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Endocrinology Vol. 140, No. 7 3025-3033
Copyright © 1999 by The Endocrine Society

ARTICLES

{alpha}-Latrotoxin Stimulates Inward Current, Rise in Cytosolic Calcium Concentration, and Exocytosis in at Pituitary Gonadotropes1

Frederick W. Tse2 and Amy Tse2

Department of Pharmacology and Division of Neuroscience, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

Address all correspondence and requests for reprints to: Dr. Frederick W. Tse, Department of Pharmacology, 9–70 Medical Science Building, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. E-mail: fred.tse{at}ualberta.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
{alpha}-Latrotoxin (LTX) from the black widow spider venom, stimulates neurotransmitter release from neuronal cells via Ca2+-dependent as well as Ca2+-independent mechanisms. In some peptide-secreting endocrine cells, however, LTX stimulates hormone release mainly via a Ca2+-independent mechanism. Here we investigated the action of LTX in rat pituitary gonadotropes that secrete the peptide, LH. Using the patch-clamp technique in conjunction with the fluorescent Ca2+ indicator (indo-1) to simultaneously measure the cytosolic Ca2+ concentration ([Ca2+]i) and ionic current, we showed that LTX elicited bursts of inward current that were accompanied by [Ca2+]i elevations. In the presence of a physiological concentration of extracellular Ca2+, the unitary conductance of the LTX-induced current was about 300 pS, and only about 6.4% of the current was carried by Ca2+. The LTX-induced current was occasionally followed by intracellular Ca2+ release. At [Ca2+]i of 1 µM or more, exocytosis (detected by membrane capacitance measurement) was consistently triggered, and it was frequently followed by endocytosis. Thus, LTX triggers Ca2+-dependent exocytosis in gonadotropes via extracellular Ca2+ entry as well as intracellular Ca2+ release. In approximately 25% of the cells, LTX could also trigger a slow exocytosis in the absence of [Ca2+]i elevation. Therefore, LTX has both Ca2+-dependent and Ca2+-independent actions in gonadotropes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A PURIFIED fraction (130 kDa) of the black widow spider venom, {alpha}-latrotoxin (LTX), is known to stimulate massive neurotransmitter release via Ca2+-dependent as well as Ca2+-independent actions and has been extensively employed in physiological studies of neuroexocytosis (1). Biochemical studies have identified two types of LTX receptors: the Ca2+-dependent receptor, neurexin 1{alpha}, that is postulated to interact with synaptotagmin (2, 3), and the Ca2+-independent receptor (CIRL/latrophilin) that resembles member of the secretin family of G protein-coupled receptors (4, 5). However, detailed understanding of the relative contribution of the two receptors as well as the mechanisms underlying the LTX actions is still obscure. Multiple mechanisms have been proposed to explain the LTX-mediated Ca2+-dependent release of neurotransmitter. One mechanism involves the opening of the cation-permeable channel (6) that allows the entry of extracellular Na+ and Ca2+ (7). This extracellular Ca2+ entry has been shown to elevate the cytosolic Ca2+ concentration ([Ca2+]i) and triggers Ca2+-dependent exocytosis in rat chromaffin cells (8, 9). A second mechanism involves the LTX-induced extracellular Na+ entry. In bovine chromaffin cells, Na+ influx is postulated to stimulate the Na+ extrusion mode of the Na+-Ca2+ exchanger, thus causing a local elevation of [Ca2+] near the plasma membrane that can, in turn, trigger secretion (10). A third mechanism proposed that LTX increased the Ca2+ sensitivity of release. In chromaffin cells as well as rat synaptosomes, LTX has also been shown to enhance Ca2+-dependent secretion, and the involvement of CIRL/latrophilin has been implicated (11, 12). A fourth mechanism suggested that LTX acting via the CIRL/latrophilin receptor stimulated the G protein-coupled phosphoinositide pathway to trigger secretion. In rat brain synaptosomes, the LTX-stimulated Ca2+-dependent vesicular release was demonstrated to be dependent on the activation of phospholipase C and the release of Ca2+ from intracellular stores (12). However, a recent study in PC12 cells suggested that the activation of CIRL/latrophilin receptor could trigger Ca2+-dependent secretion without G protein coupling (13). The LTX-mediated Ca2+-independent transmitter release, on the other hand, is suggested to be largely nonvesicular. In rat synaptosomes, norepinephrine in the cytoplasm was postulated to leak into extracellular space via the large pores (channels) formed by LTX in the presynaptic membrane (12). Interestingly, in insulin-secreting ß-cells, the LTX-stimulated hormone secretion was also independent of Ca2+. However, in these endocrine cells, the ability of LTX to open channels appeared to be absent, as LTX failed to alter the membrane potential or the resting [Ca2+]i (14). Thus, the channel-forming activity of LTX cannot explain this Ca2+-independent stimulation of insulin release.

The above observations raise the question of whether the action of LTX in hormone-secreting endocrine cells is different from that of nerve terminals or neuronal cells. Here, we investigated the action of LTX in the reproductive hormone-secreting gonadotropes from the anterior pituitary gland. In gonadotropes, Ca2+-dependent exocytosis is tightly coupled to the phosphoinositide pathway (15). In addition, secretion in gonadotropes can be stimulated via a Ca2+-independent mechanism by directly stimulating the G proteins with nonhydrolysable guanine nucleotide analogs (16, 17). In this study, we examined the temporal relationships among the LTX-induced changes in ionic current, [Ca2+]i, and exocytosis by simultaneous application of whole cell voltage clamp, measurement of [Ca2+]i (indo-1 fluorometry) and electrical measurement of membrane capacitance. In contrast to the insulin-secreting ß-cells, our results indicate that the action of LTX in gonadotropes is mostly Ca2+ dependent. LTX causes the formation of cation-permeable channels on the plasma membrane of gonadotropes. Exocytosis is triggered either directly via extracellular Ca2+ entry through the cation channels or via intracellular Ca2+ release. Our results also demonstrate for the first time that under physiological conditions, Ca2+ carries only about 6.4% of the LTX-induced current. Preliminary data from this work have been published in abstract form (18).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell preparation
The anterior lobe of the pituitary gland was removed from male Sprague Dawley rats (aged 5–6 weeks) that had been killed with halothane in accordance with the standards of the Canadian Council on Animal Care. Anterior pituitary glands were dissociated enzymatically using collagenase and trypsin as previously described (19). Single gonadotropes were identified from the heterogeneous population by reverse hemolytic plaque assay (20), using polyclonal antibodies to LH (provided by Dr. J. D. Neill, University of Alabama, Birmingham, AL). The procedures were similar to those described previously (19). The cells were maintained under standard culture conditions in DMEM supplemented with 10% (vol/vol) horse serum, 50 U/ml penicillin G, and 50 µg/ml streptomycin. Recordings were performed on cells cultured for 2–4 days.

Solutions
The standard bath solution contained 150 mM NaCl, 10 mM Na-HEPES, 8 mM glucose, 2.5 mM KCl, 2 mM CaCl2, and 1 mM MgCl2 (pH 7.4). In experiments in which extracellular Ca2+ was removed, Ca2+ was omitted from the standard bath solution, and 1 or 3 mM MgCl2 and 1 mM Na-EGTA were added. In all experiments shown here, apamin (0.4 µM) was included in the standard bath solution to inhibit the SK type Ca2+-activated K+ current (21). The standard pipette solution contained 120 mM K-aspartate, 20 mM KCl, 20 mM K-HEPES, 1 mM MgCl2, 2 mM Na2ATP, 0.1 mM Na4GTP, and 0.1 mM indo-1 (pH 7.4).

The application of LTX (Alomone Laboratories, Jerusalem, Israel; Calbiochem, La Jolla, CA; or gift from Dr. A. G. Petrenko, New York University, New York, NY) was similar to that described by Liu and Misler (9). Briefly, 10 or 20 µl LTX stock solution (0.3 or 0.6 µM in Tris buffer) were injected into the recording chamber (volume, 400–800 µl) from a micropipetter, giving a final equilibrium concentration of 4–30 nM. As LTX is a large molecule (130 kDa), the time for diffusion was slow, particularly when the tip of the micropipetter was farther away from the recording pipette (distance of ~5–10 mm). This probably contributed to the variability in the onset of LTX-induced current (10 sec to ~3 min). Indo-1 (Calbiochem) and apamin (Sigma Chemical Co., St. Louis, MO) were kept as stock solution in distilled water at -20 C.

Measurement of [Ca2+]i
[Ca2+]i was measured fluorometrically by dialyzing the Ca2+ indicator, indo-1, via the whole cell patch pipette. Details of the instrumentation and procedures of the [Ca2+]i measurement were described previously (22). [Ca2+]i was calculated from the ratio (R) of fluorescence at 405 and 500 nm, using the equation previously reported (23):[Ca2+]i = K1(R - Rmin)/(Rmax - R) (Eq I), where Rmin is the fluorescence ratio of the Ca2+-free indicator, Rmax is the ratio of the Ca2+-bound indicator, and K1 is a constant that was determined empirically. Calibrations were determined from groups of single gonadotropes (n = 5–8) dialyzed with one of the three pipette solutions. Rmin was measured in cells loaded with 52 mM K-aspartate, 50 mM K-EGTA, 10 mM KCl, 0.1 mM indo-1, and 50 mM K-HEPES (pH 7.4); Rmax was measured in cells loaded with 136 mM K-aspartate, 15 mM CaCl2, 0.1 mM indo-1, and 50 mM K-HEPES (pH 7.4). K1 was calculated from Eq I using R values obtained from cells loaded with 60 mM K-aspartate, 50 mM K-HEPES, 20 mM K-EGTA, 15 mM CaCl2, and 0.1 mM indo-1 (pH 7.4), which had a calculated free Ca2+ concentration of 212 nM at 24 C (24). For all indo-1 measurements reported here, the values of Rmin, Rmax, and K1 were 0.403, 5.05, and 2.62 µM, respectively.

Electrophysiological recording
Membrane current was recorded with the whole cell, gigaseal method (25) using an EPC-9 patch clamp amplifier. Cell capacitance (Cm) was measured with the capacitance-tracking feature of the EPC-9 amplifier. Values of [Ca2+]i, currents, and (NeuroData Corp., New York, NY) Cm were first recorded on VCR tapes with a NeuroData PCM recorder and later digitized. The pipettes were made from hematocrit glass (VWR Scientific Canada Ltd., London, Ontario, Canada), and the resistance was 2–4 Mohms after filling and 5–10 Mohms during whole cell recording. Recordings were made at room temperature (22–25 C). A -10 mV junction potential was corrected throughout. Values given in the text are the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LTX triggered inward current and [Ca2+]i elevation
Figure 1Go shows two typical examples of the action of LTX on ionic current and [Ca2+]i in single gonadotropes. The cells were voltage clamped at -70 mV, a potential at which most voltage-gated channels, including Ca2+ channels, were closed (15). In addition, the SK-type Ca2+-activated K+ channels were pharmacologically inhibited by 0.4 µM apamin (21). Application of a low concentration of LTX (4 nM) activated inward current that has a "flicker and burst" pattern (Fig. 1AGo). The amplitude of the inward current fluctuated between 0 and about -20 pA for many seconds, bearing strong resemblance to transient opening and closing of single channels. In Fig. 1AGo, the current amplitude increased transiently to about 40–60 pA for about 50 sec before returning to the resting level, as if multiple channels were opened simultaneously and then closed again. Such a flicker and burst pattern of the LTX-induced current was consistently observed in cells exposed to a lower concentration of LTX (4 nM; n = 10). In cells exposed to a higher concentration of LTX (30 nM), the current initially displayed the flicker and burst pattern, but quickly transformed into a large and continuous current of several hundred picoamperes (Fig. 1BGo; n = 26). Note that in both examples, current fluctuations were closely accompanied by changes in [Ca2+]i. In Fig. 1AGo, even when the burst of LTX-induced current was brief (~10 sec) and small (~20 pA), clearly measurable [Ca2+]i elevation (tens of nanomolar concentrations; also see Fig. 4Go) could be observed. During the long (~50 sec) and large (~40–60 pA) current burst, [Ca2+]i rose to about 0.8 µM. When the current returned to the resting level, [Ca2+]i gradually decayed to its basal level. On the other hand, at a high concentration of LTX (30 nM), when the current was large and continuous (Fig. 1BGo), [Ca2+]i eventually rose to several micromolar concentrations.



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  		Figure 1. LTX triggered inward current and [Ca2+]i elevation. A, Current and [Ca2+]i were measured simultaneously from a single rat gonadotrope in the whole cell configuration. Application of LTX (4 nM) activated bursts of inward current. A large and long burst of inward current elevated [Ca2+]i to about 0.8 µM. B, In another cell, LTX application (30 nM) activated bursts of current that rapidly transformed into a large and continuous current, and [Ca2+]i rose to several micromolar concentrations. Cells were voltage clamped at -70 mV in standard bath solution. The time of LTX application is denoted by the arrow.

 


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  		Figure 4. Ca2+ permeability of LTX channel. The activation of LTX current is closely correlated with rise in [Ca2+]i. The two arrows indicated the [Ca2+]i at 1 and 2 sec after the onset of this burst of LTX current. The cell was held at -70 mV in standard bath solution. LTX (4 nM) was applied 28 sec before the beginning of the recording trace shown here.

 
The initial current fluctuation in the presence of a low concentration of LTX (4 nM; e.g. Fig. 1AGo) appeared to involve opening and closing of one or more single channels. To estimate the unitary amplitude of the LTX-induced current, we constructed current amplitude histograms from segments of current records where the current fluctuated between different levels. A typical example of such a histogram is shown in Fig. 2AGo. In this plot, three major peaks could be observed. One of the peaks centered at the resting current level (around zero), presumably reflecting the background noise of the recording. The rest of the histogram could be well described by two Gaussian distributions with peaks centered at -22 and -44 pA. These data suggest that the LTX-induced current in gonadotropes has a unitary amplitude of approximately -22 pA when the membrane potential was held at -70 mV. To estimate the unitary conductance, we measured the amplitude of unitary LTX-induced current from individual cells that were held at potentials of -40, -70, and -90 mV. Figure 2BGo plots the unitary current amplitude as a function of the membrane potential. The values could be described by a linear fit with a slope of 0.325 nS and an x-intercept of -4.3 mV. Thus, the LTX current has a unitary conductance of about 0.3 nS in physiological saline, and the current reverses near 0 mV. These data are consistent with previous studies that suggested that the LTX-induced current involved openings of large, cation-permeable channels or pores (7). In brain synaptosomes (12), LTX has been reported to form pores that are permeable to relatively large molecules, such as fluorescein isothiocyanate (Mr = 389). We examined whether the LTX channels in gonadotropes are also permeable to large molecules by replacing all extracellular NaCl with an equalmolar concentration of N-methyl-D-glucamine chloride. If the LTX channels are also permeable to N-methyl-D-glucamine, the LTX-induced current should be inward when the membrane potential was held at negative potentials. However, in three cells examined, application of LTX induced an outward current (data not shown) when the membrane potential was held at -70 mV, suggesting that the LTX channels in gonadotropes are not significantly permeable to N-methyl-D-glucamine (Mr = 195.2).



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  		Figure 2. Unitary conductance of LTX current. A, The LTX-induced current involved opening of multiple unitary channels. Amplitude histogram of the LTX-induced current (from the record shown in Fig. 4Go) that appeared to have two levels of current amplitude. The two fitted Guassian distribution (curved line) have peaks centered at -22 and -44 pA. The cell was held at -70 mV. B, Current-voltage relation of the unitary current. The data points at different potentials were mean values of the unitary amplitude of LTX-induced current obtained from two to six cells. The dotted line is a linear fit with a slope of 0.325 nS and an x-intercept of -4.3 mV.

 
The Ca2+ fraction of the LTX-induced current
The results above suggest that the rise in [Ca2+]i is roughly correlated to the amplitude and duration of individual current burst. As the LTX-induced current in neurons and neuroendocrine cells is permeable to Ca2+ (7, 9), it is likely that the rise in [Ca2+]i in gonadotropes is a result of extracellular Ca2+ entry via the LTX-induced current. To test this idea, gonadotropes were bathed in Ca2+-free external solution for 2 min before application of LTX. Figure 3Go shows that in the absence of extracellular Ca2+, LTX could still trigger robust inward current. Although the LTX-induced current eventually became large and continuous, [Ca2+]i remained at the resting level. When Ca2+ was subsequently added to the external solution in the continued presence of LTX, a robust Ca2+ rise was observed. Similar results were observed in four other cells. Therefore, in gonadotropes, extracellular Ca2+ entry via the LTX channels is responsible for [Ca2+]i elevation.



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  		Figure 3. The LTX-elicited [Ca2+]i rise is dependent on extracellular Ca2+. LTX (30 nM) application (denoted by arrow) failed to trigger any [Ca2+]i elevation in the absence of extracellular Ca2+. The cell was initially bathed in a solution containing 3 mM Mg2+ and 1 mM EGTA (no added Ca2+). Note that subsequent addition of extracellular Ca2+ (2 mM) in the continued presence of LTX raised [Ca2+]i to several micromolar concentrations. The cell was voltage clamped at -70 mV.

 
The close correlation between the LTX-induced current and the corresponding [Ca2+]i elevation in gonadotropes allows estimation of the contribution of Ca2+ to the LTX-induced current. The estimation rests on the assumption that when a Ca2+ load enters the cytosol, Ca2+ rapidly equilibrates with the intracellular buffers before being slowly removed by membrane transport systems. Under this condition, the amplitude of the average rise in cytosolic [Ca2+]i is largely determined by the cytosolic Ca2+ buffers. In gonadotropes, we (22) have previously documented that during extracellular Ca2+ entry via brief depolarization (0.2–2 sec), on the average, only 1 of 228 Ca ions entering the cytosol remained free when 100 µM indo-1 was included in the whole cell pipette solution as the Ca2+ indicator. The other 227 Ca ions were bound by endogenous Ca2+ buffers as well as the Ca2+ indicator, indo-1. We have also shown that the clearance of a small Ca2+ load ([Ca2+]i, <200 nM) in gonadotropes typically has a time constant of about 5 sec. Based on these previous findings, it is possible to estimate the amount of extracellular Ca2+ entered during the opening of the cation-permeable channels as long as a correlation between the LTX-induced current and [Ca2+]i rise could be determined before significant Ca2+ extrusion had occurred. Indeed, in cells exposed to a low concentration of LTX (e.g. Fig. 4Go), the activation of a single burst of LTX-induced current allowed sufficient Ca2+ entry to cause a measurable rise in [Ca2+]i (10–70 nM) within 1–2 sec. To estimate the Ca2+ permeability of the LTX-induced current, we measured the [Ca2+]i rise at 1 or 2 sec after the initial activation of the LTX-induced current. As our experimental condition (100 µM indo-1) was identical to that used in our previous study (22), it is reasonable to assume that actual Ca2+ entry was, on the average, 228 times higher than that reported by our [Ca2+]i measurements here. Because gonadotropes vary in size, Ca2+ entry was normalized to the volume of individual cells. Cell volume was calculated by assuming that the cells are spherical, and individual cell radius was estimated from the cell membrane capacitance (1 µF/cm2). For example, in Fig. 4Go, the rises in [Ca2+]i following the first and second seconds of activation of LTX-induced current were 20 and 56 nM, and the estimated total Ca2+ entries were 1.44 and 4.03 pC, respectively. The corresponding time integrals (1 and 2 sec) of the inward current were 23.9 and 67.2 pC. In both cases, Ca2+ entry contributed only about 6% of the LTX-induced inward current. Similar analysis on the first second of current activation was performed in 10 cells, and on the average, Ca2+ carried only 6.4 ± 1.4% of the LTX-induced current.

Extracellular Ca2+ entry via the LTX-induced cation-permeable channels triggered exocytosis
To determine whether the LTX-triggered [Ca2+]i elevation can trigger secretion in gonadotropes, we monitored simultaneously current, [Ca2+]i, and changes in cell surface membrane area using the high temporal resolution capacitance measurement. Figure 5AGo shows an example of such measurements in the presence of 4 nM LTX. Initially, the LTX-induced current was small and discontinuous. [Ca2+]i stayed below 0.6 µM most of the time, and no significant increase in membrane capacitance was detected. When the LTX-induced current transformed into a long burst, [Ca2+]i rose to 1.2 µM, and it was accompanied by a slow (~1.8 fF/sec) and small cumulative increase (~90 fF) in membrane capacitance (reflecting exocytosis). This burst of exocytosis was followed by a rapid reduction in membrane capacitance, reflecting endocytosis. In Fig. 5BGo, the cell was exposed to a higher concentration of LTX (30 nM), the LTX-induced current was large (>100 pA) and continuous. [Ca2+]i transiently rose beyond 2 µM, and this was accompanied by a faster (~100 fF/s) and larger cumulative exocytosis (~1.2 pF). The LTX-triggered exocytosis appeared to be correlated with the amplitude of [Ca2+]i elevation. In five of seven cells exposed to a low concentration of LTX (4 nM), the LTX-induced [Ca2+]i elevations were less than 1 µM, and no exocytosis was triggered. In the remaining two cells, small cumulative exocytosis (<100 fF) was detected when [Ca2+]i stayed at about 0.7 and 0.8 µM. In contrast, in all cells (n = 13) exposed to a higher concentration of LTX (30 nM), the LTX-induced [Ca2+]i elevation typically ranged between 1–4 µM, and it was accompanied by robust cumulative exocytosis (0.2 to >1 pF).



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  		Figure 5. The LTX-induced [Ca2+]i elevation is accompanied by exocytosis. A, Simultaneous measurement of current, [Ca2+]i and membrane capacitance (Cm) from single gonadotrope. Small and brief bursts of LTX current elicited small [Ca2+]i elevations (<0.6 µM), and they were not accompanied by any increase in Cm. A significant increase in Cm (reflecting exocytosis) was detected only when the LTX current became a long burst, and [Ca2+]i rose from 0.4 to about 1.2 µM. Note that this increase in Cm is followed by a rapid reduction, reflecting endocytosis. The cell was voltage clamped at -70 mV. LTX (4 nM) was applied at the time indicated by the arrow. The two dashed lines denote [Ca2+]i at 0.5 and 1 µM. B, A faster and larger rise in Cm was triggered when the LTX current was large and continuous, and [Ca2+]i rose transiently to about 2 µM. The cell was voltage clamped at -70 mV. LTX (30 nM) was applied at the time indicated by thearrow.

 
Consistent with the idea that LTX-induced extracellular Ca2+ entry triggers exocytosis, Fig. 6Go shows that LTX caused little or no increase in membrane capacitance when extracellular Ca2+ was absent. LTX could still induce robust inward current in the absence of extracellular Ca2+, but it was not accompanied by any rise in [Ca2+]i. When extracellular Ca2+ was subsequently added, [Ca2+]i rose rapidly beyond 2 µM, and this was accompanied by a burst of rapid and robust exocytosis. The maximum increase in capacitance reached almost 1.5 pF, and the maximum rate of exocytosis was about 400 fF/s when [Ca2+]i was beyond 2 µM. In six of eight cells examined, LTX failed to trigger any exocytosis in the absence of extracellular Ca2+. In the remaining two cells, however, LTX triggered a slow rise in capacitance without any [Ca2+]i elevation. This phenomenon will be discussed later in the section of exocytosis that is independent of [Ca2+]i elevation.



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  		Figure 6. The LTX-triggered exocytosis is dependent on extracellular Ca2+. In the absence of extracellular Ca2+ (solution containing 1 mM EGTA and 3 mM Mg2+), the LTX-induced current was not accompanied by any [Ca2+]i elevations or changes in Cm. Subsequent addition of Ca2+ elevated [Ca2+]i to about 2 µM, and it was accompanied by a robust increase in Cm. The cell was held at -70 mV. LTX (30 nM) was applied at about 40 sec before the recording trace shown here.

 
Exocytosis triggered via intracellular Ca2+ release
The results above (e.g. Figs. 1Go, 4Go, and 5Go) show that [Ca2+]i elevation is closely correlated with the LTX-induced current. However, clear deviations from such correlation were observed in some cells. For example, in the cell shown in Fig. 7Go, an initial brief and small burst of LTX-induced current elicited a small transient elevation in [Ca2+]i (0.29 µM). At the end of the current burst, [Ca2+]i started to decay; however, shortly after the decay, [Ca2+]i rose rapidly to 2 µM (indicated by the first asterisk). Note that this large [Ca2+]i elevation was not accompanied by any bursts of inward current. When [Ca2+]i eventually returned to the basal level, another burst of LTX-induced current occurred, but it elicited only a small [Ca2+]i rise (< 0.2 µM). When the current burst eventually became larger and longer, another large [Ca2+]i elevation (~2 µM; indicated by the second asterisk) was elicited. However, a subsequent current burst of similar amplitude and duration induced only small rises of [Ca2+]i (< 0.5 µM). Note that each of these [Ca2+]i rises (indicated by asterisks) was accompanied by a burst of exocytosis. Thus, in some cells (11 of 23), a short burst of LTX-induced current was occasionally followed by a delayed secondary [Ca2+]i rise or by a [Ca2+]i elevation that was disproportionately large for the current activated at that time. As discussed below (in Discussion), these [Ca2+]i rises are probably caused by Ca2+ release from the intracellular stores.



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  		Figure 7. Exocytosis triggered via Ca2+-induced Ca2+ release from intracellular stores. The asterisks denote two cycles of intracellular Ca2+ release that were elicited after extracellular Ca2+ entry via the LTX channel. At the first asterisk, the [Ca2+]i elevation was not accompanied by any LTX current, and it occurred with a delay after a small LTX-induced [Ca2+]i rise (~0.3 µM). At the second asterisk, the [Ca2+]i elevation (>2 µM) was disproportionately large for the accompanying LTX current (~50 pA). Note that subsequent to this large [Ca2+]i rise, LTX current of similar amplitude and duration only raised [Ca2+]i to about 0.5 µM. Both cycles of intracellular Ca2+ release were accompanied by robust increases in Cm, reflecting bursts of exocytosis. The cell was held at -70 mV in standard bath solution. LTX (4 nM) was applied at approximately 150 sec before the beginning of the recording trace shown here.

 
LTX triggers a slow exocytosis that is not dependent of [Ca2+]i elevation
As described earlier, in a small percentage of cells (2 of 8), LTX could trigger a slow increase in membrane capacitance even in the absence of extracellular Ca2+. An example of this is shown in Fig. 8AGo. Interestingly, this slow increase in membrane capacitance (~4 fF/sec) actually started before the onset of the LTX-induced current. We examined whether an extremely localized increase of [Ca2+]i (that is undetected by our cytosolic [Ca2+] measurements) may contribute to the slow increase in membrane capacitance. In these experiments, [Ca2+]i was strongly buffered by including 10 mM of a fast Ca2+ buffer [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)] in the whole cell pipette solution, and the bath solution contained 2 mM Ca2+. Figure 8BGo shows that under this condition, [Ca2+]i remained below 0.1 µM even when the LTX-induced current became large (>100 pA) and continuous for minutes; yet, LTX could still elicit a slow increase in capacitance (~7 fF/sec) before the onset of the LTX-induced current. Similar results were observed in 6 of 22 cells. This result raises the possibility that in some gonadotropes, LTX indeed stimulated a slow exocytosis that was independent of any [Ca2+]i elevation. As only a small percentage of gonadotropes exhibited this Ca2+-independent response to LTX, this phenomenon was not pursued further.



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  		Figure 8. Ca2+-independent action of LTX. A, LTX triggered a small and slow increase in membrane capacitance (Cm) even in the absence of extracellular Ca2+. The cell was held at -70 mV and bathed in a solution containing 1 mM Mg2+ and 1 mM EGTA (no added Ca2+). B, In the presence of intracellular Ca2+ buffer, LTX could still trigger an increase in Cm. The cell was recorded with 10 mM BAPTA in the pipette solution, and voltage was clamped at -70 mV in a standard bath solution. Note that at the end of the recording, LTX current became very large and continuous. At this time, [Ca2+]i gradually rose to about 0.2 µM, suggesting that intracellular BAPTA was eventually saturated by the large influx of extracellular Ca2+. The time of LTX (30 nM) applications are denoted by arrows, and the two asterisks denote the onset of the LTX current.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
LTX-induced current and [Ca2+]i elevation
The present study shows that LTX causes the formation of cation-permeable channels on the cell plasma membrane in pituitary gonadotropes. This result contrasts with that using insulin-secreting cells (14) or CIRL/latrophilin-transfected COS cells (12), in which the channel-forming activity of LTX was lacking. The unitary conductance of the LTX-induced current in gonadotropes (when bathed in physiological saline) was 325 pS, similar to that reported in lipid bilayer (6) and neuroblastoma cells (7) and rat chromaffin cells (9). However, although large ions such as fluorescein isothiocyanate could pass through the LTX channels in brain synaptosomes (12), the LTX channels in gonadotropes were not significantly permeable to the smaller ion N-methyl-D-glucamine (Mr = 195.2). Thus, there may be some fundamental differences between the properties of LTX channels in neuronal cells and endocrine cells.

In neuronal cells, LTX, at nanomolar concentrations caused rapid and extensive neurotransmitter release (26, 27). This has been postulated to be at least in part due to a massive influx of Ca2+ during channel opening (28). Interestingly, our study shows that LTX at similar concentrations (4 nM) elicited only small and transient rises of [Ca2+]i in gonadotropes. Two factors probably contribute to the small and transient [Ca2+]i elevation observed here. First, Ca2+ carried only 6.4% of the LTX-induced inward current in gonadotropes (in the presence of physiological extracellular Ca2+ concentrations). This value is similar to that of the NMDA (N-methyl-D-aspartic acid) receptor channels in neurons (6.8%) (29), but approximately 2- to 5-fold larger than that reported for nicotinic acetylcholine receptor channels (2.5%) (30) and AMPA ({alpha}-amino-3-hydroxy-5-methylisoxazole-4-propionic acid)/kainate receptor channel (1.4%) (29). Second, at a low concentration of LTX (4 nM), the current frequently had a flicker and burst pattern, suggesting that channels could close transiently after they were formed. During the intermittent closure of the channels, some cytosolic Ca2+ was removed by the actions of intracellular Ca2+ pumps and other transport mechanisms, and [Ca2+]i gradually declined (22). However, in the presence of a high concentration of LTX (30 nM), the LTX current rapidly became large and continuous. This resulted in a large and persistent influx of extracellular Ca2+ that probably saturated the intracellular Ca2+ removal mechanisms, and [Ca2+] remained elevated.

In some cells, the small burst of LTX current was occasionally followed by a delayed or disproportionately large [Ca2+]i elevation. Such patterns of [Ca2+]i elevations resembled Ca2+ waves, which involved the release of Ca2+ from intracellular stores in gonadotropes (15, 22). As the caffeine-sensitive store is absent in gonadotropes (22), it is likely that the triggering of intracellular Ca2+ release in these cells is from the inositol trisphosphate (IP3)-sensitive stores. Two distinct mechanisms may be involved in this intracellular Ca2+ release. The first is a transient elevation of the concentration of IP3. It is possible that the LTX-induced [Ca2+]i elevation has a stimulatory effect on phopholipase C (12, 31) and thus causes the generation of IP3. Indeed, in the presence of extracellular Ca2+, LTX has been reported to stimulate inositol phosphate accumulation in PC12 cells (32) as well as in COS cells that were transfected with the CIRL/latrophilin receptors (5). The second mechanism involves a positive feedback on the opening of IP3 receptor channels during modest elevation of [Ca2+]i (33). We (22) have shown previously that intracellular Ca2+ release could be triggered in gonadotropes when [Ca2+]i was raised to a few hundred nanomolar concentrations by inhibition of the sarcoplasmic/endoplasmic reticulum Ca2+-adenosine triphosphatase (SERCA pumps) or by brief depolarization to activate extracellular Ca2+ entry via voltage-gated Ca2+ channels. Thus, it is also possible that the delayed or disproportionately large [Ca2+]i rise observed here was a result of extracellular Ca2+ entry (via the LTX channels) acting as a positive feedback stimulus on the release of Ca2+ from the IP3-sensitive stores.

LTX-triggered exocytosis
In gonadotropes, when the LTX-induced [Ca2+]i elevation was less than 1 µM, little or no Ca2+-dependent exocytosis was triggered. This result contrasts with that of bovine chromaffin cells, in which the large Na+ load from the LTX-induced current is postulated to raise local [Ca2+]i indirectly by driving the exchanger in Na+ extrusion mode and thus causing exocytosis (10). In this study, exocytosis was typically triggered when the LTX-induced [Ca2+]i rise was more than 1 µM. In the presence of LTX (30 nM), the cumulative exocytosis could reach more than 1000 fF. The exocytosis of an average vesicle (~250 nm) in gonadotropes should increase the membrane capacitance by about 1.9 fF (34). Thus, a 1000-fF increase in membrane capacitance corresponds to the release of about 520 vesicles. The amount of exocytosis triggered by LTX is comparable to that observed in cells during stimulation by its natural stimulating hormone, GnRH (15). Thus, LTX can be a potent stimulator of LH release in gonadotropes. In the majority of cells (~75%), LTX failed to trigger any exocytosis in the absence of extracellular Ca2+, suggesting that extracellular Ca2+ entry via the LTX channels is the major trigger for exocytosis.

Robust exocytosis was also triggered when the LTX-induced [Ca2+]i rise, in turn, elicited intracellular Ca2+ release, presumably from the IP3-sensitive stores. In gonadotropes, intracellular Ca2+ release from the IP3-sensitive stores (when stimulated via GnRH or flash photolysis of caged IP3) triggers robust exocytosis, and this had been ascribed to local Ca2+ gradients that were generated by rapid intracellular Ca2+ release near the exocytic sites (34). Consistent with this, Fig. 7Go shows that during each cycle of intracellular Ca2+ release (indicated by the asterisks), the rate of increase in [Ca2+]i was rapid. When [Ca2+]i rose to about 1 µM in the first Ca2+ wave of Fig. 7Go, the corresponding exocytosis already reached rates of hundreds of femtofarads per s. Thus, LTX can trigger robust Ca2+-dependent exocytosis in gonadotropes via at least two mechanisms. First, when the LTX-induced current is large [hundreds of picoamperes, as with the higher concentration (30 nM) of LTX], sufficient extracellular Ca2+ enter the cell to raise [Ca2+]i to several micromolar concentrations, and exocytosis with rates of up to hundreds of femtofarads per sec can be triggered. Secondly, even when the LTX-induced current is small [as with the lower concentration (4 nM) of LTX], the rise in [Ca2+] may be augmented by intracellular Ca2+ release from the IP3-sensitive store, and this, in turn, can trigger robust exocytosis.

In chromaffin cells, LTX has been reported to cause enhancement of depolarization-evoked secretion (9) as well as the secretion stimulated by 30 µM Ca2+ (11). In most gonadotropes, exocytosis is triggered only when the LTX-induced [Ca2+]i is above 1 µM. Thus, it is unlikely that LTX had a significant shift in the minimum [Ca2+] required to trigger Ca2+-dependent exocytosis. Nevertheless, in gonadotropes, LTX might have enhanced Ca2+-dependent exocytosis. For example, the three large [Ca2+]i elevations shown in Fig. 7Go were similar in amplitude (~2 µM), but the accompanying exocytosis was increasingly bigger with each successive [Ca2+]i rise. However, [Ca2+]i was changing at different rates in such experiments, and the relative contribution of intracellular Ca2+ release cannot be assessed; it is difficult to determine whether LTX modulates the Ca2+ sensitivity of exocytosis in gonadotropes. Future experiments using flash photolysis of caged Ca2+ compounds to directly elevate [Ca2+]i will be needed to clarify this.

In approximately 25% of the gonadotropes examined, LTX triggered a small and slow increase in membrane capacitance that was not accompanied by any [Ca2+]i elevation. The membrane capacitance started to rise before the opening of the LTX channels, suggesting that LTX may stimulate exocytosis that is independent of the consequences of its channel-forming activities. This LTX-triggered exocytosis also persisted in the absence of extracellular Ca2+ or when [Ca2+]i was buffered with 10 mM BAPTA. In the squid giant synapse (35), the microdomains created by voltage-gated Ca2+ entry could be significantly blunted by about 4 mM BAPTA. Therefore, under our experimental condition (10 mM BAPTA), the effect of any localized release of intracellular Ca2+ should be abolished. In the absence of extracellular Ca2+, LTX has also been reported to increase the rate of spontaneous release of transmitters in neuromuscular junction (36), central synapses (37), and synaptosomes (12, 27). In rat brain synaptosomes, the LTX-induced Ca2+-independent release of norepinephrine was postulated to be nonvesicular (12). In this study, the capacitance measurement was employed. Thus, the Ca2+-independent action of LTX in gonadotropes observed here involves vesicular release (exocytosis). Any nonvesicular release induced by LTX will not be detected by our measurements. At present, the underlying mechanism for this Ca2+-independent action of LTX in gonadotropes is not clear, but several possibilities can be considered. First, LTX has been reported to cause Ca2+-independent release of neurotransmitter in PC-12 cells, and it has been suggested that synaptotagmin I is essential for this action (38). Interestingly, pituitary gonadotropes were reported to have no detectable expression of synaptotagmin I to III (39). However, a role of synapatotagmin I in the LTX response in gonadotropes cannot be totally ruled out, as it is possible that synaptotagmin I to III may be present at very low concentration in gonadotropes. Alternatively, some other isoforms of synaptotagmin may be involved. Secondly, in insulin-secreting ß-cells, the LTX-stimulated insulin release is independent of channel formation and is postulated to involve a direct stimulation of exocytosis via the LTX receptor, CIRL/latrophilin (14). As the deduced topology of CIRL resembles G protein-coupled receptors, the LTX-mediated exocytosis in ß-cells may involve G protein activation. In gonadotropes, direct activation of G proteins indeed stimulates slow (a few femtofarads per sec) exocytosis or (16) LH release (15) that do not require [Ca2+]i elevation. Thus, it is tempting to speculate that G protein activation also underlies the Ca2+-independent action of LTX in gonadotropes. However, we have been unable to detect any significant expression of CIRL/latrophilin in the anterior pituitary gland (unpublished collaboration with Dr. A. G. Petrenko). As gonadotropes comprise about 5% of the anterior pituitary cells, and the Ca2+-independent action of LTX was observed in only a fraction of the gonadotropes, the possibility that low levels of CIRL/latrophilin are present in some gonadotropes cannot be completely excluded here.


   Acknowledgments
 
We thank Dr. J. D. Neill for the gift of antibodies, and Drs. V. G. Kransnoperov and A. G. Petrenko for discussion and the gift of LTX.


   Footnotes
 
1 This work was supported by the Canadian Medical Research Council (Grant MT-12070) and the Alberta Heritage Foundation for Medical Research. Back

2 Medical Research Council and Alberta Heritage Foundation for Medical Research scholars. Back

Received September 4, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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