5-Hydroxytryptamine stimulates Rb efflux from pancreatic β-cells

The effects of 5-hydroxytryptamine and 5-hydroxytryptophan on ⁸⁶Rb⁺ efflux from prelabelled ob/ob-mouse islets were studied to better understand the cellular mechanisms underlying the effects of 5-hydroxytryptamine and 5-hydroxytryptophan on insulin release. 5-Hydroxytryptophan (4 mM) had no effect on ⁸⁶Rb⁺ efflux either at a low (3 mM) or at a high (20 mM) d-glucose concentration, whereas 5-hydroxytryptamine (4 mM) stimulated ⁸⁶Rb⁺ efflux at both glucose concentrations. These results indicate that 5-hydroxytryptamine may reduce glucose-induced insulin release by inhibiting early steps in the β-cell stimulus-secretion coupling.     

Oxidative stress increases potassium efflux from pancreatic islets by depletion of intracellular calcium stores

Oxidative stress to B-cells is thought to be of relevance in declining B-cell function and in the process of B-cell destruction. In other tissues including heart, brain and liver, oxidative stress has been shown to elevate the intracellular free calcium concentration and to provoke potassium efflux. We studied the effect of oxidative stress on Ca²⁺ and K⁺ (Rb⁺) outflow from pancreatic islets using the thiol oxidants DIP and BuOOH. Both compounds reversibly increased ⁸⁶Rb⁺ efflux in the presence of 3 and 16.7 mmol/l glucose. Stimulation of ⁸⁶Rb⁺ efflux was also evident in the absence of calcium. DIP evoked release of ⁴⁵Ca²⁺ from the pancreatic islets both in the presence or absence of extracellular calcium. Employing inhibitors of the calcium-activated potassium channel (K_Ca) and the high conductance K⁺-channel (BK_Ca), the effect of DIP on ⁸⁶Rb⁺ efflux was slightly diminished. Tolbutamide had no effect on ⁸⁶Rb⁺ efflux in the presence of DIP. On the other hand thapsigargin, a blocker of the Ca²⁺-ATPase of the endoplasmic reticulum, completely suppressed the DIP-mediated ⁸⁶Rb⁺ outflow. The data suggest that thiol oxidant-induced potassium efflux from pancreatic islets is mainly mediated through liberation of intracellular calcium and subsequent stimulation of calcium-activated potassium efflux.     

Influence de i’acide abscissique sur le transport d’ions inorganiques chez la pomme de terre (solanum tuberosum cv. Bintje). etude comparée avec quelques autres phytohormones

Using detached leaves of potato plants (Solanum tuberosum cv. Bintje) it was shown that abscisic acid (ABA) induced an oriented transport of ions (⁸⁶Rb,³²P and^3SS) towards the hormone-treated leaflet. In order to understand the hormone effect on cell permeability, pretreatment of discs with hormonal solution was conducted. The pretreatment with ABA stimulated the uptake of K⁺ simultaneously with the decrease of efflux. The effect of ABA on ion uptake is compared with the action of other hormones, benzylaminopurine (BAP), gibberellic acid (GA₃) known to act over a long distance transport, too. Three kinds of specificity are underlined: - hormonal specificity (stimulation by ABA and inhibiton by BAP of K⁺ uptake by foliar tissues); - ionic specificity (ABA increase⁸⁶Rb influx, but inhibits³²P and^3SS influx); - tissue specificity shown by a comparative study between foliar tissues (source) and tuber tissues (sink). The autoradiography of foliar discs demonstrated that a treatment of foliar discs can be used, at least for Rb⁺ (K), to study role of ABA in long-distance transport.      

Activation,but not inhibition,by glucose of Ca2+-dependent K+ permeability in the rat pancreatic B-cell

The effect of glucose on the Ca²⁺-activated K⁺ permeability in pancreatic islet cells was investigated by measuring the rate of ⁸⁶Rb efflux, ⁴⁵Ca efflux and insulin release from perifused rat pancreatic islets exposed to step-wise increased in glucose concentration. When the glucose concentration was raised from intermediate (8.3 or 11.1 mM) to higher values, a rapid and sustained increase in ⁸⁶Rb outflow, ⁴⁵Ca outflow and insulin release was observed. Likewise, in the presence of 8.3 or 16.7 mM glucose, tolbutamide increased ⁸⁶Rb and ⁴⁵Ca efflux, as well as insulin release. In the two series of experiments, a tight correlation was found between the magnitude of the changes in ⁸⁶Rb and ⁴⁵Ca outflow, respectively. It is concluded that, at variance with current ideas, glucose does not inhibit the response to cytosolic Ca²⁺ of the Ca²⁺-sensitive modality of K⁺ extrusion. On the contrary, as a result of its effect upon Ca²⁺ handling, glucose stimulates the Ca²⁺-activated K⁺ permeability.     

Dopamine Stimulates K+ Efflux in the Chick Retina via D1 Receptors Independently of Adenylyl Cyclase Activation

Abstract: Dopamine (DA) stimulated K⁺ efflux (assessed as ⁸⁶Rb⁺ efflux) in retinal suspensions of posthatched chicken. This effect was dose dependent (EC₅₀= 22 μM), was mimicked by the D₁-selective antagonist SKF-38393, and reversed by the D₁-selective antagonist SCH-23390, indicating an involvement of D₁ receptors. Analogues of cyclic AMP (CAMP) did not mimic the DA action. Moreover, DA failed to affect cAMP levels, suggesting that adenylyl cyclase (AC) was not involved. In contrast, forskolin (FSK) stimulated both K⁺ efflux and cAMP accumulation in the retina (EC₅₀ of 10 μM for both effects). The FSK-elicited K⁺ efflux was not mimicked by 1,9-dideoxy-FSK (an analogue of FSK that does not activate AC), suggesting that FSK stimulated K⁺ efflux through the activation of AC. Both DA and FSK inhibited Na⁺,K⁺-ATPase activity in the retina. However, the DA-elicited K* efflux was independent of this inhibition, whereas the FSK effect on K⁺ efflux was largely due to the inhibitory action of the diterpene of the ion pump. A possible role of protein kinase C (PKC) in the DA action was explored. The PKC activator 4β-phorbol 12-myristate 13-acetate (4β-PMA) potently (EC₅₀= 4 nM) stimulated K⁺ efflux. This action was not mimicked by the inactive isomer 4α-PMA. When added together, DA and 4β-PMA behaved in an additive manner, suggesting separate mechanisms of action for these two drugs. Moreover, DA failed to stimulate retinal phosphoinositide hydrolysis, a well-known pathway leading to PKC activation. These data suggest that DA acting through D₁ receptors and independently of AC can modulate its target cell excitability in the chick retina by stimulating K⁺ efflux pathways. The mechanism of the DA action remains to be clarified.     

Evidence for the Induction of Repetitive Action Potentials in Synaptosomes by K+-Channel Inhibitors: An Analysis of Plasma Membrane Ion Fluxes

Abstract: The effects of four K⁺-channel inhibitors on synaptosomal free Ca²⁺ concentrations and ⁸⁶Rb⁺ fluxes are analysed. 4-Aminopyridine, α-dendrotoxin, charybdotoxin, and tetraethylammonium all increase the free Ca²⁺ concentration, although their potencies differ widely. In each case, the elevation in free Ca²⁺ concentration is reversed by the subsequent addition of tetrodotoxin. The transient ⁸⁶Rb⁺ efflux from preequilibrated synaptosomes induced with high concentrations of veratridine is partially inhibited by 4-aminopyridine and α-dendrotoxin. In contrast, when 4-aminopyridine or α-dendrotoxin is added to polarized synaptosomes, an enhanced⁸⁶Rb⁺ flux is seen, both for uptake and for efflux with no change in the total ⁸⁶Rb⁺/K⁺ content of the synaptosomes and with only a slight time-averaged plasma membrane depolarization (6.4 and 3.3 mV, respectively). The enhancements of flux by 4-aminopyridine or α-dendrotoxin are sensitive to ouabain and/or to tetrodotoxin. Furthermore, these flux changes show the same concentration dependencies as the blocked component of veratridine-stimulated ⁸⁶Rb⁺ efflux, the elevation of free Ca²⁺ concentration, and the facilitation of glutamate exocytosis that are elicited by 4-aminopyridine or α-dendrotoxin. It is concluded that these findings support the proposal of spontaneous, repetitive firing of synaptosomes evoked by K⁺-channel inhibitors and that the enhanced ⁸⁶Rb⁺ flux is a consequence of the activity of 4-aminopyridine- and α-dendrotoxin-insensitive K⁺ channels during these action potentials.     

Glucose inhibits Ca efflux from pancreatic β-cells also in the absence of Na-Ca countertransport

During perifusion with medium deprived of Ca²⁺, addition of glucose or omission of Na⁺ resulted in prompt and quantitatively similar inhibitions of ⁴⁵Ca efflux from β-cell rich pancreatic islets microdissected from ob / ob mice. Glucose had no additional inhibitory effect when Na⁺ was isoosmotically replaced by sucrose or choline⁺. When K⁺ was used as a substitute for Na⁺, the inhibitory effect of Na⁺ removal on ⁴⁵Ca efflux became additive to that of glucose. The observation that glucose can be equally effective in inhibiting ⁴⁵Ca efflux in the presence or absence of Na⁺ is difficult to reconcile with the postulate that the Na⁺-Ca²⁺ countertransport mechanism is a primary site of action for glucose.     

Aluminium interactions with K+(86Rb+) and 45Ca2+ fluxes in three cultivars of sugar beet (Beta vulgaris)

Three cultivars of sugar beet (Beta vulgaris L.), which are sensitive to aluminium (Al) in the order Primahill > Monohill > Regina, were grown in water culture for 2 weeks. Nutrients were supplied at 15% increase of amounts daily, corresponding to the nutrient demand for maximal growth. The 2.4-dinitrophenol (DNP)-sensitive (metabolic) and DNP-insensitive (non-metabolic) uptake of aluminium, phosphate. ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) in roots were measured as well as transport to shoots of intact plants. All 3 cultivars absorbed more aluminium if DNP was present during the aluminium treatment than in its absence. It is suggested that sugar beets are able to extrude aluminium activity or that they possess an active mechanism to keep Al outside the cell. The presence of Al in the medium during the 1-h experiment affected the metabolic and non-metabolic fluxes of ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) in different ways. In the presence of DNP, the influx of both ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) and the efflux of ⁴⁵Ca²⁺ were inhibited by Al in a competitive way. At inhibition of ⁴⁵Ca²⁺ influx, 2 Al ions are probably bound per Ca²⁺ uptake site in cv. Regina (Al-tolerant), but in cvs Primahill and Monohill only one Al ion is bound (more Al sensitive). Aluminium competitively inhibited the active efflux of ⁴⁵Ca²⁺ (absence of DNP) in almost the same way in the 3 cultivars. In contrast, aluminium stimulated the influx of K⁺(⁸⁶Rb⁺) in cvs Primahill, Monohill and Regina in the absence of DNP. Thus, the Al effects on active and passive K⁺(⁸⁶Rb⁺) influx are different. The total influx of K⁺(⁸⁶Rb⁺) increased in the presence of Al and might be connected to an active exclusion of Al. Regina is the least Al-sensitive cultivar, probably because Al interferes less with the Ca²⁺ fluxes and because this cultivar actively excludes phosphate in the presence of Al. Thus Al-phosphate precipitation within the plant could be avoided.     

Volume- and catecholamine-dependent regulation of Na/H antiporter and unidirectional potassium fluxes in Salmo trutta red blood cells

β-Adrenergic- and volume-dependent regulation of ²²Na influx and ⁸⁶Rb influx and efflux in erythrocytes of brown trout (Salmo trutta m. lacustris) were studied. Norepinephrine (10^-6 mol·1^-1) increased the rate of ²²Na influx 10-to 20-fold via the activation of a Na/H exchanger (ethyl isopropyl amiloride inhibited component of ²²Na influx). Unlike carp erythrocytes the activity of the Na, K-pump (ouabain-inhibited ⁸⁶Rb influx) was only slightly (25–35%) increased by norepinephrine. The norepinephrine-induced increment of Na, K-pump activity was completely abolished by ethyl isopropyl amiloride thus indicating that this effect was mediated by Na/H exchanger-induced increase of intracellular Na⁺ concentration. Cell shrinkage in hyperosmotic media resulted in a several-fold activation of the Na/H exchanger. Cell swelling in hypotonic media increased both the rate of K, Cl-cotransport [((dihydroindenyl)oxy)alcanaic acidsensitive components of ⁸⁶Rb influxe and efflux] and passive permeability (leakage) of erythrocyte membranes for Na⁺ and K⁺. No volume-dependent regulation of Na, K, 2Cl-cotransport (bumetanide-sensitive components of ⁸⁶Rb fluxes) was found. It may be concluded that the regulation of monovalent cation transport in erythrocytes of fast-moving (carnivorous) brown trout differs essentially from that in slowly moving (herbivorous) carp.     

2,4-D-Induced Changes in the K+ Uptake of Wheat Roots at Different pH Values

The effects of an auxin herbicide, 2,4-D, at a concentration of 0.01 mM, on the K⁺ uptake and efflux of excised roots of wheat (Triticum aestivum L. cv. Rannaya) were investigated at different pH values. The K⁺ movement was monitored with a K⁺ (⁸⁶Rb) tracer. In parallel experiments the ATPase activities of microsomal fractions were determined by the inorganic phosphate liberation method. 2,4-D inhibited the K⁺ uptake especially at low pH, irrespective of whether Ca²⁺ was present or not. No marked changes were observed in the K⁺ efflux properties at pH values above 4. The inhibitory effect on K⁺ uptake exhibited a correlation with the hydrocarbon solubility of the herbicide, but not with the 2,4-D-induced decrease of the ATPase activity. It is suggested that 2,4-D exerts a non-specific effect on the lipid-protein interactions, giving rise to a generalized alteration of the transport barrier properties of the plasma membrane even at as low a concentration as 0.01 mM.     

Effect of anisotonic media on cell volume and electrolyte fluxes in slices of the dogfish (Squalus acanthias) rectal gland

Summary Osmotic responses of slices of dogfish rectal gland to hypotonic (urea-free) and hypertonic media were studied. Transfer of tissue from isotonic (890 mosM) to hypotonic (550 mosM) saline produced an osmotic swelling associated with a slow net uptake of cell K⁺ (and Cl^–) and a slow, two-component efflux of urea. Media made hypertonic (1180 mosM) by addition of urea or mannitol produced osmotic shrinkage with a net loss of KCl. The cell osmotic responses in hypotonic media were lower than predicted for an ideal osmometer. No volume regulatory responses were seen subsequent to the initial osmotic effects. The cation influx in hypotonic media lacked specificity: in the presence of 0.5 mM ouabain or in K⁺-free media a net influx of Na⁺ was found. At steady state, the cell membrane potential evaluated from the Nernst potentials of K⁺ and triphenylmethyl phosphonium⁺, was independent of medium tonicity, suggesting the membrane potential as a determinant in the cellular osmotic response. Zero-time⁸⁶Rb⁺ fluxes were measured:⁸⁶Rb⁺ influx was not affected by hypotonicity, implying an unchanged operation of the Na⁺–K⁺-ATPase. On the other hand,⁸⁶Rb⁺ efflux was significantly reduced at hypotonicity; this effect was transient, the efflux returning to the control value once the new steady state of cell volume had been reached. A controlled efflux system is therefore involved in the cell osmotic response. The absence of the volume regulatory phenomenon suggests that the cells are not equipped with a volume-sensing mechanism.Abbreviations and symbols DW dry weight - E extracellular (polyethylene glycol) space - E Nernst potential - H₂O_e H₂O_i tissue water, extra- and intracellular - TPMP ⁺ triphenyl methyl phosphonium salt - WW wet weight     

The stimulus-secretion coupling of glucose-induced insulin release: effects of valinomycin upon pancreatic islet function.

In isolated rat pancreatic islets, valinomycin (0.01 to 1.0 μm) caused a dose-related facilitation of ⁸⁶Rb⁺ outflow and a dose-related inhibition of the glucose-induced changes in both outflow and net uptake of ⁸⁶Rb⁺. At high concentrations (0.1–1.0 μm), the ionophore also inhibited the oxidation of glucose and endogenous nutrients, decreased the adenylate charge, and lowered the concentration of reduced pyridine nucleotides in the islet cells. However, as little at 1.0 to 10.0 nm valinomycin caused anomalies in the handling of ⁴⁵Ca²⁺ (suppression of the early inhibitory effect of glucose upon ⁴⁵Ca²⁺ efflux, and reduction in the amount of ⁴⁵Ca²⁺ recovered in the islets after an extensive washing procedure) and inhibition of insulin release. Moreover, when the effect of glucose upon K⁺ conductance was abolished by high concentrations of valinomycin (0.1–1.0 μm), the glucose-induced secondary rise in ⁴⁵Ca²⁺ efflux was still observed. These findings suggest that the effects of glucose upon ⁸⁶Rb⁺ and ⁴⁵Ca²⁺ handling, respectively, although normally concomitant with one another, can be dissociated, in part at least, from one another. It is concluded that the glucose-induced reduction in K⁺ outflow may be unnecessary for the sugar to cause a partial remodeling of Ca²⁺ fluxes in the islet cells.     

Effects of ionic strength and relative humidity on the efflux of K+ (86Rb) and Ca2+ (45Ca) from roots of intact seedlings of cucumber, oat and wheat

Spring wheat (Triticum aestivum L. cv. Svenno), oat (Avena sativa L. cv. Brighton) and glasshouse cucumber (Cucumis sativus L. cv. Bestseller F1) were cultured for a week after germination on complete nutrient solutions of three different dilutions (1, 25 and 50% of the full strength medium). K⁺(⁸⁶Rb) and ⁴⁵Ca were present during the whole culture period. Relative humidity (RH) was 50% except during the last day, when half the material was transferred to 90% RH. Efflux of labelled ions was then followed during eight hours on unlabelled solutions of the same composition as before, and at both 50% and 90% RH in the atmosphere. – Uptake of K⁺(⁸⁶Rb) during growth tended to be saturated in the 25% medium. Contrariwise, the level of Ca²⁺ in the roots increased continuously with strength of the medium. At low concentrations cucumber roots were higher in Ca²⁺ than roots of oat or wheat, whereas all three species showed similar levels of Ca²⁺ in 50% medium. – At the lowest ionic strength, smooth efflux curves were obtained that could be resolved according to the three-compartment theory. At higher ionic strength, irregularities were observed, and more for Ca²⁺ than for K⁺; but for practical purposes compartment analysis with the same time constants could be applied as for the lowest concentration. – Discrimination between K⁺ and Rb⁺ differed between the roots, but not much between the shoots of different species. The roots of oat and wheat took up Rb⁺ preferentially over K⁺ in the 25% and 50% media; whereas K⁺ was preferred over Rb⁺ or little discrimination made in 1% medium and for cucumber. The shoots generally showed less discrimination than the roots. The main variability in discrimination between K⁺ and Rb⁺ thus appears to be localized in the tonoplasts of the roots cells. – Low RH around the shoots increased efflux of K⁺(⁸⁶Rb) from the cytoplasm and vacuoles of the root cells as compared to the efflux at high RH. DNP (2,4-dinitrophenol) in the medium had the same effect as high RH around the shoots. The signal system that must exist between shoots and roots is discussed as a response to “drought” conditions. In relation to investigations of others, it is assumed that the effect of DNP may indicate that part of the chain between roots and shoots consists of metabolically influenced sites, whose output is influenced by the rate of water transport.     

22Na+ and 86Rb+ fluxes in spermatozoa and oocytes of arbacia punctulata

The fluxes of ²²Na⁺ and ⁸⁶Rb⁺ in Arbacia sperm and oocytes were studied in order to determine how these cells carry out cation exchange with the sea environment. The uptake of these ions by serum followed a pattern of early rapid influx (initial 0.5 min) and subsequent efflux (1–3 min) followed by a gradual uptake (after 3 min). Neither the uptake nor the efflux of these cations by Arbacia sperm were affected by ouabain, suggesting that influx and efflux of ²²Na⁺ and ⁸⁶Rb⁺ in Arbacia sperm occur predominantly by passive transport. The ²²Na⁺ uptake by Arbacia oocytes showed a steady increase after an initial rapid uptake. A slight but significant inhibition of ²²Na⁺ uptake was observed with ouabain. However, ⁸⁶Rb⁺ uptake by the oocytes reached an early equilibrium and was not affected by ouabain. The uptake of Rb⁺ by Arbacia oocyte is by passive transport while that of Na⁺ is both by passive and active transport.     

Effects of acetylcholine and caerulein on 86Rb+ efflux in the mouse pancreas. Evidence for a sodium-potassium-chloride cotransport system

The effect of acetylcholine and the cholecystokinin-like peptide, caerulein on the fractional efflux of ⁸⁶Rb⁺ from preloaded isolated segments of mouse pancreas were studied. Both secretagogues evoked a marked transient increase in ⁸⁶Rb⁺ efflux. The removal of Ca²⁺ from the superfusing medium and addition of 10^?4 M EGTA, markedly reduced, but did not abolish the responses to either acetylcholine or caerulein. Furosemide ($\text{10}{}^{\mathrm{?5}}\text{?10}{}^{\mathrm{?3}}\text{M}$) or piretanide (10^?4 M) reduced the basal efflux and inhibited the secretagogue-elicited responses. Stimulus-induced ⁸⁶Rb⁺ outflow was abolished when the Cl^? component of the superfusing solution was replaced by either NO₃^?, SO₄^2? or I^? but not in case of replacement by Br^?, When Na⁺ was replaced with either Li⁺ or choline⁺ both acetylcholine and caerulein failed to elicit any detectable increase in ⁸⁶Rb⁺ outflow. However, when Tris⁺ was substituted for Na⁺, acetylcholine caused a moderate increase in ⁸⁶Rb⁺ efflux which was abolished by either furosemide (10^?4 M) or chloride depletion (nitrate substitution). The removal of extracellular K⁺ or pretreatment with 10^?3 M ouabain had little effect on secretagogue-evoked ⁸⁶Rb⁺ efflux. These results indicate the presence of a diuretic-sensitive Na⁺-K⁺-Cl^? cotransport system in the mouse pancreatic acinar cell membrane.     

A single mechanism for the stimulation of insulin release and 86Rb+ efflux from rat islets by cationic amino acids

The mechanisms by which cationic amino acids influence pancreatic B-cell function have been studied by monitoring simultaneously ⁸⁶Rb⁺ efflux and insulin release from perifused rat islets. The effects of two reference amino acids arginine and lysine were compared with those of closely related substances to define the structural requirements for recognition of these molecules as secretagogues. Arginine accelerated ⁸⁶Rb⁺ efflux and increased insulin release in the absence or in the presence of 7mm-glucose. Its effects on efflux did not require the presence of extracellular Ca²⁺ or Na⁺, but its insulinotropic effects were suppressed in a Ca²⁺-free medium and inhibited in an Na⁺-free medium. Among arginine derivatives, only 2-amino-3-guanidinopropionic acid mimicked its effects on ⁸⁶Rb⁺ efflux and insulin release; citrulline, guanidinoacetic acid, 3-guanidinopropionic acid and guanidine were inactive. Norvaline and valine also increased ⁸⁶Rb⁺ efflux, but their effect required the presence of extracellular Na⁺; they did not stimulate insulin release. Lysine as well as the shorter-chain cationic amino acids ornithine and 2,4-diaminobutyric acid accelerated ⁸⁶Rb⁺ efflux in a Ca²⁺- and Na⁺-independent manner. Their stimulation of insulin release was suppressed by Ca²⁺ omission, but only partially inhibited in an Na⁺-free medium. The uncharged glutamine and norleucine increased the rate of ⁸⁶Rb⁺ efflux in the presence of glucose, only if extracellular Na⁺ was present. Norleucine slightly increased release in a Ca²⁺- and Na⁺-dependent manner. The effects of lysine on efflux and release were not mimicked by other related substances such as 1,5-diaminopentane and 6-aminohexanoic acid. The results suggest that the depolarizing effect of cationic amino acids is due to accumulation of these positively charged molecules in B-cells. This causes acceleration of the efflux of K⁺ (⁸⁶Rb⁺) and activation of the influx of Ca²⁺ (which triggers insulin release). The prerequisite for the stimulation of B-cells by this mechanism appears to be the presence of a positive charge on the side chain of the amino acid, rather than a specific group.     

The Na+,K+,2Cl−-cotransport system in HeLa cells: Aspects of its physiological regulation

We have previously reported on the biochemical properties of a Na⁺,K⁺,2Cl^?-cotransport in HeLa cells and here we deal with aspects of its physiological regulation. Na⁺,K⁺,2Cl^?-cotransport in HeLa cells was studied by ⁸⁶Rb⁺ influx and ⁸⁶Rb⁺/²²Na⁺ efflux measurements. The effects of rat atrial natriuretic peptide (ANP), isoproterenol, and amino acids on ⁸⁶Rb⁺ flux, mediated by the bumet-anide-sensitive Na⁺, K⁺, 2Cl^?-cotransport system and the ouabain-sensitive Na⁺/K⁺-pump, were investigated. ANP reduced bumetanide-sensitive ⁸⁶Rb⁺ influx under isotonic as well as under hypertonic conditions. Similar decrease of bumetanide-sensitive ⁸⁶Rb⁺ influx was observed in the presence of 8-bromo-cGMP, while neither isoproterenol as a β-receptor agonist nor 8-bromo-cAMP-could alter bumetanide-sensitive ⁸⁶Rb⁺ influx. Furthermore, efflux of ⁸⁶Rb⁺ and ²²Na⁺ was greatly reduced in the presence of bumetanide and ANP. Together with our recent findings, showing functionally active, high affinity receptors for ANP on HeLa cells (Kort and Koch, Biochim. Biophys. Res. Commun. 168:148–154, 1990), this study indicates that ANP participates in the regulation of the Na⁺, K⁺, 2Cl^?-cotransport system in HeLa cells. Further measurements revealed that amino acids as present in the growth medium (Joklik's minimal essential medium) and the amino acid derivative α-methyl-aminoisobutyric acid (metAlB, 1 and 5 mM, respectively) also reduced Na⁺, K⁺, 2Cl^?-cotransport-mediated ⁸⁶Rb⁺ uptake and diminished the stimulatory effect of hypertonicity on the cotransporter. In addition, the Na⁺/K⁺-pump was markedly stimulated in the presence of amino acids, while neither ANP and 8-Br-cGMP nor isoproterenol and 8-Br-cAMP had a significant effect on the activity of the Na⁺/K⁺-pump.     

Incorporation of the transmembrane hydrophobic domain of glycophorin into small unilamellar phospholipid vesicles Ion Flux studies

Human erythrocyte glycophorin is one of the best characterized integral membrane proteins. Reconstitution of the membrane-spanning hydrophobic segment of glycophorin (the tryptic insoluble peptide released when glycophorin is treated with trypsin) with liposomes results in the production of freeze-fracture intrabilayer particles of 80 Å diameter (Segrest, J.P., Gulik-Krzywicki, T. and Sardet, C. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 3294–3298), with particles appearing at or above a tryptic insoluble peptide concentration of 4 mmol per mol phosphatidylcholine. In the present study, increasing concentrations of tryptic insoluble peptide were added to sonicated small unilamellar egg phosphatidylcholine vesicles and the rate of efflux of ²²Na⁺ was examined by rapid (30 s) gel filtration on Sephadex G-50. Below a concentation of 3–5 mmol tryptic insoluble peptide/mol phosphatidylcholine, ²²Na⁺ efflux occurs at a constant slow rate at given tryptic insoluble peptide concentrations. Above a concentration of 3–5 mM, the rate of efflux is biphasic at given tryptic insoluble peptide concentrations, exhibiting both an initial fast and a subsequent slow component. On the basis of graphic and computer curve-fitting analysis, with increasing tryptic insoluble peptide concentration, the rate of the slow component reaches a plateau at a tryptic insoluble peptide concentration of 3–5 mM and remains essentially constant until much higher concentrations are reached; the fast component increases linearly with increasing tryptic insoluble peptide concentration well beyond 5 mM. The most consistent interpretation of this data is as follows. The slow ²²Na⁺ efflux component is due to perturbations of small unilamellar vesicle integrity by tryptic insoluble peptide monomers. At a tryptic insoluble peptide concentration of 3–5 mmol/mol, a critical concentration is reached following which there is intrabilayer tryptic insoluble peptide self-association. The fast ²²Na⁺ efflux component is due to the increasing presence of tryptic insoluble peptide self-associated multimers the 80-Å particles seen by freeze-fracture electron microscopy) which results in a significantly larger bilayer defect than do tryptic insoluble peptide monomers. The failure of complete saturation of efflux by the fast component is ascribed to the presence of two populations of small unilamellar vesicles, some of which contain tryptic insoluble peptide multimers and some of which do not.Addition of cholesterol to the tryptic insoluble peptide/phosphatidylcholine vesicles decreases the rate of ²²Na⁺ efflux by inhibiting primarily the fast component. Freeze-fracture electron microscopy indicates that the presence of cholesterol has no effect on the size, number or distribution of 80-Å intra-bilayer particles in the tryptic insoluble peptide/phosphatidylcholine vesicles. These results are consistent with a mechanism to explain the fast Na⁺ efflux component involving protein-lipid boundary perturbations.Efflux of ⁴⁵Ca²⁺ from phosphatidylcholine vesicles is also enhanced by incorporation of tryptic insoluble peptide, but only if divalent cations (Ca²⁺ or Mg²⁺) are present in the external bathing media as well as inside the sonicated vesicles. If monovalent Na⁺ only is present in the bathing media no ⁴⁵Ca²⁺ efflux is seen. Under conditions where ⁴⁵Ca²⁺ efflux is seen, both a fast and a slow component are present, although both appear lower than corresponding rate constants for ²²Na⁺ efflux. These results suggest a coordinated mechanism for ion efflux induced by tryptic insoluble peptide and, together with the ²²Na⁺ efflux studies, may have mechanistic implications for the transbilayer phospholipid exchange (flip-flop) suggesed to be induced at glycophorin/phospholipid interfaces (de Kruiff, B., van Zoelen, E.J.J. and van Deenen, L.L.M. (1978) Biochim. Biophys. Acta 509, 537–542).     

Inhibition of 86Rb+ and 22Na+ efflux of Ehrlich lettré tumor cells by Ca2+.

The mechanism of the protective effect of Ca²⁺ on cellular K⁺ content was studied by examination of the effect of Ca²⁺ on efflux of the K⁺ analog, ⁸⁶Rb⁺, from preloaded cells with the use of compounds which interfere with monovalent cation movements. Ca²⁺ decreased ⁸⁶Rb⁺ efflux to the same extent in the presence and absence of ouabain, suggesting that Ca²⁺ did not alter the activity of the (Na⁺ + K⁺)-adenosine triphosphatase pump. Ca²⁺ exerted a similar protective effect in the presence of furosemide, an inhibitor of K⁺-K⁺ exchange, indicative that Ca²⁺ was not inhibiting this pathway. Since Ca²⁺ did not influence these pathways, it is concluded that Ca²⁺ exerts its primary effect by slowing passive diffusion. In support of this, Ca²⁺ also slowed ²²Na⁺ efflux. In addition, ethanol-induced leakage of ⁸⁶Rb⁺ was reversed by extracellular Ca²⁺, suggestive of a Ca²⁺-membrane phospholipid interaction.     

Distinct effects of acetylcholine and glucose on 45calcium and 86rubidium efflux from mouse pancreatic islets

The similarities between the effects of acetylcholine and glucose on phospholipid metabolism in pancreatic islet cells prompted the comparison of their effects on ionic fluxes. Acetylcholine (1 μM) consistently increased ⁴⁵Ca²⁺ efflux from mouse islets, whereas glucose increased it in the presence, but decreased it in the absence of extracellular Ca²⁺. Acetylcholine consistently accelerated ⁸⁶Rb⁺ efflux, and this effect was augmented by Ca²⁺ omission. On the other hand, glucose markedly inhibited ⁸⁶Rb⁺ efflux, except when its concentration was raised from 10 to 15 mM in the presence of Ca²⁺. Unlike their effects on phospholipid metabolism, the ionic effects of the two insulin-secretagogues are thus very different.