Creatine kinase is required for swelling-activated K-Cl cotransport in dog red blood cells.

K-Cl cotransport in resealed dog red cell ghosts requires the incorporation of creatine phosphate before resealing; incorporation of ATP has no effect [Colclasure and Parker. Am. J. Physiol. 265 (Cell Physiol. 34): C1648-C1652, 1993]. A role for creatine kinase (CK) in swelling-activated K-Cl cotransport was investigated. 2,4-Dinitrofluorobenzene (DNFB), an inhibitor of CK, inhibited K-Cl cotransport in intact red blood cells and resealed ghosts from DNFB-treated cells. Incorporation of exogenous CK into ghosts of DNFB-treated cells restored K-Cl cotransport. Therefore DNFB inhibits CK and not the cotransporter. Inhibition of native CK in ghosts by DNFB and the incorporation of CK into the ghosts were demonstrated in electrophoretic gels. In a dose-response experiment, approximately 770 molecules CK/ghost restored 50% of control cotransport. Since creatine phosphate is a substrate only for CK, CK provides ATP to a site inaccessible to cytoplasmic ATP. The nature of this site and its role in K-Cl cotransport are uncertain, but an essential function for CK is established.

ATP dependence of K-Cl cotransport in dog red blood cells.

Swelling-induced K-Cl cotransport in resealed dog red blood cell ghosts requires the presence of an ATP-generating system (G. C. Colclasure and J. C. Parker. J. Gen. Physiol. 100: 1-10, 1992). The present study shows that the endogenous adenine nucleotide present in the dog ghosts is sufficient to activate K-Cl cotransport, provided that creatine phosphate is incorporated in them. Creatine kinase is not required, because dog red blood cells, unlike those of humans, possess this enzyme. Although some ATP appears to be required for K-Cl cotransport by dog ghosts, an excess of this nucleotide is inhibitory. Creatine phosphate appears to play a special role in generating the ATP required for activation of K-Cl cotransport. If ghost ATP content is manipulated in the absence of creatine phosphate, by simply adding ATP to the hemolysate, no stimulation of K-Cl cotransport occurs. On the other hand, when creatine phosphate is present, K-Cl cotransport is activated. The results are discussed in relation to current views regarding the role of ATP in activation of K-Cl cotransport and the concept of the "phosphocreatine shuttle."

Model for the role of macromolecular crowding in regulation of cellular volume.

A simple model is proposed to account for large increases in transporter-mediated ion flux across cell membranes that are elicited by small fractional changes of cell volume. The model is based upon the concept that, as a result of large excluded volume effects in cytoplasm (macromolecular crowding), the tendency of soluble macromolecules to associate with membrane proteins is much more sensitive to changes in cell water content than expected on the basis of simple considerations of mass action. The model postulates that an ion transporter may exist in either an active dephosphorylated state or an inactive phosphorylated state and that the steady-state activity of the transporter reflects a balance between the rates of phosphatase-catalyzed activation and kinase-catalyzed inactivation. Cell swelling results in the inhibition of kinase relative to phosphatase activity, thereby increasing the steady-state concentration of the active form of the transporter. Calculated volume-dependent stimulation of ion flux is comparable to that observed experimentally.

Macromolecular crowding and volume perception in dog red cells.

To differentiate whether the primary volume signal in dog red cells arises from a change in cell configuration or the concentration and dilution of cell contents, we prepared resealed ghosts that had the same surface area and hemoglobin concentration as intact cells but less than 1/3 their volume. Shrinkage of both intact cells and resealed ghosts triggered Na/H exchange. Activation of this transporter in the two preparations correlated closely with cytosolic protein concentration but not at all with volume. The Na/H exchanger was more sensitive to shrinkage in albumin-loaded resealed ghosts than in intact cells or ghosts containing only hemoglobin. Similar results were obtained for the swelling-induced [K-Cl] cotransporter. We believe perception of cell volume originates with changes in cytoplasmic protein concentration. We think the kinases and phosphatases that control the activation of membrane transporters in response to cell swelling or shrinkage are regulated by the mechanism of macromolecular crowding.

Cytosolic protein concentration is the primary volume signal for swelling-induced [K-Cl] cotransport in dog red cells.

Chloride-dependent K transport ([K-Cl] cotransport) in dog red cells is activated by cell swelling. Whether the volume signal is generated by a change in cell configuration or by the dilution of some cytosolic constituent is not known. To differentiate between these two alternatives we prepared resealed ghosts that, compared with intact red cells, had the same surface area and similar hemoglobin concentration, but a greatly diminished volume. Swelling-induced [K-Cl] cotransport was activated in the ghosts at a volume (20 fl) well below the activation volume for intact cells (70 fl), but at a similar hemoglobin concentration (30-35 g dry solids per 100 g wet weight). Ghosts made to contain 40% albumin and 60% hemoglobin showed activation of [K-Cl] cotransport at a concentration of cell solids similar to intact cells or ghosts containing only hemoglobin. [K-Cl] cotransport in the resealed ghosts became quiescent at a dry solid concentration close to that at which shrinkage-induced Na/H exchange became activated. These results support the notion that the primary volume sensor in dog red cells is cytosolic protein concentration. We speculate that macromolecular crowding is the mechanism by which cells initiate responses to volume perturbation.

Actions of thiocyanate and N-phenylmaleimide on volume-responsive Na and K transport in dog red cells.

Two sets of observations suggest a linkage between volume-responsive Na and K transport systems in dog red blood cells. 1) The lyotropic anion thiocyanate inhibits shrinkage-induced Na-H exchange and stimulates swelling-induced K-Cl cotransport. 2) The effect of a brief incubation with N-phenylmaleimide (NPM) on Na and K transport depends on the volume of the cells at the time of exposure to the sulfhydryl reagent. Cells shrunken during the NPM incubation and then brought back to normal volume behave as though they were still shrunken, i.e., they show an increased Na flux and a decreased K flux. Cells incubated with NPM in a swollen state retain fluxes characteristic of swollen cells when returned to a normal volume. The electrophoretic mobility of the membrane-associated enzyme glyceraldehyde-3-phosphate dehydrogenase is influenced by the cell volume at the time of NPM exposure. These findings point to the existence of a system in cells that perceives volume changes and coordinates the responses of membrane transporters.

Coordinated regulation of shrinkage-induced Na/H exchange and swelling-induced [K-Cl] cotransport in dog red cells. Further evidence from activation kinetics and phosphatase inhibition.

Hypertonic shrinkage of dog red cells caused rapid activation of Na/H exchange and rapid deactivation of [K-Cl] cotransport. Hypotonic swelling caused delayed deactivation of Na/H exchange and delayed activation of [K-Cl] cotransport. Okadaic acid stimulated shrinkage-induced Na/H exchange and inhibited swelling-induced [K-Cl] cotransport. The data are compatible with the kinetic model of Jennings and Al-Rohil (1990. J. Gen. Physiol. 95:1021-1040) for volume regulation of [K-Cl] cotransport in rabbit red cells and suggest that in dog red cells Na/H exchange and [K-Cl] cotransport are controlled by a common regulatory system. The proposal of Jennings and Schulz (1991. J. Gen. Physiol. 96:799-817) that activation/deactivation of volume-sensitive transport involves phosphorylation/dephosphorylation of a regulatory protein is supported by these observations.

Cytosolic protein concentration is the primary volume signal in dog red cells.

It is not known whether the activation of Na/H exchange by shrinkage in dog red cells is due to the packing of cell contents or a change in cell configuration. To make this distinction we prepared resealed ghosts that resembled intact cells in hemoglobin concentration and surface area, but had one-third their volume. A shrinkage-induced, amiloride-sensitive Na flux in the ghosts was activated at a much smaller volume in the ghosts than in the intact cells, but at the same concentration (by weight) of dry solids in both preparations. Na/H exchange in ghosts containing a mixture of 40% albumin and 60% hemoglobin (weight/weight) was activated by osmotic shrinkage at a dry solid concentration similar to that of intact cells or of ghosts containing only hemoglobin. We conclude that the process of Na/H exchange activation by cell shrinkage originates with an increase in the concentration of intracellular protein and not with a change in membrane configuration or tension. The macromolecular crowding that accompanies the reduction in cell volume probably alters the activities of key enzymes that in turn modulate the Na/H exchanger.

Influence of Ca/Na exchange and diamide on membrane partitioning of glyceraldehyde-3-phosphate dehydrogenase in dog red cells.

1. Ca/Na exchange in dog red cells is greatly stimulated by pretreatment of the cells with diamide, but only if the diamide preincubation is carried out in a Na-free, Ca-containing medium. 2. Membranes prepared from dog red cells that had been pre-exposed to diamide in solutions containing various combinations of Ca and Na were subjected to polyacrylamide gel electrophoresis and Western blotting. 3. The band representing glyceraldehyde-3-phosphate dehydrogenase was selectively increased under the very same ionic conditions that result in a diamide-induced stimulation of Ca/Na exchange. 4. Glyceraldehyde-3-phosphate dehydrogenase may participate in the modulation by diamide of Ca/Na exchange in dog red cells.

Human serum alpha 2HS-glycoprotein modulates in vitro bone resorption.

We previously isolated a family of bone-resorbing proteins from human cancer ascites fluid and established that the three purified bone-resorbing proteins were chemically and immunochemically related to each other and to alpha-2HS glycoprotein (alpha 2HS). After this finding we purified the normal human serum counterpart of these ascites proteins and studied its effects on bone resorption. The bone-resorbing properties of normal human serum alpha 2HS were examined in vitro over a wide dose range. This normal human serum glycoprotein had a biphasic effect on 45Ca2+ release from bone. More specifically, this protein stimulated bone resorption at the lower concentrations tested, with a maximum effect [treated over control ratio of 2.5 +/- 0.30 (+/- SE); P less than 0.01] at 40 micrograms/mL. In contrast, at doses above 40 micrograms/mL, a sharp decline in calcium mobilization occurred, with a return to baseline occurring above 80 micrograms/mL. These results suggest that serum alpha 2HS may participate in the regulation of bone metabolism in vivo.