Na+ transport by mammalian stomach.

Gastric mucosas from newborn pigs (0--20 days) and rabbits (0--20 days) were used for in vitro investigation of active Na+ transport during resting (no HCl secretion) conditions. As measured with 22Na+, these tissues actively absorb Na+ from the mucosal-to serosal (m-t-s) bathing solution during both open-circuit and short-circuit current (Is) conditions. In the nonsecreting state, net Na+ transport accounts for 40--60% of Isc. The remaining current is provided by net s-to-m flux of Cl-. Amiloride (2-5 X 10(-5) M) in the mucosal solution abolishes this active Na+ transport by inhibiting m-to-s fluxes of Na+ (JNams). In vivo-in vitro experiments showed that active Na+ transport is a normal function of the resting mammalian stomach. Decreasing pH of the mucosal solution below pH 5 reversibly causes decreased current-generating capability of the tissue. Pretreatment of the tissue with amiloride abolishes this pH effect. The implication is that the low pH affects the Na+-entry step into cells. "Titration curves" of current vs. pH had an apparent pK approximately 4.0. Ouabain and K+-free solutions both cause decreases in active Na+ and Cl- current. Calculations indicate that a shunt may account for only a small (less than 30%) percentage of total transepithelial conductance.     

Fluid movements across rabbit ileum coupled to passive paracellular ion movements.

1. Theophylline (10 mM) and choleragen (1 x 10(-6) g ml.-1) abolish net fluid absorption by everted sacs of rabbit ileum. Triaminopyrimidine (20 mM) and ethacrynate (0.1 mM) prevent this inhibition of net fluid movement. Replacing Ringer Cl- with isethionate prevents the theophylline-dependent decrease in fluid absorption also. 2. Ouabain (0.1 mM) abolishes net fluid movements in both control and theophylline-treated tissue. 3. With ouabain present, hypertonic NaCl (200 mM) in the mucosal solution causes net fluid secretion (serosal-mucosal flux). With theophylline added to both the mucosal and serosal solution, net fluid absorption (mucosal-serosal flux) is observed (P less than 0.001). Triaminopyrimidine (20 mM), or ethacrynate (0.1 mM), or replacement of Ringer Na+ with choline, or Ringer Cl- with isethionate all prevent the theophylline-induced reversal of osmotic flow. 4. Theophylline increases passive net flux of Na+ and Cl- from mucosal solution containing hypertonic (200 mM) NaCl+ ouabain (0.1 mM) across sheets of ileum into serosal solution containing mannitol Ringer + ouabain. The increased passive Na+ flux is blocked by triaminopyrimidine and the increased Na+ and Cl- fluxes are blocked by ethacrynate (0.1 mM). 5. The suggested route of increased NaCl leakage is via the paracellular pathway as it is inhibited by triaminopyrimidine. The increase, itself, is a consequence of the increased passive permeability of the mucosal border to Cl-, induced by theophylline or choleragen. Water is apparently electro-osmotically coupled to the paracellular Na+ leakage (100 mole water mole-1 Na+), hence increased passive leakage reverses osmotic flow. In active tissue the lateral intercellular space contains hypertonic NaCl, and hence increased leakage of NaCl across the tight-junction in theophylline or choleragen-treated tissue gives rise to net fluid secretion.     

Factors affecting ammonium uptake by C11 clone of MDCK cells

In several tissues ammonium ions are able to use the transport pathways of other ions, particularly of K+. We investigated this possibility in the C11 clone of MDCK cells, thought to represent intercalated cells, in control and 0 Cl- conditions. Cell pH was measured by ratiometric fluorescence microscopy using the pH indicator BCECF. After preincubating the cells for 10 min in control or 0 Cl- (substituted by gluconate) Ringer, an ammonium pulse was applied to induce cell acidification. The magnitude of the initial alkalinization (DeltapH) was 0.24+/-0.03 ( n=28) pH units in controls, which fell to 0.023+/-0.01 ( n=12) in 0 Cl-, suggesting uptake of NH4+ balancing the alkalinization by NH3. Addition of 10(-3) M bumetanide or furosemide to the 0 Cl- medium, or 10(-4 )M hexamethylene amiloride, did not alter DeltapH. However, with 5 mM Ba+, DeltapH increased to 38% of control. When 2.5x10(-4) M ouabain, an inhibitor of Na+-K+ ATPase, was used, DeltapH increased to 46% of control. Inhibition of H+-K+ ATPase by SCH28080 or by omeprazol caused significant increase in DeltapH. In 0 Cl- solution, these cells underwent a mean volume reduction (-d V) of -10.24+/-1.96% per 10 min as measured by confocal microscopy. To investigate if NH4+ influx was regulated by cell volume or by cell Cl-, volume reduction was avoided by two procedures. When preincubating with NPPB, a Cl- channel blocker, in 0 Cl-, volume reduction was inhibited (d V=-2.12% per 10 min), and DeltapH was 0.24+/-0.04 ( n=5). When the cells were preincubated in hypotonic 0 Cl- (260 mosmol/l), cell volume reduction was abolished (d V=+2.6% per 10 min) and DeltapH was 0.52+/-0.07 ( n=7). Thus, activation of NH4+ influx by several transporters was due to volume reduction rather than to [Cl-] alteration.     

Noise analysis of inward and outward Na+ currents across the apical border of ouabain-treated frog skin

The passive Na+ transport across the apical membrane of frog skin (Rana catesbeiana) was studied under the following circumstances: (1) control conditions (sulfate Ringer's, K+ depolarised serosal membranes); (2) after blocking the active transport step with ouabain; (3) with an outward oriented Na+ current. The amiloride-induced Na+ current fluctuations were analysed to calculate the density of amiloride blockable channels and the current through one single channel. Despite the large reduction of the macroscopic current by ouabain, the single channel current remained unchanged, while the number of amiloride blockable Na+ channels was reduced by a factor of eight. It is concluded from these observations that the earlier described reduction of the permeability of the apical membrane is caused by a decrease of the number of electrically conductive Na+ channels. The outward oriented single channel currents were less than 50% of the currents in the opposite direction. After ouabain, the number of Na+ channels was independent from the current direction.     

Chloride transport in glands of frog skin

The effects of beta-adrenergic stimulation on the bidirectional fluxes of Na+ and Cl- across the frog skin glands were determined. Isoproterenol elicited net serosal-to-mucosal fluxes of both Na+ (JNanet) and Cl- (JClnet) equal to 0.19 +/- 0.05 (SE) and 0.57 +/- 0.05 mueq X cm-2 X h-1, respectively. The residual current (JClnet - JNanet) of 0.38 +/- 0.05 mueq X cm-2 X h-1 closely approximates the isoproterenol-induced short-circuit current of 0.30 +/- 0.04 mueq X cm-2 X h-1. Furosemide added to the serosal side prior to isoproterenol inhibited the isoproterenol-induced net fluxes of both Na+ and Cl-. The addition of dibutyryl cAMP and 3-isobutyl-1-methylxanthine to the serosal side mimicked the action of isoproterenol by stimulating glandular short-circuit current. We conclude that an active Cl(-)-transport mechanism resides in the frog skin glands and is 1) stimulated by a beta-adrenergic agonist (its action is mimicked by cAMP) and 2) inhibited by the loop diuretic furosemide.     

Evidence for Na+ independent active secretion of K+ and HCO 3 − by rat salivary duct epithelium

In order to elucidate whether or not active secretion of potassium and bicarbonate by the rat submaxillary duct epithelium operates independently of sodium reabsorption, Na+ transport was blocked by amiloride, which is known to inhibit Na+ entry from lumen into cell. With 10(-4) M amiloride in HCO - 3 -Ringer at the luminal side, the transepithelial electrical potential difference approached zero, the Na+ conductance of the luminal cell membrane was drastically reduced, and the K+ conductance was significantly reduced. Net K+ secretion was reduced by 80%, whereas net HCO - 3 secretion was significantly increased. The remaining 20% of net K+ secretion proceeded at zero net Na+ transport and in the absence of significant chemical and electrical potential differences between lumen and interstitium of the duct. This active component of net K+ secretion was accompanied by an equal rate of active HCO - 3 secretion. These findings confirm the independence of this active secretion of K+ and HCO - 3 from Na+ transport. They indicate an electrically neutral secretion of K+ and HCO - 3, probably by the postulated luminal K+ -H+ -exchange mechanism. The 80% of net K+ secretion, which were abolished by amiloride together with Na+ reabsorption, seem to be functionally coupled with Na+ transport. The linkage of K+ -to- Na+ is probably mediated by a luminal carrier exchanging Na+ for K+ and H+.     

Muscarinic down-regulation of cAMP-stimulated potassium ion secretion by rabbit distal colon

The sustained effects of the cholinergic agonist carbachol (CCh) on electrolyte transport across the isolated, short-circuited rabbit distal colon were examined in the absence and presence of secretagogue (di-butyryl cyclic-adenosine monophosphate, dB-cAMP). Steady-state, basal absorption of 22Na+, 42K+ (86Rb+), and 36Cl- were not significantly altered by addition of the CCh (10(-4) mmol/l) to the serosal reservoir. Stimulation with dB-cAMP (1.0 mmol/l, serosal) promoted K+ (or Rb+) and Cl- secretion across the colon, without significantly affecting the unidirectional or net fluxes of Na+. Serosal (but not mucosal) addition of CCh to dB-cAMP-stimulated tissues reduced the serosal to mucosal flux of Rb+ (J(Rb)SM) in a concentration-dependent manner with a half-maximum concentration approximately equal 5 micromol/l. Pretreatment with CCh (100 micromol/l, serosal) inhibited dB-cAMP-induced K+ secretion, but had no significant effect on the steady-state unidirectional fluxes of Na+ or Cl-. Serosal histamine (20 micromol/l) also inhibited J(Rb)SM in dB-cAMP-stimulated tissues. Serosal epinephrine (10 micromol/l) promoted a decrease in short-circuit current (Isc) and transepithelial potential (VT) that was mirrored by increases in J(Rb)SM. Both Isc, and VT became more positive and J(Rb)SM was reduced when CCh was added to the epinephrine-stimulated tissues. Serosal muscarine (50 micromol/l) mimicked the CCh-induced inhibition of J(Rb)SM, but serosal nicotine (50 micromol/l) had no effect. In atropine-treated tissues (1 micromol/l, serosal), CCh failed to block dB-cAMP-stimulated increases in J(Rb)SM. The inhibitory action of CCh was observed in tissues that had been pretreated with 50 micromol/l serosal hexamethonium (a ganglionic transmission blocker) or 2 micromol/l serosal tetrodotoxin (a voltage-gated Na+ channel blocker), indicating that the inhibitory action of this cholinergic agonist does not depend on remnant enteric neural pathways. Rubidium ion transport across confluent monolayers of T84 cells was similarly affected by dB-cAMP and CCh, supporting the notion that enteric neural pathways are not required. Serosal charybdotoxin (20 nmol/l) mitigated the inhibitory action of CCh on J(Rb)SM in dB-cAMP-stimulated tissues, suggesting a role for basolateral, Ca2+-dependent K+ channels in the actions of CCh. It is concluded that basolateral muscarinic receptors (and possibly other Ca2+-dependent receptor pathways) of secretory colonocytes mediate the down-regulation of potassium ion secretion by rabbit distal colon, possibly by increasing basolateral membrane K+ conductance.     

Deoxycholic acid (DOC) affects the transport properties of distal colon

Secondary bile acids can induce diarrhea. In the present study we examined the effects of deoxycholic acid (DOC) on equivalent short-circuit current (Isc) in rabbit colon and the cellular mechanisms involved in DOC action (rabbit and rat). Luminal DOC inhibited amiloride-sensitive Na+ absorption. In the presence of amiloride luminal DOC had a concentration dependent effect on Isc. Low concentrations (1-10 micromol/l) induced a lumen-positive current (51+/-3 microA/cm2, 10 micromol/l, n=7) which was inhibited by luminal Ba2+ suggesting the activation of a luminal K+ conductance. Higher luminal concentrations induced a lumen-negative current (-76+/-9 microA/cm2, 100 micromol/l, n=11). Basolateral application of DOC, also in the presence of amiloride, only induced lumen-negative Isc, (-58+/-10 microA/cm2, 100 micromol/l, n=6, EC50= 3 micromol/l). This current could be abolished completely by the K+ channel blocker 293B, a selective inhibitor of cAMP-dependent Cl- secretion. This action of DOC on Isc was additive to the effect of carbachol (CCH) but not additive to that of cAMP. In intact rat colon mucosa pre-treated with DOC a significant increase in cAMP production was observed. Fura-2 measurements of cytosolic Ca2+ activity ([Ca2+]i) in isolated colonic crypts (rabbit and rat) showed that 100 micromol/l DOC induced a weak [Ca2+]i increase. Whole-cell measurements of membrane voltage in isolated rat colonic crypts revealed a hyperpolarization by DOC (4.9+/-0.8 mV, 100 micromol/l, n=8) but a depolarization by prostaglandin E2 (PGE2, via cAMP) (24+/-7 mV, n=8). The present data show that DOC acts at more than one target in the colon: in the intact mucosa it activates luminal K+ channels and Cl- secretion and this is paralleled by an increase in cAMP production. In isolated crypts DOC probably activates a Ca(2+)-regulated K+ conductance but has no effect on cAMP. Hence DOC probably activates ion channels or channel-regulating factors in colonocytes and acts on non-epithelial cells to activate Cl- secretion indirectly.     

Electrophysiological characterization of rat type II pneumocytes in situ

Optimal aeration of the lungs is dependent on an alveolar fluid clearance, a process that is governed by Na+ and Cl- transport. However, the specific contribution of various ion channels in different alveolar cell types under basal or stimulated conditions is not exactly known. We established a novel functional model of rat lung slices suitable for nystatin-perforated whole-cell patch-clamp experiments. Lung slices retained a majority of live cells for up to 72 hours. Type II pneumocytes in situ had a mean capacitance of 8.8 +/- 2.5 pF and a resting membrane potential of -4.4 +/- 1.9 mV. Bath replacement of Na+ with NMDG+ decreased inward whole-cell currents by 70%, 21% and 52% of which were sensitive to 10 microM and 1 mM of amiloride, respectively. Exposure of slices to 0.5 microM dexamethasone for 1 hour did not affect ion currents, while chronic exposure (0.5 microM, 24-72 h) induced an increase in both total Na+-entry currents and amiloride-sensitive currents. Under acute exposure to 100 microM cpt-cAMP, Type II cells in situ rapidly hyperpolarized by 25-30 mV, due to activation of whole-cell Cl- currents sensitive to 0.1 mM of 5-Nitro-2-(3-phenylpropylamino)benzoic acid. In addition, in the presence of cpt-cAMP, total sodium currents and currents sensitive to 10 microM amiloride increased by 32% and 70%, respectively. Thus, in Type II pneumocytes in situ: (1) amiloride-sensitive sodium channels contribute to only half of total Na+-entry and are stimulated by chronic exposure to glucocorticoids; (2) acute increase in cellular cAMP content simultaneously stimulates the entry of Cl- and Na+ ions.     

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

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.     

Maxi K+ channels co-localised with CFTR in the apical membrane of an exocrine gland acinus: possible involvement in secretion

The primary secretion formed in various exocrine glands has a [K+] 2-5 times that of plasma. In this study we measured the transepithelial flux of 36Cl-, 22Na+ and 42K+ across the frog skin and applied the single-channel patch-clamp technique to the apical membrane of frog skin gland acini to investigate the pathway taken by K+ secreted by the glands. Transepithelial K+ secretion was active and was driven by a larger force than the secretion of Na+. When driving Na+ through the epithelium by clamping the transepithelial potential to 100 mV (apical solution reference), blockers of cellular secretion (apical 5-nitro-2-(3-phenylpropylamino)benzoate or basolateral quinine or furosemide) decreased K+ secretion but left Na+ secretion unaffected. We conclude that K+ follows a transcellular pathway across the epithelium. Patch-clamp analysis of the apical membrane of microdissected gland acini revealed a population of voltage- and calcium-activated K+ channels of the maxi K+ type. In cell-attached patches these channels were activated by membrane potential depolarisation or exposure to prostaglandin E2 and had a permeability of 3.6 +/- 0.3 x 10(-13) cm3 s-1, giving a calculated conductance of 170 pS with 125 mM K+ on both sides of the membrane. In inside-out patches the channels were activated by increasing intracellular [Ca2+] from 10(-7) to 10(-6) M and were blocked by Ba2+ added to the cytoplasmic side. Exposure of inside-out patches containing the maxi K+ channel to ATP on the inside activated cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, confirming that both channels are co-localised to the apical membrane. We interpret these findings in terms of a model where transepithelial NaCl secretion can be supported in part by an apical K+ conductance.     

Amphotericin B and K+ transport across excised toad urinary bladder.

The transmural electric PD of bladders bathed by Na2SO4 Ringer was not affected by amphotericin (5 x 10(-6) M, mucosal) but the PD followed the direction for K+ diffusion in the presence of a transmural K+ gradient. Increases in bathing solution K+ increased conductance. Ouabain pretreatment did not affect drug-induced changes in PD or conductance. Unidirectional fluxes of radiolabeled Na+ and K+ but not SO42- across the short-circuited bladder were increased by amphotericin. Ninety percent of the rise in the serosal-to-mucosal flow of Na+ disappeared when mucosal Na+ was replaced by choline. Amphotericin induced a 20-fold increase in mucosal-to-serosal K+ flux but K+ serosal-to-mucosal flow increased 200-fold. This flux asymmetry persisted for 110 min, was abolished by pre- or posttreatment with ouabain, and was immeasurable when bathing solution K+ was increased from 2.4 to 59 meq/liter. With 2.4 meq K+/liter the ratio of active Na+ reabsorption to K+ secretion was 8 to 1, but K+ secretion was not closely linked to Na+ transport. The results suggest that amphotericin induces a paracellular K+-selective path, Na+ isotope exchange, and K+ secretion.     

The effect of amiloride and benzimidazoleguanidine added to the inside medium on electrolyte pathways in the frog skin glands

Serosal amiloride inhibits Na+, K+ and Cl- efflux and reduces short-circuit current and transepithelial conductance in noradrenaline-stimulated frog skin in which the sodium channels in the apical membrane are blocked by amiloride. BIG (benzimidazoleguanidine) inhibits Na+ and K+ efflux and reduces the short-circuit current. In some skins BIG decreased and in others it increased Cl- efflux and conductance. The variable response appears to be due not only to inhibition of salt extrusion from the gland cells but also to activation of Cl- transport through the mitochondria-rich cells, since it was shown that BIG could increase Cl- efflux in gland-free preparations. Different target mechanisms in the gland cells where the two substances may exert their effect are discussed.     

Kinetic analysis of sodium and chloride influxes across the gills of the trout in fresh water.

1. Na and Cl intake through the gill of the perfused head of trout were studied in fresh water with (10(-5)M) or without adrenaline in the perfusing solution. 2. Ionic influxes occur exclusively across the lamellae in fresh water while in the sea-water adapted trout part of salt entry is extralamellar. 3. In absence of adrenaline, Na and Cl enter the gills at the same rate (respectively 6.9 +/- 1.30 and 6.6 +/- 1.55 muequiv/hr. 100 g). Adrenaline (10(-5)M) increased the Na influx to 47.8 +/- 4.12 muequiv/hr. 100 g, a value similar to that observed in vivo. The Cl influx remains unchanged however (6.3 +/- 2.40 muequiv/hr. 100 g), a value much smaller than that found in vivo. 4. Radioactive loading experiments coupled with unloading experiments allowed the determination of the relative permeabilities of the serosal and mucosal barrier for Na+ and Cl-. For both ions, the basal membrane is less permeable. Adrenaline by increasing the Na permeability across the apical barrier enhances the active Na pumping through the basal membrane. 5. Intracellular Na and Cl exchangeable pools were calculated. They represent less than 1% of the total ionic content of the epithelium. Adrenaline increased by sixfold the Na pool without modifying the Cl pool.     

Ouabain, acetazolamide, and Cl-flux in isolated frog skin: evidence for two distinct active Cl-transport mechanisms.

Two distinctly different mechanisms for active Cl- transport in epithelia may exist: one, ouabain-sensitive and cation-dependent, and the other, acetazolamide-sensitive and cation-independent. As a test of this hypothesis the three active Cl- transport systems in isolated short-circuited skin of Rana pipiens were examined. Sensitivity to ouabain (10(-4) M) and acetazolamide (5 X 10(-3) M) and dependence on Na+ and K+ in the medium were ascertained. The first system, net chloride influx in ordinary Ringer, exhibited specific ouabain sensitivity and acetazolamide insensitivity. As we have previously shown this system to be clearly dependent on Na+ on the cis and K+ on the trans side, cation dependence was not re-studied. The second system, isoproterenol-stimulated net Cl- outflux, was also ouabain-sensitive and acetazolamide-insensitive. It was dependent on the presence of Na+ on the cis side, but the K+ dependence was less clear. In contrast to the first two, the third system (net influx in low Cl- medium sulfate Ringer containing 2.4 mM Cl-) was largely ouabain-insensitive, completely acetazolamide-sensitive and independent of both Na+ and K+. Thus, the hypothesis of two distinct mechanisms seems to hold for the three active Cl- transport systems in frog skin. Data from various other Cl- transporting epithelia are examined, and the general applicability of such a scheme of categorization for active Cl- transport mechanisms is discussed.     

Transport and electrical phenomena in resting and secreting piglet gastric mucosa.

1. Gastric mucosae were isolated from piglets (0-5 days old) and mounted in a chamber where electrical properties and secretory function could be measured. Unlike many previously reported mammalian in vitro preparations, pig gastric mucosae were stable and physiologically responsive for many hours after isolation. 2. With similar Ringer solutions bathing both surfaces, the isolated piglet gastric mucosa maintained a p.d. with the mucosal surface 30-35 mV negative with respect to the serosal surface. Limitation of access of Na+ from the mucosal bathing solution to the tissue (e.g. replacement of Na+ on mucosal side with choline or treatment with 10- minus 5 M amiloride) produced a decrease in p.d. and increase in mucosal resistance consistent with an hypothesis of Na+ transport from mucosa to serosa. 3. Isotopic flux measurements (36Cl and 24Na) and net H+ secretory rate were performed during open and short-circuit conditions, while the tissue was at rest and after stimulation of HCl secretion by 6 times 10- minus 5 M histamine. Up to 90% of the respective short-circuit current for resting or secreting mucosae was accounted for as the algebraic sum of Cl minus, H+ or Na+ fluxes. 4. The net transport of Na+ which occurred from mucosa to serosa during rest (ca. 4-7 muequiv/cm2.hr) was somewhat reduced during HCl secretion (ca. 2-7 muequiv/cm2.hr). This active transport of Na+ was more resistant to anaerobiosis than was H+ or Cl minus transport. 5. An active transport component of Cl minus from serosa to mucosa was clearly demonstrable in the non-secreting preparations (ca. 3-9 muequiv/cm2.hr). Active Cl minus transport was stimulated three- to fourfold after H+ secretion was stimulated by histamine. Anaerobiosis promptly reduced Cl minus and H+ transport. An exchange diffusion component was demonstrated for Cl minus which appeared to be prominent during H+ secretory activity and was considerably diminished in resting mucosae. 6. Large changes in mucosal resistance were associated with conditions of rest, histamine stimulation and anaerobic conditions; mean values were 113, 74 and 197 omega.cm2, respectively. Electrical conductance of the isolated gastric mucosa was due primarily to partial ionic conductance of Cl minus (60-65%) and Na+ (10-15%). The partial conductance of H+ was extremely low. The observed increase in tissue conductance associated with H+ secretory activity and the changes in the long-time constant p.d. transient to a current pulse are discussed in terms of the relative contribution of the serosal and mucosal plasma membrane surfaces.     

Effects of ouabain and amiloride on Na pathways in turtle bladders.

In a Na-rich bathing system, addition of amiloride to the mucosal fluid of turtle bladders produces decreased in the transepithelial potential difference (PD), short-circuiting current (I-sc), and conductance. Removal of amiloride results in complete reversal of these changes; and this reversibility is incomplete in amiloride-blocked bladders exposed to ouabain. In a Na-free bathing system, step increased in mucosal [Na] evoke rapid initial spikes in PD, Isc, and conductance, the magnitudes of which are independent of prior ouabain treatment. After these spikes, PD and Isc in the ouabain-treated bladder rapidly decay, while conductance remains unchanged and high. This unchanging conductance, plus the fact that ouabain inhibits half the microsomal (Na plus K). ATPase of this tissue within 1 min, suggests that ouabain inhibits Na pumping without changing tissue conductance. The delayed decrease in conductance (beginning 30 min after ouabain addition), a nonspecific and secondary effect of ouabain, is due to a concomitant collapse of the intercellular channels.     

Dependence of oxygen consumption on Cl- transport and metabolic substrates in the early distal tubule of the Triturus kidney

The early distal tubule (eDT) and the proximal tubule (PT) were dissected as tissue samples from the Triturus kidney. The oxygen consumption (QO2) of these tubules was measured and the effects on QO2 by transport inhibitors and metabolic substrates were studied. In eDT, complete Cl- replacement with NO3- or an addition of furosemide (2 X 10(-5) M) reduced Qo2 from 8.4 +/- 1.7 to 4.4 +/- 0.8 or from 8.3 +/- 1.6 to 4.4 +/- 0.6 nl X min-1 X mg-1 (dry weight), respectively. Also Na+ replacement with choline or with an addition to ouabain (1 X 10(-3) M) reduced QO2 from 5.9 +/- 0.8 to 3.4 +/- 0.4 or from 13.2 +/- 1.8 to 4.7 +/- 0.3 nl X min-1 X mg-1 (dry weight), respectively. In PT, complete Cl- replacement with NO3- or the addition of furosemide did not affect QO2 significantly, but Na+ replacement with choline or the addition of ouabain reduced it from 8.1 +/- 1.0 to 5.4 +/- 0.7 or from 10.8 +/- 0.9 to 7.4 +/- 0.8 nl X min-1 X mg-1 (dry weight), respectively. The data indicate that about a half of the QO2 in eDT is linked to active Cl- transport, in PT, Cl- is not transported actively and is not affected by furosemide. Effects on QO2 by various metabolic substrates, including TCA-cycle intermediates and metabolizable amino acids and carboxylic acids were tested. Among them, succinate, L-glutamate, L-aspartate, citrate, and acetate were found to effectively increase the QO2. The substrate-dependencies of QO2 in eDT were very similar to those in PT, in kinds of effective substrates and in the extent of increase. These findings suggest that, in eDT and PT, the uptake mechanisms for substrates and their coupling with aerobic metabolism are very similar.     

Different types of potassium transport linked to carbachol and gamma-aminobutyric acid actions in rat sympathetic neurons

Carbachol and gamma-aminobutyric acid depolarize mammalian sympathetic neurons and increase the free extracellular K+-concentration. We have used double-barrelled ion-sensitive microelectrodes to determine changes of the membrane potential and of the free intracellular Na+-, K+- and Cl- -concentrations ( [Na+]i, [K+]i and [Cl-]i) during neurotransmitter application. Experiments were performed on isolated, desheathed superior cervical ganglia of the rat, maintained in Krebs solution at 30 degrees C. Application of carbachol resulted in a membrane depolarization accompanied by an increase of [Na+]i, a decrease of [K+]i and no change in [Cl-]i. Application of gamma-aminobutyric acid also induced a membrane depolarization which, however, was accompanied by a decrease of [K+]i and [Cl-]i, whereas [Na+]i remained constant. Blockade of the Na+/K+-pump by ouabain completely inhibited both the reuptake of K+ and the extrusion of Na+ after the action of carbachol, and also the post-carbachol undershoot of the free extracellular K+-concentration. On the other hand, in the presence of ouabain, no changes in the kinetics of the reuptake of K+ released during the action of gamma-aminobutyric acid could be observed. Furosemide, a blocker of K+/Cl- -cotransport, inhibited the reuptake of Cl- and K+ after the action of gamma-aminobutyric acid. In summary, the data reveal that rat sympathetic neurons possess, in addition to the Na+/K+-pump, another transport system to regulate free intracellular K+-concentration. This system is possibly a K+/Cl- -cotransport.