Thyrotropin induces the acidification of the secretory granules of parafollicular cells by increasing the chloride conductance of the granular membrane.

Secretory granules of sheep thyroid parafollicular cells contain serotonin, a serotonin-binding protein, and calcitonin. Parafollicular cells, isolated by affinity chromatography, were found to secrete serotonin when activated by thyrotropin (TSH) or elevated [Ca2+]e. TSH also induced a rise in [Ca2+]i. We studied the effect of these secretogogues on the pH difference (delta pH) across the membranes of the secretory granules of isolated parafollicular cells. The trapping of the weak bases, acridine orange or 3-(2,4 dinitro anilino)-3'-amino-N-methyl dipropylamine (DAMP), within the granules was used to evaluate delta pH. In contrast to lysosomes, which served as an internal control, the secretory granules of resting parafollicular cells displayed a limited and variable ability to trap either acridine orange or 3-(2,4 dinitro anilino)-3'-amino-N-methyl dipropylamine; however, when parafollicular cells were stimulated with TSH or elevated [Ca2+]e, the granules acidified. Weak base trapping was also used to evaluate the ATP-driven H+ translocation into isolated parafollicular granules. The isolated parafollicular granules did not acidify in response to addition of ATP unless their transmembrane potential was collapsed by the K+ ionophore, valinomycin. Secretory granules isolated from TSH-treated parafollicular cells had a high chloride conductance than did granules isolated similarly from untreated cells. Furthermore, ATP-driven H+ translocation into parafollicular granules isolated from TSH-stimulated parafollicular cells occurred even in the absence of valinomycin. These results demonstrate that secretogogues can regulate the internal pH of the serotonin-storing secretory granules of parafollicular cells by opening a chloride channel associated with the granule membrane. This is the first demonstration that the pH of secretory vesicles may be modified by altering the conductance of a counterion for the H+ translocating ATPase.

Abstract. Secretory granules of sheep thyroid parafollicular cells contain serotonin, a serotonin-binding protein, and calcitonin. Parafollicular cells, isolated by affinity chromatography, were found to secrete serotonin when activated by thyrotropin (TSH) or elevated [Ca2÷]~. TSH also induced a rise in [Ca2÷]~. We studied the effect of these secretogogues on the pH difference (ApH) across the membranes of the secretory granules of isolated parafollicular cells. The trapping of the weak bases, acridine orange or 3-(2,4 dinitro anilino)-Y-amino-N-methyl dipropylamine (DAMP), within the granules was used to evaluate ApH. In contrast to lysosomes, which served as an internal control, the secretory granules of resting parafollicular cells displayed a limited and variable ability to trap either acridine orange or 3-(2,4 dinitro anilino)-3'-amino-N-methyldipropylamine; however, when parafollicular cells were stimulated with TSH or elevated [Ca2+],, the granules acidified. Weak base trapping was also used to evaluate the ATP-driven H ÷ translocation into isolated parafollicular granules. The isolated parafollicular granules did not acidify in response to addition of ATP unless their transmembrane potential was collapsed by the K ÷ ionophore, valinomycin. Secretory granules isolated from TSH-treated parafollicular cells had a high chloride conductance than did granules isolated similarly from untreated cells. Furthermore, ATP-driven H + translocation into parafollicular granules isolated from TSH-stimulated parafollicular cells occurred even in the absence of valinomycin. These results demonstrate that secretogogues can regulate the internal pH of the serotonin-storing secretory granules of parafollicular cells by opening a chloride channel associated with the granule membrane. This is the first demonstration that the pH of secretory vesicles may be modified by altering the conductance of a counterion for the H ÷ translocating ATPase.
T HE parafollicular cell of the mammalian thyroid gland is a neural crest derivative (Le Douarin et al., 1974;Polak et al., 1974) that remains capable of expressing neural characteristics when exposed to nerve growth factor (Barasch et al., 1987a). Parafollicular cells also produce the neurotransmitter, 5-hydroxytryptamine (5-HT) I , which they costore in granules with the peptide hormone calcitonin (Pearse, 1966;Bussolati and Pearse, 1967;Falck and Owman, 1968;Jaim-Etchevery and Zeiher, 1968;Gershon and Nunez, 1973;Nunez and Gershon, 1978a;Barasch et al., 1987b). The secretion of these hormones is stimulated by elevated [Ca+2]e (Hirsch and Munson, 1969;Gershon et al., 1978). In addition to calcitonin and 5-HT, parafollicular cell granules contain a serotonin-binding protein (SBP) specific to 5-HT-storing cells of neurectodermal origin (Barasch et al., 1987b). Although the function of SBP in the granules of parafollicular cells or in the synaptic vesicles of serotonergic neurons has yet to be established, SBP binds 5-HT best at neutral or alkaline pH (Tamir et al., 1976). Since many secretory granules are known to maintain an acid pH interior (Johnson et al., 1982;Catty et al., 1980;Russell and Holz, 1981;Orci et al., 1986), we investigated the mechanisms that control the internal pH of the secretory granules of parafollicular cells.
The interior of lysosomes (Ohkuma et al., 1982) as well as that of secretory granules is acidified by a H + translocating ATPase that can generate a transmembrane H + gradient (ApH) and a membrane potential (AW). The extent to which the H + translocating ATPase generates either gradient is dependent on the conductance of the organelle membrane to counterions, which permit charge compensation. In fact, the fractional contribution that ApH and A¥ make to the proton gradient can be experimentally manipulated by altering the concentration of permeant ions in suspensions of isolated vesicles Glickman et al., 1983). Chromaffin granule (Pazoles et al., 1980;Njus, 1983) and lysosomal (Schneider, 1981;Dell' Antone, 1979) membranes are sufficiently permeable to CI-that they rapidly acidify when treated with ATP in a C1--containing buffer. When C1-is absent, however, these vesicles fail to generate a ApH and instead form a A~.
In intact cells, a variation has been found in the internal pH of endocytic and secretory vesicles. This has been demonstrated for organelles of the endocytic pathway with fluorescein-labeled macromolccules  and for organelles of the secretory pathway by weak base trapping (Anderson et al., 1984). The cause of the heterogeneity in intravesicular pH is unkown. Mechanisms accounting for variations in ApH may include differing conductances of the membranes of different vesicles to ions, including H +, or the presence of electrogenic ion translocating ATPases, in addition to the H ÷ ATPase (Fuchs et al., 1988). The current study was done to test the hypothesis that the differences in the pH inside different granules result from a physiological regulation of the conductance of individual granules for counterions. In this paper, we demonstrate that granules of thyroid parafoUicular cells can acidify in response to stimulation by secretagogues. Furthermore, sccretagogues increase the granule membrane conductance to CI-, which would be expected to collapse the potential across the granular membrane, thereby allowing acidification of the granule interior. In this way, these cells can maximize the granule ApH by reducing the granule A~; therefore, individual granules containing a H + translocating ATPase can vary in internal pH by varying the chloride conductance of their limiting membrane.

Isolation of ParafoUicular Cells
Parafollicular cells were isolated by an affinity chromatographic method (Bernd et al., 1981) as modified by Barasch et al. (1987a,b). Essentially, sheep throid glands were dissociated with trypsin. The resultant suspension of thyroid cells (consisting of 85 % red blood cells, 13.5 % follicular cells, and 1.5 % parafollicular cells) was treated with thyroid stimulating hormone (thyrotropin [TSH]; 5 mU/ml; Armour Pharmaceutical Co., Tarrytown, NY) and then loaded onto a column of Sepharose 6-MB beads (Pharmacia Fine Chemicals, Piscataway, NJ) at 37°C to which thyroglobnlin (Sigma Chemical Co., St. Louis, MO) had been coupled. Follicular cells become phagocytic in the presence of TSH and "attempt" to phagocytize the beads; thus, they are retained on the column while parafollicular cells pass through. Parafollicular cells eluting from the columns were further purified by centrifugation (10 rain at 800 g) on a 10% Ficoll (Pharmacia Fine Chemicals) cushion made in MEM. This step allowed separation of smaller particles such as red blood cells, bacteria, and yeast, from parafollicular cells. The purified parafollicular cells were washed and then plated onto plastic dishes and cultured for 2-3 d (Barasch et al., 1987a); 97% of the cells in the final suspension were parafollicular cells. In most experiments, parafollicular cells were resuspended in Hepes-buffered MEM (10 ram, pH 7.4) containing neither bicarbonate nor phosphate.
Cells were fixed at 37oc for 3 h with a mixture of 4 % formaldehyde (from paraformaldehyde), 0.5% glutaraldehyde, 3% sucrose in 0.1 M KPO4 buffer (pH 7.4). Fixed tissues were washed and excess aldehydes were quenched with 50 mM NI-hC1 in buffer for 15 min. The cells were again washed, dehydrated, and then embedded in a hydrophilic resin (type LR White; Ernest E Fullam, Inc., Schenectady, NY). Thin sections of DAMPtreated cells were picked up on Formvar-coated nickel grids and treated with 10% horse serum in a solution containing 0.1 M Tris-HCI, 0.9% NaCI, and 0.125 % Triton X-I00 (TBS-X) for 30 min. The sections were then incubated overnight at 4°C with a mouse monoclonal antibody to dinitrophenol (50 ttg/ml) that cross reacts with DAMP (clone HDP-1, No. 15, generously supplied by Dr. Anderson, University of Texas, Health Science Center, Dallas, TX) diluted in TBS-X with 4% horse serum. The sites where DAMP was concentrated were visualized with an anti-mouse secondary antibody coupled to colloidal gold (Janssen Pharmaceutica, Bcarse, Belgium). Sections were post fixed in 2.5 % glutaraldebyde after immunocytochemical processing and examined in a JEOLCO JEM 1200 EX electron microscope.

Analysis of Acridine Orange Trapping in Parafollicular Cells
Chromatographically purified parafollicular cells were incubated with acridine orange (6 tiM) for 5 rain and were either examined by fluorescence microscopy or loaded into a fluorescence-activated cell sorter (FACS/Id, Becton Dickinson & Co., Mountain View, CA). For fluorescence microscopy, cells were excited by light at 488 um and emitted light was passed through a 520-nm-long band pass filter. For analyses with the FACS (Consort 400 Program) a single population of cells was selected by forward, narrow angle light scatter (a reflection of particle size), and was excited with light at 488 nm from an argon gas laser at 300--400 mwatts. The red fluorescence emission intensity (a function of the concentration of acridine orange) was coUcctcd through a 650 + 50-urn-wide band pass filter) and plotted as a function of cell number. Parafollicular cells, suspended in Hepes-buffered MEM, were treated with TSH (5-30 mU/ml), CaCl2 (5-15 raM), valinomycin (1.0-2.5 tiM) or with nigericin 0.5 I~M) for 0.5-30.0 rain at 37°C, incubated with acridine orange (6 tiM) for 5 rain, and then introduced into the FACS. When the effects of brief exposures to these substances were studled, cells were incubated with acridine orange for 5 rain before addition of the experimental substance and analyzed in the FACS immediately thereafter. To confirm the homogeneity of the selected cells and to obtain ultrastructural evidence that the analyzed cells remained viable in the FACS, the cells were sorted, collected in a fixative containing 4% glutaraldehyde in 0.1 M KPO4 buffer, and then processed for examination by transmission electron microscopy. Cells were sorted at 1-2,000 cells/s and coincident fluorescent events were not allowed to exceed 15%.

Proton Transport Assay: Uptake of Acridine Orange by Isolated Parafollicular Cell Granules
ATP-dependent transport of H + into isolated parafollicular cell granules was evaluated by measuring granular accumulation of acridine orange in response to the addition of ATE Acridine orange trapping in parafollicular cell granules was measured by dual wavelength absorbance spectroscopy (492-540 nm; Glickman et al., 1983;(}luck and AI-Awqati, 1984). Parafollicular cell granules were isolated on two sequential discontinuous metrizamide gradients as described by Barasch et al. (1987b), except with the addition of 1 mM dithiothreitol (DTT) to all solutions. Aliquots of the granular fractions of each gradient (0.2-0.4 mg protein) were diluted in 150 mM KCI, 6 mM MgCI2, 2 mM Tris, 2 mM morpholino ethane sulfonic acid, and 1 mM DTT, 1.5 ml (transport buffer) incubated with 6 l~M acridine orange and placed in a stirred cuvette at room temperature. H* transport was initiated by adding Tris-ATP (0.33 raM, fitrated to pH 7.0) after a steady baseline had been achieved, when the granules were equilibrated with acridine orange.
ATP-dcpondent transport of H + into parafollicular cell granules isolated from TSH-treated cells was evaluated by measuring the granular accumulation of acridine orange. Thyroid glands were dissociated with trypsin in MEM (see above) and aliquots of suspended cells were treated with TSH (30 mU/ml) for 30 rain. Treated and unstimulated cells were diluted into 3 vol of 0.33 M sucrose (4°C) and granules were then rapidly isolated on the first metrizamide gradient (see above). The trapping of acridine orange in granules treated with ATP was measured by fluorometry (490 excitation, 520 emission) in a stirred cuvette at room temperature, as described above.

5-HT Secretion by Isolated ParafoUicular Cells
Plated, chromatographically purified parafollicular cells were treated with the monoamine oxidase inhibitor, nialamide (10 lxM), for 3 h. The ceils (1.3 × 106 cells/ml) were then suspended in Hepes-butfered MEM without glutamine and aliquots were incubated at T/°C for 1-35 rain with the secretogogues, TSH (30 mU/ml), or CaCI2 (7.5 mM). The incubation was terminated by rapidly chilling the cells to 4°C and gentle centrifugation (800 g, 15 min). The supernatant was acidified to 0.1 N HCI and then analyzed by reverse phase HPLC (Reinhard et al., 1980;Barasch et al., 1987b) for 5-HT release. Secretogogue-stimulated release was reported as the difference in the 5-HT concentration in supernatants of control and treated cells.

Measurement of Intracellular Ca ÷z in Isolated ParafoUicular Cells
The intracellular concentration of free Ca +2 and the effect of TSH on this concentration was estimated from the fluorescence of the dye, Fura-2 (Grynkiewicz et al., 1985). Chromatographically purified parafollicular cells (Bernd et al., 1981) were loaded with Fura-2 by incubation at 3"/*C with 20 I~M Fura-2 AM (the permeant ester derivative of the dye; Molecular Probes, Inc., Junction City, OR) for 30 rain. Fura-2 AM is hydrolyzed by cytoplasmic esterases to yield free Fura-2, which is trapped intracellularly. The Fura-2-1oaded cells were washed, suspended in Hepes-buffered MEM solution, and loaded into a stirred cuvette. Cells were excited alternately with light at 380 and 340 nm and the intensity oftbe emitted light was measured at 505 am. A calibration curve was constructed with 2.5 IxM Fura-2 acid in a solution containing 150 mM KCI, 1.0 mM MgCI2, 1.0 mM EGTA, and Ca +2 titrated with a calcium electrode in a range of 0.1 laM to 10.0 mM. Highly purified bovine TSH (25 U/mg protein) was obtained from Dr. B. Erlanger at Columbia University (New York).

Measurement of Cytoplasmic pH in Isolated Parafollicular Cells
The pH of the cytosol of parafollicular cells was estimated from the fluorescence of the pH-sensitive dye, BCECF (Thomas et al., 1979). Chromatographically purified parafoilicular cells were loaded with the permeant ester BCECF-AM (12.1 laM; Molecular Probes, Inc., Junction City, OR) for 30 win at 37°C. BCECF-AM is hydrolyzed by cytosolic esterases to yield free BCECE which is trapped in the cytosol. BCECF-loaded cells were washed, resuspended in Hepes-buffered MEM and loaded into a stirred cuvette (2 × 10 ~ cellstml). Cells were excited alternately with light at 460 and 490 nun and the intensity of emitted light was measured at 530 nm. A calibration curve was constructed by cleaving the BCECF ester (1.2 raM) with porcine liver esterase (Sigma Chemical Co.; 2 ~tl/ml) for 2 h in a solution consisting of 140 mM KCI, 5 mM NaCI, 1 mM MgCI2, 10 m Hepes, at pH 7.95.

Measurement of Chloride EJ~ux from Isolated ParafoUicular Cell Granules
ParafoUicular cell granules were isolated on metrizamide gradients (Barasch et al., 1987b) and were loaded with 36CI-by freezing overnight in a buffer containing 130 mM KCI, 6 mM MgCi2, 10 mM imidazole (pH 7) with 36C1-(16 mCi/g CI-; New England Nuclear, Boston, MA). Granules were then thawed and diluted 63-fold into the same salt solution at 4°C, with 2.5 ~tM valinomycin. Valinomycin was added to abolish a membrane potential. Aliquots were rapidly collected on Fiberglas GFC filters (Whatman International, Ltd., Maidstone, England) under a vacuum, and the filters washed with 2 ml of 250 mM sucrose (Landry et al., 1987). The effect of extragranular CI-on 36C1-efflux was analyzed by diluting aliquots of granules 63-fold in Cl--free, gluconate salts (130 mM K gluconate, 6 mM Mg gluconate, 10 mM imidazole, pH 7) with 2.5 ~tM valinomycin, and collecting granules on filters. In both sets of experiments, the amount of 36CI-remaining associated with granules over time was expressed as a fraction of the retained radioactivity, 30 s after resuspension.

ATP-dependent Transport of H ÷ by Isolated Parafollicular Cell Granules
We assayed ATP-driven acidification of parafollicular cell granules by measuring the accumulation of the weak base acridine orange in purified granules. Using a previously characterized technique (Barasch et al., 1987b) with two sequential discontinuous metrizamide gradients, we isolated a granule fraction that is enriched 20-fold over the homogenate in the granule marker, 5-HT, but depleted in rnitochondrial and lysosomal markers. In this fraction the granules, identified by electron microscopy, occupy '~63 % of the particulate volume (Barasch et al., 1987b). When we added ATP (0.33 mM) to granules suspended in a KC1 containing "transport buffer" there was no trapping of acridine orange. However, when we preincubated the granules with 2 I~M valinomycin, a K+-selective ionophore, the addition of ATP stimulated the initial rate of acridine orange trapping (from 0 to 8 + 2.5 x 10 -3 A absorbancz/min per mg protein; Fig.  1). Proton-conducting ionophores such as 2 lxM nigericin (a H/K exchanger) or 5 I~M CCCP (a H + carrier) released the trapped acridine orange, implying that the uptake of acridine orange was due to the development of a transmembrane pH difference (Fig. 1). Furthermore, N-ethylmaleimide (10 IxM), which inhibits many nonmitochondrial ATPases, abolished the valinomycin/ATP-induced trapping of acridine orange.
Since the induction of a K + conductance in the granule membrane by valinomycin should collapse a membrane potential and thereby stimulate H ÷ transport, these data suggest that H + transport, and thus acridine orange trapping, in isolated granules is limited by a membrane potential gen-  brahe potential difference could only arise if the granule membrane had little or no conductance to counterions such as K + or C1 +.

Acidification of ParafoUicular Cell Granules in Intact Cells
To examine whether granule acidification in intact parafollicular cells is limited in a manner analogous to that encountered in isolated granules, we compared the ApH of individual granules with that of follicular cell and neutrophil lysosomes by visualizing the subeelhlar distribution of the weak base DAMP with an electron microscopic immunogold technique (Anderson et al., 1984;Anderson and Pathak, 1985). When dissociated thyroid cells were incubated with DAMP for 15 rain and fixed without pelleting, <1% of parafollicular cell granules showed concentration of the tracer (Fig. 2 A). In contrast, we found labeling of almost all lysosomes of follicular cells and of a subset of granules in neutrophils (Fig. 2, B and C). When we incubated thyroid cells for 30 min with higher concentrations of DAMP (by washing the ceils free of albumin, to which DAMP binds) we found a variable concentration of DAMP immunoreactivity in gran-ules ( Fig. 3 A). Follicular cell lysosomes, however, were uniformly and more intensely labeled than parafollicular granules. These labeling patterns suggest that ApH varies from granule to granule in parafollicular cells and is generally less than that across lysosomal membranes; however, when we treated thyroid cells with valinomycin (10 gM) for 1 min before fixation of DAMP, we found an increased percentage of parafollicular cell granules labeled per cell (Fig. 4) and increased numbers of immunogold particles over granules (Fig.  3 B). Valinomycin treatment therefore maximized the ApH across the membranes of parafollicular cell granules in intact cells, just as it did in isolated granules. This observation suggests that in contrast to lysosomes, H + transport by parafollicular cell granules in situ is limited by a membrane potential. The addition of nigericin (7.5 lxM) virtually eliminated all DAMP immunoreactivity from the granules (Fig. 3 C), indicating that accumulation of DAMP in granules reflects the ApH across the granular membrane. The effect of nigericin was apparent after brief exposure of cells to the ionophore (0.5 min).
To examine weak base trapping in a large population of cells, we incubated chromatographically purified parafollicular cells with acridine orange and examined them by fluorescence microscopy (Allison and Young, 1964). We estimated the transmembrane H ÷ gradient of intracellular granules by observing red shifts in the wavelengths of emitted light that parallel the intragranular concentration of the dye. The cells were heterogeneous in their ability to trap acridine orange; some cells showed a punctate orange-red fluorescence while others did not trap acridine orange at all  . 5). To evaluate this range in concentration of the dye quantitatively, we incubated purified parafollicular cells with acridine orange (6 ~tM, 5 min) and analyzed the intensity of red fluorescence of individual cells with a FACS. Cells were selected for analysis by size using forward angle light scatter. In one experiment these cells were collected by cell sorting and processed for electron microscopy. This population of cells was entirely composed of parafollicular cells (,x,97 %; Fig. 6), indicating that the sorted cells were viable and that the intracellular distribution of acridine orange probably refleets its accumulation in granules. The ceils varied over a 3.5-fold range in the intensity of red fluorescence of acridine orange with a large standard deviation equal to 84 + 8 % of the mean red fluorescence. When we added valinomycin (2.5 I~M, 1 min), the mean intensity of red fluorescence increased to 122.4 + 6.8% of control (p < 0.01; Fig. 7).
These results suggest that the membrane conductance of granules must be low in intact cells as it is in isolated granules thereby preventing the granules from maximally acidifying. The heterogeneity of the internal pH of granules may result from physiological regulation of the conductance of individual granules for counterions.

Secretagogue Induced 5-HT Secretion from ParafoUicular Cells
We incubated chromatographically purified parafoUicular cells with TSH or Ca +2 for varying periods of time. The concentration of 5-HT in the supernatant was analyzed to detect the release of endogenous 5-HT (Fig. 8). The maximum rate of 5-HT secretion induced by increased [Ca+2]e (5.2 + Figure 5. Chromatographically purified parafollicular ceils were incubated with acridine orange (6.0 ItM) and examined by fluorescence microscopy. Cells were initially photographed in color and then rephotographed on panchromatic black and white film through a red filter. The brightness of the image reflects the intensity of red fluorescence. Note the heterogeneity between cells in accumulation of acridine orange. Bars, 10.0 lam.
2.1 x |0 -19 mol/ceU per min) was faster than that induced by TSH (0.4 + 0.2 x 10 -19 mol/cell per rain; p < 0.05). Moreover, the effect of increased [Ca+2]e peaked within 5 min while the amount of 5-HT released by TSH continued to increase for up to 20 rain.
We investigated whether TSH affects the intracellular concentration of free Ca +2 ([Ca+t]0 in parafollicular ceils by measuring the fluorescence of the calcium-sensitive probe, Fura-2. To validate the Fura-2 technique in parafollicular cells we added the calcium ionophore, ionomycin (2 lxM; extracellular Ca +2 = 1.8 mM) to cells loaded with Fura-2. Ionomycin increased the fluorescence signal of the cells identified in a fluorescence microscope (Fig. 9 A). Subsequent chelation of [Ca+2]¢ with EGTA reduced the fluores-cence ratio, suggesting that this method can measure changes in [Ca+2]i in parafollicular ceils. We found that the [Ca+2]i was 176 + 6 nM (n = 13) in isolated cells analyzed in a spectrofluorometer. The addition of a purified preparation (0.75 U/ml) or of a commercial preparation (30 mU/ml; Armour Pharmaceutical Co.) of TSH caused an immediate increase in [Ca+2]i to 211 + 7 nM (Fig. 9 B; p < 0.001; n = 8). The effect of TSH on Fura-2 fluorescence lasted 7 + 1 rain (n = 8).  (56 mM for 5 min), and were examined by electron microscopic immunocytochemistry (as described above)• With each of these treatments, the percentage of granules per cell labeled by DAMP increased (Fig. 10), as did the number of immunogold particles over granules (Fig. 11). In a similar manner, when we incubated isolated, acridine orange-loaded parafollicular cells with these same agents and analyzed the fluorescence with a FACS, we found an increased intensity of fluorescence emission. Elevation of the [Ca+2]o (>5 mM; Fig. 12 A) was maximally effective when cells were incubated for ,x,1 min (113 + 4% of control; p < 0.005), and longer incubations failed to further change the intensity of  Figure 8. Secretion of 5-HT induced by Ca +2 or TSH. Chromatographically purified parafollicular cells were exposed to elevated extracellular Ca ÷2 (7.5 mM) or TSH (30 mU/ml) and the 5-HT concentration in the suspending medium was measured. Both agents stimulated the cells to release 5-HT, but Ca ÷2 did so more rapidly. ==, calcium; n, TSH.

Effect of Secretagogues on the Transmembrane A pH of ParafoUicular Cell Granules
the red fluorescence. TSH (30 mU/ml; Fig. 12 B) was maximally effective after 10 rain of treatment (134 + 10 % of control; p < 0.025), an effect that persisted with continued incubation with TSH to 35 min. Finally, elevation of [K÷]o (56 mM; 1 min) rapidly increased the red fluorescence (Fig 12  C), approximately to the same extent as did TSH. The increased labeling of individual granules with DAMP or of populations of cells with acridine orange, therefore, demonstrates that secretogogues increase the transmembrane pH gradient of granules as does valinomycin. An increase in the ApH across granular membranes must result from either acidification of the parafoUicular cell granules or alkalinization of its cytoplasm. To ascertain whether secretogogues and valinomycin stimulate alkalinization of the cell cytosol, we loaded purified cells with the pH sensitive dye, BCECF, and measured the pH of the cytosol. The addition of NI-hCI to BCECF-loaded cells increased the cell pH while nigericin caused a rapid acidification (probably by exchanging K ÷ for H ÷ at the plasma membrane) validating the use of BCECF as a probe of intracellular pH in parafollicular ceils (Fig. 13 B). We found that the initial cytoplasmic pH waS 6.81 + 0.26 (n = 24 experiments) 2. The addition of TSH caused an immediate acidification by 0.13 + 0.02 pH units (p < 0.001; n = 13 experiments) that persisted with further incubation (Fig. 13, A and B). In contrast to TSH, neither the addition of Ca 2+ (n = 5 experiments), nor valinomycin (n = 5 experiments) induced a change in the cytosolic pH. These results indicate that secretogogues, as well as valinomycin, stimulate the trapping of weak bases in granules by inducing granular acidification rather than by inducing cytoplasmic alkalinization.
2. Calibration of BCECF was done by measuring the fluorescence of the dye in buffered solutions of different pH in a cuvette. Intracellular calibrations of fluorescein dyes show an alkaline shift in the pK, of the dye by 0.3-0.4 pH U (van Adeisberg and AI-Awqati, 1986). The recorded intracellular pH of parafollicular cells therefore is a minimum value.

Effect of TSH on the Chloride Permeability of ParafoUicular Cell Granule Membranes
Many intraceUular vesicles such as Golgi and clathrin-coated vesicles, whose contents are acidified by a H + translocating ATPase, have a chloride channel in parallel to the ATPase (Glickman et al., 1983). Removal of CI-(or closure of the C1-channel) reduces the pH gradient of these vesicles in vitro. Since valinomycin, an electrogenic ionophore, stimulated granule acidification, secretogogues might similarly change the internal pH of granules by altering the conductance of the granular membrane. We investigated the C1conductance of granules purified from dissociated thyroid cells that had been treated with TSH, by loading the isolated granules with 36C1-in a KCl-buffered solution (pH 7). We then measured tracer efttux from granules at 4°C upon dilution into 63 vol of the same buffer by collecting aliquots of granules on filters over time after resuspension. We found that granules isolated from cells treated with TSH showed a rapid efllux of 36C1-(reaching background in 3 min), while the 36C1-efflux from granules of untreated cells was very slow (Fig. 14). To test whether 36Cl-efflux was due to a 36C1-/C1-exchanger, we resuspended 36Cl--loaded granules in a buffered gluconate salt solution and collected aliquots over time. Again, we found that granules from TSHstimulated cells showed 36C1-efl]ux while control granules did not. This suggests that external CI-does not drive 36C1etttux and suggests that the tracer diffuses from the granules through a channel rather than by C1/CI exchange. These results demonstrate that TSH induces the opening of a CIchannel, a modification that is stable during the period of granule isolation,

Granules Isolated from TSH-treated Cells Acidify Their Contents
To investigate whether the increased chloride conductance of granules from TSH-treated cells results in increased granule acidification, we measured ATP-dependent proton transport in granules from TSH-stimulated cells. Thyroid cells were dissociated and aliquots were stimulated with TSH for 30 min. Granules were then rapidly prepared on the first metrizamide gradient. We found that the addition of ATP to these granules resulted in a rapid uptake of acridine orange. Addition of valinomycin did not enhance the rate of uptake. Granules isolated from untreated cells of the same preparation, however, showed little trapping of acridine orange. Unlike the granules from TSH-stimulated cells, valinomycin increased the rate of H + transport by the control granules at least threefold ( Fig. 15; cf. Fig. 1). Since these results were obtained with a partially purified fraction, we can not exclude the possibility that other organelles, in addition to the granules, increased their proton pumping after TSH stimulation. The acidification of granules (and perhaps other organelles) from TSH-treated cells was due to the opening of a chloride channel since these granules failed to acidify in C1--free, gluconate media (not shown). Granules from TSHtreated cells, unlike granules from unstimulated cells, can therefore maximally acidify their contents in the absence of an electrogenic ionophore. This result implies that the granule membrane conductance no longer limits H-transport. TSH-induced CI-channel opening (as demonstrated above) would collapse a membrane potential and enhance granule acidification.

Discussion
In this paper we demonstrate that TSH has multiple effects on parafollicular cells, including the induction of secretion of 5-HT (see also Nunez and Gershon, 1983). This is the first direct demonstration that TSH is a parafollicular cell secretogogue. 5-HT is taken up by follicular ceils (Gershon and Nunez, 1976) where it stimulates follicular hormone secretion (Nunez and Gershon, 1978b;Melander and Sundler, 1972), while calcitonin has distant effects on osteoclasts. Thus, two cells of distinct embryological lineage both respond to TSH. It is possible that parafollicular cells regulate follicular cell activity through the release of 5-HT (Nunez and Gershon, 1978b). Whether follicular cell hormones influence parafollicular cell metabolism remains to be determined.
We found that parafollicular cell secretogogues (TSH and elevated [Ca+2]o) induce the acidification of 5-HT storage granules. Secretogogues have been found to induce acidification of other organelles. For example, glucose acidifies 13 cell granules of pancreatic islets (Pace and Sachs, 1982). Moreover, the intracellular canaliculi of parietal cells also acidify in response to histamine (DiBona et al., 1979;Berglindh et al., 1980) and microsomes from histamine-treated parietal cells have an increased membrane conductance for K-and for C1- (Cuppoletti and Sachs, 1984). We demonstrated that TSH induces granule acidification by opening a C1-channel in the membranes of secretory granules. Opening of a C1-channel would allow charge compensation for electrogenic H-pumping, thereby stimulating the H-translocating ATPase and inducing granule acidification. Furthermore, we found that substitution of K ÷ gluconate for KCI reduced the rate of acidification of granules isolated from TSH-treated cells, suggesting that CI-, but not K-, acts as the primary counterion.  Figure 14. The effect of TSH on the granule membrane conductance for CI-, measured by 36C1-elilux from purified granules. Dissociated thyroid cells were treated with TSH and granules isolated by density gradient centrifugation. Isolated granules were loaded with 36C1-, then diluted and rapidly collected on filters. Data are expressed as a fraction of the initial content of 36C1-. Granules isolated from TSH-treated ceils show rapid efflux of the tracer, while control granules show little loss of the tracer over time.
since increased membrane conductance for chloride was present in the isolated granules hours after the cells were stimulated by TSH, modification of the channel was probably covalent rather than by direct Ca ~÷ activation. TSH is known to increase the cAMP concentration in follicular cells (Bastomsky and McKenzie, 1967) and thus a cAMP-dependent kinase may be involved in the opening of the chloride channel in granular membranes. In fact, Schoumacher et al. (1987) has demonstrated that epithelial chloride channels are opened by cAMP-dependent protein kinases. Alternatively, the increased [Ca+2]i that follows stimulation by TSH, may trigger Ca÷Lactivated kinases to induce the covalent modification. Although we have not examined the chloride conductance of granules isolated from KCl-depolarized -TSH ÷TSH A~~ " L AIIVA~ Figure 15. Acridine orange uptake into parafollicular cell granules isolated from TStt-treated cells and from unstimulated thyroid cells measured by fluorometry. In the control vesicles there is little ATP driven uptake of acridine orange; however, pretreatment with valinomycin stimulates the inward movement of acridine orange (downward deflection). In contrast, the addition of ATP to granules isolated from TSH-treated cells causes the rapid uptake of acridine orange, which is not further enhanced by treatment with valinomycin.
cells or from cells stimulated by elevated [Ca÷2]e, a similar mechanism might also occur in these cells. Variation in chloride conductance can also explain the heterogeneity in the internal pH of different organdies. Endosomes are less acidic than lysosomes (Murphy et al., 1984; and differences in pH distinguish at least two types of endocytic vesicles involved in the recycling of receptors . Moreover, individual members of a single class of organelle can vary in pH, as occurs with maturing beta cell granules (Orci et al., 1986). Vesicles that generate a large ApH such as lysosomes (Schneider, 1981;Dell' Antone, 1979;Okuma et al., 1982) and chromaffin granules Njus, 1983) are highly permeable to C1-. In contrast, Golgi vesicles that maintain a small transmembrane pH gradient must conduct C1-poorly, since valinomycin increases proton pumping in these vesicles, even in the presence of 150 mM CI- (Glickman et al., 1983). This suggests that Golgi vesicles and lysosomes maintain their CI-conductances at different levels. Variation in conductance may be regulated by membrane-associated kinases and phosphatases that can produce stable modifications in CI-channels. Indeed, recent studies by Landry et al. (1987) demonstrated that the chloride conductance of vesicles enriched in Golgi membranes was reduced by treatment with ATP or ATP-y-S, but not by nonhydrolysable ATP analogues. This suggests that these vesicles contain a membrane-associated kinase that can close the channel. Heterogeneity of organellar pH can not result from the regulation of the number or activity of the vesicular H ÷ ATPase, since a single H ÷ ATPase molecule with a turnover number of 50-100 ions/s would maximally acidify a granule (radius = 0.2 ltm) within 1 min.
The role that the proton electrochemical gradient plays in the function of these granules remains to be determined. The electrical potential generated by the H ÷ ATPase in the resting cell might act as a driving force for 5-HT accumulation in parafollicular granules. It is not clear, at present, what the function of secretogogue-induced granule acidification is in the physiology of parafollicular cells; however, it is possible that granule acidification may facilitate secretion of 5-HT by enhancing the dissociation of 5-HT from SBP before exocytosis. Upon fusion of granules with the plasma membrane and exposure of SBP to alkaline extracellular pH, rebinding of 5-HT would be prevented since SBP has a reduced binding capacity for 5-HT in the relatively high [Na÷]o and [Ca÷2]e .