Cl- secretion by cultured shark rectal gland cells. II. Effects of forskolin on cellular electrophysiology

1991 ◽  
Vol 260 (4) ◽  
pp. C824-C831 ◽  
Author(s):  
W. M. Moran ◽  
J. D. Valentich
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Electrical Potential ◽  
Ringer Solution ◽  
Rectal Gland ◽  
Electrical Potential Difference ◽  
Electrophysiological Properties ◽  
Shark Rectal Gland ◽  
Membrane Electrical Potential ◽  
Cl Conductance

Employing microelectrode techniques we have assessed the cellular electrophysiological properties of shark rectal gland (SRG) cells in primary culture. In the absence of secretagogues a 10-fold reduction in the Cl- concentration of the apical superfusate shark Ringer solution had little effect on either apical membrane electrical potential difference (Va) or fractional resistance (fRa), indicating little, if any, apical membrane Cl- conductance. Superfusing the basolateral surface with high-K+ shark Ringer solution (K+ increased 10-fold) depolarized the basolateral membrane electrical potential difference (Vb) by 43 mV, indicating that this barrier is largely K+ conductive. In addition, basolateral Ba2+ (5 mM) depolarized Vb by 12 mV and reduced fRa from 0.92 to 0.58, results consistent with a K(+)-conductive basolateral membrane in unstimulated SRG cells. Basolateral forskolin (10(-6) M) depolarized Va by 25 mV and caused a dramatic reduction in fRa from 0.97 to approximately 0.10. Under these conditions, a 10-fold decrease in apical superfusate Cl- concentration depolarized Va by 37 mV, revealing an adenosine 3',5'-cyclic monophosphate-induced apical membrane Cl- conductance. The time course of the forskolin-induced changes in Va and Vb suggests that the basolateral membrane K+ conductance increased and maintained the driving force for apical Cl- exit, as in other Cl(-)-secreting epithelia. These electrophysiological properties compare favorably with those of the perfused SRG tubule and indicate that SRG primary cultures are a suitable model for Cl(-)-secreting epithelia.

Cl- secretion by cultured shark rectal gland cells. III. Ca2+ regulation of apical membrane Cl- conductance

1993 ◽  
Vol 265 (3) ◽  
pp. C641-C649 ◽  
Author(s):  
W. M. Moran ◽  
J. D. Valentich
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Electrical Potential ◽  
Potential Difference ◽  
Rectal Gland ◽  
Electrical Potential Difference ◽  
Apical Surface ◽  
Cl Secretion ◽  
Shark Rectal Gland ◽  
Cl Conductance

Calcium ionophores (ionomycin and A-23187) were employed to assess the effects of increased intracellular Ca2+ concentration ([Ca2+]i) on apical membrane Cl- conductance (GaCl) and rate of transepithelial Cl- secretion in cultured shark rectal gland (SRG) cells. Apical 2 microM ionomycin induced dramatic changes in cellular electrophysiological properties: the apical membrane electrical potential difference (V(a)) depolarized from -66 mV to -46 mV, the fractional resistance of the apical membrane (fRa) decreased from 0.88 to 0.23, and the transepithelial electrical potential difference (Vab) increased slightly from +1.2 mV to +1.4 mV. These effects result from increased GaCl because apical low-Cl- shark Ringer (SR) depolarized V(a) by 32 mV and increased fRa from 0.23 to 0.36. Ionomycin-stimulated Vab or short-circuit current (Isc) results largely from increased Cl- secretion because approximately 80% of the increase in Isc is Cl- dependent. Establishing the Ca2+ dependence of ionophore activation of GaCl was confounded because apical low-Ca2+ SR [Ca2+ concentration ([Ca2+]) 1 mM to 0.1 microM] alone activated this conductive pathway. To establish the Ca2+ dependence of ionophore action, we assessed the effect of ionomycin on Isc in low-Ca2+ SR ([Ca2+] = 0.1 microM) and in SR. In low-Ca2+ SR, apical ionomycin stimulated Isc by 14.0 microA/cm2. In SR (normal [Ca2+]), ionomycin increased Isc further by 27.0 microA/cm2. Superfusing the basolateral surface with 2 microM ionomycin for 8-16 min failed to activate GaCl. In every case, subsequent superfusion of the apical surface with ionophore for 1.5-2 min activated GaCl. Bilateral 4 microM indomethacin (45-min superfusion) failed to block the ionomycin-induced GaCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Potassium transport by flounder intestinal mucosa

1984 ◽  
Vol 246 (6) ◽  
pp. F946-F951 ◽  
Author(s):  
R. A. Frizzell ◽  
D. R. Halm ◽  
M. W. Musch ◽  
C. P. Stewart ◽  
M. Field
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Electrical Potential ◽  
Electrical Potential Difference ◽  
Cell Type ◽  
K Secretion ◽  
Single Cell Type ◽  
Free Media ◽  
Membrane Electrical Potential ◽  
K Transport

We studied the mechanisms of K transport across an epithelium in which NaCl absorption is mediated primarily by Na/K/Cl cotransport at the apical membrane. Rubidium served as a reliable K substitute; under control conditions, both K and Rb were actively secreted. During secretion, K (Rb) enters across the basolateral membrane via the Na/K pump and exits across the apical membrane through K conductance pathways, since serosal ouabain or mucosal barium abolished K secretion, mucosal furosemide or Cl-free media blocked K secretion by interfering with access of Na to the pump, and elevated mucosal solution [K] or [Rb] depolarized the apical membrane electrical potential difference. Mucosal Ba unmasked active Rb absorption that could be blocked by mucosal furosemide. These findings illustrate active K absorption and secretion across an epithelium that comprises a single cell type in which opposing K fluxes across the apical membrane are mediated by Na/K/Cl cotransport entry and conductive K exit. The direction of transepithelial K transport is determined by the relative activities of these pathways.

Intracellular chloride activities in the isolated perfused shark rectal gland

1983 ◽  
Vol 245 (5) ◽  
pp. F640-F644
Author(s):  
M. J. Welsh ◽  
P. L. Smith ◽  
R. A. Frizzell
Keyword(s):  
Basolateral Membrane ◽  
Apical Cell ◽  
Electrical Potential ◽  
Electrochemical Potential ◽  
Potential Difference ◽  
Rectal Gland ◽  
Electrical Potential Difference ◽  
Apical Cell Membrane ◽  
Shark Rectal Gland ◽  
Electrochemical Equilibrium

The isolated, perfused shark rectal gland secretes Cl when stimulated with adenosine 3',5'-cyclic monophosphate (cAMP). To investigate the mechanism of secretion, we used Cl-selective and conventional (KCl-filled) microelectrodes to measure the intracellular Cl activity (aClc). Under nonsecreting conditions, the electrical potential difference across the basolateral membrane (psi b) was -78 m V and aClc was 57 mM, a value seven times greater than predicted for electrochemical equilibrium across the basolateral membrane. When theophylline and 8-bromo-cAMP were added to the perfusate, the transglandular electrical potential difference doubled and the rate of fluid secretion increased 20-fold; however, neither psi b nor aClc changed. During both nonsecreting and secreting conditions the intracellular accumulation of Cl results in an electrochemical potential difference favoring Cl exit across the apical cell membrane. The constancy of aClc despite the variation in secretion rate suggests that stimulation is associated with an equivalent enhancement of net Cl movement across both the apical and basolateral membranes. When stimulated glands were perfused with Na-free (choline) Ringer, secretion was abolished and aClc fell toward the value predicted for electrochemical equilibrium. These findings suggest that the "uphill" step in Cl secretion lies at the basolateral membrane, where cellular Cl accumulation probably involves secondary active transport; i.e., Cl entry is driven by an inwardly directed electrochemical potential difference for Na.

Bicarbonate reabsorption and electrophysiology of proximal tubules in uninephrectomized rats

1991 ◽  
Vol 81 (2) ◽  
pp. 141-146 ◽  
Author(s):  
Dirce M. Zanetta Limongi ◽  
Antonio Carlos Cassola ◽  
Viktoria Woronik ◽  
Gerhard Malnic
Keyword(s):  
Basolateral Membrane ◽  
Electrical Potential ◽  
Ringer Solution ◽  
Proximal Tubules ◽  
Potential Difference ◽  
Protonmotive Force ◽  
Electrical Potential Difference ◽  
Analogue Model ◽  
Significant Difference ◽  
Membrane Electrical Potential

1. The kinetics of acidification of luminal fluid in hypertrophied proximal tubules after unilateral nephrectomy was studied by stationary microperfusion and continuous measurement of luminal pH with antimony microelectrodes. 2. Trans-epithelial and basolateral membrane electrical potential differences were measured in order to detect modifications in electrogenic transport mechanisms under these conditions. 3. The values of stationary pH and HCO−3 concentration were significantly lower, the mean acidification half-time was not different and net reabsorptive HCO−3 fluxes in proximal tubules were significantly increased in uninephrectomized rats. According to an electrical analogue model, these results suggest (a) a reduction in the internal series resistance of the H+ pump, caused perhaps by an increased density of pump sites, and (b) an increase in the protonmotive force of the pump. 4. The trans-epithelial electrical potential difference measured in free flow conditions was significantly more lumen-positive in uninephrectomized rats. The trans-epithelial electrical potential difference measured during intraluminal perfusion with Ringer solution containing 30 mmol/l HCO−3 was significantly more negative in all groups studied. In uninephrectomized rats treated with acetazolamide, the trans-epithelial electrical potential difference was more lumen-negative than that in untreated uninephrectomized rats. These results are compatible with a steeper transepithelial Cl− gradient as well as with electrogenic, active H+ secretion. 5. There was no significant difference in the basolateral electrical potential difference between control and uninephrectomized rats. 6. In conclusion, our data show an increase in the transport rates of HCO−3 in the proximal tubule of uninephrectomized rats, which may be due to an increase in the density of transporters in the brush-border membrane, and an increased ability of the transport mechanism to create H+ gradients.

Electrophysiological evidence for Cl secretion in shark renal proximal tubules

1985 ◽  
Vol 248 (2) ◽  
pp. F282-F295 ◽  
Author(s):  
K. W. Beyenbach ◽  
E. Fromter
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Proximal Tubules ◽  
In Vitro Perfusion ◽  
Electrophysiological Properties ◽  
Electrophysiological Responses ◽  
Tubule Lumen ◽  
Transepithelial Voltage ◽  
Cl Conductance

The electrophysiology of shark proximal tubules (Squalus acanthias) was investigated using conventional microelectrodes and cable analysis. Under in vitro perfusion with symmetrical Ringer solutions, tubule transepithelial resistance was 36.3 +/- 2.3 omega X cm2 (means +/- SE, n = 44). Other electrophysiological variables varied widely under control conditions. In unstimulated tubules (n = 16) the transepithelial voltage (VT,o) was lumen positive (1.2 +/- 0.2 mV), the basolateral membrane potential (Vbl,x) was -61.3 +/- 1.6 mV, and the fractional resistance of the apical membrane (fRa) was 0.67 +/- 0.02. Spontaneously stimulated tubules (n = 28) had lumen-negative VT,o values (-1.5 +/- 0.4 mV), low Vbl,x values (-41.3 +/- 1.7 mV), and low fRa values (0.30 +/- 0.02). The stimulated state can be induced in unstimulated tubules via treatment with cAMP. Multiple microelectrode impalements in a single tubule revealed epithelial cells sharing similar electrophysiological properties. Selective ion substitutions in the tubule lumen and peritubular bath uncovered an increased Cl conductance in the apical membrane of spontaneously and cAMP-stimulated tubules. Anthracene-9-carboxylic acid tended to reverse the stimulated state, and furosemide hyperpolarized Vbl,x. These results constitute the first evidence for secretory Cl transport in a renal proximal tubule. The electrophysiological responses to ion substitutions, stimulators, and inhibitors are strikingly similar to those of known Cl-transporting epithelia.

Furosemide blocks basolateral membrane Cl- permeability in gallbladder epithelium

1990 ◽  
Vol 258 (6) ◽  
pp. C1150-C1158 ◽  
Author(s):  
J. S. Stoddard ◽  
G. A. Altenberg ◽  
M. L. Ferguson ◽  
L. Reuss
Keyword(s):  
Anion Exchange ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Ringer Solution ◽  
Combined Effect ◽  
Gallbladder Epithelium ◽  
K Permeability ◽  
Cl Conductance ◽  
Stimulation Of ◽  
Cl Permeability

In Necturus gallbladders bathed in a NaCl Ringer solution buffered with 10 mM HCO3(-)-1% CO2, furosemide (added to the serosal solution) caused a concentration-dependent hyperpolarization of both cell membranes that was slow and reversible. At 10(-3) M furosemide, the basolateral membrane voltage (Vcs) increased significantly from -71 +/- 3 to -85 +/- 3 mV, the depolarization of Vcs elicited by a 10-fold rise in serosal [K+] increased from 34 +/- 4 to 50 +/- 1 mV, the depolarization elicited by lowering serosal [Cl-] from 98 to 8.1 mM was reduced from 15 +/- 1 to 1 +/- 1 mV, and the depolarization in response to lowering serosal [HCO3-] from 10 to 1 mM was reduced from 13 +/- 1 to 5 +/- 0.4 mV. Furosemide could in principle decrease the basolateral membrane Cl- conductance (Gcl), increase the basolateral membrane K+ conductance, or have a combined effect. To distinguish among these possibilities, we estimated the resistance of the basolateral membrane (Rb) by means of two-point intraepithelial cable analysis experiments. Furosemide increased Rb by 22%, which indicates that furosemide reduces basolateral membrane Gcl. The effect cannot be attributed to inhibition of apical membrane anion exchange by serosal addition of furosemide, because base secretion from cells to lumen is unchanged. We conclude that furosemide blocks reversibly basolateral membrane electrodiffusive Cl- permeability. A concomitant stimulation of basolateral membrane electrodiffusive K+ permeability is also possible.

Sugar-stimulated ion absorption is not different in seahare and vertebrate intestine

1988 ◽  
Vol 255 (4) ◽  
pp. R583-R590
Author(s):  
W. M. Moran ◽  
L. T. Garretson
Keyword(s):  
Small Intestine ◽  
Apical Membrane ◽  
Short Circuit ◽  
Electrical Potential ◽  
Short Circuit Current ◽  
Electrical Potential Difference ◽  
Histological Findings ◽  
Ion Absorption ◽  
Ion Substitution ◽  
Membrane Electrical Potential

We have reexamined the notion that sugars stimulate ion absorption differently in invertebrate and vertebrate intestine. In the seahare intestine, mucosal sugar presumably increases the rate of transcellular Na+ and Cl- absorption, whereas only transcellular Na+ absorption is increased in the vertebrate small intestine. Our data indicate that the seahare intestine responds to mucosal D-galactose like the vertebrate small intestine: namely, the apical membrane electrical potential difference depolarizes, the ratio of the mucosal to serosal membrane resistances decreases, and the short-circuit current (Isc) increases. Because mucosal substitution of tetramethylammonium for Na+ abolished the increased Isc, this stimulation resulted from an increase in rheogenic Na+ absorption. Unidirectional transepithelial Cl- fluxes indicate that mucosal D-galactose had no effect on the net Cl- flux under short-circuit conditions. Further, ion substitution experiments indicate that the apical membrane is K+ conductive rather than Cl- conductive as previously reported. These electrophysiological as well as parallel histological findings indicate that studies previously reported on the seahare intestine were in fact conducted on the esophagus.

Resolution of apical from basolateral membrane of shark rectal gland

1986 ◽  
Vol 251 (5) ◽  
pp. C721-C726 ◽  
Author(s):  
W. P. Dubinsky ◽  
L. B. Monti
Keyword(s):  
Membrane Fraction ◽  
Sucrose Gradient ◽  
Apical Membrane ◽  
Specific Activity ◽  
Basolateral Membrane ◽  
Anion Transport ◽  
Squalus Acanthias ◽  
Rectal Gland ◽  
Crude Homogenate ◽  
Shark Rectal Gland

Membrane fractions were isolated from the rectal gland of Squalus acanthias using differential centrifugation and a sucrose gradient run in the presence of 1 M KBr. Using the basolateral membrane marker Na+-K+-ATPase, we obtained a sixfold purification with the most highly purified fraction from the gradient (sp act = 336 +/- 37 mumol X mg protein-1 X h-1). Electrogenic Br- transport was used as a marker activity of the apical membrane, which enabled the identification and purification of a membrane fraction that is highly resolved from the basolateral membrane. The most active fraction was purified approximately 50-fold compared with the crude homogenate. In this fraction, the specific activity of electrogenic anion transport was 296 +/- 87 nmol X mg protein-1 X min-1, whereas the ATPase was only 17.6 +/- 5.7 mumol X mg protein-1 X h-1, representing about a 4-5% contamination of the apical fraction with the basolateral membrane.

Effect of L-alanine and ouabain on membrane conductances and apical membrane potential in Aplysia intestine

1995 ◽  
Vol 268 (4) ◽  
pp. R1050-R1059 ◽  
Author(s):  
S. R. Gabbard ◽  
W. M. Moran
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Electrical Potential ◽  
Membrane Conductance ◽  
Short Circuit Current ◽  
Electrical Potential Difference ◽  
Control Value ◽  
Na Pump ◽  
Proximal Intestine

The proximal intestine of Aplysia californica was employed to assess the effect of alanine absorption on apical membrane K+ conductance (GKa) and basolateral membrane conductance (Gb) and the role of the electrogenic Na(+)-K(+)-adenosinetriphosphatase (Na+ pump) in the repolarization of apical membrane electrical potential difference (Va) after alanine-induced depolarization. Addition of 50 mM L-alanine (isosmotic substitution for mannitol) to the apical superfusate depolarized Va, reduced the ratio of apical to basolateral membrane resistances (Ra/Rb), and stimulated short-circuit current (Isc). Following these initial events, Va repolarized, Ra/Rb increased, and there was a slight decline in Isc. Apical high-K+ artificial seawater revealed an alanine-induced increase in GKa. Washout of alanine from the apical solution increased Ra/Rb above the prealanine control value. Thus alanine absorption is accompanied by an increase in Gb. Basolateral 0.1 mM ouabain abolished alanine-stimulated Isc but had little effect on Va ( < 3 mV depolarization) either before or after exposure to alanine. The repolarization of Va was not affected in tissues superfused with 0.1 mM basolateral ouabain for approximately 3 min even though the alanine-stimulated increase in Isc was abolished. Therefore, the electrogenic Na+ pump contributes minimally to the repolarization of Va in sea hare intestine. The origin of the hyperpolarization of Va resides therefore, at least in part, in the increase in GKa, which restores the driving force for Na(+)-alanine cotransport and prevents K+ accumulation in the enterocytes.

Dibutyryl cAMP activates bumetanide-sensitive electrolyte transport in Malpighian tubules

1991 ◽  
Vol 261 (3) ◽  
pp. C521-C529 ◽  
Author(s):  
J. L. Hegarty ◽  
B. Zhang ◽  
T. L. Pannabecker ◽  
D. H. Petzel ◽  
M. D. Baustian ◽  
...  
Keyword(s):  
Yellow Fever ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Fluid Secretion ◽  
Ringer Solution ◽  
Malpighian Tubules ◽  
Membrane Voltage ◽  
Dibutyryl Camp ◽  
K Secretion ◽  
Transepithelial Voltage

The effects of dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and bumetanide (both 10(-4) M) on transepithelial Na+, K+, Cl-, and fluid secretion and on tubule electrophysiology were studied in isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti. Peritubular DBcAMP significantly increased Na+, Cl-, and fluid secretion but decreased K+ secretion. In DBcAMP-stimulated tubules, bumetanide caused Na+, Cl-, and fluid secretion to return to pre-cAMP control rates and K+ secretion to decrease further. Peritubular bumetanide significantly increased Na+ secretion and decreased K+ secretion so that Cl- and fluid secretion did not change. In bumetanide-treated tubules, the secretagogue effects of DBcAMP are blocked. In isolated Malpighian tubules perfused with symmetrical Ringer solution, DBcAMP significantly hyperpolarized the transepithelial voltage (VT) and depolarized the basolateral membrane voltage (Vbl) with no effect on apical membrane voltage (Va). Total transepithelial resistance (RT) and the fractional resistance of the basolateral membrane (fRbl) significantly decreased. Bumetanide also hyperpolarized VT and depolarized Vbl, however without significantly affecting RT and fRbl. Together these results suggest that, in addition to stimulating electroconductive transport, DBcAMP also activates a nonconductive bumetanide-sensitive transport system in Aedes Malpighian tubules.

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