K-free Solution Research Articles

Cooling-induced contraction of guinea pig tracheal smooth muscle.

Cooling of isolated guinea pig tracheal smooth muscle from 38 to 28 degrees C over 2.25 min produced a transient contraction followed by sustained relaxation. The cooling-induced contraction was blocked either by pretreatment with ouabain at concentrations of 10(-5) M or greater or by substitution of normal physiological salt solution with K-free solution. In contrast, the contractile response to cooling was not inhibited by pretreatment with phentolamine (10(-5) M), atropine (10(-5) M), tetrodotoxin (3 X 10(-7) M), diphenhydramine (10(-5) M), cromolyn sodium (10(-3) M), indomethacin (3 X 10(-7) M), nifedipine (10(-7) M), or verapamil (3 X 10(-6) M). Addition of NaHCO3 to the bath during cooling, preventing a change in pH of the physiological salt solution, did not affect the cooling-induced contraction. It is concluded that cooling of isolated guinea pig trachea produces a transient ouabain-sensitive contraction, and that the data suggest the contraction is mediated by inhibition of Na-K-ATPase in the smooth muscle rather than through neuronal stimulation or chemical mediator release.

Spontaneous [3H]noradrenaline release from the main pulmonary artery of the rabbit induced by sodium-pump inhibition.

Inhibition of Na pump either by ouabain (10(-4) M) or by K removal increased the [3H]noradrenaline [( 3H]NA) release from the isolated main pulmonary artery of the rabbit in the presence of neuronal (cocaine, 3 X 10(-5) M) and extraneuronal (corticosterone, 5 X 10(-5) M) uptake blockers. The ouabain-evoked [3H]NA release began after a delay of about 30 min and peaked after 66 min of ouabain application. Both times were shortened by omission of K from the external medium. About 90% of ouabain-evoked [3H]NA release proved to be external Ca concentration ([Ca]o) dependent and the peak effect was delayed by about 80 min in Ca-free (+ 1 mM EGTA) solution. In the presence of external Ca (2.5 mM) the [3H]NA-releasing effect of 'K-free' treatment was much less pronounced than that of 10(-4) M ouabain, the initial delay in transmitter release was shorter (10-15 min) and the peak effect developed earlier (at 42 min). On readmission of K the [3H]NA release recovered quickly to the original value. Ca removal did not antagonize the transmitter release observed in K-free solution, but the peak release was delayed by about 90 min. A low concentration of ouabain (10(-5) M) failed to produce transmitter release in the presence of normal external K, but markedly increased the release in K-free solution. The release was much bigger than the sum of their separate effects, and the rate of rise was faster than when 10(-4) M ouabain was applied in normal solution. Excess Ca (7.5; 15 mM) inhibited the [3H]NA release observed in K-free solution. 7.5 mM-Ca also delayed the transmitter-releasing action of 10(-4) M ouabain, an effect antagonized by omission of K from the external medium. The mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10(-5) M) significantly increased the [3H]NA release in Ca-free, 1 mM EGTA-containing solution, and enhanced the effects of ouabain (10(-4) M). The Ca ionophore A23187 (10(-5) M) also significantly increased the [3H]NA release in the absence of external Ca and in the presence of 1 mM EGTA. Again, in the presence of A23187 the effects of 10(-4) M ouabain in releasing neurotransmitter were enhanced. When CCCP and A23187 were applied together in Ca-free, EGTA solution the [3H]NA releasing action of ouabain was still apparent. Veratridine (10(-4) M) enhanced the transmitter release in the absence of external Ca in a tetrodotoxin (TTX)-sensitive manner.(ABSTRACT TRUNCATED AT 400 WORDS)

The quantitative relationship between twitch tension and intracellular sodium activity in sheep cardiac Purkinje fibres.

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.

Perturbations in the membrane potential of cultured heart cells: role of calcium.

Intracellular recordings were obtained from spheroidal aggregates of 7-day embryonic chick heart cells after 3 days in gyratory culture. Three types of perturbations in the membrane potential were observed under experimental conditions expected to increase intracellular calcium: 1) multiple oscillations (of 5-20 mV peak-to-peak amplitude) during diastole in aggregates exposed to 10-15 mM Ca, 5 microM strophanthidin, or K-free solutions; 2) less periodic spontaneous voltage fluctuations (of less than 1.5 mV peak-to-peak amplitude) in aggregates exposed to solutions containing 22% of the normal [Na], and 3) depolarizing afterpotentials (DAPs), following repolarization of the action potential, in aggregates treated with 20-50 microM A23187, a Ca ionophore, or 5-10 mM caffeine. The oscillations were reduced markedly by 0.03-3.0 microM tetrodotoxin (TTX) and were blocked by 5-10 mM caffeine. Spontaneous voltage fluctuations were increased by raising external Ca, were unaffected by 30 microM TTX, and were blocked by 5 mM caffeine. DAPs were not blocked by 5 mM caffeine or by 0.1 microM TTX and 1 microgram/ml D 600, concentrations that greatly reduced the action potential upstroke velocity and plateau, respectively. Two intracellular electrodes were employed to test for electrotonic coupling between cells within an aggregate. An electrotonic response in one cell could be recorded when current was injected into another cell during recordings of each of the perturbations but was somewhat less during spontaneous voltage fluctuations. Possible ionic mechanisms for the perturbations and for concomitant changes in the configuration of the action potential are discussed.

Evidence for sodium-calcium exchange in the guinea-pig ureter.

The effects of high-K and low-Na solutions on the smooth muscle of the guinea-pig ureter have been examined in both normal tissues, and tissues in which the Na pump had been blocked by exposure to K-free solutions or ouabain (high-Na tissues). Tension recording, membrane potential measurements and ion analysis were used. High-K solutions depolarize normal tissues, leading to action potential generation and phasic contractions followed, at concentrations greater than 20-30 mM, by cessation of action potentials and the development of a biphasic contracture which declines slowly during continuous exposure. The contracture is abolished by Ca-antagonist drugs, procaine and Ca-free solutions. Short exposures of normal tissues to Na-free solutions do not result in tension development. Longer exposures may initiate tension, depending on the Na substitute used. Sucrose causes depolarization of the cells and spike development associated with phasic contractions, superimposed on a small contracture; Li depolarizes the cells but causes no tension generation; Tris hyperpolarizes the cells and a small increase in basal tone may be seen. On exposure to K-free solutions or ouabain, the tissues do not develop significant tone but their response to short application of high-K solutions grows with time. The tissues also develop the ability to contract on short applications of low-Na solutions. The low-Na contractures are resistant to concentrations of Ca antagonists that abolish the K responses of normal tissues, but are abolished in Ca-free solutions. The ability of the tissues to contract in Na-free solutions is accompanied by an increase in intracellular Na and loss of intracellular K. Even after several hours' exposure to ouabain, however, the tissues still contain significant amounts of K and the membrane potential is the same as, or more negative than that in normal tissues. Therefore it appears that another mechanism, apart from the Na pump, can regulate intracellular Na. On continuous exposure to Na-free solutions, the contracture declines rapidly. The decline is associated with a loss of intracellular Na. The Na-free contracture is larger when K rather than Tris is used as the substitute. This difference persists in the presence of a concentration of Mn that abolishes the K contracture of normal tissues but is abolished by high concentrations (10 mM) of procaine.(ABSTRACT TRUNCATED AT 400 WORDS)

Diminished arterial smooth muscle response to adenosine during Na-K pump inhibition.

The possibility of a role of the sarcolemmal Na-K pump of arterial smooth muscle in the mechanism of action of adenosine was explored in this study. Isolated helical strips of rabbit coronary and femoral arteries were suspended in organ baths with physiological salt solution, and isometric contractions were recorded. Concentration-dependent relaxations produced by adenosine were attenuated during Na-K pump inhibition. Ouabain (1 X 10(-5) M) significantly increased the adenosine ED50 values from 9.6 X 10(-8) M to 2.9 X 10(-7) M and from 9.4 X 10(-7) M to 8.4 X 10(-6) M in coronary and femoral artery strips, respectively. Similar results were obtained by substitution of LiCl for NaCl in the bathing solution and with a K-free solution. These effects were not attributable to augmented initial active tension, time-dependent changes in adenosine response or the release of norepinephrine from adrenergic nerve terminals. In addition, adenosine enhanced potassium-induced relaxation, an index of Na-K pump activity in isolated vascular strips. The data suggest that sarcolemmal Na-K pump activity may contribute directly or indirectly to the mechanism of action of adenosine.

Effects of Palytoxin on Na, K and ATP Contents of Vascular Smooth Muscle of Rabbit Aorta

Paiytoxin (PTX) at concentrations higher than 10−9 M increased tissue Na and decreased tissue K contents in the smooth muscle of rabbit aorta. The decrease in the tissue K content induced by PTX (10−8 M) was complete within 1 hr. Saponin (1 mg/ml) and Triton X-100 (0.1% wt./vol.) also rapidly decreased the tissue K content. On the other hand, a high concentration of ouabain (10−3 M) did not change the tissue K content within 1 hr of application, and the maximum loss of K was obtained after 6 hr. Loss of tissue Na into Na- and K-free solution from Na loaded muscle was accelerated by PTX (10−8 M). The PTX-induced increase in loss of Na was inhibited in proportion to the decrease in the temperature from 37 to 10°C, while the loss of Na in the absence of PTX was almost completely inhibited at 24°C. Decrease in the wet weight of the muscle induced by hyperosmotic solution was inhibited by pretreatment with PTX (10−8 M) or saponin (1mg/ml) for 1 hr. PTX (10−9 and 10−8 M) had no effect on the ATP content of the muscle. However, PTX at concentrations above 10−7 M reduced the ATP content, and a significant amount of ATP was detected in the incubation medium. Saponin (1 mg/ml) and Triton X-100 (0.2% wt./vol.) induced a release of Na from liposomes prepared with synthesized lecithin or total lipids of rabbit red blood cells. However, no Na leak was induced by PTX (10−8-10−6 M) in these liposomes. These results suggest that PTX at low concentrations (10−9-10−8 M) increases the membrane permeability of vascular smooth muscle cells to Na and K ions. At higher concentrations (10−7-10−6 M), PTX seems to form pores which are permeable to a larger molecule like ATP. The results further suggest that the mode of action of PTX is different from that of saponin or detergent.

Diminished arterial smooth muscle response to sodium nitroprusside during Na-K pump inhibition.

Helical strips of rabbit coronary and femoral arteries were used to determine whether Na-K pump inhibition would diminish relaxation in response to sodium nitroprusside. Ouabain (10(-5) M) decreased relaxation by 10(-7) M sodium nitroprusside from 87.0 +/- 5.4 to 65.2 +/- 9.1% in coronary, and from 62.0 +/- 6.9 to 32.8 +/- 3.3% in femoral artery strips. A low Na solution and a K-free solution also decreased relaxation by sodium nitroprusside. Relaxation in response to K+, an index of Na-K pump activity in arterial smooth muscle, was enhanced in the presence of sodium nitroprusside. These results may reflect an interaction between sodium nitroprusside and the sarcolemmal Na-K pump of arterial smooth muscle.

Effects of palytoxin on Na, K and ATP contents of vascular smooth muscle of rabbit aorta.

Palytoxin (PTX) at concentrations higher than 10(-9) M increased tissue Na and decreased tissue K contents in the smooth muscle of rabbit aorta. The decrease in the tissue K content induced by PTX (10(-8)M) was complete within 1 hr. Saponin (1 mg/ml) and Triton X-100 (0.1% wt./vol.) also rapidly decreased the tissue K content. On the other hand, a high concentration of ouabain (10(-3)M) did not change the tissue K content within 1 hr of application, and the maximum loss of K was obtained after 6 hr. Loss of tissue Na into Na- and K-free solution from Na loaded muscle was accelerated by PTX (10(-8)M). The PTX-induced increase in loss of Na was inhibited in proportion to the decrease in the temperature from 37 to 10 degrees C, while the loss of Na in the absence of PTX was almost completely inhibited at 24 degrees C. Decrease in the wet weight of the muscle induced by hyperosmotic solution was inhibited by pretreatment with PTX (10(-8)M) or saponin (1 mg/ml) for 1 hr. PTX (10(-9) and 10(-8)M) had no effect on the ATP content of the muscle. However, PTX at concentrations above 10(-7)M reduced the ATP content, and a significant amount of ATP was detected in the incubation medium. Saponin (1 mg/ml) and Triton X-100 (0.2% wt./vol.) induced a release of Na from liposomes prepared with synthesized lecithin or total lipids of rabbit red blood cells. However, no Na leak was induced by PTX (10(-8)-10(-6)M) in these liposomes. These results suggest that PTX at low concentrations (10(-9)-10(-8)M) increases the membrane permeability of vascular smooth muscle cells to Na and K ions. At higher concentrations (10(-7)-10(-6)M). PTX seems to form pores which are permeable to a larger molecule like ATP. The results further suggest that the mode of action of PTX is different from that of saponin or detergent.

Activation of feline acetylcholine synthesis in the absence of release: dependence on sodium, calcium and the sodium pump.

Following a 15 min inhibition of the Na pump in the cat superior cervical ganglion by perfusion with K-free Locke solution, a 10 min recovery period in normal Locke produced a 51% increase in acetylcholine stores. The increase in acetylcholine stores occurred without increase in acetylcholine release. Thus this procedure of pump inhibition followed by recovery selectively activates acetylcholine synthesis. The increase in acetylcholine stores occurred entirely during the 10 min recovery period in which the Na pump was re-activated. This increase represents a rate of synthesis of acetylcholine of 5.1% of stores per minute, which equals the maximum rate that can be achieved during high-frequency preganglionic nerve stimulation. The increase in stores was not affected by substituting isethionate for all but 8 mM-Cl in the perfusion fluids. The increase in stores was prevented by reducing the Na concentration of the K-free Locke to 25 mM. The increase in stores was only 17% when Ca was omitted from the K-free Locke. Omission of Ca from the perfusion fluid in the recovery period as well as in the period of pump inhibition prevented any increase in stores. It is concluded that the selective activation of acetylcholine synthesis following the pause in Na pumping was a direct result of an increase in Na pump rate and an increase in internal Ca in the nerve terminals. It is proposed that similar changes in Na pump rate and internal Ca produced by repetitive nerve impulse activity likewise activate acetylcholine synthesis independently of release of transmitter or depletion of stores.

Sodium/calcium exchange in mammalian ventricular muscle: a study with sodium-sensitive micro-electrodes.

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.

Activation of potassium transport induced by secretagogues in superfused submaxillary gland segments of rat and mouse.

In order to investigate the actions of acetylcholine (ACh), catecholamines and substance P on K transport in the submaxillary gland, measurements of net K flux to and from the gland tissue using flame photometry, Na efflux from the tissue using radioactive 22Na, and membrane potential and input resistance using micro-electrodes were carried out on isolated superfused segments of rat and mouse submaxillary glands. ACh (5.5 X 10(-8) to 5.5 X 10(-4) M), phenylephrine (5 X 10(-7) to 5 X 10(-4) M) or substance P (10(-9) to 10(-5) M) stimulation for 5 min induced a transient K release followed by a small K uptake after the cessation of stimulation. The K release was markedly enhanced by the simultaneous addition of ouabain (10(-3) M). On the other hand, isoprenaline (2.5 X 10(-9) to 2.5 X 10(-5) M) induced a transient K uptake without any preceding K release. The K uptake was completely blocked by the addition of ouabain. Noradrenaline induced only K uptake at a low concentration (3 X 10(-7) M), but induced transient K release followed by marked K uptake at higher concentrations (3 X 10(-6) to 3 X 10(-4) M). The K release induced by noradrenaline was suppressed by the addition of phentolamine (10(-5) M), while the K uptake was suppressed by propranolol (5 X 10(-6) M). The K release induced by ACh, phenylephrine, noradrenaline or substance P was severely reduced by Ca omission from the superfusing solution and restored by the re-admission of Ca. The isoprenaline- or noradrenaline-induced K uptake was, however, little affected by Ca omission. Application of isoprenaline (2.5 X 10(-6) M) induced an increase in 22 Na efflux. The increase in 22Na efflux was completely abolished in the presence of ouabain. Local application to the tissue bath of isoprenaline (4.7 X 10(-13) to 4.7 X 10(-12) mole) or noradrenaline (5.7 X 10(-12) to 5.7 X 10(-11) mole) in the presence of phentolamine (10(-5) M) induced membrane hyperpolarization without any appreciable change in input resistance. The hyperpolarization was abolished in the presence of ouabain (10(-3) M) or propranolol (5 X 10(-6) M) or in a K-free or low Na solution. Higher doses of both agonists, however, induced depolarization or biphasic responses (initial depolarization followed by hyperpolarization). The depolarizations were accompanied by a moderate reduction in input resistance. It is concluded that in the rat and mouse submaxillary gland acinar cells cholinergic, alpha-adrenergic or substance P stimulation causes K release (and perhaps Na uptake) resulting in activation of the Na-K pump, while beta-adrenergic receptor stimulation might directly activate the Na-K pump resulting in K uptake, or might cause Na uptake resulting in activation of the Na-K pump.

The role of intracellular sodium activity in the anti‐arrhythmic action of local anaesthetics in sheep Purkinje fibres.

The effects of lidocaine have been examined on the arrhythmogenic transient inward current (ITI) in voltage-clamped sheep cardiac Purkinje fibres. Tension and intracellular Na activity (aiNa) were measured simultaneously. The addition of lidocaine (200-300 microM) produced an immediate decrease of inward holding current and a gradual fall of aiNa. The relative magnitudes of the changes of current and aiNa were shown to be consistent with the outward shift of current representing principally a reduction of inward Na current. The Na pump was inhibited by reducing the external Rb concentration in a K-free solution. This produced an after-contraction and transient inward current (ITI) along with a rise of aiNa. The subsequent addition of lidocaine decreased the magnitude of ITI and the after-contraction while decreasing aiNa. Tetrodotoxin (TTX) had qualitatively similar effects to lidocaine on inward holding current, aiNa, ITI and the after-contraction. When aiNa was changed by (i) lidocaine, (ii) TTX or (iii) small changes of external Rb concentration, a hysteresis was seen in the relationship between aiNa and ITI or after-contraction. The hysteresis was similar to that previously found between aiNa and contraction (Eisner, Lederer & Vaughan-Jones, 1981). Despite this hysteresis, neither lidocaine nor TTX affected the relationship between magnitudes of ITI and the after-contraction. It is suggested that the fall of aiNa is a major factor in the reduction of ITI by lidocaine. These results are discussed in relation to the anti-arrhythmic actions of lidocaine.

The basis for the membrane potential of quiescent cells of the canine coronary sinus.

During prolonged periods of quiescence, the membrane potential of cells in the isolated canine coronary sinus, exposed to normal Tyrode solution containing 4 mM-K, declines to about -60 mV. The nature of the resting potential was investigated, in small strips of coronary sinus tissue mounted in a fast-flow system, by recording the membrane potential responses to sudden changes in the extracellular ionic environment. At extracellular K concentrations ([K]o) from 0 to 64 mM the resting potential was little affected by replacing all but 1 mM of external Cl ions with isethionate and methylsulphate ions. At [K]o levels from 4 to 150 mM the resting potential was reasonably well described by the Goldman-Hodgkin-Katz equation on the assumption that the intracellular K concentration ([K]i) was 155 mM and that the ratio of membrane permeability coefficients for Na and K, PNa/PK, was 0.07. In the presence of a high concentration of acetylcholine or carbachol (greater than or equal to 1 microM), the resting potentials at [K]o levels from 1 to 150 mM approximated K equilibrium potentials (EK) calculated on the assumption that [K]i was 155 mM. At [K]o levels less than or equal to 8 mM replacing most of the external Na with sucrose or Tris caused a substantial hyperpolarization, whereas application of 1-2 microM-tetrodotoxin caused only slight hyperpolarization. A transient hyperpolarization, due to enhanced electrogenic Na extrusion, was recorded on switching back to 4 mM-K following brief exposures to K-free solution; no transient hyperpolarization was recorded in the presence of 5 microM-acetylstrophanthidin. The acetylstrophanthidin itself caused a rapid depolarization of several millivolts. Preliminary conductance measurements made with two micro-electrodes in some smaller preparations indicate that the steady-state current-voltage relationship is N-shaped. We conclude that the low membrane potential of quiescent coronary sinus cells reflects not a low [K]i but rather a relatively high ratio PNa/PK, of about 0.07: the Na ions flow into the cells via predominantly TTX-insensitive pathways and are extruded by the electrogenic Na/K exchange pump, which thereby makes a substantial contribution to the resting potential.

Noradrenaline hyperpolarizes cells of the canine coronary sinus by increasing their permeability to potassium ions.

The mechanism of the noradrenaline-induced hyperpolarization was investigated in small strips of coronary sinus tissue mounted in a fast-flow system. The recorded hyperpolarization was negligibly small in response to 10 nM-noradrenaline but was maximal at 10 microM (average amplitude 23 mV, in 4 mM-K solution). The hyperpolarization was unaffected by 1 microM-phentolamine but was abolished by 10 microM-propranolol and so is presumably mediated via beta-adrenoceptors. The noradrenaline-induced hyperpolarization became smaller when the extracellular K concentration ([K]o) was raised or when the extracellular Na concentration was lowered. These results are consistent with two general mechanisms: noradrenaline might cause hyperpolarization by stimulating the Na/K pump to generate more outward current, as previously suggested for other cell types. Alternatively, noradrenaline might lower the permeability ratio, PNa/PK, by reducing the permeability coefficient for Na (PNa) and/or increasing that for K (PK). The noradrenaline-induced hyperpolarization is not diminished during exposure to 5 microM-acetylstrophanthidin, or to K-free solution, or to K-free solution containing acetylstrophanthidin. We conclude that the hyperpolarization does not reflect enhanced electrogenic pump activity. Conductance measurements using two micro-electrodes in very small preparations revealed that, like the muscarinic agonist carbachol, noradrenaline caused an increase in membrane slope conductance. Steady-state current-voltage curves obtained in the presence of noradrenaline, in the presence of carbachol, and in the absence of both drugs all crossed each other at about the same level of membrane potential. During the maintained injection of sufficiently large hyperpolarizing current, application of either noradrenaline or carbachol causes depolarization instead of hyperpolarization. The cross-over or 'reversal' potentials of current-voltage curves, determined with and without the drugs, vary with [K]o approximately as does the K equilibrium potential calculated assuming the intracellular K concentration to be 155 mM. We conclude that, like carbachol and acetylcholine, noradrenaline causes a specific increase in the K permeability of coronary sinus cells.

Omission of luminal potassium reduces cellular chloride in early distal tubule of amphibian kidney.

Experiments in the amphibian early distal tubule have shown that Cl transport is secondarily active, coupled to the flux of Na and dependent on the presence of luminal K. Omission of luminal K results in a decrease of cellular Na, a finding that suggests inhibition of luminal Na entry. In the present study intracellular chloride activity (Cli) and peritubular cell membrane potentials (PDpt) were evaluated before and after omission of luminal K. Furthermore, the effect of inhibition of the luminal K conductance by barium on the electrochemical gradient of Cl (E te Cl ) across the distal epithelium was determined at static head conditions. Experiments were performed in early distal segments of the isolated perfused kidney ofAmphiuma andRana esculenta. Cli and PDpt were measured simultaneously in single cells by double barreled Cl sensitive microelectrodes in the presence and absence of luminal K. E te Cl was determined at zero net flux conditions with single barreled electrodes in control tubules, in the presence of barium (3·10−3 mol/l) and in the presence of furosemide (5·10−5 mol/l). In 26 individual cellular impalements omission of luminal K hyperpolarized PDpt from 72.5±1.2 to 90.0±1.9 mV (cell interior negative). Concomitantly, Cli fell from 8.5±0.4 to 5.4±0.3 mmol/l. Both effects occurred within seconds and were fully reversible. Addition of barium to the luminal fluid diminished E te Cl (directed lumen positive) from a control value of 39.5±1.4 mV to 28.5±2.5mV. E te Cl could be further diminished to 14.1±2.1 mV and to 1.3±0.5 mV after application of barium on both sides and after luminal application of furosemide, respectively. The experiments indicate that active Cl uptake across the luminal cell membrane depends critically on the presence of luminal K. Omission of luminal K achieved either by perfusing the lumen with K-free solutions or by inhibition of K back flux from the cell interior into the lumen by barium reduces Cl reabsorption. Together with previous data on the K dependence of the Na uptake the present experiments support the hypothesis of a common transport system for K, Na, Cl located in the luminal cell membrane.

Effects of palytoxin on the membrane permeability of vascular smooth muscle of rabbit aorta and liposomes

Palytoxin (Palythoa tuberculosa)(PTX) in concentration higher than 10 M increased tissue Na and decreased tissue K contents in smooth muscle of rabbit aorta. Thegexchange of extracellular Na with intracellular K induced by PTX (10 M) was completed within Ih. Saponin (1mg/ml) and TritonX-100(0.1%wt/vol) also rapidly decreased tissue K content. On the other hand,high concentration of ouabain (10-3 M) did not change the tissue K content within one hour of application, and the maximum loss of K was obtained after 6h. Lgss of tissue Na into Na- and K-free solution was accelerated by PTX (10 M). The PTX-induced increase in loss of Na was inhibited in proportion to decrease in the temperature from 37 to 10 °C, while the loss of Na in the absence of PTX was almost completely inhibited at 24 °C. Decrease in the wet weight of the muscle induced by hyperosmotic solution was inhabited by pretreatment with PTX (10 M). PTX in concentration above 10 M reduced the ATP content, and significant amount of ATP was detected in the incubation medium. Saponin (lmg/ml) and TritonX-100 (0.1%) induced a release of Na from liposome prepared with synthesized lecithin or total lipids of^rabbit red blood cells. However, no Na leak was induced by PTX (10 -10 M) in ^hesegliposomes. These results suggest that PTX at low concentration (10 -10 M) increases membrane permeability to Na and K_i,ons_gf the vascular smooth muscle cells. At higher concentration (10 -10 M), it seems to form pores which are permeable to a larger molecule like ATP. The effects might not be mediated through the interaction with membrane lipids.

Potentiating and depressive effects of ouabain and potassium-free solutions on rat mesenteric resistance vessels.

We have investigated the in vitro effects of ouabain and K-free solutions on some pharmacological and electrophysiological properties of rat mesenteric resistance vessels (internal diameter approximately 190 micrometers). Vessels were mounted as ring preparations on a myograph capable of measuring their isometric wall tension. In normal saline solutions, vessels did not exhibit any tone and had a membrane potential of -54 mV. Both 1 mM ouabain and K-free solutions caused a transient depolarization of 5-8 mV; thereafter the membrane slowly depolarized to about -45 mV after 30 minutes. There was no mechanical response to ouabain, but K-free solutions caused a transient development of tension which could be inhibited by phentolamine (1 microM). In norepinephrine-activated vessels, exposure to ouabain or K-free solutions caused a small depolarization and an increase in tension. Long-term (30-minute) exposure to 1 mM ouabain or K-free solutions reduced the amplitude of norepinephrine responses and, for the lower (but not the higher) norepinephrine concentrations, the membranes were about 14 mV more depolarized than control. The mechanical responses to a cocktail of norepinephrine in a high potassium solution were, however, unaffected. Re-exposure to normal saline solution produced a transient hyperpolarization and transiently eliminated the norepinephrine response, but thereafter the membrane potential and response returned to normal. The results indicate that ouabain and K-free solutions can have both short-term potentiating and long-term depressive effects on the mechanical response of rat mesenteric resistance vessels to norepinephrine.

Studies of calcium channels in rat clonal pituitary cells with patch electrode voltage clamp.

1. The properties of the Ca channel in tissue cultured clonal cells (GH(3)) isolated from a rat anterior pituitary tumour were studied with the patch electrode voltage-clamp technique.2. To isolate the current through the Ca channel, the currents through the Na channel, the delayed K channel and the Ca(2+) induced K channel were minimized by replacing the external Na(+) with TEA(+) and adding EGTA to the K-free solution inside the patch electrode.3. The selectivity ratios through the Ca channel with different cations were 2.7 (Ba(2+)):1.6 (Sr(2+)):1.0 (Ca(2+)) and the m(2) form of the activation kinetics and the relationships between the time constant and the membrane potential were common to the three divalent cations.4. The amplitude of the Ba(2+) current increased linearly with [Ba(2+)](o) up to 25 mM and thereafter tended to show saturation.5. The current-voltage relation showed a positive shift along the voltage axis as [Ba(2+)](o) increased, probably due to the screening effect of Ba(2+) on the negative surface charges.6. The time constant of activation as a function of the membrane potential showed a parallel shift as [Ba(2+)](o) was increased, suggesting that the activation kinetics were independent of the permeant ion concentration.7. The time constant of the tail current was consistent with m(2) kinetics for opening and closing of the Ca channel.8. The extrapolated ;instantaneous' tail current rapidly increased as the activating membrane potential became more positive and reached an apparent saturation at membrane potentials substantially more positive than the potential that gave the maximum peak inward current, and suggested that the single channel has a sigmoidal current-voltage relationship.9. The power density spectrum obtained during the steady-state inward Ba(2+) current had a cut-off frequency which was nearly voltage independent; this is expected if the fluctuation of the current originates from m(2) activation kinetics.10. The results of noise analysis suggest that the amplitude of the single Ca channel current was about 0.2 pA at 25 mM-Ba(2+) and 0.7 pA at 100 mM-Ba(2+) for membrane potentials in the vicinity of the maximum inward current.

Fluctuations in membrane current driven by intracellular calcium in cardiac Purkinje fibers

Spontaneous oscillatory fluctuations in membrane potential are often observed in heart cells, but their basis remains controversial. Such activity is enhanced in cardiac Purkinje fibers by exposure to digitalis or K-free solutions. Under these conditions, we find that voltage noise is generated by current fluctuations that persist when membrane potential is voltage clamped. Power spectra of current signals are not made up of single time-constant components, as expected from gating of independent channels, but are dominated by resonant characteristics between 0.5 and 2 HZ. Our evidence suggests that the periodicity arises from oscillatory variations in intracellular free Ca that control ion movements across the surface membrane. The current fluctuations are strongly cross-correlated with oscillatory fluctuations in contractile force, and are inhibited by removing extracellular Ca or exposure to D600. Chelating intracellular Ca with injected EGTA also abolishes the current fluctuations. The oscillatory mechanism may involve cycles of Ca (or Sr) movement between sarcoplasmic reticulum and myoplasm, as previously suggested for skinned cardiac preparations. Our experiments in intact cells indicate that changes in surface membrane potential can modulate cytoplasmic Ca oscillations in frequency and perhaps amplitude as well. A two-way interaction between surface membrane potential and intracellular Ca stores may be a common feature of heart, neuron, and other cell types.