Persistence of vanadium compounds in lungs after intratracheal instillation in rats.

Translocation and tissue distribution of two different forms of vanadium compounds, orthovanadate (soluble) and vanadium pentoxide (less soluble), were investigated. Groups of randomly selected rats were injected intratracheally with radiolabeled vanadium (48V) compounds and the animals were sacrificed at 1, 7 and 28 days after treatment. Blood, lungs and other major organs and tissues, namely liver, kidney, spleen, heart, testes, brain, muscle, and bone were sampled and the vanadium contents determined by gamma spectrometry. The less soluble form of vanadium (vanadium pentoxide) was eliminated from the lungs at a slow but exponentially linear rate, whereas the soluble form was translocated rapidly from this organ and exhibited a non-linear decline. Compared to the less soluble form, significantly less vanadium was retained in lungs 7 and 28 days after intratracheal instillation of vanadate. One day after treatment significantly higher concentrations (approximately 4 times) of vanadium after orthovanadate were observed in liver, kidney, spleen and bone compared to the pentoxide. However, tissue residues at 7 and 28 days indicated that both forms of vanadium were rapidly eliminated, except from bone and lungs. Results suggest a prolonged retention of less soluble forms of vanadium and possible health effects following repeated occupational exposure.

Pulmonary clearance of soluble and insoluble forms of manganese.

Manganese is an essential metal of toxicologic concern primarily because of exposure via inhalation. Environmental forms of Mn exist mainly as insoluble oxides, yet much of the research information available relates to the soluble salts. In the present study, adult male Sprague-Dawley rats were intratracheally instilled with either soluble MnCl2 or insoluble Mn3O4 labeled with 54Mn. Lungs and other major organs were sampled over a span of 3 mo after dosing with the respective chemicals, which were equivalent to 8 mu Ci and 1 mumol of manganese in 0.2 ml of buffer. There was rapid clearance of Mn from the lungs in the case of both chemicals; the chloride cleared at an initial rate of nearly four times that of the oxide. Despite this early difference, the amount of 54Mn remaining in the lungs after 2 wk was similar for both compounds. The level of 54Mn in the liver, kidney, spleen, and testes peaked at the 3-d sampling point in the case of the oxide, whereas the chloride peaked in these organs within 4 h. At 1 wk after administration, however, the 54Mn activity was comparable for both compounds in most organs sampled. Mn uptake in the brain was also more rapid with the chloride form, but both compounds remained at high levels for several weeks.

Toxicity of hexavalent chromium to the alveolar macrophage in vivo and in vitro.

In vivo and in vitro systems were used to evaluate hexavalent chromium toxicity to alveolar macrophages. Rat alveolar macrophages were exposed to 2 micrograms calcium chromate (CaCrO4, insoluble) or 2 micrograms chromium trioxide (CrO3, soluble) in live animals, in vivo, and in tissue culture, in vitro, collected by lavage from the lung. Chemiluminescence and oxygen consumption were measured as indicators of toxicity. Trypan blue dye exclusion was used to determine macrophage viability. In vivo exposure of the macrophage to either chromium compound showed no toxic effects at a 2-micrograms dose. Macrophages exposed in tissue culture, however, had values significantly different from controls. The untreated controls for both exposure methods were compared to evaluate differences resulting from methods alone.

Human parotid saliva protein composition: dependence on physiological factors.

The relative concentrations of the major proteins in human parotid saliva as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis varied greatly between the six individuals included in this study but were remarkably constant for a given individual. Individual relative parotid protein concentrations differed in salivas collected under unstimulated and stimulated conditions but were at least partially independent from circadian and feeding effects. In addition, the relative concentrations of certain salivary proteins decreased with continued lemon-drop stimulation. A total of 29 different bands was composited from the six subjects. Five of the bands had mobilities corresponding with those of calibration proteins selected for their known occurrence in parotid saliva. Only those proteins comprising at least 0.75% of the total protein concentration were detected. Relative protein concentrations of parotid saliva samples collected 12 mo apart from given individuals were identical.

Toxicokinetics of nickel in rats after intratracheal administration of a soluble and insoluble form.

Ninety day laboratory studies were performed to determine the whole body distribution of two chemical forms of nickel in female Wistar rats. A single injections of 15 microCi of 63Ni, either NiCl2 as a solution or NiO as a suspension, 100 nmoles in each case, was administered intratracheally. Rats were sacrificed at post-exposure intervals of 0.5, 2, and 8 hours, 1, 3, 7, 15, 30, 60 and 90 days, and major organs and tissues were analyzed for 63Ni by liquid scintillation counting technique. The soluble NiCl2 was readily distributed throughout the body, and rapidly cleared from the tissues. The insoluble NiO was distributed slowly to other organs from pulmonary tissues. The rate of decline of 63Ni from various organs in the case of NiO was similar to that of NiCl2, with notable exceptions being the lung and associated lymph nodes. After NiO administration, these organs showed a high retention of nickel after 90 days. Results indicated that Ni in soluble form was rapidly absorbed from the site of deposition following pulmonary exposure, whereas, Ni in its oxide or insoluble form was retained in lungs and related lymphatics for a considerable period. The amount of Ni in other organs following NiO exposure, though initially low in all tissues, declined in a fashion similar to organs following NiCl2 exposure. This suggests that NiO was possibly converted to a soluble form of Ni before it was translocated from lungs to other organs, and that low environmental levels of insoluble forms of nickel, which persist in the lung and lymph nodes, do have the potential for assimilation in these tissues.

ATP-induced lysis of rat parotid secretory granules: possible role of ATP in exocytotic release.

Secretory vesicles isolated from a variety of mammalian tissues are known to lyse and thereby release their secretory products when exposed to ATP. This process, which will be termed ATP-induced lysis, has been studied most extensively using adrenal chromaffin-granule preparations. We report here that ATP causes the lysis of a highly purified preparation of rat parotid secretory granules. The rate of granule lysis was measured spectrophotometrically, and ATP-induced lysis was expressed as the increase in the rate of lysis (r = % lysis per min) when ATP was added. This lytic process was characterized with respect to pH, temperature, osmolarity, and the ionic composition of the media. ATP-induced lysis of parotid granules was found to have the following properties in common with the extensively characterized chromaffin-granule process: 1. It is a saturable function of ATP with half-maximal rates observed at 0.5 +/- 0.1 mM ATP. 2. It is temperature dependent, eg, r = 6.1 +/- 2.1%/min at 30 degrees C vs 12.2 +/- 2.5%/min at 37 degrees C. 3. It is inhibited in hyperosmotic media, eg, r = 5.3 +/- 0.3%/min at 0.3 OsM vs 0.8 +/- 0.2%/min at 0.4 OsM. 4. It shows a nucleotide preference of ATP = GTP greater than ADP greater than AMP greater than CTP = ITP. 5. It has an anion requirement. The above findings, combined with reports of ATP-induced lysis of cholinergic, insulin, and posterior-pituitary vesicles, imply that ATP-induced lysis may reflect an ATP-dependent property of all secretory vesicles, and as such, this vesicle property could play a similar role in each exocytotic release process. Using a model system, Miller and Racker [22] made a surprising finding that the extent of which liposomes fuse with a black lipid membrane depends on the osmotic gradient across the vesicle membrane. In view of the osmotic dependence of ATP-induced lysis in this and other secretory-vesicle preparations, we postulate that ATP may prime secretory vesicles for fusion with the plasma membrane by inducing and/or maintaining an osmotic gradient across the vesicle membrane.

Vanadium retention in rat tissues following acute exposures to different dose levels.

Male Wistar rats were given vanadyl trichloride (48VOCl3) ip in doses of V ranging from 0.1 to 8 mg/kg and their tissues were collected 1 and 5 d after the injection. V was distributed in the order bone greater then kidney greater then liver greater than spleen greater than intestines greater than stomach greater than muscle greater than testis greater than lung greater than brain. Residues of V in tissues declined rapidly between 24 h and 5 d after administration. The tissue:blood ratios of V were greater than unity for bone, kidney, liver, and spleen and near unity for all other organs except the brain. Brain levels of V were found to be considerably lower than blood in all cases. V residues were linearly related to dose in most organs when the dose was below 2 mg/kg. At 8 mg/kg, however, liver and kidney showed consistently higher amounts than would be expected from the linear relationship at the low doses. Subcellular distribution of V in liver and kidney indicated that it was associated with nuclei, mitochondria, microsomes, and primarily with high-molecular-weight proteins in the soluble fraction of liver. The results suggest that the distribution pattern of V is dependent on exposure level.

Paramecium fusion rosettes: possible function as Ca2+ gates.

The function of a specific intramembrane particle array, "the fusion rosette," an essential requirement for exocytosis of trichocysts in Paramecium, was probed with a temperature sensitive secretory mutant (nd9). The cells were grown at 27 degrees C, the nonpermissive, nonreleasing temperature at which fusion rosettes do not assemble. Exocytosis could be triggered, nonetheless, by addition of 40 micrometer ionophore A23187 and 15 mM Ca2+ but not Mg+. Rosette function is bypassed by this procedure, suggesting that during normal release, the rosette acts as a Ca2+ channel that allows development of a site-specific increase in Ca2+, which in turn induces fusion and release.

Saturable binding to cell membranes of the presynaptic neurotoxin, beta-bungarotoxin.

Brief exposure to the protein neurotoxin, beta-bungarotoxin, is known to disrupt neuromuscular transmission irreversibly by blocking the release of transmitter from the nerve terminal. This neurotoxin also has a phospholipase A2 activity, although phospholipases in general are not very toxic. To determine if the toxicity of this molecule might result from specific binding to neural tissue, we have looked for high affinity, saturable binding using 125I-labelled toxin. At low membrane protein concentration 125I-labeled toxin binding was directly proportional to the amount of membrane; at fixed membrane concentration 125I-labeled toxin showed saturable binding. It was unlikely that iodination markedly changed the toxin's properties since the iodinated toxin had a comparable binding affinity to that of native toxin as judged by competition experiments. Comparison of toxin binding to brain, liver and red blood cell membranes showed that all had high affinity binding sites with dissociation constants between one and two nanomolar. This is comparable to the concentrations previously shown to inhibit mitochondrial function. However, the density of these sites showed marked variation such that the density of sites was 13.0 pmol/mg protein for a brain membrane preparation, 2.4 pmol/mg for liver and 0.25 pmol/mg for red blood cell membranes. In earlier work we had shown that calcium uptake by brain mitochondria is inhibited at much lower toxin concentrations than is liver mitochondrial uptake. Both liver and brain mitochondria bind toxin specifically, but the density of 125I-labeled toxin binding sites on brain mitochondrial preparations (3.3 +/- 0.3 pmol/mg) exceeded by a factor of ten the density on liver mitochondrial preparations (0.3 +/- 0.05 pmol/mg). It is also shown that labeled toxin does not cross synaptosomal membranes, suggesting that mitochondria may not be the site of action of the toxin in vivo. We conclude that beta-bungarotoxin is an enzyme which can bind specifically with high affinity to cell membranes.

The mechanism of beta-bungarotoxin action. I. Modification of transmitter release at the neuromuscular junction.

The protein, beta-bungarotoxin, a presynaptic neurotoxin isolated from the venom of the snake Bungarus multicinctus, is known to inhibit mitochondrial function. Within 30 min after adding the toxin to a rat diaphragmphrenic nerve preparation, the quantal content increased tenfold and the frequency of miniature endplate potentials increased fourfold. No increase in miniature endplate potential frequency was seen in the absence of extracellular calcium. Since mitochondria may be involved in regulating intracellular calcium levels, the rate at which the transmitter release is turned off was studied by measuring delayed release in the presence and absence of toxin. Delayed release is elevated about eightfold by the toxin. If delayed release is due to residual calcium, as has been hypothesized, these data may be explained if the toxin does not alter the amount of calcium which enters the terminal, but rather the rate at which that calcium is removed. Alternatively, a calcium-dependent modification of the release process itself might be produced. The eventual reduction in transmitter output did not appear to result from depletion of the terminal of releaseable packets of transmitter, but does require extracellular calcium.

beta-Bungarotoxin, a pre-synaptic toxin with enzymatic activity.

beta-Bungarotoxin, a pre-synaptic neurotoxin isolated from the venom of the snake Bungarus multicinctus, has been shown to modify release of neurotransmitter at the neuromuscular junction. In this communication, we demonstrate that beta-bungarotoxin is a potent phospholipase A2 (phosphatide 2-acyl hydrolase, EC 3.1.1.4), comparable in activity with purified phospholipase enzymes from Naja naja and Vipera russellii. The phospholipase activity of beta-bungarotoxin requires calcium and is stimulated by deoxycholate. When strontium replaces calcium, no phospholipase activity is detected. Since neuromuscular transmission is not blocked when calcium is replaced by strontium, it was possible to examine the effects of the toxin on neuromuscular transmission in the presence of strontium. Under these conditions, when the phospholipase activity should be inhibited, the toxin has little or no effect on neuromuscular transmission. If beta-bungarotoxin owes its toxicity in part to its enzymatic activity, then it must be placed in a different class from those toxins which produce their effect by binding passively to an appropriate receptor.