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ABSTRACT: A topic emerging roughly 30 years ago and engendering an incompletely resolved controversy is reviewed in this article: the relatively high permeability and pH independence associated with H(+)/OH(-) passive movements across lipid membranes. We summarize the expected characteristics of simple H(+)/OH(-) diffusion and those of a reaction between H(+) and OH(-) being attracted from opposite surfaces and condensing in an interfacial zone of the membrane. An interfacial H(+)/OH(-) reaction mechanism gives the experimentally observed behavior of an H(+)/OH(-) flux that is independent of the pH measurement range. This mechanism assumes that H(+) and OH(-) within the interfacial zone become electrostatically aligned on opposite sides of the hydrophobic membrane core. Electrostatic attraction and charge delocalization among a small cluster of water molecules surrounding the ions reduce the Born energy for H(+)/OH(-) insertion into lipid. This transmembrane condensation model predicts the magnitude of the experimentally determined H(+)/OH(-) flux, which is significantly greater than that of other monovalent ions. The consequences of an elevated H(+)/OH(-) permeability compared to other ions and the relative pH independence of this flux have consequences for understanding the chemical evolution of life.
Journal of Membrane Biology 09/2010; 237(1):21-30. · 1.81 Impact Factor
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ABSTRACT: ATP-dependent (45)Ca uptake in rat brain microsomes was measured in intracellular-like media containing different concentrations of PO(4) and oxalate. In the absence of divalent anions, there was a transient (45)Ca accumulation, lasting only a few minutes. Addition of PO(4) did not change the initial accumulation but added a second stage that increased with PO(4) concentration. Accumulation during the second stage was inhibited by the following anion transport inhibitors: niflumic acid (50 microM), 4,4'-dinitrostilbene-2, 2'-disulfonic acid (DNDS; 250 microM), and DIDS (3-5 microM); accumulation during the initial stage was unaffected. Higher concentrations of DIDS (100 microM), however, inhibited the initial stage as well. Uptake was unaffected by 20 mM Na, an activator, or 1 mM arsenate, an inhibitor of Na-PO(4) cotransport. An oxalate-supported (45)Ca uptake was larger, less sensitive to DIDS, and enhanced by the catalytic subunit of protein kinase A (40 U/ml). Combinations of PO(4) and oxalate had activating and inhibitory effects that could be explained by PO(4) inhibition of an oxalate-dependent pathway, but not vice versa. These results support the existence of separate transport pathways for oxalate and PO(4) in brain endoplasmic reticulum.
AJP Cell Physiology 07/2000; 278(6):C1183-90. · 3.54 Impact Factor
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ABSTRACT: Thapsigargin is a specific and potent inhibitor of sarco/endoplasmic reticulum Ca2+-ATPases. However, in whole rat brain microsomes, 1 microM thapsigargin had no significant effect on the 10-min time course of ATP-dependent Ca2+ uptake in the absence of the luminal Ca2+ chelator oxalate. In contrast, 50 mM oxalate resolved a thapsigargin-sensitive Ca2+ uptake rate (IC50 approximately 1 nM thapsigargin) five times that of a thapsigargin-insensitive rate. This remaining approximately 20% of the total ATP-dependent accumulation was insensitive to thapsigargin (up to 10 microM), slightly less sensitive to vanadate inhibition, and unresponsive to 5 microM inositol 1,4,5-trisphosphate or 10 mM caffeine. Measuring both 12-min Ca2+ uptake and initial Ca2+ uptake rates, the apparent thapsigargin sensitivity increased as oxalate concentrations increased from 10 to 50 mM, corresponding to a range of luminal free Ca2+ concentrations of approximately 300 down to 60 nM. Addition of oxalate during steady-state 45Ca accumulation rapidly resolved the aforementioned thapsigargin sensitivity. These results strongly suggest that luminal Ca2+ may protect a large portion of neuronal endoplasmic reticulum Ca2+ pumps against thapsigargin inhibition. Although high [Ca2+] has been previously shown to protect against thapsigargin inhibition in several reticular membrane preparations, our results suggest that luminal Ca2+ alone is responsible for mediating this effect in neurons.
Journal of Biological Chemistry 03/1998; 273(9):5020-5. · 4.77 Impact Factor
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ABSTRACT: Effects of increasing intraluminal Ca ([Ca]i) on the kinetics of rat brain microsomal uptake and efflux are reported here. Isolated rat brain microsomes accumulated 45Ca in an extravesicular free Ca ([Ca]o)- and ATP-dependent manner. Increased microsomal Ca load resulted in a decreased initial rate of 45Ca uptake and an increased tau, time to reach 63% of steady-state accumulation. Isolated rate brain microsomes lost 45Ca in a temperature- and [Ca]i-dependent manner. Whether preloaded with tracer 45Ca and either < or = 0.5 or 25 microM [Ca]o, the time constant of efflux was larger at 4 degrees C as compared with 37 degrees C. Additionally, increased microsomal Ca load resulted in a decreased time constant of 45Ca efflux. This shorter efflux time constant cannot explain the effect of [Ca]i on tau during uptake which was in fact longer for preloaded microsomes. Rather, these data suggest that, as Ca accumulates into unloaded microsomes, a steadily increasing [Ca]i slows unidirectional Ca influx (presumably by inhibiting the endoplasmic reticulum Ca pump) and enhances unidirectional Ca efflux, and that these combined effects ultimately shorten the time needed to achieve steady-state luminal [Ca]i.
The American journal of physiology 11/1996; 271(5 Pt 1):C1472-9.
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ABSTRACT: The study of intracellular Ca2+ regulation usually requires using calcium chelators to adjust [Ca2+]. We examined the effects of these chelators on calcium accumulation in microsomes and saponin-permeabilized synaptosomes to assess their influence on apparent transport properties. At a fixed free Ca2+ of 0.6 microM, increasing ethylene glycol-bis(beta-aminoethyl ether)-N,N,N', N'-tetraacetic acid (EGTA) and total Ca2+ enhanced ATP-dependent 45Ca sequestration in synaptosomes and microsomes. The EGTA-Ca complex did not change the maximal initial calcium uptake rate or maximal steady-state accumulation. Rather, EGTA/Ca increased the apparent affinity of the microsomal transporter for Ca2+. The presence of the organic anion transport inhibitor probenicid (2.5 mM) had no effect on 45Ca accumulation in the presence of EGTA. Replacing part of the Ca2+ with Ni2+ but maintaining [Ca2+] approximately constant reduced 45Ca uptake, suggesting that the Ni-EGTA complex did not stimulate 45Ca transport. Our results imply that EGTA is not actively transported across the endoplasmic reticulum membrane, nor does the divalent ion-bound form of EGTA change the properties of the transporter. EGTA, and other mobile calcium chelators with similar structures, e.g., 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, indo 1, and fluo 3, may increase calcium uptake by delivering more Ca2+ to its transport site.
The American journal of physiology 03/1996; 270(2 Pt 1):C628-35.
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ABSTRACT: Calcium diffusion coefficients were measured in Myxicola axoplasm and in agar controls by two independent techniques: one utilizing 45Ca, and one utilizing Ca-specific mini-electrodes. The lowest value, approximately 0.1 x 10(-6) cm2.s-1, was measured, using the mini-electrode technique, in axoplasm with intact Ca-sequestering organelles. With ATP-depleted axoplasm, diffusion coefficients of 0.5-2 x 10(-6) cm2.s-1 were obtained by both isotope and mini-electrode techniques. In organelle-free axoplasm with a protein concentration roughly half that in the intact axoplasm, diffusion coefficients of 1.4-3 x 10(-6) cm2.s-1 were measured at 0.7 microM Ca and 7 x 10(-6) cm2.s-1 at 3-5 microM Ca. When compared with measurements of the calcium buffering capacity [Al-Baldawi NF. Abercrombie RF. (1995) Cytoplasmic Ca buffer capacity determined with Nitr-5 and DM-nitrophen. Cell Calcium, 17, 409-422], these diffusion coefficients require that part of the buffer capacity be located on mobile Ca-binding sites.
Cell Calcium 07/1995; 17(6):422-30. · 3.77 Impact Factor
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ABSTRACT: We have examined intracellular calcium buffer capacity of cytoplasm from the giant axon of the marine invertebrate Myxicola infundibulum by photolytically releasing calcium from 'caged' compounds, while monitoring free calcium, [Ca2+], with Ca-sensing electrodes. In cytoplasm containing intact organelles, two features of the [Ca2+] response were seen upon light exposure: an initial spike from basal [Ca2+], followed by a slower phase recovery. Both the amplitude of the spike in [Ca2+] and the recovery were reduced by removal of MgATP. If organelles were removed from the cytoplasm, light exposure caused only a step-like change in [Ca2+] with no recovery. Apparent buffer capacities (delta bound Ca/delta free Ca) were unaffected by changing pH from 7.0 to 7.5; however, raising basal free calcium above 3 microM significantly reduced this parameter. The buffer capacity measured after the initial spike varied by as much as an order of magnitude from one giant axon to another but averaged approximately 50 in the absence and approximately 100 in the presence of 1 mM MgATP for [Ca2+] below 3 microM.
Cell Calcium 07/1995; 17(6):409-21. · 3.77 Impact Factor
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ABSTRACT: 1. 45Ca2+ accumulation into inulin-inaccessible compartments within cytoplasm from the giant axon of Myxicola infundibulum was measured as a function of free calcium, pH, and time. Accumulation reached a maximum after 1 h and remained stable for at least 3 h. 2. At 0.5, 5, and 50 microM [Ca2+], in the presence of 1 mM ATP or 5 mM succinate, steady-state calcium uptake had a bell-shaped dependence on pH with a maximum near pH 7. Uptake was abolished by the proton uncoupling reagent carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP, 4 micrograms ml-1). 3. Uptake of the membrane permeant cation, [14C]-tetraphenylphosphonium (TPP+), also had a bell-shaped dependence on pH with a maximum pH approximately 7, suggesting a pH dependence of the electrical potential of a membrane enclosed cytoplasmic compartment. Cyanide (2 mM) inhibited TPP+ uptake. 4. Inositol 1,4,5-trisphosphate (IP3, 10 microM), reduced steady-state calcium accumulation by 20-22% at 0.5 microM free calcium, pH 7 (P < 0.01, n = 16) and at 5 microM free calcium, pH 8 (P < 0.0005, n = 35). No effects of IP3 were found at other pH or calcium concentrations. 5. Neither guanosine 5'-triphosphate (GTP) nor inositol 1,3,4,5-tetrakisphosphate (IP4) had an effect on calcium uptake (5 microM [Ca2+], pH 8). 6. At 0.5 microM free calcium; vanadate (10 microM) inhibited 20-30%, of the 45Ca2+ accumulation, thapsigargin (33 nM) inhibited 20-30%, and cyanide (2 mM) plus oligomycin B (2 micrograms ml-1), or valinomycin (1 microM), inhibited 70-80%. The fraction of uptake sensitive to thapsigargin fell as the free calcium increased; however, the sensitivity of uptake to cyanide plus oligomycin B was approximately 80% for 0.5, 5.0, and 50 microM [Ca2+]. 7. Thapsigargin had no additional inhibiting effect in the presence of cyanide plus oligomycin B. IP3 had no effect in the presence of cyanide plus oligomycin B or other mitochondrial inhibitors. 8. Results suggest the presence of both mitochondrial (70-80%) and non-mitochondrial (20-30%) calcium pools in this system (at 0.5-5.0 microM Ca2+). The apparent non-mitochondrial uptake (sensitive to thapsigargin, or IP3) is not detectable in the presence of mitochondrial inhibitors. We interpret these results as evidence of functional communication between mitochondrial and non-mitochondrial calcium stores.
The Journal of Physiology 02/1993; 461:633-46. · 4.72 Impact Factor
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ABSTRACT: The apparent cytoplasmic proton diffusion coefficient was measured using pH electrodes and samples of cytoplasm extracted from the giant neuron of a marine invertebrate. By suddenly changing the pH at one surface of the sample and recording the relaxation of pH within the sample, an apparent diffusion coefficient of 1.4 +/- 0.5 x 10(-6) cm2/s (N = 7) was measured in the acidic or neutral range of pH (6.0-7.2). This value is approximately 5x lower than the diffusion coefficient of the mobile pH buffers (approximately 8 x 10(-6) cm2/s) and approximately 68x lower than the diffusion coefficient of the hydronium ion (93 x 10(-6) cm2/s). A mobile pH buffer (approximately 15% of the buffering power) and an immobile buffer (approximately 85% of the buffering power) could quantitatively account for the results at acidic or neutral pH. At alkaline pH (8.2-8.6), the apparent proton diffusion coefficient increased to 4.1 +/- 0.8 x 10(-6) cm2/s (N = 7). This larger diffusion coefficient at alkaline pH could be explained quantitatively by the enhanced buffering power of the mobile amino acids. Under the conditions of these experiments, it is unlikely that hydroxide movement influences the apparent hydrogen ion diffusion coefficient.
Biophysical Journal 07/1992; 61(6):1470-9. · 3.65 Impact Factor
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ABSTRACT: The 45Ca2+ binding properties of axoplasmic protein from the Myxicola giant axon have been investigated using a centrifugal/concentration-dialysis technique. Scatchard plot analysis of these binding data suggest that Ca2+ is attached to a site with an equilibrium dissociation constant of 7.7 +/- 0.5 microM and a capacity of 4.4 +/- 0.2 mumol/g axoplasmic protein (n = 11). Addition of other cations--Cd2+, Mn2+, Al3+, Cu2+, Ba2+, and Zn2(+)--at concentrations up to 10 microM did not displace 0.2 microM 45Ca2+ from its binding site, probably because of buffering of these cations by amino acid residues within the protein solutions. The protein could be stored at 4 degrees C for up to 16 days with no appreciable change in the number of calcium sites. Ca2+ binding equilibrium took place in less than 30 min of incubation. Increasing the incubation temperature from 4 degrees C to 37 degree C reduced the number of Ca2+ sites. The binding capacity was reduced by one-half when the protein was dialyzed with 4 M urea or high ionic strength KCl (2 M). Calcium binding was examined as a function of pH. When the protein was dialyzed overnight at different pH values and all the binding was done at pH 7.0, the apparent number of Ca2+ sites decreased as the pH of the dialysis medium was increased. When the protein was dialyzed overnight at pH 7.0 and the binding was done at different pH values, the apparent binding capacity increased as pH increased.(ABSTRACT TRUNCATED AT 250 WORDS)
Cell Calcium 09/1990; 11(7):459-67. · 3.77 Impact Factor
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ABSTRACT: Titrimetric, 45Ca dialysis, and autoradiographic methods were used to examine how axoplasmic proteins from the giant neuron of the marine annelid Myxicola infundibulum bind calcium. Following the autoradiographic method of Maruyama et al., the 150-160 kD neurofilament subunits were identified as prominent intracellular Ca-binding peptides. Using equilibrium dialysis, extracts of axoplasmic proteins (greater than 50% neurofilament subunits) were examined in 300 mM KCl at different concentrations of free Ca and Mg, and at different pH. Axoplasmic proteins showed a high affinity Ca binding site (K1/2 3-6 microM, capacity 3-7 mumole g-1 protein) at pH 6.8 or pH 7.5. Changing the Mg concentration from 0 to 5 mM had no effect on the Ca binding. Elevating the dialysis pH from 7.0 to 9.0 reduced the apparent number of binding sites for Ca. Using microelectrodes to record the free Ca, microtitrations of axoplasmic proteins were completed by adding small amounts of CaCl2 to 100 microliters volumes of protein solutions. In a medium containing ionic constituents closely resembling those of the Myxicola axon, a Ca binding capacity of 5.0 mumole g-1 protein and a K1/2 of approximately 1 microM were measured.
Cell Calcium 06/1990; 11(5):361-70. · 3.77 Impact Factor
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ABSTRACT: The free diffusion coefficient of ionic Ca was measured in isolated samples of Myxicola axoplasm by following the migration of 45Ca. When precautions were taken to minimize the sequestration and chelation of 45Ca (i.e., using inhibitors, energy deprivation, and saturation of Ca chelation sites), a diffusion coefficient of 5.3 x 10(-6) cm2 s-1 was measured. The diffusion coefficient was not appreciably changed by lowering free calcium from 100 microM to approximately 10 microM or by increasing the diffusion time from ten to twenty minutes. In untreated cytoplasm taken directly from the giant axon of Myxicola, the migration of Ca was more complex and could not be described by a single diffusion coefficient. This result is interpreted to suggest that bulk movement of Ca-buffers may occur in untreated Myxicola axoplasm, a system that contains few microtubules.
Cell Calcium 01/1988; 8(6):437-48. · 3.77 Impact Factor
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ABSTRACT: Microliter samples of cytoplasm containing mitochondria were aspirated from giant axons of the marine annelid Myxicola infundibulum into polyethylene tubes. The small molecular constituents within these cytoplasmic samples were controlled by a dialysis capillary with a 6,000 molecular weight cut off. The negative log of the calcium ion activity (pCa) (6.72 +/- 0.03, n = 40) and, in some cases, the pH (7.51 +/- 0.01, n = 7) of the samples were monitored with ion-sensitive microelectrodes. Adding 5 mM succinate or 5 mM ATP at pH 7.5 caused the Ca activity in the cytoplasm to drop from an experimentally elevated value of approximately 10 microM to below 1 microM. This decrease could be inhibited with ruthenium red, suggesting a mitochondrial mechanism. Ca uptake, following the addition of either succinate or ATP, was reversibly slowed when the cytoplasmic pH was elevated to approximately 8.3. When ruthenium red was added after mitochondria had taken up Ca, the Ca activity in the extramitochondrial cytoplasm gradually increased suggesting ongoing release of Ca from storage sites. Increasing the cytoplasmic pH to approximately 8.5 in the presence of ruthenium red did not increase the ongoing release over that found with ruthenium red alone. The apparent washout of Ca from the energy-independent, nonmitochondrial Ca buffers was only slightly affected by pH (pH 7.5-8.5). It is concluded that elevating intracellular pH to 8.3 slows the Ca uptake by mitochondria. Thus cytoplasmic pH may have a function in regulating mitochondrial Ca metabolism and/or extramitochondrial calcium activity.
The American journal of physiology 02/1987; 252(1 Pt 1):C68-76.
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ABSTRACT: Ion-selective electrodes recorded the pH (7.49 +/- 0.05, n = 8) and pCa (6.72 +/- 0.03, n = 40) in samples (approximately 1 microliter) of isolated Myxicola axoplasm mounted within 760-micron diameter plastic tubes. We determined the interactions between Ca2+ and H+ on axoplasmic buffers by microinjecting CaCl2 or HCl into the axoplasmic samples at a distance 75-125 micron from the tips of the electrodes (distance = r). When axoplasmic pH was lowered 0.97 +/- 0.095 from its resting value (measured at r = 125 micron) by injecting 4 nmol HCl, pCa dropped 0.30 +/- 0.05 (n = 6). When expressed in units of concentration, these data show that a HCl injection of approximately 4 mmol/l axoplasm increased H+ and Ca2+ activity by approximately 0.3 microM. Lowering axoplasmic pCa 2.20 +/- 0.43 (r = 75 micron) (n = 3) by injecting 40 pmol CaCl2 had only a small effect on pH. In other experiments, two Ca2+ electrodes measured the Ca2+ activity 125 and 375 micron from the site of CaCl2 injection. Evidence of Ca2+ buffering was obtained when the Ca2+ activity at these two locations was below that expected for simple Ca2+ diffusion away from the injection site. Centrifuged axoplasm (100,000 g) taken from the bottom of the centrifuge tube had a somewhat greater Ca2+ buffering capacity than that taken from the top of the tube. Electron microscopic studies of the centrifuged axoplasm showed a greater concentration of mitochondria and other axoplasmic vesicles in the bottom of the centrifuge tube. Ruthenium red (20-40 micrograms/ml) greatly reduced Ca2+ buffering. The mitochondrial inhibitors CN (2 mM) and oligomycin (a mixture of oligomycin A, B, and C, 5 micrograms/ml) also reduced Ca2+ buffering but were not as effective as ruthenium red. Axoplasm in which ATP and mitochondrial substrates were removed by dialysis was unable to lower free Ca2+ when the concentration of this ion was elevated to approximately 10 microM. In the presence of oligomycin to block mitochondrial ATPase, and with Mg2+ -ATP as the only source of energy, axoplasm lowered Ca2+ activity slowly; with succinate as the only metabolic substrate, axoplasm rapidly lowered the Ca2+ activity from approximately 10 microM to below 1 microM.
The American journal of physiology 04/1986; 250(3 Pt 1):C391-405.
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ABSTRACT: The binding and release of 45Ca by axoplasm isolated from Myxicola giant axons were examined. Two distinct components of binding were observed, one requiring ATP and one not requiring ATP. The ATP-dependent binding was largely prevented by the addition of mitochondrial inhibitors, whereas the ATP-independent component was unaffected by these inhibitors. The ATP-independent binding accounted for roughly two-thirds of the total 45Ca uptake in solutions containing an ionized [Ca2+] = 0.54 microM and was the major focus of this investigation. This fraction of bound 45Ca was released from the axoplasm at a rate that increased with increasing concentrations of Ca2+ in the incubation fluid. The ions Cd2+ and Mn2+ were also able to increase 45Ca efflux from the sample, but Co2+, Ni2+, Mg2+, and Ba2+ had no effect. The concentration-response curves relating the 45Ca efflux rate coefficients to the concentration of Ca2+, Cd2+, and Mn2+ in the bathing solution were S-shaped. The maximum rate of efflux elicited by one of these divalent ions could not be exceeded by adding a saturating concentration of a second ion. Increasing EGTA concentration in the bath medium from 100 to 200 microM did not increase 45Ca efflux; yet increasing the concentration of the EGTA buffer in the uptake medium from 100 to 200 microM and keeping ionized Ca2+ constant caused more 45Ca to be bound by the axoplasm. These results suggest the existence of high-affinity, ATP-independent binding sites for 45Ca in Myxicola axoplasm that compete favorably with 100 microM EGTA. The 45Ca efflux results are interpreted in terms of endogenous sites that interact with Ca2+, Cd2+, or Mn2+.
The Journal of General Physiology 11/1981; 78(4):413-29. · 3.84 Impact Factor
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ABSTRACT: 1. (45)Ca efflux was examined in Myxicola giant axons injected with (45)CaCl(2) or various concentrations of (45)Ca/EGTA buffers. In axons injected with (45)CaCl(2), the Ca(o)-dependent Ca efflux in 1 mM-Ca(o) was about half that in 10 mM-Ca(o).2. Axons injected with (45)Ca/EGTA buffers consistently showed two types of results: in one type (B type), K((1/2)) for Ca(o) activation was less than 1 mM-Ca(o). In the other type of result (A type), there was an additional Ca activation of Ca efflux. This additional efflux exhibited a linear dependence on Ca(o) when the Ca(o) values ranged between 1 mM-Ca(o) and 10 mM-Ca(o).3. The B-type result remained unchanged when the injected Ca/EGTA concentrations were varied. The A type result, however, changed as a function of Ca/EGTA(i) in the following way: (a) at a constant ratio of Ca/EGTA(i) = 8/10, the megnitude of the linear component of the Ca(o)-activated Ca efflux was reduced by increasing the intracellular concentration of (Ca/EGTA) buffer; and (b) at a ratio Ca/EGTA = 1/10, the linear component of the Ca(o)-activated Ca efflux appeared to acquire a slower time response to changes in Ca(o).4. Na(o) acts synergistically with Ca(o) to produce the linear component of the Ca-activated Ca efflux seen in the A type result.5. With axons containing (45)Ca/EGTA buffers (total EGTA(i) = 1 mM), changing the Ca/EGTA ratio by repetitive injections of CaCl(2) did not increase (45)Ca efflux by as great an amount as would be predicted if Ca(i) (2+) were controlled by the EGTA buffer alone.6. Ca(i) (2+) (measured by arsenazo III absorbance) is influenced by Ca(o) irrespective of the presence of 1 mM-EGTA buffer inside the axon. There was a variability in the sensitivity of Ca(i) to Ca(o) that resembled the variability found in (45)Ca efflux measurements.7. (45)Ca influx is not affected by the concentration of Ca/EGTA buffer injected into the cell and appears to be only slightly, if at all, affected by increasing ionized Ca(i) (2+) from 0.016 to 0.56 muM in the injection medium.8. These results are consistent with the interpretation that the Ca efflux system of the Myxicola giant axon, or something controlling it, can exist in more than one state. One of these states, which exhibited a large Ca(o)-dependent Ca efflux, may represent axons in which Ca(i) is poorly controlled by the natural endogenous Ca buffers.
The Journal of Physiology 10/1980; 306:175-91. · 4.72 Impact Factor
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ABSTRACT: The operation of the sodium pump of giant axons of the squid, Loligo pealei, has been studied simultaneously in two independent ways: 1) by measuring sodium efflux with 22Na, and 2) by calculating the transmembrane current generated by the pump from measurements of membrane resistance and digitalis-sensitive membrane potential. In normal, untreated axons, the effect of increasing the external potassium concentration on both sodium efflux and pump current is similar, which suggests that Na:K pump stoichiometry remains relatively constant in the range of 0-20 mM external K. The data are compatible with a 3:2 Na:K ratio. In axons whose intracellular ADP level has been elevated by injection of L-arginine, a large, electrically silent, cardiotonic steroid-sensitive sodium efflux takes place in the absence of external potassium; this suggests that pump-mediated Na:Na exchange is 1:1 or electroneutral. Finally, elevation of external potassium levels causes the appearance, in high-ADP axons, of electrogenic pumping, with little effect on sodium efflux; hence, in contrast to what is seen in normal (low-ADP) axons, the charge translocated, per sodium ion extruded, increases sharply with increasing extracellular potassium levels.
The American journal of physiology 08/1978; 235(1):C63-8.
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ABSTRACT: In microinjected Myxicola giant axons with elevated [Na]i, Na efflux was sensitive to Cao under some conditions. In Li seawater, sensitivity to Cao was high whereas in Na seawater, sensitivity to Cao was observed only upon elevation of [Ca]o above the normal value. In choline seawater, the sensitivity of Na efflux to Cao was less than that observed in Li seawater whereas Mg seawater failed to support any detectable Cao-sensitive Na efflux. Addition of Na to Li seawater was inhibitory to Cao-sensitive Na efflux, the extent of inhibition increasing with rising values of [Na]o. The presence of 20 mM K in Li seawater resulted in about a threefold increase in the Cao-activated Na efflux. Experiments in which the membrane potential, Vm, was varied or held constant when [K]o was changed showed that the augmentation of Ca-activated Na efflux by Ko was not due to changes in Vm but resulted from a direct action of K on activation by Ca. The same experimental conditions that favored a large component of Cao-activated Na efflux also caused a large increase in Ca influx. Measurements of Ca influx in the presence of 20 mM K and comparison with values of Ca-activated Na efflux suggest that the Na:Ca coupling ratio may be altered by increasing external [K]o. Overall, the results suggest that the Cao-activated Na efflux in Myxicola giant axons requires the presence of an external monovalent cation and that the order of effectiveness at a total monovalent cation concentration of 430 mM is K + Li greater than Li greater than Choline greater than Na.
The Journal of General Physiology 05/1978; 71(4):453-66. · 3.84 Impact Factor
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ABSTRACT: Several properties of the Na pump in giant axons from the marine annelid Myxicola infundibulum have been determined in an attempt to characterize this preparation for membrane transport studies. Both NaO and KO activated the Na pump of normal microinjected Myxicola axons. In this preparation, the KO activation was less and the NaO activation much greater than that found in the squid giant axon. However, when the intracellular ATP:ADP ratio of the Myxicola axon was elevated by injection of an extraneous phosphagen system, the K sensitivity of Na efflux increased to the magnitude characteristic of squid axons and the activating effect of NaO disappeared. Several axons were injected with Na2SO4 in order to determine the effect of elevated Nai on the Na efflux. Increasing Nai enhanced a component of Na efflux which was insensitive to ouabain and dependent on [Ca] in Na-free (Li) seawater. After subtracting the CaO-dependent fraction, Na efflux was related linearly to [Na]i in all solutions except in K-free (Li) seawater, where it appeared to reach saturation at high [Na]i.
The Journal of General Physiology 07/1977; 69(6):765-78. · 3.84 Impact Factor
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ABSTRACT: The efflux of Mg++ from squid axons subject to internal solute control by dialysis is a function of ionized [Mg], [Na], [ATP], and [Na]o. The efflux of Mg++ from an axon with physiological concentrations of ATP, Na, and Mg inside into seawater is of the order of 2-4 pmol/cm2s but this efflux is strongly inhibited by increases in [Na]i, by decreases in [ATP]i, or by decreases in [Na]o. The efflux of Mg++ is largely independent of [Mg]i when ATP is at physiological levels, but in the absence of ATP reaches half the value of Mg efflux in be presence of ATP when [Mg]i is about 4 mM and [Na] 40 mM. Half-maximum responses to ATP occur at about 350 micronM ATP into seawater with Na either present or absent. The Mg efflux mechanism has many similarities to the Ca efflux system in squid axons especially with respect to the effects of ATP, Nao, and Na on the flux. The concentrations of free Mg and Ca in axoplasm differ, however, by a factor of 10(5) while the observed fluxes differ by a factor of 10(2).
The Journal of General Physiology 05/1977; 69(4):389-400. · 3.84 Impact Factor
