Aldona Butkus

Purification and cloning of a corpuscles of Stannius protein from Anguilla australis

The kidneys of teleost fish are associated with tissues containing secretory granules--the corpuscles of Stannius (CS). Electron microscopy indicates that the granules are of a proteinaceous nature and may represent hormones or enzymes of unrecognized physiological and biochemical function. In the present study, two-dimensional gel electrophoresis and electroelution was used to purify the major protein to homogeneity; it is approximately 32,000 Da in the reduced form and glycosylated. From the partial NH2-terminal sequence, a 75-mer oligonucleotide probe was synthesized and used to isolate a cDNA clone from which the complete amino acid sequence of the major CS protein was deduced. Polyclonal antibodies raised against CS homogenates were specific for the CS proteins (confirmed by immunohistochemistry). Hybridization histochemistry was used to confirm the location of the mRNA encoding the isolated protein. Incubation of CS homogenate with eel plasma or ovine renin substrate did not result in any angiotensin-like peptides whereas kidney homogenate did.

Structure and expression of Leydig insulin-like peptide mRNA in the sheep.

We have used PCR to isolate and characterise Leydig insulin-like peptide (Ley I-L) mRNA from sheep ovary. The deduced amino acid sequence of sheep Ley I-L has good homology with the pig and human peptides, having 93% and 77% amino acid identity, respectively. Northern blot analysis revealed abundant expression in both ovary and testis. An examination of ovarian RNA from non-pregnant and pregnant sheep showed that pregnancy did not significantly increase Ley I-L mRNA levels. However mRNA levels did alter depending on whether ovaries contained a corpus luteum. Also ovaries were examined by hybridisation histochemistry to locate the site of expression. Abundant Ley I-L mRNA levels were found in the theca interna cells of the ovary.

Blood Pressure and Metabolic Effects of ACTH in Normotensive and Hypertensive Man

ACTH administration (0.5 mg Synacthen Depot I/M 12 hourly for 5 days) significantly increased systolic blood pressure in normotensive subjects (n=6) and mild essential hypertensives (n=6) but not in 2 Addisonian women, indicating that the pressure rise was adrenally dependent. ACTH administration was associated with urinary sodium retention, hypokalaemia, elevation of fasting blood glucose, lymphopaenia and eosinopaenia. Body weight was increased only in the normotensive subjects. Plasma renin concentration fell and renin substrate rose. Inactive renin fell in the hypertensive subjects only. Plasma cortisol, 11-deoxycortisol, corticosterone, deoxycorticosterone, 17 alpha-hydroxyprogesterone and 17-hydroxy, 20-dihydroprogesterone were all increased by ACTH treatment. Plasma aldosterone rose initially in the normotensives but then fell. ACTH administration in man produces metabolic and hormonal changes similar to those produced by ACTH in sheep but the rise in blood pressure is systolic only in man. The steroid(s) responsible for the blood pressure rise with ACTH in man have not been defined.

Distribution of glomerular peripolar cells in different mammalian species

SummaryPeripolar cells are granulated glomerular epithelial cells that form a cuff around the vascular pole of the glomerulus. Quantitation of these cells in 17 species of mammals (including man, several laboratory animals and a variety of other species) indicated that they were detectable by light microscopy in all but one of the mammals that were examined (the Australian hopping mouse). In adult mammals with detectable peripolar cells, the “peripolar cell index” (the percentage of randomly sectioned glomeruli that displayed peripolar cells in histological sections of kidney) ranged from 0.15 (for echidna) to 11.86 (for sheep). Newborn lambs and rats showed strikingly high values (23.30 and 10.76, respectively) compared with their adult counterparts. Using electron microscopy, peripolar cells were observed in all species that were examined, including the Australian hopping mouse. Morphologically, peripolar cells were similar in all species although their size and granule population varied. They showed a predominantly outer cortical glomerular distribution and a close anatomical relationship with the renin-containing myoepithelioid cells. These findings indicate that peripolar cells are present in a wide variety of species and support the view that such cells may play a significant role in the regulation of normal renal function.

Ontogeny and regulation of the AT1 and AT2 receptors in the ovine fetal adrenal gland

The expression and regulation of the receptors for angiotensin II (both AT1 and AT2) were examined in the ovine fetal adrenal gland by RNase protection assay (RPA), in situ hybridisation histochemistry, immunohistochemistry and Western blotting. Both mRNA and protein for the AT1 receptor were present in the zona glomerulosa and zona fasciculata of the cortex, but not in the medulla, from as early as these zonas were distinguishable (60 days of gestation; term is 145-150 days), and even present in the steroidogenic cells of the unzoned gland at 40 days. The mRNA for the AT2 receptor was present in the same locations (but never in the medulla) from 40-130 days, and declined to extremely low levels after 140 days. The infusion of ang II, 1 microg/h, for 3 days, at mid-gestation (76 +/- 2 days) caused a significant decrease in mRNA for AT1 but no change in AT2 levels. Thus, the biologically active receptor (in terms of aldosterone stimulation) is present in the ovine fetal adrenal from very early in development, and can be down-regulated by mid-gestation.

The renin-angiotensin system and the development of the kidney and adrenal in sheep.

1. The earliest form of the kidney, the pronephros, does not really occur in the ovine embryo; instead, a giant glomerulus forms at the anterior end of the mesonephros.

Expression of aquaporin‐1 (AQP1) in the adult and developing sheep kidney

The distributions of aquaporin-1 mRNA and protein were studied by hybridization histochemistry with a homologous riboprobe and immunohistochemistry, in the adult sheep kidney. Heaviest labelling occurred in the thin descending limb (DTL) of the loop of Henle in the inner stripe of the outer medulla, with apparent decreasing expression in the inner medulla, outer stripe of the outer medulla and cortex, but no quantitation was performed. Only proximal tubules (PT) (convoluted and straight) and DTL labelled. The glomerulus showed no labelling, consistent with the pattern in the rat but different to that in the human. During ontogeny, no labelling occurred in the mesonephros at 27 or 41 days of gestation (term = 145-150 days) but other structures did label at 27 days (heart, lung bud, blood vessels surrounding developing spinal cord). Labelling first occurred faintly in the metanephros at 41 days of gestation and increased throughout gestation consistent with morphological development of nephrons.

Studies on stanniocalcin: characterization of bioactive and antigenic domains of the hormone

Stanniocalcin (STC) decreases branchial Ca(2+)-uptake in fish. In order to determine its bioactive domain, synthetic fragments (U amino acids (aa) 1-20; V aa 103-136; W aa 202-231) of eel STC were tested for their effect on Ca2+ uptake in tilapia (Oreochromis mossambicus). Ca2+ uptake was inhibited by an N-terminal fragment but not by a midfragment nor a C-terminal fragment of the mature hormone. We provide theoretical and experimental evidence that a midportion of STC, which is included in the synthetic fragment V, is the most antigenic site of the molecule. Polyclonal antibodies against stanniocalcin are directed against this midportion although this region of STC appears not to be essential for signal transduction. These results suggest that the currently available antibodies will recognize inactive STC fragments in the circulation. We conclude that the bioactive portion of STC does not correspond with the major antigenic portion of the hormone. The results imply that studies on plasma STC levels employing a polyclonal antiserum against STC should be interpreted with care.

THE AFFINITY OF 17α‐HYDROXYPROGESTERONE AND 17α, 20α‐DIHYDROXYPROGESTERONE FOR CLASSICAL MINERALOCORTICOID OR GLUCOCORTICOID RECEPTORS

The displacement of [3H]‐aldosterone, [3H]‐dexamethasone, and [3H]‐cortisol by 17αL‐hydroxyprogesterone, 17αL, 20αL‐dihydroxyprogesterone and 17α, 20β‐dihydroxyprogesterone from ovine cytosol receptors indicates that they do not occupy classical mineralocorticoid or glucocorticoid receptors.

Processing and bioactivity of the corpuscles of Stannius protein of the Australian eel.

The primary structure of the major protein from the Corpuscles of Stannius (CS) of the Australian eel was elucidated from the cDNA sequence and was found to bear close similarity to the N-terminal amino acid sequence of the presumably homologous salmon hormone, teleocalcin (TC). The cDNA sequence predicted a preproprotein of 263 amino acids. Following removal of a 17 amino acid signal peptide, specific monobasic cleavage at an Arg-Phe bond generates the 231 amino acid mature form of the protein. The isolation and sequence determination of the prosequence confirms that the precursor contains a prosegment of 15 residues. Various fragments of the protein have been synthesized chemically and their biological activity assessed. The N-terminal 1–20 fragment of the mature protein inhibits calcium uptake in fingerling trout, the effect being similar, but not equipotent to salmon teleocalcin. Further, infusion of either the N-terminal 1–20 or the 81–94 fragment at 50 μg/h into the renal artery of conscious sheep, caused significant decreases in systemic plasma potassium concentration and in potassium excretion. The 1–20 fragment also gave rise to a small but significant increase in sodium excretion. Infusion of TC at the same rate results in a significant decrease in plasma potassium and phosphate concentration as well as a significant decrease in potassium excretion. Bovine PTH (1–34) at 100 μg/h causes a small decrease in plasma potassium and phosphate and an increase in plasma calcium concentration, and was the only peptide to cause a significant decrease in calcium excretion.