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S. W. Manley, J. R. Bourke, and G. J. Huxham

ABSTRACT

Thyrotrophin stimulated release of radioiodine from the organic iodine pool of cultured porcine thyroid cells. The response was well developed within 2–4 h of incubation. Inhibition of the Na+/K+ pump with ouabain or incubation in sodium-free medium inhibited the response to TSH. The magnesium content of cultures was reduced by ouabain, and increasing the magnesium concentration of the medium to 10 mmol/l reversed the inhibition of the TSH response by ouabain. After prolonged incubation (4–6 h), ouabain in magnesium-enriched medium stimulated release of radioiodine. Its effects were not additive with those of TSH. Incubation for 4–6 h in media of reduced sodium concentration (34 mmol/l) also stimulated release. Sodium-free medium alone did not alter basal release rates, but magnesium enrichment of sodium-free medium promoted release after 4 h of incubation. It was concluded that the previously reported inhibition of the TSH response in thyroid tissue by ouabain or sodium-free medium was due to secondary derangements of cellular function rather than to a specific blockade of the secretomotor signal. The data are consistent with the hypothesis that a reduction in the sodium ion electrochemical gradient across the cell membrane mediates the secretomotor effect of TSH.

J. Endocr. (1987) 112, 399–405

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S. W. Manley, G. J. Huxham, and J. R. Bourke

ABSTRACT

Veratridine, a sodium channel agonist, depolarized cultured thyroid cells and increased the secretion of radioiodine from the organically bound pool. These effects were similar to those of TSH. Depolarization of the cells by increasing the potassium concentration of the medium failed to promote secretion, indicating that the sodium influx, rather than the depolarization itself, mediated the response. Veratridine, like TSH, also acutely reduced the cells' iodide uptake and inhibited the iodide transport pump. Unlike TSH, however, veratridine reduced, rather than increased, the fractional exit rate of iodide anion from the unbound pool. The data are consistent with the hypothesis that a sodium influx mediates some but not all of the actions of TSH on the thyroid gland, including the stimulation of secretion of thyroid hormones.

J. Endocr. (1986) 110, 459–466

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J. R. Bourke, E. J. Cragoe Jr, G. J. Huxham, J. V. Pearson, and S. W. Manley

ABSTRACT

Confluent monolayers of cultured porcine thyroid cells transport fluid from the apical to the basal surface, forming circumscribed zones of detachment (domes) from the culture dish substrate. Stimulation of fluid transport by prostaglandin E2 (PGE2; 1 μmol/l) was associated with an increase in transepithelial potential (TEP). Intracellular potentials (equal to the potential difference across the apical membrane of the cell, Eapical) and the TEP were measured in individual domes so that the potential difference across the basal membrane of the cell (Ebasal) could be calculated from the relationship TEP = Eapical − Ebasal. The PGE2-induced increase in TEP was associated with hyperpolarization of the basal membrane, accompanied by a slight depolarization of the apical membrane. Lines of best fit by least-squares regression showed Eapical = −20·3 mV + 0·219 TEP (correlation coefficient r = 0·627; P < 0·001) and Ebasal = −20·3 mV − 0·781 TEP (r = 0·944; P < 0·001). Phenamil (1 μmol/l), a Na+ channel selective amiloride analogue, reduced the TEP from 13·25±0·58 (s.e.m.; n = 56) to 2·39±0·16 mV (n = 51; P < 0·001) and hyperpolarized the apical membrane potential from − 20·7±0·68 (n = 60) to −32·2±0·83 mV (n = 105; P < 0·001). The response of the TEP to phenamil was immediate, and was promptly reversed on washing; in contrast, addition of 5-(N-ethyl-N-isopropyl)amiloride (20 μmol/l; selective for Na+/H+ antiporters) resulted in a slow depolarization over 30 min with a slow recovery after washout. Exposure of the cultures to media of pH 7·04 (compared with the normal pH of 7·34) resulted in a reduced response to PGE2, and a reduction in magnitude of Ebasal. It was concluded that stimulation of ion transport by PGE2 in thyroid monolayers involves activation of cation transport across the basal membrane.

Journal of Endocrinology (1990) 127, 197–202

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J. R. Bourke, S. Murdoch, S. W. Manley, T. Matainaho, G. J. Huxham, and M. J. Waters

ABSTRACT

Thyrotrophin (4-256 μU/ml) promoted an increase in the rate of release of radioiodine from the organic iodine pool of cultured porcine thyroid cells in follicular formations. This action of TSH was antagonized by low concentrations of epidermal growth factor (EGF; 0·1–5 nmol/l). The maximal effect of EGF was reached by 0·5 nmol/l. EGF (0·5–5 nmol/l) also inhibited the stimulatory effect of 8-chloro cyclic AMP (0·06–1·0 nmol/l) on radioiodine turnover. Exposure of thyroid cultures to media with a calcium concentration of 17·7 μmol/l (1% of normal) resulted in a very marked increase in the rate of release of radioiodine. The effect of TSH in low-calcium media was to inhibit the increased release of radioiodine, and EGF (0·5 nmol/l) antagonized this inhibitory effect of TSH. The calcium ionophore, A23187, stimulated radioiodine release in a dose-dependent fashion, and EGF (1·7 nmol/l) inhibited this response. Fluid transport in thyroid monolayers was stimulated by prostaglandin E2 (PGE2; 1 μmol/l). EGF (5 nmol/l) also stimulated fluid transport, but antagonized the effect of PGE2 added subsequently. It was concluded that EGF exerted acute antagonistic effects on thyroid cell responses in vitro to cyclic AMP and agents promoting accumulation of cyclic AMP in time-frames too short for these inhibitory effects to be attributable to the dedifferentiative effect of the growth factor.

Journal of Endocrinology (1991) 128, 213–218

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J. R. Bourke, P. A. McGrath, G. J. Huxham, M. J. Waters, and S. W. Manley

ABSTRACT

Cultured porcine thyroid cells maintained in media containing TSH exhibited a membrane potential of −50 mV, and hyperpolarized by about 10 mV within 1 h of the addition of epidermal growth factor (EGF; 10 ng/ml). Follicle cells had depolarized to −45 mV after 4 h of exposure to EGF. Cells maintained in dibutyryl cyclic AMP (dbcAMP) did not alter their membrane potential when exposed to EGF for up to 4 h. Cultures washed to remove the TSH or dbcAMP hyperpolarized to − 75 mV within 30 min, and a reversible depolarization to − 60 mV was observed on addition of EGF. It was concluded that EGF acts as a physiological antagonist of TSH and also exerts a separate depolarizing influence on cultured thyroid cells.

J. Endocr. (1986) 109, 321–324

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J R Bourke, K C Abel, G J Huxham, O Sand, and S W Manley

Abstract

Porcine thyroid epithelial cells cultured as a monolayer with their apical membranes facing the medium are known to absorb Na+ and secrete Cl. Two types of Na+ channels were found in cell-attached patches of apical membrane. A low conductance Na+ channel (conductance g=4 picosiemens (pS)) remained open for seconds and showed a high selectivity for Na+ compared with K+. In contrast, a high conductance Na+ channel (g=10 pS) flickered rapidly and had reduced selectivity. Both types of Na+ channel became more prevalent when the cells were exposed to Na+-free medium, though only the high conductance channel increased in prevalence on addition of prostaglandin E2, a stimulator of adenylate cyclase which increases Na+ absorption in this cultured epithelium. Two minority types of channel were also found: a non-selective small conductance cation channel which had been reported previously, and an intermediate conductance channel found only in Na+-free medium. It was concluded that passage of Na+ across the apical membrane of thyroid cells is mediated by typical epithelial Na+ channels, but that the two types of channel are differentially regulated.

Journal of Endocrinology (1996) 149, 101–108

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J. R. Bourke, T. Matainaho, G. J. Huxham, and S. W. Manley

ABSTRACT

Confluent monolayer cultures of porcine thyroid cells form dome-shaped elevations by local separation from the plastic culture dish. Formation of domes by epithelial cells in culture is generally considered to be evidence of fluid transport. A computer-controlled data acquisition system was developed to quantitate fluid transport in thyroid cultures by serial measurements of dome elevation. Thyrotrophin (10 mU/ml), prostaglandin E2 (PGE2; 0-01-1 μmol/l), forskolin (1 μmol/l), 8-(4-chlorophenylthio)adenosine 3′:5′-cyclic monophosphate (0.5 mmol/l) and 3-isobutyl-1-methyl-xanthine (0.5 mmol/l) promoted increases in dome height over 5–120 min. Dome growth in the presence of PGE2 (1 μmol/l) was inhibited by amiloride (0.1–100 μmol/l), ouabain (200 μmol/l), or by removal of bicarbonate and glucose from the medium. In media of reduced bicarbonate concentration (1 mmol/l compared with the control concentration of 10 mmol/l), dome growth was inhibited by acetazolamide (0.01– 1 mmol/l). These data are consistent with cyclic AMP-stimulated transport of fluid from apical to basal pole of the cells, dependent on sodium entry through the apical pole by an Na+/H+ exchanger.

J. Endocr. (1987) 115, 19-26

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J. R. BOURKE, K. L. CARSELDINE, S. H. FERRIS, G. J. HUXHAM, and S. W. MANLEY

Thyrotrophin (TSH), cyclic AMP, cyclic GMP and 1-methyl-3-isobutyl-xanthine (MIX) promoted the reassociation of isolated porcine and human thyroid cells into follicular structures in culture and stimulated the uptake of radio-iodide. Monolayer cells were present in all cultures, but in decreasing proportions as the concentration of stimulator was increased. The resting membrane potential of porcine thyroid cells cultured for 4 days in the presence of TSH was −54 ± 3·6 (mean ± s.d.) mV for follicular cells and −31 ± 2·6 mV for monolayer cells. In the absence of TSH, only monolayer cells were present and their membrane potential was −24 ± 2·0 mV. Removal of hormone by washing resulted in hyperpolarization to −70 ± 2·9 mV (follicular cells) or −59 ± 3·4 mV (monolayer cells). Subsequent replacement of TSH, or addition of cyclic AMP, MIX, prostaglandin E1 (PGE1) or long-acting thyroid stimulator immunoglobulin resulted in depolarization of previously hyperpolarized cells, to approximately the membrane potential observed before washing. Incubation in MIX resulted in enhanced sensitivity to the depolarizing effect of TSH. Cells cultured in the absence of TSH were unresponsive to TSH or other stimulators. The membrane potential of human thyroid cells behaved similarly in response to TSH, to hormone removal and replacement, and to MIX and PGE1.

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P. A. McGrath, J. R. Bourke, G. J. Huxham, and S. W. Manley

ABSTRACT

Cultured porcine thyroid cells, maintained in the differentiated state by dibutyryl cyclic AMP, responded to serotonin (5-HT; 10 nmol/l to 1 μmol/l) with a depolarization of the membrane potential, but did not respond to histamine (100 μmol/l) or dopamine (1 μmol/l). The resting membrane potential of these cells was about − 71 mV, maximal concentrations of 5-HT (1 μmol/l) inducing a depolarization to approximately −53 mV. Methysergide or phenoxybenzamine, but not propranolol, abolished the response to 5-HT. Sensitivity to 5-HT was reduced by previous exposure of cultures to TSH, the β-adrenoceptor agonist salbutamol or 5-HT itself.

J. Endocr. (1985) 107, 397–401

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S. W. Manley, D. S. Rose, G. J. Huxham, and J. R. Bourke

ABSTRACT

The calcium ionophore A23187 (0·1–1 μmol/l) inhibited membrane electrical polarization, uptake of 125I, fluid transport and TSH-stimulated release of radioiodine from the organic pool in follicular cultures of porcine thyroid cells. At higher concentrations (1–30 μmol/l), A23187 promoted release of radioiodine from the organic pool. Stimulation of release of radioiodine from the organic pool by veratridine (a sodium channel agonist, 0·4–1 mmol/l) and A23187 was dependent on the calcium concentration of the medium, while TSH action was independent. Incubation in medium of very low calcium concentration (0·0177 mmol/l) resulted in enhanced release from the organic pool, which was inhibited by TSH (256 μU/ml), A23187 (25 μmol/l) or veratridine (0·5 mmol/l). These data therefore do not support the hypothesis that calcium acts as a mediator of the secretomotor action of TSH, but suggest the possibility of a TSH-induced increase in intracellular calcium as a regulatory negative-feedback mechanism.

J. Endocr. (1988) 116, 373–380