cAMP mediates the increase in apical membrane Na+ conductance produced in rat CCD by vasopressin

1990 ◽  
Vol 259 (5) ◽  
pp. F823-F831 ◽  
Author(s):  
J. A. Schafer ◽  
S. L. Troutman
Keyword(s):  
Arginine Vasopressin ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Transepithelial Resistance ◽  
Bathing Solution ◽  
Dibutyryl Camp ◽  
Cyclic Monophosphate ◽  
Collecting Ducts ◽  
Transepithelial Voltage ◽  
Stimulation Of

Experiments were conducted to determine if adenosine 3',5'-cyclic monophosphate (cAMP) mediates the stimulation of Na+ absorption by arginine vasopressin (AVP) in isolated perfused cortical collecting ducts (CCD) from rats treated with deoxycorticosterone pivalate (5 mg im) 5-9 days before study. AVP (220 pM) in the bathing solution hyperpolarized the transepithelial voltage (PDT) from -4.0 +/- 0.8 (SE) to -15.1 +/- 1.4 mV (n = 9, P less than 0.001) and decreased the transepithelial resistance (RT) from 40 +/- 8 to 33 +/- 6 omega.cm2 (n = 5, P less than 0.025). Bath addition of 0.2 mM dibutyryl cAMP (DBcAMP), 0.1 mM isobutylmethylxanthine (IBMX), 0.1 mM DBcAMP plus 0.1 mM IBMX, and 10 or 50 microM forskolin produced the same effects, reversibly hyperpolarizing PDT by 7.0-11.5 mV and decreasing RT by 6-12 omega.cm2. Addition of 10 microM amiloride to the luminal perfusate reduced PDT from -0.9 to +2.0 mV and increased RT in the presence or absence of any of the test agents. Addition of DBcAMP + IBMX or 50 microM forskolin to the bathing solution also reversibly depolarized the basolateral membrane voltage of principal cells by 1-2 mV and decreased the apical membrane fractional resistance from 0.82-0.84 to 0.72-0.77. Both effects were reversed by addition of amiloride to the luminal perfusate. These results demonstrate that cAMP is the intracellular mediator of the increase in apical membrane Na+ conductance produced by AVP in the rat CCD.

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.

Conductive properties of papillary surface epithelium

1994 ◽  
Vol 266 (2) ◽  
pp. F259-F265 ◽  
Author(s):  
W. B. Reeves
Keyword(s):  
Cell Membrane ◽  
Apical Membrane ◽  
Ussing Chamber ◽  
Basolateral Membrane ◽  
Transepithelial Resistance ◽  
Surface Epithelium ◽  
Renal Papilla ◽  
Transepithelial Voltage ◽  
K Concentration ◽  
Conductive Properties

The surface epithelium of rabbit renal papilla was dissected free from its supporting tissue and mounted in an Ussing chamber. The conductive properties of the epithelium and of the apical and basolateral cell membranes were examined with KCl-filled microelectrodes. The transepithelial voltage was 0.07 +/- 0.15 mV, and the transepithelial resistance was 107 +/- 15 omega.cm2 (n = 29). The fractional resistance of the apical membrane (fRa) was 0.93 +/- 0.01 (n = 103 cells, 29 tissues). The apical membrane was not conductive to Na+, K+, or Cl-. An increase in the K+ concentration of the basolateral solution from 5 to 50 mM depolarized the basolateral membrane voltage (Vb) from -59 +/- 1.6 to -31.2 +/- 2.2 mV (n = 28 cells) and increased fRa from 0.935 +/- 0.01 to 0.962 +/- 0.01 (P < 0.001, n = 21 cells). Likewise, 5 mM barium in the basolateral solution depolarized Vb from -57.7 +/- 2.0 to -29.8 +/- 2.2 mV (n = 21 cells). A tenfold decrease in the Cl- concentration of the basolateral solution caused an 8.3 +/- 1.9 mV depolarization in Vb. Thus the basolateral cell membrane is conductive to K+ and Cl-. Exposure of the apical membrane to amphotericin B demonstrated that the transepithelial resistance is determined primarily by the paracellular pathway.

Na+ transport in isolated rat CCD: effects of bradykinin, ANP, clonidine, and hydrochlorothiazide

1991 ◽  
Vol 260 (1) ◽  
pp. F86-F95 ◽  
Author(s):  
A. J. Rouch ◽  
L. Chen ◽  
S. L. Troutman ◽  
J. A. Schafer
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Potential Difference ◽  
Na Channel ◽  
Bathing Solution ◽  
Channel Blockers ◽  
Na Transport ◽  
Luminal Membrane ◽  
Collecting Ducts ◽  
Basolateral Membrane Potential

We examined the effects of bradykinin (BK), atrial natriuretic peptide (ANP), hydrochlorothiazide (HCTZ), and clonidine on Na+ transport in isolated perfused cortical collecting ducts from rats treated with deoxycorticosterone. Arginine vasopressin was present in the bathing solution at 220 pM. Clonidine (1 microM, bathing solution) depolarized transepithelial potential difference (PDT) from -11.9 +/- 2.0 (SE) to -7.4 +/- 1.7 mV (P less than 0.001), hyperpolarized basolateral membrane potential difference (PDbl) from -85 +/- 1 to -87 +/- 1 mV (P less than 0.01), and increased the fractional resistance of the apical membrane (FRa) from 0.81 +/- 0.02 to 0.86 +/- 0.02 (P less than 0.03), indicating that it inhibited the Na+ conductance of the luminal membrane. BK (1 or 10 nM) or ANP (10 nM) in the bathing solution had no effect on PDT, PDbl, or FRa. BK, ANP, or 0.1 mM luminal HCTZ also had no effect on lumen-to-bath 22Na+ flux (J1----b), whereas we showed previously that clonidine inhibits J1----b by 30% (L. Chen, M. Paris, S. K. Williams, M. C. Reif, and J. A. Schafer. Kidney Int. 37: 366, 1990). Luminal addition of Na+ channel blockers amiloride (10 microM) or benzamil (1 microM) reduced J1----b to a level not significantly different from bath-to-lumen 22Na+ flux measured previously (M. Reif, S. L. Troutman, and J. A. Schafer. J. Clin. Invest. 77: 1291-1298, 1986), and neither BK nor HCTZ had any further effect. These results show that transcellular Na+ transport occurs exclusively through the apical membrane amiloride-sensitive channel, and this conductance is inhibited by clonidine but not by BK, ANP, or HCTZ.

Electrolyte transport by alkaline gland of little skate Raja erinacea

1985 ◽  
Vol 248 (3) ◽  
pp. R346-R352
Author(s):  
P. L. Smith
Keyword(s):  
Membrane Potential ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Transepithelial Resistance ◽  
Bathing Solution ◽  
Raja Erinacea ◽  
Cl Secretion ◽  
Transepithelial Potential

Transepithelial flux studies and conventional intracellular microelectrode measurements were employed to examine the mechanisms of ion transport by the alkaline gland of the male skate, Raja erinacea. These studies reveal that the transepithelial potential is 6.9 +/- 0.6 mV, lumen reference, and that the transepithelial resistance is 140 omega . cm2. The short-circuit current across this epithelium is entirely accounted for by net secretion of Cl, whereas transepithelial active transport of Na does not appear to be present in this tissue. Cl secretion and/or short-circuit current are reduced by serosal furosemide and abolished when the bathing solution Na is replaced with choline or when ouabain is added to the serosal bathing solution. Intracellular microelectrode studies reveal that the apical membrane potential is -43 mV, cell interior negative to the mucosal bathing solution. The transepithelial resistance in these tissues was 103 +/- 12 omega . cm2 and the apparent fractional resistance, i.e., the ratio of the change in apical membrane potential to the change in transepithelial potential produced by passing current across the epithelium was 0.39 +/- 0.09. Ion substitution experiments demonstrated that the apical membrane is dominated by a large Cl conductance while the basolateral membrane contains a barium-sensitive potassium conductance. These results suggest that the mechanism of Cl secretion by the alkaline gland is similar to the mechanism described for a variety of Cl secretory epithelia.

Potassium transport in cortical collecting tubules from mineralocorticoid-treated rat

1987 ◽  
Vol 253 (1) ◽  
pp. F76-F88 ◽  
Author(s):  
J. A. Schafer ◽  
S. L. Troutman
Keyword(s):  
Arginine Vasopressin ◽  
Flux Ratio ◽  
Potassium Transport ◽  
Passive Movement ◽  
Bathing Solution ◽  
K Secretion ◽  
Transepithelial Voltage ◽  
Unidirectional Fluxes ◽  
Collecting Tubules ◽  
Stimulation Of

Unidirectional fluxes of 86Rb+ were used to examine the stimulation of K+ secretion produced by arginine vasopressin (ADH) in isolated perfused cortical collecting tubules from rats treated with desoxycorticosterone. ADH at 100 microU/ml in the bathing solution increased the bath-to-lumen flux (Jb----l; pmol X min-1 X mm-1) from 16.9 +/- 2.3 to 43.2 +/- 4.6 (n = 16). The lumen-to-bath flux (Jl----b) fell from 3.2 +/- 0.7 to 1.3 +/- 0.4 with ADH due to hyperpolarization of the transepithelial voltage from -12.6 +/- 1.3 to -39.3 +/- 2.0 mV, but the calculated Rb+ permeability was unaltered at 0.20-0.26 micron/s. Although 2 mM lumen Ba2+ inhibited Jb----l by 55 +/- 6%, the flux ratio (Jb----l/Jl----b) of 28 +/- 8 was larger than predicted for passive exchange. In the absence of ADH 2 mM Ba2+ reduced Jb----l to the level predicted for passive movement, but addition of ADH with Ba2+ still present increased Jb----l by an amount identical to that observed without Ba2+, although the absolute Jb----l was less. Simultaneous addition of 2 mM luminal and 4 mM bath Ba2+ also inhibited Jl----b for 22Na+ but not to passive levels. These results indicate either that the concentrations of Ba2+ used were insufficient to block K+ conductances completely or that K+/Rb+ secretion can occur through a Ba2+-insensitive pathway.

Stimulation by cGMP of apical Na channels and cation channels in toad urinary bladder

1991 ◽  
Vol 260 (2) ◽  
pp. C234-C241 ◽  
Author(s):  
S. Das ◽  
M. Garepapaghi ◽  
L. G. Palmer
Keyword(s):  
Urinary Bladder ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Toad Urinary Bladder ◽  
Na Channels ◽  
Serosal Side ◽  
Cyclic Monophosphate ◽  
Stimulation Of

The effects of 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) on apical membrane cation conductances in the toad urinary bladder were investigated. 8-BrcGMP (1 mM) added to the serosal solution increased the amiloride-sensitive short-circuit current (INa) after a delay of 5 min to a steady-state value 1.8 times that of controls achieved after 30 min. Similar effects were seen when the bladders were bathed on the serosal side with a normal NaCl Ringer solution and with a high-K sucrose solution to depolarize the basolateral membrane. Under the latter conditions, the amiloride-sensitive transepithelial conductance increased in parallel with the short-circuit current, indicating stimulation of apical membrane Na channels. The threshold concentration for observing the stimulation of INa was 100 microM, 10-100 times larger than the concentration of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) required to elicit an increase in INa. Currents through an outwardly rectifying Ca-sensitive cation conductance (Iout) were also increased by 1.8-fold relative to controls. This stimulatory effect occurred after a delay of 15 min and reached maximal levels 90-120 min after addition of the nucleotide. The effects of cGMP on INa were not additive with those of 8-BrcAMP or with antidiuretic hormone, an agent known to act by increasing cAMP within the cell. Addition of 1 mM 3-isobutyl-1-methylxanthine to the serosal side of the bladders stimulated INa by 1.3-fold and Iout by 2.4-fold. In both cases, subsequent addition of cGMP produced no further activation of either conductance.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Na+ pump inhibition downregulates an ATP-sensitive K+ channel in rabbit proximal convoluted tubule

1993 ◽  
Vol 264 (4) ◽  
pp. F760-F764 ◽  
Author(s):  
A. M. Hurst ◽  
J. S. Beck ◽  
R. Laprade ◽  
J. Y. Lapointe
Keyword(s):  
Basolateral Membrane ◽  
Proximal Convoluted Tubule ◽  
K Channel ◽  
Bathing Solution ◽  
Channel Activity ◽  
Open Probability ◽  
Functional Link ◽  
Tightly Coupled ◽  
Stimulation Of

In several epithelial and nonepithelial tissues a functional link between the basolateral Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) and a basolateral K+ conductance has been established. However, the nature of this link is unclear. We have previously identified a K+ channel on the basolateral membrane of the proximal convoluted tubule perfused in vitro, the activity of which is increased by stimulation of Na+ transport [J. S. Beck, A. M. Hurst, J.-Y. Lapointe, and R. Laprade. Am. J. Physiol. 264 (Renal Fluid Electrolyte Physiol. 33): F496-F501, 1993]. In the present study we investigate whether basolateral membrane K+ channel activity is tightly coupled to Na(+)-K(+)-ATPase activity. In cell-attached patches (150 mM K+ pipette), following stimulation of channel activity by addition of Na(+)-cotransported solutes to the tubule lumen, mean channel open probability (NPo) was reduced from 0.35 +/- 0.09 to 0.14 +/- 0.06 (n = 7, P < 0.05) by blocking the Na(+)-K(+)-ATPase with 100 microM strophanthidin. In excised patches the channel was reversibly blocked by 2 mM ATP from the cytosolic face of the patch, such that NPo fell to 20.1 +/- 7.0% (n = 5, P < 0.001) of control and recovered to 52.2 +/- 11.2% (n = 5, P < 0.05) after washout of ATP. Diazoxide, a putative opener of ATP-sensitive K+ channels, when added to the bathing solution of an unstimulated tubule (microperfused in the absence of Na(+)-cotransported solutes), increased NPo from 0.046 +/- 0.035 to 0.44 +/- 0.2 (n = 6, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Coupled NaCl entry into Necturus gallbladder epithelial cells

1982 ◽  
Vol 243 (3) ◽  
pp. C140-C145 ◽  
Author(s):  
A. C. Ericson ◽  
K. R. Spring
Keyword(s):  
Epithelial Cells ◽  
Cell Volume ◽  
Apical Membrane ◽  
Ionic Composition ◽  
Basolateral Membrane ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Bathing Solution ◽  
Parallel Operation ◽  
Solute Content

NaCl entry into Necturus maculosus gallbladder epithelial cells was studied by determination of the rate of fluid movement into the cell when the Na+-K+-ATPase was inhibited by 10(-4) M ouabain in the serosal bathing solution. The cell swelling was due to continuing entrance of NaCl into the cell across the apical membrane, which increased the solute content of the cell; the resultant rise in cell osmolality induced water flow and cell swelling. The rate of swelling was 4.3% of the cell volume per minute, equivalent to a volume flow across the apical membrane of 1.44 x 10(-6) cm/s, similar in magnitude to the normal rate of fluid absorption by the gallbladder. We determined the mechanism of NaCl entry by varying the ionic composition of the mucosal bath; when most of the mucosal Na+ or Cl- was replaced, cell volume did not increase during pump inhibition. The rate of NaCl entry was a saturable function of Na+ or Cl- in the mucosal bathing solution with K1/2 values of 26.6 mM for Na+ and 19.5 mM for Cl-. The mode of NaCl entry was probably not the parallel operation of Na+-H+ and Cl(-)-HCO-3 exchangers because of the lack of effect of bicarbonate removal or of the inhibitors amiloride and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. NaCl entry was reversibly inhibited by bumetanide in the mucosal bathing solution. Transepithelial NaCl and water absorption is the result of the coupled, carrier-mediated movement of NaCl into the cell across the apical membrane and the active extrusion of Na+ by the Na+-K+-ATPase in the basolateral membrane.

Forskolin-induced HCO3- current across apical membrane of the frog corneal epithelium

1990 ◽  
Vol 259 (2) ◽  
pp. C215-C223 ◽  
Author(s):  
O. A. Candia
Keyword(s):  
Corneal Epithelium ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Pump Current ◽  
Gas Composition ◽  
Apical Side ◽  
Disulfonic Stilbene ◽  
Stimulation Of

Forskolin (and other Cl- secretagogues) does not affect the very small Na(+)-originated short-circuit current (Isc) across frog corneal epithelium bathed in Cl- free solutions. However, forskolin in combination with increased PCO2 bubbling of the solutions (5-20% CO2) stimulated Isc proportionally to PCO2 to a maximum of approximately 8 microA/cm2. This current could be eliminated and reinstated by sequentially changing the gas composition of the bubbling to 100% air and 20% CO2-80% air. The same effects were observed when PCO2 changes were limited to the apical-side solution. Stroma-to-tear HCO3- movement was deemed unlikely, since the increase in Isc was observed with a HCO3(-)-free solution on the stromal side and CO2 gassing limited to the tear side. From the effects of ouabain and tryptamine, at least 80% of the Isc across the basolateral membrane can be accounted for by the Na+ pump current plus K+ movement from cell to bath. Methazolamide also inhibited Isc. Current across the apical membrane cannot be attributed to an electronegative Na(+)-HCO3- symport given the insensitivity of Isc to a disulfonic stilbene and the fact that stroma-to-tear Na+ fluxes did not increase on stimulation of Isc. The tear-to-stroma Na+ flux also remained unaltered, negating an increased apical bath-to-cell Na+ flow. The forskolin-20% CO2 manipulation produced a depolarization of the intracellular potential, a reduction in the apical-to-basolateral resistance ratio, and a decrease in transepithelial resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

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