Interaction of NT-4 and BDNF with gp145trkb receptor: effect on cellular metabolism.

In the present study a silicon microphysiometer (Cytosensor) was applied in investigating interactions of gp145(trkb), a member of the tyrosine kinase receptor family, with different neurotrophic factors. NIH-3T3 cells transfected with gp145(trkb) receptors (NIH3T3/trkB cells) were utilized in the studies. Treatment with brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4) and neurotrophin-3 (NT-3) induced changes in the metabolic rate of NIH3T3/trkB cells. In contrast, no response was observed with nerve growth factor (NGF). The effects of NT-4 and BDNF on NIH3T3/trkB cells were receptor-specific in that they did not induce metabolic rate changes in wild type NIH3T3 cells or cells transfected with either gp140(trkb) (TrkA) or gp145(trkb) (TrkC) receptors. In contrast, NT-3 induced metabolic rate changes in cells transfected with each of the three different Trk receptors. The activity of NT-4 was significantly higher than that of BDNF. K252a, a protein kinase inhibitor, reduced the NT-4- and BDNF-induced response of the NIH3T3/trkB cells. This suggests that the NT-4 and BDNF-induced metabolic rate changes are associated with autophosphorylation of the tyrosine protein kinase residues. This hypothesis is further supported by results of western blot analysis. The results show that interactions of Trk receptors with neurotrophic factors result in metabolic changes in cells expressing the receptors.

Regulation and functional characterization of a rat recombinant dopamine D3 receptor.

We stably expressed a rat D3 receptor cDNA in C6 glioma cells (C6-D3 cells), quantifying receptor expression with the radioligands [125I]epidepride (KD = 0.1 nM) and [3H]spiperone (KD = 0.7 nM). As reported previously for D2 receptors, quinpirole induced a 9-16% increase in the rate of extracellular acidification by C6-D3 cells. The acidification was inhibited by epidepride and by the Na+/H+ antiporter inhibitors, amiloride and methylisobutylamiloride, but pertussis toxin treatment had no effect on quinpirole-induced extracellular acidification. These data suggest that D3 receptor stimulation of Na+/H+ exchange in C6 glioma cells is not mediated by the pertussis toxin-sensitive G proteins, Gi or G(o). Overnight treatment of C6-D3 cells with N-propylnorapomorphine, dopamine, or quinpirole resulted in large concentration-dependent increases (up to 500%) in the density of D3 receptors on membranes prepared from the cells. Antagonists had smaller, variable effects on the density of D3 receptors in C6-D3 cells, except for domperidone, which significantly increased the density of D3 receptors. Treatment with pertussis toxin had no effect on the agonist-induced receptor up-regulation, indicating that an interaction with pertussis toxin-sensitive G proteins was not required. Densitometry analysis of Northern blots of RNA prepared from C6-D3 cells showed no significant N-propylnorapomorphine-induced increase in D3 receptor message. Treatment with cycloheximide, however, completely prevented receptor up-regulation by N-propylnorapomorphine. Pretreatment of C6-D2 cells with 10 microM DA resulted in a substantial heterologous sensitization, in which isoproterenol-stimulated adenylyl cyclase activity was enhanced more than twofold.(ABSTRACT TRUNCATED AT 250 WORDS)

Tubulin polymerization by paclitaxel (taxol) phosphate prodrugs after metabolic activation with alkaline phosphatase.

Paclitaxel (taxol) phosphate derivatives BMY46366, BMY-46489, BMS180661 and BMS180820 were used to determine the ability of alkaline phosphatase to convert these water-soluble potential prodrugs to tubulin-polymerizing metabolites (i.e., paclitaxel). Compounds were treated up to 180 min with an in vitro metabolic activation system composed of 10% bovine alkaline phosphatase in 0.2 M tris, pH 7.4, or in 0.2 M glycine, pH 8.8, plus 0.05 M MgCl2. Samples were tested (either by direct addition or after methylene chloride extraction/dimethyl-sulfoxide resuspension) in spectrophotometric tubulin polymerization assays utilizing bovine-derived microtubule protein. Pretreatment of 2'- and 7-phosphonoxyphenylpropionate prodrugs BMS180661 and BMS180820 with alkaline phosphatase for 30 to 120 min yielded relative initial slopes of about 20 to 100% at test concentrations equimolar to paclitaxel. High-performance liquid chromatography/mass spectrometry of BMS180661 treated with alkaline phosphatase confirmed the production of paclitaxel from the prodrug. In contrast, 2'- and 7-phosphate analogs BMY46366 and BMY46489 treated with alkaline phosphatase were not active in tubulin assays. None of the paclitaxel phosphate prodrugs polymerized tubulin in the absence of metabolic activation. The differences in tubulin polymerization with metabolic activation may be related both to accessibility of the phosphate group to the enzyme and to anionic charge effects. These results demonstrate that certain paclitaxel phosphate prodrugs can be metabolized by alkaline phosphatase to yield effective tubulin polymerization.

Dopamine D2 receptor stimulation of Na+/H+ exchange assessed by quantification of extracellular acidification.

A microphysiometer was used to quantify the rate of extracellular acidification by C6 glioma cells and L fibroblasts expressing recombinant dopamine D2 receptors. The dopamine D2 receptor agonist, quinpirole, accelerated the rate of acidification of the medium by C6 cells expressing either the short or long form of D2 receptors, D2(415) and D2(444), but not by wild-type cells that were not transfected with a D2 receptor cDNA. The rate of acidification increased with increasing concentrations of quinpirole up to 100 nM. Inhibition of the response by the dopamine D2 antagonist, spiperone, provided additional evidence that the enhanced extracellular acidification resulted from stimulation of D2 receptors. To test the hypothesis that D2 receptor-stimulated extracellular acidification was due to transport of protons by a Na+/H+ antiporter and reflected intracellular alkalinization, the effect of two inhibitors of Na+/H+ exchange, amiloride and methyl-isobutyl-amiloride, was determined. Both compounds inhibited quinpirole-induced extracellular acidification at concentrations that did not alter D2 receptor-mediated inhibition of adenylylcyclase or radioligand binding to D2 receptors. In addition, quinpirole-induced extracellular acidification was greatly inhibited by removal of sodium from the extracellular medium, confirming the participation of Na+/H+ exchange in the extrusion of acid. Quinpirole (100 nM) also increased the rate of extracellular acidification by L cells expressing D2(415), LZR1 cells. Treatment with pertussis toxin (100 ng/ml for 18 h) had no effect on the quinpirole-induced acid extrusion by C6D2(415) and LZR1 cells, although the same pertussis toxin treatment regimen completely prevented inhibition of adenylylcyclase. We conclude that recombinant D2 receptors accelerate Na+/H+ exchange in C6 cells and L fibroblasts by a pathway that does not involve inhibition of adenylylcyclase or pertussis toxin-sensitive G proteins.

Effects of bradykinin on PC-12 cell differentiation.

PC-12 cells are used as a model for neuronal differentiation because they assume a neuronal phenotype, including the extension of neurites, when exposed to nerve growth factor (NGF). The present results show that bradykinin (BK) also causes PC-12 cells to extend neurites. In addition, BK potentiates the neurite-extending effect of nerve growth factor (NGF), an action which is attenuated by a BK antagonist. The potentiation of neurite extension produced by the combination of BK and NGF may be mediated at the receptor level, as indicated by an NGF-induced alteration of BK binding.

Identification of 3H-bradykinin binding sites in PC-12 cells and brain.

3H-Bradykinin binding sites with the characteristics of receptors were identified in homogenates of bovine hippocampus and PC-12 cells. The characteristics of the binding were similar in both types of tissue and paralleled those of the B2 bradykinin receptor. Following exposure to nerve growth factor for 72 hours, the number of BK binding sites in PC-12 cells significantly increased, but other characteristics of the binding remained unchanged.

Association of alpha-crystallin with actin in cultured lens cells.

The nature of the beaded filaments in the lens fiber cell has been debated for some time. One explanation is that beaded filaments represent an association of alpha-crystallin with actin filaments. By using a double labelling technique that allowed us to view actin filaments and alpha-crystallin in the same cell we have demonstrated that some of the alpha-crystallin in lens cells is indeed associated with actin.

Direct mitogenic effects of insulin, epidermal growth factor, glucocorticoid, cholera toxin, unknown pituitary factors and possibly prolactin, but not androgen, on normal rat prostate epithelial cells in serum-free, primary cell culture.

Selective nutritive conditions were used to isolate normal epithelial cells from fibroblasts in primary cell cultures prepared from adult rat prostate. The pure population of normal epithelial cells proliferated at an exponential rate on a simple polystyrene substratum with doubling times of 35 to 50 hr for 10 to 12 days in the absence of high epithelial cell density, other cell types, or added extracellular matrix elements. Optimization of the nutritive environment allowed direct analysis of the hormone:growth factor requirements for sustained proliferation of the isolated epithelial cells in serum-free medium. An in situ videometric method was used to assay the effect of over 30 known hormones and growth factors on proliferation of the prostate epithelial cell population. The results revealed direct mitogenic effects of insulin, epidermal growth factor, glucocorticoid, cholera toxin, one or more unidentified factors from bovine pituitary, and possibly prolactin. No direct mitogenic effect of androgen on isolated prostate epithelial cells could be demonstrated. Radioimmunoassay of androgen in the primary cultures showed that endogenous androgen was about 34 pM on Day 1 of culture and thus probably too low to mask a response to exogenous androgen. Deletion of any single active growth factor did not reveal an androgen response. The results demonstrate a multihormonal control of normal prostate epithelial cell maintenance and proliferation without the direct participation of androgen.

Modified nutrient medium MCDB 151, defined growth factors, cholera toxin, pituitary factors, and horse serum support epithelial cell and suppress fibroblast proliferation in primary cultures of rat ventral prostate cells.

Nutrient medium WAJC 401 containing 5 micrograms/ml insulin. 10 ng/ml EGF, 10 ng/ml cholera toxin, 50 micrograms/ml pituitary extract, 1 microgram/ml prolactin, 1 microM dexamethasone, and 5% horse serum supports the rapid proliferation of rat ventral prostate epithelial cells in primary culture. The same medium suppresses the growth of prostate fibroblasts.

Prostatic binding protein, polyamine, and DNA synthesis in rat ventral prostate cells.

Prostatic binding protein (PBP), polyamine, and DNA synthesis were examined in primary cultures of rat ventral prostate cells. Soon after aggregates of prostate cells were placed in culture, PBP synthesis fell dramatically and DNA synthetic activity in the epithelial cells increased. The amount of polyamines that were labeled with [3H] from [3H] ornithine fell transiently, but rose again at or before the peak of DNA synthesis. Individual 3H-labeled polyamines in cells and medium were dansylated and separated by thin-layer chromatography. The ratio of [3H] Spermidine plus [3H] Spermine to [3H] Putrescine in the culture medium declined as DNA synthesis increased. Ornithine decarboxylase (ODC) activity fell dramatically along with PBP synthesis even as DNA synthesis and 3H-labeled polyamines increased in the prostate-cell cultures. These results support others that suggest that high ODC activity in prostate epithelial cells is a correlate of prostate epithelial cell function rather than proliferation. However, prostate epithelial cells retain the capacity to synthesize significant levels of polyamines from ornithine (especially Putrescine) during proliferation even when ODC activity is low.