Effect of cooling on intracellular transport and secretion of thyroglobulin

Cell Tissue Res (1986) 247:505 513

andTtssue R e s e a r c h �9 Springer-Verlag 1987

Effect of cooling on intracellular transport and secretion of thyroglobulin

P. Ring, U. Bjfrkman, and R. Ekholm Department of Anatomy, University of G6teborg, G6teborg, Sweden

Summary. The effect of cooling to 20~ on the intracellular transport and secretion of thyroglobulin was studied by incubating open thyroid follicles isolated from porcine thyroid tissue. Follicles were labeled with 3H_leucine or 3H_ galactose and the secretion of labeled thyroglobulin into the incubation medium was followed by chase incubations under various experimental conditions. The observations indicate that the transport of thyroglobulin is inhibited at three sites of the intracellular pathway by cooling to 20 ~ C, i.e., between the RER cisternae and the Golgi cisternae, between the latter and the exocytic vesicles, and between these vesicles and the extracellular space (corresponding to the follicle lumen). The secretion of 3H-leucine-labeled thyroglobulin decreased linearly between 37 ~ and 20 ~ C; within this temperature range the activation energy for secretion, calculated from Arrhenius plots, was found to be 37 kcal/mol. Below 20 ~ C the secretion was scarcely measur- able. It is suggested that the three transport blocks at 20 ~ C result mainly from inhibition of membrane fission and fusion due to phase transition in membrane lipids.

Key words: Thyroid gland (porcine, rat) - Thyroglobulin Intracellular transport Secretion Low temperature

Numerous observations indicate that the secretory course of thyroglobulin in the thyroid follicle cells is basically the same as for secretory proteins in exocrine glandular cells (for references, see Ekholm 1981). This implies that the thyroglobulin polypeptides are synthesized on polysomes attached to RER membranes and transferred into the RER cisternae; the primary glycosylation occurs in connection with the transmembrane transfer of the polypeptides. From the RER compartments the glycoprotein is transported to the Golgi complex where all or most of the peripheral monosaccharides are incorporated. The thyroglobulin is carried to the apical plasma membrane by exocytic vesicles and discharged into the follicle lumen by exocytosis.

The secretory course of thyroglobulin thus involves transfer of the glycoprotein through three defined compartments: RER cisternae, Golgi cisternae and follicle lumen. It is generally accepted that in exocrine glandular cells the

Send off)~rint requests to." Dr. R. Ekholm, Dept. of Anatomy, Uni- versity of G6teborg, Box 33031, S-400 33 G6teborg, Sweden

transport between the corresponding compartments is discontinuous, by means of transit vesicles (Jamieson 1978). In thyroid follicle cells transport between the Golgi com- partment and the follicle lumen is maintained by exocytic vesicles and is thus discontinuous (Bj6rkman et al. 1974; Ekholm et al. 1975). There are good reasons to assume that the thyroglobulin is also conveyed from the RER cisternae to Golgi cisternae by transit vesicles although this has not been directly observed. Consequently, since transport between compartments by vesicles implies membrane fission and fusion, it seems that such processes are involved at four sites of the secretory course of thyroglobulin, i.e., at the distal end of the RER cisternae, on both sides of the Golgi complex, and at the secretory (apical) cell surface.

Studies have shown that many cellular transport processes depending on membrane mobility and membrane fission and fusion (e. g., endocytosis, phagosome-lysosome fusion) are sensitive to moderate cooling. However, studies on such temperature-sensitive processes involved in the secretory course are few and incomplete. The present study, therefore, was undertaken to identify and localize transport steps in the secretory pathway of thyroglobulin that are inhibitable by cooling. The study was performed on a system of isolated, open thyroid follicles in which the newly synthesized thyroglobulin is secreted into the incubation medium.

Materials and methods

Porcine thyroids were obtained from the local abattoir within 10 min after slaughter. The glands were transported to the laboratory in ice-cold Tyrode salt solution saturated with oxygen, buffered with 20 mM HEPES (pH 7.4) and supplemented with 50 IU/ml benzyl-penicillin and 75 IU/ml streptomycin. Before enzymatic digestion, the glands were cut into small pieces and rinsed with a Mg-free Tyrode solution (pH 7.4).

For in vitro studies on rat thyroid lobes male Sprague- Dawley rats weighing 10(~120 g and maintained on a stan- dard pellet diet were used. The animals were anesthetized with pentobarbital and exsanguinated. The trachea with the thyroid gland attached was removed and the lobes were dissected free under a microscope.

Isolation q/fol l icles. The isolation procedure was essentially the same,as described by Denef et al. (1980). In brief, thy-

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roid tissue was cut into small pieces and digested by incubation with collagenase, 130U/ml, in Tyrode solution, pH 7.4, without Mg but containing 5 mM CaC12, 2 lag/ml DNAse and 0.2 mg/ml trypsin inhibitor. Incubation was performed for two periods of 30 min at 37 ~ C in a water incubator under constant shaking and Oz/COa (95 : 5) gass- ing. The digestion was supplemented with mechanical disso- ciation by pipetting and followed by filtration through ny- lon mesh and centrifugation. The digestion and mechanical treatment were adjusted to produce open follicles and follicle segments. Follicle samples isolated from 1 g thyroid tissue contained on an average 200 lag DNA. Before use the follicles were preincubated for I h at 37 ~ C in Tyrode solution, gassed with 02/C02 and containing amino acids (according to Eagle 1959), DNAse and 0.5% bovine serum albumin.

Incubation of isolated follicles with 3 H-leucine or 3 H-galactose. The incubation medium was Tyrode solution, pH 7.4, supplemented with amino acids, 0.2 lag/ml DNAse and 50 IU/ml benzylpenicillin. Follicles were labeled by incubation with 3 H-leucine (cold leucine omitted) or 3 H-galactose (glucose replaced by pyruvate) at 37 ~ or 20 ~ C. The total incubation volumes differed among experiments but 100 lal medium generally contained 100 laCi 3 H-leucine or 200 ~tCi 3H-galactose and follicles isolated from 2 g thyroid tissue. At the end of the labeling 3 ml medium containing 0.1% cold leucine or cold galactose was added and the samples centrifuged at 50 • g for 5 rain to separate the follicles from the incubation medium. The follicle pellets were washed twice and were then resuspended and chase-incubated.

The chase incubation was carried out in plastic test tubes containing follicles obtained from 1 g thyroid tissue in 1.5 ml medium. The various chase conditions are indicated under Results; TSH was used at a concentration of 20 mU/ml and monensin at 1 laM. After the chase incubation the follicles and medium were separated by centrifugation at 50 • for 5 min. The follicle pellets were resuspended and washed once in 1 ml medium and again centrifuged. The supernatant was added to the previously collected chase medium. The final follicle pellet was resuspended in a hypotonic Tyrode solution, frozen and thawed, and homogenized in a Potter-Elvehjem homogenizer. After centrifugation at 105000 • for 1 h the supernatant, containing the soluble proteins, was dialyzed against phosphate- buffered saline, pH 6.8. The chase medium was dialyzed in the same way.

For measurement of total protein-bound radioactivity, the proteins in the resuspended follicle pellets were precipi- tated with trichloroacetic acid (TCA, final conc. 10%). After centrifugation precipitates were washed once in 10% TCA, twice in 95% ethanol and once in ether. The residues were dissolved in 0.4 M NaOH and used for radioactivity, protein and DNA determination. Radioactivity was determined by liquid scintillation; the scintillation cocktail contained 4 g PPO, 0.1 g POPOP and 500 ml ethanol/1 toluene. Protein was estimated according to Lowry et al. (1951) and DNA according to McIntire and Sproull (1957).

Radioactivity in thyroglobulin in the chase media and in the soluble proteins of the follicle pellets was determined after immunoprecipitation. The secretion of thyroglobulin was expressed as labeled thyroglobulin in the medium in per cent of total labeled thyroglobulin (soluble labeled thyroglobulin from the follicles+labeled thyroglobulin in the medium).

Immunoprecipitation of thyroglobulin. Aliquots of chase media and the soluble follicle proteins were mixed with anti- thyroglobulin immunoglobulins, obtained from rabbits, in the predetermined antibody-excess range. The samples, containing 1% Triton X-100, were incubated for 2 h at 24 ~ C. Antiserum to rabbit immunoglobulin was added and the incubation was continued for 1 h at 24~ and then for 24 h at 4 ~ C. The precipitates were collected by centrifugation at 1500 • g for 30 min and washed once. The precipitates were solubilized in 0.5 ml 1 M NaOH and the radioactivity was determined in pellet and supernatant by liquid scintillation as described above.

Electron-microscopic autoradiography. Rat thyroid lobes were labeled by incubation for 15 min at 20~ in 200 lal Tyrode solution with amino acids (leucine omitted) and 200 laCi 3 H-leucine. The lobes were washed twice with Tyr- ode solution at 20~ containing 0.1% unlabeled leucine. Lobes were then chase-incubated for 3 h or 4 h at 37 ~ C or 20 ~ C in Tyrode solution saturated with O2 and supplemented with amino acids.

After the chase the lobes were fixed in 2.5% glutaralde- hyde buffered with 0.05 M sodium cacodylate, pH 7.4, post- fixed in 1% OsO4, dehydrated and embedded in Epon. Thin sections for electron microscopy were stained with uranyl acetate and lead citrate. Autoradiographic emulsion, Ilford L4, was applied onto the carbon-coated sections with the loop technique. Sections were examined in a Philips 300 electron microscope.

Lobes chase-incubated for 3 h were used for quantifying the distribution of autoradiographic grains. This was done on prints of electron micrographs of follicles cut close to the equator. A sector of the follicle was delineated by lines drawn at right angles to the basal and apical surfaces of the follicle cells (Ofverholm and Ericson 1984). For each sector the numbers of grains over cell and over follicle lumen were counted.

Chemicals. Collagenase (type II) was obtained from Worth- ington Biochemical Corporation, Freehold, NJ, USA; bovine pancreas DNAse, type I, and soybean trypsin inhibitor, type I-s, from Sigma Chemical Co., St. Louis, MO, USA; bovine serum albumin fraction V from Miles Bio- chemicals, Slough, England; monensin from Calbiochem- Behring Corp., La Jolla, CA, USA; bovine TSH (30 IU/mg) was a gift from National Hormone and Pituitary Program, Baltimore, MD, USA. 3H-leucine, 130-190 Ci/mmol, and 3H-galactose, 5-20 Ci/mmol, were purchased from the Radiochemical Centre, Amersham, England.

Results

The in vitro system used in the present study consists of open pig thyroid follicles and follicle segments. We have previously demonstrated that this system is suitable for the study of thyroglobulin secretion (Denef et al. 1980; Bj6rk- man and Ekholm 1982). The follicle cells, which have normal polarity and well-preserved structure, synthesize and secrete thyroglobulin into the incubation medium. Follicle samples isolated from 1 g thyroid tissue and containing ap- proximately 200 lag DNA secrete thyroglobulin at a rate of about 2 lag per h. Leakage of protein into the medium is negligible even after 6 h.

Follicles were labeled by incubation with 3 H-leucine or

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Fig. I. Release of labeled thyroglobulin from follicles into the chase-incubation medium at 20 ~ and 37 ~ C after labeling with 3H-leucine for 15 min at 20 ~ and 37 ~ C, respectively. TSH (20 mU/ml) was present in one of each pair of samples during the last 0.5 h of chase. In this and following experiments the amount of labeled thyroglobulin in the medium is expressed as a percentage of total labeled thyroglobulin (soluble thyroglobulin from the follicles + thyroglobulin in the medium). After labeling at 37 ~ C the total thyroglobulin-bound radioactivity was 3-106 DPM per sample (follicles isolated from 1 g thyroid tissue; DNA content about 200 ~tg). The corresponding figure for follicles labeled at 20 ~ C w a s 10 6 DPM per sample

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Fig. 2. Release of labeled thyroglobulin from follicles into the chase-incubation medium at 20 ~ and 37~ after labeling with 3H-galactose for 15 rain at 20 ~ and 37~ respectively. TSH (20 mU/ml) was present in one of each pair of samples during the last 0.5 h of chase. After labeling at 37 ~ C the total thyroglobulin-bound radioactivity was 8-104 DPM per sample (follicles obtained from 1 g thyroid tissue). The corresponding figure for follicles labeled at 20 ~ C was 2-104 DPM per sample

3H-ga lac tose at 37 ~ or 20 ~ C. The i n c o r p o r a t i o n rate o f the labeled c o m p o u n d s into thy rog lobu l in at 2 0 ~ was > 50% lower than at 37 ~ C. F o r this reason the secret ion o f thyrog lobu l in was calcula ted t h r o u g h o u t as the rat io between the a m o u n t o f labeled thyrog lobu l in in the m e d i u m and the total labeled thyrog lobu l in (extracel lular + intracellular); this m e t h o d makes the es t imat ion o f secret ion insen- sitive to the rate o f thy rog lobu l in synthesis.

Secretion of thyroglobulin at 20 ~ C

Pilot exper iments in which the effect o f va r ious t empera - tures below 3 7 ~ on the secret ion o f thyrog lobu l in was tested showed that lower ing the t empera tu re to 20 ~ C and below had a s t rong inhib i tory effect. In the exper iment illus- t ra ted in Fig. 1 follicles were labeled with 3H_leucine at 20 ~ o r 3 7 ~ for 15 rain and chased at 20 ~ or 3 7 ~ for per iods ranging f rom 0.5 to 4 h. A t 3 7 ~ very small a m o u n t s o f labeled thyrog lobu l in were secreted dur ing the

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Fig. 3. This experiment indicates that the inhibition of secretion induced by cooling to 20~ is reversible. Follicles were labeled with 3H-leucine for 15 min at 20 ~ or 37~ and chased for 2 h or 4 h at 20 ~ or 37 ~ C as indicated. Each bar represents the mean of duplicate values

first hour o f chase; f rom 1.5 h onwards there was a more substant ia l secret ion and the release o f labeled thyrog lobu- lin increased progress ively t h r o u g h o u t the chase period. TSH, present dur ing the last 0.5 h o f chase, s t imula ted the secret ion but the s t imula to ry effect decreased with increasing chase t ime and was no t not iceable at 4 h. In contras t , f rom follicles chased at 2 0 ~ the release o f thyrog lobu l in was minu te t h r o u g h o u t the incuba t ion period.

F igure 2 shows a s imilar exper iment in which follicles were labeled with 3 H-ga lac tose at 20 ~ or 37 ~ C for 15 min and chased at 20 ~ or 3 7 ~ for per iods ranging between 0.5 and 2 h. A t 3 7 ~ secret ion o f labeled thyrog lobu l in was manifes t within 0.5 h and, in the absence o f T S H , the a m o u n t o f secreted thyrog lobu l in increased progressively with increasing chase t ime; T S H had a s t rong s t imula tory effect at 0.5 and 1 h and a less p r o n o u n c e d effect at 1.5 and 2 h. In contras t , follicles chased at 20 ~ C released barely measurab le a m o u n t s o f labeled thyroglobul in .

The exper iment i l lustrated in Fig. 3 shows that the inhi- b i t ion o f thyrog lobul in secret ion by lower ing the t empera - ture to 20 ~ C was reversible. Foll icles were incuba ted with 3H-leucine at 20 ~ or 3 7 ~ for 15 min and chased for 2 or 4 h at 20 ~ or 37 ~ C. It is clear that there is a small difference be tween groups B and C. However , this differ-

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Fig. 4. Experiment demonstrating that exocytosis of thyroglobulin is inhibited by cooling to 20 ~ C. Follicles were labeled with 3 H- leucine at 20 ~ C for ! 5 min and then subjected to two consecutive chase incubations as indicated. The results represent one of two similar experiments, both giving essentially the same results. For details see text

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Fig. 5. Experiment showing that exit of thyroglobulin from the Golgi complex is inhibited by cooling to 20 ~ C. Follicles were labeled with 3 H-galactose at 20 ~ C for 1 h and then chase-incubated at 20 ~ or 37 ~ C for 0.5 h; monensin (1 laM) was present as indicated. Each bar represents the mean of duplicate values; the results represent one of two similar experiments, both giving essentially the same results. For details see text

ence can be r a t iona l i zed , a t least par t ia l ly , i f one a s sumes t h a t d u r i n g labe l ing a t 37 ~ C, labe led t h y r o g l o b u l i n s t a r t ed its i n t r ace l lu l a r m i g r a t i o n immed ia t e ly , w he r ea s at 2 0 ~ labe led t h y r o g l o b u l i n did n o t m o v e f rom R E R . Conse - quent ly , the effect ive chase t ime was 15 m i n longer in g r o u p C t h a n in g r o u p B.

T h e e x p e r i m e n t s i l lus t ra ted in Figs. 1 a n d 2 show t h a t the sec re t ion o f t h y r o g l o b u l i n labe led e i the r wi th 3H_leu_ cine in the R E R c i s te rnae or wi th 3 H - g a l a c t o s e in the Golg i c o m p l e x is a l m o s t comple t e ly i n h i b i t e d a t 20 ~ C. Th i s inhi - b i t i o n cou ld be due to a s ingle b l o c k loca ted apical ly to the Go lg i c o m p l e x or several b locks loca ted a t d i f fe ren t

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Fig. 6. This experiment indicates that transport of thyroglobulin out of the RER is inhibited by cooling to 20 ~ C. Follicles were labeled with 3H-leucine for 15 min at 20~ and then subjected to two consecutive chase incubations as indicated. The results represent one of two similar experiments, both giving essentially the same results. For details see text

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Fig. 7. Arrhenius plot of the rate of secretion of thyroglobulin. Follicles were labeled with 3H-leucine for 15 min at 20~ and chase-incubated for 4 h at temperatures between 20 ~ and 37 ~ C. The data obtained were submitted to linear regression analysis to determine the lines of best fit. The activation energy calculated from the slope of the line is 37 kcal/mol. The correlation coefficient is 0.99

sites o f the in t r ace l lu l a r p a t h w a y . T o e luc ida te th is q u e s t i o n the fo l lowing e x p e r i m e n t s were p e r f o r m e d .

Exocytosis of thyroglobulin

As s h o w n above , 3H_leucine_labeled t h y r o g l o b u l i n beg ins to be secre ted in s u b s t a n t i a l a m o u n t s a f te r a b o u t 1.5 h chase at 37 ~ C. In p r ev ious s tudies we have s h o w n t h a t a t th is

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Fig. 8. Electron-microscopic autoradiograph from a rat thyroid lobe labeled by incubation with 3 H-leucine for 15 min at 20~ and then chase-incubated for 4 h at 37 ~ C. Numerous silver grains are located over the follicle lumen. Many grains remain over the cells where they are more concentrated over the apical region than over other parts, x 11000

time the exocytic vesicles have a high concentration of 3 H- leucine-labeled thyroglobulin and, furthermore, that TSH has a rapid and strong stimulatory action on exocytosis of thyroglobulin (Ekholm et al. 1975; Ofverholm and Eric- son 1984). These observations were utilized in experiments on the effect of cooling on exocytosis.

In these experiments (Fig. 4) follicles were labeled with 3 H-leucine at 20 ~ C for 15 min. They were then chased (chase I) at 20 ~ or 37 ~ C for 1.5 h; at 37 ~ C this time is sufficient to concentrate labeled thyroglobulin in the exocytic vesicles. The follicles were then washed and again chased (chase II) at 20 ~ or 37~ for 0.5 h in the absence or presence of TSH. Figure 4 shows that when both chase I and chase II were run at 37~ labeled thyroglobulin was secreted and

this secretion was greatly stimulated by TSH (Fig. 4C). When chase II was carried out at 20~ after chase I at 37 ~ C both non-st imulated and stimulated secretion was inhibited by about 75% (Fig. 4B). (When both chase incubations were performed at 20 ~ C, secretion was negligible in accordance with the experiments of Fig. 1). Consequently, these experiments show that exocytosis of thyroglobulin is strongly inhibited at 20 ~ C.

Transport of thyroglobulin from the Golgi complex

In a series of experiments we utilized the N a + / K + iono- phore monensin, which arrests the intracellular transport of proteins in the Golgi complex in a variety of cells (Tar-

510

Fig. 9. Autoradiograph from a rat thyroid lobe labeled by incubation with 3H-leucine for 15 min at 20~ and then chase-incubated for 4 h at 20 ~ C. There are no grains over the follicle lumen. Grains over the follicle cell cytoplasm are predominantly located over the RER with relatively low concentration over exocytic vesicles (V) in the apical region (cf. Fig. 8). Note also the absence of grains over the Golgi area (G). x 17000

takoff 1983). Thus, we have previously observed that monensin inhibits secretion of nascent thyroglobulin but does not essentially interfere with TSH-stimulated discharge of thyroglobulin already transferred into the exocytic vesicles (Ring et al. 1983). Follicles were incubated with 3H-galac- rose for 1 h at 20 ~ C, then washed and chased at 20 ~ or 37 ~ C for 0.5 h in the presence of TSH. Figure 5 (B) shows that chase at 37~ after labeling at 20~ resulted in a substantial secretion of 3H-galactose-labeled thyroglobulin. As expected, the secretion from follicles both labeled and chased in the presence of monensin was strongly inhibited (Fig. 5C). However, the secretion from follicles labeled in the absence and chased in the presence of monensin was inhibited to a similar degree, about 85% (D). This indicates that the transfer of 3H-galactose-labeled thyroglobulin from the Golgi complex to exocytic vesicles was blocked

during the labeling period of 1 h at 20 ~ C. (When both labeling and chase were performed at 20 ~ C, secretion was almost completely blocked in agreement with the observations presented in Fig. 2.)

Transport of thyroglobulin Jhom the RER c&ternae

Follicles were labeled with 3H-leucine for 15 min at 20 ~ C and then subjected to two consecutive chase incubations of 1.5 and 0.5 h, respectively, at 20 ~ or 37 ~ C as indicated in Fig. 6. When both the first and second chase were performed at 37 ~ C considerable amounts of labeled thyroglobulin were secreted and the secretion was stimulated by TSH (Fig. 6C). These observations are in full agreement with those illustrated in Fig. 4 showing exocytosis of thyroglobulin. When the first chase was run at 20~ and the

511

second chase at 37 ~ C (Fig. 6B), the inhibition was as complete as that observed after running both chases at 20~ (Fig. 6A). Since at 37~ 0.5 h is sufficient for secretion of thyroglobulin labeled in the Golgi complex (cf. Fig. 5), this indicates that labeled thyroglobulin did not reach the Golgi complex during the 1.5 h chase I at 20 ~ C.

Arrhenius plots of secretion rates

In follicles labeled with 3H-leucine or 3H-galactose for 15 rain at 20 ~ C, secretion of labeled thyroglobulin was measured after chase incubations of 4 h and 1 h, respectively, at temperatures between 12 ~ and 37 ~ C. The secretion rates below 20 ~ C were very low and accurate measurements were not possible. Between 20 ~ and 37 ~ C the secretion rates increased linearly. In Arrhenius plots of secretion rates within this temperature range activation energies were calculated from the slope of the lines. The activation energy for the secretion of 3H-leucine-labeled thyroglobulin was found to be 37 kcal/mol (Fig. 7). For the secretion of 3 H- galactose-labeled thyroglobulin the corresponding figure was 36 kcal/mol (correlation coefficient 0.99). TSH stimulated the secretion of 3H-galactose-labeled thyroglobulin between 20 ~ and 37~ by a factor of 2; the activation energy for this secretion was calculated to be 37 kcal/mol (correlation coefficient 0.99).

Electron-microscopic autoradiography

In autoradiographs obtained from rat thyroid lobes after labeling with 3 H-leucine at 20 ~ C for 15 min and chase incubation at 37 ~ C for 4 h, large numbers of autoradiographic grains were always found over the follicle lumina. Grains were also present over the cells. These grains were fairly evenly distributed over the cytoplasm except for the apical region where the grain density generally was higher (Fig. 8).

In autoradiographs from rat lobes labeled in the same way but chase-incubated at 20~ for 4 h practically no autoradiographic grains were found over the follicle lumina. Grains were fairly evenly distributed over the cytoplasm. However, a general finding was that grains were lacking over the apical region in cells containing great numbers of exocytic vesicles in this area (Fig. 9).

Quantification of autoradiographic grains over follicle sections from lobes chase-incubated for 3 h at 37~ and 20 ~ C, respectively, gave the following results: In 14 sectors of follicles from lobes chased at 37~ 40% (SE 2.1) of the grains were found over the follicle lumen. In 15 sectors of follicles from lobes chased at 20 ~ C the corresponding fraction was 5% (SE 0.8).

Discussion

Most studies of the effect of cooling on cellular transport concern the effect on different types of endocytosis. These processes have been investigated in fibroblasts (Steinman et al. 1974), macrophages (Silverstein et al. 1977; Pratten and Lloyd 1979; Shirazi et al. 1982), hepatocytes (Dunn et al. 1980; Ose et al. 1980; Weigel and Oka 1981), smooth muscle cells (Muir and Bowyer 1983), yolk sack (Duncan and Lloyd 1978), and thyroid gland (Rocmans et al. 1978; Van Sande et al. 1978). The common result of these studies is that the rate of endocytosis is progressively decreased at temperatures below the physiological level. Several obser-

vations further indicate that fluid-phase pinocytosis differs from receptor-mediated endocytosis, phagocytosis and macropinocytosis in the respect that the rate of fluid-phase pinocytosis is directly proportional to the temperature from 2 ~ (10 ~ to 38~ (Steinman et al. 1974; Mahoney et al. 1977; Silverstein et al. 1977; Rocmans et al. 1978; Shirazi et al. 1982), whereas for the latter types ofendocytosis there appears to be a critical temperature near 20 ~ C below which endocytosis practically ceases (Silverstein et al. 1977; Roc- mans et al. 1978; Van Sande et al. 1978; Weigel and Oka 1981; Shirazi et al. 1982). However, observations disagree- ing with this general pattern have been reported (Dunn et al. 1980; Muir and Bowyer 1983). The step following endocytosis in heterophagy, the fusion between endosomes and lysosomes, has also been shown to be strongly affected by temperature, decreasing progressively between 37 ~ and 20 ~ C and completely ceasing at 15 ~ C in macrophages (Kie- lian and Cohn 1980) and at 20~ in hepatocytes (Dunn et al. 1980). In contrast to the ample literature on temperature effects on the endocytic route only a few studies have been reported on the influence of cooling on the transport steps of the secretory course. Jamieson and Palade (1968) found a progressive decrease in transport rate for secretory proteins from the RER cisternae to condensing vacuoles of the Golgi complex in the exocrine pancreas at three temperatures below 37 ~ C (27 ~ 17 ~ and 4 ~ C). Likewise, temperature dependence of the transport between RER cisternae and Golgi complex was indicated by observations on the parathyroid gland (Chu et al. 1977). It was also shown that cooling reduces the rate of exocytosis of histamine from mast cells (Lagonoff and Wan 1974).

In the present study the influence of cooling was examined on the entire secretory pathway of thyroglobulin, from the RER cisternae to extracellular space (corresponding to the follicle lumen). Our observations indicate that the transport of thyroglobulin was blocked by cooling to 20 ~ C at three sites of the intracellular pathway. The last block con- cerned emptying of the exocytic vesicles at the apical cell surface. Demonstration of the middle block was based on observations on the thyroid gland (Herscovics 1969; Whur et al. 1969; Bouchilloux et al. 1981 ; Ring et al. 1987), as well as other cell systems (Kramer and Geuze 1980; Grif- fiths et al. 1982; Roth and Berger 1982), showing that galactose is incorporated into secretory proteins in the Golgi complex, probably in the trans-Golgi cisternae (Griffiths et al. 1982). At 20~ the transfer of 3H-galactose-labeled thyroglobulin from Golgi cisternae into secretory (exocytic) vesicles was inhibited. Regarding the first block, assumed to be located between the RER cisternae and Golgi complex, the autoradiographs showed that the transport of thyroglobulin from the RER cisternae to exocytic vesicles was very much delayed since practically no autoradiographic grains were found over exocytic vesicles even 4 h after labeling with 3 H-leucine (Fig. 9); under normal conditions labeled thyroglobulin reaches the exocytic vesicles within 1 h after radioleucine labeling (Ofverholm and Ericson 1984). Furthermore, the almost complete absence of secretion of 3H-leucine-labeled thyroglobulin during a 0.5-h chase at 37~ following a 1.5-h chase at 20~ (Fig. 6) strongly indicates that labeled thyroglobulin did not reach the Golgi complex during the 1.5-h chase I at 20 ~ C. Hence, the secretion block should be located either between the RER cisternae and the transit vesicles or between the latter and the Golgi cisternae. Since it appears from the discussion

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above that thyroglobul in is retained in the compar tment where it was located when temperature was lowered, it is conceivable that cooling to 20~ can result in t rapping of newly synthesized thyroglobul in in the R E R cisternae. Thus, it seems that at all three sites where the t ranspor t of thyroglobulin was found to be inhibited at 20 ~ C membrane fissions and fusions are necessary for t ranspor t : The terminal t ranspor t step requires fusion of exocytic vesicles with the apical plasma membrane, and the middle and first steps depend on pinching off vesicles from Golgi cisternae and R E R cisternae, respectively.

Secretion rates of thyroglobul in were found to decline linearly between 37~ and 20 ~ C. Below 20~ secretion was drastically reduced. This change in the rate of decrease in secretion at about 20 ~ C seems to correspond to the sharp al terat ion in activation energy seen in Arrhenius plots near 20 ~ C for a number of biological activities, e.g., receptor- mediated endocytosis (Silverstein et al. 1977; Weigel and Oka 1981; Shirazi et al. 1982), exocytosis (Lagonoff and Wan 1974), and plasma membrane Na +, K +-ATPase activity (Barnett and Palazzot to 1976). The existence of these transit ion temperatures is ascribed to phase transi t ion in membrane lipids resulting in a drastic increase in membrane viscosity and decrease in membrane mobil i ty below the transit ion temperature (Melchior and Steim 1976). As dis- cussed above, all three t ranspor t steps in the follicle cells found to be inhibited at 20 ~ C seem to depend on membrane fission and fusion. Accordingly, it is likely that these membrane phenomena were per turbed as a consequence of a l ipid-phase transition. Clearly, it cannot be excluded that other factors of impor tance for fission and fusion of membranes and movement of t ranspor t vesicles were affected at 20 ~ C. One such possible factor is lack of metabolic energy. However, this possibili ty seems unlikely as indicated by the observat ion by Van Sande et al. (1978) that cooling to 20~ for 2 h did not decrease the ATP level in dog thyroid slices. The same authors also reported a normal number and distr ibution of microtubules in thyroid slices at 20~ suggesting that microtubule dysfunction was not an impor tant cause of the observed inhibit ion of secretion.

Below 20~ the secretion rates were too low to allow measurement precise enough for calculation of activation energy from Arrhenius plots. Between 20 ~ and 37~ the secretion rates increased linearly and act ivat ion energies of 36-37 kcal /mol were calculated from Arrhenius plots. These values are higher than those generally found for fluid-phase pinocytosis and receptor-mediated endocytosis (15-25 kcal/ mol) but higher values for receptor-mediated phagocytosis (54 kcal/mol) have been reported (Mahoney et al. 1977). The activation energies calculated in the present study concern the complete secretion course (3 H-leucine experiments) or part of the secretion course (3 H-galactose experiments). Clearly, the secretion of thyroglobulin is a complex course of events that involves a number of integrated chemical and physical processes. The activation energy should reflect the requirements of the rate-l imiting process but the nature of this process in secretion is not known. It could concern factors inherent in the membranes or factors outside the membranes. It is noteworthy, however, that modif icat ions of the membrane phosphol ipid fatty acids change the activation energy for phagocytosis (Mahoney et al. 1977). With respect to the present observations it is interesting that the activation energy for the t ranspor t of thyroglobulin from R E R cisternae to extracellular space did not differ from

the activation energy for the t ranspor t from Golgi cisternae to extracellular space. This indicates that the rate-limiting process either is located between the Golgi complex and the apical cell surface or it is a general process involved in thyroglobulin transport . It is also noteworthy that TSH, al though doubling the secretion rate, did not change the activation energy for the secretion of 3 H-galactose-labeled thyroglobulin. This indicates that TSH did not affect the nature of the rate-limiting process in secretion.

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Accepted June 16, 1986

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Effect of cooling on intracellular transport and secretion of thyroglobulin