Ten pairs of adult female C57BL/6j mice were studied. In each pair, one mouse received 2.5 mg theophylline i.p. dissolved in 0.5 mL 0.9% sterile saline, whereas its control received only saline. Twenty-hours after theophylline, 10 µCi Na¹²⁵I (ICN Biochemicals Inc., Cleveland, OH, U.S.A.) in 0.1 mL 0.9% sterile saline were injected through the tail vein to all mice. Four hours later, the mice were sacrificed, their sera collected, and thyroid glands dissected and counted in a gamma counter (Packard Instrument Co., Downers Grove, IL, U.S.A.). To determine background radioactivity, a piece of quadriceps muscle was dissected, weighed, counted and its radioactivity per weight subtracted from that of thyroid tissue. Radioiodine uptake was expressed as cpm/mg of thyroid tissue. All of the animals were maintained in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals, and all experimental procedures were approved by the institutional animal use and care committee for use of vertebrate animals in research.

The in vitro uptake of Na¹²⁵I by rat thyroid FRTL-5 cells (Interthyr Research Foundation, Baltimore, MD, U.S.A.; ATCC CRL 8305) was measured as described by Weiss et al. (7) with slight modification. Briefly, cells were grown in 24-well plates at 37℃ in 5% CO₂, for 2-3 days in Coon's modified F-12 medium containing 5% heat-treated, mycoplasmafree calf serum (Life Technologies), 1 mM non-essential amino acids (Life Technologies) supplemented with a mixture of six hormones, including 10 µg/mL insulin, 0.4 ng/mL cortisol, 5 µg/mL transferrin, 2 ng/mL glycyl-L-histidyl-L-lysine acetate, 10 ng/mL somatostatin and 10^-10 M bovine TSH. These hormones, except cortisol (Calbiochem, La Jolla, CA, U.S.A.), were obtained from Sigma (St. Louis, MO, U.S.A.). Cells were exposed for various times to different concentrations of theophylline (Sigma), either added directly to culture medium or to serum drawn from a mouse that had been injected with theophylline. After aspirating the culture medium, cells were washed with 1 mL of buffer of following composition: Hanks' balanced salt solution (Life Technologies), which contains calcium and magnesium but does not contain sodium bicarbonate, phenol red, or BSA, with 10 mM HEPES buffer (Life Technologies), pH 7.3 (HBSS). ¹²⁵I uptake was initiated by adding 0.5 mL of warm buffered HBSS containing 10 µM NaI and Na¹²⁵I (0.1 µCi/mL, to have around 100,000 cpm per well). Incubations proceeded for 45 min at 37℃ water bath, and were terminated by aspirating the radioactive medium and washing with 1 mL of ice-cold HBSS twice. To determine the amount of Na¹²⁵I associated with the cells, 1 mL of ice-cold absolute ethanol was added to each well and incubated for 3 hr at 4℃. The supernatants containing alcohol extracts were transferred into vials for gamma counting. Radioiodine uptake is expressed as cpm per optical density of DNA in each well measured in an ELISA reader (Dynatech Laboratories, Chantilly, VA, U.S.A.) after reacting the cells with diphenylamine solution (Sigma) overnight (8).

Intracellular and total cAMP levels of FRTL-5 cells were measured using a commercial cAMP enzymatic immunoassay kit (Amersham Pharmacia Biotech Inc, Piscataway, NJ, U.S.A.). Cells were plated in 96-well microtiter plates, at a concentration of 1×10⁶ cells/mL, and incubated overnight at 37℃ in 5% CO₂. After stimulating the cells with medium containing different concentrations of theophylline or serum, a lytic solution containing dodecyltrimethylammonium bromide was either added after aspirating the culture media for subsequent intracellular cAMP quantitation or directly introduced into culture media for total cAMP measurement. Lysis of the cells was performed for 10 min by shaking the plate on a microtiter plate shaker. Standard solutions and samples from each well pipetted into microtiter plates coated with donkey anti-rabbit IgG. Optical density in each plate was determined reader at 450 nm, following the procedure recommended by manufacturer.

Student's t test and one-way ANOVA were used. Differences were considered statistically significant at p<0.05.

The kinetics of radioiodine uptake in normal mice thyroid tissue and muscle after tail vein injection was first studied. Peak thyroidal uptake was observed at 4 hr after injection (Fig. 1), which led us to measure radioiodine uptake at this time point in future experiments. Within each pair of mice, net thyroidal ¹²⁵I activity was greater in all ten of the theophylline-treated mice, 440,451±383,362 vs. 196,037±235,435 cpm/mg (mean±SD, p<0.01 by Student's paired t test), even though there was considerable variation in the degree of uptake among the pairs. Overall, thyroidal ¹²⁵I activity was 3.3-fold higher in theophylline-treated mice than in their respective controls (range 1.5- to 6.7-fold increases) (Fig. 2). No mice died before sacrifice or manifested behavior suggesting theophylline toxicity.

When FRTL-5 cells were incubated with theophylline in different concentrations for more than 30 min up to 20 hr, the same exposure time employed for in vivo studies, intracellular ¹²⁵I activity was not significantly different with any theophylline concentration from that of cells incubated with medium alone (data not shown). FRTL-5 cells were then exposed to different concentrations of theophylline (1, 10, 100 and 200 µM) for 30 min. Only 1 µM theophylline significantly increased intracellular ¹²⁵I activity compared to cells incubated with medium only (43,598±3,011 vs. 31,804±3,963 cpm/DNA [O.D], means of triplicate±SD, p=0.015) (Fig. 3). However, no effect was observed with theophylline concentrations that were comparable to therapeutic serum concentrations for bronchial asthma (100 µM) and higher (200 µM).

We investigated the time course of the intracellular cAMP concentrations of FRTL-5 cells in responses to 1 µM theophylline. Intracellular cAMP increased up to 30 min and then declined significantly (159±28 at basal, 688±104 at 30 min, 306±40 pmol/well at 120 min, means of triplicate±SD, p=0.001) (Fig. 4A). FRTL-5 cells were then exposed to various concentrations of theophylline for 30 min. Intracellular cAMP concentration of the cells incubated with 1 µM theophylline was higher than that of cells incubated with medium only (528 vs. 109 pmol/well, mean of duplicates). Although no significant increase in intracellular cAMP was observed at higher theophylline concentrations (10, 100 and 200 µM), total cAMP (cellular+medium cAMP) was increased in a dose-dependent manner throughout the studied theophylline dose range (Fig. 4B).

FRTL-5 cells were incubated for 30 min with control mouse serum or serum taken 20 h after the mouse had received theophylline 2.5 mg i.p. Theophylline-treated mouse serum significantly increased radioiodine uptake by FRTL-5 cells at dilutions 1:200 and 1:100 compared to control mouse serum (46,531±4,577 vs. 36,916±3,674 cpm/DNA [O.D] at 1: 200, 48,529±4,455 vs. 35,982±4,154 cpm/DNA [O.D] at 1:100, means of triplicate±SD, p<0.05) (Fig. 5A). Serum from the theophylline-treated mouse also showed a greater intracellular concentration of cAMP within FRTL-5 cells compared to control mouse serum (303 vs. 191 pmol/well at 1:100 dilution, mean of duplicates) (Fig. 5B).