Presynaptic imidazoline receptors and non-adrenoceptor[3H]-idazoxan binding sites in human cardiovascular tissues

1. In segments of human right atrial appendages and pulmonary arteries preincubated with [³H]-noradrenaline and superfused with physiological salt solution containing desipramine and corticosterone, the involvement of imidazoline receptors in the modulation of [³H]-noradrenaline release was investigated.
2. In human atrial appendages, the guanidines aganodine and DTG (1,3-di(2-tolyl)guanidine) which activate presynaptic imidazoline receptors, inhibited electrically-evoked [³H]-noradrenaline release. The inhibition was not affected by blockade of α₂-adrenoceptors with 1 μM rauwolscine, but antagonized by extremely high concentrations of this drug (10 and/or 30 μM; apparent pA₂ against aganodine and DTG: 5.55 and 5.21, respectively).
3. In the presence of 1 μM rauwolscine, [³H]-noradrenaline release in human atrial appendages was also inhibited by the imidazolines idazoxan and cirazoline, but not by agmatine and noradrenaline. The inhibitory effects of 100 μM idazoxan and 30 μM cirazoline were abolished by 30 μM rauwolscine.
4. In the atrial appendages, the rank order of potency of all guanidines and imidazolines for their inhibitory effect on electrically-evoked [³H]-noradrenaline release in the presence of 1 μM rauwolscine was: aganodine⩾BDF 6143 [4-chloro-2-(2-imidazolin-2-yl-amino)-isoindoline]>DTG⩾clonidine>cirazoline>idazoxan (BDF 6143 and clonidine were previously studied under identical conditions). This potency order corresponded to that previously determined at the presynaptic imidazoline receptors in the rabbit aorta.
5. When, in the experiments in the human pulmonary artery, rauwolscine was absent from the superfusion fluid, the concentration-response curve for BDF 6143 (a mixed α₂-adrenoceptor antagonist/imidazoline receptor agonist) for its facilitatory effect on electrically-evoked [³H]-noradrenaline release was bell-shaped. In the presence of 1 μM rauwolscine, BDF 6143 and cirazoline concentration-dependently inhibited the evoked [³H]-noradrenaline release.
6. In human atrial appendages, non-adrenoceptor [³H]-idazoxan binding sites were identified and characterized. The binding of [³H]-idazoxan was specific, reversible, saturable and of high affinity (K_D: 25.5 nM). The specific binding of [³H]-idazoxan (defined by cirazoline 0.1 mM) to membranes of human atrial appendages was concentration-dependently inhibited by several imidazolines and guanidines, but not by rauwolscine and agmatine. In most cases, the competition curves were best fitted to a two-site model.
7. The rank order of affinity for the high affinity site (in a few cases for the only detectable site; cirazoline=idazoxan>BDF 6143>DTG⩾clonidine) is compatible with the pharmacological properties of I₂-imidazoline binding sites, but is clearly different from the rank order of potency for inhibiting evoked noradrenaline release from sympathetic nerves in the same tissue.
8. It is concluded that noradrenaline release in the human atrium and, less well established, in the pulmonary artery is inhibited via presynaptic imidazoline receptors. These presynaptic imidazoline receptors appear to be related to those previously characterized in rabbit aorta and pulmonary artery, but differ clearly from I₁ and I₂ imidazoline binding sites.

     

Agonist and antagonist modulation of [35S]-GTPγS binding in transfected CHO cells expressing the neurotensin receptor

1. The functional interaction of the cloned rat neurotensin receptor with intracellular G-proteins was investigated by studying the binding of the radiolabelled guanylyl nucleotide analogue [³⁵S]-GTPγS induced by neurotensin to membranes prepared from transfected Chinese hamster ovary (CHO) cells.
2. The agonist-induced binding of [³⁵S]-GTPγS was only detected in the presence of NaCl in the incubation buffer. However, it was also demonstrated that the binding of [³H]-neurotensin to its receptor was inhibited by NaCl. In the presence of 50 mM NaCl, the binding of the labelled nucleotide was about 2 fold increased by stimulation with saturating concentrations of neurotensin (EC₅₀ value of 2.3±0.9 nM).
3. The stimulation of [³⁵S]-GTPγS binding by neurotensin was mimicked by the stable analogue of neurotensin, JMV-449 (EC₅₀ value of 1.7±0.4 nM) and the neurotensin related peptide neuromedin N (EC₅₀ value of 21±6 nM).
4. The NT-induced [³⁵S]-GTPγS binding was competitively inhibited by SR48692 (pA₂ value of 9.55±0.28), a non-peptide neurotensin receptor antagonist. SR48692 alone had no effect on the specific binding of [³⁵S]-GTPγS.
5. The response to neurotensin was found to be inhibited by the aminosteroid U-73122, a putative inhibitor of phospholipase C-dependent processes, indicating that this drug may act at the G-protein level.
6. Taken together, these results constitute the first characterization of the exchange of guanylyl nucleotides at the G-protein level that is induced by the neuropeptide neurotensin after binding to its receptor.

     

Prostanoid receptors of the EP3 subtype mediate inhibition of evoked [3H]acetylcholine release from isolated human bronchi

1. The release of neuronal [³H]acetylcholine (ACh) from isolated human bronchi after labelling with [³H]choline was measured to investigate the effects of prostanoids.
2. A first period of electrical field stimulation (S₁) caused a [³H]ACh release of 320±70 and 200±40 Becquerel (Bq) g⁻¹ in epithelium-denuded and epithelium-containing bronchi respectively (P>0.05). Subsequent periods of electrical stimulation (S_n, n=2, 3, and 4) released less [³H]ACh, i.e. decreasing S_n/S₁ values were obtained (0.76±0.09, 0.68±0.07 and 0.40±0.04, respectively).
3. Cumulative concentrations (1–1000 nM) of EP-receptor agonists like prostaglandin E₂, nocloprost, and sulprostone (EP₁ and EP₃ selective) inhibited evoked [³H]ACh release in a concentration dependent manner with IC₅₀ values between 4–14 nM and maximal inhibition of about 70%.
4. The inhibition of evoked [³H]ACh release by prostaglandin E₂, nocloprost and sulprostone was not affected by the DP-, EP₁- and EP₂-receptor antagonist AH6809 at a concentration of 3 μM, i.e. a 3–30 times greater concentration than its affinity (pA₂ values) at the respective receptors.
5. Circaprost (IP-receptor agonist; 1–100 nM), iloprost (IP- and EP₁-receptor agonist; 10-1000 nM) and U-46619 (TP-receptor agonist; 100–1000 nM) did not significantly affect [³H]ACh release.
6. Blockade of cyclooxygenase by 3 μM indomethacin did not significantly modulate evoked [³H]ACh release in epithelium-containing and epithelium-denuded bronchi. Likewise, the combined cyclo- and lipoxygenase inhibitor BW-755C (20 μM) did not affect evoked [³H]ACh release.
7. In conclusion, applied prostanoids appear to inhibit [³H]ACh release in epithelium-denuded human bronchi under the present in vitro conditions, most likely via prejunctional prostanoid receptors of the EP₃ subtype.

     

Characterization of [3H]-(2S,2′R,3′R)-2-(2′,3′-dicarboxy- cyclopropyl)glycine ([3H]-DCG IV) binding to metabotropic mGlu2 receptor-transfected cell membranes

1. The binding of the new selective group II metabotropic glutamate receptor radioligand, [³H]-(2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine ([³H]-DCG IV), was characterized in rat mGlu₂ receptor-transfected CHO cell membranes.
2. [³H]-DCG IV binding was pH-dependent, but was not sensitive to temperature. Saturation analysis showed the presence of a single binding site, with a K_d value of 160 nM and a B_max value of 10 pmol mg⁻¹ protein. Binding was not sensitive to Na⁺-dependent glutamate uptake blockers or Cl⁻-dependent glutamate binding inhibitors. Furthermore, up to concentrations of 1 mM, the glutamate ionotropic receptor agonists, N-methyl-D-aspartic acid (NMDA), (S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate, did not affect [³H]-DCG IV binding.
3. Of the compounds observed to inhibit [³H]-DCG IV binding, the most potent were the recently described selective group II agonist, (+)-2-aminobicyclo-[3.1.0]hexane-2,6-dicarboxylate (LY 354740; K_i value 16 nM) and antagonist, 2-amino-2-(2-carboxycyclopropan-1-yl)-3-(dibenzopyran-4-yl) propanoic acid (LY 341495; K_i value 19 nM). As expected, for a G-protein-coupled receptor, guanosine-5′-O-(3-thiotriphosphate) (GTPγS) inhibited [³H]-DCG IV binding in a concentration-dependent manner, with an IC₅₀ value of 12 nM.
4. A highly significant correlation was observed between the potencies of compounds able to inhibit [³H]-DCG IV binding and potencies obtained for agonist activity in a GTPγ³⁵S binding functional assay. In addition, these studies identified a number of compounds with previously unknown activity at mGlu₂ receptors, including L(+)-2-amino-3-phosphonopropionic acid (L-AP3), L(+)-2-amino-5-phosphonopentanoic acid (L-AP5), 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (R-CPP), N-acetyl-L-aspartyl-L-glutamic acid (NAAG) and (RS)-α-methylserine-O-phosphate (MSOP).

     

Effects of clozapine on rat striatal muscarinic receptors coupled to inhibition of adenylyl cyclase activity and on the human cloned m4 receptor

1. Clozapine has recently been claimed to behave as a selective and full agonist at the cloned m4 muscarinic receptor artificially expressed in Chinese hamster ovary (CHO) cells. In the present study we have investigated whether clozapine could activate the rat striatal muscarinic receptors coupled to the inhibition of adenylyl cyclase activity, considered as pharmacologically equivalent to the m4 gene product. In addition, we have examined the effect of the drug on various functional responses following the activation of the cloned m4 receptor expressed in CHO cells.
2. In rat striatum, clozapine (1 nM–10 μM) caused a slight inhibition of forskolin-stimulated adenylyl cyclase activity, which was not counteracted by 10 μM atropine. On the other hand, clozapine antagonized the inhibitory effect of acetylcholine with a pA₂ value of 7.51. Moreover, clozapine (1 μM) failed to inhibit dopamine D₁ receptor stimulation of adenylyl cyclase activity, but counteracted the inhibitory effect of carbachol (CCh). Clozapine displaced [³H]-N-methylscopolamine ([³H]-NMS) bound to striatal M₄ receptors with a monophasic inhibitory curve and a pK_i value of 7.69. The clozapine inhibition was not affected by the addition of guanosine-5′-O-(thio)triphosphate (GTPγS).
3. In intact CHO cells, clozapine inhibited forskolin-stimulated cyclic AMP accumulation with an EC₅₀ of 31 nM. This effect was antagonized by atropine. CCh produced a biphasic effect on cyclic AMP levels, inhibiting at concentrations up to 1 μM (EC₅₀=50 nM) and stimulating at higher concentrations (EC₅₀=7 μM). Clozapine (0.3–5 μM) antagonized the CCh stimulation of cyclic AMP with a pK_i value of 7.47. Similar results were obtained when the adenylyl cyclase activity was assayed in CHO cell membranes.
4. In CHO cells pretreated with the receptor alkylating agent 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (10 μM), the maximal inhibitory effect of clozapine on cyclic AMP formation was markedly reduced, whereas the CCh inhibitory curve was shifted to the right with no change in the maximum.
5. As in rat striatum, in CHO cell membranes the displacement of [³H]-NMS binding by clozapine yielded a monophasic curve which was not affected by GTPγS.
6. Clozapine (10 nM–10 μM) had a small stimulant effect (∼20%) on the binding of [³⁵S]-GTPγS to CHO cell membranes, whereas CCh caused a 250% increase of radioligand binding. Moreover, clozapine (50 nM–5 μM) antagonized the CCh-stimulated [³⁵S]-GTPγS binding with a pA₂ value of 7.48.
7. These results show that at the striatal M₄ receptors clozapine is a potent and competitive antagonist, whereas at the cloned m4 receptor it elicits both agonist and antagonist effects. Thus, clozapine behaves as a partial agonist, rather than as a full agonist, at the m4 receptor subtype, with intrinsic activity changing as a function of the coupling efficiency of the receptor to effector molecules.

     

Interactions between loreclezole,chlormethiazole and pentobarbitone at GABAA receptors: functional and binding studies

1. Interations were investigated between loreclezole, chlormethiazole and pentobarbitone as potentiators of depolarization responses mediated by γ-aminobutyric acid_A (GABA_A) receptors on afferent nerve terminals in the rat cuneate nucleus in vitro. These drugs were also compared as modulators of [³H]-flunitrazepam (FNZ) binding to synaptic membranes prepared from rat whole brain homogenate.
2. In rat cuneate nucleus slices, the drugs shifted muscimol log dose–response lines to the left in an approximately parallel fashion with the result that 200 μM chlormethiazole potentiated muscimol responses by 0.567±0.037 log unit (mean±s.e.mean, n=4) while loreclezole gave a maximal potentiation at 10 μM of only 0.121±0.037 (n=6) log unit and 0.071±0.039 (n=22) at 50 μM.
3. While 50 μM chlormethiazole and 30 μM pentobarbitone showed no significant interactions between each other when potentiating muscimol responses in combination, 50 μM loreclezole in combination with either chlormethiazole or pentobarbitone attenuated their potentiating effects, possibly by inducing desensitization of GABA_A receptors.
4. In the [³H]-FNZ binding studies on well-washed membranes, loreclezole enhanced binding to a maximum of 47.3±2.83% of control (mean±s.e.mean, n=3) at 300 μM. Scatchard analysis revealed no change in B_max but a decrease in K_D for [³H]-FNZ from 3.9±0.29 nM to 2.7±0.10 nM (mean±s.e.mean, n=4) in the presence of 100 μM loreclezole. In contrast, 100 μM chlormethiazole caused no potentiation. A small component of the enhancement by loreclezole could be blocked by 100 μM bicuculline and could also be blocked by 100 μM chlormethiazole. It seems likely that the effects on [³H]-FNZ binding are due predominantly to direct actions of the drugs on the GABA_A receptor and are separate from the GABA-potentiating effects.
5. The results indicate distinctly different profiles of action for loreclezole, chlormethiazole and pentobarbitone on GABA_A receptors.

     

Autoradiographic visualization of group III metabotropic glutamate receptors using [3H]-L-2-amino-4-phosphonobutyrate

1. In vitro receptor autoradiography using [³H]-L-2-amino-4-phosphonobutyrate ([³H]-L-AP4) binding to sections of rat brain has been characterized and shown to most likely represent labelling of group III metabotropic glutamate receptors.
2. Specific [³H]-L-AP4 binding to rat brain sections was observed at high densities in the molecular layer of the cerebellar cortex and the outer layer of the superior colliculus. Moderate levels were observed throughout the cerebral cortex, in the molecular layer of the hippocampal dentate gyrus, in thalamus, striatum, substantia nigra and in the medial geniculate nucleus. Low levels of [³H]-L-AP4 binding were found in other regions of the hippocampal formation, in the entorhinal cortex and the granule cell layer of cerebellum.
3. Inhibitors of sodium- or calcium/chloride-dependent glutamate uptake did not displace [³H]-L-AP4 binding to rat brain sections indicating that the observed binding does not represent [³H]-L-AP4 uptake via these carriers. Furthermore, in contrast to [³H]-L-AP4 uptake into cerebellar membranes, [³H]-L-AP4 binding to brain sections was sensitive to guanosine-5′-O-(3-thio)trisphosphate-γ-S.
4. In the molecular layer of the cerebellar cortex, [³H]-L-AP4 binding showed a maximal binding density (B_max) of 0.52±0.06 pmol mg⁻¹ tissue and an affinity (K_d) of 346 nM. The rank order of affinity for displacement of [³H]-L-AP4 binding to rat brain sections was: L-AP4>L-serine-O-phosphate>glutamate>(L)-2-aminomethyl-4-phosphonobutanoate>(1S,3R)-1-aminocyclopentane-1,3-dicarboxylate which is in agreement with a group III metabotropic glutamate receptor pharmacology.

     

Validity of (−)-[3H]-CGP 12177A as a radioligand for the ‘putative β4-adrenoceptor' in rat atrium

1. We have recently suggested the existence in the heart of a ‘putative β₄-adrenoceptor'' based on the cardiostimulant effects of non-conventional partial agonists, compounds that cause cardiostimulant effects at greater concentrations than those required to block β₁- and β₂-adrenoceptors. We sought to obtain further evidence by establishing and validating a radioligand binding assay for this receptor with (−)-[³H]-CGP 12177A ((−)-4-(3-tertiarybutylamino-2-hydroxypropoxy) benzimidazol-2-one) in rat atrium. We investigated (−)-[³H]-CGP 12177A for this purpose for two reasons, because it is a non-conventional partial agonist and also because it is a hydrophilic radioligand.
2. Increasing concentrations of (−)-[³H]-CGP 12177A, in the absence or presence of 20 μM (−)-CGP 12177A to define non-specific binding, resulted in a biphasic saturation isotherm. Low concentrations bound to β₁- and β₂-adrenoceptors (pK_D 9.4±0.1, B_max 26.9±3.1 fmol mg^-1 protein) and higher concentrations bound to the ‘putative β₄-adrenoceptor'' (pK_D 7.5±0.1, B_max 47.7±4.9 fmol mg⁻¹ protein). In other experiments designed to exclude β₁- and β₂-adrenoceptors, (−)-[³H]-CGP 12177A (1–200 nM) binding in the presence of 500 nM (−)-propranolol was also saturable (pK_D 7.6±0.1, B_max 50.8±7.4 fmol mg⁻¹ protein).
3. The non-conventional partial agonists (−)-CGP 12177A (pK_i 7.3±0.2), (±)-cyanopindolol (pK_i 7.6±0.2), (−)-pindolol (pK_i 6.6±0.1) and (±)-carazolol (pK_i 7.2±0.2) and the antagonist (−)-bupranolol (pK_i 6.6±0.2), all competed for (−)-[³H]-CGP 12177A binding in the presence of 500 nM (−)-propranolol at the ‘putative β₄-adrenoceptor'', with affinities closely similar to potencies and affinities determined in organ bath studies.
4. The catecholamines competed with (−)-[³H]-CGP 12177A at the ‘putative β₄-adrenoceptor'' in a stereoselective manner, (−)-noradrenaline (pK_iH 6.3±0.3, pK_iL 3.5±0.1), (−)-adrenaline (pK_iH 6.5±0.2, pK_iL 2.9±0.1), (−)-isoprenaline (pK_iH 6.2±0.5, pK_iL 3.4±0.1), (+)-isoprenaline (pK_i<1.7), (−)-RO363 ((−)-(1-(3,4-dimethoxyphenethylamino)-3-(3,4-dihydroxyphenoxy)-2-propranol)oxalate, pK_i 5.5±0.1).
5. The inclusion of guanosine 5-triphosphate (GTP 0.1 mM) had no effect on binding of (−)-CGP 12177A or (−)-isoprenaline to the ‘putative β₄-adrenoceptor''. In competition binding studies, (−)-CGP 12177A competed with (−)-[³H]-CGP 12177A for one receptor state in the absence (pK_i 7.3±0.2) or presence of GTP (pK_i 7.3±0.2). (−)-Isoprenaline competed with (−)-[³H]-CGP 12177A for two states in the absence (pK_iH 6.6±0.3, pK_iL 3.5±0.1; % H 25±7) or presence of GTP (pK_iH 6.2±0.5, pK_iL 3.4±0.1; % H 37±6). In contrast, at β₁-adrenoceptors, GTP stabilized the low affinity state of the receptor for (−)-isoprenaline.
6. The specificity of binding to the ‘putative β₄-adrenoceptor'' was tested with compounds active at other receptors. High concentrations of the β₃-adrenoceptor agonists, BRL 37344 ((RR+SS)[4-[2-[[2-(3-chlorophenyl)-2-hydroxy - ethyl]amino]propyl]phenoxy]acetic acid, 6 μM), SR 58611A (ethyl{(7S)-7-[(2R)-2 - (3 - chlorophenyl) - 2 - hydroxyethylamino] - 5,6,7,8 - tetrahydronaphtyl2 - yloxy} acetate hydrochloride, 6 μM), ZD 2079 ((±)-1-phenyl-2-(2-4-carboxymethylphenoxy)-ethylamino)-ethan-1-ol, 60 μM), CL 316243 (disodium (R,R)-5-[2-[2-(3-chlorophenyl)-2-hydroxyethyl-amino]propyl]- 1,3-benzodioxole-2,2-dicarboxylate, 60 μM) and antagonist SR 59230A (3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-2S-2-propanol oxalate, 6 μM) caused less than 22% inhibition of (−)-[³H]-CGP 12177A binding in the presence of 500 nM (−)-propranolol. Histamine (1 mM), atropine (1 μM), phentolamine (10 μM), 5-HT (100 μM) and the 5-HT₄ receptor antagonist SB 207710 ((1-butyl-4-piperidinyl)-methyl 8-amino-7-iodo-1,4-benzodioxan-5-carboxylate, 10 nM) caused less than 26% inhibition of binding.
7. Non-conventional partial agonists, the antagonist (−)-bupranolol and catecholamines all competed for (−)-[³H]-CGP 12177A binding in the absence of (−)-propranolol at β₁-adrenoceptors, with affinities (pK_i) ranging from 1.6–3.6 log orders greater than at the ‘putative β₄-adrenoceptor''.
8. We have established and validated a radioligand binding assay in rat atrium for the ‘putative β₄-adrenoceptor'' which is distinct from β₁-, β₂- and β₃-adrenoceptors. The stereoselective interaction with the catecholamines provides further support for the classification of the receptor as ‘putative β₄-adrenoceptor''.

     

Mg2+ and ATP dependence of KATP channel modulator binding to the recombinant sulphonylurea receptor,SUR2B

1. The binding of modulators of the ATP-sensitive K⁺ channel (K_ATP channel) to the murine sulphonylurea receptor, SUR2B, was investigated. SUR2B, a proposed subunit of the vascular K_ATP channel, was expressed in HEK 293 cells and binding assays were performed in membranes at 37°C using the tritiated K_ATP channel opener, [³H]-P1075.
2. Binding of [³H]-P1075 required the presence of Mg²⁺ and ATP. MgATP activated binding with EC₅₀ values of 10 and 3 μM at free Mg²⁺ concentrations of 3 μM and 1 mM, respectively. At 1 mM Mg²⁺, binding was lower than at 3 μM Mg²⁺.
3. [³H]-P1075 saturation binding experiments, performed at 3 mM ATP and free Mg²⁺ concentrations of 3 μM and 1 mM, gave K_D values of 1.8 and 3.4 nM and B_MAX values of 876 and 698 fmol mg⁻¹, respectively.
4. In competition experiments, openers inhibited [³H]-P1075 binding with potencies similar to those determined in rings of rat aorta.
5. Glibenclamide inhibited [³H]-P1075 binding with K_i values of 0.35 and 2.4 μM at 3 μM and 1 mM free Mg²⁺, respectively. Glibenclamide enhanced the dissociation of the [³H]-P1075-SUR2B complex suggesting a negative allosteric coupling between the binding sites for P1075 and the sulphonylureas.
6. It is concluded that an MgATP site on SUR2B with μM affinity must be occupied to allow opener binding whereas Mg²⁺ concentrations ⩾10 μM decrease the affinities for openers and glibenclamide. The properties of the [³H]-P1075 site strongly suggest that SUR2B represents the drug receptor of the openers in vascular smooth muscle.

     

Binding of KATP channel modulators in rat cardiac membranes

1. The binding of [³H]-P1075, a potent opener of adenosine-5′-triphosphate-(ATP)-sensitive K⁺ channels, was studied in a crude heart membrane preparation of the rat, at 37°C.
2. Binding required MgATP. In the presence of an ATP-regenerating system, MgATP supported [³H]-P1075 binding with an EC₅₀ value of 100 μM and a Hill coefficient of 1.4.
3. In saturation experiments [³H]-P1075 binding was homogeneous with a K_D value of 6±1 nM and a binding capacity (B_max) of 33±3 fmol mg⁻¹ protein.
4. Upon addition of an excess of unlabelled P1075, the [³H]-P1075-receptor complex dissociated in a mono-exponential manner with a dissociation rate constant of 0.13±0.01 min⁻¹. If a bi-molecular association mechanism was assumed, the dependence of the association kinetics on label concentration gave an association rate constant of 0.030±0.003 nM⁻¹ min⁻¹. From the kinetic experiments the K_D value was calculated as 4.7±0.6 nM.
5. Openers of the ATP-sensitive K⁺ channel belonging to different structural classes inhibited specific [³H]-P1075 binding in a monophasic manner to completion; an exception was minoxidil sulphate where maximum inhibition was 68%. The potencies of the openers in this assay agree with published values obtained in rat cardiocytes and are on average 3.5 times lower than those determined in rat aorta.
6. Sulphonylureas, such as glibenclamide and glibornuride and the sulphonylurea-related carboxylate, AZ-DF 265, inhibited [³H]-P1075 binding with biphasic inhibition curves. The high affinity component comprised about 60% of the curves with the IC₅₀ value of glibenclamide being ≈amp;90 nM; affinities for the low affinity component were in the μM concentration range. The fluorescein derivative, phloxine B, showed a monophasic inhibition curve with an IC₅₀ value of 6 μM, a maximum inhibition of 94% and a Hill coefficient of 1.5.
7. It is concluded that binding studies with [³H]-P1075 are feasible in rat heart membranes in the presence of MgATP and of an ATP-regenerating system. The pharmacological profile of the [³H]-P1075 binding sites in the cardiac preparation, which probably contains sulphonylurea receptors (SURs) from cardiac myocytes (SUR_2A) and vascular smooth muscle cells (SUR_2B), differs from that expected for SUR_2A and SUR_2B.

     

Tolerance to μ-opioid agonists in human neuroblastoma SH-SY5Y cells as determined by changes in guanosine-5′-O-(3-[35S]-thio)triphosphate binding

1. The agonist action of morphine on membranes prepared from human neuroblastoma SH-SY5Y cells was measured by an increase in the binding of the GTP analogue [³⁵S]-GTPγS. Morphine increased the binding of [³⁵S]-GTPγS to SH-SY5Y cell membranes by 30 fmol mg⁻¹ protein with an EC₅₀ value of 76±10 nM.
2. Incubation of SH-SY5Y cells with 10 μM morphine for 48 h caused a tolerance to morphine manifested by a 2.5 fold shift to the right in the EC₅₀ value with a 31±6% decrease in the maximum stimulation of [³⁵ S]-GTPγS binding. The response caused by the partial agonist pentazocine was reduced to a greater extent.
3. Chronic treatment of the cells with the more efficacious μ-ligand [D-Ala², MePhe⁴, Gly-ol⁵]enkephalin (DAMGO, 10 μM) for 48 h afforded a greater effect than treatment with morphine. The maximal agonist effect of morphine was reduced to 58.9±6% of that seen in control cells while the maximal effect of DAMGO was reduced to 62.8±4%. There was a complete loss of agonist activity for pentazocine.
4. The development of tolerance was complete within 24 h and was blocked by naloxone and by the nonselective protein kinase inhibitor H7, but not by the putative β-adrenoceptor kinase (β-ARK) inhibitor suramin.
5. The observed tolerance effect was accompanied by a down-regulation of μ-opioid receptors determined by a decrease in the maximal binding capacity for the opioid antagonist [³H]-diprenorphine of 66±4%, but with no change in binding affinity. Binding of the agonist [³H]-DAMGO was similarly reduced.
6. The modulation of [³⁵S]-GTPγS binding in SH-SY5Y cell membranes by opioids provides a simple method for the study of opioid tolerance at a site early in the signal transduction cascade.

     

The effects of recombinant rat μ-opioid receptor activation in CHO cells on phospholipase C, [Ca2+]i and adenylyl cyclase

1. The rat μ-opioid receptor has recently been cloned, yet its second messenger coupling remains unclear. The endogenous μ-opioid receptor in SH-SY5Y cells couples to phospholipase C (PLC), increases [Ca²⁺]_i and inhibits adenylyl cyclase (AC). We have examined the effects of μ-opioid agonists on inositol(1,4,5)trisphosphate (Ins(1,4,5)P₃), [Ca²⁺]_i and adenosine 3′ : 5′-cyclic monophosphate (cyclic AMP) formation in Chinese hamster ovarian (CHO) cells transfected with the cloned μ-opioid receptor.
2. Opioid receptor binding was assessed with [³H]-diprenorphine ([³H]-DPN) as a radiolabel. Ins(1,4,5)P₃ and cyclic AMP were measured by specific radioreceptor assays. [Ca²⁺]_i was measured fluorimetrically with Fura-2.
3. Scatchard analysis of [³H]-DPN binding revealed that the B_max varied between passages. Fentanyl (10 pM–1 μM) dose-dependently displaced [³H]-DPN, yielding a curve which had a Hill slope of less than unity (0.6±0.1), and was best fit to a two site model, with pK_i values (% of sites) of 9.97±0.4 (27±4.8%) and 7.68±0.07 (73±4.8%). In the presence of GppNHp (100 μM) and Na⁺ (100 mM), the curve was shifted to the right and became steeper (Hill slope=0.9±0.1) with a pK_i value of 6.76±0.04.
4. Fentanyl (0.1 nM–1 μM) had no effect on basal, but dose-dependently inhibited forskolin (1 μM)-stimulated, cyclic AMP formation (pIC₅₀=7.42±0.23), in a pertussis toxin (PTX; 100 ng ml⁻¹ for 24 h)-sensitive and naloxone-reversible manner (K_i=1.7 nM). Morphine (1 μM) and [D-Ala², MePhe⁴, gly(ol)⁵]-enkephalin (DAMGO, 1 μM) also inhibited forskolin (1 μM)-stimulated cyclic AMP formation, whilst [D-Pen², D-Pen⁵], enkephalin (DPDPE, 1 μM) did not.
5. Fentanyl (0.1 nM–10 μM) caused a naloxone (1 μM)-reversible, dose-dependent stimulation of Ins(1,4,5)P₃ formation, with a pEC₅₀ of 7.95±0.15 (n=5). PTX (100 ng ml⁻¹ for 24 h) abolished, whilst Ni²⁺ (2.5 mM) inhibited (by 52%), the fentanyl-induced Ins(1,4,5)P₃ response. Morphine (1 μM) and DAMGO (1 μM), but not DPDPE (1 μM), also stimulated Ins(1,4,5)P₃ formation. Fentanyl (1 μM) also caused an increase in [Ca²⁺]_i (80±16.4 nM, n=6), reaching a maximum at 26.8±2.5 s. The increase in [Ca²⁺]_i remained elevated until sampling ended (200 s) and was essentially abolished by the addition of naloxone (1 μM). Pre-incubation with naloxone (1 μM, 3 min) completely abolished fentanyl-induced increases in [Ca²⁺]_i.
6. In conclusion, the cloned μ-opioid receptor when expressed in CHO cells stimulates PLC and inhibits AC, both effects being mediated by a PTX-sensitive G-protein. In addition, the receptor couples to an increase in [Ca²⁺]_i. These findings are consistent with the previously described effector-second messenger coupling of the endogenous μ-opioid receptor.

     

Characterization of A2A adenosine receptors in human lymphocyte membranes by [3H]-SCH 58261 binding

1. The present study describes for the first time the characterization of the adenosine A_2A receptor in human lymphocyte membranes with the new potent and selective antagonist radioligand, [³H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4 triazolo [1,5-c] pyrimidine, ([³H]-SCH 58261). In addition, both receptor affinity and potency of reference adenosine receptor agonists and antagonists were determined in binding and adenylyl cyclase studies.
2. Saturation experiments revealed a single class of binding sites with K_d and B_max values of 0.85 nM and 35 fmol mg⁻¹ protein, respectively. A series of adenosine receptor ligands were found to compete for the binding of 0.8 nM [³H]-SCH 58261 to human lymphocyte membranes with a rank order of potency consistent with that typically found for interactions with the A_2A-adenosine receptor. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency similar to that observed in binding experiments.
3. Thermodynamic data indicate that [³H]-SCH 58261 binding to human lymphocytes is entropy and enthalpy-driven, a finding in agreement with the thermodynamic behaviour of antagonists for rat striatal A_2A-adenosine receptors.
4. It is concluded that in human lymphocyte membranes [³H]-SCH 58261 directly labels binding sites showing the characteristic properties of the adenosine A_2A-receptor. The presence of A_2A-receptors in peripheral tissue such as human lymphocytes strongly suggests an important role for adenosine in modulating immune and inflammatory responses.

     

Possible mechanisms underlying the midazolam-induced relaxation of the noradrenaline-contraction in rabbit mesenteric resistance artery

1. The mechanisms underlying the midazolam-induced relaxation of the noradrenaline (NA)-contraction were studied by measuring membrane potential, isometric force and intracellular concentration of Ca²⁺([Ca²⁺]_i) in endothelium-denuded muscle strips from the rabbit mesenteric resistance artery. The actions of midazolam were compared with those of nicardipine, an L-type Ca²⁺-channel blocker.
2. Midazolam (30 and 100 μM) did not modify either the resting membrane potential or the membrane depolarization induced by 10 μM NA.
3. NA (10 μM) produced a phasic, followed by a tonic increase in both [Ca²⁺]_i and force. Midazolam (10–100 μM) did not modify the resting [Ca²⁺]_i, but attenuated the NA-induced phasic and tonic increases in [Ca²⁺]_i and force, in a concentration-dependent manner. In contrast, nicardipine (0.3 μM) attenuated the NA-induced tonic, but not phasic, increases in [Ca²⁺]_i and force.
4. In Ca²⁺-free solution containing 2 mM EGTA, NA (10 μM) transiently increased [Ca²⁺]_i and force. Midazolam (10–100 μM), but not nicardipine (0.3 μM), attenuated this NA-induced increase in [Ca²⁺]_i and force, in a concentration-dependent manner. However, midazolam (10 and 30 μM), had no effect on the increases in [Ca²⁺]_i and force induced by 10 mM caffeine.
5. In ryanodine-treated strips, which have functionally lost the NA-sensitive Ca²⁺- storage sites, NA slowly increased [Ca²⁺]_i and force. Nicardipine (0.3 μM) did not modify the resting [Ca²⁺]_i but partly attenuated the NA-induced increases in [Ca²⁺]_i and force. In the presence of nicardipine, midazolam (100 μM) lowered the resting [Ca²⁺]_i and further attenuated the remaining NA-induced increases in [Ca²⁺]_i and force.
6. The [Ca²⁺]_i-force relationship was obtained in ryanodine-treated strips by the application of ascending concentrations of Ca²⁺ (0.16–2.6 mM) in Ca²⁺-free solution containing 100 mM K⁺. NA (10 μM) shifted the [Ca²⁺]_i-force relationship to the left and enhanced the maximum Ca²⁺-induced force. Under these conditions, whether in the presence or absence of 10 μM NA, midazolam (10 and 30 μM) attenuated the increases in [Ca²⁺]_i and force induced by Ca²⁺ without changing the [Ca²⁺]_i-force relationship.
7. It was concluded that, in smooth muscle of the rabbit mesenteric resistance artery, midazolam inhibits the NA-induced contraction through its inhibitory action on NA-induced Ca²⁺ mobilization. Midazolam attenuates NA-induced Ca²⁺ influx via its inhibition of both nicardipine-sensitive and -insensitive pathways. Furthermore, midazolam attenuates the NA-induced release of Ca²⁺ from the storage sites. This effect contributes to the midazolam-induced inhibition of the NA-induced phasic contraction.

     

[3H]-SCH 58261 labelling of functional A2A adenosine receptors in human neutrophil membranes

1. The present study describes the direct labelling of A_2A adenosine receptors in human neutrophil membranes with the potent and selective antagonist radioligand, [³H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4 triazolo[1,5-c]pyrimidine, ([³H]-SCH 58261). In addition, both receptor affinity and potency of a number of adenosine receptor agonists and antagonists were determined in binding, adenylyl cyclase and superoxide anion production assays.
2. Saturation experiments revealed a single class of binding sites with K_d and B_max values of 1.34 nM and 75 fmol mg⁻¹ protein, respectively. Adenosine receptor ligands competed for the binding of 1 nM [³H]-SCH 58261 to human neutrophil membranes, with a rank order of potency consistent with that typically found for interactions with the A_2A adenosine receptors. In the adenylyl cyclase and in the superoxide anion production assays the same compounds exhibited a rank order of potency identical to that observed in binding experiments.
3. Thermodynamic data indicated that [³H]-SCH 58261 binding to human neutrophils is entropy and enthalpy-driven. This finding is in agreement with the thermodynamic behaviour of antagonists binding to rat striatal A_2A adenosine receptors.
4. It was concluded that in human neutrophil membranes, [³H]-SCH 58261 directly labels binding sites with pharmacological properties similar to those of A_2A adenosine receptors of other tissues. The receptors labelled by [³H]-SCH 58261 mediated the effects of adenosine and adenosine receptor agonists to stimulate cyclic AMP accumulation and inhibition of superoxide anion production in human neutrophils.

     

Pharmacological characterization of type 1α metabotropic glutamate receptor-stimulated [35S]-GTPγS binding

1. The activation of G proteins by type 1α metabotropic glutamate receptors (mGluRs) in membranes from recombinant baby hamster kidney cells expressing the cloned rat mGluR1α receptor has been studied by use of a [³⁵S]-guanosine 5′-[γ-thio]triphosphate ([³⁵S]-GTPγS) binding assay.
2. L-Glutamate increased the rate of [³⁵S]-GTPγS binding in a concentration-dependent manner (−logEC₅₀ (M) 5.25±0.07), with an optimal (62.4±1.6%) increase over basal binding being observed following 60 min incubation at 30°C with 70 pM [³⁵S]-GTPγS, 1 μM GDP, 10 mM MgCl₂, 100 mM NaCl and 100 μg membrane protein ml⁻¹. The L-glutamate (100 μM)-stimulated increase in [³⁵S]-GTPγS binding was totally prevented in the presence of the group I mGluR antagonist (S)-4-carboxy-3-hydroxyphenylglycine (300 μM).
3. Quantitative analysis of the affinity and number of G proteins activated by a maximally effective concentration of L-glutamate revealed an equilibrium dissociation constant (K_D) for [³⁵S]-GTPγS binding of 0.76±0.20 nM and a maximal number of GTPγS-liganded G proteins (B_max) of 361±30 fmol mg⁻¹ protein.
4. Metabotropic glutamate receptor agonists, quisqualate (−logEC₅₀ (M) 6.74±0.06), 1S,3R-ACPD (4.64±0.08) and (S)-3,5-dihydroxyphenylglycine (5.16±0.23) also increased [³⁵S]-GTPγS binding in a concentration-dependent manner, with the latter two agents behaving as partial agonists.
5. (+)-α-Methylcarboxyphenylglycine (300 μM) caused a parallel rightward shift of the L-glutamate concentration-effect curve for [³⁵S]-GTPγS binding, allowing an antagonist equilibrium dissociation constant (K_D) of 34.0±7.8 μM to be calculated for this mGluR antagonist.
6. Pretreatment of BHK-mGluR1α cells with a concentration of pertussis toxin (PTX) shown to be maximally effective (100 ng ml⁻¹, 24 h) before membrane preparation resulted in a marked decrease in agonist-stimulated [³⁵S]-GTPγS binding (by 66.0±0.9%), and an altered concentration-effect relationship for agonist-stimulated [³⁵S]-GTPγS binding by the residual PTX-insensitive G-protein population.
7. The modulation of [³⁵S]-GTPγS binding by agonists and antagonists in membranes from recombinant cells provides an excellent system in which to study mGluR interactions with PTX-sensitive and -insensitive G proteins.

     

Effect of acute and chronic tramadol on [3H]-5-HT uptake in rat cortical synaptosomes

1. Tramadol hydrochloride is a centrally acting opioid analgesic, the efficacy and potency of which is only five to ten times lower than that of morphine. Opioid, as well as non-opioid mechanisms, may participate in the analgesic activity of tramadol.
2. [³H]-5-hydroxytryptamine (5-HT) uptake in rat isolated cortical synaptosomes was studied in the presence of tramadol, desipramine, fluoxetine, methadone and morphine. Methadone and tramadol inhibited synaptosomal [³H]-5-HT uptake with apparent K_is of 0.27±0.04 and 0.76±0.04 μM, respectively. Morphine essentially failed to inhibit [³H]-5-HT uptake (K_i 0.50±0.30 M).
3. Methadone, morphine and tramadol were active in the hot plate test with ED₅₀s of 3.5, 4.3 and 31 mg kg⁻¹, respectively. At the highest tested dose (80 mg kg⁻¹) tramadol produced only 77±5.3% of the maximal possible effect.
4. When [³H]-5-HT uptake was examined in synaptosomes prepared from rats 30 min after a single dose of morphine, methadone or tramadol, only tramadol (31 mg kg⁻¹, s.c., equal to the ED₅₀ in the hot plate test) and methadone (35 mg kg⁻¹, s.c., equal to the ED₉₀ in the hot plate test) decreased uptake.
5. Animals were chronically treated for 15 days with increasing doses of tramadol or methadone (5 to 40 mg kg⁻¹ and 15 to 120 mg kg⁻¹, s.c., respectively). Twenty-four hours after the last drug injection, a challenge dose of methadone (35 mg kg⁻¹, s.c.) or tramadol (31 mg kg⁻¹, s.c.) was administered. [³H]-5-HT uptake was not affected in synaptosomes prepared from rats chronically-treated with methadone, whereas chronic tramadol was still able to reduce this parameter by 42%.
6. Rats chronically-treated with methadone showed a significant increase in [³H]-5-HT uptake (190%) 72 h after drug withdrawal. In contrast, [³H]-5-HT uptake in rats chronically-treated with tramadol (110%) did not differ significantly from control animals.
7. These results further support the hypothesis that [³H]-5-HT uptake inhibition may contribute to the antinociceptive effects of tramadol. The lack of tolerance development of [³H]-5-HT uptake, together with the absence of behavioural alterations after chronic tramadol treatment, suggest that tramadol has an advantage over classical opioids in the treatment of pain disorders.

     

Tachykinin receptors in the guinea-pig isolated oesophagus: a complex system

1. The tachykinin receptors mediating contraction of isolated longitudinal strips of the guinea-pig oesophageal body were characterized with substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) as well as the analogues, [Sar⁹,Met(O₂)¹¹]SP, [Nle¹⁰]NKA(4–10) and [MePhe⁷]NKB, selective for NK₁, NK₂ and NK₃, receptors, respectively. Experiments were performed both in the absence and presence of a cocktail of peptidase inhibitors, captopril (1 μM), thiorphan (1 μM) and amastatin (20 μM), in order to determine whether membrane bound proteases are important in the metabolism of tachykinins in this preparation.
2. All agonists produced concentration-dependent contractile effects. The presence of the peptidase inhibitors shifted the concentration-response curves of SP, [Nle¹⁰]NKA(4–10) and [MePhe⁷]NKB significantly leftwards and the concentration-response curve of NKB was shifted significantly rightwards. However, the EC₅₀ values were significantly different only for [Nle¹⁰]NKA(4–10) and NKB.
3. In the presence of the peptidase inhibitors, the EC₅₀ values of the selective agonists, [MePhe⁷]NKB (0.6 nM) and [Nle¹⁰]NKA(4–10) (66 nM) indicated the presence of both tachykinin NK₃ and NK₂ receptors. [MePhe⁷]NKB produced less than 50% of the maximal response obtained with the other agonists. Since [Sar⁹,Met(O₂)¹¹]SP produced a small response in the nanomolar concentration range in about 30% of the preparations tested, it is possible that some NK₁ receptors were also present.
4. Assuming competitive antagonism, the NK₂-selective antagonist SR 48,968 (30 nM) gave apparent pK_B values of 8.13 and 8.65 for [Nle¹⁰]NKA(4–10) in the absence and presence of peptidase inhibitors, respectively, supporting the presence of NK₂ receptors.
5. The NK₃-selective antagonist SR 142,801 (0.1 μM), suppressed responses to low (0.1–10 nM) concentrations of [MePhe⁷]NKB. These contractile responses to [MePhe⁷]NKB were also abolished by atropine (0.6 μM) suggesting that this response was mediated via cholinergic nerves.
6. It is concluded that the guinea-pig oesophagus is a complex system which has both NK₂ and NK₃ receptors and possibly some NK₁ receptors as well.