THE ELEVATION OF CEREBRAL HISTAMINE-N-AND CATECHOL-O-METHYL TRANSFERASE ACTIVITIES BY l-METHIONINE-dl- SULFOXIMINE1

Abstract— The administration of the convulsant, l -methionine-dl-sulfoximine (MSO), increased histamine N-methyl transferase (E.C. 2.1.1.8) (HMT) activity in rat and mouse brain and, to a lesser extent, catechol-O-methyl transferase (E.C. 2.1.1.6) (COMT) activity in rat brain. The duration of this effect was shortened by co-administration of l -methionine. The increased HMT activity was seen in 5 or 7 rat brain regions tested. l -Methionine administration had no effect on the activity of either enzyme. Partially purified HMT preparations from rat or guinea-pig brain exhibited no alterations in activity after the in vitro addition of MSO or l -methionine over a wide range of histamine and S-adenosyl-l -methionine concentrations. Rat brain COMT was equally unaffected by MSO and l -methionine. The in vitro inhibition of HMT and COMT by S-adenosyl-l -homocysteine was the same whether tested on preparations derived from MSO-treated or control animals. The data are discussed with respect to the possible involvement of aberrant methylation processes in the MSO-induced seizure.     

Rat Kidney Histamine N-Methyltransferase: Purification and Partial Characterization

Abstract

Histamine N-methyltransferase (HMT, EC 2.1.1.8) was purified 8,420-fold In 44% yield from rat kidney. The basic steps in the purification included differential centrlfugation, calcium phosphate adsorption, DEAE cellulose chromatography, and affinity chromatography on an S-adenosylhomocysteine-agarose matrix. The resulting protein was homogeneous as determined by gel electrophoresis and was stable for at least five months at ?80°C. The apparent molecular weight of the enzyme was found to be 31,500 as determined by gel filtration through Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Isoelectric point of the enzyme was determined to be 5.4. The K_m's for histamine and S-adenosyl-L-methionine were 12.4 ± 1.3 μM and 10.2 ±0.5 μM, respectively. When S-adenosyl-L-methionlne was the variable substrate, the K₁'s for S-adenosyl-L-homocysteine and S-adenosyl-D-homocys-teine were 31.9 ± 3.4 μM and 32.0 ± 3.5 μM, respectively. When histamine was the variable substrate, the K₁ for S-adenosyl-L-homocysteine was 11.8 ± 0.6 μM. Comparison of physico-chemical and catalytic properties of the rat kidney and the guinea pig enzymes suggest that these proteins have similar structural and catalytic characteristics.     

Ovostatin,an endogenous trypsin inhibitor of sea urchin eggs: Purification and characterization of ovostatin from eggs of the sea urchin,Strongylocentrotus intermedius

A trypsin inhibitor, termed ovostatin, has been purified approximately 265-fold with 82% yield, from unfertilized eggs of the sea urchin Strongylocentrotus intermedius, using trypsin coupled Sepharose 4B as an affinity column for chromatography. The isolated ovostatin is homogeneous in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 20K–21.5K. Ovostatin inhibits preferentially trypsin-like endogenous protease purified from the eggs of the same species and bovine pancreatic trypsin and also bovine pancreatic chymotrypsin. Values of IC₅₀ (amount causing 50% inhibition of enzymes) for trypsin-like protease purified from eggs of the same species, bovine pancreatic trypsin, and bovine pancreatic chymotrypsin, are 0.91 ± 0.13 μg/ml (4.55 ± 0.65 × 10^?8 M), 3.0 ± 0.28 μg/ml (1.5 ± 0.14 × 10^?7 M), and 4.8 ± 0.2 μg/ml (2.4 ± 0.1 × 10^?7 M), respectively, in the experimental condition used. Kinetic studies indicate that ovostatin is a noncompetitive inhibitor of trypsin. The inhibitor is relatively heat labile. NaCl (0.025–0.01 M) enhances the inhibitor activity, whereas KCl is inhibitory. Ovostatin requires a low concentration of Ca²⁺ for activity. The activity is higher in unfertilized eggs than in fertilized eggs; total activity and specific activity in unfertilized eggs is about 1.67-fold and 1.85-fold higher than those in fertilized eggs, respectively. We believe that ovostatin may regulate the function of the cortical granule protease and other trypsin-like proteases that are activated in sea urchin eggs during fertilization.     

Identification, Development, and Regional Distribution of Ribonucleotide Reductase in Adult Rat Brain

The development and regional distribution of ribonucleotide reductase (EC 1.17.4.1) were determined in rat brain. Ribonucleotide reductase was partially purified by ammonium sulfate fractionation (20-40% saturation). Enzyme activity was measured by a specific radiochemical assay. This method involved the reduction of [¹⁴C]cytidine diphosphate (CDP) to [¹⁴C]deoxy-cytidine diphosphate with subsequent hydrolysis and separation of the product ([¹⁴C]deoxycytidine) from substrate ([¹⁴C]cytidine) by Dowex-1-borate ion-exchange chro-matography. The specific activity of ribonucleotide reductase in whole brain of newborn rats was 3.78 ± 0.55 units (pmol/h)/mg protein (SEM; n = 6) and declined to 0.17 ± 0.01 units/mg protein (n = 7) at 10-12 weeks of age, with a further decline to 0.11 ± 0.01 units/mg protein (n = 3) at 1 year. Ribonucleotide reductase activity in rat liver decreased from 4.58 ± 0.62 units/mg protein (n = 3) in newborn animals to 0.06 ± 0.01 units/mg protein (n = 7) at 10-12 weeks and was present at trace levels at 6 months of age. The decline in specific activity with age was not due to a change in the K_m for CDP. The K_m for CDP in brain of newborn and adult rats was 80-90 μM. In 10- to 12-week-old rats, the specific activity of ribonucleotide reductase was similar in the various regions of the brain tested except for the brainstem, which had 50% lower specific activity than the whole brain. These results indicate that ribonucleotide reductase activity is present and widely distributed in adult rat brain.     

SUBUNIT STRUCTURE AND KINETIC PROPERTIES OF 4-AMINOBUTYRATE-2-KETOGLUTARATE TRANSAMINASE PURIFIED FROM MOUSE BRAIN

Abstract— The effects of divalent metal ions, sulfhydryl reagents, carbonyl trapping reagents, substrate analogs, and organic solvents on purified mouse brain 4-aminobutyrate-2-ketoglutarate transaminase (EC 2.6.1.19) and the subunit structure of this enzyme were studied. Of the metal ions tested, Hg²⁺ was found to be the most potent inhibitor inhibiting the enzyme 50 percent at a concentration of 0-7 μM. The order of decreasing inhibitory potency for the divalent metal ions was: Hg²⁺± Cd²⁺± Zn²⁺± Cu²⁺± Co²⁺± Ba²⁺± Sr²⁺± Ni²⁺± Mn²⁺± Ca²⁺± Mg²⁺. p-Chloromercuribenzoale was the most potent inhibitor among the sulfhydryl reagents tested inhibiting the enzyme to the extent of 50 per cent at 0-5 μM 3-Mercaptopropionic acid was found to be a competitive inhibitor for GABA and non-competitive for 2-ketoglutarate. The K_i, value was estimated to be 13 μM. Aminooxyacetic acid was the most potent inhibitor of the carbonyl trapping agents with a K, value of 0-06 μM. being competitive with GABA and non-competitive with 2-ketoglutarate. Hydroxylamine and hydrazine were the next most potent compounds in this group. Of a series of substrate analogs and metabolites tested, only acetic acid, propionic acid, butyric acid, glutamic acid, adipic acid, pimelic acid and 2-ketoadipic acid inhibited the enzyme to a significant extent. Dioxan inhibited the enzyme 50 per cent at a concentration of 5 per cent (v/v) whereas methanol and ethanol only inhibited 5-10 per cent at 10 per cent (v/v) concentration. A spectrum of the native enzyme at pH 7-2 showed maxima at 278 nm. 330 nm and 411 nm. Treatment of the enzyme with aminooxyacetic acid or 3-mercaptopropionic acid caused the maximum at 411 nm to disappear. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the enzyme revealed two protein bands. The molecular weights of these two subunits were determined to be 53.000 and 58,000, respectively.     

Inhibitors of Histamine Methylation in Brain Promote Formation of Imidazoleacetic Acid, Which Interacts with GABA Receptors

Abstract: In brain, the precursor of imidazoleacetic acid (IAA), a GABA_A agonist but a GABA_C antagonist, is not known. In the periphery, IAA derives from oxidation of histamine. But in brain, histamine is thought to be metabolized solely by histamine methyltransferase (HMT), forming tele -methylhistamine (t-MH) and tele -methylimidazoleacetic acid (t-MIAA). We showed that [³H]histamine (intracerebroventricularly) could be converted to IAA in brains of rats, a process increased by inhibition of HMT. This demonstrated that brain can oxidize histamine and suggested that endogenous histamine might also be oxidized if HMT activity were reduced. We examined, in rat cerebral cortex, effects of the following HMT inhibitors (mg/kg i.p.): metoprine (10), tacrine (10), velnacrine (10, 30), and physostigmine (1, 2). Tacrine was a potent inhibitor ( K _i∼ 22 n M ). To measure histamine in tissue that contained HMT inhibitors, we developed a gas chromatography-mass spectrometry method. After 2 h, all drugs reduced endogenous levels of t-MH and t-MIAA and increased levels of histamine and IAA. Our results show that inhibition of HMT promotes oxidation of histamine in brain, probably by shunting histamine to an alternative metabolic pathway. Formation of IAA provides a novel interaction between histaminergic and GABAergic systems in brain. Accumulation of IAA should be considered when inhibitors of HMT are used to probe brain histamine function.     

METHYLASES OF MYELIN BASIC PROTEIN AND HISTONE IN RAT BRAIN

—The two enzymes methylating myelin basic protein and histone were purified 170- and 250-fold respectively from the cell sap fraction of rat brain. These enzymes methylated only arginine residues of the two proteins. The enzyme activities were present in all organs tested. Testis has the highest, brain a moderate and liver the lowest activity. Most of the activities were present in the cell sap fraction in brain, liver and testis. Methylation of myelin basic protein and histone was examined in both the cell sap and solubilized nuclear fraction of rat brain during life span after birth. The myelin basic protein methylating activity in the cell sap fraction increased during myelination. Histone methylase from the nuclear fraction was highest at birth and dropped rapidly thereafter. The other activities remained unchanged. The natural occurrence of N^G-mono- and N^G,N^G-dimethylarginine residues in histones prepared from rabbit liver was demonstrated.     

Regionaland Interspecies Differences in Brain Progesterone Metabolism

Metabolites of [³H]progesterone were studied in slices prepared from different brain regions of male rat, mouse, and monkey. The major metabolites were 5α-dihydroprogesterone (5α-DHP) and 3α,5α-tetrahydroprogesterone (3α,5α-THP) in rat brain slices, 5α-DHP and 20α- dihydroprogesterone (20α-DHP) in mouse brain slices, and 20α-DHP in monkey brain slices. In rat olfactory bulb slices, 5α-DHP represented 25.2 ± 3.3% of total radioactivity and 3α,5α-THP 17.5 ± 2.8%, whereas in rat medulla oblongata slices, 5α-DHP was 31.3 ± 3.5% and 3α,5α- THP 5.4 ± 1.5% of total radioactivity. In slices from other rat brain regions, both metabolites represented 12–20% of total radioactivity.-The highest metabolite content in mouse brain was also detected in olfactory bulb slices, where 5α-DHP represented 16.6 ± 4.6% and 20α-DHP 9.5 ± 2.3% of total radioactivity. In cortical and corpus callosum slices of monkey brain, 26.8 ± 4.4% and 2.4 ± 0.5% of total radioactivity, respectively, were converted to 20α-DHP, and less than 3% of total radioactivity could be attributed to any of the other metabolites detected. The 3α,α-THP content in both rat and monkey brain was below 1 nM, but increased in rat brain to 6.7 ± 2.5 nM after electroshock. Endogenous 3α,5α-THP might play an important role in the regulation of rat behavior through the modulation of GABA action on the GABA_A receptor. The significant interspecies differences in the brain progesterone metabolism should be considered in evaluating the functional role of neurosteroids in various species.     

Suppression of histamine-induced relaxation of rat aorta and calcium signaling in endothelial cells by two-pore channel blocker trans-NED19 and hydrogen peroxide

The blocker of two-pore channels trans-NED19 and hydrogen peroxide were found to inhibit histamine-induced relaxation of rat aorta. The degree of inhibition depended on histamine concentration. The relaxation in response to 1 µM histamine of rat aorta preconstricted with 30 mM KCl, serotonin, or endothelin-1, was completely abolished by 30 µM trans-NED19. On the other hand, trans-NED19 decreased the relaxation of the aorta in the presence of 10 µM histamine only by 2.1to 2.4-fold, and there was almost no inhibition by trans-NED19 of the relaxation induced by 100 µM histamine. Relaxation of preconstricted with serotonin aorta in response to 10 and 100 µM histamine was reduced by hydrogen peroxide (200 µM) by 10and 2.5-fold, respectively. Suppression of aorta relaxation by trans-NED19 and H₂O₂ correlated with their inhibitory effect on the histamine-induced increase in the cytoplasmic free calcium concentration in human umbilical vein endothelial cells. With the use of a fluorescent probe LysoTracker, the cis-NED19 binding sites were demonstrated to be localized in endolysosomes of the endothelial cells. These data indicate that twopore calcium channels participate in the histamine-induced endothelium-dependent relaxation of rat aorta. Furthermore, their functional role is exhibited much more clearly at low histamine concentrations. We suggest that hydrogen peroxide evokes depletion of intracellular calcium depots thereby suppressing the response to histamine.     

Effects of Melatonin on Carbonic Anhydrase from Human Erythrocytes In Vitro and from Rat Erythrocytes In Vivo

The in vitro effects of melatonin (N-acetyl-5-methoxytryptamine) on human carbonic anhydrase isozymes (HCA-I and HCA-II) from human erythrocytes and in vivo effects on rat erythrocytes carbonic anhydrase (CA) were determined. Human erythrocyte carbonic anhydrase isozymes were purified by haemolysate preparation and Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. The HCA-I enzyme, having a specific activity of 7337.5?EU/mg protein, was purified 843-fold with a yield of 60% and the HCA-II enzyme, having a specific activity of 17067?EU/mg protein, was purified 1962-fold with a yield of 22.7%. For in vitro experiments, the enzyme activity was minimal at 2×10-4?M melatonin concentration and increased above this concentration. Ten mg?kg-1 melatonin was administered intraperitoneally and showed a stimulatory effect on the enzyme. Time-dependent in vivo studies were conducted for melatonin in Sprague–Dawley type rats. It was found that CA activity in the rat erythrocytes was decreased by the melatonin after 1 and 3 hours to 2500±500.0 and 1875±239.4 respectively which were statistically significant (p<0.05) differences to the control (2660±235.8). However, CA activity was restored to its normal level after 6?h (2666±235.7) (p>0.05) probably due to metabolism of the melatonin. The findings indicate that melatonin may be pharmacologically useful in some diseases.     

FORMAMIDASE IN RAT BRAIN

Kynurenine formamidase (aryl-formylamine amidohydrolase, EC 3.5.1.9) was found to be present in rat brain and was partially purified and characterized. The partially purified enzyme catalysed the hydrolysis of 5-hydroxyformyl-dl -kynurenine to 5-hydroxy-dl -kynurenine and that of formyl-l -kynurenine to l -kynurenine at similar rates. The apparent K_m values of the enzyme for 5-hydroxyformyl-dl -kynurenine and formyl-l -kynurenine were 4.0 ± 10^?4 and 1.8 ± 10^?4m , respectively. The enzyme was active over a wide pH range (5.5–8.5). The activity was inhibited by low concentrations of Ag⁺ and Hg²⁺. The physiological significance of the enzyme is discussed.     

INHIBITION OF ACETYLCHOLINESTERASE IN VITRO BY PENTYLENETETRAZOL1

Abstract—The effect of pentylenetetrazol (PTZ) on acetylcholinesterase (E.C.3.1.1.7) was studied in vitro. The kinetics of the reaction were studied on AChE in crude homogenates of rat brain and in purified preparations from Electrophorus electricus. The K_m for rat brain AChE was 1·22 × 10^-4m, with a V_max of 1·37 μmol/g/min whereas the K₄ for competitive inhibition of the enzyme by PTZ was 4·7 × 10^-3m. The commercially purified enzyme exhibited a K_m of 1·73 × 10^-4m and a K_i of 1·00 × 10^-3m.     

Purification and characterization of L-mimosine synthase from Leucaena leucocephala

L-Mimosine synthase has been isolated from Leucaena leucocephala seedlings and purified 280-fold by heat treatment, ammonium sulphate fractionation, gel filtration and ion-exchange chromatography. The enzyme was shown to be homogeneous by gel electrophoresis (MW 64 000±2000) and to consist of two identical subunits with MWs of 32 000±2000. The purified enzyme has a K_m value of 6.25 x 10^?3 M for O-acetyl-L-serine and 5.0 x 10^?3 M for 3,4-dihydroxypyridine. In these and other properties, the enzyme differs from β-(pyrazol-1-yl)-L-alanine synthase from Citrullus vulgaris seedlings.     

Antagonism of alpha- and beta-adrenergic-mediated accumulations of cyclic AMP in rat cerebral cortical slices by the beta-antagonist (-)alprenolol.

(?)Alprenolol, a compound reported to bind with a high degree of specificity and stereoselectivity to β-adrenergic receptors from rat cerebral cortex completely inhibited the accumulations of cyclic AMP elicited by maximally effective concentrations of norepinephrine and epinephrine at antagonist concentrations as low as 10^?5M. Other β-adrenergic antagonists such as (?)propranolol, (±)sotalol, and (+)alprenolol only partially antagonized accumulations of cyclic AMP elicited by these catecholamines even at 10-fold higher concentrations. α-Adrenergic antagonists such as phentolamine, phenoxybenzamine and clonidine only partially antagonized inhibited the accumulation of cyclic AMP elicited by methoxamine, a compound shown to stimulate the accumulation of cyclic AMP by interaction with α-adrenergic receptors. The results indicate that in brain tissue containing a mixed population of α- and β- adrenergic linked cyclic AMP generating systems, (?)alprenolol does not exhibit absolute specificity for β-receptors.     

THE BINDING OF [3H]MEPYRAMINE TO HISTAMINE H1 RECEPTORS IN GUINEA-PIG BRAIN

The promethazine-sensitive binding of [³H]mepyramine to a membrane fraction from guinea-pig whole brain is saturable with a dissociation constant of 1.7 × 10^-9M. The maximum amount of [³H]mepyramine binding varied widely between preparations, range 122–365 pmol/g protein, with a mean value of 227 ± 52 pmol/g protein. The inhibition of [³H]mepyramine binding by a number of drugs correlated closely with their potency as histamine H₁ antagonists. (+) Chlorpheniramine was 240-fold more potent as an inhibitor of [³H]mepyramine binding than (-)-chlorpheniramine. All antagonists inhibited the binding of [³H]mepyramine to the same extent, but the Hill coefficients characterising the inhibition curves did not all approximate to unity, the value expected for a simple antagonist-receptor equilibrium. The distribution of histamine H₁ receptors, defined by the promethazine-sensitive binding of [³H]mepyramine, in 11 different brain regions was uneven with the largest amounts in cerebellum, superior and inferior colliculus and hypothalamus and the smallest in caudate nucleus, brain stem and spinal cord.     

Inhibition of Lipid Peroxidation by Dihydroquinoline-Type Antioxidant (CH 402)

The in vitro effect of a non-toxic, water soluble, low molecular weight, stable dihydroquinoline-type antioxidant, CH 402 (Na (2,2-dimethyl-1,2-dihydroquinoline-4-yl) –- methane sulphonic acid) was studied on free radical reactions in brain subcellular fractions. Experiments were performed using rat and mouse brain homogenate and microsomal fractions. Non … enzymatically induced lipid peroxidation by ascorbic acid was studied in correlation with ascorbic acid and CH 402 concentrations and incubation time. Malondialdehyde production during lipid peroxidation was measured by the thiobarbituric acid test. In a concentration range of 10^?2–10^?5 M CH 402 dose - dependently inhibited the ascorbic acid induced in vitro lipid peroxidation in mouse and rat brain subcellular fractions.     

CARRIER MEDIATED BLOOD-BRAIN BARRIER TRANSPORT OF CHOLINE AND CERTAIN CHOLINE ANALOGS

Blood-brain barrier (BBB) transport of choline and certain choline analogs was studied in adult and suckling rats, and additionally compared in the paleocortex and neocortex of adult rats. Saturable uptake was characterized by a single kinetic system in all cases examined, and in adult rat forebrains we determined a K_m= 442 ± 60 μM and V_max= 10.0 ± 0.6 nmol min^-1 g^-1. In 14–15-day-old suckling forebrains a similar K_m (= 404 ± 88 μM) but higher V_max (= 12.5 ± 1.5 nmol min^-1 g^-1) was determined. When choline uptake was compared in two regions of the forebrain, similar Michaelis-Menten constants were determined but a higher uptake velocity was found in the neocortex (i.e. neocortex K_m= 310 ± 103 μM and V_max= 12.6 ± 2.8 nmol min^-1g^-1; paleocortex K_m= 217 ± 76 μM and V_max= 7.2 ± 1.5 nmol min^-1 g^-1). Administration of radiolabelled choline at low (5 μM) and high (100 μM) concentrations, followed by microwave fixation 60 s later and chloroform-methanol-water separations of the homogenized brain did not suggest a relationship between concentration and the appearance of label in lipid or aqueous fractions as observed in another in-vitro study elaborating two-component kinetics of choline uptake. It was observed that 60s after carotid injection 12–14% of the radiolabel in the ipsilateral cortex was found in the chloroform-soluble fraction. Hemicholinium-3 (K_i= 111 μM), dimethylaminoethanol (K_i= 42 μM), tetraethyl ammonium chloride, tetramethyl ammonium chloride, 2-hydroxyethyl triethylammonium iodide, carnitine, normal rat serum, and to a lesser extent lithium and spermidine all inhibited choline uptake in the BBB. Unsubstituted ammonium chloride and imipramine did not inhibit choline uptake. No difference was observed in blood-brain barrier choline uptake of unanesthetised, carotid artery-catheterized animals, and comparable sodium pentobarbital-anesthetized controls.     

Histamine stimulates human lung fibroblast migration

Histamine is a potent mediator in allergic inflammatory processes and is released by basophils and mast cells. The aim of this study was to investigate the effect of histamine on in vitro migration of human fetal lung fibroblasts (HFL-1) to human plasma fibronectin (HFn), a chemoattractant. Using the blindwell chamber technique, histamine alone had no chemotactic activity. However, histamine augmented HFn-induced HFL-1 migration at concentrations ranging between 0 and 10^?7 M (290.6 ± 20.8%) (P < 0.05). The concentration-response was bell-shaped. The effect of histamine increased with time. The stimulatory effect of histamine on HFL-1 migration was inhibited by an H4 receptor antagonist, JNJ7777120 (10^?5 M). Histamine’s effect was also inhibited by pertussis toxin (50 ng/ml), showing that the effect was mediated by the H4 receptor. This study demonstrated that histamine has the potential to stimulate human lung fibroblast migration, and thus may contribute to regulation of wound healing and the development of fibrotic disorders of the lung.     

Characterization of Exchange Inhibitory Peptide Effects on Na+/Ca2+ Exchange in Rat and Human Brain Plasma Membrane Vesicles

Abstract: The inhibitory effects of Na⁺/Ca²⁺ exchange inhibitory peptide (XIP), which corresponds to residues 219–238 of the Na⁺/Ca²⁺ exchange protein from canine heart, were studied in both rat and human brain plasma membrane vesicles. XIP had very high potency with respect to the inhibition of the initial velocity of intravesicular Na⁺-dependent Ca²⁺ uptake in both rat brain [IC₅₀ = 3.05 ± 0.69 µM (mean ± SE)] and human brain (IC₅₀ = 3.58 ± 0.58 µM). The maximal inhibition seen in rat brain vesicles was ～80%, whereas human brain vesicles were inhibited 100%. XIP also inhibited extravesicular Na⁺-dependent Ca²⁺ release, and the inhibitory effect was enhanced by increasing the extravesicular Na⁺ concentration. In contrast, the inhibitory effect of bepridil was competitive with respect to extravesicular Na⁺. When XIP was added at steady state (5 min after the initiation of intravesicular Na⁺-dependent Ca²⁺ uptake), it was found that the intravesicular Ca²⁺ content declined with time. Analysis of unidirectional fluxes for Ca²⁺ at steady state showed that 50 µM XIP inhibited Ca²⁺ influx and efflux ～85 and 70%, respectively. This result suggested that XIP inhibited both Na⁺/Ca²⁺ exchange and Ca²⁺/Ca²⁺ exchange but had no effect on the passive release pathway for Ca²⁺. The results suggest structural homology among cardiac, rat, and human brain exchangers in the XIP binding domain and that the binding of Na⁺ or other monovalent cations, e.g., K⁺, is required for XIP to have its inhibitory effect on Ca²⁺ transport.     

Imidazole derivatives as a novel class of hybrid compounds with inhibitory histamine N-methyltransferase potencies and histamine hH3 receptor affinities

In this study, a novel series of imidazole-containing compounds with dual properties, that is, inhibitory potency at the enzyme histamine N(tau)-methyltransferase (HMT) and antagonist potency at histamine H(3) receptors was designed and synthesized. Pharmacologically, these new hybrid drugs were evaluated in functional assays for their inhibitory potencies at rat kidney HMT and for their antagonist activities on synaptosomes of rat cerebral cortex. For selected compounds, binding affinities at recombinant human histamine H(3) receptors were determined. The first compounds (1-10) of the series proved to be H(3) receptor ligands of high potency at rat synaptosomes or of high binding affinity at human H(3) receptors, respectively, but of only moderate activity as inhibitors of rat kidney HMT. In contrast, aminoquinoline- or tetrahydroacridine-containing derivatives 11-17 also displayed HMT inhibitory potency in the nanomolar concentration range. Preliminary data from molecular modeling investigations showed that the imidazole derivative 15 and the HMT inhibitor quinacrine possess identical binding areas. The most interesting compound (14) is simultaneously a highly potent H(3) receptor ligand (K(i)=4.1nM) and a highly potent HMT inhibitor (IC(50)=24nM), which makes this derivative a valuable pharmacological tool for further development.