Membrane polarity of the Na(+)-K+ pump in primary cultures of Xenopus retinal pigment epithelium

The retinal pigment epithelium is a transporting epithelium that helps regulate the volume and composition of the subretinal space surrounding photoreceptor outer segments. The capacity of the RPE to actively transport Na+ and K+ between the retina and the blood supply depends on the localization of the Na+, K(+)-ATPase to the apical membrane, but in culture this polar distribution can be lost. Using primary cultures of Xenopus RPE, we examined the anatomical and functional polarity of this electrogenic pump. Confluent monolayers were established on Matrigel-coated microporous filters and cultured for 2-4 weeks in serum-free defined medium. Electrogenic pump activity at the apical and basolateral membranes was assayed by mounting the monolayer and filter in an Ussing chamber and exposing one or the other surface to ouabain while recording the apical (Vap) and basolateral (Vba) membrane potentials with an intracellular microelectrode. The addition of 0.2 mM ouabain to the apical bath caused Vap to rapidly depolarize by about 4 mV, consistent with the inhibition of a hyperpolarizing pump current at that membrane. When ouabain was added to the basal bath, however, it had no effect on Vba, suggesting the absence of a functional Na(+)-K+ pump on the basolateral membrane. To confirm these electrophysiological results, we examined the distribution of the Na+, K(+)-ATPase catalytic component using an antiserum specific for the bovine kidney alpha subunit. Antibody labeling of cultures was highly polarized, with strong reaction present on the apical microvilli, but not the basolateral cell surfaces. The findings of this study indicate that the Na(+)-K+ pump in monolayers of Xenopus RPE, as in native RPE, is located mainly in the apical membrane, providing evidence of a functionally intact transport pathway in these primary cultures.     

Proton secretion in the upper part of the descending limb of long-looped nephron from hamsters

The proton transport processes in the upper part of the descending limb of the long-looped nephron (LDLu) from hamsters were studied using a fluorescent dye, 2',7'-bis(carboxyethyl)carboxyfluorescein (BCECF) in microperfused single nephron preparations. Intracellular pH (pHi), as assessed by the measurement of the fluorescence of BCECF trapped in the cytoplasm, was 7.23 +/- 0.05 (n = 18) under nominally HCO3--free conditions. Ouabain, when added to the bath, decreased pHi by 0.22 units. After an NH4Cl prepulse, the initial proton extrusion rate was 1.23 +/- 0.26 (n = 9) pH units/min, and was retarded in the presence of 1 mM amiloride either in the bath or in the lumen. pHi failed to recover when Na+ was eliminated from ambient solutions. These observations suggest that Na+/H+ antiporters exist both in the apical and basolateral cell membranes. By measuring tubular fluid pH (pHt) under stopped flow conditions, we examined whether the hamster LDLu has the capacity to generate and maintain a transmural H+ gradient. After the tubular outflow was obstructed, the luminal fluid was rapidly acidified, reaching a steady-state pH of 6.84 +/- 0.09 (n = 7). The steady-state pH was influenced by bath pH. Tubular fluid acidification was not observed in the absence of Na+ and was prevented by ouabain. We conclude that the hamster LDLu has the capability to generate and maintain a transmural proton gradient by proton secretion via a luminal Na+/H+ antiporter which is secondarily driven by the Na+-K+ ATPase in the basolateral membrane.     

A Ba(2+)-insensitive K+ conductance in the basolateral membrane of rabbit cortical thick ascending limb cells

The nature of the K+ exit across the basolateral membrane of microperfused rabbit cortical thick ascending limbs (cTALs) was investigated using the transepithelial and transmembrane potential difference (PDte, PDbl) and conductance measurements. An increase in bath K+ concentration from 4 to 10, 25, 50 mmol/l depolarized the basolateral membrane in a concentration-dependent manner, accompanied by a decrease in the fractional resistance of the basolateral membrane (FRbl). The Cl- channel blocker, 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid (NPPB), did not prevent these effects. The effect of Ba2+ on PDbl was bimodally distributed: paradoxically, in the tubules in which Ba2+ largely depolarized, the effects on PDbl of the bath K+ concentration increases were not inhibited by extracellular Ba2+, in tubules in which Ba2+ moderately depolarized, Ba2+ partially inhibited the K+ concentration increase-induced depolarization of the basolateral membrane. However, the parallel decrease in FRbl was Ba2+ insensitive, indicating that the K+ channel of the basolateral membrane was not modified by extracellular Ba2+. The Ba(2+)-induced depolarizations were prevented by furosemide suggesting that Ba2+ acts by inhibiting basolateral KCl extrusion. Finally, the K+ concentration increase-induced depolarizations were insensitive to tetraethylammonium, charybdotoxin, apamin and verapamil. In conclusion, the present study provides evidence that, in addition to a Ba(2+)-sensitive KCl cotransport system, the basolateral membrane of rabbit cTAL cells possesses a K+ conductance which is insensitive to extracellular Ba2+.     

Isolated perfused rabbit colon crypts: stimulation of Cl- secretion by forskolin

The aim of this study was to characterize ion conductances and carrier mechanisms of isolated in vitro perfused rabbit colonic crypts. Crypts were isolated from rabbit colon mucosa and mounted on a pipette system which allowed controlled perfusion of the lumen. In non-stimulated conditions basolateral membrane voltage (Vbl) was -65 +/- 1 mV (n = 240). Bath Ba2+ (1 mmol/l) and verapamil (0.1 mmol/l) depolarized Vbl by 21 +/- 2 mV (n = 7) and 31 +/- 1 (n = 4), respectively. Lowering of bath Cl- concentration hyperpolarized Vbl from -69 +/- 3 to -75 +/- 3 mV (n = 9). Lowering of luminal Cl- concentration did not change Vbl. Basolateral application of loop diuretics (furosemide, piretanide, bumetanide) had no influence on Vbl in non-stimulated crypts. Forskolin (10(-6) mol/l) in the bath depolarized Vbl by 29 +/- 2 mV (n = 54) and decreased luminal membrane resistance. In one-third of the experiments a spontaneous partial repolarization of Vbl was seen in the presence of forskolin. During forskolin-induced depolarization basolateral application of loop diuretics hyperpolarized Vbl significantly and concentration dependently with a potency sequence of bumetanide > piretanide > or = furosemide. Lowering bath Cl- concentration hyperpolarized Vbl. Lowering of luminal Cl- concentration from 120 to 32 mmol/l during forskolin-induced depolarization led to a further depolarization of Vbl by 7 +/- 2 mV (n = 10). We conclude that Vbl of rabbit colonic crypt cells is dominated by a K+ conductance. Stimulation of the cells by forskolin opens a luminal Cl- conductance. Basolateral uptake of Cl- occurs via a basolateral Na+:2Cl-:K+ cotransport system.     

Carbachol-induced increase of Na+/H+ antiport and recruitment of Na+,K(+)-ATPase in rabbit lacrimal acini

Parallel arrays of Na+/H+ and Cl-/HCO3- antiporters are believed to catalyze the first step of transepithelial electrolyte secretion in lacrimal glands by coupling Na+ and Cl- influxes across acinar cell basolateral membranes. Tracer uptake methods were used to confirm the presence of Na+/H+ antiport activity in membrane vesicles isolated from rabbit lacrimal gland fragments. Outwardly-directed H+ gradients accelerated 22Na+ uptake, and amiloride inhibited 96% of the H+ gradient-dependent 22Na+ flux. Amiloride-sensitive 22Na+ influx was half-maximal at an extravesicular Na+ concentration of 14 mM. In vitro stimulation of isolated lacrimal acini with 10 microM carbachol for 30 min increased Na+/H+ antiport activity of a subsequently isolated basolateral membrane sample 2.5-fold, but it did not significantly affect Na+/H+ antiport activity measured in intracellular membrane samples. The same treatment increased basolateral membrane Na+,K(+)-ATPase activity 1.4-fold; this increase could be accounted for by decreases in the Na+,K(+)-ATPase activities of intracellular membranes. Thus, it appears that cholinergic stimulation causes recruitment of additional Na+,K(+)-ATPase pump units to the acinar cell basolateral plasma membrane. The mechanistic basis of the increase in basolateral membrane Na+/H+ antiport activity remains unclear.     

Potential role of luminal potassium in tubuloglomerular feedback

Transport through the Na+-2Cl(-)-K+ cotransporter in the luminal membrane of macula densa cells is considered critical for tubuloglomerular feedback (TGF). Although various studies could support the importance of luminal Na+ and Cl-, the role of luminal K+ in TGF has not been thoroughly addressed. The study presented here examines this issue in nephrons with superficial glomeruli of anesthetized male Munich-Wistar-Fr?mter rats. Ambient Na+ concentration in early distal tubular fluid was approximately 22 mM, suggesting collection sites relatively close to the macula densa segment. First, it was found that ambient early distal tubular K+ concentration is approximately 1.3 mM, i.e., close to the K+ affinity of the Na+-2Cl(-)-K+ cotransporter in the thick ascending limb. Second, it was observed that a change in late proximal tubular flow rate, i.e., a maneuver that is known to induce a TGF response, significantly alters early distal tubular K+ concentration. Third, previous experiments failed to show an inhibition in TGF response during retrograde perfusion of the macula densa with K+-free solutions. Because of a potential K+ influx into the lumen between the perfusion site and the macula densa, however, the K+ channel blocker U37883A was added to the K+-free perfusate. TGF response was assessed as the fall in nephron filtration rate in response to retrograde perfusion of the macula densa segment from early distal tubular site. It was observed that luminal U37883A (100 microM) significantly attenuated TGF. Because adding 5 mM KCl to the perfusate restored TGF in the presence of U37883A and because the inhibitory action of U37883A on tubular K+ secretion was confirmed, the effect of U37883A on TGF was most likely caused by inhibition of K+ influx into the perfused segment, which decreased luminal K+ concentration at the macula densa. The present findings support a potential role for luminal K+ in TGF, which is in accordance with a transmission of the TGF signal across the macula densa via Na+-2Cl(-)-K+ cotransporter.     

Transcellular chloride pathways in ambystoma proximal tubule

The transport mechanisms of Ambystoma proximal tubule that mediate transcellular Cl- absorption linked to Na+ were investigated in isolated perfused tubules using Cl--selective and voltage-recording microelectrodes. In control solutions intracellular activity of Cl- (aiCl) is 11.3 +/- 0.5 mm, the basolateral (V1), apical (V2), and transepithelial (V3) potential differences are -68 +/- 1.2 mV, +62 +/- 1.2 mV and -6.4 +/- 0.3 mV, respectively. When Na+ absorption is decreased by removal of organic substrates from the lumen, aiCl falls by 1.3 +/- 0.3 mm and V2 hyperpolarizes by +11.4 +/- 1.7 mV. Subsequent removal of Na+ from the lumen causes aiCl to fall further by 2.3 +/- 0.4 mm and V2 to hyperpolarize further by +15.3 +/- 2.4 mV. The contribution of transporters and channels to the observed changes of aiCl was examined using ion substitutions and inhibitors. Apical Na/Cl or Na/K/2Cl symport is excluded because bumetanide, furosemide or hydrochlorothiazide have no effect on aiCl. The effects of luminal HCO-3 removal and/or of disulfonic stilbenes argue against the presence of apical Cl-base exchange such as Cl-HCO3 or Cl-OH. The effects of basolateral HCO-3 removal, of basolateral Na+ removal and/or of disulfonic stilbenes are compatible with presence of basolateral Na-independent Cl-base exchange and Na-driven Cl-HCO3 exchange. Several lines of evidence favor conductive Cl- transport across both the apical and basolateral membrane. Addition of the chloride-channel blocker diphenylamine-2-carboxylate to the lumen or bath, increases the aiCl by 2.4 +/- 0.6 mm or 2.9 +/- 1.0 mm respectively. Moreover, following inhibition by DIDS of all anion exchangers in HCO-3-free Ringer, the equilibrium potential for Cl- does not differ from the membrane potential V2. Finally, the logarithmic changes in aiCl in various experimental conditions correlate well with the simultaneous changes in either basolateral or apical membrane potential. These findings strongly support the presence of Cl- channels at the apical and basolateral cell membranes of the proximal tubule.     

Calbindin D-28k and monoamine oxidase A immunoreactive neurons in the nucleus basalis of Meynert in senile dementia of the Alzheimer type and Parkinson''s disease

The lungs must be kept "dry" for efficient gas exchange. The mechanisms that contribute to clear alveoli from fetal lung fluid at birth are still present during adult life and allow recovery from alveolar flooding. It has recently been shown with the use of different approaches in vitro, as well as in vivo, that alveolar epithelium performs solute-coupled fluid transport. Fluid absorption from alveoli occurs chiefly as a result of active transepithelial Na+ transport. The mechanisms of Na+ transport have been partly elucidated; Na+ enters alveolar cells through apical Na+ channels and Na(+)-coupled solute transporters and is pumped out at the basolateral membrane by a Na(+)-K(+)-adenosinetriphosphatase (ATPase). Transepithelial Na+ transport and fluid absorption are stimulated by beta-adrenergic agonists, with adenosine 3',5'-cyclic monophosphate being the likely intracellular second messenger. K+ is probably secreted into alveoli because its concentration in the epithelial lining fluid is larger than expected for passive distribution. K+ channels have been described that, in conjunction with Na(+)-K(+)-ATP-ase, might provide pathways for active transport. Active proton secretion or bicarbonate absorption have been reported, which may explain the low pH of the alveolar epithelial lining fluid. It is probable that active solute transports are the main determinants of epithelial lining fluid depth and composition. A challenge for the future is to understand how this homeostasis is achieved.     

Mediation by intracellular calcium-dependent signals of hypoxic hyperpolarization in rat hippocampal CA1 neurons in vitro

In response to oxygen deprivation, CA1 pyramidal neurons show a hyperpolarization (hypoxic hyperpolarization), which is associated with a reduction in neuronal input resistance. The role of extra- and intracellular Ca2+ ions in hypoxic hyperpolarization was investigated. The hypoxic hyperpolarization was significantly depressed by tolbutamide (100 microM); moreover, the response was reversed in its polarity in medium containing tolbutamide (100 microM), low Ca2+ (0.25 mM), and Co2+ (2 mM), suggesting that the hypoxic hyperpolarization is mediated by activation of both ATP-sensitive K+ (KATP) channels and Ca(2+)-dependent K+ channels. The hypoxic depolarization in medium containing tolbutamide, low Ca2+, and Co2+ is probably due to inhibition of the electrogenic Na(+)-K+ pump and concomitant accumulation of interstitial K+. Hypoxic hyperpolarizations were depressed in either low Ca2+ (0.25 or 1.25 mM) or high Ca2+ (5 or 7.5 mM) medium (control: 2.5 mM), indicating that there is an optimal extracellular Ca2+ concentration required to produce the hypoxic hyperpolarization. Bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)-AM (50-100 microM), procaine (300 microM), or ryanodine (10 microM) significantly depressed the hypoxic hyperpolarization, suggesting that Ca2+ released from intracellular Ca+ stores may have an important role in the generation of hypoxic hyperpolarization. The high-affinity calmodulin inhibitor N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonomide hydrochloride (W-7) (5 microM) completely blocked, whereas the low-affinity calmodulin inhibitor N-(6-aminohexyl)-1-naphthalenesulfonomide hydrochloride (W-5) (50 microM) did not affect, the hypoxic hyperpolarization. The calmodulin inhibitor trifluoperazine (50 microM) also suppressed the hypoxic hyperpolarization. In addition, calcium/ calmodulin kinase II inhibitor 1-[N,O-bis (1,5-isoquinol-inesulfonyl)-N-methyl-L-tyrosyl]-4-phenyl-pip erazine (KN-62) (10 microM) markedly depressed the amplitude and net outward current of the hypoxic hyperpolarization without affecting the reversal potential. In contrast, neither the myosin light chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexa-hydro-1,4-diazepin hydrochloride (ML-7) (10 microM) nor the protein kinase A inhibitor N-[2-(p-bromocinnamyl-amino) ethyl]-5-isoquinolinesulfonamide (H-89) (1 microM) significantly altered the hypoxic hyperpolarization. These results suggest that calmodulin kinase II, which is activated by calmodulin, may contribute to the generation of the hypoxic hyperpolarization. In conclusion, the present study indicates that, in the majority of hippocampal CA1 neurons, the hypoxic hyperpolarization is due to activation of both KATP channels and Ca(2+)-dependent K+ channels.     

Effects of maternal uninephrectomy on the development of fetal rat kidney: numerical and volumetric changes of the glomerulus and formation of the anionic site in the glomerular basement membrane

1. K+ and Cl- conductances and their putative regulation have been characterized in the rat colonic epithelium by Ussing-chamber experiments, whole-cell and single-channel patch-clamp recordings. 2. The apical Cl- conductance is under the control of intracellular cAMP. An increase in the concentration of this second messenger induces transepithelial Cl- secretion due to the activation of an apical 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB)- and glibenclamide-sensitive Cl- conductance. 3. In addition to the apical Cl- conductance, the basolateral membrane is equipped with Cl- channels. They are stimulated by cell swelling and play a role in cell volume regulation and transepithelial Cl- absorption. 4. The basolateral K+ conductance is under the dominant control of intracellular Ca2+. An increase in the cytosolic Ca2+ concentration leads to the opening of basolateral K+ channels, which causes a hyperpolarization of the cell membrane, indirectly supporting Cl- secretion owing to an increase in the driving force for Cl- exit. The predominant effect of cAMP on the basolateral K+ conductance is an inhibitory one, probably due to a decrease in the intracellular Ca2+ concentration. 5. The apical K+ conductance, which is involved in transepithelial K+ secretion, is stimulated by an increase in the intracellular Ca2+ concentration. 6. The differential regulation of apical and basolateral ion conductances in the epithelium of the rat distal colon provides an interesting example for the mechanisms underlying vectorial transport of ions across polarized cells.     

Active sodium-urea counter-transport is inducible in the basolateral membrane of rat renal initial inner medullary collecting ducts

Rat inner medullary collecting ducts (IMCD3s) possess a luminal Na+-dependent, active urea secretory transport process, which is upregulated by water diuresis. In this study of perfused IMCDs microdissected from base (IMCD1), middle (IMCD2), or tip (IMCD3) of the inner medulla, we tested whether furosemide diuresis alters active urea transport. Rats received furosemide (10 mg/d s.c. for 3-4 d) and were compared with pair-fed control rats. Furosemide significantly decreased urine osmolality and urea clearance, and increased blood urea nitrogen. IMCD3s from furosemide-treated rats had significantly lower rates of active urea secretion than IMCD3s from control rats. IMCD2s showed no active urea transport in control or furosemide-treated rats. IMCD1s from control rats had no active urea transport, but IMCD1s from furosemide-treated rats expressed significant rates of active urea reabsorption. In IMCD1s, this active urea reabsorptive transport process was inhibited by: (i) 0. 25 mM phloretin (bath); (ii) 1 mM ouabain (bath); and (iii) replacing bath Na+ with NMDG+; it was stimulated by 10 nM bumetanide (bath). In summary, we found that furosemide decreased active urea secretion in IMCD3s and induced active urea reabsorption in IMCD1s. The new Na+- dependent, active urea reabsorptive transport process may be a basolateral Na+-urea antiporter.     

Relation of exocytotic release of gamma-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the gamma-aminobutyric acid (GABA) carrier, NNC-711, (1-[(2-diphenylmethylene)amino]oxyethyl)- 1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride, blocks the Ca(2+)-independent release of [3H]GABA from rat brain synaptosomes induced by 50 mM K+ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca(2+)-dependent release of [3H]GABA in the total absence of carrier-mediated release. Reversal of the Na+/Ca2+ exchanger was used to increase the intracellular free Ca2+ concentration ([Ca2+]i) to test whether an increase in [Ca2+]i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca2+]i may rise to values above 400 nM, as a result of Na+/Ca2+ exchange, without inducing release of [3H]GABA, but subsequent K+ depolarization immediately induced [3H]GABA release. Thus, a rise of only a few nanomolar Ca2+ in the cytoplasm induced by 50 mM K+ depolarization, after loading the synaptosomes with Ca2+ by Na+/Ca2+ exchange, induced exocytotic [3H]GABA release, whereas the rise in cytoplasmic [Ca2+] caused by reversal of the Na+/Ca2+ exchanger was insufficient to induce exocytosis, although the value for [Ca2+]i attained was higher than that required for exocytosis induced by K+ depolarization. The voltage-dependent Ca2+ entry due to K+ depolarization, after maximal Ca2+ loading of the synaptosomes by Na+/Ca2+ exchange, and the consequent [3H]GABA release could be blocked by 50 microM verapamil. Although preloading the synaptosomes with Ca2+ by Na+/Ca2+ exchange did not cause [3H]GABA release under any conditions studied, the rise in cytoplasmic [Ca2+] due to Na+/Ca2+ exchange increased the sensitivity to external Ca2+ of the exocytotic release of [3H]GABA induced by subsequent K+ depolarization. Thus, our results show that the vesicular release of [3H]GABA is rather insensitive to bulk cytoplasmic [Ca2+] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca2+ entry through Ca2+ channels near the active zones.     

Contribution of outward currents to spike-frequency adaptation in hypoglossal motoneurons of the rat

Contribution of outward currents to spike-frequency adaptation in hypoglossal motoneurons of the rat. J. Neurophysiol. 78: 2246-2253, 1997. Spike-frequency adaptation has been attributed to the actions of several different membrane currents. In this study, we assess the contributions of two of these currents: the net outward current generated by the electrogenic Na+-K+ pump and the outward current that flows through Ca2+-activated K+ channels. In recordings made from hypoglossal motoneurons in slices of rat brain stem, we found that bath application of a 4-20 microM ouabain solution produced a partial block of Na+-K+ pump activity as evidenced by a marked reduction in the postdischarge hyperpolarization that follows a period of sustained discharge. However, we observed no significant change in either the initial, early, or late phases of spike-frequency adaptation in the presence of ouabain. Adaptation also has been related to increases in the duration and magnitude of the medium-duration afterhyperpolarization (mAHP) mediated by Ca2+-activated K+ channels. When we replaced the 2 mM Ca2+ in the bathing solution with Mn2+, there was a significant decrease in the amplitude of the mAHP after a spike. The decrease in mAHP amplitude resulted in a decrease in the magnitude of the initial phase of spike-frequency adaptation as has been reported previously by others. However, quite unexpectedly we also found that reducing the mAHP resulted in a dramatic increase in the magnitude of both the early and late phases of adaptation. These changes could be reversed by restoring the normal Ca2+ concentration in the bath. Our results with ouabain indicate that the Na+-K+ pump plays little, if any, role in the three phases of adaptation in rat hypoglossal motoneurons. Our results with Ca2+ channel blockade support the hypothesis that initial adaptation is, in part, controlled by conductances underlying the mAHP. However, our failure to eliminate initial adaptation completely by blocking Ca2+ channels suggests that other membrane mechanisms also contribute. Finally, the increase in both the early and late phases of adaptation in the presence of Mn2+ block of Ca2+ channels lends further support to the hypothesis that the initial and later (i.e., early and late) phases of spike-frequency adaptation are mediated by different cellular mechanisms.     

Self-assembled alpha-hemolysin pores in an S-layer-supported lipid bilayer

The mammalian distal colon, which is composed of different cell types, actively transports Na, K and Cl in absorptive and K and Cl in secretory directions. To further characterize the K absorption process and to identify the cells involved in K absorption, unidirectional Rb fluxes and luminal Rb uptake into different epithelial cell types were determined in isolated guinea-pig distal colon. Net Rb absorption (1.5-2.5 micromol.h-1.cm-2) was not influenced by inhibition of Na transport with amiloride or by incubating both sides of the epithelium with Na-free solutions, but was almost completely abolished by luminal ouabain, ethoxzolamide or by incubating both sides of the epithelium with Cl-free solutions. Luminal Rb uptake, blockable by luminal ouabain, preferentially occurred in columnar surface and neck cells, to a lesser extent in surface goblet cells and to an insignificant degree in lower crypt cells. Employing a luminal Rb-Ringer (5.4 mM Rb) the Rb concentration increased within 10 min in columnar surface and neck, surface goblet and lower crypt cells to 70, 32 and about 10 mmol. kg-1 wet weight, respectively. The presence of 5.4 mM K in the luminal incubation solution reduced Rb uptake almost completely indicating a much higher acceptance of the luminal H-K-ATPase for K than for Rb. The increase in Na and decrease in K concentrations in surface and neck cells induced by luminal ouabain might indicate inhibition of the basolateral Na-K-ATPase or drastic enhancement of cellular Na uptake by the Na-H exchanger. Bilateral Na-free incubation did not alter Rb uptake, but bilateral Cl-free incubation drastically reduced it. Inhibition of net Rb absorption by ethoxzolamide and inhibition of both Rb absorption and Rb uptake by bilateral Cl-free incubation support the notion that cellular CO2 hydration is a necessary prerequisite for K absorption and that HCO3 leaves the cell via a Cl-HCO3 exchanger. Since ouabain-inhibitable transepithelial Rb flux and luminal Rb uptake rate by surface and neck cells were about the same, Rb(K) absorption seems to be accomplished mainly by columnar surface cells.     

Ion transport mechanisms responsible for K+ secretion and the transepithelial voltage across marginal cells of stria vascularis in vitro

It has long been accepted that marginal cells of stria vascularis are involved in the generation of the endocochlear potential and the secretion of K+. The present study was designed to provide evidence for this hypothesis and for a cell model proposed to explain K+ secretion and the generation of the endocochlear potential. Stria vascularis from the cochlea of the gerbil was isolated and mounted into a micro-Ussing chamber such that the apical and basolateral membrane of marginal cells could be perfused independently. In this preparation, the transepithelial voltage (Vt) and resistance (Rt) were measured across marginal cells and the resulting equivalent short circuit current (Isc) was calculated (Isc = Vt/Rt). Further, K+ secretion (JK+,probe) was measured with a K(+)-selective vibrating probe in the vicinity of the apical membrane. In the absence of extrinsic chemical driving forces, when both sides of the marginal cell epithelium were bathed with a perilymph-like solution, Vt was 8 mV (apical side positive), Rt was 10 ohm-cm2 and Isc was 850 microA/cm2 (N = 27). JK+,probe was outwardly directed from the apical membrane and reversibly inhibited by basolateral bumetanide, a blocker of the Na+/Cl-/K+ cotransporter. On the basolateral but not apical side, oubain and bumetanide each caused a decline of Vt and an increase of Rt suggesting the presence of the Na,K-ATPase and the Na+/Cl-/K+ cotransporter in the basolateral membrane. The responses to [Cl-] steps demonstrated a significant Cl- conductance in the basolateral membrane and a small Cl- conductance in the paracellular pathway or the apical membrane. The responses to [Na+] steps demonstrated no significant Na+ conductance in the basolateral membrane and a small Na+ or nonselective cation conductance in the apical membrane or paracellular pathway. The responses to [K+] steps demonstrated a large K+ conductance in the apical membrane. Apical application of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and basolateral elevation of K+ caused an increase in Vt and a decrease in Rt consistent with stimulation of the apical K+ conductance. Similar observations have been made in vestibular dark cells, which suggest that strial marginal cells and vestibular dark cells are homologous and transport ions by the same pathways. Taken together, these observations are incompatible with a model for the generation of the endocochlear potential which ascribes the entire potential to the strial marginal cells [Offner et al. (1987) Hear. Res. 29, 117-124].(ABSTRACT TRUNCATED AT 400 WORDS)     

K+ fluxes mediated by Na(+)-K(+)-Cl- cotransport and Na(+)-K(+)-ATPase pumps in renal tubule cell lines transformed by wild-type and temperature-sensitive strains of Simian virus 40

The relative contributions of Na(+)-K(+)-ATPase pumps and Na(+)-K(+)-Cl- cotransport to total rubidium (Rb+) influx into primary cultures of renal tubule cells (PC.RC) and cells transformed either with the wild-type or a temperature-sensitive mutant of the simian virus 40 (SV40), were measured under various growth conditions. The Na(+)-K(+)-ATPase-mediated component represented 74% and 44-48% of total Rb+ influx into PC.RC and SV40-transformed cells, respectively. Proliferating transformed cells showed substantial ouabain-resistant bumetanide-sensitive (Or-Bs) Rb+ influx (41-45% of total) which indicated the presence of a Na(+)-K(+)-Cl- cotransport. The Or-Bs component of Rb+ influx was greatly reduced when temperature-sensitive transformed renal cells (RC.SVtsA58) grown in Petri dishes or on permeable filters were shifted from the permissive (33 degrees C) to the restrictive temperature (39.5 degrees C) to arrest cell growth. The ouabain-sensitive Rb+ influx mediated by the Na(+)-K(+)-ATPase, the total and amiloride-sensitive Na+ uptakes were not modified following inhibition of cell proliferation. A similar fall in the Or-Bs influx was obtained when renal tubule cells transformed by the wild-type SV40 (RC.SV) were incubated with the K+ channel blocker, tetraethylammonium (TEA) ion, which we had previously shown to arrest cell growth without affecting cell viability (Teulon et al.: J. Cell. Physiol., 151:113-125, 1992). Reinitiation of cell growth by removal of TEA or return to 33 degrees C of the temperature-sensitive cells restored the Or-Bs component of Rb influx. Taken together, these results indicate that the Na(+)-K(+)-Cl- cotransport activity is critically dependent on cell growth conditions.     

The mechanism of Na+ transport by rabbit urinary bladder

The mechanism of Na+ transport in rabbit urinary bladder has been studied by microelectrode techniques. Of the three layers of epithelium, the apical layer contains virtually all the transepithelial resistance. There is radial cell-to-cell coupling within this layer, but there is no detectable transverse coupling between layers. Cell coupling is apparently interrupted by intracellular injection of depolarizing current. The cell interiors are electrically negative to the bathing solutions, but the apical membrane of the apical layer depolarizes with increasing Isc. Voltage scanning detects no current sinks at the cell junctions or elsewhere. The voltage-divider ratio, alpha, (ratio of resistance of apical cell membrane, Ralpha, to basolateral cell membrane, Rb) decreases from 30 to 0.5 with increasing Isc, because of the transport-related conductance pathway in the apical membrane. Changes in effective transepithelial capacitance with Isc are predicted and possibly observed. The transepithelial resistance, Rt, has been resolved into Ra, Rb, and the junctional resistance, Rj, by four different methods: cable analysis, resistance of uncoupled cells, measurements of pairs of (Rt, alpha) values in the same bladder at different transport rates, and the relation between Rt and Isc and between alpha and Isc. Rj proves to be effectively infinite (nominally 300 k omega muF) and independent of Isc, and Ra decreases from 154 to 4 omega muF with increasing Isc. In the resulting model of Na+ transport in "tight" epithelia, the apical membrane contains an amiloride-inhibited and Ca++-inhibited conductance pathway for Na+ entry; the basolateral membrane contains a Na+--K+-activated ATPase that extrudes Na+; intracellular (Na+) may exert negative feedback on apical membrane conductance; and aldosterone acts to stimulate Na+ entry at the apical membrane via the amiloride-sensitive pathway.     

Outward potassium current oscillations in macrophage polykaryons: extracellular calcium entry and calcium-induced calcium release

Outward current oscillations associated with transient membrane hyperpolarizations were induced in murine macrophage polykaryons by membrane depolarization in the absence of external Na+. Oscillations corresponded to a cyclic activation of Ca(2+)-dependent K+ currents (IKCa) probably correlated with variations in intracellular Ca2+ concentration. Addition of external Na+ (8 mM) immediately abolished the outward current oscillations, suggesting that the absence of the cation is necessary not only for their induction but also for their maintenance. Oscillations were completely blocked by nisoldipine. Ruthenium red and ryanodine reduced the number of outward current cycles in each episode, whereas quercetin prolonged the hyperpolarization 2- to 15-fold. Neither low molecular weight heparin nor the absence of a Na+ gradient across the membrane had any influence on oscillations. The evidence suggests that Ca2+ entry through a pathway sensitive to Ca2+ channel blockers is elicited by membrane depolarization in Na(+)-free medium and is essential to initiate oscillations, which are also dependent on the cyclic release of Ca2+ from intracellular Ca(2+)-sensitive stores; Ca2+ ATPase acts by reducing intracellular Ca2+, thus allowing slow deactivation of IKCa. Evidence is presented that neither a Na+/Ca2+ antiporter nor Ca2+ release from IP3-sensitive Ca2+ stores participate directly in the mechanism of oscillation.     

Relations among transepithelial sodium transport, potassium exchange, and cell volume in rabbit ileum

The relation between active transepithelial Na transport across rabbit ileum and 42K exchange from the serosal solution across the basolateral membranes has been explored. Although 42K influx across the basolateral membranes is inhibited by ouabain and by complete depletion of cell Na, it is not affected when transepithelial Na transport is abolished (i.e. in the presence of an Na-free mucosal solution) or stimulated (i.e. when glucose or alanine is added to the mucosal solution). We are unable to detect any relation between the ouabain-sensitive Na-K exchange mechanism responsible for the maintenance of intracellular Na and K concentrations and active transcellular Na transport. In addition, the maintenance of cell volume (water content) does not appear to be dependent upon transepithelial Na transport or the ouabain-sensitive Na-K exchange pump. Although the results of these studies cannot be considered conclusive, they raise serious questions regarding the role of the Na-K exchange pump, located at the basolateral membranes, in active transepithelial Na transport and the maintenance of cell volume.     

Ion transport and membrane potential in CNS myelinated axons. II. Effects of metabolic inhibition

Compound resting membrane potential was recorded by the grease gap technique (37 degrees C) during glycolytic inhibition and chemical anoxia in myelinated axons of rat optic nerve. The average potential recorded under control conditions (no inhibitors) was -47 +/- 3 (SD) mV and was stable for 2-3 h. Zero glucose (replacement with sucrose) depolarized the nerve in a monotonic fashion to 55 +/- 10% of control after 60 min. In contrast, glycolytic inhibition with deoxyglucose (10 mM, glucose omitted) or iodoacetate (1 mM) evoked a characteristic voltage trajectory consisting of four distinct phases. A distinct early hyperpolarizing response (phase 1) was followed by a rapid depolarization (phase 2). Phase 2 was interrupted by a second late hyperpolarizing response (phase 3), which led to an abrupt reduction in the rate of potential change, causing nerves to then depolarize gradually (phase 4) to 75 +/- 9% and 55 +/- 6% of control after 60 min, in deoxyglucose and iodoacetate, respectively. Pyruvate (10 mM) completely prevented iodoacetate-induced depolarization. Effects of glycolytic inhibitors were delayed by 20-30 min, possibly due to continued, temporary oxidative phosphorylation using alternate substrates through the tricarboxylic acid cycle. Chemical anoxia (CN- 2 mM) immediately depolarized nerves, and phase 1 was never observed. However a small inflection in the voltage trajectory was typical after approximately 10 min. This was followed by a slow depolarization to 34 +/- 4% of control resting potential after 60 min of CN-. Addition of ouabain (1 mM) to CN--treated nerves caused an additional depolarization, indicating a minor glycolytic contribution to the Na+-K+-ATPase, which is fueled preferentially by ATP derived from oxidative phosphorylation. Phases 1 and 3 during iodoacetate exposure were diminished under nominally zero Ca2+ conditions and abolished with the addition of the Ca2+ chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA; 5 mM). Tetraethylammonium chloride (20 mM) also reduced phase 1 and eliminated phase 3. The inflection observed with CN- was eliminated during exposure to zero-Ca2+/EGTA. A Ca2+-activated K+ conductance may be responsible for the observed hyperpolarizing inflections. Block of Na+ channels with tetrodotoxin (TTX; 1 microM) or replacement of Na+ with the impermeant cation choline significantly reduced depolarization during glycolytic inhibition with iodoacetate or chemical anoxia. The potential-sparing effects of TTX were less than those of choline-substituted perfusate, suggesting additional, TTX-insensitive Na+ influx pathways in metabolically compromised axons. The local anesthetics, procaine (1 mM) and QX-314 (300 microM), had similar effects to TTX. Taken together, the rate and extent of depolarization of metabolically compromised axons is dependent on external Na+. The Ca2+-dependent hyperpolarizing phases and reduction in rate of depolarization at later times may reflect intrinsic mechanisms designed to limit axonal injury during anoxia/ischemia.