Segregation of Na(+)-channel gene expression during neuronal-glial branching of a rat PNS-derived stem cell line, RT4-AC.

RT4 is a family of cell lines isolated from an ethylnitrosourea-induced rat peripheral neurotumor. RT4-AC cells express both excitable membrane and glial cell properties. In a process called cell-type conversion, RT4-AC cells segregate these properties to generate three distinct derivative cell types which have been classified as either neuronal (RT4-E and RT4-B) or glial (RT4-D). In this report we demonstrate that: (1) upon cell-type conversion, Na(+)-channel mRNA expression segregates primarily with the RT4 neuronal derivatives, (2) the SkM2 Na(+)-channel gene, which was originally isolated from rat muscle cDNA libraries, is the predominant gene expressed by the RT4 neuronal derivatives, (3) the three rat brain Na(+)-channel genes I, II, and III and the muscle-derived SkM1 gene are not the principal Na(+)-channel genes involved in the segregation, although very low levels of message of these genes are detected, and (4) the RT4 glial derivative expresses slightly higher levels of message from rat brain genes I and II than the neuronal derivatives. Since the RT4 cell lines were derived from a peripheral neurotumor these results present the possibility that the SkM2 gene may be important in vivo in the rat peripheral nervous system.     

Clonal sublines of rat neurotumor RT4 and cell differentiation: IV. Cell surface proteins

There are stem cells in RT4 neurotumor that undergo spontaneous differentiation into three distinct cell types in culture (cell type conversion). Stem cells (RT4-AC) and one of the differentiated cell types (RT4-D) are tumorigenic and synthesize glial-specific S100 protein, while the others (RT4-B and RT4-E) are nontumorigenic and demonstrate some neuronal function. Interrelationships between cell surface proteins and differentiation of RT4 cells were analyzed. The surface proteins are radioiodinated by a lactoperoxidase method, separated by gel electrophoresis in two dimensions, visualized by autoradiography, and quantitated according to the relative radioactivity associated with each protein species. Integral surface proteins are compared in the present study which remain associated with plasma membrane after the extraction of radioiodinated cells with 0.1 N NaOH. The extraction also helps to remove serum proteins in the medium which are absorbed on cell surfaces and may be artifactually recognized as surface proteins. All RT4 cell types are complex in cell surface protein composition and nearly 100 integral surface proteins have been identified when all RT4 cell types are combined. Many unique proteins as well as common proteins have been identified. Considerable similarity exists between RT4-AC and RT4-D and between RT4-B and RT4-E. The two cell types of each pair are distinct yet share some common neurological and tumorigenic characteristics. In contrast, little similarity exists in other combinations of the two cell types, e.g., RT4-AC and RT4-E, etc. The results support the notion that the pattern of cell surface protein expression is a stable differentiation property and a characteristic set of proteins corresponds to each stage of differentiation.     

Regulation of the S100 protein and GFAP genes is mediated by two common mechanisms in RT4 neuro-glial cell lines

RT4-AC cells express both neuronal and glial properties and undergo cell-type conversion in culture to three distinct derivatives, described as either neuronal-like or glial-like. A coordinate induction of glial fibrillary acidic protein (GFAP) and S100 protein and GFAP gene expression is coordinately induced by cAMP. In addition, for the first time we provide direct evidence that the ability to express both the S100 and GFAP genes is conserved with cell-type conversion to the glial derivative cell types, but is coordinately lost with conversion to the neuronal derivative cell types. These results make it highly likely that the GFAP and S100 genes are regulated by two common mechanisms in RT4-AC cells: (1) cAMP-mediated control of gene expression; and (2) a mechanism that allows these two genes to be coordinately expressed or not expressed as a consequence of cell-type conversion.     

The sodium channel in non-impulsive cells. Interaction with specific neurotoxins.

The cell line C9 used in this paper has a resting potential of --50 mV (+/- 10 mV) but is unable to generate an action potential upon electrical stimulation. The cell membrane has receptors for the selectivity filter toxin tetrodotoxin as well as for the gating system toxins, veratridine, scorpion toxin and sea anemone toxin. The Na+ channel which remains silent to electrical stimulation in the absence of toxins can be chemically activated by the gating system toxins. This has been demonstarted by electrophysiological techniques and by 22Na+ flux studies. The electrophysiological approach has shown that the sea anemone toxin is able to induce a spontaneous slow-wave activity inhibited by tetrodotoxin. 22Na+ influx analyses have shown that veratridine and the sea anemone toxin produce an important increase of the initial rate of 22Na+ influx into the C9 cell. The stimulation of 22Na+ entry by these gating system toxins is similar to that found using spiking neuroblastoma cells. Veratridine and the sea anemone toxin on one hand as well as veratridine and the scorpion toxin on the other hand are synergistic in their action to stabilize an open and highly permeable form of the sodium channel. Stimulation of 22Na+ entry into the cell through the sodium channel maintained open by the gating system neurotoxins is completely suppressed by tetrodotoxin.     

Clonal sublines of rat neurotumor RT4 and cell differentiation. VI. Chromosome analysis

The RT4 neurotumor cell system consists of clonally derived cell lines where a stem cell type segregates in vitro into three biochemically and morphologically different cell types, one glial and two neuronal types. This process has been termed cell-type conversion (M. Imada and N. Sueoka, 1978, Dev. Biol. 66, 97-108). Detailed cytogenetic analysis of the RT4 cell lines are described. Giemsa-banding analysis of 12 independent clonal isolates of the four different RT4 cell types showed a relatively stable karyotype. The stem cell line, RT4-AC, is diploid and most stable, and it has one 4q+ marker chromosome in place of a normal No. 4. This 4q+ marker was identified in all cell types of the RT4 system and was not observed in other cell lines of BDIX origin. The 4q+, therefore, is a chromosomal marker of the RT4 system. Consistent chromosome rearrangement was not found in any one of the cell-type conversions of the RT4-AC cells into the three derivative cell types. The relative stability of the karyotype of the different clonal isolates gives the RT4 system an advantage in studies of genetic regulation and expression of cell-type conversion in vitro. Also the 4q+ marker can be used to identify RT4 cells in coculture experiments or to distinguish RT4 cells in cases of suspected cell-line contamination.     

Sodium-potassium exchange in sea urchin egg. II. Ionic events stimulating the Na+-K+ pump activity at fertilization

The preceding paper (Ciapa et al., 1984) provided biochemical and kinetic characterization of the Na+-K+ exchange in Paracentrotus lividus eggs. The present work is a study of the ionic events involved in the stimulation of the Na+-K+ transporter after fertilization. Fertilization in low Na+-external medium containing amiloride (0.1 mM) suppresses the stimulation of the net efflux of H+ and 86Rb uptake. Activation of eggs with the ionophore A23187 leads to stimulation of both Na+-H+ exchange and ouabain-sensitive 86Rb influx. When eggs were activated with A23187 in artificial seawater, 86Rb uptake and 24Na influx showed similar saturable kinetics with respect to the external Na+. A23187 treatment of eggs in Na+-free artificial seawater did not stimulate the Na+-K+ exchange until 10 mEq Na+ was added. Activation of eggs by NH4Cl (5 mM) stimulated 86Rb influx and Na+ exit; both fluxes were ouabain sensitive. Monensin increased cell Na+ of unfertilized eggs without any significant increase in intracellular pH: a condition in which 86Rb influx was not markedly stimulated. Addition of 10 mEq Na+ to unfertilized eggs in Na+-free artificial seawater stimulated 86Rb uptake but to a lower extent that did 10 mEq Na+ plus sperm. It is concluded that (1) the stimulation of the Na+-K+ pump at fertilization has an absolute requirement for the Na+-H+ exchange; (2) the alkalinization of eggs resulting from the acid efflux is a prerequisite for the enhancement of the Na+-K+ pump; (3) the amount of Na+ entering eggs at fertilization determines the intensity of the Na+-K+ exchange; (4) early events of fertilization such as exocytosis and calcium release which may be involved in the stimulation of the Na+-K+ pump must necessarily be coupled to cell alkalinization.     

Cation selectivity characteristics of the reconstituted voltage-dependent sodium channel purified from rat skeletal muscle sarcolemma

In this report, the alkali metal cation selectivity of the purified, voltage-dependent sodium channel from rat skeletal muscle is described. Isolated sodium channel protein (980-2840 pmol of saxitoxin binding/mg of protein) was reconstituted into egg phosphatidylcholine vesicles, and channels were subsequently activated by either batrachotoxin (5 X 10(-6) M) or veratridine (5 X 10(-4) M). Activation of the reconstituted sodium channel by batrachotoxin permitted rapid specific influx of cations into channel-containing vesicles. Quenched flow kinetic techniques were adapted to allow resolution of the kinetics of cation movement. Uptake rates for 42K+, 86Rb+, and 137Cs+ were measured directly and half-times for equilibration at 18 degrees C were determined to be 350 ms, 2.5 s, and 10 s, respectively, in this vesicle population. 22Na+ equilibration occurred within the mimimum quenching time of the apparatus (90 ms) but an upper limit of 50 ms at 18 degrees C could be assigned to its half-time. Based on this upper estimate for Na+, cation selectivity ratios of the batrachotoxin-activated channel were Na+ (1):K+ (0.14):Rb+ (0.02):Cs+ (0.005). Toxin-stimulated influx could be blocked by saxitoxin with a Ki of approximately 5 X 10(-9) M at 18 degrees C. Rates of cation movement through veratridine-activated channels were much slower, with half-times of 1.0, 1.2, 2.0, and 2.6 min at 36 degrees C for Na+, K+, Rb+, and Cs+, respectively. The temperature dependences of batrachotoxin and veratridine-stimulated cation uptake were markedly different. The activation energies for 86Rb+ and 137Cs+ movement into batrachotoxin-activated vesicles were 7.6 and 6.1 kcal/mol, respectively, while comparable measurements for these two cations in veratridine-activated vesicles yielded activation energies of 31 kcal/mol. Measurements of cation exchange with batrachotoxin-activated channels may reflect characteristics of an open sodium channel while the process of channel opening itself may be rate-limiting when veratridine is used for activation.     

The effect of nerve growth factor on the development of sodium channels in PC12 cells

We have studied the development of the action potential Na+ channels in PC12 cells, an established line that has been useful as a model for neuronal differentiation. In continuous culture PC12 cells, although electrically inexcitable, nevertheless have a low level of Na+ channels as judged by the increase in 22Na+ uptake in the presence of veratridine and scorpion toxin. These two neurotoxins have been shown to promote activation of Na+ channels in a variety of electrically excitable cells. Following treatment with nerve growth factor (NGF), conditions which induce differentiation to an electrically excitably neuronal-cell type, the neurotoxin-activated 22Na+ uptake increases approximately 12-fold, on a per cell basis, reaching a maximum in 12-16 days. The dose-response curves for veratridine and scorpion toxin are unchanged by NGF treatment (K0.5 for veratridine, 18-14 microM; K0.5 for scorpion toxin, 120-96 nM). Na+ channels in both undifferentiated and differentiated cells are tetrodotoxin sensitive and NGF treatment has no effect on the inhibition constant (Ki, 10-12 nM). Na+ channel sites were measured directly by the specific binding of [3H]saxitoxin. In NGF-treated cells, the saxitoxin receptor density reaches 154 fmol/mg protein (Kd, 1.3 nM), a level comparable to other excitable cells. Levels in control cells were too low to measure accurately. These findings show that NGF treatment of PC12 cells leads to a substantial increase in the expression of neurotoxin-sensitive Na+ channels. Furthermore, these channels are pharmacologically similar, if not identical, to those which exist in undifferentiated cells and therefore do not appear to result from the conversion of preexisting channels.     

Lysophosphatidic acid-LPA1 receptor-Rho-Rho kinase-induced up-regulation of Nav1.7 sodium channel mRNA and protein in adrenal chromaffin cells: enhancement of 22Na+ influx, 45Ca2+ influx and catecholamine secretion

In cultured bovine adrenal chromaffin cells, chronic (> or = 24 h) treatment with lysophosphatidic acid (LPA) augmented veratridine-induced 22Na+ influx via Na(v)1.7 by approximately 22% (EC(50) = 1 nmol/L), without changing nicotine-induced 22Na+ influx via nicotinic receptor-associated channel. LPA enhanced veratridine (but not nicotine)-induced 45Ca2+ influx via voltage-dependent calcium channel and catecholamine secretion. LPA shifted concentration-response curve of veratridine for 22Na+ influx upward, without altering the EC(50) of veratridine. Ptychodiscus brevis toxin-3 allosterically enhanced veratridine-induced 22Na+ influx by twofold in non-treated and LPA-treated cells. Whole-cell patch-clamp analysis showed that peak Na+ current amplitude was greater by 39% in LPA (100 nmol/L for 36 h)-treated cells; however, I-V curve and steady-state inactivation/activation curves were comparable between non-treated and LPA-treated cells. LPA treatment (> or = 24 h) increased cell surface [3H]saxitoxin binding by approximately 28%, without altering the K(d) value; the increase was prevented by cycloheximide, actinomycin D, or Ki16425, dioctylglycerol pyrophosphate 8:0 (two inhibitors of LPA(1) and LPA3 receptors), or botulinum toxin C3 (Rho inhibitor), Y27632 (Rho kinase inhibitor), consistent with LPA(1) receptor expression in adrenal chromaffin cells. LPA raised Nav1.7 mRNA level by approximately 37%. Thus, LPA-LPA(1) receptor-Rho/Rho kinase pathway up-regulated cell surface Nav1.7 and Nav1.7 mRNA levels, enhancing veratridine-induced Ca2+ influx and catecholamine secretion.     

Phorbol esters and gene expression: the role of rapid changes in K+ transport in the induction of ornithine decarboxylase by 12-O- tetradecanoylphorbol-13-acetate in BALB/c 3T3 cells and a mutant cell line defective in Na+K+Cl- cotransport

A BALB/c 3T3 preadipose cell line defective in Na+K+Cl- cotransport (3T3-E12a cells) has been used to study the relationship between phorbol ester-induced rapid changes in cation fluxes and changes in expression of a gene known to be modulated by this agent. In contrast to its effect on parental 3T3 cells, 12-O-tetradecanoylphorbol-13-acetate (TPA) did not inhibit either furosemide-sensitive 86Rb+ influx or the rate of 86Rb+ efflux from preloaded mutant cells. TPA-induced changes in intracellular K+ content were diminished in 3T3-E12a cells as compared with parental cells. Thus, mutation of the Na+K+Cl- cotransport system renders overall potassium transport in mutant cells largely insensitive to modulation by TPA. The morphological and functional responses of 3T3 and 3T3-E12a cells to TPA were also compared. In contrast to the extensive and long-lasting changes in morphology of 3T3 cells after 0.16 microM TPA addition, only slight and shorter-lived morphological effects of TPA were observed in 3T3-E12a cells. The transport properties of mutant cells were not totally unresponsive to TPA since hexose transport (2-deoxyglucose uptake) could be stimulated in both cell types. To establish a possible link between early changes in cation fluxes and activation of gene expression by TPA, the induction of the enzyme ornithine decarboxylase (ODC) was studied in detail. Addition of fresh medium containing serum or exposure to hypoosmotic conditions resulted in the induction of ODC in both 3T3 and 3T3-E12a cells. However, TPA failed to cause an increase in ODC activity in mutant cells, although a substantial induction of the enzyme was seen in parental cells. These results suggest that rapid changes in ion fluxes mediated by the Na+K+Cl- cotransport system are necessary for at least one of the phorbol ester-induced changes in gene expression in responsive cells.