Noncompetitive Amine Uptake Inhibition by the New Antidepressant Pridefine

Abstract: Pridefine (AHR-1118) is a pyrrolidine derivative with clinically established antidepressant efficacy. Previous work from this laboratory indicates that pridefine is a reuptake blocker of catecholamines and serotonin with weak releasing activity. This study characterized the mode of amine uptake inhibition by pridefine as noncompetitive. The uptake experiments were performed utilizing ouabain instead of zero-degree controls to differentiate between the passive and active components of uptake. Furthermore, the passive component was resolved into diffusion and binding of substrate. Correction was made for the effects of ouabain on binding. Kinetic constants determined from Lineweaver-Burk plots were: K_m= 3 × 10^?7 M for NE, K_m= 9 × 10^?8 M for DA, and K_m= 3 × 10^?8 M for 5-HT. Dixon analyses of uptake at various pridefine concentrations indicated noncompetitive inhibition with K_i= 2.5 × 10^?6 M for NE uptake, K_i= 2.0 × 10^?6 M for DA uptake, and K_i= 1 × 10^?5 M for 5-HT uptake. These constants compare well with IC₅₀ values for the same transmitters: NE, IC₅₀= 2.4 × 10^?6 M; DA, IC₅₀= 2.8 × 10^?6 M; 5-HT, IC₅₀= 1.0 × 10^?5 M. The in vitro results indicate that pridefine is relatively specific as a catecholamine uptake blocker. It differs from tricyclic antidepressants which are reportedly competitive inhibitors of monoamine uptake. The possible mechanisms by which pridefine acts as a noncompetitive inhibitor are discussed.     

Inhibition by β-carbolines of monoamine uptake into a synaptosomal preparation: Structure-activity relationships

The potency of a series of β-carboline compounds to inhibit ³H-serotonin (³H-5-HT) uptake into a synaptosomal suspension from mouse brain was studied. The $\text{in vitro}$ structure-activity study showed the tetrahydro-β-carbolines to be the most potent inhibitors compared to unsaturated β-carbolines. $\text{In vitro}$ inhibition of ³H-norepinephrine (³H-NE) and ³H-dopamine (³H-DA) uptake was determined for some tetrahydro-β-carbolines, and the degree of inhibition of uptake of these amines was less than that for ³H-5-HT (EC50s being 5–13 times those for ³H-5-HT). The tetrahydro-β-carbolines were also found to effectively inhibit ³H-5-HT but not ³H-NE or ³H-DA uptake when they were administered intra-peritoneally. These results suggest that the behavioral effects of the tetrahydro-β-carbolines which have been reported previously may be due to a relatively selective involvement of the serotonergic neurotransmitter system.     

The effects of prostaglandins E2 and F2α on synaptosomal accumulation and release of 3H-norepinephrine

Prostaglandins (PG) of both the E and F series may serve as modulators of norepinephrine (NE) release from peripheral sympathetic neurons. We have studied the effects of PGE₂ and PGF_2α on the accumulation and release of ³H-NE in the CNS using synaptosomes isolated from rat hypothalami.The release of ³H-NE from synaptosomes superfused with Krebs-Ringer bicarbonate buffer was multiphasic with an initial fast release phase followed by a slower release. Raising KC1 concentration of the superfusion medium to 56mM during the slow release phase is known to stimulate ³H-NE release. PGE₂ (1 × 10^?6M) attenuated ³H-NE release during the fast phase and reduced the amount of ³H-NE released due to KC1 stimulation. At lower concentrations of PGE₂ there was no change in the release profile. PGF_2α was without effect on ³H-NE release at all concentrations tested.The accumulation of ³H-NE was significantly diminished by PGE₂ at a concentration of 1 × 10^?6M, while a lower concentration (1 × 10^?7M) was ineffective. PGF_2α had no effect on ³H-NE accumulation at all concentrations investigated.     

Regulation of dopamine release by presynaptic nicotinic receptors in rat striatal slices: Effect of nicotine in a low concentration

Rat striatal slices were continuously superfused with ³H-tyrosine to study the effect of a low concentration of nicotine on the spontaneous release of newly synthesized ³H-dopamine (³H-DA). Nicotine (10^?6M) stimulated the calcium-dependent spontaneous release of ³H-DA. This effect was prevented by nicotinic blockers such as pempidine (10^?5M) and d-tubocurarine (5×10^?6M). The stimulatory effect of nicotine and its blockade by pempidine were still observed in the presence of tetrodotoxin (5×10^?7M). This demonstrates for the first time that a low concentration of nicotine is effective in releasing DA by acting on presynaptic nicotinic receptors located on terminals of the nigro-striatal dopaminergic neurons.     

Adult development of the hippocampal-serotonin system of C57BL/6N mice; analysis of high-affinity uptake of 3H-5HT in slices and synaptosomes

The saturable and specific high-affinity uptake of [³H]serotonin ([³H]5HT) (5 × 10^?8 M) was studied in slices from the hippocampus, parietal cortex, septum-preoptic area, and hypothalamus of male 2, 6, 12 and 24–32 month old C57BL/6N mice. Hippocampal [³H]5-HT uptake showed a significant biphasic relationship to age, with lower values in the 2 and 24–32 month old mice compared to 6 month old mice. No significant age effects were seen in the other regions, or in [³H]norepinephrine high-affinity uptake in the hippocampus.Studies of the high-affinity uptake mechanism in synaptosomal preparations were made in a subgroup of 12 and 24 month old mice. A micro-assay using a tissue-harvester measured high-affinity uptake on 8–30 μl of the P₂ suspension (crude-synaptosomal preparation). The high-affinity uptake was linear for 4 min at 37°C and inhibited in both the adult and aged tissue by 10^?5 M cold 5-HT (83 and 78% respectively), 10^?5 M fluoxetine (85 and 82% respectively) and 10^?3 M NaCN (57 and 57% respectively). Kinetic analysis of the [³H]5HT high-affinity uptake in the hippocampus (3 min, 37°C) revealed the same apparent K_m for serotonin at both ages (6.7 x 10^?8 M), but a 44% decrease in V_max in the aged hippocampal synaptosomal high-affinity uptake compared to adults (120 vs 215 pmol of 5-HT/g-tissue/3 min).These results are discussed in relationship to the reported age effects on the intrinsic neurons of the hippocampus.     

Inhibition of AChE by malathion and some structurally similar compounds

Inhibition of bovine erythrocyte acetylcholinesterase (free and immobilized on controlled pore glass) by separate and simultaneous exposure to malathion and malathion transformation products which are generally formed during storage or through natural or photochemical degradation was investigated. Increasing concentrations of malathion, its oxidation product malaoxon, and its isomerisation product isomalathion inhibited free and immobilized AChE in a concentration-dependent manner. K_I, the dissociation constant for the initial reversible enzyme inhibitor-complex, and k₃, the first order rate constant for the conversion of the reversible complex into the irreversibly inhibited enzyme, were determined from the progressive development of inhibition produced by reaction of native AChE with malathion, malaoxon and isomalathion. K_I values of 1.3 × 10^{? 4} M^{? 1}, 5.6 × 10^{? 6} M^{? 1} and 7.2 × 10^{? 6} M^{? 1} were obtained for malathion, malaoxon and isomalathion, respectively. The IC₅₀ values for free/immobilized AChE, (3.7 ± 0.2) × 10^{? 4} M/(1.6 ± 0.1) × 10^{? 4}, (2.4 ± 0.3) × 10^{? 6}/(3.4 ± 0.1) × 10^{? 6} M and (3.2 ± 0.3) × 10^{? 6} M/(2.7 ± 0.2) × 10^{? 6} M, were obtained from the inhibition curves induced by malathion, malaoxon and isomalathion, respectively. However, the products formed due to photoinduced degradation, phosphorodithioic O,O,S-trimethyl ester and O,O-dimethyl thiophosphate, did not noticeably affect enzymatic activity, while diethyl maleate inhibited AChE activity at concentrations > 10 mM. Inhibition of acetylcholinesterase increased with the time of exposure to malathion and its inhibiting by-products within the interval from 0 to 5 minutes. Through simultaneous exposure of the enzyme to malaoxon and isomalathion, an additive effect was achieved for lower concentrations of the inhibitors (in the presence of malaoxon/isomalathion at concentrations 2 × 10^{? 7} M/2 × 10^{? 7} M, 2 × 10^{? 7} M/3 × 10^{? 7} M and 2 × 10^{? 7} M/4.5 × 10^{? 7} M), while an antagonistic effect was obtained for all higher concentrations of inhibitors. The presence of a non-inhibitory degradation product (phosphorodithioic O,O,S-trimethyl ester) did not affect the inhibition efficiencies of the malathion by-products, malaoxon and isomalathion.     

Influence of quipazine,a potential anti-parkinsonian agent on the uptake of 3H-dopamine and 3H-serotonin into rat striatal tissue in vitro

Quipazine (2-(1-piperazinyl)quinoline maleate), an agent with anti-tremorine and serotonin-like activity, was found to inhibit the uptake of ³H-dopamine and ³H-serotonin into rat striatal tissue in vitro. Quipazine was shown to be three times more effective as an inhibitor of serotonin uptake than dopamine uptake, the IC₅₀'s being 2.98 × 10^?5M and 1.00 × 10^?4M, respectively. These data suggest that quipazine exerts serotonergic and dopaminergic effects in the central nervous system.     

Greatly extended viability of rat brain storage vesicles in an intracellular medium based upon a non-permeant polyanion

The MgATP-stimulated accumulation of (-)-³H-nor- epinephrine (NE) by rat brain neuronal storage vesicles has been characterized in a new medium based upon polyacrylic acid (avg. MW 5,000). The medium allows careful regulation of K⁺ concentration (140 mM), has a large buffer capacity, and is non-permeant to membranes. Light scattering measurements have confirmed the osmotic stability of vesicles suspended in this medium. Vesicular accumulation of (-)-³ H-NE (Km 1 × 10^?6 M) in this system (37°) was examined under saturating (10^?5 M) and non-saturating (2 × 10^?7 M) concentrations of NE. At 10^?5 M NE, uptake saturated at 5 min and remained stable for periods up to one hour, with maximal uptake levels (pmol/mg protein) of 15.7±0.30 (37°), 3.0±0.49 (0°), 4.4±0.22 (reserpine pretreated $\text{in}$$\text{vivo}$) and 6.0±0.79 (without MgATP). At 2×10^?7 M NE uptake was biphasic with maximal uptake levels (pmol/mg protein) of 4.04±0.14 (37°), 0.19±0.01 (0°), 0.95±0.01 (reserpine) and 0.83±0.08 (without MgATP). Vesicle preparations refrigerated in this medium for 24 hrs displayed properties quite similar to those measured acutely (NE = 2.2x10^?7 M).     

A serotonin sensitive guanylate cyclase associated with specific neurotransmitter binding sites on isolated synaptic membranes from mature rat brain.

Results from this study indicate that adult rat brain posesses guanylate cyclase activity sensitive to serotonin (5-HT) and localized in the synaptic plasma membrane. The enzyme appears to have multiple activation sites for 5-HT with specific activity maxima at the 5-HT concentrations of 5 × 10^?10M and 7 × 10^?8M respectively. The rates of guanosine-3′:5′-monophosphate (cyclic GMP) formation at these concentrations of 5-HT are, respectively, 170% and 307% above the endogenous or basal production rate of 2.7±0.3picomoles/minute/milligram of synaptosomal membrane protein. We have also been able to identify $\text{four}$ distinct types (Type #1, #2, #3, and #4) of high affinity, specific binding sites for 5-HT on isolated synaptosomal membranes from rat brain. Dissociation constants of 2.6 × 10^?10M, 2.5 × 10^?9M, 7.0 × 10^?9M, and 4.6 × 10^?8M, characterize the binding of 5-HT to our sites of Type #1 through Type #4 respectively. The specific, high affinity binding was saturated at 5-HT concentrations of 5 × 10^?10M for the Type #1 sites, 5 × 10^?9M for our Type #2 sites, 1 × 10^?8M for our Type #3 sites, and 7 × 10^?8M for our Type #4 sites. The 5-HT concentrations producing saturation of our specific binding sites of Type #1 and Type #4 are virtually identical to those that elicit the two maxima of 5-HT stimulated cyclic GMP production, indicating that a membrane-bound guanylase cyclase may be closely associated with certain 5-HT receptors and/or re-uptake sites.     

Effect of muscarinic agonists on the release of 3H-norepinephrine and 3H-dopamine by potassium and electrical stimulation from rat brain slices

The effect of acetylcholine (ACh) on the release of ³H-norepinephrine (NE) from the cerebellum and ³H-dopamine (DA) from the striatum following the administration of potassium chloride or electrical field stimulation was studied in superfused brain slices. ACh in conc. of 10^?6 and 10^?5M significantly inhibited the release of ³H-NE from cerebellar slices and ³H-DA from striatal slices following 2 min infusion of 50mM potassium chloride. In addition ACh produced a dose-dependent inhibition of the release of ³H-DA from striatal slices following electrical stimulation. The results obtained in the present study are quite consistent with the concept that a muscarinic inhibitory mechanism may be operative on noradrenergic and dopaminergic neurons in the brain.     

Novel nonclassical inhibitors of glycinamide ribonucleotide formyltransferase: 10-Formyl and 10-hydroxymethyl derivatives of 5,8,10-trideazapteroic acid

Several new 10-formyl and 10-hydroxymethyl derivatives of 5,8,10-trideazapteroic acid have been synthesized by a novel and convenient enamine alkylation procedure. Two of these compounds (10a and 10b) were shown to be very powerful inhibitors of L. casei (10a, IC₅₀ = 8 × 10⁻⁶ M ; 10b, IC₅₀ = 7 × 10⁻⁶ M ) and recombinant mouse (10a, IC₅₀ = 3.4 × 10⁻⁵ M ; 10b, IC₅₀ = 2.8 × 10⁻⁵ M ) glycinamide ribonucleotide formyltransferase (GARFT). These IC₅₀ values are comparable to the classical GARFT inhibitor (6R)-DDATHF (IC₅₀, L. casei 2.3 × 10⁻⁶M ; recombinant mouse 2.3 × 10⁻⁵ M ) under identical assay conditions. For both compounds, the inhibition of L. casei GARFT increased with time of incubation, but not markedly with the recombinant mouse enzyme. Due to their potential ability to interfere with purine biosynthesis and to penetrate microbial cells the new nonclassical GARFT inhibitors reported here may be useful for the treatment of infections caused by microorganisms that are sensitive and resistant to conventional antimicrobial agents.     

SR 140333, a Novel, Selective, and Potent Nonpeptide Antagonist of the NK1 Tachykinin Receptor: Characterization on the U373MG Cell Line

The effects of a novel nonpeptide NK1 tachy-kinin receptor antagonist, SR 140333, on the functional consequences of NK1 receptor activation in a human astrocytoma cell line, U373MG, were investigated. Radioligand binding conducted with ¹²⁵l-Bolton-Hunter substance P revealed a competitive inhibition by SR 140333 and its R enantiomer SR 140603 with K_i values of 0.74 and 7.40 nM, respectively. The NK1-selective agonist, [Sar⁹,Met(O₂)¹¹]-substance P, stimulated the formation of inositol phosphates with an EC₅₀ of 3.8 × 10^?9M. SR 140333 blocked the stimulatory effect of this agonist (10^?7M) with an IC₅₀ of 1.6 × 10^?9M,whereas the effect of another NK1 agonist, septide (EC₅₀= 1.5 × 10^?8M)was antagonized with an IC₅₀ of 2.1 × 10^?10M.Enhancement of [³H]taurine release by [Sar⁹,Met(O₂)¹¹]-substance P (EC₅₀= 7.4 × 10^?9M) was also inhibited by SR 140333 with an IC₅₀ of 1.8 × 10^?9 M. SR 140603 was 10-fold less potent than SR 140333 in inhibiting inositol monophosphate formation and [³H]taurine release. The calcium mobilization induced by [Sar⁹,Met(O₂)¹¹]-substance P (10^?8M) was totally prevented by 10^?8MSR 140333. Patchclamp experiments showed that SR 140333 depressed the outward current evoked by 5 × 10^?8M [Sar⁹, Met(O₂)¹¹]-substance P with an IC₅₀ of 1.3 × 10^?9M. The expression of c-fos was stimulated by [Sar⁹,Met(O₂)¹¹]-substance P with an EC₅₀ of 2.5 × 10^?10M, an effect that was also inhibited by SR 140333 with an IC₅₀ of 1.1 × 10^?9M. The present results illustrate the sequential events of the response elicited by NK1 agonists, which were antagonized by SR 140333, demonstrating its powerful NK1 antagonist activity on a functional basis.     

A serotonin sensitive adenylate cyclase in mature rat brain synaptic membranes

Results from this study have indicated serotonin-sensitive adenylate cyclase activity in adult rat brain. The enzyme is localized in the synaptosomal plasma membrane and apparently has multiple activation sites for serotonin with specific activity maxima occuring at serotonin concentrations of 5 × 10^?10, 5 × 10^?9, 1 × 10^?8, and 5 × 10^?8 moles/liter. The production of cyclic AMP at these sites appears to be unaffected by 1 × 10^?5M fluphenazine, while 1 × 10^?5M tryptamine, methysergide, and ergonovine decreased the stimulatory effect of 1 × 10^?8M 5-HT by 30 percent, 80 percent, and 57 percent respectively.     

Larval metamorphosis of the mussel Mytilus galloprovincialis Lamarck, 1819 in response to neurotransmitter blockers and tetraethylammonium

The metamorphic response of pediveliger larvae of Mytilus galloprovincialis to the neurotransmitter blockers chlorpromazine, amitriptyline, rauwolscine, idazoxan, atenolol and butoxamine, and to tetraethylammonium chloride (TEA) was investigated through a series of bioassays. Chlorpromazine, amitriptyline and idazoxin inhibited larval metamorphosis induced by 10^?4 M epinephrine. The concentration that inhibited metamorphosis by 50% (IC₅₀) for chlorpromazine and amitriptyline was 1.6 × 10^?6 M and 6.6 × 10^?5 M, respectively. Idazoxan was less effective with an IC₅₀ of 4.4 × 10¹³ M. Moreover, these three inhibitors showed no toxicity at any of the concentrations tested. The larval metamorphic response to K⁺ was not inhibited by 10^?3 M tetraethylammonium chloride after 96 h. Thus, the neurotransmitter blockers chlorpromazine and amitriptyline are inhibitors of larval metamorphosis, and will be useful tools for antifouling studies.     

Effects of amiridin and tacrine, drugs effective in Alzheimer's disease, on synaptosomal uptake of neuromediators]

The study of the drugs effective in the treatment of cognitive deficits and memory loss associated with senile dementia of the Alzheimer's type--tacrine and amiridin, acetylcholinesterase inhibitor physostigmine and nootrop piracetam on uptake of 3H-serotonin (3H-5-HT), 3H-adrenaline (3H-AD), 3H-noradrenaline (3H-HA), 2H-dopamine (3H-DA), 3H-gamma-aminobutyric acid (3H-GABA), 3H-glutamic acid (3H-GLU), 3H-aspartic acid (3H-ASP) and 3H-glycine (3H-GLI) showed that tacrine and amiridin (5 x 10(-5) M) statistically significantly (P less than 0.05) inhibited the uptake of 3H-DA and 3H-5-HT. Physostigmine at concentration 5 x 10(-4) M statistically significantly (P less than 0.05) inhibited uptake of 3H-5-HT only. Piracetam at concentration range 1-5 x 10(-3) M had no effect on uptake of all investigated neurotransmitters. The above finding suggest that the uptake of neurotransmitter in nerve terminals is not the main target of amiridin and tacrine.     

Effects of estradiol and progesterone on uterine prostaglandin levels in the pregnant rat

Prostaglandin D₂ was found to be a potent inhibitor of B-16 melanoma cell replication $\text{in vitro}$. The inhibition was dose-dependent between 3×10^?9M and 3×10^?6M (IC_50～ 0.3 μM after 6 days). On a molar basis, PGD₂ was a better inhibitor than PGA₂ or 16,16-dimethyl-PGE₂-methyl ester (di-M-PGE₂) and in higher concentrations (10^?6?10^?7M), comparable to retinoic acid. In higher concentrations, PGD₂ inhibited DNA, RNA and protein synthesis. The B-16 melanoma cell line which we used synthesized arachidonic acid metabolites which comigrated with PGA₂, PGD₂, PGE₂ and PGF_2α on a thin layer chromatography system.     

Kinetics of phosphate uptake in excised maize root

The short term uptake of phosphate involving 10 min absorption followed by 5 min desorption, both at 30 °C, in the concentration range 1.0×10^?9 to 7.5×10^?2 M KH₂PO₄ by fresh and washed maize (Zea mays L. cv. Ganga Safed-2) roots can be described by a single isotherm having five phases (0 and I–IV) with regularly spaced kinetic constants. Almost identical kinetics were observed in both fresh and washed maize roots. The kinetics of phase 0 in the concentration range 1.0×10^?9–3.0×10^?5 M. was sigmoidal in fresh maize roots, however, in washed tissue exhibited 2 phases termed here as 0a and 0b. 0a covered the concentration range 1.0×10^?9–5.0×10^?6 M and 0b 6.0×10^?6–3.0×10^?5 M. In the concentration range 1.0×10^?4–7.5×10^?2 M four distinct phases, termed as I, II, III and IV were evident in both fresh and washed maize roots. Each phase obeyed Michaelis—Menten kinetics. The values of K_m and V_max have been estimated for each phase. The uptake isotherm was accompanied by discontinuous transitions.     

Membrane transport of L-triiodthyronine by human red cell ghosts.

L-T₃ transport has been investigated in human red cell ghosts. Determination of initial T₃ uptake revealed two separate saturable uptake systems, one with a K_m of 1.6 × 10^?8M, the other with a K_m of 3.3 × 10^?6M. Binding experiments resulted in two dissociation constants, 1.4 × 10^?7M.and 2.6 × 10^?6M. Uptake was dependent on the ghost volume, indicating an intravesicular location of T₃. The T₃ was concentrated 6 times by the ghosts. Ouabain reduced the uptake by the low K_m system, but was without effect on the high K_m system. Thus evidence is provided both of binding of T₃ to the ghost membrane and of its uphill transport across the membrane.     

A specific prostaglandin I2 synthetase inhibitor, 3-hydroperoxy-3-methyl-2-phenyl-3H-indole.

Inhibitory effects of 3-hydroperoxy-3-methyl-2-phenyl-3H-indole(HPI) on prostaglandin endoperoxide synthase(EC 1.14.99.1) and prostaglandin I₂(PGI₂) synthetase were compared with those of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid, namely, 15-hydroperoxyarachidonic acid(15-HPAA) and tranylcypromine (TCP). Sheep seminal vesicle microsomes were used as a source of prostaglandin endoperoxide synthase and bovine aortic microsomes as that of PGI₂ synthetase. 15-HPAA and HPI inhibited PGI₂ synthetase with IC₅₀s of 5 × 10^?7 and 3.5 × 10^?6 M, respectively, whereas neither compound had effect on prostaglandin endoperoxide synthase at the concentration inhibiting PGI₂ synthetase by 90%. TCP was a weak(IC₅₀ = 5 × 10^?4M) PGI₂ synthetase inhibitor with low specificity.     

Effects of papaverine on basal and on isoproterenol-stimulated renin secretion from rat kidney slices

Papaverine inhibited the basal renin secretion of rat kidney slices incubated in a physiological salt solution at 37°C. Inhibition was concentration-dependent; secretion was 99 ± 0.2 % inhibited by 5 × 10^?4 M papaverine, and 8 × 10^?5 M was the estimated ED₅₀. In contrast, 2 × 10^?4 M IBMx (3-isobutyl-1-methyl-xanthine) increased the rate of secretion from 215 ± 17 to 366 ± 30 ng hr^?1mg^?1/20 min (p < 0.001). Isoprotenol (4 × 10^?7, 8 × 10^?7, and 5 × 10^?6 M) stimulated renin secretion in a concentration-dependent manner; the stimulatory effects were antagonized by papaverine but unaffected by IBMx. Thus, two known inhibitors of phosphodiesterase--IBMx and papaverine--produce sharply contrasting effects on basal and on isoproterenol-stimulated renin secretion from rat kidney slices.