Circadian rhythm of the B max of [3H]-imipramine binding in rabbit platelets

Summary [³H]-imipramine binding was measured in rabbit blood platelet membranes on a 24 h cycle. Animals were kept on a 14 h light (L) 10 h dark (D) schedule, and blood samples were collected at L + 2, L + 8, D + 2, D + 8 and L – 2 h on a following cycle. Significant differences were found for B_max values of [³H]-imipramine binding, with highest values during the dark phase and lowest during the light phase. No significant differences were found in K _d values. These results suggest the existence of a circadian rhythm for the B_max of [³H]-imipramine binding in blood platelets. Send offprint requests to S. Z. Langer     

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.     

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.     

Interaction of antihistaminics with muscarinic receptor (III)

The muscarinic antagonist 1-[benzilic 4,4′-³H]quinuclidinyl benzilate ([³H] QNB) bound to a single class of muscarinic receptors with high affinity in rabbit ileal membranes. The K _D and B _max values for [³H]QNB calculated from analysis of saturation isotherms were 52.5 pM and 154 fmol/mg, respectively. Chlorpheniramine (CHP), histamine H₁ blocker, increased K _D value for [³H]QNB without affecting the binding site concentrations and Hill coefficient. The K _i value of CHP for inhibition of [³H]QNB binding in ileal membranes was 1.44μM and the pseudo-Hill coefficient for CHP was close to unit. In the functional assay carbachol, muscarinic agonist, increased the contractile force of ileum with ED₅₀ value of 0.11μM. CHP caused the rightward shift of the dose-response curve to carbachol. The pA₂ value of CHP determined from Schild analysis of carbachol-induced contraction was 5.77 and the slope was unity indicating competitive antagonism with carbachol. The dissociation constant (K _i ) of CHP obtained in competitive experiments with [³H]QNB was similar to the K _A value (1.69μM) of CHP as inhibitor of carbachol-induced contraction in rabbit ileum. This result suggests that the binding of H₁ blocker, CHP, vs [³H]QNB to muscarinic receptors in ileal membranes represents an interaction with a receptor of physiological relevance.     

Binding studies with [3H]cis-methyldioxolane in different tissues

Summary Special conditions - tricine buffer containing Ca²⁺ and Mg²⁺, 22°C (TCM) — allow to label a much higher proportion of muscarinic receptors by [³H]cis-methyldioxolane (CD) than hitherto described (Vickroy et al. 1984 a). Taking the maximum number of binding sites, B _max, of [³H]QNB as 100%, B _max of [³H]CD amounts to 83% in the rat heart instead of the reported 17%, 33% in the cerebral cortex instead of 6%, 20% in hippocampus and 55% in pons/medulla. In the salivary glands specific binding was negligible. The affinities of a number of muscarinic agonists and antagonists to [³H]CD and [³H]QNB binding sites in different tissues of the rat are compared. Apparent affinities of agonists are much higher in the [³H]CD system, affinities of antagonists are slightly higher in the [³H]QNB system. In both assay systems receptors of heart and pons/ medulla membranes seem to have similar drug specificity. They differ somewhat from those in the cortex. Receptors in the salivary glands, however, seem to be completely different from those in the other three tissues. In the heart [³H]CD binding can be abolished almost completely by GppNHp. In the cortex about half of the [³H]CD binding is susceptible to GppNHp. The reduction of binding in the cortex is due to a change in B _max and not in the dissociation constant K _D. Competition of unlabelled pirenzepine with [³H]CD: In heart and pons/medulla only low affinity sites for pirenzepine (M₂-receptors) are labelled by [³H]CD. In regions rich in M₁ receptors like hippocampus (80% M₁ receptors) or cortex (65–70% M₁ receptors) the proportion of M₁ receptors labelled by [³H]CD is smaller than expected considering the concentration of M₁ receptors present in these tissues. Thus [³H]CD, under the conditions described in this paper, seems to label preferentially but not exclusively M₂ receptors in their agonist high affinity form. Send offprint requests to A. Closse at the above address     

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.     

Platelet 5-HT uptake sites in depression: three concurrent measures using [3H] imipramine and [3H] paroxetine

Platelet 5-HT uptake sites were measured in 40 depressed patients and 40 controls using [³H] imipramine binding, defined with desmethylimipramine (DMI) and Na⁺ dependence, and [³H] paroxetine binding. In control subjects the B_max of DMI defined [³H] imipramine binding was significantly higher than both Na⁺ dependent [³H] imipramine (by 30%) and [³H] paroxetine binding (by 22%). The B_max of Na⁺ dependent [³H] imipramine and [³H] paroxetine binding did not differ significantly. The K_d of Na⁺ dependent [³H] imipramine binding was significantly lower than the K_d of DMI defined [³H] imipramine binding. The binding of DMI defined and Na⁺ dependent [³H] imipramine and [³H] paroxetine did not differ significantly between depressed patients and controls in the total group, in those depressed patients who had never taken antidepressants or in those depressed patients who had been recently with-drawn from antidepressants. This study provides no support for the view that the number of platelet 5-HT uptake sites are reduced 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     

[3H]ICS 205-930 labels 5-HT3 recognition sites in membranes of cat and rabbit vagus nerve and superior cervical ganglion

Summary The binding characteristics of [³H]ICS 205-930, a 5-hydroxytryptamine 5-HT₃ receptor antagonist, were investigated in membranes prepared from cat and rabbit vagus nerve (VN) and superior cervical ganglion (SCG). The autoradiographic localisation of 5-HT₃ recognition sites was also assessed using [³H]ICS 205-930 in slices from cat medulla oblongata, nodose ganglion and vagus nerve.[³H]ICS 205-930 bound to a homogeneous population of high affinity recognition sites in cat VN: B_max = 201 ± 43 fmol/mg protein, pK_D = 9.26 ± 0.17 and SCG: B_max = 291 ± 40 fmol/mg, pK_D = 9.35 ± 0.80 (n = 3). Competition experiments performed in membranes from cat VN and SCG with agonists and antagonists suggested the presence of a homogeneous population of [³H]ICS 205-930 recognition sites. Competition curves were steep and monophasic and were best fitted by a 1 receptor site model. The following rank order of affinity for [³H]ICS 205-930 binding sites was observed with antagonists: SDZ 206-830 = ICS 205-930 > BRL 43694 > SDZ 206–792 > quipazine > MDL 72222 > metoclopramide > mCPP and agonists: 2-methyl-5-HT = 5-HT > phenylbiguanide. A similar profile was observed for a limited series of compounds in rabbit membranes. Drugs acting at 5-HT₁, 5-HT₂ and dopamine receptors (domperidone, spiperone and metergoline) showed very low affinities for [³H]ICS 205-930 recognition sites. The sites labelled with [³H]ICS 205-930 in vagus nerve and superior cervical ganglion of both species displayed the pharmacological profile of a 5-HT₃ receptor. There was a significant correlation between the rank order of affinity of the tested compounds for [³H]ICS 205-930 recognition sites in cat and rabbit membranes and their rank order of affinity for 5-HT₃ receptors from neuroblastoma-glioma NG 108-15 cells. Autoradiographic studies suggest that [³H]ICS 205-930 binding sites are present over and around the nodose ganglion cell somata, along certain fibers of the vagus nerve and in the terminal areas of this nerve in the medullar nucleus of the vagus.The present data demonstrate that [³H]ICS 205-930 identifies 5-HT₃ receptors in preparations of cat and rabbit vagus nerve and superior cervical ganglion.Send offprint requests to D. Hoyer at the above addressThe present results have been presented in part at the Winter Meeting of the British Pharmacological Society, London, December 20–22, 1988 (Hoyer et al. 1989)     

[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.     

Increased [3H] raclopride binding sites in postmortem brains from schizophrenic violent suicide victims

The specific binding of the D₂-dopamine receptor antagonist radioligand [³H] raclopride was quantitated in the postmortem caudate and frontal cortex from schizophrenic suicide victims and control subjects. In schizophrenic suicides the density of binding sites (B_max) was higher (40%,P<0.05) in the caudate, whereas it did not change in the cortex as compared to those in controls. The apparent dissociation constants (K _d ) were also found increased both in caudate (24%) and cortex (75%) from schizophrenics, but these apparent decreases in receptor affinity did not reach statistical significance. The mean B_max value in drug-free schizophrenic suicides (n=3) did not differ from the B_max value in neuroleptic drug-treated schizophrenics (n=7) but it was found increased in respect to control subjects (n=9). No differences in [³H] raclopride binding were observed between non-schizophrenic suicide victims (n=4) and matched controls (n=4), suggesting that the modifications of D₂-dopamine receptors in schizophrenia are not related to suicide.     

Acute ethanol intoxication may not alter α2-adrenoceptors in the human brain

The specific binding of the ₂-adrenoceptor agonists [³H]clonidine and [³H]bromoxidine (UK 14304) was measured in the postmortem brain of ethanol intoxicated nonalcoholic subjects (blood ethanol concentration: 1.37±0.30 g/l) and matched controls. In the frontal cortex, the density of binding sites for [³H]clonidine (B_max=58±7 fmol/mg protein) and [³H]bromoxidine (UK 14304) (B_max=49±7 fmol/mg protein) in ethanol intoxicated subjects did not differ from those in the control groups (B_max=68±4 and 56±8 fmol/mg protein for the respective radioligand). The dissociation constants (K_D) were also similar in both groups. The binding capacities (B_max) and K_D values for both radioligands also were found unchanged in the hypothalamus, amygdala, head of caudate, hippocampus and cerebellum. The results demonstrate that, contrary to the -adrenoceptor, the presence of ethanol in the human brain does not alter the high-affinity state of the ₂-adrenoceptor in the frontal cortex and possibly also in other brain regions.     

Effects of the endogenous cannabinoid anandamide on voltage-dependent sodium and calcium channels in rat ventricular myocytes

BACKGROUND AND PURPOSE

The endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA) exerts negative inotropic and antiarrhythmic effects in ventricular myocytes.

EXPERIMENTAL APPROACH

Whole-cell patch-clamp technique and radioligand-binding methods were used to analyse the effects of anandamide in rat ventricular myocytes.

KEY RESULTS

In the presence of 1–10 μM AEA, suppression of both Na⁺ and L-type Ca²⁺ channels was observed. Inhibition of Na⁺ channels was voltage and Pertussis toxin (PTX) – independent. Radioligand-binding studies indicated that specific binding of [³H] batrachotoxin (BTX) to ventricular muscle membranes was also inhibited significantly by 10 μM metAEA, a non-metabolized AEA analogue, with a marked decrease in B_max values but no change in K_d. Further studies on L-type Ca²⁺ channels indicated that AEA potently inhibited these channels (IC₅₀ 0.1 μM) in a voltage- and PTX-independent manner. AEA inhibited maximal amplitudes without affecting the kinetics of Ba²⁺ currents. MetAEA also inhibited Na⁺ and L-type Ca²⁺ currents. Radioligand studies indicated that specific binding of [³H]isradipine, was inhibited significantly by metAEA. (10 μM), changing B_max but not K_d.

CONCLUSION AND IMPLICATIONS

Results indicate that AEA inhibited the function of voltage-dependent Na⁺ and L-type Ca²⁺ channels in rat ventricular myocytes, independent of CB₁ and CB₂ receptor activation.     

[3H]MDL 100,907: a novel selective 5-HT2A receptor ligand

In studies using standard radioligands, unlabeled MDL 100,907 (R-(+)--(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidinemethanol) has been shown to have a high degree of selectivity for the 5-HT_2A receptor. The present study was undertaken to investigate the receptor binding characteristics of [³H]MDL 100,907 in rat cortical homogenates. [³H]MDL 100,907 was found to reach equilibrium at 37°C after 15 min. Saturation experiments indicated binding to a single site with a K_D of 0.56 nM, Hill slope of 1.15, and a B_max of 512 fmol/mg protein. In parallel experiments with the standard 5-HT_2A receptor radioligand, [³H]ketanserin, with prazosin added to block ₁ receptors, a similar Hill slope and B_max was noted but a two-fold higher K_D was found. In competition binding studies using 0.5 nM [³H]MDL 100,907, some 19 standard ligands to various receptors including the 5HT_1A, D₂, ₁, and receptors resulted in estimated K_I values that were consistent with [³H]MDL 100,907 selectively binding to the 5-HT_2A receptor. A comparison of the K_I values for 17 standard 5-HT_2A receptor agonists and antagonists displacing [³H]MDL 100,907 versus [³H]ketanserin resulted in a highly significant linear correlation (R² = 0.96, P<0.001). Taken together these results suggest that [³H]MDL 100,907 is binding to the 5-HT_2A receptor with a sub-nanomolar affinity without the use of secondary blocking agents.     

Is upregulation of benzodiazepine receptors a compensatory reaction to reduced GABAergic tone in the brain of stressed mice?

Effects of various forms of stress on the GABA_A receptor-chloride ionophore complex in the brain of NMRI mice were investigated. Male albino mice were subjected to stress by placing them on small platforms (SP; 3.5 cm diameter) surrounded by water for 24 h. This experimental model contains several stress factors like rapid eye movement (REM) sleep deprivation, isolation, immobilization, falling into water and soaking. As additional stress control groups we used animals subjected to isolation, large platform (9.0 cm diameter) and repeated swimming stress. SP stress induced an increase in the number of cortical benzodiazepine (BDZ) receptors and a reduction in the GABA-stimulated ³⁶C1^– uptake by brain microsacs, whereas none of these changes could be observed in animals exposed to isolation, swimming or large platform stresses. Furthermore, the amount of GABA-induced stimulation of [³H]flunitrazepam binding was reduced in cortical brain membranes of SP-stressed animals, an effect due to fact that these animals dispayed an increase in the basal [³H]flunitrazepam binding, whereas the absolute level of maximally enhanced BDZ binding in the presence of GABA did not differ from those found in controls. Neither basal [³H]muscimol binding or thiopentone sodium-induced stimulation of [³H]flunitrazepam binding were changed in any group of stressed mice. It is proposed that the observed upregulation in the number (B _max ) of cortical BDZ receptors in SP-stressed mice may represent a compensatory response to a stress-induced attenuation of GABAergic neurotransmission.     

A pharmacological comparison of [3H]-granisetron binding sites in brain and peripheral tissues of the mouse

The affinities of a range of structurally diverse 5-HT₃ receptor agonists and antagonists for [³H]-granisetron binding sites have been measured in membrane homogenates prepared from central and peripheral tissues of the mouse. By comparing the affinities of compounds across these tissues, the question of whether intea-species 5-HT₃ receptor subtypes exist in the mouse has been addressed.In entorhinal cortex and brainstem, [³H]-granisetron bound to a single high affinity saturable binding site (K_d 0.47 ± 0.14 and 0.60 ± 0.05 nM; B _max 20 ± 6 and 7 ± 2 fmol (mg protein)^–1 respectively; mean ±SEM; n = 3). In distal and proximal colon, the specific binding of [³H]-granisetron was best fitted to a 2-site model. K_d values obtained for the high affinity site were similar to those obtained in brain tissue (distal colon: 0.47 ± 0.09 nM, n = 4; proximal colon: 0.39 ± 0.09 nM, n = 4). In salivary gland, 2-sites were evident in 2 out of 4 experiments. The K_d value (calculated from the high affinity site in the 2-site model) was approximately 10-fold less than in brain or colon (3.3 ± 1.1 nM, n = 4). B _max values were 7 ± 2, 4 ± 1 and 71 ± 16 fmol (mg protein)^–1 for distal colon, proximal colon and salivary gland respectively. For all tissues the estimated affinity of the low affinity site was variable, and B _max values could not be reliably calculated.Extensive comparative studies performed with 17 different 5-HT₃ receptor agonists and antagonists in the five tissues did not reveal differences in affinity for any compound between the entorhinal cortex and the brainstem nor between the two regions of the colon. However, MDL72222, R-zacopride, d-tubocurarine, and GR80284 apparently had significantly lower affinity for colon than brain binding sites. Also, MDL72222, 2-methyl-5-HT, GR80284, 1-(m-chlorophenyl)-biguanide, metoclopramide, and granisetron had significantly lower affinity for the salivary gland binding sites than the brain binding sites. In an attempt to replicate these observations, we conducted a second study using the compounds which had shown the largest inter-tissue differences in affinity keeping as many variables as possible constant. Simultaneous comparative assays on entorhinal cortex, colon and salivary gland homogenates taken from the same mice showed that the differences that were apparent in the initial comparative study were not maintained. In conclusion, we can find no clear evidence for the existence of tissue-specific subtypes of the 5-HT₃ high affinity binding site for [³H]-granisetron in the mouse in the tissues tested. However, a low affinity binding site for [³H]-granisetron was detected in peripheral tissues.     

CHARACTERIZATION OF BINDING SITES FOR [3H]-IDAZOXAN, [3H]-P-AMINOCLONIDINE AND [3H]-RAUWOLSCINE IN THE KIDNEY OF THE DOG

1. We characterized the binding of [³H]-rauwolscine, [³H]-p-aminoclonidine and [³H]-idazoxan in a dog kidney membrane preparation. Our aim was to determine the pharmacological nature of the α₂-adrenoceptor- and imidazoline-preferring binding sites in this organ. 2. [³H]-Rauwolscine bound to an apparent single site with an affinity (K_D) of 2.2 nmol/ L and a maximum density (B_max) of 58.5 fmol/mg protein, when 10 μmol/L idazoxan defined non-specific binding. However displacement studies demonstrated that a number of compounds, including prazosin, inhibited [³H]-rauwolscine binding in a complex manner consistent with displacement from two distinct binding sites. The majority (69%) of the [³H]-rauwolscine binding sites had a relatively low affinity for prazosin (K_I= 398 nmol/L), while the remainder had a relatively high affinity for prazosin (K_I= 7.9 nmol/ L). 3. [³H]-p-Aminoclonidine bound to an apparent single site (K_D= 5.2 nmol/L; B_max= 72.4 fmol/mg protein), when 10 μmol/L phentolamine defined non-specific binding. When 1 μmol/L of the potent and selective α₂-adrenoceptor antagonist 2-methoxyidazoxan was included in the incubate, no specific binding was detected. We therefore conclude that under the conditions of this experiment [³H]-p-aminoclonidine binds only to α₂-adrenoceptors in the dog kidney. 4. [³H]-Idazoxan bound to two sites, with a higher (K_D= 0.95 nmol/L; B_max= 43.9 fmol/mg protein) and lower (K_D= 9.1 nmol/L; B_max= 93.8 fmol/mg protein) affinity, respectively, when 1 mmol/L phentolamine defined non-specific binding. When 10 μmol/ L GTPγS was included in the incubate, the low affinity site was unaffected but the maximum binding at the higher affinity site was reduced by 79%. 2-Methoxyidazoxan displaced [³H]-idazoxan in a monophasic manner and with low potency (IG₅₀=11.5 μmol/L). Yohimbine, efaroxan, clonidine, rilmenidine, guanabenz and idazoxan itself displaced [³H]-idazoxan in a complex manner; the slope of the displacement curves being less than unity. 5. We conclude that the dog kidney contains a heterogeneous population of α₂-adrenoceptors that can be labelled either with [³H]-rauwolscine or [³H]-p-aminoclonidine. The dog kidney also contains a heterogeneous population of non-adrenoceptor imidazoline-preferring binding sites of the I₂-subtype, that can be labelled with [³H]-idazoxan. The binding site for which [³H]-idazoxan has the highest affinity appears to be coupled to a guanine nucleotide binding regulatory protein.     

Characterization of the [3H]-desipramine binding site of the bovine adrenomedullary plasma membrane

Summary The specific (i.e. nisoxetine-sensitive) binding of [³H]desipramine was studied in membranes prepared from bovine adrenal medullae. (1) [³H]desipramine bound reversibly and with high affinity (K _D = 2.8 nmol/l) to a single class of non-interacting binding sites (Hill coefficient = 0.96); the maximal number of binding sites (B_max) was 2.1 pmol/mg protein. (2) Binding of [³H]desipramine was dependent on [Na⁺] and [Cl^–]. Increasing the concentrations of these ions increased binding. (3) Substrates and inhibitors of the neuronal noradrenaline transport system (uptake,) inhibited binding of [³H]desipramine with a rank order of potency typical for an interaction with the uptake, carrier.The characteristics of [³H]desipramine binding remained essentially unchanged after solubilization of adrenomedullary membranes with the non-ionic detergent digitonin.The results indicate that the plasma membrane of bovine adreno-medulary cells is endowed with the neuronal uptake₁ transporter. Correspondence to: H. Bönisch     

Platelet 5-HT uptake sites,labelled with [3H] paroxetine,in controls and depressed patients before and after treatment with fluoxetine or lofepramine

Platelet [³H] paroxetine binding was measured in 73 depressed patients and in 64 healthy volunteers. No differences were found in B_max or K_d either overall, or when the 61 depressed subjects who had never received psychotropic drugs were analysed separately. Within the depressed group, no differences in B_max or K_d were found between subgroups divided on the basis of endogenicity, suicidal thoughts or severity of depression. None of the subgroups differed significantly from controls. Forty of the depressed subjects were retested after 6 weeks' treatment with fluoxetine (n=22) or lofepramine (n=18). Treatment was not associated with any change in B_max but a similar and significant increase in K_d was noted following treatment with either antidepressant. Neither pre- nor post-treatment platelet binding parameters appeared to relate to clinical response to treatment.     

Drug inhibition indicates a single-site model of the 5-HT uptake site/antidepressant binding site in rat and human brain

Drug inhibition against [³H]paroxetine binding to rat cortex and human putamen was investigated in saturation experiments. The addition of 5-HT, imipramine, citalopram and clomipramine all produced changes in apparent binding affinity (K_d) without changes in the number of binding sites (B_max). These data suggest that there is no heterogeneity of specific [³H]paroxetine binding, supporting a single site model of the 5-HT uptake site and antidepressant binding site.