The adenosine 2A receptor agonist GW328267C improves lung function after acute lung injury in rats.

There is a significant unmet need for treatments of patients with acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS). The primary mechanism that leads to resolution of alveolar and pulmonary edema is active vectorial Na(+) and Cl(-) transport across the alveolar epithelium. Several studies have suggested a role for adenosine receptors in regulating this fluid transport in the lung. Furthermore, these studies point to the A(2A) subtype of adenosine receptor (A(2A)R) as playing a role to enhance fluid transport, suggesting that activation of the A(2A)R may enhance alveolar fluid clearance (AFC). The current studies test the potential therapeutic value of the A(2A)R agonist GW328267C to accelerate resolution of alveolar edema and ALI/ARDS in rats. GW328267C, at concentrations of 10(-5) M to 10(-3) M, instilled into the airspaces, increased AFC in control animals. GW328267C did not increase AFC beyond that produced by maximal ß-adrenergic stimulation. The effect of GW328267C was inhibited by amiloride but was not affected by cystic fibrosis transmembrane conductance regulator inhibition. The drug was tested in three models of ALI, HCl instillation 1 h, LPS instillation 16 h, and live Escherichia coli instillation 2 h before GW328267C instillation. After either type of injury, GW328267C (10(-4) M) decreased pulmonary edema formation and restored AFC, measured 1 h after GW328267C instillation. These findings show that GW328267C has beneficial effects in experimental models of ALI and may be a useful agent for treating patients with ALI or prophylactically to prevent ALI.

Phosphatidylinositol 4,5-bisphosphate stimulates alveolar epithelial fluid clearance in male and female adult rats.

Cell membrane phospholipids, like phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], can regulate epithelial Na channel (ENaC) activity. Gender differences in lung ENaC expression have also been demonstrated. However, the effects in vivo on alveolar fluid clearance are uncertain. Thus PI(4,5)P(2) effects on alveolar fluid clearance were studied in male and female rats. An isosmolar 5% albumin solution was intrapulmonary instilled; alveolar fluid clearance was studied for 1 h. Female rats had a 37 ± 19% higher baseline alveolar fluid clearance than male rats. Bilateral ovariectomy attenuated this gender difference. Compared with controls, PI(4,5)P(2) instillation (300 µM) increased alveolar fluid clearance by ∼93% in both genders. Amiloride or the specific αENaC small-interfering RNA inhibited baseline and PI(4,5)P(2)-stimulated alveolar fluid clearance in both genders, indicating a dependence on amiloride-sensitive pathways. The fraction of amiloride inhibition was greater in PI(4,5)P(2)-instilled rats (male: 64 ± 10%; female: 70 ± 11%) than in controls (male: 30 ± 6%; female: 44 ± 8%). PI(4,5)P(2) instillation lacked additional alveolar fluid clearance stimulation above that of terbutaline, nor did propranolol inhibit alveolar fluid clearance after PI(4,5)P(2) instillation, indicating that PI(4,5)P(2) stimulation was not secondary to endogenous ß-adrenoceptor activation. PI(4,5)P(2) amine instillation resulted in an intermediate alveolar fluid clearance stimulation, suggesting that, to reach maximal alveolar fluid clearance stimulation, PI(4,5)P(2) must reside in cell membranes. In summary, PI(4,5)P(2) instillation upregulated in vivo alveolar fluid clearance similar to short-term ß-adrenoceptor upregulation of alveolar fluid clearance. PI(4,5)P(2) stimulation was mediated partly by increased amiloride-sensitive Na transport. There exist important gender-related effects suggesting a female advantage that may have clinical implications for resolution of acute lung injury.

Fetal lung epithelial ion channels relocate in the cell membrane during late gestation.

Near the end of gestation, the direction of ion and fluid flow across the alveolar epithelium rapidly changes from secretion to absorption. Thus, the relative cell membrane location of epithelial Na channels (ENaCs) and cystic fibrosis transmembrane regulator (CFTR) Cl channels during late fetal lung development and after maternal interleukin-1ß (IL-1ß) pretreatment was the focus of our study. Western blot analysis after sucrose gradient separation of caveolin-1-(Cav-1)-rich membrane regions (CRR) and Cav-1-poor membrane (non-CRR) revealed primary CRR ENaC localization at gestation day (GD) 61 in guinea pigs. Correlating with the natural induction of distal lung fluid absorption, ENaC appeared in the non-CRR cell membrane regions at GD68. Conversely, CFTR was present in the non-CRR cell membrane regions at GD61 and in the CRRs at GD68. IL-1ß-induced conversion to distal lung fluid absorption at GD61 was associated with ENaC non-CRR presence and CFTR CRR presence, suggesting that relative ENaC and CFTR locations induced distal lung fluid absorption and decreased fluid secretion. Instilling fetal lungs with the CRR-disrupting agent methyl-ß-cyclodextrin resulted in the conversion from lung fluid secretion to absorption and ENaC non-CRR presence at GD61. Coimmunoprecipitation of Cav-1 with α- and ß-ENaC demonstrated reduced coimmunoprecipitation with increased GD and after IL-1ß pretreatment. On the other hand, coimmunoprecipitation of Cav-1 with CFTR demonstrated increased coimmunoprecipitation with increasing GD and after IL-1ß pretreatment. This concept may provide novel molecular mechanisms for the rapid transition from fetal distal lung fluid secretion to absorption in near-term lungs.

Anthocyanin-rich black currant (Ribes nigrum L.) extract affords chemoprevention against diethylnitrosamine-induced hepatocellular carcinogenesis in rats.

Anthocyanins are known to possess potent anticarcinogenic properties against several cancers thus demonstrating potential for cancer prevention. Black currant (Ribes nigrum L., Grossulariaceae) fruits have a high anthocyanin content. This "superfruit" is known to possess various pharmacological effects including alleviation of chronic oxidative stress and inflammation. In contrast to a large volume of literature on the health benefits of black currant, limited evidence on antitumor effects of black currant exists with virtually no data on the prevention of experimental carcinogenesis. In the current study, we have investigated the chemopreventive effects of an anthocyanin-rich black currant skin extract (BCSE) utilizing our well-characterized model of rat liver carcinogenesis. Initiation of hepatocarcinogenesis was done by intraperitoneal injection of diethylnitrosamine (DENA) followed by promotion with phenobarbital. The rats were exposed to dietary BCSE for 4 weeks prior to initiation, and the treatment was continued for 22 consecutive weeks. BCSE dose-dependently decreased the incidence, total number, multiplicity, size and volume of preneoplastic hepatic nodules. The antihepatocarcinogenic effect of BCSE was confirmed by histopathological examination of liver sections. Immunohistochemical analysis of proliferating cell nuclear antigen and DNA fragmentation revealed BCSE-mediated inhibition of abnormal cell proliferation and induction of apoptosis in DENA-induced rat liver tumorigenesis respectively. Mechanistic studies revealed that BCSE-mediated proapototic signal during experimental hepatocarcinogenesis may be propagated via the up-regulation of Bax and down-regulation of Bcl-2 expression at the translational level. These results along with a safety profile of BCSE encourage the development of black currant bioactive constituents as chemopreventive agents for human liver cancer.

Lung fluid absorption is induced in preterm guinea pigs ventilated with low tidal volumes.

The objective of this study was to determine if low tidal volume (V(t)) ventilation was beneficial when ventilating preterm fetuses. The authors ventilated preterm guinea pig fetuses at gestation day (GD) 67, 3 days before birth, newborn, and 10-day-old (PD10) guinea pigs with low V(t) (6 mL/kg body weight [bw]) and compared them to age-matched fetuses/animals ventilated with higher potentially injurious V(t) (12 mL/kg bw). Lung fluid absorption was measured after intratracheal instillation of 5% albumin in 0.9% NaCl. Low V(t) ventilation stimulated lung fluid absorption when compared to higher V(t) in all groups. The increased lung fluid absorption in low V(t)-ventilated fetuses was associated with increased α epithelial Na channel (αEnaC) mRNA. However, αENaC and ßENaC protein was unchanged over the 1-hour study. Because stretch induces mitogen-activated protein (MAP) kinase expression and MAP kinases may affect lung fluid absorption, the authors investigated if MAP kinase (MAPK) expression was affected by V(t). Extracellular signal-regulated kinase (ERK) and MAPK/ERK kinase (MEK) were phosphorylated in the higher V(t)-ventilated guinea pig fetuses. This suggested that a reduced activation of MAP kinases might explain the increased lung fluid absorption in the low V(t)-ventilated fetuses. Thus these data suggest that low V(t) ventilation increases fetal lung fluid absorption and thus may be preferential to use clinically.

Beta-adrenoceptor stimulation of alveolar fluid clearance is increased in rats with heart failure.

The alveolar epithelium plays a critical role in resolving pulmonary edema. We thus hypothesized that its function might be upregulated in rats with heart failure, a condition that severely challenges the lung's ability to maintain fluid balance. Heart failure was induced by left coronary artery ligation. Echocardiographic and cardiovascular hemodynamics confirmed its development at 16 wk postligation. At that time, alveolar fluid clearance was measured by an increase in protein concentration over 1 h of a 5% albumin solution instilled into the lungs. Baseline alveolar fluid clearance was similar in heart failure and age-matched control rats. Terbutaline was added to the instillate to determine whether heart failure rats responded to beta-adrenoceptor stimulation. Alveolar fluid clearance in heart failure rats was increased by 194% after terbutaline stimulation compared with a 153% increase by terbutaline in control rats. To determine the mechanisms responsible for this accelerated alveolar fluid clearance, we measured ion transporter expression (ENaC, Na-K- ATPase, CFTR). No significant upregulation was observed for these ion transporters in the heart failure rats. Lung morphology showed significant alveolar epithelial type II cell hyperplasia in heart failure rats. Thus, alveolar epithelial type II cell hyperplasia is the likely explanation for the increased terbutaline-stimulated alveolar fluid clearance in heart failure rats. These data provide evidence for previously unrecognized mechanisms that can protect against or hasten resolution of alveolar edema in heart failure.

Delineation of VEGF-regulated genes and functions in the cervix of pregnant rodents by DNA microarray analysis.

BACKGROUND: VEGF-regulated genes in the cervices of pregnant and non-pregnant rodents (rats and mice) were delineated by DNA microarray and Real Time PCR, after locally altering levels of or action of VEGF using VEGF agents, namely siRNA, VEGF receptor antagonist and mouse VEGF recombinant protein. METHODS: Tissues were analyzed by genome-wide DNA microarray analysis, Real-time and gel-based PCR, and SEM, to decipher VEGF function during cervical remodeling. Data were analyzed by EASE score (microarray) and ANOVA (Real Time PCR) followed by Scheffe's F-test for multiple comparisons. RESULTS: Of the 30,000 genes analyzed, about 4,200 genes were altered in expression by VEGF, i.e., expression of about 2,400 and 1,700 genes were down- and up-regulated, respectively. Based on EASE score, i.e., grouping of genes according to their biological process, cell component and molecular functions, a number of vascular- and non-vascular-related processes were found to be regulated by VEGF in the cervix, including immune response (including inflammatory), cell proliferation, protein kinase activity, and cell adhesion molecule activity. Of interest, mRNA levels of a select group of genes, known to or with potential to influence cervical remodeling were altered. For example, real time PCR analysis showed that levels of VCAM-1, a key molecule in leukocyte recruitment, endothelial adhesion, and subsequent trans-endothelial migration, were elevated about 10 folds by VEGF. Further, VEGF agents also altered mRNA levels of decorin, which is involved in cervical collagen fibrillogenesis, and expression of eNO, PLC and PKC mRNA, critical downstream mediators of VEGF. Of note, we show that VEGF may regulate cervical epithelial proliferation, as revealed by SEM. CONCLUSION: These data are important in that they shed new insights in VEGF's possible roles and mechanisms in cervical events near-term, including cervical remodeling.

RNA interference for CFTR attenuates lung fluid absorption at birth in rats.

BACKGROUND: Small interfering RNA (siRNA) against alphaENaC (alpha-subunit of the epithelial Na channel) and CFTR (cystic fibrosis transmembrane conductance regulator) was used to explore ENaC and CTFR function in newborn rat lungs. METHODS: Twenty-four hours after trans-thoracic intrapulmonary (ttip) injection of siRNA-generating plasmid DNA (pSi-0, pSi-4, or pSi-C2), we measured CFTR and ENaC expression, extravascular lung water, and mortality. RESULTS: alphaENaC and CFTR mRNA and protein decreased by approximately 80% and approximately 85%, respectively, following alphaENaC and CFTR silencing. Extravascular lung water and mortality increased after alphaENaC and CFTR-silencing. In pSi-C2-transfected isolated DLE cells there were attenuated CFTR mRNA and protein. In pSi-4-transfected DLE cells alphaENaC mRNA and protein were both reduced. Interestingly, CFTR-silencing also reduced alphaENaC mRNA and protein. alphaENaC silencing, on the other hand, only slightly reduced CFTR mRNA and protein. CONCLUSION: Thus, ENaC and CFTR are both involved in the fluid secretion to absorption conversion around at birth.

Involvement of {alpha}ENaC and Nedd4-2 in the conversion from lung fluid secretion to fluid absorption at birth in the rat as assayed by RNA interference analysis.

To explore interactions between the epithelial Na channel (ENaC) and neural precursor expressed, developmentally downregulated protein 4-2 (Nedd4-2) at the conversion of the rat lung from fluid secretion to absorption at birth, we used small-interfering RNA (siRNA) against alphaENaC and Nedd4-2. siRNA-generating plasmid DNA (pDNA) was administered via trans-thoracic intrapulmonary (ttip) injection 24 h before ENaC and Nedd4-2 expression, extravascular lung water, and mortality were measured. alphaENaC mRNA and protein were specifically reduced by approximately 65% after pSi-4 injection. Nedd4-2 mRNA and protein were reduced by approximately 60% after pSi-N1 injection. Interestingly, alphaENaC and betaENaC mRNA and protein expression were increased after Nedd4-2 silencing. Extravascular lung water was significantly increased after alphaENaC silencing and reduced after Nedd4-2 silencing. alphaENaC silencing resulted in a fourfold increase in newborn mortality, whereas silencing Nedd4-2 did not affect mortality. We also isolated distal lung epithelial (DLE) cells after in vivo alphaENaC or Nedd4-2 silencing and measured alphaENaC or Nedd4-2 expression in freshly isolated DLE cells. In these DLE cells, there were attenuated alphaENaC or Nedd4-2 mRNA and protein, thus demonstrating that alphaENaC and Nedd4-2 silencing occurred in alveolar epithelial cells after ttip injection. We also looked for pDNA by PCR to determine pDNA presence in the lungs and found strong evidence for pDNA presence in both lungs. Thus we provide evidence that ENaC and Nedd4-2 are involved in the transition from lung fluid secretion to fluid absorption near term and at birth.

Congenital diaphragmatic hernia prevents absorption of distal air space fluid in late-gestation rat fetuses.

We hypothesized that congenital diaphragmatic hernia (CDH) may decrease distal air space fluid absorption due to immaturity of alveolar epithelial cells from a loss of the normal epithelial Na+ transport, as assessed by amiloride and epithelial Na+ channel (ENaC) and Na-K-ATPase expression, as well as failure to respond to endogenous epinephrine as assessed by propranolol. Timed-pregnant dams were gavage fed 100 mg of nitrofen at 9.5-day gestation to induce CDH in the fetuses, and distal air space fluid absorption experiments were carried out on 22-day gestation (term) fetuses. Controls were nitrofen-exposed fetuses without CDH. Absorption of distal air space fluid was measured from the increase in 131I-albumin concentration in an isosmolar, physiological solution instilled into the developing lungs. In controls, distal air space fluid absorption was rapid and mediated by beta-adrenoceptors as demonstrated by reversal to fluid secretion after propranolol. Normal lung fluid absorption was also partially inhibited by amiloride. In contrast, CDH fetuses continued to show lung fluid secretion, and this secretion was not affected by either propranolol or amiloride. CDH lungs showed a 67% reduction in alpha-ENaC and beta-ENaC expression, but no change in alpha1-Na-K-ATPase expression. These studies demonstrate: 1) CDH delays lung maturation with impaired distal air space fluid absorption secondary to inadequate Na+ uptake by the distal lung epithelium that results in fluid-filled lungs at birth with reduced capacity to establish postnatal breathing, and 2) the main stimulus to lung fluid absorption in near-term control fetuses, elevated endogenous epinephrine levels, is not functional in CDH fetuses.

RNA interference for alpha-ENaC inhibits rat lung fluid absorption in vivo.

We used siRNA against the alpha-ENaC (epithelial Na channel) subunit to investigate ENaC involvement in lung fluid absorption in rats by the impermeable tracer technique during baseline and after beta-adrenoceptor stimulation by terbutaline. Terbutaline stimulation of lung fluid absorption increased fluid absorption by 165% in pSi-0-pretreated rat lungs (irrelevant siRNA-generating plasmid). Terbutaline failed to increase lung fluid absorption in rats given the specific alpha-ENaC siRNA-generating plasmid (pSi-4). pSi-4 pretreatment reduced baseline lung fluid absorption by approximately 30%. alpha-ENaC was undetectable in pSi-4-pretreated lungs, regardless of condition but was normal in pSi-0-pretreated lungs. We carried out a dose-response analysis where rats were given 0-200 microg/kg body wt pSi-4, and alpha-ENaC mRNA and protein expressions were analyzed. To reach IC(50) for alpha-ENaC mRNA expression, 32 microg/kg body wt pSi-4 was needed, and to reach IC(50) for alpha-ENaC protein expression, 59 microg/kg body wt pSi-4 was needed. We tested for lung tissue specificity and found no changes in beta-ENaC expression, at either mRNA or protein level, as well as no changes in alpha(1)-Na-K-ATPase protein expression. We isolated alveolar epithelial type II cells 24 h after in vivo pSi-4 pretreatment. In these cells, alpha-ENaC mRNA was undetectable, demonstrating that alveolar epithelial ENaC expression was attenuated after intratracheal alpha-ENaC siRNA-generating plasmid DNA instillation. We tested for organ specificity and found no changes in kidney alpha- and beta-ENaC mRNA and protein expression. Thus we provide conclusive evidence that beta-adrenoceptor stimulation of lung fluid absorption is critically ENaC dependent, whereas baseline lung fluid absorption seemed less ENaC dependent.

PKA delivery to the distal lung air spaces increases alveolar liquid clearance after isoproterenol-induced alveolar epithelial PKA desensitization.

Isoproterenol (Iso) infusion for 48 h in rats decreases the ability of beta-adrenoceptor (beta-AR) agonists to increase alveolar liquid clearance (ALC). An impairment in protein kinase A (PKA) function appears to be critical in producing the desensitized ALC response. To test this hypothesis, we used a novel protein delivery reagent (Chariot, Active Motif) to deliver either the PKA catalytic subunit or the PKA holoenzyme to the distal lung epithelium of Iso-infused rats (400 microg.kg(-1).h(-1), 48 h). After this infusion, ALC was measured by mass balance over 2 h. ALC in Iso-infused rats was 27.9% (SD 5.8) of instilled volume absorbed. Delivery of the catalytic PKA subunit to Iso-infused rats increased ALC to 47.7% (SD 8.9) (P < 0.05). ALC in Iso-infused rats delivered the inactive PKA holoenzyme [29.6% (SD 2.5)] was not increased above baseline values. Subsequent holoenzyme activation by intravenous infusion of the stable cAMP analog Sp-8-Bromo-cAMPS increased ALC to 41.7% (SD 8.8) (P < 0.05). Immunohistochemical localization of Chariot-delivered PKA revealed staining in the alveolar and distal airway epithelium. These data indicate that protein delivery reagents can be used to rapidly deliver biologically active proteins to the distal lung epithelium and that PKA desensitization may be an important rate-limiting event in the development of Iso-induced desensitization of the alveolar epithelial beta-AR signaling pathway.

IL-1beta stimulates alveolar fluid absorption in fetal guinea pig lungs via the hypothalamus-pituitary-adrenal gland axis.

We tested the hypothesis that interleukin (IL)-1beta-induced cortisol synthesis stimulates alveolar fluid clearance in preterm fetuses. IL-1beta was administered subcutaneously daily to timed-pregnant guinea pigs for 3 days with and without simultaneous cortisol synthesis inhibition by metyrapone. Fetuses were obtained by abdominal hysterotomy at 61 and 68 days gestation and instilled with isosmolar 5% albumin in the lungs, and alveolar fluid movement was measured over 1 h from the change in alveolar protein concentration. Alveolar fluid clearance was induced at 61 days gestation and stimulated at 68 days gestation by IL-1beta, which both were attenuated by cortisol synthesis inhibition. Plasma ACTH and cortisol concentrations were increased by IL-1beta at both gestational ages, and metyrapone reduced cortisol concentrations. IL-1beta was mostly low or undetectable in both fetal and maternal blood. Prenatal alveolar fluid clearance, when present as well as IL-1beta induced, was always propranolol and amiloride sensitive, suggesting that beta-adrenoceptor stimulation and amiloride-sensitive Na+ channels were critical for fluid absorption. IL-1beta increased lung beta-adrenoceptor density at gestation day 61, and cortisol synthesis inhibition attenuated the increased beta-adrenoceptor density. Epithelial Na+ channel and Na+-K+-ATPase subunit expressions were both increased by IL-1beta and attenuated by cortisol synthesis inhibition. These results may explain why babies delivered preterm after intrauterine inflammation have a reduced risk of developing severe respiratory distress.

Postreceptor defects in alveolar epithelial beta-adrenergic signaling after prolonged isoproterenol infusion.

We previously found that prolonged isoproterenol (Iso) infusion in rats impaired the ability of beta-adrenoceptor (beta-AR) agonists to increase alveolar liquid clearance (ALC). Here, we determined if postreceptor defects in beta-AR signaling contribute to this impairment. Iso was infused using subcutaneous miniosmotic pumps (4, 40, or 400 microg. kg-1. h-1) in rats for 48 h. At this time, forskolin-stimulated ALC was measured by mass balance. Forskolin-stimulated ALC [33.4 +/- 2.1%/h (mean +/- SE) in vehicle-infused rats] was reduced by 25 and 38%, respectively, after the 40 and 400 microg. kg-1. h-1 Iso infusions. The ability of forskolin to increase cAMP was reduced by 70% in alveolar type II (ATII) cells isolated from rats infused with 400 microg. kg-1. h-1 Iso. Additionally, the ability of the stable cAMP analog 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Sp-isomer, to increase ALC (48.7 +/- 3.0% in vehicle-infused rats) was reduced by 25 and 51%, respectively, after the 40 and 400 microg. kg-1. h-1 infusions. Finally, the ability of cAMP to increase protein kinase A activity was eliminated in ATII cells isolated from rats infused with Iso at 400 microg. kg-1. h-1. These data demonstrate that prolonged beta-AR agonist exposure can impair alveolar epithelial beta-AR signaling downstream of the beta-AR.

Lung edema clearance: 20 years of progress: invited review: alveolar edema fluid clearance in the injured lung.

Resolution of pulmonary edema involved active transepithelial sodium transport. Although several of the cellular and molecular mechanisms involved are relatively well understood, it is only recently that the regulation of these mechanisms in injured lung are being evaluated. Interestingly, in mild-to-moderate lung injury, alveolar edema fluid clearance is often preserved. This preserved or enhanced alveolar fluid clearance is mediated by catecholamine-dependent or -independent mechanisms. This stimulation of alveolar liquid clearance is related to activation or increased expression of sodium transport molecules such as the epithelial sodium channel or the Na(+)-K(+)-ATPase pump and may also involve the cystic fibrosis transmembrane conductance regulator. When severe lung injury occurs, the decrease in alveolar liquid clearance may be related to changes in alveolar permeability or to changes in activity or expression of sodium or chloride transport molecules. Multiple pharmacological tools such as beta-adrenergic agonists, vasoactive drugs, or gene therapy may prove effective in stimulating the resolution of alveolar edema in the injured lung.

Lung epithelial fluid transport and the resolution of pulmonary edema.

The discovery of mechanisms that regulate salt and water transport by the alveolar and distal airway epithelium of the lung has generated new insights into the regulation of lung fluid balance under both normal and pathological conditions. There is convincing evidence that active sodium and chloride transporters are expressed in the distal lung epithelium and are responsible for the ability of the lung to remove alveolar fluid at the time of birth as well as in the mature lung when pathological conditions lead to the development of pulmonary edema. Currently, the best described molecular transporters are the epithelial sodium channel, the cystic fibrosis transmembrane conductance regulator, Na+-K+-ATPase, and several aquaporin water channels. Both catecholamine-dependent and -independent mechanisms can upregulate isosmolar fluid transport across the distal lung epithelium. Experimental and clinical studies have made it possible to examine the role of these transporters in the resolution of pulmonary edema.

Ca(2+)-dependent stimulation of alveolar fluid clearance in near-term fetal guinea pigs.

We investigated the importance of changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) for amiloride-sensitive alveolar fluid clearance (AFC) in late-gestational guinea pigs. Fetal guinea pigs of 61, 68, and 69 days (term) gestation were investigated under normal conditions and after oxytocin-induced preterm labor. AFC or alveolar fluid secretion was measured using an impermeable tracer technique. At 61 days gestation there was net secretion of fluid into the lungs, and at birth the lungs cleared 49 +/- 7% of the instilled fluid volume over 1 h. Induction of preterm labor with oxytocin induced AFC at 61 days gestation. When present, AFC was inhibited or reversed to net fluid secretion by amiloride (10(-3) M). Inhibition of membrane Ca(2+) channels by verapamil (10(-4) M) or depletion of intracellular Ca(2+) by thapsigargin (10(-5) M) reduced AFC when net AFC was evident. Amiloride lacked an inhibitory effect on AFC when instilled with verapamil or thapsigargin. The results indicate that AFC via amiloride-sensitive pathways develops during late gestation, and that inducing preterm labor precociously may activate such pathways. Our results suggest that Ca(2+) may act as a second messenger in mediating catecholamine-stimulated AFC.

Prolonged isoproterenol infusion impairs the ability of beta(2)-agonists to increase alveolar liquid clearance.

We determined if prolonged isoproterenol (Iso) infusion in rats impaired the ability of the beta(2)-adrenergic agonist terbutaline to increase alveolar liquid clearance (ALC). We infused rats with Iso (at rates of 4, 40, or 400 microg.kg(-1).h(-1)) or vehicle (0.001 N HCl) for 48 h using subcutaneously implanted miniosmotic pumps. After this time, the rats were anesthetized, and ALC was determined (by mass-balance after instillation of Ringer lactate containing albumin into the lungs) under baseline conditions and after terbutaline administration. Baseline and terbutaline-stimulated ALC in vehicle-infused rats averaged, respectively, 19.6 +/- 1.2% (SE) and 44.7 +/- 1.5%/h. The ability of terbutaline to increase ALC was eliminated at 400 microg.kg(-1).h(-1)Iso, inhibited by 26% at 40 microg.kg(-1).h(-1) Iso, and was not affected by 4 microg.kg(-1).h(-1) Iso. beta-adrenergic receptor (betaAR) density of freshly isolated alveolar epithelial type II (ATII) cells from Iso-infused rats was reduced by the 40 and 400 microg.kg(-1).h(-1) infusion rates. These data demonstrate that prolonged exposure to beta-agonists can impair the ability of beta(2)-agonists to stimulate ALC and produce ATII cell betaAR downregulation.