Rank Order Of Inhibition Research Articles

Magnetite (Fe3O4) Nanoparticles Alleviate Growth Inhibition and Oxidative Stress Caused by Heavy Metals in Young Seedlings of Cucumber (Cucumis Sativus L)

Accumulation of heavy metals in the ecosystem and their toxic effects through food chain can cause serious ecological and health problems. In the present study, experiments were performed to understand how the addition of magnetite (Fe3 O4 ) nanoparticles reduces the toxicity caused by Cd, Pb, Cu, and Zn in cucumber plants. Plant growth parameters, lipid peroxidation, and antioxidant enzymes were measured in seedling samples treated with either metals or metals supplemented with Fe3 O4 to demonstrate the reduction in metal-induced oxidative stress conferred by Fe3 O4 . Results showed that the toxic effect of metals on seedling growth parameters can be arranged in the rank order of inhibition as follows: Cu > Cd > Zn > Pb. Exposure to metals significantly decreased the seedlings growth, the activities of superoxide dismutase (SOD) and peroxidases (POD), while the malondialdehyde (MDA) content significantly increased in cucumber seedlings. The reducing activity of nano-Fe3 O4 against heavy metals stresses was confirmed in this study by the decrease in MDA content. The correlation between the decrease of MDA concentration and the increase in SOD and POD activities in the presence of nano-Fe3 O4 suggest that the MDA reduction in the tested seedlings can result from the increased enzyme activity.

Effect of voriconazole and other azole antifungal agents on CYP3A activity and metabolism of tacrolimus in human liver microsomes

Azole antifungal agents are known to inhibit cytochrome P450 3A (CYP3A) enzymes. Limited information is available regarding the effect of voriconazole on CYP3A activity. We examined the effect of voriconazole on CYP3A activity in human liver microsomes as measured by the formation of 6β-hydroxytestosterone from testosterone. We also evaluated the interaction between voriconazole and tacrolimus, an immunosuppressive drug, using human liver microsomes. The effect of voriconazole on CYP3A activity and tacrolimus metabolism was compared to that of other azole antifungal agents.CYP3A4 activity and the metabolism of tacrolimus were measured in the absence and in the presence of various concentrations of voriconazole (0–1.43 mM), fluconazole (0–1.63 mM), itraconazole (0–14 µM) and ketoconazole (0–0.19 µM). At a concentration of 21.2 ± 15.4 µM and 29.8 ± 12.3 µM, voriconazole inhibited the formation of 6β-hydroxytestosterone from testosterone and the metabolism of tacrolimus by 50%, respectively.The rank order of inhibition of 6β-hydroxytestosterone formation from testosterone and the metabolism of tacrolimus, is ketoconazole > itraconazole > voriconazole > fluconazole.Our observations suggest that voriconazole at clinically relevant concentrations will inhibit the hepatic metabolism of tacrolimus and increase the concentration of tacrolimus more than two-fold. Close monitoring of the blood concentrations and adjustment in the dose of tacrolimus are warranted when transplant patients receiving tacrolimus are treated with voriconazole.

To B or not to B: Fat is the question

To B or not to B: Fat is the question

Aldosterone glucuronidation by human liver and kidney microsomes and recombinant UDP‐glucuronosyltransferases: Inhibition by NSAIDs

To characterize: i) the kinetics of aldosterone (ALDO) 18beta-glucuronidation using human liver and human kidney microsomes and identify the human UGT enzyme(s) responsible for ALDO 18beta-glucuronidation and ii) the inhibition of ALDO 18beta-glucuronidation by non-selective NSAIDs. Using HPLC and LC-MS methods, ALDO 18beta-glucuronidation was characterized using human liver (n= 6), human kidney microsomes (n= 5) and recombinant human UGT 1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15, 2B17 and 2B28 as the enzyme sources. Inhibition of ALDO 18beta-glucuronidation was investigated using alclofenac, cicloprofen, diclofenac, diflunisal, fenoprofen, R- and S-ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, S-naproxen, pirprofen and tiaprofenic acid. A rank order of inhibition (IC(50)) was established and the mechanism of inhibition investigated using diclofenac, S-ibuprofen, indomethacin, mefenamic acid and S-naproxen. ALDO 18beta-glucuronidation by hepatic and renal microsomes exhibited Michaelis-Menten kinetics. Mean (+/-SD) K(m), V(max) and CL(int) values for HLM and HKCM were 509 +/- 137 and 367 +/- 170 microm, 1075 +/- 429 and 1110 +/- 522 pmol min(-1) mg(-1), and 2.36 +/- 1.12 and 3.91 +/- 2.35 microl min(-1) mg(-1), respectively. Of the UGT proteins, only UGT1A10 and UGT2B7 converted ALDO to its 18beta-glucuronide. All NSAIDs investigated inhibited ALDO 18beta-G formation by HLM, HKCM and UGT2B7. The rank order of inhibition (IC(50)) of renal and hepatic ALDO 18beta-glucuronidation followed the general trend: fenamates > diclofenac > arylpropionates. A NSAID-ALDO interaction in vivo may result in elevated intra-renal concentrations of ALDO that may contribute to the adverse renal effects of NSAIDs and their effects on antihypertensive drug response.

Abstract A33: Superior efficacy of dietary 3,3’diindolylmethane (DIM) amongst several indoles to inhibit prostate cancer growth via induction of the p75NTR tumor suppressor protein

Abstract A33 A comparison of the indoles etodolac, indomethacin, 5-methylindole-3-acetic acid, indole-3-carbinol, 3,3’diindolylmethane (DIM) and the related ketorolac molecule showed superior efficacy of DIM, a dietary indole, to induce expression of the p75NTR tumor suppressor and inhibit survival of the PC-3 prostate cancer cell line. Each indole that induced p75NTR expression exhibited the same rank-order for inhibition of prostate cell survival. Comparison of the activity of DIM, the most potent of the indoles, between several cell lines, showed that DIM induced p75NTR at low concentrations in prostate cells (PC-3 , DU-145), followed by bladder (T24), with breast (MCF-7) and fibroblast cells (3T3) being the least responsive to DIM. DIM induction of p75NTR levels in the various cell lines occurred in the same rank-order as inhibition of cell survival. Since the consumption of cruciferous vegetables has been reported to reduce the risk of prostate cancer through the release of indole-3-carbinol (I3C) which is subsequently metabolized in the acidic environment of the stomach to DIM, the superior activity of DIM to induce expression of the p75NTR tumor suppressor protein and inhibit cell survival suggests a mechanism of action for the anticancer activity of DIM in the prevention of prostate cancer. In order to demonstrate a cause and effect between DIM and p75NTR inhibition of survival, we used a dominant negative of p75NTR to rescue DIM induced loss of cell survival. In order to investigate the mechanism of action by which p75NTR transduces a biochemical signaling cascade leading to cell death of prostate cancer cells we utilized knockdown with p38 MAPK siRNA to rescue DIM induction of p75NTR thereby implicating p38 MAPK in DIM induction of cell death. Moreover, DIM treatment induced phosphorylation of p38 MAPK within one minute, suggesting the target of DIM is proximal to p38 MAPK. Hence, these results suggest that the anti-cancer activity of cruciferous vegetables may be mediated in part by DIM and that the mechanism of action occurs, in part, by activation of the p38 MAPK pathway leading to expression of the p75NTR death receptor with subsequent apoptosis of prostate cancer cells. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A33.

Effect of O-superfamily conotoxin SO3 on synchronized spontaneous calcium spikes in cultured hippocampal networks

SO3 belongs to the O-superfamily of conotoxins and is known to have analgesic effects in experimental animals. In order to explore the mechanism of its potential pharmacological actions, the effect of SO3 on synchronized spontaneous calcium spikes was examined in cultured hippocampal networks by calcium imaging. Spontaneous oscillations of intracellular concentrations of calcium (Ca(2+)) in the form of waves and spikes are found in cultured hippocampal networks. Exposure to increasing concentrations of SO3 resulted in a progressive decrease in synchronized spontaneous calcium spikes. The higher concentrations (0.1 micromol/L and 1 micromol/L) of SO3 showed the strongest inhibition. The rank order of inhibition was 1 micromol/L > 0.1 micromol/L > 10 micromol/L > 0.01 micromol/L. This action of SO3 in reducing synchronized calcium spikes suggests a possible application for therapeutic treatment of epilepsy.

Characterization of the Tritium-Labeled Analog of L-threo-β-Benzyloxyaspartate Binding to Glutamate Transporters

L-Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Termination of glutamate receptor activation and maintenance of low extracellular glutamate concentrations are primarily achieved by glutamate transporters (excitatory amino acid transporters 1-5, EAATs1-5) located on both the nerve endings and the surrounding glial cells. To identify the physiological roles of each subtype, subtype-selective EAAT ligands are required. In this study, we developed a binding assay system to characterize EAAT ligands for all EAAT subtypes. We recently synthesized novel analogs of threo-beta-benzyloxyaspartate (TBOA) and reported that they blocked glutamate uptake by EAATs 1-5 much more potently than TBOA. The strong inhibitory activity of the TBOA analogs suggested that they would be suitable to use as radioisotope-labeled ligands, and we therefore synthesized a tritiated derivative of (2S,3S)-3-{3-[4-ethylbenzoylamino]benzyloxy}aspartate ([3H]ETB-TBOA). [3H]ETB-TBOA showed significant high-affinity specific binding to EAAT-transfected COS-1 cell membranes with each EAAT subtype. The Hill coefficient for the Na+-dependence of [3H]ETB-TBOA binding revealed a single class of noncooperative binding sites for Na+, suggesting that Na+ binding in the ligand binding step is different from Na+ binding in the substrate uptake process. The binding was displaced by known substrates and blockers. The rank order of inhibition by these compounds was consistent with glutamate uptake assay results reported previously. Thus, the [3H]ETB-TBOA binding assay will be useful to screen novel EAAT ligands for all EAAT subtypes.

IN VITRO P-GLYCOPROTEIN INHIBITION ASSAYS FOR ASSESSMENT OF CLINICAL DRUG INTERACTION POTENTIAL OF NEW DRUG CANDIDATES: A RECOMMENDATION FOR PROBE SUBSTRATES

Because modulation of P-glycoprotein (Pgp) through inhibition or induction can lead to drug-drug interactions by altering intestinal, central nervous system, renal, or biliary efflux, it is anticipated that information regarding the potential interaction of drug candidates with Pgp will be a future regulatory expectation. Therefore, to be able to utilize in vitro Pgp inhibition findings to guide clinical drug interaction studies, the utility of five probe substrates (calcein-AM, colchicine, digoxin, prazosin, and vinblastine) was evaluated by inhibiting their Pgp-mediated transport across multidrug resistance-1-transfected Madin-Darby canine kidney cell type II monolayers with 20 diverse drugs having various degrees of Pgp interaction (e.g., efflux ratio, ATPase, and calcein-AM inhibition). Overall, the rank order of inhibition was generally similar with IC(50) values typically within 3- to 5-fold of each other. However, several notable differences in the IC(50) values were observed. Digoxin and prazosin were the most sensitive probes (e.g., lowest IC(50) values), followed by colchicine, vinblastine, and calcein-AM. Inclusion of other considerations such as a large dynamic range, commercially available radiolabel, and a clinically meaningful probe makes digoxin an attractive probe substrate. Therefore, it is recommended that digoxin be considered as the standard in vitro probe to investigate the inhibition profiles of new drug candidates. Furthermore, this study shows that it may not be necessary to generate IC(50) values with multiple probe substrates for Pgp as is currently done for cytochrome P450 3A4. Finally, a strategy integrating results from in vitro assays (efflux, inhibition, and ATPase) is provided to further guide clinical interaction studies.

Toxicity comparison of some possible toxic metals (Cd, Cu, Pb, Se, Zn) on young seedlingsof Sinapis alba L.

The chronic ecotoxicological effects of Cd, Cu, Pb, Se and Zn were determined within this study. Mustard seedlings (Sinapis alba L.) were used as a model subject for the tests. The phytotoxicity was determined through the root growth inhibition and photosynthetic pigments production (chlorophyll a, b, total carotenoids). The metal accumulation in the roots and cotyledons was also determined using the AAS methods. On the basis of IC<sub>50</sub> values and their 95% confidence intervals (CI) for the root elongation of S. alba seedlings, metals can be arranged in the rank order of inhibition as follows: Cu > Se > Cd > Zn >> Pb. When the photosynthetic pigments production was determined, Se demonstrated the lowest inhibitory effect on all pigment types and Pb did the highest. The accumulated amount of metals from hydroponic solution into the roots and cotyledons decreased in the order Cd > Zn > Se > Pb > Cu.

Inhibition of human cytochrome P450 2E1 by nicotine, cotinine, and aqueous cigarette tar extract in vitro.

Cigarette smoke is a complex mixture containing, among other chemicals, pyridine alkaloids and N-nitrosamines. Carcinogenic tobacco-specific N-nitrosamines, N-nitrosodimethylamine (NDMA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are both activated by cytochrome P450 (CYP) 2E1 in rats. Previous reports indicate that nicotine and the main nicotine metabolite, cotinine, reduce the mutagenicity of both NNK and NDMA in Salmonella typhimurium. To study the mechanism of this effect, we examined inhibition of CYP 2E1 activity, as assessed by p-nitrophenol (pNP) hydroxylation, by nicotine, cotinine, and an aqueous cigarette tar extract (ACTE) in human 2E1-expressing microsomes. At all substrate concentrations (0-1.25 mM) nicotine was a significantly more potent inhibitor of CYP 2E1 activity compared to cotinine. Estimated Ki values for nicotine and cotinine (both at 10 mM) were 13 mM (2 mg/ml) and 308 mM (54 mg/ml) respectively. The Ki for ACTE was 0.2 mg/ml at a concentration of 0.32 mg/ml. This rank order for inhibition was also seen when the data was expressed as IC(50). When compared on a mass/vol basis, ACTE was a significantly more potent CYP 2E1 inhibitor relative to nicotine and cotinine. Double-reciprocal plots indicated that nicotine and ACTE inhibited by a competitive, while cotinine inhibited CYP 2E1 by an uncompetitive mechanism. Although the contribution of nicotine to ACTE-mediated 2E1 inhibition is probably modest, pyridine alkaloid-mediated CYP 2E1 inhibition is a possible mechanism for the observed inhibition of NNK and NDMA mutagenicity by nicotine and cotinine in vitro.

Differential binding of triglyceride-rich lipoproteins to lipoprotein lipase

In comparison to very low density lipoprotein (VLDL), chylomicrons are cleared quickly from plasma. However, small changes in fasting plasma VLDL concentration substantially delay postprandial chylomicron triglyceride clearance. We hypothesized that differential binding to lipoprotein lipase (LPL), the first step in the lipolytic pathway, might explain these otherwise paradoxical relationships. Competition binding assays of different lipoproteins were performed in a solid phase assay with purified bovine LPL at 4°C. The results showed that chylomicrons, VLDL, and low density lipoprotein (LDL) were able to inhibit specific binding of 125I-labeled VLDL to the same extent (85.1% ± 13.1, 100% ± 6.8, 90.7% ± 23.2% inhibition, P = NS), but with markedly different efficiencies. The rank order of inhibition (Ki) was chylomicrons (0.27 ± 0.02 nm apoB) > VLDL (12.6 ± 3.11 nm apoB) > LDL (34.8 ± 11.1 nm apoB). By contrast, neither triglyceride (TG) liposomes, high density lipoprotein (HDL), nor LDL from patients with familial hypercholesterolemia were efficient at displacing the specific binding of 125I-labeled VLDL to LPL (30%, 39%, and no displacement, respectively). Importantly, smaller hydrolyzed chylomicrons had less affinity than the larger chylomicrons (Ki = 2.34 ± 0.85 nm vs. 0.27 ± 0.02 nm apoB respectively, P < 0.01). This was also true for hydrolyzed VLDL, although to a lesser extent. Chylomicrons from patients with LPL deficiency and VLDL from hypertriglyceridemic subjects were also studied.▪Taken together, our results indicate an inverse linear relationship between chylomicron size and Ki whereas none was present for VLDL. We hypothesize that the differences in binding affinity demonstrated in vitro when considered with the differences in particle number observed in vivo may largely explain the paradoxes we set out to study.—Xiang, S-Q., K. Cianflone, D. Kalant, and A. D. Sniderman. Differential binding of triglyceride-rich lipoproteins to lipoprotein lipase. J. Lipid Res. 1999. 40: 1655–1662.

Identification of Inhibitors of Prohormone Convertases 1 and 2 Using a Peptide Combinatorial Library

A positional scanning synthetic peptide combinatorial library containing approximately 52 million hexapeptides was used to identify potential inhibitory peptides for recombinant mouse prohormone convertase 1 (PC1) and PC2 and to provide information on the specificity of these enzymes. The library surveys revealed that a P6 Leu, a P4 Arg, a P2 Lys, and a P1 Arg were most inhibitory against PC1, and a P6 Ile and a P4 Arg were most inhibitory against PC2. Using information derived from the library surveys, hexapeptide sets were synthesized and screened for inhibition of PC1 and PC2. The data obtained revealed the preference of both enzymes for a P3 Val. At P5, many substitutions were well tolerated. PC1 and PC2 proved to differ mainly in the selectivity of their S6 subsites. In PC1, this subsite displayed a strong preference toward occupation by Leu; the Ki value for peptide Ac-Leu-Leu-Arg-Val-Lys-Arg-NH2 was 28 times lower than that for peptide Ac-Ile-Ile-Arg-Val-Lys-Arg-NH2. In contrast, PC2 discriminated little between Leu and Ile at P6, as evidenced by the small (1.5-fold) difference in Ki values for these two peptides. Several hexapeptides synthesized as a result of the screen were found to represent potent inhibitors of PC2 (with Ki values in the submicromolar range) and, particularly, of PC1 (with Ki values in the low nanomolar range). The most potent inhibitor, Ac-Leu-Leu-Arg-Val-Lys-Arg-NH2, proved to be the same peptide for both enzymes and inhibited PC1 and PC2 in a competitive, fast-binding manner with Ki values of 3.2 and 360 nM, respectively. The four most potent peptide inhibitors of PC1 and PC2 were also tested against soluble human furin and found to exhibit a different rank order of inhibition; for example, Ac-Leu-Leu-Arg-Val-Lys-Arg-NH2 was 440-fold less potent against furin than against PC1, with a Ki of 1400 nM.

Functional characterization of ion permeation pathway in the N-type Ca2+ channel.

Multiple types of high-voltage-activated Ca2+ channels, including L-, N-, P-, Q- and R-types have been distinguished from each other mainly employing pharmacological agents that selectively block particular types of Ca2+ channels. Except for the dihydropyridine-sensitive L-type Ca2+ channels, electrophysiological characterization has yet to be conducted thoroughly enough to biophysically distinguish the remaining Ca2+ channel types. In particular, the ion permeation properties of N-type Ca2+ channels have not been clarified, although the kinetic properties of both the L- and N-type Ca2+ channels are relatively well described. To establish ion conducting properties of the N-type Ca2+ channel, we examined a homogeneous population of recombinant N-type Ca2+ channels expressed in baby hamster kidney cells, using a conventional whole cell patch-clamp technique. The recombinant N-type Ca2+ channel, composed of the alpha1B, alpha2a, and beta1a subunits, displayed high-voltage-activated Ba2+ currents elicited by a test pulse more positive than -30 mV, and were strongly blocked by the N-type channel blocker omega-conotoxin-GVIA. In the presence of 110 mM Ba2+, the unitary current showed a slope conductance of 18.2 pS, characteristic of N-type channels. Ca2+ and Sr2+ resulted in smaller ion fluxes than Ba2+, with the ratio 1.0:0. 72:0.75 of maximum conductance in current-voltage relationships of Ba2+, Ca2+, and Sr2+ currents, respectively. In mixtures of Ba2+ and Ca2+, where the Ca2+ concentration was steadily increased in place of Ba2+, with the total concentration of Ba2+ and Ca2+ held constant at 3 mM, the current amplitude went through a clear minimum when 20% of the external Ba2+ was replaced by Ca+2. This anomalous mole fraction effect suggests an ion-binding site where two or more permeant ions can sit simultaneously. By using an external solution containing 110 mM Na+ without polyvalent cations, inward Na+ currents were evoked by test potentials more positive than -50 mV. These currents were activated and inactivated in a kinetic manner similar to that of Ba2+ currents. Application of inorganic Ca2+ antagonists blocked Ba2+ currents through N-type channels in a concentration-dependent manner. The rank order of inhibition was La3+ >/= Cd2+ >> Zn2+ > Ni2+ >/= Co2+. When a short strong depolarization was applied before test pulses of moderate depolarizing potentials, relief from channel blockade by La3+ and Cd2+ and subsequent channel reblocking was observed. The measured rate (2 x 10(8) M-1 s-1) of reblocking approached the diffusion-controlled limit. These results suggest that N-type Ca2+ channels share general features of a high affinity ion-binding site with the L-type Ca2+ channel, and that this site is easily accessible from the outside of the channel pore.

Subcellular localization and molecular pharmacology of distinct populations of [3H]‐AMPA binding sites in rat hippocampus

1. The subcellular distributions of [3H]-alpha-amino-3-hydroxy-5- methylisoxazolepropionate ([3H]-AMPA) and [3H]-kainate binding sites in rat hippocampus were investigated by cell fractionation techniques. 2. Two major populations of [3H]-AMPA sites were detected with the majority of binding located intracellularly in the microsomal (P3) fraction. Most of the remaining sites were in the synaptosomal membrane fraction but some were also present in the nuclear fraction. In contrast, essentially all of the [3H]-kainate binding sites were in the synaptosomal membrane fraction. 3. Saturation binding analysis yielded KD and Bmax values for [3H]-AMPA of 147 nM and 2.6 pmol mg-1 protein respectively for the synaptosomal membrane-associated sites and 129 nM and 5.3 pmol mg-1 protein respectively for the microsomal sites. 4. Both main populations of [3H]-AMPA sites displayed the same rank order of inhibition by competitive ligands, the apparent Mr values of GluR1 subunits were equivalent, suggesting the same degree of post-translational modification and the hydrodynamic properties of the receptor complexes were identical. 5. These data are consistent with the hypothesis that the movement of AMPA receptors between cellular compartments in the postsynaptic neurone could constitute one mechanism underlying long-term potentiation in the hippocampus.

Inhibition Kinetics of Theophylline Metabolism by Mexiletine and Its Metabolites.

To further characterize the mode of drug interaction between theophylline (TP) and mexiletine (ME), in vitro kinetic studies were carried out using rat liver microsomes and 9000 x g supernatant. The kinetic study revealed that the Km value and Vmax/Km ratio for the metabolic conversion of TP to 1,3-dimethyluric acid (1,3-DMU) were the second lowest and the highest, respectively, of four metabolic pathways. Thus, the rank of efficiency of the oxidative metabolism by microsomal cytochrome P-450 (P-450) isozymes was TP to 1,3-DMU > TP to 1-methylxanthine (1-MX) > TP to 3-MX > 1,3-DMU to 1-methyluric acid, suggesting that the isozyme metabolizing TP would have a higher affinity for the oxidation at the 8-position in TP molecules than at the 1- and 3-positions. Lineweaver-Burk plots showed that the conversion of TP to 3-MX and to 1,3-DMU was inhibited competitively by ME and its metabolites, and that the pathway of TP to 1-MX was inhibited noncompetitively. In consideration of the Ki values calculated, it seems probable that deamino-p-hydroxy ME (DApHME) might be the most potent inhibitor of the metabolic pathways of TP, and that the rank order of inhibition is approximately DApHME > p-hydroxy ME > deamino-hydroxymethyl ME > ME > hydroxymethyl ME, with some exceptions. The mechanism of the interaction between TP and ME is probably due to the metabolic antagonism in the liver, and TP, ME and their metabolites share some of the same metabolic pathways, mediated by P-450 isozymes.

Otenzepad shows two populations of binding sites in human gastric smooth muscle.

Cholinergic agonists and antagonists frequently used for gastrointestinal motility disorders often produce adverse effects. A possible explanation for this is the presence of similar muscarinic receptor subtypes on smooth muscle from different gastrointestinal organs. The aim of this study was to characterize muscarinic receptor subtypes in human gastric smooth muscle with receptor binding methods. N-[3H]Methylscopolamine ([3H]NMS) saturation experiments showed a homogeneous population of noninteracting binding sites (KD = 0.76 +/- 0.07 nM, Bmax = 46.94 +/- 3.69 fmol/mg of tissue protein, nH = 0.99 +/- 0.01). The rank order of inhibition of [3H]NMS binding by nonlabelled compounds was atropine >> otenzepad >> pirenzepine. Atropine and pirenzepine bound to a homogeneous population of binding sites. The inhibition of [3H]NMS binding by otenzepad showed two populations of receptors (nH < 1, p < 0.01), whose apparent Ki1 of 298 +/- 40 nM and apparent Ki2 of 3.463 +/- 0.62 mM were similar to those reported for the M2 and M3 muscarinic receptor subtypes. The M2 subtype was the more abundant of the two, representing 79.12 +/- 5.48% of the total population. We conclude that two muscarinic receptor subpopulations similar to the M2 and M3 subtypes are present in human gastric smooth muscle and that the M2-like receptor is the more abundant of the two.

Occupancy of P 2 purinoceptors with unique properties modulates the function of human platelets

This report demonstrates that plateletes possess P 2 purinoceptors with unique properties that distinguish them from the ADP (P 2T) receptor. Extracellular ATP, and its poorly hydrolyzable analogues, inhibit collagen- and U46619 (a thromboxane mimetic)-induced platelet aggregations. Adenosine deaminase was without effect on ATP action while reversing the inhibitory effect of adenosine. A unique aspect of the P 2 receptor is the sensitivity to UTP and CTP and insensitivity to GTP. The rank order of inhibition by βγ-methylene ATP, αβ-methylene ATP > ATP indicates that a P 2x-like receptor is present on the platelet membrane. This conclusion is further supported by the nearly complete desensitization to ATP by pre-exposure of platelets to αβ-methylene-ATP. However, unlike previously described P 2x purinoceptors, the inhibition of platelet aggregation by extracellular ATP appears to result, at least in part, from the ATP-induced increase of intracellular cyclic AMP levels apparently coupled through a G s protein. The combined addition of iloprost (0.14 to 1.39 nM) and ATP (18 μM) or ATP (20–40 μM) and the phosphodiesterase inhibitor theophylline (0.5 to 1 mM) synergistically inhibited platelet aggregation implying a common interactive site with adenylate cyclase. This is further substantiated by the ability of the adenylate cyclase inhibitor, 2′,5′-dideoxyadenosine, to abrogate the inhibitory effects of ATP. The protein kinase A (PKA) inhibitor H1004 blocks ATP inhibition of platelet aggregation while the protein kinase C inhibitor H7 did not. This implies that the generation of cyclic AMP, with the subsequent activation of PKA and phosphorylation of selected proteins is required, in part, for the action of ATP.

Inhibition of interleukin-1-alpha-induced nitric oxide synthase in vascular smooth muscle and full reversal of interleukin-1-alpha-induced hypotension by N omega-amino-L-arginine.

Interleukin-1-alpha (IL-1) is a cytokine with potentially therapeutic immunoproliferative and tumoricidal activities. Preliminary clinical studies suggest that use of IL-1 may be restricted by dose-limiting hypotension. The purpose of this study was to investigate the role of nitric oxide (NO.) as a possible mediator of this hypotension. Cytokine-treated rat aortic smooth muscle cells were assayed for nitrite production, a stable breakdown product of nitric oxide. Nitric oxide synthase from smooth muscle cells was partially characterized in cytosol preparations using a novel Fe(2+)-myoglobin method to test for nitric oxide production. To determine the role of NO. on the immunorestorative and antineoplastic activity of IL-1, N omega-amino-L-arginine (NAA) or N omega-monomethyl-L-arginine (NMA), inhibitors of nitric oxide synthase, were added to either cultures of IL-1-dependent T cells or A375 melanoma cells exposed to IL-1. To investigate the effects of NAA in vivo, pentobarbital anesthetized dogs, which were made hypotensive by administration of IL-1, received a single intravenous bolus dose of NAA. The effects of NAA were then reversed by the administration of L-arginine. Our results show that cultured IL-1-activated rat aortic smooth muscle cells synthesize nitric oxide, a potent vasodilator. Induction of nitric oxide synthase is augmented by interferon-gamma and blocked by IL-1 receptor antagonist and by inhibitors of RNA or protein synthesis. Nitric oxide synthesis by IL-1-activated smooth muscle cells is inhibited by NAA, NMA, and N omega-nitro-L-arginine (NNA) with ED50 (i.e., effective dose for 50% inhibition) values of 20, 60, and 1000 microM, respectively; this rank order of inhibition is characteristic of an agonist-unregulated, inducible isoform of nitric oxide synthase. In smooth muscle cells, inhibition of NO. synthesis by NAA is reversed by excess L-arginine. Consistent with the induction of unregulated NO. synthesis in vascular smooth muscle in vivo, administration of IL-1 (50 micrograms/kg) to dogs caused a 33.5% decrease in systemic vascular resistance and a 28% decrease in blood pressure within 3 hours. Subsequent administration of NAA (20 mg/kg) rapidly and completely reversed the hypotension and increased systemic vascular resistance; these effects of NAA were reversed by L-arginine. Neither the immunoproliferative nor the tumoricidal activity of IL-1 was diminished by NAA. Our results indicate that (a) vascular smooth muscle is a likely source as well as a target of IL-1-induced NO. synthesis, causing vasodilatation and hypotension, (b) nitric oxide synthase inhibitors can fully reverse this hypotension, and (c) the therapeutically useful properties of IL-1 are not diminished by nitric oxide synthase inhibitors. Administration of inhibitors of nitric oxide synthase can reverse the pathological cardiovascular effects of IL-1 at concentrations that do not interfere with the potentially useful immunoproliferative or tumoricidal effects of this cytokine. In the context of the current clinical trials of IL-1, this finding would represent a very significant advantage.

Evidence for more than One Binding Site for Sulfonylureas in Insulin-secreting Cells

Specific binding of both [3H]glibenclamide and [3H]gliquidone has been observed in a particulate fraction of insulin-secreting rat tumour (RIN m5F) cells. The binding of both the labels was time-dependent, of high affinity (including a low affinity binding site), saturable and reversible. The rank order of inhibition of [3H]glibenclamide binding was glibenclamide greater than gliquidone greater than AG-EE 388 = AG-EE 86 = AG-EE 319 greater than AG-EE 436 (AG coded drugs are benzoic acid derivatives which lack the sulfonylurea moiety of sulfonylureas). The Kds of high affinity binding for glibenclamide and gliquidone were 0.08 and 1.3 nM, respectively. When [3H]gliquidone was used as the labelled compound this rank order of binding and the affinities of drugs were different, e.g. glibenclamide was less potent than gliquidone. The Kd values of high affinity binding to the [3H]gliquidone binding site were 810 and 79 nM with respect to glibenclamide and gliquidone. The binding site labelled by [3H]gliquidone, in contrast to that labelled by [3H]glibenclamide, was not able to discriminate between the two enantiomers AG-EE 319 and AG-EE 436. The data indicate that there are different binding sites for glibenclamide and gliquidone in RIN m5F cells. In extension to data of other groups it is speculated that there exists more than one specific binding site for sulfonylureas and other related compounds, e.g. benzoic acid derivatives and that sulfonylureas behave differently not only in quantitative but in qualitative terms as well.

Inhibition of histamine N-methyltransferase (HNMT) in vitro by neuromuscular relaxants

There have been reports of hypotension and flushing following vecuronium administration. The etiology of these symptoms, which are similar to those of histamine release, is not clear. The steroidal neuromuscular relaxants (NMRs), unlike muscle relaxants structurally similar to curare, have been shown not to cause histamine release after the administration of typical clinical doses. Histamine levels in plasma reflect a balance between release and catabolism. In humans, histamine N-methyl-transferase (HNMT) is the enzyme primarily degrading for histamine. Therefore, we performed in vitro kinetic studies of purified HNMT to determine the effects of the steroidal and curare-like NMRs and also of gallamine on histamine catabolism. We demonstrated that all NMRs tested were inhibitors of HNMT in vitro. The inhibition was competitive with respect to the cosubstrate S- adenosyl-L-[ 3 H-methyl] methionine, and noncompetitive with respect to histamine. The rank order of inhibition was vecuronium>pancuronium>gallamine> d-tubocurarine > metocurine > atracurium > pipecuronium, with K i values ranging from 1.2 to 44.8 μM. Our data suggest that HNMT-based radioenzymatic assays for histamine should be susceptible to inhibition by concurrent use of NMRs, particularly vecuronium.