Jean-Pierre Gascard

Prostanoid receptors involved in the relaxation of human pulmonary vessels

To characterize the prostanoid receptors on human pulmonary smooth muscle involved in vasodilatations, isolated arteries and veins were contracted with norepinephrine (10 μM) and vessels were subsequently challenged with different prostanoid‐receptor agonists in the absence or presence of selective antagonists. 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. 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. 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. In human pulmonary venous preparations, the PGE2‐induced relaxations were neither modified by treatment with TP/EP4‐receptor antagonist, AH23848B (10 and 30 μM, n=6), nor by the DP/EP1/EP2‐receptor antagonist, AH6809 (3 μM, n=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.

Prostanoid receptors involved in the relaxation of human bronchial preparations.

Iloprost and cicaprost (IP‐receptor agonists) induced relaxations in the histamine‐ (50 μM) contracted human bronchial preparations (pD2 values, 6.63±0.12 and 6.86±0.08; Emax values, 90±04 and 65±08% of the papaverine response for iloprost (n=6) and cicaprost (n=3), respectively). Prostaglandin E2 (PGE2) and misoprostol (EP‐receptor agonist) relaxed the histamine‐contracted human bronchial preparations (pD2 values, 7.13±0.07 and 6.33±0.28; Emax values, 67±04 and 57±08% of the papaverine response for PGE2 (n=14) and misoprostol (n=4), respectively). In addition, both relaxations were inhibited by AH6809 (DP/EP1/EP2‐receptor antagonist; 3 μM; n=5–6). The PGE2‐induced relaxations of human bronchial preparations were not modified by treatment with AH23848B (TP/EP4‐receptor antagonist; 30 μM; n=4). The contracted human bronchial preparations were significantly relaxed by prostaglandin D2 (PGD2) or by BW245C a DP‐receptor agonist. However, these responses did not exceed 40% of the relaxation induced by papaverine. In addition, the relaxations induced by PGD2 were significantly inhibited by treatment with a DP‐receptor antagonist BWA868C (0.1 μM; n=3). These data suggest that the relaxation of human isolated bronchial preparations induced by prostanoids involved IP‐, EP2‐ and to a lesser extent DP‐receptors but not EP4‐receptor.

Pharmacological evidence for a novel cysteinyl‐leukotriene receptor subtype in human pulmonary artery smooth muscle

To characterize the cysteinyl‐leukotriene receptors (CysLT receptors) in isolated human pulmonary arteries, ring preparations were contracted with leukotriene C4 (LTC4) and leukotriene D4 (LTD4) in either the absence or presence of the selective CysLT1 receptor antagonists, ICI 198615, MK 571 or the dual CysLT1/CysLT2 receptor antagonist, BAY u9773. Since the contractions induced by the cysteinyl‐leukotrienes (cysLTs) in intact preparations failed to attain a plateau response over the concentration range studied, the endothelium was removed and the tissue treated continuously with indomethacin (Rubbed+INDO). In these latter preparations, the pEC50 for LTC4 and LTD4 were not significantly different (7.61±0.07, n=20 and 7.96±0.09, n=22, respectively). However, the LTC4 and LTD4 contractions were markedly potentiated when compared with data from intact tissues. Leukotriene E4 (LTE4) did not contract human isolated pulmonary arterial preparations. In addition, treatment of preparations with LTE4 (1 μM; 30 min) did not modify either the LTC4 or LTD4 contractions. Treatment of preparations with the S‐conjugated glutathione (S‐hexyl‐GSH; 100 μM, 30 min), an inhibitor of the metabolism of LTC4 to LTD4, did not modify LTC4 contractions. The pEC50 values for LTC4 were significantly reduced by treatment of the preparations with either ICI 198615, MK 571 or BAY u9773 and the pKB values were: 7.20, 7.02 and 6.26, respectively. In contrast, these antagonists did not modify the LTD4 pEC50 values. These findings suggest the presence of two CysLT receptors on human pulmonary arterial vascular smooth muscle. A CysLT1 receptor with a low affinity for CysLT1 antagonists and a novel CysLT receptor subtype, both responsible for vasoconstriction. Activation of this latter receptor by LTC4 and LTD4 induced a contractile response which was resistant to the selective CysLT1 antagonists (ICI 198615 and MK 571) as well as the non‐selective (CysLT1/CysLT2) antagonist, BAY u9773.

Prostacyclin release and receptor activation: differential control of human pulmonary venous and arterial tone

In human pulmonary vascular preparations, precontracted arteries were more sensitive to the relaxant effect of acetylcholine (ACh) than veins (pD2 values: 7.25±0.08 (n=23) and 5.92±0.09 (n=25), respectively). Therefore, the role of prostacyclin (PGI2) was explored to examine whether this mediator may be responsible for the difference in relaxation. In the presence of the cyclooxygenase (COX) inhibitor, indomethacin (INDO), the ACh relaxations were reduced in arteries but not in veins. On the contrary, an inhibitor (L‐NOARG) of the nitric oxide synthase blocked preferentially the relaxation in veins. A greater release of 6‐keto‐PGF1α, the stable metabolite of PGI2, was observed in arterial preparations than in venous preparations when stimulated with either ACh or arachidonic acid (AA). Exogenous PGI2 produced a reduced relaxant effect in the precontracted vein when compared with the artery. In the presence of the EP1‐receptor antagonist AH6809, the PGI2 relaxation of veins was similar to arteries. In veins, AA (0.1 mM) produced a biphasic response, namely, a contraction peak (0.4–0.5 g) followed by a relaxation. These contractions in venous preparations were abolished either in the absence of endothelium or in the presence of INDO or an EP1‐receptor antagonist (AH6809, SC19220). In the arterial preparations AA induced only relaxations. In both vascular preparations, COX‐1 but not the COX‐2 protein was detected in microsomal preparations derived from homogenized tissues or freshly isolated endothelial cells. The differential vasorelaxations induced by ACh may be explained, in part, by a more pronounced production and release of PGI2 in human pulmonary arteries than in the veins. In addition, while PGI2 induced relaxation by activation of IP‐receptors in both types of vessels, a PGI2 constrictor effect was responsible for masking the relaxation in the veins by activation of the EP1‐receptor.

Cholinesterase activity in human pulmonary arteries and veins

Human isolated pulmonary vessels were treated with cholinesterase (ChE) inhibitors to determine the role of these enzymes in regulating vascular muscle tone. In addition, kinetic parameters were determined for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in human pulmonary vessel homogenates. Carbachol (CCh) and acetylcholine (ACh) were equipotent contractile agonists in human pulmonary arteries (pD2 values, 5.28±0.05 and 5.65±0.16; Emax, 0.91±0.26 and 0.98±0.30 g wt. for CCh and ACh, respectively; n=7). In venous preparations, ACh was ineffective and CCh induced small contractions (Emax, 0.08±0.04 g wt.; n=13). In human pulmonary arteries following pretreatment with tetraisopropylpyrophosphoramide (iso‐OMPA, 100 μM), an increased sensitivity to the contractile agonist ACh was observed (pD2 values, 5.80±0.13 and 6.37±0.19 for control and treated preparations, respectively; n=5). This pretreatment had no effect on the CCh concentration response curve. In contrast, human pulmonary veins pretreated with iso‐OMPA failed to elicit a contractile response to ACh. Neither Iso‐OMPA nor neostigmine elicited concentration‐dependent contractions in human isolated pulmonary arteries or veins. These results suggest the absence of sufficient spontaneous release of ACh to modulate human pulmonary vessel basal tone. CCh was less potent than ACh in relaxing precontracted human isolated pulmonary arteries (pD2 value, CCh: 6.55±0.15 and ACh: 7.16±0.13, n=4) and veins (pD2 value, CCh: 4.95±0.13; n=5 and ACh: 5.56±0.17; n=6). Pretreatment of vessels with either iso‐OMPA or neostigmine did not modify ACh relaxant responses in either type of preparation. In human pulmonary veins, the ChE activity was two fold greater than in arteries (n=6). Vmax for AChE was 1.73±0.24 and 3.36±0.26 miu mg−1 protein in arteries and veins, respectively, whereas Vss for BChE was 1.83±0.22 and 4.71±0.17 miu mg−1 protein, in these respectively. In human pulmonary arteries, BChE activity may play a role in the smooth muscle contraction but not on the smooth muscle endothelium‐dependent relaxation induced by ACh. A role for ChE activity in the control of venous tone is presently difficult to observe, even though this tissue contains a greater amount of enzyme than the artery.

Basal Secretion of Lysozyme from Human Airways in Vitro

The aim of this study was to examine the basal release of lysozyme from isolated human lung tissues. Measurements of lysozyme in the fluids derived from lung preparations were performed using a rate-of-lysis assay subsequent to acidification of the biological samples. Lysozyme released from bronchial preparations into fluids was greater than that observed for parenchymal tissues. The lysozyme quantities detected in bronchial fluids were not modified by removal of the surface epithelium. Furthermore, the quantities of lysozyme in bronchial fluids was correlated with the size of the bronchial preparations. These results suggest that the lysozyme was principally secreted by the human bronchi (submucosal layer) rather than by parenchyma tissues and that a greater release was observed in the proximal airways.

Antigen stimulation of human pulmonary smooth muscle: An in vitro model of inflammation

Human airways in vitro contract when stimulated by anti-IgE, whereas human pulmonary vessels relax. Leukotriene D4 (LTD4) induced a contractile response in the airways, while in pulmonary vessels both contractions and relaxations were observed. The LTD4 contractions in airways were blocked by cysLT1 receptor antagonists (MK 571, ICI 198615, and BAY x7195). In contrast none of the compounds affected the LTD4 contractions of pulmonary veins. These results suggest that the leukotrienes which are released during antigen challenge of airways and pulmonary vessels may be acting at distinct receptors in the human lung.

The contraction of the human pulmonary artery by LTC4 is resistant to cysLT1 antagonists and counteracted by prostacyclin release.

The role of cysteinyl leukotrienes (cysLTs; LTC4LTD4 and LTE4) in asthma has been well established (Drazen et al., 1999). However, cysLTs also contract isolated human pulmonary vascular preparations (Schellenberg and Foster, 1984; Bourdillat et al., 1987; Labat et al., 1992; Ortiz et al., 1995) and have been proposed to be involved also in vascular pathologies, for example pulmonary hypertension (Stenmark et al., 1983). This suggestion has received support from animal studies (Davidson and Drafta, 1992) but the precise function of cysLTs in the pulmonary circulation remains to be established. Previous studies have raised support for at least two different receptors for cysLTs (Labat et al., 1992; Coleman et al., 1995; Back et al., 1996; Dahlen, 1998). Human bronchi contain CysLTI receptors (Buckner et al., 1986) that are targets for the clinically used anti-asthmatic CysLTI antagonists (Drazen et al., 1999). The receptor present on the human pulmonary venous smooth muscle is however resistant to CysLTI antagonists and

Anti-IgE Response in Human Airways: Relative Contribution of Inflammatory Mediators

Heman airway preparations at resting tone were relaxed with either the leukotriene synthesis inhibitor BAY x1005 (3 μM), chlorpheniramine (1 μM) or the thromboxane receptor antagonist BAY u3405 (0.1 μM). The response to anti-IgE (1:1000) was 58 ± 8% of acetylcholine pre-contraction (2.19 ± 0.28 g). Indomethacin (3 μM) enhanced the anti-IgE-induced contraction by 28%. The anti-IgE maximal response was not modified by either chlorpheniramine, BAY x1005 or BAY u3405. When the tissues were treated with either BAY xl005/indomethacin or BAY x1005/chlorpheniramine, the anti-IgE-induced contraction was reduced. In addition, in presence of BAY xl005/indomethacin/chlorpheniramine the response was completely blocked. These results suggest that mediatots released during anti-IgE challenge cause airway contraction which may mask the evaluation of the leukotriene component.

MUC5AC mucin release from human airways in vitro: Effects of indomethacin and Bay X1005

BACKGROUND: Increased secretion of mucus is a hallmark of many respiratory diseases and contributes significantly to the airflow limitation experienced by many patients. While the current pharmacological approach to reducing mucus and sputum production in patients is limited, clinical studies have suggested that drugs which inhibit the cyclooxygenase and/or 5-lipoxygenase enzymatic pathways may reduce secretory activity in patients with airway disease. AIM: This study was performed to investigate the effects of indomethacin (cyclooxygenase inhibitor) and Bay x 1005 (5-lipoxygenase inhibitor) on MUC5AC release from human airways in vitro. METHODS: An immunoradiometric assay was used to determine the quantities of MUC5AC present in the biological fluids derived from human airways in vitro. The measurements were made with a mixture of eight monoclonal antibodies (MAbs; PM8) of which the 21 M1 MAb recognized a recombinant M1 mucin partially encoded by the MUC5AC gene. RESULTS: The quantities of MUC5AC detected in the biological fluids derived from human bronchial preparations were not modified after treatment with indomethacin (cyclooxygenase inhibitor) and/or an inhibitor of the 5-lipoxygenase metabolic pathway (BAY x 1005). CONCLUSION: These results suggest that the cyclooxygenase and 5-lipoxygenase metabolic pathways play little or no role in the release of MUC5AC from human airways.