Abstract
Carbonic anhydrase (CA) inhibitors lower the rate of aqueous humor (AH) secretion into the eye. Different CA isozymes might play different roles in the response. Here we have studied the effects of carbonic anhydrase inhibitors on cytoplasmic pH (pH(i)) regulation, using a dextran-bound CA inhibitor (DBI) to selectively inhibit membrane-associated CA in a cell line derived from rabbit NPE. pH(i) was measured using the fluorescent dye BCECF and the pHi responses to the cell permeable CA inhibitor acetazolamide (ACTZ) and DBI were compared, ACTZ markedly inhibited the rapid pHi changes elicited by bicarbonate/CO2 removal and readdition but DBI was ineffective in this respect, consistent with the inability of DBI to enter the cell and inhibit cytoplasmic CA isozymes. Added alone, ACTZ and DBI caused a similar reduction (0.2 pH units) of baseline pH(i). We considered whether CA-IV might facilitate H+ extrusion via Na-H exchange. The Na-H exchanger inhibitor amiloride (1 mM) reduced pH(i) 0.52 ± 0.10 pH units. In the presence of DBI, the magnitude of pH(i) reduction caused by amiloride was significantly (P < 0.05) reduced to 0.26 ± 0.09 pH units. ACTZ similarly reduced the magnitude of the pHi reduction. DBI also reduced by ~40% the rate of pHi recovery in cells acidified by an ammonium chloride (20 mM) prepulse; a reduction in pHi recovery rate was also caused by ACTZ and amiloride. DBI failed to alter the pH(i) alkalinization response caused by elevating external potassium concentration, a response insensitive to amiloride but sensitive to ACTZ. These observations are consistent with a reduction in Na-H exchanger activity in the presence of DBI or ACTZ. We suggest that the CA-IV isozyme might catalyze rapid equilibration of H+ and HCO3/- with CO2 in the unstirred layer outside the plasma membrane, preventing local accumulation of H+ which competes with sodium for the same external Na-H exchanger binding site. Inhibition of CA-IV could produce pH(i) changes that might alter the function of other ion transporters and channels in the NPE.
Original language | English (US) |
---|---|
Pages (from-to) | 31-38 |
Number of pages | 8 |
Journal | The Journal of Membrane Biology |
Volume | 162 |
Issue number | 1 |
DOIs | |
State | Published - Mar 1 1998 |
Externally published | Yes |
Fingerprint
Keywords
- Acetazolamide
- Carbonic anhydrase
- Ciliary epithelium
- pH
ASJC Scopus subject areas
- Biophysics
- Physiology
- Cell Biology
Cite this
Cytoplasmic pH responses to carbonic anhydrase inhibitors in cultured rabbit nonpigmented ciliary epithelium. / Wu, Q.; Pierce, W. M.; Delamere, Nicholas A.
In: The Journal of Membrane Biology, Vol. 162, No. 1, 01.03.1998, p. 31-38.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Cytoplasmic pH responses to carbonic anhydrase inhibitors in cultured rabbit nonpigmented ciliary epithelium
AU - Wu, Q.
AU - Pierce, W. M.
AU - Delamere, Nicholas A
PY - 1998/3/1
Y1 - 1998/3/1
N2 - Carbonic anhydrase (CA) inhibitors lower the rate of aqueous humor (AH) secretion into the eye. Different CA isozymes might play different roles in the response. Here we have studied the effects of carbonic anhydrase inhibitors on cytoplasmic pH (pH(i)) regulation, using a dextran-bound CA inhibitor (DBI) to selectively inhibit membrane-associated CA in a cell line derived from rabbit NPE. pH(i) was measured using the fluorescent dye BCECF and the pHi responses to the cell permeable CA inhibitor acetazolamide (ACTZ) and DBI were compared, ACTZ markedly inhibited the rapid pHi changes elicited by bicarbonate/CO2 removal and readdition but DBI was ineffective in this respect, consistent with the inability of DBI to enter the cell and inhibit cytoplasmic CA isozymes. Added alone, ACTZ and DBI caused a similar reduction (0.2 pH units) of baseline pH(i). We considered whether CA-IV might facilitate H+ extrusion via Na-H exchange. The Na-H exchanger inhibitor amiloride (1 mM) reduced pH(i) 0.52 ± 0.10 pH units. In the presence of DBI, the magnitude of pH(i) reduction caused by amiloride was significantly (P < 0.05) reduced to 0.26 ± 0.09 pH units. ACTZ similarly reduced the magnitude of the pHi reduction. DBI also reduced by ~40% the rate of pHi recovery in cells acidified by an ammonium chloride (20 mM) prepulse; a reduction in pHi recovery rate was also caused by ACTZ and amiloride. DBI failed to alter the pH(i) alkalinization response caused by elevating external potassium concentration, a response insensitive to amiloride but sensitive to ACTZ. These observations are consistent with a reduction in Na-H exchanger activity in the presence of DBI or ACTZ. We suggest that the CA-IV isozyme might catalyze rapid equilibration of H+ and HCO3/- with CO2 in the unstirred layer outside the plasma membrane, preventing local accumulation of H+ which competes with sodium for the same external Na-H exchanger binding site. Inhibition of CA-IV could produce pH(i) changes that might alter the function of other ion transporters and channels in the NPE.
AB - Carbonic anhydrase (CA) inhibitors lower the rate of aqueous humor (AH) secretion into the eye. Different CA isozymes might play different roles in the response. Here we have studied the effects of carbonic anhydrase inhibitors on cytoplasmic pH (pH(i)) regulation, using a dextran-bound CA inhibitor (DBI) to selectively inhibit membrane-associated CA in a cell line derived from rabbit NPE. pH(i) was measured using the fluorescent dye BCECF and the pHi responses to the cell permeable CA inhibitor acetazolamide (ACTZ) and DBI were compared, ACTZ markedly inhibited the rapid pHi changes elicited by bicarbonate/CO2 removal and readdition but DBI was ineffective in this respect, consistent with the inability of DBI to enter the cell and inhibit cytoplasmic CA isozymes. Added alone, ACTZ and DBI caused a similar reduction (0.2 pH units) of baseline pH(i). We considered whether CA-IV might facilitate H+ extrusion via Na-H exchange. The Na-H exchanger inhibitor amiloride (1 mM) reduced pH(i) 0.52 ± 0.10 pH units. In the presence of DBI, the magnitude of pH(i) reduction caused by amiloride was significantly (P < 0.05) reduced to 0.26 ± 0.09 pH units. ACTZ similarly reduced the magnitude of the pHi reduction. DBI also reduced by ~40% the rate of pHi recovery in cells acidified by an ammonium chloride (20 mM) prepulse; a reduction in pHi recovery rate was also caused by ACTZ and amiloride. DBI failed to alter the pH(i) alkalinization response caused by elevating external potassium concentration, a response insensitive to amiloride but sensitive to ACTZ. These observations are consistent with a reduction in Na-H exchanger activity in the presence of DBI or ACTZ. We suggest that the CA-IV isozyme might catalyze rapid equilibration of H+ and HCO3/- with CO2 in the unstirred layer outside the plasma membrane, preventing local accumulation of H+ which competes with sodium for the same external Na-H exchanger binding site. Inhibition of CA-IV could produce pH(i) changes that might alter the function of other ion transporters and channels in the NPE.
KW - Acetazolamide
KW - Carbonic anhydrase
KW - Ciliary epithelium
KW - pH
UR - http://www.scopus.com/inward/record.url?scp=0032031735&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0032031735&partnerID=8YFLogxK
U2 - 10.1007/s002329900339
DO - 10.1007/s002329900339
M3 - Article
C2 - 9516235
AN - SCOPUS:0032031735
VL - 162
SP - 31
EP - 38
JO - Journal of Membrane Biology
JF - Journal of Membrane Biology
SN - 0022-2631
IS - 1
ER -