| Alveolar fluid clearance, an important function of the alveolar epithelium, plays an important role in recovery from pulmonary edema. The basic mechanisms that drive the removal of edema fluid from the distal airspaces of the lung have been established over the past two decades. There is a close relationship between the alveolar fluid clearance capacity and resolution of pulmonary edema. An impairment of alveolar fluid clearance may predispose a patient to a critical condition due to pulmonary edema. Experimental studies have shown that intact alveolar epithelial fluid transport function is critical for resolution of pulmonary edema and acute lung injury. In addition, the increase in alveolar fluid clearance can accelerate the resolution of pulmonary edema fluid. Therefore, the study to elucidate the mechanisms responsible for alveolar fluid clearance has clinical importance in the treatment of pulmonary edema. However, it is uncertain if an a - or a β3 - adrenergic agonist increases alveolar fluid clearance. In addition, there is no study in which all α- , β1- , β2 - and β3 - adrenergic agonists were used under the same experimental preparation. Therefore, the first objective of this study was to determine if α- , β1- ,β2 ~ and β3 ~ adrenergic agonists stimulated alveolar fluid clearance in the isolated rat lungs and to determine the mechanism responsible for the effects of adrenergic agonists. There is a close relationship between the alveolar fluid clearance capacity and resolution of pulmonary edema. The increase in alveolar fluid clearance can accelerate the resolution of pulmonary edema fluid. Alveolar edema is cleared by active trans-port of salt and water from the alveoli into the lung interstitium by complex cellular mechanisms. Lung epithelial ion transport promotes salt and water movement across the lung epithelium. The mechanism is dependent on basolateral membrane Na+ - K+ - ATPase and the apical membrane Cl" and Na+ channels. Numberous studies suggest that active sodium transport is the primary mechanism that drives alveolar fluid clearance. However, the contribution of Cl" movement across the alveolar epithelium and the effects of cystic fibrosis trans-membrance conductance regulator ( CFTR) on alveolar fluid clearance have not been fully understood. CFTR and the epithelium sodium channel are two membrane proteins expressed in the apical membrane of several epithelia. They have been shown to influence each other, in the event Na+ absorptive pathways are damaged, transepithelial Cl" secretion and the consequent intra - alveolar fluid influx may be upregulated. Experimental studies have shown that |3 -r adrenergic agonists can increase active sodium transport and Na + - K+ - ATPase via the stimulation of both {Jj - and (32 — adrengic receptors. But the relationship between CFTR and (3 - adrenergic receptors is not known. So the second objective of this study was to determine the role of Cl" and CFTR in active salt and water transport across the distal airspaces in the isolated rat lungs and the interaction of CFTR and j3t - adrenergic agonist.MethodsMaterialsDenopamine ( a (3j -adrenergic agonist) and NS004 (a CFTR opener) were obtained from Tanabe Pharmaceutical Co. , Ltd. (Tokyo, Japan). Pheny-lephrine ( an a - adrenergic agonist) , prazosin (an ^ - adrenergic antagonist) , yohimbine (an a2 -adrenergic antagonist) , terbutaline (a p2 -adrenergic agonist) , BRL - 37344 ( a (33 - adrenergic agonist) , atenolol ( a §x - adrenergic antagonist), ICI - 118551 (a p2 - adrenergic antagonist), SR -59230A (a p3 -adrenergic antagonist) and glibenclamide(a CFTR blocker) were obtained from Sigma (St Louis, MO, USA). CFTRinh - 172 was kindly given by Professor Matthay MA (University of California, San Francisco, USA)Experimental ProtocolAll rats received humane care and this study was approved by the Committee for Animal Experiments at Kanazawa Medical University. Alveolar fluid clearance was measured in the isolated rat lungs in the absence of pulmonary perfusion or ventilation. Briefly, male Sprague -Dawley rats (250 ~330 g, Japan SLC, Inc. , Hamamatsu, Japan) were anesthetized with intraperitioneal pentobarbital sodium (50 mg/kg). An endotracheal tube was inserted through a tracheostomy. The rats were exsanguinated via the abdominal aorta and the trachea, lungs and heart were taken out en bloc. Isotonic saline solution (2 ml, 37°C ) containing 5% bovine albumin was instilled into the both lungs, followed by 4 ml oxygen to deliver all the instilled solution into the alveolar spaces. The lungs were placed in a humidified incubator at 37 °C and inflated with 100% oxygen at an airway pressure of 7 cm H2 0. Alveolar fluid was aspirated after 1 hour of incubation.Effects of different pharmacological agents on alveolar fluid clearanceIsotonic 5% albumin solutions containing different pharmacological agents were instilled into the rat lungs. Alveolar fluid was aspirated after 1 hour of incubation.Alveolar fluid clearanceAlbumin concentration was measured with the pyrogallol red protein dye -binding method (SRL Inc. , Tokyo, Japan) , and the alveolar fluid clearance (AFC) was calculated by the change of albumin concentration. AFC = [ ( Vi Vf)/Vi] 100, Vf = Vi Pi/Pf. V means the volume of the instilled albumin solution (i) and the final alveolar fluid ( f). P means the concentration of protein in the instilled albumin solution (i) and the final alveolar fluid (f).StatisticsData are summarized as the mean and standard deviation. The data were analyzed by a one - way analysis of variance ( ANOVA) with the Student - Newman - Keuls port hoc test. Differences with a p value of <0. 05 were regarded as significant.ResultsPhenylephrine (an a - adrenergic agonist) increased alveolar fluid clearance signifantly (AFC was 17. 1% ±1. 3% and was 2. 09 times of the basal AFC). Prazosin (a selective a! -adrenergic antagonist) , Atenolol (a ^l -adrenergic antagonist) and ICI - 118551 (a (32 ~ adrenergic antagonist) significantly inhibited the increase in alveolar fluid clearance caused by phenylephrine (the AFC were decreased to 38. 7% , 44. 4% and 46. 5% , respectively) , whereas yohimbine ( an a2 - adrenergic antagonist) did not( P > 0.05 ).Denopamine (a fjj -adrenergic agonist) , terbutaline (a p2 - adrenergic agonist) , and BRL -37344 ( a j33 - adrenergic agonist) increased alveolar fluid clearance (AFC wrere 17.1% ±2.4% ,19. 5% ± 1. 2% and 19. 9% ±2. 5% , respectively) . Atenolol (a p! -adrenergic antagonist) abolished the effect of denopamine (AFC was 6. 1% ±0.9%), but did not inhibit the effects of terbutaline and BRL -37344. ICI -118551 abolished the effect of terbutaline and BRL - 37344 ( AFC were 5. 7% ± 0. 6% and 7. 8% ± 2. 6% , respectively) , and also inhibited the effects of denopamine in part ( AFC was 12. 7% ±1. 8% ). SR - 59230A ( a p3 - adrenergic antagonist) inhibited the effects of BRL -37344 and terbutaline in part (AFC were 13. 8% ±3.1% and 14. 5% ±3. 4% ,respectively) , but did not change the effect of denopamine.Denopamine increased alveolar fluid clearance. Atenolol, a specific Pi -adrenergic antagonist, abolished the effect of denopamine to stimulate alveolar fluid clearance. Glibenclamide and CFTR - inh 172 inhibited the effect of denopamine — stimulated alveolar fluid clearance in part. Denopamine increased alveolar fluid clearance significantly ( AFC was 17.7% ±3.7%, P <0.001). Atenolol ( a {Sj - adrenergic antagonist) abolished the effects of denopamine (AFC was 7. 8% ±2. 36% ). Glibenclamide and CFTRinh - 172, both CFTR inhibitors inhibited the effects of denopamine in part (AFC were 10. 7% ±0. 9% and 12. 9% ± 1. 1% , respectively. P < 0. 05). The opener of CFTR, NS004, increased alveolar fluid clearance significantly (AFC was 13. 8% ±1. 2% , P <0.05). The inhibitor of Na+ channel, amiloride and glibenclamide orCFTRinh - 172, both CFTR inhibitors abolished the effects of NS004 ( AFC were 7.5% ±1.2% , 7.9% ±2.13% and 7.6% ±1.0%, respectively. P <0. 05). Amiloride also abolished the effects of denopamine (AFC was 8.0% ±1. 9%, P<0.001).DiscussionPulmonary edema is a life - threatening condition resulting from an imbalance between forces driving fluid into the airspaces and biological mechanisms for its removal. The basic mechanisms that drive the removal of edema fluid from the distal airspaces of the lung have been established over the past two decades. Alveolar fluid clearance is one of the important effects of alveolar epithelium. Intact alveolar fluid clearance is correlated with less histological injury, rapid resolution of hypoxaemia, and a trend towards a shorter duration of mechanical ventilation and a shorter intensive care stay and so on.The major finding of this study is that ct - and (3 - adrenergic agonists are potent stimulators of alveolar fluid clearance in the isolated rat lungs.Phenylephrine (an a - adrenergic agonist) increased alveolar fluid clear-anc. The effect was inhibited by a, - , p, - and f$2 ~ adrenergic antagonists . So the effect of phenylephrine may be mediated via o^ - , (3j - and 32 - adre-noceptors.Denopamine (a (3j -adrenergic agonist) , Terbutaline (a p2 -adrenergic agonist) , BRL - 37344 ( a (33 - adrenergic agonist) increased alveolar fluid clearance. Atenolol (a (3i - adrenergic antagonist) abolished the effect of Denopamine, but did not inhibit the effects of Terbutaline and BRL -37344. ICI -118551 (a p2 -adrenergic antagonist) abolished the effect of Terbutaline and BRL - 37344, and also inhibited the effects of Denopamine in part. SR -59230A (a p3 - adrenergic antagonist) inhibited the effects of BRL -37344 and Terbutaline in part, but did not change the effects of Denopamine. Therefore, it is likely that atenolol played a role as a selective j3, - adrenergic antagonist, and ICI -118551 played a role as a predominant |32 - adrenergic antagonist and a role as a (^ - adrenergic antagonist in part. Terbutaline mainly stimulated j32 -adrenoceptors and also stimulated $x - adrenoceptors in part. The effect of BRL - 37344 on alveolar fluid clearance was mediated via |32 - and p$3 - adrenoceptors.In summary, phenylephrine, denopamine, terbutaline and BRL - 37344 are potent stimulators of alveolar fluid clearance in the rat lungs. The effect of phenylephrine is mediated via qlx - , Pi - and (32 ~ adrenoceptors. The effects of denopamine and terbutaline are mediated via |3j - and pj2 - adrenoceptors, respectively. The effect of BRL - 37344 may be mediated via (32 ~ adrenoceptors.Clearance of pulmonary edema fluid is accomplished by active ion transport. There are various ion pumps and channels on the alveolar epithelium, which in turn can drive the movement of lung edema fluid out of the airspaces. During the past 20 years, the mechanisms of alveolar ion and fluid clearance have studied and the importance of alveolar active sodium in the clearance of alveolar edema has been clearly established. Several in vivo as well as some in vitro studies indicate that vectorial sodium transport drives fluid clearance across the alveolar epithelium.The CFTR is a c AMP - dependent protein kinase and ATP - regulated Cl ~ channel, the activity of which determines the rate of electrolyte and fluid transport in a variety of epithelial tissues.We found that denopamine increased alveolar fluid clearance significantly. The effect was abolished by Atenolol ( a specific (3, - adrenergic antagonist) and amiloride ( an inhibitor of Na * channel). Glibenclamide and CFTRinh - 172, both CFTR inhibitors, inhibited the effect of denopamine in part. The effects of denopamine may be mediated in part by CFTR . In addition, Atenolol, glibenclamide and CFTRinh - 172 have no effects on basal alveolar fluid clearance. However, inhibition of sodium transport by amiloride, an inhibitor of apical sodium uptake, can decrease basal fluid clearance.NS004 ( a CFTR opener) also increased alveolar fluid clearance significantly in the isolated rat lungs. Two kinds of the inhibitors of CFTR and amiloride a-bolished the effects NS004. The effects of NS004 may be mediated in part by Na + channel.Our experiment demonstrated that denopamine — stimulated alveolar fluid... |
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