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Myo-inositol trisphosphate-mediated calcium release from internal stores of Entamoeba histolytica
MOLECULAR AND
ELSEVIER
Molecular and Biochemical Parasitology 65 (1994) 63-71
BIOCHEMICAL PARASITOLOGY
Myo-inositol trisphosphate-mediated calcium release from internal stores of Entamoeba histolytica Sanghamitra Raha a, Basudeb Dalal b, Susweta Biswas b, Birendra B. Biswas a'b'* aDepartment of Biophysics, Molecular Biology and Genetics, Calcutta University, 92, A.P.C. Road, Calcutta 700 009, India, bDepartment of Biochemistry, Bose Institute, Centenary Building, P-1/12, C.I.T. Scheme VII-M, Calcutta 700 054. India
Received 24 September 1993; accepted 2 February 1994
Abstract
Calcium mobilisation from internal stores of the parasitic protozoan Entamoeba histolytica was studied by fluorescence measurements of the calcium indicator quin 2 and 45Ca2+ incorporation studies in saponin-permeabilised amoebae. Prior energy-dependent calcium sequestration was found to be necessary for subsequent release of calcium by inositol 1,4,5-trisphosphate (Ins(l,4,5)P3). Both Ins(l,4,5)P3 and inositol 2,4,5-trisphosphate (Ins(2,4,5)P3) could release calcium equally well from permeabilised E. histolytica with similar ECs0 (concentration which produced half maximal release) values for calcium release. Ins(1,4,5)P3-mediated calcium release occurred from a vesicular store, was sensitive to prior treatment by heparin and was attenuated by prior addition of a lower concentration of Ins(1,4,5)P3. cAMP failed to influence inositol trisphosphate induced calcium release, indicating the absence of control mechanisms through cAMP-dependent phosphorylation. GTP neither induced calcium release nor could potentiate inositol trisphosphate mediated calcium mobilisation. A saturating concentration of Ins(l,4,5)P3 could release 50% of radiolabelled calcium sequestered by energy-dependent mechanisms in E. histolytica. The energy-dependent calcium sequestration was inhibited by vanadate and the calcium antagonist Diltiazem but not by dicyclohexylcarbodiimide (DCCD), suggesting the involvement of an endoplasmic reticulum-like structure in calcium storage. Binding studies showed specific association of [3H]Ins(1,4,5)P3 to crude membrane fractions of E. histolytica, which was significantly inhibited by heparin in a dose-dependent manner. IC50 (concentration which produced half-maximal inhibition) values for displacement of radiolabelled Ins(1,4,5)P3 binding by unlabelled Ins(l,4,5)P3 and Ins(2,4,5)P3 were estimated to be 0.99 #M for both isomers. Our results suggested that Ins(1,4,5)P3-mediated calcium release from internal stores of E. histolytica most probably occurred in an inositol trisphosphate receptor-dependent manner. Key words." Calcium; Inositol trisphosphate; Entamoeba histolytica
~ponding author. Fax: (91) (33) 34-3886; Tel.: (91) (33) 37-9219/9416/9544. Abbreviations: [Ca2+], ambient free Ca 2+ concentration; pCMB, p-chloromercurobenzoic acid; DCCD, N-N'-dicyclo-
0166-6851/94/$7.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 6 - 6 8 5 1 ( 9 4 ) 0 0 0 4 5 - O
hexylcarbodiimide; GTP~S, guanosine 5'-0'-(3-thiotriphosphate); InsP3, inositol trisphosphate; EC5o, concentration which produced half-maximal release; IC5o, concentration which produced half-maximal inhibition; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; Ins(2,4,5)P3, inositol 2,4,5-trisphosphate; TMB, 8-3,4,5-trimethoxybenzoic acid.
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S. Raha et al./Molecular and Biochemical Parasitology 65 (1994) 63-71
1. Introduction
The parasitic protozoan Entamoeba histolytica invades human tissue by a multi-step process, initiated by recognition and contact of target cells with subsequent release of proteolytic enzymes from storage granules. The final step in this invasive event is the lysis of target cells and extracellular matrix [1]. Several recent reports have emphasised the role of intraparasitic calcium and protein kinase C in the cytolytic activities of E. histolytica [2,3]. 3,4,5Trimethoxybenzoic acid (TMB-8), an intracellular calcium antagonist, caused significant reduction in vesicle exocytosis by this parasite [2]. Furthermore, calmodulin has been demonstrated in E. histolytica and strong inhibition of the release of proteolytic enzymes from E. histolytica was seen when the parasite was treated with calmodulin antagonists [4]. More recently, calmodulin redistribution during electron-dense granule secretion was documented by immunofluorescence methods [5]. However, very little else is currently known about calcium homeostasis in this organism. Elevation of internal calcium levels is a necessary event associated with cellular functions such as secretion, contraction and movement [6]. Intracellular calcium stores, which may be mobilised to increase free calcium levels and resequestered, are vital components of cellular calcium homeostatic mechanisms. Energy-dependent calcium resequestration is in operation in most cell types, leading to termination of a calcium signal, along with the refilling of releasable pools [7]. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) has been identified as an important second messenger, responsible for release of calcium from intracellular stores in various eukaryotic cells. Ins(1,4,5)P3 is formed by the action of phospholipase C on phosphatidyl-inositol bisphosphate in response to agonists [8]. Although phosphatidylinositol is present in E. histolytica [9], neither the presence of inositol phosphates nor the existence of Ins(1,4,5)P3-mediated calcium release mechanisms has been demonstrated in this parasite. Cells as diverse as the slime mould Dictyostelium [10] and mammalian pancreatic cells [11] contain Ins(1,4,5)P3-sensitive calcium stores. However,
two recent studies reported the absence of Ins(1,4,5)P3-sensitive calcium stores in parasitic protozoa Trypanosoma brucei and Trypanosoma cruzi [12,13]. Our object is to characterise some of the mechanisms by which E. histolytica may store and release intracellular calcium and to identify the role inositol trisphosphate (InsP3) may play as a second messenger in this protozoan parasite.
2. Materials and methods
Materials. Quin 2, calcium ionophore A23187, Ins(l,4,5)P3, inositol(1)monophosphate (Ins(1)P~), inositol(2)monophosphate (Ins(2)P1), inositol(4,5)bisphosphate (Ins(4,5)P2), inositol hexakisphosphate (InsP6), heparin, cAMP, GTP, guanosine 5'-0'-(3-thiotriphosphate) (GTP yS) and ATP were obtained from Sigma Chemicals, USA. Inositol 2,4,5-trisphosphate (Ins(2,4,5)P3) was from Boehringer Mannheim. Saponin was from Loba Chemicals, Bombay, India. [3H]Ins(1,4,5)P3 (100 Ci mmol - l ) and the kit for detection of Ins(1,4,5)P3 were purchased from Amersham International. Diltiazem was a generous gift of P.C. Sen, Department of Chemistry, Bose Institute, Calcutta. 45Ca2+ (56 mCi g-~) was purchased from BARC, Bombay, India. Cell culture. E. histolytica (EC22) trophozoites were obtained from Kothari Medical Centre for Gastroenterology, Calcutta, where they were isolated from patients and axenised. Trophozoites were maintained in complete TYI-S-33 medium. The TYI-S-33 medium (Trypticase, Yeast extract, Iron, Serum) consisted of a nutrient broth (TYI), a vitamin/Tween 80 mixture and bovine serum. Late log phase cultures (72 h) were harvested. Preparation of cells. E. histolytica trophozoites were harvested from culture by centrifugation (300 × g, l0 rain) at room temperature and finally resuspended in an 'intracellular' type of buffer (100 mM KC1/ 2 mM MgCl2/ 10 mM NaCl/ 10 mM piperazine N,N'-bis 2-ethanesulphonic acid (Pipes)/ 10 mM glucose, pH 7.2) for permeabilisation. Cell suspensions were kept in capped tubes to
L. Biswas et al./Molecular and Biochemical Parasitology 65 (1994) 63-71
avoid direct exposure to air. Aliquots (1 ml) of the cell suspension (106 ml-1) were permeabilised by the addition of 20 #g 1-a saponin for 1 min (32°C) directly in the spectrofluorimeter cuvette immediately before the addition of test substances. Permeabilised cells were tested for trypan blue inclusion and found to be 95% positive for trypan blue.
S_pectrofluorimetric measurements. The intracellular Ca 2+ mobilisation in saponin-permeabilised amoebae was monitored by measuring changes in quin 2 fluorescence in a Hitachi F-3000 Spectrofluorimeter. Excitation and emission wavelengths were set at 339 nm and 492 nm respectively. The ambient free Ca 2+ concentration [Ca2+] was calculated using the equation: [Ca 2+ ] = Kd(F- Fmin)/(Fmax- F), where Kd = 115 nm and F is the fluorescence in arbitrary units, Fmax was calculated in the presence of the cell suspension, 2 mM CaCI2 and 0.1% Triton X-100 and Fmi n w a s measured with 5 mM ethylene glycol bistetraacetic acid (EGTA). [Ca 2+] measured in this system was the amount of free Ca 2+ in the permeabilised preparation. Quin 2 was used both as a fluorescent calcium indicator and a buffer for free calcium [14]. Changes in total calcium (buffered plus free) was determined by the addition of known amounts of CaCI2 [14]. Autofluorescence of A23187 was measured in the absence of quin 2 under exactly similar conditions used for quin 2 measurements. Autofluorescence of calcium ionophore A23187 was subtracted from all measurements made with this compound. After 1 min incubation in the presence of 100 #M quin 2 and (20 #g m l - l ) saponin, 2 mM ATP was added to initiate calcium sequestration by permeabilised cells. Fluorescence was monitored for 3 min when a steady-state level of calcium uptake was achieved. Subsequently, InsP3 and all other substances, tested for their calcium releasing properties, were added. For inhibition studies, heparin (100 /~g ml -l) and p-chloromercurobenzoic acid (pCMB) (1 mM) were added to the cell suspension simultaneously with quin 2 and saponin. The
65
ATPase inhibitors or calcium antagonists were allowed to incubate with the permeabilised cells for 1 n'fin prior to the initiation of calcium sequestration by ATP. Peak [Ca 2+ ] was taken as the maximum [Ca2÷ ] reached within the first 25 s after addition of the test substance. Basal [Ca2÷] was that measured just before the addition of the test substance. When two additions were made, basal values for the second addition were taken as [Ca 2+] before the first addition. Data from Ins(1,4,5)P3 and Ins(2,4,5)P3 dose-response experiments were fitted to the Hill equation with the equation:
1 + (g/[s])h
where Vm is the Ca 2+ release response at maximal [InsP3], v is the Ca 2+ release given by different InsP3 concentrations [S], K is the ECso (concentration which produced half maximal release) value for InsP3, and h is the Hill coefficient [14].
uptake and release in permeabilised E. histolytica trophozoites. E. histolytica trophozoites
45Ca 2+
(107m1-1) were suspended in loading buffer (100 mM KCI/ 20 mM N a C I / 2 mM MgCI2/ 80 mM Sucrose/ 1 mM EGTA/ 390 /zM CaCIz/ 20 mM Pipes, pH 7.0) [15] and permeabilised by the addition of 20 #g ml-~ saponin for 2 min at 37°C. 45Ca2+ (10/.tCi m1-1) and 2 mM ATP were subsequently added. The cells were kept at 37°C for 5 min for the 45Ca2÷ content to reach a steady state and then diluted 10-fold with loading medium in the presence or absence of 2 #M Ins(l,4,5)P3. Termination of incubation after 60 s was obtained by addition of ice-cold termination buffer (310 mM sucrose/ 1 mM EGTA/ 1 mM Hepes, pH 7.0 and subsequent filtration through GF/C filters. The filters were solubilised in scintillation cocktail and counted in a Beckman scintillation counter.
Binding of [SH]Ins(1,4,5)P3 to the crude membrane fraction from E. histolytica. Crude membrane fraction was obtained by homogenising the cells in a buffer containing 0.1 M Tris-HC1/ 1 mM EDTA, pH 9.0 with a glass/Teflon homogeniser
66
S. Raha et al./Molecular and Biochemical Parasitology 65 (1994) 63-71
for 10 min followed by centrifugation of the homogenate at 1000 x g for 15 min. The supernatant was centrifuged at 35 000 x g for 15 rain. Binding assay with E. histolytica crude membrane fraction was performed at 4°C for 10 min in the presence of 10 nM [3H]Ins(1,4,5)P3. Bound Ins(l,4,5)P3 was separated from the free by centrifugation. Non-specific binding was determined in the presence of 10/~M unlabelled Ins(1,4,5)P3 and this value was subtracted from the total binding to obtain specific binding. Displacement of binding was observed in the presence of 0.04/~M to 4 #M unlabelled Ins(1,4,5)P3 or Ins(2,4,5)P3. The concentration used for displacement of binding by other inositol phosphates was 5-80 #M. For inhibition experiments, heparin concentrations in the range of 1-20/~g ml-1 were used in the binding assay.
Ins(1,4,5)P3 was detected in protein-free extracts of E. histolytica ceils by a competition binding assay provided in the Amersham detection kit for Ins(1,4,5)P3.
3. Results
InsP3-induced calcium release in permeabilised E. histolytica. The low-calcium, high-potassium buffer used for preparing the permeabilised cell suspension was buffered by 100 #M quin 2 free acid. Initial free calcium concentrations in the permeabilised cell preparations varied between 150 and 200 nM. In this range, changes observed with added calcium were linear. It is apparent from Fig. 1A that Ins(1,4,5)P3 was effective in calcium mobilisation in permeabi-
A.
B.
200
300
150 r
c
+
%
+
~ 100
~50
L.
50 ~
A23187
|
1
1
0.2)JM InsP3
2-O~M Ins P3 3
3
I
/, 5 (rain)
I
|
I
G
7
3
8
7
~SP3 I
~ (rain)
I
I
5
6
Fig. 1. ATP-induced calcium sequestration and subsequent release by Ins(1,4,5)P3 and A23187 from saponin-permeabilised E. histolytica trophozoites. Cells were permeabilised for 1 min with 20/~g m l - i saponin. Calcium sequestration was initiated by the addition of 2 mM ATP. 3 min after the addition of ATP, (A) 2 #M Ins(l,4,5)P3 was added which was followed 2 min later by 5 #g m1-1 A23187. In (B) after calcium sequestration with ATP, 0.2/~M Ins(1,4,5)P3 was added first, followed 2 min later by 2 #M Ins(l,4,5)P3 with a final addition of 5/~g ml - I A23187 another 2 min later. The calibration bar in the figure represents 1 nmol ml - l Ca 2+ . Results are representative of 3 ~ individual experiments. Free and total calcium were estimated from fluorescence of the calcium indicator dye quin 2 as described in Materials and methods. Autofluorescence of calcium ionophore A23187 was subtracted from the peak fluorescence value given by this compound. The traces showing A23187 induced calcium release are drawn with the dotted lines to indicate corrections. Solid lines are used for original traces.
67
L. Biswas et al./Molecular and Biochemical Parasitology 65 (1994) 63-71
lised amoebae. Calcium ionophore A23187 induced a calcium release higher than that achieved by Ins(1,4,5)P3 and A23187 could release calcium after Ins(1,4,5)P3 treatment (Fig. 1A,B). In contrast, prior A23187 treatment resulted in abolition of InsP3 induced calcium release (data not shown). Also, prior treatment with a lower concentration (0.2/~M) of InsP3 reduced release by 2 #M InsP3 (Fig. 1B). Ins(1,4,5)P3 released intracellular calcium from permeabilised E. histolytica trophozoites in a dosedependent manner (Fig. 2). Similar patterns of dose-dependence was also observed with Ins(2,4,5)P3 (data not shown). The ECs0 values estimated for Ins(1,4,5)P3 and Ins(2,4,5)P3 were 0.174+0.120 /~M and 0.288+0.119 #M respectively ( P > 0.05). 45Ca2+ uptake and release studies confirmed the results obtained by fluorimetric measurements. 2.46_+0.50 nmol 4 5 C a 2 + w e r e loaded into the intracellular pools in an energy-dependent manner by 106 cells. About 50% of this labelled pool was released by 2 #M Ins(1,4,5)P3 (1.22_+0.31 nmol 10 - 6 cells). Calcium ionophore A23187 (5 #g m l - ] ) released 70.46% of the accumulated calcium label (1.73___0.70 nmoll0 -6
Comparison of various inositol phosphates and G TP as stimulators of calcium release in permeabilised E. histolytica. A saturating concentration of Ins(1,4,5)P3 (2 #M) produced a slightly higher but statistically insignificant (P> 0.05) calcium increase than that mediated by equal concentration of Ins(2,4,5)P3 (Table 1). Significant reduction ( P < 0.05) was observed in Ins(1,4,5)P3-induced calcium release by pretreatment with a lower concentration of Ins(l,4,5)P3. A23187 mobilised a significantly higher ( P < 0.05) amount of calcium than 2 #M Ins(1,4,5)P3. About 63% of the A23187 releasable calcium pool could be mobilised by 2 #M Ins(1,4,5)P3 as measured by quin 2 fluorescence. Other inositol phosphates failed to produce any change (P> 0.05) in Ca 2+. Heparin (100 #g ml - l ) significantly (P< 0.05) inhibited Ins(1,4,5)P3-mediated calcium release (Table 1). Significant potentiation of InsP3-mediated calcium release was not observed by simultaneous application of GTP, which is known to regulate calcium movements from InsP3-insensitive to InsP3-sensitive stores in mammalian cells [16,17]. Moreover, GTP (10 #M and 100 #M) or the nonTable 1 Calcium release by various inositol phosphates and calcium ionophore A23187 in permeabilised E. histolytica trophozoites
I.L 1-2
cells). 2 #M Ins(1,4,5)P3 could release about 70% of the ionophore-releasable calcium.
i
Inducer
Concentration
0.8
~
!o~
Calcium release (Peak/Basal)
~
i
~
ILO
[Ins (I,~.,5) P3~(~ M ) Fig. 2. Dose-dependent Ca 2+ release by Ins(1,4,5)P3 from permeabilised E. histolytica trophozoites. Experimental conditions are similar to those described for Fig. 1 and in Materials and methods. Free and total calcium were estimated from fluorescence m e a s u r e m e n t s o f the calcium indicator dye quin 2 as described in Materials a n d methods. Results are expressed as the m e a n of 2-6 individual experiments.
Ins(1,4,5)P3 2 gM Ins(1,4,5)P3 (added 2 rain 2 #M after 0 . 2 / t M Ins(1,4,5)P3) Ins(1,4,5)P3 (plus 100 #g ml -l 2 g M heparin) Ins(2,4,5)P3 2 #M Ins(l)monophosphate 10 # M Inositol hexakis phosphate 10 # M Calcium ionophore A23187 5 #g ml - [
2.73 + 0.36 1.86 + 0.54 1.66 + 0.36 2.39 1.10 1.00 4.31
___ 0.29 _ 0.10 _ 0.05 ___ 1.78
Experimental conditions are similar to those described in Fig. 1 and Materials and methods. Peak [Ca 2+] was taken as the m a x i m u m Ca 2+ level reached within the first 25 s after the addition of the test substance. Basal [Ca 2+ ] was that measured just before the addition of the first test substance. Data are presented as mean + S.D. o f 3-6 experiments.
68
S. Raha et aL/Molecular and Biochemical Parasitology 65 (1994) 63-71
hydrolysable GTP analogue, GTP 7S (10 /~M) alone, was unable to release any calcium from permeabilised amoebae (data not shown). Influence of calcium transport inhibitors on calcium sequestration and subsequent calcium release by Ins(1,4,5)P3. Presence of 500/tM vanadate during ATP-dependent calcium sequestration produced a very strong inhibition ( P < 0.01) of calcium uptake and subsequent relaese by Ins(1,4,5)P3 (Table 2). Neither cAMP which is known to have influence on the mammalian InsP3 receptor [18,19], nor Dicyclohexylcarbodiimide (DCCD - an inhibitor of the vacuolar proton translocating ATPase) had any effect on either of these processes. However, Diltiazem, a calcium antagonist, significantly ( P < 0.01) inhibited both calcium sequestration and subsequent release. In the absence of ATP-dependent calcium sequestartion, subsequent calcium release by Ins(1,4,5)P3 was inhibited very strongly (91.25% inhibition). pCMB, a sulphhydryl disrupting agent [14], also significantly ( P < 0.01) inhibited calcium uptake and subsequent Ins(1,4,5)Pa-mediated calcium release.
Table 2 Influence of calcium transport inhibitors on calcium sequestration and subsequent release by Ins(l,4,5)P3 in permeabilised E.
histolytica Compound
Ca 2+ sequestration (% inhibition)
Sodium orthovanadate (500 # M ) c A M P (10/tM) D C C D (20/~M) Diltiazem (10 #M)
76.85 + 22.39
para-Chloromercuroben-
9.44 6.47 71.08 98.02
+_ _ _+ _
6.45 11.21 12.42 10.10
Ins(1,4,5)P3 induced release (% inhibition) 67.86 _ 8.77 4.67 +_ 6.43 7.18 _ 3.20 93.50 _ 7.78 100
zoic acid (pCMB) (1 m M ) Experimental conditions are similar to those described in Fig. 1 and Materials a n d methods. Inhibitors were added to the cell suspension 1 min before the initiation of ATP-dependent calcium sequestration. Values obtained in the absence o f inhibitors are taken as 100%. D a t a are shown as m e a n s _ S.D. of 3 experiments.
100
75 \
~
x
\,,.
~ 50 un
-
I
!
I
0
1
2
log Ins P3(xlM) Fig. 3. Inhibition of specific[3H]Ins(1,4,5)P3binding by unlabelled Ins(1,4,5)P3 and Ins(2,4,5)Pa in the crude membrane fraction from E. histolytica trophozoites. 0, Ins(l,4,5)P3; A, Ins(2,4,5)P3. Binding assays contained 10 nM [3H]Ins(1,4,5)P3,0.1 mg of membraneprotein and indicated amounts of unlabelledIns(1,4,5)P3 and Ins(2,4,5)P3. Data are representativeof 2-3 experiments. Ins(1,4,5)P3 binding to crude membrane preparation from E. histolytica. Specific binding of [3H]Ins(1,4,5)P3 was observed in E. histolytica crude membrane fraction. [3H]Ins(1,4,5)P3 binding was increasingly displaced by the presence of 0.04 pM to 4 /zM unlabelled Ins(1,4,5)P3 and Ins(2,4,5)P3 (Fig. 3). Unspecific binding was on the average 25% of specific binding. Scatchard transformation of the isotope dilution of [3H]Ins(1,4,5)P3 binding data revealed a straight line indicative of a single binding site (data not shown). IC50 (concentration which produced halfmaximal inhibition) values were estimated from the following equation [20] which describes displacement of a trace amount of bound [3H]Ins(1,4,5)P3 by addition of unlabelled InsP3 assuming a single binding site:
L. Biswas et al./Molecular and Biochemical Parasitology 65 (1994) 63-71 Table 3 Effect of displacing agents and antagonists on the specific [3H]Ins(1,4,5)P3 binding in E. histolytica crude membrane fractions Compound
Estimated ICso
Ins(l,4,5)P3 Ins(2,4,5)P3 Ins(4,5)bisphosphate Ins(l)monophosphate Ins(2)monophosphate Inositol hexakisphosphate Heparin
0.99 0.99 12.58 17.23 15.26 19.42 0.77
_ _ _ _ + + _
0.13 0.29 4.63 4.36 2.46 2.00 0.18
Crude membrane fractions were isolated from E. histolytica trophozoites as described in the Methods section. Assay contained 100 #g of crude membrane protein, 10 nM [3H]Ins(1,4,5)P3 and various concentrations of displacing agents or the antagonist, heparin. IC5o was estimated as described in the text. IC5o values are given as/~M for inositol phosphates and as/~g ml-1 for heparin.
[InsP3]
1 + 1C5o
where B* is the amount of labelled InsP3 bound, Bo* is the amount of labelled InsP3 in the absence of unlabelled InsP3, [InsP3] is the concentration of unlabelled InsP3. The IC5o values (Table 3) for displacement of binding of [3H]InsP3 by unlabelled Ins(1,4,5)P3 and Ins(2,4,5)P3 were 0.99 #M for both isomers. On the other hand, IC5o values for other inositol phosphates were markedly larger (13-19 #M). Heparin strongly inhibited [3H]Ins(1,4,5)P3 binding in crude membrane fractions of E. histolytica in a dose-dependent manner (IC5o = 0.77 #g m l - 1). Basal intracellular Ins(1,4,5)P3 concentration was estimated to be 55.0-t-32.79 pmol (10 -6 cells) (n = 3).
4. Discussion
Adhesion and subsequent secretion of proteolytic enzymes leading to cytolysis of target cells are two major steps for a successful invasion of the host by E. histolytica. Ravdin et al. [21] have de-
69
monstrated that the adhesion of this parasite to target cells does not increase total internal calcium levels but results in regional changes in free calcium concentration. In another report, the same group of investigators [2] have emphasised the role of internal calcium, calmodulin and the cytoskeleton in the secretion of parasitic granule contents. Ravdin et al. [22] also suggested that either the entry of extracellular calcium or the release of internal calcium mediated the secretory process. We have observed the importance of calcium homeostasis in E. histolytica, as A23187 treatment of intact amoebae or Ins(1,4,5)P3 treatment of permeabilised amoebae resulted in increased motility and pseudopod formation [23]. The present study documented in E. histolytica, the release of internal calcium by the calcium mobilising second messenger Ins(1,4,5)P3 and specific binding of [3H]Ins(1,4,5)P3 to membranes of E. histolytica. The well-known InsP3 receptor antagonist heparin [24] significantly inhibited both of these processes. The ECs0 of Ins(1,4,5)P3-induced calcium release (0.1-0.75 #M) reported for permeabilised mammalian cells [6,11] seems compatible with our finding in E. histolytica (EC50-0.17 #M), indicating similar sensitivity to Ins(1,4,5)P3. In contrast, however, was the almost similar effectiveness of Ins(2,4,5)P3, which is known to be less potent in releasing calcium in mammalian cells [25,26]. Furthermore, the IC50 values demonstrated by these two isomers for displacement of labelled Ins(1,4,5)P3 binding are also similar (Table 3). In this context, the findings of two other studies [20,27] revealing equal responsiveness of the InsP3 receptor in Neurospora crassa [27], and catfish olfactory cilia [20] to Ins(1,4,5)P3 analogues could be relevant. The affinity of Ins(1,4,5)P3 for the E. histolytica binding site is 30-125 times lower than the values reported for the receptor in mammalian tissues [25,28,29]. Interestingly, the IC50 value for Ins(1,4,5)P3 in E. histolytica (0.99/zM) is very similar to that reported for catfish olfactory cilia (1.1 #M) which possesses a binding site of different molecular mass from the cerebellar Ins(1,4,5)P3 receptor [20]. Our 45Ca2+ release studies demonstrated that
70
s. Raha et al./Molecular and Biochemical Parasitology 65 (1994) 63-71
about 50% of the intracellular 45Ca2+ label was released by 2 #M Ins(1,4,5)P3. This could compare favourably with the value of 30-50% reported for m a m m a l i a n cells [8]. This finding underscores the importance of Ins(1,4,5)P3 releasable calcium pool in this parasite. Calcium ionophore A23187 failed to release all of the sequestered 45Ca2 + from E. histolytica. Similar observations about the existence of a calcium pool unresponsive to ionomycin alone have been made in PC 12 cells [30]. The presence of vacuolar structures in E. histolytica is known from microscopic studies [31]. Absence o f inhibition on calcium sequestration and subsequent InsP3-induced release by D C C D , an inhibitor of vacuolar proton translocating ATPases [32-34], demonstrated that this type of ATPase m a y not be involved in calcium uptake into InsP3-sensitive pools in E. histolytica (Table 2). Vanadate is an inhibitor of both plasma membrane and microsomal Ca 2÷ p u m p ATPase [35,36]. Our observation o f a high degree of inhibition by vanadate of the ATP-dependent Ca 2+ uptake and subsequent InsPa-induced calcium release (Table 2) strongly suggested that the InsP3-releasable pool is located in an endoplasmic reticulumlike structure. Ultrastructural studies [31] have revealed the presence of smooth endoplasmic reticulum and other endoplasmic reticulum-like membranous structures in E. histolytica. Diltiazem, an inhibitor of calcium channels in excitable tissue [37], has very recently been described [38] to block calcium entry into internal stores and also to inhibit stimulus-induced calcium release from intracellular sites in neutrophils. Inhibition of calcium uptake and subsequent release in E. histolytica by Diltiazem indicate that a therapeutically used calcium antagonist m a y be able to disrupt calcium homeostasis in this parasite. Our findings in E. histolytica were not in agreement with recent reports [12,13] on other parasitic protozoa Trypanosoma brucei and Trypanosoma cruzi. In these organisms, in spite of the presence of intracellular inositol phosphates, especially Ins(1,4,5)P3, no calcium release mediated by InsP3 was detected. We demonstrated (1,4,5)P3-mediated calcium
release and [3H]Ins(1,4,5)P3 binding in E. histolytica, most probably through an InsP3 receptor mediated mechanism. Further characterisation of the cellular signalling mechanisms in this enteric parasite m a y have implications for the development of antiparasitic therapy aimed at these vital pathways.
Acknowledgements We would like to thank the Kothari Medical Centre, Calcutta for generously providing us with the E. histolytica strain. S.R. was supported in part by the Council of Scientific and Industrial Research, India as a scientific pool officer.
References [1] Talamas-Rohana, P. and Meza, I. (1988) Interaction between amebas and fibronectin: Substrate degradation and changes in cytosolic organization. J. Celi Biol. 106, 1787-1794. [2] Ravdin, J.I., Murphy, C.F. and Schlesinger, P.H. (1988) The cellular regulation of vesicleexocytosis by Entamoeba histolytica. J. Protozool. 35, 159-163. [3] Weikel, C.S., Murphy, C.F., Orozco, E. and Ravdin, J.I. (1988) Phorbol esters specifically enhance the cytosolic activity of Entamoeba histolytica. Infect. Immun. 56, 14851491. [4] Munoz, M de L., Moreno, M.A., Perez-Garcia, J.M., Tovar, G.R. and Hernandez, V.I. (1991) Possible role of calmodulin in the secretion of Entamoeba histolytica electron-dense granules containing collagenase. Mol. Microbiol. 5, 1707-1714. [5] Munoz, M de L., O'Shea-Alvarez, M de S., Perez-Garcia, J., Weinbach, E.C., Moreno, M.A., DeLa Torri, M., Magos, M.A. and Tovar, R. (1992) Purification and biochemical properties of calmodulin in Entamoeba histolytica and its distribution during secretion of electron dense granules. Comp. Biochem. Physiol. 103B, 517-521. [6] Berridge, M.J. (1987) Inositol trisphosphate and diacyl glycerol: Two interacting second messengers. Annu. Rev. Biochem. 56, 159-193. [7] Carafoli, E. (1987) Intracellular calcium homeostasis. Annu. Rev. Biochem. 56, 395-433. [8] Berridge, M.J. and lrvine, R.F. (1989) Inositol phosphates and cell signalling. Nature 341, 197-205. [9] Aust-Kettis, A., Thorstensson, R. and Utter, G. (1983) Antigenecity of Entamoeba histolytica strain NIH-200: a survey of clinically relevant antigenic components. Am. J. Trop. Med. Hyg. 32, 512 522.
L. Biswas et al./Molecular and Biochemical Parasitology 65 (1994) 63-71
[10] Europe-Finner, G.N. and Newell, P.C. (1986) Inositol 1,4,5 trisphosphate induces calcium release from a nonmitochondrial pool in amoebae of Dictyostelium. Biochim. Biophys. Acta 887, 335-340. [11] Streb, H., Irvine, R,F., Berridge, M.J. and Schultz, I. (1983) Release of Ca 2+ from a non-mitochondrial intracellular store in pancreatic acinar cells by inositol 1,4,5 trisphosphate. Nature 306, 67-68. [12] Moreno, S.N.J,, Docampo, R. and Vercesi, A.E. (1992) Calcium homeostasis in procyclic and blood stream forms of Trypanosorna brucei: Lack of inositol 1,4,5 trisphosphate-sensitive Ca 2+ release. J. Biol. Chem. 267, 60206026. [13] Moreno, S.N.J., Vercesi, A.E., Pignatoro, O.P. and Docampo, R. (1992) Calcium homeostasis in Trypanosoma cruzi amastigotes: Presence of inositol phosphates and lack of an inositol 1,4,5 trisphosphate-sensitive calcium pool. Mol. Biochem. Parasitol. 52, 251-262. [14] Renard, D.C., Seitz, M.B. and Thomas, A.P. (1992) Oxidized glutathione causes sensitization of a calcium release to inositol 1,4,5 trisphosphate in permeabilized bepatocytes. Biochem. J. 234, 507-512. [15] Oldershaw, K.A. and Taylor, C.W. (1993) Luminal Ca 2+ increases the affinity of inositol (1,4,5) trisphosphate for its receptor. Biochem. J. 292, 631-633. [16] Ghosh, T.K., Mullaney, J.M., Tarazi, F.I. and Gill, D.L. (1989) GTP-activated communication between distinct inositol 1,4,5-trisphosphate-sensitive and -insensitive calcium pools. Nature(London) 340, 236-239. [17] Koshiyama, H. and Tashjian, A.H. Jr. (1991) Evidence for multiple intracellular calcium pools in GH4 CI cells: Investigations using thapsigargin. Biochem. Biophys. Res. Commun. 177, 551-558. [18] Supattapone,S., Danoff, S.K., Theibert, A., Joseph, S.K., Steiner, J. and Snyder, S.H. (1988) Cyclic AMP-dependent phosphorylation of a brain inositol trisphosphate receptor decreases its release of calcium. Proc. Natl. Acad. Sci. USA 85, 8747-8750. [19] Ferris, C.D., Cameron, A.M., Bredt, D.S., Huganir, R.L. and Snyder, S.H. (1991) Inositol 1,4,5-trisphosphate receptor is phosphorylated by cyclic AMP-dependent protein kinase at serines 1755 and 1789. Biochem. Biophys. Res. Commun. 175, 192-198. [20] Kalinoski, D.L., Aldinger, S.B., Boyle, A.G., Huque, T., Marecek, J.F., Prestwich, G.D. and Restrepo, D. (1992) Characterization of a novel inositol 1,4,5-trisphosphate receptor in isolated olfactory cilia. Biochem. J. 281, 449456. [21] Ravdin, J.I., Moreau, F., Sullivan, J.A., Petri, W.A. Jr. and Mandell, G.L. (1988) Relationship of free intracellular calcium to the cytosolic activity of Entamoeba histolytica. Infect. Immun. 56, 1505-1512. [22] Ravdin, J.I., Murphy, C.F., Guerrant, R.L. and LongKrug, S.A. (1985) Effects of antagonists of calcium and phospholipase A on the cytopathogenecity of Entamoeba histolytica. J. Infect. Dis. 152, 542-549. [23] Raha,S., Dalal,B., Biswas,S. and Biswas,B.B. (1994) Myoinositol trisphosphate sensitive calcium stores in Entamoe-
71
ba histolytica. Curr. Sci. 66, 232-234. [24] Ghosh, T.K., Eis, P.S., Mullaney, J.M., Ebert, C.L. and Gill, D.L. (1988) Competitive, reversible and potent antagonism of inositol 1,4,5 trisphosphate-activated calcium release by beparin. J. Biol. Chem. 263, 1107511079. [25] Nunn, D.L. and Taylor, C.W. (1990) Liver inositol 1,4,5trisphosphate-binding sites are the Ca 2+-mObilizing receptors. Biocbem J. 270, 227-232. [26] Tegge, W., Denis, G.V. and Ballou, C.E.(1991) Synthesis and calcium release activity of D and L myoinositol 2,4,5 trisphosphate and D and L chiroinositol 1,3,4 trisphosphate. Carbohydr. Res. 217, 107-116. [27] Schultz, G., Gebauer, G., Metschies, T., Rensing, L. and Jastroff, B. (1990) Cis, Cis-cyclobexane 1,3,5-triol polyphosphates release calcium from Neurospora crassa via an unspecific Ins(1,4,5)P3 receptor. Biochem. Biophys. Res. Commun. 166, 1319-1327. [28] Challiss, R.A.J., Smith, S.M., Potter, B.V.L. and Nahorski, S.R. (1991) D-[HSs(u)] Inositol 1,4,5-tris phosphorothioate, a novel radioligand for the inositol 1,4,5trisphosphate receptor. FEBS Lett. 281, 101-104. [29] Nunn, D.L., Potter, B.V.L. and Taylor, C.W. (1990) Molecular target sizes of inositol 1,4,5-triphosphate receptors in liver and cerebellum. Biochem. J. 265, 393398. [30] Fasolato, C., Zottini, M., Clementi, E., Zaccbelti, D., Meldolesi, J. and Pozzan, T., (1991) Intracellular Ca 2÷ pools in PC12 cells. Three intracellular pools are distinguished by their turnover and mechanisms of Ca 2÷ accumulation, storage and release. J. Biol. Chem. 266, 20159-20167. [31] Reeves, R.E. (1984) Metabolism of Entamoeba histolytica Schauddin, 1903. Adv. Parasitol. 23, 106-143. [32] Schumaker, K.S. and Sze, H. (1986) Calcium transport into the vacuole of oat roots. Characterization of H+/ Ca 2÷ exchange activity. J. Biol. Chem. 261, 12172-12178. [33] Schumaker, K.S. and Sze, H. (1987) Inositol 1,4,5trisphosphate releases Ca 2+ from vacuolar membrane vesicles of oat roots. J. Biol. Chem. 262, 3944-3946. [34] Solioz, M., (1984) Dicyclohexylcarbodiimide as a probe for proton translocating enzymes. Trends Biochem. Sci.9, 309-312. [35] Caroni, P. and Carafoli, E. (1981) The Ca 2+ pumping ATPase of heart sarcolemma. Characterization, Calmodulin dependence and partial purification. J. Biol. Chem. 256, 3263-3270. [36] Gill, D.L. and Chueh, S.-H. (1985) An intracellular (ATP + Mg2+)-dependent calcium pump within the N1E-115 neuronal cell line. J. Biol. Chem. 260, 9289-9297. [37] Campbell, K.P., Leung, A.T. and Sharp, A.H. (1988) The biochemistry and molecular biology of the dihydropyridine-sensitive Calcium channel. Trends Neurosci. 11,425430. [38] Rosales, C. and Brown, E.J. (1992) Calcium channel blockers nifedipine and diltiazem inhibit Ca 2+ release from intracellular stores in neutrophils. J. Biol. Chem. 267, 1443-1 448.
ELSEVIER
Molecular and Biochemical Parasitology 65 (1994) 63-71
BIOCHEMICAL PARASITOLOGY
Myo-inositol trisphosphate-mediated calcium release from internal stores of Entamoeba histolytica Sanghamitra Raha a, Basudeb Dalal b, Susweta Biswas b, Birendra B. Biswas a'b'* aDepartment of Biophysics, Molecular Biology and Genetics, Calcutta University, 92, A.P.C. Road, Calcutta 700 009, India, bDepartment of Biochemistry, Bose Institute, Centenary Building, P-1/12, C.I.T. Scheme VII-M, Calcutta 700 054. India
Received 24 September 1993; accepted 2 February 1994
Abstract
Calcium mobilisation from internal stores of the parasitic protozoan Entamoeba histolytica was studied by fluorescence measurements of the calcium indicator quin 2 and 45Ca2+ incorporation studies in saponin-permeabilised amoebae. Prior energy-dependent calcium sequestration was found to be necessary for subsequent release of calcium by inositol 1,4,5-trisphosphate (Ins(l,4,5)P3). Both Ins(l,4,5)P3 and inositol 2,4,5-trisphosphate (Ins(2,4,5)P3) could release calcium equally well from permeabilised E. histolytica with similar ECs0 (concentration which produced half maximal release) values for calcium release. Ins(1,4,5)P3-mediated calcium release occurred from a vesicular store, was sensitive to prior treatment by heparin and was attenuated by prior addition of a lower concentration of Ins(1,4,5)P3. cAMP failed to influence inositol trisphosphate induced calcium release, indicating the absence of control mechanisms through cAMP-dependent phosphorylation. GTP neither induced calcium release nor could potentiate inositol trisphosphate mediated calcium mobilisation. A saturating concentration of Ins(l,4,5)P3 could release 50% of radiolabelled calcium sequestered by energy-dependent mechanisms in E. histolytica. The energy-dependent calcium sequestration was inhibited by vanadate and the calcium antagonist Diltiazem but not by dicyclohexylcarbodiimide (DCCD), suggesting the involvement of an endoplasmic reticulum-like structure in calcium storage. Binding studies showed specific association of [3H]Ins(1,4,5)P3 to crude membrane fractions of E. histolytica, which was significantly inhibited by heparin in a dose-dependent manner. IC50 (concentration which produced half-maximal inhibition) values for displacement of radiolabelled Ins(1,4,5)P3 binding by unlabelled Ins(l,4,5)P3 and Ins(2,4,5)P3 were estimated to be 0.99 #M for both isomers. Our results suggested that Ins(1,4,5)P3-mediated calcium release from internal stores of E. histolytica most probably occurred in an inositol trisphosphate receptor-dependent manner. Key words." Calcium; Inositol trisphosphate; Entamoeba histolytica
~ponding author. Fax: (91) (33) 34-3886; Tel.: (91) (33) 37-9219/9416/9544. Abbreviations: [Ca2+], ambient free Ca 2+ concentration; pCMB, p-chloromercurobenzoic acid; DCCD, N-N'-dicyclo-
0166-6851/94/$7.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 6 - 6 8 5 1 ( 9 4 ) 0 0 0 4 5 - O
hexylcarbodiimide; GTP~S, guanosine 5'-0'-(3-thiotriphosphate); InsP3, inositol trisphosphate; EC5o, concentration which produced half-maximal release; IC5o, concentration which produced half-maximal inhibition; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; Ins(2,4,5)P3, inositol 2,4,5-trisphosphate; TMB, 8-3,4,5-trimethoxybenzoic acid.
64
S. Raha et al./Molecular and Biochemical Parasitology 65 (1994) 63-71
1. Introduction
The parasitic protozoan Entamoeba histolytica invades human tissue by a multi-step process, initiated by recognition and contact of target cells with subsequent release of proteolytic enzymes from storage granules. The final step in this invasive event is the lysis of target cells and extracellular matrix [1]. Several recent reports have emphasised the role of intraparasitic calcium and protein kinase C in the cytolytic activities of E. histolytica [2,3]. 3,4,5Trimethoxybenzoic acid (TMB-8), an intracellular calcium antagonist, caused significant reduction in vesicle exocytosis by this parasite [2]. Furthermore, calmodulin has been demonstrated in E. histolytica and strong inhibition of the release of proteolytic enzymes from E. histolytica was seen when the parasite was treated with calmodulin antagonists [4]. More recently, calmodulin redistribution during electron-dense granule secretion was documented by immunofluorescence methods [5]. However, very little else is currently known about calcium homeostasis in this organism. Elevation of internal calcium levels is a necessary event associated with cellular functions such as secretion, contraction and movement [6]. Intracellular calcium stores, which may be mobilised to increase free calcium levels and resequestered, are vital components of cellular calcium homeostatic mechanisms. Energy-dependent calcium resequestration is in operation in most cell types, leading to termination of a calcium signal, along with the refilling of releasable pools [7]. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) has been identified as an important second messenger, responsible for release of calcium from intracellular stores in various eukaryotic cells. Ins(1,4,5)P3 is formed by the action of phospholipase C on phosphatidyl-inositol bisphosphate in response to agonists [8]. Although phosphatidylinositol is present in E. histolytica [9], neither the presence of inositol phosphates nor the existence of Ins(1,4,5)P3-mediated calcium release mechanisms has been demonstrated in this parasite. Cells as diverse as the slime mould Dictyostelium [10] and mammalian pancreatic cells [11] contain Ins(1,4,5)P3-sensitive calcium stores. However,
two recent studies reported the absence of Ins(1,4,5)P3-sensitive calcium stores in parasitic protozoa Trypanosoma brucei and Trypanosoma cruzi [12,13]. Our object is to characterise some of the mechanisms by which E. histolytica may store and release intracellular calcium and to identify the role inositol trisphosphate (InsP3) may play as a second messenger in this protozoan parasite.
2. Materials and methods
Materials. Quin 2, calcium ionophore A23187, Ins(l,4,5)P3, inositol(1)monophosphate (Ins(1)P~), inositol(2)monophosphate (Ins(2)P1), inositol(4,5)bisphosphate (Ins(4,5)P2), inositol hexakisphosphate (InsP6), heparin, cAMP, GTP, guanosine 5'-0'-(3-thiotriphosphate) (GTP yS) and ATP were obtained from Sigma Chemicals, USA. Inositol 2,4,5-trisphosphate (Ins(2,4,5)P3) was from Boehringer Mannheim. Saponin was from Loba Chemicals, Bombay, India. [3H]Ins(1,4,5)P3 (100 Ci mmol - l ) and the kit for detection of Ins(1,4,5)P3 were purchased from Amersham International. Diltiazem was a generous gift of P.C. Sen, Department of Chemistry, Bose Institute, Calcutta. 45Ca2+ (56 mCi g-~) was purchased from BARC, Bombay, India. Cell culture. E. histolytica (EC22) trophozoites were obtained from Kothari Medical Centre for Gastroenterology, Calcutta, where they were isolated from patients and axenised. Trophozoites were maintained in complete TYI-S-33 medium. The TYI-S-33 medium (Trypticase, Yeast extract, Iron, Serum) consisted of a nutrient broth (TYI), a vitamin/Tween 80 mixture and bovine serum. Late log phase cultures (72 h) were harvested. Preparation of cells. E. histolytica trophozoites were harvested from culture by centrifugation (300 × g, l0 rain) at room temperature and finally resuspended in an 'intracellular' type of buffer (100 mM KC1/ 2 mM MgCl2/ 10 mM NaCl/ 10 mM piperazine N,N'-bis 2-ethanesulphonic acid (Pipes)/ 10 mM glucose, pH 7.2) for permeabilisation. Cell suspensions were kept in capped tubes to
L. Biswas et al./Molecular and Biochemical Parasitology 65 (1994) 63-71
avoid direct exposure to air. Aliquots (1 ml) of the cell suspension (106 ml-1) were permeabilised by the addition of 20 #g 1-a saponin for 1 min (32°C) directly in the spectrofluorimeter cuvette immediately before the addition of test substances. Permeabilised cells were tested for trypan blue inclusion and found to be 95% positive for trypan blue.
S_pectrofluorimetric measurements. The intracellular Ca 2+ mobilisation in saponin-permeabilised amoebae was monitored by measuring changes in quin 2 fluorescence in a Hitachi F-3000 Spectrofluorimeter. Excitation and emission wavelengths were set at 339 nm and 492 nm respectively. The ambient free Ca 2+ concentration [Ca2+] was calculated using the equation: [Ca 2+ ] = Kd(F- Fmin)/(Fmax- F), where Kd = 115 nm and F is the fluorescence in arbitrary units, Fmax was calculated in the presence of the cell suspension, 2 mM CaCI2 and 0.1% Triton X-100 and Fmi n w a s measured with 5 mM ethylene glycol bistetraacetic acid (EGTA). [Ca 2+] measured in this system was the amount of free Ca 2+ in the permeabilised preparation. Quin 2 was used both as a fluorescent calcium indicator and a buffer for free calcium [14]. Changes in total calcium (buffered plus free) was determined by the addition of known amounts of CaCI2 [14]. Autofluorescence of A23187 was measured in the absence of quin 2 under exactly similar conditions used for quin 2 measurements. Autofluorescence of calcium ionophore A23187 was subtracted from all measurements made with this compound. After 1 min incubation in the presence of 100 #M quin 2 and (20 #g m l - l ) saponin, 2 mM ATP was added to initiate calcium sequestration by permeabilised cells. Fluorescence was monitored for 3 min when a steady-state level of calcium uptake was achieved. Subsequently, InsP3 and all other substances, tested for their calcium releasing properties, were added. For inhibition studies, heparin (100 /~g ml -l) and p-chloromercurobenzoic acid (pCMB) (1 mM) were added to the cell suspension simultaneously with quin 2 and saponin. The
65
ATPase inhibitors or calcium antagonists were allowed to incubate with the permeabilised cells for 1 n'fin prior to the initiation of calcium sequestration by ATP. Peak [Ca 2+ ] was taken as the maximum [Ca2÷ ] reached within the first 25 s after addition of the test substance. Basal [Ca2÷] was that measured just before the addition of the test substance. When two additions were made, basal values for the second addition were taken as [Ca 2+] before the first addition. Data from Ins(1,4,5)P3 and Ins(2,4,5)P3 dose-response experiments were fitted to the Hill equation with the equation:
1 + (g/[s])h
where Vm is the Ca 2+ release response at maximal [InsP3], v is the Ca 2+ release given by different InsP3 concentrations [S], K is the ECso (concentration which produced half maximal release) value for InsP3, and h is the Hill coefficient [14].
uptake and release in permeabilised E. histolytica trophozoites. E. histolytica trophozoites
45Ca 2+
(107m1-1) were suspended in loading buffer (100 mM KCI/ 20 mM N a C I / 2 mM MgCI2/ 80 mM Sucrose/ 1 mM EGTA/ 390 /zM CaCIz/ 20 mM Pipes, pH 7.0) [15] and permeabilised by the addition of 20 #g ml-~ saponin for 2 min at 37°C. 45Ca2+ (10/.tCi m1-1) and 2 mM ATP were subsequently added. The cells were kept at 37°C for 5 min for the 45Ca2÷ content to reach a steady state and then diluted 10-fold with loading medium in the presence or absence of 2 #M Ins(l,4,5)P3. Termination of incubation after 60 s was obtained by addition of ice-cold termination buffer (310 mM sucrose/ 1 mM EGTA/ 1 mM Hepes, pH 7.0 and subsequent filtration through GF/C filters. The filters were solubilised in scintillation cocktail and counted in a Beckman scintillation counter.
Binding of [SH]Ins(1,4,5)P3 to the crude membrane fraction from E. histolytica. Crude membrane fraction was obtained by homogenising the cells in a buffer containing 0.1 M Tris-HC1/ 1 mM EDTA, pH 9.0 with a glass/Teflon homogeniser
66
S. Raha et al./Molecular and Biochemical Parasitology 65 (1994) 63-71
for 10 min followed by centrifugation of the homogenate at 1000 x g for 15 min. The supernatant was centrifuged at 35 000 x g for 15 rain. Binding assay with E. histolytica crude membrane fraction was performed at 4°C for 10 min in the presence of 10 nM [3H]Ins(1,4,5)P3. Bound Ins(l,4,5)P3 was separated from the free by centrifugation. Non-specific binding was determined in the presence of 10/~M unlabelled Ins(1,4,5)P3 and this value was subtracted from the total binding to obtain specific binding. Displacement of binding was observed in the presence of 0.04/~M to 4 #M unlabelled Ins(1,4,5)P3 or Ins(2,4,5)P3. The concentration used for displacement of binding by other inositol phosphates was 5-80 #M. For inhibition experiments, heparin concentrations in the range of 1-20/~g ml-1 were used in the binding assay.
Ins(1,4,5)P3 was detected in protein-free extracts of E. histolytica ceils by a competition binding assay provided in the Amersham detection kit for Ins(1,4,5)P3.
3. Results
InsP3-induced calcium release in permeabilised E. histolytica. The low-calcium, high-potassium buffer used for preparing the permeabilised cell suspension was buffered by 100 #M quin 2 free acid. Initial free calcium concentrations in the permeabilised cell preparations varied between 150 and 200 nM. In this range, changes observed with added calcium were linear. It is apparent from Fig. 1A that Ins(1,4,5)P3 was effective in calcium mobilisation in permeabi-
A.
B.
200
300
150 r
c
+
%
+
~ 100
~50
L.
50 ~
A23187
|
1
1
0.2)JM InsP3
2-O~M Ins P3 3
3
I
/, 5 (rain)
I
|
I
G
7
3
8
7
~SP3 I
~ (rain)
I
I
5
6
Fig. 1. ATP-induced calcium sequestration and subsequent release by Ins(1,4,5)P3 and A23187 from saponin-permeabilised E. histolytica trophozoites. Cells were permeabilised for 1 min with 20/~g m l - i saponin. Calcium sequestration was initiated by the addition of 2 mM ATP. 3 min after the addition of ATP, (A) 2 #M Ins(l,4,5)P3 was added which was followed 2 min later by 5 #g m1-1 A23187. In (B) after calcium sequestration with ATP, 0.2/~M Ins(1,4,5)P3 was added first, followed 2 min later by 2 #M Ins(l,4,5)P3 with a final addition of 5/~g ml - I A23187 another 2 min later. The calibration bar in the figure represents 1 nmol ml - l Ca 2+ . Results are representative of 3 ~ individual experiments. Free and total calcium were estimated from fluorescence of the calcium indicator dye quin 2 as described in Materials and methods. Autofluorescence of calcium ionophore A23187 was subtracted from the peak fluorescence value given by this compound. The traces showing A23187 induced calcium release are drawn with the dotted lines to indicate corrections. Solid lines are used for original traces.
67
L. Biswas et al./Molecular and Biochemical Parasitology 65 (1994) 63-71
lised amoebae. Calcium ionophore A23187 induced a calcium release higher than that achieved by Ins(1,4,5)P3 and A23187 could release calcium after Ins(1,4,5)P3 treatment (Fig. 1A,B). In contrast, prior A23187 treatment resulted in abolition of InsP3 induced calcium release (data not shown). Also, prior treatment with a lower concentration (0.2/~M) of InsP3 reduced release by 2 #M InsP3 (Fig. 1B). Ins(1,4,5)P3 released intracellular calcium from permeabilised E. histolytica trophozoites in a dosedependent manner (Fig. 2). Similar patterns of dose-dependence was also observed with Ins(2,4,5)P3 (data not shown). The ECs0 values estimated for Ins(1,4,5)P3 and Ins(2,4,5)P3 were 0.174+0.120 /~M and 0.288+0.119 #M respectively ( P > 0.05). 45Ca2+ uptake and release studies confirmed the results obtained by fluorimetric measurements. 2.46_+0.50 nmol 4 5 C a 2 + w e r e loaded into the intracellular pools in an energy-dependent manner by 106 cells. About 50% of this labelled pool was released by 2 #M Ins(1,4,5)P3 (1.22_+0.31 nmol 10 - 6 cells). Calcium ionophore A23187 (5 #g m l - ] ) released 70.46% of the accumulated calcium label (1.73___0.70 nmoll0 -6
Comparison of various inositol phosphates and G TP as stimulators of calcium release in permeabilised E. histolytica. A saturating concentration of Ins(1,4,5)P3 (2 #M) produced a slightly higher but statistically insignificant (P> 0.05) calcium increase than that mediated by equal concentration of Ins(2,4,5)P3 (Table 1). Significant reduction ( P < 0.05) was observed in Ins(1,4,5)P3-induced calcium release by pretreatment with a lower concentration of Ins(l,4,5)P3. A23187 mobilised a significantly higher ( P < 0.05) amount of calcium than 2 #M Ins(1,4,5)P3. About 63% of the A23187 releasable calcium pool could be mobilised by 2 #M Ins(1,4,5)P3 as measured by quin 2 fluorescence. Other inositol phosphates failed to produce any change (P> 0.05) in Ca 2+. Heparin (100 #g ml - l ) significantly (P< 0.05) inhibited Ins(1,4,5)P3-mediated calcium release (Table 1). Significant potentiation of InsP3-mediated calcium release was not observed by simultaneous application of GTP, which is known to regulate calcium movements from InsP3-insensitive to InsP3-sensitive stores in mammalian cells [16,17]. Moreover, GTP (10 #M and 100 #M) or the nonTable 1 Calcium release by various inositol phosphates and calcium ionophore A23187 in permeabilised E. histolytica trophozoites
I.L 1-2
cells). 2 #M Ins(1,4,5)P3 could release about 70% of the ionophore-releasable calcium.
i
Inducer
Concentration
0.8
~
!o~
Calcium release (Peak/Basal)
~
i
~
ILO
[Ins (I,~.,5) P3~(~ M ) Fig. 2. Dose-dependent Ca 2+ release by Ins(1,4,5)P3 from permeabilised E. histolytica trophozoites. Experimental conditions are similar to those described for Fig. 1 and in Materials and methods. Free and total calcium were estimated from fluorescence m e a s u r e m e n t s o f the calcium indicator dye quin 2 as described in Materials a n d methods. Results are expressed as the m e a n of 2-6 individual experiments.
Ins(1,4,5)P3 2 gM Ins(1,4,5)P3 (added 2 rain 2 #M after 0 . 2 / t M Ins(1,4,5)P3) Ins(1,4,5)P3 (plus 100 #g ml -l 2 g M heparin) Ins(2,4,5)P3 2 #M Ins(l)monophosphate 10 # M Inositol hexakis phosphate 10 # M Calcium ionophore A23187 5 #g ml - [
2.73 + 0.36 1.86 + 0.54 1.66 + 0.36 2.39 1.10 1.00 4.31
___ 0.29 _ 0.10 _ 0.05 ___ 1.78
Experimental conditions are similar to those described in Fig. 1 and Materials and methods. Peak [Ca 2+] was taken as the m a x i m u m Ca 2+ level reached within the first 25 s after the addition of the test substance. Basal [Ca 2+ ] was that measured just before the addition of the first test substance. Data are presented as mean + S.D. o f 3-6 experiments.
68
S. Raha et aL/Molecular and Biochemical Parasitology 65 (1994) 63-71
hydrolysable GTP analogue, GTP 7S (10 /~M) alone, was unable to release any calcium from permeabilised amoebae (data not shown). Influence of calcium transport inhibitors on calcium sequestration and subsequent calcium release by Ins(1,4,5)P3. Presence of 500/tM vanadate during ATP-dependent calcium sequestration produced a very strong inhibition ( P < 0.01) of calcium uptake and subsequent relaese by Ins(1,4,5)P3 (Table 2). Neither cAMP which is known to have influence on the mammalian InsP3 receptor [18,19], nor Dicyclohexylcarbodiimide (DCCD - an inhibitor of the vacuolar proton translocating ATPase) had any effect on either of these processes. However, Diltiazem, a calcium antagonist, significantly ( P < 0.01) inhibited both calcium sequestration and subsequent release. In the absence of ATP-dependent calcium sequestartion, subsequent calcium release by Ins(1,4,5)P3 was inhibited very strongly (91.25% inhibition). pCMB, a sulphhydryl disrupting agent [14], also significantly ( P < 0.01) inhibited calcium uptake and subsequent Ins(1,4,5)Pa-mediated calcium release.
Table 2 Influence of calcium transport inhibitors on calcium sequestration and subsequent release by Ins(l,4,5)P3 in permeabilised E.
histolytica Compound
Ca 2+ sequestration (% inhibition)
Sodium orthovanadate (500 # M ) c A M P (10/tM) D C C D (20/~M) Diltiazem (10 #M)
76.85 + 22.39
para-Chloromercuroben-
9.44 6.47 71.08 98.02
+_ _ _+ _
6.45 11.21 12.42 10.10
Ins(1,4,5)P3 induced release (% inhibition) 67.86 _ 8.77 4.67 +_ 6.43 7.18 _ 3.20 93.50 _ 7.78 100
zoic acid (pCMB) (1 m M ) Experimental conditions are similar to those described in Fig. 1 and Materials a n d methods. Inhibitors were added to the cell suspension 1 min before the initiation of ATP-dependent calcium sequestration. Values obtained in the absence o f inhibitors are taken as 100%. D a t a are shown as m e a n s _ S.D. of 3 experiments.
100
75 \
~
x
\,,.
~ 50 un
-
I
!
I
0
1
2
log Ins P3(xlM) Fig. 3. Inhibition of specific[3H]Ins(1,4,5)P3binding by unlabelled Ins(1,4,5)P3 and Ins(2,4,5)Pa in the crude membrane fraction from E. histolytica trophozoites. 0, Ins(l,4,5)P3; A, Ins(2,4,5)P3. Binding assays contained 10 nM [3H]Ins(1,4,5)P3,0.1 mg of membraneprotein and indicated amounts of unlabelledIns(1,4,5)P3 and Ins(2,4,5)P3. Data are representativeof 2-3 experiments. Ins(1,4,5)P3 binding to crude membrane preparation from E. histolytica. Specific binding of [3H]Ins(1,4,5)P3 was observed in E. histolytica crude membrane fraction. [3H]Ins(1,4,5)P3 binding was increasingly displaced by the presence of 0.04 pM to 4 /zM unlabelled Ins(1,4,5)P3 and Ins(2,4,5)P3 (Fig. 3). Unspecific binding was on the average 25% of specific binding. Scatchard transformation of the isotope dilution of [3H]Ins(1,4,5)P3 binding data revealed a straight line indicative of a single binding site (data not shown). IC50 (concentration which produced halfmaximal inhibition) values were estimated from the following equation [20] which describes displacement of a trace amount of bound [3H]Ins(1,4,5)P3 by addition of unlabelled InsP3 assuming a single binding site:
L. Biswas et al./Molecular and Biochemical Parasitology 65 (1994) 63-71 Table 3 Effect of displacing agents and antagonists on the specific [3H]Ins(1,4,5)P3 binding in E. histolytica crude membrane fractions Compound
Estimated ICso
Ins(l,4,5)P3 Ins(2,4,5)P3 Ins(4,5)bisphosphate Ins(l)monophosphate Ins(2)monophosphate Inositol hexakisphosphate Heparin
0.99 0.99 12.58 17.23 15.26 19.42 0.77
_ _ _ _ + + _
0.13 0.29 4.63 4.36 2.46 2.00 0.18
Crude membrane fractions were isolated from E. histolytica trophozoites as described in the Methods section. Assay contained 100 #g of crude membrane protein, 10 nM [3H]Ins(1,4,5)P3 and various concentrations of displacing agents or the antagonist, heparin. IC5o was estimated as described in the text. IC5o values are given as/~M for inositol phosphates and as/~g ml-1 for heparin.
[InsP3]
1 + 1C5o
where B* is the amount of labelled InsP3 bound, Bo* is the amount of labelled InsP3 in the absence of unlabelled InsP3, [InsP3] is the concentration of unlabelled InsP3. The IC5o values (Table 3) for displacement of binding of [3H]InsP3 by unlabelled Ins(1,4,5)P3 and Ins(2,4,5)P3 were 0.99 #M for both isomers. On the other hand, IC5o values for other inositol phosphates were markedly larger (13-19 #M). Heparin strongly inhibited [3H]Ins(1,4,5)P3 binding in crude membrane fractions of E. histolytica in a dose-dependent manner (IC5o = 0.77 #g m l - 1). Basal intracellular Ins(1,4,5)P3 concentration was estimated to be 55.0-t-32.79 pmol (10 -6 cells) (n = 3).
4. Discussion
Adhesion and subsequent secretion of proteolytic enzymes leading to cytolysis of target cells are two major steps for a successful invasion of the host by E. histolytica. Ravdin et al. [21] have de-
69
monstrated that the adhesion of this parasite to target cells does not increase total internal calcium levels but results in regional changes in free calcium concentration. In another report, the same group of investigators [2] have emphasised the role of internal calcium, calmodulin and the cytoskeleton in the secretion of parasitic granule contents. Ravdin et al. [22] also suggested that either the entry of extracellular calcium or the release of internal calcium mediated the secretory process. We have observed the importance of calcium homeostasis in E. histolytica, as A23187 treatment of intact amoebae or Ins(1,4,5)P3 treatment of permeabilised amoebae resulted in increased motility and pseudopod formation [23]. The present study documented in E. histolytica, the release of internal calcium by the calcium mobilising second messenger Ins(1,4,5)P3 and specific binding of [3H]Ins(1,4,5)P3 to membranes of E. histolytica. The well-known InsP3 receptor antagonist heparin [24] significantly inhibited both of these processes. The ECs0 of Ins(1,4,5)P3-induced calcium release (0.1-0.75 #M) reported for permeabilised mammalian cells [6,11] seems compatible with our finding in E. histolytica (EC50-0.17 #M), indicating similar sensitivity to Ins(1,4,5)P3. In contrast, however, was the almost similar effectiveness of Ins(2,4,5)P3, which is known to be less potent in releasing calcium in mammalian cells [25,26]. Furthermore, the IC50 values demonstrated by these two isomers for displacement of labelled Ins(1,4,5)P3 binding are also similar (Table 3). In this context, the findings of two other studies [20,27] revealing equal responsiveness of the InsP3 receptor in Neurospora crassa [27], and catfish olfactory cilia [20] to Ins(1,4,5)P3 analogues could be relevant. The affinity of Ins(1,4,5)P3 for the E. histolytica binding site is 30-125 times lower than the values reported for the receptor in mammalian tissues [25,28,29]. Interestingly, the IC50 value for Ins(1,4,5)P3 in E. histolytica (0.99/zM) is very similar to that reported for catfish olfactory cilia (1.1 #M) which possesses a binding site of different molecular mass from the cerebellar Ins(1,4,5)P3 receptor [20]. Our 45Ca2+ release studies demonstrated that
70
s. Raha et al./Molecular and Biochemical Parasitology 65 (1994) 63-71
about 50% of the intracellular 45Ca2+ label was released by 2 #M Ins(1,4,5)P3. This could compare favourably with the value of 30-50% reported for m a m m a l i a n cells [8]. This finding underscores the importance of Ins(1,4,5)P3 releasable calcium pool in this parasite. Calcium ionophore A23187 failed to release all of the sequestered 45Ca2 + from E. histolytica. Similar observations about the existence of a calcium pool unresponsive to ionomycin alone have been made in PC 12 cells [30]. The presence of vacuolar structures in E. histolytica is known from microscopic studies [31]. Absence o f inhibition on calcium sequestration and subsequent InsP3-induced release by D C C D , an inhibitor of vacuolar proton translocating ATPases [32-34], demonstrated that this type of ATPase m a y not be involved in calcium uptake into InsP3-sensitive pools in E. histolytica (Table 2). Vanadate is an inhibitor of both plasma membrane and microsomal Ca 2÷ p u m p ATPase [35,36]. Our observation o f a high degree of inhibition by vanadate of the ATP-dependent Ca 2+ uptake and subsequent InsPa-induced calcium release (Table 2) strongly suggested that the InsP3-releasable pool is located in an endoplasmic reticulumlike structure. Ultrastructural studies [31] have revealed the presence of smooth endoplasmic reticulum and other endoplasmic reticulum-like membranous structures in E. histolytica. Diltiazem, an inhibitor of calcium channels in excitable tissue [37], has very recently been described [38] to block calcium entry into internal stores and also to inhibit stimulus-induced calcium release from intracellular sites in neutrophils. Inhibition of calcium uptake and subsequent release in E. histolytica by Diltiazem indicate that a therapeutically used calcium antagonist m a y be able to disrupt calcium homeostasis in this parasite. Our findings in E. histolytica were not in agreement with recent reports [12,13] on other parasitic protozoa Trypanosoma brucei and Trypanosoma cruzi. In these organisms, in spite of the presence of intracellular inositol phosphates, especially Ins(1,4,5)P3, no calcium release mediated by InsP3 was detected. We demonstrated (1,4,5)P3-mediated calcium
release and [3H]Ins(1,4,5)P3 binding in E. histolytica, most probably through an InsP3 receptor mediated mechanism. Further characterisation of the cellular signalling mechanisms in this enteric parasite m a y have implications for the development of antiparasitic therapy aimed at these vital pathways.
Acknowledgements We would like to thank the Kothari Medical Centre, Calcutta for generously providing us with the E. histolytica strain. S.R. was supported in part by the Council of Scientific and Industrial Research, India as a scientific pool officer.
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