Bradykinin antagonists in human systems: correlation between receptor binding, calcium signalling in isolated cells, and functional activity in isolated ileum.

The determination of the relationship between ligand affinity and bioactivity is important for the understanding of receptor function in biological systems and for drug development. Several physiological and pathophysiological functions of bradykinin (BK) are mediated via the B2 receptor. In this study, we have examined the relationship between B2 receptor (soluble and membrane-bound) binding of BK peptidic antagonists, inhibition of calcium signalling at a cellular level, and in vitro inhibition of ileum contraction. Only human systems were employed in the experiments. Good correlations between the studied activities of BK antagonists were observed for a variety of different peptidic structures. The correlation coefficients (r) were in the range of 0.905 to 0.955. In addition, we analyzed the effect of the C-terminal Arg9 removal from BK and its analogs on B2 receptor binding. The ratios of binding constants (Ki(+Arg)/Ki(-Arg)) for the Arg9 containing compounds and the corresponding des-Arg9 analogs varied from about 10 to 250,000. These ratios strongly depend on the chemical structures of the compounds. The highest ratios were observed for two natural agonist pairs, BK/des-Arg9-BK and Lys0-BK/des-Arg9-Lys0-BK.

Evidence of a bradykinin B1 receptor in human ileum: pharmacological comparison to the rabbit aorta B1 receptor.

Bradykinin B1 receptors have been identified in a limited number of human tissues and may have implications in pathological states of chronic inflammation. In the present study, longitudinal strips of postmortem human ileum displayed a strong contractile response to the B2 receptor agonist, bradykinin (EC50 = 7.0 nM). Noninduced ileum strips contracted only to high concentrations (1 and 10 microM) of the B1 receptor agonists, des-Arg9-BK and Lys0des-Arg9-BK. After incubation overnight at 37 degrees C the potency of des-Arg9-BK and Lys0des-Arg9-BK dramatically increased (EC50 = 183 and 13.2 nM, respectively). The increase in B1 agonist potency was inhibited by the protein synthesis inhibitor, puromycin. Similarly, rabbit aorta strips displayed a protein synthesis-dependent induction of the B1 agonist response. Incubated human ileum and rabbit aorta exhibited a reproducible response to des-Arg9-BK over time, whereas responses to Lys0des-Arg9-BK were not reproducible, having reduced potency and magnitude over time. Lys0[Leu8]des-Arg9-BK was a more potent antagonist at the B1 receptor in both tissues compared with [Leu8]des-Arg9-BK. The B2 antagonist, HOE-140, was a very weak inhibitor of the B1 response in human ileum and inactive in rabbit aorta. In conclusion, incubation of isolated human ileum overnight induces expression of a B1 receptor through a mechanism that depends on de novo protein synthesis. The potency profile of selected B1 agonists and antagonists indicates pharmacological similarities between the inducible B1 receptor in both the human ileum and rabbit aorta.

Vagal stimulation augments pulmonary anaphylaxis in the guinea pig lung.

The effect of bilateral vagal stimulation on aerosolized antigen-induced responses was examined in the sensitized, perfused guinea pig lung. Vagal stimulation in the sensitized, perfused lung resulted in bronchoconstriction (peak response 160 +/- 18% above baseline) that was unaffected by either atropine (1 microM), a muscarinic receptor antagonist, or CP 96,345 (1 microM), a NK-1 receptor antagonist, but was transiently augmented in the presence of physostigmine (1 microM), a cholinesterase inhibitor, through an atropine-sensitive mechanism. However, SR 48968 (1 microM), a NK-2 receptor antagonist, and SR 48968 + CP 96,345 reduced by approximately 50 and 90%, respectively, vagally mediated increases in intratracheal pressure in the perfused lung. Simultaneous challenge with vagal stimulation and aerosolized antigen in the sensitized perfused lung resulted in a significant (p < 0.01) increase in intratracheal pressure (Pi), pulmonary arterial pressure (Ppa), and lung weight (LW) compared with either vagal stimulation or aerosolized antigen alone. Increases in Pi, Ppa, and LW in response to vagal stimulation + aerosolized antigen were associated with elevated venous effluent concentrations of thromboxane A2 (TXA2), prostacyclin, leukotriene C4, and histamine. Vagally mediated potentiation of aerosolized antigen-induced increases in Pi, Ppa, and LW was unaffected by atropine or CP 96,345 but was inhibited by the NK-2 receptor antagonist, SR 48968. These data suggest that vagally mediated (predominantly NK-2) potentiation of aerosolized antigen-induced increases in Pi, Ppa, and LW is characterized by elevated venous effluent concentrations of eicosanoids and histamine.

Ro 25-1553: a novel, long-acting vasoactive intestinal peptide agonist. Part II: Effect on in vitro and in vivo models of pulmonary anaphylaxis.

Studies were conducted to compare the effect of native vasoactive intestinal peptide (VIP), Ro 25-1553 (a cyclic peptide analog of VIP) and salbutamol (a beta2-adrenoceptor agonist) on antigen-induced pathophysiological effects in the guinea pig. Ro 25-1553 and salbutamol (0.01-1.0 microM) prevented antigen-induced contractions of the guinea pig trachea in vitro with IC50 values of 0.07 and 0.05 microM, respectively. VIP (0.01-1.0 microM) had no effect on antigen-induced tracheal contractions. Aerosolized Ro 25-1553 and salbutamol were equipotent in preventing antigen-induced increases in guinea pig lung resistance (IC50 value = 0.0001%), whereas aerosolized VIP (0.1%) was ineffective. Ro 25-1553 (0.1-100 micrograms), instilled intratracheally 2 min before the antigen challenge of buffer-perfused lungs from sensitized guinea pigs, produced a dose-dependent inhibition of bronchoconstrictor, vasoconstrictor and edemagenic responses, whereas intratracheal VIP (100 micrograms) had no effect. Intratracheal salbutamol (0.1-100 micrograms) inhibited antigen-induced responses in a manner comparable to Ro 25-1553. Lung inflammation was assessed as leukocyte accumulation in bronchoalveolar lavage fluid after the antigen provocation. Aerosolized antigen-induced bronchoalveolar lavage eosinophilia (13-fold increase over saline controls) at 6 hr after challenge was prevented in a concentration-dependent manner by pretreatment with nebulized Ro 25-1553 and salbutamol, but not by pretreatment with native VIP. These results indicate that Ro 25-1553 suppresses various pathophysiological features associated with pulmonary anaphylaxis and asthma, including airway reactivity, edema formation and granulocyte accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Ro 25-1553: a novel, long-acting vasoactive intestinal peptide agonist. Part I: In vitro and in vivo bronchodilator studies.

Ro 25-1553, a cyclic peptide analog of vasoactive intestinal peptide (VIP), was designed to overcome many of the deficiencies inherent in this natural neuropeptide. On isolated guinea pig tracheal smooth muscle, Ro 25-1553 produces concentration-dependent relaxation of contractile responses to a number of different spasmogens. Depending on the contractile stimulus, Ro 25-1553 is 24 to 89 times more potent than VIP as a relaxant of guinea pig trachea. The high potency of Ro 25-1553 extends to studies on isolated, histamine-contracted, human bronchial smooth muscle, where Ro 25-1553 exhibits a 390-fold enhancement over native VIP and is more potent than other bronchodilating drugs, such as the beta 2-adrenoceptor agonists isoproterenol and salbutamol. Ro 25-1553 was shown to displace the radioligand 125I-VIP from rat forebrain membranes with an IC50 value of 4.98 nM, thereby demonstrating that it acts at a VIP receptor. In addition, when tested in a battery of 40 other binding assays (e.g., muscarinic, histamine, LTs, Ca++, TxA2, endothelin, alpha and beta adrenergic, platelet-activating factor, neurokinins, etc.) at concentrations as high as 10 microM, Ro 25-1553 was found to be inactive; thus it appears to be specific for VIP receptors. The potent smooth muscle relaxant activity exhibited in vitro by Ro 25-1553 is also evident after in vivo intratracheal administration or aerosolization of the compound. Pulmonary responses evoked by histamine, leukotriene D4, platelet-activating factor and acetylcholine are inhibited dose-dependently by intratracheally instilled Ro 25-1553 with nearly identical potency (ED50 values ranging from 0.07 micrograms to 0.26 micrograms).(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacologic modulation of antigen-induced pulmonary responses in the perfused guinea pig lung.

The effect of various enzyme inhibitors and receptor antagonists on antigen (ovalbumin)-induced changes in pulmonary hemodynamics (arterial pressure, capillary pressure, and arterial and venous resistance), fluid filtration, and airway reactivity were monitored for 60 min in recirculating Ringer's-perfused, actively sensitized lungs. Bolus ovalbumin (30 micrograms) injection into the pulmonary artery produced initial (3 min postovalbumin) increases in pulmonary arterial pressure of 68 +/- 9% above baseline, which were followed by secondary increases (143 +/- 45% above baseline) at 30 min postovalbumin. Ovalbumin challenge also caused initial increases in pulmonary capillary pressure, arterial resistance, and venous resistance within 3 min after administration (100 +/- 34%, 51 +/- 10%, and 221 +/- 77% above baseline, respectively), which were further elevated at the end of the 60-min experimental period (292 +/- 74%, 66 +/- 29%, and 559 +/- 61% above baseline, respectively). Ovalbumin-induced increases in intratracheal pressure (771 +/- 142% above baseline) peaked at 3 min postchallenge and gradually returned towards baseline. Ovalbumin-induced changes in lung weight increased gradually over the perfusion period (3.5 +/- 1.0 g above baseline at 60 min postovalbumin). Antigen-induced changes in pulmonary arterial pressure, intratracheal pressure, and lung weight were abolished by pretreatment with the histamine1-receptor antagonist, pyrilamine (1 microM). The cyclooxygenase inhibitor, indomethacin (1 microM), potentiated antigen-induced secondary increases in pulmonary arterial pressure, intratracheal pressure, and lung weight.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacology of a potent platelet-activating factor antagonist: Ro 24-4736.

Ro 24-4736, (5-(3-[4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f] [1,2,4]triazolo[4,3-a][1,4]diazepin-2-yl]-2-propynyl)phenanthri din- 6(5H)-one), has been identified as a potent, selective, p.o.-active platelet-activating factor (PAF) antagonist with a long duration of action. In vitro, Ro 24-4736 competes with [3H]PAF for its receptor site on dog platelets with an IC50 of 9.8 +/- 1.0 nM and selectively inhibits PAF-induced aggregation of guinea pig, dog and human platelets with concentration dependence. Ro 24-4736 dose-dependently inhibits in vivo bronchoconstriction (ID50 of 0.006-mg/kg p.o.) and ex vivo platelet aggregation (ID50 of 0.004 mg/kg p.o.) induced by PAF in guinea pigs. Time course studies show complete blockade of PAF-induced platelet aggregation (ex vivo) up to 8 hr after a single p.o. dose of 0.03 mg/kg as well as a long duration of action in vivo (30 hr). The in vivo PAF antagonistic activity is specific because, even at high p.o. doses (up to 10 mg/kg), Ro 24-4736 shows no inhibitory activity toward the bronchoconstrictor effects of leukotriene D4 or histamine. In comparison with other PAF antagonists evaluated in this guinea pig model, Ro 24-4736 is markedly superior in terms of p.o. potency, bioavailability and p.o. duration of action. Studies were also performed with Ro 24-4736 in additional in vivo models. When administered p.o. to sensitized guinea pigs, the drug attenuates inhaled antigen-induced airway hyper-reactivity without effect on bronchoalveolar lavage leukocyte accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Airway eosinophils from actively sensitized guinea pigs exhibit enhanced superoxide anion release in response to antigen challenge.

Antigen challenge of actively sensitized guinea pigs produces airway eosinophilia, airway hyperreactivity, and late-phase bronchoconstriction. The recruited eosinophils are thought to be important cells in the development of the airway hyperreactivity and the late-phase bronchoconstriction. However, the functional abilities of these eosinophils have not been determined in response to antigen challenge. The purpose of this study was to describe the characteristics of superoxide anion release from airway eosinophils obtained 24 h after ovalbumin challenge of actively sensitized guinea pigs. Eosinophils were collected by bronchoalveolar lavage. The total bronchoalveolar lavage eosinophil count was 17- to 27-fold greater in sensitized, ovalbumin-challenged guinea pigs (9.30 +/- 0.11 x 10(6)/guinea pig) than in unsensitized guinea pigs (0.35 +/- 0.07 x 10(6)/guinea pig) or sensitized, saline-challenged guinea pigs (0.56 x 10(6)/guinea pig; n = 2). The increase in eosinophils was due to increased lavage leukocyte count and increased eosinophil differential. Eosinophils were isolated on a Percoll-plasma discontinuous gradient. Two populations of eosinophils were collected, one at the 1.093 g/ml gradient step and one at the 1.107 g/ml gradient step. Unstimulated or phorbol myristate acetate (PMA)-stimulated superoxide anion release was measured by the reduction of ferricytochrome c. Unstimulated superoxide anion release from both eosinophil populations of challenged guinea pigs (4.50 +/- 2.37 and 4.07 +/- 1.48 nmol from 1.093 and 1.107 g/ml eosinophils, respectively) was 6- to 7-fold greater than superoxide anion release from eosinophils of control guinea pigs (0.74 +/- 0.43 and 0.56 +/- 025 nmol from 1.093 and 1.107 g/ml eosinophils, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipase A2-induced pulmonary and hemodynamic responses in the guinea pig. Effects of enzyme inhibitors and mediators antagonists.

The effect of phospholipase A2 (Naja naja) PLA2) on mean arterial blood pressure and intratracheal pressure was examined in anesthetized guinea pigs. Intracheally administered PLA2 (1 to 10 U) produced acute, dose-dependent increases in mean arterial blood pressure and intracheal pressure. However, Intravenously administered PLA2 (doses as large as 1,000 U) did not alter monitored variables. Acute PLA2-induced morphologic alterations were characterized by airway constriction, airway/alveolar cell damage, and pulmonary sequestration of both leukocytes and platelets. PLA2-induced increases in both mean arterial blood pressure and intratracheal pressure were attenuated to varying degrees by pretreating intravenously with indomethacin (10 mg/kg), a cyclooxygenase inhibitor, and WEB 2086 (0.1 mg/kg), a platelet-activating factor antagonist. Both ICI 198,615 (1 mg/kg), a leukotriene D4, receptor antagonist given intravenously, and dexamethasone (50 mg/kg), a steroidal anti-inflammatory agent given intraperitoneally as a 2-day pretreatment, reduced PLA2-induced increases in intratracheal pressure. Pyrilamine (2 mg/kg), a histamine1-receptor antagonist given intravenously, did not modify PLA2-induced pathophysiologic responses. Guinea pigs exposed to aerosolized PLA2 (100 U/ml) exhibited evidence of increased bronchoalveolar lavage macrophage, leukocyte, and lymphocyte accumulation at 24 h post-PLA2. These studies suggest that in vivo PLA2-induced pathophysiologic changes in the guinea pig involve alterations in resident airway cell populations as well as sequestration and infiltration of inflammatory cells. Both eicosanoids and platelet-activating factor appear to contribute to these PLA2-induced pathophysiologic effects.

Pathophysiologic changes induced by phospholipase A2 in the isolated, perfused guinea pig lung.

Pathophysiologic changes induced by phospholipase A2 (PLA2), a lipolytic enzyme implicated in a variety of pulmonary diseases, were examined in the isolated, perfused guinea pig lung. Thirty microliters of vehicle (saline) or 30 units of PLA2 suspended in saline was given as a bolus injection into either the trachea or pulmonary artery, and the lungs were examined by combined light and transmission electron microscopy at 1, 10, and 30 min following administration. Pulmonary morphologic and physiologic changes were observed only in animals that received PLA2 via the trachea. Initially, morphologic evidence of airway constriction was accompanied by a significant increase in intratracheal pressure. No morphologic change was observed in alveolar parenchymal cell populations at this time. Later there was marked swelling of airway epithelial cells accompanied by pronounced blebbing of the apical cytoplasm with a subsequent decrease in airway lumen size. Interalveolar septa were multifocally thickened by edema and alveolar spaces contained sparse fibrogranular material 10 min after intratracheal PLA2. Significant increases in lung weight were observed at this time. Morphologic features of vasoconstriction, characterized by undulating and convoluted endothelium with prominent cytoplasmic processes resulting in decreased luminal size were observed in small caliber vessels and occurred in conjunction with significant increases in pulmonary arterial pressure. There was pronounced injury to type I alveolar epithelium, including marked cell swelling and fragmentation, accompanied by lesser injury in type II alveolar epithelium. This study demonstrates that the airway epithelium was the initial cell population having morphologic change following intratracheal PLA2, and that the airway epithelium and type I alveolar epithelium were the cell populations most severely affected following intratracheal PLA2. Results further suggest that normal circulating levels of blood-formed elements are not essential for the development of PLA2-induced morphologic change in the isolated, perfused guinea pig lung.

Phospholipase A2-induced pathophysiologic changes in the guinea pig lung.

The pathophysiology of lung injury induced by phospholipase A2 (PLA2), a lipolytic enzyme implicated in a variety of pulmonary diseases, was examined in the guinea pig. One hundred microliters of saline or 10 units of PLA2 suspended in saline was given as a bolus injection into either the trachea or jugular vein. Intratracheal pressure and mean arterial blood pressure were continuously monitored. The lungs were examined by light and transmission electron microscopy at 1, 10, and 30 minutes after administration. Pulmonary morphologic and physiologic changes were only observed in animals that received PLA2 via the trachea. Significant increases in peak intratracheal pressure occurred as early as 1 minute after intratracheal PLA2 administration. Morphologic evidence of airway constriction, accompanied by blebbing of the apical cytoplasm of airway epithelium, was also observed at this time. A transient increase in mean arterial blood pressure occurred 5 minutes after challenge. At 10 minutes after intratracheal PLA2, there was marked swelling of airway epithelial cells, pronounced blebbing of the apical cytoplasm, and a resultant decrease in size of the airway lumen. Morphologic changes in alveolar cell populations were initially observed 10 minutes after intratracheal PLA2. Interalveolar septa were hypercellular and multifocally thickened. There was prominent perivascular edema and alveolar spaces contained abundant proteinaceous material and occasional hemorrhage. Ultrastructurally, there was marked cell swelling and fragmentation of type I alveolar epithelium resulting in a denuded basal lamina. Sequestration of neutrophils and eosinophils, many of which lacked secretory granules, within alveolar capillaries was accompanied by aggregates of platelets and was observed in close proximity to injured endothelium. Morphologic changes indicative of cell injury were also observed in type II alveolar epithelium. Similar, but more frequent and severe, morphologic injury occurred 30 minutes after intratracheal PLA2. It is concluded that PLA2 induces pronounced morphologic and physiologic changes in the guinea pig and that the route of administration is important in the development of PLA2-induced lung injury.

Pulmonary responses to phospholipase A2 in the perfused guinea pig lung.

We examined the effect of phospholipase A2 (PLA2; Naja naja) challenge on pulmonary hemodynamics, airway constriction, and fluid filtration in isolated Ringer-perfused guinea pig lungs. Intratracheal PLA2 (10-100 U) produced dose-dependent increases in pulmonary arterial pressure, intratracheal pressure, and lung weight, although intravenous PLA2 administration had no effect on monitored variables. Morphological features indicative of airway constriction and pulmonary edema were observed by light microscopy. PLA2-induced increases in intratracheal pressure and/or lung weight were attenuated to varying degrees by pretreatment with indomethacin (1 microM, a cyclooxygenase inhibitor), ICI-198,615 (1 microM, a leukotriene D4 receptor antagonist), and WEB 2086 (1 microM, a platelet-activating factor antagonist). PLA2-induced increases in pulmonary arterial pressure and intratracheal pressure were also reduced in lungs removed from animals pretreated with dexamethasone (50 mg/kg ip for 2 days; a steroidal antiinflammatory agent). Pyrilamine (1 microM, a histamine1-receptor antagonist) and Takeda AA861 (1 microM, a delta 5-lipoxygenase inhibitor) did not produce significant inhibitory effects on PLA2-induced pathophysiological changes. Intratracheal instillation of high-dose platelet-activating factor (50 micrograms) or lysophosphatidylcholine (100 micrograms) produced gradual increases in intratracheal pressure and lung weight, but these changes were not as large as those induced by PLA2. Thus these studies suggest that resident cell populations associated with airways may play an important role in PLA2-induced pathophysiological changes in the perfused guinea pig lung. These PLA2-induced effects are most likely partially mediated by generation of eicosanoids and platelet-activating factor.

Antigen-induced edema formation, bronchoconstriction, and pulmonary vasospasm in the isolated perfused guinea pig lung. Evidence for a secondary edemagenic response.

The effect of antigen (ovalbumin) challenge on pulmonary hemodynamics, bronchoconstriction, and fluid filtration was investigated in Ringer's-perfused (non-recirculating) lungs that had been passively sensitized in vitro. Bolus ovalbumin injection (30 micrograms) produced immediate increases in pulmonary arterial pressure, peak intratracheal pressure, and lung weight within 1 min and secondary marked increases in intratracheal pressure and lung weight from 120 to 200 min. Electron microscopy of antigen-challenged isolated lungs showed evidence of both septal and intraalveolar edema. Ionophore A23187 (100 micrograms) challenge of nonsensitized lungs produced immediate pulmonary responses similar to antigen, whereas secondary increases in lung weight were smaller. Arachidonic acid pretreatment (1 microM) potentiated immediate antigen-induced increases in intratracheal pressure but did not affect pulmonary responses to ionophore challenge. Putative mediators of anaphylaxis including histamine, leukotrienes B4, C4, D4, and E4, platelet-activating factor, and substance P produced immediate changes in pulmonary arterial and/or intratracheal pressure similar to antigen challenge. Only platelet-activating factor and substance P partially mimicked the secondary edema formation noted following antigen challenge. Thus, antigen challenge in in vitro sensitized guinea pig lungs produced both immediate and secondary responses characterized by increases in vascular pressure, airway pressure, and edema formation. This occurred in the absence of circulating blood-formed elements and without a massive influx of cells. Synergism between mediators such as histamine, the leukotrienes, platelet-activating factor, and substance P released following antigen challenge may be necessary to produce the complete pathophysiological sequelae associated with antigen challenge in the perfused guinea pig lung.

Pulmonary microvascular response to LTB4: effects of perfusate composition.

We examined the effects of leukotriene B4 (LTB4) on pulmonary hemodynamics and vascular permeability using isolated perfused guinea pig lungs and cultured monolayers of pulmonary arterial endothelial cells. In lungs perfused with Ringer solution, containing 0.5 g/100 ml albumin (R-alb), LTB4 (4 micrograms) transiently increased pulmonary arterial pressure (Ppa) and capillary pressure (Pcap). Pulmonary edema developed within 70 min after LTB4 injection despite a normal Pcap. The LTB4 metabolite, 20-COOH-LTB4 (4 micrograms), did not induce hemodynamic and lung weight changes. In lungs perfused with autologous blood hematocrit = 12 +/- 1%; protein concentration = 1.5 +/- 0.2 g/100 ml), the increases in Ppa and Pcap were greater, and both pressures remained elevated. The lung weight did not increase in blood-perfused lungs. In lungs perfused with R-alb (1.5 g/100 ml albumin) to match the blood perfusate protein concentration, LTB4 induced similar hemodynamic changes as R-alb (0.5 g/100 ml) perfusate, but the additional albumin prevented the pulmonary edema. LTB4 (10(-11)-10(-6) M) with or without the addition of neutrophils to the monolayer did not increase endothelial 125I-albumin permeability. Therefore LTB4 induces pulmonary edema when the perfusate contains a low albumin concentration, but increasing the albumin concentration or adding blood cells prevents the edema. The edema is not due to increased endothelial permeability to protein and is independent of hemodynamic alterations. Protection at higher protein-concentration may be the result of LTB4 binding to albumin.

Monohydroxyeicosatetraenoic acids (5-HETE and 15-HETE) induce pulmonary vasoconstriction and edema.

5-, 15-, and 12-HETE (monohydroxyeicosatetraenoic acids) are products of the lipoxygenation of arachidonic acid. We investigated their role as possible mediators of pulmonary vasoactivity and pulmonary edema. Pulmonary artery pressure (Ppa), capillary pressure (Pcap), the change in lung wet weight (delta wt) from baseline, and capillary filtration coefficient (Kf) (as a measure of vascular permeability) were determined following an intravenous injection of each mono-HETE in lungs perfused at constant flow with either a phosphate-buffered Ringer's-albumin solution (PBR) or diluted blood. Injection of 2 micrograms of each compound into the pulmonary artery of lungs perfused with either PBR or diluted blood did not produce any effect. However, in PBR-perfused lungs, 4 micrograms 15-HETE induced increases in Ppa, Pcap, and lung wet weight (p less than 0.05), which were greater than the increases observed after 4 micrograms 5-HETE. Kf increased following both 5- and 15-HETE. The pulmonary vasoconstrictor and edemagenic responses were attenuated by increasing perfusate albumin concentration from 0.5 to 1.5 g%. In contrast, 12-HETE (4 micrograms) had no effect on these parameters. In blood-perfused lungs, the pulmonary vascular responses to all HETE compounds (4 micrograms) were attenuated. In both Ringer's-albumin-perfused and blood-perfused lungs, the relative magnitude of the hemodynamic and fluid filtration responses to each mono-HETE were as follows: 15-HETE greater than 5-HETE greater than 12-HETE. In conclusion, the pulmonary vasoconstrictor and edemagenic effects of 5- and 15-HETE occur independently of blood-formed elements. 15-HETE causes greater pulmonary vasoconstriction and edema than 5-HETE. Both 5- and 15-HETE induce pulmonary edema, probably as a result of increased lung vascular permeability. The results indicate that 5- and 15-HETE are potent pulmonary inflammatory mediators.

Substance P-induced pulmonary vasoreactivity in isolated perfused guinea pig lung.

We examined the effects of the neuropeptide substance P on pulmonary hemodynamic and transvascular fluid filtration in isolated Ringer's-perfused and blood-enriched Ringer's-perfused guinea pig lung and on albumin flux across bovine pulmonary artery endothelial monolayer. Mean pulmonary artery, left atrial, and capillary pressures were determined and used to calculate arterial and venous resistances, and lung weight was continuously monitored. Substance P (0.01-1.0 microM) caused marked increases in pulmonary arterial pressure, capillary pressure, venous resistance, and lung weight within 3-5 minutes after administration. These responses remained elevated above baseline at the end of the 30-minute experimental period in the Ringer's-perfused lungs but not in the blood-enriched Ringer's-perfused lungs. Substance P did not alter the capillary filtration coefficient in isolated lungs and transendothelial albumin permeability in the endothelial monolayer. Substance P resulted in an increase in venous effluent thromboxane B2 concentrations in perfused lungs but had no effect on 6-keto-prostaglandin F1 alpha concentrations. Papaverine (0.27 mM) (a smooth-muscle relaxant) abolished the pulmonary microvascular response to substance P in Ringer's-perfused lungs, and meclofenamate (0.15 mM) (a cyclooxygenase inhibitor) attenuated the pulmonary vasoconstriction and lung weight increase. Pyrilamine (1.0 microM) (a histamine1-receptor antagonist) did not alter the responses to substance P. In conclusion, substance P does not affect pulmonary vascular permeability to water and protein. Substance P induces an intense pulmonary vasoconstriction (due to greater constriction of postcapillary vessels) and an elevation in pulmonary capillary pressure that increases net transvascular fluid filtration.(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonin-induced pulmonary responses in the perfused guinea pig lung: evidence for 5HT2 receptor-mediated pulmonary vascular and airway smooth muscle constriction.

The isolated perfused guinea pig lung was used to investigate pharmacologic characteristics of serotonin receptors in pulmonary vascular and airway smooth muscle. Serotonin caused a marked dose-related increase in pulmonary arterial pressure and peak intratracheal pressure when injected into the pulmonary artery. In contrast to serotonin, 5-carboxamidotryptamine, a 5HT1A and 5HT1B receptor agonist, and 2-methylserotonin, a 5HT3 receptor agonist produced only weak vascular and airway responses. Furthermore, vasoconstriction and bronchoconstriction elicited by serotonin were antagonized by the potent 5HT2 receptor antagonists, LY53857, ketanserin, and ritanserin. Antagonist specificity for 5HT2 receptors was demonstrated in the perfused guinea pig lung since similar responses induced by histamine were not blocked. High concentrations of serotonin were tachyphylactic on vascular but not airway constriction. Tachyphylaxis of vascular responses was not observed to an equipotent concentration of leukotriene D4 (LTD4). Thus, both the pulmonary vascular and airway constriction to serotonin were predominantly due to 5HT2 receptor activation. Furthermore, tachyphylaxis of vascular responses to serotonin might prove useful to differentiate 5HT receptor systems in pulmonary blood vessels from those in airways.