1 School of Optometry, Indiana University, Bloomington, Indiana 47405; and 2 Department of Pharmacology, Weill Medical College of Cornell University, New York, New York 10021
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ABSTRACT |
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cAMP-dependent activation of the
cystic fibrosis transmembrane conductance regulator (CFTR) regulates
fluid transport in many tissues. Secretion by the corneal endothelium
is stimulated by cAMP and dependent on HCO permeability by 78% relative to
HCO
permeability was reduced 60% by 20 mM NaHSO3 (a weak
agonist of sAC). NaHSO3 alone increased apical
Cl
permeability by only 13%. The
HCO
permeability was reduced 57% in the presence of 50 µM Rp-adenosine 3',5'-cyclic monophosphorothioate, and 86% by 50 µM
5-nitro-2-(3-phenylpropyl-amino)benzoic acid but unaffected by 200 µM
apical H2DIDS. CFTR phosphorylation was increased 23, 150, and 32% by 20 mM HSO
permeability by 5 µM genistein was increased
synergistically by HCO
chloride transport; cAMP; CFTR phosphorylation
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INTRODUCTION |
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THE CORNEAL ENDOTHELIUM
MAINTAINS the hydration and optical transparency of the
cornea by continuously secreting fluid in opposition to a leak driven
by tissue swelling pressure generated by stromal glycosaminoglycans.
Endothelial fluid secretion is dependent on the presence of
HCO
(34, 47) and is slowed in the presence of carbonic
anhydrase inhibitors (18, 19, 27). Tracer flux studies
have shown that net basal to apical HCO
(21) transport can
contribute to the small (
0.5 mV) apical-side negative transepithelial
potential; however, the relative contribution of each anion is unclear.
HCO are taken up by corneal
endothelial cells via basolateral
Na+-2HCO
(NKCC1; Refs. 13, 23) cotransporters,
respectively. Both intracellular Cl
concentration
([Cl
], 40 mM; Ref. 6) and
[HCO
and HCO
One possible source of basal AC activity is the
HCO
In this study, we show that sAC is expressed in corneal endothelium and
that the presence of HCO
permeability and increase phosphorylation of CFTR. These findings indicate that, in addition to being a component of net anion transport, HCO
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MATERIALS AND METHODS |
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Cells.
Primary cultures of bovine corneal endothelial cells (BCEC) obtained
from fresh cow eyes and CHO cells were grown in DMEM (44 mM
HCO
sAC mRNA expression. A pair of sAC primers was constructed on the basis of the published cDNA sequence (10). The sAC sense primer was 5'-CCTGGAATAACCTGTTCAAG-3', and the sAC antisense primer was 5'-TCTGGTCCTTGAGCCACAG-3'. The expected length of PCR products for sAC was 544 bp.
Total RNA was extracted from cultured BCEC with TRIzol reagent (Invitrogen). The RNA was DNase treated with an RNase-free DNase set (Qiagen). Reverse transcription was performed with the Superscript cDNA synthesis system (Invitrogen) and oligo(dT) primers as previously described (43, 44). PCR amplifications used the High Fidelity TaKaRa Ex Taq PCR System kit (TaKaRa Shuzo) with denaturation at 94°C for 3 min for 1 cycle, 35 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 45 s, and a final extension for 1 cycle at 72°C for 10 min. The PCR products were loaded onto 1% agarose gels, electrophoresed, and stained with 0.5 µg/ml ethidium bromide. PCR products were purified with a 1% low-melting-point agarose gel, inserted into pCR 4-TOPO vector (Invitrogen, San Diego, CA), and sequenced as previously described (43, 44).Indirect immunofluorescence.
Confluent cultured BCEC on coverslips were fixed and immunostained with
rabbit -middle polyclonal sAC antisera (1:100 dilution; Ref.
50) and secondary antibody conjugated to Oregon green
(1:500; Molecular Probes), as previously described (42,
43). Fluorescence was observed with a standard epifluorescence
microscope equipped with a cooled charge-coupled device (CCD) camera.
Negative controls (no primary antibody) were included in all experiments.
Intracellular cAMP assay.
Culture medium was removed from confluent BCEC and replaced with
HEPES-buffered, air-equilibrated, HCO,
-methyleneadenosine
5'-diphosphate (AMP-CP, an ectonucleotidase inhibitor, to reduce
A2B receptor stimulation and thus background [cAMP]).
Parallel controls were performed in HEPES-buffered DMEM (0 HCO
CFTR phosphorylation.
Confluent cultured BCEC were incubated in HCO
Apical Cl permeability.
Relative changes in apical Cl
permeability were assessed
with the halide-sensitive fluorescent dye
6-methoxy-N-ethylquinolinium iodide (MEQ). Confluent BCEC,
grown on Anodiscs, were loaded with MEQ by exposure to diH-MEQ for 10 min (42). Anodiscs were placed in a double-sided
microscope perfusion chamber, and apical and basolateral compartments
were independently perfused at 37°C. MEQ fluorescence (excitation:
365 ± 10 nm; emission: 420-450 nm) was measured as
previously described (42). Anodiscs were initially perfused with a Cl
- and HCO
, and 5 glucose, pH 7.5), and the apical side was briefly pulsed with
Cl
-rich Ringer solution (equimolar replacement of 118 NaNO3 with NaCl). HCO
-free Ringer solution was then introduced (prepared by
equimolar substitution of 28.5 NaNO3 with
NaHCO3; gassed with 5% CO2, pH 7.5). The
apical side was then pulsed with Cl
-rich,
HCO
permeability between control and experimental
conditions in the same cells were determined by comparing the percent
change in MEQ fluorescence (F/F0) after addition of
Cl
, where F0 is the fluorescence in the
absence of Cl
. The maximum slope of fluorescence change
was determined by calculating the first derivative with Felix software (PTI).
Intracellular pH. Intracellular pH (pHi) was measured with the pH-sensitive fluorescent dye BCECF as previously described (2, 5, 43).
Statistics. All data are expressed as means ± SE, and Student's paired t-test was used for statistical analysis at P < 0.05.
Reagents. Oligonucleotides were obtained from Invitrogen (Carlsbad, CA). MEQ, BCECF-AM, and H2DIDS were obtained from Molecular Probes (Eugene, OR). Genistein and calyculin A were obtained from LC laboratories (Woburn, MA). All other reagents were obtained from Sigma (St. Louis, MO.).
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RESULTS |
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PCR amplification was performed on first-strand cDNA synthesized
from cultured BCEC total RNA with specific sAC primers. Figure 1A shows that RT-PCR produced
a clear, specific band at the predicted size. Sequencing analysis
verified the identity as sAC. Figure 1, B and C,
shows indirect immunofluorescence of sAC protein in cultured BCEC,
demonstrating cytoplasmic localization and suggesting numerous focal
microdomains. A previous study (28) showed that BCEC
cytoplasmic extracts contain HCO
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Compared with typical tmAC agonists (e.g., forskolin or -adrenergic
agonists), the increase in total [cAMP] in response to HCO
pulses.
Both apical and basolateral sides were initially perfused with
Cl
- and HCO
was added to the apical side for 90 s, a small,
slow decrease in MEQ fluorescence was observed. Both sides were then
bathed in Cl
-free, HCO
was applied to the
apical side in the presence of HCO
permeability of BCEC was significantly increased by
78% in the presence of HCO
permeability in cells that do not express apical
cAMP-dependent Cl
channels, the same experiments were
performed with CHO cells, which do not express apical CFTR. Figure 2,
C and D, shows that apical Cl
permeability in CHO cells is not affected by the presence of HCO
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Other than CFTR, an obvious candidate for increased apical
Cl permeability of BCEC in the presence of
HCO
permeability (data
not shown). Another possibility is stimulation of a
swelling-activated Cl
channel (SACC) (38,
39); however, this is unlikely because the volume changes were
small (average increased F/F0 = 1.6 ± 2%) and
RVD was complete. One property of SACCs is rapid inactivation as RVD
progresses (29, 30, 40). In three trials, we pulsed apical
Cl
after addition of HCO
permeability was sustained while in HCO
permeability (see Table 1).
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To test whether the changes in apical Cl permeability
induced by HCO
uptake by only 5% in
nonstimulated BCEC, indicating that the inhibitory effect of NPPB in
the presence of HCO
permeability.
The sensitivity of HCO
permeability to NPPB and the insensitivity to
H2DIDS are consistent with activation of CFTR.
Activation of CFTR by a HCO permeability. Although the physiological change is
smaller than the relative change in phosphorylation, the overall effect
of increasing [HCO
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Forskolin (10 µM) was previously shown to produce a four- to
fivefold increase in apical Cl
permeability in BCEC in
the absence of HCO
permeability is caused by cAMP production
from sAC. In paired experiments, we found that forskolin stimulation in
the presence of HCO
Another signature feature of CFTR is that it can also be activated by
the isoflavone genistein (1, 17, 42). Previous studies
showed that activation by low [genistein] (<10 µM) is more
effective if CFTR is already phosphorylated by PKA (22). Because HCO permeability due to 5 µM genistein in the
presence and absence of HCO
- and HCO
was added
on the apical side, there was a small drop in MEQ fluorescence, which
was significantly accelerated by the addition of 5 µM genistein. After a 10-min wash with Cl
-free and
HCO
pulse on the apical side, leading to a
significantly faster drop in MEQ fluorescence compared with that in the
absence of HCO
influx by a factor of 2 greater than the sum of the increased Cl
flux due to genistein and HCO
permeability via activation of CFTR.
If HCO
permeability is produced by stimulating sAC, then the weak agonist HSO
was applied to the apical side in the presence of
HCO
entry (Fig.
4B, inset), consistent with a weak stimulation of sAC and the relatively small increase in CFTR phosphorylation (Fig.
3A). These results, summarized in Fig. 4B,
demonstrate that HSO
permeability by 59%,
consistent with increased apical Cl
permeability from
activation of sAC. Similarly, Fig. 4C shows that when
Cl
was applied on the apical side in the presence of
HCO
permeability (Fig.
4D, inset). These results, summarized in Fig. 4D, demonstrate that Rp-cAMP[S] inhibited
HCO
permeability by
~57%, consistent with a PKA-mediated process.
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DISCUSSION |
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This is the first demonstration that activation of sAC by
HCO
We show that sAC message is present in BCEC and the enzyme is
distributed throughout the cytoplasm. Increased rates of cAMP formation
in BCEC were [HCO
In the presence of HCO permeability that is inhibited by NPPB, but not
H2DIDS, and is inconsistent with anion exchange or SACCs;
2) inhibition of HCO
permeability by HSO
permeability with increasing
[HCO
entry during the apical
Cl
pulses, so our estimates of relative permeability in
HCO
permeability (Table 1).
These results indicate that in corneal endothelium,
HCO
, contributing to net transendothelial flux of both anions.
Corneal hydration changes little over the course of a day, so corneal
endothelial fluid secretion in vivo must be continuous and relatively
constant. Our results suggest that sAC-HCO
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In conclusion, we have demonstrated in corneal endothelial cells that
sAC expression and activation by HCO permeability. Because the corneal
endothelium is not innervated and is shielded from humoral agonists,
this form of sustained autocrine stimulation may be important for the
small (4 µl · cm
2 · h
1)
but constant fluid secretion of this epithelium. In addition to
HCO
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ACKNOWLEDGEMENTS |
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This study was supported by National Institutes of Health Grants EY-08834 (J. A. Bonanno), HD-38722 (L. R. Levin), GM-62328 (J. Buck), and HD-42060 (J. Buck).
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. A. Bonanno, School of Optometry, Indiana University, 800 E. Atwater Ave., Bloomington, IN 47405 (E-mail: jbonanno{at}indiana.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 8, 2003;10.1152/ajpcell.00400.2002
Received 30 August 2002; accepted in final form 30 December 2002.
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