Laurence Walch

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.

Prostanoid EP1- and TP-receptors involved in the contraction of human pulmonary veins

To characterize the prostanoid receptors (TP, FP, EP1 and/or EP3) 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. The stable thromboxane A2 mimetic, U46619, was a potent constrictor agonist on human pulmonary veins (pEC50=8.60±0.11 and Emax=4.61±0.46 g; n=15). The affinity values for two selective TP‐antagonists (BAY u3405 and GR32191B) versus U46619 were BAY u3405: pA2=8.94±0.23 (n=3) and GR32191B: apparent pKB=8.25±0.34 (n=3), respectively. These results are consistent with the involvement of TP‐receptor in the U46619 induced contractions. The two EP1‐/EP3‐ agonists (17‐phenyl‐PGE2 and sulprostone) induced contraction of human pumonary veins (pEC50=8.56±0.18; Emax=0.56±0.24 g; n=5 and pEC50=7.65±0.13; Emax=1.10±0.12 g; n=14, respectively). The potency ranking for these agonists: 17‐phenyl‐PGE2>sulprostone suggests the involvement of an EP1‐receptor rather than EP3. In addition, the contractions induced by sulprostone, 17‐phenyl‐PGE2 and the IP‐/EP1‐ agonist (iloprost) were blocked by the DP‐/EP1‐/EP2‐receptor antagonist (AH6809) as well as by the EP1 antagonist (SC19220). PGF2α 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. These data suggest that the contractions induced by prostanoids involved TP‐ and EP1‐receptors in human pulmonary venous smooth muscle.

M1 and M3 muscarinic receptors in human pulmonary arteries

1 Acetylcholine (ACh) and the M1 agonists (McN‐A‐343 or PD142505) relaxed human isolated pulmonary arteries which were pre‐contracted with noradrenaline (10 μm). In preparations where the endothelium had been removed ACh induced a contractile response whereas the M1 agonists (McN‐A‐343 or PD142505) had no effect. 2 ACh‐ and McN‐A‐343‐induced relaxations were abolished after treatment of endothelium‐intact preparations with the drug combination NG‐nitro‐L‐arginine (L‐NOARG: 0.1 mM) and indomethacin (1.7 μm). 3 The affinity (pKB value) for pirenzepine was higher in human pulmonary arteries when tissues were relaxed with McN‐A‐343 as compared with ACh (pKB values, 7.71 ± 0.30 (n = 4) and 6.68 ± 0.15 (n = 8), respectively). In addition, the affinity for pFHHSiD against McN‐A‐343‐ and ACh‐induced relaxations was 6.86 ± 0.13 (n = 3) and 7.35 ± 0.11 (n = 9), respectively. 4 The low affinities for methoctramine in human isolated pulmonary arteries with the endothelium either intact or removed, suggested the lack of involvement of M2 and M4 receptors in the ACh responses. 5 Phenoxybenzamine (3 μm: 30 min) abolished both ACh contraction and relaxation in human pulmonary artery. The ACh contraction was present when the phenoxybenzamine treatment was preceded by incubation with pFHHSiD (2 μm) but not with pirenzepine (1 μm). In addition, the ACh relaxation was present when preparations were treated with either pFHHSiD (2 μm) or pirenzepine (1 μm), before exposure to phenoxybenzamine. 6 These results in human isolated pulmonary arteries support the notion that only M3 receptors, on smooth muscle, mediate the ACh‐induced contraction whereas M3 and M1 receptors are involved in the endothelium‐dependent ACh‐induced relaxation.

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.

Evidence for a M1-muscarinic receptor on the endothelium of human pulmonary veins.

To characterize the muscarinic receptors on human pulmonary veins associated with the acetylcholine (ACh)‐induced relaxation, isolated venous and arterial preparations were pre‐contracted with noradrenaline (10 μM) and were subsequently challenged with ACh in the absence or presence of selective muscarinic antagonists. ACh relaxed venous preparations derived from human lung with a pD2 value of 5.82±0.09 (n=16). In venous preparations where the endothelium had been removed, the ACh relaxations were abolished (n=4). ACh relaxed arterial preparations with a pD2 value of 7.06±0.14 (n=5). Atropine (1 μM), the non selective antagonist for muscarinic receptors, inhibited ACh‐induced relaxations in human pulmonary veins. The affinity value (pKB value) for atropine was: 8.64±0.10 (n=5). The selective muscarinic antagonists (darifenacin (M3), himbacine (M2,M4), methoctramine (M2) and pFHHSiD (M1,M3)) also inhibited ACh‐induced relaxations in venous preparations. The pKB values obtained for these antagonists were not those predicted for the involvement of M2–5 receptors in the ACh‐induced relaxation in human pulmonary veins. The pKB value for darifenacin (1 μM) was significantly greater in human pulmonary arterial (8.63±0.14) than in venous (7.41±0.20) preparations derived from three lung samples. In human pulmonary veins, the pKB values for pirenzepine (0.5 and 1 μM), a selective antagonist for M1 receptors, were: 7.89±0.24 (n=7) and 8.18±0.22 (n=5), respectively. In the venous preparations, the pKB values derived from the functional studies with all the different muscarinic antagonists used were correlated (r=0.89; P=0.04; slope=0.78) with the affinity values (pKi values) previously published for human cloned m1 receptors in CHO cells. These results suggest that the relaxations induced by ACh are due to the activation of M1 receptors on endothelial cells in isolated human pulmonary veins.

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.

Cholinesterase activity in pig airways and epithelial cells

Summary— Acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) activities were detected in bronchial and bronchial epithelial cell homogenates of the pig. In the bronchial homogenates, the maximal upstroke velocity (Vmax) of AChE and the maximal velocity after second substrate fixation (Vss) of BChE were 5.70 ± 0.46 and 7.87 ± 0.81 mU/mg protein, respectively. In the epithelial cell homogenates, a smaller amount of cholinesterase (ChE) was found: Vmax was 0.62 ± 0.29 and Vss was 1.56 ± 0.33 mU/mg protein for AChE and BChE, respectively. AChE activity was increased by 21 ± 5% in the bronchial homogenates and by 54 ± 14% in the epithelial cell homogenates, when intact bronchial segments were incubated with a cyclooxygenase inhibitor, indomethacin (INDO). These results suggest that prostanoids may be involved in the regulation of AChE activity in pig airways.

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