Venlafaxine induces P-glycoprotein in human Caco-2 cells.

PURPOSE: The objective of this study was to evaluate the effect of a treatment with venlafaxine on the expression of multidrug resistance-associated protein (MRP) gene and multidrug resistance-related proteins (MDR) in human colon carcinoma cells (Caco-2) compared to a known P-glycoprotein (PGY1) inducer, rifampine. METHODS: Caco-2 cells were treated with venlafaxine (50 microM, 100 microM, 250 microM, and 500 microM) and rifampin (25 microM and 50 microM) to test the possible induction of MRP and MDR expression. The treatment times used were 1.5, 3, 6, 12, 24, 48, and 72 h. RNA was isolated from the cells, and MDR and MRP genes were amplified using PCR. RESULTS: Both venlafaxine and rifampine had the most dramatic effect at the 50 microM concentration. There was an increase in MDR and MRP expression in Caco-2 cells after the acute treatment (1.5, 3, and 6 h) with venlafaxine. These results were similar to those with rifampine. CONCLUSIONS: PGY1 contributes to renal and biliary elimination of drugs by transporting the drug out of the cell and back into the intestinal lumen, where drugs may be further metabolized by intestinal enzymes such as Cytochrome P (CYP)-450 3A4. Its function is to limit the bioavailability of orally administered compounds. Due to the increase in MDR and MRP gene expression seen after the acute treatment with venlafaxine, there could be a potential drug-drug interaction with other medications that are metabolized via CYP450-3A4 when coadministered with venlafaxine.

Liquid chromatographic method with electrochemical detection for determination of cisapride in serum.

A sensitive liquid chromatographic (LC) method using electrochemical detection was developed for the identification and quantitation of cisapride in serum. The serum samples were deproteinized by a simple acetonitrile precipitation technique followed by n-hexane extraction. Cisapride in the deproteinized serum was separated by an isocratic elution with an ODS Hypersil LC column (150 x 4.6 mm) using a mobile phase consisting of 0.05M Na2HPO4-acetonitrile (60 + 40), pH 8.4. Cisapride eluted from the column was detected by a Coulochem II electrochemical detector. The precision of this assay method was determined by intra- and inter-day analyses of cisapride-free fetal bovine serum samples that were spiked with 25, 50, and 100 ng/mL cisapride. For the intra-day assay, recoveries were 94.3 +/- 1.4, 90.1 +/- 2.9, and 103.2 +/- 9.2%, respectively. This electrochemical detection LC method could be very useful in monitoring plasma levels of cisapride.

Effect of systemic lupus erythematosus (SLE) treatment drugs on GI-101A breast tumor cell growth.

The effect of systemic lupus erythematosus (SLE) treatment drugs on PKC (protein kinase C) activity and cell growth was studied using GI-101A breast tumor cells. Both hydroxychloroquine (HCQ) and prednisone treatments significantly increased the PKC activity in GI-101A cells after 60 min. Treatment of cells with a combination of HCQ/prednisone also produced the highest increase in PKC activity following 60 min incubation. When the GI-101A cells were treated with the same drugs, HCQ (10 ng/ml) prednisone (10 ng/ml) and HCQ/prednisone combination (10 ng/ml of each), for 72 hr the total PKC activity in the cells was significantly elevated and consequently the GI-101A cell growth was stimulated. As a result of drug induced cell growth stimulation the total number of cells in the treatment groups increased significantly compared to the non-treated controls. Interestingly HCQ and prednisone treatment induced cell growths were completely blocked by PKC specific inhibitor chelerythrine (50 microM). Our results suggest that HCQ and prednisone treatment can induce GI-101A cell growth via activating PKC.

Expression of vascular endothelial growth factor mRNA in GI-101A and HL-60 cell lines.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that has a strong association with growth and metastasis of various cancers. We analyzed the expression of VEGF mRNA levels in human breast-tumor derived GI-101A cells and in human promyelocytic leukemia derived HL-60 cells using RT-PCR technique. During our RT-PCR analysis we detected the expression of three splice variants of VEGF mRNA at 400, 520 and, 650 bp lengths, which were amplified by a single set of VEGF specific forward and reverse primers. The three RT-PCR products detected by us in these cells correspond to the mRNA splice variants coding for the three isoforms of VEGF respectively, VEGF(121), VEGF(165), and VEGF(189). Treatment of GI-101A and HL-60 cells with phorbol 12, 13-dibutyrate (PDB) or diethylstilbestrol (DES) resulted in a significant increase of VEGF mRNA levels in a dose dependent manner. Both treatments increased the levels of all three splice variants of VEGF mRNA and a maximum increase was detected with 10 microM concentrations of PDB or DES treatments after 2 h. Interestingly, both PDB and DES mediated stimulation of VEGF mRNA expression was completely blocked by the PKC inhibitor chelerythrine. Quantitation of VEGF levels by ELISA technique confirmed that changes seen in mRNA levels following different treatments altered the release of VEGF. Our results suggest that PDB and DES mediated effects on VEGF expression in GI-101A and HL-60 cells occur at the gene transcription level.

Biodegradable microparticles of influenza viral vaccine: comparison of the effects of routes of administration on the in vivo immune response in mice.

The objective of this study was to investigate the comparative immune response following administration of biodegradable microparticles loaded with influenza viral vaccine using subcutaneous and oral routes. Influenza viral vaccine was entrapped in poly(d,l-lactide-co-glycolide) (PLG) and poly(isobutylcyanoacrylate) (PIBCA) microparticles. Stability and immunogenicity of entrapped antigen were retained, as evaluated by SDS-PAGE and immunoblot. Microparticles in the size range of <11 microm were evaluated for protein loading and in vitro antigen release. The mice were immunized with microparticle loaded antigen and IgG levels in blood and IgA levels in saliva and gastric secretions were monitored by ELISA method. When the mice were immunized with microparticle suspensions, IgG levels were higher if administered by subcutaneous primed by oral route compared to oral primed by subcutaneous route or subcutaneous or oral route. The IgA level in saliva and gastric secretions were also found to be higher when subcutaneous immunization was given followed by oral booster than oral priming followed by subcutaneous booster. The polymer types of the microparticles had effects on both IgG and IgA levels. This study provided insights into the design of microparticles of influenza vaccine for subcutaneous administration followed by an unlimited oral boosting, which will have high cost-effectiveness and patient compliance.

Expression of mdm-2 oncoprotein in the primary and metastatic sites of mammary tumor (GI-101) implanted athymic nude mice.

The expression of mdm-2 oncoprotein (p90) was determined in a human breast tumor xenograft line (GI-101) that was derived from a 57 year old female cancer patient with recurrent, infiltrating ductal adenocarcinoma (Stage IIIa, T3N2MX). Immunoprecipitation coupled western blot analysis of the primary tumors that have been obtained from xenograft implanted athymic nude mice, using mdm-2 (Ab-1) mouse monoclonal antibody, primarily revealed high level expression of a 90 kD full length mdm-2 protein. In the GI-101 tumor the level of full length mdm-2 (p90) protein expression increased with the increase in the size of the tumor (100 to 2,000 mm(3)) and a maximum expression was detected in 2,000 mm(3) size tumors. In addition to the expression in the primary site, a significantly high level expression of mdm-2 protein (p90) was detected in the lung and liver tissues also, which are the known metastatic sites for GI-101 xenograft tumors. However, the level of mdm-2 protein expression was undetectable in the lung and liver tissues obtained from control mice. A cell line (GI-101A) derived from the GI-101 xenograft tumor also showed a high level expression of mdm-2 protein after several generations of cell passage. When the GI-101A cells were treated with DES (Diethylstilbestrol) the mdm-2 protein expression increased after 10 min treatment and reached a peak level at 40 min. Interestingly, DES (10 and 20 microM) treatment increased the total cell number also after 96 hr treatment compared to the non-treated cells. It appears that mdm-2 (p90) may have a significant role in supporting the tumor cell growth as well as the metastatic process of the GI-101A cells.

Effect of cisplatin on dopamine release from PC12 cells.

The effect of cisplatin, carboplatin and cyclophosphamide treatments on the neurotransmitter release process and the changes in intracellular Ca2+ level was determined by using PC12 cell experimental model. The neurotransmitter releasing ability of selected anticancer drugs was assessed by estimating the amount of dopamine release induced by them from PC12 cells. Among the three anticancer drugs tested in this study, cisplatin induced a maximum release of dopamine from PC12 cells. In addition, cisplatin pretreatment significantly increased the dopamine release stimulated by carbachol (0.5 mM) and KCl (55 mM). The additive effect exerted by cisplatin over KCl (55 mM) induced dopamine release was effectively blocked by verapamil (10 microM). The intracellular Ca2+ studies performed with cisplatin, carbachol and KCl (55 mM) treatments indicated that cisplatin does not induce any changes in the intracellular Ca2+ levels on its own, however it potentiates the effect of carbachol and KCl. Our study suggests that PC12 cells can be used as an experimental model to test the neurotransmitter releasing ability of emetic anticancer drugs.

High-performance liquid chromatography using electrochemical detection for the determination of prazosin in biological samples.

For the quantitation of prazosin a sensitive high-performance liquid chromatographic (HPLC) method was developed. This HPLC analysis method uses an electrochemical detection technique for the identification and quantitation of prazosin. In this assay the serum samples were deproteinized by using a simple acetonitrile precipitation technique that was followed by n-hexane extraction. Prazosin in the deproteinized serum sample was separated by an isocratic elution with an ODS Hypersil HPLC column (150 x 4.6 mm) using a mobile phase consisting of 0.05 M Na2HPO4-acetonitrile (60:40), pH 8.4. Prazosin that was eluted from the column was detected using a Coulochem II electrochemical detector. The precision of this assay method was assessed by performing inter- and intra-assay analyses by spiking prazosin free fetal bovine serum samples with 20 and 40 ng/ml concentrations of prazosin. In the intra-assay the recovery was 95.40 +/- 4.82% and 97.80 +/- 3.40%, respectively, for 20 and 40 ng/ml concentrations of prazosin that were used to spike the serum samples. This electrochemical detection HPLC assay method could be very useful in monitoring plasma levels of prazosin.

Accumulation of labeled cyanide in neuronal tissue.

Since cyanide is a reactive chemical substance and has the potential of forming a variety of adducts in biological systems, the rate of accumulation of labeled cyanide was studied in neural tissue, a major target for the toxic action of cyanide. Accumulation of 14CN in mouse brain slices and in rat pheochromocytoma (PC12) cells was about five times more rapid in the first few minutes than at later times. In both tissues, incubation at low temperature (4 degrees C) decreased the secondary phase without affecting the initial phase. In PC12 cells, determination of the subcellular distribution of cyanide revealed that the cytoplasmic fraction was the least sensitive to temperature and therefore may represent the initial rapid phase of cyanide accumulation. Accumulation of cyanide in mitochondrial and microsomal fractions was temperature sensitive. Cyanide accumulation is proportional to the concentration in the medium (0.1-1 mM) in both mouse brain and PC12 cells suggesting that the cyanide sinks in neural tissue have a large capacity and are nonspecific. Cyanide interaction with neural tissue is not uniform since cyanide accumulated more in hypothalamus than in other brain areas. Despite this, mitochondrial enzyme activity was no greater in hypothalamus than in other brain areas. No increase in thiocyanate, the main metabolite of cyanide, could be detected in brain tissue incubated 30 min with 1 mM cyanide, showing that accumulated 14C is not in the form of thiocyanate. Cyanide appears to equilibrate rapidly across the plasma membrane and then slowly accumulates in mitochondria and membrane elements of the neuronal cell. Competition for cyanide among subcellular elements may be a factor in the toxic action of cyanide.

Cyanide induces protein kinase C translocation: blockade by NMDA antagonists.

Activation and translocation of protein kinase C (PKC) during KCN-induced histotoxic hypoxia was studied in rat brain slices prepared from cerebellum, hippocampus, and cortex. Treatment with 1-10 mM KCN produced a significant increase in PKC translocation and enzyme activity in the particulate fraction of cerebellar and hippocampal slices. In cortical slices, PKC activity was not affected by cyanide treatment. The membrane-associated PKC activity reached a maximum 30 minutes after incubation with KCN and remained elevated up to 60 minutes in both the hippocampus and cerebellum. Pretreatment with MK-801 and APV, specific NMDA receptor antagonists, blocked the cyanide-stimulated translocation in the hippocampus and cerebellum, whereas CNQX, an AMPA/kainate receptor antagonist, did not alter the response. These results demonstrate that cyanide stimulates PKC activation and translocation from the cytosol to membranes in select brain areas and NMDA receptor activation mediates this process.

MK-801 prevents cyanide-induced changes of Fos levels in rat brain.

The effect of acute cyanide intoxication on levels of transcriptional regulatory proteins Fos and c-Jun in rat cortex, hippocampus, cerebellum and brain stem was studied. Western blot analysis showed a differential effect of cyanide on Fos levels in the selected brain areas. The most prominent changes were seen 60 min. following ip. injection of KCN in all brain areas except the brain stem, which showed the maximal change 120 min. following cyanide. Fos levels were doubled in cortex and cerebellum and decreased to below 70% of the control levels in hippocampus. Levels of c-Jun were not altered 60 min. following cyanide treatment. Pretreatment with the NMDA receptor antagonist, MK-801, prevented the cyanide-induced changes of Fos. The differential effect of cyanide on Fos levels in different brain areas and the blockade of these changes by MK-801 suggest involvement of multiple neuronal pathways, including the excitatory amino acid (EAA) neurotransmitter system. It is concluded that cyanide alters levels of the transcriptional regulatory protein Fos through activation of the EAA neurotransmitter system and, thus, may affect gene expression in neuronal or glia cells.

Activation of a calcium- and pH-dependent phospholipase A2 by cyanide in PC12 cells.

Previous studies suggested that alterations in phospholipid composition of plasma membranes may contribute to neuronal injury associated with cyanide-induced histotoxic hypoxia. This prompted a study of the effects of KCN on phospholipase A2 (PLA2), an enzyme which catalyzes breakdown of membrane phospholipids. PLA2 activity was measured by quantitating the release of [3H]arachidonic acid ([3H]AA) from rat pheochromocytoma (PC12) cells. KCN produced a time (1-15 min)- and concentration (0.5-10 mM)-dependent release of [3H]AA from the cells. When cells were incubated in Ca(2+)-free buffer, KCN (5 mM) was still able to release [3H]AA. In cells loaded with BAPTA, an intracellular Ca2+ chelator, cyanide-induced release of [3H]AA was blocked, indicating that mobilization of intracellular Ca2+ can activate the enzyme. The PLA2 inhibitors dibucaine (50 microM) and mepacrine (50 microM) inhibited KCN-mediated [3H]AA release. Incubation of PC12 cells in an extracellular pH of 6.50 reduced the KCN effect, whereas incubation at pH 7.90 enhanced [3H]AA release. These data indicate that in PC12 cells KCN activates a Ca(2+)- and pH-dependent PLA2 which may contribute to cyanide-induced cell damage.

Interaction of cyanide with a dopamine metabolite: formation of a cyanohydrin adduct and its implications for cyanide-induced neurotoxicity.

Incubation of rat pheochromocytoma (PC12) cells with cyanide (1-10 mM) for 30 min produced a compound which eluted between dopamine (DA) and 3,4-dihydroxyphenylacetic acid in HPLC-EC analysis. Generation of the compound was rapid, concentration-dependent and blocked by pretreatment with clorgyline, a monoamine oxidase A inhibitor. In cell free incubation, the compound formed rapidly when cyanide was added to a mixture of monoamine oxidase and DA, but was not detected when cyanide was included with DA alone. The compound was radiolabelled when 14C-KCN was added to the incubation mixture. Based on these results, it is proposed that the deaminated metabolite of DA, 3,4-dihydroxyphenylacetaldehyde (DOPAL), reacts non enzymatically with cyanide to form the cyanohydrin adduct 2-hydroxy-3-(3,4-dihydroxyphenyl) propionitrile (HPN). HPN was confirmed by spectral analysis and co-elution with synthetic HPN. Incubation of mouse brain slices with cyanide (1 mM) generated 0.98 ng HPN/100 mg wet wt. over a 10 min period and HPN was detected in brains of mice after injection of cyanide (15.6 micrograms) into the lateral brain ventricle. Repeated doses of KCN (6 mg/kg, s.c., five times) produced 0.14 +/- 0.03 ng/100 mg of tissue in striatum. Incubation of PC12 cells for 60 min with 500 microM HPN killed 23% of the cells and increased DA release from the cells by 39.8% over untreated cells. Uptake of HPN into the cells was partially blocked by the catecholamine uptake inhibitor imipramine. These results indicate cyanide reacts with the DA metabolite DOPAL to generate a biologically active cyanohydrin adduct which may contribute to the neurotoxic response to cyanide.

Lysosomal delivery of ANP receptors following internalization in PC12 cell.

Internalization and intracellular processing of ANP-B and C receptors play an important role in regulating cell responsiveness to atrial natriuretic peptides (ANP). Receptor internalization was indirectly monitored with 125I labelled ligand. When 125I-ANP(99-126) was internalized by the cells at 37 degrees C, 55% of the internalized radioactivity was localized in the lysosomal fraction. When receptors were affinity-labelled with 125I-ANP(99-126) and allowed to internalize for varying time periods, two radiolabelled proteins in the m.wt range of 56 and 52 KDa were detected in the cytosolic extract. These proteins appear to be the hydrolytic products of the ANP-C receptor expressed on the plasma membrane. In addition to lysosomal delivery, shedding of the ANP-C receptor from the cell surface was detected following incubation of cells with 125I-ANP(99-126). The dual processes may function to clear exogenous ANP from the extracellular compartments.

Interaction of cadmium with atrial natriuretic peptide receptors: implications for toxicity.

Atrial natriuretic peptide (ANP) is a diuretic and vascular smooth muscle relaxant which plays a pivotal role in cardiovascular regulation. Since cadmium (Cd) produces cardiovascular toxicity and alters ANP levels in atria and hypothalamus, its effect on ANP receptors were studied in rats and in PC12 cells exposed to Cd. Male rats were injected with CdCl2 (0.01, 0.1, 0.5 or 1.0 mg/kg, i.p.) twice a day for 7 days and then maintained for a period of 30 days. On experimental day 37 ANP receptor binding in the adrenal cortex, aorta and kidney cortex was studied by saturation isotherm analysis. In Cd-treated animals a non-dose related decrease in receptor affinity and density was observed in the kidney and aorta with the aortic ANP receptors being the most sensitive. Cellular regulation of the receptor was studied in PC12 cells, a cell line that expresses functional ANP receptors. Incubation of PC12 cells with Cd reduced both the affinity of the receptor for ANP and decreased the number of binding sites on the cell plasma membrane. The ratio of ligand-receptor complex internalized in the cell to ligand bound to the plasma membrane was significantly decreased following Cd pretreatment (500 microM). A significant decrease in the internalization rate of [125I]ANP was observed in cells incubated concurrently with Cd and ligand. In photoaffinity labelling studies with [125I]ANP, binding of ANP to B and C receptors subtypes was decreased following treatment of either intact cells or plasma membranes with Cd. It was concluded that Cd produces significant alterations in the ANP receptor, both in in vitro and in vivo models and it is proposed these effects play a role in the cardiovascular toxicity of this heavy metal.

Differential internalization and processing of atrial-natriuretic-factor B and C receptor in PC12 cells.

PC12 cells express two atrial-natriuretic-factor-(ANF)-receptor subtypes with molecular masses of 130,000 (B receptor) and 70,000 (C receptor). The B-receptor subtype constitutes 65% of the cell-surface receptor population, and the remaining 35% are C receptors as determined by saturation binding studies in the presence of C-ANF, a C-receptor-selective analogue. ANF-(99-126)-peptide [ANF(99-126)], which can bind to both B- and C-receptor subtypes, was rapidly internalized into the cells after incubation at 37 degrees C. Internalization of 125I-ANF(99-126) was used as an index of the receptor-mediated endocytosis and to quantify receptor internalization. In the presence of a saturating concentration of C-ANF, receptor-mediated internalization of 125I-ANF(99-126) was reduced by 24%, indicating B receptor mediate 76% of ligand internalization. Incubation of cells with 10 microM-ANF at 37 degrees C down-regulated both receptor subtypes as reflected by decreased surface binding. Time-dependent studies suggest that B- and C-receptor subtypes undergo differential down-regulation. Incubation of down-regulated cells for 120 min in ANF-free medium produced a recovery of 35% of the original cell-surface binding. Affinity cross-linking of 125I-ANF to the receptors on the plasma membrane in re-incubated (up-regulated) cells demonstrated expression of predominantly the B-receptor subtype. Monensin blocked 72% of receptor up-regulation, whereas cycloheximide inhibited 43%, suggesting an active recycling mechanism involved in mediating up-regulation of the B receptors. The present study demonstrates a rapid internalization and intracellular recycling mechanism for B receptors in PC12 cells. C receptors also undergo internalization and down-regulation, but recycling of this receptor subtype into the plasma membrane occurs at a lower rate and to a lesser extent than is the case for the B receptor.

Regulation of ANF receptor internalization: involvement of extracellular calcium.

Receptor mediated internalization of 125I-ANF (99-126) and the underlying mechanism was studied in PC12 cells. Phosphorylation of PC12 cell plasma membrane proteins at 0 degrees C or 37 degrees C was not altered in presence of ANF (99-126) or c-ANF (4-23). Exposure of cells to phorbol 12-myristate 13-acetate (PMA, 100 ng/ml) did not alter the endocytic rate or extent of 125I-ANF (99-126) internalization. When cells were treated with a combination of PMA and the calcium ionophore A23187, internalization was not stimulated. Incubation with A23187 (10 microM) alone decreased 125I-ANF (99-126) internalization by 22% in Ca2+ containing medium. Cell surface binding increased 10% in the presence of Ca2+ compared to Ca2+ free medium, irrespective of the presence of A23187. Ca2+ appears to play an important role in the binding of ANF to the receptor and initiation of ligand-receptor complex internalization. Activation of protein kinase C or receptor phosphorylation is not an essential step in initiating ANF receptor internalization.

Production of a monoclonal antibody against C terminus of atrial natriuretic factor by in vitro immunization.

Spleen cells, sensitized in vitro with ANF-KLH conjugate, were fused with a non-producing mouse myeloma cell line, X63-Ag8.653. One of the isolated hybridomas secreted a monoclonal antibody which exhibited an association constant of 7.25 X 10(-9) M for ANF and recognized an epitope at the C-terminal of the synthetic rat ANF(101-126). Cross reactivity experiments with Auriculin B, Atriopeptin I and Atriopeptin II suggests Phe124-Arg125-Tyr126 in the epitope recognized by this antibody. The epitope appears to be similar to the portion of the hormone molecule essential for ANF binding to the B-receptor (high m.wt. receptor). This antibody may be useful for the generation of anti-idiotypic antibodies for the B-receptors.

High affinity receptors for atrial natriuretic factor in PC12 cells.

Specific receptors for atrial natriuretic factor (ANF) are characterized on PC12 cells (rat pheochromocytoma cell line). Radioiodinated synthetic ANF (Rat, 8-33) bound to a single class of high affinity binding sites with the Kd value of 6.7 X 10(-10) M. The Bmax value was 29 fmol/10(5) cells and receptor density was calculated as 194,000 +/- 20,000/cell. Photoaffinity labelling of ANF receptor specifically labelled two protein bands with apparent m.wt of 70,000 and 130,000. When the cells were incubated with the labelled ligand at 37 degrees C the ligand was internalized. The rate of internalization increased in the presence of increased ligand concentration. ANF receptors on PC12 cells are reported for the first time which would provide a unique model for study of ANF-receptor interaction.