Latrotoxin Stimulates Secretion in Permeabilized Cells by Regulating an Intracellular Ca2+- and ATP-dependent Event

α-Latrotoxin, a component of black widow spider venom, stimulates transmitter release from nerve terminals and intact chromaffin cells and enhances secretion from permeabilized chromaffin cells already maximally stimulated by Ca2+. In this study we demonstrate that chromaffin cells contain a protein antigenically similar to the cloned Ca2+-independent receptor for α-latrotoxin. Although this receptor has homology to the secretin family of G-protein-linked receptors, pertussis toxin has no effect on the ability of α-latrotoxin to enhance secretion, suggesting that neither Gi nor Go is involved in the response. Furthermore, in the absence of Ca2+, α-latrotoxin does not stimulate polyphosphoinositide-specific phospholipase C. α-Latrotoxin specifically enhances ATP-dependent secretion in permeabilized cells. An in situ assay for protein kinase C reveals that α-latrotoxin augments the activation of protein kinase C by Ca2+, and use of protein kinase inhibitors demonstrates that this activation is important for the toxin's enhancing effect. This enhancement of secretion requires Ca2+ concentrations above 3 μm and is not supported by Ba2+ or nonhydrolyzable guanine nucleotides, which do not stimulate protein kinase C. We conclude that α-latrotoxin stimulates secretion in permeabilized cells by regulating a Ca2+- and ATP-dependent event involving protein kinase C.

␣-Latrotoxin, a component of black widow spider venom, stimulates transmitter release from nerve terminals and intact chromaffin cells and enhances secretion from permeabilized chromaffin cells already maximally stimulated by Ca 2؉ . In this study we demonstrate that chromaffin cells contain a protein antigenically similar to the cloned Ca 2؉ -independent receptor for ␣-latrotoxin. Although this receptor has homology to the secretin family of G-protein-linked receptors, pertussis toxin has no effect on the ability of ␣-latrotoxin to enhance secretion, suggesting that neither G i nor G o is involved in the response. Furthermore, in the absence of Ca 2؉ , ␣-latrotoxin does not stimulate polyphosphoinositidespecific phospholipase C. ␣-Latrotoxin specifically enhances ATP-dependent secretion in permeabilized cells. An in situ assay for protein kinase C reveals that ␣-latrotoxin augments the activation of protein kinase C by Ca 2؉ , and use of protein kinase inhibitors demonstrates that this activation is important for the toxin's enhancing effect. This enhancement of secretion requires Ca 2؉ concentrations above 3 M and is not supported by Ba 2؉ or nonhydrolyzable guanine nucleotides, which do not stimulate protein kinase C. We conclude that ␣-latrotoxin stimulates secretion in permeabilized cells by regulating a Ca 2؉ -and ATP-dependent event involving protein kinase C.
␣-Latrotoxin (␣-Ltx), 1 a component of black widow spider venom, is a potent stimulus for transmitter release from nerve terminals (1)(2)(3)(4) and intact chromaffin cells (5,6). The toxin binds to cell surface proteins in both a Ca 2ϩ -dependent and Ca 2ϩ -independent manner (7), with distinct proteins responsible for each type of binding. The first ␣-Ltx-binding protein to be cloned, neurexin I␣, interacts with ␣-Ltx in the presence of Ca 2ϩ (8 -10). Synaptosomes prepared from mice that lack the gene for neurexin I␣ do not exhibit Ca 2ϩ -dependent ␣-Ltx binding and secrete less neurotransmitter in response to ␣-Ltx than synaptosomes from control mice (11).
More recently, a Ca 2ϩ -independent receptor for ␣-Ltx, termed CIRL (6) or latrophilin (12,13), has been cloned from rat and bovine brain. The protein sequence of CIRL shows significant homology to the secretin family of G-protein-linked receptors (6). Expression of this receptor in chromaffin cells (6,14) or HIT-T15 cells (13) increases the sensitivity of the cells to ␣-Ltx, demonstrating that the protein is functional in coupling to Ca 2ϩ influx and secretion. Experiments with mutant receptors truncated at the COOH terminus indicate that the intracellular domains of both neurexins and CIRL are unnecessary in mediating ␣-Ltx binding and subsequent neurotransmitter release in intact cells (15)(16)(17). This is consistent with the finding that neurexin 1␣ and full-length and truncated CIRL all facilitate ␣-Ltx-induced channel formation (18). Ca 2ϩ influx through the toxin-induced pore is sufficient to support secretion, even in the absence of receptor signaling.
Although the effects of ␣-Ltx in intact cells appear not to require receptor signaling, we have identified a second receptor-mediated effect of ␣-Ltx which cannot be attributed to changes in Ca 2ϩ permeability of the plasma membrane. Besides stimulating transmitter release in intact cells, ␣-Ltx enhances Ca 2ϩ -dependent secretion in digitonin-permeabilized cells (14,19), which are freely accessible to Ca 2ϩ in the medium. The enhancement occurs when permeabilized cells are already maximally stimulated by Ca 2ϩ , indicating a function for ␣-Ltx at a step after Ca 2ϩ entry.
In this study we investigated the effects of ␣-Ltx in chromaffin cells in more detail. We report a major new finding: ␣-Ltx stimulates secretion in permeabilized cells by regulating a Ca 2ϩ -and ATP-dependent event that is mediated by the action of protein kinase C (PKC). These experiments provide the first indications of a signaling pathway for ␣-Ltx distinct from its ability to induce a Ca 2ϩ permeability in intact cells.

EXPERIMENTAL PROCEDURES
Cell Culture and Transfection-Bovine adrenal chromaffin cells were prepared and maintained in culture as described previously (20), except that in some experiments the medium used was Dulbecco's modified Eagle's medium/Ham's F-12 rather than Eagle's minimal essential medium. HEK293 cells were plated at a density of 2.4 ϫ 10 5 cells/well in 24-well plates and transfected with 0.25 ml of serum-and antibioticfree Dulbecco's modified Eagle's medium/well containing 2 l of Lipo-fectAMINE and 0.55 g of DNA. Medium containing 10% serum was added after 5 h and replaced by complete medium containing antibiotics and 10 M cytosine arabinoside after 24 h. Cells were harvested after an additional 24 h. The plasmid encoding CIRL (pCDR7) has been described previously (6).
Assays-Experiments were conducted with several ␣-Ltx preparations from two sources whose apparent optimal concentrations to enhance secretion in permeabilized cells were 50 pM, 0.4 nM, and 10 nM. Although the potencies of these toxin batches differed, experiments done with these preparations yielded comparable results. Physiological salt solution (PSS) contained 145 mM NaCl, 5.6 mM KCl, 5.6 mM glucose, 0.5 mM ascorbate, 15  Inositol 1,4,5-trisphosphate (IP 3 ) production was measured using a radioreceptor binding assay kit from NEN Life Science Products. PKC was measured in situ in permeabilized cells as described previously (21) using a specific peptide substrate (VRKRTLRRL) based on the epidermal growth factor receptor.
Calcium Measurements-Measurements of intracellular free Ca 2ϩ were made using fura-2 and dual-wavelength microspectrofluorometry. Cells were loaded with fura-2 by incubation in PSS containing 1 M fura-2 acetoxymethyl ester (fura-2/AM) in dimethyl sulfoxide (0.1% final concentration) for 30 min at 37°C, followed by a 20-min wash in PSS without fura-2. The standard extracellular solution (PSS) contained 145 mM NaCl, 5 mM KCl, 10 mM glucose, 10 mM HEPES (pH 7.3), 2 mM CaCl 2 , and 1 mM MgCl 2 . Ca 2ϩ -free PSS for these experiments was prepared by omitting CaCl 2 and including 1 mM EGTA. Drugs were dissolved in Ca 2ϩ -free PSS and applied by pressure injection from fused-silica tubing (Poly Micro Technologies, Phoenix, AZ) positioned immediately adjacent to the cell being recorded. Fura-2 emission signals at alternating excitation wavelengths of 340 and 380 nm were monitored at 500 nm using a photomultiplier-based SPEX Industries AR-CM system (Edison, NJ).
Materials-Reagents were received from the following sources. ␣-Ltx was from V. Krasnoperov

The Enhancement of Secretion by ␣-Ltx in Permeabilized Chromaffin Cells Is Mediated by an Extracellular Protein
Receptor-We have reported previously that ␣-Ltx enhances secretion from permeabilized chromaffin cells in a dose-dependent manner (14). Because the effect of ␣-Ltx on permeabilized cells was readily observed after a brief incubation of intact cells with the toxin, toxin binding to an extracellular receptor rather than toxin entry seemed likely to mediate the enhancement of secretion. We confirmed the importance of the extracellular receptor in mediating the effects of ␣-Ltx in permeabilized cells by using two inhibitors of ␣-Ltx binding, trypsin and concanavalin A.
Binding of ␣-Ltx to its receptors can be prevented by incubating tissues or membrane preparations with trypsin prior to exposure to ␣-Ltx (22). If the effects of ␣-Ltx in permeabilized chromaffin cells are mediated by receptors similar to those of the neuromuscular junction or in PC-12 cells, one would predict that trypsin should prevent the enhancement of secretion by the toxin. As expected, prior treatment of cells with trypsin specifically inhibited secretion stimulated by ␣-Ltx when Ltx was added before permeabilization (Fig. 1A). Incubation of cell monolayers with the indicated concentrations of trypsin for 10 min prior to incubation with ␣-Ltx, followed by permeabilization and stimulation by Ca 2ϩ , caused a dose-dependent decrease in the effects of ␣-Ltx on Ca 2ϩ -dependent secretion (Fig.  1A). The enhancement of secretion by ␣-Ltx was completely inhibited by a concentration of trypsin (500 g/ml), which had no effect on secretion in the absence of ␣-Ltx.
In the experiment in Fig. 1A, cells were exposed to ␣-Ltx before permeabilization. It is possible that the function of the plasma membrane receptor is to allow the internalization of the toxin so that the toxin may interact with an intracellular target. In that case, adding ␣-Ltx to permeabilized cells should bypass the need for the extracellular receptor and restore to cells treated with trypsin the ability to respond to ␣-Ltx. Fig.  1B demonstrates that adding ␣-Ltx after the trypsinized cells were permeabilized did not restore the ␣-Ltx effect.
The lectin concanavalin A has also been reported to prevent binding of ␣-Ltx to membranes (22,23). Indeed, a recently cloned Ca 2ϩ -independent receptor for ␣-Ltx (6) is extensively glycosylated (24,25). Preincubating intact chromaffin cells with as little as a 100 nM concentration of the lectin abolished secretion stimulated by 150 pM ␣-Ltx (not shown). We asked whether a prior incubation of cells with lectin could prevent the effects of ␣-Ltx in permeabilized chromaffin cells when ␣-Ltx was added before (Fig. 1C) or after (Fig. 1D) permeabilization. 250 nM concanavalin A completely prevented the enhancement of secretion by ␣-Ltx in permeabilized cells (Fig. 1C). Again, adding ␣-Ltx to cells after permeabilization did not restore the enhancing effect of the toxin (Fig. 1D). The data indicate that a functional extracellular receptor is required to mediate the enhancement by ␣-Ltx of secretion in permeabilized cells.

FIG. 1. Preincubation of intact cells with trypsin (panels A and B) or concanavalin A (panels C and D) blocks subsequent effects of ␣-Ltx on secretion from permeabilized chromaffin cells. Chromaffin cells were labeled with [ 3 H]NE, rinsed, and incubated with or without trypsin (panel
A receptor that binds ␣-Ltx in the absence of Ca 2ϩ , termed CIRL or latrophilin, has recently been cloned from rat (6) and bovine (12) brain. The receptor is comprised of two subunits: an extracellular domain containing the ␣-Ltx binding site (p120), and a membrane-spanning and intracellular domain (p85). An antibody generated against a peptide from the intracellular COOH terminus of p85 (CEGPGPDGDGQMQLVTSL) was used to probe a blot of chromaffin cell proteins (Fig. 2). The antibody recognized a protein in bovine brain synaptosomes (lane 1), bovine chromaffin cells (lane 2), and HEK293 cells transiently transfected with a plasmid for CIRL (lane 4). Lanes 3 and 5 contained chromaffin and transfected HEK293 cells, respectively, probed with antibody that had been preblocked with peptide, demonstrating the specificity of the antibody binding. Thus, chromaffin cells possess a protein of slightly lesser mobility than the brain isoform of CIRL, which is also recognized specifically by an antibody to the COOH terminus of CIRL.
Lack of Effect of G Protein Reagents-The cloned receptor for ␣-Ltx (6) is predicted to have seven membrane-spanning domains homologous to membrane-spanning domains of the secretin receptor family (27), suggesting that CIRL may be a G-protein-linked receptor. ADP-ribosylation of G ␣i or G ␣o by pertussis toxin effectively uncouples G i -or G o -linked receptors from their downstream effects. We asked whether the effects of ␣-Ltx in permeabilized or intact cells might be inhibited by pertussis toxin. Cultured chromaffin cells were incubated for 12 h with or without pertussis toxin, at concentrations sufficient to fully ADP-ribosylate G ␣i and G ␣o (28). Pertussis toxin treatment did not inhibit subsequent ␣-Ltx-induced secretion in either intact or permeabilized cells (not shown). Thus, if the effects of ␣-Ltx in permeabilized cells are mediated through a G-protein, the G-protein is not a pertussis toxin-sensitive member of the G i or G o families. One possible effector system for G-protein-linked receptors is the activation of polyphosphoinositide-specific phospholipase C, leading to the production of IP 3 and diacylglycerol. A complicating factor is that chromaffin cells possess a Ca 2ϩ -activated phospholipase C, which can be stimulated by micromolar Ca 2ϩ in permeabilized cells (29), or by Ca 2ϩ influx through voltage-sensitive Ca 2ϩ channels (30). Therefore, to ensure that any effect was caused by a direct activation of the ␣-Ltx receptor and not secondary to Ca 2ϩ influx through a toxin-induced pore, the experiment in Fig. 3A was conducted in the absence of extracellular Ca 2ϩ with 1 mM EGTA. ␣-Ltx (200 pM) had no effect on IP 3 levels at either 10 s or 2 min, whereas angiotensin II (Ang II, 100 nM, 10 s) increased IP 3 by 3-fold (Fig. 3A). In other experiments, ␣-Ltx had no effect at other times (1-4 min, not shown).
Because changes in IP 3 levels were only determined at selected time points, this result was confirmed by examining one of the consequences of IP 3 generation, the release of Ca 2ϩ from intracellular stores. Chromaffin cells loaded with fura-2/AM were incubated with 50 pM ␣-Ltx, in PSS without Ca 2ϩ , and with 1 mM EGTA. In the absence of extracellular Ca 2ϩ , ␣-Ltx was unable to stimulate a rise in intracellular free Ca 2ϩ (Fig. 3, B and C). Binding of ␣-Ltx to its receptor was verified by the fact that addition of 2 mM Ca 2ϩ to the bath resulted in an immediate and profound rise in intracellular Ca 2ϩ (Fig. 3B). In addition, the intracellular Ca 2ϩ stores were competent to undergo release because adding Ang II after ␣-Ltx elicited the expected Ca 2ϩ response (Fig. 3C). This result is in full agreement with the direct measurement of IP 3 levels above. There is no evidence to suggest that ␣-Ltx interaction with the endogenous chromaffin cell receptor directly activates a G-proteinlinked phospholipase C.
ATP Dependence of the Enhancing Effects of ␣-Ltx in Permeabilized Cells-When chromaffin cells were pretreated with ␣-Ltx and then permeabilized for 4 min before stimulation with Ca 2ϩ , the degree of enhancement was not uniform over the time course of the incubation with Ca 2ϩ (Fig. 4A). Little or no effect was seen at 2 or 4 min, but at 8 and 18 min, secretion was enhanced by 49% and 101%, respectively. In permeabilized cells, the rate of secretion typically declines with time, even in the presence of a continuing Ca 2ϩ stimulus (20). ␣-Ltx had no effect on the initial rates of secretion but markedly prolonged the secretory response.
We have demonstrated previously that during the first few minutes of a Ca 2ϩ stimulus, two components of secretion are present. One component does not require the presence of MgATP, and has been termed "primed" secretion (20, 31). Such primed secretion probably reflects the prior action of ATP in 3), bovine brain synaptosomes (lane 1), and HEK293 cells transiently expressing CIRL (lanes 4 and 5) were analyzed by SDS-polyacrylamide gel electrophoresis followed by protein blotting. 8 M urea was added to the sample buffer and SDS-electrophoresis system to denature the proteins without boiling (6). The blots were probed with rabbit anti-p85 antibody (6) raised against a peptide comprising the 18-amino acid C terminus of CIRL (lanes 1, 2, and 4) or with anti-p85 that was preincubated with 25 M peptide to block specific binding (lanes 3 and 5), followed by HRP-labeled secondary antibody, which was then detected by enhanced chemiluminescence. cells before permeabilization (31). In addition, MgATP is required to maintain secretion after the first few minutes (the second component). The time course of ␣-Ltx enhancement of secretion in permeabilized cells revealed that ␣-Ltx had little effect initially, but it prolonged the secretory response. Based on this observation, one would predict that ␣-Ltx might selectively enhance the ATP-dependent component of secretion while having little effect on secretion that was already primed. This was indeed the case. When cells pretreated with ␣-Ltx were then permeabilized and stimulated with Ca 2ϩ in the absence of MgATP, ␣-Ltx had no effect on secretion (Fig. 4B, open bars). The enhancement by ␣-Ltx was seen only when MgATP was included in the incubation with Ca 2ϩ (Fig. 4B, filled bars). In this experiment, ␣-Ltx doubled the ATP-dependent secretion (the difference between secretion in the presence of ATP (filled bars) and secretion in the absence of ATP (open bars), from 9.0 to 18.1%.

FIG. 2. An antibody to the COOH terminus of CIRL recognizes a protein in chromaffin cells. Cultured chromaffin cells (lanes 2 and
PKC Is Involved in the Enhancement of Secretion by ␣-Ltx-The ability of ␣-Ltx to enhance specifically the ATP-dependent component of secretion suggests the possibility that ␣-Ltx affects an ATP-requiring reaction that modulates the secretory pathway. We found that the effect of ␣-Ltx could be inhibited by a nonspecific kinase inhibitor, staurosporine (Fig. 5A). When cells were incubated with 2 M staurosporine before permeabilizing and stimulating with ␣-Ltx and Ca 2ϩ , the effect of ␣-Ltx was inhibited strongly, whereas Ca 2ϩ -dependent secretion in the absence of ␣-Ltx was inhibited by 24%.
One kinase that has been particularly well characterized in its effects on secretion is PKC (32)(33)(34). We asked whether PKC might be involved in the effects of ␣-Ltx. Preincubation with Ro31-8220 (an inhibitor of PKC which blocks the ATP binding site) inhibited by 63% the effect of ␣-Ltx to enhance secretion (Fig. 5B). Chelerythrine (another PKC inhibitor that blocks the substrate binding site) similarly inhibited the effect of ␣-Ltx (Fig. 5C). The inhibition of ␣-Ltx effects on secretion by Ro31-8220 was partial rather than complete, under treatment conditions that abolished the phosphorylation of an exogenous substrate (discussed below, Fig. 6). It is likely that these inhibitors are simply more effective at blocking phosphorylation of an exogenous rather than an endogenous substrate. Indeed, Ro31-8220 was similarly unable to block fully the enhancement of secretion by phorbol ester (not shown). Alternatively, a mechanism in addition to PKC activation could contribute to ␣-Ltx effects on secretion.
In contrast to the effects of PKC inhibitors, the enhancement of secretion by ␣-Ltx was not inhibited by H-89 (an inhibitor of cAMP-dependent protein kinase) or calmodulin-dependent protein kinase inhibitors KN-62, KN-93, and calmidazolium (Table I). The result suggested that PKC activation is required for the enhancement of secretion in permeabilized cells by ␣-Ltx. If this were the case, then one would predict that preactivation of PKC with phorbol ester (which itself enhances secretion) might prevent an additional enhancement of secretion by ␣-Ltx. In the absence of 12-O-tetradecanoylphorbol 13-acetate (TPA), ␣-Ltx caused a substantial enhancement of secretion (Fig. 5D). The reaction was stopped by the addition of trichloroacetic acid to each well to a final concentration of 5%. The acidified solutions were spotted on phosphocellulose paper and washed as described (21). The filter papers were then dried, and the radioactivity was determined by liquid scintillation spectrometry. n ϭ 4 wells/group. However, incubation of cells with 100 nM TPA for 20 min before the addition of ␣-Ltx completely prevented the enhancement of secretion by the toxin.
If PKC is involved in mediating the effects of ␣-Ltx, then loss of the enzyme by down-regulation should prevent the enhancement by ␣-Ltx. Incubation of cells for 18 h with 1 M TPA, a treatment that depletes cellular PKC by 90 -95% (33), completely abolished the ability of ␣-Ltx to enhance secretion in permeabilized cells (not shown). The data implicated PKC in regulation of the enhancement of secretion in permeabilized cells by ␣-Ltx. Inhibiting the enzyme or reducing its levels by prolonged activation blocked the enhancement of secretion by ␣-Ltx. Similarly, activating PKC before the addition of ␣-Ltx prevents the toxin's effects.
We thus investigated whether PKC was activated by ␣-Ltx using an in situ assay for the enzyme. PKC activity was measured directly in permeabilized cells by determining the extent of phosphorylation of an exogenous peptide substrate (21). Chromaffin cells were permeabilized for 4 min in KGEP containing digitonin and then stimulated with or without 30 M Ca 2ϩ and with or without 250 pM ␣-Ltx with 3 mM MgATP (80 Ci/ml [␥-32 P]ATP) and 300 M peptide substrate. Ca 2ϩ alone strongly stimulated PKC activity from 5.2 pmol of 32 P incorporated/well to 90 pmol of 32 P incorporated/well (Fig. 6). ␣-Ltx enhanced the PKC activity by an additional 40%, to 128 pmol of 32 P incorporated/well. ␣-Ltx did not stimulate PKC activity in the absence of Ca 2ϩ . In a typical experiment, phosphorylation ( 32 P incorporated/ well) in the absence of Ca 2ϩ was 5.6 Ϯ 0.3 pmol (250 pM ␣-Ltx) compared with 6.0 Ϯ 1.0 pmol (no ␣-Ltx).
Phosphorylation of the peptide substrate was specific for PKC. Inclusion of the PKC inhibitor Ro31-8220 during the permeabilization step completely abolished both Ca 2ϩ -and ␣-Ltx-stimulated phosphorylation, whereas inhibitors of cAMPdependent protein kinase (H-89) and calmodulin-dependent protein kinase (KN-93, in a separate experiment, not shown) had no effect. We conclude that PKC activation is involved in the ability of ␣-Ltx to enhance secretion in permeabilized cells.
Ca 2ϩ Dependence of the ␣-Ltx Effect in Permeabilized Cells-The Ca 2ϩ sensitivity for secretion in permeabilized chromaffin cells incubated with or without ␣-Ltx is shown in Fig. 7A. The enhancement of secretion by ␣-Ltx occurred between 10 and 1,000 M Ca 2ϩ and was similar in magnitude over this wide range of Ca 2ϩ concentrations. ␣-Ltx had no effect on secretion from permeabilized cells stimulated by Ca 2ϩ concentrations less than or equal to 3 M.
Ba 2ϩ , although somewhat less potent than Ca 2ϩ , is also an effective secretagogue in permeabilized chromaffin cells (35). However, when we compared the effects of ␣-Ltx in the presence of optimal concentrations of these two cations (30 M Ca 2ϩ and 1 mM Ba 2ϩ ), we found that, unlike Ca 2ϩ , Ba 2ϩ did not support an effect of ␣-Ltx in permeabilized cells (Fig. 7B).
Nonhydrolyzable guanine nucleotides stimulate a modest amount of secretion from permeabilized chromaffin cells which is entirely Ca 2ϩ -independent (28). This pathway, like that stimulated by Ca 2ϩ and Ba 2ϩ , is sensitive to tetanus toxin, indicating a role for synaptobrevin and probably other SNARE proteins. The experiment in Fig. 7C asked whether ␣-Ltx was able to enhance guanine nucleotide-stimulated secretion. Cells were preincubated with or without ␣-Ltx in PSS without Ca 2ϩ and then permeabilized and incubated with or without 200 M GppNHp. Again, unlike its effects on Ca 2ϩ -dependent secretion, ␣-Ltx was unable to enhance secretion stimulated by GppNHp. Either ␣-Ltx requires Ca 2ϩ for its action, or the toxin selectively enhances only the secretory pathway stimulated by Ca 2ϩ .

DISCUSSION
␣-Ltx has two effects on chromaffin cells. The toxin interacts with an endogenous receptor to increase Ca 2ϩ influx in intact cells, thus stimulating secretion. This effect occurs via the production of a high conductance channel (18) rather than through the activation of G-protein-coupled signaling pathway because it occurs with truncated receptors lacking transmembrane and intracellular domains (15,17). This paper focuses on a second effect of ␣-Ltx and presents a major new finding: ␣-Ltx, through its interaction with its extracellular receptor, enhances secretion in permeabilized chromaffin cells by a mechanism involving PKC. We have studied this phenomenon in permeabilized cells because the interpretation of ␣-Ltx effects on secretion in intact cells is complicated by the toxin's effects on Ca 2ϩ permeability. The result reported here is the first indication of an intracellular signaling pathway for the Ca 2ϩ -independent ␣-Ltx receptor, a G-protein-linked receptor whose endogenous ligand remains unknown. We additionally demonstrate that the enhancement of secretion is entirely Ca 2ϩ -and ATP-dependent and is not observed when Ba 2ϩ or guanine nucleotides are used to stimulate secretion. These results are discussed below.
An Endogenous Plasma Membrane Receptor Mediates ␣-Ltx Effects in Permeabilized Cells-We reported previously that chromaffin cells exhibit ␣-Ltx binding, the majority of which is Ca 2ϩ -independent (14). Preincubation of intact cells with ␣-Ltx without divalent cations and with EGTA led to enhanced secretion in cells subsequently permeabilized and stimulated by Ca 2ϩ . Here we show that the effect of ␣-Ltx is blocked by prior incubation of intact cells with trypsin and concanavalin A (Fig.  1), indicating a role for an endogenous glycoprotein receptor for ␣-Ltx. If, as has been suggested recently (36), the function of the plasma membrane receptor is to facilitate toxin entry into the cytoplasm, then permeabilization with digitonin should reverse the effects of agents which prevent ␣-Ltx binding, by allowing ␣-Ltx access to the cell's interior. However, ␣-Ltx had no effect following trypsin or concanavalin A treatment, even when the toxin was added after permeabilization (Fig. 1, B and  D). This result underscores the importance of the plasma membrane receptor in mediating the effects of ␣-Ltx in permeabilized as well as intact cells.
The enhancement of secretion in permeabilized cells is mediated by a cloned, Ca 2ϩ -independent receptor for ␣-Ltx, whose transient expression increases the sensitivity of both intact and permeabilized cells to stimulation by ␣-Ltx (14). The effects of high ␣-Ltx concentrations acting on endogenous receptors in nontransfected cells are virtually identical to those of very low concentrations of ␣-Ltx acting on transiently expressed CIRL. Moreover, an 85-kDa protein in nontransfected chromaffin cells is recognized by an antibody to the COOH terminus of CIRL. Taken together, the data indicate that the effects of ␣-Ltx on intact and permeabilized cells are mediated by an endogenous receptor identical to or closely resembling CIRL.
Although CIRL appears to be a G-protein-linked receptor, the effects of ␣-Ltx were insensitive to pertussis toxin, suggesting that neither G i nor G o is required for the stimulatory effects of ␣-Ltx. Furthermore, binding of ␣-Ltx to its receptor does not directly activate phospholipase C (Fig. 3), as determined by both production of IP 3 and release of Ca 2ϩ from intracellular stores. The inability of ␣-Ltx to elicit Ca 2ϩ release from intracellular stores is consistent with its inability to stimulate secretion in the absence of extracellular Ca 2ϩ (14).
The Role of PKC in Mediating the Effects of ␣-Ltx in Permeabilized Cells-The ␣-Ltx-induced enhancement occurred late in the time course of secretion (Fig. 4A), with ␣-Ltx having no effect during the first minutes of the Ca 2ϩ stimulus. This is consistent with a requirement for ATP (Fig. 4B) because the later phase of secretion is completely ATP-dependent (20,31). The ATP dependence (and the inability to enhance secretion that has already been primed) suggests a requirement for newly primed vesicles or implicates another ATP-dependent reaction (e.g. phosphorylation). Experiments with kinase inhibitors demonstrated that only those compounds that inhibit PKC inhibited the enhancement by ␣-Ltx (Fig. 5); inhibitors of cAMP-dependent and calmodulin-dependent protein kinases were ineffective (Table I). Measurements of PKC activity in situ revealed that ␣-Ltx was able to increase the already substantial activation of the enzyme by 30 M Ca 2ϩ (Fig. 6) while having no effect in the absence of Ca 2ϩ . This result is the first indication of an intracellular signaling pathway for ␣-Ltx. The inability of ␣-Ltx to activate phospholipase C directly (Fig. 3) and thus generate diacylglycerol suggests that there may be some other mechanism by which ␣-Ltx enhances the activation of PKC.
We noted that, in combination with ␣-Ltx, short term activation of PKC by TPA (itself an enhancer of secretion, Fig. 5D) not only prevented the further enhancement of secretion by ␣-Ltx, but in fact permitted less secretion than ␣-Ltx alone (Fig.  5D). This suggests the possibility that PKC may have dual effects, both mediating and subsequently moderating the effect of ␣-Ltx.
The Mode of PKC Activation Influences Its Effect on Secretion-In these experiments, several means were used to acti-vate PKC (preincubating intact cells with phorbol ester, incubating permeabilized cells with 30 M Ca 2ϩ , and incubating permeabilized cells with ␣-Ltx and Ca 2ϩ ) which have differing effects on subsequent secretion. Phorbol ester effects on secretion are not immediate; they require preincubation of the cells before stimulation with Ca 2ϩ (21). This preactivated PKC increases the number of primed granules (34,37,38) without influencing the later rates of secretion (34). In permeabilized cells, PKC activated by Ca 2ϩ in the absence of phorbol ester or ␣-Ltx is without substantial effect on secretion because virtually complete inhibition of this activity by Ro31-8220 (Fig. 6) did not alter the secretory response (Fig. 5B). In striking contrast, the additional PKC activation by ␣-Ltx in the presence of Ca 2ϩ led to enhanced secretion, and inhibition of the ␣-Ltx-dependent PKC inhibited this enhancement. Thus, the PKC activated by ␣-Ltx seems to be closely integrated with the secretory pathway, phosphorylating substrates that are able to modulate the ongoing secretory response. This might reflect localization of the ␣-Ltx-activated PKC close to secretory sites and/or specificity for substrates intimately involved in secretion.
Calcium Requirement for ␣-Ltx Effects in Permeabilized Cells-The involvement of PKC provides an explanation for what might otherwise have been a puzzling phenomenon, the inability of ␣-Ltx to enhance secretion stimulated by Ba 2ϩ or guanine nucleotides. A major difference between these secretagogues lies in their ability to stimulate PKC. In chromaffin cells, Ba 2ϩ activates neither phospholipase C (35) nor PKC. 2 Both Ca 2ϩ and guanine nucleotides activate phospholipase C, but only Ca 2ϩ activates PKC. Thus, the inability of ␣-Ltx to enhance secretion stimulated by Ba 2ϩ and guanine nucleotides corroborates a role for PKC in mediating the toxin's effects in permeabilized cells. Moreover, the sensitivity of the ␣-Ltx effect to Ca 2ϩ is not identical to the Ca 2ϩ sensitivity of the regulated pathway. ␣-Ltx was unable to enhance secretion at Ca 2ϩ concentrations below 10 M, despite the fact that this secretion is almost entirely ATP-dependent. The data suggest that Ca 2ϩ itself is specifically required in the signaling pathway by which ␣-Ltx effects are mediated.
␣-Ltx-induced secretion in intact cells depends strongly upon the ability of the toxin to facilitate divalent cation entry through ion channel formation. We conclude that ␣-Ltx effects in permeabilized cells occur through an ␣-Ltx-enhanced activation of PKC which requires both high micromolar Ca 2ϩ that directly enters permeabilized cells and a separate receptor-2 D. R. TerBush and R. W. Holz, unpublished results. mediated event. This receptor-mediated effect is likely to be present in intact cells, but it is experimentally difficult to distinguish its contribution to the secretory response. In one study using patch-clamped rat adrenal chromaffin cells, low doses of ␣-Ltx which did not produce channel activity or a rise in intracellular Ca 2ϩ were able to enhance depolarization-induced exocytosis (26). The receptor-mediated action of ␣-Ltx may reflect the physiological function of the still unknown endogenous ligand for CIRL.