Somatostatin receptors in the Rhesus monkey brain: localization and pharmacological characterization

To characterize the nature and distribution of somatostatin (SRIF) receptors, radioligand binding studies and in vitro receptor autoradiography were performed in Rhesus monkey brain using either [¹²⁵I]LTT-SRIF-28 ([Leu⁸,D-Trp²²,¹²⁵I-Tyr²⁵]SRIF-28) alone or in the presence of 3 nM seglitide (to block sst₂ sites), [¹²⁵1]Tyr³-octreotide or [¹²⁵I] CGP 23996 (c[Asu-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Tyr-Thr-Ser]) in buffer containing either 120 mM Na⁺ or 5 mM Mg²⁺. [¹²⁵I]Tyr³-octreotide labelled an apparently homogeneous population of sites in cerebral and cerebellar cortex (B _max = 27.3±2.8 fmol/mg protein and 52.6±8.6 fmol/mg protein, pK_d = 9.46±0.03 and 9.93±0.03, respectively). The pharmacological profile of these sites correlated highly significantly with that of human recombinant sst₂ receptors (r = 0.996), but not or much less with that of human recombinant sst₃ and sst₅ receptors (r = 0.12 and 0.45, respectively). [¹²⁵I]CGP 23996 (in Na⁺-buffer) also labelled an apparently homogeneous population of sites in Rhesus monkey cerebral cortex membranes (B _max = 3.1±0.3 fmol/mg protein, pKd = 10.57±0.08), the pharmacological profile of which was highly significantly correlated with the profiles of human recombinant sst₁ and sst₄ receptors (r = 0.98 and 0.96, respectively).Using receptor autoradiography, high levels of [¹²⁵I]LTT-SRIF-28 and [¹²⁵I]Tyr³-octreotide recognition sites were found in basal ganglia, molecular and granular layers of the cerebellum and layers III, V and VI of entorhinal cortex. In these regions, the addition of 3 nM seglitide produced a marked decrease of [¹²⁵I]LTT-SRIF-28 binding. Low levels of [¹²⁵I]LTT-SRIF-28 binding were observed in subiculum, pituitary and choroid plexus. By contrast, [¹²⁵I]CGP 23996 labelling in the presence of Mg²⁺ as well as Na⁺ ions was highest in pituitary and choroid plexus. However, [¹²⁵I]CGP 23996 binding was diversely affected by these ionic conditions in several regions of hippocampus and cerebral cortex. Displacement of [¹²⁵I]CGP 23996 (in Mg²⁺-buffer) with seglitide in the molecular layer of the cerebellum, deep layers of the entorhinal cortex, layers I, II and V of the insular cortex and frontal pole yielded complex competition curves suggesting the presence of two populations of SRIF receptors. By contrast, [¹²⁵I]CGP 23996 binding (in Mg²⁺-buffer) in the choroid plexus, hilus of the dentate gyrus and stratum oriens and radiatum of the CA3 field of hippocampus was not affected by seglitide up to 10 M, suggesting only sst₁ and/or sst₄ sites which have a negligible affinity for seglitide to be present in these structures.Taken together, these results suggest that [¹²⁵I]CGP 23996 (in the presence of Na⁺) labels exclusively SRIF-2 receptors (sst₁ and/or sst₄), whereas in the presence of Mg²⁺ ions, [¹²⁵I]CGP 23996 labels both SRIF-2 and SRIF-1 receptors (sst₂, sst₃ and sst₅). The present study also demonstrates the presence and differential distribution of sst₂ and sst₁/sst₄ receptors in the Rhesus monkey brain.     

Localization and pharmacological characterization of somatostatin sst2 sites in the rat cerebellum

Radioligand binding studies were performed in membranes of rat cerebellum using [¹²⁵I]-[Tyr³]octreotide ([¹²⁵I]204-090) to characterize the nature of cerebellar somatostatin receptors. Saturation experiments suggest the presence of a single class of binding sites with high affinity, pK_d = 9.53 ± 0.11, but low receptor density, B _max = 12.7 ± 1.0 fmol/mg protein. The pharmacological profile of [¹²⁵I]204-090 sites in cerebellar membranes was established using a range of ligands known to interact with SSTR-2 (now called sst₂) and other somatostatin (SRIF) receptors. SRIF analogues such as octreotide (SMS 201-995), seglitide (MK 678) and somatuline (BIM 23014) displayed very high affinity for cerebellar [¹²⁵I]204-090 binding sites. The data were compared to results obtained using the same ligand in rat cerebral cortex membranes known to represent sst₂ binding. The pharmacological characteristics of the cerebellar sites were in close correlation with those of the cerebral cortex (r = 0.976, n = 19, p < 0.001) and CHO-cells expressing human recombinant sst₂ receptor (r = 0.977, n = 19, p < 0.001). By contrast, there was very little correlation between cerebellar binding and published affinities for rat sst₅ receptors (r = 0.465), for which octreotide has also high affinity. In vitro autoradiographic studies performed in cerebellar slices using [¹²⁵I]204-090 demonstrated the presence of binding sites in the molecular layer of the rat cerebellum. In situ hybridization studies using sst₂ receptor mRNA selective oligoprobes confirmed the presence of sst₂ receptor mRNA in the rat cerebellum. Together, the present data demonstrate the presence of a low density of SRIF receptors in the molecular layer of the adult rat cerebellum which are best characterized as sst₂. This is the first pharmacological characterization and localization of sst₂ receptors in the adult rat cerebellum.     

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.     

Vanilloid receptors in the urinary bladder: regional distribution,localization on sensory nerves,and species-related differences

Summary Using selective surgical ablations we have investigated the localization of vanilloid receptors (specific [³H]resiniferatoxin binding sites) on terminals of the pelvic, hypogastric, and pudendal nerves in the rat urinary bladder. Pelvic and hypogastric nerve resections resulted in 90%6 and 25% loss of specific [³H]resiniferatoxin (RTX) binding sites, respectively, whilst pudendic nerve resection had no measurable effect on the binding. In control animals, the density of vanilloid receptors was 1.7-fold higher in the neck than in the dome of the urinary bladder; the B_max values were 57±8 and 34±7 fmol/mg protein, respectively. The binding characteristics of the vanilloid receptor were similar in the urinary bladder of the rat and mouse: K_d values were 87±15 and 61±11 pM, B_max values were 37±2 and 60±10 fmol/mg protein, respectively. In contrast to the findings for the rat and mouse, in the urinary bladder of the guinea pig and the hamster the low level of specific [³H]RTX binding prevented the detailed characterization of vanilloid receptors. Nonetheless, at a fixed (60 pM) concentration of [³H]RTX, specific binding both in the guinea pig and hamster urinary bladder was approximately 20% of that in the rat urinary bladder. In the urinary bladder of newborn rats, as in adults, a single class of specific [³H]RTX binding sites was found which bound RTX with an affinity of 110±20 pM and with a maximal binding capacity of 30±5 fmol/mg protein. We conclude that, in accord with the physiological findings, the majority of vanilloid receptors are located on terminals of the pelvic nerve in the rat urinary bladder with higher receptor density in the bladder neck as compared to the bladder dome. Whereas the comparably high density of vanilloid receptors in the rat and mouse urinary bladder and the low receptor density in the hamster are mirrored by the in vivo vanilloid-sensitivity of these species, the low level of vanilloid receptors in the urinary bladder of the guinea pig contrasts to the marked sensitivity of this species to capsaicin. We conclude that the level of vanilloid receptors is an important but not exclusive determinant of vanilloid-sensitivity.Correspondence to A. Szallasi at the above address     

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.     

[125I]Tyr10-cortistatin14 labels all five somatostatin receptors

The recently cloned rat preprocortistatin, which shows homology to the preprosomatostatin peptide, is thought to be enzymatically cleaved to cortistatin₁₄ (CST₁₄) similarly to somatostatin₁₄ (SRIF₁₄). High structural similarity of cortistatin₁₄ compared to SRIF₁₄ suggested binding properties to somatostatin receptors similar to SRIF₁₄. In the present study, we expressed stably the five human somatostatin receptor subtypes (hsst₁–hsst₅) in CCL39 cells (Chinese hamster lung fibroblast cells). The receptors were labelled with an iodinated analogue of CST₁₄ ([¹²⁵I]Tyr¹⁰-cortistatin₁₄, [¹²⁵I]Tyr¹⁰-CST) to establish the pharmacological profile of hsst₁–hsst₅ sites labelled with [¹²⁵I]Tyr¹⁰-CST. In parallel, [Leu⁸,D-Trp²²,¹²⁵I-Tyr²⁵]-SRIF₂₈ ([¹²⁵I]LTT-SRIF₂₈) was used as a control at the five recombinant SRIF receptors stably expressed in CCL39 cells. High affinity [¹²⁵I]Tyr¹⁰-CST binding could be demonstrated to all five recombinant somatostatin receptor subtypes. The pK_d (–log mol/l) and B_max values (fmol/mg) for hsst_1–5 receptors were: 10.02±0.04, 220±30; 9.45±0.09, 340±70; 10.06±0.11, 340±50; 9.67±0.14, 340±110 and 10.33±0.03, 5630±330, respectively. The pharmacological profiles determined with [¹²⁵I]Tyr¹⁰-CST and [¹²⁵I]LTT-SRIF₂₈ were very similar at every receptor studied. These data suggest that cortistatin and somatostatin have similar high affinity for SRIF receptors. None of the receptors showed marked selectivity for either CST₁₄/CST₁₇ or the somatostatins. In conclusion, the data show that cortistatin and somatostatin have very similar high affinity to all five recombinant somatostatin receptors. It remains to be seen whether there are specific receptors which bind only somatostatins or cortistatins. Received: 7 October 1997 / Accepted: 11 February 1998     

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.     

Pharmacological characterisation of human cerebral cortex somatostatin SRIF1 and SRIF2 receptors

Radioligand binding studies were performed in membranes of human cerebral cortex using [¹²⁵I]Tyr³-octreotide in the presence of 5 mM MgCl₂, [¹²⁵I]SRIF-14 ([¹²⁵I]Tyr¹¹-SRIF-14) and [¹²⁵I]CGP 23996 ([¹²⁵I]c[Asu-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Tyr-Thr-Ser]) both in the presence of 120 mM NaCl, to characterise the nature of the somatostatin (SRIF) receptors. The pharmacological profile of human brain SRIF recognition sites was compared with that of recombinant human SRIF₁ (sst₂-sst₃-sst₅) or SRIF₂ receptors (sst₁-sst₄) and with that of native rat sst₁, sst₂ and sst₄ receptors. [¹²⁵I]Tyr³-octreotide labelled binding sites in human cerebral cortex: B _max = 238 ± 36 fmol/mg protein and pK_d = 9.73 ± 0.08. The pharmacological profile of [¹²⁵I]Tyr³-octreotide labelled sites correlated very significantly with that of recombinant human sst₂ receptors (r = 0.98) and much less with those of recombinant human sst₃ (r = 0.65) or sst₅ receptors (r = 0.72). The correlation between [¹²⁵I]Tyr³-octreotide binding to native sst₂ receptors in human and rat cerebral cortex was also highly significant (r = 0.97). [¹²⁵I]SRIF-14 and [¹²⁵I]CGP 23996 binding (performed in the presence of 120 mM NaCl) in the human cerebral cortex identified very similar populations of sites B _max = 44 ± 7 and 36 ± 5 fmol/mg protein and pK_d = 9.44 ± 0.08 and 9.48 ± 0.10, respectively. The pharmacological profiles of the sites labelled with [¹²⁵I]SRIF-14 and [¹²⁵I]CGP 23996 correlated highly significantly with those of recombinant human sst₁ (r = 0.97–0.99) or sst₄ receptors (r = 0.91–0.94). Similarly, the correlations between [¹²⁵I]SRIF-14 or [¹²⁵I]CGP 23996 binding in human cortex and [¹²⁵I]SRIF-14 binding to native sst₁ sites in rat cerebral cortex were also highly significant (r = 0.97 and 0.94, respectively). Finally, the pharmacological profile of native rat lung sst₄ sites determined with [¹²⁵I]LTT-SRIF-28 ([Leu⁸,D-Trp²²,¹²⁵I-Tyr²⁵]SRIF-28) correlated with [¹²⁵I]SRIF-14 and [¹²⁵I]CGP 23996 binding in human cortex; r = 0.91 and 0.87, respectively. The present data show that in human cerebral cortex, [¹²⁵I]Tyr³-octreotide labels SRIF₁ receptor sites which are best characterised as of the sst₂ type, whereas [¹²⁵I]SRIF-14 and [¹²⁵I]CGP 23996 (both in the presence of 120 mM NaCl), label sites which fit almost equally well with sst₁ or sst₄ receptors and therefore are best described as of the SRIF₂ type. Under the conditions used, there was no evidence that either of these ligands would label sst₃ or sst₅ receptors in human cerebral cortex. Received: 8 August 1996 / Accepted: 8 November 1996     

Endothelin receptors in the human coronary artery,ventricle and atrium

Summary In the present experiments we investigated endothelin (ET) receptors in the human coronary artery, and in ventricular and atrial muscle using quantitative receptor autoradiography. Displacement of [¹²⁵I]Sf6b (Sarafotoxin S6b) (30 pM)- and [¹²⁵I]ET-1 (30 pM)-labeled binding sites was studied using ET-1, the ET_A receptor selective ligand BQ-123 (cyclo[D-Asp-L-Pro-D Val-L-Leu-D-Trp-]), and the ETB receptor selective ligand [Ala^1,3,11,15]ET-1.Specific binding was more dense in atrium and coronary artery (relative optical density (r.o.d.): 0.14±0.01 and 0.16±0.01, respectively) than in ventricular muscle (r.o.d.: 0.10±0.01). In the coronary artery, binding was especially dense in the media. ET-1 displaced [¹²⁵I]ET-1 and [¹²⁵I]Sf6b monophasically in atrium, ventricle and coronary artery. [Ala^1,3,11,15]ET 1 and BQ-123 displaced [¹²⁵I]ET-1 and [¹²⁵I]Sf6b-labeled sites biphasically in the ventricle and in the atrium. In the human coronary artery, [Ala^1,3,11,15]ET-1 and BQ-123 displaced [¹²⁵I]ET-1-labeled sites monophasically (pIC₅₀): ET-1 (9.72±0.12) > BQ-123 (6.84±0.08) > [Ala^1,3,11,15]ET-1 (6.40±0.12). In contrast, [Ala^1,3,11,15]ET-1 and BQ-123 displaced [¹²⁵I] Sf6b-labeled coronary artery sites biphasically (high affinity pIC₅₀: BQ-123, 9.03±0.25;[Ala^1,3,11,15]ET-1, 8.40±0.14; low affinity pIC₅₀: BQ-123, 7.24±0.14; [Ala^1,3,11,15]ET-1, 6.99±0.09).These data indicate that both [¹²⁵I]ET-1 and [¹²⁵I] Sf6b-labeled ET_A and ET_B binding sites in human ventricular and atrial muscle. In the human coronary artery, both radioligands labeled ET_A binding sites, but [¹²⁵I] Sf6b also labeled a non-ET_A, non-ET_B binding site with relatively high affinity for both BQ-123 and [Ala^1,3,11,15] ET-1. Correspondence to W. A. Bax, at the above address     

[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.     

Characterization of a B2-bradykinin receptor in rat renal mesangial cells

The specific binding of bradykinin (BK) was investigated using membrane fractions from mesangial cells in primary culture, a cloned cell line, and in intact adherent cells with three different radiolabelled BK analogues: ¹²⁵I-[Tyr⁰]BK, ¹²⁵I-[Tyr⁵]BK and ¹²⁵I-[Tyr⁸]BK. The best radioligand was ¹²⁵I-[Tyr⁰]BK, and assay conditions were determined to ensure maximal stable binding. Binding appeared to be reversible and not to be inhibited by a wide variety of protease inhibitors including converting enzyme inhibitor and phosphoramidon. The maximum density of binding sites (B_max) was about 88 ± 18 fmol/mg protein, which is equivalent to about 6000 sites/cell, and the dissociation constant averaged 2 nM. No significant difference in B_max was observed between membranes from cells in primary culture and those from cloned cells. Of the BK analogues tested, unmodified BK exhibited the highest inhibition constant (close to 10⁻¹⁰ M). No displacement of ¹²⁵I-[Tyr⁰]BK was observed in the presence of the B₁ agonist des-Arg⁹-BK or several unrelated peptides, including atrial natriuretic factor and angiotensin I and II, whereas 50% inhibition of binding was achieved with the B₂ antagonist [D-Arg,Hyp³,D-Phe⁷]BK (10⁻⁹ M). Addition of BK for 3 min to the incubation medium of cloned mesangial cells induced a dose- and time-dependent increase in PGE₂ unlike des-Arg⁹-BK, which showed no such effect. The secretion was strongly inhibited by prior incubation with the B₂ antagonist [D-Arg,Hyp³,D-Phe⁷]BK. The pharmacological profile of the binding site determined with various BK agonists and antagonists, and the stimulating effect of binding site activation on prostaglandin release strongly suggest that B₂-kinin-like receptors are present in rat mesangial cells.     

Quantitative [3H] dipyridamole autoradiography: evidence for adenosine transporter heterogeneity in guinea pig brain

Summary [³H] Dipyridamole binding in guinea pig brain slices has been characterized. Binding of [³H] dipyridamole to guinea pig forebrian slices was found to be rapid, reversible and saturable. Saturation experiments revealed a class of high affinity binding sites with a B _max value of 592 ± 118 fmol/mg protein and K _d value of 10.8 nM ± 2.1 nM in the analysed concentration range. In competition experiments, the adenosine transport inhibitors hexobendine and dipyridamole itself were the most potent displacers (inhibition constants of 4.6 nM ± 1 nM and 11.5 nM ± 3 nM) with pseudo-Hill coefficients close to 1. Competition curves with nitrobenzylthioinosine, another adenosine transport inhibitor, however, showed a biphasic profile with a pseudo-Hill coefficient of 0.33 ± 0.04. Just 42% ± 4% of [³H] dipyridamole binding were inhibited by nanomolar concentrations of nitrobenzylthioinosine and only micromolar concentrations displaced the remainder. Subsequent quantitative autoradiography demonstrated regional differences in the inhibition of [³H] dipyridamole binding by submicromolar concentrations of nitrobenzylthioinosine. While in cortical areas of cerebrum and cerebellum 500 nM nitrobenzylthioinosine displaced binding of [³H] dipyridamole to only about one-third of its sites (in the Purkinje cell layer less than 10%), it showed similar potency as dipyridamole in various areas of the brainstem and hypothalamus. This biphasic and regionally heterogenous interaction of nitrobenzylthioinosine with [³H] dipyridamole binding sites in guinea pig brain slices strongly suggests heterogeneity of adenosine transporters.     

[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)     

Binding of endomorphin‐2 to μ‐opioid receptors in experimental mouse mammary adenocarcinoma

Abstract: Endomorphin‐2 (Tyr‐Pro‐Phe‐Phe‐NH₂) binds with high affinity and selectivity to the μ‐opioid receptor. In the present study, [¹²⁵I]endomorphin‐2 has been used to characterize μ‐opioid‐binding sites on transplantable mouse mammary adenocarcinoma cells. Cold saturation experiments performed with [¹²⁵I]endomorphin‐2 (1 nm ) show biphasic binding curves in Scatchard coordinates. One component represents high affinity and low capacity (K_d = 18.79 ± 1.13 nm , B_max = 635 ± 24 fmol/mg protein) and the other shows low affinity and higher capacity (K_d = 7.67 ± 0.81 μm , B_max = 157 ± 13 pmol/mg protein) binding sites. The rank order of agonists competing for the [¹²⁵I]endomorphin‐2 binding site was [d ‐1‐Nal³]morphiceptin > endomorphin‐2 ? [d ‐Phe³]morphiceptin > morphiceptin > [d ‐1‐Nal³]endomorphin‐2, indicating binding of these peptides to μ‐opioid receptors. The uptake of ¹³¹I‐labeled peptides administered intraperitoneally to tumor‐bearing mice was also investigated. The highest accumulation in the tumor was observed for [d ‐1‐Nal³]morphiceptin, which reached the value of 8.19 ± 1.14% dose/g tissue.     

Autoradiographic analysis of somatostatin SRIF1 and SRIF2 receptors in the human brain and pituitary

The distribution of somatostatin (SRIF) receptor sites was studied by in vitro receptor autoradiography in the human brain and pituitary using the SRIF₁ (sst₂) receptor selective [¹²⁵I]Tyr³-octreotide, the non-subtype selective [¹²⁵I]LTT-SRIF-28 ([Leu⁸,D-Trp²²,¹²⁵I-Tyr²⁵]SRIF-28) and the SRIF₂-receptor selective [¹²⁵I]CGP 23996 (c[Asu-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Tyr-Thr-Ser]) in buffer containing 120 mM Na⁺. SRIF receptor autoradiography was compared with mRNA expression of somatostatin receptors sst_1–5 as studied by in situ hybridisation in human brain. High levels of [¹²⁵I]LTT-SRIF-28 and [¹²⁵I]Tyr³-octreotide recognition sites were found in the deep layers of cerebral cortex and molecular layer of cerebellum of the human brain. The hypothalamus, choroid plexus, most areas of the brainstem and dentate nucleus were associated with low levels of binding. In contrast to [¹²⁵I]LTT-SRIF-28 and [¹²⁵I]Tyr³-octreotide, no difference was observed for [¹²⁵I]CGP 23996 labelling in the various layers of cerebral cortex. The choroid plexus, substantia nigra and molecular layer of the cerebellum presented high densities of [¹²⁵I]CGP 23996 binding sites whereas no binding was observed in the hypothalamus and locus coeruleus using this radioligand. Both lobes of the human pituitary displayed low levels of [¹²⁵I]LTT-SRIF-28 and [¹²⁵I]Tyr³-octreotide binding. By contrast, the anterior lobe of the pituitary displayed very high levels of [¹²⁵I]CGP 23996 labelled sites whereas intermediate levels were found in the posterior lobe. There was a partial overlap between sst₂ receptor mRNA and [¹²⁵I]Tyr³-octreotide binding, although the distribution of the binding sites was much wider than that of receptor mRNA. The same observation was made for sst₁ and/or sst₄ receptor mRNA and [¹²⁵I]CGP 23996 labelled sites. The present data show that SRIF₁ and SRIF₂ receptors are present in the human brain with different distributions, especially in the cerebral cortex and the pituitary. The very similar distribution of sites labelled with [¹²⁵I]LTT-SRIF-28 and [¹²⁵I]Tyr³-octreotide suggests (i) that sst₂ receptors are predominant within the SRIF₁ family in the human brain and (ii) that [¹²⁵I]LTT-SRIF-28 under the conditions used in the present study, does not significantly label SRIF₂ sites. Received: 8 August 1996 / Accepted: 8 November 1996     

Pharmacological characterization of unique prazosin-binding sites in human kidney

In human kidney, we found unique prazosin-binding sites that were insensitive to phentolamine and were thus unlikely to be ₁-adrenoceptors.As the binding of [³H]prazosin to phentolamine-insensitive sites was prevented by 100 M guanabenz, the insensitive sites were evaluated by subtracting [³H]prazosin binding in the presence of 100 M guanabenz from that in the presence of 10 M phentolamine. [³H]Prazosin bound to the phentolamine-insensitive sites monophasically with a high affinity (pK_d; 9.1±0.08, n=8), and the B_max value (814±204 fmol mg^–1 protein, n=8) was more than ten times that of the phentolamine-sensitive ₁-adrenoceptor (pK_d=9.9±0.13, B_max=66±23 fmol mg^–1 protein, n=7). The phentolamine-insensitive sites in human kidney were highly sensitive to other quinazoline derivatives such as terazosin and doxazosin. However, other ₁-adrenoceptor antagonists (tamsulosin, WB4101 and corynanthine) did not inhibit the binding at a range of concentrations that generally exhibit ₁-adrenoceptor antagonism, and noradrenaline, rauwolscine and propranolol were without effect on the [³H]prazosin binding. On the other hand, ligands for the renal Na⁺-transporter (amiloride and triamterene) and for imidazoline recognition sites (guanabenz, guanfacine and agmatine) displaced the binding of [³H]prazosin to phentolamine-insensitive sites at micromolar concentrations. Photoaffinity labeling with [¹²⁵I]iodoarylazidoprazosin showed phentolamine-insensitive labeling at around 100 kDa, a molecular size larger than that of human _1a- and _1b-adrenoceptors expressed in 293 cells (50–60 and 70–80 kDa, respectively) on electrophoresis. In contrast, there was no detectable phentolamine-insensitive binding site but were phentolamine-sensitive ₁-adrenoceptors in human liver (pK_d=10.0±0.06, B_max=44±6 fmol mg^–1 protein, n=3). Phentolamine-insensitive prazosin binding sites were also detected in rabbit kidney (approximately 50% of specific binding sites) but were minor in rat kidney (less than 20%).In conclusion, there are unique prazosin-binding sites in human kidney, the pharmacological profiles of which were distinct from those of known adrenoceptors.     

Autoradiographic characterisation and localisation of 5-HT1D compared to 5-HT1B binding sites in rat brain

Summary The regional distribution and the pharmacology of the binding sites labelled with the novel 5-hydroxytryptamine (serotonin) 5-HT_1B/1D selective radioligand serotonin-O-carboxy-methyl-glycyl-[¹²⁵I]tyrosinamide (abbreviated [¹²⁵I]GTI for the sake of simplicity) was determined using quantitative autoradiography in rat brain. The distribution of [¹²⁵I]GTI binding sites was largely comparable to that of [¹²⁵I] iodocyanopindolol ([¹²⁵I] ICYP) which labels 5-HT_1B binding sites (in the presence of 8-OH-DPAT (8-hydroxy-[2N-dipropylamino]tetralin) and isoprenaline, to prevent binding to 5-HT_1A and -adrenoceptor binding sites), although a detailed analysis revealed differences.The pharmacology of the [¹²⁵I]GTI binding sites was analysed using compounds known to display high affinity for and/or distinguish between 5-HT_1B and 5-HT_1D sites: 5-carboxamidotryptamine (5-CT), sumatriptan, CP 93129 (5-hydroxy-3(4-1,2,5,6-tetrahydropyridyl)-4-azaindole), (–)pindolol, PAPP (4[2-[4-[3-(trifluoromethyl)phenyl]-1-piperazinyl]ethyl] benzeneamine), rauwolscine, and 8-OH-DPAT. The displacement of [¹²⁵I]GTI by 5-CT was monophasic. By contrast, the selective 5-HT_1B compound CP 93129 and (–)pindolol produced biphasic curves showing a majority of high affinity sites in the globus pallidus and the substantia nigra, whereas PAPP and sumatriptan (which are somewhat 5-HT_1D selective) produced biphasic curves indicating a minority of high affinity sites in these areas. In addition, by blocking the 5-HT_1B sites with 100 nM CP 93129, the remaining population of [¹²⁵I]GTI binding sites could be studied and was found to have high affinity for PAPP, rauwolscine and 8-OH-DPAT. The pharmacological profile of the major binding component was typical of the 5-HT_1B type: 5-CT > CP 93129 (–)pindolol > sumatriptan >/ PAPP > rauwolscine. The profile of the minor component of [¹²⁵I] GTI binding is best characterised as that of a 5-HTID site: 5-CT > PAPP sumatriptan > rauwolscine > (–)pindolol CP 93129.The localisation of the non 5-HT_1B [¹²⁵I]GTI binding sites was characterised by blocking the 5-HT_1B receptors with 100 nM CP 93129. Low densities of the 5-HT_1D recognition sites were found to be present in globus pallidus, ventral pallidum, caudate-putamen, subthalamic nucleus, entopeduncular nucleus, substantia nigra (reticular part), nuclei of the (normal and accessory) optic tract, different nuclei of the geniculate body and frontoparietal cortex, although higher densities of 5-HT_1B sites were always observed in the same structures. Thus, in agreement with the recent cloning of a rat 5-HT_1D receptor cDNA, the presence and the distribution of 5-HT_1D sites could be documented in rat brain. However, when compared to 5-HT_1B sites, 5-HT_1D sites represent only a minor component of the [¹²⁵I]GTI binding in the rat brain structures studied.Correspondence to: D. Hoyer at the above address     

Direct demonstration of dopamine D1-like receptor sites in the ciliary body of the rabbit eye by light microscope autoradiography

Summary The pharmacological characteristics and the anatomical localization of [³H]-SCH 23390 in sections of the ciliary body of the rabbit eye were analyzed using a radioreceptor assay and autoradiographic techniques. [³H]-SCH 23390 was bound to sections of rabbit ciliary body in a manner consistent with the labelling of D₁-like receptor sites. The dissociation constant (K _d) was 0.62 nmol/l, while the maximum binding capacity (B_max) was 117 ± 9 fmol/mg tissue.Light microscope autoradiography revealed [³H]-SCH 23390 binding sites within the epithelium of the ciliary processes, which is the ocular structure involved in the secretion of aqueous humor. No specific accumulation of silver grains was noticeable within the iridocorneal angle, which is the structure involved in the outflow of aqueous humor. These findings suggest that the rise in intraocular pressure caused by D₁ receptor agonists is probably mediated by an increase of aqueous humor formation rather than by an inhibition of the outflow of aqueous humor.     

Cannabidiol is an allosteric modulator at mu- and delta-opioid receptors

The mechanism of action of cannabidiol, one of the major constituents of cannabis, is not well understood but a noncompetitive interaction with mu opioid receptors has been suggested on the basis of saturation binding experiments. The aim of the present study was to examine whether cannabidiol is an allosteric modulator at this receptor, using kinetic binding studies, which are particularly sensitive for the measurement of allosteric interactions at G protein-coupled receptors. In addition, we studied whether such a mechanism also extends to the delta opioid receptor. For comparison, (-)-Δ⁹-tetrahydrocannabinol (THC; another major constituent of cannabis) and rimonabant (a cannabinoid CB₁ receptor antagonist) were studied. In mu opioid receptor binding studies on rat cerebral cortex membrane homogenates, the agonist ³H-DAMGO bound to a homogeneous class of binding sites with a K_D of 0.68±0.02 nM and a B_max of 203±7 fmol/mg protein. The dissociation of ³H-DAMGO induced by naloxone 10 μM (half life time of 7±1 min) was accelerated by cannabidiol and THC (at 100 μM, each) by a factor of 12 and 2, respectively. The respective pEC₅₀ values for a half-maximum elevation of the dissociation rate constant k_off were 4.38 and 4.67; ³H-DAMGO dissociation was not affected by rimonabant 10 μM. In delta opioid receptor binding studies on rat cerebral cortex membrane homogenates, the antagonist ³H-naltrindole bound to a homogeneous class of binding sites with a K_D of 0.24±0.02 nM and a B_max of 352±22 fmol/mg protein. The dissociation of ³H-naltrindole induced by naltrindole 10 μM (half life time of 119±3 min) was accelerated by cannabidiol and THC (at 100 μM, each) by a factor of 2, each. The respective pEC₅₀ values were 4.10 and 5.00; ³H-naltrindole dissociation was not affected by rimonabant 10 μM. The present study shows that cannabidiol is an allosteric modulator at mu and delta opioid receptors. This property is shared by THC but not by rimonabant.     

Clozapine decreases [<Superscript>3</Superscript>H] CP 55940 binding to the cannabinoid<Subscript>1</Subscript> receptor in the rat nucleus accumbens

Antipsychotic drugs are effective in the treatment of cannabis-induced psychosis, but only clozapine appears effective in the treatment of comorbid schizophrenia and cannabis use. The unique effects of clozapine on cannabis use could, therefore, be due to an as yet unidentified interaction between clozapine and the endogenous cannabinoid system. To address this hypothesis, we used in situ radioligand binding and quantitative autoradiography with the selective cannabinoid CB₁ receptor agonist, (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (side chain-2,3,4(N)-³H) ([³H]CP 55940) to measure the density of the CB₁ receptor in frontal cortex, hippocampus, nucleus accumbens and striatum from rats treated with a variety of antipsychotic drugs. Clozapine significantly decreased [³H]CP 55940 binding in the nucleus accumbens compared with vehicle after 1 (35.0±14.0 vs. 71.2±8.5 fmol/mg estimated tissue equivalent (ete); P=0.03) and 3 months (42.3±4.0 vs. 71.1±16.3 fmol/mg ete; P<0.04) of treatment, an effect not observed with haloperidol, chlorpromazine or olanzapine. In rats treated with clozapine for 3 months and then left for 1 month without treatment, [³H]CP 55940 binding was not different in the nucleus accumbens (100.5±22.2 vs. 100.9±25.4 fmol/mg ete; P>0.10). By contrast, there were significant increases in accumbal [³H]CP 55940 binding in rats treated with haloperidol (136.5±14.2 fmol/mg ete; P<0.05), chlorpromazine (137.4±12.7 fmol/mg ete; P<0.05) and olanzapine (144.7±10.1 fmol/mg ete; P<0.01). These data indicate that in the nucleus accumbens clozapine differs from other antipsychotic drugs in its effects on [³H]CP 55940 binding. If these results can be extrapolated into humans, then this effect of clozapine on the CB₁ receptor may be a mechanism that makes it uniquely effective in schizophrenia and comorbid cannabis use.