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Nitric oxide enhances thyroid peroxidase activity in primary human thyrocytes
Life Sciences, Vol. 63, No. 26, pp. PL 373-380,1998 copyright 0 1998 Ekvicr science Inc. Printed in the USA. All rights rescrvcd OOH-3205/98 $19.00 t .oo
ELSEVIER
PI1 50024-3205(98)00527-X
PhXRMACOLOGY LETTERS Accelerated Communication
NITRIC OXIDE ENHANCES
THYROID PEROXIDASE HUMAN THYROCYTES
ACTIVITY
IN PRIMARY
Lesley J. Millatt”2, Alan P. Johnstone, and Guy StJ. Whitley Department
of Cellular and Molecular Sciences, St. George’s Hospital Medical School, Cranmer Terrace, London SW1 7 ORE, United Kingdom (Submitted July 6, 1998; accepted August 24, 1998; received in final form October 13, 1998)
Abstract. Recent studies have demonstrated the production of the multi-functional messenger molecule nitric oxide (NO) by the thyroid gland. To examine a possible role for NO in thyroid function, we studied the acute and chronic effect of NO donors on thyroid peroxidase (TPO) activity in monolayer cultures of primary human thyrocytes, using a calorimetric assay technique. The presence of either S-nitrosoglutathione (GS-NO) or sodium nitroprusside (SNP) (10-6-10a M) at the time of the assay caused a significant increase in TPO activity. Pre-incubation of thyrocytes with 1Q5 M GS-NO for 3 days had no effect on the level of TPO activity when the assay was performed in the absence of NO donors. However, GS-NO pre-incubation significantly enhanced the acute stimulatory effect of GS-NO and SNP on TPO activity. These results suggest a possible role for NO in the regulation of TPO activity and thus thyroid hormone synthesis. Q 1998 Elsevier Science Inc. Key Words: thyroid peroxidase, nitric oxide, thyrocyte
Introduction The thyroid gland is a highly vascularized organ, in which an extensive network of blood capillaries is intimately associated with the thyroid follicles. The abundant blood supply of the gland ensures adequate exposure of the thyrocytes to iodide, which is essential for thyroid hormone formation. Upon uptake by thyrocytes, iodide is oxidized and coupled to thyroglobulin by the heme-containing enzyme thyroid peroxidase (TPO), in a series of reactions which requires the oxidizing agent hydrogen peroxide. In recent years, increasing evidence has emerged to suggest a role for the inter- and intra-cellular mediator nitric oxide (NO) in thyroid gland function. Nitric oxide is synthesized from L-arginine by a wide variety of cell types, in a reaction catalyzed by the NO synthase (NOS) family of enzymes (1). Three distinct isoforms of NOS have been identified, Brain (Type I) NOS (2) and endothelial (Type III) NOS (3,4) isoforms are constitutively expressed Ca”-cahnodulin-dependent enzymes, which release small amounts of NO for short periods of time. The inducible (Type II) ‘Present address: Department of Anesthesiology, University of Virginia, Charlottesville, VA 22906-0010, USA. ‘Corresponding author. Tel: +l-804-982-3969; Fax: +l-804-982-0020 E-mail: lim6n@,virginia.edu
PO Box
10010,
PL-374
NO Enhances Thyroid Peroxidase Activity
Vol. 63, No. 26, 1998
NOS isoform (5) is a Ca”-calmodulin-independent enzyme, which releases large amounts of NO for sustained periods in response to stimulation by inflammatory cytokines and/or bacterial endotoxin (6). Many of the actions of NO are mediated via its interaction with the heme moiety of the enzyme soluble guanylate cyclase, resulting in increased production of the second messenger cyclic guanosine 3’,5’-monophosphate (cGMP) (7). We have previously reported that NO stimulates the production of cGMP in human thyrocytes (8). In addition, several groups have presented evidence for an endogenous production of NO by the thyroid gland. Estevez et al. (9) reported the existence of a constitutive NOS activity in dog thyroid slices, Kasai et al. (10) demonstrated a cytokine-inducible production of NO by cultured human thyrocytes, and Colin et al. (11) showed the expression of mRNAs encoding all three NOS isoforms in the rat thyroid. However, little is known about the functional role of NO within the thyroid gland. We now report that NO donors acutely cause an increase in TPO activity in primary human thyrocytes. We demonstrate this using a calorimetric assay technique for the measurement of the oxidase component of TPO enzyme activity. Materials and Methods Materials. Bovine thyroid-stimulating hormone (TSH) was obtained from Armour Pharmaceutical Company, Eastboume, East Sussex, UK. Ham’s FlO medium and heat-inactivated newborn calf serum (NCS) were obtained from ICN Pharmaceuticals, Thame, Oxon., UK. Dispase type II was obtained from Boehringer Mannheim, Mannheim, Germany. The TPO assay substrate 3,3’,5,5’tetramethylbenzidine (TMB) was obtained from Serva, Heidelberg, Germany, and was prepared as a 6 mg/ml stock solution in DMSO. The NO donor S-nitrosoglutathione (GS-NO) was a kind gift from Dr. H. Hodson (Wellcome Research Laboratories, Beckenham, Kent, UK). All other chemicals were obtained fkom BDH, Poole, Dorset, UK, or Sigma Chemical Company, Poole, Dorset, UK. Ceil culture. Approval for the use of human thyroid tissue was obtained from the Local Ethical Committee. Primary human thyrocytes were prepared by dispase digestion of surgical specimens from patients with multinodular goiter or Graves’ disease, as previously described (12). The resulting primary thyrocytes were grown in monolayer culture in Ham’s FlO medium containing 5% (v/v) NCS and hormone supplements (5H medium) (13). Thyrocytes were either seeded directly into 96-well tissue culture plates following isolation from thyroid tissue, or were detached from 9 cm dishes upon reaching confluence, and transferred to 96-well plates at a density of 10’ cells/ml (100 @/well). Following seeding into 96-well plates, passaged and unpassaged tbyrocytes were cultured in 5H medium until they reached confluence. For passaged thyrocytes, 10 mu/ml TSH was added to the culture medium the day after passage, since TPO activity was found to be virtually undetectable in paasaged cells not exposed to TSH. Primary unpassaged thyrocytes were, for some experiments, cultured in the presence of 10” M GS-NO for three days prior to the performance of TPO assays. TPO Assay. The TPO activity of confluent primary thyrocytes grown in 96-well plates was determined using an adaptation of an assay for neutrophil and eosinophil peroxidase activity (14). Culture medium was aspirated, and cells were washed with 100 pi/well of reaction buffer (140 mM NaCl, 8 mM NaJ-IPO,, 1.5 mM KH,PO,, 5 mM KCl, 0.5 r&l CaCl,, 1.2 mM MgCl,, plus 0.1% (w/v) glucose, 0.2% (w/v) bovine serum albumin, pH 7.4). This buffer was then replaced with 70 pi/well of fresh reaction buffer, in the absence or presence of 10” to lo4 M GS-NO or
NO Enhances Thyroid Peroxidase Activity
Vol. 63, No. 26, 1998
PL375
sodium nitroprusside (SNP). The stock solution of TMH was diluted immediately prior to use in 10 mM sodium acetate, pH 4.2, and 70 @/well added to the cells, to a final concentration of 1.15 mM. Hydrogen peroxide (H,Od solution (4.2 mM) was then added (60 @/well) to initiate the peroxidase reaction, and plates were incubated at 37’C for 15 min. Reactions were terminated by the addition of 2.5 M H,SO, (50 @/well), and absorbance at 405 mn was measured with a Titertek Multiscan MC plate-reader. Quality control and statistical analysis. Control wells were included in all TPO assays. Negative controls consisted of TPO assays performed in the absence of cells or of the substrate TMB. Positive control wells contained 100 pU/well purified horseradish peroxidase (Sigma), t?eshly prepared in reaction buffer. Relative TPO activity for each well was calculated by dividing the absorbance reading at 405 nm by the absorbance reading for cells assayed in the absence of NO donors. Results are expressed as means f S.E.M. of observations performed in quintuplicate. Statistical significance was assessed using the Student’s unpaired two-tailed t-test, and differences were considered significant where PcO.05. Results and Discussion The effect of NO donors on TPO activity was initially determined using unpassaged primary human thyrocytes and the NO donors GS-NO and SNP at concentrations of 10” to lo4 M. Figure 1 shows that these two chemically diverse NO donors both caused a significant increase in TPO activity, which occurred over the range of concentrations tested. The maximal increase in TPO activity caused by both NO donors (approx. 70% increase over baseline) occurred at a concentration of 1u5 M. Therefore, this concentration was chosen for all subsequent studies.
Acute efict of NO donors on TPO activity.
2.0 **
= e
18*
> 8
1.8.
* *
,@ ‘3 $jr!
**
1.0'
lOA 10-5 10-4 GS-NO (M)
10-6 10-5 10” SNP (M)
Fig. 1 The acute effect of NO donors on the TPO activity of unpassaged primary human thyrocytes. NO donors at the indicated concentrations were added to the cells at the time of the assay. Results show the relative TPO activity in the presence of NO donors compared to activity in their absence (control). Results are means f SEM, and are representative of 3 experiments performed with separate thyrocyte isolates. *PcO.O5, **PcO.Ol compared to control.
PL-376
Vol. 63, No. 26, 1998
NO Enhances Thyroid Peroxidase Activity
Figure 2 shows the effect of 10.’ M GS-NO or SNP on the acute TPO activity of passaged human thyrocytes. It may be seen that the effect of NO donors on TPO activity was less marked than for unpassaged thyrocytes, resulting in an average 20% increase in enzyme activity over baseline. The smaller increase observed with passaged thyrocytes was not due to substrate limitation, since passaged thyrocytes exhibited a lower level of TPO activity than unpassaged thyrocytes (mean absorbance values in the absence of NO donors were 0.253 f 0.022 absorbance units for unpassaged thyrocytes and 0.102 f 0.027 absorbance units for passaged thyrocytes). This suggests that the passage of primary human thyrocytes leads to a reduction in TPO activity, which is not completely restored by culture in the presence of TSH, and that the resultant TPO has reduced sensitivity to the stimulatory effect of NO.
*
*. .
-I............ ........ ... . .. . ~~:.:.:.:.:.:.:.:.:.:,:,:.: ::::::::::j::::::::::::::::: :.):.:.:.)~~:.:.:.:.:.:.: ::::::::::::::.:::::::j:::.: ;:::::j::j::::::;:-::: ~:.:.:.:.:.:.:.:.~:.:.:.:.: ::::::i::::::::::j:::::::::
::::::j:::::::::::::::::::::
:::~~:::::::::::::~::tlj l:~i:~~:j:::::::::::::::::
C
GS-NO
SNP
Fig. 2 The acute effect of NO donors on the TPO activity of passaged human thyrocytes. Cells were cultured in the presence of 10 mu/ml TSH for 3 days prior to TPO assay. NO donors at 10s5M were added to the cells at the time of the assay. Results show the relative TPO activity in the presence of NO donors compared to activity in their absence (C). Results are means f SEM, and are representative of 3 experiments performed with separate thyrocyte isolates. *P
PL377
NO Enhances Throid Peroxidase Activity
Vol. 63, No. 26, 1998
indicating that the stimulatory effmt of NO donors on the TPO activity of human thyrocytes is not a general phenomenon of all peroxidase enzymes.
0.8 0.8
-.-
Control
- .v. - GS-NO
-m-.SNP 0.0
0.2
0.4
0.8
0.8
1.0
HRP (mu/ml) Fig. 3 The acute effect of NO donors on the enzyme activity of purified horseradish peroxidase. Peroxidase assay was performed with the indicated concentrations of horseradish peroxidase (HRP), in the absence or presence of lo-’ M GS-NO or SNP. Enzyme activity is expressed in absorbance units at 405 nm. Results are means f S.E.M., and are representative of 3 experiments performed. Error bars are encompassed by the symbols. The NO donor SNP is known to release nitrosonium ions (NO’) upon decomposition in solution (15), while the NO donor GS-NO releases NO free radicals (16). Nitrosonium ion reacts with H,O, to form the strong oxidant peroxynitrite (ONOO), and NO itself can react with superoxide free radicals to produce peroxynitrite (17). It was thus possible that the increased oxidation of TMB observed in the presence of NO donors was due to a direct oxidative effect of the donors and Therefore, their breakdown products, rather than an effect of NO on TPO enzyme activity. experiments were performed to eliminate the possibility of a direct oxidative effect of NO on the substrate TMB. The stock TMB solution was diluted to 1.15 mM in reaction buffer alone, or buffer containing either GS-NO or SNP (10” M), and incubations were performed in the absence or presence of H,O,. The mixtures were incubated at 37°C for 60 min, followed by the measurement of absorbance at 405 nm. No increase in absorbance was observed in the presence of NO donors either in the absence or the presence of H,O, (data not shown), indicating that NO has no direct oxidative effect on TMB.
Effect of chronic GS-NO pre-incubation on TPO activity. Experiments
were then performed to determine the effect of pre-incubating thyrocytes with a NO donor prior to assay for TPO activity. Primary unpassaged thyrocytes were incubated in the absence or presence of GS-NO (10” M) for 3 days, followed by TPO assay in the absence or presence of 10” M GS-NO or SNP. Figure 4 shows that GS-NO pre-incubation had no effect on the basal level of TPO activity in the absence of NO donor at the time of the assay. This suggests that NO does not affect the level of expression of the TPO protein. Expression of TPO is known to be increased in response to TSH stimulation
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NO Enhances Thyroid Peroxidase Activity
Vol. 63, No. 26, 1998
of thyrocyte cyclic adenosine 3’,5’-monophosphate (CAMP) production (18, 19). Since we have previously shown that NO has no effect on CAMP production in primary human thyrocytes (8), it was not unexpected that NO pre-incubation did not affect TPO expression in the present study.
+
m T
T
I
BASAL
GS-NO
SNP
Fig. 4 The effect of chronic GS-NO pre-incubation on basal and NO donor-stimulated TPO activity of unpassaged human thyrocytes. Cells were cultured for 3 days in the absence (open bars) or presence (solid bars) of 10.’ M GS-NO. TPO assays were then performed in the absence of NO donors (basal) or in the presence of 10.’ M GS-NO or SNP, as indicated. Results show the relative TPO activity in the presence of NO donors compared to activity in their absence. Results are means + SEM, and are representative of 3 experiments performed with separate thyrocyte isolates. *P
Vol. 63, No. 26, 1998
NO Enhances Thyroid Peroxidase Activity
PL379
formed. While the stimulatory effect of NO donors on TPO activity observed in the present study was essentially instantaneous, the inhibitory effects of NO donors on the pathway of thyroid hormone synthesis observed by others occurred after incubations of 2-4 h (lo,2 1). Therefore, the overall effect of NO on thyroid function may vary depending on the length of exposure to NO, and will be the sum of the effects of NO on each of the individual components of the pathway of thyroid hormone synthesis. In a recent study which showed the first immunohistochemical localization of NOS within the human thyroid gland, the endothelial Type III NOS isoform was detected in both the vascular endothelial cells and the thyroid follicular cells (22). Moreover, Type III NOS staining within the follicular cells was predominantly located at the apical pole, indicating a co-localization of NOS and TPO. Due to the intimate proximity of these two enzymes, it may be hypothesized that fluctuations in the amount of NO released by Type III NOS within thyrocytes will rapidly result in similar changes in the level of TPO activity. In contrast, longer-term changes in thyroid NO production, perhaps mediated by the inducible Type II NOS isoform, which is expressed by thyrocytes in response to cytokine treatment (lo), may lead to inhibition of thyroid hormone synthesis due to effects on iodide uptake and organification. In summary, the present study has suggested a potential role for NO in the regulation of the activity of the TPO enzyme of primary human thyrocytes. Thus, the release of NO from the endothelial cells of the highly vascularized thyroid gland, and from the thyrocytes themselves, may play both a paracrine and an autocrine role in the control of thyroid hormone synthesis. Acknowledgments The authors wish to thank Dr. Alessandra Knowles for advice on the development of the TPO assay. LJM was supported by the Medical Research Council and the Special Trustees of St. George’s Hospital Medical School. References 1. S. MONCADA, R.M.J. PALMER and E.A. HIGGS, Pharmacol. Rev. 43 109-142 (1991). 2. D.S. BREDT, P.M. HWANG, C.E. GLATT, C. LOWENSTEIN, R.R. REED and S.H. SNYDER, Nature 351 714-718 (1991). 3. S. LAMAS, P.A. MARSDEN, P.K.G. LI, P. TEMPST and T. MICHEL, Proc. Natl. Acad. Sci. USA 89 6348-6352 (1992). 4. W.C. SESSA, J.K. HARRISON, C.M. BARBER, D. ZENG, M.E. DURIEUX, D.D. D’ANGELO, K.R. LYNCH and M.J. PEACH, J. Biol. Chem. 267 14519-14522 (1992). 5. CR. LYONS, G.J. ORLOFF and J.M. CUNNINGHAM, J. Biol. Chem. 267 6370-6374 (1992). 6. C. NATHAN and Q. XIE, J. Biol. Chem. 269 13725-13728 (1994). 7. L.J. IGNARRO, B. BALLOT and K.S. WOOD, J. Biol. Chem. 259 6201-6207 (1984). 8. L.J. MILLATT, R. JACKSON, B.C. WILLIAMS and GStJ. WHITLEY, J. Mol. Endocrinol. 10 163-169 (1993). 9. R.Z. ESTEVEZ, J. VAN SANDE and J.E. DUMONT, Mol. Cell. Endocrinol. 90 Rl-R3 (1992). 10. K. KASAI, Y. HATTORI, N. NAKANISHI, K. MANAKA, N. BANBA, S. MOTOHASHI and S.-I. SHIMODA, Endocrinology 136 4261-4270 (1995).
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11. I.M. COLIN, E. NAVA, D. TOUSSAINT, D.M. MAITER, M.-F. VAN DENHOVE, T.F. LUSCHER, J.-M. KETELSLEGERS, J.-F. DENEF and J.L. JAMESON, Endocrinology 136 5283-5290 (1995). 12. S.R. PAGE, A.H. TAYLOR, W. DRISCOLL, M. BAINES, R. THORPE, A.P. JOHNSTONE, S.S. NUSSEY and G.StJ. WHITLEY, J. Endocrinol. 127 333-340 (1990). 13. F.S. AMBESI-IMPIOMBATO, L.A.M. PARKS and H.G. COON, Proc. Natl. Acad. Sci. USA 77 3455-3459 (1980). 14. R. MENEGAZZI, G. ZABUCCHI, A. KNOWLES, R. CRAMER and P. PATRIARCA, J. Leukoc. Biol. 52 619-624 (1992). 15. J.N. BATES, M.T. BAKER, R.Jr. GUERRA and D.G. HARRISON, Biochem. Pharmacol. 42 S157-S165 (1991). 16. E.A. KOWALUK and H.L. FUNG, J. Pharmacol. Exp. Ther. 255 1256-1264 (1990). 17. J.S. STAMLER, D.J. SINGEL and J. LOSCALZO, Science 258 1898-1902 (1992). 18. G. DAMANTE, G. CHAZENBALK, D. RUSSO, B. RAPOPORT, D. FOTI and S. FILETTI, Endocrinology 124 2889-2894 (1989). 19. Y. NAGAYAMA, S. YAMASHITA, H. HIRAYU, M. IZUMI, T. UGA, N. ISHIKAWA, K. IT0 and S. NAGATAKI, J. Clin. Endocrinol. Metab. 68 1155-l 159 (1989). 20. L. XU, J.P. EU, G. MEISSNER and J.S. STAMLER, Science 279 234-237 (1998). 21. L.V. BOCANERA, L. KRAWIEC, D. SILBERSCHMIDT, 0. PIGNATARO, G.J. JUVENAL, L.B. PREGLIASCO and M.A. PISAREV, J. Endocrinol. 155451-457 (1997). 22. I.M. COLIN, P. KOPP, J. ZBAREN, A. HABERLI, W.E. GRIZZLE and J.L. JAMESON, Eur. J. Endocrinol. 136 649-655 (1997).
ELSEVIER
PI1 50024-3205(98)00527-X
PhXRMACOLOGY LETTERS Accelerated Communication
NITRIC OXIDE ENHANCES
THYROID PEROXIDASE HUMAN THYROCYTES
ACTIVITY
IN PRIMARY
Lesley J. Millatt”2, Alan P. Johnstone, and Guy StJ. Whitley Department
of Cellular and Molecular Sciences, St. George’s Hospital Medical School, Cranmer Terrace, London SW1 7 ORE, United Kingdom (Submitted July 6, 1998; accepted August 24, 1998; received in final form October 13, 1998)
Abstract. Recent studies have demonstrated the production of the multi-functional messenger molecule nitric oxide (NO) by the thyroid gland. To examine a possible role for NO in thyroid function, we studied the acute and chronic effect of NO donors on thyroid peroxidase (TPO) activity in monolayer cultures of primary human thyrocytes, using a calorimetric assay technique. The presence of either S-nitrosoglutathione (GS-NO) or sodium nitroprusside (SNP) (10-6-10a M) at the time of the assay caused a significant increase in TPO activity. Pre-incubation of thyrocytes with 1Q5 M GS-NO for 3 days had no effect on the level of TPO activity when the assay was performed in the absence of NO donors. However, GS-NO pre-incubation significantly enhanced the acute stimulatory effect of GS-NO and SNP on TPO activity. These results suggest a possible role for NO in the regulation of TPO activity and thus thyroid hormone synthesis. Q 1998 Elsevier Science Inc. Key Words: thyroid peroxidase, nitric oxide, thyrocyte
Introduction The thyroid gland is a highly vascularized organ, in which an extensive network of blood capillaries is intimately associated with the thyroid follicles. The abundant blood supply of the gland ensures adequate exposure of the thyrocytes to iodide, which is essential for thyroid hormone formation. Upon uptake by thyrocytes, iodide is oxidized and coupled to thyroglobulin by the heme-containing enzyme thyroid peroxidase (TPO), in a series of reactions which requires the oxidizing agent hydrogen peroxide. In recent years, increasing evidence has emerged to suggest a role for the inter- and intra-cellular mediator nitric oxide (NO) in thyroid gland function. Nitric oxide is synthesized from L-arginine by a wide variety of cell types, in a reaction catalyzed by the NO synthase (NOS) family of enzymes (1). Three distinct isoforms of NOS have been identified, Brain (Type I) NOS (2) and endothelial (Type III) NOS (3,4) isoforms are constitutively expressed Ca”-cahnodulin-dependent enzymes, which release small amounts of NO for short periods of time. The inducible (Type II) ‘Present address: Department of Anesthesiology, University of Virginia, Charlottesville, VA 22906-0010, USA. ‘Corresponding author. Tel: +l-804-982-3969; Fax: +l-804-982-0020 E-mail: lim6n@,virginia.edu
PO Box
10010,
PL-374
NO Enhances Thyroid Peroxidase Activity
Vol. 63, No. 26, 1998
NOS isoform (5) is a Ca”-calmodulin-independent enzyme, which releases large amounts of NO for sustained periods in response to stimulation by inflammatory cytokines and/or bacterial endotoxin (6). Many of the actions of NO are mediated via its interaction with the heme moiety of the enzyme soluble guanylate cyclase, resulting in increased production of the second messenger cyclic guanosine 3’,5’-monophosphate (cGMP) (7). We have previously reported that NO stimulates the production of cGMP in human thyrocytes (8). In addition, several groups have presented evidence for an endogenous production of NO by the thyroid gland. Estevez et al. (9) reported the existence of a constitutive NOS activity in dog thyroid slices, Kasai et al. (10) demonstrated a cytokine-inducible production of NO by cultured human thyrocytes, and Colin et al. (11) showed the expression of mRNAs encoding all three NOS isoforms in the rat thyroid. However, little is known about the functional role of NO within the thyroid gland. We now report that NO donors acutely cause an increase in TPO activity in primary human thyrocytes. We demonstrate this using a calorimetric assay technique for the measurement of the oxidase component of TPO enzyme activity. Materials and Methods Materials. Bovine thyroid-stimulating hormone (TSH) was obtained from Armour Pharmaceutical Company, Eastboume, East Sussex, UK. Ham’s FlO medium and heat-inactivated newborn calf serum (NCS) were obtained from ICN Pharmaceuticals, Thame, Oxon., UK. Dispase type II was obtained from Boehringer Mannheim, Mannheim, Germany. The TPO assay substrate 3,3’,5,5’tetramethylbenzidine (TMB) was obtained from Serva, Heidelberg, Germany, and was prepared as a 6 mg/ml stock solution in DMSO. The NO donor S-nitrosoglutathione (GS-NO) was a kind gift from Dr. H. Hodson (Wellcome Research Laboratories, Beckenham, Kent, UK). All other chemicals were obtained fkom BDH, Poole, Dorset, UK, or Sigma Chemical Company, Poole, Dorset, UK. Ceil culture. Approval for the use of human thyroid tissue was obtained from the Local Ethical Committee. Primary human thyrocytes were prepared by dispase digestion of surgical specimens from patients with multinodular goiter or Graves’ disease, as previously described (12). The resulting primary thyrocytes were grown in monolayer culture in Ham’s FlO medium containing 5% (v/v) NCS and hormone supplements (5H medium) (13). Thyrocytes were either seeded directly into 96-well tissue culture plates following isolation from thyroid tissue, or were detached from 9 cm dishes upon reaching confluence, and transferred to 96-well plates at a density of 10’ cells/ml (100 @/well). Following seeding into 96-well plates, passaged and unpassaged tbyrocytes were cultured in 5H medium until they reached confluence. For passaged thyrocytes, 10 mu/ml TSH was added to the culture medium the day after passage, since TPO activity was found to be virtually undetectable in paasaged cells not exposed to TSH. Primary unpassaged thyrocytes were, for some experiments, cultured in the presence of 10” M GS-NO for three days prior to the performance of TPO assays. TPO Assay. The TPO activity of confluent primary thyrocytes grown in 96-well plates was determined using an adaptation of an assay for neutrophil and eosinophil peroxidase activity (14). Culture medium was aspirated, and cells were washed with 100 pi/well of reaction buffer (140 mM NaCl, 8 mM NaJ-IPO,, 1.5 mM KH,PO,, 5 mM KCl, 0.5 r&l CaCl,, 1.2 mM MgCl,, plus 0.1% (w/v) glucose, 0.2% (w/v) bovine serum albumin, pH 7.4). This buffer was then replaced with 70 pi/well of fresh reaction buffer, in the absence or presence of 10” to lo4 M GS-NO or
NO Enhances Thyroid Peroxidase Activity
Vol. 63, No. 26, 1998
PL375
sodium nitroprusside (SNP). The stock solution of TMH was diluted immediately prior to use in 10 mM sodium acetate, pH 4.2, and 70 @/well added to the cells, to a final concentration of 1.15 mM. Hydrogen peroxide (H,Od solution (4.2 mM) was then added (60 @/well) to initiate the peroxidase reaction, and plates were incubated at 37’C for 15 min. Reactions were terminated by the addition of 2.5 M H,SO, (50 @/well), and absorbance at 405 mn was measured with a Titertek Multiscan MC plate-reader. Quality control and statistical analysis. Control wells were included in all TPO assays. Negative controls consisted of TPO assays performed in the absence of cells or of the substrate TMB. Positive control wells contained 100 pU/well purified horseradish peroxidase (Sigma), t?eshly prepared in reaction buffer. Relative TPO activity for each well was calculated by dividing the absorbance reading at 405 nm by the absorbance reading for cells assayed in the absence of NO donors. Results are expressed as means f S.E.M. of observations performed in quintuplicate. Statistical significance was assessed using the Student’s unpaired two-tailed t-test, and differences were considered significant where PcO.05. Results and Discussion The effect of NO donors on TPO activity was initially determined using unpassaged primary human thyrocytes and the NO donors GS-NO and SNP at concentrations of 10” to lo4 M. Figure 1 shows that these two chemically diverse NO donors both caused a significant increase in TPO activity, which occurred over the range of concentrations tested. The maximal increase in TPO activity caused by both NO donors (approx. 70% increase over baseline) occurred at a concentration of 1u5 M. Therefore, this concentration was chosen for all subsequent studies.
Acute efict of NO donors on TPO activity.
2.0 **
= e
18*
> 8
1.8.
* *
,@ ‘3 $jr!
**
1.0'
lOA 10-5 10-4 GS-NO (M)
10-6 10-5 10” SNP (M)
Fig. 1 The acute effect of NO donors on the TPO activity of unpassaged primary human thyrocytes. NO donors at the indicated concentrations were added to the cells at the time of the assay. Results show the relative TPO activity in the presence of NO donors compared to activity in their absence (control). Results are means f SEM, and are representative of 3 experiments performed with separate thyrocyte isolates. *PcO.O5, **PcO.Ol compared to control.
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Vol. 63, No. 26, 1998
NO Enhances Thyroid Peroxidase Activity
Figure 2 shows the effect of 10.’ M GS-NO or SNP on the acute TPO activity of passaged human thyrocytes. It may be seen that the effect of NO donors on TPO activity was less marked than for unpassaged thyrocytes, resulting in an average 20% increase in enzyme activity over baseline. The smaller increase observed with passaged thyrocytes was not due to substrate limitation, since passaged thyrocytes exhibited a lower level of TPO activity than unpassaged thyrocytes (mean absorbance values in the absence of NO donors were 0.253 f 0.022 absorbance units for unpassaged thyrocytes and 0.102 f 0.027 absorbance units for passaged thyrocytes). This suggests that the passage of primary human thyrocytes leads to a reduction in TPO activity, which is not completely restored by culture in the presence of TSH, and that the resultant TPO has reduced sensitivity to the stimulatory effect of NO.
*
*. .
-I............ ........ ... . .. . ~~:.:.:.:.:.:.:.:.:.:,:,:.: ::::::::::j::::::::::::::::: :.):.:.:.)~~:.:.:.:.:.:.: ::::::::::::::.:::::::j:::.: ;:::::j::j::::::;:-::: ~:.:.:.:.:.:.:.:.~:.:.:.:.: ::::::i::::::::::j:::::::::
::::::j:::::::::::::::::::::
:::~~:::::::::::::~::tlj l:~i:~~:j:::::::::::::::::
C
GS-NO
SNP
Fig. 2 The acute effect of NO donors on the TPO activity of passaged human thyrocytes. Cells were cultured in the presence of 10 mu/ml TSH for 3 days prior to TPO assay. NO donors at 10s5M were added to the cells at the time of the assay. Results show the relative TPO activity in the presence of NO donors compared to activity in their absence (C). Results are means f SEM, and are representative of 3 experiments performed with separate thyrocyte isolates. *P
PL377
NO Enhances Throid Peroxidase Activity
Vol. 63, No. 26, 1998
indicating that the stimulatory effmt of NO donors on the TPO activity of human thyrocytes is not a general phenomenon of all peroxidase enzymes.
0.8 0.8
-.-
Control
- .v. - GS-NO
-m-.SNP 0.0
0.2
0.4
0.8
0.8
1.0
HRP (mu/ml) Fig. 3 The acute effect of NO donors on the enzyme activity of purified horseradish peroxidase. Peroxidase assay was performed with the indicated concentrations of horseradish peroxidase (HRP), in the absence or presence of lo-’ M GS-NO or SNP. Enzyme activity is expressed in absorbance units at 405 nm. Results are means f S.E.M., and are representative of 3 experiments performed. Error bars are encompassed by the symbols. The NO donor SNP is known to release nitrosonium ions (NO’) upon decomposition in solution (15), while the NO donor GS-NO releases NO free radicals (16). Nitrosonium ion reacts with H,O, to form the strong oxidant peroxynitrite (ONOO), and NO itself can react with superoxide free radicals to produce peroxynitrite (17). It was thus possible that the increased oxidation of TMB observed in the presence of NO donors was due to a direct oxidative effect of the donors and Therefore, their breakdown products, rather than an effect of NO on TPO enzyme activity. experiments were performed to eliminate the possibility of a direct oxidative effect of NO on the substrate TMB. The stock TMB solution was diluted to 1.15 mM in reaction buffer alone, or buffer containing either GS-NO or SNP (10” M), and incubations were performed in the absence or presence of H,O,. The mixtures were incubated at 37°C for 60 min, followed by the measurement of absorbance at 405 nm. No increase in absorbance was observed in the presence of NO donors either in the absence or the presence of H,O, (data not shown), indicating that NO has no direct oxidative effect on TMB.
Effect of chronic GS-NO pre-incubation on TPO activity. Experiments
were then performed to determine the effect of pre-incubating thyrocytes with a NO donor prior to assay for TPO activity. Primary unpassaged thyrocytes were incubated in the absence or presence of GS-NO (10” M) for 3 days, followed by TPO assay in the absence or presence of 10” M GS-NO or SNP. Figure 4 shows that GS-NO pre-incubation had no effect on the basal level of TPO activity in the absence of NO donor at the time of the assay. This suggests that NO does not affect the level of expression of the TPO protein. Expression of TPO is known to be increased in response to TSH stimulation
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of thyrocyte cyclic adenosine 3’,5’-monophosphate (CAMP) production (18, 19). Since we have previously shown that NO has no effect on CAMP production in primary human thyrocytes (8), it was not unexpected that NO pre-incubation did not affect TPO expression in the present study.
+
m T
T
I
BASAL
GS-NO
SNP
Fig. 4 The effect of chronic GS-NO pre-incubation on basal and NO donor-stimulated TPO activity of unpassaged human thyrocytes. Cells were cultured for 3 days in the absence (open bars) or presence (solid bars) of 10.’ M GS-NO. TPO assays were then performed in the absence of NO donors (basal) or in the presence of 10.’ M GS-NO or SNP, as indicated. Results show the relative TPO activity in the presence of NO donors compared to activity in their absence. Results are means + SEM, and are representative of 3 experiments performed with separate thyrocyte isolates. *P
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formed. While the stimulatory effect of NO donors on TPO activity observed in the present study was essentially instantaneous, the inhibitory effects of NO donors on the pathway of thyroid hormone synthesis observed by others occurred after incubations of 2-4 h (lo,2 1). Therefore, the overall effect of NO on thyroid function may vary depending on the length of exposure to NO, and will be the sum of the effects of NO on each of the individual components of the pathway of thyroid hormone synthesis. In a recent study which showed the first immunohistochemical localization of NOS within the human thyroid gland, the endothelial Type III NOS isoform was detected in both the vascular endothelial cells and the thyroid follicular cells (22). Moreover, Type III NOS staining within the follicular cells was predominantly located at the apical pole, indicating a co-localization of NOS and TPO. Due to the intimate proximity of these two enzymes, it may be hypothesized that fluctuations in the amount of NO released by Type III NOS within thyrocytes will rapidly result in similar changes in the level of TPO activity. In contrast, longer-term changes in thyroid NO production, perhaps mediated by the inducible Type II NOS isoform, which is expressed by thyrocytes in response to cytokine treatment (lo), may lead to inhibition of thyroid hormone synthesis due to effects on iodide uptake and organification. In summary, the present study has suggested a potential role for NO in the regulation of the activity of the TPO enzyme of primary human thyrocytes. Thus, the release of NO from the endothelial cells of the highly vascularized thyroid gland, and from the thyrocytes themselves, may play both a paracrine and an autocrine role in the control of thyroid hormone synthesis. Acknowledgments The authors wish to thank Dr. Alessandra Knowles for advice on the development of the TPO assay. LJM was supported by the Medical Research Council and the Special Trustees of St. George’s Hospital Medical School. References 1. S. MONCADA, R.M.J. PALMER and E.A. HIGGS, Pharmacol. Rev. 43 109-142 (1991). 2. D.S. BREDT, P.M. HWANG, C.E. GLATT, C. LOWENSTEIN, R.R. REED and S.H. SNYDER, Nature 351 714-718 (1991). 3. S. LAMAS, P.A. MARSDEN, P.K.G. LI, P. TEMPST and T. MICHEL, Proc. Natl. Acad. Sci. USA 89 6348-6352 (1992). 4. W.C. SESSA, J.K. HARRISON, C.M. BARBER, D. ZENG, M.E. DURIEUX, D.D. D’ANGELO, K.R. LYNCH and M.J. PEACH, J. Biol. Chem. 267 14519-14522 (1992). 5. CR. LYONS, G.J. ORLOFF and J.M. CUNNINGHAM, J. Biol. Chem. 267 6370-6374 (1992). 6. C. NATHAN and Q. XIE, J. Biol. Chem. 269 13725-13728 (1994). 7. L.J. IGNARRO, B. BALLOT and K.S. WOOD, J. Biol. Chem. 259 6201-6207 (1984). 8. L.J. MILLATT, R. JACKSON, B.C. WILLIAMS and GStJ. WHITLEY, J. Mol. Endocrinol. 10 163-169 (1993). 9. R.Z. ESTEVEZ, J. VAN SANDE and J.E. DUMONT, Mol. Cell. Endocrinol. 90 Rl-R3 (1992). 10. K. KASAI, Y. HATTORI, N. NAKANISHI, K. MANAKA, N. BANBA, S. MOTOHASHI and S.-I. SHIMODA, Endocrinology 136 4261-4270 (1995).
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11. I.M. COLIN, E. NAVA, D. TOUSSAINT, D.M. MAITER, M.-F. VAN DENHOVE, T.F. LUSCHER, J.-M. KETELSLEGERS, J.-F. DENEF and J.L. JAMESON, Endocrinology 136 5283-5290 (1995). 12. S.R. PAGE, A.H. TAYLOR, W. DRISCOLL, M. BAINES, R. THORPE, A.P. JOHNSTONE, S.S. NUSSEY and G.StJ. WHITLEY, J. Endocrinol. 127 333-340 (1990). 13. F.S. AMBESI-IMPIOMBATO, L.A.M. PARKS and H.G. COON, Proc. Natl. Acad. Sci. USA 77 3455-3459 (1980). 14. R. MENEGAZZI, G. ZABUCCHI, A. KNOWLES, R. CRAMER and P. PATRIARCA, J. Leukoc. Biol. 52 619-624 (1992). 15. J.N. BATES, M.T. BAKER, R.Jr. GUERRA and D.G. HARRISON, Biochem. Pharmacol. 42 S157-S165 (1991). 16. E.A. KOWALUK and H.L. FUNG, J. Pharmacol. Exp. Ther. 255 1256-1264 (1990). 17. J.S. STAMLER, D.J. SINGEL and J. LOSCALZO, Science 258 1898-1902 (1992). 18. G. DAMANTE, G. CHAZENBALK, D. RUSSO, B. RAPOPORT, D. FOTI and S. FILETTI, Endocrinology 124 2889-2894 (1989). 19. Y. NAGAYAMA, S. YAMASHITA, H. HIRAYU, M. IZUMI, T. UGA, N. ISHIKAWA, K. IT0 and S. NAGATAKI, J. Clin. Endocrinol. Metab. 68 1155-l 159 (1989). 20. L. XU, J.P. EU, G. MEISSNER and J.S. STAMLER, Science 279 234-237 (1998). 21. L.V. BOCANERA, L. KRAWIEC, D. SILBERSCHMIDT, 0. PIGNATARO, G.J. JUVENAL, L.B. PREGLIASCO and M.A. PISAREV, J. Endocrinol. 155451-457 (1997). 22. I.M. COLIN, P. KOPP, J. ZBAREN, A. HABERLI, W.E. GRIZZLE and J.L. JAMESON, Eur. J. Endocrinol. 136 649-655 (1997).