Tricyclic antidepressants inhibit voltage-dependent calcium channels and Na(+)-Ca2+ exchange in rat brain cortex synaptosomes

We have used a resting (5 mM K+) or depolarizing (60 mM K+) choline-based medium, and a nondepolarizing sodium-based or choline-based medium, to characterize the inhibitory potential of tricyclic antidepressants against the voltage-dependent calcium channels or the Na(+)-Ca2+ exchange process, respectively, in synaptosomes from rat brain cortex. Imipramine, desipramine, amitriptyline, and clomipramine inhibited net K(+)-induced 45Ca uptake with similar IC50 values (26-31 microM), and this uptake was also inhibited by diltiazem with an IC50 of 36 microM; these results indicate an inhibition of voltage-dependent calcium channels by tricyclic antidepressants. The net uptake of 45Ca induced by Na(+)-Ca2+ exchange was also inhibited by the four tricyclic antidepressants tested, but not by diltiazem; imipramine (IC50 = 94 microM) was a more potent inhibitor of this process than desipramine (IC50 = 151 microM), and the IC50 values of amitriptyline (107 microM) and clomipramine (97 microM) were similar to that of imipramine. Some degree (approximately 25%) of brain calcium channel blockade could be present at the steady-state concentrations of tricyclic antidepressants expected to occur therapeutic use of these compounds to treat depression or panic disorder.     

Different relevance of inactivation and F468 residue in the mechanisms of hEag1 channel blockage by astemizole, imipramine and dofetilide

The relevance of a point mutation at the C-terminal end of the S6 helix (F468) and the introduction of C-type inactivation in the blockage of hEag1 channels by astemizole, imipramine and dofetilide was tested. C-type inactivation decreased block by astemizole and dofetilide but not imipramine, suggesting different binding sites in the channel. F468C mutation increased IC(50) for astemizole and imipramine but in contrast to HERG channels, only slightly for dofetilide. Together with measurements on recovery of blocking, our observations indicate that the mechanism of hEag1 blockage by each of these drugs is different, and suggest relevant structural differences between hEag1 and HERG channels.     

High- and Low-Affinity Binding of [3H]Imipramine in Rat Hypothalamus

In the rat hypothalamus [3H]imipramine binding is inhibited by tricyclic and nontricyclic antidepressant drugs in a complex manner, with biphasic curves and Hill coefficients less than 1.0. 5-Hydroxytryptamine (serotonin) inhibited with high affinity a decreasing proportion of the [3H]imipramine binding sites as the [3H]imipramine concentration was raised. In the absence of sodium ions, IC50 values for the inhibition by tricyclic and nontricyclic antidepressants were increased by approximately 1,000-fold, and the inhibition curves became classically monophasic with Hill coefficients close to 1.0. These data are interpreted as suggesting that [3H]imipramine binds to two independent sites in the rat hypothalamus. One site is sodium-dependent with a high affinity for the drugs tested; the other is sodium-independent and has a low affinity for these drugs.     

GBR-12909 and fluspirilene potently inhibited binding of [3H] (+)3-PPP to sigma receptors in rat brain

Fluspirilene and GBR-12909, two compounds structurally similar to BMY-14802 and haloperidol, were assessed for their ability to interact with sigma receptors. Fluspirilene, an antipsychotic agent that interacts potently with dopamine receptors, inhibited the binding of [3H]-(+) 3-PPP (IC50 = 380 nM) more potently than rimcazole, a putative sigma antagonist that was tested clinically for antipsychotic activity. GBR-12909, a potent dopamine uptake blocker, also inhibited the binding of [3H]-(+) 3-PPP with an IC50 of 48 nM. However, other compounds that block the re-uptake of catecholamines, such as nomifensine, desipramine, imipramine, xylamine, benztropine and cocaine, were much weaker than GBR-12909 as sigma ligands. Thus, GBR-12909 and fluspirilene, compounds structurally similar to BMY-14802, are potent sigma ligands.     

A study of the mechanism by which some amphiphilic drugs affect human erythrocyte acetylcholinesterase activity.

The effects of the local anaesthetics procaine, tetracaine and lidocaine and of the antidepressant imipramine on human erythrocyte acetylcholinesterase were investigated. All four amphiphilic drugs inhibited enzymic activity, the IC50 (the concentration causing 50% inhibition) being about 0.40 mM for procaine, 0.05 mM for tetracaine, 0.70 mM for imipramine and 7.0 mM for lidocaine. Procaine and tetracaine inhibited acetylcholinesterase activity competitively at concentrations at which they did not perturb the physical state of the membrane lipid environment, as assessed by steady-state fluorescence polarization, whereas lidocaine and imipramine displayed mixed inhibition kinetics at concentrations at which they induced an enhancement of membrane fluidity. The question was addressed as to whether membrane integrity is a prerequisite for imipramine and lidocaine action. Membrane solubilization by 1% Triton X-100 and a decrease, by dilution, in the detergent concentration to 0.05% [which is above the Triton X-100 critical micelle concentration (c.m.c.)] did not substantially affect the inhibitory potency of the two amphiphilic drugs at their IC50; in the presence of increasing detergent concentrations the inhibitory potency of imipramine was gradually decreased, but not abolished, whereas the inhibitory effect of lidocaine was only slightly diminished, even at 1% Triton X-100. It is suggested that neither competitive nor mixed inhibition kinetics arise from conformational changes of the protein driven by a modification of the physical state of the lipid environment, but from a direct interaction of the amphiphilic drugs with acetylcholinesterase. In particular, the partial loss of the inhibitory potency of imipramine and lidocaine that is observed upon increasing Triton X-100 concentration well above its c.m.c. could be explained in terms of amphiphile partition in detergent micelles and, in turn, of a decreased effective concentration of the two inhibitors in the aqueous phase.     

Lack of correlation between in vitro susceptibility to Benznidazole and phylogenetic diversity of Trypanosoma cruzi, the agent of Chagas disease

Chagas disease remains an important health problem in Central and South America. Nitroimidazole derivative drugs like Benznidazole are commonly used to treat Trypanosoma cruzi infection. Natural variation of drug susceptibility between various T. cruzi stocks has been proposed as a possible explanation of treatment failure. Thus, the aim of this work was to determine potential correlations between in vitro Benznidazole susceptibility of different T. cruzi stocks and their genetic diversity. For this purpose, 16 natural stocks representing the overall genetic diversity of the parasite were analysed. Genetic characterisation was assessed by both random amplified polymorphic DNA (RAPD) and multilocus enzyme electrophoresis (MLEE) analyses. Drug activity was determined by two complementary methods, the MTT-PMS micro-method and FACs analysis. The 50% inhibitory concentrations (IC(50)s) were determined. Important variation of IC(50) values (7.3-16.9 microM) among stocks belonging to different discrete typing units (DTUs) was recorded. Further, correlation analysis showed that natural susceptibility to Benznidazole in T. cruzi expressed as IC(50) level was not related with its genetic structure represented by the different DTUs. These results are discussed in relation with the proposed hypothesis establishing a link between genetic diversity and biological behaviour in T. cruzi.     

Mechanism of block of hEag1 K+ channels by imipramine and astemizole

Ether à go-go (Eag; KV10.1) voltage-gated K+ channels have been detected in cancer cell lines of diverse origin and shown to influence their rate of proliferation. The tricyclic antidepressant imipramine and the antihistamine astemizole inhibit the current through Eag1 channels and reduce the proliferation of cancer cells. Here we describe the mechanism by which both drugs block human Eag1 (hEag1) channels. Even if both drugs differ in their affinity for hEag1 channels (IC50s are approximately 2 microM for imipramine and approximately 200 nM for astemizole) and in their blocking kinetics, both drugs permeate the membrane and inhibit the hEag1 current by selectively binding to open channels. Furthermore, both drugs are weak bases and the IC50s depend on both internal an external pH, suggesting that both substances cross the membrane in their uncharged form and act from inside the cell in their charged forms. Accordingly, the block by imipramine is voltage dependent and antagonized by intracellular TEA, consistent with imipramine binding in its charged form to a site located close to the inner end of the selectivity filter. Using inside- and outside-out patch recordings, we found that a permanently charged, quaternary derivative of imipramine (N-methyl-imipramine) only blocks channels from the intracellular side of the membrane. In contrast, the block by astemizole is voltage independent. However, as astemizole competes with imipramine and intracellular TEA for binding to the channel, it is proposed to interact with an overlapping intracellular binding site. The significance of these findings, in the context of structure-function of channels of the eag family is discussed.     

5-Methoxytryptoline, a Competitive Endocoid Acting at [3H]Imipramine Recognition Sites in Human Platelets

5-Methoxytryptoline potently inhibits [3H]imipramine binding to membranes from the cerebral cortex and platelets. Since 5-methoxytryptoline, which appears to occur endogenously with particularly high levels in the human pineal gland, also inhibits 5-hydroxytryptamine (5-HT, serotonin) uptake, it should be considered as a putative endogenous ligand modulating 5-HT transport. As the 5-HT transporter complex comprises the imipramine and the substrate recognition sites, which interact allosterically, it was essential to define the mechanism of inhibition of [3H]imipramine binding by 5-methoxytryptoline. Human platelets show an active and saturable uptake of 5-HT and tryptamine. The uptake of both substrates appears to be mediated by the same carrier and it is inhibited by 5-methoxytryptoline at submicromolar concentrations. 5-HT and tryptamine inhibit [3H]imipramine binding in human platelets with a Hill slope for inhibition close to unity and IC50 values of 3,265 and 3,475 nM, respectively. This inhibition is, however, not competitive because both 5-HT and tryptamine significantly decrease the rate of [3H]imipramine-receptor dissociation. Although 5-methoxytryptoline potently inhibits [3H]imipramine binding (IC50 = 44 nM) in human platelets with a Hill slope of unity, it does not affect the receptor-ligand dissociation rate of [3H]imipramine even at concentrations up to 100 microM. The present experiments show that 5-methoxytryptoline, in spite of its chemical similarity to the indoleamine transporter substrates, interacts with the imipramine receptor through a mechanism of competitive inhibition. This conclusion is supported by a selective effect of 5-methoxytryptoline on the Kd of [3H]imipramine binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antimalarial properties of imipramine and amitriptyline

Dietary riboflavin deficiency is known to diminish malarial parasitemia. In this study, we determined whether imipramine and amitriptyline, drugs which inhibit riboflavin metabolism, have antimalarial efficacy. In addition, we evaluated whether these drugs, like other antimalarial agents, increase the hemolytic response to ferriprotoporphyrin IX (FP). The growth of Plasmodium falciparum (FCR3) in the absence and presence of these drugs (10 to 75 microM) was measured by determining (3H)hypoxanthine uptake by intra-erythrocytic parasites for 48 h in RPMI 1640 medium. The uptake of (3H)hypoxanthine was significantly reduced in a dose-dependent manner by both imipramine and amitriptyline. The IC50 values of imipramine and amitriptyline at 48 h were 56 and 45 microM, respectively. Both drugs enhanced hemolysis induced by FP (10 or 20 microM). No hemolysis by these drugs was detected in the absence of FP. It is concluded that the tricyclic antidepressants, imipramine and amitriptyline, possess substantial antimalarial properties.     

"Specific" Binding of [3H]Imipramine to Protease-Sensitive and Protease-Resistant Sites

A number of 5-hydroxytryptamine (5-HT) uptake inhibitors have been shown to displace the binding of [3H]imipramine to rat cortical membranes in a complex manner with Hill slopes less than unity. Norzimeldine displaced the binding of [3H]imipramine in a biphasic manner with IC50 values for the two components of about 30 nM and 30 microM. This latter site alone was found in tissues that had been treated with a protease. Binding to both of these sites was displaced by 10 microM desipramine. The protease-sensitive [3H]imipramine binding sites were found to be saturable, high-affinity binding sites with a KD of 8 nM. The number of these sites varied between brain regions and was positively correlated with the regional distribution of [14C]5-HT but not [3H]noradrenaline uptake. This was not the case however for the protease-resistant but desipramine-displaceable binding sites. Since most previous [3H]imipramine binding studies have been performed with high concentrations of desipramine (10 microM) to define "specific binding," these data would suggest that either protease-sensitivity or displacability by 1 microM norzimeldine would give more reliable estimates of the specific binding.     

Effects of imipramine on ion channels and proliferation of IGR1 melanoma cells

Human IGR1 cells are a model for malignant melanoma. Since progression through the cell cycle is accompanied by transient cell hyperpolarization, we studied the properties of potassium and chloride ion channels and their impact on cell growth. The major potassium current components were mediated by outward rectifying ether à go-go (hEAG) channels and Ca2+-activated channels (KCa) of the IK/SK type. The major chloride channel component was activated by osmotic cell swelling (Clvol). To infer about the contribution of these channels to proliferation, specific inhibitors are required. Since there is no specific blocker for hEAG available, we used the tricyclic antidepressant imipramine, which blocked all channels mentioned, in combination with blockers for KCa (charybdotoxin) and Clvol (DIDS and pamoic acid). Incubation of IGR1 cells for 48 hr in 10-15 mM imipramine reduced DNA synthesis and metabolism without significant effects on apoptosis. hEAG channels were most sensitive to imipramine (IC50: 3.4 microM at +50 mV), followed by KCa (13.8 microM at +50 mV) and Clvol (12 microM at -100 mV), indicating that hEAG expression may be of importance for proliferation of melanoma cells. The contribution of KCa channels could be excluded, as 500 nM charybdotoxin, which completely blocked KCa, had no effect on proliferation. The impact of Clvol also seems to be minor, because 500 microM pamoic acid, which completely blocked Clvol, did not affect proliferation either.     

Evidence for an imipramine-sensitive serotonin transporter in human placental brush-border membranes

Serotonin is actively transported into brush-border membrane vesicles isolated from normal human term placentas and an inward-directed NaCl gradient provides the driving force for this process. Uptake is negligible if Na+ is replaced by Li+, K+, Rb+, Cs+ or choline. The presence of Cl- seems necessary for the maximal activity of this Na+-dependent uptake system. Intravesicular K+ (20-40 mM) stimulates serotonin uptake, the stimulation being considerably greater at pH 7.5 than at pH 6.5. But, in the absence of K+, uptake at pH 6.5 was twice the uptake at pH 7.5. Unlabeled serotonin and dopamine inhibit the uptake of radiolabeled serotonin and the IC50 values are 70 nM and 20 microM, respectively. Histamine and 5-hydroxytryptophan do not significantly interact with the system (IC50 greater than 1 mM). Kinetic analysis reveals that serotonin uptake in these vesicles occurs via a single, saturable, high affinity system (Kt = 51 +/- 2 nM; Vmax = 6.4 +/- 0.1 pmol/mg of protein/15 s). The transporter is highly sensitive to inhibition by imipramine (IC50 = 32 nM) and desipramine (IC50 = 160 nM) but relatively insensitive to reserpine and hydralazine.     

Purification and properties of a human plasma endogenous modulator for the platelet tricyclic binding/serotonin transport complex

An endogenous modulator for the site labeled by [3H]imipramine which is putatively coupled to the serotonin transporter in human platelets was isolated and purified from plasma. Procedures included sequential chromatography on Cibacron blue-Sepharose 4B, concanavalin A-Sepharose 4B, Mono Q HR 10/10 anion exchange, DuPont GF-250 gel permeation and Mono S HR 5/5 cation exchange columns. The purified modulator is a protein of Mr 45,000 with a very acidic pK (less than 3) and sensitive to various proteinases but heat- and acid-stable. This protein inhibited [3H]imipramine binding to platelet membranes competitively (IC50 approximately 6 microM) and enhanced serotonin uptake in fresh human platelets (EC50 approximately 7 microM). Various physicochemical properties, including chromatographic, electrophoretic and immunological as well as amino acid composition analysis revealed that the isolated protein is most probably the human alpha 1-acid glycoprotein.     

High-affinity choline uptake in regions of rat brain and the effect of antidepressants.

The effect of tricyclic antidepressants, chlorpromazine, and some monoamine oxidase inhibitors on the accumulation of [14C]choline by crude synaptosomal (P2) fraction from different regions of rat brain (cortex, striatum, and hippocampus) was investigated. Analysis of choline uptake kinetics resulted in high- and low-affinity components with different Michaelis constants. All tricyclic antidepressants tested inhibited in a dose-dependent manner the high-affinity choline uptake in the three regions, amitriptyline being the most potent. The IC50 values correlated significantly with the relative potencies of imipramine congeners in binding to muscarinic receptors in the brain. Neither tranylcypromine nor pargyline in concentrations up to 0.1 mM had any effect on choline transport. Concentrations of tricyclic antidepressants effective in inhibiting the uptake of choline failed to influence significantly the activity of choline acetyltransferase in brain regions examined. The results suggest that the effect of imipramine congeners on high-affinity choline uptake may be reflected in the anticholinergic properties of these compounds.     

Allosteric Interaction Between the Site Labeled by [3H]Imipramine and the Serotonin Transporter in Human Platelets

The nature of interaction between the site labeled by [3H]imipramine (IMI) and the 5-hydroxytryptamine (5-HT, serotonin) transporter in human platelets was examined. The sulfhydryl characterizing agent N-ethylmaleimide (NEM) differentially affected [3H]5-HT uptake and [3H]IMI binding in human platelet preparations. Concentrations of NEM that completely abolished [3H]5-HT uptake only minimally reduced [3H]IMI binding. Examining the effect of IMI on the kinetics of human platelet [3H]5-HT uptake revealed significant reductions in maximal velocity (Vmax) without altering affinity (Km). IC50 values for selected uptake blockers on [3H]IMI binding and [3H]5-HT uptake were determined. IC50 values of these compounds for uptake and binding revealed that agents such as IMI, chlorpromazine, amitriptyline, and nisoxetine were preferential inhibitors of [3H]IMI binding whereas fluoxetine, CL 216, 303, pyrilamine, and bicifadine were preferential [3H]5-HT uptake blockers. 5-HT was a weak displacer of [3H]IMI binding (IC25 = 3.0 microM) and exhibited a rather low Hill coefficient (nH app = 0.46). Results reported herein support the notion of an allosteric interaction between the [3H]IMI binding site and the 5-HT transporter complex in human platelets.     

High- and Low-Affinity Binding Components for [3H]Imipramine in Rat Cerebral Cortex

The present study demonstrates that [3H]imipramine binds to both high- and low-affinity imipramine binding components on membranes prepared from rat cerebral cortex. Scatchard and computer analyses of saturation experiments using a wide range of [3H]imipramine concentrations (0.5 nM-50 nM) revealed the presence of two binding components. Inhibition experiments in which membranes were incubated with [3H]imipramine and various concentrations of unlabelled imipramine gave shallow inhibition curves with a Hill coefficient of 0.60 +/- 0.04. When dissociation rates of imipramine were studied, biphasic dissociation curves were obtained with apparent half-times of dissociation of 2.5 +/- 0.4 min and 18.5 +/- 2.5 min. Thus analysis of saturation, competition, and dissociation experiments indicate that [3H]imipramine binds to low as well as high-affinity binding sites in rat cortex.     

Structure–Activity Relationships for Six Ketolide Antibiotics

Six structurally related 3-keto-substituted macrolide antibiotics (ketolides) were compared for concentration-dependent inhibitory effects on growth rate, viable cell number, and protein synthesis rates in Staphylococcus aureus cells. Inhibitory effects on 50S ribosomal subunit formation were also examined, as this is a second target for these antibiotics. A concentration range of 0.01 to 0.1 microg/ml was tested. An IC50 for inhibition of translation and 50S synthesis was measured for each compound, to relate structural features to inhibitory activity. ABT-773 was the most effective of the six compounds tested with an IC50 = 0.035 microg/ml. HMR 3004 was almost as effective with an IC50 = 0.05 microg/ml. Two 2-fluoroketolides (HMR 3562 and HMR 3787) were equivalent in their inhibitory activity with an IC50 = 0.06 microg/ml. Telithromycin (HMR 3647) had an IC50 = 0.08 microg/ml, and HMR 3832 was least effective with an IC50 = 0.11 microg/ml. Each antibiotic had an equivalent inhibitory effect on translation and 50S subunit formation. These results indicate specific structural features of these antimicrobial agents, which contribute to defined inhibitory activities against susceptible organisms.     

Comparative study of the inhibitory effect of antidepressants on cholinesterase activity in Bungarus sindanus (krait) venom, human serum and rat striatum

Cholinesterases are divided into two classes based on differences in their substrate specificity and tissue distribution: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). These enzymes may be inhibited by several compounds, such as antidepressants. The antidepressants paroxetine, imipramine, clomipramine and sertraline inhibited both venom AChE as well as human serum BChE in a concentration-dependent manner but had no effect on AChE in the rat brain striatum. The IC(50) of venom calculated for imipramine was 0.3 mM, paroxetine 0.38 mM, clomipramine 0.34 mM and sertraline 0.35 mM. Analysis of kinetic data indicated that the inhibition caused by sertraline and paroxetine was mixed, i.e. K(m) values increased and V(max) decreased in a concentration dependent manner. Imipramine and clomipramine exhibited competitive inhibition, i.e. K(m) values increased and V(max) remained constant. The present results suggest that these therapeutic agents used for depression can also be considered as inhibitors of snake venom and human serum cholinesterase.     

In vitro effect of pesticides (dichlofluanid, endosulfan, simazine, tolylfluanid and triallate) on proliferative activity of animal derived cell cultures

In this study pesticides with different chemical structures (dichlofluanid, endosulfan, simazine, tolylfluanid and triallate) were examined for their potential cytotoxic effect on proliferative activity of cell cultures of mammalian origin. Cell lines Madin-Darby Bovine Kidney (MDBK), Rabbit Kidney (RK13), Porcine Kidney (PK15), and semicontinual line of Bovine Embryonic Pulmonary Cells (BEPC) were used in the study. From these cell cultures cell proliferative activity was suppressed most intensively in PK15 culture by endosulfan (10(-1) - 10(-6) M). The least effect on cell proliferation in all cell cultures tested, with the exception PK 15 (10(-1) - 10(-2) M), was recorded after simazine exposure. On the basis of IC50 values the cytotoxic effect was: dichlofluanid (IC50 = 10(-3.94) M) > tolylfluanid (IC50 = 10(-3.69) M) > endosulfan (IC50 = 10(-3.24) M) > triallate (IC50 = 10(-3.12) M) > simazine (IC50 = 10(-1.78) M). The comparison of average IC50 values of cell cultures revealed that the most sensitive cell lines were PK15 (IC50 = 10(-3.27) M) and RK13 (IC50 = 10(-3.21) M), whereas MDBK (IC50 = 10(-2.55) M) and BEPC (IC50 = 10(-2.52) M) were less sensitive to pesticide exposure.     

The 5-HT1A receptor probe [3H]8-OH-DPAT labels the 5-HT transporter in human platelets

The present study characterizes a serotonin (5-HT) binding site on human platelet membranes, using [3H]8-OH-DPAT as the radioligand. [3H]8-OH-DPAT binds specifically and saturably to a site on human platelet membranes with an average KD of 43 nM and Bmax of 1078 fmol/mg protein. Determinations of IC50 values for various serotonergic characterizing agents in platelets for displacement of [3H]8-OH-DPAT were performed. For example, 8-OH-DPAT 5HT1A had an IC50 of 117 nM; TFMPP 5HT1B (2.3 microM0 and PAPP 1A + 5HT2 (9 microM); ipsapirone 5HT1A (21.1 microM) and buspirone 5HT1A (greater than 100 microM); ketanserin 5HT2 (greater than 100 microM); 5-HT uptake inhibitors: paroxetine (13 nM); chlorimipramine (73 nM) and fluoxetine (653 nM). The pharmacological inhibitory profile of the platelet 8-OH-DPAT site is not consistent with profiles reported for brain. 8-OH-DPAT does not inhibit [3H]imipramine binding, however, it does inhibit [3H]5-HT uptake in human platelets near 5-HT's Km value (IC50 = 2-4 microM). These results suggest that the human platelet site labeled by [3H]8-OH-DPAT is pharmacologically different from the neuronal site and probably is a component of the 5-HT transporter.