Division of Perinatal Medicine, Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06510
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ABSTRACT |
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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- (PLC-
) and protein kinase C (PKC) isomers and
of the
-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-
, -
I, -
II, -
, -
, -
, -
, and -µ, PLC-
3, and
Gq
in adult and newborn type II
cells. PKC-
, -
, and -
and PLC-
1, -
2, and -
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-
,
-
I, -
II, and -
in newborn type II cells were only 43-57%
those of adult cells. The level of PKC-
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-
, -
, and -µ, PLC-
3,
and Gq
. 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
,
I,
II, or
PKC isoforms.
P2Y2 purinoceptor; adenosine
receptors; phospholipase C-; adenosine 5'-triphosphate; Gq
; protein kinase C
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INTRODUCTION |
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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:
-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
-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 ,
, and
types
(22). PLC-
, of which there are four isoforms,
1,
2,
3, and
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-
isoform(s) in the type II cell is not known.
Eleven PKC isomers have been identified:
,
I,
II,
,
,
,
,
,
,
, and µ (20). PKC-
, -
, -
, -
, and
-
were identified in type II cells, and the surfactant secretagogue
12-O-tetradecanoylphorbol 13-acetate
(TPA) was reported to activate PKC-
, -
, -
, and -
(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- 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-
isomers and of
Gq
in adult and newborn type II
cells. In addition, we measured expression of adenosine and
-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).
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MATERIALS AND METHODS |
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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,
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
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
1, and
5'-ACCTCCTTCTTGCCTATCC-3' and
5'-TGATGATGCCTAAAGTCTTG-3' for the
2 receptors.
Rabbit polyclonal antibodies against PKC and PLC- isomers and
Gq/11
were obtained from Santa
Cruz Biotechnology (Santa Cruz, CA). The antibodies were directed
against carboxy sequences except for the PKC-
antibody, which was
directed against amino end sequences. The PKC and PLC-
antibodies
were isoform specific. The
Gq/11
antibody was directed
against a sequence common to the
q and
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/11). 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/11
) 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.
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RESULTS |
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Expression of purinoceptor and -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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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
1- and
2-adrenergic receptor genes
were also expressed in lung tissue and type II cells. With the
exception of the
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 ,
I,
II,
,
,
,
, and µ PKC isoforms were all
present in adult lung and type II cells (Figs. 2 and 3). PKC-
was
detected in lung tissue but not in type II cells, whereas PKC-
and
PKC-
were detected in neither (Fig. 4). The PKC-
, -
II, -
,
-
, and -
isoforms in lung and type II cells were present as
single bands that corresponded to those in brain. The PKC-
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
I,
,
,
and µ blots. The upper
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-
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-
, 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-
I (4), PKC-
(4, 26), and PKC-
(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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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) ,
I,
II, and
PKCs in newborn than in adult type II cells (Table
1). On the other hand the levels of
,
, and µ in adult and newborn cells were not significantly
different (12-24% less in newborn cells). Although the level of
PKC-
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-
.
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The PLC- isomers detected in lung tissue and type II cells are shown
in Fig. 5. PLC-
3 was present in both
lung tissue and type II cells. PLC-
1 was present in the lung but not
in type II cells, and PLC-
2 and -
4 were not present in either.
There was no difference between adult and newborn type II cells in the amounts of PLC-
3.
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The Gq/11-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).
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DISCUSSION |
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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 ,
I,
II, and
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-
3 and the
-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- 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-, -
, -
, and -
in adult rat type II cells (17).
The current finding that the amounts of PKC-
, -
I, and -
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-
and -
, which are also activated by TPA, are
the same in newborn and adult cells (Fig. 2), whereas there is less
PKC-
, 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-
and possibly PKC-
may be particularly important in
the P2Y2 signaling pathway.
As in the present study, Linke et al. (17) reported the presence of
PKC-, -
, -
, -
, and -
as well as expression of their genes in adult rat type II cells. They also failed to detect PKC-
and -
by immunoblotting, although the PKC-
gene was expressed in
their study. Whereas we observed low levels of PKC-
in adult type II
cells, Linke et al. did not detect its expression by either RT-PCR or
immunoblotting. PKC-µ and PKC-
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-
,
which we detected in lung tissue but not in type II cells. With the
exception of PKC-
and -
, the pattern of PKC isomers that we found
in rat lung tissue was the same as that recently reported in human lung
(34). PKC-
and -
were not detected by immunoblotting in human
lung, although the PKC-
gene was expressed as determined by RT-PCR
(34). However, PKC-
and -
have previously been reported in animal
lungs. PKC-
was reported in rat lung tissue (34) and in rat, canine,
and bovine trachealis (4, 34). PKC-
was reported in canine (4) and
bovine (34) trachealis, and a low level of expression of the PKC-
gene was reported in mouse lung (21).
There are no previous reports on the identity of the PLC- isomers in
the type II cell. Our data show that PLC-
1 and PLC-
3 are present
in whole lung, whereas type II cells contain only PLC-
3. The absence
of PLC-
2 and PLC-
4 in the lung is in agreement with the
restricted expression of these isomers to hematopoietic cells and
retina, respectively (6). Because
3 was the only PLC-
isomer in
type II cells, it must be the one involved in the
P2Y2 signal-transduction pathway.
As in many other systems (6), PLC-3 is probably activated by
-subunits of Gq in the type II
cell. The
-subunits of Gi
have also been shown to activate PLC-
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-
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
1- and
2-adrenergic receptor genes are
expressed in lung tissue and type II cells. In the type II cell,
expression of the
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
-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
-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.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-31175.
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FOOTNOTES |
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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.
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