Apical nonspecific cation channels in everted collecting tubules of potassium-adapted ambystoma.

We observed intermediate conductance channels in approximately 20% of successful patch-clamp seals made on collecting tubules dissected from Ambystoma adapted to 50 mm potassium. These channels were rarely observed in collecting tubules taken from animals which were maintained in tap water. Potassium-adaptation either leads to an increase in the number of channels present or activates quiescent channels. In cell-attached patches the conductance averaged 30.3 +/- 2.4 (9) pS. Since replacement of the chloride in the patch pipette with gluconate did not change the conductance, the channel carries cations, not anions. Notably, channel activity was observed at both positive and negative pipette voltages. When the pipette was voltage clamped at 0 mV or positive voltages, the current was directed inward, consistent with the movement of sodium into the cell. The pipette voltage at which the polarity of the current reversed (movement of potassium into the pipette) was -29.6 +/- 6.5(9) mV. Open probability at 0 mV pipette voltage was 0.08 +/- 0.03 and was unaffected when the apical membrane was exposed to either 2 x 10(-6) or 2 x 10(-5) m of amiloride. Exposure of the basolateral surface of the tubule to a saline containing 15 mm potassium caused a significant increase (P less than 0.001) in the open probability of these channels to 0.139 +/- 0.002 without affecting the conductance of the apical channel. These data illustrate the presence of an intermediate conductance, poorly selective, amiloride-insensitive cation channel in native vertebrate collecting tubule. We postulate that, at least in amphibia, this channel may be used to secrete potassium.

Elevation of basolateral K+ induces K+ secretion by apical maxi K+ channels in Ambystoma collecting tubule.

We previously reported that exposure of aquatic-phase Ambystoma tigrinum to a solution containing 50 mM K+ (K+ adaptation) caused a nearly 10-fold increase in the number of detectable maxi K+ channels on the apical membrane of their initial collecting tubules. In apparent contradiction to the notion that maxi K+ channels contribute to K+ secretion, these channels were not routinely active at the resting membrane potential (0 mV voltage clamp). To test the possibility that hyperkalemia yields maxi K+ channels that are secreting K+ (i.e., active at 0 mV), we patch-clamped the apical membranes of initial collecting tubules under conditions of elevated basolateral K+ (15 mM). Seven patches containing maxi K+ channels were studied. Six of the seven patches showed maxi K+ channel activity when voltage was clamped at 0 mV. Open probability and unitary current averaged 0.059 +/- 0.016 and 1.65 +/- 0.50 pA, respectively. This activity, together with the high density of channels observed (1.06 channels/micrometer2), indicates that after K+ adaptation, maxi K+ channels contribute to the ability of the late distal nephron of amphibians to secrete K+.

Environmental KCl causes an upregulation of apical membrane maxi K and ENaC channels in everted Ambystoma collecting tubule.

Patch clamp methods were used to characterize the channels on the apical membrane of initial collecting ducts from Ambystoma tigrinum. Apical membranes were exposed by everting and perfusing fragments of the renal tubule in vitro. Tubules were dissected from two groups of animals; one maintained in tap water, and the other kept in a solution of 50 mM KCl from seven to nineteen days. Patches of apical membranes on tubules taken from animals exposed to tap water expressed low-conductance amiloride sensitive sodium channels (ENaC) in 22 of 49 patches. Only three maxi K channels were observed in this group. In animals exposed to KCl, low-conductance amiloride sensitive sodium channels, 3.7 +/- 0.2 pS (36 of 45 patches) and high-conductance 98.3 +/- 5.0 pS (19 of 45 patches) potassium channels were observed. The estimated density of apical maxi K channels increased dramatically from 0.08 to 0.76 channels/mu 2 in tubules taken from animals exposed to KCl. All but four of nineteen patches which contained maxi K channels also expressed the low conductance sodium channels. Therefore, at least 85% of the maxi K channels studied were in principal cells. We speculate that the increase in maxi K channel activity may represent a mechanism for enhancing the potassium secretory capacity of the initial collecting duct. As expected, exposure of the animals to 50 mM KCl prior to dissection of the initial collecting ducts also increased the estimated density of ENaC from 0.99 to 3.89 channels/mu 2. This upregulation of sodium channel activity is presumably related to the widely recognized effect of potassium loading to increase the plasma aldosterone level.

Effects of vasopressin and aldosterone on the lateral mobility of epithelial Na+ channels in A6 renal epithelial cells.

We have previously demonstrated that apical Na+ channels in A6 renal epithelial cells are associated with spectrin-based membrane cytoskeleton proteins and that the lateral mobility of these channels, as determined by fluorescence photobleach recovery (FPR) analysis, is severely restricted by this association (Smith et al., 1991. Proc. Natl. Acad. Sci. USA 88:6971-6975). Recent data indicate that the actin component of the cytoskeleton may play a role in modulating Na+ channel activity (Cantiello et al., 1991. Am. J. Physiol. 261:C882-C888); however, it is unknown if the Na+ channel's linkage to the spectrin-based membrane cytoskeleton is also involved in regulating channel activity. In this study, we have used FPR to examine if the linkage of the Na+ channels to the membrane cytoskeleton is a site for modulation of Na+ channel activity in filter grown A6 cells by vasopressin and aldosterone. We hypothesized that if the linkage of the Na+ channels to the membrane cytoskeleton is a site for regulation of Na+ channel activity by vasopressin and aldosterone, then hormone-mediated changes in either the membrane cytoskeleton or the affinity of the Na+ channel for the membrane cytoskeleton, should be reflected in changes in the lateral mobility and/or mobile fraction of Na+ channels on the cell surface. FPR revealed that although the rates of lateral mobility were not affected, there was a twofold increase in mobility fraction (f) of apical Na+ channels in aldosterone-treated (16 hr) monolayers (f = 32.31 +/- 5.42%) when compared to control (unstimulated) (f = 14.2 +/- 0.77%) and vasopressin-treated (20 min) (f = 12.7 +/- 2.4%) monolayers. The twofold increase in mobile fraction of Na+ channels corresponds to the average increase in Na+ transport in response to aldosterone in A6 cells. The aldosterone-induced increase in Na+ transport and mobile fraction can be inhibited by the methylation inhibitor, 3-deazaadenosine, consistent with the hypothesis that a methylation event is involved in aldosterone induced upregulation of Na+ transport. We propose that the membrane cytoskeleton is involved in the aldosterone-mediated activation of epithelial Na+ channels.

Effect of basolateral or apical hyposmolarity on apical maxi K channels of everted rat collecting tubule.

We are able to evert and perfuse rat cortical collecting tubules (CCT) at 37 degrees C. Patch-clamp techniques were used to study high-conductance potassium channels (maxi K) on the apical membrane. Under control conditions (150 mM Na+ and 5 mM K+ in pipette and bathing solutions), the slope conductance averaged 109.8 +/- 6.6 pS (12 channels), and reversal potential (expressed as pipette voltage) was +26.3 +/- 2.4 mV. The percent of time the channel spends in the open state and unitary current when voltage was clamped to 0 mV were 1.4 +/- 0.7% and 3.12 +/- 0.42 pA, respectively. In six patches voltage clamped to 0 mV, the isosmotic solution perfused through the everted tubule (basolateral surface) was exchanged for one made 70 mosmol/kgH2O hyposmotic to the control saline. Open probability increased from 0.019 to 0.258, an increase of 0.239 +/- 0.065 (P < 0.005). In four patches where a maxi K channel was evident, no increase in open probability was observed when a hyposmotic saline was placed on the apical surface. However, when vasopressin was present on the basolateral surface, apical application of hyposmotic saline resulted in a series of bursts of channel activity. The average increase in open probability during bursts was (0.055 +/- 0.017, P < 0.005). We conclude that one function of the maxi K channel located in the apical membrane of the rat CCT may be to release intracellular solute (potassium) during a volume regulatory decrease induced by placing a dilute solution on the basolateral surface or when the apical osmolarity is reduced in the presence of vasopressin. These data are consistent with the hypothesis that the physiological role of the channel is to regulate cell volume during water reabsorption.

Amiloride-sensitive apical membrane sodium channels of everted Ambystoma collecting tubule.

Patch clamp methods were used to characterize sodium channels on the apical membrane of Ambystoma distal nephron. The apical membranes were exposed by everting and perfusing initial collecting tubules in vitro. In cell-attached patches, we observed channels whose mean inward unitary current averaged 0.39 +/- 0.05 pA (9 patches). The conductance of these channels was 4.3 +/- 0.2 pS. The unitary current approached zero at a pipette voltage of -92 mV. When clamped at the membrane potential the channel expressed a relatively high open probability (0.46). These characteristics, together with observation that doses of 0.5 to 2 microM amiloride reversibly inhibited the channel activity, are consistent with the presence of the high amiloride affinity, high sodium selectivity channel reported for rat cortical collecting tubule and cultured epithelial cell lines. We used antisodium channel antibodies to identify biochemically the epithelial sodium channels in the distal nephron of Ambystoma. Polyclonal antisodium channel antibodies generated against purified bovine renal, high amiloride affinity epithelial sodium channel specifically recognized 110, 57, and 55 kDa polypeptides in Ambystoma and localized the channels to the apical membrane of the distal nephron. A polyclonal antibody generated against a synthetic peptide corresponding to the C-terminus of Apx, a protein associated with the high amiloride affinity epithelial sodium channel expressed in A6 cells, specifically recognized a 170 kDa polypeptide. These data corroborate that the apically restricted sodium channels in Ambystoma are similar to the high amiloride affinity, sodium selective channels expressed in both A6 cells and the mammalian kidney.

The everted renal tubule: a methodology for direct assessment of apical membrane function.

A technique is described whereby it is possible to evert a 0.5- to 1.0-mm segment of an amphibian renal tubule and perfuse it in vitro. Consequently, the apical membranes of an intact nephron fragment are directly accessible for electrophysiological study. Viability of the cells of everted diluting segments taken from Ambystoma kidney was indicated by 1) failure of the cells to take up trypan blue and 2) the existence of an apical membrane voltage (average 66 mV, cell negative), which decreased predictably in the presence of either 5 mM barium or elevated potassium in the luminal bathing solution. The utility of the everted tubule to patch clamp studies was tested. A large conductance channel that appeared to be selective for potassium could be demonstrated in a cell-attached patch of the apical membrane of an everted initial collecting tubule. The everted tubule preparation not only provides large quantities of apical membrane for patch clamp studies but, more importantly, allows the investigator to control the solutions bathing each membrane surface independently. The application of patch clamp techniques to perfused, everted tubules may then serve to more completely describe the role of the apical membrane in transcellular ion transport.

Flow-dependent potassium secretion by rabbit cortical collecting tubule in vitro.

Cortical collecting tubules (CCT) dissected from rabbits fed a diet designed to stimulate potassium transport secreted potassium in direct proportion to the flow rate in the range of 0.4-3 nl/min (r = 0.79). This relationship was also evident in tubules from rabbits maintained on standard laboratory chow (r = 0.80). The slope of the line relating the two parameters was almost six times greater in tubules from animals fed the special diet. When the range of flow rates was expanded, potassium secretion in nine CCTs appeared to peak at 5-6 nl/min and then failed to increase despite further elevation of flow to nearly 15 nl/min. We investigated the effects of the electrical and chemical gradients on flow-dependent potassium secretion. Because transepithelial voltage was unaffected by changes in axial flow, we conclude that the flow-dependent fraction of potassium secretion is not explained by the electrical gradient. To evaluate the role of luminal potassium concentration on flow-dependent potassium secretion, 11 CCTs were perfused with both 5 and 50 mM potassium solutions at two flow rates (approximately 1.5 and 4.0 nl/min). Increases in both potassium secretion (15.6 +/- 3.9 peq.mm-1.min-1) and sodium reabsorption (11.9 +/- 5.2 peq.mm-1.min-1) were evident in the tubules perfused with 5 mM potassium. Potassium secretion was not reduced by 50 mM luminal potassium at the low flow rate when the largest chemical gradients opposing net secretion were generated. When 50 mM potassium was present in the lumen, increasing flow did not stimulate potassium secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Bicarbonate transport by initial collecting tubule of aquatic- and land-phase amphibia.

The rate of bicarbonate reabsorption (JHCO3) by in vitro initial collecting tubules (ICT) from aquatic- and land-phase Ambystoma tigrinum was measured. These animals intrinsically have different levels of plasma bicarbonate, 17.6 and 27.1 mM, respectively. ICT from both phases reabsorbed bicarbonate at 11 pmol X mm-1 X min-1 when perfused in solutions equilibrated with 3% CO2. The induction of metabolic acidosis or alkalosis had no effect on JHCO3 by in vitro ICT. JHCO3 was abolished when ICT from aquatic animals were perfused with solutions equilibrated with 1% CO2 and returned toward the control value when 3% CO2 was reintroduced. The effect of plasma CO2 partial pressure on JHCO3 of the ICT may explain, at least in part, the observed difference in plasma bicarbonate of the aquatic- and land-phase animals. JHCO3 was reduced to 22% of control with amiloride and to 42% of control when a Na+-free perfusate was used, but was insensitive to ethoxzolamide. These data are consistent with the presence of a sodium-proton exchanger on the apical membrane and a carbonic anhydrase-independent mechanism of H+ secretion.

Bicarbonate transport by amphibian nephron.

To determine the site of bicarbonate reabsorption, tubular fragments from five different segments of the salamander nephron (Ambystoma) were perfused in vitro. Bicarbonate contents (total CO2) of tubular fluid were determined by microcalorimetry. Bicarbonate was not transported by the diluting segment or the mid-distal tubule. Although proximal tubule fragments did appear to reabsorb bicarbonate (10.8 +/- 3.7 pmol X mm-1 X min-1, P less than 0.01, n = 14), the chemical gradients observed were small with respect to the sensitivity of our methods. In the late distal tubule (LDT), bicarbonate reabsorption averaged 28.9 +/- 8.2 pmol X mm-1 X min-1 (P less than 0.01, n = 16). Transport in this segment was inhibited by ethoxzolamide or by perfusing with a sodium-free solution. Ethoxzolamide had no effect on the trans-epithelial voltage. Thus bicarbonate is reabsorbed by the LDT of amphibia via a mechanism that is dependent on carbonic anhydrase and the presence of luminal sodium. The 13 initial collecting tubules (ICT) studied did not appear to reabsorb bicarbonate. On the other hand, some ICT developed substantial bicarbonate gradients. Therefore this nephron segment has the capacity to reabsorb bicarbonate.

The movement of solutes and water across the vertebrate distal nephron.

The similarity (or lack of) of mechanisms for ion and water transport across segments of the distal nephron of the various vertebrate classes is considered. Except for the reptilian distal nephron, the early distal nephrons from vertebrates of different classes appear to share certain morphological and functional characteristics. Among these are a relative impermeability of the tubule to water and the ability to preferentially reabsorb solute. This osmodilution of the luminal contents seems to be attributed to the presence of a sodium-chloride cotransport system located in the lumen membrane. The characteristics of solute and water transport of the late distal tubule are also considered. The available data suggest that there are striking similarities for the solute transport characteristics of the various vertebrate classes which have been studied. On the other hand, not all vertebrates appear to have developed the hydroosmotic response to antidiuretic hormone that has been observed in the mammals.

Direct measurement of potassium leak from single 3 M KCl microelectrodes.

Potassium loss was measured from individual microelectrodes filled with 3 M KCl. The potassium content of a distilled-water droplet into which a microelectrode was inserted for a known time interval was analyzed by helium-glow photometry. The rate of potassium loss from single microelectrodes (18-97 M omega) was within the detectable limits of this method. The rate of potassium loss appeared to decrease with increasing input resistance (determined in 0.16 M NaCl) for electrodes having a low resistance (18-50 M omega). Higher resistance electrodes (greater than 50 M omega) appeared to achieve a uniform rate of potassium loss of 3.5 fmol X s-1. Estimates of the rate of potassium loss calculated from the rate of swelling of impaled cells were identical to those measured directly on the same group of electrodes. We conclude the electrolyte loss not only occurs into a distilled-water drop but also during intracellular recording. An alternative method for fabrication of microelectrodes was tested. Of these electrodes, the ones having an input resistance greater than 50 M omega lost potassium at rates below 1 fmol X s-1.

Heterogeneity in the distal nephron of the salamander (Ambystoma tigrinum): a correlated structure function study of isolated tubule segments.

Studies on isolated perfused tubules of the tiger salamander (Ambystoma tigrinum) have shown that the distal nephron is heterogeneous with respect to function (Stoner, 1977). In this study, the initial portion of the distal tubule (diluting segment) exhibited a voltage, positive in the lumen, and a net absorption of chloride. Since the chloride was transported against an electro-chemical gradient, its transport was active. More distad, the junctional segment exhibited a lumen-negative voltage and sodium, rather than chloride, was transported actively. More recently Delaney and Stoner (1981) have demonstrated in vitro that the collecting duct of this species also has a lumen-negative voltage which is probably associated with active sodium reabsorption. The primary objective of the present paper was to correlate the morphology of the diluting and junctional segments of the Ambystoma distal tubules with the physiologic data from the same isolated perfused tubules. The results indicate that the morphological heterogeneity previously demonstrated in distal tubules of Necturus exists with respect to both structure and function in Ambystoma. The cell types found in the amphibian distal nephron appear to be homologous to those seen in the mammalian nephron.

Na+ transport properties of the peritubular membrane of cortical collecting tubule.

The effects of varying endogenous aldosterone levels on the passive and active properties of the peritubular membrane were studied. Rabbits that were fed either a low Na+ (normal K+) diet or a high Na+, low K+ diet increased or decreased plasma aldosterone, respectively. Tubules were dissected, filled with oil, and incubated in 0 K+ medium to increase intracellular sodium. Cellular sodium and potassium content was measured by helium-glow photometry. The degree to which cells accumulate sodium and lose potassium is a function not only of time of exposure but also of diet. Tubules from animals on a low Na+ diet are about 6 times more permeable to sodium than those from animals fed a high Na+ diet. When tubules were loaded with sodium and returned to a normal (5 mM K+) bathing medium, net sodium efflux and potassium influx occurred. The rate of sodium efflux by cortical collecting tubules dissected from animals on the low Na+ diet was 2.3 times greater than the efflux rate of tubules from animals on the high Na+ diet. These data suggest that high levels of endogenous aldosterone enhance sodium transport measured in vitro across the peritubular membrane of cortical collecting tubule.

Effects of MK-196 and furosemide on rat medullary thick ascending limbs of Henle in vitro.

The effects of the diuretic agents, MK-196 (6,7-dichloro-2-methyl-1-oxo-2-phenyl-5-indanyloxy acetic acid) and furosemide on function of medullary thick ascending limb (tALH) isolated from the rat kidney were studied. tALH were dissected from whole kidneys of male Sprague-Dawley rats which were first perfused in vitro in a recirculating system for 10 to 15 min with a solution containing collagenase. Standard techniques for dissection and perfusion of tALH were followed. Control transepithelial voltage averaged +5.5 +/- 0.5 mV (n = 25). Drug concentration causing 50% inhibition of the voltage (EC50) was determined. EC50 for both MK-196 and furosemide in rat tALH was 8 X 10(-6) M (EC50 for MK-196 in rabbit tALH ws 7 X 10(-5) M). In other experiments, net chloride transport (JCl) in rat tALH averaged 142 +/- 19 pmol.mm-1.min-1 (n = 12), a value nearly twice that reported for the rabbit. In the rat, MK-196 (2 X 10(-4) M) reduced the voltage by 79 +/- 2% and JCl by 76 +/- 13% (n = 6). The same concentration of furosemide inhibited the voltage by 78 +/- 8% and the JCl by 104 +/- 4%(n = 5). Results indicate that: 1) rat tALHs dissected from collagenase-treated kidneys have functional characteristics similar to those reported for the rabbit tALH; and 2) the rat tALH is equally sensitive to MK-196 and furosemide, drugs which act to selectively inhibit active chloride transport.

A single-cell technique for the measurement of membrane potential, membrane conductance, and the efflux of rapidly penetrating solutes in Amphiuma erythrocytes.

We describe a single-cell technique for measuring membrane potential, membrane resistance, and the efflux of rapidly penetrating solutes such as Cl and H2O. Erythrocytes from Amphiuma means were aspirated into a Sylgard (Dow Corning Corp.)-coated capillary. The aspirated cell separated a solution within the capillary from a solution in the bath. Each of these two solutions was contiguous with approximately 5% of the total membrane surface. Microelectrodes placed concentrically within the capillary permit the measurement of intracellular voltage, specific membrane resistance, and the electrical seal between the two solutions. The intracellular voltage averaged -17.7 mV (pH 7.6) and changed as either intra- or extracellular chloride was varied. The average specific membrane resistance measured by passing current across the exposed membrane surface was 110 ohm-cm2. 36Cl and tritiated H2O fluxes (0.84 +/- 0.05 x 10(-6) M . cm-2 . min-1 and 6.4 +/- 1.5 x 10(-3) M . cm-2 . min-1, respectively) were determined by noting the rate at which isotope leaves the cell and crosses the membrane exposed to the bath. Our measured values for the flux of 36Cl and tritiated H2O approximate reported values for free-floating cells. 36Cl efflux, in addition, is inhibited by 4-acetamido-4'-isothiocyano-stilbene 2,2'-disulfonic acid (SITS) and furosemide, known inhibitors of the anion exchange mechanism responsible for the rapid anion fluxes of red blood cells. One can also demonstrate directly that > 89% of 36Cl efflux is "electrically silent" by analyzing the flux in the presence of an imposed transcellular voltage.

The effects of pressure on the water permeability of the descending limb of Henle's loops of rabbits.

Descending limbs of Henle's loops from rabbits were perfused in vitro. Using techniques where the collecting pipets permitted cannulation of the tubule, we were able to maintain reasonable flow rates at lower perfusion reservoir heights than are required with a conventional "Sylgard seal" pipet. The bath was either isosmotic to the perfusate, or was made 300 mOsm hyperosmotic using urea. Net water reabsorption did not occur in tubules perfused at low pressure (average reservoir height = 26 cm H2O) even when the bath was hyperosmotic: delta Jv = -0.06 +/- 0.18 nl/min (n = 7). Observed increases in sodium concentration and osmolality of collected fluid, when the bath was made hyperosmotic, were 16 +/- 8 mM (n - 7) and 254 +/- 38 mOsm (n = 7), respectively. Presumably the large increase in osmolality of the collected fluid was due to entrance of urea. When the "Sylgard seal" collecting end was utilized higher perfusion reservoir heights had to be used to maintain flow (mean height 66 cm H2O). These tubules were highly permeable to water as reported by others for this tubule segment. In the presence of a hyperosmotic bath water extrusion resulted in a dramatic increase in the osmolality of the collected fluid (312 +/- 5 mOsm; 7 tubules) which was almost completely accounted for by an increase in sodium concentration (153 +/- 8 mmole/l; 6 tubules). The 14C urea permeability (measured lumen to bath) of descending limbs in a 300 mOsm bath was 0.64 x 10(-7) cm2 . s-1 +/- 0.23 x 10(-7) (11 tubules). When the bath was made hyperosmotic using urea or raffinose the 14C urea permeability increased significantly.

Calcium transport across segments of the rabbit distal nephron in vitro.

The mechanism of Ca2+ transport by various segments of the distal nephron was studied in vitro using the isolated perfused tubule technique. Calcium absorption in the distal convoluted tubule (DCT) and the granular portion of the cortical collecting duct (CCTg) was significantly enhanced in the presence of parathyroid hormone (PTH), 3 X 10(-2) U/ml. Na+ was absorbed from and K+ was secreted into the lumen of the DCT. The presence of amiloride (5 X 10(-5) M) or furosemide (5 X 10(-5) M) in the perfusate of DCT each caused a partial inhibition of Na+ but not Ca2+ absorption. The foregoing result with Na+ is consistent with the heterogeneous nature of DCT. Net Na+ absorption and K+ secretion also occurred in the CCTg; both processes were completely inhibited by amiloride. Ca2+ absorption occurred in the thick ascending limb of Henle's loop; it was not enhanced by PTH, and the results were consistent with passive movement. No net Ca2+ movement was observed in the nongranular (light) segment of the cortical collecting tubule in the presence or absence of PTH or dibutyryl cyclic adenosine monophosphate.