Stobadine,(-)-cis-2,8-dimethyl-2,3,4,4a,5,9b-hexahydro-1H-pyrido [4,3-b] indole dihydrochloride was kindly supplied by S. Stolc of the Institute of Experimental Pharmacology, SAS, Bratislava. Aminosteroid, U-74500A and vitamine E analogue U 83836 E were obtained from Upjohn Company, Kalamazoo, MI. All other chemicals were of reagent grade from commercial sources.

Preparation of synaptosomes and ER membranes. Synaptosomes and ER membranes were isolated from rabbit or gerbil whole forebrain according to the method of Edelman. Protein concentration was determined by the Lowry method. Oxidative stress was induced by incubating synaptosomes (2 mg of protein/ml) or ER membranes (7 mg of protein/ml) at 37 oC for 30 min with 0.185 mmol of H2O2 / mg of protein with subsequent addition of 0.185 mmol FeSO4 -EDTA /mg of protein to generate hydroxyl radicals. Lipid peroxidation was initiated by 0.185 mmol FeSO4-EDTA /mg of protein. To study the effect of antioxidants, membranes were preincubated with antioxidant for 15 min at 37 oC prior to the treatment with oxidants.

Measurement of Na, K- ATPase activity. Na, K- ATPase activity was measured as described previously (15) using the coupled enzyme system assay. Na, K- ATPase activity is reported as the total activity minus the activity obtained in the presence of 0.5 mM ouabain. Measurement of Ca-transport. Na-dependent Ca uptake was measured as decribed by Reeves. Synaptosomes preloaded with NaCl were rapidly diluted in Ca uptake choline medium. Samples were then filtered through glass fiber filters and radioactivity was measured. The velocity of Na dependent Ca uptake was determined by linear regression analysis from slopes of the linear parts of curves relating Ca2+ uptake to time. ATP dependent Ca uptake by microsomes was measured by rapid filtration technique. The assay of Ca uptake was supplemented by the measurement of Ca dependent ATP hydrolysis by a coupled enzyme assay as was described previously. Passive Ca permeability of ER membranes was determined as the apparent first-order rate constant for net Ca efflux from Ca preloaded ER vesicles.

Measurement of membrane fluidity and production of conjugated dienes. Labeling of membranes with diphenyl hexatriene (DPH) was performed by Shinitzki and Barenholz. Steady state fluorescence anisotropy was measured at excitation (360 nm) and emission wavelenghts (430 nm), respectively. Fluorescence anisotropy and index of membrane viscosity was calculated as previously described. The quantity of conjugated dienes was determined by monitoring the absorbance at 233 and 215 nm of microsomes dispensed in 10 mM phosphate bufer pH = 7.1 containing 1 percent (w/w) Lubrol PX.

Data analysis. Results are presented as mean ±S.E.M. One way ANOVA was first carried out to test for differences between all groups. Student`s t-test was used to determine differences between the means of individual values. A value of p<0.05 or lower was considered to be statistically significant and are indicated by asterisks.

Incubation of synatosomes isolated from gerbil forebrain with Fe/EDTA for 30 min at 37 °C decreased Na, K- ATPase activity to 50.7 per cent in comparison to control(Table1). Stobadine at a concentration of 0.1 mM significantly protected the activity to 73.3 percent of control values. However, an increase of ST concentration up to 1 mM did not lead to a further improvement of the ATPase activity. A 30 min incubation of synaptosomes in the presence of Fe/EDTA decreased the rate of Ca uptake measured in choline medium to 46.7 percent as compared to controls incubated without RGS. In contrast to Na, K- ATPase activity, the activity of Na,Ca- exchanger was fully protected by ST at a concentration of 0.05 mM (Table1). In addition, synaptosomes incubated with Fe/EDTA showed a significant increase in fluorescence anisotropy of DPH, which corresponds to a decrease in membrane fluidity. Preincubation of synaptosomes with 0.5 mM ST prior to the treatment with oxidant completly protected the membranes against oxidative stress-induced changes (Table 2).

The ratio of A233/A215 is a commonly used parameter to evaluate the extent of lipid peroxidation. We compared changes in this parameter to those in microsomal Ca transport. Incubation of microsomes with both RGS at 37 oC for 30 min led to the loss of membrane efficiency to sequester Ca2+ to 21.85 percent and 3.6 percent of control values (Table3). However, incubation of microsomes under the same condition produced only nonsignificant A233/A215 parameter changes and significant increase were observed after 2 hours of incubation with both oxidant systems (Table 2). On the other hand, both RGS significantly increased the fluorescence anisotropy, e.g. decreased membrane fluidity (Table2). In the absence of RGS, none of the antioxidants changed the fluorescence parameters. In both systems, 0.2 mM ST completely protected the ER membranes from the changes in the fluorescence parameters induced by free radicals. Since the Ca accumulation is mediated by Ca-ATPase, the effect of RGS on Ca -ATPase activity was also investigated. Incubation with both RGS caused a decrease in Ca dependent ATP hydrolysis uncoupled from Ca transport only to 79.6 percent and 62.1 percent of the control values (Table 4).

TABLE 1

The effect of stobadine on Na+, K+-ATPase activity (µmol/mg/min) (A) and Na+ dependent Ca2+ uptake in choline medium (nmol/mg/min) (B) in the presence or absence of Fe2+. Values are given as mean ± SEM of 4-5 experiments, measured in triplicate.

-----------------------------------------------------
              A                         B
-------------------------   --------------------------
Stobadine mM Control Fe2+   Stobadine µM Control Fe2+
------------------------------------------------------
0            2.0     0.98   0             2.31   1.17
0.1          2.15    1.51   10            2.07   1.25
0.5          1.97    1.53   50            2.01   2.07
1.0          2.09    1.49   100           1.99   2.15
------------------------------------------------------

TABLE 2

Effect of oxidative stress on biophysical parameters of neuronal membranes. ST- stobadine

----------------------------------------------------------------------
                   Anis.of syn.  Anis.of micros.  A233/A215 of micros.
----------------------------------------------------------------------
cont.                0. 210           0.217          0.274
cont.+0.2 mM ST      n. d.            0.217          n. d.
cont+0.5 mM ST       0.211            n. d.          n. d.
Fe                   0.227            0.227          0.269
Fe+0.2 mM ST         n. d.            0.217          n. d.
Fe+0.5 mM ST         0.213            n. d.          n. d.
Fe/H2O2              n. d.            0.228          0.279
Fe/H2O+ 0.2 mM ST    n. d.            0.220          n. d.
Fe2+2 h inc.         n. d.            n. d.          0.311
Fe/H2O2+2h inc.      n. d.            n. d.          0.324
----------------------------------------------------------------------

Since the changes in Ca uptake values did not correlate with those observed in Ca-ATPase activities, we supposed that oxidative stress produced changes in membrane lipid component manifested in the increase of passive permeability. In fact, incubation of microsomes with both RGS for 30 min led to a significant increase of Ca2+ permeability to 125.1 and 124.3percent, respectively, in comparison to control (Table4). The antioxidant effect of stobadine was compared with the effect of commonly used membrane soluble chain breaking. antioxidant BHT, less potent tocopherol acetate and aminosteroid U-74500A and of vitamine E derivative U-83836E (Table 3). In contrast to ST, ATP dependent Ca accumulation preincubated with 1 mM tocopherol acetate remained significantly depressed in both RGS. The values reached only 38.7percent and 28.4 percent, repectively. Preincubation of membrane with other membrane-soluble antioxidants protected ER membranes against depression of Ca uptake values and Ca-ATPase activities.

TABLE 3

Effect of Fe and Fe/H2O2 -induced lipid peroxidation on Ca uptake. Values are given as mean±SEM of 4 experiments. 0- no antioxidants, ST- stobadine, E- U 83836E, A- U-74500A, TA- ? tocopherol acetate all used at concentration 0.2 mM, BHT- 0.06 mM.

-------------------------------------------------
Antioxidant   Control      Fe2+/H2O2       Fe2+
-------------------------------------------------
O             6.01±0.05    0.025±0.005  1.12±0.03
ST            6.67±0.12     3.87±0.08   6.69±0.09
E             6.25±0.11     3.94±0.07   6.45±0.12
A             6.08±0.09     4.33±0.12   6.11±0.11
TA            5.33±0.06     1.33±0.09   1.98±0.05
BHT           5.87±0.07     1.09±0.04   5.03±0.08
--------------------------------------------------

TABLE 4

Effect of Fe and Fe/H2O2 on Ca-ATPase uncoupled from Ca transport (nmol P/mg/min) (A) and Ca efflux (min-1) (B). Values are given as mean± SEM of 4 experiments. 0- no antioxidants, ST- stobadine, BHT- butylated hydroxy toluene, both at concentration 0.2 mM.

====================================================
A
Antioxidant   Control      Fe2+/H2O2   Fe2+
--------------------------------------------
0             175±16       111±7       145±7
ST            173±11       167±11      165±9
BHT           188±21       159±9       194±11
----------------------------------------------------
B
Antioxidant   Control      Fe2+/H2O2    Fe2+
----------------------------------------------------
0            0.022±0.005   0.027±0.009  0.027±0.007
ST           0.024±0.006   0.023±0.007  0.0245±0.006
=====================================================

TABLE 5

Effect of antioxidants on the Fe- induced inhibition of Ca-ATPase (nmol P/mg/min). 0- no antioxidants, 0.1ST, 0.2ST- stobadine concetration in mM, 0.1GS, 0.2GS- glutathione concetration in mM, GST1- 0.04 mM stobadine + 0.16 mM glutathione, GST2- both antioxidants with 0.1mM concentration.

-----------------------------------
Antioxidant    Control      Fe2+
-----------------------------------
0              173±15        55±5
0.1 ST         198±11        93±7
0.2 ST         191±9        134±11
1 GST          188±11       136±9
2 GST          181±12       174±7
0.1 GS         180±11        81±9
0.2 GS         172±15        52±7
-----------------------------------

The extent of the protection is dependent on the experimental conditions and on the dose and nature of the antioxidant used. While in the case of Fe/EDTA all antioxidants were fully effective, significant differences were observed in the H2O2/Fe/EDTA system. Antioxidants were only partially effective in the prevention of Ca2+ uptake depression. U-74500A, U-83836E and ST protected the Ca2+ uptake value to 60.4 percent, 70.1percent, and 55.7percent of the control values, respectively. Inclusion of 0.06 mM BHT yielded only weak recovery of the Ca uptake. In addition, ST fully recovered the increasead Ca permeability induced by Fe/EDTA as well as fully protected Ca -ATPase against Fe2+ induced inhibition (Table 4). Incubation of microsomes with Fe/EDTA led to the inhibition of Ca-ATPase to 34.8percent of control. 0.2 mM ST partially prevented the inhibition of Ca -ATPase, however the effect of 0.1 mM ST in combination with 0.1 mM glutathione was significantly higher than the effect of 0.2 mM ST (Table 5). The effect of 0.04 mM ST in combination with 0.16 mM glutathione was comparable to the effect of 0.2 mM stobadine. Both 0.1 mM ST and 0.1 mM of glutathione led to the increase of Ca-ATPase activity. However, these changes were not statistically significant. Glutathione, a water soluble antioxidant, at a concentration of 0.2 mM appeared to be almost ineffective.

Iron mediated damage to neuronal cells is thought to be involved in the pathogenesis of ischemia-reperfusion injury, Parkinson`s disease and Alzheimer`s disease. The deleterious effect of iron is supposed to be mediated by the potency of Fe ions to generate ROS via their reaction with oxygen or hydrogen peroxide. In this study, we investigated the effect of oxidative stress in vitro induced by two RGS (Fe/EDTA and Fe-EDTA plus H2O2) on synaptosomal and microsomal ion transport systems as well as on the membrane fluidity. Incubation of gerbil synaptosomes with Fe/EDTA resulted in an inhibition of Na,K-ATPase activity and of Na-dependent Ca uptake, as well as in a decrease of membrane fluidity. A ROS induced inhibition of Na, K-ATPase activity has been shown in several studies. Although the exact mechanism of iron-mediated inhibition is not known, inhibition via lipid peroxidation of the membrane was proposed by several authors. Another possibility involves the modification of protein molecules either by direct oxidation or by modification mediated by products of lipid peroxidation.In our study, lipid-soluble antioxidants only partially protected Na, K-ATPase activity from oxidative stress, indicating a more complex mechanism of inhibition of this protein. While in the case of Na, K-ATPase stobadine was only partially effective in its protection against iron induced inhibition, the activity of Na,Ca-exchange was fully protected by ST. Little is known about the effect of ROS on Na,Ca-exchange in brain tissue. In contrast to Na,K-ATPase, Na,Ca-exchange was not affected by amyloid beta peptide a lipid peroxidation iniciator). Direct incubation of synaptosomes with Fe/ascorbate only slightly decreased Na-dependent Ca uptake. However, the initial rate was not shown and cannot be compared with our results.

As in our previous study, the rate of microsomal Ca accumulation was the most affected parameter of ROS- induced damage to the membrane. These results indicate that both modifications of the polypeptide chain and decrease of membrane fluidity and the increase of passive permeability play a role in the process of inhibition. The assumption that the membrane is a primary target of ROS was supported by the fact that the ATPase activity was fully protected exclusively by membrane soluble antioxidants and ST fully recovered the increase in Ca2+ permeability. Although we did not determine the significant changes in the ratio of A233/A215, it was shown that the potency of two RGS to depress Ca accumulation correlated well with the potencies to decrease membrane fluidity. The efficacy of ST to prevent Ca transport changes was higher in comparison to that observed in the case of inhibition red blood cell Ca-ATPase by t-butyl hydroperoxide. In our experiments, ST seems to be at least as effective as BHT, which is considered to be a good chain-breaking antioxidant. In contrast to ST, tocopherol acetate was less potent to confer the defence against ROS- initiated changes. The protective effect of ST on membrane structures is consistent with its antioxidant activity, which was demonstrated on liposomes, microsomal membrane and erythrocytes.

Although the preincubation of microsomes with ST partially protected the Ca-ATPase activity, the efficacy of prevention was dependent also on the presence of glutathione, a water soluble antioxidant. While in heart and skeletal muscle thiol compounds protected Ca-ATPase from ROS damage, in this study inclusion of 0.1 mM reduced glutathione alone had no protective effect. On the other hand, Rohn et aldocumented an ability of dithiotreitol to protect Ca-ATPase from t-butyl peroxide induced inhibition. Although other processes leading to the amino acid modifications may also play a role in Fe induced inhibition of Ca-ATPase, our results using a combination of ST and glutathione support the view that probably membrane is the primary site of Fe attack. Inclusion of a glutathione-ST mixture protects the membrane even at such Fe concentration at which ST alone was ineffective (data not shown). It seems that inhibition of Ca-ATPase is not based on direct protein modifications, but rather by changes in membrane fluidity and protein-lipid interactions.