Effects of tachykinin receptor agonists and antagonists on the guinea-pig isolated oesophagus

1. Vagal nerve stimulation of the guinea-pig isolated oesophagus produced a triphasic tetrodotoxin (TTX)-sensitive contractile response. The third phase, which was resistant to ganglion blocking drugs, was selectively abolished by capsaicin, suggesting the involvement of one or more neuropeptides released from afferent neurons. Receptors on cholinergic neurons were subsequently activated because the response was atropine sensitive. Contractile responses resulting from exogenous substance P were abolished by atropine and TTX and enhanced by physostigmine. These findings suggest that the third phase may be mediated by the action of a substance P-like neuropeptide released from sensory nerve endings that subsequently activated cholinergic neurons. 2. The tachykinin receptors in the body of the guinea-pig oesophagus were characterized by determining the relative agonist potencies of natural tachykinins as well as tachykinin receptor-selective analogues. Antagonist affinities were also determined. The results indicated the presence of both NK2 and NK3 receptors. In addition, the effects of a cocktail of peptidase inhibitors (captopril, thiorphan and amastatin) on responses to various tachykinins and synthetic analogues were determined. The results indicate that one or more peptidases are present in this preparation. 3. Experiments using various tachykinin receptor antagonists were performed to determine whether the activation of tachykinin receptors played a role in the mediation of the third phase of the response to vagal nerve stimulation. While this response was unaffected by NK1 and NK2 receptor-selective antagonists, it was only partially inhibited (23%) by the NK3 receptor antagonist SR 142801. Thus, in the guinea-pig oesophagus, it appears that NK3 receptors play only a minor role in mediating a contractile response when afferent neurons are excited by vagal nerve stimulation.     

Characterization of receptors mediating contraction induced by tachykinins in the guinea-pig isolated common bile duct

1. We studied the effect of the natural tachykinins and of synthetic agonists selective for the tachykinin NK₁, NK₂ and NK₃ receptors, on the motility of guinea-pig isolated common bile duct longitudinally-oriented smooth muscle.
2. All the tachykinins tested (both natural and synthetic) produced a concentration-dependent contractile response of the guinea-pig isolated common bile duct: these effects underwent a marked tachyphylaxis, especially the responses elicited by NK₁ and NK₃ receptor-selective agonists.
3. Among the natural tachykinins neurokinin B (EC₅₀=3.2 nM; 95% c.l.=2.0–5.1; n=4) was the most potent, being about 40 and 25 fold more potent than substance P (EC₅₀=121.6 nM; 95% c.l.=94–157; P<0.01; n=4) and neurokinin A (EC₅₀=83.4 nM; 95% c.l.=62–112; P<0.01; n=4), respectively. Among the synthetic analogues the NK₃ receptor-selective agonist senktide (EC₅₀=1.1 nM; 95% c.l.=0.7–1.8; n=8) was the most potent, being about 120, 110 and 20 fold more potent than [Sar⁹]substance P sulfone (NK₁ receptor-selective) (EC₅₀=130.4 nM; 95% c.l.=99–172; P<0.01; n=8), [βAla⁸]NKA (4–10) (NK₂ receptor-selective) (EC₅₀=120.1 nM; 95% c.l.=95–151; P<0.01; n=8) and septide (NK₁ receptor-selective) (EC₅₀=22.6 nM; 95% c.l.=18–28; P<0.01; n=8), respectively. All tachykinins (natural or synthetic receptor agonists) produced a similar E_max, averaging about 50% of that produced by KCl (80 mM).
4. Atropine (1 μM) did not affect the responses to either NK₁ or NK₂ receptor-selective agonists, whereas it reduced the E_max of senktide by about 50%, without affecting its potency (EC₅₀). Tetrodotoxin (1 μM) totally blocked senktide-induced contractions, as did the combined pretreatment with atropine plus the tachykinin NK₁ and NK₂ receptor-selective antagonists GR 82334 and MEN 11420 (1 μM each), respectively.
5. GR 82334 (1 μM) blocked with apparent competitive kinetics septide- (apparent pK_B=7.46±0.10; n=5) and [Sar⁹]substance P sulfone- (apparent pK_B=6.80±0.04; n=4) induced contractions. MEN 11420 (30–300 nM), a novel potent NK₂ receptor antagonist, potently antagonized [βAla⁸]NKA (4–10), with competitive kinetics (pK_B=8.25±0.08; n=12: Schild plot slope=−0.90; 95% c.l.=−1.4; −0.35). The NK₃ receptor-selective antagonist SR 142801 (30 nM) produced insurmountable antagonism of the senktide-induced contractions (E_max inhibited by 64%). None of the above antagonists, tested at the highest concentrations employed against tachykinins, affected the concentration–response curve to methacholine (0.1–300 μM).
6. We conclude that tachykinins produce contraction of the guinea-pig isolated common bile duct by stimulating NK₁, NK₂ and NK₃ receptors. The responses obtained by activating NK₁ and NK₂ receptors are atropine-resistant. The contraction obtained by stimulating NK₃ receptors is totally neurogenic, being mediated by the release of endogenous acetylcholine and tachykinins; the latter act, in turn, on postjunctional tachykinin NK₁/NK₂ receptors. The role of the NK₃ receptor as prejunctional mediator of the excitatory transmission operated by tachykinins is discussed.

     

In vitro characterization of tachykinin NK2-receptors modulating motor responses of human colonic muscle strips

1. Human in vitro preparations of transverse or distal colonic circular smooth muscle were potently and dose-dependently contracted by neurokinin A (EC₅₀, 4.9 nM), the tachykinin NK₂-receptor selective agonist [β-Ala⁸]neurokinin A (4–10) ([β-Ala⁸]NKA (4–10)) (EC₅₀, 5.0 nM), neurokinin B (EC₅₀, 5.3 nM) and substance P (EC₅₀, 160 nM), but not by the tachykinin NK₁-receptor selective agonist [Sar⁹Met(O₂)¹¹] substance P, or the NK₃-receptor selective agonists, senktide and [MePhe⁷] neurokinin B. No regional differences between transverse and distal colon were observed in response to [β-Ala⁸]NKA (4–10).
2. Atropine (1 μM) and tetrodotoxin (1 μM) did not significantly inhibit responses to [β-Ala⁸]NKA (4–10), neurokinin A, substance P or neurokinin B.
3. The newly developed non-peptide antagonists for tachykinin NK₂-receptors SR 48968, SR 144190 and its N-demethyl (SR 144743) and N,N-demethyl (SR 144782) metabolites, were used to challenge agonist responses, as appropriate. SR 144190 and the metabolites all potently and competitively antagonized the response to [β-Ala⁸]NKA (4–10), with similar potency (Schild plot pA₂ values 9.4, 9.4 and 9.3, slope=1). SR 48968 antagonism was not competitive: the Schild plot slope was biphasic with a high (X intercept∼9.3) and a low (X intercept 8.4, slope 1.6) affinity site. Co-incubation of SR 48968 (10, 100 nM) and SR 144782 (10 nM) produced additive effects; in this experimental condition, SR 48968 apparent affinity (pK_B) was 8.2. In addition, SR 144782 (0.1 μM) antagonized responses to neurokinin A, substance P and neurokinin B, with pK_B consistent with its affinity for tachykinin NK₂-receptors. The potent and selective NK₁ and NK₃-receptor antagonists, SR 140333 and SR 142801 (both 0.1 μM), failed to inhibit contractions induced by SP or NKB.
4. In conclusion, the in vitro mechanical responses of circular smooth muscle preparations from human colon are strongly consistent with the presence of non-neuronal tachykinin NK₂-receptors, but not tachykinin NK₁- or NK₃-receptors. Our findings with SR 48968 suggest the existence of two tachykinin NK₂-receptor subtypes, that it seems to distinguish, unlike SR 144190 and its metabolites. However, the precise nature of SR 48968 allotopic antagonism remains to be elucidated, since allosteric effects at the tachykinin NK₂-receptor might well account for the complexity of the observed interaction.

     

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 action potential-triggered [Ca2+]i transients in single smooth muscle cells of guinea-pig ileum

1. To characterize increases in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) associated with discharge of action potentials, membrane potential and [Ca²⁺]_i were simultaneously recorded from single smooth muscle cells of guinea-pig ileum by use of a combination of nystatin-perforated patch clamp and fura-2 fluorimetry techniques.
2. A single action potential in response to a depolarizing current pulse elicited a transient rise in [Ca²⁺]_i. When the duration of the current pulse was prolonged, action potentials were repeatedly discharged during the early period of the pulse duration with a progressive decrease in overshoot potential, upstroke rate and repolarization rate. However, such action potentials could each trigger [Ca²⁺]_i transients with an almost constant amplitude.
3. Nicardipine (1 μM) and La³⁺ (10 μM), blockers of voltage-dependent Ca²⁺ channels (VDCCs), abolished both the action potential discharge and the [Ca²⁺]_i transient.
4. Charybdotoxin (ChTX, 300 nM) and tetraethylammonium (TEA, 2 mM), blockers of large conductance Ca²⁺-activated K⁺ channels, decreased the rate of repolarization of action potentials but increased the amplitude of [Ca²⁺]_i transients.
5. Thapsigargin (1 μM), an inhibitor of SR Ca²⁺-ATPase, slowed the falling phase and somewhat increased the amplitude, of action potential-triggered [Ca²⁺]_i transients without affecting action potentials. In addition, in voltage-clamped cells, the drug had little effect on the voltage step-evoked Ca²⁺ current but exerted a similar effect on its concomitant rise in [Ca²⁺]_i to that on the action potential-triggered [Ca²⁺]_i transient.
6. Similar action potential-triggered [Ca²⁺]_i transients were induced by brief exposures to high-K⁺ solution. They were not decreased, but rather increased, after depletion of intracellular Ca²⁺ stores by a combination of ryanodine (30 μM) and caffeine (10 mM) through an open-lock of Ca²⁺-induced Ca²⁺ release (CICR)-related channels.
7. The results show that action potentials, discharged repeatedly during the early period of a long membrane depolarization, undergo a progressive change in configuration but can each trigger a constant rise in [Ca²⁺]_i. Intracellular Ca²⁺ stores have a role, especially in accelerating the falling phase of the action potential-triggered [Ca²⁺]_i transients by replenishing cytosolic Ca²⁺. No evidence was provided for the involvement of CICR in the action potential-triggered [Ca²⁺]_i transient.

     

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.

     

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.

     

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.

     

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.

     

Evidence for inverse agonism of SR141716A at human recombinant cannabinoid CB1 and CB2 receptors

The cannabinoid receptor antagonist SR141716A has been suggested to be an inverse agonist at CB₁ receptors in some isolated intact tissues. We found that the basal incorporation of [³⁵S]-GTPγS in Chinese hamster ovary cells expressing human recombinant CB₁ and CB₂ receptors was inhibited by SR141716A (mean pEC₅₀s 8.26 and 6.00, respectively), whereas cannabinol (10 μM) had no significant effect at hCB₁ receptors but inhibited the binding at hCB₂ receptors. As cannabinol had no effect on basal [³⁵S]-GTPγS binding at hCB₁ at a concentration 100 fold higher than its binding affinity (K_i=0.1 μM), we conclude that endogenous cannabinoid receptor agonists are not a confounding factor and suggest the actions of SR141716A at the hCB₁ receptor, and the actions of SR141716A and cannabinol at the hCB₂ receptor, are due to inverse agonism.     

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.

     

Further insights into the anti-aggregating activity of NMDA in human platelets

1. In the present study the effect of N-methyl-D-aspartate (NMDA) on thromboxane B₂ synthesis and on [Ca²⁺]_i was studied in human platelets.
2. NMDA (10⁻⁷ M) completely inhibited the synthesis of thromboxane B₂ from exogenous arachidonic acid (AA), while it did not interfere with the aggregating effect of the thromboxane A₂ receptor agonist U-46619.
3. NMDA (0.1 μM–10 μM) dose-dependently increased intracellular calcium in washed platelets pre-loaded with fura 2 AM, and this effect was not additive with that of AA.
4. NMDA shifted the dose-response curve of AA to the right. At the highest AA concentrations platelet aggregation was not inhibited.
5. The antiaggregating effect of NMDA was not antagonized by N^G-monomethyl-L-arginine (L-NMMA), a nitric oxide synthase (NOS) inhibitor.
6. Finally, NMDA (0.01 nM–100 nM) associated with either aspirin or indomethacin significantly potentiated the antiaggregating activity of both cyclo-oxygenase inhibitors.
7. It was concluded that NMDA is a potent inhibitor of platelet aggregation and thromboxane B₂ synthesis in human platelet rich plasma (PRP).

     

PYY-preferring receptor in the dorsal vagal complex and its involvement in PYY stimulation of gastric acid secretion in rats

1. Microinjection of peptide YY (PYY, 7–46 pmol) into the dorsal vagal complex (DVC) stimulated gastric acid secretion in urethane-anaesthetized rats. Using a variety of neuropeptide Y (NPY) and PYY derivatives, we characterized the pharmacological profile of the receptor mediating the acid secretory response to PYY.
2. [Pro³⁴]rat(r)/porcine(p)PYY and [Pro³⁴]human(h)PYY (23–117 pmol), microinjected unilaterally into the DVC resulted in a similar maximal increase in net acid secretion reaching 68±11 and 89±31 μmol 90 min⁻¹ respectively.
3. Rat/hNPY and pNPY (47 pmol) microinjected into the DVC induced a similar net gastric acid secretion (27±8 and 23±8 μmol 90 min⁻¹ respectively) and a higher dose (116 pmol) tended to reduce the response.
4. Pancreatic polypeptide (PP, 4–46 pmol), [Leu³¹,Pro³⁴]r/hNPY (47 and 117 pmol) and the Y2 selective agonists, hPYY_3-36, pNPY_5-36 and pNPY_13-36 (25–168 pmol) microinjected into the DVC failed to influence basal gastric acid secretion.
5. The rank order of potency of PYY⩾[Pro³⁴]r/pPYY=[Pro³⁴]hPYY>r/hNPY=pNPY to stimulate gastric acid secretion upon injection into the DVC and the ineffectiveness of PP, [Leu³¹,Pro³⁴]NPY and C-terminal NPY/PYY fragments suggest that a PYY-preferring receptor subtype may be involved in mediating the stimulating effect.

     

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.

     

G-protein activation at 5-HT1A receptors by the 5-ht1F ligand LY334370 in guinea-pig brain sections and recombinant cell lines

1. G-protein activation by the 5-ht_1F receptor agonist 5-(4-fluorobenzoyl)amino-3-(1-methylpiperidin-4-yl)-1H-indole fumarate ({"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370) was investigated by use of autoradiography of receptor-activated G-proteins in guinea-pig brain sections and [³⁵S]-GTPγS binding responses in cell lines stably expressing human 5-HT_1A (h 5-HT_1A) receptors.
2. {"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370 (10 μM) caused little or no stimulation of [³⁵S]-GTPγS binding in guinea-pig brain regions enriched in 5-ht_1F binding sites (e.g., claustrum, caudate/putamen and thalamic nuclei), as identified by labelling with 10 nM [³H]-sumatriptan plus 10 nM 5-carboxamidotryptamine (5-CT).
3. Application of {"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370 (10 μM) to guinea-pig brain sections resulted in an increase of [³⁵S]-GTPγS binding in hippocampus (123±17%), lateral septum (58±14%), dorsal raphe (57±10%), entorhinal (37±11%) and cingulate cortex (28±10%). This distribution fits with the G-protein activation mediated by 5-HT_1A receptors as found with lisuride (10 μM), and labelling of 5-HT_1A receptors by 140 pM [¹²⁵I]-4-(2′-methoxy-phenyl)-1-[2′-(n-2′′-pyridinyl)-p-iodobenzamido]-ethyl-piperazine (p-MPPI).
4. The {"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370-mediated [³⁵S]-GTPγS response was antagonized by the selective, silent 5-HT_1A receptor antagonist N-[2-[4-(2-methoxyphenyl)1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide (WAY100635, 1 μM) in each of the brain structures investigated. The distribution pattern of the [³⁵S]-GTPγS binding response and the antagonist profile suggest that the {"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370-induced response in guinea-pig brain is mediated by 5-HT_1A receptors.
5. The maximal {"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370-induced [³⁵S]-GTPγS binding response (83 to 94%) in membranes of recombinant C6-glial/h 5-HT_1A and HeLa/h 5-HT_1A cells was close to that of 5-HT, suggesting {"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370 to exert high intrinsic activity at h 5-HT_1A receptors.
6. In conclusion, in guinea-pig brain sections and recombinant cell lines the 5-ht_1F receptor agonist {"type":"entrez-nucleotide","attrs":{"text":"LY334370","term_id":"1257380864","term_text":"LY334370"}}LY334370 causes G-protein activation that is mediated by 5-HT_1A receptors. Caution should be taken when employing this ligand as a putative selective 5-ht_1F agonist.

     

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.

     

Evidence for involvement of neuropeptide Y receptors in the regulation of food intake: studies with Y1-selective antagonist BIBP3226

1. Experiments were conducted to evaluate the effects of the novel non-peptide neuropeptide Y Y₁ receptor antagonist, BIBP3226 (N²-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]-D-arginine amide) on spontaneous, fasting-induced and NPY-induced food intake in rats. In addition to consumption of regular chow, the effects of BIBP3226 on consumption of highly palatable sweetened mash were monitored in a 1 h test on first exposure and after familiarization with novel food.
2. BIBP3226 (10.0 nmol, i.c.v.) had no effect on the consumption of regular chow, but reduced significantly the intake of highly palatable diet and the food intake stimulated by fasting (24 h). Neuropeptide Y (NPY, 1.0 nmol, i.c.v.) significantly increased the consumption of regular rat chow. This orexigenic effect of NPY was blocked by BIBP3226 (10.0 nmol, administered i.c.v. 5 min before NPY) at 30  min and 4  h, but not at 1 and 2  h. When animals were pretreated with diazepam (0.5 mg kg⁻¹, i.p., 20 min before NPY), BIBP3226 failed to suppress NPY-induced feeding.
3. An NPY Y₁ and Y₃ receptor agonist, [Leu³¹,Pro³⁴]NPY and a Y₅ receptor agonist human peptide YY_3–36 (hPYY_3–36, both 30 pmol), microinjected into the paraventricular nucleus of the hypothalamus (PVN) increased the consumption of regular rat chow. BIBP3226 (0.4 nmol, into the PVN) completely blocked the stimulatory effect of [Leu³¹,Pro³⁴]NPY but not that of hPYY_3–36. BIBP3226 (0.4 nmol) alone failed to modify the consumption of the regular chow. Higher doses of BIBP3226 (1.0 and 2.0 nmol) injected into the vicinity of the PVN reduced the consumption of the sweetened mash.
4. These results suggest that both the NPY Y₁ and Y₅ receptors in the PVN are involved in the regulation of food intake. The stimulatory effect of exogenous NPY is probably mediated through an NPY receptor subtype that is not identical with the Y₁ receptor (possibly Y₅ receptor). However, the NPY Y₁ receptors may mediate the effect of endogenous NPY in conditions of increased energy demand or on intake of highly palatable diets.