Dopamine inhibits cell swelling-induced prolactin secretion in MMQ cells by blocking Ca2+ influx

Molecular and Cellular Endocrinoiogy, 82 (1991) 99-106

99

0 1991 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/91/$03.50 MOLCEL 02633

Dopamine inhibits cell swelling-induced prolactin secretion in MMQ cells by blocking Ca”’ influx Noriyuki Sato, Xiangbing Wang and Monte A. Greer Section

of ~~d~cr~~~lo~,

~e~art~~e?tt of Medicine, Oregon Health Sciences U~~ii~e~s~~, Portland, OR 97201, USA.

(Received 29 May 1991: accepted 24 July 1991)

Key words: Thyrotropin;

Profactin; Calcium channel: MMQ cell; Secretion; Osmolar concentration

Summary To evaluate the role of Ca2+ influx on hormone secretion induced by cell swelling, we have utilized a prolactin (PRL)-secreting rat tumor cell line, MMQ, which has plasmalemma dopamine receptors. Medium hyposmolarity or osmotically equivalent isotonic urea caused prompt cell swelling and a rise in both [Ca2+li and PRL secretion in a dose-dependent manner. Dopamine inhibited the induced increase in both [Ca2+li and PRL secretion in a dose-dependent manner but the maximum inhibition was only 50%. This effect of dopamine was prevented by haloperidol. Depletion of medium CaZf or blocking Ca’+ influx with nifedipine completely abolished the osmotically induced rise in both [Ca2’li and PRL secretion. These data indicate that Ca* f influx through nifedipine-sensitive Ca*‘- channels is an essential component of PRL secretion induced by osmotic cell swelling in MMQ cells and that a dopaminergic receptor-linked mechanism influences the opening of these channels.

Introduction Recently the concept that cell volume changes play an important direct role in the regulation of cell function has been gaining strength (Seeman et al., 1969; K&berg, 1987; Young et al., 1987; Kazilek et al., 1988; Baquet et al., 1990; Corasanti et al., 1990; Gleeson et al., 1990; Watford, 1990; Wirtz and Dobbs, 1990). Cell swelling is a powerful stimulator of hormone secretion (Greer et al., 1983, 1985; Sato et al., 1990d) while cell shrink-

Address for correspondence: Monte A, Greer, M.D., Section of Endo~rinolo~. Department of Medicine, Oregon Health Sciences University, Portland, OR 97201, U.S.A. Tel. (503) 494-8484; Fax (503) 494-6990.

age inhibits hormone secretion (Brown et al., 1978; Hampton and Holz, 1983; ChazenbaIk et al., 1988; Sato et al., 1991b). In pituitary tumorderived GH,C, cells there is a Ca2+ influx-related transduction mechanism between cell volume changes and hormone secretion {Wang and Chase, 1986; Sato et al., 1990d). Ca*’ plays an important role in the regulation of both cell volume and hormone secretion (Sato et al., 1990b, c>. Although Ca2+ influx through dihydropyridine-sensitive Ca2+ channels is required for cell swelling to induce prolactin (PRL) secretion in GH,C, cells (Sat0 et al., 1990a, d), extracellular CaZf has a negative influence on osmotically induced secretion in normal rat adenohypophyseal ceils (Greer et al., 1990; Sato et al., 1991a). The mechanism by which cell swelling opens Cal+ channels is unknown.

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Another PRL-secreting clonal pituitary line that, in contrast to GH,C, cells, expresses functional dopamine receptors has been developed and designated the MMQ line (Judd et al., 1988). The homogenei~ of MMQ cells presents a unique opportunity to further our understanding of the biochemical responses to receptor activation (Login et al., 1990a). In this study we have evaluated the effect of dopamine on cell swelling-induced Ca’+ influx and hormone secretion in this line of tumor-derived cells. Materials and methods MMQ cell preparation and stimulation media MMQ cells were kindly provided by Dr. Robert MacLeod and were incubated in RPMI-1640 medium (Gibco, Grand Island, NY, U.S.A.) supplemented with 7.5% horse serum and 2.5% fetal caitf serum at 37 o C for 2-3 days in 60 mm culture dishes (1-2 x lOh cells/dish) for cell volume experiments, on glass coverslips (10’ cells/slip) for monitoring [Caz’li, in 60 mm Petri dishes with 10 mg sterile Cytodex-3 beads (3 x 10h cells/dish) for column perifusion experiments (Sat0 et al., 1990d), and in 24-well Multiwell tissue culture plates (10s cells/well) (Joniau et al., 1990). To adhere cells on glass coverstips and the bottom of 24-well Multiwell tissue culture plates, we coated their surfaces with polyiysine (Sigma, St. Louis, MO, U.S.A.). The standard medium utilized (pH = 7.3, 300 was Krebs-Ringer Hepes buffer mOsm) (( + Ca2+)KRBH); composition in mM: NaCl (140), KC1 (5), MgSO, (l.Z), KH,PO, (1.2), glucose (61, Hepes (lo), CaCI, (1.51, and 0.1% bovine serum albumin (BSA). Caz+-depleted KRBH (( - Ca*+)-KRBH) was made with the same composition as KRBH except NaCl was 142.25 mM, CaCI, was omitted, and 0.03 mM EGTA was added. The Ca2+ concentration in (-Ca”‘)KRBH was always < 2 I*_M(range, 0.7-1.81, as determined from calibration of fura- fluorescence in the solutions (Sato et al., 1990~). The hyposmolar solution was made by diluting the medium with deionized distilled water. For the isotonic urea or mannitol solutions, each substance (lo-120 mM) was dissolved in a hyposmolar solution so that the final osmolarity was iso-

tonic (300 mOsm). Medium osmolarity was measured with an Advanced DigiMatic Osmometer model 303, with a range of O-2000 mOsm and a sensitivity of 1 mOsm; the observed osmolarity of KRBH, a 27% hypotonic solution, isotonic 80 mM urea (either (+Ca’+) or (- CaZ+)), and isotonic 80 mM mannitol was 99.3 f 1.1% (mean $ SE, tz = 12) of the predicted osmolarity. For the hypertonic urea solution, urea was dissolved in KRBH so that the final osmolarity was additive. Cell colume measurements Dynamics of cell volume changes were measured with a Coulter counter (model ZM, Coulter EIectronics, Hialeah, FL, U.S.A.) with the dual threshold analysis technique previously described (Sato et al.. 1991a). Briefly, cells were split from culture dishes and suspended in (+Ca”)KRBH at 1 X 10h cells/ml. Each experiment was started by placing 100 E_LI of cells in a vial containing 20 ml of test solution and counting the cells in a Coulter counter every 30 s. Control experiments demonstrated that the cell volume distribution curve of latex bead standards was unaffected by 27% hyposmolarity, isotonic 80 mM urea, or hypertonic SO mM urea solutions.

[Ca”+li dynamics were monitored by dual excitation microfluorimetry (FIuoroPlex III fl uorimeter, Tracer Northern, WI, U.S.A.) after loading cells with fura- by exposure to 2 PM fura-2/AM (Molecular Probes, Eugene, OR, U.S.A.) in (+ Ca2+)KRBH for 30 min at 37 o C (Sato et al., 1990d). The coverslip with attached cells was placed in a 0.5 ml superfusion chamber. Fluorescence from optically isolated 20-30 MMQ cells was monitored through a fixed circular diaphragm. Photon counts were sampled with a time constant of 2 s. [Ca*‘], was calculated from the ratio (R = F,,,,/F,,,,) of the fluorescence (Grynkiewicz et al., 1985). hormone secretion Hormone secretion was measured with column perifusion for analyzing the PRL secretory dynamics (Sato et al., 1990d) or with static culture for analyzing the total amount of secretion (Wang et al., 1991). In the column perifusion experi-

ments, MMQ cells were layered in a 0.2 ml plastic chamber containing a thin layer of Cytodex-3 beads on a polycarbonate membrane with 8 pm pores; the rest of the chamber was then packed with Cytodex-3 beads. The flow rate was 0.6 ml,/min and fractions were collected every 30 s. Two parallel columns were simultaneously perifused. In the static culture experiments, 30 min before the experiments culture media were changed from RPMI-1640 to (+Ca’+)KRBH. Cells were preincubated with control medium or medium containing dopamine (DA) and/or haloperidol (HP) (both drugs were purchased from Sigma, St. Louis, MO, U.S.A.) for 10 min before incubation with experimental medium for the time indicated detailed in each experiment. Samples were frozen at -20 “C until analysis for PRL by radioimmunoassay using protocols and specific reagents supplied by the National Hormone and Pituitary Program. Experimental design and statistical analysis Data are expressed as the mean + SE of the numbers of experiments described. To check the effect of lowering the concentration of the various medium components on the measured variables (cell volume, [Ca2+li, and hormone secretion), since both hyposmolarand isotonic urea solutions were made by diluting the medium, we used the impermeant molecule mannitol instead of urea. Isotonic mannitor (80 mM) did not affect any of the variables (data not shown except for the secretion data in Fig. 7). Statistical analysis was performed with Student’s t-test. Results Cell llolume changes When MMQ cells were suspended in the osmotically equivalent 80 mM isotonic urea or 27% hyposmolar (+ Ca’+)KRBH, the cells expanded to a maximum volume within 2-2.5 min then shrank toward their original cell volume (regulatory volume decrease (RVD)) in spite of continuous exposure to the cell expanders (Fig. 1A). Hypertonic urea did not induce an increase in cell volume.

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Fig. 1. Cell volume changes of MMQ cells in response to the osmotically equivalent isotonic 80 mM urea or 27% medium hyposmolarity started at 0 min (indicated by the arrow) and continued through the period shown. Relative cell volume changes were calculated as described in Materials and methods. Mean and SE of replicated determinations in 4-6 experiments are shown. A: Effect of 27% hyposmolarity (HzO), 80 mM isotonic urea (UREA), or 80 mM hypertonic urea (UREA * ). There were significant differences between H,O and urea stimulation 75-105 s after stimulation (P < 0.05). At every point after stimulation, there were significant differences between isotonic and hypertonic urea (P < 0.05). B: Effects of removal of medium Ca” or 10 FM dopamine on basal and 27% medium hyposmolarity-induced cell volume changes. Removal of medium Ca’+ significantly enhanced hyposmolarity-induced cell volume changes after 45 s of stimulation (P < 0.05). Dopamine did not significantly affect hyposmolarity-induced cell volume changes (P > 0.05). (- )Ca’+; H,O (solid squares) = medium Ca’+ depletion + 27% hyposmolarity: (+ )DA; Hz0 (solid triangles) = 10 PM dopamine + 27% hyposmolarity; H,O (open circles) = 27% hyposmolarity; (+ )DA (open triangles) = 10 /*M dopamine; (- )Ca’+ (open squares) = medium Ca’+ depletion.

Depletion of medium Cal+ enhanced the hyposmolarity(Fig. 1B) or isotonic urea- (data not shown) induced cell swelling and inhibited RVD. 10 PM DA did not change the hyposmolarity-induced cell volume changes significantly (Fig. 1 B). [Ca’ ‘1, changes Depolarizing 30 mM K+, 27% hypsmolarity, or 80 mM isotonic urea all caused a striking rise in [Ca’ ‘Ii. The cell-swelling inducers (hyposmolarity and isotonic urea> produced slow increases in [Ca2+li which did not peak until 100 s, while K+ caused a steep rise in [Ca2+li, reaching a zenith within 10 s (Fig. 2A). The [Ca2’li changes in-

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duced by either medium hyposmolarity or isotonic urea were stimulus intensity-dependent (Fig. 2B). Hypertonic urea did not induce a significant rise in [Ca2+li (Fig. 2A and B). 10 PM DA inhibited the increase in [Ca2’li induced by either 80 mM isotonic urea or 27% medium hyposmolarity. This effect of DA was not due to cell damage since the induced [Ca2+], responses rapidly returned after washing out DA (Fig. 3A and B). Although DA inhibited the rise in [Ca2+li induced by either X0 rnM isotonic urea or 27% medium hyposmolarity in a dose-dependent fashion, maximum DA suppression was only about 50% (Fig. 3C and L>). HP, a D,-receptor dopamine antagonist active in MMQ cells (Login et al., 1990a), prevented DA inhibition of the [Ca2+Ii rise induced by cell swelling (Fig. 4A). Furthermore, either removal of medium Ca2+ or addition of nifedipine almost completely abolished the induced rise in [Ca”+]i (Fig. 4A and C). PRL secretion

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Fig. 3. Effect of dopamine (DA) on [C’a” 1, changes in MMO cells in response to 80 mM isotonic urea (UREA) and 27%’ medium hyposmolarity (H20). The dynamics of the induced changes in [Ca2’li are shown in the A panel (urea) and E panel (hyposmolarity). The horizontal hatched and solid bars indicate the duration (3 min) of urea and hyposmolar stimulation, respectively. The cells were exposed to each stimulator 3 times, as indicated by 1, 2. and 3 at the ~ttom of the B panel. Cells were perifused with normal KRBH for 15 min between each stimulation. At the ‘2’ exposure, cells were perifused with 10 FM DA beginning 5 min before stimulation as indicated by dashed hoxes. The C and D panels show the dose-response to DA on changes in [Ca’+ 1, in response to X0 mM isotonic urea (open circles, C panel) or 27% medium hyposmolarity (closed circles, D panel) shown as percent inhibition. Each mark indicates the mean of two experiments.

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tonic urea induced significant PRL secretion (Fig. 5A). The dynamics of the PRL response to each agonist resembled the relative response in [Ca2’]i dynamics, with K’ inducing the steepest and greatest rise in both PRL secretion and iCa2+ji. The slight ‘off secretory response in secretion after stopping the hypertonic urea perifusion is characteristic of returning cells from a hyperosmolar to isosmolar medium (Greer et al., 1985). The lag time between the peak of [Ca’+]i and PRL secretion induced by K’, hyposmolarity, or isotonic urea was constant (l-1.5 min). The cell swelling-induced PRL secretion was stimulus intensity-dependent (Fig. 5B). 10 FM DA inhibited PRL secretion induced by either 27% medium hyposmolarity or 80 mM isotonic urea and this dopaminergic inhibition was prevented by 1 PM HP (Fig. 6A and B). Although DA suppressed cell swelling-induced

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Fig. 4. Effect of dopamine (DA), haloperidol (HP), depletion of medium Ca2+ (f - )Ca”’ ), and nifedipine (NIF) on [Ca’+ Ii changes in response to 27% medium hyposmolarity (H,O). MMQ cells were perifused with normal KRBH and H,O as indicated in Fig. 3. A: Effect of 10 FM DA with or without 1 PM HP. B: Effect of depletion of medium Ca2+. C: Effect of 2 yM NIF. All experiments were performed at least 3 times with essentially identical results.

sponse curves indicate the IC,,, of nifedipine is 20 nM (Sat0 et al., 199Oc, d). The first step in osmotically induced secretion is an expansion of cell volume achieved by an osmoIar difference between the cytosol and the extracellular space. We used two different kinds of inducers of cell swelling, medium hyposmolarity (producing relative extracellular hyposmolarity) and isotonic urea (producing relative intracellular hyperosmolarity). Isotonic urea presumably caused cell swelling by increasing intracellular osmotic pressure as a result of urea’s rapid diffusion through the plasmalemma into the cytosol (Seeman et al., 1969). Hypertonic urea was presumably ineffective in inducing cell swelling because extracellular hyperosmolarity balanced the increased intracellular osmotic pressure caused by urea entry (Wang et al., 1989; Sato et al., 1990b). The second step in this phenomenon appears to be Ca2+ influx through nifedipine-sensitive Ca” channels and a rise in [Ca2+li. Cell swelling induced by medium hyposmolarity, molecules permeant to the plasmalemma, or mechanical stretch cause Ca2+ influx in many different cell

PRL secretion in a dose-dependent manner, the maximum inhibition achieved was only approximately 50%, the same as the maximum dopaminergic suppression of the [Ca2’li changes (Fig. 6A and B). Removal of medium Ca2+ or addition of 1 PM nifedipine almost completely abolished PRL secretion induced by any of the stimuli (Fig. 7). Discussion

This study provides further support for our hypothesis that in tumor-derived rat pituitary cells cell swelling stimulates hormone secretion by inducing a rise in Ca2+ influx through nifedipinesensitive Ca2+ channels (Sato et al., 1990b, c, d). Although the nature of the Ca2+ channel involved was not exhaustively studied in the MMQ cells, we have previously shown in tumor-derived GH,C, cells that both Ca2+ influx and PRL secretion induced by osmotic cell swelling are blocked by verapamil and nifedipine; dose-re-

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Fig. 5. PRL secretion by MMQ cells in response to 30 mM depolarizing K+ (K+ ), 27% medium hyposmolarity (H,O), 80 mM isotonic urea (UREA), or 80 mM hypertonic urea (UREA *). The horizontal solid bar indicates the duration (3 min) of each stimulation. A: Comparison of the dynamics of PRL secretion from perifused cells; the mean and SE of replicated determinations of 4-6 experiments are shown. B: Dose effect of hyposmolarity (open squares), isotonic urea (closed circles), and hypertonic urea (open circles) on PRL secretion measured in static cultures. The mean and SE of four experiments are shown.

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types, e.g. lung epithelium (Wirtz and Dobbs, 1990), renal epithelium (Jessop et al., 1990), lymphoma (Kazilek et al., 19881, urinary bladder epithelium (Seeman et al., 19691, intestinal epithelium (Foskett and Spring, 19851, venous endothelium (Kullberg, 1987), osteosarcoma cells (Young et al., 19871, and GH,C, cells (Sat0 et al., 1990b, d). Thus, Ca’+ influx induced by cell swelling and the consequent rise in [Ca”+], may be a general cellular phenomenon. However, the mechanism responsible for the Ca2+ influx is unclear. The present data demonstrate that dopamine partially suppressed the cell swellinginduced rise in [Ca”‘.li which depends on Ca2+ influx through nifedipine-sensitive Ca’+ channels

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without affecting cell volume changes induced by osmolar stimuli. This is the first evidence that dopamine inhibits the opening of Ca2+ channels induced by cells swelling. D, dopamine receptors expressed in MMQ cells (Judd et al., 1988) suppress Ca2+ influx induced by Ca2+ channel agonists (BAY K8644 and maitotoxin ) and depolarizing K’ (Login et al.. 1990a). Pretreatment with phorbol esters inhibits the rise in [Ca”li induced by either Ca2+ channel agonists (Login et al., 1990a) or osmotic cell swelling (unpublished data). Thus, it is possible that Ca2+ channels opened by cell swelling are the same as those opened by Ca*+ channel agonists or membrane depolarization. l-iowever, further studies (including electrophysiological evaluation) are required to delineate the mechanism by which cell swelling opens Ca*+ channels. The mechanism may be complicated since the maximum suppressive effect of dopamine on cell swelling-induced changes in our

105

studies was only 50% although it abolished the increases in both [Cazfli and PRL secretion induced by Ca2+ channel agonists (Judd et al., 1988; Login et al., 1988, 1990a, b). Although dopamine can affect components of the secretory mechanism in addition to Ca2+ influx, its effect for on the Ca 2+ channel seems to be responsible the inhibition of PRL secretion in MMQ cells since dopamine does not depress cell swelling-induced PRL secretion in normal cells (unpublished data), in which Ca*+ influx plays an inhibitory instead of obligatory role in osmotically induced secretion (Greer et al., 1990). The next step in cell swelling-induced secretion is triggering of PRL secretion by the increase in [Ca”],. Our data document that the relative dynamics of cell swelling-induced PRL secretion are very similar to those of [Ca”],. Changes in cell volume, [Ca2+li, and PRL secretion induced by medium hyposmolarity were clearly faster and higher than those induced by an osmotically equivalent concentration of isotonic urea (Sat0 et al., 1990~). This may correspond to the difference in membrane permeability coefficients (H,O > urea> (Sate et al., 1990~). A preceding rise in [Ca2+li due to Ca*+ influx through Ca*+ channels thus appears to be a critical factor in PRL secretion induced by cell swelling in MMQ cells, as previously reported in GH,C, cells (Sato et al., 1990b, d). Although Ca2+ influx is necessary for cell swelling-induced PRL secretion in tumor-derived pituitary cells (GH,C, and MMQ cells), Ca2+ influx is not required for this phenomenon in normal adenohypophyseal cells; in fact, medium Ca*- has a significant negative influence on osmotically induced secretion in normal adenohypophyseal (Greer et al., 1990; Sato et al., 1991a) and non-adenohypophyseal endocrine cells (Skott, 1986, 1988; Greenwald et al., 1989; Watanabe et al., 1989). Furthermore, dopamine does not suppress cell swelling-induced PRL and thyrotropin secretion by normal adenohypophyseal cells (unpublished data). The explanation for these dissimilarities may involve fundamental differences in second messenger systems between normal and malignant cells. Cell swelling may thus be a useful tool to investigate differences between normal and malignant cells.

Acknowledgements Supported by Research Grant DK-01447 from the NIDDK, National Institutes of Health. We are indebted to Dr. Robert MacLeod for the gift of the MMQ cells, to Susan Greer and Staci McAdams for expert technical assistance, to Elizabeth Allen for her expert secretarial skills, and on the National Hormone and Pituitary Program, NIDDK for the gift of reagents for hormone measurement. References Baquet, A., Hue, L., Meijer, A.J., van Woerkom, G.M. and Plomp, P.J. (1990) J. Biol. Chem. 265, 955-959. Brown, E.M., Pazoles, C.J., Creutz. C.E., Aurbach, G.D. and Pollard, H.B. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 8766880. Chazenbalk, G.D., Valsecchi, R.M., Krawiec, L., Burton, G., Juvenal, G.J., Monteagudo, E., Chester, H.A. and Pisarev, M.A. (1988) Prostaglandins 36, 163-172. Corasanti. J.G., Gleeson, D. and Boyer, J.L. (1990) Am. J. Physiol. 258, G290-G298. Foskett, J.K. and Spring, K.R. (19851 Am. J. Physiol. 248, C27-C36. Gleeson, D.. Corasanti, J.G. and Boyer, J.L. (1990) Am. J. Physiol. 258, G299-G307. Greenwald, J.E., Apkon, M., Hruska, K.A. and Needleman, P. (19891 J. CIin. Invest. 83, 1061-1065. Greer, M.A., Greer, S.E., Opsahl, Z., McCafferty, L. and Maruta, S. (1983) Endocrinology 113, 1531-1533. Greer, M.A., Greer. S.E., Opsahl, Z. and Maruta, S. (1985) Proc. Sot. Exp. Biol. Med. 178, 24-28. Greer, M.A., Greer, S.E. and Maruta, S. (19901 Proc. Sot. Exp. Biol. Med. 193, 203-209. Grynkiewicz, G., Poenie, M. and Tsien, R.Y. (1985) J. Biol. Chem. 260, 3440-3450. Hampton, R.Y. and Holz, R.W. (19831 J. Cell Biol. 96, 10821088. Jessop, D., Sidhu, R. and Lightman, S.L. (1990) Brain Res. 516, 41-45. Joniau, M., Coudijzer, K. and De Cuyper, M. (1990) Anal. Biochem. 184, 325-329. Judd, A.M., Login, I.S., Kovacs, K., Ross, P.C., Spangelo, B.L., Jarvis, W.D. and MacLeod, R.M. (19881 Endocrinology 123. 2341-2350. Kazilek, C.J., Merkle, C.J. and Chandler, D.E. (1988) Am. J. Physiol. 254, C709-C718. Kullberg, R. (1987) Trends Neurosci. IO, 387-388. Login, I.S., Judd, A.M. and MacLeod, R.M. (1988) Biochem. Biophys. Res. Commun. 151, 913-918. Login, IS., Kuan, S.I., Judd, A.M. and MacLeod, R.M. (1990a) Cell Calcium 11, 525-530. Login, I.S., Kuan, S.I., Judd, A.M. and MacLeod, R.M. (1990bI Endocrinology 127, 194881955.

IOh Sam, N., Wang, X. and Cheer, M.A. (1990a) Biochem. Biophya. Res. Commun. 170, 968-972. Sato, N., Wang, X., Greer, M.A., Greer. SE. and McAdams. S. (199ObJ Mol. Cell. Endocrinol. 70, 2733270. Sate, N., Wang. X., Grew. M.A., Greer, SE. and McAdams, S. (1990~) Endocrinology 127, 307993086. Sate, N., Wang. X.. Greer, MA.. Greer, SE.. McAdams, S. and Oshima. T. (199Od) Endocrinology 127. 9S7-Yh4. Sam, N., Wang. X. and Greer, M.A. (1991a) Am. J. Physioi. (in press). Sam. N.. Wang, X. and Greer, M.A. (1991bJ Mol. Cell. Endocrinol. 77. 193-198. Seeman. P., Kwant, W.O., Sauks, T. and Argent, W. (1969) B&him. Biophys. Acta 1X3, 490-498.

Skott, 0. (198h) Pfliig. Arch. 406. 485-491. Skott, 0. (1988) Am. J. Physiol. 255, Fl-FlO. Wang, X., Sato, N., Greer, M.A., Greer, S.E. and McAdams, S. (1989) Biochem. Biophys. Res. Commun. 163, 471-745. Wang. X., Sato. N.. Greer, M.A. Greer, SE. and McAdams. S. (1992 J J. Pharmaol. Exp. Ther. 256, 13% 140. Watanabe. T., Oki, Y. and Orth, D.N. (1989) End(~crinology 125, 1921-1931. Watford. M. (1990) Trends. Biochem. Sci. 15, 329-330. Wirtz. H.R. and Dobbs, L.G. (1990) Science 250, 1266-1269. Wong, S.M.E. and Chase. Jr., H.S. (1986) Am. J. Physiol. 250. C841-CN52. Young, W.S., Warden. M. and Mezey, E. (lY87) Neuroendocrinology 46, 4399444.