Intracellular potassium activity in epithelial cells of frog fundic gastric mucosa

Microelectrodes were used to measure membrane potential and intracellular potassium activity in surface epithelial cells (SEC) of frog (Rana esculenta) fundic gastric mucosa in vitro. Separate measurements were carried out by applying fine-tipped, single barrelled, KCl filled non-selective electrodes and liquid K⁺-selective electrodes. Membrane potentials with respect to the mucosal and serosal surfaces, measured with non-selective electrodes, were –54.5±1.0 S.E. mV (n=59) and –73.0±1.1 S.E. mV (n=59) respectively. The electrical potential difference referred to the mucosal surface, when measured with K⁺-sensitive electrodes, was +21.2±0.8 S.E. mV (n=35), and intracellular K⁺ activity was 98.5 mmol/l. Assuming that intracellular and extracellular K⁺ activity coefficients are equal (_K=_K), the K⁺ concentration is 135.0 mmol/l. The K⁺ equilibrium potential,E _K, was calculated as –90.0 mV i.e. more negative than both membrane potentials. This result indicates active potassium accumulation in the SEC and provides direct evidence of the presence of an active K⁺ pump in either both or in only one of the cell membranes.     

Effect of sodium concentration and of atropine on the contractile response of the guinea-pig ileum to potassium ions

The possibility that the guinea-pig ileum's contractile response to K⁺-rich solutions is partly mediated by acetylcholine release from the intramural nervous tissue was examined by studying the inhibition of that response by atropine at different values of [Na⁺]₀. In a medium in which the NaCl was replaced by iso-osmotic glucose, the response to high [K⁺]₀ was not greatly affected, while the responses to acetylcholine and to other agonists were significantly reduced. In control medium (149 mM Na⁺), atropine (10^–7 M) partly inhibited the responses to K⁺-rich solutions and to agonists such as histamine, 5-hydroxytryptamine and bradykinin. When [Na⁺]₀ was reduced to 12 mM, by iso-osmotic substitution of glucose for NaCl, the response to high K⁺ was no longer inhibited by atropine, which still partly inhibited the contractions elicited by the three agonists and totally blocked the response to acetylcholine. It is proposed that atropine's inhibition of the response to high K⁺ and to agonists is not due to its specific anti-muscarinic effect, but to an unspecific action, which in the case of the agonists is independent of [Na⁺]₀. In addition, the inhibition of the response to high K⁺ would result from a different Na⁺-dependent mechanism, possibly involving the stimulation of the Na-K pump by atropine. This is supported by the observation that this drug partly relaxed ileum preparations that were contracted by ouabain.     

The sodium-activated potassium channel Slack is modulated by hypercapnia and acidosis

Slack (Slo 2.2), a member of the Slo potassium channel family, is activated by both voltage and cytosolic factors, such as Na(+) ([Na(+)](i)) and Cl(-) ([Cl(-)](i)). Since the Slo family is known to play a role in hypoxia, and since hypoxia/ischemia is associated with an increase in H(+) and CO(2) intracellularly, we hypothesized that the Slack channel may be affected by changes in intracellular concentrations of CO(2) and H(+). To examine this, we expressed the Slack channel in Xenopus oocytes and the Slo 2.2 protein was allowed to be inserted into the plasma membrane. Inside-out patch recordings were performed to examine the response of Slack to different CO(2) concentrations (0.038%, 5%, 12%) and to different pH levels (6.3, 6.8, 7.3, 7.8, 8.3). In the presence of low [Na(+)](i) (5 mM), the Slack channel open probability decreased when exposed to decreased pH or increased CO(2) in a dose-dependent fashion (from 0.28+/-0.03, n=3, at pH 7.3 to 0.006+/-0.005, n=3, P=0.0004, at pH 6.8; and from 0.65+/-0.17, n=3, at 0.038% CO(2) to 0.22+/-0.07, n=3, P=0.04 at 12% CO(2)). In the presence of high [Na(+)](i) (45 mM), Slack open probability increased (from 0.03+/-0.01 at 5 mM [Na(+)](i), n=3, to 0.11+/-0.01, n=3, P=0.01) even in the presence of decreased pH (6.3). Since Slack activity increases significantly when exposed to increased [Na(+)](i), even in presence of increased H(+), we propose that Slack may play an important role in pathological conditions during which there is an increase in the intracellular concentrations of both acid and Na(+), such as in ischemia/hypoxia.     

Potassium and sodium ions in the nerve tissue of emotionally overstrained rabbits

Measurement of K⁺ and Na⁺ concentrations in samples of individual brain nuclei and in ganglia of the autonomic nervous system from rabbits subjected to severe emotional stress (ES) through aperiodic stimulation of ventromedial hypothalamic nuclei and electrocutaneous stimulation revealed significantly altered levels of these ions in locus ceruleus samples from animals predisposed to ES-induced cardiovascular disorders and in samples of neurons of the caudal part of the brainstem from those resistant to such disorders. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, No 8, pp. 129–131, August, 1994 Presented by K. V. Sudakov, Member of the Russian Academy of Medical Sciences     

A potassium current activated by lemakalim and metabolic inhibition in rabbit mesenteric artery

K⁺ channels which are inhibited by intracellular ATP (ATP_i) (K_ATP channels) are thought to be the physiological target site of the K⁺ channel opening drugs (2) and to underlie a variety of physiological phenomena including hypoxia induced vasodilation (3). However, electrophysiological evidence for ATP_i-regulated K⁺ currents in smooth muscle is scarce. We, therefore, investigated the effects of one K⁺ channel opener, lemakalim, and metabolic inhibition on the membrane conductance of freshly dissociated rabbit mesenteric artery smooth muscle cells, using the perforated-patch whole cell recording technique (6). The cells were metabolically inhibited with 1 mM iodoacetic acid and 50 M dinitrophenol. Both lemakalim (0.1–3 M) and metabolic inhibition activated a time-independent and glyburide sensitive K⁺ current at physiological membrane potentials. The similarities between the lemakalim and metabolic inhibition activated currents suggest that a single class of channels underlies both currents. These results are the first whole-cell current recordings to demonstrate the activation of a smooth muscle membrane conductance by metabolic inhibition, lending support to the view that hypoxia induced vasodilation arises from the activation of K_ATP channels.     

Dependence of the electrogenic pump current of Xenopus oocytes on external potassium

The membrane potential of Xenopus oocytes showed a variable response to an increase of the K⁺ concentration in the bathing solution, [K⁺]_e, from 2.5 mM to 20 mM. In 54% of the cases (n=52) the cells hyperpolarized (by max. 70 mV). In the presence of 10^–5 M ouabain, all cells depolarized suggesting that the hyperpolarization was caused by an electrogenic Na⁺/K⁺ pump. In cells stored overnight in a Na⁺-free solution the transition from 2.5 to 20 mM [K⁺]_e always caused depolarization indicating that the stimulation of the pump requires high internal sodium, [Na⁺]_i. Cells stored overnight in a Na⁺-rich solution had a [Na⁺]_i of 30.7±7 mM, i.e. the Na⁺/K⁺ pump was saturated with sodium (Lafaire and Schwarz 1986). With 9 such cells we determined the K⁺ activation of the Na⁺/K⁺ pump. The activation follows Hill kinetics with I_max=90.5 nA, K_s=2.3 mM, and n=1.68.     

Contribution of potassium accumulation in narrow extracellular spaces to the genesis of nicorandil-induced large inward tail current in guinea-pig ventricular cells

The mechanism of nicorandil-induced large inward tail current (I _tail) in single guinea-pig ventricular cells was investigated using the whole-cell patch-clamp technique. In the presence of 0.5–1.0 mM nicorandil, an activator of adenosine 5-triphosphate (ATP)-sensitive K⁺ current (I _KATP), a depolarization pulse causing a large outward current was followed by a large inward I _tail on the repolarization step to the holding potential at-85 mV. The larger the outward current, the greater the I _tail. The amplitude of I _tail increased as a single exponential function (=74.9 ms) as the duration of preceding depolarization was prolonged. Both the outward current and I _tail were inhibited nearly completely after application of glibenclamide (1 M), a specific blocker of I _KATP. Substitution of K⁺ with Cs⁺ in both the external and internal solutions resulted in a virtual elimination of I _tail. I _tail was well preserved under the condition where Ca²⁺ entry during the preceding depolarization was largely inhibited or where external Na⁺ was replaced by Li⁺. A transient positive shift of reversal potential for the net current was observed at the peak of I _tail. At 30 mM external K⁺ concentration, I _tail was almost eliminated. From these findings, it is concluded that the I _tail is a K⁺ current associated with an alteration of the K⁺ equilibrium potential (E _K) following a substantial K⁺ efflux. This E _K change is most likely explained by an accumulation of K⁺in transverse tubules (T-tubules) since I _tail was not induced in atrial cells in which T-tubules are poorly developed.     

Sodium-activated potassium current in sensory neurons: a comparison of cell-attached and cell-free single-channel activities

Single-channel currents from Na⁺-dependent K⁺ channels (K_Na) were recorded from cell-attached and inside-out membrane patches of cultured avian trigeminal ganglion neurons by means of the patchclamp technique. Single-channel properties, such as the high elementary conductance and the occurrence of subconductance levels, were unchanged after the patches had been excised from the cells, indicating that they are not under the control of soluble cytoplasmic factors. In cellattached recordings at the cell resting potential the degree of K_Na activity, measured as the probability of the channel being open, P _o, was low in most cases (around 0.01) and similar to that observed in the inside-out configuration when the bath solution contained concentrations of Na⁺ around 30 mM and of K⁺ close to the physiological intracellular levels. However, in some cell-attached patches P _o was high (around 0.2) and comparable to the values measured in cell-free recordings with high Na⁺ concentrations in the bath (100 mM). The excision of a highactivity patch in the presence of 30 mM Na⁺ resulted in a fall of P _o in about 20 s, which is consistent with the wash-out of a soluble cytoplasmic molecule. After the excision all K_Na displayed a similar Na⁺ sensitivity, irrespective of the degree of activation observed in the cellattached mode. In inside-out patches the P _o values observed in the presence of either low or high concentrations of Na⁺ in bath solutions were not modified by internal Ca²⁺ (0.8–8.5 M). The variable degree of K_Na activation observed in cell-attached recordings suggests that either internal Na⁺ concentrations reach very high levels close to the membrane, or soluble factor(s) are involved in the modulation of K_Na activity: under such conditions, the Na⁺-activated K⁺ current may contribute to the maintenance of the resting membrane potential and to control neuronal membrane excitability.     

Submandibular salivary secretion in the cat and associated potassium movements: Dependence on temperature and perfusate flow rate

Cat submandibular glands were perfused with Locke solution in a thermostated chamber and intermittently stimulated with 10^–5 M acetylcholine (ACh). In one series of experiments the perfusion pressure was varied within the range 90–60 mm Hg, and secretory flow rate, active K⁺-reuptake, passive K⁺-release, and resting and ACh-induced venous flow rates were measured. The ACh-induced secretory flow rate and the maximal K⁺-fluxes were related to the simultaneous ACh-induced venous flow rates. A proportionality was found between the maximal rate of ACh-induced K⁺-release and ACh-induced venous flow rates below 8 ml/min, while at higher flow rates the K⁺-release leveled off. The maximal rate of the post-stimulatory K⁺-reuptake increased proportionally to the ACh-induced perfusate flow rate throughout the range studied. The secretory flow rate was much less affected by changes in ACh-induced perfusate flow rate. In another series of experiments the gland temperature was varied within the range 12–37°C, and the same parameters were measured. All parameters decreased with cooling being reduced to 50% of their 37°C values at: 24°C for secretion, 19°C for K⁺-reuptake, and 14°C for K⁺-release. It is concluded: that 1) the rate of ACh-induced K⁺-release is limited by the ACh-induced perfusate flow rate (within the physiological range), 2) the capacity of the K⁺-reuptake mechanism is at least one order of magnitude larger than the maximal rate of K⁺-reuptake in vivo, 3) the marked temperature sensitivity of the secretory flow rate reflects the high complexity of the mechanisms involved.     

Patch-clamp study of rubidium and potassium conductances in single cation channels from mammalian exocrine acini

Single-channel current recordings were carried out on excised inside-out patches of baso-lateral plasma membrane from exocrine acinar cells. The mouse pancreas and submandibular gland as well as the pig pancreas were investigated.In the mouse pancreas the voltage-insensitive Ca²⁺-activated cation channel was studied. Single-channel current-voltage (i/v) relationships were studied in symmetrical Rb⁺-rich solutions and in asymmetrical Rb⁺/Na⁺ and Na⁺/Rb⁺ solutions. In all cases the i/v relations were linear and had the same slope representing a single-channel conductance of about 33 pS which is identical to that previously obtained with symmetrical Na⁺ solutions or asymmetrical Na⁺/K⁺ solutions.In the mouse submandibular gland and the pig pancreas the voltage and Ca²⁺-activated K⁺ channel was studied. The outward currents observed after depolarization in the presence of quasi-physiological Na⁺/K⁺ gradients were immediately abolished when all the K⁺ in the bath fluid was replaced by Rb⁺ (bath fluid in contact with inside of plasma membrane). This effect was immediately and fully reversible upon return to the high K⁺ solution.The voltage and Ca²⁺-activated K⁺ channel was also studied in asymmetrical K⁺/Rb⁺ and Rb⁺/K⁺ solutions. In the first case inward (K⁺) currents could be observed but not outward (Rb⁺) currents, while in the other case inward (Rb⁺) currents could not be seen whereas outward (K⁺) currents were measured. The current-voltage relationships were approximately linear and the null potential was close to 0 mV in both situations. In contrast the null potential for current through the K⁺ channel in the presence of asymmetrical Na⁺/K⁺ or Li⁺/K⁺ solutions was about –70 mV and with reversed gradients about +60 mV.Outward K⁺ currents of reduced size (through the voltage and Ca²⁺-activated K⁺ channel) could be observed when the bath fluid contained 75 mM K⁺ and 75 mM Rb⁺, but not (in the same membrane patches) when 150 mM Rb⁺ and no K⁺ was present.It is concluded that the large voltage- and Ca²⁺-activated K⁺ channel has an extremely low Rb⁺ conductance. It is possible, however, that the permeability for Rb⁺ may be about the same as for K⁺. The voltage-insensitive Ca²⁺-activated cation channel does not discriminate between K⁺ and Rb⁺.     

Deliberate modification of the specific activity of leukemic cells

A comparative study was made of the efficacy of various types of deliberate modification of the leukemogenic activity of a transplantable line of bone marrow cells obtained from animals infected with Rauscher leukemia virus. Treatment of the leukemic cells with neuraminidase, or culturing them at a supraoptimal temperature led to complete loss of their leukemogenic activity, as shown by survival of 100% of the experimental animals and the absence of splenomegaly. Meanwhile, treatment of the cells with concanavalin A and 5-bromodeoxyuridine delayed the development of splenomegaly and lowered the mortality among the recipient animals by 70 and 20%, respectively. The results suggest that these methods of action on leukemogenic cells can be used in order to obtain material for subsequent immunization.Department of Virology, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. D. Solov'ev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 4, pp. 448–449, April, 1977.     

Extracellular free calcium and potassium during paroxysmal activity in the cerebral cortex of the cat

Summary Extracellular calcium and potassium activities (a_Ca and a_K) as well as neuronal activity were simultaneously recorded with ion-sensitive electrodes in the somatosensory cortex of cats. Baseline a_Ca was 1.2–1.5 mM/1, baseline a _k 2.7–3.2 mM/1. Transient decreases in a_Ca and simultaneous increases in a_K were evoked by repetitive stimulation of the contralateral forepaw, the nucleus ventroposterolateralis thalami and the cortical surface. Considerable decreases in a_Ca (by up to 0.7 mM/1) were found during seizure activity. A fall in a_Ca preceded the onset of paroxysmal discharges and the rise in a_K after injection of pentylene tetrazol. The decrease in a_Ca led also the rise in a_K during cyclical spike driving in a penicillin focus. It is concluded that alterations of Ca⁺⁺ dependent mechanisms participate in the generation of epileptic activity.     

Transient changes in the size of the extracellular space in the sensorimotor cortex of cats in relation to stimulus-induced changes in potassium concentration

Summary The time course of local changes of the extracellular space (ES) was investigated by measuring concentration changes of repeatedly injected tetramethylammonium (TMA⁺) and choline (Ch⁺) ions for which cell membranes are largely impermeable. After stimulus-induced extracellular [K⁺] elevations the [TMA⁺] and [Ch⁺] signals recorded with nominally K⁺-selective liquid ion-exchanger microelectrodes increased by up to 100%, thus indicating a reduction of the ES down to one half of its initial size. The shrinkage was maximal at sites where the K⁺ release into the ES was also largest. At very superficial and deep layers, however, considerable increases in extracellular K⁺ concentration were not accompanied by significant reductions in the ES. These findings can be explained as a consequence of K⁺ movement through spatially extended cell structures. Calculations based on a model combining the spatial buffer mechanism of Kuffler and Nicholls (1966) to osmolarity changes caused by selective K⁺ transport through primarily K⁺ permeable membranes support this concept.Following stimulation additional iontophoretically induced [K⁺]_o rises were reduced in amplitude by up to 35%, even at sites where maximal decreases of the ES were observed. This emphasizes the importance of active uptake for K⁺ clearance out of the ES.This investigation was supported by DFG grants Lu 158/10 and He 1128/1     

Determination of intracellular K+ activity in rat kidney proximal tubular cells

The intracellular K⁺ activity of rat kidney proximal tubular cells was determined in vivo, using intracellular microelectrodes. In order to minimize damage from the impaling electrodes, separate measurements on separate cells, were performed with single-barrelled KCl-filled non-selective electrodes and single-barrelled, K⁺-sensitive microelectrodes, which were filled with a liquid K⁺-exchanger resin that has also a small sensitivity to Na⁺. Both electrodes had tip diameters of 0.2 m or below. The proper intracellular localization of the electrodes was ascertained by recording the cell potential response to intermittent luminal perfusions with glucose. The membrane potential measured with the non-selective microelectrodes was –76.3±8.1 mV (n=81) and the potential difference measured with the K⁺-sensitive microelectrode was –7.2±5.8 mV (n=32). Based on the activity of K⁺ in the extracellular fluid of 3 mmol/l the intracellular K⁺ activity was estimated to be 82 mmol/l. Assuming equal K⁺-activity coefficients to prevail inside and outside the cell, this figure suggests that the intracellular K⁺ concentration is 113 mmol/l which must be considered as a lower estimate, however. The data indicate that the K⁺-ion distribution between cytoplasm and extracellular fluid is not in equilibrium with the membrane potential, but that K⁺ is actively accumulated inside the cell. This result provides direct evidence for the presence of an active K⁺ pump in the tubular cell membranes, which in view of other observations, must be envisaged as a (not necessarily electroneutral) Na⁺/K⁺-exchange pump which operates in the peritubular cell membrane and is eventually responsible for the major part of the tubular solute and water absorption.     

Effect of asphyxia on adenylate cyclase activity in cat brain cortex

Effect of 1–5-min asphyxia on adenylate cyclase activity in cat brain cortex is studied. Adenylate cyclase activity is measured in cortex specimens obtainedex vivo after 1, 2.5, and 5 min of asphyxia, and 30 and 60 min of reoxygenation by radioassay. Stimulating effects of norepinephrine and NaF on adenylate cyclase activity are assessed. Five-min asphyxia induces phasic changes in adenylate cyclase activity: on the 1st min basal activity of the enzyme increases by 97%, after 2.5 min it returns to the initial level, and increases again by 55% on the 5th min of asphyxia. On the 30th and 60th min of reoxygenation after 2.5- and 5-min asphyxia, basal adenylate cyclase activity does not differ from the initial activity. The stimulating effect of norepinephrine and NaF on enzyme activity is weakened after 5 min of asphyxia and 30 min of reoxygenation after 2.5- and 5-min asphyxia. Even short-term asphyxia affects adenylate cyclase activity and modifies the mechanisms of adrenergic signal transduction in the brain cortex in response to oxygen deficiency and probably to hypercapnia as well as during the early reoxygenation period. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 8, pp. 131–134, August, 1997     

Enhancement of transmission at the frog neuromuscular junction by adenosine deaminase: evidence for an inhibitory role of endogenous adenosine on neuromuscular transmission

Adenosine deaminase reversibly increased the amplitude and the quantum content of the end-plate potentials (EPPs) recorded from superficial muscle fibers of frog sartorius preparations in which twitches have been prevented with high-magnesium solutions. Adenosine deaminase prevented the inhibitory effect of exogenously applied adenosine but not that of 2-chloroadenosine on the amplitude of EPPs. The effect of adenosine deaminase was abolished by erythro-9(2-hydroxy-3-nonyl)adenine (EHNA). The results suggest that endogenous adenosine exerts an inhibitory 'tone' over neuromuscular transmission.     

The effect of sodium ions on the light-induced86Rb release from the isolated crayfish retina

The effect of low external Na⁺ concentrations on the light-induced K⁺ release from crayfish photoreceptor cells was tested by labelling intracellular K⁺ with the isotope⁸⁶Rb. The amount of isotope released per light stimulus is roughly proportional to the external Na⁺ concentration if the osmolarity is kept constant by replacing Na⁺ with Tris, choline or sucrose. When sucrose is used to replace the depleted Na⁺ the light-induced K⁺ release is a linear function of the external Na⁺ concentration and is reduced by approx. 95% at an external Na⁺ concentration of 5 mmol/l. For choline and Tris substitutions the relationships are less clear but at Na⁺ concentrations 56 mmol/l it seems that in comparison with sucrose the light-induced K⁺ release is smaller in a Tris solution and larger in a choline solution. It is suggested that the light-induced K⁺ release is due mainly to an activation of voltage sensitive K⁺ channels.