Edward F. Voelkel

Thyrocalcitonin: hypocalcemic hypophosphatemic principle of the thyroid gland.

A factor that lowers serum calcium and inorganic phosphate in rats has been purified 500-fold from 0.1N HCl extracts of hog thyroid glands. It is distinct from thyroxine and triiodothyronine and appears to be a polypeptide.

Characterization of beta-adrenergic receptors on rat and human osteoblast-like cells and demonstration that beta-receptor agonists can stimulate bone resorption in organ culture

We have shown by receptor-binding analyses that the beta-2 adrenergic receptor is present on rat ROS 17/2.8 osteoblast-like cells. This was confirmed by PCR amplification of cDNA copied from the mRNA. The beta-1 adrenoreceptor subtype was absent and its mRNA was not detectable, even at the level of sensitivity afforded by PCR analysis. The beta-adrenergic receptors present on ROS 17/2.8 cells were functional as measured by ligand-induced enhancement of cAMP production. We investigated whether adrenergic agonists could mimic the action of PTH to stimulate bone resorption in neonatal mouse calvariae in organ culture. PTH induced a large increase in cAMP while norepinephrine and isoproterenol induced a small but significant increase. In the presence of a phosphodiesterase inhibitor and an antioxidant, norepinephrine consistently stimulated bone resorption. In order to determine whether functional beta-adrenergic receptors were unique to ROS 17/2.8 cells, human SaOS-2 osteoblast-like cells were also examined for enhancement of cAMP production by norepinephrine, and essentially the same results were obtained. Thus, adrenergic agonists efficiently activate beta-receptors on two osteoblast-like cells and can stimulate bone resorption in intact mouse calvariae.

Hypercalcemia and tumor-prostaglandins: The VX2 carcinoma model in the rabbit☆☆☆

The VX2 carcinoma produces profound hypercalcemia (17-22 mg/100 ml) in the rabbit about 3-4 wk after transplantation. A bone resorption-stimulation factor (assayed in vitro with mouse calvaria in culture) has been extracted with diethyl ether from the tumor tissue and from the medium of a clonal strain of VX2 cells grown in culture. Serologic methods reveal that the tumors contain 294 plus or minus 51 ng/g fresh weight (mean plus or minus SE, 25 tumors) of prostaglandin E2 (PGE2), a potent bone resorption-stimulating agent. VX2 cells in culture produce 0.5-3.0 mug PGE2 per mg cell protein per 24 hr. The production of bone resorption-stimulating activity and PGE2 by VX2 cells in culture were both inhibited by indomethacin (100 ng/ml). Tumors from normocalcemic, indomethacin-treated rabbits (10-40 mg/rabbit/24 hr) contained little or no bone resorption-stimulating activity nor PGE2. Tumor-bearing rabbits receiving indomethacin continuously did not develop hypercalcemia, however, following cessation of indomethacin administration, hypercalcemia developed rapidly and was again reversed by reinstitution of indomethacin feeding. In untreated, hypercalcemic, tumor-bearing rabbits, initiation of indomethacin treatment was followed by a rapid return of the plasma calcium to the normal range. Systemic venous plasma from hypercalcemic tumor-bearing plasma contained higher concentrations of PGE2 than plasma from normocalcemic control rabbits. Venous drainage of the tumor contained even higher plasma PGE2 concentrations than systemic venous plasma in hypercalcemic animals; plasma PGE2 concentrations locally and in systemic plasma were unmeasurable (less than 70 pg/ml) in normocalcemic, indomethacin-treated, tumor-bearing rabbits. We conclude that PGE2 is a bone resorption-stimulating factor produced by VX2 tumor cells, and that secretion of PGE2 by the tumor in vivo may well be responsible for the hypercalcemia observed in tumor-bearing rabbits.

Prostaglandin production by mouse fibrosarcoma cells in culture: Inhibition by indomethacin and aspirin

Abstract Five clonal strains of mouse tumor cells (HSDM1) synthesize and secrete large quantities (0.70-2.0 μg/mg cell protein/24 hr) of prostaglandin E2. Five lines of control cells did not synthesize significant amounts of prostaglandins. HSDM1 cells produce prostaglandin E2 during both the logarithmic and stationary phases of the cell growth cycle. Prostaglandin production was inhibited by aspirin-like drugs; for example, 50% inhibition was obtained with as little as 3 × 10−9 M indomethacin. We conclude that the HSDM1 cell system will serve as a useful model system to study prostaglandin synthesis and secretion.

Successful treatment of hypercalcemia by indomethacin in mice bearing a prostaglandin-producing fibrosarcoma

Abstract Mice bearing the HSDM 1 fibrosarcoma have elevated concentrations of calcium and prostaglandin E 2 in serum. Extracts of tumor tissue contain high concentrations of bone resorption-stimulating activity and PGE 2 . Administration of indomethacin to tumor-bearing mice lowers serum calcium and PGE 2 levels, reduces in parallel tumor bone resorption-stimulating activity and PGE 2 content, and diminishes tumor size.

Cyclooxygenase products of arachidonic acid metabolism by mouse bone in organ culture

The products of endogenous and exogenous arachidonic acid metabolism via the cyclooxygenase pathway in mouse bone in organ culture were identified and quantitated by the use of high performance liquid chromatography and radioimmunoassay. Production of prostaglandins E2, F2 alpha, and I2 from endogenous substrate was stimulated by incubation of bone with epidermal growth factor and the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. Addition of arachidonic acid to the culture medium not only resulted in the accumulation of prostaglandins E2, F2 alpha, and I2 but also thromboxane. Bone metabolized prostaglandins E2 and F2 alpha to their respective 13,14-dihydro-15-keto-derivatives. Prostaglandin I2, measured as 6-keto-prostaglandin F1 alpha was synthesized by bone, and metabolic products of prostaglandin I2 or 6-keto-prostaglandin F1 alpha, either 6,15-diketo-prostaglandin F1 alpha or 13,14-dihydro-6,15-diketo-prostaglandin F1 alpha, were also detected in the culture media. Formation of cyclooxygenase products of endogenous and exogenous arachidonic acid metabolism (both basal and stimulated) and bone resorption were inhibited by indomethacin. Bone as a tissue responded biochemically not only to exogenous prostaglandins and agents that enhance endogenous prostaglandin production but also to exogenous arachidonic acid by biosynthesis of prostaglandins, prostacyclin and thromboxane. Furthermore, bone metabolized these cyclooxygenase products to their more stable metabolites.

Human calcitonin: Immunologic assay, cytologic localization and studies on medullary thyroid carcinoma

Abstract Current assay methods do not define the absolute concentrations of calcitonin in plasma of normal man. In our experience, the concentration was

Plasma concentrations of 13,14-dihydro-15-keto-prostaglandin E2 in rabbits bearing the VX2 carcinoma: effects of hydrocortisone and indomethacin.

In rabbits bearing the prostaglandin-producing VX2 carcinoma, the plasma concentration of 13,14-dihydro-15-keto-PGE2 (PGE2-M) was elevated within one week after tumor implantation and preceded the development of hypercalcemia. Both the rate of rise and magnitude of the increase were greater for the metabolite than for PGE2; at the time of peak hyercalcemia (about 4 to 5 weeks after tumor implantation), the increase over basal in plasma PGE2-M was about 75 fold whereas it was previously shown that the increase in PGE2 was less than 2 fold. Indomethacin, which inhibits PGE2 synthesis in VX2 cells in culture, lowered in parallel plasma calcaium and PGE2-M in tumor-bearing rabbits. Administration of hydrocortisone to rabbits bearing the VX2 tumor prevented the development of hypercalcemia when given at the time of tumor implantation and reversed the elevated plasma calcium in previously untreated animals; the steroid hormone also lowered plasma concentrations of PGE2-M. These findings are consistent with our hypothesis that the hypercalcemic syndrome in VX2 tumor-bearing rabbits is due to the secretion of PGE2 by the tumor.

Effects of hydrocortisone on the hypercalcemia and plasma levels of 13,14-dihydro-15-keto-prostaglandin E2 in mice bearing the HSDM1 fibrosarcoma

Abstract In mice bearing the prostaglandin-producing HSDM1 fibrosarcoma, the plasma concentration of 13,14-dihydro-15-keto-PGE2 was elevated before the development of hypercalcemia, and the magnitude of the rise was greater than that of PGE2. When hydrocortisone, which inhibits synthesis of PGE2 by HSDM1 cells in culture, was administered to tumor-bearing mice, the steroid hormone prevented the rises in plasma PGE2 metabolite and calcium concentrations. At the dose levels used, hydrocortisone did not inhibit the calcium-mobilizing action of parathyroid hormone in vivo or the bone resorption-stimulating activity of PGE 2 in vitro . These findings are consistent with our hypothesis that the hypercalcemic syndrome in HSDM1 tumor-bearing mice is due to the secretion of PGE2 by the tumor.

Dietary menhaden oil lowers plasma prostaglandins and calcium in mice bearing the prostaglandin-producing HSDM1 fibrosarcoma.

The omega 3 class of polyunsaturated fatty acids, particularly eicosapentaenoic acid (EPA, 20:5), has been shown to alter the patterns of arachidonic acid (20:4) metabolism in both in vitro and in vivo systems. To examine further the role of arachidonic acid conversion to prostaglandins (PG) in hypercalcemic mice bearing the PG-producing HSDM1 fibrosarcoma, we have performed experiments in which control and tumor-bearing animals were fed diets either low (0.1-0.2% of total fatty acid) or high (17%) in EPA. In all five experiments performed, tumor-bearing mice eating control diets had markedly elevated (average sixfold above control) plasma concentrations of 13,14-dihydro-15-keto-PGE2 (PGE2-M), while in mice bearing HSDM1 tumors and eating the EPA-enriched menhaden oil diet, the elevation was reduced to only twice control values. The increase in plasma calcium concentration (approximately 2.5 mg/dl above control) in tumor-bearing animals was also reduced significantly (P less than 0.05) to only 1.3 mg/dl above control in mice eating the diet enriched in EPA. Plasma immunoreactive hydroxy fatty acids (i12-HETE) and sulfidopeptide leukotrienes (iSRS) were not elevated in tumor-bearing mice and were unaffected by diet. The contents of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha were lower in tumor tissue from animals eating the diet high in EPA, whereas the tissue contents of i12-HETE and iSRS were not altered by diet. Fatty acid analysis of liver and tumor tissue revealed marked increases in certain omega 3 fatty acids (20:5, 22:5, and 22:6) from animals eating the enriched diet. Body weights, tumor weights, and tumor histology were not significantly altered by diet. To determine whether dietary calcium played a role in the elevation of plasma calcium in mice bearing the HSDM1 tumor and the reduction of plasma calcium in animals fed EPA, we compared results in mice fed diets containing 0.80% (normal) and 0.015% (deficient) calcium. The increases in plasma calcium and PGE2-M observed in tumor-bearing mice were the same on both normal and very low calcium intakes. We conclude, in mice of the Swiss albino strain bearing the HSDM1 fibrosarcoma, that consumption of a diet enriched in EPA reduces the production of cyclooxygenase products of arachidonic acid metabolism and thereby reduces the elevation of plasma calcium concentration. Dietary enrichment with EPA did not alter the production of serologically determined lipoxygenase products of arachidonic acid.