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R DiPolo

Publications and source records attributed to R DiPolo.

At least 55 records · Page 3Linked to original sources
PubMed1984

Direct measurement of intracellular free magnesium in frog skeletal muscle using magnesium-selective microelectrodes.

Mg2+-selective microelectrodes have been used to measure the intracellular free Mg2+ concentration in frog skeletal muscle fibers. Glass capillaries with a tip diameter of less than 0.4 micron were backfilled with the Mg2+ sensor, ETH 1117. In the absence of interfering ions, they gave Nernstian responses between 1 and 10 mM free Mg2+. In the presence of an ionic environment resembling the myoplasm, the microelectrode response was sub Nernstian (18-24 mV) but still useful. The electrodes were calibrated before and after muscle-fiber impalements . In quiescent fibers from sartorius muscle (Rana pipiens), with resting membrane potentials not less than -82 mV, the intracellular free Mg2+ concentration was 3.8 +/- 0.41 (S.E.) mM (n = 58) at 22 degrees C. No significant change in the intracellular free Mg2+ was observed following extensive (approx. 6 h) incubation in Mg2+-free media. Increasing the external concentration of magnesium from 4 to 20 mM (approx. 15 min) produced a slow and small enhancement (1.8 mM) of [Mg2+]i, which was fully reverted when the divalent cation was removed from the bathing solution. No change in ionic magnesium resting concentration was observed when the muscle fibers were treated either with caffeine 3 mM or with Na+-free solutions. In depolarized muscle fibers (-23 +/- 2.7 mV) treated with 100 mM K+, the myoplasmic [Mg2+] was 3.7 +/- 0.45 (S.E.) mM, n = 6, immediately after the spontaneous relaxation of the contracture. Similar determinations in muscle fibers during stimulation at low frequency (5 Hz), and after fatigue development, showed no changes in the concentration of free cytosolic Mg2+. These results point out that [Mg2+]i is not modified under these three different experimental conditions.

Animals↗
PubMed1984

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.

Animals↗
PubMed1984

Interactions of physiological ligands with the Ca pump and Na/Ca exchange in squid axons.

We have studied the interaction of physiological ligands other than Nai and Cai with the Ca pump and Na/Ca exchange in internally dialyzed squid axons. The results show the following. (a) Internal Mg2+ is an inhibitor of the Nao-dependent Ca efflux. At physiological Mg2+i (4 mM), the inhibition amounts to approximately 50%. The inhibition is partial and noncompetitive with Cai, and is not affected by Nai or ATP. The ATP-dependent uncoupled efflux is unaffected by Mgi up to 20 mM. Both components of the Ca efflux require Mg2+i for their activation by ATP. (b) At constant membrane potential, Ki is an important cofactor for the uncoupled Ca efflux. (c) Orthophosphate (Pi) activates the Nao-dependent Ca efflux without affecting the uncoupled component. Activation by Pi occurs only in the presence of Mg-ATP or hydrolyzable ATP analogues. Pi under physiological conditions has no effect on the uncoupled component; nevertheless, at alkaline pH, it inhibits the Ca pump, probably by product inhibition. (d) ADP is a potent inhibitor of the uncoupled Ca efflux. The Nao-dependent component is inhibited by ADP only at much higher ADP concentrations. These results indicate that (a) depending on the concentration of Ca2+i, Na+i Mg2+i, and Pi, the Na/Ca carrier can operate under a low- or high-rate regime; (b) the interactions of Mg2+i, Pi, Na+i, and ATP with the carrier are not interdependent; (c) the effect of Pi on the carrier-mediated Ca efflux resembles the stimulation of the Nao-dependent Ca efflux by internal vanadate; (d) the ligand effects on the uncoupled Ca efflux are of the type seen in the Ca pump in red cells and the sarcoplasmic reticulum.

Adenosine Diphosphate↗
PubMed1983

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.

Animals↗
PubMed1983

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+.

Adenosine Triphosphate↗
PubMed1983

Determination of ionic calcium in frog skeletal muscle fibers.

Ionic calcium concentrations were measured in frog skeletal muscle fibers using Ca-selective microelectrodes. In fibers with resting membrane potentials more negative than -85 mV, the mean pCa value was 6.94 (0.12 microM). In fibers depolarized to -73 mV with 10-mM K the mean pCa was 6.43 (0.37 microM). This increase in the intracellular [Ca2+] could be related to the higher oxygen consumption and heat production (Solandt effect) reported to occur under these conditions. Caffeine, 3 mM, also produced an increase in the free ionic calcium to a pCa of 6.52 (0.31 microM) without changes in the membrane potential. Lower caffeine concentrations, 1 and 2 mM, did not change the fiber pCa. Lower Ca concentrations in the external medium effectively reduced the internal ionic calcium to an estimated pCa of 7.43 (0.03 microM).

Animals↗
PubMed1983

Voltage-dependent calcium channel in the squid axon.

A voltage-dependent inward calcium current insensitive to tetrodotoxin has been measured in internally perfused or dialyzed squid giant axons. Sodium conductance was blocked by tetrodotoxin, and potassium conductance was irreversibly destroyed by the lack of potassium in the external and internal medium. Chloride conductance was eliminated by replacement of chloride with methylsulfonate. The calcium current was activated at about -40 mV and it peaked at about 0 mV. The peak inward current was 3 microA/cm2 when external Ca was 80 mM and 0.9 microA/cm2 in 8 mM Ca. In 80 mM Ca, the calcium current is turned on in less than 10 msec, and it does not decay appreciably for pulses up to 70 msec in duration. Barium can replace calcium, and cadmium blocks the calcium current.

Animals↗
PubMed1983

The calcium pump and sodium-calcium exchange in squid axons.

Calcium membrane extrusion in squid axons is mediated by two mechanisms able to regulate the Ca2+ with different degrees of efficiency. The ATP driven uncoupled Ca pump with its high affinity for Ca2+i will work primarily to balance the physiological Ca leak, hence controlling the [Ca2+]i at rest. The Na-Ca exchange system, although less important in the maintenance of the resting Ca2+i owing to its large capacity of transport (outward and inward), will certainly be important in physiological and unphysiological conditions in which relatively high [Ca2+]i concentrations are attained.

Animals↗
PubMed1982

Ca entry at rest and during prolonged depolarization in dialyzed squid axons.

Ca influx has been studied in squid axons under internal dialysis control. In axons dialyzed with "normal" physiological conditions (Nai = 40-50 mM, Cai2+ = 0.06-0.1 microM, ATP = 2 mM, Ki = 310 mM), 70% of the resting Ca influx is sensitive to external TTX (K0.5 congruent to 5 nM), 20% of it can be accounted by the reversal of the Na-Ca exchange, and the remaining fraction (10%) is insensitive to TTX, D-600, and Nai. The Ca antagonic drug D-600 (50-100 microM) has an inhibitory effect on the resting Ca influx. This compound was found to affect both the TTX sensitive and the Nai-dependent Ca influx components. In the presence of Nai and ATP, Cai2+ activates the carrier mediated Ca entry (Nai-dependent Ca influx). Most of the activation occurs in the submicromolar range of Cai2+ concentrations (K0.5 congruent to 0.6 microM). In the absence of Nai and/or ATP, no activation of Ca influx by Cai2+ was found up to about 5 microM Cai2+. Prolonged depolarization with high Ko causes an increase in Ca influx sustained for long time (minutes). Depolarizing the axons by removing Ki causes the same effect. This depolarization-induced Ca entry was only observed in axons containing Nai. In the absence of Nai, Ca influx decreases with increasing Ko. The activation of the carrier mediated Ca entry (electrogenic Na/Ca exchange) by membrane depolarization was found to be markedly dependent on the magnitude of Ca2+ i. Increasing the magnitude of Ca2+ i from 0.1 to 0.6 microM causes a ten fold increase in the extra Ca influx induced by a K-depolarization.

Animals↗
PubMed1981

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.

Adenosine Triphosphate↗
PubMed1981

A (Ca2+, Mg2+)-ATPase activity in plasma membrane fragments isolated from squid nerves.

A (Ca2+, Mg2+)-ATPase activity and a (Ca2+, Mg2+)-dependent phosphorylation from ATP have been found in plasma membrane fragments from squid optical nerves under conditions where contamination by intracellular organelles is unlikely. The properties of this (Ca2+, Mg2+)-ATPase activity are almost identical to those of the ATP-dependent uncoupled Ca2+ efflux observed in dialyzed squid giant axons. This gives further support to the notion that the mechanism responsible for maintaining the low levels of ionized Ca concentration in nerves at rest is not a Na+-Ca2+ exchange system but an ATP-driven uncoupled Ca2+ pump.

Adenosine Triphosphatases↗
PubMed1981

An ATP-dependent sodium-sodium exchange in strophanthidin poisoned dialysed squid giant axons.

1. Dialysed giant axons from the squid have been used to study some of the properties of the Na+ fluxes when the Na+ pump is fully inhibited by strophanthidin. 2. In axons which had been depleted of ATP, strophanthidin had no effect on Na+ efflux. Similar negative results were obtained in axons dialysed with and without internal or external K+, and with or without 100 microM-internal Ca2+. 3. In the presence of 60 mM-internal Na+, 440 mM-external Na+ and strophanthidin, the fluxes of Na+ had the following characteristics. (i) ATP stimulated an efflux and an influx of Na+ of similar magnitude. The K1/2 for ATP, measured from its effect on Na+ efflux, was about 200 microM. (ii) The non-hydrolysable ATP analogue adenylyl(beta, gamma-methylene)-diphosphonate (AMP-PCP), at 2 mM concentration, either alone or in combination with 2 mM-internal phosphate, failed to stimulate any efflux of Na+. (iii) The ATP-dependent Na+ efflux was not affected by removal of internal or external K+, or external Mg2+ or Ca2+, and was not dependent on internal Ca2+. (iv) within the resolution of the method, all the ATP-dependent Na+ influx required internal Na+, and all the ATP-dependent Na+ efflux required external Na+. From the magnitude of the unidirectional Na+ fluxes the stoichiometry seemed to be a 1 to 1 Na+--Na+ exchange. 4. The ATP-internal Na+-dependent influx of Na+ in the presence of strophanthidin was not affected by 1 mM-vandate in the dialysis solution, a concentration which fully inhibits the Na+ efflux through the Na+ pump that is activated by external K+. 5. In the presence of external Na+, the external K+ sites of the Na+ pump are completely saturated with 100 mM-external K+. In unpoisoned axons incubated with 100 mM-external K+, replacement of external Na+ with Tris+ produced no change in the efflux of Na+. However, in axons poisoned with 50 microM-strophanthidin, replacement of external Na+ with Tris+ resulted in a reversible inhibition of Na+ efflux. This could suggest that strophanthidin poisoning might induce Na+ (cations?) fluxes which are not present in normal conditions.

Adenosine Triphosphate↗
PubMed1980

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.

Adenosine Triphosphate↗
PubMed1979

Vanadate inhibits uncoupled Ca efflux but not Na--Ca exchange in squid axons.

Nerve cells can maintain a very low intracellular calcium concentration ([Ca2+]i) against large Ca2+ electrochemical gradients (see ref. 1 for review). The properties of the calcium efflux from these cells depend on [Ca2+]i (ref. 2), and within the physiological range, most Ca efflux depends on ATP (which stimulates with high affinity) and is insensitive to Na1, Na0 and Ca0 (uncoupled Ca efflux). When the [Ca2+]i is well above the physiological range, Ca efflux becomes only partially dependent on ATP (acting now with low affinity), is inhibited by Nai and is stimulated by Na0 and Ca0 (Na--Ca exchange). Orthovanadate, a powerful inhibitor of the (Na+ + K+)ATPase and the Na pump, also inhibits the Ca-stimulated ATPase activity, which is the enzymatic basis for the uncoupled Ca pump, in human red cells. The experiments reported here show that in squid axons the ATP-dependent uncoupled Ca efflux can be fully and reversibly inhibited by vanadate, whereas concentrations of vanadate 10 times higher have no effect on the Na--Ca exchange. This is another indication that the uncoupled Ca efflux represents an ATP-driven Ca pump, and supports the suggestion that the uncoupled Ca efflux and Na--Ca exchange are mediated by different mechanisms.

Adenosine Triphosphatases↗
PubMed1979

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.

Adenosine Triphosphate↗
PubMed1979

Calcium influx in internally dialyzed squid giant axons.

A method has been developed to measure Ca influx in internally dialyzed squid axons. This was achieved by controlling the dialyzed segment of the axon exposed to the external radioactive medium. The capacity of EGTA to buffer all the Ca entering the fiber was explored by changing the free EGTA at constant [Ca++]i. At a free [EGTA]i greater than 200 microM, the measured resting Ca influx and the expected increment in Ca entry during electrical stimulation were independent of the axoplasmic free [EGTA]. To avoid Ca uptake by the mitochondrial system, cyanide, oligomycin, and FCCP were included in the perfusate. Axons dialyzed with a standard medium containing: [ATP] = 2 mM, [Ca++]i = 0.06 microM, [Ca++]o = 10 mM, [Na+]i = 70 mM, and [Na+]o = 465 mM, gave a mean Ca influx of 0.14 +/- 0.012 pmol.cm-2.s-1 (n = 12. Removal of ATP drops the Ca influx to 0.085 +/- 0.007 pmol.cm-2.s-1 (n = 12). Ca influx increased to 0.35 pmol.cm-2,s-1 when Nao was removed. The increment was completely abolished by removing Nai+ and (or) ATP from the dialysis medium. At nominal zero [Ca++]i, no Nai-dependent Ca influx was observed. In the presence of ATP and Nai [Ca++]i activates the Ca influx along a sigmoid curve without saturation up to 1 microM [Ca++]i. Removal of Nai+ always reduced the Ca influx to a value similar to that observed in the absence of [Ca++]i (0.087 +/- 0.008 pmol.cm-2.s-1; n = 11). Under the above standard conditions, 50-60% of the total Ca influx was found to be insensitive to Nai+, Cai++, and ATP, sensitive to membrane potential, and partially inhibited by external Co++.

Adenosine Triphosphate↗