vivanum of the Drug Research Institute in Modra, Slovakia, by the same procedure. Dog led cells were, however, used for experiments on the same day.
Gen Physiol Biophys (1997), 16, 339—357
339
Ca 2 + -activated K + Channel and t h e Activation of C a 2 + Influx in Vanadate-treated R e d Blood Cells Ľ
VAREČKA1,
E
PETERAJOVÁ2
AND J
ŠEVČIK3
1 Department of Biochemistry and Microbiology Slovak University of Technology, Bratislava Slovakia 2 Pmel Hospital Pezmok Slovakia 3 Department of Analytical Chemistry Palacky University Olomouc Czech Republic A b s t r a c t . The mechanism by which K + inhibits vanadate-induced 1 5 C a " + influx by human led blood cells (RBC) was studied using seveial independent approaches The following results weie found 1 The inhibitor effect of K + was absent when RBC weie loaded with a 2+ Ca -chelator This treatment at the same time inhibited the vanadate-mduced K + efflux, and the membiane hypeipolarization induced by C a 2 + in vanadatetieated cells 2 The potency of K + , R b + , and C s + to inhibit vanadate-induced C a 2 + in flux conesponded to then ability to depolanze the RBC membiane via the Ca 2 + activated K + channel (K(Ca)) 3 Inhibition of the vanadate-induced 4 5 C a 2 + influx bv a protonophore pro ceeded in parallel with the inhibition of the vanadate-plus-Ca 2+ -mduced membiane hypei polarization 4 Yahnomvcin m pait released the inhibition of the vanadate-induced C a 2 + influx by known K(Ca) inhibitors (quinine, oligomycm, 4-aminopyiidine) but not by inhibitois of the C a 2 + channel ( C u 2 + , HS-reagents organic C a 2 + channel blockers) + 2+ 5 K did not inhibit the vanadate-induced C a influx in dog RBC which + have K(Ca) but no transmembrane K giadient The inhibition of the vanadate-mduced C a 2 + influx by external K + appeals to be due to the elimination of the electrical component of the C a 2 + - m o t n e force imposed by opening of the K(Ca) This implies that the C a 2 + carrier mediating the influx of C a 2 + m the piesence of vanadate is of uniport tvpe, and that the activity of K(Ca) may sei\e as a supporting element foi C a 2 + influx Correspondence to Ľudovít Varečka, Department of Biochemistrv and Microbio logy, Slovak University of Technology, Radlinského 9, 812 37 Bratislava Slovakia E-mail varecka@checdek chtf stuba sk
Varečka et al
340 2+
K e y w o r d s : Red blood cells — C a influx — Membrane potential — + 2+ activated K channel — C a - m o t i v e force — Vanadate Abbreviations: DTNB
5,j'-dithiobis-(dmitrobenzoic acid), F C C P -
boirvl cyanophenylhydiazone, K ( C a ) mycm, RBC
led blood cells, T P P
tetiachloiosahcvlamlide, VM
2+
Ca -activated K
+
2+
-
fluoiocar-
channel OM
tetraphenvlphosphonium, TCS
Ca
oligo
3 3'4' 5
vahnomycin
Introduction Although the lole of the C a 2 T a c t u a t e d Iv + channels ( k ( C a ) ) in excitable cells is wideh iecogni7ed, t h e n piesence and function in non-excitable cells is much less midoistood
In led blood cells (RBC) the actiwťs
of the Iv(Ca) was disco\eitd
\>\ Gaidos as Ca~^-sensitne K + efflux (fuithei icfeiied to as the Gaidos effct^ iiom metabolic all}
poisoned ied cells se\eiat decades ago (Gaidos 1958)
then it has been t h o i o u g h h studied (see Lew an 1 f e i i e n a 1978
Since
Schwaitz and
Passow 1983 ioi ie\iew) and shown that the Gaidos effect is closeh linked to the C , r ' + influx K
+
as suggested b) the concomnntaiit a p p e a i a n c e of C < r + influx
and
efflux using difteient c x p e i n n t n t a l pioceduies such as ATP-depletion (Gaidos
1958 F e n e n a and Lew 1977) tieatment with piopianolol (Mannmen 1970 and Gaidos 1974
Szas/ et al
1977)
Szas/
-vanadate (\aiecka and Caiatoh 1982)
oi
menadione ( F u h i m a n n et al 1985) On the othei hand, the C a 2 + influx into R B C loaded with C a 2 + chelatois like B A P T A (Lew et al 1982), or quin-2 (McNamara and Vviley 1986, P o k u d m and Oilo\ 1986), has not been íeported so fai to bt accompanied b) RfCa^ activation, and bv the G á i d o s effect The mechamsm(s) of activation of the C a 2 + influx obseived d u n n g the ac m a t i o n of the G a i d o s effect
and the pioperties of the C a
2+
influx
pathway(s)
obseived by the above methods aie mostly obscure and difficult to compaie be cause no compiehcnsive d a t a to be compared aie available
It seems probable t h a t
C a 2 + influx induced b> ATP-depletion, vanadate, oi C a 2 + chelatoi e n t i a p m e n t is mediated b\ a e a r n e r although its detailed descnption could onh be obtained in \ anadate-treated cells(Vaiéčka and Caiafoli 1982 \ a i e c k a e t a l
1986 Varečka and
Peteiajova 1990) The link of C a 2 4 influx t o K + efflux w Inch is the crucial aspec t of the activation of the Gaidos effect, does not seem to exist if the C a 2 + influx is induced by the entiapment of a C a 2 + chelatoi This is indicated b> the fact t h a t the C a 2 + influx induced by C a 2 + chelatoi e n t i a p m e n t is insensitive to the ionic composition of the suspension medium ( M c N a m a i a and Wilej 1986 P o k u d m and Oilo\ 1986)
unlike
that induced b> A T P depletion ( F e n en a and Lew 1977), oi vanadate (Varečka and Carafoh 1982), where it was found to be inhibited by increasing concentrations or by dissipation of the K + gradient by íonophores T h e difference in the sensitivity to
Red Cell Ca
+
Homeostasis
341
+
extracellular K , if compared with the different capabilities of the above methods to activate K(Ca), indicates that K(Ca) activation is a prerequisite for the inhibition of 2+ + C a influx by extracellular K This implies that it is the specific action of K(Ca) 2+ which contnbutes to the driving force of the C a influx m vanadate-treated, or 2+ ATP-depleted RBC, and thereby promotes the influx of C a into the cytoplasm There is evidence for this suggestion in propranolol-treated cells (Szász et al 1977) and in ATP-depleted cells (Gárdos et al 1980, Szász et al 1981) We used vanadatetieated RBC as a model to test the above suggestion and found evidence to support it Materials and Methods Red blood cell suspension Blood from healthy volunteers of both sexes was withdrawn by venipuncture into EDTA-contammg anticoagulant (5 mmol/1), and was used withm 3 days, stored at 0-4°C Red blood cells (RBC) were isolated after centnfugation of the blood (10 mm at 600 x g) and aspiration of the supernatant with the buffy coat, and three fold washing with and, finally, suspending into a medium containing (in mmol/1) 20 Tns-HCl, pH 7 3, 130 NaCl, 5 KC1, 10 glucose (further referred to as the suspension medium), to the final haematocnt of 30%, and immediately used for expenment Dog blood was withdrawn by venipunctuie from non-medicated dogs raised in the vivanum of the Drug Research Institute in Modra, Slovakia, by the same procedure Dog led cells were, however, used for experiments on the same day Red cell loading with permeant Ca chelators The 30% suspension was loaded with 75 /xmol/1 (if not indicated otherwise in the Figures) BAPTA/AM (oi qum-2/AM) (and controls with 0 5% DMSO) for 30 mm at 37°C m the presence of 0 2 mmol/1 EGTA, cooled to 25°C, centnfuged, and adjusted to the same volume of medium of desired composition Test and control suspensions were handled as pairs in individual experiments Vanadate-mduced Ca2+
influx
The influx of C a 2 + was measured with the íadionuchde 4 5 Ca, after repetitive washing to íemove extracellular radioactivity, as described previously (Varečka and Carafoh 1982) Aliquots of 30% suspension were premcubated with 1 mmol/1 N a V 0 3 for 15 mm at 25 °C, and 4 5 CaCl 2 (2 5 mmol/1) was added and incubated for 60 mm at the same temperature unless indicated differently The incubation was stopped by addition of the same volume of the stopping medium containing (in mmol/1) 20 Tns-HCl, pH 7 3, 75 KC1, 60 NaCl, 10 glucose, and 1 EDTA (further lefened to as the stopping medium), and by rapid centnfugation of the sample m
Varečka et al.
342
a microcentrifuge. The supernatant was sucked off, and the pellet was washed with the stopping medium three more times. Finally, the pellet was precipitated with 10% trichloroacetic acid (TCA) containing 20 mmol/1 LaCl 3 , the precipitate was centrifuged and the pellet was taken for liquid scintillation counting. Control cells without vanadate were treated in parallel. When inhibitors were tested, the same volume of solvent (DMSO, methanol, max. 0.5% V/V) was added to the control samples. All samples were run in duplicates, and the average value of parallel sam ples (+/— standard error) is given in the Figures. The standard error is indicated by bars when exceeding the size of the symbols. Experiments illustrated in the Figures were typical of at least two (as a rule, three) separate experiments. Measurement of wCa2+-induced
A5
Ca2+
efflux
The total volume of the RBC suspension to be used in the experiment was pre loaded with 4 5 C a 2 + in the presence of vanadate as described above, for 60 min, chilled on ice, and kept in an ice-water mixture until complete removal of external radioactivity by repetitive washing with the stopping medium (two washings) and two washings in the suspension medium containing 0.2 mmol/1 EGTA. Finally, the suspensions were centrifuged at 0-4°C for 5 min at 2000 x g, the supernatants were quantitatively aspirated, and the pellets were adjusted to the original volume with the suspension medium containing 0.2 mmol/1 EGTA. Aliquots of this suspension kept on ice were pipetted into test tubes pre-warmed to 25 °C for exactly 5 min and 4 0 CaCl 2 was added to the test suspensions. No addition was made to the control suspensions. At the time shown (usually 0, 2, 5, 10, 20 and 50 min), 0.5 ml aliquots were withdrawn and immediately centrifuged through a silicone oil layer. The radioactivity of both the supernatants and the pellets was measured after TCA precipitation of proteins. All measurements were done, and. the results processed as described in the preceding paragraph. Measurement of the Gárdos effect The Gárdos effect was monitored either by measuring the net K + efflux by flame photometry, or by the release of 8 6 R b + from cells pre-equilibrated with it. Net K + efflux was measured in the vanadate-treated cell suspension as described above. At time zero, 40 CaCl2 (2.5 mmol/1) was added, and aliquots of the suspension were withdrawn after a 50 min incubation at 25°C, and after spinning down RBC through a silicone oil layer the supernatant was used for flame photometry. Control test tubes were treated in parallel. When 8 e Rb was used as tracer, 2 MBq of the carrier-free radionuclide was incubated with 7.5 ml of whole blood overnight at 0°C, and RBC were isolated as described previously. Other steps were identical with those used for the measurement of net K + efflux except that radioactivity was measured in the supernatant. Values shown in Figures correspond to what is described in the preceding paragraphs.
Red Cell Ca
+
343
Homeostasis
Measurement of changes in 3H-TPP+
distribution
EGTA (0 2 mmol/1) was added to the suspension of RBC (pre-treated with BAPTA/AM, or controls with DMSO, as indicated m the Figure) Aliquots of the 20% suspension were supplemented with 1 mmol/1 NaV0 3 and 25 ^mol/1 3 HTPP"1" (approx 20,000 cpm/assay), and were equilibrated for 25 mm At time zero, aliquots weie withdiawn and centrifuged immediately through a layei of dibutylphthalate (DBP), and the phases were separated quantitatively from each other immediately aftei the centnfugation At 5 mm, CaCb (2 7 mmol/1) was added Aftei 13 mm and 55 mm aliquots were withdrawn and treated as above Control cells were tieated similarly The íadioactivity from pellets was extracted into ethanol, and separated from the cell debris by centnfugation Correction of medium and pellet radioactivity foi the íadioactivity trapped m dibutylphthalate did not sig nificantly influence the obtained values The radioactivity of the supernatants was measured m paiallel, and this is presented m the Figures Chemicals 3
H-Tetraphenyl phosphonmm chlonde fiom Radiochemical Centre, Amersham (Bucks, England), 4 5 CaCl 2 fiom Radiochemical Works (Swierk, Poland), 8 6 RbCl fiom Isocommeiz (Dresden, Germany) valmomvcm, íonomycm, qum-2/AM, and BAPTA/ AM from Calbiochem (Luzern, Switzerland), quinine and oligomycm from Sigma (St Louis USA), 4-ammopyridme from Fluka (Buchs, Switzerland), Tns base, FCCP, DTNB and dibutylphthalate from Serva (Heidelberg, Germany), 3,3',4',5tetiachloro-sahcylanilide (TCS) from Eastman-Kodak Comp (Rochestei, USA), and the methyl-phenyl silicone oil from Lučební závody Kolín (Czech Republic) N a V 0 3 was from Reachim, (Moscow, Russia) Other chemicals (all of analytical grade) were purchased from Lachema. (Brno, Czech Republic) Results Ca2+ chelator entrapment prevents the inhibition of Ca2+ influx by K+, and simul taneously blocks the Gardos effect and accompanying membrane hyperpolarization Fig 1 shows the effect of the substitution of N a + ions for K + on the vanadatemduced 4 5 C a 2 + influx which was observed earlier (Vaiečka and Carafoh 1982), and its modification by cytoplasmic Ca buffering This was brought about by preincu bation of cells with tetraacetoxymethyl esters of Ca chelators BAPTA/AM or qum -2/AM, as described by other authors (Lew et al 1982, McNamara and Wiley 1986, Pokudm and Orlov 1986) Treatment of RBC with 75 /[xmol/l qum-2/AM 45 2+ abolished the inhibition of the vanadate-induced C a influx by high extracel + lular K (Fig lA) When the cells were treated with lower concentrations of the permeant chelator some inhibition of C a 2 + influx by K + was observed (not shown)
Varečka et al
344
1
O
1
20
1
1
40 [K + ]
1
1
60 mmol/1
.
1
80
1
1
O
i
1
20
1
1
40
1
1
60
1
1
80
[BAPTA/AM], M mol/l
F i g u r e 1. Effects of C a " + chelator t r e a t m e n t on t h e (A) K + gradient d e p e n d e n c e of t h e v a n a d a t e - m d u c e d 4 5 C a 2 + influx, (B) v a n a d a t e a n d C a 2 + - i n d u c e d K + efflux ( t h e Gár dos effect) A Cells loaded with 75 /imol/1 qum-2/AM (triangles), control cells w i t h o u t chelator (0 5% v/v D M S O ) (circles) 1 mmol/1 NaVC>3 (closed symbols), controls with o u t v a n a d a t e (open symbols) B R B C pre-treated with B A P T A / A M a n d w i t h 1 mmol/1 N a V 0 3 (closed symbols) 4 0 C a 2 + (2 5 mmol/1) added (circles) Controls w i t h o u t C a 2 + (squares) and w i t h o u t NaVC>3 (open circles)
Treatment with the permeant Ca chelator stimulated the influx both in the absence, and in the presence of vanadate The increment of the influx caused by the presence of the Ca chelator in the presence of vanadate exceeded that in the control with out vanadate (Fig I A) Under the same conditions, Ca2+-buffermg significantly 2+ inhibited the vanadate plus Ca -mduced Gárdos effect (Fig IB) The highest + concentration of BAPTA/AM used (75 /imol/1) completely inhibited the K ef 2+ flux In order to localize the site of the action of the Ca -chelator, the effect of the BAPTA/AM treatment on membrane potential changes induced by the open ing of K(Ca) was investigated Membrane potential changes were monitored by 3 3 changes of H-labelled tetraphenyl-phosphomum ( H-TPP+) distribution These indicated hyperpolanzation of the RBC membrane triggered by C a 2 + in vanadatetreated cells as compared with control without vanadate, and its inhibition by the BAPTA/AM treatment (Fig 2A) This was not due to a decrease of Ca 2 + -ATPase
R e d Cell C a
1 0
+
345
Homeostasis
1 10
1
j 20
1
TIME, mm
1 30
1
1 40
0-|—i—|—i—|—i—|—i—|—i—|—i—|—i 0 5 10 15 20 25 30 TIME, mm
F i g u r e 2 . Effects of B A P T A / A M t r e a t m e n t on t h e (A) 3 H - T P P + influx d u r i n g t h e G á r d o s effect, a n d (B) 4 0 C a 2 + - i n d u c e d 4 5 C a 2 + - e f f l u x from v a n a d a t e - l o a d e d h u m a n R B C A R B C loaded with B A P T A / A M (triangles), controls with D M S O (circles) a n d supple m e n t e d w i t h N a V 0 3 a n d 3 H - T P P + 2 7 mmol/1 4 0 C a 2 + a d d e d at 5 m m (closed symbols), no a d d i t i o n in controls (open symbols) O n l y radioactivity of s u p e r n a t a n t s is p r e s e n t e d B R B C p r e m c u b a t e d with B A P T A / A M (triangles), or D M S O (circles) a n d s u b s e q u e n t l y loaded w i t h 4 5 C a 2 + by m e a n s of N a V O s , a n d washed out of extracellular r a d i o a c t i v i t y as described in Materials a n d M e t h o d s 4 0 C a 2 + a d d e d (closed symbols) or no a d d i t i o n m a d e (open symbols) Only t h e s u p e r n a t a n t r a d i o a c t i v i t y is shown
inhibition which was not always complete in our expenmental conditions (Varečka and Carafoh 1982, Vaiečka et al 1986) by the Ca chelator The corresponding evidence was provided by the measurement of 4 0 Ca 2 + -induced 4 5 C a 2 + efflux from 45 Ca 2 + -loaded RBC This showed that the radioactivity pumped out of the cells m the initial phase of the exchange process was not significantly different m either vanadate or vanadate plus BAPTA-loaded RBC (Fig 2B) The loss of sensitivity of the vanadate-induced C a 2 + influx to the increase of extracellular K + concentra tion upon the buffering of cytoplasmic C a 2 + suggests that the rise of cytoplasmic C a 2 + concentration and the subsequent opening of K(Ca) are prerequisite foi the inhibition of the C a 2 + influx by K + In Older to corroborate this suggestion by independent evidence, we studied the effect of the experimental varying of the membrane potential during the vanadate-mduced 4 5 Ca 2 4 " influx and the use of
346
Varečka et al
"mutant" RBC species lacking N a + , K + ATPase Alkali metal cations influence the vanadate-induced 45CVz2+ influx and the mem brane potential in correlation with their selectivity for K(Ca) The lelative permeabilities of human RBC K(Ca) to monovalent cations are R b + (1 0), K+ (0 67), Cs+ (0 05) (Simmons 1976) We assumed that íeplacement of Na+ ions with othei alkali metal cations display different degrees of inhibition as compaied with K + , and that the efficiency of the inhibition can be expected to correlate with the permeability of the individual ions through K(Ca) if then inhibitory effect is due to the change of the membrane potential Such an assumption seems to be justified because m isotonic solutions, the net transport of KCl aftei the opening of K(Ca) was shown to be limited by chlonde efflux (Schubert and Sarkádi 1977) In oui expenments the concentration of chloride was kept constant This assump tion is valid for human RBC which have a negligible activity of the Ca 2 + -activated Na + /H+ exchanger (Escobales and Canessa 1985) and no known active N a + chan nel as compaied with other cells As shown in Fig 3.4, the ordei of potency of K + , R b + and Cs + m inhibiting the vanadate-induced C a 2 + tiansport follows the ordei of then known permeabilities in K(Ca) (Simmons 1976), unlike with impermeant choline which stimulated C a 2 + influx (Fig 3.4) The measurement of the 3 H - T P P + distnbution changes induced by C a 2 + in vanadate-treated RBC in media with dif ferent degrees of substitution of N a + with K + , R b + , and Cs + , respectively, showed that depolarization was maximal in R b + media followed by K + media and, finally, by Cs + media (Fig 2>B) These results aie in accordance with the notion that the influence of alkali metal ions is mediated by a K(Ca)-imposed membiane potential Uncoupler inhibits the vanadate-induced Ca2+ influx and dissipates the membrane potential in a Ca2+ -dependent manner It was found previously (Varečka and Caiafoli 1982) that the uncoupler, FCCP, stiongly inhibited the vanadate-mduced C a 2 + influx and stimulated the Gárdos effect Because FCCP and derivatives are known to interact with HS-gioups (Drob2+ nica and Šturdík 1979), and the vanadate-induced C a influx is sensitive to HSmhibitois (Vaiečkaet al 1986), another inhibitor, 3,3',4',5-tetrachlorosahcylanilide (TCS) was used in these experiments As shown in Fig 4A, TCS also suppressed 2+ the C a influx when piesent in a concentration of 10 /zmol/1 At the same time, it also strongly (but not completely) inhibited hyperpolanzation of the membrane potential induced by C a 2 + and vanadate (Fig 4 5 ) Thus, effects of TCS on both vanadate-induced 4 5 C a 2 + influx and membrane potential changes suggest that to a significant extent the inhibition of 4 5 C a 2 + influx is due to the collapse of the membrane potential change induced by the opening of K(Ca)
Red Cell Ca
+
Homeostasis
T 20
I
• I • I '
347
1 80
I ' I
40 6 0 8 0 100 120 140 [Me + ] o , mmol/1
[Me + ] 0 , mmol/1
F i g u r e 3. The substitution of N a + by other monovalent cations affects similarly the vanadate-induced 4 5 C a 2 + influx (A), and change of the 3 H - T P P + distribution (B) A RBC (90 % haematocnt) pretreated with 2 5 mmol/1 NaV03 transferred by a positive displacement pipette into test tubes containing media with increasing concentrations of KCl (open circles), RbCl (open triangles), CsCl (closed triangles) and choline chloride (closed circles) instead of NaCl (final concentrations indicated in the Figure, 5 mmol/1 KCl was present in all test tubes), and the isotonicity was kept constant Controls without vanadate were treated as above, their values did not differ from each other, and weie less than 1 /imol/lceiis B RBC treated with N a V 0 3 and transferred into KCl (open circles), RbCl (open triangles), and CsCl (closed triangles) media (final concentrations indicated) 2 7 mmol/1 4 0 C a 2 + added 3 H - T P P + activity in the supernatant after 8 mm incubation after addition of Ca + is shown No differences caused by substituents were found in controls without vanadate (not shown)
The inhibition of vanadate-induced Ca
nflux by K(Ca)
inhibitors is released by
vahnomycin It is conceivable that the inhibition of K(Ca) by its specific inhibitor should elim inate the shift of the membrane potential induced by K(Ca) activation
It was
found previously that qumidine, an inhibitor of the K(Ca) (Armando-Hardy et al 1975), inhibited the vanadate-induced
45
C a 2 + influx (Varečka and Carafoh 1982)
This may be a consequence of the inhibition of either K(Ca) oi of the C a 2 + car-
Varečka et al
348
9-1 B
X
E a. o 0. Q_
NaVO, 1-
Control 0
5 [TCS], umol/l
10
O
10
20
30
TIME, min
Figure 4. The effects of uncoupler on the vanadate-mduced 4 5 C a 2 + influx (A), and on the vanadate plus Ca 2 + -mduced membrane potential changes (B) A The 4 5 C a 2 + influx in the suspension medium in the presence of TCS (3,3,4,5 - tetrachloro sahcyl anihde) with 1 mmol/1 NaV0 3 (closed circles) Controls without N a V 0 3 (open circles) B The 3 H - T P P + activity in the medium induced by C a 2 + in vanadate-treated cells in the presence of 3 Mmol/1 TCS (triangles), or methanol (0 5% v/v) (circles) 4 0 C a 2 + (2 7 mmol/1) added at 5 mm (closed symbols), controls without Ca + (open symbols)
ner, because the specificity of quimdme has not been yet tested Later, also othei compounds known as inhibitors of K(Ca), such as oligomycm (Blum and Hoff man 1972), or 4-ammopyridme (Thomsen and Wilson 1983, Chnste et al 1995) weie tested Both suppressed the vanadate-induced 4 5 C a 2 + influx (and also the vanadate-mduced Gárdos effect) It was assumed that the addition of the K + specific íonophore valmomycm (VM) to cells containing K(Ca) inhibitors would restore the membiane potential and reveises the inhibition piovided that it has been caused by the inhibition of the K(Ca) but not of the C a 2 + carrier, and that the C a 2 + carnei is uniporter As control substances, C a 2 + channel blockers, such as verapamil, or nifedipm, or divalent ions, as well as HS-reagents which were found to inhibit the vanadate-mduced 4 5 C a 2 + influx (Varečka et al 1986), were tested It was found that valmomycm never reversed the inhibition of the vanadate-mduced 45 C a 2 + influx in the group of 4 5 C a 2 + influx inhibitors but increased it m the group
Red Cell C a
+
Homeostasis
349
T a b l e 1. Inhibition of v a n a d a t e -induced 45 C !a + influx by inhibitors of C a 2 + channels and of K ( C a ) , and reversal of t h e in hibition by •valmomyci n 45
C a 2 + influx, (^mol/Leiis)
Control
Inhibitor
36 1 ± 0 6 34 7 ± 1 6
80±05 14 9 ± 0 6
4 - a m m o p y n d i n e , 10 m m o l / 1 (n = 2) Control 24 1 ± 1 1 + VM (1 / i g / m l ) 20 0 ± 1 0
11 1 ± 1 0 148±0 7
O h g o m y c m 10 / i g / m l (?) = 4) Control + VM (1 /eg/ml)
29 8 ± 0 9 27 6 ± 0 5
109±0 7 15 0 ± 0 4
Nifedipm, 60 /xmol/1 (n = 4) Control + VM (1 jug/rnl)
19 9 ± 1 9 170±15
50±02 47±05
C u 2 + 10 /./mol/1 (n = 3) Control + VM (1 /Jg/ml)
27 0 ± 1 4 23 0 ± 0 7
13 3 ± 0 0 12 3 ± 0 6
D T N B 25 /nnol/1 (n = 1) Control + VM (1 /ug/ml)
34 9 ± 1 6 26 8 ± 0 8
12 9 76
55 49
18 1 5
Quinine 1 5 mmol/1 (n = 3) Control + V M (1 / i g / m l )
Verapamil 140 /imol/1 (n = 1) Control + VM (1 /xg/ml)
Presented are results from typical experiments (the total n u m b e r of e x p e r i m e n t s foimed is shown in parentheses) where several concentrations of inhibitors were used effects of verapamil and D T N B were tested in pilot experiments only Each value average from duplicate assays ± S E except in t h e experiment with D T N B where single assays were done m t h e presence of t h e inhibitor
perThe is an only
of inhibitois of the K(Ca) (Table 1) Valmomycm slightly inhibited the influx in contiol test tubes (Table 1), although on several occasions also a small (about 10%) stimulation was obseived (not shown) In some experiments no stimulatory effect of VM was observed even in the presence of K(Ca) inhibitors but no stimulation of the influx by VM inhibited by the Ca 2 + channel inhibitois was ever
Varečka et al
350 3000-
•O-i
A
Ca' +
9-
K +NaVO
2500-
»2000o 1.1500
' 7 c h o l i n e + N a V 0 3 Jj ~ 0. Q. +
(0
O '1000
500
*-=&=
*-
I • l ' I •I •I •I ' I ' I ' 0 20 40 60 80 100 120 140 [SUBSTITUENT], mmol/l
i 40 TIME, min
F i g u r e 5. T h e 4 5 C a 2 + influx induced by v a n a d a t e in dog R B C in m e d i a with various ionic compositions (A) a n d t h e changes of t h e 3 H - T P P + d i s t r i b u t i o n induced by C a 2 + (B) A R B C pre-loaded with 1 mmol/1 N a V O s in t h e N a + - m e d i u m which was subsequently replaced by K + (closed circles), or choline"1" m e d i u m (closed triangles) containing 1 mmol/1 N a V 0 3 so t h a t t h e i r final concentration was as indicated Control suspensions (open symbols) were t r e a t e d in parallel B R B C suspended (20% h a e m a t o c n t ) m t h e N a + - n c h (circles) or K + - r i c h m e d i u m (triangles) a n d t h e suspensions were s u p p l e m e n t e d with 0 2 mmol/1 E G T A V a n a d a t e (0 1 mmol/1) a d d e d t o all samples followed by 3 H - T P P + 4 0 C a 2 + (2 7 mmol/1) was a d d e d at 8 m m (closed symbols)
obseived Thus, the dual effect of VM on the 4 5 C a 2 + influx inhibited by both C a 2 + channel inhibitois and inhibitors of K(Ca) supports the possibility that the K(Ca) inhibitors eliminate a part of the Ca 2 + -motive force dunng their actions, which could be released by valmomycm Open Ca2+ -activated K+ channel without K+ gradient cannot mediate the inhibi tion of the vanadate-induced A5Ca2+ influx with K+ Dog RBC do not possess Na + ,K + -ATPase in then membrane and have no tiansmembiane giadients of monovalent ions (Paiker 1977) but vanadate was able to induce 4 5 C a 2 + influx also in these cells Its potency in dog RBC exceeded that in human RBC by about one order of magnitude At 1 mmol/1 vanadate, i e , undei conditions used m experiments with human RBC, the influx was so massive
Red Cell Ca
+
Homeostasis
351
that it almost led to an equilibration of C a 2 + across the membrane (Fig 5A) The diffeience could not be ascnbed to an) expenmentallj introduced factoi, and it lepiesents a true inteispecies diffeience Also the 4 5 C a 2 + influx was found to be satuiatable with respect to C a 2 + and perhaps, like in human R B C , is mediated by a earner (not shown) The \anadate-mduced 4 5 C a 2 + influx m dog R B C was not leduced when N a + were stoichiometncall} substituted with either K + , 01 choline"1" In contiast, it was stimulated regardless of the substituent (Fig 5 4) When most of the N a"1" weie substituted the influx also mcieased in controls without \ a n a d a t e added (Fig 5 1) Probably, both íesponses aie due to the leveisal of the N a + / C a 2 + antipoitei a c t n i t y known to be piesent in the dog R B C membrane The same íesponse of dog RBC was obtained when 0 1 mmol/1 N a V 0 3 was used as inducer with the extent of the influx o n h being about 2 0 / (not shown) No changes in the transmembrane distribution of *H T P P + induced b> Ca2J~ in \ a n a d a t e t i e a t t d cells and m contiols in N a + iich medium (i e suspension medium) weie found (Fig 5B) H o w e \ u in a K + nch medium the lelcase of 3 H T P P ^ in the piesence and to some cxtc nt also m the absence of \aiiadate was o b s e n e d Tins could be e> plained by the pioposal that K(Ca) is also activated in dog RBC by the mcicase of the cytoplasmic C r + concentration Dnect e\idence supporting this proposal was
NaVQ3+Ca2+
.o
NaVQ3+EGTA
DC
TIME, mm
Figure 6. The effect of vanadate and Ca 2 + on the 8 0 R b + efflux from dog RBC Whole blood was loaded with 86 RbCl and RBC were isolated as described in Materials and Methods, and were supplemented with 0 2 mmol/1 EG1A 0 1 mmol/1 vanadate (closed symbols), no vanadate (open symbols) After 15 mm at 25CC, 4 0 Ca 2 + (2 7 mmol/1) (circles), and EGTA (2 5 mmol/1) (triangles) were added Radioactivity in the external medium is shown
Varečka et al
352 86
+
+
+
obtained using R b as a tracer of the K movement In the Na -nch medium + where the K concentration was approximately equal on both sides of the mem 86 + 2+ brane, R b release was observed if both C a and vanadate were present in the 2+ suspension, but not in controls with vanadate and EGTA and with C a in the absence of vanadate (Fig 6) Thus, the expenments with dog RBC show that the opening of K(Ca) without subsequent membrane hyperpolarization is not sufficient to mediate the inhibition of the vanadate-mduced 4 5 C a 2 + influx by high K + and that a steep transmembrane K4" gradient is indispensable for this effect to occui Discussion The entiapment of the Ca chelator m RBC cytoplasm changed profoundly the piopeities of vanadate-mduced 4 5 C a 2 + influx (Fig 1.4) First, the 4 5 C a 2 + influx mcieased both m the piesence and m the absence of vanadate The mciease of the influx could be explained eithei by an increase in the concentration (osmotic) component of the Ca 2 + -dnvmg force due to the buffering of the cytoplasmic C a 2 + concentiation below the testing level, oi by an increase of the passive permeability during the treatment with permeant chelator Assuming that the minimum concentration of trapped chelator of 100 /miol/1 (present in oui conditions accoidmg to Pokudm and Orlov (1986) lowers the cytoplasmic C a 2 + concentration down to about 100 200 nmol/1 (as computed by the Bound and Detei mined progiamm of Biooks and Stoiey (1992)), a value which is Inghei than that in the íestnig state if data by Lew (1990) are adopted, but it is approximately equal with the data published by Simmons (1976) Thus, the chelatoi loading seems to deciease the ,iestmg" cytoplasmic C a 2 + concentiation only maigmally and therefore, the mciease of the 4 5 C a 2 + influx in the chelator-treated cells might be better explained by an mciease of membrane peimeabihty Remarkably, the increment of the 4 5 C a 2 + influx induced by the qum-2 loading in the presence of vanadate was greater than in controls without vanadate (Fig 1.4) Because both tests were performed under otheiwise identical conditions, this may indicate that the vanadate treatment also 2+ induced an increase of passive C a peimeabihty This possibility is supported bj expeiimental evidence (Vaiečka et al 1997) Second, the 4 5 C a 2 + influx became insensitive to external K + concentration changes This fact and the paiallel inhi bition of both the Gárdos effect and the membrane hyperpolari/ition suggest that it is opening of the K(Ca) which is a prerequisite foi the obtaining the 4 5 C a 2 + influx inhibition by K + The involvement of Iv(Ca) m this process could also be suppoited bv the evidence obtained m experimental conditions where K(Ca) was open, i c , m experiments with monovalent ions and TCS (Fig 3, 4), by a paialklism between the inhibition of i 5 C a 2 + influx and the depolarization potency The mhibitoiy effects of both monovalent ions and TCS on the vanadate-induced 45 C a 2 + influx could be explained by their influence on membrane potential
Red Cell Ca
+
Homeostasis
353
The opening of K(Ca) without generating a membrane potential does not + 45 2+ seem to cause any inhibitory effect of K on the vanadate-induced C a influx + + This could be demonstrated in experiments with dog RBC which lack the N a , K ATPase (Paiker 1977) but possess the K(Ca) (Richhardt et al 1979) (Figs 5,6) 45 2+ Howevei, the ability of these cells to take up C a in the presence of vanadate has not been impaired by this fact, and was apparently driven by the osmotic component of the Ca 2 + -motive force mediated by a high-capacity transporting mechanism The íesults show that either the prevention of K(Ca) opening (Figs 1,2) or the elimination of membrane hyperpolanzation induced previously by the K(Ca) opening (Figs 3,4) inhibit 4 5 C a 2 + influx, and this implies that K(Ca) blockade could mimic inhibition of the C a 2 + carrier The action of valmomycm on 4 5 C a 2 + influx (Table 1) confirmed this notion, and diffeientiated between inhibitors of K(Ca) and inhibitors of the C a 2 + earner which could also inhibit the Gáidos effect induced by vanadate (Varečka et al 1986) Valmomycm could not be used as a tool in this íespect in experimental models where the voltage activated C a 2 + channel represent the Ca 2 4 " influx pathways The experimental approaches used to explain the mechanism of the vanadatemduced 4 5 C a 2 + influx inhibition in human RBC by external K + brought results which deny the role of the osmotic component of the electiochemical potential of K + and identify the elimination by K + of membrane hyperpolanzation imposed by opening of K(Ca) as the causative factor of the inhibition Such a model implies that a C a 2 + uniporter is a tiansporting species operating in our experimental conditions, (i e , in the piesence of vanadate) and that the membiane potential change elicited by the opening of K(Ca) increases the total Ca 2 + -motive force acioss the RBC membrane Thus, K(Ca) activity (and the subsequent Gárdos effect) participate in facilitating the C a 2 + influx in our experimental model Our results support the earlier suggestion of Szász et al (1981) and Gáidos et al (1980) based on data obtained with ATP-depleted cells, or La 3 + -treated RBC (Gárdos et al 1980), or RBC treated with propranolol (Szász et al 1977, Gárdos et al 1980) These authois used inhibition of the anion channel by the stilbene derivative SITS 45 2+ or dipyndamol (Gárdos et al 1980) which stimulated C a influx as a tool This appioach yielded similar results also in vanadate-tieated RBC (Varečka and Carafoh 1982) but could not identify the component of the K + electrochemical potential effective in the inhibition of the 4 5 C a 2 + influx by extracellular K + The inhibition of the anion channel also blocks the Gáidos effect and preserves the K + gradient Consequently, the stimulation of the 4 5 C a 2 + influx could be due to the increase of the membrane potential (if the C a 2 + carrier is a uniporter) or to the maintaining of the K + giadient (if the C a 2 + carrier is a C a 2 + / 2 K + antiporter) Our íesults, however, seem to resolve this ambiguity In our previous paper (Varečka and Caiafoli 1982) we suggested that the dia-
354
Varečka et al +
matíc shift m [K ] at both sides of the R B C membrane caused by t h e Gárdos effect which led to the dissipation of the K
+
gradient acts as negative feedback
mechanism preventing the oveiload of cytoplasm by C a
2+
Presented results are 2+
not in contiadiction with this suggestion T h e stimulation of the C a in the eailv phase aftei K(Ca) opening (few minutes after C a
2+
influx occurs
addition) when
the membrane hyperpolanzation is maximal (Fig 2.4) T h e membrane potential change fades íapidly (Fig 2A) whereas [ K + ] 0 increases gradually and reaches t h e steady-state after 30-45 mm (Vaiečka and Caiafoh 1982) Thus, both effects aie tempoiall} sepaiated
Such a dual effect of Iv + has been recently described in
synaptic processes (Matyushkin et al
1995)
T h e suggestion t h a t the activity of K(Ca) piomotes the
45
C a 2 + influx and
theieby c o n t n b u t e s t o the total C a 2 + - m o t i v e foice contains a contradiction T h e Iv(Ca) could contribute to the total C a 2 + - m o t i v e foice only aftei C a 2 + accumu late m the cvtoplasm but the accumulation is only piomoted after opening of t h e K(Ca)
This contiadiction could be explained by pioposmg that a tiansient pe
n o d o c t u i s which s t a i t s aftei the inhibition of the C a 2 + the opening of t h e K ( C a ) D u n n g tins p e n o d C a íesponsible foi t h e íestmg C a 2 + cvcling
2+
ATPase and ends aftei
accumulate by a mechanism
Anothei possibility could be that vana
d a t e exeits a dual (oi multiple) effect on the R B C membiane, affecting both the C a " + influx mechanism and K(Ca) m a cooidmate fashion
Oui results published
m the accompanying papei (\aiecka et al , 1997) support the second possibility Finallv oui íesults convincingly explain the loss in t h e sensitivity of t h e C a 2 + m flux to the medium composition obseived when Ca-chelatoi t i a p p m g pioceduies were used ( M c N a m a i a and Wiley 1986, P o k u d m and Orlov 1986)
These pioce
duies pievented t h e K ( C a ) opening and theieby t h e membrane potential change and t h e Gaidos effect
The RBC m e m b i a n e letamed its extremeh low and almost
identical K + and N a + peimeabihty (Lew and Beauge 1979) which precluded t h e "sensing" of the changes m the medium composition unlike other procedures such as vanadate t i e a t m e n t (Vaiečka and Caiafoh 1982) or A T P depletion (Ferreira and Lew 1977, Szasz et al 1977, 1981, Lew and F e r r e n a 1978), which aie accompanied by t h e Gaidos effect T h e stimulating íole of K(Ca) opening on C a 2 + influx is not restricted to RBC
Recently, a similar phenomenon was obseived during an analysis of the
lmmunoglobulin-induced
45
C a 2 + influx by basophilic leukemia cells (Labrecque et
al 1991) Othei obseivations have been made in a variety of cells where t h e C a 2 + influx induced by receptoi agonists was inhibited by a decrease of t h e m e m b r a n e potential (Oettgen et al 1985 Sage and Rink 1986 DiVirgiho et al 1987, Mohr and Fewtrell 1987, Penner et al 1988 Savage et al 1989 Luckhoff and Busse 1990, P i t t e t et al 1990) However, the role of the K(Ca) has not yet been experimentally investigated
It may be interesting t o mention t h a t M a c a r a and Gray (1987) m a d e
this obseivation also in vanadate-treated A431 epidermal carcinoma cells
These
355
R e d Cell C a 2 + Homeostasis 2+
data suggest that vanadate may mimic some Ca -mediated receptor agonist action(s) in the cell membranes which frequently occui in cell membranes (Račay and Lehotský 1996) This is not necessarily true for other inducers of the Gáidos effect For example, vanadate and fluoride tngger the Gárdos effect probably by 45 2+ diffeient mechanisms as indicated by the extent of C a influx and changes in + N a permeability elicited by these agents (Varečka et al 1994, 1995) A c k n o w l e d g e m e n t s . T h e major p a r t of this work described in this m a n u s c r i p t was performed d u r i n g t h e stay of all a u t h o r s m t h e former M e n t a l H e a l t h Research C e n t e r at t h e P s v c h i a t n c Hospital Pezmok T h e skilled technical assistance by Ms E Kovačičová a n d Ms E Píšová is acknowledged T h e a u t h o r s wish t o t h a n k Prof Dr Jozef P o g á d v for general s u p p o r t A minor p a r t of t h e work was s u p p o r t e d by t h e Science G r a n t Agency V E G A ( G r a n t No 1/4203/97)
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