Influence of divalent cations Ca²⁺ and Zn²⁺ on the activation of posthypertonic hemolysis of human erythrocytes
Human erythrocytes (RBC) were incubated for various time at 37 °C in hypertonic solutio,ts of NaCl (1.5 M) or sucrose (1.2 M) and then rehydrated in isotonic NaCl or sucrose media in the presence or absence of divalent cations Ca²⁺ and Zn²⁺. After equilibration in hypertonic sucrose both cations sig...
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Інститут молекулярної біології і генетики НАН України
1999
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| Цитувати: | Influence of divalent cations Ca²⁺ and Zn²⁺ on the activation of posthypertonic hemolysis of human erythrocytes / S.V. Patelaros // Биополимеры и клетка. — 1999. — Т. 15, № 1. — С. 43-48. — Бібліогр.: 15 назв. — англ. |
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Patelaros, S.V. 2019-06-17T15:28:40Z 2019-06-17T15:28:40Z 1999 Influence of divalent cations Ca²⁺ and Zn²⁺ on the activation of posthypertonic hemolysis of human erythrocytes / S.V. Patelaros // Биополимеры и клетка. — 1999. — Т. 15, № 1. — С. 43-48. — Бібліогр.: 15 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000504 https://nasplib.isofts.kiev.ua/handle/123456789/155919 Human erythrocytes (RBC) were incubated for various time at 37 °C in hypertonic solutio,ts of NaCl (1.5 M) or sucrose (1.2 M) and then rehydrated in isotonic NaCl or sucrose media in the presence or absence of divalent cations Ca²⁺ and Zn²⁺. After equilibration in hypertonic sucrose both cations significantly increased the extent of hemolysis at concentration dependent manner, but show more complex response after equilibration in hypertonic NaCl media. In thin case both Ca²⁺ and Zn²⁺ ions lose their activation ability during rehydration in isotonic NaCl media, and Ca2+ did so in isotonic sucrose. Dehydration of cells in hypertonic sucrose solutions in the presence of 100 mM of cations Na⁺, K⁺ and Mg²⁺ leads to decrease the extent of lysis after rehydration but dues not influence activation ability of Ca²⁺ and Zn²⁺, whereas addition of Ca²⁺ to hypertonic sucrose solution completely abolishes activation effect of both ions. The model of posthypertonic lysis is presented according to which action of cations Ca²⁺ and Zn²⁺ may be explained by their specific binding to activatory and inhibitory membrane sites, regulating membrane permeability during reswelling from hypertonic salines. Еритроцити людини інкубували в різні строки при температурі 37 °С у гіпертонічних розчинах 1,5 М NaCl і 1,2 М сахарози та регідратували в ізотонічних середовищах NaCl і сахарози в присутності та відсутності дивалентних катіонів Са²⁺ і Zn²⁺ . Встановлено, що останні здатні активувати постгіпертонічний лізис (ПЛ) еритроцитів, якщо початкова дегідратація здійснюється в сахарозному середовищі. Такої активації не спостерігається у випадку лізису, індукованого різними гемолітичними агентами (мелітин, тритон Х-100 і т. і.), де ці катіони проявляють лшае пригнічуючий ефект. Попередня інкубація клітин у гіпертонічному розчині NaCl згаданий ефект усуває. Обробка дегідратованих еритроцитів катіонами Na⁺ , К⁺, Mg²⁺ призводить до пригнічену гемолізу, однак не впливає на активуючу здатність іонів Са²⁺ і Zn²⁺ . У випадку дегідратації клітин у присутності іонів Са²⁺ катіони втрачають здатність активувати ПЛ. При переносі еритроцитів із гіпертонічних в ізотонічні середовища відбувається формування мембранних гемолітичних пор, до структури яких входять два активуючих іоноспецифічних і один блокуючий сайти для дивалентних катіонів. Вказані сайти здатні регулювати формування мембранних поиікоджень при взаємодії з катіонами Са²⁺ і Zn²⁺ . ритроциты человека инкубировали в разное время при температуре 37 С в гипертонических растворах 1,5 М NaCl и 1,2 М сахарозы и регидратировали в изотонических средах NaCl и сахарозы.ув присутствии или отсутствие дивалентных катионов Са²⁺ и Zn²⁺ . Установлено, что последние способны активировать постгипертонический лизис (ПЛ) эритроцитов если начальная дегидратация происходит в сахарозной среде. Данной активации не наблюдается в случае лизиса, индуцированного разными гемолитическими агентами (мелиттин, тритон Х-100 и т. д.), где эти катионы оказывают только ингибирующий эффект. Предварительная инкубация клеток в гипертоническом растворе NaCl указанный эффект устраняет. Обработку дегидратированных эритроцитов катионами Na⁺ , К⁺ , Mg²⁺ приводит к ингибированию гемолиза, однако не влияет на активирующую способность ионов Са²⁺ и Zn²⁺ . В случае дегидратации клеток в присутствии ионов Са²⁺ катионы утрачивают способность активировать ПЛ. При переносе эритроцитов из гипертонических в изотонические среды происходит формирование мембранных гемолитических пор, в структуру которых входят два активирующих ионоспецифических и один блокирующий сайты для дивалентных катионов. Указанные сайты способны регулировать формирование ме^ибранн^іх повреждений при взаимодействии с катионами Са²⁺ и Zn²⁺ . en Інститут молекулярної біології і генетики НАН України Биополимеры и клетка Клеточная биология Influence of divalent cations Ca²⁺ and Zn²⁺ on the activation of posthypertonic hemolysis of human erythrocytes Активуючий вплив дивалентния катіонів Ca²⁺ і Zn²⁺ на постгіпертонічний лізис еритроцитів людини Активирующее влияние дивалентных катионов Са²⁺ и Zn²⁺ на пост гипертонический лизис эритроцитов человека Article published earlier |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
| title |
Influence of divalent cations Ca²⁺ and Zn²⁺ on the activation of posthypertonic hemolysis of human erythrocytes |
| spellingShingle |
Influence of divalent cations Ca²⁺ and Zn²⁺ on the activation of posthypertonic hemolysis of human erythrocytes Patelaros, S.V. Клеточная биология |
| title_short |
Influence of divalent cations Ca²⁺ and Zn²⁺ on the activation of posthypertonic hemolysis of human erythrocytes |
| title_full |
Influence of divalent cations Ca²⁺ and Zn²⁺ on the activation of posthypertonic hemolysis of human erythrocytes |
| title_fullStr |
Influence of divalent cations Ca²⁺ and Zn²⁺ on the activation of posthypertonic hemolysis of human erythrocytes |
| title_full_unstemmed |
Influence of divalent cations Ca²⁺ and Zn²⁺ on the activation of posthypertonic hemolysis of human erythrocytes |
| title_sort |
influence of divalent cations ca²⁺ and zn²⁺ on the activation of posthypertonic hemolysis of human erythrocytes |
| author |
Patelaros, S.V. |
| author_facet |
Patelaros, S.V. |
| topic |
Клеточная биология |
| topic_facet |
Клеточная биология |
| publishDate |
1999 |
| language |
English |
| container_title |
Биополимеры и клетка |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Активуючий вплив дивалентния катіонів Ca²⁺ і Zn²⁺ на постгіпертонічний лізис еритроцитів людини Активирующее влияние дивалентных катионов Са²⁺ и Zn²⁺ на пост гипертонический лизис эритроцитов человека |
| description |
Human erythrocytes (RBC) were incubated for various time at 37 °C in hypertonic solutio,ts of NaCl (1.5 M) or sucrose (1.2 M) and then rehydrated in isotonic NaCl or sucrose media in the presence or absence of divalent cations Ca²⁺ and Zn²⁺. After equilibration in hypertonic sucrose both cations significantly increased the extent of hemolysis at concentration dependent manner, but show more complex response after equilibration in hypertonic NaCl media. In thin case both Ca²⁺ and Zn²⁺ ions lose their activation ability during rehydration in isotonic NaCl media, and Ca2+ did so in isotonic sucrose. Dehydration of cells in hypertonic sucrose solutions in the presence of 100 mM of cations Na⁺, K⁺ and Mg²⁺ leads to decrease the extent of lysis after rehydration but dues not influence activation ability of Ca²⁺ and Zn²⁺, whereas addition of Ca²⁺ to hypertonic sucrose solution completely abolishes activation effect of both ions. The model of posthypertonic lysis is presented according to which action of cations Ca²⁺ and Zn²⁺ may be explained by their specific binding to activatory and inhibitory membrane sites, regulating membrane permeability during reswelling from hypertonic salines.
Еритроцити людини інкубували в різні строки при температурі 37 °С у гіпертонічних розчинах 1,5 М NaCl і 1,2 М сахарози та регідратували в ізотонічних середовищах NaCl і сахарози в присутності та відсутності дивалентних катіонів Са²⁺ і Zn²⁺ . Встановлено, що останні здатні активувати постгіпертонічний лізис (ПЛ) еритроцитів, якщо початкова дегідратація здійснюється в сахарозному середовищі. Такої активації не спостерігається у випадку лізису, індукованого різними гемолітичними агентами (мелітин, тритон Х-100 і т. і.), де ці катіони проявляють лшае пригнічуючий ефект. Попередня інкубація клітин у гіпертонічному розчині NaCl згаданий ефект усуває. Обробка дегідратованих еритроцитів катіонами Na⁺ , К⁺, Mg²⁺ призводить до пригнічену гемолізу, однак не впливає на активуючу здатність іонів Са²⁺ і Zn²⁺ . У випадку дегідратації клітин у присутності іонів Са²⁺ катіони втрачають здатність активувати ПЛ. При переносі еритроцитів із гіпертонічних в ізотонічні середовища відбувається формування мембранних гемолітичних пор, до структури яких входять два активуючих іоноспецифічних і один блокуючий сайти для дивалентних катіонів. Вказані сайти здатні регулювати формування мембранних поиікоджень при взаємодії з катіонами Са²⁺ і Zn²⁺ .
ритроциты человека инкубировали в разное время при температуре 37 С в гипертонических растворах 1,5 М NaCl и 1,2 М сахарозы и регидратировали в изотонических средах NaCl и сахарозы.ув присутствии или отсутствие дивалентных катионов Са²⁺ и Zn²⁺ . Установлено, что последние способны активировать постгипертонический лизис (ПЛ) эритроцитов если начальная дегидратация происходит в сахарозной среде. Данной активации не наблюдается в случае лизиса, индуцированного разными гемолитическими агентами (мелиттин, тритон Х-100 и т. д.), где эти катионы оказывают только ингибирующий эффект. Предварительная инкубация клеток в гипертоническом растворе NaCl указанный эффект устраняет. Обработку дегидратированных эритроцитов катионами Na⁺ , К⁺ , Mg²⁺ приводит к ингибированию гемолиза, однако не влияет на активирующую способность ионов Са²⁺ и Zn²⁺ . В случае дегидратации клеток в присутствии ионов Са²⁺ катионы утрачивают способность активировать ПЛ. При переносе эритроцитов из гипертонических в изотонические среды происходит формирование мембранных гемолитических пор, в структуру которых входят два активирующих ионоспецифических и один блокирующий сайты для дивалентных катионов. Указанные сайты способны регулировать формирование ме^ибранн^іх повреждений при взаимодействии с катионами Са²⁺ и Zn²⁺ .
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0233-7657 |
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https://nasplib.isofts.kiev.ua/handle/123456789/155919 |
| citation_txt |
Influence of divalent cations Ca²⁺ and Zn²⁺ on the activation of posthypertonic hemolysis of human erythrocytes / S.V. Patelaros // Биополимеры и клетка. — 1999. — Т. 15, № 1. — С. 43-48. — Бібліогр.: 15 назв. — англ. |
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ISSN 0233-7657. Биополимеры и клетка. 1999. Т. 15. № 1
Influence of divalent cations Ca24 and Zn2
on the activation of posthypertonic hemolysis
of human erythrocytes
S . V. P a t e l a r o s
Institute of Problems of Cryobiology and Cryoraedicine of National Academy of Sciences of Ukraine
23 Pereyasiavska str., Kharkiv, 310015, Ukraine
Human erythrocytes (RBC) were incubated for various time at 37 °С in hypertonic solutions of NaCl
(1.5 M) or sucrose (1.2 M) and then rehydrated in isotonic NaCl or sucrose media in the presence or
absence of divalent cations Ca2* and Zn*. After equilibration in hypertonic sucrose both cations
significantly increased the extent of hemolysis at concentration dependent manner, but show more complex
response after equilibration in hypertonic NaCl media In this case both Ca2+ and Zn1* ions love their
activation ability during rehydration in isotonic NaCl media, and Ca"* did so in isotonic sucrose.
Dehydration of cells in hypertonic sucrose solutions in the presence of 100 mM of cations Na+, fC and
Mg leads to decrease the extent of lysis after rehydration but does not influence activation ability of Ca2*
and Zn+, whereas addition of Ca2 to hypertonic sucrose solution completely abolishes activation effect of
both ions. The model of posthypertonic lysis is presented according to which action of cations Ctf + and
Zn2+ may be explained by their specific binding to activatory and inhibitory membrane sites, regulating
membrane permeability during reswelling from hypertonic salines.
Introduction. It is well known that an attempt to
transfer red blood cells from hypertonic media to
isotonic one bring about RBC membrane damage and
hemolysis which is called posthypertonic hemolysis
[1 ]. Data, obtained by several authors [1—4 ] suggest
a significant role of composition of hypertonic solu-
tion, time of exposure, tonicity of the solution after
dilution, cell volume and initial tonicity after which
RBC were subjected to hypertonic treatment by
dialysis [51 or freezing and subsequent rehydration
or thawing [2 ]. It is still remain unclear why RBC can
not restore their initial volume after dehydration
without membrane damage.
Divalent cation such as Ca2" and Zn2* are effe-
ctive protectors against membrane damage inflicted by
variety of hemolytic agents, including viruses, bacte-
rial and animal toxins, and detergents [3, 6—9[ as
well as hypotonic conditions [101. We have shown
that this is also true for posthypertonic hemolysis
[11]. These common features suggest an universality
of the mechanism of pore formation in RBC mem-
© S. V. PATELAROS, 1999
brane, which is partially independent on hemolytic
stimuli. In this study we show that divalent cations
Ca2" and Zn2+ together with their inhibiting effect
[11] exhibit additional property to activate post-
hypertonic hemolysis under conditions where cells
were rehydrated in the cation contained media This
property demonstrates distinction between posthy-
pertonic membrane pores and those initiated by
action of other lysins thus suggesting differences in
the mechanism of their formation and molecular
structure.
Materials and Methods. Human RBC were ob-
tained from a clinical blood bank. They were washed
3 times in a 10-fold excess of tris-buffered saline
(TBS = 150 mM NaCl, 20 mM tris-HCl, pH 7.4)
using 2.000 g for 3 min on each occasion. 0.1 tnl of
RBC were incubated in 0.90 ml of 1.2 M sucrose or
1.5 M NaCl both with 5 rnM phosphate buffer at pH
7.4 at 37 °С and then 10 μ\ of erythrocyte suspension
were transferred into 1 ml of isotonic media of various
composition at room temperature. Mter 5 т і й in-
cubation the cell suspension was centrifuged (2.000 g,
3 min) and hemoglobin content in the supernatant
43
PATIiLAROS S. V.
was determined by absorption at 415 nm. The per-
centage hemolysis was calculated from a comparison
with the optical density of a fully lysed sample.
The distribution of cell volumes in the samples
was measured using a Coulter-type apparatus with an
orifice 50 μχη long * 5 μ\Ά in diameter [12, 13] using
a current < 0,3 mA. 2-Ю'5 cells were sampled in each
measurement and the mean linear velocity through
the orifice was < 1 m/sec: the cells were not sig-
nificantly deformed [12].
Results. Fig. 1 shows the time dependence of
posthypertonic hemolysis of RBC, dehydrated in
hypertonic NaCl and sucrose media, and rehydrated
in isotonic electrolyte (NaCl) and non-electrolyte
(sucrose) media in the presence or absence of divalent
cations. Prolonged cell incubation under hypertonic
conditions results in a gradual increase in the extent
of hemolysis.
The effect of cations depends on the type of both
dehydrative and rehydrative media. Zn2" and Ca
cations increased lysis by 2- and 3-fold, respectively,
relative to control value after dehydration in sucrose
hypertonic medium, independently of the type of
rehydrative medium. However after dehydration in
NaCl hypertonic medium Zn2* cations fail to produce
activation of hemolysis during rehydration in elec-
trolyte medium, whereas Ca2+ ions inhibit it (Fig. 1,
A). There was no appreciable effect of Ca2" і non-
electrolyte rehydrative medium though it slightly
activated hemolysis at short-term exposure, and Zn "
ions stimulated lysis (Fig. 1, C). Stimulation of
posthypertonic lysis by divalent cations was a function
of cation concentration.
Fig. 2 and 3 show the dependence of post-
hypertonic hemolysis on the concentration of Ca2" and
Zn2+ ions during rehydration in NaCl and sucrose
isotonic media. In both type of rehydrative medium
Ca2+ ions activated posthypertonic hemolysis of cells,
dehydrated in hypertonic sucrose, while poorly af-
fected hemolysis of RBC, dehydrated in hypertonic
NaCl and rehydrated in isotonic sucrose. The extent
of posthypertonic hemolysis of these RBCs was con-
siderably reduced during rehydration in sucrose-
containing media (Fig. 2, B). In contrast to Ca2", Znz*
ions lose their stimulatory ability only subsequent
rehydration in NaCl medium (Fig. 3, A). Moreover,
the concentration of Zn2" ions which is required for
activating lysis, has shown to be three order of
magnitude lower then the corresponding concentration
of Ca2" ions (compare Fig. 2 and 3). These data show
that treatment of cells in hypertonic NaCl solution
followed by rehydration in isotonic NaCl solution
eliminates the effect of posthypertonic hemolysis
activation caused by both Ca2" and Zn2" ions. Hence,
Fig. 1. Effect of divalent cations on post-
hypertonic hemolysis. RBC were incubated
selected time in hypertonic media of 1.5 M
NaCI (A, C) or 1.2 M sucrose (B, D) at
37 °С following by dehydration ir. isotonic
NaCi (A, B) or sucrose (C, D) solutions in
the absence U) or presence (2) of 100 mM
Ca2+ or 0,1 mM Zn2+ (3)
44
INFLUENCE OF Ca2+ AND Zj>2+ ON THE ACTIVATION OF HEMOLYSIS
0 20
Concentration, mM
Fig . 2. E f f e c t s of Ca 2 + ions on
p o s t h y p e r t o n i c hemolysis . KBC were
incubated 15 min in hypertonic solutions of
1.5 M NaCi </) and 1.2 M sucrose (2) and
then transferred to isotonic Natn (Л) and
sucrose (B) media contained various amount
cf Ca2+
the alteration occuring in the membrane and cyto-
plasm of cells, dehydrated in saline medium, differ
from those in non-electrolyte medium (sucrose). This
may be due to different changes in transmembrane
potential and effluxes of cations and anions, resulting
in various alterations in the intracellular content and
pH.
Hypertonic non-electrolyte medium causes a dec-
rease in the content of K~ and СГ ions in cytoplasm,
accompanied by an increase in pH [12]. In hypertonic
electrolyte medium leakage of K+ cations will not
result in the corresponding loss of СГ anions due to
inward СГ gradient. Hence, hypertonic treatment
leads to significantly altered Cl" contents in cells
treated by non-electrolyte solutions. To assess the
role of electrolytes in the mechanism of posthyper-
tonic hemolysis activation by Ca2+ and Zn2+ cations,
RBC were dehydrated in 1.2 M sucrose in the
presence of 100 mM Na+, K+, Mg2+ and Ca2+ salts.
The data represented in Fig. 4 show that dehydration
in sucrose medium in the presence of Na+, Kh and
Mg2+ reduces the level of posthypertonic hemolysis by
2—3-fold, and does not influence the ability of Ca2+
and Zn2+ ions to activate posthypertonic hemolysis as
they do in the case when dehydration was performed
in pure non-electrolyte medium. In contrast, Cazv ions
when present in dehydrative medium, completely
eliminate the posthypertonic hemolysis activation,
effect is not due to simple rise in ionic strength This
is also supported by the analysis of volume distri-
bution of rehydrated RBC, obtained after dehydration
in electrolyte and non-electrolyte media (Fig. 5) .
Activation of posthypertonic hemolysis by Zn2+
ions after cell dehydration in hypertonic sucrose is
accompanied by an increased in amount of cells and
ghosts in the swollen subpopulation indicating that
under these conditions a larger number of cells
undergo hemolysis. This finding differs from corres-
ponding volume distributions, obtained for Ca2" ions,
where reduced modal volume of swollen subpopulation
Fig. 3. Effects of Zn ions on
p o s t h y p e r t o n i c h e m o l y s i s .
Conditions and definitions of
symbols are the same as for Fig.
2
45
PATIiLAROS S. V.
Fig. 4. Effects of preincubation of RBC і-,
hypertonic sucrose media in the presence of
mono and divalent cations in the concen-
tration 100 mM on the extent of posthyper-
tonic hemolysis. RBC were incubated for 30
min at 37 °С and then rehydrated in isotonic
sucrose solutions which contain no cations
( / ) or 0,1 mM Zn2+ (2), or 100 mM Ca2+
(.3)
was found. The later supports the view that Ca2" ions
facilitate formation of large membrane pores which
restrict colloidal-osmotic swelling of cells, resulting in
the formation of ghost without cells acquiring critical
hemolytic volume. Zn2" ions do not affect volume
distributions (Fig. 5, A) as well as hemolysis of
rehydrated RBC after dehydration in NaCl medium
(Fig. 3) On the other hand, the inhibiting action of
Ca2" ions in this case resulted in a reduced swelling
of the cells which also correlated with their influence
on hemolysis. The revealed peculiarities in the Ca2+
and Zn2+ ions action on posthypertonic hemolysis
activation and volume distributions after cell rehyd-
ration demonslrate critical role of shrinking in the
development of cell injury during rehydration. At this
stage a prerequisites for irreversible changes in cei!s
are formed. They are subsequently manifested at the
stage of rehydration and may be modulated by
divalent cations. Hemolysis activation is due to the
interaction between cations: and membrane sites,
regulating membrane permeability [11]. Though the
nature of these site remains unknown the data
obtained permit to conclude: on some of their pro-
perties.
Activating sites are formed in cells under hyper-
tonic conditions, since Ca2+ cations are incapable of
activating hypotonic lysis of RBC, which, alike post-
hypertonic hemolysis, results in the swelling of cells.
Ca2" is known to inhibit hypotonic hemolysis [10!
Sites are cation specific. This is supported by the fact
that posthypertonic hemolysis is activated by only
Ca2+ and Zn2" cations. Other cations such as Mg2",
Na+, K", Ch" are capable of inhibiting but not
activating posthypertonic hemolysis [11]. This may
be due to electrostatic screening of charges in the
mouth of a hemolytic pore [8 ]. Despite the con-
centration of Ca2+ ions which is required for activation
is high enough (>20 mM), their action is no
accounted for by the screening effect, because only
incubation of RBC in hypertonic medium in the
presence of 100 mM Ca2" completely eliminates the
activating effect of Ca2" and Zn2" on pcsthypertoni
hemolysis. A similar incubation in the presence of the
other ions, though reducing the level of posihyper
tonic hemolysis, nevertheless, does not affect the
activating properties of Ca2+ and Zn2+ ν Fig. 4). Fi-
nally, the concentration of Zn2+ ions (~ 30 μΜ)
required for hemolysis activation is by three order of
magnitude lower than that of Ca2" (50 mM). Ob-
viously, the effect of Zn2+ ions in such low con-
centrations may be due solely to specific binding with
membrane components. Whether or not one and the
same structure of lipid or protein nature with differing
ionic specifity and affinity to Ca2+ and Zn2 cations is
46
INFLUENCE OF Ca2+ AND Zn2+ ON THB ACTIVATION OF HEMOLYSIS
Fig. 5. Volume distributions of RBC
after 15 min dehydration in hy-
pertonic NaCl (A) and sucrose (B)
media followed by rehydration in
isotonic NaCI solution in the absence
and presence of 0.1 mM Zn2+ and
100 mM Ca2+. Contr-volume. distri-
bution of control erythrocytes in iso-
tonic NaCl medium
responsible for posthypertonic hemolysis activating
effect of both Ca + and Zn2+. Such an ability demon-
strates the specific action of Ca2+ on the membrane
and/or cytoplasmic structures which are responsible
for activation remains unclear. Parallelism was shown
in the activating action of Ca2+ and Zn2+ for cell
incubated in hypertonic sucrose (Fig 1, B, D). A
failure to detect a corresponding parallelism following
RBC incubation in hypertonic NaCl, after which
posthypertonic hemolysis is not longer affected by
Ca2 ions during rehydration in both isotonic solutions
(Fig. 1, A, C), as well as the maintenance of Zn2+
activating properties following rehydration in non-
electrolyte isotonic medium (Fig. 3, B), suggest that
sites for Ca2+ and Zn2+ ions may be different.
The notion that modes of Ca2+ and Zn2+ activation
action are different is additionally supported by the
data on the volume distributions of the mixed popu-
lation of cells and ghosts, obtained after rehydration
in media in the presence of these ions (Fig. 5). The
volumes of ghosts in the presence of Ca2+ (right pick
of distribution) are considerably lower than those in
the presence of Zn2+, which testifies to larger dimen-
sions of pores. Earlier we have shown that both
monovalent and divalent ions, as well as EDTA and
sucrose, inhibit posthypertonic hemolysis after addi-
tion into the medium a short while after onset of
hemolysis [11]. Since such an influence was also
ion-specific and could not be accounted for by the
screening effect, it has been suppose that cations
specifically bind to blocking sites on the external
membrane surface, and close hemolytic pores in
membranes. Here we observe the activating effect of
the same Zn2+ and Ca2+ cations (but not Mg2+) when
the ions are originally present in the rehydrative
medium. The data reported elsewhere [3, 6—9 ]
demonstrate that divalent and monovalent cations
inhibit membrane defects, produced by the action of
hemolytic agents of various origin. Their activating
influence has also been shown for RBC lysis induced
by melittin [14].
However, phenomenon of lysis activation by di-
valent cations were not observed on model lipid
systems (liposomes), with composition close to RBC
membranes, [14]. Hence, activation seems Io require
some protein components of RBC membrane. The
latter is also supported by the fact that inhibition of
lysis in lipid vesicles systems requires Zn2 con-
centrations to be by 10—100-fold higher than those
for RBC [14, 15]. Hence, one may conclude that
reswelling of RBC from hypertonic media leads to
formation of composite pores in their membranes. The
structure of the pores involves at least two activating
ion-specific and one blocking site for divalent cations.
It is probably that membrane proteins participate in
pore formation, while the exact structure of the pore
and the ability of sites to interact with divalent
cations depends on electrolyte and non-electrolyte
composition of dehydrative and rehydrative media.
47
PATIiLAROS S. V.
С. її. Пателарос
2+ 2+
Активуючий вплив дивалентних катіонів Ca" і Zn
на постгіггертонічний лізис еритроцитів людини
Резюме
Еритроцити людини інкубували з різні строки при темпера-
турі 37 °С у гіпертонічних розчинах 1,5 M NaCl і 1,2 M
сахарози та регідратували в ізотонічних середовищах NaCl і
сащюзи в присутності та відсутності дивалентних катіонів
Ca" і Zn . Вспиіновлено, що останні здатні активувати
постгіпертонічний лізис (TlJi) еритроцитів, якщо початкова
дегідратація здійснюється в сахарозному середовищі. Такої
активації не спостерігається у випадку лізису, індукованого
різними гемолітичними агентами (мелітин, тритон X-IOO і
т. 1-А де ці катіони проявляють лише пригнічуючий ефект.
Попередня інкубація клітин у гіпертонічному розчині NaCl
згаданий ефект усуває. ^Обробка дегідратованих еритроцитів
катіонами Na , K , Mg призводить до пригніченню гемолізу,
однак не впливає на активуючу здатність іонів Ca 2+ і Zn . У
випадку дегідратації клітин у присутності іонів Ca катіони
втрачають здатність активувати ПЛ. При переносі еритро-
цитів із гіпертонічних в ізотонічні середовища відбувається
формування мембранних гемолітичних пор, до структури
яких входять два активуючих іоноспецифічних і один блокую-
чий сайти для дивалентних катіонів. Вказані сайти здатні
регулювати формування мембранних пошкоджень при взає-
модії з катіонами Ca і Zn .
С. В. Пателарос
Активирующее влияние дивалентных катионов Ca2+ и Zn~+ на
постгипертонический лизис эритроцитов человека
Резюме
Эритроциты человека инкубиромши в разное время при тем-
пературе 37 °С в гипертонических растворах 1,5 M NaCl и 1,2
M с,гхарозы и регидратировали в изотонических средах NaCl и
сахарозыприсутствии или отсутствие дивалентных кати-
онов Ca" и Zn . Установлено, что последние способны
активировать постгипертонический лизис (ПЛ) эритроци-
тов. если начальная дегидратация происходит в сахарозной
среде. Данной активации не наблюдается β случае лизиса,
индуцированного разными гемолитическими агентами (ме-
литтин, тритон X-IOO и т. д.), где эти катионы оказывают
только ингибирующий эффект Предварительная инкубация
плеток в гипертоническом растворе NaCl указанный эффект
устраняет Обработку дегидратированных эритроцитов· ка-
тш)чами Na , К , Mg приводит к ингибированию гемол/jga,
:jdngKo не влияет на активирующую способность ионов Ca 2«
Zn В случае дегидратации клеток β присутствии ионов Ca
катионы утрачивают способность активировать ПЛ. При
переносе эритроцитов из гипертонши;ских в изотоническш;
среды происходит формирование мембранных гемолитических
пор, в структуру которых входят два активирующих ионос-
пецифических и один блокирующий сайты для дивалентных
кат ионов. Указанные сайты способны регулировать формиро-
вание ме^ібраин^х повреждений при взаимодействии с катио-
нами Ca и Zn .
REFERENCES
1. Zade-Oppen Α. Μ. М. The effect of manitiol, sucrose,
raffinose and detxran posthypertonic hemolysis / / Acta Phy-
siol. Scand.—1968.—74, N 1—2.—P. 195—206.
2. Mazur P., Cole K. W. Role of unfrozen fraction, salt con
centration, and changes in cell volume in the survival of frozen
human erythrocytes / / Cryobiology.—1989.—26.—P. 1—29.
3. Pasternak C. A., Adler G. M., Bashford C. L. et al. Cell
damage by viruses, toxins and complement: common features of
pore-formation and its inhibition by Ca / / Biochem. Soc.
Symp—1985—50.—P. 247—264.
4. Woolgar A. E., Morris G. J. Same combinied effects of
hypertonic solutions and changes in temperature on post-hy
pertonic hemolysis of human red blood cells / / Cryobiology.—
1973.—10 — P. 82—86.
5. Pegg D. E., Diaper M. P. The effect on initial tonicity of
freezing/thawing injury to human red blood cells suspended is
solutions of sodium chloride / / Ibid.—1991.—28, N 1.—
P. 18—35.
6. Alder G. M., Arhold W. M., Bashford C. L.. Drake A.
Pasternak C. A., Zimmerman V. Divalent cation-sensitive
pores formed by natural and synthetic melittin and by triton
X-100 / / Biochim. et biophys. acta. —1991,—1061. -
P. 111 — 120.
7. Bashford C. L, Alder B. M., Menestrina B., Micklem K. J
Membrane damage by hemolytic viruses, toxins complement
and other cytotoxin agents / / J. Biol. Chem.—1986.—261.—
P. 9300—9308.
8. Bashford C. L., Alder G. M., Graham P. M., Menestrina G.,
Pasternak C. A. Ion modulation of membrane permiability:
effect of intact cells and on cells and phospholipid bilayers
treated with pore-forming agents / / J. Membr. Biol. —1988.—
103.—P. 79—84.
9. Bashford C. L., Rodriguez L., Pasternak C. A. Protection of
cells against membrane damage by hemolytic agents: divalent
cations and protons act at the extracellular side of the plasma
membrane / / Biochim. et biophys. acta.—1989.—983, N 1.—
P. 54—56.
10. Godin D. V., Garnett M. Perturbational effects of inorganic
cations on human erythrocyte membranes / / J. Membr. Biol.—
1976,—28,—P. 143—168"
11. Rudenko S. V., Patelaros S. K Activatory and inhibitory effect
of divalent cations on posthypertonic lysis of erythrocytes / /
Biol. Membrane.—1995.—N 4.—P. 374—384.
12. Akeson S. P., Mel H. C. Erythrocyte and ghost cytoplasmic
resivity and voltage / / Biophys J.—1983.—44, N 3
P. 397—403.
13. Richiery С. V., Mel II. C. Membrane and cytoplasmic resivity
properties of normal and sickle red blood cells / / Cell
Biophys.—1986.—8.—P. 243—259.
14. Portlock S. N., Clague M. J., Cherry R. Leakage of interna!
markers from erythrocytes and lipid vesicles induced by
melittin, gramicidin S and alamethicin: a comparative study / /
Biochim. et biophys. acta.—1990.—1030.—P. 1 — 10.
15. Kaszuba M., Hunt G. R. Protection against membrane damage:
A H-NMR investigation Iof of the effect of Zn and Ca on thi
permiability of phospholipid vesicles / / J Inorg Biochem. -
1990 — 40 — P. 217—225.
Received 07.04.98
48
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