Expression of Nitric Oxide Synthase Isoforms in Bone and Bone Cell Cultures

0013.7227/95/$03.00/O Endocrinology Copyright 0 1995 by The Endocrine Society

Vol. 136, No. 12 Printed in U.S.A.

Expression of Nitric Oxide Synthase Isoforms in the Thyroid Gland: Evidence for a Role of Nitric Oxide in Vascular Control during Goiter Formation*

IDES M. COLIN?, EDUARDO NAVA, DOMINIQUE TOUSSAINT, DOMINIQUE M. MAITER, MARIE-FRANCE VANDENHOVE, THOMAS F. LtJSCHER, JEAN-MARIE KETELSLEGERS, JEAN-FRANCOIS DENEF, AND J. LARRY JAMESON

Division of Endocrinology, Metabolism, and Molecular Medicine (I.M.C., J.L.J.), Northwestern University Medical School, Chicago, Illin.ois 60611; Department of Cardiology, Inselspital (E.N., T.F.L.), Berne, Switzerland; and the Histology (D.T., J.-F.D.), Diabetology and Nutrition (D.M.M., J.-M.K.), and Hormone and Metabolic Research Units, Institute of Cellular and Molecular Pathology (M.-F.V.), University of Louvain Medical School, Brussels, Belgium

ABSTRACT The thyroid gland is a highly vascular tissue, and its blood flow

changes dramatically in various pathological conditions. Although the mechanisms regulating these changes in vascularity and blood flow are not well understood, candidate mediators include endothelin-1 (ET-l) and nitric oxide (NO). In the present study, we used a reverse transcriptase-polymerase chain reaction assay to determine which components of these vasoregulatory pathways are present in the thyroid and to analyze changes in gene expression in an experimental model of goiter formation and involution. Expression of messenger RNAs (mRNAs) encoding ET-l, ET receptors (ET, and ET,), ET-converting enzyme, and the three nitric oxide synthase (NOS) isoforms (NOS I, NOS II, and NOS III) was readily detected in the rat thyroid. After goiter formation was induced by thiouracil and a low iodine diet, there was increased expression of the genes encoding ET-related proteins (ET-l, 3.2-fold; ET,, 2.9-fold; ET,, 3.5fold) as well as two of the three NOS isoforms (NOS I, 2.7-fold; NOS III,

4.9-fold). During iodide-induced involution, the ET-related mRNA levels remained elevated, whereas those of the two NOS isoforms returned to basal values. ET-converting enzyme, NOS II, and thyroglobulin mRNAs were minimally affected in this model, providing evidence for selective regulation ofthese genes. To assess whether NO plays a role in vascular changes during goiter formation, animals were treated with a NOS inhibitor, N-nitro-L-arginine methyl ester (NAME). NOS activity in the thyroid was inhibited by more than 75% after treatment with NAME. Thyroid hormone and TSH levels were unchanged. Although NAME had little effect on overall thyroid size, vascular expansion during goiter formation was decreased by 36%. We conclude that the thyroid gland expresses a complex network of vasoactive genes whose expression is regulated dynamically during thyroid goiter formation and involution. NO production and probably other locally produced vasoactive substances are involved in changes in thyroid vascularization. (Enclocrinology 136: 5283-5290, 1995)

T HE THYROID gland is richly endowed with blood ves- sels. In pathological conditions such as Graves’ hyper-

thyroidism, thyroid enlargement and hyperfunction are accompanied by a bruit, reflecting markedly increased blood flow. Other causes of thyroid goiter exhibit increased vascularization in addition to a proliferation of thyroid follicular cells. Experimental models of goiter have shown that endothelial cells proliferate before follicular cells, perhaps because vascularization may be an important prerequisite for sus- taining thyroid growth (1). During thyroid involution induced by iodide, there is a rapid contraction of capillary

Received June 26, 1995. Address all correspondence and requests for reprints to: Ides M.

Colin, M.D., Division of Endocrinology, Metabolism, and Molecular Medicine, Tarry 15-703, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611-3008.

* Part of this work was presented at the 68th Annual Meeting of the American Thyroid Association, Chicago, IL, September 1994, and at the international symposium: Endothelin in Endocrinology: New Advances, Florence, Italy, October 1994. This work was supported in part by Belgian National Fund for Scientific Research Grant 3.4577.90 (to J.-M.K.) and a grant from the Fund for Scientific Development, University of Louvain (to J.-F.D.).

t Recipient of grants from the Fogarty International Center (NIH) and the Belgian National Fund for Scientific Research.

vascularization, again emphasizing the rapidity of response of the thyroid vasculature (2, 3).

Despite this evidence for marked alterations in thyroid vascularity and blood flow, little is know about the factors that regulate these processes. Because a number of growth and vasoactive factors are produced in the thyroid, changes in thyroid microvasculature and blood flow probably involve the integrated actions of several different factors (4).

Endothelin-1 (ET-l) is an example of a candidate modu- lator. It is synthesized in the thyroid, and its production is highly regulated during goiter formation and involution (5, 6). ET-l binds to two major subtypes of receptors (ET, and ET,) that belong to the family of G protein-coupled receptors (7, 8). Binding studies using ET-1 are consistent with the presence of ET, receptors on cultured thyroid follicular cells (9-11). On the other hand, ET-l interactions with the ET, receptor on endothelial cells triggers the release of nitric oxide (NO), a potent vasodilator that could counteract ET- l-induced vasoconstriction (12-14). Thus, the cellular localization and relative abundance of ET, and ET, receptors could result in distinct responses to ET-l.

As noted above, NO is another substance that plays an important role in the regulation of local blood flow. NO is synthesized by NO synthase (NOS) enzymes, and at least

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5284 ROLE OF NO DURING GOITER FORMATION Endo. 1995 Vol 136 . No 12

three different isoforms have been identified. Brain (type I) and endothelial (type III) isoforms are constitutive Ca’+- calmodulin-dependent enzymes. The type II isoform is an inducible Ca*+-independent enzyme that is expressed pri- marily in immune cells, but is also found in endothelial and vascular smooth muscle cells (15). Although NO has been proposed as a signaling molecule in thyroid cells involved in cGMP accumulation (16, 17), little is known about the expression of the different NOS isoforms in the thyroid.

In the present study, we used a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay to analyze the expression of an array of vasoregulators during thyroid goiter formation and involution. We show that each of the components of the ET [ET-l, ET-converting enzyme (ECE), ET,, and ET,] and NOS (NOS I, II, and III) pathways is present in the thyroid. An inhibitor of NOS activity was used to examine the role of NO in thyroid vascularization. These studies reveal a complex and highly coordinated sys- tem of vasoregulatory factors in the thyroid and demonstrate a role for NO in the microvascular expansion that occurs during goiter formation.

Materials and Methods

Treatments to induce thyroid goiter and involution

Hyperplastic goiter was induced in adult male Wistar rats (-200 g) by feeding a low iodine diet (<20 pg I/kg; Remington diet, Animalabo, Brussels, Belgium) supplemented with 0.25% thiouracil (Sigma Chem- ical Co., St. Louis, MO) for 10 days, followed by a low iodine diet alone for 2 days to clear thiouracil from the gland. Involution was induced by the administration of a normal iodine diet (-2 mg I/kg) for 24 h in conjunction with a lOO-pg ip injection of iodide. In the indicated ex- periments, some rats were treated with 1 mg/ml of the NOS inhibitor N-nitro-L-arginine methyl ester (NAME; Sigma) in the drinking water.

TABLE 1. Primers used in PCR amplifications

Semiquantitative RT-PCR assay

Total RNA was isolated from thyroid glands by the guanidine thio- cyanate/cesium chloride method (18). Complementary DNA (cDNA) was generated by incubating 1 pg total RNA in the presence of random hexamers and 10 U AMV reverse transcriptase (Promega Corp., Mad- ison, WI) in a 20 &reaction for 30 min at 42 C. The RT reaction was ended by heating at 99 C for 5 min, and PCR was performed as previously described (19,20). RT products were serially diluted and amplified in a two-step PCR using Tuq polymerase (2.5 U; Perkin-Elmer Corp., Norwalk, CA) and primers specific for ET, 1538 base pairs (bp)], ET, (398 bp), ET-1 (406 bpl, ECE (376 bp), NOS I (560 bp), NOS II (400 bp), NOS III (809 bp), or thyroglobulin (Tg; 580 bp) cDNAs. Ribosomal protein L19 (RPLIY; 501 bp) cDNA was coamplified as an internal control. The primers used for these assays are depicted in Table 1. First step PCR was performed for 4 cycles (ET,, ET,, and ECE) or 8 cycles (ET-l, NOS I, NOS II, and NOS III; 94 C, 1 min; 55 C, 1 min; 72 C, 1 min) using only the primers of the investigated messenger RNA (mRNA). The reaction was kept at 25 C and then continued using a second 24-cvcle PCR (same conditions as in the first reaction) duringwhich the primers for RPLlY were added. Because of its relative abundance, Tg cDNA amplification was first initiated with RPL19 primers for 6’cyFles, followed by the second step PCR (18 cycles) containing primers for Tg. The identities of the PCR products for ET-l, ET,, ET,, ECE, NOS I, NOS II, and RPL19 were confirmed by subcloning and DNA sequencing using an Applied Biosystems model 373A DNA sequencer (Applied Biosys- terns, Foster City, CA). [?‘]Deoxy-CTP (1.25 &i) was included for detection of the PCR products that were separated by electrophoresis through 6% polyacrylamide gels. Bands were quantified using a Fuji BASlOOO PhosphorImager (Fuji Medical Systems, Stamford, CT). All mRNA samples were analyzed for genomic DNA contamination by demonstrating the absence of PCR products when RT was omitted from the reaction. For each cDNA, the conditions of reaction were optimized to assure that the product amplification remained in the exponential range (Fig. 1). Calculations of inter- and intraassay variability were made using ET-l mRNA levels. They revealed an interassay coefficient of variation of 14% and an intraassay coefficient of variation of 5%.

Measurement of NOS activity

In all experimental groups, each thyroid was removed with the tra- chea in one piece. Both lobes were rapidly isolated under a dissecting

Gene

ETA

ET,

ET-l

ECE

NOS I

NOS II

NOS III

Oligonucleotide sequences

Forward: 5’-ATTTTCATCGTGGGAATGGTG-3’ Reverse: 5’.CCAAGGGCATGCAGAAGTAGA-3’

Forward: 5’-AATACATCAACACGATTGTAT-3’ Reverse: 5’-AGCCAGAACCACAGAGACCAC-3’

Forward: 5’-GCTCCAGAAACAGCTGTCTT-3’ Reverse: 5’-CCAGCTTGGGACAGGGTTTT-3’

Forward: 5’-ACGCTGGACGAAGAGGATCTG-3’ Reverse: 5’-TGAAGGTCCCCCAGCGTGAGT-3’

Forward: 5’-CCCCGTCCTTTGAATACCAG-3’ Reverse: 5’-CCGAGAGCCGAGGCCGAACA-3’

Forward: 5’-GTCACAAGCATCAAAATG-3’ Reverse: 5’-GTGAGCTGGTAGGTTCCTGT-3’

Forward: 5’-TACGGAGCAGCAAATCCAC-3’ Reverse: 5’-CAGGCTGCAGTCCTTTGATC-3’

Nucleotide Il0.a

268-288 805-785

299-319 696-676

52-l 457-438

25-45 400-380

2102-2121 2661-2642

137-156 536-517

NAb NAb

Product Ref. size (bp) IlO.

538 7

398 8

406 42

376 33

560 43

400 44

809 23-24

Tg Forward: 5’-CAGATATGGCAACAGAACTT-3’ 140-159 580 45 Reverse: 5’-GTTGCTCCATTCTCCTCACT-3’ 719-700

RPL19 Forward: 5’.AGTATGCTTAGGCTACAGAA-3’ Reverse: 5’-TTCCTTGGTCTTAGACCTGC-3’

a Except for Tg, the sequences are numbered from the start codon. b Not available.

4-23 501 46 504-485



ROLE OF NO DURING GOITER FORMATION 5285

A ETB

C ETB/RPL19

l/m l/10 11.5 M.5

Dilutions

FIG. 1. Characteristics of the semiquantitative RT-PCR amplification assay. A representative example is shown for ET, and RPL19 transcripts amplified from a control thyroid gland. To assure that the amplification of PCR products remained in the exponential range, the variations in the intensity of signal for ET, (A) and RPL19 (B) were analyzed after different numbers of amplification cycles. C, The linear variation in ET, (28 cycles) and RPL19 (24 cycles) signals in relation to PCR performed with four different dilutions of the RT reaction.

FIG. 2. Detection of ET-l, ET receptor, Tg, and NOS isoform mRNAs in the rat thyroid gland. RT-PCR was used to am- plify ET-l, ECE, and Tg mRNAs (A); ET, and ET, mRNAs (B), and NOS I, NOS II, and NOS III mRNAs (Cl. A 500-bp fragment of RPL19 was amplified from the same diluted RT reaction in a second step PCR. Representative autoradiograms of RT-PCR amplification from hyperplastic glands are presented for each transcript analyzed. The linear increase in the signal of the different transcripts was verified with amplifications performed with four dilutions of the same RT reaction.

RPL19+ T g -d

ET-1 + 1.’ RPL19-) s

C

ETA -e

RPL 19 +

RPL19+

ETB + I

NOSI -t

RPL 19 +

RPL 19+

NOS II -b

NOS III +

RPL 19+

microscope at 4 C, weighed, quickly frozen, and kept at -80 C until use. Thyroid tissue was homogenized in 3 vol of an ice-cold buffer containing 50 mM Tris (pH 7.0),320 mM sucrose, 1 mM EDTA, 1 mM dithiothreitol, 100 pg/ml phenylmethylsulfonylfluoride, 10 pg/ml leupeptin, 10 kg/ml soybean trypsin inhibitor, and 2 pg/ml aprotinin. The homo- genates were centrifuged at 12,000 X g for 20 min.

NOS activity was determined by measuring the conversion of L-[U- “C]arginine to L-[U-i4C]citrulline, as described by Salter et al. (21). Briefly, 20 ~1 of tissue extracts were added to lo-ml tubes (prewarmed to 37 C) containing 100 ~1 of a buffer consisting of 50 mM potassium phosphate (pH 7.0),60 mM valine, 120 PM NADPH, 1.2 mM L-citrulline, 24 PM L-arginine, 150,000 dpm L-[U-14C]arginine, 1.2 mM MgCl,, and 0.24 mM CaCl,. Samples were incubated for 10 min at 37 C before termination of the reaction by the addition of 1.5 ml resin to remove substrate. The resin mixture was diluted with 5 ml H,O and left to settle for 10 min, and the supernatant (4 ml) was removed and analyzed for [U-‘4C]citrulline by liquid scintillation counting. The activity of the Ca2+-calmodulin-dependent NOS was determined from the difference between the [U-‘4C]citrulline produced by control samples and that produced by samples containing 1 mM EGTA. The activity of the Ca*+-

independent NOS was determined from the difference between samples containing 1 mM EGTA and samples containing 1 mM EGTA and 1 mM N-monomethyl-L-arginine.

Morphological analysis

Animals (five per group) were perfused at 100 mm Hg with saline at 37 C for 1 min, followed by Bouin’s fluid for 5 min, as previously described (5, 6). Thyroid glands were dissected, treated for an ad- ditional 24 h in the same fixative, dehydrated, and embedded in Paraplast (Monoject Scientific, Kildare, Ireland). Changes in the relative volume of glandular constituents (epithelium, follicular lumina, and lumina of follicular capillaries) were measured on random he- matoxylin-eosin-stained sections of each lobe using a point-counting method on a 510 microscope at a magnification of X800. The whole section was measured, including the central part and the periphery of the gland, taking into account the intraglandular heterogeneity of the thyroid (22).



5286 ROLE OF NO DURING GOITER FORMATION Endo . 1995 Vol 136 . No 12

ET-1 mRNA LEVELS ECE mRNA LEVELS Tg mRNA LEVELS

~~ i! ;u Control HyperplaSia involution Control Hypwplasia InVOlUtiO” Control Hyperplasia InVOlUtiO”

FIG. 3. Changes in the relative mRNA levels of ET-l, ECE, and Tg in the thyroid gland during goiter formation and involution. Data are expressed as levels of specific mRNA relative to the RPL19 band and represent, for each experimental group, the mean % SEM of four samples (the mRNA of each sample being extracted from a pool of four or five thyroids). *, P < 0.05 vs. control; a, P < 0.05 us. hyperplasia.

ETA mRNA LEVELS ETB mRNA LEVELS 150, * 400 ,

Y

Control Hyperplasia Involution Control H;perplasia Involution

FIG. 4. Changes in the relative mRNA levels of ET-l receptors (ET,, ET,) in the thyroid gland during goiter formation and involution. Data are expressed as levels of specific mRNA relative to the RPL19 band and represent, for each experimental group, the mean -t- SEM of four samples (the mRNA of each sample being extracted from a pool of four or five thyroids). *, P < 0.05 vs. control; a, P < 0.05 vs. hyperplasia.

Thyroid function measurements

Plasma thyroid hormones and TSH were measured by RIA using commercially available kits: Magic T3 (Corning Medical, Medfield, MA), T4 double antibody (DPC, Humbeek, Belgium), and a specific kit for rat TSH (Amersham Corp., Arlington Heights, IL).

Statistical analysis

Data are presented as the mean !z SEM. Statistical evaluation of experimental data was performed using analysis of variance followed by Scheffe’s test to evaluate differences among control, hyperplasia, and involution. Variables for NAME exposure were introduced in the analysis when effects of the NOS inhibitor were tested. Differences were considered significant for P < 0.05.

Results

Expression of ET-related mRNAs and NOS isoform mRNAs in rat thyroid gland

RT-PCR amplification was used to assess expression of ET-l, ET,, ET,, ECE, NOS I, NOS II, NOS III, and Tg mRNAs in the thyroid gland (Fig. 2). Specific PCR products were detected for each substance, and the cDNAs for ET-l, ET,, ET,, ECE, NOS I, and NOS II mRNAs were sequenced to confirm their identities. As the sequence of rat NOS III cDNA is not yet available, primers identical to those used in two previous studies were used, and the predicted 809-bp band was obtained (23,24). These results confirm that ET-l mRNA is expressed in the thyroid gland (6,25) and provide evidence for expression of ECE and the two ET-1 receptors (ET, and ET,). Additionally, we show for the first time that the two constitutive enzymes and the inducible NOS isoforms are expressed in the thyroid gland.

Mean levels of specific mRNAs are expressed relative to those of RPL19 mRNA, which encodes a ribosomal protein that has been used previously as an internal control under conditions characterized by extensive tissue reorganization (26, 27). With the caveat that comparisons of different PCR products may not be quantitative (because different primers are used), basal expression of different mRNAs was initially analyzed in control glands. In this RT-PCR paradigm, Tg mRNA levels, which were analyzed as a control, appear to be low compared to levels of other messages. However, to avoid a rapid saturation of signal, PCR amplification of this abundant mRNA was carried out using a much lower

NOS I mRNA LEVELS NOS II mRNA LEVELS NOS Ill mRNA LEVELS

Control Hyperplasia Involution Control Hyperplasia Involution Control Hyperplasia lnvolutio”

FIG. 5. Changes in the relative mRNA levels of NOS isoforms (NOS I, NOS II, and NOS III) in the thyroid gland during goiter formation and involution. Data are expressed as levels of specific mRNA relative to the RPL19 band and represent, for each experimental group, the mean % SEM of four samples (the mRNA of each sample being extracted from a pool of four or five thyroids). *, P < 0.05 us. control; a, P < 0.05 us. hyperplasia.




number of cycles (18 cycles instead of 28-32 cycles; Fig. 3). Of the two ET-l receptors (28 cycles of amplification for each), expression of ET, was somewhat more abundant than that of ET, mRNA (Fig. 4). Among the NOS isoforms (32 cycles of amplification for each), NOS III mRNA levels were greater than those of NOS I and NOS II (Fig. 5).

Effects of thyroid hyperplasia and involution on expression of ET-related mRNAs

Changes in the expression of each of these mRNAs were analyzed during goiter formation and involution. After induction of thyroid hyperplasia, ET-1 mRNA levels increased significantly (3.2-fold VS. control; P < 0.05). Although ECE mRNA levels were 2.6-fold greater than control values, the difference did not reach significance. Tg mRNA levels were minimally modified in goitrous and involuting glands, providing further evidence that the changes in ET-related proteins were specific (28) (Fig. 3). The levels of ET, (2.9-fold IIS. control; P < 0.05) and ET, receptor mRNAs (3.5-fold VS. control; P < 0.05) were also elevated after the development of hyperplasia (Fig. 4).

Twenty-four hours after iodine refeeding to induce thyroid involution, ET-l and ET, mRNA levels were significantly reduced compared to those during hyperplasia (P < 0.05), although they remained higher than in controls (P < 0.05). ET, and ECE mRNAs showed a slight reduction that was not statistically significant (Figs. 3 and 4). Overall, these results reveal a coordinate induction of ET-l; its converting enzyme, ECE; and its receptors, ET, and ET,, during thyroid hyperplasia, with partial suppression of mRNA levels after short term treatment with iodide.

Effects of thyroid hyperplasia and involution on expression of NOS isoform mRNAs

Among the three NOS isoform mRNAs, NOS I (2.7-fold) and NOS III (4.9-fold) mRNA levels were significantly increased during thyroid hyperplasia (P < 0.05), whereas NOS II mRNA levels were unchanged and remained low, as in control thyroid tissue. During iodide-induced involution, the stimulated levels of NOS I and NOS III mRNA levels returned to control values (Fig. 5).

Effect of the NOS inhibitor (NAME) on thyroid function, goiter formation, and vascularity

NOS enzyme activity was measured in the thyroid gland using the L-[U-i4C]arginine to L-[U-‘*C]citrulline conversion assay (Fig. 6). Total NOS activity (constitutive and inducible NOS) was similar in all experimental groups. Constitutive NOS activity accounted for the major part of total enzyme activity (>85%). In rats treated with a NOS inhibitor, NAME (1 mg/ml drinking water) (29), enzyme activity in the thyroid was significantly reduced (75-85%) compared to that in animals without the NOS inhibitor.

As shown in Table 2, NAME had minimal effects on T,, T,, and TSH levels. Reflecting the effects of the low iodine diet and treatment with thiouracil, plasma T, values were significantly decreased (P < 0.05) and plasma TSH levels were increased 2.3-fold (P < 0.05) during hyperplasia. After iodide

01 I, # n -NAME

q +NAME

Control Hyperplasia Involution

FIG. 6. NOS enzyme activity in the rat thyroid gland during goiter formation and involution in the presence and absence of a NOS inhibitor. Total NOS activity was determined by measuring the conversion of [U-14Clarginine to [U-14Clcitrulline. Data are expressed as the mean 2 SEM of eight pools of thyroid glands in groups without NAME and three pools of glands from animals treated with NAME (one pool = four or five glands). #, P < 0.05 us. the group without NAME.

administration, there was a fall in plasma T, values, consistent with a Wolff-Chaikoff effect (30). However, there were no statistically significant differences in thyroid function tests in animals treated with NAME.

As expected, a hyperplastic goiter was achieved after 12 days of goitrogen treatment, with a 2.1-fold increase in thyroid weight (P < 0.05; Table 3). In the presence of NAME, thyroid weight was slightly reduced in all experimental groups, but these changes were not significantly different VS. values in animals treated without NAME. Morphometric analyses (Table 3) confirmed that after 12 days of goitrogen treatment, thyroid glands fulfilled the criteria of goiter. The relative volume of epithelium was significantly increased (P < 0.05), and follicular lumina were reduced more than 3-fold (P < 0.05). NAME had minimal effects on these parameters. After 24 h of iodine refeeding, follicular lumina were wid- ened. In this case, NAME appeared to slightly enhance the increase in follicular lumen volume.

Morphometric analyses of vascular volume indicated a pronounced effect of NAME during thyroid hyperplasia (Fig. 7). The hyperplastic response was accompanied by an increase in vascular volume from 5.6% to 17%. After treatment with NAME, the increase in vascular volume was inhibited by 36% (P < 0.05). During involution, vascular volume was decreased in the absence or presence of NAME.

Discussion

Proliferation of endothelial cells, expansion of vascular spaces, and increase in local blood flow are hallmarks of iodine-deficient goiter (1). Functional studies have shown that thyroid blood flow is highly sensitive to iodide levels; blood flow increases during iodine deficiency and decreases when the trace element is given back (31). As iodide uptake by follicular cells depends in part on the rate of glandular



5288 ROLE OF NO DURING GOITER FORMATION Endo. 1995 Vol 136. No 12

TABLE 2. Parameters of thyroid function in control, hyperplastic, and involuted groups

Groups T, (/.&l/100 ml) T, (rig/ml)

-NAME +NAME -NAME +NAME

Control 2.41 k 0.19 1.94 2 0.15 0.606 t 0.039 0.662 2 0.044 Hyperplasia 1.59 2 0.11” 1.67 i 0.23 1.134 i 0.081” 0.96 2 0.049 Involution 1.11 k 0.22” 1.16 t 0.19” 0.702 i 0.096 0.712 t 0.02

Values are the mean 2 SEM of values obtained in five sera. “P < 0.05 US. control.

TSH (rig/ml)

-NAME +NAME

5.01 2 0.38 4.40 + 0.69 11.72 t 1.73” 8.66 I! 1.15 16.10 ? 1.46" 13.80 2 1.19”

TABLE 3. Changes in the relative volume of epithelium/follicular lumina and in thyroid weight in control, hyperplastic, and involuted

groups

Groups


Epithelium (% total thyroid)” Follicular lumina (% total thyroid)” Thyroid wt (mgIb

-NAME +NAME -NAME +NAME -NAME +NAME

44.6 5 1.4 44.1 ? 0.7 27.4 i- 1.5 29.4 ? 1.5 16.02 t 1.08 14.85 -+ 1.08 52.3 +- 0.9" 56.2 z O.gcad 06.7 k 0.6 09.9 t 0.9" 33.10 i 3.49' 28.88 k 3.18 52.5 2 1.7' 49.3 2 1.8" 10.8 2 1.8" 14.0 ? 0.2',' 37.80 -c 3.20 32.30 t 3.30"

a Mean t- SEM (two sections per animal, five animals). b Mean i SEM of values obtained in five glands. ’ P < 0.05 US. control. d P < 0.05 us. same experimental group without NAME. e P < 0.05 US. goiter.

15 -

10 -

5-

o-

*

-NAME

+NAME


FIG. 7. Changes in the relative volume occupied by follicular capillaries in the rat thyroid gland during goiter formation and involution in the presence and absence of a NOS inhibitor. Data are expressed as the mean 5 SEM (two sections per animal, five animals; n = 5). *, P < 0.05 US. control; #, P < 0.05 us. the group without NAME.

blood flow, thyroid-specific enlargement of the microvasculature in periods of iodine deficiency may provide a compensatory mechanism to help safeguard thyroid hormone synthesis (31). Likewise, the vasoconstriction observed after providing iodide to iodine-deficient goiter could serve as a protective mechanism against toxic effects of excess iodine (2, 3).

Mechanisms mediating thyroid microvascular modifica- tions are not well understood. There is evidence to suggest that the systemic vascular tone is controlled in part by locally generated vasoactive substances, such as ET-l or NO. These substances may act to preserve homeostatic balance in response to other humoral and hemodynamic stimuli (13,32). ET-l is present in both epithelial and vascular compartments

of rat and porcine thyroid gland, and it has recently been shown to be produced by human and porcine thyrocytes in culture (5, 10, 25). The demonstration that ET-l mRNA is expressed in the thyroid gland combined with evidence that it inhibits Tg release and TSH- and CAMP-induced iodide uptake are consistent with the action of ET-1 as a paracrine/ autocrine factor (6, 9, 11,25). This idea is further supported by the present study, in which ECE as well as ET, and ET, mRNAs were shown to be expressed in the thyroid.

ET-1 is derived from the cleavage of a 39-amino acid pro- hormone (big ET-l) by an endopeptidase termed ECE (33, 34). Our results show that ECE is expressed in the thyroid, providing a potential mechanism for modulating the synthesis of ET-l. ECE mRNA levels were relatively high compared to other messages (e.g. ET,, ET,) amplified with the same number of cycles (28 cycles) and were minimally affected by glandular growth and involution.

The effects of ET-l are thought to be mediated by two specific receptors, ET, and ET,, each of which have been cloned (7, 8). The ET, receptor is selective for ET-l-and ET-2, whereas the ET, receptor is nonselective for different ET peptides. In vitro binding studies in porcine and human thyrocytes in culture showed the presence of saturable ET-l-binding sites with characteristics of the ET, receptor (9-11). The results presented herein indicate that both ET, and ET, receptors are expressed in the thyroid gland. The mRNA levels for the ET, and ET, receptors as well as ET-l were coordinately regulated during goiter formation and iodide-induced involution. Thus, similar mechanisms may control the expression of these genes. Although there are limitations in the ability to compare the amounts of transcripts amplified using different primers, it appeared that the relative abundance of the ET, transcript was greater than that of ET, regardless of the functional status of the gland. Thus, we did not observe an inversion in the ratio of ET,/ET, as described in placenta during pregnancy (35) or in cultured rat vascular smooth muscle cells depending on culture conditions (36). The apparent preponderance of




ET, receptor, which is known to mediate vasorelaxation effects of ET-l (12,141, could be related to the requirement for maintaining high local blood flow during iodine deficiency.

In addition to potential vasodilatory effects of ET-1 via the ET, receptor, we considered the possibility that the potential vasoconstrictive effects of ET-l (via the ET, receptor) might also be antagonized by other vasodilative substances. This hypothesis is supported by the fact that soon after their discovery, ETs appeared to have the capacity to induce endothelium-dependent vasorelaxation (37). It is now clear that vasodilative effects of ET-1 involve the release of locally acting agents, of which NO is probably the most important (13, 32). The link between NO and ET-1 involves a direct coupling between ET, receptor and NO synthesis (12, 14).

The detection of NOS activity and the identification of three NOS isoform mRNAs indicate that NO is synthesized in the thyroid. The specificity of NOS activity was confirmed by the marked reduction in enzymatic activity in rats treated with NAME, a NOS inhibitor (29). Among the three NOS isoforms, type III (endothelial isoform) exhibited the highest level of expression. This finding is in keeping with the ob- servation that most of the enzymatic activity was of the constitutive type.

The use of the NOS inhibitor, NAME, allowed analysis of morphological and functional changes after blockade of NOS enzymatic activity. Morphometric data indicated that during goiter genesis, the expansion of capillaries was significantly reduced in the presence of NAME. This ob- servation suggests that NO is involved in the goiter-specific increase in the microvasculature. Importantly, there was no clear effect of NAME on thyroid function tests, suggesting that these changes in vascularization are not secondary to alterations in thyroid hormone biosynthesis or compensatory effects of TSH. This proposed role for NO in the control of thyroid vascularity is in accord with studies in several other glandular tissues which showed that NO modulates local blood flow. For example, NO has been reported to be responsible for the increase in the pancreatic blood flow after treatment with cholecystokinin or caerulein (38, 39). In the isolated adrenal gland prep- aration, NO appears to be involved in basal vascular tone (40), and infusion of L-NAME in viva causes a significant reduction in adrenal medullary blood flow in response to splanchnic nerve stimulation (41).

In summary, our data indicate that the mRNAs encoding ET receptors, ECE, and ET-l are expressed in the rat thyroid gland and are regulated in a coordinated manner during goiter formation and involution. The recent availability of specific ET receptor agonists and antagonists will be useful for examining further the relevance of ET-l in thyroid function and microvascular changes. We also provide evidence that the mRNAs of the three NOS isoforms are expressed in the thyroid gland. The significant reduction in thyroid vascular expansion in goitrous rats treated with a NOS inhibitor strongly suggests that NO is involved at least in part in the vascular remodeling that occurs in this experimental model of goiter formation.

Acknowledgments

We thank Dr. J. Weiss for expert technical advice regarding the quantitative RT-PCR assay. Helpful comments from Dr. P. Kopp are also acknowledged.

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Expression of Nitric Oxide Synthase Isoforms in Bone and Bone Cell Cultures

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