Distribution of luminal carbonic anhydrase activity along rat inner medullary collecting duct

1991 ◽  
Vol 260 (5) ◽  
pp. F738-F748 ◽  
Author(s):  
S. M. Wall ◽  
M. F. Flessner ◽  
M. A. Knepper
Keyword(s):  
Carbonic Anhydrase ◽  
Collecting Duct ◽  
Co2 Concentration ◽  
Carbonic Anhydrase Activity ◽  
Inner Medullary Collecting Duct ◽  
Proton Source ◽  
Total Ammonia ◽  
Medullary Collecting Duct ◽  
Disequilibrium Ph ◽  
Ammonia Flux

The isolated perfused tubule technique was utilized to determine whether endogenous luminal carbonic anhydrase is present in the initial or terminal parts of the inner medullary collecting duct (IMCD) of the rat. This was accomplished by measuring the luminal disequilibrium pH in the presence of a large luminal proton source created by perfusing the lumen with a solution containing 10 mM NH4Cl. (NH3 efflux causes H+ to be released from NH+4 in the lumen). The disequilibrium pH was calculated by subtracting the equilibrium pH from the measured pH at the end of the tubule lumen. The end-luminal equilibrium pH was calculated from the total CO2 concentration in the collected fluid, as measured by microcalorimetry. The end-luminal pH was determined by measuring the fluorescent signal from the the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), which was added to the luminal perfusate in its nonesterified form. In the initial IMCD, there was no measurable disequilibrium pH. With the addition of the carbonic anhydrase inhibitor acetazolamide to the luminal fluid, a significant acidic pH disequilibrium was elicited. In the terminal IMCD under control conditions a statistically significant acidic disequilibrium pH was measured. The disequilibrium was obliterated when exogenous carbonic anhydrase was added to the luminal perfusate. These findings were verified by measuring total ammonia flux by ultramicrofluorometry. The results demonstrate endogenous luminal carbonic anhydrase activity in the initial IMCD but a lack of enzyme activity in the terminal IMCD.

scholarly journals Histochemical carbonic anhydrase in rat inner medullary collecting duct.

1992 ◽  
Vol 40 (10) ◽  
pp. 1535-1545 ◽  
Author(s):  
J G Kleinman ◽  
J L Bain ◽  
C Fritsche ◽  
D A Riley
Keyword(s):  
Carbonic Anhydrase ◽  
Collecting Duct ◽  
Light And Electron Microscopy ◽  
Cell Types ◽  
Intercalated Cells ◽  
Cell Type ◽  
Variable Intensity ◽  
Inner Medullary Collecting Duct ◽  
Majority Population ◽  
Medullary Collecting Duct

Rat inner medullary collecting duct (IMCD) secretes substantial amounts of H+. However, carbonic anhydrase (CA), a concomitant of H+ secretion, has been generally reported absent in this segment. To reexamine this problem, we investigated CA and the morphological phenotypes of cells comprising the IMCD by CA histochemistry, using a modified Hansson technique with light and electron microscopy. Throughout the medulla, tubule cells exhibit histochemical CA activity. In the initial third of the inner medulla, a small proportion have features of intercalated cells and demonstrate some degree of CA activity. However, the majority population in the early portions of the IMCD appears to consist of principal cells. These also show CA staining of widely variable intensity, both among and within cells. A third cell type, previously called "IMCD cells", appears in the middle portion of the IMCD and is the only cell type present near the papilla tip. In contrast to previous reports, these "IMCD cells" have histochemical CA staining, also of highly variable intensity. These results demonstrate that stainable carbonic anhydrase to support acidification is present throughout the rat IMCD, both in intercalated cells and in some cells clearly not of this type. Therefore, the presence of CA is not specific for the intercalated cell type and suggests that other cell types may participate in acid secretion in IMCD.

H(+)-K(+)-ATPase mediates net acid secretion in rat terminal inner medullary collecting duct

1996 ◽  
Vol 271 (5) ◽  
pp. F1037-F1044 ◽  
Author(s):  
S. M. Wall ◽  
A. V. Truong ◽  
T. D. DuBose
Keyword(s):  
Acid Secretion ◽  
Collecting Duct ◽  
Specific Inhibitor ◽  
Co2 Absorption ◽  
Atpase Inhibitor ◽  
Inner Medullary Collecting Duct ◽  
Total Ammonia ◽  
Medullary Collecting Duct ◽  
Atpase Inhibition

Studies in our laboratory have demonstrated total CO2 absorption (JtCO2) and total ammonia secretion in the terminal inner medullary collecting duct (tIMCD) perfused in vitro. The purpose of the present study was to determine whether the H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) participates in proton secretion or JtCO2 in this segment. Tubules from the middle third of the tIMCD were dissected from rats with chronic metabolic acidosis (300 mM NH4Cl, 3-4 days in drinking water) and perfused in vitro. Perfusate and bath were symmetrical solutions containing 5 mM KCl, 6 mM NH4Cl, and 25 mM NaHCO3. Bafilomycin A1 (5 nM), a specific inhibitor of the H(+)-ATPase, did not affect JtCO2 compared with baseline (JtCO2, 3.0 +/- 1.0 and 3.0 +/- 0.8; n = 6, P = not significant) or with time controls (n = 4). With removal of luminal K+, JtCO2 fell from 2.8 +/- 0.6 to 1.6 +/- 0.4 pmol.mm-1.min-1 (n = 5, P < 0.05). To further evaluate K(+)-sensitive JtCO2, the effect of H(+)-K(+)-ATPase inhibition on JtCO2 was explored using the specific H(+)-K(+)-ATPase inhibitor, Sch-28080. Addition of 10 microM Sch-28080 to the luminal perfusate decreased JtCO2 (2.7 +/- 0.4 to 1.4 +/- 0.5 pmol.mm-1. min-1; n = 5, P < 0.05) but did not alter transepithelial membrane potential. Thus luminal Sch-28080 addition, as well as luminal K+ removal, limits apical H+ exit or OH-/HCO3- entry. These results demonstrate that net acid secretion is mediated by the H(+)-K(+)-ATPase in the tIMCD.

Low pH enhances expression of carbonic anhydrase II by cultured rat inner medullary collecting duct cells

1994 ◽  
Vol 266 (2) ◽  
pp. C508-C514 ◽  
Author(s):  
G. J. Schwartz ◽  
D. Brown ◽  
R. Mankus ◽  
E. A. Alexander ◽  
J. H. Schwartz
Keyword(s):  
Carbonic Anhydrase ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Northern Analysis ◽  
Human Antibody ◽  
Inner Medullary Collecting Duct ◽  
Coding Regions ◽  
Steady State Levels ◽  
Collecting Duct Cells ◽  
Medullary Collecting Duct

Carbonic anhydrase (CA) facilitates the secretion of protons from renal epithelia by catalyzing the buffering of hydroxyl ions by CO2. We have previously found that inner medullary collecting duct (IMCD) cells cultured from rat kidney secrete protons and express CA II. Incubation of IMCD cells in acidic medium for 48 h has been shown to stimulate the secretion of protons by a protein synthesis-dependent process. To establish whether CA II might be involved in this process, IMCD cells were exposed to incubation media supplemented with 10(-7) M deoxycorticosterone acetate, pH 7.0 (acid) or pH 7.7 (control) for 48 h, and CA II mRNA and protein were quantitated. Part of the CA II cDNA was obtained by reverse transcription of total RNA from rat kidney followed by amplification using oligonucleotide primers derived from conserved areas in the coding regions of human, mouse, and chick CA II cDNAs in a polymerase chain reaction. By Northern analysis, steady-state levels of CA II mRNA from acid-incubated cells showed an increase of 80% compared with controls and 70% when expressed relative to a housekeeping mRNA, beta-actin. Western blot analysis using a human antibody to CA II showed an approximate doubling of CA II protein after acid incubation. By immunofluorescence microscopy, the domes of acid-incubated IMCD cells contained considerably more CA II-stained cells than found in control cultures. Thus incubation of IMCD cells in acid medium stimulates the expression of CA II mRNA and protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Luminal disequilibrium pH and ammonia transport in outer medullary collecting duct

1987 ◽  
Vol 253 (2) ◽  
pp. F1148-F1157 ◽  
Author(s):  
Robert A. Star ◽  
Maurice B. Burg ◽  
Mark A. Knepper
Keyword(s):  
Carbonic Anhydrase ◽  
Carbonic Acid ◽  
Collecting Duct ◽  
Ammonium Bicarbonate ◽  
Ammonia Transport ◽  
Outer Stripe ◽  
Medullary Collecting Duct ◽  
Disequilibrium Ph ◽  
Luminal Ph ◽  
The Relationship

We measured bicarbonate, ammonia, and luminal pH in segments of the rabbit outer medullary collecting duct (OMCD) to determine the relationship between luminal pH and ammonia transport. Both the inner-stripe and outer-stripe portions of the OMCD absorbed bicarbonate at high rates. The outer stripe OMCD generated an acidic pH disequilibrium that was reversibly dissipated by exogenous luminal carbonic anhydrase. In contrast, the inner stripe OMCD did not generate a spontaneous pH disequilibrium unless perfused with the carbonic anhydrase inhibitor acetazolamide. Ammonia secretion was three times more rapid in the outer stripe OMCD than in the inner stripe OMCD. We conclude the following. 1) Both the inner-stripe and outer-stripe portions of the rabbit OMCD secrete protons at substantial rates. 2) Functional luminal carbonic anhydrase is present in the inner stripe OMCD but absent from the outer stripe OMCD. 3) Ammonia secretion occurs predominantly by NH3, diffusion in both portions. 4) The luminal pH disequilibrium, which is normally present in the outer stripe OMCD, enhances ammonia secretion. ammonia; ammonium; bicarbonate; diffusion trapping; nonionic diffusion; carbonic anhydrase; disequilibrium pH; fluorescence spectroscopy; hydrogen ion secretion; carbonic acid; acetazolamide; heterogeneity Submitted on September 15, 1986 Accepted on January 15, 1987

Ammonium and bicarbonate transport in rat outer medullary collecting ducts

1992 ◽  
Vol 262 (1) ◽  
pp. F1-F7 ◽  
Author(s):  
M. F. Flessner ◽  
S. M. Wall ◽  
M. A. Knepper
Keyword(s):  
Collecting Duct ◽  
Co2 Concentration ◽  
Bicarbonate Transport ◽  
Concentration Gradients ◽  
Entry Rate ◽  
Collecting Ducts ◽  
Total Ammonia ◽  
Outer Stripe ◽  
Medullary Collecting Duct

Previous in vitro studies have demonstrated spontaneous bicarbonate absorption in the outer stripe portion of the rat outer medullary collecting duct (OMCD) and inner medullary collecting duct, but net acid transport has not been studied in the inner stripe of the rat OMCD (OMCDIS). When we perfused isolated OMCDIS segments with identical bath and perfusate solutions containing HCO-3 and NH4Cl, HCO-3 was spontaneously absorbed, and total ammonia was spontaneously secreted at rapid rates in tubules from both deoxycorticosterone (DOC)-treated and untreated rats. We next measured the NH3 flux due to imposed NH3 concentration gradients. Carbonic anhydrase (CA), when added to the lumen, enhanced the NH3 flux, implying an absence of endogenous CA. The NH3 permeability was 0.0042 +/- 0.0007 cm/s. By measuring the luminal pH in perfused OMCDIS segments with an imposed lumen-to-bath NH3 gradient, we determined the pH at the end of the lumen to be 0.23 units below the equilibrium pH calculated from the simultaneously measured total CO2 concentration in collected fluid, confirming the lack of luminal CA. These results are consistent with the view that ammonium secretion in the OMCDIS occurs predominantly by H+ secretion and parallel NH3 diffusion. A luminal disequilibrium pH due to H+ secretion in the absence of endogenous luminal CA enhances the NH3 entry rate. Spontaneous net acid secretion appears to occur more rapidly in the OMCD than in other parts of the rat collecting duct system.

pH and PCO2 profiles of the rat inner medullary collecting duct

1981 ◽  
Vol 241 (6) ◽  
pp. F659-F668 ◽  
Author(s):  
M. L. Graber ◽  
H. H. Bengele ◽  
J. H. Schwartz ◽  
E. A. Alexander
Keyword(s):  
Collecting Duct ◽  
Hydrogen Ion ◽  
Arterial Pco2 ◽  
Inner Medullary Collecting Duct ◽  
Ion Secretion ◽  
Medullary Collecting Duct ◽  
Disequilibrium Ph ◽  
Ph Profiles

To directly characterize acidification by the collecting duct, we developed pH and PCO2 microelectrodes suitable for microcatheterization of the inner medullary collecting duct (IMCD). In saline-infused control rats apparent in situ pH fell significantly along the IMCD, from 5.95 at 60% length to 5.49 at the papilla tip. Luminal PCO2 averaged 34 +/- 1 mmHg and PD averaged +3 mV. In rats acutely infused with 0.1 N HCl, apparent in situ pH also decreased significantly from 5.56 to 5.28, PD averaged +2 mV, and luminal PCO2 31 +/- 1 mmHg. The luminal PCO2 of HCl-infused rats was significantly less than controls and both levels were significantly below arterial PCO2. Corroborating the in situ pH profiles, equilibrium pH measured on collected IMCD samples also decreased significantly with percent length. In samples measured in situ and at equilibrium, a small but significant acid disequilibrium pH ws seen in both groups. We interpret these results to indicate that the IMCD actively participates in distal acidification. It is proposed that acidification by the IMCD is predominantly mediated by hydrogen ion secretion which simultaneously acidifies luminal fluid and generates a cellular sink for CO2, thereby inducing an acid disequilibrium pH by two mechanisms.

Luminal disequilibrium pH and ammonia transport in outer medullary collecting duct [corrected and issued with original paging in Am J Physiol 1987 Aug;253(2 Pt 2)]

1987 ◽  
Vol 252 (6) ◽  
pp. F1148-F1157 ◽  
Author(s):  
R. A. Star ◽  
M. B. Burg ◽  
M. A. Knepper
Keyword(s):  
Carbonic Anhydrase ◽  
Collecting Duct ◽  
Carbonic Anhydrase Inhibitor ◽  
Acidic Ph ◽  
Ammonia Transport ◽  
Outer Stripe ◽  
Medullary Collecting Duct ◽  
Disequilibrium Ph ◽  
Luminal Ph ◽  
The Relationship

We measured bicarbonate, ammonia, and luminal pH in segments of the rabbit outer medullary collecting duct (OMCD) to determine the relationship between luminal pH and ammonia transport. Both the inner-stripe and outer-stripe portions of the OMCD absorbed bicarbonate at high rates. The outer stripe OMCD generated an acidic pH disequilibrium that was reversibly dissipated by exogenous luminal carbonic anhydrase. In contrast, the inner stripe OMCD did not generate a spontaneous pH disequilibrium unless perfused with the carbonic anhydrase inhibitor acetazolamide. Ammonia secretion was three times more rapid in the outer stripe OMCD than in the inner stripe OMCD. We conclude the following. 1) Both the inner-stripe and outer-stripe portions of the rabbit OMCD secrete protons at substantial rates. 2) Functional luminal carbonic anhydrase is present in the inner stripe OMCD but absent from the outer stripe OMCD. 3) Ammonia secretion occurs predominantly by NH3 diffusion in both portions. 4) The luminal pH disequilibrium, which is normally present in the outer stripe OMCD, enhances ammonia secretion.

Water permeability of apical and basolateral cell membranes of rat inner medullary collecting duct

1990 ◽  
Vol 259 (6) ◽  
pp. F986-F999 ◽  
Author(s):  
B. Flamion ◽  
K. R. Spring
Keyword(s):  
Water Flow ◽  
Water Permeability ◽  
Cell Membranes ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Volume Regulatory Decrease ◽  
Water Permeation ◽  
Inner Medullary Collecting Duct ◽  
Medullary Collecting Duct

To quantify the pathways for water permeation through the kidney medulla, knowledge of the water permeability (Posmol) of individual cell membranes in inner medullary collecting duct (IMCD) is required. Therefore IMCD segments from the inner two thirds of inner medulla of Sprague-Dawley rats were perfused in vitro using a setup devised for rapid bath and luminal fluid exchanges (half time, t1/2, of 55 and 41 ms). Differential interference contrast microscopy, coupled to video recording, was used to measure volume and approximate surface areas of single cells. Volume and volume-to-surface area ratio of IMCD cells were strongly correlated with their position along the inner medullary axis. Transmembrane water flow (Jv) was measured in response to a variety of osmotic gradients (delta II) presented on either basolateral or luminal side of the cells. The linear relation between Jv and delta II yielded the cell membrane Posmol, which was then corrected for membrane infoldings. Basolateral membrane Posmol was 126 +/- 3 microns/s. Apical membrane Posmol rose from a basal value of 26 +/- 3 microns/s to 99 +/- 5 microns/s in presence of antidiuretic hormone (ADH). Because of amplification of basolateral membrane, the ADH-stimulated apical membrane remained rate-limiting for transcellular osmotic water flow, and the IMCD cell did not swell significantly. Calculated transcellular Posmol, expressed in terms of smooth luminal surface, was 64 microns/s without ADH and 207 microns/s with ADH. IMCD cells in anisosmotic media displayed almost complete volume regulatory decrease but only partial volume regulatory increase.

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