A view of hydrogen/hydroxide flux across lipid membranes.

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.

Mitochondria shape hormonally induced cytoplasmic calcium oscillations and modulate exocytosis.

Pituitary gonadotropes transduce hormonal input into cytoplasmic calcium ([Ca(2+)](cyt)) oscillations that drive rhythmic exocytosis of gonadotropins. Using Calcium Green-1 and rhod-2 as optical measures of cytoplasmic and mitochondrial free Ca(2+), we show that mitochondria sequester Ca(2+) and tune the frequency of [Ca(2+)](cyt) oscillations in rat gonadotropes. Mitochondria accumulated Ca(2+) rapidly and in phase with elevations of [Ca(2+)](cyt) after GnRH stimulation or membrane depolarization. Inhibiting mitochondrial Ca(2+) uptake by the protonophore CCCP reduced the frequency of GnRH-induced [Ca(2+)](cyt) oscillations or, occasionally, stopped them. Much of the Ca(2+) that entered mitochondria is bound by intramitochondrial Ca(2+) buffering systems. The mitochondrial Ca(2+) binding ratio may be dynamic because [Ca(2+)](mit) appeared to reach a plateau as mitochondrial Ca(2+) accumulation continued. Entry of Ca(2+) into mitochondria was associated with a small drop in the mitochondrial membrane potential. Ca(2+) was extruded from mitochondria more slowly than it entered, and much of this efflux could be blocked by CGP-37157, a selective inhibitor of mitochondrial Na(+)-Ca(2+) exchange. Plasma membrane capacitance changes in response to depolarizing voltage trains were increased when CCCP was added, showing that mitochondria lower the local [Ca(2+)](cyt) near sites that trigger exocytosis. Thus, we demonstrate a central role for mitochondria in a significant physiological response.

Separate entry pathways for phosphate and oxalate in rat brain microsomes.

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.

Luminal Ca2+ protects against thapsigargin inhibition in neuronal endoplasmic reticulum.

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.

Effects of the intraluminal Ca load on the kinetics of 45Ca uptake and efflux in brain microsomes.

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.

Calcium chelators enhance 45Ca accumulation in permeablized synaptosomes and in microsomes.

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.

Cytoplasmic calcium buffer capacity determined with Nitr-5 and DM-nitrophen.

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.

Calcium diffusion coefficient in Myxicola axoplasm.

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.

Calcium accumulation by organelles within Myxicola axoplasm.

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.

Cytoplasmic hydrogen ion diffusion coefficient.

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.

Properties of calcium binding by Myxicola axoplasmic protein.

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)

Ca2+ binding by Myxicola neurofilament proteins.

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.

Free diffusion coefficient of ionic calcium in cytoplasm.

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.

High cytosolic pH inhibits Ca uptake by Myxicola axon mitochondria.

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.

Calcium and proton buffering and diffusion in isolated cytoplasm from Myxicola axons.

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 intracellular pH of frog skeletal muscle: its regulation in isotonic solutions.

The behaviour of intracellular pH (pHi) was studied with micro-electrodes in frog semitendinosus muscle which was superfused with Ringer solution and with depolarizing solutions. The electrodes were introduced into the depolarized muscle about 40 min after contracture had subsided. All studies were done at external pH (pHo) of 7.35 and at 22 degrees C. The pHi in normal Ringer solution buffered with HEPES was 7.18 +/- 0.03 (S.E. of mean) (n = 10); the membrane potential, Vm, was -88 +/- 1.8 mV. When pHi was lowered to about 6.8 by replacing the HEPES by 5% CO2, 24 mM-HCO3 (constant pHo), it recovered at a very slow rate of 0.025 +/- 0.011 delta pHi h-1 (n = 6). When all the Na was replaced by N-methyl-D-glucamine (initial pHi 7.20 +/- 0.04, initial Vm -89 +/- 1.5 mV, n = 8), this slow alkalinization was converted into a slow acidification at a rate of 0.069 +/- 0.024 delta pHi h-1. In muscle depolarized in 15 mM-K (Vm approximately -50 mV), the rate of recovery from CO2 acidification was not increased above that in normal Ringer solution (2.5 mM-K). When, however, the muscle was depolarized in 50 mM-K to about -20 mV, the rate of recovery increased to 0.33 +/- 0.07 delta pHi h-1 (n = 6) when external Cl was kept constant, or to 0.21 +/- 0.03 (n = 9) when [K]. [Cl] product was kept constant. In the absence of Na, pHi recovery rate in 50 mM-K was reduced by at least 90%. Enhanced recovery from CO2-induced acidification was also observed in 2.5 mM-K when the fibres were depolarized to about -20 mV in one of two ways: (a) by previous exposure for 60 min to 50 mM-K at constant Cl, or (b) by reduction of external Cl to 5.9 mM in the presence of 0.5 mM-Ba. When pHi of depolarized fibres (50 mM-K) was lowered to about 6.8 by the weak acid dimethyl-2,4-oxazolidinedione (DMO), it recovered at a rate of 0.12 delta pHi h-1 in two experiments. In fibres depolarized in 50 mM-K and constant Cl, either 0.1 mM-SITS or 0.5 mM-amiloride slowed pHi recovery from CO2 exposure by about 50%. When the depolarization was achieved at constant [K]. [Cl] product, amiloride slowed pHi recovery by about 50%, while SITS had, at most, only a slight effect.(ABSTRACT TRUNCATED AT 400 WORDS)

The intracellular pH of frog skeletal muscle: its regulation in hypertonic solutions.

Intracellular pH (pHi) was followed with micro-electrodes in frog semitendinosus muscle, superfused at 22 degrees C with hypertonic solutions (external pH, pHo, 7.35) containing 2.5, 15 or 50 mM-K. Tonicity was doubled by addition of 250 mM-mannitol or, in a few cases, 125 mM-extra NaCl. Tripling of tonicity was accomplished by adding 500 mM-mannitol. Because of the ability of hypertonicity to minimize contracture, the course of pHi could be followed from the start of depolarization. The pHi of fibres after about 40 min in Ringer solution (2.5 mM-K, HEPES buffer) of twice normal tonicity was 7.40 +/- 0.04 (S.E. of mean) (n = 17), about 0.2 higher than at normal tonicity. The membrane potential, Vm, was -87.7 +/- 1.3 mV. When the muscle was depolarized in 50 mM-K to about -30 mV, the pHi rapidly fell by 0.3-0.5 unit (n = 9), and then promptly returned. This recovery was followed by a much slower and progressive rise to above control. Removing Na from the medium did not affect the degree of acidification, but the pHi recovered at a slightly slower rate, did not reach control value and showed no progressive rise. A less pronounced transient acidification was also observed when the muscle was depolarized in 15 mM-K to about -60 mV. When contracture was prevented either by 1-2 mM-tetracaine under isotonic conditions or by raising tonicity 3-fold, 50 mM-K produced no transient acidification. When the pHi of resting fibres in Ringer solution (2.5 mM-K) of twice normal tonicity was reduced by 5% CO2 from 7.40 to 7.12 +/- 0.07 (n = 3), it recovered at a slow rate (0.06 +/- 0.03 delta pHi h-1). Depolarization by 15 or 50 mM-K enhanced recovery rate 4-6-fold. These solutions of twice normal tonicity, as compared to those of normal tonicity, shifted the curve relating pHi recovery rate and membrane potential along the potential axis in the direction of hyperpolarization. This shift may be due to increased ionic shielding of fixed negative charges at the inner membrane surface. At twice normal tonicity, the very slow pHi recovery of resting fibres from CO2-induced acidification, as well as the more rapid recovery in depolarized fibres, could be abolished by 1 mM-amiloride or by removing Na. The application of amiloride during pHi recovery in 50 mM-K was not associated with an observable change in Vm. SITS had no significant effect on recovery.(ABSTRACT TRUNCATED AT 400 WORDS)

Uptake and release of 45Ca by Myxicola axoplasm.

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+.

Calcium efflux from Myxicola giant axons: effects of extracellular calcium and intracellular EGTA.

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.

Electric current generated by squid giant axon sodium pump: external K and internal ADP effects.

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.