Potassium and the photoreceptor-dependent pigment epithelial hyperpolarization

Light-evoked changes in pigment epithelial cell membrane potentials and retinal extracellular potassium ion concentration, [K+]0, were measured in an in vitro frog retina-pigment epithelium-choroid preparation. Light stimuli hyperpolarized the apical membrane of the pigment epithelium. Through an electrical shunt pathway connecting the apical and basal membranes, the basal membrane also hyperpolarized, but to a lesser degree than the apical membrane. This differential hyperpolariation of the two membranes increased the transepithelial potential (TEP). This increase in TEP was shown to be the major voltage source of the c-wave of the electroretinogram (ERG). Direct measurement of [K+]0 in the distal retina, made with K+-specific microelectrodes, showed a light-evoked decrease in [K+]0 having an identical time course to the apical hyperpolarization. There was a linear relationship between the light-evoked change in TEP and the logarithm of [K+]0. This exact relationship was also found when the apical membrane was perfused directly with solutions of varying [K+]0. The change in TEP associated with the ERC c-wave, therefore, was explained solely by the response of the pigment epithelium to the light-evoked decrease in [K+]0 in the distal retina.     

Changes in apical [K+] produce delayed basal membrane responses of the retinal pigment epithelium in the gecko

We describe here a new retinal pigment epithelium (RPE) response, a delayed hyperpolarization of the RPE basal membrane, which is initiated by the light-evoked decrease of [K+]o in the subretinal space. This occurs in addition to an apical hyperpolarization previously described in cat (Steinberg et al., 1970; Schmidt and Steinberg, 1971) and in bullfrog (Oakley et al., 1977; Oakley, 1977). Intracellular and extracellular potentials and measurements of subretinal [K+]o were recorded from an in vitro preparation of neural retina-RPE-choroid from the lizard Gekko gekko in response to light. Extracellularly, the potential across the RPE, the transepithelial potential (TEP), first increased and then decreased during illumination. Whereas the light- evoked decrease in [K+]o predicted the increase in TEP, the subsequent decrease in TEP was greater than predicted by the reaccumulation of [K+]o. Intracellular RPE recordings showed that a delayed hyperpolarization generated at the RPE basal membrane produced the extra TEP decrease. At light offset, the opposite sequence of membrane potential changes occurred. RPE responses to changes in [K+]o were studied directly in the isolated gecko RPE-choroid. Decreasing [K+]o in the apical bathing solution produced first a hyperpolarization of the apical membrane, followed by a delayed hyperpolarization of the basal membrane, a sequence of membrane potential changes identical to those evoked by light. Increasing [K+]o produced the opposite sequence of membrane potential changes. In both preparations, the delayed basal membrane potentials were accompanied by changes in basal membrane conductance. The mechanism by which a change in extracellular [K+] outside the apical membrane leads to a polarization of the basal membrane remains to be determined.     

Effects of hypoxia on potassium homeostasis and pigment epithelial cells in the cat retina

Intracellular recordings show that light-evoked hyperpolarizations of the apical and basal membranes of the cat retinal pigment epithelium (RPE) are altered by mild hypoxia. RPE cells, like glia, have a high K+ conductance, and measurements with K+-sensitive microelectrodes show that the hypoxic changes in the RPE cell are largely the result of changes in extracellular [K+] in the subretinal space [( K+]o) rather than direct effects on RPE cells. During hypoxia, light-evoked [K+]o responses and membrane responses have longer times to peak, slower and less complete recovery during illumination, and larger amplitudes. In addition to the effects on light-evoked responses, hypoxia causes a depolarization of first the apical and then the basal membranes of RPE cells under dark-adapted conditions. The basal depolarization is accompanied by a decrease in basal membrane resistance. These depolarizations appear to be caused by a rapid increase in [K+]o at the onset of hypoxia, which is maximal in dark adaptation, and smaller if the retina is subjected to maintained illumination. All of the effects are graded with the severity of hypoxia and can be observed at arterial oxygen tensions as high as 65 mmHg, although the threshold may be even higher. We argue that the origin of hypoxic [K+]o changes is probably an inhibition of the photoreceptors' Na+/K+ pump. This work then suggests that photoreceptors are more sensitive to hypoxia than previously believed, and that the high oxygen tension normally provided by the choroidal circulation is necessary for normal photoreceptor function.     

Na+-K+ pump activation inhibits endothelium-dependent relaxation by activating the forward mode of Na+/Ca2+ exchanger in mouse aorta

The effect of Na+-K+ pump activation on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) was examined in mouse aorta and mouse aortic endothelial cells (MAECs). The Na+-K+ pump was activated by increasing extracellular K+ concentration ([K+]o) from 6 to 12 mM. In aortic rings, the Na+ ionophore monensin evoked EDR, and this EDR was inhibited by the Na+/Ca2+ exchanger (NCX; reverse mode) inhibitor KB-R7943. Monensin-induced Na+ loading or extracellular Na+ depletion (Na+ replaced by Li+) increased [Ca2+]i in MAECs, and this increase was inhibited by KB-R7943. Na+-K+ pump activation inhibited EDR and [Ca2+]i increase (K+-induced inhibition of EDR and [Ca2+]i increase). The Na+-K+ pump inhibitor ouabain inhibited K+-induced inhibition of EDR. Monensin (>0.1 microM) and the NCX (forward and reverse mode) inhibitors 2'4'-dichlorobenzamil (>10 microM) or Ni2+ (>100 microM) inhibited K+-induced inhibition of EDR and [Ca2+]i increase. KB-R7943 did not inhibit K+-induced inhibition at up to 10 microM but did at 30 microM. In current-clamped MAECs, an increase in [K+]o from 6 to 12 mM depolarized the membrane potential, which was inhibited by ouabain, Ni2+, or KB-R7943. In aortic rings, the concentration of cGMP was significantly increased by acetylcholine and decreased on increasing [K+]o from 6 to 12 mM. This decrease in cGMP was significantly inhibited by pretreating with ouabain (100 microM), Ni2+ (300 microM), or KB-R7943 (30 microM). These results suggest that activation of the forward mode of NCX after Na+-K+ pump activation inhibits Ca2+ mobilization in endothelial cells, thereby modulating vasomotor tone.     

Electrophysiological effects of basolateral [Na+] in Necturus gallbladder epithelium

In Necturus gallbladder epithelium, lowering serosal [Na+] ([Na+]s) reversibly hyperpolarized the basolateral cell membrane voltage (Vcs) and reduced the fractional resistance of the apical membrane (fRa). Previous results have suggested that there is no sizable basolateral Na+ conductance and that there are apical Ca(2+)-activated K+ channels. Here, we studied the mechanisms of the electrophysiological effects of lowering [Na+]s, in particular the possibility that an elevation in intracellular free [Ca2+] hyperpolarizes Vcs by increasing gK+. When [Na+]s was reduced from 100.5 to 10.5 mM (tetramethylammonium substitution), Vcs hyperpolarized from -68 +/- 2 to a peak value of -82 +/- 2 mV (P less than 0.001), and fRa decreased from 0.84 +/- 0.02 to 0.62 +/- 0.02 (P less than 0.001). Addition of 5 mM tetraethylammonium (TEA+) to the mucosal solution reduced both the hyperpolarization of Vcs and the change in fRa, whereas serosal addition of TEA+ had no effect. Ouabain (10(-4) M, serosal side) produced a small depolarization of Vcs and reduced the hyperpolarization upon lowering [Na+]s, without affecting the decrease in fRa. The effects of mucosal TEA+ and serosal ouabain were additive. Neither amiloride (10(-5) or 10(-3) M) nor tetrodotoxin (10(-6) M) had any effects on Vcs or fRa or on their responses to lowering [Na+]s, suggesting that basolateral Na+ channels do not contribute to the control membrane voltage or to the hyperpolarization upon lowering [Na+]s. The basolateral membrane depolarization upon elevating [K+]s was increased transiently during the hyperpolarization of Vcs upon lowering [Na+]s. Since cable analysis experiments show that basolateral membrane resistance increased, a decrease in basolateral Cl- conductance (gCl-) is the main cause of the increased K+ selectivity. Lowering [Na+]s increases intracellular free [Ca2+], which may be responsible for the increase in the apical membrane TEA(+)-sensitive gK+. We conclude that the decrease in fRa by lowering [Na+]s is mainly caused by an increase in intracellular free [Ca2+], which activates TEA(+)-sensitive maxi K+ channels at the apical membrane and decreases apical membrane resistance. The hyperpolarization of Vcs is due to increase in: (a) apical membrane gK+, (b) the contribution of the Na+ pump to Vcs, (c) basolateral membrane K+ selectivity (decreased gCl-), and (d) intraepithelial current flow brought about by a paracellular diffusion potential.     

Interaction between the basolateral K+ and apical Na+ conductances in Necturus urinary bladder

Experimental modulation of the apical membrane Na+ conductance or basolateral membrane Na+-K+ pump activity has been shown to result in parallel changes in the basolateral K+ conductance in a number of epithelia. To determine whether modulation of the basolateral K+ conductance would result in parallel changes in apical Na+ conductance and basolateral pump activity, Necturus urinary bladders stripped of serosal muscle and connective tissue were impaled through their basolateral membranes with microelectrodes in experiments that allowed rapid serosal solution changes. Exposure of the basolateral membrane to the K+ channel blockers Ba2+ (0.5 mM/liter), Cs+ (10 mM/liter), or Rb+ (10 mM/liter) increased the basolateral resistance (Rb) by greater than 75% in each case. The increases in Rb were accompanied simultaneously by significant increases in apical resistance (Ra) of greater than 20% and decreases in transepithelial Na+ transport. The increases in Ra, measured as slope resistances, cannot be attributed to nonlinearity of the I-V relationship of the apical membrane, since the measured cell membrane potentials with the K+ channel blockers present were not significantly different from those resulting from increasing serosal K+, a maneuver that did not affect Ra. Thus, blocking the K+ conductance causes a reduction in net Na+ transport by reducing K+ exit from the cell and simultaneously reducing Na+ entry into the cell. Close correlations between the calculated short-circuit current and the apical and basolateral conductances were preserved after the basolateral K+ conductance pathways had been blocked. Thus, the interaction between the basolateral and apical conductances revealed by blocking the basolateral K+ channels is part of a network of feedback relationships that normally serves to maintain cellular homeostasis during changes in the rate of transepithelial Na+ transport.     

Delayed basal hyperpolarization of cat retinal pigment epithelium and its relation to the fast oscillation of the DC electroretinogram

Previous work has shown that the cat retinal pigment epithelium (RPE) is the source of two potential changes that follow the absorption of light by photoreceptors: a hyperpolarization of the apical membrane, peaking in 2-4 s, which leads to the RPE component of the electroretinogram (ERG) c-wave, and a depolarization of the basal membrane, peaking in 5 min, which leads to the light peak. This paper describes a new basal membrane response of intermediate time course, called the delayed basal hyperpolarization. Isolation of this response from other RPE potentials showed that with maintained illumination the hyperpolarization begins approximately 2 s after light onset, peaks in 20 s, and slowly ends as the membrane repolarizes over the next 60 s. The delayed basal hyperpolarization is very small for stimuli less than 4 s in duration and grows with duration, becoming approximately 15% as large as the preceding apical hyperpolarization with stimuli longer than 20 s. Extracellularly, this response contributes to the transepithelial potential (TEP) across the RPE. In response to light the TEP first rises to a peak, the c-wave, as the apical membrane hyperpolarizes. For stimuli longer than approximately 4 s, the decline of the TEP from the peak of the c-wave results partly from the recovery of apical membrane potential and partly from the delayed basal hyperpolarization. For long periods of illumination (300 s) the delayed basal hyperpolarization leads to a trough in the TEP between the c-wave and light peak. This trough is largely responsible for a corresponding trough in vitreal recordings, which has been called the "fast oscillation." The term "fast oscillation" has also been used to denote the sequence of potential changes resulting from repeated stimuli approximately 1 min in duration. In addition to the delayed basal hyperpolarization, such responses also contain a basal off-response, a delayed depolarization.     

Phospholemman overexpression inhibits Na+-K+-ATPase in adult rat cardiac myocytes: relevance to decreased Na+ pump activity in postinfarction myocytes.

Messenger RNA levels of phospholemman (PLM), a member of the FXYD family of small single-span membrane proteins with putative ion-transport regulatory properties, were increased in postmyocardial infarction (MI) rat myocytes. We tested the hypothesis that the previously observed reduction in Na+-K+-ATPase activity in MI rat myocytes was due to PLM overexpression. In rat hearts harvested 3 and 7 days post-MI, PLM protein expression was increased by two- and fourfold, respectively. To simulate increased PLM expression post-MI, PLM was overexpressed in normal adult rat myocytes by adenovirus-mediated gene transfer. PLM overexpression did not affect the relative level of phosphorylation on serine68 of PLM. Na+-K+-ATPase activity was measured as ouabain-sensitive Na+-K+ pump current (Ip). Compared with control myocytes overexpressing green fluorescent protein alone, Ip measured in myocytes overexpressing PLM was significantly (P < 0.0001) lower at similar membrane voltages, pipette Na+ ([Na+]pip) and extracellular K+ ([K+]o) concentrations. From -70 to +60 mV, neither [Na+]pip nor [K+]o required to attain half-maximal Ip was significantly different between control and PLM myocytes. This phenotype of decreased V(max) without appreciable changes in K(m) for Na+ and K+ in PLM-overexpressed myocytes was similar to that observed in MI rat myocytes. Inhibition of Ip by PLM overexpression was not due to decreased Na+-K+-ATPase expression because there were no changes in either protein or messenger RNA levels of either alpha1- or alpha2-isoforms of Na+-K+-ATPase. In native rat cardiac myocytes, PLM coimmunoprecipitated with alpha-subunits of Na+-K+-ATPase. Inhibition of Na+-K+-ATPase by PLM overexpression, in addition to previously reported decrease in Na+-K+-ATPase expression, may explain altered V(max) but not K(m) of Na+-K+-ATPase in postinfarction rat myocytes.     

Reaccumulation of [K+]o in the toad retina during maintained illumination

Using K+-selective microelectrodes, [K+]o was measured in the subretinal space of the isolated retina of the toad, Bufo marinus. During maintained illumination, [K+]o fell to a minimum and then recovered to a steady level that was approximately 0.1 mM below its dark level. Spatial buffering of [K+]o by Müller (glial) cells could contribute to this reaccumulation of K+. However, superfusion with substances that might be expected to block glial transport of K+ had no significant effect upon the reaccumulation of K+. These substances included blockers of gK (TEA+, Cs+, Rb+, 4-AP) and a gliotoxin (alpha AAA). Progressive slowing of the rods' Na+/K+ pump (perhaps caused by a light-evoked decrease in [Na+]i) also could contribute to this reaccumulation of K+ by reducing the uptake of K+ from the subretinal space. As evidence for a major contribution by this mechanism, treatments designed to prevent such slowing of the pump reversibly blocked reaccumulation. These treatments included superfusion with 2 microM ouabain, or lowering [K+]o, PO2, or temperature. It is likely that such treatments inhibit the pump, increase [Na+]i, and attenuate any light-evoked decrease in [Na+]i. The results are consistent with the following hypothesis. At light onset, the decrease in rod gNa will reduce the Na+ influx and the resulting rod hyperpolarization will reduce the K+ efflux. In combination with these reduced passive fluxes, the continuing active fluxes will lower both [K+]o and [Na+]i, which in turn will inhibit the pump. In support of this hypothesis, the solutions to a pair of coupled differential equations that model changes in both [K+]o and [Na+]i match quantitatively the time course of the observed changes in [K+]o during and after maintained illumination for all stimuli examined.     

Alpha-1-adrenergic modulation of K and Cl transport in bovine retinal pigment epithelium.

Intracellular microelectrode techniques were used to characterize the electrical responses of the bovine retinal pigment epithelium (RPE)-choroid to epinephrine (EP) and several other catecholamines that are putative paracrine signals between the neural retina and the RPE. Nanomolar amounts of EP or norepinephrine (NEP), added to the apical bath, caused a series of conductance and voltage changes, first at the basolateral or choroid-facing membrane and then at the apical or retina-facing membrane. The relative potency of several adrenergic agonists and antagonists indicates that EP modulation of RPE transport begins with the activation of apical alpha-1-adrenergic receptors. The membrane-permeable calcium (Ca2+) buffer, amyl-BAPTA (1,2-bis(o-aminophenoxy)-ethane-N,N,N',N' tetraacetic acid) inhibited the EP-induced voltage and conductance changes by approximately 50-80%, implicating [Ca2+]i as a second messenger. This conclusion is supported by experiments using the Ca2+ ionophore A23187, which mimics the effects of EP. The basolateral membrane voltage response to EP was blocked by lowering cell Cl, by the presence of DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) in the basal bath, and by current clamping VB to the Cl equilibrium potential. In the latter experiments the EP-induced conductance changes were unaltered, indicating that EP increases basolateral membrane Cl conductance independent of voltage. The EP-induced change in basolateral Cl conductance was followed by a secondary decrease in apical membrane K conductance (approximately 50%) as measured by delta [K]o-induced diffusion potentials. Decreasing apical K from 5 to 2 mM in the presence of EP mimicked the effect of light on RPE apical and basolateral membrane voltage. These results indicate that EP may be an important paracrine signal that provides exquisite control of RPE physiology.     

Potassium modulation of taurine transport across the frog retinal pigment epithelium

Net taurine transport across the frog retinal pigment epithelium-choroid was measured as a function of extracellular potassium concentration, [K+]o. The net rate of retina-to-choroid transport increased monotonically as [K+]o increased from 0.2 mM to 2 mM on the apical (neural retinal) side of the tissue. No further increase was observed when [k+]o was elevated to 5 mM. The [K+]o changes that modulate taurine transport approximate the light-induced [K+]o changes that occur in the extracellular space separating the photoreceptors and the apical membrane of the pigment epithelium. The taurine-potassium interaction was studied by using rubidium as a substitute for potassium and measuring active rubidium transport as a function of extracellular taurine concentration. An increase in apical taurine concentration, from 0.2 mM to 2 mM, produced a threefold increase in active rubidium transport, retina to choroid. Net taurine transport can also be altered by relatively large, 55 mM, changes in [Na+]o. Apical ouabain, 10(-4) M, inhibited active taurine, rubidium, and potassium transport; in the case of taurine, this inhibition is most likely due to a decrease in the sodium electrochemical gradient. In sum, these results suggest that the apical membrane contains a taurine, sodium co-transport mechanism whose rate is modulated, indirectly, through the sodium pump. This pump has previously been shown to be electrogenic and located on the apical membrane, and its rate is modulated, indirectly, by the taurine co-transport mechanism.     

Identification and functional characterization of a dual GABA/taurine transporter in the bullfrog retinal pigment epithelium

Intracellular microelectrodes, fluorescence imaging, and radiotracer flux techniques were used to investigate the physiological response of the retinal pigment epithelium (RPE) to the major retinal inhibitory neurotransmitter, gamma-aminobutyric acid (GABA). GABA is released tonically in the dark by amphibian horizontal cells, but is not taken up by the nearby Muller cells. Addition of GABA to the apical bath produced voltage responses in the bullfrog RPE that were not blocked nor mimicked by any of the major GABA-receptor antagonists or agonists. Nipecotic acid, a substrate for GABA transport, inhibited the voltage effects of GABA. GABA and nipecotic acid also inhibited the voltage effects of taurine, suggesting that the previously characterized beta- alanine sensitive taurine carrier also takes up GABA. The voltage responses of GABA, taurine, nipecotic acid, and beta-alanine all showed first-order saturable kinetics with the following Km's: GABA (Km = 160 microM), beta-alanine (Km = 250 microM), nipecotic acid (Km = 420 microM), and taurine (Km = 850 microM). This low affinity GABA transporter is dependent on external Na, partially dependent on external Cl, and is stimulated in low [K]o, which approximates subretinal space [K]o during light onset. Apical GABA also produced a significant conductance increase at the basolateral membrane. These GABA-induced conductance changes were blocked by basal Ba2+, suggesting that GABA decreased basolateral membrane K conductance. In addition, the apical membrane Na/K ATPase was stimulated in the presence of GABA. A model for the interaction between the GABA transporter, the Na/K ATPase, and the basolateral membrane K conductance accounts for the electrical effects of GABA. Net apical-to-basal flux of [3H]-GABA was also observed in radioactive flux experiments. The present study shows that a high capacity GABA uptake mechanism with unique pharmacological properties is located at the RPE apical membrane and could play an important role in the removal of GABA from the subretinal space (SRS). This transporter could also coordinate the activities of GABA and taurine in the SRS after transitions between light and dark.     

Aging augments interstitial K+ concentrations in active muscle of rats.

Skeletal muscle performance declines with advancing age, and the underlying mechanism is not completely understood. A large body of convincing evidence has demonstrated a crucial role for interstitial K+ concentration ([K+]o) in modulating contractile function of skeletal muscle. The present study tested the hypothesis that during muscle contraction there is a greater accumulation of [K+]o in aged compared with adult skeletal muscle. Twitch muscle contraction was induced by electrical stimulation of the sciatic nerves of 8- and 32-mo-old Fischer 344 x Brown Norway rats. Levels of [K+]o were measured continuously by a microdialysis technique with the probes inserted into the gastrocnemius muscle. Stimulation at 1, 3, and 5 Hz elevated muscle [K+]o by 52, 64, and 88% in adult rats, and by 78, 98, and 104% in aged rats, respectively, and the increase was significantly higher in aged than in adult rats. Recovery for [K+]o, as measured by the time for [K+]o to recover by 20 and 50% from peak response after stimulation, was slower in aged rats. Ouabain (5 mM), a specific inhibitor of the Na+-K+ pump, was added in the perfusate to inhibit the reuptake of K+ into the cells to assess the role of the pump in the overall K+ balance. Ouabain elevated muscle [K+]o at rest, and the effect was significantly attenuated in aged animals. The present data demonstrated an augmented [K+]o in aged skeletal muscle compared with adult skeletal muscle, and the data suggested that an alteration in the function of the Na+-K+ pump may contribute, in part, to the deficiency in K+ balance in skeletal muscle of aged rats.     

Potassium-dependent volume regulation in retinal pigment epithelium is mediated by Na,K,Cl cotransport

Changes in retinal pigment epithelial (RPE) cell volume were measured by monitoring changes in intracellular tetramethylammonium (TMA) using double-barreled K-resin microelectrodes. Hyperosmotic addition of 25 or 50 mM mannitol to the Ringer of the apical bath resulted in a rapid (approximately 30 s) osmometric cell shrinkage. The initial cell shrinkage was followed by a much slower (minutes) secondary shrinkage that is probably due to loss of cell solute. When apical [K+] was elevated from 2 to 5 mM during or before a hyperosmotic pulse, the RPE cell regulated its volume by reswelling towards control within 3-10 min. This change in apical [K+] is very similar to the increase in subretinal [K+]o that occurs after a transition from light to dark in the intact vertebrate eye. The K-dependent regulatory volume increase (RVI) was inhibited by apical Na removal, Cl reduction, or the presence of bumetanide. These results strongly suggest that a Na(K),Cl cotransport mechanism at the apical membrane mediates RVI in the bullfrog RPE. A unique aspect of this cotransporter is that it also functions at a lower rate under steady-state conditions. The transport requirements for Na, K, and Cl, the inhibition of RVI by bumetanide, and thermodynamic calculations indicate that this mechanism transports Na, K, and Cl in the ratio of 1:1:2.     

Effect of taurine on the isolated retinal pigment epithelium of the frog: Electrophysiologic evidence for stimulation of an apical,electrogenic Na+−K+ pump

Summary The apical surface of the retinal pigment epithelium (RPE) faces the neural retina whereas its basal surface faces the choroid. Taurine, which is necessary for normal vision, is released from the retina following light exposure and is actively transported from retina to choroid by the RPE. In these experiments, we have studied the effects of taurine on the electrical properties of the isolated RPE of the bullfrog, with a particular focus on the effects of taurine on the apical Na⁺–K⁺ pump.Acute exposure of the apical, but not basal, membrane of the RPE to taurine decreased the normally apical positive transepithelial potential (TEP). This TEP decrease was generated by a depolarization of the RPE apical membrane and did not occur when the apical bath contained sodium-free medium. With continued taurine exposure, the initial TEP decrease was sometimes followed by a recovery of the TEP toward baseline. This recovery was abolished by strophanthidin or ouabain, indicating involvement of the apical Na⁺–K⁺ pump.To further explore the effects of taurine on the Na⁺–K⁺ pump, barium was used to block apical K⁺ conductance and unmask a stimulation of the pump that is produced by increasing apical [K⁺] ₀ . Under these conditions, increasing [K⁺] ₀ hyperpolarized the apical membrane and increased TEP. Taurine reversibly doubled these responses, but did not change total epithelial resistance or the ratio of apical-to-basal membrane resistance, and ouabain abolished these responses.Collectively, these findings indicate the presence of an electrogenic Na⁺/taurine cotransport mechanism in the apical membrane of the bullfrog RPE. They also provide direct evidence that taurine produces a sodium-dependent increase in electrogenic pumping by the apical Na⁺–K⁺ pump.     

Ion transport mechanisms in the mesonephric collecting duct system of the toad Bufo bufo: microelectrode recordings from isolated and perfused tubules

It is not clear how and whether terrestrial amphibians handle NaCl transport in the distal nephron. Therefore, we studied ion transport in isolated perfused collecting tubules and ducts from toad, Bufo bufo, by means of microelectrodes. No qualitative difference in basolateral cell membrane potential (Vbl) was observed between tubules and ducts in response to ion substitutions, inhibitor and agonist applications. Cl- substitution experiments indicated a small Cl- conductance in the basolateral membrane. The apical membrane did not have a significant Cl- conductance. Luminal [Na+] steps and amiloride application showed a small apical Na+ conductance. Arginine vasotocin depolarized Vbl. The small apical Na+ conductance indicates that the collecting duct system contributes little to NaCl reabsorption when compared to aquatic amphibians. In contrast, Vbl rapidly depolarized upon lowering of [Na+] in the bath, demonstrating the presence of a Na+-coupled anion transporter. [HCO3-] steps revealed that this transporter is not a Na+-HCO3- cotransporter. Together, our results indicate that a major task of the collecting duct system in B. bufo is not conductive NaCl transport but rather K+ secretion, as shown by our previous studies. Moreover, our results indicate the presence of a novel basolateral Na+-coupled anion transporter, the identity of which remains to be elucidated.     

Inhibition of endothelium-dependent vasorelaxation by extracellular K(+): a novel controlling signal for vascular contractility

The effects of extracellular K+ on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) were examined in mouse aorta, mouse aorta endothelial cells (MAEC), and human umbilical vein endothelial cells (HUVEC). In mouse aortic rings precontracted with prostaglandin F2alpha or norepinephrine, an increase in extracellular K+ concentration ([K+]o) from 6 to 12 mM inhibited EDR concentration dependently. In endothelial cells, an increase in [K+]o inhibited the agonist-induced [Ca2+]i increase concentration dependently. Similar to K+, Cs+ also inhibited EDR and the increase in [Ca2+]i concentration dependently. In current-clamped HUVEC, increasing [K+]o from 6 to 12 mM depolarized membrane potential from -32.8 +/- 2.7 to -8.6 +/- 4.9 mV (n = 8). In voltage-clamped HUVEC, depolarizing the holding potential from -50 to -25 mV decreased [Ca2+]i significantly from 0.95 +/- 0.03 to 0.88 +/- 0.03 microM (n = 11, P < 0.01) and further decreased [Ca2+]i to 0.47 +/- 0.04 microM by depolarizing the holding potential from -25 to 0 mV (n = 11, P < 0.001). Tetraethylammonium (1 mM) inhibited EDR and the ATP-induced [Ca2+]i increase in voltage-clamped MAEC. The intermediate-conductance Ca2+-activated K+ channel openers 1-ethyl-2-benzimidazolinone, chlorozoxazone, and zoxazolamine reversed the K+-induced inhibition of EDR and increase in [Ca2+]i. The K+-induced inhibition of EDR and increase in [Ca2+]i was abolished by the Na+-K+ pump inhibitor ouabain (10 microM). These results indicate that an increase of [K+]o in the physiological range (6-12 mM) inhibits [Ca2+]i increase in endothelial cells and diminishes EDR by depolarizing the membrane potential, decreasing K+ efflux, and activating the Na+-K+ pump, thereby modulating the release of endothelium-derived vasoactive factors from endothelial cells and vasomotor tone.     

Deprivation of Na+, Ca2+ and Mg2+ from the extracellular solution increases cytosolic Ca2+ and stimulates catecholamine secretion from cultured bovine adrenal chromaffin cells

We report here that exposing cultured chromaffin cells to a low ionic strength medium (with sucrose in place of NaCl to maintain osmolarity) can induce a marked elevation in cytosolic Ca2+ concentration ([Ca2+]i) and catecholamine (CA) release. To determine the underlying mechanism, we first studied the effects of low [Na+]o on single cell [Ca2+]i (using fluo-3 as Ca2+ indicator) and CA release from many cells. In a Mg2+ and Ca2+-deficient medium, lowering the external concentration of Na2+ ([Na+]o) evoked CA secretion preceded by a transitory [Ca2+]i rise, the amplitude of which was inversely related to [Na+]o. By contrast, in the presence of either [Ca2+]o (2 mM) and [Mg2+]o (1.4 mM) or [Mg2+]o alone (3.4 mM), lowering the ionic strength was without effect. Furthermore, in a physiologic [Na+]o, [Ca2+]o and [Mg2+]o medium, two or three consecutive applications of the cholinergic agonist oxotremorine-M (oxo-M) consistently evoked a substantial [Ca2+]i rise. By contrast, consecutive applications of oxo-M in a Ca2+-deficient medium failed to evoke a rise in [Ca2+]i after the first exposure to the agonist. To clarify the underlying mechanism, we measured and compared the effects of low [Na+]o and the cholinergic agonists nicotine and oxo-M on changes in [Ca2+]i; we studied the effects of these agonists on both membrane potential, Vm (under current clamp conditions), and [Ca2+]i by single cell microfluorimetry (indo-1 as Ca2+ indicator). We observed that, in the presence of [Ca2+]o and [Mg2+]o, lowering [Na+]o had no effect on Vm. In a Ca2+-deficient medium, lowering [Na+]o depolarized the membrane from ca. –60 to –10 mV. As expected, we found that nicotine (10 M) depolarized the membrane (from ca. –60 to –20 mV) and simultaneously evoked a substantial [Ca2+]i rise that was [Ca2+]o-dependent. However, contrary to our expectations, we found that the muscarinic agonist oxo-M (50 M) also depolarized the membrane and induced an elevation in [Ca2+]i. Furthermore, both signals were blocked by D-tubocurarine, insinuating the nicotinic character of oxo-M in adrenal chromaffin cells from bovine. These results suggest that both nicotine and oxo-M stimulate Ca2+ entry, probably through voltage-gated Ca2+-channels. We also show here that oxo-M (and not low [Na+]o) stimulates phosphoinositide turnover.     

The cellular mechanism of active chloride secretion in vertebrate epithelia: studies in intestine and trachea

The cellular mechanism of active chloride secretion, as it is manifested in the intestine and trachea, appears to possess the following elements: (1)NaCl cl-transport across the basolateral membrane; (2) Cl- accumulation in the cell above electrochemical equilibrium due to the Na+ gradient; (3) a basolateral Na+-K+ pump that maintains the Na+ gradient; (4) a hormone-regulated Cl- permeability in the apical membrane; (5) passive Na/ secretion through a paracellular route, driven by the transepithelial potential difference; and (6) an increase in basolateral membrane K+ permeability occurring in conjunction with an increase in Na+-K+ pump rate. Electrophysiological studies in canine trachea support this model. Adrenalin, a potent secretory stimulus in that tissue, increases apical membrane conductance through a selective increase in Cl- permeability. Adrenalin also appears to increase basolateral membrane K+ permeability. Whether or not adrenalin also increases paracellular Na+ permeability is unclear. Some of the testable implications of the above secretion model are discussed.     

Access channel model for the voltage dependence of the forward-running Na+/K+ pump

The voltage dependence of steady state current produced by the forward mode of operation of the endogenous electrogenic Na+/K+ pump in Na(+)- loaded Xenopus oocytes has been examined using a two-microelectrode voltage clamp technique. Four experimental cases (in a total of 18 different experimental conditions) were explored: variation of external [Na+] ([Na]o) at saturating (10 mM) external [K+] ([K]o), and activation of pump current by various [K]o at 0, 15, and 120 mM [Na]o (tetramethylammonium replacement). Ionic current through K+ channels was blocked by Ba2+ (5 mM) and tetraethylammonium (20 mM), thereby allowing pump-mediated current to be measured by addition or removal of external K+. Control measurements and corrections were made for pump current run-down and holding current drift. Additional controls were done to estimate the magnitude of the inwardly directed pump-mediated current that was present in K(+)-free solution and the residual K(+)- channel current. A pseudo two-state access channel model is described in the Appendix in which only the pseudo first-order rate coefficients for binding of external Na+ and K+ are assumed to be voltage dependent and all transitions between states in the Na+/K+ pump cycle are assumed to be voltage independent. Any three-state or higher order model with only two oppositely directed voltage-dependent rate coefficients can be reduced to an equivalent pseudo two-state model. The steady state current-voltage (I-V) equations derived from the model for each case were simultaneously fit to the I-V data for all four experimental cases and yielded least-squares estimates of the model parameters. The apparent fractional depth of the external access channel for Na+ is 0.486 +/- 0.010; for K+ it is 0.256 +/- 0.009. The Hill coefficient for Na+ is 2.18 +/- 0.06, and the Hill coefficient for K+ (which is dependent on [Na]o) ranges from 0.581 +/- 0.019 to 1.35 +/- 0.034 for 0 and 120 mM [Na]o, respectively. The model provides a reasonable fit to the data and supports the hypothesis that under conditions of saturating internal [Na+], the principal voltage dependence of the Na+/K+ pump cycle is a consequence of the existence of an external high- field access channel in the pump molecule through which Na+ and K+ ions must pass in order to reach their binding sites.