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_2α 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_2α 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

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.

SUMMARY

The major metabolic pathways of cortisol have been studied in detail after injection of a tracer dose of [4-¹⁴C]cortisol in two patients with rheumatoid arthritis and in one with disseminated lupus erythematosus. Results are also presented from preliminary experiments in two other patients with rheumatoid arthritis.

The rates of cortisol secretion by the adrenals of all the patients studied were within the normal range and the kinetics of cortisol metabolism were also normal. No abnormalities were found in either the proportions of cortisol, cortisone and 6β-hydroxycortisol in the urinary fraction containing the unconjugated steroids or in that of tetrahydrocortisol, allo-tetrahydrocortisol, tetrahydrocortisone, cortols, cortolones, 11β-hydroxyaetiocholanolone, 11β-hydroxyandrosterone and 11-oxoaetiocholanolone in the fraction released by hydrolysis of the urinary steroid conjugates.

The excretion of 6β-hydroxycortisol in a group of patients with rheumatoid arthritis was not significantly greater than that in a similar group of normal subjects in whom the upper limit of the range was higher than values previously reported.

SUMMARY

The major metabolic pathways of cortisol in a patient with sarcoidosis have been studied after intravenous injection of a tracer dose of [4-¹⁴C]cortisol. Results from some preliminary experiments in four other patients are also presented. The effects of large doses of cortisol on its metabolism were also studied.

In all the patients studied the adrenal secretion rate of cortisol was in the low normal range. The rate of disappearance of radioactivity from the plasma fractions containing the unconjugated steroids was normal before cortisol therapy but slower than normal during therapy. During therapy there was a significant decrease in the total urinary radioactivity.

In the one patient in whom detailed studies were made, an unusually large proportion of the urinary metabolites was in the unconjugated fraction in which the amount of 6β-hydroxycortisol was greater than normal. Apart from an increased ratio of 11β-hydroxy- to their corresponding 11-oxo-metabolites there was no essential difference in the pattern of excretion of the metabolites in this patient during therapy. In this patient only 65% of the injected radioactivity was excreted in urine after 48 hr., whereas the other patients excreted a normal percentage of the injected radioactivity during this period.

There is no consensus between Authors on the definition of a replete or deficient vitamin D state. Our aim was to describe a suitable method that could be used to compare vitamin D data in subject groups with small or large numbers. Two hundred and forty indigenous asymptomatic, non-pregnant adult subjects recruited from a single-consultation outpatient attendance with normal biochemistry, represented a sample of our inner city district population. 25-hydroxyvitamin D (25,OHD3) levels were measured to illustrate the effects of season, sex and ethnic group on vitamin D levels and subjected to distribution analysis. This method quantifies as a percentage the distribution of 25,OHD3 concentrations (observed concentration, OC) in pooled group data. The data can be expressed as distribution frequency domains or cumulative frequency ogives (0-100%) or transformed into discrete linear probits, amenable to regression analysis. An estimate of the OC50 (mid-point) and upper (either OC75 or OC95) or lower (either OC25 or OC5) range or at any other frequency between subject groups can be compared. A marked difference in 25,OHD3 levels between Asian and non-Asian asymptomatic adult subjects was seen during both seasons. 25,OHD3 deficiency was defined as at or below the OC25 for the non-Asian group (for both sexes: winter < 13.36 ng/ml, summer <13.38 ng/ml). The majority of Asians of both sexes were 25,OHD3 deficient (winter 94%, summer 82%). The distribution analysis provides an easy technique to compare 25,OHD3 status of different subject groups, allowing the description of populations using either longitudinal or cross-sectional data. This method may offer a way of describing 25,OHD3 deficiency between observers worldwide.

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 [Nle^8,21,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

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.

Few studies have comprehensively examined amphibian granular gland secretions for novel insulinotropic peptides. This study involved isolation and characterisation of biologically active peptides from the skin secretions of Rana palustris frogs. Crude secretions obtained by mild electrical stimulation from the dorsal skin surface were purified by reversed-phase HPLC on a semipreparative Vydac C18 column, yielding 80 fractions. These fractions were assayed for insulin-releasing activity using glucose-responsive BRIN-BD11 cells. Acute 20 min incubations were performed in Krebs Ringer bicarbonate buffer supplemented with 5.6 mmol/l glucose in the absence (control) and presence of various fractions. Fractions 29-54 and fractions 68-75 showed significant 2.0-6.5-fold increases in insulin-releasing activity (P<0.001). The fractions showing most prominent insulinotropic activity were further purified to single homogeneous peaks, which, on testing, evoked 1.5-2.8-fold increases in insulin release (P<0.001). The structures of the purified peptides were determined by mass spectrometry and N-terminal amino acid sequencing. Electrospray ionisation ion-trap mass spectrometry analysis revealed molecular masses of 2873.5-8560.4 Da. Sufficient material was isolated to determine the primary amino acid sequence of the 2873.5 Da peptide, revealing a 27 amino acid sequence, ALSILRGLEKLAKMGIALTNCKATKKC, repressing palustrin-1c. The database search for this peptide showed a 48% homology with brevinin-1, an antimicrobial peptide isolated from various Rana species, which itself stimulated insulin release from BRIN-BD11 cells in a concentration-dependent manner. In conclusion, the skin secretions of R. palustris frogs contain a novel class of peptides with insulin-releasing activity that merit further investigation.

Skin secretions of Rana saharica were evaluated for the isolation and characterisation of novel insulinotropic peptides. Crude secretions obtained from young adult frogs by mild electrical stimulation of the dorsal skin surface were purified by reverse phase HPLC yielding 80 fractions. In acute 20-min incubations with glucose responsive BRIN-BD11 cells, fractions 36–43, 46–54 and 57–63 significantly stimulated insulin release by 2- to 8-fold compared with 5.6 mM glucose alone. Pooled fractions in the latter two bands were rechromatographed to reveal 9 homogenous peaks, which elicited significant 1.3- to 3.5-fold increases in insulin release (P < 0.05). Structural analysis of the most potent non-toxic peptides was performed by mass spectrometry and automated Edman degradation. This revealed four major insulin-releasing peaks with molecular masses of 2676.9 Da, 3519.3 Da, 4920.4 Da and 4801.2 Da respectively. These peptides were found to be identical to brevinin-1E, brevinin-2EC, esculentin-1 and esculentin-1B, which belong to the group of antimicrobial peptides isolated from skin secretions of various Rana frog species. Preliminary studies on the mechanism underlying the insulinotropic actions of esculentins-1 and -1B suggested possible involvement of both cyclic AMP–protein kinase A and –C-dependent G-protein sensitive pathways. These data indicate that the skin secretions of Rana saharica frogs contain bioactive molecules with significant insulin-releasing activity. Relatives of the brevinin/esculentin peptide family merit further investigation as novel insulin secretagogues.

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.