Adenosine triphosphatase activity in the membranes of the squid nerve fiber.

This investigation deals with the localization of sites of ATPase activity, especially of transport ATPase, in nerve fibers of the squid Doryteuthis plei, at the subcellular level. Splitting of ATP liberates inorganic phosphate which reacts with lead to form a precipitate in the tissue. The reaction was made on nerve fibers fixed with glutaraldehyde. Frozen slices were incubated in Wachstein-Meisel medium containing ATP and Pb(NO(3))(2). Deposits of reaction product were found in the axolemma (towards its axoplasmic side), Schwann cell membranes (mainly at the channels crossing the layer), and mitochondria. Control experiments revealed that no deposits were observed in nerve fibers fixed in osmium tetroxide prior to incubation in the medium containing ATP, or in nerve fibers incubated without substrate or with adenosine monophosphate, adenosine diphosphate, glycerophosphate, or guanosine triphosphate as substrate. For evaluation of transport ATPase activity, these findings were compared with results obtained with nerve fibers treated with G-strophanthin or K-strophanthoside before or after glutaraldehyde fixation. The cardiac glycosides produced a disappearance or diminution of the deposits. The largest inhibitory effect was observed in the axolemma. The findings indicate that the highest ATPase activity is localized in the axolemma and may be due primarily to transport ATPase.

Calcium-induced calcium release in cerebellar Purkinje cells.

Depolarization-induced intracellular Ca2+ rises were measured in fura-2-loaded, voltage-clamped Purkinje cells. The peak Ca2+ rise increased more than linearly with voltage step duration, suggesting the presence of Ca(2+)-induced Ca2+ release. In cells from young animals, in which Ca2+ currents could be satisfactorily recorded, a supralinear relation was also found between peak Ca2+ rise and Ca2+ current integral. Responses to long pulses were inhibited in cells dialyzed with 20 microM ruthenium red and potentiated in cells bathed in the presence of 20 microM ryanodine. Upon repetitive depolarization, increasing Ca2+ rises were elicited by successive voltage pulses, probably because of a potentiating effect of residual Ca2+. Altogether, the results indicate an important contribution of Ca(2+)-induced Ca2+ release to Ca2+ signals of Purkinje cells.

The squid preparation as a general model for ionic and metabolic Na+/Ca2+ exchange interactions: physiopathological implications.

We propose an integrated kinetic model for the squid nerve Na+/Ca2+ exchanger based on experimental evidences obtained in dialyzed axons. This model satisfactorily explains the interrelationship between ionic (Na+(i)-H+(i)-Ca2+(i)) and metabolic (ATP, phosphoarginine (PA)) regulation of the exchanger. Data in dialyzed axons show that the Ca(i)-regulatory site located in the large intracellular loop plays a central role in the modulation by ATP by antagonizing the inhibitory Na+(i)-H+(i) synergism. We have used the Na(o)/Na(i) exchange mode to unequivocally measure the affinity of the Ca(i)-regulatory site. This allowed us to separate Ca(i)-regulatory from Ca(i)-transport sites and to estimate their respective affinities. In this work we show for the first time that under conditions of saturation of the Ca(i)-regulatory site (10 microM Ca2+(i), pH(i) 8.0), ATP have no effect on the Ca(i)-transport site. In addition, we have expanded our equilibrium kinetic model of ionic and metabolic interactions to a complete exchange cycle (circular model). This model, in which the Ca(i)-regulatory site plays a central role, accounts for the decrease in Na(i) inactivation, at high pH(i), high Ca2+(i,) and MgATP. Furthermore, the model also predicts the net Ca2+ movements across the exchanger based on the exchanger complexes redistribution both during physiological and pathological conditions (ischemia).

In squid axons, ATP modulates Na+-Ca2+ exchange by a Ca2+i-dependent phosphorylation.

In squid axons ATP stimulates both the forward and reverse modes of the Na+-Ca2+ exchange by changing the affinity of the carrier towards Na+ and Ca2+ ions. Whether ATP activates the Na+-Ca2+ antiporter allosterically or is hydrolyzed during activation is still debated. The hypothesis that ATP modulates the Na+-Ca2+ exchange through phosphorylation has been tested by means of [gamma-S]ATP, an ATP analog that can act as a substrate for kinases but not for ATPases. Steady-state Ca2+ efflux was measured in squid axons dialyzed without ATP and containing either 0.7 or 100 microM Ca2+i. Addition of 1 mM [gamma-S]ATP markedly increases the Na+o-dependent component of the Ca2+ efflux. The activation by [gamma-S]ATP: requires the presence of Mg2+i, is partially reversible upon removing the analog, is greater than that caused by ATP and only operates on the exchange system since no activation of the ATP-dependent uncoupled Ca2+ efflux (Ca2+ pump) can be detected. 22Na+ experiments were used to monitor the Cao-dependent Na+ efflux (reverse Na+-Ca2+ exchange). Without Ca2+i and ATP, Na+ efflux is very small ('leak'). [gamma-S]ATP does not activate the efflux of Na+ in the absence of Ca2+i. In the presence of Ca2+i the ATP analog stimulates both the Cao- and Nao-dependent Na+ efflux components. Interestingly, neither the Na+ pump, Ca2+i-independent Na+-Na+ exchange, Nai+-Mg2+i exchange or Na+/K+/Cl- cotransport are affected by [gamma-S]ATP. The experiments indicate that a Ca2+i-dependent phosphorylation occurs during the activation of the Na+-Ca2+ exchange by ATP.

An ATP-dependent Na+/Mg2+ countertransport is the only mechanism for Mg extrusion in squid axons.

The components of magnesium efflux in squid axons have been studied under internal dialysis and voltage clamp conditions. The present report rules out the existence of an ATP-dependent, Nao- and Mgo-independent Mg2+ efflux (ATP-dependent Mg2+ pump) leaving the Mg2+-Na+ exchange system as the only mechanism for Mg2+ extrusion. The main features of the Mg2+ efflux are: (1) The efflux is completely dependent on ATP. (2) The efflux can be activated either by external Na+ (forward Mg2+-Na+ exchange) or external Mg2+ (Mg2+-Mg2+ exchange). (3) The mobility of the Mg2+ exchanger in the Na+o-loaded form is greater than that in the Mg2+-loaded one. (4) In variance with the Na+-Ca2+ exchange mechanism, Mg2+-Mg2+ exchange is not activated by external monovalent cations. (5) ATP gamma S replaces ATP in activating Mg2+-Na+ exchange suggesting that a phosphorylation/dephosphorylation process regulates this transport mechanism.

In squid axons the Ca2+i regulatory site of the Na+/Ca2+ exchanger is drastically modified by sulfhydryl blocking agents. Evidences that intracellular Ca2+i regulatory and transport sites are different.

We have explored the effect of the sulfhydryl group blocker p-chloromercuryphenylsulfonic acid (PCMBS) on Ca2+ and Na+ interactions with the Na+/Ca2+ exchanger in squid giant nerve fibers. Steady-state Na+o-dependent Ca2+ efflux (forward) and Na+i-dependent Ca2+ influx (reverse) were measured in internally dialyzed, voltage clamped squid axons. External PCMBS (0.5 mM, for 25-35 min) has no effect on the activation of Ca2+ efflux by Na+o, and Ca2+o or on the activatory external monovalent cation site. In contrast, when applied internally it drastically reduces the affinity of the Na+/Ca2+ exchanger towards Ca2+i ions without affecting its maximal rate of transport; in the presence of MgATP the K0.5 for Ca2+i activation of forward Na+/Ca2+ exchange increases from 1.5 microM to 95 microM; likewise the apparent affinity of the Ca2+i stimulation of the reversal exchange decreases 100-fold. Interestingly, no effect of PCMBS was found on the interactions between Na+i and Ca2+i ions with the internal transport site(s) (inhibition of Na+2o and Ca2+o-dependent Ca2+ efflux by Na+i). On the other hand, Na+i ions do not modify the interactions of Ca2+i with that site. Two important characteristics of the Ca2+i regulatory site are uncover in this work: (i) sulfhydryl groups are important in maintaining the integrity of the Ca2+ binding domain of the Ca2+i regulatory site and (ii) Na+i and Ca2+i regulatory, or Na+i and Ca2+i transporting sites, are different entities.

The effect of pH on Ca2+ extrusion mechanisms in dialyzed squid axons.

The effect of internal and external pH, on the components of the Ca2+ efflux have been investigated in internally displayed squid axons. (1) Internal pH: a fall in intracellular pH (below 7.3) inhibited both the ATP-dependent uncoupled (Ca2+ pump) (50% at pHi 6.3) and the Na+o-dependent Ca2+ efflux (forward Na+/Ca2+ exchange) (50% at pHi 6.8). Internal alkalinization of pH 8.8 had no effect on the uncoupled component but markedly increased (4-fold) the Na+o-dependent Ca2+ efflux. (2) External pH: altering the external pH from 7.3 to 9.0 had no effect on the Na+o-dependent Ca2+ efflux mechanism. In the absence of Ca2+o, alkalinization to pHo 8.8 caused a reduction in the magnitude of the uncoupled Ca2+ pump. This inhibition is markedly enhanced by the presence of Ca2+ in the external medium. As for the case of the sarcoplasmic reticulum Ca2+-ATPase, this combined inhibitory effect of high pHo and Ca2+o is most probably related to a reversal of the cycle of the ATP driven Ca2+ pump. The marked differences in the pH dependence of the components of the Ca2+ efflux support the model of two separate mechanisms of Ca2+ extrusion in squid axons: Ca2+ pump and Na+/Ca2+ exchange.

A new internal perfusion method for transport studies in squid giant axons.

A new internal perfusion method has been developed which allows control of the internal solute composition in squid axons. The superiority of this technique compared to the old perfusion methods is shown by the experiments performed which have reproduced, both qualitatively and quantitatively, the Na+ and Ca2+ fluxes observed in intact and dialyzed axons. Compared with the internal dialysis, the perfusion method has the advantage that the permeability barrier give by the porous capillary has been eliminated. This allows the introduction into the axon of solutes with very high molecular weight, at the same time that a fast and reliable internal control can be achieved.

Vanadate selectively inhibits the Ko+-activated Na+ efflux in squid axons.

The effects of internally applied 1 mM vanadate on the Na+ efflux in dialysed squid axons were found to depend on the presence of external K+. In K+-free artificial sea water, vanadate did not produce any change in the rate of Na+ efflux, whereas in the presence of 10 mM K+ the Na+ efflux was reduced to values even lower than those observed in the absence of K+ (inversion of the K+-free effect). In vanadate-poisoned axons, K+ and NH+4 at low concentrations activated Na+ efflux, but at high concentrations both cations were inhibitory. However, NH+4 was always a better activator and a poorer inhibitor than K+.

Effect of internal and external K+ on Na+-Ca2+ exchange in dialyzed squid axons under voltage clamp conditions.

The effect of external and internal K+ on Na+o-dependent Ca2+ efflux was studied in dialyzed squid axons under constant membrane potential. With axons clamped at their resting potentials, external K+ (up to 70 mM) has no effect on Na+-Ca2+ exchange. Removal of Ki+ causes a marked inhibition in the Na+o-dependent Ca2+ efflux component. Internal K+ activates the Na+-Ca2+ exchange with low affinity (K 1/2 = 90 mM). Activation by Ki+ is similar in the presence or in the absence of Na+i, thus ruling out a displacement of Na+i from its inhibitory site. Axons dialyzed with ATP also show a dependency of Ca2+ efflux on Ki+. The present results demonstrate that Ki+ is an important cofactor (partially required) for the proper functioning of the forward Na+-Ca2+ exchange.

The effects of vanadate on calcium transport in dialyzed squid axons. Sidedness of vanadate-cation interactions.

(1) Vanadate (VO3-) fully inhibits the ATP-dependent uncoupled Ca efflux (Ca pump) in dialyzed squid axons. (2) Vanadate inhibits with high affinity. The mean apparent affinity (K 1/2) obtained was 7 microM. (3) Inhibition by vanadate is dependent on Cao. External Ca lead to a release of the inhibitory effect. (K 1/2 congruent to 3 mM). This antagonistic effect can be reverted by increasing the vanadate concentration. Internal K+ increases the affinity of the intracellular vanadate binding site. External K+ has no effect on the inhibition. (4) Vanadate has no effect on the Nao-dependent Ca efflux component (forward Na-Ca exchange) in the absence of ATP. In axons containing ATP vanadate modified this component.

The effect of ATP on calcium efflux in dialyzed barnacle muscle fibres.

Calcium efflux has been studied in barnacle muscle fibres under internal dialysis conditions. Prolonged dialysis of these fibres, with a medium free of ATP and containing 2 mM cyanide and 1 mM iodoacetate, causes the ATP in the perfusion effluent to fall to less than 20 micrometer. The mean calcium efflux from fibres dialyzed with EGTA buffered solution containing 0.3 micrometer ionized Ca and and no ATP is 0.6 pmol-cm-2-s-1. A two-fold stimulation of the calcium efflux is observed when ATP is added to fibres previously dialyzed with an ATP-free medium. Withdrawal of Na+ and Ca2+ from the external medium causes a marked drop in the Ca2+ efflux in the presence of internal ATP.

Na+-Ca2+ exchange in squid optic nerve membrane vesicles is activated by internal calcium.

The role of intracellular Ca2+ as essential activator of the Na+-Ca2+ exchange carrier was explored in membrane vesicles containing 67% right-side-out and 10% inside-out vesicles, isolated from squid optic nerves. Vesicles containing 100 microM free calcium exhibited a 2-fold increase in the initial rate of Na+i-dependent Ca2+ uptake as compared with vesicles where intravesicular calcium was chelated by 2 mM EGTA or 10 mM HEDTA. The activatory effect exerted by intravesicular Ca2+ on the reverse mode of Na+-Ca2+ exchange (i.e. Na+i-Ca2+o exchange) is saturated at about 100 microM Ca2+i and displays an apparent K 1/2 of 12 microM. Intravesicular Ca2+ produced activation of Na+i-Ca2+i exchange activity rather than an increase in Ca2+ uptake due to Ca2+-Ca2+ exchange. The presence of Ca2+i was essential for the Na+i-dependent Na+ influx, a partial reaction of the Na+-Ca2+ exchanger. In fact, the Na+ influx levels in vesicles loaded with 2 mM EGTA were close to those expected from diffusional leak while in vesicles containing Ca2+i an additional Na+-Na+ exchange was measured. The results suggest that in nerve membrane vesicles Ca2+ at the inner aspect of the membrane acts as an activator of the Na+-Ca2+ exchange system.

ATP-dependent calcium pump and Na+-Ca2+ exchange in plasma membrane vesicles from squid optic nerve.

Purified plasma membrane vesicles from the optic nerve of the squid Sepiotheutis sepioidea accumulate calcium in the presence of Mg2+ and ATP. Addition of the Ca2+ ionophore A23187 to vesicles which have reached a steady state of calcium-active uptake induces complete discharge of the accumulated cation. Kinetic analysis of the data indicates that the apparent Km for free Ca2+ and ATP are 0.2 muM and 21 muM, respectively. The average Vmax is 1 nmol Ca2+/min per mg protein at 25 degrees C. This active transport is inhibited by orthovanadate in the micromolar range. An Na+-Ca2+ exchange mechanism is also present in the squid optic nerve membrane. When an outwardly directed Na+ gradient is imposed on the vesicles, they accumulate calcium in the absence of Mg2+ and/or ATP. This ability to accumulate Ca2+ is absolutely dependent on the Na+ gradient: replacement of Na+ by K+, or passive dissipation of the Na+ gradient, abolishes transport activity. The apparent Km for Ca2+ of the Na+-Ca2+ exchange is more than 10-fold higher than that of the ATP-driven pump (app. Km=7.5 muM). While the apparent Km for Na+ is 74 mM, the Vmax of the exchanger is 27 nmol Ca2+/min per mg protein at 25 degrees C. These characteristics are comparable to those displayed by the uncoupled Ca pump and Na+-Ca2+ exchange previously described in dialyzed squid axons.

Currents related to the sodium-calcium exchange in squid giant axon.

We report the measurement of a Cai-activated membrane current in dialyzed squid axon under membrane potential control with a low-noise voltage clamp. Two additional voltage clamp systems were used to clamp the external guard plates to a value that prevented the establishment of potential differences between the central and lateral compartments of the experimental chamber. This reduced to a minimum the contribution of membrane currents generated at the axon ends to the current measured in the central pool. This latter current was reduced by using internal and external solutions designed to diminish at a maximum membrane currents, while maintaining the conditions for optimal operation of the Na+-Ca2+ exchange. Thus TTX was used to block Na+ channels and prolonged exposure to K+-free media was used to eliminate K+ conductance. The maximum concentration of external sodium was 200 mM. The addition of fixed amounts of free ionic calcium to the internal solution, activated a current whose direction and magnitude depended on the thermodynamic driving forces for calcium and sodium. When the experimental conditions determined an inwardly directed current, this depended on the presence of external sodium, and lithium could not substitute for it. The Cai-activated current, was blocked by external lanthanum and showed a high temperature dependence. In experiments in which the reversal potential was measured for the Cai-activated current, it was found to be strikingly similar to the value calculated according to Er = 3ENa - 2ECa, suggesting that the current is the electrical manifestation of the Na+-Ca2+ exchange operating with an stoichiometry of 3Na+:1Ca2+.

In dialyzed squid axons Ca2+i activates Ca2+o-Na+i and Na+o-Na+i exchanges in the absence of Ca chelating agents.

We used internally dialyzed squid axons to explore whether the reported activatory effect of Ca2+i on the partial reactions of the Na+-Ca2+ exchange (essential activator) is secondary to the presence of Ca2+ chelating agents in the internal medium. The effect of Ca2+i pulses on both the reverse (Ca2+o-dependent Na+ efflux) and Na+-Na+ exchange (Na+o-dependent Na+ efflux) modes of the Na+-Ca2+ exchange was studied in axons dialyzed without EGTA. For these experiments a substantial inhibition of the Ca2+ buffer capacity of the axoplasm was achieved by the use of Ruthenium red (10-20 microM), cyanide (1 mM) and vanadate (1 mM) in the dialysis solution. Our results indicate that the Ca2+i requirement of the reverse and Na+-Na+ exchange can not be explained by a direct inhibition of the Na+-Ca2+ exchanger by EGTA. In fact, both modes of operation of the exchanger can be activated by internal Ca2+ ions in the complete absence of Ca2+ chelating agents thus indicating that the 'catalytic' effect of Ca2+i on the Na+-Ca2+ exchanger is a real phenomenon.

In squid axons intracellular Mg2+ is essential for ATP-dependent Na+ efflux in the absence and presence of strophanthidin.

The effect on Na+ efflux of removal of intracellular Mg2+ was studied in squid giant axons dialyzed without internal Ca2+. In the absence of Mg2i+, ATP was unable to stimulate any efflux of Na+ above the baseline of about 1 pmol . cm-2 . s-1. This behavior was observed in otherwise normal axons and in axons poisoned with 50 microM strophanthidin in the sea water. Reinstatement of 4 mM MgCl2 in excess to ATP in the dialysis solution brought about the usual response of Na+ efflux to ATP, external K+ and strophanthidin. The present experiments show that, regardless of the mechanism for the ATP-dependent Na+ efflux in strophanthidin-poisoned axons, this type of flux shares with the active Na+ extrusion the need for the simultaneous presence of intracellular ATP and Mg2+.

Partial purification and characterization of the (Ca2+ + Mg2+)-ATPase from squid optic nerve plasma membrane.

A membrane fraction enriched in axolemma was obtained from optic nerves of the squid (Sepiotheutis sepioidea) by differential centrifugation and density gradient fractionation. The preparation showed an oligomycin- and NaN3-insensitive (Ca2+ + Mg2+)-ATPase activity. The dependence of the ATPase activity on calcium concentration revealed the presence of two saturable components. One had a high affinity for calcium (K1 1/2 = 0.12 microM) and the second had a comparatively low affinity (K2 1/2 = 49.5 microM). Only the high-affinity component was specifically inhibited by vanadate (K1 = 35 microM). Calmodulin (12.5 micrograms/ml) stimulated the (Ca2+ + Mg2+)-ATPase by approx. 50%, and this stimulation was abolished by trifluoperazine (10 microM). Further treatment of the membrane fraction with 1% Nonidet P-40 resulted in a partial purification of the ATPase about 15-fold compared to the initial homogenate. This (Ca2+ + Mg2+)-ATPase from squid optic nerve displays some properties similar to those of the uncoupled Ca2+-pump described in internally dialyzed squid axons, suggesting that it could be its enzymatic basis.

Sidedness of the ATP-Na+-K+ interactions with the Na+ pump in squid axons.

Using dialysed squid axons we have been able to control internal and external ionic compositions under conditions in which most of the Na+ efflux goes through the Na+ pump. We found that (i) internal K+ had a strong inhibitory effect on Na+ efflux; this effect was antagonized by ATP, with low affinity, and by internal Na+, (ii) a reduction in ATP levels from 3 mM to 50 microM greatly increased the apparent affinity for external K+, but reduced its effectiveness compared with other monovalent cations, as an activator of Na+ efflux, and (iii) the relative effectiveness of different K+ congeners as external activator of the Na+ efflux, though affected by the ATP concentration, was not affected by the Na+/K+ ratio inside the cells. These results are consistent with the idea that the same conformation of the (Na+ + K+)-ATPase can be reached by interaction with external K+ after phosphorylation and with internal K+ before rephosphorylation. They also stress a nonphosphorylating regulatory role of ATP.

Measurements of intracellular ionized calcium in squid giant axons using calcium-selective electrodes.

Ca2+-selective electrodes have been used to measure free intracellular Ca2+ concentrations in squid giant axons. Electrodes made of glass cannulas of about 20 microns in diameter, plugged with a poly(vinyl chloride) gelled sensor were used to impale the axons axially. They showed a Nernstian response to Ca2+ down to about 3 microM in solutions containing 0.3 M K+ and 0.025 M Na+. Sub-Nernstian but useful responses were obtained up to pCa 8. The electrodes showed adequate selectivity to Ca2+ over Mg2+, H+, K+ and Na+. To calibrate them properly, a set of standard solutions were prepared using different Ca2+ buffers (EGTA, HEEDTA, nitrilotriacetic acid) after carefully characterizing their apparent Ca2+ association constants under conditions resembling the axoplasmic environment. In fresh axons incubated in artificial seawater containing 4 mM Ca2+, the mean resting intracellular ionized calcium concentration was 0.106 microM (n = 15). The Ca2+-electrodes were used to investigate effects of different experimental procedures on the [Ca2+]i. The main conclusions are: (i) intact axons can extrude calcium ions at low [Ca2+]i levels by a process independent of external Na+; (ii) poisoned axons can extrude calcium ions at high levels of [Ca2+]i by an external Na+-dependent process. The level of free intracellular Ca attained at these latter conditions is about an order to magnitude greater than the resting physiological value.