Concentration and distribution of apolipoproteins A-I and E in normolipidemic, WHHL and diet-induced hyperlipidemic rabbit sera.

Two sandwich-type enzyme immunoassays have been developed to measure apolipoproteins A-I and E in rabbit serum. Specific goat antibodies were purified by affinity chromatography and used both for coating and for preparing antibody-peroxydase conjugates. The sensitivity of these assays is sufficient to allow studies of apo A-I and E distribution in lipoproteins fractionated by gel filtration from 50 microliters of serum. In WHHL rabbits, apo A-I is 5-fold lower (5.2 +/- 2.5 mg/dl) and apo E is 8-fold higher (9.9 +/- 3.5 mg/dl) than in normolipidemic rabbits (29 +/- 4.3 mg/dl and 1.3 +/- 0.5 mg/dl, respectively). In hyperlipidemic rabbits, fed 2 months on a 0.5% cholesterol diet, the apo A-I level was similar (32 +/- 12 mg/dl) to that of normolipidemic rabbits, but the apo E level is 12-fold higher (15.1 +/- 5.5 mg/dl). In addition, HDL particles were enriched with cholesterol and apo E. The bulk of apo E and cholesterol is located in large beta-VLDL in diet-induced hyperlipidemia, whereas they are mainly located in smaller size beta-VLDL in WHHL rabbits. In normolipidemic rabbits apo E occurs mainly in HDL, and cholesterol is distributed in the main three lipoprotein fractions VLDL, LDL and HDL. Interestingly, HDL of WHHL rabbit are deficient in apo A-I. These results are compatible with profound perturbations of lipoprotein composition and metabolism in atherogenic hyperlipidemia.     

Synthesis of [D-Ala2,4'-125I-Phe3,Glu4]deltorphin and characterization of its delta opioid receptor binding properties.

Both [D-Ala2,Glu4]Deltorphin and [D-Ala2,4'-I-Phe3,Glu4]Deltorphin are highly selective ligands for delta, relative to mu, opioid receptors. Radiolabeled [D-Ala2, 4'-125I-Phe3,Glu4]Deltorphin ([125I]Deltorphin) was prepared with a specific activity of 2200 Ci/mmol from [D-Ala2, 4'-NH2-Phe3, Glu4]Deltorphin through a diazonium salt intermediate. The inhibition of [125I]Deltorphin binding to rat brain membranes by ligands selective for mu, delta, and kappa opioid receptors is consistent with binding by the radioligand to a single site having the properties of a delta opioid receptor. The results of these studies are in good agreement with those obtained by structurally different delta opioid receptor ligands. The similarity between the delta receptor site labeled by [125I]Deltorphin and those labeled by other delta receptor agonists, in contrast to differences seen by in vivo studies of their analgesic effects, is discussed.     

Pharmacological characterization of a novel muscarinic partial agonist, YM796, in transfected cells expressing the m1 or m2 muscarinic receptor gene.

To investigate the pharmacological effect of a novel compound YM796, we performed radioligand binding experiments and correlative biochemical experiments using the transfected murine fibroblast B82 cells which expressed the m1 and m2 muscarinic receptor genes (cloned cell lines designated as LK3-3 and M2LKB2-2, respectively). [3H](-)methyl-3-quinuclidinyl benzilate [( 3H](-)MQNB) binding in these transfected cell lines was inhibited by different optical isomers of YM796 and other muscarinic drugs, atropine, pirenzepine, AF-DX 116, as well as selected agonists. (-)YM796, (+)YM796 and (+/-)YM796 inhibited [3H](-)MQNB binding in LK3-3 cells with Ki values of 16.4 microM, 30.1 microM and 21.8 microM and in M2LKB2-2 cells with Ki values of 52.0 microM, 108 microM and 77.1 microM, respectively. From functional assays we found the two isomers, (-)YM796 and (+)YM796 had different intrinsic activities for the M1 and M2 muscarinic receptors. (-)YM796 revealed agonistic activity: stimulation of [3H]IP1 accumulation in LK3-3 cells with an EC50 value of 26.5 microM, which was less efficacious (the Emax value was 5.6 times basal) than carbachol, a full agonist (the Emax value was 17.2 times basal). Interestingly, (-)YM796 did not show significant inhibition of cAMP formation in M2LKB2-2 cells except at extremely high concentrations (greater than 1mM). (+)YM796 exhibited no significant efficacy for the M1 and M2 muscarinic receptors. These results suggest that (-)YM796 represents a muscarinic partial agonist with functional selectivity for the M1 muscarinic receptors whereas (+)YM796 shows no efficacy for either M1 or M2 muscarinic receptors in these transfected cells.     

Lipase modification by various synthetic polymers for use in chloroform

Summary Lipase was modified with several hydrophilic and hydrophobic synthetic polymers. The modified lipase was solubilized into chloroform by. The catalytic esterification activity of modified lipase increased linearly with the increase of its solubility in chloroform.     

Excretion of peroxidase from horseradish hairy root in combination with ion supplementation

Summary The effect of ion-supplemented medium on peroxidase excretion from horseradish (Armoracia rusticana) hairy roots was studied. Supplementation of mannitol instead of ions revealed that the excretion was stimulated not by osmotic pressure in the medium but by ionic properties. Extracellular peroxidase activity per dry cell was proportionally correlated with the ionic strength of the cations. CaCl₂ or MgCl₂ was found to be the most effective agent for excretion among other combinations. CaCl₂ supplementation at the beginning of the culture caused higher peroxidase production in the medium without a significant loss of final cell mass compared with CaCl₂ addition during the culture. Repeated batch culture with 50 mM CaCl₂ supplementation allowed a continuous retention of cell viability over 149 days and produced a great amount of extracellular peroxidase, 12-fold higher than that achieved in a 40-day-old batch culture with 50 mM CaCl₂ supplementation. Correspondence to: T. Kobayashi     

Discrimination of Multiple [3H]5-Hydroxytryptamine Binding Sites by the Neuroleptic Spiperone in Rat Brain

Certain neuroleptic drugs, such as spiperone and (+) butaclamol, can discriminate between two populations of [³H]5-hydroxytryptamine ([³H]5-HT) binding sites in rat brain. The butyrophenone neuroleptic spiperone shows the greatest selectivity for these two binding sites, having at least a 3000-fold difference between its dissociation constants (2-12 nM versus 35,000 nM) for the high- and low-affinity sites, respectively. Inhibition of [³H]5-HT binding by spiperone in rat frontal cortex and corpus striatum yields distinctly biphasic inhibition curves with Hill slopes significantly less than unity. Results from nonlinear regression analysis of these inhibition studies were consistent with a two-site model in each brain region. In the frontal cortex the high-affinity neuroleptic sites comprised about 60% of the total [^3/H]5-HT binding sites whereas in the corpus striatum they accounted for only 20% of the sites. Furthermore, saturation studies of [³H]5-HT binding assayed in the absence or presence of 1 μM-spiperone (a concentration that completely blocks the high-affinity site while having minimal activity at the low-affinity site) reveal a parallel shift in the Scatchard plot with no change in the dissociation constant of [³H]5-HT, but a significant decrease (64% in frontal cortex or 28% in corpus striatum) in the number of specific binding sites. These observations are consistent with the existence of at least two populations of [³H]5-HT binding sites having a differential regional distribution in rat brain.     

Multiple benzodiazepine receptors and their regulation by gamma-aminobutyric acid

The interaction of propyl β-carboline-3-carboxylate (PCC) with benzodiazepine receptors in the cerebral cortex of the rat was investigated by direct measurements of [³H]PCC binding and by competitive inhibition of [³H]flunitrazepam (FLU) binding. Initial experiments showed that [³H]PCC binding exhibited characteristics of saturability, stereospecificity and a pharmacological specificity remarkably similar to that of [³H]FLU binding. Analysis of [³H]PCC binding isotherms and PCC/[³H]PCC competition curves revealed the presence of a small population of super high affinity PCC binding sites (K_SH = 30–100 pM) which represents approximately 3–6% of the total sites. When measured by competitive inhibition of [³H]FLU binding, receptor occupancy by PCC was generally consistent with that determined by direct measurements of [³H]PCC binding. Analysis of the PCC/[³H]FLU competition curve revealed the presence of two major populations of high and low affinity PCC binding sites with dissociation constants of 0.54 and 10 nM and relative abundances of 52 and 45%, respectively. Collectively, the results of the [³H]PCC binding isotherm, PCC/[³H]PCC competition curve and PCC/[³H]FLU competition curve are internally consistent when rationalized in terms of three populations of benzodiazepine receptors - super high, high, and low affinity - each having different affinities for PCC and equal affinity for FLU. The effects of γ-aminobutyric acid (GABA) on PCC and FLU binding were investigated, and it was observed that GABA enhanced the binding of FLU to the various receptor subtypes whereas no significant effect of GABA on the binding of PCC was detected.     

Muscarinic receptor binding in rat brain using the agonist, [3H]cis methyldioxolane

Muscarinic receptor binding was measured in rat forebrain preparations using the muscarinic agonist, [³H]cis methyldioxolane ([³H]CD). The results of equilibrium binding studies using [³H]CD concentrations between 0.5–64 nM showed that [³H]CD binding did not saturate in this concentration range, although the binding isotherm was concave downward. Nonlinear regression analysis of the binding data revealed the presence of two populations of muscarinic receptors having dissociation constants of 1.83 and 123 nM and binding capacities of 85 and 1320 fmol/mg protein, respectively. Competitive inhibition experiments showed that [³H]CD binding was readily displaced by several muscarinic agonists and antagonists. The stereospecificity of [³H]CD binding was demonstrated in competitive inhibition experiments using the stereoisomers of benzetimide and acetyl-β-methylcholine. Dexetimide was 10,000 times more potent than levetimide and l-acetyl-β-methylcholine was 520 times more potent than d-acetyl-β-methylcholine. A variety of nonmuscarinic cholinergic drugs were not effective at inhibiting [³H]CD binding at a concentration of 10 μM.     

Alterations in central and peripheral adrenergic receptors in deoxycorticosterone/salt hypertensive rats

The treatment of uninephrectomized rats with deoxycorticosterone (DOCA) and salt for 6 weeks caused a significant systolic hypertension and cardiac and renal hypertrophy. There was a significant decrease in the density of cardiac α₁- and β-, and renal α₁-adrenoceptors in DOCA/salt hypertensive rats, as compared to uninephrectomized salt loaded control rats. In contrast, the cerebral cortex, corpus striatum, hippocampus and hypothalamus/thalamus of hypertensive rats showed a significant increase in adrenoceptor binding in these hypertensive rats. In contrast, muscarinic cholinergic receptors and [³H]yohimbine binding sites were not altered in most tissues of the hypertensive rats. The present study suggests an important role for central and peripheral α₁- and β-adrenoceptors in the pathogenesis of hypertension.