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

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

SUMMARY

Incubation, with [4-¹⁴C]progesterone, of adrenocortical tissue (posterior cardinal vein preparations) from the eel yielded ¹⁴C-labelled cortisol and cortisone. These two hormones, containing both ¹⁴C and ³H, appeared when similar preparations were incubated simultaneously with [4-¹⁴C]progesterone and [16-³H]pregnenolone. In addition, the following intermediaries were isolated: [¹⁴C, ³H]progesterone; [¹⁴C, ³H]17α-hydroxyprogesterone; [³H]17α-hydroxypregnenolone and [¹⁴C, ³H]21-deoxycortisol. Analysis of the isotope content of the end products and isolated intermediaries seemed to indicate that the transformation of pregnenolone to corticosteroids proceeded to a large extent through intermediaries other than progesterone. In none of these experiments could the formation of aldosterone be demonstrated. Similarly, incubation of eel adrenocortical tissue with a mixture of [4-¹⁴C]-progesterone and [1,2-³H]corticosterone failed to yield detectable aldosterone.

A further search for aldosterone, using a large amount of eel adrenocortical tissue with [4-¹⁴C]progesterone and [16-³H]pregnenolone as substrates with added angiotensin, also gave negative results. In similar preparations, [1-¹⁴C]sodium acetate was not transformed to any recognizable corticosteroids.

Prior research indicates that growth hormone (GH) cell differentiation can be induced prematurely by treatment with glucocorticoids in vitro and in vivo. However, the nature of these responses has not been fully characterized. In this study, the time course of corticosterone induction of GH-secreting cells in cultures of chicken embryonic pituitary cells, responsiveness of differentiated somatotrophs to GH secretagogues, localization of somatotroph precursor cells within the pituitary gland, and the effect of corticosterone on GH gene expression were determined to better define the involvement of glucocorticoids in somatotroph recruitment during development. Anterior pituitary cells from embryonic day 12 chicken embryos were cultured in 10(-9) M corticosterone for 4 to 48 h and were then subjected to reverse haemolytic plaque assays (RHPAs) for GH. Corticosterone treatment for as short as 16 h increased the percentage of GH cells compared with the control. When corticosterone was removed after 48 h and cells were cultured for an additional 3 days in medium alone, the percentage of GH secretors decreased but remained greater than the proportion of somatotrophs among cells that were never treated with corticosterone. To determine if prematurely differentiated somatotrophs were responsive to GH secretagogues, cells were exposed to corticosterone for 48 h and then subjected to GH RHPAs in the presence or absence of GH-releasing hormone (GHRH) or thyrotropin-releasing hormone (TRH). Approximately half of the somatotrophs induced to differentiate with corticosterone subsequently released more GH in response to GHRH and TRH than in their absence. The somatotroph precursor cells were localized within the anterior pituitary by culturing cells from the caudal lobe and cephalic lobe of the anterior pituitary separately. Corticosterone induction of GH cells was substantially greater in cultures derived from the caudal lobe of the anterior pituitary, where somatotroph differentiation normally occurs. GH gene expression was evaluated by ribonuclease protection assay and by in situ hybridization. Corticosterone increased GH mRNA in cultured cells by greater than fourfold. Moreover, corticosterone-induced somatotroph differentiation involved GH gene expression in cells not expressing GH mRNA previously, and the extent of somatotroph differentiation was augmented by treatment with GHRH in combination with corticosterone. We conclude that corticosterone increases the number of GH-secreting cells within 16 h, increases GH gene expression in cells formerly not expressing this gene, confers somatotroph sensitivity to GHRH and TRH, and induces GH production in a precursor population found primarily in the caudal lobe of the anterior pituitary, a site consistent with GH localization in adults. These findings support the hypothesis that glucocorticoids function to induce the final stages in the differentiation of fully functional somatotrophs from cells previously committed to this lineage.