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Stephen P. Bidey, Nicholas J. Marshall and Roger P. Ekins

Abstract.

The thyrotrophin (TSH)-dependent and time-related release of cyclic AMP has been characterised in primary monolayer cultures of normal human thyroid cells. Accumulation of cyclic AMP within the incubation medium was detectable within 1 h of exposure of cultures to 5 mU TSH/ml, and increased throughout of subsequent 15 h incubation period, final levels attained being consistently in excess of the corresponding intracellular cyclic AMP levels. Accumulation of cyclic AMP in the incubation medium was dependent on TSH dose, for both short (1 h) and prolonged (16 h) incubations. Moreover, after incubation for 16 h, cyclic AMP levels in the incubation medium were significantly (P < 0.01) in excess of intracellular levels for each dose of TSH tested above 0.2 mU/ml. In the absence of TSH, accumulation of cyclic AMP in the incubation medium remained low, after both 1 h and 16 h incubation periods.

A consideration of these observations suggests that a bioassay based upon the cyclic AMP content of incubation medium samples should provide a more precise detection system for thyroid stimulators than those measuring the intracellular cyclic AMP response, and this has been demonstrated for a cell preparation in which the intracellular response to TSH was minimal.

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Stephen P. Bidey, Nicholas J. Marshall and Roger P. Ekins

Abstract.

The cyclic AMP response to thyrotrophin (TSH) has been investigated in cells prepared from human thyroid tissue obtained during surgery for sub-total laryngectomy, and maintained under in vitro conditions as primary monolayer cultures.

When cells were incubated with 1.0 mU TSH/ml, a maximal level of intracellular cyclic AMP was reached after 20 min of incubation in the presence of 0.5 mm 3-isobutyl-1-methyl xanthine (MIX). This level of cyclic AMP was sustained for at least 2 h. Half-maximal stimulation of cyclic AMP was produced by TSH doses of between 1 and 5 mU/ml. In a study of a series of eight groups of monolayer cultures, each derived from a single, different thyroid gland, the mean stimulation of cyclic AMP given by 50 mU TSH/ml was 37.8-fold greater than in non-stimulated cell monolayers.

Significant stimulation to 50 μU TSH/ml was observed in some monolayers and the precision of measurement of TSH was better than 15% over the TSH dose range 0.2–1.0 mU/ml. The magnitude of the cyclic AMP response to TSH was unaffected by the presence in the incubation medium of 20% (v/v) normal human serum. A cyclic AMP response to TSH was still demonstrable in cells that had been maintained for a period of 22 days in monolayer culture, although the response was reduced in comparison with that given by 4–5 day old cultures.

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Stephen P. Bidey, Nicholas J. Marshall and Roger P. Ekins

Abstract.

Slice preparations of normal human thyroid tissue were incubated in vitro with TSH. The cyclic AMP contents of slices were determined at intervals up to 120 min, and cyclic AMP in the incubation medium was also estimated for each incubation period. Slice cyclic AMP levels were related both to incubation time and TSH dose. In response to 10 mU TSH/ml, slice cyclic AMP levels were maximal within 60 min, and were not significantly changed at 120 min. Cyclic AMP was detectable in the medium within 10 min of slice exposure to TSH, and increased throughout the initial 60 min of incubation. Cyclic AMP release during this period was dependent on both TSH dose and incubation time. Between 60–120 min, however, cyclic AMP release partially lost its TSH dose-dependency, and 0.5–5.0 mU TSH/ml were equipotent with respect to the final medium cyclic AMP level attained. Slices incubated without TSH released only small amounts of cyclic AMP, and maximal levels were attained within 20 min. In contrast to the adenylate cyclase response of thyroid membrane preparations, which was stimulated by NaF, suggesting that cyclic AMP release was not a result of the stimulation of damaged cells.

These findings demonstrate the importance of cyclic AMP release from human thyroid slices, following in vitro exposure to TSH, and suggest that, after incubation periods such as are used for the functional biodetection of thyroid stimulators, the magnitude of cyclic AMP release may be of quantitative significance.

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Patricia A. Ealey, Leonard D. Kohn, Nicholas J. Marshall and Roger P. Ekins

Abstract. Forskolin, from the roots of the Indian medicinal plant Coleus forskohlii, has recently been shown to be a potent stimulator of adenylate cyclase in many systems, including endocrine tissues such as the thyroid gland. We describe forskolin activation of β-naphthylamidase activity in guinea pig thyroid tissue using the cytochemical bioassay (CBA) for thyroid stimulators. This CBA is the most sensitive bioassay for TSH and LATS-B currently available, being able to detect stimulation by doses as low as 10−5 mU TSH/l and 10−9 mU LATS-B/l. The dose-response curve to forskolin was bell-shaped (as is seen with TSH and LATS-B) with the ascending limb of the curve produced by 10−13 m to 10−12 m forskolin after a 3 min exposure time. Maximal stimulation was observed with 10−12 m forskolin. However, the dose-response curve to forskolin was not parallel to that given by TSH, the slope of the ascending limb being much greater. It has been suggested that stimulation of β-naphthylamidase activity in the CBA is via cAMP. We report that dibutyryl cAMP at doses from 10−16 m to 10−11 m produces a bell-shaped dose-response curve with a very broad peak response, again not parallel to that produced by TSH. Forskolin activation of β-naphthylamidase in the CBA is unaffected by a 1:106 dilution of 11E8, a monoclonal antibody raised against solubilised TSH receptors, which binds to the TSH receptor and inhibits TSH stimulation. Although the precise location of forskolin action is not known, this is further evidence that forskolin acts at a post-surface receptor site.

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Mehul T Dattani, Andrew P Winrow, Anatoly Tuil'Pakov, P Jane Pringle, Peter C Hindmarsh, Charles GD Brook and Nicholas J Marshall

Dattani MT, Winrow AP, Tuil'Pakov A, Pringle PJ, Hindmarsh PC, Brook CGD, Marshall NJ. Evaluation of growth hormone (GH) responses to pulsed GH-releasing hormone administration using the MTT–ESTA bioassay. Eur J Endocrinol 1996;135:87–95. ISSN 0804–4643

We compared the immunoactivity of human growth hormone (hGH) with its bioactivity after stimulation of hGH release into the circulation by the administration of growth hormone-releasing hormone [GHRH(1–29)-NH2] according to a pre-determined protocol to four normal adult volunteers. We used the Hybritech immunoradiometric assay to measure the immunoactive GH concentrations. Bioactive GH concentrations were measured using the highly quantitative and precise eluted stain bioassay system (ESTA). The high sample capacity of the ESTA bioassay permitted us to monitor the bioactivities in closely timed sequential samples, and in far greater detail than has previously been possible. Two pulses of GHRH(1–29)-NH2 were administered intravenously to the four adult male volunteers (aged 24–37 years) on a weekly basis over a 4-week period. Two different doses of GHRH(1–29)-NH2 (0.1 and 1.0 μg/kg) were tested. These were separated by specified time intervals (60 or 120 min). Responses in the four individuals were variable. However, although the immunoand bioactivities generally agreed well, there was a systematic and progressive increase in the bioactivity/immunoactivity (B/I) ratios as half of the response peaks were approached. After these peak concentrations, the B/I ratios subsequently returned to values that were close to unity. The enhanced bioactivity of the peak samples from the two volunteers in whom the largest magnitudes of response were observed was found to be labile after long-term storage at −20°C. We suggest that the preferential rise in GH bioactivity, as opposed to immunoactivity, in response to GHRH(1–29)-NH2 was due to progressive changes in the concentrations of isoforms of GH that are not detectable in the Hybritech immunoassay.

NJ Marshall, Division of Molecular Pathology, University College London, The Windeyer Building, 46 Cleveland Street, London W1P 6DB, UK