Isolation and characterization of a low molecular weight cadmium-binding protein from Syrian hamster lung

A low molecular weight cadmium-binding protein has been isolated from Syrian hamster lung. The protein elutes from a Sephadex G-75 column with $\text{V}{}_{\mathrm{<mtext>e</mtext>}}\text{V}{}_0$ (1.64 – 1.98) characteristic of metallothionein-like proteins and has an estimated molecular weight of 15,000. The Cd-binding protein does not appear to bind Cr³⁺, Ni²⁺, or Se⁴⁺. A low molecular weight Cd-binding protein in lungs of this species may be responsible for the prolonged retention of Cd in lung following pulmonary exposure of hamsters to CdCl₂.     

Nitrate formation in rats exposed to nitrogen dioxide

Sprague-Dawley rats exposed to atmospheres containing low levels of nitrogen dioxide (NO₂) for 24 hr had increased levels of nitrate in their urine on the day of exposure and on the 3 subsequent days. The total increase in urinary nitrate was linearly related to the nitrogen dioxide concentration administered. We recovered in urine 8.4 ± 1.1 μmol nitrate/ppm $\text{NO}{}_2\text{24}\text{-hr}$ exposure (slope ± 95% confidence limits) for 185-g rats. Both the linearity and magnitude of this effect imply that reaction with respiratory tract water is not a major pathway of NO₂ absorption in the lung. Instead, our observations support the hypothesis that the major interaction of NO₂ in the lung is with readily oxidizable tissue components to form nitrite. We estimate that 9.6 μmol of nitrite is formed in the respiratory tract of the rat per ppm NO₂ per 24-hr exposure. We also estimate that humans breathing air containing 0.1 ppm NO₂ have about 3.6 mg of nitrite formed in their respiratory tract per day.     

An orally effective, long-acting dopaminergic prodrug: (-)-10,11-methylenedioxy-N-propylnoraporphine

In rat vas deferens, (±)-170 150 proved to be an α₂-adrenoceptor blocking agent with a high $\text{α}{}_2\text{α}{}_1$ selectivity ratio In rat cerebrocortical membranes, (±-170 150 was seven times more potent than yohimbine for inhibiting specific [³H]clonidine binding. However, (±)-170 150 appeared to be 3.5 times less selective for α₂-adrenoceptors sites than yohimbine. These results indicate that (±)-170 150 is a potent preferential α₂-adrenoceptor blocking agent, as had been shown by in vivo experiments.     

Muscarinic receptors in lung and trachea: Autoradiographic localization using [3H]quinuclidinyy benzilate

[³H]Quinuclidinyl benzilate binding to slide-mounted frozen sections of ferret lung was of high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{63 ± 14}\text{pM}\text{,}\text{mean}\text{±}\text{S.E.}\text{,}\text{n}\text{= 4}$), and characteristic of interaction with muscarinic cholinergic receptors. Light microscopic autoradiography showed muscarinic receptors to be localized predominantly to smooth muscle of trachea and intrapulmonary cartilaginous airways, and to submucosal glands. There was much less labelling of bronchiolar smooth muscle, airway epithelium and vascular smooth muscle and no labelling of alveoli. This distribution of receptors parallels that of cholinergic innervation.     

Interaction of [3H]flunitrazepam with the benzodiazepine receptor: Evidence for a ligand-induced conformation change

The interaction of [³H]flunitrazepam with benzodiazepine receptors in rat brain homogenates was studied in the presence of 2 μM endogenous GABA at 0° at pH 7.2. Equilibrium binding experiments showed a dominant component of high affinity with an equilibrium dissociation constant K = 0.86 ± 0.07 nM which accounted for 75% of total binding and another component of lower affinity (K ? 30 nM). The dissociation kinetics of the [³H]flunitrazepam complex at the high affinity site were strictly monophasic with a rate constant k_off = (7.7 ± 0.3) × 10^?4/sec. The association kinetics with the high affinity sites were studied with ligand concentrations [L]₀ in large excess over binding sites. The kinetics were in accordance with a single exponential with a reaction rate τ^?1. In the higher concentration range [L]₀ ? 10 nM, τ^?1 as a function of [L]₀ deviated from linearity and started to level off. The data are compatible with a two-step mechanism where R and L rapidly combine to form a pre-complex RL which then slowly isomerizes to the final complex C:

where $\text{K}{}_1\text{= (}\text{[}\text{R}\text{][}\text{L}\text{]}\text{([}\text{RL}\text{]}\text{)}$ and $\text{[}\text{RL}\text{]}\text{[}\text{C}\text{]}\text{=}\text{k}{}_{\mathrm{?2}}\text{k}{}_2\text{= k}{}_2$. Nonlinear parameter estimation yielded K₁24.2 ± 7.1 nM, k₂ = (2.8 ± 0.5) × 10^?2/sec and k_?2 = (9 ± 2) × 10^?4/sec. The isomerization step might reflect a ligand-induced conformation change of the high affinity site which is involved in the potentiation of GABA-ergic transmission produced by the benzodiazepines.     

Acute effects of cadmium on proximal tubular function in rabbits

Acute renal failure was studied in rabbits 3 days after a single iv injection of a CdCl₂-mercaptoethanol mixture (10 μmol Cd and 350 μmol mercaptoethanol/kg). Glomerular filtration rate (GFR), p-aminohippurate clearance (C_PAH), and extraction of p-aminohippurate (E_PAH) were severely depressed by poisoning. The lower C_PAH and E_PAH in poisoned rabbits reflected, at least in part, a cytotoxic action of cadmium on cortical tissue. The filtration fraction, defined as GFR/renal plasma flow (calculated as $\text{C}{}_{\mathrm{PAH}}\text{E}{}_{\mathrm{PAH}}$) also fell. Reabsorption of all amino acids measured was strongly inhibited during the stage of acute renal failure. The site of this inhibition was the luminal membrane of tubule cells, as deduced from the identity of mean artery-to-urine transit times of [³H]inulin and ¹⁴C-labeled amino acids. In contrast to these tubular dysfunctions, the maximum tubular ability to reabsorb filtered glucose was unaltered, when corrected for changes in GFR, indicating that the effects of cadmium were relatively specific.     

Isolation of a soluble cadmium-binding protein from pulmonary macrophages

A soluble cadmium-binding protein, with properties similar to metallothionein, has been isolated from rabbit alveolar macrophages. The macrophages were cultured in Medium 199 with Earle's salts for 24 hr in the presence of 10 μm CdCl₂ and carrier-free ¹⁰⁹Cd as a tracer. The isolation procedure began with application of a 100,000 g cell supernatant to a column of Sephadex G-75 Fine. The fraction containing the greatest amount of cadmium was eluted at a relative elution volume, $\text{V}{}_{\mathrm{<mtext>e</mtext>}}\text{V}{}_{\mathrm{<mtext>o</mtext>}}$, of 1.87. A molecular weight determination performed following Sephadex chromatography indicated that the apparent molecular weight of the impure protein was approximately 11,000. The fractions containing cadmium were pooled and purification procedures were applied, including acetone fractionation, DEAE-cellulose chromatography, and polyacrylamide gel electrophoresis. DEAE-Cellulose chromatography following acetone fractionation indicated the presence of two forms of metalloprotein as has been demonstrated previously in the isolation of cadmium-thionein from liver and kidney. The two forms of metalloprotein were subjected to polyacrylamide gel electrophoresis and, although separation was incomplete, bands obtained corresponded to those typically observed in rat liver.     

Rat uterine contractility and the activities of uterine adenyl cyclase and phosphodiesterase during the estrus cycle

A study has been made of the activities of rat uterus adenyl cyclase and phosphodiesterase, and the inhibitory effect of theophylline on uterine contractions at various stages of the estrus cycle. The activities of adenyl cyclase and phosphodiesterase at proestrus were found to be 2.52 ± 0.28 pmoles/min/mg of protein and 1.90 ± 0.30 nmoles/min/mg of protein, respectively. Activities of both the enzymes increased from proestrus to peak values at metestrus (early metestrus for adenyl cyclase and late metestrus for phosphodiesterase) and then fell until the following proestrus. Theophylline inhibition of oxytocin-induced maximum uterine contractions was found to be greatest at early metestrus. Similarly, the oxytocin concentration producing 40 per cent maximum contraction in the presence and absence of the theophylline

$\text{Oxytocin}\text{(E}{}_{40}\text{)+}\text{theophylline}\text{Oxytocin}\text{(E}{}_{40}\text{)}$

was also highest at early metestrus. These findings indicate that, at early metestrus, cellular turnover of cyclic AMP may be high and that the exaggerated inhibition of oxytocin- induced maximum contraction and the high value of

$\text{Oxytocin}\text{(E}{}_{40}\text{)+}\text{theophylline}\text{Oxytocin}\text{(E}{}_{40}\text{)}$

could be due to extensive accumulation of cyclic AMP produced from theophylline inhibition of phosphodiesterase.     

Mechanism of action of 5'-methylthioadenosine in S49 cells

5'-Deoxy-5'-methylthioadenosine, a naturally occurring co-product of polyamine biosyn-thesis, has been shown to inhibit a variety of biological processes. To investigate the mode of action of this nucleoside and to assess the involvement of cAMP in this action, the effect of methylthioadenosine on S49 wild type and two cAMP-related mutant cells was examined. The sulfur-containing nucleoside potently inhibited the growth of the parental strain ($\text{IC}{}_{50}\text{= 50 μ}\text{M}$), whereas nearly 10-fold greater resistance was demonstrated by S49 adenylate cyclase deficient ($\text{IC}{}_{50}\text{= 420 μ}\text{M}$) and S49 cAMP-dependent protein kinase deficient ($\text{IC}{}_{50}\text{= 520 μ}\text{M}$) mutant cells. Methylthioadenosine was shown to competitively inhibit the S49-derived high-affinity cAMP phosphodiesterase (K_i = 62 μM) in vitro, whereas methylthioadenosine phosphorylase activity was equivalent in all three cell types. The intracellular levels of the regulatory nucleotide, cAMP, increased dramatically in the wild type (17-fold) and protein kinase deficient (6-fold) strains in response to 100 μM concentrations of the drug. It is concluded that the growth arrest produced by 5'-methylthioadenosine in S49 cells is primarily due to the inhibition of cAMP phosphodiesterase and the subsequent increase in cAMP levels that result.     

The compressibility and compactibility of powder systems

This paper investigates the problem of compressibility and compactibility of powder systems. The term ‘compressibility’ is defined as the ability of a powder to decrease in volume under pressure, and the term ‘compactibility’ is defined as the ability of the powdered material to be compressed into a tablet of specified strength (i.e. radial tensile strength or deformation hardness). A novel approach leads to the following equation, which describes the deformation hardness P of the compact as a function of the applied pressure σ_c and the relative density ρ_r:

$\text{P = P}{}_{\max }\text{(1 ?}\text{exp}\text{( ? γσ}{}_{\mathrm{<mtext>c</mtext>}}\text{ρ}{}_{\mathrm{<mtext>r</mtext>}}\text{))}$

The parameter P_max is equal to the theoretically maximal possible deformation hardness at σ_cρ_r → ∞.P_max describes the compactibility and the parameter γ, termed the compression susceptibility, describes indirectly the compressibility. The equation is valid for pure substances as well as for binary mixtures An attempt is made to develop ‘additivity rules’ for the parameters P_max and γ in case of binary mixtures. For the general case it is necessary to introduce an interaction term, which can be explained qualitatively.     

The effects of cadmium, manganese and aluminium on sodium-potassium-activated and magnesium-activated adenosine triphosphatase activity and choline uptake in rat brain synaptosomes

The effects of Cd²⁺, Mn²⁺ and Al³⁺ on rat brain synaptosomal sodium-potassium-activated and magnesium-activated adenosine triphosphatase (Na-K-ATPase and Mg-ATPase) activity and choline uptake were studied. All three types of metal ions inhibited Na-K-ATPase activity more markedly than Mg-ATPase activity. The rank order of inhibition of Na-K-ATPase was: $\text{Cd}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 5.4 μ}\text{M}\text{) >}\text{Mn}{}_{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 955 μ}\text{m}\text{) >}\text{Al}{}^{\mathrm{3+}}\text{(}\text{ic}{}_{50}\text{= 8.3}\text{mM}$). The rank order of inhibition of Mg- was:$\text{Cd}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 316 μ}\text{M}\text{>}\text{Mn}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 5.5}\text{mM}\text{>}\text{Al}{}^{\mathrm{3+}}\text{(}\text{ic}{}_{50}\text{= 21.9}\text{mM}\text{).}\text{Al}{}^{\mathrm{3+}}$ was most potent in inhibiting synaptosomal choline uptake ($\text{ic}{}_{50}\text{= 24μ}\text{M}$ in the absence of Ca²⁺ and 123 μ.M in the presence of 1 mM Ca²⁺). $\text{Cd}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 363 μ}\text{M}\text{)}$ was a more effective inhibitor of choline uptake than $\text{Mn}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 1.2?1.5}\text{mM}\text{)}$ . The presence of 1 mM Ca²⁺ did not alter choline uptake, nor did it antagonize the inhibitory actions of the three metals. Our observations that Cd²⁺ and Al³⁺ inhibited synaptosomal choline uptake, but did not show parallel inhibitory effects on Na-K-ATPase activity directly contradicts the ionic gradient hypothesis. These results are also discussed in relation to the in vivo neurotoxicity of cadmium, manganese and aluminium.     

A quantitative study of the γ-aminobutyric acid (GABA) dose/conductance relationship at the lobster inhibitory neuromuscular junction

GABA-evoked changes in membrane conductance of lobster muscle fibres were measured using the ‘short’ cable equations. In 79 fibres, the mean resting conductance $\text{(?}{}_{\mathrm{m}}\text{L)}$ was 12.9μmho (S.D. ± 5.6 μmho). In the presence of GABA (5 μM to 640 μM) the membrane conductance increased with little change in resting potential and with no visible decline in response during exposure. In contrast with the non-cumulative GABA dose/conductance curves, cumulatively established curves attained an apparent maximum at GABA concentrations exceeding 80 μM.The non-cumulative dose/conductance curve was better fitted by a two independent binding-site receptor model than by a two-site high facilitation model or a single-site model. Values for the apparent GABA/receptor dissociation constant estimated by ‘least squares’ analysis (on the basis of the independent model) were similar to those estimated by double reciprocal plot (ca. 30 μM).     

Similar activities of nerve growth factor and its homologue proinsulin in intracellular hydrogen peroxide production and metabolism in adipocytes: Transmembrane signalling relative to insulin-mimicking cellular effects

Generation of hydrogen peroxide in adipocyte plasma membrane and its intracellular metabolism and regulatory role have been shown by Mukherjee and co-workers to be a major effector system for insulin [Fedn Proc.35, 1694 (1976); Archs Biochem. Biophys.184, 69 (1977); Biochem. Pharmac.27, 2589 (1978); Fedn Proc.37, 1689 (1978); and Biochem. Pharmac.29, 1239 (1980)]. The possible involvement of this mechanism in the action of structurally similar polypeptides having some insulin-like metabolic effects was investigated. The β-subunit of nerve growth factor (2.5 S NGF, mol. wt 13,500) which has a striking structural homology with proinsulin and has been reported to exert certain insulin-like metabolic effects in its own target tissues (e.g. growing neurites and sympathetic ganglia), and the insulin-derived polypeptides, desalanine-insulin and desoctapeptide-insulin, as well as proinsulin, were examined for their effects on rat adipocytes, employing the technique of formate oxidation. Both NGF and proinsulin caused increased [¹⁴C]formate oxidation, showing similar intrinsic activities, up to a maximum of 140–160% of the basal rate; insulin increased the rate to 190–210% of the basal rate. The relative potencies of the hormones toward H₂O₂ formation and stimulation of the pentose phosphate pathway activity were: insulin $\text{(}\text{EC}{}_{50}\text{: 2.5 × 10}{}^{\mathrm{?11}}\text{M}\text{)}$, desalanine-insulin $\text{(}\text{EC}{}_{50}\text{: 2.5 × 10}{}^{\mathrm{?10}}\text{M}\text{)}$ , proinsulin $\text{(}\text{EC}{}_{50}\text{: 8 × 10}{}^{\mathrm{?9}}\text{M}\text{)}$, and NGF $\text{(}\text{EC}{}_{50}\text{: 10}{}^{\mathrm{?9}}\text{M}\text{)}$. The biologically inactive derivative, desoctapeptide-insulin, did not stimulate glucose oxidation, although it caused a small increase in formate oxidation, with an $\text{EC}{}_{50}\text{of}\text{5 × 10}{}^{\mathrm{?7}}\text{M}$, indicating a suboptimal level of H₂O₂ formation in the elevation of the hexose monophosphate shunt activity. 3-Amino-1,2,4,-triazole (50 mM), which irreversibly decomposes the peroxidatic compound II of the catalase: H₂O₂ complex, inhibited formate oxidation to a greater extent in the hormone-treated cells than in the control cells, whereas sodium azide, an inhibitor of the hemoprotein, catalase, completely inhibited it. The abilities of the polypeptides to stimulate H₂O₂ formation correlated with their abilities to promote lipogenesis from [U-¹⁴C]-D-glucose, as expected of insulin. The cellular GSH/GSSG ratio increased concomitantly with the stimulation of glucose oxidation via the shunt, indicating a tight coupling between these processes. The results confirm that the hydrogen peroxide production is a common basis of the metabolic actions of growth-promoting polypeptide hormones or mitogens beyond their respective receptors.     

Determination of extraction constant,true partition coefficient and formation constant of ion-pair complexes of quaternary ammonium salts,tetrabutylammonium bromide and isopropamide iodide.with some organic anions by a solvent extraction technique

A new method of determining the extraction constant (K_e, the true partition coefficient (TPC) and the formation constant (K_f) of ion-pairs, was developed by the solvent extraction technique. K_e and TPC were estimated from the reciprocals of the intercept and the slope of the regression line obtained by plotting

$\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{APC ? dA}\text{vs}\text{B}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{dA}\text{APC ? dA}\text{+}\text{A}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{+}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}$

in the following equation.

$\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{APC ? dA}\text{=}\text{1}\text{K}{}_{\mathrm{e}}\text{+}\text{B}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{dA}\text{APC ? dA}\text{+}\text{A}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{+}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{x}\text{1}\text{TPC}$

where [A^T_W] and [B^T_W] are the total concentrations of the cationic compound A and that of the anionic compound B in the aqueous phase respectively, APC is the apparent partition coefficient of A, dA is the partition coefficient of cation A⁺. K_f, which is expressed by K_e/TPC, was then calculated. These constants were determined for the ion-pair extraction of tetrabutylammonium bromide and isopropamide iodide with 4 organic anions, i.e. benzoic acid, p-toluenesulfonic acid, salicylic acid and taurodeoxycholic acid. This new method might be applicable to other ion-pairs without further assumptions except that the molar ratio of the ion-pair formation be 1 : 1.     

Kinetic studies on the metabolism of ethylmorphine by isolated hepatocytes from adult rats

Hepatocytes of adult rats were isolated by infusion of a hyaluronidase collagenase mixture. High yields of cells excluding trypan blue were obtained. These cells, in Hank's buffer containing rat serum and 0.1% glucose, $\text{N-}\text{demethylate}\text{[}{}^3\text{H-CH}{}_3\text{-N]}$ethylmorphine. The formaldehyde initially formed is further metabolized to tritiated water. Fifteen per cent of the original metabolic activity was observed after 21 hr at 37° in 5% CO₂-air, and cumulative metabolism is linear for up to 90 min under these conditions. The K_m for the N-demethylation of [³H-CH₃-N]ethylmorphine is 50 μM, 20 per cent of the value observed for this reaction by musomal preparations. An active transport of the substrate into the cell is postulated to account for this difference.