Action of compounds with effective in vivo mineralocorticoid activity on ion transport in leucocytes.

We have studied the in vitro short-term effects of aldosterone (1.0-1000 nmol l-1), cortisol (0.5-5.0 mumol l-1), fludrocortisone (1.0-10 nmol l-1) and carbenoxolone (0.5-3 mmol l-1) on 86rubidium influx (a model for potassium), 22sodium efflux, and [3H]-ouabain binding capacity in intact human leucocytes. No effect of aldosterone (at concentrations present in Conn's syndrome) or fludrocortisone could be demonstrated on cation fluxes or [3H]-ouabain binding compared to controls. No significant effect of cortisol, at concentrations either physiological or present in Cushing's syndrome, could be demonstrated on cation fluxes or [3H]-ouabain binding compared to controls. Carbenoxolone significantly increased 86Rb influx and 22Na efflux at concentrations known to cause hypokalaemia in man. The effect was not blocked by propranolol. No effect could be demonstrated for [3H]-ouabain binding.     

Increase in Na+/K+ pump numbers in vivo in healthy volunteers taking oral lithium carbonate and further upregulation in response to lithium in vitro.

1. We have measured [3H]-ouabain binding to lymphocyte membranes in eight healthy volunteers before and after they had taken lithium carbonate for 14 days in doses which maintained the serum lithium concentration in the range 0.5-1.0 mmol 1-1. 2. There was a statistically significant increase in the [3H]-ouabain binding capacity of the lymphocyte membranes (reflecting the number of Na+, K+-ATPase molecules) after 14 days of lithium administration in vivo. This suggests that a failure to increase pump numbers after similar exposure to lithium in vivo in patients with manic-depressive psychosis is a primary abnormality associated with the disease. 3. In vivo lithium administration did not alter the normal adaptive (upregulatory) response of lymphocyte Na+, K+-ATPase to standard pharmacological challenges, involving in vitro incubation for 3 days with lithium chloride (8 mmol 1-1) or sodium ethacrynate (1 mumol 1-1). 4. We have previously found that there is an impaired response of the Na+, K+-ATPase to these in vitro stimuli in patients with manic-depressive psychosis, and our present data suggest that this abnormality is attributable to the disease itself and not to in vivo lithium therapy. 5. The data also suggest that the increase in vivo Na+/K+ pump activity which we have previously described in healthy volunteers after 21 days of lithium administration is at least partly due to an increase in Na+/K+ pump numbers.     

Sodium load and high affinity ouabain binding in rat and guinea-pig cardiac tissue.

An estimation of the actual Na/K-ATPase transport activity in intact cardiac cells was made by measuring the binding of [3H]-ouabain to rat and guinea-pig ventricular strips. At the low [3H]-ouabain concentration of 1 nM equilibrium binding was hardly obtained after an incubation time of five hours. Different procedures known to alter the sodium load of the cardiac preparations influenced [3H]-ouabain binding: the sodium ionophore monensin enhanced [3H]-ouabain binding, the local anaesthetic dibucaine and a reduction of external sodium ion concentration diminished [3H]-ouabain binding; [3H]-ouabain binding was similarly affected by these procedures in the rat and guinea-pig. Since [3H]-ouabain binding occurred predominantly at the high-affinity binding sites of rat myocardium under the applied experimental conditions, it was concluded that these binding sites represent Na/K-ATPase molecules involved in sodium ion transport.     

The characteristics of [3H]-ouabain binding to human lymphocytes

The kinetic characteristics of [3H]-ouabain binding to human lymphocytes and mixtures of mononucleated cells, and the maximum [3H]-ouabain binding capacities of these cells were studied. The [3H]-ouabain binding was compatible with a single class of receptors with a high affinity for the drug. No signs of positive or negative cooperativity could be demonstrated. In six experiments with pure lymphocyte preparations, the association and dissociation rate constants were 3.08 +/- 0.34 X 10(4)/M/S and 1.58 +/- 0.50 X 10(-4)/S. The dissociation constant derived from equilibrium studies on lymphocytes was 0.68 +/- 0.21 X 10(-8) M, which was identical to that of mononucleated cells. In healthy subjects the maximum [3H]-ouabain binding capacities, which reflect the number of sodium/potassium pump sites were 43154 +/- 8037 molecules/cell (n = 25) in lymphocytes and 75474 +/- 6764 (n = 9) molecules/cell in mixtures of mononucleated cells. Direct determination of the [3H]-ouabain binding capacity of lymphocytes can be performed with acceptable accuracy and precision using 30 ml whole blood. Provided high cell purity, this method may be useful, when studying sodium/potassium pump function in clinical settings.     

Increase in plasma potassium concentration following indomethacin administration: absence of a role for membrane Na-K ATPase

To elucidate whether indomethacin-induced hyperkalaemia is due to an inhibition of Na-K ATPase in the membranes, indomethacin (25 mg t.d.s.) was administered to 7 normal subjects for 7 days. This resulted in an increase in plasma potassium concentrations in all 7 subjects: median (range) for the entire group increased from 4.19 (3.98-4.79) mmol/l to 4.29 (4.13-4.87) mmol/l. Leucocytes prepared from these subjects prior to and after indomethacin were tested for 86Rb influx and [3H]-ouabain binding (an index of Na-K ATPase sites). Neither 86Rb influx (total, ouabain sensitive and ouabain insensitive) nor [3H]-ouabain binding changed significantly following indomethacin. We conclude that (a) indomethacin-induced hyperkalaemia is not due to alterations in potassium influx into cells and (b) the modulation of Na-K ATPase sites/activity is in leucocytes not dependent upon prostaglandins.     

Measurement of specific [3H]-ouabain binding to different types of human leucocytes.

We have studied the specific binding of [3H]-ouabain to intact mononuclear leucocytes (82% lymphocytes) and polymorphonuclear leucocytes. In both types of cells [3H]-ouabain binding was saturable, confined to a single site of high affinity, slow to reach equilibrium, slow to reverse, temperature-dependent, competitively antagonized by potassium, and facilitated by the presence of divalent cations. The equilibrium dissociation constants were 2.4 +/- 0.7 nmol/l (polymorphs) and 2.4 +/- 0.4 nmol/l (mononuclear cells) (NS). The values of maximal specific ouabain binding, measured by Scatchard analysis of concentration vs binding curves (Bmax), were 33.9 +/- 6.0 fmol/10(6) cells (polymorphs) and 59.3 +/- 11.6 fmol/10(6) cells (mononuclear cells) (P less than 0.02). The corresponding numbers of sites per cell were 20415 +/- 3616 and 35712 +/- 6986 respectively (P less than 0.02). When the numbers of binding sites were expressed per square micron of cell surface area the difference between the two cell types was proportionately greater (83 and 186 sites per micron 2 respectively). We conclude that the [3H]-ouabain binding sites on mononuclear and polymorphonuclear leucocytes are similar in nature, but different in both number and density on the cell surface. Measurements of Bmax in mixed cell populations should therefore take account of cell type as well as cell size and number.     

Stimulation of specific [3H]-ouabain binding to microsomal preparations from rat heart and skeletal muscle by thyroid hormones: effects of 6-hydroxydopamine.

1. Surgical thyroidectomy decreased specific [3H]-ouabain binding to heart ventricular microsomes by 43% and gastrocnemius muscle microsomes by 34%. Administration of triiodothyronine to euthyroid rats enhanced specific [3H]-ouabain binding to heart and skeletal muscle membrane by 60% and 33% respectively. 2. Treatment of thyroidectomized rats with triiodothyronine increased specific [3H]-ouabain binding by 44% in skeletal muscle membrane preparation and 428% in cardiac microsomes. 3. Specific [3H]-ouabain binding decreased by 55% in heart and 53% in gastrocnemius muscle preparations following chemical sympathectomy with 6-hydroxydopamine. 4. Treatment with triiodothyronine of euthyroid rats which had been sympathectomized did not significantly alter specific [3H]-ouabain binding to heart or skeletal muscle membrane preparations. 5. Administration of triiodothyronine to thyroidectomized and sympathectomized rats increased specific [3H]-ouabain binding by 80% in heart and 83% in skeletal muscle membrane preparations. 6. These results suggest that triiodothyronine may influence specific [3H]-ouabain binding to thyroid hormone nonresponsive tissue such as sympathetic nerve endings. Therefore, the present observations are incompatible with the hypothesis that induction of (Na+ +K+)-adenosine triphosphatase of skeletal muscle membrane is the molecular mechanism for the calorigenic actions of thyroid hormones.     

Potassium changes the relationship between receptor occupancy and the inotropic effect of cardiac glycosides in guinea-pig myocardium.

K+ (2.4-15.6 mmol l-1) antagonized the positive inotropic effect of dihydro-ouabain. The concentration-effect curves became steeper with the shift to higher concentrations of the glycoside. At 1.2 mmol l-1 Ca2+, an increase in K+ from 2.4 to 12 mmol l-1 required tenfold higher concentrations of dihydro-ouabain to produce equal inotropic effects. This factor was reduced to four at 3.2 mmol l-1 Ca2+. The same change in K+ concentration, at 1.2 mmol l-1 Ca2+, diminished the inotropic effect of ouabain on rested-state contractions by a factor of six. The positive inotropic effect of Ca2+ was also antagonized by K+ (1.2-12 mmol l-1). Reduction of Na+ from 140 to 70 mmol l-1 abolished the antagonistic action of K+ (1.2-8.0 mmol l-1). Moreover the inotropic effect of Ca2+ was enhanced. Reduction of Na+, from 140 to 70 mmol l-1, antagonized the positive inotropic effect of dihydro-ouabain more at low (2.4 mmol l-1) than at high (8.0 mmol l-1) K+. Accordingly, the extent of the dihydro-ouabain-K+ antagonism was reduced. When the K+ concentration was increased from 2.4 to 12 mmol l-1, [3H]-ouabain binding was reduced by a factor of three. This is less than the reduction in the inotropic effectiveness of ouabain or dihydro-ouabain. Reduction of stimulation frequency from 1 to 0.1215 Hz did not significantly alter the antagonistic effect of K+. Diminution of Vmax of the action potential was observed only at K+ concentrations greater than 5.9 mmol l-1, whereas the resting membrane potential was continuously depolarized over the entire range of K+ concentrations. The results support the view that the reduction in receptor affinity cannot be the sole cause of the antagonism between the glycoside and K+. Impairment of passive Na+ influx during diastole, due to the K+-dependent depolarization of the resting membrane potential, contributed to about one half of the glycoside-K+ antagonism.     

3H]-ouabain binding to denervated and innervated skeletal muscle.

In cat skeletal muscles, the major part of specific [3H]-ouabain binding was found in the sympathetic nerve endings of red, slow muscle fibres. Skeletal muscle denervation increased specific [3H]-ouabain binding to muscle membrane preparation. This increase may be involved in the development of spontaneous fibrillation in the denervated muscle.     

Influence of quinidine on the binding of [3H]-ouabain and [3H]-digoxin by human lymphocytes

Summary To explore the molecular basis of the glycoside-quinidine interaction, the in vitro effect of quinidine on the binding of [³H]-ouabain and [³H]-digoxin to Na⁺ K⁺ ATPase receptors on human mononuclear cells was investigated. The maximum [³H]-ouabain binding capacity was 45.7±9.4×10³ molecules/cell in pure lymphocyte preparations (n=8) and 75.5±7.3×10³ molecules/cell in mixtures of mononuclear cells (n=8). These parameters were not influenced by 10⁻⁵ M quinidine. In eight equilibrium experiments with pure lymphocytes, the dissociation constant of [³H]-ouabain increased from 0.79±0.26×10⁻⁸ M in the absence of 10⁻⁵ M quinidine to 1.56±0.74×10⁻⁸ M in its presence (p<0.01), indicating that the affinity of the drug was decreased. Similar findings were observed using mixed mononuclear cells. In five uptake and release experiments, quinidine decreased the association rate constant of [³H]-ouabain from 3.15±0.36×10⁴ M⁻¹×s⁻¹ to 2.01±0.37×10⁴ M⁻¹ s⁻¹ (p<0.01), whereas the dissociation rate constant was not affected. A therapeutic concentration of quinidine does not affect the number of glycoside receptors on lymphocytes, but it does appear to reduce fractional receptor occupancy by both [³H]-ouabain and [³H]-digoxin at lower tracer concentrations. This finding is compatible with the clinical observation that quinidine reduces the distribution volume of digoxin.     

Evidence for two different Na+-dependent [3H]-ouabain binding sites of a Na+-K+-ATPase of guinea-pig hearts.

1. The influence of various Na+ concentrations on [3H]-ouabain binding was studied in experiments on a microsomal Na+-K+-adenosine triphosphatase (ATPase) from guinea-pig hearts. 2. The ATP-independent cardiac glycoside binding was not influenced by increasing Na+ concentrations. However, a good correlation was found between the ATP-dependent [3H]-ouabain binding and Na+ concentration. 3. A more detailed analysis of these results according to Hofstee (1952) revealed two distinct processes involved in this interaction: one ouabain binding process was activated at rather low Na+ concentrations, (K0.5 = 4.5 mM); this type of [3H]-ouabain binding was strongly correlated to the Na+ concentration necessary for half maximum phosphorylation (K0.5 = 1 mM). The other ouabain binding process was predominant at high Na+ concentrations (K0.5 = 69 mM). 4. On the basis of the commonly accepted ATPase reaction cycle a model for the interaction of cardiac glycosides with the Na+-K+-ATPase is proposed, assuming two different binding sites for cardiac glycosides (E2-P and E1-P) and involving a translocation of these drugs from an outer to an inner compartment of the cell membrane.     

Inhibition of noradrenaline release by omega-conotoxin GVIA in the rat tail artery.

1. The perivascular nerves of isolated tail arteries from Wistar rats were stimulated with field pulses (1 Hz, 2 pulses, every 2 min). omega-Conotoxin 10 nmol l-1 depressed neurogenically mediated contractions, but did not influence the contractions to noradrenaline 0.1-0.3 mumol l-1. 2. The inhibitory effect of omega-conotoxin was concentration-dependent (IC50 = 3.8 nmol l-1). It did not reach a steady-state during 30 min incubation and could not be reversed upon subsequent washout for another 60 min. 3. A gradual increase in the Ca2+ concentration of the medium from 1.25 mmol l-1 to 10 mmol l-1 enhanced vasoconstriction and attenuated the action of omega-conotoxin 10 nmol l-1. When a low stimulation intensity (120 mA) was used at high external Ca2+ (10 mmol l-1), similar contractile responses were obtained as under normal conditions (200 mA current, 2.5 mmol l-1 Ca2+). However, the inverse relationship between the effect of the toxin and external Ca2+ remained unchanged. 4. The time-course and degree of the inhibition by omega-conotoxin 3 nmol l-1 was identical in tail arteries of spontaneously hypertensive rats (SHR) and their normotensive controls (WKY). 5. When tail arteries of Wistar rats were preincubated with [3H]-noradrenaline, field stimulation (0.4 Hz, 24 pulses, every 16 min) evoked tritium overflow and vasoconstriction. omega-Conotoxin 30 nmol l-1 inhibited both responses to a similar extent. 6. Our results suggest that omega-conotoxin selectively blocks Ca2+ channels in the terminals of perivascular nerves and thereby reduces the release, but not the contractile effect of the sympathetic transmitter.     

Effects of monovalent cations on neostriatal dopamine D2 receptors labeled with [3H]raclopride

Specific [3H]raclopride binding to dopamine D2 receptors in the rabbit neostriatum was investigated in the presence of the monovalent cations sodium, lithium and potassium. NaCl and LiCl produced concentration-dependent elevations in specific [3H]raclopride binding with sodium inducing approximately 50% more binding than lithium. Inhibition of [3H]raclopride binding by the antagonist (+)-butaclamol was unaffected by the presence of sodium or lithium in the incubation medium. In contrast, the potency of dopamine to compete with [3H]raclopride was decreased by these two ions. This effect was more pronounced in the presence of sodium than lithium and was observed for both the high- and low-affinity states of the D2 receptor. The guanine nucleotide derivative 5'-guanylylimidodiphosphate (Gpp(NH)p) reduced the potency of dopamine to compete with [3H]raclopride binding in both the presence and absence of cations; however, this effect of Gpp(NH)p was a shift of the D2 receptors from a high to a lower affinity state. Saturation binding curves in the presence of sodium or lithium were compared with experiments carried out in the absence of monovalent cations (sucrose) and demonstrated that these ions increased the affinity (judged by the equilibrium dissociation constant Kd) of the neostriatal [3H]raclopride binding sites. While NaCl produced a significantly greater change in the Kd of [3H]raclopride binding as compared to LiCl, no differences were apparent in the maximum binding capacity (Bmax) values determined in the presence of these two cations. In conclusion, the results indicate that [3H]raclopride binding to rabbit neostriatal membranes exhibits a sensitivity to monovalent cations that is consistent with the ionic regulatory properties of the D2 receptor. Moreover, although lithium and sodium influence specific [3H]raclopride binding in a similar manner, there appear to be quantitative differences between these two ions.     

[H]TYRAMINE UPTAKE BY RAT LIVER SLICES

Rat liver slices were employed as experimental model to characterise the system involved in the transport process which participates in liver tyramine uptake. The uptake of 0.4 micromol l-1of [3H]tyramine by rat liver slices was linear from 5 min up to the end of incubation. At 15 min the uptake was 4.58+/-0.18 pmol mg-1protein. The accumulation of [3H]tyramine was sensitive to temperature (69. 3+/-4.0% inhibition at 0 degrees C, P<0.001), to sodium omission replaced by 150 mmol l-1Tris or 110 mmol l-1Tris+40 mmol l-1choline (27.6+/-6.0%, P<0.01, and 24.6+/-3.8% inhibition, P<0.01, respectively), and the inhibition of Na+-K+-adenosine triphosphatase by 150 micromol l-1ouabain (20.4+/-2.6% decrease, P<0.01). Uptake of [3H]tyramine was cocaine- (10 micromol l-1) and desipramine- (1 micromol l-1) dependent (32.2+/-6.4%, P<0.05, and 31.6+/-4.0% inhibition, P<0.05, respectively). Uptake of [3H]tyramine in rat liver slices was not modified by 30 micromol l-1isoprenaline, 30 micromol l-1corticosterone, 30 micromol l-1normetanephrine and noradrenaline up to 4 micrometers at higher noradrenaline concentrations tyramine transport was diminished (P<0.05). Results achieved by incubation with increasing tyramine concentrations indicate that at the micromolar level hepatic uptake occurs by a combined passive diffusion and transport-mediated mechanism, whereas at greater tyramine concentrations passive transport predominates. These results suggest that both simple diffusion and a transport-mediated mechanism are involved in this uptake from hepatocytes, which presents features similar to those described for type 1 non-neuronal uptake systems.     

Correlation between enhancement of [3H]flunitrazepam binding and suppression of pentylenetetrazol-induced seizures by L-lysine

L-Lysine enhanced the specific [3H]flunitrazepam (FTZ) binding of bovine brain membranes in vitro. Inhibition of specific [3H]FTZ binding to brain membranes in vitro by pentylenetetrazol (PTZ) at concentrations 0.46 mM and below was reversed by increasing L-lysine concentrations in the incubation mixture; further increase of L-lysine concentration enhanced this binding. However, inhibition of [3H]FTZ binding by PTZ higher than 2.3 mM was reversed only partially by L-lysine. L-Lysine enhanced specific [35S]t-butylbicyclophosphorothionate (TBPS) binding on mouse brain membranes in a dose-dependent manner (EC approximately 5 microM). This enhancement was inhibited by PTZ dose dependently. Inhibition of [35S]TBPS binding by PTZ was attenuated slightly by L-lysine. L-Lysine enhanced [3H]FTZ binding in intact mice in a dose- or concentration-dependent manner with an ED50 of 6 mmol/kg body weight or EC50 of 3 mumol/g brain tissue, respectively. Similar effect was observed for L-lysine in ex vivo [3H]FTZ binding study when [3H]FTZ was incubated in vitro with an ED50 of 1 mmol/kg mouse or EC50 of 0.7 mumol/g brain. PTZ not only induced seizures, but also inhibited specific [3H]FTZ binding to brain membranes in a dose-dependent manner. L-Lysine, in a dose-dependent manner, suppressed seizures caused by PTZ at 50 or 60 mg/kg, or prolonged the time of seizure onset (seizure latency) caused by higher doses of PTZ (90 or 100 mg/kg). Pretreatment with L-lysine at 1, 5, 10 or 20 mmol/kg not only reversed the inhibition of the specific [3H]FTZ binding caused by PTZ at 50, 90 or 100 mg/kg, but also enhanced this binding above control level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Guanine nucleotide and cation regulation of the binding of [3H]cyclohexyladenosine and [3H]diethylphenylxanthine to adenosine A1 receptors in brain membranes

Guanine nucleotides, divalent cations, and sodium differentially regulate agonist and antagonist binding to adenosine A1 receptors in brain membranes. Guanine nucleotides decrease the binding of the adenosine A1 receptor agonist [3H]N6-cyclohexyladenosine ([3H]CHA) to guinea pig and bovine brain membranes by about 50% at 1--3 microM, while not affecting binding of the antagonist [3H]1,3-diethyl-8-phenylxanthine ([3H]DPX) to A1 receptors in bovine brain. GTP decreases the potency of agonists competing for [3H]DPX binding by 3--6 times, without altering the potency of antagonists. This effect can be used to grade experimental substances along an adenosine agonist-antagonist continuum. The 66% inhibition of [3H]CHA binding by 1 mM EDTA, with no change in [3H]DPX binding, suggests that endogenous divalent cations may regulate adenosine receptor interactions. Removal of endogenous divalent cations by EDTA treatment greatly increases the enhancement of [3H]CHA binding by divalent cations. Specific binding of [3H]CHA to guinea pig brain is increased 150--170% by 0.3--1.0 mM Mn2+, Mg2+, and Ca2+ following EDTA preincubation, secondary to an increase in apparent affinity and receptor number. Sodium ions also selectively regulate the binding of [3H]CHA. Sodium decreases [3H]CHA binding 40%, whereas lithium and potassium are ineffective. Sodium does not affect [3H]DPX binding.     

Potentiation of quinazoline antifolate (CB3717) toxicity by dipyridamole in human lung carcinoma, A549, cells

A potent quinazoline antifolate inhibitor of thymidylate synthase, CB3717, inhibits the growth of A549 human lung carcinoma cells: ID50 2.74 +/- 0.53 microM. The toxic effects of thymidylate synthase inhibition may be prevented by salvage of exogenous thymidine. The nucleoside transport inhibitor, dipyridamole, at the non-toxic concentration of 1 microM, inhibited [3H]thymidine uptake/incorporation by more than 95% and significantly reduced the ID50 of CB3717 to 0.98 +/- 0.28 microM. Elimination of salvageable thymidine by the use of dialysed serum also enhanced CB3717 toxicity. Since dipyridamole was equally effective in the presence or absence of dialysed serum and was more effective than dialysed serum alone, inhibition of nucleoside efflux may be an important aspect of its potentiation. Efflux of [5-3H]deoxyuridine was inhibited by 89% and [3H]thymidine efflux by 61% in the presence of 1 microM dipyridamole. Inhibition of thymidylate synthase increases the deoxyuridine nucleotide/thymidine nucleotide pool ratio. Dipyridamole could exacerbate the nucleotide pool imbalance caused by CB3717, thereby potentiating its toxicity.     

Are dopamine receptors present on human lymphocytes?

The characteristics of [3H] spiperone binding to human lymphocytes have been examined. The haloperidol displaceable component of [3H] spiperone binding to human lymphocytes was not saturable, and stereoselective displacement by the isomers of butaclamol was not observed. These was no correlation between the ability of known dopamine active drug to cause displacement of the ligand and their rank order of potency. Chloroquine, a drug with no dopaminergic action, was the most potent displacing agent examined. Fragmentation of the cells caused a marked decrease in the haloperidol displaceable component of [3H] spiperone binding, and saturable binding to the lymphocyte membrane fragments was not observed. lysis of cells after equilibrium incubation with [3H] spiperone caused a marked reduction in the haloperidol displaceable component of ligand binding. Association of [3H] spiperone with lymphocytes was unaffected by the metabolic inhibitor iodoacetate or by replacement of sodium ions by lithium ions in the incubation medium, suggesting that such association did no involve an active process. We cannot confirm the existence of dopamine receptors on the surface of human lymphocytes. We suggest that the apparent association of [3H] spiperone with lymphocytes is due to some passive uptake process causing accumulation of the ligand within the cells.     

In vitro binding of the very potent and selective D-2 dopamine agonist, [3H]N-0437 to calf caudate membranes

N-0437, a non-catecholic aminotetralin has recently been described as a very potent and selective dopamine D-2 agonist. In this study the in vitro binding of [3H]N-0437 (specific activity 80.6 Ci/mmol) to calf caudate membranes is described. It was found that [3H]N-0437 binds with a high affinity (KD = 0.17 nM) and a low proportion of non-specific binding. Moreover the binding was saturable with a high number of binding sites (Bmax = 703 +/- 28 fmol/mg protein) and reversible (dissociation half-time = 68 min). Pharmacological analysis of [3H]N-0437 binding showed that it was selective for dopamine receptors and that it was also stereoselective for D-2 receptors. Non-dopaminergic drugs were without exception very poor displacers. Taken together the results suggest that [3H]N-0437 labels dopamine D-2 receptors with a high selectivity in the calf brain, and thus, that it should be a useful tool in studies of central dopamine receptors.