Somatostatin receptors in Neuro2A neuroblastoma cells: operational characteristics

1. We have used somatostatin (SRIF) receptor subtype-selective ligands to determine some of the operational characteristics of somatostatin receptors in Neuro2A mouse neuroblastoma cells. The potent SRIF₁-receptor selective ligand, BIM-23027, was able to displace completely the specific binding of radioiodinated somatostatin, [¹²⁵I]-Tyr¹¹-SRIF-14, with a pIC₅₀ of 10.3, suggesting that Neuro2A cells contain predominantly receptors of the SRIF₁ receptor group. The rank order of affinities for several somatostatin analogues tested in competition studies, together with the high affinity of BIM-23027, indicate that the majority of receptors in Neuro2A cells are of the sst₂ subtype.
2. The stable radioligand, [¹²⁵I]-BIM-23027, bound with high affinity (K_d=13 pM, B_max=0.2 pmol mg⁻¹ protein) to Neuro2A cell membranes, but its binding was only partially reversible at room temperature and below. Thus at 4°C, only 36% of the bound ligand dissociated within 2 h. In contrast, 60% of the ligand dissociated at 15°C and 89% of the ligand dissociated at 37°C.
3. Equilibrium binding of [¹²⁵I]-BIM-23027 was partially (25%) inhibited by 10 μM GTP, and by 120 mM NaCl (42% inhibition) but this inhibition was increased to 75% when sodium chloride and GTP were added together. This effect of GTP and sodium chloride was also seen in dissociation experiments. After incubation to equilibrium with [¹²⁵I]-BIM-23027, dissociation was initiated with excess unlabelled ligand in the presence of GTP (10 μM) and sodium chloride (120 mM). Under these conditions 67% of the ligand dissociated at 4°C, 81% at 15°C and 93% at 37°C. Binding was totally inhibited by pretreatment of cells with pertussis toxin.
4. Functionally, BIM-23027 inhibited forskolin-stimulated cyclic AMP accumulation in a concentration-dependent manner with an IC₅₀ of 1.0 nM and a maximal inhibition of 37%. This effect was abolished by pretreatment of the cells with pertussis toxin. However, unlike in studies reported with the recombinant sst₂ receptor, no rise in intracellular calcium concentration was observed with SRIF-14.
5. We conclude that Neuro2A cells provide a stable neuronal cell line for the study of functionally coupled endogenous somatostatin receptors of the sst₂ type. In addition, we have found that activation of the receptor is associated with ligand-receptor internalisation.

     

Somatostatin receptors in Neuro2A neuroblastoma cells: ligand internalization

1. Receptor-dependent internalization of somatostatin (SRIF) agonists has been a matter of controversy probably because [¹²⁵I]-Tyr¹¹-SRIF-14 is rapidly degraded. We have studied the internalization of a stable somatostatin analogue, [¹²⁵I]-BIM-23027, in a neuronal cell line, Neuro2A, which natively expresses somatostatin sst₂ receptors.
2. Incubation of Neuro2A cells with [¹²⁵I]-BIM-23027 at 37°C resulted in a time-dependent internalization of the ligand, which reached a maximum at 30 min. Acid-washing showed that cell-surface binding of the ligand accounted for only 34% of total binding at this time. internalization was dramatically reduced at 15°C.
3. internalization of [¹²⁵]-BIM-23027 was prevented by inclusion of unlabelled somatostatin receptor agonists in a concentration-dependent manner. The IC₅₀ values for inhibition of [¹²⁵I]-BIM-23027 internalization were approximately 100 fold lower than for inhibition of [¹²⁵I]-BIM-23027 binding to membrane homogenates but followed the same rank order of potencies.
4. Disruption of G-protein coupling by treatment with pertussis toxin caused a 60% reduction in internalization of ligand. A combination of antimycin (50 nM) and deoxyglucose (50 mM) pretreatment, which leads to a depletion of cellular ATP, decreased internalization of [¹²⁵I]-BIM-23027 by 66% of control and increased the proportion of surface-bound ligand. Hypertonic sucrose, which prevents clathrin-mediated endocytosis, reversibly abolished the internalization of ligand without increasing the proportion bound at the cell surface.
5. After internalization of [¹²⁵I]-BIM-23027, approximately half of the ligand was recycled back to the extracellular medium within 20 min at 37°C. This finding suggests that the intracellular content of [¹²⁵I]-BIM-23027 reaches a steady state which is determined by the rates of both internalization and recycling of the ligand. In contrast to studies in which the internalization of [¹²⁵I]-Tyr¹¹-SRIF-14 was examined, neither internalized nor recycled [¹²⁵I]-BIM-23027 was degraded to its component amino acids.
6. These findings indicate that the somatostatin agonist, [¹²⁵I]-BIM-23027, is internalized in a receptor-dependent manner which involves clathrin-coated pits in Neuro2A cells. Furthermore, much of the internalized ligand is rapidly recycled back to the extracellular medium without undergoing significant degradation.

     

G Proteins endogenously expressed in Sf 9 cells: interactions with mammalian histamine receptors

Expression of functionally active mammalian histamine H₁- and H₂-receptors was recently demonstrated in Sf9 cells. Either receptor elicited phosphoinositide degradation leading to an increased cytoplasmic calcium concentration. In the present study we focussed on identifying the Sf9 guanine nucleotide-binding proteins (G proteins) involved. Immunodetection of Sf9 membranes showed expression of Gα isoforms belonging to all four G protein subfamilies. During prolonged baculovirus infection of Sf9 cells, binding of guanosine 5’-o-(3-thiotriphosphate) as well as the intensities of G protein immunoreactivity, pertussis toxin-mediated ADP-ribosylation, GTP azidoanilide labelling of Gα, and phosphate-labelling of Gβ declined in cell membranes. Some 48h after infection with mammalian histamine receptor-encoding viruses virtually no functional coupling of ligand-activated receptors to insect G proteins was observed despite a high level of expressed receptors. In contrast, Sf9 cells infected only for 28h allowed studies on histamine-induced G protein coupling. In membranes obtained from H₁-receptor-expressing cells, histamine increased incorporation of GTP azidoanilide into G _q/11-like proteins whereas in membranes containing H₂-receptors histamine enhanced GTP azidoanilide-labelling of G _q/11-like and G_s-like proteins. In fura-loaded H₁- and H₂-receptor-expressing cells histamine induced the release of calcium from intracellular stores. This study shows firstly that Sf9 G proteins couple to mammalian histamine receptors and secondly that H₁-receptors activate only G_q/11, whereas H₂-receptors activate G_q/11 and G_s, but neither receptor couples to G_i/o or G₁₂. Finally, the time following baculovirus infection is critical for studying the functional coupling between recombinantly expressed and endogenous signal transduction components. Received: 24 January 1997 /Accepted: 14 April 1997     

[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.

     

Ca2+ signals evoked by histamine H1 receptors are attenuated by activation of prostaglandin EP2 and EP4 receptors in human aortic smooth muscle cells

Background and Purpose

Histamine and prostaglandin E₂ (PGE₂), directly and via their effects on other cells, regulate the behaviour of vascular smooth muscle (VSM), but their effects on human VSM are incompletely resolved.

Experimental Approach

The effects of PGE₂ on histamine-evoked changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and adenylyl cyclase activity were measured in populations of cultured human aortic smooth muscle cells (ASMCs). Selective ligands of histamine and EP receptors were used to identify the receptors that mediate the responses.

Key Results

Histamine, via H₁ receptors, stimulates an increase in [Ca²⁺]_i that is entirely mediated by activation of inositol 1,4,5-trisphosphate receptors. Selective stimulation of EP₂ or EP₄ receptors attenuates histamine-evoked Ca²⁺ signals, but the effects of PGE₂ on both Ca²⁺ signals and AC activity are largely mediated by EP₂ receptors.

Conclusions and Implications

Two important inflammatory mediators, histamine via H₁ receptors and PGE₂ acting largely via EP₂ receptors, exert opposing effects on [Ca²⁺]_i in human ASMCs.     

The expression and function of histamine H3 receptors in pancreatic beta cells

BACKGROUND AND PURPOSE

Histamine and its receptors in the CNS play important roles in energy homeostasis. Here, we have investigated the expression and role of histamine receptors in pancreatic beta cells, which secrete insulin.

EXPERIMENTAL APPROACH

The expression of histamine receptors in pancreatic beta cells was examined by RT-PCR, Western blotting and immunostaining. Insulin secretion assay, ATP measurement and calcium imaging studies were performed to determine the function and signalling pathway of histamine H₃ receptors in glucose-induced insulin secretion (GIIS) from MIN6 cells, a mouse pancreatic beta cell line. The function and signalling pathway of H₃ receptors in MIN6 cell proliferation were examined using pharmacological assay and Western blotting.

KEY RESULTS

Histamine H₃ receptors were expressed in pancreatic beta cells. A selective H₃ receptor agonist, imetit, and a selective inverse H₃ receptor agonist, JNJ-5207852, had inhibitory and facilitatory effects, respectively, on GIIS in MIN6 cells. Neither imetit nor JNJ-5207852 altered intracellular ATP concentration, or intracellular calcium concentration stimulated by glucose and KCl, indicating that GIIS signalling was affected by H₃ receptor signalling downstream of the increase in intracellular calcium concentration. Moreover, imetit attenuated bromodeoxyuridine incorporation in MIN6 cells. The phosphorylation of cAMP response element-binding protein (CREB), which facilitated beta cell proliferation, was inhibited, though not significantly, by imetit, indicating that activated H₃ receptors inhibited MIN6 cell proliferation, possibly by decreasing CREB phosphorylation.

CONCLUSIONS AND IMPLICATIONS

Histamine H₃ receptors were expressed in mouse beta cells and could play a role in insulin secretion and, possibly, beta cell proliferation.     

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.

     

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.

     

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.

     

Characteristics of recombinantly expressed rat and human histamine H3 receptors

Human and rat histamine H(3) receptors were recombinantly expressed and characterized using receptor binding and a functional cAMP assay. Seven of nine agonists had similar affinities and potencies at the rat and human histamine H(3) receptor. S-alpha-methylhistamine had a significantly higher affinity and potency at the human than rat receptor, and for 4-[(1R*,2R*)-2-(5,5-dimethyl-1-hexynyl)cyclopropyl]-1H-imidazole (Perceptin) the preference was the reverse. Only two of six antagonists had similar affinities and potencies at the human and the rat histamine H(3) receptor. Ciproxifan, thioperamide and (1R*,2R*)-trans-2-imidazol-4 ylcyclopropyl) (cyclohexylmethoxy) carboxamide (GT2394) had significantly higher affinities and potencies at the rat than at the human histamine H(3) receptor, while for N-(4-chlorobenzyl)-N-(7-pyrrolodin-1-ylheptyl)guanidine (JB98064) the preference was the reverse. All antagonists also showed potent inverse agonism properties. Iodoproxyfan, Perceptin, proxyfan and GR175737, compounds previously described as histamine H(3) receptor antagonists, acted as full or partial agonists at both the rat and the human histamine H(3) receptor.     

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.

     

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.

     

Functional pharmacology of H1 histamine receptors expressed in mouse preoptic/anterior hypothalamic neurons

BACKGROUND AND PURPOSE

Histamine H₁ receptors are highly expressed in hypothalamic neurons and mediate histaminergic modulation of several brain-controlled physiological functions, such as sleep, feeding and thermoregulation. In spite of the fact that the mouse is used as an experimental model for studying histaminergic signalling, the pharmacological characteristics of mouse H₁ receptors have not been studied. In particular, selective and potent H₁ receptor agonists have not been identified.

EXPERIMENTAL APPROACH

Ca²⁺ imaging using fura-2 fluorescence signals and whole-cell patch-clamp recordings were carried out in mouse preoptic/anterior hypothalamic neurons in culture.

KEY RESULTS

The H₁ receptor antagonists mepyramine and trans-triprolidine potently antagonized the activation by histamine of these receptors with IC₅₀ values of 0.02 and 0.2 μM respectively. All H₁ receptor agonists studied had relatively low potency at the H₁ receptors expressed by these neurons. Methylhistaprodifen and 2-(3-trifluoromethylphenyl)histamine had full-agonist activity with potencies similar to that of histamine. In contrast, 2-pyridylethylamine and betahistine showed only partial agonist activity and lower potency than histamine. The histamine receptor agonist, 6-[2-(4-imidazolyl)ethylamino]-N-(4-trifluoromethylphenyl)heptanecarboxamide (HTMT) had no agonist activity at the H₁ receptors H1 receptors expressed by mouse preoptic/anterior hypothalamic neurons but displayed antagonist activity.

CONCLUSIONS AND IMPLICATIONS

Methylhistaprodifen and 2-(3-trifluoromethylphenyl)histamine were identified as full agonists of mouse H₁ receptors. These results also indicated that histamine H₁ receptors in mice exhibited a pharmacological profile in terms of agonism, significantly different from those of H₁ receptors expressed in other species.     

Functional coupling of a recombinant Human 5-HT5A receptor to G-proteins in HEK-293 cells

1. We have cloned, expressed and pharmacologically characterized the Human 5-HT_5A receptor.
2. We have shown that ligand activation of the Human 5-HT_5A receptor results in functional coupling to G-proteins in HEK-293 cells.
3. Stimulation of the receptor with 5-CT (5-carboxamidotryptamine) resulted in a dose-dependent increase in the % [³⁵S]-GTPγS binding over the basal level. This is the first study to describe such G-protein activation for the Human 5-HT_5A receptor in any cell.
4. A dose-dependent inhibition of cyclic AMP accumulation was observed in the recombinant Human 5-HT_5A receptor cell line, suggesting a functional coupling to a Gαi, G-protein in the HEK-293 cell line.
5. A ligand-stimulated reduction in the detectable level of the catalytic domain of protein kinase A (PKA) in nuclear extracts isolated from Human 5-HT_5A expressing cells was observed. This observation was consistent with the reduction in the level of cyclic AMP accumulation, in response to receptor activation.

     

Endothelin receptor subtypes and their functional relevance in human small coronary arteries

1. The potent constrictor peptide endothelin (ET) has been implicated in various cardiovascular disorders including myocardial infarction and atherosclerosis. We have investigated the nature of ET receptor subtypes present on human small coronary arteries.
2. Small coronary arteries were mounted in a wire-myograph for in vitro pharmacology. To investigate the ET receptor subtypes present in different segments of the coronary vascular tree, arteries were grouped according to internal diameter. Responses in arteries with small internal diameters (mean 316.7±7.9 μm; Group B) were compared to those in larger arteries (mean 586.2±23.1 μm; Group A).
3. ET-1 consistently and potently contracted arteries from Group A and B, with EC₅₀ values of 1.7 (0.9–3.2) nM (n=15) and 2.3 (1.4–4.2) nM (n=14), respectively. No correlation was observed between ET-1 potency and internal diameter. The response to ET-1 was potently antagonized by the selective ET_A receptor antagonist PD156707 in both Group A and Group B, yielding pA₂ values of 8.60±0.12 (n=4–6) and 8.38±0.17 (n=4–6), respectively. Slopes from Schild regression were not significantly different from unity.
4. In contrast to ET-1, individual responses to ET-3 were variable. While all arteries from Group A responded to ET-3 (EC₅₀∼69 (23–210) nM) (n=12), no response was obtained in 5 of the 14 tested in Group B. Of those responding, many failed to reach a maximum at concentrations up to 1 μM. ET-1 was more potent than ET-3 in all arteries tested. A biphasic ET-3 response was observed in 8 arteries suggesting that a small ET_B population was also present in some patients. The selective ET_B receptor agonist sarafotoxin S6c had little or no effect up to 10 nM (n=4–6).
5. Responses to ET-1 and ET-3 were unaffected by removal of the endothelium in arteries from both groups suggesting a lack of functional, relaxant ET_B receptors on endothelial cells (n=5).
6. Using autoradiography, specific high density binding of the non-selective, ET_A/ET_B ligand [¹²⁵I]-ET-1 and selective ET_A ligand [¹²⁵I]-PD151242 was detected on the vascular smooth muscle layer of small intramyocardial coronary arteries (n=5). In contrast, little or no binding of the selective ET_B receptor ligand [¹²⁵I]-BQ3020 was observed (n=5). Similarly, [¹²⁵I]-ET-1 binding to vascular smooth muscle was absent in the presence of the selective ET_A receptor antagonist PD156707.
7. We conclude that human small epi- and intramyocardial coronary arteries express predominantly ET_A receptors and it is these receptors which mediate ET-induced contractions. A constrictor ET_B receptor population may exist in some patients. However, these receptors may have a limited role as contractions to ET-1 can be blocked fully by the selective ET_A receptor antagonist PD156707.

     

Pharmacological characterization of muscarinic receptors in the uterus of oestrogen-primed and pregnant rats

1. Radioligand binding and contractility studies were undertaken to determine the subtype/s of muscarinic receptors present in uteri of oestrogen-treated and late pregnant rats.
2. Competition binding studies with uterine membrane preparations and [³H]-QNB (quinuclidinyl benzilate) provided negative log dissociation constants (pK_i) for each antagonist as follows; oestrogen-treated – atropine (7.98)⩾himbacine (7.83)>methoctramine (7.52)⩾hexahydrosiladiphenidol (HHSiD; 7.32)⩾5,11-dihydro-11-[[[2-[2 - [(dipropylamino)methyl] - 1piperidinyl]ethyl]amino] - carbonyl] - 6H-pyrido- [2,3 - b][1,4] - benzodiazepin - 6-one (AF - DX 384; 7.10)>11 - [[2 - [(diethylamino)methyl]-1-piperidinyl]- acetyl]5,11-dihydro-6H-pyridol]2,3,-b][1,4]benzodiazepin-6-one (AF-DX 116, 6.77)>pirenzepine (6.17); late pregnant – atropine (8.05)⩾methoctramine (7.95)⩾himbacine (7.71)⩾HHSiD (7.52)⩾AF-DX 384 (7.34)>AF-DX 116 (6.72)>pirenzepine (6.18).
3. The potency of carbachol in causing uterine contraction was similar in preparations from pregnant and non-pregnant animals (pD₂=5.57 and 5.46, respectively). Each muscarinic antagonist caused parallel, rightward shifts of carbachol concentration-response curves. The pA₂ estimates were: oestrogen-treated – atropine (9.42)>himbacine (8.73)⩾HHSiD (8.68)⩾methoctramine (8.49)⩾AF-DX 384 (7.91)⩾AF-DX 116 (7.36)⩾pirenzepine (7.26); late pregnant – atropine (9.48)>himbacine (8.37)⩾HHSiD (8.22)⩾methoctramine (8.01)⩾AF-DX 116 (7.73)⩾AF-DX 384 (7.44)⩾pirenzepine (6.92).
4. The relative pK_i estimates for antagonists obtained in membrane preparations from oestrogen-treated rats suggest the presence of muscarinic M₂ subtypes. In functional studies pA₂ values indicated the additional presence of muscarinic M₃ receptor or, possibly an atypical receptor subtype. The similarity between pK_i and pA₂ estimates obtained in uteri from oestrogen-treated and pregnant animals, respectively, indicates that pregnancy does not affect myometrial muscarinic receptors in the rat.

     

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.

     

Identification of endothelial H1, vascular H2 and cardiac presynaptic H3 receptors in the pithed rat

Summary In pithed and vagotomized rats the effects of the H₃ receptor agonist R-(–)--methylhistamine, the H₁ receptor agonist 2-(2-thiazolyl)ethylamine and the H₂ receptor agonist dimaprit on basal diastolic blood pressure, basal heart rate and the electrically induced rise in heart rate were examined.Basal diastolic blood pressure was not altered by low, but increased by high doses of R-(–)--methylhistamine; the latter effect was not affected by selective H₁, H₂ or H₃ receptor antagonists and by prazosin, but was attenuated by rauwolscine. Rauwolscine also unmasked a vasodepressor response to R-(–)--methylhistamine not affected by the H₃ receptor antagonist thioperamide, but counteracted by the H₁ receptor antagonist dimetindene or the H₂ receptor antagonist ranitidine. The vasodepressor responses to 2-(2-thiazolyl)ethylamine and dimaprit were antagonized by dimetindene and ranitidine, respectively. The vasodepressor response to 2-(2-thiazolyl)ethylamine was not altered by indomethacin, but reduced by an inhibitor of endothelial nitric oxide synthase, N-nitro-L-arginine methyl ester (which, by itself, markedly increased blood pressure). Both drug tools did not alter the effect of dimaprit. Basal heart rate was not affected by 2-(2-thiazolyl)ethylamine (examined after administration of propranolol), dimaprit and R-(–)--methylhistamine. The electrically induced increase in heart rate (studied in animals which had received rauwolscine) was decreased by R-(–)--methylhistamine but not affected by 2-(2-thiazolyl)ethylamine and dimaprit. The effect of R-(–)--methylhistamine was abolished by thioperamide. R-(–)--methylhistamine did not influence the increase in heart rate produced by isoprenaline.In conclusion, the pithed rat offers the opportunity to study cardiac presynaptic H₃ receptors, endothelial H₁ receptors and vascular H₂ receptors in the same experimental model. Cardiac presynaptic H₁ and H₂ receptors as well as postsynaptic H₃ receptors in the heart and in the resistance vessels were not found. R-(–)--methylhistamine is a weak agonist at ₂, H₁ and H₂ receptors.Correspondence to E. Schlicker at the above address