PKC isoforms and other signaling proteins involved in surfactant secretion in developing rat type II cells

Laurice I. Gobran, Zhi-Xin Xu, and Seamus A. Rooney

Division of Perinatal Medicine, Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06510

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We previously reported that there is a developmental increase in surfactant secretion in response to P2Y2 purinoceptor agonists. UTP does not stimulate secretion in type II cells from 1- or 2-day-old rats; there is a small response to UTP in cells from 4-day-old animals, and the response increases with increasing age thereafter. Second messenger formation in response to P2Y2 agonists has a similar developmental pattern. We have investigated whether the failure to respond to P2Y2 agonists is due to a deficiency in the P2Y2 receptor or in downstream signaling factors. We compared type II cells from adult and 1- to 2-day-old rats with respect to expression of the P2Y2 receptor gene and the levels of phospholipase C-beta (PLC-beta ) and protein kinase C (PKC) isomers and of the alpha -subunit of the GTP-binding protein Gq. We measured gene expression by reverse transcriptase-polymerase chain reaction and protein levels by immunoblotting. We identified PKC-alpha , -beta I, -beta II, -delta , -eta , -zeta , -theta , and -µ, PLC-beta 3, and Gqalpha in adult and newborn type II cells. PKC-epsilon , -gamma , and -lambda and PLC-beta 1, -beta 2, and -beta 4 were not present in adult or newborn type II cells. Expression of the P2Y2 receptor gene was essentially the same in newborn and adult cells. However, the levels of PKC-alpha , -beta I, -beta II, and -zeta in newborn type II cells were only 43-57% those of adult cells. The level of PKC-theta also tended to be lower in the newborn cells. There was little difference between newborn and adult type II cells in the levels of PKC-delta , -eta , and -µ, PLC-beta 3, and Gqalpha . These data suggest that the lack of response of early newborn type II cells to P2Y2 agonists is not due to a lack of expression of the receptor gene but possibly to insufficient amounts of one or more of the alpha , beta I, beta II, or zeta PKC isoforms.

P2Y2 purinoceptor; adenosine receptors; phospholipase C-beta ; adenosine 5'-triphosphate; Gqalpha ; protein kinase C

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

SURFACTANT PHOSPHOLIPID secretion in isolated type II cells can be stimulated by a variety of agonists acting via at least three signal-transduction mechanisms (24). Although the physiological regulation of surfactant secretion is not well understood, at least three in vitro secretagogues are potential regulators in vivo: beta -adrenergic agonists, adenosine, and ATP. ATP is one of the most effective surfactant secretagogues in vitro and increases phosphatidylcholine secretion as much as five- to sixfold in isolated type II cells. ATP acts via both a P2Y2 purinoceptor on the type II cell (10, 23) and an unidentified receptor that is coupled to adenylate cyclase activation (9). UTP, another P2Y2 receptor agonist (13), also stimulates surfactant secretion in type II cells, but it does not increase cAMP formation (10). The effect of UTP is therefore mediated entirely by the P2Y2 receptor.

There is a developmental increase in the response of type II cells to surfactant secretagogues (9, 12). The response of type II cells isolated from fetal and early newborn rats to several surfactant secretagogues is considerably less than that of cells isolated from adults (12). In particular, UTP does not stimulate surfactant secretion in type II cells from 1- or 2-day-old rats, whereas ATP does, albeit to a lesser extent than in adult cells (9). However, ATP does stimulate cAMP formation in both adult and newborn type II cells (9). The lack of response to UTP and the diminished response to ATP is therefore likely due to a developmental delay in expression of one or more components of the P2Y2 signaling mechanism.

The P2Y2 purinoceptor is generally coupled to phosphoinositide-specific phospholipase C (PLC) via alpha -subunits of the GTP-binding protein (G protein) Gq (13). PLC activation leads to the formation of inositol trisphosphate and diacylglycerols (20). Diacylglycerols activate protein kinase C (PKC), and it in turn phosphorylates a protein or proteins. Protein phosphorylation is believed to initiate surfactant secretion. PLC and PKC are therefore key components in the signaling mechanism mediating surfactant secretion in response to P2Y2 agonists.

Ten mammalian PLC isoforms have been identified to date (22). Based on amino acid sequence, they are divided into beta , gamma , and delta  types (22). PLC-beta , of which there are four isoforms, beta 1, beta 2, beta 3, and beta 4, is the only one activated by G protein-coupled receptors (5, 22) and is therefore likely the type involved in surfactant secretion. The identity of the PLC-beta isoform(s) in the type II cell is not known. Eleven PKC isomers have been identified: alpha , beta I, beta II, gamma , delta , epsilon , lambda , eta , theta , zeta , and µ (20). PKC-alpha , -beta , -delta , -eta , and -zeta were identified in type II cells, and the surfactant secretagogue 12-O-tetradecanoylphorbol 13-acetate (TPA) was reported to activate PKC-alpha , -beta , -delta , and -eta (17). The PKC isomer(s) activated by P2Y2 agonists in the type II cell is not known.

The goals of this study were to identify the PLC-beta and PKC isomers in adult and newborn type II cells and to determine if a deficiency in the amounts of the P2Y2 receptor or downstream signaling factors could account for the lack of response to P2Y2 agonists in type II cells from newborn rats. We therefore measured expression of the P2Y2 receptor gene and the amounts of PKC and PLC-beta isomers and of Gqalpha in adult and newborn type II cells. In addition, we measured expression of adenosine and beta -adrenergic receptors, as activation of those receptors also stimulates surfactant secretion (24) and the response to such agonists is considerably less in newborn than in adult type II cells (12).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Chemicals and reagents. Rat IgG was obtained from Accurate (Westbury, NY); amplification grade DNase I, SuperScript II reverse transcriptase (RT), and TRIzol reagent were from GIBCO-BRL (Grand Island, NY); Taq polymerase was from Boehringer Mannheim (Indianapolis, IN); deoxynucleotide triphosphates (dNTPs) were from New England BioLabs (Beverly, MA); SeaKem LE and NuSieve GTG agarose were from FMC Bioproducts (Rockland, ME); horseradish peroxidase-conjugated goat anti-rabbit IgG was from Sigma (St. Louis, MO); and Western Blot Chemiluminescence Reagent was from Du Pont-New England Nuclear (Boston, MA). The surfactant protein C (SP-C) cDNA (31) and other reagents for molecular biology (35) and isolation and culture of type II cells (9) were obtained from previously indicated sources.

Oligonucleotides were synthesized by the DNA Synthesis Laboratory at Yale Medical School or obtained from GIBCO-BRL. Polymerase chain reaction (PCR) primers for amplification of the P2Y2, beta 2-adrenergic, and adenosine A3 receptor mRNAs were based on GenBank sequences. SP-C (33), the adenosine A1, A2A, and A2B receptor (3), and the beta 1-adrenergic receptor (19) primers were as published. Forward and reverse primer sequences were 5'-ATCGTGGTTGTGGTGGTAGTCC-3' and 5'-CCCAGAAGAATCAGAATCGG-3' for SP-C and 5'-TCTACATCTTCCTGTGCC-3' and 5'-GTAGAAAAGGAAACGCAC-3' for the P2Y2, 5'-CTCCATTCTGGCTCTGCTCG-3' and 5'-ACACTGCCGTTGGCTCTCC-3' for the A1, 5'-CCATGCTGGGCTGGAACA-3' and 5'-GAAGGGGCAGTAACACGAACG-3' for the A2A, 5'-TGGCGCTGGAGCTGGTTA-3' and 5'-GCAAAGGGGATGGCGAAG-3' for the A2B, 5'-GTCCTTTCTGGTGGGACTGA-3' and 5'-AGGGTTCATCATGGAGTTCG-3' for the A3, 5'-CGCTCACCAACCTCTTCATCATGTCC-3' and 5'-CAGCACTTGGGGTCGTTGTAGCAGC-3' for the beta 1, and 5'-ACCTCCTTCTTGCCTATCC-3' and 5'-TGATGATGCCTAAAGTCTTG-3' for the beta 2 receptors.

Rabbit polyclonal antibodies against PKC and PLC-beta isomers and Gq/11alpha were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies were directed against carboxy sequences except for the PKC-lambda antibody, which was directed against amino end sequences. The PKC and PLC-beta antibodies were isoform specific. The Gq/11alpha antibody was directed against a sequence common to the alpha q and alpha 11 subunits of Gq.

Animals. Sprague-Dawley rats were used. Timed pregnant dams were obtained from Camm (Wayne, NJ) and allowed to deliver naturally. Newborns remained with the mothers until killed 1-14 days later. Adult and 30-day-old male rats were obtained from Charles River (Kingston, NY). Based on age versus weight charts supplied by the vendor, the adults were ~60 days old. We previously reported that there was no sex difference in the response of rat type II cells to surfactant secretagogues (12).

Cell isolation and culture. Type II cells were isolated from newborn and adult rats as described previously (12). This method involves digestion of the blood-free lungs with elastase and separation of type II cells from contaminating cells by panning on bacteriological dishes coated with IgG. The freshly isolated cells were cultured on plastic dishes in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and antibiotics for 18-20 h at 37°C as previously described (9). We previously reported that >88% of the attached cells were identifiable as type II pneumocytes after the overnight culture (12). All newborns from one to two litters or three to four adult rats were used for each preparation of type II cells.

Human leukemia (HL-60) cells were cultured in RPMI 1640 medium (GIBCO-BRL) containing 10% fetal bovine serum.

mRNA analysis. mRNA content was assessed by RT-PCR. RNA was extracted from lung tissue and type II cells with TRIzol reagent as described previously (31, 35). Total RNA was treated with DNase I for 15 min at room temperature, mRNAs were annealed with oligo(dT)15 at 70°C for 10 min, and cDNAs were synthesized by incubation with RT at 42°C for 1 h.

In each experiment, all PCR incubations were carried out simultaneously in separate tubes and with equal aliquots of the same cDNA preparation. The reaction mixture (20 µl) contained KCl (50 mM), MgCl2 (1.5 mM), the four dNTPs (0.2 mM each), primers (0.5 µM each), Taq polymerase (0.5 U), and the cDNA preparation (2 µl) in 10 mM Tris · HCl (pH 8.3). The samples were overlaid with mineral oil and incubated in a thermal cycler using Touchdown conditions (MJ Research, Watertown, MA). The incubation conditions were 1 min at 94°C, 20 cycles of 92°C for 30 s followed by 40 s with temperature reduction from 70 to 60°C at 0.5°C per cycle, 30 cycles of 92°C for 30 s followed by 60°C with increasing time of incubation from 40 to 70 s at 1 s per cycle, and a final 5 min at 72°C. PCR products were subjected to electrophoresis on agarose gels (1% SeaKem and 1% NuSieve) in 40 mM Tris acetate containing 1 mM EDTA and visualized by ethidium bromide staining. The gels were photographed with Polaroid 55 film and quantitated by scanning densitometry of the negatives (Personal Densitometer SI and Image QuaNT software; Molecular Dynamics, Sunnyvale, CA). The amounts of the individual receptor products were normalized to that of SP-C.

The SP-C mRNA content of developing postnatal lung was measured by Northern analysis and normalized to 28S rRNA as described previously (31).

Western blotting. Type II and HL-60 cell monolayers were washed three times with DMEM at 37°C, ice-cold 10 mM HEPES buffer (pH 7.4) containing NaCl (140 mM), KCl (5 mM), Na2HPO4 (2.5 mM), and glucose (6 mM) was added, and the cells were removed with a rubber policeman and centrifuged at 200 g for 10 min. The pellet was suspended in 20 mM Tris · HCl buffer (pH 7.5) containing EDTA, EGTA, dithiothreitol, and phenylmethylsulfonyl fluoride (1 mM each) and aprotinin, pepstatin, and leupeptin (1 µg/ml each). The cells were disrupted by sonication and centrifuged at 800 g for 10 min. Rat tissues (lung, brain, and hindleg muscle) were homogenized in the same buffer with a Potter-Elvehjem homogenizer, filtered through cheese cloth, and centrifuged at 800 g for 10 min. The cell-free supernates were used for Western analysis.

Proteins (5-30 µg) were separated by SDS-PAGE under denaturing conditions (16) on Tris-glycine Ready Gels (Bio-Rad, Hercules, CA). Gels consisting of a 7.5% resolving gel with a 4% stacking gel overlay were used (10% for Gq/11alpha ). Resolved proteins were transferred to polyvinylidene difluoride membranes (0.2 µm) as previously described (36). The membranes were incubated in methanol for 20 min, washed with 20 mM Tris buffer containing 0.05% Tween 20 and 150 mM NaCl (TTBS), and incubated for 2 h in TTBS containing 5% nonfat dry milk. The membranes were then sequentially incubated in TTBS containing 4% nonfat dry milk with the primary antibody (1:100 dilution for PKCs and 1:200 to 1:500 dilution for PLCs and Gq/11alpha ) for 1 h, peroxidase-conjugated goat anti-rabbit IgG (1:15,000 dilution) for 1 h, and the chemiluminescence reagent for 1 min. The blots were exposed to X-ray film (Reflection; DuPont), and the autoradiographs were quantitated by scanning densitometry as described for the DNAs. The type II cell values were normalized to those of adult rat lung. Each protein was analyzed on two to six Western blots with two to five different exposures of each, and the data were meaned to yield a single value. The blots were stripped (using a protocol described by DuPont for the Western Blot Chemiluminescence Reagent) and re-probed with a different antibody. Blots were probed with four to six antibodies. Data from fresh and stripped blots were the same.

Other. Protein was measured by a Coomassie blue binding method using an assay kit supplied by Bio-Rad.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Expression of purinoceptor and beta -adrenergic receptor genes in lung tissue and type II cells is shown in Fig. 1. SP-C was included to normalize the other PCR products. SP-C expression is confined to the type II cell (2), and the levels of SP-C in fetal rat lung were reported to have reached those of adults by 21 days gestation (27). We confirmed by Northern analysis that there were no significant differences between 1- to 30-day-old newborn and adult rat lungs in SP-C mRNA content. SP-C mRNA levels were 2,217 ± 328, 1,785 ± 326, 2,048 ± 211, and 2,286 ± 383 (densitometric units, SP-C/28S rRNA) in 1-day-old, 2-day-old, 30-day-old, and adult animals, respectively (means ± SE; 3 groups of 1-5 animals at each age).


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Fig. 1.   RT-PCR analysis of surfactant protein C (SP-C), beta -adrenergic receptor, P2Y2 purinoceptor, and adenosine receptor mRNAs in adult rat lung tissue and in type II cells from 1-day-old and adult rats. RT-PCR was carried out on total RNA, and the products were subjected to electrophoresis on agarose gels and visualized by staining with ethidium bromide. The SP-C and the adenosine A2B, beta 1-adrenergic, P2Y2, beta 2-adrenergic, adenosine A1, adenosine A2A, and adenosine A3 receptor product sizes were 404, 160, 376, 179, 346, 207, 150, and 427 bp, respectively. The data are from 1 of 2 experiments with similar results.

RT-PCR established that the P2Y2 receptor gene is expressed in both lung tissue and type cells, as previously shown by Northern analysis (10), and that its expression is no greater in adult than in newborn type II cells (Fig. 1). The adenosine A1, A2A, A2B, A3 and the beta 1- and beta 2-adrenergic receptor genes were also expressed in lung tissue and type II cells. With the exception of the beta 1 gene, which was somewhat lower in the newborn cells, there was no discernible difference between adult and newborn type II cells in the expression of those receptor genes either by visual inspection (Fig. 1) or by densitometry (data not shown). These data showed that the developmental delay in the response to UTP is not due to lack of expression of the P2Y2 receptor gene. We therefore measured the amounts of downstream signaling proteins.

The PKC isoforms detected in rat lung tissue and isolated type II cells are shown in Figs. 2-4. The alpha , beta I, beta II, delta , eta , zeta , theta , and µ PKC isoforms were all present in adult lung and type II cells (Figs. 2 and 3). PKC-epsilon was detected in lung tissue but not in type II cells, whereas PKC-gamma and PKC-lambda were detected in neither (Fig. 4). The PKC-alpha , -beta II, -delta , -theta , and -epsilon isoforms in lung and type II cells were present as single bands that corresponded to those in brain. The PKC-theta band in the lung samples corresponded to that in muscle, and the same band was also present in brain. Two bands were identified in the beta I, eta , zeta , and µ blots. The upper beta I band was predominant in brain, the lower band was predominant in the lung, and both were equally expressed in adult type II cells. The two PKC-zeta bands were present in equal amounts in brain, whereas the lower band was predominant in the lung and type II cells. Both bands of PKC-eta , which is considered relatively lung specific and is not expressed in brain (1, 14), were present in lung tissue and type II cells but not in brain. The upper PKC-µ band was predominant in brain and the lower in all lung samples. Two bands were also reported in PKC-beta I (4), PKC-zeta (4, 26), and PKC-eta (4, 17) immunoblots in other studies. Multiple PKC bands may be due to differences in phosphorylation states (14, 29). All PKC bands, with the exception of PKC-µ, were in the 77-90 kDa range, in agreement with other studies (4, 28, 29). PKC-µ had a molecular mass of 110 kDa, similar to the 115 kDa reported in other studies (18, 30).


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Fig. 2.   The alpha , beta I, zeta , theta , and µ isoforms of protein kinase C (PKC) in adult rat lung tissue and in type II cells from 1- to 2-day-old and adult rats. Aliquots (30 µg protein except 5 µg brain protein in the alpha , beta I, and zeta  blots) of lung, brain, and muscle homogenates and type II cell sonicates were fractionated on SDS-PAGE gels, transferred to polyvinylidene difluoride membranes, and exposed to PKC polyclonal antibodies. The type II cell samples are from different preparations.


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Fig. 3.   The beta II, delta , and eta  isoforms of PKC in adult rat lung tissue and in type II cells from 1- to 2-day-old and adult rats. Aliquots of lung homogenates and type II cell sonicates (30 µg protein) and brain homogenate (5 µg protein) were analyzed by Western analysis as in Fig. 2.


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Fig. 4.   The epsilon , gamma , and lambda  isoforms of PKC in adult rat lung tissue and in type II cells from 1- to 2-day-old and adult rats. Aliquots (30 µg protein unless otherwise indicated) of lung and brain homogenates and type II cell sonicates were analyzed by Western analysis as in Fig. 2. Two different adult and newborn type II samples were analyzed. Brain samples in lane on left contained 5 µg protein, and those in lane on right contained either 1 µg (epsilon  and gamma  blots) or 30 µg (lambda  blot).

The Western blots showed that some PKC isoforms were expressed to a lesser extent in type II cells from newborn rats than in those from adults (Figs. 2 and 3). Densitometry revealed that there was significantly less (43-57% less) alpha , beta I, beta II, and zeta  PKCs in newborn than in adult type II cells (Table 1). On the other hand the levels of delta , eta , and µ in adult and newborn cells were not significantly different (12-24% less in newborn cells). Although the level of PKC-theta also tended to be lower in newborn than in adult cells (Fig. 2), those blots were not quantitated because of the low content of PKC-theta .

                              
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Table 1.   Quantitation of PKC isomers in type II cells from adult and newborn rat lungs

The PLC-beta isomers detected in lung tissue and type II cells are shown in Fig. 5. PLC-beta 3 was present in both lung tissue and type II cells. PLC-beta 1 was present in the lung but not in type II cells, and PLC-beta 2 and -beta 4 were not present in either. There was no difference between adult and newborn type II cells in the amounts of PLC-beta 3.


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Fig. 5.   Phospholipase C-beta (PLC-beta ) isomers and Gq/11alpha in adult rat lung tissue and in type II cells from 1- to 2-day-old and adult rats. Aliquots of lung and brain homogenates and type II and HL-60 cell sonicates were analyzed by Western analysis as in Fig. 2. PLC-beta isomers were 130-150 kDa, and Gq/11alpha was 44 kDa in size. A: all lanes contained 30 µg protein except brain, which had 5 µg. Lane 1, brain; lanes 2-4, adult type II cells; lane 5, adult lung; lanes 6 and 7, newborn type II cells. B: all lanes contained 20 µg protein. Lanes 1 and 8, HL-60 cells; lanes 2-4, adult type II cells; lane 5, adult lung; lanes 6 and 7, newborn type II cells. C: all lanes contained 22 µg protein. Lanes 1 and 7, brain; lanes 2 and 3, adult type II cells; lane 4, adult lung; lanes 5 and 6, newborn type II cells.

The Gq/11alpha -subunit was present in lung tissue and type II cells, and there was little difference between adult and newborn type II cells in its level of expression (Fig. 5).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We previously reported that there is a developmental increase in surfactant secretion in type II cells in response to P2Y2 purinoceptor agonists (9). UTP does not stimulate secretion in type II cells from 1- or 2-day-old rats. There is a small response to UTP in cells from 4-day-old animals, and the response increases with increasing age thereafter. Second messenger formation in response to UTP and ATP has a similar developmental pattern (9). The failure of P2Y2 agonists to stimulate secretion in newborn type II cells (9) is clearly not due to insufficient expression of the receptor gene, as the current data show similar P2Y2 receptor mRNA levels in type II cells from both 1-day-old and adult rats. However, because we did not measure actual receptor levels, we cannot eliminate the possibility that there is a developmental delay in message translation or in posttranslational modification of the protein. In contrast to receptor expression, there is a significant difference between newborn and adult cells in the amounts of P2Y2 signaling proteins. Levels of the alpha , beta I, beta II, and zeta  isomers of PKC in the newborn type II cells are approximately one-half those in the adult. There is no difference between newborn and adult cells in the levels of two other signaling proteins, PLC-beta 3 and the alpha -subunit of Gq. The deficiency in PKCs is, therefore, likely to be at least in part responsible for the lack of response of newborn type II cells to P2Y2 agonists.

It is possible that there is also a developmental delay in expression of other components of the P2Y2 signaling pathway. Phospholipase D (PLD) activity in type II cells is activated by ATP, UTP, and other surfactant secretagogues that activate PKC (25). Activation of PLD results in formation of larger amounts of diacylglycerols than are generated by PLC-beta and thereby sustains PKC activation and surfactant secretion (24). A number of PLD isozymes are known to exist (6), but reagents for measurement of PLD levels and gene expression are currently not available. Insufficient expression of PLD could contribute to the developmental delay in the response of newborn type II cells to P2Y2 agonists. The delayed response could also be due to a deficiency in the protein(s) phosphorylated by PKC. It is less likely that other, distal, signaling steps (24) are responsible as they are probably common to many signaling pathways, and such pathways are activated by surfactant secretagogues in type II cells from early newborn rats although in some instances to a lesser extent than in adult cells (12).

TPA, a well-established activator of PKC, stimulates surfactant secretion in type II cells (24). Similar to ATP and UTP, there is a developmental increase in surfactant secretion and PLD activation in response to TPA (9). Unlike UTP, however, TPA stimulates secretion in 1- and 2-day-old rat type II cells although its effect is significantly less in newborn than in adult cells (9). TPA has been reported to activate PKC-alpha , -beta , -delta , and -eta in adult rat type II cells (17). The current finding that the amounts of PKC-alpha , -beta I, and -beta II are significantly less in newborn than in adult type II cells may account for the developmental delay in the response to TPA. On the other hand, the amounts of PKC-delta and -eta , which are also activated by TPA, are the same in newborn and adult cells (Fig. 2), whereas there is less PKC-zeta , an isomer not activated by TPA (17), in the newborn cells (Figs. 2 and 3). It is therefore likely that TPA and P2Y2 agonists activate a different set of PKC isomers, and that may account for the difference between TPA and UTP in stimulating surfactant secretion in early newborn type II cells. PKC-zeta and possibly PKC-theta may be particularly important in the P2Y2 signaling pathway.

As in the present study, Linke et al. (17) reported the presence of PKC-alpha , -beta , -delta , -eta , and -zeta as well as expression of their genes in adult rat type II cells. They also failed to detect PKC-epsilon and -gamma by immunoblotting, although the PKC-epsilon gene was expressed in their study. Whereas we observed low levels of PKC-theta in adult type II cells, Linke et al. did not detect its expression by either RT-PCR or immunoblotting. PKC-µ and PKC-lambda were not examined in the study of Linke et al. We found that the profile of PKC isomers was the same in both type II cells and lung tissue, with the exception of PKC-epsilon , which we detected in lung tissue but not in type II cells. With the exception of PKC-delta and -theta , the pattern of PKC isomers that we found in rat lung tissue was the same as that recently reported in human lung (34). PKC-delta and -theta were not detected by immunoblotting in human lung, although the PKC-delta gene was expressed as determined by RT-PCR (34). However, PKC-delta and -theta have previously been reported in animal lungs. PKC-delta was reported in rat lung tissue (34) and in rat, canine, and bovine trachealis (4, 34). PKC-theta was reported in canine (4) and bovine (34) trachealis, and a low level of expression of the PKC-theta gene was reported in mouse lung (21).

There are no previous reports on the identity of the PLC-beta isomers in the type II cell. Our data show that PLC-beta 1 and PLC-beta 3 are present in whole lung, whereas type II cells contain only PLC-beta 3. The absence of PLC-beta 2 and PLC-beta 4 in the lung is in agreement with the restricted expression of these isomers to hematopoietic cells and retina, respectively (6). Because beta 3 was the only PLC-beta isomer in type II cells, it must be the one involved in the P2Y2 signal-transduction pathway.

As in many other systems (6), PLC-beta 3 is probably activated by alpha -subunits of Gq in the type II cell. The beta gamma -subunits of Gi have also been shown to activate PLC-beta s in a pertussis toxin-sensitive manner (6). However, as the response of type II cells to ATP is inhibited only 22-40% by pertussis toxin (11, 25, 32), it is less likely that this is the mechanism of PLC-beta 3 activation in the type II cell.

All four adenosine receptor genes are expressed in the type II cell (Fig. 1). There is considerable functional evidence that the stimulatory effects of adenosine and adenosine analogs on surfactant secretion are mediated by the A2B receptor (24), and there is evidence that activation of the A1 receptor results in inhibition of surfactant secretion (8). Functions, if any, of the A2A and A3 receptors in the type II cell remain to be determined. We found that both beta 1- and beta 2-adrenergic receptor genes are expressed in lung tissue and type II cells. In the type II cell, expression of the beta 2 gene is predominant, in agreement with previous pharmacological (7) and molecular (15) data. Although there is less surfactant secretion in response to adenosine and beta -receptor agonists in type II cells from 1-day-old rats than in those from adults (12), expression of the genes is essentially the same at both ages. Therefore, it is likely that the developmental increase in the response to these agonists is also due to a deficiency in signaling factors distal to receptor activation.

In summary, we have shown that the lack of response of type II cells from early newborn rats to P2Y2 purinoceptor agonists and the diminished response to adenosine and beta -adrenergic receptor agonists is not due to insufficient expression of the receptor genes. The lack of response to P2Y2 agonists may be due, at least in part, to a developmental delay in expression of selected PKC isomers.

    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-31175.

    FOOTNOTES

Address for reprint requests: S. A. Rooney, Dept. of Pediatrics, Yale Univ. School of Medicine, PO Box 208064, New Haven, CT 06520-8064.

Received 30 October 1997; accepted in final form 19 February 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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