Inhibition of angiotensin converting enzyme activity in cultured endothelial cells by hypoxia.

Endothelial cells in tissue culture degrade bradykinin and convert angiotensin I to angiotensin II. These are both functions of a single dipeptidyl hydrolase, angiotensin converting enzyme. Monolayer cultures were prepared from human, rabbit, pig, and calf vessels. Angiotensin converting enzyme activity was assessed by adding either bradykinin or angiotensin I to the cells in culture flasks, and measuring residual peptide over time by radioimmunoassay. Peptide degradation was inhibited by the specific converting enzyme inhibitor, SQ 20881. The flasks were equilibrated with varying hypoxic gas mixtures: hypoxia rapidly (less than 2 min) decreased enzyme activity and room air restored it as rapidly. The extent to which activity was reduced was a direct function of PO2 (r = 0.93, P less than 0.001), and there was no enzyme activity below a PO2 of 30 mm Hg. Four preparations were studied with respect to decrease in enzyme activity by hypoxia: (a) intact cells in monolayer, (b) sonicated cells, (c) sonicated cells from which converting enzyme was partially dissolved by a detergent, and (d) purified converting enzyme. Hypoxia had progressively less of an inhibiting effect on the enzyme activity of the preparations as the degree of cell integrity decreased. Hypoxia inhibits angiotensin converting enzyme activity in cultured endothelial cells, but the effect of hypoxia is not on the enzyme per se, but appears to be a unique characteristic of the endothelial cell.

Bradykinin production and increased pulmonary endothelial permeability during acute respiratory failure in unanesthetized sheep.

To investigate mechanisms of pulmonary edema in respiratory failure, we studied unanesthetized sheep with vascular catheters, pleural balloons, and chronic lung lymph fistulas. Animals breathed either a hypercapnic-enriched oxygen (n = 5) or a hypercapnic-hypoxic (n = 5) gas mixture for 2 h. Every 15 min blood gases, pressures, cardiac output, lymph flow (Qlym), plasma and lymph albumin (mol wt, 70,000), IgG (mol wt, 150,000), IgM (mol wt, 900,000), and blood bradykinin concentrations were determined. In both groups, cardiac output and pulmonary arterial pressures increased, whereas left atrial pressures were unchanged. Acidosis alone (arterial pH = 7.16, PaCO(2) = 81 mm Hg, PaO(2) = 250 mm Hg) resulted in a doubling of lymph flow, a small increase in protein flux, and a decrease in lymph to plasma protein concentration (L/P) ratio for all three proteins. Acidotic-hypoxic animals (arterial pH = 7.16, PaCO(2) = 84 mm Hg, PaO(2) = 48 mm Hg) tripled Qlym. In these animals the increase in lymphatic flux of albumin, IgG, and IgM was significantly (P < 0.05) greater than that seen in either the acidosis alone group or in animals where left atrial pressures were elevated (n = 5; P < 0.05). Also, their percent increase in flux of the large protein (IgM) was greater than for the small protein (albumin) (P < 0.05). With acidosis alone, only pulmonary arterial bradykinin concentration increased (1.27+/-0.25 ng/ml SE), whereas acidosis plus hypoxia elevated both pulmonary arterial bradykinin concentrations (4.83+/-1.14 ng/ml) and aortic bradykinin concentration (2.74+/-0.78 ng/ml). These studies demonstrate that hypercapnic acidosis stimulates in vivo production of bradykinin. With superimposed hypoxia, and therefore decreased bradykinin degradation, there is an associated sustained rise in Qlym with increased lung permeability to proteins.

Impaired angiotensin conversion and bradykinin clearance in experimental canine pulmonary emphysema.

Chronic hypoxic lung diseases are associated with abnormal blood pressure regulation. Because the lung is the principal site of angiotensin conversion and because hypoxia decreases converting enzyme activity, we examined whether angiotensin converting enzyme activity was impaired in lung disease. 12 dogs received a 6 wk course of aerosolized and intratracheal papain that produced moderate panlobular emphysema. These dogs and 24 control dogs were anesthetized and sampling catheters were placed under fluoroscopic control. Angiotensin conversion was measured by a blood pressure response bioassay. Pulmonary converting enzyme activity was also assessed by infusing bradykinin (BK) and using radioimmunoassay to measure the instantaneous clearance of BK and the concentration of BK in the pulmonary artery which first produced spillover of BK into left atrial blood. Angiotensin conversion was reduced in the emphysematous dogs to 81.1% (13.2 SD) from 92% (6 SD) in the control dogs (P < 0.01). Instantaneous clearance of BK in the emphysematous dogs was only slightly reduced (93%), despite reduction in their Pao(2) to 75 mm Hg, indicating that the greatest proportion of the perfused vascular bed was exposed to alveolar Po(2) of >90 mm Hg. However, the barrier to BK passage provided by the lung, and measured by the spillover level, was reduced (1/4) to (1/2) that observed in control animals. That the defect was promptly corrected by supplemental oxygen indicates that regional pulmonary vascular converting enzyme activity had been impaired by regional alveolar hypoxia, which permitted some peptide to pass through the lungs unmetabolized. Determination of peptide metabolism in the lungs may provide a useful measure of regional alveolar hypoxia and may lead to new ways of assessing lung injury.

Modulation of converting enzyme activity by hypoxia and its physiological effects.

To explore the haemodynamic consequences of the reduction in converting enzyme activity by acute alveolar hypoxia we made sequential haemodynamic observations in seven saline-infused and 12 bradykinin-infused anaesthetized, catheterized dogs. They were ventilated initially with room air and then for 50 minutes with hypoxic gas mixtures. Within two minutes after starting hypoxic ventilation, converting enzyme activity decreased, arterial angiotensin II concentrations dropped, and, in the bradykinin-infused dogs, arterial bradykinin concentrations rose. Both groups of dogs experienced a rise in systemic and pulmonary arterial blood pressure in response to hypoxia, but by different mechanisms. In the saline-infused (control) dogs there was increased systemic (+40%) and pulmonary (+90%) vascular resistance while cardiac output was unchanged or slightly reduced. Bradykinin-infused dogs demonstrated reduced systemic vascular resistance (-40%), no increase in pulmonary vascular resistance and a 100% increase in cardiac output. Return to room air breathing restored converting enzyme activity, releasing high concentrations of angiotensin II. Oxygen tension thus regulates converting enzyme activity and hence the circulating levels of angiotensin II and bradykinin.

Inhibition of converting enzyme activity by acute hypoxia in dogs.

We studied the effect of a change in oxygen tension on converting enzyme activity in anesthetized, paralyzed, catheterized dogs ventilated with room air, 100% O2, and hypoxic gas mixtures. Bradykinin was continuously infused into the femoral vein and simultaneous samples drawn from the pulmonary artery and left atrium; bradykinin was extracted into ethanol and measured by radioimmunoassay. Clearance of bradykinin by lung converting enzyme decreased from 96% at PaO2 levels above 95 Torr to 0% below 26 Torr. Inhibition of enzyme activity was rapid in onset (less than 2 min), closely correlated with PaO2 (r = 0.92, P less than 0.001), and reversible within 2 min after return to room air breathing. Converting enzyme activity of the systemic vascular bed was also inhibited by hypoxia; kininase I activity was unaffected by oxygen tension. Although arterial bradykinin concentrations in the range of 0.5 ng/ml produced hypotension in normoxic animals, elevations to 30 ng/ml had no hypotensive effect in hypoxic dogs. During acute hypoxia, venous bradykinin will pass through the lung unmetabolized, and local levels of angiotensin II and bradykinin will vary in vascular beds with different oxygen tensions, providing a finely-graded mechanism for blood flow regulation.

Increased circulating bradykinin during hypothermia and cardiopulmonary bypass in children.

To determine whether cold could activate the kallikrein-kinin system in vivo as it does in vitro, the circulating systemic concentrations of bradykinin were serially measured in 10 cyildren with congenital diseases of the heart undergoing corrective cardiac surgery. Bradykinin was measured by radioimmunoassay in blood samples obtained before, during and after profound hypothermia (to 18 degrees C) and cardiopulmonary bypass. The circulating concentrations of bradykinin increased significantly as body temperature decreased during surface cooling. The increase in circulating bradykinin was associated with a decrease in the circulating level of bradykininogen, the precursor of bradykinin. With the onset of cardiopulmonary bypass and hence, removal of the lung and pulmonary converting enzyme from the circulation, there was a further rise in the already elevated concentrations of bradykinin. This is the first in vivo demonstration that hypothermia leads to an increase in the circulating concentrations of bradykinin.

Gestational changes in pulmonary converting enzyme activity in the fetal rabbit.

Changes in angiotensin-converting enzyme were measured in the lungs of fetal rabbits isolated and perfused in situ at varying ages from 22 days gestation to 7 days of age under controlled conditions of flow, pH, and temperature. Enzyme activity was assessed by infusing bradykinin or angiotensin I in Krebs-Henseleit solution and measuring residual peptide in the effluent by radioimmunoassay. The levels of substrate studied were below those required for enzyme saturation. Lungs of 22 day gestation fetuses removed only one-third of either peptide. The activity at term and in neonatal life resulted in more than 80% peptide removal. The time of the greatest rise in the percent substrate cleared occurs earlier than the time of the greatest increase in lung and body weight. The lower percentage of substrate cleared in early gestation appears to result in part from a limited surface area for enzyme activity in the primitive fetal pulmonary microvascular bed, since morphological studies with fluorescein-tagged anticonverting enzyme antibody demonstrated the presence of enzyme in the lung as early as 17 days of gestation. Electron micrographs of the pulmonary endothelial cell surface reveal that the degree of surface infolding and hence surface area increases with gestation. The higher percentage of substrate cleared in later gestation closely parallels the structural and ultrastructural development of the vascular bed. The presence of converting enzyme in the placenta by the second third of gestation and the large size of the placenta suggest that this organ may be a major locus of converting enzyme activity in the fetus.