J. Endocrinol. Invest. 6: 161, 1983

Effects of trypan blue on thyroid secretion:localization of trypan blue within the colloid spaceand phagolysosomes of thyroid follicles

R.L. Peake*, A.F. Payer**, and C.L. Battle**Departmentof Medicine,and**Departmentof Anatomy, Universityof Texas Medical Branch,Galveston,Texas, 77550, USA

ABSTRACT. Trypan blue was previously shown to directly inhibit thyroid secretion following TSHstimulation. Inhibition of both colloid droplet formation and thyroglobulin proteolysis was dem­onstrated. By observing the characteristic bright red fluorescence of the dye-protein complex, wehave demonstrated that trypan blue rapidly enters the colloid space and combines with thyroglo­bulin. In addition, the dye in association with thyroglobulin has been demonstrated within phago­somes and phagolysosomes by centrifugation of the lysosomal (P1S) fraction on both sucrose andPercoll density gradients. Lability or latency of the dye with the phagolysosomal contents wasdemonstrated and the dye was found in association with thyroglobulin by column chromato­graphy. It is proposed that the complexing of trypan blue to thyroglobulin alters its attachment tospecific follicular cell receptors, inhibits pinocytosis, and, thus, thyroid hormone secretion.

INTRODUCTIONIn earlier studies.(1 ) trypan blue was shown to directlyinhibit TSH- induced thyroid secretion whether the dyewas introduced in vitro or in vivo. Two possible mecha­nisms of inhibition were suggested: the inhibition of thy­roglobulin proteolysis and the inhibition of pinocytosis(colloid droplet formation). Both of these possibilitieshave been further explored.The inhibition of thyroglobulin proteolysis was studiedby examining the effects of trypan blue on the activity ofpurified bovine thyroidal cathepsin D (EC 3.4. 23.5). Itwas demonstrated that the dye did competitively inhibitthe release of iodoamino acids from 1251-thyroglobulin.Inhibition was observed if the dye was allowed to pre­bind to either enzyme or substrate (2).The inhibition of colloid droplet formation. in response toTSH was studied by comparing the ultrastructuralchanges in thyroid follicular cells of mice after TSH, inthe presence and absence of in vivo trypan blue (3).The dye markedly attenuated and abbreviated the re­sponse to TSH, Le.,pseudopod formation was marked­ly suppressed and colloid droplet formation wasmarkedly diminished at 20 min to two hours after TSHadministration. Fusion of colloid droplets with Iyso­somes did not appear to be affected, except for the

Key-words: Colloid droplet, lysosome, phagolysosome, thyroid follicle, trypan blue,pinocytosis.

Correspondence: Dr. R.L.Peake, Department of Internal Medicine, Endocrinologyand Metabolism, The University of Texas Medical Branch, Galveston, Texas77550, USA.

Received June 25, 1982; accepted October 22,1982.

161

marked decrease in numbers of phagosomes (colloiddroplets). In contrast to previous studies employing thechick embryo yolk sac (4), it was not possible to visual­ize trypan blue within the colloid space or phagolyso­somes by electron microscopy.The present studies employing fluorescence micros­copy and isolated phagolysosomal cell fractions weredesigned to determine whether trypan blue entered thecolloid space and / or thyroid follicular cell phagolyso­somes.

MATERIALS AND METHODS

Trypan blue localization by fluorescence microscopy

Thyroid tissues were prepared for fluorescence mi­croscopy using minor modifications of the methods ofDavis and Sauter (5). Whole thyroid glands from mice,or bovine thyroid slices (see below), were incubated forvarying periods of time in Earle's solution (6) containing1mM trypan blue. For in vivo studies, mice were inject­ed ip with 500 mg/Kg trypan blue at 16 and 2h prior tosacrifice. The thyroid glands from all experiments werequick frozen in liquid nitrogen and dried overnight usinga Virtis lyophilizer. The lobes (or slices) were vapor­fixed over 4% paraformaldehyde at 51C for 2 h,then rely­ophilized for 4 h.After vacuum-embedding in soft paraf­fin, tissue was sectioned at 7 Jim; mounted on cleanglass slides using only enough water to flatten sectionsand dried at 45 C for 2 h. Sections were examined forred fluorescence characteristic of trypan blue-proteincomplex using the Leitz Orthoplan fluorescence mic­roscope with incident ultraviolet illumination (KP490excitation filter) and K515 suppression filter.

R.L. Peake, A.F. Payer, and C.L. Battle

Trypan blue desaltingThe trypan blue employed in this study was a new stock(Sigma) and was heavily contamined (65%) with saltsin contrast to the Matheson ,Coleman and Bell prepara­tion employed in our previous studies . It was necessaryto dialyze this material extensively utilizing a Spec­tra /Par HF(Spectrum).After lyophilization the resultingdye was 93% pure as determined by weight recoveryand A590 quantitation (2).

Bovine thyroid slice incubation and fractionationBovine thyroids were obtained fresh from slaughter­house (Doreck's) and maintained on ice until returnedto the laboratory. Fat and connective tissue capsulewas removed and lobes were sliced 1-2 mm thick usinga Stadie-Riggs tissue slicer (A.H. Thomas Co.). Sliceswere incubated under 95% 02-5% C02 in-Earle's solu­tion (6) containing glucose, 5 mg/ml; bovine serumalbumin, 2 mg/ml; penicillin-G, 0.05 mg/ml; strepto­mycin, 0.05 mg/ml; and 0.3jlCi Na1251/ml; in a volumeof 20 ml/g thyroid. Trypan blue was added to somesamples (1mM). Following 2 h incubation at 37C inorder to 1251-label the thyroglobulin, Thytropar (bovine

thyroid stimulating hormone, Armour) was added tosome samples at a concentration of 2-10 mU/ml, for 1h further incubation. Sample flasks were cooled in anice bath and tissue slices removed .After three rinses at4C (1 x Earle's + 2 x 0.25M sucrose), slices wereblotted dry with filter paper and quickly weighed, thenchopped in 0.25 M sucrose and homogenized with aPolytron (Brinkman) for 20 seconds, at number 5 set­ting. Utilizing previous methods of differential centrifu­gation (1, 7, 8) the 800-15,000 x g or Iysosomal-phag­olysosomal fractions (P15) and crude thyroglobUlinfractions (S15) were obtained . Portions of the Pwfrac­tion were treated by repeated freezing and thawing , orwith 0.1 % Triton X-l00, as previously described (8), torupture or dissolve phagolysosomal membranes.

Gradient fractionation

P15 fractions were diluted to the equivalent of 1 g thy ­roid/3.5 ml of 0.25 M sucrose (8.6% w/w) and layeredon discontinuous sucrose gradients composed of 3 mlof 55.5% (Specific gravity = 1.27) and 3.5 mleach of38.5%(Specific gravity =1.17) and 20% (Specific grav­ity = 1.08) sucrose (w/w); then centrifuged at 250,000

Flg.1 - Fluorescence micrographs of mouse thyroid (x 650): a. Incubated In vitro With TSH only; b. incubated in vitro with TSH andtrypan blue for 10 min; c. incubated in vitro with TSH and trypan blue for 20 min; d. from animal treated in vivo with TSH (1 h) andtrypan bfue (16 h).

162

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a. (upper) Shows sedimentationof P15 following incubation ofbovine thyroid slices in vitro with TSHonly (10 mU/ mt),and b.(lower) shows sedimentation of P15 following incubation ofthyroidslices in vitro with TSHas aboveand 1mM trypanblue;note that protein quantitation (A210) is not shown on lowerfigure or on subsequent figures as measurements were con­sistently the same as in Figure 2a.1Symbols and quantitation procedures used for this and all subsequent

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RESULTSTrypan blue localization by fluorescence microscopyExamination of thyroid tissue from both control andTSH-treated animals revealed pale green autofluores­cence when examined under the fluorescence micro­scope (Fig. 1a). Autofluorescence was limited to follicu­lar cell epithelium, the colloid space appeared black.When the mouse thyroid lobes were incubated in 1 mMtrypan blue or when animals injected with this dye in .vivo,bright red fluorescence was noted over the colloidspace. Follicular cells did not show this red fluores­cence but had a more golden color (Figs. 1b, 1c and1d).Connective tissue and the capsule of the thyroid dem­onstrated an even brighter red fluorescence. The timecourse of fluorescence was studied in vitro and palered fluorescence was first noted in the most peripheralfollicles of the lobe following 10 min of incubation,appearing first around the edges of the follicle (Fig. 1b).At later times (20 min to 2 h of incubation), brightred fluorescence was noted throughout the colloidspace of all follicles (Fig. 1c). No attempt was made todetermine the time course of in vivo labeling and allanimals were examined after two injections of trypanblue (see Materials and Methods). In these animals, redfluorescence was uni.formly distributed throughout thecolloid space of all follicles (Fig. 1d).It was never possible to demonstrate the presence oftrypan blue complexed to protein (by red fluores­cence) within colloid droplets or phagolysosomes, fol­lowing either in vitro or in vivo administration of TSH attimes from 10 min to 1 h.

Biogel 1.5A fractionationTo determine if trypan blue was primarily attached tothyroglobulin, P15(after Triton X-100) and S15 fractionswere chromatographed on a 75 x 1.5 cm column ofBiogel 1.5A (Bio-Rad) equilibrated with 0.01 M tris­HC1, pH 7.4 and eluted with the same buffer. Fractionswere analysed for 1251, protein, trypan blue and pro­tease as above.

x g for 45 min in a vertical rotor (Sorval OTO-65 ultra­centrifuge). Ten drop fractions (0.7 ml) were collectedby bottom puncture, treated with 75 J.l11 % Triton X-100,and assayed for 1251-cpm, protein (A21o), (9), proteaseactivity (hemoglobulin assay) (2) and trypan blue (A590)(2).

The above experiments were repeated on discontinu­ous Percoll (Pharmacia) gradients employing 2 ml of50%,3 ml of 35%,4 ml of 20% and 2 ml of 5% Percollsolution which contained the P15-fraction. Gradientswere centrifuged in a 23.5° fixed-angle rotor (SorvalT-865.1 ) at 4800 x g for 20 min. Duplicate gradientscontaining density-marker beads (Pharmacia) wereused for density determination. Ten-drop fractionswere handled in an identical fashion to those above.

163

R.L. Peake, A.F. Payer, and C.L. Battle

g) fraction, containing phagolysosomes and colloiddroplets, from prelabeled bovine thyroid slices, wasisolated by differential centrifugation (7). Fractionsfrom slices preincubated with and without trypan blueand subsequently stirnulateo with TSH were applied todiscontinuous sucrose gradients. Identification of aphagolysosomal fraction was made by the cosedimen­tation of 1251-thyroglobulin (mol wt 660,000) and thyroi­dal protease (mol wt 40,000), at the interface between38.5% and 55.5% sucrose. Free thyroglobulin and pro­tease were sedimented above the 20% sucrose layerof these gradients (Fig. 2). Sedimentation of fractionsfrom tissue preincubated with trypan demonstrated thedye in this same membrane-bound fraction, along withthyroglobulin and protease, indicating more dye con­centration within colloid droplets, Iysosomes or phago­Iysosomes than was associated with free thyroglobUlin(Fig. 2b). As a control, trypan blue was added to thethyroid homogenate or to the isolated P15. No specificlabeling of the phagolysosomal peak was demonstrat­ed and most of the dye remained free (not attached toprotein or membrane structures) (not shown). Whenfractions from thyroid slices treated with trypan blueand TSH (Fig. 2b) were compared to fractions fromtissue treated with TSH only (Fig. 2a), membrane­bound thyroglobulin (at the lower interface) wasmarkedly less in dye-treated P15, indicating inhibition ofcolloid droplet formation. The graph of protease activityoff the gradient was usually smooth and correspondedidentically with that of the thyroglobulin, dye and proteinas in Figure 2a. The jagged appearance of proteaseshown in Figure 2b was not observed in duplicateexperiments. (This experiment was chosen for the illus­trations because this same P15 was employed for thelysosomal lability studies discussed below).

Demonstration that trypan blue is contained withinmembranes and attached to thyroglobulinTwo common means of demonstrating the lability of thecontents of Iysosomes and phagolysosomes has beenexposure to repeated treezinq and thawing and treat­ment with Triton X-100 (8, 10). Both of these methodswere employed in the present experiments with somedifference in results. When Pis-tracticns from TSH +trypan blue-treated thyroid slices were subjected torepeated freezing and thawing, the thyroidal proteasewas completely released and sedimented at the inter­face between sample volume (8.6% sucrose) and 20%sucrose (Fig. 3a). In contrast, most of the 1251-thyroglo­bulin remained at the lower interface showing that itwas not released from membrane-bound structures bythis process. Trypan blue appeared to have been p~r­

tially releas·ed and appeared as protein bound and freedye. When the P15-fractions were treated with Triton­100, virtually all the phagolysosomal contents werereleased, thyroglobulin and protease being recoveredas free or soluble proteins (Fig. 3b). The trypan bluerecovered as protein bound had a relative concentra-

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Identification of trypan blue within thyroid follicular cellsBecause of the inability to visualize trypan blue-proteincomplex within follicular cells by fluorescence micros­copy, isolated subcellular fractions were examined bydensity gradient centrifugation. The P15 (800-15,000 x

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Fig. 3 - Profiles of gradient fractionation of P15, after in vitroincubation of bovine thyroid slices with TSH and trypan blueas in Figure 2b: a. (upper) showing fractionation of P15 afterfreezing and thawing three times to disrupt membrane-boundstructures, solubilizing only the protease; b. (lower) showingfractionation of P15 after treatment with 0.1% Triton X-100 todissolve membrane and solubilize or release all the contents,i.e. thyroglobulin and trypan blue as well as protease.

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tion that was less than before release by Triton. Thepellet from this gradient centrifugation remained blueindicating that some of the dye was attached to mem­brane fragments (not shown). These same experi­ments were repeated employing Percoll gradients (notshown) and the results were identical; 1251-thyroglobul­in, protease and trypan blue were concentrated withina phagolysosome peak on gradients and their contentswere releasable by repeated freezing and thawing, orTriton X-100 treatment. By employing marker beads(color coded for density) it was possible to identify therelative density in Percoll for the phagolysosomes(1.04) (1.1 ) and for soluble proteins, thyroglobulin andprotease (1.02). The separation was not as definite asthat on discontinuous sucrose gradients.To further explore whether trypan blue was specificallyattached to 1251-thyroglobulin or to the lysosomal pro­tease, similar T8H + trypan blue-fractions were treatedwith Triton X-1DO, recentrifuged and the supernatantapplied to a 75 cm Biogel 1.5A column. Trypan bluerepeatedly eluted in the leading edge of the 12SI-thyro­globulin peak (Fig. 4). No dye could be demonstratedwhere the protease eluted or at any other area of thecolumn eluate. Free or non-protein bound dye is ad­sorbed to the gel and does not elute (2). This indicatedthat the dye previously observed within the P1s-fractionwas predominantly associated with the thyroglobulin(see above).Column chromatoqraphy of 815 or soluble thyroglobulinfrom these same slices eluted with this same pattern(not shown), trypan blue eluting with the 1251-thyroglo­bulin peak.

DISCUSSIONTrypan blue, a water-soluble bisazo dye derived fromnaphthalene sulfonic acid (4), has been employed as avital dye since 1909 (12) and it is most commonly used

165

Trypan blue localization in thyroid follicles

to demonstrate that cells are alive (4). Viable cellsexclude the dye while non-viable cells are stained. Thedye also produces a brilliant red fluorescence when it isallowed to complex with soluble and tissue proteinswhile the pure dye does not demonstrate this property(5, 13, 14). Fluorescence of the dye-protein complexhas been employed to study selective permiability ofthe blood brain barrier, and chemical and physicaldamage to this barrier (13, 14).This property has also been employed to study thepenetration of dye into avian and mammalian yolk sacsand to explore the mechanism of the teratogenic prop­erties of trypan blue (5, 12).In previous experiments we were unable to visualizetrypan blue within the colloid space or follicular cells bylight or electron microscopy (1, 3). Lloyd, Beck et al.were also unable to visualize the dye within the thyroid,liver or yolk sac by light microscopy. Blue droplets werenoted within renal tubular epithelium by light micros­copy and dense granules of dye were visualized in yolksac phagolysosomes of trypan blue treated animals byelectron microscopy (4). Only by employing the fluo­rescent property of the dye can the time course anddistribution of trypan blue within the yolk sacs be dem­onstrated (5, 12). Uptake of the dye by the visceralendoderm of embryos was also demonstrated (5).In the present studies we have again demonstrated thelack of fluorescence of trypan blue alone' and haveemployed it to demonstrate that the dye very rapidly«10 min) enters intact thyroid follicles (whole mousethyroid in vitro) and combines with the protein, thyro­globulin (see below), in the colloid space. Following invitro incubations of 20 min or longer the dye-proteincomplex was found throughout all follicles and washomogenously distributed throughout· the colloid ofeach follicle. Although no studies were performed as tothe mechanism of transport, it was presumed that thewater-soluble dye entered the colloid space by diffu­sion transcellular or along the intercellular spaces andattached to the most proximate thyroglobulin mole­cules. Dye was not visualized within cells or dropletsthat might have represented transport through the cells.Other areas of the thyroid were even more intenselystained, i.e., the thyroid capsule and tissues surround­ing arterioles demonstrated brilliant red fluorescence(not shown). The follicular cells no longer demonstrat­ed the typical green autofluorescence, but instead as­sumed a more golden color in the presence of trypanblue. This may represent the effect of the intense colorof the colloid space or it may represent binding tofollicular cell membranes.It was not possible to demonstrate fluorescence withincolloid droplets or phagolysosomes afterTSH in vitro orin vivo. This was due to the inhibition of colloid dropletformation induced by the dye (1 , 3), the thickness of thesections (7 pm), the resolving power of our microscope(1000 x), and the small size of these organelles (0.1-3pm) (15-17).

R.L. Peake, A.F. Payer, and C.L. Battle

Because we were unable to visualize the trypan blue­thyroglobulin complex within colloid droplets andphagolysosomes under the microscope, attentionturned to isolation of the Iysosome-phagolysosomefraction by differential centrifugation. First, in vivoexperiments were attempted using mice and dogs but itwas not possible to inject enough dye to be able toidentify it in tissue fractions with the spectrophotometer(A590) although it had been possible to visualize it underthe fluorescence microscope (see above). To increasethe concentration and amount of dye bound, bovinethyroid slices were employed. Following in vitro incuba­tion, the 800-15,000 x g (P15) fraction, that had pre­viously been shown (7, 8, 10) to contain colloid dro­plets, Iysosomes and phagolysosomes, was isolatedand the cosedimentation of labeled thyroglobulin, thy­roidal protease and trypan blue within membranebound structures was demonstrated separate fromfree or soluble proteins, enzymes and dye. The specificdensities for the these membrane bound elementswere between 1.17 and 1.27 for sucrose gradients and1.04 on Percoll gradients in agreement with those ofIysosomes as published by Pertoft and Laurent (11).The differences in the values by the two gradient sys­tems is due to the hyperosmolarity of sucrose as ex­plained in this reference.To further demonstrate that the membrane-bound iso­lates were indeed phagolysosomes, we examined theproperty of lability or latency. We examined sedimenta­tion profiles of fractions following repeated freezing andthawing or Triton X-100 treatment to dissolve mem­brane. Different results were obtained, i.e., both me­thods released most of the protease, but much of thethyroglobulin and trypan blue remained in the phagoly­some isolated after repeated freezing and thawing.Treatment with Triton X-100 released all the phagolys­osomal contents. These experiments indicated thatTriton X-100 treatment more completely dissolved ordestroyed the phagolysosomal membrane whereastreezlnq and thawfng only ruptured the membrane withpartial release of contents, i.e. protease. Similar obser­vations on relative release of the various lysosomalenzymes have indicated this same difference betweenthe two methods of demonstrating lability (18).The experiments indicated that trypan blue was boundto thyroglobulin or the phagolysosome membrane,rather than to lysosomal protease. When P15 contentsreleased by Triton X-100, were applied to Biogel1.5A,trypan blue eluted with thyroglobulin and was not dem­onstrated in the area of elution of protease. Binding ofsmall amounts of dye to this enzyme were not totallyexcluded but the major portion of dye was associatedwith the larger molecule. That some of the dye wasassociated with or concentrated within the phagolyso­somal membrane appeared likely because the relativeconcentration of dye in the gradient phagolysosomefraction was always much higher than that associatedwith soluble thyroglobulin in the P15-fractions. In addi-

166

tion, the membrane pellet sedimenting to the bottom ofthe gradient tube after Triton treatment remained blueindicating that not all the dye was released with thyro­globulin and protease.It has been demonstrated that trypan blue can anddoes rapidly enter the colloid space and attaches to thestored thyroglobulin. It also was demonstrated that thedye entered follicular cells and was concentrated in thephagosomes. It is proposed that the complexing oftrypan blue with thyroglobulin interferes with the pino­cytosis of colloid and colloid droplet formation possiblyby altering its recognition and binding by specific thy­roglobulin receptors on follicular cell membranes (19,20). Ongoing work in our laboratory with isolated follicu­lar cell membranes indicates that trypan blue doesindeed inhibit specific thyroglobulin binding (manus­cript in preparation). Similar to the conclusions of Lloyd,Beck and co-workers studying yolk sac pinocytosis of1251-albumin (21), the major effect of the dye in inhibit­ing thyroid hormone secretion may be by limiting pino­cytosis. That the dye also inhibits the proteolysis ofthyrogiobulin by lysosomal cathepsin 0 has been dem­onstrated (2) but this may be a less important effect inthe process of TSH-induced thyroid hormone. secre­tion.

REFERENCES1. Peake R.L., Battle C.L., Harrison R.L., Payer A.F.

Direct effects of trypan blue on thyroid secretion.Horm. Res. 10: 191, 1979.

2. Battle C.L., Payer A.F., Peake R.L.Effects of trypan blue on thyroid secretion Inhibition ofpurified cathepsin 0 from bovine thyroid.Biochim. Biophys. Acta 660: 8, 1981 .

3. Payer A.F., Battle C.L., Peake R.L.Ultrastructural and cytochemical effects of trypan blueon TSH stimulation of thyroid follicular cells.Cell tissue Res. 218: 547, 1981.

4. Lloyd J.B., Beck F., Griffiths A., Parry I.M.The mechanism of action of acid bisazo dyesIn: Campbell P.N. (Ed.), The interaction of drugs andsubcellular components in animal cells.Little Brown and Co., Boston, 1968, p. 171.

5. Davis H.W., Sauter R.W.Fluorescence of trypan blue in frozen-dried embryos ofthe rat.Histochemistry 54: 177,1977.

6. Williams J.A., Wolff J.Thyroid secretion in vitro: Multiple actions of agentsaffecting secretion.Endocrinology 88: 206, 1971.

7. Balasubramaniam K., Deiss W.P., Jr.Characteristics of thyroid lysosomal cathepsin.Biochim. Biophys. Acta 110: 564, 1965.

8. Peake R.L., Cates R.J., Deiss W.P., Jr.Thyroglobulin degradation: particulate intermediatesproduced in vivo.Endocrinology 87: 494, 1970.

9. Bailey J.L.Estimation of proteinTechniques in protein chemistry, 2nd Ed.,Elsevier, Ams­terdam, 1967, p. 340.

10. De Duve C.Lysosomes, a new group of cytoplasmic particlesIn: Hayashi T. (Ed.), Subcellular particles.Ronald Press, New York, 1959, p. 128.

11. Pertoft H., Laurent T.C.Isopycnic separation of cells and cell organelles bycentrifugation in modified colloidal silica gradients.In: Catsimpoolas N., (Ed.), Methods of cell separation.Plenum Press, New York, 1977, Vol. 1, p. 53.

12. Callebaut M., Harrisson F., Vakaet L.Peripheral avian yolk assemblage and its persistance inthe blastoderm, studied by trypan blue-induced fluores­cence.Anal. Embryol. (Berl~) 163: 55, 1981.

13. Hamberger A., Hamberger B.Uptake of catecholamines and penetration of trypanblue after blood-brain barrier lesions.Z. Zellforsch. 70: 386, 1966.

14. Steinwall 0., Klatzo I.Selective vulnerability of the blood-brain barrier in chem­ically induced lesions.J. Neuropathol. Exp. Neurol. 25: 542, 1966.

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Trypan blue localization in thyroid follicles

15. Ericson L.E.Exocytosis and endocytosis in the thyroid follicle cell.Mol. Cell. Endocrinol. 22: 1, 1981.

16. Seljelid R.Endocytosis _in thyroid follicle cells III. An electron mi­croscopic study of the cell surface and related struc­tures.J. Ultrastruct. Res. 18: 1, 1967.

17. Seljelid R.Endocytosis in thyroid follicle cells IV.On the acid phos­phatase activity in thyroid follicle cells, with special ref­erence to the quantitative aspects.J. Ultrastruct. Res. 18: 237, 1967.

18. Wattiaux R., De Duve C.Release of bound hydrolases by means of Triton X-100.Biochem. J. 63: 606, 1956.

19. Hashizume K., Fenzi G.F., DeGroot L.Thyroglobulin inhibition of thyrotropin binding to thyroidplasma membrane.J. Clin. Endocrinol.Metab. 46: 679, 1978.

20. Consiglio E., Salvatore G., Rail J., Kohn L.Thyroglobulin interactions with thyroid plasma mem­branes.J. BioI. Chem. 254: 5065, 1979.

21. Williams K., Roberts G., Kidston M.E., Beck F., Lloyd J.Inhibition of pinocytosis in rat yolk sac by trypan blue.Teratology 14: 343,1976.