Effects of several agents on the calcium transport in the human RBC membrane
Calcium has profound effects on the numerous cellular functions. Among them, Ca**++ plays an essential role in excitation-contraction coupling in the cardiac and skeletal muscle. Therefore, during the past decade it has been an interest to understand of calcium movement across the cellular membrane.
Calcium is not at electrochemical equilibrium in cells and its concentration gradient between intra-and extracellular fluid is large. Neverthless, calcium enters the cell by passive process and actively extrudes from the cell against electrochemical gradient. On this phenomenon. "Calcium pump" mechanism was suggested(Router and Seitz, 1968: Barker et al., 1969).
The energy-linked transport of Ca**++ in mitochondria and sarcoplasmic reticulum has been established during the past decade(Hasselbach and Makinose, 1961 : Hasselbach, 1964: Brierly et al., 1964; Engstrom and De Luca, 1964: Lee et al.,1966). However, little was known about the regulation of this process until the active ATP-dependent Ca**++ transport mechanism was demonstrated in RBC ghosts(Schatzmann, 1966). Schatzmann and Vincenzi (1969) showed the Ca**++ -activated ATPase located in the red cell membrane was intimately related to this active transport of Ca**++ and it wart confirmed subsequently (Schatzmann and Rossi, 1971 : Cha et al., 1971).
The present study was undertaken to investigate the effect of Ni**++, La**+++ , and prenylamine lactate, which were known to influence Ca**++ transport across cardiac cell membrane (Nayler and Anderson, 1965: Sanborn and Langer, 1970; Ong and Bailey, 1973: Kloot, 1973), on the calcium transport and the Ca**++ -activated ATPase activity of the red cell membrane. And also, the effect of p-nitrophenyl phosphate(p-NPP), which wart demonstrated as a energy source of the Ca**++ transport in sarcoplasmic reticulum (Inesi, 1971), was examined on the active Ca**++ transport in the human RBC membrane.
A) Measurement of Ca**++ -activated Mg**++ ATPase: Fresh human blood were obtained and the RBC membrane fragments (RBCMF) were isolated by the method described by Dodge et al., (Dodge et al., 1963) and protein was measured by the method of Lowry et al. (1951).
This RBCMF suspension was preincubated in the incubation mixture which consisted of 5mM MgCl^^2 , 120mM KCI, 0.5 mM CaCl^^2 , and 30mM Tris buffer(pH 7.4) at 37℃ for 10 minutes. The ratio of suspension to incubation medium was 1:1. Reaction wag started by adding 1.7mM ATP to the mixture. 30 minutes after the incubation at 37℃, the reaction was stopped by adding 1ml of 30% trichloroacetic acid. Then the mixture was centrifuged and the amount of inorganic acid (Pi) in the supernatant was measured by the method of Fiske and Subbarow (1929).
To observe the effect of Ni**++ , La**+++ , and prenylamine lactate on the Ca**++ -activated ATPase activity, each of 25μM, 60μM, and 100μM of these substances were independently added in the incubation mixture.
B) Measurement of Ca**45 -transport from the resealed RBC : Citrated where blood was washed 4 times with saline solutions, hemolyzed. and resealed according to the method of Schatzmann(1966).
Incubation of the resealed RBC was started at 37℃ and samplings were done at the time of 0, 1, 2, 5, 10, and 20 minutes. The specimens were centrifuged for 10 minutes. 0.1ml of supernatant was obtained from the each specimen and they were
introduced into counting vials. Ca**45 extruded from the resealed cell into the medium was counted by a Tri-Carb liquid scintillation counter (Model 3320, Parkard Tri-Carb Instrument Co., Inc.).
During the process of resealing RBC, Ni**++ (0, 0.2mM, 0.5mM. and 1.OmM), La**+++ (0, 0.125mM, 0.25mM, and 0.5mM), and prenylamine lactate(0, 0.5mM, and 1.OmM) were independently included in the lysing solution. To observe the effect of these
substances on the Ca**++ transport when they were placed outside of resealed RBC. each of 1.0 mM Ni**++ , 2mM La**+++ and 2mM prenylamine lactate were also simultaneously added to the incubation solution.
C) Measurement of Ca**++ binding to the RBCMF: RBCMF was obtained as described in the preparation of Ca**++ -activated ATPase in red cell membrane. Membrane fragment (0.5mg protein) was spreaded over a cover glass and dried in desiccator at 4℃.
These cover glasses were introduced into a plexiglass rack by the method described by Kang(Kang, 1969).
The racks were immersed at room temperature for 10 minutes in 2mM Tris(pH 7.0) and then incubated at room temperature for 30 minutes in 50mM Tria (pH 7.4) solution containing 0.5, 1.0, 5.0, and 10.0mM Ca**++ with 0.05μci/ml Ca**45 . After incubation, the rack was washed in 2mM Tria solution (pH 7.0) dipping 6 times for 2 seconds each. Then the cover glass was taken out and dried in room temperature. Each cover class was introduced into a counting vial with a counting solution and the radioactivity remaining on the cover class was counted.
To investigate the effect of Ni**++ , La**+++, and prenylamine lactate on Ca**++ binding to the RBCMF, Ni**++ (0, 2.0 mM), La**+++ (0, 0.5 mM), and prenylamine lactate(0, 0.5 mM, 1.0 mM) were seperately mixed into the incubation solution.
Results and conclusions:
1) The effect of p-NPP on the Ca**45 transport in the resealed RBC; The amounts of Ca**45 extruded from the resealed cell into the medium were determined under the presence of 2mM ATP, 5mM p-NPP, and substrate free group.
The amount of Ca**45 extruded from the resealed RBC was increased as incubation progressed. The most significant increase was observes in the ATP added group.
There was a only modest raise of Ca**45 in the substrate free control group. Endogenous ATP in RBC membrane might be responsible for this raise. However, there was no significant difference between the substrate free and p-NPP added group.
2) The effects of Ni**++ , La**+++ , and prenylamine lactate on the Ca**++ -activated Mg**++ ATPase activity; Ca**++ -activated ATPase activity was inhibited by these substances when they were added into the incubation mixtures. The inhibitory effects of these materials on this enzyme system were highly dependent
on concentration of the agents. La**+++ displayed the most prominent inhibitory effect among them.
3) Effects of Ni**++ , La**+++ , and prenylamine lactate on the transport of Ca**45 in the resealed RBC ; These substances when resealed in RBC ghosts as described abode displayed inhibitory effects on the active Ca**45 transport process, and the effects were also concentration dependent. The amounts of Ca**45 extruded from the resealed cell were significantly decreased when the each
substance wan simultaneously added in the outside of RBC. From this finding, it seems to be obvious that Ni**++ , La**+++ , and prenylamine lactate can block not only active Ca**++ transport but also passive diffusion of Ca**++ .
4) The effects of Ni**++ , La**+++ , and prenylamine lactate on Ca**++ binding to RBCMF; In the presence of 0, 0.5mM, and 1.OmM of prenylamine, Ca**++ binding to RBCMF was inhibited in a range of 0.5-1.0mM of calcium. Lineweaver-Burk plot showed a typical competition of prenylamine lactate to calcium binding. Similar results were demonstrated when Ni**++ , and La**+++ were independently mixed into the medium.