Hiroyoshi Tsubochi

Single dexamethasone injection increases alveolar fluid clearance in adult rats.

ObjectiveEpithelial Na+ channels and Na+/K+-adenosine triphosphatase (ATPase) in alveolar epithelium have a very important role in the absorption of excessive fluid from the alveolar space. We examined whether single dexamethasone injection at therapeutic doses would modulate lung epithelial Na+ channels and Na+/K+-ATPase and increase alveolar fluid clearance in adult rats. DesignControlled laboratory study. SettingUniversity research laboratory. SubjectsAdult male Sprague-Dawley rats (n = 138). InterventionsRats were intraperitoneally injected with dexamethasone at a dose ranging from 0.02 to 2.0 mg/kg, and allowed free access to food and water. Measurements and Main ResultsAlveolar fluid clearance was determined by measuring the increase in albumin concentration in the lung instillate solution. We discovered a significant increase in alveolar fluid clearance at 48 and 72 hrs after dexamethasone treatment. The effect of dexamethasone was dose dependent. In addition, increased alveolar fluid clearance was associated with a faster recover from hypoxemia, which was induced by filling the alveolar space with instillate solution. The dexamethasone-induced increase in alveolar fluid clearance was inhibited by amiloride and ouabain. Quantitative reverse transcriptase-polymerase chain reaction showed that dexamethasone treatment increased lung &bgr;-epithelial Na+ channel mRNA levels. The expression of &ggr;-epithelial Na+ channel mRNA was also increased slightly. In contrast, &agr;-epithelial Na+ channel mRNA levels did not differ from control levels. There was no change in &agr;1- or &bgr;1-Na+/K+-ATPase mRNA levels over 72 hrs after dexamethasone treatment. However, we found that lung Na+/K+-ATPase hydrolytic activity, determined by monitoring the ouabain-sensitive ATPase hydrolysis, was increased at 48 and 72 hrs after dexamethasone treatment. ConclusionsSingle dexamethasone injection at therapeutic doses is capable of modulating lung epithelial Na+ channels and Na+/K+-ATPase and increase alveolar fluid clearance, thereby accelerating recovery from pulmonary edema.

MODULATION OF TRANSALVEOLAR FLUID ABSORPTION BY ENDOGENOUS ALDOSTERONE IN ADULT RATS

We examined whether transalveolar fluid transport is modulated by aldosterone in adult rats. Because colocalization of mineralocorticoid receptors (MR) with 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) is important for aldosterone specific action, we first determined the immunohistochemical distribution of MR and 11betaHSD2 in the lung. We found that alveolar epithelial cells express both MR and 11betaHSD2. Reverse transcriptase polymerase chain reaction (RT-PCR) demonstrated that rat alveolar type II epithelial cells express both MR and 11betaHSD2. We then measured alveolar fluid clearance in rats treated with chronic low-sodium diet. A low-sodium diet (0.1% NaCl for 12 to 14 days) caused hyperaldosteronism accompanied by hypokalemia, whereas serum corticosterone and adrenaline levels remained normal. We found that hyperaldosteronism was associated with significantly higher alveolar fluid clearance and that this increase was related to the amiloride-sensitive component. In addition, the increase in alveolar fluid clearance was inhibited by spironolactone. Our results show that aldosterone is able to stimulate Na+ channels of alveolar epithelial cells. We conclude that alveolar epithelium is a physiological target tissue for aldosterone and transalveolar fluid absorption could in part be modulated by endogenous aldosterone acting via MR.

Immunohistochemical localization of Ca2+/calmodulin-dependent protein kinase type IV in the peripheral ganglia and paraganglia of developing and mature rats

The immunohistochemical localization of Ca2+/calmodulin-dependent protein kinase type IV (CaM kinase IV) was examined in rat peripheral ganglia and paraganglia as well as brain. In sensory ganglia including the trigeminal and dorsal root ganglia, small- to medium-sized neurons were intensely immunoreactive. In the spinal cord, immunoreactive small neurons were seen in superficial laminae of the dorsal horn, whereas motoneurons were immunonegative. In autonomic ganglia including the superior cervical, celiac, and submandibular ganglia, almost all neurons were intensely immunoreactive for CaM kinase IV. In the small intestine, immunoreactive neurons were seen in the submucosal and myenteric ganglia. In all immunoreactive neurons, the immunoreactivity was localized predominantly in cell nuclei, whereas nucleoli and nerve fibers were completely free from immunoreaction. From the wide distribution and predominant nuclear localization of CaM kinase IV, it is suggested that CaM kinase IV might be involved in the modulation of gene transcription through the nuclear Ca(2+)-signaling in the peripheral as well as central nervous system.

Ca2+/calmodulin-dependent protein kinase type IV in dorsal root ganglion : colocalization with peptides, axonal transport and effect of axotomy

Using the indirect immunofluorescence technique, the distribution of Ca2+/calmodulin-dependent protein kinase IV (CaM kinase IV) was studied in dorsal root ganglia (DRGs) and the sciatic nerve under normal circumstances and after axotomy and nerve ligation. CaM kinase IV-like immunoreactivity (-LI) was observed mainly in small DRG neurons but also in some large ones with the immunoreactivity mainly confined to the cell nuclei and with varying levels in the cytoplasm. CaM kinase IV-LI was present in around 1/4 of all CGRP-positive neurons and in the vast majority of the somatostatin-positive neurons. The enzyme levels decreased markedly after axotomy. The enzyme was also observed in axons in the sciatic nerve and accumulated both proximal and distal to a ligation. The present results suggest that CaM kinase is not of direct importance for upregulation of neuropeptides in DRG neurons after nerve injury. In addition to a nuclear function it may also play a role in the peripheral processes of DRG neurons.

Increased expression of 11β‐hydroxysteroid dehydrogenase type 2 in the lungs of patients with acute respiratory distress syndrome

We  examined  the  immunohistochemical  distribution  of 11β‐hydroxysteroid dehydrogenase type 2 (11β‐HSD2), the enzyme responsible for the conversion of bioactive glucocorticoids to their receptor‐inactive forms, in lung tissue obtained at autopsy from 14 patients who had died due to acute respiratory distress syndrome (ARDS). We found positive immunoreactivity for 11β‐HSD2 in 13 cases. The cells expressing 11β‐HSD2 in the alveolar wall were positive for surfactant apoprotein‐A as well as cytokeratin. Immunoreactivity for 11β‐HSD2 was also detected in the CD68+ cells, which were found in the alveolar spaces. All patients had been treated with glucocorticoids for ARDS and/or the underlying diseases. There was no statistically significant correlation between the use of glucocorticoids and 11β‐HSD2 immunoreactivity in the alveolar wall (P = 0.0729). However, expression of grade + + was found in three out of five patients who received dexamethasone pulse therapy at relatively large doses, as well as in three other patients treated with prednisolone for a long period of time for the underlying disease. An increase in the expression of 11β‐HSD2 may result in faster glucocorticoid breakdown in lung cells in patients with ARDS. Impaired glucocorticoid availability in the lungs of such patients may explain, in part, the fact that glucocorticoid therapy does not always rescue patients with ARDS.

Effects of EP4 solution and LPD solution vs Euro-Collins solution on Na+/K+-ATPase activity in rat alveolar type II cells and human alveolar epithelial cell line A549 cells

BACKGROUND Intact alveolar epithelial Na(+)/K(+)- adenosinetriphosphatase (ATPase) function is important in preventing alveolar fluid accumulation after lung transplantation. We examined whether the type of preservation solution used influences Na(+)/K(+)-ATPase activity in alveolar epithelial cells. METHODS Rat alveolar type II cells were preserved with EP4, low-potassium dextran (LPD), or Euro-Collins solution at 7 degrees C for 5 and 20 hours. To assess cell toxicity, we measured cell viability and lactate dehydrogenase release. Na(+)/K(+)-ATPase activity was measured as ouabain-sensitive ATPase hydrolysis. We also examined the effect of terbutaline (10(-3) mol/liter) and dibutyryl cyclic adenosine monophosphate (dbcAMP) (10(-3) mol/liter) on Na(+)/K(+)-ATPase activity in A549 cells preserved for 5 hours. RESULTS All solutions caused significant damage of rat alveolar type II cells at 20 hours. However, Na(+)/K(+)-ATPase activity was preserved at normal levels with EP4 and LPD over 20 hours. Terbutaline and dbcAMP significantly increased Na(+)/K(+)-ATPase activity in A549 cells preserved with EP4 and LPD solutions for 5 hours. However, we observed no activation in the cells preserved with Euro-Collins solution. We found no significant difference in intracellular cAMP levels after terbutaline challenge among the types of preservation solution. CONCLUSIONS We conclude that extracellular-type solutions such as EP4 and LPD may be preferable for maintaining not only the basal activity but also the ability to activate Na(+)/K(+)-ATPase in response to beta-adrenergic agonists, in alveolar epithelial cells.

DIFFERENCE IN THE EFFECT OF PHLORIDZIN ON ALVEOLAR FLUID ABSORPTION IN ANESTHETIZED RATS AND IN EX VIVO RAT LUNGS

We reexamined the effect of phloridizin on alveolar fluid absorption by utilizing ex vivo rat lungs, which are considered to be a useful tool to investigate electrolyte and fluid transport across alveolar epithelium. Alveolar fluid absorption was almost completely reduced by 10(-3) M phloridzin with 10(-4) M amiloride as reported previously. However, we found that phloridzin alone was also able to significantly reduce alveolar fluid absorption. We then examined the effect of phloridzin on lung metabolism and compared the data with those determined in the presence of iodoacetic acid (IAA) and NaCN. Phloridzin reduced alveolar glucose uptake with no decrease in lung ATP content. Both IAA and NaCN decreased lung ATP content significantly. Our data indicate that the effect of phloridzin on alveolar fluid absorption in ex vivo rat lungs is not the secondary effect to the alteration of lung energy metabolism. Therefore our data support the current concept that Na(+)-glucose cotransport is involved with transalveolar active Na+ transport, which is a separated pathway from amiloride-sensitive Na+ channels.

Effects of intraalveolar oxygen concentration on alveolar fluid absorption and metabolism in isolated rat lungs

We evaluated the effects of intraalveolar oxygen concentration on alveolar fluid absorption and metabolism in isolated rat lungs. Alveolar fluid absorption was determined by measuring increase in albumin concentration in the instillate solution during 2 h of incubation. Oxidative phosphorylation was assessed by gas analysis of the solution. Glycolysis was assessed by determining glucose escape and lactate release in the solution. We found that alveolar fluid absorption did not change under hyperoxic and hypoxic experimental environments (range 100-10% oxygen). Glycolysis was reduced under hyperoxia and stimulated under hypoxia, however, lung ATP content did not change. When oxidative phosphorylation was inhibited by NaCN, both alveolar fluid absorption and lung ATP content were reduced. Our data indicate that isolated rat lungs maintain optimal energy production for alveolar fluid absorption by stimulating glycolysis, even though glycolysis alone is not enough. We conclude that alveolar fluid absorption determined in isolated rat lungs is not influenced by intraalveolar oxygen concentration in the range above 10% oxygen.

Intact alveolar epithelial permeability and transalveolar fluid absorption after thoracic irradiation in rats.

We have addressed the question of how the alveolar space stays relatively free of fluid when thoracic irradiation injures the pulmonary capillary endothelium and plasma fluid leaks into the interstitium. A single dose of 15 Gy to the thorax of rats significantly increased the pulmonary capillary filtration coefficient and the lung wet/dry weight ratio 2 h after irradiation. However, there was no significant increase in the release of lactose dehydrogenase or leaking of Evans blue dye into the alveolar space, indicating that alveolar epithelial permeability remained intact. We found no significant difference in the basal alveolar fluid clearance between control and irradiated animals. There was also no significant difference in blockage of alveolar fluid clearance by amiloride. This indicates that the function of the alveolar epithelial Na(+) channels is not impaired and that alveolar epithelium absorbs fluid normally. Examination of lung tissue by light microscopy demonstrated accumulation of fluid in the perivascular region but not in the alveolar space. Our data appear to indicate that the alveolar epithelial barrier function is more resistant to radiation than that of the pulmonary capillary endothelium. We conclude that intact alveolar epithelial permeability and normal transalveolar epithelial fluid absorption ability are of critical importance in keeping the alveolar space relatively free of fluid during acute radiation lung injury.