Is clozapine selective for the dopamine D4 receptor?

Binding of ³H-spiperone and ³H-raclopride to membranes of cells stably-transfected with a human dopamine D₂ receptor clone was investigated, as was that of ³H-spiperone to those stably-transfected with a human D₄ receptor clone. ³H-spiperone and ³H-raclopride labeled the same number of sites in the D₂ receptor preparation. The inhibition of binding by clozapine, spiperone, (−) eticlopride, haloperidol and the novel substituted benzamide 1192U90 was also investigated. Clozapine and 1192U90 showed greater inhibition of ³H-raclopride binding than ³H-spiperone binding to the D₂ receptor. Comparison with inhibition of ³H-spiperone binding to the D₄ receptor revealed that clozapine and 1192U90 displayed apparent selectivity (as assessed by K_i ratios) for the D₄ receptor when compared with binding of ³H-spiperone, but not ³H-raclopride, to the D₂ receptor.     

Cation specificity of 3H-sulpiride binding involves alteration in the number of striatal binding sites

Specific ³H-sulpiride binding to rat striatal membranes shows an absolute requirement for the presence of sodium ions in the incubation buffer. Potassium, rubidium and caesium ions were unable to initiate specific ³H-sulpiride binding in a sodium free buffer, and lithium ionscould only partially replace sodium ions. Specific ³H-spiperone binding was unaffected by variation of the cation content of the incubation buffer. The alteration in ³H-sulpiride binding caused by sodium and lithium ions was due predominantly to an increase in the number of available binding sites, rather than to altered receptor affinity. Sodium ions may be essential for the accessability of ³H-sulpiride to a single site labelled also by ³H-spiperone. However, the Ki value for sulpiride displacement of ³H-spiperone in the presence of sodium ions was 20 times greater than the K_D value for ³H-sulpiride binding. So, ³H-sulpiride may interact with a highly sodium dependent binding site distinct from that labelled by ³H-spiperone.     

Purification and kinetic properties of ATP: citrate oxaloacetate lyase from rat brain.

Abstract— A method for a partial purification of ATP:citrate oxaloacetate lyase from rat brain is described. The Lineweaver–Burk plots of velocity vs citrate concentration are biphasic in the presence of fixed concentrations of MgCl₂. Therefore two values of K_m, corresponding to low and high concentrations of citrate, can be determined. When MgCl₂ is added in equimolar concentrations with citrate, a monophasic plot with one K_m of 0.13 mm is obtained. The K_m value for MgATP^2- was independent of citrate concentration, being equal to 0.40–0.43 mm. The K_m for CoA was 0.0007 mm. ADP and P_i are competitive inhibitors with respect to ATP. K_i for MgADP⁻ is equal to 0.13 mm. dl -isocitrate and cis-aconitate are partially competitive inhibitors with respect to citrate with K_i values of 5.8 and 4.8 mm, respectively. α-Ketoglutarate and pyruvate are noncompetitive inhibitors with respect to ATP and citrate, with K_i values equal to 9 and 45 mm, respectively. The physiological significance of these effectors for the regulation of citrate lyase activity in brain is discussed.     

Effects of ouabain on proliferation of human endothelial cells correlate with Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity and intracellular ratio of Na<Superscript>+</Superscript> and K<Superscript>+</Superscript>

Side-by-side with inhibition of the Na⁺,K⁺-ATPase ouabain and other cardiotonic steroids (CTS) can affect cell functions by mechanisms other than regulation of the intracellular Na⁺ and K⁺ ratio ([Na⁺]_i/[K⁺]_i). Thus, we compared the doseand time-dependences of the effect of ouabain on intracellular [Na⁺]_i/[K⁺]_i ratio, Na⁺,K⁺-ATPase activity, and proliferation of human umbilical vein endothelial cells (HUVEC). Treatment of the cells with 1-3 nM ouabain for 24-72 h decreased the [Na⁺]_i/[K⁺]_i ratio and increased cell proliferation by 20-50%. We discovered that the same ouabain concentrations increased Na⁺,K⁺-ATPase activity by 25-30%, as measured by the rate of ⁸⁶Rb⁺ influx. Higher ouabain concentrations inhibited Na⁺,K⁺-ATPase, increased [Na⁺]_i/[K⁺]_i ratio, suppressed cell growth, and caused cell death. When cells were treated with low ouabain concentrations for 48 or 72 h, a negative correlation between [Na⁺]_i/[K⁺]_i ratio and cell growth activation was observed. In cells treated with high ouabain concentrations for 24 h, the [Na⁺]_i/[K⁺]_i ratio correlated positively with proliferation inhibition. These data demonstrate that inhibition of HUVEC proliferation at high CTS concentrations correlates with dissipation of the Na⁺ and K⁺ concentration gradients, whereas cell growth stimulation by low CTS doses results from activation of Na⁺,K⁺-ATPase and decrease in the [Na⁺]_i/[K⁺]_i ratio.     

Zero extracellular K+ and prostaglandin E release in the guinea-pig taenia coli

Prostaglandin E release rates from isolated strips of guinea-pig taenia coli increased during exposure to zero K⁺ bathing fluid, from control values of $\text{0.78 ± 0.11}\text{ng/g}$ per min to levels as high as 29.2 ng/per min. Release rates increased for 40–50 min and then remained constant or fell despite progressive increases in intracellular sodium [Na_i⁺] or fall in intracellular potassium [K_i⁺]. Readmittance of K⁺ to the bathing solution resulted in rapid reversal of elevated prostaglandin E release rates. [Na⁺_i] and [K⁺_i] were markedly more abnormal in strips exposed to zero K⁺ for 70–201 min compared to 30-min exposures. Upon the readdition of K⁺ after long zero K⁺ exposure, the rate of prostaglandin E release fell long before [Na⁺_i] and [K⁺_i] returned to control levels. After K⁺ was readded to the bathing solution, the ion concentration of tissues exposed to zero K⁺ for 30 min returned to normal much more quickly than did those of tissues exposed for the longer time periods, yet the exponential rate constants for fall of prostaglandin E release rate after K⁺ was added were not significantly different after short or long zero K⁺ exposure. Thus there was a dissociation between the return of [Na⁺_i] and [K⁺_i] and the fall of prostaglandin E release rate to control levels. Ouabain augmented prostaglandin E release under conditions where [K⁺_i] could not fall. Addition of known neurotransmitters present in this tissue to the bathing fluid did not augment prostaglandin E release. Guinea-pig taenia coli strips that had been incubated with [³H]arachidonic acid, constantly released [³H]arachidonic acid and [³H]prostaglandin E and a prostaglandin which cochromatographed with prostaglandin E but could not be converted to prostaglandin B by alkali and was shown to be 6-ketoprostaglandin F_1α. Release of [³H]arachidonic acid and [³H]prostaglandin E plus 6-[³H]ketoprostaglandin F_1α was increased when strips were exposed to zero K⁺. Data obtained in this study suggest the augmented prostaglandin E release seen during zero K⁺ or ouabain is related to increased availability of unbound arachidonic acid at the site of cyclooxygenase in the cell. Augmented prostaglandin E release is apparently not related to alterations in intracellular electrolyte concentrations or release of known neurotransmitters.     

Reaction mechanism of phosphoribulokinase from a cyanobacterium,Synechococcus PCC7942

The dependence of the activity of phosphoribulokinase isolated from a cyanobacterium, Synechococcus PCC7942, on Mg²⁺ showed that its real substrates were Mg-ATP and free D-ribulose 5-phosphate. On the basis of results of kinetic inhibition studies and previously reported result of affinity chromatography, an ordered bi bi mechanism in which Mg-ATP binds before ribulose 5-phosphate is proposed. The K_m values for ATP and D-ribulose 5-phosphate were 0.09 and 0.27 mM, respectively. K_i values of ADP and D-ribulose 1,5-bisphosphate were 0.32 and 10.0 mM, respectively. Inhibition constants K_i1 and K_i2 for 6-phosphogluconate were 9.3 and 0.49 mM. K_ia was 0.13 mM. New kinetics on PRK gave higher control coefficient than the kinetics on Spinach PRK did in the model with PRK activity from 175 to 1000 µmol min^–1 mg^–1 chl.     

Cytosolic free calcium concentrations in synaptosomes during histotoxic hypoxia

Altered cytosolic free calcium concentrations ([Ca²⁺]_i) accompany impaired brain metabolism and may mediate subsequent effects on brain function and cell death. The current experiments examined whether hypoxia-induced elevations in [Ca²⁺]_i are from external or internal sources. In the absence of external calcium, neither KCl depolarization, histotoxic hypoxia (KCN), nor the combination changed [Ca²⁺]_i. However, with external CaCl₂ concentrations as small as 13 M, KCl depolarization increased [Ca²⁺]_i instantaneously while hypoxia gradually raised [Ca²⁺]_i. The combination of KCN and KCl was additive. Increasing external calcium concentrations up to 2.6 mM exaggerated the effects of K⁺ and KCN on [Ca²⁺]_i, but raising medium calcium to 5.2 mM did not further augment the rise. Diminishing the sodium in the media, which alters the activity and perhaps the direction of the Na/Ca exchanger, reduced the increase in [Ca²⁺]_i due to hypoxia, but enhanced the KCl response. The changes in ATP following K⁺ depolarization, KCN or their combination in the presence of physiological calcium concentrations did not parallel alterations in [Ca²⁺]_i, which suggests that diminished activity of the calcium dependent ATPase does not underlie the elevation in [Ca²⁺]_i. Valinomycin, an ionophore which reduces the mitochondrial membrane potential, elevated [Ca²⁺]_i and the effects were additive with K⁺ depolariration in a calcium dependent manner that paralleled the effects of hypoxia. Together these results suggest that hypoxia-induced elevations of synaptosomal [Ca²]_i are due to an inability of the synaptosome to buffer entering calcium.     

Effects of potassium on the anion and cation contents of primary cultures of mouse astrocytes and neurons

Inastrocytes, as [K⁺]_o was increased from 1.2 to 10 mM, [K⁺]_i and [Cl^–]_i were increased, whereas [Na⁺]_i was decreased. As [K⁺]_o was increased from 10 to 60 mM, intracellular concentration of these three ions showed no significant change. When [K⁺]_o was increased from 60 to 122 mM, an increase in [K⁺]_i and [Cl^–]_i and a decrease in [Na⁺]_i were observed.Inneurons, as [K⁺]_o was increased from 1.2 to 2.8 mM, [Na⁺]_i and [Cl^–]_i were decreased, whereas [K⁺]_i was increased. As [K⁺]_o was increased from 2.8 to 30 mM, [K⁺]_i, [Na⁺]_i and [Cl^–]_i showed no significant change. When [K⁺]_o was increased from 30 to 122 mM, [K⁺]_i and [Cl^–]_i were increased, whereas [Na⁺]_i was decreased. Inastrocytes, pH_i increased when [K⁺]_o was increased. Inneurons, there was a biphasic change in pH_i. In lower [K⁺]_o (1.2–2.8 mM) pH_i decreased as [K⁺]_o increased, whereas in higher [K⁺]_o (2.8–122 mM) pH_i was directly related to [K⁺]_o. In bothastrocytes andneurons, changes in [K⁺]_o did not affect the extracellular water content, whereas the intracellular water content increased as the [K⁺]_o increased. Transmembrane potential (E_m) as measured with Tl-204 was inversely related to [K⁺]_o between 1.2 and 90 mM, a ten-fold increase in [K⁺]_o depolarized the astrocytes by about 56 mV and the neurons about 52 mV. The E_m values measured with Tl-204 were close to the potassium equilibrium potential (E_k) except those in neurons at lower [K⁺]_o. However, they were not equal to the chloride equilibrium potential (E_Cl) at [K⁺]_o lower than 30 mM in both astrocytes and neurons. Results of this study demonstrate that alteration of [K⁺]_o produced different changes in [K⁺]_i, [Na⁺]_i, [Cl^–]_i, and pH_i in astrocytes and neurons. The data show that astrocytes can adapt to alterations in [K⁺]_o, in such a way to maintain a more suitable environment for neurons.     

Glyceraldehyde 3-Phosphate:NADP Reductase of Spinach Leaves : Steady State Kinetics and Effect of Inhibitors

The steady state kinetics of glyceraldehyde 3-phosphate:NADP⁺ oxidoreductase (GNR) (EC 1.2.1.9) have been investigated. The enzyme exhibits hyperbolic behavior over a wide range of substrate concentrations. Double-reciprocal plots are nearly parallel or distantly convergent with limiting K_m values of 2 to 5 micromolar for NADP⁺ and 20 to 40 micromolar for D-glyceraldehyde 3-phosphate (G3P). The velocity response to NADP⁺ as the varied substrate is however sigmoidal if G3P concentration exceeds 10 micromolar, whereas the response to G3P may show inhibition above this concentration. This `G3P-inhibited state' is alleviated by saturating amounts of NADP⁺ or NADPH. Product inhibition patterns indicate NADPH as a potent competitive inhibitor to NADP⁺ (K_i 30 micromolar) and mixed inhibitor towards G3P, and 3-phosphoglycerate (3PGA) as mixed inhibitor to both NADP⁺ and G3P (K_i 10 millimolar). The data, and those obtained with dead-end inhibitors, are consistent with a nonrapid equilibrium random mechanism with two alternative kinetic pathways. Of these, a rapid kinetic sequence (probably ordered with NADP⁺ binding first and G3P binding as second substrate) is dominant in the range of hyperbolic responses. A reverse reaction with 3PGA and NADPH as substrates is unlikely, and was not detected. Of a number of compounds tested, erythrose 4-phosphate (K_i 7 micromolar) and Pi (K_i 2.4 millimolar) act as competitive inhibitors to G3P (uncompetitive towards NADP⁺) and are likely to affect the in vivo activity. Ribose 5-phosphate, phosphoenolpyruvate, ATP, and ADP are also somewhat inhibitory. Full GNR activity in the leaf seems to be allowed only under high photosynthesis conditions, when levels of several inhibitors are low and substrate is high. We suggest that a main function of leaf GNR is to supply NADPH required for photorespiration, the reaction product 3PGA being cycled back to chloroplasts.     

Modulation of gating of a metabolically regulated,ATP-dependent K+ channel by intracellular pH in B cells of the pancreatic islet

Summary Membrane-permeant weak acids and bases, when applied to the bath, modulate the resting membrane potential and the glucose-induced electrical activity of pancreatic B cells, as well as their insulin secretion. These substances alter the activity of a metabolite-regulated. ATP-sensitive K⁺ channel which underlies the B-cell resting potential. We now present several lines of evidence indicating that the channel may be directly gated by pH _i . (1) The time course of K⁺(ATP) channel activity during exposure to and washout of NH₄Cl under a variety of experimental conditions, including alteration of the electrochemical gradient for NH₄Cl entry and inhibition of the Na _o ⁺ H _i ⁺ exchanger, resembles the time course of pH _i measured in other cell types that have been similarly treated. (2) Increasing pH _o over the range 6.25–7.9 increases K⁺(ATP) channel activity in cell-attached patches where the cell surface exposed to the bath has been permeabilized to H⁺ by the application of the K⁺/H⁺ exchanger nigericin. (3) Increasing pH _i over a similar range produces similar effects on K⁺(ATP) channels in inside-out excised patches exposed to small concentrations of ATP _i . The physiological role of pH _i in the metabolic gating of this channel remains to be explored.     

Properties of the phosphate and phosphite transport systems of Phytophthora palmivora

Germlings of Phytophthora palmivora possess at least two systems for the uptake of inorganic phosphate (P_i). The first is synthesized on germination in medium containing 50 M P_i and has a K_m of approx. 30 M (V_max=7–9 nmol P_i/h·10⁶ cells). The second is synthesized under conditions of P_i-deprivation and has a higher affinity for P_i (K_m=1–2 M), but a lower V_max (0.5–2 nmol P_i/h·10⁶ cells). The fungicide phosphite likewise enters the germlings via two different transport systems, the synthesis of which also depends on the concentration of P_i in the medium. The K_m of the lower affinity system is 3 mM (V_max=20 nmol phosphite/h·10⁶ cells) and that of the higher affinity system is 0.6 mM (V_max=12 nmol/h·10⁶ cells). P_i and phosphite are competitive inhibitors for each other's transport in both systems. However, whereas mM concentrations of phosphite are necessary to inhibit P_i transport, only M concentrations of P_i are required to inhibit phosphite transport. A third system of uptake for P_i also exists, since when phosphate-deprived cells are presented with mM concentrations of P_i, they transport the anion at a very high rate (around 100 nmol/h·10⁶ cells). High rates of transport of phosphite are also observed when these cells are presented with mM concentrations of this anion.     

ARA290 Improves Insulin Release and Glucose Tolerance in Type 2 Diabetic Goto-Kakizaki Rats

Effects of ARA290 on glucose homeostasis were studied in type 2 diabetic Goto-Kakizaki (GK) rats. In GK rats receiving ARA290 daily for up to 4 wks, plasma glucose concentrations were lower after 3 and 4 wks, and hemoglobin A_1c (Hb A_1c) was reduced by ~20% without changes in whole body and hepatic insulin sensitivity. Glucose-stimulated insulin secretion was increased in islets from ARA290-treated rats. Additionally, in response to glucose, carbachol and KCl, islet cytoplasmic free Ca²⁺ concentrations, [Ca²⁺]_i, were higher and the frequency of [Ca²⁺]_i oscillations enhanced compared with placebo. ARA290 also improved stimulus–secretion coupling for glucose in GK rat islets, as shown by an improved glucose oxidation rate, ATP production and acutely enhanced glucose-stimulated insulin secretion. ARA290 also exerted an effect distal to the ATP-sensitive potassium (K_ATP) channel on the insulin exocytotic pathway, since the insulin response was improved following islet depolarization by KCl when K_ATP channels were kept open by diazoxide. Finally, inhibition of protein kinase A completely abolished effects of ARA290 on insulin secretion. In conclusion, ARA290 improved glucose tolerance without affecting hematocrit in diabetic GK rats. This effect appears to be due to improved β-cell glucose metabolism and [Ca²⁺]_i handling, and thereby enhanced glucose-induced insulin release.     

K+ channels of stomatal guard cells: Abscisic-acid-evoked control of the outward rectifier mediated by cytoplasmic pH

The activation by abscisic acid (ABA) of current through outward-rectifying K⁺ channels and its dependence on cytoplasmic pH (pH_i) was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with multibarrelled and H⁺-selective microelectrodes to record membrane potentials and pH_i during exposures to ABA and the weak acid butyrate. Potassium channel currents were monitored under voltage clamp and, in some experiments, guard cells were loaded with pH buffers by iontophoresis to suppress changes in pH_i. Following impalements, stable pH_i values ranged between 7.53 and 7.81 (7.67±0.04, n = 17). On adding 20 M ABA, pH_i rose over periods of 5–8 min to values 0.27±0.03 pH units above the pH_i before ABA addition, and declined slowly thereafter. Concurrent voltage-clamp measurements showed a parallel rise in the outward-rectifying K⁺ channel current (I_{K, out}) and, once evoked, both pH_i and I_{K, out} responses were unaffected by ABA washout. Acid loads, imposed with external butyrate, abolished the ABA-evoked rise in I_{K, out}. Butyrate concentrations of 10 and 30 mM (pH₀ 6.1) caused pH_i to fall to values near 7.0 and below, both before and after adding ABA, consistent with a cytoplasmic buffer capacity of 128±12 mM per pH unit (n = 10) near neutrality. Butyrate washout was characterised by an appreciable alkaline overshoot in pH_i and concomitant swell in the steady-state conductance of I_{K, out}. The rise in pH_i and i_{K, out} in ABA were also virtually eliminated when guard cells were first loaded with pH buffers to raise the cytoplasmic buffer capacity four- to sixfold; however, buffer loading was without appreciable effect on the ABA-evoked inactivation of a second, inward-rectifying class of K⁺ channels (I_{K, in}). The pH_i dependence of I_{K, out} was consistent with a cooperative binding of at least 2H⁺ (apparent pK_a = 8.3) to achieve a voltage-independent block of the channel. These results establish a causal link previously implicated between cytoplasmic alkalinisation and the activation of I_{K, out} in ABA and, thus, affirm a role for H⁺ in signalling and transport control in plants distinct from its function as a substrate in H⁺-coupled transport. Additional evidence implicates a coordinate control of I_{K, in} by cytoplasmic-free [Ca²⁺] and pH_i.Abbreviations ABA abscisic acid - [Ca²⁺]_i cytoplasmic free [Ca²⁺]_i - E_K K⁺ equilibrium potential - I_{K, out}, I_{K, in} outward-, inward-rectifying K⁺ channel (current) - I-V current-voltage (relation) - Mes 2-(N-morpholino)ethanesulfonic acid - pH_i cytoplasmic pH - Tes 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-amino}ethanesulfonic acid - V_m membrane potential We are grateful to G. Thiel (Pflanzenphysiologisches Institut, Universität Göttingen, Germany) for helpful discussions. This work was possible with equipment grants-in-aid from the Gatsby Charitable Foundation, the Royal Society and the University of London Central Research Fund. F.A. holds a Sainsbury Studentship.     

Vinyl functionalized 5,6-dimethylbenzimidazolium salts: Synthesis and biological activities

A series of vinyl functionalized 5,6-dimethylbenzimidazolium salts are synthesized. All compounds were fully characterized by elemental analyses, MS, ¹H-NMR, ¹³C-NMR, and IR spectroscopy techniques. Enzyme inhibition is a very active area of research in drug design and development. In this study, the synthesized novel benzimidazolium salts were evaluated toward the human erythrocyte carbonic anhydrase I (hCA I), and II (hCA II) isoenzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. They demonstrated highly potent inhibition ability against hCA I with K_i values of 484.8 ± 62.6–1389.7 ± 243.2 nM, hCA II with K_i values of 298.9 ± 55.7–926.1 ± 330.0 nM, α-glycosidase with K_i values of 170.3 ± 27–760.1 ± 269 μM, AChE with K_i values of 27.1 ± 3–77.6 ± 1.7 nM, and BChE with K_i values of 21.0 ± 5–61.3 ± 15 nM. As a result, novel vinyl functionalized 5,6-dimethylbenzimidazolium salts (1a–g) exhibited effective inhibition profiles toward studied metabolic enzymes. Therefore, we believe that these results may contribute to the development of new drugs particularly to treat some global disorders including glaucoma, Alzheimer's disease, and diabetes.     

Reference Phase Analysis of Free and Bound Intracellular Solutes: II. Isothermal and Isotopic Studies of Cytoplasmic Sodium, Potassium, and Water

The intracellular reference phase (RP) method and ultra-low temperature micro-dissection were used for isothermal and isotopic phase distribution studies of Na⁺, K⁺, and water in amphibian oocyte cytoplasm. One-third of the cytoplasmic water is available as solvent for [³H]sucrose. This fraction, designated c1, quantitatively coincides with the water volume in which Na⁺ and K⁺ are freely diffusible. Two-thirds of the cytoplasmic water is inaccessible to sucrose and is designated c2. The Na⁺ and K⁺ associated with c2 are extremely slowly exchanging (bound) and at different concentrations than in c1. The cations in c1 are in mass-action equilibria with those in c2, each described by an equation of the form

C^c_i = C^c1_i + C^c2_i = q_i·C^RP_i + ^maxC^c2_i·f(C^RP_i

in which C^c_i is the cytoplasmic Na⁺ or K⁺ concentration, C^c1_i is the free, and C^c2_i the bound cation concentration averaged over the cytoplasmic water. q_i is the fractional free solute space, C^RP_i the RP concentration, ^maxC^c2_i the concentration of binding sites, and the function f is satisfied by the Langmuir isotherm. Numerical values for the variables of the isotherm are determined. Activity coefficients are calculated from RP data and provide a basis for generalizing the oocyte results to other cells. The conclusion is drawn that both c1 and c2 are widely distributed in cells, and that cellular ionic activities involve two distinct systems: the cell-membrane system and an adsorbed water ion-exchange-like buffering system. Alternative explanations for the two-component cytoplasm are considered. A model is proposed in which c1 is a normal intracellular aqueous phase controlled by the plasma membrane, whereas c2 consists of water and ions adsorbed in hydrate crystalline structures. In oocytes these structures are identified with yolk platelets.

     

Development of selective and reversible pyrazoline based MAO-A inhibitors: Synthesis,biological evaluation and docking studies

3,5-Diaryl pyrazolines analogs were synthesized and evaluated for their monoamine oxidase (MAO) inhibitory activity. The compounds were found reversible and selective towards MAO-A with selectivity index in the magnitude of 10³–10⁵. The docking studies were carried out to gain further structural insights of the binding mode and possible interactions with the active site of MAO-A. Interestingly, the theoretical (K_i) values obtained by molecular docking studies were in congruence with their experimental (K_i) values.     

The role of calmodulin on Ca2+-dependent K+ transport regulation in the human red cell

Several lipophilic calmodulin antagonists (phenotiazines, butyrophenones and diphenylbutylpiperidines) inhibited Ca²⁺-induced loss of KCl from human red cells. However, the K_i values for this effect did not bear good correlation with the K_i values reported for well-known calmodulin-dependent systems. In addition, the inhibition was strongly dependent on the haematocrit and valinomycin-induced KCl fluxes were also affected. Added calmodulin did not have any effect on Ca²⁺-dependent ⁸⁶Rb uptake by inside-out vesicles derived from red cell membranes whereas stimulation of Ca²⁺-dependent ATPase was apparent. Lipophilic anticalmodulins at high doses had all kinds of effects on ⁸⁶Rb uptake by inside-out vesicles: increase, decrease or no change of the fraction of activated vesicles reached at submaximal Ca²⁺ concentrations, with or without modification of the relative rate of ⁸⁶Rb uptake. The hydrophylic compound 48/80 decreased the fraction of activated vesicles reached at submaximal Ca²⁺ concentrations without affecting the relative rate of ⁸⁶Rb uptake, but this effect took place only at concentrations 10-fold higher than the reported K_i for calmodulin-dependent systems. These results suggest that Ca²⁺-dependent K⁺ channels of red cells are not regulated by calmodulin.     

Changes in free-calcium levels and pH in synaptosomes during transmitter release

The free cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) in rat brain synaptosomes estimated by the indicator quin 2 is 104±8 nM (S.D.) in artificial cerebrospinal fluid (1.2 mM Ca²⁺), but decreases at lower Ca²⁺ concentrations in the medium. The presence of quin 2 in the synaptosomes does not affect either the spontaneous release of transmitter (γ-aminobutyric acid) or the release induced by K⁺ depolarisation. In quin 2-loaded synaptosomes, depolarisation by K⁺ causes an abrupt increase in [Ca]_i (less than 2-fold) that is approximately proportional to the extent of depolarisation, whereas depolarisation by veratrine alkaloids produces a slow rise in [Ca]_i. The increase in [Ca]_i produced by K⁺ depolarisation does not occur in the absence of Ca²⁺ in the medium. The data are consistent with a direct correlation between [Ca_i] and transmitter release in functional synaptosomes. The pH in synaptosomes estimated by the indicator quene 1 is 7.04±0.07 and is stable in media containing 5 mM bicarbonate. The pH in synaptosomes was decreased by protoveratrine but not by K⁺ depolarisation.     

MEMBRANE THIAMINE TRTPHOSPHATASE FROM RAT BRAIN: INHIBITION BY ATP AND ADP

—The hydrolysis of ThTP by rat brain membrane-bound ThTPase is inhibited by nucleoside diphosphates and triphosphates. ATP and ADP are most effective, reducing hydrolysis by 50% at concentrations of 2 × 10^?5m and 7·5 × 10^?5m respectively. Nucleoside monophosphates and free nuclcosides as well as P_i have no effect on enzyme activity. ThMP and ThDP also fail to inhibit hydrolysis in concentrations up to 5 × 10^?3m . Non-hydrolysable methylene phosphate analogs of ATP and ADP were used in further kinetic studies with the ThTPase. The mechanism of inhibition by these analogs is shown to be of mixed non-competitive nature for both compounds. An observed K_i, of 4 × 10^?5m for the ATP analog adenosine-PPCP and 9 × 10^?5m for the ADP analog adenosine-PCP is calculated at pH 6·5. Formation of the true enzyme substrate, the [Mg²⁺. ThTP] complex, is not significantly affected by concentrations of analogs producing maximal (>95%) inhibition of enzyme activity. Likewise the relationships between pH and observed K_m and pH and V_max are not shifted by the presence of similar concentrations of inhibitor.