The microsporidian spore invasion tube. IV. Discharge activation begins with pH-triggered Ca2+ influx.

The microsporidian spore extrusion apparatus activates with a calcium influx from Spraguea lophii spore wall/plasma membrane; this influx requires preconditioning with an extrasporular shift in medium pH to the alkaline in the presence of the polyanions mucin or polyglutamate. Undischarged S. lophii spores display calcium bound to the wall/plasma membrane with a characteristic calcium-chlorotetracycline fluorescence; this fluorescence declines significantly during spore discharge. S. lophii spores do not discharge when spore wall/plasma membrane calcium is removed with EGTA. Extrasporular mucin or polyglutamate and a pH shift to the alkaline appear to be necessary preconditions for the triggering of the influx of spore wall/plasma membrane-bound 45Ca2+. Ionophore A-23187 also effectively activates spore discharge without other extrasporular polyanions. Micromolar concentrations of the calcium antagonists lanthanum or verapamil prevent spore discharge, and micromolar concentrations of calmodulin inhibitors chlorpromazine and trifluroperazine prevent spore discharge. Calmodulin, visualized with a calmodulin antibody and a peroxidase conjugate, is localized particularly on the plasma membrane and the polaroplast membranes of the extrusion apparatus.

trifluoperazine (TFP) and chlorpromazine (Sigma Chemical Co.). Stock solutions were made up in deionized water. The spores were immersed in a range of concentrations of these antagonists in deionized water for 5 min. After this incubation, spores were immersed in DM and after 5 rain, the spores were lightly fixed with glutaraldehyde, and the incidence of discharge was scored.
Reversibility tests were done to determine whether the antagonists had a permanent effect on spore discharge. Spores were first immersed in antagonist medium (in distilled water) for 60 min, thoroughly washed free of antagonist with distilled water, and resuspended in DM. The incidence of spore discharge was then scored.
Calcium Probe, Chlorotetracycline (CTC): S Iophiisporeswere placed in a medium with I-5 ul of freshly prepared unbuffered glucose with 2 mM CTC, yielding a final concentration of 100-150 ~M CTC. Spores were mounted on wet-mount slides and examined for fluorescence. Fluorescence was monitored with Leitz epifluorescence optics with oil at 1,200x (E. Leitz, Inc., Rockleigh, N J). The CTC fluorescence was induced by light excitation from a mercury lamp, and emission was limited by a 520-nm barrier filter.
Radiochromatography: Five controlled experiments (A-E) were performed with l0 g S. lophii spores in each. Spores were washed in deionized water before incubations. In experiment A, spores were immersed in 0.2 M cold CaCl2, washed in deionized water, and transferred to 0.5 ml of 4SCaCl_, that had a specific activity of 5.0 ~Ci for 10 min. In experiment B, spores were placed in 0.15 mM EGTA, washed in deionized water, and immersed with 4~CaCI2 that had a specific activity of 5.0 #Ci for l0 rain. In experiment C, spores were placed in 0.15 mM EGTA, washed in deionized water, and exposed to 4~CaCl2 with 0.5/zl of 15 mM lonophore A-23187, but the spores remained undischarged since the medium was at pH 7.0. In experiment D, spores were treated with 0.15 mM EGTA, washed in deionized water, and placed in 0.5 ml of 45CaCb that had a specific activity of 5.0 uCi for l0 min, then in DM at pH 9.0 that contained 0.1 ul of ionophore A-23187 stock solution. The pH of 9 induced spore discharge in the presence of 45CaCl2. Experiment E was the same as D except that porcine mucin (0.2%) was used instead ofA-23187. The spores from experiments A-E were washed in deionized water and transferred to Millex (Millipore Corp., Bedford, MA) 0.22-um disposable filters. Circular cutouts (8.0 mm) of the filters were scanned with a 7230 radiochromatogram scanner (Packard Instrument Co., Inc., Downers Grove, IL).
Immunoperoxidase: The binding of antibody to calmodulin was detected by using the indirect immunoperoxidase method described elsewhere (4). Spores were fixed for 5 min in 0.12 M cacodylate-buffered 0.5% glutaraldehyde and washed thoroughly. The frozen pellet was cryostat-sectioned at 5um thickness. This sectioned spore material was washed in Ringer's solution and immersed in sheep anti-calmodulin (provided by CAABCO, Inc., Houston, TX) diluted 1:25 in Ringer's solution and incubated at 30°C for 50 mira cells were washed in Ringer's solution at 4°C and exposed to rabbit anti-sbeep lgG conjugated to peroxidase (provided by Cappel Laboratories, Inc., Cochranville, PA) for 20 min, washed in 0.1 M cacodylate buffer, and fixed in 1.5% glutaraldehyde-buffered cacodylate (pH 7.2) for 45 rain. Cells were then washed in 0.1 M cacodylate buffer and exposed to diaminobenzidine working medium (1) for 15 min. The cells were washed in 0.1 M cacodylate buffer, postfixed in 1% osmium in distilled water, and processed for transmission electron microscopy.

RESULTS
Microsporidian spore anatomy is presented as a background to our observations on the role of calcium and associated factors in spore discharge activation. The major parts of the spores of S. lophii are the sporoplasm, extrusion apparatus, and spore wall (Fig. 1). The sporoplasm is the infective cell that typically discharges with subsecond velocity after activation of the extrusion apparatus. The extrusion apparatus consists of an aperture, polar filament, membrane-rich polaroplast, and a posterior vacuole (Fig. l).

Polyanions and oH on S. Iophii Spore Extrusion
Spores of S. lophii discharged in glycylglycine or carbonate buffer during the switch from neutral (or acid) to alkaline pH in the presence of mucin or polyglutamate (Fig. 2). The polyanions and the shift to the alkaline pH were required protocol for spore discharge. No discharge took place when spores were immersed into medium with polyanions but at neutral pH. Conversely, no spore discharge occurred when spores were placed in alkaline medium without the polyanions polyglutamate or mucin (Fig. 2).

CTC As a Probe for Membrane-associated Ca 2÷ during Spore Discharge
Calcium-chlorotetracycline (Ca-CTC) fluorescence was apparent at the spore boundary before extrusion (Fig. 3 a). After discharge, much of this fluorescence was extinguished (Fig. 3, a and b). Spores pretreated with EGTA showed little Ca-CTC fluorescence.

EGTA and Spore Discharge
When spores were washed in 0.2 M CaCI2, rinsed thoroughly with distilled water, and immersed in DM (pH 9.0), a 70% frequency of spore discharge took place; however, when spores were immersed in CaCI2, washed in distilled water, and then transferred for 10 min in 0.07 mM EGTA, there was only a 5% incidence of spore discharge. No spore discharge occurred when the EGTA concentration was raised to 0.25 mM. When the EGTA-treated spores were then washed thoroughly in distilled water, incubated in glycylglycine buffer overnight (pH 7.0), exposed to CaC12, washed with distilled water, and immersed in DM, there was a return to normal spore discharge frequency. However, if the EGTA incubation was at concentrations above 0.15 mM, there was a reduced capability for spores to regain a discharge capacity.

Activation of S. Iophii Spores with Ionophore A-23187
S. Iophii spores in glycylglycine buffer (0.1 M) with A-23187 (but without polyanionic mucin or polyglutamate) showed a 70% frequency of spore discharge with a pH shift of the extrasporular medium to 9.0; however, the incidence of spore discharge remained near zero if the pH of the medium was kept at 7.0. Polyglutamate or mucin effected a similar 70% frequency of spore discharge; this discharge frequency

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THE JOURNAL OF CELL BIOLOGY • VOLUME 100, 1985 did not increase when A-23187 was added to the medium with polyglutamate or mucin. S. lophii spores pretreated with CaCI2, washed in distilled water, and then incubated in 0.15 mM EGTA would not discharge when placed in DM with A-23187. The calcium channel antagonist verapamil prevented S. lophii spore extrusion in DM at concentrations as low as 20 #M (Fig. 4); this apparent inhibition was reversed with verapamil removal (six washes in distilled water), spore incubation overnight in glycylglycine buffer at pH 7, and the spore immersion in DM (Fig. 4). Lanthanum, a noted Ca 2+ antagonist, prevented S. lophii spore discharge at concentrations of 50 and 100 ~M (Fig. 5); this inhibition did not occur when lanthanum was removed from the extrasporular medium and the spores were transferred to the standard DM (Fig. 5).

Radiochromatography of 45Ca2+ Influx into S. Iophii Spores
Calcium influx into S. lophii spores was monitored by radiochromatography. The A spores (which were not placed in DM and thus remained undischarged) were immersed in cold CaC12 and then transferred to 45CaCl:. These spores showed little 4SCa2+ uptake (Fig. 6, A). The B spores, immersed in EGTA before 45CaC12 incubation, showed slight amounts of 45Ca2+ uptake (Fig. 6, B). The C spores were washed in EGTA, incubated in 45CaC12, and immersed in A-23187 but at a pH of 7 (therefore, the spores remained undischarged). The C spores showed a slight amount of 45Ca2+ uptake (Fig.  6, C). The D spores were washed in EGTA, incubated in 45CaC12 and transferred to A-23187 in DM at pH 9. These spores displayed a high level of discharge and showed a significant increase in the 45Ca2÷ uptake (Fig. 6, D). The E spores were washed in EGTA, incubated in 45CAC12, and induced to discharge with mucin in the extrasporular medium with a pH of 9. The level of incorporation of 45Ca2+ was similar to that observed for the D spores (Fig. 6, E).

Calmodulin Inhibitors, Anti-Calmodulin, and S. Iophii Spore Discharge
The calmodulin antagonists, TFP and chlorpromazine, were used to determine whether extrusion apparatus activa-    Figs. 7 and 8). The phenothiazine-treated spores regained the capacity to discharge after the drug was removed from the extrasporular medium (by thorough washing in distilled water) and the spores were resuspended in fresh DM. However, incubations of the spores in 80 #M chlorpromazine had an obvious permanent negative effect on spore discharge; these spores showed little capacity to regain a discharge capability when the spores were washed and placed in DM (Fig. 8) .¢: Radiochromatogram scan of 4SCaCI2 uptake into S. /ophii spores. The A spores were exposed to CaCIz, washed in deionized distilled water, and incubated in 4SCaCI2; the B spores were treated with EGTA, washed, and incubated in 4SCaCI2; the C spores were treated with EGTA, washed, and incubated in 4SCaCI2 with A-23187 at pH 7; the D spores were pretreated with EGTA, washed, incubated in 4SCaCI2 with glycylglycine buffer with A-23187 at pH 9; and the E spores were pretreated with EGTA, washed, and incubated in 4SCaCI2 with glycylglycine buffer with 0.2% mucin at pH 9. Counts of final wash supernatant for each sample are depicted in a-e. Results report ISD as compared with control samples without chlorpromazine (+). i-I, results of reversibility tests of chlorpromazineinhibited spores washed and transferred to fresh discharge medium without chlorpromazine. Note that spores in 80/~M chlorpromazine did not respond well to reversibility test. the calmodulin within cryostat-sectioned spores. Presumptive anti-calmodulin peroxidase conjugates were mostly found near the membranes of the spore extrusion apparatus (Fig. 9,  a and b). Control spores exposed to peroxidase but without anti-calmodulin showed no reaction (Fig. 9 c).

DISCUSSION
The results indicate that three extrasporular conditions are required to activate S. lophii spore invasion tube discharge: (a) a pH shift from acid/neutral to the alkaline; (b) the presence of calcium bound to the spore wall/plasma membrane; and (c) the presence of polyanions (such as polyglutamate or mucin). The following evidence indicates that a calcium influx is a primary signal for S. lophii spore discharge.
First, the calcium chelator EGTA blocked spore discharge; the CTC fluorescence studies indicate that the EGTA removed the bound Ca 2÷ from the spore wall/plasma membrane. The work of Wolniak et al. indicates that Ca-CTC fluorescence is clearly distinctive when associated with hydrophobic regions (13). Secondly, A-23187 induced a C a 2+ influx immediately before spore discharge. The following evidence supports Hagiwara and Byerly's criteria for possible calcium channels in S. lophii plasma membranes: (a) The organic channel antagonist verapamil strongly blocked S. lophii spore discharge at concentrations typical for inhibiting calcium channels (2,5); as expected, the verapamil-treated spores could effectively activate and discharge after the removal of the verapamil. (b) The inorganic channel antagonist lanthanum blocked S. lophii spore discharge. The lanthanumblocked spores still could be activated and discharged after removal of the lanthanum from the extrasporular medium.
(c) Finally, the phenothiazine inhibitors calmodulin, chlorpromazine, and TFP also blocked spore discharge. These antagonists have a strong affinity for calmodulin (3). The interference caused by the phenothiazines may be due to their hydrophobic affinities; however, it is unclear whether the blocking action is due to a binding to the specific ports in the membrane, or whether the molecules complex directly with the hydrophobic regions on the calmodulin molecule (3,6). The anti-calmodulin peroxidase conjugates seem to indicate that calmodulin is in the vicinity of the spore extrusion apparatus membrane. The evidence indicates a good probability for a calcium-calmodulin mediated system involved in the initiation of spore discharge.
Polyanions are likely involved in the activation of the calcium influx into S. lophii spores. The sialic acid residues on the mucin molecule may be partly responsible for effecting spore activation since other anionic groups (such as polyglutamate) also affect the triggering of the calcium influx. Indeed, substrate-associated SiO3-groups on the surfaces of glass slides could activate S. lophii spores charged with calcium and shifted to an alkaline pH; this activity was reduced to zero when the slide surface was covered with polylysine. Probably mucin serves as a natural stimulator for S. lophii spore discharge along the mucosal lining of the host animal intes-tine. The mucin may create an ion exchange phenomenon during the shift of the extrasporular medium from acid/ neutral to alkaline pH. Such a phenomenon fits the Spray et al. model for the triggering of the opening of calcium channels in plasma membranes (8). This model indicates that under low free hydrogen conditions (alkaline), calcium channels are open; however, a shift to a free hydrogen medium (slightly acid) closes the calcium channels. Perhaps the polyanions in the extrasporular environs create a buildup in the ion potential so that when a pH shift to the alkaline occurs, a sudden influx of calcium with the opening of the channels is permitted. Examples of cationic influxes are believed to be common phenomena along the mucosal surface of the intestine (7).