Activation of endothelin ETA receptors masks the constrictor role of endothelin ETB receptors in rat isolated small mesenteric arteries

1. Endothelin-1 (ET-1) produces constriction of the rat mesenteric vascular bed in vivo via ET_A and ET_B receptor subtypes. The aim of this study was to investigate the relative roles of these receptor subtypes in rat isolated, endothelium-denuded, small mesenteric arteries, under pressure, by use of ET-1; the ET_A receptor antagonist, BQ-123; the ET_B receptor selective agonist, sarafotoxin S6c (SRTX S6c); the ET_B receptor selective antagonist, BQ-788; and the ET_A/ET_B antagonist, TAK-044.
2. In 3rd generation mesenteric arteries, ET-1 (10⁻¹³10⁻⁷ M) produced concentration-dependent contractions (pD₂ 9.86). SRTX S6c (10⁻¹²10⁻⁷ M) also induced concentration-dependent contractions in 53% of arteries studied, although the E_max was much less than that obtained with ET-1 (10.7±2.9% vs 101.9±2.6% of the 60 mM KCl-induced contraction).
3. Neither ET_B receptor desensitization, by a supra-maximal concentration of SRTX S6c (10⁻⁷ M), nor incubation with BQ-788 (3×10⁻⁸ M), had any significant effect on the ET-1 concentration-response curve, although both treatments tended to enhance rather than inhibit responses to ET-1.
4. In the presence of BQ-123 (10⁻⁶ M), responses to low concentrations of ET-1 (up to 10⁻¹⁰ M) were unaffected but responses to concentrations of ET-1 above 10⁻¹⁰ M were significantly inhibited.
5. SRTX S6c desensitization followed by incubation with BQ-123 (10⁻⁶ M) or co-incubation with BQ-788 (3×10⁻⁸ M) and BQ-123 caused inhibition of responses to all concentrations of ET-1, resulting in a rightward shift of the ET-1 concentration-response curve. The same effect was obtained by incubation with TAK-044 (10⁻⁸ M and 3×10⁻⁷ M).
6. Thus, responses of rat small mesenteric arteries to ET-1 are mediated by both ET_A and ET_B receptors. The relative role of ET_B receptors is greater than that predicted by the small responses to SRTX S6c or by resistance of ET-1-induced contraction to ET_B receptor desensitization or BQ-788. The effect of ET_B receptor desensitization or blockade is only revealed in the presence of ET_A receptor blockade, suggesting the presence of a ‘crosstalk'' mechanism between the receptors. These results support the concept that dual receptor antagonists, like TAK-044, may be required to inhibit completely constrictor responses to ET-1.

     

Different role of endothelin ETA and ETB receptors and endothelial modulators in diabetes-induced hyperreactivity of the rabbit carotid artery to endothelin-1

The influence of diabetes on regulatory mechanisms and specific receptors implicated in the contractile response of isolated rabbit carotid arteries to endothelin-1 was examined. Endothelin-1 induced a concentration-dependent contraction that was greater in arteries from diabetic rabbits than in arteries from control rabbits. Endothelium removal or N(G)-nitro-L-arginine enhanced contractions in response to endothelin-1 only in control arteries, without modifying the endothelin-1 response in diabetic arteries. Indomethacin, furegrelate (thromboxane A(2) inhibitor), or cyclo-(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123; endothelin ET(A) receptor antagonist) inhibited the contractions in response to endothelin-1, the inhibition being greater in diabetic arteries than in control arteries. 2,6-Dimethylpiperidinecarbonyl-gamma-methyl-Leu-N(in)-(methoxycarbonyl)-D-Trp-D-Nle (BQ-788; endothelin ET(B) receptor antagonist) enhanced the contraction elicited by endothelin-1 in control arteries and displaced to the right the contractile curve for endothelin-1 in diabetic arteries. In summary, diabetes induces hyperreactivity of the rabbit carotid artery to endothelin-1 by a mechanism that at least includes: (1) enhanced activity of muscular endothelin ET(A) receptors; (2) impairment of endothelin ET(B) receptor-mediated nitric oxide (NO) release; and (3) enhancement of the production of thromboxane A(2).     

Endothelin receptors in the human coronary artery,ventricle and atrium

Summary In the present experiments we investigated endothelin (ET) receptors in the human coronary artery, and in ventricular and atrial muscle using quantitative receptor autoradiography. Displacement of [¹²⁵I]Sf6b (Sarafotoxin S6b) (30 pM)- and [¹²⁵I]ET-1 (30 pM)-labeled binding sites was studied using ET-1, the ET_A receptor selective ligand BQ-123 (cyclo[D-Asp-L-Pro-D Val-L-Leu-D-Trp-]), and the ETB receptor selective ligand [Ala^1,3,11,15]ET-1.Specific binding was more dense in atrium and coronary artery (relative optical density (r.o.d.): 0.14±0.01 and 0.16±0.01, respectively) than in ventricular muscle (r.o.d.: 0.10±0.01). In the coronary artery, binding was especially dense in the media. ET-1 displaced [¹²⁵I]ET-1 and [¹²⁵I]Sf6b monophasically in atrium, ventricle and coronary artery. [Ala^1,3,11,15]ET 1 and BQ-123 displaced [¹²⁵I]ET-1 and [¹²⁵I]Sf6b-labeled sites biphasically in the ventricle and in the atrium. In the human coronary artery, [Ala^1,3,11,15]ET-1 and BQ-123 displaced [¹²⁵I]ET-1-labeled sites monophasically (pIC₅₀): ET-1 (9.72±0.12) > BQ-123 (6.84±0.08) > [Ala^1,3,11,15]ET-1 (6.40±0.12). In contrast, [Ala^1,3,11,15]ET-1 and BQ-123 displaced [¹²⁵I] Sf6b-labeled coronary artery sites biphasically (high affinity pIC₅₀: BQ-123, 9.03±0.25;[Ala^1,3,11,15]ET-1, 8.40±0.14; low affinity pIC₅₀: BQ-123, 7.24±0.14; [Ala^1,3,11,15]ET-1, 6.99±0.09).These data indicate that both [¹²⁵I]ET-1 and [¹²⁵I] Sf6b-labeled ET_A and ET_B binding sites in human ventricular and atrial muscle. In the human coronary artery, both radioligands labeled ET_A binding sites, but [¹²⁵I] Sf6b also labeled a non-ET_A, non-ET_B binding site with relatively high affinity for both BQ-123 and [Ala^1,3,11,15] ET-1. Correspondence to W. A. Bax, at the above address     

Flavan-3-ol esters: new agents for exploring modulatory sites on GABA(A) receptors

BACKGROUND AND PURPOSE

Enhancement of GABAergic function is the primary mechanism of important therapeutic agents such as benzodiazepines, barbiturates, neurosteroids, general anaesthetics and some anticonvulsants. Despite their chemical diversity, many studies have postulated that these agents may bind at a common or overlapping binding site, or share an activation domain. Similarly, we found that flavan-3-ol esters act as positive modulators of GABA_A receptors, and noted that this action resembled the in vitro profile of general anaesthetics. In this study we further investigated the interactions between these agents.

EXPERIMENTAL APPROACH

Using two-electrode voltage clamp electrophysiological recordings on receptors of known subunit composition expressed in Xenopus oocytes, we evaluated positive modulation by etomidate, loreclezole, diazepam, thiopentone, 5α-pregnan-3α-ol-20-one (THP) and the flavan-3-ol ester 2S,3R-trans 3-acetoxy-4′-methoxyflavan (Fa131) on wild-type and mutated GABA_A receptors.

KEY RESULTS

The newly identified flavan, 2S,3S-cis 3-acetoxy-3′,4′-dimethoxyflavan (Fa173), antagonized the potentiating actions of Fa131, etomidate and loreclezole at α1β2 and α1β2γ2L GABA_A receptors. Furthermore, Fa173 blocked the potentiation of GABA responses by high, but not low, concentrations of diazepam, but did not block the potentiation induced by propofol, the neurosteroid THP or the barbiturate thiopental. Mutational studies on ‘anaesthetic-influencing’ residues showed that, compared with wild-type GABA_A receptors, α1M236Wβ2γ2L and α1β2N265Sγ2L receptors are resistant to potentiation by etomidate, loreclezole and Fa131.

CONCLUSIONS AND IMPLICATIONS

Fa173 is a selective antagonist that can be used for allosteric modulation of GABA_A receptors. Flavan-3-ol derivatives are potential ligands for etomidate/loreclezole-related binding sites at GABA_A receptors and the low-affinity effects of diazepam are mediated via the same site.     

Adenosine negatively regulates duodenal motility in mice: role of A(1) and A(2A) receptors

BACKGROUND AND PURPOSE

Adenosine is considered to be an important modulator of intestinal motility. This study was undertaken to investigate the role of adenosine in the modulation of contractility in the mouse duodenum and to characterize the adenosine receptor subtypes involved.

EXPERIMENTAL APPROACH

RT-PCR was used to investigate the expression of mRNA encoding for A₁, A_2A, A_2B and A₃ receptors. Contractile activity was examined in vitro as changes in isometric tension.

KEY RESULTS

In mouse duodenum, all four classes of adenosine receptors were expressed, with the A_2B receptor subtype being confined to the mucosal layer. Adenosine caused relaxation of mouse longitudinal duodenal muscle; this was antagonized by the A₁ receptor antagonist and mimicked by N⁶-cyclopentyladenosine (CPA), selective A₁ agonist. The relaxation induced by A₁ receptor activation was insensitive to tetrodotoxin (TTX) or N^ω-nitro-l-arginine methyl ester (l-NAME). Adenosine also inhibited cholinergic contractions evoked by neural stimulation, effect reversed by the A₁ receptor antagonist, but not myogenic contractions induced by carbachol. CPA and 2-p-(2-carboxyethyl) phenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride hydrate (CGS-21680), A_2A receptor agonist, both inhibited the nerve-evoked cholinergic contractions. l-NAME prevented only the CGS-21680-induced effects. S-(4-Nitrobenzyl)-6-thioinosine, a nucleoside uptake inhibitor, reduced the amplitude of nerve-evoked cholinergic contractions, an effect reversed by an A_2A receptor antagonist or l-NAME.

CONCLUSIONS AND IMPLICATIONS

Adenosine can negatively regulate mouse duodenal motility either by activating A₁ inhibitory receptors located post-junctionally or controlling neurotransmitter release via A₁ or A_2A receptors. Both receptors are available for pharmacological recruitment, even if only A_2A receptors appear to be preferentially stimulated by endogenous adenosine.

LINKED ARTICLE

This article is commented on by Antonioli et al., pp. 1577–1579 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01529.x     

Renal protective effect of YM598, a selective endothelin ET(A) receptor antagonist,against diabetic nephropathy in OLETF rats

We have investigated the effect of potassium (E)-N-[6-methoxy-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl) pyrimidin-4-yl]-2-phenylenthenesulfonamidate (YM598), a selective endothelin ET(A) receptor antagonist, on renal function in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an animal model of type II diabetes. YM598 (0.1 or 1 mg kg(-1)), enalapril (5 mg kg(-1)), an angiotensin-converting enzyme inhibitor, or vehicle was administered once daily by gastric gavage to 22-week-old male Otsuka Long-Evans Tokushima Fatty rats for 32 weeks. Enalapril but not YM598 mildly lowered blood pressure in the diabetic rats. YM598 blunted the development of albuminuria in a dose-dependent manner. High dose of YM598 reduced albuminuria comparable to enalapril. Urinary endothelin-1 excretion was greater in the diabetic than in the control rats, and was not substantially influenced by the agents. These data suggest that endothelin is involved in the progression of diabetic nephropathy in Otsuka Long-Evans Tokushima Fatty rats, and an endothelin ET(A) receptor antagonist may be useful for the treatment of diabetic nephropathy.     

2'-Methoxy-6-methylflavone: a novel anxiolytic and sedative with subtype selective activating and modulating actions at GABA(A) receptors

BACKGROUND AND PURPOSE

Flavonoids are known to have anxiolytic and sedative effects mediated via actions on ionotropic GABA receptors. We sought to investigate this further.

EXPERIMENTAL APPROACH

We evaluated the effects of 2′-methoxy-6-methylflavone (2′MeO6MF) on native GABA_A receptors in new-born rat hippocampal neurons and determined specificity from 18 human recombinant GABA_A receptor subtypes expressed in Xenopus oocytes. We used ligand binding, two-electrode voltage clamp and patch clamp studies together with behavioural studies.

KEY RESULTS

2′MeO6MF potentiated GABA at α2β1γ2L and all α1-containing GABA_A receptor subtypes. At α2β2/3γ2L GABA_A receptors, however, 2′MeO6MF directly activated the receptors without potentiating GABA. This activation was attenuated by bicuculline and gabazine but not flumazenil indicating a novel site. Mutation studies showed position 265 in the β1/2 subunit was key to whether 2′MeO6MF was an activator or a potentiator. In hippocampal neurons, 2′MeO6MF directly activated single-channel currents that showed the hallmarks of GABA_A Cl^- currents. In the continued presence of 2′MeO6MF the single-channel conductance increased and these high conductance channels were disrupted by the γ2(381–403) MA peptide, indicating that such currents are mediated by α2/γ2-containing GABA_A receptors. In mice, 2′MeO6MF (1–100 mg·kg⁻¹; i.p.) displayed anxiolytic-like effects in two unconditioned models of anxiety: the elevated plus maze and light/dark tests. 2′MeO6MF induced sedative effects at higher doses in the holeboard, actimeter and barbiturate-induced sleep time tests. No myorelaxant effects were observed in the horizontal wire test.

CONCLUSIONS AND IMPLICATIONS

2′MeO6MF will serve as a tool to study the complex nature of the activation and modulation of GABA_A receptor subtypes.     

Role of endothelin receptors,calcium and nitric oxide in the potentiation by endothelin-1 of the sympathetic contraction of rabbit ear artery during cooling

1. To examine further the potentiation by endothelin-1 on the vascular response to sympathetic stimulation, we studied the isometric response of isolated segments, 2 mm long, from the rabbit central ear artery to electrical field stimulation (1–8 Hz), under different conditions, at 37°C and during cooling (30°C).
2. Electrical stimulation produced frequency-dependent contraction, which was reduced (about 63% for 8 Hz) during cooling. At 30°C, but not at 37°C, endothelin-1 (1, 3 and 10 nM) potentiated the contraction to electrical stimulation in a dose-dependent way (from 43±7% to 190±25% for 8 Hz).
3. This potentiation by endothelin-1 was reduced by the antagonist for endothelin ET_A receptors BQ-123 (10 μM) but not by the antagonist for endothelin ET_B receptors BQ-788 (10 μM). The agonist for endothelin ET_B receptors IRL-1620 (0.1 μM) did not modify the contraction to electrical stimulation.
4. The blocker of L-type Ca²⁺ channels verapamil (10 μM l⁻¹) reduced (about 72% for 8 Hz) and the unspecific blocker of Ca²⁺-channels NiCl₂ (1 mM) practically abolished (about 98%), the potentiating effects of endothelin-1 found at 30°C.
5. Inhibition of nitric oxide synthesis with N^G-nitro-L-arginine (L-NOARG, 0.1 mM) increased the contraction to electrical stimulation at 30°C more than at 37°C (for 8 Hz, this increment was 297±118% at 30°C, and 66±15% at 37°C). Endothelium removal increased the contraction to electrical stimulation at 30°C (about 91% for 8 Hz) but not at 37°C. Both L-NOARG and endothelium removal abolished the potentiating effects of endothelin-1 on the response to electrical stimulation found at 30°C.
6. These results in the rabbit ear artery suggest that during cooling, endothelin-1 potentiates the contraction to sympathetic stimulation, which could be mediated at least in part by increasing Ca²⁺ entry after activation of endothelin ET_A receptors. This potentiating effect of endothelin-1 may require the presence of an inhibitory tone due to endothelial nitric oxide.

     

Expression and functional role of adenosine receptors in regulating inflammatory responses in human synoviocytes

Background and purpose:

Adenosine is an endogenous modulator, interacting with four G-protein coupled receptors (A₁, A_2A, A_2B and A₃) and acts as a potent inhibitor of inflammatory processes in several tissues. So far, the functional effects modulated by adenosine receptors on human synoviocytes have not been investigated in detail. We evaluated mRNA, the protein levels, the functional role of adenosine receptors and their pharmacological modulation in human synoviocytes.

Experimental approach:

mRNA, Western blotting, saturation and competition binding experiments, cyclic AMP, p38 mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB activation, tumour necrosis factor α (TNF-α) and interleukin-8 (IL-8) release were assessed in human synoviocytes isolated from patients with osteoarthritis.

Key results:

mRNA and protein for A₁, A_2A, A_2B and A₃ adenosine receptors are expressed in human synoviocytes. Standard adenosine agonists and antagonists showed affinity values in the nanomolar range and were coupled to stimulation or inhibition of adenylyl cyclase. Activation of A_2A and A₃ adenosine receptors inhibited p38 MAPK and NF-κB pathways, an effect abolished by selective adenosine antagonists. A_2A and A₃ receptor agonists decreased TNF-α and IL-8 production. The phosphoinositide 3-kinase or G_s pathways were involved in the functional responses of A₃ or A_2A adenosine receptors. Synoviocyte A₁ and A_2B adenosine receptors were not implicated in the inflammatory process whereas stimulation of A_2A and A₃ adenosine receptors was closely associated with a down-regulation of the inflammatory status.

Conclusions and implications:

These results indicate that A_2A and A₃ adenosine receptors may represent a potential target in therapeutic modulation of joint inflammation.     

The role of kinin B1 and B2 receptors in the scratching behaviour induced by proteinase-activated receptor-2 agonists in mice

Background and purpose:

Activation of the proteinase-activated receptor-2 (PAR-2) induces scratching behaviour in mice. Here, we have investigated the role of kinin B₁ and B₂ receptors in the pruritogenic response elicited by activators of PAR-2.

Experimental approach:

Scratching was induced by an intradermal (i.d.) injection of trypsin or the selective PAR-2 activating peptide SLIGRL-NH₂ at the back of the mouse neck. The animals were observed for 40 min and their scratching response was quantified.

Key results:

I.d. injection of trypsin or SLIGRL-NH₂ evoked a scratching behaviour, dependent on PAR-2 activation. Mice genetically deficient in kinin B₁ or B₂ receptors exhibited reduced scratching behaviour after i.d. injection of trypsin or SLIGRL-NH₂. Treatment (i.p.) with the non-peptide B₁ or B₂receptor antagonists SSR240612 and {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657, respectively, prevented the scratching behaviour caused by trypsin or SLIGRL-NH₂. Nonetheless, only treatment i.p. with the peptide B₂receptor antagonist, Hoe 140, but not the B₁receptor antagonist (DALBK), inhibited the pruritogenic response to trypsin. Hoe 140 was also effective against SLIGRL-NH₂-induced scratching behaviour when injected by i.d. or intrathecal (i.t.) routes. Also, the response to SLIGRL-NH₂ was inhibited by i.t. (but not by i.d.) treatment with DALBK. Conversely, neither Hoe 140 nor DALBK were able to inhibit SLIGRL-NH₂-induced scratching behaviour when given intracerebroventricularly (i.c.v.).

Conclusions and implications:

The present results demonstrated that kinins acting on both B₁ and B₂ receptors played a crucial role in controlling the pruriceptive signalling triggered by PAR-2 activation in mice.     

Role of endothelin ET(B) receptors in the renal hemodynamic and excretory responses to big endothelin-1

We determined the role of endothelin ET(B) receptor in the renal hemodynamic and excretory responses to big endothelin-1, using A-192621, a selective endothelin ET(B) receptor antagonist and the spotting-lethal (sl) rat, which carries a naturally occurring deletion in the endothelin ET(B) receptor gene. An intravenous injection of big endothelin-1 produced a hypertensive effect, which is greater in wild-type (+/+) rats pretreated with A-192621 and in homozygous (sl/sl) rats. Big endothelin-1 markedly increased urine flow, urinary excretion of sodium and fractional excretion of sodium in wild-type rats treated with the vehicle. These excretory responses to big endothelin-1 were markedly reduced by pharmacological endothelin ET(B) receptor blockade. On the other hand, big endothelin-1 injection to the endothelin ET(B) receptor-deficient homozygous animals resulted in a small diuretic effect. When renal perfusion pressure was protected from big endothelin-1-induced hypertension by an aortic clamp, the excretory responses in vehicle-treated wild-type rats were markedly attenuated. In homozygous or A-192621-treated wild-type rats, there was a small but significant decreasing effect in urine flow. In addition, big endothelin-1 significantly elevated nitric oxide (NO) metabolite production in the kidney of wild-type rats but not in the homozygous rats. We suggest that the diuretic and natriuretic responses to big endothelin-1 consist of pressure-dependent and pressure-independent effects and that the increased NO production via the activation of endothelin ET(B) receptors in the kidney is closely related to the big endothelin-1-induced excretory responses.     

Chronic blockade of cannabinoid CB2 receptors induces anxiolytic-like actions associated with alterations in GABA(A) receptors

BACKGROUND AND PURPOSE

The aim of this study was to explore the effects of CB₂ receptor agonist and antagonist in the regulation of anxiety-like behaviours.

EXPERIMENTAL APPROACHES

Effects of acute and chronic treatment with the CB₂ receptor agonist JWH133 and CB₂ receptor antagonist AM630 were evaluated in the light-dark box (LDB) and elevated plus maze (EPM) tests in Swiss ICR mice. CB₂ receptor, GABA_Aα₂ and GABA_Aγ₂ gene and protein expression in the cortex and amygdala of mice chronically treated with JWH133 or AM630 were examined by RT-PCR and Western blot. Effects of chronic AM630 treatment were evaluated in spontaneously anxious DBA/2 mice in LDB.

KEY RESULTS

Acute JWH133 treatment failed to produce any effect. Acute AM630 treatment increased anxiety and was blocked by pre-treatment with JWH133. Chronic JWH133 treatment increased anxiety-like behaviour whereas chronic AM630 treatment was anxiolytic in LDB and EPM tests. Chronic AM630 treatment increased gene and reduced protein expression of CB₂ receptors, GABA_Aα₂ and GABA_Aγ₂ in cortex and amygdala. Chronic JWH133 treatment resulted in opposite gene and protein alterations. In addition, chronic AM630 administration decreased the anxiety of DBA/2 mice in the LDB test.

CONCLUSIONS AND IMPLICATIONS

The opposing behavioural and molecular changes observed after chronic treatment with AM630 or JWH133 support the key role of CB₂ receptors in the regulation of anxiety. Indeed, the efficacy of AM630 in reducing the anxiety of the spontaneously anxious DBA/2 strain of mice strengthens the potential of the CB₂ receptor as a new target in the treatment of anxiety-related disorders.     

Effects of tumour necrosis factor-α on the coronary circulation of the rat isolated perfused heart: a potential role for thromboxane A2 and sphingosine

1. The actions of tumour necrosis factor-α (TNF-α) on the coronary circulation were investigated in the rat isolated heart, perfused under constant flow, recirculating conditions.
2. An early increase in coronary perfusion pressure (CPP) was observed upon treatment with TNF-α (increase in CPP 10 min after TNF-α treatment: 45±12 mmHg vs control: 15±4 mmHg, P<0.05). The role of sphingosine, prostanoids and endothelins, in this coronary constrictor action, was investigated with the use of pharmacological inhibitors and antagonists.
3. The TNF-α induced increase in coronary tone was blocked by indomethacin, 10 μM (increase in CPP after 10 min: 13±4 mmHg vs TNF-α alone, P<0.05).
4. The thromboxane receptor antagonist GR32191, 10 μM, attenuated the TNF-α induced coronary constriction (12±2 mmHg vs TNF-α alone, P<0.05), as did the joint thromboxane A₂ synthesis inhibitor and receptor antagonist ZD1542, 10 μM (8±1 mmHg vs TNF-α alone, P<0.05).
5. The ceramidase inhibitor N-oleoylethanolamine (NOE), 1 μM, also blocked the TNF-α induced response (8±4 mmHg vs TNF-α alone, P<0.05).
6. In contrast, the coronary constrictor action of TNF-α was not inhibited by the endothelin_A/B receptor antagonist bosentan, 3 μM (38±9 mmHg vs TNF-α, P=NS).
7. These data indicated that the early coronary vasoconstriction induced by TNF-α was mediated by both thromboxane A₂ and sphingosine, suggesting an interaction between both the sphingomyelinase and phospholipase A₂ metabolic pathways.

     

Facilitation of noradrenaline release by adenosine A(2A) receptors in the epididymal portion and adenosine A(2B) receptors in the prostatic portion of the rat vas deferens

The adenosine-receptor modulation of noradrenaline release was compared in prostatic and epididymal portions of rat vas deferens. In both portions, tritium overflow elicited by electrical stimulation (100 pulses/8 Hz) was reduced by the adenosine A(1) receptor agonist, N(6)-cyclopentyladenosine, and enhanced by the nonselective receptor agonist, 5'-N-ethylcarboxamidoadenosine, in the presence of the adenosine A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 20 and 100 nM). The adenosine A(2A) receptor agonist, 2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxamidoadenosine, increased tritium overflow, but only in the epididymal portion. The enhancement caused by NECA was prevented by the adenosine A(2A) receptor 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; 20 nM), in the epididymal and by the adenosine A(2B) receptor antagonist, alloxazine (1 microM), in the prostatic portion. Inhibition of adenosine uptake enhanced tritium overflow in both portions, an effect blocked by ZM 241385 in the epididymal and by alloxazine in the prostatic portion. The results indicate that adenosine exerts an adenosine A(1) receptor-mediated inhibition, in both portions, and facilitation mediated by adenosine A(2A) receptors in the epididymal and by A(2B) receptors in the prostatic portion.     

Inhibition of tumour necrosis factor and reversal of endotoxin-induced shock by U-83836E,a 'second generation' lazaroid in rats

1. Antioxidants can exert protective effects in endotoxic shock by either a reduction of the oxidant damage or attenuation of Tumour Necrosis Factor (TNF-α) production.
2. Lazaroids are a family of compounds that inhibit lipid peroxidation. Besides, they can also reduce TNF-α. U-83836E is a new lazaroid lacking the glucocorticoid ring.
3. Aim of our study was to investigate the effect of U-83836E on TNF-α production either in vivo or in vitro. Endotoxic shock was produced in male rats by a single intravenous (i.v.) injection of 20 mg kg⁻¹ of S. enteritidis lipopolysaccharide (LPS). LPS administration reduced survival rate (0% survival, 72 h after endotoxin administration), decreased mean arterial blood pressure, increased serum and macrophage TNF-α and enhanced plasma malonylaldehyde (MAL) levels. Furthermore aortic rings from shocked rats showed a marked hyporeactivity to phenylephrine (PE 1 nM–10 μM).
4. Treatment with U-83836E (7.5, 15 and 30 mg kg⁻¹, i.v.) 5 min after endotoxin challenge significantly protected against LPS induced lethality (90% survival rate and 80% survival rate 24 h and 72 h after LPS injection respectively, following the highest dose of the drug), reduced hypotension, blunted plasma MAL, decreased serum and macrophage TNF-α and restored the hyporeactivity of aortic rings to control values. In vitro LPS stimulation (50 μg ml⁻¹ for 4 h) significantly increased cytokine production in macrophages (MΦ) harvested from untreated normal rats. Pretreatment with pertussis toxin (PT; 0.1, 1 and 10 ng ml⁻¹ 4 h before LPS) significantly increased TNF-α production. PT effects on these LPS responses were correlated with a PT mediated ADP ribosylation of a 41 kDa protein. U-83836E (50 μM) reduced, in a dose dependent manner, LPS induced TNF-α production and inhibited the PT effects on cytokine production and on ADP ribosylation of the protein.
5. Our data suggest that lazaroids may affect the early events associated with LPS receptor mediated activation of a G protein in LPS induced TNF-α production. These molecular events may explain, at least in part, the in vivo inhibition of cytokine production and reversal of endotoxic shock.

     

A study of subunit selectivity,mechanism and site of action of the delta selective compound 2 (DS2) at human recombinant and rodent native GABAA receptors

Background and Purpose

Most GABA_A receptor subtypes comprise 2α, 2β and 1γ subunit, although for some isoforms, a δ replaces a γ-subunit. Extrasynaptic δ-GABA_A receptors are important therapeutic targets, but there are few suitable pharmacological tools. We profiled DS2, the purported positive allosteric modulator (PAM) of δ-GABA_A receptors to better understand subtype selectivity, mechanism/site of action and activity at native δ-GABA_A receptors.

Experimental Approach

Subunit specificity of DS2 was determined using electrophysiological recordings of Xenopus laevis oocytes expressing human recombinant GABA_A receptor isoforms. Effects of DS2 on GABA concentration–response curves were assessed to define mechanisms of action. Radioligand binding and electrophysiology utilising mutant receptors and pharmacology were used to define site of action. Using brain-slice electrophysiology, we assessed the influence of DS2 on thalamic inhibition in wild-type and δ^0/0 mice.

Key Results

Actions of DS2 were primarily determined by the δ-subunit but were additionally influenced by the α, but not the β, subunit (α4/6βxδ > α1βxδ >> γ2-GABA_A receptors > α4β3). For δ-GABA_A receptors, DS2 enhanced maximum responses to GABA, with minimal influence on GABA potency. (iii) DS2 did not act via the orthosteric, or known modulatory sites on GABA_A receptors. (iv) DS2 enhanced tonic currents of thalamocortical neurones from wild-type but not δ^0/0 mice.

Conclusions and Implications

DS2 is the first PAM selective for α4/6βxδ receptors, providing a novel tool to investigate extrasynaptic δ-GABA_A receptors. The effects of DS2 are mediated by an unknown site leading to GABA_A receptor isoform selectivity.     

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     

Cardiovascular effects of activation of central alpha7 and alpha4beta2 nAChRs: a role for vasopressin in anaesthetized rats

BACKGROUND AND PURPOSE: Central application of nicotine causes the release of vasopressin and affects blood pressure. Involvement of the 5 neuronal nicotinic receptor groups, alpha2(*)-alpha7(*) in these effects is unknown. The availability of selective agonists for alpha7 (PSAB-OFP) and alpha4beta2 (TC-2559) nACh receptors allowed their role to be investigated. EXPERIMENTAL APPROACH: Recordings were made of arterial blood pressure, heart rate and renal sympathetic nerve activity in anaesthetized male rats with neuromuscular blockade and artificial respiration. Effects of the agonists, PSAB-OFP (1-10 micromol kg(-1)) and TC-2559 (1-10 micromol kg(-1)) on these variables given intracerebroventricularly (i.c.v.) and intracisternally (i.c.) in the presence or absence of the antagonists, DhbetaE (10 micromol kg(-1)) and MLA (0.5 micromol kg(-1)), for the appropriate nicotinic receptor subtypes, respectively, and a V(1) receptor antagonist, given i.v. or centrally, were investigated. KEY RESULTS: Both agonists given i.c.v. caused a delayed rise in blood pressure and renal nerve activity which could be blocked only with the appropriate antagonist. The agonists had an earlier onset of action when given i.c., favouring the brainstem as the major site of action. The effects of these agonists were also attenuated by the V(1) receptor antagonist given i.v. and blocked when this antagonist was given centrally. Antagonists had no effect on baseline variables.CONCLUSIONS AND IMPLICATIONS: Activation of alpha4beta2 and alpha7 receptors in the brainstem is mainly responsible for the cardiovascular effects of activating these receptors, which have a similar profile of action. These actions, although independent, are mediated by the central release of vasopressin.     

Effects of L-NG-nitro-arginine on noradrenaline induced contraction in the rat anococcygeus muscle

1. The influence of L-N^G-nitro-arginine (L-NOARG, 30 μM) on contractile responses to exogenous noradrenaline was studied in the rat anococcygeus muscle.
2. Noradrenaline (0.1–100 μM) contracted the muscle in a concentration-dependent manner. L-NOARG (30 μM) had no effect on noradrenaline responses.
3. Phenoxybenzamine (Pbz 0.1 μM) depressed by 46% (P<0.001) the maximum response and shifted to the right (P<0.001) the E/[A] curve to noradrenaline (pEC₅₀ control: 6.92±0.09; pEC₅₀ Pbz: 5.30±0.10; n=20).
4. The nested hyperbolic null method of analysing noradrenaline responses after phenoxybenzamine showed that only 0.61% of the receptors need to be occupied to elicit 50% of the maximum response, indicating a very high functional receptor reserve.
5. Contractile responses to noradrenaline after partial α₁-adrenoceptor alkylation with phenoxybenzamine (0.1 μM) were clearly enhanced by L-NOARG.
6. The potentiating effect of L-NOARG on noradrenaline responses after phenoxybenzamine was reversed by (100 μM) L-arginine but not by (100 μM) D-arginine.
7. These results indicate that spontaneous release of NO by nitrergic nerves can influence the α₁-adrenoceptor-mediated response to exogenous noradrenaline.

     

Peripheral kinin B(1) and B(2) receptor-operated mechanisms are implicated in neuropathic nociception induced by spinal nerve ligation in rats

The kinin system can contribute distinctly to the sensory changes associated with different models of nerve injury-induced neuropathic pain. This study examines the roles of kinin B(1) and B(2) receptor-operated mechanisms in alterations in nociceptive responses of rats submitted to unilateral L5/L6 spinal nerve ligation (SNL) injury. Behavioural responses to ipsilateral hind paw stimulation with acetone (evaporation-evoked cooling), radiant heat (Hargreaves method) or von Frey hairs revealed that SNL rats developed long-lasting cold allodynia (from Days 3 to 40 post-surgery, peak on Day 6), heat hyperalgesia (stable peak from Days 9 to 36) and tactile allodynia (stable peak from Days 3 to 51). SNL rats manifested nocifensive responses to intraplantar injections on Day 12 of the selective B(1) receptor agonist des-Arg(9)-bradykinin (DABK) and augmented responses to the selective B(2) receptor agonist bradykinin (BK; each at 0.01-1nmol/paw). Systemic treatment of SNL rats with des-Arg(9)-Leu(8)-BK or HOE 140 (peptidic B(1) and B(2) receptor antagonists, respectively; 0.1-1mumol/kg, i.p.) selectively blocked responses triggered by DABK and BK (1nmol/paw) and alleviated partially and transiently established cold allodynia, heat hyperalgesia and (to a lesser extent) tactile allodynia. Western blot analysis revealed enhanced expression of kinin B(1) and B(2) receptor protein in ipsilateral L4-L6 spinal nerve and hind paw skin samples collected on Day 12 after SNL surgery. These results indicate that peripheral pronociceptive kinin B(1) and B(2) receptor-operated mechanisms contribute significantly to the maintenance of hind paw cold and mechanical allodynia and heat hyperalgesia induced by L5/L6 SNL in rats.