The testis isoform of the phosphorylase kinase catalytic subunit (PhK-gammaT) plays a critical role in regulation of glycogen mobilization in developing lung.

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-T) was detectable in fetal lung. In situ hybridization analyses indicated that expression of PhK-T RNA in developing lung tissue was widespread and not restricted to Type II epithelial cells; PhK-T protein expression was temporally and spatially correlated with expression of PhK-T RNA. PhK-T RNA and protein expression was also characterized in the PhK-deficient glycogen storage disease (gsd) rat. PhK-T RNA levels were similar in Type II cells isolated from wild type and gsd/gsd fetuses; in contrast, PhK-T protein was virtually undetectable in gsd/gsd Type II cells and enzyme activity was very low. These results suggest that PhK-T 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-T RNA or unstable protein.

The epithelium of the fetal respiratory tree is characterized by the accumulation of glycogen during the last part of gestation. Glycogen mobilization is both temporally and spatially regulated such that catabolism in the proximal airways proceeds at or close to birth, whereas catabolism in the distal airway occurs earlier coinciding with maturation of the Type II epithelial cell (1). On day 17 of gestation, the respiratory epithelium of the fetal rat lung consists of undifferentiated columnar cells that contain small amounts of glycogen; lamellar bodies, the intracellular storage granules for surfactant, are not detectable (2). On day 19 of gestation, the epithelium consists of cuboidal cells which contain large pools of glycogen and a few lamellar bodies. By day 21 of gestation, the epithelium approximates that observed in postnatal life consisting of thin Type I cells and cuboidal Type II cells which are devoid of glycogen and contain numerous lamellar bodies. Kikkawa and co-workers (3) noted the inverse relationship between glycogen and lamellar bodies and speculated that glycogenolysis might be associated with surfactant synthesis. A direct link between the breakdown of glycogen on day 20 of gestation and surfactant synthesis was demonstrated by Farrell and Bourbon (4) who showed that glucose, originating from glycogen, provided substrate for fatty acid and glycerol synthesis which, in turn, were incorporated into surfactant phosphatidylcholine. Both biochemical and morphological studies therefore support the hypothesis that glycogen breakdown may be an important event in prenatal surfactant phospholipid synthesis.
The initial step in glycogen catabolism is the phosphorolysis of glycogen to glucose 1-phosphate by phosphorylase a, the activated form of glycogen phosphorylase. Glycogen phosphorylase enzyme activity has been shown to increase in late gestation fetal lung concomitant with initiation of glycogen degradation (5). In contrast to the developmentally regulated increase in enzyme activity, the level of RNA encoding brain glycogen phosphorylase, the predominant isoform of glycogen phosphorylase in developing rabbit lung, does not change during the perinatal period (6), suggesting that enzyme activity is regulated by allosteric activation and/or by post-translational phosphorylation. 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, ultimately 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.
Muscle PhK is a hexadecameric protein (␣ 4 ␤ 4 ␥ 4 ␦ 4 ) with a molecular mass of 1.3 ϫ 10 6 Da (for review, see Ref. 9). Calmodulin, the ␦ subunit, is responsible for the Ca 2ϩ -dependent activity of this enzyme; the ␣ and ␤ subunits regulate the activity of PhK in response to phosphorylation by cAMP-dependent protein kinase A. Glycogen phosphorylase is activated following phosphorylation by the catalytic subunit, ␥, a 387amino acid protein. cDNAs encoding muscle PhK␥ have been cloned and characterized by a number of laboratories (10 -12). RNAs of 2.4 and 1.6 kilobases encoding muscle PhK␥ were * This work was supported by National Institutes of Health Grant HD20748 (to T. E. W.) and the American Heart Association (to S. R. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Children's Hospital Medical Center, Div. of Pulmonary Biology, TCHRF, 3333 Burnet Ave., Cincinnati, OH 45229-3039. Tel.: 513-559-7223; Fax: 513-559-7868. detected in mouse skeletal muscle, brain, and cardiac tissues but not in liver, consistent with tissue-specific expression of the muscle enzyme (10). 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 PhK-␥T cDNA detected an abundant 2.0-kilobase RNA in testis which was expressed at much lower levels in most other tissues including lung. PhK-␥T RNA was not detected prior to 2 months of age, indicating both temporal as well as a tissuespecific regulation of expression. 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.

MATERIALS AND METHODS
Animals and Breeding-Rats used in these studies were from the NZR/Mh strain derived from a random bred Wistar line found to be homozygous for liver phosphorylase b kinase deficiency (gsd/gsd). Normal time-dated pregnant Wistar controls were obtained from Charles River Laboratories. Control and gsd/gsd animals were bred at the same time so each strain could be compared on the same experimental day. There was no difference in the length of gestation between control Wistars or the gsd/gsd strain; therefore, specific metabolic differences could be distinguished from the effects of gestational age.
Isolation of Cells-Lungs from 2-3 litters of a single gestational age (days 19 -21) were removed and placed in sterile 1 ϫ Hanks' balanced salts (without calcium or magnesium), minced into 1-mm cubes, and disrupted in a solution containing 2 mg/ml collagenase (Sigma, Type IV) and 0.15 mg/ml bovine pancreas DNase I (Calbiochem). Cells were then filtered through 160-m nylon mesh, centrifuged, and resuspended in Joklik's modified Eagle's medium containing 10% fetal bovine serum. Mixed cells were plated on 100-mm tissue culture plates and incubated at 37°C to promote differential adherence of fibroblasts over a 2-h interval. Type II cells were removed by panning and loaded onto Percoll gradients with density steps of 1.095 and 1.045; cells were collected from the interface. Isolation of adult Type II cells was performed as described previously (15). Cell pellets were divided and sonicated separately for independent phosphorylase and phosphorylase kinase assays when yield permitted.
Cloning and Sequencing of PK␥ Isozyme in Fetal Lung Tissue-The cDNA sequences encoding rat muscle (16) and rat testis (14) PhK␥ subunits were aligned and degenerate PCR primer pairs selected on the basis of conserved regions: forward primer L, 5Ј-CT(CG)TTTGAC-TA(CT)CTCAC(TA)GAG-3Ј, nucleotides (nt) 685-705 (all nucleotide numbering in this study conforms to the reported sequence for PhK-␥T cDNA, GenBank accession number M73808); forward primer N, 5Ј-AACATTGT(ACG)CATCG(GA)GACCTG-3Ј, nt 784 -804, and reverse primer S, 5Ј-TCATCCCACTC(CTG)GGTGA-3Ј, nt 1119 -1102. The LS and NS primer pairs were used to optimize conditions for amplification of PhK␥ using the muscle PhK␥ cDNA (the kind gift of J. S. Chamberlain, Baylor College of Medicine) or the testis PhK␥ cDNA (the kind gift of S. Hanks, Vanderbilt University Medical School) as templates for PCR (Fig. 1). Optimal PCR conditions and degenerate primer pairs were subsequently used to amplify PhK␥ in cDNA pools that were reversed transcribed from day 19 fetal rat lung poly(A) ϩ RNA prepared with the FastTrack RNA isolation kit (Invitrogen, San Diego, CA). PCR products were size-fractionated by agarose gel electrophoresis, cloned into pCRscript SK (Stratagene, La Jolla, CA), and sequenced in both directions by the method of Sanger (17).
Analysis of PhK-␥T RNA Expression-PhK-␥T RNA levels in lung tissue and isolated Type II epithelial cells were assessed by ribonuclease protection assays using the protocol described by the supplier (Ambion, Austin, TX). For generation of antisense probes, the regions encoding nucleotides 1567-1834 (3Ј-untranslated sequence) and 1464 -1643 (3Ј coding sequences) of rat PhK-␥T and the comparable regions of rat muscle PhK␥ were cloned into pCRscript SK. 32 P-Labeled antisense probes were synthesized with T7 or T3 RNA polymerase, gel-purified, and hybridized with 10 g of total RNA, isolated by the method of Chomczynski (18). Following an overnight incubation at 42°C, unhybridized RNA was digested with ribonuclease and the protected fragments analyzed by SDS-PAGE/autoradiography. PhK-␥T RNA levels were quantitated by PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA). Since actin levels varied during development, a probe for rat ribosomal protein L32 (GenBank accession number X06483) was used as an internal control for gel loading and transfer.
Spatial expression of PhK-␥T RNA was assessed by in situ hybridization. Sense and antisense probes were generated as described above, using 35 S-labeled UTP, and hybridized with cryosections of lung tissues as we have previously described (19).
Analysis of PhK␥ Protein Levels-The entire coding sequence of rat PhK-␥T (nt 343-1563) was amplified by PCR, cloned into the bacterial expression vector pET21 (Novagen, Madison, WI), and transfected into Escherichia coli BL21(DE3) cells. Following induction with 0.1 mM isopropyl-␤-D-thiogalactoside for 4 h, recombinant PhK␥ was recovered by extraction of the bacterial pellet with 6 M guanidine in 20 mM Tris (pH 7.9), 500 mM NaCl followed by chromatography on NTA-agarose, as described by the supplier (Quiagen, Chatsworth, CA). Recombinant rat PhK␥ eluted from the NTA column was dialyzed against TBS (20 mM Tris, pH 7.4, 150 mM NaCl) and injected into rabbits to generate polyclonal antisera as described previously (20). Affinity-purified antiserum was prepared by chromatography over recombinant PhK-␥T immobilized on Aminolink resin (Pierce Chemical Co.). The column was eluted with 5 mM glycine (pH 2.3), 150 mM NaCl, 0.1% bovine serum albumin and the affinity purified antiserum immediately neutralized with 0.1 volume of 0.5 M Na 2 HPO 4 .
For Western blot analyses, lung tissues or Type II epithelial cell pellets were homogenized in 10 mM Tris (pH 7.5), 0.25 M sucrose, 1 mM EDTA, 5 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride, and 10 g/ml each of pepstatin A, aprotinin, antipain, leupeptin, and chymostatin followed by centrifugation at 140 ϫ g for 10 min (4°C); a cytosolic fraction was prepared by centrifugation of the supernatant at 100,000 ϫ g for 30 min (4°C). Equal amounts of cytosolic protein, estimated by the method of Lowry (21), were dissolved in electrophoresis sample buffer, subjected to SDS-PAGE followed by electrophoretic transfer to nitrocellulose membrane, and PhK identified by Western blotting (22). The specificity of the immunoreaction was verified by prior incubation of the antiserum with 100 g of recombinant PhK␥ or by immunoblotting with preimmune serum.
Immunocytochemistry was performed on paraffin-embedded lung tissues exactly as described by Vorbroker et al. (23). Tissue sections were incubated with either affinity-purified PhK␥ antisera or antiserum directed against the SP-C proprotein (23). The specificity of the immunoreaction was verified as described above.
Analysis of PhK and Phosphorylase Enzyme Activity-Phosphorylase a and total phosphorylase were measured in the supernatant fraction of lung or cells by the reverse reaction where [ 14 C]glucose 1-phosphate incorporation into glycogen was determined in the absence or presence of 5 mM AMP as described previously (8,24). One unit of activity represents the conversion of 1 mol of glucose 1-phosphate into glycogen per minute. Phosphorylase b kinase activity was determined by incubating cell or tissue extracts with rabbit muscle phosphorylase b, followed by assays for phosphorylase a and total phosphorylase as above. One unit of activity catalyzes the formation of 1 mol of phosphorylase b per minute, assuming a molecular weight for phosphorylase b of 97,400 and that the activity of phosphorylase a in the absence of AMP ϭ 70% of the activity in the presence of AMP (25). Activities are expressed per wet tissue or cell weight.

Identification of PhK␥ in Developing
Lung-The cDNA sequences encoding rat muscle (16) and rat testis (14) PhK were aligned and degenerate PCR primer pairs designed based on the highly conserved catalytic domain encompassing subdomains VIb-X (26). PCR conditions were adjusted such that the predicted 434-bp fragment could be amplified from both the muscle and testis cDNA template with equal efficiency and degenerate primer pairs were subsequently used to amplify cDNA reverse transcribed from day 19 fetal rat lung poly(A) ϩ RNA (Fig. 1A). Fragments of 434 and 509 bp were amplified, cloned, and sequenced. Sequence analyses of cDNA clones from multiple amplification reactions indicated that the 434-bp fragment corresponded exactly to the reported sequence for rat PhK-␥T (14) (Fig. 1B); cDNA clones encoding muscle PhK␥ were not detected and this result was subsequently confirmed by RNase protection assays (see Fig. 2). Sequence analyses of cDNA clones encoding the 509-bp fragment indicated that this fragment also corresponded to rat PhK-␥T but contained a 75-bp insert at position 736. The 75-bp insert did not exhibit significant homology to any sequence in the GenBank data base and was detected in Type II cell RNA isolated from both fetal and adult rat lung (see Fig. 8B). Amplification of rat genomic DNA, with primers flanking the insert, followed by sequence analysis confirmed that this sequence represented an unspliced intron (Fig. 1C). No other forms of PhK␥ were detected with these or a distinct set of degenerate primers (NS primer pair, not shown) suggesting that the testis isozyme was the major or only form of the catalytic subunit expressed in fetal lung tissue.
Expression of PhK-␥T RNA in Developing Lung-PhK␥ RNA levels were assessed in developing lung tissues by RNase protection using radiolabeled probes generated from the unique 3Ј-untranslated regions of rat testis or muscle PhK␥. Consistent with previous reports (13,14) testis PhK␥ was readily detected in adult testis and was expressed at much lower levels in adult skeletal muscle, adult lung, and fetal lung; in contrast, muscle PhK␥ expression was detected in skeletal muscle but not lung or testis (Fig. 2). The level of testis PhK␥ RNA in lung tissue remained relatively constant from day 17 of gestation through the postnatal period and in the adult (Fig. 3) and was similar in isolated fetal (day 20 of gestation) and adult Type II epithelial cells. In situ hybridization with antisense probes indicated that PhK-␥T RNA was expressed predominantly in the epithelium of developing airways on day 17 of gestation (Fig. 4A); however, by day 19 of gestation virtually all cells in the lung expressed PhK-␥T RNA and this expression profile was maintained postnatally (Fig. 4). Overall the results of these studies suggest that the temporal-spatial expression of PhK-␥T RNA did not vary significantly during the perinatal period.
Hybridization with control sense probes resulted in significant signal in the bronchiolar epithelium and blood vessel walls of fetal (not shown) and postnatal lung (Fig. 4F). The specificity of PhK-␥T antisense RNA expression was confirmed by: 1) in situ hybridization with two separate sense probes from the 3Ј-untranslated or 3Ј-coding region of PhK-␥T; 2) by in situ hybridization with irrelevant sense probes (cdk-4 and cdk-1); and 3) by RNase protection assays with labeled PhK-␥T sense probes (Fig. 5). Antisense PhK-␥T RNA was consistently detected in both fetal and adult lung but not in adult testis or skeletal muscle (Fig. 5). These results suggest that PhK-␥T antisense RNA is expressed in lung in a spatially restricted manner.
Expression of PhK-␥T Protein in Developing Lung-The en-   figure) were generated from the 3Ј-untranslated region of the rat testis PhK␥ (PKT) cDNA (see Fig. 1B) or the rat muscle PhK␥ (PKM) cDNA. Labeled probes were hybridized with 10 g of total RNA isolated from adult rat testis (T), muscle (M), lung (AL), or day 19 fetal lung (FL). Following an overnight incubation at 42°C, unhybridized RNA was digested with ribonuclease and the protected fragments were analyzed by SDS-PAGE/autoradiography. A 268-base fragment is protected by PKT in all four tissues, although expression in lung and muscle is very low relative to testis; in contrast, PKM protects RNA only in muscle tissue. tire coding sequence of rat PhK-␥T was expressed in bacteria and the full-length recombinant protein purified by chromatography on NTA-agarose (Fig. 6A). Rabbit polyclonal antiserum generated against recombinant PhK-␥T detected purified muscle PhK (Sigma) by Western blotting (Fig. 6B). Affinity-purified immune serum detected a protein, M r approximately 41,000, in Western blots of cytosol from adult testis or skeletal muscle and immunoreactivity was competed by prior incubation of the antiserum with recombinant PhK-␥T (not shown). Western analyses of fetal, postnatal, and adult lung cytosols indicated that levels of PhK-␥T protein were similar at all time points; similar results were obtained with cytosols prepared from isolated fetal and adult Type II epithelial cells. Expression of PhK-␥T protein was localized by immunocytochemistry to Type II epithelial cells and to the bronchiolar epithelium in rat and mouse lung although lower level expression was detectable throughout the lung (Fig. 7); this pattern of expression was similar in both pre-and postnatal tissues. Surprisingly PhK-␥T protein levels in adult testis were comparable to those in lung despite much higher levels of PhK-␥T RNA in testis (not shown).
Expression of PhK-␥T in gsd/gsd Rats-The glycogen storage disease (gsd/gsd) rat is incapable of mobilizing liver glycogen due to deficiency of phosphorylase kinase activity (for review, see Ref. 7) and Rannels et al. (8) have demonstrated that no net glycogenolysis occurred in the lungs of gsd/gsd fetuses. In order to determine if the testis isoform of PhK␥ plays an important role in regulation of pulmonary glycogen mobilization, expression of PhK-␥T RNA and protein was characterized in the lungs and isolated Type II epithelial cells of gsd/gsd and wild type fetuses. Quantitation of PhK-␥T RNA by RNase protection assays indicated no difference in PhK-␥T RNA levels in Type II epithelial cells isolated from gsd/gsd and control fetuses (Fig. 8A); furthermore, the alternatively spliced form of PhK-␥T was also detected in both fetal and adult gsd/gsd Type II cells (Fig. 8B). In contrast to these results, PhK-␥T protein was virtually undetectable in Western blots of gsd/gsd lungs or isolated fetal and adult Type II epithelial cells (Fig. 8C). This finding was confirmed by immunocytochemistry which demonstrated readily detectable PhK-␥T protein in Type II epithelial cells of control but not gsd/gsd fetuses (not shown).
PhK Enzyme Activity in Developing Lung-PhK enzyme activity in freshly isolated fetal Type II epithelial cells (day 20 of gestation) was reduced 8-fold in gsd/gsd Type II cells (Table I). The decline in gsd/gsd PhK activity was accompanied by a 12-fold reduction in phosphorylase a activity but only a 2-fold decrease in total phosphorylase activity. These results are consistent with a major role for PhK in activation of phosphorylase in the fetal Type II cell. The corresponding decreases in PhK In situ hybridization with sense probes resulted in relatively little signal in day 17 gestation lung (panel E); however, significant signal was observed in the epithelium of some airways in day 21 gestation lung (panel F). Sense signal was not detected with irrelevant sense probes and was also detected with a second sense probe generated from the 3Ј end of the coding sequence of the rat PK␥ cDNA (not shown).

FIG. 5. Expression of antisense PhK-␥T RNA in fetal and adult
lung. 32 P-Labeled sense PK-␥T RNA probes were generated from the 3Ј-untranslated region of the rat PK␥ cDNA and hybridized with 10 g of total RNA isolated from fetal (day 17 and 21 of gestation) rat lung, adult rat lung (L), adult rat testis (T), and adult rat muscle (M). Following overnight incubation at 42°C, unhybridized RNA was digested with RNase and the protected fragments were analyzed by SDS-PAGE/autoradiography. enzyme activity, PhK␥ immunoreactive protein and glycogen degradation in gsd/gsd Type II cells strongly implicate the testis isoform of PhK␥ in the regulation of glycogen degradation during fetal lung development. PhK enzyme activity in isolated adult Type II cells was increased approximately 3-fold over activity in comparable fetal cells (Table I). Despite the fact that immunoreactive PhK␥ protein was virtually undetectable in isolated gsd/gsd adult Type II cells PhK activity was reduced only 1.4-fold relative to wild type cells. These results, coupled with the presence of significant phosphorylase a activity in adult gsd/gsd Type II cells (approximately 7-fold higher than in comparable fetal cells), suggest that a novel form of PhK␥ may be expressed in the adult Type II cell. DISCUSSION 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. PCR conditions were designed to identify both known and closely related novel isoforms of PhK␥. The collective results of PCR, RNase protection, and in situ hybridization studies indicate that the testis isoform was the only detectable form of PhK␥ in fetal lung tissue. Furthermore, PhK-␥T RNA was readily detected in fetal Type II epithelial cells and was present at comparable levels in wild type and gsd/gsd fetuses; however, in contrast to wild type animals, PhK-␥T protein and enzyme activity were significantly decreased in gsd/gsd fetal Type II cells. Given these results, it is likely that the lack of PhK-␥T protein is directly responsible for impaired glycogen mobilization in gsd/ gsd Type II cells during the perinatal period.
It is unclear if the small amount of enzyme activity detected in fetal gsd/gsd Type II cells is due to residual testis PhK isozyme, to contamination of the Type II cell preparation with some other cell types, or to the presence of a minor, novel PhK isozyme. Support for a novel PhK isozyme comes from the unexpected finding that PhK activity is significantly increased in adult Type II cells and is only slightly decreased in adult gsd/gsd Type II cells. PhK␥ in adult Type II cells is immunologically distinct and is sufficiently divergent in nucleic acid sequence from the muscle and testis isoforms such that it is not detected in fetal Type II cells by PCR with degenerate primers. The identity and function of this putative novel PhK isozyme remains unknown but is particularly intriguing given the paucity of glycogen in adult Type II epithelial cells.
Apart from modulation of ␣ and ␤ regulatory subunit expression, PhK-␥T enzyme activity in developing lung may be regulated at several levels. Differential splicing of the PhK-␥T gene gives rise to a least one alternatively spliced RNA transcript which was detected in fetal and adult Type II cells from both wild type and gsd/gsd animals. The alternatively spliced PhK-␥T transcript encodes a 75-bp in-frame insertion which corresponds positionally to the E intron of the rat muscle PhK␥ . SP-C expression is restricted to alveolar Type II cells, whereas expression of PK␥ is detected in both Type II cells as well as bronchiolar epithelial cells.
FIG. 8. Expression of PhK-␥T RNA and protein in Type II epithelial cells isolated from gsd/gsd rats. A, Type II epithelial cells were isolated from adult rat lungs and from wild type or gsd/gsd fetal lungs on days 20 and 21 of gestation. Total RNA was isolated from each group of Type II cells and RNase protection assays performed with PhK-␥T antisense probes, as described in the legends to Figs. 2 and 3. PhK-␥T RNA was detected at similar levels in Type II cells isolated from wild type and gsd/gsd fetuses. B, Type II cells were isolated from gsd/gsd fetuses (lanes 1-3), or from wild type fetuses on day 20 of gestation (lanes 4 and 5) and from adult rat lungs (lane 6). RNA was isolated from each Type II cell preparation and analyzed by reverse transcriptase-PCR, using the LS primer pair, as described in the legend to Fig. 1. Alternatively spliced forms of PhK-␥T RNA were detected in wild type and gsd/gsd fetal Type II cells as well as in adult Type II cells. C, 125 g of protein from lung homogenates (lanes 1-3) and Type II cell lysates (lanes 4 -6) were analyzed by Western blotting as described in the legend to Fig. 6. PhK-␥T protein, M r approximately 41,000, was detected in wild type tissues (lanes 1, 4, and 5) but not in Type II cells or lung tissues from gsd/gsd fetuses (lanes 2, 3, and 6). gene (27). Insertion of 25 amino acids into the catalytic domain of PhK-␥T could conceivably reduce enzyme activity or alter substrate specificity. Differential splicing resulting in deletion, replacement, or insertion of specific sequences has previously been reported for muscle ␣ and ␤ and liver ␣ subunits (28,29) and may play a role in regulating PhK enzyme activity.
Spatial distribution of PhK-␥T may be regulated in part by antisense RNA expression. Antisense transcripts were detected by RNase protection and were localized by in situ hybridization to portions of the bronchiolar epithelium and the blood vessel wall. There was no detectable change in antisense expression in fetal and adult lung; antisense expression was not detected in testis or muscle, consistent with a tissue-specific response. However, it was not clear from these studies if PhK-␥T protein and/or enzyme activity was reduced at the site of antisense expression.
The PhK-␥T RNA transcript contains a number of elements consistent with translational control of expression. First there is an AUG located at position Ϫ82, upstream from the major open reading frame, which is followed by an in-frame STOP codon at position 27-29 within the coding sequence of PhK-␥T. The sequence context of the upstream AUG is similar to the authentic start codon and may therefore be recognized by the ribosome resulting in inhibition of translation (for review, see Ref. 30). Second, an RNA folding algorithim (31) predicts stable secondary structures within the 5Ј leader sequence with the potential to inhibit ribosomal scanning (32). It is possible that alternate sites of PhK-␥T transcription initiation (14) or differential splicing of the primary transcript may lead to loss of inhibitory elements in the leader region resulting in more efficient translation. In this regard, translational control may account for similar levels of PhK-␥T protein in lung and testis despite the fact that PhK-␥T RNA levels are approximately 100-fold higher in testis.
The testis isoform of PhK␥ is the major source of PhK activity in the fetal Type II epithelial cells. The importance of PhK-␥T in glycogen mobilization in the prenatal lung is underscored by parallel changes in PhK-␥T protein, enzyme activity, and glycogen catabolism in gsd/gsd fetal Type II cells. The temporo-spatial regulation of PhK-␥T expression in fetal lung is likely to be very complex and may involve differential splicing, antisense expression, and translational controls in addition to interaction with various regulatory subunits. Despite a clear role for PhK-␥T in fetal lung, the function of this enzyme and the identity of other PhK␥ isozyme(s) in postnatal lung remains unknown.