Co-operative signalling through DP(1) and DP(2) prostanoid receptors is required to enhance leukotriene C(4) synthesis induced by prostaglandin D(2) in eosinophils

BACKGROUND AND PURPOSE

Prostaglandin (PG) D₂ has emerged as a key mediator of allergic inflammatory pathologies and, particularly, PGD₂ induces leukotriene (LT) C₄ secretion from eosinophils. Here, we have characterized how PGD₂ signals to induce LTC₄ synthesis in eosinophils.

EXPERIMENTAL APPROACH

Antagonists and agonists of DP₁ and DP₂ prostanoid receptors were used in a model of PGD₂-induced eosinophilic inflammation in vivo and with PGD₂-stimulated human eosinophils in vitro, to identify PGD₂ receptor(s) mediating LTC₄ secretion. The signalling pathways involved were also investigated.

KEY RESULTS

In vivo and in vitro assays with receptor antagonists showed that PGD₂-triggered cysteinyl-LT (cysLT) secretion depends on the activation of both DP₁ and DP₂ receptors. DP₁ and DP₂ receptor agonists elicited cysLTs production only after simultaneous activation of both receptors. In eosinophils, LTC₄ synthesis, but not LTC₄ transport/export, was activated by PGD₂ receptor stimulation, and lipid bodies (lipid droplets) were the intracellular compartments of DP₁/DP₂ receptor-driven LTC₄ synthesis. Although not sufficient to trigger LTC₄ synthesis by itself, DP₁ receptor activation, signalling through protein kinase A, did activate the biogenesis of eosinophil lipid bodies, a process crucial for PGD₂-induced LTC₄ synthesis. Similarly, concurrent DP₂ receptor activation used Pertussis toxin-sensitive and calcium-dependent signalling pathways to achieve effective PGD₂-induced LTC₄ synthesis.

CONCLUSIONS AND IMPLICATIONS

Based on pivotal roles of cysLTs in allergic inflammatory pathogenesis and the collaborative interaction between PGD₂ receptors described here, our data suggest that both DP₁ and DP₂ receptor antagonists might be attractive candidates for anti-allergic therapies.

LINKED ARTICLE

This article is commented on by Mackay and Stewart, pp. 1671–1673 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01236.x     

Receptors involved in the modulation of guinea pig urinary bladder motility by prostaglandin D2

Background and Purpose

We have described a urothelium-dependent release of PGD₂-like activity which had inhibitory effects on the motility of guinea pig urinary bladder. Here, we have pharmacologically characterized the receptors involved and localized the sites of PGD₂ formation and of its receptors.

Experimental Approach

In the presence of selective DP and TP receptor antagonists alone or combined, PGD₂ was applied to urothelium-denuded diclofenac-treated urinary bladder strips mounted in organ baths. Antibodies against PGD₂ synthase and DP₁ receptors were used with Western blots and for histochemistry.

Key Results

PGD₂ inhibited nerve stimulation -induced contractions in strips of guinea pig urinary bladder with estimated pIC₅₀ of 7.55 ± 0.15 (n = 13), an effect blocked by the DP₁ receptor antagonist BW-A868C. After blockade of DP₁ receptors, PGD₂ enhanced the contractions, an effect abolished by the TP receptor antagonist SQ-29548. Histochemistry revealed strong immunoreactivity for PGD synthase in the urothelium/suburothelium with strongest reaction in the suburothelium. Immunoreactive DP₁ receptors were found in the smooth muscle of the bladder wall with a dominant localization to smooth muscle membranes.

Conclusions and Implications

In guinea pig urinary bladder, the main effect of PGD₂ is an inhibitory action via DP₁ receptors localized to the smooth muscle, but an excitatory effect via TP receptors can also be evoked. The urothelium with its suburothelium might signal to the smooth muscle which is rich in PGD₂ receptors of the DP₁ type. The results are important for our understanding of regulation of bladder motility.     

Selectivity profiling of the novel EP2 receptor antagonist,PF-04418948, in functional bioassay systems: atypical affinity at the guinea pig EP2 receptor

BACKGROUND AND PURPOSE

Understanding the role of the EP₂ receptor has been hampered by the lack of a selective antagonist. Recently, a selective EP₂ receptor antagonist, PF-04418948, has been discovered. The aim of this study was to demonstrate the selectivity profile of PF-04418948 for the EP₂ receptor over other EP receptors using a range of isolated tissue systems.

EXPERIMENTAL APPROACH

PF-04418948 was profiled on a range of isolated tissues to assess its EP receptor potency and selectivity: ONO-DI-004-induced contraction of guinea pig trachea (EP₁); ONO-AE1-259 and PGE₂- induced relaxation of mouse and guinea pig trachea (EP₂); PGE₂-induced depolarization of guinea pig isolated vagus (EP₃); PGE₂-induced relaxation of human and rat trachea (EP₄). PF-04418948 was also profiled in functional murine TP, IP, DP and FP receptor assays.

KEY RESULTS

In bioassay systems, where assessment of potency/selectivity is made against the ‘native’ receptor, PF-04418948 only acted as an antagonist of EP₂ receptor-mediated events. PF-04418948 competitively inhibited relaxations of murine and guinea pig trachea induced by ONO-AE1-259 and PGE₂ respectively. However, the affinity of PF-04418948 was not equal in the two preparations.

CONCLUSIONS AND IMPLICATIONS

Using a wide range of bioassay systems, we have demonstrated that PF-04418948 is a selective EP₂-receptor antagonist. Interestingly, an atypically low affinity was found on the guinea pig trachea, questioning its utility as an EP₂ receptor assay system. Nevertheless, this compound should be an invaluable tool for investigating the biological activity of PGE₂ and the role of EP₂ receptors in health and disease.     

Dual bronchodilatory and pulmonary anti-inflammatory activity of RO5024118, a novel agonist at vasoactive intestinal peptide VPAC2 receptors

BACKGROUND AND PURPOSE

Vasoactive intestinal peptide is expressed in the respiratory tract and induces its effects via its receptors, VPAC₁ and VPAC₂. RO5024118 is a selective VPAC₂ receptor agonist derived via chemical modification of an earlier VPAC₂ agonist, RO0251553. In the present studies, we characterized the pharmacological activity of RO5024118.

EXPERIMENTAL APPROACH

Stability of RO5024118 to human neutrophil elastase was assessed. Bronchodilatory activity of RO5024118 was investigated in guinea pig and human isolated airway smooth muscle preparations and in a guinea pig bronchoconstriction model. Pulmonary anti-inflammatory activity of RO5024118 was investigated in a lipopolysaccharide mouse model and in a porcine pancreatic elastase (PPE) rat model.

KEY RESULTS

RO5024118 demonstrated increased stability to neutrophil elastase compared with RO0251553. In human and guinea pig isolated airway preparations, RO5024118 induced bronchodilatory effects comparable with RO0251553 and the long-acting β-agonist salmeterol and was significantly more potent than native vasoactive intestinal peptide and the short-acting β-agonist salbutamol. In 5-HT-induced bronchoconstriction in guinea pigs, RO5024118 exhibited inhibitory activity with similar efficacy as, and longer duration than, RO0251553. In a lipopolysaccharide-mouse model, RO5024118 inhibited neutrophil and CD8⁺ cells and myeloperoxidase levels. In rats, intratracheal instillation of PPE induced airway neutrophilia that was resistant to dexamethasone. Pretreatment with RO5024118 significantly inhibited PPE-induced neutrophil accumulation.

CONCLUSIONS AND IMPLICATIONS

These results demonstrate that RO5024118 induces dual bronchodilatory and pulmonary anti-inflammatory activity and may be beneficial in treating airway obstructive and inflammatory diseases.     

Pharmacological characterization of the first potent and selective antagonist at the cysteinyl leukotriene 2 (CysLT2) receptor

Background and purpose:

Cysteinyl leukotrienes (CysLTs) have been implicated in the pathophysiology of inflammatory and cardiovascular disorders. Their actions are mediated by CysLT₁ and CysLT₂ receptors. Here we report the discovery of 3-({[(1S,3S)-3-carboxycyclohexyl]amino}carbonyl)-4-(3-{4-[4-(cyclo-hexyloxy)butoxy]phenyl}propoxy) benzoic acid (HAMI3379), the first potent and selective CysLT₂ receptor antagonist.

Experimental approach:

Pharmacological characterization of HAMI3379 was performed using stably transfected CysLT₁ and CysLT₂ receptor cell lines, and isolated, Langendorff-perfused, guinea pig hearts.

Key results:

In a CysLT₂ receptor reporter cell line, HAMI3379 antagonized leukotriene D₄- (LTD₄-) and leukotriene C₄- (LTC₄-) induced intracellular calcium mobilization with IC₅₀ values of 3.8 nM and 4.4 nM respectively. In contrast, HAMI3379 exhibited very low potency on a recombinant CysLT₁ receptor cell line (IC₅₀ > 10 000 nM). In addition, HAMI3379 did not exhibit any agonistic activity on both CysLT receptor cell lines. In binding studies using membranes from the CysLT₂ and CysLT₁ receptor cell lines, HAMI3379 inhibited [³H]-LTD₄ binding with IC₅₀ values of 38 nM and >10 000 nM respectively. In isolated Langendorff-perfused guinea pig hearts HAMI3379 concentration-dependently inhibited and reversed the LTC₄-induced perfusion pressure increase and contractility decrease. The selective CysLT₁ receptor antagonist zafirlukast was found to be inactive in this experimental setting.

Conclusions and implications:

HAMI3379 was identified as a potent and selective CysLT₂ receptor antagonist, which was devoid of CysLT receptor agonism. Using this compound, we showed that the cardiac effects of CysLTs are predominantly mediated by the CysLT₂ receptor.     

A selective antagonist of histamine H4 receptors prevents antigen-induced airway inflammation and bronchoconstriction in guinea pigs: involvement of lipocortin-1

Background and Purpose

Among the pathogenic mechanisms of asthma, a role for oxidative/nitrosative stress has been well documented. Recent evidence suggests that histamine H₄ receptors play a modulatory role in allergic inflammation. Here we report the effects of compound JNJ 7777120 (JNJ), a selective H₄ receptor antagonist, on antigen-induced airway inflammation, paying special attention to its effects on lipocortin-1 (LC-1/annexin-A1), a 37 kDA anti-inflammatory protein that plays a key role in the production of inflammatory mediators.

Experimental Approach

Ovalbumin (OA)-sensitized guinea pigs placed in a respiratory chamber were challenged with antigen. JNJ (5, 7.5 and 10 mg·kg⁻¹) was given i.p. for 4 days before antigen challenge. Respiratory parameters were recorded. Bronchoalveolar lavage (BAL) fluid was collected and lung specimens taken for further analyses 1 h after antigen challenge. In BAL fluid, levels of LC-1, PGD₂, LTB₄ and TNF-α were measured. In lung tissue samples, myeloperoxidase, caspase-3 and Mn-superoxide dismutase activities and 8-hydroxy-2-deoxyguanosine levels were measured.

Key Results

OA challenge decreased LC-1 levels in BAL fluid, induced cough, dyspnoea and bronchoconstriction and increased PGD₂, LTB₄ and TNF-α levels in lung tissue. Treatment with JNJ dose-dependently increased levels of LC-1, reduced respiratory abnormalities and lowered levels of PGD₂, LTB₄ and TNF-α in BAL fluid.

Conclusions and Implications

Antigen-induced asthma-like reactions in guinea pigs decreased levels of LC-1 and increased TNF-α and eicosanoid production. JNJ pretreatment reduced allergic asthmatic responses and airway inflammation, an effect associated with LC-1 up-regulation.

Linked Articles

This article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2013.170.issue-1     

A role for endogenous peptide YY in tachykinin NK2 receptor-triggered 5-HT release from guinea pig isolated colonic mucosa

BACKGROUND AND PURPOSE

The colon-derived peptide hormone, peptide YY (PYY), regulates colonic motility, secretion and postprandial satiety; but little is known about the influence of endogenous PYY on 5-HT release from colonic mucosa. Tachykinin NK₂ receptor-selective agonist, βAla-NKA-(4-10) induces 5-HT release from guinea pig colonic mucosa via NK₂ receptors on the mucosal layer. The present study was designed to determine the influence of endogenous PYY on 5-HT release from guinea pig colonic mucosa, evoked by the NK₂ receptor agonist, βAla-NKA-(4-10).

EXPERIMENTAL APPROACH

Muscle layer-free mucosal preparations of guinea pig colon were incubated in vitro and the outflow of PYY or 5-HT and its metabolite, 5-HIAA, from these preparations were determined by enzyme immunoassays or HPLC with electrochemical detection respectively.

KEY RESULTS

βAla-NKA-(4-10) produced a tetrodotoxin-resistant sustained increase in the outflow of PYY and 5-HT from the mucosal preparations. The βAla-NKA-(4-10)-evoked 5-HT outflow was partially inhibited by Y₁ receptor antagonist, BIBO3304, and Y₂ receptor antagonist, BIIE0246, but with less potency. Exogenously-applied PYY also produced a sustained increase in the outflow of 5-HT that was inhibited by Y₁ blockade but not Y₂ blockade.

CONCLUSION AND IMPLICATIONS

Our findings support the view that the NK₂ receptor-selective agonist, βAla-NKA-(4-10) produces a long-lasting PYY release from guinea pig colonic mucosa via NK₂ receptors on L cells and βAla-NKA-(4-10)-evoked 5-HT release is in part mediated by endogenously released PYY, acting mainly on Y₁ receptors on EC cells. The PYY-containing L cells appear to play a role in controlling the release of 5-HT from colonic EC cells.     

Melatonin inhibits tachykinin NK₂ receptor-triggered 5-HT release from guinea pig isolated colonic mucosa

BACKGROUND AND PURPOSE

Melatonin is involved in the regulation of colonic motility, and sensation, but little is known about the influence of melatonin on 5-hydroxytryptamine (5-HT) release from colonic mucosa. A tachykinin NK₂ receptor-selective agonist, [β-Ala⁸]-neurokinin A_4-10[βAla-NKA-(4-10)] can induce 5-HT release from guinea pig colonic mucosa via NK₂ receptors on the mucosal layer. The present study was designed to determine the influence of melatonin on 5-HT release from guinea pig colonic mucosa, evoked by the NK₂ receptor agonist, βAla-NKA-(4-10).

EXPERIMENTAL APPROACH

The effect of melatonin was investigated on the outflow of 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) from muscle layer-free mucosal preparations of guinea pig colon, using high-performance liquid chromatography with electrochemical detection.

KEY RESULTS

Melatonin caused a sustained decline in the βAla-NKA-(4-10)-evoked 5-HT outflow from the muscle layer-free mucosal preparations, but failed to affect its metabolite 5-HIAA outflow. The specific MT₃ receptor agonist, 5-methoxycarbonylamino-N-acetyltryptamine mimicked the inhibitory effect of melatonin on βAla-NKA-(4-10)-evoked 5-HT outflow. A MT₃ receptor antagonist prazosin shifted the concentration-response curve of melatonin to the right in a concentration-dependent manner and depressed the maximum effect, but neither a combined MT₁/MT₂ receptor antagonist luzindole, nor a MT₂ receptor antagonist N-pentanoyl-2-benzyltryptamine modified the concentration–response curve to melatonin.

CONCLUSIONS AND IMPLICATIONS

Melatonin inhibits NK₂ receptor-triggered 5-HT release from guinea pig colonic mucosa by acting at a MT₃ melatonin receptor located directly on the mucosal layer, without affecting 5-HT degradation processes. Possible contributions of MT₁/MT₂ melatonin receptors to the inhibitory effect of melatonin appear to be negligible. Melatonin may act as a modulator of excess 5-HT release from colonic mucosa.     

Novel blockers of hyperpolarization-activated current with isoform selectivity in recombinant cells and native tissue

BACKGROUND AND PURPOSE

Selective hyperpolarization activated, cyclic nucleotide-gated channel (HCN) blockers represent an important therapeutic goal due to the wide distribution and multiple functions of these proteins, representing the molecular correlate of f- and h-current (I_f or I_h). Recently, new compounds able to block differentially the homomeric HCN isoforms expressed in HEK293 have been synthesized. In the present work, the electrophysiological and pharmacological properties of these new HCN blockers were characterized and their activities evaluated on native channels.

EXPERIMENTAL APPROACH

HEK293 cells expressing mHCN1, mHCN2 and hHCN4 isoforms were used to verify channel blockade. Selected compounds were tested on native guinea pig sinoatrial node cells and neurons from mouse dorsal root ganglion (DRG) by patch-clamp recordings and on dog Purkinje fibres by intracellular recordings.

KEY RESULTS

In HEK293 cells, EC18 was found to be significantly selective for HCN4 and MEL57A for HCN1 at physiological membrane potential. When tested on guinea pig sinoatrial node cells, EC18 (10 µM) maintained its activity, reducing I_f by 67% at −120 mV, while MEL57A (3 µM) reduced I_f by 18%. In contrast, in mouse DRG neurons, only MEL57A (30 and 100 µM) significantly reduced I_h by 60% at −80 mV. In dog cardiac Purkinje fibres, EC18, but not MEL57A, reduced the amplitude and slowed the slope of the spontaneous diastolic depolarization.

CONCLUSIONS

Our results have identified novel and highly selective HCN isoform blockers, EC18 and MEL57A; the selectivity found in recombinant system was maintained in various tissues expressing different HCN isoforms.     

Characterization of biosynthesis and modes of action of prostaglandin E2 and prostacyclin in guinea pig mesenteric lymphatic vessels

Background and purpose:

Rhythmical transient constrictions of the lymphatic vessels provide the means for efficient lymph drainage and interstitial tissue fluid balance. This activity is critical during inflammation, to avoid or limit oedema resulting from increased vascular permeability, mediated by the release of various inflammatory mediators. In this study, we investigated the mechanisms by which prostaglandin E₂ (PGE₂) and prostacyclin modulate lymphatic contractility in isolated guinea pig mesenteric lymphatic vessels.

Experimental approach:

Quantitative RT-PCR was used to assess the expression of mRNA for enzymes and receptors involved in the production and action of PGE₂ and prostacyclin in mesenteric collecting lymphatic vessels. Frequency and amplitude of lymphatic vessel constriction were measured in the presence of these prostaglandins and the role of their respective EP and IP receptors assessed.

Key results:

Prostaglandin E₂ and prostacyclin decreased concentration-dependently the frequency, without affecting the amplitude, of lymphatic constriction. Data obtained in the presence of the EP₄ receptor antagonists, GW627368x (1 µM) and AH23848B (30 µM) and the IP receptor antagonist CAY10441 (0.1 µM) suggest that PGE₂ predominantly activates EP₄, whereas prostacyclin mainly stimulates IP receptors. Inhibition of responses to either prostaglandin with H89 (10 µM) or glibenclamide (1 µM) suggested a role for the activation of protein kinase A and ATP-sensitive K⁺ channels.

Conclusions and implications:

Our findings characterized the inhibition of lymphatic pumping induced by PGE₂ or prostacyclin in guinea pig mesenteric lymphatics. This action is likely to impair oedema resolution and to contribute to the pro-inflammatory actions of these prostaglandins.     

Etanercept prevents airway hyperresponsiveness by protecting neuronal M2 muscarinic receptors in antigen-challenged guinea pigs

Background and purpose

Increased tumour necrosis factor-α (TNF-α) is associated with airway hyperreactivity in antigen-challenged animals. In human asthmatics, TNF-α is increased and blocking it prevents airway hyperreactivity in some asthmatic patients. However, the mechanisms by which TNF-α mediates hyperreactivity are unknown. Airway hyperreactivity can be caused by dysfunction of neuronal M₂ muscarinic receptors that normally limit acetylcholine release from parasympathetic nerves. Here we test whether blocking TNF-α receptors with etanercept prevents M₂ receptor dysfunction and airway hyperreactivity in antigen-challenged guinea pigs.

Experimental approach

Ovalbumin-sensitized guinea pigs were challenged by inhalation of antigen. Some animals received etanercept (3 mg kg⁻¹ i.p.) 3 h before challenge. 24 h after challenge, airway hyperreactivity and M₂ receptor function were tested. Inflammatory cells in bronchoalveolar lavage, blood and lung were counted. TNF-α and its receptors were detected by real-time RT-PCR and immunocytochemistry in parasympathetic nerves from humans and guinea pigs and in human neuroblastoma cells.

Key results

Antigen-challenged animals were hyperreactive to vagal stimulation and neuronal M₂ receptors were dysfunctional. Both M₂ receptor dysfunction and airway hyperreactivity were prevented by etanercept. Etanercept reduced eosinophils around airway nerves, and in blood, bronchoalveolar lavage and airway smooth muscle. Also, TNF-α decreased M₂ receptor mRNA in human and guinea pig parasympathetic neurons.

Conclusions and implications

Tumour necrosis factor-α may contribute to M₂ receptor dysfunction and airway hyperreactivity directly by decreasing receptor expression and indirectly by promoting recruitment of eosinophils, containing major basic protein, an M₂ antagonist. This suggests that etanercept may be beneficial in treatment of allergic asthma.     

Differential actions of urocortins on neurons of the myenteric division of the enteric nervous system in guinea pig distal colon

Background and purpose:

Urocortins (Ucns) 1, 2 and 3 are corticotropin-releasing factor (CRF)-related neuropeptides and may be involved in neural regulation of colonic motor functions. Nevertheless, details of the neural mechanism of action for Ucns have been unclear. We have, here, tested the hypothesis that Ucns act in the enteric nervous system (ENS) to influence colonic motor behaviour.

Experimental approach:

We used intracellular recording with ‘sharp’ microelectrodes, followed by intraneuronal injection of biocytin, and immunohistochemical localization of CRF₁ and CRF₂ receptors in guinea pig colonic tissue.

Key results:

Application of Ucn1 depolarized membrane potentials and elevated excitability in 58% of AH-type and 60% of S-type colonic myenteric neurons. In most of the neurons tested, depolarizing responses evoked by Ucn-1 were suppressed by the CRF₁ receptor antagonist NBI 27914, but were unaffected by the CRF₂ receptor antagonist antisauvagine-30. The selective CRF₂ receptor agonists, Ucn2 and Ucn3, evoked depolarizing responses in 12 and 8% of the AH-type myenteric neurons, respectively, and had no effect on S-type neurons. Antisauvagine-30, but not NBI 27914, suppressed these Ucn2- and Ucn3-evoked responses. Immunohistochemical staining identified CRF₁ as the predominant CRF receptor subtype expressed by ganglion cell somas, while CRF₂-immunoreactive neuronal somas were sparse. Ucns did not affect excitatory synaptic transmission in the ENS.

Conclusions and implications:

The results suggest that Ucns act as neuromodulators to influence myenteric neuronal excitability. The excitatory action of Ucn1 in myenteric neurons was primarily at CRF₁ receptors, and the excitatory action of Ucn2 and Ucn3 was at CRF₂ receptors.     

Pharmacological characterization of GSK1004723, a novel, long-acting antagonist at histamine H(1) and H(3) receptors

BACKGROUND AND PURPOSE

Preclinical pharmacological characterization of GSK1004723, a novel, dual histamine H₁ and H₃ receptor antagonist.

EXPERIMENTAL APPROACH

GSK1004723 was characterized in vitro and in vivo using methods that included radioligand binding, intracellular calcium mobilization, cAMP production, GTPγS binding, superfused human bronchus and guinea pig whole body plethysmography.

KEY RESULTS

In cell membranes over-expressing human recombinant H₁ and H₃ receptors, GSK1004723 displayed high affinity, competitive binding (H₁ pKi = 10.2; H₃ pKi = 10.6). In addition, GSK1004723 demonstrated slow dissociation from both receptors with a t_1/2 of 1.2 and 1.5 h for H₁ and H₃ respectively. GSK1004723 specifically antagonized H₁ receptor mediated increases in intracellular calcium and H₃ receptor mediated increases in GTPγS binding. The antagonism exerted was retained after cell washing, consistent with slow dissociation from H₁ and H₃ receptors. Duration of action was further evaluated using superfused human bronchus preparations. GSK1004723 (100 nmol·L⁻¹) reversed an established contractile response to histamine. When GSK1004723 was removed from the perfusate, only 20% recovery of the histamine response was observed over 10 h. Moreover, 21 h post-exposure to GSK1004723 there remained almost complete antagonism of responses to histamine. In vivo pharmacology was studied in conscious guinea pigs in which nasal congestion induced by intranasal histamine was measured indirectly (plethysmography). GSK1004723 (0.1 and 1 mg·mL⁻¹ intranasal) antagonized the histamine-induced response with a duration of up to 72 h.

CONCLUSIONS AND IMPLICATIONS

GSK1004723 is a potent and selective histamine H₁ and H₃ receptor antagonist with a long duration of action and represents a potential novel therapy for allergic rhinitis.     

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.     

Agonist-specific patterns of β2-adrenoceptor responses in human airway cells during prolonged exposure

Background and purpose:

β₂-Adrenoceptor agonists (β₂-agonists) are important bronchodilators used in the treatment of asthma and chronic obstructive pulmonary disease. At the molecular level, β₂-adrenergic agonist stimulation induces desensitization of the β₂-adrenoceptor. In this study, we have examined the relationships between initial effect and subsequent reduction of responsiveness to restimulation for a panel of β₂-agonists in cellular and in vitro tissue models.

Experimental approach:

β₂-Adrenoceptor-induced responses and subsequent loss of receptor responsiveness were studied in primary human airway smooth muscle cells and bronchial epithelial cells by measuring cAMP production. Receptor responsiveness was compared at equi-effective concentrations, either after continuous incubation for 24 h or after a 1 h pulse exposure followed by a 23 h washout. Key findings were confirmed in guinea pig tracheal preparations in vitro.

Key results:

There were differences in the reduction of receptor responsiveness in human airway cells and in vitro guinea pig trachea by a panel of β₂-agonists. When restimulation occurred immediately after continuous incubation, loss of responsiveness correlated with initial effect for all agonists. After the 1 h pulse exposure, differences between agonists emerged, for example isoprenaline and formoterol induced the least reduction of responsiveness. High lipophilicity was, to some extent, predictive of loss of responsiveness, but other factors appeared to be involved in determining the relationships between effect and subsequent loss of responsiveness for individual agonists.

Conclusions and implications:

There were clear differences in the ability of different β₂ agonists to induce loss of receptor responsiveness at equi-effective concentrations.     

Interaction of prostanoid EP₃ and TP receptors in guinea-pig isolated aorta: contractile self-synergism of 11-deoxy-16,16-dimethyl PGE₂

BACKGROUND AND PURPOSE

Surprisingly high contractile activity was reported for 11-deoxy-16,16-dimethyl prostaglandin E₂ (DX-DM PGE₂) on pig cerebral artery when used as a selective EP₃ receptor agonist. This study investigated the selectivity profile of DX-DM PGE₂, focusing on the interaction between its EP₃ and TP (thromboxane A₂-like) agonist activities.

EXPERIMENTAL APPROACH

Contraction of guinea-pig trachea (EP₁ system) and aorta (EP₃ and TP systems) was measured in conventional organ baths.

KEY RESULTS

Strong contraction of guinea-pig aorta to sulprostone and 17-phenyl PGE₂ (EP₃ agonists) was only seen under priming with a second contractile agent such as phenylephrine, histamine or U-46619 (TP agonist). In contrast, DX-DM PGE₂ induced strong contraction, which on the basis of treatment with (DG)-3ap (EP₃ antagonist) and/or BMS-180291 (TP antagonist) was attributed to self-synergism arising from co-activation of EP₃ and TP receptors. EP₃/TP self-synergism also accounted for contraction induced by PGF_2α and its analogues (+)-cloprostenol and latanoprost-FA. DX-DM PGE₂ also showed significant EP₁ agonism on guinea-pig trachea as defined by the EP₁ antagonists SC-51322, (ONO)-5-methyl-1 and AH-6809, although AH-6809 exhibited poor specificity at concentrations ≥3 µM.

CONCLUSIONS AND IMPLICATIONS

EP₃/TP self-synergism, as seen with PGE/PGF analogues in this study, may confound EP₃ agonist potency comparisons and the characterization of prostanoid receptor systems. The competitive profile of a TP antagonist may be distorted by variation in the silent/overt contraction profile of the EP₃ system in different studies. The relevance of self-synergism to in vivo actions of natural prostanoid receptor agonists is discussed.     

Antidepressant-like effects of a novel 5-HT3 receptor antagonist 6z in acute and chronic murine models of depression

Aim:

To investigate the antidepressant-like effects of a novel 5-HT₃ receptor antagonist N-(benzo[d]thiazol-2-yl)-3-methoxyquinoxalin-2-carboxamide (6z) in acute and chronic murine models of depression.

Methods:

5-HT₃ receptor antagonism was examined in guinea pig ileum in vitro. A tail suspension test (TST) was used as acute depression model to evaluate the antidepressant-like behavior in mice treated with 6z (0.5–2 mg/kg, ip). In chronic depression model, mice were exposed to a 4-week chronic unpredictable stress (CUS) protocol, and treated with 6z (0.5–2 mg·kg⁻¹·d⁻¹, po) or a positive drug fluoxetine (10 mg·kg⁻¹·d⁻¹, po) in the last 2 weeks, followed by behavioral and biochemical assessments.

Results:

The 5-HT₃ receptor antagonism of 6z (pA₂=7.4) in guinea pig ileum was more potent than that of a standard 5-HT₃ receptor antagonist ondansetron (pA₂=6.9). In acute depression model, 6z administration significantly decreased the immobility duration. In chronic depression model, 6z administration reversed CUS-induced depressive-like behavior, as evidenced by increased immobility duration in the forced swim test and sucrose preference in the sucrose preference test. Furthermore, chronic administration of 6z prevented CUS-induced brain oxidative stress, with significant reduction of pro-oxidant markers and elevation of antioxidant enzyme activity. Moreover, chronic administration of 6z attenuated CUS-induced hypothalamic-pituitary-adrenal axis hyperactivity, as shown by reduced plasma corticosterone levels. Similar results were observed in the fluoxetine-treated group.

Conclusion:

6z is a novel 5-HT₃ receptor antagonist with potential antidepressant-like activities, which may be related to modulating hypothalamic-pituitary-adrenal axis and attenuating brain oxidative damage.     

Novel selective cannabinoid CB(1) receptor antagonist MJ08 with potent in vivo bioactivity and inverse agonistic effects

Aim:

To characterize the biological profiles of MJ08, a novel selective CB₁ receptor antagonist.

Methods:

Radioligand binding assays were performed using rat brain and spleen membrane preparations. CB₁ and CB₂ receptor redistribution and intracellular Ca²⁺ ([Ca²⁺]_i) assays were performed with IN CELL Analyzer. Inverse agonism was studied using intracellular cAMP assays, and in guinea-pig ileum and mouse vas deferens smooth muscle preparations. In vivo pharmacologic profile was assessed in diet-induced obesity (DIO) mice.

Results:

In radioligand binding assay, MJ08 selectively antagonized CB₁ receptor (IC₅₀=99.9 nmol/L). In EGFP-CB₁_U2OS cells, its IC₅₀ value against CB₁ receptor activation was 30.23 nmol/L (SR141716A: 32.16 nmol/L). WIN 55,212-2 (1 μmol/L) increased [Ca²⁺]_i in the primary cultured hippocampal neuronal cells and decreased cAMP accumulation in CHO-hCB₁ cells. MJ08 (10 nmol/L–10 μmol/L) blocked both the WIN 55,212-2-induced effects. Furthermore, MJ08 reversed the inhibition of electrically evoked twitches of mouse vas deferens by WIN 55,212-2 (pA₂=10.29±1.05). MJ08 and SR141716A both showed an inverse agonism activity by markedly promoting the contraction force and frequency of guinea pig ileum muscle. MJ08 significantly increased the cAMP level in CHO-hCB₁ cells with an EC₅₀ value of 78.6 nmol/L, which was lower than the EC₅₀ value for SR141716A (159.2 nmol/L). Besides the more potent pharmacological effects of cannabinoid CB₁ receptor antagonism in DIO mice, such as reducing food intake, decreasing body weight, and ameliorating dyslipidemia, MJ08 (10 mg/kg) unexpectedly raised the fasted blood glucose in vivo.

Conclusion:

MJ08 is a novel, potent and selective CB₁ receptor antagonist/inverse agonist with potent bioactive responses in vitro and in vivo that may be useful for disclosure the versatile nature of CB₁ receptors.     

Selective desensitization of the 5-HT4 receptor-mediated response in pig atrium but not in stomach

Background and purpose:

The time dependency of the effect of 5-HT₄ receptor agonists depends on many specific regulatory mechanisms, which vary between tissues. This has important implications with regard to the effects of endogenous 5-HT, as well as to the clinical use of 5-HT₄ receptor agonists, and might contribute to tissue selectivity of agonists.

Experimental approach:

The progression and desensitization of 5-HT₄ receptor-mediated responses were evaluated in an organ bath set-up using two, clinically relevant, porcine in vitro models: gastric cholinergic neurotransmission and atrial contractility.

Key results:

Exposure of gastric tissue to 5-HT or to the selective 5-HT₄ receptor agonists prucalopride and M0003 results in a sustained non-transient effect during exposure; after washout, the response to a subsequent challenge with 5-HT shows no clear desensitization. Incubation of left atrial tissue with 5-HT resulted in a transient response, leading after washout to a marked desensitization of the subsequent response to 5-HT. The selective 5-HT₄ receptor agonists prucalopride and M0003 induce only very weak atrial responses whereas they are very effective in desensitizing the atrial response to 5-HT. The observations also suggest that the properties of prucalopride and M0003 to bind to and/or activate the 5-HT₄ receptor differ from those of 5-HT. This difference might have contributed to the observed desensitization.

Conclusions and implications:

The high potency of prucalopride and M0003 in desensitizing the response to 5-HT together with their low efficacy in the atrium emphasizes the cardiac safety of this class of 5-HT₄ receptor agonists.