Synaptotagmin I increases the probability of vesicle fusion at low [Ca2+] in pituitary cells

M. Kreft, V. Kuster, S. Grilc, M. Rupnik, I. Milisav, and R. Zorec

Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Medical Faculty, 1000 Ljubljana; and Celica Biomedical Science Center, 1000 Ljubljana, Slovenia


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Synaptotagmin I (Syt I), a low-affinity Ca2+-binding protein, is thought to serve as the Ca2+ sensor in the release of neurotransmitter. However, functional studies on the calyx of Held synapse revealed that the rapid release of neurotransmitter requires only approximately micromolar [Ca2+], suggesting that Syt I may play a more complex role in determining the high-affinity Ca2+ dependence of exocytosis. Here we tested this hypothesis by studying pituitary cells, which possess high- and low-affinity Ca2+-dependent exocytic pathways and express Syt I. Using patch-clamp capacitance measurements to monitor secretion and the acute antisense deletion of Syt I from differentiated cells, we have shown that the rapid and the most Ca2+-sensitive pathway of exocytosis in rat melanotrophs requires Syt I. Furthermore, stimulation of the Ca2+-dependent exocytosis by cytosol dialysis with solutions containing 1 µM [Ca2+] was completely abolished in the absence of Syt I. Similar results were obtained by the preinjection of antibodies against the CAPS (Ca2+-dependent activator protein for secretion) protein. These results indicate that synaptotagmin I and CAPS proteins increase the probability of vesicle fusion at low cytosolic [Ca2+].

rat melanotrophs; exocytic module; membrane capacitance; calcium sensor


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SYNAPTOTAGMINS constitute a family of vesicle membrane proteins (35) that play a role in various aspects of vesicular trafficking, such as endocytosis (19, 58), but most notably in regulated exocytosis. Genetic studies in Drosophila, Caenorhabditis elegans, and mice have implicated synaptotagmin (Syt) I as an essential component of rapid Ca2+-dependent neurotransmitter release (11, 14, 15, 24, 25, 34). Biochemical properties of Syt I, a low-affinity Ca2+-binding protein (10), are consistent with the view that Syt I may serve as a major Ca2+ sensor for exocytosis (5, 10, 14, 15, 23, 24). However, physiological studies of the Ca2+ requirement in transmitter release from the calyx of Held revealed that the apparent Ca2+ affinity of the exocytic apparatus is much higher than that of Syt I (4, 42). This may indicate that Syt I is not the only Ca2+ sensor but may play a part in a more complex mechanism of high-affinity Ca2+ sensing, for example, by interacting with other molecular partners (14, 23, 45). If this is the case, then in the absence of Syt I it should be possible to record a secretory response devoid of the high-affinity Ca2+-dependent exocytosis. Moreover, one needs to show that the presence of Syt I is required to elicit secretory activity even at very low [Ca2+], at which binding to Syt I in vitro appears to be negligible (10).

An ideal approach to study such a hypothesis would be to use a secretory cell type in which multiple Ca2+-dependent pathways of exocytosis are present, such as the pituitary melanotrophs, which secrete pro-opiomelanocortin-derived peptides via dense-core vesicle exocytosis (27). Previous work has shown that melanotrophs possess a high- and a low-affinity Ca2+-dependent exocytic pathway (38). Here we used an acute strategy to block the expression of Syt I by transfection of an antisense construct into differentiated neuroendocrine cells to study the impact of selective deletion of Syt I on the Ca2+-triggered vesicle exocytosis. Secretory activity was monitored by membrane capacitance (Cm) measurements (31) combined with flash photolysis to deliver rapid and spatially homogeneous steps in cytosolic [Ca2+] ([Ca2+]i), (32), which elicit multiple kinetic components in secretory activity (17, 20, 32, 33, 38, 46, 49, 50, 57). We report that the rapid but not the slow component of exocytosis (38) is blocked in the absence of Syt I, indicating a role of Syt I in increasing the probability of vesicle fusion at low [Ca2+]i. Furthermore, secretory activity stimulated by dialysis of pipette solutions containing 1 µM [Ca2+] was abolished in Syt I-depleted cells and by preinjection of the CAPS (Ca2+-dependent activator protein for secretion) antibody. These data show directly that Syt I and CAPS increase the probability of vesicle fusion at low [Ca2+]i in pituitary cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Isolation of mRNA, cell culture, tissue preparation, and RT-PCR. To isolate mRNAs of Syt isoforms, cell cultures or tissue of Wistar rats (200-300 g, male) were used. Tissue mRNA from hippocampus, cerebellum (one hemisphere), and spinal cord (thoracic region) were prepared from homogenized samples (~0.1 g). Cell cultures from the pars intermedia and anterior lobe were prepared as described (39). Cells (~107) were washed in Earl's balanced salt solution and sedimented before RNA extraction. The QuickPrep micro mRNA purification kit (Pharmacia Biotech) was used to isolate polyadenylated RNA. For the synthesis of first-strand cDNA, 6-12 ng of poly(A) mRNA were incubated for 10 min at 25°C and then for 60 min at 42°C in the presence of 5 µM random hexamers (Pharmacia Biotech), 500 µM mixed deoxynucleotide triphosphates (Pharmacia Biotech), 3 U/µl RNase inhibitor (RNasin; Promega), 10 mM dithiothreitol (Sigma), 10 U/µl reverse transcriptase (SuperScript II; GIBCO), and 1× First Strand buffer (GIBCO) in a reaction volume of 10 µl. PCR was carried out in Eppendorf Mastercycler Gradient (Eppendorf, Hamburg, Germany) thermal cyclers. The 100-µl amplification mixture consisted of 1 µl of the sample from the RT, 2 mM MgCl2, 1× GeneAmp PCR buffer II and 2.5 U AmpliTaq Gold polymerase (Perkin Elmer), 200 µM mixed deoxynucleotides (Pharmacia Biotech), and 2 µM primers (Table 1). A 9-min pre-PCR heat step at 95°C was performed. PCR cycling times and temperatures were as follows: 1 min at 94°C, 1 min at 57.5°C, and 1 min at 72°C; 42-48 cycles were used. The final elongation step was at 72°C for 5 min. The RT-PCR products were electrophoresed on ethidium bromide-stained (1.4%) agarose gels.

                              
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Table 1.   Primers used to amplify Syt reverse transcribed DNA (Syt I-VII)

Introduction of oligonucleotides and plasmid DNA into mammalian cells. An antisense Syt I oligonucleotide (5'-TCA-GGA-TGA-CTG-GCA-CTC-ACC-3') and a sense Syt I oligonucleotide (5'-GGT-GAG-TGC-CAG-TCA-TCC-TGA-3') were introduced into 1- to 2-day-old cells with the plasmid DNA encoding EGFP (pEGFP-C1; Clontech) by cationic lipids (Lipofectamine Plus reagent; Life Technologies), according to the manufacturer's instructions. We used 0.5 µg of EGFP plasmid and 0.5 µg of oligonucleotides dissolved in 100 µl of culture medium without serum (transfection medium). To this solution, we added 6 µl of Plus reagent and incubated for 15 min at room temperature. Lipofectamine reagent (4 µl) was then added to the transfection medium and incubated for another 15 min. This mixture was then added to the cells for 3-5 h at 37°C, after which the procedure was stopped by the addition of 30 µl of serum. After 24 h, cells were observed under the microscope and patch-clamp examined or prepared for immunocytochemistry. These conditions yielded 2-6% of cells (around 60 cells per coverslip) fluorescing in green. Immunocytochemistry using an anti-Syt I antibody was used to verify that the transfection of cells with antisense oligonucleotides affected the level of Syt I protein levels in a cell. When the antisense oligonucleotide was used together with the EGFP plasmid, the level of anti-Syt I signal was significantly reduced in all green-fluorescing cells (see Fig. 2), indicating the inhibition of expression of Syt I in these cells by the antisense procedure.

Immunocytochemistry and confocal microscopy. Melanotrophs were washed with PBS and then fixed for 15 min in 4% paraformaldehyde in PBS. For the following 10 min, cells were kept in fixative containing 0.1% Triton X-100 and then washed four times with PBS. Nonspecific staining was reduced by incubation in 3% bovine serum albumin (BSA) and 10% normal goat serum in PBS. Cells were then incubated with anti-Syt I primary antibodies for 2 h at 37°C (21, 46), diluted 1:2,000 in PBS containing 3% BSA. Cells were then washed and incubated in PBS containing Cy3-labeled anti-mouse secondary antibodies (Molecular Probes, Leiden, The Netherlands) and 3% BSA for 45 min, washed, and mounted in Light Antifade (Molecular Probes). Stained cells were observed by the confocal microscope (Zeiss LSM 510; oil-immersion lens ×63, NA 1.4). EGFP was excited with the argon laser (488 nm) and Cy3 with the helium-neon laser (543 nm). The EGFP emission signal was filtered at 505-530 nm, and the Cy3 emission signal was long-pass filtered at 560 nm.

Electrophysiology, flash photolysis, and [Ca 2+]i measurements. A SWAM IIB patch-clamp/lock-in amplifier (Celica, Ljubljana, Slovenia) was used for Cm measurements (31, 59). Uncompensated Cm measurements were used to monitor slow changes in Cm, as described previously (39), by applying a sine wave voltage [1 mV (RMS), 1.6 kHz] to the holding potential. Direct current (DC), holding potential, and real and imaginary admittance signals (low pass 10 Hz, -3 dB, 6-pole Bessel) were digitized by a CED 1381 analog-to-digital converter (Cambridge, UK) and analyzed with the CAP3 software (J. Dempster, Univ. of Strathclyde, Strathclyde, UK), which computed Cm, access (Ga), and membrane conductance (Gm). The recording bath solution consisted of (in mM) 131.8 NaCl, 1.8 CaCl2, 5 KCl, 2 MgCl2, 10 HEPES/NaOH, 10 D-glucose, 0.5 NaH2PO4 · 2H2O, and 5 NaHCO3 (pH 7.2). The pipette solution consisted of (in mM) 150 KCl, 2 MgCl2, 10 HEPES/KOH, 0.65 EGTA, 4.35 Ca2+-saturated EGTA ([Ca2+]i = 1 µM), 2 Na2ATP (pH 7.2). [Ca2+]i was calculated by assuming an apparent dissociation constant (Kd) for the Ca-EGTA complex of 150 nM (16) and assuming that the cytosol equilibrates with the pipette solution upon the establishment of the whole cell recording. Total EGTA concentration was 5 mM, which exceeds the buffering capacity of melanotrophs (48). Cells were voltage clamped at a holding potential of -70 mV. The reversal potential of the whole cell current was -50 mV and did not change during recordings. Fast changes in Cm were measured by using the compensated method [111 mV (RMS), 1.6 kHz] (38). Upon establishment of the whole cell configuration, Cm and Ga were compensated by Cslow and Ga controls. The phase angle setting was determined by a 1-pF pulse and by monitoring the projection of the pulse from the C (proportional to Cm) to the G output of the lock-in amplifier. These two signals were stored unfiltered (C-DAT4; Cygnus) for off-line analysis. Simultaneously, we recorded filtered (300 Hz, 4-pole Bessel) C and G signals, the fluorescence intensity from a C660 photon counter (Thorn EMI), and DC (0-10 Hz, low pass). The PhoCal program (LSR, Cambridge, UK) was used to acquire signals every 5 ms. For high temporal resolution measurements of Cm, the records on DAT were played back and a 10-s epoch of the signal enveloping each flash was digitized at 50 kHz using the CDR program (J. Dempster). Signals were digitally filtered (1 kHz, 2-way 150th-order FIR filter; MATLAB) and resampled at 10 kHz. The pipette solution contained (in mM) 110 KCl, 10 TEA-Cl, 38 KOH/HEPES, 2 Na2ATP, 2 MgCl2, 4 K4-nitrophenyl-EGTA, 3.6 CaCl2, and 0.5 furaptra (pH 7.2). The bath contained (in mM) 131.8 NaCl, 1.8 CaCl2, 5 KCl, 2 MgCl2, 10 HEPES/NaOH, 10 D-glucose, 0.5 NaH2PO4 · 2H2O, and 5 NaHCO3 (pH 7.2). All salts were obtained from Sigma. Recordings were made at room temperature with pipette resistance between 1 and 4 MOmega (in KCl-rich solution), giving Ga of >80 nS. The pipette and bath solutions were of similar osmolarity (within 5%) measured by freezing point depression (Camlab, Cambridge, UK). We used nitrophenyl-EGTA (Molecular Probes, Eugene, OR) to manipulate [Ca2+ ]i (13). A UV flash from a xenon arc flash lamp (38) was delivered to cells through a ×38 fluor oil-immersion objective of a Nikon Diaphot microscope. The same optical pathway as in flash photolysis was used to illuminate the fluorescent [Ca2+ ]i indicator furaptra (Molecular Probes) as described previously (7, 38).

Microinjection. Transjector 4657 with a micromanipulator (Eppendorf, Germany) was used. Pulses (8-12 hPa, 0.3-1 s) were applied with a compensation pressure (1 hPa). Microinjection solution consisted of (in mM) 150 K-gluconate, 2 MgCl2, 10 HEPES, 6 mg/ml RITC, and 2-20 mg/ml antibody. The anti-CAPS antibody was affinity purified, and the control antibodies (preimmune antibody) were purified by using protein A-Sepharose, to prepare immunoglobulin fractions (29, 55). Pipettes were prepared with a horizontal puller (P-87; Sutter Instruments, city, CA). Microinjected cells were identified with coinjection of rhodamine-labeled dextran (25 kDa; Sigma). Injection of dextran marker alone did not affect secretory responses per se. Botulinum neurotoxin A, B, or E (a gift from Dr. Das Gupta, Madison, WI) was microinjected into cells (20 ng/µl) together with the dextran marker. Before the patch-clamp experiments were continued, the cells were allowed to rest for 1-2 h at 37°C.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Syt I, III, IV, V, and VII are expressed in rat melanotrophs. Previous work indicated that Syt I transcript is present in the rat pituitary (28), but it is not clear which of the other Syt isoforms are expressed. We used RT-PCR to examine the presence of Syt transcripts (I-VII) in the pituitary pars intermedia and anterior lobe, hippocampus, cerebellum, and spinal cord. Poly(A) mRNA was isolated from the tissues, and then cDNAs for Syt isoforms were amplified by specific primer sets (Table 1). Figure 1 depicts the expression analyses of the Syt transcripts from the pituitary pars intermedia. Contamination of cDNA with genomic DNA was tested by performing parallel amplifications on mRNA; contamination from extraneous sources was checked by replacing cDNA templates with water (not shown). Two to three separately isolated mRNA samples were analyzed for the presence of Syt isoforms, and the fragments obtained by RT-PCR were verified by nucleotide sequencing. Figure 1 shows that the transcripts for Syt I, III (a/b; see Table 1), IV, V (IX; see Ref. 44), and VII are present in the pituitary pars intermedia and all other tissues tested (not shown). Syt II was present in cerebellum and spinal cord, and Syt VI was present in cerebellum, spinal cord, and the anterior lobe of pituitary (not shown). In conclusion, the Syt isoforms I, III, IV, V, and VII are expressed in the pituitary pars intermedia.


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Fig. 1.   PCR analysis of synaptotagmins (Syt) in the pituitary. Agarose gel electrophoresis of PCR products were obtained by RT-PCR of poly(A) RNA from melanotrophs of the rat pars intermedia for Syt isoforms (Syt I-VII). PCR product for Syt III was obtained with the primers yielding an expected product size of 263 bp (see Table 1). A 100-bp DNA ladder (GIBCO) was used as a molecular size marker (M).

Antisense strategy to block the expression of Syt I. To block the expression of Syt I from differentiated melanotrophs, we introduced an antisense oligonucleotide complementary to the sequence at the 5'-end of Syt I. The transfected cells were labeled by cotransfection of the antisense or sense oligonucleotides with the expression plasmid for EGFP. The content of Syt I was checked by immunocytochemistry, using monoclonal antibodies against Syt I with a Cy3-labeled secondary antibodies.

When the sense probe and EGFP plasmid were introduced into cells, the successfully transfected cells were fluorescing green and were immunocytochemically positive for Syt I (Fig. 2A). The detected amounts of Syt I were comparable in transfected and untransfected cells. Transfected cells had therefore retained Syt I in the presence of EGFP. In contrast, when the cells were transfected with the antisense probe alongside the EGFP plasmid, the green-fluorescing cells contained greatly reduced amounts of Syt I, because the signal reporting the presence of Syt I was faint or negative (Fig. 2B). This finding demonstrates a significant reduction of the Syt I levels in the antisense-treated cells.


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Fig. 2.   Confocal images of melanotrophs treated with sense and antisense Syt I probes. Confocal images show melanotrophs transfected with sense (A) or antisense (B) Syt I probes together with EGFP plasmid and stained by the anti-Syt I monoclonal antibody labeled with Cy3 (red). Left: fluorescence images excited by both excitation lights (488 and 543 nm); middle: images excited by 543-nm light; right: images excited by 488-nm light only. In the presence of the sense Syt I probe (A), the cells are fluorescing in red, because Syt I is expressed in these cells. The antisense probe significantly reduced the Syt I expression (B, middle). Note that these cells, which were not successfully transfected (not green), emit red fluorescence because of the Syt I expression. Bar, 10 µm.

Rapid exocytosis is diminished in the absence of Syt I. With the use of Syt I null mutant mice, it was shown that in chromaffin cells, Syt I expression is required for the rapid exocytosis, whereas the slow exocytosis was not affected (54). If many isoforms of a protein exist in a cell, the deletion of one member of the family may be compensated during embryogenesis by other isoforms in the knockout animals (8, 41). The family of synaptotagmins consists of at least 13 members (43), therefore one has to consider that the physiology of neuroendocrine cells in Syt I null animals may be a result of a complex embryogenetic corrective response induced by the loss of Syt I. To this end, we performed an acute block of the expression of Syt I in differentiated cells. Figure 3 (middle traces) shows the exocytic response of a control cell (sense probe) and of a cell without Syt I (antisense probe) to a flash-induced rise in [Ca2+]i to ~50 µM (top traces). In control cells, the increase in Cm over the first 800 ms consisted of two components, a rapid and a slow sustained rise in Cm, whereas with the antisense probe the rise in Cm appeared to be devoid of the rapid component (Fig. 3, bottom traces). The two kinetic components of Cm increase in control cells represent two populations of vesicles that enter distinct exocytic pathways (36, 38). Figure 4 shows the results of measurements of the amplitude of the Cm increase at two time points. The amplitude A (rapid exocytosis) was measured as the positive peak value in Cm (relative to the Cm value preceding the flash) during the first 100 ms after the flash, whereas the amplitude B of the slow exocytosis was determined as the positive peak value in Cm after the time point of 100 ms. The control cells, pretreated with the sense probe, exhibited a biphasic response in Cm as described previously (38). In these cells, the amplitude A of the rapid component was indistinguishable from responses measured in untreated cells (not shown). In contrast, cells treated with the antisense probe exhibited a significantly reduced amplitude A, indicating the reduction of the rapid component of exocytosis (Fig. 3, bottom; Fig. 4C). The amplitude of the slow component was similar in cells treated with the sense and the antisense probe (Fig. 4B). Although the inspection of Fig. 3 (middle traces) appears to indicate that the treatment of cells by the antisense probe did affect endocytosis measured as the reduction in Cm 20 s after the peak positive value (Fig. 3, amplitude C), statistical analysis shows that this is unlikely because of relatively large variability (Fig. 4A). These results indicate that Syt I is required for the rapid exocytosis in neuroendocrine cells and is not affecting endocytosis, as reported previously (54).


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Fig. 3.   Time-dependent changes in membrane capacitance (Cm) evoked by a UV photolysis-induced rise in cytosolic Ca2+ concentration ([Ca2+]i). Representative records are shown of [Ca2+]i (top) and changes elicited by photolysis of caged Ca2+ compound nitrophenyl-EGTA, Cm (middle), and portions of these latter traces are shown at a shorter time scale (bottom). Data at right represent records obtained on a melanotroph cell that was pretreated by a sense Syt I probe and the EGFP plasmid, whereas data at left were obtained from a cell pretreated with an antisense Syt I probe. Both records were obtained 24-26 h after the transfection procedure. Note that the amplitude of the rapid component of Cm (amplitude A) is reduced in the antisense-treated cell. See Fig. 4 legend for detailed descriptions of amplitudes B and C.



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Fig. 4.   Syt I is required for the rapid component of exocytosis in melanotrophs. Rapid flash-induced component of Cm increase is blocked in the absence of Syt I. A: mean amplitude of Cm reduction due to endocytosis (see Fig. 3, amplitude C). B: mean amplitude of the slow kinetic component of Cm (see Fig. 3, amplitude B). C: mean amplitude of the rapid component of Cm. D: average peak [Ca2+] elicited by UV flash photolysis of Ca2+-loaded NP/EGTA in cells treated with an antisense or a sense Syt I probe.

Syt I and the CAPS protein are required for secretory activity at low [Ca2+]i. The requirement of Syt I in the rapid secretory component (Figs. 3 and 4) may indicate that Syt I increases the probability of vesicle fusion at low [Ca2+]i. Therefore, we stimulated secretory activity by cytosol dialysis with pipette solutions containing 1 µM [Ca2+]. The increase in Cm, after 200 s from the start of recording, was in control cells around 15% relative to the resting Cm (Fig. 5), determined at the start of the whole cell recording. Responses in Cm were completely abolished in antisense probe-treated cells (Fig. 5, A and D). This effect was specific, because responses in Cm were not affected by the transfection of EGFP only (not shown) or by the pretreatment with the sense probe and the EGFP plasmid (Fig. 5D) compared with untreated cells (Fig. 5, C and D). These results support the view that Syt I augments the probability of vesicle fusion at low [Ca2+] in rat melanotrophs. Moreover, cytosol dialysis with solutions containing low [Ca2+] activate a pathway that corresponds to the rapid, Syt I-dependent response recorded after flash photolysis.


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Fig. 5.   Dialysis-evoked secretory activity with 1 µM [Ca2+] is blocked in the absence of Syt I. Time-dependent changes in Cm (Delta Cm) induced by dialysis of cytosol with a solution containing 1 µM Ca2+ are shown in control (A), in a cell pretreated with the sense Syt I probe and the EGFP plasmid (B), and in a cell pretreated with an antisense Syt I probe and the EGFP plasmid (C). Numbers adjacent to traces indicate resting Cm recorded at the time of whole cell recording establishment. D: mean amplitudes of %Delta Cm measured relative to the resting Cm 200 s after the establishment of the whole cell recording in control cells, cells pretreated with the antisense Syt I or sense probe, cells preinjected with a neutralizing CAPS antibody (CAPS immune), cells preinjected with a preimmune serum, and cells preinjected with botulinum neurotoxins A, B, and E (BontA, BontB, BontE) or boiled botulinum neurotoxin E (BontE boiled). Values are means + SE; numbers adjacent to error bars indicate number of cells tested. * Statistical significance (P > 0.01) between samples and controls.

It was shown previously that the rapid secretory component displays a high-affinity Ca2+-dependent exocytosis that requires the CAPS protein (38). To further verify that cytosol dialysis with pipette solutions containing low [Ca2+] stimulates the high-affinity Ca2+-dependent exocytosis, we preinjected cells with a CAPS-neutralizing antibody (47). Figure 5D shows that preinjection of cells with the CAPS-neutralizing antibody completely blocked the response in Cm, whereas injection of the preimmune serum had no effect on Cm compared with noninjected cells (Fig. 5). These results indicate that cytosol dialysis stimulates the high-affinity Ca2+-dependent changes in Cm and that CAPS may play a role similar to that of Syt I in augmenting the probability of vesicular fusion at low [Ca2+]i.

The SNARE [soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor] proteins synaptobrevin/VAMP (vesicle-associated membrane protein), syntaxin, and SNAP-25 are essential constituents for exocytosis in neuroendocrine cells and constitute the molecular targets for the clostridial neurotoxin proteases (6). To determine whether the dialysis-evoked ([Ca2+] = 1 µM) changes in Cm are due to SNARE-dependent vesicular exocytosis, botulinum neurotoxins were injected before patch-clamp determinations. Injection of botulinum neurotoxin B, which cleaves VAMP, completely blocked Ca2+-induced changes in Cm. Similar results were obtained by the injection of botulinum neurotoxin E and A, which cleave SNAP-25 (Fig. 5D). These results show that the high-affinity Ca2+-dependent elevation in Cm represents a SNARE-dependent vesicle fusion.

Slow endocytosis requires Syt I. Syt I binds also to the clathrin assembly protein complex AP-2 and may serve as a receptor (58) or regulator (18) in endocytosis. Although rapid endocytosis after the delivery of a UV flash was not affected by the absence of Syt I (Figs. 3 and 4), we examined whether slow endocytosis (time scale of minutes) may require Syt I. If melanotrophs are dialyzed by practically zero [Ca2+]i, Cm is characterized by a slow, steady decline that is dominated by endocytosis (Fig. 6B) (39, 40). In the antisense-treated cells, the rate of decline in Cm is not negative but becomes positive. The slopes of regression lines in Fig. 6B are significantly different, indicating that endocytosis is blocked in the absence of Syt I. This response is specific, because changes in Cm are not affected in EGFP or in the sense probe- and EGFP-treated cells (Fig. 6C). These results show that some, but not all, endocytic pathways require Syt I. 


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Fig. 6.   Slow endocytosis requires the expression of Syt I. Time-dependent changes in Cm were measured every 100 s and averaged. Values are means ± SE. A: untreated control cells and 7 cells pretreated with the antisense Syt I probe and EGFP plasmid (+ antisense, +EGFP) were dialyzed with a pipette solution containing 1 µM Ca2+ (high Ca2+). Regression lines have the form Cm (%) = 0.069 ± 0.012 (s) for controls and Cm (%) = 0.001 ± 0.004 (s) for the antisense probe-treated cells. Slopes of lines are significantly different (P < 0.01). B: control cells and cells pretreated with the antisense Syt I probe and EGFP were dialyzed with a pipette solution practically free of Ca2+. The lines drawn through the points represent regression lines of the form Cm (%) = -0.016 ± 0.008 (s) for control and Cm (%) = 0.01 ± 0.005 (s) for the antisense-treated cells. The slopes are significantly different (P < 0.01). C: mean amplitude of Cm relative to the resting Cm measured 500 s after the establishment of whole cell recording in control cells dialyzed with a Ca2+-free pipette solution, after lipofection of the EGFP plasmid (+EGFP), and after lipofection of the antisense or sense Syt I probes (+antisense or +sense) with the EGFP plasmid.


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ABSTRACT
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The distribution of Syt isoforms (I-VII) in pituitary pars intermedia (Fig. 1), hippocampus, cerebellum, and spinal cord (not shown) is consistent with previous reports (22, 28, 30, 51, 52). In anterior lobe, the presence of Syt isoforms is similar to that in the pars intermedia except that Syt VI transcript was also detected (not shown). With in situ hybridization, Syt I was detected in abundance in pars intermedia and at a lower level in the anterior lobe, but Syt II was not detected (28), as found in this work (Fig. 1). We detected Syt III in both examined parts of the pituitary, which contrasts with a previous report (28). However, Syt III was detected in the whole pituitary (30). Different techniques used and/or animals of different developmental stages may account for the discrepancies. In addition to Syt I and III, rat pars intermedia also expresses Syt IV, V, and VII. Syt IV was previously reported to be present in the whole pituitary (53). Note that two different Syt sequences have been named Syt V. In our work, the Syt V isoform is that reported by Craxton and Goedert (9), corresponding to Syt IX as listed by Südhof and Rizo (44).

In transgenic mice, the Syt I-insensitive component of secretory response that remained in the absence of Syt I displayed a Ca2+ dependence similar to that of the Syt I-dependent exocytosis (54). Similar Ca2+ dependence of secretion in the Syt I null mutants and in controls may be due to a compensatory mechanism that occurs during embryogenesis, where proteins other than Syt I, such as CAPS (55), may partially correct for the loss of Syt I function (see Ref. 37). Here we used an acute deletion of Syt I to test the hypothesis that Syt I plays a role in determining the high-affinity Ca2+ sensing by the exocytic apparatus. We blocked the expression of a protein by introducing an antisense DNA probe into cells (26). The transfection of EGFP, a marker of lipofection, did not alter the properties of recorded functional parameters (Figs. 4 and 6; see Ref. 3). In the presence of the antisense probe, green-fluorescing cells were devoid of Syt I (Fig. 2). Compared with the control sense probe-treated cells, antisense probe-treated cells revealed that the flash-induced rapid component of Cm was significantly diminished (Figs. 3 and 4). This finding indicates a role of Syt I in the rapid exocytosis of neuroendocrine cells, as reported previously (54). The rapid exocytosis, characterized by a high sensitivity to Ca2+, was also diminished by the CAPS-neutralizing antibodies (38). Thus Syt I and CAPS may increase the probability of vesicle fusion at low [Ca2+]i. Therefore, we stimulated secretory responses by cytosol dialysis with solutions containing low [Ca2+] and found that secretory responses were completely blocked in the antisense-treated cells (Fig. 5). Furthermore, changes in Cm were also completely blocked by the CAPS antibody (1, 47), whereas responses in cells treated with the sense Syt I probe or preinjected with the preimmune serum were not significantly different from the controls (Fig. 5D). These results show that Syt I and CAPS act in the high-affinity Ca2+-dependent exocytosis, probably by increasing the probability of vesicle fusion at low [Ca2+]i.

Syt I was proposed to act as the receptor for AP-2 in endocytosis (58). The decline in Cm following the flash-induced peak in Cm was insensitive to the absence of Syt I (Fig. 4A). In contrast, slow endocytosis that dominates the time course of Cm for several minutes in the cytosol dialysis experiments is sensitive to the absence of Syt I (Fig. 6). These results indicate that certain types of endocytosis in melanotrophs require Syt I, which is consistent with the existence of multiple pathways of endocytosis (2). Furthermore, these results support the proposal that in melanotrophs there are distinct cycles of exo-endocytosis (36). Multiple exo-endocytosis cycles appear to have distinct biochemical characteristics. Exocytosis that is sensitive to Syt I appears to be coupled to Syt I-sensitive endocytosis (Fig. 6). On the other hand, Syt I-insensitive exocytosis is coupled to the Syt I-insensitive endocytosis (Fig. 4). Distinct exo-endocytosis mechanisms appear to cluster into distinct functional modules, termed exocytic modules.

It was proposed that functional roles of multiple Syt isoforms are based on their in vitro biochemical properties (44, 45), grouped into three classes. Class A, represented by Syt I and II, requires high [Ca2+] for syntaxin binding. Class B, represented by Syt III and VII, requires low [Ca2+] for syntaxin binding. Class C includes synaptotagmins that do not bind syntaxin as a function of [Ca2+] [Syt IV, V (IX), and VI]. Although all of these classes are expressed in melanotrophs (Fig. 1), our results on single cells show that Syt I may play a role in the high-affinity Ca2+-dependent exocytosis, whereas the low-affinity Ca2+-dependent mechanism of secretion appears to be Syt I insensitive (Fig. 3). We propose that Syt I plays a role in Ca2+ sensing by increasing the probability of vesicle fusion at low [Ca2+]i. CAPS may function in a similar way, because functional neutralization of this protein results in the attenuation of the rapid, high-affinity Ca2+-dependent exocytosis in neuroendocrine cells (38) (see also Fig. 5D). Moreover, the genetic deletion of CAPS homolog resulted in a reduced Ca2+ sensitivity of the exocytic apparatus in both Drosophila neurons (37) and neuroendocrine cells (38). Finally, CAPS and Syt I both appear to affect the stability of the fusion pore dwell time (12, 56). We propose that CAPS and the low-affinity Ca2+-binding protein Syt I are essential elements of the exocytic apparatus and act by augmenting the probability of vesicle fusion at low [Ca2+]i. This mechanism may involve interactions with other molecular partners in the exocytic module.


    ACKNOWLEDGEMENTS

We thank Dr. M. Seagar (Univ. de la Méditerranée, Marseille, France) for the antibody against Syt I and Dr. T. F. J. Martin (Univ. of Wisconsin, Madison, WI) for the antibody against CAPS.


    FOOTNOTES

This work was supported by the Ministry of Education, Sciences, and Sports of the R. Slovenia Fund P3 521 0381.

Present address of M. Rupnik: European Neuroscience Institute, 37073 Göttingen, Germany.

Address for reprint requests and other correspondence: R. Zorec, Medical Faculty, Institute of Pathophysiology, Zaloska 4, 1000 Ljubljana, Slovenia (E-mail: Robert.Zorec{at}mf.uni-lj.si).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published October 16, 2002;10.1152/ajpcell.00333.2002

Received 17 July 2002; accepted in final form 15 October 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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