S. W. Manley

Amiloride analogues induce responses in isolated rat cardiovascular tissues by inhibition of Na+/Ca2+ exchange

SummaryThe role of inhibition of Na+/Ca2+ exchange in the positive inotropic, negative chronotropic and vasorelaxant responses to amiloride and some of its analogues was investigated in isolated cardiovascular tissues from female Wistar rats. The compounds tested were amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA, a potent inhibitor of Na+/H+ exchange), phenamil and 2′,4′-dimethylbenzamil (DMB), both potent Na+ channel inhibitors with activity against Na+/Ca2+ exchange, and 5-(N-4-chlorobenzyl)-2′,4′-dimethylbenzamil (CBDMB), a potent inhibitor of Na+/Ca2+ exchange with reduced activity against Na+ channels compared with its parent compound DMB.Phenamil, DMB and CBDMB increased the force of contraction of right ventricular papillary muscles with similar potencies (-log EC50 values: 4.77 ± 0.06, 5.09 ± 0.09, 4.97 ± 0.17 respectively), while amiloride and EIPA gave small negative inotropic responses. All compounds gave negative chronotropic responses at similar concentrations to those which exerted inotropic effects. Inhibition of KCl contraction of endothelium-free aortic rings was observed with all compounds tested. Phenamil, DMB and CBDMB but not amiloride or EIPA showed a shift to the left of the concentration-response curves in the presence of intact endothelium.These results provide further evidence for positive inotropic and endothelium-dependent vasorelaxant effects of amiloride analogues mediated by inhibition of Na+/Ca2+ exchange.

Membrane transport of thyroid hormone in the human choriocarcinoma cell line, JAR

We studied uptake of L-triiodothyronine (T3) by the human choriocarcinoma cell line, JAR. Uptake was time dependent with a half-time of 56.2 +/- 7.2 min (mean +/- SEM, n = 4). A non-saturable component accounted for about 24% of total uptake. We found a single saturable uptake mechanism with a calculated Michaelis constant (Km) of 586 +/- 206 nM (n = 9) and a corresponding maximum velocity of 17.0 +/- 5.7 pmol/min per mg protein (n = 9), values similar to those we have described recently in cultured normal human trophoblast cells. Uptake was dependent on temperature and intracellular energy, being reduced at lower temperatures and in the presence of potassium cyanide. It was independent of the Na+ gradient across the cell membrane and the presence of Na+ in the external medium, but was affected by the cell membrane potential.

Dedifferentiation of cultured thyroid cells by epidermal growth factor: some insights into the mechanism

Epidermal growth factor (EGF) has been shown to enhance both the proliferation and dedifferentiation of thyroid cells in culture, leading to a maintained dedifferentiated state, even in the presence of thyrotropin (TSH). Since this maintained loss of differentiated function is not seen with other mitogens, it may relate to a regulatory role for EGF in thyroid function. Therefore, we have examined the loci affected by the dedifferentiative actions of EGF using porcine thyroid cells in culture. EGF (10 ng/ml) induces a loss of thyrotropin (TSH) receptors with a time course identical to the loss in ability to transport iodide. This could account for the difference in extent of iodide uptake and morphological dedifferentiation seen between TSH- and cAMP-supported cells, although the fact that cAMP-supported cells also dedifferentiate implies a lesion distal to the cyclase. Reciprocal plot analysis of iodide uptake in control and EGF-treated cells shows that EGF increases the Km for iodide transport, corresponding to a decreased affinity of iodide pump sites for iodide. These effects on iodide pump affinity and TSH receptor number may result from reversal of thyroid cell polarity in monolayer culture, or they may be the result of more specific actions of EGF at these loci. It has been possible to discriminate between the proliferative and dedifferentiating actions of EGF using amiloride, a non-specific inhibitor of the Na+/H+ antiporter. An optimum concentration of amiloride (0.1 mM) was able to block EGF-stimulated incorporation of [3H]thymidine into DNA without preventing the blockade of iodide uptake, which implies that dedifferentiation is not a consequence of proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of cell polarity and epithelial junctions in the choriocarcinoma cell line, JAR

In the placenta the trophoblast cell layer separates maternal and fetal circulations and is involved in the active transport of selected substances across this barrier. We have used the JAR choriocarcinoma cell line to study aspects of trophoblast membrane transport. To determine whether JAR cells could be used in studies of vectorial transepithelial transport it was necessary to determine whether these cells were polarized and assembled tight junctions. In the present study we investigated JAR cells using a range of markers for specific cell surface domains combined with confocal laser scanning microscopy. Freshly isolated cells initially formed a confluent epithelial monolayer with recruitment of a tight junction-associated protein, ZO-1, and a cell adhesion molecule, E-cadherin, to the surface at sites of cell-cell contact. They did not, however, display cell surface polarization, as NaK-ATPase was not segregated in the basolateral domain, and a differentiated apical cell surface was not assembled. The monolayer stage was also unstable, as continued proliferation resulted in the formation of multilayered aggregates where ZO-1 and E-cadherin were lost from the cell surface. These results suggest that the JAR cell line is unlikely to be a suitable model for studies of transepithelial transport in the placenta.

Microtubule integrity is essential for apical polarization and epithelial morphogenesis in the thyroid

In this study, we examined the contribution of microtubules to epithelial morphogenesis in primary thyroid cell cultures. Thyroid follicles consist of a single layer of polarized epithelial cells surrounding a closed compartment, the follicular lumen. Freshly isolated porcine thyroid cells aggregate and reorganize to form follicles when grown in primary cultures. Follicular reorganization is principally a morphogenetic process that entails the assembly of biochemically distinct apical and basolateral membrane domains, delimited by tight junctions. The establishment of cell surface polarity during folliculogenesis coincided with the polarized redistribution of microtubules, predominantly in the developing apical poles of cells. Disruption of microtubule integrity using either colchicine or nocodazole caused loss of defined apical membrane domains, tight junctions and follicular lumina. Apical membrane and tight junction markers became randomly distributed at the outer surfaces of aggregates. In contrast, the basolateral surface markers, E-cadherin and Na(+),K(+)-ATPase, remained correctly localized at sites of cell-cell contact and at the free surfaces of cell aggregates. These findings demonstrate that microtubules play a necessary role in thyroid epithelial morphogenesis. Specifically, microtubules are essential to preserve the correct localization of apical membrane components within enclosed cellular aggregates, a situation that is also likely to pertain where lumina must be formed from solid aggregates of epithelial precursors.

UTP‐preferring P2 receptor mediates inhibition of sodium transport in porcine thyroid epithelial cells

The effects of adenosine 5′‐triphosphate (ATP), uridine 5′‐triphosphate (UTP) and analogues on forskolin‐stimulated absorption of Na+ by porcine thyroid epithelial cells were analysed in cultures grown as confluent monolayers on permeable supports in Transwell Ussing chambers. 85% of the forskolin (10u2003μM)‐stimulated short‐circuit current was inhibited by phenamil (1u2003μM), which is a selective antagonist for epithelial type Na+ channels. Phenamil‐sensitive current was inhibited in a dose dependent manner by nucleotides added to the apical compartment of Ussing chambers. In contrast, the phenamil‐resistant current, previously shown to represent anion secretion, was unaffected by nucleotides. The order of potency (with EC50 values given in μM) was UTP (0.08)>>ATP (6.3)=uridine 5′‐diphosphate (UDP) (6.6)>2methyl‐thio‐adenosine‐5′‐triphosphate (2MeSATP) (84.5)>adenosine 5′‐diphosphate (ADP) (147.8)>α,β‐methylene ATP (>150)>>adenosine (>1000). P2 receptors mediating inhibition of sodium absorption were present on the apical membrane of the cells since addition of UTP (1–1000u2003μM) to the basal compartment of the Ussing chambers had little effect while subsequent addition to the apical compartment produced a normal response. Cibachron blue (Reactive blue 2) (1–100u2003μM), an antagonist at some P2 receptor subtypes, inhibited phenamil sensitive current in a dose dependent manner with half maximal inhibition occurring at 14.25u2003μM. Suramin (100u2003μM), pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) (100u2003μM) and pyridoxal 5′‐phosphate (P5P) (100u2003μM) showed only slight competitive antagonism against the response to UTP. These results indicate that a UTP‐preferring P2 receptor located on the apical membrane of thyroid epithelial cells mediates inhibition of Na+ absorption.

Chloride conductance of apical membrane in cultured porcine thyroid cells activated by cyclic AMP

The thyroid epithelium transports fluid bidirectionally using active transport of Na+ ions from apical to basal poles and active transport of Cl- in the reverse direction. In these studies we sought evidence for cyclic AMP activated Cl- channels on the apical membranes of thyroid cells in monolayer culture. A Cl(-)-dependent basal-positive short-circuit current (ISC) was demonstrated in bicameral chambers after blocking Na+ transport with phenamil, and responded to prostaglandin (PG) E2 with a spike of 5-10 min duration followed by a plateau. The onset of the spike coincided with an increase in the conductance of the epithelium. Application of an external Cl- concentration gradient, by replacing the medium in the apical compartment with Cl(-)-free medium, resulted in an increase in ISC after, but not before, addition of PGE2. Forskolin and thyroid-stimulating hormone (TSH), but not A23187, also stimulated Cl- transport. In conjunction with previous observations that Cl- transport was mediated by a bumetanide-sensitive NaKCl2 symporter on the basal membrane, these observations indicated the presence of a cyclic AMP activated Cl- conductance in the apical membrane of thyroid cells.

Regulation of thyroid follicular volume by bidirectional transepithelial ion transport

Previous studies have shown that thyroid cells in monolayer culture exhibit bidirectional ion transport comprising apical-to-basal amiloride-sensitive Na+ transport and oppositely directed bumetanide-sensitive Cl- transport. We have now investigated the role of ion transport in the regulation of thyroid follicular size using follicular primary porcine thyroid cell cultures. Bumetanide (10 microM) added at the beginning of culture inhibited the formation of follicular lumina and caused a fall in follicle height when added to 3-day-old cultures. In contrast, phenamil (1 microM; an amiloride analog) increased follicle size both in freshly isolated and 3-day-old cultures. The effect of bumetanide was prevented by the prior addition of phenamil. Micropuncture studies showed that follicles had a lumen-negative, basal-positive transepithelial potential difference which was progressively reduced in magnitude by the serial addition of bumetanide (10 microM) and phenamil (1 microM). We conclude that thyroid follicles possess a bidirectional ion transport system which transports Na+ in an apical-to-basal direction and Cl- in the opposite direction. The balance between these two processes determines net solute flux and hence follicular size. A physiological role of ion transport in the thyroid may be to regulate follicular volume suggesting that abnormalities of ion transport may be responsible for disorders of follicular size.

Inflammatory peptide in spinal cord: evidence that the mediator of antidromic vasodilatation is not substance P.

Extracts from rat and bovine spinal cord were found to have 300-1000 times more cutaneous oedema-inducing activity than could be attributed to their substance P-like activity estimated by assay on guinea-pig ileum. The activity on both assay systems was reduced in cord extracts from rats pretreated as neonates with capsaicin. Results of assays of fractions obtained from gel-filtration chromatography indicated that an agent, in the molecular size range for peptides, was present in spinal cord extracts, which possessed some of the properties of substance P but was not identical to it.

The thyrotrophin receptor

ConclusionTwo decades of investigation of the TSH receptor as an autoantigen have led not only to elucidation of the pathogenesis of Graves disease but also to a general theory of autoimmunity. The H-gene theory [10] indicates a strategy for the treatment of autoimmune disease by intervention in the network of paratope-idiotope interactions controlling clonal deletions [54].In the past 10 years, investigation of the hormone-binding function of the receptor has demonstrated the mechanism of action of the thyroid-stimulating autoantibodies, and provided the basis for the convenient radioreceptor assay of TSaab. This technique has yet to achieve its apparent potential, but the widely-used methodology has been essentially unchanged since 1973 [80]. Recent work has shown improvements in receptor abundance, affinity, and specificity are achievable by manipulation of ionic strength [100], observations which may provide the key to eliminating the major failings of the radioreceptor assay, namely sensitivity and the nonspecific effect of normal control globulins.Since the amounts of TSH receptor in thyroid tissue are minute, it is unlikely that purification to homogeneity will be achieved in the near future. Nevertheless, the recent demonstrations of receptor heterogeneity [37, 79] and the possible role of gangliosides of known simple structure in receptor function suggest that new and important insights into hormone-receptor interaction may still be forthcoming from the study of TSH receptors.