Effects of trypan blue on thyroid secretion: localization of trypan blue within the colloid space and phagolysosomes of thyroid follicles
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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 dem-onstrated. 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 thyroglo-bulin. In addition, the dye in association with thyroglobulin has been demonstrated within phago-somes 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 chromato-graphy. 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 mecha-nisms of inhibition were suggested: the inhibition of thy-roglobulin 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 pre-bind 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 re-sponse to TSH, Le.,pseudopod formation was marked-ly suppressed and colloid droplet formation wasmarkedly diminished at 20 min to two hours after TSHadministration. Fusion of colloid droplets with Iyso-somes 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.
marked decrease in numbers of phagosomes (colloiddroplets). In contrast to previous studies employing thechick embryo yolk sac (4), it was not possible to visual-ize trypan blue within the colloid space or phagolyso-somes by electron microscopy.The present studies employing fluorescence micros-copy and isolated phagolysosomal cell fractions weredesigned to determine whether trypan blue entered thecolloid space and / or thyroid follicular cell phagolyso-somes.
MATERIALS AND METHODS
Trypan blue localization by fluorescence microscopy
Thyroid tissues were prepared for fluorescence mi-croscopy 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 inject-ed 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 vapor-fixed over 4% paraformaldehyde at 51C for 2 h,then rely-ophilized for 4 h.After vacuum-embedding in soft paraf-fin, 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 mic-roscope 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 prepara-tion employed in our previous studies . It was necessaryto dialyze this material extensively utilizing a Spec-tra /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 slaughter-house (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 solu-tion (6) containing glucose, 5 mg/ml; bovine serumalbumin, 2 mg/ml; penicillin-G, 0.05 mg/ml; strepto-mycin, 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 set-ting. Utilizing previous methods of differential centrifu-gation (1, 7, 8) the 800-15,000 x g or Iysosomal-phag-olysosomal fractions (P15) and crude thyroglobUlinfractions (S15) were obtained . Portions of the Pwfrac-tion were treated by repeated freezing and thawing , orwith 0.1 % Triton X-l00, as previously described (8), torupture or dissolve phagolysosomal membranes.
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 grav-ity = 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).
Trypan blue localizatjon in thyroid follicles
~ ~ ~ ~ ~. . . . coI ~ I ~ I C;; I ~ I cxi I
O' I I I I I I 01 5 10 15 20 25
Bottom Gradient Fractions Top
Fig.2 - Profilesof sucrose gradient fractionation (0.7ml/frac-tion) of 800-15,000 xg Iysosomal-phagolysosomal(P15) frac-tions from 125 iodine-prelabeledbovine thyroidslices,showingdistribution of cpm(--), protease activity by hemoglobulinassay (11111111), quantitation by spectrophotometric mea-surement of protein,A21 0 ( ), and trypanblue,A590 (- --). The protein peak in the lower fractions (at the 38.5%-55.5% sucrose interface) represents cosedimentation ofcomponents in membrane-bound cytoplasmic particles,while the peak near the top of the gradient within the 20%sucrose layer represents labile or soluble components1.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 con-sistently the same as in Figure 2a.1Symbols and quantitation procedures used for this and all subsequent
figures are the same.
20-1 Concentration (w/w)of Sucrose in Gradient
T T1 .!
I Io 0,- ,-)( )(
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~ ~ FreeThyro-globulin
RESULTSTrypan blue localization by fluorescence microscopyExamination of thyroid tissue from both control andTSH-treated animals revealed pale green autofluores-cence when examined under the fluorescence micro-scope (Fig. 1a). Autofluorescence was limited to follicu-lar 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 fluores-cence but had a more golden color (Figs. 1b, 1c and1d).Connective tissue and the capsule of the thyroid dem-onstrated 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 fluores-cence) within colloid droplets or phagolysosomes, fol-lowing 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 tris-HC1, pH 7.4 and eluted with the same buffer. Fractionswere analysed for 1251, protein, trypan blue and pro-tease as above.
x g for 45 min in a vertical rotor (Sorval OTO-65 ultra-centrifuge). 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 discontinu-ous 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.
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 cosedimen-tation of 1251-thyroglobulin (mol wt 660,000) and thyroi-dal protease (mol wt 40,000), at the interface between38.5% and 55.5% sucrose. Free thyroglobulin and pro-tease 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 con-centration within colloid droplets, Iysosomes or phago-Iysosomes 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 demonstrat-ed 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), membrane-bound 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 illus-trations 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 treat-ment 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 inter-face between sample volume (8.6% sucrose) and 20%sucrose (Fig. 3a). In contrast, most of the 1251-thyroglo-bulin remained at the lower interface showing that itwas not released from membrane-bound structures bythis process. Trypan blue appeared to have been p~rtially released and appeared as protein bound and freedye. When the P15-fractions were treated with Triton-100, 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-
~ T 1.c N:0 Io 0g. ~e i~ ~
T T_ 25 ;:;- ..!..
I I NThyroglobulin ~ bb 20 / Iodide and - ~ ~~ lodoamino acids ~ JE 15 I:J~ ~o 0- ~~ 10 20120
5j i ~\--J ~ ,\., 1IO20 40 60 80 100 120 140 160
MI eluted from Biogel1.5A
Fig. 4 - Biogel 1.5A chromatography of Triton-treated P15fraction following in vitro incubation of bovine thyroid sliceswith TSH and trypan blue, showing elution of trypan blue alongwith thyroglobulin and indicating attachment of the two mole-cules.
tion that was less than before release by Triton. Thepellet from this gradient centrifugation remained blueindicating that some of the dye was attached to mem-brane fragments (not shown). These same expe...