Abstracts from the Summer Meeting, July 1993 239

stimulatory and direct inhibitory (1) (107-111) effects on osteoclasts in r&o and stimulates adenylate cyclase activity in osteoblasts. In this study the expression and production of PTHrP by osteoblasts derived from human trabecular bone has been investigated. mRNA was extracted from the cells and reverse transcriptaslinked polymerase chain reaction (RT-PCR) used to identify PTHrP transcripts. PTHrP PCR products were detected in 13 of 17 samples. In addition immunoreactive PTHrP, secreted into osteoblast conditioned medium, was measured using an IRMA (Nichols Inst.) and was detected in 9 of the 16 samples assayed. The highest levels of mRNA and immunoreactive protein were observed in children (~4 yrs.) and in elderly patients. Patients in t‘he range 9-44 ym. expressed lower levels df immunoreactive orotein and PTHrP mRNA. The effects of the glucocorticoid p;ednisolone on PTHrP secretion by human osteoblasts was also investigated. Secretion of immunoreactive PTHrP was suppressed by 1OOOpM doses of prednisolone. Southern blot analysis of PCR products showed a decrease in lTHrP mRNA in these cultures. Our data indicate that F’l’HrP expression by human osteoblasts may be important in regulation of bone remodelling and in glucocorticoid-induced osteoporosis. (1) Fenton AJ et al, Endocrinol 1291762.

P37. Molecular proceaaing of PTH-related protein(pTHRp) by a pancreatic endocrine tumour WA Rat&&, SJ Bowden, F Dunne’, J Emly, JK Pye**, JT Baker+, CP Williams++ Wolfson Research L&oratories and l Department of Medicine, Queen Elizabeth Medical Centre, Birmingham and **Departments of Surgery, +Medicine and ++Pathology, Wre.vham Mnelor Hospital

Surgical resection of a pancreatic tumour in a 39 year old female with hypercalcaemia (corr calcium 4.17 mmol/l) and elevated plasma PTHRPl-86 (13 pmol/l) resulted in normalisation of serum calcium, although PTHRP remained elevated (0.3-1.4 pmol/l). Histological examination showed a tumour of endocrine cell type and immunohistochemistry for insulin, glucagon, somatostatin, pancreatic polypeptide, and gastrin were negative. Both PTHRP mRNA and PTHRP peptide were identified in tumour tissue by in situ hybridisation and immunohistochemistry respectively. PTHRP quantitated in an extract of the tumour using three region-specific immunoassays (PTHRPl-86 12.0 pmol/g; PTHRPl-34 27.6 pmol/g; PTHRP37-67 42.5 pmol/g] indicated a molar excess of amino-terminal and mid-region- immunsoreactivity. On chromatography of the extract on Bionel 1’100 3 peaks of immunoreactivity were detected: a spe~&~ of 22 kDa*contained equimolar amou& of l- 86, 1-34 and 37-67 activity; a major species of 16 kDa contained only 37-67 activity; and a species of 6 kDa contained only 1-34 activity. Recombinant PTHRPI-141 behaves as 22-25 kDa on gel chromatography. We conclude that the tumour contained native or intact PIXIRP together with two major subfragments containing l-34 and 37-67 immunoreactivity respectively, consistent with proteolytic processing at a site between the epitopes of the antibodies, ie residues 17-27 and 50-61.

P38. Stimulation of secretion of calcitonin by inrulin AD Care, C. Strimbu, L Gozariu, SK Abbas, CW Cooper Uniuersity of Wales, Aberystruyth, Wales, UK; University of Medician and Pharmacy, Cluj, Romania; University of Texas Medical Branch, Galveston, USA

This shidy was designed to determine whether insulin stimulates secretion of calcitonin (a) from the thyroid gland. The rationale was that the osteopenia observed in some patients with Type 1 diabetes mellitus might result from a fall in calcitonin secretion associated with a reduction in circulating insulin. In a preliminary experiment in rats made diabetic with

streptozotocin, Cl’ was measured (human CT RIA) in jugular vein blood close to the thyroid, and plasma CT concentration was lower in untreated diabetic rather than in diabetic rats treated with insulin to normalize blood glucose. To examine further the potential effect of insulin on ff release, we studied 5 young pigs (20~30kg b.w.) in which the thyroid was surgically isolated and perfused in situ at a fixed flow rate with autologous blood. Thyroid venous blood was completely collected, and CT in the throid venous plasma was measured by an homologous RIA for porcine CT. Porcine insulin, added to the perfusing blood at concentrations ranging from 1.2 - 427 r&ml, caused a 15 - 1350% increase in the secretion rate of CT - ie secretion rose from 0.6 - 5.4 ng/min during the control perfusion period to 2.5 - 45 ng/min during perfusion with insulin. Plasma Ca2+ remained normal throughout each experiwnt except at the end when it was deliberately elevated. In each case, hypercalcaemia stimulated CT release markedly. These studies show that insulin can act directly on the thyroid gland to enhance CT release. The findings imply that insulin may play a role in CT secretion.

P39. The effects of pcptides of the calcitonin-gene family on human tibmblast colony fonning cella (CFU-Fs) PA Davies, BO Oyajobi, ROC Oreffo*, D Johnstone*, RGG Russell Department of Human Metabolism and Clinical Biochemistry, Uniwrsity of Sheffield Medical School,, Sheffield SIO 2RX and ‘Zeneca Pharmaceuticals, VIM ResearchDepartment, Mereside, Alder@ Park, Cheshire, SK20 4TG

Calcitonin, calcitonin gene-related peptide (CGRP) and amylin are members of a related gene family and all inhibit osteoclast- mediated bone resorption. Calcitonin and CGRP also have effects on signal transduction and cellular mechanisms in osteoblastic cells. We analysed the effects of these peptides on the proliferation, differentiation and signal,transduction in cultures of human CFW-Fs. Marrow cells were plated at 2 x 10s cells/cm2 maintained in MEM + IOIFCS. After seven days cultures were fixed, colony numbers counted and alkaline phosphatase (ALP) expression determined. Signal transduction effects were examined in terms of adenylate cyclase activity. Human calcitonin (1.4 x 102~6M increased colony formation 132% f 4.3% of control (~~0.05). Both human CGRP and amylin also increased colony formation at 1.3 x lO-7M and 1.3 x 10-6M, with maximal increases of 149% f 9.9% (~~0.05) and 141% f 4.9% (p&01) of control, at 1.3 x IO-6M CGRP and amylin respectively. This stimulation was blocked in the presence of a calcitonin antagonist, calcitoningg2 (1.8 x l&SM). The percentage of ALP positive colonies remained unchanged in the presence of anv of the anents examined. Of the three altents. onlv CGRP (1.3 x 1017M) inckased CAMP production (56% i 65b of control, ETOOl&;)d this was blocked by a CGRP antagonist, CGRPa37,

. x This suggests the possible presence of both CGRP and calcitonin receptors on human CFU-Fs. However the high concentration of peptides required to produce these effects has to be considered.

P40. PDGF l xplrasion in normally healing human frachtrea JG Andrew, JA Hoyland*, AJ Freemonr, DR Marsh Departments of Orthopaedics and ‘Pathological Sciences, Munchester University Medical School, Manchester, Ml3 9PT

Platelet derived growth factor (PDGF) has potent effects on connective tissue cells and may be important in soft tissue and bone repair. We Performed an inununohistochemical and in situ hybrldisation study of PDGF expression in normally healing h-n fractures. Biopy specimens from 16 fractures were examined. PDGF A