Parathyroid hormone (PTH)-related protein, PTH, and 1,25-dihydroxyvitamin D in dogs with cancer-associated hypercalcemia.

Circulating N-terminal PTH-related protein (PTHrP), N-terminal PTH, and 1,25-dihydroxyvitamin D [1,25-(OH)2D] concentrations were measured in normal dogs and dogs with cancer-associated hypercalcemia (CAH), parathyroid adenomas, and miscellaneous tumors. PTHrP was undetectable (less than 1.8 pM) in normal dogs and increased in dogs with CAH due to adenocarcinomas derived from apocrine glands of the anal sac (44.9 +/- 27 pM), lymphoma (8.3 +/- 4.4 pM), and miscellaneous carcinomas (13.3 +/- 11.4 pM). The PTHrP concentration decreased in dogs with lymphoma and anal sac adenocarcinomas after successful treatment of CAH. The PTHrP concentration had a significant linear correlation with total serum calcium in dogs with anal sac adenocarcinomas and hypercalcemia, but not in dogs with lymphoma and hypercalcemia. Serum N-terminal PTH concentrations were usually in the normal range (12-34 pg/ml) for all groups of dogs except dogs with parathyroid adenomas (83 +/- 38 pg/ml). The serum PTH concentration increased after successful treatment of CAH. Serum 1,25-(OH)2D concentrations were decreased, normal, or increased in dogs with CAH, and 1,25-(OH)2D levels decreased after treatment of CAH. In summary, circulating concentrations of PTHrP are consistently increased in dogs with CAH, and PTHrP appears to play an important role in the induction of hypercalcemia.

Sequences of the cDNAs encoding canine parathyroid hormone-related protein and parathyroid hormone.

The cDNA clones encoding canine parathyroid-hormone-related protein (cPTHrP) and parathyroid hormone (cPTH) have been isolated and sequenced. The predicted amino-acid sequences of the mature canine homologs have a high degree of homology to human PTHrP (hPTHrP) and PTH (hPTH), especially in the biologically active regions. The cPTHrP cDNA is unique, since it has homology to exon 1A of hPTHrP which suggests that dogs utilize a promoter similar to P1 of hPTHrP which has not been demonstrated in other species.

Altered parathyroid hormone-related protein secretion and mRNA expression in squamous carcinoma cells in vitro.

Parathyroid hormone-related protein (PTHrP), a major factor in the pathogenesis of humoral hypercalcemia of malignancy, is produced by many squamous carcinoma cells (SCCs). Two SCC lines were grown in multilayered culture systems and compared to cells grown as monolayers to evaluate the effects of cell proliferation, confluence and differentiation on PTHrP secretion and mRNA expression. Well-differentiated (SCC 2/88) and poorly differentiated (SCC-A253) SCCs were grown as monolayer and three-dimensional cultures on collagen-coated membranes to compare the regulation of PTHrP expression and secretion by the cell lines in vitro. Parathyroid hormone-related protein secretion was evaluated by radioimmunoassay and immunohistochemistry. Messenger RNA expression was analyzed by RNase protection assay and in situ hybridization. Secretion of PTHrP was greatest in preconfluent SCC 2/88 cells in monolayer culture and decreased after confluence, which was the result of decreased PTHrP mRNA expression. In contrast, PTHrP secretion in cultures of SCC-A253 cells reached maximal levels after confluence. In multilayered cultures, total PTHrP secretion and mRNA expression remained high in both SCC 2/88 and SCC-A253 cells, and secretion by the multi-layered cultures was principally in the basal direction. Parathyroid hormone-related protein was present in all cell layers in three-dimensional cultures as determined by immunohistochemistry. These results indicated that multilayered cultures of SCCs produced PTHrP continuously, whereas decreased proliferation in monolayer cultures was associated with decreased PTHrP production. Multilayered cultures represent a better system to investigate PTHrP secretion and mRNA expression in vitro and PTHrP secretion by SCCs in vivo may be greatest by proliferating cells.

Studies on chicken polyclonal anti-peptide antibodies specific for parathyroid hormone-related protein (1-36).

Chicken polyclonal antibodies were prepared against a synthetic peptide corresponding to the first 36 N-terminal amino acids of parathyroid hormone-related protein (PTHrP) by immunizing laying hens. Significant increases of antibodies to PTHrP were first detected after the second immunization. Production of anti-PTHrP egg yolk antibodies peaked 1-2 weeks after the second through sixth immunizations and declined over a period of 2-4 weeks. Polyclonal IgG (IgY) to PTHrP was purified from the egg yolks with high levels of PTHrP specific binding. The anti-PTHrP IgG was used to develop a radioimmunoassay for PTHrP that was able to detect 100 pg PTHrP ml-1 (23 pM) in conditioned cell culture medium. The anti-PTHrP IgG was bound to a solid phase and utilized to immunopurify iodinated [Tyr36]-PTHrP (1-36). Anti-PTHrP IgG inhibited the in vitro biologic activity of PTHrP as demonstrated by the inhibition of adenylate cyclase stimulation in a rat osteoblast-like cell line (ROS 17/2.8). The anti PTHrP IgG was immunopurified and utilized for immunohistochemical localization of PTHrP in canine skin. Chickens were advantageous in producing large amounts of high affinity, neutralizing antibodies to a highly conserved mammalian protein such as PTHrP. The antibodies will be useful to investigate the function and metabolism of PTHrP in vivo and in vitro.

Humoral hypercalcemia of malignancy associated with ameloblastoma in a horse.

Humoral hypercalcemia of malignancy was evident in a horse that had a locally invasive ameloblastoma of the left hemimandible. Surgical removal of the neoplasm resulted in prompt return of serum calcium and parathyroid hormone concentrations to within reference limits. The tumor contained parathyroid hormone-related protein, as demonstrated by immunohistochemistry and western blot analysis. It is likely that production of this protein by the neoplasm was important in the pathogenesis of the hypercalcemia. The case represented a sporadic form of humoral hypercalcemia of malignancy attributable to an uncommon epithelial neoplasm, and indicated that humoral hypercalcemia of malignancy can develop with neoplasms in horses.

Parathyroid hormone-related protein in normal and neoplastic canine tissues: immunohistochemical localization and biochemical extraction.

Two polyclonal antibodies, directed against N-terminal amino acids (1-36) or the midregion (amino acids 34-53) of parathyroid hormone-related protein (PTHrP), were used to localize PTHrP in a variety of normal and neoplastic canine tissues. Parathyroid hormone (PTH) immunoreactivity was demonstrated using anti-bovine PTH (amino acids 14-34). The following tissues (among others) stained strongly positive for PTHrP: all layers of epidermal keratinocytes, with the most intense staining of the basal layer; hair follicle keratinocytes; myoepithelial cells of dermal apocrine glands, mammary glands, and apocrine glands of the anal sac; anal sac epithelium; mammary duct epithelium; and thyroid C cells. Adenocarcinomas of the anal sac stained moderately positive (5/22 dogs), weakly positive (11/22 dogs), or did not stain (6/22 dogs). Most parathyroid gland adenomas stained moderately (2/6 dogs) or weakly positive (3/6 dogs) for PTHrP. Squamous cell carcinomas (6/6 dogs) stained strongly positive. Lymphomas stained weakly positive (2/10 dogs) or did not stain (8/10 dogs). There was no consistent relationship between the staining intensity of the tumors and serum calcium concentrations of the dogs. The anti-PTH antibodies stained only parathyroid chief cells strongly positive. Concentrations of PTHrP were measured by radioimmunoassay in protein extracts from an adenocarcinoma derived from the apocrine glands of the anal sac, pancreas, kidney, liver, heart, thyroid, adrenal, and parathyroid glands. PTHrP concentrations varied from undetectable up to 150 pg/mg in normal tissues as compared with 2,000 pg/mg in apocrine adenocarcinoma of the anal sac.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum calcitriol and parathyroid hormone levels following prolonged infusion of calcitriol in vitamin D replete and depleted rabbits.

The objectives of this study are (1) to determine if serum calcitriol levels resulting from calcitriol infusion by subcutaneously implanted osmotic pumps are in proportion to dose, (2) to determine if such serum levels remain steady during the "pump-life" and (3) to determine if these increased calcitriol levels have an effect on serum parathyroid hormone (PTH) levels. A general objective of the study is to define a model whereby agents (calcitriol, and parathyroid hormone) suspected of enhancing absorption of metals other than calcium from the gastrointestinal tract could be evaluated individually by the levels of these hormones present in serum at a series of intervals during calcitriol infusion by osmotic pump. Calcitriol was infused into rabbits by subcutaneously implanted osmotic pumps at 3 different dosages (30, 60, and 100 IU/day) for 7 days and one dose (60 IU/day) for a 28 day period. The serum calcitriol levels initially rose markedly in all experimental rabbits in proportion to infused dosage and peaked at 3 days. The quantitative relationship between infused calcitriol and serum calcitriol at 3-5 days showed a correlation coefficient of 0.977 (P less than 0.005) for rabbits receiving the 7 day pumps with the 3 different dosages. There was subsequent decline of serum calcitriol which continued for the remainder of the pump-life, suggesting acceleration of degradation mechanisms. There was a sharp reduction in serum parathyroid hormone (PTH) levels 3-5 days after starting the calcitriol infusion, which was interpreted to result from direct suppression of PTH synthesis by calcitriol and/or the feedback effect of enhanced intestinal calcium absorption. Rabbits which had been depleted of vitamin D prior to implantation of the 28 day osmotic pumps showed a similar pattern of serum calcitriol and PTH. Multiple analyses within a single rabbit showed a reciprocal relationship between the serum calcitriol and PTH during the 28 days. There was a statistically significant negative correlation between these parameters (r = 0.635, P less than 0.05). The data indicate that despite a constant rate of infusion of calcitriol by osmotic pump, the levels of serum calcitriol and parathyroid hormone do not remain constant, but rather undergo marked changes. These findings demonstrate the necessity of monitoring blood levels, even in studies in which animals receive a constant infusion of calcitriol.

Effect of calcitriol infusions on serum aluminum in vitamin D-depleted rabbits fed an aluminum-supplemented ration.

Three dosages of calcitriol (10, 30 and 60 IU/day) were given to rabbits by subcutaneously implanted osmotic pumps. The purpose was to compare the dosages with regard to their putative effect in elevating serum aluminum levels by mechanisms such as enhancing intestinal absorption, diminishing renal excretion, or others. To establish uniform levels of endogenous calcitriol and its precursors, all rabbits had been depleted of vitamin D. The depletion was demonstrated by their serum calcidiol and calcitriol levels declining to unmeasurable levels, following the regimen of a vitamin D-free diet. The 8 rabbits were then placed on an aluminum-supplemented (aluminum lactate) ration. The amount of feed (and aluminum) consumed was determined at daily intervals. Serum aluminum levels were determined at intervals during the 7 days on this regimen. In a second test, the same 8 rabbits received the same regimen but in addition were infused with 10, 30 or 60 IU calcitriol per day. It was found that the aluminum-fed rabbits receiving 60 IU/day and 30 IU/day calcitriol infusions showed statistically significantly elevated serum aluminum levels as compared to their levels without calcitriol (p = 0.0208 and p = 0.434, respectively). Rabbits receiving pumps delivering 10 IU/day while receiving the aluminum-supplemented ration showed no rise in serum aluminum with time or treatment during the 7 day study. Likewise rabbits receiving aluminum-supplemented rations without calcitriol showed only an early minimal rise in mean serum aluminum which returned to the pre-test level by the end of a week in spite of continued consumption of aluminum-supplemented rations.

Comparative effects of calcitriol and parathyroid hormone on serum aluminum in vitamin D-depleted rabbits fed an aluminum-supplemented diet.

Under normal circumstances, the body barriers effectively limit the entry and retention of dietary aluminum. However, both parathyroid hormone (PTH) and calcitriol (physiologically active hormonal form of vitamin D3) have been reported to produce elevation of serum aluminum in animals fed an aluminum-supplemented ration. To compare the effects of calcitriol with those of PTH with reference to their putative effect to enhance aluminum absorption, an experiment was designed wherein the serum levels of both PTH and calcitriol would be changing markedly during a short time-frame. To condition the rabbits used for this comparison, they were fed a vitamin D-free diet, which caused the level of calcitriol and its precursors to decline rapidly. The calcitriol deficit together with the ensuing lack of calcium absorption resulted in a state of secondary hyperparathyroidism. Vitamin D-depletion was shown to be complete by the high level of serum PTH and a low (unmeasurable) level of serum calcitriol. To enable comparison of PTH with calcitriol, exogenous calcitriol infusion (60 IU/day) was started by osmotic pump simultaneously with the beginning of an aluminum (aluminum lactate) supplemented diet. Aliquots were collected for both serum PTH and serum calcitriol at intervals during the 7 day study. A rising serum aluminum level was highly correlated with the rising serum calcitriol level in the rabbits (r = 0.903, p = 0.036) during the first 4 days of the infusion. The mean serum aluminum levels rose nearly 13 parts per billion (ppb) in the 7 day period. Declining serum PTH (due to feedback mechanisms of calcitriol suppressing PTH synthesis) showed a negative correlation of serum aluminum and serum PTH (r = -0.959, p = < 0.01) during the first 4 days of infusion. Control rabbits (vitamin-D depleted) fed aluminum-supplemented rations have shown only a minimal transient rise in serum aluminum level which returned to the pre-test level by the end of the week. To test for any effect of PTH on serum aluminum in the absence of calcitriol, five rabbits were implanted with osmotic pumps infusing PTH (mean 6.0 U/hr) and started on an aluminum supplemented diet. These rabbits, having previously been depleted of vitamin D were already in a state of nutritional secondary hyperparathyroidism as shown by their elevated pretest PTH levels. During the 7 day infusion, the serum aluminum rose only a mean of approximately 1 part per billion (ppb).(ABSTRACT TRUNCATED AT 400 WORDS)

Age-related differences in phosphonoformate-induced bone toxicity in cats.

Phosphonoformate (PFA), a monophosphonate pyrophosphate analog, caused plasma biochemical and bone histomorphologic abnormalities in cats given 1,000 mg/kg/day as a continuous intravenous infusion for 14 days. Plasma biochemical alterations observed in young cats (10 weeks old) treated with PFA included increased calcium and decreased phosphorus, alkaline phosphatase, and calcitriol. Young cats treated with PFA developed rickets-like lesions characterized by widened growth plates, increased osteoid, and failure of mineralization. In addition, area of mineralized trabecular bone was decreased. Osteoclast size was increased whereas osteoclast perimeter and number were unaffected in young PFA-treated cats. Plasma alkaline phosphatase was decreased in adult cats (greater than or equal to 1 year old) treated with PFA but changes in calcium, calcitriol, and immunoreactive parathyroid hormone were highly variable and not significantly different. Adult cats treated with PFA exhibited osteomalacia characterized by increased osteoid area, perimeter, and width with failure of mineralization. In addition, static resorption indices were increased in PFA-treated adult cats but area of mineralized trabecular bone was not decreased. The monophosphonate PFA inhibited bone mineralization in young and adult cats similar to bisphosphonate treatment in other species. Because PFA is currently in phase I trials for use in AIDS, results of this study suggest a need to evaluate patients treated with PFA for metabolic bone disease.

Identification of parathyroid hormone-related protein in canine apocrine adenocarcinoma of the anal sac.

The presence of parathyroid hormone-related protein (PTHrP) in the apocrine adenocarcinoma tumor line (CAC-8) derived from a hypercalcemic dog was demonstrated by western and northern blot analyses. Western blots of CAC-8 tumor extracts revealed a major protein with a molecular weight of approximately 18,000 daltons that cross-reacted with antiserum to human PTHrP. Northern blots demonstrated multiple-sized messenger RNA transcripts in CAC-8 that hybridized to a full-length cDNA probe to human PTHrP. Adenocarcinomas derived from apocrine glands of the anal sac also were stained immunohistochemically for antigens that cross-react with antiserum to human PTHrP. The tumor line (CAC-8) maintained in nude mice stained positively for PTHrP in 13 of 24 tumors. Three of ten apocrine adenocarcinomas from dogs with hypercalcemia stained for PTHrP, whereas zero of ten tumors were positive from normocalcemic dogs. Normal canine epidermal keratinocytes and areas of squamous metaplasia in a perianal gland carcinoma also were positive for PTHrP. These data demonstrated that canine tissues contained a homologue to human PTHrP that likely is important in the pathogenesis of humoral hypercalcemia of malignancy.