Abstract
In order to identify the form of phosphorylase kinase catalytic subunit expressed in developing lung, degenerate polymerase chain reaction primers were designed based on conserved domains of the two known catalytic subunits, expressed primarily in muscle and testis. Amplification of cDNA from day 19 fetal rat lung followed by cloning and sequence analyses indicated that only the testis isoform of phosphorylase kinase (PhK-gammaT) was detectable in fetal lung. In situ hybridization analyses indicated that expression of PhK-gammaT RNA in developing lung tissue was widespread and not restricted to Type II epithelial cells; PhK-gammaT protein expression was temporally and spatially correlated with expression of PhK-gammaT RNA. PhK-gammaT RNA and protein expression was also characterized in the PhK-deficient glycogen storage disease (gsd) rat. PhK-gammaT RNA levels were similar in Type II cells isolated from wild type and gsd/gsd fetuses; in contrast, PhK-gammaT protein was virtually undetectable in gsd/ gsd Type II cells and enzyme activity was very low. These results suggest that PhK-gammaT plays a critical role in mobilization of glycogen during fetal lung development and that failure to catabolize glycogen in the gsd/gsd rat is related to an untranslatable PhK-gammaT RNA or unstable protein.
Highlights
The epithelium of the fetal respiratory tree is characterized by the accumulation of glycogen during the last part of gestation
Sequence analyses of cDNA clones from multiple amplification reactions indicated that the 434-bp fragment corresponded exactly to the reported sequence for rat phosphorylase kinase (PhK)-␥T [14] (Fig. 1B); cDNA clones encoding muscle PhK␥ were not detected and this result was subsequently confirmed by RNase protection assays
The present study focused to the identification and characterization of PhK␥ expression in lung and, in particular, the Type II epithelial cell during the perinatal period
Summary
The epithelium of the fetal respiratory tree is characterized by the accumulation of glycogen during the last part of gestation. Activation of glycogen phosphorylase by phosphorylation is regulated by phosphorylase kinase (PhK).1 The importance of this enzyme in glycogen mobilization is demonstrated by the gsd/gsd rat [7] which lacks detectable PhK activity in developing lung tissue [8]. Phosphorylase kinase deficiency in gsd/gsd fetuses leads to an accumulation of the inactive form glycogen phosphorylase and failure to mobilize pulmonary glycogen, resulting in reduced phosphatidylcholine synthesis [8] These results suggest that posttranslational phosphorylation of glycogen phosphorylase by PhK is a critical step in the regulation of pulmonary glycogen catabolism. The tissue-specific expression of PhK may be related to the presence of distinct PhK isozymes Consistent with this hypothesis, human and rat testis PhK␥ (PhK-␥T) cDNAs have been isolated and shown to be 67% identical to rabbit skeletal muscle PhK␥ at the nucleic acid level [13, 14]. The purpose of this study was to identify the PhK␥ isozyme expressed in the fetal Type II epithelial cell and characterize its temporo-spatial pattern of expression during the perinatal period
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