Inhibition of the calcitonin-induced outward current in identifiedAplysia neurons by interleukin-1 and interleukin-2

Summary 1. The effects of bath-applied recombinant human interleukin-1 (rhIL-1) and interleukin-2 (rhIL-2) on the calcitonin (CT)-induced outward current recorded from identified neurons (R9–R12) ofAplysia kurodai were investigated with conventional voltage-clamp and pressure ejection techniques.2. Micropressure ejection of CT onto the soma of the neuron induced a slow outward current [I _o(CT); 4–6 nA in amplitude, 30–40 sec in duration] associated with a decrease in input membrane conductance.3.I _o(CT) was increased by hyperpolarization.4. The extrapolated reversal potential was +10 mV. Additionally,I _o(CT) was sensitive to changes in (Na⁺)_o but not to changes in (K⁺)_o, (Ca²⁺)_o, and (Cl^–)_o.5. Micropressure-ejected forskolin produced a slow outward current similar to that induced by CT.6. Bath-applied rhIL-1 and rhIL-2 (10–40 U/ml) reduced the CT-induced current in identifiedAplysia neurons without affecting the resting membrane conductance or the holding current.7. The inhibitory effects of both cytokines on the current were completely reversible. Heat-inactivated rhIL-1 and rhIL-2 were without effect.8. These results suggest that the immunomodulators, IL-1 and IL-2, can modulate the CT-induced outward current associated with a decrease in Na⁺ conductance in the nervous system ofAplysia. Therefore, the study suggests that these cytokines may also serve as neuromodulators.     

Role of the furosemide-sensitive Na+/K+ transport system in determining the steady-state Na+ and K+ content and volume of human erythrocytesin vitro andin vivo

Summary To study the physiological role of the bidirectionally operating, furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes, the effect of furosemide on red cell cation and hemoglobin content was determined in cells incubated for 24 hr with ouabain in 145mm NaCl media containing 0 to 10mm K⁺ or Rb⁺. In pure Na⁺ media, furosemide accelerated cell Na⁺ gain and retarded cellular K⁺ loss. External K⁺ (5mm) had an effect similar to furosemide and markedly reduced the action of the drug on cellular cation content. External Rb⁺ accelerated the Na⁺ gain like K⁺, but did not affect the K⁺ retention induced by furosemide. The data are interpreted to indicate that the furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes mediates an equimolar extrusion of Na⁺ and K⁺ in Na⁺ media (Na⁺/K⁺ cotransport), a 1:1 K⁺/K⁺ (K⁺/Rb⁺) and Na⁺/Na⁺ exchange progressively appearing upon increasing external K⁺ (Rb⁺) concentrations to 5mm. The effect of furosemide (or external K⁺/Rb⁺) on cation contents was associated with a prevention of the cell shrinkage seen in pure Na⁺ media, or with a cell swelling, indicating that the furosemide-sensitive Na⁺/K⁺ transport system is involved in the control of cell volume of human erythrocytes. The action of furosemide on cellular volume and cation content tended to disappear at 5mm external K⁺ or Rb⁺. Thein vivo red cell K⁺ content was negatively correlated to the rate of furosemide-sensitive K⁺ (Rb⁺) uptake, and a positive correlation was seen between mean cellular hemoglobin content and furosemide-sensitive transport activity. The transport system possibly functions as a K⁺ and waterextruding mechanism under physiological conditiosin vivo. The red cell Na⁺ content showed no correlation to the activity of the furosemide-sensitive transport system.     

Metabolic control of the K+ channel of human red cells

Summary The effects of cAMP, ATP and GTP on the Ca²⁺-dependent K⁺ channel of fresh (1–2 days) or cold-stored (28–36 days) human red cells were studied using atomic absorption flame photometry of Ca²⁺-EGTA loaded ghosts which had been resealed to monovalent cations in dextran solutions. When high-K⁺ ghosts were incubated in an isotonic Na⁺ medium, the rate constant of Ca²⁺-dependent K⁺ efflux was reduced by a half on increasing the theophylline concentration to 40mm. This effect was observed in ghosts from both fresh and stored cells, but only if they were previously loaded with ATP. The inhibition was more marked when Mg²⁺ was added together with ATP, and it was abolished by raising free Ca²⁺ to the micromolar level. Like theophylline, isobutyl methylxanthine (10mm) also affected K⁺ efflux. cAMP (0.2–0.5mm), added both internally and externally (as free salt, dibutyryl or bromide derivatives), had no significant effect on K⁺ loss when the ghost free-Ca²⁺ level was below 1 m, but it was slightly inhibitory at higher concentrations. The combined presence of cAMP (0.2mm) plus either theophylline (10mm), or isobutyl methylxanthine (0.5mm), was more effective than cAMP alone. This inhibition showed a strict requirement for ATP plus Mg²⁺ and it, was not overcome by raising internal Ca²⁺. Ghosts from stored cells seemed more sensitive than those from fresh cells, to the combined action of cAMP and methylxanthines. Loading ATP into ghosts from fresh or stored cells markedly decreased K⁺ loss. Although this effect was observed in the absence of added Mg²⁺ (0.5mm EDTA present), it was potentiated upon adding 2mm Mg²⁺. The K⁺ efflux from ATP-loaded ghosts was not altered by dithio-bis-nitrobenzoic acid (10mm) or acridine orange (100 m), while it was increased two-to fourfold by incubating with MgF₂ (10mm), or MgF₂ (10mm)+theophylline (40mm), respectively. By contrast, a marked efflux reduction was obtained by incorporating 0.5mm GTP into ATP-containing ghosts. The degree of phosphorylation obtained by incubating membranes with (-³²P)ATP under various conditions affecting K⁺ channel activity, was in direct correspondence to their effect on K⁺ efflux. The results suggest that the K⁺ channel of red cells is under complex metabolic control, via cAMP-mediated and nonmediated mechanisms, some which require ATP and presumably, involve phosphorylation of the channel proteins.     

Ca2+-activated K+ permeability in human erythrocytes: Modulation of single-channel events

Elevated levels of intracellular Ca²⁺ activate a K⁺-selective permeability in the membrane of human erythrocytes. Currents through single channels were analysed in excised inside-out membrane patches. The effects of several ions that are known to inhibit K⁺ fluxes are described with respect to the single-channel events. The results suggest that the blocking ions can partly move into the channels (but cannot penetrate) and interact with other ions inside the pore. The reduction of single-channel conductance by Cs⁺, tetraethylammonium and Ba²⁺ and of single-channel activity by quinine and Ba²⁺ is referred to different rates of access to the channel. The concentration- and voltage-dependent inhibition by ions with measurable permeability (Na⁺ and Rb⁺) can be explained by their lower permeability, with single-file movement and ionic interactions inside the pore.     

Characterization of K+-dependent and K+-independentp-nitrophenylphosphatase activity of synaptosomes

These experiments examined effects of several ligands on the K⁺ p-nitrophenylphosphatase activity of the (Na⁺,K⁺)-ATPase in membranes of a rat brain cortex synaptosomal preparation. K⁺-independent hydrolysis of this substrate by the synaptosomal preparation was studied in parallel; the rate of hydrolysis in the absence of K⁺ was approximately 75% less than that observed when K⁺ was included in the incubation medium. The response to the H⁺ concentrations was different: K⁺-independent activity showed a pH optimum around 6.5–7.0, while the K⁺-dependent activity was relatively low at this pH range. Ouabain (0.1 mM) inhibited K⁺-dependent activity 50%; a concentration 10 times higher did not produce any appreciable effect on the K⁺-independent activity. Na⁺ did not affect K⁺-independent activity at all, while the same ligand concentration inhibited sharply the K⁺-dependent activity; this inhibition was not competitive with the substrate,p-nitrophenyl phosphate. K⁺-dependent activity was stimulated by Mg²⁺ with low affinity (millimolar range), and 3 mM Mg²⁺ produced a slight stimulation of the activity in absence of K⁺, which could be interpreted as Mg²⁺ occupying the K⁺ sites. Ca²⁺ had no appreciable effect on the activity in the absence of K⁺. However, in the presence of K⁺ a sharp inhibition was found with all Ca²⁺ concentrations studied. ATP (0.5 mM) did not affect the K⁺-independent activity, but this nucleotide behaved as a competitive inhibitor top-nitrophenylphosphate. Pi inhibited activity in the presence of K⁺, competively to the substrate, so it could be considered as the second product of the reaction sequence.Abbreviations used p-NPP p-nitrophenylphosphate - p-NPPase rho-nitrophenylphosphatase activity     

Beta-Adrenergic modulation of K+ current in human T lymphocytes

Summary The whole-cell voltage-clamp technique was employed to study the -adrenergic modulation of voltage-gated K⁺ currents in CD8⁺ human peripheral blood lymphocytes. The -receptor agonist, isoproterenol, decreased the peak current amplitude and increased the rate of inactivation of the delayed rectifier K⁺ current. In addition, isoproterenol decreased the voltage dependence of steady-state inactivation and shifted the steady-state inactivation curve to the left. Isoproterenol, on the other hand, had no significant effect on the steady-state parameters of current activation. The isoproterenol-induced decrease in peak current amplitude was inhibited by the -blocker propranolol. Bath application of dibutyryl cAMP (1mm) mimicked the effects of isoproterenol on both K⁺ current amplitude and time course of inactivation. Furthermore, the reduction in the peak current amplitude in response to isoproterenol was attenuated when PKI_5–24 (2–5 m), a synthetic peptide inhibitor of cAMP-dependent protein kinase, was present in the pipette solution. The increase in the rate of inactivation of the K⁺ currents in response to isoproterenol was mimicked by the internal application of GTP--S (300 m) and by exposure of the cell to cholera toxin (1 g/ml), suggesting the involvement of a G protein. These results demonstrate that the voltage-dependent K⁺ conductance in T lymphocytes can be modulated by -adrenergic stimulation. The effects of -agonists, i.e., isoproterenol, appear to be receptor mediated and could involve cAMP-dependent protein kinase as well as G proteins. Since inhibition of the delayed rectifier K⁺ current has been found to decrease the proliferative response in T lymphocytes, the -adrenergic modulation of K⁺ current may well serve as a feedback control mechanism limiting the extent of cellular proliferation.     

Evidence for two K+ currents activated upon hyperpolarization ofParamecium tetraurelia

Summary Hyperpolarization of voltage-clampedParamecium tetraurelia in K⁺ solutions elicits a complex of Ca²⁺ and K⁺ currents. The tail current that accompanies a return to holding potential (–40 mV) contains two K⁺ components. The tail current elicited by a step to –110 mV of 50-msec duration contains fast-decaying (3.5 msec) and slow-decaying (20 msec) components. The reversal potential of both components shifts by 55–57 mV/10-fold change in external [K⁺], suggesting that they represent pure K⁺ currents. The dependence of the relative amplitudes of the two tail currents on duration of hyperpolarization suggests that the slow K⁺ current activates slowly and is sustained, whereas the fast current activates rapidly during hyperpolarization and then rapidly inactivates. Iontophoretic injection of a Ca²⁺ chelator, EGTA, specifically reduces slow tail-current amplitude without affecting the fast tail component. Both K⁺ currents are inhibited by extracellular TEA⁺ in a concentration-dependent, noncooperative manner, whereas the fast K⁺ current alone is inhibited by 0.7mm quinidine.     

K+ current stimulation by Cl- in the midgut epithelium of tobacco hornworm (Manduca sexta)

Summary Chloride-stimulated K⁺ secretion by Manduca sexta midgut (5th-instar larvae) was measured as K⁺-carried short-circuit current of the tissue mounted in an Ussing chamber. Microscopic parameters, such as single-channel current and channel density for the rate-determining passive transport step across the basolateral goblet cell membrane (i.e. K⁺ channels), were estimated by means of current-fluctuation analysis of the K⁺ channel blockade by haemolymph-side Ba²⁺ ions. Ba²⁺ was equally effective with Cl^- or gluconate (Glu^-) as the principal ambient anion. The Ba²⁺-induced K⁺ channel conduction noise is reflected by a Lorentzian, or relaxation, noise component in the power spectrum of the K⁺ current fluctuations. A reduced Lorentzian plateau value, but an unchanged corner frequency, were observed when Cl^- was replaced by Glu^-. The results from the analysis of a two-state model of K⁺ channel block by Ba²⁺, with respect to the anion-replacement effects, suggest that the observed changes in K⁺ current and Lorentzian plateau value mirror a complex change of the underlying parameters: Cl^- omission reduces single channel current but increases channel density so that the product of single channel current and channel density is smaller in Glu^- than in Cl^-. It seems likely that basolateral K⁺ channels (1) are subject to anionic gating ligands, and (2) depend on anions with respect to the rate of K⁺ transfer through and open K⁺ channel.Abbreviations a.c. alternating current - single-channel conductance - E _K K⁺ Nernst potential - f frequency contained in current noise - f _c corner frequency - Glu^- gluconate - G _t transepithelial conductance - I _sc short-circuit current - I _K K⁺ current - I _K(max) maximal K⁺ current - i single-channel current - K _Ba barium inhibition constant - K _m Michaelis constant of saturating K⁺ current - k ₀₁ and k ₁₀ barium association and dissociation rate constant, respectively - M K⁺ channel density - S _f power density - S _o Lorentzian plateau value - P _o channel-open probability - P _K K⁺ permeability - V _sc cellular potential at short-circuit These results have already been presented in part, at the 1989 joint meeting of the German and Israel Physiological Societies in Jerusalem (Zeiske et al. 1990).     

An endogenous factor which interacts with synaptosomal membrane Na+, K+-ATPase activation by K+

In previous papers, the isolation of brain soluble fractions able to modify neuronal Na⁺, K⁺-ATPase activity has been described. One of those fractions-peak I-stimulates membrane Na⁺, K⁺-ATPase while another-peak II-inhibits this enzyme activity, and has other ouabain-like properties. In the present study, synaptosomal membrane Na⁺, K⁺-ATPase was analyzed under several experimental conditions, using ATP orp-nitrophenylphosphate (p-NPP) as substrate, in the absence and presence of cerebral cortex peak II. Peak II inhibited K⁺-p-NPPase activity in a concentration dependent manner. Double reciprocal plots indicated that peak II uncompetitively inhibits K⁺-p-NPPase activity regarding substrate, Mg²⁺ and K⁺ concentration. Peak II failed to block the known K⁺-p-NPPase stimulation caused by ATP plus Na⁺. At various K⁺ concentrations, percentage K⁺-p-NPPase inhibition by peak II was similar regardless of the ATP plus Na⁺ presence, indicating lack of correlation with enzyme phosphorylation. Na⁺, K⁺-ATPase activity was decreased by peak II depending on K⁺ concentration. It is postulated that the inhibitory factor(s) present in peak II interfere(s) with enzyme activation by K⁺.     

Redox modulation of A-type K+ currents in pain-sensing dorsal root ganglion neurons

Redox modulation of fast inactivation has been described in certain cloned A-type voltage-gated K⁺ (Kv) channels in expressing systems, but the effects remain to be demonstrated in native neurons. In this study, we examined the effects of cysteine-specific redox agents on the A-type K⁺ currents in acutely dissociated small diameter dorsal root ganglion (DRG) neurons from rats. The fast inactivation of most A-type currents was markedly removed or slowed by the oxidizing agents 2,2′-dithio-bis(5-nitropyridine) (DTBNP) and chloramine-T. Dithiothreitol, a reducing agent for the disulfide bond, restored the inactivation. These results demonstrated that native A-type K⁺ channels, probably Kv1.4, could switch the roles between inactivating and non-inactivating K⁺ channels via redox regulation in pain-sensing DRG neurons. The A-type channels may play a role in adjusting pain sensitivity in response to peripheral redox conditions.     

Ba2+ uptake and the inhibition by Ba2+ of K+ flux into rat liver mitochondria

Summary Rapid uptake of Ba²⁺ by respiring rat liver mitochondria is accompanied by a transient stimulation of respiration. Following accumulation of Ba²⁺, e.g. at a concentration of 120 nmol per mg protein, the mitochondria exhibit reduced rates of state 3 and uncoupler-stimulated respiration. ADP-stimulated respiration is inhibited at a lower concentration of Ba²⁺ than is required to affect uncoupler-stimulated respiration, suggesting a distinct effect of Ba²⁺ on mechanisms involved in synthesis of ATP. Ba²⁺, which has an ionic radius similar to that of K⁺, inhibits unidirectional K⁺ flux into respiring rat liver mitochondria. This effect on K⁺ influx is observable at concentrations of Ba²⁺, e.g. 23 to 37 nmol per mg protein, which cause no significant change in state 4 or uncoupler-stimulated respiration. The accumulated Ba²⁺ decreases the measuredV _max of K⁺ influx, while having little effect on the apparentK _m for K⁺. The inhibition of K⁺ influx by Ba²⁺ is seen in the presence and absence of mersalyl, an activator of K⁺ influx. In contrast, under the conditions studied, Ba²⁺ has no apparent effet on the rate of unidirectional K⁺ efflux. These data are consistent with the idea that K⁺ may enter and leave mitochondria via spearate mechanisms.     

Kinetics of K+-p-Nitrophenyl Phosphatase Stimulation by a Brain Soluble Fraction

We have already described the separation of two brain soluble fractions by Sephadex G-50, one of which stimulates (peak I) and the other inhibits (peak II) Na⁺, K⁺-ATPase and K⁺-p-nitrophenylphosphatase (K⁺-p-NPPase) activities. Here we examine the features of synaptosomal membrane p-NPPase activity in the presence and absence of brain peak I. It was observed that stimulation of Mg²⁺, K⁺-p-NPPase activity by peak I was concentration dependent, The ability of peak I to stimulate p-NPPase activity was lost by heat treatment followed by brief centrifugation. Pure serum albumin also stimulated enzyme activity. K⁺-p-NPPase stimulation by peak I proved dependent on K⁺ concentration but independent of Mg²⁺ and substrate p-nitrophenylphosphate concentrations. Since our determinations were performed in a non-phosphorylating condition reflecting the Na⁺, K⁺-ATPase Na⁺ site, it is suggested that peak I may stimulate the Na⁺-dependent enzyme phosphorylation known to take place from the internal cytoplasmic side.     

Activation by Ca2+ and block by divalent ions of the K+ channel in the membrane of cytoplasmic drops fromChara australis

Summary Patch-clamp studies of cytoplasmic drops from the charophyteChara australis have previously revealed K⁺ channels combining high conductance (170 pS) with high selectivity for K⁺, which are voltage activated. The cation-selectivity sequence of the channel is shown here to be: K⁺>Rb⁺>NH ₄ ⁺ Na⁺ and Cl^–. Divalent cytosolic ions reduce the K⁺ conductance of this channel and alter its K⁺ gating in a voltage-dependent manner. The order of blocking potency is Ba²⁺>Sr²⁺>Ca²⁺>Mg²⁺. The channel is activated by micromolar cytosolic Ca²⁺, an activation that is found to be only weakly voltage dependent. However, the concentration dependence of calcium activation is quite pronounced, having a Hill coefficient of three, equivalent to three bound Ca²⁺ needed to open the channel. The possible role of the Ca²⁺-activated K⁺ channel in the tonoplast ofChara is discussed.     

Voltage dependence of the Ca2+-activated K+ conductance of human red cell membranes is strongly dependent on the extracellular K+ concentration

Summary The conductance of the Ca²⁺-activated K⁺ channel (g _K(Ca)) of the human red cell membrane was studied as a function of membrane potential (V _m ) and extracellular K⁺ concentration ([K⁺]_ex). ATP-depleted cells, with fixed values of cellular K⁺ (145mm) and pH (7.1), and preloaded with 27 m ionized Ca were transferred, with open K⁺ channels, to buffer-free salt solutions with given K⁺ concentrations. Outward-current conductances were calculated from initial net effluxes of K⁺, correspondingV _m , monitored by CCCP-mediated electrochemical equilibration of protons between a buffer-free extracellular and the heavily buffered cellular phases, and Nernst equilibrium potentials of K ions (E _K) determined at the peak of hyperpolarization. Zero-current conductances were calculated from unidirectional effluxes of⁴²K at (V _m –E _K)0, using a single-file flux ratio exponent of 2.7. Within a [K⁺]_ex range of 5.5 to 60mm and at (V _m –E _K) 20 mV a basic conductance, which was independent of [K⁺]_ex, was found. It had a small voltage dependence, varying linearly from 45 to 70 S/cm² between 0 and –100 mV. As (V _m –E _K) decreased from 20 towards zero mVg _K(Ca) increased hyperbolically from the basic value towards a zero-current value of 165 S/cm². The zero-current conductance was not significantly dependent on [K⁺]_ex (30 to 156mm) corresponding toV _m (–50 mV to 0). A further increase ing _K(Ca) symmetrically aroundE _K is suggested as (V _m –E _K) becomes positive. Increasing the extracellular K⁺ concentration from zero and up to 3mm resulted in an increase ing _K(Ca) from 50 to 70 S/cm². Since the driving force (V _m –E _K) was larger than 20 mV within this range of [K⁺]_ex this was probably a specific K⁺ activation ofg _K(Ca). In conclusion: The Ca²⁺-activated K⁺ channel of the human red cell membrane is an inward rectifier showing the characteristic voltage dependence of this type of channel.     

Glutathione-gated K+ channels of Escherichia coli carry out K+ efflux controlled by the redox state of the cell

The kinetics of K⁺ efflux across the membranes of i) wild-type Escherichia coli poisoned by the thiol reagent N-ethylmaleimide, ii) K⁺ retention mutants and iii) glutathione-deficient mutants, have revealed a common K⁺ leaky phenotype; it is characterized by a very high rate of K⁺ efflux. The results suggest that the products of kefB and kefC genes could encode two K⁺ channels, both gated by glutathione. The possible function of these K⁺ channels seems to be a K⁺ exit controlled by the redox state of the cell; indeed, it can be inferred from the effects of several oxidants and reductants that turning on and off of the K⁺ efflux mediated by the channels can be correlated with the redox state of glutathione.     

Whole-cell K+ current activation in response to voltages and carbachol in gastric parietal cells isolated from guinea pig

Summary Patch-clamp studies of whole-cell ionic currents were carried out in parietal cells obtained by collagenase digestion of the gastric fundus of the guinea pig stomach. Applications of positive command pulses induced outward currents. The conductance became progressively augmented with increasing command voltages, exhibiting an outwardly rectifying current-voltage relation. The current displayed a slow time course for activation. In contrast, inward currents were activated upon hyperpolarizing voltage applications at more negative potentials than the equilibrium potential to K⁺ (E _K). The inward currents showed time-dependent inactivation and an inwardly rectifying current-voltage relation. Tail currents elicited by voltage steps which had activated either outward or inward currents reversed at nearE _K, indicating that both time-dependent and voltagegated currents were due to K⁺ conductances. Both outward and inward K⁺ currents were suppressed by extracellular application of Ba²⁺, but little affected by quinine. Tetraethylammonium inhibited the outward current without impairing the inward current, whereas Cs⁺ blocked the inward current but not the outward current. The conductance of inward K⁺ currents, but not outward K⁺ currents, became larger with increasing extracellular K⁺ concentration. A Ca²⁺-mobilizing acid secretagogue, carbachol, and a Ca²⁺ ionophore, ionomycin, brought about activation of another type of outward K⁺ currents and voltage-independent cation currents. Both currents were abolished by cytosolic Ca²⁺ chelation. Quinine preferentially inhibited this K⁺ current. It is concluded that resting parietal cells of the guinea pig have two distinct types of voltage-dependent K⁺ channels, inward rectifier and outward rectifier, and that the cells have Ca²⁺-activated K⁺ channels which might be involved in acid secretion under stimulation by Ca²⁺-mobilizing secretagogues.     

Inositol 1,3,4,5-tetrakisphosphate is essential for sustained activation of the Ca2+-dependent K+ current in single internally perfused mouse lacrimal acinar cells

Summary We have examined the effects of various inositol polyphosphates, alone and in combination, on the Ca²⁺-activated K⁺ current in internally perfused, single mouse lacrimal acinar cells. We used the patch-clamp technique for whole-cell current recording with a set-up allowing exchange of the pipette solution during individual experiments so that control and test periods could be directly compared in individual cells. Inositol 1,4,5-trisphosphate (Ins 1,4,5 P₃) (10–100 m) evoked a transient increase in the Ca²⁺-sensitive K⁺ current that was independent of the presence of Ca²⁺ in the external solution. The transient nature of the Ins 1,4,5 P₃ effect was not due to rapid metabolic breakdown, as similar responses were obtained in the presence of 5mm 2,3-diphosphoglyceric acid, that blocks the hydrolysis of Ins 1,4,5 P₃, as well as with the stable analoguedl-inositol 1,4,5-trisphosphorothioate (Ins 1,4,5 P(S)₃) (100 m). Ins 1,3,4 P₃ (50 m) had no effect, whereas 50 m Ins 2,4,5 P₃ evoked responses similar to those obtained by 10 m Ins 1,4,5 P₃. A sustained increase in Ca²⁺-dependent K⁺ current was only observed when inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5 P₄) (10 m) was added to the Ins 1,4,5 P₃ (10 m)-containing solution and this effect could be terminated by removal of external Ca²⁺. The effect of Ins 1,3,4,5 P₄ was specifically dependent on the presence of Ins 1,4,5 P₃ as it was not found when 10 m concentrations of Ins 1,3,4 P₃ or Ins 2,4,5 P₃ were used. Ins 2,4,5 P₃ (but not Ins 1,3,4 P₃) at the higher concentration of 50 m did, however, support the Ins 1,3,4,5 P₄-evoked sustained current activation. Ins 1,3,4 P₃ could not evoke sustained responses in combination with Ins 1,4,5 P₃ excluding the possibility that the action of Ins 1,3,4,5 P₄ could be mediated by its breakdown product Ins 1,3,4 P₃. Ins 1,3,4,5 P₄ also evoked a sustained response when added to an Ins 1,4,5 P(S)₃-containing solution. Ins 1,3,4,5,6 P₅ (50 m) did not evoke any effect when administered on top of Ins 1,4,5 P₃. In the absence of external Ca²⁺, addition of Ins 1,3,4,5 P₄ to an Ins 1,4,5 P₃-containing internal solution evoked a second transient K⁺ current activation. Readmitting external Ca²⁺ in the continued presence internally of Ins 1,4,5 P₃ and Ins 1,3,4,5 P₄ made the response reappear. We conclude that both Ins 1,4,5 P₃ and Ins 1,3,4,5 P₄ play crucial and specific roles in controlling intracellular Ca²⁺ homeostasis.     

Macroscopic Ca2+ -Na+ and K+ currents in single heart and aortic cells

Summary The whole-cell voltage clamp technique was used to study the slow inward currents and K⁺ outward currents in single heart cells of embryonic chick and in rabbit aortic cells. In single heart cells of 3-day-old chick embryo three types of slow inward Na⁺ currents were found. The kinetics and the pharmacology of the slow I_Na, were different from those of the slow Ica in older embryos. Two types of slow inward currents were found in aortic single cells of rabbit; angiotensin 11 increased the sustained type and d-cAMP and d-cGMP decreased the slow transient component. Two types of outward K⁺ currents were found in both aortic and heart cells. Single channel analysis demonstrated the presence of a high single K⁺ channel conductance in aortic cells. In cardiac and vascular smooth muscles, slow inward currents do share some pharmacological properties, although the regulation of these channels by cyclic nucleotides and several drugs seems to be different.     

Interactions of K+ ATP channel blockers with Na+/K+-ATPase

Two K⁺ _ATP channel blockers, 5-hydroxydecanoate (5-HD) and glyburide, are often used to study cross-talk between Na⁺/K⁺-ATPase and these channels. The aim of this work was to characterize the effects of these blockers on purified Na⁺/K⁺-ATPase as an aid to appropriate use of these drugs in studies on this cross-talk. In contrast to known dual effects (activating and inhibitory) of other fatty acids on Na⁺/K⁺-ATPase, 5-HD only inhibited the enzyme at concentrations exceeding those that block mitochondrial K⁺ _ATP channels. 5-HD did not affect the ouabain sensitivity of Na⁺/K⁺-ATPase. Glyburide had both activating and inhibitory effects on Na⁺/K⁺-ATPase at concentrations used to block plasma membrane K⁺ _ATP channels. The findings justify the use of 5-HD as specific mitochondrial channel blocker in studies on the relation of this channel to Na⁺/K⁺-ATPase, but question the use of glyburide as a specific blocker of plasma membrane K⁺ _ATP channels, when the relation of this channel to Na⁺/K⁺-ATPase is being studied.