Effects of voltage clamping on epithelial cell composition in toad urinary bladder studied with X-ray microanalysis

Toad urinary bladder epithelial cells were incubated in Na Ringer's with the serosal surface of the epithelium clamped at either +50 mV, O mV (short-circuited) or –50 mV with respect to the mucosal surface. Following incubation, portions of tissue were coated with an external albumin standard and rapidly frozen. Cryosections were freeze-dried and cell composition determined by x-ray microanalysis. Cell water and ion contents were unaffected when tissues were short-circuited rather than clamped close to their open-circuit potential difference (+50 mV). Incubation with vasopressin at +50 mV, and under short-circuit conditions, caused Na uptake without cell swelling or gain in Cl. Clamping at –50 mV resulted in uptake of water and ions, with considerable variation from cell to cell. These variations in cell composition were exacerbated by vasopressin. The greater the increase in water content, the greater the rise in cell Cl. However, there was no consistent pattern to the associated changes in cation contents. Most cells gained some Na. In some cells, this gain was accompanied by an increase in K. In others, the gain of Na was predominant and cell K content actually fell. At –50 mV with ouabain, many of the cells also gained water. As was found in our earlier study with ouabain under short circuit conditions (Bowler et al., 1991), there was considerable variation in the extent of the Na gain and K loss; some cells were largely depleted of K while in others the K content remained relatively normal. These results indicate differences between granular cells in the availabilities in the plasma membranes of ion pathways, either as a consequence of differences in the numbers of such pathways or in their control.This work was supported by a grant from the Health Research Council of New Zealand. Purchase of the equipment was made possible through grants from the Medical Research Council of New Zealand, the Medical Distribution Committee of the Lottery Board, the University Grants Committee, the Telford Trust, the New Zealand Neurological Foundation and the National Heart Foundation. We are grateful for the excellent technical assistance of Ms. S. Zellhuber-McMillan.     

Biology and mode of action of pure antioestrogens

The properties of a series of 7 alpha-alkyl analogues of oestradiol are described. Studies of chemical structure and activity in the immature rat uterotrophic/antiuterotrophic assay revealed that molecules containing a terminal functional group (acid, alcohol, amine, amide) linked to the steroid by a decamethylene bridge possess both oestradiol agonist and antagonist activity. However, certain amides, exemplified by the compound ICI 164,384 [N-n-butyl-11-(3,17 beta-dihydroxyoestra-1,3,5(10)-trien-7 alpha-yl)-N-methylundecanamide], were devoid of oestrogenic activity but possessed potent antioestrogenic activity. Comparison of receptor binding and biological potency of steroid 7 alpha- and 7 beta-isomers showed that activity is confined largely to the 7 alpha-isomer. Comparison of the effects of tamoxifen and ICI 164,384 on progesterone receptor (PR) concentration in the rat uterus showed that, unlike tamoxifen, ICI 164,384 did not induce PR and blocked induction of PR by oestradiol. Chronic treatment of mature female rats with ICI 164,384 led to an ovariectomy-like regression of the uterus without affecting LH secretion or the rate of growth. ICI 164,384 was also an effective antitumour agent in rats bearing carcinogen-induced mammary tumours.     

Interspin distances in spin-labeled metmyoglobin variants determined by saturation recovery EPR

Saturation recovery (SR) electron paramagnetic resonance was used to determine the distance between iron and nitroxyl for spin-labeled metmyoglobin variants in low-spin and high-spin states of the Fe(III). The interspin distances were measured by analyzing the effect of the heme iron on the spin-lattice relaxation rates of the nitroxyl spin label using the modified Bloembergen equation for low-spin species, and an analogue of the Bloembergen equation for high-spin species. Insight simulations of the spin-labeled protein structures also were used to determine the interspin distances. The distances obtained by SR for high-spin and low-spin complexes with 15-20 A interspin distances, for low-spin CN(-) and high-spin formate adducts at distances up to about 30 A, and results from Insight calculations were in good agreement. For variants with 25-30 A interspin distances, the distances obtained by SR for the fluoride adducts were shorter than observed for the CN(-) or formate adducts or predicted by Insight simulations. Of the heme axial ligands examined (CN(-), imidazole, F(-), and formate), CN(-) is the best choice for determination of iron-nitroxyl distances in the range of 15-30 A.     

The action of inhibitors of protein kinases on fluid and ion secretion by Malpighian tubules of Locusta migratoria,L

Fluid production in Locusta Malpighian tubules was stimulated by corpora cardiaca extract (c. 100%) and dibutyryl cAMP (c. 50%). Chelerythrine and staurosporine (Protein kinase C, PKC inhibitors) inhibited it in the range 0.07-60&mgr;M (IC(50)3&mgr;M), whereas Rp-cAMP (Protein kinase A, PKA inhibitor) caused inhibition over the concentration range 10-1000&mgr;M (IC(50)264&mgr;M). The protein phosphatase inhibitor, okadaic acid, was also inhibitory over the concentration range 0.1-1000nM (IC(50) 91nM). CC extract stimulation increased fluid [Na(+)] from 41 to 59mM and decreased [K(+)] from 127 to 107mM; stimulation with cAMP had no such effect. The PKC inhibitors reduced the [K(+)] in the secreted fluid from 126 to 107mM but had no effect on the [Na(+)]. Subsequent addition of CC extract stimulated fluid production and caused an increase in [Na(+)] from 41 to about 50mM. The addition of Rp-cAMP reduced fluid production but caused a decrease in [Na(+)] from 37 to 28mM and an increase in its [K(+)] from 124 to 148mM. Fluid production by Rp-cAMP inhibited tubules was not stimulated by corpora cardiaca extract or cAMP, but [Na(+)] rose to 36mM. Protein phosphorylation plays a role in the regulation of fluid production probably via the apical and basal membrane cation transporters.     

Signaling in human osteoblasts by extracellular nucleotides. Their weak induction of the c-fos proto-oncogene via Ca2+ mobilization is strongly potentiated by a parathyroid hormone/cAMP-dependent protein kinase pathway independently of mitogen-activated protein kinase.

Extracellular nucleotides acting through specific P2 receptors activate intracellular signaling cascades. Consistent with the expression of G protein-coupled P2Y receptors in skeletal tissue, the human osteosarcoma cell line SaOS-2 and primary osteoblasts express P2Y1 and P2Y2 receptors, respectively. Their activation by nucleotide agonists (ADP and ATP for P2Y1; ATP and UTP for P2Y2) elevates [Ca2+]i and moderately induces expression of the c-fos proto-oncogene. A synergistic effect on c-fos induction is observed by combining ATP and parathyroid hormone, a key bone cell regulator. Parathyroid hormone elevates intracellular cAMP levels and correspondingly activates a stably integrated reporter gene driven by the Ca2+/cAMP-responsive element of the human c-fos promoter. Nucleotides have little effect on either cAMP levels or this reporter, instead activating luciferase controlled by the full c-fos promoter. This induction is reproduced by a stably integrated serum response element reporter independently of mitogen-activated protein kinase activation and ternary complex factor phosphorylation. This novel example of synergy between the cAMP-dependent protein kinase/CaCRE signaling module and a non-mitogen-activated protein kinase/ternary complex factor pathway that targets the serum response element shows that extracellular ATP, via P2Y receptors, can potentiate strong responses to ubiquitous growth and differentiative factors.     

Adult Non-Cystic Fibrosis Bronchiectasis Is Characterised by Airway Luminal Th17 Pathway Activation

Background

Non-cystic fibrosis (CF) bronchiectasis is characterised by chronic airway infection and neutrophilic inflammation, which we hypothesised would be associated with Th17 pathway activation.

Methods

Th17 pathway cytokines were quantified in bronchoalveolar lavage fluid (BALF), and gene expression of IL-17A, IL-1β, IL-8 and IL-23 determined from endobronchial biopsies (EBx) in 41 stable bronchiectasis subjects and 20 healthy controls. Relationships between IL-17A levels and infection status, important clinical measures and subsequent Pseudomonas aeruginosa infection were determined.

Results

BALF levels of all Th17 cytokines (median (IQR) pg/mL) were significantly higher in bronchiectasis than control subjects, including IL-17A (1.73 (1.19, 3.23) vs. 0.27 (0.24, 0.35), 95% CI 1.05 to 2.21, p<0.0001) and IL-23 (9.48 (4.79, 15.75) vs. 0.70 (0.43, 1.79), 95% CI 4.68 to 11.21, p<0.0001). However, BALF IL-17A levels were not associated with clinical measures or airway microbiology, nor predictive of subsequent P. aeruginosa infection. Furthermore, gene expression of IL-17A in bronchiectasis EBx did not differ from control. In contrast, gene expression (relative to medians of controls) in bronchiectasis EBx was significantly higher than control for IL1β (4.12 (1.24, 8.05) vs 1 (0.13, 2.95), 95% CI 0.05 to 4.07, p = 0.04) and IL-8 (3.75 (1.64, 11.27) vs 1 (0.54, 3.89), 95% CI 0.32 to 4.87, p = 0.02) and BALF IL-8 and IL-1α levels showed significant relationships with clinical measures and airway microbiology. P. aeruginosa infection was associated with increased levels of IL-8 while Haemophilus influenzae was associated with increased IL-1α.

Conclusions and Clinical Relevance

Established adult non-CF bronchiectasis is characterised by luminal Th17 pathway activation, however this pathway may be relatively less important than activation of non-antigen-specific innate neutrophilic immunity.