Saturable Binding Component Research Articles

Pharmacokinetic and Pharmacodynamic (PKPD) Modeling to Determine the Andexanet Alfa Dose to Reverse the Anticoagulant Activity of Direct Fxa Inhibitors

IntroductionAndexanet alfa (AnXa) is a specific antidote under development for direct and indirect factor Xa (fXa) inhibitors. AnXa is a recombinant, engineered version of human fXa that is catalytically inactive but retains high affinity for direct fXa inhibitors. AnXa binds fXa inhibitors, sequestering them in plasma, thereby reducing the free inhibitor concentration, reversing fXa inhibition, and normalizing thrombin generation. The decreased unbound concentrations of the direct fXa inhibitors (apixaban, rivaroxaban and edoxaban) cause a portion of the extravascular fXa inhibitors to move into the vasculature. Reversal of fXa inhibitor-induced anticoagulation requires a molar excess of AnXa relative to the total amount of fXa inhibitor in the blood - i.e., the initial fXa inhibitor plasma concentration plus the redistributed amount. We report a PKPD model that accounts for this extravascular-intravascular redistribution and allows determination of the appropriate AnXa dose to reverse the anti-fXa activity for each approved direct fXa inhibitor.MethodsA model for rivaroxaban was constructed first by jointly analyzing AnXa concentrations, total and unbound rivaroxaban concentrations, and anti-fXa activity for 5 different doses of AnXa (each administered at the peak plasma level of 20 mg QD rivaroxaban at steady state). Model parameters were estimated by maximum likelihood using nonlinear regression (NONMEM, v. 7.2.0). The model was used to simulate the potential level of reversal of anti-fXa activity after 2.5-40 mg/day rivaroxaban (QD and BID) for different bolus doses of AnXa and to predict maintenance of reversal by various follow-on infusion rates. A similar model was developed to characterize the interaction between AnXa and apixaban. Learnings from the rivaroxaban and apixaban models were used to develop a universal model to predict the level of reversal of anticoagulation for 60 mg edoxaban after a particular dose of AnXa. The model accurately predicted the degree of reversal of edoxaban anticoagulation. The final model was used to construct a nomogram of AnXa doses that could be used for each fXa inhibitor at each approved dose based on timing of the last dose of the inhibitor.ResultsThe final PKPD model was a three-compartment PK model for the fXa inhibitors, including one central and two tissue compartments. The two tissue compartments included one that rapidly equilibrated with the central compartment and a second that equilibrated slowly. The combined volume of the central and rapidly equilibrating compartments was similar to the volume of central compartment of the fXa inhibitor PK models in the absence of AnXa.The AnXa PK was best described by a two-compartment PK model with an additional saturable binding component. The AnXa-fXa inhibitor complex moved to a "sequestration" compartment. Upon release from AnXa, the fXa inhibitor was able to return to the central compartment. The fXa inhibitor did not clear with AnXa. The binding between AnXa and free fXa inhibitor was characterized by a reversible binding equilibrium. There was a direct PKPD linear relationship between free fXa inhibitor concentrations and anti-fXa activity.Simulations were used to estimate the best dosing regimen to reverse anticoagulation for each direct fXa inhibitor. A bolus dose of 800 mg of AnXa followed by an 8 mg/min infusion was sufficient to fully reverse the peak level of anti-fXa activity after the 20 mg QD dose of rivaroxaban. This represents a >90% decrease in anti-fXa activity compared to pre-AnXa administration. Sustained reversal of peak anti-fXa levels for the 5 mg BID dose of apixaban required a 400 mg bolus AnXa dose with a follow-on infusion of 4 mg/min. The peak anticoagulant activity of the 60 mg dose of edoxaban could be reversed by an 800 mg bolus dose of AnXa followed by an 8 mg/min infusion.Conclusions•A PKPD model was constructed that accurately predicted the AnXa dose necessary to reverse coagulation inhibition of each direct fXa inhibitor. The model incorporated the PK of AnXa, the anti-fXa activity, unbound and total levels of the fXa inhibitor, and the redistribution of each fXa inhibitor between the extravascular and intravascular compartments.•Simulations were used to predict the AnXa dose necessary to reverse each approved dose of each direct fXa inhibitor and for different times after the last dose taken of the fXa inhibitor. DisclosuresJanet:Portola Pharmaceuticals, Inc.: Employment. Lu:Portola Pharmaceuticals, Inc.: Employment. Curnutte:3-V Biosciences: Equity Ownership; Sea Lane Biotechnologies: Consultancy; Portola Pharmaceuticals, Inc.: Employment. Conley:Portola Pharmaceuticals, Inc.: Employment. Mandema:Portola Pharmaceuticals, Inc.: Consultancy.

Pharmacological Characterization of [3H]CHIBA-3007 Binding to Glycine Transporter 1 in the Rat Brain

Glycine transporter-1 (GlyT-1) in glial cells regulates extracellular levels of glycine, which acts as an obligatory co-agonist at the N-methyl-D-aspartate (NMDA) receptors in the brain. In the present study, we developed a novel radioligand, [3H]3-chloro-N-((S)-((R)-1-methylpiperidin-2-yl)(thiophen- 3-yl)methyl)-4- (trifluoromethyl)picolinamide ([3H]CHIBA-3007), for studying GlyT-1 in the brain. The presence of a single saturable high-affinity binding component for [3H]CHIBA-3007 binding to the rat brain membranes was detected. Scatchard analysis revealed an apparent equilibrium dissociation constant (Kd) of 1.61±0.16 nM and a maximal number of binding sites (Bmax) of 692.8±22.8 fmol/mg protein (mean ± SEM, n = 3). The specific binding of [3H]CHIBA-3007 was inhibited by a number of GlyT-1 inhibitors, such as CHIBA-3007, desmethyl-CHIBA-3007, CHIBA-3008, SSR504734, NFPS/ALX5407, LY2365109 and Org24598, consistent with the pharmacological profiles of GlyT-1 inhibitors. Interestingly, the potency of eight GlyT-1 inhibitors (CHIBA-3007, desmethyl-CHIBA-3007, NFPS/ALX5407, LY2365109, Org24598, SSR504734, sarcosine, and glycine) for blocking in vitro specific binding of [3H]CHIBA-3007 was significantly correlated with the potency of these inhibitors for inhibiting [14C]glycine uptake in the rat brain membranes. In contrast, the GlyT-2 inhibitor ALX1393 exhibited very weak for [3H]CHIBA-3007 binding. Furthermore, the regional distribution of [3H]CHIBA-3007 binding in the rat brain was similar to the previously reported distribution of GlyT-1. The present findings suggest that [3H]CHIBA-3007 would be a useful new radioligand for studying GlyT-1 in the brain.

Pharmacological characterization of [ 125I]CHIBA-1006 binding, a new radioligand for α7 nicotinic acetylcholine receptors, to rat brain membranes

The α7 nicotinic acetylcholine receptors (nAChRs) play an important role in the pathophysiology of neuropsychiatric diseases such as schizophrenia and Alzheimer's disease. However, there are currently no suitable small molecule radioligands for imaging α7 nAChRs in the brain. In this study, we synthesized the novel radioligand [ 125I]4-iodophenyl 1,4-diazaicyclo[3.2.2]nonane-4-carboxylate ([ 125I]CHIBA-1006), a iodine-derivative of the selective α7 nAChR agonist SSR180711, and studied the characterization of [ 125I]CHIBA-1006 binding to rat brain membranes. The assays of [ 125I]CHIBA-1006 binding to rat brain membranes were performed at 4 °C. The presence of a single saturable high-affinity binding component for [ 125I]CHIBA-1006 in the rat brain was shown. Scatchard analysis revealed an apparent equilibrium dissociation constant ( K d) of 88.2 ± 21.4 nM and a maximal number of binding sites ( B max) of 65.4 ± 6.8 fmol/mg protein (mean ± SEM, n = 4). The specific binding of [ 125I]CHIBA-1006 was inhibited by a number of α7 nAChR-selective ligands (e.g., unlabeled CHIBA-1006, SSR180711, CHIBA-1001, MG624 and A844606), suggesting a similarity among α7 nAChR pharmacological profiles. In contrast, α-bungarotoxin, MLA, and nicotine showed very weak affinity for [ 125I]CHIBA-1006 binding. The regional distribution of [ 125I]CHIBA-1006 binding to crude membranes from dissected regions of the rat brain was different from that of [ 125I]α-bungarotoxin binding, suggesting that [ 125I]CHIBA-1006 binding sites may not be identical to [ 125I]α-bungarotoxin binding sites in the rat brain. The present findings suggest that [ 125I]CHIBA-1006 would be a useful new small molecule radioligand for α7 nAChRs in the brain.

Cadmium accumulation, gill Cd binding, acclimation, and physiological effects during long term sublethal Cd exposure in rainbow trout

Juvenile rainbow trout, on 3% of body weight daily ration, were exposed to 0 (control), 3, and 10 μg l −1 Cd (as Cd(NO 3) 2 · 4H 2O) in moderately hard (140 mg l −1 as CaCO 3), alkaline (95 mg l −1 as CaCO 3, pH 8.0) water for 30 days. Particular attention focused on acclimation, and on whether a gill surface binding model, originally developed in dilute softwater, could be applied in this water quality to fish chronically exposed to Cd. Only the higher Cd concentration caused mortality (30%, in the first few days). The costs of acclimation, if any, in our study were subtle since no significant effects of chronic Cd exposure were seen in growth rate, swimming performance (stamina and U Crit), routine O 2 consumption, or whole body ion levels. Substantial acclimation occurred in both exposure groups, manifested as 11- to 13-fold increases in 96-h LC 50 values. In water quality regulations, which are based on toxicity tests with non-acclimated fish only, this remarkable protective effect of acclimation is not taken into account. Cd accumulated in a time- and concentration-dependent fashion to 60–120× (gills), 8–20× (liver), 2–7× (carcass), and 5–12× (whole bodies) control levels by 30 days. Chronically accumulated gill Cd could not be removed by ethylenediaminetetraacetic acid (EDTA) challenge. These gill Cd concentrations were 20- to 40-fold greater than levels predicted by the gill-binding model to cause mortality during acute exposure. In short-term gill Cd-binding experiments (up to 70 μg l −1 exposures for 3 h), gill Cd burden increased as predicted in control fish, but was not detectable against the high background concentrations in acclimated fish. In light of these results, Cd uptake/turnover tests were performed using radioactive 109Cd to improve sensitivity. With this approach, a small saturable binding component was seen, but could not be related to toxic response in acclimated fish. Acclimated trout internalized less 109Cd than control fish, but interpretation was complicated by the possibility of radioisotopic exchange and specific activity dilution in the large `cold' Cd pool on the gills. We conclude that gill Cd burden is not predictive of mortality in acclimated fish, that the present gill modelling approach does not work in acclimated fish, and that longer term 109Cd turnover studies are needed for this purpose.

Mode of action of CryIIA: a Bacillus thuringiensis delta-endotoxin

CryIIA is an effective insecticidal delta-endotoxin produced by several strains of Bacillus thuringiensis. Unlike CryI and CryIIIA-toxins that demonstrate some degree of saturable binding on the brush border of susceptible insects, neither saturable binding nor a saturable binding component was found for CryIIA on the midgut brush border of Helicoverpa zea. CryIIA did not dilute and block CryIA(c) binding, however, CryIA(c) effectively diluted CryIIA and stopped the initial binding of CryIIA to the brush border. These observations suggest that CryIIA and CryIA(c) toxins share a common component for binding on the midgut brush border. CryIIA formed voltage-dependent and not highly cation-selective channels in planar lipid bilayers unlike CryIA(c) and CryIIIA. Both CryIA(c) and CryIIA were stable in the digestive fluids of H. zea, but CryIIA was significantly less soluble than CryIA(c). Despite this difference in solubility, CryIIA arrested the feeding of third instar H. zea as rapidly as did CryIA(c), however, the onset of acute morbidity was delayed for CryIIA. Differences in solubility, binding, and ion channels formed by CryIIA toxin, resulted in reduced bioactivity against H. zea when compared with CryIA(c) but represent a unique mode of action among the delta endotoxins.

ELISA estimation of the binding of epidermolytic toxin to neonatal mouse skin

B. P. Lockhart and C. J. Bailey. ELISA estimation of the binding of epidermolytic toxin to neonatal mouse skin. Toxicon 31, 569–576, 1993.— A sandwich ELISA, with antisera from rat and rabbit, was used to determine epidermolytic toxin (ET) to a limit of about 0.01 ng at 0.1 ng/ml. The binding of ET to the epidermis of skin discs was measured in vitro. The ability of the assay to discriminate between the two forms of the toxin was used to demonstrate that there was a saturable component of toxin binding to the epidermis. The rate of uptake, the effect of the inhibitor EGTA and comparative experiments with the inactive nitrated toxin confirmed that the observed binding is associated with toxigenesis. From measurements at toxin concentrations from 0.25 μg/ml to 100 μg/ml, it was calculated that the saturable binding component has a K d of about 2 μg/ml (approximately 60 nM) and a capacity of 0.5 ng per skin disc (1 ng per cm 2 of epidermis).

Characterization and subtype identification of the Na+-H+exchanger in bovine corneal epithelium

Amiloride analogues with N5-alkyl substitutions are specific high-affinity ligands for the Na(+)-H+ exchanger in various tissues. As a means to characterize the Na(+)-H+ exchanger in the bovine corneal epithelium, we determined the binding properties of [3H] methylisobutylamiloride (MIA) to a fraction enriched in plasma membrane from this tissue. [3H]MIA bound to these membranes in a time, -a temperature-, and -a pH-dependent manner. The binding was optimal at 4 degrees C and at pH 8.5 and it reached equilibrium at 60 min. Under these conditions, specific binding, which was inhibitable by excess unlabeled MIA, was about 85%. Scatchard analysis of this specific binding revealed a single saturable binding component with a Kd of 61 nM and a Bmax of 271 pmoles/mg protein. Inhibition of [3H]MIA specific binding by amiloride analogues showed the following order of potency: MIA > dimethylamiloride (DMA) > benzamil > amiloride. Na+ did not compete with MIA for binding. The effectiveness of clonidine, an alpha 2 agonist, and cimetidine, an H2 receptor antagonist, as inhibitors of Na(+)-H+ exchange activity was also determined because these compounds are used to distinguish between the exchanger subtypes. At concentrations higher than those needed for receptor interaction, clonidine was more effective than cimetidine in decreasing MIA binding. The activity of Na(+)-H+ exchanger, which was measured as the uptake of 22Na+ in the presence of an outwardly directly H+ gradient, was also inhibited by DMA, benzamil and amiloride with the same order of potency as obtained in the binding studies.(ABSTRACT TRUNCATED AT 250 WORDS)

19F-NMR spin-spin relaxation (T2) method for characterizing volatile anesthetic binding to proteins. Analysis of isoflurane binding to serum albumin.

This paper characterizes the low-affinity ligand binding interactions of a fluorinated volatile anesthetic, isoflurane (CHF2OCHClCF3), with bovine serum albumin (BSA) using 19F-NMR transverse relaxation (T2). 19F-NMR spectra of isoflurane in aqueous BSA reveal a single isoflurane trifluoromethyl resonance, indicative of rapid exchange of isoflurane between protein-bound and aqueous (free) environments. The exchange is slow enough, however, that the chemical shift difference between bound and free isoflurane (delta omega = 0.545 ppm) contributes to the observed isoflurane T2. The contribution of delta omega to T2 can be minimized by shortening the interval between 180 degrees refocusing pulses in the Carr-Purcell-Meiboom-Gill pulse sequence used to monitor T2. Analysis of the dependence of T2 on interpulse interval additionally allows determination of the T2 (6.2 ms) and the average lifetime (tau b = 187 microseconds) of bound isoflurane molecules. By use of a short interpulse interval (less than 100 microseconds), T2 measurements can readily be used to analyze equilibrium binding of isoflurane to BSA. This analysis revealed a discrete saturable binding component with a KD = 1.4 mM that was eliminated either by coincubation with oleic acid (6 mol/mol of BSA) or by conversion of BSA to its "expanded" form by titration to pH 2.5. The binding was independently characterized using a gas chromatographic partition analysis (KD = 1.4 mM, Bmax = 3-4 sites). In summary, this paper describes a method whereby T2 measurements can be used to characterize equilibrium binding of low-affinity ligands to proteins without the confounding contributions of chemical shift.(ABSTRACT TRUNCATED AT 250 WORDS)

Demonstration of binding components specific for 7,8-disubstituted guanine ribonucleosides in murine B lymphocytes.

7,8-Disubstituted guanine ribonucleosides are known to be potent intracellular modulators of immune responses. These compounds trigger and modulate a wide variety of lymphocyte responses including effects exerted directly on B cells. However, little is known about their mechanism of action. The current paper describes studies undertaken to evaluate whether binding components specific for these bioactive molecules exist in splenic B lymphocytes. After exposure of cells to labeled nucleoside, two different pools of nucleoside can be distinguished: a rapidly exchangeable nucleoside pool and a slowly exchangeable pool. The material in the latter pool consists of authentic unaltered nucleoside that is complexed to a relatively hydrophobic cellular component with an apparent Mr of 30,000-40,000; binding appears to interfere with free interaction of the nucleoside's cis hydroxyls with a boronate affinity resin. The slowly exchangeable nucleoside pool is seen to localize predominantly to the nucleus in electron microscopic autoradiographs. This pool is maximally bound by 30 min of incubation. Specific, saturable binding is demonstrable, with an apparent Kd of approximately 7 microM. This value correlates well with concentrations at which half-maximal biological activity occurs and suggests that the binding component likely mediates antigen-dependent immunomodulatory activity. Splenic B cells express approximately 2 x 10(4) binding sites/cell, whereas thymic lymphocytes, which do not respond functionally to nucleosides, do not display a measurable number of nucleoside binding sites. Ligand specificity of the binding interaction is confirmed by binding inhibition studies, in which binding inhibitory activity of unlabeled agonistic structural analogs recapitulate their degree of immunobiological activity. These data are most consistent with the existence of a saturable binding component with apparent specificity for 7,8-disubstituted guanine ribonucleosides in splenic B cells.

High-affinity binding of [ 3H]6-nitroquipazine to 5-hydroxytryptamine transporter in human platelets

The characteristics of the binding [ 3H]6-nitroquipazine, a very potent and selective inhibitor of 5-hydroxytryptamine (5-HT; serotonin) uptake, to human platelet membranes were studied at a physiological temperature of 37°C. The presence of a single saturable high-affinity binding component for [ 3H]6-nitroquipazine was demonstrated. Non-specific binding was estimated in the presence of 1 μM paroxetine. Scatchard analysis revealed an apparent equilibrium dissociation constant (K d) of 0.450 ± 0.04 nM and a maximal number of binding sites (B max) of 2508 ± 360 fmol/mg protein (mean ± S.D., n = 4). The kinetically derived dissociation constant (K d) was 0.431 nM. [ 3H]6-Nitroquipazine binding was inhibited selectively by 5-HT uptake inhibitors, and the potency of various drugs to inhibit [ 3H]6-nitroquipazine binding closely correlated with their inhibitory effects on [ 3H]5-HT uptake into synaptosome. Moreover, K i values for drug inhibition of [ 3H]6-nitroquipazine binding to human platelet membranes were significantly correlated with the corresponding K i values for inhibition of [ 3H]paroxetine binding at 37°C. The present results suggest that the binding sites for [ 3H]6-nitroquipazine are associated with the 5-HT transporter in human platelets.

High-affinity [ 3H]6-nitroquipazine binding sites in rat brain

6-Nitroquipazine is a very potent and selective inhibitor of neuronal 5-hydroxytryptamine (5-HT; serotonin) uptake. We have characterized the specific binding of [ 3H]6-nitroquipazine to rat brain membranes at 22°C. The present results indicate the presence of a single saturable high-affinity binding component for [ 3H]6-nitroquipazine. Scatchard analysis revealed an apparent equilibrium dissociation constant (K d) of 93.0 ± 2.23 pM, and a maximal number of binding sites (B max) of 831.7 ± 18.7 fmol/mg protein (mean ± S.D., n = 4). The kinetically derived dissociation constant was 74.5 pM. [ 3H]6-Nitroquipazine binding was inhibited selectively by 5-Ht uptake inhibitors, and a good correlation was demonstrated between the potency of various drugs to inhibit [ 3H]6-nitroquipazine binding and [ 3H]5-HT uptake. The highest densities of [ 3H]6-nitroquipazine binding were obtained in the hypothalamus and midbrain, intermediate binding was observed in the striatum, hippocampus, medulla oblongata and cortex, and the lowest binding was observed in the cerebellum. Lesioning of 5-HT neurons with p-chloroamphetamine resulted in a 72% reduction in [ 3H]6-nitroquipazine binding compared to controls. These results indicate that the binding site specifically labelled by [ 3H]6-nitroquipazine is associated with the neuronal 5-HT transporter complex. [ 3H]6-Nitroquipazine is an excellent radioligand for the study of the 5-HT uptake system.

Different binding of propranolol enantiomers to human alpha 1-acid glycoprotein

The binding of propranolol enantiomers to human alpha 1-acid glycoprotein was studied using high performance liquid chromatography in order to provide insight into binding models and to describe individual binding parameters of both enantiomers. The binding of (−)-propranolol was shown to be saturable with one major binding site ( n = 0.81, k = 2.73 × 10 5/ M). The saturation process achieved its upper asymptotic value at drug/protein molar ratio of approximately 1. In the case of the opposite (+)- enantiomer the binding isotherm did not show evidence of saturation even at higher drug/protein molar ratios (up to 50). The individual binding parameters for (+)-enantiomer were n = 0.38, k = 3.4 × 10 6/M and n′ k′ = 1.39 × 10 4/M for the saturable and nonsaturable binding component, respectively. At drug/protein molar ratio 2 the circular dichroism measurements confirmed the existence of different binding models for individual propranolol enantiomers.

Phorbol esters potentiate glucocorticoid-induced cytotoxicity in CEM-C7 human T-leukemia cell line

Phorbol ester tumor promoter, such as 12- O-tetradecanoylphorbol-13-acetate (TPA), is synergistic with dexamethasone to cause growth inhibition of CEM-C7 human T-leukemia cell line. A specific saturable binding component which may mediate the phorbol ester effects has been identified by using [20- 3H]phorbol 12,13-dibutyrate in a whole cell binding assay. Saturation of the specific binding occurs at a concentration (approx. 100 nM) consistent with causing maximal cytotoxicity. Scatchard analysis of the binding after 15 min at 37°C demonstrates a single class of binding sites. The number is 194,000 sites per cell. Other phorbol esters are also cytotoxic to CEM-C7 cell in the presence of 30 nM dexamethasone in an approximate proportion to their activity in competing for [20- 3H]phorbol 12,13-dibutyrate binding. Phorbol 12, 13-didecanoate, 4- O-methyl PMA, 4- β-phorbol do not compete for specific binding. The synergism of phorbol esters and dexamethasone on CEM-C7 cells is reversible by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine(H-7). However, by treating CEM-C7 cells with TPA for 48 h there is not any increase in the affinity or levels of glucocorticoid receptor. It is tentatively concluded that phorbol esters may play an important role linked to the glucocorticoid-induced growth inhibition in CEM-C7 human T-leukemic cell.

Estimation of the number and turnover rate of Na+/H+ exchangers in lymphocytes. Effect of phorbol ester and osmotic shrinking.

Rat thymic lymphocytes possess an amiloride-sensitive Na+/H+ exchanger in their plasma membrane. Kinetic studies revealed that 5-(N-methyl-N-isobutyl)amiloride (MIA) was a more potent inhibitor of the antiport than amiloride (cf. apparent Ki of 174 nM and 6 microM, respectively). Inhibition by MIA was rapid (less than 5 s) and readily reversible. [3H]MIA binding to whole cells was assayed by rapid centrifugation following short (5 s) incubations to minimize nonspecific binding. A saturable binding component (Kd approximately equal to 170 nM) which was displaced by amiloride was detected. In contrast, there was no significant amiloride-displaceable binding to human erythrocytes, which have comparatively little Na+/H+ exchange activity. In lymphocytes, the ability of amiloride and several of its analogs to displace [3H]MIA correlated with their potency as inhibitors of the antiport. Both kinetic and binding studies revealed that extracellular H+, but not Na+, inhibited the interaction of MIA with its receptor(s). Taken together, these data suggest that [3H]MIA binds to the Na+/H+ exchanger. Scatchard analysis revealed that [3H]MIA bound to a maximum of 8000 high affinity sites/cell. Activation of Na+/H+ exchange by osmotic shrinking or by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate was not accompanied by a significant change in [3H]MIA binding. Given an upper limit of 8000 functional sites/thymocyte, we estimate that the turnover number of each maximally activated exchanger is at least 2000 cycles/s.

Substance P mechanisms of the spinal cord related to vasomotor tone in the spontaneously hypertensive rat

This report deals with substance P (SP) mechanisms involved in regulation of vasomotor tone at the spinal cord levels in normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Our results indicate the following. (1) Intrathecal injections of the SP antagonist, d-Pro 4, d-Trp 7.9-SP 4–11 cause dose-dependent decreases in mean arterial pressure and heart rate in Sprague-Dawley rats, WKYs and SHRs; the maximal blood pressure decreases are equal to those seen after cervical spinal cord transection. (2) Intrathecal injections of this antagonist into the L1 spinal level in WKYs or SHRs that had previously had their C8 spinal cords transected caused a rise in blood pressure and heart rate, suggesting that intrinsic spinal SP mechanisms are probably not involved in vasomotor tone. (3) The intermediolateral cell column region (IML) of 16-week-old WKYs and SHRs has a single high affinity and saturable binding component with approximately the same dissociation constant ( K d = 1.21nM for WKYs; K d = 1.25nM for SHRs); the SHRs showed a higher number of sites (B max= 24.5fmol/mg protein) than WKY rats (B max= 19.9fmol/mg protein). The K d and B max obtained from IML sections from 4-week-old WKYs and SHRs exhibit no differences, although their binding values with 2 nM [ 3H]SP are higher than those obtained from the 16-week-old animals. (4) d-Pro 4, d-Trp 7,9-SP 4–11 has the same relatively low (micromolar range) potency for displacing [ 3H]SP binding in the IML of WKYs and SHRs ((5) SHRs (16 weeks old) contain 20% more SP immunoreactivity in the IML than WKY rats (834 ± 36pg/mg protein vs694 ± 50pg/mg protein); 4-week-old rats do not show such differences. The potential significance of these results is discussed in relation to the control of vasomotor tone.

Effects of apamin, quinine and neuromuscular blockers on calcium-activated potassium channels in guinea-pig hepatocytes.

The bee venom peptide, apamin, has been radiolabelled with 125I, the monoiodinated derivative purified, and its binding to intact guinea-pig liver cells studied. At 37 degrees C 125I-monoiodoapamin associated with, and dissociated from, guinea-pig hepatocytes remarkably rapidly. The association and dissociation rate constants were 1.4 X 10(8) M-1 s-1 and 0.035 s-1 respectively. Equilibrium binding studies demonstrated a saturable binding component compatible with 1:1 binding to a single class of site and having an equilibrium dissociation constant (KL) of 390 pM. The maximal binding capacity was 1.1 fmol mg-1 dry wt. of tissue. Unlabelled apamin displaced bound 125I-monoiodoapamin with a KI of 380 pM, which is consistent with the concentration of apamin required to inhibit Ca2+-activated K+ permeability (PK(Ca) ) in these cells. Inhibitable binding of 125I-monoiodoapamin to rat hepatocytes was much less than to guinea-pig hepatocytes and could not be reliably quantified. Neither was there any discernible inhibitable binding to human erythrocytes. This is in keeping with the reported lack of apamin-sensitive Ca2+-activated K+ channels in these cell types. Various agents were tested for their ability to inhibit monoiodoapamin binding to, and Ca2+-mediated K+ efflux from, guinea-pig hepatocytes. All compounds tested which inhibited binding also blocked K+ efflux at similar concentrations. TEA and quinine affected hepatocytes only at high concentration (KI = 5.8 and 0.51 mM respectively). 9-aminoacridine, quinacrine and chloroquine were slightly more effective (KI = 70-180 microM). By far the most active compounds (apart from apamin) were the neuromuscular blocking agents; tubocurarine, pancuronium and atracurium (KI = 7.5, 6.8 and 4.5 microM respectively). Gallamine was slightly less effective (KI = 14 microM) and decamethonium and hexamethonium much less so (KI = 620 and 760 microM respectively). 3,4-diaminopyridine, alpha-bungarotoxin and tetrodotoxin were among several compounds which showed little or no affinity for apamin binding sites or inhibition of K+ efflux in guinea-pig hepatocytes. The saturable binding of 125I-monoiodoapamin to guinea-pig hepatocytes corresponds to about 1700 sites per cell. Assuming, tentatively, that binding sites correspond to channels the rate of K+ loss observed following agonist action can readily be explained if these channels have unitary conductances in the range reported for PK(Ca) in other tissues.

Synaptic binding sites in brain for [ 3H]β-bungarotoxin — A specific probe that perturbs transmitter release

A neurotoxic, [ 3H]labelled derivative of β-bungarotoxin, known to inhibit neurotransmitter release and to be free of phospholipase activity, was used to demonstrate autoradiographically the distribution and ultrastructural location of its saturable binding component in brain. Light-microscope autoradiography of rat cerebellum and hippocampus showed that it resides primarily in synaptic-rich areas, with much lower densities of sites in other regions containing cell bodies; also, little binding was associated with myelinated tracts. Ultrastructural localisation and sub-fractionation studies on purified cerebrocortical synaptosomes showed that [ 3H]toxin binding sites are located predominantly on brain synaptosomal membranes, consistent with their possible association with transmitter release.

Characterization and measurement of cytoplasmic and nuclear oestradiol-17 beta-receptor proteins in benign hypertrophied human prostate.

The demonstration and partial characterization of a high-affinity saturable binding component for oestradiol-17 beta in the cytosol and nuclear extract of the benign hypertrophied human prostate is reported. This binding component was found to precipitate with protamine sulphate and to exhibit marked specificity for oestradiol and diethylstilboestrol (DES) but not dihydrotestosterone (DHT) or progesterone. Analysis of oestradiol-labelled cytosol on low ionic strength glycerol gradients revealed a binding component with a sedimentation coefficient of 4S which was inhibited with DES but not DHT and destroyed either by preheating labelled cytosol at 45 degrees C for 30 min or by treatment with the sulphydryl blocking agent, parachloromercureobenzoate. The oestradiol-binding complex precipitated by ammonium sulphate was found to have an isoelectric point (pI) of 6.3 as analysed on polyacrylamide-gel plates. The oestradiol-binding component in the nuclear extract also exhibited similar steroid specificity and sedimented with a coefficient of 2.8S on high ionic strength glycerol gradients. Isoelectric focusing of the heparin-extracted nuclear oestradiol-binding complex revealed a pI of 6.0. The dissociation constants (Kd) and the concentrations of the cytoplasmic and nuclear oestradiol-binding sites in 19 samples of benign hypertrophied prostates were estimated by Scatchard plot analyses. The mean Kd and concentration of binding sites in the cytosol were 2.9 +/- 2.6 (S.D.) nmol/l and 11.7 +/- 8.4 fmol/mg protein respectively. The corresponding values for nuclear extract were 4.1 +/- 7.5 nmol/l and 347.1 +/- 415.5 fmol/mg DNA respectively. There was no significant difference between the mean concentration of oestradiol-binding sites in the two cellular fractions.

Localization of sites for 125I-labelled botulinum neurotoxin at murine neuromuscular junction and its binding to rat brain synaptosomes

Botulinum neurotoxin, purified to homogeneity from Clostridium botulinum (Type A), was found to be highly neurotoxic (>8 x 10 7 mouse ld 50/mg protein). Labelling of this pure neurotoxin with 125I-iodine to high specific radioactivity was achieved without appreciable loss of biological activity. This was used to demonstrate saturable binding sites for this toxin at the neuromuscular junction, following in vivo administration into mice. A demonstrable inhibitory effect of the neurotoxin on release of acetylcholine from rat cerebrocortical synaptosomes indicates that it affects synapses in the central nervous system. Kinetic studies on the binding of 125I-labelled neurotoxin to brain synaptosomes yielded an association rate constant of 2.3 x 10 5M −1s −1; dissociation plots were biphasic and the predominant species showed a rate constant of 1.2 x 10 −4s −1. The saturable binding component is heatsensitive and inactivated by trypsin. Preliminary studies showed that botulinum neurotoxin associates with plasma membrane fractions of synaptosomes and that binding does not result in any gross structural changes, at least in the majority of the toxin molecules.

Benzamil binding to kidney cell membranes

Tile binding of [ 3H]benzamil ( N-benzylamidino-3,5-diamino-6-chloropyrazine carboxamide), a blocker of passive sodium channels, was investigated in tissues from several species (rat, guinea-pig, rabbit, chicken, frog and two species of fish). Tissues used were kidney, colon epithelium, cardiac and skeletal muscle, brain, liver, epididymis and red cell membranes. Displaceable binding was detected principally in target tissues, and particularly in the kidney. In rat and rabbit kidney considerably more binding activity was present in the cortex than in the medulla. In rat kidney cortex, a saturable binding component (2 nmoles g −1 protein) with high affinity (approx. 5 × 10 7 M −1) was detected. Other agents which are known to interact with sodium channels competed with [ 3H]benzamil for the binding site. The affinity of amiloride detected in this way was 10 5 M −1. Neither adrenalectomy nor pretreatment with aldosterone affected the amount of benzamil binding activity. Sodium had no effect on either the affinity or the capacity of binding. The binding activity appeared not to be associated with the base carrier known to be present in the proximal convoluted tubule. Binding activity was increased in both crude plasma membrane fractions and in fractions enriched in brush border membranes. The possible physiological role of the diuretic receptor is discussed.