Pharmacological profile of a novel cyclic AMP-linked P2 receptor on undifferentiated HL-60 leukemia cells

1. Extracellular ATP (EC₅₀=146±57 μM) and various ATP analogues activated cyclic AMP production in undifferentiated HL-60 cells.
2. The order of agonist potency was: ATPγS (adenosine 5′-O-[3-thiotriphosphate])⩾BzATP (2′&3′O-(4-benzoylbenzoyl)-adenosine-5′-triphosphate)⩾dATP>ATP. The following agonists (in order of effectiveness at 1 mM) were all less effective than ATP at concentrations up to 1 mM: β,γ methylene ATP⩾2-methylthioATP>ADP⩾Ap4A (P¹, P⁴-di(adenosine-5′) tetraphosphate)⩾Adenosine>UTP. The poor response to UTP indicates that P2Y₂ receptors are not responsible for ATP-dependent activation of adenylyl cyclase.
3. Several thiophosphorylated analogs of ATP were more potent activators of cyclic AMP production than ATP. Of these, ATPγS (EC₅₀=30.4±6.9 μM) was a full agonist. However, adenosine 5′-O-[1-thiotriphosphate] (ATPαS; EC₅₀=45±15 μM) and adenosine 5′-O-[2-thiodiphosphate] (ADPβS; EC₅₀=33.3±5.0 μM) were partial agonists.
4. ADPβS (IC₅₀=146±32 μM) and adenosine 5′-O-thiomonophosphate (AMPS; IC₅₀=343±142 μM) inhibited cyclic AMP production by a submaximal concentration of ATP (100 μM). Consistent with its partial agonist activity, ADPβS was estimated to maximally suppress ATP-induced cyclic AMP production by about 65%. AMPS has not been previously reported to inhibit P2 receptors.
5. The broad spectrum P2 receptor antagonist, suramin (500 μM), abolished ATP-stimulated cyclic AMP production by HL-60 cells but the adenosine receptor antagonists xanthine amine congener (XAC; 20 μM) and 8-sulpho-phenyltheophylline (8-SPT; 100 μM) were without effect.
6. Extracellular ATP also activated protein kinase A (PK-A) consistent with previous findings that PK-A activation is involved in ATP-induced differentiation of HL-60 cells (Jiang et al., 1997).
7. Taken together, the data indicate the presence of a novel cyclic AMP-linked P2 receptor on undifferentiated HL-60 cells.

     

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.

     

Effects of adrenomedullin and calcitonin gene-related peptide on contractions of the rat aorta and porcine coronary artery

1. Effects of adrenomedullin and α-calcitonin gene-related peptide (CGRP) on the contractions and cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) of the rat aorta and porcine coronary artery were investigated. Characteristics of the receptors mediating the effects of adrenomedullin and α-CGRP were also investigated.
2. Adrenomedullin and α-CGRP caused a concentration-dependent relaxation in the rat aorta contracted with noradrenaline. The IC₅₀ values for adrenomedullin and α-CGRP were 2.4 nM and 4.0 nM, respectively. The relaxant effects of these peptides were abolished by removal of the endothelium and significantly attenuated by an inhibitor of nitric oxide synthase, N^G-monomethyl-L-arginine (L-NMMA, 100 μM), but not by a cyclo-oxygenase inhibitor, indomethacin (10 μM).
3. Adrenomedullin and α-CGRP increased the endothelial [Ca²⁺]_i in the rat aorta with endothelium, whereas they did not change [Ca²⁺]_i in the smooth muscle.
4. An antagonist of the CGRP₁ receptor, CGRP (8–37), antagonized the relaxant effects of α-CGRP and the β-isoform of CGRP (β-CGRP) but not those of adrenomedullin in the rat aorta.
5. In the porcine coronary artery contracted with U46619, adrenomedullin and α-CGRP caused a concentration-dependent relaxation with an IC₅₀ of 27.6 and 4.1 nM, respectively. Removal of the endothelium altered neither the IC₅₀ values nor the maximal relaxations induced by adrenomedullin or α-CGRP. When the artery was contracted with high K⁺ solution (72.7 mM), these peptides caused a small relaxation.
6. Adrenomedullin and α-CGRP increased cyclic AMP content and decreased the smooth muscle [Ca²⁺]_i in the porcine coronary artery.
7. CGRP (8–37) significantly antagonized the relaxant effects of adrenomedullin and α-CGRP in the porcine coronary artery. However, it had little effect on the relaxations induced by the β-isoform of CGRP (β-CGRP).
8. These results suggest that in the rat aorta, adrenomedullin and α-CGRP increase the endothelial [Ca²⁺]_i, activate nitric oxide synthase and release nitric oxide, without a direct inhibitory action on smooth muscle. In the porcine coronary artery, in contrast, adrenomedullin and α-CGRP directly act on smooth muscle, increase cyclic AMP content, decrease the smooth muscle [Ca²⁺]_i and inhibit contraction. The rat aortic endothelium seems to express the CGRP receptor which is sensitive to α-CGRP, β-CGRP and CGRP (8–37) and the adrenomedullin specific receptor. The porcine coronary smooth muscle, in contrast, seems to express two types of CGRP receptor; one of which is sensitive to α-CGRP, CGRP (8–37) and adrenomedullin and the other is sensitive only to β-CGRP.

     

Characterization of the Ca2+ responses evoked by ATP and other nucleotides in mammalian brain astrocytes

1. This study was aimed at characterizing ATP-induced rises in cytosolic free calcium ion, [Ca²⁺]_i, in a population of rat striatal astrocytes loaded with the fluorescent Ca²⁺ probe Fura2, by means of fluorescence spectrometry.
2. ATP triggered a fast and transient elevation of [Ca²⁺]_i in a concentration-dependent manner. The responses of the purine analogues 2-methylthio-ATP (2-meSATP), adenosine-5′-O-(2-thiodiphosphate) (ADPβS), as well as uridine-5′-triphosphate (UTP) resembled that of ATP, while α,β-methylene-ATP (α,β-meATP) and β,γ-methylene-ATP (β,γ-meATP) were totally ineffective.
3. Suramin (50 μM) had only a minor effect on the ATP response, whereas pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (5 μM) significantly depressed the maximum response.
4. Extracellular Ca²⁺ did not contribute to the observed [Ca²⁺]_i rise: removing calcium from the extracellular medium (with 1 mM EGTA) or blocking its influx by means of either Ni²⁺ (1 mM) or Mn²⁺ (1 mM) did not modify the nucleotide responses.
5. Furthermore, after preincubation with 10 μM thapsigargin, the nucleotide-evoked [Ca²⁺]_i increments were completely abolished. In contrast, 10 mM caffeine did not affect the responses, suggesting that thapsigargin-, but not caffeine/ryanodine-sensitive stores are involved.
6. Both application of the G-protein blocker guanosine-5′-O-(2-thiodiphosphate) (GDPβS) (1 mM) and preincubation with pertussis toxin (PTx) (350 ng ml⁻¹) partially inhibited the nucleotide-mediated responses. Moreover, the phospholipase C (PLC) inhibitor U-73122, but not its inactive stereoisomer U-73343 (5 μM), significantly reduced the ATP-evoked [Ca²⁺]_i rise.
7. In conclusion, our results suggest that, in rat striatal astrocytes, ATP-elicited elevation of [Ca²⁺]_i is due solely to release from intracellular stores and is mediated by a G-protein-linked P2Y receptor, partially sensitive to PTx and coupled to PLC.

     

The effect of pretreatment with a δ2-opioid receptor antisense oligodeoxynucleotide on the recovery from acute antinociceptive tolerance to δ2-opioid receptor agonist in the mouse spinal cord

1. An intrathecal (i.t.) injection of a selective δ₂-opioid receptor agonist, [D-Ala²]deltorphin II, produced an acute antinociceptive tolerance to the antinociceptive effect of a subsequent i.t. challenge of [D-Ala²]deltorphin II. This acute tolerance lasted 3 to 9 h and completely subsided by 12 h. The experiments were designed to examine the effect of pretreatment with an antisense oligodeoxynucleotide to δ₂-opioid receptor mRNA (δ-AS oligo) on the recovery from tolerance to [D-Ala²]deltorphin II-induced antinociception in male ICR mice.
2. Pretreatment with δ-AS oligo (1.63 to 163 pmol, i.t.), but not mismatched oligo (MM oligo) (163 pmol), prevented the recovery from acute tolerance to [D-Ala²]deltorphin II-induced antinociception in a dose-dependent manner. However, treatment with δ-AS oligo (163 pmol) did not prevent the recovery from tolerance to either the μ-opioid receptor agonist [D-Ala²,NMePhe⁴,Gly(ol)⁵]enkephalin (DAMGO) or the κ-opioid receptor agonist U50,488H, indicating subtype specificity in the mechanism by which δ-AS oligo inhibits recovery from δ₂-opioid tolerance.
3. Treatment with [D-Ala²]deltorphin II (i.t.) significantly reduced the binding of [tyrosyl-3,5-³H(N)]-Tyr-D-Ser-Gly-Phe-Leu-Thr ([³H]-DSLET), a δ₂-opioid receptor agonist ligand, in the spinal cord 3 h after treatment, but binding returned to control levels by 24 h after treatment. However, [³H]-DSLET binding in the spinal cord remained significantly reduced at 24 h if δ-AS oligo (163 pmol) was coadministered with [D-Ala²]deltorphin II (6.4 nmol).
4. Based on these findings, it is concluded that a single stimulation of spinal cord δ₂-opioid receptors by intrathecally-administered [D-Ala²]deltorphin II induces a long-lasting desensitization of δ₂-opioid receptors to [D-Ala²]deltorphin II. Recovery from δ₂-opioid receptor-mediated antinociceptive tolerance apparently depends on replenishment by newly synthesized δ₂-opioid receptor protein rather than immediate reversal of δ₂-opioid receptors.

     

Involvement of bradykinin B1 and B2 receptors in relaxation of mouse isolated trachea

1. The aim of the present study was to investigate the effects of bradykinin and [des-Arg⁹]-bradykinin and their relaxant mechanisms in the mouse isolated trachea.
2. In the resting tracheal preparations with intact epithelium, bradykinin and [des-Arg⁹]-bradykinin (each drug, 0.01–10 μM) induced neither contraction nor relaxation. In contrast, bradykinin (0.01–10 μM) induced concentration-dependent relaxation when the tracheal preparations were precontracted with methacholine (1 μM). The relaxation induced by bradykinin was inhibited by the B₂ receptor antagonist, D-Arg⁰-[Hyp³,Thi⁵,D-Tic⁷,Oic⁸]-bradykinin (Hoe 140, 0.01–1 μM) in a concentration-dependent manner whereas the B₁ receptor antagonist, [des-Arg⁹,Leu⁸]-bradykinin (0.01–1 μM), had no inhibitory effect on bradykinin-induced relaxation. [des-Arg⁹]-bradykinin (0.01–10 μM) also caused concentration-dependent relaxation after precontraction with methacholine. The relaxation induced by [des-Arg⁹]-bradykinin was concentration-dependently inhibited by the B₁ receptor antagonist, [des-Arg⁹,Leu⁸]-bradykinin (0.01–1 μM), whereas the B₂ receptor antagonist, Hoe 140 (0.01–1 μM) was without effect.
3. In the presence of the cyclo-oxygenase inhibitor, indomethacin (0.01–1 μM), the relaxations induced by bradykinin and [des-Arg⁹]-bradykinin were inhibited concentration-dependently.
4. Two nitric oxide (NO) biosynthesis inhibitors N^G-nitro-L-arginine methyl ester (L-NAME, 100 μM) and N^G-nitro-L-arginine (L-NOARG, 100 μM) had no inhibitory effects on the relaxations induced by bradykinin and [des-Arg⁹]-bradykinin. Neither did the selective inhibitor of the soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 μM) inhibit the relaxations induced by bradykinin and [des-Arg⁹]-bradykinin.
5. Prostaglandin E₂ (PGE₂, 0.01–33 μM) caused concentration-dependent relaxation of the tracheal preparations precontracted with methacholine. Indomethacin (1 μM) and ODQ (10 μM) exerted no inhibitory effects on the relaxation induced by PGE₂.
6. The NO-donor, sodium nitroprusside (SNP; 0.01–100 μM) also caused concentration-dependent relaxation of the tracheal preparations precontracted with methacholine. ODQ (0.1–1 μM) concentration-dependently inhibited the relaxation induced by SNP.
7. These data demonstrate that bradykinin and [des-Arg⁹]-bradykinin relax the mouse trachea precontracted with methacholine by the activation of bradykinin B₂-receptors and B₁-receptors, respectively. The stimulation of bradykinin receptors induces activation of the cyclo-oxygenase pathway, leading to the production of relaxing prostaglandins. The NO pathway is not involved in the bradykinin-induced relaxation. The relaxation caused by NO-donors in the mouse trachea is likely to be mediated via activation of soluble guanylate cyclase.

     

Differential activation of the epithelial and smooth muscle NK1 receptors by synthetic tachykinin agonists in guinea-pig trachea

1. The presence of tachykinin NK₁ receptors have been shown in the epithelium and smooth muscle of guinea-pig airways. Previous data showed that substance P (SP), and the NK₁ receptor agonist, [Sar⁹, Met (O₂)¹¹]-SP, relax guinea-pig tracheal tube preparations by stimulation of epithelial NK₁ receptors and via nitric oxide (NO) release. However, the selective tachykinin NK₁ receptor agonist, septide, was unable to produce this effect. The aim of the present study was to investigate the ability of a series of SP analogues to stimulate NK₁ receptors of guinea-pig airway epithelium.
2. Isometric tension was recorded in isolated tracheal tube preparations in which compounds were administered intraluminally in the presence of phosphoramidon, indomethacin (both 1 μM) and the tachykinin NK₂ receptor antagonist, SR 48,968 ((S)-N-methyl N-(4-acetyl-amino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl)benzamide) (0.1 μM). Cumulative concentration-response curves were obtained in preparations under resting tone or in preparations precontracted with acetylcholine (ACh, 10 μM).
3. Contractile responses to low concentrations (0.1–10 nM) of substance P (SP) and the selective agonist of NK₁ receptors, [Pro⁹]-SP, in non precontracted tracheae were higher in preparations pretreated with the NO-synthase inhibitor, N^G-monomethyl L-arginine (L-NMMA, 100 μM) than in preparations pretreated with its inactive enantiomer D-NMMA (100 μM). Tracheal tube preparations precontracted with ACh and pretreated with D-NMMA were relaxed by low concentrations of SP and [Pro⁹]-SP (0.1–10 nM). In contrast, after pretreatment with L-NMMA, SP and [Pro⁹]-SP contracted tracheae at all the concentrations tested.
4. Concentration-response curves to the NK₁ receptor agonists, SP methyl ester, [Apa^9–10]-SP and [pGlu⁶] SP (6–11) obtained in non-precontracted tracheae were similar in the presence of either D-NMMA or L-NMMA. SP methyl ester, [Apa^9–10]-SP and [pGlu⁶] SP (6–11) did not produce any relaxation, but instead, cause contractions in tracheal tube preparations precontracted with ACh and pretreated with D-NMMA. Concentration-response curves produced by all these agonists were similar in preparations precontracted with ACh and pretreated with L-NMMA or D-NMMA.
5. In guinea-pig tracheal tube preparations two groups of NK₁ receptor agonists can be distinguished: one group, including [Pro⁹]-SP, stimulator epithelial NK₁ receptors, the other group, including SP methyl ester, [Apa^9–10]-SP and [pGlu⁶] SP (6–11), does not. One possible explanation for these findings and for the existence of compounds with a peculiar ‘septide-like'' pharmacological profile in the guinea-pig trachea could be the recently proposed phenomenon referred to as ‘agonist-directed receptor trafficking''.

     

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.

     

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.

     

Characterization of the P2 receptors on the human umbilical vein endothelial cell line ECV304

1. To characterize the P2 receptors present on the human umbilical vein endothelial-derived cell line, ECV304, cytosolic Ca²⁺, ([Ca²⁺]_c), responses were recorded in single cells and in cell suspensions to a series of nucleotides and nucleotide agonists.
2. Concentration response curves were obtained in fura-2-loaded ECV304 cell suspensions, with EC₅₀ values of 4.2 μM for ATP, 2.5 μM for UTP and 14 μM for adenosine-5′-O-(3-thio)triphosphate (ATPγS). EC₅₀ values for 2-methylthioATP, ADP, adenosine-5′-O-(2-thio)diphosphate (ADPβS) and AMP were 0.5 μM, 3.5 μM, 15 μM and 4.7 μM respectively, but maximal [Ca²⁺]_c responses were less than those produced by a maximal addition of ATP/UTP. ECV304 cells were unresponsive to UDP and β,γ,methyleneATP.
3. Cross-desensitization studies on ECV304 cells suggested that ATP and UTP recognized the same receptor. However, ADP recognized a receptor distinct from the UTP-sensitive receptor and AMP recognized a third distinct receptor.
4. ECV304 [Ca²⁺]_c responses to 2-methylthioATP were inhibited in the presence of 30 μM pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), whereas [Ca²⁺]_c responses to UTP were unaffected by this treatment.
5. ECV304 cells responded to the diadenosine polyphosphate Ap₃A with rises in [Ca²⁺]_c. Apparent responses to Ap₄A, Ap₅A and Ap₆A, were shown to be due to a minor nucleotide contaminant that could be removed by pre-treatment of the diadenosine samples with either alkaline phosphatase or apyrase.
6. ECV304 cells display a pharmacology consistent with the presence of at least two P2 receptors; a P2Y₂ receptor insensitive to the diadenosine polyphosphates and a P2Y₁ receptor sensitive to Ap₃A. In addition, ECV304 cells respond to AMP with increases in [Ca²⁺]_c via an as yet uncharacterized receptor.

     

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.

     

Potentiation of adenosine A1 receptor-mediated inositol phospholipid hydrolysis by tyrosine kinase inhibitors in CHO cells

1. The effect of protein tyrosine kinase inhibitors on human adenosine A₁ receptor-mediated [³H]-inositol phosphate ([³H]-IP) accumulation has been studied in transfected Chinese hamster ovary cells (CHO-A1) cells.
2. In agreement with our previous studies the selective adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA) stimulated the accumulation of [³H]-IPs in CHO-A1 cells. Pre-treatment with the broad spectrum tyrosine kinase inhibitor genistein (100 μM; 30 min) potentiated the responses elicited by 1 μM (199±17% of control CPA response) and 10 μM CPA (234±15%). Similarly, tyrphostin A47 (100 μM) potentiated the accumulation of [³H]-IPs elicited by 1 μM CPA (280±32%).
3. Genistein (EC₅₀=13.7±1.2 μM) and tyrphostin A47 (EC₅₀=10.4±3.9 μM) potentiated the [³H]-IP response to 1 μM CPA in a concentration-dependent manner.
4. Pre-incubation with the inactive analogues of genistein and tyrphostin A47, daidzein (100 μM; 30 min) and tyrphostin A1 (100 μM; 30 min), respectively, had no significant effect on the accumulation of [³H]-IPs elicited by 1 μM CPA.
5. Genistein (100 μM) had no significant effect on the accumulation of [³H]-IPs produced by the endogenous thrombin receptor (1 u ml⁻¹; 100±10% of control response). In contrast, tyrphostin A47 produced a small augmentation of the thrombin [³H]-IP response (148±13%).
6. Genistein (100 μM) had no effect on the [³H]-IP response produced by activation of the endogenous G_q-protein coupled CCK_A receptor with the sulphated C-terminal octapeptide of cholecystokinin (1 μM CCK-8; 96±6% of control). In contrast, tyrphostin A47 (100 μM) caused a small but significant increase in the response to 1 μM CCK-8 (113±3% of control).
7. The phosphatidylinositol 3-kinase inhibitor LY 294002 (30 μM) and the MAP kinase kinase inhibitor PD 98059 (50 μM) had no significant effect on the [³H]-IP responses produced by 1 μM CPA and 1 μM CCK-8.
8. These observations suggest that a tyrosine kinase-dependent pathway may be involved in the regulation of human adenosine A₁ receptor mediated [³H]-IP responses in CHO-A1 cells.

     

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.

     

Cyclothiazide and AMPA receptor desensitization: analyses from studies of AMPA-induced release of [3H]-noradrenaline from hippocampal slices

1. Responses in brain produced by the activation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype of ionotropic receptor for L-glutamate are often rapidly desensitizing. AMPA-induced desensitization and its characteristics, and the potentiating effect of cyclothiazide were investigated in vitro by analysing AMPA-induced release of [³H]-noradrenaline from prisms of rat hippocampus.
2. AMPA (1–1000 μM) stimulated the release of [³H]-noradrenaline in a concentration-dependent manner that was both calcium-dependent and tetrodotoxin-sensitive, and attenuated by the AMPA-selective antagonists, NBQX (1 and 10 μM), LY 293558 (1 and 10 μM) and GYKI 52466 (10 and 30 μM).
3. By use of an experimental procedure with consecutive applications of AMPA (100 μM, 28 min apart), the second response was reduced, indicative of receptor desensitization, and was reversed by cyclothiazide in a concentration-dependent manner (1–300 μM). The concentration-response curve for AMPA-induced release of [³H]-noradrenaline was shifted leftwards, but the reversal by cyclothiazide of the desensitized response was partial and failed to reach the maximal response of the first stimulus.
4. Observations made with various schedules of cyclothiazide application indicated that the initial AMPA-evoked response was already partially desensitized (150% potentiation by 100 μM cyclothiazide) and that the desensitization was not likely to be due to a time-dependent diminution and was long-lasting (second application of cyclothiazide was ineffective).
5. Co-application of a number of drugs with actions on second messenger systems, in association with the second AMPA stimulus, revealed significant potentiation of the AMPA-induced release of [³H]-noradrenaline: forskolin (10 μM, +78%), Rp-cAMPS (100 μM, +65%), Ro 31-8220 (10 μM, + 163%) and thapsigargin (100 μM, +161%).
6. The AMPA receptor-mediated response regulating the release of [³H]-noradrenaline from rat hippocampal slices was desensitized and cyclothiazide acted to reverse partially the desensitization in a concentration-dependent manner. Since the time-course of desensitization was longer lasting than that noted in previous electrophysiological studies, multiple events may be involved in the down-regulation of AMPA receptor activity including receptor phosphorylation and depletion of intracellular Ca²⁺ stores.

     

Modulation of spasmogen-stimulated Ins(1,4,5)P3 generation and functional responses by selective inhibitors of types 3 and 4 phosphodiesterase in airways smooth muscle

1. The effects of isoenzyme-selective inhibitors of phosphodiesterases PDE3 and PDE4 on cyclic AMP concentration, two indices of phosphoinositide hydrolysis, and contractile responses to spasmogens have been investigated in bovine tracheal smooth muscle (BTSM).
2. Neither the PDE3-selective inhibitor ORG 9935, nor the PDE4-selective inhibitor rolipram increased cyclic AMP levels in BTSM. However, rolipram addition in the presence of PDE3 inhibition (ORG 9935; 1 μM) concentration-dependently (−log EC₅₀ (M), 6.55±0.15; n=3) increased cyclic AMP levels to about 70% of the maximal response to the β-adrenoceptor agonist isoprenaline.
3. Rolipram per se inhibited histamine-stimulated [³H]-inositol (poly)phosphate ([³H]-InsP_X) accumulation by >80% (−log EC₅₀ (M), 6.92±0.11; n=3). Although ORG 9935 (1 μM) had little effect on histamine-stimulated [³H]-InsP_X accumulation alone it greatly facilitated the inhibitory action of rolipram (−log EC₅₀ (M), 8.82±0.39; n=3). The effects of PDE3 and/or PDE4 inhibition on [³H]-InsP_X accumulation stimulated by muscarinic acetylcholine (mACh) receptor activation were less marked. However, combined PDE3/4 inhibition significantly decreased this response at a submaximal concentration of mACh receptor agonist (carbachol; 1 μM).
4. The greater-than-additive effect of combined PDE3/4 inhibition was also observed at the level of contractile responses to histamine and carbachol. In experiments designed to investigate the effects of PDE3 and/or 4 inhibitors on the carbachol-mediated phasic contraction, additions of rolipram (10 μM) or ORG 9935 (1 μM) were without effect, whereas added together the inhibitors caused a significant (P<0.01) 40% reduction in the peak phasic contractile response.
5. The effect on contraction correlated with a substantial inhibitory effect of PDE3/4 inhibition on the initial increase in inositol 1,4,5-trisphosphate (InsP₃) accumulation stimulated by spasmogen. Thus, in the presence of ORG 9935 (1 μM) rolipram concentration-dependently inhibited carbachol-stimulated InsP₃ accumulation by ⩾50% (−log EC₅₀ (M), 6.77±0.21; n=4).
6. Carbachol (100 μM) addition caused a rapid decrease (by 67% at 10 s) in BTSM cyclic AMP level in the presence of PDE3/4 inhibition. However, omission of Ca²⁺ from the incubation medium prevented the carbachol-evoked decrease in cyclic AMP and this coincided with a greater inhibition (⩾80%) of the carbachol-stimulated InsP₃ response.
7. These data indicate that combined PDE3 and PDE4 inhibition has greater-than-additive effects on second messenger and functional responses to spasmogens in BTSM. Furthermore, the ability of PDE3/4 inhibition significantly to attenuate mACh receptor-mediated contractile responses, may be, at least in part, attributed to an effect exerted at the level of InsP₃ generation.

     

Effect of oxytocin as a partial agonist at vasoconstrictor vasopressin receptors on the human isolated uterine artery

• The effect of oxytocin on endothelium-intact and endothelium-denuded segments of the human uterine artery rings was investigated.
• In both types of preparation oxytocin induced contraction of human uterine artery with similar potency and efficacy (pEC₅₀ values: 6.95±0.05 vs 7.06±0.01; maximal response values: 61±4.1% vs 63±5.1% for arteries with and without endothelium, respectively).
• In contrast, human uterine arteries, both intact and denuded of endothelium, did not respond to the addition of the selective oxytocin receptor agonist, [Thr⁴, Gly⁷]oxytocin (10 nM–1 μM).
• The vasopressin receptor antagonists, [d(CH₂)₅Tyr(Me)]AVP (10–100 nM) and [d(CH₂)₅,D-Ile²,Ile⁴]AVP (300 nM–3 μM) produced parallel rightward shifts of the curves for oxytocin. The Schild plots constrained to a slope of unity gave the following −log K_B values: [d(CH₂)₅Tyr(Me)] AVP vs [d(CH₂)₅,D-Ile²,Ile⁴] AVP 9.24 vs 6.91 and 9.26 vs 6.84 for human uterine artery with intact and those denuded of endothelium, respectively. In contrast, in both types of preparations the oxytocin receptor antagonist, [d(CH₂)₅Tyr(OMe), ²Orn⁸]vasotocin (1 μM), did not significantly affect oxytocin-induced contractions.
• The calculated pK_A values for oxytocin itself also did not differ between preparations: 6.56 and 6.43 for human uterine artery with and without endothelium, respectively. In both types of preparations, the receptor reserve (K_A/EC₅₀) was close to unity (intact vs denuded: 3.9 vs 3.0).
• It is concluded that, in human uterine artery, oxytocin induces contractions that are not modulated by the endothelium. It is likely that oxytocin acts as a partial agonist on human uterine artery, regardless of the endothelial condition. On the basis of differential antagonists affinity and affinity of oxytocin itself, it is probable that receptors involved in oxytocin-induced contraction in human uterine arteries belong to the V_1A vasopressin receptors.

     

A1 and A2 adenosine receptor modulation of contractility in the cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon phenylephrine-stimulated contractility and [³H]-cyclic adenosine monophosphate ([³H]-cyclic AMP) accumulation in the cauda epididymis of the guinea-pig were investigated. The α₁-adrenoceptor agonist, phenylephrine elicited concentration dependent contractile responses from preparations of epididymis. In the absence or presence of the L-type Ca²⁺ channel blocker, nifedipine (10 μM) the non-selective adenosine receptor agonist, 5′-N-ethylcarboxamido-adenosine (NECA, 1 μM) shifted phenylephrine concentration-response curves to the left (4 and 5 fold respectively). Following the incubation of preparations with pertussis toxin (200 ng ml⁻¹ 24 h) NECA shifted phenylephrine concentration-response curves to the right (5.7±0.9 fold).
2. In the presence of phenylephrine (1 μM), NECA and the A₁ adenosine receptor selective agonists, N⁶-cyclopentyladenosine (CPA) and (2S)-N⁶-[2-endo-norbornyl]adenosine ((S)-ENBA) elicited concentration-responses dependent contractions from preparations of epididymis (pEC₅₀ values 8.18±0.19, 7.79±0.29 and 8.15±0.43 respectively). The A₃ adenosine receptor agonists N⁶-iodobenzyl-5′-N-methyl-carboxamido adenosine (IBMECA) and N⁶-2-(4-aminophenyl) ethyladenosine (APNEA) mimicked this effect (but only at concentrations greater than 10 μM). In the presence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 30 nM) CPA concentration-response curves were shifted, in parallel to the right (apparent pK_B 8.75±0.88) and the maximal response to NECA was reduced.
3. In the presence of DPCPX (100 nM) the adenosine agonist NECA and the A_2A adenosine receptor selective agonist, CGS 21680 (2-p-(2-carboxyethyl)-phenethylamino-N-ethylcarboxamido adenosine), but not CPA, inhibited phenylephrine (20 μM) stimulated contractions (pIC₅₀ 7.15±0.48). This effect of NECA was blocked by xanthine amine congener (XAC, 1 μM) and the A_2A adenosine receptor-selective antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; 30 nM).
4. (S)-ENBA (in the absence and presence of ZM 241385, 100 nM), but not NECA or CPA inhibited the forskolin (30 μM)-stimulated accumulation of [³H]-cyclic AMP in preparations of the epididymis of the guinea-pig (by 17±6% of control). In the presence of DPCPX (100 nM) NECA and CGS 21680, but not (S)-ENBA, increased the accumulation of [³H]-cyclic AMP in preparations of epididymis (pEC₅₀ values 5.35±0.35 and 6.42±0.40 respectively), the NECA-induced elevation of [³H]-cyclic AMP was antagonised by XAC (apparent pK_B 6.88±0.88) and also by the A_2A adenosine receptor antagonist, ZM 241385 (apparent pK_B 8.60± 0.76).
5. These studies are consistent with the action of stable adenosine analogues at post-junctional A₁ and A₂ adenosine receptors in the epididymis of the guinea-pig. A₁ Adenosine receptors potentiate α₁-adrenoceptor contractility, an effect blocked by pertussis toxin, but which may not be dependent upon an inhibition of adenylyl cyclase. The epididymis of the guinea-pig also contains A₂ adenosine receptors, possibly of the A_2A subtype, which both inhibit contractility and also stimulate adenylyl cyclase.

     

Regulation of phosphoinositide turnover in neonatal rat cerebral cortex by group I- and II- selective metabotropic glutamate receptor agonists

1. The interactive effects of different metabotropic glutamate (mGlu) receptor subtypes to regulate phosphoinositide turnover have been studied in neonatal rat cerebral cortex and hippocampus by use of agonists and antagonists selective between group I and II mGlu receptors.
2. The group II-selective agonist 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC; 100 μM) had no effect on basal total inositol phosphate ([³H]-InsP_x) accumulation (in the presence of Li⁺) in myo-[³H]-inositol pre-labelled slices, but enhanced the maximal [³H]-InsP_x response to the group I-selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) by about 100% in both hippocampus and cerebral cortex. In cerebral cortex the enhancing effect of 2R,4R-APDC occurred with respect to the maximal responsiveness and had no effect on EC₅₀ values for DHPG (-log EC₅₀ (M): control, 5.56±0.05; +2R,4R-APDC, 5.51±0.08). 2R,4R-APDC also caused a significant enhancement of the DHPG-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) mass response over an initial 0–300 s time-course.
3. The enhancing effects of 2R,4R-APDC on DHPG-stimulated [³H]-InsP_x accumulation were observed in both the presence and nominal absence of extracellular Ca²⁺, and irrespective of whether 2R,4R-APDC was added before, simultaneous with, or subsequent to DHPG. Furthermore, increasing the tissue cyclic AMP concentration up to 100 fold had no effect on DHPG-stimulated Ins(1,4,5)P₃ accumulation in the absence or presence of 2R,4R-APDC.
4. 2R,4R-APDC and (2S, 1′R, 2′R, 3′R)-2-(2,3-dicarboxylcyclopropyl)glycine (DCG-IV), the latter agent in the presence of MK-801 to prevent activation of NMDA-receptors, each inhibited forskolin-stimulated cyclic AMP accumulation by about 50%, with respective EC₅₀ values of 1.3 and 0.04 μM (-log EC ₅₀ (M): 2R,4R-APDC, 5.87±0.09; DCG-IV, 7.38±0.05). In the presence of DHPG (30 μM), 2R,4R-APDC and DCG-IV also concentration-dependently increased [³H]-InsP_x accumulation with respective EC₅₀ values of 4.7 and 0.28 μM (-log EC₅₀ (M): 2R,4R-APDC, 5.33±0.04; DCG-IV, 6.55±0.09) which were 3–7 fold rightward-shifted relative to the adenylyl cyclase inhibitory responses.
5. The group II-selective mGlu receptor antagonist {"type":"entrez-nucleotide","attrs":{"text":"LY307452","term_id":"1257780057","term_text":"LY307452"}}LY307452 (30 μM) caused parallel rightward shifts in the concentration-effect curves for inhibition of forskolin-stimulated adenylyl cyclase, and enhancement of DHPG-stimulated [³H]-InsP_x accumulation, by 2R,4R-APDC yielding similar equilibrium dissociation constants (K_ds, 3.7±1.1 and 4.1±0.4 μM respectively) for each response.
6. The ability of 2R,4R-APDC to enhance receptor-mediated [³H]-InsP_x accumulation appeared to be agonist-specific; thus although DHPG (100 μM) and the muscarinic cholinoceptor agonist carbachol (10 μM) stimulated similar [³H]-InsP_x accumulations, only the response to the former agonist was enhanced by co-activation of group II mGlu receptors.
7. These data demonstrate that second messenger-generating phosphoinositide responses stimulated by group I mGlu receptors are positively modulated by co-activation of group II mGlu receptors in cerebral cortex and hippocampus. The data presented here are discussed with respect to the possible mechanisms which might mediate the modulatory activity, and the physiological and pathophysiological significance of such crosstalk between mGlu receptors.

     

Heterologous expression of rat epitope-tagged histamine H2 receptors in insect Sf9 cells

1. Rat histamine H₂ receptors were epitope-tagged with six histidine residues at the C-terminus to allow immunological detection of the receptor. Recombinant baculoviruses containing the epitope-tagged H₂ receptor were prepared and were used to infect insect Sf9 cells.
2. The His-tagged H₂ receptors expressed in insect Sf9 cells showed typical H₂ receptor characteristics as determined with [¹²⁵I]-aminopotentidine (APT) binding studies.
3. In Sf9 cells expressing the His-tagged H₂ receptor histamine was able to stimulate cyclic AMP production 9 fold (EC₅₀=2.1±0.1 μM) by use of the endogenous signalling pathway. The classical antagonists cimetidine, ranitidine and tiotidine inhibited histamine induced cyclic AMP production with K_i values of 0.60±0.43 μM, 0.25±0.15 μM and 28±7 nM, respectively (mean±s.e.mean, n=3).
4. The expression of the His-tagged H₂ receptors in infected Sf9 cells reached functional levels of 6.6±0.6 pmol mg⁻¹ protein (mean±s.e.mean, n=3) after 3 days of infection. This represents about 2×10⁶ copies of receptor/cell. Preincubation of the cells with 0.03 mM cholesterol-β-cyclodextrin complex resulted in an increase of [¹²⁵I]-APT binding up to 169±5% (mean±s.e.mean, n=3).
5. The addition of 0.03 mM cholesterol-β-cyclodextrin complex did not affect histamine-induced cyclic AMP production. The EC₅₀ value of histamine was 3.1±1.7 μM in the absence of cholesterol-β-cyclodextrin complex and 11.1±5.5 μM in the presence of cholesterol-β-cyclodextrin complex (mean±s.e.mean, n=3). Also, the amount of cyclic AMP produced in the presence of 100 μM histamine was identical, 85±18 pmol/10⁶ cells in the absence and 81±11 pmol/10⁶ cells in the presence of 0.03 mM cholesterol-β-cyclodextrin complex (mean±s.e.mean, n=3).
6. Immunofluorescence studies with an antibody against the His-tag revealed that the majority of the His-tagged H₂ receptors was localized inside the insect Sf9 cells, although plasma membrane labelling could be identified as well.
7. These experiments demonstrate the successful expression of His-tagged histamine H₂ receptors in insect Sf9 cells. The H₂ receptors couple functionally to the insect cell adenylate cyclase. However, our studies with cholesterol complementation and with immunofluorescent detection of the His-tag reveal that only a limited amount of H₂ receptor protein is functional. These functional receptors are targeted to the plasma membrane.