Transport and control of Ca2+ by pigeon erythrocytes. III. A 'paradoxical' expulsion of Ca2+ induced by a low dose of A23187 at 0 degrees C.

Pigeon erythrocytes expelled preloaded 45Ca2+ in response to a low dose of A23187 at 0 degrees C. We call this phenomenon 'paradoxical' expulsion. Within the first minute, 1.85 +/- 0.38 mumol/l cell water was expelled; after that the internal 45Ca2+ began to rise. The rises in Ca2+ uptake with and without A23187 addition were essentially paralleled. No premonitory rise of 45Ca2+ upon the addition of A23187 was observed. Expulsion of 45Ca2+ in response to A23187 was probably by the action of the Ca2+ pump and not by Na+-Ca2+ exchange since vanadate inhibited, but K+ replacement of Na+ in the medium had no effect. Lysophosphatidylcholine (lysoPC) caused an abrupt increase in 45Ca2+ influx by cells at 0 degrees C and was dose dependent. However, a very low dose of lysoPC induced expulsion of preloaded 45Ca2+ similar to that by A23187, the response was fast and transitory, without any premonitory rise in 45Ca2+ uptake. The results lend support to the suggestion that the signal to which cells respond may be a sudden change in Ca2+ influx per se rather than a change in internal Ca2+ concentration. These features of 'paradoxical' 45Ca2+ expulsion induced by A23187 and lysoPC are not expected from mass-action equilibria but, instead, agree with the characteristics of an energy-dissipating control mechanism.

Comparison of human and pigeon erythrocyte membrane proteins by one- and two-dimensional gel electrophoresis.

Pigeon and human bands 1 and 2 (spectrin) and 5 (actin) are conserved. Band 3 anion porters have similar SDS positions, but the pigeon porter has a higher isoelectric point. Both anion porters are inhibited by similar doses of pyridoxal phosphate. Many differences are apparent in minor bands.

Lysolecithin-induced Ca2+ uptake by pigeon red cells.

Lysolecithin (LPC) induced a rapid but transitory increase in 45Ca2+ influx, and an abrupt 45Ca2+ net uptake by pigeon red cells. The effect on uptake was seen with doses of added lysolecithin calculated to be 2.1-8.4% of the cytoplasmic membrane phospholipid, well within the reported physiological range of 1-9%. Lysolecithin effects were not due to prelytic membrane changes nor action as a platelet activating factor agonist. Changes in the amino acids in the medium mimicking human homocysteinuria, an atherosclerosis risk factor, increased the lysolecithin effect. The findings are consistent with a role for lysolecithin in atherosclerosis.

An isoelectric focusing procedure for erythrocyte membrane proteins and its use for two-dimensional electrophoresis.

Procedures are described and evaluated for one-dimensional isoelectric focusing of erythrocyte membrane dissolved in lysine, urea, and Triton X-100 without using sodium dodecyl sulfate (SDS) and for two-dimensional electrophoresis with SDS in the second dimension. The membrane was completely dissolved, most of the proteins including the anion porter(s) entered the focusing gel, and complex, well-resolved patterns were seen. Ampholines, 2-mercaptoethanol, or SDS in the applied sample each seriously reduced focusing resolution and phenylmethylsulfonyl fluoride blurred the patterns. The two-dimensional patterns showed more and sharper spots than did patterns obtained from membrane initially dissolved with SDS. Anion porter spots were seen with both procedures. However, major cytoskeletal proteins were much less well recovered with the former procedure than with the latter.

Evidence that lysolecithin is an important causal agent of atherosclerosis.

We examine the hypothesis of Portman et al. (1970) that lysolecithin is a causal agent of atherosclerosis. Four lines of argument support this hypothesis. (1) Lysolecithin is present, taken up and can act. Large amounts of lysolecithin are formed in plasma concomitant with triglyceride transport and it is readily taken up by arteries and retained for some time. Lysolecithin in aortic intima increases several-fold early in the induction of atherosclerosis in animals. From model system studies it is plausible that physiological doses of lysolecithin have physiologically significant effects. (2) At least one plausible mechanism of action can be formulated: stimulation of smooth muscle cell division due to lysolecithin-increased Ca2+ uptake. (3) The hypothesis is consistent with, and rationalizes, many literature observations in that inferred lysolecithin levels or production rates are appropriately correlated with a variety of positive and negative risk factors to a degree highly unlikely by chance. We found no data contradicting the hypothesis and only one piece weakening it. (4) A mechanism is outlined showing that low density lipoprotein receptor deficiency, the severest known risk factor, should cause the delivery of very high lysolecithin doses to artery walls. We conclude that the evidence, although indirect, is strong enough to give direct tests a high priority.

Transport and control of Ca2+ by pigeon erythrocytes. I. Survey of some cell responses to a range of A23187 doses in the presence of Ca2+.

Cells were subjected to a range of 45Ca2+ influx loads with A23187. We measured cell 45Ca2+ with time and A23187 dose, and the apparent 45Ca2+ influxes (identical to "J(in,app)") at Ca2+ steady state. We also measured endogeneous exchangeable and total cell Ca2+, which were 50 and 17-220 microM respectively. The effects of A23187 and Ca2+ on cell ATP, swelling, net Cl- permeability, and cell morphology were measured. These were modest and do not affect our conclusions. J(in,app) congruent to 3 X 10(-4) [A23187]2.9 X [Ca2+(o)]mumoles/l X min with 92-552 microM [Ca2+(o)] (identical to external Ca2+ concentration) and 0-7 microM A23187. J(in,app) was increased an order of magnitude by vanadate and is probably much less than the true influx. The least unlikely explanation found for the high [A23187] exponent, 2.9, was that most of the Ca2+ crossing the membrane is expelled by the pump before it can move deeper into the cell. Calcium pumping increased rapidly in response to increased influx, but the steady state cell 45Ca2+ was approximately proportional to J(in,app) rather than approximately constant between 10 and 120 mumoles/l X min with 184 microM [Ca2+(o)]. This is not the result expected from a simple feedback mechanism. At high A23187 doses the pump appears fully activated resulting in a linear relation between cell/medium 45Ca2+ and [A23187]-2. From the plot we calculated alpha identical to free/total exchangeable Ca2+ = 0.38 +/- 0.08 (n = 3) and a maximum pump rate, "Pmax" = 78 mumole/l X min. Pmax is underestimated insofar as J(in,app) is less than the true influx.

Transport and control of Ca2+ by pigeon erythrocytes. II. Evidence against a simple feedback control of cell Ca2+ and evidence for the involvement of more than one pool.

Pigeon erythrocytes did not behave as expected from simple feedback mechanisms. The pool size for exchangeable cell Ca2+ was approximately proportional to the A23187-induced apparent 45Ca2+ influx ("J(in,app)") from 0.4 to 14 mumoles/min X l cell water at 184 microM external [Ca2+]. From earlier data, total cell 45Ca2+ was approximately proportional to J(in,app) from 10 to 120 mumoles/l X min. Thus there was no influx range where cell 45Ca2+ was held approximately constant. External [Ca2+] affected Ca2+ pool size independently of its effect of J(in,app). Trifluoperazine did not increase cell 45Ca2+ with or without A23187. In the presence of A23187, 45Ca2+ entered a pool early in the incubation which later became inaccessible to 45Ca2+ entry and exit. Lysolecithin addition produced an abrupt rise in cell 45Ca2+, much of which occupied a pool that quickly became inaccessible. The increased 45Ca2+ influx induced by lysolecithin dropped quickly and markedly with time. It is hard to explain inaccessible pool(s), especially in the presence of A23187 by membrane-bounded compartments. We suggest that nonexchangeable 45Ca2+ might be held by an energy-dependent binding protein(s).

Rapid stopping of A23187 action by phosphatidylcholine.

The action on pigeon erythrocytes of the Ca2+ ionophore, A23187, can be nearly completely stopped within 30 s by treatment with a phosphatidylcholine dispersion at 39 degrees C. A time-limited 45Ca2+ uptake pulse can be produced by treating cells sequentially with A23187 and lipid, and the time-course of expulsion of this 45Ca2+ uptake pulse can be easily determined.

Active Ca2+ transport by membrane vesicles from pigeon erythrocytes. Stimulation by amino acids, ATP, GTP, Pi and some other cell constituents.

Pretreatment of pigeon erythrocyte membrane vesicles with amino acids, ATP, GTP, Pi and some other simple cell constituents (singly and in combination) causes an increase in ATP-dependent Ca2+-uptake activity of vesicles upon subsequent incubation with 45Ca2+ after removal of the above agents from the 'i' face. Amino acids augment the stimulation by all stimulatory agents and are required for stimulation by Pi. The effects of amino acids, ATP, GTP and Pi all occur at physiological concentrations. Many if not all of the effects of the mixture of amino acids that occur naturally in the cells can be accounted for by the group transported by the 'ASC' transport system of Christensen (Christensen, H.N. (1975) Biological Transport, 2nd edn., W.A. Benjamin, Inc., Reading, MA), but not by any single amino acid at its physiological concentration. The effects of ATP and GTP are not mimicked by their non-hydrolysable beta,gamma-imido analogues not by the corresponding 3',5'-cyclic monophosphates. None of the effects described appears to involve calmodulin. We suggest that amino acid transport plays a role in metabolic regulation through effects on cell [Ca2+]. Analogous effects on cell [Ca2+] may be involved in the action of the many hormones which augment amino acid accumulation by the 'A' amino acid transport system.

Calcium transport by pigeon erythrocyte membrane vesicles.

Membrane vesicles from pigeon erythrocytes show a rapid, ATP-dependent accumulation of 45Ca2+.Ca2+ accumulation ratios greater than or approximately equal to 10(4) are readily attained. For ATP-dependent Ca2+ uptake, V is 1.5 mmol.1(-1).min(-1) at 27 degrees C (approx. 0.9 nmol.mg-1 protein.min-1), [Ca2+]1/2 is 0.18 microM, [ATP]1/2 is 30--60 microM, the Ca2+ uptake rate depends on [Ca2+]2 and the dependence of uptake rate on ATP concentration implies strong ATP-ATP cooperativity. The Arrhenius activation energy is 19.1 +/- 1.4 kcal/mol and the pH optimum is approx. 6.9.

Active Ca2+ transport by vesicles reconstituted from Triton X-100-solubilized pigeon erythrocyte membrane.

Pigeon erythrocyte membrane was solubilized partially, but relatively unselectively by Triton X-100. Vesicles were reconstituted from mixtures of Triton-solubilized membrane and lipid (phosphatidylcholine plus phosphatidyl-ethanolamine plus cholesterol) by addition of bovine high-density lipoprotein. This efficiently removed the Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electropherograms of reconstituted vesicles showed band patterns resembling those of the original membrane. The reconstituted vesicles showed ATP-dependent active accumulation of 45Ca2+. ATP-dependent 45Ca2+ uptake by the reconstituted vesicles resembled the corresponding activity of the original membrane vesicles; in both preparations the Ca2+ uptake rate depended on the square of the Ca2+ concentration and had similar [Ca2+]1/2 values, 0.16 microM and 0.18 microM, respectively.

Hybridization by cosonication of pigeon erythrocyte membrane with exogenous lipid vesicles.

Concentrated mixtures of lipid vesicles and pigeon erythrocyte membrane were cosonicated in order to produce functional hybrid vesicles. From the properties of the resulting material, we conclude that hybrids were very probably formed. These properties were as follows: (i) The presence of membrane increased the sonic fragmentability of lipid vesicles. Sonic fragmentability was assessed by measuring sonication-induced release of previously trapped [14C]-choline and trapping of external [3H]-choline. (ii) Space enclosed by lipid was served by the membrane-like properties of 36Cl- permeability and ATP-dependent 45Ca++ uptake activity. (iii) 36Cl-permeability was more readily and fully induced into the more easily fragmented lipid vesicles. Further sonication caused loss of the induced 36Cl--permeability. This loss was less rapid with the less easily fragmented lipid vesicles; i.e., less easily fragmented lipids protected 36Cl--permeability better. (iv) Glycine uptake activity was partially protected from sonic damage by the presence of lipid vesicles. (v) On centrifugation in bovine serum albumin density gradients, cosonicated material showed lipid properties (enclosed choline and 32Pi space and [3H]-cholesterol) and membrane properties (36Cl--permeability and ATP-dependent 45Ca2+ uptake) coinciding at a density intermediate between those reached by separately sonicated membrane and lipid vesicles. (vi) Electron micrographs showed the disappearance of pure membrane-like structures and the appearance of large amounts of new vesicles whose appearance is consistent with a hybrid structure.

A method for demonstrating the heterogeneity of pigeon red cell membrane vesicles based on their glycine transport activity.

A procedure is described which is capable of detecting the changes in size and/or density of small membrane vesicles resulting from solute uptake. Vesicles which have taken up solute sediment more slowly in a density gradient, the ratio of glycine uptake/vesicle-trapped space is not uniform in the vesicle population, and vesicles with higher uptake/space ratios are preferentially retarded upon centrifugation. Alanine transport activity is associated with glycine transport activity in that retardation of vesicles due to glycine uptake equally retards vesicles possessing alanine uptake activity.

Glycine transport by hemolysed and restored pigeon red cells. Effects of a Donnan-induced electrical potential on entry and exit kinetics.

The influence of a Donnan effect on the transport of glycine by hemolysed and restored pigeon red cells was examined. The Donnan effect was produced by replacing Cl- with 2,4-toluenedisulfonate or glutamate. The effects of the associated membrane potential and inside-outside pH difference on glycine entry and exit rates were examined. The effects of pH on entry and exit rates in the absence of a Donnan effect were also examined. In the absence of a Donnan effect, Na+-dependent glycine entry requires the protonated form of a group with a pKapp of 7.9 and the deprotonated form of another group with a pKapp of 6.8. Neither of these are required for exit but the deprotonated form of a group(s) with a pKapp of 6.2 is required. The pK 7.9 group and pK 6.2 group probably react with H+ at the inner face of the membrane and the pK 6.8 group probably reacts at the outer face. The V for glycine entry was determined for cells with their Cl- largely replaced by toluenedisulfonate and without such replacement. Between pH 6.1 and 7, the ratio of the respective V values, VT/VC1, was 1.5-1.7. VT/VC1 rose above pH 7 to near 4 at pH 8.3. At pH 6.9, with glutamate replacing cell Cl-, the analogous ratio (VGlu/VC1) was 1.7. The increase of VT/VC1 above pH 7 could be quantitatively accounted for by the increase in cell [H+]/medium [H+] caused by the Donnan effect together with the assumption that the pK 7.9 group reacts with H+ at the inner face of the membrane. When cell Cl- was replaced by toluenedisulfonate or glutamate there was a drop in the term in the glycine Km describing Na+ dependence of glycine entry. When cell Cl- was replaced by toluenedisulfonate therewas a rise in the Na+-independent term in the glycine entry Km. By replacing varying amounts of cell Cl- with either toluenedisulfonate or glutamate, plots were obtained of entry rates vs. the cell [Cl-]/ medium [Cl-] ratio consistent with the assumption that the Donnan-induced membrane potential acts on a "moving" charge. Glycine exit was only slightly accelerated by trans-toluenedisulfonate. The ratio, exit rate into toluenedisulfonate medium/exit rate into Cl- medium rose with decreasing pH. This rise could be accounted for by a Donnan-induced inside-outside pH difference which affects a pKapp 6.2 group reacting with internal H+. The observed influences of the Donnan effect on V (glycine entry), on both components of Km (glycine entry), on the shape of the plot of glycine entry rate vs. the cell [Cl-]/medium [Cl-] ratio and on glycine exit all fit the assumptions that when the empty porter reorients, one unit of negative charge accompanies it "across" the membrane and that no other steps involve charge movement. The properties of the system seem inconsistent with a translational ("ferry boat") mobile carrier.