K-free Solution Research Articles

Co-existence of a fast tetrodotoxin-sensitive low voltage-activated non selective Na/Ca/K channel and of T-type Ca channels in rat isolated pulmonary vein cardiomyocytes

Ectopic foci in pulmonary veins (PV) are involved in the onset of atrial fibrillation. Norepinephrine (NE) induces an automatic activity occurring in bursts in rat PV cardiomyocytes (CM) which depends on Ca (upstroke) and Na (inter-burst) channels. To characterize low voltage-activated (LVA) Ca currents in rat PVCM susceptible to trigger ectopic foci. Whole-cell ICa (5 mM Ca) was recorded from -100 mV with classical Na- and K-free solutions at room temperature in enzymatically isolated PVCM from male Wistar rats. A fast LVA ICa (FLVA-ICa; −1.5 ± 0.2 pA/pF, n = 45; 26% of peak ICa, max recorded at + 20 mV) was activated between −55 and −20 mV in ∼56% of PVCM and blocked by 10 μM tetrodotoxin (TTX). In that, it was similar to the reported TTX-sensitive Ca current. However, it was markedly increased by addition of NaCl (1 or 3 mM) or KCl (5 or 10 mM). Permeability ratios P’Ca/PNa and P’Ca/PK calculated for bi-ionic conditions were respectively 2.25 ± 0.51 (n = 6) and 1.88 ± 0.25 (n = 14), and not different from a value of 2. NE (10 μM, n = 13) increased FLVA-ICa and negatively shifted its activation threshold, an effect partially reversed by 5 μM Atenolol. It was partially blocked by 5 μM Nifedipine (n = 5) and increased by 300 nM BayK8644 (n = 6), but not blocked by Ranolazine (10 μM, n = 6). NiCl2 (40 μM, n = 9) and TTA-A2 (10 or 100 nM, n = 11) increased FLVA-ICa. Neither Ba nor Sr alone could permeate the FLVA channel or block Ca influx but revealed a large slower activating and inactivating LVA Ca current (SLVA-ICa), present in 10 out of 80 PVCM and absent in LACM. SLVA-ICa was partially blocked by 100 nM TTA-A2. The ionic channel underlying FLVA-ICa is likely a fast voltage-gated non-selective channel permeable to Na/Ca/K with a dihydropyridine binding site. SLVA-ICa might correspond to Ca influx through Cav3.1 channels. The respective role of these channels to triggering ectopic foci in the PV myocardial sleeve remained to be determined.

Modulation of L-Type Calcium Current by Calcium-Dependent Mechanism in Trout Ventricular Myocytes

In cardiac myocytes, Ca has a dual role upon of L-type calcium current (ICa) by either inactivating it (calcium-dependent inactivation, CDI) or facilitating it (calcium-dependent facilitation, CDF). In fish cardiac myocytes, it is unclear whether both Ca modulations exist. This study addresses this point. Trout ventricular myocytes were enzymatically isolated. ICa was recorded using whole cell patch clamp with Na- and K-free solutions to avoid contaminating currents. With a low concentration of a slow Ca buffer (EGTA 2mM) in the pipette solution, ICa inactivated slowly (compared to mammalian cardiac myocytes): the time to reach 37% of peak current (T37) was 26.2± 2.4 ms (mean±SEM, n=14). CDF was absent in all cells studied. When a fast Ca buffer (BAPTA 10 mM) was present in the pipette solution, ICa decay was similar to the decay in the presence of EGTA (T37: 25.4±1.5 ms, NS, t-test, n=9) and CDF was absent (n=9). We quantified the relative contribution of CDI and sarcoplasmic reticulum (SR) CDI according to our published method, and estimated that CDI represents ∼39% of total ICa inactivation, and that SR Ca release causes ∼12% of CDI. We conclude that in fish myocytes CDI play a role in ICa modulation but CDF is absent.Supported by the Welcome Trust.

Quantification Of L-type Ca Current Inactivation Mechanisms In Trout Ventricular Myocytes.

Inactivation of L-type calcium current (ICa) is due to two mechanisms: voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). In fish cardiac myocytes, it is unknown whether Ca release from the sarcoplasmic reticulum (SR) participates in CDI of ICa. This study assesses the relative contribution of different inactivation mechanisms of ICa in trout ventricular myocytes. Trout ventricular myocytes were enzymatically isolated. ICa was recorded using whole cell patch clamp with Na- and K-free solutions to avoid contaminating currents. With a low concentration of a slow Ca buffer (EGTA 2mM) in the pipette solution, ICa inactivated slowly (compared to mammalian cardiac myocytes): the time to reach 37% of peak current (T37) was 26.2±2.4 ms (mean±SEM, n=14). When a fast Ca buffer (BAPTA 10 mM) was present in the pipette solution ICa decay was similar to the decay in the presence of EGTA (T37: 25.4±1.5 ms, NS, t-test, n=9). When barium was used as a charge carrier, IBa inactivates mainly via VDI and T37 was significantly increased (43.7±3.1 ms, n=9, p<0.05, t-test), albeit T37 is twice faster than in mammalian cardiac myocytes. We quantified the relative contribution of VDI and CDI according to the method previously described [Brette et al. (2004). Circ Res; 95; e1-7]. We measured the fraction of current remaining 20 ms after its peak (IR20). IR20 was 0.39±0.03 in EGTA, 0.42±0.02 in BAPTA and 0.64±0.03 in barium. We estimated that CDI represents ∼39% of total fast ICa inactivation, and that SR Ca release causes only ∼12% of CDI. We conclude that the main inactivation mechanisms in the trout myocyte are due to VDI and CDI from Ca entering the cell via ICa and not SR Ca release.Supported by the Wellcome Trust and the BBSRC.

Intracellular and extracellular renin have opposite effects on the regulation of heart cell volume. Implications for myocardial ischaemia

The influence of intracellular renin plus angiotensinogen (Ao) as well as angiotensin (Ang) II on cell volume was investigated in myocytes isolated from the heart of four-month-old cardiomyopathic hamsters (TO-2) and normal hamsters (F1B). Measurements of cell width and cell length were performed on quiescent cells using a Px-it imaging and computer system. The cell volume was calculated assuming the cells as elliptical cylinders and taking the cell depth equal to one third of cell width. For measurements of sodium pump current, the cells were voltage clamped (holding potential -40 mV) using the whole cell configuration. Cells were exposed to K-free solution to inhibit the pump and then to normal Krebs solution to reactivate the pump. In other experiments the cells were voltage clamped (holding potential -40 mV) and changes in the background current elicited by renin plus Ao or by Ang II were monitored. The results indicated that: a) intracellular dialysis of renin (128 pmol Ang I/ml) plus Ao (110 pmol Ang I generated by renin by exhaustion) decreased the cell volume concurrently with the activation of the sodium pump; b) intracellular losartan (10(-)8 M) or extracellular ouabain (10(-8) M) abolished the effect of renin plus Ao on cell volume; c) intracellular Ang II (10(-8) M), by itself, reduced cell volume and increased the pump current density; d) extracellular administration of renin plus Ao, at the same concentration used intracellularly, increased cell volume and inhibited the sodium pump. This increase of cell volume elicited by extracellular renin plus Ao was related to the activation of the Na-K-2Cl cotransporter; e) intracellular Ang II (10(-8) M) reversed cell swelling induced by hypotonic solutions. Intracellular and extracellular renin plus Ao have opposite effects on sodium pump and cell volume regulation in the failing heart. Both effects of renin plus Ao are dependent upon the formation of Ang II. Since intracellular Ang II counteracted the cell swelling induced by hypotonic solution, it is reasonable to think that the activation of the intracrine renin-angiotensin system might play a protective role during myocardial ischaemia by reducing cell volume.

Ouabain-sensitive and insensitive acetylcholine receptors on the membrane of the same neuron in Helix pomatia

Using internally dialyzed neurons of Helix pomatia as a model, the effect of structural analogs of acetylcholine (ACh) were investigated for their cholinomimetic properties on A- and B-types of ACh-responses. Specifically we analyzed choline esters of N- para- and ortho-alkoxybenzoyl-β-alanines, (CH 3O-, C 2H 5O-, C 3H 7O-, iso-C 3H 7O-, C 4H 9O-, iso-C 4H 9O-, C 5H 11O-, iso-C 5H 11O-), (in all, 16 combinations). The compounds evoked differing sensitivities of response to factors de-activating the Na–K-pump (ouabain and K-free solution). Most compounds resembled ACh: ionic currents caused by these compounds were inhibited by Na–K pump blockers in the case of A-type responses — B-type responses were insensitive to these factors. Ionic currents induced by choline esters of p-, o-propoxy- and iso-propoxybenzoyl-β-alanines were insensitive to ouabain and K-free solution in the case of A- and B-type responses. Ionic currents induced by the choline ester of p-butoxybenzoyl-β-alanine were inhibited by ouabain and K-free solution on both types of neuron. The results allow us to classify choline esters of p-, o-propoxy- and iso-propoxybenzoyl-β-alanines as N-cholinomimetics, while the choline ester of p-butoxybenzoyl-β-alanine can be considered as M-cholinomimetic. We conclude that both M- and N-type ACh receptors exist on the membrane of the same neurons.

The relation between low K +/Na + ratio and salt-tolerance in the wild tomato species Lycopersicon pennellii

The cultivated tomato Lycopersicon esculentum cultivar M82 and its wild salt-tolerant relative L. pennellii accession Atico were analyzed for: (a) dry weight, content of Na+ and K+ ions (in young and old leaves), and dry weight/potassium ratio (in young leaves) in plants grown in standard Hoagland and K-free solutions, without or with 100 mmol L−1 NaCl; and (b) dry weight in plants grown in K-free medium, in which K+ was substituted by the same concentration (5 mmol L−1) of Na+. Plants of L. pennellii, as expected: (a) responded better than the cultivated species to the high salinity in the standard solution with respect to biomass production; and (b) accumulated less K+ and more Na+ onder this condition. Furthermore, L. pennellii plants were characterized by: (a) a more efficient substitution of K+ function by Na+, as expressed by the fact that Na+ added to the K-free medium caused a greater increase of dry weight in the wild species; (b) a higher retranslocation of K+ from old to young leaves, and consequently (c) a higher K-efficiency (dry weight/K+) ratio. It is suggested that. (a) the use of less energy in L. pennellii, as compared with the cultivated species, to exclude Na+ and to accumulate K+ under salt stress contributes to the mechanism of salt tolerance in that species, and (b) the higher Na+ in the wild species is used as a cheap osmoticum in the vacuole and, possibly, as a partial substitute for K+ in some of its functions, and the lower K+ is compensated, at least in part, by the higher K-efficiency ratio.

Electrical responses of gastric smooth muscles in streptozotocin-induced diabetic rats.

Electrical responses of gastric smooth muscles produced by transmural nerve stimulation, acetylcholine, norepinephrine, or K-free solution were investigated in streptozotocin-induced diabetic rats, using intracellular microelectrode techniques. In muscles from diabetic rats, 1) the resting membrane potential remained unchanged, 2) slow waves disappeared or were markedly reduced in amplitude, 3) the excitatory junction potential was absent, and in most cases only an inhibitory junction potential of reduced amplitude was elicited, 4) the amplitude of the hyperpolarization generated after superfusion with K-free solution was reduced, 5) the sensitivity of the acetylcholine-induced membrane depolarization was increased, and 6) the norepinephrine-induced hyperpolarization was reduced because of functional loss of alpha- and beta-adrenoceptors. Thus diabetes mellitus caused functional impairment of neuromuscular transmission, reduced the maximum activity of the electrogenic pump, increased the sensitivity of muscarinic receptors, reduced the sensitivity of adrenoceptors, and reduced the myogenic activity in gastric smooth muscles. These alterations in the properties of smooth muscle may be involved in the diabetes-induced gastroparesis.

Effects of Na-K-2Cl cotransport inhibition on myocardial Na and Ca during ischemia and reperfusion.

In the context of the "pump-leak" hypothesis (37), changes in myocardial intracellular Na (Nai) during ischemia and reperfusion have historically been interpreted to be the result of changes in Na efflux via the Na-K pump. We investigated the alternative hypothesis that changes in Nai during ischemia are the result of changes in the Na "leak" rather than changes in the pump. More specifically, we hypothesize that the increase in Nai during ischemia is in part the result of increased Na uptake mediated by Na/H exchange. Furthermore, we present data consistent with the interpretation that the Na-K-2Cl cotransporter is active (or, alternatively, displaced from equilibrium) during ischemia and may contribute an additional Na efflux pathway during reperfusion. Thus inhibition of Na efflux via Na-K-2Cl cotransport during ischemia and reperfusion could result in increased Nai and therefore decreased force driving Ca efflux via Na/Ca exchange and ultimately increased intracellular Ca concentration ([Ca]i). Nai (in meq/kg dry wt) and [Ca]i (in nM) were measured in isolated Langendorff-perfused rabbit hearts using nuclear magnetic resonance spectroscopy. Except, during the 65 min of ischemia, hearts were perfused with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered Krebs-Henseleit solution equilibrated with 100% O2 at 23 degrees C and pH 7.4 +/- 0.05. During ischemia, Nai rose from 16.6 +/- 0.3 to 62.9 +/- 5.1 (delta Nai approximately 46) meq/kg dry wt and decreased during subsequent reperfusion (mean +/- SE, n = 3 hearts). To measure Na uptake ("leak") in the absence of efflux via the Na-K pump, in all of the protocols described below, the perfusate was nominally K-free solution containing 1 mM ouabain for 10 min before ischemia and during the 30-min reperfusion. After K-free perfusion, Nai rose from 20.2 +/- 0.5 to 79.1 +/- 5.3 (delta Nai approximately 59) meq/kg dry wt (n = 3) during ischemia and decreased during K-free reperfusion. When amiloride (1 mM) was added to the K-free perfusate to inhibit Na/H exchange, Nai rose from 16.3 +/- 0.9 to 44.7 +/- 5.1 (delta Nai approximately 28) meq/kg dry wt (n = 3) during ischemia; i.e., amiloride decreased Na uptake. When bumetanide (20 microM) was added to the nominally K-free perfusate to inhibit Na-K-2Cl contransport, Nai rose from 22.5 +/- 3.9 to 83.8 +/- 13.9 (delta Nai approximately 61 meq/kg dry wt (n = 3) during ischemia and did not decrease during reperfusion; i.e., bumetanide inhibited Na recovery during reperfusion (P < 0.05 compared with bumetanide free). For the same protocol, the presence of bumetanide resulted in increased [Ca]i during ischemia and reperfusion (P < 0.05); these increases in [Ca]i are interpreted to be the result of increased Nai. Thus the results are consistent with the hypotheses.

Hyperpolarization induced by sodium removal in rabbit sinoatrial node cells: Possible role of electrogenic sodium-calcium exchange

Spontaneously active rabbit sinoatrial node (SAN) cells were bathed in K-free solution or in K-free ouabain (20 μM)-containing solution to depress the electrogenic Na + K + pump activity. In SAN cells exposed to K-free solution, the automatic action potentials ceased with gradual depolarization, followed by an eventual steady-state membrane potential of −32 ± 1 mV. Under conditions where the Na + K + pump was blocked, removal of external Na + produced a large and rapid hyperpolarization in the membrane potential and the membrane was hyperpolarized by 23 ± 0.5 mV. When the external Na + was lowered, Na + was replaced by Li +. The Na-free hyperpolarization was not affected by applications of verapamil (4 μM), lidocaine (1 mM), and quinidine (50 μM), but was inhibited by either quinacrine (50 μM) or Cd 2+ (10 mM), which are blockers of Na + Ca 2+ exchange. In the absence of external K +, replacement of external NaCl by sucrose produced a hyperpolarization similar to that seen in the replacement of external Na + by Li +. In the K-free ouabain (20 μM)-containing solution, removal of external Na + also produced a hyperpolarization, and the membrane potential dropped from −29 ± 1 to −48 ± 1 mV. The intracellular acidification due to NH 4Cl removal after exposure to NH 4Cl (20 mM) produced a decrease in Na-free hyperpolarization, which, in the presence of ouabain was inhibited by the application of Cd 2+ (10 mM). Removal of external Ca 2+ nearly completely blocked Na-free hyperpolarization. It can be concluded that Na-free hyperpolarizations are related to the functioning of an electrogenic Na + Ca 2+ exchange mechanism.

Action of niflumic acid on evoked and spontaneous calcium‐activated chloride and potassium currents in smooth muscle cells from rabbit portal vein

1. The action of niflumic acid was studied on spontaneous and evoked calcium-activated chloride (ICl(Ca)) and potassium (IK(Ca)) currents in rabbit isolated portal vein cells. 2. With the nystatin perforated patch technique in potassium-containing solutions at a holding potential of -77 mV (the potassium equilibrium potential), niflumic acid produced a concentration-dependent inhibition of spontaneous transient inward current (STIC, calcium-activated chloride current) amplitude. The concentration to reduce the STIC amplitude by 50% (IC50) was 3.6 x 10(-6) M. 3. At -77 mV holding potential, niflumic acid converted the STIC decay from a single exponential to 2 exponential components. In niflumic acid the fast component of decay was faster, and the slow component was slower than the control decay time constant. Increasing the concentration of niflumic acid enhanced the decay rate of the fast component and reduced the decay rate of the slow component. 4. The effect of niflumic acid on STIC amplitude was voltage-dependent and at -50 and +50 mV the IC50 values were 2.3 x 10(-6) M and 1.1 x 10(-6) M respectively (cf. 3.6 x 10(-6) M at -77 mV). 5. In K-free solutions at potentials of -50 mV and +50 mV, niflumic acid did not induce a dual exponential STIC decay but just increased the decay time constant at both potentials in a concentration-dependent manner. 6. Niflumic acid, in concentrations up to 5 x 10(-5) M, had no effect on spontaneous calcium-activated potassium currents. 7. Niflumic acid inhibited noradrenaline- and caffeine-evoked IO(Ca) with an ICM50 of 6.6 x 10-6 M, i.e.was less potent against evoked currents compared to spontaneous currents. In contrast niflumic acid(2 x 10-6 M-5 x 105 M) increased noradrenaline- and caffeine-induced IK(ca).8. The results are discussed with respect to the mechanism of block of ICl(Ca) by niflumic acid and its suitability as a pharmacological tool for assessing the role of Ic(ca) in physiological mechanisms.

Charge movement by the Na/K pump in Xenopus oocytes.

Pre-steady-state transient currents (1986. Nakao, M., and D. C. Gadsby. Nature [Lond.]. 323:628-630) mediated by the Na/K pump were measured under conditions for Na/Na exchange (K-free solution) in voltage-clamped Xenopus oocytes. Signal-averaged (eight times) current records obtained in response to voltage clamp steps over the range -160 to +60 mV after the addition of 100 microM dihydroouabain (DHO) or removal of external Na (control) were subtracted from test records obtained before the solution change. A slow component of DHO- or Na-sensitive difference current was consistently observed and its properties were analyzed. The quantity of charge moved was well described as a Boltzmann function of membrane potential with an apparent valence of 1.0. The relaxation rate of the current was fit by the sum of an exponentially voltage-dependent reverse rate coefficient plus a voltage-independent forward rate constant. The quantity of charge moved at the on and off of each voltage pulse was approximately equal except at extreme negative values of membrane potential where the on charge tended to be less than the off. The midpoint voltage of the charge distribution function (Vq) was shifted by -24.8 +/- 1.7 mV by changing the external [Na] in the test condition from 90 to 45 mM and by +14.7 +/- 1.7 mV by changing the test [Na] from 90 to 120 mM. A pseudo three-state model of charge translocation is discussed in which Na+ is bound and occluded at the internal face of the enzyme and is released into an external-facing high field access channel (ion well). The model predicts a shift of the charge distribution function to more hyperpolarized potentials as extracellular [Na] is lowered; however, several features of the data are not predicted by the model.

Effects of the supply of K, Ca, and Mg on the absorption and assimilation of ammonium- and nitrate-nitrogen in tomato plants

Abstract Forty-two-day-old tomato (Lycopersicon esculentum Mill. cv. Fukuju Nigo) plants were treated with complete, K-free, Ca-limited, and Mg-free nutrient solutions for 10 days using 2 mM NH4NO3 as the N source, which was replaced with 4 niM 15NH4Cl or Na15NO3 for the following 2 days to examine the absorption, translocation, and assimilation of inorganic N in relation to the supply of K, Ca, and Mg. The changes in the leaf nitrate reductase activity (NRA) were examined in tomato plants grown in nutrient solutions in which the form of N source and the supply of K and Ca were varied. Leaf contents of K, Ca, and Mg decreased to 38, 45, and 67% of those of the control plants in the low-K plants, the low-Ca plants and the low-Mg plants, respectively. Tomato plants absorbed NO3-N more efficiently than NH4-N. A larger amount of labelled N from NO3-N than from NH4-N was translocated to shoot. Removal of K from the nutrient solution slightly enhanced the absorption of NH4-N, but suppressed the absorption, tran...

Some properties of the H-ATPase activity present in root plasmalemma of Avena sativa L. Two different enzymes or one enzyme with two ATP sites?

The effects of Mg 2+, K + and ATP on a H-ATPase activity from a native plasmalemma fraction of oat roots were explored at 20°C and pH 6.5. In the presence of 3 mM ATP and no K +, H-ATPase activity vs. [Mg 2+] approached a monotonic activation but it became biphasic, with a decline above 3 mM Mg 2+, in the presence of 20 mM K +. Mg 2+ inhibition occurred also in K-free solutions when [ATP] was lowered to 0.05 mM. Also, an apparent monotonic H-ATPase activation by [K +] at 3.0 mM ATP was transformed in biphasic (inhibition by high [K +]) when [ATP] was reduced to 0.05 mM. The best fits of the ATP stimulation curves of hydrolysis satisfied the sum of two Michaelian functions where that with higher affinity had lower V max. Taking into consideration all conditions of activity assay, the high-affinity component (1) had a K m about 11–16 μM and a V max around 0.14–0.28 μmol P i/mg per min whereas that with lower affinity (2) had a K m of 220–540 μM and a V max of 0.5–1.0 μmol P i/mg per min. K m2 was markedly affected by the [K +] and [Mg 2+]; at optimal concentrations of these cations (1 mM Mg 2+ and 10 mM K +) it had a value of 235±24 μM which was increased to 540±35 μM at 20 mM [Mg 2+] and 60 mM [K +]. In addition, V mx1 was reduced to about a half when the concentrations of Mg 2+ and K + were increased to inhibitory levels. These results could be explained by the existence of two different enzymes or one enzyme with two ATP sites. In the second case, we could not tell at this stage if both are catalytic or one is regulatory.

Net basolateral potassium flux and short-circuit current in ouabain-treated frog skin

A new technique has been developed to correlate K loss from cells (JK) across the basolateral membrane into a K-free ouabain Ringer solution and short-circuit current (Isc) for a model Na-transporting epithelium, the frog skin. Distinct differences were observed when the tissue was bathed in sulfate or chloride Ringer. In sulfate Ringer, K-free ouabain treatment caused both JK and Isc to decline in a nearly parallel fashion with time. JK-Isc was approximately 1 microA/cm2. In sulfate Ringer, isoproterenol caused parallel increases, whereas amiloride (apical side) caused parallel decreases in JK and Isc. In chloride Ringer, K-free ouabain treatment caused Isc to decline at a slightly faster rate than JK.JK-Isc was approximately 8 microA/cm2. Bumetanide decreased JK with very little effect on Isc. Barium caused small parallel changes in both Isc and JK. Amiloride decreased Isc with very little effect on JK. These experiments show that after ouabain treatment changes in JK from the cells across the basolateral membrane can largely account for changes in Isc. However, JK also occurs via neutral mechanisms and perhaps from cells not related to the transport pathway, demonstrating that there is not always a tight coupling of K loss at the basolateral membrane with Na entry across the apical membrane.

Evidence for two calcium-dependent potassium conductances in lizard motor nerve terminals

Action potentials and afterpotentials were recorded with a microelectrode inserted into lizard motor axons within a few millimeters of their motor terminals. In the presence of 1 mM 4-aminopyridine (4-AP), the duration of the action potential recorded near motor terminals was Ca-sensitive: repolarization was more rapid when bath [Ca] was elevated, and became slower when bath [Ca] was removed or when 0.1-1 mM Mn was added. Repolarization was also slowed following addition of 3-10 nM charybdotoxin or 100-300 microM tetraethylammonium (TEA) to the bath, and following intra-axonal injection of the Ca buffer BAPTA. These results, in agreement with published extracellular recordings, indicate that the motor nerve terminal membrane contains rapidly activating, Ca-activated K channels. When these (and other) K channels were blocked by 10 mM TEA, the action potential recorded near motor terminals was followed by Ca-dependent depolarizing afterpotentials, followed in turn by a slow hyperpolarizing afterpotential (h.a.p.) that lasted several seconds. This slow h.a.p. was also Ca-sensitive: it became larger with increasing bath [Ca] and was abolished by removal of bath [Ca] and by addition of 1 mM Mn. Intra-axonal injection of BAPTA reduced the amplitude of the slow h.a.p., and prolonged injections promoted repetitive discharge. The slow h.a.p. following single action potentials was observed in 100 microM ouabain and in K-free solutions and thus is pharmacologically distinct from the hyperpolarization that follows tetanic stimulation. The slow h.a.p. was selectively inhibited by 100 nM apamin, but persisted in 100 nM charybdotoxin. This afterpotential was enhanced by 0.1-1 mM 4-AP and by the dihydropyridine Bay K 8644 (0.1-1 microM). These results suggest that the slow h.a.p. in lizard motor nerve terminals is mediated by Ca-activated K channels that can be activated near the resting potential and are pharmacologically distinct from the Ca-activated K channels that contribute to action potential repolarization. The slow h.a.p. was enhanced by 0.1-1 mM caffeine and inhibited by 100 microM procaine, raising the possibility that this afterpotential may be activated not only by Ca entering via the plasma membrane, but also by Ca released from intra-terminal stores.

Na/K pump current in aggregates of cultured chick cardiac myocytes.

Spontaneously beating aggregates of cultured embryonic chick cardiac myocytes, maintained at 37 degrees C, were voltage clamped using a single microelectrode switching clamp to measure the current generated by the Na/K pump (Ip). In resting, steady-state preparations an ouabain-sensitive current of 0.46 +/- 0.03 microA/cm2 (n = 22) was identified. This current was not affected by 1 mM Ba, which was used to reduce inward rectifier current (IK1) and linearize the current-voltage relationship. When K-free solution was used to block Ip, subsequent addition of Ko reactivated the Na/K pump, generating an outward reactivation current that was also ouabain sensitive. The reactivation current magnitude was a saturating function of Ko with a Hill coefficient of 1.7 and K0.5 of 1.9 mM in the presence of 144 mM Nao. The reactivation current was increased in magnitude when Nai was increased by lengthening the period of time that the preparation was exposed to K-free solution prior to reactivation. When Nai was raised by 3 microM monensin, steady-state Ip was increased more than threefold above the resting value to 1.74 +/- 0.09 microA/cm2 (n = 11). From these measurements and other published data we calculate that in a resting myocyte: (a) the steady-state Ip should hyperpolarize the membrane by 6.5 mV, (b) the turnover rate of the Na/K pump is 29 s-1, and (c) the Na influx is 14.3 pmol/cm2.s. We conclude that in cultured embryonic chick cardiac myocytes, the Na/K pump generates a measurable current which, under certain conditions, can be isolated from other membrane currents and has properties similar to those reported for adult cardiac cells.

Endothelium-dependent relaxation and hyperpolarization of canine coronary artery smooth muscles in relation to the electrogenic Na-K pump.

1 In the smooth muscle cells of canine coronary artery, acetylcholine (ACh) produced a transient, endothelium-dependent hyperpolarization of the membrane. A similar hyperpolarization was also elicited by exposure to Krebs solution after incubation of the artery in K-free solution for 30 min. 2 A hyperpolarization of reproducible amplitude was generated when ACh was applied at intervals greater than 30 min. Repetitive application of ACh at 15 min intervals caused a successive reduction in the amplitude of hyperpolarization. 3 The reduction in the amplitude of relaxation during five successive applications of ACh at 15 min intervals was less than 10% of the first relaxation. 4 The ACh-induced hyperpolarization was blocked by atropine but not by ouabain, whereas the K-free induced hyperpolarization was blocked by ouabain. In low Na (Li-substituted) solution, ACh still induced a hyperpolarization but the K-free induced hyperpolarization was absent. 5 In coronary artery precontracted by high-K solution, ACh produced an endothelium-dependent relaxation, without membrane hyperpolarization. The associated relaxation was resistant to ouabain but sensitive to atropine. 6 It is concluded that in the canine coronary artery, the electrogenic Na-K pump does not contribute to the endothelium-dependent hyperpolarization or relaxation. The results are consistent with the release of two different inhibitory factors from the vascular endothelium.

Acidosis depresses delayed afterdepolarization in guinea pig myocardium.

We examined the relationship among intracellular sodium activity (aiNa), intracellular pH (pHi), and delayed afterdepolarization (DAD) in guinea pig ventricular papillary muscle fibers exposed to K-free solution in vitro, using Na+- and H+-selective microelectrodes. In unstimulated papillary muscles, exposure to K-free solution caused a rapid increase in aiNa of 16.2-22 mM at the end of a 20-min superfusion period with rates of 0.51-0.73 mM/min. This was paralleled by a decrease in intracellular pH of 0.10-0.15 units. DADs were induced after a train of driven action potentials 5-15 min after superfusion with K-free solution at the elevated aiNa from the steady-state level. Prevention of intracellular acidosis associated with the increased aiNa in K-free solution by external alkalosis at pH 8.0 enhanced the amplitude of the DAD, whereas restoration of external pH to 7.4 and a further acidification of external pH to 6.4 suppressed DADs. We concluded that intracellular acidosis associated with the increased aiNa per se increased the likelihood for inhibition of sarcoplasmic reticulum Ca2+ release in spite of increased cytosolic Ca2+.

Contractile responses of the guinea-pig vas deferens to the combination of vanadium ions with ouabain

1. 1. Vanadate and vanadyl ions (10 −5–10 −2M) induced dose-dependent rhythmic contractions of the vas deferens of reserpine-treated guinea-pigs. The Na, K-ATPase blocker ouabain (10 −5–10 −3M) induced similar, though smaller, effects. Experiments were performed to verify if these effects are due to an interaction with the same receptor population. 2. 2. Ouabain caused a striking potentiation of vanadium effects, which was also observed in denervated organs, indicating that a release of neuronal substances is not involved in potentiation. Similar potentiations were observed by combining vanadium with K-free solutions instead of ouabain, corroborating the involvement of the latter drug with Na, K, ATPase. 3. 3. From the analysis of time-response and concentration-response curves, there are at least three indications that vanadium and ouabain interact with different sites: (a) the combined effect of both agonists was several times higher than the corresponding isolated effects; (b) the combined effect, expected to be independent of the order of addition of the agonist, was higher if vanadium was added before, than after ouabain; (c) the combined effect depended on the time elapsed between the addition of the two agonists, being higher if an interval of at least 10 min was allowed between vanadium and ouabain additions. 4. 4. In conclusion, our results do not support the hypothesis that vanadium compounds and ouabain have a similar mechanism of action for the contraction induced in guinea-pig vas deferens.

Voltage dependence of Na/K pump current in Xenopus oocytes.

Stage V and VI (Dumont, J.N., 1972, J. Morphol. 136:153-180) oocytes of Xenopus laevis were treated with collagenase to remove follicular cells and were placed in K-free solution for 2 to 4 days to elevate internal [Na]. Na/K pump activity was studied by restoring the eggs to normal 3 mM K Barth's solution and measuring membrane current-voltage (I-V) relationships before and after the addition of 10 microM dihydroouabain (DHO) using a two-microelectrode voltage clamp. Two pulse protocols were used to measure membrane I-V relationships, both allowing membrane currents to be determined twice at each of a series of membrane potentials: (i) a down-up-down sequence of 5 mV, 1-sec stair steps and (ii) a similar sequence of 1-sec voltage pulses but with consecutive pulses separated by 4-sec recovery periods at the holding potential (-40 mV). The resulting membrane I-V relationships determined both before and during exposure to DHO showed significant hysteresis between the first and second current measurements at each voltage. DHO difference curves also usually showed hysteresis indicating that DHO caused a change in a component of current that varied with time. Since, by definition, the steady-state Na/K pump I-V relationship must be free of hysteresis, the presence of hysteresis in DHO difference I-V curves can be used as a criterion for excluding such data from consideration as a valie measure of the Na/K pump I-V relationship. DHO difference I-V relationships that did not show hysteresis were sigmoid functions of membrane potential when measured in normal (90 mM) external Na solution. The Na/K pump current magnitude saturated near 0 mV at a value of 1.0-1.5 microA cm-2, without evidence of negative slope conductance for potentials up to +55 mV. The Na/K pump current magnitude in Na-free external solution was approximately voltage independent. Since these forward-going Na/K pump I-V relationships do not show a region of negative slope over the voltage range -110 to +55 mV, it is not necessary to postulate the existence of more than one voltage-dependent step in the reaction cycle of the forward-going Na/K pump.