Application Of Ouabain Research Articles

Phorbol esters inhibit smooth muscle contractions through activation of Na+ ‐K+ ‐ATPase

1. The role of protein kinase C (PKC) in agonist-induced contractions of guinea-pig ileum longitudinal smooth muscle has been investigated. 2. The phorbol esters, phorbol 12,13-dibutyrate (PDBu), phorbol 12,13-diacetate (PDA) and phorbol 12-myristate 13-acetate (PMA), relaxed tissues precontracted by submaximal concentrations of carbachol, histamine or substance P. 3. This inhibitory action of the phorbol esters was reversed following the application of ouabain, a specific inhibitor of Na(+)-K(+)-ATPase. Similarly, pretreatment with ouabain inhibited the ability of phorbol esters to relax tissues precontracted by the above agonists. 4. The slow relaxation of the tonic component of contraction induced by submaximal concentrations of carbachol and histamine, and all concentrations of substance P, was abolished in the presence of ouabain. 5. In Na(+)-loaded tissues, PDBu and carbachol caused a concentration-dependent increase of Na(+)-K(+)-ATPase activity, assessed by ouabain-sensitive 86Rb(+)-uptake. Extrusion of Na+, assessed by the cellular content of the ion, was also stimulated by PDBu (the effect of carbachol was not investigated). 6. We conclude that phorbol esters inhibit the tonic component of contractions induced by submaximal concentrations of these agonists through activation of Na(+)-K(+)-ATPase. We suggest that PKC may exert feedback control over the tonic component of agonist contractions through stimulation of the pump.

Role of Na-K pump potassium regulation and IPSPs in seizures and spreading depression in immature rabbit hippocampal slices

1. Using the immature (8-12 days postnatal) rabbit hippocampal slice preparation, we investigated regional extracellular potassium concentration [( K+]o) changes that occur during spontaneous and evoked spreading depression (SD) episodes. We report here a difference between the CA1 and CA3 cell populations in the immature hippocampus with regard to 1) resting [K+]o, 2) magnitude of the [K+]o change during seizurelike events and SDs, and 3) susceptibility to SD episodes. Experiments were also performed to elucidate the roles that the Na-K pump and synaptic inhibition play in controlling SD onset, duration, and recovery. We demonstrated a major role for potassium regulation by the Na-K pump and a lesser modulatory role for inhibitory postsynaptic potentials (IPSPs) in preventing SD in the CA3 region. 2. Simultaneous intra- and extracellular recordings were made in the CA1 and CA3 regions of the immature rabbit hippocampus during spontaneous or evoked SD, while potassium ion-sensitive microelectrodes (K-ISMs) monitored changes in [K+]o. The CA1 region had 1) a higher frequency of spontaneous SD episodes than CA3, 2) a lower threshold to potassium-triggered SD, 3) a longer duration SD episode, and 4) smaller post-SD membrane potential and [K+]o undershoots (below the original resting membrane potential and resting [K+]o). 3. During the onset of a SD episode in the CA1 region, the local [K+]o rose either before or at the same time as the membrane potential depolarization. 4. In the CA3 region, spontaneous ictallike events consisting of tonic cell depolarization with repetitive activity followed by clonic afterdischarges were more likely to occur than SD episodes. During these ictallike episodes, [K+]o rose above the 10- to 12-mM ceiling level reported for adult CNS tissue during seizures. Increases in [K+]o evoked by repetitive stimulation were regulated at a lower level in CA3 (average [K+]o rise to 11.4 mM) than in CA1 (average [K+]o rise to 18.3 mM). 5. In CA3, bath application of 10 microM bicuculline or 3.4 mM penicillin did not change the frequency of spontaneously occurring SDs or the SD response threshold to local pressure ejection of 2 M KCl. However, blockade of IPSPs did lead to lower thresholds for SD or seizurelike episodes elicited by stimulation of the mossy fibers. 6. A single application of ouabain (10 microM) to CA3 by local pressure ejection caused a slow rise in local [K+]o measured with K-ISMs. The ouabain treatment also increased the frequency of spontaneous postsynaptic potential activity and decreased the amplitude and duration of CA3 pyramidal cell afterhyperpolarizations (AHPs).(ABSTRACT TRUNCATED AT 400 WORDS)

Ouabain reverses conduction disturbances in single demyelinated nerve fibers.

Symptoms in patients with demyelinating disorders are associated with 3 important conduction abnormalities: complete conduction block, rate-dependent conduction block, and slowed impulse conduction. The recent observation that the electrogenic Na/K pump causes the nerve membrane hyperpolarization responsible for rate-dependent conduction block in demyelinated axons raises the possibility that focal inhibition of the pump may reverse the rate-dependent block. Since the pump is also responsible for maintaining part of the resting membrane potential, pump inhibition might have an additional effect of reversing the complete conduction block by reducing the threshold. We have demonstrated that inhibition of the pump by topical application of ouabain, a short-acting cardiac glycoside, reverses the conduction abnormalities in acutely demyelinated single rat ventral root axons by reducing the threshold of transmission. Ouabain or other cardiac glycosides show potential promise as agents for trial in the symptomatic treatment of patients with MS and other demyelinating disorders.

Sodium transfer from endolymph through a luminal amiloride-sensitive channel

An in vitro preparation of frog semicircular canal was devised to study the mechanisms of Na transport across the labyrinthine epithelium. When the lumen of the semicircular canal was filled with perilymph-like solution, the structure was able to secrete K into and to absorb Na from the lumen and to generate a lumen-positive transepithelial potential. When the lumen of the semicircular canal was filled with endolymph-like solution, the electrochemical composition of the luminal fluid was partly maintained up to 2 h. In this last experimental condition net and unidirectional fluxes were calculated in absence or presence of transport inhibitors, separately for the ampulla and for the nonampullar part of the canal. Amiloride (10(-5) M) but not dimethyl amiloride (10(-5) M) inhibited 60% of the unidirectional Na efflux out of endolymph; this Na efflux decrease resulted in an increase of the inward net Na flux. The net Na flux was also increased after abluminal application of ouabain (10(-3) M), furosemide (10(-4) M), and bumetanide (10(-6) M). This study validates this isolated preparation as a suitable tool for the study of endolymph secretion, confirms that the secretion of endolymph is achieved in the ampulla, and provides evidence for an apical amiloride-sensitive Na channel through which Na is transferred out of endolymph along an electrochemical gradient provided by the activity of the abluminal Na+-K+-ATPase.

Calcium-dependent and ouabain-resistant oxygen consumption in the rat neurohypophysis

To analyze rapid changes in energy metabolism in the neurohypophysis, pO 2 was measured in the tissue in vitro with a miniature O 2 electrode (tip diameter< 100 μm, 90%response time< 3s). Electrical stimulation (20 Hz, 5 s) evoked immediate pO 2 decreases by93.4 ± 10.5mm Hg(mean±S.E.M., n = 12) which lasted for about 1 min and were blocked by tetrodotoxin (1 μM) or sodium cyanide (1 mM). Replacement of Ca 2+ in the perifusing medium with Mn 2+ reduced the pO 2 decreases to23.1 ± 4.9% (n = 5) of the value before the replacement. In normal medium, ouabain application (1 mM, 3 min) suppressed the electrically evoked pO 2 decreases only slightly to82.6 ± 6.5% (n = 5). In the Mn 2+ medium, the same ouabain application suppressed the pO 2 changes to28.8 ± 1.4%. High K + (70 mM) evoked pO 2 decreases by175.8 ± 14.9mm Hg(n = 5) within 1–2 min. These pO 2 changes were reduced to35.6 ± 3.8% in an Mn 2+ medium. Veratridine (100 μM) evoked pO 2 decreases by204.8 ± 36.3mm Hg(n = 5). During the pO 2 decreases, the effects of electrical or high K + stimulation on pO 2 were blocked. These results indicate that O 2 consumption was evoked by electrical stimulation, and probably that high K + or veratridine application in the neurohypophysis is mainly dependent on extracellular calcium and resistant to ouabain. The relationship between O 2 consumption and exotytotic release is discussed.

Components of resting membrane electrogenesis in Lepidopteran skeletal muscle

The partial contributions of K +, Na +, Cl −, Mg 2+ and an electrogenic, metabolic pump to resting membrane electrogenesis in skeletal muscles of Chilo partellus (Lepidoptera) have been quantified. The contributions of the ions were evaluated by ionic substitution experiments, whilst the role of the pump was investigated by cooling and application of ouabain. In response to changes in extracellular K +, Na + and Cl − activities, membrane potentials changed in rapid, graded, sustained and reversible steps. For a 10-fold change in the extracellular activity, the maximal changes in the membrane potentials were 31 mV (K +), 2 mV (Na +) and 17 mV (Cl −). In constrast, changing the extracellular Mg 2+ concentration had no immediate effect on resting potentials. Cooling preparations by 14°C from room temperature resulted in rapid membrane depolarizations of some 12 mV. However, application of 1 mM ouabain in Ringers containing varying concentrations of K + had no effect on the membrane potential. It followed, therefore, that a metabolic mechanism was also involved in electrogenesis, but this was unlikely to be an ouabain-sensitive (Na +K +) ATPase. The contributions of K +, Na +, Cl − and the metabolic pump were sufficient to account quantitatively for the prevailing resting membrane potential (mean, −46 mV) with an error margin of some 7%.

Effects of Na + and Ca 2+ gradients on intracellular free Ca 2+ in voltage-clamped Aplysia neurons

Selected neurons of the abdominal ganglion of Aplysia californica were voltage-clamped and intracellular free Ca ([Ca 2+] i) and Na ([Na +] i) concentrations were monitored with ion selective microelectrodes. Reducing [Na +] o from 500 mM (normal seawater, NSW) to 5 mM resulted in a decrease of the potential measured by the Ca electrode ( V Ca). Increasing [Ca 2+] o from 10 to 50 mM increased [Ca 2+] i two-fold, keeping [Ca 2+] o at 50 mM and decreasing [Na +] o to 5 mM still led to a decrease in V Ca. With 100 mM [Ca 2+] o, which also increased [Ca 2+] i, decreasing [Na +] o increased V Ca in two of the eight cells tested. This indicates that in normal or moderately high resting [Ca 2+] i, Ca 2+ extrusion by Na/Ca exchange (forward mode) is not essential for [Ca 2+] i buffering. [Na +] i was12.9 ± 3.6mM (S.E.M., n = 7) in NSW; reducing [Na +] o to 5 mM decreased [Na +] i to2.0 ± 1.1mM (S.E.M.). Keeping [Na +] o at 5 mM and increasing [Ca 2+] o from 10 to 20 mM further decreased [Na +] i to about 1.0 mM, evidence of Na/Ca exchange operating in the reverse mode. Attempts to increase [Ca 2+] i by bath application of the Ca ionophores A23187, X537A, ionomycin or ETH 1001 resulted in no measurable change of the resting [Ca 2+] i. Application of Ouabain caused an apparent increase in [Ca 2+] i in two of the six cells tested. In cells injected with the metallochromic indicator arsenazo III (AIII), the rate of the falling phase of the AIII absorbance increase, following a voltage-clamp pulse, was significantly slower in 5 mM [Na +] o. This indicates that in its forward mode Na/Ca exchange is active in clearing large submembrane increases in [Ca 2+] i.

Follicle‐stimulating hormone induces hyperpolarization of immature rat Sertoli cells in monolayer culture.

1. The effect of FSH on the membrane potential of Sertoli cells has been investigated by intracellular microelectrode recording from monolayer cultures of cells, isolated from immature rat testes by enzymatic treatment. 2. In standard Earle's solution, the membrane potential of unstimulated cells in a 3-day-old monolayer was -21.6 +/- 0.2 mV (n = 300). The recorded potentials were unchanged on varying the culture period from 3 to 7 days or by removal of chloride or calcium from the media. However, they were decreased in a low-sodium or potassium-rich medium. 3. Ovine FSH caused hyperpolarization of the cell in a dose-dependent manner. Maximal values were obtained with concentrations ranging between 2.9 and 5.9 micrograms/ml (-37.0 +/- 0.2 mV; n = 310). Dibutyryl cyclic AMP (1 mM) produced a similar effect to FSH. Under similar conditions human chorionic gonadotrophin (hCG) had no effect on the membrane potential. 4. The FSH-induced hyperpolarization was unchanged by chloride or bicarbonate replacement. It was decreased by low-sodium media (9 mM) (-29.6 +/- 0.3 mV; n = 110), by removal of calcium (-32.4 +/- 0.6 mV; n = 128) and by an increase in the potassium concentration of the bathing medium. A tenfold increase in potassium concentration depolarized the cell by 29.5 mV against 16 mV for unstimulated cells. 5. FSH-induced hyperpolarization was decreased by application of ouabain (10(-4) M), quinidine (1.4 x 10(-4) M) and cobalt (10(-3) M) (respectively: -28.7 +/- 0.3 mV, n = 124; -26.6 +/- 0.3 mV, n = 52 and -24.8 +/- 0.3 mV, n = 68) but was unchanged by amiloride (10(-4) M) and TTX (3 x 10(-6) M). None of these drugs modified the membrane potential of unstimulated cells. 6. All these results suggest that FSH stimulation of Sertoli cells in monolayer culture involves the modification of their membrane permeabilities.

Role of sodium pump in membrane potential gradient of canine proximal colon.

A large gradient in membrane potential exists through the thickness of the circular layer in canine colonic muscles. This study tested the effects of several experimental manipulations known to block electrogenic sodium pumping on the resting potentials of colonic muscles. Membrane potentials were recorded with microelectrodes from cells through the circular muscle layer. In cells adjacent to the submucosal surface of the circular layer, application of ouabain (10(-6) to 10(-5) M) caused an average membrane depolarization of 36 mV. Removal of the external K+ resulted in depolarizations similar to the effect of ouabain. Readmission of K+ (5.9 mM) produced repolarization and an additional hyperpolarization that averaged 13 mV beyond the resting potential. When exposed to 15 mM K+, cells hyperpolarized well beyond the estimated potassium equilibrium potential (EK). Ouabain blocked the repolarization in response to reintroduction of external K+. Lowering the bath temperature to 20 degrees C rapidly depolarized membrane potential; rewarming repolarized cells. Ouabain and K+-free solutions blocked the repolarization response to rewarming. Cells also depolarized when exposed to solutions in which the NaCl was replaced with LiCl. Membrane potentials of cells within the bulk of the circular layer decreased as a function of distance from the submucosal border. Cells at the myenteric border of the circular muscle were not significantly affected by ouabain and K+-free solution, but these treatments abolished the gradient in membrane potential across the circular layer.(ABSTRACT TRUNCATED AT 250 WORDS)

Release of Prostaglandins during the Contraction of Rat Aorta Induced by Ouabain and K+-Free Solution

Contractile effects of ouabain and K+-free solution on rat aortic strips were investigated. In the aorta without endothelium, application of ouabain or K+-free solution produced, after a latency period, a slow contraction reaching the maximum after 80-100 min. Pretreatment of the muscle with either indomethacin (20 μM) or verapamil (1 μM) decreased the maximum level of these contractions, whereas verapamil, but not indomethacin, prolonged the latency period. Simultaneous application of these inhibitors showed additive inhibitory effects. In the presence of endothelium, the latency period slightly increased without changing the maximum contractile tension. Methylene blue (5 μM) shortened the latency period only in the aorta with endothelium. These results suggest that the contractions of rat aorta induced by ouabain and K+-free solution are due not only to membrane depolarization and Na+-Ca2+ exchange but also to the release of prostaglandins. Endothelium-derived relaxing factor seems to inhibit a part of these contractions.

Two differential mechanisms of automaticity in diseased human atrial fibers.

The ionic mechanisms of automaticity in spontaneously active preparations (n = 38) from "diseased" human atria were investigated. The cycle length (CL) of the automatic action potential (AAP) ranged from 0.6 to 4.5 s (mean +/- S.D.: 2.0 +/- 1.0 s). The AAP from preparations obtained from non-digitalized patients (group 1, n = 29) showed various CLs, in that 16 patients had a CL longer than 2.0 s (slow-type AAP) while in tissues from the other 13 patients, the CL was 2.0 s or less (fast-type AAP). On the other hand, all preparations obtained from the digitalized patients prior to cardiac surgery (group 2, n = 9) had the fast-type AAP (CL less than or equal to 2.0 s). The slow-type AAP was accelerated and changed to the fast one after application of ouabain (1 microM) or by perfusing with 50% [Na+]o Tyrode solution. Ryanodine (1 microM), a specific inhibitor of calcium release from the sarcoplasmic reticulum (SR), markedly lengthened the CL of fast-type AAP, without affecting the slow-type AAP. In contrast, caffeine (15 mM) shortened the CL of the slow-type AAP and remarkably lengthened the CL of the fast-type AAP, as is the case in ryanodine. The rate of slow-type AAP was enhanced with an increase in [Ca2+]o and depressed with a decrease in [Ca2+]o or with application of diltiazem, a Ca2+ channel blocker. The slow-type AAP was changed to the fast-type AAP by stretching the preparation by about 20%. In vitro preparations excised from patients with dilated atria revealed a shorter CL of AAPs. We conclude that in "diseased" human atrial preparations, the ionic mechanism responsible for generation of slow- and fast-type automaticity differs. Slow automaticity (CL greater than 2.0 s) perhaps relates to activation of the slow inward Ca2+ current, while the fast-type automaticity (CL less than or equal to 2.0 s) is linked to cyclic increases in [Ca2+]i.

An investigation of sodium‐calcium exchange in the smooth muscle of guinea‐pig ureter.

1. After application of ouabain (10(-4) M), the intracellular Na+ activity (alpha iNa) of smooth muscle cells in the guinea-pig ureter stabilizes at a relatively low level which can be rapidly lowered by reduction of external Na+ (Na+o) or elevation of Ca2+o. Both these procedures also elicit a transient contracture. These observations have previously been interpreted as evidence for Na+-Ca2+ exchange. The presence of such an exchange mechanism has now been further investigated by measurements of alpha iNa, tension, ion analysis and 22Na efflux. 2. Ion analysis demonstrated that tissues were able to maintain a high cellular K+ content in the presence of ouabain, but slowly lost K+ and gained Na+ if K+o was also removed, as expected for an infinite outward gradient for K+ and a fully inhibited Na+ pump. 3. Tissues were only able to maintain a low cellular Na+ and high cellular K+ in the presence of ouabain if Ca2+ was present in the bathing solution. Reduction of Ca2+o to very low levels also caused a continual slow rise in alpha iNa in the presence of ouabain, provided that the prolonged depolarization caused by these low levels was prevented by elevation of Mg2+o. Alteration of the membrane potential by changing K+o at constant Na+o showed that alpha iNa decreased by about 1.2 mM for a 10 mV depolarization, within the range from -70 to -30 mV. 4. A small Ca2+o-activated 22Na efflux was observed in ouabain-treated tissues in the absence of Na+o. 40 mM-Ca2+ was not more effective at activating this efflux than was 2.5 mM-Ca2+, while 40 mM-Mg2+ was ineffective. Restoration of the normal Na+o caused a large increase in the rate of 22Na loss. 5. Application of Mn2+ in the presence of ouabain caused a slow rise in alpha iNa and a small decline in resting tension. The fall in alpha iNa on reduction of Na+o was slowed by the presence of Mn2+ (mean half-time increased from 1.7 to 5.0 min) and the concomitant contracture was almost abolished. These results are consistent with a Mn2+-induced inhibition of Na+-Ca2+ exchange. However, the fall in alpha iNa induced by elevation of Ca2+o was unaffected by the presence of Mn2+ and the attendant contracture was, if anything, enhanced. 6. Observation of changes in alpha iNa and tension at various Mn2+ and Ca2+ concentrations demonstrated a competitive interaction between the two divalent cations.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of ion conductance changes and of the sodium‐pump in adrenaline‐induced hyperpolarization of rat diaphragm muscle fibres

The ionic mechanism of membrane hyperpolarization induced by adrenaline in rat diaphragm muscle fibres was studied. Removal of the extracellular K+ ([K+]o) from Krebs-Ringer solution initially increased the resting membrane potential and then caused an increase in the intracellular Na+ activity ([Na+]i) and a decrease in the intracellular K+ activity ([K+]i). All the changes were maintained for more than 3 h. Application of ouabain (0.1 mM) or lowering the temperature rapidly reduced the resting potential by about 10 mV in the K+-free solution. It then produced further progressive decreases in resting potential and in [K+]i and a progressive increase in [Na+]i. These observations indicate that an electrogenic Na-pump operates in the K+-free solution. Removal of most of the Cl- in the K+-free solution did not affect the resting potential or the magnitude of the initial decrease produced by ouabain, despite an increased input resistance; this result implies a passive distribution of Cl-. Adrenaline (30-60 microM) either added to the bathing solution or applied to the membrane by ionophoresis produced a hyperpolarization (3-10 mV: adrenaline hyperpolarization), the amplitude of which was decreased with a rise in [K+]o and increased with a reduction in [K+]o, but unaffected by the removal of Cl-. Adrenaline produced an increase in input resistance, the relative magnitude (17-18%) of which was constant whether external K+ or Cl- was removed. In contrast, a conditioning membrane hyperpolarization hardly affected the resistance. Ouabain (0.1 mM) or low temperature (8-10 degrees C) abolished both the hyperpolarization and the increased input resistance induced by adrenaline. The [K+]i, [Na+]i and the peak of the action potential remained unchanged after a 20 min exposure to adrenaline (30 microM). The hyperpolarization induced by the replacement of all Na+ with Tris (Tris-hyperpolarization) in the K+-free solution was depressed by 39% during the early period (4-31 min) of exposure to adrenaline (30 microM), while it was enhanced by 26% during the later period (80-130 min). The initial depression suggested a decrease in the ratio of the membrane permeability for Na+ (PNa) to that for K+ (PK). These results suggest that the adrenaline hyperpolarization is generated largely by a decrease in PNa/PK, which is associated with the activity of the Na-pump.

Investigation of factors affecting the intracellular sodium activity in the smooth muscle of guinea‐pig ureter.

1. The intracellular Na+ activity (aNai) of the smooth muscle cells from guinea-pig ureter has been measured using double-barrelled Na+-sensitive micro-electrodes. aiNa in modified Krebs solution at 35 degrees C was of a mean 7.4 +/- 2.9 mM (n = 32, S.D. of an observation), equivalent to a Na+ equilibrium potential (ENa) of +66.7 mV. Membrane potential (Em) was of a mean -50.8 +/- 4.6 mV. 2. Inhibition of the Na+ pump by application of ouabain or removal of external K+ (K+o) resulted in a restricted rise of aNai. The rate of rise was faster in the presence of ouabain (10(-4) M) but the stabilized aNai was not significantly different from that observed after the prolonged absence of K+o. The mean aiNa recorded after prolonged Na+ pump inhibition was 20.6 +/- 5.5 mM (n = 28), equivalent to an ENa of +39.6 mV. Neither removal of K+o after aNai had stabilized in the presence of ouabain nor application of ouabain after aNai had stabilized in K+-free solution caused a rise in aiNa, suggesting that the Na+ pump was fully inhibited by either procedure. 3. Reduction of Na+o resulted in a rapid fall in aiNa against the electrochemical gradient, both before and after Na+ pump inhibition. At each level of Na+o, aNai stabilized such that ENa remained approximately constant in either condition. Readdition of Na+o resulted in a rapid recovery of aNai. 4. Elevation of Ca2+o (at constant Na+o) caused a fall in aNai of much the same time course as that observed on reduction of Na+o, both before and after Na+ pump inhibition. The extent of the fall was dependent upon the initial aNai. Reduction of Ca2+o resulted in a rise in aNai. 5. Elevation of the external divalent cation concentration with Mn2+ or, to a lesser extent, Mg2+ reduced aiNa in the presence of a functional Na+ pump. But after prolonged exposure to ouabain or K+-free solution, elevation of Mg2+o had no effect on aiNa while application of Mn2+o caused a slow rise. These results suggest that Ca2+o affects aiNa in two ways. One is mimicked by Mg2+ and Mn2+ and is probably due to alteration of the Na+ leak. The other is a specific effect, revealed by Na+ pump inhibition. 6. It is concluded that aiNa can be maintained far from equilibrium in the absence of a functional Na+ pump. Several lines of evidence are discussed which indicate the participation of Na+-Ca2+ exchange in Na+ extrusion in this condition.

The mechanism of electrodiffusive K+ transport in leaky epithelia and some of its consequences for anion transport.

The ventricular membrane of the epithelium from the choroid plexus of Necturus maculosus was probed with double-barrelled ion-selective microelectrodes while the ventricular bathing solution was changed abruptly. The transient states induced by increasing the external concentration of K+ or by the application of ouabain showed that the passive movements of K+ across the ventricular membrane were electro-diffusive and could be described by the Goldmann equation and one constant permeability (PK) of 24 X 10(-6) cm s-1. The passive efflux was balanced by a ouabain-sensitive influx. PK was different in different steady states; when the cell was acidified by half a pH unit by increasing CO2 in the bathing solutions from 1 to 5%, then PK decreased to 13 X 10(-6) cm s-1. Removal of Cl- from the bathing solution increased PK by 50% and reduction of Cl- transport by furosemide did not alter PK, consequently major movements of K+ were independent of Cl- movements. Depolarizations of the cell caused an increase in the cellular HCO-3 concentration due to an electrodiffusive permeability (PHCO3), the value of which was estimated to 17 X 10(-6) cm s-1.

Factors related to the low resting membrane potentials of diseased human atrial muscles.

We studied the ionic mechanism of low resting potential (RP) of quiescent "diseased" human atrial fibers. The RP was -49.7 +/- 0.8 mV (n = 179) in normal Tyrode's solution (5.4 mM [K]o, 36 degrees C). The changes in RP measured at various levels of [K]o appeared to fit the RP-[K]o relationship predicted by the Goldman-Hodgkin-Katz equation, assuming PNa/PK ratio (alpha) to be 0.102 and [K]i to be 131.9 mM. The alpha far exceeded the normal value (about 0.01) by a factor of 10. Acetylcholine (ACh, 10 microM) led to marked increases in the RP. An application of tetrodotoxin (TTX, 6 microM) and perfusion with low [Na]o (10% of the control) media in the presence and absence of ACh produced considerable hyperpolarizations of the RP. These findings indicate that increased alpha value is due to a combination of decreased PK and increased PNa. Applications of ouabain (5 microM) and a cooling procedure (12.3 degrees C) depolarized the membrane, whereas epinephrine (1 microM) hyperpolarized it. Transient hyperpolarization, which exceeded the steady state levels of RP at 5.4 mM [K]o, was observed with perfusing of 5.4 mM [K]o media following perfusion with K-free media. These findings suggest that electrogenic Na pump current plays a significant role in the maintenance of the RP. In conclusion, partial depolarization of "diseased" human atrial fibers was attributed to both decreases in membrane K+ conductance and increases in Na+ conductance. The electrogenic outward pump current seemed to protect the fibers from severe depolarization produced by the conductance abnormality (increased PNa/PK).

Electrophysiologic properties differ in the ventricular endocardium and epicardium of the Japanese monkey

We measured action potential duration (APD) from the endocardium (Endo) and epicardium (Epi) of the left ventricular free wall in Japanese monkey hearts and found that the APD of Endo is significantly longer than that of Epi at a stimulus cycle length of 1500 msec in normal Tyrode solution (control condition). We then hypothesized that shorter APD of Epi results from greater outward pump current and that the difference in the current may be due to a difference in membrane Na,K-ATPase activity between Endo and Epi. If this were the case, interventions which alter the Na,K-pump activity should alter electrophysiologic characteristics in the endocardium and the epicardium to different degrees. An application of ouabain (10(-6) M), an inhibitor of Na,K-ATPase, produced greater shortening of APD and greater depolarization of the resting potential in Endo as compared with Epi. Shortening of stimulus cycle length shortened the APD more markedly in Endo than Epi, resulting in a significantly longer APD in Epi than Endo at a stimulus cycle length of 200 msec. The hyperpolarization and the APD shortening produced when the tissues were returned to 5.4 mM K+ Tyrode solution from K+-free medium were also more marked in Endo than in Epi. Such findings suggest that endocardial cells are more liable to accumulate Na ions intracellularly and K ions extracellularly when the Na, K-pump is suppressed by ouabain or K+-free perfusion, presumably due to lower activity of Na, K-ATPase in Endo compared to Epi.(ABSTRACT TRUNCATED AT 250 WORDS)

Mucus and intestinal ion exchanges in the sea-water adapted eel,Anguilla anguilla L.

1. Scanning electron microscopy of the intestinal epithelium in the cel showed an important mucus layer over the middle intestine. 2. Ion-selective microelectrodes (Na+, Cl−, K+) demonstrated several standing gradients of ion activities in the mucus layer. 3. The Na+ and Cl− gradients were identical and related to transepithelial NaCl absorption. They were cancelled by ouabain application on the serosal side. 4. Symmetrical substitution of Na+ or Cl− in the luminal and serosal Ringer solution gave results in agreement with the presence of an apical Na+−K+−2Cl− cotransport system, and of K+ channels. 5. The intestinal mucus appeared, at least in the middle part of the intestine, to act as a diffusion barrier essential to K+ recycling.

Ion activities and potassium uptake mechanisms of glial cells in guinea‐pig olfactory cortex slices.

1. Double-barrelled ion-sensitive micro-electrodes were used to measure changes in the intracellular activities of K+, Na+ and Cl- (aiK, aiNa, aiCl) in glial cells of slices from guinea-pig olfactory cortex during repetitive stimulation of the lateral olfactory tract. 2. Base-line levels of aiK, aiNa and aiCl were about 66, 25 and 6 mM, respectively, for cells with resting potentials higher than -80 mV. During stimulation, intraglial aiK and aiCl increased, whereas aiNa decreased. Within about 2 min after stimulation the ion activities returned to their base-line levels. 3. The Cl- equilibrium potential was found to be close to the membrane potential (Em). There was also a strong correlation between changes of Em and aiCl. These observations indicate a high Cl- conductance of the glial cell membrane. 4. In the presence of Ba2+, the usual depolarizing response of the glial cells to a rise of the extracellular K+ activity (aeK) reversed into a membrane hyperpolarization. Furthermore, Ba2+ strongly reduced the stimulus-related rise of intraglial aiK. An additional application of ouabain blocked both the membrane hyperpolarization as well as the remaining rise of aiK. 5. In conclusion, our data show that glial cells in guinea-pig olfactory cortex slices possess at least two mechanisms of K+ accumulation. One mechanism is sensitive to the K+ channel blocker Ba2+ and might be a passive KCl influx. The other appears to be the electrogenic Na+/K+ pump, which can be activated by excess extracellular K+.

Effects of ouabain and temperature on cell membrane potentials in isolated perfused straight proximal tubules of the mouse kidney.

In isolated perfused segments of the mouse proximal tubule, the potential difference across the basolateral cell membrane (PDbl) was determined with conventional microelectrodes. Under control conditions with symmetrical solutions it amounted to -62 +/- 1 mV (n = 118). The potential difference across the epithelium (PDte) was -1.7 +/- 0.1 mV (n = 45). Transepithelial resistance amounted to 1.82 +/- 0.09 k omega cm (n = 28), corresponding to 11.4 +/- 0.6 omega cm2. Increasing bath potassium concentration from 5 to 20 mmol/l depolarized PDbl by +24 +/- 1 mV (n = 103), and PDte by +1.6 +/- 0.1 mV (n = 19). Thus, the basolateral cell membrane is preferably conductive to potassium. Rapid cooling of the bath perfusate from 38 degrees C to 10 degrees C led to a transient hyperpolarization of PDbl from -60 +/- 1 to -65 +/- 1 mV (n = 21) within 40 s followed by gradual depolarization by +18 +/- 1% (n = 14) within 5 min. The transepithelial resistance increased significantly from 1.78 +/- 0.11 k omega cm to 2.20 +/- 0.21 k omega cm (n = 15). Rapid rewarming of the bath to 38 degrees C caused a depolarization from -61 +/- 2 mV (n = 17) to -43 +/- 2 mV (n = 16) within 15 s followed by a repolarization to -59 +/- 2 mV (n = 10) within 40 s. Ouabain invariably depolarized PDbl. During both, sustained cooling or application of ouabain, the sensitivity of PDbl to bath potassium concentration decreased in parallel to PDbl pointing to a gradual decrease of potassium conductance.(ABSTRACT TRUNCATED AT 250 WORDS)