Characterization of calcium involvement in the Clostridium perfringens type A enterotoxin-induced release of 3H-nucleotides from Vero cells

Microbial Pathogenesis 1989 ; 6 : 17-28

Characterization of calcium involvement in the Clostridium perfringens type A enterotoxin-induced release of 3H-nucleotides from Vero cells Bruce A . McClane Department of Microbiology, Biochemistry and Molecular Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, U .S .A . (Received June 16, 1988; accepted in revised form September 6, 1988)

McClane, B . A. (Dept . of Microbiology, Biochemistry and Molecular Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, U .S .A .) . Characterization of calcium involvement in the Clostridium perfringens type A enterotoxin- induced release of 3 H-nucleotides from Vero cells . Microbial Pathogenesis 1989 ; 6: 17-28 . This report characterizes the involvement of Call in the release of nucleotides from Vero cells caused by Clostridium perfringens enterotoxin (CPE) . A positive linear correlation was observed between increased CPE-induced nucleotide-release and increased extracellular calcium over the range 0 .01 to 10 mm calcium . Above 5 mm Call, CPE-specific lysis (i .e . disintegration of cells as monitored by light microscopy) was observed . Addition of 1 .7 MM C a ll to Vero cells previously CPE-treated in Ca 2 '-free buffer rapidly increased nucleotide-release, even when cells had been previously incubated for 1 h at 37°C in Ca l '-free buffer . Withdrawal of Ca t ', even after the onset of nucleotide-release, halted further CPE-induced nucleotide-release . These results indicate that Ca 21 must be continuously present for significant CPE-induced nucleotiderelease . However, withdrawal of Ca 2' did not reverse membrane bleb formation by CPE . This differentiates bleb formation and nucleotide-release (both Ca l '-dependent CPE effects) and suggests that nucleotide-release does not result simply from bleb formation . Lastly, it was shown that other ions besides physiologic Ca 21 (1 .7 mm) are required for CPE-induced nucleotide-release . Interestingly, a role for other ions (but not physiologic Ca 21) is also shown for 86 Rb-release by CPE (an early Ca 2 '-independent CPE effect) . This indicates that extracellular 21 21 ions other than physiologic Ca can be required for both Ca -independent and Ca"-dependent CPE effects . Key words: enterotoxin ; Clostridium perfringens ; calcium; membrane permeability .

Introduction

Clostridium perfringens enterotoxin (CPE) causes symptoms associated with C .

perfringens food poisoning and may also be involved in other gastrointestinal diseases .' Recent studies 2-9 have indicated the existence of both Ca2 '-independent and Ca 2+ dependent steps in CPE action . Ca2+ -dependent CPE effects are known to include 2'3'S membrane bleb formation, release of noradrenaline by neurosecretory cells,' and membrane permeability alterations for larger molecules," such as CPE-induced release of intracellular nucleotides . Currently there is considerable interest in the involvement of calcium in the action of membrane-active agents, including bacterial toxins . 10 Relatively little is known about the involvement of calcium in Ca 2+ -dependent CPE effects (see below) . The importance of Ca"-dependent effects in CPE action in vivo and in vitro also remains 0882-4010/89/010017+12$03 .00/0

© 1989 Academic Press Limited



18

B . A. McClane

unclear . However, these Ca"-dependent CPE effects may contribute to disease in vivo . For example, CPE-induced morphologic damage (a Ca t +-dependent CPE effect 2 .3 .5 ) is believed to contribute to, if not directly cause, diarrhea associated with C. perfringens food poisoning ." Previous studies 2' 3 ' 5 have partially characterized the involvement of Ca 21 in CPEinduced membrane bleb formation in Vero cells . Our current study now characterizes Ca 21 involvement in nucleotide-release, which is another Ca t+ -dependent CPE effect .' These studies will facilitate direct comparisons of two Ca t+ -dependent CPE effects in the same cell culture system . This information should increase understanding of calcium involvement in Ca t+ -dependent CPE effects . For example, do all Ca Z+ dependent effects have similar Ca t+ requirements under all conditions? Secondly, this study may also help to determine whether a simple cause/effect relationship exists between bleb formation and nucleotide-release . It is currently unclear whether bleb formation directly causes nucleotide-release .' To date, it is clear only that conditions which inhibit bleb formation also inhibit nucleotide- release .' Z ' 13 However, this does not necessarily prove a direct relationship between these effects . The putative relationship between these CPE effects is studied further in this report .

Results

Effects of Ca'* concentrations on CPE-induced (3H) nucleotide release A previous study' has reported that CPE-induced ( 3 H) nucleotide-release is much stronger when Vero cells are CPE-treated in the presence of physiologic Ca" (1 .7 mm) than when CPE treatment is conducted with Ca"-free solutions . The relationship of Ca" concentration to CPE-induced ( 3 H) nucleotide loss is now studied over a broad range of Ca" concentrations (Fig . 1) since variations in extracellular Ca" concentrations are known to proportionally affect the expression of action of other membrane-active agents . 1 ' ,15 The degree of CPE-induced nucleotide-release showed a positive linear correlation

I 0

1

1

5 10 Calcium ImMI Fig . 1 . Effects of Cat' concentration on enterotoxin-induced ( 3 H) nucleotide release . Vero cells were treated for 30 min with 5,ug CPE/2 ml in test buffer containing the specified Ca r ' concentrations (the Ca 2, concentration in HBSS is usually 1 .7 mm) . Mg t + is present throughout at 1 .8 mm but similar results also are observed in Mgt -free buffer (data not shown) . Results shown are from duplicate samples in three experiments . Maximal release averged 1 .9 x 105 cpm/culture in these experiments . Spontaneous release was similar under all calcium conditions (range of means was 1 .5-2 .1 x 10' cpm/culture) .



19

Ca" involvement in CPE-induced nucleotide release

Table 1 High Ca' levels specifically stimulate enterotoxininduced nucleotide-release Enterotoxin-induced percent of maximal release Treatment solution' (HBSS with) : 1 xCa2 1 XMg 2+b 10xCa 2 +, 1 xMg 2+ 10xCa 2 + (Mg 2 +-free) 1 xCa2+ , 10xMg2+ 10xMg 2 ± (Ca 2 +-free)

Enterotoxin (µg/2 ml) : 2 .5 5 8 .8±1 .4 36 .3±1 .8 31 .7±2 .6 9 .1+2 .8 0 .7±0 .4

17 .6±1 .4`'d 52 .4±2 .0 44 .7±2 .4 17 .8±2 .7 2 .0±1 .1

Morphologic damage` + + + + -

'Cells were treated with enterotoxin for 30 min at 37°C in the specified solutions . b Concentrations (1 x) of Ca2+ and Mg 2 ' were 1 .7 mm and 1 .8 mm, respectively . `Values are given as mean + S .E . (n _> 3) . 'There were no significant differences (range of means from 13 940±510 cpm to 15220±1160 cpm) in spontaneous release from control cells incubated in any of the test solutions without CPE . Maximal release averaged 1 .8x105 cpm/culture in these experiments . 'Bleb formation 2,1,1,11 was apparent in >50% of cells after 30 min (+) or damage was absent (-) .

with increasing extracellular Ca 2+ concentration over the range of 0 .01 to 10 mm calcium (Fig . 1) . This effect was similar whether Mg 2+ (1 .8 mm) was present or absent from test buffer (data not shown) . High Ca 2 + and Mg 2 + concentrations did not affect spontaneous release (no CPE) from control cells (Table 1) . The promotional effect of high Ca 21 concentrations on CPE-induced ( 3 H) nucleotide loss was not a nonspecific consequence of increased osmolarity since high Mg 2 + levels did not promote CPE action in this assay (Table 1) . Interestingly, high (>5 mm) Ca 21 concentrations promoted CPE-specific cell lysis (i .e . disintegration of cells visible by light microscopy) within 30 min in this assay system . CPE-specific membrane bleb formation was consistently apparent within 1530 min at Ca 21 concentrations >-0 .5 mm, although bleb formation was apparent in some cells in some experiments at even lower Ca 21 concentrations . These results are + similar to earlier morphologic observations .2,3,1 Control cells (no CPE) in high Ca 2 concentrations for 30 min did not form membrane blebs or have altered cell shape visible by light microscopy . If both Ca 2 + and Mg 2 + were omitted from the incubation buffers, control Vero cells started to change from a spindle shape to a round shape after 20-30 min . However, control cells incubated in 1 .8 mm Mg 2 +-containing buffer (but no Ca 2+) showed no change in cell shape or bleb formation within 30 min . Demonstration that Ca" increases CPE-induced (3 H) nucleotide-release after preincubation of cells in Ca Z+ -free buffer Experiments were performed to determine whether addition of calcium could increase CPE-induced ( 3 H) nucleotide-release in cells previously CPE-treated and incubated for prolonged periods at 37°C in Ca 2+ -free HBSS (Table 2) . These studies clearly demonstrate that CPE-induced loss of ( 3 H) nucleotides can be significantly increased 2+, by later incubation with Ca even after 60 min of incubation of CPE-treated cells in Ca 2+ -free buffers.* * Student's t-test was used to compare control and toxin-treated cultures for each incubation condition . Cultures incubated in Ca2+ -containing test solution compared with the appropriate culture receiving Ca2+ free test solution for the same incubation period showed significant (P < 0 .05) differences in CPE-induced nucleotide-release under all incubation conditions and times tested .



20

B . A . McClane

Table 2 Late addition of Ca 2+ enhances enterotoxin-induced nucleotide-release in cells previously incubated in Ca 2 +-free buffer Cells were labeled for 2 h with ( 3 H) uridine and then washed twice with 0 .32 M sucrose containing 10 mm Hepes, pH 7 .4 . Enterotoxin (5 pg/2 ml) dissolved in Ca t '-free HBSS (the 'binding' solution) was then added to these cells for 7 .5 min and the cells were washed again to remove unbound CPE . After washing, the cells were incubated for 30 or 60 min in Ca t '-free HBSS and washed again . At this time, the appropriate test solution was added and the cells were incubated for 30 min and then assayed as described in the Materials and methods section . Mgt, was present throughout the experiment at 1 .8 mm . When present, Ca t ' was 1 .7 mm . All steps were performed at 37°C . Maximal release averaged 1 .6x10 5 cpm/culture in these experiments . c 0

m y

0

m dt d= o~ am

d

c .o

cc =m

y` 2C VX (F yy A

=

M

2 hr

CP CFT' CF CFT CF CFT CF CFT

00

Zu.

I -

y6 O2 U V

H~ 2

N

Uy

Q,

N yJU,

U y)~1-,

!

++

I. 1.

I

'Binding' solution

y

ym

7.5 min

30-60 min

Incubation period in CF following binding and washing (min)

Test solution

CPM released

30 30 30 30 60 60 60 60

CF CF C° C CF CF C C

9070+1420° 18010+ _ 1790 8220+ _ 2210 29220+ _ 2580 9980+870 15410+ _ 1840 7080+750 23890+2870

30 min

Percent of maximal release

Morphologic damage'

5 .2%±1 .0% 12 .2%+1 .5% 3 .2%+1 .1% 9 .8%±2 .1%

aCF=Ca t . -free HBSS . 'CPM and maximal release values are mean ±S .E . (n >_ 3) . 'CFT= Cat'-free HBSS with CPE (5 pg CPE/2 ml solution) . °C=CaZ'-containing HBSS . 2 .3,5,18 was apparent (+) or absent (-) in >50% of cells . Note : e Bleb formation typical of CPE treatment cells incubated in CF buffer for 60 min started to round, but no blebs typical of CPE treatment were apparent in these cells (with or without CPE) .

Addition of Ca 2, to cells previously CPE-treated in the absence of Ca 2+ resulted in rapid membrane bleb formation characteristic of CPE-induced morphologic damage in all experiments described in this report . This effect has been previously reported ."' Also as reported ,2,3,5 bleb formation was absent from cells CPE-treated and continuously incubated in Ca 2 +-free solutions (Tables 2 and 3) . Studies to further examine the role of Ca" dependency in CPE action Results presented in Tables 2 and 3 confirm previous 125 1-CPE binding studies which indicated that Ca 2+ need not be present for enterotoxin binding .' Significant CPEinduced ( 3 H) nucleotide loss or bleb formation was observed in Tables 2 and 3 whether Ca 21 was present or absent in the binding buffer (as long as Ca 21 was later



Ca" involvement in CPE-induced nucleotide release

21

Table 3 Effects of Ca" withdrawal or addition on CPE-induced ( 3 H) nucleotide-release from Vero cells Vero cells were ( 3 H) nucleotide-labeled as described in the Materials and methods section and washed twice with 0 .32 M sucrose containing 10 mm Hepes, pH 7 .4 . CPE (5 µg/2 ml) dissolved in the appropriate 'binding' solution was then incubated with cells for 7 .5, 12 .5 or 20 min . After washing to remove unbound toxin, the desired 'test' solution was added and cells were incubated for 30 min prior to assay as described in the Materials and methods section . Mg` was present throughout the experiment at 1 .8 mm . When present, Ca21 was 1 .7 mm . All procedures were performed at 3TC . N X C C 0 7 N O

x V)

rn

C

om oC xm

v

0 U) C 1-m O

~~

mC =

O

3O

C

I 2 hr

.C

N N

++

j

7.5-20 min

30 min

Enterotoxin-induced percent of maximal release Binding solution CFT' CFT CT' CT

Test solution CFb Cd

CF C

Min in binding solution : 7 .5 12 .5 20 2 .7+0 .8` 16 .3+1 .0 2 .1+0 .2 9 .4+0 .7

1 .9+0 .2 21 .0+1 .7 3 .5+0 .4 16 .1+1 .0

3 .1+0 .2 27 .1+2 .8 4 .0+0 .2 22 .7+2 .9

Morphologic damage' + + +

CFT = Ca Z +-free HBSS with CPE (5 µg CPE/2 ml solution) . CF = Ca2+ -free HBSS . `Values are given as mean +S .E . (n >_ 3) . Spontaneous release was similar under all experimental conditions (range 6-8 x 103 cpm/culture) . Maximal release averaged 1 .5 x 105 cpm/culture in these experiments . IC =Ca t +-containing HBSS . a CT = Ca t +-containing HBSS with CPE (5 µg CPE/2 ml solution) . 'Bleb formation typical of CPE treatment 2,3.5,18 was apparent (+) or absent (-) in ->50% of cells after binding (7 .5-20 min) and 30 min incubation in 'test' solution . ± indicates morphologic damage was apparent after 30 min in test solution in cells CPE-treated in binding solution for 20 min but not for 7 .5 min (note : bleb formation occurred in Ca 2+ containing binding solution after 10-15 min) . a

present in the assay buffer) . The reason for the apparent increase in CPE-induced ( 3 H) nucleotide loss after CPE binding in Ca t+ -free conditions (followed by readdition of Ca 21) observed in Table 3 is not clear, but this effect is observed following similar experimental procedures in Fig . 2 also . Preincubation of cells in a Ca Z+ -free milieu for 15 min before CPE addition did not increase sensitivity to CPE (data not shown) . Also, incubation of control cells under either Ca"-containing or Ca t+ -free conditions described in Tables 2 and 3 had no effect on spontaneous release (data not shown) . Approximately equivalent low levels of CPE-induced ( 3 H) nucleotide release were measured from cells in CaZ+-free 'test' buffer, whether enterotoxin 'binding' occurred



22

B . A. McClane

0

10

20

30

Minutes Fig . 2 . Kinetics of CPE-induced ( 3 H) nucleotide release after addition of Ca 2 . ( 3 H) nucleotide-labeled Vero cells were washed twice with 0 .32 M sucrose containing 10 mm Hepes, pH 7 .4 and treated for 7 .5 min with CPE (5 µg/2 ml) dissolved in HBSS with or without Ca t (i .e . binding solution) . After 7 .5 min in binding solution (HBSS±Ca 2+ ), cultures treated with CPE showed no significant (
in the presence or absence of Ca" (Table 3) . It should be noted that this effect was observed even in cells incubated with CPE for 12 .5 or 20 min in Ca 21 -containing 'binding' solution . Note that cells incubated for 12 .5 or 20 min with CPE in Ca 2 'containing buffer are actively releasing ( 3 H) nucleotides (8 .5%±0 .6%and 12 .8%±0 .9% of maximal release of nucleotides occurred after 12 .5 or 20 min, respectively, of CPE treatment in the presence of Ca t+ ) . After 7 .5 min of CPE treatment in the presence of Ca", CPE-induced nucleotide-release was negligible (<11% of maximal release) . These results show that removal of Ca t + even after the onset of CPE-induced ( 3 H) nucleotide loss reduces subsequent ( 3 H) nucleotide release to levels equivalent to cells never exposed to Ca t+ during CPE binding or assay incubation . CPE-induced membrane bleb formation was detectable after 10-15 min in binding solution containing Ca 2+, similar to previous results . 2,3,5,16 Interestingly, CPE-induced membrane blebs, which require extracellular Ca 21 for formation, 2,3,5 did not disappear when Ca 2+ was removed (Table 3) . To further examine the time or stage at which Ca` is required for CPE-induced



Ca

21 involvement in CPE-induced nucleotide release

23

nucleotide-release, additional kinetic studies were performed (Fig . 2) . These studies show that once CPE is bound (whether in Ca"-containing or Ca 2 +-free buffer), there need be little or no lag for the expression of Ca 2 +-dependent CPE-induced ( 3 H) nucleotide-release . As in Table 2, Fig . 2 shows that cells treated with CPE in Ca 2+ free solutions (followed by Ca 2' addition) release more nucleotides than cells continuously incubated in the presence of Ca t + . Membrane bleb formation was also rapid (occurring within 5 min) after Ca 2' addition to cells previously CPE-treated in Ca2+ -free solutions, as noted previously . 3 .5 Evidence that Ca" is not the sole ion mediator of CPE action It has become clear from this and previous studies 2-9 that Ca" has a role in some CPE effects . Previous studies (see McClane et al.' for review) have also suggested that CPE affects the overall osmotic balance of the cell . Therefore, studies were conducted to further examine the importance of Ca 21 versus other ions in both 'Ca 21 -dependent' and 'Ca2 +-independent' CPE effects . The CPE-induced release of nucleotides in Table 4 is clearly Ca 21 -dependent, as noted earlier in this report and briefly in a previous report . 7 Interestingly, no nucleotiderelease caused by CPE is noted in the presence of 0 .32 M sucrose [present either as 0 .32 M sucrose-10 mm Hepes buffer or 0 .32 M sucrose added to HBSS (Hanks Balanced Salts Solution)] even if Ca 2+ is present at physiologic concentrations . Results presented in Table 4 strongly suggest that Ca 21 is not the sole ion mediator of CPEinduced nucleotide-release in physiologic solutions (e .g . HBSS) . The inhibition of CPE-induced nucleotide-release when 0 .32 M sucrose is added to HBSS containing Ca 2 + has been noted previously ." 86 Rb-release has been previously reported to be a Ca 2 +-independent CPE effects •' 16 Results presented in Table 4 agree with this . Rb-release occurs to an equal extent in the presence or absence of 1 .7 mm calcium, as long as other ions were present . However, no 86 Rb-release occurred in 0 .32 M sucrose-10 mm Hepes although 86 RbTable 4 Demonstration of a requirement for extracellular ions (other than physiologic Ca 21) for both Ca 2 +-independent and Ca 2 +-dependent CPE-induced membrane permeability alterations Cells were labeled for 2 h with either (3 H) uridine or 86 RbCl (see Methods) and then washed twice with 0 .32 M sucrose containing 10 mm Hepes, pH 7 .4 . Enterotoxin (5 ug/2 ml) dissolved in the specified treatment solution was then added to the cells for 30 min at 37°C . Release of ( 3 H)-nucleotides or 86 Rb was then assayed as described in the Materials and methods section . When present, Mg` was 1 .8 mm and Ca 21 was 1 .7 mm . Maximal release in these experiments averaged 1 .7x105 cpm/culture for nucleotide-release and 6 x 103 cpm/culture for 86 Rb-release . Spontaneous release was similar under all experimental conditions (range : 9 .7-11 x 103 cpm/culture for nucleotide-release and 1-1 .5x 103 cpm/culture for 86 Rb-release) . Hepes was added to all treatment solutions to 10 mm, pH 7 .4 . Enterotoxin-induced percent' of maximal release Treatment solution HBSS with Ca", Mgt' HBSS with Mg t ', without Ca 2+ HBSS with Ca21, Mg t ' and 0 .32 M sucrose HBSS with Mgt± and 0 .32 M sucrose, without Ca 2± 0 .32 M sucrose 0 .32 M sucrose with Ca 2 ±

Nucleotide-release 19 .5±1 .6' 3 .3±1 .3 3 .6±0 .8 0 .8±0 .1 1 .4+0 .7 3 .2±0 .7

S

6 Rb-release

Morphologic' damage

98 .4±1 .7 97 .2±2 .0 95 .1+2 .1 97 .6±2 .3 1 .8+1 .3 3 .5±2 .1

+ -

'Values are given as mean +S .E. (n >_ 3) . b Bleb formation 2'3'5'18 typical of CPE treatment was present (+) or absent (-) . Some cells (-<25% in most experiments) CPE-treated in Ca 2 +-containing sucrose or HBSS with Ca 2+, Mgt' and 0 .32 M sucrose did round but no membrane blebs were apparent .



24

B . A . McClane

release was noted in HBSS containing 0 .32 M sucrose-10 mm Hepes, as reported previously . 13 This indicates that 86 Rb-release by CPE also requires the presence of extracellular ions other than physiologic Ca z + . Bleb formation occurred only in Ca 2+ -containing HBSS . No bleb formation was noted under any Ca 2 +-free conditions or any solution containing 0 .32 M sucrose . The prevention of bleb formation by sucrose was noted previously . 2,3 .5 .12,13 Cells CPEtreated in sucrose-Hepes buffer did not form membrane blebs but some (usually <25%) CPE-treated cells did change from a spindle shape to a round morphology by 30 min in the presence of 1 .7 mm Ca z+ . Discussion This report provides a number of novel observations on CPE action . Included among these observations are detailed results describing the relationship of Ca 21 to CPEinduced nucleotide-release, differentiation of CPE-induced nucleotide-release from bleb formation in Vero cells and information indicating the involvement of ions other than physiologic Ca 21 in some Ca 2 +-independent and Ca 2 +-dependent CPE effects . Each of these results will be discussed further with emphasis on their importance to current understanding of CPE action . Recently, we reported' that C PE- induced nucleotide- release occurred in the presence of 1 .7 mm Ca2' but that this CPE effect was significantly lower in Ca 2+ -free solutions . A direct positive relationship between Ca 21 concentration and nucleotide-release by CPE is shown in the current report. Importantly, we now show that addition of Ca 21 beyond physiologic levels (1 .7 mm Ca 2+ ) results in a continued increase in nucleotiderelease and that very high Ca 21 levels also promote visible lysis of Vero cells . These results hold importance for two reasons . First, they argue against a low 'threshold' requirement for Ca z + in CPE action, at least for nucleotide-release . If extracellular Ca 21 is required only at a threshold value, then nucleotide-release should be constant after attainment of the threshold concentration . The demonstration of increasing nucleotiderelease beyond 1 .7 mm Ca 21 contrasts CPE-induced nucleotide-release in Vero cells with CPE-induced noradrenaline release by neurosecretory cells .' Noradrenalinerelease by CPE is also Ca 2 +-dependent but this effect is fully expressed at physiologic Ca 2+ and plateaus with further increases in Ca 21 concentration .' This indicates that there are differences in Ca 2+ requirements for different Ca 2+ -dependent CPE effects, an observation to be expanded on later in the Discussion . Secondly, the observation that high Ca 21 concentrations beyond physiologic levels increases at least some CPE effects in some cells is interesting since staphylococcal x toxin is inhibited by high Ca z + levels.' 0,15 CPE and staphylococcal a toxin are of similar size and both toxins are membrane-active and disrupt the osmotic barrier of cells . 10 '12,15 However, the opposite response of these toxins to high Ca 21 levels indicates that there is also a fundamental difference in their action(s) . Further, Basford etal. have reported 10 that high Ca 21 concentrations decrease permeability alterations for many diverse membrane-active agents and they have proposed that many membrane-active agents share common mechanisms . From our current report, CPE clearly has a distinguishable effect from these other agents with respect to high Ca 21 sensitivity, indicating that not all membrane-active agents have similar effects and actions . It was also observed in this report that addition of Ca 21 to cells CPE-treated for an hour at 37°C in Ca 2+ -free solutions strongly increased both CPE-induced ( 3 H) nucleotide-release and bleb formation (a result similar to a previous morphology study3 ) . These results may suggest that Vero cells have little ability to repair CPEinduced membrane damage . We have reported previously' that specifically bound CPE



Ca" involvement in CPE-induced nucleotide release

25

is physically very stable (up to at least 1 h) and resistant to degradation in membranes . Our current results and results from a previous morphology study' strongly suggest that CPE is also functionally very stable in membranes, even after prolonged incubation at 37°C . 21 contributes to CPE effects on It remains unclear where or how extracellular Ca cells . However, this report does provide valuable information which should facilitate 21 from CPEfuture studies on this problem . First, it was noted that removal of Ca treated cultures results in a rapid shut-off of further ( 3 H) nucleotide-release . This is 21 the first report of the reversibility of any CPE effect and indicates that Ca must be continuously present to obtain CPE-induced nucleotide-release . However, CPEinduced membrane bleb formation was not reversible by Ca t+ removal . These important results differentiate for the first time these two Ca t+ -dependent CPE effects which occur in the same cells . All previous studies have shown (see McClane et al.' for review) both bleb formation and nucleotide-release to be virtually identical with respect to kinetics of onset, Ca t +-dependency and sensitivity to inhibition by osmotic 21 is withdrawn, stabilizers . The observation that nucleotide-release ceases when Ca although blebs do not disappear, also indicates for the first time that there is not a simple direct cause and effect relationship between bleb formation and nucleotiderelease, i .e ., these important results indicate that bleb formation alone does not necessarily produce nucleotide-release . The current study has clearly described experimental manipulations which can differentiate bleb formation from nucleotide-release in Vero cells . As mentioned above, we also have differentiated the response of CPE-induced nucleotide-release in Vero cells from noradrenaline release in neurosecretory cells with respect to response to increasing Ca 21 concentration . Interestingly, it was reported' that neurosecretory cells 21) did not form blebs (even in the presence of Ca or have generalized membrane permeability alterations following CPE treatment (the authors hypothesized that noradrenaline result may involve increased vesicular secretion following CPE treat21 ment) . Considered collectively, these results indicate that all known Ca -dependent CPE effects are now distinguishable and strongly suggest that there may be multiple mechanisms involved in the interaction(s) between cells, Ca t ' and CPE . It was also observed that addition of Ca 21 to cultures CPE-treated in Ca 21 -free solutions results in rapid nucleotide-release (this study) or bleb formation (this study and Sugimoto et al .') . This indicates that the Cat+ -sensitive cell component(s) responsible for nucleotide-release and bleb formation which are affected by CPE treatment must be sensitive to rapid changes in extracellular calcium levels . It is known that CPE rapidly affects intracellular Ca 21 concentrations, presumably by rapidly increasing Ca 21 influx .', ' These results also strongly suggest that the significant delay (usually about 10-15 min) observed for CPE-induced ( 3 H) nucleotide-release 1,3,5,11 (this report) is probably due (McClane et al .' and this report) or bleb formation to a Ca t +-independent step(s) since onset of Ca t +-dependent steps can be very rapid (<4 min) when Ca 21 is added to cells previously treated with CPE in Ca t +-free buffer . We also observed in Fig . 2 and Table 3 that cells CPE-treated in Ca t +-free solution and then incubated in the presence of calcium release more nucleotides than cells treated with CPE and incubated continuously in Ca 21 containing buffer . This is an interesting but currently unexplainable effect . From 125 1-CPE binding studies,' it is unlikely that this effect results from greater CPE binding in Ca t+ -free solutions . Perhaps it reflects a synergistic response of cells to simultaneous exposure to two deleterious 21 stresses (Ca 2+ -deprivation and CPE treatment) . Further understanding of how Ca participates in nucleotide-release by CPE is necessary to provide mechanistic explanation of this phenomenon .



26

B . A. McClane

With regard to how and where Ca 21 is involved in Ca t+ -dependent CPE effects, there are two promising targets . First, CPE could cause rapid influx of Ca t+ and produce consequent high intracellular Ca 21 levels," resulting in Ca 21 interaction with a cellular structural component . The cytoskeleton is known to be highly sensitive to intracellular Ca t+ and high intracellular Ca t ` levels can cause rapid collapse of the cytoskeleton ." This effect would be consistent with the 'rounding' of CPE-treated cells noted previously . 18 The cytoskeleton is known to be involved in proper membrane function ." CPE-induced morphologic damage to the cytoskeleton might lead to permeability alterations for larger molecules, if permissive conditions (e .g . proper ionic conditions, see below) exist . Alternatively, CPE may cause Ca"-dependent effects by activating a Ca"-dependent metabolic cascade . 20 Precedent for this exists since staphylococcal a toxin can activate the Cat+ -dependent arachidonic acid cascade and this activation can occur rapidly . 21 Further studies will be required to examine these possibilities, which are not mutuallyexclusive . Since this report provides information differentiating the three known Ca 2 dependent CPE effects, it is very possible that different mechanisms may be involved in various Ca t+ -dependent CPE effects . Lastly, important new information is presented which indicates (1) Ca 21 is not the sole ion required for nucleotide-release and (2) extracellular ions (other than Ca t- ) are required to obtain CPE-induced 86 Rb-release . If Ca 21 is the sole ion mediator of Ca t+ -dependent CPE effects, then full CPE activity should have been obtained in isoosmotic medium containing sucrose and physiologic Ca t ' but free of other ions . Results in Table 4 do not support this possibility . The lack of nucleotide-release in sucrose-containing solution cannot be attributed to a blockage of CPE binding by sucrose since CPE-induced 86 Rb-release occurred in the presence of sucrose, provided other ions were present as noted previously ." Further, a previous study" also reported that CPE-induced 22 Na fluxes, as well as 86 Rb-release occurred in the presence of sucrose . It should be noted that in our previous study, sucrose was always tested for effects on CPE-induced 86 Rb-release in the presence of physiologic ion concentrations . The current study clearly shows that little or no 86 Rb-release occurs if cells are CPEtreated in buffer without most extracellular ions (despite the presence of physiologic Ca 21) . Our current study now provides important direct results which suggest that other ions besides physiologic Ca t ` are involved in both some Ca"-dependent and Ca t+ independent CPE effects, including at least some very early effects of CPE ( 86 Rbrelease occurs rapidly after CPE binding') . This interesting conclusion raises numerous questions which require further study . For example, are all Ca 21_ independent CPE steps' dependent on the presence of extracellular ions? Which extracellular ions, at what concentration, are important? What are the role(s) of these extracellular ion(s) in CPE action? Preliminary studies' suggest that ion requirements for CPE-induced bleb formation may be relatively nonspecific (other than for Ca t+ ) but more detailed analysis is required . Studies are now underway to further address this problem .

Materials and methods Enterotoxin . C. perfringens enterotoxin was prepared and assayed for biologic activity as described previously ." The specific activity of CPE preparations varied from 500000 to 1000000 plating efficiency units/mg protein ." Enterotoxin was dissolved in double-distilled water and 60 pl aliquots were stored frozen at -70°C until used . Aliquots were used within 2 weeks of their preparation . Culture of Vero ce//s . Vero (African green monkey kidney) cells were grown as monolayer



Ca" involvement in CPE-induced nucleotide release

27

cultures in Medium 199 (Flow) as described previously .' The cells were seeded in 16 mm wells in tissue culture cluster dishes (Co-Star) at a density of 5 x 104 cells per well . After 3 days, fresh medium was added . (3H) Nucleotide labeling of Vero cells . Cells were nucleotide-labeled by methods previously reported .' Confluent 6-day-old monolayers were washed with Medium 199 and labeled by the addition of 2 ml Medium 199 containing 2 µCi ( 3 H) uridine (35-50 Ci/mmol, ICN Radiochemicals) . After 2 h, the labeling medium was removed . 86Rb-labeling of Vero cells . Cells were 86 Rb-labeled as described previously .' Confluent monolayers were washed with Medium 199 and labeled by the addition of 2 ml Medium 199 containing 4 µCi 86 RbCI (1 .2 mCi/mg, DuPont-New England Nuclear) . After 2 h, the labeling medium was removed . Treatment of (3H) nucleotide-labeled Vero cultures with enterotoxin . Unless otherwise specified, prior to enterotoxin treatment ( 3 H) nucleotide-labeled or 86 Rb-labeled cultures were washed twice with 0 .32 M sucrose containing 10 mm Hepes, pH 7 .4 . Precise treatment conditions are specified for each experiment in the appropriate figure or table legend . All experimental procedures were performed at 37°C . After the desired incubation period for each assay, the culture supernatant was gently removed, vortex-mixed, and centrifuged at 4°C for 3 min in an Eppendorf microcentrifuge . Radioactivity was measured in the supernatant . Determination of percent of maximal release . Released radioactivity was calculated as described by Thelestam and Mollby : 24 toxin-induced release-spontaneous release of maximal release=

x 100

maximal release-spontaneous release

Spontaneous release (i .e . background release occurring in the absence of CPE) after 30 min at 37°C was usually less than 10% of maximal release for nucleotide-release in all experimental protocols . Spontaneous release of 86 Rb was less than 25% of maximal release after 15 minutes at 37°C . As described previously,' the maximal release of cytoplasmic label represents total cytoplasmic radioactivity at the end of radiolabeling (i .e . before CPE addition) . Maximal release was determined' after cell membrane rupture by the addition of 1 ml of 1 M citric acid and 1 ml of 0 .5% saponin buffer per well . Maximal release was 1-2x10 5 cpm/culture for nucleotidelabeled cultures and 5-8x 10 3 cpm culture for 86 Rb-labeled cultures . Approximately 77% of the maximal release radioactivity (i .e . total cytoplasmic radioactivity at the start of the assay) is in a nucleotide form in nucleotide-labeled cultures ." Measurement of radioactivity . Samples (0 .2 ml) from ( 3 H) nucleotide-labeled cultures were counted in a Searle Mark III liquid scintillation spectrophotometer . 86 Rb-containing samples (1 ml) were counted in a Packard y counter . This investigation was supported by Public Health Service grant Al 19844-06 from the National Institute of Allergy and Infectious Diseases . We are grateful to Barbara Baum and Betty Ann Rooney for typing of the manuscipt .

References 1 . McClane BA, Hanna PC, Wnek AP . Clostridium perfringens enterotoxin : a mini-review . Microbial Pathogenesis 1988 ; 4 : 317-23 . 2 . Matsuda M, Sugimoto N . Calcium-independent and dependent steps in action of Clostridium perfringens enterotoxin on HeLa and Vero cells . Biochem Biophys Res Commun 1979 ; 91 : 629-36 . 3 . Sugimoto N, Ozutsumi K, Matsuda M . Morphological alterations and changes in cellular cations induced by Clostridium perfringens type A enterotoxin in tissue culture cells . Eur J Epidemiol 1985; 1 : 264-73 . 4. 5.

Granum PE . The effect of Ca" and Mg t+ on the action of Clostridium perfringens enterotoxin on Vero cells . Acta Pathol Microbial Immunol Scand [B] 1985 ; 93 : 41-8 . Matsuda M, Ozutsumi K, Iwahasi H, Sugimoto N . Primary action of Clostridium perfringens type A enterotoxin on HeLa and Vero cells in the absence of extracellular calcium : rapid and characteristic changes in membrane permeability . Biochem Biophys Res Commun 1986 ; 141 : 704-10 .



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6 . Horiguchi Y, Uemura T, Kozaki S, Sakaguchi G . Effects of Ca 21 and other cations on the action of Clostridium perfringens enterotoxin . Biochim Biophys Acta 1986 ; 889 : 65-71 . 7 . McClane BA, Wnek AP, Hulkower KI, Hanna PC . Divalent cation involvement in the action of Clostridium perfringens type A enterotoxin : early events in enterotoxin action are divalent cationindependent . J Biol Chem 1988; 263 : 2423-35 . 8 . Ozutsumi K, Sugimoto N, Matsuda M . Clostridium perfringens type A enterotoxin induces release of noradrenaline from the neurosecretory PC12 cell line . Biochem Biophys Res Commun 1987 ; 144 : 21723 . 9 . Hulkower KI, McClane BA . The effects of Clostridium perfringens enterotoxin on intracellular levels or transport of uridine, thymidine and leucine do not fully explain enterotoxin-induced inhibition of macromolecular synthesis in Vero cells . Biochem Biophys Res Commun 1988 ; 153 : 699-707 . 10 . Bashford CL, Alder GM, Menestrina G, Micklem KJ, Murphy JJ, Pasternak CA . Membrane damage by hemolytic viruses, toxins, complement and other cytotoxic agents . A common mechanism blocked by divalent cations. J Biol Chem 1986 ; 261 : 9300-8 . 11 . McDonel JL . Toxins of Clostridium perfringens types A, B, C, D, and E . In : Dorner F, Drews J, eds . Pharmacology of bacterial toxins . Oxford : Pergamon Press 1986 : 477-517 . 12 . McClane BA, McDonel JL. Protective effects of osmotic stabilizers on morphological and permeability alterations induced in Vero cells by Clostridium perfringens enterotoxin . Biochim Biophys Acta 1981 ; 641 : 401-9 . 13 . McClane BA . Osmotic stabilizers differentially inhibit permeability alterations induced in Vero cells by Clostridium perfringens enterotoxin . Biochim Biophys Acta 1984 ; 777 : 99-106 . 14 . Sandvig K, Olsnes S . Entry of the toxic proteins abrin, modeccin, ricin, and diphtheria toxin into cells : 1 . Requirement for calcium . J Biol Chem 1982 ; 257 : 7495-503 . 15 . Bashford CL, Alder GM, Patek K, Pasternak CA . Common action of certain viruses, toxins, and activated complement : pore formation and its prevention by extracellular Ca t ' . Biosci Rep 1984 ; 4: 797-805 . 16 . McClane BA, McDonel JL . Characterization of membrane permeability alterations in Vero cells by Clostridium perfringens enterotoxin . Biochim Biophys Acta 1980 ; 600 : 974-85 . 17 . Glenney JR, Glenney P . Comparison of Ca t '-regulated events in the intestinal brush border . J Cell Biol 1985; 100 : 754-63 . 18 . McClane BA, McDonel JL . The effects of Clostridium perfringens enterotoxin on morphology, viability and macromolecular synthesis in Vero cells . J Cell Physiol 1979 ; 99 : 191-200 . 19 . Mooseker MS, Bonder EM, Conzelman KA, Fishkind DJ, Howe CL, Keller TCS . The cytoskeletal apparatus of the intestinal brush border . In : Donowitz M, Sharp GWG, eds . Mechanisms of intestinal electrolyte transport and regulation by calcium . New York : AR Liss, 1984 : 287-307 . 20 . Powell DW, Berschneider HM, Lawson LD, Martens H . Regulation of water and ion movement in intestine. In : Evered D, Whelan J, eds . Microbial toxins and diarrheal disease . London : Pitman, 1985 : 14-27 . 21 . Suttorp N, Seeger W, Dewein E, Bhakdi S, Roka L . Staphylococcal x toxin-induced PGI 2 production in endothelial cells role of calcium . Am J Physiol 1985 ; 248 : C127-34 . 22 . McDonel JL, McClane BA. Production, purification and assay of Clostridium perfringens enterotoxin . In : Harshman S, ed . Microbial toxins : tools in enzymology . Methods in enzymology, vol . 165 . New York : Academic Press, 1988 : 94-103 . 23 . McDonel JL, McClane BA . Highly sensitive assay for Clostridium perfringens enterotoxin that uses inhibition of plating efficiency of Vero cells grown in culture. J Clin Microbiol 1981 ; 13 : 940-6 . 24 . Thelestam M, Mollby R . Sensitive assay for detection of toxin-induced damage to the cytoplasmic membrane of human diploid fibroblasts . Infect Immun 1975 ; 12 : 225-32 .