Protease-activated receptor-2 peptides activate neurokinin-1 receptors in the mouse isolated trachea

Protective roles for protease-activated receptor-2 (PAR(2)) in the airways including activation of epithelial chloride (Cl(-)) secretion are based on the use of presumably PAR(2)-selective peptide agonists. To determine whether PAR(2) peptide-activated Cl(-) secretion from mouse tracheal epithelium is dependent on PAR(2), changes in ion conductance across the epithelium [short-circuit current (I(SC))] to PAR(2) peptides were measured in Ussing chambers under voltage clamp. In addition, epithelium- and endothelium-dependent relaxations to these peptides were measured in two established PAR(2) bioassays, isolated ring segments of mouse trachea and rat thoracic aorta, respectively. Apical application of the PAR(2) peptide SLIGRL caused increases in I(SC), which were inhibited by three structurally different neurokinin receptor-1 (NK(1)R) antagonists and inhibitors of Cl(-) channels but not by capsaicin, the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP(8-37), or the nonselective cyclooxygenase inhibitor indomethacin. Only high concentrations of trypsin caused an increase in I(SC) but did not affect the responses to SLIGRL. Relaxations to SLIGRL in the trachea and aorta were unaffected by the NK(1)R antagonist nolpitantium (SR 140333) but were abolished by trypsin desensitization. The rank order of potency for a range of peptides in the trachea I(SC) assay was 2-furoyl-LIGRL > SLCGRL > SLIGRL = SLIGRT > LSIGRL compared with 2-furoyl-LIGRL > SLIGRL > SLIGRT > SLCGRL (LSIGRL inactive) in the aorta relaxation assay. In the mouse trachea, PAR(2) peptides activate both epithelial NK(1)R coupled to Cl(-) secretion and PAR(2) coupled to prostaglandin E(2)-mediated smooth muscle relaxation. Such a potential lack of specificity of these commonly used peptides needs to be considered when roles for PAR(2) in airway function in health and disease are determined.     

Influence of dexamethasone on protease-activated receptor 2-mediated responses in the airways

Stimulants of protease-activated receptor (PAR)(2) promote the generation of the bronchoprotective prostanoid prostaglandin (PG) E(2) by airway epithelial cells. In contrast, glucocorticoids reduce the levels of PGE(2) in airway epithelial cell cultures by concomitantly inhibiting pathways required for PGE(2) synthesis and facilitating pathways involved in PGE(2) inactivation. The aim of this study was to determine whether glucocorticoids inhibited PAR(2)-mediated, PGE(2)-dependent responses in epithelial cell cultures, in intact airway preparations, and in whole animals. In cultures of A549 cells, a PAR(2)-activating peptide SLI-GRL-NH(2) produced concentration and time-dependent increases in PGE(2) levels, which were significantly enhanced after exposure to lipopolysaccharide (LPS). However, SLIGRL-NH(2)-induced increases in PGE(2) levels were abolished by pretreatment of cells with the glucocorticoid, dexamethasone. In mouse isolated tracheal preparations, SLIGRL-NH(2) and PGE(2) induced concentration-dependent relaxation responses that were unaffected by dexamethasone, irrespective of whether dexamethasone exposure occurred in vitro or in vivo. Intranasal administration of LPS produced a pronounced increase in the numbers of neutrophils recovered from the bronchoalveolar lavage fluid of BALB/c mice. Numbers of recovered neutrophils were 40 to 60% lower in mice that received f-LIGRL-NH(2) (PAR(2)-activating peptide, 30 microg intranasally), PGE(2) (10 mugintranasally), or dexamethasone (1 mg/kg i.p.). In the combined presence of dexamethasone and f-LIGRL-NH(2) or dexamethasone and PGE(2), the number of neutrophils was suppressed further (80-83% lower). Thus, although dexamethasone abolished PAR(2)-mediated generation of PGE(2) in A549 cells, neither the smooth muscle relaxant nor the anti-inflammatory effects of PAR(2)-activating peptides (and PGE(2)) were diminished by in vitro or in vivo exposure to dexamethasone.     

Intrathecal protease-activated receptor stimulation produces thermal hyperalgesia through spinal cyclooxygenase activity

Activation of protease-activated receptors (PARs) in non-neural tissue results in prostaglandin production. Because PARs are found in the spinal cord and increased prostaglandin release in the spinal cord causes thermal hyperalgesia, we hypothesized that activation of these spinal PARs would stimulate prostaglandin production and cause a cyclooxygenase-dependent thermal hyperalgesia. PARs were activated using either thrombin or peptide agonists derived from the four PAR subtypes, delivered to the lumbar spinal cord. Dialysis experiments were conducted in conscious, unrestrained rats using loop microdialysis probes placed in the lumbar intrathecal space. Intrathecal thrombin stimulated release of prostaglandin E (PGE)(2) but not aspartate or glutamate. Intrathecal delivery of the PAR 1-derived peptide SFLLRN-NH(2) and the PAR 2-derived peptide SLIGRL both stimulated PGE(2) release; PAR 3-derived TFRGAP and PAR 4-derived GYPGQV were inactive. Intrathecal thrombin had no effect upon formalin-induced flinching or tactile sensitivity but resulted in a thermal hyperalgesia. Intrathecal SFLLRN-NH(2) and SLIGRL both produced thermal hyperalgesia. Consistent with their effects on spinal PGE(2), hyperalgesia from these peptides was blocked by pretreatment with the cyclooxygenase inhibitor ibuprofen. SLIGRL-induced hyperalgesia was also blocked by the selective inhibitors SC 58,560 [5-(4-fluorophenyl)-1-[4-(methylsulfonyl)phenyl]-3-(trifluoromethyl)-1H-pyrazole; cyclooxygenase (COX) 1] and SC 58,125 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole; COX 2]. These data indicate that activation of spinal PAR 2 and possibly PAR 1 results in the stimulation of the spinal cyclooxygenase cascade and a prostaglandin-dependent thermal hyperalgesia.     

Mechanism of cytokine-induced modulation of beta-adrenoceptor responsiveness in airway smooth muscle.

To elucidate the role of specific proinflammatory cytokines in regulating airway responsiveness, we examined the effects and mechanisms of action of IL-1beta, TNF-alpha, and IL-2 on the beta-adrenoceptor- and postreceptor-coupled transmembrane signaling mechanisms regulating relaxation in isolated rabbit tracheal smooth muscle (TSM) segments. During half-maximal isometric contraction of the tissues with acetylcholine, relaxation responses to isoproterenol, PGE2, and forskolin were separately compared in control (untreated) TSM and tissues incubated for 18 h with IL-1beta (10 ng/ml), TNF-(alpha (100 ng/ml), or IL-2 (200 ng/ml). Relative to controls, IL-1beta- and TNF-alpha-treated TSM, but not IL-2-treated tissues, depicted significant attenuation of their maximal relaxation and sensitivity (i.e., -log dose producing 50% maximal relaxation) to isoproterenol (P < 0.001) and PGE2 (P < 0.05); whereas the relaxation responses to direct stimulation of adenylate cyclase with forskolin were similar in the control and cytokine-treated tissues. Further, the attenuated relaxation to isoproterenol and PGE2 was ablated in the IL-1beta-treated TSM that were pretreated with either the muscarinic M2-receptor antagonist, methoctramine (10(-6) M), or pertussis toxin (100 ng/ml). Moreover, Western immunoblot analysis demonstrated that: (a) Gi protein expression was significantly enhanced in membrane fractions isolated from IL-1beta-treated TSM; and (b) the latter was largely attributed to induced enhanced expression of the Gi alpha2 and Gi alpha3 subunits. Collectively, these observations provide new evidence demonstrating that IL-lbeta and TNF-alpha induce impaired receptor-coupled airway relaxation in naive TSM, and that the latter effect is associated with increased muscarinic M2-receptor/Gi protein-coupled expression and function.     

Interaction of prostaglandin A2 and prostaglandin B2 on vascular smooth muscle tone, vascular reactivity and electrolyte transport.

The effects of prostaglandin A2 (PGA2) and prostaglandin B2 (PGB2) on vascular smooth muscle tone, electrolyte movements and responses to vasoactive stimuli were evaluated with superfused canine tibial arteries. PGA2 and PGB2 constricted superfused tibial arteries. PGB2 was 10.7 (8.3-14.1) times more potent as a constrictor than PGA2. PGA2 and PGB2-induced vasoconstriction was associated with a decrease in 22Na efflux and a tendency toward an increase in cellular sodium (expressed as micromoles per gram of wet weight). These prostaglandins also decreased the total potassium content of tibial arteries. 45Ca exchange was enhanced by PGA2 and PGB2. The time course of PG-induced increases in 45Ca efflux was similar to the temporal increase in force produced by PGA2 and PGB2. The duration of the contractile response to barium chloride was greatly prolonged during superfusion with both PGA2 and PGB2. These effects were probably not mediated by PG-induced alterations in the resting membrane potential of tibial arteries since presumed depolarization by both high potassium and zero-potassium physiologic saline solutions did not mimic the effects of these prostaglandins on vascular smooth muscle tone or responses to barium chloride. These data suggest that PGA2 and PGB2 may increase tone of vascular smooth muscle by inhibition of those processes involved in sequestration of calcium ion, i.e., the relaxation process, rather than acting on the contractile process.     

Autocrine role of interleukin 1beta in altered responsiveness of atopic asthmatic sensitized airway smooth muscle.

The role of IL-1beta in regulating altered airway responsiveness in the atopic/asthmatic sensitized state was examined in isolated rabbit tracheal smooth muscle (TSM) tissue and cultured cells passively sensitized with sera from atopic asthmatic patients or nonatopic/nonasthmatic (control) subjects. During half-maximal isometric contraction of the tissues with acetylcholine, relative to control TSM, the atopic sensitized TSM exhibited significant attenuation of both their maximal relaxation (P < 0.001) and sensitivity (i.e., -log dose producing 50% maximal relaxation) to isoproterenol and PGE2 (P < 0.05), whereas the relaxation responses to direct stimulation of adenylate cyclase with forskolin were similar in both tissue groups. The impaired relaxation responses to isoproterenol and PGE2 were ablated in sensitized TSM that were pretreated with either the IL-1 recombinant human receptor antagonist or an IL-1beta-neutralizing antibody. Moreover, extended studies demonstrated that, in contrast to their respective controls, both passively sensitized rabbit TSM tissue and cultured cells exhibited markedly induced expression of IL-1beta mRNA at 6 h after exposure to the sensitizing serum, a finding similar to that also obtained in passively sensitized human bronchial smooth muscle tissue. Finally, unlike their respective controls, passively sensitized TSM tissue and cultured cells also displayed progressively enhanced release of IL-1beta protein into the culture media for up to 24 h after exposure to atopic/asthmatic serum. Collectively, these observations provide new evidence demonstrating that the altered responsiveness of atopic/asthmatic sensitized airway smooth muscle is largely attributed to its autologously induced expression and autocrine action of IL-1beta.     

Furosemide directly stimulates prostaglandin E2 production in the thick ascending limb of Henle's loop

Studies were conducted to investigate direct effects of loop diuretics on prostaglandin E2 (PGE2) production using microdissected nephron segments. At first, the effect of indomethacin on the diuretic response to furosemide was re-evaluated in anesthetized rats. Indomethacin significantly attenuated the diuretic, natriuretic and chloruretic effects of furosemide without significantly affecting inulin and p-aminohippurate clearance or filtration fraction. But, in nondiuretic states, indomethacin had no significant effects on these parameters. Furosemide, ethacrynic acid and bumetanide significantly increased PGE2 production in cortical and medullary thick ascending limbs of Henle's loop (P less than .001), but not PGE2 production in the cortical and outer medullary collecting tubules. The effect of furosemide on PGE2 production in CTAL was dose-dependent, and higher concentrations of of furosemide than 10(-6) M significantly increased PGE2 production. On the other hand, chlorothiazide showed no PGE2 productive stimulation in these four nephron segments. This study demonstrates that the enhanced PGE2 production in the thick ascending limb of Henle's loop by furosemide and other loop diuretics is one possible mechanism of these drugs.     

Influence of prostaglandins E1 and F2alpha on pulmonary vascular resistance, isolated lobar vessels and cyclic nucleotide levels.

The effects of prostaglandins E1 (PGE1) and F2alpha) on the pulmonary vascular bed were studied in the intact dog under conditions of controlled pulmonary blood flow. PGF2alpha increased lobar arterial and venous pressure when injected or infused into the lobar, artery. The pressor response was dose-related and doses as low as 0.03 and 0.1 mug, which established concentrations of 0.1 to 0.3 ng/ml in lobar arterial blood, increased pulmonary vascular resistance. PGF2alpha also increased airway resistance in the left lower lobe. However, the effects of this substance on the vascular bed were not related to its effects on bronchomotor tone since similar pressor responses were observed in normal and nonrespiring lobes, PGE1 decreased pressure in the lobar artery and vein when infused into the lobar artery and the effects of PGE1 and PGF2alpha on the pulmonary vascular bed were similar when the lung was perfused with dextran or with blood. PGF2alpha increased isometric tension in isolated helical segments of lobar vein 3 to 5 mm in diameter but was without effect on arterial segments of the same diameter. The increase in isometric tension in the venous segments with PGF2alpha was associated with a significant increase in intracellular levels of guanosine 3',5'-monophosphate (cGMP) but no change in adenosine 3',5'-monophosphate (cAMP) levels. PGE1 decreased isometric tension in both arterial and venous segments and the decrease in tension was accompanied by a significant elevation in smooth muscle cAMP levels and a small but significant reduction in vein cGMP. Results of the present study indicate that PGF2alpha increases pulmonary resistance by constricting lobar veins and to a lesser extent vessels upstream in the precapillary bed whereas PGE1 dilates lobar veins and upstream vessels. These results suggest that PGE1-induced vasodilation may be mediated by an increase in cAMP levels while PGF2alpha-induced venoconstriction may be related to increased smooth muscle levels of cGMP.     

Potentiation of nociceptive responses to low pH injections in humans by prostaglandin E2.

Inflammation and trauma lead to tissue acidification and release of inflammatory mediators, including prostaglandin E2 (PGE2). Protons can evoke pain through acid-sensing ion channels (ASICs) and TRPV1 receptors. In this study, we examined whether PGE2 can potentiate proton-induced nociception in humans on injection into skin and muscle. Psychophysical and vascular responses to microinjections of protons (pH 6.0 and 6.5), PGE2 (10-6 and 10-7 M) and their combinations into forearm skin (30 microL) or anterior tibial muscle (50 microL) were assessed in 16 male subjects. Pain intensity, axon reflex erythema, and heat pain thresholds were recorded after skin challenge; pain intensity and thresholds for pressure-evoked pain were recorded after intramuscular injections. Intradermal or intramuscular injections of PGE2 induced very low levels of pain similar to saline. Administration of low pH caused moderate pain within 5 seconds that declined rapidly over 15 to 20 seconds. In comparison, coinjection of low pH with PGE2 led to a biphasic profile of the pain response. Combined pH + PGE2 stimulation provoked significantly increased pain in the second phase after injections (20 to 100 seconds) both in skin and muscle, whereas the initial injection pain was not enhanced. Heat pain thresholds were reduced after PGE2 and combined pH + PGE2, whereas flare responses were rather attenuated on coadministration of low pH with PGE2. Intriguingly, when compared with skin, muscle pain was significantly lower in the initial phase (0 to 15 seconds) but significantly higher in the second phase (20 to 100 seconds after injection). PERSPECTIVE: PGE2 can potentiate nociceptor activation by protons in human skin and muscle, indicated by increased sustained pain ratings. This can be best explained by TRPV1 sensitization in the presence of PGE2, a mechanism potentially relevant for inflammatory and injury-induced pain.     

Complement activation in acetaminophen-induced liver injury in mice

The principal aim of the study was to determine the influence of influenza A virus infection on capsaicin-induced relaxation responses in mouse isolated tracheal segments and clarify the underlying mechanisms. Anesthetized mice were intranasally inoculated with influenza A/PR-8/34 virus (VIRUS) or vehicle (SHAM), and 4 days later tracheal segments were harvested for isometric tension recording and biochemical and histologic analyses. Capsaicin induced dose-dependent relaxation responses in carbachol-contracted SHAM trachea (e.g., 10 μM capsaicin produced 66 ± 4% relaxation; n = 11), which were significantly inhibited by capsazepine [transient receptor potential vanilloid type 1 (TRPV1) antagonist], (2S,3S)-3-{[3,5-bis(trifluoromethyl)phenyl]methoxy}-2-phenylpiperidine hydrochloride (L-733,060) [neurokinin 1 (NK?) receptor antagonist], indomethacin [cyclooxygenase (COX) inhibitor], and the combination of 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH6809) and 7-[5α-([1S,1α(Z)-biphenyl]-4-ylmethoxy)-2β-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid, calcium salt, hydrate (AH23848) [E-prostanoid (EP)? and EP? receptor antagonists, respectively], indicating that capsaicin-induced relaxation involved the TRPV1-mediated release of substance P (SP), activation of epithelial NK? receptors, and production of COX products capable of activating relaxant EP?/EP? receptors. Consistent with this postulate, capsaicin-induced relaxation was associated with the significant release of SP and prostaglandin E? (PGE?) from mouse tracheal segments. As expected, influenza A virus infection was associated with widespread disruption of the tracheal epithelium. Tracheal segments from VIRUS mice responded weakly to capsaicin (7 ± 3% relaxation) and were 25-fold less responsive to SP than tracheas from SHAM mice. In contrast, relaxation responses to exogenous PGE? and the β-adrenoceptor agonist isoprenaline were not inhibited in VIRUS trachea. Virus infection was associated with impaired capsaicin-induced release of PGE?, but the release of SP was not affected. In summary, influenza A virus infection profoundly inhibits capsaicin- and SP-induced relaxation responses, most likely by inhibiting the production of PGE?.     

三维立体培养人胚肺成纤维母细胞前列腺素E_2释放过程中蛋白酶激活受体的调节作用

背景：前列腺素E2是蛋白酶激活受体信号转导通路中关键递质之一,在人胚肺成纤维母细胞的收缩、增殖和趋化运动中发挥重要的调节作用。但成纤维细胞三维立体培养过程中,蛋白酶激活受体对前列腺素E2释放的调节作用尚不清楚。目的：观察在三维立体培养的人胚肺成纤维母细胞过程中,蛋白酶激活受体1和蛋白酶激活受体2对前列腺素E2释放的调节作用。方法：自大鼠尾腱提取Ⅰ型胶原。体外培养人胚肺成纤维母细胞,应用免疫印迹法测定蛋白酶激活受体1、蛋白酶激活受体2的表达。将人胚肺成纤维母细胞包裹在含培养基的Ⅰ型胶原中漂浮培养作为体外组织重构模型,即人胚肺成纤维母细胞三维立体培养,分别应用蛋白酶激活受体1及蛋白酶激活受体2受体激动剂凝血酶、胰蛋白酶、SLIGKV-NH2、TFLLR-NH2刺激人胚肺成纤维母细胞,ELISA法测定培养上清液中前列腺素E2的含量。结果与结论：蛋白酶激活受体1和蛋白酶激活受体2表达于三维立体培养的人胚肺成纤维母细胞。基础状态下三维立体培养的人胚肺成纤维母细胞可释放前列腺素E2;蛋白酶激活受体1非选择性受体激动剂凝血酶、蛋白酶激活受体2非选择性受体激动剂胰蛋白酶和蛋白酶激活受体1选择性受体激动剂SLIGKV-NH2均显著刺激前列腺素E2产生（P〈0.01）,而蛋白酶激活受体2选择性受体激动剂TFLLR-NH2呈剂量依赖地抑制前列腺素E2的释放（P〈0.01）。结果提示蛋白酶激活受体1和蛋白酶激活受体2参与调节三维立体培养的人胚肺成纤维母细胞前列腺素E2的释放。     

Platelet-activating factor-acether-induced relaxation of guinea pig airway muscle: role of prostaglandin E2 and the epithelium

A fixed concentration of paf-acether (platelet-activating factor; 4 microM) relaxed isolated guinea pig tracheal preparations which had been contracted with histamine (50 microM), serotonin (1 microM) or leukotriene D4 (0.1 microM). The relaxations were approximately 43, 100 and 57%, respectively. We did not observe any relaxant effect of paf-acether (4 microM) in tissues contracted with acetylcholine (50 microM). Both lyso paf-acether (10 microM) and bovine serum albumin (25 micrograms/ml) were without effect on histamine-contracted preparations. In the presence of indomethacin (1.7 microM; 30 min) or aspirin (0.1 mM; 30 min) the relaxant effect of paf-acether (4 microM) in tissues contracted with histamine was significantly reduced to approximately 10 and 12%, respectively. When paf-acether (4 microM) was added to histamine-contracted tracheal preparations in the presence of noradrenaline (0.1 microM) or prostaglandin E2 (PGE2, 10 nM) the relaxations were 62 and 82%, respectively. Noradrenaline and PGE2 alone had only a slight relaxant effect in these tissues (7 and 14%, respectively). In the presence of indomethacin (1.7 microM) the synergistic effect of paf-acether and PGE2 was still observed. The basal production of PGE2 in isolated guinea pig tracheal preparations was 4.6 +/- 1.4 pg/mg of tissue. In the presence of paf-acether (4 microM) increased levels of this prostanoid were detected (11.2 +/- 2.4 pg/mg of tissue). Isolated guinea pig tracheal preparations when contracted with histamine released PGE2 (17.6 +/- 4.1 pg/mg of tissue). In the presence of histamine and paf-acether there was a significant increase in detectable levels of PGE2 (48.6 +/- 13.2 pg/mg of tissue).(ABSTRACT TRUNCATED AT 250 WORDS)     

Rationale for the combination of PGE(1) and S-nitroso-glutathione to induce relaxation of human penile smooth muscle

Many men with erectile dysfunction have been successfully treated with intracavernosal injection of prostaglandin E(1) (PGE(1)) but this treatment is ineffective in 30 to 40% of patients. The goals of this study were to characterize PGE(1)-induced relaxation of isolated human penile smooth muscle (penile arteries and trabecular strips), correlating this in vitro response with the clinical response to this drug, and to evaluate the effects of the combination of PGE(1) with S-nitrosoglutathione (SNO-Glu) on relaxation of isolated human penile smooth muscle. Large variability in the EC(50) and maximal relaxation induced by PGE(1) was observed between tissues of different patients. Patients with poor clinical response to intracavernosal alprostadil (PGE(1)) had significantly larger EC(50) values and smaller maximal relaxation compared with patients with partial or complete clinical response to this drug. SNO-Glu consistently produced complete or near complete relaxation of human corpus cavernosum strips and penile arteries, even when the tissue responded poorly to PGE(1). In trabecular strips, the combination of PGE(1) and SNO-Glu in a 1:100 ratio demonstrated a synergistic relaxation effect. The combination of PGE(1) and SNO-Glu simultaneously increased the levels of both cAMP and cGMP in human corpus cavernosum tissue. Our results suggest that the clinical effectiveness of intracavernosal administration of PGE(1) is related to the variability of the relaxation responses of human trabecular tissue and penile arteries to this drug. The synergistic interaction of PGE(1) and SNO-Glu makes this combination an effective method to cause penile smooth muscle relaxation, a necessary step to initiate and maintain penile erection.     

Activation of an epithelial neurokinin NK-1 receptor induces relaxation of rat trachea through release of prostaglandin E2.

We studied the type of neurokinin (NK) receptor involved in the epithelium-dependent substance P (SP)-induced relaxation of rat trachea precontracted with serotonin (5-HT). We first compared the relaxant effects of different agonists to the three NK receptors on rat trachea in the presence (E+) and absence (E-) of the epithelium. The three agonists to the NK-1 receptor, SP, SP-O-methylester and [beta Ala4, Sar9, Met(O2)] SP(4-11), at a concentration of 1 microM induced a relaxation of 40 +/- 5, 33 +/- 4 and 31 +/- 6%, respectively in E+ segments. They had weak and nonsignificant effects in E- segments. In addition, (+/-)CP-96,345 (1 microM), the NK-1-selective non-peptide antagonist, inhibited the SP-induced relaxation by 45%. Conversely, the three NK-2 receptor agonists, NKA, NKA(4-10) and [Nle10]NKA(4-10), and the two NK-3 receptor agonists, neurokinin B (NKB) and [MePhe7]NKB(4-10), had no effect on E+ or E- tracheal segments. The N-terminal SP fragment SP(1-9) was also inactive. These results suggest that SP-induced relaxation is mediated through activation of epithelial NK-1 receptors. Preincubation with the cyclooxygenase inhibitor, indomethacin (2.8 microM), abrogated the relaxant effect of the three NK-1 receptor agonists on E+ tracheas. We measured in additional experiments prostaglandin E2 (PGE2), PGF2 alpha, 6-keto PGF1 alpha and thromboxane B2. SP (1 microM) induced a 6.1-fold increase in PGE2 production (from 13 pg after 5-HT to 78 pg) in E+ segments, whereas only a 1.5-fold increase occurred in E- preparations.(ABSTRACT TRUNCATED AT 250 WORDS)     

M2 muscarinic receptors inhibit isoproterenol-induced relaxation of canine airway smooth muscle.

Classification of muscarinic receptors in the central airways has revealed the coexistence of M2 and M3 muscarinic receptors in this tissue, with the M2 subtype being predominant. Although M3 muscarinic receptors have been linked to airway smooth muscle contraction, a functional role for the M2 subtype in this tissue has been unclear. In nonairway smooth muscle, stimulation of the M2 muscarinic receptor has been shown to be associated with inhibition of adenylyl cyclase. In the present study, characterization of muscarinic receptors in canine tracheal smooth muscle confirmed that the majority of these muscarinic receptors were of the M2 subtype (89 +/- 3%), with a minor population of M3 receptors (11 +/- 3%). In functional studies, both isoproterenol and forskolin cause a dose-dependent relaxation of precontracted airway smooth muscle. In tissues precontracted with methacholine, 11-([[2-(diethylamino)methyl]-1-piperidinyl]acetyl)5,11- dihydro-6H-pyrido[2,3-6][1,4]benzodiazepine-6-one (AF-DX 116), a selective M2 antagonist, shifted dose-response curves to both isoproterenol and forskolin significantly to the left. In contrast, AF-DX 116 did not alter relaxation induced by the K+ channel opener BRL 38227. Furthermore, the ability of AF-DX 116 to enhance isoproterenol-induced relaxation appears to be limited to smooth muscle precontracted with muscarinic agonists because AF-DX 116 had no effect on isoproterenol dose-response curves in muscle strips precontracted with histamine. Hexahydrosiladifenidol (HHSiD), a selective antagonist for M3 receptors, did not shift the isoproterenol dose-response curve in muscle precontracted with methacholine. This study demonstrates that stimulation of M2 muscarinic receptors in canine airway smooth muscle plays an important role in functional antagonism by reducing the relaxation caused by agents such as isoproterenol and forskolin.     

Signal transduction for proteinase-activated receptor-2-triggered prostaglandin E2 formation in human lung epithelial cells

We investigated proteinase-activated receptor-2 (PAR(2))-triggered signal transduction pathways causing increased prostaglandin E(2) (PGE(2)) formation in human lung-derived A549 epithelial cells. The PAR(2) agonist, SLIGRL-NH(2) (Ser-Leu-Ile-Gly-Arg-Leu-amide), evoked immediate cytosolic Ca(2+) mobilization and delayed (0.5-3 h) PGE(2) formation. The PAR(2)-triggered PGE(2) formation was attenuated by inhibition of the following signal pathway enzymes: cyclooxygenases 1 and 2 (COX-1 and COX-2, respectively), cytosolic Ca(2+)-dependent phospholipase A(2) (cPLA(2)), the mitogen-activated protein kinases (MAPKs), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) and p38 MAPK, Src family tyrosine kinase, epidermal growth factor (EGF) receptor tyrosine kinase (EGFRK), and protein kinase C (PKC), but not by inhibition of matrix metalloproteinases. SLIGRL-NH(2) caused prompt (5 min) and transient ERK phosphorylation, blocked in part by inhibitors of PKC and tyrosine kinases but not by an EGFRK inhibitor. SLIGRL-NH(2) also evoked a relatively delayed (15 min) and persistent (30 min) phosphorylation of p38 MAPK, blocked by inhibitors of Src and EGFRK but not by inhibitors of COX-1 or COX-2. SLIGRL-NH(2) elicited a Src inhibitor-blocked prompt (5 min) and transient phosphorylation of the EGFRK. SLIGRL-NH(2) up-regulated COX-2 protein and/or mRNA levels that were blocked by inhibition of p38 MAPK, EGFRK, Src, and COX-2 but not MEK-ERK. SLIGRL-NH(2) also caused COX-1-dependent up-regulation of microsomal PGE synthase-1 (mPGES-1). We conclude that PAR(2)-triggered PGE(2) formation in A549 cells involves a coordinated up-regulation of COX-2 and mPGES-1 involving cPLA(2), increased cytosolic Ca(2+), PKC, Src, MEK-ERK, p38 MAPK, Src-mediated EGF receptor trans-activation, and also metabolic products of both COX-1 and COX-2.     

Enhanced thromboxane receptor-mediated responses and impaired endothelium-dependent relaxation in human corpus cavernosum from diabetic impotent men: role of protein kinase C activity

We have evaluated the influence of protein kinase C (PKC) activity on penile smooth muscle tone in tissues from diabetic and nondiabetic men with erectile dysfunction. Human corpus cavernosum (HCC) strips were obtained from impotent diabetic and nondiabetic men at the time of penile prosthesis implantation and studied in organ chambers. Contractility responses to a prostanoid precursor, to prostanoids, and to the endothelium-dependent vasodilator acetylcholine were studied. Arachidonic acid (AA; 100 microM) caused cyclooxygenase-dependent relaxation of HCC. This relaxation was impaired in diabetic tissues and normalized by blocking thromboxane (TP) receptors with 20 nM [1S-[1alpha,2alpha(Z),3alpha,4alpha]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (SQ29548). Diabetes did not affect prostaglandin (PG)E(1)-induced relaxation, but it reduced relaxation induced by the PGE(1) metabolite PGE(0). This effect was related to an interaction of PGE(0) with TP receptors. Diabetic tissues had reduced endothelium-dependent relaxation, which was partially improved by SQ29548 and completely normalized by the PKC inhibitor 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF109203X; 1 microM). In HCC from nondiabetic patients, treatment with the PKC activator phorbol-12,13-dibutyrate (0.3 microM) significantly attenuated endothelium-dependent relaxation, an effect prevented by coadministration of GF109203X. Tissues from diabetic patients had enhanced sensitivity to the contractile effects of the TP receptor agonist 9,11-dideoxy-9alpha,11alpha-epoxymethano PGF(2alpha) (U46619) (EC(50) = 0.65 +/- 0.42 and 6.01 +/- 2.28 nM in diabetic and nondiabetic patients, respectively). Inhibition of PKC with 1 microM GF109203X, prevented diabetes-induced hypersensitivity to U46619-induced contractions (EC(50) = 8.55 +/- 3.12 microM). Overactivity of PKC in diabetes is responsible for enhanced contraction and reduced endothelium-dependent relaxation of HCC smooth muscle. Such alterations can result in erectile dysfunction.     

Mast cell tryptase and chymase reverse airway smooth muscle relaxation induced by vasoactive intestinal peptide in the ferret

Recent evidence suggests that nonadrenergic airway relaxation may be controlled by vasoactive intestinal peptide (VIP). The magnitude and duration of smooth muscle relaxation in response to VIP may be influenced by rates of peptide degradation after release from efferent peptidergic neurons. To explore the potential role of mast cell mediators in modulating neural control of airway tone, we studied the effect of the mast cell proteases tryptase and chymase on airway smooth muscle relaxation induced by VIP in ferret airway. Tracheal rings precontracted by serotonin (10(-6) M) in a muscle bath were relaxed by VIP (10(-7) M). We found that protease-rich supernatant obtained by degranulation of dog mastocytoma cells reversed VIP-induced relaxation, as did highly purified tryptase and chymase incubated with the tracheal rings. Either enzyme completely reversed the effect of VIP, but tryptase was more potent than chymase, paralleling previous test tube observations on the relative rates of VIP cleavage by the two enzymes. Inhibitors of mast cell tryptase and chymase preincubated with the supernatant or with the purified proteases prevented reversal of VIP-induced relaxation. Mast cell proteases did not reverse the tracheal relaxation caused by the nonpeptide adrenergic agonist isoproterenol. These findings show that mast cell proteases tryptase and chymase counteract the smooth muscle relaxant effects of VIP in ferret trachea and suggest a potential role for the mast cell proteases in the modulation of nonadrenergic neural control of airway tone by VIP.     

Evidence for distinct prostaglandin I2 and D2 receptors in human platelets.

Incubation of human platelet-rich plasma with prostaglandin I2 (PGI2), results in a marked increase in adenosine 3':5'-monophosphate (cAMP) that persists for at least 60 min. The persistent stimulation of cAMP levels by PGI2 can be rapidly reversed by the addition of either prostaglandin E1 or E2 (PGE1, PGE2), but not by prostaglandin D2 (PGD2). Studies of agonist-specific desensitization of cAMP accumulation show that PGE1 or PGE2 can desensitize for subsequent PGE or PGI2 activation, and that subthreshold levels of PGI2 desensitize for subsequent PGE1 stimulation. PGD2 desensitizes for consequent PGD2 activation, but not for PGE1, PGE2 or PGI2, and PGE compounds and PGI2 do not desensitize for subsequent PGD2 activation. Agonist-specific desensitization for PGI2 is not dependent on cAMP accumulation, but appears to be a consequence of receptor occupation. Support of the desensitization experiments was obtained through the use of the prostaglandin antagonist N-0164 [sodium-p-benzyl-4-[-oxo-2-(4-chlorobenzyl)-3-phenyl-propyl]phenyl phosphonate). This compound proved to be a potent antagonist of PGD2 and a weak antagonist of PGI2-stimulated cAMP accumulation. These data indicate that human platelets have distinct pharmacological receptors for both PGI2 and PGD2, and that PGE compounds may actually interact with a PGI2 receptor.