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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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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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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
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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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ABSTRACT
The membrane potential of cultured porcine thyroid follicular cells depolarized by up to 20 mV from the resting value of about − 73 mV on exposure to β-adrenoceptor agonists. A similar response was induced by TSH or dibutyryl cyclic AMP. α-Adrenoceptor agonists were without effect. The receptor subtype was shown to be (at least predominantly) β2 by the order of potency for β-agonists (isoprenaline ≅ fenoterol⪢adrenaline >noradrenaline) and by the relative potency of selective β-antagonists (ICI 118,551 ⪢atenolol). The α-agonist phenylephrine had no effect on the TSH response but weakly inhibited the β-agonist response. Rather than a physiological antagonism between α- and β-adrenoceptor-mediated responses, this effect was shown to be due to the weak β-antagonist effect of phenylephrine since the α-antagonist phentolamine failed to potentiate the depolarizing response to the mixed agonist nor-adrenaline, and also failed to block the inhibitory action of phenylephrine on the β-agonist effect. Sensitivity to β-agonist was enhanced by omission of serum from the culture medium and reduced by exposure to β-agonists or a high concentration of TSH or dibutyryl cyclic AMP.
J. Endocr. (1985) 107, 23–30
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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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ABSTRACT
Cultured porcine thyroid cells exhibit a resting membrane potential of about − 73 mV and depolarize to about − 54 mV on exposure to TSH. The depolarizing response to TSH was preserved in a medium consisting only of inorganic salts and buffers, but was abolished in sodium-free medium, demonstrating dependence on an inward sodium current. Increasing the potassium concentration of the medium resulted in a reduction in the resting membrane potential of 60 mV per tenfold change in potassium concentration, and a diminished TSH response. A hyperpolarizing TSH response was observed in a sodium- and bicarbonate-free medium, indicating that a hyperpolarizing ion current (probably carried by potassium) was also enhanced in the presence of TSH. Tetrodotoxin blocked the TSH response. We conclude that the response of the thyroid cell membrane to TSH involves increases in permeability to sodium and potassium, and that the thyroid membrane ion channels bear some similarity to the voltage-dependent sodium channels of excitable tissues, despite the absence of action potentials in the thyroid.
J. Endocr. (1986) 108, 225–230
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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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Abstract
Porcine thyroid epithelial cells cultured as a monolayer with their apical membranes facing the medium are known to absorb Na+ and to secrete the anions Cl− and HCO3 −. Chloride channels were found in the apical membrane, and displayed a reversal potential close to the resting membrane potential, linear current–voltage relationships, a conductance at physiological temperature of 6·5 pS, and a small but significant permeability to HCO3 −. Stimulation of ion transport with prostaglandin E2 or 8-(4-chlorophenylthio) adenosine 3′:5′-cyclic monophosphate promoted activation of Cl− channels in cell-attached patches, and excised patches were reactivated by exposure of their cytoplasmic surface to protein kinase A and ATP. Physiological temperatures were necessary for activation of Cl− channels in cell-attached patches. The channels exhibited sub-states with a conductance exactly half that of the full unit conductance, suggesting a dual-barrelled channel structure. It is concluded that the apical membrane of thyroid epithelial cells contains cyclic AMP-activated Cl− channels controlling anion transport.
Journal of Endocrinology (1995) 147, 441–448
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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
