Clinical and Experimental Pharmucology and Physiology zyxwvuts (1998) 25,750-752 Proceedings of the Satellite Symposium for the Australian Physiological and Pharmacological Society: The Evolution of Physiological Processes zy PARATHYROID HORMONE-RELATED PROTEIN IN LOWER VERTEBRATES JA Danks," MK Trivett," DM Power: AVM Canario: T J Martin" and PM Ingletod *St Vincent's Institute zyxwvuts of Medical Research, St Vincent zyxwv S Hospital, Fitzroy, Victoria, Australia, tUniversidade do Algawe, Campus de Gambelas, Faro, Portugal and 'Institute of Endocrinology, University of Shefield Medical School, Shefield, zyxw UK SUMMARY 1. Parathyroid hormone-related protein (PTHrP) is an im- portant mediator of humoral hypercalcaemiaof malignancy in humans. Normal human subjects have very low levels of PTHrP in their circulation. 2. Parathyroid hormone-related protein has recently been demonstratedin high levels in the circulation and tissues of the sea bream and the dogfish, leading to the hypothesisthat PTHrP may be a 'classical' hormone in fish. 3. Immunohistochemistry and in zyxwvutsr situ hybridization were per- formed to investigate the evolutionary history of PTHrP. Tissues were examined from a number of lower vertebrates, including lungfish, lamprey and several species of bony and cartilaginous fish. Parathyroid hormone-relatedprotein was localized to the skin and to kidney tubules in all animals studied. In the develop- ing lungfish, PTHrP was observed in the notochord, developing brain and skeletal muscle layers. These results suggest that PTHrP is of ancient origin and has a basic and fundamental function in vertebrates. Key words: calcium regulation, comparative endocrinology, osmoregulation,parathyroid hormone-relatedprotein. INTRODUCTION Since Albright's description of humoral hypercalcaemia of malig- nancy (HHM) in 1941,' this condition was thought to be due to ectopic secretion of parathyroid hormone (PTH) because the bio- chemical features of HHM are similar to hyperparathyroidism.' However, increased levels of circulating PTH in humans could not be found unequivocally and it was only the preparation of tumour extracts, with PTH-like bioactivity but lacking PTH immuno logical characteristics, that established the existence of a distinct factor. The factor was isolated and cloned from tumour tissues in zyxwvut ~~ ~~ ~~~~ ~ ~~~~ ~~ Correspondence: Janine A Danks, zyxwvutsrq St Vincent's Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia. Email: <j.danks @ medicine.unimelb.edu.auz Presented at the Australian Physiological and Pharmacological Society Satellite Symposium on the Evolution of Physiological Processes. Received 2 April 1998; revision 4 May 1998; accepted 11 May 1998. 1987.3-6 Analysis of the peptide showed homology with PTH only at the N terminus, where eight of the first 13 amino acids are identical. This limited sequence homology, together with structural homology in the 14-34 is sufficient to allow interactions at the PTH receptors, principally in bone and kidney, which lead to development of HHM9.'' and also explains the cross-reactivity with some antibodies to PTH. The factor was named parathyroid hormone-related protein (PTHrP) because of its limited homology with PTH (it has also been known as PTH-like peptide). We decided to examine lower vertebrates for the presence of PTHrP to study its evolutionary history and uncover the non- malignant roles of PTHrP. PARATHYROID HORMONE-RELATED PROTEIN AND TELEOSTS Fish are quite different from mammals in that they do not have a parathyroid gland and they are not likely to use their skeleton as a calcium reservoir. Some studies in the 1970s and 1980s suggested that PTH was present in the plasma and tissues of fish, but this re- mains uncertain." In 1993, PTHrP was identified in the tissues and circulation of a teleost (Sparus aurutu; sea bream)." Using a rabbit polyclonal antisera to human PTHrP( 1-14) and a peroxidase- anti-peroxidase method, PTHrP antigen was found in the pituitary'* as well as in kidney tubules, epidermis (J Danks, unpubl. obs., 1998) and saccus vas~ulosus.'~ Immunohistochemistry with a rabbit poly- clonal antiserum to human PTH(1-34) was performed on all sea bream tissues and there was no positive staining in any of the tissues." A section of human parathyroid gland was used as a posi- tive control in this assay. Western blot analysis of pituitary extracts showed two immunoreactive isoforms, one of 29 kDa and the other of 26 kDa. Single pituitaries were incubated for up to 14 days in Krebs'-Ringer bicarbonate and three isoforms of immunode- tectable PTHrP were released into the medium, two of them corre- sponding to the 26 and 29 kDa molecular forms, but there was an additional band at 24 kDa. Radioimmunoassay using antisera to human PTHrP( 1-40) found that the concentration of PTHrP in sea bream plasma was 12.4 pmolL, comparable with levels observed in human subjects with HHM. The circulating levels in normal human subjects is less than 2 pmolk. Circulating levels of PTHrP were initially determined on neat plasma from individual fish. The authenticity of circulating PTHrP was confirmed by the subsequent