File Name: SLS1_05a.DOC

EXPERIMENT SLS-1-05A

ERYTHROCYTE METABOLISM AND MEMBRANE STATE

[PIs]{.underline}

[Russia:]{.underline}

S. M. Ivanova, Institute of Biomedical Problems, Moscow

[USA:]{.underline}

A. Merrill, Emory University, Atlanta, Georgia

[Co-Investigators:]{.underline}

N. E. Spitsyna, Institute of Biomedical Problems, Moscow

O. I. Labetskaya, Institute of Biomedical Problems, Moscow

[Introduction]{.underline}

The rat experiments on Cosmos-1667 demonstrated an enhanced rate of
energy metabolism in red blood cells suggestive of both higher
compensatory potential of the cell and possible rejuvenation of blood
cells in response to space flight effects \[4\]. The rat experiments on
Cosmos-1887 and Cosmos-2044 also showed changes in the metabolic status
of erythrocytes indicating metabolic shifts at the cellular and membrane
level.

The present investigation was a continuation of the above experiments
the purpose of which was to study the effects of weightlessness and
return to the Earth\'s gravity on erythrocyte metabolism, as evaluated
by energy forming and redox systems, as well as to measure Na+, K+ and
Ca++-dependent ATPases, lipids and phospholipids in the membrane.

[Materials and Methods]{.underline}

The above parameters were measured in frozen erythrocytes of SLS-1 rats.
The rats were subdivided into 5 groups, 5 animals each: BC - basal
control, R+0 - flight rats dissected immediately after recovery, C+0 -
controls for R+0 rats, R+9 - flight rats dissected 9 days after
recovery, and C+9 - controls for R+9 rats.

Metabolic parameters, i.e., content of adenosine triphosphate (ATP) and
lactate, activity of lactate dehydrogenase (LDH), hexokinase (HK), level
of reduced glutathione and activity of glucose-6-phosphate dehydrogenase
(G6PD), were measured spectrophotometrically \[8\]. Transport ATPase was
measured in terms of inorganic phosphate increase which was built up as
a result of ATP dephosphorylation during incubation (for 1 hour at 38
oC) \[5\]. Lipids and phospholipids in erythrocyte membranes were
detected by means of thin-layer chromatography \[6\]. The results
obtained were calculated per g hemoglobin (Hb) which was determined,
using the cyan methemoglobin method \[2\].

Analysis of the data from the three control groups (BC, C+0 and C+9) did
not reveal any significant differences between them. Due to this, the
results obtained from the flight rats R+0 and R+9 were compared with the
pooled control data (C) derived from 15 animals.

[Results and Discussion]{.underline}

The data presented in Table 1 show changes in the metabolic parameters
under study: slight increase of ATP, significant increase of reduced
glutathione and decrease

of lactate. Almost all the metabolic parameters of the flight rats were
identical to the controls 9 days after flight.

Increase in intracellular ATP can be produced by both enhancement of the
energy forming process, i.e., glycolysis, and reduction in the
utilization of this nucleotide. The latter can be associated with a
decline in the allosteric ATP - hemoglobin interaction \[3\] and decline
of energy-dependent processes at the membrane level, viz., ion transport
and phosphorylation of protein and lipid components \[1\].

Study of the parameters characterizing the membrane state (Table 2)
revealed a decline in Na+ and K+-dependent ATPases in the R+0 rats and a
significant increase in Ca++-dependent ATPases in the R+0 and R+9 rats.
At R+9 day Na+ and K+-dependent ATPases increased, exceeding the value
recorded in the controls.

Study of membrane lipids did not detect any significant changes in
either flight group as compared to the controls. However, the R+0 rats
showed a trend toward lower phospholipids and higher triglycerides.

Study of phospholipid fractions revealed more noticeable changes. The
R+0 rats showed a significant decline of sphyngomyelin and an increase
of lysophosphatidyl choline. This is suggestive of a potential
development of destructive processes at the membrane level. These
changes were accompanied by a significant increase of reduced
glutathione which plays a major part in the elimination of free radical
oxidation reactions and in the maintenance of reduced SH-groups of the
protein moiety of hemoglobin, membrane structural proteins and enzymes
\[8\], which can be regarded as a cellular compensatory response. It is
very probable that increase of Ca++-dependent ATPase in the R+0 and R+9
rats is of similar type: it is to enhance the Ca pump in order to
eliminate excessive amounts of Ca++ in the membrane.

In summary, the above findings indicate that the metabolic status of the
cell underwent changes, which were probably induced by changes in the
structure and function of the cellular membrane. The changes were
adaptive since 9 days after recovery the parameters under study returned
to the norm.

[Table 1]{.underline}

Metabolic parameters in red blood cells

  ----------------- -----------------------------------------------------
  Parameter         Rat group

  ----------------- -----------------------------------------------------

  ----------------- ----------------- ----------------- -----------------
                    C (n = 15)        R+0 (n = 5)       R+9 (n = 5)

  ----------------- ----------------- ----------------- -----------------

  ----------------- --------------------- --------------------- ---------------------
  ATP, μmol/g Hb    2.47[+]{.underline}   3.36[+]{.underline}   2.93[+]{.underline}
                    0.33                  0.46                  0.33

  ----------------- --------------------- --------------------- ---------------------

  ----------------- ----------------- ----------------- -----------------
  Lactate, μmol/g   13.17             5.04              10.53
  Hb                [+]{.underline}   [+]{.underline}   [+]{.underline}
                    2.04              0.62\*            0.77

  ----------------- ----------------- ----------------- -----------------

  ----------------- ----------------- ----------------- -----------------
  LDH, μmol NADH/g  34.64             33.56             34.40
  Hb/min            [+]{.underline}   [+]{.underline}   [+]{.underline}
                    1.52              2.66              1.52

  ----------------- ----------------- ----------------- -----------------

  ----------------- ----------------- ----------------- -----------------
  G6PD, μmol        7.53              6.59              6.51
  NADPH/g Hb/min    [+]{.underline}   [+]{.underline}   [+]{.underline}
                    0.51              0.62              0.62

  ----------------- ----------------- ----------------- -----------------

  ----------------- ----------------- ----------------- -----------------
  HK, μmol NADPH/g  1.13              1.09              1.31
  Hb/min            [+]{.underline}   [+]{.underline}   [+]{.underline}
                    0.17              0.10              0.04

  ----------------- ----------------- ----------------- -----------------

  ------------------- ----------------- ----------------- -----------------
  Reducedglutathion   6.34              9.74              6.57
  μmol/g Hb           [+]{.underline}   [+]{.underline}   [+]{.underline}
                      0.69              0.85\*            1.00

  ------------------- ----------------- ----------------- -----------------

Note: \* stands for p \< 0.01

[Table 2]{.underline}

Parameters characterizing membrane status

  ----------------- -----------------------------------------------------
  Parameter         Rat group

  ----------------- -----------------------------------------------------

  ----------------- ----------------- ----------------- -----------------
                    C (n = 15)        R+0 (n = 5)       R+9 (n = 5)

  ----------------- ----------------- ----------------- -----------------

  -----------------------------------------------------------------------
  Activity, μg Pi/g Hb erythrocytes/hour:

  -----------------------------------------------------------------------

  ----------------- ----------------- ----------------- -----------------
  Na+ , K+ - ATPase 114\. 45          42.00             178.10
                    [+]{.underline}   [+]{.underline}   [+]{.underline}
                    14.1              10.40\*           13.2\*\*

  ----------------- ----------------- ----------------- -----------------

  ----------------- ----------------- ----------------- -----------------
  Ca++ - ATPase     713.00            982.00            985.00
                    [+]{.underline}   [+]{.underline}   [+]{.underline}
                    63.0              23.00\*           56.0\*\*

  ----------------- ----------------- ----------------- -----------------

  -----------------------------------------------------------------------
  Membrane lipids, %

  -----------------------------------------------------------------------

  ----------------- ----------------- ----------------- -----------------
  PL                21.44             18.29             20.51
                    [+]{.underline}   [+]{.underline}   [+]{.underline}
                    0.93              3.45              0.90

  ----------------- ----------------- ----------------- -----------------

  ----------------- ----------------- ----------------- -----------------
  FC                34.66             35.64             33.06
                    [+]{.underline}   [+]{.underline}   [+]{.underline}
                    1.29              3.81              2.58

  ----------------- ----------------- ----------------- -----------------

  ----------------- ----------------- ----------------- -----------------
  TG                17.32             19.70             17.90
                    [+]{.underline}   [+]{.underline}   [+]{.underline}
                    0.48              1.25              0.71

  ----------------- ----------------- ----------------- -----------------

  ----------------- ----------------- ----------------- -----------------
  NEFA              11.54             11.26             12.62
                    [+]{.underline}   [+]{.underline}   [+]{.underline}
                    1.79              1.20              1.36

  ----------------- ----------------- ----------------- -----------------

  ----------------- ----------------- ----------------- -----------------
  ECS               15.03             15.11             15.90
                    [+]{.underline}   [+]{.underline}   [+]{.underline}
                    0.75              1.02              0.47

  ----------------- ----------------- ----------------- -----------------

  -----------------------------------------------------------------------
  Membrane phospholipids, %

  -----------------------------------------------------------------------

  ----------------- ----------------- ----------------- -----------------
  LPC               1.28              3.88              1.18
                    [+]{.underline}   [+]{.underline}   [+]{.underline}
                    1.07              1.55              2.85

  ----------------- ----------------- ----------------- -----------------

  ----------------- ----------------- ----------------- -----------------
  SPM               20.28             13.63             14.49
                    [+]{.underline}   [+]{.underline}   [+]{.underline}
                    1.78              0.73\*\*          1.18

  ----------------- ----------------- ----------------- -----------------

  ----------------- ----------------- ----------------- -----------------
  PC                42.42             44.41             38.63
                    [+]{.underline}   [+]{.underline}   [+]{.underline}
                    2.68              6.17              1.88

  ----------------- ----------------- ----------------- -----------------

  ----------------- ----------------- ----------------- -----------------
  PEA               26.86             28.21             31.97
                    [+]{.underline}   [+]{.underline}   [+]{.underline}
                    2.83              1.52              1.73

  ----------------- ----------------- ----------------- -----------------

  ----------------- ----------------- ----------------- -----------------
  NFP-1             7.17              9.85              13.72
                    [+]{.underline}   [+]{.underline}   [+]{.underline}
                    3.59              3.56              2.86

  ----------------- ----------------- ----------------- -----------------

Designations: \* stands for p \< 0.001, \*\* for p \< 0.01

PL - phospholipids, FC - free cholesterol, TG - triglycerides, NEFA -
nonesterified fatty acids, ECS - cholesterol esters, LPC -
lysophosphatidyl choline, SPM - sphyngomyelin, PC - phosphatidyl
choline, PEA - phosphatidyl ethanolamine, NFP-1 - nonidentified
fractions of phosphatide acid.

[References]{.underline}

1\. Boldyrev, A. S. Introduction into Membrane Biochemistry. Vysch.
Shkola, Moscow, 1986, 111 pp. (in Russian).

2\. Derviz, G. V. Measurement of Hemoglobin in Blood. Laborator. Delo,
1973,

No. 2, pp. 67-72 (in Russian).

3\. Irzhak, L. I. Respiratory Function of Blood in the Course of
Individual Development of Mammals. Nauka, Moscow, 1974, 183 pp. (in
Russian).

4\. Ushakov, A. S., Ivanova, A. M., Brantova, S. S. et al. Metabolic
Processes in Erythrocytes of Rats Flown on Cosmos-1667. Abstr. XXI
Meeting of Intercosmos Joint Working Group on Space Biology and
Medicine, Poland, 1988, p. 30 (in Russian).

5\. Yakusheva, A. I., Orlova, L. I. A Method of Measuring ATPases in
Human Blood Hemolysates. Laborator. Delo, 1970, No. 8, pp. 479-501 (in
Russian).

6\. Stal, E. Thin-Layer Chromatography. Mir, Moscow, 1965, 508 pp. (in
Russian).

7\. Beutler, M. D. Red Cell Metabolism. A Manual of Biochemical Methods.
New York - London, 1971, pp. 11-45, 96-100.

8\. Kosower, N. J., Kosower, E. M. The Significance of Intracellular
Glutathione. In: Red Cell Structure and Metabolism. Acad. Press, New
York - London, 1971, 161 pp.

[Summary]{.underline}

The present investigation demonstrated changes in cellular metabolism,
probably, caused by alterations in the structure and function of
cellular membranes. Those latter included changes in phospholipids and
enhanced rate of the Na+ , K+, Ca++-pump.The above changes were required
to preserve cell integrity. They were adaptive in nature because 9 day
after recovery they returned to the norm.