Effect of aminophylline on the pulmonary and systemic hemodynamic response to group B beta-hemolytic Streptococcus and leukotriene D4 in newborn lambs.

Aminophylline, a methyl xanthine, has been used for many years in the treatment of apnea of prematurity and bronchospasm. Aminophylline relaxes smooth muscle through several proposed mechanisms. We hypothesized that aminophylline might be effective in relaxing preconstricted pulmonary vascular smooth muscle and would be ideally suited for clinical trial in babies with pulmonary hypertension. To test this hypothesis, the haemodynamic response of chronically instrumented newborn lambs to injections of heat-killed Group B beta-hemolytic Streptococcus (GBS) and leukotriene (LT) D4, potent pulmonary vasoconstrictors was compared before and after pretreatment with a clinically therapeutic dose of intravenous aminophylline. GBS (10(9)cfu) significantly increased pulmonary arterial pressure 130%. LTD4 (1.0 microgram/kg) significantly increased pulmonary arterial pressure 142% and systemic arterial pressure 23% and decreased cardiac output 47%. Aminophylline did not significantly affect the baseline variables or alter the pulmonary or systemic haemodynamic response to either stimuli. Therefore, it is unlikely that aminophylline will be clinically useful in the treatment of babies with persistent pulmonary hypertension whose etiology is infectious or leukotriene-mediated.     

Action of PGI2 analogs on the pulmonary and systemic circulations in the conscious newborn lamb

Previous work (Lock et al., J. Pharm . Exp. Ther. 215:156, 1980) has shown that conventional screening procedures for vasoactive PGI2 analogs were little value in predicting pulmonary vasodilator activity in the newborn lamb. To gain a better insight into the structural requirements for pulmonary vasoactivity and possibly identify useful compounds for the management of neonatal pulmonary hypertensive disorders, we have tested the following PGI2 analogs in normoxic and hypoxic newborn lambs: 15(S)-9-deoxy-15-methyl-9 alpha,6- nitrilo -PGF1 (analog I); 9-deoxy-9 alpha,5- nitrilo -PGF1 (analog II); (6S, 15S)-15-methyl-PGI2 (analog III); and ( 6R , 15S)-15-methyl-PGI1 (analog IV). A prostaglandin analog mimicking PGI2 (compound BW245C ; (+/-)-5-(6- carboxyhexyl )-1-(3-cyclohexyl-3-hydroxypropyl)hydantoin ) was tested as well. Compounds were injected into a branch pulmonary artery and any local pulmonary effect could be assessed from the change in the ratio of blood flow to the injected lung over total flow. None of the analogs tested proved to be a selective pulmonary dilator. BW245C was a potent peripheral vasodilator (threshold around 0.5 microgram/kg) and indirectly lowered pulmonary vascular resistance through its systemic effects. Analog I also dilated the systemic circulation, but only at the highest dose tested (100 micrograms/kg). The latter finding is surprising because it was previously shown that the parent, non-methylated compound is a fairly potent and selective pulmonary vasodilator. Analog II and IV were inactive at a dose up to, respectively, 30 and 20 micrograms/kg. Analog III, on the other hand, weakly constricted the systemic circulation at a dose of 10 micrograms/kg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of leukotriene D4 on arterial pressure and gastrointestinal electrical activity in the conscious piglet.

In 6 conscious weaned piglets with implanted electrodes in the corpus and antrum of the stomach, the duodenum, jejunum, ileum and caecum the influence of intravenous infusion of leukotriene (LT)D4, 0.1 and 1 microgram kgmin for 10 min, on mean arterial pressure and gastrointestinal electrical activity was examined. LTD4 induced a significant increase in arterial pressure. Gastrointestinal electrical activity, however, was little influenced, since only the antrum pylori revealed a transient decrease.     

Relationship of leukotriene C4 and D4 to hypoxic pulmonary vasoconstriction in dogs

Leukotrienes C4 and D4 have been implicated as possible mediators of hypoxic pulmonary vasoconstriction. To test this hypothesis, the relationship between pulmonary leukotriene (LT) synthesis in response to hypoxia and alterations in pulmonary hemodynamics was evaluated in pentobarbital sodium-anesthetized, neuromuscular-blocked, male, mongrel dogs. A reduction in the fraction of inspired O2 (FIO2) in vehicle-treated animals (n = 12) from 0.21 to 0.10 was associated with increases in LTC4 and LTD4 in bronchoalveolar lavage fluid (BALF). After 30 min of continuous hypoxia, LTC4 and LTD4 increased from control values of 59.4 +/- 10.4 and 91.7 +/- 18.1 ng/lavage to 142.7 +/- 31.8 (P less than 0.05) and 156.3 +/- 25.3 (P less than 0.01) ng/lavage, respectively. Concomitantly, mean pulmonary arterial pressure (Ppa) and pulmonary vascular resistance (PVR) were increased over control by 67 +/- 7 (P less than 0.001) and 62 +/- 7% (P less than 0.001), respectively. In contrast, in animals treated with diethylcarbamazine (n = 5), a leukotriene A4 synthase inhibitor, identical reductions in FIO2 were not associated with increases in LTC4 and LTD4 in BALF, although at the same time period, Ppa and PVR were increased over control by 60 +/- 13 (P less than 0.05) and 112 +/- 31% (P less than 0.05), respectively. These results, therefore, do not support the contention that leukotrienes mediate hypoxic pulmonary vasoconstriction in dogs.     

Action of PGI2 analogs on the pulmonary and systemic circulations in the conscious newborn lamb

Previous work (Lock , J. Pharm. Exp. Ther. :156, 1980) has shown that conventional screening procedures for vasoactive PGI₂ analogs have little value in predicting pulmonary vasodilator activity in the newborn lamb. To gain a better insight into the structural requirements for pulmonary vasoactivity and possibly identify useful compounds for the management of neonatal pulmonary hypertensive disorders, we have tested the following PGI₂ analogs in normoxic and hypoxic newborn lambs: 15(S)-9-deoxy-15-methyl1–9α, 6-nitrilo-PGF₁ (analog I); 9-deoxy-9α, 5-nitrilo-PGF₁ (analog II); (6S, 15S)-15-methyl-PG1₁ (analog III); and (6R, 15S)-15-methyl-PGI₁ (analog IV). A prostaglandin analog mimicking PGI₂ (compound BW245C; (±)-5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl)hydantoin) was tested as well. Compounds were injected into a branch pulmonary artery and any local pulmonary effect could be assessed from the change in the ratio of blood flow to the injected lung over total flow. None of the analogs tested proved to be a selective pulmonary dilator. BW245C was a potent peripheral vasodilator (threshold around 0.5 μg/kg) and indirectly lowered pulmonary vascular resistance through its systemic effects. Analog I also dilated the systemic circulation, but only at the highest dose tested (100 μg/kg). The latter finding is surprising because it was previously shown that the parent, non-methylated compound is a fairly potent and selective pulmonary vasodilator. Analog II and IV were inactive at a dose up to, respectively, 30 and 20 μg/kg. Analog III, on the other hand, weakly constricted the systemic circulation at a dose of 10 μg/kg. These findings suggest that the neonatal pulmonary vasculature is endowed with specific receptor sites which can discriminative between closely related PGI₂ analogs.     

The effect of leukotriene D4 on the isolated stomach and colon of the rat

The effect of synthetic leukotriene D4 (LTD4) was evaluated on isolated gastric longitudinal or circular smooth muscle and distal colon of the rat. The concentrations of LTD4, 2.5 X 10(-10)M to 5 X 10(-7)M, evoked minimal to maximal contractile responses. In addition, selected prostaglandins were used to identify the mediator of LTD4-induced contraction of gastric smooth muscle. FPL 55712 inhibited LTD4-induced contractions of gastric longitudinal or circular muscle. Indomethacin inhibited only LTD4-induced contractions of the longitudinal muscle. A combination of FPL 55712 and indomethacin produced greater inhibition of LTD4-induced contractions of longitudinal muscle than either agent alone. However, the same combination of inhibitors showed no greater effect than FPL 55712 alone on LTD4-induced contractions of circular smooth muscle. Unlike PGI2, PGF2, PGA2, or PGD2, PGE2 evoked contraction of the longitudinal muscle and relaxation of the circular muscle of the stomach. The dissimilar effect of PGE2 in the two smooth muscle layers of the rat stomach may signify that PGE2 is the prostaglandin released by LTD4 from the longitudinal and circular gastric muscle. However, the opposing pharmacologic effects following LTD4-induced release of prostaglandins in the circular muscle of the stomach would preclude the appearance of an inhibitory effect of indomethacin in this tissue. In contrast, PGE2 and other prostaglandins contract gastric longitudinal muscle in response to LTD4. Thus, these studies clearly suggest that LTD4 has both a direct and indirect effect on gastric smooth muscle of the rat. Unlike the stomach, LTD4-induced contraction of the distal colon was not inhibited by indomethacin while FPL 55712 antagonized contractions. Thus, these findings indicate a differential mechanism of stimulation of rat gastrointestinal tissue by LTD4.     

Generation of leukotriene B4 and leukotriene E4 from porcine pulmonary artery

When chopped porcine pulmonary arteries were incubated with calcium ionophore A23187 (1) in the presence of indomethacin there was a time dependent generation of a substance which produced contractions of superfused strips of guinea-pig ileum smooth muscle (GPISM) which were indistinguishable from those induced by LTD4. This material however had a different retention time from LTD4 when subjected to HPLC and co-chromatographed with synthetic LTE4. In addition to LTE4 a substance which had properties indistinguishable from those of LTB4 when assayed on a combination of guinea-pig lung parenchymal strips (GPP) and GPISM (2) was generated from the pulmonary artery. This substance co-chromatographed with synthetic LTB4. The adventitia and intima were the richest source of LTE4, the adventitia releasing slightly more than the intima. The output of LTB4 and LTE4 was inhibited by 6,9-deepoxy-6,9-(phenylimino)-delta 6,8 prostaglandin I (U-60,257). Nordihydroguaiaretic acid (NDGA) inhibited the generation of LTE4.     

Studies on the leukotriene D4-metabolizing enzyme of rat leukocytes, which catalyzes the conversion of leukotriene D4 to leukotriene E4

Leukotriene D4-metabolizing enzyme was studied using rat neutrophils, lymphocytes and macrophages. These leukocyte sonicates converted leukotriene D4 to leukotriene E4. However, the leukotriene D4-metabolizing activity varied with cell type, and macrophages showed the highest activity among these leukocytes. The subcellular localization of the leukotriene D4-metabolizing enzyme of macrophages was examined, and the leukotriene D4-metabolizing activity was found to be present in the membrane fraction, but not in the nuclear, granular and cytosol fractions. When macrophages were modified chemically with diazotized sulfanilic acid, a poorly permeant reagent which inactivates cell-surface enzymes selectively, the leukotriene D4-metabolizing activity of macrophages decreased significantly (about 95%) without any inhibition of marker enzymes of microsome, cytosol, lysosome and mitochondria. When neutrophils and lymphocytes were modified with diazotized sulfanilic acid, the leukotriene D4-metabolizing activity was also inhibited about 90% by the modification. Among various enzyme inhibitors used, o-phenanthroline, a metal chelator, remarkably inhibited the leukotriene D4-metabolizing activity of leukocytes, and the o-phenanthroline-inactivated enzyme activity was fully reactivated by Co2+ and Zn2+. These findings seem to indicate that rat neutrophils, lymphocytes and macrophages possess the leukotriene D4-metabolizing metalloenzyme which converts leukotriene D4 to leukotriene E4, on the cell surface, although macrophages have a higher enzyme activity than the other two.     

A fetal-instrumented model to study age-specific responses to leukotriene D4 and prostaglandin D2 in unsedated newborn lambs

Sequential studies of the pulmonary vascular response to leukotriene D4 (LTD4) and prostaglandin D2 (PGD2) in the immediate newborn period were performed in lambs, instrumented in utero and delivered vaginally. Compounds were tested in fully conscious 1.5-day-old lambs and the study was repeated 1 week later. Bolus injections of PGD2 (0.05-2.0 micrograms/kg) or LTD4 (0.01-1.0 micrograms/kg) were made into the main pulmonary artery or aorta while pulmonary blood flow and aortic, pulmonary artery, and left and right atrial pressures were monitored continuously. PGD2 was a systemic constrictor regardless of age. In lambs 1.5 days of age, it decreased pulmonary vascular pressure and resistance by 6% (p less than 0.05) and 15% (p less than 0.05), respectively, while 1 week later it increased pulmonary vascular resistance by 18% (p less than 0.05). In contrast, LTD4 was a pulmonary and systemic vasoconstrictor in both the early and late newborn, the threshold dose being between 0.01 and 0.05 micrograms/kg at either age. The decrease in pulmonary blood flow and the increase in pressure and resistance were greater in older animals. In lambs 1.5 days of age, LTD4 (1 micrograms/kg) increased pulmonary vascular resistance by 66.1% (p less than 0.05) and 1 week later by 210% (p less than 0.001). These sequential observations in the same animal indicate that unlike PGD2, LTD4 is a pulmonary vasoconstrictor regardless of age, and its effectiveness increases significantly with age. These results support previous reports that PGD2 action in the pulmonary circulation changes shortly after birth from dilation to constriction.     

Increased leukotriene E4 excretion in systemic mastocytosis

Cysteinyl leukotrienes such as LTE₄ are produced by mast cells, neutrophils, eosinophils, and macrophages. LTE₄ levels have not been reported in systemic mastocytosis, a disorder with a large increase in mast cell numbers. Urinary LTE₄ from patients referred for symptoms potentially due to mast cell degranulation or systemic mastocytosis was measured by a commercial cysteinyl leukotriene enzyme immunoassay kit. The diagnosis of systemic mastocytosis was established using current World Health Organization criteria. Compared with a control group of patients with various potential mast cell–related symptoms (e.g., “spells”), patients with systemic mastocytosis had a significant (P = .01) increase in urinary LTE₄ excretion, whether expressed as LTE₄ ng/g creatinine or as LTE₄ ng/24 h. There was a moderate correlation of LTE₄ ng/24 h with excretion of N-methyl histamine and serum tryptase but not with urinary 11β-prostaglandin F_2α (11β-PGF_2α) excretion. LTE₄ excretion is increased in patients with systemic mastocytosis and potentially contributes to clinical symptoms.     

Production of early and late pulmonary responses with inhaled leukotriene D4 in allergic sheep

Some allergic sheep respond to inhalation of Ascaris suum antigen with both immediate and late increases in airflow resistance (late response). The mechanism of the late response is unknown but recent evidence suggests that the initial generation of slow-reacting substance of anaphylaxis (SRS-A) immediately after antigen challenge is a necessary pre-requisite for the physiologic expression of this late response. Based on this evidence we hypothesized that airway challenge with leukotriene D4 (LTD4), an active component of SRS-A would produce acute and late airway responses in allergic sheep similar to those observed with antigen. In five allergic sheep with documented early and late pulmonary responses to Ascaris suum antigen, inhalation of leukotriene D4 aerosol (delivered dose (mean +/- SE) 0.55 +/- 0.08 ug) resulted in significant early and late increases in specific lung resistance (SRL). In three allergic sheep which only demonstrated acute responses to antigen, LTD4 aerosol (delivered dose 0.59 +/- 0.09 ug) only produced an acute increase in SRL. In the late responders pretreatment with aerosol cromolyn sodium (1 mg/kg) did not affect the acute response but blunted the late increase in SRL. Pretreatment with aerosol FPL-57231 (1% w/v solution) completely blocked both the acute and late responses. These data support the hypothesis that initial release of LTD4 in the airways of sensitive animals is important for the physiologic expression of the late response.     

The effect of oxidative stress on nucleotide-excision repair in colon tissue of newborn piglets

Nucleotide-excision repair (NER) is important for the maintenance of genomic integrity and to prevent the onset of carcinogenesis. Oxidative stress was previously found to inhibit NER in vitro, and dietary antioxidants could thus protect DNA not only by reducing levels of oxidative DNA damage, but also by protecting NER against oxidative stress-induced inhibition. To obtain further insight in the relation between oxidative stress and NER activity in vivo, oxidative stress was induced in newborn piglets by means of intra-muscular injection of iron (200 mg) at day 3 after birth. Indeed, injection of iron significantly increased several markers of oxidative stress, such as 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) levels in colon DNA and urinary excretion of 8-oxo-7,8-dihydroguanine (8-oxoGua). In parallel, the influence of maternal supplementation with an antioxidant-enriched diet was investigated in their offspring. Supplementation resulted in reduced iron concentrations in the colon (P = 0.004) at day 7 and a 40% reduction of 8-oxodG in colon DNA (P = 0.044) at day 14 after birth. NER capacity in animals that did not receive antioxidants was significantly reduced to 32% at day 7 compared with the initial NER capacity on day 1 after birth. This reduction in NER capacity was less pronounced in antioxidant-supplemented piglets (69%). Overall, these data indicate that NER can be reduced by oxidative stress in vivo, which can be compensated for by antioxidant supplementation.     

Visualization and comparison of molecular dynamics simulations of leukotriene C4, leukotriene D4, and leukotriene E4

Molecular dynamics simulations of leukotriene C₄ (LTC₄), leukotriene D₄ (LTD₄), and leukotriene E₄ (LTE₄) were carried out, and the data were visualized in an animated video format. Three-dimensional ghost images show the positions of the heavy atoms of all three molecules throughout the simulations. The ghost images can be superimposed to give a single three-dimensional image in which the shapes of the most populated conformers of each molecule are apparent and can be compared. Leukotriene D₄ was found to occupy mostly T-shaped conformations, while LTC₄ occupied mostly cup-shaped conformations, and LTE₄ occupied a wide range of conformations spanning the LTD₄ and LTC₄ types. Digital filtering and graphing of the internal geometries of the molecules as a function of time revealed differences in dynamic behavior. The results are discussed in light of current knowledge about leukotriene receptors.     

The excretion of leukotriene E4 into urine following inhalation of leukotriene D4 by human individuals

Healthy volunteers underwent bronchial challenge with increasing doses of nebulized leukotriene D4 (0.007 - 200 nmol) at 15 min intervals. Total amounts of 200 nmol (females) and 400 nmol (males) were inhaled, corresponding to approximately 100 nmol and 200 nmol deposited in the lung, respectively. Of the latter amounts 3 +/- 1% (mean +/- S.E.M., n = 5) was found to be excreted as leukotriene E4 into the urine within 12 h. No further excretion after this period was observed. Approximately 50% of the total urinary leukotriene E4 was excreted during the first 2 h. These results suggest that a possible formation of sulfidopeptide leukotrienes in the lung in vivo can be monitored by measuring leukotriene E4 excretion into the urine.     

Pharmacology of aerosol leukotriene C4- and D4-induced alteration of pulmonary mechanics in anesthetized cynomolgus monkeys

Aerosol administration of solutions of 900 micrograms/ml of leukotriene C4 (LT) or D4 to cynomolgus monkeys produced dose-dependent, equipotent increases in pulmonary resistance (Rp) and decreases in dynamic lung compliance (Cdyn). Time to peak response was, in part, related to dose and ranged from 4 to 20 min. Both LTC4 and LTD4 were less potent than histamine. Aerosol pretreatment with the cyclooxygenase inhibitor indomethacin had no significant effect on either LTC4 or LTD4 dose-response curves; however, at the highest doses of these agonists a notable, nonsignificant inhibition of effects on both Rp and Cdyn was seen. Intravenous dl-propranolol had no effect on responses to LTD4. Aerosol pretreatment with FPL 55712 significantly (P less than 0.05) inhibited airway responses to both LTC4 and D4. In contrast, an intravenous infusion of FPL 55712 failed to block the bronchospastic activity of LTD4. In conclusion, cynomolgus monkeys are responsive to aerosol administration of LTC4 and LTD4, and the pharmacology of their responses appears to resemble that of man.