Role of extracellular electrolytes in the activation of ribosomal protein S6 kinase by epidermal growth factor, insulin-like growth factor 1, and insulin in ZR-75-1 cells

Activation of ribosomal protein S6 kinase by epidermal growth factor (EGF), insulin, and insulin-like growth factor 1 (IGF1) was studied in the human mammary tumor cell line ZR-75-1 in isotonic buffers. In contrast to growth factor-dependent S6 phosphorylation which is strongly dependent on extracellular pH (Chambard, J. C., and J. Pouyssegur. 1986. Exp. Cell Res. 164:282-294.) preincubation of cells in buffers with different pH values ranging from 7.5 to 6.5 had no effect on basal or EGF-stimulated S6 kinase activity. Replacement of extracellular Na+ with choline or replacement of extracellular Ca++ with EGTA also did not inhibit stimulation of S6 kinase by EGF. When intracellular Ca++ was buffered with the permeable Ca++ chelator quin2, EGF stimulation was reduced 50%. A similar inhibition of the EGF response was observed when cells were incubated in buffers with high K+ concentrations or in the presence of the K+ ionophore valinomycin. Insulin and IGF1 stimulation of S6 kinase were also inhibited by high K+ concentrations and by buffering intracellular Ca++. In contrast to the responses to EGF, insulin- and IGF1-activation of S6 kinase was enhanced when glucose was present and depended on the presence of bicarbonate in the medium. The results indicate that ionic signals generated by growth factors and insulin, such as increases in intracellular pH or Na+, do not seem to be involved in the activation of S6 kinase. However, effects of growth factors or insulin on membrane potential and/or K+ fluxes and redistribution of intracellular Ca++ may play a role in the activation process. Furthermore, the mechanism of insulin activation of S6 kinase is distinct from the growth factors by its dependency on extracellular bicarbonate.     

Influence of sodium-calcium exchange on calcium current rundown and the duration of calcium-dependent chloride currents in pituitary cells, studied with whole cell and perforated patch recording

The whole cell patch-clamp technique, in both standard and perforated patch configurations, was used to study the influence of Na+-Ca++ exchange on rundown of voltage-gated Ca++ currents and on the duration of tail currents mediated by Ca++-dependent Cl- channels. Ca++ currents were studied in GH3 pituitary cells; Ca++-dependent Cl- currents were studied in AtT-20 pituitary cells. Na+-Ca++ exchange was inhibited by substitution of tetraethylammonium (TEA+) or tetramethylammonium (TMA+) for extracellular Na+. Control experiments demonstrated that substitution of TEA+ for Na+ did not produce its effects via a direct interaction with Ca++-dependent Cl- channels or via blockade of Na+-H+ exchange. When studied with standard whole cell methods, Ca++ and Ca++-dependent Cl- currents ran down within 5-20 min. Rundown was accelerated by inhibition of Na+-Ca++ exchange. In contrast, the amplitude of both Ca++ and Ca++-dependent Cl- currents remained stable for 30-150 min when the perforated patch method was used. Inhibition of Na+-Ca++ exchange within the first 30 min of perforated patch recording did not cause rundown. The rate of Ca++-dependent Cl- current deactivation also remained stable for up to 70 min in perforated patch experiments, which suggests that endogenous Ca++ buffering mechanisms remained stable. The duration of Ca++-dependent Cl- currents was positively correlated with the amount of Ca++ influx through voltage-gated Ca++ channels, and was prolonged by inhibition of Na+-Ca++ exchange. The influence of Na+-Ca++ exchange on Cl- currents was greater for larger currents, which were produced by greater influx of Ca++. Regardless of Ca++ influx, however, the prolongation of Cl- tail currents that resulted from inhibition of Na+-Ca++ exchange was modest. Tail currents were prolonged within tens to hundreds of milliseconds of switching from Na+- to TEA+-containing bath solutions. After inhibition of Na+-Ca++ exchange, tail current decay kinetics remained complex. These data strongly suggest that in the intact cell, Na+-Ca++ exchange plays a direct but nonexclusive role in limiting the duration of Ca++-dependent membrane currents. In addition, these studies suggest that the perforated patch technique is a useful method for studying the regulation of functionally relevant Ca++ transients near the cytoplasmic surface of the plasma membrane.     

Interactions of sodium transport, cell volume, and calcium in frog urinary bladder

The volume of individual cells in intact frog urinary bladders was determined by quantitative microscopy and changes in volume were used to monitor the movement of solute across the basolateral membrane. When exposed to a serosal hyposmotic solution, the cells swell as expected for an osmometer, but then regulate their volume back to near control in a process that involves the loss of KCl. We show here that volume regulation is abolished by Ba++, which suggests that KCl movements are mediated by conductive channels for both ions. Volume regulation is also inhibited by removing Ca++ from the serosal perfusate, which suggests that the channels are activated by this cation. Previously, amiloride was observed to inhibit volume regulation: in this study, amiloride-inhibited, hyposmotically swollen cells lost volume when the Ca++ ionophore A23187 was added to Ca++-replete media. We attempted to effect volume changes under isosmotic conditions by suddenly inhibiting Na+ entry across the apical membrane with amiloride, or Na+ exit across the basolateral membrane with ouabain. Neither of these Na+ transport inhibitors produced the expected results. Amiloride, instead of causing a decrease in cell volume, had no effect, and ouabain, instead of causing cell swelling, caused cell shrinkage. However, increasing cell Ca++ with A23187, in both the absence and presence of amiloride, caused cells to lose volume, and Ca++-free Ringer's solution (serosal perfusate only) caused ouabain-blocked cells to swell. Finally, again under isosmotic conditions, removal of Na+ from the serosal perfusate caused a loss of volume from cells exposed to amiloride. These results strongly suggest that intracellular Ca++ mediates cell volume regulation by exerting a negative control on apical membrane Na+ permeability and a positive control on basolateral membrane K+ permeability. They also are compatible with the existence of a basolateral Na+/Ca++ exchanger.     

Ca2+ gradient and drugs reveal different binding sites for Pi and Mg2+ in phosphorylation of the sarcoplasmic reticulum ATPase.

The first step towards ATP synthesis by the Ca2-ATPase of sarcoplasmic reticulum is the phosphorylation of the enzyme by Pi. Phosphoenzyme formation requires both Pi and Mg2+. At 35 degrees C, the presence of a Ca2+ gradient across the vesicle membrane increases the apparent affinity of the ATPase for Pi more than 10-fold, whereas it had no effect on the apparent affinity for Mg2+. In the absence of a Ca2+ gradient, the phosphorylation reaction is inhibited by both K+ and Na+ at all Mg2+ concentrations used. However, in the presence of 1 mM Mg2+ and of a transmembrane Ca2+ gradient, the reaction is still inhibited by Na+, but the inhibition promoted by K+ is greatly decreased. When the Mg2+ concentration is raised above 2 mM, the enzyme no longer discriminates between K+ and Na+, and the phosphorylation reaction is equally inhibited by the two cations. Trifluoperazine, ruthenium red and spermidine were found to inhibit the phosphorylation reaction by different mechanisms. In the absence of a Ca2+ gradient, trifluoperazine competes with the binding to the enzyme of both Pi and Mg2+, whereas spermidine and ruthenium red were found to compete only with Mg2+. The data presented suggest that the enzyme has different binding sites for Mg2+ and for Pi.     

Rapid release of 45Ca from an occluded state of the Na,K-pump

45Ca is bound to the occluded state of the Na,K-pump, apparently at K+ sites. Only one 45Ca ion is bound in place of two K+ ions, with an affinity approximately 0.08 mM; K+ competes with an apparent affinity approximately 0.04 mM. 45Ca is released rapidly from Na,K-ATPase in the presence of ATP or ADP, presumably to the intracellular medium. The rate constant of 45Ca release with ATP is greater than 100 s-1 at 20 degrees C, more than twice as fast as the rate of release of 42K from the occluded state. Phosphorylation of Na,K-ATPase with MgPi, which would lead to release of occluded K+ or Rb+ to the extracellular face of the membrane, stabilizes occluded 45Ca. 45Ca release is slower immediately after exposure to MgPi than after a rinse in the absence of Pi indicating that in the former circumstance the rate of 45Ca release is limited by dephosphorylation; 45Ca release is even slower after exposure to Mg2+ arsenate, consistent with dearsenylation being slower than dephosphorylation. When limited by dephosphorylation, the rate of 45Ca release is dependent on the species of monovalent cation present, increasing in the order N-methylglucamine less than Cs+ less than Li+ less than Na+ less than Rb+ less than K+. When the 45Ca occluded state is exposed to K + Mg + Pi and then to Na+ + Mg2+ + ATP, the exposure to K+ is "remembered," indicating simultaneous occlusion of 45Ca and K+. The apparent affinity for K+ in formation of this state is 10-50 mM, and the rate of release of K+ is approximately 2 s-1. Ca2+ has effects on the release of 86Rb from the occluded state: With ATP, Ca2+ acts like Mg2+ by stimulating 86Rb release at low concentrations and inhibiting at high concentrations; with MgPi, Ca2+ inhibits 86Rb release, presumably by preventing phosphorylation. Thus, Ca2+ has two actions on the Na,K-pump as studied here: one as a Mg2+ congener, and another as a K+ congener at transport sites. In the latter role Ca2+ is unusual in that it appears to be able to bind to the transport sites from the intracellular face of the pump and to become occluded, but unable to be released from extracellular sites.     

Active Uptake of Ca++ and Ca++-Activated Mg++ ATPase in Red Cell Membrane Fragments

Isolated human red blood cell membrane fragments (RBCMF) were found to take up Ca⁺⁺ in the presence of ATP.¹ This ATP-dependent Ca⁺⁺ uptake by RBCMF appears to be the manifestation of an active Ca⁺⁺ transport mechanism in the red cell membrane reported previously (Schatzmann, 1966; Lee and Shin, 1969). The influences of altering experimental conditions on Ca⁺⁺-stimulated Mg⁺⁺ ATPase (Ca⁺⁺ ATPase) and Ca⁺⁺ uptake of RBCMF were studied. It was found that pretreatment of RBCMF at 50°C abolished both Ca⁺⁺ ATPase and Ca⁺⁺ uptake. Pretreatment of RBCMF with phospholipases A and C decreased both Ca⁺⁺ ATPase and Ca⁺⁺ uptake, whereas pretreatment with phospholipase D did not significantly alter either Ca⁺⁺ ATPase or Ca⁺⁺ uptake. Both Ca⁺⁺ ATPase and Ca⁺⁺ uptake had ATP specificity, similar optimum pH's, and optimum incubation temperatures. From these results, it was concluded that Ca⁺⁺ uptake is intimately linked to Ca⁺⁺ ATPase.     

Regulation of Na+-K+-ATPase: effect of Mg and Ca ions

The participation of Mg2+ and Ca2+ in complicated mechanisms of Na+, K(+)-ATPase regulation is discussed in the survey. The regulatory actions of Mg2+ on Na+, K(+)-ATPase such as its participation in phosphorylation and dephosphorylation of the enzyme, ADP/ATP-exchange inhibition, cardiac glycosides and vanadate binding with the enzyme, conformational changes induction during ATPase cycle are reviewed in detail. Some current views of mechanisms of above mentioned Mg2+ regulatory effects are discussed. The experimental evidence of Ca2+ immediate influence on the functional activity of Na+, K(+)-ATPase (catalytic, transport and glycoside-binding) are given. It's noted that these effects are based on the conformational changes in the enzyme and also on the phase transition in membrane induced by Ca2+. Unimmediate action of Ca2+ on Na+, K(+)-ATPase is also discussed, especially due to its effect on other membrane systems functionally linked with Na(+)-pump (for instance, due to Na+/Ca(+)-exchanger activation). It's concluded that Mg2+ and Ca2+ as "universal regulators" of the cell effectively influence the functional activity and conformational states of Na+, K(+)-ATPase.     

Ca2+-activated Na+ fluxes in human red cells. Amiloride sensitivity

The effect of Ca2+ on the ouabain- and bumetanide-resistant Na+ fluxes in intact red cells was studied at relatively constant internal Ca2+, membrane potential, and cell volume. The red cell calcium concentration was modified using the ionophore A23187. In fresh red cells, the Na+ influx and efflux (1.2 +/- 0.13 and 0.26 +/- 0.07 mmol/liter cells x h, respectively) were not affected by amiloride (1 mM). When external Ca2+ was raised from 0 to 150 microM, in the presence of A23187, both the Na+ influx and efflux were stimulated (about 3.5-fold). The Ca2+-activated Na+ efflux and influx had an apparent Km for activation by Ca2+o of about 25 microM. The Ca2+-dependent Na+ transport was inhibited 30-60% by amiloride (ID50 = 17.3 +/- 8 microM). Amiloride, however, had no effect on the Ca2+-dependent K+ influx. The amiloride-sensitive (AS) transport pathway was a linear function of the Na+o concentration in the range from 0 to 75 mM. The Ca2+i activation seems to depend on the metabolic integrity of red cells. 1) It does not take place in ATP-depleted red cells; 2) ATP-repletion of ATP-depleted red cells fully restored AS Na influx; and 3) ATP-enrichment (ATP-red cells) enhanced the AS Na influx by about 100%. The Ca2+-activated AS Na+ influx was not affected by either DIDS or trifluoperazine. The present results indicate that in human erythrocytes an increase in internal Ca2+ activates on otherwise silent AS Na+-transport system, which is dependent on the metabolic integrity of the red cells.     

The reaction of Mg2+ with the Ca2+-ATPase from human red cell membranes and its modification by Ca2+

Media prepared with CDTA and low concentrations of Ca2+, as judged by the lack of Na+-dependent phosphorylation and ATPase activity of (Na+ +K+)-ATPase preparations are free of contaminant Mg2+. In these media, the Ca2+-ATPase from human red cell membranes is phosphorylated by ATP, and a low Ca2+-ATPase activity is present. In the absence of Mg2+ the rate of phosphorylation in the presence of 1 microM Ca2+ is very low but it approaches the rate measured in Mg2+-containing media if the concentration of Ca2+ is increased to 5 mM. The KCa for phosphorylation is 2 microM in the presence and 60 microM in the absence of Mg2+. Results are consistent with the idea that for catalysis of phosphorylation the Ca2+-ATPase needs Ca2+ at the transport site and Mg2+ at an activating site and that Ca2+ replaces Mg2+ at this site. Under conditions in which it increases the rate of phosphorylation, Ca2+ is without effect on the Ca2+-ATPase activity in the absence of Mg2+ suggesting that to stimulate ATP hydrolysis Mg2+ accelerates a reaction other than phosphorylation. Activation of the E1P----E2P reaction by Mg2+ is prevented by Ca2+ after but not before the synthesis of E1P from E1 and ATP, suggesting that Mg2+ stabilizes E1 in a state from which Mg2+ cannot be removed by Ca2+ and that Ca2+ stabilizes E1P in a state insensitive to Mg2+. The response of the Ca2+-ATPase activity to Mg2+ concentration is biphasic, activation with a KMg = 88 microM is followed by inhibition with a Ki = 9.2 mM. Ca2+ at concentration up to 1 mM acts as a dead-end inhibitor of the activation by Mg2+, and Mg2+ at concentrations up to 0.5 mM acts as a dead-end inhibitor of the effects of Ca2+ at the transport site of the Ca2+-ATPase.     

Chagasic IgG modifies the activity of sarcolemmal ATPases through a beta adrenergic mechanism

The effect of anti beta adrenoceptor IgG from chagasic sera upon Ca2+-ATPase and Na++K+-ATPase of myocardial membrane was studied. Chagasic IgG stimulated Ca2+-ATPase and inhibited Na++K+-ATPase activities. Both enzymatic effects of the IgG could be prevented after beta adrenoceptor blockade or after the absorption of chagasic IgG with turkey red blood cells. Isoproterenol acted similarly. These results provide information concerning to the biochemical mechanism, by which an antibody, known to activate adenylate cyclase system coupled to cardiac beta adrenoceptor, produces stimulation of myocardial contractility.     

The calcium dependence of pigment translocation in freshwater shrimp red ovarian chromatophores

The roles of calcium in cell signaling consequent to chromatophorotropin action and as an activator of mechanochemical transport proteins responsible for pigment granule translocation were investigated in the red ovarian chromatosomes of the freshwater shrimp Macrobrachium olfersii. Chromatosomes were perfused with known concentrations of free Ca++ (10(-3) to 10(-9) M) prepared in Mg(++)-EGTA-buffered physiological saline after selectively permeabilizing with 25 microM calcium ionophore A23187 or with 10(-8) M red pigment concentrating hormone (RPCH). The degree of pigment aggregation and the translocation velocity of the leading edges of the pigment mass were recorded in individual chromatosomes during aggregation induced by RPCH or A23187 and dispersion induced by low Ca++. Aggregation is Ca++ dependent, showing a dual extracellular and intracellular requirement. After perfusion with reduced Ca++ (10(-4) to 10(-9) M), RPCH triggers partial aggregation (approximately 65%), although the maximum translocation velocities (approximately 16.5 microns/min) and velocity profiles are unaffected. After aggregation induced at or below 10(-5) M Ca++, spontaneous pigment dispersion ensues, suggesting a Ca++ requirement for RPCH coupling to its receptor, or a concentration-dependent, Ca(++)-induced Ca(++)-release mechanism. The Ca(++)-channel blockers Mn++ (5 mM) and verapamil (50 microM) have no effect on RPCH-triggered aggregation. An intracellular Ca++ requirement for aggregation was demonstrated in chromatosomes in which the Ca++ gradient across the cell membrane was dissipated with A23187. At free [Ca++] above 10(-3) M, aggregation is complete; at 10(-4) M, aggregation is partial, followed by spontaneous dispersion; below 10(-5) M Ca++, pigments do not aggregate but disperse slightly. Aggregation velocities diminish from 11.6 +/- 1.2 microns/min at 5.5 mM Ca++ to 7.4 +/- 1.3 microns/min at 10(-4) M Ca++. Half-maximum aggregation occurs at 3.2 x 10(-5) M Ca++ and half-maximum translocation velocity at 4.8 x 10(-5) M Ca++. Pigment redispersion after 5.5 mM Ca(++)-A23187-induced aggregation is initiated by reducing extracellular Ca++: slight dispersion begins at 10(-7) M, complete dispersion being attained at 10(-9) M Ca++. Dispersion velocities increase from 0.6 +/- 0.2 to 3.1 +/- 0.5 microns/min. Half-maximum dispersion occurs at 7.6 x 10(-9) M Ca++ and half-maximum translocation velocity at 2.9 x 10(-9) M Ca++. These data reveal an extracellular and an intracellular Ca++ requirement for RPCH action, and demonstrate that the centripetal or centrifugal direction of pigment movement, the translocation velocity, and the degree of pigment aggregation or dispersion attained are calcium-dependent properties of the granule translocation apparatus.     

The plasma membrane calcium pump: regulation by a soluble Ca2+ binding protein

The Ca2+ pump of the plasma membrane of human red blood cells is associated with the activity of a (Ca2+ + Mg2+)-ATPase. Both the ATPase and the pump are stimulated above basal activities by calmodulin, an ubiquitous Ca2+-binding protein. Calmodulin isolated from human red blood cells was shown to be equipotent and equieffective with that isolated from beef brain. Half-maximal activation of ATPase (isolated red blood cell membranes, 37 C) and transport (inside-out red blood cell membrane vesicles, 25 C) were obtained with 2.5 and 4.4 nM calmodulin, respectively. Ca2+ dependence of Ca2+ transport was measured in the absence and in the presence of 50 nM calmodulin. At all Ca2+ concentrations above 2 X 10(-7) M Ca2+, the rate of transport was greater in the presence of calmodulin. The results implicate calmodulin in the regulation of the plasma membrane Ca2+ pump, but the mechanism(s) remain to be elucidated.     

The Ca++ permeability of the apical membrane in neuromast hair cells

The mechanosensitivity of eel (Anguilla anguilla) neuromasts was measured by the impulse responses of single afferent nerve fibers to mechanical stimuli. It is dependent on the potential across the skin and on the ions in the water outside the apical membrane of the sensory cells. The mechanosensitivity decreases to zero when the skin is polarized by 10-100 mV cathodal DC (skin surface negative); it increases with increasing (10-60 mV) anodal DC and remains remarkably constant with higher polarization (Fig. 1). The mechanosensitivity increases with increasing concentrations of Ca++ outside the apical membrane of the sensory cells. Na+ and K+ have no influence. Addition of La , Co++, Mg++, D 600 and A-QA 39 inhibits the mechanosensitivity; the degree of inhibition varies with the inhibitor and the ratio [Ca++]/[inhibitor], indicating that the inhibition is competitive (Figs. 2, 3). We conclude that the apical membrane is specifically permeable to Ca++ ('late Ca channel') and that the inward receptor current through the apical membrane is carried by Ca++. Streptomycin also inhibits mechanosensitivity by competing with Ca++. With streptomycin, however, anodal polarization reduces, rather than increases, the mechanosensitivity (Fig. 4).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ionic blockage of the light-regulated sodium channels in isolated rod outer segments.

We have investigated, with osmotic techniques, the light-regulated Na+ channels in rod outer segments (ROS) and ROS fragments freshly isolated from the frog retina. Values of Na+ permeability (PNa) similar to those observed electrophysiologically in the retina were observed using the osmotic technique (continuous flow) described by Korenbrot and Cone. In the other osmotic techniques that we explored, PNa was greatly diminished, if not completely suppressed; however, we found with these techniques that antioxidant conditions (N2 atmosphere or EDTA) significantly increased PNa, suggesting that the Na+ channels are highly sensitivive to membrane oxidation. Using the continuous flow technique, we investigated the H+ and Ca++ dependence of the Na+ channels and found that both of these ions, at micromolar activities, can block the channels. Raising the external H+ activity decreases PNa (reversibly) in a single "sigmoidal" response with an apparent pKa of 5.8. Similarly, in the presence of the ionophores X537A or A23187 which allow equilibration of Ca++ across membranes, the Na+ channels are blocked when the external Ca++ activity is increased from 10(-7) to 10(-5) M. This high sensitivity to both H+ and Ca++ ions suggests that high field strength anionic sites may exist in or near the Na+ channels and that the channels are blocked when these sites bind H+ or Ca++ ions.     

Depolarizing response of rat parathyroid cells to divalent cations

Membrane potentials were recorded from rat parathyroid glands continuously perfused in vitro. At 1.5 mM external Ca++, the resting potential averages -73 +/- 5 mV (mean +/- SD, n = 66). On exposure to 2.5 mM Ca++, the cells depolarize reversibly to a potential of -34 +/- 8 mV (mean +/- SD). Depolarization to this value is complete in approximately 2-4 min, and repolarization on return to 1.5 mM Ca++ takes about the same time. The depolarizing action of high Ca++ is mimicked by all divalent cations tested, with the following order of effectiveness: Ca++ greater than Sr++ greater than Mg++ greater than Ba++ for alkali-earth metals, and Ca++ greater than Cd++ greater than Mn++ greater than Co++ greater than Zn++ for transition metals. Input resistance in 1.5 mM Ca++ was 24.35 +/- 14 M omega (mean +/- SD) and increased by an average factor of 2.43 +/- 0.8 after switching to 2.5 mM Ca++. The low value of input resistance suggests that cells are coupled by low-resistance junctions. The resting potential in low Ca++ is quite insensitive to removal of external Na+ or Cl-, but very sensitive to changes in external K+. Cells depolarize by 61 mV for a 10- fold increase in external K+. In high Ca++, membrane potential is less sensitive to an increase in external K+ and is unchanged by increasing K+ from 5 to 25 mM. Depolarization evoked by high Ca++ may be slowed, but is unchanged in amplitude by removal of external Na+ or Cl-. Organic (D600) and inorganic (Co++, Cd++, and Mn++) blockers of the Ca++ channels do not interfere with the electrical response to Ca++ changes. Our results show remarkable parallels to previous observations on the control of parathormone (PTH) release by Ca++. They suggest an association between membrane voltage and secretion that is very unusual: parathyroid cells secrete when fully polarized, and secrete less when depolarized. The extraordinary sensitivity of parathyroid cells to divalent cations leads us to hypothesize the existence in their membranes of a divalent cation receptor that controls membrane permeability (possibly to K+) and PTH secretion.     

Properties of vagal excitation transfer to the heart atrium]

A mathematical model of pre- and postganglionic parasympathetic nerve fiber excitation transfer is developed. This model gives a measure Q of acetylcholine (ACh) release from presynaptic preganglionic boutons and postganglionic varicosities. When increasing Ca++ the measure Q increases too. Na-ions exert a competitive inhibition. The relationship between Q and the Ca/Na2-quotient is a hyperbolic one. Mn++ inhibits the release of ACh non-competitively. Q increases both by excess potassium and Cs+ depolarization. The ACh release is diminished by Mg++. Ba++ cannot replace the effect of Ca++ on ACh release in Ca++ depleting conditions. Q increases with decreasing pH-level. The ACh release is not significantly influenced by increasing pH, Verapamil (4 mg/l), prostaglandins E2 and F2alpha (20 ng/ml) and substitution of nonpermeable anions for Cl-.     

Interactions of K+ with (Na,K)-ATPase orientation of K+-phosphatase sites studied with inside-out red cell membrane vesicles

Inside-out membrane vesicles from human red cells were used to investigate the side specificity of K+ interactions with the K+-activated phosphatase, a partial reaction of the (Na, K)-ATPase. In the absence of Na+ and ATP, K+ at moderate affinity sites at the extravesicular surface (cytoplasmic K+) stimulates activity, whereas intravesicular K+ (K+ normally at the extracellular surface) is without effect. In contrast, under conditions of phosphorylation of (Na, K)-ATPase (Na+ and ATP present), K+ ions acting at high affinity sites at both surfaces are required. It is concluded that an enzyme x K complex is involved in K+-activated phosphatase activity and that it is formed either by interaction of cytoplasmic K+ with the dephosphoenzyme, or as a consequence of extracellular K+ binding and dephosphorylation of the phosphoenzyme formed in the presence of Na+ plus ATP.     

Properties of tetraethylammonium ion-resistant K+ channels in the photoreceptor membrane of the giant barnacle

After the offset of illumination, barnacle photoreceptors undergo a large hyperpolarization that lasts seconds or minutes. We studied the mechanisms that generate this afterpotential by recording afterpotentials intracellularly from the medial photoreceptors of the giant barnacle Balanus nubilus. The afterpotential has two components with different time-courses: (a) an earlier component due to an increase in conductance to K+ that is not blocked by extracellular tetraethylammonium ion (TEA+) or 3-aminopyridine (3-AP) and (b) a later component that is sensitive to cardiac glycosides and that requires extracellular K+, suggesting that it is due to an electrogenic Na+ pump. The K+ conductance component increases in amplitude with increasing CA++ concentration and is inhibited by extracellular Co++; the Co++ inhibition can be overcome by increasing the Ca++ concentration. Thus, the K+ conductance component is Ca++ dependent. An afterpotential similar to that evoked by a brief flash of light is generated by depolarization with current in the dark and by eliciting Ca++ action potentials in the presence of TEA+ in the soma, axon, or terminal regions of the photoreceptor. The action potential undershoot is generated by an increase in conductance to K+ that is resistant to TEA+ and 3-AP and inhibited by Co++. The similarity in time-course and pharmacology of the hyperpolarization afterpotentials elicited by (a) a brief flash of light, (b) depolarization with current, and (c) an action potential indicates that Ca++-dependent K+ channels throughout the photoreceptor membrane are responsible for all three hyperpolarizing events.     

Presence of a low-affinity nucleotide binding site on the (K+ + H+)-ATPase phosphoenzyme

The effects of Mg2+ and nucleotides on the dephosphorylation process of the (K+ + H+)-ATPase phosphoenzyme have been studied. Phosphorylation with [gamma-32P]ATP is stopped either by addition of non-radioactive ATP or by complexing of Mg2+ with EDTA. The dephosphorylation process is slow and monoexponential when dephosphorylation is initiated with ATP. When phosphorylation is stopped by complexing of Mg2+ the dephosphorylation process is fast and biexponential. The discrepancy could be explained by a nucleotide mediated inhibition of the dephosphorylation process. The I0.5 for ATP for this inhibition is 0.1 mM and that for ADP is 0.7 mM, suggesting that a low-affinity binding site is involved. When Mg2+ is present in millimolar concentrations in addition to the nucleotides the dephosphorylation process is enhanced. Evidence has been obtained that Mg2+ acts through lowering the affinity for ATP. In contrast to K+, Mg2+ does not stimulate dephosphorylation in the absence of nucleotides. Mg2+ and nucleotides show the same interaction in the dephosphorylation process of a phosphoenzyme generated from inorganic phosphate. These findings suggest the presence of a low-affinity nucleotide binding site on the phosphoenzyme, as is found in the (Na+ + K+)-ATPase phosphoenzyme. This low-affinity binding site may function as a feed-back mechanism in proton transport.     

Inositol phosphates turnover, cytosolic Ca++ and pH: putative signals for the control of cell growth

The signals that induce a cell to divide are usually external and in the form of a binding of growth factors. We focussed our attention in defining the sequence of events which occurs after the binding of the mitogens to their surface receptors. One of the early membrane events stimulated by growth factors is a Na+ flux coupled to a H+ efflux that is typically inhibited by amiloride. The importance of this event and of the consequent cytoplasmic alkalinization for the cell proliferation is discussed. Recent data indicate that mitogens increase intracellular Ca++ levels and activate protein kinase C by inducing the hydrolysis of membrane phosphoinositides. A role for Ca++ and protein kinase C in activating Na+/H+ A role for Ca++ and protein kinase C in activating Na+/H+ exchange system is discussed. Finally a model is presented that illustrates the first membrane events stimulated by the growth factors. The model reveals an intimate interconnections between phosphoinositide metabolism, cytosolic Ca++ rise, protein kinase C and cytoplasmic alkalinization.