Two types of secretory vesicle are represented in synaptic terminals. One is the small (50 nm) synaptic vesicle (SV) ()that stores small molecule neurotransmitters(1) . SVs can be replenished locally by endocytic recycling from the plasma membrane (2, 3, 4) and thus are derived from endosomes(5, 6) . The other type of vesicle, which is also found in neuroendocrine cells, is a large, dense cored vesicle that contains proteins and/or peptides packaged in the trans-Golgi network and transported to terminals for regulated secretion(7) . Paraneurons, such as thyroid parafollicular (PF) cells, are endocrine cells that are closely related to neurons(8) . They are derived embryologically from the neural crest (9) and can be induced by nerve growth factor to assume a neuronal phenotype in vitro(10) . The secretory vesicles of PF cells share characteristics of both the large trans-Golgi network-derived, peptide-containing dense cored vesicles and the small, endosome-derived, low molecular weight neurotransmitter-containing SVs. PF secretory vesicles resemble large, dense cored vesicles in their size, initial formation in the trans-Golgi network(11, 12) , and content of calcitonin and other peptides(13) ; however, like SVs, PF secretory vesicles contain a small molecule neurotransmitter, 5-HT(14, 15, 16) , and they recycle(17) . In contrast to the peptides that are added to vesicles at the time of their formation in the trans-Golgi network, 5-HT is loaded into PF vesicles by transmembrane transport from the cytosol(15, 18) . As is generally true of monoamines in SVs, this transport is mediated by a transporter protein in the vesicular membrane (19) and driven by a transmembrane proton gradient (18) that is established by the vesicular H-ATPase(20) . SV proteins, including the synaptotagmin I, synaptophysin, and synaptobrevin(1) , are also present in the membrane of PF secretory vesicles(13, 48) .

PF secretory vesicles exhibit properties that have not yet been detected either in neuronal SVs or large, dense cored vesicles. The internal environment of PF secretory vesicles is regulated by the same stimuli that initiate secretion(18, 22, 48) . The interior of the secretory vesicles of PF cells becomes acidic only when the cells are stimulated by a secretogogue, such as increased extracellular Ca ([Ca]). Because the membranes of PF vesicles are not permeable to Cl under resting conditions, influx of H is limited by the generation of a transmembrane potential gradient ()(18, 22) . In response to stimulation of PF cells with a secretogogue, a Cl channel is opened in the vesicular membrane(18) . The opening of this channel dissipates the and permits Cl to enter vesicles as a counterion (48) so that transport of H is no longer electrogenic (18) and acidification of vesicles can proceed(22) .

The mechanism responsible for stimulus acidification coupling has not yet been determined. A 64-kDa protein (p64), which is identical to a Cl channel that has been cloned from epithelial cells (23) , is present in the membranes of PF secretory vesicles(48) . This protein has potential phosphorylation sites, and stimulation of PF cells with a secretogogue increases the phosphorylation of vesicular p64(48) . Acidification of vesicles, moreover, is inhibited by compounds that antagonize a variety of protein kinases or phosphatases (48) . These observations are consistent with the following working hypotheses: (i) p64 is the Cl channel in vesicular membranes that permits the secretion-induced entry of Cl into vesicles; (ii) the state and site of phosphorylation of p64 determine whether the channel is open or closed; (iii) signal transduction pathways (still to be identified) couple plasmalemmal secretogogue receptors to protein kinases and/or phosphatases that determine the level of phosphorylation of the channel protein. Because stimulus acidification and stimulus secretion coupling can be dissociated(48) . The signal transduction pathways responsible for each are not identical.

Because the acidification of secretory vesicles is evoked by exposing PF cells to secretogogues, both acidification and secretion could conceivably be mediated either by a cell surface Ca receptor or by a channel that is directly sensitive to the [Ca]. Recently, each of the [Ca]-sensitive endocrine cells, parathyroid chief cells(24) , and PF cells(49) , have been found to express a plasmalemmal calcium receptor (CaR). The CaR is a heptahelical integral membrane protein that when expressed in oocytes, couples to a pertussis toxin-sensitive G protein(24) . Because [Ca] evokes secretion in PF cells and inhibits secretion in parathyroid cells, it is possible that the CaR in each cell type is coupled to a different G protein. The current experiments were undertaken to test the hypothesis that the PF cell CaR is responsible for mediating the effects of [Ca] on vesicle acidification. Our data demonstrate that the acidification of the secretory vesicles in PF cells is mediated via activation of the CaR and that the CaR is coupled via G and to a cascade of second messengers, including inositol trisphosphate (IP), cytosolic free Ca, NO, cGMP, and protein kinase G, resulting in the phosphorylation and gating of the Cl channel in the membranes of PF secretory vesicles.

MATERIALS AND METHODS

Isolation of PF Cells

Determination of Cytosolic Ca Using Fura-2/AM

Acidification of PF Vesicles

Immunocytochemical Detection of NO Synthase

[S]GTPS Binding to G Proteins and Immunoprecipitation

Pertussis Toxin

In order to test the effect of PTx on the binding of [S]GTPS in response to stimulation by ligand, membranous fraction obtained from PF cells (50 µg in 100 µl) was preincubated with activated PTx (50 µg/ml) in the solubilization buffer containing NAD (100 µM), ATP (2 mM), EDTA (1 mM), and dithiothreitol (1 mM) at 37 °C for 60 min. Membranes were washed and incubated with 2.0 nM [S]GTPS. Immunoprecipitation of the G subunits were then investigated in control and PTx-treated fractions.

Drugs and Chemicals

RESULTS

The CaR Agonists, Ca and Gd, Cause the Secretory Vesicles of PF Cells to Acidify

Figure 1: Acidification of PF vesicles in response to CaR agonists is concentration-dependent. A, the concentration effect relationship for the [Ca]-induced acidification of vesicles. Note that the response does not saturate in the absence of the calcium channel blocker nimodipine but does so in its presence. B, the concentration effect relationships for the acidification of vesicles induced by Gd and [Ca]+ nimodipine. Note that Gd is a more potent agonist than [Ca] and that the effect of Gd saturates. The percentage of cells acidified in controls was 22.3 ± 4.5 % (n = 20). The percentage of cells where the color was not clear was 5 ± 2 % (n = 20); these cells were not counted. Dead cells were not fluorescent and appeared as ghosts.

Stimulation of PF Cells by the CaR Agonists, Ca or Gd, Induces the Binding of [S]GTPS to G and G

PF cell membranes were isolated and stimulated with Ca or Gd. Isolated PF cell membranes were incubated with [S]GTPS and exposed to Ca (1 mM) or Gd (250 µM). The radioactivity immunoprecipitated by antibodies that react specifically with G, G, or all known G subunits was determined. Baseline values were also ascertained for control preparations, in which membranes were incubated without agonists and in the presence of EGTA. The data were expressed as the ratio of the radioactivity immunoprecipitated from membranes incubated in the presence of agonists to that of the corresponding controls in the same experiment. The effectiveness and specificity of the antibodies were evaluated by immunoblotting (30) (Fig. 2). The antibodies we used for immunoprecipitation (anti-Gi and anti-Gq) were specific in that anti-Gi immunoprecipitated G but not G, and anti-Gq immunoprecipitated G but not G. Antibodies to the membrane Cl channel, p64, failed to immunoprecipitate either G or G. Both Ca and Gd were found to stimulate the binding of [S]GTPS to G and, to a much lesser extent, also to G (Fig. 3A and B) (p < 0.01 for Gd; p < 0.05 for Caversus control). The concentrations of the agonists used in these experiments were each supramaximal with respect to the acidification of vesicles (see Fig. 1B). At these concentrations, the degree of stimulation of [S]GTPS binding to G and G evoked by Ca was not significantly different from that evoked by Gd. The sum of the radioactivity immunoprecipitated by antibodies to G and G was equal to that immunoprecipitated by antibodies that react with all known G subunits, suggesting that no additional G proteins are stimulated by Ca or Gd. When membranes were treated with PTx, the Ca-induced binding of [S]GTPS to G was abolished (Fig. 3B); however, the stimulation by Ca of the binding of [S]GTPS to G was not affected by PTx (Fig. 3B).

Figure 2: Western blots showing the specificity of antibodies used to immunoprecipitate G and G. Lane 1, immunoprecipitate obtained with anti-Gq and probed with antibodies to G; lane 2, immunoprecipitate obtained with anti-Gq and probed with antibodies to G; lane 3, immunoprecipitate obtained with anti-Gi and probed with antibodies to G; lane 4, immunoprecipitate obtained with anti-p64 (membrane Cl channel) and probed with antibodies to G.

Figure 3: Gd and [Ca] activate G and G. Membranes isolated from PF cells were incubated with Gd (250 µM) or Ca (1.0 mM) in the presence of [S]GTPS. A, radioactive G and G were immunoprecipitated from membranes stimulated with Gd. The data are expressed as the experimental to control (no agonist) ratio, so that a value of 1.00 (dotted line) indicates no effect. Note that far more [S]G than [S]G was immunoprecipitated. B, [S]G and [S]G were immunoprecipitated from membranes stimulated with Ca. Again, as with membranes stimulated by Gd, [S]G [S]G. The activation of G but not that of G was prevented by preincubation of membranes with PTx. The experimental to control ratio for G stimulation by Ca was 1.26, which is a very small change but significantly different from 1.00 (t = 2.479; p < 0.05). 1590 ± 250 cpm were obtained in controls; following activation, the radioactivity associated with the same amount of protein of G was 6757 ± 1050 and 5962 ± 925 cpm for Gd and Ca, respectively. The radioactivity associated with G following activation was 2170 ± 100 and 2105 ± 50 cpm for Gd and Ca, respectively.

The CaR Agonists, Ca and Gd, Mobilize Ca from an Internal Source

Figure 4: Gd mobilizes Ca from an intracellular compartment in a concentration-dependent manner. Cells were loaded with fura-2 and exposed to Gd in a Ca-free medium. The [Ca] was determined by fluorescence. Note that there is good agreement between the ED for Gd-induced acidification and that for Gd release of [Ca].

Figure 5: [Ca] can increase [Ca] independently of Ca entry through nimodipine-sensitive Ca channels. Cells were loaded with fura-2 and exposed to Ca (10 µM-10 mM). The [Ca]was determined by fluorescence as in Fig. 3. The solid line shows the [Ca] measured as a function of [Ca] in the absence of nimodipine, whereas the dashed line shows the [Ca] as a function of [Ca] in the presence of nimodipine.

The Increase in [Ca] Participates in CaR-induced Vesicle Acidification

Figure 6: Both the mobilization of [Ca] and the acidification of secretory vesicles are inhibited by pretreatment of cells with thapsigargin. A, the [Ca] was measured in fura-2-loaded cells ± thapsigargin pretreatment as a function of time following challenge with Gd. The rise in [Ca] was prevented by thapsigargin. B, the [Ca] is shown as a function of [Ca]. Nimodipine was present to inhibit the entry of Ca through L-type calcium channels. [Ca] is lower than control in thapsigargin-treated cells; however, at [Ca]above 1 mM, a rise in [Ca] is seen despite the presence of nimodipine, possibly through nonvoltage-gated cation channels. C, both Gd- and [Ca]-induced acidification of vesicles is antagonized by thapsigargin (Gd > [Ca]).

Neither ryanodine (10 µM) nor caffeine (10 mM) increased [Ca] or acidified the vesicles of PF cells (data not illustrated). These observations suggest that PF cells lack ryanodine receptors and are consistent with the idea that the CaR mobilizes Ca from an IP-sensitive source. This hypothesis was tested. The CaR was stimulated with either Gd (250 µM) or a low concentration of Ca (1.0 mM), vesicle acidification was measured, and the ability of U 73122, a specific inhibitor of PI-phospholipase C, to antagonize the response was determined. U 73122 (10 µM) but not U 73343 (10 µM), an inactive congener of U 73122, blocked the Gd-induced acidification of PF vesicles (Table 2). U 73122 also inhibited the Gd-induced increase in [Ca] (Table 2). These data confirm that PI-phospholipase C and thus IP mediate the increase in [Ca] that follows stimulation of the CaR; moreover, the observations also provide additional support for the idea that the rise in [Ca] is a critical step in the signal transduction pathway that leads to vesicle acidification.

NO and cGMP Participate in CaR Signal Transduction

Figure 7: NO synthase immunoreactivity can be detected in PF cells by immunocytochemistry. A, isolated PF cells show NO synthase immunoreactivity. B, PF cells show no immunostaining when antibodies to NO synthase are omitted and nonimmune serum is substituted. Bars, 15 µm.

Figure 8: NO appears to participate in signaling for Gd- and [Ca]-induced vesicle acidification. A, Gd, [Ca], the NO donor, sodium nitroprusside (1.0 mM), and dibutyryl cGMP (1.0 mM) all induce vesicle acidification. Imino-ornithine (10 µM), a NO synthase inhibitor, blocks the response to Gd and [Ca] but not that to sodium nitroprusside or dibutyryl cGMP. Shaded bars, no inhibitor; bars with dots, imino-ornithine. B, LY 83583 (10 µM), a guanylyl cyclase inhibitor, antagonizes vesicle acidification in response to Gd, [Ca], and sodium nitroprusside but does not affect the response to dibutyryl cGMP. Rp-cGMPS (10 µM), an inhibitor of protein kinase G, antagonizes vesicle acidification induced by Gd and [Ca]. Lightly shaded bars, no inhibitor; hatched bars, LY 83583; darkly shaded bars, Rp-8-(4-chlorophenylthio)-cGMPS.

PKC Promotes Vesicle Acidification

Figure 9: PKC participates in mediating calcium channel-dependent but not calcium channel-independent vesicle acidification. A, the PKC activator, PMA (10 nM) induces vesicle acidification equivalent to that induced by Gd (250 µM). The response to PMA, but not that to Gd is blocked by the PKC inhibitor, staurosporine, or by down-regulation of PKC. B, vesicle acidification induced by a low concentration of [Ca] (1.0 mM), which is channel-independent (see Fig. 1A), is unaffected by staurosporine (10 nM) or down-regulation of PKC (PMA, 10 nM; overnight). In contrast, vesicle acidification induced by a high concentration of [Ca] (5.0 mM), which is channel-dependent, is antagonized by staurosporine or down-regulation of PKC. Note that the degree of acidification induced by 5.0 mM [Ca] in the presence of staurosporine or after PKC down-regulation is roughly equivalent to that evoked by 1.0 mM [Ca]. Shaded bars, no inhibitor; bars with thin hatching lines, staurosporine; bars with thick hatching lines, down-regulation.

Because studies with nimodipine indicated that an increment in vesicle acidification above that induced by the CaR occurred when Ca entered cells through L-type calcium channels (see Fig. 1A), we tested the possibility that PKC contributes to the Ca channel-related acidification of vesicles. PF cells were exposed to 5.0 mM [Ca] (rather than 1.0 mM) in the absence of nimodipine, so that Ca would enter cells through calcium channels. When this was done, vesicles acidified strongly (Fig. 9B). Both staurosporine and down-regulation of PKC now significantly antagonized vesicle acidification (Fig. 9B); however, neither of these inhibitory treatments totally prevented acidification of vesicles. Instead, the level of acidification induced by 5.0 mM Ca in the presence of staurosporine or after PKC down-regulation was reduced to a level that approximated that induced by 1.0 mM Ca (Fig. 9B). These data are consistent with the idea that although PKC does not mediate the basal acidification of vesicles induced by the CaR, it is responsible for the Ca channel-related increment in vesicle acidification.

Activation of the CaR by Ba Leads to Vesicle Acidification

Figure 10: Ba induces vesicle acidification in nimodipine-pretreated PF cells. A, in the absence of nimodipine, Ba (2.5 mM) does not induce a significant degree of vesicle acidification. In contrast, in the presence of nimodipine, Ba induces vesicle acidification equivalent to that evoked by Ca (1.0 mM; ± nimodipine) or Gd (250 µM). Black bars, nimodipine absent; hatched bars, nimodipine present. B, 2.5 mM Ba strongly inhibits acidification in response to 5.0 mM Ca but not that in response to 250 µM Gd. In fact, vesicle acidification in response to Gd and Ba is greater than that evoked by either ion alone, suggesting that the effects of Gd and Ba on vesicle acidification are additive. Black bars, Ba absent; hatched bars, Ba present.

DISCUSSION

The current study was undertaken to determine whether the CaR is responsible for the phenomenon of secretogogue-evoked acidification of secretory vesicles in PF cells and if so, to characterize the signal transduction pathway. It was, however, first necessary to distinguish effects mediated by the CaR from those resulting from the entry of Ca through plasmalemmal calcium channels. Gd was particularly valuable for this purpose. Gd is both an efficient ligand at the CaR (8, 24) and a highly effective calcium channel blocker(39) ; therefore, when Gd is used to activate the CaR, responses are not complicated by the influx of Ca through calcium channels. Gd was found to be an agonist that induced vesicle acidification in a concentration-dependent manner. A similar, but more complicated response was elicited by [Ca]. At low concentrations of [Ca] ( 1.0 mM), vesicle acidification was unaffected by the addition of calcium channel blockers and the concentration effect curve was roughly parallel to that of Gd, although it was shifted to the right. These data suggest that vesicle acidification in response to low concentrations of [Ca] and Gd is the result of an action at a common receptor and that Gd is a more potent agonist. At higher concentrations of [Ca], however, an increment in acidification occurred that was beyond that induced by Gd. This increment was blocked by the L-type calcium channel blocker, nimodipine. It is possible that this increment represents the influx of Ca in a subpopulation of cells that does not respond to low concentrations of Ca. These observations suggest that acidification of PF vesicles can be induced by mechanisms that are both independent of and dependent on plasma membrane calcium channels.

A third CaR agonist, Ba, provided further insight into the nature of calcium channel-independent vesicle acidification. By itself, Ba causes PF cells to secrete, but it does not induce vesicle acidification(48) . This observation provided the initial evidence that acidification of vesicles is not a requirement for secretion by PF cells and that the transduction mechanisms responsible for vesicle acidification and secretion are not identical. When added together with nimodipine, however, Ba caused vesicles to acidify; moreover, the response to Ba was synergistic with that of Gd. In contrast, in the absence of nimodipine, Ba inhibited the vesicle acidification induced by low concentrations of [Ca]. These observations suggest that Ba, like Gd and [Ca], can cause vesicles to acidify through a mechanism that is independent of calcium channels. This effect of Ba, however, is only manifested when Ba is prevented from entering PF cells. If Ba enters cells, as it does in the absence of calcium, channel blockade can evidently inhibit signal transduction from a plasmalemmal receptor. This intracellular action accounts for the ability of Ba to inhibit the action of [Ca] when Ba is allowed to enter PF cells. In the absence of nimodipine, therefore, the plasmalemmal action of Ba is probably masked by a counteracting intracellular effect. The intracellular inhibitory action of Ba, however, is not manifest when Ba is applied together with Gd, which prevents the entry of Ba through calcium channels. The observation that each of three CaR agonists, Ca, Gd, and Ba can induce vesicle acidification by a plasmalemmal action that is independent of calcium channels, supports the idea that the CaR is responsible for their common effect. The nimodipine-dependent increment in vesicle acidification induced by concentrations of [Ca] above 1.0 mM suggests that Ca entry through L-type calcium channels can also contribute to vesicle acidification through a process that does not involve the CaR.

If the CaR is responsible for agonist-induced vesicle acidification, then stimulus-acidification coupling would be expected to be mediated by a signal transduction pathway that involves a G protein(8, 24) . The identification of such a pathway would thus simultaneously provide evidence for a role of the CaR in vesicle acidification and also provide important insight into the responsible mechanism. We thus tested the roles played by G proteins, likely second messengers, and effectors in mediating vesicle acidification in response to CaR agonists (Gd or a low concentration of [Ca] in the presence of nimodipine). Vesicle acidification induced by Gd and Ca was abolished by PTx, and these agonists also activated G (as determined by measuring the binding of [S]GTPS to immunoprecipitated G). Although the agonists were also found to activate G, this effect was not, like that of the activation of G, inhibited by PTx. The ability of Ca to activate Gq was slight but significant (p < 0.05 when each sample was compared with its own control). It thus seems likely that coupling of the CaR to G is a critical step in stimulus acidification coupling. The coupling of the CaR to G in PF cells is similar to the coupling of the receptor expressed in oocytes(24) . Coupling of the CaR to G may be involved in other responses of PF cells to stimulation of the receptor. Stimulus-induced acidification, for example, is distinct from stimulus-induced secretion, which is resistant to PTx(48) .

Several observations supported the idea that the G protein activated by the CaR stimulates PI-phospholipase C. Both Gd and low concentrations of [Ca] mobilized Ca from an internal pool that was sensitive to thapsigargin. Thapsigargin inhibits the calcium pumps of the ER, preventing the reuptake of Ca, which leads to an irreversible depletion of IP-mobilizable internal Ca stores(32) . Because ryanodine did not mobilize Ca, PF cells probably lack ryanodine receptors; therefore, cyclic ADP-ribose, which acts through these receptors(40) , is not likely to be involved in the CaR-stimulated mobilization of Ca from internal stores. More direct evidence for the participation of PI-phospholipase C was obtained with the PI-phospholipase C inhibitor, U 73122(4) , which specifically blocked both the mobilization of [Ca] and the acidification of vesicles in response to Gd or [Ca]. These data suggest that PI-phospholipase C activated secondary to the coupling of the CaR to G causes the release of IP, which mobilizes [Ca]. Although the ability of the subunits of the G family of G proteins to activate PI-phospholipase C has been demonstrated directly(3) , it has been difficult to demonstrate PI-phospholipase C activation by the subunits of PTx-sensitive G proteins(5, 41, 42) ; nevertheless, receptor-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate is blocked by PTx in many different types of cell(2, 41, 43) . Many reports have indicated that the subunits, rather than the subunits, are the components of PTx-sensitive G proteins, including G, which activate PI-phospholipase C(41, 42, 44) . It is thus possible that the subunits released from G following its coupling to the CaR are responsible for the CaR-dependent activation of PI-phospholipase C. A role for G, however, cannot be ruled out. Both the and the subunits of G have been found to participate in the adenosine A1 receptor-mediated activation of PI-phospholipase C(45) .

Thapsigargin not only antagonized the agonist-induced increase in [Ca], but also antagonized stimulus acidification coupling, suggesting that the PI-phospholipase C-mediated increase in [Ca] is a critical component of the transduction pathway. It seems likely that the effect of the mobilization of [Ca] is mediated by activation of NO synthase. PF cells were found by immunocytochemistry to contain NO synthase (and NADPH diaphorase activity), which is known to be activated by Ca/calmodulin(46) ; moreover, the NO synthase inhibitor, imino-ornithine, blocked the vesicle acidification induced by Gd or Ca but not that induced by the NO generator, sodium nitroprusside. In addition, as expected for an effect mediated by NO synthase(28) , Gd- and Ca-induced vesicle acidification were antagonized by an inhibitor of guanylyl cyclase (LY 83583, which also blocked the effects of sodium nitroprusside) and G kinase (Rp-cGMPS). The involvement of NO synthase in stimulus acidification could account for the previous observation that inhibition of calcineurin, a Ca/calmodulin-dependent protein phosphatase, antagonizes Ca-stimulated vesicle acidification(48) . Phosphorylated NO synthase is a substrate for calcineurin, and phosphorylation of NO synthase decreases its catalytic activity(47) . The proposed signal transduction pathway for CaR-induced vesicle acidification is shown in Fig. 11.

Figure 11: A model showing the proposed signal transduction pathway leading to the acidification of parafollicular vesicles in response to extracellular Ca or Gd.

Although the evidence outlined above supports the hypothesis that the CaR is coupled to vesicle acidification, stimulation of this receptor is not the only means by which vesicle acidification is controlled. It is probable that the entry of Ca through nimodipine-sensitive L-type calcium channels also causes vesicles to acidify, that this effect is independent of the CaR, and that the Ca channel-related and CaR-induced components of vesicle acidification are additive. Thus, a nimodipine-inhibitable increment in acidification is seen when Ca is present at concentrations above that needed to stimulate the CaR. The magnitude of the CaR-mediated component of acidification can be estimated either from the maximal response to Gd or from that to Ca in the presence of nimodipine, which is about the same. The observation that the calcium channel-related component of vesicle acidification, but not that which appears to be stimulated by the CaR, is blocked by inhibitors of PKC (staurosporine and calphostin C) and by PKC down-regulation confirms that independent mechanisms are responsible for the two components. The diacyl glycerol that is generated by the action of PI-phospholipase C activated in response to CaR stimulation could activate PKC. Diacyl glycerol activation, however, seems to be insufficient for vesicle acidification. The data are consistent with the idea that the component of vesicle acidification related to calcium channels is mediated by PKC, whereas that related to the CaR is mediated by a G kinase. In both cases, phosphorylation of p64, the chloride channel in the membranes of PF vesicles could be responsible for increasing the Cl conductance that underlies acidification(48) .

The role played by the CaR in normal homeostasis remains to be determined. [Ca] was observed to activate the receptor in concentrations that are below those normally found in extracellular fluid. The effect of such low concentrations of [Ca], however, were only detected in comparison to cells incubated in media containing EGTA, which are almost Ca-free. It is possible that the CaR is partially desensitized under physiological conditions and thus responds in vivo only to higher than normal concentrations of [Ca]. If so, it is likely that secretogogue-induced vesicle acidification will be mediated both by the CaR and the entry of Ca through L-type calcium channels, which was found to occur at concentrations of >1.0 mM. PF cells are depolarized by exposure to greater than resting concentrations of [Ca](21) . It is conceivable that stimulation of the CaR contributes to the [Ca]-induced depolarization of PF cells. Whether or not this is so, the depolarization is probably linked to the opening of L-type calcium channels, which are voltage-dependent, and thus to the activation of the parallel PKC-dependent pathway of vesicle acidification.

The function of the phenomenon of stimulus-induced vesicle acidification also needs to be clarified. The uptake of 5-HT from the cytosol, where it is synthesized, into the vesicles in which it is stored is driven more by the proton gradient than by the potential difference across the vesicular membrane(18) . Most of the vesicles of nonstimulated PF cells are not acidic because the development of a membrane potential difference limits acidification until the vesicular Cl channel opens in response to stimulation. It thus seems likely that the loading of 5-HT into vesicles is episodic and greatest after stimulus-induced vesicle acidification. The potential-driven uptake of 5-HT into the vesicles of resting cells is probably small in comparison with the pH gradient-driven uptake into the vesicles of secretogogue-stimulated cells. In fact, this enhancement of 5-HT uptake by secretogogue stimulation has actually been demonstrated(18) . In contrast, the stability of 5-HT within vesicles may be decreased and its osmotic activity may be increased by acidification. PF vesicles contain a matrix protein, serotonin-binding protein, that binds 5-HT in a neutral but not acidic medium(16, 22) . Acidification would thus be expected to facilitate the secretion of 5-HT. Serotonin-binding protein is retained by secretory vesicles, even though they recycle(17) . Serotonin-binding protein, therefore, probably remains membrane-bound during exocytosis and is recaptured with the vesicular membrane at endocytosis. Although exposure to secretogogues does not cause the fusion of all secretory vesicles with the plasma membrane, it does cause virtually all vesicles to become acidic(22) . Vesicles can thus load episodically with 5-HT, which is retained during the intersecretory period. Retention may be facilitated by binding to serotonin-binding protein or other matrix components. Vesicle acidification may also contribute to the processing of calcitonin, somatostatin, or other products within vesicles(25) .

FOOTNOTES

*

This study was supported in part by National Institutes of Health Grant DK19743. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Division of Neuroscience, New York State Psychiatric Inst., New York, NY 10032. Tel.: 212-960-5837; Fax: 212-740-5329.

()

The abbreviations used are: SV, synaptic vesicle; AO, acridine orange; [Ca], elevated external Ca concentration; [Ca], elevated intracellular free Ca concentration; CaR, plasmalemmal Ca receptor; GTPS, guanosine 5`-O-(3-thiotriphosphate); 5-HT, 5-hydroxytryptamine, (serotonin); imino-ornithine, L-N-(1-iminoethyl)ornithine; PF, parafollicular cells; PKC, protein kinase C; PMA, phorbol 12-myristate, 13-acetate; IP, inositol trisphosphate; PI, phosphatidylinositol; PTx, pertussis toxin.

REFERENCES

1. Jahn, R., and Sudhof, T. C. (1994) Annu. Rev. Neurosci. 17, 219-246 [CrossRef][Medline] [Order article via Infotrieve]
2. Fain, J. N. (1990) Biochim. Biophys. Acta 1053, 81-88 [Medline] [Order article via Infotrieve]
3. Smrcka, A. V., and Sternweis, P. C. (1993) J. Biol. Chem. 268, 9667-9674 [Abstract/Free Full Text]
4. Yule, D. I., and Williams, J. A. (1992) J. Biol. Chem. 267, 13830-13835 [Abstract/Free Full Text]
5. Wu, D., Lee, C. H., Rhee, S. G., and Simon, M. I. (1992) J. Biol. Chem. 267, 1811-1817 [Abstract/Free Full Text]
6. Linstedt, A. D., and Kelly, R. B. (1991) Neuron 7, 309-317 [CrossRef][Medline] [Order article via Infotrieve]
7. Thureson-Klein, A. K., and Klein, R. L. (1990) Int. Rev. Cytol. 121, 67-126 [Medline] [Order article via Infotrieve]
8. Nemeth, E. F. (1995) News Physiol. Sci. 10, 1-5 [Abstract/Free Full Text]
9. Le Douarin, N. M. (1982) The Neural Crest , Cambridge University Press, Cambridge
10. Barasch, J. M., Mackey, H., Tamir, H., Nunez, E. A., and Gershon, M. D. (1987) J. Neurosci. 7, 2874-2883 [Abstract]
11. Nunez, E. A., and Gershon, M. D. (1978) Int. Rev. Cytol. 52, 1-80 [Medline] [Order article via Infotrieve]
12. Amara, S. G., Jonas, V., and Rosenfeld, M. G. (1982) Nature 298, 240-244 [CrossRef][Medline] [Order article via Infotrieve]
13. Weiler, R., Cidon, S., Gershon, M. D., Tamir, H., Hogue-Angeletti, R., and Winkler, H. (1989) FEBS Lett. 257, 457-459 [CrossRef][Medline] [Order article via Infotrieve]
14. Nunez, E. A., and Gershon, M. D. (1976) Am. J. Anat. 147, 375-392 [CrossRef][Medline] [Order article via Infotrieve]
15. Nunez, E. A., and Gershon, M. D. (1972) Endocrinology 90, 1008-1024 [Abstract/Free Full Text]
16. Barasch, J. M., Tamir, H., Nunez, E. A., and Gershon, M. D. (1987) J. Neurosci. 7, 4017-4033 [Abstract]
17. Tamir, H., Liu, K. P., Hsiung, S. C., Adlersberg, M., and Gershon, M. D. (1994) J. Neurochem. 63, 97-107 [Medline] [Order article via Infotrieve]
18. Cidon, S., Tamir, H., Nunez, E. A., and Gershon, M. D. (1991) J. Biol. Chem. 266, 4392-4400 [Abstract/Free Full Text]
19. Liu, Y., Peter, D., Roghani, A., Schuldiner, S., Prive, G. G., Eisenberg, D., Brecha, N., and Edwards, R. H. (1992) Cell 70, 539-551 [CrossRef][Medline] [Order article via Infotrieve]
20. Moriyama, Y., and Nelson, N. (1989) J. Biol. Chem. 264, 18445-18450 [Abstract/Free Full Text]
21. Hsiung, S. C., Tamir, H., and McGhee, D. S. (1993) Neuroscience Abs. 19, 1333
22. Barasch, J., Gershon, M. D., Nunez, E. A., Tamir, H., and Al-Awqati, Q. (1988) J. Cell Biol. 107, 2137-2147 [Abstract/Free Full Text]
23. Landry, D., Sullivan, S., Nicolaides, M., Redhead, C., Edelman, A., Field, M., Al-Awqati, Q., and Edwards, J. (1993) J. Biol. Chem. 268, 14948-14955 [Abstract/Free Full Text]
24. Brown, E. M., Gamba, G., Riccardi, D., Lombardi, M., Butters, R., Kifor, O., Sun, A., Hediger, M. A., Lytton, J., and Hebert, S. C. (1993) Nature 366, 575-580 [CrossRef][Medline] [Order article via Infotrieve]
25. Shields, D. (1994) Biochimie (Paris) 76, 257-264
26. Bernd, P., Gershon, M. D., Nunez, E. A., and Tamir, H. (1981) J. Cell Biol. 88, 499-508 [Abstract/Free Full Text]
27. Tamir, H., Liu, K. P., Hsiung, S. H., Adlersberg, M., Nunez, E. A., and Gershon, M. D. (1990) J. Neurosci. 10, 3743-3753 [Abstract]
28. Moncada, S., Palmer, R. M. J., and Higgs, E. A. (1991) Pharmacol. Rev. 43, 109-142 [Medline] [Order article via Infotrieve]
29. Gluck, S., and Al-Awqati, Q. (1984) J. Clin. Invest. 73, 1704-1710
30. Wang, H. Y., Eberle-Wang, K., Simansky, K. J., and Friedman, E. (1993) J. Pharmacol. Exp. Ther. 267, 1002-1010 [Abstract/Free Full Text]
31. Hess, P. (1990) Annu. Rev. Neurosci. 13, 337-356 [CrossRef][Medline] [Order article via Infotrieve]
32. Ghosh, T. K., Bian, J., Short, A. D., Rybak, S. L., and Gill, D. L. (1991) J. Biol. Chem. 266, 24690-24697 [Abstract/Free Full Text]
33. Hope, B., Michael, G., Knigge, K., and Vincent, S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2811-2814 [Abstract/Free Full Text]
34. Bert, D., and Snyder, S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 9030-9033 [Abstract/Free Full Text]
35. McCall, T., Feelish, R., Palmer, M., and Moncada, S. (1991) Brit. J. Pharmacol. 102, 234-239 [Medline] [Order article via Infotrieve]
36. Young, H., McConalogue, K., Furness, J., and DeVente, J. (1993) Neuroscience 55, 583-596 [CrossRef][Medline] [Order article via Infotrieve]
37. Pandol, S. J., and Schoeffield-Payne, M. S. (1990) J. Biol. Chem. 265, 12846-12853 [Abstract/Free Full Text]
38. Przywara, D. A., Chwdhury, P. S., Bhave, S. V., Wakade, T. D., and Wakade, A. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 557-561 [Abstract/Free Full Text]
39. Racké, K., and Schwörer, H. (1993) Naunyn-Schmiedeberg's Arch. Pharmacol. 347, 1-8
40. Berridge, M. J. (1993) Nature 361, 315-325 [CrossRef][Medline] [Order article via Infotrieve]
41. Park, D., Jhon, D. Y., Lee, C. W., Lee, K. H., and Rhee, S. G. (1993) J. Biol. Chem. 268, 4573-4576 [Abstract/Free Full Text]
42. Blank, J. L., Brattain, K. A., and Exton, J. H. (1991) J. Biol. Chem. 267, 23069-23075 [Abstract/Free Full Text]
43. Cockcroft, S. (1987) Trends Biochem. Sci. 12, 75-78
44. Dickenson, J. M., Camps, M., Gierschik, P., and Hill, S. J. (1995) Eur. J. Pharmacol. 15, 393-398
45. Murthy, K. S., and Makhlouf, G. M. (1995) Mol. Pharmacol. 47, 1172-1179 [Abstract]
46. Dawson, T. M., Bredt, D. S., Fotuhi, M., Hwang, P. M., and Synder, S. H. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7797-7801 [Abstract/Free Full Text]
47. Dawson, T. M., Steiner, J. P., Dawson, V. L., Dinerman, J. L., Uhl, G. R., and Snyder, S. H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9808-9812 [Abstract/Free Full Text]
48. Tamir, H, Piscopo, I., Liu, K. P., Hsiung, S. C., Adlersberg, M., Nicolaides, M., Al-Awqati, Q., and Gershon, M. D. (1994) Endocrinology 135, 2045-2057 [Abstract]
49. Garrett, J. E., Tamir, H., Kifor, O., Simin, R. T., Rogers, K. V., Mithal, A., Gagel, R. F., and Brown, E. M. (1995) Endocrinology 136, 5202-5211 [Abstract]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				K.-p. Liu, A. F. Russo, S.-c. Hsiung, M. Adlersberg, T. F. Franke, M. D. Gershon, and H. Tamir Calcium Receptor-Induced Serotonin Secretion by Parafollicular Cells: Role of Phosphatidylinositol 3-Kinase-Dependent Signal Transduction Pathways J. Neurosci., March 15, 2003; 23(6): 2049 - 2057. [Abstract] [Full Text] [PDF]

				E. N Pothos, E. Mosharov, K.-P. Liu, W. Setlik, M. Haburcak, G. Baldini, M. D Gershon, H. Tamir, and D. Sulzer Stimulation-dependent regulation of the pH, volume and quantal size of bovine and rodent secretory vesicles J. Physiol., July 15, 2002; 542(2): 453 - 476. [Abstract] [Full Text] [PDF]

				E. M. Brown and R. J. MacLeod Extracellular Calcium Sensing and Extracellular Calcium Signaling Physiol Rev, January 1, 2001; 81(1): 239 - 297. [Abstract] [Full Text] [PDF]

				T. Shimizu, S. Morishima, and Y. Okada Ca2+-sensing receptor-mediated regulation of volume-sensitive Cl- channels in human epithelial cells J. Physiol., November 1, 2000; 528(3): 457 - 472. [Abstract] [Full Text] [PDF]

				K.-p. Liu, S.-c. Hsiung, M. Adlersberg, T. Sacktor, M. D. Gershon, and H. Tamir Ca2+-Evoked Serotonin Secretion by Parafollicular Cells: Roles in Signal Transduction of Phosphatidylinositol 3'-kinase, and the gamma and zeta Isoforms of Protein Kinase C J. Neurosci., February 15, 2000; 20(4): 1365 - 1373. [Abstract] [Full Text] [PDF]

				L. Gama, L. M. Baxendale-Cox, and G. E. Breitwieser Ca2+-sensing receptors in intestinal epithelium Am J Physiol Cell Physiol, October 1, 1997; 273(4): C1168 - C1175. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tamir, H. Articles by Gershon, M. D. Search for Related Content PubMed PubMed Citation Articles by Tamir, H. Articles by Gershon, M. D. Social Bookmarking                      What's this?