Characterization of the outer pore region of the apamin-sensitive Ca2+-activated K+ channel rSK2.

We have studied the interaction between the SK2 channel and different scorpion toxins in order to find similarity and differences to other K⁺ channels. Beside apamin, ScTX is a high affinity blocker of the SK2 channel, whereas CTX is unable to block current through SK2. In order to prove that the ScTX affinity can be explained by the character of the different residues in the outer pore of the SK channels we introduced point mutations that render SK2 K⁺ channel SK1 and SK3 like. Directed by the results of the toxin receptor on the ShakerK⁺ channel, we changed single amino acids of the SK2 K⁺ channel that should render it sensitive to other peptide toxins like CTX a blocker of the IK channel, or KTX a blocker of the voltage-dependent channel Kv1.1 and Kv1.3. Amino acids V342G, S344E, and G384D of SK2 were changed to amino acids known from ShakerK⁺ channel to improve Shaker K⁺ channel CTX sensitivity. Interestingly SK2 V342G became CTX sensitive with a K_d of 19 nM and was also KTX sensitive K_d=97 nM. SK2 S344E (K_dCTX=105 nM,K_dKTX=144 nM) and G348D (K_dCTX=31 nM,Kd KTX=89 nM) became also CTX and KTX sensitive with a lower affinity. The mutant channels SK V342G, SK2 S344E and SK2 G348D showed reduced ScTX sensitivity (K_d=6 nM,K_d=48 nM, and K_d=12 nM). Because the exchange of a single residue could create a new high affinity binding site for CTX and KTX we concluded that the outer vestibule around position V342, S344, and G348 of the SK2 K⁺ channel pore is very similar to those of voltage-gated K⁺ channels such as the Shaker K⁺ channel, Kv1.1 and Kv1.3 channels and also to the prokaryotic KcsA channel. From mutant cycle analysis of KTX position H34 and SK2 position V342G, S344E, and G348D we could deduce that KTX binds in a similar way to SK2 channel mutant pore than to the Kv1.1 pore.     

Gastrointestinal-tract models and techniques for use in safety pharmacology

Introduction: The human ether-a-go-go-related gene (hERG) encodes a potassium channel responsible for the cardiac delayed rectifier current (I_Kr) involved in ventricular repolarization. Drugs that block hERG have been associated with QT interval prolongation and serious, sometimes fatal, cardiac arrhythmias (including torsade de pointes). While displacement of [³H]dofetilide, a potent methanesulfonanilide hERG blocker, from cells heterologously expressing hERG has been suggested as a screening assay, questions have been raised about its predictive value.Methods: To validate the utility of this assay as a screening tool, we performed a series of saturation and competition binding studies using [³H]dofetilide as ligand and either intact cells or membrane preparations from HEK 293 cells stably transfected with hERG K⁺ channels. The object of these experiments was to (1) compare binding K_i values for 22 hERG blockers using intact cells or membrane homogenates to determine whether maintaining cell integrity enhanced assay reliability; (2) evaluate the ability of different K⁺ concentrations (2, 5, 10, 20, and 60 mM) to modulate hERG binding; and (3) to establish the predictive value of the assay by comparing K_i values from binding studies at 5 and 60 mM [K⁺]_o to functional IC₅₀ values for hERG current block using 56 structurally diverse drugs.Results: We found (a) comparable K_i values in the intact cell and isolated membrane binding assays, although there were some differences in rank order; (b) increasing [K⁺]_o lowered the K_d and increased the B_max for [³H]dofetilide, particularly in the membrane assay; and (c) good correlation between binding K_i values and functional IC₅₀ values for hERG current block.Discussion: In conclusion, increasing K⁺ concentrations results in an increase in both [³H]dofetilide affinity for hERG and available binding sites, particularly when using membrane homogenates. There are no meaningful differences between K_i values when comparing intact cell versus membrane assay, neither are there meaningful trends with increasing [K⁺]_o within assays. There is good correlation between binding K_i values and functional (whole-cell patch clamp) IC₅₀ values at both 5 and 60 mM K⁺ concentrations (R² values of .824 and .863, respectively). The simplicity, predictability, and adaptability to high-throughput platforms make the [³H]dofetilide membrane binding assay a useful tool for screening and ranking compounds for their potential to block the hERG K⁺ channel.     

The influence of pH, temperature and buffers on the degradation kinetics of cefetamet pivoxil hydrochloride in aqueous solutions

The first-order hydrolysis kinetics of cefetamet pivoxil (CP) were investigated as a function of pH, temperature and buffers. The degradation was followed by HPLC. Buffer catalysis was observed in acetate and phosphate buffers. The pH–rate profiles for hydrolysis of cefetamet pivoxil were obtained at 333, 343, 353 and 363 K. The pH–rate expression was k_pH=k_H⁺a_H⁺+k_H2Ok_OH⁻a_OH⁻, where k_H⁺ and k_OH⁻ are the second-order rate constants (mol⁻¹ l s⁻¹) for hydrogen ion activity and for hydroxyl ion activity respectively, and k_H2O is the pseudo-first-order rate constant (s⁻¹) for spontaneous reaction under the influence of water. The pH–rate profile was characteristically U-shaped. Maximum stability was observed in the pH region from 3 to 5.     

Drug permeability in 16HBE14o- airway cell layers correlates with absorption from the isolated perfused rat lung

The permeability of the lung is critical in determining the disposition of inhaled drugs and the respiratory epithelium provides the main physical barrier to drug absorption. The 16HBE14o- human bronchial epithelial cell line has been developed recently as a model of the airway epithelium. In this study, the transport of 10 low molecular weight compounds was measured in the 16HBE14o- cell layers, with apical to basolateral (absorptive) apparent permeability coefficients (P_app) ranging from 0.4 × 10⁻⁶ cm s⁻¹ for Tyr-D-Arg-Phe-Phe-NH₂ to 25.2 × 10⁻⁶ cm s⁻¹ for metoprolol. Permeability in 16HBE14o- cells was found to correlate with previously reported P_app in Caco-2 cells and absorption rates in the isolated perfused rat lung (k_a,lung) and the rat lung in vivo (k_{a,in vivo}) [Tronde, et al., 2003. J. Pharm. Sci. 92, 1216–1233; Tronde, et al., 2003. J. Drug Target. 11, 61–74]. Log linear relationships were established between P_app in 16HBE14o- cells and P_app in Caco-2 cells (r² = 0.82), k_a,lung (r² = 0.78) and k_{a,in vivo} (r² = 0.68). The findings suggest that permeability in 16HBE14o- cells may be useful to predict the permeability of compounds in the lung, although no advantage of using the organ-specific cell line 16HBE14o- compared to Caco-2 cells was found in this study.     

Electrochemical study on the behavior of Morin and its interaction with DNA

Voltammetric behavior of Morin was studied in 0.1 M HAc–NaAc + 50 mM KCl (pH 3.4) solution at glassy carbon electrode (GCE) using cyclic voltammetry (CV). Morin showed an irreversible anodic peak at 0.720 V in CV which was involving two electrons and two protons. Also, the interaction of Morin with double-stranded calf thymus DNA (ctDNA) was studied by CV at GCE with an irreversible electrochemical equation. As a result of reaction with ctDNA, the voltammetric peak of Morin was a position shift and the peak current decreased. The diffusion coefficients of both free and binding Morin (D_f = 1.1086 × 10⁻⁷ cm² s⁻¹ and D_b = 8.2544 × 10⁻⁹ cm² s⁻¹), binding constant (K = 1.7765 × 10⁷ cm³ mol⁻¹), and binding site size (s = 0.8510) of the Morin–DNA complex were obtained simultaneously by non-linear fit analysis. The results demonstrate that Morin can bind to ctDNA in 0.1 M HAc–NaAc + 50 mM KCl (pH 3.4) solution and the ring B of Morin intercalates between the DNA base pairs.     

Similarities of the in vivo and in vitro effects of mercuric chloride on H]ouabain binding and potassium activation of Na/K-ATPase in isolated rat cerebral microvessels

A previous study revealed that a single i.p. administration of 6 mg/kg body wt. of mercuric chloride (MC) durably inhibits the rat cerebral microvascular Na⁺/K⁺-ATPase activity [1]. In this study, cerebral microvessels isolated 18 h after MC treatment were compared to those obtained from control rats and subsequently treated or not treated with MC in vitro, with regard to: (a) ³H]ouabain binding to, and (b) K⁺-activation kinetics of, the Na⁺/K⁺-ATPase. Microvessels from MC-treated rats showed a decrease of ³H]ouabain binding down to 62% of the control binding, and the same degree of inhibition was attained in microvessels treated in vitro with 5 μM MC. Analysis of the K⁺-activation kinetics of Na⁺/K⁺-ATPase revealed a decrease of V_max from the control value of 13.1 to 7.67 μmol/mg/h in microvessels from MC-treated rats and 6.07 μmol/mg/h in microvessels treated in vitro with 5 μM MC, with no change in K_m in either case. The similarity of the effects of in vivo and in vitro treatments suggests that the inhibition of the cerebromicrovascular Na⁺/K⁺ATPase following in vivo administration of MC results from a direct interaction of Hg⁺ with the enzyme.     

Enhanced platelet serotonin 5-HT2A receptor binding in anorexia nervosa and bulimia nervosa.

Some evidence exists to suggest that serotonin 5-HT_2A receptor function is altered in anorexia nervosa and bulimia nervosa. In order to further investigate the 5-HT_2A receptor in eating disorders, platelet [³H]lysergic acid diethylamide ([³H]LSD) binding was studied in ten patients with anorexia nervosa, 23 patients with bulimia nervosa and 33 healthy controls. At admission, B_max for platelet [³H]LSD binding was significantly higher both in the anorexia nervosa group (30.6±4.2 fmol/mg protein; mean±S.D.) and in the bulimia nervosa group (30.8±7.6 fmol/mg protein) than in the control group (23.5 ±6.3 fmol/mg protein; p=0.01 and p=0.003, respectively). K_d was borderline significantly higher among anorexics (median 1.45 nM) and significantly higher among bulimics (median 1.66 nM) than among controls (median 0.95 nM; p=0.05 and 0.003, respectively). The Global Assessment of Functioning score and the body mass index were both significantly negatively correlated to K_d (r=−0.40; p=0.03 and r=−0.41 p=0.03, respectively), but not to B_max. The present study indicates that patients with anorexia nervosa as well as patients with bulimia nervosa have an enhanced 5-HT_2A receptor binding and provides further evidence for a serotonergic dysfunction in eating disorders.     

Uptake and transport of the ACE-inhibitor ceronapril (SQ 29852) by monolayers of human intestinal absorptive (Caco-2) cells in vitro

The angiotensin-converting enzyme (ACE)-inhibitor ceronapril (SQ 29852) is shown to be a substrate of the intestinal dipeptide transporter. Uptake by Caco-2 cells, grown as confluent monolayers, follows a major saturable pathway (K_m, 0.91 ± 0.11 mM; 90% at 1 mM) together with a minor passive component (k_J, 32.3 ± 6.6 ng (10⁶ cells)⁻¹ (20 min)⁻¹. Uptake was inhibited by competition with dipeptides such as l-AIa-l-Pro (K_i, 2.96 mM) and l-Phe-Gly (K_i, 3.84 mM) but not by cephalosporins such as cephalexin. In contrast, transport was non-saturable, flux increased linearly with concentration and data were consistent with a passive transepithelial transport mechanism. Transport profiles showed a biphasic dependence upon time with an initial flux of 0.83 ± 0.02 ng insert⁻¹ min⁻¹ (k₁) and a terminal value of 1.65 ± 0.08 ng insert⁻¹ min⁻¹ ((k₂) at 100 μM. It is concluded that the basolateral efflux is retarded so that the passive paracellular transport controls the overall transepithelial transport characteristics in the Caco-2 model. Carrier-mediated uptake into intestinal enterocytres, followed by rate-limiting basolateral efflux, may explain the extended t_max in vivo following oral administration.     

Mechanism of interaction between human serum albumin and N-alkyl phenothiazines studied using spectroscopic methods

The binding characteristics of human serum albumin (HSA) with N-alkyl phenothiazines derivatives (NAP) viz., levomepromazine monomaleate (LMM) and propericiazine (PPC) have been studied by employing fluorescence, absorption, circular dichroism and FT-IR techniques. The Stern–Volmer quenching constant, K_SV values were found to decrease with increase in temperature thereby indicating the presence of static quenching mechanism in the interactions of NAP with HSA. The number of binding sites, n and the binding constant, K were noticed to be, respectively, 1.11 and (5.188 ± 0.034) × 10⁴ M⁻¹ for LMM and 1.06 and (4.436 ± 0.066) × 10⁴ M⁻¹ for PPC at 298 K. The negative value of enthalpy change and positive value of entropy change in the present study indicated that the hydrophobic forces played a major role in the binding of NAP to HSA. The circular dichroism and FT-IR spectral data revealed the conformational changes in the structure of protein upon its interaction with NAP. The binding distances and the energy transfer efficiency between NAP and protein were determined based on Förster's theory of energy transfer. The decreased binding constants of HAS–LMM and HAS–PPC systems in presence of common ions indicated the availability of higher concentration of free drug in plasma.     

Selective labelling of 5-HT7 receptor recognition sites in rat brain using [H]5-carboxamidotryptamine

The aim of the present study was to establish a radioligand binding assay to selectively label the native 5-HT₇ receptor expressed in rat brain. In rat whole brain (minus cerebellum and striatum) homogenate, (±)-pindolol (10 μM)-insensitive [³H]5-CT ([³H]5-carboxamidotryptamine; 0.5 nM) specific binding (defined by 5-HT, 10 μM) displayed a pharmacological profile similar to the recombinant 5-HT₇ receptor, although the Hill coefficients for competition curves generated by methiothepin, ritanserin, sumatriptan, clozapine and pimozide were significantly less than unity. In homogenates of rat hypothalamus, (±)-pindolol (10 μM)-insensitive [³H]5-CT recognition sites also resembled, pharmacologically, the 5-HT₇ receptor, although pimozide still generated Hill coefficients significantly less than unity. Subsequent studies were performed in the additional presence of WAY100635 (100 nM) to prevent [³H]5-CT binding to residual, possibly, 5-HT_1A sites. Competition for this [³H]5-CT binding indicated the labelling in whole rat brain homogenate of a homogenous population of sites with the pharmacological profile of the 5-HT₇ receptor. Saturation studies also indicated that (±)-pindolol (10 μM)/WAY 100635 (100 nM)-insensitive [³H]5-CT binding to homogenates of whole rat brain was saturable and to an apparently homogenous population of sites which were labelled with nanomolar affinity (B_max=33.2±0.7 fmol mg⁻¹ protein, pK_d=8.78±0.05, mean±S.E.M., n=3). The development of this 5-HT₇ receptor binding assay will aid investigation of the rat native 5-HT₇ receptor.     

Cellular disposition of arabinogalactan in primary cultured rat hepatocytes

To characterize a targeting property of arabinogalactan (AG) as a carrier to the liver, we examined cellular disposition, such as binding and internalization in primary cultured rat hepatocytes, comparing them to those of asialofetuin (AF). A tyramine derivative of AG was synthesized to allow labeling with ¹²⁵I. Binding of AG to the cells was concentration-dependent and saturable. The number of binding sites (n) of AG on the cell surface was 4.0 × 10⁵ ± 0.1 × 10⁵ sites per cell which was about similar to that of AF. The value of K_a of AG was 2.2 × 10⁸ ± 0.1 × 10⁸ M⁻¹ being seven-fold higher than that of AF. The binding of AG was competitively inhibited by AF and was decreased by calcium depletion. These results indicate that AG can bind strongly to hepatocytes probably through the recognition by the asialoglycoprotein receptor (ASGP-R). Both ¹²⁵I-labeled AG and fluorescein-labeled AG were internalized into the cells. The rate of internalization of AG was faster than that of AF, indicating that AG is effectively endocytosed. Microscopic observations showed that FITC labeled AG accumulated in granules within the primary cultured rat hepatocytes. Subcellular fractionation indicated that the internalized AG was mainly associated with the lysosomal fraction. However, the internalized AG seemed to remain intact in the hepatocytes. In conclusion, we found that AG is effectively internalized in primary cultured rat hepatocytes. Although AG seems a good candidate for targeting to the liver due to its high affinity binding and rapid internalization, it remains to be established whether the apparent lack of biodegradation will result in cytotoxic effects at chronic administration in vivo.     

Characterization of insulin-resistance: role of receptor alteration in insulin-dependent diabetes mellitus, essential hypertension and cardiac hypertrophy

Insulin-resistance is associated with a number of disease states such as diabetes, syndrome X, and hypertension. These situations may be coupled to insulin-resistance through the insulin signaling system as a common pathway. The purpose of this study was to investigate the receptor binding alterations in streptozotocin-induced diabetic rats, spontaneously hypertensive rats and aortocaval shunted rats (eccentric cardiac hypertrophy). A physical model describing a 1:1 stoichiometry of ligand binding with its receptor is proposed describing reversible binding of [¹²⁵I]insulin or [¹²⁵I]IGF-1 at the microvascular endothelial as well as with the cardiac myocytes after CHAPS-treatment. Analysis of the collected effluents are curve-fitted with a conservation equation and a first-order Bessel function which allowed the calculation of the forward binding constants (k_n), the reversible constants (k_−n), the dissociation constants (k_d) and the residency time constants (τ). The results showed that streptozotocin-induced diabetic rats showed insulin-resistance through alterations in the kinetics of insulin receptor binding. The normotensive controls of the spontaneously hypertension rats (SHR) carry themselves insulin-resistant receptors whose binding to insulin worsens in the hypertensive SHR. Negative cooperativity between insulin-like growth factor IGF-1 and insulin receptors could be a causative factor predisposing for insulin-resistance in the aortocaval shunted rats to insulin resistance. The defects may be occurring at the receptor level in insulin-dependent diabetes mellitus, Wistar-Kyoto rats and spontaneously hypertensive rats. In conclusion, alterations in the kinetics of insulin binding to its receptor seem to play a central role for the initiation of insulin-resistance during the various pathophysiological states.     

[H]ATPA: a high affinity ligand for GluR5 kainate receptors

The pharmacological properties of [³H]ATPA ((RS)-2-amino-3(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid) are described. ATPA is a tert-butyl analogue of AMPA (-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) that has been shown to possess high affinity for the GluR5 subunit of kainate receptors. [³H]ATPA exhibits saturable, high affinity binding to membranes expressing human GluR5 (GluR5) kainate receptors (K_d13 nM). No specific binding was observed in membranes expressing GluR2 and GluR6 receptors. Several compounds known to interact with the GluR5 kainate receptor inhibited [³H]ATPA binding with potencies similar to those obtained for competition of [³H]kainate binding to GluR5. Saturable, high affinity [³H]ATPA binding (K_d4 nM) was also observed in DRG neuron (DRG) membranes isolated from neonatal rats. The rank order potency of compounds to inhibit [³H]ATPA binding in rat DRG and GluR5 membranes were in agreement. These finding demonstrate that [³H]ATPA can be used as a radioligand to examine the pharmacological properties of GluR5 containing kainate receptors.     

Effects of Fe on ion channels: Na channel, delayed rectified and transient outward K channels

The effects of Fe²⁺ on the properties of three types of ion channels were studied in acutely dissociated rat hippocampal pyramidal neurons from area CA1 at postnatal ages of 7–14 days using the whole cell patch clamp technique. The results indicated that: (1) in the existence of Fe²⁺, the activation voltage threshold of transient outward K⁺ currents (I_A) was decreased. The normalized current-voltage curves of activation were well fitted with a single Boltzmann function, and the V_1/2 was 2.44±1.14 mV (n=15) in control, whereas 1.79±1.53 (n=15), −2.96±0.92 (n=14), −5.11±1.31 (n=13), −9.05±1.64 mV (n=12) in 1, 10, 100 and 1000 μ Fe²⁺, respectively. Differences between two groups were significant (P<0.05, n=12–15), except for that between the control and 1 μ (P>0.05, n=15). (2) Fe²⁺ caused a left shift of the current–voltage curves of steady-state inactivation of I_A in a concentration-dependent manner. The curves were well fitted with a single Boltzmann function with similar slope (P>0.05, n=10–13). The V_1/2 were −70.71±1.23 (n=13), −71.14±1.37 (n=13), −78.21±1.17 (n=11), −84.61±1.34 (n=12), and −89.68±2.59 mV (n=10) in control, 1, 10, 100 and 1000 μ Fe²⁺, respectively. Fe²⁺ also shifted the current–voltage curves of Na⁺ channel steady-state inactivation to more negative depolarization potentials in parallel, with V_1/2, −67.37±1.33 mV (n=12) in control, and −67.52±1.28 mV (n=12), −68.24±1.61 mV (n=10), −71.58±1.45 mV (n=10), −76.65±1.76 mV (n=9) in 1, 10, 100 and 1000 μ Fe²⁺ solutions, respectively. (3) In Fe²⁺ solutions, the recovery from inactivation of I_A was slowed. (4) With application of different concentrations of Fe²⁺, the voltage threshold of activation of delayed rectified outward K⁺ currents (I_K) was decreased, while Fe²⁺ showed a little inhibition at more positive depolarization. Briefly, the results demonstrated that Fe²⁺ is a dose- and voltage-dependent, reversible modulator of I_A, I_K and Na⁺ channels. The results will be helpful to explain the mechanism of Fe²⁺ physiological function and Fe²⁺ intoxication in the central nervous system.     

A comparison of the affinities of rat (Na + K)-ATPase isozymes for cardioactive steroids, role of lactone ring, sugar moiety and KCl concentration

Binding experiments at equilibrium were performed to study pharmacological properties of isozymes of (Na⁺ + K⁺)-ATPase from rat tissues. Experiments were performed on brain (₃ isozyme), kidney (₁ isozyme) and heart microsomes (₁ and ₂ isozymes). Affinity of series of ouabain and digoxin derivatives was studied in competition experiments. It was observed that: (i) ouabain and digoxin had higher affinity (P < 0.01) for ₃ isozyme (K_d of 0.071 ± 0.004 and 0.066 ± 0.001 μM, respectively) than for ₁ isozyme (K_d of 15.9 ± 0.8 and 1.78 ± 0.46 μM, respectively) and ₂ isozyme (K_d of 0.26 ± 0.0 and 0.15 ± 0.06 μM, respectively); (ii) saturation of the C₂₀---C₂₂ bond on the C_17β lactone ring present in ouabain and digoxin markedly decreased the drug affinity for all isozymes (P < 0.01); and (iii) suppression of the C_3β osidic chain decreased the affinity of ouabain and digoxin to a higher extent for ₂ and ₃ than for ₁ (P < 0.01). The presence of 10 mM KCl in the incubation medium decreased ouabain affinity for the ₁ isozyme to a much higher extent (K_d increase of about 20-fold) than for the other isozymes (K_d increase of about 2-fold). The results show that the isozymes of (Na⁺ + K⁺)-ATPase from rat tissue are differently sensitive to changes in the substituents of the cardioactive steroids and to the presence of 10 mM KCl.     

Effect of a phospholipase A2 with cardiotoxin-like properties, from Bungarus fasciatus snake venom, on calcium-modulated potassium currents

, , , and . Effect of a phospholipase A₂ with cardiotoxin-like properties, from Bungarus fasciatus snake venom, on calcium-modulated potassium currents. Toxicon 27, 1339–1349, 1989.—The action of a 16,300mol. wt phospholipase A₂ with cardiotoxin-like properties from Bungarus fasciatus venom on membrane electrical properties of two human cell types was examined in vitro by using tight-seal whole-cell recording methods. Epithelial cells exhibited a voltage-and Ca²⁺-activated K⁺current; the sensitivity for voltage activation of the K⁺ current was enhanced by increasing free Ca²⁺ in the recording pipette from 10⁻⁸ M to 2 × 10⁻⁶ M. In contrast, peripheral blood lymphocytes possessed voltage-activated K⁺ currents that were inhibited by increasing intracellular Ca²⁺.

Exposure of either preparation to B.fasciatus toxin (0.2–5 × 10⁻⁶ M) for up to 30 min in the bath did not alter membrane leakage current, as judged by the maintenance of low pre-treatment values over the range of − 140mV to − 40mV. However, the sensitivity for voltage activation of the K⁺ current was enhanced in the epithelial cells even at the lowest concentrations tested. In contrast to the results with epithelial cells, toxin exposure inhibited the activation of voltage-activated K⁺ currents in human lymphocytes, suggesting a specific increase in intracellular Ca²⁺ levels in both cell types.

The fluorescent probe indo-1/AM was used to monitor cytoplasmic Ca²⁺ levels. Exposure of either lymphocytes or epithelial cells to toxin (10⁻⁶ M) resulted in a transient increase in Ca²⁺. However, while the Ca²⁺ response to toxin was transient, K-channel modulation by the toxin appeared to be irreversible over the experimental time course. The longer-lasting modulation of Ca²⁺-regulated K⁺ channels may reflect an irreversible action of the B.fasciatus phospholipase A₂ on a Ca²⁺-dependent regulatory process.     

A surface plasmon resonance method for detecting multiple modes of DNA-ligand interactions

A simple and general surface plasmon resonance (SPR) based method has been developed to detect and quantitate binding of low molecular weight compounds (200–1200 Da) to double stranded DNA. Several compounds were chosen to probe three different modes of binding interactions, intercalation, minor groove binding and electrostatic interactions. Ethidium bromide (MW 390 Da), a probe of intercalative binding, was tested by plotting the steady state SPR responses measured on a DNA modified surface versus ethidium bromide concentration. The best fit of the binding isotherm gave a K_eq of 1.8×10⁵ M⁻¹. Co-solvents such as DMSO are often used in activity assays to increase the solubility of poorly water-soluble drugs. The effect of DMSO on the ethidium bromide/DNA interaction was also tested by measuring binding in the presence of 0, 1 and 5% DMSO. No effect on the measured K_eq was observed at these DMSO concentrations. The binding of actinomycin (MW 1255 Da), an antibiotic known to bind DNA through intercalation and minor groove binding, was also tested. The K_eq estimated from the steady state responses on a DNA surface was 1.9×10⁶ M⁻¹. DAPI (MW 350 Da) (4′,6-diamidino-2-phenylindole) a fluorescent probe which binds the minor groove of DNA was also tested and gave a K_eq of 1.8×10⁶ M⁻¹ measured by SPR. Finally, spermine (MW 202) a compound known to bind DNA through ionic interactions gave the weakest K_eq of 1.7×10⁴ M⁻¹. All the K_eq values measured by SPR and reported for these compounds were in good agreement with literature values measured by other techniques.     

Prednisolone-3,20-bisguanylhydrazone: The mode of interaction with rat brain sosium and potassium-activated adenosine triphosphatase

The mechanism of interaction between prednisolone-3,20-bisguanylhydrazone (PBGH) and Na⁺,K⁺-ATPase (ATP phosphohydrolase, EC 3.6.1.3) was studied using partially purified rat brain enzyme preparations. PBGH inhibited Na⁺,K⁺-ATPase rapidly and reversibly. The enzyme-inhibiting action of PBGH was competitively antagonized by potassium. PBGH inhibited Na⁺, Mg²⁺ and ATP-supported binding of (³H)-ouabain to the enzyme. When PBGH was added to the incubation mixture at the time when the (³H)-ouabain binding was close to its equilibrium state, the concentration of (³H)-ouabain enzyme complex was rapidly reduced and shifted to a lower equilibrium state. A double reciprocal plot analysis of the (³H)-ouabain binding data indicates that the inhibition of ouabain binding by PBGH is apparently competitive. Binding of (³H)-ouabain in the presence of Tris-phosphate and Mg²⁺ was also inhibited by unlabeled PBGH. Thus, it appears that the binding of PBGH precludes the bindings of ouabain to Na⁺,K⁺-ATPase.     

Investigation of the inhibitory effect of broussochalcone A on respiratory burst in neutrophils

Broussochalcone A, a prenylated chalcone isolated from Broussonetia papyrifera (L.) VENT. (Moraceae), inhibited O₂ consumption in formylmethionyl-leucyl-phenylalanine (fMLP)- and phorbol 12-myristate 13-acetate (PMA)-stimulated rat neutrophils in a concentration-dependent manner with IC₅₀ values of 70.3±4.9 and 63.9±7.1 μM, respectively. Broussochalcone A did not affect the fMLP-induced increase of cellular inositol trisphosphate (IP₃) and [Ca²⁺]_i. However, the enzyme activity of neutrophil cytosolic protein kinase C was effectively suppressed by broussochalcone A. Broussochalcone A had no effect on either [³H]phorbol 12,13-dibutyrate ([³H]PDB) binding to neutrophil cytosolic protein kinase C or on PMA-induced membrane translocation of protein kinase C-β in neutrophils. Broussochalcone A suppressed the enzyme activity of trypsin-treated rat brain protein kinase C in a concentration-dependent manner. In PMA-activated neutrophil particulate NADPH oxidase, broussochalcone A attenuated superoxide anion radical (O₂⁻) generation with an IC₅₀ value of 61.8±5.4 μM. These results show that the inhibitory effect of broussochalcone A on respiratory burst in neutrophils is not mediated by the reduction of phospholipase C activity, but is mediated partly by the suppression of protein kinase C activity through interference with the catalytic region and by the attenuation of O₂⁻ generation from the NADPH oxidase complex.     

Characterization of [(3)H]ucb 30889 binding to synaptic vesicle protein 2A in the rat spinal cord

The novel antiepileptic drug levetiracetam ((2S-(2-oxo-1-pyrrolidinyl)butanamide, KEPPRA®) possesses a specific binding site in brain, which has very recently been identified as the synaptic vesicle protein SV2A. The aim of this study was to evaluate the presence of a levetiracetam binding site in the spinal cord and compare its properties to that in rat brain. We used [³H]ucb 30889 ((2S)-2-[4-(3-azidophenyl)-2-oxopyrrolidin-1-yl]butanamide), a levetiracetam analogue, to perform binding assays, photoaffinity labelling and autoradiography experiments, and revealed the presence of SV2A by Western-blot analysis. [³H]ucb 30889 binding kinetics at 4 °C were biphasic and saturation binding curves were compatible with the labelling of a homogenous population of binding sites with a K_d similar to that in brain. Competition curves with ligands known to interact with levetiracetam binding sites and photolabelling experiments indicated that [³H]ucb 30889 labels the same 90 kDa protein in both spinal cord and brain. Levetiracetam binding site was localised in the grey matter of the spinal cord and its expression was not modified in a model of neuropathic pain. This study demonstrates the presence of a specific levetiracetam binding site in the rat spinal cord, which is similar to that found in rat brain.