Modulation of airway smooth muscle tone by protease activated receptor-1,-2,-3 and -4 in trachea isolated from influenza A virus-infected mice

Relaxant and contractile effects of the tethered ligand domain sequences of murine PAR-1, PAR-2, PAR-3 and PAR-4, and of the proteases thrombin and trypsin were examined in mouse isolated tracheal preparations. The epithelium- and cyclo-oxygenase-dependence of these effects and the potential modulatory effects of respiratory tract viral infection were also investigated. In carbachol-contracted preparations, trypsin, thrombin, and the tethered ligand domain sequences of murine PAR-1 (SFFLRN-NH(2)), PAR-2 (SLIGRL-NH(2)) and PAR-4 (GYPGKF-NH(2)), but not PAR-3 (SFNGGP-NH(2)), induced transient, relaxant responses that were abolished by the cyclo-oxygenase inhibitor indomethacin. Repeated administration of SFFLRN-NH(2), SLIGRL-NH(2) or GYPGKF-NH(2) (30 microM) was associated with markedly diminished relaxation responses (homologous desensitization), although there was no evidence of cross-desensitization between these peptides. The tethered ligand domain sequences for PAR-1 and PAR-4 induced a rapid, transient contractile response that preceded the relaxant response. Contractions were not inhibited by indomethacin and were not induced by either thrombin or trypsin. Influenza A virus infection did not significantly affect the responses induced by either the proteases or peptides. Furthermore, epithelial disruption caused by mechanical rubbing had no significant effect on responses to these PAR activators in preparations from either virus- or sham-infected mice. In summary, the proteases trypsin and thrombin, and peptide activators of PAR-1, PAR-2 and PAR-4 induced relaxant responses of mouse isolated tracheal smooth muscle preparations, which were mediated by a prostanoid, probably PGE(2). Interestingly, PAR-mediated relaxations were not significantly diminished following acute damage to the epithelium caused by mechanical rubbing and/or the respiratory tract viral pathogen, influenza A. British Journal of Pharmacology (2000) 129, 63 - 70.     

Pharmacological characterization of pre- and postsynaptic prostanoid receptors in pig gastric fundus

This study characterized the subtype of prostanoid receptors on the cholinergic neurones and smooth muscle cells in circularly oriented muscle strips of the pig gastric fundus. Tissues were electrically stimulated (40 V, 4 Hz, 0.25 ms, 2 min) to induce tritium outflow after incubation with [3H]-choline. Indomethacin increased the electrically induced tritium outflow, suggesting an inhibitory effect of endogenous prostanoids. In the presence of indomethacin, PGE2 > PGF2alpha >PGI2 inhibited tritium release while the TP-receptor agonist U-46619 and PGD2 had no effect. The EP2-receptor agonist butaprost had no effect while the EP1- and EP3-receptor agonist sulprostone mimicked the effect of PGE2. The effect of sulprostone was not affected by AH 6809, that antagonizes EP1- and EP2-receptors, suggesting the presence of presynaptic EP3-receptors on the cholinergic nerve endings. All prostanoid receptor agonists, except butaprost, contracted the tissues concentration-dependently; the rank order of potency (U-46619 > sulprostone > PGE2 > PGF2alpha > PGD2 = PGI2) suggests the presence of TP- and EP1- and EP3-receptors on the circular smooth muscle cells.     

Prostaglandin E2 activates EP2 receptors to inhibit human lung mast cell degranulation

The prostanoid, PGE2, is known to inhibit human lung mast cell activity. The aim of the present study was to characterize the EP receptor that mediates this effect. PGE2 (pEC(50), 5.8+/-0.1) inhibited the IgE-mediated release of histamine from mast cells in a concentration-dependent manner. Alternative EP receptor agonists were studied. The EP2-selective agonist, butaprost (pEC50, 5.2+/-0.2), was an effective inhibitor of mediator release whereas the EP1/EP3 receptor agonist, sulprostone, and the EP1-selective agonist, 17-phenyl-trinor-PGE2, were ineffective. The DP agonist PGD2, the FP agonist PGF(2alpha), the IP agonist iloprost and the TP agonist U-46619 were ineffective inhibitors of IgE-mediated histamine release from mast cells. PGE2 induced a concentration-dependent increase in intracellular cAMP levels in mast cells. The effects of the EP1/EP2 receptor antagonist, AH6809, and the EP4 receptor antagonist, AH23848, on the PGE2-mediated inhibition of histamine release were determined. AH6809 (pK(B), 5.6+/-0.1) caused a modest rightward shift in the PGE2 concentration-response curve, whereas AH23848 was ineffective. Long-term (24 h) incubation of mast cells with either PGE2 or butaprost (EP2 agonist), but not sulprostone (EP1/EP3 agonist), caused a significant reduction in the subsequent ability of PGE2 to inhibit histamine release. Collectively, these data suggest that PGE2 mediates effects on human lung mast cells by interacting with EP2 receptors.     

Characterization of the prostaglandin E2 sensitive (EP)-receptor in the rat isolated trachea.

1. Using a range of natural and synthetic prostanoid receptor agonists and antagonists, we have shown that the rat isolated trachea contains a heterogeneous population of prostaglandin receptor sub-types mediating both relaxation and contraction of the smooth muscle. Prostaglandin E2 (PGE2) elicits smooth muscle relaxation of pre-contracted preparations, the responses being well defined, with a mean potency (p[A50]) of 7.81 +/- 0.05. 2. 11-deoxy PGE1 16,16-dimethyl PGE2 and misoprostol were all full agonists at this receptor, whilst AH13205 was a low potency agonist, and sulprostone was inactive. 3. The EP1 receptor antagonist, AH6809 (5 microM), and the selective DP receptor antagonist, BW A868C (0.1 microM), had no significant effect on the concentration-effect (E/[A]) curves to PGE2. 4. The putative EP4-receptor antagonist, AH23848B, produced non-competitive antagonism of the PGE2 response curves; pA2 values of 5.07 +/- 0.15 and 5.24 +/- 0.19 were obtained at concentrations of 30 microM and 100 microM respectively. 5. The synthetic thromboxane A2 mimetic, U46619, caused smooth muscle contractions, with a mean p[A50] of 6.90 +/- 0.11. This response was antagonized by the TP receptor antagonist, GR32191B, yielding a mean pA2 of 8.31. 6. At concentrations of 1 microM and above, prostaglandin D2 (PGD2) and the IP-receptor agonist, cicaprost, generally elicited concentration-dependent relaxations of the rat trachea. Prostaglandin F2 alpha (PGF2 alpha) was without affinity or efficacy. 7. These data suggest that the rat isolated trachea contains EP-receptors, TP-receptors, and few, if any DP, IP or FP-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification in human airways smooth muscle cells of the prostanoid receptor and signalling pathway through which PGE2 inhibits the release of GM-CSF

1. The prostanoid receptor(s) on human airways smooth muscle (HASM) cells that mediates the inhibitory effect of PGE(2) on interleukin (IL)-1 beta-induced granulocyte/macrophage colony-stimulating factor (GM-CSF) release has been classified. 2. IL-1 beta evoked the release of GM-CSF from HASM cells, which was suppressed by PGE(2), 16,16-dimethyl PGE(2) (nonselective), misoprostol (EP(2)/EP(3)-selective), ONO-AE1-259 and butaprost (both EP(2)-selective) with pIC(50) values of 8.61, 7.13, 5.64, 8.79 and 5.43, respectively. EP-receptor agonists that have selectivity for the EP(1)-(17-phenyl-omega-trinor PGE(2)) and EP(3)-receptor (sulprostone) subtypes as well as cicaprost (IP-selective), PGD(2), PGF(2 alpha) and U-46619 (TP-selective) were poorly active or inactive at concentrations up to 10 microM. 3. AH 6809, a drug that can be used to selectively block EP(2)-receptors in HASM cells, antagonised the inhibitory effect of PGE(2), 16,16-dimethyl PGE(2) and ONO-AE1-259 with apparent pA(2) values of 5.85, 6.09 and 6.1 respectively. In contrast, the EP(4)-receptor antagonists, AH 23848B and L-161,982, failed to displace to the right the concentration-response curves that described the inhibition of GM-CSF release evoked by PGE(2) and ONO-AE1-259. 4. Inhibition of GM-CSF release by PGE(2) and 8-Br-cAMP was abolished in cells infected with an adenovirus vector encoding an inhibitor protein of cAMP-dependent protein kinase (PKA) but not by H-89, a purported small molecule inhibitor of PKA. 5. We conclude that prostanoid receptors of the EP(2)-subtype mediate the inhibitory effect of PGE(2) on GM-CSF release from HASM cells by recruiting a PKA-dependent pathway. In addition, the data illustrate that caution should be exercised when using H-89 in studies designed to assess the role of PKA in biological processes.     

Prostanoid EP(1)- and TP-receptors involved in the contraction of human pulmonary veins.

1. To characterize the prostanoid receptors (TP, FP, EP(1) and/or EP(3)) involved in the vasoconstriction of human pulmonary veins, isolated venous preparations were challenged with different prostanoid-receptor agonists in the absence or presence of selective antagonists. 2. The stable thromboxane A(2) mimetic, U46619, was a potent constrictor agonist on human pulmonary veins (pEC(50)=8.60+/-0.11 and E(max)=4.61+/-0.46 g; n=15). The affinity values for two selective TP-antagonists (BAY u3405 and GR32191B) versus U46619 were BAY u3405: pA(2)=8.94+/-0.23 (n=3) and GR32191B: apparent pK(B)=8.25+/-0.34 (n=3), respectively. These results are consistent with the involvement of TP-receptor in the U46619 induced contractions. 3. The two EP(1)-/EP(3)- agonists (17-phenyl-PGE(2) and sulprostone) induced contraction of human pumonary veins (pEC(50)=8.56+/-0.18; E(max)=0.56+/-0.24 g; n=5 and pEC(50)=7.65+/-0.13; E(max)=1.10+/-0.12 g; n=14, respectively). The potency ranking for these agonists: 17-phenyl-PGE(2) > sulprostone suggests the involvement of an EP(1)-receptor rather than EP(3). In addition, the contractions induced by sulprostone, 17-phenyl-PGE(2) and the IP-/EP(1)- agonist (iloprost) were blocked by the DP-/EP(1)-/EP(2)-receptor antagonist (AH6809) as well as by the EP(1) antagonist (SC19220). 4. PGF(2alpha) induced small contractions which were blocked by AH6809 while fluprostenol was ineffective. These results indicate that FP-receptors are not implicated in the contraction of human pulmonary veins. 5. These data suggest that the contractions induced by prostanoids involved TP- and EP(1)-receptors in human pulmonary venous smooth muscle.     

Prostanoid receptors involved in the relaxation of human pulmonary vessels

1. To characterize the prostanoid receptors on human pulmonary smooth muscle involved in vasodilatations, isolated arteries and veins were contracted with norepinephrine (10 microM) and vessels were subsequently challenged with different prostanoid-receptor agonists in the absence or presence of selective antagonists. 2. Prostaglandin D2 (PGD2) and the selective DP-receptor agonist, BW245C, induced relaxations in the contracted human pulmonary venous preparations. The pD2 values were: 6.88+/-0.11 (n=17) and 7.31+/-0.12 (n=5), respectively. The relaxant responses induced by PGD2 were reduced by the selective DP-receptor antagonist, BWA868C, and the estimated pA2 value was 7.84+/-0.16 (n=4). PGD2 and BW245C did not relax contracted human pulmonary arteries. 3. The selective IP-receptor agonists, iloprost and cicaprost, both induced relaxations in the contracted human vascular preparations. The pD2 values for iloprost were: 7.84+/-0.08 (n=6) and 8.25+/-0.06 (n=4) and for cicaprost: 8.06+/-0.12 (n=5) and 8.11+/-0.09 (n=5) in arteries and veins respectively. 4. Prostaglandin E2 (PGE2) and the EP2/EP3-receptor agonist, misoprostol, partially relaxed the contracted venous preparations and the pD2 values were: 8.10+/-0.15 (n=15) and 6.24+/-0.33 (n=3), respectively. These relaxations suggest the presence of an EP receptor in the human pulmonary veins. The contracted human pulmonary arteries did not relax when challenged with PGE2. 5. In human pulmonary venous preparations, the PGE2-induced relaxations were neither modified by treatment with TP/EP4-receptor antagonist, AH23848B (10 and 30 microM, n=6), nor by the DP/EP1/EP2-receptor antagonist, AH6809 (3 microM, n=6). 6. These data suggest that the relaxation induced by prostanoids involved DP-, IP-receptors and to a lesser extent an EP-receptor on human pulmonary venous smooth muscle. In contrast, only the IP-receptor is involved in the prostanoid induced relaxations on human pulmonary arterial smooth muscle.     

Partial agonism of taprostene at prostanoid IP receptors in vascular preparations from guinea-pig, rat, and mouse

This study investigates whether incomplete relaxation of vascular smooth muscle preparations induced by the prostacyclin analogue taprostene is due to partial agonism at prostanoid IP receptors. In the presence of the prostanoid EP4 receptor antagonist AH 23848, 3 microM taprostene induced 45% relaxation of phenylephrine-contracted guinea-pig saphenous vein rings and displaced log concentration-response curves for the prostacyclin analogues AFP-07, TEI-9063, and cicaprost to the right, parallel to their predicted addition curves. In contrast, taprostene interacted additively with prostaglandin E2 (PGE2), ONO-AE1-259 (selective EP2 agonist), and acetylcholine. Similarly, on rat tail artery contracted with phenylephrine, 3 microM taprostene (20% relaxation) opposed AFP-07- but not PGE2-induced relaxation. However, under U-46619-induced tone (AH 23848 absent), taprostene antagonized AFP-07 and cicaprost more than TEI-9063, suggesting that the latter has more than one relaxation mechanism. The presence of a sensitive EP3 contractile system in mouse aorta interfered with IP receptor-mediated relaxation. By generating tone with phenylephrine and the potent EP3 agonist sulprostone, it was possible to show that 3 microM taprostene (15% relaxation) selectively opposed relaxations induced by AFP-07, TEI-9063, and cicaprost. Our experiments indicate that taprostene is a partial agonist at prostanoid IP receptors, and may be a lead to an IP receptor antagonist.     

Characterization of receptors involved in the direct and indirect actions of prostaglandins E and I on the guinea-pig ileum.

1. A study of the effects of prostaglandin E2 (PGE2) and eleven synthetic analogues on the guinea-pig isolated ileum preparation has revealed three distinct contractile actions, each associated with a different prostaglandin E (EP-) receptor subtype. In addition, PGI2 (prostacyclin) and its stable analogues can activate prostaglandin I (IP-) receptors to elicit both contraction and relaxation of the ileum. 2. Two of the PGE actions involve direct stimulation of the smooth muscle, being unaffected by 1 microM morphine treatment. One action is blocked by AH 6809 at micromolar concentrations and ICI 80205 and 16,16-dimethyl PGE2 are particularly potent agonists. Activation of EP1-receptors appears to be involved. The second action is unaffected by AH 6809; sulprostone and MB 28767 are potent agonists. Comparison with agonist potency rankings on the guinea-pig vas deferens indicates that EP3-receptors may be involved. 3. The third PGE effect and the stimulant PGI effect are blocked by morphine, indicating enteric neurones and/or sensory nerve terminals as sites of action. EP2-receptors may be involved in the PGE action, in view of the marked effect of morphine on the contractile actions of misoprostol, 11-deoxy PGE2-1-alcohol, 11-deoxy PGE1 and butaprost, all of which show some selectivity for EP2-receptors. The PGI action is most easily studied with cicaprost (EC25 = 1.3 nM), since iloprost, carbacyclin and to a lesser extent PGI2 also have agonist activity at EP1-receptors. 4. The contractile action of 17-phenyl-omega-trinor PGE2 on the ileum is unaffected by morphine.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro characterization of prostanoid EP-receptors in the non-pregnant human myometrium.

1. Prostaglandin receptors of the PGE type have been characterized in the non-pregnant human myometrium in vitro according to the scheme of Coleman et al. (1984) by use of the agonists PGE2, sulprostone, rioprostil, AY23626, butaprost, misoprostol, 16,16-dimethylprostaglandin E2, enprostil and iloprost, and, the antagonist AH6809. 2. All prostanoids tested were active in non-pregnant human myometrium either as stimulators and/or inhibitors of spontaneous activity or both. Biphasic responses to PGE2 indicate that at least two receptor types of the EP-receptor exist, one mediating relaxation and the other mediating contraction. 3. Further evidence for the EP-receptor mediating excitation and relaxation was provided by the action of the EP2-/EP3-receptor selective prostanoids rioprostil, AY23626 and misoprostol, and the EP1-/EP2-receptor selective agonist 16,16-dimethylprostaglandin E2. 4. Butaprost, an EP2-receptor selective agonist, produced potent inhibition of spontaneous activity in the tissue which was generally longer-lasting than that evoked by the natural prostanoid PGE2. 5. The EP1-/EP3-receptor selective agonist sulprostone and the EP3-receptor agonist enprostil produced potent contractile responses supporting the presence of contractile EP3-receptors in the non-pregnant human myometrium in vitro. 6. The EP1-/IP-receptor selective agonist, iloprost, produced mixed responses in non-pregnant human myometrium. The contractile response was inhibited by the EP1-receptor antagonist AH6809. However, responses to the EP1-/EP3-receptor selective agonist sulprostone were unaffected by AH6809 which may indicate that only a small population of EP1-receptors is present. 7. Therefore it would seem that a heterogeneous population of EP-receptors is present in the non-pregnant human myometrium.     

Characterization of the prostaglandin E2 pathway in a rat model of esophageal adenocarcinoma

Accumulating evidence indicates that the cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway plays a key role in esophageal carcinogenesis. A better understanding of the pathway downstream of COX-2 may reveal novel targets for the prevention of esophageal adenocarcinoma (EAC). The objective of this study was to characterize the profile of genes involved in PGE2 metabolism and signaling in an experimental model of EAC. Esophagojejunostomy with gastric preservation was performed in wistar rats to induce gastroduodenal reflux. Rats were sacrificed 2 or 4 months after surgery. Nine non-operated rats were used to obtain normal (control) esophageal tissues. RESULTS: All rats that underwent esophagojejunostomy developed inflammation. In addition, 90% of the animals showed intestinal metaplasia; of those, 40% progressed to AC. This process was accompanied by a significant increase in esophageal PGE2 levels and the induction of both mRNA and protein levels of COX-2, COX-1, prostaglandin E synthase, 15-hydroxyprostaglandin dehydrogenase, and PGE2 receptors EP3, EP4 and especially EP2, which rose to particularly high levels in experimental rats. In addition, exposure to a selective COX-2 inhibitor (SC58125) or an EP1/EP2 antagonist (AH6809), but not an EP4 antagonist (AH23848B), significantly reduced cell proliferation of esophageal explants in 24 hour-organ culture experiments. Our data suggest that, in addition to COX-2, other components of the PGE2 pathway, including COX-1, may play important roles in the development of EAC induced by gastroduodenal reflux in the rat. Although it must be confirmed in vivo, the EP2 receptor may represent a promising selective target in the prevention of Barrett's associated AC.     

TNF alpha reduces tachykinin, PGE2-dependent, relaxation of the cultured mouse trachea by increasing the activity of COX-2

1. Chronic inflammation is a central feature of asthma. The inflammatory cytokine tumour necrosis factor alpha (TNFalpha) has been implicated in this disease, and is known to alter airway smooth muscle functionally. 2. The aim of this study was to investigate the influence of TNFalpha on tachykinin-induced airway relaxation. Mouse tracheae were cultured in the absence and presence of TNFalpha for 1 or 4 days. 3. In the absence of TNFalpha, substance P (SP) and neurokinin A (NKA) induced comparable levels of relaxation in fresh and cultured segments. Functional studies with selective antagonists/inhibitors indicated that the relaxation was mediated by the NK(1) receptor coupled to cyclooxygenase (COX)-2 activation and subsequent release of prostaglandin E(2) (PGE(2)). TNFalpha attenuated SP- and NKA-induced relaxation in a time- and concentration-dependent manner, decreasing the ability of PGE(2) to relax tissues. 4. Further studies indicated that TNFalpha elevated COX-2 activity and that concomitant inhibition of COX-2 reversed TNFalpha-attenuated PGE(2) relaxation. Culture with PGE(2) decreased SP- and PGE(2)-mediated relaxation, further implicating the activity of COX-2 in the attenuation of tachykinin signalling. 5. Gene expression analysis demonstrated that TNFalpha increased the expression of smooth muscle COX-2, PGE(2) synthase and EP(2) receptor mRNA, and decreased the expression of the EP(4) receptor. 6. Overall, these results show that NK(1) receptor-mediated relaxation induced by PGE(2) is attenuated by prolonged TNFalpha stimulation. Increased COX-2 activity induced by TNFalpha appears to be central to this process.     

Dual modulation by thrombin of the motility of rat oesophageal muscularis mucosae via two distinct protease-activated receptors (PARs): a novel role for PAR-4 as opposed to PAR-1

Since protease-activated receptors (PARs) are distributed throughout the gastrointestinal tract, we investigated the role of PARs in modulation of the motility of the rat oesophageal muscularis mucosae. Thrombin produced contraction of segments of the upper and lower part of the smooth muscle. Trypsin contracted both the muscle preparations only at high concentrations. SFLLR-NH(2) and TFLLR-NH(2) (PAR-1-activating peptides), but not the PAR-1-inactive peptide FSLLR-NH(2), evoked a marked contraction. In contrast, the PAR-2 agonist SLIGRL-NH(2) and the PAR-4 agonist GYPGKF-NH(2) caused no or only a negligible contraction. In oesophageal preparations precontracted with carbachol, thrombin produced a dual action i.e. relaxation followed by contraction. TFLLR-NH(2) further contracted the precontracted preparations with no preceding relaxation. GYPGKF-NH(2), but not the inactive peptide GAPGKF-NH(2), produced marked relaxation. Trypsin or SLIGRL-NH(2) caused no relaxation. The PAR-1-mediated contraction was completely abolished in Ca(2+)-free medium and considerably attenuated by nifedipine (1 microM) and in a low Na(+) medium. The PAR-4-mediated relaxation was resistant to tetrodotoxin (10 microM), apamin (0.1 microM), charybdotoxin (0.1 microM), L-N(G)-nitroarginine methyl ester (100 microM), indomethacin (3 microM), propranolol (5 microM) or adenosine 3', 5'-cyclic monophosphorothioate, 8-bromo, Rp-isomer (30 microM). Thus, thrombin plays a dual role in modulating the motility of the oesophageal muscularis mucosae, producing contraction via PAR-1 and relaxation via PAR-4. The PAR-1-mediated effect appears to occur largely through increased Na(+) permeability followed by activation of L-type Ca(2+) channels and subsequent influx of extracellular Ca(2+). Our data could provide evidence for a novel role of PAR-4 as opposed to PAR-1, although the underlying mechanisms are still open to question.     

Characterization of the inhibitory prostanoid receptors on human neutrophils.

1. We have evaluated the effects of various prostanoid agonists on the release of leukotriene B4 (LTB4) and superoxide anions (O2-) from human neutrophils stimulated with opsonized zymosan (OZ) and formyl-methionyl-leucyl-phenylalanine (FMLP), respectively. 2. Prostaglandin E2 (PGE2) and PGD2 inhibited both OZ-induced LTB4 release (EC50 0.72 microM and 0.91 microM respectively), and FMLP-induced O2- release (EC50 0.42 microM and 0.50 microM respectively). PGF2 alpha, the TP-receptor agonist, U46619, and the IP-receptor agonist, iloprost, were also active, but were all at least an order of magnitude less potent than PGE2 and PGD2. 3. The EP2/EP3-receptor agonist, misoprostol, and the selective EP2-agonist, AH13205, were both effective inhibitors of LTB4 release, being approximately equipotent with and 16-times less potent than PGE2, respectively. In contrast, the EP1/EP3-receptor agonist, sulprostone, had no inhibitory activity at concentrations of up to 10 microM. 4. The selective DP-receptor agonist, BW245C, inhibited LTB4 release, (EC50 0.006 microM) being approximately 50 times more potent than PGD2. BW245C also inhibited O2- release, and this inhibition was antagonized competitively by the DP-receptor blocking drug, AH6809 (pA2 6.6). 5. These data indicate the presence of both inhibitory EP- and DP-receptors on the human neutrophil. The rank order of potency of EP-receptor agonists suggest that the EP-receptors are of the EP2-subtype.     

Modulation by protease-activated receptors of the rat duodenal motility in vitro: possible mechanisms underlying the evoked contraction and relaxation

1 The present study examined effects of agonist enzymes and receptor-activating peptides for protease-activated receptors (PARs) on duodenal motility in the rat, and also investigated possible mechanisms underlying the evoked responses. 2 Thrombin at 0.03-0.1 microM and the PAR-1-activating peptide SFLLR-NH2 at 3-100 microM or TFLLR-NH2 at 10-50 microM produced a dual action, relaxation followed by contraction of the duodenal longitudinal muscle. The PAR-2-activating peptide SLIGRL-NH2 at 10-100 microM elicited only small contraction. Trypsin at 0.08 microM induced small contraction, or relaxation followed by contraction, depending on preparations. The PAR-4-activating peptide GYPGKF-NH2 at 1000 microM exhibited no effect. 3 The contractile responses of the duodenal strips to TFLLR-NH2 and to SLIGRL-NH2 were partially attenuated by the L-type calcium channel blocker nifedipine (1 microM), the protein kinase C inhibitor GF109203X (1 microM) and the tyrosine kinase inhibitor genistein (15 microM), but were resistant to indomethacin (3 microM) and tetrodotoxin (1-10 microM). 4 The relaxation of the preparations exerted by TFLLR-NH2 was unaffected by indomethacin (3 microM), propranolol (5 microM), NG-nitro-L-arginine methyl ester (100 microM) and tetrodotoxin (1-10 microM). This relaxation was resistant to either GF109203X (1 microM) or genistein (15 microM), but was, remarkably, attenuated by combined application of these two kinase inhibitors. 5 Apamin (0.1 microM), an inhibitor of calcium-activated, small-conductance potassium channels, but not charybdotoxin (0.1 microM), completely abolished the PAR-1-mediated duodenal relaxation, and significantly enhanced the PAR-1-mediated contraction. 6 These findings demonstrate that PAR-1 plays a dual role, suppression and facilitation of smooth muscle motility in the rat duodenum, while PAR-2 plays a minor excitatory role in the muscle, and that PAR-4 is not involved in the duodenal tension modulation. The results also suggest that the contractile responses to PAR-1 and PAR-2 activation are mediated, in part, by activation of L-type calcium channels, protein kinase C and tyrosine kinase, and that the relaxation response to PAR-1 activation occurs via activation of apamin-sensitive, but charybdotoxin-insensitive, potassium channels, in which both protein kinase C and tyrosine kinase might be involved synergistically.     

E-ring 8-isoprostanes are agonists at EP2- and EP4-prostanoid receptors on human airway smooth muscle cells and regulate the release of colony-stimulating factors by activating cAMP-dependent protein kinase

8-Isoprostanes are bioactive lipid mediators formed via the nonenzymatic peroxidation of arachidonic acid by free radicals and reactive oxygen species. However, their cognate receptors, biological actions, and signaling pathways are poorly studied. Here, we report the effect of a variety of E- and Falpha-ring 8-isoprostanes on the release of granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) from human airway smooth muscle (HASM) cells stimulated with interleukin-1beta (IL-1beta). The elaboration of GM-CSF and G-CSF by IL-1beta was inhibited and augmented, respectively, in a concentration-dependent manner by 8-iso-prostaglandin (PG) E(1) and 8-iso-PGE(2), but not by 8-iso-PGF(1alpha), 8-iso-PGF(2alpha), and 8-iso-PGF(3)alpha. AH 6809 (6-isopropoxy-9-oxoxanthine-2-carboxylic acid), an EP(1)-/EP(2)-/DP-receptor blocking drug, antagonized the inhibitory effect of 8-iso-PGE(1) and 8-iso-PGE(2) on GM-CSF output with an affinity consistent with an interaction at prostanoid receptors of the EP(2)-subtype. In contrast, the facilitation by 8-iso-PGE(1) and 8-iso-PGE(2) of G-CSF release was unaffected by AH 6809 and the selective EP(4)-receptor antagonist L-161,982 [4'-[3-butyl-5-oxo-1-(2-trifluoromethyl-phenyl)-1,5-dihydro-[1,2,4]triazol-4-ylmethyl]-biphenyl-2-sulfonic acid (3-methyl-thiophene-2-carbonyl)-amide]. However, when used in combination, AH 6809 and L-161,982 displaced 5-fold to the right the 8-iso-PGE and 8-iso-PGE concentration-response curves. The opposing (1)effect of E-ring (2)8-isoprostanes on GM-CSF and G-CSF release was mimicked by 8-bromo-cAMP and abolished in cells infected with an adenovirus vector encoding an inhibitor protein of cAMP-dependent protein kinase (PKA). Together, these data demonstrate that E-ring 8-isoprostanes regulate the secretion of GM-CSF and G-CSF from HASM cells by a cAMP- and PKA-dependent mechanism. Moreover, antagonist studies revealed that 8-iso-PGE(1) and 8-iso-PGE(2) act solely via EP(2) -receptors to inhibit GM-CSF release, whereas both EP(2)- and EP(4)-receptor subtypes positively regulate G-CSF output.     

Characterisation of receptors mediating the contractile effects of prostanoids in guinea-pig and human airways

Receptors mediating the contractile effects of prostanoids in the airways of guinea-pig and man have been compared using a range of selective agonists and antagonists. In guinea-pig and human lung parenchymal strips, contractile responses to all prostanoid agonists were blocked by EP092 and AH23848 (antagonists of thromboxane-sensitive receptors) but were unaffected by the EP1 receptor antagonist, AH6809. In guinea-pig tracheal rings responses to the thromboxane mimetic U46619 were antagonised by EP092 and AH23848. AH6809 was ineffective, but did antagonise responses to 16,16-dimethylPGE2 and PGF2 alpha in this tissue. In the human bronchiolar ring responses to U46619 were poorly antagonised by AH23848, EP092 and AH6809. The results suggest that prostanoid contractile agonists act through thromboxane receptors in guinea-pig and human lung parenchymal strips and through thromboxane and EP1 receptors in guinea-pig trachea. A novel prostanoid receptor type, or subtype, mediating contractile effects may be present in human bronchioles.     

Evidence that PAR-1 and PAR-2 mediate prostanoid-dependent contraction in isolated guinea-pig gallbladder

We have investigated the ability of protease-activated receptor-1 (PAR-1), PAR-2, PAR-3 and PAR-4 agonists to induce contractile responses in isolated guinea-pig gallbladder. Thrombin, trypsin, mouse PAR-1 activating (SFLLRN-NH(2)) peptide, and mouse PAR-2 activating (SLIGRL-NH(2)) and human PAR-2 activating (SLIGKV-NH(2)) peptides produced a concentration-dependent contractile response. Mouse PAR-4 activating (GYPGKF-NH(2)) peptide, the mouse PAR-1 reverse (NRLLFS-NH(2)) peptide, the mouse PAR-2 reverse (LRGILS-NH(2)) and human PAR-2 reverse (VKGILS-NH(2)) peptides caused negligible contractile responses at the highest concentrations tested. An additive effect was observed following the contractile response induced by either trypsin or thrombin, with the addition of a different PAR agonist (SFLLRN-NH(2) and SLIGRL-NH(2), respectively). Desensitization to PAR-2 activating peptide attenuated the response to trypsin but failed to attenuate the response to PAR-1 agonists, and conversely desensitization to PAR-1 attenuated the response to thrombin but failed to alter contractile responses to PAR-2 agonists. The contractile responses produced by thrombin, trypsin, SFLLRN-NH(2) and SLIGRL-NH(2) were markedly reduced in the presence of the cyclo-oxygenase inhibitor, indomethacin, whilst the small contractile response produced by NRLLFS-NH(2) and LRGILS-NH(2) were insensitive to indomethacin. The contractile responses to thrombin, trypsin, SFLLRN-NH(2) and SLIGRL-NH(2) were unaffected by the presence of: the non-selective muscarinic antagonist, atropine; the nitric oxide synthase inhibitor, L-NAME; the sodium channel blocker, tetrodotoxin; the combination of selective tachykinin NK(1) and NK(2) receptor antagonists, (S)-1-[2-[3-(3,4-dichlorphenyl)-1 (3-isopropoxyphenylacetyl) piperidin-3-yl] ethyl]-4-phenyl-1 azaniabicyclo [2.2.2] octane chloride (SR140333) and (S)-N-methyl-N-[4-acetylamino-4-phenylpiperidino-2-(3, 4-dichlorophenyl)-butyl] benzamide (SR48968), respectively. The results indicate that PAR-1 and PAR-2 activation causes contractile responses in the guinea-pig gallbladder, an effect that is mediated principally by prostanoid release, and is independent of neural mechanisms.     

E-ring 8-isoprostanes inhibit ACh release from parasympathetic nerves innervating guinea-pig trachea through agonism of prostanoid receptors of the EP3-subtype

1. In the present study, we examined the effect of E-ring 8-isoprostanes on cholinergic neurotransmission in guinea-pig trachea and identified the receptor(s) involved. As isoprostanes are isomeric with prostaglandins, PGE(2) and sulprostone (a selective EP(3)-receptor agonist) were examined in parallel. 2. 8-Iso-PGE(1), 8-iso-PGE(2) (0.1 nm-1 microM), sulprostone (1 nm-1 microM) and PGE(2) (1 microM) suppressed EFS-evoked [(3)H]ACh release from guinea-pig trachea in a concentration-dependent manner, producing 39.5, 53.9, 61.2 and 59.9% inhibition, respectively, at 1 microM. It should be noted that an established maximum effective concentration was not determined. 3. Neither SQ 29,548 (1 microm; a TP-receptor antagonist) nor AH 6809 (10 microM; an EP(1)-/EP(2)-/DP-receptor antagonist) reversed the inhibitory effect of these compounds. 4. L-798,106, a novel and highly selective EP(3)-receptor antagonist, produced a parallel shift to the right of the concentration-response curves that described the inhibitory action of sulprostone on EFS-evoked contractile responses in guinea-pig vas deferens (an established EP(3)-receptor-expressing tissue), from which a mean pA(2) of 7.48 was derived. On guinea-pig trachea, L-798,106 also antagonised sulprostone-induced inhibition of EFS-induced twitch responses, with similar potency (mean pA(2)=7.82). 5. The inhibitory effects of 8-iso-PGE(1), 8-iso-PGE(2), sulprostone and PGE(2) on EFS-induced [(3)H]ACh release was blocked by L-798,106 at a concentration (10 microM) that binds only weakly to human recombinant EP(1)-, EP(2)- and EP(4)-receptor subtypes expressed in HEK 293 cells. 6. These data suggest that E-ring 8-isoprostanes, PGE(2) and sulprostone inhibit EFS-evoked [(3)H]ACh release from cholinergic nerves innervating guinea-pig trachea, by interacting with prejunctional prostanoid receptors of the EP(3)-subtype.     

Regulation of human penile smooth muscle tone by prostanoid receptors

We have characterized the prostanoid receptors involved in the regulation of human penile arterial and trabecular smooth muscle tone. Arachidonic acid induced relaxation of human corpus cavernosum strips (HCCS) that was blocked by the cyclo-oxygenase inhibitor, indomethacin, and augmented by the thromboxane receptor (TP) antagonist, SQ29548, suggesting that endogenous production of prostanoids regulates penile smooth muscle tone. TP-receptors mediate contraction of HCCS and penile resistance arteries (HPRA), since the agonist of these receptors, U46619, potently contracted HCCS (EC50 8.3+/-2.8 nM) and HPRA (EC50 6.2+/-2.2 nM), and the contractions produced by prostaglandin F(2alpha) at high concentrations (EC50 6460+/-3220 nM in HCCS and 8900+/-6700 nM in HPRA) were inhibited by the selective TP-receptor antagonist, SQ29548 (0.02 microM). EP-receptors are responsible for prostanoid-induced relaxant effects in HCCS because only prostaglandin E1 (PGE1), prostaglandin E2 and the EP2/EP4-receptor agonist, butaprost, produced consistent relaxation of this tissue (EC50 93.8+/-31.5, 16.3+/-3.8 and 1820+/-1284 nM, respectively). In HPRA, both prostacyclin and PGE1 (EC50 60.1+/-18.4 and 109.0+/-30.9 nM, respectively) as well as the selective IP receptor agonist, cicaprost, and butaprost (EC50 25.2+/-15.2 and 7050+/-6020 nM, respectively) caused relaxation, suggesting co-existence of IP- and EP-receptors (EP2 and/or EP4). In summary, endogenous production of prostanoids may regulate penile smooth muscle contractility by way of specific receptors. TP-receptors mediate contraction in HCCS and HPRA, while the relaxant effects of prostanoids are mediated by EP2- and/or EP4-receptors in HCCS and by EP- and IP-receptors in HPRA.