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The Journal of Physiology Sep 2006Glycoside-induced cardiac inotropy has traditionally been attributed to direct Na(+)-K(+)-ATPase inhibition, causing increased intracellular [Na(+)] and consequent...
Glycoside-induced cardiac inotropy has traditionally been attributed to direct Na(+)-K(+)-ATPase inhibition, causing increased intracellular [Na(+)] and consequent Ca(2+) gain via the Na(+)-Ca(2+) exchanger (NCX). However, recent studies suggested alternative mechanisms of glycoside-induced inotropy: (1) direct activation of sarcoplasmic reticulum Ca(2+) release channels (ryanodine receptors; RyRs); (2) increased Ca(2+) selectivity of Na(+) channels (slip-mode conductance); and (3) other signal transduction pathways. None of these proposed mechanisms requires NCX or an altered [Na(+)] gradient. Here we tested the ability of ouabain (OUA, 3 microm), digoxin (DIG, 20 microm) or acetylstrophanthidin (ACS, 4 microm) to alter Ca(2+) transients in completely Na(+)-free conditions in intact ferret and cat ventricular myocytes. We also tested whether OUA directly activates RyRs in permeabilized cat myocytes (measuring Ca(2+) sparks by confocal microscopy). In intact ferret myocytes (stimulated at 0.2 Hz), DIG and ACS enhanced Ca(2+) transients and cell shortening during twitches, as expected. However, prior depletion of [Na(+)](i) (in Na(+)-free, Ca(2+)-free solution) and in Na(+)-free solution (replaced by Li(+)) the inotropic effects of DIG and ACS were completely prevented. In voltage-clamped cat myocytes, OUA increased Ca(2+) transients by 48 +/- 4% but OUA had no effect in Na(+)-depleted cells (replaced by N-methyl-d-glucamine). In permeabilized cat myocytes, OUA did not change Ca(2+) spark frequency, amplitude or spatial spread (although spark duration was slightly prolonged). We conclude that the acute inotropic effects of DIG, ACS and OUA (and the effects on RyRs) depend on the presence of Na(+) and a functional NCX in ferret and cat myocytes (rather than alternate Na(+)-independent mechanisms).
Topics: Animals; Bacterial Proteins; Calcium Signaling; Cardiac Glycosides; Cardiotonic Agents; Cats; Digoxin; Ferrets; Heart Ventricles; In Vitro Techniques; Membrane Potentials; Myocardial Contraction; Myocytes, Cardiac; Ouabain; Patch-Clamp Techniques; Ryanodine Receptor Calcium Release Channel; Sodium; Sodium-Calcium Exchanger; Streptolysins; Strophanthidin
PubMed: 16825310
DOI: 10.1113/jphysiol.2006.111252 -
The Journal of General Physiology Aug 1968The effects which alterations in the concentrations of internal sodium and high energy phosphate compounds had on the sodium influx and efflux of internally dialyzed...
The effects which alterations in the concentrations of internal sodium and high energy phosphate compounds had on the sodium influx and efflux of internally dialyzed squid axons were examined. Nine naturally occurring high energy phosphate compounds were ineffective in supporting significant sodium extrusion. These compounds were: AcP, PEP, G-3-P, ADP, AMP, GTP, CTP, PA, and UTP.(1) the compound d-ATP supported 25-50% of the normal sodium extrusion, while ATP supported 80-100%. The relation between internal ATP and sodium efflux was nonlinear, rising most steeply in the range 1 to 10 microM and more gradually in the range 10 to 10,000 microM. There was no evidence of saturation of efflux even at internal ATP concentrations of 10,000 microM. The relation between internal sodium and sodium efflux was linear in the range 2 to 240 mM. The presence of external strophanthidin (10 microM) changed the sodium efflux to about 8-12 pmoles/cm(2) sec regardless of the initial level of efflux; this changed level was not altered by subsequent dialysis with large concentrations of ATP. Sodium influx was reduced about 50 % by removal of either ATP or Na and about 70 % by removing both ATP and Na from inside the axon.
Topics: Adenine Nucleotides; Adenosine Triphosphate; Animals; Axons; Cardanolides; Cell Membrane; Dialysis; Edetic Acid; Guanine Nucleotides; Methods; Mollusca; Phosphates; Sodium; Sodium Isotopes; Sulfonic Acids; Uracil Nucleotides
PubMed: 4970418
DOI: 10.1085/jgp.52.2.181 -
The Journal of Physiology Oct 1983A method for mounting and rapidly perfusing small ventricular trabeculae (diameter around 250 micron) from either ferret or guinea-pig is described. Tension, membrane...
A method for mounting and rapidly perfusing small ventricular trabeculae (diameter around 250 micron) from either ferret or guinea-pig is described. Tension, membrane potential (Em) and intracellular Na activity (aiNa) were measured. aiNa was measured using Na-sensitive micro-electrodes. At room temperature (22-26 degrees C), [Na]o 155 mmol/l and [Ca]o 5.4 mmol/l, aiNa was 10.9 mmol/l +/- S.D. 4.2 mmol/l (n = 148). When [Na]o was reduced from 155 to 1.5 mmol/l contractures developed. These were about twitch height in guinea-pig but less than the twitch height in ferret. Associated with the development of the contracture there was a decrease in aiNa. The aiNa halved within 30 s. The decrease in aiNa was not influenced by changing pHo from 7.4 to 9.5, K-free solution or strophanthidin 50 mumol/l and was not passive since, even when the [Na]o was 1.5 mmol/l, the driving force for Na ions remained inward. The aiNa decreased if [Ca]o was increased and [Na]o decreased or vice versa. On the basis of these findings it is concluded that the decrease in aiNa is mainly due to Na/Ca exchange. Despite the large decrease in aiNa the [Ca]i, as monitored by tension changes, hardly increased. Since Ca uptake does occur in Na-free conditions in heart muscle it is proposed that the mitochondria take up Ca ions and so prevent an excessive rise in cytoplasmic Ca. Strophanthidin increased both aiNa and the withdrawal contracture, but collected results from a number of experiments showed no clear correlation between the initial aiNa and contracture amplitude. Strophanthidin may, therefore, have actions additional to increasing aiNa.
Topics: Animals; Calcium; Ferrets; Guinea Pigs; Heart; Heart Ventricles; Hydrogen-Ion Concentration; Membrane Potentials; Microelectrodes; Myocardial Contraction; Myocardium; Sodium; Strophanthidin
PubMed: 6644617
DOI: 10.1113/jphysiol.1983.sp014891 -
The Journal of Physiology Nov 19871. The contraction and relaxation of rings of rat thoracic aorta and bovine tail artery were examined as a function of changes in the Na+ electrochemical gradient in...
1. The contraction and relaxation of rings of rat thoracic aorta and bovine tail artery were examined as a function of changes in the Na+ electrochemical gradient in order to determine the role of Na-Ca exchange in the control of contractility. 2. Inhibition of the Na+ pump in rat aorta by K+-free media or a low concentration (5 x 10(-5) M) of strophanthidin reversibly increased the contractile responses to caffeine and noradrenaline. These effects were dependent upon external Ca2+ and were observed even in the presence of a Ca2+ channel blocker (10 microM-verapamil or 10 microM-diltiazem) and an alpha-receptor blocker (10 microM-phentolamine). 3. Reduction of external Na+ concentration, [Na+]o (replaced by N-methylglucamine, tetramethylammonium or Tris), also caused an external Ca2+-dependent increase in tonic tension and, in rat aorta, an increase in the response to caffeine. These effects were also observed in the presence of verapamil and phentolamine. 4. Caffeine relaxed the bovine tail artery, but increased the sensitivity of the rat aorta to reduced [Na+]o. The latter effect was presumably due to block of Ca2+ sequestration in the sarcoplasmic reticulum, so that entering Ca2+ was more effective in raising the intracellular free Ca2+ level, [Ca2+]i. 5. Relaxation from K+-free or low-Na+ contractions, in Ca2+-free media, depended upon [Na+]o. Reduction of [Na+]o to 1.2 or 7.5 mM slowed the relaxation of rat aorta (5 mM-caffeine present) 3- to 5-fold, and the relaxation of bovine tail artery (without caffeine) 5- to 10-fold. These effects were seen in the presence of verapamil and phentolamine. 6. These observations are all consistent with an Na-Ca exchange transport system that can move Ca2+ either into or out of the arterial smooth muscle cells. Ca2+ entry is enhanced by raising [Na+]i (by Na+ pump inhibition) and/or lowering [Na+]o. Ca2+ extrusion from the contracted muscles is largely dependent upon external Na+. The latter observation implies that, when [Ca2+] exceeds the contraction threshold, Ca2+ efflux is mediated primarily by the Na-Ca exchanger, rather than by the sarcolemmal ATP-driven Ca2+ pump. 7. When bovine tail artery was treated with verapamil and phentolamine, and [Na+]o was reduced from 139.2 to 43.9 mM, substitution of K+ for Na+ induced a larger external Ca2+-dependent contraction than did substitution of Tris for Na+. The amplitudes of these contractions were greatly increased when the Na+ pump was inhibited by 5 x 10(-5) M-strophanthidin, presumably because of the rise in [Na+]i.(ABSTRACT TRUNCATED AT 400 WORDS)
Topics: Animals; Aorta; Arteries; Caffeine; Calcium; Cattle; In Vitro Techniques; Ion Channels; Muscle Contraction; Muscle, Smooth, Vascular; Phentolamine; Rats; Rats, Inbred Strains; Sodium; Strophanthidin; Tail; Verapamil
PubMed: 2451733
DOI: 10.1113/jphysiol.1987.sp016800 -
Journal of Neurophysiology May 2000Interneurons innervating dentate granule cells are potent regulators of the entorhino-hippocampal interplay. Traumatic brain injury, a leading cause of death and...
Interneurons innervating dentate granule cells are potent regulators of the entorhino-hippocampal interplay. Traumatic brain injury, a leading cause of death and disability among young adults, is frequently associated with rapid neuropathological changes, seizures, and short-term memory deficits both in humans and experimental animals, indicating significant posttraumatic perturbations of hippocampal circuits. To determine the pathophysiological alterations that affect the posttraumatic functions of dentate neuronal networks within the important early (hours to days) posttraumatic period, whole cell patch-clamp recordings were performed from granule cells and interneurons situated in the granule cell layer of the dentate gyrus of head-injured and age-matched, sham-operated control rats. The data show that a single pressure wave-transient delivered to the neocortex of rats (mimicking moderate concussive head trauma) resulted in a characteristic ( approximately 10 mV), transient (<4 days), selective depolarizing shift in the resting membrane potential of dentate interneurons, but not in neighboring granule cells. The depolarization was not associated with significant changes in action potential characteristics or input resistance, and persisted in the presence of antagonists of ionotropic and metabotropic glutamate, and GABA(A) and muscarinic receptors, as well as blockers of voltage-dependent sodium channels and of the h-current. The differential action of the cardiac glycosides oubain and stophanthidin on interneurons from control versus head-injured rats indicated that the depolarization of interneurons was related to the trauma-induced decrease in the activity of the electrogenic Na(+)/K(+)-ATPase. In contrast, the Na(+)/K(+)-ATPase activity in granule cells did not change. Intracellular injection of Na(+), Ca(2+)-chelator and ATP, as well as ATP alone, abolished the difference between the resting membrane potentials of control and injured interneurons. The selective posttraumatic depolarization increased spontaneous firing in interneurons, enhanced the frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) in granule cells, and augmented the efficacy of depolarizing inputs to discharge interneurons. These results demonstrate that mechanical neurotrauma delivered to a remote site has highly selective effects on different cell types even within the same cell layer, and that the electrogenic Na(+)-pump plays a role in setting the excitability of hippocampal interneuronal networks after injury.
Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Benzoates; Bicuculline; Brain Injuries; Dentate Gyrus; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; Glycine; In Vitro Techniques; Interneurons; Membrane Potentials; Patch-Clamp Techniques; Pyrimidines; Rats; Rats, Wistar; Sodium-Potassium-Exchanging ATPase; Strophanthidin; Tetrodotoxin; Wounds, Nonpenetrating
PubMed: 10805688
DOI: 10.1152/jn.2000.83.5.2916 -
The Journal of Neuroscience : the... May 1994Astrocytes in vitro and in situ have been shown to express voltage-activated ion channels previously thought to be restricted to excitable cells, including...
Astrocytes in vitro and in situ have been shown to express voltage-activated ion channels previously thought to be restricted to excitable cells, including voltage-activated Na+, Ca2+, and K+ channels. However, unlike neurons, astrocytes do not generate action potentials, and the functional role of voltage-activated channels in astrocytes has been an enigma. In order to study the function of Na+ channels in glial cells, we carried out ion flux measurements, patch-clamp recordings, and ratiometric imaging of [Na+]i during blockade of Na+ channels on rat spinal cord astrocytes cultured for 7-10 d. Acute blockade of astrocyte Na+ channels by TTX had multiple effects: (1) TTX reduced, in a dose-dependent manner, Na+/K(+)-ATPase activity measured as unidirectional influx of 86Rb+; (2) TTX depolarized astrocyte membrane potential at a rate of approximately 1 mV/min; (3) TTX (100 microM) reduced [Na+]i; and (4) prolonged exposure to micromolar TTX induced astrocyte death. All these effects of TTX could be mimicked by ouabain or strophanthidin, specific blockers of the Na+/K(+)-ATPase. The effects of TTX and ouabain (or strophanthidin) were not additive. These results suggest that TTX-blockable Na+ channels in glial cells serve functions that do not require their participation in action potential electrogenesis; in particular, we propose that glial Na+ channels constitute a "return" pathway for Na+/K(+)-ATPase function, which permits Na+ ions to enter the cells to maintain [Na+]i at concentrations necessary for activity of the Na+/K(+)-ATPase. Since astrocyte Na+/K(+)-ATPase is believed to participate in [K+]o homeostasis in the CNS, the coupling of Na+ flux through voltage-activated Na+ channels to ATPase activity may provide a feedback loop that participates in the regulation of K+ ion levels in the extracellular space.
Topics: Animals; Animals, Newborn; Astrocytes; Astrocytoma; Cell Line; Cells, Cultured; Electrophysiology; Ganglia, Spinal; Glioma; Membrane Potentials; Models, Biological; Ouabain; Rats; Rats, Sprague-Dawley; Rubidium; Sodium; Sodium Channels; Sodium-Potassium-Exchanging ATPase; Strophanthidin; Tetrodotoxin; Time Factors; Tumor Cells, Cultured
PubMed: 8182422
DOI: 10.1523/JNEUROSCI.14-05-02464.1994 -
Journal of Applied Physiology... Sep 2001Vocal fold hydration is critical to phonation. We hypothesized that the vocal fold generates bidirectional water fluxes, which are regulated by activity of the...
Vocal fold hydration is critical to phonation. We hypothesized that the vocal fold generates bidirectional water fluxes, which are regulated by activity of the Na(+)-K(+)- ATPase. Western blots and immunohistochemistry demonstrated the presence of the alpha-subunit Na(+)-K(+)-ATPase in the canine vocal fold (n = 11). Luminal cells, basal and adjacent one to two layers of suprabasal cells within stratified squamous epithelium, were immunopositive, as well as basolateral membranes of submucosal seromucous glands underlying transitional epithelia. Canine (n = 6) and ovine (n = 14) vocal fold mucosae exhibited transepithelial potential differences of 8.1 +/- 2.8 and 9.3 +/- 1.3 mV (lumen negative), respectively. The potential difference and short-circuit current (ovine = 31 +/- 4 microA/cm(2); canine = 41 +/- 10 microA/cm(2)) were substantially reduced by luminal administration of 75 microM acetylstrophanthidin (P < 0.05). Ovine (n = 7) transepithelial water fluxes decreased from 5.1 +/- 0.3 to 4.3 +/- 0.3 microl x min(-1) x cm(-2) from the basal to luminal chamber and from 5.2 +/- 0.2 to 3.9 +/- 0.3 microl x min(-1) x cm(-2) from the luminal to basal chamber by luminal acetylstrophanthidin (P < 0.05). The presence of the Na(+)-K(+)-ATPase in the vocal fold epithelium and the electrolyte transport derived from its activity provide the intrinsic mechanisms to regulate cell volume as well as vocal fold hydration.
Topics: Animals; Biological Transport, Active; Dogs; Epithelial Cells; Membrane Potentials; Patch-Clamp Techniques; Sheep; Sodium-Potassium-Exchanging ATPase; Strophanthidin; Vocal Cords; Water
PubMed: 11509542
DOI: 10.1152/jappl.2001.91.3.1401 -
The Journal of Biological Chemistry Nov 1988The role of protons as substitutes for Na+ and/or K+ in the sodium pump reaction was examined using inside-out membrane vesicles derived from human red cells. Na+-like...
The role of protons as substitutes for Na+ and/or K+ in the sodium pump reaction was examined using inside-out membrane vesicles derived from human red cells. Na+-like effects of protons suggested previously (Blostein, R. (1985) J. Biol. Chem. 260, 829-833) were substantiated by the following observations: (i) in the absence of extravesicular (cytoplasmic) Na+, an increase in cytoplasmic [H+] increased both strophanthidin-sensitive ATP hydrolysis (nu) and the steady-state level of phosphoenzyme, EP, and (ii) as [H+] is increased, the Na+/ATP coupling ratio is decreased. K+-like effects of protons were evidenced in the following results: (i) an increase in nu, decrease in EP, and hence increase in EP turnover (nu/EP) occur when intravesicular (extracellular) [H+] is increased; (ii) an increase in the rate of Na+ influx into K+(Rb+)-free inside-out vesicles and (iii) a decrease in Rb+/ATP coupling occur when [H+] is increased. Direct evidence for H+ being translocated in place of cytoplasmic Na+ and extracellular K+ was obtained by monitoring pH changes using fluorescein isothiocyanate-dextran-filled vesicles derived from 4',4-diisothiocyano-2',2-stilbene disulfonate-treated cells. With the initial pHi = pHo = pH 6.2, a strophanthidin-sensitive decrease in pHi was observed following addition of ATP provided the vesicles contained K+. This pH gradient was abolished following addition of Na+. With alkali cation-free inside-out vesicles, a strophanthidin-sensitive increase in pH was observed upon addition of both ATP and Na+. The foregoing changes in pHi were not affected by the addition of tetrabutylammonium to dissipate any membrane potential and were not observed at pH 6.8. These ATP-dependent cardiac glycoside-sensitive proton movements indicate Na,K-ATPase mediated Na+/H+ exchange in the absence of extracellular K+ as well as H+/K+ exchange in the absence of cytoplasmic Na+.
Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Humans; Hydrogen-Ion Concentration; Potassium; Protons; Quaternary Ammonium Compounds; Rubidium; Sodium; Sodium-Potassium-Exchanging ATPase; Strophanthidin
PubMed: 2846547
DOI: No ID Found -
The Journal of Physiology Oct 1984Tension was measured in voltage clamped sheep cardiac Purkinje fibres while simultaneously measuring the intracellular Na activity (aiNa) with a recessed-tip,...
Tension was measured in voltage clamped sheep cardiac Purkinje fibres while simultaneously measuring the intracellular Na activity (aiNa) with a recessed-tip, Na-selective micro-electrode. Inhibiting the Na-K pump either by exposing the preparation to a K-free solution or by adding the cardioactive steroid strophanthidin increased both aiNa and twitch tension and resulted in the development of tonic tension, after-contractions and a transient inward current (ITI). The increase of twitch tension was present at lower aiNa than that required to produce the other phenomena. The relationship between the magnitude of the twitch tension and aiNa was always non-linear. Twitch tension increased steeply with aiNa at first but the relationship flattened off at higher aiNa and tension eventually decreased. Over the steep range, the relationship between tension and aiNa could be represented as: twitch tension = b (aiNa)y where y had a mean value of 3.2. Changing membrane potential or [Ca2+]o changed b but had little effect on y. Mn (2 mmol/l) greatly decreased twitch tension but, at least initially, had little effect on tonic tension. The steep relationship between twitch tension and aiNa was seen, irrespective of whether the Na-K pump was inhibited either by exposure to K-free solution or to strophanthidin and whether the relationship was measured either when aiNa was increasing or after it had reached a steady state. The steep dependence of twitch tension on aiNa observed in the present work means that manoeuvres which produce even small changes of aiNa will have significant effects on contraction.
Topics: Animals; Calcium; Heart Conduction System; In Vitro Techniques; Ion Channels; Manganese; Membrane Potentials; Myocardial Contraction; Potassium; Purkinje Fibers; Rubidium; Sheep; Sodium; Stimulation, Chemical; Strophanthidin
PubMed: 6092625
DOI: 10.1113/jphysiol.1984.sp015417 -
The Journal of Biological Chemistry Jan 1985Although the sodium pump normally exchanges three sodium for two potassium ions, experiments with inside-out red cell membrane vesicles show that the stoichiometry is...
Although the sodium pump normally exchanges three sodium for two potassium ions, experiments with inside-out red cell membrane vesicles show that the stoichiometry is reduced when the cytoplasmic sodium concentration is decreased to less than 1 mM. The present study was designed to gain insight into the question whether other monovalent cations, particularly protons, can act as sodium congeners in effecting pump-mediated potassium transport (ATP-dependent rubidium efflux from inside-out vesicles). The results show that at low cytoplasmic sodium concentration, an increase in proton concentration effects a further reduction in sodium:rubidium stoichiometry, to a value less than the minimal expected (1Na+:3Rb+). Furthermore, when vesicles containing 86RbCl are incubated in nominally sodium-free medium. ATP-dependent net rubidium efflux (normal influx) occurs when the pH is reduced from approximately 7.0 to 6.2 or less. This efflux is inhibited by strophanthidin and vanadate. These experiments support the notion that the sodium pump can operate as an ATP-dependent proton-activated rubidium (potassium) pump without obligatory countertransport of sodium ions.
Topics: Adenosine Triphosphate; Carrier Proteins; Erythrocyte Membrane; Humans; Hydrogen-Ion Concentration; Kinetics; Protons; Rubidium; Sodium; Sodium-Hydrogen Exchangers; Strophanthidin; Vanadates; Vanadium
PubMed: 2981847
DOI: No ID Found