Radiolabeling of dopamine uptake sites in mouse striatum: comparison of binding sites for cocaine,mazindol, and GBR 12935

Summary This study addressed the possibility of a unique binding interaction between cocaine and the dopamine transporter as compared with other blockers of dopamine uptake. Cocaine binding sites in a fresh P₂ fraction of mouse striatum were labeled with [³H]CFT, a phenyltropane analog of cocaine also known as WIN 35,428, and compared with sites labeled with [³H]mazindol or [³H]GBR 12935. Under the conditions used, homogeneous binding was observed that was inhibited monophasically by cocaine, CFT, and mazindol; the same potencies were observed with the three radioligands. Saturation analysis in the presence and in the absence of unlabeled inhibitor (CFT, mazindol, cocaine) indicated a change in the K_d but not the B_max, consonant with a competitive mechanism. Tris-HCl reduced the affinity of each radioligand and unlabeled inhibitor without changing the B_max. N-Ethylmaleimide reduced the binding of all radioligands equally and cocaine offered protection. The dissociation rate of [³H]CFT and [³H]mazindol binding was not affected by the presence of mazindol and CFT, respectively. The B_max of [³H]CFT and [³H]mazindol binding was the same; the relatively higher value for [³H]GBR 12935 binding in analyses involving varying tritiated GBR 12935 only, was due primarily to an underestimation of the specific activity of [³H]GBR 12935. All results are in agreement with a one-site model in which cocaine, CFT, mazindol, and GBR 12935 share a common binding site in mouse striatum.     

Binding of [<Superscript>3</Superscript>H]mazindol to cardiac norepinephrine transporters: kinetic and equilibrium studies

The norepinephrine transporter (NET) is the carrier that drives the neuronal norepinephrine uptake mechanism (uptake₁) in mammalian hearts. The radioligand [³H]mazindol binds with high affinity to NET. In this study, the kinetics of [³H]mazindol binding to NET were measured using a rat heart membrane preparation. Results from these studies were used to set up saturation binding assays designed to measure cardiac NET densities (B_max) and competitive inhibition assays designed to measure inhibitor binding affinities (K_I) for NET. Saturation binding assays measured NET densities in rat, rabbit, and canine hearts. Assay reproducibility was assessed and the effect of NaCl concentration on [³H]mazindol binding to NET was studied using membranes from rat and canine hearts. Specificity of [³H]mazindol binding to NET was determined in experiments in which the neurotoxin 6-hydroxydopamine (6-OHDA) was used to selectively destroy cardiac sympathetic nerve terminals in rats. Competitive inhibition studies measured K_I values for several NET inhibitors and substrates. In kinetic studies using rat heart membranes, [³H]mazindol exhibited a dissociation rate constant k_off=0.0123±0.0007 min^–1 and an association rate constant k_on=0.0249±0.0019 nM^–1min^–1. In saturation binding assays, [³H]mazindol binding was monophasic and saturable in all cases. Increasing the concentration of NaCl in the assay buffer increased binding affinity significantly, while only modestly increasing B_max. Injections of 6-OHDA in rats decreased measured cardiac NET B_max values in a dose-dependent manner, verifying that [³H]mazindol binds specifically to NET from sympathetic nerve terminals. Competitive inhibition studies provided NET inhibitor and substrate K_I values consistent with previously reported values. These studies demonstrate the high selectivity of [³H]mazindol binding for the norepinephrine transporter in membrane preparations from mammalian hearts.     

Sodium dependent [3H]cocaine binding associated with dopamine uptake sites in the rat striatum and human putamen decrease after dopaminergic denervation and in Parkinsons disease

Summary The binding of radiolabelled cocaine, an inhibitor of dopamine uptake, to the post-mortem human putamen was studied and compared to that in the rat striatum. Saturation analysis of [³H]cocaine binding to the human putamen revealed the presence of a high affinity component of binding with a K _d of 0.21 mol/l and a B _max of 1.47 pmol/mg protein. In addition a low affinity component (K _d=26.4 mol/l) was demonstrated, having a B _max of 42.2 pmol/mg protein. Also in the rat striatum [³H]cocaine binding was both of high affinity (K _d=0.36 mol/l, B _max=5.56 pmol/mg protein) and low affinity (K _d=25.9 mol/l, B _max=35.6 pmol/mg protein). A pharmacological characterisation of high affinity [³H]cocaine binding to rat striatal membranes clearly indicates an association with the neuronal dopamine transporter. The IC₅₀ values of 8 selected drugs for inhibition of [³H]cocaine binding in the rat striatum were highly significantly correlated with their potency to inhibit [³H]dopamine uptake into slices of the rat striatum. [³H]Cocaine binding was stereospecifically inhibited by (+)nomifensine and (+)diclofensine which were 50–80-fold more active than their respective (-)isomers. Drugs with dopamine releasing activity were more potent at inhibiting [³H]dopamine uptake than at competing for the high affinity site of [³H]cocaine binding. A highly significant correlation was found between IC₅₀ values for [³H]cocaine binding in the rat striatum and the human putamen. Further evidence in support of an association of [³H]cocaine binding in the rat striatum with the dopamine transporter was obtained from lesion studies. Thus, intranigral 6-hydroxydopamine administration produced a marked (67%) decrease in striatal [³H]cocaine binding. Also in the human putamen high affinity [³H]cocaine binding sites appear localized on dopaminergic nerve terminals as evidenced by a prominent decrease in binding in the putamen obtained from subjects with Parkinsons disease. It is concluded that [³H]cocaine may be a useful ligand to examine the dopamine transporter in the rat striatum and the human putamen. Therefore it offers a new and valuable approach in the study of drug effects and neuropsychiatric diseases.Preliminary results on some parts of this study have appeared previously in abstract form (Langer et al. 1984a) or as a rapid communication (Pimoule et al. 1983)     

3H]mazindol binding associated with neuronal dopamine and norepinephrine uptake sites

[3H]Mazindol labels neuronal dopamine uptake sites in corpus striatum membranes (KD = 18 nM) and neuronal norepinephrine uptake sites in cerebral cortex and submaxillary/sublingual gland membranes (KD = 4 nM). The potencies of various inhibitors of biogenic amine uptake in reducing [3H]mazindol binding in striatal membranes correlate with their potencies for inhibition of neuronal [3H]dopamine accumulation, whereas their potencies in reducing [3H]mazindol binding to cortical and salivary gland membranes correlate with their potencies for inhibition of neuronal [3H]norepinephrine accumulation. Similar to the dopamine and norepinephrine uptake systems, [3H]mazindol binding in all three tissues is dependent upon sodium (with potassium, lithium, rubidium, and Tris being ineffective substitutes) and chloride (with sulfate and phosphate being ineffective substitutes). In membranes of the cerebral cortex and salivary gland, half-maximal stimulation is observed at 50-80 mM NaCl, whereas in membranes of the corpus striatum half-maximal stimulation occurs at 240 mM NaCl. In striatal membranes NaCl increases the affinity of [3H]mazindol binding with no effect on the maximal number of sites. The enhancement of affinity is due to a selective slowing of the dissociation of the ligand from its binding site. The association of [3H]mazindol binding sites with neuronal dopamine uptake sites in the corpus striatum is further supported by the reduction of [3H]mazindol binding sites in striatal membranes following destruction of dopaminergic neurons by 6-hydroxydopamine. Similarly, the association of [3H]mazindol binding sites with neuronal norepinephrine uptake sites in cerebral cortex is supported by the reduction of [3H]mazindol binding to cortical membranes following destruction of noradrenergic neurons by N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine.     

[3H] sertraline binding to rat brain membranes

Tritiated sertraline, a radiolabeled form of a potent and selective inhibitor of serotonin uptake, was found to bind with high affinity to rat whole brain membranes. Characterization studies showed that [³H] sertraline binding occurred at a single site with the following parameters:K _d 0.57 nM,B _max 821 fmol/mg protein,n _h 1.06. This binding was reversible; the dissociation constant calculated from kinetic measurements (K _d 0.81 nM) agreed with that determined by saturation binding experiments. [³H] Sertraline binding in the presence of serotonin, paroxetine, fluoxetine or imipramine suggested competitive inhibition of binding (large increase inK _d with little change inB _max). The rank order of potency of inhibition of [³H] sertraline binding was similar to that of inhibition of serotonin uptake for known uptake inhibitors and the 1-amino-4-phenyltetralin uptake blockers. A marked decrease in ex vivo [³H] sertraline binding in the brain of rats 7 days after treatment withp-chloroamphetamine was consistent with the loss of serotonin uptake sites induced by this agent. The results of our study indicated that [³H] sertraline labels serotonin uptake sites in rat brain.     

Reduced Bmax of [3H]-imipramine binding to platelets of depressed patients free of previous medication with 5HT uptake inhibitors

The high-affinity binding sites for [³H]-imipramine (IMI) present in human platelets are associated with the neuronal uptake system for 5HT. It was recently demonstrated that previous antidepressant therapy with drugs which inhibit 5HT uptake could down-regulate [³H]-IMI binding and that this effect could persist up to 1 month after the end of treatment. We therefore re-examined the reported differences inB _max of [³H]-IMI binding in platelets between control and depressed untreated patients, to evaluate the residual influence of previous antidepressant medication. The saturation characteristics of [³H]-IMI binding were compared in platelets from 17 depressed patients care-fully selected according to previous antidepressant therapy and washout period, who were closely matched, for age and sex, with a group of control healthy volunteers. The results reveal a significant decrease by 47% in theB _max of [³H]-IMI binding in platelets of untreated depressed patients when compared with controls. There was no significant modification ofK _d values for platelet [³H]-IMI binding between the depressed and the control groups. Our results support the view that platelet [³H]-IMI binding is a useful tool as a biological marker in depression.     

Dopamine D2 receptors selectively labeled by a benzamide neuroleptic: [3H]-YM-09151-2

Summary In order to label dopamine D₂ receptors selectively we tritiated the potent benzamide neuroleptic, YM-09151-2 (26.7 Ci/mmol). The binding of [³H]-YM-09151-2 to canine striatal membranes was saturable and specific with a K _D of 57 pmol/l and B _max of 36 pmol/g tissue as determined by Scatchard analysis. The K _D, but not the B _max, of [³H]-YM-09151-2 increased 6-fold in the absence of sodium chloride. [³H]-YM-09151-2 labeled 40% more sites than [³H]-spiperone in the same tissue homogenate. [³H]-YM-09151-2 binding was inhibited by dopaminergic drugs in a concentration and stereoselective manner with the appropriate dopamine D₂ receptor profile. Thus, dopamine agonists inhibited [³H]-YM-09151-2 binding to canine striatal membranes with the following rank order of potency: (–)-N-n-propylnorapomorphine > apomorphine > (±)-6,7-dihydroxy-2-aminotetralin > (+)-N-n-propylnorapomorphine > dopamine > (–)-noradrenaline > serotonin > (–)-isoprenaline. Dopaminergic antagonists competed for [³H]-YM-09151-2 binding with the following order of potency: spiperone > (+)-butaclamol > haloperidol > clebopride > (–)-sulpiride > SCH-23390 > (–)-butaclamol. Furthermore, dopamine agonists recognized 2 states of the receptor labeled by [³H]-YM-09151-2, D ₂ ^high and D ₂ ^low . The D ₂ ^high state of the receptor could be converted to D ₂ ^low by guanine nucleotides and sodium ions as is the case for [³H]-spiperone binding to D₂ receptors. [³H]-YM-09151-2 appears to be a more selective ligand for dopamine D₂ receptors than [³H]-spiperone, since YM-09151-2 displays approximately 9-fold lower affinity than spiperone for cortical serotonergic (S₂) receptors. [³H]-YM-09151-2 may become a useful tool for the selective characterization of dopamine D₂ receptors.Abbreviations used (±)ADTN (±)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene - NPA N-n-propylnorapomorphine - Gpp(NH)p 5-guanylylimidodiphosphate     

Relationships among dopamine transporter affinities and cocaine-like discriminative-stimulus effects

Rationale: The discriminative-stimulus effects of cocaine have been reported to be mediated by indirect agonist actions initiated by the blockade of dopamine uptake, and the potencies of drugs that have discriminative-stimulus effects like cocaine are directly related to their dopamine transporter binding affinities. The binding to the dopamine transporter by cocaine and many of its analogs has been reported to fit better using a two-site model than a one-site model. Objectives: The present study examined the relationship among binding affinities of dopamine uptake inhibitors at these two sites and their potencies to produce discriminative-stimulus effects. Methods: The inhibition constants (K _i values) were derived for unlabeled dopamine uptake inhibitors for displacement of [³H]WIN 35,428 from rat caudate putamen membranes. These K _i values were related to the ED₅₀ values obtained in rats trained to discriminate 10 mg/kg cocaine from saline injections under a fixed-ratio 20 schedule of food reinforcement. Results: Among the dopamine uptake inhibitors studied, the binding data for eight compounds (WIN 35,428, nomifensine, WIN 35,981, WIN 35,065-2, methylphenidate, cocaine, cocaethylene, and bupropion) were better fit by a two-site model than a one-site model. The data for the remaining eleven compounds (RTI-31, RTI-55, RTI-121, RTI-32, LU19-005, BTCP, GBR12909, GBR12935, mazindol, LU17-133, and EXP561) were better fit by a one-site model. Of the drugs that were fit best by a two-site model, there was a higher correlation among the K _i values for the high-affinity site and the ED₅₀ values (R²=0.655; P=0.015) than there was for the low-affinity site (R²=0.543; P=0.037). Of the remaining drugs, there was a high correlation among the K _i values and the ED₅₀ values for the discriminative-stimulus effects (R²=0.523; P=0.012). Conclusions: These data suggest that the discriminative-stimulus effects of cocaine are more closely related to actions mediated by high-affinity binding to the dopamine transporter than they are to actions mediated by the low-affinity site. The further assessment of the respective contributions of high- and low-affinity binding to the behavioral effects of cocaine will be greatly enhanced with the development of pharmacological tools that have a high degree of selectivity for one of these components. Received: 24 May 1999 / Final version: 30 July 1999     

Further in vitro and in vivo studies with the putative presynaptic dopamine agonist N,N-dipropyl-7-hydroxy-2-aminotetralin

Summary The in vitro binding of the putative dopamine autoreceptor agonist [³H]DP-7-ATN to rat striatal membrane homogenates was investigated. The maximum number of binding sites B _max was 497.5 ± 50.2 fmol/mg protein and the affinity constant K _D was 8.3 ± 1.5 nM using 10 M (+) butaclamol to define non-specific binding. Lesion of the left medium forebrain bundle by 6-hydroxydopamine resulted in an almost complete loss of dopamine in the striatum but did not affect the binding of [³H]DP-7-ATN. The binding of [³H]DP-7-ATN to the homogenates of the dopaminergic cell bodies in the substantia nigra revealed a B _max of 542.4 ± 40.1 fmol/mg protein and a K _D of 11.1 ± 1.3 nM. The pharmacological profile of the binding was characterized as being to D-2 receptors. No direct in vitro evidence could be found for a selective binding to DA autoreceptors. The dopamine uptake inhibitor GBR 12909 interacted in a noncompetitive manner with the in vitro binding of [³H]DP-7-ATN and the latter compounds uptake into isolated synaptosomes was not through the specific dopamine uptake system but rather through diffusion. GBR 12909 failed to reveal any agonistic or antagonistic activity in the GBL model but was able to antagonize the hypomotility in rats induced by 0.25 mg/kg DP-7-ATN. The inhibitory effect of DP-7-ATN on DA release was also demonstrated using in vivo brain dialysis in conscious rats. Based on the above results, the possibility is discussed that the release regulating DA autoreceptors, which might be coupled to the reuptake complex, and the DA biosynthesis regulating autoreceptors, are different entities. Send offprint requests to A. S. Horn     

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

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

[3H]HOE166 defines a novel calcium antagonist drug receptor — distinct from the 1,4 dihydropyridine binding domain

Summary Benzothiazinones represent a novel class of drugs which block voltage-dependent L-type calcium channels in different tissues. [³H]HOE166 (R-(±)-3,4-dihydro-2-isopro-opyl-4-methyl-2-[2-[4-[4-[2-(3, 4, 5-trimethoxyphenyl)ethyl]piperazinyl]butoxy]phenyl]-2H -1, 4-benzothiazin-3-on-dihydrochloride; 57 Ci/mmol) a potent optically pure benzothiazinone was employed to characterize receptors associated with skeletal muscle transverse tubule calcium channels. [³H]HOE166 reversibly labels the membrane-bound calcium channels with high affinity (K_d = 0.36 ± 0.05 nM; B_max = 18.2 ± 3.3 pmol/mg of membrane protein; means ± SD, n = 13), HOE166 (K_i = 0.76 nM) is 29-fold more potent than the respective (S)-enantiomer (K_i = 22.1 nM). Binding is inhibited by divalent and trivalent cations (Cd²⁺ and La³⁺ being most potent) and other calcium channel drugs (1,4 dihydropyridines, phenylalkylamines, benzothiazepines). High affinity [³H]HOE166 binding activity is maintained (Kd = 4.5–9.0 nM) after solubilization and purification (554–1350 pmoles/mg of protein) of the calcium channel complex from transverse-tubule membranes. The following data support our recent claim (Striessnig et al. 1985, 1988) that HOE166 labels a domain on L-type calcium channels which is distinct from that defined by 1,4 dihydropyridines, phenylalkylamines or benzothiazepines: (1) All 1,4 dihydropyridine-, phenylalkylamine-and benzothiazepine-receptor-selective drugs tested are only very weak inhibitors of [³H]HOE166 binding. (2) (+)-PN200-110 only partially inhibits [³H]HOE166 binding to the purified calcium. channel complex. (3) The decay of the [³H]HOE166-receptor complex is monoexponential but the dissociation rate constants depend on the ligand concentration; (+)-PN200-100 accelerates the dissociation in the presence of unlabelled HOE166. (4) Nanomolar concentrations of HOE166 and HOE167 completely inhibit (–)-[³H]desmethoxyverapamil binding to a Drosophila phenylalkylamine receptor (which lacks a 1,4 dihydropyridine binding domain). Taken together, these results are incompatible with the view that [³H]HOE166 binds competitively to the calcium channel linked 1,4 dihydropyridine drug receptors.Abbreviations kd dissociation constant - K_i inhibition constant - k_–1,k₊₁ dissociation, association rate constant - SDS sodium dodecyl sulfate - T-tubule transverse tubule - s_20,w sedimentation coefficient Send offprint requests to H. Glossmann at the above address     

Protection by verapamil and nifedipine against ischaemia-induced loss of [3H]-(+)-PN 200-110 binding sites in the rat heart

Summary We have studied the effects of 60 min global ischaemia and 30 min of subsequent reperfusion on the binding of [³H]-(+)-PN 200–110 and [³H]-(–)-devapamil (desmethoxyverapamil or D888) in rat heart membranes. The hearts were perfused in the Langendorff-mode and pretreated with 1 mol/l verapamil, 30 nmol/l and 1 mol/l nifedipine. After 60 min of global ischaemia in the absence of drugs, we found a reduction of [³H]-(+)-PN 200-110 binding sites, without changes in the equilibrium dissociation binding constant (K _d). After the subsequent reperfusion maximum specific binding (B _max) was further reduced, whereas the K _d remained constant. [³H]-devapamil binding sites were influenced to a lower extend and showed only a decrease in B _max at reperfusion. Pretreatment with 1 mol/l verapamil completely prevented the changes which were observed for [³H]-(+)-PN 200-110. Pretreatment with a low, vasodilating concentration (30 nmol/l) of nifedipine displayed selective protection against the extra reduction in B _max which was observed during reperfusion. It is concluded that calcium antagonists show protection against the ischaemia-induced loss of dihydropyridine binding sites in relation to their negative inotropic, energy-saving activity. Furthermore, nifedipine at low, vasodilating but not negative inotropic concentrations protects against further reperfusion-induced injury, which protection may be related to an improved flow during reperfusion. Send offprint requests to J. Zaagsma at the above address     

Short-term lithium administration to healthy volunteers produces long-lasting pronounced changes in platelet serotonin uptake but not imipramine binding

Platelet [³H]-5HT uptake, [³H]-imipramine binding and endogenous 5HT levels were measured in healthy volunteers during short-term (20 days) administration of lithium, and following its withdrawal. The V _max of [³H]-5HT uptake was significantly decreased during lithium treatment. Following lithium withdrawal, platelet [³H]-5HT uptake (V _max) remained decreased and was followed by a pronounced rebound effect in some of the subjects for up to 3 months. The affinity constant (K _m) of [³H-5HT uptake was not modified. Binding of tritiated imipramine during the same period and platelet 5HT levels measured till 14 days after withdrawal was not affected by lithium treatment. As lithium is devoid of in vitro effects on both 5HT uptake and imipramine binding, it is concluded that the effects of lithium on the 5HT transporter do not reflect a direct effect on the transporter complex. Our results indicate that lithium-induced changes at the level of 5HT uptake in platelets are not correlated with concomitant variations in platelet 5HT content and can be dissociated from modifications at the level of imipramine binding sites within the macromolecular complex of the 5HT transporter. Moreover, platelet 5HT uptake is apparently modulated by lithium, with a similar pattern in healthy volunteers and in manic-depressive patients.     

Phencyclidine increases the affinity of dihydropyridine calcium channel antagonist binding in rat brain

Summary Phencyclidine (PCP) significantly reduces the apparent dissociation constant (K _D) of the dihydropyridine (DHP) calcium channel antagonist, [³H]nitrendipine, in synaptosomal membranes of rat and mouse brain without significantly effecting the maximum binding capacity (B _max). At an optimum concentration of PCP (10 M) the apparentK _D of [³H]nitrendipine was reduced from 178±9 pM to 112±9 pM in rat forebrain, a 58% increase in affinity. The structural derivatives of PCP, P-Br-PCP {1-[1-(4-bromophenyl-cyclohexyl)piperidine]}, m-NH₂-PCP {1-[1-(3-anilo)-cyclohexyl]piperidine}, (±)-PCMP [1-(1-phenyl)-cyclohexyl-3-methylpiperidine] also increased the apparent affinity of [³H]nitrendipine in the following order, p-Br-PCP PCMp>PCP>m-NH₂-PCP. Local anesthetics either reduced the apparent affinity of [³H]nitrendipine or had no effect. Kinetic analysis revealed that PCP both increased the microassociation rate constant and decreased the microdissociation rate contant of [³H]nitrendipine. The magnitude of this enhanced binding varied with the brain region studied; the greatest increase in apparent affinity of [³H]nitrendipine was observed in striatum, while no significant increase in affinity was observed in brainstem. In some brain areas, PCP was more effective in reducing theK _D in crude homogenates than in washed tissue. PCP (10 M) did not alter theK _D of [³H]nitrendipine to rat cardiac tissue. Both Ca²⁺ and Mg²⁺ inhibited the effect of PCP, while monovalent ions were ineffective in this regard. These data are consistent with an allosteric modulation of DHP calcium channel antagonist binding sites by PCP and structural derivatives that is not mediated through the brain PCP binding site. This modulation of DHP binding sites may account for some of the psychopharmacologic actions PCP and related compounds in vivo.     

Binding of3H-desipramine to the neuronal noradrenaline carrier of rat phaeochromocytoma cells (PC-12 cells)

Summary The specific (i.e., nisoxetine-sensitive) binding of³H-desipramine was studied in purified plasma membranes of PC-12 cells (rat phaeochromocytoma cells).³H-desipramine bound reversibly and with high affinity (K _D=4.5 nmol/l) to a single, non-interacting site (Hill coefficient=1.04); the maximal number of binding sites (B _max) was 19.6 pmol/mg protein.Like the uptake of noradrenaline (by uptake₁), the binding of³H-desipramine was dependent on both sodium and chloride. The stimulation of binding by chloride and sodium was characterized by a Hill coefficient of about 1 and 2, respectively. Both, chloride and sodium, slowed the rate of dissociation of bound³H-desipramine. Increasing concentrations of sodium decreased theK _D of³H-desipramine binding without altering theB _max.The binding of³H-desipramine was inhibited by tricyclic antidepressants and other noradrenaline uptake blockers. There was a highly significant correlation between the potencies of a series of drugs for the inhibition of³H-desipramine binding and for the inhibition of³H-noradrenanne uptake into intact PC-12 cells. Both, binding of³H-desipramine and uptake of³H-noradrenaline, were stereoselectively inhibited by the enantiomers of cocaine and oxaprotiline. However, for most of the substrates of uptake₁ the IC₅₀ for inhibition of³H-desipramine binding was much higher than that for inhibition of³H-noradrenaline uptake. Nevertheless, noradrenaline competitively inhibited³H-desipramine binding and unmasked dissociation of bound³H-desipramine. Thus,³H-desipramine probably binds to the substrate recognition site.From theB _max of³H-desipramine binding to PC-12 membranes and from theV _max of³H-noradrenaline uptake into PC-12 cells, a duration of about 400 ms for the transport cycle for a single noradrenaline molecule was calculated. In addition, from theB _max the maximum number of³H-desipramine binding sites (carriers) for a single PC-12 cell was calculated to be 55,000.A part of this study was presented at the IUPAHR 9th International Congress of Pharmacology (Bönisch et al. 1984)This study was supported by the Deutsche Forschungsgemeinschaft (Bo 521 and SFB 176)     

Repeated administration of sulpiride for three weeks produces behavioural and biochemical evidence for cerebral dopamine receptor supersensitivity

Administration of sulpiride (2 × 100 mg/kg i.p.) or haloperidol (5 mg/kg i.p.) to rats for 3 weeks with subsequent withdrawal for 3 or 4 days induced cerebral dopamine receptor supersensitivity. Apomorphine-induced stereotyped behaviour after drug withdrawal was enhanced by pretreatment with either haloperidol or sulpiride both of which increased the number of specific striatal binding sites (B_max) for [³H]spiperone, [³H]N,n-propylnorapomorphine and [³H]sulpiride. Neither drug altered the dissociation constant (K_D) for the ligand binding assays. Striatal dopamine sensitive adenylate cyclase activity was unaltered by such a pretreatment with either haloperidol or sulpiride. The data show that sulpiride, like haloperidol, is capable of inducing behavioural and biochemical supersensitivity of cerebral dopamine receptors.     

Inhibitory effects of clozapine and other antipsychotic drugs on noradrenaline transporter in cultured bovine adrenal medullary cells

The effects of clozapine and other antipsychotic drugs on noradrenaline (NA) transport were examined in cultured bovine adrenal medullary cells and in transfected Xenopus laevis oocytes expressing the bovine NA transporter. Incubation of adrenal medullary cells with clozapine (30–1000 ng/ml) inhibited desipramine (DMI)-sensitive uptake of [³H]NA in a concentration-dependent manner (IC₅₀=110 ng/ml or 336 nM). Other antipsychotic drugs such as haloperidol, chlorpromazine, and risperidone also decreased [³H]NA uptake (IC₅₀= 144, 220, and 210 ng/ml or 383, 690, and 512 nM, respectively). Eadie-Hofstee analysis showed that clozapine reduced V_max of uptake of [³H]NA and increased K_m. Furthermore, clozapine inhibited specific binding of [³H]DMI to plasma membranes isolated from bovine adrenal medulla (IC₅₀=48 ng/ml or 146 nM). Scatchard plot analysis of [³H]DMI binding revealed that clozapine decreased both B_max and K_d. Other antipsychotic drugs, including haloperidol, chlorpromazine, and risperidone, also reduced [³H]DMI binding to the membranes. In transfected Xenopus oocytes expressing the bovine NA transporter, clozapine inhibited [³H]NA uptake in a concentration-dependent manner similar to that observed in adrenal medullary cells. These results suggest that clozapine and haloperidol directly inhibit transport of NA by acting on the site of an NA transporter that influences both substrate transport and binding of tricyclic antidepressants. Received: 13 April 1999 / Final version: 2 November 1999     

Pharmacology of high-affinity binding of [3H](±)2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN) to bovine caudate nucleus tissue

High-affinity binding of [³H](±)2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene ([³H]-ADTN) was improved by use of a subcellular fraction (P₄) of tissue obtained from calf brain. The highest concentration of binding sites was found in caudate nucleus which was evaluated extensively. Binding of 0.5 nM [³H]-ADTN was optimal at 25° and pH 7.5 to 8.0 when a cation-free medium containing antioxidants was used and was 82–87% displaceable (“specific”). The T_{$\text{1}\text{2}$} for association was 20 min, and for dissociation 38 min, under these conditions. Analysis of association-dissociation kinetics and of ligand saturation isotherms revealed an apparent affinity (K_d) of 1–2 nM and a binding maximum (B_max) of 422 fmoles/mg protein. The process proved to be reversible by, and monophasically competitive with, several potent dopamine agonists, with Hill constants dose to unity. Stereoselectivity was found with eight isomer-pairs. Binding of [³H]-ADTN was selective for 3,4-dihydroxyphenethylamines and 10,11-dihydroxyaporphines with potent dopamine-agonist actions, but not for adrenergic catecholamines or other catechols, or blockers of dopamine uptake. Dopamine antagonists competed more weakly and in poor correspondence with their in vivo activities. There was a close correspondence between IC₅₀ values obtained for fifty of the agents tested with both [³H]-ADTN and [³H]-(?)apomorphine (r and slope > 0.9), supporting impressions of the structure-activity characteristics of dopamine agonist binding sites based on prior studies with [³H]-apomorphine.     

Ketamine interacts with the noradrenaline transporter at a site partly overlapping the desipramine binding site

Effects of the intravenous anaesthetic ketamine on the desipramine-sensitive noradrenaline transporter (NAT) were examined in cultured bovine adrenal medullary cells and in transfected Xenopus laevis oocytes expressing the bovine NAT (bNAT). Incubation (1–3 h) of adrenal medullary cells with ketamine (10–300 μM) caused an increase in appearance of catecholamines in culture medium. Ketamine (10–1000 μM) inhibited desipramine-sensitive uptake of [³H] _{noradrenaline} (NA) (IC₅₀=97 μM). Saturation analysis showed that ketamine reduced V _max of [³H]NA uptake without changing K _m, indicating a non-competitive inhibition. Other inhibitors of NAT, namely cocaine and desipramine, showed a competitive inhibition of [³H]NA uptake while a derivative of ketamine, phencyclidine, showed a mixed type of inhibition. Ketamine (10–1000 μM) also inhibited the specific binding of [³H]desipramine to plasma membranes isolated from bovine adrenal medulla. Scatchard analysis of [³H]desipramine binding revealed that ketamine increased K _d without altering B _max, indicating a competitive inhibition. In transfected Xenopus oocytes expressing the bNAT, ketamine attenuated [³H]NA uptake with a kinetic characteristic similar to that of cultured adrenal medullary cells. These findings are compatible with the idea that ketamine non-competitively inhibits the transport of NA by interacting with a site which partly overlaps the desipramine binding site on the NAT. Received: 18 December 1997 / Accepted: 17 June 1998     

Effect of ageing on the number of neuronal noradrenaline uptake sites in the rat vas deferens

Summary Previous work has shown an age-related reduction in neuronal uptake of noradrenaline in the prostatic, but not in the epididymal portion of the rat vas deferens. In the present paper, the influence of ageing on the number of [³H]desipramine binding sites and on the effect of lithium on neuronal [³H]noradrenaline uptake were studied in the prostatic and epididymal portions of vasa deferentia from 4- and 20-month-old rats. The affinity for [³H]desipramine (K _d values) in the epididymal and prostatic portions did not change with age. However, ageing reduced the maximal number of [³H]desipramine binding sites (B_max values) in the prostatic, but not in the epididymal portion. Lithium potentiated neuronal [³H]noradrenaline uptake only in the prostatic portion and this potentiation was not changed by ageing. The results showed differences in neuronal noradrenaline uptake between the two portions of the vas deferens. Furthermore, the data suggest that the age-related reduction in neuronal uptake in the prostatic portion is due to a reduction in the number of neuronal uptake sites for noradrenaline. Correspondence to R. Pekelmann Markus at the above address