INTRODUCTION

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Glucocorticoids are critical regulators of the surfactant system in late fetal life (1), and antenatal glucocorticoids given to women at risk of preterm delivery decrease respiratory distress syndrome (2). Exposure of human fetal lung explants to glucocorticoids accelerates the appearance of surfactant components (3). The essential requirement of glucocorticoids for late gestational lung maturation recently was demonstrated by the observations that fetal mice deficient for corticotropin-releasing hormone (CRH) or deficient for glucocorticoid receptors will die of respiratory failure after birth (4, 5). The lungs have an arrest in terminal bronchiolar and alveolar development from Day 15.5 postconception and inadequate surfactant function. In the adult rat, high-dose glucocorticoids selectively increase lamellar body and alveolar saturated phosphatidylcholine (Sat PC) and surfactant protein A (SP-A) pools without increases in phospholipids in other lung cell fractions or in other organs (6, 7). Although very little is known about how the normal lung regulates surfactant pools, hormones do influence surfactant metabolism. -Agonists cause surfactant secretion (8), and granulocyte-macrophage colony stimulating factor (GM-CSF) deficiency results in catabolic abnormalities (9). We hypothesized that glucocorticoids contribute to normal surfactant homeostasis. We evaluated the lung tissue and alveolar pools of Sat PC as well as radiolabeled choline incorporation in adult mice with very low plasma corticosterone levels secondary to CRH deficiency.

	METHODS

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Mice

Breeding pairs of C57B1/6J CRH/ and normal CRH+/+ mice were the kind gift of Dr. Joseph A. Majzoub, Harvard Medical School. The CRH/ mice were maintained on water containing 30 µg/ml corticosterone for the period from breeding to about 2 wk after delivery of the pups because CRH/ mice will not breed unless given supplemental glucocorticoids (4). All mice were then allowed to grow without glucocorticoid supplementation until 6 to 8 wk of age. Because Muglia and coworkers (4) found that the baseline corticosterone levels and stress levels were higher in female CRH/ mice than male CRH/ mice, we recorded the sex of each animal studied.

Surfactant Measurements

The mice were given a weight-adjusted intraperitoneal injection of 50 µCi/kg [³H]choline chloride (American Radiolabeled Chemical, St. Louis, MO), and the animals were deeply anesthetized 8 h later with intraperitoneal pentobarbital (9). The chest of each animal was opened and blood was drawn for the subsequent assay of corticosterone. The lungs then were processed as previously described (9). In brief, a 20-gauge catheter was tied into the trachea and an extensive alveolar wash was recovered for each animal. The lungs were then homogenized in saline.

Analytic Techniques

Saturated phosphatidylcholine (Sat PC) was recovered by chromatography using neutral alumina columns from chloroform:methanol (2:1) extracts of alveolar washes and lung homogenates (10). The amount of Sat PC was measured by phosphorus assay (11). Plasma corticosterone was measured using a kit from ICN (Costa Mesa, CA).

Data Analysis

All values are given as means ± SE. Differences between groups were evaluated using unpaired two-tailed t tests, with p < 0.05 considered as significant.

	RESULTS

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The six male and five female CRH/ mice weighed 25.6 ± 0.7 and 22.6 ± 1.1 g, respectively. The wild-type mice weighed 23.7 ± 1.3 g (6 male) and 22.5 ± 1.7 g (7 female). Plasma corticosterone values for wild-type male and female mice were 352 ± 5 and 334 ± 19 µg/ml, respectively. Corticosterone values for the CRH/ mice were very low and could not be reliably measured because they were at the limits of the assay.

Alveolar, lung tissue, and total lung (alveolar plus lung tissue) pool sizes of Sat PC were not different by sex for CRH/ or wild-type mice. The pool sizes also were not different between CRH/ mice and wild-type mice (Figure 1A). The total amount of [³H]choline recovered in Sat PC was not different for CRH/ mice or wild-type mice, and there were no differences based on sex (Figure 1B).

View larger version (20K): [in this window] [in a new window]   Figure 1.   Sat PC pool sizes (A) and [3H]Sat PC in mouse lungs (B). The amounts of Sat PC in alveolar washes, lung tissue, and total lungs (alveolar wash plus lung tissue) were not different for 13 wild-type and 11 CRH/ mice. The counts per minute of 3H from [3H]choline in Sat PC measured 8 h after precursor injection was similar in the total lungs for wild-type and CRH/ mice. There was less [3H] Sat PC recovered in alveolar washes from wild-type mice than from CRH/ mice (p < 0.05), and percent secretion also was lower for wild-type mice than CRH/ mice (p < 0.05).

The amount of [³H]choline-labeled Sat PC recovered by alveolar wash was lower for wild-type mice than for CRH/ mice. The percent secreted at 8 h estimated as the amount of [³H]Sat PC in the alveolar washes divided by the [³H]Sat PC in the lungs of each animal was lower for wild-type than for CRH/ mice. This difference resulted from a lower percent secretion in female wild-type mice (10.2 ± 2%) than for female CRH/ mice (20.1 ± 0.7%). Percent secretion for male wild-type mice was 15.6 ± 2.8% and for male CRH/ mice was 17.9 ± 1.8%, values that were not significantly different.

	DISCUSSION

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These experiments were based on the hypothesis that glucocorticoids would participate in surfactant homeostasis because they are essential for maturation of the developing lung (4, 5) and pharmacologic doses of glucocorticoids cause large increases in surfactant lipid and protein pools in adult rats (6, 7). The metabolism and function of the surfactant system is complex, and these experiments did not evaluate numbers of type II cells and processes such as recycling and alveolar form conversions (9). However, Sat PC pool sizes were normal in CRH/ mice, and incorporation of a precursor into Sat PC was the same for CRH/ mice and wild-type mice. The percent radiolabeled Sat PC recovered by alveolar wash also was similar for male CRH/ and wild-type mice. The only difference identified was a higher percent secretion for female CRH/ mice than for female wild-type mice. In previous experiments, normal values for secretion in female mice are similar to the values found for the female CRH/ mice (9). It is possible that there are subtle adaptive mechanisms (changes in type II cell numbers, for example) that reflect abnormalities in surfactant metabolism. However, our overall conclusion is that surfactant homeostasis is similar for CRH/ mice and wild-type mice.

The CRH/ mice had very low plasma corticosterone levels and the concentrations measured in the wild-type mice were similar to stress responses in mice reported previously (4). These high concentrations were probably the result of the stress related to terminal anesthesia. Although the CRH/ mice had received corticosterone via the mother or in the water for the first 2 wk of life, they had not received supplemental glucocorticoid for 4 to 6 wk before study. The 8-h interval from precursor administration to study was selected based on previous measurements of Sat PC labeling at multiple times after precursor administration (12). Maximal incorporation using labeled choline occurs within several hours after intraperitoneal injection, and degradation is not apparent by 8 h. Secretion also is close to maximal by 8 h (9), making this single time point for assessment a reasonable compromise.

Mechanisms regulating surfactant homeostasis are unknown. Short-term increases in alveolar surfactant occur after -agonist administration or hyperventilation (8, 13). GM-CSF cytokine or receptor deficiencies result in pulmonary alveolar proteinosis and slow catabolism of surfactant in mice and humans (14). The GM-CSF-deficient mice appear normal despite abnormal surfactant homeostasis. Other cytokines such as tumor necrosis factor alpha can acutely suppress surfactant protein B messenger RNA (SP-B mRNA) levels (15). Our results indicate that glucocorticoids are not important regulators of surfactant homeostasis in otherwise healthy adult mice.