0021-972X/92/7402-0266$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society
Vol. 74, No. 2
Printed in U.S.A.
Calcium-Regulated Secretion of Tissue Plasminogen Activator and Parathyroid Hormone from Human Parathyroid Cells* DEVI
D. BANSAL
Department
of
Anatomy
AND
RONAL
and Cell Biology,
R. MacGREGOR University
of
Kansas Medical
ABSTRACT. The effects of Ca and other agents on secretion of plasminogen activator (PA) and PTH have been examined and compared, using parathyroid cells obtained from the glands of chronic renal patients. During 2 weeks culture at different [Cal, the secretory rates of PA activity and PTH were parallel; steady-state secretion over 24-h periods was maximal at 0.5-0.9 mM Ca, minimal at 1.5-2.5 mM Ca, and the [Ca] at 50% suppression was 1.1 mM. At 2.5 mM Ca, two inhibitors of cellular proteolysis, 3-methyladenine and chloroquine, stimulated secretion of both PA activity and PTH. The results indicated that secretion of PA from human parathyroid cells is regulated sim-
P
LASMINOGEN activators (PA) are involved in numerous processes that involve tissue remodeling, including tumor growth and metastasis, angiogenesis, and the release of matrix-associated growth factors (l4). The formation of PA is stimulated by growth factors like epidermal growth factor and fibroblast growth factor in several tissues (5), and by oncogene products (6). Many cells and tissues contain and secrete PA (7, 8). In humans, two genes code for PA, one PA is a 70 kilodalton (kDa) protein called tissue PA (tPA), whereas the other is called urokinase (uPA) (1,2). The same PA is normally secreted by a particular cell type in most species, but in some cases the same cell type in two species secretes different PA; for example the ovarian granulosa cells of mice secrete uPA, whereas the corresponding rat cells secrete tPA (9). We recently reported that bovine parathyroid cells secrete a 44 kDa PA (lo), whose secretion is regulated by extracellular [Cal like that of PTH. Its characteristics Received March 1, 1991. Address reprint requests and all correspondence to: Ronal R. MacGregor, Department of Anatomy and Cell Biology, University of Kansas Medical Center, 39th and Rainbow Boulevard, Kansas City, Kansas 66103. * This work was supported by NIH Grant DK-26416 (to RRM) and bv BRSG S07-RR-05373 from the Biomedical Research Support Grant Program, Division of Research Resources, NIH. Presented-in part at the 12th Annual Meeting of the American Society for Bone and Mineral Research, and at the 30th Annual Meeting of the American Society for Cell Biology.
Center, Kansas
City, Kansas 66103
ilarly to that of PTH. The characteristics of human parathyroid PA were also examined using human parathyroid adenoma tissue. In homogenates, the highest specific activity of PA was in microsomal fractions. The M, of PA from tissue and from culture media was 70 kilodalton by sodium dodecyl sulfate gel electrophoresis followed by zymography, or by Western blotting using antisera to human tissue PA @PA). Enzyme activity was inhibited by incubation with antisera to tPA but not to urokinase. In contrast to bovine parathyroid cells that secrete a urokinase, human parathyroids apparently contain and secrete tPA. (J Clin Endocrinol Metab 74: 266-271,1992)
are of uPA. The function of PA in parathyroid tissue is unknown, but its known involvement in tissue remodeling and growth suggests that it could be involved with the hypertrophy and hyperplasia of the parathyroid that result from the conditions of chronic renal failure in humans. In this study we demonstrate that human parathyroid cells contain and secrete PA, but that the enzyme secreted from human parathyroid cells is not that secreted from bovine parathyroid cells. Materials High
M, human
uPA,
and Methods tPA,
placental
uPA
inhibitor,
and
thrombin were purchased from Calbiochem (La Jolla, CA). Human plasminogen, tPA stimulator (fibrin peptides), and substrate S-2251 were purchased from Helena Laboratories (Beaumont, TX). Bovine fibrinogen, bovine plasminogen, amiloride, chloroquine, and other general chemicals were from Sigma (St. Louis, MO) and 3-methyladenine was from Aldrich Chemical (Milwaukee, WI). Electrophoresis reagents were from Bio-Rad (Richmond, CA) and collagenase type I was purchased from Worthington (Freehold, NJ) and purified as described previously (11). Anti-human tPA rabbit antiserum was a gift from Dr. D. Loskutoff (La Jolla, CA), and anti-human uPA goat antiserum was purchased from Vector laboratories (Burlington, CA). Bovine PTH and PTH(37-84) were purified from bovine parathyroids as described previously (12,13), and Nalz51 was purchased from DuPont/New England Nuclear (Wilmington, DE).
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Ca-REGULATED Cell preparation
SECRETION
and treatments
Secretion studies were performed with parathyroid cells obtained as described earlier (14) from chronic renal patients, since their responses to Ca are more reproducible. Cells were counted with a hemocytometer. One or 2 million cells/ml were placed in sterile culture tubes which were then placed in a rotating drum (Bellco) at 37 C in air. Added to the serum-free medium (10) were BSA (1 mg/mL), bovine insulin (5 pg/mL), and bovine transferrin (5 pg/mL). This medium does not support replication of cells, based on observations of monolayers (MacGregor, R., unpublished results). Incubations of fresh cells were terminated after 5 h. If culture was continued, cells aggregated to form organoids (15) with which experiments involving steady state secretion were performed. The media were changed every 2 or 3 days, and after experimental conditions were established, the same [Cal (expressed as total [Cal) were maintained for each organoid throughout the culture periods. Samples of culture media for analysis were always removed 24 h after medium changes. Subcellular
fractionation
Parathyroid adenoma tissue was used because it was considered unsuitable for studies on secretory regulation. The tissue was homogenized with a Potter-Elvehjem homogenizer at 4 C in 0.25 M sucrose. Subsequent steps were performed at 4 C. The homogenate was centrifuged at 600 x g for 10 min to obtain the nuclear fraction. The supernatant was centrifuged at 10,000 X g for 20 min to obtain the mitochondrial fraction. The supernatant was centrifuged at 140,000 x g for 1 h to obtain the microsomal fraction and the supernatant. Assay for PA activity
Assays were carried out in 96 well plates according to Campbell et al. (16) using the plasmin substrate D-val-leu-lys-pnitroanilide (S-2251) at a final concentration of 37 pg/ml. Experimental samples or standard human tPA was added in 10 ~1; plasminogen was present at 0.2 IU/ml and 7 pg heatinactivated bovine parathyroid microsomes were present as an activator (10) in a total volume of 125 ~1. The buffer was 0.1 M Tricine, pH 8.4. Blank incubations contained culture medium or 0.25 M sucrose in place of enzyme. Differences in [Cal or the presence of chloroquine or 3-methyladenine did not alter assay results. Absorbance at 405 nm was read at 30-min or l-h intervals using a Titertek Multiscan plate reader. Activity was expressed as international units of standard human tPA (Calbiochem), or as differences in Ados. Fibrin-agarose
zymography
Samples of cell homogenates and fractions thereof, incubation and culture media, and PA standards were treated with sodium dodecyl sulfate (SDS) at 40 C and separated on 13.5% polyacrylamide gel slabs according to the method of Laemmli (17). Gels were washed with 2.5% Triton X-100 and then placed over fibrin-agarose gels containing plasminogen (18); they were incubated at 37 C, 100% humidity until clear bands representing hydrolyzed fibrin were observed. To estimate the M, of
OF PA AND PTH
267
parathyroid PA, test samples, human tPA, high M, uPA, and prestained protein standards were run on the same plate. Western blots
After electrophoresis as described above, proteins were transferred to nitrocellulose membranes using the Genie transfer apparatus (Idea Scientific Co., Orvallis, OR). Primary and secondary antisera, blocking sera and avidin-biotin horseradish peroxidase (Vector Laboratories Inc.) were diluted with 100 mM Tris, 0.9% NaCl, 0.1% Tween 20, pH 7.5. Membranes were treated with blocking buffer, anti-tPA, secondary antibody, and avidin-biotin horseradish peroxidase reagent, respectively for 30 min each at room temperature at 160% humidity. After each reaction the membrane was extensively washed with Tris buffer described above for 3 x 5 min with gentle shaking. Finally the blot was developed with 4-chloro-L-naphthol and hydrogen peroxide in PBS, pH 7.4, until the bands appeared. RIA for PTH and other methods
Medium samples were examined for PTH using an RIA called the “COOH RIA” that detects PTH and COOH-terminal PTH fragments equally (19). The COOH RIA used bovine PTH as standard, and the tracer was lz51-PTH(37-84) iodinated by the method of Hunter and Greenwood (20). Protein was estimated by the method of Hartree (21). Data are presented as averages *SE, and comparisons made using the Student’s t test.
Results Regulation of PA secretion The steady-state secretion of PTH and PA activity from parathyroid organoids was inhibited by ionized Ca (our data are expressed as total [Cal that is about 85% ionized) (Fig. 1). The sensitivities to Ca of PTH and PA secretion were identical and unchanged between 7 and 20 days of culture; the magnitudes of the secretory responses were likewise stable. Secretion of PA responded to Ca between 1 and 30 days of culture (data not shown). The [Cal at 50% inhibition of steady state secretion of both PA and PTH was 1.1 mM. After correction for the percent ionized, the [Ca”] at 50% inhibition in Fig. 1 was apprOXimately 0.94 mM [&I++].
The role of intracellular degradation in secretion of PA and PTH was examined using 3-methyladenine and chloroquine, agents previously shown to stimulate PTH and PA secretion from bovine parathyroids cells incubated at high [Cal (10, 22). These agents were used to provide information as to whether PTH and PA followed similar secretory pathways. Figure 2 shows that these agents increased the release of PA and PTH in parallel from human parathyroid cells at high [Cal. In combination, their effects on the release of PTH and PA were additive.
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BANSAL AND MacGREGOR
268
JCE & M -1992 Vol74.No2
1.2
O-O.
3
-0
FIG. 1. Steady state regulation of PA and PTH secretion by Ca. A, PA secreted during four 24-h periods of culture; B, PTH secreted during four 24-h periods of culture; C, PA and PTH after 4 days at different [Cal. From hyperplastic parathyroid tissue of one renal patient, parathyroid organoids were cultured for 7 days in M/W medium at 1.8 mM Ca, and then groups of 10 organoids were cultured further at the [Cal indicated until the end of the experiment. Media were sampled after the first 24 h of treatment (day 1 = 8 days of culture) and after 24-h periods following media changes as indicated in the figure. Data points represent averages of six assays of individual cultures for PA, and of five RIA of media from pools of two cultures each for COOH-PTH. SE are omitted for clarity in A and B; they ranged from 310% of the data values for PA, and 26% of the data values for PTH. Results represent one of three similar experiments, each performed with cells from a different patient.
day 1
O-0,
day4
A-A,
day 6
A-&day13
20
2 g
15
3 $
\
10
5 I
&is
B. 0.0
01 0.0
[Cal,
f ( 1.0
3.0
1.0
3.0
[Cal, rnM2’
rni”
O-0,
1.0
PTH --25
A- .A,
PA --20
--15
2 < z c
--10
= h
-- 5
1.0
[Co],
Characterization
of human
parathyroid
PA
The specific activity of PA in fractions of parathyroid homogenates was highest in microsomes, less in mitochondrial and nuclear fractions and almost zero in the high speed cytosol (Fig. 3). The distribution of PTH was similar except for the presence of significant immunoreactivity in the cytosol. To examine the possibility that cytosol might contain a PA inhibitor, its effect on PA activity of spent culture medium was examined. No inhibition of PA activity was detected. To identify the type of PA secreted from human parathyroid cells, medium obtained from cultured organoids was treated with anti-human tPA and anti-human high M, uPA, and then assayed for PA. Table 1 shows that anti-tPA inhibited the PA activity of authentic tPA completely, and of media by 86% and 66% at dilutions of l/1000 and l/2000, respectively. Anti-uPA inhibited authentic uPA, but not the PA in medium. The effects
2.0
LO0
total
of human placental uPA inhibitor and amiloride (23) on secreted PA were examined. Neither agent inhibited the activity (results not shown). The results of the combined inhibitor and antibody studies indicated that PA secreted by human parathyroid cells is tPA. To estimate the M, of human parathyroid PA, samples of microsomes, media, and standard PAS were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and gels were used for zymography or for transfer to Millipore Immobilon-P membranes for Western blotting. Figure 4 shows that on zymography, one band was obtained that migrated as a protein of 70 kDa. Figure 5 shows that Western blots developed with anti-tPA also showed one band 70 kDa. These results indicated that human parathyroid cells secrete only tPA. Discussion This study showed that human parathyroid cells, both freshly isolated and cultured, secrete PA in parallel with
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Ca-REGULATED
SECRETION
OF PA AND PTH
TABLE
1. Effect
of PA antisera
269 on secreted Type
Treatment
05
CO
25
JMA
25 +
2.5 f
clq
25 + +
05
25
25 +
2.5 +
25 + +
WC. 2. Effects of 3-methyladenine and chloroquine on secretion of A, ubated 5 h at 37 C PA; B, PTH. Fresh cells, 2 x 10’ per tube, WI oquine (50 PM) or in the presence of 3-methyladenine (5 mM) both. [Cal below the bars are listed as millimoiars of total Ca. Data represent averages + SE for four samples of pools from two cultures each per group for PA, and eight single RIA of eight individual cultures for PTH. All conditions are significantly different from the control group at 2.5 mM Ca, P < 0.01. Results represent one of two similar experiments, each performed with cells derived from a different patient.
300
Culture
None Anti tPA l/1000 l/2000 Nonimmune serum l/1000 l/2000
0.38 f 0.01
None Anti uPA l/100 l/200
PA activity
of PA activity tPA
High
M, uPA
0.21 f 0.01
0.54 k 0.03
0.05 + 0.001” 0.13 + 0.01”
0.00” 0.002"
0.40 + 0.02 0.49 + 0.028
0.40 f 0.030 0.39 + 0.004
0.13 + 0.002” 0.14 + 0.003”
0.49 + 0.025 0.49 + 0.031
0.38 + 0.016
0.23 + 0.005
0.43 f 0.009
0.46 + 0.005 0.47 + 0.01
0.20 iz 0.004 0.25 + 0.004
0.025 + 0.001” 0.051 + 0.002”
Samples assayed were: 10 ~1 conditioned medium from organoid culture, 0.1 IU human tPA, 0.1 IU human uPA. PA samples were incubated 30 min at 21 C with 10 ~1 assay buffer, anti-tPA, anti-uPA, or nonimmune serum in a total volume of 70 ~1, and then assayed for PA activity; the data represent the A,,, after 2 h 30 min of incubation. ’ Different from no treatment control. P < 0.01.
.E 5L& 200 Y >
E _ 100 2
0 N
Mt
MC
Cyt
N
Mt
MC
CYt
3. PA activity and PTH in centrifugal fractions of human parathyroid homogenates obtained from an adenoma. For assays of PA activity, samples were treated with 0.5% Triton X-100 at room temperature. For COOH-PTH RIA, samples were treated with 1% SDS, 15% mercaptoethanol, diluted to 10 pg protein/100 ~1, and further diluted serially for RIA. Data are expressed as averages of triplicate samples + SE. N, Nuclear fraction; Mt, mitochondrial fraction; MC, microsomal fraction; Cyt, cytosol fraction.
FIG.
PTH under several conditions. The significance of PA secretion is not yet known; it has not been demonstrated to occur from normal human parathyroid cells, but the fact of its release from normal bovine parathyroid cells supports the notion that it is a normal secretory product of the parathyroid. What is its relationship to parathyroid physiology? Recognized functions of PAS from other tissues center around extracellular interactions with the surrounding matrix (1, 2). A potentially important role for PA with clinical relevance is that the enzyme, by activating latent proteases in the matrix, could mediate in part changes that accompany adaptation to chronically high or low [Cal. Studies of wound healing and other cell-matrix interactions have suggested that growth factors are present in the extracellular matrix; these
Microsomes UPA
FIG. 4. Zymography of human tPA, 0.2 U; medium from organoid culture as in Fig. 1, 20 ~1; human parathyroid adenoma microsomes, 2 fig protein; high M, human uPA, 0.2 U. PA from the microsomes and medium along with M, markers were separated by SDS-PAGE (nonreducing) and zymograms generated as described in Materials and Methods.
could be released and/or activated by a pathway that is regulated by the rate of release of PA from the cells (4). The possible results of such activation include the direct action of growth activating or inhibiting factors on parathyroid cell replication, remodeling of the extracellular matrix to accommodate hypertrophic or atrophic responses of the parathyroid, or angiogenic stimulation (3). Elucidation of which, if any of these functions is modulated by parathyroid PA must await further progress. It has not yet been definitely established that parathy-
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BANSAL AND MacGREGOR
270
Mr Stds TPA Media Cone
Media
% 1 i
Media Microsomes UPA
5. Immunological analysis of human parathyroid PA after SDSPAGE and Western blotting. Cellular (microsomes) and secreted (media) PA, human tPA and high M, human uPA were separated by SDSPAGE and then transferred to Immobilon membrane. Samples from the top of the figure were 1) biotinylated M, standards; 2) human tPa: 40 ng; 3) human parathyroid cell culture medium from previous studies: medium after 4 days culture as monolayers; 4) medium from organoid culture as in Fig. 1, concentrated 10x on a Centricon 10 (Amicon, Beverly, MA); 5) the same medium before concentration; 6) 25 pg microsomal protein from a human parathyroid adenoma; 7) uPA, 10 ng. Anti-tPA reacted with human tPA and not with uPA. Single bands of 70 kDa were produced on reaction with tPA, cellular and secreted PA samples. One medium sample contained insufficient PA to produce a visible band in the figure, and uPA did not react in this or other analyses. FIG.
roid PA is secreted from parathyroid chief cells. Since, however, the parathyroid is one of few tissues whose secretion is negatively regulated by [Cal, and since the release of PA and PTH remain parallel in the presence of agents like 3-methyladenine and chloroquine, we tentatively conclude that PA is secreted from parathyroid cells and follows a cellular pathway similar to that of PTH. It is possible, however, that the source of PA is endothelial cells or other cell types that are isolated with the parathyroid cells from the tissue, and that the parallelism of secretion to PTH is generated by responses of those cells to PTH, chromogranin A, or other macromolecules released by parathyroid cells. In our earlier study (lo), added PTH had no effect on PA release, but the effects of chromogranin A have not been examined. Confirmation of our tentative conclusion will require immunocytochemical demonstration of PA in the same cells as PTH. From day 8 to day 20 of culture in the study of Fig. 1, the levels of secretion at each [Cal, the degree of inhibition of secretion by Ca, and the [Cal where secretion is inhibited by 50% remained unchanged. Each experimental group adapted from 1.8 mM Ca to its new [Cal in 1
JCE&M.1992 Vol74.No2
day, between days 7 and 8, and secretion rates did not change thereafter. The relative constancy of secretion at each [Cal indicates that within the 2-week period observed, secondary adaptations induced by high or low [Cal did not occur. This result is surprising in light of the demonstrated adaptation of the bovine parathyroid cell to high or low [Cal, wherein the synthesis of proPTH (24) and the cellular levels of its messenger RNA (25) are reduced at high [Cal like 2.5 mM, and increased at [Cal like 0.5 mM. In many respects, the PA of bovine and human parathyroid cells are similar. They are distributed in the same overall subfractions of tissue homogenates (10); the finding that substantial portions of PA and PTH were observed in the nuclear and mitochondrial fractions is likely due simply to the crudeness of the fractionation procedure rather than to their presence in organelles of different sedimentation coefficients. Additionally, bovine and human PA are both secreted, and their secretion is regulated by Ca and modulated by 3-methyladenine and chloroquine in manners indistinguishable from the controls of PTH secretion, suggestive of common pathways of cellular processing. In light of the similarities of the cellular distributions and secretory properties of bovine and human parathyroid PA, it was unexpected that they would be different gene products that are generally accepted to perform different physiological functions. The meaning of the differences in the forms of PA is not clear. Possibly the enzymes function inside the parathyroid cells to regulate the degradation of secreted PTH and chromogranin A; further studies may clarify the question. The purine 3-methyladenine inhibits the formation of autophagosomes in liver cells (26). In bovine parathyroid cells, where its mode of action is unknown, it partially negates the inhibitory action of high [Cal on both PTH and chromogranin A secretion. The antimalarial drug chloroquine is known to disrupt pH gradients in many cell types, notably those between the cytoplasm and interior of lysosomes (27). In bovine parathyroid cells, it increases the release of intact, bioactive PTH, presumably by inhibiting its cleavage by secretory vesicle proteases. Effects of 3-methyladenine and chloroquine are additive in bovine parathyroid cells, and their combined action negates inhibitory effects of [Cal on secretion. In addition to effects on PTH and chromogranin A in bovine parathyroid cells, both agents stimulate the release of uPA and their effects are additive. Because of these properties of the drugs, they constitute useful probes of secretory function in spite of the lack of precise knowledge of their modes of action. When tested on the parathyroid cells of human chronic renal patients in this study, they stimulated PTH and PA secretion at high [Cal in exactly the same additive way that they did in
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Ca-REGULATED
SECRETION
bovine parathyroid cells, indicative of similar secretory pathways in the parathyroid cells of both species. References 1. Dano K, Andreasen PA, Grondahl-Hansen J, Kristensen P, Nielsen LS, Skriver L. Plasminogen activators, tissue degradation and cancer. Adv Cancer Res. 1985;44:139-266. 2. Saksela 0, Rifkin DB. Cell-associated plasminogen activation: regulation and physiological functions. Ann Rev Cell Biol. 1988;4:93-126. 3. Montesano R, Vassali J-D, Baird A, Guillemin R, Orci L. Basic fibroblast growth factor induces angiogenesis in vitro. Proc Nat1 Acad Sci USA 1986;83:7297-7301. 4. Saksela 0, Rifkin DB. Release of basic tihroblast growth factorheparan sulfate complexes from endothelial cells by plasminogen activator-mediated proteolytic activity. J Cell Biol. 1990;110:767775. 5. Galway AB, Oikawa M, Ny T, Hsueh AJW. Epidermal growth factor stimulates tissue plasminogen activator activity and messenger ribonucleic acid levels in cultured rat granulosa cells: mediation by pathways independent of protein kinases-A and -C. Endocrinology. 1989;125:126-135. 6. Bell SM, Brackenbury RW, Leslie ND, Degen JL. Plasminogen activator gene expression is induced by the src oncogene product and tumor promoters. J Biol Chem. 1990;265:1333-1338. 7. Rijken DC, Wijngaards G, Welbergen J. Immunological characterization of plasminogen activator activities in human tissue and body fluids. J Lab Clin Med 1981;97:477-486. a. Rickles RJ, Strickland S. Tissue plasminogen activator mRNA in murine tissues. FEBS Lett. 1988;229:100-106. 9. Canipari R, O’Connell ML, Meyer G, Strickland S. Mouse ovarian granulosa cells produce urokinase-type plasminogen activator, whereas the corresponding rat cells produce tissue type plasminogen activator. J Cell Biol. 1987;105:977-981. 10. Bansal DD, MacGregor RR. Secretion of plasminogen activator from bovine parathyroid cells. Endocrinology. 1990;126:2245-2251. 11. Schultz GS, Sarras Jr MP, Gunther GR, Hull BE, Alicea HA, Gorelick FS, Jamieson JD. Guinea pig pancreatic acini prepared with nurified collaaenase. Exn Cell Res. 1980:130:49-62. 12. Hamilton JW, Spi&o FW, MacGregor RR, Cohn DV. Studies on the biosynthesis in vitro of parathyroid hormone. II. The effect of calcium and magnesium on synthesis of parathyroid hormone isolated from bovine parathyroid tissue and incubation medium. J
OF PA AND PTH
271
Biol Chem. 1971;246:3224-3233. 13. MacGregor RR, Hamilton JW, Kent GN, Shofstall RE, Cohn DV. The degradation of proparathormone and parathormone by parathyroid and liver cathepsin B. J Biol Chem. 1979;254:4428-4433. 14. Ridgeway RD, MacGregor RR. Opposite effects of 1,25(OHhD3 on synthesis and release of PTH compared with secretory protein I. Am J Physiol. l988,254(Endocrinoi Metab 17):E279-E286. 15. Ridgeway RD, Hamilton JW, MacGregor RR. Characteristics of bovine parathyroid cell organoids in culture. In Vitro Cell Dev Biol. 1986;22:91-99. 16. Campbell EE, Shitman MA, Lewis JG, Pasqua JJ, Pizza SV. A calorimetric assay for releasable nlasminoeen activator. Clin Chem. 1982;28:1125-1128. 17. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacterionhaae T4. Nature 1970:227:680-685. 18. Levin EG, Loskutoff DJ. Cultured bovine endothelial cells produce both urokinase and tissue-type plasminogen activators. J Cell Biol. 1982;94:631-636. 19. MacGregor RR, Cohn DV, Hamilton JW. The content of carboxylterminal fragments of parathormone in extracts of fresh bovine parathyroids. Endocrinology. 1983;112:1019-1025. 20. Hunter WM, Greenwood FC. Preparation of iodine 131 labelled human growth hormone of high specific activity. Nature. 1962;194:495-496. 21. Hartree EF. Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal Biochem. 1972;48:422-427. 22. MacGregor RR, Bansal DD. Inhibitors of cellular proteolysis cause increased secretion from parathyroid cells. Biochem Biophys Res Comun. 1989;160:1339-1343. 23. Vassali JD, Belin D. Amiloride selectively inhibits the urokinase type plasminogen activator. FEBS Lett. 1987;214:187-191. 24. MacGregor RR, Hinton DA, Ridgeway RD. Effects of calcium on synthesis and secretion of parathyroid hormone and secretory protein 1. Am J Physiol 1988;255(Endocrinol Metab 18):E299E305. 25. Brookman JJ, Farrow SM, Nicholson L, O’Riordan JLH, Hendy GN. Regulation by calcium of parathyroid hormone mRNA in cultured parathyroid tissue. J Bone Min Res. 1986;1:529-537. 26. Seglen PO, Gordon PB. Methsladenine: snecific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Nat1 Acad Sci USA. 1982 3:79:1889-1892. 27. DeDuve C, De Barsey T, Poole B, Trouet A, Tulkens P, Van Hoof F. Lysosomotropic agents. Biochem Pharmacol. 1974;23:24952531.
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Vol. 74, No. 2
Printed in U.S.A.
Calcium-Regulated Secretion of Tissue Plasminogen Activator and Parathyroid Hormone from Human Parathyroid Cells* DEVI
D. BANSAL
Department
of
Anatomy
AND
RONAL
and Cell Biology,
R. MacGREGOR University
of
Kansas Medical
ABSTRACT. The effects of Ca and other agents on secretion of plasminogen activator (PA) and PTH have been examined and compared, using parathyroid cells obtained from the glands of chronic renal patients. During 2 weeks culture at different [Cal, the secretory rates of PA activity and PTH were parallel; steady-state secretion over 24-h periods was maximal at 0.5-0.9 mM Ca, minimal at 1.5-2.5 mM Ca, and the [Ca] at 50% suppression was 1.1 mM. At 2.5 mM Ca, two inhibitors of cellular proteolysis, 3-methyladenine and chloroquine, stimulated secretion of both PA activity and PTH. The results indicated that secretion of PA from human parathyroid cells is regulated sim-
P
LASMINOGEN activators (PA) are involved in numerous processes that involve tissue remodeling, including tumor growth and metastasis, angiogenesis, and the release of matrix-associated growth factors (l4). The formation of PA is stimulated by growth factors like epidermal growth factor and fibroblast growth factor in several tissues (5), and by oncogene products (6). Many cells and tissues contain and secrete PA (7, 8). In humans, two genes code for PA, one PA is a 70 kilodalton (kDa) protein called tissue PA (tPA), whereas the other is called urokinase (uPA) (1,2). The same PA is normally secreted by a particular cell type in most species, but in some cases the same cell type in two species secretes different PA; for example the ovarian granulosa cells of mice secrete uPA, whereas the corresponding rat cells secrete tPA (9). We recently reported that bovine parathyroid cells secrete a 44 kDa PA (lo), whose secretion is regulated by extracellular [Cal like that of PTH. Its characteristics Received March 1, 1991. Address reprint requests and all correspondence to: Ronal R. MacGregor, Department of Anatomy and Cell Biology, University of Kansas Medical Center, 39th and Rainbow Boulevard, Kansas City, Kansas 66103. * This work was supported by NIH Grant DK-26416 (to RRM) and bv BRSG S07-RR-05373 from the Biomedical Research Support Grant Program, Division of Research Resources, NIH. Presented-in part at the 12th Annual Meeting of the American Society for Bone and Mineral Research, and at the 30th Annual Meeting of the American Society for Cell Biology.
Center, Kansas
City, Kansas 66103
ilarly to that of PTH. The characteristics of human parathyroid PA were also examined using human parathyroid adenoma tissue. In homogenates, the highest specific activity of PA was in microsomal fractions. The M, of PA from tissue and from culture media was 70 kilodalton by sodium dodecyl sulfate gel electrophoresis followed by zymography, or by Western blotting using antisera to human tissue PA @PA). Enzyme activity was inhibited by incubation with antisera to tPA but not to urokinase. In contrast to bovine parathyroid cells that secrete a urokinase, human parathyroids apparently contain and secrete tPA. (J Clin Endocrinol Metab 74: 266-271,1992)
are of uPA. The function of PA in parathyroid tissue is unknown, but its known involvement in tissue remodeling and growth suggests that it could be involved with the hypertrophy and hyperplasia of the parathyroid that result from the conditions of chronic renal failure in humans. In this study we demonstrate that human parathyroid cells contain and secrete PA, but that the enzyme secreted from human parathyroid cells is not that secreted from bovine parathyroid cells. Materials High
M, human
uPA,
and Methods tPA,
placental
uPA
inhibitor,
and
thrombin were purchased from Calbiochem (La Jolla, CA). Human plasminogen, tPA stimulator (fibrin peptides), and substrate S-2251 were purchased from Helena Laboratories (Beaumont, TX). Bovine fibrinogen, bovine plasminogen, amiloride, chloroquine, and other general chemicals were from Sigma (St. Louis, MO) and 3-methyladenine was from Aldrich Chemical (Milwaukee, WI). Electrophoresis reagents were from Bio-Rad (Richmond, CA) and collagenase type I was purchased from Worthington (Freehold, NJ) and purified as described previously (11). Anti-human tPA rabbit antiserum was a gift from Dr. D. Loskutoff (La Jolla, CA), and anti-human uPA goat antiserum was purchased from Vector laboratories (Burlington, CA). Bovine PTH and PTH(37-84) were purified from bovine parathyroids as described previously (12,13), and Nalz51 was purchased from DuPont/New England Nuclear (Wilmington, DE).
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Ca-REGULATED Cell preparation
SECRETION
and treatments
Secretion studies were performed with parathyroid cells obtained as described earlier (14) from chronic renal patients, since their responses to Ca are more reproducible. Cells were counted with a hemocytometer. One or 2 million cells/ml were placed in sterile culture tubes which were then placed in a rotating drum (Bellco) at 37 C in air. Added to the serum-free medium (10) were BSA (1 mg/mL), bovine insulin (5 pg/mL), and bovine transferrin (5 pg/mL). This medium does not support replication of cells, based on observations of monolayers (MacGregor, R., unpublished results). Incubations of fresh cells were terminated after 5 h. If culture was continued, cells aggregated to form organoids (15) with which experiments involving steady state secretion were performed. The media were changed every 2 or 3 days, and after experimental conditions were established, the same [Cal (expressed as total [Cal) were maintained for each organoid throughout the culture periods. Samples of culture media for analysis were always removed 24 h after medium changes. Subcellular
fractionation
Parathyroid adenoma tissue was used because it was considered unsuitable for studies on secretory regulation. The tissue was homogenized with a Potter-Elvehjem homogenizer at 4 C in 0.25 M sucrose. Subsequent steps were performed at 4 C. The homogenate was centrifuged at 600 x g for 10 min to obtain the nuclear fraction. The supernatant was centrifuged at 10,000 X g for 20 min to obtain the mitochondrial fraction. The supernatant was centrifuged at 140,000 x g for 1 h to obtain the microsomal fraction and the supernatant. Assay for PA activity
Assays were carried out in 96 well plates according to Campbell et al. (16) using the plasmin substrate D-val-leu-lys-pnitroanilide (S-2251) at a final concentration of 37 pg/ml. Experimental samples or standard human tPA was added in 10 ~1; plasminogen was present at 0.2 IU/ml and 7 pg heatinactivated bovine parathyroid microsomes were present as an activator (10) in a total volume of 125 ~1. The buffer was 0.1 M Tricine, pH 8.4. Blank incubations contained culture medium or 0.25 M sucrose in place of enzyme. Differences in [Cal or the presence of chloroquine or 3-methyladenine did not alter assay results. Absorbance at 405 nm was read at 30-min or l-h intervals using a Titertek Multiscan plate reader. Activity was expressed as international units of standard human tPA (Calbiochem), or as differences in Ados. Fibrin-agarose
zymography
Samples of cell homogenates and fractions thereof, incubation and culture media, and PA standards were treated with sodium dodecyl sulfate (SDS) at 40 C and separated on 13.5% polyacrylamide gel slabs according to the method of Laemmli (17). Gels were washed with 2.5% Triton X-100 and then placed over fibrin-agarose gels containing plasminogen (18); they were incubated at 37 C, 100% humidity until clear bands representing hydrolyzed fibrin were observed. To estimate the M, of
OF PA AND PTH
267
parathyroid PA, test samples, human tPA, high M, uPA, and prestained protein standards were run on the same plate. Western blots
After electrophoresis as described above, proteins were transferred to nitrocellulose membranes using the Genie transfer apparatus (Idea Scientific Co., Orvallis, OR). Primary and secondary antisera, blocking sera and avidin-biotin horseradish peroxidase (Vector Laboratories Inc.) were diluted with 100 mM Tris, 0.9% NaCl, 0.1% Tween 20, pH 7.5. Membranes were treated with blocking buffer, anti-tPA, secondary antibody, and avidin-biotin horseradish peroxidase reagent, respectively for 30 min each at room temperature at 160% humidity. After each reaction the membrane was extensively washed with Tris buffer described above for 3 x 5 min with gentle shaking. Finally the blot was developed with 4-chloro-L-naphthol and hydrogen peroxide in PBS, pH 7.4, until the bands appeared. RIA for PTH and other methods
Medium samples were examined for PTH using an RIA called the “COOH RIA” that detects PTH and COOH-terminal PTH fragments equally (19). The COOH RIA used bovine PTH as standard, and the tracer was lz51-PTH(37-84) iodinated by the method of Hunter and Greenwood (20). Protein was estimated by the method of Hartree (21). Data are presented as averages *SE, and comparisons made using the Student’s t test.
Results Regulation of PA secretion The steady-state secretion of PTH and PA activity from parathyroid organoids was inhibited by ionized Ca (our data are expressed as total [Cal that is about 85% ionized) (Fig. 1). The sensitivities to Ca of PTH and PA secretion were identical and unchanged between 7 and 20 days of culture; the magnitudes of the secretory responses were likewise stable. Secretion of PA responded to Ca between 1 and 30 days of culture (data not shown). The [Cal at 50% inhibition of steady state secretion of both PA and PTH was 1.1 mM. After correction for the percent ionized, the [Ca”] at 50% inhibition in Fig. 1 was apprOXimately 0.94 mM [&I++].
The role of intracellular degradation in secretion of PA and PTH was examined using 3-methyladenine and chloroquine, agents previously shown to stimulate PTH and PA secretion from bovine parathyroids cells incubated at high [Cal (10, 22). These agents were used to provide information as to whether PTH and PA followed similar secretory pathways. Figure 2 shows that these agents increased the release of PA and PTH in parallel from human parathyroid cells at high [Cal. In combination, their effects on the release of PTH and PA were additive.
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BANSAL AND MacGREGOR
268
JCE & M -1992 Vol74.No2
1.2
O-O.
3
-0
FIG. 1. Steady state regulation of PA and PTH secretion by Ca. A, PA secreted during four 24-h periods of culture; B, PTH secreted during four 24-h periods of culture; C, PA and PTH after 4 days at different [Cal. From hyperplastic parathyroid tissue of one renal patient, parathyroid organoids were cultured for 7 days in M/W medium at 1.8 mM Ca, and then groups of 10 organoids were cultured further at the [Cal indicated until the end of the experiment. Media were sampled after the first 24 h of treatment (day 1 = 8 days of culture) and after 24-h periods following media changes as indicated in the figure. Data points represent averages of six assays of individual cultures for PA, and of five RIA of media from pools of two cultures each for COOH-PTH. SE are omitted for clarity in A and B; they ranged from 310% of the data values for PA, and 26% of the data values for PTH. Results represent one of three similar experiments, each performed with cells from a different patient.
day 1
O-0,
day4
A-A,
day 6
A-&day13
20
2 g
15
3 $
\
10
5 I
&is
B. 0.0
01 0.0
[Cal,
f ( 1.0
3.0
1.0
3.0
[Cal, rnM2’
rni”
O-0,
1.0
PTH --25
A- .A,
PA --20
--15
2 < z c
--10
= h
-- 5
1.0
[Co],
Characterization
of human
parathyroid
PA
The specific activity of PA in fractions of parathyroid homogenates was highest in microsomes, less in mitochondrial and nuclear fractions and almost zero in the high speed cytosol (Fig. 3). The distribution of PTH was similar except for the presence of significant immunoreactivity in the cytosol. To examine the possibility that cytosol might contain a PA inhibitor, its effect on PA activity of spent culture medium was examined. No inhibition of PA activity was detected. To identify the type of PA secreted from human parathyroid cells, medium obtained from cultured organoids was treated with anti-human tPA and anti-human high M, uPA, and then assayed for PA. Table 1 shows that anti-tPA inhibited the PA activity of authentic tPA completely, and of media by 86% and 66% at dilutions of l/1000 and l/2000, respectively. Anti-uPA inhibited authentic uPA, but not the PA in medium. The effects
2.0
LO0
total
of human placental uPA inhibitor and amiloride (23) on secreted PA were examined. Neither agent inhibited the activity (results not shown). The results of the combined inhibitor and antibody studies indicated that PA secreted by human parathyroid cells is tPA. To estimate the M, of human parathyroid PA, samples of microsomes, media, and standard PAS were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and gels were used for zymography or for transfer to Millipore Immobilon-P membranes for Western blotting. Figure 4 shows that on zymography, one band was obtained that migrated as a protein of 70 kDa. Figure 5 shows that Western blots developed with anti-tPA also showed one band 70 kDa. These results indicated that human parathyroid cells secrete only tPA. Discussion This study showed that human parathyroid cells, both freshly isolated and cultured, secrete PA in parallel with
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Ca-REGULATED
SECRETION
OF PA AND PTH
TABLE
1. Effect
of PA antisera
269 on secreted Type
Treatment
05
CO
25
JMA
25 +
2.5 f
clq
25 + +
05
25
25 +
2.5 +
25 + +
WC. 2. Effects of 3-methyladenine and chloroquine on secretion of A, ubated 5 h at 37 C PA; B, PTH. Fresh cells, 2 x 10’ per tube, WI oquine (50 PM) or in the presence of 3-methyladenine (5 mM) both. [Cal below the bars are listed as millimoiars of total Ca. Data represent averages + SE for four samples of pools from two cultures each per group for PA, and eight single RIA of eight individual cultures for PTH. All conditions are significantly different from the control group at 2.5 mM Ca, P < 0.01. Results represent one of two similar experiments, each performed with cells derived from a different patient.
300
Culture
None Anti tPA l/1000 l/2000 Nonimmune serum l/1000 l/2000
0.38 f 0.01
None Anti uPA l/100 l/200
PA activity
of PA activity tPA
High
M, uPA
0.21 f 0.01
0.54 k 0.03
0.05 + 0.001” 0.13 + 0.01”
0.00” 0.002"
0.40 + 0.02 0.49 + 0.028
0.40 f 0.030 0.39 + 0.004
0.13 + 0.002” 0.14 + 0.003”
0.49 + 0.025 0.49 + 0.031
0.38 + 0.016
0.23 + 0.005
0.43 f 0.009
0.46 + 0.005 0.47 + 0.01
0.20 iz 0.004 0.25 + 0.004
0.025 + 0.001” 0.051 + 0.002”
Samples assayed were: 10 ~1 conditioned medium from organoid culture, 0.1 IU human tPA, 0.1 IU human uPA. PA samples were incubated 30 min at 21 C with 10 ~1 assay buffer, anti-tPA, anti-uPA, or nonimmune serum in a total volume of 70 ~1, and then assayed for PA activity; the data represent the A,,, after 2 h 30 min of incubation. ’ Different from no treatment control. P < 0.01.
.E 5L& 200 Y >
E _ 100 2
0 N
Mt
MC
Cyt
N
Mt
MC
CYt
3. PA activity and PTH in centrifugal fractions of human parathyroid homogenates obtained from an adenoma. For assays of PA activity, samples were treated with 0.5% Triton X-100 at room temperature. For COOH-PTH RIA, samples were treated with 1% SDS, 15% mercaptoethanol, diluted to 10 pg protein/100 ~1, and further diluted serially for RIA. Data are expressed as averages of triplicate samples + SE. N, Nuclear fraction; Mt, mitochondrial fraction; MC, microsomal fraction; Cyt, cytosol fraction.
FIG.
PTH under several conditions. The significance of PA secretion is not yet known; it has not been demonstrated to occur from normal human parathyroid cells, but the fact of its release from normal bovine parathyroid cells supports the notion that it is a normal secretory product of the parathyroid. What is its relationship to parathyroid physiology? Recognized functions of PAS from other tissues center around extracellular interactions with the surrounding matrix (1, 2). A potentially important role for PA with clinical relevance is that the enzyme, by activating latent proteases in the matrix, could mediate in part changes that accompany adaptation to chronically high or low [Cal. Studies of wound healing and other cell-matrix interactions have suggested that growth factors are present in the extracellular matrix; these
Microsomes UPA
FIG. 4. Zymography of human tPA, 0.2 U; medium from organoid culture as in Fig. 1, 20 ~1; human parathyroid adenoma microsomes, 2 fig protein; high M, human uPA, 0.2 U. PA from the microsomes and medium along with M, markers were separated by SDS-PAGE (nonreducing) and zymograms generated as described in Materials and Methods.
could be released and/or activated by a pathway that is regulated by the rate of release of PA from the cells (4). The possible results of such activation include the direct action of growth activating or inhibiting factors on parathyroid cell replication, remodeling of the extracellular matrix to accommodate hypertrophic or atrophic responses of the parathyroid, or angiogenic stimulation (3). Elucidation of which, if any of these functions is modulated by parathyroid PA must await further progress. It has not yet been definitely established that parathy-
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BANSAL AND MacGREGOR
270
Mr Stds TPA Media Cone
Media
% 1 i
Media Microsomes UPA
5. Immunological analysis of human parathyroid PA after SDSPAGE and Western blotting. Cellular (microsomes) and secreted (media) PA, human tPA and high M, human uPA were separated by SDSPAGE and then transferred to Immobilon membrane. Samples from the top of the figure were 1) biotinylated M, standards; 2) human tPa: 40 ng; 3) human parathyroid cell culture medium from previous studies: medium after 4 days culture as monolayers; 4) medium from organoid culture as in Fig. 1, concentrated 10x on a Centricon 10 (Amicon, Beverly, MA); 5) the same medium before concentration; 6) 25 pg microsomal protein from a human parathyroid adenoma; 7) uPA, 10 ng. Anti-tPA reacted with human tPA and not with uPA. Single bands of 70 kDa were produced on reaction with tPA, cellular and secreted PA samples. One medium sample contained insufficient PA to produce a visible band in the figure, and uPA did not react in this or other analyses. FIG.
roid PA is secreted from parathyroid chief cells. Since, however, the parathyroid is one of few tissues whose secretion is negatively regulated by [Cal, and since the release of PA and PTH remain parallel in the presence of agents like 3-methyladenine and chloroquine, we tentatively conclude that PA is secreted from parathyroid cells and follows a cellular pathway similar to that of PTH. It is possible, however, that the source of PA is endothelial cells or other cell types that are isolated with the parathyroid cells from the tissue, and that the parallelism of secretion to PTH is generated by responses of those cells to PTH, chromogranin A, or other macromolecules released by parathyroid cells. In our earlier study (lo), added PTH had no effect on PA release, but the effects of chromogranin A have not been examined. Confirmation of our tentative conclusion will require immunocytochemical demonstration of PA in the same cells as PTH. From day 8 to day 20 of culture in the study of Fig. 1, the levels of secretion at each [Cal, the degree of inhibition of secretion by Ca, and the [Cal where secretion is inhibited by 50% remained unchanged. Each experimental group adapted from 1.8 mM Ca to its new [Cal in 1
JCE&M.1992 Vol74.No2
day, between days 7 and 8, and secretion rates did not change thereafter. The relative constancy of secretion at each [Cal indicates that within the 2-week period observed, secondary adaptations induced by high or low [Cal did not occur. This result is surprising in light of the demonstrated adaptation of the bovine parathyroid cell to high or low [Cal, wherein the synthesis of proPTH (24) and the cellular levels of its messenger RNA (25) are reduced at high [Cal like 2.5 mM, and increased at [Cal like 0.5 mM. In many respects, the PA of bovine and human parathyroid cells are similar. They are distributed in the same overall subfractions of tissue homogenates (10); the finding that substantial portions of PA and PTH were observed in the nuclear and mitochondrial fractions is likely due simply to the crudeness of the fractionation procedure rather than to their presence in organelles of different sedimentation coefficients. Additionally, bovine and human PA are both secreted, and their secretion is regulated by Ca and modulated by 3-methyladenine and chloroquine in manners indistinguishable from the controls of PTH secretion, suggestive of common pathways of cellular processing. In light of the similarities of the cellular distributions and secretory properties of bovine and human parathyroid PA, it was unexpected that they would be different gene products that are generally accepted to perform different physiological functions. The meaning of the differences in the forms of PA is not clear. Possibly the enzymes function inside the parathyroid cells to regulate the degradation of secreted PTH and chromogranin A; further studies may clarify the question. The purine 3-methyladenine inhibits the formation of autophagosomes in liver cells (26). In bovine parathyroid cells, where its mode of action is unknown, it partially negates the inhibitory action of high [Cal on both PTH and chromogranin A secretion. The antimalarial drug chloroquine is known to disrupt pH gradients in many cell types, notably those between the cytoplasm and interior of lysosomes (27). In bovine parathyroid cells, it increases the release of intact, bioactive PTH, presumably by inhibiting its cleavage by secretory vesicle proteases. Effects of 3-methyladenine and chloroquine are additive in bovine parathyroid cells, and their combined action negates inhibitory effects of [Cal on secretion. In addition to effects on PTH and chromogranin A in bovine parathyroid cells, both agents stimulate the release of uPA and their effects are additive. Because of these properties of the drugs, they constitute useful probes of secretory function in spite of the lack of precise knowledge of their modes of action. When tested on the parathyroid cells of human chronic renal patients in this study, they stimulated PTH and PA secretion at high [Cal in exactly the same additive way that they did in
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Ca-REGULATED
SECRETION
bovine parathyroid cells, indicative of similar secretory pathways in the parathyroid cells of both species. References 1. Dano K, Andreasen PA, Grondahl-Hansen J, Kristensen P, Nielsen LS, Skriver L. Plasminogen activators, tissue degradation and cancer. Adv Cancer Res. 1985;44:139-266. 2. Saksela 0, Rifkin DB. Cell-associated plasminogen activation: regulation and physiological functions. Ann Rev Cell Biol. 1988;4:93-126. 3. Montesano R, Vassali J-D, Baird A, Guillemin R, Orci L. Basic fibroblast growth factor induces angiogenesis in vitro. Proc Nat1 Acad Sci USA 1986;83:7297-7301. 4. Saksela 0, Rifkin DB. Release of basic tihroblast growth factorheparan sulfate complexes from endothelial cells by plasminogen activator-mediated proteolytic activity. J Cell Biol. 1990;110:767775. 5. Galway AB, Oikawa M, Ny T, Hsueh AJW. Epidermal growth factor stimulates tissue plasminogen activator activity and messenger ribonucleic acid levels in cultured rat granulosa cells: mediation by pathways independent of protein kinases-A and -C. Endocrinology. 1989;125:126-135. 6. Bell SM, Brackenbury RW, Leslie ND, Degen JL. Plasminogen activator gene expression is induced by the src oncogene product and tumor promoters. J Biol Chem. 1990;265:1333-1338. 7. Rijken DC, Wijngaards G, Welbergen J. Immunological characterization of plasminogen activator activities in human tissue and body fluids. J Lab Clin Med 1981;97:477-486. a. Rickles RJ, Strickland S. Tissue plasminogen activator mRNA in murine tissues. FEBS Lett. 1988;229:100-106. 9. Canipari R, O’Connell ML, Meyer G, Strickland S. Mouse ovarian granulosa cells produce urokinase-type plasminogen activator, whereas the corresponding rat cells produce tissue type plasminogen activator. J Cell Biol. 1987;105:977-981. 10. Bansal DD, MacGregor RR. Secretion of plasminogen activator from bovine parathyroid cells. Endocrinology. 1990;126:2245-2251. 11. Schultz GS, Sarras Jr MP, Gunther GR, Hull BE, Alicea HA, Gorelick FS, Jamieson JD. Guinea pig pancreatic acini prepared with nurified collaaenase. Exn Cell Res. 1980:130:49-62. 12. Hamilton JW, Spi&o FW, MacGregor RR, Cohn DV. Studies on the biosynthesis in vitro of parathyroid hormone. II. The effect of calcium and magnesium on synthesis of parathyroid hormone isolated from bovine parathyroid tissue and incubation medium. J
OF PA AND PTH
271
Biol Chem. 1971;246:3224-3233. 13. MacGregor RR, Hamilton JW, Kent GN, Shofstall RE, Cohn DV. The degradation of proparathormone and parathormone by parathyroid and liver cathepsin B. J Biol Chem. 1979;254:4428-4433. 14. Ridgeway RD, MacGregor RR. Opposite effects of 1,25(OHhD3 on synthesis and release of PTH compared with secretory protein I. Am J Physiol. l988,254(Endocrinoi Metab 17):E279-E286. 15. Ridgeway RD, Hamilton JW, MacGregor RR. Characteristics of bovine parathyroid cell organoids in culture. In Vitro Cell Dev Biol. 1986;22:91-99. 16. Campbell EE, Shitman MA, Lewis JG, Pasqua JJ, Pizza SV. A calorimetric assay for releasable nlasminoeen activator. Clin Chem. 1982;28:1125-1128. 17. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacterionhaae T4. Nature 1970:227:680-685. 18. Levin EG, Loskutoff DJ. Cultured bovine endothelial cells produce both urokinase and tissue-type plasminogen activators. J Cell Biol. 1982;94:631-636. 19. MacGregor RR, Cohn DV, Hamilton JW. The content of carboxylterminal fragments of parathormone in extracts of fresh bovine parathyroids. Endocrinology. 1983;112:1019-1025. 20. Hunter WM, Greenwood FC. Preparation of iodine 131 labelled human growth hormone of high specific activity. Nature. 1962;194:495-496. 21. Hartree EF. Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal Biochem. 1972;48:422-427. 22. MacGregor RR, Bansal DD. Inhibitors of cellular proteolysis cause increased secretion from parathyroid cells. Biochem Biophys Res Comun. 1989;160:1339-1343. 23. Vassali JD, Belin D. Amiloride selectively inhibits the urokinase type plasminogen activator. FEBS Lett. 1987;214:187-191. 24. MacGregor RR, Hinton DA, Ridgeway RD. Effects of calcium on synthesis and secretion of parathyroid hormone and secretory protein 1. Am J Physiol 1988;255(Endocrinol Metab 18):E299E305. 25. Brookman JJ, Farrow SM, Nicholson L, O’Riordan JLH, Hendy GN. Regulation by calcium of parathyroid hormone mRNA in cultured parathyroid tissue. J Bone Min Res. 1986;1:529-537. 26. Seglen PO, Gordon PB. Methsladenine: snecific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Nat1 Acad Sci USA. 1982 3:79:1889-1892. 27. DeDuve C, De Barsey T, Poole B, Trouet A, Tulkens P, Van Hoof F. Lysosomotropic agents. Biochem Pharmacol. 1974;23:24952531.
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