Presence of adrenocorticotropin and β-Endorphin immunoreactivities in the magnocellular neurosecretory system of the rat hypothalamus

Summary Antisera raised against ACTH (1–39), -endorphin and the 16K proopiocortin were used, in association with the immunoperoxidase reaction, to localize positively-staining cell bodies and nerve fibres in the hypothalamus of the rat. Antigens, cross-reactive against anti-ACTH (1–39) serum were detected in a fibre system in the rostro-dorsal hypothalamus situated between the optic chiasm and the third ventricle while immunoreactive 16K-like material was present in fibres localized in the caudal hypothalamus, dorso-lateral to the arcuate nucleus. This latter system was also associated with the appearance of ACTH (1–39) and ACTH (17–39) immunoreactivity.Cells of the arcuate nucleus stained positively for ACTH (1–39), 16K antigen and -endorphin, and on examining adjacent thin sections it was observed that cells that contained 16K antigen-like material, also gave a positive immunoreaction with ACTH (1–39) and -endorphin antisera. In the magnocellular system, cells of the supraoptic (SON) and paraventricular (PVN) nuclei also gave a positive immunoreaction with anti-ACTH (1–39), 16K antigen and -endorphin serum. As in the case of the arcuate nucleus, common cells stained for these three antigens.On the basis of the precursor theory for the synthesis of ACTH, 16K antigen and -endorphin, it was not unexpected to find these three fragments of pro-opiocortin localized together in cells of the arcuate nucleus. That ACTH (1–39), 16K antigen and -endorphin-like materials are present in the magnocellular neurosecretory system would suggest that cells of the SON and PVN are not only involved in the synthesis of neurophysin and the neurohypophysial hormones, but also of some products of the pro-opiocortin molecule. Whether the biochemical nature of the ACTH and -endorphin in cells of the SON and PVN is identical to that of anterior pituitary origin remains to be established, as does the biosynthetic relationship between neurophysin and oxytocin/ vasopressin and these fragments of pro-opiocortin.Drs. M.M. Wilkes, S.S.C. Yen, G. Pelletier, B.A. Eipper and R. Walter are thanked for supplying some of the antisera and antigens used in this study. Thanks also go to Ciba-Geigy Ltd. and Organon Inc. for supplies of ACTH (17–39) and ACTH (1–24) respectively. This work was financed by The Medical Research Council of New Zealand     

Attenuation of hypotensive effect of propranolol and thiazide diuretics by indomethacin.

The effects of 100 mg indomethacin daily for three weeks on blood pressure and urinary excretion of prostaglandin F2 alpha were studied in a double-blind, placebo-controlled comparison of two groups of patients with essential hypertension, eight receiving propranolol and seven thiazide diuretics. Compared with placebo, adding indomethacin to the patients'' established antihypertensive treatment increased blood pressure by 14/5 Hg supine and 16/9 mm Hg erect in the patients receiving propranolol, and by 13/9 mm Hg supine and 16/9 mm Hg erect in the patients receiving thiazide diuretics (all p less than or equal to 0.05). The excretion of the major urinary metabolite of prostaglandin F2 alpha was reduced by 67% in the propranolol-treated patients and by 57% in those receiving a thiazide diuretic. Body weight increased by 0 . 8 kg (propranolol) and 1 . 1 kg (thiazide diuretic) when indomethacin was given, but there were no significant changes in creatinine clearance, urinary sodium excretion, or packed cell volume in either treatment group. These results suggest that products formed by the arachidonic acid cyclo-oxygenase contribute to the regulation of blood pressure during treatment with both propranolol and thiazide diuretics. Inhibition of the cyclo-oxygenase with indomethacin partially antagonises the hypotensive effect of these drugs.     

Isolation and properties of an NAD- and guanidine-dependent ADP-ribosyltransferase from turkey erythrocytes

An NAD- and guanidine-dependent ADP-ribosyltransferase has been purified more than 500,000-fold from turkey erythrocytes with an 18% yield. The enzyme in the 100,000 X g supernatant fraction was bound to phenyl-Sepharose, eluted with 50% propylene glycol, and further purified by sequential chromatographic steps on carboxymethylcellulose, NAD-agarose and concanavalin A-agarose. The transferase was specifically eluted from concanavalin A-agarose with alpha-methylmannoside. The enzymatic activity was extremely labile following the first purification step. Both propylene glycol and NaCl stabilized the transferase; significant increases in enzyme recovery were obtained by conducting the NAD- and concanavalin A-agarose chromatography in buffer containing propylene glycol. The purified protein exhibits one predominant protein band on SDS-polyacrylamide gels with an estimated molecular weight of 28,300. On Ultrogel AcA54 chromatography, single coincident peaks of ADP-ribosyltransferase activity and protein were observed. Enzyme activity was independent of DNA; the highly purified transferase was inhibited by thymidine, nicotinamide, and theophylline. The specific activity of the purified enzyme (350 mumol of ADP-ribose transferred from NAD to arginine methyl estermin-1mg-1) is comparable to that reported for purified NAD glycohydrolases and poly(ADP-ribosyl)transferases.     

Solution properties of sulfated monohydroxy bile salts. Relative insolubility of the disodium salt of glycolithocholate sulfate

Physical-chemical properties of the major sulfated monohydroxy bile salts of man are described. In general, the sulfates are significantly more water-soluble than the non-sulfated species as a result of lower critical micellar temperatures, high aqueous monomeric solubilities and critical micellar concentrations. Nevertheless, at 37 degrees C the disodium salt of glycolithocholate sulfate, the major monohydroxy bile salt of man is not more soluble than its non-sulfated form. Since aqueous solubility correlates inversely with the cholestatic potential of bile salts, our results suggest that this sulfate may be potentially hepatoxic. Micellar solubility of phosphatidylcholine and cholesterol by the majority of non-sulfated and sulfated monohydroxy bile salts is slight. Nonetheless, phosphatidylcholine is very well solubilized by taurolithocholate sulfate but cholesterol solubility is not increased appreciably. Cholesterol saturation in model bile systems of taurochenodeoxycholate and phosphatidylcholine is impaired by the addition of sulfated lithocholate conjugates but with physiological bile salt compositions this reduction is not significant.     

The synthesis of DNA by DNA-membrane complexes from irradiated E. coli B/r and Bs-1: the role of the membrane.

DNA-membrane complexes were isolated from lysed E. coli B/r and Bs-1, either by low g forces from a low salt solution, or by high g forces through a discontinuous sucrose gradient. The latter method was more gentle. Irradiation of the intact bacteria had no effect on the membrane macromolecules or on RNA components of these complexes. DNA loss was not significant after irradiation under anoxic conditions but complexes isolated from from Bs-1 irradiated in air showed an appreciable decrease in DNA content. In the presence of the appropriate nucleotide mixture, both 'free' DNA, found in the supernatant fractions, and rapidly sedimented membrane-associated DNA were able to synthesize DNA in the absence of added polymerase. DNA synthesis associated with 'free' DNA was more sensitive to radiation than that associated with DNA bound to the membrane, which appeared to moderate the effects of radiation on new DNA synthesis. It is concluded that the depression of DNA synthesis is primarily a result of irradiation-induced changes on genome-DNA. The interpretation of earlier work from our laboratories that DNA-membrane complexes contained the macromolecular structure which responded to radiation with a high o.e.r. is not supported by the evidence in this work.     

Oxytocinase-immunohistochemical demonstration in the immature and term human placenta

Summary Oxytocinase (cystine aminopeptidase) was purified from human retroplacental serum by a combination of fractional precipitation, hydroxylapatite chromatography and gel exlusion chromatography on Sephadex G-200. The purified enzyme possessed a specific activity of 980 mIU/mg using L-cystine-di-p-nitroanilide as substrate. This represented a 3200 fold concentration from the starting material in an overall yield of 12%. Antibodies against oxytocinase were raised in rabbits and the -globulin fraction labelled with fluorescein isothiocyanate prior to its use in the immunofluorescence histochemical localization of the enzyme in human placental tissue. Oxytocinase was confined to the syncytiotrophoblastic cells of normal term, and immature placentas as well as in placentas from patients suffering from severe toxaemia. Specific immunofluorescence was also present in the outer margins of the chorion and to a lesser extent in the amnion.This work was financed by a grant from The Medical Research Council of New Zealand.     

STUDIES ON THE PROPERTIES OF RETINAL ALCOHOL DEHYDROGENASE FROM THE RAT

An NAD-dependent alcohol dehydrogenase (alcohol:NAD oxidoreductase; EC 1.1.1.1) has been isolated and partially purified from the retinal cytosol of the rat. Its substrate specificity and sensitivity to inhibitors of hepatic alcohol dehydrogenase have been investigated. Ethanol, 1-propanol and 1-butanol served as substrates for this enzyme but the K_m values were more than 100-fold higher than those reported for hepatic alcohol dehydrogenase. Methanol and retinol were unreactive with this alcohol dehydrogenase. Inhibition by pyrazole was observed but the K_t was about 100-fold higher than the value observed for hepatic alcohol dehydrogenase. n-Butyraldoxime inhibited retinal alcohol dehydrogenase with a K_t of 2 μM, a value which approximates its K_t for hepatic alcohol dehydrogenase. 1, 10-Phenanthroline was ineffective as an inhibitor. Oxidation of retinol was observed in retinal homogenates in the presence of NADP but no inhibition was observed with ethanol, methanol or pyrazole. We conclude that oxidation of retinol is not catalysed by soluble retinal alcohol dehydrogenase.     

Detection of NAD:arginine ADPribosyltransferases in animal tissues using 125I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane as ADPribose acceptor

125I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane (N-guanyltyramine), previously used to assay for the bacterial toxin choleragen (Mekalanos, J.J., Collier, R.J. and Romig, W.R. (1979) J. Biol. Chem. 254, 5849-5854) was utilized to identify NAD:arginine ADPribosyltransferases in animal tissues. The use of this radiolabelled ADPribose acceptor, rather than radiolabelled NAD, would bypass the problem posed by the almost ubiquitous presence of enzymes that degrade NAD. With a homogeneous ADPribosyltransferase from turkey erythrocytes, NAD and 125I-labeled guanyltyramine as ADPribose acceptor, formation of ADPribosyl 125I-guanyltyramine was linear with time and enzyme concentration. The product was indistinguishable on both thin-layer and high-performance liquid chromatography from that formed by choleragen. Using 125I-guanyltyramine, ADPribosyltransferase activity was also demonstrated in crude turkey erythrocyte cytosolic and membrane fractions. When rat liver was fractionated, apparent activity was detected primarily in the microsomes. The NAD-dependent product of the microsomal reaction was, however, distinguished from the turkey erythrocyte transferase product by thin-layer and DEAE-Sephadex chromatography; this product had a retention time identical to that of free 125I on high-performance liquid chromatography. In addition to NAD, the microsomal deiodinase activity was supported by NADH, NADP and NADPH. Phenyl boronate selectively bound ADPribosyl 125I-guanyltyramine and other metabolites of 125I-guanyltyramine which were formed by microsomes in a NAD-dependent process. These metabolites were distinguished from ADPribosyl 125I-guanyltyramine by high-performance liquid chromatography. These results indicate that in some cases, for example, turkey erythrocyte cytosolic and membrane fractions, 125I-guanyltyramine can be used to quantify ADPribosyltransferases in crude mixtures, whereas in others, for example, rat liver microsomes, high-performance liquid chromatographic analysis must be used to identify products.     

Isolation and some observations of the properties of a bovine neurohypophysial milk-ejecting factor.

A substance possessing milk-ejecting activity has been isolated from an acetone powder preparation of bovine posterior pituitary glands by Sephadex G-25 chromatography of the neurophysin-neurohypophysial hormone complex. While the material possessed an oxytocic activity of 2.8 IU/mg as measured on the isolated rat uterus, the milk-ejecting activity was more than three fold greater, 9.6 IU/mg. The peptide had an antidiuretic activity of 0.133 IU/mg and a pressor activity of 0.083 IU/mg. Neither the uterine-stimulating action nor the pressor activity was destroyed by incubating the peptide with 0.01 M sodium thioglycollate at 65 degrees C for 5 min. The oxytocic activity was antagonized neither by 1.4 X 10(-6) M atropine nor 3.3 X 10(-7) M phenoxybenzamine.