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PubMed1997

Cycloheximide inhibition of delayed early gene expression in baculovirus-infected cells.

The baculovirus protein IE1 is required for the transactivation of many early viral genes in transient expression assays. However, cycloheximide inhibition studies have failed to reveal a dependence of early gene transcription on expression of IE1 in infected cells. We show here that synthesis of IE1 was not effectively inhibited by the addition of 100 microg/ml cycloheximide, the concentration routinely used in these studies. However, when cycloheximide was added at 250 microg/ml, IE1 synthesis was repressed to less than 5% of control levels. These more stringent conditions were used to discriminate between immediate early and delayed early genes. Transcription of three immediate early genes (ie1, ie2, and ie0) was increased by the addition of high concentrations of cycloheximide. However, transcription of three other early genes (39k, p35, and lef-3), which are known to be dependent on IE1 transactivation, was significantly reduced by the addition of 250 microg/ml cycloheximide. Immunoblot analyses also revealed a difference between the immediate and delayed early class of viral genes. Synthesis of IE1, IE2, and IE0 was resistant to cycloheximide treatment, while translation of SSB/LEF-3 and pp31 was strongly inhibited even at the lower concentration of cycloheximide. Although cycloheximide was shown to be useful in defining early temporal classes, it induced apoptosis in both uninfected and infected Sf9 cells when used at the inhibitory concentration.

Animals↗
PubMed1977

HeLa cell DNA polymerases: the effect of cycloheximide in vivo and detection of a new form of DNA polymerase alpha.

Blockage of protein synthesis in HeLa cells by cycloheximide leads to selective effects on the levels of DNA polymerases alpha, beta, and gamma in the cell. The total activity of DNA polymerase alpha remains unchanged after 7 h exposure of cells to cycloheximide but drops to 50% of its original level after 24 h. The level of the beta-polymerase falls rapidly in the cell and is reduced to less than 30% of its initial value by 7 h after treatment of the cells with cycloheximide. The gamma-polymerase level is diminished by 30--40% during the 7 h cycloheximide treatment and reaches 50% of its original level after 24 h. Cells which have been exposed to cycloheximide for 7 h will regain normal levels of the beta- and gamma-polymerases within 90 min after removal of the drug. The cycloheximide-treated cells also show the presence of a new form of the alpha-polymerase, designated alpha1, which can be clearly detected as a separate entity in column chromatography. The level of alpha1 in the nucleus increases during the period that the cells are treated and cycloheximide so that after 24 h it represents almost 50% of the nuclear DNA polymerase activity. The presence of alpha1 in the cytoplasmic fraction can also be demonstrated in both cycloheximide-treated and normal, growing cells.

Cell Nucleus↗
PubMed1984

Short term reductions in cerebral muscarinic receptor concentration of the mouse after in vivo administration of cycloheximide.

[3H]quinuclidinyl benzilate ([3H]QNB) was used to investigate the effects of administration of cycloheximide to mice on the concentration of muscarinic receptors in the cerebral cortex. A single subcutaneous (s.c.) injection of cycloheximide (3.5 mg/kg) reduced receptor concentration by 18% afer an apparent latency of 1 hr. A single intraperitoneal (i.p.) injection of 3.5 mg/kg produced a 14% reduction in muscarinic receptor concentration 20 min after injection. After both s.c. and i.p. administration the effects of cycloheximide were reversible, dose-related and maximal at a dose of 3.5 mg/kg. The maximum reductions in receptor concentrations obtained were 13-18%. Multiple doses of cycloheximide did not produce greater falls in receptor concentration than single doses and did not prevent its return towards control levels. Treatment of mice for 10 day with cycloheximide reduced muscarinic receptor concentration to 71% of the control at 3 days after stopping administration; receptor levels returned to control values 35 days after the last dose of cycloheximide. Treatment of mice with cycloheximide had no effect on the affinity of [3H]QNB for the receptor and, in vitro, 10(-4)M cycloheximide did not have any significant effect on the binding of the radiolabel.

Animals↗
PubMed1988

Abrogation of etoposide-mediated cytotoxicity by cycloheximide.

The antitumor agent etoposide interacts with DNA topoisomerase II to produce a unique form of DNA-enzyme intermediate referred to as a "cleavable complex". These drug-induced DNA strand breaks initiate poorly defined cell processes which result in lethality. To explore the mechanism of etoposide cytotoxicity, we studied the effect of protein synthesis inhibitor on Balb/C 3T3 fibroblasts and CCRF-CEM and L1210 leukemia cells by exposing these cell lines to cycloheximide for various periods of time prior to etoposide challenge. Cycloheximide alone during these periods of exposure was not cytotoxic; however, it conferred increasing cytoprotection from etoposide in a time-dependent fashion when it preceded etoposide. Although cycloheximide did cause a decrease in enzyme content and in etoposide-induced DNA cleavage of Balb/C 3T3 and the CCRF-CEM cell lines, cytoprotection by cycloheximide could not be accounted for completely by these phenomena since, in L1210 cells, cytoprotection was observed without significant change in DNA cleavage or enzyme content. Cycloheximide diminished DNA synthesis as well as protein synthesis. However, DNA synthesis resumed within 6 hr after removal of cycloheximide, in spite of the fact that cytoprotection persisted. Cycloheximide did not alter cell cycle distribution as measured by flow cytometry. Our data, therefore, clearly demonstrate that cycloheximide can diminish the cytotoxic potential of etoposide-mediated topoisomerase-DNA complexes. Elucidation of the mechanism by which cytoprotection occurs should shed light on the basis for the cytotoxic effect of topoisomerase II-active drugs.

Animals↗
PubMed1988

Cycloheximide decreases glucose transporters in rat adipocyte plasma membranes without affecting insulin-stimulated glucose transport.

This study examines the relationship between insulin-stimulated glucose transport and insulin-induced translocation of glucose transporters in isolated rat adipocytes. Adipose cells were incubated with or without cycloheximide, a potent inhibitor of protein synthesis, for 60 min and then for an additional 30 min with or without insulin. After the incubation we measured 3-O-methylglucose transport in the adipose cells, and subcellular membrane fractions were prepared. The numbers of glucose transporters in the various membrane fractions were determined by the cytochalasin B binding assay. Basal and insulin-stimulated 3-O-methylglucose uptakes were not affected by cycloheximide. Furthermore, cycloheximide affected neither Vmax. nor Km of insulin-stimulated 3-O-methylglucose transport. In contrast, the number of glucose transporters in plasma membranes derived from cells preincubated with cycloheximide and insulin was markedly decreased compared with those from cells incubated with insulin alone (10.5 +/- 0.8 and 22.2 +/- 1.8 pmol/mg of protein respectively; P less than 0.005). The number of glucose transporters in cells incubated with cycloheximide alone was not significantly different compared with control cells. SDS/polyacrylamide-gel-electrophoretic analysis of [3H]cytochalasin-B-photolabelled plasma-membrane fractions revealed that cycloheximide decreases the amount of labelled glucose transporters in insulin-stimulated membranes. However, the apparent molecular mass of the protein was not changed by cycloheximide treatment. The effect of cycloheximide on the two-dimensional electrophoretic profile of the glucose transporter in insulin-stimulated low-density microsomal membranes revealed a decrease in the pI-6.4 glucose-transporter isoform, whereas the insulin-translocatable isoform (pI 5.6) was decreased. Thus the observed discrepancy between insulin-stimulated glucose transport and insulin-induced translocation of glucose transporters strongly suggests that a still unknown protein-synthesis-dependent mechanism is involved in insulin activation of glucose transport.

3-O-Methylglucose↗
PubMed1975

Nature of the increase in renal ornithine decarboxylase activity after cycloheximide administration in the rat.

The present study was designed to determine whether the increase in rat renal ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity after cycloheximide administration was a primary effect on the kidney or was a secondary effect of adrenal or pituitary hormones released in response to the drug. Renal ornithine decarboxylase activity was reduced approximately 70% 1 hr after intraperitoneal administration of doses of cycloheximide that also inhibited renal protein synthesis by 68-95% within 1 hr. Protein synthesis began to recover by the second hour, accompanied by a rise in decarboxylase activity that reached a peak about six times greater than pretreatment values at 8 hr, then gradually declined to preinjection levels by 16 hr. Peak ornithine decarboxylase activity was directly proportional to cycloheximide doses up to 250 mug; larger doses, which almost abolished protein synthesis for 8 hr, where inhibitory. Plasma corticosterone rose rapidly after cycloheximide, reached a peak at 2 hr, then fell to baseline by 8 hr. Corticosterone response was also dose-dependent up to 250 mug, but larger doses were inhibitorymadrenalectomy did not reduce decarboxylase activity response to cycloheximide, nor did cortisol administration enhance it. Hypophysectomy greatly reduced baseline renal decarboxylase activity within 9 hr and all but abolished the increase in enzyme activity normally seen after cycloheximide administration to the intact rat. The hypophysectomized animal exhibited apparent increased sensitivity to cycloheximide, since a smaller dose of the drug caused a reduction in renal protein synthesis similar to that seen with a larger dose in the intact rat. As protein synthesis was recovering in the hypophysectomized animals, renal decarboxylase activity responded adequately to the injection of a crude pituitary extract. These data suggest that renal ornithine decarboxylase turnover is rapid, that baseline activity is.maintained by new protein synthesis, and that the increase in renal enzyme activity after cycloheximide is in larger part dependent upon pituitary hormone action.

Adrenal Glands↗
PubMed1999

A possible involvement of ion transporter in tumor necrosis factor alpha and cycloheximide-induced apoptosis of endothelial cells.

We examined the tumor necrosis factor alpha (TNFalpha)-induced apoptosis of vascular endothelial cells from the standpoint of ion channels. Cultured vascular endothelial cells from bovine carotid artery were used. Apoptosis was determined by a propidium iodide assay. Treatment of the endothelial cells with TNFalpha and cycloheximide for 6 h induced nuclear fragmentation in a TNFalpha dose-dependent manner (1-10 ng/ml). Concomitant treatment of endothelial cells with TNFalpha at a dose of 10 ng/ml and cycloheximide at a dose of 10 microg/ml elicited endothelial cell apoptosis as high as 23.4+/-4.1% at 6 h after administration. However, 10 ng/ml TNFalpha alone elicited a little apoptosis at 6 h after its administration (% apoptosis=4.1+/-0.8%). Cycloheximide (10 microg/ml) did not induce apoptosis at all. Concomitant treatment of endothelial cells with 1 mmol/l of 4,4-diisothiocyanatostilbene-2,2-disulfonic acid, which is a chloride bicarbonate exchanger blocker, partially inhibited the TNFalpha and cycloheximide-induced endothelial cell apoptosis. On the other hand, endothelial cell apoptosis due to TNFalpha and cycloheximide was completely inhibited by benzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene (50 micromol/l), an inhibitor of caspase. Moreover, pyrrolidine dithiocarbanate, an inhibitor of nuclear factor kappa B (NF-kappaB), also suppressed endothelial cell apoptosis induced by TNFalpha and cycloheximide completely. These findings suggest that the endothelial cell apoptosis induced by TNFalpha and cycloheximide is closely related to not only chloride ions, but also both NF-kappaB and caspase activation. That is to say, there is a possibility that chloride ions or bicarbonate (pH) may play an important role in signal transduction such as NF-kappaB and caspase activation in the apoptosis induced by TNFalpha and cycloheximide.

Animals↗
PubMed1984

Effects of treatment with cycloheximide at proestrus on subsequent in vitro follicular steroidogenesis in the hamster.

Follicles from hamster ovaries removed at 1000 h of proestrus were incubated for 5 h with the medium changed every hour. Proestrous hamsters were also injected at 1400 h with either 4 mg cycloheximide or saline (control), and follicles were dissected from the ovaries at 1500 h and similarly incubated. During the first hour of incubation, the 1000-h follicles produced picogram amounts of all steroids from progesterone (P4) through estradiol (E2). Thereafter E2, androstenedione (delta) and testosterone (T) accumulation in the medium were reduced by about one-half over the next 4 h, whereas 17 alpha-hydroxyprogesterone (17-OHP) increased to become the dominant steroid. The control follicles incubated at 1500 h produced steroids in nanogram amounts and the granulosa cells in vitro now produced C21 steroids, especially P4. In contrast, throughout the 5-h incubation, the follicles of cycloheximide-treated hamsters accumulated considerably reduced amounts of C21 steroids and this was reflected in granulosal production of P4 at one-half to one-tenth of control levels. The profiles for accumulation of C19 and C18 steroids by the intact follicles of both groups were superimposable over the 5-h period. Thecal production of delta was unaffected by injection of cycloheximide at 1400 h, which suggests that the theca requires the continued presence of high concentrations of cycloheximide to affect steroidogenesis. Incubation of granulosa cells from cycloheximide-exposed follicles with 10 ng of various steroid precursors restored P4, 17-OHP, E2 and estrone (E1) to the same levels as controls, indicating that the steroidogenic enzymes from 3 beta-ol-dehydrogenase through aromatase were unaltered and therefore the lack of sterol precursors was the critical step affected by cycloheximide. Incubation of cycloheximide-treated granulosa cells with 25-hydroxycholesterol led to a 3-fold increase in P4 levels which are still only one-half of control values. This points to decreased accessibility of cholesterol to mitochondrial side-chain cleavage as one of the key events blocked by cycloheximide. The experiments show that after exposure to the proestrous surge of gonadotropins, there is normally a rapid recruitment of the granulosal compartment as a source of C21 steroids and this is a protein-dependent process.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

Cycloheximide inhibits S-14 gene transcription and abolishes DNase I hypersensitive S-14 sites in the livers of euthyroid but not hypothyroid rats.

Earlier studies from our laboratory have demonstrated that cycloheximide administration to hypothyroid rats inhibited the induction of the hepatic mRNA-S14 by T3. These results suggested a role of short-lived proteins in the hormonal regulation of this gene. To define the possible mechanism of the cycloheximide effect, we examined the influence of cycloheximide on the in vitro transcription rate of the gene and its chromatin structure. Forty-five minutes after injection of cycloheximide to euthyroid rats, the in vitro transcriptional rate fell by 60% and this effect persisted for 4 h. In the same euthyroid rats, cycloheximide caused the disappearance of all four DNase I-hypersensitive sites situated in the 5'-flanking region of the gene. However, cycloheximide given to hypothyroid rats affected neither the basal transcription rate nor the chromatin structure. When cycloheximide was administered 30 min after an acute injection of T3 (200 micrograms/100 g BW) to hypothyroid animals, it completely blocked the hormone induction of the transcriptional rate. These results suggest that one or more labile proteins are required for maintenance of S14 chromatin structure in a configuration which permits hormonal regulation of gene expression. The ability of cycloheximide to block mRNA-S14 induction by T3 appears to be mediated at least in part by an inhibition of T3-stimulated transcription.

Animals↗
PubMed1993

Cycloheximide stabilizes insulin-like growth factor-binding protein-1 (IGFBP-1) mRNA and inhibits IGFBP-1 transcription in H4-II-E rat hepatoma cells.

The insulin-like growth factor-binding proteins (IGFBPs) are a family of six proteins that modulate the biological activity of IGF-I and IGF-II and determine their bioavailability to tissues. One of the IGFBPs, IGFBP-1, is distinctive in the dynamic response of its levels in human plasma to metabolic changes. Parallel changes occur in IGFBP-1 mRNA and IGFBP-1 transcription in rat liver. Using the well differentiated H4-II-E rat hepatoma cell line as a model system, we demonstrated previously that IGFBP-1 transcription is positively regulated by dexamethasone and negatively regulated by insulin. We now examine the effect of the protein synthesis inhibitor, cycloheximide, on the hormonal regulation of IGFBP-1 gene expression. Preincubation of H4-II-E cells with 10.7 microM cycloheximide for 1.5 h did not prevent the induction of IGFBP-1 mRNA and IGFBP-1 transcription (determined in nuclear run-on assays) by dexamethasone. By contrast, cycloheximide treatment abolished the decrease in IGFBP-1 mRNA induced by insulin. Insulin rapidly decreased IGFBP-1 transcription in the absence of cycloheximide (> 50% inhibition in 20 min) and caused a similar decrease in cells pretreated with cycloheximide. Cycloheximide alone also decreased IGFBP-1 transcription. Similar results were observed with a second protein synthesis inhibitor, anisomycin, which also prevented the insulin-induced decrease in IGFBP-1 mRNA without abolishing the insulin-induced inhibition of IGFBP-1 transcription. These results suggest that although insulin decreases IGFBP-1 gene transcription in the presence of protein synthesis inhibitors, IGFBP-1 mRNA levels are maintained because of stabilization of the mRNA. Stabilization was demonstrated directly in actinomycin D-treated cells, where the t1/2 of IGFBP-1 mRNA increased from approximately 2 to approximately 20 h in the presence of cycloheximide; insulin did not affect IGFBP-1 mRNA turnover. Thus, cycloheximide-sensitive labile proteins contribute to the maintenance of basal IGFBP-1 promoter activity and the rapid turnover of IGFBP-1 mRNA, which determine the dynamic regulation of IGFBP-1 gene expression.

Animals↗
PubMed2000

Characterization of the L41 gene in Cryptococcus neoformans: its application as a selectable transformation marker for cycloheximide resistance.

A transformation system using resistance to the antibiotic cycloheximide as a dominant selectable marker was developed for the pathogenic yeast Cryptococcus neoformans. A 3.5 kb DNA fragment containing a gene encoding the ribosomal protein L41 was cloned from a wild-type strain of C. neoformans which is sensitive to cycloheximide. The open reading frame of the L41 gene contains five introns and encodes a protein of 107 amino acids, which is similar to those reported for other yeasts. The cycloheximide resistance gene to be used as a marker was constructed by replacing a DNA segment of the wild-type L41 gene, which contained the amino acid proline at its 56th position with a homologous DNA segment from a mutant strain resistant to cycloheximide that contained leucine in that position. Cycloheximide resistant transformants were obtained by electroporation on YEPD plates, supplemented with 10-20 microg/ml cycloheximide, at a maximum efficiency of 300 transformants/microg plasmid DNA. While with other genes, most transformants of serotype D in C. neoformans maintain the transforming DNA as episomes, the cycloheximide-resistant transformants were all the result of ectopic genomic integration events.

Amino Acid Sequence↗
PubMed1992

Cycloheximide protection against actinomycin D cytotoxicity.

Pretreatment plus concomitant treatment with 10 micrograms/ml cycloheximide protected Chinese hamster ovary cells and Swiss 3T3 cells against the cytotoxicity of actinomycin D. The cycloheximide treatment reduced the intracellular concentration of actinomycin D by reducing the level of actinomycin D bound to the acid precipitable fraction of the cell. Levels of unbound actinomycin D were unaffected by cycloheximide, indicating that the plasma membrane permeability to AD was not reduced. Actinomycin D inhibited total transcription but did not reduce cytoplasmic levels of rRNA nor of most tested mRNA; however, cytoplasmic levels of c-myc mRNA were reduced below detectability. Cycloheximide treatment further inhibited total transcription and had no effect on cytoplasmic levels of rRNA nor of most tested mRNA. Cytoplasmic levels of c-myc were elevated by cycloheximide and remained so even in the presence of actinomycin D. These data suggested that a reduction in cytoplasmic levels of short lived, essential mRNA, such as c-myc mRNA, was one lethal lesion of actinomycin D. Furthermore, cycloheximide's protection may result, in part, from its ability to stabilize and/or elevate cytoplasmic levels of these mRNA, thus counteracting their depletion by actinomycin D. Protection may also result from the cycloheximide-induced reduction of actinomycin D bound to the acid precipitable fraction of the cells.

Animals↗
PubMed1993

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.

Cycloheximide↗
PubMed1994

Parthenogenetic development of bovine oocytes matured in vitro for 24 hr and activated by ethanol and cycloheximide.

This research was undertaken to improve development of parthenogenetic embryos following various combined treatments of ethanol and cycloheximide. In Experiment 1 in vitro matured oocytes (IVM, 24 hr) were treated with 7% ethanol for 5 min followed by incubation in 10 micrograms/ml cycloheximide in Medium 199 for 0 (control), 5, 10, and 20 hr. Development to 2-8 cells following culture for 3 days was similar among treated groups (32-41%; P > 0.05), which was higher than that of controls (6%; P < 0.05). Experiment 2 compared pre-ethanol exposures for 0, 1, 2.5, and 5 min, followed by 5 hr cycloheximide treatment on activation development. One- to 5-min groups resulted in 42-44% cleavage contrasted to 1-12% for controls (P < 0.05). Experiment 3 examined the effect on oocyte development of ethanol and different concentrations of cycloheximide (0, 1, 5, and 10 micrograms/ml). Cleavage to 2-8 cells was similar among the 5 and 10 micrograms/ml cycloheximide groups (36% and 42%, P > 0.05) but lower (P < 0.05) for the 1 micrograms/ml group (24%) and the controls (2-13%). When 5 micrograms/ml cycloheximide was used (Experiment 4), pre-exposure to ethanol (1, 2.5, and 5 min) resulted in more oocytes cleaved (38-41%) than in the cycloheximide alone group (0%) or the control (0%, P < 0.05). Experiment 5 tested blastocyst development of the activated oocytes with or without cytochalasin B treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

The soybean SAUR open reading frame contains a cis element responsible for cycloheximide-induced mRNA accumulation.

Little is known about how mRNA stability is regulated in higher plants. The SAURs (Small Auxin-Up RNAs) are a family of highly unstable mRNAs in soybean that rapidly increase in abundance after excised organs are treated with the plant hormone auxin. The SAURs are also induced by protein synthesis inhibitors, including cycloheximide, in the absence of auxin treatment and are superinduced when organs are treated with cycloheximide plus auxin. While the induction of SAURs is transcriptionally regulated by auxin, the induction by cycloheximide is posttranscriptional. Cycloheximide as well as other protein synthesis inhibitors appear to induce SAUR accumulation by increasing the stabilities of these mRNAs. To determine whether the 5'-untranslated region, the 3'-untranslated region, or the open reading frame of these unstable mRNAs is responsible for the cycloheximide inducibility, we have used chimeric genes in transgenic tobacco plants to test each of these mRNA regions. Our results show that the SAUR open reading frame within a chimeric mRNA confers cycloheximide inducibility in transgenic tobacco plants whereas chimeric mRNAs containing the SAUR 5'-untranslated region or 3'-untranslated region as isolated elements or in combination are not induced by cycloheximide. These results suggest that the SAUR open reading frame contains sequence elements that are involved in the stability of these mRNAs.

Blotting, Northern↗
PubMed1982

Initial inhibition and recovery of protein synthesis in cycloheximide-treated hepatocytes.

Previous studies conducted with intact rats had demonstrated that protein synthesis was reversibly inhibited by cycloheximide. Polysome aggregation occurred during inhibition with a return to normal during recovery. Suggesting that the block of translational activity involved termination and release of polypeptides. This study involving freshly isolated hepatocytes was undertaken to clarify the mechanism of the biphasic response to cycloheximide. Cycloheximide at 1 microM inhibited [3H]leucine incorporation into both cellular and secreted proteins by at least 86%, without having deleterious effects on membrane integrity as indicated by trypan blue uptake and lactate dehydrogenase (LDH) (EC 1.1.1.27) release. After removal of cycloheximide, incorporation of labeled amino acids into cellular protein and protein secreted into the medium returned to control levels. Kinetically, incorporation into secreted protein exhibited a lag of 30-45 min, indicating that a longer recovery period for restoration of proteosynthetic ability is required for membrane-bound polysomes. During the first 100 min of the recovery period, 30% of the cellular protein, which had been prelabeled during cycloheximide inhibition, was secreted into the medium; treated cells, however, secreted prelabeled protein at a lower initial rate. To elucidate the mechanism of action of cycloheximide, the content of the cytoplasmic ribonucleoprotein complexes (RPC), polysome size classes, and the distribution of radioactivity among the various ribosome classes were determined during inhibition and recovery. Larger size class polysomes (7+) were increased by cycloheximide treatment and remained increased during recovery. During inhibition, there was enhanced [3H]leucine labeling with increasing polysome size, implicating termination as the rate-limiting step, whereas during the recovery phase the labeled nascent polypeptides were removed from the ribonucleoprotein complex at a 3- to 4-fold greater rate than control, indicating an accelerated release of completed proteins.

Animals↗
PubMed1984

Identification of the cycloheximide-sensitive site in angiotensin-stimulated aldosterone synthesis.

We have investigated the action of a protein synthesis inhibitor on the ability of angiotensin II (AII) to stimulate steroid synthesis. Isolated bovine adrenal glomerulosa cells were incubated in the presence and absence of angiotensin and cycloheximide, and the effects of the inhibitor on six cellular processes were measured. Cycloheximide at 7 and 28 microM inhibited the ability of the hormone to stimulate aldosterone synthesis. These concentrations of cycloheximide blocked protein synthesis by 72 and 79% respectively. Cycloheximide did not block receptor binding of angiotensin, the effect of angiotensin on [32P]phosphate incorporation into phosphatidylinositol, nor the ability of the hormone to alter 45Ca2+ fluxes. Mitochondrial conversion of cholesterol to pregnenolone is thought to be the rate-determining step in corticosteroid synthesis. Mitochondria isolated from cells treated with angiotensin made pregnenolone at a higher rate than control mitochondria. Cycloheximide blocked this effect when it was present in the cell incubation medium with angiotensin. Cycloheximide added directly to mitochondria had no effect on pregnenolone synthesis. Cycloheximide also blocked AII stimulation of pregnenolone synthesis in intact cells. We propose that protein synthesis is required for angiotensin to exert its stimulatory effects at one particular locus: activation of mitochondrial pregnenolone synthesis. Protein synthesis is not required for other angiotensin-stimulated processes in bovine adrenal glomerulosa cells.

Adrenal Glands↗
PubMed1985

Effects of cycloheximide and tunicamycin on opiate receptor activities in neuroblastoma X glioma NG108-15 hybrid cells.

The molecular mechanism of opiate receptor down-regulation and desensitization was investigated by studying the effects of cycloheximide and tunicamycin on opiate receptor activities in neuroblastoma X glioma NG108-15 hybrid cells. Cycloheximide inhibited [35S]methionine and [3H]-glucosamine incorporation by hybrid cells, while tunicamycin inhibited [3H]glucosamine incorporation only. Exposing hybrid cells to these two agents did not alter the viability of the cell. Treatment of NG108-15 cells with cycloheximide or tunicamycin produced a decrease in [3H]diprenorphine binding dependent on both time and concentrations of inhibitors, with no measurable modification in the ability of etorphine to regulate intracellular cyclic AMP production. Cycloheximide attenuated [3H]-diprenorphine binding by decreasing both the number of sites, Bmax, and the affinity of the receptor, Kd. Tunicamycin treatment produced a decrease in Bmax with no apparent alteration in Kd values. Cycloheximide and tunicamycin did not potentiate the rate or magnitude of etorphine-induced down-regulation or desensitization of opiate receptor in NG108-15 cells. Furthermore, there was an apparent antagonism in cycloheximide action on receptor down-regulation. The reappearance of opiate binding sites after agonist removal was affected by these two inhibitors. Cycloheximide and tunicamycin eliminated the increase in [3H]diprenorphine binding in the chronic etorphine-treated cells after agonist removal. These two inhibitors did not alter the resensitization of hybrid cells to etorphine. Thus, the site of opiate agonist action to induce receptor down-regulation and desensitization is not at the site of protein synthesis or protein glycosylation. These data substantiate previously reported observations that receptor down-regulation and receptor desensitization are two different cellular adaptation processes.

Adenylyl Cyclase Inhibitors↗