced amnesia (5-HTP-induced amnesia). This amnesia was attenuated by ritanserin, a 5-HT2-selective antagonist, but not by pindolol, a 5-HT1-selective antagonist. Naftidrofuryl oxalate also attenuated the 5-HTP-induced amnesia. A binding study revealed that naftidrofuryl oxalate inhibited the binding of [3H]ketanserin to 5-HT2 receptors in mouse brain synaptic membrane in a dose-dependent fashion (IC50 = 1.42 x 10(-7) M), but did not inhibit that of [3H]serotonin to 5-HT1 receptors. These results suggest that naftidrofuryl oxalate may attenuate cycloheximide- and 5-HTP-induced amnesia by blocking 5-HT2 receptor subtypes.