LPS-or 8Br-cyclic AMP-induced production of T cell-activating factor(s) in macrophage tumor cell line J774.1.

The macrophage tumor cell line J774.1 replaced the function of normal macrophages in the induction of polyclonal killer T cells with 2-mercaptoethanol. J774.1 does not normally release soluble factor(s) which we have shown to be responsible for the differentiation of T cells to killer T cells. However, stimulation of J774.1 with LPS induced soluble factor(s) for T cell activation. An optimum concentration of LPS for the production of soluble factor(s) was 1 to 10 microgram/ml, which completely inhibited growth of the tumor cells. The production of soluble factor(s) was observed within 6 hr after LPS stimulation and reached its maximum level at 24 hr. Incubation of the cell line with 8Br-cyclic AMP and theophylline induced soluble factor(s), suggesting that LPS stimulation induced an increase in intracellular cyclic AMP which leads to the synthesis of soluble factor(s).     

The effect of temperature on CL 218872 and propyl beta-carboline-3-carboxylate inhibition of [3H]-flunitrazepam binding in rat brain

The competitive inhibition of [³H]-flunitrazepam binding by CL 218872 and propyl beta-carboline-3-carboxylate (PCC), non-benzodiazepine compounds that show differential affinities for benzodiazepine (BZD) receptor subtypes, was studied in the rat cerebral cortex and hippocampus at different temperatures of incubation. The potency of both inhibitors was significantly greater at 0° than at 37°C. The magnitude of temperature induced enhancement of potency may correlate with the pharmacological efficacy of compounds that interact with BZD receptors. Hill slopes for CL 218872 shifted from 0.52 to 0.97 in the cerebral cortex when incubations were performed at 0° and 37°C, respectively. Hill values for PCC changed from 0.68 to 0.93 under similar temperature conditions. These observations suggest the presence of a homogenous population of benzodiazepine receptors at physiological temperatures or the inability of CL 218872 and PCC to distinguish between receptor subtypes at 37°C.     

Inhibitory effect of mitoxantrone on activity of protein kinase C and growth of HL60 cells.

Mitoxantrone, a new anthraquinone, showed inhibitory an effect on protein kinase C (PKC) activity. Its IC50 value was 4.4 micrograms/ml (8.5 microM), which is much lower than those of the well-known anthracyclines daunorubicin and doxorubicin, the IC50 values of which are more than 100 micrograms/ml (> 170 microM). Kinetic studies demonstrated that mitoxantrone inhibited PKC in a competitive manner with respect to histone H1, and its Ki value was 6.3 microM (Ki values of daunorubicin and doxorubicin were 0.89 and 0.15 mM, respectively), and in a non-competitive manner with respect to phosphatidylserine and ATP. Inhibition of phosphorylation by mitoxantrone was observed with various substrates including S6 peptide, myelin basic protein and its peptide substrate derived from the amino-terminal region. Their IC50 values were 0.49 microgram/ml (0.95 microM), 1.8 micrograms/ml (3.5 microM), and 0.82 microgram/ml (1.6 microM), respectively. Mitoxantrone did not markedly inhibit the activity of cyclic AMP-dependent protein kinase, casein kinase I or casein kinase II, at concentrations of less than 10 micrograms/ml. On the other hand, brief exposure (5 min) of HL60 cells to mitoxantrone caused the inhibition of cell growth with an IC50 value of 52 ng/ml (0.1 microM). In HL60 cells, most of the PKC activity (about 90%) was detected in the cytosolic fraction. When HL60 cells exposed to 10 micrograms/ml mitoxantrone for 5 min were observed with fluorescence microscopy, the fluorescence elicited from mitoxantrone was detected in the extranuclear area. These results indicated that mitoxantrone is a potent inhibitor of PKC, and this inhibition may be one of the mechanisms of antitumor activity of mitoxantrone.     

Phosphorylation of histone H2A by protein kinase C and identification of the phosphorylation site.

In regenerating rat liver, nuclear protein histone H2A was shown to be phosphorylated on its amino-terminal serine residue [Sung et al. (1971) J. Biol. Chem. 246, 1358-1364], but the protein kinase which phosphorylates this residue has not been identified. To evaluate the possibility that protein kinase C can phosphorylate this residue, calf thymus histone H2A was 32P-labeled by incubation with [gamma-32P]ATP and highly purified protein kinase C from rat brain in the presence of calcium and phospholipid. About 1 mol of 32P was incorporated per mol of histone H2A and the Km and apparent Vmax of the reaction were calculated to be 2.1 microM and 0.35 mumol/min/mg, respectively. So histone H2A seemed to be a good substrate for protein kinase C. Further, the proteolytic phosphopeptides of 32P-labeled histone H2A were isolated by means of a series of column chromatographies and analyzed for their amino acid compositions. Comparison of the data with the known primary structure of histone H2A revealed their amino acid sequence as 1Ser-Gly-Arg. These data suggest that protein kinase C may be a candidate for the protein kinase which phosphorylates the amino-terminal serine residue of histone H2A during the regeneration of rat liver.     

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.     

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.