Polarity of volume-regulatory increase by Necturus gallbladder epithelium

1985 ◽  
Vol 249 (5) ◽  
pp. C471-C475 ◽  
Author(s):  
D. J. Marsh ◽  
K. R. Spring
Keyword(s):  
Volume Regulation ◽  
Apical Cell ◽  
Ringer Solution ◽  
Transport Processes ◽  
Disulfonic Acid ◽  
Bathing Solution ◽  
Hypotonic Solution ◽  
Apical Cell Membrane ◽  
Necturus Gallbladder ◽  
Volume Regulatory Increase

Necturus gallbladder epithelial cells respond to the presence of a hypertonic perfusate in either bathing solution by first shrinking due to osmotic water loss and then swelling back to their original volume (volume-regulatory increase). Previous investigations involving increases in the osmolality of the mucosal bath had suggested that volume-regulatory increase was due to the activation of ion exchangers in the apical cell membrane. In the present study the sidedness of the transport processes involved in volume-regulatory increase was investigated. The osmolality of the serosal bath was increased by 18% either in the absence of HCO3- or when an inhibitor of volume-regulatory increase, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), was added to the mucosal or serosal bath. Volume regulation was HCO3- dependent. DIDS was only effective in inhibiting volume regulation when it was added to mucosal bathing solution, suggesting that volume-regulatory increase depended on transport across the apical membrane. Volume-regulatory increase could also be activated by first swelling the cells in hypotonic solution and then returning the tissue to control Ringer solution. The volume-regulatory increase that occurred upon return to control Ringer was also shown to be sensitive to DIDS in the mucosal bath.

Volume regulation by Necturus gallbladder: apical Na+-H+ and Cl(-)-HCO-3 exchange

1982 ◽  
Vol 243 (3) ◽  
pp. C146-C150 ◽  
Author(s):  
A. C. Ericson ◽  
K. R. Spring
Keyword(s):  
Apical Membrane ◽  
Maximum Velocity ◽  
Disulfonic Acid ◽  
Bathing Solution ◽  
Fluid Absorption ◽  
Control Value ◽  
Necturus Gallbladder ◽  
Osmotic Water ◽  
Volume Regulatory Increase ◽  
Solution Volume

Necturus gallbladder epithelial cells exhibited volume regulatory swelling when exposed to a hypertonic mucosal bathing solution. The initial, osmotically induced shrinkage was followed by a rapid increase in cell volume back to the control value despite continuing hypertonicity of the mucosal perfusate. This volume regulatory increase occurred by osmotic water flow accompanying the transient cellular uptake of NaCl from the mucosal bathing solution. Volume regulatory increase required Na+ and Cl- in the mucosal bath; it was inhibited by amiloride or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid but not by bumetanide or ouabain. The K1/2 for Na+ was 2.8 mM, the K1/2 for Cl- was 1.9 mM, and maximum velocity of fluid flow into the cell for both ions was greater than 10 x 10(-6) cm/s. Both volume regulatory increase and transepithelial fluid absorption involve NaCl flux across the apical membrane into the cells, but the nature of the NaCl fluxes differ in the two processes. During volume regulatory increase NaCl enters the cells by parallel Na+-H+ and Cl(-)-HCO-3 exchanges, whereas during transepithelial fluid absorption NaCl enters the cell by the coupled flux of NaCl.

Ca2(+)-dependent inhibition of sodium transport in rabbit cortical collecting tubules

1990 ◽  
Vol 258 (3) ◽  
pp. F568-F582 ◽  
Author(s):  
G. Frindt ◽  
E. E. Windhager
Keyword(s):  
Cell Membrane ◽  
Amphotericin B ◽  
Apical Cell ◽  
Ringer Solution ◽  
Na Transport ◽  
Apical Cell Membrane ◽  
Dependent Inhibition ◽  
Collecting Tubules ◽  
Rate Limiting ◽  
Sustained Increase

Experiments were carried out to test whether maneuvers believed to increase intracellular Ca2+ concentration [( Ca2+]cell) inhibit Na transport in cortical collecting tubules (CCTs). Unidirectional Na efflux (JNa1----b) and Na influx (JNab----1) were measured isotopically in isolated perfused renal CCTs of rabbits. The animals were either untreated or pretreated with deoxycorticosterone (DOC) for 1-3 wk. To raise [Ca2+]cell, ionomycin or quinidine were added to, or [Na] reduced in, pertubular fluid. In control DOC-pretreated CCTs JNa1----b tended to saturate as luminal Na concentration was increased, reaching 22.9 +/- 1.2 pmol.cm-1.s-1 at 145 mM. In addition, in these CCTs, in contrast to non-DOC-treated tubules, the apical cell membrane was not found to be rate limiting for Na reabsorption as neither amphotericin B nor vasopressin further enhanced JNa1----b. In non-DOC-treated CCTs 10(-6) M ionomycin inhibited JNa1----b by 44.7%. When DOC-pretreated CCTs were exposed to either 10(-6)M ionomycin or 10(-4)M quinidine, JNa1----b was inhibited by 27 and 26%, respectively, while JNab----1 remained unchanged. This ionomycin-induced inhibition was Ca dependent. Exposure of DOC-pretreated CCTs to 5 mM Na-Ringer solution (Na replaced by choline or N-methyl-D-glucamine) for 30 min reduced JNa1----b by 18-30%. The inhibition of JNa1----b caused by any of the three maneuvers was fully reversed upon addition of amphotericin B to the luminal fluid. The results are consistent with the view that a sustained increase in [Ca2+]cell reduces Na transport by inhibition of the rate of Na+ entry across the apical cell membrane.

Identification of Na+/H+ exchange on the apical side of surface colonocytes using BCECF

1994 ◽  
Vol 267 (1) ◽  
pp. G119-G128 ◽  
Author(s):  
G. G. King ◽  
W. E. Lohrmann ◽  
J. W. Ickes ◽  
G. M. Feldman
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Distal Colon ◽  
Transport Processes ◽  
Acetoxymethyl Ester ◽  
Apical Cell Membrane ◽  
Apical Side ◽  
Free Media ◽  
Phi Regulation

Colonocytes must regulate intracellular pH (pHi) while they transport H+ and HCO3-. To investigate the membrane transport processes involved in pHi regulation, colonocyte pHi was measured with 2,'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) in intact segments of rat distal colon mounted on a holder that fits into a standard fluorometer cuvette and allows independent superfusion of mucosal and serosal surfaces. When NCECF-acetoxymethyl ester was in the mucosal solution only, BCECF loaded surface colonocytes with a high degree of selectivity. In HEPES-buffered solutions, basal pHi was 7.31 +/- 0.01 (n = 68), and pHi was dependent on extracellular Na+. Cells acidified in Na(+)-free solution, and pHi rapidly corrected when Na+ was returned. pHi recovered at 0.22 +/- 0.01 pH/min (n = 6) when Na+ was introduced into the mucosal solution and at 0.02 +/- 0.01 pH/min (n = 7) when Na+ was absent from the mucosal solution. The presence or absence of Na+ in the serosal solution did not affect pHi. This indicated that the Na(+)-dependent pHi recovery process is located in the apical cell membrane, but not in the basolateral membrane. Because amiloride (1 mM) inhibited Na(+)-dependent pHi recovery by 75%, Na+/H+ exchange appears to be present in the apical membrane. Because Na(+)-independent pHi recovery was not affected by K(+)-free media, 50 microM SCH-28080, 100 nM bafilomycin A1, or Cl(-)-free media, this transport mechanism does not involve a gastriclike H(+)-K(+)-ATPase, a vacuolar H(+)-ATPase, or a Cl-/base exchanger. In summary, pHi was selectively measured in surface colonocytes by this technique. In these cells, the Na+/H+ exchange activity involved in pHi regulation was detected in the apical membrane, but not in the basolateral membrane.

Electroneutral H+ secretion in distal tubule of Amphiuma

1987 ◽  
Vol 252 (4) ◽  
pp. F691-F699 ◽  
Author(s):  
B. Stanton ◽  
A. Omerovic ◽  
B. Koeppen ◽  
G. Giebisch
Keyword(s):  
Basolateral Membrane ◽  
Apical Cell ◽  
Distal Tubule ◽  
Cell Types ◽  
Disulfonic Acid ◽  
Type I ◽  
Cellular Mechanisms ◽  
Apical Cell Membrane ◽  
Membrane Type

This study examines the cellular mechanisms of acid secretion by the in vitro perfused late distal tubule of Amphiuma kidney. Acidification of tubule fluid occurred against an electrochemical gradient of 16 mV; thus H+ secretion was active. Amiloride (1 mM) or a reduction of sodium in the perfusion fluid (from 83.7 to 7.7 mM) partially reduced acidification. Amiloride, in the presence of low sodium, completely inhibited acidification. Furthermore, acetazolamide and ouabain in the bath solution (0.1 mM) also inhibited acidification. Conductive properties of the epithelium and of individual cell membranes were determined by means of cable analysis of the tubule and intracellular voltage recordings. The transepithelial voltage and resistance averaged -0.4 +/- 0.4 mV, lumen negative, and 7,147 +/- 845 omega X cm, respectively. Two functionally different cell types were identified by intracellular microelectrodes. Type I cells had a basolateral membrane voltage (Vbl) of -67.7 mV. As determined by ion substitution experiments, the basolateral membrane was conductive to K+ and Cl-. This cell also had a 4-acetamido-4'-isothiocyanostilbene-2-2'-disulfonic acid (SITS)-sensitive Na+-dependent HCO3- exit pathway in the basolateral membrane. Type II cells had a Vbl of -76.1 mV (P less than 0.05 vs. type I) and the basolateral membrane was conductive to K+ and Cl- but not to HCO3-. HCO3- movement across the basolateral membrane in this cell may occur by electroneutral Cl- -HCO3- exchange. The apical cell membrane of both cell types did not contain measurable ionic conductances, as evidenced by a high value of apical membrane fractional resistance (0.98 +/- 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

pH in principal cells of frog skin (Rana pipiens): dependence on extracellular Na+

1988 ◽  
Vol 255 (5) ◽  
pp. F930-F935 ◽  
Author(s):  
K. Drewnowska ◽  
E. J. Cragoe ◽  
T. U. Biber
Keyword(s):  
Frog Skin ◽  
Rana Pipiens ◽  
Apical Cell ◽  
Ringer Solution ◽  
Feedback Mechanism ◽  
Open Circuit ◽  
Apical Cell Membrane ◽  
Principal Cells ◽  
Apical Side ◽  
Basolateral Side

Measurements of intracellular pH (pHi) and of apical cell membrane potential (Va) were made in principal cells of frog skin (Rana pipiens) with double-barrel microelectrodes under open-circuit conditions. The tissues were pretreated with stilbenes (10(-3) M) and bathed in HCO3- -free NaCl Ringer solution that was buffered with 6 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.8). Substitution of extracellular Na+ on both sides of the epithelium with N-methyl-D-glucamine caused intracellular acidification by 0.27 pH units. Restoration of Na+ on the apical side alone or on both sides caused a pHi recovery of 0.24 and 0.28 pH units, respectively, whereas return of Na+ on the basolateral side caused no recovery. Recovery of pHi on restoration of Na+ to the apical side was prevented by 10(-5) M 5-(N-ethyl-N-isopropyl)-amiloride. In individual preparations there was no correlation between pHi recovery due to return of apical Na+ and changes in Va. The average change in pHi was several times greater than the one expected from voltage clamp-induced changes in Va at constant extracellular Na+. The results suggest the presence of a Na+-H+ exchange on the apical side of principal cells. Such a process could be part of a negative feedback mechanism for regulation of Na+ entry via apical Na+ channels into principal cells.

pH in principal cells of frog skin (Rana pipiens): effects of amiloride and potential

1988 ◽  
Vol 255 (5) ◽  
pp. F922-F929 ◽  
Author(s):  
K. Drewnowska ◽  
T. U. Biber
Keyword(s):  
Cell Membrane ◽  
Voltage Clamp ◽  
Frog Skin ◽  
Rana Pipiens ◽  
Apical Cell ◽  
Disulfonic Acid ◽  
Intracellular Acidification ◽  
Apical Cell Membrane ◽  
Principal Cells ◽  
Apical Side

Intracellular pH (pHi) and apical cell membrane potential (Va) were determined in principal cells of frog skin (Rana pipiens) with double-barrel micro-electrodes. In the Northern and Southern varieties, respectively, pHi is 0.38 and 0.26 pH units below bath pH. Amiloride, applied apically, causes reversible intracellular acidification at concentrations of 10(-5) M or higher. Voltage clamp-induced hyperpolarization and depolarization of Va result in intracellular acidification and alkalinization, respectively. This response of pHi is inhibited or abolished when the apical side is treated with 10(-3) M 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Amiloride-induced intracellular acidification is not exclusively due to the hyperpolarization of Va that accompanies amiloride treatment since 1) amiloride causes greater acidification than equivalent voltage clamp-induced hyperpolarization of Va, 2) amiloride-induced acidification persists in DIDS-treated tissues, and 3) there is no correlation between hyperpolarization of Va and intracellular acidification occurring after amiloride. We conclude that pHi is below the extracellular pH. Amiloride causes intracellular acidification that may be in part connected with hyperpolarization of Va. However, a major component of amiloride-induced acidification is due to other factors, possibly inhibition of apical Na+-H+ exchange. The inhibitory effect of apically applied DIDS suggests that the voltage dependent changes in pHi are related to movement of HCO3 (or OH) ions across the apical cell membrane.

Circadian rhythm of acidification in insect vas deferens regulated by rhythmic expression of vacuolar H+-ATPase

2002 ◽  
Vol 205 (1) ◽  
pp. 37-44
Author(s):  
Piotr Bebas ◽  
Bronislaw Cymborowski ◽  
Jadwiga M. Giebultowicz
Keyword(s):  
Circadian Rhythm ◽  
Circadian Clock ◽  
Vas Deferens ◽  
Apical Cell ◽  
Transport Processes ◽  
Daily Rhythm ◽  
Constant Darkness ◽  
Cotton Leaf ◽  
Apical Cell Membrane ◽  
Peripheral Tissues

SUMMARY Recent studies have demonstrated that the peripheral tissues of vertebrates and invertebrates contain circadian clocks; however, little is known about their functions and the rhythmic outputs that they generate. To understand clock-controlled rhythms at the cellular level, we investigated a circadian clock located in the reproductive system of a male moth (the cotton leaf worm Spodoptera littoralis) that is essential for the production of fertile spermatozoa. Previous work has demonstrated that spermatozoa are released from the testes in a daily rhythm and are periodically stored in the upper vas deferens (UVD). In this paper, we demonstrate a circadian rhythm in pH in the lumen of the UVD, with acidification occurring during accumulation of spermatozoa in the lumen. The daily rhythm in pH correlates with a rhythmic increase in the expression of a proton pump, the vacuolar H+-ATPase (V-ATPase), in the apical portion of the UVD epithelium. Rhythms in pH and V-ATPase persist in light/dark cycles and constant darkness, but are abolished in constant light, a condition that disrupts clock function and renders spermatozoa infertile. Treatment with colchicine impairs the migration of V-ATPase-positive vesicles to the apical cell membrane and abates the acidification of the UVD lumen. Bafilomycin, a selective inhibitor of V-ATPase activity, also prevents the decline in luminal pH. We conclude that the circadian clock generates a rhythm of luminal acidification by regulating the levels and subcellular distribution of V-ATPase in the UVD epithelium. Our data provide the first evidence for circadian control of V-ATPase, the fundamental enzyme that provides the driving force for numerous secondary transport processes. They also demonstrate how circadian rhythms displayed by individual cells contribute to the synchrony of physiological processes at the organ level.

Microelectrode studies of Necturus antral mucosa: electrical potentials and resistances

1983 ◽  
Vol 244 (1) ◽  
pp. G71-G75
Author(s):  
T. P. Grady ◽  
L. Y. Cheung
Keyword(s):  
Membrane Potential ◽  
Cell Membrane ◽  
Apical Cell ◽  
Cell Membrane Potential ◽  
Transepithelial Resistance ◽  
Bathing Solution ◽  
Antral Mucosa ◽  
Apical Cell Membrane ◽  
Electrical Potentials ◽  
Basolateral Cell Membrane

Intracellular microelectrode techniques were applied to Necturus antral mucosa. Stable intracellular impalements were obtained with 15-50 M omega microelectrodes filled with 3M KCl. It was possible to change rapidly the mucosal bathing solution while maintaining the microelectrode in the cell. With these techniques, we were able to measure the electrical potentials and resistances of the cell membranes and the shunt pathway. The transepithelial potential was -4.9 +/- 1.3 mV, serosal solution reference. Apical cell membrane potential was -43.9 +/- 0.6 mV, cell negative to the mucosal solution. Basolateral cell membrane potential was -48.8 +/- 1.3 mV, cell negative to serosal solution. Transepithelial resistance was 427 +/- 66 omega . cm2. The ratio of apical to basolateral membrane resistances was 3.4 +/- 0.3. The electrical resistances of the transcellular and paracellular pathway were determined by the measurement of the total transepithelial resistance and the ratio of apical to basolateral cell membrane resistances before and after blocking apical membrane sodium permeability with amiloride. The resistances of the apical cell membrane, basolateral cell membrane, and the shunt were 2,203 +/- 585, 1,296 +/- 384, and 604 +/- 81 omega . cm2, respectively (mean +/- SE). Calculations from these measurements indicate that the shunt contribution to transepithelial conductance was approximately 85%.

Membrane stretch: a physiological stimulator of Ca2+-activated K+ channels in thick ascending limb

1989 ◽  
Vol 257 (3) ◽  
pp. F347-F352 ◽  
Author(s):  
J. Taniguchi ◽  
W. B. Guggino
Keyword(s):  
Negative Pressure ◽  
Apical Cell ◽  
Bathing Solution ◽  
Thick Ascending Limb ◽  
Open Probability ◽  
Apical Cell Membrane ◽  
K Channels ◽  
Membrane Stretch ◽  
Medullary Thick Ascending Limb ◽  
Tubular Flow

The effects of membrane stretch on Ca2+-activated (maxi) K+ channels were examined in the apical membrane of cultured medullary thick ascending limb (MTAL) cells. Using cell-attached patchclamp technique, we found that negative pressure (-33 +/- 5 cmH2O) applied to the patch membrane increased fractional open probability (NPo) from 0.3 +/- 0.2 to 29.9 +/- 7.6% (n = 12) in the presence of 1.8 mM Ca2+ in the pipette. The activity returned to control on releasing the negative pressure. Reduction of extracellular osmolality from 293.2 +/- 1.6 to 219.8 +/- 1.1 mosmol/kg also activated K+ channels (NPo = 43.8 +/- 12.2%, n = 8) in cell-attached patches. Removal of Ca2+ from both pipette and bathing solution inhibited osmotic activation of K+ channels. K+ channels were shown to be Ca2+-activated K+ channels by their conductance (146 +/- 7 pS, n = 5) and Ca2+ dependence. Our data suggest that membrane stretch caused by swelling or possibly by tubular flow enhances Ca2+ entry across the apical cell membrane of MTAL cells activating maxi K+ channels.

Active potassium secretion by rabbit proximal colon

1986 ◽  
Vol 250 (4) ◽  
pp. G475-G483 ◽  
Author(s):  
S. K. Sullivan ◽  
P. L. Smith
Keyword(s):  
Short Circuit ◽  
Apical Cell ◽  
Distal Colon ◽  
Proximal Colon ◽  
Bathing Solution ◽  
Uptake Mechanism ◽  
Apical Cell Membrane ◽  
Ussing Chambers ◽  
K Concentration

Fluxes of K from mucosa to serosa or serosa to mucosa have been examined in stripped preparations of rabbit proximal and distal colon in vitro under short-circuit conditions in Ussing chambers. Results from these studies demonstrate that steady-state radioisotopic fluxes of K are achieved after 90 min and remain constant for at least 2 h. Determination of the K concentration dependence of the serosal-to-mucosal K flux revealed that this flux contains both saturable and nonsaturable components. Addition of ouabain (0.1 mM) abolished the saturable component of the serosal-to-mucosal K flux. The mucosal-to-serosal K flux is a linear function of K concentration between 1 and 20 mM under basal conditions. In paired tissues, serosal-to-mucosal K flux is always greater than mucosal-to-serosal flux under basal conditions resulting in net K secretion. However, addition of barium (2 mM) to the mucosal or serosal bathing solution had no significant effect on either unidirectional or net K fluxes. In addition, mucosal bumetanide (0.1 mM) or removal of Cl from both bathing solutions had no significant effect on unidirectional or net K fluxes. In rabbit distal colon, Cl removal from the bathing solutions significantly reduced serosal-to-mucosal K flux, resulting in net K absorption. These results indicate that rabbit proximal colon like rabbit distal colon actively secretes K. However, unlike distal colon the proximal colon does not possess an active K uptake mechanism at the apical cell membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

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