Showing papers in "Molecular Pharmacology in 1979"

Multiple Serotonin Receptors: Differential Binding of [3H]5-Hydroxytryptamine, [3H]Lysergic Acid Diethylamide and [3H]Spiroperidol

Abstract: [3H]5-Hydroxytryptamine (5-HT), [3H]lysergic acid diethylamide (LSD) and [3H]spiroperidol bind to membranes from the rat frontal cerebral cortex in a manner indicating a selective interaction with serotonin receptors. Differential drug potencies in competing for [3H]5-HT and [3H]spiroperidol binding sites suggest that these two [3H]ligands respectively label two distinct populations of receptors, while [3H]LSD labels both the [3H]5-HT and [3H]spiroperidol sites. After incubation of brain membranes with 30 nM spiroperidol, drug specificity of the residual [3H]LSD binding resembles that of receptors labeled by [3H]5-HT. Conversely, drug effects on [3H]LSD binding in the presence of 300 nM 5-HT resemble effects with [3H]spiroperidol. We propose that [3H]5-HT and [3H]-spiroperidol label distinct populations of serotonin receptors in rat brain, designated 5-HT1 and 5-HT2 receptors, respectively. [3H]LSD appears to bind to both receptors to a similar extent.

Opiate-Like Materials in the Adrenal Medulla: Evidence for Storage and Secretion with Catecholamines

Abstract: Opiate-like materials, as measured by the radioreceptor assay, were found in the adrenal gland of 7 mammalian species including man. The opiate-like materials are confined to the adrenal medulla, and followed a pattern identical to catecholamines on differential and isopycnic centrifugation. In the isolated perfused dog adrenal gland, acetylcholine stimulates a Ca2+-dependent secretion of opiate-like materials and catecholamines in the same molar ratio as they are stored within the chromaffin cell. Ba2+ also stimulates the secretion of both products from the adrenal gland. Separation of the opiate-like materials by Sephadex G-50 and reverse phase high pressure liquid chromatography indicates the presence of four peaks of opiate-like materials of molecular weights below 2,000. Two of these peaks comigrate in these systems with authentic met- and leu-enkephalins and react with the corresponding antibodies. These results indicate that enkephalins and other low molecular weight opiate-like materials are stored in and secreted from the chromaffin vesicles with catecholamines in the adrenal glands. These findings support a role for enkephalins as neurotransmitters and/or neurohormones. Also, they suggest that enkephalins and other opiate-like materials may exert important neuroendocrine functions outside the central nervous system.

Binding of Drugs to Human Serum Albumin:: XI. The Specificity of Three Binding Sites as Studied with Albumin Immobilized in Microparticles

Abstract: Human serum albumin can be immobilized in spherical, macroporous microparticles of polyacrylamide of about 1 µm in diameter with retention of its native properties. It has been shown that diazepam, digitoxin and warfarin independently bind to albumin and can conveniently be used as markers of three separate, discrete binding sites on albumin. A simple technique has been devised by which the capacity of about 140 drugs and other compounds to affect the binding of the radioactively labeled markers has been studied. Some drugs, e.g. antirheumatic drugs of the isopropionic acid-type, some antidiabetic agents, penicillin derivatives, benzodiazepines, tryptophan, dansylsarcosine, and suiphobromophthalein efficiently displace diazepam. Other drugs, e.g., some diuretics, sulpha drugs, phenytoin, salicylic acid and butazone derivatives, azapropazoe, bilirubin, and dansylamide displace warfarin. Displacement of digitoxin is less common. In some cases the binding of the markers is improved, e.g., tamoxifen increases the binding of warfarin. Both competitive and allosteric mechanisms are responsible for the changed binding of the markers. Some results suggest the presence of more than the three binding sites for drugs on the albumin surface studied with diazepam, digitoxin and warfarin.

Simultaneous Determination of Beta-1 and Beta-2-Adrenergic Receptors in Tissues Containing Both Receptor Subtypes

Abstract: A method for determining the relative concentrations and properties of β-1 and β-2-adrenergic receptors in tissues containing both receptor subtypes has been developed. Since (125I)-iodohydroxybenzylpindolol has similar affinities for β-1 and β-2-adrenergic receptors, it is possible to determine the total concentration of β-adrenergic receptors in a tissue by Scatchard analysis of specific (125I)-iodohydroxybenzylpindolol binding. In the presence of GTP, inhibition of specific (125I)-iodohydroxybenzylpindolol binding in rat heart, lung and five regions of rat brain by agonists and antagonists that are not specific for β-1 or β-2-adrenergic receptors yields linear Hofstee plots with Hill coefficients of approximately 1.0. On the other hand, the inhibition of specific (125I)-iodohydroxybenzylpindolol binding by drugs which have been shown to have different affinities for heart (β-1) and lung (β-2) receptors in vitro results in nonlinear Hofstee plots in each of these tissues. Two of these drugs (practolol and metoprolol) are more potent on β-1 than β-2 receptors and two of these drugs (zinterol and salmefamol) are more potent on β-2 than on β-1 receptors. The nonlinear Hofstee plots are consistent with there being two types of binding sites in each of the tissues with different affinities for the drugs. The relative number of each type of binding site and the affinity of each drug for each of the two types of site has been calculated using a computer based iterative procedure. Using this method, the relative percentages of the two receptor subtypes in rat heart, lung, cerebral cortex, caudate, cerebellum, hippocampus and diencephalon were determined. In each tissue, the use of four different drugs with different in vitro selectivity (two β-1 selective and two β-2 selective) resulted in approximately the same calculated β-1/β-2 ratio. This suggests that the assumption that the nonlinear Hofstee plots are composed of only two components is correct. In addition, the calculated affinity of each drug for β-1 and β-2 receptors was quantitatively similar in each of the seven tissues examined. The calculated ratios of β-l:β-2-adrenergic receptors are: heart 83:17; lung 15:85; cortex 81:19; caudate 76:24; cerebellum 15:85; hippocampus 81:19; and diencephalon 71:29. The absolute concentrations of β-1-adrenergic receptors in the brain regions examined varied by almost 20-fold. However, the absolute concentration of β-2-adrenergic receptors varied less than 3-fold. This suggests that β-2-adrenergic receptors in rat brain are associated with a more homogeneously distributed cellular element than are β-1-adrenergic receptors.

Multiple Opiate Receptors: Different Regional Distribution in the Brain and Differential Binding of Opiates and Opioid Peptides

Abstract: In rat brain membrane preparations, the parenterally and orally active peptide, [D-Ala2, MePhe4, Met(O)5-ol]-enkephalin, binds to morphine receptor sites ([3H]naloxone or [3H]dihydromorphine binding sites) with an affinity higher than that for enkephalin receptor sites ([125I] [D-Ala2, D-Leu5]-enkephalin binding sites). [125I] [D-Ala2, MePhe4, Met(O)5-ol]-enkephalin binds to morphine receptor sites stereospecifically, in a saturable manner and with characteristics similar to that of [3H]dihydromorphine; this ligand can be used as an 125I-labeled probe to measure specific binding to morphine receptor sites. Na+ decreases and Mn2+ increases the binding capacity with a concomitant reduction of affinity for [125I] [D-Ala2, MePhe4, Met(O)5-ol]-enkephalin. This peptide does not bind to neuroblastoma cells with high affinity. The brain regional distribution of binding of [125I] [D-Ala2, MePhe4, Met(O)5-ol]-enkephalin or [3H]naloxone and [125I] [D-Ala2, D-Leu5]-enkephalin are different. The differential potency of binding of opiate agonists, antagonists, mixed agonist-antagonists, enkephalins and enkephalin analogues is studied by competition of binding of [3H]naloxone or [125I] [D-Ala2, MePhe4, Met(O)5-ol]-enkephalin (morphine receptor) and of [125I] [D-Ala2, D-Leu5]-enkephalin sites (enkephalin receptor). All of these results support the contention that there are multiple opiate receptors with differing characteristics.

Multiple apparent alpha-noradrenergic receptor binding sites in rat brain: effect of 6-hydroxydopamine.

Abstract: [3H]Clonidine (26.7 Ci/mmole) binds with high affinity to sites on rat brain membranes with properties of alpha -noradrenergic receptors. [3H]Clonidine binding shows a biphasic pattern in kinetic and saturation experiments. Fifty percent of the specific binding of 0.4 nM [3H]clonidine is dissociated rapidly in 2 min by excess norepinephrine or clonidine, and the remaining 50% dissociates at a 10-fold slower rate. When slowly-dissociating [3H]clonidine binding is examined in isolation, saturation curves show a single population of high-affinity sites with a KD of 0.5 nM and B max of 1.3-1.6 pmoles/g tissue in cerebral cortex. Rapidly-dissociating [3H]clonidine binding, estimated from the difference between total binding and high-affinity binding, involves a single population of lower affinity sites with a KD of 3.0 nM and B max of 9-10 pmoles/g cerebral cortex tissue. Alpha agonists are in general more potent at the high affinity [3H]clonidine site, while alpha antagonists are more potent at the low affinity site. α-Methylnorepinephrine is less potent than norepinephrine at the high affinity site, but more potent at the low affinity site. Neither [3H]-clonidine binding site resembles the alpha receptor site labeled by [3H]WB-4101. The distribution of high and low affinity [3H]clonidine binding throughout rat central nervous system is different. High affinity binding levels vary 14 fold between lowest values in corpus striatum and cerebellum, and highest values in cerebral cortex. Low affinity binding varies less regionally, with highest levels in hypothalamus. 6-Hydroxydopamine treatment doubles the number of high affinity [3H]clonidine sites in the cortex, but does not alter the number of low affinity sites. 6-Hydroxydopamine increases high affinity binding much more than low affinity binding throughout the brain. 6-Hydroxydopamine also increases by 50% the number of [3H]WB-4101 and [3H]epinephrine alpha receptor sites in the cerebral cortex. ACKNOWLEDGMENTS We thank Mrs. Lynda Hester for excellent technical assistance, and Pam Morgan and Billie Jean Little for manuscript preparation.

Spin trapping of superoxide.

Abstract: Due to conflicting reports in the literature, the spin trapping of superoxide by the nitrone, DMPO, has been re-investigated. We found that superoxide can indeed be trapped and that the DMPO-OOH and DMPO-OH adducts are distinguishable, in agreement with the original work of Harbour et al. (10). In contrast to their work, we found that DMPO-OOH was highly unstable and that it decomposed into DMPO-OH. We observed that TMPO, a spin trap structurally similar to DMPO, but lacking a β-hydrogen, formed a stable nitroxide upon reaction with superoxide. Rate constants for the reaction of superoxide with DMPO and TMPO were measured and indicated that spin trapping was an extremely inefficient method for the detection of superoxide. The new spin trap described by Janzen et al. (18), 4-POBN, was also capable of distinguishing the difference between superoxide and hydroxyl radical. The nitroxide formed upon the reaction of superoxide with 4-POBN (4-POBN-OOH) was found to be unstable. However, unlike DMPO-OOH, 4-POBN-OOH does not decompose into the hydroxynitroxide, 4-POBN-OH.

Specific Binding of [3H]Kainic Acid to Receptor Sites in Rat Brain

Abstract: The binding of [3H]kainic acid to washed membranes from rat forebrain was saturable with dissociation constants at 2° of 4-16 nM and 27-66 nM for high and low affinity binding, respectively. The Hill coefficients for binding to high and low affinity sites were 0.99 and 0.85, respectively. At 2° [3H]kainic acid receptor binding reached equilibrium by 60 minutes. Dissociation of the ligand-receptor complex at 2° was biphasic. The kinetically-determined dissociation constant for the high affinity binding site corresponded well with the value obtained in equilibrium studies. Of the substances tested, kainic acid and quisqualic acid were the most potent in competing for [3H]kainic acid binding sites. L-glutamic acid was 40-fold less potent than kainic acid itself but 2500 times more effective than D-glutamic acid; dihydrokainic acid was 200-fold less potent than kainic acid at the low affinity site and 1000-fold less active in competing for binding to the high affinity site. Among the poor displacers of [3H]kainic acid (IC50 > 0.1 mM at 50 nM [3H]kainic acid) were D- and L-aspartic acids as well as several proposed glutamate antagonists. In brain, there was considerable variation in the regional distribution of high affinity binding sites: striatum > frontal cortex = hippocampus > cerebellum > medulla-pons; more than 90% of the total specific binding in the cerebellum and medulla-pons was to low affinity sites. Thus, on the basis of the kinetics of binding, differential affinity for competitors and differences in regional distribution in brain, the high and low affinity binding sites represent independent entities. Negligible specific binding was obtained (< 1 fmole/mg tissue) in liver, lung, kidney and intestine. Ablation of the intrinsic neurons of the caudate nucleus by injecting 10 nmole of kainic acid 28 days before sacrifice was associated with a 57-64% reduction in high and low affinity binding in this region. ACKNOWLEDGMENTS We thank A. Hunter Thompson for providing excellent technical assistance and Victoria Rhodes, Princie Campbell and Carol Kenyon for preparing the manuscript. We are also grateful to Dr. Morley Hollenberg for his helpful comments and Dr. Kathleen Biziere for her contributions.

The pharmacological specificity of beta-1 and beta-2 adrenergic receptors in rat heart and lung in vitro.

Abstract: The potency and selectivity of a variety of agonists and antagonists were determined for β-1 and β-2 adrenergic receptors on membranes prepared from rat ventricular muscle and lung. Activation or inhibition of β-adrenergic receptor stimulated adenylate cyclase activity and inhibition of specific (125I)-iodohydroxybenzylpindolol binding were used as in vitro measurements of receptor occupancy. With both assays the relative potencies of isoproterenol, epinephrine and norepinephrine with cardiac membranes was approximately 1:10:10, indicating a population of mainly β-1 adrenergic receptors. With membranes from lung the order of potency of these compounds was approximately 1:10:100, indicating a population mainly of β-2 adrenergic receptors. Several drugs previously reported to be β-2 selective agonists (salbutamol, soterenol, salmefamol, zinterol and fenoterol) activated adenylate cyclase in the lung but not in the heart. These compounds turned out to be partial agonists and isoproterenol-stimulated adenylate cyclase activity was inhibited by them in both tissues. Several compounds previously reported to be either β-1 (dobutamine) or β-2 (terbutaline and metaproterenol) selective agonists had similar potencies for stimulation of adenylate cyclase from both tissues. A series of compounds reported to be β-1 selective antagonists were also investigated. Metoprolol and practolol were 10-fold, and atenolol was 3-fold more potent in the heart than the lung. Butoxamhe, a β-2 antagonist, was 2-4 fold more potent in the lung than the heart, while H35/25 showed no specificity. The ability of antagonists to inhibit [125I]-iodohydroxybenzylpindolol binding to membranes prepared from the heart and lung agreed well with their effects on adenylate cyclase. The β-2 selective agonists zinterol and salmefamol also showed a 10-50 fold greater potency in inhibiting [125I]-iodohydroxybenzylpindolol binding in the lung than the heart. However salbutamol, soterenol and fenoterol, which selectively activated adenylate cyclase in the lung, inhibited [125I]-iodohydroxybenzylpindolol binding in the two tissues with equal potency. This apparent discrepancy appears to be due to the fact that these drugs which are partial agonists in the lung are competitive antagonists in the heart, and that the Ki values in the heart are very similar to the K act values in the lung.

The effects of some organic "calcium antagonists" on calcium influx in presynaptic nerve terminals.

Abstract: The actions of the organic "Ca antagonists" verapamil and D-600 were tested on pinched-off presynaptic nerve terminals (synaptosomes) from rat brain, and on the frog neuromuscular junction. 45Ca uptake was measured in control media, and in depolarizing media containing either 75 mM potassium or veratridine, an alkaloid that opens sodium channels. The extra uptake induced by depolarizing media appears to be mediated by voltage-sensitive Ca channels. Synaptosome depolarization was indirectly determined with the voltage-sensitive fluorescent dye, di-pentyl oxacarbocyanine. Verapamil or D-600 (100 µM) inhibited the K+-induced 45Ca uptake by about two thirds, but had no effect on the K+-induced synaptosome depolarization; this inhibition of Ca uptake is, presumably, due to block of Ca-channels. Veratridine-induced 45Ca influx was more than 80% inhibited by verapamil or D-600 (100 µM), and veratridine-induced depolarization was almost completely blocked. These observations indicate that Na channels as well as Ca channels are inhibited by verapamil and D-600. Recordings of miniature end-plate potentials were used to evaluate the actions of verapamil and D-600 at the frog neuromuscular junction, after miniature end-plate potential frequency had been made sensitive to changes in the bathing Ca concentration by raising the external K+. Miniature end-plate potential frequency was not affected by verapamil(40-50 µM) or D-600 (10 µM) but was significantly reduced by Mn2+ (0.2 mM), a known blocker of Ca channels. Although verapamil and D-600 appear to be very potent antagonists of Ca currents in heart and smooth muscle, we conclude that Ca channels in vertebrate neurons are much less sensitive to these drugs.

Quantitative resolution of beta-adrenergic receptor subtypes by selective ligand binding: application of a computerized model fitting technique.

Abstract: Frog myocardium appears to possess both beta1 and beta2 receptors, based on the potency order of several adrenergic agonists to compete for [3H]dihydroalprenolo1 binding. Selective beta blocking agents are able to distinguish two receptor subtypes in frog myocardium, but only one site in rat ventricle. Computer modeling using a PDP 11/45 indicates that all rat beta receptors are beta1, whereas only 15%-25% of frog ventricular beta receptors are of the beta1 subtype. Computerized curve fitting can provide a more accurate estimate of receptor parameters than currently available graphical methods of analysis.

Selective inhibitors of Ca2+-binding modulator of phosphodiesterase produce vascular relaxation and inhibit actin-myosin interaction.

Abstract: To elucidate the role of Ca 2+ -regulated activator protein (a protein modulator) of cyclic nucleotide phosphodiesterase in smooth muscle contraction, we determined the effects of various drugs that selectively inhibit a protein modulator-induced stimulation of cyclic nucleotide phosphodiesterase on contractile response of isolated rabbit aortic strip, and superprecipitation of bovine aorta smooth muscle actomyosin, considered to be an in vitro analogue of muscle contraction. Actomyosin preparation from bovine aorta smooth muscle contained approximately 200 units (2 µg) of protein modulator per mg actomyosin and superprecipitated by addition of ATP. Selective inhibitors of the stimulation of the phosphodiesterase by a protein modulator such as N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and its derivatives, or psychotropic agents such as chlorpromazine, chlorprothixene, amitriptyline and desipramine produced relaxation of isolated aortic strips contracted by various agonists such as KCl, CaCl 2 , norepinephrine, histamine, and prostaglandin F 2α . The relaxation induced by these agents was not affected by treatment with adrenergic or cholinergic blocking agents such as propranolol and atropine, suggesting that such do not work through these receptors. These drugs not only reduced the extent of actomyosin superprecipitation but also prolonged the time required to attain the maximum superprecipitation, suggesting that they inhibit the superprecipitation of aorta smooth muscle actomyosin. The addition of various amounts of protein modulator prevented in dose-dependent fashion the inhibition by these compounds of actomyosin superprecipitation. The order of the potency of the drugs was much the same in assay systems such as inhibiting the stimulation of the phosphodiesterase by a protein modulator, superprecipitation of actomyosin and producing a relaxation of aortic strip. Other vasodilating drugs such as isoproterenol, sodium nitrite and fusaric acid had no effect on smooth muscle contraction process (superprecipitation of actomyosin). Papaverine, which is a potent inhibitor of cyclic nucleotide phosphodiesterase and not a selective inhibitor, served also as the control. However, even papaverine failed to inhibit the superprecipitation. Our results provide pharmacological evidence that a protein modulator participates in the regulation in smooth muscle contraction.

Agonist-specific effects of guanine nucleotides on alpha-adrenergic receptors in human platelets.

Abstract: The effect of GTP on the binding affinity of alpha-adrenergic receptors for alpha-adrenergic agents was studied in human platelet lysates using direct ligand binding methods with [3H]dihydroergocryptine. GTP at a concentration of 0.1 mM markedly decreased the binding affinity of the agonist (-)epinephrine for the receptors (more than 10-fold) but had no effect on the binding of antagonists. The half maximal effect of GTP on epinephrine binding occurred at a concentration of 4 µM. Gpp(NH)p was as effective as GTP at the same concentration, whereas GDP was only 80% as effective. Other nucleotides such as ATP and ITP were less effective. The extent of the GTP-induced reduction in the affinity of alpha-adrenergic agents for the receptors was directly related to the intrinsic activity of these agents for inhibition of PGE1-stimulated adenylate cyclse. The effect of GTP appears to depend on the concurrent presence of Mg++. In the absence of Mg++, GTP caused only a slight decrease in the agonist binding affinity. When Mg++ was present without GTP, the binding affinity of the agonist (-)epinephrine was increased by 5-fold. GTP in the presence of Mg++ induces a state of diminished affinity of the receptor for the agonist which is lower than that induced by the nucleotide in the absence of MgCl2. The relationship between GTP and MgCl2 in the regulation of platelet alpha-adrenergic receptors which are inhibitory to adenylate cyclase activity appears to be analogous to their role in regulating beta-adrenergic receptors which are stimulatory for the enzyme in other tissues.

Ethanol-induced fluidization of brain lipid bilayers: required presence of cholesterol in membranes for the expression of tolerance.

Abstract: The effects of ethanol on fluidity of reconstituted membranes formed from lipid extracts of tolerant and control mice synaptosomal membranes were assessed by fluorescence polarization techniques. Ethanol was less able to fluidize reconstituted membranes of ethanol-tolerant mice than controls. Acute in vivo administration of ethanol did not alter ethanol-induced fluidization of the reconstituted membranes. Since increased membrane cholesterol has been suggested to account for tolerance, the cholesterol and trace nonpolar lipids from the lipid extracts were removed and then cholesterol was added back so as to equalize its level in all groups. Following removal of cholesterol, it was not possible to detect any differences between the tolerant and control groups. However, when the cholesterol was added back so that all groups had a cholesterol/phospholipid ratio of 0.54, i.e., the control group level, it was possible to measure differences between tolerant and control lipid extracts. These results suggest that changes in the lipid composition of membranes can account in part for tissue adaptation to ethanol-induced membrane fluidization. Moreover, while the presence of cholesterol in the membranes appears to be required for the expression of tolerance, the small changes in the cholesterol content of membranes observed following development of tolerance probably are not responsible for the attenuation of ethanol-induced membrane fluidization.

Effect of gangliosides on adenylate cyclase activity in rat cerebral cortical membranes.

Abstract: The addition of 50 µM mixed brain gangliosides to membrane preparations from rat cerebral cortex caused a 50-95% increase in the basal adenylate cyclase activity. The activation was rapid at all temperatures and was relatively irreversible, as evidenced by the fact that repeated washing of the membrane after exposure to gangliosides failed to restore adenylate cyclase activity to its basal level. The V max of the enzyme was increased with no apparent change in the K m for the substrate, ATP. The expression of activation of adenylate cyclase by gangliosides showed a marked temperature dependence, with maximal effects seem in the 30-40° range and no activation seen at either 10° or 50°. The activation of adenylate cyclase by brain gangliosides was additive with respect to activation by NaF and detergents, but was not additive with respect to activation by GppNHp or lysolecithin. The activation of the enzyme by gangliosides was unaffected by the presence of calcium ions, calcium-dependent activator protein, or EGTA. The addition of brain gangliosides to the membranes caused a consistent restoration of the responsiveness of the adenylate cyclase system to activation by β-adrenergic agents, an effect similar in magnitude to that observed by addition of GppNHp to the membrane preparations.

Immunological comparison of hepatic and extrahepatic cytochromes P-450.

Abstract: Antibodies were raised to two apparently homogeneous cytochromes P-450 prepared from liver microsomes of phenobarbital- and 3-methylcholanthrene-treated rats; microsomal fractions were prepared from the livers, lungs, kidneys, testes, spleens, hearts, small intestines, colons, stomachs, and brains of untreated and phenobarbital- and 3-methylcholanthrene-treated rats. Cross reactivity was observed in each tissue with at least one of the two antibodies as judged by immunodiffusion analysis. Inhibition of benzo(a)pyrene hydroxylase or 7-ethoxycoumarin O-deethylase activity was observed with microsomes prepared from each tissue with the exception of spleen and colon; inhibition curves were dependent upon the substrate and antibody used as well as the pretreatment of the animals from which the microsomes were prepared. As a general rule, microsomes prepared from 3-methylcholanthrene-treated rats were inhibited most readily by antibody raised to cytochrome P-450 isolated from liver microsomes prepared from 3-methylcholanthrene-treated rats, and the same general rule held for phenobarbital induction; however, several exceptions were noted. On the basis of these results, the microsomal P-450s of extrahepatic tissues are, in general, immunologically similar to the major hepatic microsomal P-450s in the rat.

Inhibition of DNA excision repair in human cells by arabinofuranosyl cytosine: effect on normal and xeroderma pigmentosum cells.

Abstract: The antineoplastic agent arabinofuranosyl cytosine (ara-C) produces an inhibition of the pyrimidine dimer excision system of human DNA repair. Alkaline sucrose gradient analysis of DNA from normal human skin fibroblasts exposed to 20 J/m/sup 2/ of ultraviolet radiation (254 nm) shows an accumulation of DNA single-strand breaks when DNA repair is attempted in the presence of 10 ..mu..M ara-C. Cells from complementaion groups of xeroderma pigmentosum that are defective in early steps of excision repair show reduced numbers of DNA single strand breaks/10/sup 8/ daltons when compared with normal cells. Cesium chloride gradient analysis of radioactive precurser uptake during repair replication indicates that ara-C causes a 6 to 56% reduction in the number of nucleotide bases inserted into the DNA at concentrations of 1 and 10 ..mu..M, respectively. These concentrations result in the substitution for deoxycytidine (dCyd) by ara-C of 40 and 100%, respectively, in repaired regions. Repair inhibition is reversed by 50% upon removal of ara-C and by > 95% with the addition of 100 ..mu..M dCyd. Chromatography of digested DNA shows that incorporated ara-C is not removed during dCyd reversal, suggesting that ara-C incorporation per se does not play a significant role in the inhibition of repair synthesis.more » The repair inhibition observed here is dependent on 2 mM hydroxyurea, presumably due to reduction in the intracellular pool of dCyd. The overall results suggest the possibility that ara-C is a weak competitive inhibitor of DNA polymerases associated with ultraviolet-induced excision repair.« less

Inhibition of calcium-dependent regulator-stimulated phosphodiesterase activity by neuroleptic drugs is unrelated to their clinical efficacy.

Abstract: It has previously been suggested that some of the pharmacological and therapeutic effects of certain neuroleptic drugs depend upon their ability to inhibit calcium-dependent regulator-(CDR)-stimulated phosphodiesterase activity. To further test this hypothesis, clinically active and inactive isomers of a number of neuroleptics were examined as CDR inhibitors. Both active and inactive isomers of all the drugs investigated were equally inhibitory in this assay. The IC50 values for CDR inhibition obtained for these and other neuroleptics correlated well with their octanol:water partition coefficients. It is concluded, therefore, that the inhibitory effects of these drugs on the CDR protein are unrelated to their clinical efficacy and may be due to nonstereospecific hydrophobic interactions with the CDR protein.

Identification of a Local Anesthetic Binding Site in Nicotinic Post-Synaptic Membranes Isolated from Torpedo marmorata Electric Tissue

Abstract: Studies are presented of the interaction of aromatic amine noncompetitive antagonists with the receptor-rich (post-synaptic) membranes isolated from Torpedo marmorata electric organ When present at micromolar concentrations, methyl iodide quaternary derivatives of the potent non-competitive antagonists proadifen and dimethisoquin increase the affinity of the membrane-bound nicotinic receptor for [ 3 H]acetylcholine and [ 14 C]dimethyltubocurarine The equilibrium binding of [ 14 C]meproadifen (2-(diethylmethylamino)ethyl-2,2-diphenyl valerate) to the receptor-rich membranes is characterized by an ultracentrifugatiom assay When all acetylcholine binding sites are occupied by the agonist carbamylcholine or the antagonist tubocurarine, [ 14 C]meproadifen is bound with a dissociation constant K D = 05 µM to a number of sites equal to one-quarter the number of α-neurotoxin binding sites That high affinity binding site is found in the receptor-rich membranes and not in other membrane fractions isolated from Torpedo electric organ Other non-competitive aromatic amino antagonists including dimethisoquin, proadifen, and prilocaine displace [ 14 C]meproadifen, as does perhydrohistrionicotoxin It is concluded that there is a specific site of binding for the aromatic amine non-competitive antagonists in the isolated nicotinic post-synaptic membrane that is distinct from the site of binding of acetylcholine Analysis of the interaction of [ 14 C]meproadifen with the receptor-rich membranes in the absence of cholinergic ligands indicates that under these circumstances the ligand binds weakly to both the anesthetic binding site and the acetylcholine binding site ( K D = 5 µM) The functional significance of the specific binding site for the aromatic amine non-competitive antagonists is discussed in terms of its possible relation to the site of ion translocatiom and to the mechanism of receptor desensitization

Adenylate cyclase assembled in vitro: cholera toxin substrates determine different patterns of regulation by isoproterenol and guanosine 5'-triphosphate.

Abstract: Genetic and biochemical evidence indicates that hormone-sensitive adenylate cyclase consists of at least three separable components: hormone receptor (R), a catalytic unit (C) that synthesizes cAMP from ATP, and one or more regulatory factors, which we have termed N, that are required for functional coupling of R and C and also for cyclase stimulation by guanyl nucleotides, NaF, and cholera toxin. The functional absence of N in a S49 lymphoma variant clone, cyc-, allows in vitro assembly of hormone-sensitive adenylate cyclase, using cyc- membranes and N components in detergent extracts from membranes of other cells (Ross, E. M. and Gilman, A. G. Proc. Nat. Acad. Sci., USA 74,3715-3719, 1977). Such cyclase systems, assembled in vitro using N components from wild type S49 cells and turkey erythrocytes, are functionally distinguishable, each resembling—in responses to guanyl nucleotides and isoproterenol—the cyclase system from which the N component was derived. Thus, the N component determines certain functional characteristics of the response to guanine nucleotides and isoproterenol. Human erythrocyte membranes, which are virtually devoid of catalytic adenylate cyclase, also contain the functional N component. Donor extracts of human and turkey erythrocytes and S49 cells contain cholera toxin substrates, by two criteria: 1. The extracts transmit effects of toxin on donor membranes to adenylate cyclase assembled in vitro using cyc- membranes; 2. Incubation with toxin plus [32P]NAD+ specifically radiolabels substrates in each type of membrane, including a peptide of Mr = 42,000, common to all three. The results are consistent with the hypothesis that this peptide participates directly in multiple functions of N.

Structure/activity studies of flavonoids as inhibitors of cyclic AMP phosphodiesterase and relationship to quantum chemical indices.

Abstract: A series of 45 flavonoids and related compounds were tested as inhibitors of liver fluke cyclic nucleotide phosphodiesterase. Many were found to be potent inhibitors; seven were as potent or more potent than any inhibitor previously tested. The kinetics of six compounds spanning a wide range of activities were investigated and found to be competitive. The most potent inhibitors, cyanidin chloride and quercetin, had Ki values of 10 ± 3 µM and 13 ± 6 µM, respectively, approaching the Km for CAMP (8 µM). Structure/ activity studies showed that adding exocyclic substituents to the basic flavonoid skeleton affected activity only slightly, while changing the planarity of the heterocyclic ring greatly decreased activity. This observation, taken with the competitive kinetics, suggests that flavonoids compete with cAMP for a nucleotide binding site at which stacking occurs, perhaps similar to the binding sites of bovine pancreatic ribonuclease A and lobster glyceraldehyde-3-phosphate dehydrogenase. Quantum chemical calculations further suggest that the competition arises from the mimicking of the pyrimidine ring in cAMP by the pyranone ring of the flavonoids. If the flavonoids are comparably potent inhibitors of other phosphodiesterases, several reported pharmacological effects of the flavonoids might be explained.

Dopamine Receptor Binding Regulated by Guanine Nucleotides

Abstract: Guanosine triphosphate (GTP) and dighosphate nucleotides decrease the binding of the agonist ligands [3H]apomorphine and [3H]ADTN to dopamine receptors in the rat corpus striatum with half maximal reduction of binding at 5 µM. These nucleotides also reduce agonist inhibition of the antagonist [3H]spiroperidol binding to dopamine receptors without affecting total [3H]spiroperidol binding. Guanosine monophosphate and adenine nucleotides display negligible influence on dopamine receptor binding. GTP reduces the affinity of [3H]apomorphine binding with no effect on the maximal numbers of binding sites.

Local anesthetics, chlorpromazine and propranolol inhibit stimulus-activation of phospholipase A2 in human platelets.

Abstract: Stimulation of platelets by various agents such as thrombin, collagen and A23187 leads to a rapid Ca2+ - dependent, phospholipase A2-mediated mobilization of arachidonic acid from certain phospholipids and the biotransformation of this fatty acid to prostaglandin endoperoxides, thromboxanes A2 and B2, prostaglandins, hydroxyacids and malonyldialdehyde (MDA). Local anesthetics (dibucaine, tetracaine, benzocaine, and QX572), chlorpromazine and propranolol are very effective inhibitors of MDA formation induced by thrombin, collagen and A23187, but they do not block conversion of exogenous arachidonate to MDA. This result indicates that these drugs do not affect the enzyme prostaglandin synthetase responsible for the initial steps in arachidonate conversion to PG endoperoxides and thromboxanes. Utilizing platelets whose phospholipids were prelabeled with 14C-arachidonate and then stimulated with thrombin or A23187 we have found that local anesthetics, chlorpromazine and propranolol block: (a) formation of 14C-thromboxane B2, 14C-prostaglandins and 14C-hydroxyacids (12-L-hydroxy-5,8,10,14-eicosatetraenoic acid and 12-L-hydroxy-5,8,10-heptadecatrienoic acid) from endogenous (phospholipid) 14C-archidonate; and (b) block the fall in the 14C-arachidonate content of platelet phospholipids, especially phosphatidyl choline. Local anesthetics, chlorpromazine and propranolol did not inhibit the conversion of 14C-arachidonate to l4C-l2-L-hydroxy-5,8,10,14-eicosatetraenoic acid by a soluble fraction from platelets containing lipoxygenase activity. These results conclusively demonstrate that local anesthetics, chlorpromazine and propranolol inhibit the activation of PLA2 by stimuli, which is the initial and rate-limiting step in arachidonate metabolism. To some extent the ability of local anesthetics, chlorpromazine and propranolol to inhibit platelet aggregation and the release reaction (secretion of ADP, serotonin, etc.) can undoubtedly be attributed to their effect on PLA2 activation which prevents formation of the aggregating agents PG endoperoxides and thromboxane A2. However, other actions of these drugs, notably their ability to block aggregation induced by thromboxane A2 and by other aggregating agents, under conditions which are independent of PG endoperoxide and thromboxane A2 production, indicates additional mechanisms of action which can prevent cellular adhesion and exocytosis. These other actions may be related to non-arachidonate mediated effects on Ca2+-dependent phospholipases, antagonism of Ca2+-dependent phenomena necessary for membrane adhesion and fusion, or interference with the process of intracellular Ca2+ release by stimuli. ACKNOWLEDGMENT We gratefully acknowledge the technical assistance of Mrs. Carol Fraser.

Characterization of Muscarinic Acetylcholine Receptors from Mouse Brain: Evidence for Regional Heterogeneity and Isomerization

Abstract: High affinity binding of tritium labeled N-methyl-4-piperidyl benzilate to homogenates from various regions of mouse brain is used to characterize the binding mechanism to specific muscarinic sites at 25°. Binding experiments at equilibrium reveal differences in the affinity of various muscarinic agonists and antagonists for the binding sites in the various regions: cortex, putamen, hippocampus, medulla pons, and cerebellum. The density of muscarinic binding sites is also different in these regions. The regional heterogeneity is further investigated by kinetic experiments which measure the rates and the mechanisms of association and dissociation of [3H]-N-methyl-4-piperidyl benzilate. These experiments suggest that while each region contains a homogeneous population of binding sites, the association and dissociation of the ligands do not follow a simple first order mechanism. The simplest model which fits the experimental findings and which is compatible with previously published models, consists of a fast binding step followed by a slow isomerization process of the receptor-ligand complex. ACKNOWLEDGMENTS Enlightening discussions on this subject with N. Zisapel, H. Weinstein and D. Michaelson, are gratefully acknowledged. Mrs. Ronit Galron provided excellent technical assistance.

Serotonin Receptor Binding Sites Affected Differentially by Guanine Nucleotides

Abstract: Guanosine triphosphate (GTP) and diphosphate (GDP) decrease the binding of the agonist [3H]serotonin (5-HT) to serotonin receptors in mammalian brain membranes. Binding of the antagonist [3H]spiroperidol is not affected by guanine nucleotides while the mixed agonist-antagonist [3H]lysergic acid diethylamide ([3H]LSD) is affected in an intermediate manner. GTP lowers the apparent affinity of [3H]5-HT for its receptors without changing the number of binding sites. Both the association and dissociation rates of [3H]5-HT are increased by GTP. The potency of 5-HT agonists in competing for [3H]5-HT but not [3H]LSD or [3H]spiroperidol binding sites is decreased by GTP. Competition by partial agonists and antagonists for [3H]5-HT binding sites is not affected by GTP.

Gonadotropin Releasing Hormone Stimulation of Cultured Pituitary Cells Requires Calcium

Abstract: The requirement for Ca2+ during luteinizing hormone (LH) release stimulated by gonadotropin-releasing hormone (GnRH) was studied in dispersed cultured rat pituicytes. With maximal GnRH stimulation, LH release required 1 mM Ca2+ for optimal stimulation, although basal production was not calcium dependent. Stimulated release was inhibited when cells were incubated in media lacking Ca2+, and by LaCl3 (a potent inhibitor of Ca2+ action) which inhibited GnRH-induced LH release in the presence of Ca2+. The requirement for Ca2+ is specific, since Mg2+ is not as effective, even at elevated concentrations. Two agents that block Ca2+ movement into and within cells, Ruthenium Red and D-600 (methoxy-verapamil), also blocked the release of LH from stimulated pituicytes. The present results demonstrate a specific dependence on calcium for GnRH stimulation of LH release from cultured gonadotrophs. Our observations satisfy some of the criteria require for a postulated role of Ca2+ in stimulus-secretion coupling in this system.

Superoxide radical as an intermediate in the oxidation of hydroxylamines by mixed function amine oxidase.

Abstract: Previous observations have shown that mixed function amine oxidase (MFAO), a flavo-protein, is responsible for the oxidation of hydroxylamines to nitroxides. We had assumed that the hydroperoxyflavin directly attacked the hydroxylamine giving the hydroxylamine oxide as an intermediate which rapidly decomposed to yield a nitroxide and a hydroxyl radical. A kinetic investigation showed that MFAO does not directly attack the hydroxylamine but suggested that MFAO releases an oxidizing agent that is responsible for hydroxylamine oxidation. The Haber-Weiss reaction, known to produce hydroxyl radicals, was shown not to be involved in the formation of nitroxide free radicals. It was found that superoxide is solely responsible for MFAO mediated nitroxide formation. ACKNOWLEDGMENTS We thank Dr. Dan. Ziegler, University of Texas, for his most generous gift of purified mixed function amine oxidase. We are grateful to Dr. Henry Fisher of the Environmental Protection Agency for the use of his Varian Associates E-9 spectrometer.

Poly (L-Lysine) and Poly (D-Lysine) Conjugates of Methotrexate: Different Inhibitory Effect on Drug Resistant Cells

Abstract: Conjugation of methotrexate to poly(L-lysine) markedly increases its cellular uptake and offers a new way to overcome drug resistance related to deficient transport. Conjugates using poly(L-lysines) of molecular weights varying from 3,100 to 130,000 have comparable effects on a drug resistant CHO cell line. Conjugates using poly(D-lysine), however, have no effect on either resistant or normal CHO cells. Both the L- and D-isomeric conjugates are taken up by cells in comparable fashion. Since even the biologically active conjugates are poor inhibitors of dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase; EC 1.5.1.3) in vitro, it is concluded that the conjugate must give rise in the cell to a pharmacologically active breakdown product. Such a product can be detected in cells exposed to the L-isomeric, but not in cells exposed to the D-isomeric conjugates. Excess of poly(L-lysine) only moderately decreases methotrexate-poly(L-lysine) uptake but markedly decreases its inhibitory effect on cell growth.

Anomalous Properties of [3H]Spiperone Binding Sites in Various Areas of the Rat Limbic System

Abstract: [3H]Spiperone, a neuroleptic/dopamine receptor ligand, binds with high affinity (Kd 0.15 nM) to a single specific site on rat corpus striatum membranes. The "specific" binding represents about 80% of the total binding and is displaced by dopamime, apomorphine, and stereospecifically by neuroleptics such as butaclamol and flupenthixol. However, in contrast to the striatum, only 30-40% of the binding of [3H]spiperone to limbic forebrain membranes is displaced stereospecifically by butaclamol or flupenthixol, whereas dopamine and certain spiperone analogues compete with high affinity for about 70% of the labeled sites. These additional sites are saturable, reversible, and of high affinity. Kinetic analysis of association and dissociation rates yields a Kd value (1.5 nM) in agreement with equilibrium saturation data for these sites. They also possess a precise distribution, with high amounts being found in the hippocampus, septum, and nucleus accumbens, but they are completely absent in areas such as the corpus striatum, olfactory tubercles and hypothalamus. Moreover, these [3H]spiperone binding sites show strict structure-affinity relationships in that only spirodecanone butyrophenone derivatives and dopamine are capable of displacing this binding with relatively high affinities. The results emphasize the complex nature of neuroleptic/dopamine "receptor" binding sites in brain and the need for precise definition of such "specific" binding sites.

Ligand specificity of state transitions in the cholinergic receptor: behavior of agonists and antagonists.

Abstract: Several cholinergic ligands, including a series of methylene-linked bisquaternary ammonium compounds, have been examined for their ability to induce a state transition in the membrane-associated cholinergic receptor where there is a slow increase in affinity for the associating ligand. All of the agonists tested, as well as certain antagonists, notably the metaphiic antagonists, convert the receptor to a higher affinity state. Moreover, the rate of receptor conversion appears to be independent of the ligands’ capacity to activate the receptor. By applying the two-state cyclic scheme used to explain desensitization to these affinity increases, the apparent binding constants of each ligand for the R and R’ receptor states were determined. Full agonists have a 300-fold higher affinity for the R’ state while the classical antagonists (i.e., d-tubocurarine and gallamine) have the same affinity for both receptor states. Apparent negative cooperativity is observed for the antagonists. Partial agonists, which are represented by the methylene-linked bisquaternary series have ratios of affinities for the two states which lie between these extremes. The metaphilic antagonists, which appear to be unique in situ in showing enhanced antagonism for receptors previously conditioned by agonist, will in themselves effect a conversion in receptor state. The structural specificity for the ligands’ capacity to effect a conversion in receptor state lends further support to the contention that the conversion from the low to high affinity state in vitro and receptor desensitization involve the same molecular transition.