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C N Gillis

Publications and source records attributed to C N Gillis.

At least 37 records · Page 2Linked to original sources
PubMed1989

In situ iodination of angiotensin-converting enzyme and other pulmonary endothelial membrane proteins.

Biochemical responses of endothelial cells in culture to pharmacological or physiological stimuli are often extrapolated to define the behavior of the vascular endothelium in vivo. However, culture conditions cannot recreate the environment of endothelial cells in vivo. To compare cell functions in vivo and in vitro, we iodinated endothelial membrane proteins of both the perfused rabbit lung and cultured rabbit lung endothelial cells. Endothelial cell protein 125I-labeling in the perfused intact lung was catalyzed by lactoperoxidase and glucose oxidase immobilized on 3-10 microns polyacrylamide beads (Enzymobeads, Bio-Rad). Changes in 5-hydroxytryptamine uptake, angiotensin converting enzyme activity and perfusion pressure made before, during and/or after iodination were small, suggesting that the procedure does not grossly injury the lung. As confirmed by tissue autoradiography, iodination was confined to the vascular space. A subcellular "membrane" fraction of the whole homogenate was enriched for several iodinated proteins. Lectin binding further purified a library of putative iodinated endothelial membranes proteins, one of which was angiotensin-converting enzyme as shown by immunoprecipitation with goat anti-rabbit antibody to angiotensin-converting enzyme. Iodinated proteins of similar molecular weights were also isolated from cultured rabbit lung endothelium iodinated under the same conditions, thus confirming the endothelial lineage of proteins iodinated in the intact lung. We conclude that this technique labels endothelial surface proteins in the intact lung without causing observable tissue injury and thus should be valuable in the study of the physiology and pathophysiology of the vascular lining in vivo.

Animals↗
PubMed1989

Resolution of impaired pulmonary function and pulmonary hypertension after phorbol ester administration in rabbits.

To assess metabolic functions of the pulmonary circulation during lung injury and subsequent recovery from injury, we measured angiotensin-converting enzyme (ACE) activity by means of benzoyl-phenylalanyl-alanyl-proline (BPAP) hydrolysis and 5-hydroxytryptamine (5-HT) removal in vivo in three groups of anesthetized rabbits. One group was treated with 30 micrograms/kg/day phorbol myristate acetate (PMA) intravenously 10 times over 14 days (PMA group). A second group received the same PMA treatment but was not studied until 14 days after the last treatment (PMA/recovery group). A third group was treated with vehicle alone. At the end of PMA treatment, rabbits had an elevated pulmonary artery pressure and depressed ACE activity, expressed as the ratio Vmax/Km. Decreased Vmax/Km for ACE was due to a significant reduction in apparent Vmax for BPAP (control = 235 +/- 37, PMA = 139 +/- 12 nmol/s). Km was unchanged (control = 25 +/- 4, PMA = 31 +/- 7 microM). Uptake of 5-HT was unaffected by PMA treatment. After 2 wk of recovery (PMA/recovery group), pulmonary hypertension had resolved. In this group, Vmax for BPAP hydrolysis was not significantly different from control (280 +/- 18 nmol/s), but Km was significantly increased (48 +/- 5 microM). We conclude that repeated exposure of rabbits to PMA results in lung injury manifested as depressed pulmonary ACE activity and pulmonary artery hypertension. Although much of these alterations were reversible within 2 wk after discontinuing PMA, an increase in apparent Km of ACE may be a more persistent alteration in vascular endothelial cell dysfunction.

Animals↗
PubMed1989

Roles of platelet-activating factor and thromboxane in group B Streptococcus-induced pulmonary hypertension in piglets.

Platelet-activating factor causes pulmonary hypertension, shock, hypoxemia, neutropenia, and increased pulmonary vascular permeability; some of its effects are due to thromboxane A2 release. Evidence for a possible role of these mediators in the genesis of group B Streptococcus (GBS)-induced pulmonary hypertension was sought using specific receptor antagonists for PAF and thromboxane A2 (TxA2) in anesthetized, ventilated piglets (less than or equal to 12 d of age; n = 22). Infusion of 1 X 10(8) GBS/kg/min for one hour resulted in a sustained and significant increase in pulmonary artery pressure (PPA) from 17 +/- 1 to 35 +/- 3 torr. Pretreatment with the TxA2 antagonist SQ 29548 (0.75 mg/kg intravenous), completely inhibited the effect of GBS on PPA. Pretreatment with either platelet-activating factor antagonists SRI 63072 (3 mg/kg intravenous) or SRI 63441 (1 mg/kg) did not affect the pulmonary hypertension due to GBS infusion. GBS-induced pulmonary hypertension could be reversed by SQ 29548; SRI 63072 did not affect PPA when administered to pigs with GBS-induced elevation in PPA. Inasmuch as prevention and reversal of GBS-induced pulmonary hypertension are accomplished with the TxA2 antagonist but not with PAF antagonists, these data suggest that TxA2, rather than PAF, is responsible for the early pulmonary hypertension in this model of neonatal GBS sepsis. Therefore, TxA2 antagonists may be clinically useful in treating pulmonary hypertension related to GBS sepsis.

Animals↗
PubMed1988

Pulmonary angiotensin-converting enzyme kinetics after acute lung injury in the rabbit.

Depression of lung endothelial cell metabolic function may be an early and sensitive indicator of lung damage. When such functions are measured in vivo, substrates injected usually must be limited to "trace" doses due to the significant hemodynamic effects of high doses of substrate. Under first-order conditions (i.e., trace doses) the enzyme or transport system rate constant Vmax/Km may be calculated, but independent estimates of each variable (Vmax and Km) are not available. We therefore used multiple indicator-dilution methods and higher substrate concentrations to apply a mathematical model, based on saturable kinetics that yield independent estimates of the apparent kinetic parameters Vmax and Km for pulmonary angiotensin-converting enzyme (ACE). We used the ACE substrate, [3H]benzoyl-phenylalanyl-alanyl-proline ([3H]BPAP) and made these measurements and also estimates of serotonin [5-hydroxytryptamine (5-HT)] removal, before and after acute lung injury induced by intratracheal administration of phorbol myristate acetate (PMA). PMA significantly depressed the percent 5-HT removal (62 +/- 3 to 44 +/- 4%) and BPAP percent metabolism (74 +/- 2 to 66 +/- 2), when trace amounts of either compound were injected as a bolus.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Propranolol and serotonin removal in lung injury.

Indicator dilution technique was used to study effects of reduced vascular volume or acute injury on removal of low doses of [3H]propranolol and [14C]serotonin (5-hydroxytryptamine, 5-HT) by perfused rabbit lung. Glass-bead (500 micron) embolization doubled pulmonary arterial pressure (Ppa) at flow rates of 20, 50, and 100 ml/min, decreased volume of distribution by approximately 50%, and increased pulmonary vascular resistance by at least 60%. Before embolization, (flow rate 20 ml/min) removal of [3H]propranolol and [14C] 5-HT was 89 +/- 2 and 75 +/- 5%, respectively, and was unaltered by changes in flow rate. However, after embolization, [3H]propranolol and [14C]5-HT removal decreased in a flow-dependent manner, reaching 28 +/- 4 and 1 +/- 3% (P less than 0.05), respectively, at a flow rate of 100 ml/min. When phorbol myristate acetate (PMA, 200 nM) was perfused (50 ml/min) through the lungs for 15 min, Ppa increased from 13 +/- 1 to 25 +/- 2 cmH2O (P less than 0.05), whereas [3H]propranolol removal decreased from 92 +/- 1 to 75 +/- 5% (P less than 0.05) and [14C]5-HT removal decreased from 73 +/- 3 to 46 +/- 8% (P less than 0.05). The PMA also caused vasoconstriction, which could be partially blocked by adding papaverine (500 microM) to the perfusion medium. Under the latter conditions, Ppa increased to 19 +/- 1 cmH2O and [3H]propranolol removal was unaffected. However, the combination of PMA and papaverine reduced [14C]5-HT removal from 64 +/- 4 to 19 +/- 3%.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Antagonism of acetylcholine-mediated relaxation of rabbit pulmonary arteries by phorbol myristate acetate.

We studied the effect of 4-phorbol 12-myristate 13-acetate (PMA) on endothelium-dependent relaxation of rabbit isolated lobar pulmonary arteries contracted with phenylephrine. After full development of relaxation in response to 1.0 microM acetylcholine, addition of 10.0 nM PMA reversed relaxation rapidly and completely. This effect of PMA on acetylcholine-induced relaxation was unchanged when catalase was included in the medium. In separate groups of experiments, lobar pulmonary arteries were preincubated with 10.0 nM PMA for 20 min and then challenged with acetylcholine without washout of PMA. In these experiments PMA did not block completely the relaxation in response to addition of 1.0 microM acetylcholine. Log-concentration response curves for 0.01 to 31.6 microM acetylcholine vs. relaxation as percentage of phenylephrine-induced tone exhibited rightward shifts and depression of maxima after pretreatment with PMA. The EC50 for acetylcholine-induced relaxation was increased from 57 +/- 9.0 to 377 +/- 6.30 nM (values are mean +/- S.E.). Relaxation of lobar pulmonary arteries induced by 10.0 nM A23187 was much less sensitive to treatment with PMA than relaxation induced by acetylcholine. At least for the low concentration of PMA used here, these data are consistent with the following: 1) PMA blocks acetylcholine-induced relaxation by disrupting the link between acetylcholine receptor occupancy and the formation or release of the endothelium-derived relaxing factor, 2) PMA does not directly block the relaxant action of endothelium-derived relaxing factor on vascular smooth muscle and 3) the effect of PMA on acetylcholine-induced relaxation is not mediated by hydrogen peroxide or derived oxygen radicals.

Acetylcholine↗
PubMed1987

Measurement of endothelial metabolic functions in vivo.

Lung metabolic functions include the interaction of microvascular endothelium with blood-borne substances such as physiologically important amine, eicosanoid, and peptide hormones and drugs. This activity is mediated by endothelial transport systems and enzymes which either synthesize or degrade these substances. Because they can alter the hormone content of aortic blood, these functions play a role in homeostasis, and their disturbance has been implicated in the pathogenesis of several forms of lung injury and disease. Both steady-state infusion and single injection, multiple indicator dilution techniques have been applied to measure endothelial metabolic functions in the intact lung. Considerable progress has been made in development of mathematical models for the processes, and their application has been tested both under normal conditions and also when the lung is perturbed experimentally. Unique experimental challenges are presented by measurement of metabolic functions in vivo, when steady-state infusion techniques cannot be used because systemic toxicity could result. In this case, the bolus injection approach has been used, with some success, both in laboratory animals and man. Although major challenges remain, their solution is essential if we are to apply knowledge of endothelial cell function in vitro to understanding lung microvascular physiology and pathophysiology in the intact animal.

Animals↗
PubMed1987

Effect of nafazatrom and indomethacin on pulmonary removal of prostaglandin E1 after endotoxin in rabbits.

1 We compared the effects of endotoxin on pulmonary prostaglandin E1 (PGE1) removal in groups of rabbits pretreated with the cyclo-oxygenase inhibitor, indomethacin, or nafazatrom (Bay g 6575), which has been shown to increase plasma prostacyclin concentrations. 2 In untreated animals, endotoxin transiently decreased pulmonary removal of [3H]-PGE1, caused pulmonary hypertension, systemic hypotension and increased plasma concentrations of PGE2 and 6-keto-PGF1 alpha. 3 Indomethacin pretreatment prevented the transient decrease in pulmonary removal of [3H]-PGE1 in response to endotoxin, prevented the haemodynamic effects and inhibited prostaglandin synthesis. Pretreatment with nafazatrom did not affect the decreased pulmonary removal of [3H]-PGE1, exacerbated the haemodynamic response, reduced survival and potentiated the increase in circulating 6-keto-PGF1 alpha. 4 We conclude that indomethacin acts to prevent the depression of pulmonary [3H]-PGE1 removal by eliminating surface area changes associated with endotoxin-induced pulmonary vasoconstriction. 5 These data suggest that nafazatrom treatment results in exacerbation of the endotoxin-induced systemic hypotension presumably due to its effect on increased plasma prostacyclin during the later phase of endotoxaemia.

Alprostadil↗
PubMed1987

Effect of flow and surface area on angiotensin-converting enzyme activity in rabbit lungs.

Pulmonary angtiotensin-converting enzyme (ACE) is located on the luminal surface of pulmonary microvasculature. Multiple indicator-dilution techniques have been used to measure pulmonary ACE activity in vivo and in isolated lungs. These studies suggest that ACE activity is depressed in several forms of acute lung injury. Depression of ACE activity may reflect impaired substrate delivery to enzyme sites because of flow-related reduction of perfused surface area. To assess the role of altered microvascular flow and surface area in the measurement of ACE activity, we utilized similar techniques to estimate the apparent Km and Vmax of pulmonary ACE in isolated, Krebs-perfused rabbit lungs. Km is an estimate of the affinity of a synthetic ACE substrate, [3H]benzoyl-phenyl-alanyl-alanyl-proline ([3H]BPAP), for ACE and should not be influenced by the rate of substrate delivery to luminal enzyme sites. Conversely, Vmax is an index of the number of ACE sites and should be influenced by perfusion changes that alter the number of perfused sites (recruitment or derecruitment). When isolated lungs were subjected to physiological maneuvers designed to increase or decrease perfused surface area, apparent Vmax increased or decreased respectively. Apparent Km was not altered by these maneuvers. Km and Vmax were independent of changes in perfusion rate when surface area was held constant. Thus these parameters should be useful in evaluating perfusion changes in normal and injured lungs.

Animals↗
PubMed1987

In-line measurement of pulmonary metabolic function in the anesthetized rabbit.

Quantitative assessment of lung metabolic function is thought to provide biochemical information reflecting integrity of the pulmonary microcirculation. Although multiple indicator-dilution techniques are useful in such pharmacokinetic studies, the need for fractionation and subsequent processing of blood samples greatly prolongs data generation. Accordingly, we designed and tested an in-line system which rapidly can quantify single-pass disposition of photon-emitting substances in the pulmonary circulation of intact animals. The nuclear detection system consisted of a phoswich scintillation probe optically coupled to a photomultiplier tube. Pulses were discriminated for height and shape and counts recorded in a counter-timer, the output of which was interfaced with a personal computer. A mixture of an intravascular reference substance (99mTc-sulfur colloid) and an inhibitor of angiotensin-converting enzyme, N-[1(S)-carboxy-(4-OH-3-[125I]-phenyl) ethyl]-L-alanyl-L-proline (125I-CPAP), was injected as a bolus in the right heart of anesthetized ventilated rabbits and arterial blood was diverted through a flow-cell cuvette directly apposed to the phoswich detector. Single-pass extraction of 125I-CPAP was 39 +/- 3% (mean +/- SE; n = 20) and was depressed in a dose-dependent fashion by the addition of unlabeled CPAP (1-10 micrograms/kg) to the injection. These data indicate that we can now quantify, in the intact animal, saturable binding of an inhibitor to angiotensin-converting enzyme expressed on the surface of the pulmonary microvascular endothelium. Furthermore, such data can be obtained rapidly.

Animals↗
PubMed1987

Rapid increases in respiratory epithelial permeability occur after intratracheal instillation of PMA.

We measured pulmonary epithelial permeability by quantifying the disappearance of two water-soluble compounds, [14C]mannitol and [3H]inulin, after their instillation, with and without phorbol myristate acetate (PMA), into gas-filled perfused (50 ml/min) rabbit lungs in situ. Both tracers disappeared in a monoexponential fashion over 30 min with calculated first-order rate constants (control; n = 11) of 0.0008 +/- 0.0002 and 0.0027 +/- 0.0008 min-1 for inulin and mannitol, respectively. The ratio of the rate constants (3.1 +/- 0.5) was not significantly different from the ratio of diffusivities of mannitol:inulin (3.7). Addition of PMA (250 micrograms) significantly (n = 9, P less than 0.05) increased the rate constants for both inulin and mannitol to 0.0024 +/- 0.0007 and 0.0087 +/- 0.0025 min-1, respectively, while not affecting their ratio (4.3 +/- 0.5). Addition of human leukocytes (4-8 X 10(8)/l) to the perfusate did not exacerbate the effect of 250 micrograms PMA (n = 3). The addition of catalase (n = 7) completely inhibited the effect of 250 micrograms PMA. PMA (250 micrograms) did not significantly affect perfusion pressure but increased wet-to-dry weight ratios. Light microscopic histology showed damage to epithelial and endothelial cells after 250 micrograms PMA which was not seen after coinstillation of catalase. Catalase sensitivity of functional and structural effects of PMA suggests that the effect was secondary to production of hydrogen peroxide. Since this effect was noted in lungs not perfused with neutrophils and addition of leukocytes did not exacerbate the increase in permeability, we hypothesize that an undetermined pulmonary cell type was the source of hydrogen peroxide. Finally, we found no evidence for restrictive pores with radii of 0.4-1.4 nm.

Animals↗
PubMed1987

Removal and release of recirculating atrial natriuretic peptide (ANP) by rabbit lungs perfused in situ.

The finding that radioiodinated, synthetic ANP (125I-ANP), analogous to the ANP secreted by the right atrium, binds to specific sites of endothelial cells, smooth muscle, adrenals and lungs (1-3) raised the importance of analyzing lung-ANP interactions. Rabbit lungs were accordingly loaded with 125I-ANP, 2000 Ci/mM (2.5 pM) in Krebs-albumin recirculating perfusion medium at a rate of 20 ml/min, over 27 min. 125I-ANP binding was estimated by taking samples at 2-min intervals from the perfusate. Fourteen min after recirculation, uptake was maximal (66.4 +/- 5.2%, N = 8) (X +/- SEM) and not different from that observed after a single passage through the lungs and was dose-dependent, antagonized by unlabelled ANP. The system was then changed to single-pass washout and efflux curves were obtained, collecting the entire efflux every 6 s per sample, over 15 min. Efflux curves after 125I-ANP loading fitted a bi-exponential decay model (Y(to) = Y(t)e-b11t + Y(t)e-b2t). Lung ANP uptake/release may be involved in regulating systemic concentrations and hence renal and other actions of ANP.

Animals↗
PubMed1987

Evidence for the role of endothelium-derived relaxing factor in acetylcholine-induced vasodilatation in the intact lung.

The vascular effects of acetylcholine were evaluated in blood-free lungs of rabbits perfused in situ at a constant flow rate of 15 ml/min with Kreb's-bicarbonate solution containing 3% dextran. Infusion of 1 microM acetylcholine normally elicited vasoconstriction. However, acetylcholine (3.0 nM-1.0 microM) produced concentration-dependent vasodilatation when administered to lungs in which pulmonary artery pressure (i.e., perfusion pressure) was elevated and cyclooxygenase activity was blocked, respectively, by infusing the endoperoxide analog U46619 (2.0-10.0 nM) and 30.0 microM indomethacin. Three additional groups of lungs treated with indomethacin and preconstricted with U46619 were challenged twice with 1.0 microM acetylcholine. Two of the groups were equilibrated with an infusion of either 20.0 microM quinacrine or 10.0 microM ferrous hemoglobin before and during their second challenges with acetylcholine and a control group received no treatment between trials. Neither quinacrine nor hemoglobin altered base-line pulmonary artery pressure, but both agents reversed the effect of acetylcholine from vasodilatation to vasoconstriction. This study agrees with previous reports of a pulmonary vasoconstrictor action of acetylcholine dependent on an intact cyclooxygenase pathway, but also provides new evidence for a vasodilator action of acetylcholine in the intact lungs of rabbits. Quinacrine and hemoglobin are known to antagonize the endothelium-dependent relaxation of vascular smooth muscle elicited by acetylcholine; therefore, this study provides indirect evidence supporting a role for the endothelium-derived relaxing factor in the regulation of pulmonary vascular tone.

Acetylcholine↗
PubMed1986

Removal of atrial natriuretic peptide by perfused rabbit lungs in situ.

Removal of iodinated 28-amino acid atrial natriuretic peptide ([125I]ANP) by rabbit lungs was measured by indicator-dilution methods. After bolus injection of 6.5 pmoles of [125I]ANP, 66.9 +/- 2.9% was removed in a single pass through the lungs. Removal was unaltered by a kininase II inhibitor but was reversibly decreased by unlabelled ANP. Thus the lungs can remove ANP from the pulmonary circulation by a mechanism that does not involve hydrolysis by kininase II. Lungs therefore may be involved in regulating systemic concentrations and hence renal and other actions of ANP.

Animals↗
PubMed1986

Depressed prostaglandin E1 and 5-hydroxytryptamine removal in patients with adult respiratory distress syndrome.

Because experimental lung injury is associated with decreased removal of 3H-prostaglandin E1 (3H-PGE1) and 14C-5-hydroxytryptamine (14C-5-HT), we questioned whether a similar reduction would be evident in patients with the adult respiratory distress syndrome (ARDS). Accordingly, we measured, by indicator dilution techniques, pulmonary removal of 3H-PGE1 and 14C-5-HT in 11 patients undergoing cardiopulmonary bypass surgery in whom respiratory function was essentially normal, and compared them with similar measurements in 9 patients who had ARDS. In addition, we made 5 successive measurements of lung removal functions in the bypass group of patients during the 48-h period after the first measurement. These measurements were made before and 4 times (within 48 h) after the first measurement. Before bypass, removal of 3H-PGE1 and 14C-5-HT was 78 +/- 2 SEM and 89 +/- 2%, respectively; these did not change during the subsequent 48 h. Therefore, we compared prebypass values in this group with measurements made in patients with ARDS. The latter group had significantly decreased removal of 3H-PGE1 and 14C-5-HT (values were 66 +/- 3 and 72 +/- 5%, respectively). We suggest that these changes reflect a diffuse functional injury to the endothelium similar to that seen after acute lung injury in laboratory animals.

Adult↗
PubMed1986

Uptake of N-[1 (S)-carboxy-(4-OH-3-125I-phenyl)ethyl]-L-Ala-L-Pro, an inhibitor of angiotensin-converting enzyme by rabbit lungs in situ.

Single pass extraction of a new iodinated inhibitor of angiotensin-converting enzyme (ACE) was measured by means of indicator-dilution techniques applied to rabbit lungs, perfused in situ at 20 ml/min with Krebs bicarbonate solution containing 3% bovine serum albumin. A bolus containing the inhibitor, N-[1(S)-carboxy-(4-OH-3-125I-phenyl)ethyl]-L-Ala-L-Pro (CPAP), and an intravascular marker, [14C]dextran, was injected and extraction calculated at the peak of the resulting venous outflow-time curve. In 13 of 21 lungs used, a synthetic substrate for ACE, [3H]benzoyl-phenylalanyl-alanyl-proline (BPAP), was added to the bolus and appearance of its hydrolysis product, [3H]benzoyl-Phe, measured in effluent samples. When low amounts (0.15 nmol) of [125I]CPAP were injected, pulmonary extraction (E) of CPAP was 67 +/- 14% (X +/- S.D; n = 21) and metabolism (M) of BPAP was 56 +/- 9% (n = 13). Addition of unlabeled CPAP (3, 34 or 340 nmol) to the Addition of unlabeled CPAP (3, 34 or 340 nmol) to the injected bolus caused dose-dependent reduction of E(CPAP) and M(BPAP) that was no longer evident 10 min after the largest dose of CPAP. Coadministration of the ACE inhibitor, captopril (3, 6, 8 and 28 nmol), also caused dose-dependent, reversible depression of both E(CPAP) and M(BPAP). Accordingly, extraction of CPAP by perfused rabbit lung is saturable. Inasmuch as CPAP inhibits ACE activity (as reflected by BPAP metabolism) and CPAP uptake is inhibited by captopril (which also inhibits BPAP hydrolysis), it appears that a large portion of this saturable process probably reflects binding to vascular ACE.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin-Converting Enzyme Inhibitors↗