Transformation of human osteoblast cells to the tumorigenic phenotype by depleted uranium-uranyl chloride.

Depleted uranium (DU) is a dense heavy metal used primarily in military applications. Although the health effects of occupational uranium exposure are well known, limited data exist regarding the long-term health effects of internalized DU in humans. We established an in vitro cellular model to study DU exposure. Microdosimetric assessment, determined using a Monte Carlo computer simulation based on measured intracellular and extracellular uranium levels, showed that few (0.0014%) cell nuclei were hit by alpha particles. We report the ability of DU-uranyl chloride to transform immortalized human osteoblastic cells (HOS) to the tumorigenic phenotype. DU-uranyl chloride-transformants are characterized by anchorage-independent growth, tumor formation in nude mice, expression of high levels of the k-ras oncogene, reduced production of the Rb tumor-suppressor protein, and elevated levels of sister chromatid exchanges per cell. DU-uranyl chloride treatment resulted in a 9.6 (+/- 2.8)-fold increase in transformation frequency compared to untreated cells. In comparison, nickel sulfate resulted in a 7.1 (+/- 2.1)-fold increase in transformation frequency. This is the first report showing that a DU compound caused human cell transformation to the neoplastic phenotype. Although additional studies are needed to determine if protracted DU exposure produces tumors in vivo, the implication from these in vitro results is that the risk of cancer induction from internalized DU exposure may be comparable to other biologically reactive and carcinogenic heavy-metal compounds (e.g., nickel).

Membrane current underlying muscarinic cholinergic excitation of motoneurons in lobster cardiac ganglion.

1. We studied the effect of cholinergic agonists on motoneurons of the lobster cardiac ganglion under voltage clamp. 2. In unclamped neurons, acetylcholine (ACh) caused a depolarization and increase in burst potential frequency. By the use of nicotinic and muscarinic agonists, we determined that both types of receptors are present on the neurons. We therefore used specific muscarinic agonists to further study ionic mechanisms underlying the muscarinic cholinergic current (Imch). 3. Muscarinic agonists produced detectable inward current at doses above 10(-6) M, and maximum effect was seen at doses above 10(-3) M. 4. Imch was voltage-dependent. When the membrane holding potential was shifted to levels negative to the resting potential, the response declined, nulling but not reversing at -80 to -100 mV. The response enlarged with membrane depolarization, reaching a maximum at between -30 and -10 mV. With further depolarization, the response declined and then reversed at potentials around +20 mV. 5. The muscarinic response varied as a function of extracellular Na+ concentration and was completely blocked in Na+-free solutions. The relationship between response amplitude and external Na+ was well described by the electrodiffusion equation for Na+ driving force. 6. Imch amplitude also varied as a function of extracellular potassium concentration, becoming larger with low external K+ and smaller at higher concentrations. Shifting the Cl- equilibrium potential did not affect the properties of the Imch. 7. Tetrodotoxin (TTX) had no effect on Imch. In concentrations of 1-10 mM, such K+-channel blocking agents as Ba2+, Cs+, 4-aminopyridine (4-AP), or tetraethylammonium (TEA), and such Ca2+-channel blockers as Co2+ or Mn2+, when applied externally, did not suppress Imch. Above 30 mM, TEA did inhibit the response, and combinations of K+-channel blocking agents, each at concentrations insufficient alone to block the current, also inhibited Imch. 8. Current-voltage (I-V) curves obtained during muscarinic agonist perfusion consistently crossed the control I-V curves at a mean membrane potential of +24 mV. The reversal potential shifted to a more negative value in low extracellular Na+. 9. Although no reversal of Imch was seen when agonists were applied to cells clamped at negative holding potentials, the averaged curve of Imch, obtained by subtracting control ramp I-V curves from those obtained in the presence of agonist, did show a small net outward current at membrane potentials negative to -100 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Coupling ratio of the Na-K pump in the lobster cardiac ganglion.

The electrogenic Na-K pump coupling ratio in the large neurons of the lobster cardiac ganglion was determined by two different electrophysiological techniques. A graphical analysis plotting exp(EmF/RT) vs. [K]o after the pump was blocked by ouabain was used to determine values for [K]i, PNa/PK, and the pump coupling ratio. These measurements were made 4-8 h after the cells were penetrated with microelectrodes, and thus represent non-Na-loaded steady state values. The value obtained for the pump coupling ratio under these conditions was 1.44 +/- 0.06 (n = 9) or close to 3 Na for 2 K. The second technique used to measure the coupling ratio was to iontophoretically inject Na ions into the neuron. Neurons were penetrated with three microelectrodes, two of which were filled with 2 M Na-citrate; the third electrode contained either 2 M K-citrate or 3 M KCl. By passing current between the Na salt-containing electrodes, Na was injected into the cell soma. The injection system was calibrated by injecting 24Na-citrate into counting vials from representative microelectrodes (calculated 24Na transport = 0.92). By knowing the Na load injected into the cells, and by measuring the time-current area produced by the Na activation of the Na-K pump, the coupling ratio was calculated to be 1.54 +/- 0.05 (n = 19), which is not significantly different from the value obtained by the first method. This value represents a Na-loaded experimental situation. When Na was removed from the external bathing solution, the coupling ratio shifted to 2 Na to 1 K (2.0 +/- 0.07, n = 4). These results suggest that the pump normally operates with a 3:2 ratio both in steady state and under Na load but that in the absence of external Na, it can operate with less than a full complement (2) of K on the external surface of the pump.

Demonstration of an electrogenic Na+-K+ pump in mouse spleen macrophages.

The effect of ouabain on the membrane potential of cultured mouse spleen macrophages was examined. Ouabain (10(-3) M) induced a membrane depolarization (6.6 mV) in 18 of 19 cells studied that occurred within several minutes after exposure and was not associated with significant changes in current-voltage relationships. In related studies, cells were placed in K+-free medium in the cold for 2 h to block pump activity. Subsequent exposure to K+ at 37 degrees C resulted in a membrane hyperpolarization. However, addition of K+ also enhanced inward rectification. To differentiate between the effect of K+ on the Na+-K+ pump and its action on inward rectification, two types of experiments were done. Studies performed in the presence of barium, which blocks inward rectification, demonstrated a K+-induced hyperpolarization with no changes in rectification. Additional studies examined the effects of rubidium on Na+-loaded cells. Rubidium, which blocks inward rectification but substitutes for K+ in activating the Na+-K+ pump, induced membrane hyperpolarizations that were reversed by addition of ouabain. These data indicate that macrophages exhibit an electrogenic Na+-K+ pump, which probably contributes to the resting membrane potential under steady-state conditions and can be activated under conditions designed to Na+ load the cells. In addition, they demonstrate that increasing extracellular K+ enhances inward rectification in macrophages.

Fast axonal transport in permeabilized lobster giant axons is inhibited by vanadate.

We have developed a method for permeabilizing axons and reactivating the fast transport of microscopically visible organelles. Saltatory movements of organelles in motor axons isolated from lobster walking legs were observed using Nomarski optics and time-lapse video microscopy. In the center of the axon most of the particles and mitochondria moved in the retrograde direction, but immediately below the axolemma the majority moved in the anterograde direction. When axons were permeabilized with 0.02% saponin in an adenosine 5'-triphosphate (ATP)-free "internal" medium, all organelle movement ceased. Saltatory movements resembling those in intact axons immediately reappeared upon the addition of MgATP. Very slight movement could be detected with ATP concentrations as low as 10 microM, and movement appeared to be maximal with 1 to 5 mM ATP. Vanadate, which does not affect axonal transport in intact axons, inhibited the reactivated organelle movements in permeabilized axons. Movement was rapidly and reversibly inhibited by 50 to 100 microM sodium orthovanadate. The effects of vanadate, including the time course of inhibition, its reversibility, and its concentration dependence, are consistent with the hypothesis that a dyneinlike like molecule may play a role in the mechanism of fast axonal transport.

Alterations in spontaneous transmitter release by divalent cations after treatment of the neuromuscular junction with beta-bungarotoxin.

1. Spontaneous transmitter release was studied at the frog sartorius neuromuscular junction in the presence of a variety of cations before and after treatment with the specific presynaptic neurotoxin, beta-bungarotoxin (beta-BuTX). 2. Treatment with beta-BuTX produced a maintained increase in spontaneous release, as indicated by the miniature end-plate potential (m.e.p.p.) frequency. It was demonstrated that the m.e.p.p. frequency remained dependent on the extracellular calcium concentration. 3. A 30 mM increase in extracellular sodium chloride produced a reversible increase in frequency only after beta-BuTX treatment, indicating that beta-BuTX had increased the permeability of the presynaptic terminal. 4. Furthermore, several divalent cations other than calcium were shown to either maintain or greatly increase the m.e.p.p. frequency after beta-BuTX treatment (before toxin treatment replacement of calcium by these divalent cations produced only small changes in frequency). The relative effectiveness of the divalent cations tested in increasing spontaneous transmitter release after toxin treatment was Co2+ congruent to Ni2+ greater than Mg2+ greater than Ca2+ congruent to Sr2+ greater than Mn2+. The effect of cobalt, which increased the m.e.p.p. frequency 6.5 times after toxin treatment, was studied in detail.

Uptake and release of 45Ca by Myxicola axoplasm.

The binding and release of 45Ca by axoplasm isolated from Myxicola giant axons were examined. Two distinct components of binding were observed, one requiring ATP and one not requiring ATP. The ATP-dependent binding was largely prevented by the addition of mitochondrial inhibitors, whereas the ATP-independent component was unaffected by these inhibitors. The ATP-independent binding accounted for roughly two-thirds of the total 45Ca uptake in solutions containing an ionized [Ca2+] = 0.54 microM and was the major focus of this investigation. This fraction of bound 45Ca was released from the axoplasm at a rate that increased with increasing concentrations of Ca2+ in the incubation fluid. The ions Cd2+ and Mn2+ were also able to increase 45Ca efflux from the sample, but Co2+, Ni2+, Mg2+, and Ba2+ had no effect. The concentration-response curves relating the 45Ca efflux rate coefficients to the concentration of Ca2+, Cd2+, and Mn2+ in the bathing solution were S-shaped. The maximum rate of efflux elicited by one of these divalent ions could not be exceeded by adding a saturating concentration of a second ion. Increasing EGTA concentration in the bath medium from 100 to 200 microM did not increase 45Ca efflux; yet increasing the concentration of the EGTA buffer in the uptake medium from 100 to 200 microM and keeping ionized Ca2+ constant caused more 45Ca to be bound by the axoplasm. These results suggest the existence of high-affinity, ATP-independent binding sites for 45Ca in Myxicola axoplasm that compete favorably with 100 microM EGTA. The 45Ca efflux results are interpreted in terms of endogenous sites that interact with Ca2+, Cd2+, or Mn2+.

Inward rectification in mouse macrophages: evidence for a negative resistance region.

The electrical properties of cultured mouse thioglycollate-induced peritoneal macrophages were investigated using intracellular recording techniques. Thirty-five percent of the cells studied had membrane potentials ranging from -65 to -95 mV and exhibited S-shaped, steady-state current-voltage (I-V) relationships containing a transitional region. Analysis of currents in the transitional region from the rate of rise and fall of the voltage responses to current pulses indicated the presence of a negative resistance region in this area. Tetrodotoxin (3 X 10(-5) M), cobalt chloride (3 mM), 4-aminopyridine (4 mM), and tetraethylammonium chloride (8 mM) did not eliminate the transitional region of the I-V curves, whereas addition of barium chloride (4 mM) and rubidium chloride (3 mM) did. Increasing the external concentration of potassium shifted the I-V relationship horizontally along the current axis but did not eliminate the transitional region. These data indicate that the inward rectification and the negative resistance region probably result from a voltage-dependent potassium conductance.

Nonlinear current-voltage relationships in cultured macrophages.

Intracellular recordings of cultured mouse thioglycolate-induced peritoneal exudate macrophages reveal that these cells can exhibit two different types of electrophysiological properties characterized by differences in their current-voltage relationships and their resting membrane potentials. The majority of cells had low resting membrane potentials (-20 to -40 mV) and displayed current-voltage relationships that were linear for inward-going current pulses and rectifying for outward-going pulses. Small depolarizing transients, occurring either spontaneously or induced by current pulses, were seen in some cells with low resting membrane potentials. A second smaller group of cells exhibited more hyperpolarized resting membrane potentials (-60 to -90 mV) and S-shaped current-voltage relationships associated with a high-resistance transitional region. Cells with S-shaped current-voltage relationships sometimes exhibited two stable states of membrane potential on either side of the high-resistance transitional region. These data indicate that macrophages exhibit complex electrophysiological properties often associated with excitable cells.

Blockade of neuromuscular transmission by enzymatically active and inactive beta-bungarotoxin.

beta-Bungarotoxins have been shown to be presynaptic blockers of neuromuscular transmission. This paper reports experiments using the most positively charged beta-bungarotoxin that elutes from a CM-Sephadex C-25 column. The toxin is shown to be a single polypeptide with a molecular weight of approximately 11,000 and has phospholipase A2 activity. The application of the enzymatically active toxin to the frog sciatic nerve-sartorius muscle preparation results in an initial decrease in the average endplate potential amplitude followed by a temporary rebound in endplate potential amplitude, and finally a complete inhibition of endplate potentials. Similarly, minature endplate potential frequency is initially reduced upon toxin application but then increases dramatically. After the phospholipase A2 of the toxin is inactivated, treatment with the toxin results in only the initial decrease in transmitter release. There results suggest that this beta-bungarotoxin acts in two functionally separate steps: (i) by binding to a specific presynaptic site possibly associated with calcium entry, and (ii) by perturbing the presynaptic membrane by its enzyme action, which results in an increase and then a failure in transmitter release.

Characterization of a depolarizing dopamine response in a vertebrate neuronal somatic cell hybrid.

The physiology and pharmacology of a depolarizing dopamine response was studied in the vertebrate neuronal somatic cell hybrid TCX11. The average resting membrane potential was -50 mV (S.D.=+/-7) with a membrane resistance of 40.5 mOhms (S.D.=+/-8) as determined from intracellular recordings. Depolarizing current pulses did not elicit an action potential. Cells displayed a linear current-voltage relationship when artificially depolarized up to +30 mV. Iontophoretically applied dopamine elicited a depolarizing response with a conductance increase and a reversal potential of -15 mV (S.D.=+/-4.7). Experiments altering medium ion concentrations demonstrated the conductance increase was to sodium and most likely potassium. The dopamine agonist ET495 (Piribedil) and the analogue epinine mimicked dopamine, while closely related biogenic amines, with the exception of noradrenaline, elicited no response. Apomorphine also elicited a depolarizing response but was much less efficacious than Piribedil. Noradrenaline was less potent than dopamine and appeared to act at the dopamine receptor. Methylation (3-methoxytyramine) or absence of the 3-hydroxy group (tyramine) of dopamine resulted in total loss of activity. The dopamine antagonists chlorpromazine, trifluoperazine, promazine, and bulbocapnine reversibly blocked the response to dopamine at medium concentrations less than 5 micronM. The adrenergic antagonist phentolamine blocked the response while phenoxybenzamine only reduced the response at higher concentrations. The acetylcholine antagonists alpha-bungarotoxin, hexamethonium, and scopolamine did not block the dopamine response. Both d-tubocurarine and atropine acted as antagonists. Collectively, these results demonstrate the presence of a receptor on a cultured cell line that is specific for dopamine, mediates a depolarizing and conductance increase response to dopamine, and displays the pharmacology most closely associated with dopamine receptors.

Correlation of transmitter release with membrane properties of the presynaptic fiber of the squid giant synapse.

Depolarization of the presynaptic terminal by current produced a postsynaptic potential (PSP) which increased with increasing presynaptic polarization and then reached a plateau. Iontophoretic injection of tetraethylammonium ions (TEA) into the presynaptic axon near the terminal produced a prolonged presynaptic spike. The resulting PSP is increased in size and its time course closely followed that of the presynaptic spike. The presynaptic fiber no longer exhibited rectification and strong depolarizations revealed that the PSP reached a maximum with about 110 mv depolarization. Further depolarization produced a decrease in PSP amplitude and finally transmission was blocked. However, a PSP then always appeared on withdrawal of the depolarizing current. Under the conditions of these experiments, the PSP could be considered a direct measure of transmitter release. Bathing the TEA-injected synapse with concentrations of tetrodotoxin (TTX) sufficient to block spike activity in both pre- and postsynaptic axons did not greatly modify postsynaptic electrogenesis. However, doubling TTX concentration reversibly blocked PSP. Thus the permeability changes to Na and K accompanying the spike do not appear necessary for transmitter release. Some other processes related to the level of presynaptic polarization must be involved to explain the data. The inhibition of transmitter release by strong depolarizations appears to be related to Ca action. A membrane Ca current may also be necessary for normal transmitter release.

Tetraethylammonium ions: effect of presynaptic injection on synaptic transmission.

Tetraethylammonium ions were injected into the presynaptic axon of the squid giant synapse. Injection of these ions caused prolongation of the action potential with decreased out ward current. The prolonged spike was associated with increased release and prolonged activity of the transmitter substance. Although the amplitude of the postsynaptic potential increased with presynaptic depolarization, strong depolarization blocked transmitter re lease. In the injected presynaptic axon, transmitter release was blocked by 10(-6) gram of tetrodotoxin per milliliter. Transmitter release appears to be under control of presynaptic potential levels.