Guinea Pig Cardiac Membranes Research Articles

Organophosphorus Pesticides Decrease M2 Muscarinic Receptor Function in Guinea Pig Airway Nerves via Indirect Mechanisms

BackgroundEpidemiological studies link organophosphorus pesticide (OP) exposures to asthma, and we have shown that the OPs chlorpyrifos, diazinon and parathion cause airway hyperreactivity in guinea pigs 24 hr after a single subcutaneous injection. OP-induced airway hyperreactivity involves M2 muscarinic receptor dysfunction on airway nerves independent of acetylcholinesterase (AChE) inhibition, but how OPs inhibit neuronal M2 receptors in airways is not known. In the central nervous system, OPs interact directly with neurons to alter muscarinic receptor function or expression; therefore, in this study we tested whether the OP parathion or its oxon metabolite, paraoxon, might decrease M2 receptor function on peripheral neurons via similar direct mechanisms.Methodology/Principal FindingsIntravenous administration of paraoxon, but not parathion, caused acute frequency-dependent potentiation of vagally-induced bronchoconstriction and increased electrical field stimulation (EFS)-induced contractions in isolated trachea independent of AChE inhibition. However, paraoxon had no effect on vagally-induced bradycardia in intact guinea pigs or EFS-induced contractions in isolated ileum, suggesting mechanisms other than pharmacologic antagonism of M2 receptors. Paraoxon did not alter M2 receptor expression in cultured cells at the mRNA or protein level as determined by quantitative RT-PCR and radio-ligand binding assays, respectively. Additionally, a biotin-labeled fluorophosphonate, which was used as a probe to identify molecular targets phosphorylated by OPs, did not phosphorylate proteins in guinea pig cardiac membranes that were recognized by M2 receptor antibodies.Conclusions/SignificanceThese data indicate that neither direct pharmacologic antagonism nor downregulated expression of M2 receptors contributes to OP inhibition of M2 function in airway nerves, adding to the growing evidence of non-cholinergic mechanisms of OP neurotoxicity.

Species comparison of adenosine A 1 receptors in isolated mammalian atrial and ventricular myocardium

Various species have been used as models to study the effects of adenosine (ADO) on atrial and ventricular myocardium, but few direct tissue comparisons between species have been made. This study further characterizes adenosine A 1 receptor binding, adenylate cyclase activity and direct and indirect A 1 receptor-mediated functional activity in atrial and ventricular tissue from Sprague-Dawley rats and Hartley guinea pigs. Rat right atria (RA) were found to be significantly more sensitive to cyclopentyladenosine (CPA), while guinea pig left atria (LA) were more sensitive to CPA. After the addition of isoproterenol (ISO), the reduction of CPA response in rat RA was significantly greater than in guinea pig; however, after ISO treatment, the guinea pig LA was more sensitive to CPA than the rat. Adenylate cyclase inhibition by CPA was significantly greater in atria and ventricles obtained from guinea pig than rat. In competition binding experiments, guinea pig RA had significantly more high affinity sites than rat, but the K is were not significantly different. There were no significant differences between guinea pig LA and rat LA. Guinea pig ventricular tissue had fewer high affinity sites than rat without any differences in their K i values. In antagonist saturation experiments, the density and affinity of A 1 receptors in guinea pig cardiac membranes were significantly greater than in rat. Our results indicate definite species differences as well as tissue differences between rat and guinea pig. These differences must be considered when interpreting studies using rat and guinea pig tissue as models for cardiac function.

Molecular rigidity and potency of bispyridinium type allosteric modulators at muscarinic M 2-receptors

Several bispyridinium compounds have been shown to be potent allosteric modulators of ligand binding to muscarinic M 2-receptors. “Uno compounds” are benzyl derivatives of the bispyridinium “TMB4” (trimethylene-bis-[4-hydroxy-iminomethyl-pyridinium]). To gain more insight into structure activity relationships, eleven derivatives with varying structure of the oxime-linked aromatic substituent were tested for their ability to inhibit the equilibrium-binding of [ 3H]N-methylscopolamine ([ 3H]NMS) in guinea pig cardiac membranes and to retard [ 3H]NMS-dissociation allosterically. At a concentration of 3 μM, all compounds reduced [ 3H]NMS-binding to about 40 % of the control level, indicating a similar potency to inhibit the association of [ 3H]NMS. Allosteric retardation of [ 3H]NMS-dissociation required higher concentrations. Comparing the effects of the compounds at 30 and 300 μM, respectively, revealed considerable differences in potency. Therefore, the concentration-dependency of the delay of [ 3H]NMS-dissociation was determined for selected compounds. The results indicate that introduction of a benzyl-moiety into TMB4 leads to a 20-fold increase in allosteric potency. A further increment by a factor of 10 is obtained with the 2,6-dichlorobenzyl-substitution and with the naphthyl-derivative. The other compounds were less potent. An inverse correlation was found between the rotational freedom of the aromatic substituent and the allosteric potency. In conclusion, the aromatic moiety of non-symmetric bispyridinium-type modulators does not seem to be part of the pharmacophore involved in the inhibitory effect on the association of [ 3H]NMS. In contrast, a rigid aromatic lateral moiety appears to be essential for the interaction with the recognition site mediating the allosteric delay of [ 3H]NMS dissociation from muscarinic M 2-receptors.

Chronic dipyridamole administration downregulates [3H]nitrobenzylthioinosine binding site affinity in guinea pig kidney but not heart and brain.

Specific binding of the nucleoside transporter probe, [3H]nitrobenzylthioinosine, ([3H]NBMPR) was measured in washed guinea pig cardiac, renal and forebrain membranes after 14 days of treatment with dipyridamole (37.5 mg/kg, s.c., b.i.d.) or vehicle. When compared to values in vehicle-treated animals, a 100 percent increase in equilibrium dissociation constant (Kd) was observed in the kidney of dipyridamole-treated animals (0.51 +/- 0.04 to 1.0 +/- 0.06, p < 0.01). The maximal binding capacity (Bmax) was unaltered. No changes were observed in the heart or forebrain. The increase in Kd suggests that chronic dipyridamole treatment decreases the apparent binding affinity of NBMPR for kidney nucleoside transporters. Cardiac and brain nucleoside transporters may be either less susceptible to chronic dipyridamole administration or have a different adaptive mechanism.

A stable and highly potent hexamethonium derivative which modulates muscarinic receptors allosterically in guinea-pig hearts.

W84 (N,N,N',N'-tetramethyl-N,N'-bis-(3-phthalimidopropyl)-N,N'-hexane- 1,6- diyl-bis-ammonium dibromide) is a potent stabilizer of antagonist binding to muscarinic receptors; however, W84 hydrolyses in aqueous buffered medium. The synthesis of the stable derivative CHIN3/6 is presented containing a 2-phenyl-quinazolinone instead of the labile phthalimide substituent. This compound retarded [3H]N-methylscopolamine-dissociation in guinea-pig cardiac membranes with slightly higher potency than W84, the EC50 values amounting to 7.5 x 10(-7) and 13 x 10(-7) M, respectively. Molecular modelling revealed differences in the electron density of the substituents and in their molecular shape. It is suggested that the derivatives use partially different sites of attachment when occupying the allosteric binding site of the receptor protein.

C14-Aminosteroid LNF 209, An Agent with Positive Inotropic and Antimuscarinic Activity

LNF 209 is a cardioactive steroid containing at position C14 a protonized amino group. It was tested whether LNF 209 has antimuscarinic properties besides its cardiotonic effect, which was also quantified. In guinea pig left atria (3 Hz, 0.9 mM Ca2+, 5.4 mM K+), LNF 209 increased the force of contraction at concentrations > or = 1 microM, thus having a tenfold lower potency than ouabain. Correspondingly, cardiac glycoside binding sites in guinea pig cardiac membranes labeled with [3H]ouabain were occupied by LNF 209 (Ki = 1.7 microM) at concentrations tenfold higher compared with ouabain (Kd = 0.14 microM). The negative inotropic effect of the muscarinic agonist oxotremorine was antagonized by LNF 209, but not by ouabain; in the Schild plot, the data for LNF 209 could be connected by a line with a slope of unity and a pA2 = 7.5. The binding of [3H]N-methylscopolamine ([3H]NMS) to the M2-cholinoceptors in guinea pig cardiac membranes was inhibited by LNF 209 with a Ki = 0.5 microM; LNF reduced the affinity of [3H]NMS binding without affecting the number of receptor sites. In isolated segments of guinea pig ileum, LNF 209 antagonized the effect of oxotremorine with a pA2 = 7.4. It is concluded that LNF 209 is a competitive antagonist at M-cholinoceptors without preference for the cardiac M2-cholinoceptors. Whether the antimuscarinic property is of significance in species highly sensitive to digitalis remains to be established.

Further evidence that propentofylline (HWA 285) influences both adenosine receptors and adenosine transport.

We have examined the actions of a novel xanthine derivative, propentofylline (HWA 285), that has been shown to protect against ischemic brain damage in rats and gerbils, on adenosine receptors (A1 and A2), and on adenosine transporters using several techniques, cells and tissues. Propentofylline and its hydroxylated metabolite A 72 0287 were about 20 times less potent than theophylline in displacing A1-agonist binding to membranes from rat cortex, and A1-antagonist binding to whole DDT, MF-2 smooth muscle cells. A1-agonist binding to adenosine A1-receptors in several brain structures was inhibited in a concentration-dependent manner by A 72 0287 and propentofylline as judged by quantitative autoradiography (IC50-values 300-600 microM in eg striatum and in cortex layer IV). In two functional assays, A1-receptor mediated effects were blocked by propentofylline. A1-receptor-mediated inhibition of cyclic AMP accumulation was virtually abolished by 100 microM propentofylline. The A1-receptor-mediated inhibition of evoked acetylcholine release was also reduced by propentofylline, but in this case the effect is not due exclusively to adenosine receptor antagonism but also to another action since the presynaptic inhibitory effect of carbachol was also inhibited. Adenosine A2-receptors were also antagonized by propentofylline as judged by a concentration-dependent antagonism of A2-agonist-induced cAMP accumulation in human T-leukemia cells (possessing putative A2b-receptors; pA2-value 180 microM compared to 0.26 microM for 8-cpt), and in PC-12 cells (possessing putative A2a-receptors, Ki-value 365 microM). Finally, adenosine transporters were affected by propentofylline and A 72 0287. Thus, [3H]-nitrobenzylthioinosine-binding to guinea-pig cardiac membranes was blocked by propentofylline or A 72 0287 (Ki 270 microM). The present results show that propentofylline and its hydroxylated metabolite can influence adenosine mechanisms in a multitude of ways. How these different actions may contribute to the ability of propentofylline to reduce the magnitude of ischemic damage is discussed.

Specific binding of 3H-ICI 198, 615, a potent LTD 4 antagonist, to guinea pig cardiac ventricular membranes

Peptido-leukotrienes (LTs) elicit myocardial depression in several mammalian species, and radioligand binding assays with 3H-LTC 4 and 3H-LTD 4 have provided evidence of putative receptor sites on guinea pig cardiac ventricular membranes (GPCVM). Our objective was to characterize specific binding of 3H-ICI 198,615, a potent and selective LTD 4 antagonist, to the 155,000 X g pellet of GPCVM. 3H-ICI 198,615 (0.01 –3.8 nM) showed high specific binding (85 – 90% of total), which was protein dependent, saturable (Bmax = 4914 ± 706 fmol/mg protein, n = 3), of high affinity (Kd = 4.3 ± 0.8 nM, n = 3) and without cooperativity. Equilibrium binding was achieved by 20 minutes and could be rapidly reversed by addition of excess unlabeled ICI 198,165 or FPL55712. Competition studies with 3H-ICI 198,615 against several LTD 4 antagonists produced an order of potency: ICI 198,615 > SKF102922 > FPL55712 ≥ LY171883. Addition of divalent cations caused a concentration dependent decrease in specific binding apparently due to a reduction in affinity. Binding was not influenced by the guanine nucleotide analogs GTPγS and Gpp(NH)p, EDTA, or a multitude of diverse non-LT receptor agonists and antagonists. These data provide evidence supporting the existence of specific and high affinity binding sites for 3H-ICI 198,615 in GPCVM.

Adenosine transporters in vascular smooth muscle and endothelium: Multiple [3H]Nitrobenzylthioinosine binding sites in human umbilical vein endothelium

AbstractCultured vascular smooth muscle and endothelial cells may be useful models for studying the cardiovascular adenosine transport system and metabolism. We have quantified the nucleoside transporter elements of cultured primate vascular smooth muscle, bovine aortic endothelial, and human umbilical vein endothelial cells by radioligand binding and by using membrane preparations of these cells and the nucleoside transporter probe nitrobenzylthioinosine ([3H]NBMPR), a potent and tightly bound inhibitor of nucleoside transport. The binding was rapid, reversible, saturable, and site‐specific. Scatchard analysis of the saturation data showed that [3H]NBMPR bound to high and low affinity binding sites in human umbilical vein endothelial cell membranes with apparent binding affinities (KD) of 0.093±0.01 nM 1.92±0.1 nM, and binding site densities (Bmax values) of 13.48±1.2 and 69+5.3 fmol/mg protein, respectively. In contrast, the binding to primate vascular smooth muscle and bovine aortic endothelial cell membranes occurred to an apparently high affinity single class of binding sites at which the KD was 1.4±0.3nM and 0.28±0.05nM, respectively, and which had Bmax values of 1,977±163 and 1,284±267 fmol/mg protein, respectively. Scatchard analysis of the binding inhibition by dipyridamole showed a mixed type inhibition, while NBMPR inhibited the binding competitively. Several recognized nucleoside transport inhibitors and vasodilators inhibited the binding with an order of potency similar to that observed for the inhibition of [3H]NBMPR binding to guinea pig cardiac membranes.

LY249933

Compound LY249933 and its component diastereomers, (RR) and (SR), were studied for their vascular and cardiac effects in vitro and in vivo. In guinea pig cardiac ventricular membranes, LY249933, (RR), and (SR) potently displaced bound [3H]nitrendipine (Kd values = 2-6 nM). In isolated guinea pig right ventricular strips, LY249933 produced a small but significant increase in contraction, whereas (RR) substantially increased (-log EC50 (M) = 4.6 +/- 0.8) and (SR) decreased contraction (-log EC50 (M) = 4.1 +/- 0.8). In isolated canine cephalic vein, contracted with 80 mM KCl, an increase in contraction was produced by (RR), whereas relaxation was produced by LY249933 (-log EC50 (M) = 5.9 +/- 0.9) and (SR) (-log EC50 (M) = 6.0 +/- 0.7). At 20 mM KCl, (RR) increased, (SR) decreased, but LY249933 did not alter contraction. In anesthetized dogs, LY249933 (200 micrograms/kg/min, i.v.) increased dP/dt60, decreased heart rate, but did not change vascular resistance or rate pressure product. At the same dose, (RR) and (SR) both tended to increase dP/dt60 nonsignificantly, whereas (RR) increased and (SR) decreased vascular resistance. Both (RR) and (SR) tended to decrease heart rate nonsignificantly, whereas (RR) did not change and (SR) decreased rate pressure product. Thus, LY249933 produced potentially beneficial cardiovascular changes resulting from the combined actions of its (RR) and (SR) diastereomers that are postulated to be calcium agonist and antagonist, respectively.

Interaction of the Cardiotonic Agent DPI 201-106 with Cardiac Ca2+ Channels

The cardiotonic agent DPI 201-106 was investigated for its effects on (a) contractile force in guinea pig left atria, (b) membrane currents in isolated guinea pig cardiac myocytes, and (c) [3H]nitrendipine binding in guinea pig cardiac membranes. The compound elicited a positive inotropic effect in normally polarized (5.9 mM extracellular KCl) and a negative inotropic effect in partially depolarized (20 mM KCl) isolated, electrically stimulated left atria. This decrease in contractile force was probably caused by inactivation of the fast Na+ inward current and concomitant blockade of the inward Ca2+ current. The blocking effect on Ca2+ channels was directly shown in voltage-clamp experiments using isolated cardiocytes. Further evidence for interaction of DPI 201-106 with Ca2+ channels was obtained from the [3H]nitrendipine binding studies. Thus, Ca2+ antagonism contributes to the complex pharmacologic profile of DPI 201-106, and is probably responsible for the bradycardia and lowering of systemic vascular resistance observed in vivo.

Inhibition of myocardial Ca2+ channels by three dihydropyridines with different structural features: potential‐dependent blockade by Ro 18–3981

Inhibition of myocardial Ca2+ channels was investigated for three dihydropyridines with different structural features: Ro 18-3981, darodipine (PY 108-068) and nifedipine. Ro 18-3981 contains a sulphamoyl acetyl side-chain. In voltage-clamps experiments with isolated cardiac myocytes of guinea-pig, Ro 18-3981 caused a concentration-dependent inhibition of the Ca2+ current, which was influenced by the membrane holding potential. A markedly greater inhibition by Ro 18-3981 was observed when myocytes were depolarized (to +10 mV) from a holding potential (Vh) of -20 mV (IC50 = 2.3 nm) than at -50 mV (IC50 = 100 nM). The three dihydropyridines caused a concentration-dependent reduction in contractile force of isolated, electrically-stimulated left atria of the guinea-pig. Elevation of the extracellular K+ concentration from 5.9 to 24 mM resulted in a significant reduction in negative inotropic IC50 values for Ro 18-3981 (137 fold), darodipine (8 fold) and nifedipine (20 fold). The affinity of these drugs for the high-affinity (+)-[3H]-PN 200-110 binding site was determined in guinea-pig cardiac membranes. The KD value of Ro 18-3981 (1.0 nM) was similar to the IC50 value for blockade of ICa at a Vh of -20 mV (2.3 nM), i.e. at a level of near-maximal depolarization. Thus, structurally-different dihydropyridines exert potential-dependent inhibition of myocardial Ca2+ channel activity consistent with the modulated receptor hypothesis. These results demonstrate that blockade of myocardial excitation-contraction coupling by Ca2+ entry blockers is also potential-dependent.

Ouabain binding and inotropy in acute potassium depletion in guinea pigs.

In hypokalaemia the incidence of cardiac toxicity with digitalis is increased, possibly through changes in the affinity or capacity of the digitalis receptor in the heart. Previous studies have reported an increased ouabain binding capacity or Na+-K+ ATPase activity after hypokalaemia in human erythrocytes and rabbit and guinea pig hearts and no change or decreases in rabbit or rat skeletal muscles with no changes in ouabain affinity. The present study determined (a) the effect of potassium on 3H-ouabain binding to normokalaemic guinea pig cardiac cell membranes, (b) the effect of acute hypokalaemia induced by a potassium deficient diet for 14-18 days in guinea pigs on 3H-ouabain binding to erythrocytes and cardiac and skeletal muscle homogenates, and (c) ouabain induced inotropy in isolated contracting guinea pig left atria, right ventricular papillary muscles, and soleus muscle strips from normokalaemic and hypokalaemic guinea pigs. 3H-ouabain binds to cardiac cell membranes in the presence of magnesium and inorganic phosphate with an affinity (KD value) of 1.13 X 10(-7) mol X litre-1. Potassium decreased this affinity without changing the binding capacity. Erythrocytes and heart muscle homogenates showed the same affinity as cardiac cell membranes, whereas soleus muscle homogenates had a higher affinity for ouabain (KD 5.1 X 10(-8) mol X litre-1). After hypokalaemia, the ouabain affinity did not change in any tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

The peripheral high affinity benzodiazepine binding site is not coupled to the cardiac Ca 2+ channel

Ro 5-4864, the prototype ligand of the peripheral benzodiazepine binding site caused a decrease of the action potential duration in isolated guinea-pig cardiac myocytes. Voltage-clamp experiments showed that, at concentrations below 3 X 10(-6) M, Ro 5-4864 caused a parallel outward shift of the membrane current elicited by depolarization to + 10 mV from a holding potential of -50 mV. The peak inward Ca2+ current (ICa) and the inwardly rectifying K+ current were not affected. ICa was reduced by Ro 5-4864 at concentrations above 3 X 10(-6) M. At these concentrations, Ro 5-4864 also caused a negative inotropic effect in isolated guinea-pig papillary muscles, reduced K+ depolarization-induced contractures of the isolated rat aorta and inhibited [3H]nitrendipine binding to guinea-pig cardiac membranes. These data provide no evidence that the peripheral high affinity benzodiazepine binding site is coupled to the cardiac Ca2+ channel. The possibility cannot be excluded that a postulated micromolar affinity benzodiazepine receptor is associated with the Ca2+ channel.

Affinity of calcium channel inhibitors, benzodiazepines, and other vasoactive compounds for the nucleoside transport system.

There is evidence to suggest that several different groups of drugs including the so-called coronary vasodilators, benzodiazepines, and calcium channel inhibitors may owe their vasoactivity, in part, to the potentiation of the vasorelaxant effects of endogenous adenosine. To measure the affinity of some of these agents for the membrane-located nucleoside transport system, competition binding assays have been performed using the high-affinity radioligand [3H]nitrobenzylthioinosine (NBMPR). Experiments were performed on human erythrocytes and cardiac membranes from guinea pigs and rats. Recognized nucleoside transport inhibitors had high affinity (less than 50 nM) for NBMPR recognition sites associated with the nucleoside transporter complex in human erythrocytes, whereas calcium channel inhibitors and benzodiazepines had predominantly low affinity (greater than 1 microM). Although some recognized transport inhibitors, such as dipyridamole, show marked differences in affinity for NBMPR sites in guinea pig and rat tissues, benzodiazepines and calcium channel blockers displayed no such species selectivity and had low affinity (greater than 1 microM) for NBMPR sites in both guinea pig and rat cardiac membranes. Consequently, it is unlikely that agents such as benzodiazepines and calcium channel inhibitors cause significant inhibition of adenosine transport, and hence potentiate adenosine actions, at the concentrations required to induce effects through occupation of their respective, specific high-affinity sites.

Photoaffinity labeling of adenosine transporter in cardiac membranes with nitrobenzylthioinosine.

The kinetic and molecular properties of the adenosine transporter in guinea pig cardiac membranes were studied using nitrobenzylthioinosine (NBMPR), a potent and specific inhibitor of nucleoside transport. [3H]-NBMPR bound tightly but reversibly to guinea pig cardiac membranes (apparent dissociation constant 0.24 +/- 0.07 nM; maximum binding capacity 1.24 +/- 0.45 pmol of NBMPR bound/mg protein). Reversible high-affinity [3H]NBMPR binding was inhibited in an apparent competitive manner by adenosine (apparent inhibition constant 0.14 mM). L-N-phenylisopropyladenosine (L-PIA) had no effect on NBMPR binding. Exposure of cardiac membranes in the presence of [3H]-NBMPR and dithiothreitol, a free-radical scavenger, to ultraviolet light resulted in covalent incorporation of 3H into polypeptides of apparent molecular weight 66,000-50,000. Covalent attachment of [3H]NBMPR under equilibrium binding conditions was inhibited by adenosine, nitrobenzylthioguanosine , and dipyridamole but was unaffected by the adenosine receptor agonist L-PIA. These data suggest that the photolabeled molecular weight protein (apparent mol wt 66,000-50,000) is involved in adenosine permeation by guinea pig cardiac membranes.

Two binding sites for ouabain in cardiac cell membranes.

Cardiac glycoside receptors were defined by simultaneous measurement of 3H-ouabain binding and its effects on cardiac cell membranes, contracting cardiac muscle and cultured cardiac cells. These measurements show that: Rat and guinea pig cardiac cell membranes have two specific ouabain binding sites. In both species, ouabain binding to the high affinity site on cell membranes correlates with the positive inotropic effect in contracting cardiac muscle. Inhibition of (Na+ + K+)-ATPase activity corresponds to binding to the low affinity site. This questions the hypothesis that (Na+ + K+)-ATPase inhibition is necessary for ouabain-induced positive inotropy. K+ may induce an heterogeneity in the ouabain binding sites of the digitalis-sensitive cat and human heart.

Effects of temperature and allosteric modulators on [3H] nitrendipine binding: methods for detecting potential Ca2+ channel blockers.

The effects of incubation temperature and allosteric modulators were studied on [3H]nitrendipine binding to guinea-pig cardiac membranes. Incubation temperature only slightly affected the ability of nifedipine and verapamil derivatives to inhibit binding. By contrast, the Ca2+ channel blockers d-cis-diltiazem and fostedil (KB-944) stimulated [3H]nitrendipine binding in a temperature-dependent manner (37 degrees greater than 25 degrees greater than 4 degrees C). The stimulatory effect of fostedil could be related to a decrease (2.3-fold at 37 degrees C) in the rate of radioligand binding site dissociation, without significant effects on association kinetics. Both fostedil and d-cis-diltiazem caused a shift to the right of the concentration-inhibition curve of tiapamil, a negative allosteric modulator of [3H]nitrendipine binding. Neither compound affected the ability of nifedipine, a competitive antagonist, to inhibit radioligand binding. This selective effect of fostedil or d-cis-diltiazem may be useful for testing whether potential Ca2+ channel blockers interact in a competitive as opposed to allosteric manner with the dihydropyridine site. Varying the incubation temperature may also be useful in detecting compounds which act as positive allosteric modulators (stimulators) of dihydropyridine binding.

Saturable, high affinity binding of the nucleoside transport inhibitor, nitrobenzylthioinosine, to guinea pig cardiac membranes

The site-specific binding of the potent nucleoside transport inhibitor, [ 3H]nitrobenzylthionosine ([ 3H]NBMPR), to guinea pig cardiac membranes was rapid, reversible and saturable. [ 3H]NBMPR bound with high affinity to a single class of sites at which the K D was 0.23 ± 0.07 nM and which had a B max of 1700 ± 290 fmol/mg protein. Several recognized nucleoside transport inhibitors and benzodiazepines inhibited the binding of [ 3H]NBMPR with an order of potency similar to that observed for the inhibition of the binding of [ 3H]NBMPR to human erythrocytes and guinea pig synaptosomes.