Involvement of tropomyosin in the sensitivity of Na+ + K+ ATPase to ouabain

The Na⁺/K⁺ ATPase sensitivity to ouabain was shown to be increased by 300 to 1000-fold after treatment of the plasma membrane by EDTA. Addition of proteins detached from the plasma membrane with Ca²⁺ ions to EDTA treated membranes reconstituted the original Na⁺/K⁺ ATPase resistance to ouabain inhibition. Tropomyosin with Ca²⁺ ions (not with Mg²⁺ ions) induced the same effect. When suboptimal doses of tropomyosin were used for such a reconstitution, the dose-response curve indicated a full reconstitution of a given percentage of enzyme molecules. This observation led us to assume a direct or indirect effect of tropomyosin on Na⁺/K⁺ ATPase functions.     

Effect of ethanol on the kinetics of rat brain (Na+ + K+) ATPase and K+-dependent phosphatase with different alkali ions.

(Na⁺ + K⁺)-dependent ATPase [(Na + K)-ATPase] and K⁺-dependent p-nitrophenyl phosphatase [pNPPase] activities in rat brain heavy microsomal fractions were studied in the presence of 120 mM Na⁺ and varied concentrations of K⁺, Rb⁺, Cs⁺, Li⁺ or NH₄⁺. Scatchard and Hill plots indicated non-hyperbolicity (cooperativity) with all except Li⁺, which supported a considerably lower activity than any of the other ions tested. Addition of 0.22 M ethanol to the incubation mixtures produced a formally competitive inhibition of ATPase activity with K⁺, Rb⁺ and Cs⁺, a non-competitive inhibition with Li⁺, and a mixed inhibition with NH₄⁺. The changes in pNPPase activity generally followed a similar but less clear-cut pattern. The values of the Hill constants were not changed for either enzyme activity. The findings are interpreted as evidence that ethanol inhibits ATPase activity by inducing conformational changes which alter the consequences of ion binding to the various receptor sites.     

Synthesis and characterization of Pyrene/Quinoline based Zn2+ selective fluorescent sensor

We have synthesized a new Pyrene/Quinoline-based Zn²⁺ selective fluorescent sensor and characterized metal ion sensing properties. The fluorescent Zn²⁺ sensor, Py2, was synthesized by reaction of pyrene-1-carbaldehyde with quinoline derivative in high yield. We found that Py2 showed a good selectivity toward Zn²⁺ over other metal ions in the mixed H₂O-ethanol solution. In the absence of Zn²⁺, fluorescence was observed at 420 nm upon 340 nm photo-excitation. Upon addition of 5 equiv of tested metal ions, only Zn²⁺ leads to the appearance of new fluorescence band at 500 nm. Since Py2 exhibited fluorescence changes upon the addition of Zn²⁺ with high selectivity, it could be applicable in a variety of area such as environment monitoring and diagnostic analysis.     

Structural stability of β-lactoglobulin in the presence of kosmotropic salts A kinetic and thermodynamic study

The thiol group of β-lactoglobulin reacted very sluggishly with dithio-bis-nitro-benzoic acid as compared to that of glutathione at pH 6.85. The pK_app value of the thiol group of the protein was 9.35. In the presence of 3 M urea, the thiol group reacted completely with dithio-bis-nitrobenzoic acid at pH 6.85. Heating (from 50° to 80°) increased the exposure of the thiol by dissociating the dimer unit. From the pseudo-first order rate constants of heat-exposure of thiol, thermodynamic activation parameters, ΔG⁺₊, ΔH⁺₊, and ΔS⁺₊, for the heat-dissociation of β-lactoglobulin dimer were estimated to be 23 290 cal/mol, 31 160cal/mol, and 22.9 e.u. (at 70°), respectively. Addition of kosmotropic salts, chloride, tartrate, sulfate, phosphate, and citrate (0.2 m ) decreased the heat-induced exposure of the thiol group (at 70°), probably by decreasing the dissociation of the dimer at pH 6.85. The relative change in free energy of activation for the dissociation of the dimer, Δ(ΔG⁺_+dimer), in the presence of the salts was positive, suggesting that these additives increase the stability of the dimer against heat. These salts also increased the conformational stability of β-lactoglobulin as revealed by an increase in - Δ(ΔG⁰_conf) values in their presence. Both Δ(ΔG⁺_+dimer) and - Δ(ΔG⁰_conf) values followed the order, chloride < tartrate < sulfate < phosphate < citrate. These salts seem to manifest their structure-stabilizing effect by increasing both inter- and intramolecular hydrophobic interactions via changes in structure of water.     

Characteristics of palytoxin-induced cation currents and Ca2+ mobilization in smooth muscle cells of rabbit portal vein

We examined the nature of the palytoxin (PTX)-induced channel and its relevance to the Ca²⁺ mobilizing effect of the toxin on smooth muscle cells isolated from rabbit portal vein using whole-cell voltage-clamp and microfluorimetric techniques. PTX (1 nM) induced a sustained, irreversible inward current at a holding potential of –40 mV. The PTX-induced current reversed at 0.5 ± 0.6 mV, and the PTX-induced channel permitted the passage of Na⁺, K⁺, Cs⁺ and, to a lesser extent, Li⁺, but not choline⁺ or Ca²⁺. During the sustained phase of the current, superfusion of Ni²⁺ (5 mM), La³⁺ (0.5 mM) or 2,4-dichlorobenzamil (2,4-DCB, 25 μM) reduced the current amplitude and decreased the slope conductance without changing the reversal potential. In 5 of 7 experiments, ouabain transiently increased the PTX-induced inward current and shifted the reversal potential in a positive direction. Subsequently, ouabain inhibited the current in every cell. PTX (10 nM) induced a sustained rise in cytosolic Ca²⁺ ([Ca²⁺]_i), which was resistant to verapamil but suppressed by omission of extracellular Ca²⁺. When external Na⁺ was replaced by choline⁺, PTX did not increase [Ca²⁺]_i. Pretreatment with 2,4-DCB prevented the elevation of [Ca²⁺]_i due to PTX. These results suggest that PTX does not directly stimulate Ca²⁺ entry but induces entry through Na⁺-Ca²⁺ exchange as a consequence of increased cytosolic Na⁺. Ni²⁺, La³⁺, 2,4-DCB and ouabain were shown to act as blockers of the PTX-induced channel. Ouabain may also inhibit Na⁺ pump current activated by cytosolic Na⁺. Received: 15 May 1996 / Accepted: 28 August 1996     

Enzyme modifications that alter interactions of K+ and cardioactive steroids with (Na+ + K+)-dependent ATPase

Treatment of a purified (Na ⁺ + K ⁺-dependent ATPase preparation from dog kidney medulla with acetic anhydride or trinitrobenzene sulfonate (TNBS) produced a dose-dependent irreversible inactivation of the (Na ⁺ + K ⁺)-ATPase and K ⁺-phosphatase reactions catalyzed by the enzyme. Both K ⁺ and the cardioactive steroid strophanthidin protected against inactivation. Prior treatment with concentrations of acetic anhydride or TNBS that produced only partial inactivation also modified the residual activity of the enzyme, as manifested by an increase in the K_0.5 for K ⁺ as activator of the phosphatase reaction (mediated through the moderate-affinity α-sites for K ⁺) but not in the K_0.5, for K ⁺ as activator of the ATPase reaction (mediated through the high-affinity β-sites for K ⁺): correspondingly. the I ₅₀ values (concentrations required to produce 50 per cent inhibition) for ouabain and strophanthidin as inhibitors of the ATPase reaction were increased, and the binding of [³H]ouabain was decreased, by such treatment. Ouabain activated the phosphatase reaction in the absence of K ⁺ and. alter similar treatment, the apparent affinity for ouabain as activator was also decreased. These experiments demonstrate interactions between cardioactive steroids at their sites on the extracellular face of this transmembrane enzyme and K ⁺ at its moderate-affinity α-sites on the intracellular face. and further indicate that K ⁺ can modulate enzyme-drug interactions at such sites, as well as through K ⁺ -sites on the extracellular face of the enzyme.     

Design of peptides with αβ-dehydro residues: Synthesis,crystal structure and molecular conformation of N-Boc-L-Ile-ΔPhe-L-Trp-OCH3

The dehydro-peptide Boc-L-Ile-ΔPhe-L-Trp-OCH₃ was synthesized by the azlactone method in the solution phase. The peptide was crystallized from methanol in an orthorhombic space group P2₁2₁2₁ with a = 10.777(2), b= 11.224(2), c= 26.627(10) Å. The structure was determined by direct methods and refined to an R value of 0.069 for 3093 observed reflections [l≥ 2σ(l)].The peptide failed to adopt a folded conformation with backbone torsion angles: φ₁, = 90.8(8)°, ψ₁= -151.6(6)°, φ₂= 89.0(8)°, ψ₂= 15.9(9)°, φ₃= 165.7(7)°, ψ^T₃= -166.0(7)°. A general rule derived from earlier studies indicates that a three-peptide unit sequence with a ΔPhe at the (i+ 2) position adopts a β-turn II conformation. Because the branched β-carbon residues such as valine and isoleucine have strong conformational preferences, they combine with the ΔPhe residue differently to generate a unique set of conformations in such peptides. The presence of β-branched residues simultaneously at both (i+ 1) and (i+ 3) positions induces unfolded conformations in tetrapeptides, but a β-branched residue substituted only at (i+ 3) positron can not prevent the formation of a folded β-turn II conformation. On the other hand, the present structure shows that a β-branched residue substituted at the (i+ 1) position prevents the formation of a β-turn II conformation. These observations indicate that a β-branched residue at the (i+ 1) position prevents a folded conformation whereas it cannot generate the same degree of effect from the (i+ 3) position. This may be because of the trans disposition of the planar ΔPhe side-chain with respect to the C=O group in the residue. The molecules are packed in an anti-parallel manner to generate N₂-H₂…O₂ (-x,y-1/2, -z+ 3/2) and N^ε1₃-H^ε1₃…O₁(-x,y -1/2, -z+ 3/2) hydrogen bonds.     

The versatility of polysorbate 80 (tween 80) as an ionophore

A number of experiments were performed to illustrate the unusual versatility of Polysorbate 80 (Tween 80) as an ionophore. New ions shown to be transported by it from and to water layers through a model membrane (CH₂Cl₂) include H₃O⁺, Li⁺, Pb²⁺, Co²⁺, piperidinium ion, guanidinium ion, and Paraquat, while two complex ions resisted transport under the conditions used. “Reverse” transport of lipophilic guests (azobenzene, azulene, ferrocene) from and to organic solvents through water was also promoted by Tween 80, but C₆₀ was not carried. Three water molecules were transported per molecule of KSCN by the Tween.     

Lanthanides inhibit the human noradrenaline, 5-hydroxytryptamine and dopamine transporters

Transporters for the monoamine neurotransmitters, including noradrenaline, 5-hydroxytryptamine [5-HT] and dopamine, have twelve transmembrane spanning regions and cotransport Na⁺ and Cl^– ions. Another family of Na⁺-dependent transporters is that containing the Na⁺/glucose and Na⁺/proline cotransporters that are found in the epithelial cells of renal and intestinal brush border membranes. It has been shown that various trivalent lanthanides can substitute for Na⁺ for transport of glucose and proline. The aim of this study was to determine the effects of lanthanides on the activities of the human noradrenaline, 5-HT and dopamine transporters. Cultured cells were incubated for 2min with 10nM ³H-noradrenaline (SK-N-SH-SY5Y human neuroblastoma cells), ³H-5-HT (JAR human placental choriocarcinoma cells) or ³H-dopamine (COS-7 cells transfected with the cDNA of the human dopamine transporter). Specific amine uptake was determined as the difference between accumulation of the amine in the cells in the absence and presence of a corresponding uptake inhibitor. Under both isotonic (150mM NaCl or LiCl or 90mM lanthanide salt) and hypertonic (150mM NaCl + 100mM LiCl, 250mM LiCl or 150mM lanthanide salt) conditions, replacement of Na⁺ by Li⁺, La³⁺, Eu³⁺ or Sm³⁺ abolished the specific uptake of noradrenaline in SK-N-SH-SY5Y cells and replacement of Na⁺ by Li⁺ or Eu³⁺ decreased the specific uptake of 5-HT in JAR cells by 94–100% and that of dopamine in transfected COS-7 cells by 95–99%. The direct effects of Eu³⁺ (with Na⁺ present) on the human noradrenaline transporter in SK-N-SH-SY5Y cells were also examined. Eu³⁺ inhibited noradrenaline uptake into the cells (IC₅₀ 2.6mM) and nisoxetine binding to crude membranes of SK-N-SH-SY5Y cells (IC₅₀ 4.7mM) with similar potencies. Further experiments showed that 4.5mM EuCl₃ in the presence of 150mM Na⁺ caused a 3.5-fold increase in the K_m of noradrenaline and no change in the maximal rate of noradrenaline uptake. EuCl₃ (4.5mM) also caused a pronounced inhibition of the Na⁺-dependent stimulation of noradrenaline uptake by SK-N-SH-SY5Y cells. It can be concluded from these data that, in contrast with the Na⁺/glucose and Na⁺/proline cotransporters, the lanthanides cannot substitute for Na⁺ in the transport of substrates by the monoamine neurotransmitter transporters and that the lanthanides inhibit the latter transporters by interacting with sites of the transporters involved in amine and Na⁺ binding. Received: 15 July 1996 / Accepted: 28 January 1997     

Enhancing the potency of lithospermate B for inhibiting Na+/K+-ATPase activity by forming transition metal ion complexes

Aim:

To determine whether replacing Mg²⁺ in magnesium lithospermate B (Mg-LSB) isolated from danshen (Salvia miltiorrhiza) with other metal ions could affect its potency in inhibition of Na⁺/K⁺-ATPase activity.

Methods:

Eight metal ions (Na⁺, K⁺, Mg²⁺, Cr³⁺, Mn²⁺, Co²⁺, Ni²⁺, and Zn²⁺) were used to form complexes with LSB. The activity of Na⁺/K⁺-ATPase was determined by measuring the amount of inorganic phosphate (Pi) liberated from ATP. Human adrenergic neuroblastoma cell line SH-SY5Y was used to assess the intracellular Ca²⁺ level fluctuation and cell viability. The metal binding site on LSB and the binding mode of the metal-LSB complexes were detected by NMR and visible spectroscopy, respectively.

Results:

The potencies of LSB complexed with Cr³⁺, Mn²⁺, Co²⁺, or Ni²⁺ increased by approximately 5 times compared to the naturally occurring LSB and Mg-LSB. The IC₅₀ values of Cr-LSB, Mn-LSB, Co-LSB, Ni-LSB, LSB, and Mg-LSB in inhibition of Na⁺/K⁺-ATPase activity were 23, 17, 26, 25, 101, and 128 μmol/L, respectively. After treatment of SH-SY5Y cells with the transition metal-LSB complexes (25 μmol/L), the intracellular Ca²⁺ level was substantially elevated, and the cells were viable for one day. The transition metals, as exemplified by Co²⁺, appeared to be coordinated by two carboxylate groups and one carbonyl group of LSB. Titration of LSB against Co²⁺ demonstrated that the Co-LSB complex was formed with a Co²⁺:LSB molar ratio of 1:2 or 1:1, when [Co²⁺] was less than half of the [LSB] or higher than the [LSB], respectively.

Conclusion:

LSB complexed with Cr³⁺, Mn²⁺, Co²⁺, or Ni²⁺ are stable, non-toxic and more potent in inhibition of Na⁺/K⁺-ATPase. The transition metal-LSB complexes have the potential to be superior substitutes for cardiac glycosides in the treatment of congestive heart failure.     

Inhibition of N-methyl-d-aspartate (NMDA) and l-glutamate-induced noradrenaline and acetylcholine release in the rat brain by ethanol

Summary The influence of ethanol on stimulation-evoked ³H-transmitter release was examined in slices of the rat brain cortex and corpus striatum preincubated with ³H-noradrenaline and ³H-choline, respectively. ³H-Transmitter release was stimulated by NMDA, l-glutamate, electrical impulses, reintroduction of Ca²⁺ ions (Ca²⁺-evoked release; after superfusion with Ca²⁺-free, K⁺-rich solution) or veratridine. In cortical slices preincubated with ³H-noradrenaline and superfused with Mg²⁺-free, otherwise physiologically composed salt solution, ethanol inhibited the NMDA- or l-glutamate-induced tritium overflow (IC₅₀ 45 and 37 mmol/l, respectively). In contrast, the tritium overflow in response to electrical stimulation, reintroduction of Ca²⁺ ions or veratridine was not affected by ethanol at concentrations up to 320 mmol/l; these experiments were carried out in cortical slices superfused with solution containing a physiological Mg²⁺ concentration. Ethanol also failed to inhibit Ca²⁺-evoked release in the absence of Mg2⁺ ions. In the presence of 1 mol/l veratridine, but not in its absence, NMDA induced tritium overflow even when cortical slices were superfused with salt solution containing a physiological Mg²⁺ concentration; again, ethanol inhibited this NMDA-evoked tritium overflow (IC₅₀ 73 mmol/l). In striatal slices preincubated with ³H-choline and superfused with Mg²⁺-free physiological salt solution, the NMDA-evoked tritium overflow was also, although at lower potency, inhibited by ethanol (IC₅₀ 192 mmol/l).In spite of the differences between the IC₅₀ values of ethanol determined for the inhibition of cortical noradrenaline and striatal acetylcholine release, it may be concluded that the NMDA receptor-ion channel complex is one of the sites of action underlying the ethanol-induced inhibition of neurotransmitter release. Since in the brain cortex the NMDA-induced ³H-noradrenaline release appears to be mediated by an excitatory interneurone activated by NMDA, this neuronal system may be involved in the cortical actions of ethanol.     

Kinetics of catecholamine sensitive Na+-K+ ATPase activity in mouse brain synaptosomes.

The effects of several neurotransmitters on mouse brain synaptosomal ATPase activities were determined in vitro. Both dopamine and norepinephrine activated Na⁺-K⁺ and Mg²⁺ ATPase activities in a dose-dependent manner. Na⁺-K⁺ ATPase was more sensitive to the catecholamines than was Mg²⁺ ATPase activity. Acetylcholine, γ-aminobutyric acid, L-glutamic acid and serotonin were without effect up to 10^?3 M concentration. Chlorpromazine, an antipsychotic agent, which has been shown to block the dopamine-receptor site, totally inhibited the dopamine-stimulated Na⁺-K⁺ and Mg²⁺ ATPase activities in mouse brain synaptosomes. Further, catecholamine-sensitive ATPase activities from mouse brain synaptosomal preparation were determined in relation to the substrate, pH and ionic concentrations in the reaction medium. The results indicate that Na⁺-K⁺ and Mg²⁺ ATPases were activated by dopamine (DA) and norepinephrine (NE) at different concentrations of ATP. Lineweaver-Burk plots reveal that Na⁺-K⁺ ATPase was activated non-competitively to ATP with a K_m value of 5.5 × 10^?4 M, whereas Mg²⁺ ATPase exhibited a mixed type of activation in that K_m was decreased and V_max increased in the presence of DA or NE. A maximum stimulation occurred by catecholamines at the optimum pH of 7.5 for Na⁺-K⁺ and 8.0 for Mg²⁺ ATPase activities. Both catecholamines increased the Na⁺-K⁺ ATPase activity in the presence of Na⁺ and K⁺ in the reaction medium. However, in the absence of Na⁺ ions the K⁺ -ATPase activity was stimulated by DA and NE but in the absence of K⁺ ions the Na⁺ ATPase, was not activated by DA or NE, indicating that the ATPase activity was more sensitive to catecholamines in the presence of K⁺ than Na⁺.     

Human β-endorphin

¹H spectra at 270MHz of the β_h-endorphin glycyl residues in aqueous solution are reported. The chemical shifts, coupling constants and temperature coefficients are compared with those of the glycyl residues in Met-enkephalin and in a random coil model peptide. The local conformation of Tyr-Gly-Gly-Phe-segment observed in Met-enkephalin is maintained in β_h-endorphin.     

Influence of alkali metal counterions on the glass transition temperature of amorphous indomethacin salts

Purpose. To test the hypothesis that the choice of counterion in salt formation would generally result in a change in the glass transition temperature, T_g, in relation to the nature of the interaction between the ionized parent structure and its counterion. Methods. Various alkali metal salts of indomethacin (IMC), lithium (Li⁺), sodium (Na⁺), potassium (K⁺), rubidium (Rb⁺), and cesium (Cs⁺) IMC were prepared as amorphous solid powders by lyophilization. The T_g was measured using differential scanning calorimetry or modulated-temperature differential scanning calorimetry. The spectroscopic properties of these salts were analyzed using Fourier transform-Raman and Fourier transform-infrared spectroscopy. Results. A comparison of the T_g values of the five salts showed T_g values in the order of Li⁺ > Na⁺ > K⁺ > Rb⁺ > Cs⁺, which is consistent with an order of decreasing ionic radius, and hence an increased charge density and an increased electrostatic interaction energy between the carboxylate ion and the alkali metal cation. Spectroscopic data support this interpretation by showing that the different salts exhibit spectral differences only in the region of the carboxylate group. Conclusions. Changes in T_g due to salt formation for alkali metal salts of IMC mainly result from differences in ionic interaction between the oppositely charged ions that appear to be related to the size/charge ratio of the counterion.     

Interaction of two sulfhydryl reagents with a cation recognition site on the neuronal dopamine carrier evidences small differences between [3H]GBR 12783 and [3H]cocaine binding sites

We have compared the effect of treating rat striatal cell membranes with ionic hydrophilic sulfhydryl reagents on the specific bindings of [³H]cocaine and of [³H]GBR 12783 (1-[2-(diphenylmethoxy)ethyl]4-(3-phenyl-2-[1-³H]propenyl)-piperazine) to the neuronal transporter of dopamine. Treatment with 1 mmol/1 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) resulted in similar time-and concentration-dependent reductions of the specific binding of both radioligands. None of the uptake blockers tested afforded any protection against 1 mmol/1 DTNB. Addition of (sub)millimolar concentrations of CaCl₂ or MgCl₂, or 250 mmol/1 KCl to a treatment medium containing 10 mmol/l Na + significantly increased the DTNB-induced reduction of the specific binding of both radioligands. Cations were likely to be responsible for this effect since ions in combination with DTNB induced similar reductions in binding when either 1 mmol/l CaCl₂ or 50–250 mmol/l NaCl were added. Effects of cations on the DTNB-induced inhibition of binding were generally more marked on [³H]GBR 12783 than on [³H]cocaine binding. When added to a medium containing 10 mmol/1 Na⁺ 1 mmol/1 DTNB induced a reduction in the B_max of the specific binding of both radioligands. Addition of 1 mmol/l Ca²⁺ maintained or increased this B_max reduction and elicited a decrease in affinity which was significant for [³H]GBR 12783 binding.Treatment of membranes with the sodium salt of p-hydroxymercurybenzenesulfonate (pHMBS) induced time-and concentration-dependent decreases in [³H]GBR 12783 binding which were significantly greater than decreases in [³H]cocaine binding. However, 50mol/lpHMBS produced a similar decrease in the B_max of the specific binding of both radioligands. The pHMBS-induced reduction of [³H]GBR 12783 binding was not reversed by drugs whose action is purely that of uptake inhibition or by substrates of the dopamine carrier. Some of these drugs (100 mol/l dopamine, 1 mol/l mazindol or 100 mol/l cocaine) protected the specific binding of [³H]cocaine against the effects of pHMBS, whereas 1 mmol/1 p-tyramine, 10 mol/l nomifensine and 10 nmol/l GBR 12783 were ineffective. Addition of 120 mmol/l Na⁺, 1 mmol/l Ca²⁺ or 10 mmol/l Mg²⁺ to a treatment medium containing 10 mmol/l Na⁺ significantly reduced the effects of pHMBS on the specific binding of both radioligands. When striatal cell membranes were treated in a medium containing 130 mmol/1 Na⁺, there was a general decrease in the effects of ions on the reductions of specific binding produced by DTNB or pHMBS. Cation concentrations which interfered with the actions of DTNB and pHMBS were approximately those which blocked the specific binding of [³H]GBR 12783 when they were present during association of the radioligand (K⁺, Ca ²⁺, Mg²⁺), or, in the case of Na⁺, which are effective in reducing this blockade (Bonnet et al. 1988).The present data are consistent with the existence of mutually exclusive binding sites for [³H]GBR and [³H]cocaine on the neuronal dopamine transporter. The hypothesis of a cation recognition site which could gate admission of uptake inhibitors or carrier substrates to their binding domain on the transporter is discussed.     

Studies on the mechanism of dog heart (Na+ + K+)-ATPase inhibition by D,L-aspartic acid and its K+ and Mg2+ salts.

d,l-aspartic acid as well as its K⁺ and Mg²⁺ salts have been found to be inhibitory to Na⁺ + K⁺)-ATPase activity of the dog heart microsomal fraction enriched with sarcolemma. According to intensity of the inhibitory effect (noncompetitive) they form the following sequel: mono-K⁺-aspartate $\text{?}\text{d,l}\text{-aspartic acid}\text{<}\text{mono-Mg}{}^{\mathrm{2+}}\text{-}\text{aspartate}\text{<}\text{mixture}$ of K⁺ and Mg²⁺-aspartate in a molar ratio of 1:1 (K⁺, Mg²⁺-ASP). The latter mixture is widely used as an agent in cardiac failure. For (Na⁺ + K⁺)-ATPase the salient effects of d,l-aspartic acid and/or its K⁺ and Mg²⁺ salts were: (i) decrease in V for ATP as substrate with unchanged K_m; (ii) for Na⁺ as an allosteric modifier of (Na⁺ + K⁺)-ATPase activity a decrease in V without any alteration in n as a measure of cooperativity between activating sites; (iii) for K⁺ a decrease in V and n as well as an increase in K_0.5. In the presence of Na⁺ and ATP the high affinity of the enzyme for K⁺ became reduced by d,l-aspartic acid, lowering at the same time the K_0.5 value.Effects like these have also been described for ouabain. The present data show that K⁺ and Mg²⁺ salts of d,l-aspartic acid act at a similar locus as does ouabain.     

The dissociation constant of verapamil estimated from its effect on Ca concentration-tension curves in guinea-pig tracheal muscle

Concentration-tension curves for calcium ions (Ca²⁺) were studied in indomethacin-treated guinea-pig tracheal muscle in the presence of different concentrations of carbachol in media containing 5.9 mM K⁺ or 40 mM K⁺. The effect of verapamil was investigated taking into account the steepness (the Hill coefficient) of the Ca²⁺ curve. When carbachol (1 μM) was added to 40 mM K⁺ solution, the Ca²⁺ concentration to produce half maximum tension (EC₅₀) was reduced from 0.2 mM to 0.08 mM and the Hill coefficient was increased from 1.4 to 2.0, respectively. In the presence of carbachol (1 μM), the Ca²⁺ concentration-tension curve was not much influenced by increasing the K⁺ concentration from 5.9 to 40 mM K⁺. Verapamil (0.5 μM) shifted the Ca²⁺ concentration-tension curve to the right in a parallel manner under all experimental conditions, the shift being greater with curves having a smaller Hill coefficient. The dissociation constant of verapamil was not altered by carbachol when estimated from the shift of the curve if the Hill coefficient is taken into consideration. It is concluded that the relatively low susceptibility of carbachol-induced contractions to verapamil in the presence of 40 mM K⁺, compared with these produced by K⁺ alone, is not due to a decreased verapamil affinity but to improved Ca²⁺-response coupling.     

Occupation of brain receptors by benzodiazepines and β-carbolines: Multiple mechanisms and responses

High affinity for the brain benzodiazepine receptor can no longer be considered predictive of benzodiazepine-like pharmacology in vivo. Studies of ³H-diazepam (³H-DZ) and ³H-propyl-β-carboline-3-carboxylate (³H-PrCC) binding were performed to investigate how occupation of the benzodiazepine receptor by these two high-affinity ligands leads to their significantly different pharmacological effects. In mouse hypothalamus, there are four times as many ³H-DZ binding sites as ³H-PrCC binding sites (B_max values = 1,025 and 265 fmol/mg protein, respectively) and both types of site form a distinct anterior-to-posterior gradient in this tissue. Physiological Ca⁺² concentrations do not regulate ³H-ligand binding in Ca⁺²-depleted whole mouse brain membranes, but in the presence of increasing concentrations of guanosine 5′-triphosphate (GTP), Ca⁺² stimulates ³H-DZ binding up to 25%. In contrast, ³H-PrCC binding is unaffected by Ca⁺² and GTP. Diazepam (DZ) and ethyl β-carboline-3-carboxylate (βCCE) both stimulate ³H-muscimol binding to GABA receptors in whole mouse brain membranes (DZ = +57%, βCCE = +27%), and βCCE partially blocks the effect of the benzodiazepine. Moreover, alkyl β-carboline-3-carboxylates prevent a temperature-induced thermodynamic transition in the benzodiazepine receptor that occurs in the presence of GABA. Studies of 8-anilino-1-naphthalene sulfonic acid fluorescence (ANS) perturbation have also suggested that occupation of the receptor by a benzodiazepine results in a conformational change. Although benzodiazepines and β-carbolines are high-affinity ligands for the benzodiazepine receptor, these results suggest that their different pharmacological profiles may result from significant differences in binding mechanisms and receptor responses.     

Sodium-Calcium exchange: A possible target for drug development

The Na⁺-Ca²⁺ exchange system is a carrier-mediated transport process which couples the transmembrane movement of Ca²⁺ ions to the movement of Na⁺ ions in the opposite direction. It functions primarily as a Ca²⁺ extrusion process in cardiac cells and is thought to be an important mechanism for altering myocardial contractility through changes in intracellular [Na⁺]. We khave investigated the properties of the cardiac Na⁺-Ca²⁺ exchange system using a subcellular preparation of membrane vesicles derived from the cardiac sarcolemma. Vesicle studies have been useful in investigating the kinetics of Na⁺-Ca²⁺ exchange activity and in establishin the stoichiometry of the exchange process as 3 Na⁺ per Ca²⁺. The kinetec results are most easily interpreted in terms of a model for the Na⁺-Ca²⁺ exchange carrier which features two types of cation binding sites: a divalent site for which Ca²⁺ and 1-2 Na⁺ ions compete and a second, monovalent site which binds the third Na⁺ involved in Na⁺-Ca²⁺ exchange. Na⁺-Ca²⁺ exchange activity in vesicles is stimulated by a variety of agents or treatments, including limited proteolysis, phospholipase treatment, redox reagents, anionic amphiphiles, and intravesicular Ca²⁺, all of which lower the apparent K_m for Ca²⁺. The physiological significance of these modes of regulation of exchange activity is at present uncertain. Progress in identifying and purifying the exchange carrier has been hampered by the lack of specific high-affinity probes that could be used in labelling studies. Indirect estimates of the number of exchange carriers in sarcolemmal vesicles suggest that there are 10-20 pmol of exchanger per mg of membrane protein and that the exchange system has a maximal turnover of approximately 10³ sec^?1. Studies are under way in several laboratories to clone a cDNA for the exchange carrier using oocytes of Xenopus laevis as an expression system.     

Synthesis and conformational study in solution and in solid state of oligopeptides containing l-leucine and glycine

The synthesis and conformational studies of the oligopeptide N-tert.-butyloxycarbonyl-l -Leu-(l -Leu-Gly)_n-OBzl (n = 1, 3, 5) and N-tert.-butyloxycarbonyl-(l -Leu-Gly)₂-OBzl are described. The peptides were synthesized by stepwise and fragment condensation techniques using dicyclohexylcarbodiimide as the condensing agent in solution. The conformational study of the oligopeptides was carried out using CD, u.v. and i.r. spectra. The conformation in solution was examined in trifluoroethanol, hexafluoroisopropanol. hexafluoroacetone trihydrate, and methanol. CD spectra in trifluoroethanol exhibited a gradual variation with increasing peptide chain length. This can be interpreted as a formation of an ordered structure which is already present in the heptapeptide and, to a greater extent, in the undecapeptide. The results obtained from the CD profiles and i.r. spectra showed the presence of β structure with antiparallel chains in the heptapeptide and undecapeptide. Finally, CD spectra revealed in trifluoroethanol-water solution the binding of Ca²⁺ to heptapeptide and undecapeptide together with a contemporaneous conformational change. This change is probably due to the formation of β-turns. No change in the CD profiles was obtained by using Mg²⁺, K⁺, Na⁺, and Li⁺ ions instead of Ca²⁺.