Rapid acid extrusion response triggered by alpha-1a adrenoceptor in CHO cells]

Using a microphysiometer with synchronized valve switching, we investigated real-time acid extrusion from CHO cells, in which human alpha-1a adrenoceptor (AR) is stably expressed, in response to noradrenaline (NA). The time course of the extracellular acidification rate after stimulation had two phases: in the first phase, it transiently reached a rate several times greater than the base rate with a peak at around 10 s, and in the second, it reached to 2 times the base rate and reached a plateau in 2 min. Both phases showed concentration-dependent increase of acidification rates in response to NA, but had distinct pEC50 values: 5.6 for the transient phase and 7.2 for the steady phase. HOE642, an inhibitor of Na(+)-H+ exchanger (NHE) 1, inhibited the acid extrusion response in a concentration-dependent manner. HOE642 had high pIC50 values (7.3) for inhibition of the transient phase response. In contrast, it revealed the presence of two components in the steady phase response: one had high pIC50 values (8.2) and the other had low pIC50 values (6.0). As Ca2+ was depleted, the transient phase disappeared, while the steady phase was not affected. These results suggest that alpha-1a AR drives two acid extrusion systems in CHO cells upon stimulation: one elicits the transient response that is largely mediated by a HOE642-sensitive and Ca(2+)-dependent NHE, presumably NHE1, and the other induces the steady acid extrusion that is mediated by NHE1 and another NHE that has low sensitivity to HOE642.     

DNA and protein synthesis inhibition in Chinese hamster ovary cells by dichlorophenoxyacetic acid

The effects of the herbicide dichlorophenoxyacetic acid (2,4-D) on DNA and protein synthesis were investigated in chinese hamster ovary cells (CHO) employing two different methods. The results showed that the herbicide affects DNA and protein synthesis depending on the stage of growth and method of treatment. 2,4-D action appears to concentrate the cells mainly in the G1/S boundary of the cell cycle. The effect is expressed as an inhibition of DNA and protein synthesis. This effect was revealed not only by the chemical determination of DNA and protein synthesis but also by experiments using autoradiography, using the labelling index to detect the incorporation of [3H]thymidine into the cells. Labelling of the cell nucleus was reduced markedly when cells treated with 2,4-D were in confluency for 4 days after reaching plateau growth.     

Effects of 2,4-dichlorophenoxyacetic acid on polyamine synthesis in Chinese hamster ovary cells

It was found that 1 mM 2,4-dichlorophenoxyacetate (2,4-D) inhibited DNA and protein synthesis in Chinese hamster ovary (CHO) cells. When the possible relationship of this phenomenon to the presence of polyamines in the culture medium was investigated, it was found that: (a) the pesticide inhibited ornithine decarboxylase activity; (b) when the concentration of polyamines present in cells treated with the pesticide was determined, the putrescine concentration did not change, and the spermine and spermidine concentration decreased; (c) the addition of spermidine and spermine to CHO cells grown in the presence of 2,4-D normalized DNA and protein synthesis. Putrescine did not have any effect.     

Changes in 2-aminoisobutyric acid and cycloleucine uptake produced by 2,4-dichlorophenoxyacetic acid in Chinese hamster ovary cells

The effect of dichlorophenoxyacetic acid on the transport of two non-metabolizable amino acids, 2-aminoisobutyric acid (AIB) and cycloleucine (CL) was studied in chinese hamster ovary (CHO) cells. The herbicide did not exert any direct effect on the AIB transport. However, when the pesticide was in contact with the cells for 24 h an inhibition of the uptake was observed. Removal of the pesticide from the culture medium restored the influx of the amino acids which reached maximum values 1 h before cell division. The transport kinetics showed changes in Vmax but no variations in Km. These results may indicate that 2,4-dichlorophenoxyacetic acid produces a decrease in the carrier number but without modification of the affinity.     

Apoptosis induced by different doses of caffeine on Chinese hamster ovary cells

Caffeine has been investigated for its potential mutagenic activity to bacteria, fungi and mammalian cells in culture, and at high concentrations it is also an inducer of apoptosis. Caffeine can exert acute cellular toxicity, including inhibition of cell growth and cell death, in Chinese hamster ovary cells.The aim of this study was to evaluate the cell survival and apoptotic or non-apoptotic effects of caffeine to different concentrations in Chinese hamster ovary cells (CHO-K1). These effects were evaluated by measuring cell viability, caspase 8 activity and fragmented DNA. This study suggests that the concentration of caffeine is of critical importance because high doses of caffeine induce apoptosis and low concentrations can act as an antioxidant. Previously, the cytotoxicity of caffeine was evaluated using a wide range of concentrations by the neutral red test. From this screening, adequate doses were selected to perform the caspase activity and fragmentation DNA studies. The potential antioxidant effect of caffeine was studied using tert-butyl-hydroperoxide as a free-radical generator. The repeatability was checked through three separate tests with the same concentration.     

Bcl-2蛋白对环匹阿尼酸诱导CHO细胞凋亡的保护作用

Hoechst 33258染色和DNA电泳分析揭示了内质网钙泵抑制剂环匹阿尼酸(CPA)呈浓度依赖性地诱导CHO细胞凋亡, 表现出染色体固缩和DNA片断形成的典型特征. Bcl-2的过度表达对CPA诱导的CHO细胞凋亡具有保护作用. 采用Fura-2技术测定细胞内游离钙浓度(［Ca²⁺］_i)变化，观察到Bcl-2对CPA触发的细胞Ca²⁺库Ca²⁺释放无任何影响. 结果表明Bcl-2蛋白对CPA诱导的CHO细胞凋亡的保护作用不是通过抑制CPA诱导的Ca²⁺释放，而是作用于［Ca²⁺］_i升高的下游途径.     

Changes of glycolipids and gangliosides in 2,4-dichlorophenoxyacetic acid treated Chinese hamster ovary cells in monolayer culture

The effects of 2,4-dichlorophenoxyacetic acid (2,4-D) on the growth of Chinese hamster ovary cells were studied. 2,4-D (10^?3M) had a cell growth inhibitory effect on CHO cells in monolayer cultures. When 2,4-D was removed after four days of treatment, a rapid resumption of cell growth rate occurred. At 10^?5M 2,4-D, only a delay of growth was observed, and glycolipid and ganglioside contents were increased in both logarithmic and stationary phases of culture, while no changes in protein, cholesterol, and phospholipids were observed.     

Blockade by N-3 polyunsaturated fatty acid of the Kv4.3 current stably expressed in Chinese hamster ovary cells.

1. The Kv4.3 gene is believed to encode a large proportion of the transient outward current (Ito), responsible for the early phase of repolarization of the human cardiac action potential. There is evidence that this current is involved in the dispersion of refractoriness which develops during myocardial ischaemia and which predisposes to the development of potentially fatal ventricular tachyarrhythmias. 2. Epidemiological, clinical, animal, and cellular studies indicate that these arrhythmias may be ameliorated in myocardial ischaemia by n-3 polyunsaturated fatty acids (n-3 PUFA) present in fish oils. 3. We describe stable transfection of the Kv4.3 gene into a mammalian cell line (Chinese hamster ovary cells), and using patch clamp techniques have shown that the resulting current closely resembles human Ito. 4. The current is rapidly activating and inactivating, with both processes being well fit by double exponential functions (time constants of 3.8 +/- 0.2 and 5.3 +/- 0.4 ms for activation and 20.0 +/- 1.2 and 96.6+/-6.7 ms for inactivation at +45 mV at 23 degrees C). Activation and steady state inactivation both show voltage dependence (V1/2 of activation= -6.7+/-2.5 mV, V1,2 of steady state inactivation= -51.3+/-0.2 mV at 23 degrees C). Current inactivation and recovery from inactivation are faster at physiologic temperature (37 degrees C) compared to room temperature (23 degrees C). 5. The n-3 PUFA docosahexaenoic acid blocks the Kv4.3 current with an IC50 of 3.6 micromol L(-1). Blockade of the transient outward current may be an important mechanism by which n-3 PUFA provide protection against the development of ventricular fibrillation during myocardial ischaemia.     

Drynariae Rhizoma promotes osteoblast differentiation and mineralization in MC3T3-E1 cells through regulation of bone morphogenetic protein-2, alkaline phosphatase, type I collagen and collagenase-1

In a previous study (Jeong et al., 2003, Inhibition of Drynariae Rhizoma extracts on bone resorption mediated by processing of cathepsin K in cultured mouse osteoclasts. International Immunopharmacology 3, 1685–1697), treatment of osteoclasts-containing long bone cells with Drynariae Rhizoma (DR) extract prevented the intracellular maturation of cathepsin K and thus, it was considered that DR is a pro-drug of a potent bone resorption inhibitor. To further clarify the role of DR in ossification, we investigated the effects of DR on the proliferation and differentiation of osteoblastic cell lines in vitro. In this study, the bone effect of DR is studied. We assessed the effects of DR on osteoblastic differentiation in nontransformed osteoblastic cells (MC3T3-E1) and rat bone marrow cells. DR enhanced alkaline phosphatase (ALP) activity and mineralization in a dose- and time-dependent fashion. This stimulatory effect of the DR was observed at relatively low doses (significant at 50–150 μg/ml and maximal at 150 μg/ml). Northern blot analysis showed that the DR (100 μg/ml) increased in bone morphogenetic protein-2 as well as ALP mRNA concentrations in MC3T3-E1 cells. DR (60 μg/ml) slightly increased in type I collagen mRNA abundance throughout the culture period, whereas it markedly inhibited the gene expression of collagenase-1 between days 15 and 20 of culture. These results indicate that DR has anabolic effects on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of common metabolic bone diseases such as osteoporosis.     

Suppression of sodium arsenite-potentiated cytotoxicity of ultraviolet light by cycloheximide in Chinese hamster ovary cells

Post-treatment with sodium arsenite synergistically increased the cytotoxicity of ultraviolet (UV) light. The potentiation of UV cytotoxicity by sodium arsenite was apparently suppressed by cycloheximide (CHM), a protein synthesis inhibitor. The protective effect of CHM against sodium arsenite-potentiated UV cytotoxicity was well correlated to its activity in inhibiting the synthesis of stress proteins, particularly a small polypeptide with a molecular weight of 8500 dalton. This small stress protein was demonstrated as ubiquitin by immunoprecipitation. Our results also showed that neither ubiquitin induction nor potentiation of UV cytotoxicity by post-treatment with sodium arsenite was observed in the stationary cells. Thus, we suggested that ubiquitin is possibly involved in the action of arsenite in potentiating UV-induced cell killing.     

Transfected D2 dopamine receptors mediate the potentiation of arachidonic acid release in Chinese hamster ovary cells.

A rat D2L dopamine receptor, a splice variant of the D2 receptor, has recently been cloned. When transfected into and stably expressed in Chinese hamster ovary cells, these receptors mediate the inhibition of both basal and forskolin-stimulated cAMP production, as previously described. We examined what role this receptor might play in the production of the second messenger arachidonic acid. The calcium ionophore A23187 stimulated the release of arachidonic acid, and this release of arachidonic acid was potentiated by dopamine in a concentration-dependent manner. Dopamine alone, however, had no effect on arachidonic acid release. Quinpirole, a D2-selective agonist, augmented A23187-stimulated arachidonic acid release, and sulpiride, a D2-selective antagonist, blocked this augmentation. cAMP analogs and agents that activate adenylyl cyclase were utilized in an attempt to overcome this dopamine effect. Forskolin, prostaglandin E2, dibutyryl-cAMP, 8-(4-chlorophenylthio)-cAMP, and pertussis toxin all had no appreciable effect on either A23187-stimulated arachidonic acid release or the dopamine enhancement. Inhibition of protein kinase C using long term phorbol ester desensitization and pharmacological inhibitors diminished the dopamine potentiation of arachidonic acid release. These results suggest that the D2 receptor may be increasing the release of arachidonic acid by a mechanism involving protein kinase C but independent of the D2 receptor's inhibition of adenylyl cyclase.     

Induction of apoptosis in cultured Chinese hamster ovary cells by Ukrain and its synergistic action with etoposide

The induction of apoptosis by Ukrain, a novel antitumor drug, was studied in Chinese hamster ovary (CHO) cells bearing multiple copies of recombinant human erythropoietin gene incorporated into their genome (cell lines CHO-k38 and -k38/12). Ukrain was found to be capable of the in vitro induction of apoptosis in the cell lines studied. The effect was less expressed in cells with type I multiple drug resistance (k38/12). Ukrain acted synergistically with etoposide, i.e., the combined effect of both agents was evident at significantly reduced concentrations. This suggests that pharmacological compositions of the drugs may reduce the effective doses used in chemotherapy and thus significantly diminish its toxic side effects. Ukrain was found to exert an unusual effect, manifested as the inhibition of protein secretion by target cells. This phenomenon may be used for the express determination of cell sensitivity to colchicine-like cytostatics, including Ukrain.     

Detection of DNA single-strand breaks induced by procarcinogens in Chinese hamster ovary cells cocultured with rat hepatocytes

DNA single-strand breaks induced by procarcinogens were detected in Chinese hamster ovary (CHO) cells cocultured with adult rat hepatocytes. Freshly isolated adult rat hepatocytes were added to the CHO cell culture prelabeled with [3H]thymidine. After allowing the hepatocytes to attach on or near the CHO cells, aflatoxin B1 or benzo[a]pyrene was added to the culture and incubated for the desired time. DNA single-strand breaks in CHO cells were measured by the alkaline elution technique. Aflatoxin B1 induced some DNA single-strand breaks in CHO cells cultured alone, but in coculture system with hepatocytes the number of DNA single-strand breaks increased greatly. The magnitude of the increase was related to the dose and the time of exposure to aflatoxin B1. Addition of proteinase-K to the cell lysates increased the elution of DNA compared to that of samples without proteinase-K. Benzo[a]pyrene did not induce any DNA single-strand breaks in CHO cells in the absence of liver cells, but a significant number of single-strand breaks were detected in the coculture system.     

Influence of receptor number on the cAMP response to forskolin in Chinese hamster ovary cells transfected with human beta2-adrenoceptor

We examined the basal adenosine 3',5'-cyclic monophosphate (cAMP) levels and forskolin-induced cAMP accumulation in cultured Chinese hamster ovary cells (CHO) expressing different levels of human beta(2)-adrenoceptors. Both the basal and forskolin-induced cAMP accumulation in the cells that express higher density of beta(2)-adrenoceptors (CHO-beta(2)/H; 560 fmol/mg protein) were larger than those in the cells that express lower density of beta(2)-adrenoceptors (CHO-beta(2)/L; 270 fmol/mg protein). In addition, isoproterenol-induced cAMP accumulation was also augmented as the number of beta(2)-adrenoceptors was increased. ICI 118,551, a selective beta(2)-adrenoceptor antagonist with inverse agonist properties, decreased all the levels of cAMP observed in both cell lines. These results suggest that the agonist-independent (constitutive) activity of beta(2)-adrenoceptors plays a key role in the control of forskolin-induced cAMP accumulation.     

Independent coupling of the human tachykinin NK2 receptor to phospholipases C and A2 in transfected Chinese hamster ovary cells

The human tachykinin NK₂ receptor stably expressed in Chinese hamster ovary cells (CHO-hNK₂R cells) was characterized by studying the effect of neurokinin A (NKA), the preferred natural ligand, and that of other agonists and antagonists in both binding experiments and functional assays. Competition experiments using [¹²⁵I]NKA showed that CHO-hNK₂R cells express binding sites which have high affinity for NKA (K _i=3.4±0.9 nM), GR 64349 (K _i=12±3 nM) and [βAla⁸]NKA(4–10) (K _i=21±8 nM) and for the antagonists MEN 10627 (K _i=0.55±0.2 nM), and MEN 11420 (K _i=2.4±0.8 nM). In contrast, the tachykinin NK₁ and NK₃ receptor agonists [Sar⁹,Met(O₂)¹¹]SP and senktide, respectively, were recognized with low affinity (K _i>10 μM). NKA (EC₅₀=68±18 nM) induced a rapid and concentration-dependent increase in the intracellular level of inositoltrisphosphate (IP₃). The concentration-response curve to GR 64349 (EC₅₀=155±14 nM) was close to that of NKA, whereas [βAla⁸]NKA(4–10) (EC₅₀=445±78 nM) and SP (EC₅₀=3197±669 nM) were 7- and 50-fold less potent, respectively. In addition, NKA stimulated the release of arachidonic acid and the production of prostaglandin E₂ (PGE₂) in a concentration-dependent manner. Also in this assay, NKA was found to be more potent than the other agonists tested (the EC₅₀ values were 3±0.3, 9±3, 7.8±0.9 and 217±37 nM for NKA, GR 64349, [βAla⁸]NKA(4–10) and SP, respectively). MEN 10627 and MEN 11420 were potent and competitive antagonists in blocking NKA-induced IP₃ formation and PGE₂ release: MEN 10627 and MEN 11420 displayed comparable potencies in blocking the two functional responses initiated by occupancy of the NK₂ receptor by NKA. Pretreatment of the cells with pertussis toxin (500 ng/ml for 18 h) did not significantly modify the basal or stimulated phosphatidylinositol turnover but reduced the basal and NKA-induced PGE₂ release by about 35%. The phospholipase C inhibitor U-73122 (10 μM) prevented the NKA-induced formation of IP₃ but did not affect PGE₂ release. Conversely, the phospholipase A₂ inhibitor quinacrine (100 μM) blocked the release of arachidonic acid and PGE₂ without affecting the NKA-stimulated formation of IP₃. Chelation of extracellular calcium with 3 mM EGTA inhibited the NKA-induced PGE₂ release by 81% but was without effect on basal and NKA-stimulated IP₃ production. The calcium channel blockers verapamil (10 μM) and ω-conotoxin GVIA (0.1 μM) did not modify the basal PGE₂ production and had no significant effect on the response to tachykinins while the blocker of non-selective cation channels, SKF-96365 (10 μM), inhibited the response to NKA by about 74%. SKF-96365 did not affect the basal or the NKA-induced IP₃ formation. In conclusion, our data demonstrate that the human tachykinin NK₂ receptor expressed in CHO cells displays binding affinity and functional properties which are those of a native NK₂ receptor. No pharmacological evidence for heterogeneity of the human NK₂ receptor was obtained in this study. Our findings indicate that the human tachykinin NK₂ receptor is independently coupled to both PLC and PLA₂ signaling pathways. Activation of the PLA₂ pathway may be linked to the opening of a voltage-independent cation channel which activates a Ca²⁺-dependent PLA₂. Received: 7 April 1998 / Accepted: 17 July 1998     

Effect of diisopropanolamine upon choline uptake and phospholipid synthesis in Chinese hamster ovary cells.

Aminoalcohols differ in mammalian toxicity at least in part based upon their ability to alter the metabolism of phospholipids and to cause depletion of the essential nutrient choline in animals. This study examined the incorporation of diisopropanolamine (DIPA) into phospholipids (PLs) and effects of DIPA upon choline uptake and phospholipid synthesis in Chinese hamster ovary (CHO) cells. Results were compared to those of a related secondary alcohol amine, diethanolamine (DEA), whose systemic toxicity is closely associated with its metabolic incorporation into PLs and depletion of choline pools. DIPA caused a dose-related inhibition of (3)H-choline uptake by CHO cells that was approximately 3-4 fold less potent, based upon an IC50, than that reported for DEA. DIPA, in contrast to DEA, did not cause changes in the synthesis rates of (33)P-phosphatidylethanolamine, (33)P-phosphatidylcholine or (33)P-sphingomyelin at either non-toxic or moderately toxic concentrations. Only approximately 0.004%, of administered (14)C-DIPA was metabolically incorporated into PLs, over 30-fold less than the incorporation of (14)C-DEA under similar conditions. Overall, these data and previous pharmacokinetic and toxicity data obtained in vivo suggests that DIPA is distinct from DEA and lacks significant choline and PL metabolism related toxicity in animals.     

Usnic acid attenuates genomic instability in Chinese hamster ovary (CHO) cells as well as chemical-induced preneoplastic lesions in rat colon

Usnic acid (UA) is one of the pharmacologically most important compounds produced by several lichen species. To better understand the mechanism of action (MOA) of this important substance, this study examined the genotoxicity attributed to UA and its influence on mutagens with varying MOA using the micronucleus (MN) test in Chinese hamster ovary cells (CHO). Additional experiments were conducted to investigate the effect of UA on colon carcinogenesis in Wistar rats employing the aberrant crypt focus (ACF) assay. In vitro studies showed a significant increase in the frequency of MN in cultures treated with the highest UA concentration tested (87.13 µM). In contrast, UA concentrations of 10.89, 21.78, or 43.56 µM produced an approximate 60% reduction in chromosomal damage induced by doxorubicin, hydrogen peroxide, and etoposide, indicating an antigenotoxic effect. In the ACF assay, male Wistar rats treated with different UA doses (3.125, 12.5, or 50 mg/kg b.w.) and with the carcinogen 1,2-dimethylhydrazine exhibited a significantly lower incidence of neoplastic lesions in the colon than animals treated only with the carcinogen. Data suggest that the MOA responsible for the chemopreventive effect of UA may be related to interaction with DNA topoisomerase II and/or the antioxidant potential of the compound.     

Characterization of mexiletine as an antagonist of beta-adrenoceptor in Chinese hamster ovary cells expressing cloned human beta-adrenoceptors

We characterized the beta-adrenoceptor-blocking property of mexiletine, a class Ib antiarrhythmic drug, on Chinese hamster ovary (CHO) cells stably expressing cloned human beta1-, beta2-, and beta3-adrenoceptors. In radioligand binding experiments, mexiletine (10 microM-1 mM) concentration-dependently displaced the specific binding of [125I]cyanopindolol to human beta1- and beta2-adrenoceptors in the membrane fraction of the cells. High concentration (100 microM-1 mM) of mexiletine partially displaced the specific binding of [125I]cyanopindolol to human beta3-adrenoceptor. On the other hand, high concentration (300 microM and 1 mM) of lidocaine, another class Ib antiarrhythmic drug, partially displaced the specific binding of [125I]cyanopindolol to human beta1-adrenoceptor, whereas it did not affect the specific binding of [125I]cyanopindolol to human beta2- and beta3-adrenoceptors. Mexiletine (5, 50, and 500 microM) reduces basal adenosine 3',5'-cyclic monophosphate (cAMP) level and isoprenaline-induced cAMP accumulation on CHO cells stably expressing cloned human beta1- and beta2-adrenoceptors. Lidocaine (10 and 100 microM and 1 mM) tend to reduce basal cAMP level on CHO cells stably expressing cloned human beta1-adrenoceptors, whereas the drug did not reduce the isoprenaline-induced cAMP accumulation on CHO cells stably expressing cloned human beta1-, beta2-, and beta3-adrenoceptors. Mexiletine and lidocaine have no effect on forskolin (0.1, 1, and 3 microM)-induced cAMP accumulation. These results demonstrate that mexiletine blocks the binding of agonists to beta1- and beta2-adrenoceptors, and thereby attenuates the agonist-induced cAMP accumulation, and that the action of mexiletine as an antagonist of beta1- and beta2-adrenoceptors is independent of its antiarrhythmic property.     

Induction of sister chromatid exchanges in Chinese hamster cells by the reducing agents bisulfite and ascorbic acid

Sister chromatid exchanges (SCEs) are induced in Chinese hamster cells by a 2--3-h exposure to ascorbic acid or bisulfite in the concentration range 10(-4)--10(-2) M. This activity of these 2 chemicals was intensified when cell cultures were exposed to each agent for longer time periods (24 h). The divalent metal cation, Cu2+, was effective in potentiating the ability of ascorbic acid to induce SCEs and toxicity, suggesting that the autooxidation of ascorbic acid was involved in this action. The ability of sodium bisulfite to induce SCEs was not affected by variation in the concentration of 5'-bromodeoxyuridine used as a labelling compound. This was interpreted as supporting the view that bisulfite, not a synergistic reaction between bisulfite and BrdU, was responsible for the elevated SCE levels.