The effect of nitric oxide on cytokine-induced release of PGE2 by human cultured astroglial cells

1. The role of the L-arginine-nitric oxide (NO) pathway on the formation of prostaglandin E₂ (PGE₂) by human cultured astroglial cells incubated with interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α) was investigated.
2. Incubation of T 67 astroglial cell line with IL-β (10 ng ml⁻¹) and TNF-α (500 u ml⁻¹) produced a significant (P<0.05) increase of both nitrite (the breakdown product of NO), cyclic GMP and PGE₂ levels in cell supernatants. N^ω-nitro-L-arginine methyl ester (L-NAME; 20–300 μM), an inhibitor of NO synthase (NOS), inhibited the increase of cyclic GMP and nitrite levels found in supernatants of cytokine-treated astroglial cells and reduced the release of PGE₂. The latter effect showed that the enhanced arachidonic acid (AA) metabolism subsequent to stimulation of astroglial cells with IL-1β and TNF-α was, at least in part, induced by NO. This occurred also when sodium nitroprusside (SNP; 120 μM), an NO donor, was incubated with astroglial cells, an effect antagonized by oxyhaemoglobin (OxyHb; 10 μM).
3. The inhibition elicited by L-NAME on PGE₂-release by cytokine-treated astroglial cells was reversed by adding AA (40 μM), showing that the effect of NO on cytokine-dependent PGE₂ release occurred at the cyclo-oxygenase (COX) level. Furthermore, the release of PGE₂ in cytokine-treated astroglial cells was inhibited by indomethacin (10 μM), a COX inhibitor as well as by preincubating cells with dexamethasone (20 μM), an inhibitor of inducible enzymes, showing that the inducible isoform of COX (COX-2) was involved.
4. On the other hand, pretreating astroglial cells with methylene blue (MB; 10 μM), an inhibitor of NO biological activity acting at the guanylate cyclase level, failed to affect PGE₂ release in cytokine-treated astroglial cells, leading to the conclusion that cyclic GMP changes related to NO formation are not involved in the generation of AA metabolites.
5. The present experiments demonstrated that the release of PGE₂ by astroglial cells pretreated with IL-1β and TNF-α is due to enhanced COX-2 activity via activation of the L-arginine-NO pathway, and this may be relevant to the understanding of the pathophysiological mechanisms underlying neuroimmune disorders.

     

Evidence for a dilator function of 8-iso prostaglandin F2α in rat pulmonary artery

1. 8-Iso prostaglandin F_2α (8-iso PGF_2α) is one of a series of prostanoids formed independently of the cyclo-oxygenase pathway. It has been shown to be upregulated in many conditions of oxidant stress where its formation is induced by free radical-catalysed actions on arachidonic acid. As 8-iso PGF_2α is formed in vivo in diseases in which oxidant stress is high such as septic shock, we have assessed the relative potency and efficacy of this compound in pulmonary arteries from control and lipopolysaccharide (LPS)-treated rats.
2. Several studies have characterized the contractile actions of 8-iso PGF_2α on various smooth muscle preparations, but its potential dilator actions have not been addressed. Thus these studies examined both the contractile and dilator actions of 8-iso PGF_2α in rat pulmonary artery rings. The thromboxane mimetic U46619, PGE₂ sodium nitroprusside (SNP) and acetyl choline (ACh) were used for comparison. Each prostanoid had to be dissolved in ethanol to a maximum concentration of 1×10⁻² M. At high concentrations, ethanol directly contracted pulmonary vessels. We were therefore limited by the actions of the vehicle such that we were unable to add prostanoids at concentrations higher than 1×10⁻⁴ M. In some cases this meant that maximum responses were not achieved and in these cases the E_max and pD₂ values are apparent estimates.
3. The following rank order of potency was obtained from contractile studies; U46619>8-iso PGF_2α>PGE₂, each prostanoid producing concentration-dependent contractions (10⁻¹⁰3×10⁻⁴ M, 10⁻⁹10⁻⁴ M, 10⁻⁸10⁻⁴ M, respectively). As has been shown previously for other smooth muscle preparations, the thromboxane receptor (TP) antagonist ICI 192605, (1×10⁻⁶, 1×10⁻⁵ and 1×10⁻⁴ M), inhibited the contractions of 8-iso PGF_2α in a concentration-dependent fashion.
4. The nitric oxide synthase inhibitor, N^G-nitro-L-arginine methyl ester (L-NAME; 1×10⁻⁴ M), enhanced the contractile function of both 8-iso PGF_2α and PGE₂, but had no effect on that caused by U46619. Similarly, L-NAME inhibited the dilator function of all agents tested except the exogenous nitric oxide (NO) donor SNP, indicating that PGE₂ and 8-iso PGF_2α like ACh, act through the release of NO. The specificity of the effects of L-NAME were confirmed in studies with the inactive enantiomer D-NAME (1×10⁻⁴ M), which did not affect the contractile or the dilator actions of 8-iso PGF_2α. Furthermore, ICI 192605 enhanced the dilator actions of 8-iso PGF_2α, suggesting that the dilator component of 8-iso PGF_2α was achieved via activation of a non-TP receptor.
5. Isoprostanes may modulate vascular tone by a direct action on TP receptors to cause contraction and via a distinct receptor leading to the release of NO to cause dilatation.

     

Early effects of acute γ-radiation on vascular arterial tone

1. To determine the acute effects of irradiation on the functionality of vessel, rat aortic rings were mounted in an organ bath for isometric tension measurements and irradiated (⁶⁰Co, 1 Gy min⁻¹, 15 min).
2. Irradiation, which is without effect on non-contracted or endothelium-denuded vessels, led to an immediate and reversible increase in vascular tone on (−)-phenylephrine (1 μM)-precontracted aortic rings. The tension reached a plateau about 5 min after the beginning of irradiation.
3. The maximal radiation-induced contraction occurred on aortic rings relaxed by acetylcholine (ACh) (1 μM). In this condition, the addition of catalase (1000 u ml⁻¹), which reduces hydrogen peroxide, and DMSO (0.1% v/v), which scavenges hydroxyl radical, had no influence on tension level while superoxide dismutase (SOD) (100 u ml⁻¹), a superoxide anion scavenger, reduced the observed contraction. A similar result was obtained in the presence of indomethacin (10 μM), a cyclo-oxygenase blocker.
4. Pretreatment of rings with the nitric oxide synthase inhibitor, N_ω-nitro-L-arginine methyl ester (L-NAME) (10–100 μM) inhibited the radiation-induced contraction.
5. This effect was dose rate-dependent and even occurred for a very low dose rate (0.06 Gy min⁻¹).
6. The present results indicate that γ-radiation induces an instantaneous vascular tone increase that is endothelium and dose rate-dependent. This effect is (i) maximal when nitric oxide (NO) is produced, (ii) greatly reduced by SOD and (iii) inhibited by L-NAME, suggesting a major involvement of complexes between NO and superoxide anion.

     

Loss of endothelium-derived nitric oxide in rabbit aorta by oxidant stress: restoration by superoxide dismutase mimetics

1. Structurally distinct superoxide dismutase (SOD) mimetics were examined for their ability to protect nitric oxide (NO) from destruction by oxidant stress in rabbit aorta.
2. These were the spin traps, PTIYO (4-phenyl-2,2,5,5-tetramethyl imidazolin-1-yloxy-5-oxide), tempol (4-hydroxy 2,2,6,6,-tetramethylpiperidine-1-oxyl) and tiron (4,5-dihydroxy-1,3-benzene-disulphonic acid), the metal salts, CuSO₄ and MnCl₂, and the metal-based agents CuDIPS (Cu (II)-[diisopropylsalicylate]₂) and MnTMPyP (Mn (III) tetrakis [1-methyl-4-pyridyl]porphyrin).
3. Oxidant stress was generated in isolated aortic rings by inactivating endogenous Cu/Zn SOD with diethyldithiocarbamate (DETCA; 60 min) either alone at 3 mM or at 0.3 mM in combination with superoxide generation using xanthine oxidase (XO; 4.8 mu ml⁻¹) and hypoxanthine (HX; 0.1 mM).
4. Acetylcholine (ACh)-induced relaxation was inhibited by DETCA (3 mM, 60 min) and was not restored by exogenous SOD (250 u ml⁻¹), suggesting the oxidant stress was intracellular. MnTMPyP (600 μM and 1 mM) and MnCl₂ (100 μM) were the only agents to reverse the blockade of ACh-induced relaxation.
5. Addition of XO/HX to DETCA (0.3 mM)-treated tissues powerfully impaired ACh-induced relaxation and exogneous SOD (250 u ml⁻¹) fully reversed the blockade, suggesting the oxidant stress was extracellular. CuDIPS (0.1–3 μM), CuSO₄ (0.3–3 μM), MnCl₂ (1–100 μM) and MnTMPyP (100–600 μM) also reversed blockade powerfully, tempol (30 μM–1 mM) and tiron (0.3–10 mM) reversed blockade weakly and PTIYO (10–300 μM) enhanced the blockade.
6. Thus, MnTMPyP was the only SOD mimetic to restore NO-dependent relaxation in conditions of both extracellular and intracellular oxidant stress. This agent may, therefore, provide a lead in the development of SOD mimetics for the treatment of pathologies associated with oxidant stress.

     

Interaction between superoxide anion and nitric oxide in the regulation of vascular endothelial function

1. Nitric oxide (NO)-mediated, endothelium-dependent vasodilator function in rat aortic smooth muscle was investigated in an in vitro model of endogenous vascular superoxide anion stress, generated by pretreatment with the Cu/Zn superoxide dismutase (SOD, EC 1.15.1.1) inhibitor, diethyldithiocarbamate (DETCA).
2. Contraction to noradrenaline (NA, 1 nM–1 μM) in endothelium-intact vessels was augmented after a 30 min pretreatment with DETCA (10 mM) followed by 30 min washout. This effect was abolished by N^G-nitro-L-arginine methyl ester (L-NAME, 0.3 mM) and removal of the endothelium and partially reversed by exogenous Cu/Zn SOD (200 u ml⁻¹).
3. Endothelium- and basal NO-dependent vasorelaxation to the phosphodiesterase (PDE) type V inhibitor ONO-1505 (4-[2-(2-hydroxyethoxy)ethylamino]-2-(1H-imidazol-1-yl)-6-methoxyquinazoline methanesulphonate) (0.1–10 μM) was inhibited after DETCA (10 mM) pretreatment. In addition, the ability of L-NAME (0.3 mM) to enhance established contractile tone was effectively absent.
4. In contrast, DETCA pretreatment did not significantly affect vasorelaxation to acetylcholine (ACh, 1 nM–3 μM) or S-nitroso-N-acetyl penicillamine (SNAP, 0.03–30 μM). However, L-NAME (0.3 mM) unmasked an inhibitory effect of DETCA pretreatment on vasorelaxation to SNAP in endothelium-intact vessels while markedly potentiating vasorelaxation to SNAP in control tissue.
5. L-NAME (0.3 mM)- and exogenous catalase (200 u ml⁻¹)-sensitive vasorelaxation to exogenous Cu/Zn SOD (200 u ml⁻¹) was greater after DETCA (10 mM) pretreatment in endothelium-intact aortic rings. This difference was abolished by catalase (200 u ml⁻¹).
6. In conclusion, tissue Cu/Zn SOD inhibition elicited a selective lesion in basal endothelial function in rat isolated aortic smooth muscle, consistent with the inactivation of basal NO by superoxide anion. The resulting leftward shift in nitrovasodilator reactivity, due to the loss of the tonic depression by basal NO, is likely to mask the inhibitory effect of superoxide anion on agonist-stimulated endothelial function and nitrovasodilator-derived NO, thereby accounting for the differential pattern of endothelial dysfunction after DETCA pretreatment.

     

Differential effect of L-NAME and S-methyl-isothiourea on leukocyte emigration in carrageenin-soaked sponge implants in rat

1. The role of nitric oxide (NO) in leukocyte (polymorphonuclear cells, monocytes and lymphocytes) emigration was studied in a model of carrageenin-sponge implants in rats.
2. The subcutaneous implantation of 1% (w/v) of λ-carrageenin-soaked sponges elicited an inflammatory response that was characterized by a time-related increase in leukocyte infiltration in the sponges and increased levels of nitrite in the exudate. Total leukocyte infiltration and nitrite production were maximal at 24 h and decreased after 48 and 96 h. The mononuclear cell influx was maximal at 48 h (21% of the total leukocytes). Therefore, this time point was used in the successive experiments.
3. Polymorphonuclear cell (PMN) and lymphocyte infiltration in the sponges significantly increased when rats were treated with the non-specific NO-synthase (NOS) inhibitor, N^G-nitro-L-arginine methylester (L-NAME) (1 mg ml⁻¹ in drinking water ad libitum). Monocyte emigration was not affected by L-NAME treatment. The nitrite levels in the exudate of L-NAME-treated rats were significantly reduced. The concomitant ingestion of L-arginine (30 mg ml⁻¹) resulted in a reversion of the L-NAME effect, while D-arginine (30 mg ml⁻¹) had no effect, indicating the involvement of the L-arginine: NO pathway.
4. Administration of L-NAME resulted also in an increased release of tumour necrosis factor-α (TNF-α) and prostacyclin (measured as the stable metabolite, 6-keto-PGF_1α). L-NAME had no effect on monocyte chemoattractant protein-1 (MCP-1) release in the exudate.
5. Since L-NAME may have effects on the local blood flow, phenylephrine (0.034 mg ml⁻² in drinking water) was used as it has an effect on the local blood flow similar to L-NAME. Phenylephrine had no effect on either leukocyte emigration, or on nitrite, TNF-α, prostacyclin or MCP-1 accumulation in the exudate.
6. In contrast, the more selective iNOS inhibitor S-methyl-isothiourea (SMT) (10 μg ml⁻¹ in drinking water) significantly reduced PMNs and lymphocyte influx in the sponge, having no effect on monocyte influx. Moreover, SMT decreased nitrite production in the exudate to a comparable extent as L-NAME.
7. Administration of SMT significantly reduced MCP-1 release in the exudate, without an effect on TNF-α or prostacyclin production. Moreover SMT did not produce any changes in local blood flow.
8. Our results show that a different outcome of the inflammatory process can be obtained depending on the types of NOS inhibitor used.

     

Modulation of IL-1-induced cartilage injury by NO synthase inhibitors: a comparative study with rat chondrocytes and cartilage entities

1. Nitric oxide (NO) is produced in diseased joints and may be a key mediator of IL-1 effects on cartilage. Therefore, we compared the potency of new [aminoguanidine (AG), S-methylisothiourea (SMT), S-aminoethylisothiourea (AETU)] and classical [N^ω-monomethyl-L-arginine (L-NMMA), N^ω-nitro-L-arginine methyl ester (L-NAME)] NO synthase (NOS) inhibitors on the inhibitory effect of recombinant human interleukin-1β (rhIL-1β) on rat cartilage anabolism. Three different culture systems were used: (1) isolated chondrocytes encapsulated in alginate beads; (2) patellae and (3) femoral head caps.
2. Chondrocyte beads and cartilage entities were incubated in vitro for 48 h in the presence of rhIL-1β with a daily change of incubation medium to obtain optimal responses on proteoglycan synthesis and NO production. Proteoglycan synthesis was assessed by incorporation of radiolabelled sodium sulphate [Na₂³⁵SO₄] and NO production by cumulated nitrite release during the period of study.
3. Chondrocytes and patellae, as well as femoral head caps, responded concentration-dependently to IL-1β challenge (0 to 250 U ml⁻¹ and 0 to 15 U ml⁻¹ respectively) by a large increase in nitrite level and a marked suppression of proteoglycan synthesis. Above these concentrations of IL-1β (2500 U ml⁻¹ and 30 U ml⁻¹ respectively), proteoglycan synthesis plateaued whereas nitrite release still increased thus suggesting different concentration-response curves.
4. When studying the effect of NOS inhibitors (1 to 1000 μM) on NO production by cartilage cells stimulated with IL-1β (25 U ml⁻¹ or 5 U ml⁻¹), we observed that: (i) their ability to reduce nitrite level decreased from chondrocytes to cartilage samples, except for L-NMMA and AETU; (ii) they could be roughly classified in the following rank order of potency: AETU>L-NMMA⩾SMT>AG⩾L-NAME and (iii) AETU was cytotoxic when used in the millimolar range.
5. When studying the effect of NOS inhibitors on proteoglycan synthesis by cartilage cells treated with IL-1β, we observed that: (i) they had more marked effects on proteoglycan synthesis in chondrocytes than in cartilage samples; (ii) they could be roughly classified in the following rank order of potency: L-NAME⩾L-NMMA>>AG>SMT>>AETU and (iii) potentiation of the IL-1 effect by AETU was consistent with cytotoxicity in the millimolar range.
6. D-isomers of L-arginine analog inhibitors (1000 μM) were unable to correct nitrite levels or proteoglycan synthesis in IL-1β treated cells. L-arginine (5000 μM) tended to reverse the correcting effect of L-NMMA (1000 μM) on proteoglycan synthesis, thus suggesting a NO-related chondroprotective effect. However, data with L-NAME and SMT argued against a general inverse relationship between nitrite level and proteoglycan synthesis.
7. Dexamethasone (0.1 to 100 μM) (i) failed to inhibit NO production in femoral head caps and chondrocytes beads whilst reducing it in patellae (50%) and (ii) did not affect or worsened the inhibitory effect of IL-1β on proteoglycan synthesis. Such results suggested a corticosteroid-resistance of rat chondrocyte iNOS. Data from patellae supported a possible contribution of subchondral bone in NO production.
8. In conclusion, our results suggest that (i) NO may account only partially for the suppressive effects of IL-1β on proteoglycan synthesis, particularly in cartilage samples; (ii) the chondroprotective potency of NOS inhibitors can not be extrapolated from their effects on NO production by joint-derived cells and (iii) L-arginine analog inhibitors are more promising than S-substituted isothioureas for putative therapeutical uses.

     

Coexistence of purino- and pyrimidinoceptors on activated rat microglial cells

1. Nucleotide-induced currents in untreated (proliferating) and lipopolysaccharide (LPS; 100 ng ml⁻¹) treated (non-proliferating) rat microglial cells were recorded by the whole-cell patch-clamp technique. Most experiments were carried out on non-proliferating microglial cells. ATP (100 nM–1 mM), ADP (10 nM–10 mM) and UTP (1 μM–100 mM), but not uridine (100 μM–10 mM) produced a slow outward current at a holding potential of 0 mV. The effect of UTP (1 mM) did not depend on the presence of extracellular Mg²⁺ (1 mM). The outward current response to UTP (1 mM) was similar in non-proliferating and proliferating microglia.
2. In non-proliferating microglial cells, the ATP (10 μM)-induced outward current was antagonized by suramin (300 μM) or reactive blue 2 (50 μM), whereas 8-(p-sulphophenyl)-theophylline (8-SPT; 100 μM) was inactive. By contrast, the current induced by UTP (1 mM) was increased by suramin (300 μM) and was not altered by reactive blue 2 (50 μM) or 8-SPT (100 μM).
3. The current response to UTP (1 mM) disappeared when K⁺ was replaced in the pipette solution by an equimolar concentration of Cs⁺ (150 mM). However, the effect of UTP (1 mM) did not change when most Cl⁻ was replaced with an equimolar concentration of gluconate⁻ (145 mM). The application of 4-aminopyridine (1 mM) or Cs⁺ (1 mM) to the bath solution failed to alter the UTP (1 mM)-induced current. UTP (1 mM) had almost no effect in a nominally Ca²⁺-free bath medium, or in the presence of charybdotoxin (0.1 μM); the inclusion of U-73122 (5 μM) or heparin (5 mg ml⁻¹) into the pipette solution also blocked the responses to UTP (1 mM). By contrast, the effect of ATP (10 μM) persisted under these conditions.
4. I-V relations were determined by delivering fast voltage ramps before and during the application of UTP (1 mM). In the presence of extracellular Cs⁺ (1 mM) and 4-aminopyridine (1 mM) the UTP-evoked current crossed the zero current level near−75 mV. Omission of Ca²⁺ from the Cs⁺ (1 mM)- and 4-aminopyridine (1 mM)-containing bath medium or replacement of K⁺ by Cs⁺ (150 mM) in the pipette solution abolished the UTP current.
5. Replacement of GTP (200 μM) by GDP-β-S (200 μM) in the pipette solution abolished the current evoked by UTP (1 mM).
6. When the pipette solution contained Cs⁺ (150 mM) instead of K⁺ and in addition inositol 1,4,5,-trisphosphate (InsP₃; 10 μM), an inward current absolutely dependent on extracellular Ca²⁺ was activated after the establishment of whole-cell recording conditions. This current had a typical delay, a rather slow time course and did not reverse its amplitude up to 100 mV, as measured by fast voltage ramps.
7. A rise of the internal free Ca²⁺ concentration from 0.01 to 0.5 μM on excised inside-out membrane patches produced single channel activity with a reversal potential of 0 mV in a symmetrical K⁺ solution. The reversal potential was shifted to negative values, when the extracellular K⁺ concentration was decreased from 144 to 32 mM. By contrast, a decrease of the extracellular Cl⁻ concentration from 164 to 38 mM did not change the reversal potential.
8. Purine and pyrimidine nucleotides act at separate receptors in rat microglial cells. Pyrimidinoceptors activate via a G protein the enzyme phospholipase C with the subsequent release of InsP₃. The depletion of the intracellular Ca²⁺ pool appears to initiate a capacitative entry of Ca⁺ from the extracellular space. This Ca²⁺ then activates a Ca²⁺-dependent K⁺ current.

     

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

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

     

Modulation by general anaesthetics of rat GABAA receptors comprised of α1β3 and β3 subunits expressed in human embryonic kidney 293 cells

1. Radioligand binding and patch-clamp techniques were used to study the actions of γ-aminobutyric acid (GABA) and the general anaesthetics propofol (2,6-diisopropylphenol), pentobarbitone and 5α-pregnan-3α-ol-20-one on rat α1 and β3 GABA_A receptor subunits, expressed either alone or in combination.
2. Membranes from HEK293 cells after transfection with α1 cDNA did not bind significant levels of [³⁵S]-tert-butyl bicyclophosphorothionate ([³⁵S]-TBPS) (<0.03 pmol mg⁻¹ protein). GABA (100 μM) applied to whole-cells transfected with α1 cDNA and clamped at −60 mV, also failed to activate discernible currents.
3. The membranes of cells expressing β3 cDNAs bound [³⁵S]-TBPS (∼1 pmol mg⁻¹ protein). However, the binding was not influenced by GABA (10 nM–100 μM). Neither GABA (100 μM) nor picrotoxin (10 μM) affected currents recorded from cells expressing β3 cDNA, suggesting that β3 subunits do not form functional GABA^A receptors or spontaneously active ion channels.
4. GABA (10 nM–100 μM) modulated [³⁵S]-TBPS binding to the membranes of cells transfected with both α1 and β3 cDNAs. GABA (0.1 μM–1 mM) also dose-dependently activated inward currents with an EC₅₀ of 9 μM recorded from cells transfected with α1 and β3 cDNAs, clamped at −60 mV.
5. Propofol (10 nM–100 μM), pentobarbitone (10 nM–100 μM) and 5α-pregnan-3α-ol-20-one (1 nM–30 μM) modulated [³⁵S]-TBPS binding to the membranes of cells expressing either α1β3 or β3 receptors. Propofol (100 μM), pentobarbitone (1 mM) and 5α-pregnan-3α-ol-20-one (10 μM) also activated currents recorded from cells expressing α1β3 receptors.
6. Propofol (1 μM–1 mM) and pentobarbitone (1 mM) both activated currents recorded from cells expressing β3 homomers. In contrast, application of 5α-pregnan-3α-ol-20-one (10 μM) failed to activate detectable currents.
7. Propofol (100 μM)-activated currents recorded from cells expressing either α1β3 or β3 receptors reversed at the C1⁻ equilibrium potential and were inhibited to 34±13% and 39±10% of control, respectively, by picrotoxin (10 μM). 5α-Pregnan-3α-ol-20-one (100 nM) enhanced propofol (100 μM)-evoked currents mediated by α1β3 receptors to 1101±299% of control. In contrast, even at high concentration 5α-pregnan-3α-ol-20-one (10 μM) caused only a modest facilitation (to 128±12% of control) of propofol (100 μM)-evoked currents mediated by β3 homomers.
8. Propofol (3–100 μM) activated α1β3 and β3 receptors in a concentration-dependent manner. For both receptor combinations, higher concentrations of propofol (300 μM and 1 mM) caused a decline in current amplitude. This inhibition of receptor function reversed rapidly during washout resulting in a ‘surge'' current on cessation of propofol (300 μM and 1 mM) application. Surge currents were also evident following pentobarbitone (1 mM) application to cells expressing either receptor combination. By contrast, this phenomenon was not apparent following applications of 5α-pregnan-3α-ol-20-one (10 μM) to cells expressing α1β3 receptors.
9. These observations demonstrate that rat β3 subunits form homomeric receptors that are not spontaneously active, are insensitive to GABA and can be activated by some general anaesthetics. Taken together, these data also suggest similar sites on GABA_A receptors for propofol and barbiturates, and a separate site for the anaesthetic steroids.

     

Evidence that the substance P-induced enhancement of pacemaking in lymphatics of the guinea-pig mesentery occurs through endothelial release of thromboxane A2

1. In vitro studies were performed to examine the mechanisms underlying substance P-induced enhancement of constriction rate in guinea-pig mesenteric lymphatic vessels.
2. Substance P caused an endothelium-dependent increase in lymphatic constriction frequency which was first significant at a concentration of 1 nM (115±3% of control, n=11) with 1 μM, the highest concentration tested, increasing the rate to 153±4% of control (n=9).
3. Repetitive 5 min applications of substance P (1 μM) caused tachyphylaxis with tissue responsiveness tending to decrease (by an average of 23%) and significantly decreasing (by 72%) for application at intervals of 30 and 10 min, respectively.
4. The competitive antagonist of tachykinin receptors, spantide (5 μM) and the specific NK₁ receptor antagonist, WIN51708 (10 μM) both prevented the enhancement of constriction rate induced by 1 μM substance P.
5. Endothelial cells loaded with the Ca²⁺ sensing fluophore, fluo 3/AM did not display a detectable change in [Ca²⁺]_i upon application of 1 μM substance P.
6. Inhibition of nitric oxide synthase by N^G nitro-L-arginine (L-NOARG; 100 μM) had no significant effect on the response induced by 1 μM substance P.
7. The enhancement of constriction rate induced by 1 μM substance P was prevented by the cyclo-oxygenase inhibitor, indomethacin (3 μM), the thromboxane A₂ synthase inhibitor, imidazole (50 μM), and the thromboxane A₂ receptor antagonist, SQ29548 (0.3 μM).
8. The stable analogue of thromboxane A₂, U46619 (0.1 μM) significantly increased the constriction rate of lymphangions with or without endothelium, an effect which was prevented by SQ29548 (0.3 μM).
9. Treatment with pertussis toxin (PTx; 100 ng ml⁻¹) completely abolished the response to 1 μM substance P without inhibiting either the perfusion-induced constriction or the U46619-induced enhancement of constriction rate.
10. Application of the phospholipase A₂ inhibitor, antiflammin-1 (1 nM) prevented the enhancement of lymphatic pumping induced by substance P (1 μM), without inhibiting the response to either U46619 (0.1 μM) or acetylcholine (10 μM).
11. The data support the hypothesis that the substance P-induced increase in pumping rate is mediated via the endothelium through NK₁ receptors coupled by a PTx sensitive G-protein to phospholipase A₂ and resulting in generation of the arachidonic acid metabolite, thromboxane A₂, this serving as the diffusible activator.

     

Effects of bradykinin on signal transduction,cell proliferation,and cytokine,prostaglandin E2 and collagenase-1 release from human corneal epithelial cells

1. We recently demonstrated the presence of phospholipase C-coupled bradykinin (BK) B₂-receptors in human primary and SV40 virus-immortalized corneal epithelial (CEPI) cells.
2. The aims of the present studies were to demonstrate the specific binding of [³H]-BK to CEPI cell membranes and to study its pharmacological characteristics. In addition, we wished to study the functional coupling of the BK receptors to various physiological and pathological mechanisms in the CEPI cells, including phosphoinositide (PI) turnover, intracellular Ca²⁺-mobilization ([Ca²⁺]_i), cell proliferation (via [³H]-thymidine incorporation), and the release of various cytokines, collagenase-1 (matrix metalloproteinase-1) and prostaglandin E₂ (PGE₂).
3. Specific [³H]-BK binding comprised 83±2% of the total binding, and was of high affinity (K_d=1.66±0.52 nM, n=5), saturable (B_max=640±154 fmol g⁻¹ wet weight) and reversible. Competition studies yielded the following affinity values for BK and a number of BK-related peptides: Hoe-140 (D-Arg-[Hyp³,Thi⁵,D-Tic⁷,Oic⁸]BK; icatibant): K_i=0.17±0.07 nM; BK: K_i=1.0±0.11 nM; [Tyr⁸]-BK: K_i=12.9±2.3 nM; [des-Arg⁹]-BK: K_i>9,200 nM (all n=3–5)).
4. BK potently stimulated PI turnover (EC₅₀=2.3±0.3 nM; n=7) and [Ca²⁺]_i mobilization (EC₅₀=8–20 nM) in CEPI cells and both responses were inhibited in a concentration-dependent manner by 100 nM–10 μM Hoe-140, a selective B₂-receptor antagonist, and also inhibited by the selective phospholipase C (PLC) inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) (IC₅₀=3.0±1.6 μM). BK-induced [Ca²⁺]_i mobilization was reduced by about 30% in the presence of 4 mM EGTA, but was not significantly affected by 100 nM nifedipine.
5. BK (0.1 nM–10 μM) significantly (P<0.05–0.001) stimulated [³H]-thymidine incorporation into CEPI cellular DNA. However, while interleukin-1α (IL-1α; 10 ng ml⁻¹) potently stimulated the release of IL-6, IL-8 and granulocyte macrophage colony-stimulating factor from CEPI cells, BK (0.1 nM–10 μM) was without effect.
6. Whilst phorbol-12-myristate-13-acetate (PMA; 3 μg ml⁻¹) and 10% foetal bovine serum (positive control agents) significantly stimulated the release of both MMP-1 and PGE₂ from CEPI cells, BK (0.1 nM–10 μM) was without any significant effect under these conditions.
7. In conclusion, these data indicate that the CEPI cells express high-affinity [³H]-BK binding sites representing B₂-subtype BK receptors coupled to PI turnover and [Ca²⁺]_i mobilization which appear to stimulate [³H]-thymidine incorporation into cellular DNA. In contrast, BK failed to elicit the release of PGE₂, various cytokines and MMP-1 from CEPI cells. These results suggest that BK may have a potential role in corneal epithelium wound healing by stimulating cell proliferation.

     

Regulation of RANTES and IL-8 production in normal human dermal fibroblasts by active vitamin D3 (tacalcitol)

1. The production of chemokines, RANTES and IL-8 in cultured human dermal fibroblasts and the effects of tacalcitol (1α,24(R)-dihydroxyvitamin D₃) were studied using an enzyme-linked immunosorbent assay.
2. In the unstimulated condition, RANTES and IL-8 were at a trace level in the culture supernatant. On stimulation with TNF-α alone for 24 h, RANTES and IL-8 production were induced. Tacalcitol suppressed RANTES and IL-8 production dose-dependently at concentrations between 10⁻¹² M and 10⁻⁷ M.
3. When the cells were treated with TNF-α and IFN-γ in combination, RANTES production was enhanced, but IL-8 production was not changed, compared to TNF-α-treated cells. Tacalcitol decreased IL-8 production dose-dependently as observed in the TNF-α-treated cells. On the other hand, RANTES production was enhanced by 10⁻¹¹ M and 10⁻¹⁰ M of tacalcitol, and dose-dependently suppressed by tacalcitol concentrations higher than 10⁻⁹ M.
4. Active vitamin D₃ compounds, betamethasone valerate and cyclosporin A were compared with respect to their effects on chemokine production. Three active vitamin D₃ compounds, tacalcitol, 1α,25-dihydroxyvitamin D₃ and MC903 (calcipotriol), inhibited the production of RANTES and IL-8, with very similar potencies. Betamethasone valerate also inhibited these chemokine productions, but with greater potency than active vitamin D₃ compounds. Cyclosporin A significantly stimulated RANTES production at 10⁻⁶ M and IL-8 production at 10⁻⁷ M and 10⁻⁶ M.
5. The results of this study suggest that active vitamin D₃ compounds exert some beneficial effects in the treatment of inflammatory skin diseases via regulation of the production of chemokines by dermal fibroblasts.

     

Pharmacological characterization of thromboxane and prostanoid receptors in human isolated urinary bladder

1. Cumulative concentration-response curves (CRC) to prostaglandin E₁ (PGE₁), PGE₂, PGD₂ and PGF_2α (0.01–30 μM) and to the thromboxane A₂ (TXA₂) receptor agonist U-46619 (0.01–30 μM) were constructed in human isolated detrusor muscle strips both in basal conditions and during electrical field stimulation.
2. All the agonists tested contracted the detrusor muscle. The rank order of agonist potency was: PGF_2α>U-46619>PGE₂ whereas weak contractile responses were obtained with PGD₂ and PGE₁. Any of the agonists tested was able to induce a clear plateau of response even at 30 μM.
3. The selective TXA₂ antagonist, GR 32191B (vapiprost), antagonized U-46619-induced contractions with an apparent pK_B value of 8.27±0.12 (n=4 for each antagonist concentration). GR 32191B (0.3 μM) did not antagonize the contractile responses to PGF_2α and it was a non-surmountable antagonist of PGE₂ (apparent pK_B of 7.09±0.04; n=5). The EP receptor antagonist AH 6809 at 10 μM shifted to the right the CRC to U-46619 (apparent pK_B value of 5.88±0.04; n=4).
4. Electrical field stimulation (20 Hz, 70 V, pulse width 0.1 ms, trains of 5 s every 60 s) elicited contractions fully sensitive to TTX (0.3 μM) and atropine (1 μM). U-46619 (0.01–3 μM) potentiated the twitch contraction in a dose-dependent manner and this effect was competitively antagonized by GR 32191B with an estimated pK_B of 8.54±0.14 (n=4 for each antagonist concentration). PGF_2α in the range 0.01–10 μM (n=7), but not PGE₂ and PGE₁ (n=3 for each), also potentiated the twitch contraction of detrusor muscle strips (23.5±0.3% of KCl 100 mM-induced contraction) but this potentiation was unaffected by 0.3 μM GR 32191B (n=5).
5. Cumulative additions of U-46619 (0.01–30 μM) were without effect on contractions induced by direct smooth muscle excitation (20 Hz, 40 V, 6 ms pulse width, trains of 2 s every 60 s, in the presence of TTX 1 μM; n=3). Moreover, pretreatment of the tissue with 0.3 μM U-46619 did not potentiate the smooth muscle response to 7 μM bethanecol (n=2).
6. We concluded that TXA₂ can induce direct contraction of human isolated urinary bladder through the classical TXA₂ receptor. Prostanoid receptors, fully activated by PGE₂ and PGF_2α are also present. All these receptors are probably located post-junctionally. The rank order of agonist potency and the fact that GR32191B, but not AH6809, antagonized responses to PGE₂ seem to indicate the presence of a new EP receptor subtype. Moreover, we suggest the presence of prejuctional TXA₂ and FP receptors, potentiating acetylcholine release from cholinergic nerve terminals.

     

Pharmacological characterization of CGRP receptors mediating relaxation of the rat pulmonary artery and inhibition of twitch responses of the rat vas deferens

1. CGRP receptors mediating vasorelaxation of the rat isolated pulmonary artery and inhibition of contractions of the rat isolated prostatic vas deferens were investigated using CGRP agonists, homologues and the antagonist CGRP_8-37.
2. In the pulmonary artery, human (h)α-CGRP-induced relaxation of phenylephrine-evoked tone was abolished either by removal of the endothelium or by N^G-nitro-L-arginine (10⁻⁵ M). The inhibitory effect of N^G-nitro-L-arginine was stereoselectively reversed by L- but not by D-arginine (10⁻⁴ M). Thus, CGRP acts via nitric oxide released from the endothelium.
3. In the endothelium-intact artery, hα-CGRP, hβ-CGRP and human adrenomedullin (10⁻¹⁰–3×10⁻⁷ M), dose-dependently relaxed the phenylephrine-induced tone with similar potency. Compared with hα-CGRP, rat amylin was around 50 fold less potent, while [Cys(ACM^2,7)] hα-CGRP (10⁻⁷–10⁻⁴ M) was at least 3000 fold less potent. Salmon calcitonin was inactive (up to 10⁻⁴ M).
4. Human α-CGRP_8-37 (3×10⁻⁷–3×10⁻⁶ M) antagonized hα-CGRP (pA₂ 6.9, Schild plot slope 1.2±0.1) and hβ-CGRP (apparent pK_B of 7.1±0.1 for hα-CGRP_8-37 10⁻⁶ M) in the pulmonary artery. Human β-CGRP_8-37 (10⁻⁶ M) antagonized hα-CGRP responses with a similar affinity (apparent pK_B 7.1±0.1). Human adrenomedullin responses were not inhibited by hα-CGRP_8-37 (10⁻⁶ M).
5. In the prostatic vas deferens, hα-CGRP, hβ-CGRP and rat β-CGRP (10⁻¹⁰–3×10⁻⁷ M) concentration-dependently inhibited twitch responses with about equal potency, while rat amylin (10⁻⁸–10⁻⁵ M) was around 10 fold less potent and the linear analogue [Cys(ACM^2,7)] hα-CGRP was at least 3000 fold weaker. Salmon calcitonin was inactive (up to 10⁻⁴ M).
6. The antagonist effect of hα-CGRP_8-37 (10⁻⁵–3×10⁻⁵) in the vas deferens was independent of the agonist, with pA₂ values against hα-CGRP of 6.0 (slope 0.9±0.1), against hβ-CGRP of 5.8 (slope 1.1±0.1), and an apparent pK_B value of 5.8±0.1 against both rat β-CGRP and rat amylin. Human β-CGRP_8-37 (3×10⁻⁵–10⁻⁴ M) competitively antagonized hα-CGRP responses (pA₂ 5.6, slope 1.1±0.2). The inhibitory effect of hα-CGRP on noradrenaline-induced contractions in both the prostatic and epididymal vas deferens was antagonized by hα-CGRP_8-37 (pA₂ 5.8 and 5.8, slope 1.0±0.2 and 1.0±0.3, respectively).
7. The effects of hα-CGRP and hα-CGRP_8-37 in both rat pulmonary artery and vas deferens were not significantly altered by pretreatment with peptidase inhibitors (amastatin, bestatin, captopril, phosphoramidon and thiorphan, all at 10⁻⁶ M). The weak agonist activity of [Cys(ACM^2,7)] hα-CGRP in the vas deferens was not increased by peptidase inhibitors.
8. These data demonstrate that two different CGRP receptors may exist in the rat pulmonary artery and vas deferens, a CGRP₁ receptor subtype in the rat pulmonary artery (CGRP_8-37 pA₂ 6.9), while the lower affinity for CGRP_8-37 (pA₂ 6.0) in the vas deferens is consistent with a CGRP₂ receptor.

     

A comparative study of the effects of three guanylyl cyclase inhibitors on the L-type Ca2+ and muscarinic K+ currents in frog cardiac myocytes

1. To investigate the participation of guanylyl cyclase in the muscarinic regulation of the cardiac L-type calcium current (I_Ca), we examined the effects of three guanylyl cyclase inhibitors, 1H-[1,2,4]oxidiazolo[4,3-a]quinoxaline-1-one (ODQ), 6-anilino-5,8-quinolinedione (LY 83583), and methylene blue (MBlue), on the β-adrenoceptor; muscarinic receptor and nitric oxide (NO) regulation of I_Ca and on the muscarinic activated potassium current I_K,ACh, in frog atrial and ventricular myocytes.
2. ODQ (10 μM) and LY 83583 (30 μM) antagonized the inhibitory effect of an NO-donor (S-nitroso-N-acetylpenicillamine, SNAP, 1 μM) on the isoprenaline (Iso)-stimulated I_Ca which was consistent with their inhibitory action on guanylyl cyclase. However, MBlue (30 μM) had no effect under similar conditions.
3. In the absence of SNAP, LY 83583 (30 μM) potentiated the stimulations of I_Ca by either Iso (20 nM), forskolin (0.2 μM) or intracellular cyclic AMP (5–10 μM). ODQ (10 μM) had no effect under these conditions, while MBlue (30 μM) inhibited the Iso-stimulated I_Ca.
4. LY 83583 and MBlue, but not ODQ, reduced the inhibitory effect of up to 10 μM acetylcholine (ACh) on I_Ca.
5. MBlue, but not LY 83583 and ODQ, antagonized the activation of I_K,ACh by ACh in the presence of intracellular GTP, and this inhibition was weakened when I_K,ACh was activated by intracellular GTPγS.
6. The potentiating effect of LY 83583 on Iso-stimulated I_Ca was absent in the presence of either DL-dithiothreitol (DTT, 100 μM) or a combination of superoxide dismutase (150 u ml⁻¹) and catalase (100 u ml⁻¹).
7. All together, our data demonstrate that, among the three compounds tested, only ODQ acts in a manner which is consistent with its inhibitory action on the NO-sensitive guanylyl cyclase. The two other compounds produced severe side effects which may involve superoxide anion generation in the case of LY 83583 and alteration of β-adrenoceptor and muscarinic receptor-coupling mechanisms in the case of MBlue.

     

Pharmacologically distinct GABAB receptors that mediate inhibition of GABA and glutamate release in human neocortex

1. The release of endogenous γ-aminobutyric acid (GABA) and glutamic acid in the human brain has been investigated in synaptosomal preparations from fresh neocortical samples obtained from patients undergoing neurosurgery to reach deeply located tumours.
2. The basal outflows of GABA and glutamate from superfused synaptosomes were largely increased during depolarization with 15 mM KCl. The K⁺-evoked overflows of both amino acids were almost totally dependent on the presence of Ca²⁺ in the superfusion medium.
3. The GABA_B receptor agonist (−)-baclofen (1, 3 or 10 μM) inhibited the overflows of GABA and glutamate in a concentration-dependent manner. The inhibition caused by 10 μM of the agonist ranged from 45–50%.
4. The effect of three selective GABA_B receptor antagonists on the inhibition of the K⁺-evoked GABA and glutamate overflows elicited by 10 μM (−)-baclofen was investigated. Phaclofen antagonized (by about 50% at 100 μM; almost totally at 300 μM) the effect of (−)-baclofen on GABA overflow but did not modify the inhibition of glutamate release. The effect of (−)-baclofen on the K⁺-evoked GABA overflow was unaffected by 3-amino-propyl (diethoxymethyl)phosphinic acid (CGP 35348; 10 or 100 μM); however, CGP 35348 (10 or 100 μM) antagonized (−)-baclofen (complete blockade at 100 μM) at the heteroreceptors on glutamatergic terminals. Finally, [3-[[(3,4-dichlorophenyl) methyl]amino]propyl] (diethoxymethyl) phosphinic aid (CGP 52432), 1 μM, blocked the GABA_B autoreceptor, but was ineffective at the heteroreceptors. The selectivity of CGP 52432 was lost at 30 μM, as the compound, at this concentration, inhibited completely the (−)-baclofen effect both on GABA and glutamate release.
5. It is concluded that GABA and glutamate release evoked by depolarization of human neocortex nerve terminals can be affected differentially through pharmacologically distinct GABA_B receptors.

     

Induction of cyclo-oxygenase-2 by cytokines in human cultured airway smooth muscle cells: novel inflammatory role of this cell type

1. Cyclo-oxygenase (COX) is the enzyme that converts arachidonic acid to prostaglandin H₂ (PGH₂) which can then be further metabolized to prostanoids which modulate various airway functions. COX exists in at least two isoforms. COX-1 is expressed constitutively, whereas COX-2 is expressed in response to pro-inflammatory stimuli. Prostanoids are produced under physiological and pathophysiological conditions by many cell types in the lung. However, the regulation of the different COX isoforms in human airway smooth muscle (HASM) cells has not yet been determined.
2. COX-1 and COX-2 protein were measured by Western blot analysis with specific antibodies for COX-1 and COX-2. COX-2 mRNA levels were assessed by Northern blot analysis by use of a COX-2 cDNA probe. COX activity was determined by measuring conversion of either endogenous or exogenous arachidonic acid to three metabolites, PGE₂, thromboxane B₂ or 6-ketoPGF_1α by radioimmunoassay.
3. Under control culture conditions HASM cells expressed COX-1, but not COX-2, protein. However, a mixture of cytokines (interleukin-1β (IL-1β), tumour necrosis factor α (TNFα) and interferon γ (IFNγ) each at 10 ng ml⁻¹) induced COX-2 mRNA expression, which was maximal at 12 h and inhibited by dexamethasone (1 μM; added 30 min before the cytokines). Furthermore, COX-2 protein was detected 24 h after the cytokine treatment and the expression of this protein was also inhibited by dexamethasone (1 μM) and cyclohexamide (10 μg ml⁻¹; added 30 min before the cytokines).
4. Untreated HASM cells released low or undetectable amounts of all COX metabolites measured over a 24 h period. Incubation of the cells with the cytokine mixture (IL-1β, TNFα, IFNγ each at 10 ng ml⁻¹ for 24 h) caused the accumulation of PGE₂ and 6-keto-PGF_1α.
5. In experiments where COX-2 metabolized endogenous stores of arachidonic acid, treatment of HASM cells with IL-1β in combination with TNFα caused a similar release of PGE₂ to that when the three cytokines were given in combination.
6. In other experiments designed to measure COX-2 activity directly, cells were treated with cytokines for 24 h before fresh culture medium was added containing exogenous arachidonic acid (30 μM for 15 min) after which PGE₂ was measured. IL-1β and TNFα increased COX-2 activity and an additional small increase was produced by the three cytokines in combination.
7. These findings suggest that the increased expression of COX-2 is intimately involved in the exaggerated release of prostanoids from HASM cells exposed to pro-inflammatory cytokines. These data indicate a role for airway smooth muscle cells, in addition to their contractile function, as inflammatory cells involved in the production of mediators which may contribute to the inflammatory response seen in diseases such as asthma.

     

The effects of recombinant rat μ-opioid receptor activation in CHO cells on phospholipase C, [Ca2+]i and adenylyl cyclase

1. The rat μ-opioid receptor has recently been cloned, yet its second messenger coupling remains unclear. The endogenous μ-opioid receptor in SH-SY5Y cells couples to phospholipase C (PLC), increases [Ca²⁺]_i and inhibits adenylyl cyclase (AC). We have examined the effects of μ-opioid agonists on inositol(1,4,5)trisphosphate (Ins(1,4,5)P₃), [Ca²⁺]_i and adenosine 3′ : 5′-cyclic monophosphate (cyclic AMP) formation in Chinese hamster ovarian (CHO) cells transfected with the cloned μ-opioid receptor.
2. Opioid receptor binding was assessed with [³H]-diprenorphine ([³H]-DPN) as a radiolabel. Ins(1,4,5)P₃ and cyclic AMP were measured by specific radioreceptor assays. [Ca²⁺]_i was measured fluorimetrically with Fura-2.
3. Scatchard analysis of [³H]-DPN binding revealed that the B_max varied between passages. Fentanyl (10 pM–1 μM) dose-dependently displaced [³H]-DPN, yielding a curve which had a Hill slope of less than unity (0.6±0.1), and was best fit to a two site model, with pK_i values (% of sites) of 9.97±0.4 (27±4.8%) and 7.68±0.07 (73±4.8%). In the presence of GppNHp (100 μM) and Na⁺ (100 mM), the curve was shifted to the right and became steeper (Hill slope=0.9±0.1) with a pK_i value of 6.76±0.04.
4. Fentanyl (0.1 nM–1 μM) had no effect on basal, but dose-dependently inhibited forskolin (1 μM)-stimulated, cyclic AMP formation (pIC₅₀=7.42±0.23), in a pertussis toxin (PTX; 100 ng ml⁻¹ for 24 h)-sensitive and naloxone-reversible manner (K_i=1.7 nM). Morphine (1 μM) and [D-Ala², MePhe⁴, gly(ol)⁵]-enkephalin (DAMGO, 1 μM) also inhibited forskolin (1 μM)-stimulated cyclic AMP formation, whilst [D-Pen², D-Pen⁵], enkephalin (DPDPE, 1 μM) did not.
5. Fentanyl (0.1 nM–10 μM) caused a naloxone (1 μM)-reversible, dose-dependent stimulation of Ins(1,4,5)P₃ formation, with a pEC₅₀ of 7.95±0.15 (n=5). PTX (100 ng ml⁻¹ for 24 h) abolished, whilst Ni²⁺ (2.5 mM) inhibited (by 52%), the fentanyl-induced Ins(1,4,5)P₃ response. Morphine (1 μM) and DAMGO (1 μM), but not DPDPE (1 μM), also stimulated Ins(1,4,5)P₃ formation. Fentanyl (1 μM) also caused an increase in [Ca²⁺]_i (80±16.4 nM, n=6), reaching a maximum at 26.8±2.5 s. The increase in [Ca²⁺]_i remained elevated until sampling ended (200 s) and was essentially abolished by the addition of naloxone (1 μM). Pre-incubation with naloxone (1 μM, 3 min) completely abolished fentanyl-induced increases in [Ca²⁺]_i.
6. In conclusion, the cloned μ-opioid receptor when expressed in CHO cells stimulates PLC and inhibits AC, both effects being mediated by a PTX-sensitive G-protein. In addition, the receptor couples to an increase in [Ca²⁺]_i. These findings are consistent with the previously described effector-second messenger coupling of the endogenous μ-opioid receptor.