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D. R. FERGUSON
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The investigation of naturally occurring neurohypophysial hormones demands methods of separation applicable to microgram quantities. The first effective method developed was the paper chromatographic procedure of Heller & Lederis (1958). Thin-layer chromatography is quicker, its reliability is much less affected by environmental factors and it has excellent resolving power. It has therefore replaced paper chromatography for many purposes and an attempt was therefore made to use it for the separation of neurohypophysial peptides.

The adsorbent was cellulose powder (Macherey, Nagel and Co., No. MN 300, free of calcium sulphate). The plates were prepared by mixing 1·5 g. dry cellulose powder with 9 ml. of water, until a smooth slurry resulted. The slurry was spread on the glass plate by means of a Perspex plate, 10·2 × 7·5 cm. in size, with a recess of 8·2 cm. × 0·5 mm. machined in one margin. The plate was dried at 105° for

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D. R. FERGUSON
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B. R. TWITE
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SUMMARY

The results support previous findings that vasopressin produces dephosphorylation of a toad bladder membrane protein of 50000 mol. wt. At vasopressin concentrations of 50 mu./ml phosphorylation of this protein was 60 ± 7% of the control level, and maximal natriferic and hydro-osmotic responses were observed in intact bladders. However, at concentrations of vasopressin of 10 mu./ml when a maximal natriferic response but no significant hydro-osmotic response were observed, there was no significant difference in phosphorylation of the proteins from control or vasopressin-treated bladders.

We conclude that the 50000 mol. wt membrane protein is likely to be associated with the hydro-osmotic response of the toad bladder to vasopressin.

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D. R. FERGUSON
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M. W. SMITH
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SUMMARY

A technique is described by which the unidirectional influx of sodium into toad bladder mucosal cells can be measured over short periods.

The uptake of sodium, which is linear over a 60 s contact period, changes in a non-linear way when the external sodium concentration is varied from 5 to 115mm.

Vasotocin applied to the serosal surface increases significantly the influx of sodium; amiloride applied to the mucosal surface inhibits influx. This is true whether high or low concentrations of sodium are used to bathe the mucosal surface.

The kinetics for the unidirectional influx of sodium provide direct evidence that the passage of sodium into this tissue is by some means other than simple diffusion.

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P. J. BENTLEY
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D. R. FERGUSON
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Anuran Amphibia (frogs and toads) sitting in tap water increase in weight when injected with neurohypophysial hormones (Brunn, 1921). This response has been called the 'Brunn' or 'water-balance' effect (Heller, 1941) and reflects water retention by the animal. The effect results from an action of the hormones at three sites: the skin, which becomes more permeable to water, the kidney, which produces less urine, and the bladder, from which urinary stored water can be reabsorbed (see Bentley, 1963). Urodele Amphibia (newts and salamanders) also retain water when injected with neurohypophysial hormones, but in the species so far examined the response is due either to an action on the kidney as in Triturus (Bentley & Heller, 1964), or on the urinary bladder as in Salamandra maculosa (Bentley & Heller, 1965). The relative importance of these organs in water retention by anuran Amphibia has not been examined previously and this is the

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N. P. Lewis
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D. R. Ferguson
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ABSTRACT

Basolateral membranes were prepared from rat renal cortex by density gradient centrifugation. Their purity was confirmed by electron microscopy and by marker enzyme enrichment. The basolateral membrane preparation was shown to be derived predominantly from the proximal renal tubule by measurement of hormone-stimulated adenylate cyclase; marked stimulation of adenylate cyclase was found with parathyroid hormone, but not with isoprenaline, antidiuretic hormone or calcitonin. A single class of specific high-affinity [3H]angiotensin II-binding site was identified in the basolateral membrane preparation which, after correction of results for tracer degradation, showed equilibrium dissociation constant of 0·23 nmol/l and binding site concentration of 485·8 fmol/mg protein.

Binding sites for [3H]angiotensin II were measured in basolateral membranes prepared from rats fed diets with a low, normal or high sodium content. A trend of increased binding site density with reduced sodium intake was found which did not reach statistical significance. No effect on affinity was demonstrated. Treatment of rats on a low-sodium diet with captopril (500 mg/l drinking water) caused a significant reduction in binding site density; no effect on affinity was demonstrated.

These findings suggest that the density of angiotensin II receptors at this site is altered by the activity of the renin-angiotensin system.

Journal of Endocrinology (1989) 122, 499–507

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