IBMS/ECTS 2001 - PROGRAM and ABSTRACTS
ABSTRACTS
IIIrd INTERNATIONAL SYMPOSIUM ON
COMPARATIVE ENDOCRINOLOGY OF CALCIUM METABOLISM
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symposium program
SUBCELLULAR CA HOMEOSTASIS DURING TRANSEPITHELIAL CA FLUX ASSOCIATED WITH CRUSTACEAN MOLTING: WHAT ROLE DO SERCAS AND PMCAS PLAY?
M. Wheatly1, Y. Gao1, Z. Zhang1, D. Chen1, F. Zanotto2, M. Hubbard1
1Wright State University, Dayton, OH, USA
2 University of Sao Paulo, Brazil
The crustacean molting cycle is used as a model to study regulation of expression of Ca translocating proteins. Postmolt mineralization is associated with massive unidirectional influx of Ca across transporting epithelia (gills, kidney, liver) that is in stark contrast to baseline intermolt Ca balance. Calcium ATPases on plasma membranes (PMCAs) and endomembranes of the ER/SR (SERCAs) are involved in cellular Ca homeostasis that is challenged during increased epithelial flux. Work in our lab has focused on physiological and molecular characterization of PMCAs and SERCAs, and how their regulation may be coordinated during the stages of molting. Vesicles made from basolateral and SR membranes have been employed to study kinetics of PMCAs and SERCAs. Their relative roles in transporting epithelia have been compared. Two full length cDNAs for SERCA have been cloned and sequenced from axial abdominal and cardiac muscle. Expression decreased in pre- and postmolt compared with intermolt. Partial sequences of PMCA1, 2, 3 and 4 were successfully obtained from the following tissues: egg, gill, kidney, heart, liver and axial muscle. In gills PMCA expression increased in postmolt commensurate with massive unidirectional Ca influx. From preliminary data it would appear that SERCA and PMCA are inversely regulated. Understanding the interrelation between these pumps at different cellular locations will be key to comprehending subcellular homeostasis.
Funded by NSF grants IBN 9603723 and 00076035 to MGW
BIOLOGICAL EFFECT OF THE WATER-SOLUBLE MATRIX EXTRACT FROM THE NACRE OF THE BIVALVE MOLLUSC PINCTADA MAXIMA ON VERTEBRATE CELL PROLIFERATION AND DIFFERENTIATION.
L. Pereira, C. Milet, M. J. Almeida, M. Rousseau, F. Robichon, E. Lopez
Laboratoire de Physiologie GZnZrale et ComparZe. UMR CNRS 8572. MusZum National d'Histoire Naturelle, 7, Rue Cuvier, 75 231 PARIS, Cedex 05, France.
In vivo and in vitro studies point out that the nacre (mother-of-pearl) from the giant oyster Pinctada maxima possess in its organic matrix water-soluble molecules that exhibit osteogenic properties in Vertebrates. The molecules present in the nacre Water-Soluble Matrix (WSM) act on proliferation and differentiation of human fibroblastic cells (MRC-5) and rat osteoprogenitor cells from bone marrow. In both cellular populations, one of the first markers of osteoblastic differentiation, the Alkaline Phosphatase (ALP) activity, is significantly stimulated by the WSM. In order to confirm the osteogenic activity of these water-soluble molecules, we have analysed the production of ALP, collagen type I and II and osteocalcin mRNAs of cells exposed to WSM, using RT-PCR. Moreover we are investigating the Mitogen Activated Protein (MAP) kinase transduction pathways to understand the signaling mechanisms mediating the biological activity of nacre WSM. Recent information suggests distinct roles for 2 different MAP kinase pathways in regulating the activity of osteoblast-like cells: one involved in cell proliferation and the other in cell differentiation resulting in the stimulation of ALP activity. The potential role of the MAP kinase pathway in the biological activity of WSM will be presented using kinetic analysis with:
1) the specific SAPK2/p38 inhibitor SB 202190 and the ERK inhibitor PD 98059
2) immunoblotting with antibodies turned against the activated form of MAP kinases.
The implication in Mollusc biomineralization of the macromolecules present in the shell organic matrix is widely described, even if their characterization and function are still not well established. Nevertheless, new data on protein structure are now available in literature. The remarkable result presented here is that we demonstrate that nacre organic matrix (WSM) contain signal molecules which are able to stimulate the osteogenic pathway in Mammalian Vertebrates.
Supported by grant PRAXIS XXI/BD/20023/99, from the Science and Technology Foundation of Portugal
CALCIUM REGULATING FACTORS IN BONY AND CARTILAGINOUS FISH
J. A. Danks
St Vincent's Institute of Medical Research, Fitzroy, 3065, Australia
Numerically, fish constitute the largest group of vertebrates with estimates of total species varying from 25,000-35000. They are highly successful with evolved physiologies ensuring survival in water of high or low ionic strength and, in some species, with the ability to adapt to both conditions. Today there are two major groups, the jawed bony fish and the jawed cartilaginous fish, including sharks and rays. Originally, all fish were marine and would have been more dependent on hypocalcemic factors such as stanniocalcin and calcitonin. During evolution, as fish colonised the freshwater environment, hypercalcemic agents would have been essential for survival in water with lower calcium.
A hypercalcemic agent may have developed from a factor already in marine fish but fulfilling a different role. Parathyroid hormone-related hormone (PTHrP) could be one such factor. Over the last eight years we have demonstrated the presence of PTHrP in representatives from the three extant groups of fish, i.e. bony, cartilaginous and jawless fish. The PTHrP gene has been isolated and cloned from two bony fish (Spartus aurata and Fugu rubripes). As yet, parathyroid hormone (PTH) has not been isolated from fish and these animals lack an encapsulated parathyroid gland. The question arises as to whether this hormone is present in fish. Studies on the evolution of the vertebrate genome have provided indirect evidence for the presence of the PTH gene in fish. Three zebrafish PTH receptors have been isolated and cloned, but fish PTH that binds to these receptrors and is presumably homologous to the tetrapod PTH has not been identified. Until the PTH gene is isolated and cloned from fish PTHrP remains the only potential hypercalcemic PTH-like factor in fish.
THE ROLE OF SCALES IN CALCIUM METABOLISM Oreochromis mossambicus
G. Flik
Department of Animal Physiology, Faculty of Science, University of Nijmegen, Toernooiveld 1 6525 ED Nijmegen, The Netherlands
A tilapia weighing 20 g has concentrated around 6.5 mmol calcium in its body, of which around 1.5 mmol (23%) is associated with the scale compartment. The net uptake of calcium from the water via the gills is around 450 nmol per hour and this rate is similar to the growth-related Ca accumulation rate. In other words, waterborne calcium suffices as a source for growth and by its essentially infinite size would provide a buffer in times of extreme need for calcium. The question then rises whether fish have internal calcium compartments that act as buffers for calcium homeostasis. As scales in many fish form the calcium compartment with the highest variability in calcium content (and thus seem to behave as buffer compartments), the scale of tilapia was analysed for mineral content and ionic exchange in vivo and in vivo. For a 20 g fish , a single scale taken from the fish’s side contains 3.4 :mol Ca (content around 5 :mol/mg dry weight). The Ca/P ratio in scales ranges between 1.55 – 1.67, values indicative of (defective) apatites and whitlockites. In a normally growing tilapia, the growth-related net inflow of calcium into the scale is about 200 pmol per h. The unidirectional inflow of calcium in vitro in such a scale determined by 45Ca uptake at plasma [Ca2+] is 18 nmol per h and thus a considerable efflux and exchange of calcium must occur. Compartmental analysis shows that in a scale containing 3.5 :mol calcium, 51 nmol (1.5%) of the total is readily exchangeable. Experiments with 32P show that the scale has a similarly sized readily exchangeable phosphate pool and that the Ca/P-ratio of this pool is 1, indicative of brushite. When scales are removed from the skin experimentally, news scales are formed and these regenerating scales have Ca/P ratios of 1 after 4 days and 1.53 after 10 days. We conclude that the scale compartment represents a very active and significant internal buffer compartment for calcium and phosphorus in fish.
THE IMPORTANCE OF CALCIUM AND CALCIOTROPIC HORMONES IN THE AVIAN SKELETON
C. Farquharson
Bone Biology Group, Roslin Institute, Roslin, Midlothian EH25 9PS, Scotland
The majority of calcium (Ca) in birds is present within their bones. Together with phosphorous (P) and other minerals it forms hydroxyapatite crystals and thereby provides structural support to the skeleton. Most of the remaining Ca (1%) is cellular and acts as a secondary messenger to regulate a multitude of essential biological processes. Under normal conditions there is a stable balance between the amount of Ca deposited within bones and the amount released by osteoclastic resorption into the birds circulation. However, under conditions in which Ca homeostasis is compromised e.g. nutritional hypocalcaemia, eggshell formation etc, there is increased bone resorption and a net loss of skeletal Ca to the circulation.
This review will detail our work and interest in the role of both Ca and calciotropic hormones in the maintenance and development of the avian skeleton. Their relevance to important avian skeletal disorders such as tibial dyschondroplasia and osteoporosis will also be discussed. In brief:
The Ca and P requirement for good bone health in the modern meat-type chicken is unclear. A number of bone related parameters including plasma1,25(OH)2D3, Ca2+, Pi, and bone turnover markers together with bone mineral contents and mechanical properties have been studied to identify the optimum dietary Ca and P contents for maximum skeletal health.
Tibial dyschondroplasia results from a defect in chondrocyte maturation. Numerous in vivo and in vitro studies have been completed to investigate the mechanisms by which its development and severity in thechicken is influenced by dietary Ca and 1,25(OH)2D3 levels. Results will also be presented on the impact of nutrition, such as the provision of particulate calcium in the diet, on bone quality and the severity of osteoporosis in the laying hen.
Calciotropic hormones such as vitamin D metabolites, PTH and PTHrP acting through their respective growth plate chondrocyte receptors strongly influence the rate of longitudinal bone growth. Using novel techniques we have been able to further dissect the role and interactions of both 1,25(OH)2D3 and PTHrP in pacing the rate of chondrocyte differentiation in the post-embryonic avian growth plate.
BIOCHEMICAL AND CELLULAR ASPECTS OF OSTEOPOROSIS IN LAYING HENS SELECTED FOR DIVERGENT SKELETAL CHARACTERISTICS
R. H. Fleming, H. A. McCormack, C. C. Whitehead
Roslin Institute, Edinburgh, Scotland, UK
Osteoporosis in humans and hens alike involves nutritional, environmental and genetic mechanisms, although increased oestrogen in hens appears responsible for bone loss, in contrast to humans. At sexual maturity in the hen, raised circulating oestrogen triggers woven medullary bone (MB) formation. Lamellar bone apposition ceases during the 50 week laying period whilst osteoclasts vigorously resorb MB for eggshell calcification and also remove structural bone, causing osteoporosis and fractures.Avian osteoporosis has a strong genetic component (Bishop et al, 2000). A restricted selection index applied to six generations of hens (G1-G6) has created two distinct lines (high and low bone index [BI]) with different fracture incidences.
These lines were used to examine possible divergent expression in biochemical markers of bone metabolism. In G4 birds, plasma carboxylated osteocalcin (Carb.Oc.) levels were determined at growth (6 weeks), pre-lay (15 weeks) and end-of-lay (70 weeks). In G6 birds, measurements were made at 70 weeks of tibiotarsal breaking strength, (TSTR) plasma oestradiol and serum pyridinoline (serum Pyd). Transverse tibiotarsal sections were reacted for tartrate resistant acid phosphatase (TRAP) to identify active osteoclasts. TRAP+ve osteoclasts were expressed as a proportion of MB area, (TRAP+veOc./MB).
At 6 weeks mean Carb. Oc. increased in G4 high BI hens (285ng/ml) compared to low BI hens (230ng/ml), (P=0.10). This slight elevation may reflect differences in structural bone formation between the lines. Carb. Oc. levels decreased in both lines at 15 weeks (high BI = 145ng/ml, low BI = 153ng/ml) to very low levels (both <10ng/ml) at 70 weeks, even though MB turnover continues during lay. This implies that non-collagenous proteins may be unimportant in relatively transient MB.
At 70 weeks G6 high BI hens had TSTR values 37% greater than low BI hens (P<0.001). Oestradiol levels were 467pg/ml for high BI and 438pg/ml for low BI hens, (P=0.09). Serum Pyd levels were significantly raised (P<0.05) in low BI hens (0.46pmol/microl.) compared to high BI hens (0.39pmol/microl.) suggesting increased resorption in low BI birds. There were more TRAP+veOc/MB in low BI hens (1229±73/mm2MB) compared to high BI hens (1063±101/mm2MB, P=0.10). These biochemical and cellular changes are consistent with the hypothesis that low BI hens have poorer bone formation during growth, and increased bone resorption during the laying period.
Reference: BISHOP, S.C., et al, (2000) British Poultry Science, 41:33-40.
A SEARCH FOR GENES INVOLVED IN AVIAN EGGSHELL CALCIFICATION: IDENTIFICATION, LOCALIZATION AND REGULATION
I. Lavelin, N. Meiri, M. Pines
Institute of Animal Science, The Volcani Center
Avian eggshell formation represents a unique biological system in which the calcification process takes place in a circadian fashion. The eggshell is formed during the passage of the egg through the oviduct in which the different layers are assembled sequentially. Approximately 5g of calcium are deposited into the shell during the 20h that the egg resides within the eggshell gland (ESG), which makes eggshell formation one of the most rapid biomineralization processes known. It is widely accepted that various proteins play a major role in the control of calcification. Some of them have been identified by protein extraction from shells and others by searching for proteins known to be involved in bone calcification.
We used RNA fingerprinting to search for genes involved in eggshell formation. ESG gene expression was compared at two physiological stages: A- no egg in the ESG, no calcification occurring; B- egg residing the ESG, at the peak of calcification. We isolated and sequenced 14 cDNAs that were induced during calcification. Some were known chicken genes (e.g. Na-K-ATPase), some exhibited high homology to mammalian genes (e.g. glypican-4), and others were new, unknown genes.
All studied genes were expressed in a circadian manner. Expression was initiated only after the egg entered the ESG; it peaked during the period of rapid shell formation and decreased before oviposition, at the phase of eggshell completion. The site of expression differed for different genes: the osteopontin gene was expressed in the pseudostratified epithelium (PE) facing the lumen, whereas calbindin and glypican-4 were expressed in the glandular epithelium (GE). The Na-K-ATPase gene was expressed by both epithelial types but with different time courses. The genes expressed in the PE were regulated by mechanical stress, whereas those expressed in the GE were regulated by calcium flux. Looking for endocrine regulation, we found that the PTH/PTHrP receptor gene is also expressed in the ESG in a circadian fashion. These results, together with evidence that PTHrP is synthesized in the isthmus, raise the possibility of local PTHrP regulation of genes involved in the eggshell calcification process.
LOCALISATION AND SEMIQUANTITATIVE ANALYSIS OF ESROGEN RECEPTOR-ALPHA AND BETA mRNAs IN AVIAN MEDULLARY BONE
T. Sugiyama1, E. Minagawa2, T. Imamura1, S. Kusuhara1
1Department of Animal Science, Nigata University, Niigata 950-2181, Japan
2Graduate School of Science Technology, Niigata University, Niigata 950-2181, Japan
Medullary bone is specific to female birds and plays an important role as a calcium reservoir for egg-shell formation. On the surface of medullary bone, osteoblasts and osteoclasts alternate the bone formation and resorption in relation to the position of the egg in the oviduct. Early formation of this medullary bone is known to be induced by estrogen. In fact, medullary bone is easily formed by estrogen treatment and is reduced by estrogen antagonist such as tamoxifen. These effects of estrogen are generally supposed to be mediated by classical estrogen receptor (estrogen receptor-alpha). However, a novel estrogen receptor subtype, estrogen receptor-beta, has been recently cloned, and the presence, and role of, this novel estrogen receptor in medullary bone is still unknown. Accordingly, we observed the expression of estrogen receptor-alpha and -beta in the hen medullary bone by semiquantitative RT-PCR and in situ hybridization. The total cellular RNAs were prepared from hen medullary bone developed in bone marrow cavities of femurs with RNAzol B, and then the expression of estrogen receptor-alpha and -beta was detected with semiquantitative RT-PCR. Secondly, the localization of the estrogen receptors in medullary bone was observed with in situ hybridization in which the digoxigenine (DIG)-labeled RNA probes were prepared with pGEM-T Easy Vector system (Promega) and DIG RNA labeling kit (Boerhringer Mannheim). As a result, the expression of estrogen receptor-alpha mRNA was higher than that of estrogen receptor-beta. Also, estrogen receptor-alpha mRNA was intensively expressed in osteoblasts on the medullary bone surface. These results suggest that estrogen receptor-alpha plays an important role for medullary bone formation. Taken together from the present study and the previous reports that estrogen receptor-beta is expressed in cortical bone-derived osteogenic cells at high levels similar to estrogen receptor-alpha, it is possible that these differential expression of estrogen receptor-alpha and -beta might determine specific characters of avian medullary bone such as the acid mucopolysaccharide-rich matrix, the rapid bone formation and resorption.
PLASMA MEMBRANE CALCIUM ATPASE, SODIUM CALCIUM EXCHANGER AND PTHrP RECEPTOR IN OSTEOBLASTS FROM YOUNG AND AGED RAT LONG BONE.
C. V. Gay, Y. Zeng, A. M. Mastro
The Pennsylvania State University, University Park, PA, USA
Plasma membrane Ca-ATPase (PMCA) and sodium-calcium exchanger (NCX) are responsible for Ca++ efflux; the former is ubiquitous, the latter is present mainly in excitable cells and osteoblasts. The discrete and opposing locations of these two transmembrane proteins in osteoblasts suggest distinct roles. PMCA is present on the marrow-facing side of osteoblasts, whereas NCX is on the matrix secreting side of the cell. Blocking the exchanger results in diminished delivery of Ca++ to sites of mineralization of matrix in an in vitro model. Since PMCA in osteoblasts is confined to the signalling surface of the cells it is likely responsible for restoring intracellular [Ca++] to basal levels following hormone stimulation. We compared expression of PMCA and NCX at both mRNA and protein levels in osteoblasts isolated from the periosteum of 4 mo and 15 mo old rat long bone. PMCA mRNA expression did not change with donor age relative to 18 S RNA, nor did PMCA protein expression relative to actin, indicating that this aspect of Ca++ signalling remains intact as cell donors aged. For NCX, both mRNA expression (relative to 18 S) and protein (relative to actin) was reduced. PTHrP receptor message levels were also diminished as a function of age. The results indicate that aging may affect the capacity of osteoblasts to respond to a key calcium regulatory hormone. Also delivery of Ca++ to sites of mineralization may be impaired.
GESTATIONAL AND PUERPERAL HIGH RESORPTION OF BONE IS MEDIATED BY CYTOKININS RELEASED BY PERIPHERAL BLOOD MONONUCLEAR CELLS AND BY SOLUBLE IL-6 RECEPTOR RESPECTIVELY
H. Fukuoka, M. Haruna
Department of Developmental Medical Sciences, the University of Tokyo
During pregnancy, high levels of estrogens are thought to protect the maternal bone. The occurence of idiopathic pregnancy-induced osteoporosis is however, increasing, suggesting that pregnancy might induce bone loss. To evaluate the maternal bone metabolism, we studied the metabolic turnover of bone cytokine production by peripheral blood mononuclear cell (PBMC) and serum soluble IL-6 receptor (sIL-6R), with informed patient consent.
Urinary secretion of free deoxypyridinoline (D-Pyr) and serum tartrate-resistant acid phosphatase (TrACP) were analyzed as resorption markers, and intact osteocalcin (I-OC) and bone specific alkaline phosphatase (BALP) as formation markers.
PBMC separated from heparinized blood were incubated for 48 hours, and secreted cytokines, TNF-a and sIL-6R, were measured with a two-site ELISA. During pregnancy, both P-Pyr and TrACP levels continuously increased until term (about 1.6 times higher compared with non-pregnancy), and after delivery remained higher for 6 months when they returned to the basal level. Intact osteocalcin, and bone specific alkaline phosphatase levels however, decreased to about half of non-pregnant levels in the 2nd trimester and returned to the baseline near term, and after delivery both of them increased and kept higher until about one year postpartum. At term, PBMC released higher amounts of TNF-a, compared with those from non-pregnant women, but after delivery these promptly returned to basal levels. After delivery, however, high levels of bone resorption continued for 6 months. The gestational level of sIL-6R remained high with no change until 6 months post-partum, and after that it tended to decrease.
Following these results, we conclude that pregnancy with high estrogen levels paradoxically might stimulate secretion of TNF-a in PBMC and thus induce the specific uncoupling of bone with high resorption and low formation and resultant physiological osteopenea, while high level of sIL-6R might induce the high resorption of the bone in early puerperium which will be followed by increased bone formation.
MOLECULAR CLONING OF FELINE PREPROPARATHYROID HORMONE
R. E. Toribio, C. W. Kohn, D. J. Chew, G. W. Leone, C. C. Capen, T. J. Rosol
The Ohio State University, Columbus, OH, USA
Several pathological conditions of the cat (e.g., hyperparathyroidism, hypoparathyroidism, malignancy, post-parturient hypocalcemia, chronic renal failure, idiopathic chronic hypercalcemia, urolithiasis, urethral obstruction, and sepsis) result in disturbances of calcium metabolism. Measurement of serum parathyroid hormone (PTH) is essential in these conditions. Feline PTH is measured using human-specific immunoassays. However, there is no information on the nucleotide and amino acid sequence of feline PTH. Therefore, we cloned and sequenced the cDNA encoding for feline preproparathyroid hormone. A feline parathyroid gland library was constructed in Lamda ZAP (Stratagene). The library was screened with a 399 bp probe generated by PCR of canine PTH cDNA. Two clones with the cDNA sequence for PTH were isolated. Feline preproPTH cDNA (737bp) consisted of a 63 bp 5' untranslated region, a 348 bp coding region, and a 326 bp 3' untranslated region, including an 18 bp poly (A+) tail. The 348 bp coding region encoded for a 115 aa peptide (preproPTH, MW=12,921 Da). Feline PTH consisted of 84 amino acids (MW=9449 Da). Feline preproPTH and PTH nucleotide and amino acid sequences were compared to sequences of other species using the BLOSUM 62 scoring matrix in Align Plus. Feline mature PTH, PTH (1-84), is preceded by a 6-amino acid ‘pro’ sequence and a 25- amino acid ‘pre’ sequence (signal or leader sequence). Feline PTH has >80% homology to canine, bovine, swine, equine, human, and monkey PTH (approximately 70% to rat and mouse PTH, and 44% to chicken PTH). N-terminal feline PTH (1-34), responsible for the calcium-regulating hormonal activity, has >80% homology amongst mammals and 64% with chicken PTH. Within the PTH (1-34) domain is the principal binding domain, PTH (25-34), which has 90% homology to known forms of mammalian PTH, and 60% homology to chicken PTH. The feline activation domain, PTH (1-6), SVSEIQ, responsible for activation of receptor signal transduction, has 100% homology to canine, swine, equine, human, and monkey, and 83% to bovine, rat, mouse, and chicken PTH (1-6). We concluded that feline PTH is a highly conserved peptide, with the highest homology to canine PTH (89%), and the lowest to mouse PTH (69%). Sequence information was submitted to the NCBI with accession number AF309967.
CLONING OF EQUINE CALCITONIN, CALCITONIN GENE-RELATED PEPTIDE –I, AND CALCITONIN GENE-RELATED PEPTIDE –II cDNAs
R. E. Toribio, C. W. Kohn, G. W. Leone, C. C. Capen, T. J. Rosol
The Ohio State University, Columbus, OH, USA
Increased serum concentrations of calcitonin (CT) and procalcitonin (PCT) are associated with hypocalcemia in patients with sepsis or endotoxemia. As in human beings, the role of CT, PCT, and calcitonin gene-related peptide (CGRP) in the pathogenesis of hypocalcemia in horses with sepsis or endotoxemia associated with enterocolitis is unknown. The cDNAs encoding for equine calcitonin, and equine CGRP-I and CGRP-II were cloned from an equine thyroid gland cDNA library constructed in Lambda TriplEx phage. We also describe a divergent form of CGRP (CGRP-I). Four cDNAs were isolated for equine preprocalcitonin (preproCT), preproCGRP-I and preproCGRP-II. The nucleotide sequence for equine preproCT (835bp) consisted of a 102 bp 5' UTR, a 423 bp coding region for a 140-amino acid protein, and a 310 bp 3' UTR. The predicted amino acid sequence for equine calcitonin (32 aa) has a high degree of homology (>85%) to human, rat and mouse calcitonin. The nucleotide sequence for equine preproCGRP-I (702 bp), consisted of a 102 bp 5' UTR, a 384 bp coding region for a 127 amino acid protein, and a 216 bp 3' UTR. The deduced aa sequence for equine CGRP-I (37 aa) has a low homology (<59%) to CGRPs of other species. The signal and terminal peptides for equine calcitonin and CGRP-I were identical, indicating that these peptides are encoded by a gene equivalent to the human CALC-I gene. The nucleotide sequence for equine preproCGRP-II (960 bp), consisted of a 69 bp 5' UTR, a 390 bp coding region for a 129 amino acid protein, and a 501 bp 3' UTR. The deduced amino acid for equine CGRP-II (37 aa) has a high homology (>80%) to chicken, human, rat, ovine, swine, and bovine CGRPs. The homology between equine CGRP-I and CGRP-II is low (56%). The signal and N-terminal peptides of equine preproCGRP-II has 65% homology with those of equine preproCT and preproCGRP-I, suggesting that CGRP-II is encoded by a gene similar to the human CALC-II gene. The high homology of equine CGRP-II, and the low homology of CGRP-I to other species CGRPs (and equine CGRP-II) were unexpected findings, because the homology of all known CGRP peptides was reported to be high across mammalian. Sequence information was submitted to the NCBI with accession numbers AF249307 (calcitonin), AF257470 (CGRP-II), and AF257471 (CGRP-I). These data will be used to develop horse-specific immunoassays for CT, PCT, and CGRP to investigate their role in the pathogenesis of hypocalcemia associated with enterocolitis.
PARATHYROID POLYHORMONE ACTION ON OVIDUCT MOTILITY IN BIRDS AND MAMMALS
M. Francis, M. Arkle, L. Martin, T. M. Butler, M. C. Cruz, J. F. Brown, C. G. Dacke
Division of Pharmacology, University of Portsmouth, St Michael’s Building, White Swan Road, Portsmouth, PO1 2DT, UK
We have investigated the in vitro effects of parathyroid hormone (bPTH) and parathyroid hormone related peptide (bPTHrP) on the motility of oviduct tissues taken from egglaying Japanese quail (10-15 hours post ovulation) and 4 day pregnant mice. Oviducal/ myometrial tissues from both species showed high levels of contractility when incubated in organ baths with De Jalon’s solution, containing moderate to high levels (0.3-2.5 mM) of Ca++. N-terminal PTH (1-34) and PTHrP(1-40), caused dose related inhibition of contractions over a dose range of 10-9-10-6 M. C-terminal peptides were devoid of such activity. PTH and PTHrP had similar potencies in both avian and 4 day pregnant mouse tissues, however in tissues derived from 9 day pregnant mice, the IC50 for PTH (10-7 M) is approximately three times more sensitive than for PTHrP(1-40) (3x10-7 M). Repeated dosing with the peptides produced marked tachyphyllaxis. The IC50 responses were partially inhibited by a PTH/PTHrP antagonist, PTHrP (7-34) amide. The responses were completely inhibited by the nitric oxide synthase inhibitor Nw-nitro-L-arginine methyl ester (L-NAME, 1mM) in the presence of L-arginine and by 0.1mM L-NAME in he absence of L-arginine. Similar results were obtained with 100 mM and 1 mM concentrations respectively of 7-nitroindazole. We conclude that the relaxant activities of PTH polyhormones are well conserved and involve nitric oxide linked mechanisms.
SKELETAL GROWTH, MODELLING REMODELLING AND METABOLISM DURING MAMMALIAN REPRODUCTION
S. C. Miller
University of Utah, Salt Lake City, UT, USA 84108
Remarkable changes occur in the mammalian skeleton before, during and after the reproductive cycle. Skeletal changes occur with ovarian maturation and initiation of menses and estrus in adolescence, which may result in a greater accumulation of skeletal mineral in the adolescent female vs. the male skeleton. Additionally, an excess skeletal mass is observed in female experimental animals. Some cyclic changes in mineral metabolism are also associated with estrus and menstrual cycles. In early pregnancy growth, modeling and perhaps suppressed remodeling promote the accumulation of calcium. Some changes may also occur with the transition from pituitary to placental control of the pregnancy. In later pregnancy, increase bone turnover appears to coincide with fetal skeletal mineralization. The transition from pregnancy to lactation is acute as the mammary gland rather than the uterus draws on maternal calcium stores. Endochondral growth almost ceases during lactation and envelope-specific bone modeling and remodeling are greatly increased. This is generally associated with a loss of skeletal mass and density, more apparent at sites with less of a mechanical role (e.g. central metaphysis, endocortex). The post-lactational period is profoundly 'anabolic' with substantial increases in bone formation, but blunted resorption at almost all skeletal envelopes. Skeletal mass increases and skeletal structure and mechanics are greatly improved. The resumption of estrus or menses may provide a favorable endocrine milieu.
Important observations include: 1) The nulliparous animal appears to have an excess skeletal mass, perhaps to compensate for maternal metabolic inefficiency of the first reproductive cycle. 2) Changes in growth, modeling and remodeling occur at different times and at different skeletal envelopes during the reproductive cycle. These site-specific, temporal changes appear to be adaptations that facilitate the use of skeletal mineral while preserving mechanical competence. 3) After the first reproductive cycle, modeling and remodeling optimize the existing skeletal mass for the prevailing mechanical environment. 4) Remodeling 'transients' may play important roles during the cycle. 5) The post-lactational period is profoundly anabolic, likely the most anabolic period in the life-history of the adult female skeleton, and may provide new strategies for preservation of skeletal mass when reproductive capacity ceases.
SERUM PARATHYROID HORMONE, IONIZED CALCIUM AND FRACTIONAL URINARY CLEARANCE OF CALCIUM AND PHOSPHORUS IN HORSES WITH ENTEROCOLITIS AND HYPOCALCEMIA
R. E. Toribio, C. W. Kohn, D. J. Chew, R. A. Sams, T. J. Rosol
The Ohio State University, Columbus, OH, USA
Many horses with severe enterocolitis (diarrhea) are hypocalcemic. We hypothesized that horses with enterocolitis and hypocalcemia would have increased serum PTH concentrations. For reasons not yet understood, in humans and animals with certain pathologic conditions (endotoxemia, sepsis, severe burn injury), serum Ca2+ concentration decreases, often resulting in an increase in serum PTH concentrations that restore serum calcium to the physiological range. We investigated the serum concentrations of total calcium (tCa), ionized calcium and magnesium, phosphorus (P) and parathyroid hormone (PTH), and the fractional clearance of calcium (FCa) and phosphorus (FP) in horses with enterocolitis. We compared the use of a human immunochemiluminometric assay (ICMA) with the validated human immunoradiometric assay (IRMA) to measure PTH in horses. Sixty-four horses with enterocolitis and 62 healthy horses were studied. The FCa and FP were determined in 20 normal healthy and 20 horses with enterocolitis. PTH was measured in 40 animals by both IRMA and ICMA. Most horses with enterocolitis had decreased serum tCa, Ca2+ and Mg2+, and increased P concentrations. Horses with hypocalcemia had a wide range of serum PTH concentrations and were divided into 3 groups (nonresponders, n=15, PTH range 0.6 – 21.9 pM; mid-responders, n=26, PTH 23.3 – 121 pM; and high-responders, n=10, PTH 184 – 290 pM). The FCa was decreased and the FP was increased in horses with enterocolitis (P<0.05). There was agreement between the ICMA and the IRMA for measurement of PTH in horses. In conclusion, enterocolitis in horses is often associated with hypocalcemia, and 80% of horses in this study had ionized hypocalcemia. In addition, horses of this study had low FCa, and it is therefore unlikely that renal calcium loss was the cause of hypocalcemia. We believe that impaired calcium mobilization from bone or sequestration/loss of calcium in the gastrointestinal tract, as well as failure of the parathyroid gland to secrete adequate PTH in some horses, are the most likely causes of hypocalcemia in horses with enterocolitis.
EFFECTS OF SWEAT LOSS ON CALCIUM AND MAGNESIUM HOMEOSTASIS IN HORSES
D. Weiss1, D. Burger2, A. Fakler3, U. E. Spichiger3, R. Forrer4, M. Weishaupt5, M. Wanner1, J. L. Riond1
1 Institute of Animal Nutrition, University of Zurich
2 National Studfarm, Avenches
3 Department of Pharmacy, ETH, Zurich
4 Department of Vet. Int. Med., University of Zurich
5 Department of Vet. Surgery, University of Zurich
Prolonged exercise in horses is associated with a major loss of water and electrolytes. Dietary supplementation of Ca and Mg may be required.
Eleven adult stallions underwent on a treadmill a prolonged exercise test which included a short walk followed by 6 trot intervals of each 20 min. Blood samples were collected one hour before the test, after each trot interval and one and two hours during recovery.
The body weight loss was between 19 and 25 kg. Plasma total Ca decreased during the exercise test and increased during recovery whereas ionized Ca remained unchanged. Plasma total Mg decreased continuously during exercise and the first recovery period. Mean plasma total Mg was below the normal range during the last trot interval and the first two hours of recovery. Erythrocyte Mg concentration decreased continuously during the last two trot intervals and during the recovery period. The concentrations of Ca and Mg loss with the sweat was reflected in the significant decrease of plasma total Ca, total Mg and erythrocyte Mg. The decrease was not pronounced most likely because Ca and Mg are mobilized from body stores implying that dietary supplementation is not required. However, repeated long-term exercise could more severely deplete Ca and Mg body stores.
INFLUENCE OF A VEGETARIAN VERSUS A DIET WITH FISH-MEAL ON BONE IN PIGS
A. Liesegang1, E. Bürgi2, M-L. Sassi3, J. Risteli3, M. Wanner1
1 Institute of Animal Nutrition, Zurich, Switzerland
2 Unit for swine medicine, University of Zurich, Zurich, Switzerland
3 Department of Clinical Chemistry, Oulu, Finland
The role of phosphorus and calcium on bone turnover and bone growth is not negligible. Dietary requirements have thus to be properly adjusted in order to ensure optimal bone health. The purpose of this study was to examine if substantial bone loss occurs in weaned pigs by feeding a vegetarian diet in comparison to a diet with fish-meal.
Twelve weaned pigs were assigned to 2 groups: group V (vegetarian diet; 0.66% P and 0.79% Ca in dry matter (DM) until 25 kg and 0.49% P and 1.04% Ca until the end of fattening period), group F (diet with fish-meal; 0.66% P (0.79% Ca) in DM until 25 kg and 0.49% P (1.04% Ca) until the end of fattening period). These two diets were fed to the groups for a period of 6 weeks. Blood samples were collected once a week. Feces were collected three times a week every week. Carboxyterminal telopeptide of type I collagen (ICTP) in serum was used as a marker of bone resorption. Osteocalcin (OC) and bone-specific alkaline phosphatase (bAP) were used as bone formation markers. Bone mineral density (BMD) and content (BMC) were determined by peripheral quantitative computer tomography (pQCT) in the metatarsus and phalanx. In addition, 1,25-dihydroxyvitamin D and parathyroid hormone were measured in serum and the apparent digestitbiliy was calculated.
Significant changes in bAP activities and in OC concentrations did occur with time during the six weeks. There were significant differences between the two groups in bAP activities, but not in OC concentrations. Interestingly, also 1,25-dihydroxyvitamin D as well as PTH concentrations showed significant differences between the groups and with time. In contrast, ICTP concentrations did not increase significantly with time in both groups, but showed significant differences between the two groups. BMC in metatarsus and BMD in phalanx significantly decreased in group V.
In conclusion, the results show that a vegetarian diet induces a minor loss of bone in group V and a compensatory bone formation. Phosphorus and calcium dietary requirements in pigs should be more precisely defined for a better understanding of its effect on bone.
