Effect of the ionophores A23187 and X-537A (Lasalocid) and of the divalent cations Ca2+, Mg2+, Ba2+ and Mn2+ on transformation in Leishmania donovani

Camp.Btochem.Physiol.Vol.69A, pp. 65 to 72. 1981 Printed in Great Brttain.All rights reserved

0~9629/81/690065~g~2.~/0 Copyright 0 1981 Pergamon Press Ltd

A23187 AND X-537A OF THE IONOPHORES ~LASALOCID) AND OF THE DIVALENT CATIONS Ca2+, Mg2 +, Ba2+ AND Mn’ f ON TRANSFORMATION IN LElSHMANIA DONOT/ANI*

EFFECT

D. MORROW’, BARBARAFLORY-GRANGER’ and STUART M. KRASSNER~ ‘Microbiology and Immunolo~, School of Medicine, University of California, Los Angeles, CA 90024 and ~Developmental and Cell Biology, University of California, Irvine CA 92717 U.S.A.

CASEY

(Received 2 September 1980) Abstract-l.

Concentrations of the carboxylic ionophores A23187 and X-537A (Lasalocid) 2 lo-’ M inhibited amastigote to promastigote transformation of Leishmnnia donouani when Ca2+ and Mg” were deleted from the culture medium. 2. Normally, an exogenous source of these ions is not required for transformation. 3. Ionophore concentrations greater than 5 x 10e6 M were lethal. 4. Inhibition of transformation by A23187 (10e6 M) was reduced by either Ca*+ (10e3 M), Ba*+ (10e3 M), Mn’+ (lo-’ M) or ADP (lo-’ M) but not by Mg*+. 5. Ion concentrations greater than 10-s M were lethal. 6. Inhibition of transformation by X-537A was not reduced by addition of Ca’+. Mg*+, Mn*+ or ADP to the culture medium. 7. Cells lost their sensitivity to A23187 within 6-hr tr~sformation. 8. These results indicate that intracellular Ca’+, Ba*+, and possibly MnZf may be required for at least the first few hours of L. donooani amastigote to promastigote transformation.

MATERIALS AND

INTRODUCTION

Ionophores are lipid soluble compounds that transport polar cations (e.g. K+, Na+, Ca2+ and Mg’+) across biological membranes (Reed & Lardy, 1972a). Originally used as antibiotics, ionophores have more recently served as valuable tools for illustrating membrane models, investigating mitochondrial respiration and for perturbing biological systems (Pressman, 1973). Two intensively studied ionophores are A23187 and X-537A (Lasalocid); these carboxylic acids promote exchange diffusion of divalent cations. Divalent cations are probably of great importance in protistan culture and cytodifferentiation (Hutner, 1972) and divalent cation ionophores would be of value in studying the roles played by these ions in the above processes. Transformation in hemoflagellates is a fundamental cytodifferentiation which involves changes in their biochemistry, physiology, antigenicity, morphology and infectivity (Bowman & Flynn, 1976; Simpson, 1968; Vickerman & Preston, 1976). Since cations probably play key roles in transformation, we decided to study the effect of A23187 and X-537A on the transformation of Leish~n~ff donova~~ amastigotes to promastigotes, a relatively well-characterized cytodifferentiation process (Brun et a[., 1976; Brun & Krassner, 1976; Dwyer et al., 1974; Rudzinska et al., 1964; Simpson, 1968; Morrow et al., 1980; Krassner et al., 1980). * This investigation was supported by a U.C. Irvine President’s Undergraduate Fellowship (CDM) and by Research Grant AI 14824 from the Public Health Service (SMK). 65

METHODS

Parasites

Leishmanin donovani, Malakal area Sudan strain (is), obtained originally from the late Professor L. A. Stauber, Rutgers University, were passaged in &S-week old 70-100 g male golden hamsters (Mesocricetus auratus) (Simonson Laboratories, Inc., Gilroy, CA) by intrasplenic injection of l-5 x 10’ amastigotes liberated from infected hamster spleens or livers. Infected hamsters were ready for harvesting of parasites in 6-10wk. Animals were killed with ether, the spleens excised using sterile procedures and isolation steps done in the cold to retard spontaneous transformation. Spleens were homogenized in lOm1 cold Tris (0.2 M)-sucrose (0.25 M) buffer, pH 7.8 in a Ten Broeck glass homogenizer to liberate amastigotes from host cells (lO@-300mg spleen/ mi buffer). Homogenates were centrifuged at t50rr for 10 mm, the pellet discarded and the re&tant supernatant fluid centrifuged at 1000~ for 20 min. The nellet famastigotes) was resuspended in cold Tris-sucrose buffer and the washing procedure repeated twice. Final concentrations of amastigotes were determined by counting the cells in a Petroff-Hauser counting chamber. Amastigotes (5-7 x 10’) were permitted to transform at 2627°C in 0.5 ml Mg’+-and Ca’+-free HO-MEM medium (Berens et al., 1976) without fetal calf serum (fcs) in 16 x 125 mm screw-capped plastic test tubes. Exogenous fcs, Mg’+ and Car+ are not required for L. donovani amastigote to promastigote transformation; their presence in the culture medium led to equivocal results in ionophore experiments and they were deleted except where noted. Neutral red was added to all media to allow monitoring of pH. Osmolarity was periodically measured to ensure that any inhibition of transformation found was not the result of a change in tonicity (all cultures were _ 300 mOsm).

CASEY D. MORROWet nl.

66

Several morphological intermediate stages were noted during amastigote to promastigote transformation (Brun et al., 1976). These are shown in Fig. 1. The oval-shaped amastigote enlarges about ten-fold after about 8 hr (1st intermediate stage) (Fig. lb). Within 8 hr after parasite enlargement, the cell elongates into the second intermediate stage (Fig. lc). Flagella formation and further elongation occurs during the subsequent 2-4 hr. This is the promastigote stage (Fig. Id). Therefore transformation of a population of L. ~o~o~a~~amastigotes normally results in the first appearance of flagellates after _ 18-20 hr incubation in culture medium (Brun et al., 1976; Brun & Krassner, 1976). All cells capable of developmental transition to promastigotes ( 5 60--80:1; of total amastigote population) complete cytodifferentiation within approximately 48 hr (Brun et al., 1976; Brun & Krassner, 1976). We use the terms inhibition or “blocking activity” when a test material delayed transformation; time of blocking refers to the difference in time between control and test cells when flagellate cells (promastigotes) are first seen. Therefore when we state that ionophore “blocked” or delayed transformation for 12 hr, promastigotes in the test population were first observed 12 hr after they had appeared in control populations (w/o ionophore) and 100% of the cells was inhibited for that 12-hr period. In the current study we used a 4%hr test period; amastigote populations that did not give rise to promastigotes within the test period were considered not transformed even though a few promastigotes were sometimes found after 48 hr. We also determined the percent of the most advanced stage observed at each test time as a further measure of ionophore and cation activity. Cultures were tested at various time intervals by removing samples with a sterile bacteriological loop and observing the parasite stages in 50 fields at 400 x using phase contrast optics. All inhibition and other experiments were done in duplicate a minimum of three times. Reagents

A23187 (fermentation product of Streptomyces clrartreusis NRRL 3882) was received as a gift from Dr R. L. Hamill, Lilly Research Laboratories, In~~apolis, Indiana. X-537A (Lasalocid sodium, Ro2-2985) was a gift from Dr W. E. Scott, Hoffmann-La Roche, Nutley, New Jersey. The ionophores were dissolved in 959; ethanol to a final concentration of IO-’ M; subsequent dilutions were made with distilled water. Control solutions containing the volume of ethanol used for tests had no effect upon the parasites. CaCI, (anhydrous), MgCI,.6Hz0, MnCI,‘4H,O and Ba(OH), were AR grade (Mallinkrodt, St. Louis, Missouri). ADP (sodium dihydrate) and ATP were obtained from Calbiochem (San Diego, California). RESULTS Ionophore

inhibition

of tr~ns~orm~tio~~

The effects of A23187 and of X-537A (concentration range 1O-5--lO-7 M) on transformation of L. donov~?~~ were followed for 48 hr. Ionophore con~ntrations above 5 x 10e6 M were lethal for the cells. As stated in Materials and Methods the blocking activity of ionophores was more consistent and more obvious when fcs, Ca2+ and Mg’+ were deleted from the culture media. Summarized observations on the effects of 10-7-10-6 M ionophore on transformation are shown in Figs 2 and 3. In addition, tests with EDTA showed no effect by this chelating agent upon transformation. There was a dose-response by amastigotes to the ionophores. When 5 x 10e6 M A23187 was added, development of second stage intermediate forms was delayed more than 12 hr and promastigote appear-

ance was inhibited for almost 24 hr as compared with control cells. Inhibition was decidedly less when cells were incubated with 10s6 M A23187; the delay in promastigote appearance was < 10-12 hr as compared with control cells. Cells incubated with lo- ’ M A23187 gave rise to flagellates within 24 hr; this was a delay of less than 4 hr. Thus the time of blocking of transformation by A23187 was in proportion to ionophore concentration. In the case of X-537A. 5 x IO-” M ionophore delayed the appearance of second intermediate stage cells and of promastigotes by more than 24 hr when compared with control cells. A dose-response was also noted with this ionophore; inhibition of transformation time was lower when 10m6 M X-537A was added and even less with lo-’ M X-537A. Equimolar concentrations of X-537A seemed to result in less pronounced inhibition of transformation when compared with A23187. Since A23187 complexes most readily with Ca” (Haynes et nl., 1974) various concentrations of the cation were added to see if ionophore inhibition of transformation could be reduced. Reduction of ionophore inhibition was found with 10~3-10~4 M Ca” ; this was most dramatic at lo-’ M. Ca’+ ion concentrations > 1O-3 M were toxic for the amastigotes. A summary of our results with 10e3 M Ca” is shown in Figs 2 and 3. Addition of 10m3 M Ca2’ resulted in no reduction in the delay of transformation by 5 x 10-h M A23187. -4-hr reduction in the delay due to 10e6 M A23187 and complete reversal of inhibition by lo-’ M ionophore. Two divalent cations readily complexed by A23187 are Ba2+ and Mn”: these were tested for their ability to modulate ionophore inhibition of transformation. Both ions reversed ionophore inhibition of transformation (see Fig. 2). Reduction of the X-537A induced inhibition of transformation by 10e3 M Ca2+ or by 10m3 M Mn2’ was not striking (Fig. 3) although Ca’+ appeared to be better than Mn”‘. These results are not surprising because X-537A is not as specific a cation ionophore for Ca’+ and Mn2+ as is A23187; it may even complex with monovalent ions (Pressman, 1973). In some of the experiments Mg’+ was added to the culture media; Mg ” did not reduce the transformation blocking activity by A23187 or X-537A. Concentrations of Mg’ + > lo-” M were toxic. These results suggest that Ca’+, Ba2+ and possibly Mn2+ (presumably endogenous) were required for transformation of L. ~offo~~~~~j amastigotes to promastigotes. Time course effects of ionop~ore.~

It was not clear if the parasites were sensitive to perturbation of endogenous ion levels by ionophores during the entire transformation period. Previous studies in our laboratory had shown significant metabolic changes in L. donocani during the first 6 hr of transformation (Morrow et al., 1980, Krassner et al., 1980). Therefore a series of experiments were done in which ionophores were added at different times after initiation of transformation. The results of experiments using 5 x lOm6 M A23187 and X-537A are summarized in Table I. The parasites began to show decreased sensitivity

Ionophore inhibition of L.

donovani

transformation

67

Fig. 1. Phase contrast micrographs (400 x ) of the different stages of L. donocani amastigote to promastigote transformation. a. Amastigotes freshly isolated from infected hamster spleen (amastigote isolation procedures as in Materials and Methods). b. First intermediate stage; these cells show _ IO-fold increase in size (enlarged stage) and first appear z 8 hr after initiation of transformation. c. Second intermediate stage; these elongated cells (elongate stage) are found within 8 hr after development of the first intermediate stage. d. Promastigote stage; these flagellate cells are first seen after _ 18-20 hr incubation. All cells enlarged 4800 x ; arrows point to the stages described in legend.

to A23187 within a few hr of transformation. After 5-hr transformation the cells had lost much of their sensitivity to A23187. Similar results were found when 1V6 M A23187 was tested except that loss of sensitivity to the ionophore appeared earlier (within 2 hr) and was more complete (no delay in transformation to promastigotes when ionophore was added after 6 hr). An interesting result was noted in the experiments with 5 x 10m6 M X-537A. Amastigotes were prevented from transforming into promastigotes even when ionophore was added 8 hr after the start of transformation (Table I). However, ceils did differentiate more readily into the 2nd intermediate stage when X-537A was added after 3 hr of culture. Similar resuits were found when 10m6 M X-537A was tested except that flagellates were found after 4%hr culture even when ionophore was added at 0 time, Since Ca’+, Mn’+ and Mg2+ do not reduce the inhibitory activity of X-537A (see Fig. 3), the above results suggested that additional ions were required for L. donouani to develop from the 2nd intermediate stage to the promastigote stage.

Other workers have shown that A23187 uncouples oxidative phosphoryfation by severely depleting intramitochondrial Ca2+ (Reed 81 Lardy, 1972a). It is possible that one effect of this ionophore upon transforming L. danooani amastigotes is a drop in intracellular ATP. We therefore added 10e2 M ATP to cells cultured with A23187. Reduction of ionophore inhibition was noted only with the lowest concentration (lo-.’ M) of ionophore; cells given ATP in addition to A23187 started to transform into promastigotes after 21 hr whereas cells without ATP differentiated only as far as the 2nd intermediate stage at that time. Since most ceiIs are believed to be relatively impermeable with respect to ATP, ADP (lo-* M) was added to transforming cells exposed to ionophore because ADP is more permeable and can serve as an energy source. The results of these experiments are summarized in Pig. 4. ADP was able to reduce inhibition of transformation by A231 87 but not the blocking activity exerted by X-537A.

68

CASEY

r

D.

MORROW

et ai

Cantm I

60%

1

t F I 1% 30%

1

2”d &e f% 30% I” s&e 1

l&i 100% A ._

TRANSFORMATION

TIME

(-=

16-48

hours)

Fig. 2. Effect of ionophore A23187 and of 10S3 M Ca’+, Ba2+ and MI?’ on L. donowni amastigote to promastigote transformation. The range of percent most advanced stage found is shown plotted vs the transformation time. Maximum values of 300/, are depicted for 1st and 2nd intermediate stages because once the 300/, level is attained, a few cells of the next stage are always found. Transformation times shown are from 18 to 48 hr (- = 18-48 hr). Amastigote isotation procedures, culture methods and determination of stages observed as in Materials and Methods. All tests were done in duplicate a minimum of three times. A-amastigote stage; 1st stage-ist intermediate (enlarged) stage; 2nd stage-2nd intermediate (elongated) stage; f-promastigote (flagellate) stage. Inonophore concentration: l-5 x 1O-6 M; 2-10-6 M; 3-lo-’ M.

DISCUSSION

an extremely broad spectrum of ions: Cs+ > Rb+ % K+ > Na+ > Li; Ba’+ > Si*+ > Ca*+ > Mg*+ (Pressman, 1976). This ionophore induces contraction of aortic strips, increases the rate and contractility of isolated perfused rabbit heart and, like A23187, releases Ca2+ from energy loaded vesicles derived from muscle sarcoplasmic reticulum (Pressman, 1976). The monocarboxylic acid, A23187 (mol. wt 5231, is

Ionophores are compounds of moderate molecular weight ( + 200-2000) that form lipid soluble complexes with polar cations. Their transport turnover numbers across biological membranes attain values exceeding the turnover rates of most enzymes (Haynes et al., 1974). X-537A (Lasalocid) is one of the smallest carboxylic ionophores (mol. wt 590) and complexes with l-

Imtra

-

X-537A

600,

0

I

38

3

I

2

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122

33 1

‘I- 1

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TRANSFORMATION

TIME I-=

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Is’ stage

_

2

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2”6 stogf 1s 35”

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Co” + X-537A Mn*+

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18-24

hours)

Fig. 3. Effect of ionophore X-537A and 10S3 M Ca2+ and Mn’+ on L.donovani amastigote to promastigote transformation. The range of percent most advanced stage found is plotted vs the transformation time. Isolation procedures, culture conditions and determinations of stages as in Materials and Methods. All tests were done in duplicate a minimum of three times. Abbreviations as in Fig. 2.

Ionophore

inhibition

of L. donovani transformation

69

a unique ionophore because it is predominantly selective for divalent over monovalent ions: Mn’+ % Ca2’ > Mgzc > Sr*+ > Ba2+ (Pressman, 1976). It has the highest affinity for Cazi and somewhat less for Mgzc at neutral pH (Reed & Lardy, 1972a). This ionophore apparently acts as a freely mobile carrier across biological membranes to catalyze equilibrium of divalent cations between external medium and organelle or cell interior (Reed & Lardy, 1972b). In the absence of exogenous divalent cations, it releases endogenous Ca*+ and Mg2+ from erythrocytes and sperm (Reed & Lardy, 1972b). A23187 is capable of stimulating various Ca’+dependent biological reactions without disturbing pre-existing balances of Na+ and K+ (Pressman, 1973). AIthough this ionophore transfers Iv@+, selgradients of Mg2+ across biological membrane dom participate in biological control (Pressman, 1976). A23187 stimulates mitochondrial respiration, induces morphologic blast formation, induces DNA synthesis and mitosis in human lymphocytes when Ca’+ is present in external medium (Luckasen et al., 1974), activates DNA synthesis in sea urchin egg (Steinhart & Epel, 1974) and activates secretion in platelets (Feinman & Detwiler, 1974), and mast cells (Cochrane & Douglas, 1974; Foreman et al., 1973). When rat mitochondria are incubated with A23187 and EDTA, endogenous Ca*+ and Mgzf is discharged, producing simultaneous uncoupling of oxidative phosphoryIation and inhibition of ATPase (Reed & Lardy, 1972b). In the absence of EDTA, rat liver mitochondrial Ca*+ remains constant because mitochondria actively take up Ca’ ’ (Lehninger et al., 1967; Vanio et al., 1970). Reed & Lardy (1972b) have proposed that A23187-induced uncoupling of oxidative phosphorylation in the presence of EDTA is due to a cyclic energy-dissipating flux of mitochondrial Ca2+. In our study, addition of EDTA had no effect on transformation. Intracellular Ca*+ is thought to play a number of roles in addition to influencing mitochondrial activity. Many enzymes are activated or inhibited by Ca2+ (Rasmussen, 1975). An interesting proposal is the idea that ions such as CaZf may act as “secondary messengers” in much the way cyclic nucleotides, such as cyclic adenosine monophosphate (CAMP), behave in a number of eukaryotic systems (Rasmussen, 1975). In many processes where CAMP is implicated as the secondary messenger, Ca2+ is so intimately involved that the messenger role is really filled by a CAMP-Ca’+ system (Rasmussan, 1975). McMahon (1974) has suggested that CAMP changes the intracellular concentration of cations by liberating them from a sequestered pool; these cations in turn may then activate or inhibit enzymes such as adenylate cyclase, CAMP phosphodiesterase, and CAMP-dependent protein kinase (McMahon, 1974). Barth & Barth (1972) have found that Ca*’ and Li+ channel the differentiation of ectodermal cells into neurons and pigment cells by lowering the cellular concentration of CAMP. In the blowfly CAMP regulates Ca2+ release from mitochondria and the released Ca2+ in turn regulates CAMP production by inactivating adenylate cyclase (Rasmussen 1975). Phytohemagglutin added to peripheral lymphocytes leads to a Ca*+-dependent rise in

70

CASEY D. MORROW

Control +ADP VW

A23187

et ~1.

A23187 + ADP

X-537A

X-537A +ADP

60%

1F

0

II

I 1% 30%

1 Q 0

__

_,

, 2 1:

00

2 3 11111 2 33

~ 3?

*$ 3

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, -,-,-I

-I-,_

(18--48) (18+48) TRANSFORMATION TIME

-m,+,---w,-b,_, (18-+38) (hours)

(18-38)

Fig. 4. Effect of ADP (lo-’ M) on transformation-inhibiting activity against L. donoooni by phores A23187 and X-537A. The range of percent most advanced stage found is plotted vs formation time (in these experiments some of the samples were followed for only 38 hr). All done in duplicate a minimum of three times. Procedures used and abbreviations as in Figs

cGMP concentration early in the lymphocyte activation response (Rasmussen, 1975). The current report (see discussion below) suggests that Ca’+ may play a similar role in the differentiation of L. donouani amastigotes to promastigotes. Of interest here are unpublished results in our laboratory indicating that cGMP (10e3 M) (but not CAMP) inhibits amastigote to promastigote transformation. Stickler & Patton (1975) have found that intracellular pools of cyclic nucleotides in another hemoflagellate, Trypanosoma lewisi show significant changes during ablastin-induced transformation. In another protistan system, aggregation of the slime mold, Dictyostelium discoides, depends upon both production of CAMP by the amoebas and low concentrations (10-6-10-4 M) of intracellular Ca’+ (Mason, 1971). The amoebas cannot respond to CAMP in the absence of Ca”. Although the current and earlier reports show that changes in cyclic nucleotides and Ca2+ are important in hemoflagellate transformation, it is not as yet clear whether the roles played by these two factors are related. Relatively few studies have been done on the role of Ca2+ in protozoans and even less has been done on Ba2+ and Mn2+. As pointed out by Hutner (1972) a clear-cut requirement for Ca2+ has been rarely demonstrated in microbes; it is a ubiquitous contaminant and abundantly released from glassware. This was a major reason for the use of plastic glassware in our study. Ca ‘+ is weakly chelated so that it is readily displaced from complexes by other metals and thus is readily available even in highly chelated media (Berens et ul., 1976). Growth of Crithidia ,fascicu/ata at pH 4.0 was secured by increasing Mg2+ and Ca” as well as some Kreb cycle and amino acids (Tamburro et (I/., 1971). Trager (1955) found that Ca*+ was essential for in vitro culture of Plasmodium lophurae; Bishop &

the ionothe transtests were 2 and 3.

McConnachie (1960), however, suggested that Ca2 + was not essential for culture of P. gallinaceum. Much of the current work on the role of Cazf in protozoa deals with the response of cilia and flagella to this ion. Contractility and ciliary reversal may be brought about by changes in intracellualar Ca2+ (Eckert & Naitoh, 1972). Ca2+ seems to be required for ATPase activity of isolated Euglena gracilis flagella (Pacini & Albergoni, 1973). Protozoan cilia and flagella are activated by divalent cations such as Mg2+ and Ca2+ (Pacini & Albergoni, 1973). It is interesting to note that spermatozoan Ca2+ uptake induced by A23187 depresses motility (Luckasen et al., 1974). Finally, Cronkite (1976) has reported a role for Ca2+ in the chemical induction of mating in Purumecium tetruureliu.

The current report underlines Hutner’s statements (1972) concerning the difficulty in demonstrating clear-cut requirements for Ca2 +. A23187 had to be added to our cells in order to show that Ca2+ was involved in transformation. We assume that the required Ca2 + comes from internal stores as in other systems (Rasmussen, 1975). Our results might have been even more dramatic if EGTA, a specific chelator of Ca2+ that does not significantly affect Mg2+ ion concentration (Balk, 1971), had been added to our preparations. Ba2+ is a divalent cation closely related to Ca2’ ; once inside cells it may mimic the action of Ca2+ on secretory processes although its effects on membrane processes differ in some ways from those of Ca2+ (Rubin, 1974). Mg2+ and Mn2’ are inhibitors of Ca*+ movement across cell membranes and Mn2+ acts as an antagonist to Ca2+ in many biological processes (Rubin, 1974). The time course results are important in light of other studies in our laboratory involving L. donovafli amastigote to promastigote transformation. Intra-

Ionophore inhibition of L. donouani transformation cellular polyamine levels, particularly spermine, changed drastically during the first 6 hr of transformation (Morrow et al., 1980) and parasite sensitivity to infected hamster spleen transformation blocking factor(s) decreases within 6 hr of transformation (Krassner et al., 1980). All of these changes occur before any gross morphological change [i.e. differentiation of amastigotes to 1st intermediate (enlarged) stage] takes place. Some of the changes may be mediated by the parasite membrane and future studies on cell membrane changes during the early stages of transformation are of great importance. It is not unreasonable to invoke a “secondary messenger” role for Ca’+ in L. donouani transformation; certainly there is support for this role in other differentiating systems (Rasmussen, 1975). The ADP experiments, however, show that energy depletion resulting from perturbation of Ca2+ levels by A23187 could also be important in our system. Ca’+ levels are probably related to energy metabolism and addition of A23187 affects those levels thereby influencing energy production. Addition of ADP modified the perturbating activity of ionophore possibly by providing the energy necessary to maintain proper endogenous Ca2’ levels. At this time it is not possible to conclusively state the key role(s) played by Ca*+ in hemoflagellate transformation. Ca2+ may be a secondary messenger, a critical ion for mitochondriaidependent energy production, or it could act in a, as yet undetermined, completely different capacity necessary for cytodifferentiation (e.g. microtubule development). Our experiments have demonstrated the value of ionophores for determining ion requirements of L. donooani and as specific probes for following biochemical changes during transformation. Preliminary work in our laboratory with valinomycin, a K+-specifit ionophore, suggests that K+ is also required for amastigote to promastigote development. Valinomycin inhibits transformation and K+, but not Na+, can reverse this inhibition. Unfortunately these studies have been hindered by the extreme toxicity of valinomycin for L. donouani and further work is necessary to substantiate these preliminary observations. Acknowledgements-We are grateful to Drs R. L. Hamill (Lilly Research Laboratories) and W. E. Scott (Hoffman-La Rouche) for their generous gifts of A23187 and X-537A, to S. Peterson (U.C. Irvine) for his aid in the photomicrography, and to R. Wrightsman and R. Lee for technical assistance. REFERENCES BALK S. D. (1971) Calcium as a regulator of the proliferation of normal but not transformed, chicken fibroblasts in a plasma-containing medium. Proc. natn. Acnd. Sci. USA. 68, 271-5. BARTH L. G. & BARTH L. J. (1972) “Sodium and 45Calcium uptake during embryonic induction in Rana pipiens. Deal Biol. 28, 18-34. BERENS R. L., BRUN R. & KRASSNER S. M. (1976) A simple monophasic medium for axenic culture of hemoflagellates. J. Parasir. 62, 36G-5. BISHOP A. & MCCONNACHIE E. W. (1960) Further observation on the in vitro development of the gametocytes of Plasmodium gallinaceum. Parasitology 50, 431-48.

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Note added in proof-Tests with EGTA, a specific chelator of Ca *+ that does not significantly affect showed no effect upon amastigote to promastigote transformation even when Mg ‘+ ion concentration, added with A23187. This further supports the view that, in addition to Ca*+, other ions are required to complete transformation.