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C. G. Dacke
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A. J. Shaw
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ABSTRACT

The rapid effects of parathyroid hormones and a variety of prostaglandins on net uptake of 45Ca into the skeleton have been investigated in chicks and, in a limited parallel study, in immature rats.

Intravenous injection of bovine (b) parathyroid hormone(1–34) bPTH(1–34)) or 16,16-dimethyl prostaglandin E2 (16,16-dimethyl PGE2) in a 45Ca-labelled vehicle, combined with subsequent microwave fixation of tissue isotope levels, resulted in rapid (3–15 min) net inhibition of 45Ca uptake into endochondral bone (femur) in chicks (12 days old) and rats (4 weeks old). Use of 125I-labelled albumin and [14C]mannitol indicated that these responses were not a reflection of gross changes in tissue vascular or extracellular space. In rats, bPTH(1–84) also caused significant net inhibition of 45Ca uptake into femur at 10 min. Both bPTH(1–34) and 16,16-dimethyl PGE2 produced generally smaller decreases in 45Ca uptake into chick dermal bone (calvarium) at 3–15 min. In rat calvarium, however, these agents stimulated net uptake of 45Ca at these times. When microwave fixation was omitted, inhibitory responses were reduced or disappeared, while the stimulatory response in rat calvarium was enhanced. Responses to natural prostaglandins (PGE1, PGE2, PGF and PGI2) in chicks at 3 min were similar but less marked than those to 16,16-dimethyl PGE2; 45Ca uptake into femur and, to a lesser extent in calvarium, being inhibited. In rats, PGE1, PGE2 and PGF showed a tendency to decrease 45Ca uptake into femur while PGE1 and PGE2 both increased 45Ca uptake into calvarium.

J. Endocr. (1987) 115, 369–377

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A.J. Shaw
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C.G. Dacke
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ABSTRACT

Mechanisms of initial hypercalcaemic responses to parathyroid hormone (PTH) and 16, 16-dimethyl prostaglandin (PG) E2 have been investigated in 10-to 12-day-old chicks in vivo using a combination of acute 45Ca injection and microwave fixation to stabilize tissue isotope levels.

Single i.v. injection of 16, 16-dimethyl PGE2 (20 μg/100 g body wt) caused an approximately 100% increase in soft tissue 45Ca levels compared with vehicle control injected chicks at 30 min. 45Ca levels were lowered in calvarium by 26% and in femur by 60% with this treatment. Bovine PTH (1-34) (3.3 μg/100 g body wt) had no effect on soft tissue 45Ca levels, but in calvarium it had a similar effect to the PG. In femur this dose of PTH lowered 45Ca by 19%. When expressed on an absolute basis (c.p.m./ 100 mg tissue wt), responses to the PG in soft tissue were only 3 and 10% respectively of those in femur and calvarium.

The duration of inhibitory responses in bone were examined and those to PTH found to be transient (< 45 min) compared with the responses to the PG (> 135 min). Dose-response curves for PTH- and PG-induced inhibition of 45Ca uptake into femur at 15 min were essentially parallel and indicated that the lowest doses of PTH and PG used (0.74 μg and 1.1 μg/100 body wt respectively) produced significant responses.

In a separate experiment it was found that inhibition of 45Ca uptake into femur was evident as early as 3 min following PTH or PG injection.

The experiments described in this paper collectively indicate a higherto unrecognized action of PTH and PGE in the regulation of Ca metabolism in chicks which is to inhibit entry of Ca into bone.

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A. J. Shaw
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M. Z. Mughal
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M. J. A. Maresh
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C. P. Sibley
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ABSTRACT

Two human parathyroid hormone-related protein (hPTHrP) fragments were tested for effects on maternofetal transfer of 45Ca and Mg across the in-situ perfused rat placenta at 21 days of gestation (term = 23 days). The fetal placental circulation was perfused with a Mg-free Krebs–Ringer solution and the unidirectional maternofetal clearance (K mf) of 45Ca and Mg compared with that of 51Cr-EDTA, the latter being employed as a paracellular diffusional marker. Placental perfusion with hPTHrP(1–34) (100 ng/ml) or hPTHrP(75–86)amide (50 ng/ml) did not significantly alter the K mf of 45Ca or that of Mg. In separate rats, however, hPTHrP(1–34) but not hPTHrP(75–86)amide stimulated marked placental cyclic AMP (cAMP) release, the peak response of 63±7 pmol/min occurring 10 min after the beginning of the peptide perfusion. A lower dose of hPTHrP(1–34) (4 ng/ml) produced a similar peak release of cAMP, as did [Nle8,21,Tyr34]-rPTH(1–34)amide (4 ng/ml) and the adenylate cyclase agonist forskolin (17 μmol/l). Forskolin also rapidly increased the K mf of 45Ca but not that of Mg or 51Cr-EDTA. The present study indicates that hPTHrP does not acutely affect maternofetal transfer of Ca or Mg across the perfused rat placenta. The data also question the role played by cAMP in the stimulatory actions of forskolin on placental Ca transport.

Journal of Endocrinology (1991) 129, 399–404

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C. H. GRAY
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J. M. GREENAWAY
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N. J. HOLNESS
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D. A. SHAW
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SUMMARY

The metabolism of [4-14C]cortisol in a patient with Cushing's syndrome has been studied by the isolation, identification and measurement of the specific radioactivities of the major metabolites.

The results show that the metabolism of cortisol was not abnormal in the aspects studied. The biological half-lives of cortisol and of the tetrahydrocorticosteroid metabolites were found to be normal. Data obtained on excretion rates of metabolites indicated that the metabolic pathways of cortisol were normal. There was no evidence for an increased conversion of cortisol to 6β-hydroxycortisol when the excretion of the latter was expressed as a fraction of the cortisol production.

The overall pattern was one of an abnormally high secretion of cortisol by the adrenals, resulting in a proportionally high excretion of tetrahydrocortisone, tetrahydrocortisols, cortolones, cortols, 11-oxygenated 17-oxosteroids, 6β-hydroxycortisol, cortisone and cortisol. Apart from an increased ratio of 11β-hydroxy-metabolites to 11-oxo-metabolites, each metabolite, expressed as a fraction of the cortisol secreted, was excreted in a normal proportion.

Hence, in spite of the grossly elevated cortisol secretion rate, the major pathways available for cortisol metabolism were not overloaded and there was no evidence of increased metabolism via minor pathways.

Evidence for an increased secretion of corticosterone by the adrenals was obtained by the isolation of abnormal amounts of tetrahydrocorticosterone.

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A Alidibbiat
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C E Marriott Diabetes Research Group, School of Pharmacy and Biomolecular Sciences, Department of Diabetes, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, UK

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K T Scougall
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S C Campbell
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G C Huang Diabetes Research Group, School of Pharmacy and Biomolecular Sciences, Department of Diabetes, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, UK

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W M Macfarlane Diabetes Research Group, School of Pharmacy and Biomolecular Sciences, Department of Diabetes, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, UK

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J A M Shaw
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Generation of new β-cells from the adult pancreas or the embryonic stem cells is being pursued by research groups worldwide. Success will be dependent on confirmation of true β-cell phenotype evidenced by capacity to process and store proinsulin. The aim of these studies was to robustly determine endocrine characteristics of the AR42J rat pancreatic acinar cell line before and after in vitro transdifferentiation. β-cell phenotypic marker expression was characterised by RT-PCR, immunostaining, western blotting, ELISA and in human preproinsulin transgene over-expression studies in wild-type AR42J cells and after culture on Matrigel basement membrane matrix with and without growth/differentiation factor supplementation. Pancreatic duodenal homeobox 1 (PDX1), forkhead box transcription factor a2 (Foxa2), glucokinase, pancreatic polypeptide and low-level insulin gene transcription in wild-type AR42J cells were confirmed by RT-PCR. Culture on Matrigel-coated plates and supplementation of medium with glucagon-like peptide 1 induced expression of the β-cell Glut 2 with maintained expression of insulin and PDX1. Increased biosynthesis and secretion of proinsulin were confirmed by immunocytochemical staining and sensitive ELISA. Absence of the regulated secretory pathway was demonstrated by undetectable prohormone convertase expression. In addition, inability to process and store endogenous proinsulin or human proinsulin translated from a constitutively over-expressed preproinsulin transgene was confirmed. The importance of robust phenotypic characterisation at the protein level in attempted β-cell transdifferentiation studies has been confirmed. Rodent and human sensitive/specific differential proinsulin/insulin ELISA in combination with human preproinsulin over-expression enables detailed elucidatation of core endocrine functions of proinsulin processing and storage in putative new β-cells.

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C. N. A. TROTMAN
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I. J. S. FIDDES
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E. R. GRUND
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S. McHANWELL
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D. J. SANDERS
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CATHERINE SANDERSON
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B. SHAW
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SUMMARY

Immunoreactive gastrin was measured in subcellular fractions of rat gastric mucosa. The sedimentational properties of subcellular gastrin-containing structures were distinct from those of mitochondria. After centrifugation in sucrose density gradients using a zonal rotor, the peak of immunoreactive gastrin was found in 1·17–1·18 g cm−3 density sucrose (1·35 m; 39·5%, w/w). A thermolabile component with 125I-labelled gastrin-binding activity present in gastric mucosal homogenates and fractions was not associated with the gastrin storage vesicles sedimenting in density gradients.

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Eileen I Chang Department of Pediatrics, University of Colorado School of Medicine, Perinatal Research Center, Aurora, Colorado, USA

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Paul J Rozance Department of Pediatrics, University of Colorado School of Medicine, Perinatal Research Center, Aurora, Colorado, USA

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Stephanie R Wesolowski Department of Pediatrics, University of Colorado School of Medicine, Perinatal Research Center, Aurora, Colorado, USA

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Leanna M Nguyen Department of Pediatrics, University of Colorado School of Medicine, Perinatal Research Center, Aurora, Colorado, USA

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Steven C Shaw Department of Pediatrics, University of Colorado School of Medicine, Perinatal Research Center, Aurora, Colorado, USA

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Robert A Sclafani Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado, USA

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Kristen K Bjorkman Department of Molecular, Cellular and Developmental Biology and BioFrontiers Institute, University of Colorado Boulder, Boulder, Colorado, USA

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Angela K Peter Department of Molecular, Cellular and Developmental Biology and BioFrontiers Institute, University of Colorado Boulder, Boulder, Colorado, USA

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William W Hay Jr Department of Pediatrics, University of Colorado School of Medicine, Perinatal Research Center, Aurora, Colorado, USA

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Laura D Brown Department of Pediatrics, University of Colorado School of Medicine, Perinatal Research Center, Aurora, Colorado, USA

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Intrauterine growth-restricted (IUGR) fetuses are born with reduced skeletal muscle mass. We hypothesized that reduced rates of myogenesis would contribute to fewer and smaller myofibers in IUGR fetal hindlimb muscle compared to the normally growing fetus. We tested this hypothesis in IUGR fetal sheep with progressive placental insufficiency produced by exposing pregnant ewes to elevated ambient temperatures from 38 to 116 days gestation (dGA; term = 147 dGA). Surgically catheterized control (CON, n = 8) and IUGR (n = 13) fetal sheep were injected with intravenous 5-bromo-2′-deoxyuridine (BrdU) prior to muscle collection (134 dGA). Rates of myogenesis, defined as the combined processes of myoblast proliferation, differentiation, and fusion into myofibers, were determined in biceps femoris (BF), tibialis anterior (TA), and flexor digitorum superficialis (FDS) muscles. Total myofiber number was determined for the entire cross-section of the FDS muscle. In IUGR fetuses, the number of BrdU+ myonuclei per myofiber cross-section was lower in BF, TA, and FDS (P < 0.05), total myonuclear number per myofiber cross-section was lower in BF and FDS (P < 0.05), and total myofiber number was lower in FDS (P < 0.005) compared to CON. mRNA expression levels of cyclins, cyclin-dependent protein kinases, and myogenic regulatory factors were lower (P < 0.05), and inhibitors of the cell cycle were higher (P < 0.05) in IUGR BF compared to CON. Markers of apoptosis were not different in IUGR BF muscle. These results show that in IUGR fetuses, reduced rates of myogenesis produce fewer numbers of myonuclei, which may limit hypertrophic myofiber growth. Fewer myofibers of smaller size contribute to smaller muscle mass in the IUGR fetus.

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