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The mitogenic effect of elevated concentrations of zinc ions on human lymphocytes was found to be inhibited by the glucocorticoid hormone dexamethasone. The effect decreased progressively from complete block to partial inhibition when the culture period before the addition of dexamethasone was prolonged. In contrast, dexamethasone did not inhibit an induced formation of metallothionein in mononuclear cells. Furthermore, dexamethasone itself was found to induce small amounts of metallothionein. Apparently, the mitogenic effect and the induction of metallothionein by zinc ions in mononuclear cells occur by mutually independent mechanisms.

1. 1. Low mol. wt proteins with high Cd-binding capacity were found to be induced by Zn in cultured monocytes and lymphocytes. 2. 2. In T cell cultures one protein was found to be induced by 125 μM Zn for 6 days, while in monocytes and B enriched cells under these conditions two proteins were found, of which the one with higher mol. wt had similar electrophoretic mobility to the T cell protein on polyacrylamide gels. 3. 3. Mol. wt criteria and crossreactivity towards anti-metallothionein (Mt) antibody identified these proteins to be Mts of about 23 and 27 kDa mol. wt. 4. 4. Metal binding studies indicated that monocyte and lymphocyte Mts had a higher affinity to Zn compared to Cd than rat liver Mt and Mts from Cd-resistant substrains of a human epithelial and a murine fibroblast cell line. 5. 5. The presence of the T cell mitogen phytohemagglutinin (PHA) was found not to be necessary for this Mt induction by Zn.

Metallothionein (MT) is produced at high rates in isolated monocytes, and T and B lymphocytes during induction in vitro. At optimal concentrations, 125 microM for Zn and 10 microM for Cd and dexamethasone (dex), MT was demonstrated after only 2 h in some cases, and in all cell types substantial levels were measured after 1 day of exposure to all three inductors. With Cd, lower amounts of MT were found, but maximum levels were reached faster than with Zn. The same result was found for dex compared to Zn. Zn and dex in combination showed the same accumulation rate as Zn alone. If the inductors were used in lower concentrations than optimal, reduced accumulation rates occurred, particularly during the first part of the exposure period. No MT was found for concentrations below 5 microM Zn, 1 microM Cd or 0.5 microM dex. The constitutive levels of MT (mean +/- S.E.M.) were 0.11 +/- 0.05, 0.54 +/- 0.3, 0.06 +/- 0.05 and 0.15 +/- 0.08 nmol Cd bound/5 x 10(6) unseparated mononuclear cells (MNC), monocytes, T lymphocytes and B lymphocytes, respectively. Monocytes accumulated 19 times and B lymphocytes 6 times more MT than T lymphocytes after 2 days of exposure to 125 microM Zn. Despite these differences in accumulated amounts of MT, the fold accumulation values were rather similar between the cell types, reflecting corresponding variations in background MT levels. After exposure of unseparated MNC to 125 microM Zn for 2 days, removal of the metal caused constitutive MT levels to be reestablished in 5 days. Five different MT forms, all capable of Cd complexation, were demonstrated in these cells. These forms had kinetically different behaviour during Zn exposure among the cell types, and the response to Cd was different from the Zn response. The results indicate metals to be closely controlled in MNC and emphasize a role for multiple MT forms in the process of regulation.

1. 1. Frog brain acetylcholinesterase (AChE) was more resistant to inhibition by organophosphorus compounds than chicken and rat brain AChE. The differences in inhibition rates were 2 times for soman, 10 times for sarin, tabun and Tx-60, 100 times for DFP, dimethylphosphorylfluoride and paraoxon and 1000 times for diethylphosphorylfluoride. 2. 2. Physostigmine and neostigmine inhibited chicken and rat brain AChE about 100 times faster than frog brain AChE. 3. 3. Chicken and rat brain AChE were inhibited only slightly faster by methylsulphonylfluoride than the frog brain AChE. The inhibition was accelerated by tetraethylammonium ions and retarded by tensilon in all cases. 4. 4. Tetraethylammonium ions, however, slightly retarded the inhibition rate of chicken and rat brain AChE by dimethylphosphorylfluoride. No significant effect was observed for the frog brain AChE. 5. 5. The species differences in inhibition rates were probably not due to differences in phosphorylation rates or conformational changes, but rather to the steric arrangement in the active site of the enzyme molecules.

1. The contractile behaviour of rabbit and man bronchial musculature has been tested in response to some commonly used substrates for cholinesterase, histamine and adrenaline by the kymograph technique. 2. The sensitivity of the smooth bronchial musculature from both rabbit and man was found to be highest for acetylcholine and acetyl-β-methylcholine (6.3 × 10 −8 M) and lowest for butyrylcholine (6.3 × 10 −6 M). 3. The smooth bronchial musculature of man was slightly more sensitive to histamine and adrenaline than that of the rabbit. 4. The results indicate that the contractile behaviour in the smooth bronchial musculature of rabbit and man is remarkably similar.

Radiolabelled paraquat (bis-N-(14C)methyl-4,4'-bipyridylium chloride) was administered intravenously to pregnant rats and guinea-pigs and examined by the whole body autoradiography method. Male rats were examined for comparison with the same method. The results showed that the radiolabelled compound was rapidly distributed throughout the tissues, with the highest concentrations in the cartilage, kidney, lung, spleen, salivary gland and skin. High concentrations were found in the placenta and throughout the fetuses 0.5 hr after the administration.

1. Diisopropoxyphosphorylfluoride and soman caused rapid decline of the cholinesterase (ChE) activity levels in earthworm, chicken, guinea pig, mouse and rat tissues. 2. The onset of recovery of ChE activity was well established within 5 days in all species except for the earthworm whose whole body ChE activity level remained low for at least 15 days. 3. Plasma ChE of the rat recovered to the normal activity level already in 6 days. After that, however, the activity level still continued to increase. This overshoot effect was much more pronounced in females than in males. 4. In females the activity level reached 50% above normal values after about 7 days. The overshoot of plasma ChE in the female rat still persisted 15 days after inhibition. 5. The overshoot of ChE activity was also observed in mouse and rat liver after inhibition. In the chicken ChE overshoot was not found. 6. The recovery of erythrocyte and brain ChE activity of the rat was observed to be slower than that of plasma and liver. Likewise the recovery of brain ChE activity of the mouse seemed to be slower than that of liver. Similar results were obtained for the chicken. Generally soluble ChE seem to recover faster than membrane bound. 7. In the case of erythrocyte and brain ChE the results indicated a similar ChE turnover as generally observed for other proteins in brain tissue. 8. In brain and retina of chicken as well as in rat brain and in guinea pig iris two distinct forms of ChE were found with marked different molecular weights. 9. In the chicken the activity of the form with a low molecular weight was found to predominate during the early recovery stages following ChE inhibition. At later stages the activity of a heavy weight form dominated. 10. In rat brain and retina as well as in guinea pig iris the activity of the form with the higher molecular weight was always predominating. 11. The results may, however, be interpreted such that the synthesis of the form of ChE with the lower molecular weight proceeds the appearance of the higher molecular weight form. 12. Administration of l -anserine nitrate, l -homocarnosine sulphate, l -carnosine and thyrotropin releasing hormone to the rats as well as primobolan to the mouse did not affect the recovery of activity and molecular forms of ChE following irreversible ChE inhibition. In the rats hypophysectomy was not found to affect the recovery either.

1. The high affinity uptake of GABA in optic tectum was found to be about 40% higher in frogs kept in complete darkness for 3 weeks, than in frogs in the normal condition. 2. No effects were obtained in uptake of glutamate and activity of GAD in the optic tectum. 3. The isoenzyme composition of cholinesterases in frog optic tectum and retina was not affected by dark adaptation either.

1. 1. Dexamethasone induced thionein in rat liver cross reacts with antibody made towards Cd-thionein from rat liver. 2. 2. Dex-thionein binds Cd. 3. 3. Compared to Cd-thionein, dex-thionein behaves differently on SDS polyacrylamide gels. 4. 4. On molecular filtration on Sephadex G-75 columns, dex-thionein elutes slightly more towards the low molecular weight region than Cd-thionein. 5. 5. Dex-thionein elutes in one peak with two shoulders after DEAE-Sephadex A-50 ion exchange chromatography, while Cd-thionein elutes in two peaks.

A vascularly perfused phrenic nerve-hemidiaphragm preparation from the rat was developed to study effects of physostigmine and some organophosphate inhibitors on the synthesis and release of endogenous and deuterium-labelled (choline--D9) acetylcholine (ACh) as well as the presynaptic uptake of choline. Choline and ACh were determined by combined gas chromatography/mass spectrometry. Without stimulation the endogenous levels of ACh were 320 pmole/hemidiaphragm for unlabelled and less than 1 pmole/hemidiaphragm of deuterium-labelled ACh. After stimulation at 15 Hz for 1 hr, 460 pmole/hemidiaphragm of unlabelled and 15 pmole/hemidiaphragm of deuterium-labelled ACh were found. Without stimulation the release of unlabelled ACh was 6 pmole/min/hemidiaphragm and for deuterium-labelled 0.2 pmole/min/hemidiaphragm. Evoked release (15 Hz, 1 hr) was 22 pmole/min/hemidiaphragm for unlabelled and 1.8 pmole/min/hemidiaphragm for deuterium labelled ACh. During stimulation and treatment with high concentrations (10(-5)-10(-4) M) of soman, DFP and Vx the level of unlabelled endogenous ACh increased, but the level of deuterium labelled ACh decreased in the diaphragm. During stimulation and treatment with these inhibitors the release of both unlabelled and labelled ACh decreased. During treatment with high concentrations (10(-5)-10(-4) M) of sarin and physostigmine there were no changes in endogenous levels or release of unlabelled or deuterium labelled ACh. The different effects of cholinesterase inhibitors are probably linked to the synthesis and release mechanism of ACh rather than to the choline uptake mechanism.

1. 1. Brain cholinesterase (ChE) levels in Wistar rats from Mollegarden, Denmark (Mol: WIST) were found to be higher than in Wistar rats from Charles River, France (Cre: (WI)BR). 2. 2. Serum ChE levels were considerably higher in females than in males of both Wistar stocks. 3. 3. This difference disappeared after hypophysectomy such that male rat serum ChE levels increased while those in females decreased.

1. 1. The cholinesterase (ChE) of frog brain and retina could be easily solubilized. About 10% of the brain and 20% of the retina ChE were found to be soluble in 0.05 M phosphate buffer. After treatment with 0.5% (v/v) Triton X-100, about 30% of the total ChE activity of the brain and only 10% for retina was left particle bound. NaCl by itself did not solublize ChE. Use of higher NaCl concentrations in combination with Triton X-100 as well as higher detergent concentrations alone seemed to cause an inhibiting effect of the solubilized ChE from retina. 2. 2. The solubilized ChE from brain as well as retina were electrofocused as one main activity peak, corresponding to isoelectric points of pH 6.1 and 6.0, respectively. A second molecular form at pH 5.9 was distinguishable for the brain, but not for retina ChE. 3. 3. Sucrose gradient centrifugation indicated that the ChE solubilized from the brain and retina consists of two molecular forms exhibiting S values of 5.1 ± 0.24, 10.9 ± 0.33 and 6.1 ± 0.30, 10.9 ± 0.43, respectively. After solubilization by higher Triton X-100 concentrations the soluble extracts from brain and retina seemed to contain the activity of these forms in different proportions. 4. 4. Polyacrylamide gel electrophoresis separated three molecular forms of the brain ChE. One of these forms was found to have a molecular weight of 394,000 ± 20,000. The others were found to have an identical molecular weight of 550,000 ± 10,000. Two molecular forms exhibiting molecular weights of 292,000 ± 10,000 and 470,000 ± 10,000, could be separated for retina. The high as well as the low molecular weight form of brain and retina seemed to differ with respect to charge. 5. 5. The results found clearly indicate that the molecular forms of the frog ChE exhibit a high degree of lability. 6. 6. Substrate specificity, substrate inhibition, K m values and inhibition rates were examined without finding any significant differences between ChE solubilized from the brain and retina of the frog ( Rana temporaria ).

Publisher Summary Four quaternary nitrogen compounds, one of them an irreversible cholinesterase inhibitor (2-MPAM-ES), the remaining three cholinesterase reactivators—namely,P2S, Tx 10, and Toxogonin— are used in this chapter, in a study of the permeability of the blood-brain barrier (BBB) and the blood-retinal barrier (BRB), the latter being, from an ontogenetic point of view, a part of the former. In contrast to other parts of the BBB, which have been found in earlier work to show slight mutual differences with regard to permeability, the BRB is found to be more permeable to the reactivators than was the BBB proper. Both the BBB and the BRB are impermeable to 2-MPAM-ES. This impermeability is not influenced by high X-ray doses (5,000 rad), nor by toxic doses of 2,4-dinitrophenol or ouabain. No marked differences are found among the cholinesterase reactivators with regard to penetration of either barrier.

1. 1. There are considerable differences between acetylcholinesterase (AChE) from different species with regard to their reactions with organophosphorous anticholinesterases. AChE from chicken brain was inhibited 20 times as fast with 2-MPAM-ES as rat and mouse brain AChE. In contrast AChE from frog brain was inhibited 1000 times slower with 2-MPAM-ES, 100 times slower with DFP and 10 times slower with 2-MPA-ES than rat brain AChE. 2. 2. The differences in inhibition rates were similar for membrane-bound enzymes and enzymes solubilized by Triton X-100. 3. 3. Both chicken and frog brain AChE were inhibited to the same extent by quaternary ammonium salts. 4. 4. There were only small species differences with regard to reactivation of DFP-inhibited enzymes by 2-PAM. 5. 5. When AChE from the different species were inhibited by DFP, the enzyme from frog brain “aged” 10 times slower than in the other enzymes. 6. 6. Substrate specificity studies showed that chicken brain enzyme exhibited its highest activity towards propionylcholine whereas frog enzyme had the highest activity towards acetylcholine.