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R Dipolo

Publications and source records attributed to R Dipolo.

15 recordsLinked to original sources
PubMed2004

A genistein-sensitive Na+/Ca2+ exchange is responsible for the resting [Ca2+]i and most of the Ca2+ plasma membrane fluxes in stimulated rat cerebellar type 1 astrocytes.

The differential role of Na+/Ca2+ exchange in the regulation of intracellular ionized calcium ([Ca2+]i) in immunological and pharmacologically identified type 1 astrocytes and Purkinje cells was studied in rat cerebellar culture, using Ca2+ (Fluo-3, Fura-2) and Na+ (SBFI) fluorescence measurements. The mean resting [Ca2+]i was significantly higher (191 +/- 8 nM, n=25) in type 1 astrocytes than in Purkinje cells (92 +/- 2.5 nM, n=35). In contrast to Purkinje cells, in unstimulated cerebellar type 1 astrocytes, forward and reverse Na+/Ca2+ modes operate under resting physiological conditions, being responsible for most of the total Ca2+ transplasma membrane fluxes. Four observations support this hypothesis: (1) under resting conditions of temperature and ionic composition, Na+o removal causes a remarkable increase in [Ca2+]i, being inhibited by 2',4' dichlorobenzamil (DCB), and 2-[2-[4-(nitrobenzilloxiphenyl ethyl] isothiourea metanesulfonate (KB-R7943); (2) Ca2+o removal in the presence of Na+o causes an important drop in [Ca2+]i, which is absent in Li+o or NMG+o (N-methyl-D-glucamine) containing medium; (3) the reverse mode exchange inhibitor KB-R7943 mimics the removal of Ca2+o only in the presence of Na+o; and (4) under loaded [Na+]i conditions (ouabain or the activation of taurine-Na+-cotransport), reverse mode exchange increases in both astrocytes and Purkinje cells. In type 1 astrocytes stimulated with endothelin-3 (ET-3), the recovery of the Ca2+i signal occurs largely through the Na+/Ca2+ exchanger. Genistein, a tyrosine kinase inhibitor, completely and reversibly blocks all exchange activity, but not its inactive analogue daidzein, thus suggesting that the Na+/Ca2+ exchanger of cerebellar type 1 astrocytes may be modulated by phosphorylation. Our main conclusion is that in rat cerebellar type 1 astrocytes under resting physiological conditions, most of the total transplasma membrane Ca2+ fluxes take place through the Na+/Ca2+ exchanger, thus accounting for the resting [Ca(2+)]i.

Animals↗
PubMed1989

Measurement of Na-K pump current in acinar cells of rat lacrimal glands.

Isolated cells from rat lacrimal glands were voltage clamped using the tight-seal whole-cell recording technique. The intracellular solution contained ATP and an elevated Na concentration (70 mM). Removing external K ions elicited an inward current shift. Ouabain (0.5 mM) induced an inward current shift of identical amplitude, but with slower kinetics. In the presence of ouabain, removal of K ions did not alter the cell current. The potassium- and ouabain-sensitive current was outward between -120 and +20 mV, and its amplitude decreased below -60 mV. This current was highly sensitive to temperature, and was not affected by blockers of the K channels which are present in these cells. It was attributed to an inhibition of the Na-K pump. The Na-K pump current was estimated to be 15 pA for an average acinar cell at physiological temperature, with 70 mM internal Na ions and 20 mM external K ions. Implications of this value in terms of electrolyte secretion are discussed.

Animals↗
PubMed1989

Modulation of K channels in dialyzed squid axons. ATP-mediated phosphorylation.

In squid axons, internally applied ATP potentiates the magnitude of the potassium conductance and slows down its activation kinetics. This effect was characterized using internally dialyzed axons under voltage-clamp conditions. Both amplitude potentiation and kinetic slow-down effects are very selective towards ATP, other nucleotides like GTP and ITP are ineffective in millimolar concentrations. The current potentiation Km for ATP is near 10 microM with no further effects for concentrations greater than 100 microM. ATP effect is most likely produced via a phosphorylative reaction because Mg ion is an obligatory requirement and nonhydrolyzable ATP analogues are without effect. In the presence of ATP, the K current presents more delay, resembling a Cole-Moore effect due to local hyperpolarization of the channel. ATP effect induces a 10-20 mV shift in both activation and inactivation parameters towards more depolarized potentials. As a consequence of this shift, conductance-voltage curves with and without ATP cross at approximately -40 mV. This result is consistent with the hyperpolarization observed with ATP depletion, which is reversed by ATP addition. At potentials around the resting value, addition of ATP removes almost completely K current slow inactivation. It is suggested that a change in the amount of the slow inactivation is responsible for the differences in current amplitude with and without ATP, possibly as a consequence of the additional negative charge carried by the phosphate group. However, a modification of the local potential is not enough to explain completely the differences under the two conditions.

Adenosine Triphosphate↗
PubMed1985

Voltage dependence of the Na/Ca exchange in voltage-clamped, dialyzed squid axons. Na-dependent Ca efflux.

A combination of the voltage-clamp and the intracellular dialysis techniques has been used to study the membrane potential dependence of the Nao-dependent Ca efflux in squid giant axons. In order to improve axon survival, experiments were carried out using internal solutions prepared with large impermeant organic anions and cations, which did not affect the operation of the Na/Ca exchange mechanism. In axons dialyzed with solutions prepared without internal Na, the Nao-dependent Ca efflux had a small sensitivity to membrane potential changes. For a 25-mV membrane displacement in the hyperpolarizing direction, the basal Ca efflux increased by only 7.4% (n = 13). When the dialysis medium contained Na (from 20 to 55 mM), the efflux increased 32.3% (n = 25) for the same membrane potential change. The K1/2 for this effect is approximately 5 mM Na, and saturation appears to occur at a Na concentration above 20 mM. Adding ATP to the dialysis medium increased the magnitude of the Nao-dependent Ca efflux without changing its voltage sensitivity. Wide changes in the intracellular ionized Ca concentration (from 0.1 to 230 microM) did not modify the voltage sensitivity of the exchange system. Elimination of the reversal of Na/Ca exchange (Nai-dependent Ca influx) by removing Cao did not modify the voltage sensitivity of the Nao-dependent Ca efflux. When the axon membrane potential was submitted to prolonged changes, the corresponding changes in the Ca efflux were not sustained, but declined exponentially to intermediate values. This effect may indicate a slow inactivation process in the Na/Ca exchange mechanism. Voltage-clamp pulse experiments revealed: (a) the absence of a fast inactivation process in the Na/Ca exchange, and (b) that the activation of the carrier for hyperpolarizing pulses occurs as rapidly as 1 ms.

Adenosine Triphosphate↗
PubMed1982

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.

Adenosine Triphosphate↗
PubMed1979

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+.

Ammonia↗
PubMed1978

Contractile activation phenomena in voltage-clamped barnacle muscle fiber.

Tension development in voltage-clamped barnacle muscle fibers occurs with depolarizing pulses so small as not to activate the potassium and calcium conductance systems. Peak tension and the tension time integral appear to be graded by both amplitude and duration of the depolarizing pulses. Subthreshold depolarizing conditioning pulses shorter than 500 ms potentiate the response to a given test pulse. This effect diminishes and reverts when the duration of the conditioning pulse is increasingly prolonged. The relationship between fiber membrane potential and tension developed in response to depolarizing pulses is described by an S-shaped curve. The tension saturates at a membrane potential of about +10 mV (inside positive). For a given pulse duration the saturation value remains constant even when the fiber interior reaches a value of +230 mV, which is well above what may be estimated to be the equilibrium potential of calcium ions (Eca = +120). In the presence of 5 mM external procaine, the shape of the tension-potential curve changes; the maximum value tension besides being diminished is not sustained by falls when the potential approaches the estimated value for Eca. These results suggest that under physiological conditions the contractile activator is probably released from an internal store, and that the calcium entering the fiber as inward current does not play a direct major role in contractile activation.

Action Potentials↗
PubMed1976

Ionized calcium concentrations in squid axons.

Values for ionized [Ca] in squid axons were obtained by measuring the light emission from a 0.1-mul drop of aequorin confined to a plastic dialysis tube of 140-mum diameter located axially. Ionized Ca had a mean value of 20 x 10(-9) M as judged by the subsequent introduction of CaEGTA/EGTA buffer (ratio ca. 0.1) into the axoplasm, and light measurement on a second aequorin drop. Ionized Ca in axoplasma was also measured by introducing arsenazo dye into an axon by injection and measuring the Ca complex of such a dye by multichannel spectrophotometry. Values so obtained were ca. 50 x 10(-9) M as calibrated against CaEGTA/EGTA buffer mixtures. Wth a freshly isolated axon in 10 mM Ca seawater, the aequorin glow invariably increased with time; a seawater [Ca] of 2-3 mM allowed a steady state with respect to [Ca]. Replacement of Na+ in seawater with choline led to a large increase in light emission from aequorin. Li seawater partially reversed this change and the reintroduction of Na+ brought light levels back to their initial value. Stimulation at 60/s for 2-5 min produced an increase in aequorin glow about 0.1% of that represented by the known Ca influx, suggesting operationally the presence of substantial Ca buffering. Treatment of an axon with CN produced a very large increase in aequorin glow and in Ca arsenazo formation only if the external seawater contained Ca.

Aequorin↗
PubMed1976

The influence of nucleotides on calcium fluxes.

The effect of ATP and other nucleotides on calcium efflux was studied in squid axons dialyzed with Ca:ethylene glycol tetraacetic acid buffers to control the internal ionized calcium concentration. In the virtual absence of internal ATP (ca. 1 muM) a significant level of calcium efflux occurs which could be increased by the addition of internal ATP. At low concentrations of ionized calcium (ca. 200 nM), efflux increased 10-fold. At high levels of ionized calcium (ca. 100 muM), the increase was only twofold. This stimulation of efflux by ATP requires internal sodium. Conversely, ATP renders the calcium efflux insensitive to internal sodium and prevents the inhibition of calcium efflux produced by internal sodium in the absence of ATP. Of 12 nucleotides tested, only ATP, deoxy-ATP and alpha, beta-methylene ATP significantly stimulated calcium efflux. The data are interpreted as indicating that ATP induces an affinity in change in the carrier system binding calcium to the internal site, possibly by a phosphorylating step.

Adenosine Diphosphate↗
PubMed1974

Effect of ATP on the calcium efflux in dialyzed squid giant axons.

Dialysis perfusion technique makes it possible to control the internal composition of squid giant axons. Calcium efflux has been studied in the presence and in the virtual absence (<5 microM) of ATP. The mean calcium efflux from axons dialyzed with 0.3 microM ionized calcium, [ATP](i) > 1,000 microM, and bathed in artificial seawater (ASW) was 0.24 +/- 0.02 pmol.cm(-2).s(-1) (P/CS) (n = 8) at 22 degrees C. With [ATP](i) < 5 microM the mean efflux was 0.11 +/- 0.01 P/CS (n = 15). The curve relating calcium efflux to [ATP](i) shows a constant residual calcium efflux in the range of 1-100 microM [ATP](i). An increase of the calcium efflux is observed when [ATP](i) is >100 microM and saturates at [ATP](i) > 1,000 microM. The magnitude of the ATP-dependent fraction of the calcium efflux varies with external concentrations of Na(+), Ca(++), and Mg(++). These results suggest that internal ATP changes the affinity of the calcium transport system for external cations.

Adenosine Triphosphate↗
PubMed1973

Calcium efflux from internally dialyzed squid giant axons.

Calcium efflux has been studied in squid giant axons under conditions in which the internal composition was controlled by means of a dialysis perfusion technique. The mean calcium efflux from axons dialyzed with 0.3 microM calcium and 5 mM ATP was 0.26 pmol/cm(2).s at 22 degrees C. The curve relating the Ca efflux with the internal Ca concentration had a slope of about one for [Ca](i) lower than 0.3microM and a slope smaller than one for higher concentrations. Under the above conditions replacement of [Na](o) and [Ca](o) by Tris and Mg causes an 80% fall in the calcium efflux. When the axons were dialyzed with a medium free of ATP and containing 2 mM cyanide plus 5microg/ml oligomycin, analysis of the perfusion effluent gave values of 1-4 microM ATP. Under this low ATP condition, replacement of external sodium and calcium causes the same drop in the calcium efflux. The same effect was observed at higher [Ca](i), (80 microM). These results suggest that the Na-Ca exchange component of the calcium efflux is apparently not dependent on the amounts of ATP in the axoplasm. Axons previously depleted of ATP show a significant transient drop in the calcium efflux when ATP is added to the dialysis medium. This effect probably represents the sequestering of calcium by the mitochondrial system. The consumption of calcium by the mitochondria of the axoplasm in dialyzed axons was determined to be of the order of 6.0 x 10(-7) mol Ca(++)/mg of protein with an initial rate of 2.6 x 10(-8) mol Ca(++)/min.mg of protein. Axons dialyzed with 2 mM cyanide after 8-10-min delays show a rise in the calcium efflux in the presence of "normal" amounts of exogenous ATP. This effect seems to indicate that cyanide, per se, can release calcium ions from internal sources.

Adenosine Triphosphate↗
PubMed1973

Ionic diffusion delays in the transverse tubules of frog twitch muscle fibres.

1. The possibility of retarding the diffusional processes in the transverse tubules of frog single muscle fibres was explored by increasing the viscosity of the bathing medium. The viscosity was increased by adding Dextran 15 to the experimental solutions.2. The criterion used to test this possibility was the speed of repolarization of muscle fibre membranes produced by sudden reduction in [K](o) in media of different viscosity.3. In fibres equilibrated in media containing 15% Dextran 15, and that had been depolarized with 80 mM-K for more than 5 sec, the repolarization caused by lowering the [K](o) to 40 mM proceeded more slowly than in fibres in Dextran-free media.4. On the basis of the above results, it was deduced that high viscosity solutions would delay the diffusion of Na ions in the transverse tubules. Thus the effect of sudden changes in [Na](o) on the twitch tension of single fibres could be studied.5. The recovery of twitches when the fibres were exposed from a solution containing no Na, to one that contained 46 mM-Na, occurred more slowly in high viscosity media.6. This result can be explained assuming that a Na regenerative process occurs at the level of the membrane of the transverse tubules.

Action Potentials↗
PubMed1972

Effect of temperature on membrane potential and ionic fluxes in intact and dialysed barnacle muscle fibres.

1. The temperature-dependent component of the resting potential in intact, cannulated and dialysed fibres from the muscle of the barnacle Balanus nubilus was studied under a variety of different experimental conditions. A decrease in temperature from 22 to 12 degrees C produced a mean depolarization of 10 mV.2. Neither addition of strophanthidin, nor replacement of external sodium by lithium affect the voltage shift induced by temperature. However, the magnitude of the voltage shift depends on the external chloride and potassium concentration.3. The dialysis technique was applied to measure the potassium, chloride and sodium fluxes as a function of temperature. The Q(10) for the passive fluxes of these ions was 1.9, 1.7, and 1.4 respectively.4. The temperature-dependent changes in the passive ionic fluxes combined with the inability of inhibitors of the sodium pump to alter the temperature dependence of the resting potential suggest that the change induced by temperature on the resting potential is primarily caused by a change in the passive permeability ratios, and is not related to active ion transport.

Animals↗
PubMed1968

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.

Adenosine Triphosphatases↗