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D. J. Woods
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J. Soden
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S. Tomlinson
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S. P. Bidey
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ABSTRACT

Using the fluorescent pH indicator 2′7′-bis(2-carboxyethyl)-5′-(6′)-carboxyfluorescein to monitor intracellular pH (pHi), we have investigated whether transmembrane Na+/H+ exchange, as measured by experimental changes in pHi under bicarbonate-free incubation conditions, may be involved in the early growth-promoting actions of insulin-like growth factor-I (IGF-I) on the rat thyroid cell stain FRTL-5. In initial studies to characterize Na+/H+ exchange in FRTL-5 cell suspensions, the recovery of a resting pHi in acid-loaded cells was shown to be dependent upon the presence of extracellular Na+, was enhanced by the presence of the sodium ionophore monensin and was abolished by amiloride, an antagonist of Na+/H+ antiport activity.

Unlike TSH, which was without effect on the pHi of FRTL-5 cells for up to 15 min after addition, IGF-I (1000 μg/1) caused a rapid and sustained increase within 3 min, which was abolished in medium in which Na+ had been replaced with an iso-osmotic level of choline chloride. The change in pHi in response to IGF-I was mimicked by phorbol 12-myristate 13-acetate (PMA; 100 nmol/1), an activator of thyroid cell proliferation. In the presence of TSH, exposure of cells to IGF-I or PMA had no additional effect on the cytoplasmic alkalinization induced by either of these two agonists alone. However, blockade of transmembrane Na+/H+ exchange with amiloride inhibited both the individual actions of IGF-I and PMA on [methyl-3H]thymidine incorporation, and the synergistic interaction between TSH and IGF-I. These findings are consistent with a differential mode of action of TSH and IGF-I on the early events associated with FRTL-5 cell proliferation, and suggest involvement of an amiloride-sensitive transmembrane Na+/H+ exchange in mediating the early cellular response to the latter. Furthermore, the later actions of PMA, which are also dependent upon early transmembrane Na+/H+ exchange, differ from those of IGF-I with respect to interaction with the growth-promoting actions of TSH.

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D. J. Woods
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J. Soden
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S. P. Bidey
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ABSTRACT

Using the fluorescent indicators 2′,7′-bis(2-carboxyethyl)-5′-(6′)-carboxyfluorescein and Oxonol V to monitor intracellular pH (pHi) and cell membrane potential respectively, we have investigated the involvement of H+-dependent ATPase and H+-dependent K+ channels in the recovery of the rat thyroid cell strain FRTL-5 from experimentally induced cytosolic acidification and membrane hyperpolarization events. Following exposure of cells to the weak acid sodium butyrate (24mmol/l) under bicarbonate-free incubation conditions, cytoplasmic acidification was maximal after 3 min, attaining a pHi of 6.42. The subsequent recovery of pHi was unimpaired by the absence of extracellular K+, but was reduced in the presence of the Na+ antagonist amiloride (1 mmol/l), recovering by 0.11±0.003 units, compared with 0.27±0.02 units under amiloride-free conditions. In the presence of the H+-dependent ATPase antagonist N,N′-dicyclohexylcarbodiimide (DCC), the pHi recovery observed in amiloride-containing, K+-free buffer was abolished.

The recovery of pHi in Na+- and K+-containing buffer was accompanied by hyperpolarization of the cell membrane, the later stage of which was reduced after blockade of K+ channels with BaCl2, implying a major contribution of transmembrane K+ movement to such events. In contrast to its attenuating effect on pHi recovery, DCC was ineffective in reducing butyrate-dependent membrane hyperpolarization, suggesting that H+-dependent ATPase may not be a major contributory factor to this event. However, when K+ channels were blocked by addition of BaCl2, addition of DCC abolished the butyrate-induced membrane depolarization. These findings are consistent with the presence of two independent hyperpolarizing transport processes in the FRTL-5 cell membrane which appear to involve (i) a H+-dependent ATPase, activated in response to cytosolic acidification, and allowing partial recovery of pHi in the absence of extracellular Na+ and HCO3 , and (ii) H+-dependent K+ channels which, while contributing to membrane hyperpolarization, may not play a major role in the normal maintenance of pHi.

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J. M. Johnston
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D. F. Wood
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E. A. Bolaji
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D. G. Johnston
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ABSTRACT

Some pituitary tumours respond to dopamine by decreasing the release of prolactin and/or GH and by inhibition of tumour growth. Certain tumours are unresponsive. Dopamine D2 receptor high-affinity binding is impaired in these tumours, and the rat GH3 cell line behaves in a similar way. The hypothesis that the dopamine-binding defect results from impaired D2 receptor gene expression has been tested in the present study. On Northern blots, D2 receptor mRNA was present in both normal rat pituitary cells and in GH3 cells. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis identified a putative D2 receptor protein in normal and GH3 cell membranes. The lack of effect of dopamine in GH3 cells does not reflect the absence of D2 receptor gene expression.

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P J Lowry
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S C Koerber
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R J Woods
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S Baigent
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S Sutton
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D P Behan
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W Vale
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J Rivier
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ABSTRACT

As the association of corticotrophin-releasing factor (CRF) with its binding protein (BP) to form a dimer complex (CRF2/BP2) appears to be dependent on the nature of the ligand we have compared the circular dichroism difference spectra after association of the BP with ovine (o) CRF, human (h) CRF and the α-helical CRF(9–41) antagonist. All three ligands caused a negative change in molar ellipticity above 210 nm, with oCRF having the least and hCRF the greatest effect. Below 210 nm there was a marked divergence of difference spectra, with the reaction with the natural peptides, hCRF and oCRF, resulting in a positive change in ellipticity, whilst that with the antagonist produced a negative change. In view of the BP spectrum indicating predominantly β-sheet and the peptides showing mainly α-helix these results were interpreted as the changes above 210 nm being due to dimerization and below 210 nm to a change in the conformation of ligand on binding. The opposite change in α-helicity of the antagonist observed on binding compared with the two natural CRF peptides could have fundamental pharmacological implications.

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