The Effects Of Beta-adrenergic Agonists On Alveolar Fluid Clearance In Injured Rat Lungs

PrefaceThe alveolar epithelial barrier plays an essential role in regulating fluid and protein balance under both normal and abnormal conditions. In earlier studies, some injury may be damage the alveolar epithelial barrier and result in pulmonary edema. However, it is not very clear about the relationship between the morphological changes and the role in regulating fluid in injured lung.Experimental studies have demonstrated that Na⁺ enters the apical membranes of alveolar typeâ…¡cells through amiloride-sensitive ion channels and is actively transported across the basolateral membranes of these cells by the ouabain-sensitive Na⁺-K⁺-ATPase. The stimulation of alveolar fluid clearance (AFC) accelerated the resolution of pulmonary edema. Recently, it was reported thatβ₂-agonists increase AFC in physiologic and pathologic conditions. However, it is unknown whether selectiveβ₁-adrenergic agonist-andβ₃-adrenergic agonists increase AFC in rats with lung injury.Therefore, the first objective of this study was to study the morphological changes occurring during lung injury. The second objective was to determine whether selectiveβ₁-adrenergic agonist andβ₃-adrenergic agonists stimulate AFC in injured rat lungs.Materials & MethodsThe specific-pathogen-free male Wistar rats weighing 300-380g were supplied by animal center, Second Affiliated Hospital, China Medical University. Denopamine (Tanabe Pharmaceutical Co., Ltd., Tokyo); BRL-37344, CGP-12177, SR-59230A, atenolol, terbutaline, ICI-118551, amiloride, ouabain, colchicine, andβ₁umicolchicine (Sigma, St. Louis, MO, USA).Bovine serum albumine(Beijing superior chemical & instruments CO.LTD).General Protocol: Hyperoxic exposure: For the hyperoxia experiments, rats were exposed to normobaric hyperoxia for up to 72h in a specially designed chamber (80x60x50cm), which ,was continuously flooded with oxygen at 10L/min. When the oxygen concentration reached 90% and then the gas fl0w was maintained at 2-3L/min. The oxygen concentration were continuously monitored with an oxygen analyzer. Carbon dioxide was trapped by soda lime granules in the box. Animals were allowed free access to food and water. The procedure was the same for control rats, except that the chamber was ventilated with 21% oxygen.Hypoxic exposure: For the hypoxia experiments, rats were continuously flooded with nitrogen at 10L/min. When the oxygen concentration reached 10% and then the gas flow was maintained at 0.5-1.5L/min. The oxygen concentration were continuously monitored with an oxygen analyzer. Carbon dioxide was trapped by soda lime granules in the box. Animals were allowed free access to food and water.Measurement of AFC: Rats were anesthetized by intraperitoneal administration of chloral hydrate (0.03ml/10g). An endotracheal tube was inserted through a tracheostomy. The rats were exsanguinated through the abdominal aorta. The trachea, lungs, and heart were excised. They were placed in a humidified incubator at 37℃. The lungs were ventilated with 100% oxygen (100% nitrogen in hypoxic rats). Physiological saline solution (5ml/kg) containing 5% albumin and Evans blue dye (0.15mg/ml) was instilled into the alveolar spaces through the endotracheal tube. After instillation, the lungs were inflated with 100% oxygen (100% nitrogen in hypoxic rats) at an airway pressure of 7cmH₂O. Alveolar fluid was aspirated 1h after instillation. The concentration of Evans blue-labeled albumin in the instilled and aspirated solutions were measured by a spectrophotometer at a wavelength of 621 nm. AFC was calculated as follows: AFC=[(Vi- Vf)/Vi] x 100, Vf= Vi xPi/Pf. where Vi is the volume of instilled albumin solution, and Vf is the volume of final alveolar fluid. Pi is the concentration of Evans blue in the instilled albumin solution and Pf is the concentration in the final alveolar fluid.Measurement of TLW : The TLW of the right lung was measured by drying the lungs to a constant weight at 60℃for 72h. The TLW was measured as Noble method: TLW= (wet lung weight-dry lung weight)/ dry lung weight.Specific protocols: Rats were randomly allocated with ten animals each.Tissue samples for electron microscopy and light microscopy were taken from left lungs of rats.Effects ofβ-adrenergic agonists on AFC: Isomolar albumin solution in the presence of different drugs were instilled into the alveolar spaces in rats.Statistical analysis: The data were summarized as mean and standard deviation. The data were analyzed by one-way analysis of variance (ANOVA) with the Student-Newman-Keuls post hoc test when multiple comparisons were needed. When comparisons were made between two experimental groups, an unpaired Students t-test was used. Differences with a P-value＜0.05 were regarded as significant.ResultsMorphometric studies. At hyperoxic 48h, the Typeâ…¡epithelial cells were observed to have no marked structural change; The capillary endothelial cell membrane became thickened, the mitochondria showed vacuole-like changes and the cytoplasm was edematous. At hypoxic 48h, Other than microvillus inversion lying-down and irregular arrays, the alveolar Typeâ…¡epithelial cells had no marked structural change.Effects of hypoxia on AFC and TLW: The TLW significantly increased in rats exposed to hypoxia up to 48h or hypoxia up to 24h.However, AFC did not change at the same time.Effects ofβ₁-adrenergic agonist on AFC: 10^-5mol/L denopamine significantly increased AFC in control rats and rats exposed to hypoxia for 24h, 48h and 72h. Atenolol inhibited the increase in AFC by denopamine. However, ICI-118551 did not inhibit the increase in AFC by denopamine. Amiloride inhibited AFC by 35% and ouabain ,inhibited AFC by 53%. Colchicine inhibited the stimulatory effect of denopamine on AFC in rats exposed to hypoxia for 48h, whereas the isomer-β-1umicolchicine did not affect the effect of denopamine on AFC.Effects ofβ₃-adrenergic agonists on AFC: After the rats were exposed to 90% oxygen concentration at 24h and 48h, the level of AFC showed no marked difference with that of normal rats. The increase in AFC by BRL-37344 was inhibited by ICI-118551 and SR-59230A. The increase in AFC by CGP-12177 was only inhibited by SR-59230A. Amiloride inhibited AFC stimulated by CGP-12177 by 33% and ouabain inhibited AFC by 51%, and amiloride inhibited AFC stimulated by BRL-37344 by 43% and ouabain inhibited AFC by 46%.DiscussionHypoxia is a common feature of many physiologic and pathologic processes. Hypoxia may activate some lung cells to release a wide variety of mediators potentially capable of impairing alveolar cells. Oxygen is needed in many hypoxic diseases, and lung is often injured by hyperoxia. Hypoxic and hyperoxic lung injury depends on exposure time as well as oxygen concentration. In the present experiment, the rats were exposed to 10% and 90% oxygen concentration. At 24h in hypoxic rats and 24h,48h in hyperoxic rats, TLW increased, but AFC did not change significantly. It illustrates that AFC is not synchronous with TLW.Meanwhile, the capillary endothelial cell showed structural changes while the alveolar epithelial cell had no marked change. It illustrates that, as compared with the endothelial cell, the alveolar epithelial cell has a stronger anti-injury capacity. The results of the present experiment demonstrate that, at hypoxic 48h, as compared with the normal control group, AFC decreased markedly. But the alveolar epithelial cell had no marked structural change. It illustrates that the normal structural; morphology and the integrity of the tight junction of alveolar epithelium constitute the material foundations of maintaining the normal alveolar clearance capacity. But the decrease of AFC will not necessarily bring about marked changes to the morphological structures of epithelial cell.The main regulating function of alveolar epithelial fluid transport is catecholamine-dePendent regulatory mechanism. Denopamine acts through stimulatingβ₁ adrenergic receptor. And the effect comparison of 10^-5mol/L terbutaline and 10^-5mol/L denopamine revealed no significant difference. This result proves that the distribution ratio ofβ₁ receptor andβ₂ receptor in typeâ…¡alveolar epithelial cell is basically comparable. The results of the present study also demonstrate that denopamine could increase markedly AFC in control rats and hypoxic rats. Denopamine could elevate the level of AFC back to normal in 48h. And in 72h hypoxic rats, the level of AFC remained markedly lower than normal despite under the action of denopamine. It can be proved that denopamine has a certain limitation of regulating the post-injury alveolar epithelial fluid transport.Na⁺-K⁺-ATPase exists in intracellular pools and in response to specific signals can rapidly recruited via cell microtubular transport into the plasma membrane. Therefore we studied whether inhibition of cell microtubular transport of Na⁺-K⁺-ATPase from intracellular pools to the plasma membrane by colchicines could inhibit the stimulatory effects of denopamine on active Na⁺ transport. The results suggest that denopamine stimulation of AFC is probably mediated by recruitment of ion-transporting proteins from inner pools to the plasma membrane in the alveolar epithelium.It was demonstrated that the rate of short-term AFC was not adversely affected by the absence of perfusion or ventilation to the lung .AFC is maintained for 1h in isolated rat lung,and also over 4h in ex vivo human lung,so we studied AFC under the ex vivo condition. For lack of participation of pulmonary circulation, the ex vivo model has some advantage. Since there is no blood perfusion and a reduced inflow of a large quantity of protein fluid during the lung injury, this model should be better for short-term studing of AFC. Both BRL-37344 and CGP-12177 are all beta-adrenergic agonists. However, the effects of stimulation AFC are not equal. BRL-37344 acts through stimulatingβ₂ adrenergic receptor andβ₃ adrenergic receptor, whereas CGP-12177 acts through stimulatingβ₃ adrenergic receptor mainly. Both amiloride and ouabain can inhibit the stimulated effect of BRL-37344 and CGP-12177.It illuminated that BRL-37344 and CGP-12177 stimulating AFC is probably mediated by amiloride- sensitive ion channels and Na⁺-K⁺-ATPase in the alveolar epithelium.Beta-adrenergic agonists regulate the AFC capacity in injury lung mainly through promoting the Na⁺ transport. These drugs can accelerate the resolution of pulmonary edema and thus provide more options for treating pulmonary edema in clinical practice.